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Sample records for synaptic scaffolding protein

  1. Molecular Recognition within Synaptic Scaffolds

    DEFF Research Database (Denmark)

    Erlendsson, Simon

    -length structural model of the PICK1 dimer in-solution. We found the PICK1 BAR dimer to resemble an elongated crescent-shaped structure, spanning ~160 Å, with the PICK1 PDZ domains loosely attached to the BAR domain. This finding is in contrast to previous findings for other BAR domain proteins, where adjacent......Scaffolding proteins are abundant participants and regulators of the extensive intracellular framework required for maintaining cellular functions such as cellular adhesion and signal transduction cascades. In excitatory neuronal synapses these scaffolding proteins often contain one or more PDZ...... domains, responsible for tethering their respective synaptic protein ligands. Therefore, understanding the specificity and binding mechanisms of PDZ domain proteins is essential to understand regulation of synaptic plasticity. PICK1 is a PDZ domain-containing scaffolding protein predominantly expressed...

  2. Shank synaptic scaffold proteins: keys to understanding the pathogenesis of autism and other synaptic disorders.

    Science.gov (United States)

    Sala, Carlo; Vicidomini, Cinzia; Bigi, Ilaria; Mossa, Adele; Verpelli, Chiara

    2015-12-01

    Shank/ProSAP proteins are essential to synaptic formation, development, and function. Mutations in the family of SHANK genes are strongly associated with autism spectrum disorders (ASD) and other neurodevelopmental and neuropsychiatric disorders, such as intellectual disability (ID), and schizophrenia. Thus, the term 'Shankopathies' identifies a number of neuronal diseases caused by alteration of Shank protein expression leading to abnormal synaptic development. With this review we want to summarize the major genetic, molecular, behavior and electrophysiological studies that provide new clues into the function of Shanks and pave the way for the discovery of new therapeutic drugs targeted to treat patients with SHANK mutations and also patients affected by other neurodevelopmental and neuropsychiatric disorders. Shank/ProSAP proteins are essential to synaptic formation, development, and function. Mutations in the family of SHANK genes are strongly associated with autism spectrum disorders (ASD) and other neurodevelopmental and neuropsychiatric disorders, such as intellectual disability (ID), and schizophrenia (SCZ). With this review we want to summarize the major genetic, molecular, behavior and electrophysiological studies that provide new clues into the function of Shanks and pave the way for the discovery of new therapeutic drugs targeted to treat patients with SHANK mutations. © 2015 International Society for Neurochemistry.

  3. Stochastic lattice model of synaptic membrane protein domains

    Science.gov (United States)

    Li, Yiwei; Kahraman, Osman; Haselwandter, Christoph A.

    2017-05-01

    Neurotransmitter receptor molecules, concentrated in synaptic membrane domains along with scaffolds and other kinds of proteins, are crucial for signal transmission across chemical synapses. In common with other membrane protein domains, synaptic domains are characterized by low protein copy numbers and protein crowding, with rapid stochastic turnover of individual molecules. We study here in detail a stochastic lattice model of the receptor-scaffold reaction-diffusion dynamics at synaptic domains that was found previously to capture, at the mean-field level, the self-assembly, stability, and characteristic size of synaptic domains observed in experiments. We show that our stochastic lattice model yields quantitative agreement with mean-field models of nonlinear diffusion in crowded membranes. Through a combination of analytic and numerical solutions of the master equation governing the reaction dynamics at synaptic domains, together with kinetic Monte Carlo simulations, we find substantial discrepancies between mean-field and stochastic models for the reaction dynamics at synaptic domains. Based on the reaction and diffusion properties of synaptic receptors and scaffolds suggested by previous experiments and mean-field calculations, we show that the stochastic reaction-diffusion dynamics of synaptic receptors and scaffolds provide a simple physical mechanism for collective fluctuations in synaptic domains, the molecular turnover observed at synaptic domains, key features of the observed single-molecule trajectories, and spatial heterogeneity in the effective rates at which receptors and scaffolds are recycled at the cell membrane. Our work sheds light on the physical mechanisms and principles linking the collective properties of membrane protein domains to the stochastic dynamics that rule their molecular components.

  4. Stochastic lattice model of synaptic membrane protein domains.

    Science.gov (United States)

    Li, Yiwei; Kahraman, Osman; Haselwandter, Christoph A

    2017-05-01

    Neurotransmitter receptor molecules, concentrated in synaptic membrane domains along with scaffolds and other kinds of proteins, are crucial for signal transmission across chemical synapses. In common with other membrane protein domains, synaptic domains are characterized by low protein copy numbers and protein crowding, with rapid stochastic turnover of individual molecules. We study here in detail a stochastic lattice model of the receptor-scaffold reaction-diffusion dynamics at synaptic domains that was found previously to capture, at the mean-field level, the self-assembly, stability, and characteristic size of synaptic domains observed in experiments. We show that our stochastic lattice model yields quantitative agreement with mean-field models of nonlinear diffusion in crowded membranes. Through a combination of analytic and numerical solutions of the master equation governing the reaction dynamics at synaptic domains, together with kinetic Monte Carlo simulations, we find substantial discrepancies between mean-field and stochastic models for the reaction dynamics at synaptic domains. Based on the reaction and diffusion properties of synaptic receptors and scaffolds suggested by previous experiments and mean-field calculations, we show that the stochastic reaction-diffusion dynamics of synaptic receptors and scaffolds provide a simple physical mechanism for collective fluctuations in synaptic domains, the molecular turnover observed at synaptic domains, key features of the observed single-molecule trajectories, and spatial heterogeneity in the effective rates at which receptors and scaffolds are recycled at the cell membrane. Our work sheds light on the physical mechanisms and principles linking the collective properties of membrane protein domains to the stochastic dynamics that rule their molecular components.

  5. Mutations in the gene encoding the synaptic scaffolding protein SHANK3 are associated with autism spectrum disorders

    Science.gov (United States)

    Durand, Christelle M.; Betancur, Catalina; Boeckers, Tobias M.; Bockmann, Juergen; Chaste, Pauline; Fauchereau, Fabien; Nygren, Gudrun; Rastam, Maria; Gillberg, I Carina; Anckarsäter, Henrik; Sponheim, Eili; Goubran-Botros, Hany; Delorme, Richard; Chabane, Nadia; Mouren-Simeoni, Marie-Christine; de Mas, Philippe; Bieth, Eric; Rogé, Bernadette; Héron, Delphine; Burglen, Lydie; Gillberg, Christopher; Leboyer, Marion; Bourgeron, Thomas

    2007-01-01

    SHANK3 (also known as ProSAP2) regulates the structural organization of dendritic spines and is a binding partner of neuroligins; genes encoding neuroligins are mutated in autism and Asperger syndrome. Here, we report that a mutation of a single copy of SHANK3 on chromosome 22q13 can result in language and/or social communication disorders. These mutations concern only a small number of individuals, but they shed light on one gene dosage-sensitive synaptic pathway that is involved in autism spectrum disorders. PMID:17173049

  6. Olfactory receptor signaling is regulated by the post-synaptic density 95, Drosophila discs large, zona-occludens 1 (PDZ) scaffold multi-PDZ domain protein 1.

    LENUS (Irish Health Repository)

    Dooley, Ruth

    2009-12-01

    The unique ability of mammals to detect and discriminate between thousands of different odorant molecules is governed by the diverse array of olfactory receptors expressed by olfactory sensory neurons in the nasal epithelium. Olfactory receptors consist of seven transmembrane domain G protein-coupled receptors and comprise the largest gene superfamily in the mammalian genome. We found that approximately 30% of olfactory receptors possess a classical post-synaptic density 95, Drosophila discs large, zona-occludens 1 (PDZ) domain binding motif in their C-termini. PDZ domains have been established as sites for protein-protein interaction and play a central role in organizing diverse cell signaling assemblies. In the present study, we show that multi-PDZ domain protein 1 (MUPP1) is expressed in the apical compartment of olfactory sensory neurons. Furthermore, on heterologous co-expression with olfactory sensory neurons, MUPP1 was shown to translocate to the plasma membrane. We found direct interaction of PDZ domains 1 + 2 of MUPP1 with the C-terminus of olfactory receptors in vitro. Moreover, the odorant-elicited calcium response of OR2AG1 showed a prolonged decay in MUPP1 small interfering RNA-treated cells. We have therefore elucidated the first building blocks of the putative \\'olfactosome\\

  7. Stochastic single-molecule dynamics of synaptic membrane protein domains

    Science.gov (United States)

    Kahraman, Osman; Li, Yiwei; Haselwandter, Christoph A.

    2016-09-01

    Motivated by single-molecule experiments on synaptic membrane protein domains, we use a stochastic lattice model to study protein reaction and diffusion processes in crowded membranes. We find that the stochastic reaction-diffusion dynamics of synaptic proteins provide a simple physical mechanism for collective fluctuations in synaptic domains, the molecular turnover observed at synaptic domains, key features of the single-molecule trajectories observed for synaptic proteins, and spatially inhomogeneous protein lifetimes at the cell membrane. Our results suggest that central aspects of the single-molecule and collective dynamics observed for membrane protein domains can be understood in terms of stochastic reaction-diffusion processes at the cell membrane.

  8. Metabolic Turnover of Synaptic Proteins: Kinetics, Interdependencies and Implications for Synaptic Maintenance

    Science.gov (United States)

    Cohen, Laurie D.; Zuchman, Rina; Sorokina, Oksana; Müller, Anke; Dieterich, Daniela C.; Armstrong, J. Douglas; Ziv, Tamar; Ziv, Noam E.

    2013-01-01

    Chemical synapses contain multitudes of proteins, which in common with all proteins, have finite lifetimes and therefore need to be continuously replaced. Given the huge numbers of synaptic connections typical neurons form, the demand to maintain the protein contents of these connections might be expected to place considerable metabolic demands on each neuron. Moreover, synaptic proteostasis might differ according to distance from global protein synthesis sites, the availability of distributed protein synthesis facilities, trafficking rates and synaptic protein dynamics. To date, the turnover kinetics of synaptic proteins have not been studied or analyzed systematically, and thus metabolic demands or the aforementioned relationships remain largely unknown. In the current study we used dynamic Stable Isotope Labeling with Amino acids in Cell culture (SILAC), mass spectrometry (MS), Fluorescent Non–Canonical Amino acid Tagging (FUNCAT), quantitative immunohistochemistry and bioinformatics to systematically measure the metabolic half-lives of hundreds of synaptic proteins, examine how these depend on their pre/postsynaptic affiliation or their association with particular molecular complexes, and assess the metabolic load of synaptic proteostasis. We found that nearly all synaptic proteins identified here exhibited half-lifetimes in the range of 2–5 days. Unexpectedly, metabolic turnover rates were not significantly different for presynaptic and postsynaptic proteins, or for proteins for which mRNAs are consistently found in dendrites. Some functionally or structurally related proteins exhibited very similar turnover rates, indicating that their biogenesis and degradation might be coupled, a possibility further supported by bioinformatics-based analyses. The relatively low turnover rates measured here (∼0.7% of synaptic protein content per hour) are in good agreement with imaging-based studies of synaptic protein trafficking, yet indicate that the metabolic load

  9. Systematic Prediction of Scaffold Proteins Reveals New Design Principles in Scaffold-Mediated Signal Transduction

    Science.gov (United States)

    Hu, Jianfei; Neiswinger, Johnathan; Zhang, Jin; Zhu, Heng; Qian, Jiang

    2015-01-01

    Scaffold proteins play a crucial role in facilitating signal transduction in eukaryotes by bringing together multiple signaling components. In this study, we performed a systematic analysis of scaffold proteins in signal transduction by integrating protein-protein interaction and kinase-substrate relationship networks. We predicted 212 scaffold proteins that are involved in 605 distinct signaling pathways. The computational prediction was validated using a protein microarray-based approach. The predicted scaffold proteins showed several interesting characteristics, as we expected from the functionality of scaffold proteins. We found that the scaffold proteins are likely to interact with each other, which is consistent with previous finding that scaffold proteins tend to form homodimers and heterodimers. Interestingly, a single scaffold protein can be involved in multiple signaling pathways by interacting with other scaffold protein partners. Furthermore, we propose two possible regulatory mechanisms by which the activity of scaffold proteins is coordinated with their associated pathways through phosphorylation process. PMID:26393507

  10. Recombinant protein scaffolds for tissue engineering

    International Nuclear Information System (INIS)

    Werkmeister, Jerome A; Ramshaw, John A M

    2012-01-01

    New biological materials for tissue engineering are now being developed using common genetic engineering capabilities to clone and express a variety of genetic elements that allow cost-effective purification and scaffold fabrication from these recombinant proteins, peptides or from chimeric combinations of these. The field is limitless as long as the gene sequences are known. The utility is dependent on the ease, product yield and adaptability of these protein products to the biomedical field. The development of recombinant proteins as scaffolds, while still an emerging technology with respect to commercial products, is scientifically superior to current use of natural materials or synthetic polymer scaffolds, in terms of designing specific structures with desired degrees of biological complexities and motifs. In the field of tissue engineering, next generation scaffolds will be the key to directing appropriate tissue regeneration. The initial period of biodegradable synthetic scaffolds that provided shape and mechanical integrity, but no biological information, is phasing out. The era of protein scaffolds offers distinct advantages, particularly with the combination of powerful tools of molecular biology. These include, for example, the production of human proteins of uniform quality that are free of infectious agents and the ability to make suitable quantities of proteins that are found in low quantity or are hard to isolate from tissue. For the particular needs of tissue engineering scaffolds, fibrous proteins like collagens, elastin, silks and combinations of these offer further advantages of natural well-defined structural scaffolds as well as endless possibilities of controlling functionality by genetic manipulation. (topical review)

  11. Arc protein: a flexible hub for synaptic plasticity and cognition.

    Science.gov (United States)

    Nikolaienko, Oleksii; Patil, Sudarshan; Eriksen, Maria Steene; Bramham, Clive R

    2017-09-07

    Mammalian excitatory synapses express diverse types of synaptic plasticity. A major challenge in neuroscience is to understand how a neuron utilizes different types of plasticity to sculpt brain development, function, and behavior. Neuronal activity-induced expression of the immediate early protein, Arc, is critical for long-term potentiation and depression of synaptic transmission, homeostatic synaptic scaling, and adaptive functions such as long-term memory formation. However, the molecular basis of Arc protein function as a regulator of synaptic plasticity and cognition remains a puzzle. Recent work on the biophysical and structural properties of Arc, its protein-protein interactions and post-translational modifications have shed light on the issue. Here, we present Arc protein as a flexible, multifunctional and interactive hub. Arc interacts with specific effector proteins in neuronal compartments (dendritic spines, nuclear domains) to bidirectionally regulate synaptic strength by distinct molecular mechanisms. Arc stability, subcellular localization, and interactions are dictated by synaptic activity and post-translational modification of Arc. This functional versatility and context-dependent signaling supports a view of Arc as a highly specialized master organizer of long-term synaptic plasticity, critical for information storage and cognition. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  12. Protein Scaffolding for Small Molecule Catalysts

    Energy Technology Data Exchange (ETDEWEB)

    Baker, David [Univ. of Washington, Seattle, WA (United States)

    2014-09-14

    We aim to design hybrid catalysts for energy production and storage that combine the high specificity, affinity, and tunability of proteins with the potent chemical reactivities of small organometallic molecules. The widely used Rosetta and RosettaDesign methodologies will be extended to model novel protein / small molecule catalysts in which one or many small molecule active centers are supported and coordinated by protein scaffolding. The promise of such hybrid molecular systems will be demonstrated with the nickel-phosphine hydrogenase of DuBois et. al.We will enhance the hydrogenase activity of the catalyst by designing protein scaffolds that incorporate proton relays and systematically modulate the local environment of the catalyticcenter. In collaboration with DuBois and Shaw, the designs will be experimentally synthesized and characterized.

  13. BDNF-induced local protein synthesis and synaptic plasticity.

    Science.gov (United States)

    Leal, Graciano; Comprido, Diogo; Duarte, Carlos B

    2014-01-01

    Brain-derived neurotrophic factor (BDNF) is an important regulator of synaptic transmission and long-term potentiation (LTP) in the hippocampus and in other brain regions, playing a role in the formation of certain forms of memory. The effects of BDNF in LTP are mediated by TrkB (tropomyosin-related kinase B) receptors, which are known to be coupled to the activation of the Ras/ERK, phosphatidylinositol 3-kinase/Akt and phospholipase C-γ (PLC-γ) pathways. The role of BDNF in LTP is best studied in the hippocampus, where the neurotrophin acts at pre- and post-synaptic levels. Recent studies have shown that BDNF regulates the transport of mRNAs along dendrites and their translation at the synapse, by modulating the initiation and elongation phases of protein synthesis, and by acting on specific miRNAs. Furthermore, the effect of BDNF on transcription regulation may further contribute to long-term changes in the synaptic proteome. In this review we discuss the recent progress in understanding the mechanisms contributing to the short- and long-term regulation of the synaptic proteome by BDNF, and the role in synaptic plasticity, which is likely to influence learning and memory formation. This article is part of the Special Issue entitled 'BDNF Regulation of Synaptic Structure, Function, and Plasticity'. Copyright © 2013 Elsevier Ltd. All rights reserved.

  14. Synaptic proteins and receptors defects in autism spectrum disorders

    Directory of Open Access Journals (Sweden)

    Jianling eChen

    2014-09-01

    Full Text Available Recent studies have found that hundreds of genetic variants, including common and rare variants, rare and de novo mutations, and common polymorphisms have contributed to the occurrence of autism spectrum disorders (ASDs. The mutations in a number of genes such as neurexin, neuroligin, postsynaptic density protein 95 (PSD-95, SH3 and multiple ankyrin repeat domains 3 (SHANK3, synapsin, gephyrin, cadherin (CDH and protocadherin (PCDH, thousand-and-one-amino acid 2 kinase (TAOK2, and contactin (CNTN, have been shown to play important roles in the development and function of synapses. In addition, synaptic receptors, such as gamma-aminobutyric acid (GABA receptors and glutamate receptors, have also been associated with ASDs. This review will primarily focus on the defects of synaptic proteins and receptors associated with ASDs and their roles in the pathogenesis of ASDs via synaptic pathways.

  15. Isoform Specificity of Protein Kinase Cs in Synaptic Plasticity

    Science.gov (United States)

    Sossin, Wayne S.

    2007-01-01

    Protein kinase Cs (PKCs) are implicated in many forms of synaptic plasticity. However, the specific isoform(s) of PKC that underlie(s) these events are often not known. We have used "Aplysia" as a model system in order to investigate the isoform specificity of PKC actions due to the presence of fewer isoforms and a large number of documented…

  16. Membrane-mediated interaction between strongly anisotropic protein scaffolds.

    Directory of Open Access Journals (Sweden)

    Yonatan Schweitzer

    2015-02-01

    Full Text Available Specialized proteins serve as scaffolds sculpting strongly curved membranes of intracellular organelles. Effective membrane shaping requires segregation of these proteins into domains and is, therefore, critically dependent on the protein-protein interaction. Interactions mediated by membrane elastic deformations have been extensively analyzed within approximations of large inter-protein distances, small extents of the protein-mediated membrane bending and small deviations of the protein shapes from isotropic spherical segments. At the same time, important classes of the realistic membrane-shaping proteins have strongly elongated shapes with large and highly anisotropic curvature. Here we investigated, computationally, the membrane mediated interaction between proteins or protein oligomers representing membrane scaffolds with strongly anisotropic curvature, and addressed, quantitatively, a specific case of the scaffold geometrical parameters characterizing BAR domains, which are crucial for membrane shaping in endocytosis. In addition to the previously analyzed contributions to the interaction, we considered a repulsive force stemming from the entropy of the scaffold orientation. We computed this interaction to be of the same order of magnitude as the well-known attractive force related to the entropy of membrane undulations. We demonstrated the scaffold shape anisotropy to cause a mutual aligning of the scaffolds and to generate a strong attractive interaction bringing the scaffolds close to each other to equilibrium distances much smaller than the scaffold size. We computed the energy of interaction between scaffolds of a realistic geometry to constitute tens of kBT, which guarantees a robust segregation of the scaffolds into domains.

  17. SynProt: A Database for Proteins of Detergent-Resistant Synaptic Protein Preparations

    Science.gov (United States)

    Pielot, Rainer; Smalla, Karl-Heinz; Müller, Anke; Landgraf, Peter; Lehmann, Anne-Christin; Eisenschmidt, Elke; Haus, Utz-Uwe; Weismantel, Robert; Gundelfinger, Eckart D.; Dieterich, Daniela C.

    2012-01-01

    Chemical synapses are highly specialized cell–cell contacts for communication between neurons in the CNS characterized by complex and dynamic protein networks at both synaptic membranes. The cytomatrix at the active zone (CAZ) organizes the apparatus for the regulated release of transmitters from the presynapse. At the postsynaptic side, the postsynaptic density constitutes the machinery for detection, integration, and transduction of the transmitter signal. Both pre- and postsynaptic protein networks represent the molecular substrates for synaptic plasticity. Their function can be altered both by regulating their composition and by post-translational modification of their components. For a comprehensive understanding of synaptic networks the entire ensemble of synaptic proteins has to be considered. To support this, we established a comprehensive database for synaptic junction proteins (SynProt database) primarily based on proteomics data obtained from biochemical preparations of detergent-resistant synaptic junctions. The database currently contains 2,788 non-redundant entries of rat, mouse, and some human proteins, which mainly have been manually extracted from 12 proteomic studies and annotated for synaptic subcellular localization. Each dataset is completed with manually added information including protein classifiers as well as automatically retrieved and updated information from public databases (UniProt and PubMed). We intend that the database will be used to support modeling of synaptic protein networks and rational experimental design. PMID:22737123

  18. Interactome of invadopodia scaffold protein TKS5

    Directory of Open Access Journals (Sweden)

    Kropyvko S. V.

    2015-12-01

    Full Text Available TKS5 is a scaffold protein that takes part in invadopodia functioning and reactive oxygen species (ROS production. TKS5 is a critical component of invadopodia as its absence results in the loss of cancer cells ability to form these invasive structures. TKS5 is phosphorylated by SRC kinase and consequently interacts with the membrane phosphatidylinositol phosphates launching the invadopodia formation process. At later stages TKS5 regulates the actin cytoskeleton reorganization and extracellular matrix degradation. TKS5 also regulates the production of ROS, which are the important signal regulators of different cellular functions.

  19. Soy Protein Scaffold Biomaterials for Tissue Engineering and Regenerative Medicine

    Science.gov (United States)

    Chien, Karen B.

    Developing functional biomaterials using highly processable materials with tailorable physical and bioactive properties is an ongoing challenge in tissue engineering. Soy protein is an abundant, natural resource with potential use for regenerative medicine applications. Preliminary studies show that soy protein can be physically modified and fabricated into various biocompatible constructs. However, optimized soy protein structures for tissue regeneration (i.e. 3D porous scaffolds) have not yet been designed. Furthermore, little work has established the in vivo biocompatibility of implanted soy protein and the benefit of using soy over other proteins including FDA-approved bovine collagen. In this work, freeze-drying and 3D printing fabrication processes were developed using commercially available soy protein to create porous scaffolds that improve cell growth and infiltration compared to other soy biomaterials previously reported. Characterization of scaffold structure, porosity, and mechanical/degradation properties was performed. In addition, the behavior of human mesenchymal stem cells seeded on various designed soy scaffolds was analyzed. Biological characterization of the cell-seeded scaffolds was performed to assess feasibility for use in liver tissue regeneration. The acute and humoral response of soy scaffolds implanted in an in vivo mouse subcutaneous model was also investigated. All fabricated soy scaffolds were modified using thermal, chemical, and enzymatic crosslinking to change properties and cell growth behavior. 3D printing allowed for control of scaffold pore size and geometry. Scaffold structure, porosity, and degradation rate significantly altered the in vivo response. Freeze-dried soy scaffolds had similar biocompatibility as freeze-dried collagen scaffolds of the same protein content. However, the soy scaffolds degraded at a much faster rate, minimizing immunogenicity. Interestingly, subcutaneously implanted soy scaffolds affected blood

  20. Engineering protein scaffolds for protein separation, biocatalysis and nanotechnology applications

    Science.gov (United States)

    Liu, Fang

    Globally, there is growing appreciation for developing a sustainable economy that uses eco-efficient bio-processes. Biotechnology provides an increasing range of tools for industry to help reduce cost and improve environmental performance. Inspired by the naturally evolved machineries of protein scaffolds and their binding ligands, synthetic protein scaffolds were engineered based on cohesin-dockerin interactions and metal chelating peptides to tackle the challenges and make improvements in three specific areas: (1) protein purification, (2) biofuel cells, and (3) nanomaterial synthesis. The first objective was to develop efficient and cost-effective non-chromatographic purification processes to purify recombinant proteins in an effort to meet the dramatically growing market of protein drugs. In our design, the target protein was genetically fused with a dockerin domain from Clostridium thermocellum and direct purification and recovery was achieved using thermo-responsive elastin-like polypeptide (ELP) scaffold containing the cohesin domain from the same species. By exploiting the highly specific interaction between the dockerin and cohesin domain and the reversible aggregation property of ELP, highly purified and active dockerin-tagged proteins, such as endoglucanase CelA, chloramphenicol acetyl transferase (CAT) and enhanced green fluorescence protein (EGFP), were recovered directly from crude cell extracts in a single purification step with yields achieving over 90%. Incorporation of a self-cleaving intein domain enabled rapid removal of the affinity tag from the target proteins by another cycle of thermal precipitation. The purification cost can be further reduced by regenerating and recycling the ELP-cohesin capturing scaffolds. However, due to the high binding affinity between cohesin and dockerin domains, the bound dockerin-intein tag cannot be completely disassociated from ELP-cohesin scaffold after binding. Therefore, a truncated dockerin with the calcium

  1. Roles for Regulator of G Protein Signaling Proteins in Synaptic Signaling and Plasticity.

    Science.gov (United States)

    Gerber, Kyle J; Squires, Katherine E; Hepler, John R

    2016-02-01

    The regulator of G protein signaling (RGS) family of proteins serves critical roles in G protein-coupled receptor (GPCR) and heterotrimeric G protein signal transduction. RGS proteins are best understood as negative regulators of GPCR/G protein signaling. They achieve this by acting as GTPase activating proteins (GAPs) for Gα subunits and accelerating the turnoff of G protein signaling. Many RGS proteins also bind additional signaling partners that either regulate their functions or enable them to regulate other important signaling events. At neuronal synapses, GPCRs, G proteins, and RGS proteins work in coordination to regulate key aspects of neurotransmitter release, synaptic transmission, and synaptic plasticity, which are necessary for central nervous system physiology and behavior. Accumulating evidence has revealed key roles for specific RGS proteins in multiple signaling pathways at neuronal synapses, regulating both pre- and postsynaptic signaling events and synaptic plasticity. Here, we review and highlight the current knowledge of specific RGS proteins (RGS2, RGS4, RGS7, RGS9-2, and RGS14) that have been clearly demonstrated to serve critical roles in modulating synaptic signaling and plasticity throughout the brain, and we consider their potential as future therapeutic targets. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  2. Deletion of PTEN produces autism-like behavioral deficits and alterations in synaptic proteins

    Directory of Open Access Journals (Sweden)

    Joaquin N Lugo

    2014-04-01

    Full Text Available Many genes have been implicated in the underlying cause of autism but each gene accounts for only a small fraction of those diagnosed with autism. There is increasing evidence that activity-dependent changes in neuronal signaling could act as a convergent mechanism for many of the changes in synaptic proteins. One candidate signaling pathway that may have a critical role in autism is the PI3K/AKT/mTOR pathway. A major regulator of this pathway is the negative repressor phosphatase and tensin homolog (PTEN. In the current study we examined the behavioral and molecular consequences in mice with neuron subset-specific deletion of PTEN.The knockout (KO mice showed deficits in social chamber and social partition test. KO mice demonstrated alterations in repetitive behavior, as measured in the marble burying test and hole-board test. They showed no changes in ultrasonic vocalizations emitted on postnatal day 10 or 12 compared to wildtype (WT mice. They exhibited less anxiety in the elevated-plus maze test and were more active in the open field test compared to WT mice. In addition to the behavioral alterations, KO mice had elevation of phosphorylated AKT, phosphorylated S6, and an increase in S6K. KO mice had a decrease in mGluR but an increase in total and phosphorylated fragile x mental retardation protein. The disruptions in intracellular signaling may be why the KO mice had a decrease in the dendritic potassium channel Kv4.2 and a decrease in the synaptic scaffolding proteins PSD-95 and SAP102. These findings demonstrate that deletion of PTEN results in long-term alterations in social behavior, repetitive behavior, activity, and anxiety. In addition, deletion of PTEN significantly alters mGluR signaling and many synaptic proteins in the hippocampus. Our data demonstrates that deletion of PTEN can result in many of the behavioral features of autism and may provide insights into the regulation of intracellular signaling on synaptic proteins.

  3. Sleep and protein synthesis-dependent synaptic plasticity: impacts of sleep loss and stress

    Science.gov (United States)

    Grønli, Janne; Soulé, Jonathan; Bramham, Clive R.

    2014-01-01

    Sleep has been ascribed a critical role in cognitive functioning. Several lines of evidence implicate sleep in the consolidation of synaptic plasticity and long-term memory. Stress disrupts sleep while impairing synaptic plasticity and cognitive performance. Here, we discuss evidence linking sleep to mechanisms of protein synthesis-dependent synaptic plasticity and synaptic scaling. We then consider how disruption of sleep by acute and chronic stress may impair these mechanisms and degrade sleep function. PMID:24478645

  4. Comparing development of synaptic proteins in rat visual, somatosensory, and frontal cortex

    Science.gov (United States)

    Pinto, Joshua G. A.; Jones, David G.; Murphy, Kathryn M.

    2013-01-01

    Two theories have influenced our understanding of cortical development: the integrated network theory, where synaptic development is coordinated across areas; and the cascade theory, where the cortex develops in a wave-like manner from sensory to non-sensory areas. These different views on cortical development raise challenges for current studies aimed at comparing detailed maturation of the connectome among cortical areas. We have taken a different approach to compare synaptic development in rat visual, somatosensory, and frontal cortex by measuring expression of pre-synaptic (synapsin and synaptophysin) proteins that regulate vesicle cycling, and post-synaptic density (PSD-95 and Gephyrin) proteins that anchor excitatory or inhibitory (E-I) receptors. We also compared development of the balances between the pairs of pre- or post-synaptic proteins, and the overall pre- to post-synaptic balance, to address functional maturation and emergence of the E-I balance. We found that development of the individual proteins and the post-synaptic index overlapped among the three cortical areas, but the pre-synaptic index matured later in frontal cortex. Finally, we applied a neuroinformatics approach using principal component analysis and found that three components captured development of the synaptic proteins. The first component accounted for 64% of the variance in protein expression and reflected total protein expression, which overlapped among the three cortical areas. The second component was gephyrin and the E-I balance, it emerged as sequential waves starting in somatosensory, then frontal, and finally visual cortex. The third component was the balance between pre- and post-synaptic proteins, and this followed a different developmental trajectory in somatosensory cortex. Together, these results give the most support to an integrated network of synaptic development, but also highlight more complex patterns of development that vary in timing and end point among the

  5. Comparing development of synaptic proteins in rat visual, somatosensory, and frontal cortex.

    Science.gov (United States)

    Pinto, Joshua G A; Jones, David G; Murphy, Kathryn M

    2013-01-01

    Two theories have influenced our understanding of cortical development: the integrated network theory, where synaptic development is coordinated across areas; and the cascade theory, where the cortex develops in a wave-like manner from sensory to non-sensory areas. These different views on cortical development raise challenges for current studies aimed at comparing detailed maturation of the connectome among cortical areas. We have taken a different approach to compare synaptic development in rat visual, somatosensory, and frontal cortex by measuring expression of pre-synaptic (synapsin and synaptophysin) proteins that regulate vesicle cycling, and post-synaptic density (PSD-95 and Gephyrin) proteins that anchor excitatory or inhibitory (E-I) receptors. We also compared development of the balances between the pairs of pre- or post-synaptic proteins, and the overall pre- to post-synaptic balance, to address functional maturation and emergence of the E-I balance. We found that development of the individual proteins and the post-synaptic index overlapped among the three cortical areas, but the pre-synaptic index matured later in frontal cortex. Finally, we applied a neuroinformatics approach using principal component analysis and found that three components captured development of the synaptic proteins. The first component accounted for 64% of the variance in protein expression and reflected total protein expression, which overlapped among the three cortical areas. The second component was gephyrin and the E-I balance, it emerged as sequential waves starting in somatosensory, then frontal, and finally visual cortex. The third component was the balance between pre- and post-synaptic proteins, and this followed a different developmental trajectory in somatosensory cortex. Together, these results give the most support to an integrated network of synaptic development, but also highlight more complex patterns of development that vary in timing and end point among the

  6. SynProt: A Comprehensive Database for Proteins of the Detergent-Resistant Synaptic Junctions Fraction

    Directory of Open Access Journals (Sweden)

    Rainer ePielot

    2012-06-01

    Full Text Available Chemical synapses are highly specialized cell-cell contacts for communication between neurons in the CNS characterized by complex and dynamic protein networks at both synaptic membranes. The cytomatrix at the active zone (CAZ organizes the apparatus for the regulated release of transmitters from the presynapse. At the postsynaptic side, the postsynaptic density constitutes the machinery for detection, integration and transduction of the transmitter signal. Both pre- and postsynaptic protein networks represent the molecular substrates for synaptic plasticity. Their function can be altered both by regulating their composition and by post-translational modification of their components. For a comprehensive understanding of synaptic networks the entire ensemble of synaptic proteins has to be considered. To support this, we established a comprehensive database for synaptic junction proteins (SynProt database primarily based on proteomics data obtained from biochemical preparations of detergent-resistant synaptic junctions. The database currently contains 2,788 non-redundant entries of rat, mouse and some human proteins, which mainly have been manually extracted from twelve proteomic studies and annotated for synaptic subcellular localization. Each dataset is completed with manually added information including protein classifiers as well as automatically retrieved and updated information from public databases (UniProt and PubMed. We intend that the database will be used to support modeling of synaptic protein networks and rational experimental design.

  7. Thermoreversible protein hydrogel as cell scaffold.

    Science.gov (United States)

    Yan, Hui; Saiani, Alberto; Gough, Julie E; Miller, Aline F

    2006-10-01

    A thermoreversible fibrillar hydrogel has been formed from an aqueous lysozyme solution in the presence of dithiothreitol (DTT). Its physical properties and potential as a tissue engineering scaffold have been explored. Hydrogels were prepared by dissolving 3 mM protein in a 20 mM DTT/water mixture, heating to 85 degrees C and cooling at room temperature. No gel was observed for the equivalent sample without DTT. The elastic nature of the gel formed was confirmed by rheology, and the storage modulus of our gel was found to be of the same order of magnitude as for other cross-linked biopolymers. Micro differential scanning calorimetry (microDSC) experiments confirmed that the hydrogel was thermally reversible and that gelation and melting occurs through a solid-liquid-like first-order transition. Infrared spectroscopy of the hydrogel and transmission electron microscopy studies of very dilute samples revealed the presence of beta-sheet-rich fibrils that were approximately 4-6 nm in diameter and 1 mum in length. These fibrils are thought to self-assemble along their long axes to form larger fibers that become physically entangled to form the three-dimensional network observed in both cryo-scanning electron microscopy (cryo-SEM) and small-angle neutron scattering (SANS) studies. The hydrogel was subsequently cultured with 3T3 fibroblasts and cells spread extensively after 7 days and stretched actin filaments formed that were roughly parallel to each other, indicating the development of organized actin filaments in the form of stress fibers in cells.

  8. Novel blood protein based scaffolds for cardiovascular tissue engineering

    Directory of Open Access Journals (Sweden)

    Kuhn Antonia I.

    2016-09-01

    Full Text Available A major challenge in cardiovascular tissue engineering is the fabrication of scaffolds, which provide appropriate morphological and mechanical properties while avoiding undesirable immune reactions. In this study electrospinning was used to fabricate scaffolds out of blood proteins for cardiovascular tissue engineering. Lyophilised porcine plasma was dissolved in deionised water at a final concentration of 7.5% m/v and blended with 3.7% m/v PEO. Electrospinning resulted in homogeneous fibre morphologies with a mean fibre diameter of 151 nm, which could be adapted to create macroscopic shapes (mats, tubes. Cross-linking with glutaraldehyde vapour improved the long-term stability of protein based scaffolds in comparison to untreated scaffolds, resulting in a mass loss of 41% and 96% after 28 days of incubation in aqueous solution, respectively.

  9. Scaffolding proteins in membrane trafficking : the role of ELKS

    NARCIS (Netherlands)

    Yu, K.L.

    2015-01-01

    Intracellular membrane trafficking is an essential cellular process that involves cooperation of many factors such as scaffolding proteins, GTPases and SNAREs. These proteins work together to ensure proper delivery of different membrane-enclosed cargoes to specific cellular destinations. In this

  10. Using Protein Dimers to Maximize the Protein Hybridization Efficiency with Multisite DNA Origami Scaffolds

    Science.gov (United States)

    Verma, Vikash; Mallik, Leena; Hariadi, Rizal F.; Sivaramakrishnan, Sivaraj; Skiniotis, Georgios; Joglekar, Ajit P.

    2015-01-01

    DNA origami provides a versatile platform for conducting ‘architecture-function’ analysis to determine how the nanoscale organization of multiple copies of a protein component within a multi-protein machine affects its overall function. Such analysis requires that the copy number of protein molecules bound to the origami scaffold exactly matches the desired number, and that it is uniform over an entire scaffold population. This requirement is challenging to satisfy for origami scaffolds with many protein hybridization sites, because it requires the successful completion of multiple, independent hybridization reactions. Here, we show that a cleavable dimerization domain on the hybridizing protein can be used to multiplex hybridization reactions on an origami scaffold. This strategy yields nearly 100% hybridization efficiency on a 6-site scaffold even when using low protein concentration and short incubation time. It can also be developed further to enable reliable patterning of a large number of molecules on DNA origami for architecture-function analysis. PMID:26348722

  11. Using Protein Dimers to Maximize the Protein Hybridization Efficiency with Multisite DNA Origami Scaffolds.

    Directory of Open Access Journals (Sweden)

    Vikash Verma

    Full Text Available DNA origami provides a versatile platform for conducting 'architecture-function' analysis to determine how the nanoscale organization of multiple copies of a protein component within a multi-protein machine affects its overall function. Such analysis requires that the copy number of protein molecules bound to the origami scaffold exactly matches the desired number, and that it is uniform over an entire scaffold population. This requirement is challenging to satisfy for origami scaffolds with many protein hybridization sites, because it requires the successful completion of multiple, independent hybridization reactions. Here, we show that a cleavable dimerization domain on the hybridizing protein can be used to multiplex hybridization reactions on an origami scaffold. This strategy yields nearly 100% hybridization efficiency on a 6-site scaffold even when using low protein concentration and short incubation time. It can also be developed further to enable reliable patterning of a large number of molecules on DNA origami for architecture-function analysis.

  12. UNC-16/JIP3 regulates early events in synaptic vesicle protein trafficking via LRK-1/LRRK2 and AP complexes.

    Directory of Open Access Journals (Sweden)

    Bikash Choudhary

    2017-11-01

    Full Text Available JIP3/UNC-16/dSYD is a MAPK-scaffolding protein with roles in protein trafficking. We show that it is present on the Golgi and is necessary for the polarized distribution of synaptic vesicle proteins (SVPs and dendritic proteins in neurons. UNC-16 excludes Golgi enzymes from SVP transport carriers and facilitates inclusion of specific SVPs into the same transport carrier. The SVP trafficking roles of UNC-16 are mediated through LRK-1, whose localization to the Golgi is reduced in unc-16 animals. UNC-16, through LRK-1, also enables Golgi-localization of the μ-subunit of the AP-1 complex. AP1 regulates the size but not the composition of SVP transport carriers. Additionally, UNC-16 and LRK-1 through the AP-3 complex regulates the composition but not the size of the SVP transport carrier. These early biogenesis steps are essential for dependence on the synaptic vesicle motor, UNC-104 for axonal transport. Our results show that UNC-16 and its downstream effectors, LRK-1 and the AP complexes function at the Golgi and/or post-Golgi compartments to control early steps of SV biogenesis. The UNC-16 dependent steps of exclusion, inclusion and motor recruitment are critical for polarized distribution of neuronal cargo.

  13. GluR2 protein-protein interactions and the regulation of AMPA receptors during synaptic plasticity.

    OpenAIRE

    Duprat, Fabrice; Daw, Michael; Lim, Wonil; Collingridge, Graham; Isaac, John

    2003-01-01

    AMPA-type glutamate receptors mediate most fast excitatory synaptic transmissions in the mammalian brain. They are critically involved in the expression of long-term potentiation and long-term depression, forms of synaptic plasticity that are thought to underlie learning and memory. A number of synaptic proteins have been identified that interact with the intracellular C-termini of AMPA receptor subunits. Here, we review recent studies and present new experimental data on the roles of these i...

  14. Diurnal rhythms in neurexins transcripts and inhibitory/excitatory synapse scaffold proteins in the biological clock.

    Science.gov (United States)

    Shapiro-Reznik, Mika; Jilg, Anje; Lerner, Hadas; Earnest, David J; Zisapel, Nava

    2012-01-01

    The neurexin genes (NRXN1/2/3) encode two families (α and β) of highly polymorphic presynaptic proteins that are involved in excitatory/inhibitory synaptic balance. Recent studies indicate that neuronal activation and memory formation affect NRXN1/2/3α expression and alternative splicing at splice sites 3 and 4 (SS#3/SS#4). Neurons in the biological clock residing in the suprachiasmatic nuclei of the hypothalamus (SCN) act as self-sustained oscillators, generating rhythms in gene expression and electrical activity, to entrain circadian bodily rhythms to the 24 hours day/night cycles. Cell autonomous oscillations in NRXN1/2/3α expression and SS#3/SS#4 exons splicing and their links to rhythms in excitatory/inhibitory synaptic balance in the circadian clock were explored. NRXN1/2/3α expression and SS#3/SS#4 splicing, levels of neurexin-2α and the synaptic scaffolding proteins PSD-95 and gephyrin (representing excitatory and inhibitory synapses, respectively) were studied in mRNA and protein extracts obtained from SCN of C3H/J mice at different times of the 24 hours day/night cycle. Further studies explored the circadian oscillations in these components and causality relationships in immortalized rat SCN2.2 cells. Diurnal rhythms in mNRXN1α and mNRXN2α transcription, SS#3/SS#4 exon-inclusion and PSD-95 gephyrin and neurexin-2α levels were found in the SCN in vivo. No such rhythms were found with mNRXN3α. SCN2.2 cells also exhibited autonomous circadian rhythms in rNRXN1/2 expression SS#3/SS#4 exon inclusion and PSD-95, gephyrin and neurexin-2α levels. rNRXN3α and rNRXN1/2β were not expressed. Causal relationships were demonstrated, by use of specific siRNAs, between rNRXN2α SS#3 exon included transcripts and gephyrin levels in the SCN2.2 cells. These results show for the first time dynamic, cell autonomous, diurnal rhythms in expression and splicing of NRXN1/2 and subsequent effects on the expression of neurexin-2α and postsynaptic scaffolding proteins

  15. Synapse associated protein 102 (SAP102 binds the C-terminal part of the scaffolding protein neurobeachin.

    Directory of Open Access Journals (Sweden)

    Juliane Lauks

    Full Text Available Neurobeachin (Nbea is a multidomain scaffold protein abundant in the brain, where it is highly expressed during development. Nbea-null mice have severe defects in neuromuscular synaptic transmission resulting in lethal paralysis of the newborns. Recently, it became clear that Nbea is important also for the functioning of central synapses, where it is suggested to play a role in trafficking membrane proteins to both, the pre- and post-synaptic sites. So far, only few binding partners of Nbea have been found and the precise mechanism of their trafficking remains unclear. Here, we used mass spectrometry to identify SAP102, a MAGUK protein implicated in trafficking of the ionotropic glutamate AMPA- and NMDA-type receptors during synaptogenesis, as a novel Nbea interacting protein in mouse brain. Experiments in heterologous cells confirmed this interaction and revealed that SAP102 binds to the C-terminal part of Nbea that contains the DUF, PH, BEACH and WD40 domains. Furthermore, we discovered that introducing a mutation in Nbea's PH domain, which disrupts its interaction with the BEACH domain, abolishes this binding, thereby creating an excellent starting point to further investigate Nbea-SAP102 function in the central nervous system.

  16. Synaptic vesicle proteins under conditions of rest and activation: analysis by 2-D difference gel electrophoresis.

    Science.gov (United States)

    Burré, Jacqueline; Beckhaus, Tobias; Corvey, Carsten; Karas, Michael; Zimmermann, Herbert; Volknandt, Walter

    2006-09-01

    Synaptic vesicles are organelles of the nerve terminal that secrete neurotransmitters by fusion with the presynaptic plasma membrane. Vesicle fusion is tightly controlled by depolarization of the plasma membrane and a set of proteins that may undergo post-translational modifications such as phosphorylation. In order to identify proteins that undergo modifications as a result of synaptic activation, we induced massive exocytosis and analysed the synaptic vesicle compartment by benzyldimethyl-n-hexadecylammonium chloride (BAC)/SDS-PAGE and difference gel electrophoresis (DIGE) followed by MALDI-TOF-MS. We identified eight proteins that revealed significant changes in abundance following nerve terminal depolarization. Of these, six were increased and two were decreased in abundance. Three of these proteins were phosphorylated as detected by Western blot analysis. In addition, we identified an unknown synaptic vesicle protein whose abundance increased on synaptic activation. Our results demonstrate that depolarization of the presynaptic compartment induces changes in the abundance of synaptic vesicle proteins and post-translational protein modification.

  17. Programmable DNA scaffolds for spatially-ordered protein assembly

    Science.gov (United States)

    Chandrasekaran, Arun Richard

    2016-02-01

    Ever since the notion of using DNA as a material was realized, it has been employed in the construction of complex structures that facilitate the assembly of nanoparticles or macromolecules with nanometer-scale precision. Specifically, tiles fashioned from DNA strands and DNA origami sheets have been shown to be suitable as scaffolds for immobilizing proteins with excellent control over their spatial positioning. Supramolecular assembly of proteins into periodic arrays in one or more dimensions is one of the most challenging aspects in the design of scaffolds for biomolecular investigations and macromolecular crystallization. This review provides a brief overview of how various biomolecular interactions with high degree of specificity such as streptavidin-biotin, antigen-antibody, and aptamer-protein interactions have been used to fabricate linear and multidimensional assemblies of structurally intact and functional proteins. The use of DNA-binding proteins as adaptors, polyamide recognition on DNA scaffolds and oligonucleotide linkers for protein assembly are also discussed.Ever since the notion of using DNA as a material was realized, it has been employed in the construction of complex structures that facilitate the assembly of nanoparticles or macromolecules with nanometer-scale precision. Specifically, tiles fashioned from DNA strands and DNA origami sheets have been shown to be suitable as scaffolds for immobilizing proteins with excellent control over their spatial positioning. Supramolecular assembly of proteins into periodic arrays in one or more dimensions is one of the most challenging aspects in the design of scaffolds for biomolecular investigations and macromolecular crystallization. This review provides a brief overview of how various biomolecular interactions with high degree of specificity such as streptavidin-biotin, antigen-antibody, and aptamer-protein interactions have been used to fabricate linear and multidimensional assemblies of structurally

  18. Dystrophin complex functions as a scaffold for signalling proteins.

    Science.gov (United States)

    Constantin, Bruno

    2014-02-01

    Dystrophin is a 427kDa sub-membrane cytoskeletal protein, associated with the inner surface membrane and incorporated in a large macromolecular complex of proteins, the dystrophin-associated protein complex (DAPC). In addition to dystrophin the DAPC is composed of dystroglycans, sarcoglycans, sarcospan, dystrobrevins and syntrophin. This complex is thought to play a structural role in ensuring membrane stability and force transduction during muscle contraction. The multiple binding sites and domains present in the DAPC confer the scaffold of various signalling and channel proteins, which may implicate the DAPC in regulation of signalling processes. The DAPC is thought for instance to anchor a variety of signalling molecules near their sites of action. The dystroglycan complex may participate in the transduction of extracellular-mediated signals to the muscle cytoskeleton, and β-dystroglycan was shown to be involved in MAPK and Rac1 small GTPase signalling. More generally, dystroglycan is view as a cell surface receptor for extracellular matrix proteins. The adaptor proteins syntrophin contribute to recruit and regulate various signalling proteins such as ion channels, into a macromolecular complex. Although dystrophin and dystroglycan can be directly involved in signalling pathways, syntrophins play a central role in organizing signalplex anchored to the dystrophin scaffold. The dystrophin associated complex, can bind up to four syntrophin through binding domains of dystrophin and dystrobrevin, allowing the scaffold of multiple signalling proteins in close proximity. Multiple interactions mediated by PH and PDZ domains of syntrophin also contribute to build a complete signalplex which may include ion channels, such as voltage-gated sodium channels or TRPC cation channels, together with, trimeric G protein, G protein-coupled receptor, plasma membrane calcium pump, and NOS, to enable efficient and regulated signal transduction and ion transport. This article is part

  19. Protein kinase A regulates AKAP250 (gravin) scaffold binding to the β2-adrenergic receptor

    OpenAIRE

    Tao, Jiangchuan; Wang, Hsien-yu; Malbon, Craig C.

    2003-01-01

    A-kinase-anchoring protein 250 (AKAP250; gravin) acts as a scaffold that binds protein kinase A (PKA), protein kinase C and protein phosphatases, associating reversibly with the β2-adrenergic receptor. The receptor-binding domain of the scaffold and the regulation of the receptor–scaffold association was revealed through mutagenesis and biochemical analyses. The AKAP domain found in other members of this superfamily is essential for the scaffold–receptor interactions. Gravin constructs lackin...

  20. A Pareto-optimal refinement method for protein design scaffolds.

    Science.gov (United States)

    Nivón, Lucas Gregorio; Moretti, Rocco; Baker, David

    2013-01-01

    Computational design of protein function involves a search for amino acids with the lowest energy subject to a set of constraints specifying function. In many cases a set of natural protein backbone structures, or "scaffolds", are searched to find regions where functional sites (an enzyme active site, ligand binding pocket, protein-protein interaction region, etc.) can be placed, and the identities of the surrounding amino acids are optimized to satisfy functional constraints. Input native protein structures almost invariably have regions that score very poorly with the design force field, and any design based on these unmodified structures may result in mutations away from the native sequence solely as a result of the energetic strain. Because the input structure is already a stable protein, it is desirable to keep the total number of mutations to a minimum and to avoid mutations resulting from poorly-scoring input structures. Here we describe a protocol using cycles of minimization with combined backbone/sidechain restraints that is Pareto-optimal with respect to RMSD to the native structure and energetic strain reduction. The protocol should be broadly useful in the preparation of scaffold libraries for functional site design.

  1. A Pareto-optimal refinement method for protein design scaffolds.

    Directory of Open Access Journals (Sweden)

    Lucas Gregorio Nivón

    Full Text Available Computational design of protein function involves a search for amino acids with the lowest energy subject to a set of constraints specifying function. In many cases a set of natural protein backbone structures, or "scaffolds", are searched to find regions where functional sites (an enzyme active site, ligand binding pocket, protein-protein interaction region, etc. can be placed, and the identities of the surrounding amino acids are optimized to satisfy functional constraints. Input native protein structures almost invariably have regions that score very poorly with the design force field, and any design based on these unmodified structures may result in mutations away from the native sequence solely as a result of the energetic strain. Because the input structure is already a stable protein, it is desirable to keep the total number of mutations to a minimum and to avoid mutations resulting from poorly-scoring input structures. Here we describe a protocol using cycles of minimization with combined backbone/sidechain restraints that is Pareto-optimal with respect to RMSD to the native structure and energetic strain reduction. The protocol should be broadly useful in the preparation of scaffold libraries for functional site design.

  2. Interaction between the glutamate transporter GLT1b and the synaptic PDZ domain protein PICK1

    DEFF Research Database (Denmark)

    Bassan, Merav; Liu, Hongguang; Madsen, Kenneth L

    2008-01-01

    Synaptic plasticity is implemented by the interaction of glutamate receptors with PDZ domain proteins. Glutamate transporters provide the only known mechanism of clearance of glutamate from excitatory synapses, and GLT1 is the major glutamate transporter. We show here that GLT1 interacts...... expressing PICK1 and GLT1b. In addition, expression of GLT1b in COS7 cells changed the distribution of PICK1, bringing it to the surface. GLT1b and PICK1 co-localized with each other and with synaptic markers in hippocampal neurons in culture. Phorbol ester, an activator of protein kinase C (PKC), a known...

  3. SynGAP regulates protein synthesis and homeostatic synaptic plasticity in developing cortical networks.

    Directory of Open Access Journals (Sweden)

    Chih-Chieh Wang

    Full Text Available Disrupting the balance between excitatory and inhibitory neurotransmission in the developing brain has been causally linked with intellectual disability (ID and autism spectrum disorders (ASD. Excitatory synapse strength is regulated in the central nervous system by controlling the number of postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs. De novo genetic mutations of the synaptic GTPase-activating protein (SynGAP are associated with ID and ASD. SynGAP is enriched at excitatory synapses and genetic suppression of SynGAP increases excitatory synaptic strength. However, exactly how SynGAP acts to maintain synaptic AMPAR content is unclear. We show here that SynGAP limits excitatory synaptic strength, in part, by suppressing protein synthesis in cortical neurons. The data presented here from in vitro, rat and mouse cortical networks, demonstrate that regulation of translation by SynGAP involves ERK, mTOR, and the small GTP-binding protein Rheb. Furthermore, these data show that GluN2B-containing NMDARs and the cognitive kinase CaMKII act upstream of SynGAP and that this signaling cascade is required for proper translation-dependent homeostatic synaptic plasticity of excitatory synapses in developing cortical networks.

  4. The Roles of Protein Expression in Synaptic Plasticity and Memory Consolidation

    Directory of Open Access Journals (Sweden)

    Tali eRosenberg

    2014-11-01

    Full Text Available The amount and availability of proteins are regulated by their synthesis, degradation, and transport. These processes can specifically, locally, and temporally regulate a protein or a population of proteins, thus affecting numerous biological processes in health and disease states. Accordingly, malfunction in the processes of protein turnover and localization underlies different neuronal diseases. However, as early as a century ago, it was recognized that there is a specific need for normal macromolecular synthesis in a specific fragment of the learning process, memory consolidation, which takes place minutes to hours following acquisition. Memory consolidation is the process by which fragile short-term memory is converted into stable long-term memory. It is accepted today that synaptic plasticity is a cellular mechanism of learning and memory processes. Interestingly, similar molecular mechanisms subserve both memory and synaptic plasticity consolidation. In this review, we survey the current view on the connection between memory consolidation processes and proteostasis, i.e., maintaining the protein contents at the neuron and the synapse. In addition, we describe the technical obstacles and possible new methods to determine neuronal proteostasis of synaptic function and better explain the process of memory and synaptic plasticity consolidation.

  5. Role for a novel Usher protein complex in hair cell synaptic maturation.

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    Marisa Zallocchi

    Full Text Available The molecular mechanisms underlying hair cell synaptic maturation are not well understood. Cadherin-23 (CDH23, protocadherin-15 (PCDH15 and the very large G-protein coupled receptor 1 (VLGR1 have been implicated in the development of cochlear hair cell stereocilia, while clarin-1 has been suggested to also play a role in synaptogenesis. Mutations in CDH23, PCDH15, VLGR1 and clarin-1 cause Usher syndrome, characterized by congenital deafness, vestibular dysfunction and retinitis pigmentosa. Here we show developmental expression of these Usher proteins in afferent spiral ganglion neurons and hair cell synapses. We identify a novel synaptic Usher complex comprised of clarin-1 and specific isoforms of CDH23, PCDH15 and VLGR1. To establish the in vivo relevance of this complex, we performed morphological and quantitative analysis of the neuronal fibers and their synapses in the Clrn1-/- mouse, which was generated by incomplete deletion of the gene. These mice showed a delay in neuronal/synaptic maturation by both immunostaining and electron microscopy. Analysis of the ribbon synapses in Ames waltzer(av3J mice also suggests a delay in hair cell synaptogenesis. Collectively, these results show that, in addition to the well documented role for Usher proteins in stereocilia development, Usher protein complexes comprised of specific protein isoforms likely function in synaptic maturation as well.

  6. Elliptical structure of phospholipid bilayer nanodiscs encapsulated by scaffold proteins

    DEFF Research Database (Denmark)

    Skar-Gislinge, Nicholas; Simonsen, Jens Bæk; Mortensen, Kell

    2010-01-01

    neutron scattering in combination with variable-temperature studies of synchrotron small-angle X-ray scattering on nanodiscs in solution, we show that the fundamental nanodisc unit, consisting of a lipid bilayer surrounded by amphiphilic scaffold proteins, possesses intrinsically an elliptical shape......Phospholipid bilayers host and support the function of membrane proteins and may be stabilized in disc-like nanostructures, allowing for unprecedented solution studies of the assembly, structure, and function of membrane proteins (Bayburt et al. Nano Lett. 2002, 2, 853-856). Based on small-angle...... the experimental scattering profile from nanodiscs. The model paves the way for future detailed structural studies of functional membrane proteins encapsulated in nanodiscs....

  7. Long lasting protein synthesis- and activity-dependent spine shrinkage and elimination after synaptic depression.

    Directory of Open Access Journals (Sweden)

    Yazmín Ramiro-Cortés

    Full Text Available Neuronal circuits modify their response to synaptic inputs in an experience-dependent fashion. Increases in synaptic weights are accompanied by structural modifications, and activity dependent, long lasting growth of dendritic spines requires new protein synthesis. When multiple spines are potentiated within a dendritic domain, they show dynamic structural plasticity changes, indicating that spines can undergo bidirectional physical modifications. However, it is unclear whether protein synthesis dependent synaptic depression leads to long lasting structural changes. Here, we investigate the structural correlates of protein synthesis dependent long-term depression (LTD mediated by metabotropic glutamate receptors (mGluRs through two-photon imaging of dendritic spines on hippocampal pyramidal neurons. We find that induction of mGluR-LTD leads to robust and long lasting spine shrinkage and elimination that lasts for up to 24 hours. These effects depend on signaling through group I mGluRs, require protein synthesis, and activity. These data reveal a mechanism for long lasting remodeling of synaptic inputs, and offer potential insights into mental retardation.

  8. Survival motor neuron protein in motor neurons determines synaptic integrity in spinal muscular atrophy.

    Science.gov (United States)

    Martinez, Tara L; Kong, Lingling; Wang, Xueyong; Osborne, Melissa A; Crowder, Melissa E; Van Meerbeke, James P; Xu, Xixi; Davis, Crystal; Wooley, Joe; Goldhamer, David J; Lutz, Cathleen M; Rich, Mark M; Sumner, Charlotte J

    2012-06-20

    The inherited motor neuron disease spinal muscular atrophy (SMA) is caused by deficient expression of survival motor neuron (SMN) protein and results in severe muscle weakness. In SMA mice, synaptic dysfunction of both neuromuscular junctions (NMJs) and central sensorimotor synapses precedes motor neuron cell death. To address whether this synaptic dysfunction is due to SMN deficiency in motor neurons, muscle, or both, we generated three lines of conditional SMA mice with tissue-specific increases in SMN expression. All three lines of mice showed increased survival, weights, and improved motor behavior. While increased SMN expression in motor neurons prevented synaptic dysfunction at the NMJ and restored motor neuron somal synapses, increased SMN expression in muscle did not affect synaptic function although it did improve myofiber size. Together these data indicate that both peripheral and central synaptic integrity are dependent on motor neurons in SMA, but SMN may have variable roles in the maintenance of these different synapses. At the NMJ, it functions at the presynaptic terminal in a cell-autonomous fashion, but may be necessary for retrograde trophic signaling to presynaptic inputs onto motor neurons. Importantly, SMN also appears to function in muscle growth and/or maintenance independent of motor neurons. Our data suggest that SMN plays distinct roles in muscle, NMJs, and motor neuron somal synapses and that restored function of SMN at all three sites will be necessary for full recovery of muscle power.

  9. Fragile X mental retardation protein regulates trans-synaptic signaling in Drosophila

    Directory of Open Access Journals (Sweden)

    Samuel H. Friedman

    2013-11-01

    Fragile X syndrome (FXS, the most common inherited determinant of intellectual disability and autism spectrum disorders, is caused by loss of the fragile X mental retardation 1 (FMR1 gene product (FMRP, an mRNA-binding translational repressor. A number of conserved FMRP targets have been identified in the well-characterized Drosophila FXS disease model, but FMRP is highly pleiotropic in function and the full spectrum of FMRP targets has yet to be revealed. In this study, screens for upregulated neural proteins in Drosophila fmr1 (dfmr1 null mutants reveal strong elevation of two synaptic heparan sulfate proteoglycans (HSPGs: GPI-anchored glypican Dally-like protein (Dlp and transmembrane Syndecan (Sdc. Our recent work has shown that Dlp and Sdc act as co-receptors regulating extracellular ligands upstream of intracellular signal transduction in multiple trans-synaptic pathways that drive synaptogenesis. Consistently, dfmr1 null synapses exhibit altered WNT signaling, with changes in both Wingless (Wg ligand abundance and downstream Frizzled-2 (Fz2 receptor C-terminal nuclear import. Similarly, a parallel anterograde signaling ligand, Jelly belly (Jeb, and downstream ERK phosphorylation (dpERK are depressed at dfmr1 null synapses. In contrast, the retrograde BMP ligand Glass bottom boat (Gbb and downstream signaling via phosphorylation of the transcription factor MAD (pMAD seem not to be affected. To determine whether HSPG upregulation is causative for synaptogenic defects, HSPGs were genetically reduced to control levels in the dfmr1 null background. HSPG correction restored both (1 Wg and Jeb trans-synaptic signaling, and (2 synaptic architecture and transmission strength back to wild-type levels. Taken together, these data suggest that FMRP negatively regulates HSPG co-receptors controlling trans-synaptic signaling during synaptogenesis, and that loss of this regulation causes synaptic structure and function defects characterizing the FXS disease state.

  10. A tunable protein-based scaffold for the study of central nervous system regeneration

    Science.gov (United States)

    Straley, Karin

    Central nervous system (CNS) injuries pose a significant and potentially debilitating health problem in society today and, to date, no successful clinical repair strategies have been advanced. The development of effective treatments is severely hindered by the quick formation of a complex, inhibitory scar at the site of CNS injury. This scar both physically blocks and chemically suppresses nerve regeneration. It has been hypothesized that combinatorial approaches involving biomaterial scaffolds, cell transplantation, and pro-survival factors, which provide a more permissive growth environment, have the highest chance of stimulating regeneration. The work completed in this thesis focuses on the design and characterization of a biomimetic hydrogel scaffold constructed from chemically crosslinked recombinant proteins. This protein-based scaffold has been designed to offer a flexible platform for the systematic optimization of key scaffold design parameters, such as mechanical strength, degradation, cellular interaction, molecule delivery, and topography. Specifically, a collection of proteins containing sequences previously shown to enhance cell adhesion, to promote neurite extension, and to exhibit varying susceptibility to cleavage by neurite-secreted proteases were synthesized to serve as the polymer backbone for the scaffold. Experiments were conducted to analyze the capacity of scaffolds, constructed from single proteins or mixtures of proteins, to independently control cell behavior, scaffold degradation properties, and scaffold mechanical properties based upon differences in the primary protein sequence and crosslinking conditions. In addition, composite scaffolds constructed by layered spatial deposition of chemically crosslinked, protease-degradable proteins were applied to the formation of dynamic internal, three-dimensional scaffold patterns that can be directly coupled to molecule delivery. Overall, this work demonstrates the tunable and bio

  11. Neuronal signaling modulates protein homeostasis in Caenorhabditis elegans post-synaptic muscle cells.

    Science.gov (United States)

    Garcia, Susana M; Casanueva, M Olivia; Silva, M Catarina; Amaral, Margarida D; Morimoto, Richard I

    2007-11-15

    Protein homeostasis maintains proper intracellular balance by promoting protein folding and clearance mechanisms while minimizing the stress caused by the accumulation of misfolded and damaged proteins. Chronic expression of aggregation-prone proteins is deleterious to the cell and has been linked to a wide range of conformational disorders. The molecular response to misfolded proteins is highly conserved and generally studied as a cell-autonomous process. Here, we provide evidence that neuronal signaling is an important modulator of protein homeostasis in post-synaptic muscle cells. In a forward genetic screen in Caenorhabditis elegans for enhancers of polyglutamine aggregation in muscle cells, we identified unc-30, a neuron-specific transcription factor that regulates the synthesis of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). We used additional sensors of protein conformational states to show that defective GABA signaling or increased acetylcholine (ACh) signaling causes a general imbalance in protein homeostasis in post-synaptic muscle cells. Moreover, exposure to GABA antagonists or ACh agonists has a similar effect, which reveals that toxins that act at the neuromuscular junction are potent modifiers of protein conformational disorders. These results demonstrate the importance of intercellular communication in intracellular homeostasis.

  12. Synaptic proteins and receptors defects in autism spectrum disorders

    OpenAIRE

    Chen, Jianling; Yu, Shunying; Fu, Yingmei; Li, Xiaohong

    2014-01-01

    Recent studies have found that hundreds of genetic variants, including common and rare variants, rare and de novo mutations, and common polymorphisms have contributed to the occurrence of autism spectrum disorders (ASDs). The mutations in a number of genes such as neurexin, neuroligin, postsynaptic density protein 95 (PSD-95), SH3 and multiple ankyrin repeat domains 3 (SHANK3), synapsin, gephyrin, cadherin (CDH) and protocadherin (PCDH), thousand-and-one-amino acid 2 kinase (TAOK2), and conta...

  13. Studying Protein Synthesis-Dependent Synaptic Changes in Tuberous Sclerosis

    Science.gov (United States)

    2013-04-01

    and infantile spasm in TSC is treatable with Vigabatrin, a GABA transaminase inhibitor. This indicates that abnormal protein synthesis may skew the...microscopic duplication of 22q13 including the Shank3 gene manifested with infantile hypotonia, developmental delay and growth deficiency (6). Shank1...deviation toward hyperexcitability was not corrected at least 30 minute after rapamycin treatment (100 µM). This may be because the effect of

  14. Botulinum neurotoxin D uses synaptic vesicle protein SV2 and gangliosides as receptors.

    Directory of Open Access Journals (Sweden)

    Lisheng Peng

    2011-03-01

    Full Text Available Botulinum neurotoxins (BoNTs include seven bacterial toxins (BoNT/A-G that target presynaptic terminals and act as proteases cleaving proteins required for synaptic vesicle exocytosis. Here we identified synaptic vesicle protein SV2 as the protein receptor for BoNT/D. BoNT/D enters cultured hippocampal neurons via synaptic vesicle recycling and can bind SV2 in brain detergent extracts. BoNT/D failed to bind and enter neurons lacking SV2, which can be rescued by expressing one of the three SV2 isoforms (SV2A/B/C. Localization of SV2 on plasma membranes mediated BoNT/D binding in both neurons and HEK293 cells. Furthermore, chimeric receptors containing the binding sites for BoNT/A and E, two other BoNTs that use SV2 as receptors, failed to mediate the entry of BoNT/D suggesting that BoNT/D binds SV2 via a mechanism distinct from BoNT/A and E. Finally, we demonstrated that gangliosides are essential for the binding and entry of BoNT/D into neurons and for its toxicity in vivo, supporting a double-receptor model for this toxin.

  15. Synaptic adhesion molecule IgSF11 regulates synaptic transmission and plasticity

    Science.gov (United States)

    Shin, Hyewon; van Riesen, Christoph; Whitcomb, Daniel; Warburton, Julia M.; Jo, Jihoon; Kim, Doyoun; Kim, Sun Gyun; Um, Seung Min; Kwon, Seok-kyu; Kim, Myoung-Hwan; Roh, Junyeop Daniel; Woo, Jooyeon; Jun, Heejung; Lee, Dongmin; Mah, Won; Kim, Hyun; Kaang, Bong-Kiun; Cho, Kwangwook; Rhee, Jeong-Seop; Choquet, Daniel; Kim, Eunjoon

    2016-01-01

    Summary Synaptic adhesion molecules regulate synapse development and plasticity through mechanisms including trans-synaptic adhesion and recruitment of diverse synaptic proteins. We report here that the immunoglobulin superfamily member 11 (IgSF11), a homophilic adhesion molecule preferentially expressed in the brain, is a novel and dual-binding partner of the postsynaptic scaffolding protein PSD-95 and AMPAR glutamate receptors (AMPARs). IgSF11 requires PSD-95 binding for its excitatory synaptic localization. In addition, IgSF11 stabilizes synaptic AMPARs, as shown by IgSF11 knockdown-induced suppression of AMPAR-mediated synaptic transmission and increased surface mobility of AMPARs, measured by high-throughput, single-molecule tracking. IgSF11 deletion in mice leads to suppression of AMPAR-mediated synaptic transmission in the dentate gyrus and long-term potentiation in the CA1 region of the hippocampus. IgSF11 does not regulate the functional characteristics of AMPARs, including desensitization, deactivation, or recovery. These results suggest that IgSF11 regulates excitatory synaptic transmission and plasticity through its tripartite interactions with PSD-95 and AMPARs. PMID:26595655

  16. Repetitive transcranial magnetic stimulation effectively facilitates spatial cognition and synaptic plasticity associated with increasing the levels of BDNF and synaptic proteins in Wistar rats.

    Science.gov (United States)

    Shang, Yingchun; Wang, Xin; Shang, Xueliang; Zhang, Hui; Liu, Zhipeng; Yin, Tao; Zhang, Tao

    2016-10-01

    Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive technique, by which cognitive deficits can be alleviated. Furthermore, rTMS may facilitate learning and memory. However, its underlying mechanism is still little known. The aim of this study was to investigate if the facilitation of spatial cognition and synaptic plasticity, induced by rTMS, is regulated by enhancing pre- and postsynaptic proteins in normal rats. Morris water maze (MWM) test was performed to examine the spatial cognition. The synaptic plasticity, including long-term potentiation (LTP) and depotentiation (DEP), presynaptic plasticity paired-pulse facilitation (PPF), from the hippocampal Schaffer collaterals to CA1 region was subsequently measured using in vivo electrophysiological techniques. The expressions of brain-derived neurotrophic factor (BDNF), presynaptic protein synaptophysin (SYP) and postsynaptic protein NR2B were measured by Western blot. Our data show that the spatial learning/memory and reversal learning/memory in rTMS rats were remarkably enhanced compared to that in the Sham group. Furthermore, LTP and DEP as well as PPF were effectively facilitated by 5Hz-rTMS. Additionally, the expressions of BDNF, SYP and NR2B were significantly increased via magnetic stimulation. The results suggest that rTMS considerably increases the expressions of BDNF, postsynaptic protein NR2B and presynaptic protein SYP, and thereby significantly enhances the synaptic plasticity and spatial cognition in normal animals. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Evaluating protein incorporation and release in electrospun composite scaffolds for bone tissue engineering applications.

    Science.gov (United States)

    Briggs, Tonye; Matos, Jeffrey; Collins, George; Arinzeh, Treena Livingston

    2015-10-01

    Electrospun polymer/ceramic composites have gained interest for use as scaffolds for bone tissue engineering applications. In this study, we investigated methods to incorporate Platelet Derived Growth Factor-BB (PDGF-BB) in electrospun polycaprolactone (PCL) or PCL prepared with polyethylene oxide (PEO), where both contained varying levels (up to 30 wt %) of ceramic composed of biphasic calcium phosphates, hydroxyapatite (HA)/β-tricalcium phosphate (TCP). Using a model protein, lysozyme, we compared two methods of protein incorporation, adsorption and emulsion electrospinning. Adsorption of lysozyme on scaffolds with ceramic resulted in minimal release of lysozyme over time. Using emulsion electrospinning, lysozyme released from scaffolds containing a high concentration of ceramic where the majority of the release occurred at later time points. We investigated the effect of reducing the electrostatic interaction between the protein and the ceramic on protein release with the addition of the cationic surfactant, cetyl trimethylammonium bromide (CTAB). In vitro release studies demonstrated that electrospun scaffolds prepared with CTAB released more lysozyme or PDGF-BB compared with scaffolds without the cationic surfactant. Human mesenchymal stem cells (MSCs) on composite scaffolds containing PDGF-BB incorporated through emulsion electrospinning expressed higher levels of osteogenic markers compared to scaffolds without PDGF-BB, indicating that the bioactivity of the growth factor was maintained. This study revealed methods for incorporating growth factors in polymer/ceramic scaffolds to promote osteoinduction and thereby facilitate bone regeneration. © 2015 Wiley Periodicals, Inc.

  18. Expression of the scaffolding subunit A of protein phosphatase 2A during rat testicular development

    NARCIS (Netherlands)

    van den Ham, R.; van Dissel-Emiliani, F. M. F.; van Pelt, A. M. M.

    2003-01-01

    Previously, we found that the poly(A)+ RNA of the scaffolding subunit A (alpha isoform) of protein phosphatase 2A (PP2A-Aalpha) was clearly expressed by fetal gonocytes but weakly expressed by adult single (As), paired (Apr), and aligned (Aal) A spermatogonia. The scaffolding subunit A of PP2A

  19. Fibrous scaffolds loaded with protein prepared by blend or coaxial electrospinning.

    NARCIS (Netherlands)

    Ji, W.; Yang, F.; Beucken, J.J.J.P van den; Bian, Z.; Fan, M.; Chen, Z.; Jansen, J.A.

    2010-01-01

    The aim of the present study was to fabricate polycaprolactone-based nanofibrous scaffolds with incorporated protein via either the blend or coaxial electrospinning technique. Both techniques were compared with respect to processing set-up and scaffold characteristics as well as the release kinetics

  20. CCL2-ethanol interactions and hippocampal synaptic protein expression in a transgenic mouse model

    Directory of Open Access Journals (Sweden)

    Donna eGruol

    2014-04-01

    Full Text Available Chronic exposure to ethanol produces a number of detrimental effects on behavior. Neuroadaptive changes in brain structure or function underlie these behavioral changes and may be transient or persistent in nature. Central to the functional changes are alterations in the biology of neuronal and glial cells of the brain. Recent data show that ethanol induces glial cells of the brain to produce elevated levels of neuroimmune factors including CCL2, a key innate immune chemokine. Depending on the conditions of ethanol exposure, the upregulated levels of CCL2 can be transient or persistent and outlast the period of ethanol exposure. Importantly, results indicate that the upregulated levels of CCL2 may lead to CCL2-ethanol interactions that mediate or regulate the effects of ethanol on the brain. Glial cells are in close association with neurons and regulate many neuronal functions. Therefore, effects of ethanol on glial cells may underlie some of the effects of ethanol on neurons. To investigate this possibility, we are studying the effects of chronic ethanol on hippocampal synaptic function in a transgenic mouse model that expresses elevated levels of CCL2 in the brain through enhanced glial expression, a situation know to occur in alcoholics. Both CCL2 and ethanol have been reported to alter synaptic function in the hippocampus. In the current study, we determined if interactions are evident between CCL2 and ethanol at level of hippocampal synaptic proteins. Two ethanol exposure paradigms were used; the first involved ethanol exposure by drinking and the second involved ethanol exposure in a paradigm that combines drinking plus ethanol vapor. The first paradigm does not produce dependence on ethanol, whereas the second paradigm is commonly used to produce ethanol dependence. Results show modest effects of both ethanol exposure paradigms on the level of synaptic proteins in the hippocampus of CCL2 transgenic mice compared with their non

  1. Temporal requirements of the fragile X mental retardation protein in modulating circadian clock circuit synaptic architecture

    Directory of Open Access Journals (Sweden)

    Cheryl L Gatto

    2009-08-01

    Full Text Available Loss of fragile X mental retardation 1 (FMR1 gene function is the most common cause of inherited mental retardation and autism spectrum disorders, characterized by attention disorder, hyperactivity and disruption of circadian activity cycles. Pursuit of effective intervention strategies requires determining when the FMR1 product (FMRP is required in the regulation of neuronal circuitry controlling these behaviors. In the well-characterized Drosophila disease model, loss of the highly conserved dFMRP causes circadian arrhythmicity and conspicuous abnormalities in the circadian clock circuitry. Here, a novel Sholl Analysis was used to quantify over-elaborated synaptic architecture in dfmr1-null small ventrolateral neurons (sLNvs, a key subset of clock neurons. The transgenic Gene-Switch system was employed to drive conditional neuronal dFMRP expression in the dfmr1-null mutant background in order to dissect temporal requirements within the clock circuit. Introduction of dFMRP during early brain development, including the stages of neurogenesis, neuronal fate specification and early pathfinding, provided no rescue of dfmr1 mutant phenotypes. Similarly, restoring normal dFMRP expression in the adult failed to restore circadian circuit architecture. In sharp contrast, supplying dFMRP during a transient window of very late brain development, wherein synaptogenesis and substantial subsequent synaptic reorganization (e.g. use-dependent pruning occur, provided strong morphological rescue to reestablish normal sLNvs synaptic arbors. We conclude that dFMRP plays a developmentally restricted role in sculpting synaptic architecture in these neurons that cannot be compensated for by later reintroduction of the protein at maturity.

  2. Fluorescence imaging preparation methods for tissue scaffolds implanted into a green fluorescent protein porcine model.

    Science.gov (United States)

    Smith, Sarah E; White, Richard A; Grant, David A; Grant, Sheila A

    2015-10-01

    Green fluorescent protein (GFP) animal models have become increasingly popular due to their potential to enhance in vivo imaging and their application to many fields of study. We have developed a technique to observe host tissue integration into scaffolds using GFP expressing swine and fluorescence imaging. Current fluorescence imaging preparation methods cannot be translated to a full GFP animal model due to several challenges and limitations that are investigated here. We have implanted tissue scaffolds into GFP expressing swine and have prepared explanted scaffolds for fluorescence imaging using four different methods including formalin fixation and paraffin embedding, vapor fixation, freshly prepared paraformaldehyde fixation, and fresh frozen tissue. Explanted scaffolds and tissue were imaged using confocal microscopy with spectral separation to evaluate the GFP animal model for visualization of host tissue integration into explanted scaffolds. All methods except fresh frozen tissue induced autofluorescence of the scaffold, preventing visualization of detail between host tissue and scaffold fibers. Fresh frozen tissue preparation allowed for the most reliable visualization of fluorescent host tissue integration into non-fluorescent scaffolds. It was concluded that fresh frozen tissue preparation is the best method for fluorescence imaging preparation when using scaffolds implanted into GFP whole animal models.

