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Sample records for bioaminergic neuronal cells

  1. The cellular prion protein interacts with the tissue non-specific alkaline phosphatase in membrane microdomains of bioaminergic neuronal cells.

    Directory of Open Access Journals (Sweden)

    Myriam Ermonval

    Full Text Available BACKGROUND: The cellular prion protein, PrP(C, is GPI anchored and abundant in lipid rafts. The absolute requirement of PrP(C in neurodegeneration associated to prion diseases is well established. However, the function of this ubiquitous protein is still puzzling. Our previous work using the 1C11 neuronal model, provided evidence that PrP(C acts as a cell surface receptor. Besides a ubiquitous signaling function of PrP(C, we have described a neuronal specificity pointing to a role of PrP(C in neuronal homeostasis. 1C11 cells, upon appropriate induction, engage into neuronal differentiation programs, giving rise either to serotonergic (1C11(5-HT or noradrenergic (1C11(NE derivatives. METHODOLOGY/PRINCIPAL FINDINGS: The neuronal specificity of PrP(C signaling prompted us to search for PrP(C partners in 1C11-derived bioaminergic neuronal cells. We show here by immunoprecipitation an association of PrP(C with an 80 kDa protein identified by mass spectrometry as the tissue non-specific alkaline phosphatase (TNAP. This interaction occurs in lipid rafts and is restricted to 1C11-derived neuronal progenies. Our data indicate that TNAP is implemented during the differentiation programs of 1C11(5-HT and 1C11(NE cells and is active at their cell surface. Noteworthy, TNAP may contribute to the regulation of serotonin or catecholamine synthesis in 1C11(5-HT and 1C11(NE bioaminergic cells by controlling pyridoxal phosphate levels. Finally, TNAP activity is shown to modulate the phosphorylation status of laminin and thereby its interaction with PrP. CONCLUSION/SIGNIFICANCE: The identification of a novel PrP(C partner in lipid rafts of neuronal cells favors the idea of a role of PrP in multiple functions. Because PrP(C and laminin functionally interact to support neuronal differentiation and memory consolidation, our findings introduce TNAP as a functional protagonist in the PrP(C-laminin interplay. The partnership between TNAP and PrP(C in neuronal cells may

  2. Neuronal Differentiation of Human Mesenchymal Stem Cells Using Exosomes Derived from Differentiating Neuronal Cells

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    Takeda, Yuji S.; Qiaobing Xu

    2015-01-01

    Exosomes deliver functional proteins and genetic materials to neighboring cells, and have potential applications for tissue regeneration. One possible mechanism of exosome-promoted tissue regeneration is through the delivery of microRNA (miRNA). In this study, we hypothesized that exosomes derived from neuronal progenitor cells contain miRNAs that promote neuronal differentiation. We treated mesenchymal stem cells (MSCs) daily with exosomes derived from PC12 cells, a neuronal cell line, for 1...

  3. Human embryonic stem cell-derived neuronal cells form spontaneously active neuronal networks in vitro.

    Science.gov (United States)

    Heikkilä, Teemu J; Ylä-Outinen, Laura; Tanskanen, Jarno M A; Lappalainen, Riikka S; Skottman, Heli; Suuronen, Riitta; Mikkonen, Jarno E; Hyttinen, Jari A K; Narkilahti, Susanna

    2009-07-01

    The production of functional human embryonic stem cell (hESC)-derived neuronal cells is critical for the application of hESCs in treating neurodegenerative disorders. To study the potential functionality of hESC-derived neurons, we cultured and monitored the development of hESC-derived neuronal networks on microelectrode arrays. Immunocytochemical studies revealed that these networks were positive for the neuronal marker proteins beta-tubulin(III) and microtubule-associated protein 2 (MAP-2). The hESC-derived neuronal networks were spontaneously active and exhibited a multitude of electrical impulse firing patterns. Synchronous bursts of electrical activity similar to those reported for hippocampal neurons and rodent embryonic stem cell-derived neuronal networks were recorded from the differentiated cultures until up to 4 months. The dependence of the observed neuronal network activity on sodium ion channels was examined using tetrodotoxin (TTX). Antagonists for the glutamate receptors NMDA [D(-)-2-amino-5-phosphonopentanoic acid] and AMPA/kainate [6-cyano-7-nitroquinoxaline-2,3-dione], and for GABAA receptors [(-)-bicuculline methiodide] modulated the spontaneous electrical activity, indicating that pharmacologically susceptible neuronal networks with functional synapses had been generated. The findings indicate that hESC-derived neuronal cells can generate spontaneously active networks with synchronous communication in vitro, and are therefore suitable for use in developmental and drug screening studies, as well as for regenerative medicine.

  4. General overview of neuronal cell culture.

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    Gordon, Jennifer; Amini, Shohreh; White, Martyn K

    2013-01-01

    In this introductory chapter, we provide a general overview of neuronal cell culture. This is a rapidly evolving area of research and we provide an outline and contextual framework for the different chapters of this book. These chapters were all contributed by scientists actively working in the field who are currently using state-of-the-art techniques to advance our understanding of the molecular and cellular biology of the central nervous system. Each chapter provides detailed descriptions and experimental protocols for a variety of techniques ranging in scope from basic neuronal cell line culturing to advanced and specialized methods.

  5. Murine Mueller cells are progenitor cells for neuronal cells and fibrous tissue cells

    International Nuclear Information System (INIS)

    Mammalian Mueller cells have been reported to possess retinal progenitor cell properties and generate new neurons after injury. This study investigates murine Mueller cells under in vitro conditions for their capability of dedifferentiation into retinal progenitor cells. Mueller cells were isolated from mouse retina, and proliferating cells were expanded in serum-containing medium. For dedifferentiation, the cultured cells were transferred to serum-replacement medium (SRM) at different points in time after their isolation. Interestingly, early cell passages produced fibrous tissue in which extracellular matrix proteins and connective tissue markers were differentially expressed. In contrast, aged Mueller cell cultures formed neurospheres in SRM that are characteristic for neuronal progenitor cells. These neurospheres differentiated into neuron-like cells after cultivation on laminin/ornithine cell culture substrate. Here, we report for the first time that murine Mueller cells can be progenitors for both, fibrous tissue cells and neuronal cells, depending on the age of the cell culture

  6. Prospects for Replacement of Auditory Neurons by Stem Cells

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    Shi, Fuxin; Edge, Albert S. B.

    2013-01-01

    Sensorineural hearing loss is caused by degeneration of hair cells or auditory neurons. Spiral ganglion cells, the primary afferent neurons of the auditory system, are patterned during development and send out projections to hair cells and to the brainstem under the control of largely unknown guidance molecules. The neurons do not regenerate after loss and even damage to their projections tends to be permanent. The genesis of spiral ganglion neurons and their synapses forms a basis for regene...

  7. Genetic strategies to investigate neuronal circuit properties using stem cell-derived neurons

    Directory of Open Access Journals (Sweden)

    Isabella eGarcia

    2012-12-01

    Full Text Available The mammalian brain is anatomically and functionally complex, and prone to diverse forms of injury and neuropathology. Scientists have long strived to develop cell replacement therapies to repair damaged and diseased nervous tissue. However, this goal has remained unrealized for various reasons, including nascent knowledge of neuronal development, the inability to track and manipulate transplanted cells within complex neuronal networks, and host graft rejection. Recent advances in embryonic stem cell (ESC and induced pluripotent stem cell (iPSC technology, alongside novel genetic strategies to mark and manipulate stem cell-derived neurons now provide unprecedented opportunities to investigate complex neuronal circuits in both healthy and diseased brains. Here, we review current technologies aimed at generating and manipulating neurons derived from ESCs and iPSCs towards investigation and manipulation of complex neuronal circuits, ultimately leading to the design and development of novel cell-based therapeutic approaches.

  8. Neuron-glia cell adhesion molecule interacts with neurons and astroglia via different binding mechanisms

    OpenAIRE

    1988-01-01

    The neuron-glia cell adhesion molecule (Ng-CAM) is present in the central nervous system on postmitotic neurons and in the periphery on neurons and Schwann cells. It has been implicated in binding between neurons and between neurons and glia. To understand the molecular mechanisms of Ng-CAM binding, we analyzed the aggregation of chick Ng- CAM either immobilized on 0.5-micron beads (Covaspheres) or reconstituted into liposomes. The results were correlated with the binding of these particles t...

  9. Cell cycle markers have different expression and localization patterns in neuron-like PC12 cells and primary hippocampal neurons.

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    Negis, Yesim; Unal, Aysegul Yildiz; Korulu, Sirin; Karabay, Arzu

    2011-06-01

    Neuron-like PC12 cells are extensively used in place of neurons in published studies. Aim of this paper has been to compare mRNA and protein expressions of cell cycle markers; cyclinA, B, D, E; Cdk1, 2 and 4; and p27 in post-mitotic primary hippocampal neurons, mitotically active PC12 cells and NGF-differentiated post-mitotic PC12 cells. Contrary to PC12 cells, in neurons, the presence of all these markers was detected only at mRNA level; except for cyclinA, cyclinE and Cdk4, which were detectable also at protein levels. In both NGF-treated PC12 cells and neurons, cyclinE was localized only in the nucleus. In NGF-treated PC12 cells cyclinD and Cdk4 were localized in the nucleus while, in neurons cyclinD expression was not detectable; Cdk4 was localized in the cytoplasm. In neurons, cyclinA was nuclear, whereas in NGF-treated PC12 cells, it was localized in the cell body and along the processes. These results suggest that PC12 cells and primary neurons are different in terms of cell cycle protein expressions and localizations. Thus, it may not be very appropriate to use these cells as neuronal model system in order to understand neuronal physiological activities, upstream of where may lie cell cycle activation triggered events.

  10. Closing the Phenotypic Gap between Transformed Neuronal Cell Lines in Culture and Untransformed Neurons

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    Myers, Tereance A.; Nickerson, Cheryl A.; Kaushal, Deepak; Ott, C. Mark; HonerzuBentrup, Kerstin; Ramamurthy, Rajee; Nelman-Gonzales, Mayra; Pierson, Duane L.; Philipp, Mario T.

    2008-01-01

    Studies of neuronal dysfunction in the central nervous system (CNS) are frequently limited by the failure of primary neurons to propagate in vitro. Neuronal cell lines can be substituted for primary cells but they often misrepresent normal conditions. We hypothesized that a dimensional (3-D) cell culture system would drive the phenotype of transformed neurons closer to that of untransformed cells. In our studies comparing 3-D versus 2-dimensional (2-D) culture, neuronal SH-SY5Y (SY) cells underwent distinct morphological changes combined with a significant drop in their rate of cell division. Expression of the proto-oncogene N-myc and the RNA binding protein HuD was decreased in 3-D culture as compared to standard 2-D conditions. We observed a decline in the anti-apoptotic protein Bcl-2 in 3-D culture, coupled with increased expression of the pro-apoptotic proteins Bax and Bak. Moreover, thapsigargin (TG)-induced apoptosis was enhanced in the 3-D cells. Microarray analysis demonstrated significantly differing mRNA levels for over 700 genes in the cells of each culture type. These results indicate that a 3-D culture approach narrows the phenotypic gap between neuronal cell lines and primary neurons. The resulting cells may readily be used for in vitro research of neuronal pathogenesis.

  11. Neuronal inhibition of astroglial cell proliferation is membrane mediated

    OpenAIRE

    1987-01-01

    Previously we have used a microwell tissue culture assay to show that early postnatal mouse cerebellar astroglia have a flattened morphology and proliferate rapidly when they are cultured in the absence of neurons, but develop specific cell-cell contacts and undergo morphological differentiation when they are co-cultured with purified granule neurons (Hatten, M. E., 1985, J. Cell Biol., 100:384-396). In these studies of cell binding between neurons and astroglia, measurement with light and fl...

  12. Multiple Modes of Communication between Neurons and Oligodendrocyte Precursor Cells

    NARCIS (Netherlands)

    Maldonado, Paloma P; Angulo, María Cecilia

    2015-01-01

    The surprising discovery of bona fide synapses between neurons and oligodendrocytes precursor cells (OPCs) 15 years ago placed these progenitors as real partners of neurons in the CNS. The role of these synapses has not been established yet, but a main hypothesis is that neuron-OPC synaptic activity

  13. Prospects for replacement of auditory neurons by stem cells.

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    Shi, Fuxin; Edge, Albert S B

    2013-03-01

    Sensorineural hearing loss is caused by degeneration of hair cells or auditory neurons. Spiral ganglion cells, the primary afferent neurons of the auditory system, are patterned during development and send out projections to hair cells and to the brainstem under the control of largely unknown guidance molecules. The neurons do not regenerate after loss and even damage to their projections tends to be permanent. The genesis of spiral ganglion neurons and their synapses forms a basis for regenerative approaches. In this review we critically present the current experimental findings on auditory neuron replacement. We discuss the latest advances with a focus on (a) exogenous stem cell transplantation into the cochlea for neural replacement, (b) expression of local guidance signals in the cochlea after loss of auditory neurons, (c) the possibility of neural replacement from an endogenous cell source, and (d) functional changes from cell engraftment. PMID:23370457

  14. Cortical cell and neuron density estimates in one chimpanzee hemisphere.

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    Collins, Christine E; Turner, Emily C; Sawyer, Eva Kille; Reed, Jamie L; Young, Nicole A; Flaherty, David K; Kaas, Jon H

    2016-01-19

    The density of cells and neurons in the neocortex of many mammals varies across cortical areas and regions. This variability is, perhaps, most pronounced in primates. Nonuniformity in the composition of cortex suggests regions of the cortex have different specializations. Specifically, regions with densely packed neurons contain smaller neurons that are activated by relatively few inputs, thereby preserving information, whereas regions that are less densely packed have larger neurons that have more integrative functions. Here we present the numbers of cells and neurons for 742 discrete locations across the neocortex in a chimpanzee. Using isotropic fractionation and flow fractionation methods for cell and neuron counts, we estimate that neocortex of one hemisphere contains 9.5 billion cells and 3.7 billion neurons. Primary visual cortex occupies 35 cm(2) of surface, 10% of the total, and contains 737 million densely packed neurons, 20% of the total neurons contained within the hemisphere. Other areas of high neuron packing include secondary visual areas, somatosensory cortex, and prefrontal granular cortex. Areas of low levels of neuron packing density include motor and premotor cortex. These values reflect those obtained from more limited samples of cortex in humans and other primates.

  15. Hyperexcitable neurons and altered non-neuronal cells in the compressed spinal ganglion

    Institute of Scientific and Technical Information of China (English)

    Robert H. LaMotte; Chao MA

    2008-01-01

    The cell body or soma in the dosal root ganglion (DRG) is normally excitable and this excitability can increase and persist after an injury of peripheral sensory neurons. In a rat model of radicular pain, an intraforaminal implantation of a rod that chronically compressed the lumbar DRG ("CCD" model) resulted in neuronal somal hyperexcitability and spontaneous activity that was accom-panied by hyperalgesia in the ipsilateral hind paw. By the 5th day after onset of CCD, there was a novel upregulation in neuronal expression of the chemokine, monocyte chemoattractant protein-1 (MCP- 1 or CCL2) and also its receptor, CCR2. The neurons developed, in response to topically applied MCP-1, an excitatory response that they normally do not have. CCD also activated non-neuronal cells including, for example, the endothelial cells as evidenced by angiogenesis in the form of an increased number of capillaries in the DRG after 7 days. A working hypothesis is that the CCD induced changes in neurons and non-neuronal cells that may act together to promote the survival of the injured tissue. The release of ligands such as CCL2, in addition to possibly activating nociceptive neurons (maintaining the pain), may also act to preserve injured cells in the face of ischemia and hypoxia, for example, by promoting angiogenesis. Thus, somal hyperexcitability, as often said of inflammation, may represent a double edged sword.

  16. Reconstruction of phrenic neuron identity in embryonic stem cell-derived motor neurons.

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    Machado, Carolina Barcellos; Kanning, Kevin C; Kreis, Patricia; Stevenson, Danielle; Crossley, Martin; Nowak, Magdalena; Iacovino, Michelina; Kyba, Michael; Chambers, David; Blanc, Eric; Lieberam, Ivo

    2014-02-01

    Air breathing is an essential motor function for vertebrates living on land. The rhythm that drives breathing is generated within the central nervous system and relayed via specialised subsets of spinal motor neurons to muscles that regulate lung volume. In mammals, a key respiratory muscle is the diaphragm, which is innervated by motor neurons in the phrenic nucleus. Remarkably, relatively little is known about how this crucial subtype of motor neuron is generated during embryogenesis. Here, we used direct differentiation of motor neurons from mouse embryonic stem cells as a tool to identify genes that direct phrenic neuron identity. We find that three determinants, Pou3f1, Hoxa5 and Notch, act in combination to promote a phrenic neuron molecular identity. We show that Notch signalling induces Pou3f1 in developing motor neurons in vitro and in vivo. This suggests that the phrenic neuron lineage is established through a local source of Notch ligand at mid-cervical levels. Furthermore, we find that the cadherins Pcdh10, which is regulated by Pou3f1 and Hoxa5, and Cdh10, which is controlled by Pou3f1, are both mediators of like-like clustering of motor neuron cell bodies. This specific Pcdh10/Cdh10 activity might provide the means by which phrenic neurons are assembled into a distinct nucleus. Our study provides a framework for understanding how phrenic neuron identity is conferred and will help to generate this rare and inaccessible yet vital neuronal subtype directly from pluripotent stem cells, thus facilitating subsequent functional investigations.

  17. Phenazopyridine induces and synchronizes neuronal differentiation of embryonic stem cells.

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    Suter, David M; Preynat-Seauve, Olivier; Tirefort, Diderik; Feki, Anis; Krause, Karl-Heinz

    2009-09-01

    Embryonic stem (ES) cells are powerful tools to understand mechanisms of neuronal differentiation and to engineer neurons for in vitro studies and cell therapy. We developed a screening approach to identify small organic molecules driving neuronal differentiation of ES cells. For this purpose, we used a lentivector carrying a dual luciferase reporter system to engineer an ES cell line which allowed us to screen for small organic molecules enhancing neuronal differentiation. One of them, phenazopyridine, was further analysed in human ES cells. Phenazopyridine: (i) enhanced neuronal differentiation, (ii) increased cell survival, (iii) decreased the amount of non-neuronal and undifferentiated cells and (iv) synchronized the cellular differentiation state. Phenazopyridine allowed the development of a differentiation protocol compatible with the generation of clinical grade neural precursors, which were able differentiate into different neuronal subtypes, astrocytes and oligodendrocytes. In summary, we describe a powerful approach to identify small molecules directing stem cell differentiation. This led to the establishment of a new application for an old drug and the development of a novel clinical grade protocol for neuronal differentiation of ES cells. PMID:20196783

  18. Expression of polysialylated neural cell adhesion molecules on adult stem cells after neuronal differentiation of inner ear spiral ganglion neurons

    International Nuclear Information System (INIS)

    Highlights: • PolySia expressed on neurons primarily during early stages of neuronal development. • PolySia–NCAM is expressed on neural stem cells from adult guinea pig spiral ganglion. • PolySia is a biomarker that modulates neuronal differentiation in inner ear stem cells. - Abstract: During brain development, polysialylated (polySia) neural cell adhesion molecules (polySia–NCAMs) modulate cell–cell adhesive interactions involved in synaptogenesis, neural plasticity, myelination, and neural stem cell (NSC) proliferation and differentiation. Our findings show that polySia–NCAM is expressed on NSC isolated from adult guinea pig spiral ganglion (GPSG), and in neurons and Schwann cells after differentiation of the NSC with epidermal, glia, fibroblast growth factors (GFs) and neurotrophins. These differentiated cells were immunoreactive with mAb’s to polySia, NCAM, β-III tubulin, nestin, S-100 and stained with BrdU. NSC could regenerate and be differentiated into neurons and Schwann cells. We conclude: (1) polySia is expressed on NSC isolated from adult GPSG and on neurons and Schwann cells differentiated from these NSC; (2) polySia is expressed on neurons primarily during the early stage of neuronal development and is expressed on Schwann cells at points of cell–cell contact; (3) polySia is a functional biomarker that modulates neuronal differentiation in inner ear stem cells. These new findings suggest that replacement of defective cells in the inner ear of hearing impaired patients using adult spiral ganglion neurons may offer potential hope to improve the quality of life for patients with auditory dysfunction and impaired hearing disorders

  19. Expression of polysialylated neural cell adhesion molecules on adult stem cells after neuronal differentiation of inner ear spiral ganglion neurons

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    Park, Kyoung Ho [Department of Otolaryngology Head and Neck Surgery, College of Medicine, Catholic University, Seoul (Korea, Republic of); Yeo, Sang Won, E-mail: swyeo@catholic.ac.kr [Department of Otolaryngology Head and Neck Surgery, College of Medicine, Catholic University, Seoul (Korea, Republic of); Troy, Frederic A., E-mail: fatroy@ucdavis.edu [Department of Biochemistry and Molecular Medicine, University of California, School of Medicine, Davis, CA 95616 (United States); Xiamen University, School of Medicine, Xiamen City (China)

    2014-10-17

    Highlights: • PolySia expressed on neurons primarily during early stages of neuronal development. • PolySia–NCAM is expressed on neural stem cells from adult guinea pig spiral ganglion. • PolySia is a biomarker that modulates neuronal differentiation in inner ear stem cells. - Abstract: During brain development, polysialylated (polySia) neural cell adhesion molecules (polySia–NCAMs) modulate cell–cell adhesive interactions involved in synaptogenesis, neural plasticity, myelination, and neural stem cell (NSC) proliferation and differentiation. Our findings show that polySia–NCAM is expressed on NSC isolated from adult guinea pig spiral ganglion (GPSG), and in neurons and Schwann cells after differentiation of the NSC with epidermal, glia, fibroblast growth factors (GFs) and neurotrophins. These differentiated cells were immunoreactive with mAb’s to polySia, NCAM, β-III tubulin, nestin, S-100 and stained with BrdU. NSC could regenerate and be differentiated into neurons and Schwann cells. We conclude: (1) polySia is expressed on NSC isolated from adult GPSG and on neurons and Schwann cells differentiated from these NSC; (2) polySia is expressed on neurons primarily during the early stage of neuronal development and is expressed on Schwann cells at points of cell–cell contact; (3) polySia is a functional biomarker that modulates neuronal differentiation in inner ear stem cells. These new findings suggest that replacement of defective cells in the inner ear of hearing impaired patients using adult spiral ganglion neurons may offer potential hope to improve the quality of life for patients with auditory dysfunction and impaired hearing disorders.

  20. Adipose tissue-derived stromal cells express neuronal phenotypes

    Institute of Scientific and Technical Information of China (English)

    杨立业; 刘相名; 孙兵; 惠国桢; 费俭; 郭礼和

    2004-01-01

    Background Adipose tissue-derived stromal cells (ADSCs) can be greatly expanded in vitro, and induced to differentiate into multiple mesenchymal cell types, including osteogenic, chondrogenic, myogenic, and adipogenic cells. This study was designed to investigate the possibility of ADSCs differentiating into neurons.Methods Adipose tissue from rats was digested with collagenase, and adherent stromal cells were cultured. A medium containing a low concentration of fetal bovine serum was adopted to induce the cells to differentiate. ADSCs were identified by immunocytochemistry, and semi-quantitative RT-PCR was applied to detect mRNA expression of neurofilament 1 (NF1), nestin, and neuron-specific enolase (NSE).Results Nestin-positive cells were found occasionally among ADSCs. ADSCs were found to express NSE mRNA and nestin mRNA, but not NF1 mRNA. ADSCs could differentiate into neuron-like cells in a medium composed of a low concentration of fetal bovine serum, and these differentiated cells displayed complicated neuron-like morphologies.Conclusions The data support the hypothesis that adipose tissue contains stem cells capable of differentiating into neurons. These stem cells can overcome their mesenchymal commitment, and may represent an alternative autologous stem cell source for CNS cell transplantation.

  1. Mouse bone marrow stromal cells differentiate to neuron-like cells upon inhibition of BMP signaling.

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    Saxena, Monika; Prashar, Paritosh; Yadav, Prem Swaroop; Sen, Jonaki

    2016-01-01

    Bone marrow stromal cells (BMSCs) are a source of autologous stem cells that have the potential for undergoing differentiation into multiple cell types including neurons. Although the neuronal differentiation of mesenchymal stem cells has been studied for a long time, the molecular players involved are still not defined. Here we report that the genetic deletion of two members of the bone morphogenetic protein (Bmp) family, Bmp2 and Bmp4 in mouse BMSCs causes their differentiation into cells with neuron-like morphology. Surprisingly these cells expressed certain markers characteristic of both neuronal and glial cells. Based on this observation, we inhibited BMP signaling in mouse BMSCs through a brief exposure to Noggin protein which also led to their differentiation into cells expressing both neuronal and glial markers. Such cells seem to have the potential for further differentiation into subtypes of neuronal and glial cells and thus could be utilized for cell-based therapeutic applications.

  2. AUDITORY HAIR CELL EXPLANT CO-CULTURES PROMOTE THE DIFFERENTIATION OF STEM CELLS INTO BIPOLAR NEURONS

    OpenAIRE

    Coleman, B.; Fallon, J. B.; Gillespie, L.N.; Silva, M.G.; Shepherd, R.K.

    2006-01-01

    Auditory neurons, the target neurons of the cochlear implant, degenerate following a sensorineural hearing loss. The goal of this research is to direct the differentiation of embryonic stem cells (SCs) into bipolar auditory neurons that can be used to replace degenerating neurons in the deafened mammalian cochlea. Successful replacement of auditory neurons is likely to result in improved clinical outcomes for cochlear implant recipients. We examined two post-natal auditory co-culture models w...

  3. Specification of region-specific neurons including forebrain glutamatergic neurons from human induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Hui Zeng

    Full Text Available BACKGROUND: Directed differentiation of human induced pluripotent stem cells (hiPSC into functional, region-specific neural cells is a key step to realizing their therapeutic promise to treat various neural disorders, which awaits detailed elucidation. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed neural differentiation from various hiPSC lines generated by others and ourselves. Although heterogeneity in efficiency of neuroepithelial (NE cell differentiation was observed among different hiPSC lines, the NE differentiation process resembles that from human embryonic stem cells (hESC in morphology, timing, transcriptional profile, and requirement for FGF signaling. NE cells differentiated from hiPSC, like those from hESC, can also form rostral phenotypes by default, and form the midbrain or spinal progenitors upon caudalization by morphogens. The rostrocaudal neural progenitors can further mature to develop forebrain glutamatergic projection neurons, midbrain dopaminergic neurons, and spinal motor neurons, respectively. Typical ion channels and action potentials were recorded in the hiPSC-derived neurons. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that hiPSC, regardless of how they were derived, can differentiate into a spectrum of rostrocaudal neurons with functionality, which supports the considerable value of hiPSC for study and treatment of patient-specific neural disorders.

  4. Sensory neurons do not induce motor neuron loss in a human stem cell model of spinal muscular atrophy.

    Science.gov (United States)

    Schwab, Andrew J; Ebert, Allison D

    2014-01-01

    Spinal muscular atrophy (SMA) is an autosomal recessive disorder leading to paralysis and early death due to reduced SMN protein. It is unclear why there is such a profound motor neuron loss, but recent evidence from fly and mouse studies indicate that cells comprising the whole sensory-motor circuit may contribute to motor neuron dysfunction and loss. Here, we used induced pluripotent stem cells derived from SMA patients to test whether sensory neurons directly contribute to motor neuron loss. We generated sensory neurons from SMA induced pluripotent stem cells and found no difference in neuron generation or survival, although there was a reduced calcium response to depolarizing stimuli. Using co-culture of SMA induced pluripotent stem cell derived sensory neurons with control induced pluripotent stem cell derived motor neurons, we found no significant reduction in motor neuron number or glutamate transporter boutons on motor neuron cell bodies or neurites. We conclude that SMA sensory neurons do not overtly contribute to motor neuron loss in this human stem cell system.

  5. Target cell-specific modulation of neuronal activity by astrocytes

    OpenAIRE

    Kozlov, A. S.; Angulo, M. C.; Audinat, E.; Charpak, S

    2006-01-01

    Interaction between astrocytes and neurons enriches the behavior of brain circuits. By releasing glutamate and ATP, astrocytes can directly excite neurons and modulate synaptic transmission. In the rat olfactory bulb, we demonstrate that the release of GABA by astrocytes causes long-lasting and synchronous inhibition of mitral and granule cells. In addition, astrocytes release glutamate, leading to a selective activation of granule-cell NMDA receptors. Thus, by releasing excitatory and inhibi...

  6. Citalopram increases the differentiation efficacy of bone marrow mesenchymal stem cells into neuronal-like cells

    OpenAIRE

    Verdi, Javad; Mortazavi-Tabatabaei, Seyed AbdolReza; Sharif, Shiva; Verdi, Hadi; Shoae-Hassani, Alireza

    2014-01-01

    Several studies have demonstrated that selective serotonin reuptake inhibitor antidepressants can promote neuronal cell proliferation and enhance neuroplasticity both in vitro and in vivo. It is hypothesized that citalopram, a selective serotonin reuptake inhibitor, can promote the neuronal differentiation of adult bone marrow mesenchymal stem cells. Citalopram strongly enhanced neuronal characteristics of the cells derived from bone marrow mesenchymal stem cells. The rate of cell death was d...

  7. Sambucus williamsii induced embryonic stem cells differentiated into neurons.

    Science.gov (United States)

    Liu, Shih-Ping; Hsu, Chien-Yu; Fu, Ru-Huei; Huang, Yu-Chuen; Chen, Shih-Yin; Lin, Shinn-Zong; Shyu, Woei-Cherng

    2015-01-01

    The pluripotent stem cells, including embryonic stem cells (ESCs), are capable of self-renewal and differentiation into any cell type, thus making them the focus of many clinical application studies. However, the efficiency of ESCs differentiated into neurons needs to improve. In this study, we tried to increase efficiently to a neural fate in the presence of various transitional Chinese medicines through a three-step differentiation strategy. From extracts of 10 transitional Chinese medicine candidates, we determined that Sambucus williamsii (SW) extract triggers the up-regulation of Nestin and Tuj1 (neuron cells markers) gene expression levels. After determining the different concentrations of SW extract, the number of neurons in the 200 μg/ml SW extract group was higher than the control, 50, 100, and 400 μg/ml SW extract groups. In addition, the number of neurons in the 200 μg/ml SW extract group was higher and higher after each time passage (three times). We also detected the Oct4, Sox2 (stem cells markers), Tuj1, and Nestin genes expression levels by RT-PCR. In the differentiated process, Oct4 and Sox2 genes decreased while the Tuj1 and Nestin genes expression levels increased. In summary, we demonstrated that SW could induce pluripotent stem cells differentiated into neurons. Thus, SW might become a powerful material for neurons-differentiating strategies.

  8. Citalopram increases the differentiation efifcacy of bone marrow mesenchymal stem cells into neuronal-like cells

    Institute of Scientific and Technical Information of China (English)

    Javad Verdi; Seyed Abdolreza Mortazavi-Tabatabaei; Shiva Sharif; Hadi Verdi; Alireza Shoae-Hassani

    2014-01-01

    Several studies have demonstrated that selective serotonin reuptake inhibitor antidepressants can promote neuronal cell proliferation and enhance neuroplasticity both in vitro and in vivo. It is hypothesized that citalopram, a selective serotonin reuptake inhibitor, can promote the neuronal differentiation of adult bone marrow mesenchymal stem cells. Citalopram strongly enhanced neuronal characteristics of the cells derived from bone marrow mesenchymal stem cells. The rate of cell death was decreased in citalopram-treated bone marrow mesenchymal stem cells than in control cells in neurobasal medium. In addition, the cumulative population doubling level of the citalopram-treated cells was signiifcantly increased compared to that of control cells. Also BrdU incorporation was elevated in citalopram-treated cells. These ifndings suggest that citalopram can improve the neuronal-like cell differentiation of bone marrow mesenchymal stem cells by increasing cell proliferation and survival while maintaining their neuronal characteristics.

  9. Directed neuronal differentiation of human embryonic stem cells

    Directory of Open Access Journals (Sweden)

    Noggle Scott A

    2003-10-01

    Full Text Available Abstract Background We have developed a culture system for the efficient and directed differentiation of human embryonic stem cells (HESCs to neural precursors and neurons. HESC were maintained by manual passaging and were differentiated to a morphologically distinct OCT-4+/SSEA-4- monolayer cell type prior to the derivation of embryoid bodies. Embryoid bodies were grown in suspension in serum free conditions, in the presence of 50% conditioned medium from the human hepatocarcinoma cell line HepG2 (MedII. Results A neural precursor population was observed within HESC derived serum free embryoid bodies cultured in MedII conditioned medium, around 7–10 days after derivation. The neural precursors were organized into rosettes comprised of a central cavity surrounded by ring of cells, 4 to 8 cells in width. The central cells within rosettes were proliferating, as indicated by the presence of condensed mitotic chromosomes and by phosphoHistone H3 immunostaining. When plated and maintained in adherent culture, the rosettes of neural precursors were surrounded by large interwoven networks of neurites. Immunostaining demonstrated the expression of nestin in rosettes and associated non-neuronal cell types, and a radial expression of Map-2 in rosettes. Differentiated neurons expressed the markers Map-2 and Neurofilament H, and a subpopulation of the neurons expressed tyrosine hydroxylase, a marker for dopaminergic neurons. Conclusion This novel directed differentiation approach led to the efficient derivation of neuronal cultures from HESCs, including the differentiation of tyrosine hydroxylase expressing neurons. HESC were morphologically differentiated to a monolayer OCT-4+ cell type, which was used to derive embryoid bodies directly into serum free conditions. Exposure to the MedII conditioned medium enhanced the derivation of neural precursors, the first example of the effect of this conditioned medium on HESC.

  10. Direct reprogramming of somatic cells into neural stem cells or neurons for neurological disorders

    Institute of Scientific and Technical Information of China (English)

    Shaoping Hou; Paul Lu

    2016-01-01

    Direct reprogramming of somatic cells into neurons or neural stem cells is one of the most important fron-tier ifelds in current neuroscience research. Without undergoing the pluripotency stage, induced neurons or induced neural stem cells are a safer and timelier manner resource in comparison to those derived from induced pluripotent stem cells. In this prospective, we review the recent advances in generation of induced neurons and induced neural stem cellsin vitro andin vivo and their potential treatments of neurological disorders.

  11. D609 induces vascular endothelial cells and marrow stromal cells differentiation into neuron-like cells

    Institute of Scientific and Technical Information of China (English)

    Nan WANG; Chun-qing DU; Shao-shan WANG; Kun XIE; Shang-li ZHANG; Jun-ying MIAO

    2004-01-01

    AIM: To investigate the effect of tricyclodecane-9-yl-xanthogenate (D609) on cell differentiation in vascular endothelial cells (VECs) and marrow stromal cells (MSCs). METHODS: Morphological changes were observed under phase contrast microscope. Electron microscope and immunostaining were used for VECs identification. The expressions of neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) were examined by immunohistochemistry. RESULTS: After 6 h of induction with D609, some VECs showed morphological changes characteristic of neurones. 9 h later, more VECs became neuron-like cells. About 30.8 % of VECs displayed positive NSE (P<0.01), while the expression of GFAP was negative. When MSCs were exposed to D609, the cells displayed neuronal morphologies, such as pyramidal cell bodies and processes formed extensive networks at 3 h. 6 h later, almost all of the cells exhibited a typical neuronal appearance, and 85.6 % of MSCs displayed intensive positive NSE, but GFAP did not express. CONCLUSION: D609 induces VECs and MSCs differentiation into neuron-like cells.

  12. An experimental electronic model for a neuronal cell

    International Nuclear Information System (INIS)

    Over the last two decades, the study of information transmission in living beings has acquired great relevance, because it regulates and conducts the functioning of all of the organs in the body. In information transmission pathways, the neuron plays an important role in that it receives, transmits, and processes electrical signals from different parts of the human body; these signals are transmitted as electrical impulses called action potentials, and they transmit information from one neuron to another. In this work, and with the aim of developing experiments for teaching biological processes, we implemented an electronic circuit of the neuron cell device and its mathematical model based on piecewise linear functions. (paper)

  13. Strategies for metabolic exchange between glial cells and neurons.

    Science.gov (United States)

    Deitmer, J W

    2001-12-01

    The brain is a major energy consumer and dependent on carbohydrate and oxygen supply. Electrical and synaptic activity of neurons can only be sustained given sufficient availability of ATP. Glial cells, which have long been assigned trophic functions, seem to play a pivotal role in meeting the energy requirements of active neurons. Under conditions of high neuronal activity, a number of glial functions, such as the maintenance of ion homeostasis, neurotransmitter clearance from synaptic domains, the supply of energetic compounds and calcium signalling, are challenged. In the vertebrate brain, astrocytes may increase glucose utilization and release lactate, which is taken up and consumed by neurons to generate ATP by oxidative metabolism. The CO(2) produced is processed primarily in astrocytes, which display the major activity of carboanhydrase in the brain. Protons and bicarbonate in turn may contribute to drive acid/base-coupled transporters. In the present article a scenario is discussed which couples the transfer of energy and the conversion of CO(2) with the high-affinity glutamate uptake and other transport processes at glial and neuronal cell membranes. The transporters can be linked to glial signalling and may cooperate with each other at the cellular level. This could save energy, and would render energy exchange processes between glial cells and neurons more effective. Functions implications and physiological responses, in particular in chemosensitive brain areas, are discussed. PMID:11738647

  14. Strategies for metabolic exchange between glial cells and neurons.

    Science.gov (United States)

    Deitmer, J W

    2001-12-01

    The brain is a major energy consumer and dependent on carbohydrate and oxygen supply. Electrical and synaptic activity of neurons can only be sustained given sufficient availability of ATP. Glial cells, which have long been assigned trophic functions, seem to play a pivotal role in meeting the energy requirements of active neurons. Under conditions of high neuronal activity, a number of glial functions, such as the maintenance of ion homeostasis, neurotransmitter clearance from synaptic domains, the supply of energetic compounds and calcium signalling, are challenged. In the vertebrate brain, astrocytes may increase glucose utilization and release lactate, which is taken up and consumed by neurons to generate ATP by oxidative metabolism. The CO(2) produced is processed primarily in astrocytes, which display the major activity of carboanhydrase in the brain. Protons and bicarbonate in turn may contribute to drive acid/base-coupled transporters. In the present article a scenario is discussed which couples the transfer of energy and the conversion of CO(2) with the high-affinity glutamate uptake and other transport processes at glial and neuronal cell membranes. The transporters can be linked to glial signalling and may cooperate with each other at the cellular level. This could save energy, and would render energy exchange processes between glial cells and neurons more effective. Functions implications and physiological responses, in particular in chemosensitive brain areas, are discussed.

  15. Neuroprotection Signaling of Nuclear Akt in Neuronal Cells

    OpenAIRE

    Ahn, Jee-Yin

    2014-01-01

    Akt is one of the central kinases that perform a pivotal function in mediating survival signaling in a wide range of neuronal cell types in response to growth factor stimulation. The recent findings of a number of targets for Akt suggest that it prohibits neuronal death by both impinging on the cytoplasmic cell death machinery and by regulating nuclear proteins. The presence of active Akt in the nuclei of mammalian cells is no longer debatable, and this has been corroborated by the finding of...

  16. Ultrastructure of neuronal-like cells differentiated from adult adipose-derived stromal cells

    Institute of Scientific and Technical Information of China (English)

    Changqing Ye; Xiaodong Yuan; Hui Liu; Yanan Cai; Ya Ou

    2010-01-01

    β-mercaptoethanol induces in vitro adult adipose-derived stromal cells (ADSCs) to differentiate into neurons. However, the ultrastructural features of the differentiated neuronal-like cells remain unknown. In the present study, inverted phase contrast microscopy was utilized to observe β-mercaptcethanol-induced differentiation of neuronal-like cells from human ADSCs, and immunocytochemistry and real-time polymerase chain reaction were employed to detect expression of a neural stem cells marker (nestin), a neuronal marker (neuron-specific enolase), and a glial marker (glial fibrillary acidic protein). In addition, ultrastructure of neuronal-like cells was observed by transmission election microscopy. Results revealed highest expression rate of nestin and neuron-specific enolase at 3 and 5 hours following induced differentiation; cells in the 5-hour induction group exhibited a neuronal-specific structure, i.e., Nissl bodies. However, when induction solution was replaced by complete culture medium after 8-hour induction, the differentiated cells reverted to the fibroblast-like morphology from day 1. These results demonstrate that β-mercaptoethanol-induced ADSCs induced differentiation into neural stem cells, followed by morphology of neuronal-like cells. However, this differentiation state was not stable.

  17. Optical Imaging for Stem Cell Differentiation to Neuronal Lineage

    Energy Technology Data Exchange (ETDEWEB)

    Hwang, Do Won; Lee, Dong Soo [Seoul National Univ., Seoul (Korea, Republic of)

    2012-03-15

    In regenerative medicine, the prospect of stem cell therapy hold great promise for the recovery of injured tissues and effective treatment of intractable diseases. Tracking stem cell fate provides critical information to understand and evaluate the success of stem cell therapy. The recent emergence of in vivo noninvasive molecular imaging has enabled assessment of the behavior of grafted stem cells in living subjects. In this review, we provide an overview of current optical imaging strategies based on cell or tissue specific reporter gene expression and of in vivo methods to monitor stem cell differentiation into neuronal lineages. These methods use optical reporters either regulated by neuron-specific promoters or containing neuron-specific microRNA binding sites. Both systems revealed dramatic changes in optical reporter imaging signals in cells differentiating a yeast GAL4 amplification system or an engineering-enhanced luciferase reported gene. Furthermore, we propose an advanced imaging system to monitor neuronal differentiation during neurogenesis that uses in vivo multiplexed imaging techniques capable of detecting several targets simultaneously.

  18. Information in a Network of Neuronal Cells: Effect of Cell Density and Short-Term Depression

    OpenAIRE

    Onesto, Valentina; Cosentino, Carlo; Di Fabrizio, Enzo; Cesarelli, Mario; Amato, Francesco; Gentile, Francesco

    2016-01-01

    Neurons are specialized, electrically excitable cells which use electrical to chemical signals to transmit and elaborate information. Understanding how the cooperation of a great many of neurons in a grid may modify and perhaps improve the information quality, in contrast to few neurons in isolation, is critical for the rational design of cell-materials interfaces for applications in regenerative medicine, tissue engineering, and personalized lab-on-a-chips. In the present paper, we couple an...

  19. Neural Progenitor Cells Derived from Human Embryonic Stem Cells as an Origin of Dopaminergic Neurons

    Directory of Open Access Journals (Sweden)

    Parinya Noisa

    2015-01-01

    Full Text Available Human embryonic stem cells (hESCs are able to proliferate in vitro indefinitely without losing their ability to differentiate into multiple cell types upon exposure to appropriate signals. Particularly, the ability of hESCs to differentiate into neuronal subtypes is fundamental to develop cell-based therapies for several neurodegenerative disorders, such as Alzheimer’s disease, Huntington’s disease, and Parkinson’s disease. In this study, we differentiated hESCs to dopaminergic neurons via an intermediate stage, neural progenitor cells (NPCs. hESCs were induced to neural progenitor cells by Dorsomorphin, a small molecule that inhibits BMP signalling. The resulting neural progenitor cells exhibited neural bipolarity with high expression of neural progenitor genes and possessed multipotential differentiation ability. CBF1 and bFGF responsiveness of these hES-NP cells suggested their similarity to embryonic neural progenitor cells. A substantial number of dopaminergic neurons were derived from hES-NP cells upon supplementation of FGF8 and SHH, key dopaminergic neuron inducers. Importantly, multiple markers of midbrain neurons were detected, including NURR1, PITX3, and EN1, suggesting that hESC-derived dopaminergic neurons attained the midbrain identity. Altogether, this work underscored the generation of neural progenitor cells that retain the properties of embryonic neural progenitor cells. These cells will serve as an unlimited source for the derivation of dopaminergic neurons, which might be applicable for treating patients with Parkinson’s disease.

  20. Characterization of PrP(Sc) transmission from immune cells to neuronal cells.

    Science.gov (United States)

    Tanaka, Yufuko; Sadaike, Tetsuji; Inoshima, Yasuo; Ishiguro, Naotaka

    2012-10-01

    We investigated PrP(Sc) transmission in neuronal cells, spleen cells and several immune cells using an in vitro cell-to-cell transmission system. The transmission of PrP(Sc) in the supernatant of PrP(Sc)-infected neuronal cells was also investigated. We found that PrP(Sc) transmission was more efficient in the cell-to-cell transmission system than in the supernatant-mediated system. PrP(Sc) was more efficiently transmitted from adherent spleen cells to neuronal cells than from floating spleen cells. The adherent spleen cells were composed of macrophages (80%), dendritic cells (8%) and follicular dendritic cells (3%), indicating that macrophages play an important role in PrP(Sc) transmission from immune cells to neuronal cells. Although PrP(Sc) in the immune cells used as donor cells was gradually degraded, the PrP(Sc) transmitted to neuronal cells was observed by Western blot analysis. Investigation of the mechanism of PrP(Sc) transmission between cells represents an important step towards understanding the pathogenesis of prion diseases. PMID:23246505

  1. Neuron-NG2 Cell Synapses: Novel Functions for Regulating NG2 Cell Proliferation and Differentiation

    Directory of Open Access Journals (Sweden)

    Qian-Kun Yang

    2013-01-01

    Full Text Available NG2 cells are a population of CNS cells that are distinct from neurons, mature oligodendrocytes, astrocytes, and microglia. These cells can be identified by their NG2 proteoglycan expression. NG2 cells have a highly branched morphology, with abundant processes radiating from the cell body, and express a complex set of voltage-gated channels, AMPA/kainate, and GABA receptors. Neurons notably form classical and nonclassical synapses with NG2 cells, which have varied characteristics and functions. Neuron-NG2 cell synapses could fine-tune NG2 cell activities, including the NG2 cell cycle, differentiation, migration, and myelination, and may be a novel potential therapeutic target for NG2 cell-related diseases, such as hypoxia-ischemia injury and periventricular leukomalacia. Furthermore, neuron-NG2 cell synapses may be correlated with the plasticity of CNS in adulthood with the synaptic contacts passing onto their progenies during proliferation, and synaptic contacts decrease rapidly upon NG2 cell differentiation. In this review, we highlight the characteristics of classical and nonclassical neuron-NG2 cell synapses, the potential functions, and the fate of synaptic contacts during proliferation and differentiation, with the emphasis on the regulation of the NG2 cell cycle by neuron-NG2 cell synapses and their potential underlying mechanisms.