  3. Protein kinase A regulates AKAP250 (gravin) scaffold binding to the beta2-adrenergic receptor.

    Science.gov (United States)

    Tao, Jiangchuan; Wang, Hsien-Yu; Malbon, Craig C

    2003-12-15

    A-kinase-anchoring protein 250 (AKAP250; gravin) acts as a scaffold that binds protein kinase A (PKA), protein kinase C and protein phosphatases, associating reversibly with the beta(2)-adrenergic receptor. The receptor-binding domain of the scaffold and the regulation of the receptor-scaffold association was revealed through mutagenesis and biochemical analyses. The AKAP domain found in other members of this superfamily is essential for the scaffold-receptor interactions. Gravin constructs lacking the AKAP domain displayed no binding to the receptor. Metabolic labeling studies in vivo demonstrate agonist-stimulated phosphorylation of gravin and enhanced gravin-receptor association. Analysis of the AKAP domain revealed two canonical PKA sites phosphorylated in response to elevated cAMP, blocked by PKA inhibitor, and essential for scaffold-receptor association and for resensitization of the receptor. The AKAP appears to provide the catalytic PKA activity responsible for phosphorylation of the scaffold in response to agonist activation of the receptor as well as for the association of the scaffold with the receptor, a step critical to receptor resensitization.

  4. Structural Insights for Engineering Binding Proteins Based on Non-Antibody Scaffolds

    OpenAIRE

    Gilbreth, Ryan N.; Koide, Shohei

    2012-01-01

    Engineered binding proteins derived from non-antibody scaffolds constitute an increasingly prominent class of reagents in both research and therapeutic applications. The growing number of crystal structures of these “alternative” scaffold-based binding proteins in complex with their targets illustrate the mechanisms of molecular recognition that are common among these systems and those unique to each. This information is useful for critically assessing and improving/expanding engineering stra...

  5. Nanodisc Films for Membrane Protein Studies by Neutron Reflection: Effect of the Protein Scaffold Choice

    OpenAIRE

    Bertram, Nicolas; Laursen, Tomas; Barker, Robert; Bavishi, Krutika; M?ller, Birger Lindberg; C?rdenas, Marit?

    2015-01-01

    Nanodisc films are a promising approach to study the equilibrium conformation of membrane bound proteins in native-like environment. Here we compare nanodisc formation for NADPH-dependent cytochrome P450 oxidoreductase (POR) using two different scaffold proteins, MSP1D1 and MSP1E3D1. Despite the increased stability of POR loaded MSP1E3D1 based nanodiscs in comparison to MSP1D1 based nanodiscs, neutron reflection at the silicon?solution interface showed that POR loaded MSP1E3D1 based nanodisc ...

  6. Minireview: Role of Intracellular Scaffolding Proteins in the Regulation of Endocrine G Protein-Coupled Receptor Signaling

    Science.gov (United States)

    Walther, Cornelia

    2015-01-01

    The majority of hormones stimulates and mediates their signal transduction via G protein-coupled receptors (GPCRs). The signal is transmitted into the cell due to the association of the GPCRs with heterotrimeric G proteins, which in turn activates an extensive array of signaling pathways to regulate cell physiology. However, GPCRs also function as scaffolds for the recruitment of a variety of cytoplasmic protein-interacting proteins that bind to both the intracellular face and protein interaction motifs encoded by GPCRs. The structural scaffolding of these proteins allows GPCRs to recruit large functional complexes that serve to modulate both G protein-dependent and -independent cellular signaling pathways and modulate GPCR intracellular trafficking. This review focuses on GPCR interacting PSD95-disc large-zona occludens domain containing scaffolds in the regulation of endocrine receptor signaling as well as their potential role as therapeutic targets for the treatment of endocrinopathies. PMID:25942107

  7. Crystallization and preliminary crystallographic characterization of an SH3 domain from the IB1 scaffold protein

    DEFF Research Database (Denmark)

    Dar, Imran; Bonny, Christophe; Pedersen, Jan Torleif

    2003-01-01

    IB1 is a mammalian scaffold protein that interacts with components of the c-Jun N-terminal kinase (JNK) signal-transduction pathway mainly via its protein-protein interaction domains. Crystallization of the key Src homology 3 (SH3) domain of IB1 has been achieved. Crystallization experiments with...

  8. The human synaptic vesicle protein, SV2A, functions as a galactose transporter in Saccharomyces cerevisiae.

    Science.gov (United States)

    Madeo, Marianna; Kovács, Attila D; Pearce, David A

    2014-11-28

    SV2A is a synaptic vesicle membrane protein expressed in neurons and endocrine cells and involved in the regulation of neurotransmitter release. Although the exact function of SV2A still remains elusive, it was identified as the specific binding site for levetiracetam, a second generation antiepileptic drug. Our sequence analysis demonstrates that SV2A has significant homology with several yeast transport proteins belonging to the major facilitator superfamily (MFS). Many of these transporters are involved in sugar transport into yeast cells. Here we present evidence showing, for the first time, that SV2A is a galactose transporter. We expressed human SV2A in hexose transport-deficient EBY.VW4000 yeast cells and demonstrated that these cells are able to grow on galactose-containing medium but not on other fermentable carbon sources. Furthermore, the addition of the SV2A-binding antiepileptic drug levetiracetam to the medium inhibited the galactose-dependent growth of hexose transport-deficient EBY.VW4000 yeast cells expressing human SV2A. Most importantly, direct measurement of galactose uptake in the same strain verified that SV2A is able to transport extracellular galactose inside the cells. The newly identified galactose transport capability of SV2A may have an important role in regulating/modulating synaptic function. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Inhibition of protein kinase C affects on mode of synaptic vesicle exocytosis due to cholesterol depletion

    Energy Technology Data Exchange (ETDEWEB)

    Petrov, Alexey M., E-mail: fysio@rambler.ru; Zakyrjanova, Guzalija F., E-mail: guzik121192@mail.ru; Yakovleva, Anastasia A., E-mail: nastya1234qwer@mail.ru; Zefirov, Andrei L., E-mail: zefiroval@rambler.ru

    2015-01-02

    Highlights: • We examine the involvement of PKC in MCD induced synaptic vesicle exocytosis. • PKC inhibitor does not decrease the effect MCD on MEPP frequency. • PKC inhibitor prevents MCD induced FM1-43 unloading. • PKC activation may switch MCD induced exocytosis from kiss-and-run to a full mode. • Inhibition of phospholipase C does not lead to similar change in exocytosis. - Abstract: Previous studies demonstrated that depletion of membrane cholesterol by 10 mM methyl-beta-cyclodextrin (MCD) results in increased spontaneous exocytosis at both peripheral and central synapses. Here, we investigated the role of protein kinase C in the enhancement of spontaneous exocytosis at frog motor nerve terminals after cholesterol depletion using electrophysiological and optical methods. Inhibition of the protein kinase C by myristoylated peptide and chelerythrine chloride prevented MCD-induced increases in FM1-43 unloading, whereas the frequency of spontaneous postsynaptic events remained enhanced. The increase in FM1-43 unloading still could be observed if sulforhodamine 101 (the water soluble FM1-43 quencher that can pass through the fusion pore) was added to the extracellular solution. This suggests a possibility that exocytosis of synaptic vesicles under these conditions could occur through the kiss-and-run mechanism with the formation of a transient fusion pore. Inhibition of phospholipase C did not lead to similar change in MCD-induced exocytosis.

  10. The quantum physics of synaptic communication via the SNARE protein complex.

    Science.gov (United States)

    Georgiev, Danko D; Glazebrook, James F

    2018-01-31

    Twenty five years ago, Sir John Carew Eccles together with Friedrich Beck proposed a quantum mechanical model of neurotransmitter release at synapses in the human cerebral cortex. The model endorsed causal influence of human consciousness upon the functioning of synapses in the brain through quantum tunneling of unidentified quasiparticles that trigger the exocytosis of synaptic vesicles, thereby initiating the transmission of information from the presynaptic towards the postsynaptic neuron. Here, we provide a molecular upgrade of the Beck and Eccles model by identifying the quantum quasiparticles as Davydov solitons that twist the protein α-helices and trigger exocytosis of synaptic vesicles through helical zipping of the SNARE protein complex. We also calculate the observable probabilities for exocytosis based on the mass of this quasiparticle, along with the characteristics of the potential energy barrier through which tunneling is necessary. We further review the current experimental evidence in support of this novel bio-molecular model as presented. Copyright © 2018 Elsevier Ltd. All rights reserved.

  11. Orthogonal dual-modification of proteins for the engineering of multivalent protein scaffolds

    Directory of Open Access Journals (Sweden)

    Michaela Mühlberg

    2015-05-01

    Full Text Available To add new tools to the repertoire of protein-based multivalent scaffold design, we have developed a novel dual-labeling strategy for proteins that combines residue-specific incorporation of unnatural amino acids with chemical oxidative aldehyde formation at the N-terminus of a protein. Our approach relies on the selective introduction of two different functional moieties in a protein by mutually orthogonal copper-catalyzed azide–alkyne cycloaddition (CuAAC and oxime ligation. This method was applied to the conjugation of biotin and β-linked galactose residues to yield an enzymatically active thermophilic lipase, which revealed specific binding to Erythrina cristagalli lectin by SPR binding studies.

  12. Self-assembly of nanoscale particles with biosurfactants and membrane scaffold proteins.

    Science.gov (United States)

    Faas, Ramona; Pohle, Annelie; Moß, Karin; Henkel, Marius; Hausmann, Rudolf

    2017-12-01

    Nanodiscs are membrane mimetics which may be used as tools for biochemical and biophysical studies of a variety of membrane proteins. These nanoscale structures are composed of a phospholipid bilayer held together by an amphipathic membrane scaffold protein (MSP). In the past, nanodiscs were successfully assembled with membrane scaffold protein 1D1 and 1,2-dipalmitoyl- sn -glycero-3-phosphorylcholine with a homogeneous diameter of ∼10 nm. In this study, the formation of nanoscale particles from MSP1D1 and rhamnolipid biosurfactants is investigated. Different protein to lipid ratios of 1:80, 1:90 and 1:100 were used for the assembly reaction, which were consecutively separated, purified and analyzed by size-exclusion chromatography (SEC) and dynamic light scattering (DLS). Size distributions were measured to determine homogeneity and confirm size dimensions. In this study, first evidence is presented on the formation of nanoscale particles with rhamnolipid biosurfactants and membrane scaffold proteins.

  13. Fusion to a homo-oligomeric scaffold allows cryo-EM analysis of a small protein.

    Science.gov (United States)

    Coscia, Francesca; Estrozi, Leandro F; Hans, Fabienne; Malet, Hélène; Noirclerc-Savoye, Marjolaine; Schoehn, Guy; Petosa, Carlo

    2016-08-03

    Recent technical advances have revolutionized the field of cryo-electron microscopy (cryo-EM). However, most monomeric proteins remain too small (protein is genetically fused to a homo-oligomeric scaffold protein and the junction optimized to allow the target to adopt the scaffold symmetry, thereby generating a chimeric particle suitable for cryo-EM. To demonstrate the concept, we fused maltose-binding protein (MBP), a 40 kDa monomer, to glutamine synthetase, a dodecamer formed by two hexameric rings. Chimeric constructs with different junction lengths were screened by biophysical analysis and negative-stain EM. The optimal construct yielded a cryo-EM reconstruction that revealed the MBP structure at sub-nanometre resolution. These findings illustrate the feasibility of using homo-oligomeric scaffolds to enable cryo-EM analysis of monomeric proteins, paving the way for applying this strategy to challenging structures resistant to crystallographic and NMR analysis.

  14. Ethanol extract of the seed of Zizyphus jujuba var. spinosa potentiates hippocampal synaptic transmission through mitogen-activated protein kinase, adenylyl cyclase, and protein kinase A pathways.

    Science.gov (United States)

    Jo, So Yeon; Jung, In Ho; Yi, Jee Hyun; Choi, Tae Joon; Lee, Seungheon; Jung, Ji Wook; Yun, Jeanho; Lee, Young Choon; Ryu, Jong Hoon; Kim, Dong Hyun

    2017-03-22

    As the seed of Zizyphus jujuba var. spinosa (Bunge) Hu ex H.F. Chow (Rhamnaceae) has been used to sleep disturbances in traditional Chinese and Korean medicine, many previous studies have focused on its sedative effect. Recently, we reported the neuroprotective effect of the effect of Z. jujuba var. spinosa. However, its effects on synaptic function have not yet been studied. In this project, we examined the action of ethanol extract of the seed of Z. jujuba var. spinosa (DHP1401) on synaptic transmission in the hippocampus. To investigate the effects of DHP1401, field recordings were conducted using hippocampal slices (400µm). Object recognition test was introduced to examine whether DHP1401 affect normal recognition memory. DHP1401 (50μg/ml) induced a significant increase in synaptic activity in Shaffer collateral pathway in a concentration-dependent manner. This increase of synaptic responses was blocked by NBQX, a broad spectrum α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist, but not IEM-1460, a Ca 2+ -permeable AMPAR blocker. Moreover, U0126, a mitogen-activated protein kinase inhibitor, SQ22536, an adenylyl cyclase inhibitor, and PKI, a protein kinase A inhibitor, blocked DHP1401-induced increase in synaptic transmission. Finally, DHP1401 facilitated object recognition memory. These results suggest that DHP1401 increase synaptic transmission through increase of synaptic AMPAR transmission via MAPK, AC and PAK. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

  15. The destructive effect of botulinum neurotoxins on the SNARE protein: SNAP-25 and synaptic membrane fusion

    Directory of Open Access Journals (Sweden)

    Bin Lu

    2015-06-01

    Full Text Available Synaptic exocytosis requires the assembly of syntaxin 1A and SNAP-25 on the plasma membrane and synaptobrevin 2 (VAMP2 on the vesicular membrane to bridge the two opposite membranes. It is believed that the three SNARE proteins assemble in steps along the dynamic assembly pathway. The C-terminus of SNAP-25 is known to be the target of botulinum neurotoxins (BoNT/A and BoNT/E that block neurotransmitters release in vivo. In this study, we employed electron paramagnetic resonance (EPR spectroscopy to investigate the conformation of the SNAP-25 C-terminus in binary and ternary SNARE complexes. The fluorescence lipid mixing assay shows that the C-terminal of SNAP-25 is essential for membrane fusion, and that the truncated SNAP-25 mutants cleaved by BoNT/A and BoNT/E display different inhibition effects on membrane fusion: SNAP-25E (Δ26 abolishes the fusion activity of the SNARE complex, while SNAP-25A (Δ9 loses most of its function, although it can still form a SDS-resistant SNARE complex as the wild-type SNAP-25. CW-EPR spectra validate the unstable structures of the SNARE complex formed by SNAP-25 mutants. We propose that the truncated SNAP-25 mutants will disrupt the assembly of the SNARE core complex, and then inhibit the synaptic membrane fusion accordingly.

  16. Expression of synaptic proteins in the hippocampus and spatial learning in chicks following prenatal auditory stimulation.

    Science.gov (United States)

    Chaudhury, Sraboni; Jain, Suman; Wadhwa, Shashi

    2010-07-01

    Prenatal auditory stimulation by species-specific sound influences the expression and levels of calcium-binding proteins in the chick hippocampus, which is important to learning and memory. Stimulation by sitar music additionally produces structural changes in the hippocampus. Synapse density, which influences the synaptic plasticity, is also increased following both types of sound stimulation. Here we report the expression of mRNA as well as levels of synaptic proteins (synaptophysin, synapsin I and PSD-95) in the hippocampus of developing chicks subjected to prenatal auditory stimulation. Further, to evaluate the behavioral outcome following acoustic stimulation, posthatch day 1 (PH1) chicks were analyzed by T-maze test for spatial learning. Fertilized zero day eggs were incubated under normal conditions and subjected to patterned sounds of species-specific or sitar music at 65 dB levels for 15 min/h over 24 h at a frequency range of 100-6,300 Hz for a period of 11 days from embryonic day (E) 10 until hatching. Following both types of prenatal acoustic stimulation, a significant increase in the levels of synaptophysin mRNA and protein was found from E12, whereas that of synapsin I and PSD-95 was observed from E16, suggesting early maturation of the excitatory synapse. A significant decrease in the time taken to reach the target over the 3 trials in both sound-stimulated groups indicates improved spatial learning. In the music-stimulated group, however, the time taken to reach the target was reduced from the very first trial, which may point to an involvement of other behavioral attributes in facilitating spatial navigation. Copyright 2010 S. Karger AG, Basel.

  17. Structural insights for engineering binding proteins based on non-antibody scaffolds.

    Science.gov (United States)

    Gilbreth, Ryan N; Koide, Shohei

    2012-08-01

    Engineered binding proteins derived from non-antibody scaffolds constitute an increasingly prominent class of reagents in both research and therapeutic applications. The growing number of crystal structures of these 'alternative' scaffold-based binding proteins in complex with their targets illustrate the mechanisms of molecular recognition that are common among these systems and those unique to each. This information is useful for critically assessing and improving/expanding engineering strategies. Furthermore, the structural features of these synthetic proteins produced under tightly controlled, directed evolution deepen our understanding of the underlying principles governing molecular recognition. Copyright © 2012 Elsevier Ltd. All rights reserved.

  18. Guanylate kinase domains of the MAGUK family scaffold proteins as specific phospho-protein-binding modules.

    Science.gov (United States)

    Zhu, Jinwei; Shang, Yuan; Xia, Caihao; Wang, Wenning; Wen, Wenyu; Zhang, Mingjie

    2011-11-25

    Membrane-associated guanylate kinases (MAGUKs) are a large family of scaffold proteins that play essential roles in tissue developments, cell-cell communications, cell polarity control, and cellular signal transductions. Despite extensive studies over the past two decades, the functions of the signature guanylate kinase domain (GK) of MAGUKs are poorly understood. Here we show that the GK domain of DLG1/SAP97 binds to asymmetric cell division regulatory protein LGN in a phosphorylation-dependent manner. The structure of the DLG1 SH3-GK tandem in complex with a phospho-LGN peptide reveals that the GMP-binding site of GK has evolved into a specific pSer/pThr-binding pocket. Residues both N- and C-terminal to the pSer are also critical for the specific binding of the phospho-LGN peptide to GK. We further demonstrate that the previously reported GK domain-mediated interactions of DLGs with other targets, such as GKAP/DLGAP1/SAPAP1 and SPAR, are also phosphorylation dependent. Finally, we provide evidence that other MAGUK GKs also function as phospho-peptide-binding modules. The discovery of the phosphorylation-dependent MAGUK GK/target interactions indicates that MAGUK scaffold-mediated signalling complex organizations are dynamically regulated.

  19. Disruption of Axonal Transport Perturbs Bone Morphogenetic Protein (BMP) - Signaling and Contributes to Synaptic Abnormalities in Two Neurodegenerative Diseases

    Science.gov (United States)

    Kang, Min Jung; Hansen, Timothy J.; Mickiewicz, Monique; Kaczynski, Tadeusz J.; Fye, Samantha; Gunawardena, Shermali

    2014-01-01

    Formation of new synapses or maintenance of existing synapses requires the delivery of synaptic components from the soma to the nerve termini via axonal transport. One pathway that is important in synapse formation, maintenance and function of the Drosophila neuromuscular junction (NMJ) is the bone morphogenetic protein (BMP)-signaling pathway. Here we show that perturbations in axonal transport directly disrupt BMP signaling, as measured by its downstream signal, phospho Mad (p-Mad). We found that components of the BMP pathway genetically interact with both kinesin-1 and dynein motor proteins. Thick vein (TKV) vesicle motility was also perturbed by reductions in kinesin-1 or dynein motors. Interestingly, dynein mutations severely disrupted p-Mad signaling while kinesin-1 mutants showed a mild reduction in p-Mad signal intensity. Similar to mutants in components of the BMP pathway, both kinesin-1 and dynein motor protein mutants also showed synaptic morphological defects. Strikingly TKV motility and p-Mad signaling were disrupted in larvae expressing two human disease proteins; expansions of glutamine repeats (polyQ77) and human amyloid precursor protein (APP) with a familial Alzheimer's disease (AD) mutation (APPswe). Consistent with axonal transport defects, larvae expressing these disease proteins showed accumulations of synaptic proteins along axons and synaptic abnormalities. Taken together our results suggest that similar to the NGF-TrkA signaling endosome, a BMP signaling endosome that directly interacts with molecular motors likely exist. Thus problems in axonal transport occurs early, perturbs BMP signaling, and likely contributes to the synaptic abnormalities observed in these two diseases. PMID:25127478

  20. Disruption of axonal transport perturbs bone morphogenetic protein (BMP)--signaling and contributes to synaptic abnormalities in two neurodegenerative diseases.

    Science.gov (United States)

    Kang, Min Jung; Hansen, Timothy J; Mickiewicz, Monique; Kaczynski, Tadeusz J; Fye, Samantha; Gunawardena, Shermali

    2014-01-01

    Formation of new synapses or maintenance of existing synapses requires the delivery of synaptic components from the soma to the nerve termini via axonal transport. One pathway that is important in synapse formation, maintenance and function of the Drosophila neuromuscular junction (NMJ) is the bone morphogenetic protein (BMP)-signaling pathway. Here we show that perturbations in axonal transport directly disrupt BMP signaling, as measured by its downstream signal, phospho Mad (p-Mad). We found that components of the BMP pathway genetically interact with both kinesin-1 and dynein motor proteins. Thick vein (TKV) vesicle motility was also perturbed by reductions in kinesin-1 or dynein motors. Interestingly, dynein mutations severely disrupted p-Mad signaling while kinesin-1 mutants showed a mild reduction in p-Mad signal intensity. Similar to mutants in components of the BMP pathway, both kinesin-1 and dynein motor protein mutants also showed synaptic morphological defects. Strikingly TKV motility and p-Mad signaling were disrupted in larvae expressing two human disease proteins; expansions of glutamine repeats (polyQ77) and human amyloid precursor protein (APP) with a familial Alzheimer's disease (AD) mutation (APPswe). Consistent with axonal transport defects, larvae expressing these disease proteins showed accumulations of synaptic proteins along axons and synaptic abnormalities. Taken together our results suggest that similar to the NGF-TrkA signaling endosome, a BMP signaling endosome that directly interacts with molecular motors likely exist. Thus problems in axonal transport occurs early, perturbs BMP signaling, and likely contributes to the synaptic abnormalities observed in these two diseases.

  1. Dynamic Control of Synaptic Adhesion and Organizing Molecules in Synaptic Plasticity

    Energy Technology Data Exchange (ETDEWEB)

    Rudenko, Gabby (Texas-MED)

    2017-01-01

    Synapses play a critical role in establishing and maintaining neural circuits, permitting targeted information transfer throughout the brain. A large portfolio of synaptic adhesion/organizing molecules (SAMs) exists in the mammalian brain involved in synapse development and maintenance. SAMs bind protein partners, formingtrans-complexes spanning the synaptic cleft orcis-complexes attached to the same synaptic membrane. SAMs play key roles in cell adhesion and in organizing protein interaction networks; they can also provide mechanisms of recognition, generate scaffolds onto which partners can dock, and likely take part in signaling processes as well. SAMs are regulated through a portfolio of different mechanisms that affect their protein levels, precise localization, stability, and the availability of their partners at synapses. Interaction of SAMs with their partners can further be strengthened or weakened through alternative splicing, competing protein partners, ectodomain shedding, or astrocytically secreted factors. Given that numerous SAMs appear altered by synaptic activity, in vivo, these molecules may be used to dynamically scale up or scale down synaptic communication. Many SAMs, including neurexins, neuroligins, cadherins, and contactins, are now implicated in neuropsychiatric and neurodevelopmental diseases, such as autism spectrum disorder, schizophrenia, and bipolar disorder and studying their molecular mechanisms holds promise for developing novel therapeutics.

  2. Versatile protein recognition by the encoded display of multiple chemical elements on a constant macrocyclic scaffold

    Science.gov (United States)

    Li, Yizhou; De Luca, Roberto; Cazzamalli, Samuele; Pretto, Francesca; Bajic, Davor; Scheuermann, Jörg; Neri, Dario

    2018-03-01

    In nature, specific antibodies can be generated as a result of an adaptive selection and expansion of lymphocytes with suitable protein binding properties. We attempted to mimic antibody-antigen recognition by displaying multiple chemical diversity elements on a defined macrocyclic scaffold. Encoding of the displayed combinations was achieved using distinctive DNA tags, resulting in a library size of 35,393,112. Specific binders could be isolated against a variety of proteins, including carbonic anhydrase IX, horseradish peroxidase, tankyrase 1, human serum albumin, alpha-1 acid glycoprotein, calmodulin, prostate-specific antigen and tumour necrosis factor. Similar to antibodies, the encoded display of multiple chemical elements on a constant scaffold enabled practical applications, such as fluorescence microscopy procedures or the selective in vivo delivery of payloads to tumours. Furthermore, the versatile structure of the scaffold facilitated the generation of protein-specific chemical probes, as illustrated by photo-crosslinking.

  3. Supplementary Material for: A new mode of SAM domain mediated oligomerization observed in the CASKIN2 neuronal scaffolding protein

    KAUST Repository

    Smirnova, Ekaterina

    2016-01-01

    Abstract Background CASKIN2 is a homolog of CASKIN1, a scaffolding protein that participates in a signaling network with CASK (calcium/calmodulin-dependent serine kinase). Despite a high level of homology between CASKIN2 and CASKIN1, CASKIN2 cannot bind CASK due to the absence of a CASK Interaction Domain and consequently, may have evolved undiscovered structural and functional distinctions. Results We demonstrate that the crystal structure of the Sterile Alpha Motif (SAM) domain tandem (SAM1-SAM2) oligomer from CASKIN2 is different than CASKIN1, with the minimal repeating unit being a dimer, rather than a monomer. Analytical ultracentrifugation sedimentation velocity methods revealed differences in monomer/dimer equilibria across a range of concentrations and ionic strengths for the wild type CASKIN2 SAM tandem and a structure-directed double mutant that could not oligomerize. Further distinguishing CASKIN2 from CASKIN1, EGFP-tagged SAM tandem proteins expressed in Neuro2a cells produced punctae that were distinct both in shape and size. Conclusions This study illustrates a new way in which neuronal SAM domains can assemble into large macromolecular assemblies that might concentrate and amplify synaptic responses.

  4. Modulation of Synaptic Plasticity by Exercise Training as a Basis for Ischemic Stroke Rehabilitation.

    Science.gov (United States)

    Nie, Jingjing; Yang, Xiaosu

    2017-01-01

    In recent years, rehabilitation of ischemic stroke draws more and more attention in the world, and has been linked to changes of synaptic plasticity. Exercise training improves motor function of ischemia as well as cognition which is associated with formation of learning and memory. The molecular basis of learning and memory might be synaptic plasticity. Research has therefore been conducted in an attempt to relate effects of exercise training to neuroprotection and neurogenesis adjacent to the ischemic injury brain. The present paper reviews the current literature addressing this question and discusses the possible mechanisms involved in modulation of synaptic plasticity by exercise training. This review shows the pathological process of synaptic dysfunction in ischemic roughly and then discusses the effects of exercise training on scaffold proteins and regulatory protein expression. The expression of scaffold proteins generally increased after training, but the effects on regulatory proteins were mixed. Moreover, the compositions of postsynaptic receptors were changed and the strength of synaptic transmission was enhanced after training. Finally, the recovery of cognition is critically associated with synaptic remodeling in an injured brain, and the remodeling occurs through a number of local regulations including mRNA translation, remodeling of cytoskeleton, and receptor trafficking into and out of the synapse. We do provide a comprehensive knowledge of synaptic plasticity enhancement obtained by exercise training in this review.

  5. A new biocompatible delivery scaffold containing heparin and bone morphogenetic protein 2

    Directory of Open Access Journals (Sweden)

    Thanyaphoo Suphannee

    2016-09-01

    Full Text Available Silicon-substituted calcium phosphate (Si-CaP was developed in our laboratory as a biomaterial for delivery in bone tissue engineering. It was fabricated as a 3D-construct of scaffolds using chitosan-trisodium polyphosphate (TPP cross-linked networks. In this study, heparin was covalently bonded to the residual -NH2 groups of chitosan on the scaffold applying carbodiimide chemistry. Bonded heparin was not leached away from scaffold surfaces upon vigorous washing or extended storage. Recombinant human bone morphogenetic protein 2 (rhBMP-2 was bound to conjugated scaffolds by ionic interactions between the negatively charged SO42- clusters of heparin and positively charged amino acids of rhBMP-2. The resulting scaffolds were inspected for bone regenerative capacity by subcutaneous implanting in rats. Histological observation and mineralization assay were performed after 4 weeks of implantation. Results from both in vitro and in vivo experiments suggest the potential of the developed scaffolds for bone tissue engineering applications in the future.

  6. A new mode of SAM domain mediated oligomerization observed in the CASKIN2 neuronal scaffolding protein

    KAUST Repository

    Smirnova, Ekaterina

    2016-08-22

    Background: CASKIN2 is a homolog of CASKIN1, a scaffolding protein that participates in a signaling network with CASK (calcium/calmodulin-dependent serine kinase). Despite a high level of homology between CASKIN2 and CASKIN1, CASKIN2 cannot bind CASK due to the absence of a CASK Interaction Domain and consequently, may have evolved undiscovered structural and functional distinctions.

  7. Engineering a recyclable elastin-like polypeptide capturing scaffold for non-chromatographic protein purification.

    Science.gov (United States)

    Liu, Fang; Chen, Wilfred

    2013-01-01

    Previously, we reported a non-chromatographic protein purification method exploiting the highly specific interaction between the dockerin and cohesin domains from Clostridium thermocellum and the reversible aggregation property of elastin-like polypeptide (ELP) to provide fast and cost-effective protein purification. However, the bound dockerin-intein tag cannot be completely dissociated from the ELP-cohesin capturing scaffold due to the high binding affinity, resulting in a single-use approach. In order to further reduce the purification cost by recycling the ELP capturing scaffold, a truncated dockerin domain with the calcium-coordinating function partially impaired was employed. We demonstrated that the truncated dockerin domain was sufficient to function as an effective affinity tag, and the target protein was purified directly from cell extracts in a single binding step followed by intein cleavage. The efficient EDTA-mediated dissociation of the bound dockerin-intein tag from the ELP-cohesin capturing scaffold was realized, and the regenerated ELP capturing scaffold was reused in another purification cycle without any decrease in the purification efficiency. This recyclable non-chromatographic based affinity method provides an attractive approach for efficient and cost-effective protein purification. © 2013 American Institute of Chemical Engineers.

  8. Changes in the expression of collapsin response mediator protein-2 during synaptic plasticity in the mouse hippocampus.

    Science.gov (United States)

    Kadoyama, Keiichi; Matsuura, Kenji; Nakamura-Hirota, Tooru; Takano, Masaoki; Otani, Mieko; Matsuyama, Shogo

    2015-11-01

    We have previously reported that nicotine application to the adult mouse causing long-term potentiation-like facilitation in vivo in the hippocampus can serve as a model of synaptic plasticity. The present study clarifies the involvement of collapsin response mediator protein-2 (CRMP2) in synaptic plasticity. CRMP2 was detected in hippocampal neurons of adult mice. The levels of CRMP2 mRNA and protein were increased 2-24 hr and 4-24 hr, respectively, after application of nicotine (3 mg/kg, i.p.), finally returning to the basal level by 48 hr. Furthermore, the ratio of phosphorylated CRMP2 (pCRMP2) at Thr514 residue, an inactive form, to total CRMP2 levels was not changed during synaptic plasticity expressed by nicotine, indicating an enhanced level of non-pCRMP2. This increase of CRMP2 was inhibited by blockade of nicotinic acetylcholine receptors (nAChRs) and required activation of both α4β2 and α7 nAChRs. Although the level of ubiquitinated CRMP2 was increased 8 hr after nicotine treatment, the ratio of ubiquitinated CRMP2 to total CRMP2 protein was similar for nicotine-treated and nontreated mice. This study demonstrates that the expression of CRMP2 increases in hippocampal neurons during synaptic plasticity and that the increment is due mainly to mRNA expression. We propose that CRMP2, particularly non-pCRMP2, could contribute to long-lasting synaptic plasticity. © 2015 Wiley Periodicals, Inc.

  9. Controlled release of recombinant human cementum protein 1 from electrospun multiphasic scaffold for cementum regeneration.

    Science.gov (United States)

    Chen, Xiaofeng; Liu, Yu; Miao, Leiying; Wang, Yangyang; Ren, Shuangshuang; Yang, Xuebin; Hu, Yong; Sun, Weibin

    2016-01-01

    Periodontitis is a major cause for tooth loss, which affects about 15% of the adult population. Cementum regeneration has been the crux of constructing the periodontal complex. Cementum protein 1 (CEMP1) is a cementum-specific protein that can induce cementogenic differentiation. In this study, poly(ethylene glycol) (PEG)-stabilized amorphous calcium phosphate (ACP) nanoparticles were prepared by wet-chemical method and then loaded with recombinant human CEMP1 (rhCEMP1) for controlled release. An electrospun multiphasic scaffold constituted of poly(ε-caprolactone) (PCL), type I collagen (COL), and rhCEMP1/ACP was fabricated. The effects of rhCEMP1/ACP/PCL/COL scaffold on the attachment proliferation, osteogenic, and cementogenic differentiations of human periodontal ligament cells, (PDLCs) were systematically investigated. A critical size defect rat model was introduced to evaluate the effect of tissue regeneration of the scaffolds in vivo. The results showed that PEG-stabilized ACP nanoparticles formed a core-shell structure with sustained release of rhCEMP1 for up to 4 weeks. rhCEMP1/ACP/PCL/COL scaffold could suppress PDLCs proliferation behavior and upregulate the expression of cementoblastic markers including CEMP1 and cementum attachment protein while downregulating osteoblastic markers including osteocalcin and osteopontin when it was cocultured with PDLCs in vitro for 7 days. Histology analysis of cementum after being implanted with the scaffold in rats for 8 weeks showed that there was cementum-like tissue formation but little bone formation. These results indicated the potential of using electrospun multiphasic scaffolds for controlled release of rhCEMP1 for promoting cementum regeneration in reconstruction of the periodontal complex.

  10. Immune labeling and purification of a 71-kDa glutamate-binding protein from brain synaptic membranes

    International Nuclear Information System (INIS)

    Chen, J.W.; Cunningham, M.D.; Galton, N.; Michaelis, E.K.

    1988-01-01

    Immunoblot studies of synaptic membranes isolated from rat brain using antibodies raised against a previously purified glutamate-binding protein (GBP) indicated labeling of an ∼ 70-kDa protein band. Since the antibodies used were raised against a 14-kDa GBP, the present studies were undertaken to explore the possibility that the 14-kDa protein may have been a proteolytic fragment of a larger M/sub r/ protein in synaptic membranes. The major protein enriched in the most highly purified fractions was a 71-kDa glycoprotein, but a 63-kDa protein was co-purified during most steps of the isolation procedure. The glutamate-binding characteristics of these isolated protein fractions were very similar to those previously described for the 14-kDa GBP, including estimated dissociation constants for L-glutamate binding of 0.25 and 1 + M, inhibition of glutamate binding by azide and cyanide, and a selectivity of the ligand binding site for L-glutamate and L-aspartate. The neuroexcitatory analogs of L-glutamate and L-aspartate, ibotenate, quisqualate, and D-glutamate, inhibited L[ 3 H]glutamate binding to the isolated proteins, as did the antagonist of L-glutamate-induced neuronal excitation, L-glutamate diethylester. On the basis of the lack of any detectable glutamate-related enzyme activity associated with the isolated proteins and the presence of distinguishing sensitivities to analogs that inhibit glutamate transport carriers in synaptic membranes, it is proposed that the 71-kDa protein may be a component of a physiologic glutamate receptor complex in neuronal membranes

  11. Changes in hippocampal synaptic functions and protein expression in monosodium glutamate-treated obese mice during development of glucose intolerance.

    Science.gov (United States)

    Sasaki-Hamada, Sachie; Hojo, Yuki; Koyama, Hajime; Otsuka, Hayuma; Oka, Jun-Ichiro

    2015-05-01

    Glucose is the sole neural fuel for the brain and is essential for cognitive function. Abnormalities in glucose tolerance may be associated with impairments in cognitive function. Experimental obese model mice can be generated by an intraperitoneal injection of monosodium glutamate (MSG; 2 mg/g) once a day for 5 days from 1 day after birth. MSG-treated mice have been shown to develop glucose intolerance and exhibit chronic neuroendocrine dysfunction associated with marked cognitive malfunctions at 28-29  weeks old. Although hippocampal synaptic plasticity is impaired in MSG-treated mice, changes in synaptic transmission remain unknown. Here, we investigated whether glucose intolerance influenced cognitive function, synaptic properties and protein expression in the hippocampus. We demonstrated that MSG-treated mice developed glucose intolerance due to an impairment in the effectiveness of insulin actions, and showed cognitive impairments in the Y-maze test. Moreover, long-term potentiation (LTP) at Schaffer collateral-CA1 pyramidal synapses in hippocampal slices was impaired, and the relationship between the slope of extracellular field excitatory postsynaptic potential and stimulus intensity of synaptic transmission was weaker in MSG-treated mice. The protein levels of vesicular glutamate transporter 1 and GluA1 glutamate receptor subunits decreased in the CA1 region of MSG-treated mice. These results suggest that deficits in glutamatergic presynapses as well as postsynapses lead to impaired synaptic plasticity in MSG-treated mice during the development of glucose intolerance, though it remains unknown whether impaired LTP is due to altered inhibitory transmission. It may be important to examine changes in glucose tolerance in order to prevent cognitive malfunctions associated with diabetes. © 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  12. Elliptical structure of phospholipid bilayer nanodiscs encapsulated by scaffold proteins: casting the roles of the lipids and the protein.

    Science.gov (United States)

    Skar-Gislinge, Nicholas; Simonsen, Jens Bæk; Mortensen, Kell; Feidenhans'l, Robert; Sligar, Stephen G; Lindberg Møller, Birger; Bjørnholm, Thomas; Arleth, Lise

    2010-10-06

    Phospholipid bilayers host and support the function of membrane proteins and may be stabilized in disc-like nanostructures, allowing for unprecedented solution studies of the assembly, structure, and function of membrane proteins (Bayburt et al. Nano Lett. 2002, 2, 853-856). Based on small-angle neutron scattering in combination with variable-temperature studies of synchrotron small-angle X-ray scattering on nanodiscs in solution, we show that the fundamental nanodisc unit, consisting of a lipid bilayer surrounded by amphiphilic scaffold proteins, possesses intrinsically an elliptical shape. The temperature dependence of the curvature of the nanodiscs prepared with two different phospholipid types (DLPC and POPC) shows that it is the scaffold protein that determines the overall elliptical shape and that the nanodiscs become more circular with increasing temperature. Our data also show that the hydrophobic bilayer thickness is, to a large extent, dictated by the scaffolding protein and adjusted to minimize the hydrophobic mismatch between protein and phospholipid. Our conclusions result from a new comprehensive and molecular-based model of the nanodisc structure and the use of this to analyze the experimental scattering profile from nanodiscs. The model paves the way for future detailed structural studies of functional membrane proteins encapsulated in nanodiscs.

  13. Fragile X mental retardation protein controls synaptic vesicle exocytosis by modulating N-type calcium channel density

    Science.gov (United States)

    Ferron, Laurent; Nieto-Rostro, Manuela; Cassidy, John S.; Dolphin, Annette C.

    2014-04-01

    Fragile X syndrome (FXS), the most common heritable form of mental retardation, is characterized by synaptic dysfunction. Synaptic transmission depends critically on presynaptic calcium entry via voltage-gated calcium (CaV) channels. Here we show that the functional expression of neuronal N-type CaV channels (CaV2.2) is regulated by fragile X mental retardation protein (FMRP). We find that FMRP knockdown in dorsal root ganglion neurons increases CaV channel density in somata and in presynaptic terminals. We then show that FMRP controls CaV2.2 surface expression by targeting the channels to the proteasome for degradation. The interaction between FMRP and CaV2.2 occurs between the carboxy-terminal domain of FMRP and domains of CaV2.2 known to interact with the neurotransmitter release machinery. Finally, we show that FMRP controls synaptic exocytosis via CaV2.2 channels. Our data indicate that FMRP is a potent regulator of presynaptic activity, and its loss is likely to contribute to synaptic dysfunction in FXS.

  14. Templated self-assembly of quantum dots from aqueous solution using protein scaffolds

    Energy Technology Data Exchange (ETDEWEB)

    Blum, Amy Szuchmacher [Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, 4555 Overlook Avenue SW, Washington, DC 20375 (United States); Soto, Carissa M [Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, 4555 Overlook Avenue SW, Washington, DC 20375 (United States); Wilson, Charmaine D [Geo-Centers, Incorporated, Newton, MA 02459 (United States); Whitley, Jessica L [Geo-Centers, Incorporated, Newton, MA 02459 (United States); Moore, Martin H [Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, 4555 Overlook Avenue SW, Washington, DC 20375 (United States); Sapsford, Kim E [George Mason University, 10910 University Boulevard, Manassas, VA 20110 (United States); Lin, Tianwei [Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 (United States); Chatterji, Anju [Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 (United States); Johnson, John E [Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 (United States); Ratna, Banahalli R [Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, 4555 Overlook Avenue SW, Washington, DC 20375 (United States)

    2006-10-28

    Short, histidine-containing peptides can be conjugated to lysine-containing protein scaffolds to controllably attach quantum dots (QDs) to the scaffold, allowing for generic attachment of quantum dots to any protein without the use of specially engineered domains. This technique was used to bind quantum dots from aqueous solution to both chicken IgG and cowpea mosaic virus (CPMV), a 30 nm viral particle. These quantum dot-protein assemblies were studied in detail. The IgG-QD complexes were shown to retain binding specificity to their antigen after modification. The CPMV-QD complexes have a local concentration of quantum dots greater than 3000 nmol ml{sup -1}, and show a 15% increase in fluorescence quantum yield over free quantum dots in solution.

  15. PSD-95 and PSD-93 Play Critical but Distinct Roles in Synaptic Scaling Up and Down

    Science.gov (United States)

    Sun, Qian; Turrigiano, Gina G.

    2011-01-01

    Synaptic scaling stabilizes neuronal firing through the homeostatic regulation of postsynaptic strength, but the mechanisms by which chronic changes in activity lead to bidirectional adjustments in synaptic AMPAR abundance are incompletely understood. Further, it remains unclear to what extent scaling up and scaling down utilize distinct molecular machinery. PSD-95 is a scaffold protein proposed to serve as a binding “slot” that determines synaptic AMPAR content, and synaptic PSD-95 abundance is regulated by activity, raising the possibility that activity-dependent changes in the synaptic abundance of PSD-95 or other MAGUKs drives the bidirectional changes in AMPAR accumulation during synaptic scaling. We found that synaptic PSD-95 and SAP102 (but not PSD-93) abundance were bidirectionally regulated by activity, but these changes were not sufficient to drive homeostatic changes in synaptic strength. Although not sufficient, the PSD-95-MAGUKs were necessary for synaptic scaling, but scaling up and down were differentially dependent on PSD-95 and PSD-93. Scaling down was completely blocked by reduced or enhanced PSD-95, through a mechanism that depended on the PDZ1/2 domains. In contrast scaling up could be supported by either PSD-95 or PSD-93 in a manner that depended on neuronal age, and was unaffected by a superabundance of PSD-95. Taken together, our data suggest that scaling up and down of quantal amplitude is not driven by changes in synaptic abundance of PSD-95-MAGUKs, but rather that the PSD-95 MAGUKs serve as critical synaptic organizers that utilize distinct protein-protein interactions to mediate homeostatic accumulation and loss of synaptic AMPAR. PMID:21543610

  16. Calcium Phosphate Scaffolds Combined with Bone Morphogenetic Proteins or Mesenchymal Stem Cells in Bone Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Han Sun

    2015-01-01

    Full Text Available Objective: The purpose of this study was to review the current status of calcium phosphate (CaP scaffolds combined with bone morphogenetic proteins (BMPs or mesenchymal stem cells (MSCs in the field of bone tissue engineering (BTE. Date Sources: Data cited in this review were obtained primarily from PubMed and Medline in publications from 1979 to 2014, with highly regarded older publications also included. The terms BTE, CaP, BMPs, and MSC were used for the literature search. Study Selection: Reviews focused on relevant aspects and original articles reporting in vitro and/or in vivo results concerning the efficiency of CaP/BMPs or CaP/MSCs composites were retrieved, reviewed, analyzed, and summarized. Results: An ideal BTE product contains three elements: Scaffold, growth factors, and stem cells. CaP-based scaffolds are popular because of their outstanding biocompatibility, bioactivity, and osteoconductivity. However, they lack stiffness and osteoinductivity. To solve this problem, composite scaffolds of CaP with BMPs have been developed. New bone formation by CaP/BMP composites can reach levels similar to those of autografts. CaP scaffolds are compatible with MSCs and CaP/MSC composites exhibit excellent osteogenesis and stiffness. In addition, a CaP/MSC/BMP scaffold can repair bone defects more effectively than an autograft. Conclusions: Novel BTE products possess remarkable osteoconduction and osteoinduction capacities, and exhibit balanced degradation with osteogenesis. Further work should yield safe, viable, and efficient materials for the repair of bone lesions.

  17. RACK1, A Multifaceted Scaffolding Protein: Structure and Function

    LENUS (Irish Health Repository)

    Adams, David R

    2011-10-06

    Abstract The Receptor for Activated C Kinase 1 (RACK1) is a member of the tryptophan-aspartate repeat (WD-repeat) family of proteins and shares significant homology to the β subunit of G-proteins (Gβ). RACK1 adopts a seven-bladed β-propeller structure which facilitates protein binding. RACK1 has a significant role to play in shuttling proteins around the cell, anchoring proteins at particular locations and in stabilising protein activity. It interacts with the ribosomal machinery, with several cell surface receptors and with proteins in the nucleus. As a result, RACK1 is a key mediator of various pathways and contributes to numerous aspects of cellular function. Here, we discuss RACK1 gene and structure and its role in specific signaling pathways, and address how posttranslational modifications facilitate subcellular location and translocation of RACK1. This review condenses several recent studies suggesting a role for RACK1 in physiological processes such as development, cell migration, central nervous system (CN) function and circadian rhythm as well as reviewing the role of RACK1 in disease.

  18. Optimization of protein cross-linking in bicomponent electrospun scaffolds for therapeutic use

    Energy Technology Data Exchange (ETDEWEB)

    Papa, Antonio [Institute for Polymers, Composites and Biomaterials, National Research Council of Italy (IPCB-CNR), V.le Kennedy 54, Naples 80125 (Italy); IMAST SCaRL, Piazza Bovio 22, 80133 Naples (Italy); Guarino, Vincenzo, E-mail: vincenzo.guarino@cnr.it; Cirillo, Valentina; Oliviero, Olimpia; Ambrosio, Luigi [Institute for Polymers, Composites and Biomaterials, National Research Council of Italy (IPCB-CNR), V.le Kennedy 54, Naples 80125 (Italy)

    2015-12-17

    Bio-instructive electrospun scaffolds based on the combination of synthetic polymers, such as PCL or PLLA, and natural polymers (e.g., collagen) have been extensively investigated as temporary extracellular matrix (ECM) analogues able to support cell proliferation and stem cell differentiation for the regeneration of several tissues. The growing use of natural polymers as carrier of bioactive molecules is introducing new ideas for the design of polymeric drug delivery systems based on electrospun fibers with improved bioavailability, therapeutic efficacy and programmed drug release. In particular, the release mechanism is driven by the use of water soluble proteins (i.e., collagen, gelatin) which fully degrade in in vitro microenvironment, thus delivering the active principles. However, these protein are generally rapidly digested by enzymes (i.e., collagenase) produced by many different cell types, both in vivo and in vitro with significant drawbacks in tissue engineering and controlled drug delivery. Here, we aim at investigating different chemical strategies to improve the in vitro stability and mechanical strength of scaffolds against enzymatic degradation, by modifying the biodegradation rates of proteins embedded in bicomponent fibers. By comparing scaffolds treated by different cross-linking agents (i.e., GC, EDC, BDDGE), we have provided an extensive morphological/chemical/physical characterization via SEM and TGA to identify the best conditions to control drug release via protein degradation from bicomponent fibers without compromising in vitro cell response.

  19. Optimization of protein cross-linking in bicomponent electrospun scaffolds for therapeutic use

    International Nuclear Information System (INIS)

    Papa, Antonio; Guarino, Vincenzo; Cirillo, Valentina; Oliviero, Olimpia; Ambrosio, Luigi

    2015-01-01

    Bio-instructive electrospun scaffolds based on the combination of synthetic polymers, such as PCL or PLLA, and natural polymers (e.g., collagen) have been extensively investigated as temporary extracellular matrix (ECM) analogues able to support cell proliferation and stem cell differentiation for the regeneration of several tissues. The growing use of natural polymers as carrier of bioactive molecules is introducing new ideas for the design of polymeric drug delivery systems based on electrospun fibers with improved bioavailability, therapeutic efficacy and programmed drug release. In particular, the release mechanism is driven by the use of water soluble proteins (i.e., collagen, gelatin) which fully degrade in in vitro microenvironment, thus delivering the active principles. However, these protein are generally rapidly digested by enzymes (i.e., collagenase) produced by many different cell types, both in vivo and in vitro with significant drawbacks in tissue engineering and controlled drug delivery. Here, we aim at investigating different chemical strategies to improve the in vitro stability and mechanical strength of scaffolds against enzymatic degradation, by modifying the biodegradation rates of proteins embedded in bicomponent fibers. By comparing scaffolds treated by different cross-linking agents (i.e., GC, EDC, BDDGE), we have provided an extensive morphological/chemical/physical characterization via SEM and TGA to identify the best conditions to control drug release via protein degradation from bicomponent fibers without compromising in vitro cell response

  20. Optimization of protein cross-linking in bicomponent electrospun scaffolds for therapeutic use

    Science.gov (United States)

    Papa, Antonio; Guarino, Vincenzo; Cirillo, Valentina; Oliviero, Olimpia; Ambrosio, Luigi

    2015-12-01

    Bio-instructive electrospun scaffolds based on the combination of synthetic polymers, such as PCL or PLLA, and natural polymers (e.g., collagen) have been extensively investigated as temporary extracellular matrix (ECM) analogues able to support cell proliferation and stem cell differentiation for the regeneration of several tissues. The growing use of natural polymers as carrier of bioactive molecules is introducing new ideas for the design of polymeric drug delivery systems based on electrospun fibers with improved bioavailability, therapeutic efficacy and programmed drug release. In particular, the release mechanism is driven by the use of water soluble proteins (i.e., collagen, gelatin) which fully degrade in in vitro microenvironment, thus delivering the active principles. However, these protein are generally rapidly digested by enzymes (i.e., collagenase) produced by many different cell types, both in vivo and in vitro with significant drawbacks in tissue engineering and controlled drug delivery. Here, we aim at investigating different chemical strategies to improve the in vitro stability and mechanical strength of scaffolds against enzymatic degradation, by modifying the biodegradation rates of proteins embedded in bicomponent fibers. By comparing scaffolds treated by different cross-linking agents (i.e., GC, EDC, BDDGE), we have provided an extensive morphological/chemical/physical characterization via SEM and TGA to identify the best conditions to control drug release via protein degradation from bicomponent fibers without compromising in vitro cell response.

  1. Structural basis for recognition of synaptic vesicle protein 2C by botulinum neurotoxin A

    Science.gov (United States)

    Benoit, Roger M.; Frey, Daniel; Hilbert, Manuel; Kevenaar, Josta T.; Wieser, Mara M.; Stirnimann, Christian U.; McMillan, David; Ceska, Tom; Lebon, Florence; Jaussi, Rolf; Steinmetz, Michel O.; Schertler, Gebhard F. X.; Hoogenraad, Casper C.; Capitani, Guido; Kammerer, Richard A.

    2014-01-01

    Botulinum neurotoxin A (BoNT/A) belongs to the most dangerous class of bioweapons. Despite this, BoNT/A is used to treat a wide range of common medical conditions such as migraines and a variety of ocular motility and movement disorders. BoNT/A is probably best known for its use as an antiwrinkle agent in cosmetic applications (including Botox and Dysport). BoNT/A application causes long-lasting flaccid paralysis of muscles through inhibiting the release of the neurotransmitter acetylcholine by cleaving synaptosomal-associated protein 25 (SNAP-25) within presynaptic nerve terminals. Two types of BoNT/A receptor have been identified, both of which are required for BoNT/A toxicity and are therefore likely to cooperate with each other: gangliosides and members of the synaptic vesicle glycoprotein 2 (SV2) family, which are putative transporter proteins that are predicted to have 12 transmembrane domains, associate with the receptor-binding domain of the toxin. Recently, fibroblast growth factor receptor 3 (FGFR3) has also been reported to be a potential BoNT/A receptor. In SV2 proteins, the BoNT/A-binding site has been mapped to the luminal domain, but the molecular details of the interaction between BoNT/A and SV2 are unknown. Here we determined the high-resolution crystal structure of the BoNT/A receptor-binding domain (BoNT/A-RBD) in complex with the SV2C luminal domain (SV2C-LD). SV2C-LD consists of a right-handed, quadrilateral β-helix that associates with BoNT/A-RBD mainly through backbone-to-backbone interactions at open β-strand edges, in a manner that resembles the inter-strand interactions in amyloid structures. Competition experiments identified a peptide that inhibits the formation of the complex. Our findings provide a strong platform for the development of novel antitoxin agents and for the rational design of BoNT/A variants with improved therapeutic properties.

  2. Protein scaffolds for selective enrichment of metal ions

    Science.gov (United States)

    He, Chuan; Zhou, Lu; Bosscher, Michael

    2016-02-09

    Polypeptides comprising high affinity for the uranyl ion are provided. Methods for binding uranyl using such proteins are likewise provided and can be used, for example, in methods for uranium purification or removal.

  3. The Diverse Roles of Arrestin Scaffolds in G Protein-Coupled Receptor Signaling.

    Science.gov (United States)

    Peterson, Yuri K; Luttrell, Louis M

    2017-07-01

    The visual/ β -arrestins, a small family of proteins originally described for their role in the desensitization and intracellular trafficking of G protein-coupled receptors (GPCRs), have emerged as key regulators of multiple signaling pathways. Evolutionarily related to a larger group of regulatory scaffolds that share a common arrestin fold, the visual/ β -arrestins acquired the capacity to detect and bind activated GPCRs on the plasma membrane, which enables them to control GPCR desensitization, internalization, and intracellular trafficking. By acting as scaffolds that bind key pathway intermediates, visual/ β -arrestins both influence the tonic level of pathway activity in cells and, in some cases, serve as ligand-regulated scaffolds for GPCR-mediated signaling. Growing evidence supports the physiologic and pathophysiologic roles of arrestins and underscores their potential as therapeutic targets. Circumventing arrestin-dependent GPCR desensitization may alleviate the problem of tachyphylaxis to drugs that target GPCRs, and find application in the management of chronic pain, asthma, and psychiatric illness. As signaling scaffolds, arrestins are also central regulators of pathways controlling cell growth, migration, and survival, suggesting that manipulating their scaffolding functions may be beneficial in inflammatory diseases, fibrosis, and cancer. In this review we examine the structure-function relationships that enable arrestins to perform their diverse roles, addressing arrestin structure at the molecular level, the relationship between arrestin conformation and function, and sites of interaction between arrestins, GPCRs, and nonreceptor-binding partners. We conclude with a discussion of arrestins as therapeutic targets and the settings in which manipulating arrestin function might be of clinical benefit. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

  4. The brain-tumor related protein podoplanin regulates synaptic plasticity and hippocampus-dependent learning and memory.

    Science.gov (United States)

    Cicvaric, Ana; Yang, Jiaye; Krieger, Sigurd; Khan, Deeba; Kim, Eun-Jung; Dominguez-Rodriguez, Manuel; Cabatic, Maureen; Molz, Barbara; Acevedo Aguilar, Juan Pablo; Milicevic, Radoslav; Smani, Tarik; Breuss, Johannes M; Kerjaschki, Dontscho; Pollak, Daniela D; Uhrin, Pavel; Monje, Francisco J

    2016-12-01

    Podoplanin is a cell-surface glycoprotein constitutively expressed in the brain and implicated in human brain tumorigenesis. The intrinsic function of podoplanin in brain neurons remains however uncharacterized. Using an established podoplanin-knockout mouse model and electrophysiological, biochemical, and behavioral approaches, we investigated the brain neuronal role of podoplanin. Ex-vivo electrophysiology showed that podoplanin deletion impairs dentate gyrus synaptic strengthening. In vivo, podoplanin deletion selectively impaired hippocampus-dependent spatial learning and memory without affecting amygdala-dependent cued fear conditioning. In vitro, neuronal overexpression of podoplanin promoted synaptic activity and neuritic outgrowth whereas podoplanin-deficient neurons exhibited stunted outgrowth and lower levels of p-Ezrin, TrkA, and CREB in response to nerve growth factor (NGF). Surface Plasmon Resonance data further indicated a physical interaction between podoplanin and NGF. This work proposes podoplanin as a novel component of the neuronal machinery underlying neuritogenesis, synaptic plasticity, and hippocampus-dependent memory functions. The existence of a relevant cross-talk between podoplanin and the NGF/TrkA signaling pathway is also for the first time proposed here, thus providing a novel molecular complex as a target for future multidisciplinary studies of the brain function in the physiology and the pathology. Key messages Podoplanin, a protein linked to the promotion of human brain tumors, is required in vivo for proper hippocampus-dependent learning and memory functions. Deletion of podoplanin selectively impairs activity-dependent synaptic strengthening at the neurogenic dentate-gyrus and hampers neuritogenesis and phospho Ezrin, TrkA and CREB protein levels upon NGF stimulation. Surface plasmon resonance data indicates a physical interaction between podoplanin and NGF. On these grounds, a relevant cross-talk between podoplanin and NGF as well

  5. Mechanical spectroscopy of retina explants at the protein level employing nanostructured scaffolds.

    Science.gov (United States)

    Mayazur Rahman, S; Reichenbach, Andreas; Zink, Mareike; Mayr, Stefan G

    2016-04-14

    Development of neuronal tissue, such as folding of the brain, and formation of the fovea centralis in the human retina are intimately connected with the mechanical properties of the underlying cells and the extracellular matrix. In particular for neuronal tissue as complex as the vertebrate retina, mechanical properties are still a matter of debate due to their relation to numerous diseases as well as surgery, where the tension of the retina can result in tissue detachment during cutting. However, measuring the elasticity of adult retina wholemounts is difficult and until now only the mechanical properties at the surface have been characterized with micrometer resolution. Many processes, however, such as pathological changes prone to cause tissue rupture and detachment, respectively, are reflected in variations of retina elasticity at smaller length scales at the protein level. In the present work we demonstrate that freely oscillating cantilevers composed of nanostructured TiO2 scaffolds can be employed to study the frequency-dependent mechanical response of adult mammalian retina explants at the nanoscale. Constituting highly versatile scaffolds with strong tissue attachment for long-term organotypic culture atop, these scaffolds perform damped vibrations as fingerprints of the mechanical tissue properties that are derived using finite element calculations. Since the tissue adheres to the nanostructures via constitutive proteins on the photoreceptor side of the retina, the latter are stretched and compressed during vibration of the underlying scaffold. Probing mechanical response of individual proteins within the tissue, the proposed mechanical spectroscopy approach opens the way for studying tissue mechanics, diseases and the effect of drugs at the protein level.

  6. Synaptic Plasticity and Translation Initiation

    Science.gov (United States)

    Klann, Eric; Antion, Marcia D.; Banko, Jessica L.; Hou, Lingfei

    2004-01-01

    It is widely accepted that protein synthesis, including local protein synthesis at synapses, is required for several forms of synaptic plasticity. Local protein synthesis enables synapses to control synaptic strength independent of the cell body via rapid protein production from pre-existing mRNA. Therefore, regulation of translation initiation is…

  7. Cleft palate reconstruction using collagen and nanofiber scaffold incorporating bone morphogenetic protein in rats.

    Science.gov (United States)

    Mostafa, Nesrine Z; Talwar, Reena; Shahin, Mostafa; Unsworth, Larry D; Major, Paul W; Doschak, Michael R

    2015-01-01

    Absorbable collagen sponge (ACS) loaded with bone morphogenetic protein-2 (BMP-2) is approved for selected clinical applications; however, burst release limits its widespread use. Therefore, nanofiber (NF)-based scaffold with ACS backbone was developed to sustain release of loaded BMP-2 to improve the outcomes of bone grafting in a rodent model of cleft palate. BMP-2 was loaded on ACS scaffold and then NF hydrogel with different densities (1-2%) was added to sustain the BMP-2 release. The release profiles of BMP-2 from constructs with different NF densities were evaluated in vitro to explore the optimum NF density that could recapitulate physiological bone healing process. Subsequently, scaffold with the appropriate NF density was implanted into a rodent model of cleft palate. Wistar rats, with surgically induced maxillary cleft defects, were then assigned to one of the following groups (n=6/group): no scaffold (control), ACS, ACS+BMP-2, NF+ACS, and NF+ACS+BMP-2. Micro-computed tomography (μCT) was utilized to evaluate percent bone filling (%BF) at defect site as well as changes in anteroposterior and transverse dimensions of the maxilla at weeks 0, 4, and 8. Histological assessment of bone healing was performed at week 8. In vitro release experiments showed that scaffolds containing 2% NF exhibited a release profile conducive to the natural stages of bone healing and, hence, it was utilized for subsequent in vivo studies. Bone healing occurred at the defect margins leaving a central bone void in the control, ACS, and NF+ACS groups over the 8-week study period. BMP-2-treated groups demonstrated higher %BF as compared with other groups at week 8 (pscaffold when compared with the ACS+BMP-2 group. NF+ACS+BMP-2 constructs exhibited osteoinductive properties together with preparation simplicity, which makes it a novel approach for BMP-2 delivery for cleft palate reconstruction.

  8. Reward memory relieves anxiety-related behavior through synaptic strengthening and protein kinase C in dentate gyrus.

    Science.gov (United States)

    Lei, Zhuofan; Liu, Bei; Wang, Jin-Hui

    2016-04-01

    Anxiety disorders are presumably associated with negative memory. Psychological therapies are widely used to treat this mental deficit in human beings based on the view that positive memory competes with negative memory and relieves anxiety status. Cellular and molecular processes underlying psychological therapies remain elusive. Therefore, we have investigated its mechanisms based on a mouse model in which food reward at one open-arm of the elevated plus-maze was used for training mice to form reward memory and challenge the open arms. Mice with the reward training showed increased entries and stay time in reward open-arm versus neutral open-arm as well as in open-arms versus closed-arms. Accompanying with reward memory formation and anxiety relief, glutamatergic synaptic transmission in dentate gyrus in vivo and dendritic spines in granule cells became upregulated. This synaptic up-regulation was accompanied by the expression of more protein kinase C (PKC) in the dendritic spines. The inhibition of PKC by chelerythrine impaired the formation of reward memory, the relief of anxiety-related behavior and the up-regulation of glutamate synapses. Our results suggest that reward-induced positive memory relieves mouse anxiety-related behavior by strengthening synaptic efficacy and PKC in the hippocampus, which imply the underlying cellular and molecular processes involved in the beneficial effects of psychological therapies treating anxiety disorders. © 2015 Wiley Periodicals, Inc.

  9. pH induced protein-scaffold biosynthesis of tunable shape gold nanoparticles

    Science.gov (United States)

    Zhang, Xiaorong; He, Xiaoxiao; Wang, Kemin; Ren, Fang; Qin, Zhihe

    2011-09-01

    In this paper, a pH-inductive protein-scaffold biosynthesis of shape-tunable crystalline gold nanoparticles at room temperature has been developed. By simple manipulation of the reaction solution's pH, anisotropic gold nanoparticles including spheres, triangles and cubes could be produced by incubating an aqueous solution of sodium tetrachloroaurate with Dolichomitriopsis diversiformis biomasses after immersion in ultrapure Millipore water overnight. A moss protein with molecular weight of about 71 kDa and pI of 4.9 was the primary biomolecule involved in the biosynthesis of gold nanoparticles. The secondary configuration of the proteins by CD spectrum implied that the moss protein could display different secondary configurations including random coil, α-helix and intermediate conformations between random coil and α-helix for the experimental pH solution. The growth process of gold nanoparticles further showed that the moss protein with different configurations provided the template scaffold for the shape-controlled biosynthesis of gold nanoparticles. The constrained shape of the gold nanoparticles, however, disappeared in boiled moss extract. The gold nanoparticles with designed morphology were successfully reconstructed using the moss protein purified from the gold nanoparticles. Structural characterizations by SEM, TEM and SAED showed that the triangular and cubic gold nanoparticles were single crystalline.

  10. pH induced protein-scaffold biosynthesis of tunable shape gold nanoparticles

    International Nuclear Information System (INIS)

    Zhang Xiaorong; He Xiaoxiao; Wang Kemin; Ren Fang; Qin Zhihe

    2011-01-01

    In this paper, a pH-inductive protein-scaffold biosynthesis of shape-tunable crystalline gold nanoparticles at room temperature has been developed. By simple manipulation of the reaction solution's pH, anisotropic gold nanoparticles including spheres, triangles and cubes could be produced by incubating an aqueous solution of sodium tetrachloroaurate with Dolichomitriopsis diversiformis biomasses after immersion in ultrapure Millipore water overnight. A moss protein with molecular weight of about 71 kDa and pI of 4.9 was the primary biomolecule involved in the biosynthesis of gold nanoparticles. The secondary configuration of the proteins by CD spectrum implied that the moss protein could display different secondary configurations including random coil, α-helix and intermediate conformations between random coil and α-helix for the experimental pH solution. The growth process of gold nanoparticles further showed that the moss protein with different configurations provided the template scaffold for the shape-controlled biosynthesis of gold nanoparticles. The constrained shape of the gold nanoparticles, however, disappeared in boiled moss extract. The gold nanoparticles with designed morphology were successfully reconstructed using the moss protein purified from the gold nanoparticles. Structural characterizations by SEM, TEM and SAED showed that the triangular and cubic gold nanoparticles were single crystalline.

  11. Role of the ubiquitin ligase E6AP/UBE3A in controlling levels of the synaptic protein Arc

    Science.gov (United States)

    Kühnle, Simone; Mothes, Benedikt; Matentzoglu, Konstantin; Scheffner, Martin

    2013-01-01

    Inactivation of the ubiquitin ligase E6 associated protein (E6AP) encoded by the UBE3A gene has been associated with development of the Angelman syndrome. Recently, it was reported that in mice, loss of E6AP expression results in increased levels of the synaptic protein Arc and a concomitant impaired synaptic function, providing an explanation for some phenotypic features of Angelman syndrome patients. Accordingly, E6AP has been shown to negatively regulate activity-regulated cytoskeleton-associated protein (Arc) and it has been suggested that E6AP targets Arc for ubiquitination and degradation. In our study, we provide evidence that Arc is not a direct substrate for E6AP and binds only weakly to E6AP, if at all. Furthermore, we show that down-regulation of E6AP expression stimulates estradiol-induced transcription of the Arc gene. Thus, we propose that Arc protein levels are controlled by E6AP at the transcriptional rather than at the posttranslational level. PMID:23671107

  12. Nanofibrous yet injectable polycaprolactone-collagen bone tissue scaffold with osteoprogenitor cells and controlled release of bone morphogenetic protein-2

    Energy Technology Data Exchange (ETDEWEB)

    Subramanian, Gayathri; Bialorucki, Callan [Department of Bioengineering, College of Engineering, University of Toledo, Toledo, OH 43606 (United States); Yildirim-Ayan, Eda, E-mail: eda.yildirimayan@utoledo.edu [Department of Bioengineering, College of Engineering, University of Toledo, Toledo, OH 43606 (United States); Department of Orthopaedic Surgery, University of Toledo Medical Center, Toledo, OH 43614 (United States)

    2015-06-01

    In this work, we developed a nanofibrous, yet injectable orthobiologic tissue scaffold that is capable of hosting osteoprogenitor cells and controlling kinetic release profile of the encapsulated pro-osteogenic factor without diminishing its bioactivity over 21 days. This innovative injectable scaffold was synthesized by incorporating electrospun and subsequently O{sub 2} plasma-functionalized polycaprolactone (PCL) nanofibers within the collagen type-I solution along with MC3T3-E1 cells (pre-osteoblasts) and bone morphogenetic protein-2 (BMP2). Through changing the PCL nanofiber concentration within the injectable scaffolds, we were able to tailor the mechanical strength, protein retention capacity, bioactivity preservation, and osteoinductive potential of the scaffolds. The nanofibrous internal structure of the scaffold allowed us to use a low dose of BMP2 (200 ng/ml) to achieve osteoblastic differentiation in in vitro culture. The osteogenesis capacity of the injectable scaffolds were evaluated though measuring MC3T3-E1 cell proliferation, ALP activity, matrix mineralization, and early- and late-osteoblast specific gene expression profiles over 21 days. The results demonstrated that the nanofibrous injectable scaffold provides not only an osteoinductive environment for osteoprogenitor cells to differentiate, but also a suitable biomechanical and biochemical environment to act as a reservoir for osteogenic factors with controlled release profile. - Highlights: • Injectable nanofibrous scaffold with osteoprogenitor cells and BMP2 was synthesized. • PCL nanofiber concentration within collagen scaffold affected the BMP2 retention and bioactivity. • Optimal PCL concentration was identified for mechanical stability, injectability, and osteogenic activity. • Scaffolds exhibited long-term osteoinductive capacity for bone repair and regeneration.

  13. Scaffolds, levers, rods and springs: diverse cellular functions of long coiled-coil proteins.

    Science.gov (United States)

    Rose, A; Meier, I

    2004-08-01

    Long alpha-helical coiled-coil proteins are involved in a variety of organizational and regulatory processes in eukaryotic cells. They provide cables and networks in the cyto- and nucleoskeleton, molecular scaffolds that organize membrane systems, motors, levers, rotating arms and possibly springs. A growing number of human diseases are found to be caused by mutations in long coiled-coil proteins. This review summarizes our current understanding of the multifaceted group of long coiled-coil proteins in the cytoskeleton, nucleus, Golgi and cell division apparatus. The biophysical features of coiled-coil domains provide first clues toward their contribution to the diverse protein functions and promise potential future applications in the area of nanotechnology. Combining the power of fully sequenced genomes and structure prediction algorithms, it is now possible to comprehensively summarize and compare the complete inventory of coiled-coil proteins of different organisms.

  14. Expressions of visual pigments and synaptic proteins in neonatal chick retina exposed to light of variable photoperiods.

    Science.gov (United States)

    Jha, Kumar Abhiram; Nag, Tapas C; Wadhwa, Shashi; Roy, Tara Sankar

    2016-12-01

    Light causes damage to the retina, which is one of the supposed factors for age-related macular degeneration in human. Some animal species show drastic retinal changes when exposed to intense light (e.g. albino rats). Although birds have a pigmented retina, few reports indicated its susceptibility to light damage. To know how light influences a cone-dominated retina (as is the case with human), we examined the effects of moderate light intensity on the retina of white Leghorn chicks (Gallus g. domesticus). The newly hatched chicks were initially acclimatized at 500 lux for 7 days in 12 h light: 12 h dark cycles (12L:12D). From posthatch day (PH) 8 until PH 30, they were exposed to 2000 lux at 12L:12D, 18L:6D (prolonged light) and 24L:0D (constant light) conditions. The retinas were processed for transmission electron microscopy and the level of expressions of rhodopsin, S- and L/M cone opsins, and synaptic proteins (Synaptophysin and PSD-95) were determined by immunohistochemistry and Western blotting. Rearing in 24L:0D condition caused disorganization of photoreceptor outer segments. Consequently, there were significantly decreased expressions of opsins and synaptic proteins, compared to those seen in 12L:12D and 18L:6D conditions. Also, there were ultrastructural changes in outer and inner plexiform layer (OPL, IPL) of the retinas exposed to 24L:0D condition. Our data indicate that the cone-dominated chick retina is affected in constant light condition, with changes (decreased) in opsin levels. Also, photoreceptor alterations lead to an overall decrease in synaptic protein expressions in OPL and IPL and death of degenerated axonal processes in IPL.

  15. Sex differences in the effects of pre- and postnatal caffeine exposure on behavior and synaptic proteins in pubescent rats.

    Science.gov (United States)

    Sallaberry, Cássia; Ardais, Ana Paula; Rocha, Andréia; Borges, Maurício Felisberto; Fioreze, Gabriela T; Mioranzza, Sabrina; Nunes, Fernanda; Pagnussat, Natália; Botton, Paulo Henrique S; Porciúncula, Lisiane O

    2018-02-02

    Few studies have addressed the effects of caffeine in the puberty and/or adolescence in a sex dependent manner. Considering that caffeine intake has increased in this population, we investigated the behavioral and synaptic proteins changes in pubescent male and female rats after maternal consumption of caffeine. Adult female Wistar rats started to receive water or caffeine (0.1 and 0.3g/L in drinking water; low and moderate dose, respectively) during the active cycle at weekdays, two weeks before mating. The treatment lasted up to weaning and the offspring received caffeine until the onset of puberty (30-34days old). Behavioral tasks were performed to evaluate locomotor activity (open field task), anxious-like behavior (elevated plus maze task) and recognition memory (object recognition task) and synaptic proteins levels (proBDNF, BDNF, GFAP and SNAP-25) were verified in the hippocampus and cerebral cortex. While hyperlocomotion was observed in both sexes after caffeine treatment, anxiety-related behavior was attenuated by caffeine (0.3g/L) only in females. While moderate caffeine worsened recognition memory in females, an improvement in the long-term memory was observed in male rats for both doses. Coincident with memory improvement in males, caffeine increased pro- and BDNF in the hippocampus and cortex. Females presented increased proBDNF levels in both brain regions, with no effects of caffeine. While GFAP was not altered, moderate caffeine intake increased SNAP-25 in the cortex of female rats. Our findings revealed that caffeine promoted cognitive benefits in males associated with increased BDNF levels, while females showed less anxiety. Our findings revealed that caffeine promotes distinct behavioral outcomes and alterations in synaptic proteins during brain development in a sex dependent manner. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Differential regulation of synaptic and extrasynaptic α4 GABA(A) receptor populations by protein kinase A and protein kinase C in cultured cortical neurons.

    Science.gov (United States)

    Bohnsack, John Peyton; Carlson, Stephen L; Morrow, A Leslie

    2016-06-01

    The GABAA α4 subunit exists in two distinct populations of GABAA receptors. Synaptic GABAA α4 receptors are localized at the synapse and mediate phasic inhibitory neurotransmission, while extrasynaptic GABAA receptors are located outside of the synapse and mediate tonic inhibitory transmission. These receptors have distinct pharmacological and biophysical properties that contribute to interest in how these different subtypes are regulated under physiological and pathological states. We utilized subcellular fractionation procedures to separate these populations of receptors in order to investigate their regulation by protein kinases in cortical cultured neurons. Protein kinase A (PKA) activation decreases synaptic α4 expression while protein kinase C (PKC) activation increases α4 subunit expression, and these effects are associated with increased β3 S408/409 or γ2 S327 phosphorylation respectively. In contrast, PKA activation increases extrasynaptic α4 and δ subunit expression, while PKC activation has no effect. Our findings suggest synaptic and extrasynaptic GABAA α4 subunit expression can be modulated by PKA to inform the development of more specific therapeutics for neurological diseases that involve deficits in GABAergic transmission. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. The backbone of the post-synaptic density originated in a unicellular ancestor of choanoflagellates and metazoans

    Directory of Open Access Journals (Sweden)

    Manuel Michaël

    2010-02-01

    Full Text Available Abstract Background Comparative genomics of the early diverging metazoan lineages and of their unicellular sister-groups opens new window to reconstructing the genetic changes which preceded or accompanied the evolution of multicellular body plans. A recent analysis found that the genome of the nerve-less sponges encodes the homologues of most vertebrate post-synaptic proteins. In vertebrate excitatory synapses, these proteins assemble to form the post-synaptic density, a complex molecular platform linking membrane receptors, components of their signalling pathways, and the cytoskeleton. Newly available genomes from Monosiga brevicollis (a member of Choanoflagellata, the closest unicellular relatives of animals and Trichoplax adhaerens (a member of Placozoa: besides sponges, the only nerve-less metazoans offer an opportunity to refine our understanding of post-synaptic protein evolution. Results Searches for orthologous proteins and reconstruction of gene gains/losses based on the taxon phylogeny indicate that post-synaptic proteins originated in two main steps. The backbone scaffold proteins (Shank, Homer, DLG and some of their partners were acquired in a unicellular ancestor of choanoflagellates and metazoans. A substantial additional set appeared in an exclusive ancestor of the Metazoa. The placozoan genome contains most post-synaptic genes but lacks some of them. Notably, the master-scaffold protein Shank might have been lost secondarily in the placozoan lineage. Conclusions The time of origination of most post-synaptic proteins was not concomitant with the acquisition of synapses or neural-like cells. The backbone of the scaffold emerged in a unicellular context and was probably not involved in cell-cell communication. Based on the reconstructed protein composition and potential interactions, its ancestral function could have been to link calcium signalling and cytoskeleton regulation. The complex later became integrated into the evolving

  18. A-Kinase Anchoring Protein-Lbc: A Molecular Scaffold Involved in Cardiac Protection

    Directory of Open Access Journals (Sweden)

    Dario Diviani

    2018-02-01

    Full Text Available Heart failure is a lethal disease that can develop after myocardial infarction, hypertension, or anticancer therapy. In the damaged heart, loss of function is mainly due to cardiomyocyte death and associated cardiac remodeling and fibrosis. In this context, A-kinase anchoring proteins (AKAPs constitute a family of scaffolding proteins that facilitate the spatiotemporal activation of the cyclic adenosine monophosphate (AMP-dependent protein kinase (PKA and other transduction enzymes involved in cardiac remodeling. AKAP-Lbc, a cardiac enriched anchoring protein, has been shown to act as a key coordinator of the activity of signaling pathways involved in cardiac protection and remodeling. This review will summarize and discuss recent advances highlighting the role of the AKAP-Lbc signalosome in orchestrating adaptive responses in the stressed heart.

  19. Biomimetic Engineering of Nanofibrous Gelatin Scaffolds with Noncollagenous Proteins for Enhanced Bone Regeneration

    Science.gov (United States)

    Sun, Yao; Jiang, Yong; Liu, Qilin; Gao, Tian; Feng, Jian Q.; Dechow, Paul; D'Souza, Rena N.; Qin, Chunlin

    2013-01-01

    Biomimetic approaches are widely used in scaffolding designs to enhance tissue regeneration. In this study, we integrated noncollagenous proteins (NCPs) from bone extracellular matrix (ECM) with three-dimensional nanofibrous gelatin (NF-Gelatin) scaffolds to form an artificial matrix (NF-Gelatin-NCPs) mimicking both the nano-structured architecture and chemical composition of natural bone ECM. Through a chemical coupling process, the NCPs were evenly distributed over all the surfaces (inner and outer) of the NF-gelatin-NCPs. The in vitro study showed that the number of osteoblasts (MC3T3-E1) on the NF-Gelatin-NCPs was significantly higher than that on the NF-Gelatin after being cultured for 14 days. Both the alkaline phosphatase (ALP) activity and the expression of osteogenic genes (OPN, BSP, DMP1, CON, and Runx2) were significantly higher in the NF-Gelatin-NCPs than in the NF-Gelatin at 3 weeks. Von Kossa staining, backscattered scanning electron microscopy, and microcomputed tomography all revealed a higher amount of mineral deposition in the NF-Gelatin-NCPs than in the NF-Gelatin after in vitro culturing for 3 weeks. The in vivo calvarial defect study indicated that the NF-Gelatin-NCPs recruited more host cells to the defect and regenerated a higher amount of bone than the controls after implantation for 6 weeks. Immunohistochemical staining also showed high-level mineralization of the bone matrix in the NF-Gelatin-NCPs. Taken together, both the in vitro and in vivo results confirmed that the incorporation of NCPs onto the surfaces of the NF-Gelatin scaffold significantly enhanced osteogenesis and mineralization. Biomimetic engineering of the surfaces of the NF-Gelatin scaffold with NCPs, therefore, is a promising strategy to enhance bone regeneration. PMID:23469769

  20. Neto1 is a novel CUB-domain NMDA receptor-interacting protein required for synaptic plasticity and learning.

    Directory of Open Access Journals (Sweden)

    David Ng

    2009-02-01

    Full Text Available The N-methyl-D-aspartate receptor (NMDAR, a major excitatory ligand-gated ion channel in the central nervous system (CNS, is a principal mediator of synaptic plasticity. Here we report that neuropilin tolloid-like 1 (Neto1, a complement C1r/C1s, Uegf, Bmp1 (CUB domain-containing transmembrane protein, is a novel component of the NMDAR complex critical for maintaining the abundance of NR2A-containing NMDARs in the postsynaptic density. Neto1-null mice have depressed long-term potentiation (LTP at Schaffer collateral-CA1 synapses, with the subunit dependency of LTP induction switching from the normal predominance of NR2A- to NR2B-NMDARs. NMDAR-dependent spatial learning and memory is depressed in Neto1-null mice, indicating that Neto1 regulates NMDA receptor-dependent synaptic plasticity and cognition. Remarkably, we also found that the deficits in LTP, learning, and memory in Neto1-null mice were rescued by the ampakine CX546 at doses without effect in wild-type. Together, our results establish the principle that auxiliary proteins are required for the normal abundance of NMDAR subunits at synapses, and demonstrate that an inherited learning defect can be rescued pharmacologically, a finding with therapeutic implications for humans.

  1. The Human Synaptic Vesicle Protein, SV2A, Functions as a Galactose Transporter in Saccharomyces cerevisiae * ♦

    Science.gov (United States)

    Madeo, Marianna; Kovács, Attila D.; Pearce, David A.

    2014-01-01

    SV2A is a synaptic vesicle membrane protein expressed in neurons and endocrine cells and involved in the regulation of neurotransmitter release. Although the exact function of SV2A still remains elusive, it was identified as the specific binding site for levetiracetam, a second generation antiepileptic drug. Our sequence analysis demonstrates that SV2A has significant homology with several yeast transport proteins belonging to the major facilitator superfamily (MFS). Many of these transporters are involved in sugar transport into yeast cells. Here we present evidence showing, for the first time, that SV2A is a galactose transporter. We expressed human SV2A in hexose transport-deficient EBY.VW4000 yeast cells and demonstrated that these cells are able to grow on galactose-containing medium but not on other fermentable carbon sources. Furthermore, the addition of the SV2A-binding antiepileptic drug levetiracetam to the medium inhibited the galactose-dependent growth of hexose transport-deficient EBY.VW4000 yeast cells expressing human SV2A. Most importantly, direct measurement of galactose uptake in the same strain verified that SV2A is able to transport extracellular galactose inside the cells. The newly identified galactose transport capability of SV2A may have an important role in regulating/modulating synaptic function. PMID:25326386

  2. SynSysNet: integration of experimental data on synaptic protein-protein interactions with drug-target relations

    NARCIS (Netherlands)

    von Eichborn, J.; Dunkel, M.; Gohlke, B.O.; Preissner, S.C.; Hoffmann, M.F.; Bauer, J.M.J.; Armstrong, J.D.; Schaefer, M.H.; Andrade-Navarro, M.A.; Le Novere, N.; Croning, M.D.R.; Grant, S.G.N.; van Nierop, P.; Smit, A.B.; Preissner, R.

    2013-01-01

    We created SynSysNet, available online at http://bioinformatics.charite.de/ synsysnet, to provide a platform that creates a comprehensive 4D network of synaptic interactions. Neuronal synapses are fundamental structures linking nerve cells in the brain and they are responsible for neuronal

  3. Distinct expression of synaptic NR2A and NR2B in the central nervous system and impaired morphine tolerance and physical dependence in mice deficient in postsynaptic density-93 protein

    Directory of Open Access Journals (Sweden)

    Johns Roger A

    2008-10-01

    Full Text Available Abstract Postsynaptic density (PSD-93, a neuronal scaffolding protein, binds to and clusters N-methyl-D-aspartate receptor (NMDAR subunits NR2A and NR2B at cellular membranes in vitro. However, the roles of PSD-93 in synaptic NR2A and NR2B targeting in the central nervous system and NMDAR-dependent physiologic and pathologic processes are still unclear. We report here that PSD-93 deficiency significantly decreased the amount of NR2A and NR2B in the synaptosomal membrane fractions derived from spinal cord dorsal horn and forebrain cortex but did not change their levels in the total soluble fraction from either region. However, PSD-93 deficiency did not markedly change the amounts of NR2A and NR2B in either synaptosomal or total soluble fractions from cerebellum. In mice deficient in PSD-93, morphine dose-dependent curve failed to shift significantly rightward as it did in wild type (WT mice after acute and chronic morphine challenge. Unlike WT mice, PSD-93 knockout mice also showed marked losses of NMDAR-dependent morphine analgesic tolerance and associated abnormal sensitivity in response to mechanical, noxious thermal, and formalin-induced inflammatory stimuli after repeated morphine injection. In addition, PSD-93 knockout mice displayed dramatic loss of jumping activity, a typical NMDAR-mediated morphine withdrawal abstinence behavior. These findings indicate that impaired NMDAR-dependent neuronal plasticity following repeated morphine injection in PSD-93 knockout mice is attributed to PSD-93 deletion-induced alterations of synaptic NR2A and NR2B expression in dorsal horn and forebrain cortex neurons. The selective effect of PSD-93 deletion on synaptic NMDAR expression in these two major pain-related regions might provide the better strategies for the prevention and treatment of opioid tolerance and physical dependence.

  4. Comparative study of human and mouse postsynaptic proteomes finds high compositional conservation and abundance differences for key synaptic proteins.

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    Alex Bayés

    Full Text Available Direct comparison of protein components from human and mouse excitatory synapses is important for determining the suitability of mice as models of human brain disease and to understand the evolution of the mammalian brain. The postsynaptic density is a highly complex set of proteins organized into molecular networks that play a central role in behavior and disease. We report the first direct comparison of the proteome of triplicate isolates of mouse and human cortical postsynaptic densities. The mouse postsynaptic density comprised 1556 proteins and the human one 1461. A large compositional overlap was observed; more than 70% of human postsynaptic density proteins were also observed in the mouse postsynaptic density. Quantitative analysis of postsynaptic density components in both species indicates a broadly similar profile of abundance but also shows that there is higher abundance variation between species than within species. Well known components of this synaptic structure are generally more abundant in the mouse postsynaptic density. Significant inter-species abundance differences exist in some families of key postsynaptic density proteins including glutamatergic neurotransmitter receptors and adaptor proteins. Furthermore, we have identified a closely interacting set of molecules enriched in the human postsynaptic density that could be involved in dendrite and spine structural plasticity. Understanding synapse proteome diversity within and between species will be important to further our understanding of brain complexity and disease.

  5. Green fluorescent protein as a scaffold for high efficiency production of functional bacteriotoxic proteins in Escherichia coli.

    Science.gov (United States)

    Soundrarajan, Nagasundarapandian; Cho, Hye-Sun; Ahn, Byeongyong; Choi, Minkyung; Thong, Le Minh; Choi, Hojun; Cha, Se-Yeoun; Kim, Jin-Hoi; Park, Choi-Kyu; Seo, Kunho; Park, Chankyu

    2016-02-11

    The availability of simple, robust, and cost-effective methods for the large-scale production of bacteriotoxic peptides such as antimicrobial peptides (AMPs) is essential for basic and pharmaceutical research. However, the production of bacteriotoxic proteins has been difficult due to a high degree of toxicity in bacteria and proteolytic degradation. In this study, we inserted AMPs into the Green fluorescent protein (GFP) in a loop region and expressed them as insoluble proteins in high yield, circumventing the inherent toxicity of AMP production in Escherichia coli. The AMPs inserted were released by cyanogen bromide and purified by chromatography. We showed that highly potent AMPs such as Protegrin-1, PMAP-36, Buforin-2, and Bactridin-1 are produced in high yields and produced AMPs showed similar activities compared to chemically synthesized AMPs. We increased the yield more than two-fold by inserting three copies of Protegrin-1 in the GFP scaffold. The immunogold electron micrographs showed that the expressed Protegrin-1 in the GFP scaffold forms large and small size aggregates in the core region of the inclusion body and become entirely nonfunctional, therefore not influencing the proliferation of E. coli. Our novel method will be applicable for diverse bacteriotoxic peptides which can be exploited in biomedical and pharmaceutical researches.

  6. A Protein Scaffold Coordinates SRC-Mediated JNK Activation in Response to Metabolic Stress

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    Shashi Kant

    2017-09-01

    Full Text Available Obesity is a major risk factor for the development of metabolic syndrome and type 2 diabetes. How obesity contributes to metabolic syndrome is unclear. Free fatty acid (FFA activation of a non-receptor tyrosine kinase (SRC-dependent cJun NH2-terminal kinase (JNK signaling pathway is implicated in this process. However, the mechanism that mediates SRC-dependent JNK activation is unclear. Here, we identify a role for the scaffold protein JIP1 in SRC-dependent JNK activation. SRC phosphorylation of JIP1 creates phosphotyrosine interaction motifs that bind the SH2 domains of SRC and the guanine nucleotide exchange factor VAV. These interactions are required for SRC-induced activation of VAV and the subsequent engagement of a JIP1-tethered JNK signaling module. The JIP1 scaffold protein, therefore, plays a dual role in FFA signaling by coordinating upstream SRC functions together with downstream effector signaling by the JNK pathway.

  7. A Protein Scaffold Coordinates SRC-Mediated JNK Activation in Response to Metabolic Stress.

    Science.gov (United States)

    Kant, Shashi; Standen, Claire L; Morel, Caroline; Jung, Dae Young; Kim, Jason K; Swat, Wojciech; Flavell, Richard A; Davis, Roger J

    2017-09-19

    Obesity is a major risk factor for the development of metabolic syndrome and type 2 diabetes. How obesity contributes to metabolic syndrome is unclear. Free fatty acid (FFA) activation of a non-receptor tyrosine kinase (SRC)-dependent cJun NH 2 -terminal kinase (JNK) signaling pathway is implicated in this process. However, the mechanism that mediates SRC-dependent JNK activation is unclear. Here, we identify a role for the scaffold protein JIP1 in SRC-dependent JNK activation. SRC phosphorylation of JIP1 creates phosphotyrosine interaction motifs that bind the SH2 domains of SRC and the guanine nucleotide exchange factor VAV. These interactions are required for SRC-induced activation of VAV and the subsequent engagement of a JIP1-tethered JNK signaling module. The JIP1 scaffold protein, therefore, plays a dual role in FFA signaling by coordinating upstream SRC functions together with downstream effector signaling by the JNK pathway. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  8. Universal method for protein bioconjugation with nanocellulose scaffolds for increased cell adhesion.

    Science.gov (United States)

    Kuzmenko, Volodymyr; Sämfors, Sanna; Hägg, Daniel; Gatenholm, Paul

    2013-12-01

    Bacterial nanocellulose (BNC) is an emerging biomaterial since it is biocompatible, integrates well with host tissue and can be biosynthesized in desired architecture. However, being a hydrogel, it exhibits low affinity for cell attachment, which is crucial for the cellular fate process. To increase cell attachment, the surface of BNC scaffolds was modified with two proteins, fibronectin and collagen type I, using an effective bioconjugation method applying 1-cyano-4-dimethylaminopyridinium (CDAP) tetrafluoroborate as the intermediate catalytic agent. The effect of CDAP treatment on cell adhesion to the BNC surface is shown for human umbilical vein endothelial cells and the mouse mesenchymal stem cell line C3H10T1/2. In both cases, the surface modification increased the number of cells attached to the surfaces. In addition, the morphology of the cells indicated more healthy and viable cells. CDAP activation of bacterial nanocellulose is shown to be a convenient method to conjugate extracellular proteins to the scaffold surfaces. CDAP treatment can be performed in a short period of time in an aqueous environment under heterogeneous and mild conditions preserving the nanofibrillar network of cellulose. © 2013.

  9. Scaffold proteins LACK and TRACK as potential drug targets in kinetoplastid parasites: Development of inhibitors.

    Science.gov (United States)

    Qvit, Nir; Schechtman, Deborah; Pena, Darlene Aparecida; Berti, Denise Aparecida; Soares, Chrislaine Oliveira; Miao, Qianqian; Liang, Liying Annie; Baron, Lauren A; Teh-Poot, Christian; Martínez-Vega, Pedro; Ramirez-Sierra, Maria Jesus; Churchill, Eric; Cunningham, Anna D; Malkovskiy, Andrey V; Federspiel, Nancy A; Gozzo, Fabio Cesar; Torrecilhas, Ana Claudia; Manso Alves, Maria Julia; Jardim, Armando; Momar, Ndao; Dumonteil, Eric; Mochly-Rosen, Daria

    2016-04-01

    Parasitic diseases cause ∼ 500,000 deaths annually and remain a major challenge for therapeutic development. Using a rational design based approach, we developed peptide inhibitors with anti-parasitic activity that were derived from the sequences of parasite scaffold proteins LACK (Leishmania's receptor for activated C-kinase) and TRACK (Trypanosoma receptor for activated C-kinase). We hypothesized that sequences in LACK and TRACK that are conserved in the parasites, but not in the mammalian ortholog, RACK (Receptor for activated C-kinase), may be interaction sites for signaling proteins that are critical for the parasites' viability. One of these peptides exhibited leishmanicidal and trypanocidal activity in culture. Moreover, in infected mice, this peptide was also effective in reducing parasitemia and increasing survival without toxic effects. The identified peptide is a promising new anti-parasitic drug lead, as its unique features may limit toxicity and drug-resistance, thus overcoming central limitations of most anti-parasitic drugs.

  10. Decreased astrocytic thrombospondin-1 secretion after chronic ammonia treatment reduces the level of synaptic proteins: in vitro and in vivo studies.

    Science.gov (United States)

    Jayakumar, Arumugam R; Tong, Xiao Y; Curtis, Kevin M; Ruiz-Cordero, Roberto; Shamaladevi, Nagarajarao; Abuzamel, Missa; Johnstone, Joshua; Gaidosh, Gabriel; Rama Rao, Kakulavarapu V; Norenberg, Michael D

    2014-11-01

    Chronic hepatic encephalopathy (CHE) is a major complication in patients with severe liver disease. Elevated blood and brain ammonia levels have been implicated in its pathogenesis, and astrocytes are the principal neural cells involved in this disorder. Since defective synthesis and release of astrocytic factors have been shown to impair synaptic integrity in other neurological conditions, we examined whether thrombospondin-1 (TSP-1), an astrocytic factor involved in the maintenance of synaptic integrity, is also altered in CHE. Cultured astrocytes were exposed to ammonia (NH₄Cl, 0.5-2.5 mM) for 1-10 days, and TSP-1 content was measured in cell extracts and culture media. Astrocytes exposed to ammonia exhibited a reduction in intra- and extracellular TSP-1 levels. Exposure of cultured neurons to conditioned media from ammonia-treated astrocytes showed a decrease in synaptophysin, PSD95, and synaptotagmin levels. Conditioned media from TSP-1 over-expressing astrocytes that were treated with ammonia, when added to cultured neurons, reversed the decline in synaptic proteins. Recombinant TSP-1 similarly reversed the decrease in synaptic proteins. Metformin, an agent known to increase TSP-1 synthesis in other cell types, also reversed the ammonia-induced TSP-1 reduction. Likewise, we found a significant decline in TSP-1 level in cortical astrocytes, as well as a reduction in synaptophysin content in vivo in a rat model of CHE. These findings suggest that TSP-1 may represent an important therapeutic target for CHE. Defective release of astrocytic factors may impair synaptic integrity in chronic hepatic encephalopathy. We found a reduction in the release of the astrocytic matricellular proteins thrombospondin-1 (TSP-1) in ammonia-treated astrocytes; such reduction was associated with a decrease in synaptic proteins caused by conditioned media from ammonia-treated astrocytes. Exposure of neurons to CM from ammonia-treated astrocytes, in which TSP-1 is over

  11. Deletion of the Vaccinia Virus I2 Protein Interrupts Virion Morphogenesis, Leading to Retention of the Scaffold Protein and Mislocalization of Membrane-Associated Entry Proteins.

    Science.gov (United States)

    Hyun, Seong-In; Weisberg, Andrea; Moss, Bernard

    2017-08-01

    The I2L open reading frame of vaccinia virus (VACV) encodes a conserved 72-amino-acid protein with a putative C-terminal transmembrane domain. Previous studies with a tetracycline-inducible mutant demonstrated that I2-deficient virions are defective in cell entry. The purpose of the present study was to determine the step of replication or entry that is affected by loss of the I2 protein. Fluorescence microscopy experiments showed that I2 colocalized with a major membrane protein of immature and mature virions. We generated a cell line that constitutively expressed I2 and allowed construction of the VACV I2L deletion mutant vΔI2. As anticipated, vΔI2 was unable to replicate in cells that did not express I2. Unexpectedly, morphogenesis was interrupted at a stage after immature virion formation, resulting in the accumulation of dense spherical particles instead of brick-shaped mature virions with well-defined core structures. The abnormal particles retained the D13 scaffold protein of immature virions, were severely deficient in the transmembrane proteins that comprise the entry fusion complex (EFC), and had increased amounts of unprocessed membrane and core proteins. Total lysates of cells infected with vΔI2 also had diminished EFC proteins due to instability attributed to their hydrophobicity and failure to be inserted into viral membranes. A similar instability of EFC proteins had previously been found with unrelated mutants blocked earlier in morphogenesis that also accumulated viral membranes retaining the D13 scaffold. We concluded that I2 is required for virion morphogenesis, release of the D13 scaffold, and the association of EFC proteins with viral membranes. IMPORTANCE Poxviruses comprise a large family that infect vertebrates and invertebrates, cause disease in both in humans and in wild and domesticated animals, and are being engineered as vectors for vaccines and cancer therapy. In addition, investigations of poxviruses have provided insights into

  12. Reactive Oxygen Species-Mediated Loss of Synaptic Akt1 Signaling Leads to Deficient Activity-Dependent Protein Translation Early in Alzheimer's Disease.

    Science.gov (United States)

    Ahmad, Faraz; Singh, Kunal; Das, Debajyoti; Gowaikar, Ruturaj; Shaw, Eisha; Ramachandran, Arathy; Rupanagudi, Khader Valli; Kommaddi, Reddy Peera; Bennett, David A; Ravindranath, Vijayalakshmi

    2017-12-01

    Synaptic deficits are known to underlie the cognitive dysfunction seen in Alzheimer's disease (AD). Generation of reactive oxygen species (ROS) by β-amyloid has also been implicated in AD pathogenesis. However, it is unclear whether ROS contributes to synaptic dysfunction seen in AD pathogenesis and, therefore, we examined whether altered redox signaling could contribute to synaptic deficits in AD. Activity dependent but not basal translation was impaired in synaptoneurosomes from 1-month old presymptomatic APP Swe /PS1ΔE9 (APP/PS1) mice, and this deficit was sustained till middle age (MA, 9-10 months). ROS generation leads to oxidative modification of Akt1 in the synapse and consequent reduction in Akt1-mechanistic target of rapamycin (mTOR) signaling, leading to deficiency in activity-dependent protein translation. Moreover, we found a similar loss of activity-dependent protein translation in synaptoneurosomes from postmortem AD brains. Loss of activity-dependent protein translation occurs presymptomatically early in the pathogenesis of AD. This is caused by ROS-mediated loss of pAkt1, leading to reduced synaptic Akt1-mTOR signaling and is rescued by overexpression of Akt1. ROS-mediated damage is restricted to the synaptosomes, indicating selectivity. We demonstrate that ROS-mediated oxidative modification of Akt1 contributes to synaptic dysfunction in AD, seen as loss of activity-dependent protein translation that is essential for synaptic plasticity and maintenance. Therapeutic strategies promoting Akt1-mTOR signaling at synapses may provide novel target(s) for disease-modifying therapy in AD. Antioxid. Redox Signal. 27, 1269-1280.

  13. A collagen-hydroxyapatite scaffold allows for binding and co-delivery of recombinant bone morphogenetic proteins and bisphosphonates.

    Science.gov (United States)

    Murphy, Ciara M; Schindeler, Aaron; Gleeson, John P; Yu, Nicole Y C; Cantrill, Laurence C; Mikulec, Kathy; Peacock, Lauren; O'Brien, Fergal J; Little, David G

    2014-05-01

    An emerging paradigm in orthopedics is that a bone-healing outcome is the product of the anabolic (bone-forming) and catabolic (bone-resorbing) outcomes. Recently, surgical and tissue engineering strategies have emerged that combine recombinant human bone morphogenetic proteins (rhBMPs) and bisphosphonates (BPs) in order to maximize anabolism and minimize catabolism. Collagen-based scaffolds that are the current surgical standard can bind rhBMPs, but not BPs. We hypothesized that a biomimetic collagen-hydroxyapatite (CHA) scaffold would bind both agents and produce superior in vivo outcomes. Consistent with this concept, in vitro elution studies utilizing rhBMP-2 ELISA assays and scintillation counting of (14)C-radiolabeled zoledronic acid (ZA) confirmed delayed release of both agents from the CHA scaffold. Next, scaffolds were tested for their capacity to form ectopic bone after surgical implantation into the rat hind limb. Using CHA, a significant 6-fold increase in bone volume was seen in rhBMP-2/ZA groups compared to rhBMP-2 alone, confirming the ability of ZA to enhance rhBMP-2 bone formation. CHA scaffolds were found to be capable of generating mineralized tissue in the absence of rhBMP-2. This study has implications for future clinical treatments of critical bone defects. It demonstrates the relative advantages of co-delivering anabolic and anti-catabolic agents using a multicomponent scaffold system. Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.

  14. Elevated progranulin contributes to synaptic and learning deficit due to loss of fragile X mental retardation protein.

    Science.gov (United States)

    Zhang, Kun; Li, Yu-Jiao; Guo, Yanyan; Zheng, Kai-Yin; Yang, Qi; Yang, Le; Wang, Xin-Shang; Song, Qian; Chen, Tao; Zhuo, Min; Zhao, Ming-Gao

    2017-12-01

    Fragile X syndrome is an inheritable form of intellectual disability caused by loss of fragile X mental retardation protein (FMRP, encoded by the FMR1 gene). Absence of FMRP caused overexpression of progranulin (PGRN, encoded by GRN), a putative tumour necrosis factor receptor ligand. In the present study, we found that progranulin mRNA and protein were upregulated in the medial prefrontal cortex of Fmr1 knock-out mice. In Fmr1 knock-out mice, elevated progranulin caused insufficient dendritic spine pruning and late-phase long-term potentiation in the medial prefrontal cortex of Fmr1 knock-out mice. Partial progranulin knock-down restored spine morphology and reversed behavioural deficits, including impaired fear memory, hyperactivity, and motor inflexibility in Fmr1 knock-out mice. Progranulin increased levels of phosphorylated glutamate ionotropic receptor GluA1 and nuclear factor kappa B in cultured wild-type neurons. Tumour necrosis factor receptor 2 antibody perfusion blocked the effects of progranulin on GluA1 phosphorylation; this result indicates that tumour necrosis factor receptor 2 is required for progranulin-mediated GluA1 phosphorylation and late-phase long-term potentiation expression. However, high basal level of progranulin in Fmr1 knock-out mice prevented further facilitation of synaptic plasticity by exogenous progranulin. Partial downregulation of progranulin or tumour necrosis factor receptor 2/nuclear factor kappa B signalling restored synaptic plasticity and memory deficits in Fmr1 knock-out mice. These findings suggest that elevated PGRN is linked to cognitive deficits of fragile X syndrome, and the progranulin/tumour necrosis factor receptor 2 signalling pathway may be a putative therapeutic target for improving cognitive deficits in fragile X syndrome. © The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  15. Biophysical characterization and modeling of human Ecdysoneless (ECD) protein supports a scaffolding function.

    Science.gov (United States)

    Mir, Riyaz A; Lovelace, Jeff; Schafer, Nicholas P; Simone, Peter D; Kellezi, Admir; Kolar, Carol; Spagnol, Gaelle; Sorgen, Paul L; Band, Hamid; Band, Vimla; Borgstahl, Gloria E O

    2016-01-01

    The human homolog of Drosophila ecdysoneless protein (ECD) is a p53 binding protein that stabilizes and enhances p53 functions. Homozygous deletion of mouse Ecd is early embryonic lethal and Ecd deletion delays G 1 -S cell cycle progression. Importantly, ECD directly interacts with the Rb tumor suppressor and competes with the E2F transcription factor for binding to Rb. Further studies demonstrated ECD is overexpressed in breast and pancreatic cancers and its overexpression correlates with poor patient survival. ECD overexpression together with Ras induces cellular transformation through upregulation of autophagy. Recently we demonstrated that CK2 mediated phosphorylation of ECD and interaction with R2TP complex are important for its cell cycle regulatory function. Considering that ECD is a component of multiprotein complexes and its crystal structure is unknown, we characterized ECD structure by circular dichroism measurements and sequence analysis software. These analyses suggest that the majority of ECD is composed of α-helices. Furthermore, small angle X-ray scattering (SAXS) analysis showed that deletion fragments, ECD(1-432) and ECD(1-534), are both well-folded and reveals that the first 400 residues are globular and the next 100 residues are in an extended cylindrical structure. Taking all these results together, we speculate that ECD acts like a structural hub or scaffolding protein in its association with its protein partners. In the future, the hypothetical model presented here for ECD will need to be tested experimentally.

  16. Biophysical characterization and modeling of human Ecdysoneless (ECD protein supports a scaffolding function

    Directory of Open Access Journals (Sweden)

    Nicholas P. Schafer

    2016-03-01

    Full Text Available The human homolog of Drosophila ecdysoneless protein (ECD is a p53 binding protein that stabilizes and enhances p53 functions. Homozygous deletion of mouse Ecd is early embryonic lethal and Ecd deletion delays G1-S cell cycle progression. Importantly, ECD directly interacts with the Rb tumor suppressor and competes with the E2F transcription factor for binding to Rb. Further studies demonstrated ECD is overexpressed in breast and pancreatic cancers and its overexpression correlates with poor patient survival. ECD overexpression together with Ras induces cellular transformation through upregulation of autophagy. Recently we demonstrated that CK2 mediated phosphorylation of ECD and interaction with R2TP complex are important for its cell cycle regulatory function. Considering that ECD is a component of multiprotein complexes and its crystal structure is unknown, we characterized ECD structure by circular dichroism measurements and sequence analysis software. These analyses suggest that the majority of ECD is composed of α-helices. Furthermore, small angle X-ray scattering (SAXS analysis showed that deletion fragments, ECD(1–432 and ECD(1–534, are both well-folded and reveals that the first 400 residues are globular and the next 100 residues are in an extended cylindrical structure. Taking all these results together, we speculate that ECD acts like a structural hub or scaffolding protein in its association with its protein partners. In the future, the hypothetical model presented here for ECD will need to be tested experimentally.

  17. Nanoscale Molecular Reorganization of the Inhibitory Postsynaptic Density Is a Determinant of GABAergic Synaptic Potentiation.

    Science.gov (United States)

    Pennacchietti, Francesca; Vascon, Sebastiano; Nieus, Thierry; Rosillo, Christian; Das, Sabyasachi; Tyagarajan, Shiva K; Diaspro, Alberto; Del Bue, Alessio; Petrini, Enrica Maria; Barberis, Andrea; Cella Zanacchi, Francesca

    2017-02-15

    Gephyrin is a key scaffold protein mediating the anchoring of GABAA receptors at inhibitory synapses. Here, we exploited superresolution techniques combined with proximity-based clustering analysis and model simulations to investigate the single-molecule gephyrin reorganization during plasticity of inhibitory synapses in mouse hippocampal cultured neurons. This approach revealed that, during the expression of inhibitory LTP, the increase of gephyrin density at postsynaptic sites is associated with the promoted formation of gephyrin nanodomains. We demonstrate that the gephyrin rearrangement in nanodomains stabilizes the amplitude of postsynaptic currents, indicating that, in addition to the number of synaptic GABAA receptors, the nanoscale distribution of GABAA receptors in the postsynaptic area is a crucial determinant for the expression of inhibitory synaptic plasticity. In addition, the methodology implemented here clears the way to the application of the graph-based theory to single-molecule data for the description and quantification of the spatial organization of the synapse at the single-molecule level. SIGNIFICANCE STATEMENT The mechanisms of inhibitory synaptic plasticity are poorly understood, mainly because the size of the synapse is below the diffraction limit, thus reducing the effectiveness of conventional optical and imaging techniques. Here, we exploited superresolution approaches combined with clustering analysis to study at unprecedented resolution the distribution of the inhibitory scaffold protein gephyrin in response to protocols inducing LTP of inhibitory synaptic responses (iLTP). We found that, during the expression of iLTP, the increase of synaptic gephyrin is associated with the fragmentation of gephyrin in subsynaptic nanodomains. We demonstrate that such synaptic gephyrin nanodomains stabilize the amplitude of inhibitory postsynaptic responses, thus identifying the nanoscale gephyrin rearrangement as a key determinant for inhibitory

  18. A targeted library screen reveals a new inhibitor scaffold for protein kinase D.

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    Manuj Tandon

    Full Text Available Protein kinase D (PKD has emerged as a potential therapeutic target in multiple pathological conditions, including cancer and heart diseases. Potent and selective small molecule inhibitors of PKD are valuable for dissecting PKD-mediated cellular signaling pathways and for therapeutic application. In this study, we evaluated a targeted library of 235 small organic kinase inhibitors for PKD1 inhibitory activity at a single concentration. Twenty-eight PKD inhibitory chemotypes were identified and six exhibited excellent PKD1 selectivity. Five of the six lead structures share a common scaffold, with compound 139 being the most potent and selective for PKD vs PKC and CAMK. Compound 139 was an ATP-competitive PKD1 inhibitor with a low double-digit nanomolar potency and was also cell-active. Kinase profiling analysis identified this class of small molecules as pan-PKD inhibitors, confirmed their selectivity again PKC and CAMK, and demonstrated an overall favorable selectivity profile that could be further enhanced through structural modification. Furthermore, using a PKD homology model based on similar protein kinase structures, docking modes for compound 139 were explored and compared to literature examples of PKD inhibition. Modeling of these compounds at the ATP-binding site of PKD was used to rationalize its high potency and provide the foundation for future further optimization. Accordingly, using biochemical screening of a small number of privileged scaffolds and computational modeling, we have identified a new core structure for highly potent PKD inhibition with promising selectivity against closely related kinases. These lead structures represent an excellent starting point for the further optimization and the design of selective and therapeutically effective small molecule inhibitors of PKD.

  19. Identification of the antiepileptic racetam binding site in the synaptic vesicle protein 2A by molecular dynamics and docking simulations

    Science.gov (United States)

    Correa-Basurto, José; Cuevas-Hernández, Roberto I.; Phillips-Farfán, Bryan V.; Martínez-Archundia, Marlet; Romo-Mancillas, Antonio; Ramírez-Salinas, Gema L.; Pérez-González, Óscar A.; Trujillo-Ferrara, José; Mendoza-Torreblanca, Julieta G.

    2015-01-01

    Synaptic vesicle protein 2A (SV2A) is an integral membrane protein necessary for the proper function of the central nervous system and is associated to the physiopathology of epilepsy. SV2A is the molecular target of the anti-epileptic drug levetiracetam and its racetam analogs. The racetam binding site in SV2A and the non-covalent interactions between racetams and SV2A are currently unknown; therefore, an in silico study was performed to explore these issues. Since SV2A has not been structurally characterized with X-ray crystallography or nuclear magnetic resonance, a three-dimensional (3D) model was built. The model was refined by performing a molecular dynamics simulation (MDS) and the interactions of SV2A with the racetams were determined by docking studies. A reliable 3D model of SV2A was obtained; it reached structural equilibrium during the last 15 ns of the MDS (50 ns) with remaining structural motions in the N-terminus and long cytoplasmic loop. The docking studies revealed that hydrophobic interactions and hydrogen bonds participate importantly in ligand recognition within the binding site. Residues T456, S665, W666, D670 and L689 were important for racetam binding within the trans-membrane hydrophilic core of SV2A. Identifying the racetam binding site within SV2A should facilitate the synthesis of suitable radio-ligands to study treatment response and possibly epilepsy progression. PMID:25914622

  20. Identification of the antiepileptic racetam binding site in the vesicle synaptic protein 2A by molecular dynamics and docking simulations

    Directory of Open Access Journals (Sweden)

    José eCorrea-Basurto

    2015-04-01

    Full Text Available Synaptic vesicle protein 2A (SV2A is an integral membrane protein necessary for the proper function of the central nervous system (CNS and is associated to the physiopathology of epilepsy. SV2A is the molecular target of the anti-epileptic drug levetiracetam (LEV and its racetam analogues. The racetam binding site in SV2A and the non-covalent interactions between racetams and SV2A are currently unknown; therefore, an in silico study was performed to explore these issues. Since SV2A has not been structurally characterized with X-ray crystallography or nuclear magnetic resonance, a three-dimensional (3D model was built. The model was refined by performing a molecular dynamics simulation (MDS and the interactions of SV2A with the racetams were determined by docking studies. A reliable 3D model of SV2A was obtained; it reached structural equilibrium during the last 15 ns of the MDS (50 ns with remaining structural motions in the N-terminus and long cytoplasmic loop. The docking studies revealed that hydrophobic interactions and hydrogen bonds participate importantly in ligand recognition within the binding site. Residues T456, S665, W666, D670 and L689 were important for racetam binding within the trans-membrane hydrophilic core of SV2A. Identifying the racetam binding site within SV2A should facilitate the synthesis of suitable radio-ligands to study treatment response and possibly epilepsy progression.

  1. Adaptive Assembly: Maximizing the Potential of a Given Functional Peptide with a Tailor-Made Protein Scaffold.

    Science.gov (United States)

    Watanabe, Hideki; Honda, Shinya

    2015-09-17

    Protein engineering that exploits known functional peptides holds great promise for generating novel functional proteins. Here we propose a combinatorial approach, termed adaptive assembly, which provides a tailor-made protein scaffold for a given functional peptide. A combinatorial library was designed to create a tailor-made scaffold, which was generated from β hairpins derived from a 10-residue minimal protein "chignolin" and randomized amino acid sequences. We applied adaptive assembly to a peptide with low affinity for the Fc region of human immunoglobulin G, generating a 54-residue protein AF.p17 with a 40,600-fold enhanced affinity. The crystal structure of AF.p17 complexed with the Fc region revealed that the scaffold fixed the active conformation with a unique structure composed of a short α helix, β hairpins, and a loop-like structure. Adaptive assembly can take full advantage of known peptides as assets for generating novel functional proteins. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Near-atomic cryo-EM imaging of a small protein displayed on a designed scaffolding system.

    Science.gov (United States)

    Liu, Yuxi; Gonen, Shane; Gonen, Tamir; Yeates, Todd O

    2018-03-27

    Current single-particle cryo-electron microscopy (cryo-EM) techniques can produce images of large protein assemblies and macromolecular complexes at atomic level detail without the need for crystal growth. However, proteins of smaller size, typical of those found throughout the cell, are not presently amenable to detailed structural elucidation by cryo-EM. Here we use protein design to create a modular, symmetrical scaffolding system to make protein molecules of typical size suitable for cryo-EM. Using a rigid continuous alpha helical linker, we connect a small 17-kDa protein (DARPin) to a protein subunit that was designed to self-assemble into a cage with cubic symmetry. We show that the resulting construct is amenable to structural analysis by single-particle cryo-EM, allowing us to identify and solve the structure of the attached small protein at near-atomic detail, ranging from 3.5- to 5-Å resolution. The result demonstrates that proteins considerably smaller than the theoretical limit of 50 kDa for cryo-EM can be visualized clearly when arrayed in a rigid fashion on a symmetric designed protein scaffold. Furthermore, because the amino acid sequence of a DARPin can be chosen to confer tight binding to various other protein or nucleic acid molecules, the system provides a future route for imaging diverse macromolecules, potentially broadening the application of cryo-EM to proteins of typical size in the cell.

  3. Conformational coupling between receptor and kinase binding sites through a conserved salt bridge in a signaling complex scaffold protein.

    Directory of Open Access Journals (Sweden)

    Davi R Ortega

    Full Text Available Bacterial chemotaxis is one of the best studied signal transduction pathways. CheW is a scaffold protein that mediates the association of the chemoreceptors and the CheA kinase in a ternary signaling complex. The effects of replacing conserved Arg62 of CheW with other residues suggested that the scaffold protein plays a more complex role than simply binding its partner proteins. Although R62A CheW had essentially the same affinity for chemoreceptors and CheA, cells expressing the mutant protein are impaired in chemotaxis. Using a combination of molecular dynamics simulations (MD, NMR spectroscopy, and circular dichroism (CD, we addressed the role of Arg62. Here we show that Arg62 forms a salt bridge with another highly conserved residue, Glu38. Although this interaction is unimportant for overall protein stability, it is essential to maintain the correct alignment of the chemoreceptor and kinase binding sites of CheW. Computational and experimental data suggest that the role of the salt bridge in maintaining the alignment of the two partner binding sites is fundamental to the function of the signaling complex but not to its assembly. We conclude that a key feature of CheW is to maintain the specific geometry between the two interaction sites required for its function as a scaffold.

  4. Protein stabilization with a dipeptide-mimic triazine-scaffolded synthetic affinity ligand.

    Science.gov (United States)

    Sousa, I T; Lourenço, N M T; Afonso, C A M; Taipa, M A

    2013-02-01

    Protein stabilization was achieved by a novel approach based on the adsorption and establishment of affinity-like interactions with a biomimetic triazine-scaffolded ligand. A synthetic lead compound (ligand 3'/11, K(a) ≈ 10(4) M(-1)) was selected from a previously screened solid-phase library of affinity ligands for studies of adsorption and stabilization of cutinase from Fusarium solani pisi used as a model system. This ligand, directly synthesized in agarose by a well-established solid-phase synthesis method, was able to strongly bind cutinase and led to impressive half-lives of more than 8 h at 70 °C, and of approximately 34 h at 60 °C for bound protein (a 25- and 57-fold increase as compared with the free enzyme, respectively). The ligand density in the solid matrix was found to be a determinant parameter for cutinase stabilization. It is conceivable that the highly stabilizing effect observed results from the binding of more than one ligand residue to the enzyme, creating specific macromolecular configurations that lock structural mobility thus improving molecular stability. Copyright © 2013 John Wiley & Sons, Ltd.

  5. Carbohydrate particles as protein carriers and scaffolds: physico-chemical characterization and collagen stability

    Energy Technology Data Exchange (ETDEWEB)

    Peres, Ivone; Rocha, Sandra; Loureiro, Joana A.; Carmo Pereira, Maria do [University of Porto, LEPAE, Chemical Engineering Department, Faculty of Engineering (Portugal); Ivanova, Galya [Universidade do Porto, REQUIMTE, Departamento de Quimica, Faculdade de Ciencias (Portugal); Coelho, Manuel, E-mail: mcoelho@fe.up.pt [University of Porto, LEPAE, Chemical Engineering Department, Faculty of Engineering (Portugal)

    2012-09-15

    The preservation of protein properties after entrapping into polymeric matrices and the effects of drying the emulsions still remains uncertain and controversial. Carbohydrate particles were designed and prepared by homogenization of gum arabic and maltodextrin mixture, with collagen hydrolysate (CH) followed by spray-drying. The encapsulation of CH in the carbohydrate matrix was achieved with an efficiency of 85 {+-} 2 %. The morphology and the size of the particles, before (40-400 nm) and after spray-drying (<20 {mu}m), were characterized by scanning electron microscopy and dynamic light scattering. Measurements of the nuclear relaxation times and application of diffusion ordered spectroscopy, obtained through pulsed field gradient NMR experiments, have been performed to determine the structure of the CH-polysaccharide conjugates and to clarify the mechanism of CH immobilization in the polysaccharide matrix. In vitro release profiles in ultrapure water and in cellular medium reveal that the diffusion rate of CH from the polymeric matrix to the dialysis solution decreases in average 30-50 % over time, compared to free CH molecules. In cellular medium at 37 Degree-Sign C, the complete release of CH from the particles is achieved only after 24 h, demonstrating a significant decrease in the CH mass transfer process when compared with free CH. The findings of this study outline the ability of gum arabic/maltodextrin matrices to entrap and preserve CH original properties after the spray-drying process and support the potential of the polymeric scaffold for protein delivery and tissue engineering.

  6. Mechanisms of the scaffold subunit in facilitating protein phosphatase 2A methylation.

    Directory of Open Access Journals (Sweden)

    Vitali Stanevich

    Full Text Available The function of the biologically essential protein phosphatase 2A (PP2A relies on formation of diverse heterotrimeric holoenzymes, which involves stable association between PP2A scaffold (A and catalytic (C or PP2Ac subunits and binding of variable regulatory subunits. Holoenzyme assembly is highly regulated by carboxyl methylation of PP2Ac-tail; methylation of PP2Ac and association of the A and C subunits are coupled to activation of PP2Ac. Here we showed that PP2A-specific methyltransferase, LCMT-1, exhibits a higher activity toward the core enzyme (A-C heterodimer than free PP2Ac, and the A-subunit facilitates PP2A methylation via three distinct mechanisms: 1 stabilization of a proper protein fold and an active conformation of PP2Ac; 2 limiting the space of PP2Ac-tail movement for enhanced entry into the LCMT-1 active site; and 3 weak electrostatic interactions between LCMT-1 and the N-terminal HEAT repeats of the A-subunit. Our results revealed a new function and novel mechanisms of the A-subunit in PP2A methylation, and coherent control of PP2A activity, methylation, and holoenzyme assembly.

  7. Scaffold proteins LACK and TRACK as potential drug targets in kinetoplastid parasites: Development of inhibitors

    Directory of Open Access Journals (Sweden)

    Nir Qvit

    2016-04-01

    Full Text Available Parasitic diseases cause ∼500,000 deaths annually and remain a major challenge for therapeutic development. Using a rational design based approach, we developed peptide inhibitors with anti-parasitic activity that were derived from the sequences of parasite scaffold proteins LACK (Leishmania's receptor for activated C-kinase and TRACK (Trypanosoma receptor for activated C-kinase. We hypothesized that sequences in LACK and TRACK that are conserved in the parasites, but not in the mammalian ortholog, RACK (Receptor for activated C-kinase, may be interaction sites for signaling proteins that are critical for the parasites' viability. One of these peptides exhibited leishmanicidal and trypanocidal activity in culture. Moreover, in infected mice, this peptide was also effective in reducing parasitemia and increasing survival without toxic effects. The identified peptide is a promising new anti-parasitic drug lead, as its unique features may limit toxicity and drug-resistance, thus overcoming central limitations of most anti-parasitic drugs.

  8. Scaffold protein enigma homolog activates CREB whereas a short splice variant prevents CREB activation in cardiomyocytes.

    Science.gov (United States)

    Ito, Jumpei; Iijima, Masumi; Yoshimoto, Nobuo; Niimi, Tomoaki; Kuroda, Shun'ichi; Maturana, Andrés D

    2015-12-01

    Enigma Homolog (ENH1 or Pdlim5) is a scaffold protein composed of an N-terminal PDZ domain and three LIM domains at the C-terminal end. The enh gene encodes for several splice variants with opposing functions. ENH1 promotes cardiomyocytes hypertrophy whereas ENH splice variants lacking LIM domains prevent it. ENH1 interacts with various Protein Kinase C (PKC) isozymes and Protein Kinase D1 (PKD1). In addition, the binding of ENH1's LIM domains to PKC is sufficient to activate the kinase without stimulation. The downstream events of the ENH1-PKC/PKD1 complex remain unknown. PKC and PKD1 are known to phosphorylate the transcription factor cAMP-response element binding protein (CREB). We tested whether ENH1 could play a role in the activation of CREB. We found that, in neonatal rat ventricular cardiomyocytes, ENH1 interacts with CREB, is necessary for the phosphorylation of CREB at ser133, and the activation of CREB-dependent transcription. On the contrary, the overexpression of ENH3, a LIM-less splice variant, inhibited the phosphorylation of CREB. ENH3 overexpression or shRNA knockdown of ENH1 prevented the CREB-dependent transcription. Our results thus suggest that ENH1 plays an essential role in CREB's activation and dependent transcription in cardiomyocytes. At the opposite, ENH3 prevents the CREB transcriptional activity. In conclusion, these results provide a first molecular explanation to the opposing functions of ENH splice variants. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Enhancer of rudimentary homologue interacts with scaffold attachment factor B at the nuclear matrix to regulate SR protein phosphorylation.

    Science.gov (United States)

    Drakouli, Sotiria; Lyberopoulou, Aggeliki; Papathanassiou, Maria; Mylonis, Ilias; Georgatsou, Eleni

    2017-08-01

    Scaffold attachment factor B1 (SAFB1) is an integral component of the nuclear matrix of vertebrate cells. It binds to DNA on scaffold/matrix attachment region elements, as well as to RNA and a multitude of different proteins, affecting basic cellular activities such as transcription, splicing and DNA damage repair. In the present study, we show that enhancer of rudimentary homologue (ERH) is a new molecular partner of SAFB1 and its 70% homologous paralogue, scaffold attachment factor B2 (SAFB2). ERH interacts directly in the nucleus with the C-terminal Arg-Gly-rich region of SAFB1/2 and co-localizes with it in the insoluble nuclear fraction. ERH, a small ubiquitous protein with striking homology among species and a unique structure, has also been implicated in fundamental cellular mechanisms. Our functional analyses suggest that the SAFB/ERH interaction does not affect SAFB1/2 function in transcription (e.g. as oestrogen receptor α co-repressors), although it reverses the inhibition exerted by SAFB1/2 on the splicing kinase SR protein kinase 1 (SRPK1), which also binds on the C-terminus of SAFB1/2. Accordingly, ERH silencing decreases lamin B receptor and SR protein phosphorylation, which are major SRPK1 substrates, further substantiating the role of SAFB1 and SAFB2 in the co-ordination of nuclear function. © 2017 Federation of European Biochemical Societies.

  10. Emerging Synaptic Molecules as Candidates in the Etiology of Neurological Disorders

    Directory of Open Access Journals (Sweden)

    Viviana I. Torres

    2017-01-01

    Full Text Available Synapses are complex structures that allow communication between neurons in the central nervous system. Studies conducted in vertebrate and invertebrate models have contributed to the knowledge of the function of synaptic proteins. The functional synapse requires numerous protein complexes with specialized functions that are regulated in space and time to allow synaptic plasticity. However, their interplay during neuronal development, learning, and memory is poorly understood. Accumulating evidence links synapse proteins to neurodevelopmental, neuropsychiatric, and neurodegenerative diseases. In this review, we describe the way in which several proteins that participate in cell adhesion, scaffolding, exocytosis, and neurotransmitter reception from presynaptic and postsynaptic compartments, mainly from excitatory synapses, have been associated with several synaptopathies, and we relate their functions to the disease phenotype.

  11. The role of c-AMP-dependent protein kinase in spinal cord and post synaptic dorsal column neurons in a rat model of visceral pain

    OpenAIRE

    Wu, Jing; Su, Guangxiao; Ma, Long; Zhang, Xuan; Lei, Yongzhong; Lin, Qing; Nauta, Haring J.W.; Li, Junfa; Fang, Li

    2007-01-01

    Visceral noxious stimulation induces central neuronal plasticity changes and suggests that the c-AMP-dependent protein kinase (PKA) signal transduction cascade contributes to long-term changes in nociceptive processing at the spinal cord level. Our previous studies reported the clinical neurosurgical interruption of post synaptic dorsal column neuron (PSDC) pathway by performing midline myelotomy effectively alleviating the intractable visceral pain in patients with severe pain. However, the ...

  12. The Networks of Genes Encoding Palmitoylated Proteins in Axonal and Synaptic Compartments Are Affected in PPT1 Overexpressing Neuronal-Like Cells

    Directory of Open Access Journals (Sweden)

    Francesco Pezzini

    2017-08-01

    Full Text Available CLN1 disease (OMIM #256730 is an early childhood ceroid-lipofuscinosis associated with mutated CLN1, whose product Palmitoyl-Protein Thioesterase 1 (PPT1 is a lysosomal enzyme involved in the removal of palmitate residues from S-acylated proteins. In neurons, PPT1 expression is also linked to synaptic compartments. The aim of this study was to unravel molecular signatures connected to CLN1. We utilized SH-SY5Y neuroblastoma cells overexpressing wild type CLN1 (SH-p.wtCLN1 and five selected CLN1 patients’ mutations. The cellular distribution of wtPPT1 was consistent with regular processing of endogenous protein, partially detected inside Lysosomal Associated Membrane Protein 2 (LAMP2 positive vesicles, while the mutants displayed more diffuse cytoplasmic pattern. Transcriptomic profiling revealed 802 differentially expressed genes (DEGs in SH-p.wtCLN1 (as compared to empty-vector transfected cells, whereas the number of DEGs detected in the two mutants (p.L222P and p.M57Nfs*45 was significantly lower. Bioinformatic scrutiny linked DEGs with neurite formation and neuronal transmission. Specifically, neuritogenesis and proliferation of neuronal processes were predicted to be hampered in the wtCLN1 overexpressing cell line, and these findings were corroborated by morphological investigations. Palmitoylation survey identified 113 palmitoylated protein-encoding genes in SH-p.wtCLN1, including 25 ones simultaneously assigned to axonal growth and synaptic compartments. A remarkable decrease in the expression of palmitoylated proteins, functionally related to axonal elongation (GAP43, CRMP1 and NEFM and of the synaptic marker SNAP25, specifically in SH-p.wtCLN1 cells was confirmed by immunoblotting. Subsequent, bioinformatic network survey of DEGs assigned to the synaptic annotations linked 81 DEGs, including 23 ones encoding for palmitoylated proteins. Results obtained in this experimental setting outlined two affected functional modules (connected to

  13. Guanylate kinase domains of the MAGUK family scaffold proteins as specific phospho-protein-binding modules

    OpenAIRE

    Zhu, Jinwei; Shang, Yuan; Xia, Caihao; Wang, Wenning; Wen, Wenyu; Zhang, Mingjie

    2011-01-01

    Membrane-associated guanylate kinases (MAGUK) family proteins contain an inactive guanylate kinase (GK) domain, whose function has been elusive. Here, this domain is revealed as a new type of phospho-peptide-binding module, in which the GMP-binding site has evolved to accommodate phospho-serines or -threonines.

  14. Self-assembly of silk-elastinlike protein polymers into three-dimensional scaffolds for biomedical applications

    Science.gov (United States)

    Zeng, Like

    Production of brand new protein-based materials with precise control over the amino acid sequences at single residue level has been made possible by genetic engineering, through which artificial genes can be developed that encode protein-based materials with desired features. As an example, silk-elastinlike protein polymers (SELPs), composed of tandem repeats of amino acid sequence motifs from Bombyx mori (silkworm) silk and mammalian elastin, have been produced in this approach. SELPs have been studied extensively in the past two decades, however, the fundamental mechanism governing the self-assembly process to date still remains largely unresolved. Further, regardless of the unprecedented success when exploited in areas including drug delivery, gene therapy, and tissue augmentation, SELPs scaffolds as a three-dimensional cell culture model system are complicated by the inability of SELPs to provide the embedded tissue cells with appropriate biochemical stimuli essential for cell survival and function. In this dissertation, it is reported that the self-assembly of silk-elastinlike protein polymers (SELPs) into nanofibers in aqueous solutions can be modulated by tuning the curing temperature, the size of the silk blocks, and the charge of the elastin blocks. A core-sheath model was proposed for nanofiber formation, with the silk blocks in the cores and the hydrated elastin blocks in the sheaths. The folding of the silk blocks into stable cores -- affected by the size of the silk blocks and the charge of the elastin blocks -- plays a critical role in the assembly of silk-elastin nanofibers. The assembled nanofibers further form nanofiber clusters on the microscale, and the nanofiber clusters then coalesce into nanofiber micro-assemblies, interconnection of which eventually leads to the formation of three-dimensional scaffolds with distinct nanoscale and microscale features. SELP-Collagen hybrid scaffolds were also fabricated to enable independent control over the

  15. Hypoxia and Amino Acid Supplementation Synergistically Promote the Osteogenesis of Human Mesenchymal Stem Cells on Silk Protein Scaffolds

    OpenAIRE

    Sengupta, Sejuti; Park, Sang-Hyug; Patel, Atur; Carn, Julia; Lee, Kyongbum; Kaplan, David L.

    2010-01-01

    Tailoring tissue engineering strategies to match patient- and tissue-specific bone regeneration needs offers to improve clinical outcomes. As a step toward this goal, osteogenic outcomes and metabolic parameters were assessed when varying inputs into the bone formation process. Silk protein scaffolds seeded with human mesenchymal stem cells in osteogenic differentiation media were used to study in vitro osteogenesis under varied conditions of amino acid (lysine and proline) concentration and ...

  16. Scaffold protein JLP mediates TCR-initiated CD4+T cell activation and CD154 expression.

    Science.gov (United States)

    Yan, Qi; Yang, Cheng; Fu, Qiang; Chen, Zhaowei; Liu, Shan; Fu, Dou; Rahman, Rahmat N; Nakazato, Ryota; Yoshioka, Katsuji; Kung, Sam K P; Ding, Guohua; Wang, Huiming

    2017-07-01

    CD4 + T-cell activation and its subsequent induction of CD154 (CD40 ligand, CD40L) expression are pivotal in shaping both the humoral and cellular immune responses. Scaffold protein JLP regulates signal transduction pathways and molecular trafficking inside cells, thus represents a critical component in maintaining cellular functions. Its role in regulating CD4 + T-cell activation and CD154 expression, however, is unclear. Here, we demonstrated expression of JLP in mouse tissues of lymph nodes, thymus, spleen, and also CD4 + T cells. Using CD4+ T cells from jlp-deficient and jlp-wild-type mice, we demonstrated that JLP-deficiency impaired T-cell proliferation, IL-2 production, and CD154 induction upon TCR stimulations, but had no impacts on the expression of other surface molecules such as CD25, CD69, and TCR. These observed impaired T-cell functions in the jlp-/- CD4 + T cells were associated with defective NF-AT activation and Ca 2 + influx, but not the MAPK, NF-κB, as well as AP-1 signaling pathways. Our findings indicated that, for the first time, JLP plays a critical role in regulating CD4 + T cells response to TCR stimulation partly by mediating the activation of TCR-initiated Ca 2+ /NF-AT. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Protein-protein interactions in paralogues: Electrostatics modulates specificity on a conserved steric scaffold.

    Science.gov (United States)

    Ivanov, Stefan M; Cawley, Andrew; Huber, Roland G; Bond, Peter J; Warwicker, Jim

    2017-01-01

    An improved knowledge of protein-protein interactions is essential for better understanding of metabolic and signaling networks, and cellular function. Progress tends to be based on structure determination and predictions using known structures, along with computational methods based on evolutionary information or detailed atomistic descriptions. We hypothesized that for the case of interactions across a common interface, between proteins from a pair of paralogue families or within a family of paralogues, a relatively simple interface description could distinguish between binding and non-binding pairs. Using binding data for several systems, and large-scale comparative modeling based on known template complex structures, it is found that charge-charge interactions (for groups bearing net charge) are generally a better discriminant than buried non-polar surface. This is particularly the case for paralogue families that are less divergent, with more reliable comparative modeling. We suggest that electrostatic interactions are major determinants of specificity in such systems, an observation that could be used to predict binding partners.

  18. Protein-protein interactions in paralogues: Electrostatics modulates specificity on a conserved steric scaffold.

    Directory of Open Access Journals (Sweden)

    Stefan M Ivanov

    Full Text Available An improved knowledge of protein-protein interactions is essential for better understanding of metabolic and signaling networks, and cellular function. Progress tends to be based on structure determination and predictions using known structures, along with computational methods based on evolutionary information or detailed atomistic descriptions. We hypothesized that for the case of interactions across a common interface, between proteins from a pair of paralogue families or within a family of paralogues, a relatively simple interface description could distinguish between binding and non-binding pairs. Using binding data for several systems, and large-scale comparative modeling based on known template complex structures, it is found that charge-charge interactions (for groups bearing net charge are generally a better discriminant than buried non-polar surface. This is particularly the case for paralogue families that are less divergent, with more reliable comparative modeling. We suggest that electrostatic interactions are major determinants of specificity in such systems, an observation that could be used to predict binding partners.

  19. Increased signaling by the autism-related Engrailed-2 protein enhances dendritic branching and spine density, alters synaptic structural matching, and exaggerates protein synthesis.

    Directory of Open Access Journals (Sweden)

    Asma Soltani

    Full Text Available Engrailed 1 (En1 and 2 (En2 code for closely related homeoproteins acting as transcription factors and as signaling molecules that contribute to midbrain and hindbrain patterning, to development and maintenance of monoaminergic pathways, and to retinotectal wiring. En2 has been suggested to be an autism susceptibility gene and individuals with autism display an overexpression of this homeogene but the mechanisms remain unclear. We addressed in the present study the effect of exogenously added En2 on the morphology of hippocampal cells that normally express only low levels of Engrailed proteins. By means of RT-qPCR, we confirmed that En1 and En2 were expressed at low levels in hippocampus and hippocampal neurons, and observed a pronounced decrease in En2 expression at birth and during the first postnatal week, a period characterized by intense synaptogenesis. To address a putative effect of Engrailed in dendritogenesis or synaptogenesis, we added recombinant En1 or En2 proteins to hippocampal cell cultures. Both En1 and En2 treatment increased the complexity of the dendritic tree of glutamatergic neurons, but only En2 increased that of GABAergic cells. En1 increased the density of dendritic spines both in vitro and in vivo. En2 had similar but less pronounced effect on spine density. The number of mature synapses remained unchanged upon En1 treatment but was reduced by En2 treatment, as well as the area of post-synaptic densities. Finally, both En1 and En2 elevated mTORC1 activity and protein synthesis in hippocampal cells, suggesting that some effects of Engrailed proteins may require mRNA translation. Our results indicate that Engrailed proteins can play, even at low concentrations, an active role in the morphogenesis of hippocampal cells. Further, they emphasize the over-regulation of GABA cell morphology and the vulnerability of excitatory synapses in a pathological context of En2 overexpression.

  20. Increased signaling by the autism-related Engrailed-2 protein enhances dendritic branching and spine density, alters synaptic structural matching, and exaggerates protein synthesis.

    Science.gov (United States)

    Soltani, Asma; Lebrun, Solène; Carpentier, Gilles; Zunino, Giulia; Chantepie, Sandrine; Maïza, Auriane; Bozzi, Yuri; Desnos, Claire; Darchen, François; Stettler, Olivier

    2017-01-01

    Engrailed 1 (En1) and 2 (En2) code for closely related homeoproteins acting as transcription factors and as signaling molecules that contribute to midbrain and hindbrain patterning, to development and maintenance of monoaminergic pathways, and to retinotectal wiring. En2 has been suggested to be an autism susceptibility gene and individuals with autism display an overexpression of this homeogene but the mechanisms remain unclear. We addressed in the present study the effect of exogenously added En2 on the morphology of hippocampal cells that normally express only low levels of Engrailed proteins. By means of RT-qPCR, we confirmed that En1 and En2 were expressed at low levels in hippocampus and hippocampal neurons, and observed a pronounced decrease in En2 expression at birth and during the first postnatal week, a period characterized by intense synaptogenesis. To address a putative effect of Engrailed in dendritogenesis or synaptogenesis, we added recombinant En1 or En2 proteins to hippocampal cell cultures. Both En1 and En2 treatment increased the complexity of the dendritic tree of glutamatergic neurons, but only En2 increased that of GABAergic cells. En1 increased the density of dendritic spines both in vitro and in vivo. En2 had similar but less pronounced effect on spine density. The number of mature synapses remained unchanged upon En1 treatment but was reduced by En2 treatment, as well as the area of post-synaptic densities. Finally, both En1 and En2 elevated mTORC1 activity and protein synthesis in hippocampal cells, suggesting that some effects of Engrailed proteins may require mRNA translation. Our results indicate that Engrailed proteins can play, even at low concentrations, an active role in the morphogenesis of hippocampal cells. Further, they emphasize the over-regulation of GABA cell morphology and the vulnerability of excitatory synapses in a pathological context of En2 overexpression.

  1. Plasma treatment induces internal surface modifications of electrospun poly(L-lactic) acid scaffold to enhance protein coating

    International Nuclear Information System (INIS)

    Jin Seo, Hyok; Hee Lee, Mi; Kwon, Byeong-Ju; Kim, Hye-Lee; Park, Jong-Chul; Jin Lee, Seung; Kim, Bong-Jin; Wang, Kang-Kyun; Kim, Yong-Rok

    2013-01-01

    Advanced biomaterials should also be bioactive with regard to desirable cellular responses, such as selective protein adsorption and cell attachment, proliferation, and differentiation. To enhance cell-material interactions, surface modifications have commonly been performed. Among the various surface modification approaches, atmospheric pressure glow discharge plasma has been used to change a hydrophobic polymer surface to a hydrophilic surface. Poly(L-lactic acid) (PLLA)-derived scaffolds lack cell recognition signals and the hydrophobic nature of PLLA hinders cell seeding. To make PLLA surfaces more conducive to cell attachment and spreading, surface modifications may be used to create cell-biomaterial interfaces that elicit controlled cell adhesion and maintain differentiated phenotypes. In this study, (He) gaseous atmospheric plasma glow discharge was used to change the characteristics of a 3D-type polymeric scaffold from hydrophobic to hydrophilic on both the outer and inner surfaces of the scaffold and the penetration efficiency with fibronectin was investigated. Field-emission scanning electron microscope images showed that some grooves were formed on the PLLA fibers after plasma treatment. X-ray photoelectron spectroscopy data also showed chemical changes in the PLLA structure. After plasma treatment, -CN (285.76 eV) was increased in C1s and -NH 2 (399.70 eV) was increased significantly and –N=CH (400.80 eV) and –NH 3 + (402.05 eV) were newly appeared in N1s. These changes allowed fibronectin to penetrate into the PLLA scaffold; this could be observed by confocal microscopy. In conclusion, helium atmospheric pressure plasma treatment was effective in modifying the polymeric scaffold, making it hydrophilic, and this treatment can also be used in tissue engineering research as needed to make polymers hydrophilic

  2. Plasma treatment induces internal surface modifications of electrospun poly(L-lactic) acid scaffold to enhance protein coating

    Energy Technology Data Exchange (ETDEWEB)

    Jin Seo, Hyok; Hee Lee, Mi; Kwon, Byeong-Ju; Kim, Hye-Lee; Park, Jong-Chul [Cellbiocontrol Laboratory, Department of Medical Engineering, Yonsei University College of Medicine, Seoul 120-752 (Korea, Republic of); Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul 120-752 (Korea, Republic of); Jin Lee, Seung [Department of Industrial Pharmacy, College of Pharmacy, Ewha Womans University, Seoul 120-750 (Korea, Republic of); Kim, Bong-Jin; Wang, Kang-Kyun; Kim, Yong-Rok [Department of Chemistry, Yonsei University, 50 Yonsei-ro, Seodaemun-Gu, Seoul 120-749 (Korea, Republic of)

    2013-08-21

    Advanced biomaterials should also be bioactive with regard to desirable cellular responses, such as selective protein adsorption and cell attachment, proliferation, and differentiation. To enhance cell-material interactions, surface modifications have commonly been performed. Among the various surface modification approaches, atmospheric pressure glow discharge plasma has been used to change a hydrophobic polymer surface to a hydrophilic surface. Poly(L-lactic acid) (PLLA)-derived scaffolds lack cell recognition signals and the hydrophobic nature of PLLA hinders cell seeding. To make PLLA surfaces more conducive to cell attachment and spreading, surface modifications may be used to create cell-biomaterial interfaces that elicit controlled cell adhesion and maintain differentiated phenotypes. In this study, (He) gaseous atmospheric plasma glow discharge was used to change the characteristics of a 3D-type polymeric scaffold from hydrophobic to hydrophilic on both the outer and inner surfaces of the scaffold and the penetration efficiency with fibronectin was investigated. Field-emission scanning electron microscope images showed that some grooves were formed on the PLLA fibers after plasma treatment. X-ray photoelectron spectroscopy data also showed chemical changes in the PLLA structure. After plasma treatment, -CN (285.76 eV) was increased in C1s and -NH{sub 2} (399.70 eV) was increased significantly and –N=CH (400.80 eV) and –NH{sub 3}{sup +} (402.05 eV) were newly appeared in N1s. These changes allowed fibronectin to penetrate into the PLLA scaffold; this could be observed by confocal microscopy. In conclusion, helium atmospheric pressure plasma treatment was effective in modifying the polymeric scaffold, making it hydrophilic, and this treatment can also be used in tissue engineering research as needed to make polymers hydrophilic.

  3. Cerebellin 4, a synaptic protein, enhances inhibitory activity and resistance of neurons to amyloid-β toxicity.

    Science.gov (United States)

    Chacón, Pedro J; del Marco, Ángel; Arévalo, Ángeles; Domínguez-Giménez, Paloma; García-Segura, Luis Miguel; Rodríguez-Tébar, Alfredo

    2015-02-01

    Imbalances between excitatory and inhibitory transmissions in the brain anticipate the neuronal damage and death that occur in the neurodegenerative diseases like Alzheimer's disease (AD). We previously showed that amyloid-β (Aß), a natural peptide involved in the onset and development of AD, counteracts the neurotrophic activity of the nerve growth factor (NGF) by dampening the γ-aminobutyric acid (GABA)ergic connectivity of cultured hippocampal neurons. Neuronal plasticity is partly controlled by the NGF-promoted expression of the homologue of enhancer-of-split 1 (Hes1), a transcription factor that regulates the formation of GABAergic synapses. We now show that Hes1 controls the expression of cerebellin 4 (Cbln4), a member of a small family of secreted synaptic proteins, and we present the evidence that Cbln4 plays an essential role in the formation and maintenance of inhibitory GABAergic connections. Cbln4 immunoreactivity was found in the hippocampus, mostly in the dendrites and somata of pyramidal neurons. In the CA1, the hippocampal region where the first neurons degenerate in AD, Cbln4 immunoreactivity was associated with GABAergic synapses (detected by vesicular inhibitory amino acid transporter [VGAT] immunostaining), which appear to surround and embrace the somata of CA1 pyramidal neurons (basket cells). Moreover, significant decreases of Hes1, Cbln4, and VGAT immunoreactivities and messenger RNA expression were found in the hippocampus of a mouse model of AD. We also found that either the overexpression of Cbln4 in cultured hippocampal neurons or the application of recombinant Cbln4 to the cultures increased the number of GABAergic varicosities, rescuing neurons from Aß-induced death. In contrast, knockdown of Cbln4 gene in cultured neurons was followed by a large reduction of GABAergic connections. Such an effect was reverted by exogenously added Cbln4. These findings suggest a therapeutic potential for Cbln4 in the treatment of AD. Copyright

  4. Optimization of the silk scaffold sericin removal process for retention of silk fibroin protein structure and mechanical properties

    Energy Technology Data Exchange (ETDEWEB)

    Teh, Thomas K H; Toh, Siew-Lok; Goh, James C H, E-mail: dosgohj@nus.edu.s, E-mail: dostkh@nus.edu.s, E-mail: bietohsl@nus.edu.s [Division of Bioengineering, National University of Singapore (Singapore)

    2010-06-01

    In the process of removing sericin (degumming) from a raw silk scaffold, the fibroin structural integrity is often challenged, leading to mechanical depreciation. This study aims to identify the factors and conditions contributing to fibroin degradation during alkaline degumming and to perform an optimization study of the parameters involved to achieve preservation of fibroin structure and properties. The methodology involves degumming knitted silk scaffolds for various durations (5-90 min) and temperatures (60-100 {sup 0}C). Mechanical agitation and use of the refreshed solution during degumming are included to investigate how these factors contribute to degumming efficiency and fibroin preservation. Characterizations of silk fibroin morphology, mechanical properties and protein components are determined by scanning electron microscopy (SEM), single fiber tensile tests and gel electrophoresis (SDS-PAGE), respectively. Sericin removal is ascertained via SEM imaging and a protein fractionation method involving SDS-PAGE. The results show that fibroin fibrillation, leading to reduced mechanical integrity, is mainly caused by prolonged degumming duration. Through a series of optimization, knitted scaffolds are observed to be optimally degummed and experience negligible mechanical and structural degradation when subjected to alkaline degumming with mechanical agitation for 30 min at 100 {sup 0}C.

  5. Characterization of Small Molecule Scaffolds that Bind to the Shigella Type III Secretion System Protein IpaD

    Science.gov (United States)

    Dey, Supratim; Anbanandam, Asokan; Mumford, Ben E.; De Guzman, Roberto N.

    2017-01-01

    Many pathogens such as Shigella and other bacteria assemble the type III secretion system (T3SS) nanoinjector to inject virulence proteins into their target cells to cause infectious diseases in humans. The rise of drug resistance among pathogens that rely on the T3SS for infectivity, plus the dearth of new antibiotics require alternative strategies in developing new antibiotics. The Shigella T3SS tip protein IpaD is an attractive target for developing anti-infectives because of its essential role in virulence and its exposure on the bacterial surface. Currently, the only known small molecules that bind to IpaD are bile salts sterols. Here, we identified four new small molecule scaffolds that bind to IpaD based on the methylquinoline, pyrrolidin-aniline, hydroxyindole, and morpholinoaniline scaffolds. NMR mapping revealed potential hotspots in IpaD for binding small molecules. These scaffolds can be used as building blocks in developing small molecule inhibitors of IpaD that could lead to new anti-infectives. PMID:28750143

  6. Characterization of Small-Molecule Scaffolds That Bind to the Shigella Type III Secretion System Protein IpaD.

    Science.gov (United States)

    Dey, Supratim; Anbanandam, Asokan; Mumford, Ben E; De Guzman, Roberto N

    2017-09-21

    Many pathogens such as Shigella and other bacteria assemble the type III secretion system (T3SS) nanoinjector to inject virulence proteins into their target cells to cause infectious diseases in humans. The rise of drug resistance among pathogens that rely on the T3SS for infectivity, plus the dearth of new antibiotics require alternative strategies in developing new antibiotics. The Shigella T3SS tip protein IpaD is an attractive target for developing anti-infectives because of its essential role in virulence and its exposure on the bacterial surface. Currently, the only known small molecules that bind to IpaD are bile salt sterols. In this study we identified four new small-molecule scaffolds that bind to IpaD, based on the methylquinoline, pyrrolidine-aniline, hydroxyindole, and morpholinoaniline scaffolds. NMR mapping revealed potential hotspots in IpaD for binding small molecules. These scaffolds can be used as building blocks in developing small-molecule inhibitors of IpaD that could lead to new anti-infectives. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Effects of neonatal exposure to the flame retardant tetrabromobisphenol-A, aluminum diethylphosphinate or zinc stannate on long-term potentiation and synaptic protein levels in mice.

    Science.gov (United States)

    Hendriks, Hester S; Koolen, Lucas A E; Dingemans, Milou M L; Viberg, Henrik; Lee, Iwa; Leonards, Pim E G; Ramakers, Geert M J; Westerink, Remco H S

    2015-12-01

    Brominated flame retardants such as tetrabromobisphenol-A (TBBPA) may exert (developmental) neurotoxic effects. However, data on (neuro)toxicity of halogen-free flame retardants (HFFRs) are scarce. Recent in vitro studies indicated a high neurotoxic potential for some HFFRs, e.g., zinc stannate (ZS), whereas the neurotoxic potential of other HFFRs, such as aluminum diethylphosphinate (Alpi), appears low. However, the in vivo (neuro)toxicity of these compounds is largely unknown. We therefore investigated effects of neonatal exposure to TBBPA, Alpi or ZS on synaptic plasticity in mouse hippocampus. Male C57bl/6 mice received a single oral dose of 211 µmol/kg bw TBBPA, Alpi or ZS on postnatal day (PND) 10. On PND 17-19, effects on hippocampal synaptic plasticity were investigated using ex vivo extracellular field recordings. Additionally, we measured levels of postsynaptic proteins involved in long-term potentiation (LTP) as well as flame retardant concentrations in brain, muscle and liver tissues. All three flame retardants induced minor, but insignificant, effects on LTP. Additionally, TBBPA induced a minor decrease in post-tetanic potentiation. Despite these minor effects, expression of selected synaptic proteins involved in LTP was not affected. The flame retardants could not be measured in significant amounts in the brains, suggesting low bioavailability and/or rapid elimination/metabolism. We therefore conclude that a single neonatal exposure on PND 10 to TBBPA, Alpi or ZS does affect neurodevelopment and synaptic plasticity only to a small extent in mice. Additional data, in particular on persistence, bioaccumulation and (in vivo) toxicity, following prolonged (developmental) exposure are required for further (human) risk assessment.

  8. IL-1 receptor accessory protein-like 1 associated with mental retardation and autism mediates synapse formation by trans-synaptic interaction with protein tyrosine phosphatase δ.

    Science.gov (United States)

    Yoshida, Tomoyuki; Yasumura, Misato; Uemura, Takeshi; Lee, Sung-Jin; Ra, Moonjin; Taguchi, Ryo; Iwakura, Yoichiro; Mishina, Masayoshi

    2011-09-21

    Mental retardation (MR) and autism are highly heterogeneous neurodevelopmental disorders. IL-1-receptor accessory protein-like 1 (IL1RAPL1) is responsible for nonsyndromic MR and is associated with autism. Thus, the elucidation of the functional role of IL1RAPL1 will contribute to our understanding of the pathogenesis of these mental disorders. Here, we showed that knockdown of endogenous IL1RAPL1 in cultured cortical neurons suppressed the accumulation of punctate staining signals for active zone protein Bassoon and decreased the number of dendritic protrusions. Consistently, the expression of IL1RAPL1 in cultured neurons stimulated the accumulation of Bassoon and spinogenesis. The extracellular domain (ECD) of IL1RAPL1 was required and sufficient for the presynaptic differentiation-inducing activity, while both the ECD and cytoplasmic domain were essential for the spinogenic activity. Notably, the synaptogenic activity of IL1RAPL1 was specific for excitatory synapses. Furthermore, we identified presynaptic protein tyrosine phosphatase (PTP) δ as a major IL1RAPL1-ECD interacting protein by affinity chromatography. IL1RAPL1 interacted selectively with certain forms of PTPδ splice variants carrying mini-exon peptides in Ig-like domains. The synaptogenic activity of IL1RAPL1 was abolished in primary neurons from PTPδ knock-out mice. IL1RAPL1 showed robust synaptogenic activity in vivo when transfected into the cortical neurons of wild-type mice but not in PTPδ knock-out mice. These results suggest that IL1RAPL1 mediates synapse formation through trans-synaptic interaction with PTPδ. Our findings raise an intriguing possibility that the impairment of synapse formation may underlie certain forms of MR and autism as a common pathogenic pathway shared by these mental disorders.

  9. A three-dimensional multiporous fibrous scaffold fabricated with regenerated spider silk protein/poly(l-lactic acid) for tissue engineering.

    Science.gov (United States)

    Yu, Qiaozhen; Sun, Chengjun

    2015-02-01

    An axially aligned three-dimensional (3-D) fibrous scaffold was fabricated with regenerated spider silk protein (RSSP)/poly (l-lactic acid) (PLLA) through electrospinning and post treatment. The morphology, mechanical and degradation properties of the scaffold were controlled through the weight ratio of RSSP to PLLA, the thickness of the scaffold and the treatment time. The scaffold with a weight ratio of 2:3 (RSSP:PLLA) had a nanoleaves-on-nanofibers hierarchical nanostructure; the length and thickness of the nanoleaves were about 400 and 30 nm, respectively. The holes of the scaffolds ranged from hundreds of nanometers to several microns. The scaffold showed an ideal mechanical property that it was stiff when dry, but became soft once hydrated in the culture medium. Its degradation rate was very slow in the first 2 months, and then accelerated in the following 2 months. The pH values of the degradation mediums of all the samples remained in the range of 7.40-7.12 during degradation for 6 months. It had good biocompatibility with PC 12 cells. The aligned hierarchical nanostructure could guide the directions of the axon extension. This scaffold has a potential application in Tissue Engineering and controlled release. This study provides a method to produce synthetic or natural biodegradable polymer scaffold with tailored morphology, mechanical, and degradation properties. © 2014 Wiley Periodicals, Inc.

  10. Caffeine and modafinil given during 48 h sleep deprivation modulate object recognition memory and synaptic proteins in the hippocampus of the rat.