  2. Novel culturing platform for brain slices and neuronal cells

    DEFF Research Database (Denmark)

    Svendsen, Winnie Edith; Al Atraktchi, Fatima Al-Zahraa; Bakmand, Tanya;

    2015-01-01

    In this paper we demonstrate a novel culturing system for brain slices and neuronal cells, which can control the concentration of nutrients and the waste removal from the culture by adjusting the fluid flow within the device. The entire system can be placed in an incubator. The system has been te...

  3. Neuron-mediated generation of regulatory T cells from encephalitogenic T cells suppresses EAE

    DEFF Research Database (Denmark)

    Liu, Yawei; Teige, Ingrid; Birnir, Bryndis;

    2006-01-01

    Neurons have been neglected as cells with a major immune-regulatory function because they do not express major histocompatibility complex class II. Our data show that neurons are highly immune regulatory, having a crucial role in governing T-cell response and central nervous system (CNS......) inflammation. Neurons induce the proliferation of activated CD4+ T cells through B7-CD28 and transforming growth factor (TGF)-beta1-TGF-beta receptor signaling pathways, resulting in amplification of T-cell receptor signaling through phosphorylated ZAP-70, interleukin (IL)-2 and IL-9. The interaction between...... neurons and T cells results in the conversion of encephalitogenic T cells to CD25+ TGF-beta1+ CTLA-4+ FoxP3+ T regulatory (Treg) cells that suppress encephalitogenic T cells and inhibit experimental autoimmune encephalomyelitis. Suppression is dependent on cytotoxic T lymphocyte antigen (CTLA)-4 but not...

  4. Signals mediating Klotho-induced neuroprotection in hippocampal neuronal cells.

    Science.gov (United States)

    Cheng, Meng-Fu; Chen, Li-Jen; Niu, Ho-Shan; Yang, Ting-Ting; Lin, Kao-Chang; Cheng, Juei-Tang

    2015-01-01

    The erythropoietin (Epo) receptor (EpoR) is expressed in the brain and was shown to have neuroprotective effects against brain damage in animal models. A recent study indicated that EpoR and its activity are the downstream effectors of Klotho for cytoprotection in the kidney. Thus, we propose that Klotho can stimulate the expression of EpoR in neuronal cells to enhance Epo-mediated protection. H19-7 hippocampal neuronal cells were treated with recombinant Klotho. In H19-7 cells, Klotho increased the expression of both the EpoR protein and mRNA. Klotho also enhanced the transcription activity of the EpoR promoter in H19-7 cells. Moreover, Klotho augmented the Epo-triggered phosphorylation of Jak2 and Stat5 and protected H19-7 cells from hydrogen peroxide cytotoxicity. The silencing of EpoR abolished the protective effect of Klotho against peroxide-induced cytotoxicity. Finally, the silencing of GATA1 diminished the Klotho-induced increase in EpoR protein and mRNA expression as well as its promoter activity. In conclusion, Klotho increased EpoR expression in neuronal cells through GATA1, thereby enabling EpoR to function as a cytoprotective protein against oxidative injury. PMID:25856523

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  1. Photoresist Derived Carbon for Growth and Differentiation of Neuronal Cells

    Directory of Open Access Journals (Sweden)

    Tie Zou

    2007-08-01

    Full Text Available Apoptosis or necrosis of neurons in the central nervous system (CNS is thehallmark of many neurodegenerative diseases and Traumatic Brain Injury (TBI. Theinability to regenerate in CNS offers little hope for naturally repairing the damagedneurons. However, with the rapid development of new technologies, regenerative medicineoffers great promises to patients with these disorders. Among many events for furtheradvancement of regenerative medicine, extracellular matrix (ECM plays a critical role forcellular migration and differentiation. To develop a biocompatible and electricallyconductive substrate that can be potentially used to promote growth and regeneration ofneurons and to record intracellular and multisite signals from brain as a probe, a polymericprecursor – SPR 220.7 was fabricated by pyrolysis at temperatures higher than 700 oC.Human Neuroblastoma cells - SK-N-MC, SY5Y, mouse teratocarcinoma cells P-19 and ratPC12 cells were found to attach and proliferate on photoresist derived carbon film.Significantly, neuronal differentiation of PC12 cells induced by NGF was demonstrated byobserving cell shape and size, and measuring the length of neurites under SEM. Our resultsindicated that fabricated carbon could potentially be explored in regenerative medicine forpromoting neuronal growth and differentiation in CNS with neurodegeneration.

  2. TRIM32-dependent transcription in adult neural progenitor cells regulates neuronal differentiation

    OpenAIRE

    Hillje, Anna-Lena; Pavlou, Maria Angeliki; Beckmann, Elisabeth; Worlitzer, Maik; Bahnassawy, Lamiaa; Lewejohann, Lars; Palm, Thomas; Schwamborn, Jens Christian

    2013-01-01

    In the adult mammalian brain, neural stem cells in the subventricular zone continuously generate new neurons for the olfactory bulb. Cell fate commitment in these adult neural stem cells is regulated by cell fate-determining proteins. Here, we show that the cell fate-determinant TRIM32 is upregulated during differentiation of adult neural stem cells into olfactory bulb neurons. We further demonstrate that TRIM32 is necessary for the correct induction of neuronal differentiation in these cells...

  3. Cellular Programming and Reprogramming: Sculpting Cell Fate for the Production of Dopamine Neurons for Cell Therapy

    Directory of Open Access Journals (Sweden)

    Julio C. Aguila

    2012-01-01

    success of clinical applications depends on our ability to steer pluripotent stem cells towards the right neuronal identity. In Parkinson disease, the loss of dopamine neurons is more pronounced in the ventrolateral population that projects to the sensorimotor striatum. Because synapses are highly specific, only neurons with this precise identity will contribute, upon transplantation, to the synaptic reconstruction of the dorsal striatum. Thus, understanding the developmental cell program of the mesostriatal dopamine neurons is critical for the identification of the extrinsic signals and cell-intrinsic factors that instruct and, ultimately, determine cell identity. Here, we review how extrinsic signals and transcription factors act together during development to shape midbrain cell fates. Further, we discuss how these same factors can be applied in vitro to induce, select, and reprogram cells to the mesostriatal dopamine fate.

  4. Neuronal cell fate decisions:  O2 and CO2 sensing neurons require egl-13/Sox5

    DEFF Research Database (Denmark)

    Gramstrup Petersen, Jakob; Pocock, Roger David John

    2013-01-01

    We recently conducted a study that aimed to describe the differentiation mechanisms used to generate O2 and CO2 sensing neurons in C. elegans. We identified egl-13/Sox5 to be required for the differentiation of both O2 and CO2 sensing neurons. We found that egl-13 functions cell autonomously...

  5. Tff3 is Expressed in Neurons and Microglial Cells

    Directory of Open Access Journals (Sweden)

    Ting Fu

    2014-11-01

    Full Text Available Background/Aims: The trefoil factor family (TFF peptide TFF3 is typically secreted by mucous epithelia, but is also expressed in the immune system and the brain. It was the aim of this study to determine the cerebral cell types which express Tff3. Methods: Primary cultures from rat embryonic or neonatal cerebral cortex and hippocampus, respectively, were studied by means of RT-PCR and immunofluorescence. Moreover, Tff3 expression was localized by immunocytochemistry in sections of adult rat cerebellum. Results: Tff3 transcripts were detectable in neural cultures of both the cortex and the hippocampus as well as in glial cell-enriched cultures. Tff3 peptide co-localized with Map2 indicating an expression in neurons in vitro. The neuronal expression was confirmed by immunofluorescence studies of adult rat cerebellum. Furthermore, Tff3 peptide showed also a clear co-localization with Iba-1 in vitro typical of activated microglial cells. Conclusion: The neuronal expression of Tff3 is in line with a function of a typical neuropeptide influencing, e.g., fear, memory, depression and motoric skills. The expression in activated microglial cells, which is demonstrated here for the first time, points towards a possible function for Tff3 in immune reactions in the CNS. This opens a plethora of additional possible functions for Tff3 including synaptic plasticity and cognition as well as during neuroinflammatory diseases and psychiatric disorders.

  6. Bee Venom Protects against Rotenone-Induced Cell Death in NSC34 Motor Neuron Cells.

    Science.gov (United States)

    Jung, So Young; Lee, Kang-Woo; Choi, Sun-Mi; Yang, Eun Jin

    2015-09-01

    Rotenone, an inhibitor of mitochondrial complex I of the mitochondrial respiratory chain, is known to elevate mitochondrial reactive oxygen species and induce apoptosis via activation of the caspase-3 pathway. Bee venom (BV) extracted from honey bees has been widely used in oriental medicine and contains melittin, apamin, adolapin, mast cell-degranulating peptide, and phospholipase A₂. In this study, we tested the effects of BV on neuronal cell death by examining rotenone-induced mitochondrial dysfunction. NSC34 motor neuron cells were pretreated with 2.5 μg/mL BV and stimulated with 10 μM rotenone to induce cell toxicity. We assessed cell death by Western blotting using specific antibodies, such as phospho-ERK1/2, phospho-JNK, and cleaved capase-3 and performed an MTT assay for evaluation of cell death and mitochondria staining. Pretreatment with 2.5 μg/mL BV had a neuroprotective effect against 10 μM rotenone-induced cell death in NSC34 motor neuron cells. Pre-treatment with BV significantly enhanced cell viability and ameliorated mitochondrial impairment in rotenone-treated cellular model. Moreover, BV treatment inhibited the activation of JNK signaling and cleaved caspase-3 related to cell death and increased ERK phosphorylation involved in cell survival in rotenone-treated NSC34 motor neuron cells. Taken together, we suggest that BV treatment can be useful for protection of neurons against oxidative stress or neurotoxin-induced cell death. PMID:26402700

  7. Bee Venom Protects against Rotenone-Induced Cell Death in NSC34 Motor Neuron Cells

    Directory of Open Access Journals (Sweden)

    So Young Jung

    2015-09-01

    Full Text Available Rotenone, an inhibitor of mitochondrial complex I of the mitochondrial respiratory chain, is known to elevate mitochondrial reactive oxygen species and induce apoptosis via activation of the caspase-3 pathway. Bee venom (BV extracted from honey bees has been widely used in oriental medicine and contains melittin, apamin, adolapin, mast cell-degranulating peptide, and phospholipase A2. In this study, we tested the effects of BV on neuronal cell death by examining rotenone-induced mitochondrial dysfunction. NSC34 motor neuron cells were pretreated with 2.5 μg/mL BV and stimulated with 10 μM rotenone to induce cell toxicity. We assessed cell death by Western blotting using specific antibodies, such as phospho-ERK1/2, phospho-JNK, and cleaved capase-3 and performed an MTT assay for evaluation of cell death and mitochondria staining. Pretreatment with 2.5 μg/mL BV had a neuroprotective effect against 10 μM rotenone-induced cell death in NSC34 motor neuron cells. Pre-treatment with BV significantly enhanced cell viability and ameliorated mitochondrial impairment in rotenone-treated cellular model. Moreover, BV treatment inhibited the activation of JNK signaling and cleaved caspase-3 related to cell death and increased ERK phosphorylation involved in cell survival in rotenone-treated NSC34 motor neuron cells. Taken together, we suggest that BV treatment can be useful for protection of neurons against oxidative stress or neurotoxin-induced cell death.

  8. Innervation of Cochlear Hair Cells by Human Induced Pluripotent Stem Cell-Derived Neurons In Vitro

    Science.gov (United States)

    Gunewardene, Niliksha; Crombie, Duncan; Dottori, Mirella; Nayagam, Bryony A.

    2016-01-01

    Induced pluripotent stem cells (iPSCs) may serve as an autologous source of replacement neurons in the injured cochlea, if they can be successfully differentiated and reconnected with residual elements in the damaged auditory system. Here, we explored the potential of hiPSC-derived neurons to innervate early postnatal hair cells, using established in vitro assays. We compared two hiPSC lines against a well-characterized hESC line. After ten days' coculture in vitro, hiPSC-derived neural processes contacted inner and outer hair cells in whole cochlear explant cultures. Neural processes from hiPSC-derived neurons also made contact with hair cells in denervated sensory epithelia explants and expressed synapsin at these points of contact. Interestingly, hiPSC-derived neurons cocultured with hair cells at an early stage of differentiation formed synapses with a higher number of hair cells, compared to hiPSC-derived neurons cocultured at a later stage of differentiation. Notable differences in the innervation potentials of the hiPSC-derived neurons were also observed and variations existed between the hiPSC lines in their innervation efficiencies. Collectively, these data illustrate the promise of hiPSCs for auditory neuron replacement and highlight the need to develop methods to mitigate variabilities observed amongst hiPSC lines, in order to achieve reliable clinical improvements for patients. PMID:26966437

  9. Human Embryonic Stem Cell-Derived Dopaminergic Neurons Reverse Functional Deficit in Parkinsonian Rats

    OpenAIRE

    Yang, Dali; Zhang, Zhi-jian; Oldenburg, Michael; Ayala, Melvin; Zhang, Su-Chun

    2007-01-01

    We show that human embryonic stem cell-derived dopaminergic neurons survived transplantation to the neurotoxin 6-hydroxydopamine-lesioned rat striatum and, in combination with the cells newly differentiated from their progenitors, contributed to locomotive function recovery at 5 months. The animal behavioral improvement was correlated with the dopamine neurons present in the graft. Although the donor cells contained forebrain and midbrain dopamine neurons, the dopamine neurons present in the ...

  10. Comprehensive qPCR profiling of gene expression in single neuronal cells

    OpenAIRE

    Citri, Ami; Pang, Zhiping P.; Sudhof, Thomas C.; Wernig, Marius; Malenka, Robert C.

    2011-01-01

    A major challenge in neuronal stem cell biology lies in characterization of lineage-specific reprogrammed human neuronal cells, a process that necessitates the use of an assay sensitive to the single-cell level. Single-cell gene profiling can provide definitive evidence regarding the conversion of one cell type into another at a high level of resolution. The protocol we describe employs Fluidigm Biomark dynamic arrays for high-throughput expression profiling from single neuronal cells, assayi...

  11. Making Human Neurons from Stem Cells after Spinal Cord Injury

    OpenAIRE

    Jun Yan; Leyan Xu; Welsh, Annie M; Glen Hatfield; Thomas Hazel; Karl Johe; Koliatsos, Vassilis E.

    2007-01-01

    Editors' Summary Background. Every year, spinal cord injuries, many caused by road traffic accidents, paralyze about 11,000 people in the US. This paralysis occurs because the spinal cord is the main communication highway between the body and the brain. Information from the skin and other sensory organs is transmitted to the brain along the spinal cord by bundles of neurons, nervous system cells that transmit and receive messages. The brain then sends information back down the spinal cord to ...

  12. Pneumolysin causes neuronal cell death through mitochondrial damage

    OpenAIRE

    Braun, Johann S.; Hoffmann, Olaf; Schickhaus, Miriam; Freyer, Dorette; Dagand, Emilie; Bermpohl, Daniela; Mitchell, Tim J.; Bechmann, Ingo; Weber, Joerg R.

    2007-01-01

    Bacterial toxins such as pneumolysin are key mediators of cytotoxicity in infections. Pneumolysin is a pore-forming toxin released by Streptococcus pneumoniae, the major cause of bacterial meningitis. We found that pneumolysin is the pneumococcal factor that accounts for the cell death pathways induced by live bacteria in primary neurons. The pore-forming activity of pneumolysin is essential for the induction of mitochondrial damage and apoptosis. Pneumolysin colocalized with mitochondrial me...

  13. Transient maintenance in bioreactor improves health of neuronal cells.

    Science.gov (United States)

    Di Loreto, Silvia; Sebastiani, Pierluigi; Benedetti, Elisabetta; Zimmitti, Vincenzo; Caracciolo, Valentina; Amicarelli, Fernanda; Cimini, Annamaria; Adorno, Domenico

    2006-01-01

    To examine whether a neuronal cell suspension can be held in vitro for a relatively short period without compromising survival rates and functionality, we have set up an experimental protocol planning 24 h of suspension culture in a rotary wall vessel (RWV) bioreactor before plating in a conventional adherent system. Apoptosis measurement and activated caspase-8, -9, and -3 detection have demonstrated that survey of the cells was not affected. The activity of major antioxidant enzymes (AOE), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT), was significantly decreased in RWV-maintained cells. A significant decrease of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) is coupled with a level of activated nuclear factor-kappaB (NF-kappaB) protein significantly lower in RVW cells than in the control. On the contrary, the level of IL-6 expression did not change between the test and the control. A significant up-regulation of growth-associated protein-43 (GAP-43), peroxisome proliferator-activated receptor-beta/delta (PPARbeta/delta), and acyl-CoA synthetase 2 (ACS2) in RWV cells has been detected. We provide the evidence that primary neuronal cells, at an early stage of development, can be maintained in a suspension condition before adherent plating. This experimental environment does not induce detrimental effects but may have an activator role, leading cells to development and maturation in a tridimensional state.

  14. Reduced synaptic activity in neuronal networks derived from embryonic stem cells of murine Rett syndrome model

    OpenAIRE

    Lydia eBarth; Rosmarie eSütterlin; Markus eNenniger; Kaspar Emanuel Vogt

    2014-01-01

    Neurodevelopmental diseases such as the Rett syndrome have received renewed attention, since the mechanisms involved may underlie a broad range of neuropsychiatric disorders such as schizophrenia and autism. In vertebrates early stages in the functional development of neurons and neuronal networks are difficult to study. Embryonic stem cell-derived neurons provide an easily accessible tool to investigate neuronal differentiation and early network formation. We used in vitro cultures of neuron...

  15. Neuronal classification and marker gene identification via single-cell expression profiling of brainstem vestibular neurons subserving cerebellar learning

    OpenAIRE

    Kodama, Takashi; Guerrero, Shiloh; Shin, Minyoung; Moghadam, Seti; Faulstich, Michael; du Lac, Sascha

    2012-01-01

    Identification of marker genes expressed in specific cell types is essential for the genetic dissection of neural circuits. Here we report a new strategy for classifying heterogeneous populations of neurons into functionally distinct types and for identifying associated marker genes. Quantitative single-cell expression profiling of genes related to neurotransmitters and ion channels enables functional classification of neurons; transcript profiles for marker gene candidates identify molecular...

  16. Secretory phospholipase A2-mediated neuronal cell death involves glutamate ionotropic receptors

    DEFF Research Database (Denmark)

    de Turco, Elena B; Diemer, Nils Henrik; Bazan, Nicolas G;

    2002-01-01

    To define the significance of glutamate ionotropic receptors in sPLA -mediated neuronal cell death we used the NMDA receptor antagonist MK-801 and the AMPA receptor antagonist PNQX. In primary neuronal cell cultures both MK-801 and PNQX inhibited sPLA - and glutamate-induced neuronal death. [ H]A...

  17. Rapid method for culturing embryonic neuron-glial cell cocultures

    DEFF Research Database (Denmark)

    Svenningsen, Åsa Fex; Shan, Wei-Song; Colman, David R;

    2003-01-01

    A streamlined, simple technique for primary cell culture from E17 rat tissue is presented. In an attempt to standardize culturing methods for all neuronal cell types in the embryo, we evaluated a commercial medium without serum and used similar times for trypsinization and tested different surfaces...... for plating. In 1 day, using one method and a single medium, it is possible to produce robust E17 cultures of dorsal root ganglia (DRG), cerebellum, and enteric plexi. Allowing the endogenous glial cells to repopulate the cultures saves time compared with existing techniques, in which glial cells are added...... to cultures first treated with antimitotic agents. It also ensures that all the cells present in vivo will be present in the culture. Myelination commences after approximately 2 weeks in culture for dissociated DRG and 3-4 weeks in cerebellar cultures. In enteric cultures, glial wrapping of the enteric...

  18. Reinnervation of hair cells by neural stem cell-derived neurons

    Institute of Scientific and Technical Information of China (English)

    Yuan Yasheng; Wang Yang; Chi Fanglu

    2014-01-01

    Background Replacement of spiral ganglion neurons would be one prioritized step in an attempt to restore sensory neuronal hearing loss.However,the possibility that transplanted neurons could regenerate new synaptic connections to hair cells has not been explored.The objective of this study was to test whether neural stem cell (NSC)-derived neurons can form synaptic connections with hair cells in vitro.Methods NSCs were mechanically separated from the hippocampus in SD rat embryos (E12-E14) and cultured in a serum-free medium containing basic fibroblast growth factor and epidermal growth factor.Rat NSCs were co-cultured with explants of cochlea sensory epithelia obtained from postnatal Day 3 rats under transway filter membrane.Results At Day 3,the NSCs began to show chemotactic differentiation and grew toward cochlea sensory epithelia.After 9-day co-culture,neurites of NSC-derived neurons predominantly elongated toward hair cells.Immunohistochemical analyses revealed the fibers overlapped with synapsin and hair cells,indicating the formation of new synaptic connections.After 14-day culture,triple staining revealed the fibers overlapped with PSD95 (postsynaptic density) which is juxtaposed with CtBP2 (presynaptic vesicle),indicating the formation of new ribbon synapse.Conclusions NSC-derived neurons can make synaptic connections with hair cells and provide a model for studying synaptic plasticity and regeneration.Whether the newly forming synapse is functional merits further electrophysiological study.

  19. Single neuron transcriptome analysis can reveal more than cell type classification: Does it matter if every neuron is unique?

    Science.gov (United States)

    Harbom, Lise J; Chronister, William D; McConnell, Michael J

    2016-02-01

    A recent single cell mRNA sequencing study by Dueck et al. compares neuronal transcriptomes to the transcriptomes of adipocytes and cardiomyocytes. Single cell omic approaches such as those used by the authors are at the leading edge of molecular and biophysical measurement. Many groups are currently employing single cell sequencing approaches to understand cellular heterogeneity in cancer and during normal development. These single cell approaches also are beginning to address long-standing questions regarding nervous system diversity. Beyond an innate interest in cataloging cell type diversity in the brain, single cell neuronal diversity has important implications for neurotypic neural circuit function and for neurological disease. Herein, we review the authors' methods and findings, which most notably include evidence of unique expression profiles in some single neurons. PMID:26749010

  20. The Specification and Maturation of Nociceptive Neurons from Human Embryonic Stem Cells

    OpenAIRE

    Erin M. Boisvert; Engle, Sandra J; Shawn E. Hallowell; Ping Liu; Zhao-Wen Wang; Xue-Jun Li

    2015-01-01

    Nociceptive neurons play an essential role in pain sensation by transmitting painful stimuli to the central nervous system. However, investigations of nociceptive neuron biology have been hampered by the lack of accessibility of human nociceptive neurons. Here, we describe a system for efficiently guiding human embryonic stem cells into nociceptive neurons by first inducing these cells to the neural lineage. Subsequent addition of retinoic acid and BMP4 at specific time points and concentrati...

  1. Targeting single neuronal networks for gene expression and cell labeling in vivo.

    Science.gov (United States)

    Marshel, James H; Mori, Takuma; Nielsen, Kristina J; Callaway, Edward M

    2010-08-26

    To understand fine-scale structure and function of single mammalian neuronal networks, we developed and validated a strategy to genetically target and trace monosynaptic inputs to a single neuron in vitro and in vivo. The strategy independently targets a neuron and its presynaptic network for specific gene expression and fine-scale labeling, using single-cell electroporation of DNA to target infection and monosynaptic retrograde spread of a genetically modifiable rabies virus. The technique is highly reliable, with transsynaptic labeling occurring in every electroporated neuron infected by the virus. Targeting single neocortical neuronal networks in vivo, we found clusters of both spiny and aspiny neurons surrounding the electroporated neuron in each case, in addition to intricately labeled distal cortical and subcortical inputs. This technique, broadly applicable for probing and manipulating single neuronal networks with single-cell resolution in vivo, may help shed new light on fundamental mechanisms underlying circuit development and information processing by neuronal networks throughout the brain.

  2. Gene expression pattern of functional neuronal cells derived from human bone marrow mesenchymal stromal cells

    Directory of Open Access Journals (Sweden)

    Bron Dominique

    2008-04-01

    Full Text Available Abstract Background Neuronal tissue has limited potential to self-renew or repair after neurological diseases. Cellular therapies using stem cells are promising approaches for the treatment of neurological diseases. However, the clinical use of embryonic stem cells or foetal tissues is limited by ethical considerations and other scientific problems. Thus, bone marrow mesenchymal stomal cells (BM-MSC could represent an alternative source of stem cells for cell replacement therapies. Indeed, many studies have demonstrated that MSC can give rise to neuronal cells as well as many tissue-specific cell phenotypes. Methods BM-MSC were differentiated in neuron-like cells under specific induction (NPBM + cAMP + IBMX + NGF + Insulin. By day ten, differentiated cells presented an expression profile of real neurons. Functionality of these differentiated cells was evaluated by calcium influx through glutamate receptor AMPA3. Results Using microarray analysis, we compared gene expression profile of these different samples, before and after neurogenic differentiation. Among the 1943 genes differentially expressed, genes down-regulated are involved in osteogenesis, chondrogenesis, adipogenesis, myogenesis and extracellular matrix component (tuftelin, AGC1, FADS3, tropomyosin, fibronectin, ECM2, HAPLN1, vimentin. Interestingly, genes implicated in neurogenesis are increased. Most of them are involved in the synaptic transmission and long term potentialisation as cortactin, CASK, SYNCRIP, SYNTL4 and STX1. Other genes are involved in neurite outgrowth, early neuronal cell development, neuropeptide signaling/synthesis and neuronal receptor (FK506, ARHGAP6, CDKRAP2, PMCH, GFPT2, GRIA3, MCT6, BDNF, PENK, amphiregulin, neurofilament 3, Epha4, synaptotagmin. Using real time RT-PCR, we confirmed the expression of selected neuronal genes: NEGR1, GRIA3 (AMPA3, NEF3, PENK and Epha4. Functionality of these neuron-like cells was demonstrated by Ca2+ influx through glutamate

  3. Radial Glial Cell-Neuron Interaction Directs Axon Formation at the Opposite Side of the Neuron from the Contact Site.

    Science.gov (United States)

    Xu, Chundi; Funahashi, Yasuhiro; Watanabe, Takashi; Takano, Tetsuya; Nakamuta, Shinichi; Namba, Takashi; Kaibuchi, Kozo

    2015-10-28

    How extracellular cues direct axon-dendrite polarization in mouse developing neurons is not fully understood. Here, we report that the radial glial cell (RGC)-cortical neuron interaction directs axon formation at the opposite side of the neuron from the contact site. N-cadherin accumulates at the contact site between the RGC and cortical neuron. Inhibition of the N-cadherin-mediated adhesion decreases this oriented axon formation in vitro, and disrupts the axon-dendrite polarization in vivo. Furthermore, the RGC-neuron interaction induces the polarized distribution of active RhoA at the contacting neurite and active Rac1 at the opposite neurite. Inhibition of Rho-Rho-kinase signaling in a neuron impairs the oriented axon formation in vitro, and prevents axon-dendrite polarization in vivo. Collectively, these results suggest that the N-cadherin-mediated radial glia-neuron interaction determines the contacting neurite as the leading process for radial glia-guided neuronal migration and directs axon formation to the opposite side acting through the Rho family GTPases.

  4. BRCA1 May Modulate Neuronal Cell Cycle Re-Entry in Alzheimer Disease

    OpenAIRE

    Evans, Teresa A.; Raina, Arun K; Delacourte, André; Aprelikova, Olga; Lee, Hyoung-gon; Zhu, Xiongwei; Perry, George; Smith, Mark A.

    2007-01-01

    In Alzheimer disease, neuronal degeneration and the presence of neurofibrillary tangles correlate with the severity of cognitive decline. Neurofibrillary tangles contain the antigenic profile of many cell cycle markers, reflecting a re-entry into the cell cycle by affected neurons. However, while such a cell cycle re-entry phenotype is an early and consistent feature of Alzheimer disease, the mechanisms responsible for neuronal cell cycle are unclear. In this regard, given that a dysregulated...

  5. Efficient Differentiation of Embryonic Stem Cells into Neurons in Glial Cell-conditioned Medium under Attaching Conditions

    Institute of Scientific and Technical Information of China (English)

    Hai-Bin TIAN; Zeng-Liang BAI; Hong WANG; Jian-Quan CHEN; Guo-Xiang CHENG

    2005-01-01

    Embryonic stem (ES) cells can differentiate into neurons in vitro, which provides hope for the treatment of some neurodegenerative diseases through cell transplantation. However, it remains a challenge to efficiently induce ES cells to differentiate into neurons. Here, we show that murine ES cells can efficiently differentiate into neurons when cultured in glial cell- conditioned medium (GCM) under attaching conditions without the formation of embryoid bodies. In comparison with murine embryonic fibroblast-conditioned medium, we found that GCM has a positive effect on limiting the generation of non-neuronal cells, such as astrocytes. In addition, compared with suspension conditions, attaching conditions delay the differentiation process of ES cells.

  6. Non-cell autonomous influence of the astrocyte system xc− on hypoglycaemic neuronal cell death

    Directory of Open Access Journals (Sweden)

    Sandra J Hewett

    2012-02-01

    Full Text Available Despite longstanding evidence that hypoglycaemic neuronal injury is mediated by glutamate excitotoxicity, the cellular and molecular mechanisms involved remain incompletely defined. Here, we demonstrate that the excitotoxic neuronal death that follows GD (glucose deprivation is initiated by glutamate extruded from astrocytes via system xc− – an amino acid transporter that imports l-cystine and exports l-glutamate. Specifically, we find that depriving mixed cortical cell cultures of glucose for up to 8 h injures neurons, but not astrocytes. Neuronal death is prevented by ionotropic glutamate receptor antagonism and is partially sensitive to tetanus toxin. Removal of amino acids during the deprivation period prevents – whereas addition of l-cystine restores – GD-induced neuronal death, implicating the cystine/glutamate antiporter, system xc−. Indeed, drugs known to inhibit system xc− ameliorate GD-induced neuronal death. Further, a dramatic reduction in neuronal death is observed in chimaeric cultures consisting of neurons derived from WT (wild-type mice plated on top of astrocytes derived from sut mice, which harbour a naturally occurring null mutation in the gene (Slc7a11 that encodes the substrate-specific light chain of system xc− (xCT. Finally, enhancement of astrocytic system xc− expression and function via IL-1β (interleukin-1β exposure potentiates hypoglycaemic neuronal death, the process of which is prevented by removal of l-cystine and/or addition of system xc− inhibitors. Thus, under the conditions of GD, our studies demonstrate that astrocytes, via system xc−, have a direct, non-cell autonomous effect on cortical neuron survival.

  7. Non-cell autonomous influence of the astrocyte system xc- on hypoglycaemic neuronal cell death.

    Science.gov (United States)

    Jackman, Nicole A; Melchior, Shannon E; Hewett, James A; Hewett, Sandra J

    2012-02-08

    Despite longstanding evidence that hypoglycaemic neuronal injury is mediated by glutamate excitotoxicity, the cellular and molecular mechanisms involved remain incompletely defined. Here, we demonstrate that the excitotoxic neuronal death that follows GD (glucose deprivation) is initiated by glutamate extruded from astrocytes via system xc---an amino acid transporter that imports L-cystine and exports L-glutamate. Specifically, we find that depriving mixed cortical cell cultures of glucose for up to 8 h injures neurons, but not astrocytes. Neuronal death is prevented by ionotropic glutamate receptor antagonism and is partially sensitive to tetanus toxin. Removal of amino acids during the deprivation period prevents--whereas addition of L-cystine restores--GD-induced neuronal death, implicating the cystine/glutamate antiporter, system xc-. Indeed, drugs known to inhibit system xc- ameliorate GD-induced neuronal death. Further, a dramatic reduction in neuronal death is observed in chimaeric cultures consisting of neurons derived from WT (wild-type) mice plated on top of astrocytes derived from sut mice, which harbour a naturally occurring null mutation in the gene (Slc7a11) that encodes the substrate-specific light chain of system xc- (xCT). Finally, enhancement of astrocytic system xc- expression and function via IL-1β (interleukin-1β) exposure potentiates hypoglycaemic neuronal death, the process of which is prevented by removal of l-cystine and/or addition of system xc- inhibitors. Thus, under the conditions of GD, our studies demonstrate that astrocytes, via system xc-, have a direct, non-cell autonomous effect on cortical neuron survival.

  8. Stem Cells from Human-Exfoliated Deciduous Teeth Can Differentiate into Dopaminergic Neuron-Like Cells

    OpenAIRE

    Wang, Jinsong; Wang, Xuan; Sun, Zuoli; Wang, Xiaomin; Yang, Hui; Shi, Songtao; Wang, Songlin

    2010-01-01

    Stem cells from human exfoliated deciduous teeth (SHED) have been identified as a novel population of postnatal stem cells capable of differentiating into neural cells, odontogenic cells, and adipocytes. SHED were reported to differentiate into neural cells based on cellular morphology and the expression of early neuronal markers when cultured under neural inductive conditions. This study therefore investigated the therapeutic efficacy of SHED in alleviating Parkinson's disease (PD) in a rat ...

  9. Satellite glial cells can promote the extension of neuronal axons in vitro

    Institute of Scientific and Technical Information of China (English)

    Jiu-Hong Zhao; Yi-Di Huang; Xi-Nan Yi; Quan-Peng Zhang; Xian-Fang Zhang; Xu Dong; Gang Luo; Hai-Ying Zhang; Kun-Ju Wang; Mei-Li Lao

    2015-01-01

    Objective: To study the influence of satellite glial cells (SGCs) on the outgrowth of neuronal neurite and the role of Slit1 protein and the contact with neurons in this process, in vitro. Methods: Neurons culture and SGC-neuron co-culture were used as the cell models. The length of axons and dendrites were measured via immunofluorescence to observe the influence of SGCs on the outgrowth of neuronal neurite. The Slit1 protein was added into SGC-neuron co-culture model. The length of dendrites was measured via immunofluorescence at different point times. Result: The anatomical relationship between neurons and SGCs changed as culture period expand. At 12 h after culture, SGCs all surrounded neurons; by 72 h after culture, SGCs were all off neurons. SGCs can promote the growth of neuronal axos, but inhibit the growth of its dendrites; when SGCs closely contact with neurons, the effect of Slit1 on promoting the dendritic growth is not obvious, but when SGCs were off neurons, the effect of Slit1 on promoting the dendritic growth is significant. Conclusion: SGCs can promote the growth of neuronal axos, but inhibit the growth of its dendrites; Slit- Robo signaling pathways and contact with neurons play a role in this process.

  10. Activation of Divergent Neuronal Cell Death Pathways in Different Target Cell Populations during Neuroadapted Sindbis Virus Infection of Mice

    OpenAIRE

    Havert, Michael B.; Schofield, Brian; Griffin, Diane E.; Irani, David N.

    2000-01-01

    Infection of adult mice with neuroadapted Sindbis virus (NSV) results in a severe encephalomyelitis accompanied by prominent hindlimb paralysis. We find that the onset of paralysis parallels morphologic changes in motor neuron cell bodies in the lumbar spinal cord and in motor neuron axons in ventral nerve roots, many of which are eventually lost over time. However, unlike NSV-induced neuronal cell death found in the brain of infected animals, the loss of motor neurons does not appear to be a...

  11. Cloned myogenic cells can transdifferentiate in vivo into neuron-like cells.

    Directory of Open Access Journals (Sweden)

    Rachel Sarig

    Full Text Available BACKGROUND: The question of whether intact somatic cells committed to a specific differentiation fate, can be reprogrammed in vivo by exposing them to a different host microenvironment is a matter of controversy. Many reports on transdifferentiation could be explained by fusion with host cells or reflect intrinsic heterogeneity of the donor cell population. METHODOLOGY/PRINCIPAL FINDINGS: We have tested the capacity of cloned populations of mouse and human muscle progenitor cells, committed to the myogenic pathway, to transdifferentiate to neurons, following their inoculation into the developing brain of newborn mice. Both cell types migrated into various brain regions, and a fraction of them gained a neuronal morphology and expressed neuronal or glial markers. Likewise, inoculated cloned human myogenic cells expressed a human specific neurofilament protein. Brain injected donor cells that expressed a YFP transgene controlled by a neuronal specific promoter, were isolated by FACS. The isolated cells had a wild-type diploid DNA content. CONCLUSIONS: These and other results indicate a genuine transdifferentiation phenomenon induced by the host brain microenvironment and not by fusion with host cells. The results may potentially be relevant to the prospect of autologous cell therapy approach for CNS diseases.

  12. Human neuronal cells in culture: from concepts to basic methodology.

    Science.gov (United States)

    Silani, V; Pizzuti, A; Donato, M F; Falini, A; Bassani, R; Strada, O; Causarano, R I; Mariani, D; Villani, R M; Scarlato, G

    1990-01-01

    The paper reviews some conceptual and methodological aspects of the tissue culture models which, during the past three decades, demonstrated a remarkable mimicry of many important structures and functions of the mammalian Central Nervous System (CNS) and related peripheral sensory and motor elements. Emphasis is placed on an original human neuronal tissue culture model obtained from selective CNS areas. The different cell types were identified and the neurotrophic interactions preliminary characterized. Neuropathological findings suggest hypothesis that can be fully tested using in vitro human models of affected cerebral specific areas. PMID:2102114

  13. Differentiation of neuron-like cells from mouse parthenogenetic embryonic stem cells

    Institute of Scientific and Technical Information of China (English)

    Xingrong Yan; Liwen Li; Fulin Chen; Yanhong Yang; Wei Liu; Wenxin Geng; Huichong Du; Jihong Cui; Xin Xie; Jinlian Hua; Shumin Yu

    2013-01-01

    Parthenogenetic embryonic stem cells have pluripotent differentiation potentials, akin to fertilized embryo-derived embryonic stem cells. The aim of this study was to compare the neuronal differentiation potential of parthenogenetic and fertilized embryo-derived embryonic stem cells. Before differentiation, karyotype analysis was performed, with normal karyotypes detected in both parthenogenetic and fertilized embryo-derived embryonic stem cells. Sex chromosomes were identified as XX. Immunocytochemistry and quantitative real-time PCR detected high expression of the pluripotent gene, Oct4, at both the mRNA and protein levels, indicating pluripotent differentiation potential of the two embryonic stem cell subtypes. Embryonic stem cells were induced with retinoic acid to form embryoid bodies, and then dispersed into single cells. Single cells were differentiated in N2 differentiation medium for 9 days. Immunocytochemistry showed parthenogenetic and fertilized embryo-derived embryonic stem cells both express the neuronal cell markers nestin, βIII-tubulin and myelin basic protein. Quantitative real-time PCR found expression of neurogenesis related genes (Sox-1, Nestin, GABA, Pax6, Zic5 and Pitx1) in both types of embryonic stem cells, and Oct4 expression was significantly decreased. Nestin and Pax6 expression in parthenogenetic embryonic stem cells was significantly higher than that in fertilized embryo-derived embryonic stem cells. Thus, our experimental findings indicate that parthenogenetic embryonic stem cells have stronger neuronal differentiation potential than fertilized embryo-derived embryonic stem cells.

  14. Vascular Mural Cells Promote Noradrenergic Differentiation of Embryonic Sympathetic Neurons.

    Science.gov (United States)

    Fortuna, Vitor; Pardanaud, Luc; Brunet, Isabelle; Ola, Roxana; Ristori, Emma; Santoro, Massimo M; Nicoli, Stefania; Eichmann, Anne

    2015-06-23

    The sympathetic nervous system controls smooth muscle tone and heart rate in the cardiovascular system. Postganglionic sympathetic neurons (SNs) develop in close proximity to the dorsal aorta (DA) and innervate visceral smooth muscle targets. Here, we use the zebrafish embryo to ask whether the DA is required for SN development. We show that noradrenergic (NA) differentiation of SN precursors temporally coincides with vascular mural cell (VMC) recruitment to the DA and vascular maturation. Blocking vascular maturation inhibits VMC recruitment and blocks NA differentiation of SN precursors. Inhibition of platelet-derived growth factor receptor (PDGFR) signaling prevents VMC differentiation and also blocks NA differentiation of SN precursors. NA differentiation is normal in cloche mutants that are devoid of endothelial cells but have VMCs. Thus, PDGFR-mediated mural cell recruitment mediates neurovascular interactions between the aorta and sympathetic precursors and promotes their noradrenergic differentiation.

  15. Direct reprogramming of human fibroblasts into dopaminergic neuron-like cells

    Institute of Scientific and Technical Information of China (English)

    Xinjian Liu; Dabing Zhang; Timothy A Benke; John R Sladek; Nancy R Zahniser; Chuan-Yuan Li; Fang Li; Elizabeth A Stubblefield; Barbara Blanchard; Toni L Richards; Gaynor A Larson; Yujun He; Qian Huang; Aik-Choon Tan

    2012-01-01

    Transplantation of exogenous dopaminergic neuron (DA neurons) is a promising approach for treating Parkinson's disease (PD).However,a major stumbling block has been the lack of a reliable source of donor DA neurons.Here we show that a combination of five transcriptional factors Mash1,Ngn2,Sox2,Nurr1,and Pitx3 can directly and effectively reprogram human fibroblasts into DA neuron-like cells.The reprogrammed cells stained positive for various markers for DA neurons.They also showed characteristic DA uptake and production properties.Moreover,they exhibited DA neuron-specific electrophysiological profiles.Finally,they provided symptomatic relief in a rat PD model.Therefore,our directly reprogrammed DA neuron-like cells are a promising source of cell-replacement therapy for PD.

  16. Information in a Network of Neuronal Cells: Effect of Cell Density and Short-Term Depression

    KAUST Repository

    Onesto, Valentina

    2016-05-10

    Neurons are specialized, electrically excitable cells which use electrical to chemical signals to transmit and elaborate information. Understanding how the cooperation of a great many of neurons in a grid may modify and perhaps improve the information quality, in contrast to few neurons in isolation, is critical for the rational design of cell-materials interfaces for applications in regenerative medicine, tissue engineering, and personalized lab-on-a-chips. In the present paper, we couple an integrate-and-fire model with information theory variables to analyse the extent of information in a network of nerve cells. We provide an estimate of the information in the network in bits as a function of cell density and short-term depression time. In the model, neurons are connected through a Delaunay triangulation of not-intersecting edges; in doing so, the number of connecting synapses per neuron is approximately constant to reproduce the early time of network development in planar neural cell cultures. In simulations where the number of nodes is varied, we observe an optimal value of cell density for which information in the grid is maximized. In simulations in which the posttransmission latency time is varied, we observe that information increases as the latency time decreases and, for specific configurations of the grid, it is largely enhanced in a resonance effect.

  17. Information in a Network of Neuronal Cells: Effect of Cell Density and Short-Term Depression

    Science.gov (United States)

    Onesto, Valentina; Cosentino, Carlo; Di Fabrizio, Enzo; Cesarelli, Mario; Amato, Francesco; Gentile, Francesco

    2016-01-01

    Neurons are specialized, electrically excitable cells which use electrical to chemical signals to transmit and elaborate information. Understanding how the cooperation of a great many of neurons in a grid may modify and perhaps improve the information quality, in contrast to few neurons in isolation, is critical for the rational design of cell-materials interfaces for applications in regenerative medicine, tissue engineering, and personalized lab-on-a-chips. In the present paper, we couple an integrate-and-fire model with information theory variables to analyse the extent of information in a network of nerve cells. We provide an estimate of the information in the network in bits as a function of cell density and short-term depression time. In the model, neurons are connected through a Delaunay triangulation of not-intersecting edges; in doing so, the number of connecting synapses per neuron is approximately constant to reproduce the early time of network development in planar neural cell cultures. In simulations where the number of nodes is varied, we observe an optimal value of cell density for which information in the grid is maximized. In simulations in which the posttransmission latency time is varied, we observe that information increases as the latency time decreases and, for specific configurations of the grid, it is largely enhanced in a resonance effect.

  18. Information in a Network of Neuronal Cells: Effect of Cell Density and Short-Term Depression

    Directory of Open Access Journals (Sweden)

    Valentina Onesto

    2016-01-01

    Full Text Available Neurons are specialized, electrically excitable cells which use electrical to chemical signals to transmit and elaborate information. Understanding how the cooperation of a great many of neurons in a grid may modify and perhaps improve the information quality, in contrast to few neurons in isolation, is critical for the rational design of cell-materials interfaces for applications in regenerative medicine, tissue engineering, and personalized lab-on-a-chips. In the present paper, we couple an integrate-and-fire model with information theory variables to analyse the extent of information in a network of nerve cells. We provide an estimate of the information in the network in bits as a function of cell density and short-term depression time. In the model, neurons are connected through a Delaunay triangulation of not-intersecting edges; in doing so, the number of connecting synapses per neuron is approximately constant to reproduce the early time of network development in planar neural cell cultures. In simulations where the number of nodes is varied, we observe an optimal value of cell density for which information in the grid is maximized. In simulations in which the posttransmission latency time is varied, we observe that information increases as the latency time decreases and, for specific configurations of the grid, it is largely enhanced in a resonance effect.

  19. Information in a Network of Neuronal Cells: Effect of Cell Density and Short-Term Depression.

    Science.gov (United States)

    Onesto, Valentina; Cosentino, Carlo; Di Fabrizio, Enzo; Cesarelli, Mario; Amato, Francesco; Gentile, Francesco

    2016-01-01

    Neurons are specialized, electrically excitable cells which use electrical to chemical signals to transmit and elaborate information. Understanding how the cooperation of a great many of neurons in a grid may modify and perhaps improve the information quality, in contrast to few neurons in isolation, is critical for the rational design of cell-materials interfaces for applications in regenerative medicine, tissue engineering, and personalized lab-on-a-chips. In the present paper, we couple an integrate-and-fire model with information theory variables to analyse the extent of information in a network of nerve cells. We provide an estimate of the information in the network in bits as a function of cell density and short-term depression time. In the model, neurons are connected through a Delaunay triangulation of not-intersecting edges; in doing so, the number of connecting synapses per neuron is approximately constant to reproduce the early time of network development in planar neural cell cultures. In simulations where the number of nodes is varied, we observe an optimal value of cell density for which information in the grid is maximized. In simulations in which the posttransmission latency time is varied, we observe that information increases as the latency time decreases and, for specific configurations of the grid, it is largely enhanced in a resonance effect. PMID:27403421

  20. Adult adipose-derived stromal cells differentiate into neurons with normal electrophysiological functions

    Institute of Scientific and Technical Information of China (English)

    Xiaodong Yuan; Yanan Cai; Ya Ou; Yanhui Lu

    2011-01-01

    β-mercaptoethanol was used to induce in vitro neuronal differentiation of adipose-derived stromal cells. Within an 8-hour period post-differentiation, the induced cells exhibited typical neuronal morphology, and expression of microtubule-associated protein 2 and neuron-specific enolase, which are markers of mature neurons, reached a peak at 5 hours. Specific organelle Nissl bodies of neurons were observed under transmission electron microscopy. Results of membrane potential showed that fluorescence intensity of cells was greater after 5 hours than adipose-derived stromal cells prior to induction. In addition, following stimulation with high-concentration potassium solution, fluorescence intensity increased. These experimental findings suggested that neurons differentiated from adipose-derived stromal cells and expressed mature K+ channels. In addition, following stimulation with high potassium solution, the membrane potential depolarized and fired an action potential, confirming that the induced cells possessed electrophysiological functions.