    Science.gov (United States)

    Wadhwa, M; Sahu, S; Kumari, P; Kauser, H; Ray, K; Panjwani, U

    2015-11-01

    We aimed to evaluate the effect of caffeine/modafinil on sleep deprivation (SD) induced alterations in recognition memory and synaptic proteins. The data revealed a beneficial effect of caffeine/modafinil against deficit in the familiar object retrieval performance and object exploration ratio after 48 h SD. Caffeine treatment prevented the SD induced down-regulation of synaptophysin and synapsin I proteins with no change in PSD-95 protein in hippocampus. However, modafinil administration improved the down-regulation of synaptophysin, synapsin I and PSD-95 proteins in hippocampus. Hence, caffeine/modafinil can serve as counter measures in amelioration of SD induced consequences at behavioural and protein levels. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Protein growth factors loaded highly porous chitosan scaffold: A comparison of bone healing properties

    Energy Technology Data Exchange (ETDEWEB)

    Nandi, Samit K., E-mail: samitnandi1967@gmail.com [Department of Veterinary Surgery and Radiology, West Bengal University of Animal and Fishery Sciences, Kolkata (India); Kundu, Biswanath, E-mail: biswa_kundu@rediffmail.com [Bioceramics and Coating Division, CSIR—Central Glass and Ceramic Research Institute, Kolkata (India); Basu, Debabrata [Bioceramics and Coating Division, CSIR—Central Glass and Ceramic Research Institute, Kolkata (India)

    2013-04-01

    Present study aimed to investigate and compare effectiveness of porous chitosan alone and in combination with insulin like growth factor-1 (IGF-1) and bone morphogenetic protein-2 (BMP-2) in bone healing. Highly porous (85 ± 2%) with wide distribution of macroporous (70–900 μm) chitosan scaffolds were fabricated as bone substitutes by employing a simple liquid hardening method using 2% (w/v) chitosan suspension. IGF-1 and BMP-2 were infiltrated using vacuum infiltration with freeze drying method. Adsorption efficiency was found to be 87 ± 2 and 90 ± 2% for BMP-2 and IGF-1 respectively. After thorough material characterization (pore details, FTIR and SEM), samples were used for subsequent in vivo animal trial. Eighteen rabbit models were used to evaluate and compare control (chitosan) (group A), chitosan with IGF-1 (group B) and chitosan with BMP-2 (group C) in the repair of critical size bone defect in tibia. Radiologically, there was evidence of radiodensity in defect area from 60th day (initiated on 30th day) in groups B and C as compared to group A and attaining nearly bony density in most of the part at day 90. Histological results depicted well developed osteoblastic proliferation around haversian canal along with proliferating fibroblast, vascularization and reticular network which was more pronounced in group B followed by groups C and A. Fluorochrome labeling and SEM studies in all groups showed similar outcome. Hence, porous chitosan alone and in combination with growth factors (GFs) can be successfully used for bone defect healing with slight advantage of IGF-1 in chitosan samples. - Highlights: ► Fabrication and characterization of porous chitosan with or without IGF-1 and BMP-2 ► Highly porous growth factor loaded chitosan studied in animal subjects for 3 months ► Parameters studied: histopathology, radiology and fluorochrome labeling ► IGF-1 loaded porous chitosan found to be very effective for bone defect healing.

  12. Protein growth factors loaded highly porous chitosan scaffold: A comparison of bone healing properties

    International Nuclear Information System (INIS)

    Nandi, Samit K.; Kundu, Biswanath; Basu, Debabrata

    2013-01-01

    Present study aimed to investigate and compare effectiveness of porous chitosan alone and in combination with insulin like growth factor-1 (IGF-1) and bone morphogenetic protein-2 (BMP-2) in bone healing. Highly porous (85 ± 2%) with wide distribution of macroporous (70–900 μm) chitosan scaffolds were fabricated as bone substitutes by employing a simple liquid hardening method using 2% (w/v) chitosan suspension. IGF-1 and BMP-2 were infiltrated using vacuum infiltration with freeze drying method. Adsorption efficiency was found to be 87 ± 2 and 90 ± 2% for BMP-2 and IGF-1 respectively. After thorough material characterization (pore details, FTIR and SEM), samples were used for subsequent in vivo animal trial. Eighteen rabbit models were used to evaluate and compare control (chitosan) (group A), chitosan with IGF-1 (group B) and chitosan with BMP-2 (group C) in the repair of critical size bone defect in tibia. Radiologically, there was evidence of radiodensity in defect area from 60th day (initiated on 30th day) in groups B and C as compared to group A and attaining nearly bony density in most of the part at day 90. Histological results depicted well developed osteoblastic proliferation around haversian canal along with proliferating fibroblast, vascularization and reticular network which was more pronounced in group B followed by groups C and A. Fluorochrome labeling and SEM studies in all groups showed similar outcome. Hence, porous chitosan alone and in combination with growth factors (GFs) can be successfully used for bone defect healing with slight advantage of IGF-1 in chitosan samples. - Highlights: ► Fabrication and characterization of porous chitosan with or without IGF-1 and BMP-2 ► Highly porous growth factor loaded chitosan studied in animal subjects for 3 months ► Parameters studied: histopathology, radiology and fluorochrome labeling ► IGF-1 loaded porous chitosan found to be very effective for bone defect healing

  13. The RNA-binding protein, ZC3H14, is required for proper poly(A) tail length control, expression of synaptic proteins, and brain function in mice.

    Science.gov (United States)

    Rha, Jennifer; Jones, Stephanie K; Fidler, Jonathan; Banerjee, Ayan; Leung, Sara W; Morris, Kevin J; Wong, Jennifer C; Inglis, George Andrew S; Shapiro, Lindsey; Deng, Qiudong; Cutler, Alicia A; Hanif, Adam M; Pardue, Machelle T; Schaffer, Ashleigh; Seyfried, Nicholas T; Moberg, Kenneth H; Bassell, Gary J; Escayg, Andrew; García, Paul S; Corbett, Anita H

    2017-10-01

    A number of mutations in genes that encode ubiquitously expressed RNA-binding proteins cause tissue specific disease. Many of these diseases are neurological in nature revealing critical roles for this class of proteins in the brain. We recently identified mutations in a gene that encodes a ubiquitously expressed polyadenosine RNA-binding protein, ZC3H14 (Zinc finger CysCysCysHis domain-containing protein 14), that cause a nonsyndromic, autosomal recessive form of intellectual disability. This finding reveals the molecular basis for disease and provides evidence that ZC3H14 is essential for proper brain function. To investigate the role of ZC3H14 in the mammalian brain, we generated a mouse in which the first common exon of the ZC3H14 gene, exon 13 is removed (Zc3h14Δex13/Δex13) leading to a truncated ZC3H14 protein. We report here that, as in the patients, Zc3h14 is not essential in mice. Utilizing these Zc3h14Δex13/Δex13mice, we provide the first in vivo functional characterization of ZC3H14 as a regulator of RNA poly(A) tail length. The Zc3h14Δex13/Δex13 mice show enlarged lateral ventricles in the brain as well as impaired working memory. Proteomic analysis comparing the hippocampi of Zc3h14+/+ and Zc3h14Δex13/Δex13 mice reveals dysregulation of several pathways that are important for proper brain function and thus sheds light onto which pathways are most affected by the loss of ZC3H14. Among the proteins increased in the hippocampi of Zc3h14Δex13/Δex13 mice compared to control are key synaptic proteins including CaMK2a. This newly generated mouse serves as a tool to study the function of ZC3H14 in vivo. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  14. Synaptic Cell Adhesion

    OpenAIRE

    Missler, Markus; Südhof, Thomas C.; Biederer, Thomas

    2012-01-01

    Chemical synapses are asymmetric intercellular junctions that mediate synaptic transmission. Synaptic junctions are organized by trans-synaptic cell adhesion molecules bridging the synaptic cleft. Synaptic cell adhesion molecules not only connect pre- and postsynaptic compartments, but also mediate trans-synaptic recognition and signaling processes that are essential for the establishment, specification, and plasticity of synapses. A growing number of synaptic cell adhesion molecules that inc...

  15. Antagonism of brain insulin-like growth factor-1 receptors blocks estradiol effects on memory and levels of hippocampal synaptic proteins in ovariectomized rats

    Science.gov (United States)

    Nelson, Britta S.; Springer, Rachel C.; Daniel, Jill M.

    2013-01-01

    Rationale Treatment with estradiol, the primary estrogen produced by the ovaries, enhances hippocampus-dependent spatial memory and increases levels of hippocampal synaptic proteins in ovariectomized rats. Increasing evidence indicates that the ability of estradiol to impact the brain and behavior is dependent upon its interaction with insulin-like growth factor-1 (IGF-1). Objectives The goal of the current experiment was to test the hypothesis that the ability of estradiol to impact hippocampus-dependent memory and levels of hippocampal synaptic proteins is dependent on its interaction with IGF-1. Methods Adult rats were ovariectomized and implanted with estradiol or control capsules and trained on a radial-maze spatial memory task. After training, rats were implanted with intracerebroventricular cannulae attached to osmotic minipumps (flow rate 0.15 μl/hr). Half of each hormone treatment group received continuous delivery of JB1 (300 μg/ml), an IGF-1 receptor antagonist, and half received delivery of aCSF vehicle. Rats were tested on trials in the radial-arm maze during which delays were imposed between the 4th and 5th arm choices. Hippocampal levels of synaptic proteins were measured by western blotting. Results Estradiol treatment resulted in significantly enhanced memory. JB1 blocked that enhancement. Estradiol treatment resulted in significantly increased hippocampal levels of postsynaptic density protein 95 (PSD-95), spinophilin, and synaptophysin. JB1 blocked the estradiol-induced increase of PSD-95 and spinophilin and attenuated the increase of synaptophysin. Conclusions Results support a role for IGF-1 receptor activity in estradiol-induced enhancement of spatial memory that may be dependent on changes in synapse structure in the hippocampus brought upon by estradiol/IGF-1 interactions. PMID:24146138

  16. Scaffold protein enigma homolog 1 overcomes the repression of myogenesis activation by inhibitor of DNA binding 2

    International Nuclear Information System (INIS)

    Nakatani, Miyuki; Ito, Jumpei; Koyama, Riko; Iijima, Masumi; Yoshimoto, Nobuo; Niimi, Tomoaki; Kuroda, Shun'ichi; Maturana, Andrés D.

    2016-01-01

    Enigma Homolog 1 (ENH1) is a scaffold protein for signaling proteins and transcription factors. Previously, we reported that ENH1 overexpression promotes the differentiation of C2C12 myoblasts. However, the molecular mechanism underlying the role of ENH1 in the C2C12 cells differentiation remains elusive. ENH1 was shown to inhibit the proliferation of neuroblastoma cells by sequestering Inhibitor of DNA binding protein 2 (Id2) in the cytosol. Id2 is a repressor of basic Helix-Loop-Helix transcription factors activity and prevents myogenesis. Here, we found that ENH1 overcome the Id2 repression of C2C12 cells myogenic differentiation and that ENH1 overexpression promotes mice satellite cells activation, the first step toward myogenic differentiation. In addition, we show that ENH1 interacted with Id2 in C2C12 cells and mice satellite cells. Collectively, our results suggest that ENH1 plays an important role in the activation of myogenesis through the repression of Id2 activity. -- Highlights: •Enigma Homolog 1 (ENH1) is a scaffold protein. •ENH1 binds to inhibitor of DNA binding 2 (Id2) in myoblasts. •ENH1 overexpression overcomes the Id2's repression of myogenesis. •The Id2-ENH1 complex play an important role in the activation of myogenesis.

  17. Scaffold protein enigma homolog 1 overcomes the repression of myogenesis activation by inhibitor of DNA binding 2

    Energy Technology Data Exchange (ETDEWEB)

    Nakatani, Miyuki [Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Nagoya, 464-8106 (Japan); Ito, Jumpei [Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Nagoya, 464-8106 (Japan); Japan Society for the Promotion of Science, Tokyo, 102-0083 (Japan); Koyama, Riko [Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Nagoya, 464-8106 (Japan); Iijima, Masumi; Yoshimoto, Nobuo [The Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka, 567-0047 (Japan); Niimi, Tomoaki [Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Nagoya, 464-8106 (Japan); Kuroda, Shun' ichi [Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Nagoya, 464-8106 (Japan); The Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka, 567-0047 (Japan); Maturana, Andrés D., E-mail: maturana@agr.nagoya-u.ac.jp [Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Nagoya, 464-8106 (Japan)

    2016-05-27

    Enigma Homolog 1 (ENH1) is a scaffold protein for signaling proteins and transcription factors. Previously, we reported that ENH1 overexpression promotes the differentiation of C2C12 myoblasts. However, the molecular mechanism underlying the role of ENH1 in the C2C12 cells differentiation remains elusive. ENH1 was shown to inhibit the proliferation of neuroblastoma cells by sequestering Inhibitor of DNA binding protein 2 (Id2) in the cytosol. Id2 is a repressor of basic Helix-Loop-Helix transcription factors activity and prevents myogenesis. Here, we found that ENH1 overcome the Id2 repression of C2C12 cells myogenic differentiation and that ENH1 overexpression promotes mice satellite cells activation, the first step toward myogenic differentiation. In addition, we show that ENH1 interacted with Id2 in C2C12 cells and mice satellite cells. Collectively, our results suggest that ENH1 plays an important role in the activation of myogenesis through the repression of Id2 activity. -- Highlights: •Enigma Homolog 1 (ENH1) is a scaffold protein. •ENH1 binds to inhibitor of DNA binding 2 (Id2) in myoblasts. •ENH1 overexpression overcomes the Id2's repression of myogenesis. •The Id2-ENH1 complex play an important role in the activation of myogenesis.

  18. Two Mutations Preventing PDZ-Protein Interactions of GluR1 Have Opposite Effects on Synaptic Plasticity

    Science.gov (United States)

    Boehm, Jannic; Ehrlich, Ingrid; Hsieh, Helen; Malinow, Roberto

    2006-01-01

    The regulated trafficking of GluR1 contributes significantly to synaptic plasticity, but studies addressing the function of the GluR1 C-terminal PDZ-ligand domain in this process have produced conflicting results. Here, we resolve this conflict by showing that apparently similar C-terminal mutations of the GluR1 PDZ-ligand domain result in…

  19. The human Na+/H+ exchanger 1 is a membrane scaffold protein for extracellular signal-regulated kinase 2

    DEFF Research Database (Denmark)

    Hendus-Altenburger, Ruth; Pedraz Cuesta, Elena; Olesen, Christina Wilkens

    2016-01-01

    the human Na(+)/H(+) exchanger 1 (hNHE1) as a membrane scaffold protein for ERK2 and show direct hNHE1-ERK1/2 interaction in cellular contexts. Using nuclear magnetic resonance (NMR) spectroscopy and immunofluorescence analysis we demonstrate that ERK2 scaffolding by hNHE1 occurs by one of three D......-domains and by two non-canonical F-sites located in the disordered intracellular tail of hNHE1, mutation of which reduced cellular hNHE1-ERK1/2 co-localization, as well as reduced cellular ERK1/2 activation. Time-resolved NMR spectroscopy revealed that ERK2 phosphorylated the disordered tail of hNHE1 at six sites...... in vitro, in a distinct temporal order, with the phosphorylation rates at the individual sites being modulated by the docking sites in a distant dependent manner. CONCLUSIONS: This work characterizes a new type of scaffolding complex, which we term a "shuffle complex", between the disordered hNHE1-tail...

  20. Efficient Malic Acid Production in Escherichia coli Using a Synthetic Scaffold Protein Complex.

    Science.gov (United States)

    Somasundaram, Sivachandiran; Eom, Gyeong Tae; Hong, Soon Ho

    2018-04-01

    Recently, malic acid has gained attention due to its potential application in food, pharmaceutical, and medical industries. In this study, the synthetic scaffold complex strategy was employed between the two key enzymes pyruvate kinase (PykF) and malic enzyme (SfcA); SH3 ligand was attached to PykF, and the SH3 domain was attached to the C-terminus of ScfA. Synthetic scaffold systems can organize enzymes spatially and temporally to increase the local concentration of intermediates. In a flask culture, the recombinant strain harboring scaffold complex produced a maximum concentration of 5.72 g/L malic acid from 10 g/L glucose. The malic acid production was significantly increased 2.1-fold from the initial culture period. Finally, malic acid production was elevated to 30.2 g in a 5 L bioreactor from recombinant strain XL-1 blue.

  1. Periodontal ligament stem/progenitor cells with protein-releasing scaffolds for cementum formation and integration on dentin surface.

    Science.gov (United States)

    Cho, Hankyu; Tarafder, Solaiman; Fogge, Michael; Kao, Kristy; Lee, Chang H

    2016-11-01

    Purpose/Aim: Cementogenesis is a critical step in periodontal tissue regeneration given the essential role of cementum in anchoring teeth to the alveolar bone. This study is designed to achieve integrated cementum formation on the root surfaces of human teeth using growth factor-releasing scaffolds with periodontal ligament stem/progenitor cells (PDLSCs). Human PDLSCs were sorted by CD146 expression, and characterized using CFU-F assay and induced multi-lineage differentiation. Polycaprolactone scaffolds were fabricated using 3D printing, embedded with poly(lactic-co-glycolic acids) (PLGA) microspheres encapsulating connective tissue growth factor (CTGF), bone morphogenetic protein-2 (BMP-2), or bone morphogenetic protein-7 (BMP-7). After removing cementum on human tooth roots, PDLSC-seeded scaffolds were placed on the exposed dentin surface. After 6-week culture with cementogenic/osteogenic medium, cementum formation and integration were evaluated by histomorphometric analysis, immunofluorescence, and qRT-PCR. Periodontal ligament (PDL) cells sorted by CD146 and single-cell clones show a superior clonogenecity and multipotency as compared with heterogeneous populations. After 6 weeks, all the growth factor-delivered groups showed resurfacing of dentin with a newly formed cementum-like layer as compared with control. BMP-2 and BMP-7 showed de novo formation of tissue layers significantly thicker than all the other groups, whereas CTGF and BMP-7 resulted in significantly improved integration on the dentin surface. The de novo mineralized tissue layer seen in BMP-7-treated samples expressed cementum matrix protein 1 (CEMP1). Consistently, BMP-7 showed a significant increase in CEMP1 mRNA expression. Our findings represent important progress in stem cell-based cementum regeneration as an essential part of periodontium regeneration.

  2. CNTNAP2 and NRXN1 are mutated in autosomal-recessive Pitt-Hopkins-like mental retardation and determine the level of a common synaptic protein in Drosophila

    DEFF Research Database (Denmark)

    Zweier, Christiane; de Jong, Eiko K; Zweier, Markus

    2009-01-01

    Heterozygous copy-number variants and SNPs of CNTNAP2 and NRXN1, two distantly related members of the neurexin superfamily, have been repeatedly associated with a wide spectrum of neuropsychiatric disorders, such as developmental language disorders, autism spectrum disorders, epilepsy...... neuropsychiatric disorders and to severe MR as reported here, evidence for a synaptic role of the CNTNAP2-encoded protein CASPR2 has so far been lacking. Using Drosophila as a model, we now show that, as known for fly Nrx-I, the CASPR2 ortholog Nrx-IV might also localize to synapses. Overexpression of either...

  3. EDITORIAL: Synaptic electronics Synaptic electronics

    Science.gov (United States)

    Demming, Anna; Gimzewski, James K.; Vuillaume, Dominique

    2013-09-01

    Conventional computers excel in logic and accurate scientific calculations but make hard work of open ended problems that human brains handle easily. Even von Neumann—the mathematician and polymath who first developed the programming architecture that forms the basis of today's computers—was already looking to the brain for future developments before his death in 1957 [1]. Neuromorphic computing uses approaches that better mimic the working of the human brain. Recent developments in nanotechnology are now providing structures with very accommodating properties for neuromorphic approaches. This special issue, with guest editors James K Gimzewski and Dominique Vuillaume, is devoted to research at the serendipitous interface between the two disciplines. 'Synaptic electronics', looks at artificial devices with connections that demonstrate behaviour similar to synapses in the nervous system allowing a new and more powerful approach to computing. Synapses and connecting neurons respond differently to incident signals depending on the history of signals previously experienced, ultimately leading to short term and long term memory behaviour. The basic characteristics of a synapse can be replicated with around ten simple transistors. However with the human brain having around 1011 neurons and 1015 synapses, artificial neurons and synapses from basic transistors are unlikely to accommodate the scalability required. The discovery of nanoscale elements that function as 'memristors' has provided a key tool for the implementation of synaptic connections [2]. Leon Chua first developed the concept of the 'The memristor—the missing circuit element' in 1971 [3]. In this special issue he presents a tutorial describing how memristor research has fed into our understanding of synaptic behaviour and how they can be applied in information processing [4]. He also describes, 'The new principle of local activity, which uncovers a minuscule life-enabling "Goldilocks zone", dubbed the

  4. Isolation of Synaptosomes, Synaptic Plasma Membranes, and Synaptic Junctional Complexes.

    Science.gov (United States)

    Michaelis, Mary L; Jiang, Lei; Michaelis, Elias K

    2017-01-01

    Isolation of synaptic nerve terminals or synaptosomes provides an opportunity to study the process of neurotransmission at many levels and with a variety of approaches. For example, structural features of the synaptic terminals and the organelles within them, such as synaptic vesicles and mitochondria, have been elucidated with electron microscopy. The postsynaptic membranes are joined to the presynaptic "active zone" of transmitter release through cell adhesion molecules and remain attached throughout the isolation of synaptosomes. These "post synaptic densities" or "PSDs" contain the receptors for the transmitters released from the nerve terminals and can easily be seen with electron microscopy. Biochemical and cell biological studies with synaptosomes have revealed which proteins and lipids are most actively involved in synaptic release of neurotransmitters. The functional properties of the nerve terminals, such as responses to depolarization and the uptake or release of signaling molecules, have also been characterized through the use of fluorescent dyes, tagged transmitters, and transporter substrates. In addition, isolated synaptosomes can serve as the starting material for the isolation of relatively pure synaptic plasma membranes (SPMs) that are devoid of organelles from the internal environment of the nerve terminal, such as mitochondria and synaptic vesicles. The isolated SPMs can reseal and form vesicular structures in which transport of ions such as sodium and calcium, as well as solutes such as neurotransmitters can be studied. The PSDs also remain associated with the presynaptic membranes during isolation of SPM fractions, making it possible to isolate the synaptic junctional complexes (SJCs) devoid of the rest of the plasma membranes of the nerve terminals and postsynaptic membrane components. Isolated SJCs can be used to identify the proteins that constitute this highly specialized region of neurons. In this chapter, we describe the steps involved

  5. Towards the synthesis of hydroxyapatite/protein scaffolds with controlled porosities: bulk and interfacial shear rheology of a hydroxyapatite suspension with protein additives.

    Science.gov (United States)

    Maas, Michael; Bodnar, Pedro Marcus; Hess, Ulrike; Treccani, Laura; Rezwan, Kurosch

    2013-10-01

    The synthesis of porous hydroxyapatite scaffolds is essential for biomedical applications such as bone tissue engineering and replacement. One way to induce macroporosity, which is needed to support bone in-growth, is to use protein additives as foaming agents. Another reason to use protein additives is the potential to introduce a specific biofunctionality to the synthesized scaffolds. In this work, we study the rheological properties of a hydroxyapatite suspension system with additions of the proteins bovine serum albumin (BSA), lysozyme (LSZ) and fibrinogen (FIB). Both the rheology of the bulk phase as well as the interfacial shear rheology are studied. The bulk rheological data provides important information on the setting behavior of the thixotropic suspension, which we find to be faster with the addition of FIB and LSZ and much slower with BSA. Foam bubble stabilization mechanisms can be rationalized via interfacial shear rheology and we show that it depends on the growth of interfacial films at the suspension/air interface. These interfacial films support the stabilization of bubbles within the ceramic matrix and thereby introduce macropores. Due to the weak interaction of the protein molecules with the hydroxyapatite particles of the suspension, we find that BSA forms the most stable interfacial films, followed by FIB. LSZ strongly interacts with the hydroxyapatite particles and thus only forms thin films with very low elastic moduli. In summary, our study provides fundamental rheological insights which are essential for tailoring hydroxyapatite/protein suspensions in order to synthesize scaffolds with controlled porosities. Copyright © 2013 Elsevier Inc. All rights reserved.

  6. The interplay between nanostructured carbon-grafted chitosan scaffolds and protein adsorption on the cellular response of osteoblasts: structure-function property relationship.

    Science.gov (United States)

    Depan, D; Misra, R D K

    2013-04-01

    The rapid adsorption of proteins occurs during the early stages of biomedical device implantation into physiological systems. In this regard, the adsorption of proteins is a strong function of the nature of a biomedical device, which ultimately governs the biological functions. The objective of this study was to elucidate the interplay between nanostructured carbon-modified (graphene oxide and single-walled carbon nanohorn) chitosan scaffolds and consequent protein adsorption and biological function (osteoblast function). We compare and contrast the footprint of protein adsorption on unmodified chitosan and nanostructured carbon-modified chitosan. A comparative analysis of cell-substrate interactions using an osteoblast cell line (MC3T3-E1) implied that biological functions were significantly enhanced in the presence of nanostructured carbon, compared with unmodified chitosan. The difference in their respective behaviors is related to the degree and topography of protein adsorption on the scaffolds. Furthermore, there was a synergistic effect of nanostructured carbon and protein adsorption in terms of favorably modulating biological functions, including cell attachment, proliferation and viability, with the effect being greater on nanostructured carbon-modified scaffolds. The study also underscores that protein adsorption is favored in nanostructured carbon-modified scaffolds such that bioactivity and biological function are promoted. Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  7. CDC42EP4, a perisynaptic scaffold protein in Bergmann glia, is required for glutamatergic tripartite synapse configuration.

    Science.gov (United States)

    Ageta-Ishihara, Natsumi; Konno, Kohtarou; Yamazaki, Maya; Abe, Manabu; Sakimura, Kenji; Watanabe, Masahiko; Kinoshita, Makoto

    2018-01-09

    Configuration of tripartite synapses, comprising the pre-, post-, and peri-synaptic components (axon terminal or bouton, dendritic spine, and astroglial terminal process), is a critical determinant of neurotransmitter kinetics and hence synaptic transmission. However, little is known about molecular basis for the regulation of tripartite synapse morphology. Previous studies showed that CDC42EP4, an effector protein of a cell morphogenesis regulator CDC42, is expressed exclusively in Bergmann glia in the cerebellar cortex, that it forms tight complex with the septin heterooligomer, and that it interacts indirectly with the glutamate transporter GLAST and MYH10/nonmuscle myosin ΙΙB. Scrutiny of Cdc42ep4 -/- mice had revealed that the CDC42EP4-septins-GLAST interaction facilitates glutamate clearance, while the role for CDC42EP4-septins-MYH10 interaction has remained unsolved. Here, we find anomalous configuration of the tripartite synapses comprising the parallel fiber boutons, dendritic spines of Purkinje cells, and Bergmann glial processes in Cdc42ep4 -/- mice. The complex anomalies include 1) recession of Bergmann glial membranes from the nearest active zones, and 2) extension of nonactive synaptic contact around active zone. In line with the recession of Bergmann glial membranes by the loss of CDC42EP4, overexpression of CDC42EP4 in heterologous cells promotes cell spreading and partitioning of MYH10 to insoluble (i.e., active) fraction. Paradoxically, however, Cdc42ep4 -/- cerebellum contained significantly more MYH10 and N-cadherin, which is attributed to secondary neuronal response mainly in Purkinje cells. Given cooperative actions of N-cadherin and MYH10 for adhesion between neurons, we speculate that their augmentation may reflect the extension of nonactive synaptic contacts in Cdc42ep4 -/- cerebellum. Transcellular mechanism that links the absence of CDC42EP4 in Bergmann glia to the augmentation of N-cadherin and MYH10 in neurons is currently unknown

  8. Changes in cortical N-methyl-D-aspartate receptors and post-synaptic density protein 95 in schizophrenia, mood disorders and suicide.

    Science.gov (United States)

    Dean, Brian; Gibbons, Andrew S; Boer, Simone; Uezato, Akihito; Meador-Woodruff, James; Scarr, Elizabeth; McCullumsmith, Robert E

    2016-03-01

    In humans, depending on dose, blocking the N-methyl-D-aspartate receptor (NMDAR) with ketamine can cause psychomimetic or antidepressant effects. The overall outcome for drugs such as ketamine depends on dose and the number of its available binding sites in the central nervous system, and to understand something of the latter variable we measure NMDAR in the frontal pole, dorsolateral prefrontal, anterior cingulate and parietal cortices from people with schizophrenia, bipolar disorder, major depressive disorders and age/sex matched controls. We measured levels of NMDARs (using [(3)H]MK-801 binding) and NMDAR sub-unit mRNAs (GRINs: using in situ hybridisation) as well as post-synaptic density protein 95 (anterior cingulate cortex only; not major depressive disorders: an NMDAR post-synaptic associated protein) in bipolar disorder, schizophrenia and controls. Compared to controls, levels of NMDAR were lower in the outer laminae of the dorsolateral prefrontal cortex (-17%, p = 0.01) in people with schizophrenia. In bipolar disorder, levels of NMDAR binding (laminae IV-VI; -19%, p suicide completers, levels of GRIN2B mRNA were higher in parietal cortex (+20%, p suicide completion and may contribute to different responses to ketamine. © The Royal Australian and New Zealand College of Psychiatrists 2015.

  9. Control of Homeostatic Synaptic Plasticity by AKAP-Anchored Kinase and Phosphatase Regulation of Ca2+-Permeable AMPA Receptors.

    Science.gov (United States)

    Sanderson, Jennifer L; Scott, John D; Dell'Acqua, Mark L

    2018-02-13

    -dependent plasticity that control excitatory synaptic strength, including LTP/LTD that adjusts strength of individual synapses and homeostatic plasticity that adjusts overall strength of all synapses. Mechanisms controlling LTP/LTD and homeostatic plasticity were originally thought to be distinct; however, recent studies suggest that CP-AMPAR phosphorylation regulation is important during both LTP/LTD and homeostatic plasticity. Here we show that CP-AMPAR regulation by the kinase PKA and phosphatase CaN co-anchored to the scaffold protein AKAP150, a mechanism previously implicated in LTP/LTD, is also crucial for controlling synaptic strength during homeostatic plasticity. These novel findings significantly expand our understanding of homeostatic plasticity mechanisms and further emphasize how intertwined they are with LTP and LTD. Copyright © 2018 the authors.

  10. Imbalance of excitatory/inhibitory synaptic protein expression in iPSC-derived neurons from FOXG1+/− patients and in foxg1+/− mice

    Science.gov (United States)

    Patriarchi, Tommaso; Amabile, Sonia; Frullanti, Elisa; Landucci, Elisa; Lo Rizzo, Caterina; Ariani, Francesca; Costa, Mario; Olimpico, Francesco; W Hell, Johannes; M Vaccarino, Flora; Renieri, Alessandra; Meloni, Ilaria

    2016-01-01

    Rett syndrome (RTT) is a severe neurodevelopmental disorder associated with mutations in either MECP2, CDKL5 or FOXG1. The precise molecular mechanisms that lead to the pathogenesis of RTT have yet to be elucidated. We recently reported that expression of GluD1 (orphan glutamate receptor δ-1 subunit) is increased in iPSC-derived neurons obtained from patients with mutations in either MECP2 or CDKL5. GluD1 controls synaptic differentiation and shifts the balance between excitatory and inhibitory synapses toward the latter. Thus, an increase in GluD1 might be a critical factor in the etiology of RTT by affecting the excitatory/inhibitory balance in the developing brain. To test this hypothesis, we generated iPSC-derived neurons from FOXG1+/− patients. We analyzed mRNA and protein levels of GluD1 together with key markers of excitatory and inhibitory synapses in these iPSC-derived neurons and in Foxg1+/− mouse fetal (E11.5) and adult (P70) brains. We found strong correlation between iPSC-derived neurons and fetal mouse brains, where GluD1 and inhibitory synaptic markers (GAD67 and GABA AR-α1) were increased, whereas the levels of a number of excitatory synaptic markers (VGLUT1, GluA1, GluN1 and PSD-95) were decreased. In adult mice, GluD1 was decreased along with all GABAergic and glutamatergic markers. Our findings further the understanding of the etiology of RTT by introducing a new pathological event occurring in the brain of FOXG1+/− patients during embryonic development and its time-dependent shift toward a general decrease in brain synapses. PMID:26443267

  11. A novel {sup 18}F-labeled two-helix scaffold protein for PET imaging of HER2-positive tumor

    Energy Technology Data Exchange (ETDEWEB)

    Miao, Zheng; Ren, Gang; Jiang, Lei; Liu, Hongguang; Cheng, Zhen [Stanford University, Molecular Imaging Program at Stanford (MIPS), Department of Radiology, Stanford Cancer Center, Bio-X Program, Stanford, CA (United States); Webster, Jack M.; Zhang, Rong; Syud, Faisal [General Electric Company, Global Research, Niskayuna, NY (United States); Namavari, Mohammad; Gambhir, Sanjiv S. [Stanford University, MIPS, Departments of Radiology and Bioengineering, Stanford Cancer Center, Bio-X Program, Stanford, CA (United States)

    2011-11-15

    Two-helix scaffold proteins ({proportional_to} 5 kDa) against human epidermal growth factor receptor type 2 (HER2) have been discovered in our previous work. In this research we aimed to develop an {sup 18}F-labeled two-helix scaffold protein for positron emission tomography (PET) imaging of HER2-positive tumors. An aminooxy-functionalized two-helix peptide (AO-MUT-DS) with high HER2 binding affinity was synthesized through conventional solid phase peptide synthesis. The purified linear peptide was cyclized by I{sub 2} oxidation to form a disulfide bridge. The cyclic peptide was then conjugated with a radiofluorination synthon, 4-{sup 18}F-fluorobenzyl aldehyde ({sup 18}F-FBA), through the aminooxy functional group at the peptide N terminus (30% yield, non-decay corrected). The binding affinities of the peptides were analyzed by Biacore analysis. Cell uptake assay of the resulting PET probe, {sup 18}F-FBO-MUT-DS, was performed at 37 C. {sup 18}F-FBO-MUT-DS with high specific activity (20-32 MBq/nmol, 88-140 {mu}Ci/{mu}g, end of synthesis) was injected into mice xenograft model bearing SKOV3 tumor. MicroPET and biodistribution and metabolic stability studies were then conducted. Cell uptake assays showed high and specific cell uptake ({proportional_to}12% applied activity at 1 h) by incubation of {sup 18}F-FBO-MUT-DS with HER2 high-expressing SKOV3 ovarian cancer cells. The affinities (K{sub D}) of AO-MUT-DS and FBO-MUT-DS as tested by Biacore analysis were 2 and 1 nM, respectively. In vivo small animal PET demonstrated fast tumor targeting, high tumor accumulation, and good tumor to normal tissue contrast of {sup 18}F-FBO-MUT-DS. Biodistribution studies further revealed that the probe had excellent tumor uptake (6.9%ID/g at 1 h post-injection) and was cleared through both liver and kidneys. Co-injection of the probe with 500 {mu}g of HER2 Affibody protein reduced the tumor uptake (6.9 vs 1.8%ID/g, p < 0.05). F-FBO-MUT-DS displays excellent HER2 targeting ability

  12. The Scaffolding Protein Synapse-Associated Protein 97 is Required for Enhanced Signaling Through Isotype-Switched IgG Memory B Cell Receptors

    Science.gov (United States)

    Liu, Wanli; Chen, Elizabeth; Zhao, Xing Wang; Wan, Zheng Peng; Gao, Yi Ren; Davey, Angel; Huang, Eric; Zhang, Lijia; Crocetti, Jillian; Sandoval, Gabriel; Joyce, M. Gordon; Miceli, Carrie; Lukszo, Jan; Aravind, L.; Swat, Wojciech; Brzostowski, Joseph; Pierce, Susan K.

    2012-01-01

    Memory B cells are generated during an individual's first encounter with a foreign antigen and respond to re-encounter with the same antigen through cell surface immunoglobulin G (IgG) B cell receptors (BCRs) resulting in rapid, high-titered IgG antibody responses. Despite a central role for IgG BCRs in B cell memory, our understanding of the molecular mechanism by which IgG BCRs enhance antibody responses is incomplete. Here, we showed that the conserved cytoplasmic tail of the IgG BCR, which contains a putative PDZ-binding motif, associated with synapse-associated protein 97 (SAP97), a member of the PDZ domain–containing, membrane-associated guanylate-kinase family of scaffolding molecules that play key roles in controlling receptor density and signal strength at neuronal synapses. We showed that SAP97 accumulated and bound to IgG BCRs in the immune synapses that formed in response to engagement of the B cell with antigen. Knocking down SAP97 in IgG-expressing B cells or mutating the putative PDZ-binding motif in the tail impaired immune synapse formation, the initiation of IgG BCR signaling, and downstream activation of p38 mitogen-activated protein kinase. Thus, heightened B cell memory responses are encoded, in part, by a mechanism that involves SAP97 serving as a scaffolding protein in the IgG BCR immune synapse. PMID:22855505

  13. Preventive Effects of Resveratrol on Endocannabinoid System and Synaptic Protein Modifications in Rat Cerebral Cortex Challenged by Bilateral Common Carotid Artery Occlusion and Reperfusion

    Directory of Open Access Journals (Sweden)

    Gianfranca Carta

    2018-01-01

    Full Text Available This study aims to evaluate the putative roles of a single acute dose of resveratrol (RVT in preventing cerebral oxidative stress induced by bilateral common carotid artery occlusion, followed by reperfusion (BCCAO/R and to investigate RVT’s ability to preserve the neuronal structural integrity. Frontal and temporal-occipital cortices were examined in two groups of adult Wistar rats, sham-operated and submitted to BCCAO/R. In both groups, 6 h before surgery, half the rats were gavage-fed with a single dose of RVT (40 mg/per rat in 300 µL of sunflower oil as the vehicle, while the second half received the vehicle alone. In the frontal cortex, RVT pre-treatment prevented the BCCAO/R-induced increase of lipoperoxides, augmented concentrations of palmitoylethanolamide and docosahexaenoic acid, increased relative levels of the cannabinoid receptors type 1 (CB1 and 2 (CB2, and peroxisome-proliferator-activated-receptor (PPAR-α proteins. Increased expression of CB1/CB2 receptors mirrored that of synaptophysin and post-synaptic density-95 protein. No BCCAO/R-induced changes occurred in the temporal-occipital cortex. Collectively, our results demonstrate that, in the frontal cortex, RVT pre-treatment prevents the BCCAO/R-induced oxidative stress and modulates the endocannabinoid and PPAR-α systems. The increased expression of synaptic structural proteins further suggests the possible efficacy of RVT as a dietary supplement to preserve the nervous tissue metabolism and control the physiological response to the hypoperfusion/reperfusion challenge.

  14. Diacylglycerol Kinases in the Coordination of Synaptic Plasticity.

    Science.gov (United States)

    Lee, Dongwon; Kim, Eunjoon; Tanaka-Yamamoto, Keiko

    2016-01-01

    Synaptic plasticity is activity-dependent modification of the efficacy of synaptic transmission. Although, detailed mechanisms underlying synaptic plasticity are diverse and vary at different types of synapses, diacylglycerol (DAG)-associated signaling has been considered as an important regulator of many forms of synaptic plasticity, including long-term potentiation (LTP) and long-term depression (LTD). Recent evidences indicate that DAG kinases (DGKs), which phosphorylate DAG to phosphatidic acid to terminate DAG signaling, are important regulators of LTP and LTD, as supported by the results from mice lacking specific DGK isoforms. This review will summarize these studies and discuss how specific DGK isoforms distinctly regulate different forms of synaptic plasticity at pre- and postsynaptic sites. In addition, we propose a general role of DGKs as coordinators of synaptic plasticity that make local synaptic environments more permissive for synaptic plasticity by regulating DAG concentration and interacting with other synaptic proteins.

  15. Selective Allosteric Antagonists for the G Protein-Coupled Receptor GPRC6A Based on the 2-Phenylindole Privileged Structure Scaffold

    DEFF Research Database (Denmark)

    Johansson, Henrik; Boesgaard, Michael Worch; Nørskov-Lauritsen, Lenea

    2015-01-01

    G protein-coupled receptors (GPCRs) represent a biological target class of fundamental importance in drug therapy. The GPRC6A receptor is a newly deorphanized class C GPCR that we recently reported for the first allosteric antagonists based on the 2-arylindole privileged structure scaffold (e.g., 1...

  16. Changes in mGlu5 receptor-dependent synaptic plasticity and coupling to homer proteins in the hippocampus of Ube3A hemizygous mice modeling angelman syndrome.

    Science.gov (United States)

    Pignatelli, Marco; Piccinin, Sonia; Molinaro, Gemma; Di Menna, Luisa; Riozzi, Barbara; Cannella, Milena; Motolese, Marta; Vetere, Gisella; Catania, Maria Vincenza; Battaglia, Giuseppe; Nicoletti, Ferdinando; Nisticò, Robert; Bruno, Valeria

    2014-03-26

    Angelman syndrome (AS) is caused by the loss of Ube3A, an ubiquitin ligase that commits specific proteins to proteasomal degradation. How this defect causes autism and other pathological phenotypes associated with AS is unknown. Long-term depression (LTD) of excitatory synaptic transmission mediated by type 5 metabotropic glutamate (mGlu5) receptors was enhanced in hippocampal slices of Ube3A(m-/p+) mice, which model AS. No changes were found in NMDA-dependent LTD induced by low-frequency stimulation. mGlu5 receptor-dependent LTD in AS mice was sensitive to the protein synthesis inhibitor anisomycin, and relied on the same signaling pathways as in wild-type mice, e.g., the mitogen-activated protein kinase (MAPK) pathway, the phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycine pathway, and protein tyrosine phosphatase. Neither the stimulation of MAPK and PI3K nor the increase in Arc (activity-regulated cytoskeleton-associated protein) levels in response to mGlu5 receptor activation were abnormal in hippocampal slices from AS mice compared with wild-type mice. mGlu5 receptor expression and mGlu1/5 receptor-mediated polyphosphoinositide hydrolysis were also unchanged in the hippocampus of AS mice. In contrast, AS mice showed a reduced expression of the short Homer protein isoform Homer 1a, and an increased coupling of mGlu5 receptors to Homer 1b/c proteins in the hippocampus. These findings support the link between Homer proteins and monogenic autism, and lay the groundwork for the use of mGlu5 receptor antagonists in AS.

  17. Basic mechanisms for recognition and transport of synaptic cargos

    NARCIS (Netherlands)

    M.A. Schlager (Max); C.C. Hoogenraad (Casper)

    2009-01-01

    textabstractSynaptic cargo trafficking is essential for synapse formation, function and plasticity. In order to transport synaptic cargo, such as synaptic vesicle precursors, mitochondria, neurotransmitter receptors and signaling proteins to their site of action, neurons make use of molecular motor

  18. Pushing synaptic vesicles over the RIM.

    Science.gov (United States)

    Kaeser, Pascal S

    2011-05-01

    In a presynaptic nerve terminal, neurotransmitter release is largely restricted to specialized sites called active zones. Active zones consist of a complex protein network, and they organize fusion of synaptic vesicles with the presynaptic plasma membrane in response to action potentials. Rab3-interacting molecules (RIMs) are central components of active zones. In a recent series of experiments, we have systematically dissected the molecular mechanisms by which RIMs operate in synaptic vesicle release. We found that RIMs execute two critical functions of active zones by virtue of independent protein domains. They tether presyanptic Ca(2+) channels to the active zone, and they activate priming of synaptic vesicles by monomerizing homodimeric, constitutively inactive Munc13. These data indicate that RIMs orchestrate synaptic vesicle release into a coherent process. In conjunction with previous studies, they suggest that RIMs form a molecular platform on which plasticity of synaptic vesicle release can operate.

  19. Biophysical characterization of Atg11, a scaffold protein essential for selective autophagy in yeast.

    Science.gov (United States)

    Suzuki, Hironori; Noda, Nobuo N

    2018-01-01

    Autophagy is an intracellular degradation system in which the formation of an autophagosome is a key event. In budding yeast, autophagosomes are generated from the preautophagosomal structure (PAS), in which Atg11 and Atg17 function as scaffolds essential for selective and nonselective types of autophagy, respectively. Structural studies have been extensively performed on Atg17, but not on Atg11, preventing us from understanding the selective type of the PAS. Here, we purified and characterized Atg11. Biophysical analyses, including analytical ultracentrifugation and CD, showed that Atg11 behaves as an elongated homodimer abundant in α-helices in solution. Moreover, truncation analyses suggested that Atg11 has a parallel coiled-coil architecture, in contrast to the antiparallel dimeric architecture of Atg17.

  20. The Chromatin Scaffold Protein SAFB1 Renders Chromatin Permissive for DNA Damage Signaling

    DEFF Research Database (Denmark)

    Altmeyer, Matthias; Toledo Lazaro, Luis Ignacio; Gudjonsson, Thorkell

    2013-01-01

    the chromatin-associated scaffold attachment factor SAFB1 as a component of the DNA damage response and show that SAFB1 cooperates with histone acetylation to allow for efficient γH2AX spreading and genotoxic stress signaling. SAFB1 undergoes a highly dynamic exchange at damaged chromatin in a poly......Although the general relevance of chromatin modifications for genotoxic stress signaling, cell-cycle checkpoint activation, and DNA repair is well established, how these modifications reach initial thresholds in order to trigger robust responses remains largely unexplored. Here, we identify...... barriers by making chromatin permissive for DNA damage signaling, whereas the ensuing exclusion of SAFB1 may help prevent excessive signaling....

  1. Synaptically Driven Phosphorylation of Ribosomal Protein S6 Is Differentially Regulated at Active Synapses versus Dendrites and Cell Bodies by MAPK and PI3K/mTOR Signaling Pathways

    Science.gov (United States)

    Pirbhoy, Patricia Salgado; Farris, Shannon; Steward, Oswald

    2017-01-01

    High-frequency stimulation of the medial perforant path triggers robust phosphorylation of ribosomal protein S6 (rpS6) in activated dendritic domains and granule cell bodies. Here we dissect the signaling pathways responsible for synaptically driven rpS6 phosphorylation in the dentate gyrus using pharmacological agents to inhibit PI3-kinase/mTOR…

  2. Study of epithelial differentiation and protein expression of keratinocyte-mesenchyme stem cell co-cultivation on electrospun nylon/B. vulgaris extract composite scaffold.

    Science.gov (United States)

    Hosseinzadeh, Simzar; Soleimani, Masoud; Vossoughi, Manuchehr; Ranjbarvan, Parviz; Hamedi, Shokoh; Zamanlui, Soheila; Mahmoudifard, Matin

    2017-06-01

    Employing of the composite electrospun scaffold containing herbal extract in conjugation with co-culturing of cells can open up new window to the design of efficient biomaterials for skin tissue regeneration. Here, we introduce the synergistic effect of composite electrospun nanofibrous scaffold of nylon66 loaded with Beta vulgaris (B. vulgaris) (extract of beet roots, a plants whose widely used in Iranian folk medicine as wound healing medicine) and co-culture of mesenchymal stem-cells (MSCs)-human keratinocyte (H-keratino) differentiation towards epithelial lineage. In vitro biocompatibility was examined through MTT assay and epithelial differentiation checked by real-time PCR and immunocytochemistry (ICC) assay after co-culturing of MSCs and H-keratino on proposed scaffold. Significant enhancement in cell proliferation was detected after cell culturing on the composite type of electrospun scaffold containing B. vulgaris. Moreover, after 14days of co-culturing process, gene expression results revealed that both composite and non-composite nylon66 electrospun scaffold promote epithelial differentiation compared to mono-cell culturing of H-keratino in terms of several markers as Cytokeratin 10, Cytokeratin 14 and Involucrin and ICC of some dermal proteins like Cytokeratin 14 and Loricrin. To the best of our knowledge, findings of this study will introduce new way for the generation of novel biomaterials for the development of current skin tissue engineering. Copyright © 2017. Published by Elsevier B.V.

  3. Scaffold protein enigma homolog 1 overcomes the repression of myogenesis activation by inhibitor of DNA binding 2.

    Science.gov (United States)

    Nakatani, Miyuki; Ito, Jumpei; Koyama, Riko; Iijima, Masumi; Yoshimoto, Nobuo; Niimi, Tomoaki; Kuroda, Shun'ichi; Maturana, Andrés D

    2016-05-27

    Enigma Homolog 1 (ENH1) is a scaffold protein for signaling proteins and transcription factors. Previously, we reported that ENH1 overexpression promotes the differentiation of C2C12 myoblasts. However, the molecular mechanism underlying the role of ENH1 in the C2C12 cells differentiation remains elusive. ENH1 was shown to inhibit the proliferation of neuroblastoma cells by sequestering Inhibitor of DNA binding protein 2 (Id2) in the cytosol. Id2 is a repressor of basic Helix-Loop-Helix transcription factors activity and prevents myogenesis. Here, we found that ENH1 overcome the Id2 repression of C2C12 cells myogenic differentiation and that ENH1 overexpression promotes mice satellite cells activation, the first step toward myogenic differentiation. In addition, we show that ENH1 interacted with Id2 in C2C12 cells and mice satellite cells. Collectively, our results suggest that ENH1 plays an important role in the activation of myogenesis through the repression of Id2 activity. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. A flagellar A-kinase anchoring protein with two amphipathic helices forms a structural scaffold in the radial spoke complex.

    Science.gov (United States)

    Sivadas, Priyanka; Dienes, Jennifer M; St Maurice, Martin; Meek, William D; Yang, Pinfen

    2012-11-12

    A-kinase anchoring proteins (AKAPs) contain an amphipathic helix (AH) that binds the dimerization and docking (D/D) domain, RIIa, in cAMP-dependent protein kinase A (PKA). Many AKAPs were discovered solely based on the AH-RIIa interaction in vitro. An RIIa or a similar Dpy-30 domain is also present in numerous diverged molecules that are implicated in critical processes as diverse as flagellar beating, membrane trafficking, histone methylation, and stem cell differentiation, yet these molecules remain poorly characterized. Here we demonstrate that an AKAP, RSP3, forms a dimeric structural scaffold in the flagellar radial spoke complex, anchoring through two distinct AHs, the RIIa and Dpy-30 domains, in four non-PKA spoke proteins involved in the assembly and modulation of the complex. Interestingly, one AH can bind both RIIa and Dpy-30 domains in vitro. Thus, AHs and D/D domains constitute a versatile yet potentially promiscuous system for localizing various effector mechanisms. These results greatly expand the current concept about anchoring mechanisms and AKAPs.

  5. In vivo evaluation of whey protein-based biofilms as scaffolds for cutaneous cell cultures and biomedical applications

    International Nuclear Information System (INIS)

    Rouabhia, Mahmoud; Gilbert, Vanessa; Wang Hongxum; Subirade, Muriel

    2007-01-01

    This study evaluated the toxicity, biodegradability and immunogenicity of newly developed whey protein-based biofilms for possible use as biomaterials for medical applications. Biofilms were prepared using (A) a whey protein isolate plasticized with either diethylene glycol (DEG) or glycerol (GLY), and (B) β-lactoglobulin (βLGA) plasticized with DEG. The biofilms were implanted subcutaneously into Balb/c mice. Analyses were performed at various time points. At 15, 30 and 60 days post-implantation, no necrotic zones or exudates were present at the recipient sites. The biofilms began to degrade as early as 15 days post-implantation, as evidenced by erosion and crumbling. The macroscopic observations were supported by tissue analyses revealing no tissue necrosis or degradation and confirming that the biodegradation of the biofilms began as early as 15 days post-implantation and was almost complete after 60 days. The biodegradation was accompanied by significant leukocyte infiltration at 15 days which significantly decreased at 60 days. The absence of splenomagaly in the implanted mice confirms that these biofilms were not immunogenic. Whey protein-based biofilms are biocompatible and biodegradable and may be of interest for medical applications such as scaffolds for cutaneous cell cultures and skin recovery in burn patients

  6. In Vivo Host Response and Degradation of Copolymer Scaffolds Functionalized with Nanodiamonds and Bone Morphogenetic Protein 2.

    Science.gov (United States)

    Suliman, Salwa; Sun, Yang; Pedersen, Torbjorn O; Xue, Ying; Nickel, Joachim; Waag, Thilo; Finne-Wistrand, Anna; Steinmüller-Nethl, Doris; Krueger, Anke; Costea, Daniela E; Mustafa, Kamal

    2016-03-01

    The aim is to evaluate the effect of modifying poly[(l-lactide)-co-(ε-caprolactone)] scaffolds (PLCL) with nanodiamonds (nDP) or with nDP+physisorbed BMP-2 (nDP+BMP-2) on in vivo host tissue response and degradation. The scaffolds are implanted subcutaneously in Balb/c mice and retrieved after 1, 8, and 27 weeks. Molecular weight analysis shows that modified scaffolds degrade faster than the unmodified. Gene analysis at week 1 shows highest expression of proinflammatory markers around nDP scaffolds; although the presence of inflammatory cells and foreign body giant cells is more prominent around the PLCL. Tissue regeneration markers are highly expressed in the nDP+BMP-2 scaffolds at week 8. A fibrous capsule is detectable by week 8, thinnest around nDP scaffolds and at week 27 thickest around PLCL scaffolds. mRNA levels of ALP, COL1α2, and ANGPT1 are significantly upregulating in the nDP+BMP-2 scaffolds at week 1 with ectopic bone seen at week 8. Even when almost 90% of the scaffold is degraded at week 27, nDP are observable at implantation areas without adverse effects. In conclusion, modifying PLCL scaffolds with nDP does not aggravate the host response and physisorbed BMP-2 delivery attenuates inflammation while lowering the dose of BMP-2 to a relatively safe and economical level. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Design of compound libraries based on natural product scaffolds and protein structure similarity clustering (PSSC)

    NARCIS (Netherlands)

    Balamurugan, Rengarajan; Dekker, Frank J; Waldmann, Herbert; Dekker, Frans

    Recent advances in structural biology, bioinformatics and combinatorial chemistry have significantly impacted the discovery of small molecules that modulate protein functions. Natural products which have evolved to bind to proteins may serve as biologically validated starting points for the design

  8. Synaptic Determinants of Rett Syndrome

    Science.gov (United States)

    Boggio, Elena M.; Lonetti, Giuseppina; Pizzorusso, Tommaso; Giustetto, Maurizio

    2010-01-01

    There is mounting evidence showing that the structural and molecular organization of synaptic connections is affected both in human patients and in animal models of neurological and psychiatric diseases. As a consequence of these experimental observations, it has been introduced the concept of synapsopathies, a notion describing brain disorders of synaptic function and plasticity. A close correlation between neurological diseases and synaptic abnormalities is especially relevant for those syndromes including also mental retardation in their symptomatology, such as Rett syndrome (RS). RS (MIM312750) is an X-linked dominant neurological disorder that is caused in the majority of cases by mutations in methyl-CpG-binding protein 2 (MeCP2). This review will focus on the current knowledge of the synaptic alterations produced by mutations of the gene MeCP2 in mouse models of RS and will highlight prospects experimental therapies currently in use. Different experimental approaches have revealed that RS could be the consequence of an impairment in the homeostasis of synaptic transmission in specific brain regions. Indeed, several forms of experience-induced neuronal plasticity are impaired in the absence of MeCP2. Based on the results presented in this review, it is reasonable to propose that understanding how the brain is affected by diseases such as RS is at reach. This effort will bring us closer to identify the neurobiological bases of human cognition. PMID:21423514

  9. Synaptic determinants of Rett syndrome

    Directory of Open Access Journals (Sweden)

    Elena M B Boggio

    2010-08-01

    Full Text Available There is mounting evidence showing that the structural and molecular organization of synaptic connections are affected both in human patients and in animal models of neurological and psychiatric diseases. As a consequence of these experimental observations, it has been introduced the concept of synapsopathies, a notion describing brain disorders of synaptic function and plasticity. A close correlation between neurological diseases and synaptic abnormalities is especially relevant for those syndromes including also mental retardation in their symptomatology, such as Rett Syndrome (RS. RS (MIM312750 is an X-linked dominant neurological disorder that is caused, in the majority of cases by mutations in methyl-CpG-binding protein 2 (MeCP2. This review will focus on the current knowledge of the synaptic alterations produced by mutations of the gene MeCP2 in mouse models of RS and will highlight prospects experimental therapies currently in use. Different experimental approaches have revealed that RS could be the consequence of an impairment in the homeostasis of synaptic transmission in specific brain regions. Indeed, several forms of experience-induced neuronal plasticity are impaired in the absence of MeCP2. Based on the results presented in this review, it is reasonable to propose that understanding how the brain is affected by diseases such as RS is at reach. This effort will bring us closer to identify the neurobiological bases of human cognition.

  10. Quantitative Proteomics of Synaptic and Nonsynaptic Mitochondria: Insights for Synaptic Mitochondrial Vulnerability

    Science.gov (United States)

    2015-01-01

    Synaptic mitochondria are essential for maintaining calcium homeostasis and producing ATP, processes vital for neuronal integrity and synaptic transmission. Synaptic mitochondria exhibit increased oxidative damage during aging and are more vulnerable to calcium insult than nonsynaptic mitochondria. Why synaptic mitochondria are specifically more susceptible to cumulative damage remains to be determined. In this study, the generation of a super-SILAC mix that served as an appropriate internal standard for mouse brain mitochondria mass spectrometry based analysis allowed for the quantification of the proteomic differences between synaptic and nonsynaptic mitochondria isolated from 10-month-old mice. We identified a total of 2260 common proteins between synaptic and nonsynaptic mitochondria of which 1629 were annotated as mitochondrial. Quantitative proteomic analysis of the proteins common between synaptic and nonsynaptic mitochondria revealed significant differential expression of 522 proteins involved in several pathways including oxidative phosphorylation, mitochondrial fission/fusion, calcium transport, and mitochondrial DNA replication and maintenance. In comparison to nonsynaptic mitochondria, synaptic mitochondria exhibited increased age-associated mitochondrial DNA deletions and decreased bioenergetic function. These findings provide insights into synaptic mitochondrial susceptibility to damage. PMID:24708184

  11. Spontaneous Glutamatergic Synaptic Activity Regulates Constitutive COX-2 Expression in Neurons: OPPOSING ROLES FOR THE TRANSCRIPTION FACTORS CREB (cAMP RESPONSE ELEMENT BINDING) PROTEIN AND Sp1 (STIMULATORY PROTEIN-1).

    Science.gov (United States)

    Hewett, Sandra J; Shi, Jingxue; Gong, Yifan; Dhandapani, Krishnan; Pilbeam, Carol; Hewett, James A

    2016-12-30

    Burgeoning evidence supports a role for cyclooxygenase metabolites in regulating membrane excitability in various forms of synaptic plasticity. Two cyclooxygenases, COX-1 and COX-2, catalyze the initial step in the metabolism of arachidonic acid to prostaglandins. COX-2 is generally considered inducible, but in glutamatergic neurons in some brain regions, including the cerebral cortex, it is constitutively expressed. However, the transcriptional mechanisms by which this occurs have not been elucidated. Here, we used quantitative PCR and also analyzed reporter gene expression in a mouse line carrying a construct consisting of a portion of the proximal promoter region of the mouse COX-2 gene upstream of luciferase cDNA to characterize COX-2 basal transcriptional regulation in cortical neurons. Extracts from the whole brain and from the cerebral cortex, hippocampus, and olfactory bulbs exhibited high luciferase activity. Moreover, constitutive COX-2 expression and luciferase activity were detected in cortical neurons, but not in cortical astrocytes, cultured from wild-type and transgenic mice, respectively. Constitutive COX-2 expression depended on spontaneous but not evoked excitatory synaptic activity and was shown to be N-methyl-d-aspartate receptor-dependent. Constitutive promoter activity was reduced in neurons transfected with a dominant-negative cAMP response element binding protein (CREB) and was eliminated by mutating the CRE-binding site on the COX-2 promoter. However, mutation of the stimulatory protein-1 (Sp1)-binding site resulted in an N-methyl-d-aspartate receptor-dependent enhancement of COX-2 promoter activity. Basal binding of the transcription factors CREB and Sp1 to the native neuronal COX-2 promoter was confirmed. In toto, our data suggest that spontaneous glutamatergic synaptic activity regulates constitutive neuronal COX-2 expression via Sp1 and CREB protein-dependent transcriptional mechanisms. © 2016 by The American Society for Biochemistry

  12. Quantitative proteomics identifies altered O-GlcNAcylation of structural, synaptic and memory-associated proteins in Alzheimer's disease: Brain protein O-GlcNAcylation in Alzheimer's disease

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Sheng [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA USA; Yang, Feng [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA USA; Petyuk, Vladislav A. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA USA; Shukla, Anil K. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA USA; Monroe, Matthew E. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA USA; Gritsenko, Marina A. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA USA; Rodland, Karin D. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA USA; Smith, Richard D. [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA USA; Qian, Wei-Jun [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA USA; Gong, Cheng-Xin [New York State Institute for Basic Research in Developmental Disabilities, Staten Island, New York USA; Liu, Tao [Biological Sciences Division, Pacific Northwest National Laboratory, Richland WA USA

    2017-07-28

    Protein modification by O-linked beta-N-acetylglucosamine (O-GlcNAc) is emerging as an important factor in the pathogenesis of sporadic Alzheimer’s disease. Herein we report the most comprehensive, quantitative proteomics analysis for protein O-GlcNAcylation in post-mortem human brains with and without Alzheimer’s using isobaric tandem mass tags labeling, chemoenzymatic photocleavage enrichment and liquid chromatography coupled to mass spectrometry. A total of 1,850 O-GlcNAc peptides covering 1,094 O-GlcNAcylation sites were identified from 530 proteins in the human brain. 128 O-GlcNAc peptides covering 78 proteins were altered significantly in Alzheimer’s brain as compared to controls (q<0.05). Moreover, alteration of the O-GlcNAc peptide abundance could be attributed more to O-GlcNAcylation level than to protein level changes. The altered O-GlcNAcylated proteins belong to several structural and functional categories, including synaptic proteins, cytoskeleton proteins, and memory-associated proteins. These findings suggest that dysregulation of O-GlcNAcylation of multiple brain proteins may be involved in the development of sporadic Alzheimer’s disease.

  13. Structural and functional insights into the interaction between the Cas family scaffolding protein p130Cas and the focal adhesion-associated protein paxillin

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Chi; Miller, Darcie J.; Guibao, Cristina D.; Donato, Dominique M.; Hanks, Steven K.; Zheng, Jie J.

    2017-08-31

    The Cas family scaffolding protein p130Cas is a Src substrate localized in focal adhesions (FAs) and functions in integrin signaling to promote cell motility, invasion, proliferation, and survival. p130Cas targeting to FAs is essential for its tyrosine phosphorylation and downstream signaling. Although the N-terminal SH3 domain is important for p130Cas localization, it has also been reported that the C-terminal region is involved in p130Cas FA targeting. The C-terminal region of p130Cas or Cas family homology domain (CCHD) has been reported to adopt a structure similar to that of the focal adhesion kinase C-terminal focal adhesion-targeting domain. The mechanism by which the CCHD promotes FA targeting of p130Cas, however, remains unclear. In this study, using a calorimetry approach, we identified the first LD motif (LD1) of the FA-associated protein paxillin as the binding partner of the p130Cas CCHD (in a 1:1 stoichiometry with a Kd ~4.2 μM) and elucidated the structure of the p130Cas CCHD in complex with the paxillin LD1 motif by X-ray crystallography. Of note, a comparison of the CCHD/LD1 complex with a previously solved structure of CCHD in complex with the SH2-containing protein NSP3 revealed that LD1 had almost identical positioning of key hydrophobic and acidic residues relative to NSP3. Because paxillin is one of the key scaffold molecules in FAs, we propose that the interaction between the p130Cas CCHD and the LD1 motif of paxillin plays an important role in p130Cas FA targeting.

  14. Elevation of synaptic protein is associated with the antidepressant-like effects of ferulic acid in a chronic model of depression.

    Science.gov (United States)

    Liu, Ya-Min; Hu, Chun-Yue; Shen, Ji-Duo; Wu, Su-Hui; Li, Yu-Cheng; Yi, Li-Tao

    2017-02-01

    Ferulic acid is a hydroxycinnamic acid that widely presents in plant cell wall components. It has been demonstrated that ferulic acid can reverse depressive-like behaviors in both forced swimming test and tail suspension test. However, it is unclear whether chronic ferulic acid treatment can ameliorate the depressive-like behaviors in chronic unpredictable mild stress (CUMS). Because of the putative relationship between neurotrophic system and antidepressant-like activity, we also investigated the effects of chronic ferulic acid on the brain-derived neurotrophic factor (BDNF), postsynaptic protein PSD95, presynaptic protein synapsin I in both prefrontal cortex and hippocampus. The results showed that ferulic acid significantly alleviated CUMS-induced depressive-like behaviors in sucrose preference test and forced swimming test. In addition, ferulic acid significantly up-regulated the levels of BDNF, PSD95 and synapsin I in the prefrontal cortex and hippocampus. The present data indicated that ferulic acid exerted the antidepressant-like effects on behaviors by increasing neurotrophin-related synaptic protein levels in CUMS mice. Copyright © 2016. Published by Elsevier Inc.

  15. Exposure of Neonatal Mice to Tobacco Smoke Disturbs Synaptic Proteins and Spatial Learning and Memory from Late Infancy to Early Adulthood.

    Science.gov (United States)

    Torres, Larissa Helena; Garcia, Raphael C T; Blois, Anne M M; Dati, Lívia M M; Durão, Ana Carolina; Alves, Adilson Silva; Pacheco-Neto, Maurílio; Mauad, Thais; Britto, Luiz R G; Xavier, Gilberto Fernando; Camarini, Rosana; Marcourakis, Tania

    2015-01-01

    Exposure to environmental tobacco smoke (ETS) in the early postnatal period has been associated with several diseases; however, little is known about the brain effects of ETS exposure during this critical developmental period or the long-term consequences of this exposure. This study investigated the effects of the early postnatal ETS exposure on both reference and working memory, synaptic proteins and BDNF from late infancy to early adulthood (P3-P73). BALB/c mice were exposed to ETS generated from 3R4F reference research cigarettes (0.73 mg of nicotine/cigarette) from P3 to P14. Spatial reference and working memory were evaluated in the Morris water maze during infancy (P20-P29), adolescence (P37-P42) and adulthood (P67-P72). Synapsin, synaptophysin, PSD95 and brain-derived neurotrophic factor (BDNF) were assessed at P15, P35 and P65 by immunohistochemistry and immunoblotting. Mice that were exposed to ETS during the early postnatal period showed poorer performance in the spatial reference memory task. Specifically, the ETS-exposed mice exhibited a significantly reduced time and distance traveled in the target quadrant and in the platform location area than the controls at all ages evaluated. In the spatial working memory task, ETS disrupted the maintenance but not the acquisition of the critical spatial information in both infancy and adolescence. ETS also induced changes in synaptic components, including decreases in synapsin, synaptophysin, PSD95 and BDNF levels in the hippocampus. Exposure to ETS in the early postnatal period disrupts both spatial reference and working memory; these results may be related to changes in synaptogenesis in the hippocampus. Importantly, most of these effects were not reversed even after a long exposure-free period.

  16. Implication of Genetic Deletion of Wdr13 in Mice: Mild Anxiety, Better Performance in Spatial Memory Task, With Upregulation of Multiple Synaptic Proteins

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    Shiladitya Mitra

    2016-08-01

    Full Text Available WDR13 expresses from the X chromosome and has a highly conserved coding sequence. There have been multiple associations of WDR13 with memory. However, its detailed function in context of brain and behavior remains unknown. We characterized the behavioral phenotype of two months old male mice lacking the homologue of WDR13 gene (Wdr13-/0. Taking cue from analysis of its expression in the brain, we chose hippocampus for molecular studies to delineate its function. Wdr13-/0 mice spent less time in the central area of the open field test and with the novel object in novel object recognition test as compared to the wild-type. However, these mice didn’t show any significant changes in total time spent in arms or frequency of arm entries in elevated plus maze. In the absence of Wdr13, there was a significant upregulation of synaptic proteins, viz., SYN1, RAB3A, CAMK2A etc accompanied with increased spine density of hippocampal CA1 neurons and better spatial memory in mice as measured by increased time spent in target quadrant of Morris water maze during probe test. Parallel study from our lab has established c-JUN, ER α/ β and HDAC 1,3,7 as interacting partners of WDR13. WDR13 represses transcription from AP1 (c-JUN responsive and ERE (Estrogen Receptor Element promoters. We hypothesized that absence of Wdr13 would resulted in de-regulated expression of a number of genes including multiple synaptic genes leading to the observed phenotype. Knocking down Wdr13 in Neuro2a cell lines led to increased transcripts of Camk2a and Nrxn2 consistent with in-vivo results. Summarily, our data provides functional evidence for the role of Wdr13 in brain.

  17. Aberrant location of inhibitory synaptic marker proteins in the hippocampus of dystrophin-deficient mice: implications for cognitive impairment in duchenne muscular dystrophy.

    Science.gov (United States)

    Krasowska, Elżbieta; Zabłocki, Krzysztof; Górecki, Dariusz C; Swinny, Jerome D

    2014-01-01

    Duchenne muscular dystrophy (DMD) is a neuromuscular disease that arises from mutations in the dystrophin-encoding gene. Apart from muscle pathology, cognitive impairment, primarily of developmental origin, is also a significant component of the disorder. Convergent lines of evidence point to an important role for dystrophin in regulating the molecular machinery of central synapses. The clustering of neurotransmitter receptors at inhibitory synapses, thus impacting on synaptic transmission, is of particular significance. However, less is known about the role of dystrophin in influencing the precise expression patterns of proteins located within the pre- and postsynaptic elements of inhibitory synapses. To this end, we exploited molecular markers of inhibitory synapses, interneurons and dystrophin-deficient mouse models to explore the role of dystrophin in determining the stereotypical patterning of inhibitory connectivity within the cellular networks of the hippocampus CA1 region. In tissue from wild-type (WT) mice, immunoreactivity of neuroligin2 (NL2), an adhesion molecule expressed exclusively in postsynaptic elements of inhibitory synapses, and the vesicular GABA transporter (VGAT), a marker of GABAergic presynaptic elements, were predictably enriched in strata pyramidale and lacunosum moleculare. In acute contrast, NL2 and VGAT immunoreactivity was relatively evenly distributed across all CA1 layers in dystrophin-deficient mice. Similar changes were evident with the cannabinoid receptor 1, vesicular glutamate transporter 3, parvalbumin, somatostatin and the GABAA receptor alpha1 subunit. The data show that in the absence of dystrophin, there is a rearrangement of the molecular machinery, which underlies the precise spatio-temporal pattern of GABAergic synaptic transmission within the CA1 sub-field of the hippocampus.

  18. Nanoscale sensor design via in situ labeling of gold nanoparticles onto protein scaffolds.

    Science.gov (United States)

    Afrasiabi, Zahra; Shukla, Ravi; Chanda, Nripen; Bhaskaran, Sharanya; Upendran, Anandhi; Zambre, Ajit; Katti, Kattesh V; Kannan, Raghuraman

    2010-02-01

    A one-step method for synthesis of bioconjugated gold nanoparticles is reported. A non-toxic and biocompatible phosphorus based reducing agent was used for reduction of gold (III) and formation of nanoparticles. Physicochemical properties of protein-A stabilized gold nanoparticls were investigated. Result of immunoassay experiments confirmed the potential of the synthesized anti-protein-A conjugated gold nanoparticles for use as a simple and inexpensive test for quantitative screening of protein-A samples.

  19. Object-Place Recognition Learning Triggers Rapid Induction of Plasticity-Related Immediate Early Genes and Synaptic Proteins in the Rat Dentate Gyrus

    Directory of Open Access Journals (Sweden)

    Jonathan Soulé

    2008-01-01

    Full Text Available Long-term recognition memory requires protein synthesis, but little is known about the coordinate regulation of specific genes. Here, we examined expression of the plasticity-associated immediate early genes (Arc, Zif268, and Narp in the dentate gyrus following long-term object-place recognition learning in rats. RT-PCR analysis from dentate gyrus tissue collected shortly after training did not reveal learning-specific changes in Arc mRNA expression. In situ hybridization and immunohistochemistry were therefore used to assess possible sparse effects on gene expression. Learning about objects increased the density of granule cells expressing Arc, and to a lesser extent Narp, specifically in the dorsal blade of the dentate gyrus, while Zif268 expression was elevated across both blades. Thus, object-place recognition triggers rapid, blade-specific upregulation of plasticity-associated immediate early genes. Furthermore, Western blot analysis of dentate gyrus homogenates demonstrated concomitant upregulation of three postsynaptic density proteins (Arc, PSD-95, and α-CaMKII with key roles in long-term synaptic plasticity and long-term memory.

  20. The MAP kinase ERK and its scaffold protein MP1 interact with the chromatin regulator Corto during Drosophila wing tissue development.

    Science.gov (United States)

    Mouchel-Vielh, Emmanuèle; Rougeot, Julien; Decoville, Martine; Peronnet, Frédérique

    2011-03-14

    Mitogen-activated protein kinase (MAPK) cascades (p38, JNK, ERK pathways) are involved in cell fate acquisition during development. These kinase modules are associated with scaffold proteins that control their activity. In Drosophila, dMP1, that encodes an ERK scaffold protein, regulates ERK signaling during wing development and contributes to intervein and vein cell differentiation. Functional relationships during wing development between a chromatin regulator, the Enhancer of Trithorax and Polycomb Corto, ERK and its scaffold protein dMP1, are examined here. Genetic interactions show that corto and dMP1 act together to antagonize rolled (which encodes ERK) in the future intervein cells, thus promoting intervein fate. Although Corto, ERK and dMP1 are present in both cytoplasmic and nucleus compartments, they interact exclusively in nucleus extracts. Furthermore, Corto, ERK and dMP1 co-localize on several sites on polytene chromosomes, suggesting that they regulate gene expression directly on chromatin. Finally, Corto is phosphorylated. Interestingly, its phosphorylation pattern differs between cytoplasm and nucleus and changes upon ERK activation. Our data therefore suggest that the Enhancer of Trithorax and Polycomb Corto could participate in regulating vein and intervein genes during wing tissue development in response to ERK signaling.

  1. The MAP kinase ERK and its scaffold protein MP1 interact with the chromatin regulator Corto during Drosophila wing tissue development

    Directory of Open Access Journals (Sweden)

    Peronnet Frédérique

    2011-03-01

    Full Text Available Abstract Background Mitogen-activated protein kinase (MAPK cascades (p38, JNK, ERK pathways are involved in cell fate acquisition during development. These kinase modules are associated with scaffold proteins that control their activity. In Drosophila, dMP1, that encodes an ERK scaffold protein, regulates ERK signaling during wing development and contributes to intervein and vein cell differentiation. Functional relationships during wing development between a chromatin regulator, the Enhancer of Trithorax and Polycomb Corto, ERK and its scaffold protein dMP1, are examined here. Results Genetic interactions show that corto and dMP1 act together to antagonize rolled (which encodes ERK in the future intervein cells, thus promoting intervein fate. Although Corto, ERK and dMP1 are present in both cytoplasmic and nucleus compartments, they interact exclusively in nucleus extracts. Furthermore, Corto, ERK and dMP1 co-localize on several sites on polytene chromosomes, suggesting that they regulate gene expression directly on chromatin. Finally, Corto is phosphorylated. Interestingly, its phosphorylation pattern differs between cytoplasm and nucleus and changes upon ERK activation. Conclusions Our data therefore suggest that the Enhancer of Trithorax and Polycomb Corto could participate in regulating vein and intervein genes during wing tissue development in response to ERK signaling.

  2. Electrochemical Aptamer Scaffold Biosensors for Detection of Botulism and Ricin Proteins.

    Science.gov (United States)

    Daniel, Jessica; Fetter, Lisa; Jett, Susan; Rowland, Teisha J; Bonham, Andrew J

    2017-01-01

    Electrochemical DNA (E-DNA) biosensors enable the detection and quantification of a variety of molecular targets, including oligonucleotides, small molecules, heavy metals, antibodies, and proteins. Here we describe the design, electrode preparation and sensor attachment, and voltammetry conditions needed to generate and perform measurements using E-DNA biosensors against two protein targets, the biological toxins ricin and botulinum neurotoxin. This method can be applied to generate E-DNA biosensors for the detection of many other protein targets, with potential advantages over other systems including sensitive detection limits typically in the nanomolar range, real-time monitoring, and reusable biosensors.

  3. Klotho regulates CA1 hippocampal synaptic plasticity.

    Science.gov (United States)

    Li, Qin; Vo, Hai T; Wang, Jing; Fox-Quick, Stephanie; Dobrunz, Lynn E; King, Gwendalyn D

    2017-04-07

    Global klotho overexpression extends lifespan while global klotho-deficiency shortens it. As well, klotho protein manipulations inversely regulate cognitive function. Mice without klotho develop rapid onset cognitive impairment before they are 2months old. Meanwhile, adult mice overexpressing klotho show enhanced cognitive function, particularly in hippocampal-dependent tasks. The cognitive enhancing effects of klotho extend to humans with a klotho polymorphism that increases circulating klotho and executive function. To affect cognitive function, klotho could act in or on the synapse to modulate synaptic transmission or plasticity. However, it is not yet known if klotho is located at synapses, and little is known about its effects on synaptic function. To test this, we fractionated hippocampi and detected klotho expression in both pre and post-synaptic compartments. We find that loss of klotho enhances both pre and post-synaptic measures of CA1 hippocampal synaptic plasticity at 5weeks of age. However, a rapid loss of synaptic enhancement occurs such that by 7weeks, when mice are cognitively impaired, there is no difference from wild-type controls. Klotho overexpressing mice show no early life effects on synaptic plasticity, but decreased CA1 hippocampal long-term potentiation was measured at 6months of age. Together these data suggest that klotho affects cognition, at least in part, by regulating hippocampal synaptic plasticity. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

  4. The First Alcohol Drink Triggers mTORC1-Dependent Synaptic Plasticity in Nucleus Accumbens Dopamine D1 Receptor Neurons.

    Science.gov (United States)

    Beckley, Jacob T; Laguesse, Sophie; Phamluong, Khanhky; Morisot, Nadege; Wegner, Scott A; Ron, Dorit

    2016-01-20

    Early binge-like alcohol drinking may promote the development of hazardous intake. However, the enduring cellular alterations following the first experience with alcohol consumption are not fully understood. We found that the first binge-drinking alcohol session produced enduring enhancement of excitatory synaptic transmission onto dopamine D1 receptor-expressing neurons (D1+ neurons) in the nucleus accumbens (NAc) shell but not the core in mice, which required D1 receptors (D1Rs) and mechanistic target of rapamycin complex 1 (mTORC1). Furthermore, inhibition of mTORC1 activity during the first alcohol drinking session reduced alcohol consumption and preference of a subsequent drinking session. mTORC1 is critically involved in RNA-to-protein translation, and we found that the first alcohol session rapidly activated mTORC1 in NAc shell D1+ neurons and increased synaptic expression of the AMPAR subunit GluA1 and the scaffolding protein Homer. Finally, D1R stimulation alone was sufficient to activate mTORC1 in the NAc to promote mTORC1-dependent translation of the synaptic proteins GluA1 and Homer. Together, our results indicate that the first alcohol drinking session induces synaptic plasticity in NAc D1+ neurons via enhanced mTORC1-dependent translation of proteins involved in excitatory synaptic transmission that in turn drives the reinforcement learning associated with the first alcohol experience. Thus, the alcohol-dependent D1R/mTORC1-mediated increase in synaptic function in the NAc may reflect a neural imprint of alcohol's reinforcing properties, which could promote subsequent alcohol intake. Significance statement: Consuming alcohol for the first time is a learning event that drives further drinking. Here, we identified a mechanism that may underlie the reinforcing learning associated with the initial alcohol experience. We show that the first alcohol experience induces a persistent enhancement of excitatory synaptic transmission on NAc shell D1+ neurons

  5. Fabrication of Calixarene Based Protein Scaffold by Electrospin Coating for Tissue Engineering.

    Science.gov (United States)

    Cagil, Esra Maltas; Ozcan, Fatih; Ertul, Seref

    2018-08-01

    In this study, calixarene was synthesized by using different functional groups as p-tert-butyl-Calix[4]arene ester and amides. Calixarene nanofibers were produced by electrospin coating. Protein immobilization onto the calixarene nanofibers was carried out with human serum albumin (HSA). The maximum amount of binding on produced three different calixarene nanofibers (DE, 2-AMP and 3-AMP) was compared by using a fluorescence technique for protein analysis. Result showed that maximum binding amount was found to be as 177.85 mg cm-2 for 3-AMP surface. The protein binding was also characterized by using SEM, TEM, AFM and FT-IR. From obtained results, calixarene-albumin nanofiber was also fabricated by spin coating using 3-AMP which has ability max binding of protein.

  6. Synaptic Homeostasis and Its Immunological Disturbance in Neuromuscular Junction Disorders

    Directory of Open Access Journals (Sweden)

    Masaharu Takamori

    2017-04-01

    Full Text Available In the neuromuscular junction, postsynaptic nicotinic acetylcholine receptor (nAChR clustering, trans-synaptic communication and synaptic stabilization are modulated by the molecular mechanisms underlying synaptic plasticity. The synaptic functions are based presynaptically on the active zone architecture, synaptic vesicle proteins, Ca2+ channels and synaptic vesicle recycling. Postsynaptically, they are based on rapsyn-anchored nAChR clusters, localized sensitivity to ACh, and synaptic stabilization via linkage to the extracellular matrix so as to be precisely opposed to the nerve terminal. Focusing on neural agrin, Wnts, muscle-specific tyrosine kinase (a mediator of agrin and Wnts signalings and regulator of trans-synaptic communication, low-density lipoprotein receptor-related protein 4 (the receptor of agrin and Wnts and participant in retrograde signaling, laminin-network (including muscle-derived agrin, extracellular matrix proteins (participating in the synaptic stabilization and presynaptic receptors (including muscarinic and adenosine receptors, we review the functional structures of the synapse by making reference to immunological pathogenecities in postsynaptic disease, myasthenia gravis. The synapse-related proteins including cortactin, coronin-6, caveolin-3, doublecortin, R-spondin 2, amyloid precursor family proteins, glia cell-derived neurotrophic factor and neurexins are also discussed in terms of their possible contribution to efficient synaptic transmission at the neuromuscular junction.

  7. The induction of cytochrome P450 2E1 by ethanol leads to the loss of synaptic proteins via PPARα down-regulation.

    Science.gov (United States)

    Na, Shufang; Li, Jie; Zhang, Huibo; Li, Yueran; Yang, Zheqiong; Zhong, Yanjun; Dong, Guicheng; Yang, Jing; Yue, Jiang

    2017-06-15

    Ethanol, one of the most commonly abused substances throughout history, is a substrate and potent inducer of cytochrome P450 2E1 (CYP2E1). Our previous study showed that brain CYP2E1 was induced by chronic ethanol treatment and was associated with ethanol-induced neurotoxicity in rats. We therefore investigated the possible mechanism of brain CYP2E1 involvement in ethanol-induced neurodegeneration. Compared with the controls, chronic ethanol treatment (3.0g/kg, i.g., 160days) significantly increased CYP2E1 mRNA levels in the rat cortex, but the mRNA levels of peroxisome proliferator-activated receptor α (PPARα) and the pre- and post-synaptic proteins (synaptophysin, SYP, and drebrin1, DBN1) were decreased. Ethanol treatment dose-dependently induced CYP2E1 mRNA expression, and CYP2E1 overexpression exacerbated the ethanol-induced neurotoxicity. Pretreatment with p38 inhibitor (SB202190) and ERK1/2 inhibitor (U0126) attenuated the induction of CYP2E1 mRNA and protein levels by ethanol; however, no change was observed with JNK inhibitor pretreatment. Ethanol exposure or CYP2E1 overexpression significantly decreased PPARα, SYP, and DBN1 expression as indicated by the data from real-time RT-PCR, Western blotting and immunocytochemistry. The activation of PPARα by WY14643 increased the activity of the SYP and DBN1 promoters and attenuated the inhibition of these genes by ethanol. The specific siRNA for CYP2E1 significantly attenuated the ethanol-induced inhibition of PPARα, SYP and DBN1 mRNA levels. These results suggest that the induction of CYP2E1 by ethanol may be mediated via the p38 and ERK1/2 signaling pathways in neurons but not via the JNK pathway. The CYP2E1-PPARα axis may play a role in ethanol-induced neurotoxicity via the alteration of the genes related with synaptic function. Copyright © 2017. Published by Elsevier B.V.

  8. An Ipomoea batatas iron-sulfur cluster scaffold protein gene, IbNFU1, is involved in salt tolerance.

    Science.gov (United States)

    Liu, Degao; Wang, Lianjun; Liu, Chenglong; Song, Xuejin; He, Shaozhen; Zhai, Hong; Liu, Qingchang

    2014-01-01

    Iron-sulfur cluster biosynthesis involving the nitrogen fixation (Nif) proteins has been proposed as a general mechanism acting in various organisms. NifU-like protein may play an important role in protecting plants against abiotic and biotic stresses. An iron-sulfur cluster scaffold protein gene, IbNFU1, was isolated from a salt-tolerant sweetpotato (Ipomoea batatas (L.) Lam.) line LM79 in our previous study, but its role in sweetpotato stress tolerance was not investigated. In the present study, the IbNFU1 gene was introduced into a salt-sensitive sweetpotato cv. Lizixiang to characterize its function in salt tolerance. The IbNFU1-overexpressing sweetpotato plants exhibited significantly higher salt tolerance compared with the wild-type. Proline and reduced ascorbate content were significantly increased, whereas malonaldehyde (MDA) content was significantly decreased in the transgenic plants. The activities of superoxide dismutase (SOD) and photosynthesis were significantly enhanced in the transgenic plants. H2O2 was also found to be significantly less accumulated in the transgenic plants than in the wild-type. Overexpression of IbNFU1 up-regulated pyrroline-5-carboxylate synthase (P5CS) and pyrroline-5-carboxylate reductase (P5CR) genes under salt stress. The systemic up-regulation of reactive oxygen species (ROS) scavenging genes was found in the transgenic plants under salt stress. These findings suggest that IbNFU1gene is involved in sweetpotato salt tolerance and enhances salt tolerance of the transgenic sweetpotato plants by regulating osmotic balance, protecting membrane integrity and photosynthesis and activating ROS scavenging system.

  9. An Ipomoea batatas Iron-Sulfur Cluster Scaffold Protein Gene, IbNFU1, Is Involved in Salt Tolerance

    Science.gov (United States)

    Song, Xuejin; He, Shaozhen; Zhai, Hong; Liu, Qingchang

    2014-01-01

    Iron-sulfur cluster biosynthesis involving the nitrogen fixation (Nif) proteins has been proposed as a general mechanism acting in various organisms. NifU-like protein may play an important role in protecting plants against abiotic and biotic stresses. An iron-sulfur cluster scaffold protein gene, IbNFU1, was isolated from a salt-tolerant sweetpotato (Ipomoea batatas (L.) Lam.) line LM79 in our previous study, but its role in sweetpotato stress tolerance was not investigated. In the present study, the IbNFU1 gene was introduced into a salt-sensitive sweetpotato cv. Lizixiang to characterize its function in salt tolerance. The IbNFU1-overexpressing sweetpotato plants exhibited significantly higher salt tolerance compared with the wild-type. Proline and reduced ascorbate content were significantly increased, whereas malonaldehyde (MDA) content was significantly decreased in the transgenic plants. The activities of superoxide dismutase (SOD) and photosynthesis were significantly enhanced in the transgenic plants. H2O2 was also found to be significantly less accumulated in the transgenic plants than in the wild-type. Overexpression of IbNFU1 up-regulated pyrroline-5-carboxylate synthase (P5CS) and pyrroline-5-carboxylate reductase (P5CR) genes under salt stress. The systemic up-regulation of reactive oxygen species (ROS) scavenging genes was found in the transgenic plants under salt stress. These findings suggest that IbNFU1gene is involved in sweetpotato salt tolerance and enhances salt tolerance of the transgenic sweetpotato plants by regulating osmotic balance, protecting membrane integrity and photosynthesis and activating ROS scavenging system. PMID:24695556

  10. Muscle A-Kinase Anchoring Protein-α is an Injury-Specific Signaling Scaffold Required for Neurotrophic- and Cyclic Adenosine Monophosphate-Mediated Survival

    Directory of Open Access Journals (Sweden)

    Yan Wang

    2015-12-01

    Full Text Available Neurotrophic factor and cAMP-dependent signaling promote the survival and neurite outgrowth of retinal ganglion cells (RGCs after injury. However, the mechanisms conferring neuroprotection and neuroregeneration downstream to these signals are unclear. We now reveal that the scaffold protein muscle A-kinase anchoring protein-α (mAKAPα is required for the survival and axon growth of cultured primary RGCs. Although genetic deletion of mAKAPα early in prenatal RGC development did not affect RGC survival into adulthood, nor promoted the death of RGCs in the uninjured adult retina, loss of mAKAPα in the adult increased RGC death after optic nerve crush. Importantly, mAKAPα was required for the neuroprotective effects of brain-derived neurotrophic factor and cyclic adenosine-monophosphate (cAMP after injury. These results identify mAKAPα as a scaffold for signaling in the stressed neuron that is required for RGC neuroprotection after optic nerve injury.

  11. Synaptic Ribbons Require Ribeye for Electron Density, Proper Synaptic Localization, and Recruitment of Calcium Channels

    Directory of Open Access Journals (Sweden)

    Caixia Lv

    2016-06-01

    Full Text Available Synaptic ribbons are structures made largely of the protein Ribeye that hold synaptic vesicles near release sites in non-spiking cells in some sensory systems. Here, we introduce frameshift mutations in the two zebrafish genes encoding for Ribeye and thus remove Ribeye protein from neuromast hair cells. Despite Ribeye depletion, vesicles collect around ribbon-like structures that lack electron density, which we term “ghost ribbons.” Ghost ribbons are smaller in size but possess a similar number of smaller vesicles and are poorly localized to synapses and calcium channels. These hair cells exhibit enhanced exocytosis, as measured by capacitance, and recordings from afferent neurons post-synaptic to hair cells show no significant difference in spike rates. Our results suggest that Ribeye makes up most of the synaptic ribbon density in neuromast hair cells and is necessary for proper localization of calcium channels and synaptic ribbons.

  12. A Method for Reconstruction of Severely Damaged Spinal Cord using Autologous Hematopoietic Stem Cells and Platelet-rich Protein as a Biological Scaffold

    OpenAIRE

    Ammar, Ahmed Sabry; Osman, Yasser; Hendam, Ahmed Taher; Hasen, Mohammed Ahmed; Al Rubaish, Fatma Abdullah; Al Nujaidi, Danya Yaagoub; Al Abbas, Faisal Mishal

    2017-01-01

    Introduction: There have been attempts to alter the prognosis of severe spinal cord injury in different centers, but none of which have reliably altered the outcome. Some trials use stem cells (SCs) that produced widely differing results. We hereby add our experience in our center of a surgical reconstruction of the damaged spinal cord using a mixture of SCs and Platelet-Rich Protein (PRP) with fibrin coated as a biological scaffold. Materials and Methods: Four cases of severely damaged spina...

  13. In-vivo Generation of Dental Pulp-Like Tissue Using Human Pulpal Stem Cells, a Collagen Scaffold and Dentin Matrix Protein 1 Following Subcutaneous Transplantation in Mice

    Science.gov (United States)

    Prescott, Rebecca S.; Alsanea, Rajaa; Fayad, Mohamed I.; Johnson, Bradford R.; Wenckus, Christopher S.; Hao, Jianjun; John, Asha S.; George, Anne

    2008-01-01

    The presence of a perforation is known to significantly compromise the outcome of endodontic treatment. One potential use of regenerative endodontic therapy may be the repair of root canal perforations. In addition to nutrients and systemic in-situ interactions, the three main components believed to be essential for tissue regeneration are: stem cells, scaffold, and growth factors. This study investigated the role of each component of the tissue engineering triad in the organization and differentiation of Dental Pulp Stem Cells (DPSCs) in a simulated furcal perforation site using a mouse model. Collagen served as the scaffold and dentin matrix protein 1 (DMP1) was the growth factor. Materials were placed in simulated perforation sites in dentin slices. MTA was the control repair material. At six weeks, the animals were sacrificed and the perforation sites were evaluated by light microscopy and histological staining. Organization of newly derived pulp tissue was seen in the group containing the triad of DPSCs, a collagen scaffold, and DMP1. The other four groups did not demonstrate any apparent tissue organization. Under the conditions of the present study, it may be concluded that the triad of DPSCs, a collagen scaffold, and DMP1 can induce an organized matrix formation similar to that of pulpal tissue, which may lead to hard tissue formation. PMID:18358888

  14. Hypoxia and amino acid supplementation synergistically promote the osteogenesis of human mesenchymal stem cells on silk protein scaffolds.

    Science.gov (United States)

    Sengupta, Sejuti; Park, Sang-Hyug; Patel, Atur; Carn, Julia; Lee, Kyongbum; Kaplan, David L

    2010-12-01

    Tailoring tissue engineering strategies to match patient- and tissue-specific bone regeneration needs offers to improve clinical outcomes. As a step toward this goal, osteogenic outcomes and metabolic parameters were assessed when varying inputs into the bone formation process. Silk protein scaffolds seeded with human mesenchymal stem cells in osteogenic differentiation media were used to study in vitro osteogenesis under varied conditions of amino acid (lysine and proline) concentration and oxygen level. The cells were assessed to probe how the microenvironment impacted metabolic pathways and thus osteogenesis. The most favorable osteogenesis outcomes were found in the presence of low (5%) oxygen combined with high lysine and proline concentrations during in vitro cultivation. This same set of culture conditions also showed the highest glucose consumption, lactate synthesis, and certain amino acid consumption rates. On the basis of these results and known pathways, a holistic metabolic model was derived which shows that lysine and proline supplements as well as low (5%) oxygen levels regulate collagen matrix synthesis and thereby rates of osteogenesis. This study establishes early steps toward a foundation for patient- and tissue-specific matches between metabolism, repair site, and tissue engineering approaches toward optimized bone regeneration.

  15. The role of c-AMP-dependent protein kinase in spinal cord and post synaptic dorsal column neurons in a rat model of visceral pain.

    Science.gov (United States)

    Wu, Jing; Su, Guangxiao; Ma, Long; Zhang, Xuan; Lei, Yongzhong; Lin, Qing; Nauta, Haring J W; Li, Junfa; Fang, Li

    2007-04-01

    Visceral noxious stimulation induces central neuronal plasticity changes and suggests that the c-AMP-dependent protein kinase (PKA) signal transduction cascade contributes to long-term changes in nociceptive processing at the spinal cord level. Our previous studies reported the clinical neurosurgical interruption of post synaptic dorsal column neuron (PSDC) pathway by performing midline myelotomy effectively alleviating the intractable visceral pain in patients with severe pain. However, the intracellular cascade in PSDC neurons mediated by PKA nociceptive neurotransmission was not known. In this study, by using multiple experimental approaches, we investigated the role of PKA in nociceptive signaling in the spinal cord and PSDC neurons in a visceral pain model in rats with the intracolonic injection of mustard oil. We found that mustard oil injection elicited visceral pain that significantly changed exploratory behavior activity in rats in terms of decreased numbers of entries, traveled distance, active and rearing time, rearing activity and increased resting time when compared to that of rats receiving mineral oil injection. However, the intrathecal infusion of PKA inhibitor, H89 partially reversed the visceral pain-induced effects. Results from Western blot studies showed that mustard oil injection significantly induced the expression of PKA protein in the lumbosacral spinal cord. Immunofluorescent staining in pre-labeled PSDC neurons showed that mustard oil injection greatly induces the neuronal profile numbers. We also found that the intrathecal infusion of a PKA inhibitor, H89 significantly blocked the visceral pain-induced phosphorylation of c-AMP-responsive element binding (CREB) protein in spinal cord in rats. The results of our study suggest that the PKA signal transduction cascade may contribute to visceral nociceptive changes in spinal PSDC pathways.

  16. DNA origami scaffold for studying intrinsically disordered proteins of the nuclear pore complex

    NARCIS (Netherlands)

    Ketterer, Philip; Ananth, Adithya N; Laman Trip, Diederik S; Mishra, Ankur; Bertosin, Eva; Ganji, Mahipal; van der Torre, Jaco; Onck, Patrick; Dietz, Hendrik; Dekker, Cees

    2018-01-01

    The nuclear pore complex (NPC) is the gatekeeper for nuclear transport in eukaryotic cells. A key component of the NPC is the central shaft lined with intrinsically disordered proteins (IDPs) known as FG-Nups, which control the selective molecular traffic. Here, we present an approach to realize

  17. Architecture of the Yeast Mitochondrial Iron-Sulfur Cluster Assembly Machinery: THE SUB-COMPLEX FORMED BY THE IRON DONOR, Yfh1 PROTEIN, AND THE SCAFFOLD, Isu1 PROTEIN*

    OpenAIRE

    Ranatunga, Wasantha; Gakh, Oleksandr; Galeano, Belinda K.; Smith, Douglas Y.; Söderberg, Christopher A. G.; Al-Karadaghi, Salam; Thompson, James R.; Isaya, Grazia

    2016-01-01

    The biosynthesis of Fe-S clusters is a vital process involving the delivery of elemental iron and sulfur to scaffold proteins via molecular interactions that are still poorly defined. We reconstituted a stable, functional complex consisting of the iron donor, Yfh1 (yeast frataxin homologue 1), and the Fe-S cluster scaffold, Isu1, with 1:1 stoichiometry, [Yfh1]24·[Isu1]24. Using negative staining transmission EM and single particle analysis, we obtained a three-dimensional reconstruction of th...

  18. Combining modelling and mutagenesis studies of synaptic vesicle protein 2A to identify a series of residues involved in racetam binding.

    Science.gov (United States)

    Shi, Jiye; Anderson, Dina; Lynch, Berkley A; Castaigne, Jean-Gabriel; Foerch, Patrik; Lebon, Florence

    2011-10-01

    LEV (levetiracetam), an antiepileptic drug which possesses a unique profile in animal models of seizure and epilepsy, has as its unique binding site in brain, SV2A (synaptic vesicle protein 2A). Previous studies have used a chimaeric and site-specific mutagenesis approach to identify three residues in the putative tenth transmembrane helix of SV2A that, when mutated, alter binding of LEV and related racetam derivatives to SV2A. In the present paper, we report a combined modelling and mutagenesis study that successfully identifies another 11 residues in SV2A that appear to be involved in ligand binding. Sequence analysis and modelling of SV2A suggested residues equivalent to critical functional residues of other MFS (major facilitator superfamily) transporters. Alanine scanning of these and other SV2A residues resulted in the identification of residues affecting racetam binding, including Ile273 which differentiated between racetam analogues, when mutated to alanine. Integrating mutagenesis results with docking analysis led to the construction of a mutant in which six SV2A residues were replaced with corresponding SV2B residues. This mutant showed racetam ligand-binding affinity intermediate to the affinities observed for SV2A and SV2B.

  19. 5-HT(2C) serotonin receptor blockade prevents tau protein hyperphosphorylation and corrects the defect in hippocampal synaptic plasticity caused by a combination of environmental stressors in mice.

    Science.gov (United States)

    Busceti, Carla Letizia; Di Pietro, Paola; Riozzi, Barbara; Traficante, Anna; Biagioni, Francesca; Nisticò, Robert; Fornai, Francesco; Battaglia, Giuseppe; Nicoletti, Ferdinando; Bruno, Valeria

    2015-09-01

    Exposure to multimodal sensory stressors is an everyday occurrence and sometimes becomes very intense, such as during rave parties or other recreational events. A growing body of evidence suggests that strong environmental stressors might cause neuronal dysfunction on their own in addition to their synergistic action with illicit drugs. Mice were exposed to a combination of physical and sensory stressors that are reminiscent of those encountered in a rave party. However, this is not a model of rave because it lacks the rewarding properties of rave. A 14-h exposure to environmental stressors caused an impairment of hippocampal long-term potentiation (LTP) and spatial memory, and an enhanced phosphorylation of tau protein in the CA1 and CA3 regions. These effects were transient and critically depended on the activation of 5-HT2C serotonin receptors, which are highly expressed in the CA1 region. Acute systemic injection of the selective 5-HT2C antagonist, RS-102,221 (2 mg/kg, i.p., 2 min prior the onset of stress), prevented tau hyperphosphorylation and also corrected the defects in hippocampal LTP and spatial memory. These findings suggest that passive exposure to a combination of physical and sensory stressors causes a reversible hippocampal dysfunction, which might compromise mechanisms of synaptic plasticity and spatial memory for a few days. Drugs that block 5-HT2C receptors might protect the hippocampus against the detrimental effect of environmental stressors. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Incorporation of fibronectin to enhance cytocompatibility in multilayer elastin-like protein scaffolds for tissue engineering

    OpenAIRE

    Ravi, Swathi; Caves, Jeffrey M.; Martinez, Adam W.; Haller, Carolyn A.; Chaikof, Elliot L.

    2012-01-01

    Recombinant, elastin-like protein (ELP) polymers are of significant interest for the engineering of compliant, resilient soft tissues due to a wide range of tunable mechanical properties, biostability, and biocompatibility. Here, we enhance endothelial cell (EC) and mesenchymal stem cell compatibility with ELP constructs by addition of fibronectin (Fn) to the surface or bulk of ELP hydrogels. We find that cell adhesion, proliferation, and migration can be modulated by Fn addition. Adsorption ...

  1. Mitochondrial and Nucleolar Localization of Cysteine Desulfurase Nfs and the Scaffold Protein Isu in Trypanosoma brucei

    Czech Academy of Sciences Publication Activity Database

    Kovářová, Julie; Horáková, Eva; Changmai, Piya; Vancová, Marie; Lukeš, Julius

    2014-01-01

    Roč. 13, č. 3 (2014), s. 353-362 ISSN 1535-9778 R&D Projects: GA ČR(CZ) GAP305/11/2179; GA MŠk LH12104; GA MŠk(CZ) EE2.3.30.0032 Institutional support: RVO:60077344 Keywords : transfer RNA * iron sulfur protein * blood stream forms Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.820, year: 2014

  2. ROMP- and RAFT-Based Guanidinium-Containing Polymers as Scaffolds for Protein Mimic Synthesis.

    Science.gov (United States)

    Sarapas, Joel M; Backlund, Coralie M; deRonde, Brittany M; Minter, Lisa M; Tew, Gregory N

    2017-05-17

    Cell-penetrating peptides are an important class of molecules with promising applications in bioactive cargo delivery. A diverse series of guanidinium-containing polymeric cell-penetrating peptide mimics (CPPMs) with varying backbone chemistries was synthesized and assessed for delivery of both GFP and fluorescently tagged siRNA. Specifically, we examined CPPMs based on norbornene, methacrylate, and styrene backbones to determine how backbone structure impacted internalization of these two cargoes. Either charge content or degree of polymerization was held constant at 20, with diguanidinium norbornene molecules being polymerized to both 10 and 20 repeat units. Generally, homopolymer CPPMs delivered low amounts of siRNA into Jurkat T cells, with no apparent backbone dependence; however, by adding a short hydrophobic methyl methacrylate block to the guanidinium-rich methacrylate polymer, siRNA delivery to nearly the entire cell population was achieved. Protein internalization yielded similar results for most of the CPPMs, though the block polymer was unable to deliver proteins. In contrast, the styrene-based CPPM yielded the highest internalization for GFP (≈40 % of cells affected), showing that indeed backbone chemistry impacts protein delivery, specifically through the incorporation of an aromatic group. These results demonstrate that an understanding of how polymer structure affects cargo-dependent internalization is critical to designing new, more effective CPPMs. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Vaccinia Virus Immunomodulator A46: A Lipid and Protein-Binding Scaffold for Sequestering Host TIR-Domain Proteins.

    Directory of Open Access Journals (Sweden)

    Sofiya Fedosyuk

    2016-12-01

    Full Text Available Vaccinia virus interferes with early events of the activation pathway of the transcriptional factor NF-kB by binding to numerous host TIR-domain containing adaptor proteins. We have previously determined the X-ray structure of the A46 C-terminal domain; however, the structure and function of the A46 N-terminal domain and its relationship to the C-terminal domain have remained unclear. Here, we biophysically characterize residues 1-83 of the N-terminal domain of A46 and present the X-ray structure at 1.55 Å. Crystallographic phases were obtained by a recently developed ab initio method entitled ARCIMBOLDO_BORGES that employs tertiary structure libraries extracted from the Protein Data Bank; data analysis revealed an all β-sheet structure. This is the first such structure solved by this method which should be applicable to any protein composed entirely of β-sheets. The A46(1-83 structure itself is a β-sandwich containing a co-purified molecule of myristic acid inside a hydrophobic pocket and represents a previously unknown lipid-binding fold. Mass spectrometry analysis confirmed the presence of long-chain fatty acids in both N-terminal and full-length A46; mutation of the hydrophobic pocket reduced the lipid content. Using a combination of high resolution X-ray structures of the N- and C-terminal domains and SAXS analysis of full-length protein A46(1-240, we present here a structural model of A46 in a tetrameric assembly. Integrating affinity measurements and structural data, we propose how A46 simultaneously interferes with several TIR-domain containing proteins to inhibit NF-κB activation and postulate that A46 employs a bipartite binding arrangement to sequester the host immune adaptors TRAM and MyD88.

  4. Synaptic Tagging, Evaluation of Memories, and the Distal Reward Problem

    Science.gov (United States)

    Papper, Marc; Kempter, Richard; Leibold, Christian

    2011-01-01

    Long-term synaptic plasticity exhibits distinct phases. The synaptic tagging hypothesis suggests an early phase in which synapses are prepared, or "tagged," for protein capture, and a late phase in which those proteins are integrated into the synapses to achieve memory consolidation. The synapse specificity of the tags is consistent with…

  5. Development of Scaffolds for Light Harvesting and Photocatalysis from the Coat Protein of Tobacco Mosaic Virus

    Science.gov (United States)

    Dedeo, Michel Toussaint

    The utility of a previously developed TMV-based light harvesting system has been dramatically expanded through the introduction of reactive handles for the site-specific modification of the interior and exterior surfaces. Further experiments to reengineer the coat protein have produced structures with unique, unexpected, and useful assembly properties that complement the newly available surface modifications. Energy transfer from chromophores in the RNA channel of self-assembled TMV structures to the exterior was made possible by conjugation of acceptor dyes and porphyrins to the N-terminus. By repositioning the N-terminus to the pore through circular permutation, this process was repeated to create structures that mimic the light harvesting 1 complex of photosynthetic bacteria. To study and improve upon natural photosynthesis, closely packed chromophore arrays and gold nanoparticles were tethered to the pore of stabilized TMV disks through introduction of a uniquely reactive lysine. Finally, a dimeric TMV coat protein was produced to control the distribution and arrangement of synthetic groups with synergistic activity.

  6. Inter-channel scaffolding of presynaptic CaV2.2 via the C terminal PDZ ligand domain

    Directory of Open Access Journals (Sweden)

    Sabiha R. Gardezi

    2013-04-01

    Calcium entry through CaV2.2 calcium channels clustered at the active zone (AZ of the presynaptic nerve terminal gates synaptic vesicle (SV fusion and the discharge of neurotransmitters, but the mechanism of channel scaffolding remains poorly understood. Recent studies have implicated the binding of a PDZ ligand domain (PDZ-LD at the tip of the channel C terminal to a partner PDZ domain on RIM1/2, a synaptic vesicle-associated protein. To explore CaV2.2 scaffolding, we created intracellular region fusion proteins and used these to test for binding by ‘fishing’ for native CaV2.2 channels from cell lysates. Fusion proteins mimicking the distal half of the channel C terminal (C3strep reliably captured CaV2.2 from whole brain crude membrane or purified synaptosome membrane lysates, whereas channel I–II loop or the distal half of the II–III loop proteins were negative. This capture could be replicated in a non-synaptic environment using CaV2.2 expressed in a cell line. The distal tip PDZ-LD, DDWC-COOH, was confirmed as the critical binding site by block of pull-down with mimetic peptides. Pull-down experiments using brain crude membrane lysates confirmed that RIM1/2 can bind to the DDWC PDZ-LD. However, robust CaV2.2 capture was observed from synaptosome membrane or in the cell line expression system with little or no RIM1/2 co-capture. Thus, we conclude that CaV2.2 channels can scaffold to each other via an interaction that involves the PDZ-LD by an inter-channel linkage bridged by an unknown protein.

  7. Modulation of firing and synaptic transmission of serotonergic neurons by intrinsic G protein-coupled receptors and ion channels

    Directory of Open Access Journals (Sweden)

    Takashi eMaejima

    2013-05-01

    Full Text Available Serotonergic neurons project to virtually all regions of the CNS and are consequently involved in many critical physiological functions such as mood, sexual behavior, feeding, sleep/wake cycle, memory, cognition, blood pressure regulation, breathing and reproductive success. Therefore serotonin release and serotonergic neuronal activity have to be precisely controlled and modulated by interacting brain circuits to adapt to specific emotional and environmental states. We will review the current knowledge about G protein-coupled receptors and ion channels involved in the regulation of serotonergic system, how their regulation is modulating the intrinsic activity of serotonergic neurons and its transmitter release and will discuss the latest methods for controlling the modulation of serotonin release and intracellular signaling in serotonergic neurons in vitro and in vivo.

  8. Rare exonic deletions implicate the synaptic organizer Gephyrin (GPHN) in risk for autism, schizophrenia and seizures.

    Science.gov (United States)

    Lionel, Anath C; Vaags, Andrea K; Sato, Daisuke; Gazzellone, Matthew J; Mitchell, Elyse B; Chen, Hong Yang; Costain, Gregory; Walker, Susan; Egger, Gerald; Thiruvahindrapuram, Bhooma; Merico, Daniele; Prasad, Aparna; Anagnostou, Evdokia; Fombonne, Eric; Zwaigenbaum, Lonnie; Roberts, Wendy; Szatmari, Peter; Fernandez, Bridget A; Georgieva, Lyudmila; Brzustowicz, Linda M; Roetzer, Katharina; Kaschnitz, Wolfgang; Vincent, John B; Windpassinger, Christian; Marshall, Christian R; Trifiletti, Rosario R; Kirmani, Salman; Kirov, George; Petek, Erwin; Hodge, Jennelle C; Bassett, Anne S; Scherer, Stephen W

    2013-05-15

    The GPHN gene codes for gephyrin, a key scaffolding protein in the neuronal postsynaptic membrane, responsible for the clustering and localization of glycine and GABA receptors at inhibitory synapses. Gephyrin has well-established functional links with several synaptic proteins that have been implicated in genetic risk for neurodevelopmental disorders such as autism spectrum disorder (ASD), schizophrenia and epilepsy including the neuroligins (NLGN2, NLGN4), the neurexins (NRXN1, NRXN2, NRXN3) and collybistin (ARHGEF9). Moreover, temporal lobe epilepsy has been linked to abnormally spliced GPHN mRNA lacking exons encoding the G-domain of the gephyrin protein, potentially arising due to cellular stress associated with epileptogenesis such as temperature and alkalosis. Here, we present clinical and genomic characterization of six unrelated subjects, with a range of neurodevelopmental diagnoses including ASD, schizophrenia or seizures, who possess rare de novo or inherited hemizygous microdeletions overlapping exons of GPHN at chromosome 14q23.3. The region of common overlap across the deletions encompasses exons 3-5, corresponding to the G-domain of the gephyrin protein. These findings, together with previous reports of homozygous GPHN mutations in connection with autosomal recessive molybdenum cofactor deficiency, will aid in clinical genetic interpretation of the GPHN mutation spectrum. Our data also add to the accumulating evidence implicating neuronal synaptic gene products as key molecular factors underlying the etiologies of a diverse range of neurodevelopmental conditions.

  9. Rad4 mainly functions in Chk1-mediated DNA damage checkpoint pathway as a scaffold protein in the fission yeast Schizosaccharomyces pombe.

    Directory of Open Access Journals (Sweden)

    Ming Yue

    Full Text Available Rad4/Cut5 is a scaffold protein in the Chk1-mediated DNA damage checkpoint in S. pombe. However, whether it contains a robust ATR-activation domain (AAD required for checkpoint signaling like its orthologs TopBP1 in humans and Dpb11 in budding yeast has been incompletely clear. To identify the putative AAD in Rad4, we carried out an extensive genetic screen looking for novel mutants with an enhanced sensitivity to replication stress or DNA damage in which the function of the AAD can be eliminated by the mutations. Two new mutations near the N-terminus were identified that caused significantly higher sensitivities to DNA damage or chronic replication stress than all previously reported mutants, suggesting that most of the checkpoint function of the protein is eliminated. However, these mutations did not affect the activation of Rad3 (ATR in humans yet eliminated the scaffolding function of the protein required for the activation of Chk1. Several mutations were also identified in or near the recently reported AAD in the C-terminus of Rad4. However, all mutations in the C-terminus only slightly sensitized the cells to DNA damage. Interestingly, a mutant lacking the whole C-terminus was found resistant to DNA damage and replication stress almost like the wild type cells. Consistent with the resistance, all known Rad3 dependent phosphorylations of checkpoint proteins remained intact in the C-terminal deletion mutant, indicating that unlike that in Dpb11, the C-terminus of Rad4 does not contain a robust AAD. These results, together with those from the biochemical studies, show that Rad4 mainly functions as a scaffold protein in the Chk1, not the Cds1(CHK2 in humans, checkpoint pathway. It plays a minor role or is functionally redundant with an unknown factor in Rad3 activation.

  10. Zinc and the iron donor frataxin regulate oligomerization of the scaffold protein to form new Fe-S cluster assembly centers.

    Science.gov (United States)

    Galeano, B K; Ranatunga, W; Gakh, O; Smith, D Y; Thompson, J R; Isaya, G

    2017-06-21

    Early studies of the bacterial Fe-S cluster assembly system provided structural details for how the scaffold protein and the cysteine desulfurase interact. This work and additional work on the yeast and human systems elucidated a conserved mechanism for sulfur donation but did not provide any conclusive insights into the mechanism for iron delivery from the iron donor, frataxin, to the scaffold. We previously showed that oligomerization is a mechanism by which yeast frataxin (Yfh1) can promote assembly of the core machinery for Fe-S cluster synthesis both in vitro and in cells, in such a manner that the scaffold protein, Isu1, can bind to Yfh1 independent of the presence of the cysteine desulfurase, Nfs1. Here, in the absence of Yfh1, Isu1 was found to exist in two forms, one mostly monomeric with limited tendency to dimerize, and one with a strong propensity to oligomerize. Whereas the monomeric form is stabilized by zinc, the loss of zinc promotes formation of dimer and higher order oligomers. However, upon binding to oligomeric Yfh1, both forms take on a similar symmetrical trimeric configuration that places the Fe-S cluster coordinating residues of Isu1 in close proximity of iron-binding residues of Yfh1. This configuration is suitable for docking of Nfs1 in a manner that provides a structural context for coordinate iron and sulfur donation to the scaffold. Moreover, distinct structural features suggest that in physiological conditions the zinc-regulated abundance of monomeric vs. oligomeric Isu1 yields [Yfh1]·[Isu1] complexes with different Isu1 configurations that afford unique functional properties for Fe-S cluster assembly and delivery.

  11. Incorporation of fibronectin to enhance cytocompatibility in multilayer elastin-like protein scaffolds for tissue engineering.

    Science.gov (United States)

    Ravi, Swathi; Caves, Jeffrey M; Martinez, Adam W; Haller, Carolyn A; Chaikof, Elliot L

    2013-07-01

    Recombinant, elastin-like protein (ELP) polymers are of significant interest for the engineering of compliant, resilient soft tissues due to a wide range of tunable mechanical properties, biostability, and biocompatibility. Here, we enhance endothelial cell (EC) and mesenchymal stem cell compatibility with ELP constructs by addition of fibronectin (Fn) to the surface or bulk of ELP hydrogels. We find that cell adhesion, proliferation, and migration can be modulated by Fn addition. Adsorption of Fn to the hydrogel surface is more efficient than bulk blending. Surface immobilization of Fn by genipin crosslinking leads to stability without loss of bioactivity. Gels of varying mechanical modulus do not alter cell adhesion, proliferation, and migration in the range we investigate. However, more compliant gels promote an EC morphology suggesting tubulogenesis or network formation, whereas stiffer gels promote cobblestone morphology. Multilayer structures consisting of thin ELP sheets reinforced with collagen microfiber are fabricated and laminated through the culture of MSCs at layer interfaces. High cell viability in the resulting three-dimensional constructs suggests the applicability of Fn to the design of strong, resilient artificial blood vessels and other soft tissue replacements. Copyright © 2012 Wiley Periodicals, Inc.

  12. The Scaffolding Protein IQGAP1 Interacts with NLRC3 and Inhibits Type I IFN Production.

    Science.gov (United States)

    Tocker, Aaron M; Durocher, Emily; Jacob, Kimberly D; Trieschman, Kate E; Talento, Suzanna M; Rechnitzer, Alma A; Roberts, David M; Davis, Beckley K

    2017-10-15

    Sensing of cytosolic nucleotides is a critical initial step in the elaboration of type I IFN. One of several upstream receptors, cyclic GMP-AMP synthase, binds to cytosolic DNA and generates dicyclic nucleotides that act as secondary messengers. These secondary messengers bind directly to stimulator of IFN genes (STING). STING recruits TNFR-associated NF-κB kinase-binding kinase 1 which acts as a critical node that allows for efficient activation of IFN regulatory factors to drive the antiviral transcriptome. NLRC3 is a recently characterized nucleotide-binding domain, leucine-rich repeat containing protein (NLR) that negatively regulates the type I IFN pathway by inhibiting subcellular redistribution and effective signaling of STING, thus blunting the transcription of type I IFNs. NLRC3 is predominantly expressed in lymphoid and myeloid cells. IQGAP1 was identified as a putative interacting partner of NLRC3 through yeast two-hybrid screening. In this article, we show that IQGAP1 associates with NLRC3 and can disrupt the NLRC3-STING interaction in the cytosol of human epithelial cells. Furthermore, knockdown of IQGAP1 in THP1 and HeLa cells causes significantly more IFN-β production in response to cytosolic nucleic acids. This result phenocopies NLRC3-deficient macrophages and fibroblasts and short hairpin RNA knockdown of NLRC3 in THP1 cells. Our findings suggest that IQGAP1 is a novel regulator of type I IFN production, possibly via interacting with NLRC3 in human monocytic and epithelial cells. Copyright © 2017 by The American Association of Immunologists, Inc.

  13. A presynaptic role for PKA in synaptic tagging and memory

    NARCIS (Netherlands)

    Park, Alan Jung; Havekes, Robbert; Choi, Jennifer H K; Luczak, Vincent; Nie, Ting; Huang, Ted; Abel, Ted

    2014-01-01

    Protein kinase A (PKA) and other signaling molecules are spatially restricted within neurons by A-kinase anchoring proteins (AKAPs). Although studies on compartmentalized PKA signaling have focused on postsynaptic mechanisms, presynaptically anchored PKA may contribute to synaptic plasticity and

  14. Semiotic scaffolding

    DEFF Research Database (Denmark)

    Hoffmeyer, Jesper

    2015-01-01

    Life processes at all levels (from the genetic to the behavioral) are coordinated by semiotic interactions between cells, tissues, membranes, organs, or individuals and tuned through evolution to stabilize important functions. A stabilizing dynamics based on a system of semiotic scaffoldings impl...... semiotic scaffolding is not, of course, exclusive for phylogenetic and ontogenetic development, it is also an important dynamical element in cultural evolution.......Life processes at all levels (from the genetic to the behavioral) are coordinated by semiotic interactions between cells, tissues, membranes, organs, or individuals and tuned through evolution to stabilize important functions. A stabilizing dynamics based on a system of semiotic scaffoldings...... implies that genes do not control the life of organisms, they merely scaffold it. The nature-nurture dynamics is thus far more complex and open than is often claimed. Contrary to physically based interactions, semiotic interactions do not depend on any direct causal connection between the sign vehicle...

  15. Biologic Scaffolds.

    Science.gov (United States)

    Costa, Alessandra; Naranjo, Juan Diego; Londono, Ricardo; Badylak, Stephen F

    2017-09-01

    Biologic scaffold materials composed of allogeneic or xenogeneic extracellular matrix are commonly used for the repair and functional reconstruction of injured and missing tissues. These naturally occurring bioscaffolds are manufactured by the removal of the cellular content from source tissues while preserving the structural and functional molecular units of the remaining extracellular matrix (ECM). The mechanisms by which these bioscaffolds facilitate constructive remodeling and favorable clinical outcomes include release or creation of effector molecules that recruit endogenous stem/progenitor cells to the site of scaffold placement and modulation of the innate immune response, specifically the activation of an anti-inflammatory macrophage phenotype. The methods by which ECM biologic scaffolds are prepared, the current understanding of in vivo scaffold remodeling, and the associated clinical outcomes are discussed in this article. Copyright © 2017 Cold Spring Harbor Laboratory Press; all rights reserved.

  16. Using in vitro maturation and cell-free expression to explore [FeFe] hydrogenase activation and protein scaffolding requirements

    Energy Technology Data Exchange (ETDEWEB)

    Swartz, James [Stanford Univ., CA (United States)

    2017-01-25

    Final Project Report describing work to elucidate mechanisms for the activation of [FeFe]-hydrogenases and to explore the impact of the polypeptide scaffolding on the function of the Fe-S redox and catalytic centers with emphasis on improving oxygen tolerance.

  17. Cooperative stochastic binding and unbinding explain synaptic size dynamics and statistics.

    Science.gov (United States)

    Shomar, Aseel; Geyrhofer, Lukas; Ziv, Noam E; Brenner, Naama

    2017-07-01

    Synapses are dynamic molecular assemblies whose sizes fluctuate significantly over time-scales of hours and days. In the current study, we examined the possibility that the spontaneous microscopic dynamics exhibited by synaptic molecules can explain the macroscopic size fluctuations of individual synapses and the statistical properties of synaptic populations. We present a mesoscopic model, which ties the two levels. Its basic premise is that synaptic size fluctuations reflect cooperative assimilation and removal of molecules at a patch of postsynaptic membrane. The introduction of cooperativity to both assimilation and removal in a stochastic biophysical model of these processes, gives rise to features qualitatively similar to those measured experimentally: nanoclusters of synaptic scaffolds, fluctuations in synaptic sizes, skewed, stable size distributions and their scaling in response to perturbations. Our model thus points to the potentially fundamental role of cooperativity in dictating synaptic remodeling dynamics and offers a conceptual understanding of these dynamics in terms of central microscopic features and processes.

  18. Cooperative stochastic binding and unbinding explain synaptic size dynamics and statistics.

    Directory of Open Access Journals (Sweden)

    Aseel Shomar

    2017-07-01

    Full Text Available Synapses are dynamic molecular assemblies whose sizes fluctuate significantly over time-scales of hours and days. In the current study, we examined the possibility that the spontaneous microscopic dynamics exhibited by synaptic molecules can explain the macroscopic size fluctuations of individual synapses and the statistical properties of synaptic populations. We present a mesoscopic model, which ties the two levels. Its basic premise is that synaptic size fluctuations reflect cooperative assimilation and removal of molecules at a patch of postsynaptic membrane. The introduction of cooperativity to both assimilation and removal in a stochastic biophysical model of these processes, gives rise to features qualitatively similar to those measured experimentally: nanoclusters of synaptic scaffolds, fluctuations in synaptic sizes, skewed, stable size distributions and their scaling in response to perturbations. Our model thus points to the potentially fundamental role of cooperativity in dictating synaptic remodeling dynamics and offers a conceptual understanding of these dynamics in terms of central microscopic features and processes.

  19. Potentiation of electrical and chemical synaptic transmission mediated by endocannabinoids

    Science.gov (United States)

    Cachope, Roger; Mackie, Ken; Triller, Antoine; O’Brien, John; Pereda, Alberto E.

    2009-01-01

    SUMMARY Endocannabinoids are well established as inhibitors of chemical synaptic transmission via presynaptic activation of the cannabinoid type 1 receptor (CB1R). Contrasting this notion, we show that dendritic release of endocannabinoids mediates potentiation of synaptic transmission at mixed (electrical and chemical) synaptic contacts on the goldfish Mauthner cell. Remarkably, the observed enhancement was not restricted to the glutamatergic component of the synaptic response but also included a parallel increase in electrical transmission. This novel effect involved the activation of CB1 receptors and was indirectly mediated via the release of dopamine from nearby varicosities, which in turn led to potentiation of the synaptic response via a cAMP-dependent protein kinase-mediated postsynaptic mechanism. Thus, endocannabinoid release can potentiate synaptic transmission and its functional roles include the regulation of gap junction-mediated electrical synapses. Similar interactions between endocannabinoid and dopaminergic systems may be widespread and potentially relevant for the motor and rewarding effects of cannabis derivatives. PMID:18093525

  20. Mechanical properties, biological activity and protein controlled release by poly(vinyl alcohol)–bioglass/chitosan–collagen composite scaffolds: A bone tissue engineering applications

    International Nuclear Information System (INIS)

    Pon-On, Weeraphat; Charoenphandhu, Narattaphol; Teerapornpuntakit, Jarinthorn; Thongbunchoo, Jirawan; Krishnamra, Nateetip; Tang, I-Ming

    2014-01-01

    In the present study, composite scaffolds made with different weight ratios (0.5:1, 1:1 and 2:1) of bioactive glass (15Ca:80Si:5P) (BG)/polyvinyl alcohol (PVA) (PVABG) and chitosan (Chi)/collagen (Col) (ChiCol) were prepared by three mechanical freeze–thaw followed by freeze-drying to obtain the porous scaffolds. The mechanical properties and the in vitro biocompatibility of the composite scaffolds to simulated body fluid (SBF) and to rat osteoblast-like UMR-106 cells were investigated. The results from the studies indicated that the porosity and compressive strength were controlled by the weight ratio of PVABG:ChiCol. The highest compressive modulus of the composites made was 214.64 MPa which was for the 1:1 weight ratio PVABG:ChiCol. Mineralization study in SBF showed the formation of apatite crystals on the PVABG:ChiCol surface after 7 days of incubation. In vitro cell availability and proliferation tests confirmed the osteoblast attachment and growth on the PVABG:ChiCol surface. MTT and ALP tests on the 1:1 weight ratio PVABG:ChiCol composite indicated that the UMR-106 cells were viable. Alkaline phosphatase activity was found to increase with increasing culturing time. In addition, we showed the potential of PVABG:ChiCol drug delivery through PBS solution studies. 81.14% of BSA loading had been achieved and controlled release for over four weeks was observed. Our results indicated that the PVABG:ChiCol composites, especially the 1:1 weight ratio composite exhibited significantly improved mechanical, mineral deposition, biological properties and controlled release. This made them potential candidates for bone tissue engineering applications. - Graphical abstract: Mechanical properties, biological activity and protein controlled release by poly(vinyl alcohol)–bioglass/chitosan–collagen composite scaffolds: A bone tissue engineering applications. - Highlights: • Preparation of PVABG:ChiCol hybrid composites and their bioactivities • Mechanical

  1. Mechanical properties, biological activity and protein controlled release by poly(vinyl alcohol)–bioglass/chitosan–collagen composite scaffolds: A bone tissue engineering applications

    Energy Technology Data Exchange (ETDEWEB)

    Pon-On, Weeraphat, E-mail: fsciwpp@ku.ac.th [Department of Physics, Faculty of Science, Kasetsart University, Bangkok 10900 (Thailand); Charoenphandhu, Narattaphol; Teerapornpuntakit, Jarinthorn; Thongbunchoo, Jirawan; Krishnamra, Nateetip [Center of Calcium and Bone Research (COCAB), Faculty of Science, Mahidol University (Thailand); Department of Physiology, Faculty of Science, Mahidol University (Thailand); Tang, I-Ming [ThEP Center, Commission of Higher Education, 328 Si Ayutthaya Rd. (Thailand); Department of Materials Science, Faculty of Science, Kasetsart University, Bangkok 10900 (Thailand)

    2014-05-01

    In the present study, composite scaffolds made with different weight ratios (0.5:1, 1:1 and 2:1) of bioactive glass (15Ca:80Si:5P) (BG)/polyvinyl alcohol (PVA) (PVABG) and chitosan (Chi)/collagen (Col) (ChiCol) were prepared by three mechanical freeze–thaw followed by freeze-drying to obtain the porous scaffolds. The mechanical properties and the in vitro biocompatibility of the composite scaffolds to simulated body fluid (SBF) and to rat osteoblast-like UMR-106 cells were investigated. The results from the studies indicated that the porosity and compressive strength were controlled by the weight ratio of PVABG:ChiCol. The highest compressive modulus of the composites made was 214.64 MPa which was for the 1:1 weight ratio PVABG:ChiCol. Mineralization study in SBF showed the formation of apatite crystals on the PVABG:ChiCol surface after 7 days of incubation. In vitro cell availability and proliferation tests confirmed the osteoblast attachment and growth on the PVABG:ChiCol surface. MTT and ALP tests on the 1:1 weight ratio PVABG:ChiCol composite indicated that the UMR-106 cells were viable. Alkaline phosphatase activity was found to increase with increasing culturing time. In addition, we showed the potential of PVABG:ChiCol drug delivery through PBS solution studies. 81.14% of BSA loading had been achieved and controlled release for over four weeks was observed. Our results indicated that the PVABG:ChiCol composites, especially the 1:1 weight ratio composite exhibited significantly improved mechanical, mineral deposition, biological properties and controlled release. This made them potential candidates for bone tissue engineering applications. - Graphical abstract: Mechanical properties, biological activity and protein controlled release by poly(vinyl alcohol)–bioglass/chitosan–collagen composite scaffolds: A bone tissue engineering applications. - Highlights: • Preparation of PVABG:ChiCol hybrid composites and their bioactivities • Mechanical

  2. A presynaptic role for PKA in synaptic tagging and memory.

    Science.gov (United States)

    Park, Alan Jung; Havekes, Robbert; Choi, Jennifer Hk; Luczak, Vince; Nie, Ting; Huang, Ted; Abel, Ted

    2014-10-01

    Protein kinase A (PKA) and other signaling molecules are spatially restricted within neurons by A-kinase anchoring proteins (AKAPs). Although studies on compartmentalized PKA signaling have focused on postsynaptic mechanisms, presynaptically anchored PKA may contribute to synaptic plasticity and memory because PKA also regulates presynaptic transmitter release. Here, we examine this issue using genetic and pharmacological application of Ht31, a PKA anchoring disrupting peptide. At the hippocampal Schaffer collateral CA3-CA1 synapse, Ht31 treatment elicits a rapid decay of synaptic responses to repetitive stimuli, indicating a fast depletion of the readily releasable pool of synaptic vesicles. The interaction between PKA and proteins involved in producing this pool of synaptic vesicles is supported by biochemical assays showing that synaptic vesicle protein 2 (SV2), Rim1, and SNAP25 are components of a complex that interacts with cAMP. Moreover, acute treatment with Ht31 reduces the levels of SV2. Finally, experiments with transgenic mouse lines, which express Ht31 in excitatory neurons at the Schaffer collateral CA3-CA1 synapse, highlight a requirement for presynaptically anchored PKA in pathway-specific synaptic tagging and long-term contextual fear memory. These results suggest that a presynaptically compartmentalized PKA is critical for synaptic plasticity and memory by regulating the readily releasable pool of synaptic vesicles. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. The Ubiquitin-Proteasome Pathway and Synaptic Plasticity

    Science.gov (United States)

    Hegde, Ashok N.

    2010-01-01

    Proteolysis by the ubiquitin-proteasome pathway (UPP) has emerged as a new molecular mechanism that controls wide-ranging functions in the nervous system, including fine-tuning of synaptic connections during development and synaptic plasticity in the adult organism. In the UPP, attachment of a small protein, ubiquitin, tags the substrates for…

  4. Calcineurin mediates homeostatic synaptic plasticity by regulating retinoic acid synthesis.

    Science.gov (United States)

    Arendt, Kristin L; Zhang, Zhenjie; Ganesan, Subhashree; Hintze, Maik; Shin, Maggie M; Tang, Yitai; Cho, Ahryon; Graef, Isabella A; Chen, Lu

    2015-10-20

    Homeostatic synaptic plasticity is a form of non-Hebbian plasticity that maintains stability of the network and fidelity for information processing in response to prolonged perturbation of network and synaptic activity. Prolonged blockade of synaptic activity decreases resting Ca(2+) levels in neurons, thereby inducing retinoic acid (RA) synthesis and RA-dependent homeostatic synaptic plasticity; however, the signal transduction pathway that links reduced Ca(2+)-levels to RA synthesis remains unknown. Here we identify the Ca(2+)-dependent protein phosphatase calcineurin (CaN) as a key regulator for RA synthesis and homeostatic synaptic plasticity. Prolonged inhibition of CaN activity promotes RA synthesis in neurons, and leads to increased excitatory and decreased inhibitory synaptic transmission. These effects of CaN inhibitors on synaptic transmission are blocked by pharmacological inhibitors of RA synthesis or acute genetic deletion of the RA receptor RARα. Thus, CaN, acting upstream of RA, plays a critical role in gating RA signaling pathway in response to synaptic activity. Moreover, activity blockade-induced homeostatic synaptic plasticity is absent in CaN knockout neurons, demonstrating the essential role of CaN in RA-dependent homeostatic synaptic plasticity. Interestingly, in GluA1 S831A and S845A knockin mice, CaN inhibitor- and RA-induced regulation of synaptic transmission is intact, suggesting that phosphorylation of GluA1 C-terminal serine residues S831 and S845 is not required for CaN inhibitor- or RA-induced homeostatic synaptic plasticity. Thus, our study uncovers an unforeseen role of CaN in postsynaptic signaling, and defines CaN as the Ca(2+)-sensing signaling molecule that mediates RA-dependent homeostatic synaptic plasticity.

  5. Scaffolded biology.

    Science.gov (United States)

    Minelli, Alessandro

    2016-09-01

    Descriptions and interpretations of the natural world are dominated by dichotomies such as organism vs. environment, nature vs. nurture, genetic vs. epigenetic, but in the last couple of decades strong dissatisfaction with those partitions has been repeatedly voiced and a number of alternative perspectives have been suggested, from perspectives such as Dawkins' extended phenotype, Turner's extended organism, Oyama's Developmental Systems Theory and Odling-Smee's niche construction theory. Last in time is the description of biological phenomena in terms of hybrids between an organism (scaffolded system) and a living or non-living scaffold, forming unit systems to study processes such as reproduction and development. As scaffold, eventually, we can define any resource used by the biological system, especially in development and reproduction, without incorporating it as happens in the case of resources fueling metabolism. Addressing biological systems as functionally scaffolded systems may help pointing to functional relationships that can impart temporal marking to the developmental process and thus explain its irreversibility; revisiting the boundary between development and metabolism and also regeneration phenomena, by suggesting a conceptual framework within which to investigate phenomena of regular hypermorphic regeneration such as characteristic of deer antlers; fixing a periodization of development in terms of the times at which a scaffolding relationship begins or is terminated; and promoting plant galls to legitimate study objects of developmental biology.

  6. The Role of Proteases in Hippocampal Synaptic Plasticity: Putting Together Small Pieces of a Complex Puzzle.

    Science.gov (United States)

    Salazar, Ivan L; Caldeira, Margarida V; Curcio, Michele; Duarte, Carlos B

    2016-02-01

    Long-term synaptic plasticity in the hippocampus is thought to underlie the formation of certain forms of memory, including spatial memory. The early phase of long-term synaptic potentiation and synaptic depression depends on post-translational modifications of synaptic proteins, while protein synthesis is also required for the late-phase of both forms of synaptic plasticity (L-LTP and L-LTD). Numerous pieces of evidence show a role for different types of proteases in synaptic plasticity, further increasing the diversity of mechanisms involved in the regulation of the intracellular and extracellular protein content. The cleavage of extracellular proteins is coupled to changes in postsynaptic intracellular mechanisms, and additional alterations in this compartment result from the protease-mediated targeting of intracellular proteins. Both mechanisms contribute to initiate signaling cascades that drive downstream pathways coupled to synaptic plasticity. In this review we summarize the evidence pointing to a role for extracellular and intracellular proteases, with distinct specificities, in synaptic plasticity. Where in the cells the proteases are located, and how they are regulated is also discussed. The combined actions of proteases and translation mechanisms contribute to a tight control of the synaptic proteome relevant for long-term synaptic potentiation and synaptic depression in the hippocampus. Additional studies are required to elucidate the mechanisms whereby these changes in the synaptic proteome are related with plasticity phenomena.

  7. Differential regulation of synaptic AP-2/clathrin vesicle uncoating in synaptic plasticity.

    Science.gov (United States)

    Candiello, Ermes; Mishra, Ratnakar; Schmidt, Bernhard; Jahn, Olaf; Schu, Peter

    2017-11-17

    AP-1/σ1B-deficiency causes X-linked intellectual disability. AP-1/σ1B -/- mice have impaired synaptic vesicle recycling, fewer synaptic vesicles and enhanced endosome maturation mediated by AP-1/σ1A. Despite defects in synaptic vesicle recycling synapses contain two times more endocytic AP-2 clathrin-coated vesicles. We demonstrate increased formation of two classes of AP-2/clathrin coated vesicles. One which uncoats readily and a second with a stabilised clathrin coat. Coat stabilisation is mediated by three molecular mechanisms: reduced recruitment of Hsc70 and synaptojanin1 and enhanced μ2/AP-2 phosphorylation and activation. Stabilised AP-2 vesicles are enriched in the structural active zone proteins Git1 and stonin2 and synapses contain more Git1. Endocytosis of the synaptic vesicle exocytosis regulating Munc13 isoforms are differentially effected. Regulation of synaptic protein endocytosis by the differential stability of AP-2/clathrin coats is a novel molecular mechanism of synaptic plasticity.

  8. Calcium/calmodulin-dependent protein kinase II is a ubiquitous molecule in human long-term memory synaptic plasticity: A systematic review

    Directory of Open Access Journals (Sweden)

    Negar Ataei

    2015-01-01

    Conclusions: The studies have shown the most important intracellular signal of long-term memory is calcium-dependent signals. Calcium linked calmodulin can activate CaMKII. After receiving information for learning and memory, CaMKII is activated by Glutamate, the most important neurotransmitter for memory-related plasticity. Glutamate activates CaMKII and it plays some important roles in synaptic plasticity modification and long-term memory.

  9. Nerve injury-induced calcium channel alpha-2-delta-1 protein dysregulation leads to increased pre-synaptic excitatory input into deep dorsal horn neurons and neuropathic allodynia

    Science.gov (United States)

    Zhou, Chunyi; Luo, Z. David

    2015-01-01

    Background Upregulation of voltage-gated-calcium-channel α2δ1 subunit post spinal nerve ligation injury (SNL) or in α2δ1-overexpressing transgenic (Tg) mice correlates with tactile allodynia, a pain state mediated mainly by Aβ sensory fibers forming synaptic connections with deep dorsal horn neurons. It is not clear however whether dysregulated α2δ1 alters deep dorsal horn synaptic neurotransmission that underlies tactile allodynia development post nerve injury. Methods Tactile allodynia was tested in the SNL and α2δ1 Tg models. Miniature excitatory/inhibitory postsynaptic currents were recorded in deep dorsal horn (DDH) neurons from these animal models using whole cell patch clamp slice recording techniques.. Results There was a significant increase in the frequency, but not amplitude, of miniature excitatory postsynaptic currents (mEPSC) in DDH neurons that correlated with tactile allodynia in SNL and α2δ1 Tg mice. Gabapentin, an α2δ1 ligand that is known to block tactile allodynia in these models, also normalized mEPSC frequency dose-dependently in DDH neurons from SNL and α2δ1 Tg mice. In contrast, neither frequency nor amplitude of miniature inhibitory postsynaptic currents (mIPSC) was altered in DDH neurons from SNL and α2δ1 Tg mice. Conclusion Our data suggest that α2δ1 dysregulation is highly likely contributing to tactile allodynia through a pre-synaptic mechanism involving facilitation of excitatory synaptic neurotransmission in deep dorsal horn of spinal cord. PMID:25691360

  10. Tetrameric far-red fluorescent protein as a scaffold to assemble an octavalent peptide nanoprobe for enhanced tumor targeting and intracellular uptake in vivo.

    Science.gov (United States)

    Luo, Haiming; Yang, Jie; Jin, Honglin; Huang, Chuan; Fu, Jianwei; Yang, Fei; Gong, Hui; Zeng, Shaoqun; Luo, Qingming; Zhang, Zhihong

    2011-06-01

    Relatively weak tumor affinities and short retention time in vivo hinder the application of targeting peptides in tumor molecular imaging. Multivalent strategies based on various scaffolds have been utilized to improve the ability of peptide-receptor binding or extend the clearance time of peptide-based probes. Here, we use a tetrameric far-red fluorescent protein (tfRFP) as a scaffold to create a self-assembled octavalent peptide fluorescent nanoprobe (Octa-FNP) using a genetic engineering approach. The multiligand connecting, fluorophore labeling and nanostructure formation of Octa-FNP were performed in one step. In vitro studies showed Octa-FNP is a 10-nm fluorescent probe with excellent serum stability. Cellular uptake of Octa-FNP by human nasopharyngeal cancer 5-8F cells is 15-fold of tetravalent probe, ∼80-fold of monovalent probe and ∼600-fold of nulvalent tfRFP. In vivo enhanced tumor targeting and intracellular uptake of Octa-FNP were confirmed using optical imaging and Western blot analysis. It achieved extremely high contrast of Octa-FNP signal between tumor tissue and normal organs, especially seldom Octa-FNP detected in liver and spleen. Owing to easy preparation, precise structural and functional control, and multivalent effect, Octa-FNP provides a powerful tool for tumor optical molecular imaging and evaluating the targeting ability of numerous peptides in vivo.

  11. Testosterone delivered with a scaffold is as effective as bone morphologic protein-2 in promoting the repair of critical-size segmental defect of femoral bone in mice.

    Directory of Open Access Journals (Sweden)

    Bi-Hua Cheng

    Full Text Available Loss of large bone segments due to fracture resulting from trauma or tumor removal is a common clinical problem. The goal of this study was to evaluate the use of scaffolds containing testosterone, bone morphogenetic protein-2 (BMP-2, or a combination of both for treatment of critical-size segmental bone defects in mice. A 2.5-mm wide osteotomy was created on the left femur of wildtype and androgen receptor knockout (ARKO mice. Testosterone, BMP-2, or both were delivered locally using a scaffold that bridged the fracture. Results of X-ray imaging showed that in both wildtype and ARKO mice, BMP-2 treatment induced callus formation within 14 days after initiation of the treatment. Testosterone treatment also induced callus formation within 14 days in wildtype but not in ARKO mice. Micro-computed tomography and histological examinations revealed that testosterone treatment caused similar degrees of callus formation as BMP-2 treatment in wildtype mice, but had no such effect in ARKO mice, suggesting that the androgen receptor is required for testosterone to initiate fracture healing. These results demonstrate that testosterone is as effective as BMP-2 in promoting the healing of critical-size segmental defects and that combination therapy with testosterone and BMP-2 is superior to single therapy. Results of this study may provide a foundation to develop a cost effective and efficient therapeutic modality for treatment of bone fractures with segmental defects.

  12. Structural and biochemical characterization of human PR70 in isolation and in complex with the scaffolding subunit of protein phosphatase 2A.

    Directory of Open Access Journals (Sweden)

    Rebecca Dovega

    Full Text Available Protein Phosphatase 2A (PP2A is a major Ser/Thr phosphatase involved in the regulation of various cellular processes. PP2A assembles into diverse trimeric holoenzymes, which consist of a scaffolding (A subunit, a catalytic (C subunit and various regulatory (B subunits. Here we report a 2.0 Å crystal structure of the free B''/PR70 subunit and a SAXS model of an A/PR70 complex. The crystal structure of B''/PR70 reveals a two domain elongated structure with two Ca2+ binding EF-hands. Furthermore, we have characterized the interaction of both binding partner and their calcium dependency using biophysical techniques. Ca2+ biophysical studies with Circular Dichroism showed that the two EF-hands display different affinities to Ca2+. In the absence of the catalytic C-subunit, the scaffolding A-subunit remains highly mobile and flexible even in the presence of the B''/PR70 subunit as judged by SAXS. Isothermal Titration Calorimetry studies and SAXS data support that PR70 and the A-subunit have high affinity to each other. This study provides additional knowledge about the structural basis for the function of B'' containing holoenzymes.

  13. Controlled chondrogenesis from adipose-derived stem cells by recombinant transforming growth factor-β3 fusion protein in peptide scaffolds.

    Science.gov (United States)

    Zheng, Dong; Dan, Yang; Yang, Shu-hua; Liu, Guo-hui; Shao, Zeng-wu; Yang, Cao; Xiao, Bao-jun; Liu, Xiangmei; Wu, Shuilin; Zhang, Tainjin; Chu, Paul K

    2015-01-01

    Adipose-derived stem cells (ADSCs) are promising for cartilage repair due to their easy accessibility and chondrogenic potential. Although chondrogenesis of transforming growth factor-β (TGF-β) mediated mesenchymal stem cells (MSCs) is well established in vitro, clinical tissue engineering requires effective and controlled delivery of TGF-β in vivo. In this work, a self-assembled peptide scaffold was employed to construct cartilages in vivo through the chondrogenesis from ADSCs controlled by recombinant fusion protein LAP-MMP-mTGF-β3 that was transfected by lentiviral vectors. During this course, the addition of matrix metalloproteinases (MMPs) can trigger the release of mTGF-β3 from the recombinant fusion protein of LAP-MMP-mTGF-β3 in the combined scaffolds, thus stimulating the differentiation of ADSCs into chondrogenesis. The specific expression of cartilage genes was analyzed by real-time polymerase chain reaction and Western blot. The expression of chondrocytic markers was obviously upregulated to a higher level compared to the one by commonly used TGF-β3 alone. After 3 weeks of in vitro culturing, the hybrids with differentiated chondrogenesis were then injected subcutaneously into nude mice and retrieved after 4 weeks of culturing in vivo. Histological analysis also confirmed that the recombinant fusion protein was more effective for the formation of cartilage matrix than the cases either with TGF-β3 alone or without LAP-MMP-mTGF-β3 (P<0.05). This study demonstrates that controlled local delivery of the LAP-MMP-mTGF-β3 constructs can accelerate differentiation of ADSCs into the cartilage in vivo, which indicates the great potential of this hybrid in rapid therapy of osteoarthritis. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  14. Ubiquitination-dependent mechanisms regulate synaptic growth and function.

    Science.gov (United States)

    DiAntonio, A; Haghighi, A P; Portman, S L; Lee, J D; Amaranto, A M; Goodman, C S

    2001-07-26

    The covalent attachment of ubiquitin to cellular proteins is a powerful mechanism for controlling protein activity and localization. Ubiquitination is a reversible modification promoted by ubiquitin ligases and antagonized by deubiquitinating proteases. Ubiquitin-dependent mechanisms regulate many important processes including cell-cycle progression, apoptosis and transcriptional regulation. Here we show that ubiquitin-dependent mechanisms regulate synaptic development at the Drosophila neuromuscular junction (NMJ). Neuronal overexpression of the deubiquitinating protease fat facets leads to a profound disruption of synaptic growth control; there is a large increase in the number of synaptic boutons, an elaboration of the synaptic branching pattern, and a disruption of synaptic function. Antagonizing the ubiquitination pathway in neurons by expression of the yeast deubiquitinating protease UBP2 (ref. 5) also produces synaptic overgrowth and dysfunction. Genetic interactions between fat facets and highwire, a negative regulator of synaptic growth that has structural homology to a family of ubiquitin ligases, suggest that synaptic development may be controlled by the balance between positive and negative regulators of ubiquitination.

  15. Bioresorbable Scaffolds.

    Science.gov (United States)

    Panaich, Sidakpal; Schreiber, Theodore; Grines, Cindy

    2014-08-01

    Percutaneous coronary intervention (PCI) has undergone major advances including the evolution in stent technology, from bare metal stents (BMS), to their drug eluting counterparts, to the development of bioresorbable scaffolds (BRS). The primary notion of BRS was to facilitate complete vascular healing and restore normal endothelial function following the resorption of stent scaffold while providing equivalent mechanical properties of a metallic drug eluting stents (DES) in the earlier stages. BRS provide attractive physiologic advancements over the existing DES and have shown promising results in initial clinical studies albeit with small sample sizes. Their use has been primarily restricted to patients recruited in clinical trials with limited real-world applicability. Thus, data from larger randomised control trials is awaited. The major objective of this article is to review the evidence on BRS and identify their clinical applicability in current interventional practice.

  16. A Method for Reconstruction of Severely Damaged Spinal Cord using Autologous Hematopoietic Stem Cells and Platelet-rich Protein as a Biological Scaffold.

    Science.gov (United States)

    Ammar, Ahmed Sabry; Osman, Yasser; Hendam, Ahmed Taher; Hasen, Mohammed Ahmed; Al Rubaish, Fatma Abdullah; Al Nujaidi, Danya Yaagoub; Al Abbas, Faisal Mishal

    2017-01-01

    There have been attempts to alter the prognosis of severe spinal cord injury in different centers, but none of which have reliably altered the outcome. Some trials use stem cells (SCs) that produced widely differing results. We hereby add our experience in our center of a surgical reconstruction of the damaged spinal cord using a mixture of SCs and Platelet-Rich Protein (PRP) with fibrin coated as a biological scaffold. Four cases of severely damaged spinal cord have been operated for neurolysis and reconstruction of the spinal cord using SCs and platelet-rich protein (PRP) with fibrin coated harvested from the peripheral circulation of the patient. PRP serves to maintain the position of the SCs. One milliliter suspension contains an average of 2.8 × 10 6 of autologous hematopoietic SCs. Patients were intraoperatively monitored by somatosensory evoked potential, motor evoked potentials, and delta wave. They are clinically followed postoperatively and electromyogram was repeated every 2 weeks. Magnetic resonance imaging (MRI) was repeated regularly. The patients are followed up for a period between 2 and 3 years. One patient demonstrated motor and objective sensory improvement ( P = 0.05), two other patients reported subjective sensory improvement, and the fourth one remained without any improvement ( P = 0.1). None of these patients demonstrated any sign of deterioration or complication either on the surgery or on implanting of the SCs. MRI clearly proved that the inserted biological scaffold remained in place of reconstruction. SCs may play a role in restoring spinal cord functions. However, the unsolved problems of the use of SCs and related ethical issues should be addressed.

  17. Evaluation of protection induced by a dengue virus serotype 2 envelope domain III protein scaffold/DNA vaccine in non-human primates.

    Science.gov (United States)

    McBurney, Sean P; Sunshine, Justine E; Gabriel, Sarah; Huynh, Jeremy P; Sutton, William F; Fuller, Deborah H; Haigwood, Nancy L; Messer, William B

    2016-06-24

    We describe the preclinical development of a dengue virus vaccine targeting the dengue virus serotype 2 (DENV2) envelope domain III (EDIII). This study provides proof-of-principle that a dengue EDIII protein scaffold/DNA vaccine can protect against dengue challenge. The dengue vaccine (EDIII-E2) is composed of both a protein particle and a DNA expression plasmid delivered simultaneously via intramuscular injection (protein) and gene gun (DNA) into rhesus macaques. The protein component can contain a maximum of 60 copies of EDIII presented on a multimeric scaffold of Geobacillus stearothermophilus E2 proteins. The DNA component is composed of the EDIII portion of the envelope gene cloned into an expression plasmid. The EDIII-E2 vaccine elicited robust antibody responses to DENV2, with neutralizing antibody responses detectable following the first boost and reaching titers of greater than 1:100,000 following the second and final boost. Vaccinated and naïve groups of macaques were challenged with DENV2. All vaccinated macaques were protected from detectable viremia by infectious assay, while naïve animals had detectable viremia for 2-7 days post-challenge. All naïve macaques had detectable viral RNA from day 2-10 post-challenge. In the EDIII-E2 group, three macaques were negative for viral RNA and three were found to have detectable viral RNA post challenge. Viremia onset was delayed and the duration was shortened relative to naïve controls. The presence of viral RNA post-challenge corresponded to a 10-30-fold boost in neutralization titers 28 days post challenge, whereas no boost was observed in the fully protected animals. Based on these results, we determine that pre-challenge 50% neutralization titers of >1:6000 correlated with sterilizing protection against DENV2 challenge in EDIII-E2 vaccinated macaques. Identification of the critical correlate of protection for the EDIII-E2 platform in the robust non-human primate model lays the groundwork for further

  18. Infant avoidance training alters cellular activation patterns in prefronto-limbic circuits during adult avoidance learning: I. Cellular imaging of neurons expressing the synaptic plasticity early growth response protein 1 (Egr1).

    Science.gov (United States)

    Gröger, Nicole; Mannewitz, Anja; Bock, Jörg; de Schultz, Tony Fernando; Guttmann, Katja; Poeggel, Gerd; Braun, Katharina

    2017-11-01

    Both positive feedback learning and negative feedback learning are essential for adapting and optimizing behavioral performance. There is increasing evidence in humans and animals that the ability of negative feedback learning emerges postnatally. Our work in rats, using a two-way active avoidance task (TWA) as an experimental paradigm for negative feedback learning, revealed that medial and lateral prefrontal regions of infant rats undergo dramatic synaptic reorganization during avoidance training, resulting in improved avoidance learning in adulthood. The aim of this study was to identify changes of cellular activation patterns during the course of training and in relation to infant pretraining. We applied a quantitative cellular imaging technique using the immunocytochemical detection of the activity marker early growth response protein 1 (Egr1) as a candidate contributing to learning-induced synaptic plasticity. We found region-specific cellular activity patterns, which indicate that during the acquisition phase, Egr1 expression is specifically elevated in cellular ensembles of the orbitofrontal, dorsal anterior cingulate and hippocampal CA1 region. During memory retrieval Egr1 expression is elevated in cellular ensembles of the dentate gyrus. Moreover, we, for the first time, show here that TWA training during infancy alters adult learning- and memory-related patterns of Egr1 expression in these brain regions. It is tempting to speculate that during infant learning, specific Egr1-expressing cellular ensembles are "tagged" representing long-term memory formation, and that these cell ensembles may be reactivated during adult learning.

  19. Neuronal Networks on Nanocellulose Scaffolds.

    Science.gov (United States)

    Jonsson, Malin; Brackmann, Christian; Puchades, Maja; Brattås, Karoline; Ewing, Andrew; Gatenholm, Paul; Enejder, Annika

    2015-11-01

    Proliferation, integration, and neurite extension of PC12 cells, a widely used culture model for cholinergic neurons, were studied in nanocellulose scaffolds biosynthesized by Gluconacetobacter xylinus to allow a three-dimensional (3D) extension of neurites better mimicking neuronal networks in tissue. The interaction with control scaffolds was compared with cationized nanocellulose (trimethyl ammonium betahydroxy propyl [TMAHP] cellulose) to investigate the impact of surface charges on the cell interaction mechanisms. Furthermore, coatings with extracellular matrix proteins (collagen, fibronectin, and laminin) were investigated to determine the importance of integrin-mediated cell attachment. Cell proliferation was evaluated by a cellular proliferation assay, while cell integration and neurite propagation were studied by simultaneous label-free Coherent anti-Stokes Raman Scattering and second harmonic generation microscopy, providing 3D images of PC12 cells and arrangement of nanocellulose fibrils, respectively. Cell attachment and proliferation were enhanced by TMAHP modification, but not by protein coating. Protein coating instead promoted active interaction between the cells and the scaffold, hence lateral cell migration and integration. Irrespective of surface modification, deepest cell integration measured was one to two cell layers, whereas neurites have a capacity to integrate deeper than the cell bodies in the scaffold due to their fine dimensions and amoeba-like migration pattern. Neurites with lengths of >50 μm were observed, successfully connecting individual cells and cell clusters. In conclusion, TMAHP-modified nanocellulose scaffolds promote initial cellular scaffold adhesion, which combined with additional cell-scaffold treatments enables further formation of 3D neuronal networks.

  20. Protein-releasing polymeric scaffolds induce fibrochondrocytic differentiation of endogenous cells for knee meniscus regeneration in sheep

    Science.gov (United States)

    Lee, Chang H.; Rodeo, Scott A.; Fortier, Lisa Ann; Lu, Chuanyong; Erisken, Cevat

    2015-01-01

    Regeneration of complex tissues, such as kidney, liver, and cartilage, continues to be a scientific and translational challenge. Survival of ex vivo cultured, transplanted cells in tissue grafts is among one of the key barriers. Meniscus is a complex tissue consisting of collagen fibers and proteoglycans with gradient phenotypes of fibrocartilage and functions to provide congruence of the knee joint, without which the patient is likely to develop arthritis. Endogenous stem/progenitor cells regenerated the knee meniscus upon spatially released human connective tissue growth factor (CTGF) and transforming growth factor–β3 (TGFβ3) from a three-dimensional (3D)–printed biomaterial, enabling functional knee recovery. Sequentially applied CTGF and TGFβ3 were necessary and sufficient to propel mesenchymal stem/progenitor cells, as a heterogeneous population or as single-cell progenies, into fibrochondrocytes that concurrently synthesized procollagens I and IIα. When released from microchannels of 3D–printed, human meniscus scaffolds, CTGF and TGFβ3 induced endogenous stem/progenitor cells to differentiate and synthesize zone-specific type I and II collagens. We then replaced sheep meniscus with anatomically correct, 3D–printed scaffolds that incorporated spatially delivered CTGF and TGFβ3. Endogenous cells regenerated the meniscus with zone-specific matrix phenotypes: primarily type I collagen in the outer zone, and type II collagen in the inner zone, reminiscent of the native meniscus. Spatiotemporally delivered CTGF and TGFβ3 also restored inhomogeneous mechanical properties in the regenerated sheep meniscus. Survival and directed differentiation of endogenous cells in a tissue defect may have implications in the regeneration of complex (heterogeneous) tissues and organs. PMID:25504882

  1. Role of MicroRNA in Governing Synaptic Plasticity.

    Science.gov (United States)

    Ye, Yuqin; Xu, Hongyu; Su, Xinhong; He, Xiaosheng

    2016-01-01

    Although synaptic plasticity in neural circuits is orchestrated by an ocean of genes, molecules, and proteins, the underlying mechanisms remain poorly understood. Recently, it is well acknowledged that miRNA exerts widespread regulation over the translation and degradation of target gene in nervous system. Increasing evidence suggests that quite a few specific miRNAs play important roles in various respects of synaptic plasticity including synaptogenesis, synaptic morphology alteration, and synaptic function modification. More importantly, the miRNA-mediated regulation of synaptic plasticity is not only responsible for synapse development and function but also involved in the pathophysiology of plasticity-related diseases. A review is made here on the function of miRNAs in governing synaptic plasticity, emphasizing the emerging regulatory role of individual miRNAs in synaptic morphological and functional plasticity, as well as their implications in neurological disorders. Understanding of the way in which miRNAs contribute to synaptic plasticity provides rational clues in establishing the novel therapeutic strategy for plasticity-related diseases.