  1. Siah regulation of Pard3A controls neuronal cell adhesion during germinal zone exit.

    Science.gov (United States)

    Famulski, Jakub K; Trivedi, Niraj; Howell, Danielle; Yang, Yuan; Tong, Yiai; Gilbertson, Richard; Solecki, David J

    2010-12-24

    The brain's circuitry is established by directed migration and synaptogenesis of neurons during development. Although neurons mature and migrate in specific patterns, little is known about how neurons exit their germinal zone niche. We found that cerebellar granule neuron germinal zone exit is regulated by proteasomal degradation of Pard3A by the Seven in Absentia homolog (Siah) E3 ubiquitin ligase. Pard3A gain of function and Siah loss of function induce precocious radial migration. Time-lapse imaging using a probe to measure neuronal cell contact reveals that Pard3A promotes adhesive interactions needed for germinal zone exit by recruiting the epithelial tight junction adhesion molecule C to the neuronal cell surface. Our findings define a Siah-Pard3A signaling pathway that controls adhesion-dependent exit of neuronal progenitors or immature neurons from a germinal zone niche.

  2. Neuroprotective effects of Neuregulin-1 on B35 Neuronal Cells following Ischemia

    OpenAIRE

    Croslan, DaJoie R.; Schoell, Matthew C.; Ford, Gregory D.; Pulliam, John V.; Gates, Alicia; Clement, Ceilessia M.; Harris, Adalynn E.; Ford, Byron D.

    2008-01-01

    We previously showed that neuregulin-1 (NRG-1) protected neurons from death in vivo following focal ischemia. The goal of this study was to develop an in vitro rat ischemia model to examine the cellular and molecular mechanisms involved in the neuroprotective effects of NRG-1 on ischemia-induced neuronal death. Rat B-35 neuroblastoma cells differentiated by serum withdrawal, developed enhanced neuronal characteristics including, neurite extension and upregulation of neuronal markers of differ...

  3. Differentiation of Human Cord Blood and Stromal Derived Stem Cells into Neuron Cells

    Directory of Open Access Journals (Sweden)

    Özlem Pamukçu Baran

    2007-01-01

    Full Text Available The most basic properties of stem cells are the capacities to self-renew indefinitely and to differentiate into multiple cell or tissue types. Umbilical cord blood has been utilized for human hematopoietic stem cell transplantation as an alternative source to bone marrow.The experiments show that Wharton’s jelly cells are easily attainable and can be expanded in vitro, maintained in culture, and induced to differentiate into neural cells. Almost recent studies it has been discovered that the cord blood-derived cells can differantiate not only to blood cells but also to various somatic cells like neuron or muscle cell with the signals taken from the envoirenment.Interestingly, neural cells obtained from umbilical cord blood show a relatively high spontaneous differentiation into oligodendrocytes, Embryonic stem cells proliferate indefinitely and can differentiate spontaneously into all tissue types.It has been shown that embryonic stem cells can be induced to differentiate into neurons and glia by treatment with retinoic acid or basic fibroblast growth factor. It has been studied that the diseases as Motor Neuron Disease, Parkinson, Alzheimer and degeneration of medulla spinalis and also paralysises could be treated with transplantation of cord blood-dericed stem cells.

  4. Cells from the adult corneal stroma can be reprogrammed to a neuron-like cell using exogenous growth factors

    Energy Technology Data Exchange (ETDEWEB)

    Greene, Carol Ann, E-mail: carol.greene@auckland.ac.nz; Chang, Chuan-Yuan; Fraser, Cameron J.; Nelidova, Dasha E.; Chen, Jing A.; Lim, Angela; Brebner, Alex; McGhee, Jennifer; Sherwin, Trevor; Green, Colin R.

    2014-03-10

    Cells thought to be stem cells isolated from the cornea of the eye have been shown to exhibit neurogenic potential. We set out to uncover the identity and location of these cells within the cornea and to elucidate their neuronal protein and gene expression profile during the process of switching to a neuron-like cell. Here we report that every cell of the adult human and rat corneal stroma is capable of differentiating into a neuron-like cell when treated with neurogenic differentiation specifying growth factors. Furthermore, the expression of genes regulating neurogenesis and mature neuronal structure and function was increased. The switch from a corneal stromal cell to a neuron-like cell was also shown to occur in vivo in intact corneas of living rats. Our results clearly indicate that lineage specifying growth factors can affect changes in the protein and gene expression profiles of adult cells, suggesting that possibly many adult cell populations can be made to switch into another type of mature cell by simply modifying the growth factor environment. - Highlights: • Adult corneal stromal cells can differentiated into neuron-like cells. • Neuronal specification of the adult stromal cell population is stochastic. • Neuronal specification in an adult cell population can be brought about by growth factors.

  5. Synaptic network activity induces neuronal differentiation of adult hippocampal precursor cells through BDNF signaling

    Directory of Open Access Journals (Sweden)

    Harish Babu

    2009-09-01

    Full Text Available Adult hippocampal neurogenesis is regulated by activity. But how do neural precursor cells in the hippocampus respond to surrounding network activity and translate increased neural activity into a developmental program? Here we show that long-term potential (LTP-like synaptic activity within a cellular network of mature hippocampal neurons promotes neuronal differentiation of newly generated cells. In co-cultures of precursor cells with primary hippocampal neurons, LTP-like synaptic plasticity induced by addition of glycine in Mg2+-free media for 5 min, produced synchronous network activity and subsequently increased synaptic strength between neurons. Furthermore, this synchronous network activity led to a significant increase in neuronal differentiation from the co-cultured neural precursor cells. When applied directly to precursor cells, glycine and Mg2+-free solution did not induce neuronal differentiation. Synaptic plasticity-induced neuronal differentiation of precursor cells was observed in the presence of GABAergic neurotransmission blockers but was dependent on NMDA-mediated Ca2+ influx. Most importantly, neuronal differentiation required the release of brain-derived neurotrophic factor (BDNF from the underlying substrate hippocampal neurons as well as TrkB receptor phosphorylation in precursor cells. This suggests that activity-dependent stem cell differentiation within the hippocampal network is mediated via synaptically evoked BDNF signaling.

  6. Modeling chemotherapeutic neurotoxicity with human induced pluripotent stem cell-derived neuronal cells.

    Directory of Open Access Journals (Sweden)

    Heather E Wheeler

    Full Text Available There are no effective agents to prevent or treat chemotherapy-induced peripheral neuropathy (CIPN, the most common non-hematologic toxicity of chemotherapy. Therefore, we sought to evaluate the utility of human neuron-like cells derived from induced pluripotent stem cells (iPSCs as a means to study CIPN. We used high content imaging measurements of neurite outgrowth phenotypes to compare the changes that occur to iPSC-derived neuronal cells among drugs and among individuals in response to several classes of chemotherapeutics. Upon treatment of these neuronal cells with the neurotoxic drug paclitaxel, vincristine or cisplatin, we identified significant differences in five morphological phenotypes among drugs, including total outgrowth, mean/median/maximum process length, and mean outgrowth intensity (P < 0.05. The differences in damage among drugs reflect differences in their mechanisms of action and clinical CIPN manifestations. We show the potential of the model for gene perturbation studies by demonstrating decreased expression of TUBB2A results in significantly increased sensitivity of neurons to paclitaxel (0.23 ± 0.06 decrease in total neurite outgrowth, P = 0.011. The variance in several neurite outgrowth and apoptotic phenotypes upon treatment with one of the neurotoxic drugs is significantly greater between than within neurons derived from four different individuals (P < 0.05, demonstrating the potential of iPSC-derived neurons as a genetically diverse model for CIPN. The human neuron model will allow both for mechanistic studies of specific genes and genetic variants discovered in clinical studies and for screening of new drugs to prevent or treat CIPN.

  7. Strategies for Regenerating Striatal Neurons in the Adult Brain by Using Endogenous Neural Stem Cells

    Directory of Open Access Journals (Sweden)

    Kanako Nakaguchi

    2011-01-01

    Full Text Available Currently, there is no effective treatment for the marked neuronal loss caused by neurodegenerative diseases, such as Huntington's disease (HD or ischemic stroke. However, recent studies have shown that new neurons are continuously generated by endogenous neural stem cells in the subventricular zone (SVZ of the adult mammalian brain, including the human brain. Because some of these new neurons migrate to the injured striatum and differentiate into mature neurons, such new neurons may be able to replace degenerated neurons and improve or repair neurological deficits. To establish a neuroregenerative therapy using this endogenous system, endogenous regulatory mechanisms that can be co-opted for efficient regenerative interventions must be understood, along with any potential drawbacks. Here, we review current knowledge on the generation of new neurons in the adult brain and discuss their potential for use in replacing striatal neurons lost to neurodegenerative diseases, including HD, and to ischemic stroke.

  8. Cytotoxic effects of catechols to glial and neuronal cells

    Directory of Open Access Journals (Sweden)

    Ramon Santos El-Bachá

    2015-04-01

    Full Text Available Catechols are compounds that autoxidises under physiological conditions leading to the formation of reactive oxygen species (ROS, semiquinones, and quinones. These molecules can be formed in organisms because of the metabolism of exogenous aromatic substances, such as benzene. However, there are several important endogenous catechols, which have physiological functions, such as catecholamines. Furthermore, several pharmacological agents are catechols, such as apomorphine, or can be metabolised to generate these compounds. In this presentation we will show that apomorphine can unspecifically bind to proteins during its autoxidation, a phenomenon that is inhibited by thiols. Brain endothelial cells and glial cells express xenobiotic-metabolising enzymes as components of the metabolic blood-brain barrier in an attempt to protect the central nervous system against drugs. Since UDP-glucuronosyltransferases (EC 2.4.1.17 are among these enzymes, we investigated the ability of brain microsomes to conjugate catechols with glucuronate. Despite the fact that 1-naphtol could be glucuronidated in the presence of brain cortex microsomes, the same was not observed for most of catechols that were tested. Therefore, this is not the main mechanism used to protect the brain against them. Indeed, catechols may inhibit other xenobiotic-metabolising enzymes. We showed that apomorphine inhibited the cytochrome P450-dependent dealkylation activity. The production of ROS and reactive quinones, as well as their effects on protein functions, seems to be involved in the cytotoxicity of catechols. Glial cells are more resistant than neuronal cells. Apomorphine was more toxic to rat neurons than to rat C6 glioma cells. 1,2-Dihydroxybenzene (catechol killed human GL-15 cells with an EC50 of 230 uM after 72 h, a effect that was significantly inhibited by superoxide dismutase (EC 1.15.1.1. Another mechanism that we found to be involved in catechol cytotoxicity is the inhibition

  9. Human dental pulp stem cells express many pluripotency regulators and differentiate into neuronal cells

    Institute of Scientific and Technical Information of China (English)

    Behnam Ebrahimi; Mohammad Mehdi Yaghoobi; Ali Mohammadi Kamal-abadi; Maryam Raoof

    2011-01-01

    Stem cells were isolated from human dental pulp using an optimized method, in which pulp pieces were digested by enzymes and immobilized to enhance cell outgrowth. Stem cell marker expression was detected by reverse transcription-PCR (RT-PCR), and differentiation markers were detected by real-time quantitative RT-PCR and immunocytochemistry. Results showed that dental pulp stem cells actively expressed nanog, oct4, nucleostemin slain-1, jmjd1a, jmjd2c, and cyclin D1. When stem cells were induced to differentiate into neurons, nucleostemin, nanog, and cyclin D1 expres-sion significantly decreased, whereas expression of neuronal markers, such as microtubule asso-ciated protein-2 and neurofilament-heavy, significantly increased. These results suggested that stem cells exited a pluripotent state and entered a neuronal differentiation pathway. In addition, results demonstrated that human dental pulp serves as a reservoir of stem cells that express defined stem cell markers; these cells were easily isolated and were induced to differentiate towards a desired cell lineage.

  10. The Specification and Maturation of Nociceptive Neurons from Human Embryonic Stem Cells.

    Science.gov (United States)

    Boisvert, Erin M; Engle, Sandra J; Hallowell, Shawn E; Liu, Ping; Wang, Zhao-Wen; Li, Xue-Jun

    2015-11-19

    Nociceptive neurons play an essential role in pain sensation by transmitting painful stimuli to the central nervous system. However, investigations of nociceptive neuron biology have been hampered by the lack of accessibility of human nociceptive neurons. Here, we describe a system for efficiently guiding human embryonic stem cells into nociceptive neurons by first inducing these cells to the neural lineage. Subsequent addition of retinoic acid and BMP4 at specific time points and concentrations yielded a high population of neural crest progenitor cells (AP2α(+), P75(+)), which further differentiated into nociceptive neurons (TRKA(+), Nav1.7(+), P2X3(+)). The overexpression of Neurogenin 1 (Neurog1) promoted the neurons to express genes related to sensory neurons (Peripherin, TrkA) and to further mature into TRPV1(+) nociceptive neurons. Importantly, the overexpression of Neurog1 increased the response of these neurons to capsaicin stimulation, a hallmark of mature functional nociceptive neurons. Taken together, this study reveals the important role that Neurog1 plays in generating functional human nociceptive neurons.

  11. Recombinant AAV-mediated Expression of Human BDNF Protects Neurons against Cell Apoptosis in Aβ-induced Neuronal Damage Model

    Institute of Scientific and Technical Information of China (English)

    LIU Zhaohui; MA Dongliang; FENG Gaifeng; MA Yanbing; HU Haitao

    2007-01-01

    The human brain-derived neurotrophic factor (hBDNF) gene was cloned by polymerase chain reaction and the recombinant adeno-associated viral vector inserted with hBDNF gene (AAV-hBDNF) was constructed. Cultured rat hippocampal neurons were treated with Aβ25-35 and serued as the experimental Aβ-induced neuronal damage model (AD model), and the AD model was infected with AAV-hBDNF to explore neuroprotective effects of expression of BDNF. Cell viability was assayed by MTT. The expression of bcl-2 anti-apoptosis protein was detected by immunocytochemical staining. The change of intracellular free Ca ion ([Ca2+]i) was measured by laser scanning confocal microscopy. The results showed that BDNF had protective effects against Aβ-induced neuronal damage. The expression of the bcl-2 anti-apoptosis protein was raised significantly and the balance of [Ca2+]i was maintained in the AAV-hBDNF treatment group as compared with AD model group. These data suggested that recombinant AAV mediated a stable expression of hBDNF in cultured hippocampal neurons and resulted in significant neuron protective effects in AD model. The BDNF may reduce neuron apoptosis through increasing the expression of the bcl-2 anti-apoptosis protein and inhibiting intracellular calcium overload. The viral vector-mediated gene expression of BDNF may pave the way of a novel therapeutic strategy for the treatment of neurodegenerative diseases such as Alzheimer's disease.

  12. Comparison Between Supervised and Unsupervised Classifications of Neuronal Cell Types: A Case Study

    OpenAIRE

    Guerra, Luis; McGarry, Laura M.; Robles Forcada, Víctor; Bielza, Concha; Larrañaga Múgica, Pedro; Yuste, Rafael

    2010-01-01

    In the study of neural circuits, it becomes essential to discern the different neuronal cell types that build the circuit. Traditionally, neuronal cell types have been classified using qualitative descriptors. More recently, several attempts have been made to classify neurons quantitatively, using unsupervised clustering methods. While useful, these algorithms do not take advantage of previous information known to the investigator, which could improve the classification task. For neocortical ...

  13. Protoplasmic Astrocytes Enhance the Ability of Neural Stem Cells to Differentiate into Neurons In Vitro

    OpenAIRE

    Yuan Liu; Li Wang; Zaiyun Long; Lin Zeng; Yamin Wu

    2012-01-01

    Protoplasmic astrocytes have been reported to exhibit neuroprotective effects on neurons, but there has been no direct evidence for a functional relationship between protoplasmic astrocytes and neural stem cells (NSCs). In this study, we examined neuronal differentiation of NSCs induced by protoplasmic astrocytes in a co-culture model. Protoplasmic astrocytes were isolated from new-born and NSCs from the E13-15 cortex of rats respectively. The differentiated cells labeled with neuron-specific...

  14. Adolescent development of neuron structure in dentate gyrus granule cells of male Syrian hamsters

    OpenAIRE

    Zehr, Julia L.; Nichols, Liana R.; Schulz, Kalynn M.; Sisk, Cheryl L.

    2008-01-01

    Hippocampal function, including spatial cognition and stress responses, matures during adolescence. In addition, hippocampal neuron structure is modified by gonadal steroid hormones, which increase dramatically at this time. This study investigated pubertal changes in dendritic complexity of dentate gyrus neurons. Dendrites, spines, and cell bodies of Golgiimpregnated neurons from the granule cell layer were traced in pre-, mid-, and late pubertal male Syrian hamsters (21, 35, and 49 days of ...

  15. Self-contained induction of neurons from human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Tsuyoshi Okuno

    Full Text Available BACKGROUND: Neurons and glial cells can be efficiently induced from mouse embryonic stem (ES cells in a conditioned medium collected from rat primary-cultured astrocytes (P-ACM. However, the use of rodent primary cells for clinical applications may be hampered by limited supply and risk of contamination with xeno-proteins. METHODOLOGY/PRINCIPAL FINDINGS: We have developed an alternative method for unimpeded production of human neurons under xeno-free conditions. Initially, neural stem cells in sphere-like clusters were induced from human ES (hES cells after being cultured in P-ACM under free-floating conditions. The resultant neural stem cells could circumferentially proliferate under subsequent adhesive culture, and selectively differentiate into neurons or astrocytes by changing the medium to P-ACM or G5, respectively. These hES cell-derived neurons and astrocytes could procure functions similar to those of primary cells. Interestingly, a conditioned medium obtained from the hES cell-derived astrocytes (ES-ACM could successfully be used to substitute P-ACM for induction of neurons. Neurons made by this method could survive in mice brain after xeno-transplantation. CONCLUSION/SIGNIFICANCE: By inducing astrocytes from hES cells in a chemically defined medium, we could produce human neurons without the use of P-ACM. This self-serving method provides an unlimited source of human neural cells and may facilitate clinical applications of hES cells for neurological diseases.

  16. Cell-based optical assay for amyloid β-induced neuronal cell dysfunction using femtosecond-pulsed laser

    Science.gov (United States)

    Lee, Seunghee; Yoon, Jonghee; Choi, Chulhee

    2015-03-01

    Amyloid β-protein (Aβ) is known as a key molecule related to the pathogenesis of Alzheimer's disease (AD). Over time, the amyloid cascade disrupts essential function of mitochondria including Ca2+ homeostasis and reactive oxygen species (ROS) regulation, and eventually leads to neuronal cell death. However, there have been no methods that analyze and measure neuronal dysfuction in pathologic conditions quantitatively. Here, we suggest a cell-based optical assay to investigate neuronal function in AD using femtosecond-pulsed laser stimulation. We observed that laser stimulation on primary rat hippocampal neurons for a few microseconds induced intracellular Ca2+ level increases or produced intracellular ROS which was a primary cause of neuronal cell death depending on delivered energy. Although Aβ treatment alone had little effect on the neuronal morphologies and networks in a few hours, Aβ-treated neurons showed delayed Ca2+ increasing pattern and were more vulnerable to laser-induced cell death compared to normal neurons. Our results collectively indicate that femtosecond laser stimulation can be a useful tool to study neuronal dysfuction related to AD pathologies. We anticipate this optical method to enable studies in the early progression of neuronal impairments and the quantitative evaluation of drug effects on neurons in neurodegenerative diseases, including AD and Parkinson's disease in a preclinical study.

  17. Interaction of SH-SY5Y cells with nanogratings during neuronal differentiation: comparison with primary neurons.

    Science.gov (United States)

    Tonazzini, Ilaria; Cecchini, Alessandra; Elgersma, Ype; Cecchini, Marco

    2014-04-01

    Controlling neuronal cell adhesion, migration, and axonal outgrowth via contact interactions with biomaterials is a critical element for tissue engineering applications and for developing artificial neuronal interfaces. One promising approach relies on the exploitation of nanostructured surfaces. Here, the human neuroblastoma cell line SH-SY5Y is interfaced with plastic nanogratings (NGs; anisotropic topographies composed by alternating lines of grooves and ridges with sub-micrometer lateral dimension). The SH-SY5Y cells' (SHs) contact guidance is investigated under proliferating conditions and upon differentiation after treatment with retinoic acid (RA) and brain-derived neurotrophin factor (BDNF), and compared with mouse primary hippocampal neurons (HNs). Quantitative readouts are obtained by measuring changes in tubulin cytoskeleton organization and cell morphology induced by mechanotransduction. Results demonstrate that SHs effectively retrieve substrate topographical signals, in particular during differentiation. Remarkably, RA/BDNF improves SH responsiveness to NG directional cues, and significantly enhances the alignment to the NG lines. HNs behave similarly, showing a marked change in network organization if cultured on NGs. These results might help the rational engineering of neuro-regenerative scaffolds to improve peripheral nerve wound healing, as well as to investigate the basic mechanisms of neuronal wiring.

  18. Differentiation of neuronal stem cells into motor neurons using electrospun poly-L-lactic acid/gelatin scaffold.

    Science.gov (United States)

    Binan, Loïc; Tendey, Charlène; De Crescenzo, Gregory; El Ayoubi, Rouwayda; Ajji, Abdellah; Jolicoeur, Mario

    2014-01-01

    Neural stem cells (NSCs) provide promising therapeutic potential for cell replacement therapy in spinal cord injury (SCI). However, high increases of cell viability and poor control of cell differentiation remain major obstacles. In this study, we have developed a non-woven material made of co-electrospun fibers of poly L-lactic acid and gelatin with a degradation rate and mechanical properties similar to peripheral nerve tissue and investigated their effect on cell survival and differentiation into motor neuronal lineages through the controlled release of retinoic acid (RA) and purmorphamine. Engineered Neural Stem-Like Cells (NSLCs) seeded on these fibers, with and without the instructive cues, differentiated into β-III-tubulin, HB-9, Islet-1, and choactase-positive motor neurons by immunostaining, in response to the release of the biomolecules. In addition, the bioactive material not only enhanced the differentiation into motor neuronal lineages but also promoted neurite outgrowth. This study elucidated that a combination of electrospun fiber scaffolds, neural stem cells, and controlled delivery of instructive cues could lead to the development of a better strategy for peripheral nerve injury repair. PMID:24161168

  19. Molecular codes for neuronal individuality and cell assembly in the brain

    Directory of Open Access Journals (Sweden)

    Takeshi eYagi

    2012-04-01

    Full Text Available The brain contains an enormous, but finite, number of neurons. The ability of this limited number of neurons to produce nearly limitless neural information over a lifetime is typically explained by combinatorial explosion; that is, by the exponential amplification of each neuron’s contribution through its incorporation into cell assemblies and neural networks. In development, each neuron expresses diverse cellular recognition molecules that permit the formation of the appropriate neural cell assemblies to elicit various brain functions. The mechanism for generating neuronal assemblies and networks must involve molecular codes that give neurons individuality and allow them to recognize one another and join appropriate networks. The extensive molecular diversity of cell-surface proteins on neurons is likely to contribute to their individual identities. The cadherin-related neuronal receptors and clustered protocadherins (CNR/Pcdh is a large subfamily within the diverse cadherin superfamily. The CNR/Pcdh genes are encoded in tandem by three gene clusters, and are present in all known vertebrate genomes. The set of CNR/Pcdh genes is expressed in a random and combinatorial manner in each neuron. In addition, cis-tetramers composed of heteromultimeric CNR/Pcdh isoforms represent selective binding units for cell-cell interactions. Here I present the mathematical probabilities for neuronal individuality based on the random and combinatorial expression of CNR/Pcdh isoforms and their formation of cis-tetramers in each neuron. Notably, CNR/Pcdh gene products are known to play crucial roles in correct axonal projections, synaptic formation, and neuronal survival. Their molecular and biological features suggest that the diverse CNR/Pcdh molecules provide the molecular code by which neuronal individuality and cell assembly permit the combinatorial explosion of networks that supports enormous processing capability and plasticity of the brain.

  20. Olfactory ensheathing cells form the microenvironment of migrating GnRH-1 neurons during mouse development.

    Science.gov (United States)

    Geller, Sarah; Kolasa, Elise; Tillet, Yves; Duittoz, Anne; Vaudin, Pascal

    2013-04-01

    During development, GnRH-1 neurons differentiate extracerebraly from the nasal placode and migrate from the vomeronasal organ to the forebrain along vomeronasal and terminal nerves. Numerous studies have described the influence of different molecules on the migration of GnRH-1 neurons, however, the role of microenvironment cells remains poorly understood. This study used GFAP-GFP transgenic mice to detect glial cells at early developmental stages. Using nasal explant cultures, the comigration of glial cells with GnRH-1 neurons was clearly demonstrated. This in vitro approach showed that glial cells began migrating from the explants before GnRH-1 neurons. They remained ahead of the GnRH-1 migratory front and stopped migrating after the GnRH-1 neurons. The association of these glial cells with the axons combined with gene expression analysis of GFAP-GFP sorted cells enabled them to be identified as olfactory ensheathing cells (OEC). Immunohistochemical analysis revealed the presence of multiple glial cell-type markers showing several OEC subpopulations surrounding GnRH-1 neurons. Moreover, these OEC expressed genes whose products are involved in the migration of GnRH-1 neurons, such as Nelf and Semaphorin 4. In situ data confirmed that the majority of the GnRH-1 neurons were associated with glial cells along the vomeronasal axons in nasal septum and terminal nerves in the nasal forebrain junction as early as E12.5. Overall, these data demonstrate an OEC microenvironment for migrating GnRH-1 neurons during mouse development. The fact that this glial cell type precedes GnRH-1 neurons and encodes for molecules involved in their nasal migration suggests that it participates in the GnRH-1 system ontogenesis. PMID:23404564

  1. Attenuation of oxidative neuronal cell death by coffee phenolic phytochemicals

    International Nuclear Information System (INIS)

    Neurodegenerative disorders such as Alzheimer's disease (AD) are strongly associated with oxidative stress, which is induced by reactive oxygen species (ROS) including hydrogen peroxide (H2O2). Recent studies suggest that moderate coffee consumption may reduce the risk of neurodegenerative diseases such as AD, but the molecular mechanisms underlying this effect remain to be clarified. In this study, we investigated the protective effects of chlorogenic acid (5-O-caffeoylquinic acid; CGA), a major phenolic phytochemical found in instant decaffeinated coffee (IDC), and IDC against oxidative PC12 neuronal cell death. IDC (1 and 5 μg/ml) or CGA (1 and 5 μM) attenuated H2O2-induced PC12 cell death. H2O2-induced nuclear condensation and DNA fragmentation were strongly inhibited by pretreatment with IDC or CGA. Pretreatment with IDC or CGA also inhibited the H2O2-induced cleavage of poly(ADP-ribose) polymerase (PARP), and downregulation of Bcl-XL and caspase-3. The accumulation of intracellular ROS in H2O2-treated PC12 cells was dose-dependently diminished by IDC or CGA. The activation of c-Jun N-terminal protein kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) by H2O2 in PC12 cells was also inhibited by IDC or CGA. Collectively, these results indicate that IDC and CGA protect PC12 cells from H2O2-induced apoptosis by blocking the accumulation of intracellular ROS and the activation of MAPKs

  2. Attenuation of oxidative neuronal cell death by coffee phenolic phytochemicals

    Energy Technology Data Exchange (ETDEWEB)

    Cho, Eun Sun; Jang, Young Jin [Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921 (Korea, Republic of); Hwang, Mun Kyung; Kang, Nam Joo [Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921 (Korea, Republic of); Department of Bioscience and Biotechnology, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul 143-701 (Korea, Republic of); Lee, Ki Won [Department of Bioscience and Biotechnology, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul 143-701 (Korea, Republic of)], E-mail: kiwon@konkuk.ac.kr; Lee, Hyong Joo [Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921 (Korea, Republic of)], E-mail: leehyjo@snu.ac.kr

    2009-02-10

    Neurodegenerative disorders such as Alzheimer's disease (AD) are strongly associated with oxidative stress, which is induced by reactive oxygen species (ROS) including hydrogen peroxide (H{sub 2}O{sub 2}). Recent studies suggest that moderate coffee consumption may reduce the risk of neurodegenerative diseases such as AD, but the molecular mechanisms underlying this effect remain to be clarified. In this study, we investigated the protective effects of chlorogenic acid (5-O-caffeoylquinic acid; CGA), a major phenolic phytochemical found in instant decaffeinated coffee (IDC), and IDC against oxidative PC12 neuronal cell death. IDC (1 and 5 {mu}g/ml) or CGA (1 and 5 {mu}M) attenuated H{sub 2}O{sub 2}-induced PC12 cell death. H{sub 2}O{sub 2}-induced nuclear condensation and DNA fragmentation were strongly inhibited by pretreatment with IDC or CGA. Pretreatment with IDC or CGA also inhibited the H{sub 2}O{sub 2}-induced cleavage of poly(ADP-ribose) polymerase (PARP), and downregulation of Bcl-X{sub L} and caspase-3. The accumulation of intracellular ROS in H{sub 2}O{sub 2}-treated PC12 cells was dose-dependently diminished by IDC or CGA. The activation of c-Jun N-terminal protein kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) by H{sub 2}O{sub 2} in PC12 cells was also inhibited by IDC or CGA. Collectively, these results indicate that IDC and CGA protect PC12 cells from H{sub 2}O{sub 2}-induced apoptosis by blocking the accumulation of intracellular ROS and the activation of MAPKs.

  3. NMDA receptor-dependent glutamate excitotoxicity in human embryonic stem cell-derived neurons

    OpenAIRE

    Gupta, K.; Hardingham, G. E.; Chandran, S

    2013-01-01

    Thanks to the development of efficient differentiation strategies, human pluripotent stem cells (HPSC) offer the opportunity for modelling neuronal injury and dysfunction in human neurons in vitro. Critically, the effective use of HPSC-derived neural cells in disease-modelling and potentially cell replacement therapies hinges on an understanding of the biology of these cells, specifically their development, subtype specification and responses to neurotoxic signalling mediators. Here, we gener...

  4. Highly efficient and large-scale generation of functional dopamine neurons from human embryonic stem cells.

    Science.gov (United States)

    Cho, Myung Soo; Lee, Young-Eun; Kim, Ji Young; Chung, Seungsoo; Cho, Yoon Hee; Kim, Dae-Sung; Kang, Sang-Moon; Lee, Haksup; Kim, Myung-Hwa; Kim, Jeong-Hoon; Leem, Joong Woo; Oh, Sun Kyung; Choi, Young Min; Hwang, Dong-Youn; Chang, Jin Woo; Kim, Dong-Wook

    2008-03-01

    We developed a method for the efficient generation of functional dopaminergic (DA) neurons from human embryonic stem cells (hESCs) on a large scale. The most unique feature of this method is the generation of homogeneous spherical neural masses (SNMs) from the hESC-derived neural precursors. These SNMs provide several advantages: (i) they can be passaged for a long time without losing their differentiation capability into DA neurons; (ii) they can be coaxed into DA neurons at much higher efficiency than that from previous reports (86% tyrosine hydroxylase-positive neurons/total neurons); (iii) the induction of DA neurons from SNMs only takes 14 days; and (iv) no feeder cells are required during differentiation. These advantages allowed us to obtain a large number of DA neurons within a short time period and minimized potential contamination of unwanted cells or pathogens coming from the feeder layer. The highly efficient differentiation may not only enhance the efficacy of the cell therapy but also reduce the potential tumor formation from the undifferentiated residual hESCs. In line with this effect, we have never observed any tumor formation from the transplanted animals used in our study. When grafted into a parkinsonian rat model, the hESC-derived DA neurons elicited clear behavioral recovery in three behavioral tests. In summary, our study paves the way for the large-scale generation of purer and functional DA neurons for future clinical applications. PMID:18305158

  5. Differential Neuronal Plasticity of Dental Pulp Stem Cells From Exfoliated Deciduous and Permanent Teeth Towards Dopaminergic Neurons.

    Science.gov (United States)

    Majumdar, Debanjana; Kanafi, Mohammad; Bhonde, Ramesh; Gupta, Pawan; Datta, Indrani

    2016-09-01

    Based on early occurrence in chronological age, stem-cells from human exfoliated deciduous teeth (SHED) has been reported to possess better differentiation-potential toward certain cell-lineage in comparison to stem-cells from adult teeth (DPSCs). Whether this same property between them extends for the yield of functional central nervous system neurons is still not evaluated. Hence, we aim to assess the neuronal plasticity of SHED in comparison to DPSCs toward dopaminergic-neurons and further, if the difference is reflected in a differential expression of sonic-hedgehog (SHH)-receptors and basal-expressions of tyrosine-hydroxylase [TH; through cAMP levels]. Human SHED and DPSCs were exposed to midbrain-cues [SHH, fibroblast growth-factor8, and basic fibroblast growth-factor], and their molecular, immunophenotypical, and functional characterization was performed at different time-points of induction. Though SHED and DPSCs spontaneously expressed early-neuronal and neural-crest marker in their naïve state, only SHED expressed a high basal-expression of TH. The upregulation of dopaminergic transcription-factors Nurr1, Engrailed1, and Pitx3 was more pronounced in DPSCs. The yield of TH-expressing cells decreased from 49.8% to 32.16% in SHED while it increased from 8.09% to 77.47% in DPSCs. Dopamine release and intracellular-Ca(2+) influx upon stimulation (KCl and ATP) was higher in induced DPSCs. Significantly lower-expression of SHH-receptors was noted in naïve SHED than DPSCs, which may explain the differential neuronal plasticity. In addition, unlike DPSCs, SHED showed a down-regulation of cyclic adenosine-monophosphate (cAMP) upon exposure to SHH; possibly another contributor to the lesser differentiation-potential. Our data clearly demonstrates for the first time that DPSCs possess superior neuronal plasticity toward dopaminergic-neurons than SHED; influenced by higher SHH-receptor and lower basal TH expression. J. Cell. Physiol. 231: 2048-2063, 2016. © 2016

  6. Reduced synaptic activity in neuronal networks derived from embryonic stem cells of murine Rett syndrome model.

    Science.gov (United States)

    Barth, Lydia; Sütterlin, Rosmarie; Nenniger, Markus; Vogt, Kaspar E

    2014-01-01

    Neurodevelopmental diseases such as the Rett syndrome (RTT) have received renewed attention, since the mechanisms involved may underlie a broad range of neuropsychiatric disorders such as schizophrenia and autism. In vertebrates early stages in the functional development of neurons and neuronal networks are difficult to study. Embryonic stem cell-derived neurons provide an easily accessible tool to investigate neuronal differentiation and early network formation. We used in vitro cultures of neurons derived from murine embryonic stem cells missing the methyl-CpG-binding protein 2 (MECP2) gene (MeCP2-/y) and from wild type cells of the corresponding background. Cultures were assessed using whole-cell patch-clamp electrophysiology and immunofluorescence. We studied the functional maturation of developing neurons and the activity of the synaptic connections they formed. Neurons exhibited minor differences in the developmental patterns for their intrinsic parameters, such as resting membrane potential and excitability; with the MeCP2-/y cells showing a slightly accelerated development, with shorter action potential half-widths at early stages. There was no difference in the early phase of synapse development, but as the cultures matured, significant deficits became apparent, particularly for inhibitory synaptic activity. MeCP2-/y embryonic stem cell-derived neuronal cultures show clear developmental deficits that match phenotypes observed in slice preparations and thus provide a compelling tool to further investigate the mechanisms behind RTT pathophysiology.

  7. Differentiation of human adipose-derived stem cells into neuron-like cells by Radix Angelicae Sinensis

    Institute of Scientific and Technical Information of China (English)

    Qiaozhi Wang; Lile Zhou; Yong Guo; Guangyi Liu; Jiyan Cheng; Hong Yu

    2013-01-01

    Human adipose tissues are an ideal source of stem cells. It is important to find inducers that can safely and effectively differentiate stem cells into functional neurons for clinical use. In this study, we investigate the use of Radix Angelicae Sinensis as an inducer of neuronal differentiation. Primary human adipose-derived stem cells were obtained from adult subcutaneous fatty tissue, then pre-induced with 10%Radix Angelicae Sinensis injection for 24 hours, and incubated in serum-free Dulbecco’s modified Eagle’s medium/Nutrient Mixture F-12 containing 40% Radix Angelicae Si-nensis to induce its differentiation into neuron-like cells. Butylated hydroxyanisole, a common in-ducer for neuronal differentiation, was used as the control. After human adipose-derived stem cells differentiated into neuron-like cells under the induction of Radix Angelicae Sinensis for 24 hours, the positive expression of neuron-specific enolase was lower than that of the butylated hydroxyani-sole-induced group, and the expression of glial fibril ary acidic protein was negative. After they were induced for 48 hours, the positive expression of neuron specific enolase in human adipose-derived stem cells was significantly higher than that of the butylated hydroxyanisole-induced group. Our experimental findings indicate that Radix Angelicae Sinensis can induce human adipose-derived stem celldifferentiation into neuron-like cells and produce less cytotoxicity.

  8. Differentiation of Human Dental Pulp Stem Cells into Dopaminergic Neuron-like Cells in Vitro.

    Science.gov (United States)

    Chun, So Young; Soker, Shay; Jang, Yu-Jin; Kwon, Tae Gyun; Yoo, Eun Sang

    2016-02-01

    We investigated the potential of human dental pulp stem cells (hDPSCs) to differentiate into dopaminergic neurons in vitro as an autologous stem cell source for Parkinson's disease treatment. The hDPSCs were expanded in knockout-embryonic stem cell (KO-ES) medium containing leukemia inhibitory factor (LIF) on gelatin-coated plates for 3-4 days. Then, the medium was replaced with KO-ES medium without LIF to allow the formation of the neurosphere for 4 days. The neurosphere was transferred into ITS medium, containing ITS (human insulin-transferrin-sodium) and fibronectin, to select for Nestin-positive cells for 6-8 days. The cells were then cultured in N-2 medium containing basic fibroblast growth factor (FGF), FGF-8b, sonic hedgehog-N, and ascorbic acid on poly-l-ornithine/fibronectin-coated plates to expand the Nestin-positive cells for up to 2 weeks. Finally, the cells were transferred into N-2/ascorbic acid medium to allow for their differentiation into dopaminergic neurons for 10-15 days. The differentiation stages were confirmed by morphological, immunocytochemical, flow cytometric, real-time PCR, and ELISA analyses. The expressions of mesenchymal stem cell markers were observed at the early stages. The expressions of early neuronal markers were maintained throughout the differentiation stages. The mature neural markers showed increased expression from stage 3 onwards. The percentage of cells positive for tyrosine hydroxylase was 14.49%, and the amount was 0.526 ± 0.033 ng/mL at the last stage. hDPSCs can differentiate into dopaminergic neural cells under experimental cell differentiation conditions, showing potential as an autologous cell source for the treatment of Parkinson's disease.

  9. Surface strategies for control of neuronal cell adhesion: A review

    Science.gov (United States)

    Roach, P.; Parker, T.; Gadegaard, N.; Alexander, M. R.

    2010-06-01

    Material engineering methods have been used for many years to develop biomedical devices for use within the body to augment, repair or replace damaged tissues ranging from contact lenses to heart valves. Here we review the findings gathered from the wide and varied surface analytical approaches applied to study the interaction between biology and man-made materials. The key material characteristics identified to be important for biological recognition are surface chemistry, topography and compliance. Model surfaces with controlled chemistry and topography have provided insight into biological response to various types of topographical features over a wide range of length scales from nano to micrometres, along with 3D matrices that have been used as scaffolds to support cells for tissue formation. The cellular response to surfaces with localised areas of patterned chemistry and to those presenting gradually changing chemistry are discussed. Where previous reviews have been structured around specific classes of surface modification, e.g. self-assembly, or have broadly examined the response of various cells to numerous surfaces, we aim in this article to focus in particular on the tissues involved in the nervous system whilst providing a broad overview of key issues from the field of cell and protein surface interactions with surfaces. The goal of repair and treatment of diseases related to the central and peripheral nervous systems rely on understanding the local interfacial environment and controlling responses at the cellular level. The role of the protein layer deposited from serum containing media onto man-made surfaces is discussed. We highlight the particular problems associated with the repair of the nervous system, and review how neuronal attachment and axon guidance can be accomplished using various surface cues when cultured with single and multiple cell types. We include a brief glossary of techniques discussed in the body of this article aimed at the

  10. Niche-dependent development of functional neuronal networks from embryonic stem cell-derived neural populations

    Directory of Open Access Journals (Sweden)

    Siebler Mario

    2009-08-01

    Full Text Available Abstract Background The present work was performed to investigate the ability of two different embryonic stem (ES cell-derived neural precursor populations to generate functional neuronal networks in vitro. The first ES cell-derived neural precursor population was cultivated as free-floating neural aggregates which are known to form a developmental niche comprising different types of neural cells, including neural precursor cells (NPCs, progenitor cells and even further matured cells. This niche provides by itself a variety of different growth factors and extracellular matrix proteins that influence the proliferation and differentiation of neural precursor and progenitor cells. The second population was cultivated adherently in monolayer cultures to control most stringently the extracellular environment. This population comprises highly homogeneous NPCs which are supposed to represent an attractive way to provide well-defined neuronal progeny. However, the ability of these different ES cell-derived immature neural cell populations to generate functional neuronal networks has not been assessed so far. Results While both precursor populations were shown to differentiate into sufficient quantities of mature NeuN+ neurons that also express GABA or vesicular-glutamate-transporter-2 (vGlut2, only aggregate-derived neuronal populations exhibited a synchronously oscillating network activity 2–4 weeks after initiating the differentiation as detected by the microelectrode array technology. Neurons derived from homogeneous NPCs within monolayer cultures did merely show uncorrelated spiking activity even when differentiated for up to 12 weeks. We demonstrated that these neurons exhibited sparsely ramified neurites and an embryonic vGlut2 distribution suggesting an inhibited terminal neuronal maturation. In comparison, neurons derived from heterogeneous populations within neural aggregates appeared as fully mature with a dense neurite network and punctuated

  11. Development of an artificial neuronal network with post-mitotic rat fetal hippocampal cells by polyethylenimine.

    Science.gov (United States)

    Liu, Bingfang; Ma, Jun; Gao, Erjing; He, Yu; Cui, Fuzhai; Xu, Qunyuan

    2008-03-14

    The selection of appropriate surface materials that promote cellular adhesion and growth is an important consideration when designing a simplified neuronal network in vitro. In the past, extracellular matrix proteins such as laminin (LN) or positively charged substances such as poly-l-lysine (PLL) have been used. In this study, we examined the ability of another positively charged polymer, polyethyleneimine (PEI), to promote neuronal adhesion, growth and the formation of a functional neuronal network in vitro. PEI, PLL and LN were used to produce grid-shape patterns on glass coverslips by micro-contact printing. Post-mitotic neurons from the rat fetal hippocampus were cultured on the different polymers and the viability and morphology of these neurons under serum-free culture conditions were observed using fluorescent microscopy and atomic force microscopy (AFM). We show that neurons cultured on the PEI- and PLL-coated surfaces adhered to and extended neurites along the grid-shape patterns, whereas neurons cultured on the LN-coated coverslips clustered into clumps of cells. In addition, we found that the neurons on the PEI and PLL-coated grids survived for more than 2 weeks in serum-free conditions, whereas most neurons cultured on the LN-coated grids died after 1 week. Using AFM, we observed some neurosynapse-like structures near the neuronal soma on PEI-coated coverslips. These findings indicate that PEI is a suitable surface for establishing a functional neuronal network in vitro.

  12. Differentiation of fetal pancreatic stem cells into neuron-like and islet-like cells in vitro ★

    OpenAIRE

    Hua, Xiufeng; Wang, Yanwei; Lian, Peiwen; Zhang, Shouxin; Li, Jianyuan; Wang, Haiyan; Chen, Shulin; Gao, Wei

    2012-01-01

    Pancreatic stem cells were isolated and cultured from aborted human fetal pancreases of gestational age 14–20 weeks. They were seeded at a density of 1 × 104 in serum-free media for differentiation into neuron-like cells, expressing β-tubulin III and glial fibrillary acidic protein. These neuron-like cells displayed a synapse-like morphology and appeared to form a neuronal network. Pancreatic stem cells were also seeded at a density of 1 × 105 for differentiation into islet-like cells, expres...

  13. Rett syndrome induced pluripotent stem cell-derived neurons reveal novel neurophysiological alterations.

    Science.gov (United States)

    Farra, N; Zhang, W-B; Pasceri, P; Eubanks, J H; Salter, M W; Ellis, J

    2012-12-01

    Rett syndrome (RTT) is a neurodevelopmental autism spectrum disorder caused by mutations in the methyl-CpG-binding protein 2 (MECP2) gene. Here, we describe the first characterization and neuronal differentiation of induced pluripotent stem (iPS) cells derived from Mecp2-deficient mice. Fully reprogrammed wild-type (WT) and heterozygous female iPS cells express endogenous pluripotency markers, reactivate the X-chromosome and differentiate into the three germ layers. We directed iPS cells to produce glutamatergic neurons, which generated action potentials and formed functional excitatory synapses. iPS cell-derived neurons from heterozygous Mecp2(308) mice showed defects in the generation of evoked action potentials and glutamatergic synaptic transmission, as previously reported in brain slices. Further, we examined electrophysiology features not yet studied with the RTT iPS cell system and discovered that MeCP2-deficient neurons fired fewer action potentials, and displayed decreased action potential amplitude, diminished peak inward currents and higher input resistance relative to WT iPS-derived neurons. Deficiencies in action potential firing and inward currents suggest that disturbed Na(+) channel function may contribute to the dysfunctional RTT neuronal network. These phenotypes were additionally confirmed in neurons derived from independent WT and hemizygous mutant iPS cell lines, indicating that these reproducible deficits are attributable to MeCP2 deficiency. Taken together, these results demonstrate that neuronally differentiated MeCP2-deficient iPS cells recapitulate deficits observed previously in primary neurons, and these identified phenotypes further illustrate the requirement of MeCP2 in neuronal development and/or in the maintenance of normal function. By validating the use of iPS cells to delineate mechanisms underlying RTT pathogenesis, we identify deficiencies that can be targeted for in vitro translational screens.