  2. Neuron Membrane Trafficking and Protein Kinases Involved in Autism and ADHD

    Directory of Open Access Journals (Sweden)

    Yasuko Kitagishi

    2015-01-01

    Full Text Available A brain-enriched multi-domain scaffolding protein, neurobeachin has been identified as a candidate gene for autism patients. Mutations in the synaptic adhesion protein cell adhesion molecule 1 (CADM1 are also associated with autism spectrum disorder, a neurodevelopmental disorder of uncertain molecular origin. Potential roles of neurobeachin and CADM1 have been suggested to a function of vesicle transport in endosomal trafficking. It seems that protein kinase B (AKT and cyclic adenosine monophosphate (cAMP-dependent protein kinase A (PKA have key roles in the neuron membrane trafficking involved in the pathogenesis of autism. Attention deficit hyperactivity disorder (ADHD is documented to dopaminergic insufficiencies, which is attributed to synaptic dysfunction of dopamine transporter (DAT. AKT is also essential for the DAT cell-surface redistribution. In the present paper, we summarize and discuss the importance of several protein kinases that regulate the membrane trafficking involved in autism and ADHD, suggesting new targets for therapeutic intervention.

  3. Developmental Scaffolding

    DEFF Research Database (Denmark)

    Giorgi, Franco; Bruni, Luis Emilio

    2015-01-01

    . As this boundary is gradually defined during development, cells enter into new functional relationships, while, at the same time, are relieved from their physical determinism. The resulting constraints can thus become the driving forces that upgrade embryonic scaffolding from the simple molecular signalling...... to the complexity of sign recognition proper of a cellular community. In this semiotic perspective, the apparent goal directness of any developmental strategy should no longer be accounted for by a predetermined genetic program, but by the gradual definition of the relationships selected amongst the ones...

  4. Pannexin 1 Regulates Bidirectional Hippocampal Synaptic Plasticity in Adult Mice

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    Alvaro O. Ardiles

    2014-10-01

    Full Text Available The threshold for bidirectional modification of synaptic plasticity is known to be controlled by several factors, including the balance between protein phosphorylation and dephosphorylation, postsynaptic free Ca2+ concentration and NMDA receptor (NMDAR composition of GluN2 subunits. Pannexin 1 (Panx1, a member of the integral membrane protein family, has been shown to form non-selective channels and to regulate the induction of synaptic plasticity as well as hippocampal-dependent learning. Although Panx1 channels have been suggested to play a role in excitatory long-term potentiation (LTP, it remains unknown whether these channels also modulate long-term depression (LTD or the balance between both types of synaptic plasticity. To study how Panx1 contributes to excitatory synaptic efficacy, we examined the age-dependent effects of eliminating or blocking Panx1 channels on excitatory synaptic plasticity within the CA1 region of the mouse hippocampus. By using different protocols to induce bidirectional synaptic plasticity, Panx1 channel blockade or lack of Panx1 were found to enhance LTP, whereas both conditions precluded the induction of LTD in adults, but not in young animals. These findings suggest that Panx1 channels restrain the sliding threshold for the induction of synaptic plasticity and underlying brain mechanisms of learning and memory.

  5. Pannexin 1 regulates bidirectional hippocampal synaptic plasticity in adult mice.

    Science.gov (United States)

    Ardiles, Alvaro O; Flores-Muñoz, Carolina; Toro-Ayala, Gabriela; Cárdenas, Ana M; Palacios, Adrian G; Muñoz, Pablo; Fuenzalida, Marco; Sáez, Juan C; Martínez, Agustín D

    2014-01-01

    The threshold for bidirectional modification of synaptic plasticity is known to be controlled by several factors, including the balance between protein phosphorylation and dephosphorylation, postsynaptic free Ca(2+) concentration and NMDA receptor (NMDAR) composition of GluN2 subunits. Pannexin 1 (Panx1), a member of the integral membrane protein family, has been shown to form non-selective channels and to regulate the induction of synaptic plasticity as well as hippocampal-dependent learning. Although Panx1 channels have been suggested to play a role in excitatory long-term potentiation (LTP), it remains unknown whether these channels also modulate long-term depression (LTD) or the balance between both types of synaptic plasticity. To study how Panx1 contributes to excitatory synaptic efficacy, we examined the age-dependent effects of eliminating or blocking Panx1 channels on excitatory synaptic plasticity within the CA1 region of the mouse hippocampus. By using different protocols to induce bidirectional synaptic plasticity, Panx1 channel blockade or lack of Panx1 were found to enhance LTP, whereas both conditions precluded the induction of LTD in adults, but not in young animals. These findings suggest that Panx1 channels restrain the sliding threshold for the induction of synaptic plasticity and underlying brain mechanisms of learning and memory.

  6. Role of DHA in aging-related changes in mouse brain synaptic plasma membrane proteome.

    Science.gov (United States)

    Sidhu, Vishaldeep K; Huang, Bill X; Desai, Abhishek; Kevala, Karl; Kim, Hee-Yong

    2016-05-01

    Aging has been related to diminished cognitive function, which could be a result of ineffective synaptic function. We have previously shown that synaptic plasma membrane proteins supporting synaptic integrity and neurotransmission were downregulated in docosahexaenoic acid (DHA)-deprived brains, suggesting an important role of DHA in synaptic function. In this study, we demonstrate aging-induced synaptic proteome changes and DHA-dependent mitigation of such changes using mass spectrometry-based protein quantitation combined with western blot or messenger RNA analysis. We found significant reduction of 15 synaptic plasma membrane proteins in aging brains including fodrin-α, synaptopodin, postsynaptic density protein 95, synaptic vesicle glycoprotein 2B, synaptosomal-associated protein 25, synaptosomal-associated protein-α, N-methyl-D-aspartate receptor subunit epsilon-2 precursor, AMPA2, AP2, VGluT1, munc18-1, dynamin-1, vesicle-associated membrane protein 2, rab3A, and EAAT1, most of which are involved in synaptic transmission. Notably, the first 9 proteins were further reduced when brain DHA was depleted by diet, indicating that DHA plays an important role in sustaining these synaptic proteins downregulated during aging. Reduction of 2 of these proteins was reversed by raising the brain DHA level by supplementing aged animals with an omega-3 fatty acid sufficient diet for 2 months. The recognition memory compromised in DHA-depleted animals was also improved. Our results suggest a potential role of DHA in alleviating aging-associated cognitive decline by offsetting the loss of neurotransmission-regulating synaptic proteins involved in synaptic function. Published by Elsevier Inc.

  7. Synaptic Homeostasis and Restructuring across the Sleep-Wake Cycle.

    Directory of Open Access Journals (Sweden)

    Wilfredo Blanco

    2015-05-01

    Full Text Available Sleep is critical for hippocampus-dependent memory consolidation. However, the underlying mechanisms of synaptic plasticity are poorly understood. The central controversy is on whether long-term potentiation (LTP takes a role during sleep and which would be its specific effect on memory. To address this question, we used immunohistochemistry to measure phosphorylation of Ca2+/calmodulin-dependent protein kinase II (pCaMKIIα in the rat hippocampus immediately after specific sleep-wake states were interrupted. Control animals not exposed to novel objects during waking (WK showed stable pCaMKIIα levels across the sleep-wake cycle, but animals exposed to novel objects showed a decrease during subsequent slow-wave sleep (SWS followed by a rebound during rapid-eye-movement sleep (REM. The levels of pCaMKIIα during REM were proportional to cortical spindles near SWS/REM transitions. Based on these results, we modeled sleep-dependent LTP on a network of fully connected excitatory neurons fed with spikes recorded from the rat hippocampus across WK, SWS and REM. Sleep without LTP orderly rescaled synaptic weights to a narrow range of intermediate values. In contrast, LTP triggered near the SWS/REM transition led to marked swaps in synaptic weight ranking. To better understand the interaction between rescaling and restructuring during sleep, we implemented synaptic homeostasis and embossing in a detailed hippocampal-cortical model with both excitatory and inhibitory neurons. Synaptic homeostasis was implemented by weakening potentiation and strengthening depression, while synaptic embossing was simulated by evoking LTP on selected synapses. We observed that synaptic homeostasis facilitates controlled synaptic restructuring. The results imply a mechanism for a cognitive synergy between SWS and REM, and suggest that LTP at the SWS/REM transition critically influences the effect of sleep: Its lack determines synaptic homeostasis, its presence causes

  8. A fluorescent protein scaffold for presenting structurally constrained peptides provides an effective screening system to identify high affinity target-binding peptides.

    Directory of Open Access Journals (Sweden)

    Tetsuya Kadonosono

    Full Text Available Peptides that have high affinity for target molecules on the surface of cancer cells are crucial for the development of targeted cancer therapies. However, unstructured peptides often fail to bind their target molecules with high affinity. To efficiently identify high-affinity target-binding peptides, we have constructed a fluorescent protein scaffold, designated gFPS, in which structurally constrained peptides are integrated at residues K131-L137 of superfolder green fluorescent protein. Molecular dynamics simulation supported the suitability of this site for presentation of exogenous peptides with a constrained structure. gFPS can present 4 to 12 exogenous amino acids without a loss of fluorescence. When gFPSs presenting human epidermal growth factor receptor type 2 (HER2-targeting peptides were added to the culture medium of HER2-expressing cells, we could easily identify the peptides with high HER2-affinity and -specificity based on gFPS fluorescence. In addition, gFPS could be expressed on the yeast cell surface and applied for a high-throughput screening. These results demonstrate that gFPS has the potential to serve as a powerful tool to improve screening of structurally constrained peptides that have a high target affinity, and suggest that it could expedite the one-step identification of clinically applicable cancer cell-binding peptides.

  9. VPS29 is not an active metallo-phosphatase but is a rigid scaffold required for retromer interaction with accessory proteins.

    Directory of Open Access Journals (Sweden)

    James D Swarbrick

    Full Text Available VPS29 is a key component of the cargo-binding core complex of retromer, a protein assembly with diverse roles in transport of receptors within the endosomal system. VPS29 has a fold related to metal-binding phosphatases and mediates interactions between retromer and other regulatory proteins. In this study we examine the functional interactions of mammalian VPS29, using X-ray crystallography and NMR spectroscopy. We find that although VPS29 can coordinate metal ions Mn(2+ and Zn(2+ in both the putative active site and at other locations, the affinity for metals is low, and lack of activity in phosphatase assays using a putative peptide substrate support the conclusion that VPS29 is not a functional metalloenzyme. There is evidence that structural elements of VPS29 critical for binding the retromer subunit VPS35 may undergo both metal-dependent and independent conformational changes regulating complex formation, however studies using ITC and NMR residual dipolar coupling (RDC measurements show that this is not the case. Finally, NMR chemical shift mapping indicates that VPS29 is able to associate with SNX1 via a conserved hydrophobic surface, but with a low affinity that suggests additional interactions will be required to stabilise the complex in vivo. Our conclusion is that VPS29 is a metal ion-independent, rigid scaffolding domain, which is essential but not sufficient for incorporation of retromer into functional endosomal transport assemblies.

  10. Enigma homolog 1 scaffolds protein kinase D1 to regulate the activity of the cardiac L-type voltage-gated calcium channel.

    Science.gov (United States)

    Maturana, Andrés D; Wälchli, Sébastien; Iwata, Miki; Ryser, Stephan; Van Lint, Johannes; Hoshijima, Masahiko; Schlegel, Werner; Ikeda, Yasuhiro; Tanizawa, Katsuyuki; Kuroda, Shun'ichi

    2008-06-01

    In cardiomyocytes, protein kinase D1 (PKD1) plays a central role in the response to stress signals. From a yeast two-hybrid assay, we have identified Enigma Homolog 1 (ENH1) as a new binding partner of PKD1. Since in neurons, ENH1, associated with protein kinase Cepsilon, was shown to modulate the activity of N-type calcium channels, and the pore-forming subunit of the cardiac L-type voltage-gated calcium channel, alpha1C, possesses a potential phosphorylation site for PKD1, we studied here a possible role of ENH1 and PKD1 in the regulation of the cardiac L-type voltage-gated calcium channel. PKD1-interacting proteins were searched by yeast two-hybrid screening. In vivo protein interactions in cardiomyocytes isolated from heart ventricles of newborn rats were tested by co-immunoprecipitation. Small interfering RNA and a dominant negative mutant of PKD1 were delivered into cardiomyocytes by use of an adenovirus. Calcium currents were measured by the patch-clamp technique. Both ENH1 and PKD1 interact with alpha1C in cardiomyocytes. This interaction is increased upon stimulation. Silencing of ENH1 prevented the binding of PKD1 to alpha1C. Moreover, a dominant negative mutant of PKD1 or the silencing of ENH1 inhibited the alpha-adrenergic-induced increase of L-type calcium currents. We found a new binding partner, ENH1, and a new target, alpha1C, for PKD1 in neonatal rat cardiomyocytes. We propose a model where ENH1 scaffolds PKD1 to alpha1C in order to form a signalling complex that regulates the activity of cardiac L-type voltage-gated Ca(2+) channels.

  11. Free mRNA in excess upon polysome dissociation is a scaffold for protein multimerization to form stress granules.

    Science.gov (United States)

    Bounedjah, Ouissame; Desforges, Bénédicte; Wu, Ting-Di; Pioche-Durieu, Catherine; Marco, Sergio; Hamon, Loic; Curmi, Patrick A; Guerquin-Kern, Jean-Luc; Piétrement, Olivier; Pastré, David

    2014-07-01

    The sequence of events leading to stress granule assembly in stressed cells remains elusive. We show here, using isotope labeling and ion microprobe, that proportionally more RNA than proteins are present in stress granules than in surrounding cytoplasm. We further demonstrate that the delivery of single strand polynucleotides, mRNA and ssDNA, to the cytoplasm can trigger stress granule assembly. On the other hand, increasing the cytoplasmic level of mRNA-binding proteins like YB-1 can directly prevent the aggregation of mRNA by forming isolated mRNPs, as evidenced by atomic force microscopy. Interestingly, we also discovered that enucleated cells do form stress granules, demonstrating that the translocation to the cytoplasm of nuclear prion-like RNA-binding proteins like TIA-1 is dispensable for stress granule assembly. The results lead to an alternative view on stress granule formation based on the following sequence of events: after the massive dissociation of polysomes during stress, mRNA-stabilizing proteins like YB-1 are outnumbered by the burst of nonpolysomal mRNA. mRNA freed of ribosomes thus becomes accessible to mRNA-binding aggregation-prone proteins or misfolded proteins, which induces stress granule formation. Within the frame of this model, the shuttling of nuclear mRNA-stabilizing proteins to the cytoplasm could dissociate stress granules or prevent their assembly. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  12. Architecture of the Yeast Mitochondrial Iron-Sulfur Cluster Assembly Machinery: THE SUB-COMPLEX FORMED BY THE IRON DONOR, Yfh1 PROTEIN, AND THE SCAFFOLD, Isu1 PROTEIN.

    Science.gov (United States)

    Ranatunga, Wasantha; Gakh, Oleksandr; Galeano, Belinda K; Smith, Douglas Y; Söderberg, Christopher A G; Al-Karadaghi, Salam; Thompson, James R; Isaya, Grazia

    2016-05-06

    The biosynthesis of Fe-S clusters is a vital process involving the delivery of elemental iron and sulfur to scaffold proteins via molecular interactions that are still poorly defined. We reconstituted a stable, functional complex consisting of the iron donor, Yfh1 (yeast frataxin homologue 1), and the Fe-S cluster scaffold, Isu1, with 1:1 stoichiometry, [Yfh1]24·[Isu1]24 Using negative staining transmission EM and single particle analysis, we obtained a three-dimensional reconstruction of this complex at a resolution of ∼17 Å. In addition, via chemical cross-linking, limited proteolysis, and mass spectrometry, we identified protein-protein interaction surfaces within the complex. The data together reveal that [Yfh1]24·[Isu1]24 is a roughly cubic macromolecule consisting of one symmetric Isu1 trimer binding on top of one symmetric Yfh1 trimer at each of its eight vertices. Furthermore, molecular modeling suggests that two subunits of the cysteine desulfurase, Nfs1, may bind symmetrically on top of two adjacent Isu1 trimers in a manner that creates two putative [2Fe-2S] cluster assembly centers. In each center, conserved amino acids known to be involved in sulfur and iron donation by Nfs1 and Yfh1, respectively, are in close proximity to the Fe-S cluster-coordinating residues of Isu1. We suggest that this architecture is suitable to ensure concerted and protected transfer of potentially toxic iron and sulfur atoms to Isu1 during Fe-S cluster assembly. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Synaptic vesicle dynamic changes in a model of fragile X.

    Science.gov (United States)

    Broek, Jantine A C; Lin, Zhanmin; de Gruiter, H Martijn; van 't Spijker, Heleen; Haasdijk, Elize D; Cox, David; Ozcan, Sureyya; van Cappellen, Gert W A; Houtsmuller, Adriaan B; Willemsen, Rob; de Zeeuw, Chris I; Bahn, Sabine

    2016-01-01

    Fragile X syndrome (FXS) is a single-gene disorder that is the most common heritable cause of intellectual disability and the most frequent monogenic cause of autism spectrum disorders (ASD). FXS is caused by an expansion of trinucleotide repeats in the promoter region of the fragile X mental retardation gene (Fmr1). This leads to a lack of fragile X mental retardation protein (FMRP), which regulates translation of a wide range of messenger RNAs (mRNAs). The extent of expression level alterations of synaptic proteins affected by FMRP loss and their consequences on synaptic dynamics in FXS has not been fully investigated. Here, we used an Fmr1 knockout (KO) mouse model to investigate the molecular mechanisms underlying FXS by monitoring protein expression changes using shotgun label-free liquid-chromatography mass spectrometry (LC-MS(E)) in brain tissue and synaptosome fractions. FXS-associated candidate proteins were validated using selected reaction monitoring (SRM) in synaptosome fractions for targeted protein quantification. Furthermore, functional alterations in synaptic release and dynamics were evaluated using live-cell imaging, and interpretation of synaptic dynamics differences was investigated using electron microscopy. Key findings relate to altered levels of proteins involved in GABA-signalling, especially in the cerebellum. Further exploration using microscopy studies found reduced synaptic vesicle unloading of hippocampal neurons and increased vesicle unloading in cerebellar neurons, which suggests a general decrease of synaptic transmission. Our findings suggest that FMRP is a regulator of synaptic vesicle dynamics, which supports the role of FMRP in presynaptic functions. Taken together, these studies provide novel insights into the molecular changes associated with FXS.

  14. The neuroprotection of cannabidiol against MPP⁺-induced toxicity in PC12 cells involves trkA receptors, upregulation of axonal and synaptic proteins, neuritogenesis, and might be relevant to Parkinson's disease.

    Science.gov (United States)

    Santos, Neife Aparecida Guinaim; Martins, Nádia Maria; Sisti, Flávia Malvestio; Fernandes, Laís Silva; Ferreira, Rafaela Scalco; Queiroz, Regina Helena Costa; Santos, Antônio Cardozo

    2015-12-25

    Cannabidiol (CBD) is a non-psychoactive constituent of Cannabis sativa with potential to treat neurodegenerative diseases. Its neuroprotection has been mainly associated with anti-inflammatory and antioxidant events; however, other mechanisms might be involved. We investigated the involvement of neuritogenesis, NGF receptors (trkA), NGF, and neuronal proteins in the mechanism of neuroprotection of CBD against MPP(+) toxicity in PC12 cells. CBD increased cell viability, differentiation, and the expression of axonal (GAP-43) and synaptic (synaptophysin and synapsin I) proteins. Its neuritogenic effect was not dependent or additive to NGF, but it was inhibited by K252a (trkA inhibitor). CBD did not increase the expression of NGF, but protected against its decrease induced by MPP(+), probably by an indirect mechanism. We also evaluated the neuritogenesis in SH-SY5Y cells, which do not express trkA receptors. CBD did not induce neuritogenesis in this cellular model, which supports the involvement of trkA receptors. This is the first study to report the involvement of neuronal proteins and trkA in the neuroprotection of CBD. Our findings suggest that CBD has a neurorestorative potential independent of NGF that might contribute to its neuroprotection against MPP(+), a neurotoxin relevant to Parkinson's disease. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Unraveling the molecular mechanism of interactions of the Rho GTPases Cdc42 and Rac1 with the scaffolding protein IQGAP2.

    Science.gov (United States)

    Ozdemir, E Sila; Jang, Hyunbum; Gursoy, Attila; Keskin, Ozlem; Li, Zhigang; Sacks, David B; Nussinov, Ruth

    2018-03-09

    IQ motif-containing GTPase-activating proteins (IQGAPs) are scaffolding proteins playing central roles in cell-cell adhesion, polarity, and motility. The Rho GTPases Cdc42 and Rac1, in their GTP-bound active forms, interact with all three human IQGAPs. The IQGAP-Cdc42 interaction promotes metastasis by enhancing actin polymerization. However, despite their high sequence identity, Cdc42 and Rac1 differ in their interactions with IQGAP. Two Cdc42 molecules can bind to the Ex-domain and the RasGAP site of the GTPase-activating protein (GAP)-related domain (GRD) of IQGAP and promote IQGAP dimerization. Only one Rac1 molecule might bind to the RasGAP site of GRD and may not facilitate the dimerization, and the exact mechanism of Cdc42 and Rac1 binding to IQGAP is unclear. Using all-atom molecular dynamics simulations, site-directed mutagenesis, and Western blotting, we unraveled the detailed mechanisms of Cdc42 and Rac1 interactions with IQGAP2. We observed that Cdc42 binding to the Ex-domain of GRD of IQGAP2 (GRD2) releases the Ex-domain at the C-terminal region of GRD2, facilitating IQGAP2 dimerization. Cdc42 binding to the Ex-domain promoted allosteric changes in the RasGAP site, providing a binding site for the second Cdc42 in the RasGAP site. Of note, the Cdc42 "insert loop" was important for the interaction of the first Cdc42 with the Ex-domain. By contrast, differences in Rac1 insert-loop sequence and structure precluded its interaction with the Ex-domain. Rac1 could bind only to the RasGAP site of apo-GRD2 and could not facilitate IQGAP2 dimerization. Our detailed mechanistic insights help decipher how Cdc42 can stimulate actin polymerization in metastasis.

  16. Ubiquitination-Related MdBT Scaffold Proteins Target a bHLH Transcription Factor for Iron Homeostasis1[OPEN

    Science.gov (United States)

    Zhao, Qiang; Wang, Qing-Jie; Wang, Xiao-Fei; You, Chun-Xiang

    2016-01-01

    Iron (Fe) homeostasis is crucial for plant growth and development. A network of basic helix-loop-helix (bHLH) transcription factors positively regulates Fe uptake during iron deficiency. However, their up-regulation or overexpression leads to Fe overload and reactive oxygen species generation, thereby damaging the plants. Here, we found that two BTB/TAZ proteins, MdBT1 and MdBT2, interact with the MbHLH104 protein in apple. In addition, the function of MdBT2 was characterized as a regulator of MdbHLH104 degradation via ubiquitination and the 26S proteasome pathway, thereby controlling the activity of plasma membrane H+-ATPases and the acquisition of iron. Furthermore, MdBT2 interacted with MdCUL3 proteins, which were required for the MdBT2-mediated ubiquitination modification of MdbHLH104 and its degradation. In sum, our findings demonstrate that MdBT proteins interact with MdCUL3 to bridge the formation of the MdBTsMdCUL3 complex, which negatively modulates the degradation of the MdbHLH104 protein in response to changes in Fe status to maintain iron homeostasis in plants. PMID:27660166

  17. Assembly of the novel five-component apicomplexan multi-aminoacyl-tRNA synthetase complex is driven by the hybrid scaffold protein Tg-p43.

    Directory of Open Access Journals (Sweden)

    Jason M van Rooyen

    Full Text Available In Toxoplasma gondii, as in other eukaryotes, a subset of the amino-acyl-tRNA synthetases are arranged into an abundant cytoplasmic multi-aminoacyl-tRNA synthetase (MARS complex. Through a series of genetic pull-down assays, we have identified the enzymes of this complex as: methionyl-, glutaminyl-, glutamyl-, and tyrosyl-tRNA synthetases, and we show that the N-terminal GST-like domain of a partially disordered hybrid scaffold protein, Tg-p43, is sufficient for assembly of the intact complex. Our gel filtration studies revealed significant heterogeneity in the size and composition of isolated MARS complexes. By targeting the tyrosyl-tRNA synthetases subunit, which was found exclusively in the complete 1 MDa complex, we were able to directly visualize MARS particles in the electron microscope. Image analyses of the negative stain data revealed the observed heterogeneity and instability of these complexes to be driven by the intrinsic flexibility of the domain arrangements within the MARS complex. These studies provide unique insights into the assembly of these ubiquitous but poorly understood eukaryotic complexes.

  18. Crystal Structure of Bacillus subtilis Cysteine Desulfurase SufS and Its Dynamic Interaction with Frataxin and Scaffold Protein SufU.

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    Bastian Blauenburg

    Full Text Available The biosynthesis of iron sulfur (Fe-S clusters in Bacillus subtilis is mediated by a SUF-type gene cluster, consisting of the cysteine desulfurase SufS, the scaffold protein SufU, and the putative chaperone complex SufB/SufC/SufD. Here, we present the high-resolution crystal structure of the SufS homodimer in its product-bound state (i.e., in complex with pyrodoxal-5'-phosphate, alanine, Cys361-persulfide. By performing hydrogen/deuterium exchange (H/DX experiments, we characterized the interaction of SufS with SufU and demonstrate that SufU induces an opening of the active site pocket of SufS. Recent data indicate that frataxin could be involved in Fe-S cluster biosynthesis by facilitating iron incorporation. H/DX experiments show that frataxin indeed interacts with the SufS/SufU complex at the active site. Our findings deepen the current understanding of Fe-S cluster biosynthesis, a complex yet essential process, in the model organism B. subtilis.

  19. Structure-Based Scaffold Repurposing for G Protein-Coupled Receptors: Transformation of Adenosine Derivatives into 5HT2B/5HT2CSerotonin Receptor Antagonists.

    Science.gov (United States)

    Tosh, Dilip K; Ciancetta, Antonella; Warnick, Eugene; Crane, Steven; Gao, Zhan-Guo; Jacobson, Kenneth A

    2016-12-22

    Adenosine derivatives developed to activate adenosine receptors (ARs) revealed micromolar activity at serotonin 5HT 2B and 5HT 2C receptors (5HTRs). We explored the structure-activity relationship at 5HT 2 Rs and modeled receptor interactions in order to optimize affinity and simultaneously reduce AR affinity. Depending on N 6 substitution, small 5'-alkylamide modification maintained 5HT 2B R affinity, which was enhanced upon ribose substitution with rigid bicyclo[3.1.0]hexane (North (N)-methanocarba), e.g., N 6 -dicyclopropylmethyl 4'-CH 2 OH derivative 14 (K i 11 nM). 5'-Methylamide 23 was 170-fold selective as antagonist for 5HT 2B R vs 5HT 2C R. 5'-Methyl 25 and ethyl 26 esters potently antagonized 5HT 2 Rs with moderate selectivity in comparison to ARs; related 6-N,N-dimethylamino analogue 30 was 5HT 2 R-selective. 5' position flexibility of substitution was indicated in 5HT 2B R docking. Both 5'-ester and 5'-amide derivatives displayed in vivo t 1/2 of 3-4 h. Thus, we used G protein-coupled receptor modeling to repurpose nucleoside scaffolds in favor of binding at nonpurine receptors as novel 5HT 2 R antagonists, with potential for cardioprotection, liver protection, or central nervous system activity.

  20. Crystal Structure of Bacillus subtilis Cysteine Desulfurase SufS and Its Dynamic Interaction with Frataxin and Scaffold Protein SufU.

    Science.gov (United States)

    Blauenburg, Bastian; Mielcarek, Andreas; Altegoer, Florian; Fage, Christopher D; Linne, Uwe; Bange, Gert; Marahiel, Mohamed A

    2016-01-01

    The biosynthesis of iron sulfur (Fe-S) clusters in Bacillus subtilis is mediated by a SUF-type gene cluster, consisting of the cysteine desulfurase SufS, the scaffold protein SufU, and the putative chaperone complex SufB/SufC/SufD. Here, we present the high-resolution crystal structure of the SufS homodimer in its product-bound state (i.e., in complex with pyrodoxal-5'-phosphate, alanine, Cys361-persulfide). By performing hydrogen/deuterium exchange (H/DX) experiments, we characterized the interaction of SufS with SufU and demonstrate that SufU induces an opening of the active site pocket of SufS. Recent data indicate that frataxin could be involved in Fe-S cluster biosynthesis by facilitating iron incorporation. H/DX experiments show that frataxin indeed interacts with the SufS/SufU complex at the active site. Our findings deepen the current understanding of Fe-S cluster biosynthesis, a complex yet essential process, in the model organism B. subtilis.

  1. Molecular Signatures Underlying Synaptic Vesicle Cargo Retrieval

    Science.gov (United States)

    Mori, Yasunori; Takamori, Shigeo

    2018-01-01

    Efficient retrieval of the synaptic vesicle (SV) membrane from the presynaptic plasma membrane, a process called endocytosis, is crucial for the fidelity of neurotransmission, particularly during sustained neural activity. Although multiple modes of endocytosis have been identified, it is clear that the efficient retrieval of the major SV cargos into newly formed SVs during any of these modes is fundamental for synaptic transmission. It is currently believed that SVs are eventually reformed via a clathrin-dependent pathway. Various adaptor proteins recognize SV cargos and link them to clathrin, ensuring the efficient retrieval of the cargos into newly formed SVs. Here, we summarize our current knowledge of the molecular signatures within individual SV cargos that underlie efficient retrieval into SV membranes, as well as discuss possible contributions of the mechanisms under physiological conditions. PMID:29379416

  2. Novel binders derived from an albumin-binding domain scaffold targeting human prostate secretory protein 94 (PSP94)

    Czech Academy of Sciences Publication Activity Database

    Marečková, Lucie; Petroková, Hana; Osička, Radim; Kuchař, Milan; Malý, Petr

    2015-01-01

    Roč. 6, č. 10 (2015), s. 774-779 ISSN 1674-800X Institutional support: RVO:86652036 ; RVO:61388971 Keywords : prostate secretory protein * prostate cancer * oncomarker Subject RIV: EB - Genetics ; Molecular Biology; EB - Genetics ; Molecular Biology (MBU-M) Impact factor: 3.817, year: 2015

  3. Synaptic electronics: materials, devices and applications.

    Science.gov (United States)

    Kuzum, Duygu; Yu, Shimeng; Wong, H-S Philip

    2013-09-27

    In this paper, the recent progress of synaptic electronics is reviewed. The basics of biological synaptic plasticity and learning are described. The material properties and electrical switching characteristics of a variety of synaptic devices are discussed, with a focus on the use of synaptic devices for neuromorphic or brain-inspired computing. Performance metrics desirable for large-scale implementations of synaptic devices are illustrated. A review of recent work on targeted computing applications with synaptic devices is presented.

  4. Radix Puerariae modulates glutamatergic synaptic architecture and potentiates functional synaptic plasticity in primary hippocampal neurons.

    Science.gov (United States)

    Bhuiyan, Mohammad Maqueshudul Haque; Haque, Md Nazmul; Mohibbullah, Md; Kim, Yung Kyu; Moon, Il Soo

    2017-09-14

    Neurologic disorders are frequently characterized by synaptic pathology, including abnormal density and morphology of dendritic spines, synapse loss, and aberrant synaptic signaling and plasticity. Therefore, to promote and/or protect synapses by the use of natural molecules capable of modulating neurodevelopmental events, such as, spinogenesis and synaptic plasticity, could offer a preventive and curative strategy for nervous disorders associated with synaptic pathology. Radix Puerariae, the root of Pueraria monatana var. lobata (Willd.) Sanjappa&Pradeep, is a Chinese ethnomedicine, traditionally used for the treatment of memory-related nervous disorders including Alzheimer's disease. In the previous study, we showed that the ethanolic extracts of Radix Puerariae (RPE) and its prime constituent, puerarin induced neuritogenesis and synapse formation in cultured hippocampal neurons, and thus could improve memory functions. In the present study, we specifically investigated the abilities of RPE and puerarin to improve memory-related brain disorders through modulating synaptic maturation and functional potentiation. Rat embryonic (E19) brain neurons were cultured in the absence or presence of RPE or puerarin. At predetermined times, cells were live-stained with DiO or fixed and immunostained to visualize neuronal morphologies, or lysed for protein harvesting. Morphometric analyses of dendritic spines and synaptogenesis were performed using Image J software. Functional pre- and postsynaptic plasticity was measured by FM1-43 staining and whole-cell patch clamping, respectively. RPE or puerarin-mediated changes in actin-related protein 2 were assessed by Western blotting. Neuronal survivals were measured using propidium iodide exclusion assay. RPE and puerarin both: (1) promoted a significant increase in the numbers, and maturation, of dendritic spines; (2) modulated the formation of glutamatergic synapses; (3) potentiated synaptic transmission by increasing the sizes of

  5. Cdk5 is essential for synaptic vesicle endocytosis

    DEFF Research Database (Denmark)

    Tan, Timothy C; Valova, Valentina A; Malladi, Chandra S

    2003-01-01

    Synaptic vesicle endocytosis (SVE) is triggered by calcineurin-mediated dephosphorylation of the dephosphin proteins. SVE is maintained by the subsequent rephosphorylation of the dephosphins by unidentified protein kinases. Here, we show that cyclin-dependent kinase 5 (Cdk5) phosphorylates dynamin...

  6. A dimer of the Toll-like receptor 4 cytoplasmic domain provides a specific scaffold for the recruitment of signalling adaptor proteins.

    Directory of Open Access Journals (Sweden)

    Ricardo Núñez Miguel

    2007-08-01

    Full Text Available The Toll-like receptor 4 (TLR4 is a class I transmembrane receptor expressed on the surface of immune system cells. TLR4 is activated by exposure to lipopolysaccharides derived from the outer membrane of Gram negative bacteria and forms part of the innate immune response in mammals. Like other class 1 receptors, TLR4 is activated by ligand induced dimerization, and recent studies suggest that this causes concerted conformational changes in the receptor leading to self association of the cytoplasmic Toll/Interleukin 1 receptor (TIR signalling domain. This homodimerization event is proposed to provide a new scaffold that is able to bind downstream signalling adaptor proteins. TLR4 uses two different sets of adaptors; TRAM and TRIF, and Mal and MyD88. These adaptor pairs couple two distinct signalling pathways leading to the activation of interferon response factor 3 (IRF-3 and nuclear factor kappaB (NFkappaB respectively. In this paper we have generated a structural model of the TLR4 TIR dimer and used molecular docking to probe for potential sites of interaction between the receptor homodimer and the adaptor molecules. Remarkably, both the Mal and TRAM adaptors are strongly predicted to bind at two symmetry-related sites at the homodimer interface. This model of TLR4 activation is supported by extensive functional studies involving site directed mutagenesis, inhibition by cell permeable peptides and stable protein phosphorylation of receptor and adaptor TIR domains. Our results also suggest a molecular mechanism for two recent findings, the caspase 1 dependence of Mal signalling and the protective effects conferred by the Mal polymorphism Ser180Leu.

  7. Analysis of in vitro bioactivity data extracted from drug discovery literature and patents: Ranking 1654 human protein targets by assayed compounds and molecular scaffolds

    Directory of Open Access Journals (Sweden)

    Southan Christopher

    2011-05-01

    Full Text Available Abstract Background Since the classic Hopkins and Groom druggable genome review in 2002, there have been a number of publications updating both the hypothetical and successful human drug target statistics. However, listings of research targets that define the area between these two extremes are sparse because of the challenges of collating published information at the necessary scale. We have addressed this by interrogating databases, populated by expert curation, of bioactivity data extracted from patents and journal papers over the last 30 years. Results From a subset of just over 27,000 documents we have extracted a set of compound-to-target relationships for biochemical in vitro binding-type assay data for 1,736 human proteins and 1,654 gene identifiers. These are linked to 1,671,951 compound records derived from 823,179 unique chemical structures. The distribution showed a compounds-per-target average of 964 with a maximum of 42,869 (Factor Xa. The list includes non-targets, failed targets and cross-screening targets. The top-278 most actively pursued targets cover 90% of the compounds. We further investigated target ranking by determining the number of molecular frameworks and scaffolds. These were compared to the compound counts as alternative measures of chemical diversity on a per-target basis. Conclusions The compounds-per-protein listing generated in this work (provided as a supplementary file represents the major proportion of the human drug target landscape defined by published data. We supplemented the simple ranking by the number of compounds assayed with additional rankings by molecular topology. These showed significant differences and provide complementary assessments of chemical tractability.

  8. Analysis of in vitro bioactivity data extracted from drug discovery literature and patents: Ranking 1654 human protein targets by assayed compounds and molecular scaffolds.

    Science.gov (United States)

    Southan, Christopher; Boppana, Kiran; Jagarlapudi, Sarma Arp; Muresan, Sorel

    2011-05-13

    Since the classic Hopkins and Groom druggable genome review in 2002, there have been a number of publications updating both the hypothetical and successful human drug target statistics. However, listings of research targets that define the area between these two extremes are sparse because of the challenges of collating published information at the necessary scale. We have addressed this by interrogating databases, populated by expert curation, of bioactivity data extracted from patents and journal papers over the last 30 years. From a subset of just over 27,000 documents we have extracted a set of compound-to-target relationships for biochemical in vitro binding-type assay data for 1,736 human proteins and 1,654 gene identifiers. These are linked to 1,671,951 compound records derived from 823,179 unique chemical structures. The distribution showed a compounds-per-target average of 964 with a maximum of 42,869 (Factor Xa). The list includes non-targets, failed targets and cross-screening targets. The top-278 most actively pursued targets cover 90% of the compounds. We further investigated target ranking by determining the number of molecular frameworks and scaffolds. These were compared to the compound counts as alternative measures of chemical diversity on a per-target basis. The compounds-per-protein listing generated in this work (provided as a supplementary file) represents the major proportion of the human drug target landscape defined by published data. We supplemented the simple ranking by the number of compounds assayed with additional rankings by molecular topology. These showed significant differences and provide complementary assessments of chemical tractability.

  9. Frataxin directly stimulates mitochondrial cysteine desulfurase by exposing substrate-binding sites, and a mutant Fe-S cluster scaffold protein with frataxin-bypassing ability acts similarly.

    Science.gov (United States)

    Pandey, Alok; Gordon, Donna M; Pain, Jayashree; Stemmler, Timothy L; Dancis, Andrew; Pain, Debkumar

    2013-12-27

    For iron-sulfur (Fe-S) cluster synthesis in mitochondria, the sulfur is derived from the amino acid cysteine by the cysteine desulfurase activity of Nfs1. The enzyme binds the substrate cysteine in the pyridoxal phosphate-containing site, and a persulfide is formed on the active site cysteine in a manner depending on the accessory protein Isd11. The persulfide is then transferred to the scaffold Isu, where it combines with iron to form the Fe-S cluster intermediate. Frataxin is implicated in the process, although it is unclear where and how, and deficiency causes Friedreich ataxia. Using purified proteins and isolated mitochondria, we show here that the yeast frataxin homolog (Yfh1) directly and specifically stimulates cysteine binding to Nfs1 by exposing substrate-binding sites. This novel function of frataxin does not require iron, Isu1, or Isd11. Once bound to Nfs1, the substrate cysteine is the source of the Nfs1 persulfide, but this step is independent of frataxin and strictly dependent on Isd11. Recently, a point mutation in Isu1 was found to bypass many frataxin functions. The data presented here show that the Isu1 suppressor mimics the frataxin effects on Nfs1, explaining the bypassing activity. We propose a regulatory mechanism for the Nfs1 persulfide-forming activity. Specifically, at least two separate conformational changes must occur in the enzyme for optimum activity as follows: one is mediated by frataxin interaction that exposes the "buried" substrate-binding sites, and the other is mediated by Isd11 interaction that brings the bound substrate cysteine and the active site cysteine in proximity for persulfide formation.

  10. Pembuatan Scaffold Trikalsium Fosfat Berpori Menggunakan Metode Protein Foaming-Consolidation dengan Variasi Waktu dan Temperatur Pengeringan

    OpenAIRE

    Wirata, Gede Indra Ludy; Fadli, Ahmad '; Akbar, Fajril '

    2016-01-01

    Tricalcium phosphate (TCP) is a ceramics material which is widely used for bone subtitude. Protein foaming-consolidation method is a technique for production of porous tricalciumphosphate (TCP) using egg yolk as pore creating agent. The purpose of this research was to investigate the effect of time and drying temperature effect on physical, chemical and mechanical properties of porous TCP and characterizing porous TCP as bones implant. This research start by mixing TCP powder, sago powder, Da...

  11. PRRT2: from Paroxysmal Disorders to Regulation of Synaptic Function.

    Science.gov (United States)

    Valtorta, Flavia; Benfenati, Fabio; Zara, Federico; Meldolesi, Jacopo

    2016-10-01

    In the past few years, proline-rich transmembrane protein (PRRT)2 has been identified as the causative gene for several paroxysmal neurological disorders. Recently, an important role of PRRT2 in synapse development and function has emerged. Knock down of the protein strongly impairs the formation of synaptic contacts and neurotransmitter release. At the nerve terminal, PRRT2 endows synaptic vesicle exocytosis with Ca 2+ sensitivity by interacting with proteins of the fusion complex and with the Ca 2+ sensors synaptotagmins (Syts). In the postsynaptic compartment, PRRT2 interacts with glutamate receptors. The study of PRRT2 and of its mutations may help in refining our knowledge of the process of synaptic transmission and elucidating the pathogenetic mechanisms leading to derangement of network function in paroxysmal disorders. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Fragile X Proteins FMRP and FXR2P Control Synaptic GluA1 Expression and Neuronal Maturation via Distinct Mechanisms

    Directory of Open Access Journals (Sweden)

    Weixiang Guo

    2015-06-01

    Full Text Available Fragile X mental retardation protein (FMRP and its autosomal paralog FXR2P are selective neuronal RNA-binding proteins, and mice that lack either protein exhibit cognitive deficits. Although double-mutant mice display more severe learning deficits than single mutants, the molecular mechanism behind this remains unknown. In the present study, we discovered that FXR2P (also known as FXR2 is important for neuronal dendritic development. FMRP and FXR2P additively promote the maturation of new neurons by regulating a common target, the AMPA receptor GluA1, but they do so via distinct mechanisms: FXR2P binds and stabilizes GluA1 mRNA and enhances subsequent protein expression, whereas FMRP promotes GluA1 membrane delivery. Our findings unveil important roles for FXR2P and GluA1 in neuronal development, uncover a regulatory mechanism of GluA1, and reveal a functional convergence between fragile X proteins in neuronal development.

  13. Role of mental retardation-associated dystrophin-gene product Dp71 in excitatory synapse organization, synaptic plasticity and behavioral functions.

    Directory of Open Access Journals (Sweden)

    Fatma Daoud

    Full Text Available BACKGROUND: Duchenne muscular dystrophy (DMD is caused by deficient expression of the cytoskeletal protein, dystrophin. One third of DMD patients also have mental retardation (MR, likely due to mutations preventing expression of dystrophin and other brain products of the DMD gene expressed from distinct internal promoters. Loss of Dp71, the major DMD-gene product in brain, is thought to contribute to the severity of MR; however, the specific function of Dp71 is poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: Complementary approaches were used to explore the role of Dp71 in neuronal function and identify mechanisms by which Dp71 loss may impair neuronal and cognitive functions. Besides the normal expression of Dp71 in a subpopulation of astrocytes, we found that a pool of Dp71 colocalizes with synaptic proteins in cultured neurons and is expressed in synaptic subcellular fractions in adult brains. We report that Dp71-associated protein complexes interact with specialized modular scaffolds of proteins that cluster glutamate receptors and organize signaling in postsynaptic densities. We then undertook the first functional examination of the brain and cognitive alterations in the Dp71-null mice. We found that these mice display abnormal synapse organization and maturation in vitro, altered synapse density in the adult brain, enhanced glutamatergic transmission and reduced synaptic plasticity in CA1 hippocampus. Dp71-null mice show selective behavioral disturbances characterized by reduced exploratory and novelty-seeking behavior, mild retention deficits in inhibitory avoidance, and impairments in spatial learning and memory. CONCLUSIONS/SIGNIFICANCE: Results suggest that Dp71 expression in neurons play a regulatory role in glutamatergic synapse organization and function, which provides a new mechanism by which inactivation of Dp71 in association with that of other DMD-gene products may lead to increased severity of MR.

  14. Chitin and carbon nanotube composites as biocompatible scaffolds for neuron growth

    Science.gov (United States)

    Singh, Nandita; Chen, Jinhu; Koziol, Krzysztof K.; Hallam, Keith R.; Janas, Dawid; Patil, Avinash J.; Strachan, Ally; G. Hanley, Jonathan; Rahatekar, Sameer S.

    2016-04-01

    The design of biocompatible implants for neuron repair/regeneration ideally requires high cell adhesion as well as good electrical conductivity. Here, we have shown that plasma-treated chitin carbon nanotube composite scaffolds show very good neuron adhesion as well as support of synaptic function of neurons. The addition of carbon nanotubes to a chitin biopolymer improved the electrical conductivity and the assisted oxygen plasma treatment introduced more oxygen species onto the chitin nanotube scaffold surface. Neuron viability experiments showed excellent neuron attachment onto plasma-treated chitin nanotube composite scaffolds. The support of synaptic function was evident on chitin/nanotube composites, as confirmed by PSD-95 staining. The biocompatible and electrically-conducting chitin nanotube composite scaffold prepared in this study can be used for in vitro tissue engineering of neurons and, potentially, as an implantable electrode for stimulation and repair of neurons.

  15. Circadian Regulation of Synaptic Plasticity

    Directory of Open Access Journals (Sweden)

    Marcos G. Frank

    2016-07-01

    Full Text Available Circadian rhythms refer to oscillations in biological processes with a period of approximately 24 h. In addition to the sleep/wake cycle, there are circadian rhythms in metabolism, body temperature, hormone output, organ function and gene expression. There is also evidence of circadian rhythms in synaptic plasticity, in some cases driven by a master central clock and in other cases by peripheral clocks. In this article, I review the evidence for circadian influences on synaptic plasticity. I also discuss ways to disentangle the effects of brain state and rhythms on synaptic plasticity.

  16. Co-Application of Corticosterone and Growth Hormone Upregulates NR2B Protein and Increases the NR2B:NR2A Ratio and Synaptic Transmission in the Hippocampus

    Directory of Open Access Journals (Sweden)

    Ghada S. Mahmoud

    2014-10-01

    Full Text Available Objectives: This in vitro study aimed to investigate the possible mechanism underlying the protective effect of growth hormone (GH on hippocampal function during periods of heightened glucocorticoid exposure. Methods: This study was conducted between January and June 2005 at the Joan C. Edwards School of Medicine, Marshall University, in Huntington, West Virginia, USA. The effects of the co-application of GH and corticosterone (CORT were tested at different concentrations on the field excitatory postsynaptic potentials (fEPSPs of the hippocampal slices of rats in two different age groups. Changes in the protein expression of N-methyl-D-aspartate receptor (NMDAR subunits NR1, NR2B and NR2A were measured in hippocampal brain slices treated with either artificial cerebrospinal fluid (ACSF, low doses of CORT alone or both CORT and GH for three hours. Results: The co-application of CORT and GH was found to have an additive effect on hippocampal synaptic transmission compared to either drug alone. Furthermore, the combined use of low concentrations of GH and CORT was found to have significantly higher effects on the enhancement of fEPSPs in older rats compared to young ones. Both GH and CORT enhanced the protein expression of the NR2A subunit. Simultaneous exposure to low concentrations of GH and CORT significantly enhanced NR2B expression and increased the NR2B:NR2A ratio. In contrast, perfusion with CORT alone caused significant suppression in the NR1 and NR2B protein expression and a decrease in the NR2B:NR2A ratio. Conclusion: These results suggest that NMDARs provide a potential target for mediating the GH potential protective effect against stress and age-related memory and cognitive impairment.

  17. Transgenic Mice Expressing a Truncated Form of CREB-Binding Protein (CBP) Exhibit Deficits in Hippocampal Synaptic Plasticity and Memory Storage

    Science.gov (United States)

    Wood, Marcelo A.; Kaplan, Michael P.; Park, Alice; Blanchard, Edward J.; Oliveira, Ana M. M.; Lombardi, Thomas L.; Abel, Ted

    2005-01-01

    Deletions, translocations, or point mutations in the CREB-binding protein (CBP) gene have been associated with Rubinstein-Taybi Syndrome; a human developmental disorder characterized by retarded growth and reduced mental function. To examine the role of CBP in memory, transgenic mice were generated in which the CaMKII[alpha] promoter drives…

  18. Flow-induced endothelial cell alignment requires the RhoGEF Trio as a scaffold protein to polarize active Rac1 distribution.

    Science.gov (United States)

    Kroon, Jeffrey; Heemskerk, Niels; Kalsbeek, Martin J T; de Waard, Vivian; van Rijssel, Jos; van Buul, Jaap D

    2017-07-01

    Endothelial cells line the lumen of the vessel wall and are exposed to flow. In linear parts of the vessel, the endothelial cells experience laminar flow, resulting in endothelial cell alignment in the direction of flow, thereby protecting the vessel wall from inflammation and permeability. In order for endothelial cells to align, they undergo rapid remodeling of the actin cytoskeleton by local activation of the small GTPase Rac1. However, it is not clear whether sustained and local activation of Rac1 is required for long-term flow-induced cell alignment. Using a FRET-based DORA Rac1 biosensor, we show that local Rac1 activity remains for 12 h upon long-term flow. Silencing studies show that the RhoGEF Trio is crucial for keeping active Rac1 at the downstream side of the cell and, as a result, for long-term flow-induced cell alignment. Surprisingly, Trio appears to be not involved in flow-induced activation of Rac1. Our data show that flow induces Rac1 activity at the downstream side of the cell in a Trio-dependent manner and that Trio functions as a scaffold protein rather than a functional GEF under long-term flow conditions. © 2017 Kroon et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  19. Engineering a Biocompatible Scaffold with Either Micrometre or Nanometre Scale Surface Topography for Promoting Protein Adsorption and Cellular Response

    Directory of Open Access Journals (Sweden)

    Xuan Le

    2013-01-01

    Full Text Available Surface topographical features on biomaterials, both at the submicrometre and nanometre scales, are known to influence the physicochemical interactions between biological processes involving proteins and cells. The nanometre-structured surface features tend to resemble the extracellular matrix, the natural environment in which cells live, communicate, and work together. It is believed that by engineering a well-defined nanometre scale surface topography, it should be possible to induce appropriate surface signals that can be used to manipulate cell function in a similar manner to the extracellular matrix. Therefore, there is a need to investigate, understand, and ultimately have the ability to produce tailor-made nanometre scale surface topographies with suitable surface chemistry to promote favourable biological interactions similar to those of the extracellular matrix. Recent advances in nanoscience and nanotechnology have produced many new nanomaterials and numerous manufacturing techniques that have the potential to significantly improve several fields such as biological sensing, cell culture technology, surgical implants, and medical devices. For these fields to progress, there is a definite need to develop a detailed understanding of the interaction between biological systems and fabricated surface structures at both the micrometre and nanometre scales.

  20. Modification of Triclosan Scaffold in Search of Improved Inhibitors for Enoyl-Acyl Carrier Protein (ACP) Reductase in Toxoplasma gondii

    Science.gov (United States)

    Stec, Jozef; Fomovska, Alina; Afanador, Gustavo A.; Muench, Stephen P.; Zhou, Ying; Lai, Bo-Shiun; Bissati, Kamal El; Hickman, Mark R.; Lee, Patty J.; Leed, Susan E.; Auschwitz, Jennifer M.; Sommervile, Caroline; Woods, Stuart; Roberts, Craig W.; Rice, David; Prigge, Sean T.; McLeod, Rima; Kozikowski, Alan P.

    2013-01-01

    Through our focused effort to discover new and effective agents against toxoplasmosis, a structure-based drug design approach was utilized to develop a series of potent inhibitors of the enoyl-acyl carrier protein (ACP) reductase (ENR) enzyme in Toxoplasma gondii (TgENR). Modifications to positions 5 and 4′ of the well-known ENR inhibitor triclosan afforded a series of 29 new analogs. Among the resulting compounds, many showed high potency and improved physicochemical properties in comparison with the lead. The most potent compounds 16a and 16c have IC50 values of 250 nM against Toxoplasma gondii tachyzoites without apparent toxicity to the host cells. Their IC50 values against the recombinant TgENR were 43 and 26 nM, respectively. Additionally, 11 other analogs in this series had IC50 values ranging from 17 to 130 nM in the enzyme-based assay. With respect to their excellent in vitro activity as well as improved drug-like properties, the lead compounds 16a and 16c are deemed to be an excellent starting point for the development of new medicines to effectively treat Toxoplasma gondii infections. PMID:23776166

  1. Catalytic autoantibodies against myelin basic protein (MBP) isolated from serum of autistic children impair in vitro models of synaptic plasticity in rat hippocampus.

    Science.gov (United States)

    Gonzalez-Gronow, Mario; Cuchacovich, Miguel; Francos, Rina; Cuchacovich, Stephanie; Blanco, Angel; Sandoval, Rodrigo; Gomez, Cristian Farias; Valenzuela, Javier A; Ray, Rupa; Pizzo, Salvatore V

    2015-10-15

    Autoantibodies from autistic spectrum disorder (ASD) patients react with multiple proteins expressed in the brain. One such autoantibody targets myelin basic protein (MBP). ASD patients have autoantibodies to MBP of both the IgG and IgA classes in high titers, but no autoantibodies of the IgM class. IgA autoantibodies act as serine proteinases and degrade MBP in vitro. They also induce a decrease in long-term potentiation in the hippocampi of rats either perfused with or previously inoculated with this IgA. Because this class of autoantibody causes myelin sheath destruction in multiple sclerosis (MS), we hypothesized a similar pathological role for them in ASD. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Fas-associated factor 1 is a scaffold protein that promotes β-transducin repeat-containing protein (β-TrCP)-mediated β-catenin ubiquitination and degradation.

    Science.gov (United States)

    Zhang, Long; Zhou, Fangfang; Li, Yihao; Drabsch, Yvette; Zhang, Juan; van Dam, Hans; ten Dijke, Peter

    2012-08-31

    FAS-associated factor 1 (FAF1) antagonizes Wnt signaling by stimulating β-catenin degradation. However, the molecular mechanism underlying this effect is unknown. Here, we demonstrate that the E3 ubiquitin ligase β-transducin repeat-containing protein (β-TrCP) is required for FAF1 to suppress Wnt signaling and that FAF1 specifically associates with the SCF (Skp1-Cul1-F-box protein)-β-TrCP complex. Depletion of β-TrCP reduced FAF1-mediated β-catenin polyubiquitination and impaired FAF1 in antagonizing Wnt/β-catenin signaling. FAF1 was shown to act as a scaffold for β-catenin and β-TrCP and thereby to potentiate β-TrCP-mediated β-catenin ubiquitination and degradation. Data mining revealed that FAF1 expression is statistically down-regulated in human breast carcinoma compared with normal breast tissue. Consistent with this, FAF1 expression is higher in epithelial-like MCF7 than mesenchymal-like MDA-MB-231 human breast cancer cells. Depletion of FAF1 in MCF7 cells resulted in increased β-catenin accumulation and signaling. Importantly, FAF1 knockdown promoted a decrease in epithelial E-cadherin and an increase in mesenchymal vimentin expression, indicative for an epithelial to mesenchymal transition. Moreover, ectopic FAF1 expression reduces breast cancer cell migration in vitro and invasion/metastasis in vivo. Thus, our studies strengthen a tumor-suppressive function for FAF1.

  3. Endocannabinoid signaling and synaptic function

    Science.gov (United States)

    Castillo, Pablo E.; Younts, Thomas J.; Chávez, Andrés E.; Hashimotodani, Yuki

    2012-01-01

    Endocannabinoids are key modulators of synaptic function. By activating cannabinoid receptors expressed in the central nervous system, these lipid messengers can regulate several neural functions and behaviors. As experimental tools advance, the repertoire of known endocannabinoid-mediated effects at the synapse, and their underlying mechanism, continues to expand. Retrograde signaling is the principal mode by which endocannabinoids mediate short- and long-term forms of plasticity at both excitatory and inhibitory synapses. However, growing evidence suggests that endocannabinoids can also signal in a non-retrograde manner. In addition to mediating synaptic plasticity, the endocannabinoid system is itself subject to plastic changes. Multiple points of interaction with other neuromodulatory and signaling systems have now been identified. Synaptic endocannabinoid signaling is thus mechanistically more complex and diverse than originally thought. In this review, we focus on new advances in endocannabinoid signaling and highlight their role as potent regulators of synaptic function in the mammalian brain. PMID:23040807

  4. Distinct Subunit Domains Govern Synaptic Stability and Specificity of the Kainate Receptor

    Directory of Open Access Journals (Sweden)

    Christoph Straub

    2016-07-01

    Full Text Available Synaptic communication between neurons requires the precise localization of neurotransmitter receptors to the correct synapse type. Kainate-type glutamate receptors restrict synaptic localization that is determined by the afferent presynaptic connection. The mechanisms that govern this input-specific synaptic localization remain unclear. Here, we examine how subunit composition and specific subunit domains contribute to synaptic localization of kainate receptors. The cytoplasmic domain of the GluK2 low-affinity subunit stabilizes kainate receptors at synapses. In contrast, the extracellular domain of the GluK4/5 high-affinity subunit synergistically controls the synaptic specificity of kainate receptors through interaction with C1q-like proteins. Thus, the input-specific synaptic localization of the native kainate receptor complex involves two mechanisms that underlie specificity and stabilization of the receptor at synapses.

  5. Histone Deacetylase Inhibition Facilitates Massed Pattern-Induced Synaptic Plasticity and Memory

    Science.gov (United States)

    Pandey, Kiran; Sharma, Kaushik P.; Sharma, Shiv K.

    2015-01-01

    Massed training is less effective for long-term memory formation than the spaced training. The role of acetylation in synaptic plasticity and memory is now well established. However, the role of this important protein modification in synaptic plasticity induced by massed pattern of stimulation or memory induced by massed training is not well…

  6. Partially nanofibrous architecture of 3D tissue engineering scaffolds.

    Science.gov (United States)

    Wei, Guobao; Ma, Peter X

    2009-11-01

    An ideal tissue-engineering scaffold should provide suitable pores and appropriate pore surface to induce desired cellular activities and to guide 3D tissue regeneration. In the present work, we have developed macroporous polymer scaffolds with varying pore wall architectures from smooth (solid), microporous, partially nanofibrous, to entirely nanofibrous ones. All scaffolds are designed to have well-controlled interconnected macropores, resulting from leaching sugar sphere template. We examine the effects of material composition, solvent, and phase separation temperature on the pore surface architecture of 3D scaffolds. In particular, phase separation of PLLA/PDLLA or PLLA/PLGA blends leads to partially nanofibrous scaffolds, in which PLLA forms nanofibers and PDLLA or PLGA forms the smooth (solid) surfaces on macropore walls, respectively. Specific surface areas are measured for scaffolds with similar macroporosity but different macropore wall architectures. It is found that the pore wall architecture predominates the total surface area of the scaffolds. The surface area of a partially nanofibrous scaffold increases linearly with the PLLA content in the polymer blend. The amounts of adsorbed proteins from serum increase with the surface area of the scaffolds. These macroporous scaffolds with adjustable pore wall surface architectures may provide a platform for investigating the cellular responses to pore surface architecture, and provide us with a powerful tool to develop superior scaffolds for various tissue-engineering applications.

  7. Synaptic mRNAs are modulated by learning.

    Science.gov (United States)

    Ferrara, Eugenia; Cefaliello, Carolina; Eyman, Maria; De Stefano, Rosanna; Giuditta, Antonio; Crispino, Marianna

    2009-07-01

    We have recently demonstrated that brain plastic events significantly modify synaptic protein synthesis measured by the incorporation of [(35)S]methionine in brain synaptosomal proteins. Notably, in rats learning a two-way active avoidance task, the local synthesis of two synaptic proteins was selectively enhanced. Because this effect may be attributed to transcriptional modulation, we used reverse transcriptase-polymerase chain reaction methods to determine the content of discrete synaptosomal mRNAs in rats exposed to the same training protocol. Correlative analyses between behavioral responses and synaptosomal mRNA content showed that GAT-1 mRNA (a prevalent presynaptic component) correlates with avoidances and escapes in rat cerebellum, while glial fibrillary acid protein mRNA (an astrocytic component) correlates with freezings in cerebellum and cerebral cortex. These observations support the hypothesis that synaptic protein synthesis may be transcriptionally regulated. The cellular origin of synaptic transcripts is briefly discussed, with special regard to those present at large distances from neuron somas. (c) 2009 Wiley-Liss, Inc.

  8. Mandibular defect reconstruction using three-dimensional polycaprolactone scaffold in combination with platelet-rich plasma and recombinant human bone morphogenetic protein-2: de novo synthesis of bone in a single case.

    Science.gov (United States)

    Schuckert, Karl-Heinz; Jopp, Stefan; Teoh, Swee-Hin

    2009-03-01

    This publication describes the clinical case of a 71-year-old female patient. Using polycaprolactone (PCL) scaffold, platelet-rich plasma (PRP) and recombinant human bone morphogenetic protein-2 (rhBMP-2), a critical-sized defect in the anterior mandible was regenerated using de novo-grown bone. A bacterial infection had caused a periimplantitis in two dental implants leading to a large destruction in the anterior mandible. Both implants were removed under antibiotic prophylaxis. A PCL scaffold was prepared especially for this clinical case. In a second procedure with antibiotic prophylaxis, the bony defect was reopened. The PCL scaffold was fitted and charged with PRP and rhBMP-2 (1.2 mg). After complication-free wound healing, the radiological control demonstrated de novo-grown bone in the anterior mandible 6 months postoperatively. Dental implants were inserted in a third operation. A bone biopsy of the newly grown bone, as well as of the bordering local bone, was taken and histologically examined. The bone samples were identical and presented vital laminar bone.

  9. Identifying the binding mode of a molecular scaffold

    Science.gov (United States)

    Chema, Doron; Eren, Doron; Yayon, Avner; Goldblum, Amiram; Zaliani, Andrea

    2004-01-01

    We describe a method for docking of a scaffold-based series and present its advantages over docking of individual ligands, for determining the binding mode of a molecular scaffold in a binding site. The method has been applied to eight different scaffolds of protein kinase inhibitors (PKI). A single analog of each of these eight scaffolds was previously crystallized with different protein kinases. We have used FlexX to dock a set of molecules that share the same scaffold, rather than docking a single molecule. The main mode of binding is determined by the mode of binding of the largest cluster among the docked molecules that share a scaffold. Clustering is based on our `nearest single neighbor' method [J. Chem. Inf. Comput. Sci., 43 (2003) 208-217]. Additional criteria are applied in those cases in which more than one significant binding mode is found. Using the proposed method, most of the crystallographic binding modes of these scaffolds were reconstructed. Alternative modes, that have not been detected yet by experiments, could also be identified. The method was applied to predict the binding mode of an additional molecular scaffold that was not yet reported and the predicted binding mode has been found to be very similar to experimental results for a closely related scaffold. We suggest that this approach be used as a virtual screening tool for scaffold-based design processes.

  10. Proteome rearrangements after auditory learning: high-resolution profiling of synapse-enriched protein fractions from mouse brain.

    Science.gov (United States)

    Kähne, Thilo; Richter, Sandra; Kolodziej, Angela; Smalla, Karl-Heinz; Pielot, Rainer; Engler, Alexander; Ohl, Frank W; Dieterich, Daniela C; Seidenbecher, Constanze; Tischmeyer, Wolfgang; Naumann, Michael; Gundelfinger, Eckart D

    2016-07-01

    Learning and memory processes are accompanied by rearrangements of synaptic protein networks. While various studies have demonstrated the regulation of individual synaptic proteins during these processes, much less is known about the complex regulation of synaptic proteomes. Recently, we reported that auditory discrimination learning in mice is associated with a relative down-regulation of proteins involved in the structural organization of synapses in various brain regions. Aiming at the identification of biological processes and signaling pathways involved in auditory memory formation, here, a label-free quantification approach was utilized to identify regulated synaptic junctional proteins and phosphoproteins in the auditory cortex, frontal cortex, hippocampus, and striatum of mice 24 h after the learning experiment. Twenty proteins, including postsynaptic scaffolds, actin-remodeling proteins, and RNA-binding proteins, were regulated in at least three brain regions pointing to common, cross-regional mechanisms. Most of the detected synaptic proteome changes were, however, restricted to individual brain regions. For example, several members of the Septin family of cytoskeletal proteins were up-regulated only in the hippocampus, while Septin-9 was down-regulated in the hippocampus, the frontal cortex, and the striatum. Meta analyses utilizing several databases were employed to identify underlying cellular functions and biological pathways. Data are available via ProteomeExchange with identifier PXD003089. How does the protein composition of synapses change in different brain areas upon auditory learning? We unravel discrete proteome changes in mouse auditory cortex, frontal cortex, hippocampus, and striatum functionally implicated in the learning process. We identify not only common but also area-specific biological pathways and cellular processes modulated 24 h after training, indicating individual contributions of the regions to memory processing. © 2016 The

  11. Frequency of Synaptic Autoantibody Accompaniments and Neurological Manifestations of Thymoma.

    Science.gov (United States)

    Zekeridou, Anastasia; McKeon, Andrew; Lennon, Vanda A

    2016-07-01

    Thymoma is commonly recognized in association with paraneoplastic autoimmune myasthenia gravis (MG), an IgG-mediated impairment of synaptic transmission targeting the nicotinic acetylcholine receptor of muscle. Newly identified synaptic autoantibodies may expand the serological profile of thymoma. To investigate the frequency of potentially pathogenic neural synaptic autoantibodies in patients with thymoma. We retrospectively identified patients with histopathologically confirmed thymoma and serum available to test for synaptic autoantibodies (collected 1986-2014) at the Mayo Clinic Neuroimmunology Laboratory. We identified and classified 193 patients with thymoma into 4 groups: (1) lacking neurological autoimmunity (n = 43); (2) isolated MG (n = 98); (3) MG plus additional autoimmune neurological manifestations (n = 26); and (4) neurological autoimmunity other than MG (n = 26). Clinical presentation and serum profile of autoantibodies reactive with molecularly defined synaptic plasma membrane proteins of muscle, peripheral, and central nervous systems. Of the 193 patients with thymoma, mean patient age was 52 years and did not significantly differ by sex (106 women) or group. Myasthenia gravis was the most prevalent clinical manifestation (64%) followed by dysautonomia (16 patients [8%]) and encephalopathy (15 patients [8%]); 164 patients (85%) had at least 1 synaptic autoantibody, and 63 of these patients (38%) had at least 1 more. Muscle acetylcholine receptor was most frequent (78%), followed by ganglionic acetylcholine receptor (20%), voltage-gated Kv1 potassium channel-complex (13%), and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (5%). Less frequent were aquaporin-4, voltage-gated Kv1 potassium channel-complex related proteins (leucine-rich glioma-inactivated 1 and contactin-associated protein-like 2), glycine receptor, and γ-aminobutyric acid-A receptor. Synaptic autoantibodies were significantly more frequent in patients

  12. Monothiol glutaredoxin Grx5 interacts with Fe-S scaffold proteins Isa1 and Isa2 and supports Fe-S assembly and DNA integrity in mitochondria of fission yeast

    International Nuclear Information System (INIS)

    Kim, Kyoung-Dong; Chung, Woo-Hyun; Kim, Hyo-Jin; Lee, Kyung-Chang; Roe, Jung-Hye

    2010-01-01

    Mitochondrial monothiol glutaredoxins that bind Fe-S cluster are known to participate in Fe-S cluster assembly. However, their precise role has not been well understood. Among three monothiol glutaredoxins (Grx3, 4, and 5) in Schizosaccharomyces pombe only Grx5 resides in mitochondria. The Δgrx5 mutant requires cysteine on minimal media, and does not grow on non-fermentable carbon source such as glycerol. We found that the mutant is low in the activity of Fe-S enzymes in mitochondria as well as in the cytoplasm. Screening of multi-copy suppressor of growth defects of the mutant identified isa1 + gene encoding a putative A-type Fe-S scaffold, in addition to mas5 + and hsc1 + genes encoding putative chaperones for Fe-S assembly process. Examination of other scaffold and chaperone genes revealed that isa2 + , but not isu1 + and ssc1 + , complemented the growth phenotype of Δgrx5 mutant as isa1 + did, partly through restoration of Fe-S enzyme activities. The mutant also showed a significant decrease in the amount of mitochondrial DNA. We demonstrated that Grx5 interacts in vivo with Isa1 and Isa2 proteins in mitochondria by observing bimolecular fluorescence complementation. These results indicate that Grx5 plays a central role in Fe-S assembly process through interaction with A-type Fe-S scaffold proteins Isa1 and Isa2, each of which is an essential protein in S. pombe, and supports mitochondrial genome integrity as well as Fe-S assembly.

  13. Monothiol glutaredoxin Grx5 interacts with Fe-S scaffold proteins Isa1 and Isa2 and supports Fe-S assembly and DNA integrity in mitochondria of fission yeast

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Kyoung-Dong; Chung, Woo-Hyun; Kim, Hyo-Jin; Lee, Kyung-Chang [Laboratory of Molecular Microbiology, School of Biological Sciences, Institute of Microbiology, Seoul National University, Seoul 151-742 (Korea, Republic of); Roe, Jung-Hye, E-mail: jhroe@snu.ac.kr [Laboratory of Molecular Microbiology, School of Biological Sciences, Institute of Microbiology, Seoul National University, Seoul 151-742 (Korea, Republic of)

    2010-02-12

    Mitochondrial monothiol glutaredoxins that bind Fe-S cluster are known to participate in Fe-S cluster assembly. However, their precise role has not been well understood. Among three monothiol glutaredoxins (Grx3, 4, and 5) in Schizosaccharomyces pombe only Grx5 resides in mitochondria. The {Delta}grx5 mutant requires cysteine on minimal media, and does not grow on non-fermentable carbon source such as glycerol. We found that the mutant is low in the activity of Fe-S enzymes in mitochondria as well as in the cytoplasm. Screening of multi-copy suppressor of growth defects of the mutant identified isa1{sup +} gene encoding a putative A-type Fe-S scaffold, in addition to mas5{sup +} and hsc1{sup +} genes encoding putative chaperones for Fe-S assembly process. Examination of other scaffold and chaperone genes revealed that isa2{sup +}, but not isu1{sup +} and ssc1{sup +}, complemented the growth phenotype of {Delta}grx5 mutant as isa1{sup +} did, partly through restoration of Fe-S enzyme activities. The mutant also showed a significant decrease in the amount of mitochondrial DNA. We demonstrated that Grx5 interacts in vivo with Isa1 and Isa2 proteins in mitochondria by observing bimolecular fluorescence complementation. These results indicate that Grx5 plays a central role in Fe-S assembly process through interaction with A-type Fe-S scaffold proteins Isa1 and Isa2, each of which is an essential protein in S. pombe, and supports mitochondrial genome integrity as well as Fe-S assembly.

  14. Leucine-rich repeat-containing synaptic adhesion molecules as organizers of synaptic specificity and diversity.

    Science.gov (United States)

    Schroeder, Anna; de Wit, Joris

    2018-04-09

    The brain harbors billions of neurons that form distinct neural circuits with exquisite specificity. Specific patterns of connectivity between distinct neuronal cell types permit the transfer and computation of information. The molecular correlates that give rise to synaptic specificity are incompletely understood. Recent studies indicate that cell-surface molecules are important determinants of cell type identity and suggest that these are essential players in the specification of synaptic connectivity. Leucine-rich repeat (LRR)-containing adhesion molecules in particular have emerged as key organizers of excitatory and inhibitory synapses. Here, we discuss emerging evidence that LRR proteins regulate the assembly of specific connectivity patterns across neural circuits, and contribute to the diverse structural and functional properties of synapses, two key features that are critical for the proper formation and function of neural circuits.

  15. Expression of cAMP-responsive element binding proteins (CREBs) in fast- and slow-twitch muscles: a signaling pathway to account for the synaptic expression of collagen-tailed subunit (ColQ) of acetylcholinesterase at the rat neuromuscular junction.

    Science.gov (United States)

    Choi, Roy C Y; Chen, Vicky P; Luk, Wilson K W; Yung, Amanda W Y; Ng, Alice H M; Dong, Tina T X; Tsim, Karl W K

    2013-03-25

    The gene encoding the collagen-tailed subunit (ColQ) of acetylcholinesterase (AChE) contains two distinct promoters that drive the production of two ColQ mRNAs, ColQ-1 and ColQ-1a, in slow- and fast-twitch muscles, respectively. ColQ-1a is expressed at the neuromuscular junction (NMJ) in fast-twitch muscle, and this expression depends on trophic factors supplied by motor neurons signaling via a cAMP-dependent pathway in muscle. To further elucidate the molecular basis of ColQ-1a's synaptic expression, here we investigated the expression and localization of cAMP-responsive element binding protein (CREB) at the synaptic and extra-synaptic regions of fast- and slow-twitch muscles from adult rats. The total amount of active, phosphorylated CREB (P-CREB) present in slow-twitch soleus muscle was higher than that in fast-twitch tibialis muscle, but P-CREB was predominantly expressed in the fast-twitch muscle at NMJs. In contrast, P-CREB was detected in both synaptic and extra-synaptic regions of slow-twitch muscle. These results reveal, for the first time, the differential distribution of P-CREB in fast- and slow-twitch muscles, which might support the crucial role of cAMP-dependent signaling in controlling the synapse-specific expression of ColQ-1a in fast-twitch muscles. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  16. Inhibition of hippocampal synaptic transmission by impairment of Ral function

    DEFF Research Database (Denmark)

    Owe-Larsson, Björn; Chaves-Olarte, Esteban; Chauhan, Ashok

    2005-01-01

    Large clostridial cytotoxins and protein overexpression were used to probe for involvement of Ras-related GTPases (guanosine triphosphate) in synaptic transmission in cultured rat hippocampal neurons. The toxins TcdA-10463 (inactivates Rho, Rac, Cdc42, Rap) and TcsL-1522 (inactivates Ral, Rac, Ra...

  17. Scaffold preferences of mesenchymal stromal cells and adipose-derived stem cells from green fluorescent protein transgenic mice influence the tissue engineering of bone.

    Science.gov (United States)

    Wittenburg, Gretel; Flade, Viktoria; Garbe, Annette I; Lauer, Günter; Labudde, Dirk

    2014-05-01

    We have analysed the growth and differentiation of mesenchymal stromal cells (MSC) from bone marrow, and of adipose derived stem cells (ASC) from murine abdominal fat tissue, of green fluorescent protein (GFP) transgenic animals grown directly on two types of hydroxyapatite ceramic bone substitutes. BONITmatrix® and NanoBone® have specific mechanical and physiochemical properties such as porosity and an inner surface that influence cellular growth. Both MSC and ASC were separately seeded on 200mg of each biomaterial and cultured for 3 weeks under osteogenic differentiation conditions. The degree of mineralisation was assessed by alizarin red dye and the specific alkaline phosphatase activity of the differentiated cells. The morphology of the cells was examined by scanning electron microscopy and confocal microscopy. The osteoblastic phenotype of the cells was confirmed by analysing the expression of bone-specific genes (Runx2, osteocalcin, osteopontin, and osteonectin) by semiquantitative reverse transcriptase polymerase chain reaction (PCR). Comparison of BONITmatrix® and NanoBone® showed cell type-specific preferences in terms of osteogenic differentiation. MSC-derived osteoblast-like cells spread optimally on the surface of NanoBone® but not BONITmatrix® granules. In contrast BONITmatrix® granules conditioned the growth of osteoblast-like cells derived from ASC. The osteoblastic phenotype of the cultured cells on all matrices was confirmed by specific gene expression. Our results show that the in vitro growth and osteogenic differentiation of murine MSC or ASC of GFP transgenic mice are distinctly influenced by the ceramic substratum. While NanoBone® granules support the proliferation and differentiation of murine MSC isolated from bone marrow, the growth of murine ASC is supported by BONITmatrix® granules. NanoBone® is therefore recommended for use as scaffold in tissue engineering that requires MSC, whereas ASC can be combined with BONITmatrix® for

  18. Aging synaptic mitochondria exhibit dynamic proteomic changes while maintaining bioenergetic function.

    Science.gov (United States)

    Stauch, Kelly L; Purnell, Phillip R; Fox, Howard S

    2014-04-01

    Aging correlates with a progressive impairment of mitochondrial homeostasis and is an influential factor for several forms of neurodegeneration. However, the mechanisms underlying age-related alterations in synaptosomal mitochondria, a neuronal mitochondria population highly susceptible to insults and critical for brain function, remain incompletely understood. Therefore this study investigates the synaptic mitochondrial proteomic and bioenergetic alterations that occur with age. The utilization of a state of the art quantitative proteomics approach allowed for the comparison of protein expression levels in synaptic mitochondria isolated from 5 (mature), 12 (old), and 24 (aged) month old mice. During the process of aging we find that dynamic proteomic alterations occur in synaptic mitochondria. Despite direct (mitochondrial DNA deletions) and indirect (increased antioxidant protein levels) signs of mitochondrial damage in the aged mice, there was an overall maintenance of mitochondrial function. Therefore the synaptic mitochondrial proteomic changes that occur with aging correlate with preservation of synaptic mitochondrial function.

  19. GABA type a receptor trafficking and the architecture of synaptic inhibition.

    Science.gov (United States)

    Lorenz-Guertin, Joshua M; Jacob, Tija C

    2018-03-01

    Ubiquitous expression of GABA type A receptors (GABA A R) in the central nervous system establishes their central role in coordinating most aspects of neural function and development. Dysregulation of GABAergic neurotransmission manifests in a number of human health disorders and conditions that in certain cases can be alleviated by drugs targeting these receptors. Precise changes in the quantity or activity of GABA A Rs localized at the cell surface and at GABAergic postsynaptic sites directly impact the strength of inhibition. The molecular mechanisms constituting receptor trafficking to and from these compartments therefore dictate the efficacy of GABA A R function. Here we review the current understanding of how GABA A Rs traffic through biogenesis, plasma membrane transport, and degradation. Emphasis is placed on discussing novel GABAergic synaptic proteins, receptor and scaffolding post-translational modifications, activity-dependent changes in GABA A R confinement, and neuropeptide and neurosteroid mediated changes. We further highlight modern techniques currently advancing the knowledge of GABA A R trafficking and clinically relevant neurodevelopmental diseases connected to GABAergic dysfunction. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 78: 238-270, 2018. © 2017 Wiley Periodicals, Inc.

  20. Synaptic Democracy and Vesicular Transport in Axons

    Science.gov (United States)

    Bressloff, Paul C.; Levien, Ethan

    2015-04-01

    Synaptic democracy concerns the general problem of how regions of an axon or dendrite far from the cell body (soma) of a neuron can play an effective role in neuronal function. For example, stimulated synapses far from the soma are unlikely to influence the firing of a neuron unless some sort of active dendritic processing occurs. Analogously, the motor-driven transport of newly synthesized proteins from the soma to presynaptic targets along the axon tends to favor the delivery of resources to proximal synapses. Both of these phenomena reflect fundamental limitations of transport processes based on a localized source. In this Letter, we show that a more democratic distribution of proteins along an axon can be achieved by making the transport process less efficient. This involves two components: bidirectional or "stop-and-go" motor transport (which can be modeled in terms of advection-diffusion), and reversible interactions between motor-cargo complexes and synaptic targets. Both of these features have recently been observed experimentally. Our model suggests that, just as in human societies, there needs to be a balance between "efficiency" and "equality".

  1. Linking Network Activity to Synaptic Plasticity during Sleep: Hypotheses and Recent Data.

    Science.gov (United States)

    Puentes-Mestril, Carlos; Aton, Sara J

    2017-01-01

    Research findings over the past two decades have supported a link between sleep states and synaptic plasticity. Numerous mechanistic hypotheses have been put forth to explain this relationship. For example, multiple studies have shown structural alterations to synapses (including changes in synaptic volume, spine density, and receptor composition) indicative of synaptic weakening after a period of sleep. Direct measures of neuronal activity and synaptic strength support the idea that a period of sleep can reduce synaptic strength. This has led to the synaptic homeostasis hypothesis (SHY), which asserts that during slow wave sleep, synapses are downscaled throughout the brain to counteract net strengthening of network synapses during waking experience (e.g., during learning). However, neither the cellular mechanisms mediating these synaptic changes, nor the sleep-dependent activity changes driving those cellular events are well-defined. Here we discuss potential cellular and network dynamic mechanisms which could underlie reductions in synaptic strength during sleep. We also discuss recent findings demonstrating circuit-specific synaptic strengthening (rather than weakening) during sleep. Based on these data, we explore the hypothetical role of sleep-associated network activity patterns in driving synaptic strengthening. We propose an alternative to SHY-namely that depending on experience during prior wake, a variety of plasticity mechanisms may operate in the brain during sleep. We conclude that either synaptic strengthening or synaptic weakening can occur across sleep, depending on changes to specific neural circuits (such as gene expression and protein translation) induced by experiences in wake. Clarifying the mechanisms underlying these different forms of sleep-dependent plasticity will significantly advance our understanding of how sleep benefits various cognitive functions.

  2. Reactive Oxygen Species: Physiological and Physiopathological Effects on Synaptic Plasticity

    Science.gov (United States)

    Beckhauser, Thiago Fernando; Francis-Oliveira, José; De Pasquale, Roberto

    2016-01-01

    In the mammalian central nervous system, reactive oxygen species (ROS) generation is counterbalanced by antioxidant defenses. When large amounts of ROS accumulate, antioxidant mechanisms become overwhelmed and oxidative cellular stress may occur. Therefore, ROS are typically characterized as toxic molecules, oxidizing membrane lipids, changing the conformation of proteins, damaging nucleic acids, and causing deficits in synaptic plasticity. High ROS concentrations are associated with a decline in cognitive functions, as observed in some neurodegenerative disorders and age-dependent decay of neuroplasticity. Nevertheless, controlled ROS production provides the optimal redox state for the activation of transductional pathways involved in synaptic changes. Since ROS may regulate neuronal activity and elicit negative effects at the same time, the distinction between beneficial and deleterious consequences is unclear. In this regard, this review assesses current research and describes the main sources of ROS in neurons, specifying their involvement in synaptic plasticity and distinguishing between physiological and pathological processes implicated. PMID:27625575

  3. Magnetic responsive hydroxyapatite composite scaffolds construction for bone defect reparation.

    Science.gov (United States)

    Zeng, Xiao Bo; Hu, Hao; Xie, Li Qin; Lan, Fang; Jiang, Wen; Wu, Yao; Gu, Zhong Wei

    2012-01-01

    In recent years, interest in magnetic biomimetic scaffolds for tissue engineering has increased considerably. A type of magnetic scaffold composed of magnetic nanoparticles (MNPs) and hydroxyapatite (HA) for bone repair has been developed by our research group. In this study, to investigate the influence of the MNP content (in the scaffolds) on the cell behaviors and the interactions between the magnetic scaffold and the exterior magnetic field, a series of MNP-HA magnetic scaffolds with different MNP contents (from 0.2% to 2%) were fabricated by immersing HA scaffold into MNP colloid. ROS 17/2.8 and MC3T3-E1 cells were cultured on the scaffolds in vitro, with and without an exterior magnetic field, respectively. The cell adhesion, proliferation and differentiation were evaluated via scanning electron microscopy; confocal laser scanning microscopy; and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), alkaline phosphatase, and bone gla protein activity tests. The results demonstrated the positive influence of the magnetic scaffolds on cell adhesion, proliferation, and differentiation. Further, a higher amount of MNPs on the magnetic scaffolds led to more significant stimulation. The magnetic scaffold can respond to the exterior magnetic field and engender some synergistic effect to intensify the stimulating effect of a magnetic field to the proliferation and differentiation of cells.

  4. Optogenetic analysis of synaptic function

    NARCIS (Netherlands)

    Liewald, Jana F.; Brauner, Martin; Stephens, Greg J.; Bouhours, Magali; Schultheis, Christian; Zhen, Mei; Gottschalk, Alexander

    2008-01-01

    We introduce optogenetic investigation of neurotransmission (OptIoN) for time-resolved and quantitative assessment of synaptic function via behavioral and electrophysiological analyses. We photo-triggered release of acetylcholine or γ-aminobutyric acid at Caenorhabditis elegans neuromuscular

  5. The dynamics of scaffolding

    NARCIS (Netherlands)

    Van Geert, P. L. C.; Steenbeek, H.W.

    2005-01-01

    In this article we have reinterpreted a relatively standard definition of scaffolding in the context of dynamic systems theory. Our main point is that scaffolding cannot be understood outside the context of a dynamic approach of learning and (formal or informal) teaching. We provide a dynamic

  6. Synaptic AMPA receptor plasticity and behavior

    NARCIS (Netherlands)

    Kessels, Helmut W.; Malinow, Roberto

    2009-01-01

    The ability to change behavior likely depends on the selective strengthening and weakening of brain synapses. The cellular models of synaptic plasticity, long-term potentiation (LTP) and depression (LTD) of synaptic strength, can be expressed by the synaptic insertion or removal of AMPA receptors

  7. Gelatin–PMVE/MA composite scaffold promotes expansion of embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Chhabra, Hemlata [Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai (India); Gupta, Priyanka [Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai (India); IITB-Monash Research Academy, Mumbai (India); Department of Chemical Engineering, Monash University, Melbourne (Australia); Verma, Paul J. [Turretfield Research Centre, South Australian Research and Development Institute, Rosedale, South Australia (Australia); Jadhav, Sameer; Bellare, Jayesh R. [Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai (India)

    2014-04-01

    We introduce a new composite scaffold of gelatin and polymethyl vinyl ether-alt-maleic anhydride (PMVE/MA) for expansion of embryonic stem cells (ESCs) in an in vitro environment. To optimize the scaffold, we prepared a gelatin scaffold (G) and three composite scaffolds namely GP-1, GP-2, and GP-3 with varying PMVE/MA concentrations (0.2–1%) and characterized them by scanning electron microscopy (SEM), swelling study, compression testing and FTIR. SEM micrographs revealed interconnected porous structure in all the scaffolds. The permissible hemolysis ratio and activation of platelets by scaffolds confirmed the hemocompatibility of scaffolds. Initial biocompatibility assessment of scaffolds was conducted using hepatocarcinoma (Hep G2) cells and adhesion, proliferation and infiltration of Hep G2 cells in depth of scaffolds were observed, proving the scaffold's biocompatibility. Further Oct4B2 mouse embryonic stem cells (mESCs), which harbor a green fluorescence protein transgene under regulatory control of the Oct4 promotor, were examined for expansion on scaffolds with MTT assay. The GP-2 scaffold demonstrated the best cell proliferation and was further explored for ESC adherence and infiltration in depth (SEM and confocal), and pluripotent state of mESCs was assessed with the expression of Oct4-GFP and stage-specific embryonic antigen-1 (SSEA-1). This study reports the first demonstration of biocompatibility of gelatin–PMVE/MA composite scaffold and presents this scaffold as a promising candidate for embryonic stem cell based tissue engineering. - Highlights: • Composite scaffolds of gelatin and PMVE/MA were prepared by freeze-drying method. • SEM micrographs showed porous structure in all scaffolds of varying pore dimension. • GP-2 composite exhibited better cellular response in comparison to other scaffolds. • mESCs proliferated and expressed Oct-4 and SSEA-1, when cultured on GP-2 scaffold.

  8. Tumor necrosis factor alpha maintains denervation-induced homeostatic synaptic plasticity of mouse dentate granule cells

    Directory of Open Access Journals (Sweden)

    Denise eBecker

    2013-12-01

    Full Text Available Neurons which lose part of their input respond with a compensatory increase in excitatory synaptic strength. This observation is of particular interest in the context of neurological diseases, which are accompanied by the loss of neurons and subsequent denervation of connected brain regions. However, while the cellular and molecular mechanisms of pharmacologically induced homeostatic synaptic plasticity have been identified to a certain degree, denervation-induced homeostatic synaptic plasticity remains not well understood. Here, we employed the entorhinal denervation in vitro model to study the role of tumor necrosis factor alpha (TNFα on changes in excitatory synaptic strength of mouse dentate granule cells following partial deafferentation. Our experiments disclose that TNFα is required for the maintenance of a compensatory increase in excitatory synaptic strength at 3/4 days postlesion (dpl, but not for the induction of synaptic scaling at 1 - 2 dpl. Furthermore, laser capture microdissection (LMD combined with quantitative PCR (qPCR demonstrates an increase in TNFα-mRNA levels in the denervated zone, which is consistent with our previous finding on a local, i.e., layer-specific increase in excitatory synaptic strength at 3 - 4 dpl. Immunostainings for the glial fibrillary acidic protein (GFAP and TNFα suggest that astrocytes are a source of TNFα in our experimental setting. We conclude that TNFα-signaling is a major regulatory system that aims at maintaining the homeostatic synaptic response of denervated neurons.

  9. Poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) scaffolds coated with PhaP-RGD fusion protein promotes the proliferation and chondrogenic differentiation of human umbilical cord mesenchymal stem cells in vitro.

    Science.gov (United States)

    Li, Xiaoli; Chang, Huimin; Luo, Huanan; Wang, Zhenghui; Zheng, Guoxi; Lu, Xiaoyun; He, Xijing; Chen, Fang; Wang, Ting; Liang, Jianmin; Xu, Min

    2015-03-01

    Human umbilical cord blood-derived mesenchymal stem cells (hUC-MSCs) have been widely used in tissue engineering. The aim of this study is to evaluate the ability of poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) scaffolds coated with polyhydroxyalkanoate binding protein fused with arginyl-glycyl-aspartic acid (PhaP-RGD) to promote the proliferation and chondrogenic differentiation of hUC-MSCs seeded on them. The PhaP-RGD fusion protein was expressed by Escherichia coli. PHBHHx films were coated with PhaP-RGD fusion protein and the physiochemical properties were examined. hUC-MSCs were seeded on PHBHHx films with or without PhaP-RGD precoating and tested for changes in morphology, viability, and chondrogenic differentiation. We found that PhaP-RGD-coated PHBHHx films had similar surface morphology to uncoated PHBHHx. The water contact angle of the coated PHBHHx surface was lower than that of the uncoated surface (10.63° vs. 98.69°). At 7 and 14 days after seeding, the PhaP-RGD-coated PHBHHx group showed greater numbers of viable cells compared to the uncoated PHBHHx group. The expression levels of aggrecan and collagen II were enhanced in the PhaP-RGD-coated PHBHHx group relative to the uncoated PHBHHx group. Histological analysis using toluidine blue staining showed elevated formation of proteoglycan producing chondrocytes in the PhaP-RGD-coated PHBHHx group. Additionally, the synthesis of proteoglycan and collagen was significantly enhanced within the PhaP-RGD constructs. Taken together, PhaP-RGD coating promotes the proliferation and chondrogenic differentiation of hUC-MSCs seeded on PHBHHx films. PhaP-RGD-coated PHBHHx may be a useful scaffold for cartilage tissue engineering. © 2014 Wiley Periodicals, Inc.

  10. Mechanical properties, biological activity and protein controlled release by poly(vinyl alcohol)-bioglass/chitosan-collagen composite scaffolds: a bone tissue engineering applications.

    Science.gov (United States)

    Pon-On, Weeraphat; Charoenphandhu, Narattaphol; Teerapornpuntakit, Jarinthorn; Thongbunchoo, Jirawan; Krishnamra, Nateetip; Tang, I-Ming

    2014-05-01

    In the present study, composite scaffolds made with different weight ratios (0.5:1, 1:1 and 2:1) of bioactive glass (15Ca:80Si:5P) (BG)/polyvinyl alcohol (PVA) (PVABG) and chitosan (Chi)/collagen (Col) (ChiCol) were prepared by three mechanical freeze-thaw followed by freeze-drying to obtain the porous scaffolds. The mechanical properties and the in vitro biocompatibility of the composite scaffolds to simulated body fluid (SBF) and to rat osteoblast-like UMR-106 cells were investigated. The results from the studies indicated that the porosity and compressive strength were controlled by the weight ratio of PVABG:ChiCol. The highest compressive modulus of the composites made was 214.64 MPa which was for the 1:1 weight ratio PVABG:ChiCol. Mineralization study in SBF showed the formation of apatite crystals on the PVABG:ChiCol surface after 7 days of incubation. In vitro cell availability and proliferation tests confirmed the osteoblast attachment and growth on the PVABG:ChiCol surface. MTT and ALP tests on the 1:1 weight ratio PVABG:ChiCol composite indicated that the UMR-106 cells were viable. Alkaline phosphatase activity was found to increase with increasing culturing time. In addition, we showed the potential of PVABG:ChiCol drug delivery through PBS solution studies. 81.14% of BSA loading had been achieved and controlled release for over four weeks was observed. Our results indicated that the PVABG:ChiCol composites, especially the 1:1 weight ratio composite exhibited significantly improved mechanical, mineral deposition, biological properties and controlled release. This made them potential candidates for bone tissue engineering applications. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Alzheimer's disease: synaptic dysfunction and Abeta

    LENUS (Irish Health Repository)

    Shankar, Ganesh M

    2009-11-23

    Abstract Synapse loss is an early and invariant feature of Alzheimer\\'s disease (AD) and there is a strong correlation between the extent of synapse loss and the severity of dementia. Accordingly, it has been proposed that synapse loss underlies the memory impairment evident in the early phase of AD and that since plasticity is important for neuronal viability, persistent disruption of plasticity may account for the frank cell loss typical of later phases of the disease. Extensive multi-disciplinary research has implicated the amyloid β-protein (Aβ) in the aetiology of AD and here we review the evidence that non-fibrillar soluble forms of Aβ are mediators of synaptic compromise. We also discuss the possible mechanisms of Aβ synaptotoxicity and potential targets for therapeutic intervention.

  12. Quantitative proteomics identifies Gemin5, a scaffolding protein involved in ribonucleoprotein assembly, as a novel partner for eukaryotic initiation factor 4E

    DEFF Research Database (Denmark)

    Fierro-Monti, Ivo; Mohammed, Shabaz; Matthiesen, Rune

    2006-01-01

    Protein complexes are dynamic entities; identification and quantitation of their components is critical in elucidating functional roles under specific cellular conditions. We report the first quantitative proteomic analysis of the human cap-binding protein complex. Components and proteins associa...

  13. Directional Trans-Synaptic Labeling of Specific Neuronal Connections in Live Animals.

    Science.gov (United States)

    Desbois, Muriel; Cook, Steven J; Emmons, Scott W; Bülow, Hannes E

    2015-07-01

    Understanding animal behavior and development requires visualization and analysis of their synaptic connectivity, but existing methods are laborious or may not depend on trans-synaptic interactions. Here we describe a transgenic approach for in vivo labeling of specific connections in Caenorhabditis elegans, which we term iBLINC. The method is based on BLINC (Biotin Labeling of INtercellular Contacts) and involves trans-synaptic enzymatic transfer of biotin by the Escherichia coli biotin ligase BirA onto an acceptor peptide. A BirA fusion with the presynaptic cell adhesion molecule NRX-1/neurexin is expressed presynaptically, whereas a fusion between the acceptor peptide and the postsynaptic protein NLG-1/neuroligin is expressed postsynaptically. The biotinylated acceptor peptide::NLG-1/neuroligin fusion is detected by a monomeric streptavidin::fluorescent protein fusion transgenically secreted into the extracellular space. Physical contact between neurons is insufficient to create a fluorescent signal, suggesting that synapse formation is required. The labeling approach appears to capture the directionality of synaptic connections, and quantitative analyses of synapse patterns display excellent concordance with electron micrograph reconstructions. Experiments using photoconvertible fluorescent proteins suggest that the method can be utilized for studies of protein dynamics at the synapse. Applying this technique, we find connectivity patterns of defined connections to vary across a population of wild-type animals. In aging animals, specific segments of synaptic connections are more susceptible to decline than others, consistent with dedicated mechanisms of synaptic maintenance. Collectively, we have developed an enzyme-based, trans-synaptic labeling method that allows high-resolution analyses of synaptic connectivity as well as protein dynamics at specific synapses of live animals. Copyright © 2015 by the Genetics Society of America.

  14. Synaptic dysfunction in amygdala in intellectual disorder models.

    Science.gov (United States)

    Aincy, Marianne; Meziane, Hamid; Herault, Yann; Humeau, Yann

    2018-06-08

    The amygdala is a part of the limbic circuit that has been extensively studied in terms of synaptic connectivity, plasticity and cellular organization since decades (Ehrlich et al., 2009; Ledoux, 2000; Maren, 2001). Amygdala sub-nuclei, including lateral, basolateral and central amygdala appear now as "hubs" providing in parallel and in series neuronal processing enabling the animal to elicit freezing or escaping behavior in response to external threats. In rodents, these behaviors are easily observed and quantified following associative fear conditioning. Thus, studies on amygdala circuit in association with threat/fear behavior became very popular in laboratories and are often used among other behavioral tests to evaluate learning abilities of mouse models for various neuropsychiatric conditions including genetically encoded intellectual disabilities (ID). Yet, more than 100 human X-linked genes - and several hundreds of autosomal genes - have been associated with ID in humans. These mutations introduced in mice can generate social deficits, anxiety dysregulations and fear learning impairments (McNaughton et al., 2008; Houbaert et al., 2013; Jayachandran et al., 2014; Zhang et al., 2015). Noteworthy, a significant proportion of the coded ID gene products are synaptic proteins. It is postulated that the loss of function of these proteins could destabilize neuronal circuits by global changes of the balance between inhibitory and excitatory drives onto neurons. However, whereas amygdala related behavioral deficits are commonly observed in ID models, the role of most of these ID-genes in synaptic function and plasticity in the amygdala are only sparsely studied. We will here discuss some of the concepts that emerged from amygdala-targeted studies examining the role of syndromic and non-syndromic ID genes in fear-related behaviors and/or synaptic function. Along describing these cases, we will discuss how synaptic deficits observed in amygdala circuits could impact

  15. EEA1 restores homeostatic synaptic plasticity in hippocampal neurons from Rett syndrome mice.

    Science.gov (United States)

    Xu, Xin; Pozzo-Miller, Lucas

    2017-08-15

    Rett syndrome is a neurodevelopmental disorder caused by loss-of-function mutations in MECP2, the gene encoding the transcriptional regulator methyl-CpG-binding protein 2 (MeCP2). Mecp2 deletion in mice results in an imbalance of excitation and inhibition in hippocampal neurons, which affects 'Hebbian' synaptic plasticity. We show that Mecp2-deficient neurons also lack homeostatic synaptic plasticity, likely due to reduced levels of EEA1, a protein involved in AMPA receptor endocytosis. Expression of EEA1 restored homeostatic synaptic plasticity in Mecp2-deficient neurons, providing novel targets of intervention in Rett syndrome. Rett syndrome is a neurodevelopmental disorder caused by loss-of-function mutations in MECP2, the gene encoding the transcriptional regulator methyl-CpG-binding protein 2 (MeCP2). Deletion of Mecp2 in mice results in an imbalance of synaptic excitation and inhibition in hippocampal pyramidal neurons, which affects 'Hebbian' long-term synaptic plasticity. Since the excitatory-inhibitory balance is maintained by homeostatic mechanisms, we examined the role of MeCP2 in homeostatic synaptic plasticity (HSP) at excitatory synapses. Negative feedback HSP, also known as synaptic scaling, maintains the global synaptic strength of individual neurons in response to sustained alterations in neuronal activity. Hippocampal neurons from Mecp2 knockout (KO) mice do not show the characteristic homeostatic scaling up of the amplitude of miniature excitatory postsynaptic currents (mEPSCs) and of synaptic levels of the GluA1 subunit of AMPA-type glutamate receptors after 48 h silencing with the Na + channel blocker tetrodotoxin. This deficit in HSP is bidirectional because Mecp2 KO neurons also failed to scale down mEPSC amplitudes and GluA1 synaptic levels after 48 h blockade of type A GABA receptor (GABA A R)-mediated inhibition with bicuculline. Consistent with the role of synaptic trafficking of AMPA-type of glutamate receptors in HSP, Mecp2 KO neurons

  16. Exact approaches for scaffolding

    OpenAIRE

    Weller, Mathias; Chateau, Annie; Giroudeau, Rodolphe

    2015-01-01

    This paper presents new structural and algorithmic results around the scaffolding problem, which occurs prominently in next generation sequencing. The problem can be formalized as an optimization problem on a special graph, the "scaffold graph". We prove that the problem is polynomial if this graph is a tree by providing a dynamic programming algorithm for this case. This algorithm serves as a basis to deduce an exact algorithm for general graphs using a tree decomposition of the input. We ex...

  17. Purines released from astrocytes inhibit excitatory synaptic transmission in the ventral horn of the spinal cord

    DEFF Research Database (Denmark)

    Carlsen, Eva Maria Meier; Perrier, Jean-Francois Marie

    2014-01-01

    . Neurons responded to electrical stimulation by monosynaptic EPSCs (excitatory monosynaptic postsynaptic currents). We used mice expressing the enhanced green fluorescent protein under the promoter of the glial fibrillary acidic protein to identify astrocytes. Chelating calcium with BAPTA in a single...... by different neuromodulators. These substances are usually thought of being released by dedicated neurons. However, in other networks from the central nervous system synaptic transmission is also modulated by transmitters released from astrocytes. The star-shaped glial cell responds to neurotransmitters...... by releasing gliotransmitters, which in turn modulate synaptic transmission. Here we investigated if astrocytes present in the ventral horn of the spinal cord modulate synaptic transmission. We evoked synaptic inputs in ventral horn neurons recorded in a slice preparation from the spinal cord of neonatal mice...

  18. IgG Antibody Responses to Recombinant gp120 Proteins, gp70V1/V2 Scaffolds, and a CyclicV2 Peptide in Thai Phase I/II Vaccine Trials Using Different Vaccine Regimens.

    Science.gov (United States)

    Karasavvas, Nicos; Karnasuta, Chitraporn; Savadsuk, Hathairat; Madnote, Sirinan; Inthawong, Dutsadee; Chantakulkij, Somsak; Rittiroongrad, Surawach; Nitayaphan, Sorachai; Pitisuttithum, Punnee; Thongcharoen, Prasert; Siriyanon, Vinai; Andrews, Charla A; Barnett, Susan W; Tartaglia, James; Sinangil, Faruk; Francis, Donald P; Robb, Merlin L; Michael, Nelson L; Ngauy, Viseth; de Souza, Mark S; Paris, Robert M; Excler, Jean-Louis; Kim, Jerome H; O'Connell, Robert J

    2015-11-01

    RV144 correlates of risk analysis showed that IgG antibodies to gp70V1V2 scaffolds inversely correlated with risk of HIV acquisition. We investigated IgG antibody responses in RV135 and RV132, two ALVAC-HIV prime-boost vaccine trials conducted in Thailand prior to RV144. Both trials used ALVAC-HIV (vCP1521) at 0, 1, 3, and 6 months and HIV-1 gp120MNgD and gp120A244gD in alum (RV135) or gp120SF2 and gp120CM235 in MF59 (RV132) at 3 and 6 months. We assessed ELISA binding antibodies to the envelope proteins (Env) 92TH023, A244gD and MNgD, cyclicV2, and gp70V1V2 CaseA2 (subtype B) and 92TH023 (subtype CRF01_AE), and Env-specific IgG1 and IgG3. Antibody responses to gp120 A244gD, MNgD, and gp70V1V2 92TH023 scaffold were significantly higher in RV135 than in RV132. Antibodies to gp70V1V2 CaseA2 were detected only in RV135 vaccine recipients and IgG1 and IgG3 antibody responses to A244gD were significantly higher in RV135. IgG binding to gp70V1V2 CaseA2 and CRF01_AE scaffolds was higher with the AIDSVAX(®)B/E boost but both trials showed similar rates of antibody decline post-vaccination. MF59 did not result in higher IgG antibody responses compared to alum with the antigens tested. However, notable differences in the structure of the recombinant proteins and dosage used for immunizations may have contributed to the magnitude and specificity of IgG induced by the two trials.

  19. Presynaptic Active Zone Density during Development and Synaptic Plasticity.

    Science.gov (United States)

    Clarke, Gwenaëlle L; Chen, Jie; Nishimune, Hiroshi

    2012-01-01

    Neural circuits transmit information through synapses, and the efficiency of synaptic transmission is closely related to the density of presynaptic active zones, where synaptic vesicles are released. The goal of this review is to highlight recent insights into the molecular mechanisms that control the number of active zones per presynaptic terminal (active zone density) during developmental and stimulus-dependent changes in synaptic efficacy. At the neuromuscular junctions (NMJs), the active zone density is preserved across species, remains constant during development, and is the same between synapses with different activities. However, the NMJ active zones are not always stable, as exemplified by the change in active zone density during acute experimental manipulation or as a result of aging. Therefore, a mechanism must exist to maintain its density. In the central nervous system (CNS), active zones have restricted maximal size, exist in multiple numbers in larger presynaptic terminals, and maintain a constant density during development. These findings suggest that active zone density in the CNS is also controlled. However, in contrast to the NMJ, active zone density in the CNS can also be increased, as observed in hippocampal synapses in response to synaptic plasticity. Although the numbers of known active zone proteins and protein interactions have increased, less is known about the mechanism that controls the number or spacing of active zones. The following molecules are known to control active zone density and will be discussed herein: extracellular matrix laminins and voltage-dependent calcium channels, amyloid precursor proteins, the small GTPase Rab3, an endocytosis mechanism including synaptojanin, cytoskeleton protein spectrins and β-adducin, and a presynaptic web including spectrins. The molecular mechanisms that organize the active zone density are just beginning to be elucidated.

  20. Presynaptic active zone density during development and synaptic plasticity.

    Directory of Open Access Journals (Sweden)

    Gwenaëlle L Clarke

    2012-02-01

    Full Text Available Neural circuits transmit information through synapses, and the efficiency of synaptic transmission is closely related to the density of presynaptic active zones, where synaptic vesicles are released. The goal of this review is to highlight recent insights into the molecular mechanisms that control the number of active zones per presynaptic terminal (active zone density during developmental and stimulus-dependent changes in synaptic efficacy. At the neuromuscular junctions (NMJs, the active zone density is preserved across species, remains constant during development, and is the same between synapses with different activities. However, the NMJ active zones are not always stable, as exemplified by the change in active zone density during acute experimental manipulation or as a result of aging. Therefore, a mechanism must exist to maintain its density. In the central nervous system (CNS, active zones have restricted maximal size, exist in multiple numbers in larger presynaptic terminals, and maintain a constant density during development. These findings suggest that active zone density in the CNS is also controlled. However, in contrast to the NMJ, active zone density in the CNS can also be increased, as observed in hippocampal synapses in response to synaptic plasticity. Although the numbers of known active zone proteins and protein interactions have increased, less is known about the mechanism that controls the number or spacing of active zones. The following molecules are known to control active zone density and will be discussed herein: extracellular matrix laminins and voltage-dependent calcium channels, amyloid precursor proteins, the small GTPase Rab3, an endocytosis mechanism including synaptojanin, cytoskeleton protein spectrins and β-adducin, and a presynaptic web including spectrins. The molecular mechanisms that organize the active zone density are just beginning to be elucidated.

  1. Torsin A Localization in the Mouse Cerebellar Synaptic Circuitry.

    Directory of Open Access Journals (Sweden)

    Francesca Puglisi

    Full Text Available Torsin A (TA is a ubiquitous protein belonging to the superfamily of proteins called "ATPases associated with a variety of cellular activities" (AAA(+ ATPase. To date, a great deal of attention has been focused on neuronal TA since its mutant form causes early-onset (DYT1 torsion dystonia, an inherited movement disorder characterized by sustained muscle contractions and abnormal postures. Interestingly, it has been proposed that TA, by interacting with the cytoskeletal network, may contribute to the control of neurite outgrowth and/or by acting as a chaperone at synapses could affect synaptic vesicle turnover and neurotransmitter release. Accordingly, both its peculiar developmental expression in striatum and cerebellum and evidence from DYT1 knock-in mice suggest that TA may influence dendritic arborization and synaptogenesis in the brain. Therefore, to better understand TA function a detailed description of its localization at synaptic level is required. Here, we characterized by means of rigorous quantitative confocal analysis TA distribution in the mouse cerebellum at postnatal day 14 (P14, when both cerebellar synaptogenesis and TA expression peak. We observed that the protein is broadly distributed both in cerebellar cortex and in the deep cerebellar nuclei (DCN. Of note, Purkinje cells (PC express high levels of TA also in the spines and axonal terminals. In addition, abundant expression of the protein was found in the main GABA-ergic and glutamatergic inputs of the cerebellar cortex. Finally, TA was observed also in glial cells, a cellular population little explored so far. These results extend our knowledge on TA synaptic localization providing a clue to its potential role in synaptic development.

  2. Fluorapatite-modified Scaffold on Dental Pulp Stem Cell Mineralization

    Science.gov (United States)

    Guo, T.; Li, Y.; Cao, G.; Zhang, Z.; Chang, S.; Czajka-Jakubowska, A.; Nör, J.E.; Clarkson, B.H.; Liu, J.

    2014-01-01

    In previous studies, fluorapatite (FA) crystal-coated surfaces have been shown to stimulate the differentiation and mineralization of human dental pulp stem cells (DPSCs) in two-dimensional cell culture. However, whether the FA surface can recapitulate these properties in three-dimensional culture is still unknown. This study examined the differences in behavior of human DPSCs cultured on electrospun polycaprolactone (PCL) NanoECM nanofibers with or without the FA crystals. Under near-physiologic conditions, the FA crystals were synthesized on the PCL nanofiber scaffolds. The FA crystals were evenly distributed on the scaffolds. DPSCs were cultured on the PCL+FA or the PCL scaffolds for up to 28 days. Scanning electron microscope images showed that DPSCs attached well to both scaffolds after the initial seeding. However, it appeared that more multicellular aggregates formed on the PCL+FA scaffolds. After 14 days, the cell proliferation on the PCL+FA was slower than that on the PCL-only scaffolds. Interestingly, even without any induction of mineralization, from day 7, the upregulation of several pro-osteogenic molecules (dmp1, dspp, runx2, ocn, spp1, col1a1) was detected in cells seeded on the PCL+FA scaffolds. A significant increase in alkaline phosphatase activity was also seen on FA-coated scaffolds compared with the PCL-only scaffolds at days 14 and 21. At the protein level, osteocalcin expression was induced only in the DPSCs on the PCL+FA surfaces at day 21 and then significantly enhanced at day 28. A similar pattern was observed in those specimens stained with Alizarin red and Von Kossa after 21 and 28 days. These data suggest that the incorporation of FA crystals within the three-dimensional PCL nanofiber scaffolds provided a favorable extracellular matrix microenvironment for the growth, differentiation, and mineralization of human DPSCs. This FA-modified PCL nanofiber scaffold shows promising potential for future bone, dental, and orthopedic regenerative

  3. Synaptic Control of Secretory Trafficking in Dendrites

    Directory of Open Access Journals (Sweden)

    Cyril Hanus

    2014-06-01

    Full Text Available Localized signaling in neuronal dendrites requires tight spatial control of membrane composition. Upon initial synthesis, nascent secretory cargo in dendrites exits the endoplasmic reticulum (ER from local zones of ER complexity that are spatially coupled to post-ER compartments. Although newly synthesized membrane proteins can be processed locally, the mechanisms that control the spatial range of secretory cargo transport in dendritic segments are unknown. Here, we monitored the dynamics of nascent membrane proteins in dendritic post-ER compartments under regimes of low or increased neuronal activity. In response to activity blockade, post-ER carriers are highly mobile and are transported over long distances. Conversely, increasing synaptic activity dramatically restricts the spatial scale of post-ER trafficking along dendrites. This activity-induced confinement of secretory cargo requires site-specific phosphorylation of the kinesin motor KIF17 by Ca2+/calmodulin-dependent protein kinases (CaMK. Thus, the length scales of early secretory trafficking in dendrites are tuned by activity-dependent regulation of microtubule-dependent transport.

  4. Synaptic genes are extensively downregulated across multiple brain regions in normal human aging and Alzheimer’s disease

    Science.gov (United States)

    Berchtold, Nicole C.; Coleman, Paul D.; Cribbs, David H.; Rogers, Joseph; Gillen, Daniel L.; Cotman, Carl W.

    2014-01-01

    Synapses are essential for transmitting, processing, and storing information, all of which decline in aging and Alzheimer’s disease (AD). Because synapse loss only partially accounts for the cognitive declines seen in aging and AD, we hypothesized that existing synapses might undergo molecular changes that reduce their functional capacity. Microarrays were used to evaluate expression profiles of 340 synaptic genes in aging (20–99 years) and AD across 4 brain regions from 81 cases. The analysis revealed an unexpectedly large number of significant expression changes in synapse-related genes in aging, with many undergoing progressive downregulation across aging and AD. Functional classification of the genes showing altered expression revealed that multiple aspects of synaptic function are affected, notably synaptic vesicle trafficking and release, neurotransmitter receptors and receptor trafficking, postsynaptic density scaffolding, cell adhesion regulating synaptic stability, and neuromodulatory systems. The widespread declines in synaptic gene expression in normal aging suggests that function of existing synapses might be impaired, and that a common set of synaptic genes are vulnerable to change in aging and AD. PMID:23273601

  5. Synaptic proteome changes in the hypothalamus of mother rats.

    Science.gov (United States)

    Udvari, Edina Brigitta; Völgyi, Katalin; Gulyássy, Péter; Dimén, Diána; Kis, Viktor; Barna, János; Szabó, Éva Rebeka; Lubec, Gert; Juhász, Gábor; Kékesi, Katalin Adrienna; Dobolyi, Árpád

    2017-04-21

    To establish synaptic proteome changes associated with motherhood, we isolated synaptosome fractions from the hypothalamus of mother rats and non-maternal control females at the 11th postpartum day. Proteomic analysis by two-dimensional differential gel electrophoresis combined with mass spectrometric protein identification established 26 significant proteins, 7 increasing and 19 decreasing protein levels in the dams. The altered proteins are mainly involved in energy homeostasis, protein folding, and metabolic processes suggesting the involvement of these cellular processes in maternal adaptations. The decrease in a significantly altered protein, complement component 1q subcomponent-binding protein (C1qbp) was validated with Western blotting. Furthermore, immunohistochemistry showed its presence in hypothalamic fibers and terminals in agreement with its presence in synaptosomes. We also found the expression of C1qbp in different hypothalamic nuclei including the preoptic area and the paraventricular hypothalamic nucleus at the protein and at the mRNA level using immunohistochemistry and in situ hybridization histochemistry, respectively. Bioinformatical network analysis revealed that cytokines, growth factors, and protein kinases are common regulators, which indicates a complex regulation of the proteome change in mothers. The results suggest that maternal responsiveness is associated with synaptic proteins level changes in the hypothalamus, and that growth factors and cytokines may govern these alterations. The period of motherhood is accompanied with several behavioral, neuroendocrine, emotional and metabolic adaptations in the brain. Although it is established that various hypothalamic networks participate in the maternal adaptations of the rodent brain, our knowledge on the molecular background of these alterations remains seriously limited. In the present study, we first determined that the functional alterations of the maternal brain can be detected at the

  6. Network response synchronization enhanced by synaptic plasticity

    Science.gov (United States)

    Lobov, S.; Simonov, A.; Kastalskiy, I.; Kazantsev, V.

    2016-02-01

    Synchronization of neural network response on spatially localized periodic stimulation was studied. The network consisted of synaptically coupled spiking neurons with spike-timing-dependent synaptic plasticity (STDP). Network connectivity was defined by time evolving matrix of synaptic weights. We found that the steady-state spatial pattern of the weights could be rearranged due to locally applied external periodic stimulation. A method for visualization of synaptic weights as vector field was introduced to monitor the evolving connectivity matrix. We demonstrated that changes in the vector field and associated weight rearrangements underlay an enhancement of synchronization range.

  7. Synaptic Plasticity and Memory Formation

    Science.gov (United States)

    1993-06-30

    suspected of being the substrate of several forms of memory encoded by synapses in the forebrain of humans and other mammals. Work in the past year...of LTP will enhance the encoding of memory . Aniracetam , as noted, prolongs the open time of the AMPA receptor and in this way facilitates excitatory...121 t Iffw,,a" S. FUNO4NG mUMSERS Synaptic Plasticity and Memory Formation F 49620-92-0307 C (ci) b.q F Gary Lynch 7. Pf(RfO*INN ORGAMIZAMNIO NMMW(S

  8. Experience-Dependent Equilibration of AMPAR-Mediated Synaptic Transmission during the Critical Period

    Directory of Open Access Journals (Sweden)

    Kyung-Seok Han

    2017-01-01

    Full Text Available Experience-dependent synapse refinement is essential for functional optimization of neural circuits. However, how sensory experience sculpts excitatory synaptic transmission is poorly understood. Here, we show that despite substantial remodeling of synaptic connectivity, AMPAR-mediated synaptic transmission remains at equilibrium during the critical period in the mouse primary visual cortex. The maintenance of this equilibrium requires neurogranin (Ng, a postsynaptic calmodulin-binding protein important for synaptic plasticity. With normal visual experience, loss of Ng decreased AMPAR-positive synapse numbers, prevented AMPAR-silent synapse maturation, and increased spine elimination. Importantly, visual deprivation halted synapse loss caused by loss of Ng, revealing that Ng coordinates experience-dependent AMPAR-silent synapse conversion to AMPAR-active synapses and synapse elimination. Loss of Ng also led to sensitized long-term synaptic depression (LTD and impaired visually guided behavior. Our synaptic interrogation reveals that experience-dependent coordination of AMPAR-silent synapse conversion and synapse elimination hinges upon Ng-dependent mechanisms for constructive synaptic refinement during the critical period.

  9. Synaptic plasticity modulation by circulating peptides and metaplasticity: Involvement in Alzheimer's disease.

    Science.gov (United States)

    Peineau, Stéphane; Rabiant, Kevin; Pierrefiche, Olivier; Potier, Brigitte

    2018-02-06

    Synaptic plasticity is a cellular process involved in learning and memory whose alteration in its two main forms (Long Term Depression (LTD) and Long Term Potentiation (LTP)), is observed in most brain pathologies, including neurodegenerative disorders such as Alzheimer's disease (AD). In humans, AD is associated at the cellular level with neuropathological lesions composed of extracellular deposits of β-amyloid (Aβ) protein aggregates and intracellular neurofibrillary tangles, cellular loss, neuroinflammation and a general brain homeostasis dysregulation. Thus, a dramatic synaptic environment perturbation is observed in AD patients, involving changes in brain neuropeptides, cytokines, growth factors or chemokines concentration and diffusion. Studies performed in animal models demonstrate that these circulating peptides strongly affect synaptic functions and in particular synaptic plasticity. Besides this neuromodulatory action of circulating peptides, other synaptic plasticity regulation mechanisms such as metaplasticity are altered in AD animal models. Here, we will review new insights into the study of synaptic plasticity regulatory/modulatory mechanisms which could influence the process of synaptic plasticity in the context of AD with a particular attention to the role of metaplasticity and peptide dependent neuromodulation. Copyright © 2018 Elsevier Ltd. All rights reserved.

  10. Histological and radiographic evaluation of the muscle tissue of rats after implantation of bone morphogenic protein (rhBMP-2 in a scaffold of inorganic bone and after stimulation with low-power laser light

    Directory of Open Access Journals (Sweden)

    Bengtson Antonio

    2010-01-01

    Full Text Available Objective: The present study histologically and radiologically evaluates the muscle tissue of rats after implantation of bone morphogenic protein (rhBMP-2 in a natural inorganic bone mineral scaffold from a bull calf femur and irradiation with low-power light laser. Materials and Methods: The right and left hind limbs of 16 rats were shaved and an incision was made in the muscle on the face corresponding to the median portion of the tibia, into which rhBMP-2 in a scaffold of inorganic bone was implanted. Two groups of limbs were formed: control (G1 and laser irradiation (G2. G2 received diode laser light applied in the direction of the implant, at a dose of 8 J/cm2 for three minutes. On the 7th, 21st, 40th and 112th days after implantation, hind limbs of 4 animals were radiographed and their implants removed together with the surrounding tissue for study under the microscope. The histological results were graded as 0=absence, 1=slight presence, 2=representative and 3=very representative, with regard to the following events: formation of osteoid structure, acute inflammation, chronic inflammation, fibrin deposition, neovascularization, foreign-body granuloma and fibrosis. Results: There were no statistically significant differences in these events at each evaluation times, between the two groups (P > 0.05; Mann-Whitney test. Nevertheless, it could be concluded that the natural inorganic bone matrix with rhBMP-2, from the femur of a bull calf, is a biocompatible combination. Conclusions: Under these conditions, the inductive capacity of rhBMP-2 for cell differentiation was inhibited. There was a slight acceleration in tissue healing in the group that received irradiation with low-power laser light.

  11. Macromolecular multi-chromophoric scaffolding

    NARCIS (Netherlands)

    Schwartz, E.; Schwartz, Erik; Le Gac, Stephane; le Gac, Severine; Cornelissen, Jeroen Johannes Lambertus Maria; Nolte, Roeland J.M.; Rowan, Alan E.

    2010-01-01

    This critical review describes recent efforts in the field of chromophoric scaffolding. The advances in this research area, with an emphasis on rigid scaffolds, for example, synthetic polymers, carbon nanotubes (CNTs), nucleic acids, and viruses, are presented (166 references).

  12. Biomimetic Scaffolds for Osteogenesis

    Science.gov (United States)

    Yuan, Nance; Rezzadeh, Kameron S.; Lee, Justine C.

    2015-01-01

    Skeletal regenerative medicine emerged as a field of investigation to address large osseous deficiencies secondary to congenital, traumatic, and post-oncologic conditions. Although autologous bone grafts have been the gold standard for reconstruction of skeletal defects, donor site morbidity remains a significant limitation. To address these limitations, contemporary bone tissue engineering research aims to target delivery of osteogenic cells and growth factors in a defined three dimensional space using scaffolding material. Using bone as a template, biomimetic strategies in scaffold engineering unite organic and inorganic components in an optimal configuration to both support osteoinduction as well as osteoconduction. This article reviews the various structural and functional considerations behind the development of effective biomimetic scaffolds for osteogenesis and highlights strategies for enhancing osteogenesis. PMID:26413557

  13. Semiotic Scaffolding in Mathematics

    DEFF Research Database (Denmark)

    Johansen, Mikkel Willum; Misfeldt, Morten

    2015-01-01

    This paper investigates the notion of semiotic scaffolding in relation to mathematics by considering its influence on mathematical activities, and on the evolution of mathematics as a research field. We will do this by analyzing the role different representational forms play in mathematical...... cognition, and more broadly on mathematical activities. In the main part of the paper, we will present and analyze three different cases. For the first case, we investigate the semiotic scaffolding involved in pencil and paper multiplication. For the second case, we investigate how the development of new...... in both mathematical cognition and in the development of mathematics itself, but mathematical cognition cannot itself be reduced to the use of semiotic scaffolding....

  14. Cornification in reptilian epidermis occurs through the deposition of keratin-associated beta-proteins (beta-keratins) onto a scaffold of intermediate filament keratins.

    Science.gov (United States)

    Alibardi, Lorenzo

    2013-02-01

    The isolation of genes for alpha-keratins and keratin-associated beta-proteins (formerly beta-keratins) has allowed the production of epitope-specific antibodies for localizing these proteins during the process of cornification epidermis of reptilian sauropsids. The antibodies are directed toward proteins in the alpha-keratin range (40-70 kDa) or beta-protein range (10-30 kDa) of most reptilian sauropsids. The ultrastructural immunogold study shows the localization of acidic alpha-proteins in suprabasal and precorneous epidermal layers in lizard, snake, tuatara, crocodile, and turtle while keratin-associated beta-proteins are localized in precorneous and corneous layers. This late activation of the synthesis of keratin-associated beta-proteins is typical for keratin-associated and corneous proteins in mammalian epidermis (involucrin, filaggrin, loricrin) or hair (tyrosine-rich or sulfur-rich proteins). In turtles and crocodilians epidermis, keratin-associated beta-proteins are synthesized in upper spinosus and precorneous layers and accumulate in the corneous layer. The complex stratification of lepidosaurian epidermis derives from the deposition of specific glycine-rich versus cysteine-glycine-rich keratin-associated beta-proteins in cells sequentially produced from the basal layer and not from the alternation of beta- with alpha-keratins. The process gives rise to Oberhäutchen, beta-, mesos-, and alpha-layers during the shedding cycle of lizards and snakes. Differently from fish, amphibian, and mammalian keratin-associated proteins (KAPs) of the epidermis, the keratin-associated beta-proteins of sauropsids are capable to form filaments of 3-4 nm which give rise to an X-ray beta-pattern as a consequence of the presence of a beta-pleated central region of high homology, which seems to be absent in KAPs of the other vertebrates. Copyright © 2012 Wiley Periodicals, Inc.

  15. Evolution of the aging brain transcriptome and synaptic regulation.

    Directory of Open Access Journals (Sweden)

    Patrick M Loerch

    Full Text Available Alzheimer's disease and other neurodegenerative disorders of aging are characterized by clinical and pathological features that are relatively specific to humans. To obtain greater insight into how brain aging has evolved, we compared age-related gene expression changes in the cortex of humans, rhesus macaques, and mice on a genome-wide scale. A small subset of gene expression changes are conserved in all three species, including robust age-dependent upregulation of the neuroprotective gene apolipoprotein D (APOD and downregulation of the synaptic cAMP signaling gene calcium/calmodulin-dependent protein kinase IV (CAMK4. However, analysis of gene ontology and cell type localization shows that humans and rhesus macaques have diverged from mice due to a dramatic increase in age-dependent repression of neuronal genes. Many of these age-regulated neuronal genes are associated with synaptic function. Notably, genes associated with GABA-ergic inhibitory function are robustly age-downregulated in humans but not in mice at the level of both mRNA and protein. Gene downregulation was not associated with overall neuronal or synaptic loss. Thus, repression of neuronal gene expression is a prominent and recently evolved feature of brain aging in humans and rhesus macaques that may alter neural networks and contribute to age-related cognitive changes.

  16. Structure of the GH1 domain of guanylate kinase-associated protein from Rattus norvegicus

    International Nuclear Information System (INIS)

    Tong, Junsen; Yang, Huiseon; Eom, Soo Hyun; Chun, ChangJu; Im, Young Jun

    2014-01-01

    Graphical abstract: - Highlights: • The crystal structure of GKAP homology domain 1 (GH1) was determined. • GKAP GH1 is a three-helix bundle connected by short flexible loops. • The predicted helix α4 associates weakly with the helix α3, suggesting dynamic nature of the GH1 domain. - Abstract: Guanylate-kinase-associated protein (GKAP) is a scaffolding protein that links NMDA receptor-PSD-95 to Shank–Homer complexes by protein–protein interactions at the synaptic junction. GKAP family proteins are characterized by the presence of a C-terminal conserved GKAP homology domain 1 (GH1) of unknown structure and function. In this study, crystal structure of the GH1 domain of GKAP from Rattus norvegicus was determined in fusion with an N-terminal maltose-binding protein at 2.0 Å resolution. The structure of GKAP GH1 displays a three-helix bundle connected by short flexible loops. The predicted helix α4 which was not visible in the crystal structure associates weakly with the helix α3 suggesting dynamic nature of the GH1 domain. The strict conservation of GH1 domain across GKAP family members and the lack of a catalytic active site required for enzyme activity imply that the GH1 domain might serve as a protein–protein interaction module for the synaptic protein clustering

  17. Structure of the GH1 domain of guanylate kinase-associated protein from Rattus norvegicus

    Energy Technology Data Exchange (ETDEWEB)

    Tong, Junsen; Yang, Huiseon [College of Pharmacy, Chonnam National University, Gwangju 500-757 (Korea, Republic of); Eom, Soo Hyun [School of Life Sciences, Steitz Center for Structural Biology, and Department of Chemistry, Gwangju Institute of Science and Technology, Gwangju 500-712 (Korea, Republic of); Chun, ChangJu, E-mail: cchun1130@jnu.ac.kr [College of Pharmacy, Chonnam National University, Gwangju 500-757 (Korea, Republic of); Im, Young Jun, E-mail: imyoungjun@jnu.ac.kr [College of Pharmacy, Chonnam National University, Gwangju 500-757 (Korea, Republic of)

    2014-09-12

    Graphical abstract: - Highlights: • The crystal structure of GKAP homology domain 1 (GH1) was determined. • GKAP GH1 is a three-helix bundle connected by short flexible loops. • The predicted helix α4 associates weakly with the helix α3, suggesting dynamic nature of the GH1 domain. - Abstract: Guanylate-kinase-associated protein (GKAP) is a scaffolding protein that links NMDA receptor-PSD-95 to Shank–Homer complexes by protein–protein interactions at the synaptic junction. GKAP family proteins are characterized by the presence of a C-terminal conserved GKAP homology domain 1 (GH1) of unknown structure and function. In this study, crystal structure of the GH1 domain of GKAP from Rattus norvegicus was determined in fusion with an N-terminal maltose-binding protein at 2.0 Å resolution. The structure of GKAP GH1 displays a three-helix bundle connected by short flexible loops. The predicted helix α4 which was not visible in the crystal structure associates weakly with the helix α3 suggesting dynamic nature of the GH1 domain. The strict conservation of GH1 domain across GKAP family members and the lack of a catalytic active site required for enzyme activity imply that the GH1 domain might serve as a protein–protein interaction module for the synaptic protein clustering.

  18. The developmental stages of synaptic plasticity

    NARCIS (Netherlands)

    Lohmann, Christian; Kessels, Helmut W.

    2014-01-01

    The brain is programmed to drive behaviour by precisely wiring the appropriate neuronal circuits. Wiring and rewiring of neuronal circuits largely depends on the orchestrated changes in the strengths of synaptic contacts. Here, we review how the rules of synaptic plasticity change during development

  19. Magnetic responsive hydroxyapatite composite scaffolds construction for bone defect reparation

    Directory of Open Access Journals (Sweden)

    Zeng XB

    2012-07-01

    Full Text Available Xiao Bo Zeng, Hao Hu, Li Qin Xie, Fang Lan, Wen Jiang, Yao Wu, Zhong Wei GuNational Engineering Research Center for Biomaterials, Sichuan University, Chengdu, Sichuan, People's Republic of ChinaIntroduction: In recent years, interest in magnetic biomimetic scaffolds for tissue engineering has increased considerably. A type of magnetic scaffold composed of magnetic nanoparticles (MNPs and hydroxyapatite (HA for bone repair has been developed by our research group.Aim and methods: In this study, to investigate the influence of the MNP content (in the scaffolds on the cell behaviors and the interactions between the magnetic scaffold and the exterior magnetic field, a series of MNP-HA magnetic scaffolds with different MNP contents (from 0.2% to 2% were fabricated by immersing HA scaffold into MNP colloid. ROS 17/2.8 and MC3T3-E1 cells were cultured on the scaffolds in vitro, with and without an exterior magnetic field, respectively. The cell adhesion, proliferation and differentiation were evaluated via scanning electron microscopy; confocal laser scanning microscopy; and 3-(4,5-Dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT, alkaline phosphatase, and bone gla protein activity tests.Results: The results demonstrated the positive influence of the magnetic scaffolds on cell adhesion, proliferation, and differentiation. Further, a higher amount of MNPs on the magnetic scaffolds led to more significant stimulation.Conclusion: The magnetic scaffold can respond to the exterior magnetic field and engender some synergistic effect to intensify the stimulating effect of a magnetic field to the proliferation and differentiation of cells.Keywords: magnetic therapy, magnetic nanoparticles, bone repair, magnetic responsive

  20. Size distribution and radial density profile of synaptic vesicles by SAXS and light scattering

    Energy Technology Data Exchange (ETDEWEB)

    Castorph, Simon; Salditt, Tim [Institute for X-ray Physics, Goettingen (Germany); Holt, Matthew; Jahn, Reinhard [Max Plank Institute for Biophysical Chemistry, Goettingen (Germany); Sztucki, Michael [European Synchrotron Radiation Facility, Grenoble (France)

    2008-07-01

    Synaptic vesicles are small membraneous organelles within the nerve terminal, encapsulating neurotransmitters by a lipid bilayer. The transport of the neurotransmitter, the fusion at the plasma membrane, and the release of the stored neurotransmitters into the synaptic cleft are since long know as essential step in nerve conduction of the chemical synapse. A detailed structural view of these molecular mechanisms is still lacking, not withstanding the enormous progress in the field during recent years. From measurements and quantitative fitting of small angle X-ray scattering curves and dynamic light scattering the averaged structural properties of synaptic vesicles can be determined. We present SAXS measurements and fits revealing the width of the size distribution function and details of the radial scattering length profile of synaptic vesicles from rat brain. Representative values for the inner and outer radius and the size polydispersity as well as the density and width of the outer protein layer are obtained.

  1. Intrinsic cellular and molecular properties of in vivo hippocampal synaptic plasticity are altered in the absence of key synaptic matrix molecules.

    Science.gov (United States)

    Jansen, Stephan; Gottschling, Christine; Faissner, Andreas; Manahan-Vaughan, Denise

    2017-08-01

    Hippocampal synaptic plasticity comprises a key cellular mechanism for information storage. In the hippocampus, both long-term potentiation (LTP) and long-term depression (LTD) are triggered by synaptic Ca 2+ -elevations that are typically mediated by the opening of voltage-gated cation channels, such as N-methyl-d-aspartate receptors (NMDAR), in the postsynaptic density. The integrity of the post-synaptic density is ensured by the extracellular matrix (ECM). Here, we explored whether synaptic plasticity is affected in adult behaving mice that lack the ECM proteins brevican, neurocan, tenascin-C, and tenascin-R (KO). We observed that the profiles of synaptic potentiation and depression in the dentate gyrus (DG) were profoundly altered compared to plasticity profiles in wild-type littermates (WT). Specifically, synaptic depression was amplified in a frequency-dependent manner and although late-LTP (>24 hr) was expressed following strong afferent tetanization, the early component of LTP (4 hr) elicited by weaker tetanization was equivalent in WT and KO animals. Furthermore, this latter form of LTP was NMDAR-dependent in WT but not KO mice. Scrutiny of DG receptor expression revealed significantly lower levels of both the GluN2A and GluN2B subunits of the N-methyl-d-aspartate receptor, of the metabotropic glutamate receptor, mGlu5 and of the L-type calcium channel, Ca v 1.3 in KO compared to WT animals. Homer 1a and of the P/Q-type calcium channel, Ca v 1.2 were unchanged in KO mice. Taken together, findings suggest that in mice that lack multiple ECM proteins, synaptic plasticity is intact, but is fundamentally different. © 2017 Wiley Periodicals, Inc.

  2. Bone tissue engineering scaffolding: computer-aided scaffolding techniques.

    Science.gov (United States)

    Thavornyutikarn, Boonlom; Chantarapanich, Nattapon; Sitthiseripratip, Kriskrai; Thouas, George A; Chen, Qizhi

    Tissue engineering is essentially a technique for imitating nature. Natural tissues consist of three components: cells, signalling systems (e.g. growth factors) and extracellular matrix (ECM). The ECM forms a scaffold for its cells. Hence, the engineered tissue construct is an artificial scaffold populated with living cells and signalling molecules. A huge effort has been invested in bone tissue engineering, in which a highly porous scaffold plays a critical role in guiding bone and vascular tissue growth and regeneration in three dimensions. In the last two decades, numerous scaffolding techniques have been developed to fabricate highly interconnective, porous scaffolds for bone tissue engineering applications. This review provides an update on the progress of foaming technology of biomaterials, with a special attention being focused on computer-aided manufacturing (Andrade et al. 2002) techniques. This article starts with a brief introduction of tissue engineering (Bone tissue engineering and scaffolds) and scaffolding materials (Biomaterials used in bone tissue engineering). After a brief reviews on conventional scaffolding techniques (Conventional scaffolding techniques), a number of CAM techniques are reviewed in great detail. For each technique, the structure and mechanical integrity of fabricated scaffolds are discussed in detail. Finally, the advantaged and disadvantage of these techniques are compared (Comparison of scaffolding techniques) and summarised (Summary).

  3. Degeneracy in the regulation of short-term plasticity and synaptic filtering by presynaptic mechanisms.

    Science.gov (United States)

    Mukunda, Chinmayee L; Narayanan, Rishikesh

    2017-04-15

    also revealed that calcium- and release-control mechanisms were effective regulators of synaptic filters, but accomplished this without changes in terminal excitability or calcium influx. Next, to perform global sensitivity analysis, we generated 7000 randomized models spanning 15 presynaptic parameters, and computed eight different physiological measurements in each of these models. We validated these models by applying experimentally obtained bounds on their measurements, and found 104 (∼1.5%) models to match the validation criteria for all eight measurements. Analysing these valid models, we demonstrate that analogous synaptic filters emerge from disparate combinations of presynaptic parameters exhibiting weak pairwise correlations. Finally, using virtual knockout models, we establish the variable and differential impact of different presynaptic channels on synaptic filters, underlining the critical importance of interactions among different presynaptic components in defining synaptic physiology. Our results have significant implications for protein-localization strategies required for physiological robustness and for degeneracy in long-term synaptic plasticity profiles. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

  4. Development of a porcine renal extracellular matrix scaffold as a platform for kidney regeneration.

    Science.gov (United States)

    Choi, Seock Hwan; Chun, So Young; Chae, Seon Yeong; Kim, Jin Rae; Oh, Se Heang; Chung, Sung Kwang; Lee, Jin Ho; Song, Phil Hyun; Choi, Gyu-Seog; Kim, Tae-Hwan; Kwon, Tae Gyun

    2015-04-01

    Acellular scaffolds, possessing an intact three-dimensional extracellular matrix (ECM) architecture and biochemical components, are promising for regeneration of complex organs, such as the kidney. We have successfully developed a porcine renal acellular scaffold and analyzed its physical/biochemical characteristics, biocompatibility, and kidney reconstructive potential. Segmented porcine kidney cortexes were treated with either 1% (v/v) Triton X-100 (Triton) or sodium dodecyl sulfate (SDS). Scanning electron microscopy showed both treatments preserved native tissue architecture, including porosity and composition. Swelling behavior was higher in the Triton-treated compared with the SDS-treated scaffold. Maximum compressive strength was lower in the Triton-treated compared with the SDS-treated scaffold. Attenuated total reflective-infrared spectroscopy showed the presence of amide II (-NH) in both scaffolds. Furthermore, richer ECM protein and growth factor contents were observed in the Triton-treated compared with SDS-treated scaffold. Primary human kidney cell adherence, viability, and proliferation were enhanced on the Triton-treated scaffold compared with SDS-treated scaffold. Following murine in vivo implantation, tumorigenecity was absent for both scaffolds after 8 weeks and in the Triton-treated scaffold only, glomeruli-like structure formation and neovascularity were observed. We identified 1% Triton X-100 as a more suitable decellularizing agent for porcine renal ECM scaffolds prior to kidney regeneration. © 2014 Wiley Periodicals, Inc.

  5. Emergent spatial synaptic structure from diffusive plasticity.

    Science.gov (United States)

    Sweeney, Yann; Clopath, Claudia

    2017-04-01

    Some neurotransmitters can diffuse freely across cell membranes, influencing neighbouring neurons regardless of their synaptic coupling. This provides a means of neural communication, alternative to synaptic transmission, which can influence the way in which neural networks process information. Here, we ask whether diffusive neurotransmission can also influence the structure of synaptic connectivity in a network undergoing plasticity. We propose a form of Hebbian synaptic plasticity which is mediated by a diffusive neurotransmitter. Whenever a synapse is modified at an individual neuron through our proposed mechanism, similar but smaller modifications occur in synapses connecting to neighbouring neurons. The effects of this diffusive plasticity are explored in networks of rate-based neurons. This leads to the emergence of spatial structure in the synaptic connectivity of the network. We show that this spatial structure can coexist with other forms of structure in the synaptic connectivity, such as with groups of strongly interconnected neurons that form in response to correlated external drive. Finally, we explore diffusive plasticity in a simple feedforward network model of receptive field development. We show that, as widely observed across sensory cortex, the preferred stimulus identity of neurons in our network become spatially correlated due to diffusion. Our proposed mechanism of diffusive plasticity provides an efficient mechanism for generating these spatial correlations in stimulus preference which can flexibly interact with other forms of synaptic organisation. © 2016 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  6. A Novel Albumin-Based Tissue Scaffold for Autogenic Tissue Engineering Applications

    OpenAIRE

    Li, Pei-Shan; -Liang Lee, I.; Yu, Wei-Lin; Sun, Jui-Sheng; Jane, Wann-Neng; Shen, Hsin-Hsin

    2014-01-01

    Tissue scaffolds provide a framework for living tissue regeneration. However, traditional tissue scaffolds are exogenous, composed of metals, ceramics, polymers, and animal tissues, and have a defined biocompatibility and application. This study presents a new method for obtaining a tissue scaffold from blood albumin, the major protein in mammalian blood. Human, bovine, and porcine albumin was polymerised into albumin polymers by microbial transglutaminase and was then cast by freeze-drying-b...

  7. All for One But Not One for All: Excitatory Synaptic Scaling and Intrinsic Excitability Are Coregulated by CaMKIV, Whereas Inhibitory Synaptic Scaling Is Under Independent Control.

    Science.gov (United States)

    Joseph, Annelise; Turrigiano, Gina G

    2017-07-12

    Neocortical circuits use a family of homeostatic plasticity mechanisms to stabilize firing, including excitatory and inhibitory synaptic scaling and homeostatic intrinsic plasticity (Turrigiano and Nelson, 2004). All three mechanisms can be induced in tandem in cultured rat neocortical pyramidal neurons by chronic manipulations of firing, but it is unknown whether they are coinduced by the same activity-sensors and signaling pathways, or whether they are under independent control. Calcium/calmodulin-dependent protein kinase type IV (CaMKIV) is a key sensory/effector in excitatory synaptic scaling that senses perturbations in firing through changes in calcium influx, and translates this into compensatory changes in excitatory quantal amplitude (Ibata et al., 2008; Goold and Nicoll, 2010). Whether CaMKIV also controls inhibitory synaptic scaling and intrinsic homeostatic plasticity was unknown. To test this we manipulated CaMKIV signaling in individual neurons using dominant-negative (dn) or constitutively-active (ca) forms of nuclear-localized CaMKIV and measured the induction of all three forms of homeostatic plasticity. We found that excitatory synaptic scaling and intrinsic plasticity were bidirectionally coinduced by these manipulations. In contrast, these cell-autonomous manipulations had no impact on inhibitory quantal amplitude. Finally, we found that spontaneous firing rates were shifted up or down by dnCaMKIV or caCaMKIV, respectively, suggesting that uncoupling CaMKIV activation from activity generates an error signal in the negative feedback mechanism that controls firing rates. Together, our data show that excitatory synaptic scaling and intrinsic excitability are tightly coordinated through bidirectional changes in the same signaling pathway, whereas inhibitory synaptic scaling is sensed and regulated through an independent control mechanism. SIGNIFICANCE STATEMENT Maintaining stable function in highly interconnected neural circuits is essential for

  8. A versatile optical tool for studying synaptic GABAA receptor trafficking.

    Science.gov (United States)

    Lorenz-Guertin, Joshua M; Wilcox, Madeleine R; Zhang, Ming; Larsen, Mads B; Pilli, Jyotsna; Schmidt, Brigitte F; Bruchez, Marcel P; Johnson, Jon W; Waggoner, Alan S; Watkins, Simon C; Jacob, Tija C

    2017-11-15

    Live-cell imaging methods can provide critical real-time receptor trafficking measurements. Here, we describe an optical tool to study synaptic γ-aminobutyric acid (GABA) type A receptor (GABA A R) dynamics through adaptable fluorescent-tracking capabilities. A fluorogen-activating peptide (FAP) was genetically inserted into a GABA A R γ2 subunit tagged with pH-sensitive green fluorescent protein (γ2 pH FAP). The FAP selectively binds and activates Malachite Green (MG) dyes that are otherwise non-fluorescent in solution. γ2 pH FAP GABA A Rs are expressed at the cell surface in transfected cortical neurons, form synaptic clusters and do not perturb neuronal development. Electrophysiological studies show γ2 pH FAP GABA A Rs respond to GABA and exhibit positive modulation upon stimulation with the benzodiazepine diazepam. Imaging studies using γ2 pH FAP-transfected neurons and MG dyes show time-dependent receptor accumulation into intracellular vesicles, revealing constitutive endosomal and lysosomal trafficking. Simultaneous analysis of synaptic, surface and lysosomal receptors using the γ2 pH FAP-MG dye approach reveals enhanced GABA A R turnover following a bicucculine-induced seizure paradigm, a finding not detected by standard surface receptor measurements. To our knowledge, this is the first application of the FAP-MG dye system in neurons, demonstrating the versatility to study nearly all phases of GABA A R trafficking. © 2017. Published by The Company of Biologists Ltd.

  9. Models of Short-Term Synaptic Plasticity.

    Science.gov (United States)

    Barroso-Flores, Janet; Herrera-Valdez, Marco A; Galarraga, Elvira; Bargas, José

    2017-01-01

    We focus on dynamical descriptions of short-term synaptic plasticity. Instead of focusing on the molecular machinery that has been reviewed recently by several authors, we concentrate on the dynamics and functional significance of synaptic plasticity, and review some mathematical models that reproduce different properties of the dynamics of short term synaptic plasticity that have been observed experimentally. The complexity and shortcomings of these models point to the need of simple, yet physiologically meaningful models. We propose a simplified model to be tested in synapses displaying different types of short-term plasticity.

  10. Protein-RNA and Protein-Protein Recognition by Dual KH1/2 Domains of the Neuronal Splicing Factor Nova-1

    Energy Technology Data Exchange (ETDEWEB)

    M Teplova; L Malinina; J Darnell; J Song; M Lu; R Abagyan; K Musunuru; A Teplov; S Burley; et al.

    2011-12-31

    Nova onconeural antigens are neuron-specific RNA-binding proteins implicated in paraneoplastic opsoclonus-myoclonus-ataxia (POMA) syndrome. Nova harbors three K-homology (KH) motifs implicated in alternate splicing regulation of genes involved in inhibitory synaptic transmission. We report the crystal structure of the first two KH domains (KH1/2) of Nova-1 bound to an in vitro selected RNA hairpin, containing a UCAG-UCAC high-affinity binding site. Sequence-specific intermolecular contacts in the complex involve KH1 and the second UCAC repeat, with the RNA scaffold buttressed by interactions between repeats. Whereas the canonical RNA-binding surface of KH2 in the above complex engages in protein-protein interactions in the crystalline state, the individual KH2 domain can sequence-specifically target the UCAC RNA element in solution. The observed antiparallel alignment of KH1 and KH2 domains in the crystal structure of the complex generates a scaffold that could facilitate target pre-mRNA looping on Nova binding, thereby potentially explaining Nova's functional role in splicing regulation.

  11. Cortactin is a scaffolding platform for the E-cadherin adhesion complex and is regulated by protein kinase D1 phosphorylation.

    Science.gov (United States)

    Sroka, Robert; Van Lint, Johan; Katz, Sarah-Fee; Schneider, Marlon R; Kleger, Alexander; Paschke, Stephan; Seufferlein, Thomas; Eiseler, Tim

    2016-06-15

    Dynamic regulation of cell-cell adhesion by the coordinated formation and dissolution of E-cadherin-based adherens junctions is crucial for tissue homeostasis. The actin-binding protein cortactin interacts with E-cadherin and enables F-actin accumulation at adherens junctions. Here, we were interested to study the broader functional interactions of cortactin in adhesion complexes. In line with literature, we demonstrate that cortactin binds to E-cadherin, and that a posttranslational modification of cortactin, RhoA-induced phosphorylation by protein kinase D1 (PKD1; also known as PRKD1) at S298, impairs adherens junction assembly and supports their dissolution. Two new S298-phosphorylation-dependent interactions were also identified, namely, that phosphorylation of cortactin decreases its interaction with β-catenin and the actin-binding protein vinculin. In addition, binding of vinculin to β-catenin, as well as linkage of vinculin to F-actin, are also significantly compromised upon phosphorylation of cortactin. Accordingly, we found that regulation of cell-cell adhesion by phosphorylation of cortactin downstream of RhoA and PKD1 is vitally dependent on vinculin-mediated protein interactions. Thus, cortactin, unexpectedly, is an important integration node for the dynamic regulation of protein complexes during breakdown and formation of adherens junctions. © 2016. Published by The Company of Biologists Ltd.

  12. Mapping synaptic pathology within cerebral cortical circuits in subjects with schizophrenia

    Directory of Open Access Journals (Sweden)

    Robert Sweet

    2010-06-01

    Full Text Available Converging lines of evidence indicate that schizophrenia is characterized by impairments of synaptic machinery within cerebral cortical circuits. Efforts to localize these alterations in brain tissue from subjects with schizophrenia have frequently been limited to the quantification of structures that are non-selectively identified (e.g. dendritic spines labeled in Golgi preparations, axon boutons labeled with synaptophysin, or to quantification of proteins using methods unable to resolve relevant cellular compartments. Multiple label fluorescence confocal microscopy represents a means to circumvent many of these limitations, by concurrently extracting information regarding the number, morphology, and relative protein content of synaptic structures. An important adaptation required for studies of human disease is coupling this approach to stereologic methods for systematic random sampling of relevant brain regions. In this review article we consider the application of multiple label fluorescence confocal microscopy to the mapping of synaptic alterations in subjects with schizophrenia and describe the application of a novel, readily automated, iterative intensity/morphological segmentation algorithm for the extraction of information regarding synaptic structure number, size, and relative protein level from tissue sections obtained using unbiased stereological principles of sampling. In this context, we provide examples of the examination of pre- and post-synaptic structures within excitatory and inhibitory circuits of the cerebral cortex.

  13. Activity-dependent modulation of neural circuit synaptic connectivity

    Directory of Open Access Journals (Sweden)

    Charles R Tessier

    2009-07-01

    Full Text Available In many nervous systems, the establishment of neural circuits is known to proceed via a two-stage process; 1 early, activity-independent wiring to produce a rough map characterized by excessive synaptic connections, and 2 subsequent, use-dependent pruning to eliminate inappropriate connections and reinforce maintained synapses. In invertebrates, however, evidence of the activity-dependent phase of synaptic refinement has been elusive, and the dogma has long been that invertebrate circuits are “hard-wired” in a purely activity-independent manner. This conclusion has been challenged recently through the use of new transgenic tools employed in the powerful Drosophila system, which have allowed unprecedented temporal control and single neuron imaging resolution. These recent studies reveal that activity-dependent mechanisms are indeed required to refine circuit maps in Drosophila during precise, restricted windows of late-phase development. Such mechanisms of circuit refinement may be key to understanding a number of human neurological diseases, including developmental disorders such as Fragile X syndrome (FXS and autism, which are hypothesized to result from defects in synaptic connectivity and activity-dependent circuit function. This review focuses on our current understanding of activity-dependent synaptic connectivity in Drosophila, primarily through analyzing the role of the fragile X mental retardation protein (FMRP in the Drosophila FXS disease model. The particular emphasis of this review is on the expanding array of new genetically-encoded tools that are allowing cellular events and molecular players to be dissected with ever greater precision and detail.

  14. Buyang Huanwu decoction facilitates neurorehabilitation through an improvement of synaptic plasticity in cerebral ischemic rats.

    Science.gov (United States)

    Pan, Ruihuan; Cai, Jun; Zhan, Lechang; Guo, Youhua; Huang, Run-Yue; Li, Xiong; Zhou, Mingchao; Xu, Dandan; Zhan, Jie; Chen, Hongxia

    2017-03-28

    Loss of neural function is a critical but unsolved issue after cerebral ischemia insult. Neuronal plasticity and remodeling are crucial for recovery of neural functions after brain injury. Buyang Huanwu decoction, which is a classic formula in traditional Chinese medicine, can positively alter synaptic plasticity. This study assessed the effects of Buyang Huanwu decoction in combination with physical exercise on neuronal plasticity in cerebral ischemic rats. Cerebral ischemic rats were administered Buyang Huanwu decoction and participated in physical exercise after the induction of a permanent middle cerebral artery occlusion. The neurobehavioral functions and infarct volumes were evaluated. The presynaptic (SYN), postsynaptic (GAP-43) and cytoskeletal (MAP-2) proteins in the coronal brain samples were evaluated by immunohistochemistry and western blot analyses. The ultrastructure of the neuronal synaptic junctions in the same region were analyzed using transmission electron microscopy. Combination treatment of Buyang Huanwu decoction and physical exercise ameliorated the neurobehavioral deficits (p synaptic ultrastructure. Buyang Huanwu decoction facilitated neurorehabilitation following a cerebral ischemia insult through an improvement in synaptic plasticity. Graphical abstract The Buyang Huanwu decoction (BYHWD) combined with physical exercise (PE) attenuates synaptic disruption and promotes synaptic plasticity following cerebral ischemia (stroke).

  15. Dynamic expression of long noncoding RNAs and repeat elements in synaptic plasticity.

    Science.gov (United States)

    Maag, Jesper L V; Panja, Debabrata; Sporild, Ida; Patil, Sudarshan; Kaczorowski, Dominik C; Bramham, Clive R; Dinger, Marcel E; Wibrand, Karin

    2015-01-01

    Long-term potentiation (LTP) of synaptic transmission is recognized as a cellular mechanism for learning and memory storage. Although de novo gene transcription is known to be required in the formation of stable LTP, the molecular mechanisms underlying synaptic plasticity remain elusive. Noncoding RNAs have emerged as major regulatory molecules that are abundantly and specifically expressed in the mammalian brain. By combining RNA-seq analysis with LTP induction in the dentate gyrus of live rats, we provide the first global transcriptomic analysis of synaptic plasticity in the adult brain. Expression profiles of mRNAs and long noncoding RNAs (lncRNAs) were obtained at 30 min, 2 and 5 h after high-frequency stimulation of the perforant pathway. The temporal analysis revealed dynamic expression profiles of lncRNAs with many positively, and highly, correlated to protein-coding genes with known roles in synaptic plasticity, suggesting their possible involvement in LTP. In light of observations suggesting a role for retrotransposons in brain function, we examined the expression of various classes of repeat elements. Our analysis identifies dynamic regulation of LINE1 and SINE retrotransposons, and extensive regulation of tRNA. These experiments reveal a hitherto unknown complexity of gene expression in long-term synaptic plasticity involving the dynamic regulation of lncRNAs and repeat elements. These findings provide a broader foundation for elucidating the transcriptional and epigenetic regulation of synaptic plasticity in both the healthy brain and in neurodegenerative and neuropsychiatric disorders.

  16. Binding Sites for Amyloid-β Oligomers and Synaptic Toxicity

    Science.gov (United States)

    Smith, Levi M.; Strittmatter, Stephen M.

    2017-01-01

    In Alzheimer’s disease (AD), insoluble and fibrillary amyloid-β (Aβ) peptide accumulates in plaques. However, soluble Aβ oligomers are most potent in creating synaptic dysfunction and loss. Therefore, receptors for Aβ oligomers are hypothesized to be the first step in a neuronal cascade leading to dementia. A number of cell-surface proteins have been described as Aβ binding proteins, and one or more are likely to mediate Aβ oligomer toxicity in AD. Cellular prion protein (PrPC) is a high-affinity Aβ oligomer binding site, and a range of data delineates a signaling pathway leading from Aβ complexation with PrPC to neuronal impairment. Further study of Aβ binding proteins will define the molecular basis of this crucial step in AD pathogenesis. PMID:27940601

  17. The Rev1 interacting region (RIR) motif in the scaffold protein XRCC1 mediates a low-affinity interaction with polynucleotide kinase/phosphatase (PNKP) during DNA single-strand break repair.