  14. Highly efficient and large-scale generation of functional dopamine neurons from human embryonic stem cells

    OpenAIRE

    Cho, Myung Soo; Lee, Young-Eun; Kim, Ji Young; Chung, Seungsoo; Cho, Yoon Hee; Kim, Dae-Sung; Kang, Sang-Moon; Lee, Haksup; Kim, Myung-Hwa; Kim, Jeong-Hoon; Leem, Joong Woo; Oh, Sun Kyung; Choi, Young Min; Hwang, Dong-Youn; Chang, Jin Woo

    2008-01-01

    We developed a method for the efficient generation of functional dopaminergic (DA) neurons from human embryonic stem cells (hESCs) on a large scale. The most unique feature of this method is the generation of homogeneous spherical neural masses (SNMs) from the hESC-derived neural precursors. These SNMs provide several advantages: (i) they can be passaged for a long time without losing their differentiation capability into DA neurons; (ii) they can be coaxed into DA neurons at much higher effi...

  15. Efficient and cost-effective generation of mature neurons from human induced pluripotent stem cells.

    Science.gov (United States)

    Badja, Cherif; Maleeva, Galyna; El-Yazidi, Claire; Barruet, Emilie; Lasserre, Manon; Tropel, Philippe; Binetruy, Bernard; Bregestovski, Piotr; Magdinier, Frédérique

    2014-12-01

    For years, our ability to study pathological changes in neurological diseases has been hampered by the lack of relevant models until the recent groundbreaking work from Yamanaka's group showing that it is feasible to generate induced pluripotent stem cells (iPSCs) from human somatic cells and to redirect the fate of these iPSCs into differentiated cells. In particular, much interest has focused on the ability to differentiate human iPSCs into neuronal progenitors and functional neurons for relevance to a large number of pathologies including mental retardation and behavioral or degenerative syndromes. Current differentiation protocols are time-consuming and generate limited amounts of cells, hindering use on a large scale. We describe a feeder-free method relying on the use of a chemically defined medium that overcomes the need for embryoid body formation and neuronal rosette isolation for neuronal precursors and terminally differentiated neuron production. Four days after induction, expression of markers of the neurectoderm lineage is detectable. Between 4 and 7 days, neuronal precursors can be expanded, frozen, and thawed without loss of proliferation and differentiation capacities or further differentiated. Terminal differentiation into the different subtypes of mature neurons found in the human brain were observed. At 6-35 days after induction, cells express typical voltage-gated and ionotrophic receptors for GABA, glycine, and acetylcholine. This specific and efficient single-step strategy in a chemically defined medium allows the production of mature neurons in 20-40 days with multiple applications, especially for modeling human pathologies.

  16. Differentiation of fetal pancreatic stem cells into neuron-like and islet-like cells in vitro

    Institute of Scientific and Technical Information of China (English)

    Xiufeng Hua; Yanwei Wang; Peiwen Lian; Shouxin Zhang; Jianyuan Li; Haiyan Wang; Shulin Chen; Wei Gao

    2012-01-01

    Pancreatic stem cells were isolated and cultured from aborted human fetal pancreases of gestational age 14-20 weeks.They were seeded at a density of 1 × 104 in serum-free media for differentiation into neuron-like cells, expressing β-tubulin III and glial fibrillary acidic protein.These neuron-like cells displayed a synapse-like morphology and appeared to form a neuronal network.Pancreatic stem cells were also seeded at a density of 1 × 105 for differentiation into islet-like cells, expressing insulin and glucagon, with an islet-like morphology.These cells had glucose-stimulated secretion of human insulin and C-peptide.Results suggest that pancreatic stem cells can be differentiated into neuron-like and islet-like cells.

  17. Label-free distinguishing between neurons and glial cells based on two-photon excited fluorescence signal of neuron perinuclear granules

    Science.gov (United States)

    Du, Huiping; Jiang, Liwei; Wang, Xingfu; Liu, Gaoqiang; Wang, Shu; Zheng, Liqin; Li, Lianhuang; Zhuo, Shuangmu; Zhu, Xiaoqin; Chen, Jianxin

    2016-08-01

    Neurons and glial cells are two critical cell types of brain tissue. Their accurate identification is important for the diagnosis of psychiatric disorders such as depression and schizophrenia. In this paper, distinguishing between neurons and glial cells by using the two-photon excited fluorescence (TPEF) signals of intracellular intrinsic sources was performed. TPEF microscopy combined with TUJ-1 and GFAP immunostaining and quantitative image analysis demonstrated that the perinuclear granules of neurons in the TPEF images of brain tissue and the primary cultured cortical cells were a unique characteristic of neurons compared to glial cells which can become a quantitative feature to distinguish neurons from glial cells. With the development of miniaturized TPEF microscope (‘two-photon fiberscopes’) imaging devices, TPEF microscopy can be developed into an effective diagnostic and monitoring tool for psychiatric disorders such as depression and schizophrenia.

  18. Individual aggregates of amyloid beta induce temporary calcium influx through the cell membrane of neuronal cells

    Science.gov (United States)

    Drews, Anna; Flint, Jennie; Shivji, Nadia; Jönsson, Peter; Wirthensohn, David; De Genst, Erwin; Vincke, Cécile; Muyldermans, Serge; Dobson, Chris; Klenerman, David

    2016-01-01

    Local delivery of amyloid beta oligomers from the tip of a nanopipette, controlled over the cell surface, has been used to deliver physiological picomolar oligomer concentrations to primary astrocytes or neurons. Calcium influx was observed when as few as 2000 oligomers were delivered to the cell surface. When the dosing of oligomers was stopped the intracellular calcium returned to basal levels or below. Calcium influx was prevented by the presence in the pipette of the extracellular chaperone clusterin, which is known to selectively bind oligomers, and by the presence a specific nanobody to amyloid beta. These data are consistent with individual oligomers larger than trimers inducing calcium entry as they cross the cell membrane, a result supported by imaging experiments in bilayers, and suggest that the initial molecular event that leads to neuronal damage does not involve any cellular receptors, in contrast to work performed at much higher oligomer concentrations. PMID:27553885

  19. Autophagy activator promotes neuronal differentiation of adult adipose-derived stromal cells

    Institute of Scientific and Technical Information of China (English)

    Yanhui Lu; Xiaodong Yuan; Qiaoyu Sun; Ya Ou

    2013-01-01

    Preliminary research from our group found altered autophagy intensity during adipose-derived stromal cell differentiation into neuronal-like cells, and that this change was associated with morphological changes in differentiated cells. This study aimed to verify the role of rapamycin, an autophagy activator, in the process of adipose-derived stromal cell differentiation into neuronal-like cells. Immunohistochemical staining showed that expression of neuron-specific enolase and neurofilament-200 were gradually upregulated in adipose-derived stromal cells after 5 mM β-mercaptoethanol induction, and the differentiation rate gradually increased with induction time. Using transmission electron microscopy, induced cells were shown to exhibit cytoplasmic autophagosomes, with bilayer membranes, and autolysosomes. After rapamycin (200μg/L) induction for 1 hour, adipose-derived stromal cells began to extend long processes, similar to the morphology of neuronal-like cells, while untreated cells did not exhibit similar morphologies until 3 hours after induction. Moreover, the differentiation rate was significantly increased after rapamycin treatment. Compared with untreated cells, expression of LC3, an autophagy protein, was also significantly upregulated. Positive LC3 expression tended to concentrate at cell nuclei with increasing induction times. Our experimental findings indicate that autophagy can significantly increase the speed of adipose-derived stromal cell differentiation into neuronal-like cells.

  20. Non-neuronal Cells in ALS: Role of Glial, Immune cells and Blood-CNS Barriers.

    Science.gov (United States)

    Puentes, Fabiola; Malaspina, Andrea; van Noort, Johannes M; Amor, Sandra

    2016-03-01

    Neurological dysfunction and motor neuron degeneration in amyotrophic lateral sclerosis (ALS) is strongly associated with neuroinflammation reflected by activated microglia and astrocytes in the CNS. In ALS endogenous triggers in the CNS such as aggregated protein and misfolded proteins activate a pathogenic response by innate immune cells. However, there is also strong evidence for a neuroprotective immune response in ALS. Emerging evidence also reveals changes in the peripheral adaptive immune responses as well as alterations in the blood brain barrier that may aid traffic of lymphocytes and antibodies into the CNS. Understanding the triggers of neuroinflammation is key to controlling neuronal loss. Here, we review the current knowledge regarding the roles of non-neuronal cells as well as the innate and adaptive immune responses in ALS. Existing ALS animal models, in particular genetic rodent models, are very useful to study the underlying pathogenic mechanisms of motor neuron degeneration. We also discuss the approaches used to target the pathogenic immune responses and boost the neuroprotective immune pathways as novel immunotherapies for ALS. PMID:26780491

  1. Mitosis in neurons: Roughex and APC/C maintain cell cycle exit to prevent cytokinetic and axonal defects in Drosophila photoreceptor neurons.

    Directory of Open Access Journals (Sweden)

    Robert Ruggiero

    Full Text Available The mechanisms of cell cycle exit by neurons remain poorly understood. Through genetic and developmental analysis of Drosophila eye development, we found that the cyclin-dependent kinase-inhibitor Roughex maintains G1 cell cycle exit during differentiation of the R8 class of photoreceptor neurons. The roughex mutant neurons re-enter the mitotic cell cycle and progress without executing cytokinesis, unlike non-neuronal cells in the roughex mutant that perform complete cell divisions. After mitosis, the binucleated R8 neurons usually transport one daughter nucleus away from the cell body into the developing axon towards the brain in a kinesin-dependent manner resembling anterograde axonal trafficking. Similar cell cycle and photoreceptor neuron defects occurred in mutants for components of the Anaphase Promoting Complex/Cyclosome. These findings indicate a neuron-specific defect in cytokinesis and demonstrate a critical role for mitotic cyclin downregulation both to maintain cell cycle exit during neuronal differentiation and to prevent axonal defects following failed cytokinesis.

  2. Human stem cell neuronal differentiation on silk-carbon nanotube composite

    Science.gov (United States)

    Chen, Chi-Shuo; Soni, Sushant; Le, Catherine; Biasca, Matthew; Farr, Erik; Chen, Eric Y.-T.; Chin, Wei-Chun

    2012-02-01

    Human embryonic stem cells [hESCs] are able to differentiate into specific lineages corresponding to regulated spatial and temporal signals. This unique attribute holds great promise for regenerative medicine and cell-based therapy for many human diseases such as spinal cord injury [SCI] and multiple sclerosis [MS]. Carbon nanotubes [CNTs] have been successfully used to promote neuronal differentiation, and silk has been widely applied in tissue engineering. This study aims to build silk-CNT composite scaffolds for improved neuron differentiation efficiency from hESCs. Two neuronal markers (β-III tubulin and nestin) were utilized to determine the hESC neuronal lineage differentiation. In addition, axonal lengths were measured to evaluate the progress of neuronal development. The results demonstrated that cells on silk-CNT scaffolds have a higher β-III tubulin and nestin expression, suggesting augmented neuronal differentiation. In addition, longer axons with higher density were found to associate with silk-CNT scaffolds. Our silk-CNT-based composite scaffolds can promote neuronal differentiation of hESCs. The silk-CNT composite scaffolds developed here can serve as efficient supporting matrices for stem cell-derived neuronal transplants, offering a promising opportunity for nerve repair treatments for SCI and MS patients.

  3. Apoptotic neurons induce proliferative responses of progenitor cells in the postnatal neocortex.

    Science.gov (United States)

    Petrenko, Volodymyr; Mihhailova, Jevgenia; Salmon, Patrick; Kiss, Jozsef Z

    2015-11-01

    Apoptotic cell death is the leading cause of neuronal loss after neonatal brain injury. Little is known about the intrinsic capacity of the immature cerebral cortex for replacing dead cells. Here we test the hypothesis that neuronal apoptosis is able to trigger compensatory proliferation in surrounding cells. In order to establish a "pure" apoptotic cell death model and to avoid the confounding effects of broken blood-brain barrier and inflammatory reactions, we used a diphtheria toxin (DT) and diphtheria toxin receptor (DTR) system to induce ablation of layer IV neurons in the rodent somatosensory cortex during the early postnatal period. We found that DT-triggered apoptosis is a slowly progressing event lasting about for 7 days. While dying cells expressed the morphological features of apoptosis, we could not detect immunoreactivity for activated caspase-3 in these cells. Microglia activation and proliferation represented the earliest cellular responses to apoptotic cell death. In addition, we found that induced apoptosis triggered a massive proliferation of undifferentiated progenitor cell pool including Sox2 as well as NG2 cells. The default differentiation pattern of proliferating progenitors appears to be the glial phenotype; we could not find evidence for newly generated neurons in response to apoptotic neuronal death. These results suggest that mitotically active progenitor populations are intrinsically capable to contribute to the repair process of injured cortical tissue and may represent a potential target for neuronal replacement strategies.

  4. ATP released by injured neurons activates Schwann cells

    Directory of Open Access Journals (Sweden)

    Samuele eNegro

    2016-05-01

    Full Text Available Injured nerve terminals of neuromuscular junctions (NMJs can regenerate. This remarkable and complex response is governed by molecular signals that are exchanged among the cellular components of this synapse: motor axon nerve terminal (MAT, perisynaptic Schwann cells (PSCs, and muscle fibre. The nature of signals that govern MAT regeneration is ill-known. In the present study the spider toxin α-Latrotoxin has been used as tool to investigate the mechanisms underlying peripheral neuroregeneration. Indeed this neurotoxin induces an acute, specific, localized and fully reversible damage of the presynaptic nerve terminal, and its action mimics the cascade of events that leads to nerve terminal degeneration in injured patients and in many neurodegenerative conditions. Here we provide evidence of an early release by degenerating neurons of ATP as alarm messenger, that contributes to the activation of a series of intracellular pathways within SCs that are crucial for nerve regeneration: Ca2+, cAMP, ERK1/2, and CREB. These results contribute to define the cross-talk taking place among degenerating nerve terminals and PSCs, involved in the functional recovery of the NMJ.

  5. Modeling Neurological Disease by Rapid Conversion of Human Urine Cells into Functional Neurons

    Directory of Open Access Journals (Sweden)

    Shu-Zhen Zhang

    2016-01-01

    Full Text Available Somatic cells can be directly converted into functional neurons by ectopic expression of defined factors and/or microRNAs. Since the first report of conversion mouse embryonic fibroblasts into functional neurons, the postnatal mouse, and human fibroblasts, astroglia, hepatocytes, and pericyte-derived cells have been converted into functional dopaminergic and motor neurons both in vitro and in vivo. However, it is invasive to get all these materials. In the current study, we provide a noninvasive approach to obtain directly reprogrammed functional neurons by overexpression of the transcription factors Ascl1, Brn2, NeuroD, c-Myc, and Myt1l in human urine cells. These induced neuronal (iN cells could express multiple neuron-specific proteins and generate action potentials. Moreover, urine cells from Wilson’s disease (WD patient could also be directly converted into neurons. In conclusion, generation of iN cells from nonneural lineages is a feasible and befitting approach for neurological disease modeling.

  6. Thrombospondin 1 promotes synaptic formation in bone marrow-derived neuron-like cells

    Institute of Scientific and Technical Information of China (English)

    Yun Huang; Mingnan Lu; Weitao Guo; Rong Zeng; Bin Wang; Huaibo Wang

    2013-01-01

    In this study, a combination of growth factors was used to induce bone marrow mesenchymal stem cells differentiation into neuron-like cells, in a broader attempt to observe the role of thrombospondin 1 in synapse formation. Results showed that there was no significant difference in the differentiation rate of neuron-like cells between bone marrow mesenchymal stem cells with thrombospondin induction and those without. However, the cell shape was more complex and the neurites were dendritic, with unipolar, bipolar or multipolar morphologies, after induction with thrombospondin 1. The induced cells were similar in morphology to normal neurites. Immunohistochemical staining showed that the number of positive cells for postsynaptic density protein 95 and synaptophysin 1 protein was significantly increased after induction with thrombospondin 1. These findings indicate that thrombospondin 1 promotes synapse formation in neuron-like cells that are differentiated from bone marrow mesenchymal stem cells.

  7. [Glial cells are involved in iron accumulation and degeneration of dopamine neurons in Parkinson's disease].

    Science.gov (United States)

    Xu, Hua-Min; Wang, Jun; Song, Ning; Jiang, Hong; Xie, Jun-Xia

    2016-08-25

    A growing body of evidence suggests that glial cells play an important role in neural development, neural survival, nerve repair and regeneration, synaptic transmission and immune inflammation. As the highest number of cells in the central nervous system, the role of glial cells in Parkinson's disease (PD) has attracted more and more attention. It has been confirmed that nigral iron accumulation contributes to the death of dopamine (DA) neurons in PD. Until now, most researches on nigral iron deposition in PD are focusing on DA neurons, but in fact glial cells in the central nervous system also play an important role in the regulation of iron homeostasis. Therefore, this review describes the role of iron metabolism of glial cells in death of DA neurons in PD, which could provide evidence to reveal the mechanisms underlying nigral iron accumulation of DA neurons in PD and provide the basis for discovering new potential therapeutic targets for PD. PMID:27546505

  8. Reduced Neurite Density in Neuronal Cell Cultures Exposed to Serum of Patients with Bipolar Disorder

    Science.gov (United States)

    Wollenhaupt-Aguiar, Bianca; Pfaffenseller, Bianca; Chagas, Vinicius de Saraiva; Castro, Mauro A A; Passos, Ives Cavalcante; Kauer-Sant’Anna, Márcia; Kapczinski, Flavio

    2016-01-01

    Background: Increased inflammatory markers and oxidative stress have been reported in serum among patients with bipolar disorder (BD). The aim of this study is to assess whether biochemical changes in the serum of patients induces neurotoxicity in neuronal cell cultures. Methods: We challenged the retinoic acid-differentiated human neuroblastoma SH-SY5Y cells with the serum of BD patients at early and late stages of illness and assessed neurite density and cell viability as neurotoxic endpoints. Results: Decreased neurite density was found in neurons treated with the serum of patients, mostly patients at late stages of illness. Also, neurons challenged with the serum of late-stage patients showed a significant decrease in cell viability. Conclusions: Our findings showed that the serum of patients with bipolar disorder induced a decrease in neurite density and cell viability in neuronal cultures. PMID:27207915

  9. Cell-Type-Specific Sensorimotor Processing in Striatal Projection Neurons during Goal-Directed Behavior.

    Science.gov (United States)

    Sippy, Tanya; Lapray, Damien; Crochet, Sylvain; Petersen, Carl C H

    2015-10-21

    Goal-directed sensorimotor transformation drives important aspects of mammalian behavior. The striatum is thought to play a key role in reward-based learning and action selection, receiving glutamatergic sensorimotor signals and dopaminergic reward signals. Here, we obtain whole-cell membrane potential recordings from the dorsolateral striatum of mice trained to lick a reward spout after a whisker deflection. Striatal projection neurons showed strong task-related modulation, with more depolarization and action potential firing on hit trials compared to misses. Direct pathway striatonigral neurons, but not indirect pathway striatopallidal neurons, exhibited a prominent early sensory response. Optogenetic stimulation of direct pathway striatonigral neurons, but not indirect pathway striatopallidal neurons, readily substituted for whisker stimulation evoking a licking response. Our data are consistent with direct pathway striatonigral neurons contributing a "go" signal for goal-directed sensorimotor transformation leading to action initiation. VIDEO ABSTRACT.

  10. An Atomic Force Microscopy based investigation of specific biomechanical properties for various types of neuronal cells

    Science.gov (United States)

    Spedden, Elise; White, James; Kaplan, David; Staii, Cristian

    2012-02-01

    Here we describe the use of Atomic Force Microscope (AFM) based techniques to characterize and explore the influence of biochemical and biomechanical cues on the growth and interaction of neuronal cells with surrounding guidance factors. Specifically, we use AFM topography and AFM force spectroscopy measurements to systematically investigate the morphology, elasticity, and real time growth of neuronal processes in the presence of different types of extracellular matrix proteins and growth factors. We therefore create a series of systems containing specified neuron densities where the type of the underlying growth promoting protein is different from sample to sample. For each system we measure key biomechanical parameters related to neuronal growth such as height and elastic modulus at multiple growth points on several types of neurons. We show that systematic measurements of these parameters yield fundamental information about the role played by substrate-plated guidance factors in determining elastic and morphological properties of neurons during growth.

  11. Molecular heterogeneity of midbrain dopaminergic neurons--Moving toward single cell resolution.

    Science.gov (United States)

    Anderegg, Angela; Poulin, Jean-Francois; Awatramani, Rajeshwar

    2015-12-21

    Since their discovery, midbrain dopamine (DA) neurons have been researched extensively, in part because of their diverse functions and involvement in various neuropsychiatric disorders. Over the last few decades, reports have emerged that midbrain DA neurons were not a homogeneous group, but that DA neurons located in distinct anatomical locations within the midbrain had distinctive properties in terms of physiology, function, and vulnerability. Accordingly, several studies focused on identifying heterogeneous gene expression across DA neuron clusters. Here we review the importance of understanding DA neuron heterogeneity at the molecular level, previous studies detailing heterogeneous gene expression in DA neurons, and finally recent work which brings together previous heterogeneous gene expression profiles in a coordinated manner, at single cell resolution.

  12. Representation of spontaneous movement by dopaminergic neurons is cell-type selective and disrupted in parkinsonism.

    Science.gov (United States)

    Dodson, Paul D; Dreyer, Jakob K; Jennings, Katie A; Syed, Emilie C J; Wade-Martins, Richard; Cragg, Stephanie J; Bolam, J Paul; Magill, Peter J

    2016-04-12

    Midbrain dopaminergic neurons are essential for appropriate voluntary movement, as epitomized by the cardinal motor impairments arising in Parkinson's disease. Understanding the basis of such motor control requires understanding how the firing of different types of dopaminergic neuron relates to movement and how this activity is deciphered in target structures such as the striatum. By recording and labeling individual neurons in behaving mice, we show that the representation of brief spontaneous movements in the firing of identified midbrain dopaminergic neurons is cell-type selective. Most dopaminergic neurons in the substantia nigra pars compacta (SNc), but not in ventral tegmental area or substantia nigra pars lateralis, consistently represented the onset of spontaneous movements with a pause in their firing. Computational modeling revealed that the movement-related firing of these dopaminergic neurons can manifest as rapid and robust fluctuations in striatal dopamine concentration and receptor activity. The exact nature of the movement-related signaling in the striatum depended on the type of dopaminergic neuron providing inputs, the striatal region innervated, and the type of dopamine receptor expressed by striatal neurons. Importantly, in aged mice harboring a genetic burden relevant for human Parkinson's disease, the precise movement-related firing of SNc dopaminergic neurons and the resultant striatal dopamine signaling were lost. These data show that distinct dopaminergic cell types differentially encode spontaneous movement and elucidate how dysregulation of their firing in early Parkinsonism can impair their effector circuits. PMID:27001837

  13. Unidirectional signal propagation in primary neurons micropatterned at a single-cell resolution

    Science.gov (United States)

    Yamamoto, H.; Matsumura, R.; Takaoki, H.; Katsurabayashi, S.; Hirano-Iwata, A.; Niwano, M.

    2016-07-01

    The structure and connectivity of cultured neuronal networks can be controlled by using micropatterned surfaces. Here, we demonstrate that the direction of signal propagation can be precisely controlled at a single-cell resolution by growing primary neurons on micropatterns. To achieve this, we first examined the process by which axons develop and how synapses form in micropatterned primary neurons using immunocytochemistry. By aligning asymmetric micropatterns with a marginal gap, it was possible to pattern primary neurons with a directed polarization axis at the single-cell level. We then examined how synapses develop on micropatterned hippocampal neurons. Three types of micropatterns with different numbers of short paths for dendrite growth were compared. A normal development in synapse density was observed when micropatterns with three or more short paths were used. Finally, we performed double patch clamp recordings on micropatterned neurons to confirm that these synapses are indeed functional, and that the neuronal signal is transmitted unidirectionally in the intended orientation. This work provides a practical guideline for patterning single neurons to design functional neuronal networks in vitro with the direction of signal propagation being controlled.

  14. Representation of spontaneous movement by dopaminergic neurons is cell-type selective and disrupted in parkinsonism.

    Science.gov (United States)

    Dodson, Paul D; Dreyer, Jakob K; Jennings, Katie A; Syed, Emilie C J; Wade-Martins, Richard; Cragg, Stephanie J; Bolam, J Paul; Magill, Peter J

    2016-04-12

    Midbrain dopaminergic neurons are essential for appropriate voluntary movement, as epitomized by the cardinal motor impairments arising in Parkinson's disease. Understanding the basis of such motor control requires understanding how the firing of different types of dopaminergic neuron relates to movement and how this activity is deciphered in target structures such as the striatum. By recording and labeling individual neurons in behaving mice, we show that the representation of brief spontaneous movements in the firing of identified midbrain dopaminergic neurons is cell-type selective. Most dopaminergic neurons in the substantia nigra pars compacta (SNc), but not in ventral tegmental area or substantia nigra pars lateralis, consistently represented the onset of spontaneous movements with a pause in their firing. Computational modeling revealed that the movement-related firing of these dopaminergic neurons can manifest as rapid and robust fluctuations in striatal dopamine concentration and receptor activity. The exact nature of the movement-related signaling in the striatum depended on the type of dopaminergic neuron providing inputs, the striatal region innervated, and the type of dopamine receptor expressed by striatal neurons. Importantly, in aged mice harboring a genetic burden relevant for human Parkinson's disease, the precise movement-related firing of SNc dopaminergic neurons and the resultant striatal dopamine signaling were lost. These data show that distinct dopaminergic cell types differentially encode spontaneous movement and elucidate how dysregulation of their firing in early Parkinsonism can impair their effector circuits.

  15. Cell surface estrogen receptor alpha is upregulated during subchronic metabolic stress and inhibits neuronal cell degeneration.

    Directory of Open Access Journals (Sweden)

    Cristiana Barbati

    Full Text Available In addition to the classical nuclear estrogen receptor, the expression of non-nuclear estrogen receptors localized to the cell surface membrane (mER has recently been demonstrated. Estrogen and its receptors have been implicated in the development or progression of numerous neurodegenerative disorders. Furthermore, the pathogenesis of these diseases has been associated with disturbances of two key cellular programs: apoptosis and autophagy. An excess of apoptosis or a defect in autophagy has been implicated in neurodegeneration. The aim of this study was to clarify the role of ER in determining neuronal cell fate and the possible implication of these receptors in regulating either apoptosis or autophagy. The human neuronal cell line SH-SY5Y and mouse neuronal cells in primary culture were thus exposed to chronic minimal peroxide treatment (CMP, a form of subcytotoxic minimal chronic stress previously that mimics multiple aspects of long-term cell stress and represents a limited molecular proxy for neurodegenerative processes. We actually found that either E2 or E2-bovine serum albumin construct (E2BSA, i.e. a non-permeant form of E2 was capable of modulating intracellular cell signals and regulating cell survival and death. In particular, under CMP, the up-regulation of mERα, but not mERβ, was associated with functional signals (ERK phosphorylation and p38 dephosphorylation compatible with autophagic cytoprotection triggering and leading to cell survival. The mERα trafficking appeared to be independent of the microfilament system cytoskeletal network but was seemingly associated with microtubular apparatus network, i.e., to MAP2 molecular chaperone. Importantly, antioxidant treatments, administration of siRNA to ERα, or the presence of antagonist of ERα hindered these events. These results support that the surface expression of mERα plays a pivotal role in determining cell fate, and that ligand-induced activation of mER signalling exerts a

  16. In vitro differentiation of bone marrow stromal cells into neurons and glial cells and differential protein expression in a two-compartment bone marrow stromal cell/neuron co-culture system.

    Science.gov (United States)

    Qi, Xu; Shao, Ming; Peng, Haisheng; Bi, Zhenggang; Su, Zhiqiang; Li, Hulun

    2010-07-01

    This study was performed to establish a bone marrow stromal cell (BMSC)/neuron two-compartment co-culture model in which differentiation of BMSCs into neurons could occur without direct contact between the two cell types, and to investigate protein expression changes during differentiation of this entirely BMSC-derived population. Cultured BMSCs isolated from Wistar rats were divided into three groups: BMSC culture, BMSC/neuron co-culture and BMSC/neuron two-compartment co-culture. Cells were examined for neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) expression. The electrophysiological behavior of the BMSCs was examined using patch clamping. Proteins that had significantly different expression levels in BMSCs cultured alone and co-cultured with neurons were studied using a protein chip-mass spectroscopy technique. Expression of NSE and GFAP were significantly higher in co-culture cells than in two-compartment co-culture cells, and significantly higher in both co-culture groups than in BMSCs cultured alone. Five proteins showed significant changes in expression during differentiation: TIP39_RAT and CALC_RAT underwent increases, and INSL6_RAT, PNOC_RAT and PCSK1_RAT underwent decreases in expression. We conclude that BMSCs can differentiate into neurons during both contact co-culture with neurons and two-compartment co-culture with neurons. The rate at which BMSCs differentiated into neurons was higher in contact co-culture than in non-contact co-culture.

  17. miR-29a modulates neuronal differentiation through targeting REST in mesenchymal stem cells.

    Directory of Open Access Journals (Sweden)

    Ping Duan

    Full Text Available OBJECTIVE: To investigate the modulation of microRNAs (miRNAs upon the neuronal differentiation of mesenchymal stem cells (MSCs through targeting RE-1 Silencing Factor (REST, a mature neuronal gene suppressor in neuronal and un-neuronal cells. METHODS: Rat bone marrow derived-MSCs were induced into neuron-like cells (MSC-NCs by DMSO and BHA in vitro. The expression of neuron specific enolase (NSE, microtubule-associated protein tau (Tau, REST and its target genes, including synaptosomal-associated protein 25 (SNAP25 and L1 cell adhesion molecular (L1CAM, were detected in MSCs and MSC-NCs. miRNA array analysis was conducted to screen for the upregulated miRNAs after neuronal differentiation. TargetScan was used to predict the relationship between these miRNAs and REST gene, and dual luciferase reporter assay was applied to validate it. Gain and loss of function experiments were used to study the role of miR-29a upon neuronal differentiation of MSCs. The knockdown of REST was conducted to show that miR-29a affected this process through targeting REST. RESULTS: MSCs were induced into neuron-like cells which presented neuronal cell shape and expressed NSE and Tau. The expression of REST declined and the expression of SNAP25 and L1CAM increased upon the neuronal differentiation of MSCs. Among 14 upregulated miRNAs, miR-29a was validated to target REST gene. During the neuronal differentiation of MSCs, miR-29a inhibition blocked the downregulation of REST, as well as the upregulation of SNAP25, L1CAM, NSE and Tau. REST knockdown rescued the effect of miR-29a inhibition on the expression of NSE and Tau. Meanwhile, miR-29a knockin significantly decreased the expression of REST and increased the expression of SNAP25 and L1CMA in MSCs, but did not significantly affect the expression of NSE and Tau. CONCLUSION: miR-29a regulates neurogenic markers through targeting REST in mesenchymal stem cells, which provides advances in neuronal differentiation research

  18. The NG2 Protein Is Not Required for Glutamatergic Neuron-NG2 Cell Synaptic Signaling.

    Science.gov (United States)

    Passlick, Stefan; Trotter, Jacqueline; Seifert, Gerald; Steinhäuser, Christian; Jabs, Ronald

    2016-01-01

    NG2 glial cells (as from now NG2 cells) are unique in receiving synaptic input from neurons. However, the components regulating formation and maintenance of these neuron-glia synapses remain elusive. The transmembrane protein NG2 has been considered a potential mediator of synapse formation and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) clustering, because it contains 2 extracellular Laminin G/Neurexin/Sex Hormone-Binding Globulin domains, which in neurons are crucial for formation of transsynaptic neuroligin-neurexin complexes. NG2 is connected via Glutamate Receptor-Interacting Protein with GluA2/3-containing AMPARs, thereby possibly mediating receptor clustering in glial postsynaptic density. To elucidate the role of NG2 in neuron-glia communication, we investigated glutamatergic synaptic transmission in juvenile and aged hippocampal NG2 cells of heterozygous and homozygous NG2 knockout mice. Neuron-NG2 cell synapses readily formed in the absence of NG2. Short-term plasticity, synaptic connectivity, postsynaptic AMPAR current kinetics, and density were not affected by NG2 deletion. During development, an NG2-independent acceleration of AMPAR current kinetics and decreased synaptic connectivity were observed. Our results indicate that the lack of NG2 does not interfere with genesis and basic properties of neuron-glia synapses. In addition, we demonstrate frequent expression of neuroligins 1-3 in juvenile and aged NG2 cells, suggesting a role of these molecules in synapse formation between NG2 glia and neurons.

  19. Mutant SOD1 in cell types other than motor neurons and oligodendrocytes accelerates onset of disease in ALS mice

    OpenAIRE

    Yamanaka, Koji; Boillee, Severine; Roberts, Elizabeth A.; Garcia, Michael L.; McAlonis-Downes, Melissa; Mikse, Oliver R.; Cleveland, Don W.; Lawrence S B Goldstein

    2008-01-01

    Dominant mutations in ubiquitously expressed superoxide dismutase (SOD1) cause familial ALS by provoking premature death of adult motor neurons. To test whether mutant damage to cell types beyond motor neurons is required for the onset of motor neuron disease, we generated chimeric mice in which all motor neurons and oligodendrocytes expressed mutant SOD1 at a level sufficient to cause fatal, early-onset motor neuron disease when expressed ubiquitously, but did so in a cellular environment co...

  20. Bone marrow mesenchymal stem cells stimulate proliferation and neuronal differentiation of retinal progenitor cells.

    Directory of Open Access Journals (Sweden)

    Jing Xia

    Full Text Available During retina development, retinal progenitor cell (RPC proliferation and differentiation are regulated by complex inter- and intracellular interactions. Bone marrow mesenchymal stem cells (BMSCs are reported to express a variety of cytokines and neurotrophic factors, which have powerful trophic and protective functions for neural tissue-derived cells. Here, we show that the expanded RPC cultures treated with BMSC-derived conditioned medium (CM which was substantially enriched for bFGF and CNTF, expressed clearly increased levels of nuclear receptor TLX, an essential regulator of neural stem cell (NSC self-renewal, as well as betacellulin (BTC, an EGF-like protein described as supporting NSC expansion. The BMSC CM- or bFGF-treated RPCs also displayed an obviously enhanced proliferation capability, while BMSC CM-derived bFGF knocked down by anti-bFGF, the effect of BMSC CM on enhancing RPC proliferation was partly reversed. Under differentiation conditions, treatment with BMSC CM or CNTF markedly favoured RPC differentiation towards retinal neurons, including Brn3a-positive retinal ganglion cells (RGCs and rhodopsin-positive photoreceptors, and clearly diminished retinal glial cell differentiation. These findings demonstrate that BMSCs supported RPC proliferation and neuronal differentiation which may be partly mediated by BMSC CM-derived bFGF and CNTF, reveal potential limitations of RPC culture systems, and suggest a means for optimizing RPC cell fate determination in vitro.

  1. Crocin suppresses tumor necrosis factor-alpha-induced cell death of neuronally differentiated PC-12 cells.

    Science.gov (United States)

    Soeda, S; Ochiai, T; Paopong, L; Tanaka, H; Shoyama, Y; Shimeno, H

    2001-11-01

    Crocus sativus L. is used in Chinese traditional medicine to treat some disorders of the central nervous system. Crocin is an ethanol-extractable component of Crocus sativus L.; it is reported to prevent ethanol-induced impairment of learning and memory in mice. In this study, we demonstrate that crocin suppresses the effect of tumor necrosis factor (TNF)-alpha on neuronally differentiated PC-12 cells. PC-12 cells dead from exposure to TNF-alpha show apoptotic morphological changes and DNA fragmentation. These hallmark features of cell death did not appear in cells treated in the co-presence of 10 microM crocin. Moreover, crocin suppressed the TNF-alpha-induced expression of Bcl-Xs and LICE mRNAs and simultaneously restored the cytokine-induced reduction of Bcl-X(L) mRNA expression. The modulating effects of crocin on the expression of Bcl-2 family proteins led to a marked reduction of a TNF-alpha-induced release of cytochrome c from the mitochondria. Crocin also blocked the cytochrome c-induced activation of caspase-3. To learn how crocin exhibits these anti-apoptotic actions in PC-12 cells, we tested the effect of crocin on PC-12 cell death induced by daunorubicin. We found that crocin inhibited the effect of daunorubicin as well. Our findings suggest that crocin inhibits neuronal cell death induced by both internal and external apoptotic stimuli.

  2. Efficient derivation of functional dopaminergic neurons from human embryonic stem cells on a large scale.

    Science.gov (United States)

    Cho, Myung-Soo; Hwang, Dong-Youn; Kim, Dong-Wook

    2008-01-01

    Cell-replacement therapy using human embryonic stem cells (hESCs) holds great promise in treating Parkinson's disease. We have recently reported a highly efficient method to generate functional dopaminergic (DA) neurons from hESCs. Our method includes a unique step, the formation of spherical neural masses (SNMs), and offers the highest yield of DA neurons ever achieved so far. In this report, we describe our method step by step, covering not only how to differentiate hESCs into DA neurons at a high yield, but also how to amplify, freeze and thaw the SNMs, which are the key structures that make our protocol unique and advantageous. Although the whole process of generation of DA neurons from hESCs takes about 2 months, only 14 d are needed to derive DA neurons from the SNMs. PMID:19008875

  3. Role of non-neuronal cells in body weight and appetite control

    Directory of Open Access Journals (Sweden)

    Pilar eArgente-Arizón

    2015-03-01

    Full Text Available The brain is composed of neurons and non-neuronal cells, with the latter encompassing glial, ependymal and endothelial cells, as well as pericytes and progenitor cells. Studies aimed at understanding how the brain operates have traditionally focused on neurons, but the importance of non-neuronal cells has become increasingly evident. Once relegated to supporting roles, it is now indubitable that these diverse cell types are fundamental for brain development and function, including that of metabolic circuits and they may play a significant role in obesity onset and complications. They participate in processes of neurogenesis, synaptogenesis and synaptic plasticity of metabolic circuits both during development and in adulthood. Some glial cells, such as tanycytes and astrocytes, transport circulating nutrients and metabolic factors that are fundamental for neuronal viability and activity into and within the hypothalamus. All of these cell types express receptors for a variety of metabolic factors and hormones, suggesting that they participate in metabolic function. They are the first line of defense against any assault to neurons. Indeed, microglia and astrocytes participate in the hypothalamic inflammatory response to high fat diet (HFD-induced obesity, with this process contributing to inflammatory related insulin and leptin resistance. Moreover, HFD-induced obesity and hyperleptinemia modify hypothalamic astroglial morphology, which is associated with changes in the synaptic inputs to neuronal metabolic circuits. Astrocytic contact with the microvasculature is increased by HFD intake and this could modify nutrient/hormonal uptake into the brain. In addition, progenitor cells in the hypothalamus are now known to have the capacity to renew metabolic circuits and this can be affected by HFD intake and obesity. Here we discuss our current understanding of how non-neuronal cells participate in physiological and physiopathological metabolic control.

  4. Differentiation of Human Breast-Milk Stem Cells to Neural Stem Cells and Neurons

    Directory of Open Access Journals (Sweden)

    Seyed Mojtaba Hosseini

    2014-01-01

    Full Text Available Objectives. Human breast milk contains a heterogeneous population of cells that have the potential to provide a noninvasive source of cells for cell therapy in many neurodegenerative diseases without any ethical concern. The objectives of this study were to differentiate the breast milk-derived stem cells (BMDSC toward neural stem cells and then into the neurons and neuroglia. Materials and Methods. To do this, the BMDSC were isolated from human breast milk and cultured in Dulbecco’s modified Eagle medium/F12 (DMEM/F12 containing fibroblast growth factor (bFGF. The cells were then characterized by evaluation of the embryonic and stem cell markers. Then, the cells were exposed to culture medium containing 1% B27 and 2% N2 for 7–10 days followed by medium supplemented with B27, N2, bFGF 10 µg/mL, and endothelial growth factor (EGF 20 µg/mL. Then, the sphere-forming assay was performed. The spheres were then differentiated into three neural lineages by withdrawing growth factor in the presence of 5% FBS (fetal bovine serum. The immunofluorescence was done for β-tubulin III, O4, and GFAP (glial fibrillary acidic protein. Results. The results indicated that the cells expressed both embryonic and mesenchymal stem cell (MSC markers. They also showed neurospheres formation that was nestin-positive. The cells were also differentiated into all three neural lineages. Conclusion. The BMDSC can behave in the same way with neural stem cells. They were differentiated into oligodendrocytes, and astrocytes as well as neurons.

  5. Omega-3 Polyunsaturated Fatty Acids Enhance Neuronal Differentiation in Cultured Rat Neural Stem Cells

    Directory of Open Access Journals (Sweden)

    Masanori Katakura

    2013-01-01

    Full Text Available Polyunsaturated fatty acids (PUFAs can induce neurogenesis and recovery from brain diseases. However, the exact mechanisms of the beneficial effects of PUFAs have not been conclusively described. We recently reported that docosahexaenoic acid (DHA induced neuronal differentiation by decreasing Hes1 expression and increasing p27kip1 expression, which causes cell cycle arrest in neural stem cells (NSCs. In the present study, we examined the effect of eicosapentaenoic acid (EPA and arachidonic acid (AA on differentiation, expression of basic helix-loop-helix transcription factors (Hes1, Hes6, and NeuroD, and the cell cycle of cultured NSCs. EPA also increased mRNA levels of Hes1, an inhibitor of neuronal differentiation, Hes6, an inhibitor of Hes1, NeuroD, and Map2 mRNA and Tuj-1-positive cells (a neuronal marker, indicating that EPA induced neuronal differentiation. EPA increased the mRNA levels of p21cip1 and p27kip1, a cyclin-dependent kinase inhibitor, which indicated that EPA induced cell cycle arrest. Treatment with AA decreased Hes1 mRNA but did not affect NeuroD and Map2 mRNA levels. Furthermore, AA did not affect the number of Tuj-1-positive cells or cell cycle progression. These results indicated that EPA could be involved in neuronal differentiation by mechanisms alternative to those of DHA, whereas AA did not affect neuronal differentiation in NSCs.

  6. Generation of Induced Neuronal Cells by the Single Reprogramming Factor ASCL1

    Directory of Open Access Journals (Sweden)

    Soham Chanda

    2014-08-01

    Full Text Available Direct conversion of nonneural cells to functional neurons holds great promise for neurological disease modeling and regenerative medicine. We previously reported rapid reprogramming of mouse embryonic fibroblasts (MEFs into mature induced neuronal (iN cells by forced expression of three transcription factors: ASCL1, MYT1L, and BRN2. Here, we show that ASCL1 alone is sufficient to generate functional iN cells from mouse and human fibroblasts and embryonic stem cells, indicating that ASCL1 is the key driver of iN cell reprogramming in different cell contexts and that the role of MYT1L and BRN2 is primarily to enhance the neuronal maturation process. ASCL1-induced single-factor neurons (1F-iN expressed mature neuronal markers, exhibited typical passive and active intrinsic membrane properties, and formed functional pre- and postsynaptic structures. Surprisingly, ASCL1-induced iN cells were predominantly excitatory, demonstrating that ASCL1 is permissive but alone not deterministic for the inhibitory neuronal lineage.

  7. Tracking neuronal marker expression inside living differentiating cells using molecular beacons

    DEFF Research Database (Denmark)

    Ilieva, Mirolyuba; Della Vedova, Paolo; Hansen, Ole;

    2013-01-01

    Monitoring gene expression is an important tool for elucidating mechanisms of cellular function. In order to monitor gene expression during nerve cell development, molecular beacon (MB) probes targeting markers representing different stages of neuronal differentiation were designed and synthesized...

  8. Structured PDMS Chambers for Enhanced Human Neuronal Cell Activity on MEA Platforms

    Institute of Scientific and Technical Information of China (English)

    Joose Kreutzer; Laura Yl(a)-Outinen; Paula K(a)irn(a); Tiina Kaarela; Jarno Mikkonen; Heli Skottman; Susanna Narkilahti; Pasi Kallio

    2012-01-01

    Structured poly(dimethylsiloxane) (PDMS) chambers were designed and fabricated to enhance the signaling of human Embryonic Stem Cell (hESC) - derived neuronal networks on Microelectrode Array (MEA) platforms.The structured PDMS chambers enable cell seeding on restricted areas and thus,reduce the amount of needed coating materials and cells.In addition,the neuronal cells formed spontaneously active networks faster in the structured PDMS chambers than that in control chainbers.In the PDMS chambers,the neuronal networks were more active and able to develop their signaling into organized signal trains faster than control cultures.The PDMS chamber design enables much more repeatable analysis and rapid growth of functional neuronal network in vitro.Moreover,due to its easy and cheap fabrication process,new configurations can be easily fabricated based on investigator requirements.

  9. Protein hairy enhancer of split-1 expression during differentiation of muscle-derived stem cells into neuron-like cells

    Institute of Scientific and Technical Information of China (English)

    Mina Huang; Zhanpeng Guo; Kun Liu; Xifan Mei; Shiqiang Fang; Jinhao Zeng; Yansong Wang; Yajiang Yuan

    2012-01-01

    Muscle-derived stem cells were isolated from the skeletal muscle of Sprague-Dawley neonatal rats aged 3 days old.Cells at passage 5 were incubated in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum,20 μg/L nerve growth factor,20 μg/L basic fibroblast growth factor and 1% (v/v) penicillin for 6 days.Cells presented with long processes, similar to nerve cells.Connections were formed between cell processes.Immunocytochemical staining with neuron specific enolase verified that cells differentiated into neuron-like cells. Immunofluorescence cytochemistry and western blot results revealed that the expression of protein hairy enhancer of split-1 was significantly reduced.These results indicate that low expression of protein hairy enhancer of split-1 participates in the differentiation of muscle-derived stem cells into neuron-like cells.