    Science.gov (United States)

    Breslin, Claire; Mani, Rajam S; Fanta, Mesfin; Hoch, Nicolas; Weinfeld, Michael; Caldecott, Keith W

    2017-09-29

    The scaffold protein X-ray repair cross-complementing 1 (XRCC1) interacts with multiple enzymes involved in DNA base excision repair and single-strand break repair (SSBR) and is important for genetic integrity and normal neurological function. One of the most important interactions of XRCC1 is that with polynucleotide kinase/phosphatase (PNKP), a dual-function DNA kinase/phosphatase that processes damaged DNA termini and that, if mutated, results in ataxia with oculomotor apraxia 4 (AOA4) and microcephaly with early-onset seizures and developmental delay (MCSZ). XRCC1 and PNKP interact via a high-affinity phosphorylation-dependent interaction site in XRCC1 and a forkhead-associated domain in PNKP. Here, we identified using biochemical and biophysical approaches a second PNKP interaction site in XRCC1 that binds PNKP with lower affinity and independently of XRCC1 phosphorylation. However, this interaction nevertheless stimulated PNKP activity and promoted SSBR and cell survival. The low-affinity interaction site required the highly conserved Rev1-interacting region (RIR) motif in XRCC1 and included three critical and evolutionarily invariant phenylalanine residues. We propose a bipartite interaction model in which the previously identified high-affinity interaction acts as a molecular tether, holding XRCC1 and PNKP together and thereby promoting the low-affinity interaction identified here, which then stimulates PNKP directly. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Fabrication and characterization of scaffold from cadaver goat-lung tissue for skin tissue engineering applications

    Energy Technology Data Exchange (ETDEWEB)

    Gupta, Sweta K. [Department of Polymer and Process Engineering, Indian Institute of Technology, Roorkee (India); Dinda, Amit K. [Department of Pathology, All India Institute of Medical Sciences, New Delhi (India); Potdar, Pravin D. [Department of Molecular Medicine, Jaslok Hospital and Research Centre, Mumbai (India); Mishra, Narayan C., E-mail: mishrawise@gmail.com [Department of Polymer and Process Engineering, Indian Institute of Technology, Roorkee (India)

    2013-10-15

    The present study aims to fabricate scaffold from cadaver goat-lung tissue and evaluate it for skin tissue engineering applications. Decellularized goat-lung scaffold was fabricated by removing cells from cadaver goat-lung tissue enzymatically, to have cell-free 3D-architecture of natural extracellular matrix. DNA quantification assay and Hematoxylin and eosin staining confirmed the absence of cellular material in the decellularized lung-tissue. SEM analysis of decellularized scaffold shows the intrinsic porous structure of lung tissue with well-preserved pore-to-pore interconnectivity. FTIR analysis confirmed non-denaturation and well maintainance of collagenous protein structure of decellularized scaffold. MTT assay, SEM analysis and H and E staining of human skin-derived Mesenchymal Stem cell, seeded over the decellularized scaffold, confirms stem cell attachment, viability, biocompatibility and proliferation over the decellularized scaffold. Expression of Keratin18 gene, along with CD105, CD73 and CD44, by human skin-derived Mesenchymal Stem cells over decellularized scaffold signifies that the cells are viable, proliferating and migrating, and have maintained their critical cellular functions in the presence of scaffold. Thus, overall study proves the applicability of the goat-lung tissue derived decellularized scaffold for skin tissue engineering applications. - Highlights: • We successfully fabricated decellularized scaffold from cadaver goat-lung tissue. • Decellularized goat-lung scaffolds were found to be highly porous. • Skin derived MSC shows high cell viability and proliferation over the scaffold. • Phenotype of MSCs was well maintained over the scaffold. • The scaffold shows potential for applications in skin tissue engineering.

  19. Fabrication and characterization of scaffold from cadaver goat-lung tissue for skin tissue engineering applications

    International Nuclear Information System (INIS)

    Gupta, Sweta K.; Dinda, Amit K.; Potdar, Pravin D.; Mishra, Narayan C.

    2013-01-01

    The present study aims to fabricate scaffold from cadaver goat-lung tissue and evaluate it for skin tissue engineering applications. Decellularized goat-lung scaffold was fabricated by removing cells from cadaver goat-lung tissue enzymatically, to have cell-free 3D-architecture of natural extracellular matrix. DNA quantification assay and Hematoxylin and eosin staining confirmed the absence of cellular material in the decellularized lung-tissue. SEM analysis of decellularized scaffold shows the intrinsic porous structure of lung tissue with well-preserved pore-to-pore interconnectivity. FTIR analysis confirmed non-denaturation and well maintainance of collagenous protein structure of decellularized scaffold. MTT assay, SEM analysis and H and E staining of human skin-derived Mesenchymal Stem cell, seeded over the decellularized scaffold, confirms stem cell attachment, viability, biocompatibility and proliferation over the decellularized scaffold. Expression of Keratin18 gene, along with CD105, CD73 and CD44, by human skin-derived Mesenchymal Stem cells over decellularized scaffold signifies that the cells are viable, proliferating and migrating, and have maintained their critical cellular functions in the presence of scaffold. Thus, overall study proves the applicability of the goat-lung tissue derived decellularized scaffold for skin tissue engineering applications. - Highlights: • We successfully fabricated decellularized scaffold from cadaver goat-lung tissue. • Decellularized goat-lung scaffolds were found to be highly porous. • Skin derived MSC shows high cell viability and proliferation over the scaffold. • Phenotype of MSCs was well maintained over the scaffold. • The scaffold shows potential for applications in skin tissue engineering

  20. BMP-2 immobilized PLGA/hydroxyapatite fibrous scaffold via polydopamine stimulates osteoblast growth.

    Science.gov (United States)

    Zhao, Xingyu; Han, Yu; Li, Jiawei; Cai, Bo; Gao, Hang; Feng, Wei; Li, Shuqiang; Liu, Jianguo; Li, Dongsong

    2017-09-01

    Combining biomaterials scaffolds with bone morphogenetic protein-2 (BMP-2) is currently used to promote the regeneration of bone tissue. However, the traditional strategies used to add BMP-2 into the polymer scaffolds directly suffer from limitations that can result in lower growth factor loading and damage the bioactivity of growth factors. In this study, we report the fabrication of poly(lactide-co-glycolide)/hydroxyapatite (PLGA/HA) composite fibrous scaffolds via melt-spinning method to mimic native extracellular matrix (ECM). In order to effectively immobilize BMP-2 on PLGA/HA composite fibrous scaffolds, the surface of the scaffold was modified with polydopamine (PDA) (PDA-PLGA/HA). PDA was chosen as an adhesive polymeric bridge-layer between PLGA/HA fibrous scaffolds and BMP-2. Analysis of the scaffold using scanning electron microscopy, Fourier transform infrared spectroscopy and X-ray photoelectron spectroscope revealed that the PDA coating was attached to the scaffold surface. Moreover, analysis of the scaffold using water contact angle demonstrated an increased hydrophilicity via PDA modification. Furthermore, the PDA coating effectively immobilized BMP-2 on the PDA-PLGA/HA fibrous scaffold and a sustained release profile of BMP-2 was achieved in the BMP-2-immobilized PLGA/HA fibrous scaffold. In vitro experiments showed that BMP-2-immobilized PLGA/HA fibrous scaffold significantly promoted the attachment and proliferation of MC3T3-E1 cells. More importantly, the ALP activity, mRNA expression of osteosis-related genes and calcium deposition in MC3T3-E1 cells cultured on BMP-2-immobilized PLGA/HA fibrous scaffold were significantly increased. These results collectively demonstrate that the BMP-2-immobilized PLGA/HA fibrous scaffold is a promising candidate for bone regeneration. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. c-SRC mediates neurite outgrowth through recruitment of Crk to the scaffolding protein Sin/Efs without altering the kinetics of ERK activation

    DEFF Research Database (Denmark)

    Yang, Liang-Tung; Alexandropoulos, Konstantina; Sap, Jan

    2002-01-01

    moderate activation of endogenous SRC by receptor-protein-tyrosine phosphatase alpha (a physiological SRC activator). We show that such a qualitative change in the response to EGF is not accompanied by changes in the extent or kinetics of ERK induction in response to this factor. Instead, the pathway...

  2. Scaffolding students’ assignments

    DEFF Research Database (Denmark)

    Slot, Marie Falkesgaard

    2013-01-01

    learning goals) can help students structure their argumentative and communica-tive learning processes, and how various multimodal representations can give more open-ended learning possibilities for collaboration. The article presents a short introduction of the skills for 21st century learning and defines......This article discusses scaffolding in typical student assignments in mother tongue learning materials in upper secondary education in Denmark and the United Kingdom. It has been determined that assignments do not have sufficient scaffolding end features to help pupils understand concepts and build...... objects. The article presents the results of empirical research on tasks given in Danish and British learning materials. This work is based on a further development of my PhD thesis: “Learning materials in the subject of Danish” (Slot 2010). The main focus is how cognitive models (and subsidiary explicit...

  3. Molecular mechanisms of synaptic remodeling in alcoholism.

    Science.gov (United States)

    Kyzar, Evan J; Pandey, Subhash C

    2015-08-05

    Alcohol use and alcohol addiction represent dysfunctional brain circuits resulting from neuroadaptive changes during protracted alcohol exposure and its withdrawal. Alcohol exerts a potent effect on synaptic plasticity and dendritic spine formation in specific brain regions, providing a neuroanatomical substrate for the pathophysiology of alcoholism. Epigenetics has recently emerged as a critical regulator of gene expression and synaptic plasticity-related events in the brain. Alcohol exposure and withdrawal induce changes in crucial epigenetic processes in the emotional brain circuitry (amygdala) that may be relevant to the negative affective state defined as the "dark side" of addiction. Here, we review the literature concerning synaptic plasticity and epigenetics, with a particular focus on molecular events related to dendritic remodeling during alcohol abuse and alcoholism. Targeting epigenetic processes that modulate synaptic plasticity may yield novel treatments for alcoholism. Published by Elsevier Ireland Ltd.

  4. Discovery of an Unexplored Protein Structural Scaffold of Serine Protease from Big Blue Octopus (Octopus cyanea): A New Prospective Lead Molecule.

    Science.gov (United States)

    Panda, Subhamay; Kumari, Leena

    2017-01-01

    Serine proteases are a group of enzymes that hydrolyses the peptide bonds in proteins. In mammals, these enzymes help in the regulation of several major physiological functions such as digestion, blood clotting, responses of immune system, reproductive functions and the complement system. Serine proteases obtained from the venom of Octopodidae family is a relatively unexplored area of research. In the present work, we tried to effectively utilize comparative composite molecular modeling technique. Our key aim was to propose the first molecular model structure of unexplored serine protease 5 derived from big blue octopus. The other objective of this study was to analyze the distribution of negatively and positively charged amino acid over molecular modeled structure, distribution of secondary structural elements, hydrophobicity molecular surface analysis and electrostatic potential analysis with the aid of different bioinformatic tools. In the present study, molecular model has been generated with the help of I-TASSER suite. Afterwards the refined structural model was validated with standard methods. For functional annotation of protein molecule we used Protein Information Resource (PIR) database. Serine protease 5 of big blue octopus was analyzed with different bioinformatical algorithms for the distribution of negatively and positively charged amino acid over molecular modeled structure, distribution of secondary structural elements, hydrophobicity molecular surface analysis and electrostatic potential analysis. The functionally critical amino acids and ligand- binding site (LBS) of the proteins (modeled) were determined using the COACH program. The molecular model data in cooperation to other pertinent post model analysis data put forward molecular insight to proteolytic activity of serine protease 5, which helps in the clear understanding of procoagulant and anticoagulant characteristics of this natural lead molecule. Our approach was to investigate the octopus

  5. A PDZ-Like Motif in the Biliary Transporter ABCB4 Interacts with the Scaffold Protein EBP50 and Regulates ABCB4 Cell Surface Expression.

    Directory of Open Access Journals (Sweden)

    Quitterie Venot

    Full Text Available ABCB4/MDR3, a member of the ABC superfamily, is an ATP-dependent phosphatidylcholine translocator expressed at the canalicular membrane of hepatocytes. Defects in the ABCB4 gene are associated with rare biliary diseases. It is essential to understand the mechanisms of its canalicular membrane expression in particular for the development of new therapies. The stability of several ABC transporters is regulated through their binding to PDZ (PSD95/DglA/ZO-1 domain-containing proteins. ABCB4 protein ends by the sequence glutamine-asparagine-leucine (QNL, which shows some similarity to PDZ-binding motifs. The aim of our study was to assess the potential role of the QNL motif on the surface expression of ABCB4 and to determine if PDZ domain-containing proteins are involved. We found that truncation of the QNL motif decreased the stability of ABCB4 in HepG2-transfected cells. The deleted mutant ABCB4-ΔQNL also displayed accelerated endocytosis. EBP50, a PDZ protein highly expressed in the liver, strongly colocalized and coimmunoprecipitated with ABCB4, and this interaction required the QNL motif. Down-regulation of EBP50 by siRNA or by expression of an EBP50 dominant-negative mutant caused a significant decrease in the level of ABCB4 protein expression, and in the amount of ABCB4 localized at the canalicular membrane. Interaction of ABCB4 with EBP50 through its PDZ-like motif plays a critical role in the regulation of ABCB4 expression and stability at the canalicular plasma membrane.

  6. Inter-Synaptic Lateral Diffusion of GABAA Receptors Shapes Inhibitory Synaptic Currents.

    Science.gov (United States)

    de Luca, Emanuela; Ravasenga, Tiziana; Petrini, Enrica Maria; Polenghi, Alice; Nieus, Thierry; Guazzi, Stefania; Barberis, Andrea

    2017-07-05

    The lateral mobility of neurotransmitter receptors has been shown to tune synaptic signals. Here we report that GABAA receptors (GABAARs) can diffuse between adjacent dendritic GABAergic synapses in long-living desensitized states, thus laterally spreading "activation memories" between inhibitory synapses. Glutamatergic activity limits this inter-synaptic diffusion by trapping GABAARs at excitatory synapses. This novel form of activity-dependent hetero-synaptic interplay is likely to modulate dendritic synaptic signaling. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  7. The effect of sterilization methods on the physical properties of silk sericin scaffolds.

    Science.gov (United States)

    Siritientong, Tippawan; Srichana, Teerapol; Aramwit, Pornanong

    2011-06-01

    Protein-based biomaterials respond differently to sterilization methods. Since protein is a complex structure, heat, or irradiation may result in the loss of its physical or biological properties. Recent investigations have shown that sericin, a degumming silk protein, can be successfully formed into a 3-D scaffolds after mixing with other polymers which can be applied in skin tissue engineering. The objective of this study was to investigate the effectiveness of ethanol, ethylene oxide (EtO) and gamma irradiation on the sterilization of sericin scaffolds. The influence of these sterilization methods on the physical properties such as pore size, scaffold dimensions, swelling and mechanical properties, as well as the amount of sericin released from sericin/polyvinyl alcohol/glycerin scaffolds, were also investigated. Ethanol treatment was ineffective for sericin scaffold sterilization whereas gamma irradiation was the most effective technique for scaffold sterilization. Moreover, ethanol also caused significant changes in pore size resulting from shrinkage of the scaffold. Gamma-irradiated samples exhibited the highest swelling property, but they also lost the greatest amount of weight after immersion for 24 h compared with scaffolds obtained from other sterilization methods. The results of the maximum stress test and Young's modulus showed that gamma-irradiated and ethanol-treated scaffolds are more flexible than the EtO-treated and untreated scaffolds. The amount of sericin released, which was related to its collagen promoting effect, was highest from the gamma-irradiated scaffold. The results of this study indicate that gamma irradiation should have the greatest potential for sterilizing sericin scaffolds for skin tissue engineering.

  8. Frataxin Directly Stimulates Mitochondrial Cysteine Desulfurase by Exposing Substrate-binding Sites, and a Mutant Fe-S Cluster Scaffold Protein with Frataxin-bypassing Ability Acts Similarly*♦

    Science.gov (United States)

    Pandey, Alok; Gordon, Donna M.; Pain, Jayashree; Stemmler, Timothy L.; Dancis, Andrew; Pain, Debkumar

    2013-01-01

    For iron-sulfur (Fe-S) cluster synthesis in mitochondria, the sulfur is derived from the amino acid cysteine by the cysteine desulfurase activity of Nfs1. The enzyme binds the substrate cysteine in the pyridoxal phosphate-containing site, and a persulfide is formed on the active site cysteine in a manner depending on the accessory protein Isd11. The persulfide is then transferred to the scaffold Isu, where it combines with iron to form the Fe-S cluster intermediate. Frataxin is implicated in the process, although it is unclear where and how, and deficiency causes Friedreich ataxia. Using purified proteins and isolated mitochondria, we show here that the yeast frataxin homolog (Yfh1) directly and specifically stimulates cysteine binding to Nfs1 by exposing substrate-binding sites. This novel function of frataxin does not require iron, Isu1, or Isd11. Once bound to Nfs1, the substrate cysteine is the source of the Nfs1 persulfide, but this step is independent of frataxin and strictly dependent on Isd11. Recently, a point mutation in Isu1 was found to bypass many frataxin functions. The data presented here show that the Isu1 suppressor mimics the frataxin effects on Nfs1, explaining the bypassing activity. We propose a regulatory mechanism for the Nfs1 persulfide-forming activity. Specifically, at least two separate conformational changes must occur in the enzyme for optimum activity as follows: one is mediated by frataxin interaction that exposes the “buried” substrate-binding sites, and the other is mediated by Isd11 interaction that brings the bound substrate cysteine and the active site cysteine in proximity for persulfide formation. PMID:24217246

  9. Lateral regulation of synaptic transmission by astrocytes.

    Science.gov (United States)

    Covelo, A; Araque, A

    2016-05-26

    Fifteen years ago the concept of the "tripartite synapse" was proposed to conceptualize the functional view that astrocytes are integral elements of synapses. The signaling exchange between astrocytes and neurons within the tripartite synapse results in the synaptic regulation of synaptic transmission and plasticity through an autocrine form of communication. However, recent evidence indicates that the astrocyte synaptic regulation is not restricted to the active tripartite synapse but can be manifested through astrocyte signaling at synapses relatively distant from active synapses, a process termed lateral astrocyte synaptic regulation. This phenomenon resembles the classical heterosynaptic modulation but is mechanistically different because it involves astrocytes and its properties critically depend on the morphological and functional features of astrocytes. Therefore, the functional concept of the tripartite synapse as a fundamental unit must be expanded to include the interaction between tripartite synapses. Through lateral synaptic regulation, astrocytes serve as an active processing bridge for synaptic interaction and crosstalk between synapses with no direct neuronal connectivity, supporting the idea that neural network function results from the coordinated activity of astrocytes and neurons. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

  10. Changed Synaptic Plasticity in Neural Circuits of Depressive-Like and Escitalopram-Treated Rats

    Science.gov (United States)

    Li, Xiao-Li; Yuan, Yong-Gui; Xu, Hua; Wu, Di; Gong, Wei-Gang; Geng, Lei-Yu; Wu, Fang-Fang; Tang, Hao; Xu, Lin

    2015-01-01

    Background: Although progress has been made in the detection and characterization of neural plasticity in depression, it has not been fully understood in individual synaptic changes in the neural circuits under chronic stress and antidepressant treatment. Methods: Using electron microscopy and Western-blot analyses, the present study quantitatively examined the changes in the Gray’s Type I synaptic ultrastructures and the expression of synapse-associated proteins in the key brain regions of rats’ depressive-related neural circuit after chronic unpredicted mild stress and/or escitalopram administration. Meanwhile, their depressive behaviors were also determined by several tests. Results: The Type I synapses underwent considerable remodeling after chronic unpredicted mild stress, which resulted in the changed width of the synaptic cleft, length of the active zone, postsynaptic density thickness, and/or synaptic curvature in the subregions of medial prefrontal cortex and hippocampus, as well as the basolateral amygdaloid nucleus of the amygdala, accompanied by changed expression of several synapse-associated proteins. Chronic escitalopram administration significantly changed the above alternations in the chronic unpredicted mild stress rats but had little effect on normal controls. Also, there was a positive correlation between the locomotor activity and the maximal synaptic postsynaptic density thickness in the stratum radiatum of the Cornu Ammonis 1 region and a negative correlation between the sucrose preference and the length of the active zone in the basolateral amygdaloid nucleus region in chronic unpredicted mild stress rats. Conclusion: These findings strongly indicate that chronic stress and escitalopram can alter synaptic plasticity in the neural circuits, and the remodeled synaptic ultrastructure was correlated with the rats’ depressive behaviors, suggesting a therapeutic target for further exploration. PMID:25899067

  11. Changed Synaptic Plasticity in Neural Circuits of Depressive-Like and Escitalopram-Treated Rats.

    Science.gov (United States)

    Li, Xiao-Li; Yuan, Yong-Gui; Xu, Hua; Wu, Di; Gong, Wei-Gang; Geng, Lei-Yu; Wu, Fang-Fang; Tang, Hao; Xu, Lin; Zhang, Zhi-Jun

    2015-04-21

    Although progress has been made in the detection and characterization of neural plasticity in depression, it has not been fully understood in individual synaptic changes in the neural circuits under chronic stress and antidepressant treatment. Using electron microscopy and Western-blot analyses, the present study quantitatively examined the changes in the Gray's Type I synaptic ultrastructures and the expression of synapse-associated proteins in the key brain regions of rats' depressive-related neural circuit after chronic unpredicted mild stress and/or escitalopram administration. Meanwhile, their depressive behaviors were also determined by several tests. The Type I synapses underwent considerable remodeling after chronic unpredicted mild stress, which resulted in the changed width of the synaptic cleft, length of the active zone, postsynaptic density thickness, and/or synaptic curvature in the subregions of medial prefrontal cortex and hippocampus, as well as the basolateral amygdaloid nucleus of the amygdala, accompanied by changed expression of several synapse-associated proteins. Chronic escitalopram administration significantly changed the above alternations in the chronic unpredicted mild stress rats but had little effect on normal controls. Also, there was a positive correlation between the locomotor activity and the maximal synaptic postsynaptic density thickness in the stratum radiatum of the Cornu Ammonis 1 region and a negative correlation between the sucrose preference and the length of the active zone in the basolateral amygdaloid nucleus region in chronic unpredicted mild stress rats. These findings strongly indicate that chronic stress and escitalopram can alter synaptic plasticity in the neural circuits, and the remodeled synaptic ultrastructure was correlated with the rats' depressive behaviors, suggesting a therapeutic target for further exploration. © The Author 2015. Published by Oxford University Press on behalf of CINP.

  12. Nuclear Calcium Signaling Induces Expression of the Synaptic Organizers Lrrtm1 and Lrrtm2*

    Science.gov (United States)

    Hayer, Stefanie N.; Bading, Hilmar

    2015-01-01

    Calcium transients in the cell nucleus evoked by synaptic activity in hippocampal neurons function as a signaling end point in synapse-to-nucleus communication. As an important regulator of neuronal gene expression, nuclear calcium is involved in the conversion of synaptic stimuli into functional and structural changes of neurons. Here we identify two synaptic organizers, Lrrtm1 and Lrrtm2, as targets of nuclear calcium signaling. Expression of both Lrrtm1 and Lrrtm2 increased in a synaptic NMDA receptor- and nuclear calcium-dependent manner in hippocampal neurons within 2–4 h after the induction of action potential bursting. Induction of Lrrtm1 and Lrrtm2 occurred independently of the need for new protein synthesis and required calcium/calmodulin-dependent protein kinases and the nuclear calcium signaling target CREB-binding protein. Analysis of reporter gene constructs revealed a functional cAMP response element in the proximal promoter of Lrrtm2, indicating that at least Lrrtm2 is regulated by the classical nuclear Ca2+/calmodulin-dependent protein kinase IV-CREB/CREB-binding protein pathway. These results suggest that one mechanism by which nuclear calcium signaling controls neuronal network function is by regulating the expression of Lrrtm1 and Lrrtm2. PMID:25527504

  13. Nuclear calcium signaling induces expression of the synaptic organizers Lrrtm1 and Lrrtm2.

    Science.gov (United States)

    Hayer, Stefanie N; Bading, Hilmar

    2015-02-27

    Calcium transients in the cell nucleus evoked by synaptic activity in hippocampal neurons function as a signaling end point in synapse-to-nucleus communication. As an important regulator of neuronal gene expression, nuclear calcium is involved in the conversion of synaptic stimuli into functional and structural changes of neurons. Here we identify two synaptic organizers, Lrrtm1 and Lrrtm2, as targets of nuclear calcium signaling. Expression of both Lrrtm1 and Lrrtm2 increased in a synaptic NMDA receptor- and nuclear calcium-dependent manner in hippocampal neurons within 2-4 h after the induction of action potential bursting. Induction of Lrrtm1 and Lrrtm2 occurred independently of the need for new protein synthesis and required calcium/calmodulin-dependent protein kinases and the nuclear calcium signaling target CREB-binding protein. Analysis of reporter gene constructs revealed a functional cAMP response element in the proximal promoter of Lrrtm2, indicating that at least Lrrtm2 is regulated by the classical nuclear Ca(2+)/calmodulin-dependent protein kinase IV-CREB/CREB-binding protein pathway. These results suggest that one mechanism by which nuclear calcium signaling controls neuronal network function is by regulating the expression of Lrrtm1 and Lrrtm2. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Repetitive transcranial magnetic stimulation (rTMS) influences spatial cognition and modulates hippocampal structural synaptic plasticity in aging mice.

    Science.gov (United States)

    Ma, Jun; Zhang, Zhanchi; Kang, Lin; Geng, Dandan; Wang, Yanyong; Wang, Mingwei; Cui, Huixian

    2014-10-01

    Normal aging is characteristic with the gradual decline in cognitive function associated with the progressive reduction of structural and functional plasticity in the hippocampus. Repetitive transcranial magnetic stimulation (rTMS) has developed into a novel neurological and psychiatric tool that can be used to investigate the neurobiology of cognitive function. Recent studies have demonstrated that low-frequency rTMS (≤1Hz) affects synaptic plasticity in rats with vascular dementia (VaD), and it ameliorates the spatial cognitive ability in mice with Aβ1-42-mediated memory deficits, but there are little concerns about the effects of rTMS on normal aging related cognition and synaptic plasticity changes. Thus, the current study investigated the effects of rTMS on spatial memory behavior, neuron and synapse morphology in the hippocampus, and synaptic protein markers and brain-derived neurotrophic factor (BDNF)/tropomyosin-related kinase B (TrkB) in normal aging mice, to illustrate the mechanisms of rTMS in regulating cognitive capacity. Relative to adult animals, aging caused hippocampal-dependent cognitive impairment, simultaneously inhibited the activation of the BDNF-TrkB signaling pathway, reduced the transcription and expression of synaptic protein markers: synaptophysin (SYN), growth associated protein 43 (GAP43) and post-synaptic density protein 95 (PSD95), as well as decreased synapse density and PSD (post-synaptic density) thickness. Interestingly, rTMS with low intensity (110% average resting motor threshold intensity, 1Hz, LIMS) triggered the activation of BDNF and TrkB, upregulated the level of synaptic protein markers, and increased synapse density and thickened PSD, and further reversed the spatial cognition dysfunction in aging mice. Conversely, high-intensity magnetic stimulation (150% average resting motor threshold intensity, 1Hz, HIMS) appeared to be detrimental, inducing thinning of PSDs, disordered synaptic structure, and a large number of

  15. SHOP: receptor-based scaffold hopping by GRID-based similarity searches

    DEFF Research Database (Denmark)

    Bergmann, Rikke; Liljefors, Tommy; Sørensen, Morten D

    2009-01-01

    A new field-derived 3D method for receptor-based scaffold hopping, implemented in the software SHOP, is presented. Information from a protein-ligand complex is utilized to substitute a fragment of the ligand with another fragment from a database of synthetically accessible scaffolds. A GRID...

  16. Dysregulation of Elongation Factor 1A Expression is Correlated with Synaptic Plasticity Impairments in Alzheimer's Disease.

    Science.gov (United States)

    Beckelman, Brenna C; Day, Stephen; Zhou, Xueyan; Donohue, Maggie; Gouras, Gunnar K; Klann, Eric; Keene, C Dirk; Ma, Tao

    2016-09-06

    Synaptic dysfunction may represent an early and crucial pathophysiology in Alzheimer's disease (AD). Recent studies implicate a connection between synaptic plasticity deficits and compromised capacity of de novo protein synthesis in AD. The mRNA translational factor eukaryotic elongation factor 1A (eEF1A) is critically involved in several forms of long-lasting synaptic plasticity. By examining postmortem human brain samples, a transgenic mouse model, and application of synthetic human Aβ42 on mouse hippocampal slices, we demonstrated that eEF1A protein levels were significantly decreased in AD, particularly in the hippocampus. In contrast, brain levels of eukaryotic elongation factor 2 were unaltered in AD. Further, upregulation of eEF1A expression by the adenylyl cyclase activator forskolin, which induces long-lasting synaptic plasticity, was blunted in hippocampal slices derived from Tg2576 AD model mice. Finally, Aβ-induced hippocampal long-term potentiation defects were alleviated by upregulation of eEF1A signaling via brain-specific knockdown of the gene encoding tuberous sclerosis 2. In summary, our findings suggest a strong correlation between the dysregulation of eEF1A synthesis and AD-associated synaptic failure. These findings provide insights into the understanding of molecular mechanisms underlying AD etiology and may aid in identification of novel biomarkers and therapeutic targets.

  17. Nitric oxide signaling is recruited as a compensatory mechanism for sustaining synaptic plasticity in Alzheimer's disease mice.

    Science.gov (United States)

    Chakroborty, Shreaya; Kim, Joyce; Schneider, Corinne; West, Anthony R; Stutzmann, Grace E

    2015-04-29

    Synaptic plasticity deficits are increasingly recognized as causing the memory impairments which define Alzheimer's disease (AD). In AD mouse models, evidence of abnormal synaptic function is present before the onset of cognitive deficits, and presents as increased synaptic depression revealed only when synaptic homeostasis is challenged, such as with suppression of ryanodine receptor (RyR)-evoked calcium signaling. Otherwise, at early disease stages, the synaptic physiology phenotype appears normal. This suggests compensatory mechanisms are recruited to maintain a functionally normal net output of the hippocampal circuit. A candidate calcium-regulated synaptic modulator is nitric oxide (NO), which acts presynaptically to boost vesicle release and glutamatergic transmission. Here we tested whether there is a feedforward cycle between the increased RyR calcium release seen in presymptomatic AD mice and aberrant NO signaling which augments synaptic plasticity. Using a combination of electrophysiological approaches, two-photon calcium imaging, and protein biochemistry in hippocampal tissue from presymptomatic 3xTg-AD and NonTg mice, we show that blocking NO synthesis results in markedly augmented synaptic depression mediated through presynaptic mechanisms in 3xTg-AD mice. Additionally, blocking NO reduces the augmented synaptically evoked dendritic calcium release mediated by enhanced RyR calcium release. This is accompanied by increased nNOS levels in the AD mice and is reversed upon normalization of RyR-evoked calcium release with chronic dantrolene treatment. Thus, recruitment of NO is serving a compensatory role to boost synaptic transmission and plasticity during early AD stages. However, NO's dual role in neuroprotection and neurodegeneration may convert to maladaptive functions as the disease progresses. Copyright © 2015 the authors 0270-6474/15/356893-10$15.00/0.

  18. Biomimetic apatite-coated porous PVA scaffolds promote the growth of breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Ye, Mao; Mohanty, Pravansu; Ghosh, Gargi, E-mail: gargi@umich.edu

    2014-11-01

    Recapitulating the native environment of bone tissue is essential to develop in vitro models of breast cancer bone metastasis. The bone is a composite material consisting of organic matrix and inorganic mineral phase, primarily hydroxyapatite. In this study, we report the mineralization of porous poly vinyl alcohol (PVA) scaffolds upon incubation in modified Hanks' Balanced Salt Solution (HBSS) for 14 days. Scanning electron microscopy, energy dispersive X-ray analysis, and X-ray diffraction analysis revealed that the deposited minerals have composition similar to hydroxyapatite. The study demonstrated that the rate of nucleation and growth of minerals was faster on surfaces of less porous scaffolds. However, upon prolonged incubation, formation of mineral layer was observed on the surface of all the scaffolds. In addition, the study also demonstrated that 3D mineralization only occurred for scaffolds with highly interconnected porous networks. The mineralization of the scaffolds promoted the adsorption of serum proteins and consequently, the adhesion and proliferation of breast cancer cells. - Highlights: • Porous PVA scaffolds fabricated via mechanical agitation followed by freeze-drying. • Mineralization of the scaffold was carried out by utilizing biomimetic approach. • Mineralization resulted in increased protein adsorption on the scaffold. • Increased breast cancer cell growth was observed on mineralized scaffolds.

  19. Fabrication and biocompatibility of poly(L-lactic acid) and chitosan composite scaffolds with hierarchical microstructures

    Energy Technology Data Exchange (ETDEWEB)

    Lou, Tao, E-mail: taolou72@aliyun.com [College of Chemistry and Chemical Engineering, Qingdao University, Qingdao 266071 (China); Wang, Xuejun [College of Chemistry and Chemical Engineering, Qingdao University, Qingdao 266071 (China); Yan, Xu [College of Physics & Collaborative Innovation Center for Low-Dimensional Nanomaterials and Optoelectronic Devices, Qingdao University, Qingdao 266071 (China); Miao, Yu [Department of Mechanical Engineering, Columbia University, New York, NY 10027 (United States); Long, Yun-Ze, E-mail: yunzelong@163.com [College of Physics & Collaborative Innovation Center for Low-Dimensional Nanomaterials and Optoelectronic Devices, Qingdao University, Qingdao 266071 (China); Yin, Hai-Lei [Department of Osteology, No. 401 Hospital of P. L. A., Qingdao 266071 (China); Sun, Bin [College of Physics & Collaborative Innovation Center for Low-Dimensional Nanomaterials and Optoelectronic Devices, Qingdao University, Qingdao 266071 (China); Song, Guojun [College of Chemistry and Chemical Engineering, Qingdao University, Qingdao 266071 (China)

    2016-07-01

    The scaffold microstructure is crucial to reconstruct tissue normal functions. In this article, poly(L-lactic acid) and chitosan fiber (PLLA/CTSF) composite scaffolds with hierarchical microstructures both in fiber and pore sizes were successfully fabricated by combining thermal induced phase separation and salt leaching techniques. The composite scaffolds consisted of a nanofibrous PLLA matrix with diameter of 50–500 nm, and chitosan fibers with diameter of about 20 μm were homogenously distributed in the PLLA matrix as a microsized reinforcer. The composite scaffolds also had high porosity (> 94%) and hierarchical pore size, which were consisted of both micropores (50 nm–10 μm) and macropores (50–300 μm). By tailoring the microstructure and chemical composition, the mechanical property, pH buffer and protein adsorption capacity of the composite scaffold were improved significantly compared with those of PLLA scaffold. Cell culture results also revealed that the PLLA/CTSF composite scaffolds supported MG-63 osteoblast proliferation and penetration. - Highlights: • Composite scaffolds fabricated by combining thermal induced phase separation and salt leaching techniques • Hierarchical microstructure both in fiber and pore sizes • The scaffold microenvironment facilitates the protein adsorption, cell proliferation and penetration.

  20. The Role of Histone Deacetylase 6 in Synaptic Plasticity and Memory

    Directory of Open Access Journals (Sweden)

    Sarah Perry

    2017-02-01

    Full Text Available Histone deacetylases (HDACs have been extensively studied as drug targets in neurodegenerative diseases, but less is known about their role in healthy neurons. We tested zinc-dependent HDACs using RNAi in Drosophila melanogaster and found memory deficits with RPD3 and HDAC6. We demonstrate that HDAC6 is required in both the larval and adult stages for normal olfactory memory retention. Neuronal expression of HDAC6 rescued memory deficits, and we demonstrate that the N-terminal deacetylase (DAC domain is required for this ability. This suggests that deacetylation of synaptic targets associated with the first DAC domain, such as the active-zone scaffold Bruchpilot, is required for memory retention. Finally, electrophysiological experiments at the neuromuscular junction reveal that HDAC6 mutants exhibit a partial block of homeostatic plasticity, suggesting that HDAC6 may be required for the stabilization of synaptic strength. The learning deficit we observe in HDAC6 mutants could be a behavioral consequence of these synaptic defects.

  1. CDK and MAPK Synergistically Regulate Signaling Dynamics via a Shared Multi-site Phosphorylation Region on the Scaffold Protein Ste5.

    Science.gov (United States)

    Repetto, María Victoria; Winters, Matthew J; Bush, Alan; Reiter, Wolfgang; Hollenstein, David Maria; Ammerer, Gustav; Pryciak, Peter M; Colman-Lerner, Alejandro

    2018-03-15

    We report an unanticipated system of joint regulation by cyclin-dependent kinase (CDK) and mitogen-activated protein kinase (MAPK), involving collaborative multi-site phosphorylation of a single substrate. In budding yeast, the protein Ste5 controls signaling through a G1 arrest pathway. Upon cell-cycle entry, CDK inhibits Ste5 via multiple phosphorylation sites, disrupting its membrane association. Using quantitative time-lapse microscopy, we examined Ste5 membrane recruitment dynamics at different cell-cycle stages. Surprisingly, in S phase, where Ste5 recruitment should be blocked, we observed an initial recruitment followed by a steep drop-off. This delayed inhibition revealed a requirement for both CDK activity and negative feedback from the pathway MAPK Fus3. Mutagenesis, mass spectrometry, and electrophoretic analyses suggest that the CDK and MAPK modify shared sites, which are most extensively phosphorylated when both kinases are active and able to bind their docking sites on Ste5. Such collaborative phosphorylation can broaden regulatory inputs and diversify output dynamics of signaling pathways. Copyright © 2018 Elsevier Inc. All rights reserved.

  2. Selective laser sintering fabrication of nano-hydroxyapatite/poly-ε-caprolactone scaffolds for bone tissue engineering applications

    Science.gov (United States)

    Xia, Yan; Zhou, Panyu; Cheng, Xiaosong; Xie, Yang; Liang, Chong; Li, Chao; Xu, Shuogui

    2013-01-01

    The regeneration of functional tissue in osseous defects is a formidable challenge in orthopedic surgery. In the present study, a novel biomimetic composite scaffold, here called nano-hydroxyapatite (HA)/poly-ε-caprolactone (PCL) was fabricated using a selective laser sintering technique. The macrostructure, morphology, and mechanical strength of the scaffolds were characterized. Scanning electronic microscopy (SEM) showed that the nano-HA/PCL scaffolds exhibited predesigned, well-ordered macropores and interconnected micropores. The scaffolds have a range of porosity from 78.54% to 70.31%, and a corresponding compressive strength of 1.38 MPa to 3.17 MPa. Human bone marrow stromal cells were seeded onto the nano-HA/PCL or PCL scaffolds and cultured for 28 days in vitro. As indicated by the level of cell attachment and proliferation, the nano-HA/PCL showed excellent biocompatibility, comparable to that of PCL scaffolds. The hydrophilicity, mineralization, alkaline phosphatase activity, and Alizarin Red S staining indicated that the nano-HA/PCL scaffolds are more bioactive than the PCL scaffolds in vitro. Measurements of recombinant human bone morphogenetic protein-2 (rhBMP-2) release kinetics showed that after nano-HA was added, the material increased the rate of rhBMP-2 release. To investigate the in vivo biocompatibility and osteogenesis of the composite scaffolds, both nano-HA/PCL scaffolds and PCL scaffolds were implanted in rabbit femur defects for 3, 6, and 9 weeks. The wounds were studied radiographically and histologically. The in vivo results showed that both nano-HA/PCL composite scaffolds and PCL scaffolds exhibited good biocompatibility. However, the nano-HA/PCL scaffolds enhanced the efficiency of new bone formation more than PCL scaffolds and fulfilled all the basic requirements of bone tissue engineering scaffolds. Thus, they show large potential for use in orthopedic and reconstructive surgery. PMID:24204147

  3. Microporous dermal-mimetic electrospun scaffolds pre-seeded with fibroblasts promote tissue regeneration in full-thickness skin wounds.

    Directory of Open Access Journals (Sweden)

    Paul P Bonvallet

    Full Text Available Electrospun scaffolds serve as promising substrates for tissue repair due to their nanofibrous architecture and amenability to tailoring of chemical composition. In this study, the regenerative potential of a microporous electrospun scaffold pre-seeded with dermal fibroblasts was evaluated. Previously we reported that a 70% collagen I and 30% poly(Ɛ-caprolactone electrospun scaffold (70:30 col/PCL containing 160 μm diameter pores had favorable mechanical properties, supported fibroblast infiltration and subsequent cell-mediated deposition of extracellular matrix (ECM, and promoted more rapid and effective in vivo skin regeneration when compared to scaffolds lacking micropores. In the current study we tested the hypothesis that the efficacy of the 70:30 col/PCL microporous scaffolds could be further enhanced by seeding scaffolds with dermal fibroblasts prior to implantation into skin wounds. To address this hypothesis, a Fischer 344 (F344 rat syngeneic model was employed. In vitro studies showed that dermal fibroblasts isolated from F344 rat skin were able to adhere and proliferate on 70:30 col/PCL microporous scaffolds, and the cells also filled the 160 μm pores with native ECM proteins such as collagen I and fibronectin. Additionally, scaffolds seeded with F344 fibroblasts exhibited a low rate of contraction (~14% over a 21 day time frame. To assess regenerative potential, scaffolds with or without seeded F344 dermal fibroblasts were implanted into full thickness, critical size defects created in F344 hosts. Specifically, we compared: microporous scaffolds containing fibroblasts seeded for 4 days; scaffolds containing fibroblasts seeded for only 1 day; acellular microporous scaffolds; and a sham wound (no scaffold. Scaffolds containing fibroblasts seeded for 4 days had the best response of all treatment groups with respect to accelerated wound healing, a more normal-appearing dermal matrix structure, and hair follicle regeneration

  4. The Role of Co-chaperones in Synaptic Proteostasis and Neurodegenerative Disease

    Directory of Open Access Journals (Sweden)

    Erica L. Gorenberg

    2017-05-01

    Full Text Available Synapses must be preserved throughout an organism's lifespan to allow for normal brain function and behavior. Synapse maintenance is challenging given the long distances between the termini and the cell body, reliance on axonal transport for delivery of newly synthesized presynaptic proteins, and high rates of synaptic vesicle exo- and endocytosis. Hence, synapses rely on efficient proteostasis mechanisms to preserve their structure and function. To this end, the synaptic compartment has specific chaperones to support its functions. Without proper synaptic chaperone activity, local proteostasis imbalances lead to neurotransmission deficits, dismantling of synapses, and neurodegeneration. In this review, we address the roles of four synaptic chaperones in the maintenance of the nerve terminal, as well as their genetic links to neurodegenerative disease. Three of these are Hsp40 co-chaperones (DNAJs: Cysteine String Protein alpha (CSPα; DNAJC5, auxilin (DNAJC6, and Receptor-Mediated Endocytosis 8 (RME-8; DNAJC13. These co-chaperones contain a conserved J domain through which they form a complex with heat shock cognate 70 (Hsc70, enhancing the chaperone's ATPase activity. CSPα is a synaptic vesicle protein known to chaperone the t-SNARE SNAP-25 and the endocytic GTPase dynamin-1, thereby regulating synaptic vesicle exocytosis and endocytosis. Auxilin binds assembled clathrin cages, and through its interactions with Hsc70 leads to the uncoating of clathrin-coated vesicles, a process necessary for the regeneration of synaptic vesicles. RME-8 is a co-chaperone on endosomes and may have a role in clathrin-coated vesicle endocytosis on this organelle. These three co-chaperones maintain client function by preserving folding and assembly to prevent client aggregation, but they do not break down aggregates that have already formed. The fourth synaptic chaperone we will discuss is Heat shock protein 110 (Hsp110, which interacts with Hsc70, DNAJAs, and

  5. Effects of designed PLLA and 50:50 PLGA scaffold architectures on bone formation in vivo.

    Science.gov (United States)

    Saito, Eiji; Liao, Elly E; Hu, Wei-Wen; Krebsbach, Paul H; Hollister, Scott J

    2013-02-01

    Biodegradable porous scaffolds have been investigated as an alternative approach to current metal, ceramic, and polymer bone graft substitutes for lost or damaged bone tissues. Although there have been many studies investigating the effects of scaffold architecture on bone formation, many of these scaffolds were fabricated using conventional methods such as salt leaching and phase separation, and were constructed without designed architecture. To study the effects of both designed architecture and material on bone formation, this study designed and fabricated three types of porous scaffold architecture from two biodegradable materials, poly (L-lactic acid) (PLLA) and 50:50 Poly(lactic-co-glycolic acid) (PLGA), using image based design and indirect solid freeform fabrication techniques, seeded them with bone morphogenetic protein-7 transduced human gingival fibroblasts, and implanted them subcutaneously into mice for 4 and 8 weeks. Micro-computed tomography data confirmed that the fabricated porous scaffolds replicated the designed architectures. Histological analysis revealed that the 50:50 PLGA scaffolds degraded but did not maintain their architecture after 4 weeks implantation. However, PLLA scaffolds maintained their architecture at both time points and showed improved bone ingrowth, which followed the internal architecture of the scaffolds. Mechanical properties of both PLLA and 50:50 PLGA scaffolds decreased but PLLA scaffolds maintained greater mechanical properties than 50:50 PLGA after implantation. The increase of mineralized tissue helped support the mechanical properties of bone tissue and scaffold constructs between 4-8 weeks. The results indicate the importance of choice of scaffold materials and computationally designed scaffolds to control tissue formation and mechanical properties for desired bone tissue regeneration. Copyright © 2011 John Wiley & Sons, Ltd.

  6. Boron containing poly-(lactide-co-glycolide) (PLGA) scaffolds for bone tissue engineering

    Energy Technology Data Exchange (ETDEWEB)

    Doğan, Ayşegül; Demirci, Selami [Department of Genetics and Bioengineering, Faculty of Engineering and Architecture, Yeditepe University 34755 Istanbul (Turkey); Bayir, Yasin [Department of Biochemistry, Faculty of Pharmacy, Ataturk University, 25240, Erzurum (Turkey); Halici, Zekai [Department of Pharmacology, Faculty of Medicine, Ataturk University, 25240, Erzurum (Turkey); Karakus, Emre [Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Ataturk University, 25240, Erzurum (Turkey); Aydin, Ali [Department of Orthopedics and Traumatology, Faculty of Medicine, Ataturk University, 25240, Erzurum (Turkey); Cadirci, Elif [Department of Pharmacology, Faculty of Pharmacy, Ataturk University, 25240, Erzurum (Turkey); Albayrak, Abdulmecit [Department of Pharmacology, Faculty of Medicine, Ataturk University, 25240, Erzurum (Turkey); Demirci, Elif [Department of Pathology, Faculty of Medicine, Ataturk University, 25240, Erzurum (Turkey); Karaman, Adem [Department of Radiology, Faculty of Medicine, Ataturk University, 25240, Erzurum (Turkey); Ayan, Arif Kursat [Department of Nuclear Medicine, Faculty of Medicine, Ataturk University, 25240, Erzurum (Turkey); Gundogdu, Cemal [Department of Pathology, Faculty of Medicine, Ataturk University, 25240, Erzurum (Turkey); Şahin, Fikrettin, E-mail: fsahin@yeditepe.edu.tr [Department of Genetics and Bioengineering, Faculty of Engineering and Architecture, Yeditepe University 34755 Istanbul (Turkey)

    2014-11-01

    Scaffold-based bone defect reconstructions still face many challenges due to their inadequate osteoinductive and osteoconductive properties. Various biocompatible and biodegradable scaffolds, combined with proper cell type and biochemical signal molecules, have attracted significant interest in hard tissue engineering approaches. In the present study, we have evaluated the effects of boron incorporation into poly-(lactide-co-glycolide-acid) (PLGA) scaffolds, with or without rat adipose-derived stem cells (rADSCs), on bone healing in vitro and in vivo. The results revealed that boron containing scaffolds increased in vitro proliferation, attachment and calcium mineralization of rADSCs. In addition, boron containing scaffold application resulted in increased bone regeneration by enhancing osteocalcin, VEGF and collagen type I protein levels in a femur defect model. Bone mineralization density (BMD) and computed tomography (CT) analysis proved that boron incorporated scaffold administration increased the healing rate of bone defects. Transplanting stem cells into boron containing scaffolds was found to further improve bone-related outcomes compared to control groups. Additional studies are highly warranted for the investigation of the mechanical properties of these scaffolds in order to address their potential use in clinics. The study proposes that boron serves as a promising innovative approach in manufacturing scaffold systems for functional bone tissue engineering. - Highlights: • Boron containing PLGA scaffolds were developed for bone tissue engineering. • Boron incorporation increased cell viability and mineralization of stem cells. • Boron containing scaffolds increased bone-related protein expression in vivo. • Implantation of stem cells on boron containing scaffolds improved bone healing.

  7. Ca2+-permeable AMPA receptors in homeostatic synaptic plasticity

    Directory of Open Access Journals (Sweden)

    Hey-Kyoung eLee

    2012-02-01

    Full Text Available Neurons possess diverse mechanisms of homeostatic adaptation to overall changes in neural and synaptic activity, which are critical for proper brain functions. Homeostatic regulation of excitatory synapses has been studied in the context of synaptic scaling, which allows neurons to adjust their excitatory synaptic gain to maintain their activity within a dynamic range. Recent evidence suggests that one of the main mechanisms underlying synaptic scaling is by altering the function of postsynaptic AMPA receptors (AMPARs, including synaptic expression of Ca2+-permeable (CP- AMPARs. CP-AMPARs endow synapses with unique properties, which may benefit adaptation of neurons to periods of inactivity as would occur when a major input is lost. This review will summarize how synaptic expression of CP-AMPARs is regulated during homeostatic synaptic plasticity in the context of synaptic scaling, and will address the potential functional consequences of altering synaptic CP-AMPAR content.

  8. SRC Inhibition Reduces NR2B Surface Expression and Synaptic Plasticity in the Amygdala

    Science.gov (United States)

    Sinai, Laleh; Duffy, Steven; Roder, John C.

    2010-01-01

    The Src protein tyrosine kinase plays a central role in the regulation of N-methyl-d-aspartate receptor (NMDAR) activity by regulating NMDAR subunit 2B (NR2B) surface expression. In the amygdala, NMDA-dependent synaptic plasticity resulting from convergent somatosensory and auditory inputs contributes to emotional memory; however, the role of Src…

  9. Synaptic Plasticity: Cleaved Kinases and the Specificity of Erasing Traumatic Memories.

    Science.gov (United States)

    Abrams, Thomas W

    2017-09-25

    New possibilities for treating posttraumatic stress disorder and anxiety disorders involving abnormal memories are emerging from analysis of persistent protein kinase activation and mechanisms of synapse-specific modification, known as synaptic tagging. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Hsp90 chaperone inhibitor 17-AAG attenuates Aβ-induced synaptic toxicity and memory impairment.

    Science.gov (United States)

    Chen, Yaomin; Wang, Bin; Liu, Dan; Li, Jing Jing; Xue, Yueqiang; Sakata, Kazuko; Zhu, Ling-qiang; Heldt, Scott A; Xu, Huaxi; Liao, Francesca-Fang

    2014-02-12

    The excessive accumulation of soluble amyloid peptides (Aβ) plays a crucial role in the pathogenesis of Alzheimer's disease (AD), particularly in synaptic dysfunction. The role of the two major chaperone proteins, Hsp70 and Hsp90, in clearing misfolded protein aggregates has been established. Despite their abundant presence in synapses, the role of these chaperones in synapses remains elusive. Here, we report that Hsp90 inhibition by 17-AAG elicited not only a heat shock-like response but also upregulated presynaptic and postsynaptic proteins, such as synapsin I, synaptophysin, and PSD95 in neurons. 17-AAG treatment enhanced high-frequency stimulation-evoked LTP and protected neurons from synaptic damage induced by soluble Aβ. In AD transgenic mice, the daily administration of 17-AAG over 7 d resulted in a marked increase in PSD95 expression in hippocampi. 17-AAG treatments in wild-type C57BL/6 mice challenged by soluble Aβ significantly improved contextual fear memory. Further, we demonstrate that 17-AAG activated synaptic protein expression via transcriptional mechanisms through the heat shock transcription factor HSF1. Together, our findings identify a novel function of Hsp90 inhibition in regulating synaptic plasticity, in addition to the known neuroprotective effects of the chaperones against Aβ and tau toxicity, thus further supporting the potential of Hsp90 inhibitors in treating neurodegenerative diseases.

  11. The N-ethylmaleimide-sensitive factor and dysbindin interact to modulate synaptic plasticity.

    Science.gov (United States)

    Gokhale, Avanti; Mullin, Ariana P; Zlatic, Stephanie A; Easley, Charles A; Merritt, Megan E; Raj, Nisha; Larimore, Jennifer; Gordon, David E; Peden, Andrew A; Sanyal, Subhabrata; Faundez, Victor

    2015-05-13

    Dysbindin is a schizophrenia susceptibility factor and subunit of the biogenesis of lysosome-related organelles complex 1 (BLOC-1) required for lysosome-related organelle biogenesis, and in neurons, synaptic vesicle assembly, neurotransmission, and plasticity. Protein networks, or interactomes, downstream of dysbindin/BLOC-1 remain partially explored despite their potential to illuminate neurodevelopmental disorder mechanisms. Here, we conducted a proteome-wide search for polypeptides whose cellular content is sensitive to dysbindin/BLOC-1 loss of function. We identified components of the vesicle fusion machinery as factors downregulated in dysbindin/BLOC-1 deficiency in neuroectodermal cells and iPSC-derived human neurons, among them the N-ethylmaleimide-sensitive factor (NSF). Human dysbindin/BLOC-1 coprecipitates with NSF and vice versa, and both proteins colocalized in a Drosophila model synapse. To test the hypothesis that NSF and dysbindin/BLOC-1 participate in a pathway-regulating synaptic function, we examined the role for NSF in dysbindin/BLOC-1-dependent synaptic homeostatic plasticity in Drosophila. As previously described, we found that mutations in dysbindin precluded homeostatic synaptic plasticity elicited by acute blockage of postsynaptic receptors. This dysbindin mutant phenotype is fully rescued by presynaptic expression of either dysbindin or Drosophila NSF. However, neither reduction of NSF alone or in combination with dysbindin haploinsufficiency impaired homeostatic synaptic plasticity. Our results demonstrate that dysbindin/BLOC-1 expression defects result in altered cellular content of proteins of the vesicle fusion apparatus and therefore influence synaptic plasticity. Copyright © 2015 the authors 0270-6474/15/357643-11$15.00/0.

  12. scaffolds for tissue engineering application

    Indian Academy of Sciences (India)

    2017-07-27

    Jul 27, 2017 ... Moreover, the peculiar physiochemical and structural properties of the CH–CMP scaffold has proved better tensile strength and excellent .... C at a heating rate of 10. ◦. C min. −1 under nitrogen atmosphere using 3mg of the sample [21]. 2.3c SEM and particle size: CMP, CH–CMP and PVA–. CMP scaffolds ...

  13. Scaffold: a novel carrier for cell and drug delivery.

    Science.gov (United States)

    Garg, Tarun; Singh, Onkar; Arora, Saahil; Murthy, R

    2012-01-01

    Scaffolds are implants or injects, which are used to deliver cells, drugs, and genes into the body. Different forms of polymeric scaffolds for cell/drug delivery are available: (1) a typical three-dimensional porous matrix, (2) a nanofibrous matrix, (3) a thermosensitive sol-gel transition hydrogel, and (4) a porous microsphere. A scaffold provides a suitable substrate for cell attachment, cell proliferation, differentiated function, and cell migration. Scaffold matrices can be used to achieve drug delivery with high loading and efficiency to specific sites. Biomaterials used for fabrication of scaffold may be natural polymers such as alginate, proteins, collagens, gelatin, fibrins, and albumin, or synthetic polymers such as polyvinyl alcohol and polyglycolide. Bioceramics such as hydroxyapatites and tricalcium phosphates also are used. Techniques used for fabrication of a scaffold include particulate leaching, freeze-drying, supercritical fluid technology, thermally induced phase separation, rapid prototyping, powder compaction, sol-gel, and melt moulding. These techniques allow the preparation of porous structures with regular porosity. Scaffold are used successfully in various fields of tissue engineering such as bone formation, periodontal regeneration, repair of nasal and auricular malformations, cartilage development, as artificial corneas, as heart valves, in tendon repair ,in ligament replacement, and in tumors. They also are used in joint pain inflammation, diabetes, heart disease, osteochondrogenesis, and wound dressings. Their application of late has extended to delivery of drugs and genetic materials, including plasmid DNA, at a controlled rate over a long period of time. In addition, the incorporation of drugs (i.e., inflammatory inhibitors and/or antibiotics) into scaffolds may be used to prevent infection after surgery and other disease for longer duration. Scaffold also can be used to provide adequate signals (e.g., through the use of adhesion

  14. Multifunctional Thin Film Biomatrice Biosensor in a Degradable Scaffold Containing Bone Morphogenetic Protein-2 (BMP-2) for Controlled Release in Skeletal Tissue Engineering

    Science.gov (United States)

    McDaniel, Harvey; Lomax, Linda

    2001-03-01

    Bone morphonogenetic proteins (BMP-2) have been under investigation for three decades. Deminerialized bone and extracts of deminerialized bone are o steoinductive with a temporal sequence of bone induction. Native and recombi nant BMP's have shown the ability, thru growth and differentiative factors t o induce de novo bone formation both invitro and invivo. Their principle fun ction is to induce transformation of undifferentiated mesenchymal cells into osteoblasts. Native and recombinant BMP's, when purified and used without carrier disp erse after implantation and exert no effect on bone induction. The delivery system provides the missing component to successsfully applying osteogenic p roteins for clinical need. Biological and physio-chemical properties are str ictly adhered tofor a successful delivery system. The BMP delivery system ca rrier for osteo inductive payload provided; 1)non tumorgenic genecity, 2) no n immunogenecity, 3) water insoluble, 4) biosorbability with predictable enz ymatic degradation, and 5) an optimized surface for compatibility, cell migr ation and attachment with a negative surface change that encouraged target c ell attachment. Being a controlled Release System, it binded the proteins wi th predictible BMP released kinetics. Porosity with interconnecting voids pr otected the BMP from noon specific proteolysis and promoted rapid vascular a nd mesenchymal invasion. Far wide ranging clinical applications of mechanica l and biofunctional requirements were met with the BMP delivery system. Cohe sion and malleability were reqiured forcontour augmentation, and reconstruct ion of the discontinuity defects, prevented dislocation and retained the sha pe and bone replaced the system. Biological systems have elastic activity associated with them. The activi ty was current associated with a time dependant biological/biochemical react ion (enzymic activity). Bioelectric phoenomena associated with charged molec ules in a biologic structure caused

  15. Oxidative Stress, Synaptic Dysfunction, and Alzheimer's Disease.

    Science.gov (United States)

    Tönnies, Eric; Trushina, Eugenia

    2017-01-01

    Alzheimer's disease (AD) is a devastating neurodegenerative disorder without a cure. Most AD cases are sporadic where age represents the greatest risk factor. Lack of understanding of the disease mechanism hinders the development of efficacious therapeutic approaches. The loss of synapses in the affected brain regions correlates best with cognitive impairment in AD patients and has been considered as the early mechanism that precedes neuronal loss. Oxidative stress has been recognized as a contributing factor in aging and in the progression of multiple neurodegenerative diseases including AD. Increased production of reactive oxygen species (ROS) associated with age- and disease-dependent loss of mitochondrial function, altered metal homeostasis, and reduced antioxidant defense directly affect synaptic activity and neurotransmission in neurons leading to cognitive dysfunction. In addition, molecular targets affected by ROS include nuclear and mitochondrial DNA, lipids, proteins, calcium homeostasis, mitochondrial dynamics and function, cellular architecture, receptor trafficking and endocytosis, and energy homeostasis. Abnormal cellular metabolism in turn could affect the production and accumulation of amyloid-β (Aβ) and hyperphosphorylated Tau protein, which independently could exacerbate mitochondrial dysfunction and ROS production, thereby contributing to a vicious cycle. While mounting evidence implicates ROS in the AD etiology, clinical trials with antioxidant therapies have not produced consistent results. In this review, we will discuss the role of oxidative stress in synaptic dysfunction in AD, innovative therapeutic strategies evolved based on a better understanding of the complexity of molecular mechanisms of AD, and the dual role ROS play in health and disease.

  16. Synaptic maturation at cortical projections to the lateral amygdala in a mouse model of Rett syndrome.

    Directory of Open Access Journals (Sweden)

    Frédéric Gambino

    Full Text Available Rett syndrome (RTT is a neuro-developmental disorder caused by loss of function of Mecp2--methyl-CpG-binding protein 2--an epigenetic factor controlling DNA transcription. In mice, removal of Mecp2 in the forebrain recapitulates most of behavioral deficits found in global Mecp2 deficient mice, including amygdala-related hyper-anxiety and lack of social interaction, pointing a role of Mecp2 in emotional learning. Yet very little is known about the establishment and maintenance of synaptic function in the adult amygdala and the role of Mecp2 in these processes. Here, we performed a longitudinal examination of synaptic properties at excitatory projections to principal cells of the lateral nucleus of the amygdala (LA in Mecp2 mutant mice and their wild-type littermates. We first show that during animal life, Cortico-LA projections switch from a tonic to a phasic mode, whereas Thalamo-LA synapses are phasic at all ages. In parallel, we observed a specific elimination of Cortico-LA synapses and a decrease in their ability of generating presynaptic long term potentiation. In absence of Mecp2, both synaptic maturation and synaptic elimination were exaggerated albeit still specific to cortical projections. Surprisingly, associative LTP was unaffected at Mecp2 deficient synapses suggesting that synaptic maintenance rather than activity-dependent synaptic learning may be causal in RTT physiopathology. Finally, because the timing of synaptic evolution was preserved, we propose that some of the developmental effects of Mecp2 may be exerted within an endogenous program and restricted to synapses which maturate during animal life.

  17. The Hypoxia Mimetic Protocatechuic Acid Ethyl Ester Inhibits Synaptic Signaling and Plasticity in the Rat Hippocampus.

    Science.gov (United States)

    Lanigan, Sinead M; O'Connor, John J

    2018-01-15

    During hypoxia a number of physiological changes occur within neurons including the stabilization of hypoxia-inducible factors (HIFs). The activity of these proteins is regulated by O 2 , Fe 2+ , 2-OG and ascorbate-dependant hydroxylases which contain prolyl-4-hydroxylase domains (PHDs). PHD inhibitors have been widely used and have been shown to have a preconditioning and protective effect against a later and more severe hypoxic insult. In this study we have investigated the neuroprotective effects of the PHD inhibitor, protocatechuic acid ethyl ester (ethyl 3,4, dihydroxybenzoate: EDHB), as well as its effects on synaptic transmission and plasticity in the rat hippocampus using electrophysiological techniques. We report for the first time, an acute concentration-dependent and reversible inhibitory effect of EDHB (10-100 μM) on synaptic transmission in the dentate gyrus but not Cornu Ammonis 1 (CA1) region which does not affect cell viability. This effect was attenuated through the application of the NMDA or GABA A receptor antagonists, AP-5 and picrotoxin in the dentate gyrus. There were no changes in the ratio of paired responses after EDHB application suggesting a post-synaptic mechanism of action. EDHB (100 μM), was found to inhibit synaptic plasticity in both the dentate gyrus and CA1 regions. Application of exogenous Fe 2+ (100 μM) or digoxin (100 nM) did not reverse EDHB's inhibitory effect on synaptic transmission or plasticity in both regions, suggesting that its effects may be HIF-independent. These results highlight a novel modulatory role for the PHD inhibitor EDHB in hippocampal synaptic transmission and plasticity. A novel post-synaptic mechanism of action may be involved, possibly involving NMDA and GABA A receptor activation. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

  18. Cloning, purification, crystallization and preliminary X-ray studies of flagellar hook scaffolding protein FlgD from Pseudomonas aeruginosa PAO1

    International Nuclear Information System (INIS)

    Luo, Miao; Niu, Siqiang; Yin, Yibing; Huang, Ailong; Wang, Deqiang

    2009-01-01

    In order to better elucidate the functions of FlgD in flagellar hook biosynthesis, the three-dimensional structure of FlgD is being determined by X-ray crystallography. Here, the expression, purification, crystallization and preliminary crystallographic analysis of FlgD from P. aeruginosa are reported. FlgD regulates the assembly of the hook cap structure to prevent leakage of hook monomers into the medium and hook monomer polymerization and also plays a role in determination of the correct hook length, with the help of the FliK protein. In order to better elucidate the functions of FlgD in flagellar hook biosynthesis, the three-dimensional structure of FlgD is being determined by X-ray crystallography. Here, the expression, purification, crystallization and preliminary crystallographic analysis of FlgD from P. aeruginosa are reported. The crystal belonged to space group I222 and diffracted to a resolution of 2.5 Å, with unit-cell parameters a = 116.47, b = 118.71, c = 118.85 Å. The crystals are most likely to contain three molecules in the asymmetric unit, with a V M value of 2.73 Å 3 Da −1

  19. The Spacing Effect for Structural Synaptic Plasticity Provides Specificity and Precision in Plastic Changes.

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    San Martin, Alvaro; Rela, Lorena; Gelb, Bruce; Pagani, Mario Rafael

    2017-05-10

    In contrast to trials of training without intervals (massed training), training trials spaced over time (spaced training) induce a more persistent memory identified as long-term memory (LTM). This phenomenon, known as the spacing effect for memory, is poorly understood. LTM is supported by structural synaptic plasticity; however, how synapses integrate spaced stimuli remains elusive. Here, we analyzed events of structural synaptic plasticity at the single-synapse level after distinct patterns of stimulation in motoneurons of Drosophila We found that the spacing effect is a phenomenon detected at synaptic level, which determines the specificity and the precision in structural synaptic plasticity. Whereas a single pulse of stimulation (massed) induced structural synaptic plasticity, the same amount of stimulation divided in three spaced stimuli completely prevented it. This inhibitory effect was determined by the length of the interstimulus intervals. The inhibitory effect of the spacing was lost by suppressing the activity of Ras or mitogen-activated protein kinase, whereas the overexpression of Ras-WT enhanced it. Moreover, dividing the same total time of stimulation into five or more stimuli produced a higher precision in the number of events of plasticity. Ras mutations associated with intellectual disability abolished the spacing effect and led neurons to decode distinct stimulation patterns as massed stimulation. This evidence suggests that the spacing effect for memory may result from the effect of the spacing in synaptic plasticity, which appears to be a property not limited to neurons involved in learning and memory. We propose a model of spacing-dependent structural synaptic plasticity. SIGNIFICANCE STATEMENT Long-term memory (LTM) induced by repeated trials spaced over time is known as the spacing effect, a common property in the animal kingdom. Altered mechanisms in the spacing effect have been found in animal models of disorders with intellectual

  20. Unbiased View of Synaptic and Neuronal Gene Complement in Ctenophores: Are There Pan-neuronal and Pan-synaptic Genes across Metazoa?

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    Moroz, Leonid L; Kohn, Andrea B

    2015-12-01

    Hypotheses of origins and evolution of neurons and synapses are controversial, mostly due to limited comparative data. Here, we investigated the genome-wide distribution of the bilaterian "synaptic" and "neuronal" protein-coding genes in non-bilaterian basal metazoans (Ctenophora, Porifera, Placozoa, and Cnidaria). First, there are no recognized genes uniquely expressed in neurons across all metazoan lineages. None of the so-called pan-neuronal genes such as embryonic lethal abnormal vision (ELAV), Musashi, or Neuroglobin are expressed exclusively in neurons of the ctenophore Pleurobrachia. Second, our comparative analysis of about 200 genes encoding canonical presynaptic and postsynaptic proteins in bilaterians suggests that there are no true "pan-synaptic" genes or genes uniquely and specifically attributed to all classes of synapses. The majority of these genes encode receptive and secretory complexes in a broad spectrum of eukaryotes. Trichoplax (Placozoa) an organism without neurons and synapses has more orthologs of bilaterian synapse-related/neuron-related genes than do ctenophores-the group with well-developed neuronal and synaptic organization. Third, the majority of genes encoding ion channels and ionotropic receptors are broadly expressed in unicellular eukaryotes and non-neuronal tissues in metazoans. Therefore, they cannot be viewed as neuronal markers. Nevertheless, the co-expression of multiple types of ion channels and receptors does correlate with the presence of neural and synaptic organization. As an illustrative example, the ctenophore genomes encode a greater diversity of ion channels and ionotropic receptors compared with the genomes of the placozoan Trichoplax and the demosponge Amphimedon. Surprisingly, both placozoans and sponges have a similar number of orthologs of "synaptic" proteins as we identified in the genomes of two ctenophores. Ctenophores have a distinct synaptic organization compared with other animals. Our analysis of

  1. Synaptic Correlates of Working Memory Capacity.

    Science.gov (United States)

    Mi, Yuanyuan; Katkov, Mikhail; Tsodyks, Misha

    2017-01-18

    Psychological studies indicate that human ability to keep information in readily accessible working memory is limited to four items for most people. This extremely low capacity severely limits execution of many cognitive tasks, but its neuronal underpinnings remain unclear. Here we show that in the framework of synaptic theory of working memory, capacity can be analytically estimated to scale with characteristic time of short-term synaptic depression relative to synaptic current time constant. The number of items in working memory can be regulated by external excitation, enabling the system to be tuned to the desired load and to clear the working memory of currently held items to make room for new ones. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Astrocytes optimize the synaptic transmission of information.

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    Suhita Nadkarni

    2008-05-01

    Full Text Available Chemical synapses transmit information via the release of neurotransmitter-filled vesicles from the presynaptic terminal. Using computational modeling, we predict that the limited availability of neurotransmitter resources in combination with the spontaneous release of vesicles limits the maximum degree of enhancement of synaptic transmission. This gives rise to an optimal tuning that depends on the number of active zones. There is strong experimental evidence that astrocytes that enwrap synapses can modulate the probabilities of vesicle release through bidirectional signaling and hence regulate synaptic transmission. For low-fidelity hippocampal synapses, which typically have only one or two active zones, the predicted optimal values lie close to those determined by experimentally measured astrocytic feedback, suggesting that astrocytes optimize synaptic transmission of information.

  3. Monodisperse 130 kDa and 260 kDa Recombinant Human Hemoglobin Polymers as Scaffolds for Protein Engineering of Hemoglobin-Based Oxygen Carriers

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    Jacqueline F. Aitken

    2012-01-01

    Full Text Available A recombinant 130 kDa dihemoglobin which is made up of a single-chain tetra-α globin and four β globins has been expressed as a soluble protein in E. coli. The sequence of the single chain tetra-α is: αI-Gly-αII-(SerGlyGly5Ser-αIII-Gly-αIV. This dihemoglobin has been purified and characterized in vitro by size exclusion chromatography, electrospray mass spectroscopy, equilibrium oxygen binding, and analytical ultracentrifugation. The observed values of P50 and nmax for the dihemoglobin are slightly lower than those observed for the recombinant hemoglobin rHb1.1 (a “monohemoglobin” comprised of two β globins and an αI-Gly-αII diα-globin chain. Titration of the deoxy form of dihemoglobin with CO shows that all eight heme centers bind ligand. In vivo, dihemoglobin showed increased circulating halflife and a reduced pressor response in conscious rats when compared to rHb1.1. These observations suggest that dihemoglobin is an oxygen carrying molecule with desirable in vivo properties and provides a platform for an isooncotic hemoglobin solution derived solely from a recombinant source. A 260 kDa tetrahemoglobin has also been produced by chemical crosslinking of a dihemoglobin that contains a Lys16Cys mutation in the C-terminal α-globin subunit. Tetrahemoglobin also shows reduced vasoactivity in conscious rats that is comparable to that observed for dihemoglobin.

  4. Artificial Synaptic Devices Based on Natural Chicken Albumen Coupled Electric-Double-Layer Transistors.

    Science.gov (United States)

    Wu, Guodong; Feng, Ping; Wan, Xiang; Zhu, Liqiang; Shi, Yi; Wan, Qing

    2016-03-24

    Recent progress in using biomaterials to fabricate functional electronics has got growing attention for the new generation of environmentally friendly and biocompatible electronic devices. As a kind of biological material with rich source, proteins are essential natural component of all organisms. At the same time, artificial synaptic devices are of great significance for neuromorphic systems because they can emulate the signal process and memory behaviors of biological synapses. In this report, natural chicken albumen with high proton conductivity was used as the coupling electrolyte film for organic/inorganic hybrid synaptic devices fabrication. Some important synaptic functions including paired-pulse facilitation, dynamic filtering, short-term to long-term memory transition and spatial summation and shunting inhibition were successfully mimicked. Our results are very interesting for biological friendly artificial neuron networks and neuromorphic systems.

  5. Recent advances in understanding synaptic abnormalities in Rett syndrome [version 1; referees: 2 approved

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    Michael Johnston

    2015-12-01

    Full Text Available Rett syndrome is an extremely disabling X-linked nervous system disorder that mainly affects girls in early childhood and causes autism-like behavior, severe intellectual disability, seizures, sleep disturbances, autonomic instability, and other disorders due to mutations in the MeCP2 (methyl CpG-binding protein 2 transcription factor. The disorder targets synapses and synaptic plasticity and has been shown to disrupt the balance between glutamate excitatory synapses and GABAergic inhibitory synapses. In fact, it can be argued that Rett syndrome is primarily a disorder of synaptic plasticity and that agents that can correct this imbalance may have beneficial effects on brain development. This review briefly summarizes the link between disrupted synaptic plasticity mechanisms and Rett syndrome and early clinical trials that aim to target these abnormalities to improve the outcome for these severely disabled children.

  6. SMN requirement for synaptic vesicle, active zone and microtubule postnatal organization in motor nerve terminals.

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    Laura Torres-Benito

    Full Text Available Low levels of the Survival Motor Neuron (SMN protein produce Spinal Muscular Atrophy (SMA, a severe monogenetic disease in infants characterized by muscle weakness and impaired synaptic transmission. We report here severe structural and functional alterations in the organization of the organelles and the cytoskeleton of motor nerve terminals in a mouse model of SMA. The decrease in SMN levels resulted in the clustering of synaptic vesicles (SVs and Active Zones (AZs, reduction in the size of the readily releasable pool (RRP, and the recycling pool (RP of synaptic vesicles, a decrease in active mitochondria and limiting of neurofilament and microtubule maturation. We propose that SMN is essential for the normal postnatal maturation of motor nerve terminals and that SMN deficiency disrupts the presynaptic organization leading to neurodegeneration.

  7. The penumbra of learning: a statistical theory of synaptic tagging and capture.

    Science.gov (United States)

    Gershman, Samuel J

    2014-01-01

    Learning in humans and animals is accompanied by a penumbra: Learning one task benefits from learning an unrelated task shortly before or after. At the cellular level, the penumbra of learning appears when weak potentiation of one synapse is amplified by strong potentiation of another synapse on the same neuron during a critical time window. Weak potentiation sets a molecular tag that enables the synapse to capture plasticity-related proteins synthesized in response to strong potentiation at another synapse. This paper describes a computational model which formalizes synaptic tagging and capture in terms of statistical learning mechanisms. According to this model, synaptic strength encodes a probabilistic inference about the dynamically changing association between pre- and post-synaptic firing rates. The rate of change is itself inferred, coupling together different synapses on the same neuron. When the inputs to one synapse change rapidly, the inferred rate of change increases, amplifying learning at other synapses.

  8. Synaptic molecular imaging in spared and deprived columns of mouse barrel cortex with array tomography.

    Science.gov (United States)

    Weiler, Nicholas C; Collman, Forrest; Vogelstein, Joshua T; Burns, Randal; Smith, Stephen J

    2014-01-01

    A major question in neuroscience is how diverse subsets of synaptic connections in neural circuits are affected by experience dependent plasticity to form the basis for behavioral learning and memory. Differences in protein expression patterns at individual synapses could constitute a key to understanding both synaptic diversity and the effects of plasticity at different synapse populations. Our approach to this question leverages the immunohistochemical multiplexing capability of array tomography (ATomo) and the columnar organization of mouse barrel cortex to create a dataset comprising high resolution volumetric images of spared and deprived cortical whisker barrels stained for over a dozen synaptic molecules each. These dataset has been made available through the Open Connectome Project for interactive online viewing, and may also be downloaded for offline analysis using web, Matlab, and other interfaces.

  9. Heterosynaptic Plasticity Prevents Runaway Synaptic Dynamics

    Science.gov (United States)

    Chen, Jen-Yung; Lonjers, Peter; Lee, Christopher; Chistiakova, Marina; Volgushev, Maxim

    2013-01-01

    Spike timing-dependent plasticity (STDP) and other conventional Hebbian-type plasticity rules are prone to produce runaway dynamics of synaptic weights. Once potentiated, a synapse would have higher probability to lead to spikes and thus to be further potentiated, but once depressed, a synapse would tend to be further depressed. The runaway synaptic dynamics can be prevented by precisely balancing STDP rules for potentiation and depression; however, experimental evidence shows a great variety of potentiation and depression windows and magnitudes. Here we show that modifications of synapses to layer 2/3 pyramidal neurons from rat visual and auditory cortices in slices can be induced by intracellular tetanization: bursts of postsynaptic spikes without presynaptic stimulation. Induction of these heterosynaptic changes depended on the rise of intracellular calcium, and their direction and magnitude correlated with initial state of release mechanisms. We suggest that this type of plasticity serves as a mechanism that stabilizes the distribution of synaptic weights and prevents their runaway dynamics. To test this hypothesis, we develop a cortical neuron model implementing both homosynaptic (STDP) and heterosynaptic plasticity with properties matching the experimental data. We find that heterosynaptic plasticity effectively prevented runaway dynamics for the tested range of STDP and input parameters. Synaptic weights, although shifted from the original, remained normally distributed and nonsaturated. Our study presents a biophysically constrained model of how the interaction of different forms of plasticity—Hebbian and heterosynaptic—may prevent runaway synaptic dynamics and keep synaptic weights unsaturated and thus capable of further plastic changes and formation of new memories. PMID:24089497

  10. Epsin 1 Promotes Synaptic Growth by Enhancing BMP Signal Levels in Motoneuron Nuclei.

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    Phillip A Vanlandingham

    Full Text Available Bone morphogenetic protein (BMP retrograde signaling is crucial for neuronal development and synaptic plasticity. However, how the BMP effector phospho-Mother against decapentaplegic (pMad is processed following receptor activation remains poorly understood. Here we show that Drosophila Epsin1/Liquid