  10. Two forms of cerebellar glial cells interact differently with neurons in vitro

    OpenAIRE

    1984-01-01

    Specific interactions between neurons and glia dissociated from early postnatal mouse cerebellar tissue were studied in vitro by indirect immunocytochemical staining with antisera raised against purified glial filament protein, galactocerebroside, and the NILE glycoprotein. Two forms of cells were stained with antisera raised against purified glial filament protein. The first, characterized by a cell body 9 microns diam and processes 130-150 microns long, usually had two to three neurons asso...

  11. Differentiation-specific effects of LHON mutations introduced into neuronal NT2 cells.

    Science.gov (United States)

    Wong, Alice; Cavelier, Lucia; Collins-Schramm, Heather E; Seldin, Michael F; McGrogan, Michael; Savontaus, Marja-Liisa; Cortopassi, Gino A

    2002-02-15

    Inheritance of one of three primary mutations at positions 11778, 3460 or 14484 of the mitochondrial genome in subunits of Complex I causes Leber's Hereditary Optic Neuropathy (LHON), a specific degeneration of the optic nerve, resulting in bilateral blindness. It has been unclear why inheritance of a systemic mitochondrial mutation would result in a specific neurodegeneration. To address the neuron-specific degenerative phenotype of the LHON genotype, we have created cybrids using a neuronal precursor cell line, Ntera 2/D1 (NT2), containing mitochondria from patient lymphoblasts bearing the most common LHON mutation (11778) and the most severe LHON mutation (3460). The undifferentiated LHON-NT2 mutant cells were not significantly different from the parental cell control in terms of mtDNA/nDNA ratio, mitochondrial membrane potential, reactive oxygen species (ROS) production or the ability to reduce Alamar Blue. Differentiation of NT2s resulted in a neuronal morphology and neuron-specific pattern of gene expression, and a 3-fold reduction in mtDNA/nDNA ratio in both mutant and control cells; however, the differentiation protocol yielded significantly less LHON cells than controls, by 30%, indicating either a decreased proliferative potential or increased cell death of the LHON-NT2 cells. Differentiation of the cells to the neuronal form also resulted in significant increases in ROS production in the LHON-NT2 neurons versus controls, which is abolished by rotenone, a specific inhibitor of Complex I. We infer that the LHON genotype requires a differentiated neuronal environment in order to induce increased mitochondrial ROS, which may be the cause of the reduced NT2 yield; and suggest that the LHON degenerative phenotype may be the result of an increase in mitochondrial superoxide which is caused by the LHON mutations, possibly mediated through neuron-specific alterations in Complex I structure.

  12. Relationships between neuronal cell adhesion molecule and LHRH neurons in the urodele brain: a developmental immunohistochemical study

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    S Gianola

    2009-12-01

    Full Text Available Polysialic acid (PSA, a homopolymer attached to neural cell adhesion molecule (NCAM is considered a major hallmark of vertebrate cell migration. We studied the distribution of PSA-NCAM by immunohistochemistry, during brain development, in two urodele amphibians, Pleurodeles waltl and the neotenic newt Ambystoma mexicanum. In both species a gradual increase of immunolabelling was observed throughout the brain from developmental stage 30 to stage 52. At the onset of metamorphosis, some differences became evident: in Pleurodeles immunostaining was gradually restricted to the olfactory system while in Ambystoma, PSA-NCAM maintained a more extended distribution (for example throughout the telencephalic walls suggesting, for the brain of this latter species, a rather preserved neuronal plasticity. The aim of the present work was to correlate the above described PSA-NCAMimmunoreactivity (IR with the distribution of luteinizing hormone-releasing hormone (LH-RH containing neurons, which represent a well known example of neural elements migrating from the olfactory placode. LHRH-IR, undetectable till stage 30, was later found together with PSA-NCAM-IR in both the olfactory system and septo-hypothalamic areas. Such observations further support a role of PSA in providing a migration route toward the establishment of a part, at least, of the urodele LHRH system. The possible functional meaning of the LHRH-containing neurons localized between dorsal and ventral thalamus of Ambystoma, never reported before in this area, almost devoid of PSANCAM- IR, is discussed.

  13. Cholinergic properties of neurons differentiated from an embryonal carcinoma cell-line (P19).

    Science.gov (United States)

    Parnas, D; Linial, M

    1995-11-01

    P19 is a mouse-derived embryonal carcinoma cell-line capable of differentiation toward ectodermal, mesodermal and endodermal lineages. Following treatment with retinoic acid these cells differentiate into neurons, astrocytes and fibroblast-like cells. We induced P19 differentiation under conditions which lead to a homogeneous neuronal culture (> 95% neurons). Under these conditions, most cells (approximately 85%) express high levels of the cholinergic markers acetyl cholinesterase and choline acetyltransferase while approximately 10% of cells express the GABAergic marker glutamic acid decarboxylase. While the proportion of the GABAergic neurons is constant at different culture conditions, the cholinergic phenotype is suppressed at high cell densities. The cholinergic nature of P19 neurons is also evident in their ability to form contacts with a muscle cell-line--C2. At day 10 of differentiation cells are capable of depolarization-dependent acetylcholine release. The release is Ca2+ dependent, and drops to baseline levels at 0.5 mM Ca2+. The cells also respond to sub-nM levels of alpha-latrotoxin by acetylcholine release. All major proteins implicated in synapse functionality are expressed prior to day 10 at both at RNA and protein levels. However, the expression pattern of each gene is unique. The genes include cytoskeletal proteins, synaptic vesicle proteins and terminal specific proteins. We suggest that this cell-line can serve as an in-vitro model system for the study of neuronal phenotype acquisition. Under our conditions, the P19 cells can also provide a system in which to study the differentiation of functional cholinergic neurons. PMID:8787867

  14. Cooperative transcription activation by Nurr1 and Pitx3 induces embryonic stem cell maturation to the midbrain dopamine neuron phenotype

    DEFF Research Database (Denmark)

    Martinat, Cecile; Bacci, Jean-Jacques; Leete, Thomas;

    2006-01-01

    Midbrain dopamine (DA) neurons play a central role in the regulation of voluntary movement, and their degeneration is associated with Parkinson's disease. Cell replacement therapies, and in particular embryonic stem (ES) cell-derived DA neurons, offer a potential therapeutic venue for Parkinson...... to the midbrain DA neuron phenotype in murine and human ES cell cultures.......'s disease. We sought to identify genes that can potentiate maturation of ES cell cultures to the midbrain DA neuron phenotype. A number of transcription factors have been implicated in the development of midbrain DA neurons by expression analyses and loss-of-function knockout mouse studies, including Nurr1...

  15. Inhibition of HIF-prolyl-4-hydroxylases prevents mitochondrial impairment and cell death in a model of neuronal oxytosis

    NARCIS (Netherlands)

    Neitemeier, S; Dolga, A M; Honrath, B; Karuppagounder, S S; Alim, I; Ratan, R R; Culmsee, C

    2016-01-01

    Mitochondrial impairment induced by oxidative stress is a main characteristic of intrinsic cell death pathways in neurons underlying the pathology of neurodegenerative diseases. Therefore, protection of mitochondrial integrity and function is emerging as a promising strategy to prevent neuronal dama

  16. Ex vivo differentiation of human bone marrow-derived stem cells into neuronal cell-like lineages

    Directory of Open Access Journals (Sweden)

    Al-Zoubi A

    2016-06-01

    Full Text Available Adeeb Al-Zoubi,1,2 Feras Altwal,3 Farah Khalifeh,2 Jamil Hermas,4 Ziad Al-Zoubi,5 Emad Jafar,5 Mohammed El-Khateeb,6,7 1Department of Surgery, University of Illinois College of Medicine at Peoria, Peoria, IL, USA; 2Stem Cells of Arabia, Amman, Jordan; 3Department of Neuroscience, School of Graduate and Postdoctoral Studies, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA; 4Stem Cell Division, Al-Yamama Company, 5Jordan Orthopedic and Spinal Center, 6National Center for Diabetes, Endocrinology and Genetics, 7Department of Pathology, Faculty of Medicine, University of Jordan, Amman, Jordan Background: Methods to obtain safe and practical populations of stem cells (SCs at a clinical grade that are able to differentiate into neuronal cell lineages are yet to be developed. In a previous study, we showed that mouse bone marrow-derived SCs (BM-SCs differentiated into neuronal cell-like lineages when put in a neuronal-like environment, which is a special media supplemented with the necessary growth factors needed for the differentiation of SCs into neuronal cell-like lineages. Aim: In this study, we aim to assess the potentials of adult human CD34+ and CD133+ SCs to differentiate into neuronal cell-like lineages ex vivo when placed in a neuronal-like microenvironment. Methods: The neuronal-like microenvironment was created by culturing cells in nonhematopoietic expansion media (NHEM supplemented with growth factors that favor differentiation into neuronal cell lineages (low-affinity nerve growth factor [LNGF], mouse spinal cord extract [mSpE], or both. Cultured cells were assessed for neuronal differentiation by cell morphologies and by expression of GFAP. Results: Our results show that culturing unpurified human BM-derived mononuclear cells (hBM-MNCs in NHEM+LNGF+mSpE did not lead to neuronal differentiation. In contrast, culturing of purified CD34+ hBM-SCs in NHEM+LNGF+mSpE favored their differentiation into astrocyte

  17. C3G regulates cortical neuron migration, preplate splitting and radial glial cell attachment.

    Science.gov (United States)

    Voss, Anne K; Britto, Joanne M; Dixon, Mathew P; Sheikh, Bilal N; Collin, Caitlin; Tan, Seong-Seng; Thomas, Tim

    2008-06-01

    Neuronal migration is integral to the development of the cerebral cortex and higher brain function. Cortical neuron migration defects lead to mental disorders such as lissencephaly and epilepsy. Interaction of neurons with their extracellular environment regulates cortical neuron migration through cell surface receptors. However, it is unclear how the signals from extracellular matrix proteins are transduced intracellularly. We report here that mouse embryos lacking the Ras family guanine nucleotide exchange factor, C3G (Rapgef1, Grf2), exhibit a cortical neuron migration defect resulting in a failure to split the preplate into marginal zone and subplate and a failure to form a cortical plate. C3G-deficient cortical neurons fail to migrate. Instead, they arrest in a multipolar state and accumulate below the preplate. The basement membrane is disrupted and radial glial processes are disorganised and lack attachment in C3G-deficient brains. C3G is activated in response to reelin in cortical neurons, which, in turn, leads to activation of the small GTPase Rap1. In C3G-deficient cells, Rap1 GTP loading in response to reelin stimulation is reduced. In conclusion, the Ras family regulator C3G is essential for two aspects of cortex development, namely radial glial attachment and neuronal migration.

  18. Activity deprivation induces neuronal cell death: mediation by tissue-type plasminogen activator.

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    Eldi Schonfeld-Dado

    Full Text Available Spontaneous activity is an essential attribute of neuronal networks and plays a critical role in their development and maintenance. Upon blockade of activity with tetrodotoxin (TTX, neurons degenerate slowly and die in a manner resembling neurodegenerative diseases-induced neuronal cell death. The molecular cascade leading to this type of slow cell death is not entirely clear. Primary post-natal cortical neurons were exposed to TTX for up to two weeks, followed by molecular, biochemical and immunefluorescence analysis. The expression of the neuronal marker, neuron specific enolase (NSE, was down-regulated, as expected, but surprisingly, there was a concomitant and striking elevation in expression of tissue-type plasminogen activator (tPA. Immunofluorescence analysis indicated that tPA was highly elevated inside affected neurons. Transfection of an endogenous tPA inhibitor, plasminogen activator inhibitor-1 (PAI-1, protected the TTX-exposed neurons from dying. These results indicate that tPA is a pivotal player in slowly progressing activity deprivation-induced neurodegeneration.

  19. Extensive neuronal differentiation of human neural stem cell grafts in adult rat spinal cord.

    Directory of Open Access Journals (Sweden)

    Jun Yan

    2007-02-01

    Full Text Available BACKGROUND: Effective treatments for degenerative and traumatic diseases of the nervous system are not currently available. The support or replacement of injured neurons with neural grafts, already an established approach in experimental therapeutics, has been recently invigorated with the addition of neural and embryonic stem-derived precursors as inexhaustible, self-propagating alternatives to fetal tissues. The adult spinal cord, i.e., the site of common devastating injuries and motor neuron disease, has been an especially challenging target for stem cell therapies. In most cases, neural stem cell (NSC transplants have shown either poor differentiation or a preferential choice of glial lineages. METHODS AND FINDINGS: In the present investigation, we grafted NSCs from human fetal spinal cord grown in monolayer into the lumbar cord of normal or injured adult nude rats and observed large-scale differentiation of these cells into neurons that formed axons and synapses and established extensive contacts with host motor neurons. Spinal cord microenvironment appeared to influence fate choice, with centrally located cells taking on a predominant neuronal path, and cells located under the pia membrane persisting as NSCs or presenting with astrocytic phenotypes. Slightly fewer than one-tenth of grafted neurons differentiated into oligodendrocytes. The presence of lesions increased the frequency of astrocytic phenotypes in the white matter. CONCLUSIONS: NSC grafts can show substantial neuronal differentiation in the normal and injured adult spinal cord with good potential of integration into host neural circuits. In view of recent similar findings from other laboratories, the extent of neuronal differentiation observed here disputes the notion of a spinal cord that is constitutively unfavorable to neuronal repair. Restoration of spinal cord circuitry in traumatic and degenerative diseases may be more realistic than previously thought, although major

  20. Differentiation of neurons from neural precursors generated in floating spheres from embryonic stem cells

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    Forrester Jeff

    2009-09-01

    Full Text Available Abstract Background Neural differentiation of embryonic stem (ES cells is usually achieved by induction of ectoderm in embryoid bodies followed by the enrichment of neuronal progenitors using a variety of factors. Obtaining reproducible percentages of neural cells is difficult and the methods are time consuming. Results Neural progenitors were produced from murine ES cells by a combination of nonadherent conditions and serum starvation. Conversion to neural progenitors was accompanied by downregulation of Oct4 and NANOG and increased expression of nestin. ES cells containing a GFP gene under the control of the Sox1 regulatory regions became fluorescent upon differentiation to neural progenitors, and ES cells with a tau-GFP fusion protein became fluorescent upon further differentiation to neurons. Neurons produced from these cells upregulated mature neuronal markers, or differentiated to glial and oligodendrocyte fates. The neurons gave rise to action potentials that could be recorded after application of fixed currents. Conclusion Neural progenitors were produced from murine ES cells by a novel method that induced neuroectoderm cells by a combination of nonadherent conditions and serum starvation, in contrast to the embryoid body method in which neuroectoderm cells must be selected after formation of all three germ layers.

  1. Transfection of the glial cell line-derived neurotrophic factor gene promotes neuronal differentiation

    Institute of Scientific and Technical Information of China (English)

    Jie Du; Xiaoqing Gao; Li Deng; Nengbin Chang; Huailin Xiong; Yu Zheng

    2014-01-01

    Glial cell line-derived neurotrophic factor recombinant adenovirus vector-transfected bone marrow mesenchymal stem cells were induced to differentiate into neuron-like cells using inductive medium containing retinoic acid and epidermal growth factor. Cell viability, micro-tubule-associated protein 2-positive cell ratio, and the expression levels of glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43 protein in the su-pernatant were signiifcantly higher in glial cell line-derived neurotrophic factor/bone marrow mesenchymal stem cells compared with empty virus plasmid-transfected bone marrow mes-enchymal stem cells. Furthermore, microtubule-associated protein 2, glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43 mRNA levels in cell pellets were statistically higher in glial cell line-derived neurotrophic factor/bone marrow mesen-chymal stem cells compared with empty virus plasmid-transfected bone marrow mesenchymal stem cells. These results suggest that glial cell line-derived neurotrophic factor/bone marrow mesenchymal stem cells have a higher rate of induction into neuron-like cells, and this enhanced differentiation into neuron-like cells may be associated with up-regulated expression of glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43.

  2. Selective neuronal differentiation of neural stem cells induced by nanosecond microplasma agitation

    Directory of Open Access Journals (Sweden)

    Z. Xiong

    2014-03-01

    Full Text Available An essential step for therapeutic and research applications of stem cells is their ability to differentiate into specific cell types. Neuronal cells are of great interest for medical treatment of neurodegenerative diseases and traumatic injuries of central nervous system (CNS, but efforts to produce these cells have been met with only modest success. In an attempt of finding new approaches, atmospheric-pressure room-temperature microplasma jets (MPJs are shown to effectively direct in vitro differentiation of neural stem cells (NSCs predominantly into neuronal lineage. Murine neural stem cells (C17.2-NSCs treated with MPJs exhibit rapid proliferation and differentiation with longer neurites and cell bodies eventually forming neuronal networks. MPJs regulate ~75% of NSCs to differentiate into neurons, which is a higher efficiency compared to common protein- and growth factors-based differentiation. NSCs exposure to quantized and transient (~150 ns micro-plasma bullets up-regulates expression of different cell lineage markers as β-Tubulin III (for neurons and O4 (for oligodendrocytes, while the expression of GFAP (for astrocytes remains unchanged, as evidenced by quantitative PCR, immunofluorescence microscopy and Western Blot assay. It is shown that the plasma-increased nitric oxide (NO production is a factor in the fate choice and differentiation of NSCs followed by axonal growth. The differentiated NSC cells matured and produced mostly cholinergic and motor neuronal progeny. It is also demonstrated that exposure of primary rat NSCs to the microplasma leads to quite similar differentiation effects. This suggests that the observed effect may potentially be generic and applicable to other types of neural progenitor cells. The application of this new in vitro strategy to selectively differentiate NSCs into neurons represents a step towards reproducible and efficient production of the desired NSC derivatives.

  3. Nicotinamide promotes neuronal differentiation of mouse embryonic stem cells in vitro.

    Science.gov (United States)

    Griffin, Síle M; Pickard, Mark R; Orme, Rowan P; Hawkins, Clive P; Fricker, Rosemary A

    2013-12-18

    Factors controlling proliferation and differentiation are crucial in advancement of neural cell-based experimental neurodegenerative therapies. In this regard, nicotinamide has been shown to determine the fate of neural cells, enhance neuralization, and influence DNA repair and apoptosis. This study investigated whether the biologically active vitamin B3 metabolite, nicotinamide, could direct the differentiation of mouse embryonic stem cells, cultured as monolayers, into neurons at either early or late stages of development. Interestingly, we observed a dose-responsive increase in the percentage of neurons when nicotinamide was added at early stages to the cells undergoing differentiation (days 0-7). Nicotinamide (10 mM) had a significant effect on neuronal differentiation, increasing the βIII-tubulin-positive neuronal population and concomitantly decreasing the total number of cells in culture, measured by quantification of 4',6-diamidino-2-phenylindole (DAPI)-positive cells. Nicotinamide added between days 7 and 14 had no effect on neuronal induction. High levels of nicotinamide (20 mM) induced cytotoxicity and cell death. Current work is focusing on elucidating the mechanism(s) mediating neural specification by nicotinamide--that is, induction of cell-cycle exit and/or selective apoptosis in non-neural populations. Preliminary data suggest a reduction in the proportion of proliferating cells in nicotinamide-treated cultures--that is, nicotinamide enhances cell-cycle exit, thereby promoting neuronal differentiation. Future work will focus on evaluating the effect of nicotinamide on the differentiation of midbrain dopamine neurons, towards a therapy for Parkinson's disease.

  4. Mechanism of neuronal versus endothelial cell uptake of Alzheimer's disease amyloid beta protein.

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    Karunya K Kandimalla

    Full Text Available Alzheimer's disease (AD is characterized by significant neurodegeneration in the cortex and hippocampus; intraneuronal tangles of hyperphosphorylated tau protein; and accumulation of beta-amyloid (Abeta proteins 40 and 42 in the brain parenchyma as well as in the cerebral vasculature. The current understanding that AD is initiated by the neuronal accumulation of Abeta proteins due to their inefficient clearance at the blood-brain-barrier (BBB, places the neurovascular unit at the epicenter of AD pathophysiology. The objective of this study is to investigate cellular mechanisms mediating the internalization of Abeta proteins in the principle constituents of the neurovascular unit, neurons and BBB endothelial cells. Laser confocal micrographs of wild type (WT mouse brain slices treated with fluorescein labeled Abeta40 (F-Abeta40 demonstrated selective accumulation of the protein in a subpopulation of cortical and hippocampal neurons via nonsaturable, energy independent, and nonendocytotic pathways. This groundbreaking finding, which challenges the conventional belief that Abeta proteins are internalized by neurons via receptor mediated endocytosis, was verified in differentiated PC12 cells and rat primary hippocampal (RPH neurons through laser confocal microscopy and flow cytometry studies. Microscopy studies have demonstrated that a significant proportion of F-Abeta40 or F-Abeta42 internalized by differentiated PC12 cells or RPH neurons is located outside of the endosomal or lysosomal compartments, which may accumulate without degradation. In contrast, BBME cells exhibit energy dependent uptake of F-Abeta40, and accumulate the protein in acidic cell organelle, indicative of endocytotic uptake. Such a phenomenal difference in the internalization of Abeta40 between neurons and BBB endothelial cells may provide essential clues to understanding how various cells can differentially regulate Abeta proteins and help explain the vulnerability of cortical

  5. Transfection of the glial cell line-derived neurotrophic factor gene promotes neuronal differentiation

    OpenAIRE

    Du, Jie; Gao, Xiaoqing; Deng, Li; Chang, Nengbin; Xiong, Huailin; Zheng, Yu

    2014-01-01

    Glial cell line-derived neurotrophic factor recombinant adenovirus vector-transfected bone marrow mesenchymal stem cells were induced to differentiate into neuron-like cells using inductive medium containing retinoic acid and epidermal growth factor. Cell viability, microtubule-associated protein 2-positive cell ratio, and the expression levels of glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43 protein in the supernatant were significantly hig...

  6. Effect of leukemia inhibitory factor on embryonic stem cell differentiation: implications for supporting neuronal differentiation

    Institute of Scientific and Technical Information of China (English)

    Zhao HE; Jing-jing LI; Chang-hong ZHEN; Lin-ying FENG; Xiao-yan DING

    2006-01-01

    Aim: Leukemia inhibitory factor (LIF), a pleiotropic cytokine, has been used extensively in the maintenance of mouse embryonic stem cell pluripotency. In this current work, we examined the effect of the LIF signaling pathway in embryonic stem (ES) cell differentiation to a neural fate. Methods: In the presence of LIF (1000 U/mL), the production of neuronal cells derived from embryoid bodies (EB)was tested under various culture conditions. Inhibition of the LIF pathway was examined with specific inhibitors. The effects of cell apoptosis and proliferation on neural differentiation were examined. ES cell differentiation into three-gem layers was compared. Results: Under various culture conditions, neuronal differentiation was increased in the presence of LIF. Blocking the LIF-activated STAT3signaling pathway with specific inhibitors abolished the neuronal differentiation of ES cells, whereas inhibition of the LIF-activated MEK signaling pathway impaired the differentiation of ES cells toward a glial fate. LIF suppressed cell apoptosis and promoted cell proliferation during ES cell differentiation. LIF inhibited the differentiation of ES cells to both mesoderm and extraembryonic endoderm fates, but enhanced the determination of neural progenitors. Conclusion:These results suggest that LIF plays a positive role during the differentiation of ES cells into neuronal cells.

  7. Grouping and Classifying Electrophysiologically-Defined Classes of Neocortical Neurons by Single Cell, Whole-Genome Expression Profiling

    OpenAIRE

    Subkhankulova, Tatiana; Yano, Kojiro; Robinson, Hugh P. C.; Livesey, Frederick J

    2010-01-01

    The diversity of neuronal cell types and how to classify them are perennial questions in neuroscience. The advent of global gene expression analysis raised the possibility that comprehensive transcription profiling will resolve neuronal cell types into groups that reflect some or all aspects of their phenotype. This approach has been successfully used to compare gene expression between groups of neurons defined by a common property. Here we extend this approach to ask whether single neuron ge...

  8. Grouping and classifying electrophysiologically-defined classes of neocortical neurons by single cell, whole-genome expression profiling

    OpenAIRE

    Tatiana Subkhankulova; Kojiro Yano; Hugh Robinson; Livesey, Frederick J

    2010-01-01

    The diversity of neuronal cell types and how to classify them are perennial questions in neuroscience. The advent of global gene expression analysis raised the possibility that comprehensive transcription profiling will resolve neuronal cell types into groups that reflect some or all aspects of their phenotype. This approach has been successfully used to compare gene expression between groups of neurons defined by a common property. Here we extend this approach to ask whether single neuron ge...

  9. Apoptosis during β-mercaptoethanol-induced differentiation of adult adipose-derived stromal cells into neurons

    Institute of Scientific and Technical Information of China (English)

    Yanan Cai; Xiaodong Yuan; Ya Ou; Yanhui Lu

    2011-01-01

    β-mercaptoethanol can induce adipose-derived stromal cells to rapidly and efficiently differentiate into neurons in vitro. However, because of the short survival time of the differentiated cells, clinical applications for this technique are limited. As such, we examined apoptosis of neurons differentiated from adipose-derived stromal cells induced with β-mercaptoethanol in vitro using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and transmission electron microscopy. The results revealed that the number of surviving cells decreased and apoptosis rate increased as induction time extended. Taken together, these results suggest that apoptosis occurring in the process of adipose-derived stromal cells differentiating into neurons is the main cause of cell death. However, the mechanism underlying cellular apoptosis should be researched further to develop methods of controlling apoptosis for clinical applications.

  10. Neurons Differentiated from Transplanted Stem Cells Respond Functionally to Acoustic Stimuli in the Awake Monkey Brain.

    Science.gov (United States)

    Wei, Jing-Kuan; Wang, Wen-Chao; Zhai, Rong-Wei; Zhang, Yu-Hua; Yang, Shang-Chuan; Rizak, Joshua; Li, Ling; Xu, Li-Qi; Liu, Li; Pan, Ming-Ke; Hu, Ying-Zhou; Ghanemi, Abdelaziz; Wu, Jing; Yang, Li-Chuan; Li, Hao; Lv, Long-Bao; Li, Jia-Li; Yao, Yong-Gang; Xu, Lin; Feng, Xiao-Li; Yin, Yong; Qin, Dong-Dong; Hu, Xin-Tian; Wang, Zheng-Bo

    2016-07-26

    Here, we examine whether neurons differentiated from transplanted stem cells can integrate into the host neural network and function in awake animals, a goal of transplanted stem cell therapy in the brain. We have developed a technique in which a small "hole" is created in the inferior colliculus (IC) of rhesus monkeys, then stem cells are transplanted in situ to allow for investigation of their integration into the auditory neural network. We found that some transplanted cells differentiated into mature neurons and formed synaptic input/output connections with the host neurons. In addition, c-Fos expression increased significantly in the cells after acoustic stimulation, and multichannel recordings indicated IC specific tuning activities in response to auditory stimulation. These results suggest that the transplanted cells have the potential to functionally integrate into the host neural network.

  11. miR-124 promotes the neuronal differentiation of mouse inner ear neural stem cells

    Science.gov (United States)

    Jiang, Di; Du, Jintao; Zhang, Xuemei; Zhou, Wei; Zong, Lin; Dong, Chang; Chen, Kaitian; Chen, Yu; Chen, Xihui; Jiang, Hongyan

    2016-01-01

    MicroRNAs (miRNAs or miRs) act as key regulators in neuronal development, synaptic morphogenesis and plasticity. However, their role in the neuronal differentiation of inner ear neural stem cells (NSCs) remains unclear. In this study, 6 miRNAs were selected and their expression patterns during the neuronal differentiation of inner ear NSCs were examined by RT-qPCR. We demonstrated that the culture of spiral ganglion stem cells present in the inner ears of newborn mice gave rise to neurons in vitro. The expression patterns of miR-124, miR-132, miR-134, miR-20a, miR-17-5p and miR-30a-5p were examined during a 14-day neuronal differentiation period. We found that miR-124 promoted the neuronal differentiation of and neurite outgrowth in mouse inner ear NSCs, and that the changes in the expression of tropomyosin receptor kinase B (TrkB) and cell division control protein 42 homolog (Cdc42) during inner ear NSC differentiation were associated with miR-124 expression. Our findings indicate that miR-124 plays a role in the neuronal differentiation of inner ear NSCs. This finding may lead to the development of novel strategies for restoring hearing in neurodegenerative diseases.

  12. Mature neurons modulate neurogenesis through chemical signals acting on neural stem cells.

    Science.gov (United States)

    Pardal, Ricardo; López Barneo, José

    2016-06-01

    The discovery of neural stem cells has revealed a much higher structural and functional plasticity in the adult nervous system than previously anticipated. Progenitor cells are able to give rise to new neurons and glial cells when needed, thanks to their surveillance of the environment from the germinal niches. Multiple different factors define neural stem cell niches, including cellular and non-cellular components. Innervation of neurogenic centers is crucial, as it allows the functional connection between stem cell behavior and surrounding neuronal activity. Although the association between organismal behavior and neurogenesis is well documented, much less is known about the cellular and molecular mechanisms by which neurons control stem cell activity. In this review we discuss the existing data on this type of regulation from the three best characterized germinal niches in the adult nervous system: the subventricular zone, the hippocampal subgranular zone, and the carotid body. In all cases, neuronal activity modulates stem cell behavior either by neurotransmitter spillover or by synaptic-like contacts. Currently, the molecular mechanisms underlying mature neuron-stem cell interaction are being clarified. Functional consequences and potential clinical relevance of these phenomena are also discussed. PMID:27101323

  13. Relationship between seizure frequency and number of neuronal and non-neuronal cells in the hippocampus throughout the life of rats with epilepsy.

    Science.gov (United States)

    Lopim, Glauber Menezes; Vannucci Campos, Diego; Gomes da Silva, Sérgio; de Almeida, Alexandre Aparecido; Lent, Roberto; Cavalheiro, Esper Abrão; Arida, Ricardo Mario

    2016-03-01

    The relationship between seizure frequency and cell death has been a subject of controversy. To tackle this issue, we determined the frequency of seizures and the total number of hippocampal cells throughout the life of rats with epilepsy using the pilocarpine model. Seizure frequency varied in animals with epilepsy according to which period of life they were in, with a progressive increase in the number of seizures until 180 days (sixth months) of epileptic life followed by a decrease (330 days-eleventh month) and subsequently stabilization of seizures. Cell counts by means of isotropic fractionation showed a reduction in the number of hippocampal neuronal cells following 30, 90, 180 and 360 days of spontaneous recurrent seizures (SRS) in rats compared to their controls (about 25%-30% of neuronal cell reduction). In addition, animals with 360 days of SRS showed a reduction in the number of neuronal cells when compared with animals with 90 and 180 days of seizures. The total number of hippocampal non-neuronal cells was reduced in rats with epilepsy after 30 days of SRS, but no significant alteration was observed on the 90th, 180th and 360th days. The total number of neuronal cells was negatively correlated with seizure frequency, indicating an association between occurrence of epileptic seizures throughout life and neuronal loss. In sum, our results add novel data to the literature concerning the time-course of SRS and hippocampal cell number throughout epileptic life.

  14. Effects of histone deacetylation inhibition on neuronal differentiation of embryonic mouse neural stem cells

    NARCIS (Netherlands)

    Balasubramaniyan, V.; Boddeke, E.; Bakels, R.; Kust, B.; Kooistra, S.; Veneman, A.; Copray, S.

    2006-01-01

    Neural stem cells (NSCs) are multipotent cells that have the capacity for self-renewal and for differentiation into the major cell types of the nervous system, i.e. neurons, astrocytes and oligodendrocytes. The molecular mechanisms regulating gene transcription resulting in NSC differentiation and c

  15. Ontogenesis of Gonadotropin-Releasing Hormone Neurons: A Model for Hypothalamic Neuroendocrine Cell Development

    Directory of Open Access Journals (Sweden)

    Erica L Stevenson

    2013-07-01

    Full Text Available The vertebrate hypothalamo-pituitary-gonadal (HPG axis is the anatomical framework responsible for reproductive competence and species propagation. Essential to the coordinated actions of this three-tiered biological system is the fact that the regulatory inputs ultimately converge on the gonadotropin-releasing hormone (GnRH neuronal system, which in rodents primarily resides in the preoptic/hypothalamic region. In this short review we will focus on: 1 the general embryonic temporal and spatial development of the rodent GnRH neuronal system, 2 the origin(s of GnRH neurons, and 3 which transcription - and growth factors have been found to be critical for GnRH neuronal ontogenesis and cellular fate-specification. Moreover, we ask the question whether the molecular and cellular mechanisms involved in GnRH neuronal development may also be play a role in the development of other hypophyseal secreting neuroendocrine cells in the hypothalamus.

  16. APAF1 is a key transcriptional target for p53 in the regulation of neuronal cell death

    DEFF Research Database (Denmark)

    Fortin, A; Cregan, S P; MacLaurin, J G;

    2001-01-01

    p53 is a transcriptional activator which has been implicated as a key regulator of neuronal cell death after acute injury. We have shown previously that p53-mediated neuronal cell death involves a Bax-dependent activation of caspase 3; however, the transcriptional targets involved in the regulation...... of this process have not been identified. In the present study, we demonstrate that p53 directly upregulates Apaf1 transcription as a critical step in the induction of neuronal cell death. Using DNA microarray analysis of total RNA isolated from neurons undergoing p53-induced apoptosis a 5-6-fold upregulation...... of Apaf1 mRNA was detected. Induction of neuronal cell death by camptothecin, a DNA-damaging agent that functions through a p53-dependent mechanism, resulted in increased Apaf1 mRNA in p53-positive, but not p53-deficient neurons. In both in vitro and in vivo neuronal cell death processes of p53-induced...

  17. Preliminary Study on in Vitro Induced Differentiation of Embryonic Stem Cells into Neurons

    Institute of Scientific and Technical Information of China (English)

    JianGe; ShunongLi; 等

    2002-01-01

    Purpose;To study preliminarily in vitro induced differentiation of embryonic stem cells into neurons for further investigation of an alternative for the treatment of glaumatous neuropathy.Materials and methods:Supernatant of cultured Buffalo rat liver cells (buffalorat liver cell-conditioned medium,BRL-CM)was used for culturing embryonic stem cells(ES-D3 cell line)..Morphological features of undifferentiated ES cells were studied by HE staining and electron microscopy.Based on the methods used by Bain et al,we modified the methods and used retinoic,acid(RA) as an inducer to differentiate ES-D3 cells and cytosine arabinoside(Ara-C) as inhibitor of proliferative cells.The growth of the cells was observed under phase contrast microscope.Reuslts:ES-D3 cells cultured by BRL-CM grew in aggregates and remained undifferentiated.Electromicroscopy showed large nucleus and a large amount of mitochondria in undifferentiated ES cells and many processes on the surfaces.In the first day after the adding of retinoic acid,some neuron-like cells with one,two or more processes were present.In the second day after adding RA and the first day after the plus of 10μm Ara-C,a large amount of neuron-like cells appeared,with the formation of neuron-like networks.Conclusions:Combined use of RA and Ara-C can induce ES cells to different into neuron-like cells.Our present preliminary study might provide insights into an alternative for the treatment of glaucomatous neuropathy by the transplantation of embryonic stem cells.Eye Science 2000;16:1-6.

  18. Neonatal Neural Progenitor Cells and Their Neuronal and Glial Cell Derivatives Are Fully Permissive for Human Cytomegalovirus Infection▿

    OpenAIRE

    Luo, Min Hua; Philip H. Schwartz; Fortunato, Elizabeth A.

    2008-01-01

    Congenital human cytomegalovirus (HCMV) infection causes central nervous system structural abnormalities and functional disorders, affecting both astroglia and neurons with a pathogenesis that is only marginally understood. To better understand HCMV's interactions with such clinically important cell types, we utilized neural progenitor cells (NPCs) derived from neonatal autopsy tissue, which can be differentiated down either glial or neuronal pathways. Studies were performed using two viral i...

  19. Polarized Trafficking of the Sorting Receptor SorLA in Neurons and MDCK Cells

    DEFF Research Database (Denmark)

    Klinger, Stine C; Højland, Anne; Jain, Shweta;

    2016-01-01

    The sorting receptor SorLA is highly expressed in neurons and is also found in other polarized cells. The receptor has been reported to participate in the trafficking of several ligands, some of which are linked to human diseases, including the amyloid precursor protein, TrkB and lipoprotein lipase...... (LpL). Despite this, only the trafficking in non-polarized cells has been described so far. Due to the many differences between polarized and non-polarized cells, we examined the localization and trafficking of SorLA in epithelial Madin-Darby canine kidney (MDCK) cells and rat hippocampal neurons. We...

  20. Suppression of KV7/KCNQ potassium channel enhances neuronal differentiation of PC12 cells.

    Science.gov (United States)

    Zhou, Najing; Huang, Sha; Li, Li; Huang, Dongyang; Yan, Yunli; Du, Xiaona; Zhang, Hailin

    2016-10-01

    Membrane potential shift driven by electrical activity is critical in determining the cell fate of proliferation or differentiation. As such, the ion channels that underlie the membrane electrical activity play an important role in cell proliferation/differentiation. KV7/KCNQ potassium channels are critical in determining the resting membrane potentials in many neuronal cells. However, the role of these channels in cell differentiation is not well studied. In the present study, we used PC12 cells as well as primary cultured rat cortical neurons to study the role and mechanism of KV7/KCNQ in neuronal differentiation. NGF induced PC12 cell differentiation into neuron-like cells with growth of neurites showing typical growth cone-like extensions. The Kv7/KCNQ blocker XE991 promoted NGF-induced neurite outgrowth, whereas Kv7/KCNQ opener retigabine (RTG) inhibited outgrowth. M-type Kv7 channels are likely involved in regulating neurite growth because overexpression of KCNQ2/Q3 inhibited neurite growth whereas suppression of KCNQ2/Q3 with shRNA promoted neurite growth. Membrane depolarization possibly underpins enhanced neurite growth induced by the suppression of Kv7/KCNQ. Additionally, high extracellular K(+) likely induced membrane depolarization and also promoted neurite growth. Finally, T-type Ca(2+) channels may be involved in membrane-depolarization-induced neurite growth. This study provides a new perspective for understanding neuronal differentiation as well as KV7/KCNQ channel function. PMID:27450567

  1. Neuroprotective effects of neuregulin-1 on B35 neuronal cells following ischemia.

    Science.gov (United States)

    Croslan, DaJoie R; Schoell, Matthew C; Ford, Gregory D; Pulliam, John V; Gates, Alicia; Clement, Ceilessia M; Harris, Adalynn E; Ford, Byron D

    2008-05-19

    We previously showed that neuregulin-1 (NRG-1) protected neurons from death in vivo following focal ischemia. The goal of this study was to develop an in vitro rat ischemia model to examine the cellular and molecular mechanisms involved in the neuroprotective effects of NRG-1 on ischemia-induced neuronal death. Rat B-35 neuroblastoma cells differentiated by serum withdrawal, developed enhanced neuronal characteristics including, neurite extension and upregulation of neuronal markers of differentiation. When B35 neurons were subjected to oxygen glucose deprivation (OGD)/reoxygenation or glutamate, widespread neuronal death was seen after both treatments. Treatment with NRG-1 immediately after OGD significantly increased neuronal survival. NRG-1 administration also resulted in a significant decrease in annexin V, an early marker of apoptosis. However, the neurotoxic actions of glutamate were unaffected by NRG-1. The neuroprotective effects of NRG-1 were prevented by an inhibitor of the phosphatidylinositol-3-kinase/Akt pathway. These results provide a new model to gain insight into the mechanisms employed by NRG-1 to protect neurons from ischemic brain injury. PMID:18410912

  2. Neuroprotective effects of Neuregulin-1 on B35 Neuronal Cells following Ischemia

    Science.gov (United States)

    Croslan, DaJoie R.; Schoell, Matthew C.; Ford, Gregory D.; Pulliam, John V.; Gates, Alicia; Clement, Ceilessia M.; Harris, Adalynn E.; Ford, Byron D.

    2008-01-01

    We previously showed that neuregulin-1 (NRG-1) protected neurons from death in vivo following focal ischemia. The goal of this study was to develop an in vitro rat ischemia model to examine the cellular and molecular mechanisms involved in the neuroprotective effects of NRG-1 on ischemia-induced neuronal death. Rat B-35 neuroblastoma cells differentiated by serum withdrawal, developed enhanced neuronal characteristics including, neurite extension and upregulation of neuronal markers of differentiation. When B35 neurons were subjected to oxygen glucose deprivation (OGD)/reoxygenation or glutamate, widespread neuronal death was seen after both treatments. Treatment with NRG-1 immediately after OGD significantly increased neuronal survival. NRG-1 administration also resulted in a significant decrease in annexin V, an early marker of apoptosis. However, the neurotoxic actions of glutamate were unaffected by NRG-1. The neuroprotective effects of NRG-1 were prevented by an inhibitor of the phosphatidylinositol-3-kinase/Akt pathway. These results provide a new model to gain insight into the mechanisms employed by NRG-1 to protect neurons from ischemic brain injury. PMID:18410912

  3. Spontaneous Neuronal Activity in Developing Neocortical Networks: From Single Cells to Large-Scale Interactions.

    Science.gov (United States)

    Luhmann, Heiko J; Sinning, Anne; Yang, Jenq-Wei; Reyes-Puerta, Vicente; Stüttgen, Maik C; Kirischuk, Sergei; Kilb, Werner

    2016-01-01

    Neuronal activity has been shown to be essential for the proper formation of neuronal circuits, affecting developmental processes like neurogenesis, migration, programmed cell death, cellular differentiation, formation of local and long-range axonal connections, synaptic plasticity or myelination. Accordingly, neocortical areas reveal distinct spontaneous and sensory-driven neuronal activity patterns already at early phases of development. At embryonic stages, when immature neurons start to develop voltage-dependent channels, spontaneous activity is highly synchronized within small neuronal networks and governed by electrical synaptic transmission. Subsequently, spontaneous activity patterns become more complex, involve larger networks and propagate over several neocortical areas. The developmental shift from local to large-scale network activity is accompanied by a gradual shift from electrical to chemical synaptic transmission with an initial excitatory action of chloride-gated channels activated by GABA, glycine and taurine. Transient neuronal populations in the subplate (SP) support temporary circuits that play an important role in tuning early neocortical activity and the formation of mature neuronal networks. Thus, early spontaneous activity patterns control the formation of developing networks in sensory cortices, and disturbances of these activity patterns may lead to long-lasting neuronal deficits.

  4. Importance of being Nernst: Synaptic activity andfunctional relevance in stem cell-derived neurons

    Institute of Scientific and Technical Information of China (English)

    2015-01-01

    Functional synaptogenesis and network emergence aresignature endpoints of neurogenesis. These behaviorsprovide higher-order confirmation that biochemicaland cellular processes necessary for neurotransmitterrelease, post-synaptic detection and network propagation of neuronal activity have been properly expressed andcoordinated among cells. The development of synapticneurotransmission can therefore be considered a definingproperty of neurons. Although dissociated primaryneuron cultures readily form functioning synapsesand network behaviors in vitro , continuously culturedneurogenic cell lines have historically failed to meet thesecriteria. Therefore, in vitro -derived neuron models thatdevelop synaptic transmission are critically needed for awide array of studies, including molecular neuroscience,developmental neurogenesis, disease research andneurotoxicology. Over the last decade, neurons derivedfrom various stem cell lines have shown varying ability todevelop into functionally mature neurons. In this review,we will discuss the neurogenic potential of various stemcells populations, addressing strengths and weaknessesof each, with particular attention to the emergenceof functional behaviors. We will propose methods tofunctionally characterize new stem cell-derived neuron(SCN) platforms to improve their reliability as physiologicalrelevant models. Finally, we will review howsynaptically active SCNs can be applied to accelerateresearch in a variety of areas. Ultimately, emphasizingthe critical importance of synaptic activity and networkresponses as a marker of neuronal maturation is anticipatedto result in in vitro findings that better translateto efficacious clinical treatments.

  5. Rapid and efficient generation of neurons from human pluripotent stem cells in a multititre plate format.

    Science.gov (United States)

    Zhang, Miao; Schöler, Hans R; Greber, Boris

    2013-01-01

    Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are multistep procedures involving the isolation and expansion of neural precursor cells, prior to terminal differentiation. In comparison to these time-consuming approaches, we have recently found that combined inhibition of three signaling pathways, TGFβ/SMAD2, BMP/SMAD1, and FGF/ERK, promotes rapid induction of neuroectoderm from hPSCs, followed by immediate differentiation into functional neurons. Here, we have adapted our procedure to a novel multititre plate format, to further enhance its reproducibility and to make it compatible with mid-throughput applications. It comprises four days of neuroectoderm formation in floating spheres (embryoid bodies), followed by a further four days of differentiation into neurons under adherent conditions. Most cells obtained with this protocol appear to be bipolar sensory neurons. Moreover, the procedure is highly efficient, does not require particular expert skills, and is based on a simple chemically defined medium with cost-efficient small molecules. Due to these features, the procedure may serve as a useful platform for further functional investigation as well as for cell-based screening approaches requiring human sensory neurons or neurons of any type. PMID:23486189

  6. Regulation of neuroendocrine cells and neuron factors in the ovary by zinc oxide nanoparticles.

    Science.gov (United States)

    Liu, Xin-Qi; Zhang, Hong-Fu; Zhang, Wei-Dong; Zhang, Peng-Fei; Hao, Ya-Nan; Song, Ran; Li, Lan; Feng, Yan-Ni; Hao, Zhi-Hui; Shen, Wei; Min, Ling-Jiang; Yang, Hong-Di; Zhao, Yong

    2016-08-10

    The pubertal period is an important window during the development of the female reproductive system. Development of the pubertal ovary, which supplies the oocytes intended for fertilization, requires growth factors, hormones, and neuronal factors. It has been reported that zinc oxide nanoparticles (ZnO NPs) cause cytotoxicity of neuron cells. However, there have been no reports of the effects of ZnO NPs on neuronal factors and neuroendocrine cells in the ovary (in vivo). For the first time, this in vivo study investigated the effects of ZnO NPs on gene and protein expression of neuronal factors and the population of neuroendocrine cells in ovaries. Intact NPs were detected in ovarian tissue and although ZnO NPs did not alter body weight, they reduced the ovary organ index. Compared to the control or ZnSO4 treatments, ZnO NPs treatments differentially regulated neuronal factor protein and gene expression, and the population of neuroendocrine cells. ZnO NPs changed the contents of essential elements in the ovary; however, they did not alter levels of the steroid hormones estrogen and progesterone. These data together suggest that intact ZnO NPs might pose a toxic effect on neuron development in the ovary and eventually negatively affect ovarian developmental at puberty. PMID:27215404

  7. Ancylostoma caninum: the finger cell neurons mediate thermotactic behavior by infective larvae of the dog hookworm.

    Science.gov (United States)

    Bhopale, V M; Kupprion, E K; Ashton, F T; Boston, R; Schad, G A

    2001-02-01

    Bhopale, V. M., Kupprion, E. K., Ashton, F. T., Boston, R., and Schad, G. A. 2001. Ancylostoma caninum: The finger cell neurons mediate thermotactic behavior by infective larvae of the dog hookworm. Experimental Parasitology 97, 70-76. In the amphids (anteriorly positioned, paired sensilla) of the free-living nematode Caenorhabditis elegans, the so-called finger cells (AFD), a pair of neurons, each of which ends in a cluster of microvilli-like projections, are known to be the primary thermoreceptors. A similar neuron pair in the amphids of the parasitic nematode Haemonchus contortus is also known to be thermoreceptive. The hookworm of dogs, Ancylostoma caninum, has apparent structural homologs of finger cells in its amphids. The neuroanatomy of the amphids of A. caninum and H. contortus is strikingly similar, and the amphidial cell bodies in the lateral ganglia of the latter nematode have been identified and mapped. When the lateral ganglia of first-stage larvae (L1) of A. caninum are examined with differential interference contrast microscopy, positional homologs of the recognized amphidial cell bodies in the lateral ganglia of H. contortus L1 are readily identified in A. caninum. The amphidial neurons in A. caninum were consequently given the same names as those of their apparent homologs in H. contortus. It was hypothesized that the finger cell neurons (AFD) might mediate thermotaxis by the skin-penetrating infective larvae (L3) of A. caninum. Laser microbeam ablation experiments with A. caninum were conducted, using the H. contortus L1 neuronal map as a guide. A. caninum L1 were anesthetized and the paired AFD class neurons were ablated. The larvae were then cultured to L3 and assayed for thermotaxis on a thermal gradient. L3 with ablated AFD-class neuron pairs showed significantly reduced thermotaxis compared to control groups. The thermoreceptive function of the AFD-class neurons associates this neuron pair with the host-finding process of the A. caninum

  8. Controlled neuronal cell patterning and guided neurite growth on micropatterned nanofiber platforms

    International Nuclear Information System (INIS)

    Patterning neuronal cells and guiding neurite growth are important for applications such as prosthetics, cell based biosensors, and tissue engineering. In this paper, a microdevice is presented that provides neuronal cell patterning and guided neurite growth on a collagen coated gelatin/PCL nanofiber mat. The pattern consisted of a grid of polystyrene microwells/nodes to confine the cell bodies and orthogonal grooves to guide neurite growth from each node. Vacuum assisted cell seeding was used to localize cell bodies in the microwells and physically separate the cells during seeding. The electrospun nanofiber mats under the polystyrene microstructures were coated with collagen to enhance the cellular attachment and enhance differentiation. We evaluated the performance of our device using adhesion, viability, and differentiation assays of neuron-like PC12 cells compared to controls for vacuum seeding, spatial isolation and guidance, and collagen coating of the fibers. The device provided PC12 cell patterning with increased adhesion, differentiation, and guided neurite outgrowth compared to controls, demonstrating its potential for in vitro neuronal cell patterning studies. (paper)

  9. Controlled neuronal cell patterning and guided neurite growth on micropatterned nanofiber platforms

    Science.gov (United States)

    Malkoc, Veysi; Gallego-Perez, Daniel; Nelson, Tyler; Lannutti, John J.; Hansford, Derek J.

    2015-12-01

    Patterning neuronal cells and guiding neurite growth are important for applications such as prosthetics, cell based biosensors, and tissue engineering. In this paper, a microdevice is presented that provides neuronal cell patterning and guided neurite growth on a collagen coated gelatin/PCL nanofiber mat. The pattern consisted of a grid of polystyrene microwells/nodes to confine the cell bodies and orthogonal grooves to guide neurite growth from each node. Vacuum assisted cell seeding was used to localize cell bodies in the microwells and physically separate the cells during seeding. The electrospun nanofiber mats under the polystyrene microstructures were coated with collagen to enhance the cellular attachment and enhance differentiation. We evaluated the performance of our device using adhesion, viability, and differentiation assays of neuron-like PC12 cells compared to controls for vacuum seeding, spatial isolation and guidance, and collagen coating of the fibers. The device provided PC12 cell patterning with increased adhesion, differentiation, and guided neurite outgrowth compared to controls, demonstrating its potential for in vitro neuronal cell patterning studies.

  10. Differentiation of Wharton’s jelly mesenchymal stem cells into neurons in alginate scaffold

    Institute of Scientific and Technical Information of China (English)

    Seyed Mojtaba Hosseini; Attiyeh Vasaghi; Newsha Nakhlparvar; Reza Roshanravan; Tahereh Talaei-khozani; Zahra Razi

    2015-01-01

    Alginate scaffold has been considered as an appropriate biomaterial for promoting the differ-entiation of embryonic stem cells toward neuronal cell lineage. We hypothesized that alginate scaffold is suitable for culturing Wharton’s jelly mesenchymal stem cells (WJMSCs) and can pro-mote the differentiation of WJMSCs into neuron-like cells. In this study, we cultured WJMSCs in a three-dimensional scaffold fabricated by 0.25% alginate and 50 mM CaCl2 in the presence of neurogenic medium containing 10 µM retinoic acid and 20 ng/mL basic ifbroblast growth factor. These cells were also cultured in conventional two-dimensional culture condition in the presence of neurogenic medium as controls. After 10 days, immunolfuorescence staining was performed for detectingβ-tubulin (marker for WJMSCs-differentiated neuron) and CD271 (motor neuron marker).β-Tubulin and CD271 expression levels were significantly greater in the WJMSCs cultured in the three-dimensional alginate scaffold than in the conventional two-dimensional culture condition. These findings suggest that three-dimensional alginate scaffold cell culture system can induce neuronal differentiation of WJMSCs effectively.

  11. Phase II enzyme induction by a carotenoid, lutein, in a PC12D neuronal cell line

    Energy Technology Data Exchange (ETDEWEB)

    Miyake, Seiji [Laboratory of Retinal Cell Biology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Department of Ophthalmology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Wakasa Seikatsu Co., Ltd., 134 Chudoujiminami-cho, Shimogyo-ku, Kyoto 600-8813 (Japan); Kobayashi, Saori [Wakasa Seikatsu Co., Ltd., 134 Chudoujiminami-cho, Shimogyo-ku, Kyoto 600-8813 (Japan); Tsubota, Kazuo [Laboratory of Retinal Cell Biology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Ozawa, Yoko, E-mail: ozawa@a5.keio.jp [Laboratory of Retinal Cell Biology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Department of Ophthalmology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan)

    2014-04-04

    Highlights: • Lutein reduced ROS levels in a PC12D neuronal cell line. • Lutein induced mRNAs of phase II antioxidative enzymes in PC12D neuronal cells. • Lutein increased protein levels of HO-1, SOD2, and NQO-1 in PC12D neuronal cells. • Lutein had no effect on intranuclear Nrf2 levels in PC12D neuronal cells. • Lutein did not activate potential upstream Nrf2 nuclear translocation pathways. - Abstract: The mechanism by which lutein, a carotenoid, acts as an antioxidant in retinal cells is still not fully understood. Here, lutein treatment of a neuronal cell line (PC12D) immediately resulted in reduced intracellular ROS levels, implying that it has a direct role in ROS scavenging. Significantly, lutein treatment also induced phase II antioxidative enzyme expression, probably via a nuclear factor-like 2 (Nrf2) independent pathway. This latter mechanism could explain why lutein acts diversely to protect against oxidative/cytotoxic stress, and why it is physiologically involved in the human neural tissue, such as the retina.

  12. Clonal Heterogeneity in the Neuronal and Glial Differentiation of Dental Pulp Stem/Progenitor Cells

    Directory of Open Access Journals (Sweden)

    Fraser I. Young

    2016-01-01

    Full Text Available Cellular heterogeneity presents an important challenge to the development of cell-based therapies where there is a fundamental requirement for predictable and reproducible outcomes. Transplanted Dental Pulp Stem/Progenitor Cells (DPSCs have demonstrated early promise in experimental models of spinal cord injury and stroke, despite limited evidence of neuronal and glial-like differentiation after transplantation. Here, we report, for the first time, on the ability of single cell-derived clonal cultures of murine DPSCs to differentiate in vitro into immature neuronal-like and oligodendrocyte-like cells. Importantly, only DPSC clones with high nestin mRNA expression levels were found to successfully differentiate into Map2 and NF-positive neuronal-like cells. Neuronally differentiated DPSCs possessed a membrane capacitance comparable with primary cultured striatal neurons and small inward voltage-activated K+ but not outward Na+ currents were recorded suggesting a functionally immature phenotype. Similarly, only high nestin-expressing clones demonstrated the ability to adopt Olig1, Olig2, and MBP-positive immature oligodendrocyte-like phenotype. Together, these results demonstrate that appropriate markers may be used to provide an early indication of the suitability of a cell population for purposes where differentiation into a specific lineage may be beneficial and highlight that further understanding of heterogeneity within mixed cellular populations is required.

  13. Differentiation of mesenchymal stem cells into neuronal cells on fetal bovine acellular dermal matrix as a tissue engineered nerve scaffold

    Institute of Scientific and Technical Information of China (English)

    Yuping Feng; Jiao Wang; Shixin Ling; Zhuo Li; Mingsheng Li; Qiongyi Li; Zongren Ma; Sijiu Yu

    2014-01-01

    The purpose of this study was to assess fetal bovine acellular dermal matrix as a scaffold for supporting the differentiation of bone marrow mesenchymal stem cells into neural cells fol-lowing induction with neural differentiation medium. We performed long-term, continuous observation of cell morphology, growth, differentiation, and neuronal development using several microscopy techniques in conjunction with immunohistochemistry. We examined speciifc neu-ronal proteins and Nissl bodies involved in the differentiation process in order to determine the neuronal differentiation of bone marrow mesenchymal stem cells. The results show that bone marrow mesenchymal stem cells that differentiate on fetal bovine acellular dermal matrix display neuronal morphology with unipolar and bi/multipolar neurite elongations that express neuro-nal-speciifc proteins, includingβIII tubulin. The bone marrow mesenchymal stem cells grown on fetal bovine acellular dermal matrix and induced for long periods of time with neural differen-tiation medium differentiated into a multilayered neural network-like structure with long nerve ifbers that was composed of several parallel microifbers and neuronal cells, forming a complete neural circuit with dendrite-dendrite to axon-dendrite to dendrite-axon synapses. In addition, growth cones with filopodia were observed using scanning electron microscopy. Paraffin sec-tioning showed differentiated bone marrow mesenchymal stem cells with the typical features of neuronal phenotype, such as a large, round nucleus and a cytoplasm full of Nissl bodies. The data suggest that the biological scaffold fetal bovine acellular dermal matrix is capable of supporting human bone marrow mesenchymal stem cell differentiation into functional neurons and the subsequent formation of tissue engineered nerve.

  14. Glial cell-expressed mechanosensitive channel TRPV4 mediates infrasound-induced neuronal impairment.

    Science.gov (United States)

    Shi, Ming; Du, Fang; Liu, Yang; Li, Li; Cai, Jing; Zhang, Guo-Feng; Xu, Xiao-Fei; Lin, Tian; Cheng, Hao-Ran; Liu, Xue-Dong; Xiong, Li-Ze; Zhao, Gang

    2013-11-01

    Vibroacoustic disease, a progressive and systemic disease, mainly involving the central nervous system, is caused by excessive exposure to low-frequency but high-intensity noise generated by various heavy transportations and machineries. Infrasound is a type of low-frequency noise. Our previous studies demonstrated that infrasound at a certain intensity caused neuronal injury in rats but the underlying mechanism(s) is still largely unknown. Here, we showed that glial cell-expressed TRPV4, a Ca(2+)-permeable mechanosensitive channel, mediated infrasound-induced neuronal injury. Among different frequencies and intensities, infrasound at 16 Hz and 130 dB impaired rat learning and memory abilities most severely after 7-14 days exposure, a time during which a prominent loss of hippocampal CA1 neurons was evident. Infrasound also induced significant astrocytic and microglial activation in hippocampal regions following 1- to 7-day exposure, prior to neuronal apoptosis. Moreover, pharmacological inhibition of glial activation in vivo protected against neuronal apoptosis. In vitro, activated glial cell-released proinflammatory cytokines IL-1β and TNF-α were found to be key factors for this neuronal apoptosis. Importantly, infrasound induced an increase in the expression level of TRPV4 both in vivo and in vitro. Knockdown of TRPV4 expression by siRNA or pharmacological inhibition of TRPV4 in cultured glial cells decreased the levels of IL-1β and TNF-α, attenuated neuronal apoptosis, and reduced TRPV4-mediated Ca(2+) influx and NF-κB nuclear translocation. Finally, using various antagonists we revealed that calmodulin and protein kinase C signaling pathways were involved in TRPV4-triggered NF-κB activation. Thus, our results provide the first evidence that glial cell-expressed TRPV4 is a potential key factor responsible for infrasound-induced neuronal impairment. PMID:24002225

  15. Inhibition of telomerase causes vulnerability to endoplasmic reticulum stress-induced neuronal cell death.

    Science.gov (United States)

    Hosoi, Toru; Nakatsu, Kanako; Shimamoto, Akira; Tahara, Hidetoshi; Ozawa, Koichiro

    2016-08-26

    Endoplasmic reticulum (ER) stress is implicated in several diseases, such as cancer and neurodegenerative diseases. In the present study, we investigated the possible involvement of telomerase in ER stress-induced cell death. ER stress-induced cell death was ameliorated in telomerase reverse transcriptase (TERT) over-expressing MCF7 cells (MCF7-TERT cell). Telomerase specific inhibitor, BIBR1532, reversed the inhibitory effect of TERT on ER stress-induced cell death in MCF7-TERT cells. These findings suggest that BIBR1532 may specifically inhibit telomerase activity, thereby inducing cell death in ER stress-exposed cells. TERT was expressed in the SH-SY5Y neuroblastoma cell line. To analyze the possible involvement of telomerase in ER stress-induced neuronal cell death, we treated SH-SY5Y neuroblastoma cells with BIBR1532 and analyzed ER stress-induced cell death. We found that BIBR1532 significantly enhanced the ER stress-induced neuronal cell death. These findings suggest that inhibition of telomerase activity may enhance vulnerability to neuronal cell death caused by ER stress. PMID:27443785

  16. Differentiation of the immortalized adult neuronal progenitor cell line HC2S2 into neurons by regulatable suppression of the v-myc oncogene.

    OpenAIRE

    Hoshimaru, M; Ray, J.; Sah, D W; Gage, F.H.

    1996-01-01

    A regulatable retroviral vector in which the v-myc oncogene is driven by a tetracycline-controlled transactivator and a human cytomegalovirus minimal promoter fused to a tet operator sequence was used for conditional immortalization of adult rat neuronal progenitor cells. A single clone, HC2S2, was isolated and characterized. Two days after the addition of tetracycline, the HC2S2 cells stopped proliferating, began to extend neurites, and expressed the neuronal markers tau, NeuN, neurofilament...

  17. Cell dialysis by sharp electrodes can cause nonphysiological changes in neuron properties.

    Science.gov (United States)

    Hooper, Scott L; Thuma, Jeffrey B; Guschlbauer, Christoph; Schmidt, Joachim; Büschges, Ansgar

    2015-08-01

    We recorded from lobster and leech neurons with two sharp electrodes filled with solutions often used with these preparations (lobster: 0.6 M K2SO4 or 2.5 M KAc; leech: 4 M KAc), with solutions approximately matching neuron cytoplasm ion concentrations, and with 6.5 M KAc (lobster, leech) and 0.6 M KAc (lobster). We measured membrane potential, input resistance, and transient and sustained depolarization-activated outward current amplitudes in leech and these neuron properties and hyperpolarization-activated current time constant in lobster, every 10 min for 60 min after electrode penetration. Neuron properties varied with electrode fill. For fills with molarities ≥2.5 M, neuron properties also varied strongly with time after electrode penetration. Depending on the property being examined, these variations could be large. In leech, cell size also increased with noncytoplasmic fills. The changes in neuron properties could be due to the ions being injected from the electrodes during current injection. We tested this possibility in lobster with the 2.5 M KAc electrode fill by making measurements only 10 and 60 min after penetration. Neuron properties still changed, although the changes were less extreme. Making measurements every 2 min showed that the time-dependent variations in neuron properties occurred in concert with each other. Neuron property changes with high molarity electrode-fill solutions were great enough to decrease neuron firing strongly. An experiment with (14)C-glucose electrode fill confirmed earlier work showing substantial leak from sharp electrodes. Sharp electrode work should thus be performed with cytoplasm-matched electrode fills.

  18. Sequential generation of olfactory bulb glutamatergic neurons by Neurog2-expressing precursor cells

    Directory of Open Access Journals (Sweden)

    Brill Monika S

    2011-04-01

    Full Text Available Abstract Background While the diversity and spatio-temporal origin of olfactory bulb (OB GABAergic interneurons has been studied in detail, much less is known about the subtypes of glutamatergic OB interneurons. Results We studied the temporal generation and diversity of Neurog2-positive precursor progeny using an inducible genetic fate mapping approach. We show that all subtypes of glutamatergic neurons derive from Neurog2 positive progenitors during development of the OB. Projection neurons, that is, mitral and tufted cells, are produced at early embryonic stages, while a heterogeneous population of glutamatergic juxtaglomerular neurons are generated at later embryonic as well as at perinatal stages. While most juxtaglomerular neurons express the T-Box protein Tbr2, those generated later also express Tbr1. Based on morphological features, these juxtaglomerular cells can be identified as tufted interneurons and short axon cells, respectively. Finally, targeted electroporation experiments provide evidence that while the majority of OB glutamatergic neurons are generated from intrabulbar progenitors, a small portion of them originate from extrabulbar regions at perinatal ages. Conclusions We provide the first comprehensive analysis of the temporal and spatial generation of OB glutamatergic neurons and identify distinct populations of juxtaglomerular interneurons that differ in their antigenic properties and time of origin.

  19. Nanotopography induced contact guidance of the F11 cell line during neuronal differentiation: a neuronal model cell line for tissue scaffold development

    Science.gov (United States)

    Wieringa, Paul; Tonazzini, Ilaria; Micera, Silvestro; Cecchini, Marco

    2012-07-01

    The F11 hybridoma, a dorsal root ganglion-derived cell line, was used to investigate the response of nociceptive sensory neurons to nanotopographical guidance cues. This established this cell line as a model of peripheral sensory neuron growth for tissue scaffold design. Cells were seeded on substrates of cyclic olefin copolymer (COC) films imprinted via nanoimprint lithography (NIL) with a grating pattern of nano-scale grooves and ridges. Different ridge widths were employed to alter the focal adhesion formation, thereby changing the cell/substrate interaction. Differentiation was stimulated with forskolin in culture medium consisting of either 1 or 10% fetal bovine serum (FBS). Per medium condition, similar neurite alignment was achieved over the four day period, with the 1% serum condition exhibiting longer, more aligned neurites. Immunostaining for focal adhesions found the 1% FBS condition to also have fewer, less developed focal adhesions. The robust response of the F11 to guidance cues further builds on the utility of this cell line as a sensory neuron model, representing a useful tool to explore the design of regenerative guidance tissue scaffolds.

  20. Familial Dysautonomia (FD Human Embryonic Stem Cell Derived PNS Neurons Reveal that Synaptic Vesicular and Neuronal Transport Genes Are Directly or Indirectly Affected by IKBKAP Downregulation.

    Directory of Open Access Journals (Sweden)

    Sharon Lefler

    Full Text Available A splicing mutation in the IKBKAP gene causes Familial Dysautonomia (FD, affecting the IKAP protein expression levels and proper development and function of the peripheral nervous system (PNS. Here we found new molecular insights for the IKAP role and the impact of the FD mutation in the human PNS lineage by using a novel and unique human embryonic stem cell (hESC line homozygous to the FD mutation originated by pre implantation genetic diagnosis (PGD analysis. We found that IKBKAP downregulation during PNS differentiation affects normal migration in FD-hESC derived neural crest cells (NCC while at later stages the PNS neurons show reduced intracellular colocalization between vesicular proteins and IKAP. Comparative wide transcriptome analysis of FD and WT hESC-derived neurons together with the analysis of human brains from FD and WT 12 weeks old embryos and experimental validation of the results confirmed that synaptic vesicular and neuronal transport genes are directly or indirectly affected by IKBKAP downregulation in FD neurons. Moreover we show that kinetin (a drug that corrects IKBKAP alternative splicing promotes the recovery of IKAP expression and these IKAP functional associated genes identified in the study. Altogether, these results support the view that IKAP might be a vesicular like protein that might be involved in neuronal transport in hESC derived PNS neurons. This function seems to be mostly affected in FD-hESC derived PNS neurons probably reflecting some PNS neuronal dysfunction observed in FD.

  1. Regulation of progenitor cell proliferation and neuronal differentiation in enteric nervous system neurospheres.

    Directory of Open Access Journals (Sweden)

    Sokratis Theocharatos

    Full Text Available Enteric nervous system (ENS progenitor cells isolated from mouse and human bowel can be cultured in vitro as neurospheres which are aggregates of the proliferating progenitor cells, together with neurons and glial cells derived from them. To investigate the factors regulating progenitor cell proliferation and differentiation, we first characterised cell proliferation in mouse ENS neurospheres by pulse chase experiments using thymidine analogs. We demonstrate rapid and continuous cell proliferation near the neurosphere periphery, after which postmitotic cells move away from the periphery to become distributed throughout the neurosphere. While many proliferating cells expressed glial markers, expression of the neuronal markers β-tubulin III (Tuj1 and nitric oxide synthase was detected in increasing numbers of post-mitotic cells after a delay of several days. Treatment of both mouse and human neurospheres with the γ-secretase inhibitor N-[N-(3,5-Difluorophenacetyl-L-alanyl]-S-phenylglycine t-butyl ester (DAPT reduced expression of the transcription factors Hes1 and Hes5, demonstrating inhibition of Notch signaling. DAPT treatment also inhibited progenitor cell proliferation and increased the numbers of differentiating neurons expressing Tuj1 and nitric oxide synthase. To confirm that the cellular effects of DAPT treatment were due to inhibition of Notch signaling, siRNA knockdown of RBPjκ, a key component of the canonical Notch signaling pathway, was demonstrated both to reduce proliferation and to increase neuronal differentiation in neurosphere cells. These observations indicate that Notch signaling promotes progenitor cell proliferation and inhibits neuronal differentiation in ENS neurospheres.

  2. Dorsal root ganglion neurons promote proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    Pei-xun Zhang; Xiao-rui Jiang; Lei Wang; Fang-min Chen; Lin Xu; Fei Huang

    2015-01-01

    Preliminary animal experiments have conifrmed that sensory nerve ifbers promote osteoblast differentiation, but motor nerve ifbers have no promotion effect. Whether sensory neurons pro-mote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells remains unclear. No results at the cellular level have been reported. In this study, dorsal root ganglion neurons (sensory neurons) from Sprague-Dawley fetal rats were co-cultured with bone marrow mesenchymal stem cells transfected with green lfuorescent protein 3 weeks after osteo-genic differentiationin vitro, while osteoblasts derived from bone marrow mesenchymal stem cells served as the control group. The rat dorsal root ganglion neurons promoted the prolifera-tion of bone marrow mesenchymal stem cell-derived osteoblasts at 3 and 5 days of co-culture, as observed by lfuorescence microscopy. The levels of mRNAs for osteogenic differentiation-re-lated factors (including alkaline phosphatase, osteocalcin, osteopontin and bone morphogenetic protein 2) in the co-culture group were higher than those in the control group, as detected by real-time quantitative PCR. Our ifndings indicate that dorsal root ganglion neurons promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells, which pro-vides a theoretical basis forin vitro experiments aimed at constructing tissue-engineered bone.

  3. Enhanced apoptosis during early neuronal differentiation in mouse ES cells with autosomal imbalance

    Institute of Scientific and Technical Information of China (English)

    Yoshiteru Kai; Teruhiko Wakayama; Mitsuo Oshimura; Chi Chiu Wang; Satoshi Kishigami; Yasuhiro Kazuki; Satoshi Abe; Masato Takiguchi; Yasuaki Shirayoshi; Toshiaki Inoue; Hisao Ito

    2009-01-01

    Although particular chromosomal syndromes are phenotypically and clinically distinct, the majority of individuals with autosomai imbalance, such as aneuploidy, manifest mental retardation. A common abnormal phenotype of Down syndrome (DS), the most prevalent autosomal aneuploidy, shows a reduction in both the number and the density of neurons in the brain. As a DS model, we have recently created chimeric mice from ES cells containing a single human chromosome 21. The mice mimicked the characteristic phenotypic features of DS, and ES cells showed a higher incidence of apoptosis during early neuronal differentiation in vitro. In this study, we examined the induction of anomalous early neural development by aneuploidy in mouse ES cells by transferring various human chromosomes or additional mouse chromosomes. Results showed an elevated incidence of apoptosis in all autosome-aneuploid clones examined during early neuronal differentiation in vitro. Further, cDNA microarray analysis revealed a common cluster of down-regulated genes, of which eight known genes are related to cell proliferation, neurite outgrowth and differentiation. Importantly, targeting of these genes by siRNA knockdown in normal mouse ES cells led to enhanced apoptosis during early neuronal differentiation. These findings strongly suggest that autosomal imbalance is associated with general neuronal loss through a common molecular mechanism for apoptosis.

  4. Functional Properties of Human Stem Cell-Derived Neurons in Health and Disease

    Directory of Open Access Journals (Sweden)

    Jason P. Weick

    2016-01-01

    Full Text Available Stem cell-derived neurons from various source materials present unique model systems to examine the fundamental properties of central nervous system (CNS development as well as the molecular underpinnings of disease phenotypes. In order to more accurately assess potential therapies for neurological disorders, multiple strategies have been employed in recent years to produce neuronal populations that accurately represent in vivo regional and transmitter phenotypes. These include new technologies such as direct conversion of somatic cell types into neurons and glia which may accelerate maturation and retain genetic hallmarks of aging. In addition, novel forms of genetic manipulations have brought human stem cells nearly on par with those of rodent with respect to gene targeting. For neurons of the CNS, the ultimate phenotypic characterization lies with their ability to recapitulate functional properties such as passive and active membrane characteristics, synaptic activity, and plasticity. These features critically depend on the coordinated expression and localization of hundreds of ion channels and receptors, as well as scaffolding and signaling molecules. In this review I will highlight the current state of knowledge regarding functional properties of human stem cell-derived neurons, with a primary focus on pluripotent stem cells. While significant advances have been made, critical hurdles must be overcome in order for this technology to support progression toward clinical applications.

  5. Cerebellar stem cells do not produce neurons and astrocytes in adult mouse

    Energy Technology Data Exchange (ETDEWEB)

    Su, Xin; Guan, Wuqiang; Yu, Yong-Chun; Fu, Yinghui, E-mail: fuyh@fudan.edu.cn

    2014-07-18

    Highlights: • No new neurons and astrocytes are generated in adult mouse cerebellum. • Very few mash1{sup +} or nestin{sup +} stem cells exist, and most of them are quiescent. • Cell proliferation rate is diversified among cerebellar regions and decreases over time. - Abstract: Although previous studies implied that cerebellar stem cells exist in some adult mammals, little is known about whether these stem cells can produce new neurons and astrocytes. In this study by bromodeoxyuridine (BrdU) intraperitoneal (i.p.) injection, we found that there are abundant BrdU{sup +} cells in adult mouse cerebellum, and their quantity and density decreases significantly over time. We also found cell proliferation rate is diversified in different cerebellar regions. Among these BrdU{sup +} cells, very few are mash1{sup +} or nestin{sup +} stem cells, and the vast majority of cerebellar stem cells are quiescent. Data obtained by in vivo retrovirus injection indicate that stem cells do not produce neurons and astrocytes in adult mouse cerebellum. Instead, some cells labeled by retrovirus are Iba1{sup +} microglia. These results indicate that very few stem cells exist in adult mouse cerebellum, and none of these stem cells contribute to neurogenesis and astrogenesis under physiological condition.

  6. Cerebellar stem cells do not produce neurons and astrocytes in adult mouse

    International Nuclear Information System (INIS)

    Highlights: • No new neurons and astrocytes are generated in adult mouse cerebellum. • Very few mash1+ or nestin+ stem cells exist, and most of them are quiescent. • Cell proliferation rate is diversified among cerebellar regions and decreases over time. - Abstract: Although previous studies implied that cerebellar stem cells exist in some adult mammals, little is known about whether these stem cells can produce new neurons and astrocytes. In this study by bromodeoxyuridine (BrdU) intraperitoneal (i.p.) injection, we found that there are abundant BrdU+ cells in adult mouse cerebellum, and their quantity and density decreases significantly over time. We also found cell proliferation rate is diversified in different cerebellar regions. Among these BrdU+ cells, very few are mash1+ or nestin+ stem cells, and the vast majority of cerebellar stem cells are quiescent. Data obtained by in vivo retrovirus injection indicate that stem cells do not produce neurons and astrocytes in adult mouse cerebellum. Instead, some cells labeled by retrovirus are Iba1+ microglia. These results indicate that very few stem cells exist in adult mouse cerebellum, and none of these stem cells contribute to neurogenesis and astrogenesis under physiological condition

  7. Localization of West Nile Virus in monkey brain: double staining antigens immunohistochemically of neurons, neuroglia cells and West Nile Virus

    OpenAIRE

    He, Xianli; Ren, Junping; Xu, Fangling; Ferguson, Monique R; Li, Guangyu

    2009-01-01

    West Nile virus (WNV) can cause encephalitis or meningitis that affects brain tissue, which can also lead to permanent neurological damage that can be fatal. To our knowledge, no consistent double immunohistochemical staining of neurons, neuroglia cells, and WNV has yet been reported. To establish a method for performing double-label immunohistochemical detection of neurons, neuroglia cells and WNV, examining the pathological characteristics of WNV-infected neurons, neuroglia cells, and inves...

  8. Conditional induction of Math1 specifies embryonic stem cells to cerebellar granule neuron lineage and promotes differentiation into mature granule neurons.

    Science.gov (United States)

    Srivastava, Rupali; Kumar, Manoj; Peineau, Stéphane; Csaba, Zsolt; Mani, Shyamala; Gressens, Pierre; El Ghouzzi, Vincent

    2013-04-01

    Directing differentiation of embryonic stem cells (ESCs) to specific neuronal subtype is critical for modeling disease pathology in vitro. An attractive means of action would be to combine regulatory differentiation factors and extrinsic inductive signals added to the culture medium. In this study, we have generated mature cerebellar granule neurons by combining a temporally controlled transient expression of Math1, a master gene in granule neuron differentiation, with inductive extrinsic factors involved in cerebellar development. Using a Tetracyclin-On transactivation system, we overexpressed Math1 at various stages of ESCs differentiation and found that the yield of progenitors was considerably increased when Math1 was induced during embryonic body stage. Math1 triggered expression of Mbh1 and Mbh2, two target genes directly involved in granule neuron precursor formation and strong expression of early cerebellar territory markers En1 and NeuroD1. Three weeks after induction, we observed a decrease in the number of glial cells and an increase in that of neurons albeit still immature. Combining Math1 induction with extrinsic factors specifically increased the number of neurons that expressed Pde1c, Zic1, and GABAα6R characteristic of mature granule neurons, formed "T-shaped" axons typical of granule neurons, and generated synaptic contacts and action potentials in vitro. Finally, in vivo implantation of Math1-induced progenitors into young adult mice resulted in cell migration and settling of newly generated neurons in the cerebellum. These results show that conditional induction of Math1 drives ESCs toward the cerebellar fate and indicate that acting on both intrinsic and extrinsic factors is a powerful means to modulate ESCs differentiation and maturation into a specific neuronal lineage.

  9. Brain scaling in mammalian evolution as a consequence of concerted and mosaic changes in numbers of neurons and average neuronal cell size

    Directory of Open Access Journals (Sweden)

    Suzana eHerculano-Houzel

    2014-08-01

    Full Text Available Enough species have now been subject to systematic quantitative analysis of the relationship between the morphology and cellular composition of their brain that patterns begin to emerge and shed light on the evolutionary path that led to mammalian brain diversity. Based on an analysis of the shared and clade-specific characteristics of 41 modern mammalian species in 6 clades, and in light of the phylogenetic relationships among them, here we propose that ancestral mammal brains were composed and scaled in their cellular composition like modern afrotherian and glire brains: with an addition of neurons that is accompanied by a decrease in neuronal density and very little modification in glial cell density, implying a significant increase in average neuronal cell size in larger brains, and the allocation of approximately 2 neurons in the cerebral cortex and 8 neurons in the cerebellum for every neuron allocated to the rest of brain. We also propose that in some clades the scaling of different brain structures has diverged away from the common ancestral layout through clade-specific (or clade-defining changes in how average neuronal cell mass relates to numbers of neurons in each structure, and how numbers of neurons are differentially allocated to each structure relative to the number of neurons in the rest of brain. Thus, the evolutionary expansion of mammalian brains has involved both concerted and mosaic patterns of scaling across structures. This is, to our knowledge, the first mechanistic model that explains the generation of brains large and small in mammalian evolution, and it opens up new horizons for seeking the cellular pathways and genes involved in brain evolution.

  10. In vitro generation of motor neuron precursors from mouse embryonic stem cells using mesoporous nanoparticles

    DEFF Research Database (Denmark)

    Garcia-Bennett, Alfonso E; König, Niclas; Abrahamsson, Ninnie;

    2014-01-01

    Aim: Stem cell-derived motor neurons (MNs) are utilized to develop replacement strategies for spinal cord disorders. Differentiation of embryonic stem cells into MN precursors involves factors and their repeated administration. We investigated if delivery of factors loaded into mesoporous...

  11. Development of functional human embryonic stem cell-derived neurons in mouse brain

    OpenAIRE

    Muotri, Alysson R.; Nakashima, Kinichi; Toni, Nicolas; Sandler, Vladislav M.; Gage, Fred H

    2005-01-01

    Human embryonic stem cells are pluripotent entities, theoretically capable of generating a whole-body spectrum of distinct cell types. However, differentiation of these cells has been observed only in culture or during teratoma formation. Our results show that human embryonic stem cells implanted in the brain ventricles of embryonic mice can differentiate into functional neural lineages and generate mature, active human neurons that successfully integrate into the adult mouse forebrain. Moreo...

  12. Wnt signaling pathway participates in valproic acid-induced neuronal differentiation of neural stem cells

    OpenAIRE

    Wang, Li; Liu, Yuan; Li, Sen; Zai-yun LONG; Wu, Ya-min

    2015-01-01

    Neural stem cells (NSCs) are multipotent cells that have the capacity for differentiation into the major cell types of the nervous system, i.e. neurons, astrocytes and oligodendrocytes. Valproic acid (VPA) is a widely prescribed drug for seizures and bipolar disorder in clinic. Previously, a number of researches have been shown that VPA has differential effects on growth, proliferation and differentiation in many types of cells. However, whether VPA can induce NSCs from embryonic cerebral cor...

  13. The NG2 Protein Is Not Required for Glutamatergic Neuron-NG2 Cell Synaptic Signaling.

    Science.gov (United States)

    Passlick, Stefan; Trotter, Jacqueline; Seifert, Gerald; Steinhäuser, Christian; Jabs, Ronald

    2016-01-01

    NG2 glial cells (as from now NG2 cells) are unique in receiving synaptic input from neurons. However, the components regulating formation and maintenance of these neuron-glia synapses remain elusive. The transmembrane protein NG2 has been considered a potential mediator of synapse formation and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) clustering, because it contains 2 extracellular Laminin G/Neurexin/Sex Hormone-Binding Globulin domains, which in neurons are crucial for formation of transsynaptic neuroligin-neurexin complexes. NG2 is connected via Glutamate Receptor-Interacting Protein with GluA2/3-containing AMPARs, thereby possibly mediating receptor clustering in glial postsynaptic density. To elucidate the role of NG2 in neuron-glia communication, we investigated glutamatergic synaptic transmission in juvenile and aged hippocampal NG2 cells of heterozygous and homozygous NG2 knockout mice. Neuron-NG2 cell synapses readily formed in the absence of NG2. Short-term plasticity, synaptic connectivity, postsynaptic AMPAR current kinetics, and density were not affected by NG2 deletion. During development, an NG2-independent acceleration of AMPAR current kinetics and decreased synaptic connectivity were observed. Our results indicate that the lack of NG2 does not interfere with genesis and basic properties of neuron-glia synapses. In addition, we demonstrate frequent expression of neuroligins 1-3 in juvenile and aged NG2 cells, suggesting a role of these molecules in synapse formation between NG2 glia and neurons. PMID:25100858

  14. Clinical implications of the involvement of tPA in neuronal cell death.

    Science.gov (United States)

    Tsirka, S E

    1997-05-01

    Tissue plasminogen activator (tPA), the serine protease that converts inactive plasminogen to the protease plasmin, was recently shown to mediate neurodegeneration in the mouse hippocampus. Mice deficient in tissue plasminogen activator (tPA) display a dramatic resistance to a paradigm of excitotoxic neuronal death that involves intrahippocampal injection of the excitotoxin. This model is thought to reproduce the mechanism of neuronal death observed during acute (such as ischemic stroke) and degenerative (such as amyotrophic lateral sclerosis) diseases of the nervous system. The requirement for the proteolytic activity of tPA to mediate neuronal death is acute in the adult mouse. Serine protease inhibitors, specific for tPA or the tPA/plasmin proteolytic cascade, are effective in conferring extensive neuroprotection following the excitotoxic injection. These findings suggest possible new ways for interfering with the neuronal death observed in the hippocampus as a result of excitotoxicity. In addition, tPA is produced in the hippocampus primarily by microglial cells, which become activated in response to the neuronal injury. Blocking microglial activation has been shown in other injury paradigms to protect against neuronal death, therefore suggesting another way to retard neurodegeneration in the CNS. Furthermore, after the insult has been inflicted and in the presence of a compromised blood-brain barrier macrophages (cells deriving from the same lineage as microglia) migrate into the brain, where they are thought to contribute to the neuronal cell loss by secreting neurotoxic molecules. If these macrophages/microglia expressed, however, a tPA inhibitor, rather than the possibly neurotoxic tPA, they might be able to protect the neurons from dying.

  15. Synaptotagmin 1 causes phosphatidyl inositol lipid-dependent actin remodeling in cultured non-neuronal and neuronal cells

    Energy Technology Data Exchange (ETDEWEB)

    Johnsson, Anna-Karin; Karlsson, Roger, E-mail: roger.karlsson@wgi.su.se

    2012-01-15

    Here we demonstrate that a dramatic actin polymerizing activity caused by ectopic expression of the synaptic vesicle protein synaptotagmin 1 that results in extensive filopodia formation is due to the presence of a lysine rich sequence motif immediately at the cytoplasmic side of the transmembrane domain of the protein. This polybasic sequence interacts with anionic phospholipids in vitro, and, consequently, the actin remodeling caused by this sequence is interfered with by expression of a phosphatidyl inositol (4,5)-bisphosphate (PIP2)-targeted phosphatase, suggesting that it intervenes with the function of PIP2-binding actin control proteins. The activity drastically alters the behavior of a range of cultured cells including the neuroblastoma cell line SH-SY5Y and primary cortical mouse neurons, and, since the sequence is conserved also in synaptotagmin 2, it may reflect an important fine-tuning role for these two proteins during synaptic vesicle fusion and neurotransmitter release.

  16. Transcriptional profiling at whole population and single cell levels reveals somatosensory neuron molecular diversity.

    Science.gov (United States)

    Chiu, Isaac M; Barrett, Lee B; Williams, Erika K; Strochlic, David E; Lee, Seungkyu; Weyer, Andy D; Lou, Shan; Bryman, Gregory S; Roberson, David P; Ghasemlou, Nader; Piccoli, Cara; Ahat, Ezgi; Wang, Victor; Cobos, Enrique J; Stucky, Cheryl L; Ma, Qiufu; Liberles, Stephen D; Woolf, Clifford J

    2014-01-01

    The somatosensory nervous system is critical for the organism's ability to respond to mechanical, thermal, and nociceptive stimuli. Somatosensory neurons are functionally and anatomically diverse but their molecular profiles are not well-defined. Here, we used transcriptional profiling to analyze the detailed molecular signatures of dorsal root ganglion (DRG) sensory neurons. We used two mouse reporter lines and surface IB4 labeling to purify three major non-overlapping classes of neurons: 1) IB4(+)SNS-Cre/TdTomato(+), 2) IB4(-)SNS-Cre/TdTomato(+), and 3) Parv-Cre/TdTomato(+) cells, encompassing the majority of nociceptive, pruriceptive, and proprioceptive neurons. These neurons displayed distinct expression patterns of ion channels, transcription factors, and GPCRs. Highly parallel qRT-PCR analysis of 334 single neurons selected by membership of the three populations demonstrated further diversity, with unbiased clustering analysis identifying six distinct subgroups. These data significantly increase our knowledge of the molecular identities of known DRG populations and uncover potentially novel subsets, revealing the complexity and diversity of those neurons underlying somatosensation. PMID:25525749

  17. Inhibition of apoptosis blocks human motor neuron cell death in a stem cell model of spinal muscular atrophy.

    Directory of Open Access Journals (Sweden)

    Dhruv Sareen

    Full Text Available Spinal muscular atrophy (SMA is a genetic disorder caused by a deletion of the survival motor neuron 1 gene leading to motor neuron loss, muscle atrophy, paralysis, and death. We show here that induced pluripotent stem cell (iPSC lines generated from two Type I SMA subjects-one produced with lentiviral constructs and the second using a virus-free plasmid-based approach-recapitulate the disease phenotype and generate significantly fewer motor neurons at later developmental time periods in culture compared to two separate control subject iPSC lines. During motor neuron development, both SMA lines showed an increase in Fas ligand-mediated apoptosis and increased caspase-8 and-3 activation. Importantly, this could be mitigated by addition of either a Fas blocking antibody or a caspase-3 inhibitor. Together, these data further validate this human stem cell model of SMA, suggesting that specific inhibitors of apoptotic pathways may be beneficial for patients.

  18. Progenitor Cell Dynamics in the Newt Telencephalon during Homeostasis and Neuronal Regeneration

    Directory of Open Access Journals (Sweden)

    Matthew Kirkham

    2014-04-01

    Full Text Available The adult newt brain has a marked neurogenic potential and is highly regenerative. Ventricular, radial glia-like ependymoglia cells give rise to neurons both during normal homeostasis and after injury, but subpopulations among ependymoglia cells have not been defined. We show here that a substantial portion of GFAP+ ependymoglia cells in the proliferative hot spots of the telencephalon has transit-amplifying characteristics. In contrast, proliferating ependymoglia cells, which are scattered along the ventricular wall, have stem cell features in terms of label retention and insensitivity to AraC treatment. Ablation of neurons remodels the proliferation dynamics and leads to de novo formation of regions displaying features of neurogenic niches, such as the appearance of cells with transit-amplifying features and proliferating neuroblasts. The results have implication both for our understanding of the evolutionary diversification of radial glia cells as well as the processes regulating neurogenesis and regeneration in the adult vertebrate brain.

  19. Anions Govern Cell Volume: A Case Study of Relative Astrocytic and Neuronal Swelling in Spreading Depolarization.

    Directory of Open Access Journals (Sweden)

    Niklas Hübel

    Full Text Available Cell volume changes are ubiquitous in normal and pathological activity of the brain. Nevertheless, we know little about the dynamics of cell and tissue swelling, and the differential changes in the volumes of neurons and glia during pathological states such as spreading depolarizations (SD under ischemic and non-ischemic conditions, and epileptic seizures. By combining the Hodgkin-Huxley type spiking dynamics, dynamic ion concentrations, and simultaneous neuronal and astroglial volume changes into a comprehensive model, we elucidate why glial cells swell more than neurons in SD and the special case of anoxic depolarization (AD, and explore the relative contributions of the two cell types to tissue swelling. Our results demonstrate that anion channels, particularly Cl-, are intrinsically connected to cell swelling and blocking these currents prevents changes in cell volume. The model is based on a simple and physiologically realistic description. We introduce model extensions that are either derived purely from first physical principles of electroneutrality, osmosis, and conservation of particles, or by a phenomenological combination of these principles and known physiological facts. This work provides insights into numerous studies related to neuronal and glial volume changes in SD that otherwise seem contradictory, and is broadly applicable to swelling in other cell types and conditions.

  20. Distribution of Neuron Cell Bodies in the Intraspinal Portion of the Spinal Accessory Nerve in Humans.

    Science.gov (United States)

    Boehm, Karl E; Kondrashov, Peter

    2016-01-01

    The spinal accessory nerve is often identified as a purely motor nerve innervating the trapezius and sternocleidomastoid muscles. Although it may contain proprioceptive neurons found in cervical spinal levels C2-C4, limited research has focused on the histology of the spinal accessory nerve. The objective of the present study was to examine the spinal accessory nerve to determine if there are neuronal cell bodies within the spinal accessory nerve in humans. Cervical spinal cords were dissected from eight cadavers that had previously been used for dissection in other body regions. The segmental rootlets were removed to quantify the neuron cell bodies present at each spinal level. Samples were embedded in paraffin; sectioned; stained with hematoxylin and eosin; and examined using a microscope at 4×, 10×, and 40× magnification. Digital photography was used to image the samples. Neuronal cell bodies were found in 100% of the specimens examined, with non-grossly visible ganglia found at spinal levels C1-C4. The C1 spinal level of the spinal accessory nerve had the highest number of neuron cell bodies.

  1. Peripheral and central neuronal ATF3 precedes CD4+ T-cell infiltration in EAE.

    Science.gov (United States)

    Frezel, Noémie; Sohet, Fabien; Daneman, Richard; Basbaum, Allan I; Braz, Joao M

    2016-09-01

    Experimental allergic encephalomyelitis (EAE), an animal model of multiple sclerosis produced by immunization with myelin oligodendrocyte glycoprotein (MOG) and adjuvants, results from profound T-cell mediated CNS demyelination. EAE is characterized by progressive, ascending motor dysfunction and symptoms of ongoing pain and hypersensitivity, in some cases preceding or concomitant with the motor deficits. In this regard, the EAE model mimics major features of multiple sclerosis, where a central neuropathic pain state is common. Although the latter condition is presumed to arise from a CNS loss of inhibitory controls secondary to the demyelination, dysfunction of sensory neurons may also contribute. Based on our previous studies that demonstrated the utility of monitoring expression of activating transcription factor 3 (ATF3), a sensitive marker of injured sensory neurons, here we followed both ATF3 and CD4+ T cells invasion of sensory ganglia (as well as the CNS) at different stages of the EAE model. We found that ATF3 is induced in peripheral sensory ganglia and brainstem well before the appearance of motor deficits. Unexpectedly, the ATF3 induction always preceded T cell infiltration, typically in adjacent, but non-overlapping regions. Surprisingly, control administration of the pertussis toxin and/or Complete Freund's adjuvants, without MOG, induced ATF3 in sensory neurons. In contrast, T cell infiltration only occurred with MOG. Taken together, our results suggest that the clinical manifestations in the EAE result not only from central demyelination but also from neuronal stress and subsequent pathophysiology of sensory neurons. PMID:27343802

  2. Subplate cells: amplifiers of neuronal activity in the developing cerebral cortex

    Directory of Open Access Journals (Sweden)

    Heiko J Luhmann

    2009-10-01

    Full Text Available Due to their unique structural and functional properties, subplate cells are ideally suited to function as important amplifying units within the developing neocortical circuit. Subplate neurons have extensive dendritic and axonal ramifications and relatively mature functional properties, i.e. their action potential firing can exceed frequencies of 40 Hz. At earliest stages of corticogenesis subplate cells receive functional synaptic inputs from the thalamus and from other cortical and non-cortical sources. Glutamatergic and depolarizing GABAergic inputs arise from cortical neurons and neuromodulatory inputs arise from the basal forebrain and other sources. Activation of postsynaptic metabotropic receptors, i.e. muscarinic receptors, elicits in subplate neurons oscillatory burst discharges which are transmitted via electrical and chemical synapses to neighbouring subplate cells and to immature neurons in the cortical plate. The tonic nonsynaptic release of GABA from GABAergic subplate cells facilitates the generation of burst discharges. These cellular bursts are amplified by prominent gap junction coupling in the subplate and cortical plate, thereby eliciting 10 to 20 Hz oscillations in a local columnar network. Thus, we propose that neuronal networks are organized at earliest stages in a gap junction coupled columnar syncytium. We postulate that the subplate does not only serve as a transient relay station for afferent inputs, but rather as an active element amplifying the afferent and intracortical activity.

  3. In Vivo Reprogramming for CNS Repair: Regenerating Neurons from Endogenous Glial Cells.

    Science.gov (United States)

    Li, Hedong; Chen, Gong

    2016-08-17

    Neuroregeneration in the CNS has proven to be difficult despite decades of research. The old dogma that CNS neurons cannot be regenerated in the adult mammalian brain has been overturned; however, endogenous adult neurogenesis appears to be insufficient for brain repair. Stem cell therapy once held promise for generating large quantities of neurons in the CNS, but immunorejection and long-term functional integration remain major hurdles. In this Perspective, we discuss the use of in vivo reprogramming as an emerging technology to regenerate functional neurons from endogenous glial cells inside the brain and spinal cord. Besides the CNS, in vivo reprogramming has been demonstrated successfully in the pancreas, heart, and liver and may be adopted in other organs. Although challenges remain for translating this technology into clinical therapies, we anticipate that in vivo reprogramming may revolutionize regenerative medicine by using a patient's own internal cells for tissue repair. PMID:27537482

  4. Live-cell imaging of post-golgi transport vesicles in cultured hippocampal neurons

    DEFF Research Database (Denmark)

    Jensen, Camilla Stampe; Misonou, Hiroaki

    2015-01-01

    The subcellular localization of neuronal membrane signaling molecules such as receptors and ion channels depends on intracellular trafficking mechanisms. Essentially, vesicular trafficking mechanisms ensure that a large number of membrane proteins are correctly targeted to different subcellular...... compartments of neurons. In the past two decades, the establishment and advancement of fluorescent protein technology have provided us with opportunities to study how proteins are trafficked in living cells. However, live imaging of trafficking processes in neurons necessitate imaging tools to distinguish...... mechanisms by which post-Golgi vesicles are trafficked in neurons. Our protocol uniquely combines the classic temperature-block with close monitoring of the transient expression of transfected protein tagged with fluorescent proteins, and provides a quick and easy way to study protein trafficking in living...

  5. Multipotent Neural Crest Stem Cell-Like Cells from Rat Vibrissa Dermal Papilla Induce Neuronal Differentiation of PC12 Cells

    Directory of Open Access Journals (Sweden)

    Meiying Li

    2014-01-01

    Full Text Available Bone marrow mesenchymal stem cells (BMSCs transplants have been approved for treating central nervous system (CNS injuries and diseases; however, their clinical applications are limited. Here, we model the therapeutic potential of dermal papilla cells (DPCs in vitro. DPCs were isolated from rat vibrissae and characterized by immunocytofluorescence, RT-PCR, and multidifferentiation assays. We examined whether these cells could secrete neurotrophic factors (NTFs by using cocultures of rat pheochromocytoma cells (PC12 with conditioned medium and ELISA assay. DPCs expressed Sox10, P75, Nestin, Sox9, and differentiated into adipocytes, osteoblasts, smooth muscle cells, and neurons under specific inducing conditions. The DPC-conditioned medium (DPC-CM induced neuronal differentiation of PC12 cells and promoted neurite outgrowth. Results of ELISA assay showed that compared to BMSCs, DPCs secreted more brain-derived neurotrophic factor (BDNF and glial cell line-derived neurotrophic factor (GDNF. Moreover, we observed that, compared with the total DPC population, sphere-forming DPCs expressed higher levels of Nestin and P75 and secreted greater amounts of GDNF. The DPCs from craniofacial hair follicle papilla may be a new and promising source for treating CNS injuries and diseases.

  6. Activity-dependent long-term plasticity of afferent synapses on grafted stem/progenitor cell-derived neurons.

    OpenAIRE

    Toft Sörensen, Andreas; Rogelius, Nina; Lundberg, Cecilia; Kokaia, Merab

    2011-01-01

    Stem cell-based cell replacement therapies aiming at restoring injured or diseased brain function ultimately rely on the capability of transplanted cells to promote functional recovery. The mechanisms by which stem cell-based therapies for neurological conditions can lead to functional recovery are uncertain, but structural and functional repair appears to depend on integration of transplanted cell-derived neurons into neuronal circuitries. The nature by which stem/progenitor cell-derived neu...

  7. Factors inducing human umbilical cord blood-derived mesenchymal stem cells to differentiate into neuron-like cells

    Institute of Scientific and Technical Information of China (English)

    Nawei Zhang; Fengqing Ji

    2006-01-01

    OBJECTIVE:Human umbilical cord blood-derived mesenchymal stem cells (HUCB-derived MSCs)can differentiate into neuron-like cells,which can be used to treat some central nervous system(CNS)diseases.To investigate the factors,which can induce HUCB-derived MSCs to differentiate into neuron-like cells,so as to find effective methods for future clinical application.DATA SOURCES:Using the key terms"human umbilical cord blood"combined with"mesenchymal stem cells,neuron-like cells,neural cells"respectively,the relevant articles in English published during the period from January 1999 to June 2006 were searched from the Medline database.Meanwhile,relevant Chinese articles published from January 1999 to June 2006 were searched Using the same key terms.STUDY SELECTION: All articles associated with the differentiation from human umbilical cord blood into neuron-like cells were selected firstly.Then the full texts were looked up by searchling Ovid medical Journals full-text database and Elsevier Electrical Journals Full-text Database.Articles with full expeiments,enrolled in inducible factors or involved inducible mechanism were retdeved.DATA EXTRACTION:Among 119 collected correlative articles,29 were involved and 90 were excluded.DATA SYNTHESIS:The inducible factors of HUCB-derived MSCs differentiatling into neuron-like cells included renal endothelial growth factors,fibroblasts,β-mercaptoethanol,dimethyl sulfoxide,butyl hydroxyl anisol,brain-derived neurotrophic factor,Danshen,retinoic acid,sodium ferulate and so on,but its mechanism was unclear.CONCLUSION:Human umbilical cord blood-derived MSCs can differentiate into neuron-like cells,with varied inductors.

  8. Differentiation of human gingival mesenchymal stem cells into neuronal lineages in 3D bioconjugated injectable protein hydrogel construct for the management of neuronal disorder

    Science.gov (United States)

    Rao, Suresh Ranga; Subbarayan, Rajasekaran; Dinesh, Murugan Girija; Arumugam, Gnanamani; Raja, Selvaraj Thirupathi Kumara

    2016-01-01

    The success of regeneration attempt is based on an ideal combination of stem cells, scaffolding and growth factors. Tissue constructs help to maintain stem cells in a required area for a desired time. There is a need for easily obtainable cells, potentially autologous stem cells and a biologically acceptable scaffold for use in humans in different difficult situations. This study aims to address these issues utilizing a unique combination of stem cells from gingiva and a hydrogel scaffold, based on a natural product for regenerative application. Human gingival mesenchymal stem cells (HGMSCs) were, with due induction, differentiated to neuronal lineages to overcome the problems associated with birth tissue-related stem cells. The differentiation potential of neuronal lineages was confirmed with suitable specific markers. The properties of mesenchymal stem cells in encapsulated form were observed to be similar to free cells. The encapsulated cells (3D) were then subjected to differentiation into neuronal lineages with suitable inducers, and the morphology and gene expression of transient cells were analyzed. HGMSCs was differentiated into neuronal lineages as both free and encapsulated forms without any significant differences. The presence of Nissl bodies and the neurite outgrowth confirm the differentiation. The advantages of this new combination appear to make it a promising tissue construct for translational application. PMID:26869025

  9. Manganese nanoparticle activates mitochondrial dependent apoptotic signaling and autophagy in dopaminergic neuronal cells

    Energy Technology Data Exchange (ETDEWEB)

    Afeseh Ngwa, Hilary; Kanthasamy, Arthi [Department of Biomedical Sciences, Iowa Center for Advanced Neurotoxicology, Iowa State University, Ames, IA 50011 (United States); Gu, Yan; Fang, Ning [Department of Chemistry, Iowa State University, Ames, IA 50011 (United States); Anantharam, Vellareddy [Department of Biomedical Sciences, Iowa Center for Advanced Neurotoxicology, Iowa State University, Ames, IA 50011 (United States); Kanthasamy, Anumantha G., E-mail: akanthas@iastate.edu [Department of Biomedical Sciences, Iowa Center for Advanced Neurotoxicology, Iowa State University, Ames, IA 50011 (United States)

    2011-11-15

    The production of man-made nanoparticles for various modern applications has increased exponentially in recent years, but the potential health effects of most nanoparticles are not well characterized. Unfortunately, in vitro nanoparticle toxicity studies are extremely limited by yet unresolved problems relating to dosimetry. In the present study, we systematically characterized manganese (Mn) nanoparticle sizes and examined the nanoparticle-induced oxidative signaling in dopaminergic neuronal cells. Differential interference contrast (DIC) microscopy and transmission electron microscopy (TEM) studies revealed that Mn nanoparticles range in size from single nanoparticles ({approx} 25 nM) to larger agglomerates when in treatment media. Manganese nanoparticles were effectively internalized in N27 dopaminergic neuronal cells, and they induced a time-dependent upregulation of the transporter protein transferrin. Exposure to 25-400 {mu}g/mL Mn nanoparticles induced cell death in a time- and dose-dependent manner. Mn nanoparticles also significantly increased ROS, accompanied by a caspase-mediated proteolytic cleavage of proapoptotic protein kinase C{delta} (PKC{delta}), as well as activation loop phosphorylation. Blocking Mn nanoparticle-induced ROS failed to protect against the neurotoxic effects, suggesting the involvement of other pathways. Further mechanistic studies revealed changes in Beclin 1 and LC3, indicating that Mn nanoparticles induce autophagy. Primary mesencephalic neuron exposure to Mn nanoparticles induced loss of TH positive dopaminergic neurons and neuronal processes. Collectively, our results suggest that Mn nanoparticles effectively enter dopaminergic neuronal cells and exert neurotoxic effects by activating an apoptotic signaling pathway and autophagy, emphasizing the need for assessing possible health risks associated with an increased use of Mn nanoparticles in modern applications. -- Highlights: Black-Right-Pointing-Pointer Mn nanoparticles

  10. Differentiation of embryonic versus adult rat neural stem cells into dopaminergic neurons in vitro

    Institute of Scientific and Technical Information of China (English)

    Chunlong Ke; Baili Chen; Shaolei Guo; Chao Yang

    2008-01-01

    BACKGROUND: It has been reported that the conversion of neural stem cells into dopaminergic neurons in vitro can be increased through specific cytokine combinations. Such neural stem cell-derived dopaminergic neurons could be used for the treatment of Parkinson's disease. However, little is known about the differences in dopaminergic differentiation between neural stem cells derived from adult and embryonic rats.OBJECTIVE: To study the ability of rat adult and embryonic-derived neural stem cells to differentiate into dopaminergic neurons in vitro.DESIGN: Randomized grouping design.SETTING: Department of Neurosurgery in the First Affiliated Hospital of Sun Yat-sen University.MATERIALS: This experiment was performed at the Surgical Laboratory in the First Affiliated Hospital of Sun Yat-scn University (Guangzhou, Guangdong, China) from June to December 2007. Eight, adult, male,Sprague Dawley rats and eight, pregnant, Sprague Dawley rats (embryonic day 14 or 15) were provided by the Experimental Animal Center of Sun Yat-sen University.METHODS: Neural stem cells derived from adult and embryonic rats were respectively cultivated in serum-free culture medium containing epidermal growth factor and basic fibroblast growth factor. After passaging, neural stem cells were differentiated in medium containing interleukin-1 ct, interleukin-11, human leukemia inhibition factor, and glial cell line-derived neurotrophic factor. Six days later, cells were analyzed by immunocytochemistry and flow cytometry.MAIN OUTCOME MEASURES: Alterations in cellular morphology after differentiation of neural stem cells derived from adult and embryonic rats; and percentage of tyrosine hydroxylase-positive neurons in the differentiated cells.RESULTS: Neural stem cells derived from adult and embryonic rats were cultivated in differentiation medium. Six days later, differentiated cells were immunoreactive for tyrosine hydroxylasc. The percentage of tyrosine hydroxylase positive neurons was (5.6 ± 2

  11. TRANSCRIPTIONAL LANDSCAPE OF NEURONAL and CANCER STEM CELLS

    OpenAIRE

    Miele, Evelina

    2013-01-01

    Tumor mass is composed by heterogeneous cell population including a subset of “cancer stem cells” (CSC). Oncogenic signals foster CSC by transforming tissue stem cells or by reprogramming progenitor/differentiated cells towards stemness. Thus, CSC share features with cancer and stem cells (e.g. self-renewal, hierarchical developmental program leading to differentiated cells, epithelial/mesenchimal transition) and these latter are maintained by the constitutive activation of stemne...

  12. The role of NO synthase isoforms in PDT-induced injury of neurons and glial cells

    Science.gov (United States)

    Kovaleva, V. D.; Berezhnaya, E. V.; Uzdensky, A. B.

    2015-03-01

    Nitric oxide (NO) is an important second messenger, involved in the implementation of various cell functions. It regulates various physiological and pathological processes such as neurotransmission, cell responses to stress, and neurodegeneration. NO synthase is a family of enzymes that synthesize NO from L-arginine. The activity of different NOS isoforms depends both on endogenous and exogenous factors. In particular, it is modulated by oxidative stress, induced by photodynamic therapy (PDT). We have studied the possible role of NOS in the regulation of survival and death of neurons and surrounding glial cells under photo-oxidative stress induced by photodynamic treatment (PDT). The crayfish stretch receptor consisting of a single identified sensory neuron enveloped by glial cells is a simple but informative model object. It was photosensitized with alumophthalocyanine photosens (10 nM) and irradiated with a laser diode (670 nm, 0.4 W/cm2). Antinecrotic and proapoptotic effects of NO on the glial cells were found using inhibitory analysis. We have shown the role of inducible NO synthase in photoinduced apoptosis and involvement of neuronal NO synthase in photoinduced necrosis of glial cells in the isolated crayfish stretch receptor. The activation of NO synthase was evaluated using NADPH-diaphorase histochemistry, a marker of neurons expressing the enzyme. The activation of NO synthase in the isolated crayfish stretch receptor was evaluated as a function of time after PDT. Photodynamic treatment induced transient increase in NO synthase activity and then slowly inhibited this enzyme.

  13. Rhinacanthus nasutus protects cultured neuronal cells against hypoxia induced cell death.

    Science.gov (United States)

    Brimson, James M; Tencomnao, Tewin

    2011-07-26

    Rhinacanthus nasutus (L.) Kurz (Acanthaceae) is an herb native to Thailand and Southeast Asia, known for its antioxidant properties. Hypoxia leads to an increase in reactive oxygen species in cells and is a leading cause of neuronal damage. Cell death caused by hypoxia has been linked with a number of neurodegenerative diseases including some forms of dementia and stroke, as well as the build up of reactive oxygen species which can lead to diseases such as Huntington's disease, Parkinson's disease and Alzeheimer's disease. In this study we used an airtight culture container and the Mitsubishi Gas Company anaeropack along with the MTT assay, LDH assay and the trypan blue exlusion assay to show that 1 and 10 µg mL⁻¹ root extract of R. nasutus is able to significantly prevent the death of HT-22 cells subjected to hypoxic conditions, and 0.1 to 10 µg mL⁻¹ had no toxic effect on HT-22 under normal conditions, whereas 100 µg mL⁻¹ reduced HT-22 cell proliferation. We also used H₂DCFDA staining to show R. nasutus can reduce reactive oxygen species production in HT-22 cells.

  14. Rhinacanthus nasutus Protects Cultured Neuronal Cells against Hypoxia Induced Cell Death

    Directory of Open Access Journals (Sweden)

    James M. Brimson

    2011-07-01

    Full Text Available Rhinacanthus nasutus (L. Kurz (Acanthaceae is an herb native to Thailand and Southeast Asia, known for its antioxidant properties. Hypoxia leads to an increase in reactive oxygen species in cells and is a leading cause of neuronal damage. Cell death caused by hypoxia has been linked with a number of neurodegenerative diseases including some forms of dementia and stroke, as well as the build up of reactive oxygen species which can lead to diseases such as Huntington’s disease, Parkinson’s disease and Alzeheimer’s disease. In this study we used an airtight culture container and the Mitsubishi Gas Company anaeropack along with the MTT assay, LDH assay and the trypan blue exlusion assay to show that 1 and 10 µg mL−1 root extract of R. nasutus is able to significantly prevent the death of HT-22 cells subjected to hypoxic conditions, and 0.1 to 10 µg mL−1 had no toxic effect on HT-22 under normal conditions, whereas 100 µg mL−1 reduced HT-22 cell proliferation. We also used H2DCFDA staining to show R. nasutus can reduce reactive oxygen species production in HT-22 cells.

  15. Single-cell RNA-seq reveals distinct injury responses in different types of DRG sensory neurons

    Science.gov (United States)

    Hu, Ganlu; Huang, Kevin; Hu, Youjin; Du, Guizhen; Xue, Zhigang; Zhu, Xianmin; Fan, Guoping

    2016-01-01

    Peripheral nerve injury leads to various injury-induced responses in sensory neurons including physiological pain, neuronal cell death, and nerve regeneration. In this study, we performed single-cell RNA-sequencing (scRNA-seq) analysis of mouse nonpeptidergic nociceptors (NP), peptidergic nociceptors (PEP), and large myelinated sensory neurons (LM) under both control and injury conditions at 3 days after sciatic nerve transection (SNT). After performing principle component and weighted gene co-expression network analysis, we categorized dorsal root ganglion (DRG) neurons into different subtypes and discovered co-regulated injury-response genes including novel regeneration associated genes (RAGs) in association with neuronal development, protein translation and cytoplasm transportation. In addition, we found significant up-regulation of the genes associated with cell death such as Pdcd2 in a subset of NP neurons after axotomy, implicating their actions in neuronal cell death upon nerve injury. Our study revealed the distinctive and sustained heterogeneity of transcriptomic responses to injury at single neuron level, implicating the involvement of different gene regulatory networks in nerve regeneration, neuronal cell death and neuropathy in different population of DRG neurons. PMID:27558660

  16. Glia-Neuron Interactions in the Retina Can Be Studied in Cocultures of Muller Cells and Retinal Ganglion Cells

    DEFF Research Database (Denmark)

    Skytt, D. M.; Toft-Kehler, A. K.; Braendstrup, C. T.;

    2016-01-01

    Glia-neuron partnership is important for inner retinal homeostasis and any disturbances may result in retinal ganglion cell (RGC) death. Müller cells support RGCs with essential functions such as removing excess glutamate and providing energy sources. The aim was to explore the impact of Müller c...

  17. Thyroid Hormone-Otx2 Signaling Is Required for Embryonic Ventral Midbrain Neural Stem Cells Differentiated into Dopamine Neurons.

    Science.gov (United States)

    Chen, Chunhai; Ma, Qinglong; Chen, Xiaowei; Zhong, Min; Deng, Ping; Zhu, Gang; Zhang, Yanwen; Zhang, Lei; Yang, Zhiqi; Zhang, Kuan; Guo, Lu; Wang, Liting; Yu, Zhengping; Zhou, Zhou

    2015-08-01

    Midbrain dopamine (DA) neurons are essential for maintaining multiple brain functions. These neurons have also been implicated in relation with diverse neurological disorders. However, how these neurons are developed from neuronal stem cells (NSCs) remains largely unknown. In this study, we provide both in vivo and in vitro evidence that the thyroid hormone, an important physiological factor for brain development, promotes DA neuron differentiation from embryonic ventral midbrain (VM) NSCs. We find that thyroid hormone deficiency during development reduces the midbrain DA neuron number, downregulates the expression of tyrosine hydroxylase (TH) and the dopamine transporter (DAT), and impairs the DA neuron-dependent motor behavior. In addition, thyroid hormone treatment during VM NSC differentiation in vitro increases the production of DA neurons and upregulates the expression of TH and DAT. We also found that the thyroid hormone enhances the expression of Otx2, an important determinant of DA neurogenesis, during DA neuron differentiation. Our in vitro gene silencing experiments indicate that Otx2 is required for thyroid hormone-dependent DA neuron differentiation from embryonic VM NSCs. Finally, we revealed both in vivo and in vitro that the thyroid hormone receptor alpha 1 is expressed in embryonic VM NSCs. Furthermore, it participates in the effects of thyroid hormone-induced Otx2 upregulation and DA neuron differentiation. These data demonstrate the role and molecular mechanisms of how the thyroid hormone regulates DA neuron differentiation from embryonic VM NSCs, particularly providing new mechanisms and a potential strategy for generating dopamine neurons from NSCs.

  18. Automated computation of arbor densities: a step toward identifying neuronal cell types.

    Science.gov (United States)

    Sümbül, Uygar; Zlateski, Aleksandar; Vishwanathan, Ashwin; Masland, Richard H; Seung, H Sebastian

    2014-01-01

    The shape and position of a neuron convey information regarding its molecular and functional identity. The identification of cell types from structure, a classic method, relies on the time-consuming step of arbor tracing. However, as genetic tools and imaging methods make data-driven approaches to neuronal circuit analysis feasible, the need for automated processing increases. Here, we first establish that mouse retinal ganglion cell types can be as precise about distributing their arbor volumes across the inner plexiform layer as they are about distributing the skeletons of the arbors. Then, we describe an automated approach to computing the spatial distribution of the dendritic arbors, or arbor density, with respect to a global depth coordinate based on this observation. Our method involves three-dimensional reconstruction of neuronal arbors by a supervised machine learning algorithm, post-processing of the enhanced stacks to remove somata and isolate the neuron of interest, and registration of neurons to each other using automatically detected arbors of the starburst amacrine interneurons as fiducial markers. In principle, this method could be generalizable to other structures of the CNS, provided that they allow sparse labeling of the cells and contain a reliable axis of spatial reference.

  19. Cholinergic neuronal differentiation of bone marrow mesenchymal stem cells in rhesus monkeys

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    The purpose of the present study was to determine the best cholinergic neuronal differentiation method of rhesus monkey bone marrow mesenchymal stem cells(BMSCs).Four methods were used to induce differentiation,and the groups were assigned accordingly:basal inducing group(culture media,bFGF,and forskolin);SHH inducing group(SHH,inducing group);RA inducing group(RA,basal inducing group);and SHH+RA inducing group(SHH,RA,and basal inducing group).All groups displayed neuronal morphology and increased expression of nestin and neuron-specific enolase.The basal inducing group did not express synapsin,and cells from the SHH inducing group did not exhibit neuronal resting membrane potential.In contrast,results demonstrated that BMSCs from the RA and SHH+RA inducing groups exhibited neuronal resting membrane potential,and cells from the SHH+RA inducing group expressed higher levels of synapsin and acetylcholine.In conclusion,the induction of cholinergic differentiation through SHH+RA was determined to be superior to the other methods.

  20. Automated computation of arbor densities: a step toward identifying neuronal cell types

    Directory of Open Access Journals (Sweden)

    Uygar eSümbül

    2014-11-01

    Full Text Available The shape and position of a neuron convey information regarding its molecular and functional identity. The identification of cell types from structure, a classic method, relies on the time-consuming step of arbor tracing. However, as genetic tools and imaging methods make data-driven approaches to neuronal circuit analysis feasible, the need for automated processing increases. Here, we first establish that mouse retinal ganglion cell types can be as precise about distributing their arbor volumes across the inner plexiform layer as they are about distributing the skeletons of the arbors. Then, we describe an automated approach to computing the spatial distribution of the dendritic arbors, or arbor density, with respect to a global depth coordinate based on this observation. Our method involves three-dimensional reconstruction of neuronal arbors by a supervised machine learning algorithm, post-processing of the enhanced stacks to remove somata and isolate the neuron of interest, and registration of neurons to each other using automatically detected arbors of the starburst amacrine interneurons as fiducial markers. In principle, this method could be generalizable to other structures of the CNS, provided that they allow sparse labeling of the cells and contain a reliable axis of spatial reference.

  1. Induced Pluripotent Stem Cell Models of Progranulin-Deficient Frontotemporal Dementia Uncover Specific Reversible Neuronal Defects

    Directory of Open Access Journals (Sweden)

    Sandra Almeida

    2012-10-01

    Full Text Available The pathogenic mechanisms of frontotemporal dementia (FTD remain poorly understood. Here we generated multiple induced pluripotent stem cell lines from a control subject, a patient with sporadic FTD, and an FTD patient with a novel heterozygous GRN mutation (progranulin [PGRN] S116X. In neurons and microglia differentiated from PGRN S116X induced pluripotent stem cells, the levels of intracellular and secreted PGRN were reduced, establishing patient-specific cellular models of PGRN haploinsufficiency. Through a systematic screen of inducers of cellular stress, we found that PGRN S116X neurons, but not sporadic FTD neurons, exhibited increased sensitivity to staurosporine and other kinase inhibitors. Moreover, the serine/threonine kinase S6K2, a component of the phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways, was specifically downregulated in PGRN S116X neurons. Both increased sensitivity to kinase inhibitors and reduced S6K2 were rescued by PGRN expression. Our findings identify cell-autonomous, reversible defects in patient neurons with PGRN deficiency, and provide a compelling model for studying PGRN-dependent pathogenic mechanisms and testing potential therapies.

  2. The effect of cell size and channel density on neuronal information encoding and energy efficiency.

    Science.gov (United States)

    Sengupta, Biswa; Faisal, A Aldo; Laughlin, Simon B; Niven, Jeremy E

    2013-09-01

    Identifying the determinants of neuronal energy consumption and their relationship to information coding is critical to understanding neuronal function and evolution. Three of the main determinants are cell size, ion channel density, and stimulus statistics. Here we investigate their impact on neuronal energy consumption and information coding by comparing single-compartment spiking neuron models of different sizes with different densities of stochastic voltage-gated Na(+) and K(+) channels and different statistics of synaptic inputs. The largest compartments have the highest information rates but the lowest energy efficiency for a given voltage-gated ion channel density, and the highest signaling efficiency (bits spike(-1)) for a given firing rate. For a given cell size, our models revealed that the ion channel density that maximizes energy efficiency is lower than that maximizing information rate. Low rates of small synaptic inputs improve energy efficiency but the highest information rates occur with higher rates and larger inputs. These relationships produce a Law of Diminishing Returns that penalizes costly excess information coding capacity, promoting the reduction of cell size, channel density, and input stimuli to the minimum possible, suggesting that the trade-off between energy and information has influenced all aspects of neuronal anatomy and physiology.

  3. Human embryonic stem cell-derived neurons as a tool for studying neuroprotection and neurodegeneration

    OpenAIRE

    Hardingham, G. E.; Patani, R; Baxter, P.; Wyllie, David; Chandran, S

    2010-01-01

    The capacity to generate myriad differentiated cell types, including neurons, from human embryonic stem (hES) cell lines offers great potential for developing cell-based therapies and also for increasing our understanding of human developmental mechanisms. In addition, the emerging development of this technology as an experimental tool represents a potential opportunity for neuroscientists interested in mechanisms of neuroprotection and neurodegeneration. Potentially unlimited generation of w...

  4. Monosynaptic glutamatergic activation of locus coeruleus and other lower brainstem noradrenergic neurons by the C1 cells in mice.

    Science.gov (United States)

    Holloway, Benjamin B; Stornetta, Ruth L; Bochorishvili, Genrieta; Erisir, Alev; Viar, Kenneth E; Guyenet, Patrice G

    2013-11-27

    The C1 neurons, located in the rostral ventrolateral medulla (VLM), are activated by pain, hypotension, hypoglycemia, hypoxia, and infection, as well as by psychological stress. Prior work has highlighted the ability of these neurons to increase sympathetic tone, hence peripheral catecholamine release, probably via their direct excitatory projections to sympathetic preganglionic neurons. In this study, we use channelrhodopsin-2 (ChR2) optogenetics to test whether the C1 cells are also capable of broadly activating the brain's noradrenergic system. We selectively expressed ChR2(H134R) in rostral VLM catecholaminergic neurons by injecting Cre-dependent adeno-associated viral vectors into the brain of adult dopamine-β-hydroxylase (DβH)(Cre/0) mice. Most ChR2-expressing VLM neurons (75%) were immunoreactive for phenylethanolamine N-methyl transferease, thus were C1 cells, and most of the ChR2-positive axonal varicosities were immunoreactive for vesicular glutamate transporter-2 (78%). We produced light microscopic evidence that the axons of rostral VLM (RVLM) catecholaminergic neurons contact locus coeruleus, A1, and A2 noradrenergic neurons, and ultrastructural evidence that these contacts represent asymmetric synapses. Using optogenetics in tissue slices, we show that RVLM catecholaminergic neurons activate the locus coeruleus as well as A1 and A2 noradrenergic neurons monosynaptically by releasing glutamate. In conclusion, activation of RVLM catecholaminergic neurons, predominantly C1 cells, by somatic or psychological stresses has the potential to increase the firing of both peripheral and central noradrenergic neurons.

  5. Beneficial effects of BV2 cell on proliferation and neuron-differentiating of mesenchymal stem cells in the circumstance of injured PC12 cell supernatant

    Institute of Scientific and Technical Information of China (English)

    Xiao-Guang LUO; Hong WANG; Jin ZHOU; Rong YAN; Zhe WU; Chao-Dong ZHANG; Qiu-Shuang WANG

    2006-01-01

    Objective The microglias is the representative of immune cells in the brain. It plays dual roles of both repairing and damaging in injured nervous system, and works as an inevitable component of the circumstance of injured neurons. This study was aiming at the effects of the microglias on the biological activities of mesenchymal stem cells (MSCs) inthe circumstance of injured neurons. Methods MSCs were obtained by primary culture. We adopted PC12 cells (PC12) and BV2 cells (BV2) to substitute for neurons and microglias, respectively. PC12 were injured by aged Aβ1-40 and the supernatant of the injured PC12 was used to set up the circumstance of injured neurons. Transwells were used for co-culture of BV2 and MSCs, which allowed the independent detection of cells after co-culture. Immunofluorescence was used to identify MSCs and neuron-differentiating cells with CD44 and neuron specific enolase (NSE) staining, respectively. MTT assay was adopted to measure the proliferation. Results In the circumstance of both BV2 presence and injured PC12 supernatant incubation, either the proliferation or the differentiation of MSCs reached the highest, which seemed to be contradictory, but we gave our explanations. With the BV2 co-culture, the proliferation of MSCs tend to be higher, but the neuron-differentiating MSCs were similar to those incubated without BV2 co-culture either in normal or injured in PC12 supernatant. With the incubation of injured PC12 supernatant, the neuron-differentiating cells were significantly higher than that of control (P < 0.05). Conclusion In the circumstance of injured neurons, microlgias tend to promote the MSCs proliferation. Although not helpful in neuron-differentiating, microglias did not exert any negative effect either.

  6. Sleep-active neuronal nitric oxide synthase-positive cells of the cerebral cortex: a local regulator of sleep?

    OpenAIRE

    Wisor, Jonathan P.; Gerashchenko, Dmitry; Kilduff, Thomas S.

    2011-01-01

    Our recent report demonstrated that a small subset of GABAergic interneurons in the cerebral cortex of rodents expresses Fos protein, a marker for neuronal activity, during slow wave sleep (Gerashchenko et al., 2008). The population of sleep-active neurons consists of strongly immunohistochemically-stained cells for the enzyme neuronal nitric oxide synthase. By virtue of their widespread localization within the cerebral cortex and their widespread projections to other cortical cell types, cor...

  7. Stem cell therapy and cellular engineering for treatment of neuronal dysfunction in Huntington's disease.

    Science.gov (United States)

    Choi, Kyung-Ah; Hwang, Insik; Park, Hang-soo; Oh, Seung-Ick; Kang, Seongman; Hong, Sunghoi

    2014-07-01

    Huntington's disease (HD) is a fatal inherited neurodegenerative disorder characterized by progressive loss of neurons in the striatum, a sub-cortical region of the forebrain. The sub-cortical region of the forebrain is associated with the control of movement and behavior, thus HD initially presents with coordination difficulty and cognitive decline. Recent reprogramming technologies, including induced pluripotent stem cells (iPSCs) and induced neural stem cells (iNSCs), have created opportunities to understand the pathological cascades that underlie HD and to develop new treatments for this currently incurable neurological disease. The ultimate objectives of stem cell-based therapies for HD are to replace lost neurons and to prevent neuronal dysfunction and death. In this review, we examine the current understanding of the molecular and pathological mechanisms involved in HD. We discuss disease modeling with HD-iPSCs derived from the somatic cells of patients, which could provide an invaluable platform for understanding HD pathogenesis. We speculate about the benefits and drawbacks of using iNSCs as an alternative stem cell source for HD treatment. Finally, we discuss cell culture and engineering systems that promote the directed differentiation of pluripotent stem cell-derived NSCs into a striatal DARPP32(+) GABAergic MSN phenotype for HD. In conclusion, this review summarizes the potentials of cell reprogramming and engineering technologies relevant to the development of cell-based therapies for HD.

  8. Differentiation factors that influence neuronal markers expression in vitro from human amniotic epithelial cells

    Directory of Open Access Journals (Sweden)

    H Niknejad

    2010-01-01

    Full Text Available The differentiation of neural cells from embryonic stem cells is influenced by several growth factors. Amniotic epithelial cells (AECs share many of the same characteristics as embryonic stem cells, and therefore those factors may similarly affect the derivation of neural cells from AECs. In this study, we examined the differentiation of neural cells in vitro from AECs following AECs treatment with retinoic acid (RA, basic fibroblast growth factor (bFGF as well as investigation of bFGF withdrawal on neuronal differentiation. We also studied whether blocking bone morphogenetic protein (BMP signaling using its antagonist, noggin, affects the derivation of neuronal cells from AECs. The effects of serum on the rate of neural markers expression were also examined. Analysis of AECs derived neurons was performed at some neural markers expression level by immunocytochemistry. All cultures treated with noggin showed the higher levels of neural markers expression than noggin free cultures. Combined treatment with bFGF and RA showed the highest level of neural markers in all treatment groups with or without noggin. bFGF withdrawal did not promote expression of neural markers, while its maintenance increased the expression of these markers. Serum-free condition decreased the viability of cells but increased the rate of neural markers expression. These results show the capability of AECs to express neural cell markers and this ability is affected by some factors including serum, noggin, bFGF and RA.

  9. pSVPoMcat modifying Schwann cell to protect injured spinal neurons in rats

    Institute of Scientific and Technical Information of China (English)

    陈礼刚; 高立达; 朴永旭; 毛伯镛; 曾凡俊

    2002-01-01

    Objective: To investigate the protective effect of pSVPoMcat (myelin basic protein microgene)modifying Schwann cell on injured spinal neurons.Methods: A model of rat spinal cord injured by hemisection was used. One hundred and twenty healthy SD rats of both sexes weighing 250-300 g were divided into three groups: Group A (n=40, treated with implantation of pSVPoMcat modifying Schwann cell), Group B (n= 40, treated with implantation of Schwann cell only) and Group C (n=400, treated with sham operation as the control). One week after operation the rat functional recovery was observed dynamically by using combined behavioral score (CBS) and cortical somatasensory evoked potentials, the spinal cord sections were stained by Nissl, acid phosphatase enzyme histochemistry and cell apoptosis was examined by methye green, terminal deoxynucleotidyl and the dUTP Nick end labeling technique. Quantitative analysis was done by computer image analysis system.Results: In Group A the injured neurons recovered well morphologically. The imaging analysis showed a result of Group A>Group B>Group C in the size of the neurons (P<0.01). The percentage of ACP (acid phosphatase) stained area and the rate of apoptosis sequence were groups Acell implantation has protective effect on injured spinal neurons and promotes recovery of injured spinal cord function in rats.

  10. Development of Gonadotropin-Releasing Hormone-Secreting Neurons from Human Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Carina Lund

    2016-08-01

    Full Text Available Gonadotropin-releasing hormone (GnRH neurons regulate human puberty and reproduction. Modeling their development and function in vitro would be of interest for both basic research and clinical translation. Here, we report a three-step protocol to differentiate human pluripotent stem cells (hPSCs into GnRH-secreting neurons. Firstly, hPSCs were differentiated to FOXG1, EMX2, and PAX6 expressing anterior neural progenitor cells (NPCs by dual SMAD inhibition. Secondly, NPCs were treated for 10 days with FGF8, which is a key ligand implicated in GnRH neuron ontogeny, and finally, the cells were matured with Notch inhibitor to bipolar TUJ1-positive neurons that robustly expressed GNRH1 and secreted GnRH decapeptide into the culture medium. The protocol was reproducible both in human embryonic stem cells and induced pluripotent stem cells, and thus provides a translational tool for investigating the mechanisms of human puberty and its disorders.

  11. Cutting edge: neuronal recognition by CD8 T cells elicits central diabetes insipidus.

    Science.gov (United States)

    Scheikl, Tanja; Pignolet, Béatrice; Dalard, Cécile; Desbois, Sabine; Raison, Danièle; Yamazaki, Masanori; Saoudi, Abdelhadi; Bauer, Jan; Lassmann, Hans; Hardin-Pouzet, Hélène; Liblau, Roland S

    2012-05-15

    An increasing number of neurologic diseases is associated with autoimmunity. The immune effectors contributing to the pathogenesis of such diseases are often unclear. To explore whether self-reactive CD8 T cells could attack CNS neurons in vivo, we generated a mouse model in which the influenza virus hemagglutinin (HA) is expressed specifically in CNS neurons. Transfer of cytotoxic anti-HA CD8 T cells induced an acute but reversible encephalomyelitis in HA-expressing recipient mice. Unexpectedly, diabetes insipidus developed in surviving animals. This robust phenotype was associated with preferential accumulation of cytotoxic CD8 T cells in the hypothalamus, upregulation of MHC class I molecules, and destruction of vasopressin-expressing neurons. IFN-γ production by the pathogenic CD8 T cells was necessary for MHC class I upregulation by hypothalamic neurons and their destruction. This novel mouse model, in combination with related human data, supports the concept that autoreactive CD8 T cells can trigger central diabetes insipidus. PMID:22504649

  12. Development of Gonadotropin-Releasing Hormone-Secreting Neurons from Human Pluripotent Stem Cells.

    Science.gov (United States)

    Lund, Carina; Pulli, Kristiina; Yellapragada, Venkatram; Giacobini, Paolo; Lundin, Karolina; Vuoristo, Sanna; Tuuri, Timo; Noisa, Parinya; Raivio, Taneli

    2016-08-01

    Gonadotropin-releasing hormone (GnRH) neurons regulate human puberty and reproduction. Modeling their development and function in vitro would be of interest for both basic research and clinical translation. Here, we report a three-step protocol to differentiate human pluripotent stem cells (hPSCs) into GnRH-secreting neurons. Firstly, hPSCs were differentiated to FOXG1, EMX2, and PAX6 expressing anterior neural progenitor cells (NPCs) by dual SMAD inhibition. Secondly, NPCs were treated for 10 days with FGF8, which is a key ligand implicated in GnRH neuron ontogeny, and finally, the cells were matured with Notch inhibitor to bipolar TUJ1-positive neurons that robustly expressed GNRH1 and secreted GnRH decapeptide into the culture medium. The protocol was reproducible both in human embryonic stem cells and induced pluripotent stem cells, and thus provides a translational tool for investigating the mechanisms of human puberty and its disorders. PMID:27426041

  13. Translational repression determines a neuronal potential in Drosophila asymmetric cell division.

    Science.gov (United States)

    Okabe, M; Imai, T; Kurusu, M; Hiromi, Y; Okano, H

    2001-05-01

    Asymmetric cell division is a fundamental strategy for generating cellular diversity during animal development. Daughter cells manifest asymmetry in their differential gene expression. Transcriptional regulation of this process has been the focus of many studies, whereas cell-type-specific 'translational' regulation has been considered to have a more minor role. During sensory organ development in Drosophila, Notch signalling directs the asymmetry between neuronal and non-neuronal lineages, and a zinc-finger transcriptional repressor Tramtrack69 (TTK69) acts downstream of Notch as a determinant of non-neuronal identity. Here we show that repression of TTK69 protein expression in the neuronal lineage occurs translationally rather than transcriptionally. This translational repression is achieved by a direct interaction between cis-acting sequences in the 3' untranslated region of ttk69 messenger RNA and its trans-acting repressor, the RNA-binding protein Musashi (MSI). Although msi can act downstream of Notch, Notch signalling does not affect MSI expression. Thus, Notch signalling is likely to regulate MSI activity rather than its expression. Our results define cell-type-specific translational control of ttk69 by MSI as a downstream event of Notch signalling in asymmetric cell division.

  14. Neuronal chemokines : Versatile messengers in central nervous system cell interaction

    NARCIS (Netherlands)

    de Haas, A. H.; van Weering, H. R. J.; de Jong, E. K.; Boddeke, H. W. G. M.; Biber, K. P. H.

    2007-01-01

    Whereas chemokines are well known for their ability to induce cell migration, only recently it became evident that chemokines also control a variety of other cell functions and are versatile messengers in the interaction between a diversity of cell types. In the central nervous system (CNS), chemoki

  15. Tyrosinase-Expressing Neuronal Cell Line as in Vitro Model of Parkinson’s Disease

    Directory of Open Access Journals (Sweden)

    Takafumi Hasegawa

    2010-03-01

    Full Text Available Oxidized metabolites of dopamine known as dopamine quinone derivatives are thought to play a pivotal role in the degeneration of nigrostriatal dopaminergic neurons in Parkinson’s disease. Although such quinone derivatives are usually produced via the autoxidation of catecholamines, tyrosinase, which is a key enzyme in melanin biosynthesis via the production of DOPA and subsequent molecules, can potentially accelerate the induction of catecholamine quinone derivatives by its oxidase activity. We have developed neuronal cell lines in which the expression of human tyrosinase was inducible. Overexpression of tyrosinase resulted in increased intracellular dopamine content in association with the formation of melanin pigments in neuronal somata, which eventually causes apoptotic cell death. This cellular model will provide a useful tool for detailed analyses of the neurotoxicity of oxidized catechol metabolites.

  16. Effects of cerebrolysin on motor-neuron-like NSC-34 cells

    International Nuclear Information System (INIS)

    Although the peripheral nervous system is capable of regeneration, this capability is limited. As a potential means of augmenting nerve regeneration, the effects of cerebrolysin (CL) – a proteolytic peptide fraction – were tested in vitro on the motor-neuron-like NSC-34 cell line and organotypic spinal cord cultures. Therefore, NSC-34 cells were subjected to mechanical stress by changing media and metabolic stress by oxygen glucose deprivation. Afterwards, cell survival/proliferation using MTT and BrdU-labeling (FACS) and neurite sprouting using ImageJ analysis were evaluated. Calpain-1, Src and α-spectrin protein expression were analyzed by Western blot. In organotypic cultures, the effect of CL on motor neuron survival and neurite sprouting was tested by immunohistochemistry. CL had a temporary anti-proliferative but initially neuroprotective effect on OGD-stressed NSC-34 cells. High-dosed or repeatedly applied CL was deleterious for cell survival. CL amplified neurite reconstruction to limited extent, affected calpain-1 protein expression and influenced calpain-mediated spectrin cleavage as a function of Src expression. In organotypic spinal cord slice cultures, CL was not able to support motor neuron survival/neurite sprouting. Moreover, it hampered astroglia and microglia activities. The data suggest that CL may have only isolated positive effects on injured spinal motor neurons. High-dosed or accumulated CL seemed to have adverse effects in treatment of spinal cord injury. Further experiments are required to optimize the conditions for a safe clinical administration of CL in spinal cord injuries. - Highlights: • Cerebrolysin (CL) is anti-proliferative but initially neuroprotective in OGD-stressed NSC-34 cells. • CL amplified neurite reconstruction of NSC-34 cells. • CL affected calpain-1 expression and calpain-mediated spectrin cleavage as function of Src expression. • In organotypic spinal cord cultures, CL hampered motor neuron survival and

  17. Effects of cerebrolysin on motor-neuron-like NSC-34 cells

    Energy Technology Data Exchange (ETDEWEB)

    Keilhoff, Gerburg, E-mail: Gerburg.keilhoff@med.ovgu.de [Institute of Biochemistry and Cell Biology, Otto-von-Guericke University Magdeburg, Leipziger Str. 44, D-39120 Magdeburg (Germany); Lucas, Benjamin; Pinkernelle, Josephine; Steiner, Michael [Institute of Biochemistry and Cell Biology, Otto-von-Guericke University Magdeburg, Leipziger Str. 44, D-39120 Magdeburg (Germany); Fansa, Hisham [Department of Plastic, Reconstructive and Aesthetic Surgery, Hand Surgery, Klinikum Bielefeld, Teutoburger Str. 50, D-33604 Bielefeld (Germany)

    2014-10-01

    Although the peripheral nervous system is capable of regeneration, this capability is limited. As a potential means of augmenting nerve regeneration, the effects of cerebrolysin (CL) – a proteolytic peptide fraction – were tested in vitro on the motor-neuron-like NSC-34 cell line and organotypic spinal cord cultures. Therefore, NSC-34 cells were subjected to mechanical stress by changing media and metabolic stress by oxygen glucose deprivation. Afterwards, cell survival/proliferation using MTT and BrdU-labeling (FACS) and neurite sprouting using ImageJ analysis were evaluated. Calpain-1, Src and α-spectrin protein expression were analyzed by Western blot. In organotypic cultures, the effect of CL on motor neuron survival and neurite sprouting was tested by immunohistochemistry. CL had a temporary anti-proliferative but initially neuroprotective effect on OGD-stressed NSC-34 cells. High-dosed or repeatedly applied CL was deleterious for cell survival. CL amplified neurite reconstruction to limited extent, affected calpain-1 protein expression and influenced calpain-mediated spectrin cleavage as a function of Src expression. In organotypic spinal cord slice cultures, CL was not able to support motor neuron survival/neurite sprouting. Moreover, it hampered astroglia and microglia activities. The data suggest that CL may have only isolated positive effects on injured spinal motor neurons. High-dosed or accumulated CL seemed to have adverse effects in treatment of spinal cord injury. Further experiments are required to optimize the conditions for a safe clinical administration of CL in spinal cord injuries. - Highlights: • Cerebrolysin (CL) is anti-proliferative but initially neuroprotective in OGD-stressed NSC-34 cells. • CL amplified neurite reconstruction of NSC-34 cells. • CL affected calpain-1 expression and calpain-mediated spectrin cleavage as function of Src expression. • In organotypic spinal cord cultures, CL hampered motor neuron survival and

  18. Quantitative glycomics monitoring of induced pluripotent- and embryonic stem cells during neuronal differentiation

    Directory of Open Access Journals (Sweden)

    Michiyo Terashima

    2014-11-01

    Full Text Available Alterations in the structure of cell surface glycoforms occurring during the stages of stem cell differentiation remain unclear. We describe a rapid glycoblotting-based cellular glycomics method for quantitatively evaluating changes in glycoform expression and structure during neuronal differentiation of murine induced pluripotent stem cells (iPSCs and embryonic stem cells (ESCs. Our results show that changes in the expression of cellular N-glycans are comparable during the differentiation of iPSCs and ESCs. The expression of bisect-type N-glycans was significantly up-regulated in neurons that differentiated from both iPSCs and ESCs. From a glycobiological standpoint, iPSCs are an alternative neural cell source in addition to ESCs.

  19. Herpes simplex virus-mediated human hypoxanthine-guanine phosphoribosyltransferase gene transfer into neuronal cells

    Energy Technology Data Exchange (ETDEWEB)

    Palella, T.D.; Silverman, L.J.; Schroll, C.T.; Homa, F.L.; Levine, M.; Kelley, W.N.

    1988-01-01

    The virtually complete deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) results in a devastating neurological disease, Lesch-Nyhan syndrome. Transfer of the HPRT gene into fibroblasts and lymphoblasts in vitro and into hematopoietic cells in vivo has been accomplished by other groups with retroviral-derived vectors. It appears to be necessary, however, to transfer the HPRT gene into neuronal cells to correct the neurological dysfunction of this disorder. The neurotropic virus herpes simplex virus type 1 has features that make it suitable for use as a vector to transfer the HPRT gene into neuronal tissue. This report describes the isolation of an HPRT-deficient rat neuroma cell line, designated B103-4C, and the construction of a recombinant herpes simplex virus type 1 that contained human HPRT cDNA. These recombinant viruses were used to infect B103-4C cells. Infected cells expressed HPRT activity which was human in origin.

  20. ALS/FTLD-linked TDP-43 regulates neurite morphology and cell survival in differentiated neurons

    Energy Technology Data Exchange (ETDEWEB)

    Han, Jeong-Ho; Yu, Tae-Hoon; Ryu, Hyun-Hee; Jun, Mi-Hee; Ban, Byung-Kwan [Department of Biotechnology, College of Life Science and Nanotechnology, Hannam University, Dajeon 305-811 (Korea, Republic of); Jang, Deok-Jin [Department of Applied Biology, College of Ecology and Environment, Kyungpook National University, 386, Gajang-dong, Sangju-si, Kyungbuk 742-711 (Korea, Republic of); Lee, Jin-A, E-mail: leeja@hnu.kr [Department of Biotechnology, College of Life Science and Nanotechnology, Hannam University, Dajeon 305-811 (Korea, Republic of)

    2013-08-01

    Tar-DNA binding protein of 43 kDa (TDP-43) has been characterized as a major component of protein aggregates in brains with neurodegenerative diseases such as frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). However, physiological roles of TDP-43 and early cellular pathogenic effects caused by disease associated mutations in differentiated neurons are still largely unknown. Here, we investigated the physiological roles of TDP-43 and the effects of missense mutations associated with diseases in differentiated cortical neurons. The reduction of TDP-43 by siRNA increased abnormal neurites and decreased cell viability. ALS/FTLD-associated missense mutant proteins (A315T, Q331K, and M337V) were partially mislocalized to the cytosol and neurites when compared to wild-type and showed abnormal neurites similar to those observed in cases of loss of TDP-43. Interestingly, cytosolic expression of wild-type TDP-43 with mutated nuclear localization signals also induced abnormal neurtie morphology and reduction of cell viability. However, there was no significant difference in the effects of cytosolic expression in neuronal morphology and cell toxicity between wild-type and missense mutant proteins. Thus, our results suggest that mislocalization of missense mutant TDP-43 may contribute to loss of TDP-43 function and affect neuronal morphology, probably via dominant negative action before severe neurodegeneration in differentiated cortical neurons. Highlights: • The function of nuclear TDP-43 in neurite morphology in mature neurons. • Partial mislocalization of TDP-43 missense mutants into cytosol from nucleus. • Abnormal neurite morphology caused by missense mutants of TDP-43. • The effect of cytosolic expression of TDP-43 in neurite morphology and in cell survival.

  1. Functional integration of grafted neural stem cell-derived dopaminergic neurons monitored by optogenetics in an in vitro Parkinson model.

    Directory of Open Access Journals (Sweden)

    Jan Tønnesen

    Full Text Available Intrastriatal grafts of stem cell-derived dopamine (DA neurons induce behavioral recovery in animal models of Parkinson's disease (PD, but how they functionally integrate in host neural circuitries is poorly understood. Here, Wnt5a-overexpressing neural stem cells derived from embryonic ventral mesencephalon of tyrosine hydroxylase-GFP transgenic mice were expanded as neurospheres and transplanted into organotypic cultures of wild type mouse striatum. Differentiated GFP-labeled DA neurons in the grafts exhibited mature neuronal properties, including spontaneous firing of action potentials, presence of post-synaptic currents, and functional expression of DA D₂ autoreceptors. These properties resembled those recorded from identical cells in acute slices of intrastriatal grafts in the 6-hydroxy-DA-induced mouse PD model and from DA neurons in intact substantia nigra. Optogenetic activation or inhibition of grafted cells and host neurons using channelrhodopsin-2 (ChR2 and halorhodopsin (NpHR, respectively, revealed complex, bi-directional synaptic interactions between grafted cells and host neurons and extensive synaptic connectivity within the graft. Our data demonstrate for the first time using optogenetics that ectopically grafted stem cell-derived DA neurons become functionally integrated in the DA-denervated striatum. Further optogenetic dissection of the synaptic wiring between grafted and host neurons will be crucial to clarify the cellular and synaptic mechanisms underlying behavioral recovery as well as adverse effects following stem cell-based DA cell replacement strategies in PD.

  2. Functional integration of grafted neural stem cell-derived dopaminergic neurons monitored by optogenetics in an in vitro Parkinson model.

    Science.gov (United States)

    Tønnesen, Jan; Parish, Clare L; Sørensen, Andreas T; Andersson, Angelica; Lundberg, Cecilia; Deisseroth, Karl; Arenas, Ernest; Lindvall, Olle; Kokaia, Merab

    2011-03-04

    Intrastriatal grafts of stem cell-derived dopamine (DA) neurons induce behavioral recovery in animal models of Parkinson's disease (PD), but how they functionally integrate in host neural circuitries is poorly understood. Here, Wnt5a-overexpressing neural stem cells derived from embryonic ventral mesencephalon of tyrosine hydroxylase-GFP transgenic mice were expanded as neurospheres and transplanted into organotypic cultures of wild type mouse striatum. Differentiated GFP-labeled DA neurons in the grafts exhibited mature neuronal properties, including spontaneous firing of action potentials, presence of post-synaptic currents, and functional expression of DA D₂ autoreceptors. These properties resembled those recorded from identical cells in acute slices of intrastriatal grafts in the 6-hydroxy-DA-induced mouse PD model and from DA neurons in intact substantia nigra. Optogenetic activation or inhibition of grafted cells and host neurons using channelrhodopsin-2 (ChR2) and halorhodopsin (NpHR), respectively, revealed complex, bi-directional synaptic interactions between grafted cells and host neurons and extensive synaptic connectivity within the graft. Our data demonstrate for the first time using optogenetics that ectopically grafted stem cell-derived DA neurons become functionally integrated in the DA-denervated striatum. Further optogenetic dissection of the synaptic wiring between grafted and host neurons will be crucial to clarify the cellular and synaptic mechanisms underlying behavioral recovery as well as adverse effects following stem cell-based DA cell replacement strategies in PD.

  3. Human primordial germ cell-derived progenitors give rise to neurons and glia in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Teng, Yincheng [Department of Gynecology and Obstetrics, The 6th People' s Hospital, School of Medicine, Shanghai Jiao Tong University, 600 Yishan Road, Shanghai 200233 (China); Chen, Bin [Center for Developmental Biology, Xinhua Hospital, School of Medicine, Shanghai Jiao Tong University, 1665 Kong Jiang Road, Shanghai 200092 (China); Tao, Minfang, E-mail: Taomf@126.com [Department of Gynecology and Obstetrics, The 6th People' s Hospital, School of Medicine, Shanghai Jiao Tong University, 600 Yishan Road, Shanghai 200233 (China)

    2009-12-18

    We derived a cell population from cultured human primordial germ cells from early human embryos. The derivates, termed embryoid body-derived (EBD) cells, displayed an extensive capacity for proliferation and expressed a panel of markers in all three germ layers. Interestingly, EBD cells were also positive for markers of neural stem/progenitor cells, such as nestin and glial fibrillary acidic protein. When these cells were transplanted into the brain cavities of fetal sheep and postnatal NOD-SCID mice or nerve-degenerated tibialis anterior muscles, they readily gave rise to neurons or glial cells. To our knowledge, our data are the first to demonstrate that EBD cells can undergo further neurogenesis under suitable environments in vivo. Hence, with the abilities of extensive expansion, self-renewal, and differentiation, EBD cells may provide a useful donor source for neural stem/progenitor cells to be used in cell-replacement therapies for diseases of the nervous system.

  4. Transient increase in neuronal chloride concentration by neuroactive amino acids released from glioma cells

    Directory of Open Access Journals (Sweden)

    Cristina eBertollini

    2012-11-01

    Full Text Available Neuronal chloride concentration ([Cl-]i is known to be dynamically modulated and alterations in Cl- homeostasis may occur in the brain at physiological and pathological conditions, being also likely involved in glioma-related seizures. However, the mechanism leading to changes in neuronal [Cl-]i during glioma invasion are still unclear. To characterize the potential effect of glioma released soluble factors on neuronal [Cl-]i, we used genetically encoded CFP/YFP-based ratiometric Cl-Sensor transiently expressed in cultured hippocampal neurons. Exposition of neurons to glioma conditioned medium (GCM caused rapid and transient elevation of [Cl-]i, resulting in the increase of fluorescence ratio, which was strongly reduced by blockers of ionotropic glutamate receptors APV and NBQX. Furthermore, in HEK cells expressing GluR1-AMPA receptors, GCM activated ionic current with efficacy similar to those caused by glutamate, supporting the notion that GCM contains glutamate or glutamatergic agonists, which cause neuronal depolarization, activation of NMDA and AMPA/KA receptors leading to elevation of [Cl-]i. Chromatographic analysis of the GCM showed that it contained several aminoacids, including glutamate, whose release from glioma cells did not occur via the most common glial mechanisms of transport, or in response to hypoosmotic stress. GCM also contained glycine, whose action contrasted the glutamate effect. Indeed, strychnine application significantly increased GCM-induced depolarization and [Cl-]i rise. GCM-evoked [Cl-]i elevation was not inhibited by antagonists of Cl- transporters and significantly reduced in the presence of anion channels blocker NPPB, suggesting that Cl-selective channels are a major route for GCM-induced Cl- influx. Altogether, these data show that glioma released aminoacids may dynamically alter Cl- equilibrium in surrounding neurons, deeply interfering with their inhibitory balance, likely leading to physiological and

  5. Isolation and Culture of Pig Spermatogonial Stem Cells and Their in Vitro Differentiation into Neuron-Like Cells and Adipocytes

    Directory of Open Access Journals (Sweden)

    Xiaoyan Wang

    2015-11-01

    Full Text Available Spermatogonial stem cells (SSCs renew themselves throughout the life of an organism and also differentiate into sperm in the adult. They are multipopent and therefore, can be induced to differentiate into many cells types in vitro. SSCs from pigs, considered an ideal animal model, are used in studies of male infertility, regenerative medicine, and preparation of transgenic animals. Here, we report on a culture system for porcine SSCs and the differentiation of these cells into neuron-like cells and adipocytes. SSCs and Sertoli cells were isolated from neonatal piglet testis by differential adhesion and SSCs were cultured on a feeder layer of Sertoli cells. Third-generation SSCs were induced to differentiate into neuron-like cells by addition of retinoic acid, β-mercaptoethanol, and 3-isobutyl-1-methylxanthine (IBMX to the induction media and into adipocytes by the addition of hexadecadrol, insulin, and IBMX to the induction media. The differentiated cells were characterized by biochemical staining, qRT-PCR, and immunocytochemistry. The cells were positive for SSC markers, including alkaline phosphatase and SSC-specific genes, consistent with the cells being undifferentiated. The isolated SSCs survived on the Sertoli cells for 15 generations. Karyotyping confirmed that the chromosomal number of the SSCs were normal for pig (2n = 38, n = 19. Pig SSCs were successfully induced into neuron-like cells eight days after induction and into adipocytes 22 days after induction as determined by biochemical and immunocytochemical staining. qPCR results also support this conclusion. The nervous tissue markers genes, Nestin and β-tubulin, were expressed in the neuron-like cells and the adipocyte marker genes, PPARγ and C/EBPα, were expressed in the adipocytes.

  6. Death and survival of neuronal and astrocytic cells in ischemic brain injury: a role of autophagy

    Institute of Scientific and Technical Information of China (English)

    Min XU; Hui-ling ZHANG

    2011-01-01

    Autophagy is a highly regulated cellular mechanism that leads to degradation of long-lived proteins and dysfunctional organelles. The process has been implicated in a variety of physiological and pathological conditions relevant to neurological diseases. Recent studies show the existence of autophagy in cerebral ischemia, but no consensus has yet been reached regarding the functions of autophagy in this condition. This article highlights the activation of autophagy during cerebral ischemia and/or reperfusion, especially in neurons and astrocytes, as well as the role of autophagy in neuronal or astrocytic cell death and survival. We propose that physiological levels of autophagy, presumably caused by mild to modest hypoxia or ischemia, appear to be protective. However, high levels of autophagy caused by severe hypoxia or ischemia and/or reperfusion may cause self-digestion and eventual neuronal and astrocytic cell death. We also discuss that oxidative and endoplasmic reticulum (ER) stresses in cerebral hypoxia or ischemia and/or reperfusion are potent stimuli of autophagy in neurons and astrocytes. In addition, we review the evidence suggesting a considerable overlap between autophagy on one hand, and apoptosis, necrosis and necroptosis on the other hand, in determining the outcomes and final morphology of damaged neurons and astrocytes.

  7. Coupled electrophysiological recording and single cell transcriptome analyses revealed molecular mechanisms underlying neuronal maturation.

    Science.gov (United States)

    Chen, Xiaoying; Zhang, Kunshan; Zhou, Liqiang; Gao, Xinpei; Wang, Junbang; Yao, Yinan; He, Fei; Luo, Yuping; Yu, Yongchun; Li, Siguang; Cheng, Liming; Sun, Yi E

    2016-03-01

    The mammalian brain is heterogeneous, containing billions of neurons and trillions of synapses forming various neural circuitries, through which sense, movement, thought, and emotion arise. The cellular heterogeneity of the brain has made it difficult to study the molecular logic of neural circuitry wiring, pruning, activation, and plasticity, until recently, transcriptome analyses with single cell resolution makes decoding of gene regulatory networks underlying aforementioned circuitry properties possible. Here we report success in performing both electrophysiological and whole-genome transcriptome analyses on single human neurons in culture. Using Weighted Gene Coexpression Network Analyses (WGCNA), we identified gene clusters highly correlated with neuronal maturation judged by electrophysiological characteristics. A tight link between neuronal maturation and genes involved in ubiquitination and mitochondrial function was revealed. Moreover, we identified a list of candidate genes, which could potentially serve as biomarkers for neuronal maturation. Coupled electrophysiological recording and single cell transcriptome analysis will serve as powerful tools in the future to unveil molecular logics for neural circuitry functions. PMID:26883038

  8. Cell surface area regulation in neurons in hippocampal slice cultures is resistant to oxygen-glucose deprivation

    Directory of Open Access Journals (Sweden)

    Natalya Shulyakova

    2010-09-01

    Full Text Available Natalya Shulyakova1,2, Jamie Fong2, Diana Diec2, Adrian Nahirny1,2, Linda R Mills1,21Department of Physiology, University of Toronto, Toronto, ON, Canada, M5T 2S8; 2Toronto Western Hospital Research Institute, University Health Network, 11-430, 399 Bathurst St, Toronto, ON, Canada, M5T 2S8Background: Neurons swell in response to a variety of insults. The capacity to recover, ie, to shrink, is critical for neuronal function and survival. Studies on dissociated neurons have shown that during swelling and shrinking, neurons reorganize their plasma membrane; as neurons swell, in response to hypo-osmotic media, the bilayer area increases. Upon restoration of normo-osmotic media, neurons shrink, forming transient invaginations of the plasma membrane known as vacuole-like dilations (VLDs, to accommodate the decrease in the bilayer.Methods: Here we used confocal microscopy to monitor neuronal swelling and shrinking in the three-dimensional (3D environment of post-natal rat hippocampal slice cultures. To label neurons, we used biolistic transfection, to introduce enhanced green fluorescent protein (eGFP targeted to the cytoplasm; and a membrane targeted GFP (lckGFP, targeted to the plasma membrane.Results: Neurons in slice cultures swelled and shrank in response to hypo-osmotic to normo-osmotic media changes. Oxygen-glucose deprivation (OGD caused sustained neuronal swelling; after reperfusion, some neurons recovered but in others, VLD recovery was stalled. OGD did not impair neuronal capacity to recover from a subsequent osmotic challenge.Conclusion: These results suggest cell surface area regulation (SAR is an intrinsic property of neurons, and that neuronal capacity for SAR may play an important role in the brain’s response to ischemic insults.Keywords: neurons, swelling, ischemia, cell surface area, hippocampal slice culture

  9. Overexpression of glial cell line-derived neurotrophic factor induces genes regulating migration and differentiation of neuronal progenitor cells.

    Science.gov (United States)

    Pahnke, Jens; Mix, Eilhard; Knoblich, Rupert; Müller, Jana; Zschiesche, Marlies; Schubert, Beke; Koczan, Dirk; Bauer, Peter; Böttcher, Tobias; Thiesen, Hans-Jürgen; Lazarov, Ludmil; Wree, Andreas; Rolfs, Arndt

    2004-07-15

    The glial cell line-derived neurotrophic factor (GDNF) is involved in the development and maintenance of neural tissues. Mutations in components of its signaling pathway lead to severe migration deficits of neuronal crest stem cells, tumor formation, or ablation of the urinary system. In animal models of Parkinson's disease, GDNF has been recognized to be neuroprotective and to improve motor function when delivered into the cerebral ventricles or into the substantia nigra. Here, we characterize the network of 43 genes induced by GDNF overproduction of neuronal progenitor cells (ST14A), which mainly regulate migration and differentiation of neuronal progenitor cells. GDNF down-regulates doublecortin, Paf-ah1b (Lis1), dynamin, and alpha-tubulin, which are involved in neocortical lamination and cytoskeletal reorganization. Axonal guidance depends on cell-surface molecules and extracellular matrix proteins. Laminin, Mpl3, Alcam, Bin1, Id1, Id2, Id3, neuregulin1, the ephrinB2-receptor, neuritin, focal adhesion kinase (FAK), Tc10, Pdpk1, clusterin, GTP-cyclooxygenase1, and follistatin are genes up-regulated by GDNF overexpression. Moreover, we found four key enzymes of the cholesterol-synthesis pathway to be down-regulated leading to decreased farnesyl-pyrophospate production. Many proteins are anchored by farnesyl-derivates at the cell membrane. The identification of these GDNF-regulated genes may open new opportunities for directly influencing differentiation and developmental processes of neurons. PMID:15212950

  10. Somatosensory neuron types identified by high-coverage single-cell RNA-sequencing and functional heterogeneity.

    Science.gov (United States)

    Li, Chang-Lin; Li, Kai-Cheng; Wu, Dan; Chen, Yan; Luo, Hao; Zhao, Jing-Rong; Wang, Sa-Shuang; Sun, Ming-Ming; Lu, Ying-Jin; Zhong, Yan-Qing; Hu, Xu-Ye; Hou, Rui; Zhou, Bei-Bei; Bao, Lan; Xiao, Hua-Sheng; Zhang, Xu

    2016-01-01

    Sensory neurons are distinguished by distinct signaling networks and receptive characteristics. Thus, sensory neuron types can be defined by linking transcriptome-based neuron typing with the sensory phenotypes. Here we classify somatosensory neurons of the mouse dorsal root ganglion (DRG) by high-coverage single-cell RNA-sequencing (10 950 ± 1 218 genes per neuron) and neuron size-based hierarchical clustering. Moreover, single DRG neurons responding to cutaneous stimuli are recorded using an in vivo whole-cell patch clamp technique and classified by neuron-type genetic markers. Small diameter DRG neurons are classified into one type of low-threshold mechanoreceptor and five types of mechanoheat nociceptors (MHNs). Each of the MHN types is further categorized into two subtypes. Large DRG neurons are categorized into four types, including neurexophilin 1-expressing MHNs and mechanical nociceptors (MNs) expressing BAI1-associated protein 2-like 1 (Baiap2l1). Mechanoreceptors expressing trafficking protein particle complex 3-like and Baiap2l1-marked MNs are subdivided into two subtypes each. These results provide a new system for cataloging somatosensory neurons and their transcriptome databases.

  11. Neuronal Chemokines: Versatile Messengers In Central Nervous System Cell Interaction

    OpenAIRE

    de Haas, A. H.; van Weering, H. R. J.; Jong, E.K.; Boddeke, H. W. G. M.; Biber, K.P.H.

    2007-01-01

    Whereas chemokines are well known for their ability to induce cell migration, only recently it became evident that chemokines also control a variety of other cell functions and are versatile messengers in the interaction between a diversity of cell types. In the central nervous system (CNS), chemokines are generally found under both physiological and pathological conditions. Whereas many reports describe chemokine expression in astrocytes and microglia and their role in the migration of leuko...

  12. Role of lipid rafts in neuronal differentiation of dental pulp-derived stem cells.

    Science.gov (United States)

    Mattei, Vincenzo; Santacroce, Costantino; Tasciotti, Vincenzo; Martellucci, Stefano; Santilli, Francesca; Manganelli, Valeria; Piccoli, Luca; Misasi, Roberta; Sorice, Maurizio; Garofalo, Tina

    2015-12-10

    Human dental pulp-derived stem cells (hDPSCs) are characterized by a typical fibroblast-like morphology. They express specific markers for mesenchymal stem cells and are capable of differentiation into osteoblasts, adipoblasts and neurons in vitro. Previous studies showed that gangliosides are involved in the induction of early neuronal differentiation of hDPSCs. This study was undertaken to investigate the role of lipid rafts in this process. Lipid rafts are signaling microdomains enriched in glycosphingolipids, cholesterol, tyrosine kinase receptors, mono- or heterotrimeric G proteins and GPI-anchored proteins. We preliminary showed that established cells expressed multipotent mesenchymal stromal-specific surface antigens. Then, we analyzed the distribution of lipid rafts, revealing plasma membrane microdomains with GM2 and EGF-R enrichment. Following stimulation with EGF/bFGF, neuronal differentiation was observed. To analyze the functional role of lipid rafts in EGF/bFGF-induced hDPSCs differentiation, cells were preincubated with lipid raft affecting agents, i.e. [D]-PDMP or methyl-β-cyclodextrin. These compounds significantly prevented neuronal-specific antigen expression, as well as Akt and ERK 1/2 phosphorylation, induced by EGF/bFGF, indicating that lipid raft integrity is essential for EGF/bFGF-induced hDPSCs differentiation. These results suggest that lipid rafts may represent specific chambers, where multimolecular signaling complexes, including lipids (gangliosides, cholesterol) and proteins (EGF-R), play a role in hDPSCs differentiation.

  13. Cell reprogramming and neuronal differentiation applied to neurodegenerative diseases: Focus on Parkinson's disease.

    Science.gov (United States)

    Wenker, Shirley D; Casalía, Mariana; Candedo, Verónica Cavaliere; Casabona, Juan Cruz; Pitossi, Fernando J

    2015-11-14

    Adult cells from patients can be reprogrammed to induced pluripotent stem cells (iPSCs) which successively can be used to obtain specific cells such as neurons. This remarkable breakthrough represents a new way of studying diseases and brought new therapeutic perspectives in the field of regenerative medicine. This is particular true in the neurology field, where few techniques are amenable to study the affected tissue of the patient during illness progression, in addition to the lack of neuroprotective therapies for many diseases. In this review we discuss the advantages and unresolved issues of cell reprogramming and neuronal differentiation. We reviewed evidence using iPSCs-derived neurons from neurological patients. Focusing on data obtained from Parkinson's disease (PD) patients, we show that iPSC-derived neurons possess morphological and functional characteristics of this disease and build a case for the use of this technology to study PD and other neuropathologies while disease is in progress. These data show the enormous impact that this new technology starts to have on different purposes such as the study and design of future therapies of neurological disease, especially PD.

  14. Functional differentiation of stem cell-derived neurons from different murine backgrounds

    Directory of Open Access Journals (Sweden)

    Lydia eBarth

    2014-02-01

    Full Text Available Murine stem cell derived-neurons have been used to study a wide variety of neuropsychiatric diseases with a hereditary component, ranging from autism to Alzheimer’s. While a significant amount of data on their molecular biology has been generated, there is little data on the physiology of these cultures. Different mouse strains show clear differences in behavioural and other neurobiologically relevant readouts. We have studied the physiology of early differentiation and network formation in neuronal cultures derived from three different mouse embryonic stem cell lines. We have found largely overlapping patterns with some significant differences in the timing of the functional milestones. Neurons from R1 showed the fastest development of intrinsic excitability, while E14Tg2a and J1 were slower. This was also reflected in an earlier appearance of synaptic activity in R1 cultures, while E14Tg2a and J1 were delayed by up to two days. In conclusion, stem cells from all backgrounds could be successfully differentiated into functioning neural networks with similar developmental patterns. Differences in the timing of specific milestones, suggest that control cell lines and time-points should be carefully chosen when investigating genetic alterations that lead to subtle deficits in neuronal function.

  15. Cycloastragenol Is a Potent Telomerase Activator in Neuronal Cells: Implications for Depression Management

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    Fanny C.F. Ip

    2014-07-01

    Full Text Available Cycloastragenol (CAG is an aglycone of astragaloside IV. It was first identified when screening Astragalus membranaceus extracts for active ingredients with antiaging properties. The present study demonstrates that CAG stimulates telomerase activity and cell proliferation in human neonatal keratinocytes. In particular, CAG promotes scratch wound closure of human neonatal keratinocyte monolayers in vitro. The distinct telomerase-activating property of CAG prompted evaluation of its potential application in the treatment of neurological disorders. Accordingly, CAG induced telomerase activity and cAMP response element binding (CREB activation in PC12 cells and primary neurons. Blockade of CREB expression in neuronal cells by RNA interference reduced basal telomerase activity, and CAG was no longer efficacious in increasing telomerase activity. CAG treatment not only induced the expression of bcl2, a CREB-regulated gene, but also the expression of telomerase reverse transcriptase in primary cortical neurons. Interestingly, oral administration of CAG for 7 days attenuated depression-like behavior in experimental mice. In conclusion, CAG stimulates telomerase activity in human neonatal keratinocytes and rat neuronal cells, and induces CREB activation followed by tert and bcl2 expression. Furthermore, CAG may have a novel therapeutic role in depression. © 2014 S. Karger AG, Basel

  16. The role of matrix metalloproteinases in regulating neuronal and nonneuronal cell invasion into PEGylated fibrinogen hydrogels.

    Science.gov (United States)

    Sarig-Nadir, Offra; Seliktar, Dror

    2010-09-01

    Injured peripheral nerve tissue could benefit from biomaterial nerve guidance conduits (NGCs) that are designed to promote neuronal regeneration. Nerve regeneration is a complex multi-step process that involves the remodeling of the ECM surrounding the regenerating neural tissue. Hydrogel biomaterials have been used as provisional matrices to regulate this regeneration process by providing the desired physical properties and controllable degradation characteristics. The purpose of this investigation was to understand the mechanism by which nerve cells penetrate into a hydrogel made from PEGylated fibrinogen. In this context, the dorsal root ganglion (DRG) assay was used as an in vitro model to study the cellular invasion behavior of both neural and nonneuronal cells. Our hypothesis stipulated that DRG cells employ matrix metalloproteinases (MMPs) in order to degrade the dense hydrogel matrix and penetrate the biomaterial. Three dimensional (3D) DRG-hydrogel constructs were cultured with MMP inhibitors (MMPi) and the effect of the inhibitors on DRG cell outgrowth was investigated. We also examined the effect of inhibitors on two dimensional (2D) DRG cell outgrowth on PEGylated fibrinogen hydrogels and on tissue culture polystyrene (TCP). Our results demonstrate that DRG cell outgrowth into and onto PEGylated fibrinogen hydrogels was inhibited by MMPi and that the outgrowth characteristics was dependent on the type of inhibitor and its concentration. MMP-3i and MMP-8i decreased both neuronal and nonneuronal outgrowth, where MMP-3i had a stronger inhibitory effect on nonneuronal cells. MMP-2/9i, on the other hand, affected the neuronal outgrowth much more than the others. We concluded that MMPs play a central role in the process of DRG cell penetration into PEGylated fibrinogen hydrogels and may also regulate the adhesion, migration and elongation of neuronal cells on the surface of these hydrogel biomaterials. PMID:20537384

  17. Isolation and Culture of Pig Spermatogonial Stem Cells and Their in Vitro Differentiation into Neuron-Like Cells and Adipocytes

    OpenAIRE

    Xiaoyan Wang; Tingfeng Chen; Yani Zhang; Bichun Li; Qi Xu; Chengyi Song

    2015-01-01

    Spermatogonial stem cells (SSCs) renew themselves throughout the life of an organism and also differentiate into sperm in the adult. They are multipopent and therefore, can be induced to differentiate into many cells types in vitro. SSCs from pigs, considered an ideal animal model, are used in studies of male infertility, regenerative medicine, and preparation of transgenic animals. Here, we report on a culture system for porcine SSCs and the differentiation of these cells into neuron-like c...

  18. Calreticulin Binds to Fas Ligand and Inhibits Neuronal Cell Apoptosis Induced by Ischemia-Reperfusion Injury

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    Beilei Chen

    2015-01-01

    Full Text Available Background. Calreticulin (CRT can bind to Fas ligand (FasL and inhibit Fas/FasL-mediated apoptosis of Jurkat T cells. However, its effect on neuronal cell apoptosis has not been investigated. Purpose. We aimed to evaluate the neuroprotective effect of CRT following ischemia-reperfusion injury (IRI. Methods. Mice underwent middle cerebral artery occlusion (MCAO and SH-SY5Y cells subjected to oxygen glucose deprivation (OGD were used as models for IRI. The CRT protein level was detected by Western blotting, and mRNA expression of CRT, caspase-3, and caspase-8 was measured by real-time PCR. Immunofluorescence was used to assess the localization of CRT and FasL. The interaction of CRT with FasL was verified by coimmunoprecipitation. SH-SY5Y cell viability was determined by MTT assay, and cell apoptosis was assessed by flow cytometry. The measurement of caspase-8 and caspase-3 activity was carried out using caspase activity assay kits. Results. After IRI, CRT was upregulated on the neuron surface and bound to FasL, leading to increased viability of OGD-exposed SH-SY5Y cells and decreased activity of caspase-8 and caspase-3. Conclusions. This study for the first time revealed that increased CRT inhibited Fas/FasL-mediated neuronal cell apoptosis during the early stage of ischemic stroke, suggesting it to be a potential protector activated soon after IRI.

  19. Reciprocal interactions between cell adhesion molecules of the immunoglobulin superfamily and the cytoskeleton in neurons

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    Vladimir eSytnyk

    2016-02-01

    Full Text Available Cell adhesion molecules of the immunoglobulin superfamily (IgSF including the neural cell adhesion molecule (NCAM and members of the L1 family of neuronal cell adhesion molecules play important functions in the developing nervous system by regulating formation, growth and branching of neurites and establishment of the synaptic contacts between neurons. In the mature brain, members of IgSF regulate synapse composition, function and plasticity required for learning and memory. The intracellular domains of IgSF cell adhesion molecules interact with the components of the cytoskeleton including the submembrane actin-spectrin meshwork, actin microfilaments, and microtubules. In this review, we summarize current data indicating that interactions between IgSF cell adhesion molecules and the cytoskeleton are reciprocal, and that while IgSF cell adhesion molecules regulate the assembly of the cytoskeleton, the cytoskeleton plays an important role in regulation of the functions of IgSF cell adhesion molecules. Reciprocal interactions between NCAM and L1 family members and the cytoskeleton and their role in neuronal differentiation and synapse formation are discussed in detail.

  20. Reciprocal Interactions between Cell Adhesion Molecules of the Immunoglobulin Superfamily and the Cytoskeleton in Neurons.

    Science.gov (United States)

    Leshchyns'ka, Iryna; Sytnyk, Vladimir

    2016-01-01

    Cell adhesion molecules of the immunoglobulin superfamily (IgSF) including the neural cell adhesion molecule (NCAM) and members of the L1 family of neuronal cell adhesion molecules play important functions in the developing nervous system by regulating formation, growth and branching of neurites, and establishment of the synaptic contacts between neurons. In the mature brain, members of IgSF regulate synapse composition, function, and plasticity required for learning and memory. The intracellular domains of IgSF cell adhesion molecules interact with the components of the cytoskeleton including the submembrane actin-spectrin meshwork, actin microfilaments, and microtubules. In this review, we summarize current data indicating that interactions between IgSF cell adhesion molecules and the cytoskeleton are reciprocal, and that while IgSF cell adhesion molecules regulate the assembly of the cytoskeleton, the cytoskeleton plays an important role in regulation of the functions of IgSF cell adhesion molecules. Reciprocal interactions between NCAM and L1 family members and the cytoskeleton and their role in neuronal differentiation and synapse formation are discussed in detail. PMID:26909348

  1. Extra-hepatic replication and infection of hepatitis E virus in neuronal-derived cells.

    Science.gov (United States)

    Drave, S A; Debing, Y; Walter, S; Todt, D; Engelmann, M; Friesland, M; Wedemeyer, H; Neyts, J; Behrendt, P; Steinmann, E

    2016-07-01

    Hepatitis E virus (HEV) is the causative agent of hepatitis E in humans and a member of the genus Orthohepevirus in the family Hepeviridae. Infection usually leads to acute hepatitis that can become fulminant, particularly among pregnant women and in patients with preexisting liver disease, or may evolve to a chronic state, especially in immunosuppressed individuals. HEV has been shown to produce a range of extra-hepatic manifestations including aplastic anaemia, acute thyroiditis, glomerulonephritis as well as neurological disorders such as Guillain-Barré syndrome, neuralgic amyotrophy and encephalitis. The pathogenesis of these neurological injuries remains largely unknown, and it is also uncertain whether or not HEV can directly infect neuronal cells. In this study, we investigated whether HEV is capable of completing the viral life cycle in human neuronal-derived cell lines such as neuroepithelioma (SK-N-MC), desmoplastic cerebellar medulloblastoma (DAOY), glioblastoma multiforme (DBTRG), glioblastoma astrocytoma (U-373 MG) and oligodendrocytic (M03.13) cells. Following transfection of these cells with HEV Gaussia luciferase reporter virus, all tested cell lines supported HEV RNA replication. Furthermore, extra- and intracellular viral capsid was detected by an HEV antigen ELISA as a marker for virus assembly and release. Permissiveness for HEV cell entry could be demonstrated for the oligodendrocytic cell line M03.13. In conclusion, these results indicate that HEV tropism is not restricted to the liver and HEV can potentially complete the full viral life cycle in neuronal-derived tissues explaining neurologic disorders during HEV infection. PMID:26891712

  2. PEP-1-GSTpi protein enhanced hippocampal neuronal cell survival after oxidative damage

    Science.gov (United States)

    Sohn, Eun Jeong; Shin, Min Jea; Kim, Dae Won; Son, Ora; Jo, Hyo Sang; Cho, Su Bin; Park, Jung Hwan; Lee, Chi Hern; Yeo, Eun Ji; Choi, Yeon Joo; Yu, Yeon Hee; Kim, Duk-Soo; Cho, Sung-Woo; Kwon, Oh Shin; Cho, Yong-Jun; Park, Jinseu; Eum, Won Sik; Choi, Soo Young

    2016-01-01

    Reactive oxygen species generated under oxidative stress are involved in neuronal diseases, including ischemia. Glutathione S-transferase pi (GSTpi) is a member of the GST family and is known to play important roles in cell survival. We investigated the effect of GSTpi against oxidative stress-induced hippocampal HT-22 cell death, and its effects in an animal model of ischemic injury, using a cell-permeable PEP-1-GSTpi protein. PEP-1-GSTpi was transduced into HT-22 cells and significantly protected against H2O2-treated cell death by reducing the intracellular toxicity and regulating the signal pathways, including MAPK, Akt, Bax, and Bcl-2. PEP-1-GSTpi transduced into the hippocampus in animal brains, and markedly protected against neuronal cell death in an ischemic injury animal model. These results indicate that PEP-1-GSTpi acts as a regulator or an antioxidant to protect against oxidative stress-induced cell death. Our study suggests that PEP-1-GSTpi may have potential as a therapeutic agent for the treatment of ischemia and a variety of oxidative stress-related neuronal diseases. [BMB Reports 2016; 49(7): 382-387] PMID:27049109

  3. STAT3 modulation to enhance motor neuron differentiation in human neural stem cells.

    Directory of Open Access Journals (Sweden)

    Rajalaxmi Natarajan

    Full Text Available Spinal cord injury or amyotrophic lateral sclerosis damages spinal motor neurons and forms a glial scar, which prevents neural regeneration. Signal transducer and activator of transcription 3 (STAT3 plays a critical role in astrogliogenesis and scar formation, and thus a fine modulation of STAT3 signaling may help to control the excessive gliogenic environment and enhance neural repair. The objective of this study was to determine the effect of STAT3 inhibition on human neural stem cells (hNSCs. In vitro hNSCs primed with fibroblast growth factor 2 (FGF2 exhibited a lower level of phosphorylated STAT3 than cells primed by epidermal growth factor (EGF, which correlated with a higher number of motor neurons differentiated from FGF2-primed hNSCs. Treatment with STAT3 inhibitors, Stattic and Niclosamide, enhanced motor neuron differentiation only in FGF2-primed hNSCs, as shown by increased homeobox gene Hb9 mRNA levels as well as HB9+ and microtubule-associated protein 2 (MAP2+ co-labeled cells. The increased motor neuron differentiation was accompanied by a decrease in the number of glial fibrillary acidic protein (GFAP-positive astrocytes. Interestingly, Stattic and Niclosamide did not affect the level of STAT3 phosphorylation; rather, they perturbed the nuclear translocation of phosphorylated STAT3. In summary, we demonstrate that FGF2 is required for motor neuron differentiation from hNSCs and that inhibition of STAT3 further increases motor neuron differentiation at the expense of astrogliogenesis. Our study thus suggests a potential benefit of targeting the STAT3 pathway for neurotrauma or neurodegenerative diseases.

  4. Human iPSC-derived neurons and lymphoblastoid cells for personalized medicine research in neuropsychiatric disorders

    Science.gov (United States)

    Gurwitz, David

    2016-01-01

    The development and clinical implementation of personalized medicine crucially depends on the availability of high-quality human biosamples; animal models, although capable of modeling complex human diseases, cannot reflect the large variation in the human genome, epigenome, transcriptome, proteome, and metabolome. Although the biosamples available from public biobanks that store human tissues and cells may represent the large human diversity for most diseases, these samples are not always sufficient for developing biomarkers for patient-tailored therapies for neuropsychiatric disorders. Postmortem human tissues are available from many biobanks; nevertheless, collections of neuronal human cells from large patient cohorts representing the human diversity remain scarce. Two tools are gaining popularity for personalized medicine research on neuropsychiatric disorders: human induced pluripotent stem cell-derived neurons and human lymphoblastoid cell lines. This review examines and contrasts the advantages and limitations of each tool for personalized medicine research.

  5. Highly ordered large-scale neuronal networks of individual cells - toward single cell to 3D nanowire intracellular interfaces.

    Science.gov (United States)

    Kwiat, Moria; Elnathan, Roey; Pevzner, Alexander; Peretz, Asher; Barak, Boaz; Peretz, Hagit; Ducobni, Tamir; Stein, Daniel; Mittelman, Leonid; Ashery, Uri; Patolsky, Fernando

    2012-07-25

    The use of artificial, prepatterned neuronal networks in vitro is a promising approach for studying the development and dynamics of small neural systems in order to understand the basic functionality of neurons and later on of the brain. The present work presents a high fidelity and robust procedure for controlling neuronal growth on substrates such as silicon wafers and glass, enabling us to obtain mature and durable neural networks of individual cells at designed geometries. It offers several advantages compared to other related techniques that have been reported in recent years mainly because of its high yield and reproducibility. The procedure is based on surface chemistry that allows the formation of functional, tailormade neural architectures with a micrometer high-resolution partition, that has the ability to promote or repel cells attachment. The main achievements of this work are deemed to be the creation of a large scale neuronal network at low density down to individual cells, that develop intact typical neurites and synapses without any glia-supportive cells straight from the plating stage and with a relatively long term survival rate, up to 4 weeks. An important application of this method is its use on 3D nanopillars and 3D nanowire-device arrays, enabling not only the cell bodies, but also their neurites to be positioned directly on electrical devices and grow with registration to the recording elements underneath.

  6. In vivo tracking of neuronal-like cells by magnetic resonance in rabbit models of spinal cord injury

    OpenAIRE

    Zhang, Ruiping; Zhang, Kun; Li, Jianding; Liu, Qiang; Xie, Jun

    2013-01-01

    In vitro experiments have demonstrated that neuronal-like cells derived from bone marrow mesenchymal stem cells can survive, migrate, integrate and help to restore the function and behaviors of spinal cord injury models, and that they may serve as a suitable approach to treating spinal cord injury. However, it is very difficult to track transplanted cells in vivo. In this study, we injected superparamagnetic iron oxide-labeled neuronal-like cells into the subarachnoid space in a rabbit model ...

  7. Cocaine-induced oxidative stress precedes cell death in human neuronal progenitor cells.

    Science.gov (United States)

    Poon, H Fai; Abdullah, Laila; Mullan, Myles A; Mullan, Michael J; Crawford, Fiona C

    2007-01-01

    By 2003, an estimated 34 million Americans had used cocaine according to the National Survey on Drug Use & Health. About 5.9 million of those had used in the past 12 months. Chronic cocaine users often develop addiction, dependency and tolerance to the drug. The psychological and physical effects of cocaine are due to the disruption of the limbic system in the central nervous system (CNS). Increased oxidative stress reported in the frontal cortex and the striatum of rats exposed to cocaine suggests that oxidative damage plays a significant role in cocaine-induced disruption of the CNS. Although it is evident that cocaine induces oxidative stress in the CNS, little has been learned about whether such increased oxidative stress is also relevant to apoptosis in cocaine-exposed models. To gain insight into the role of cocaine-induced oxidative stress in apoptosis, we hypothesized that oxidative stress precedes cell death when cocaine is administrated. To test this hypothesis, we have monitored the oxidative stress and apoptotic effects of acute cocaine exposure in human neuronal progenitor cells (HNPC). We found that oxidative stress was significantly increased at 48h after a 30min cocaine exposure compared to control cells, and that this was followed by cell death at 72h. Using the same experimental paradigm we have previously shown that pro-inflammatory genes are up-regulated in cocaine-exposed HNPC at 24h. Therefore, we suggest that the increased oxidative stress (possibly mediated by inflammatory responses) precedes cell death in cocaine-exposed HNPC. This may have implications for the consequences of cocaine abuse in situations where antioxidant capacity is compromised, as in the aging brain. PMID:16956698

  8. Schwann cells genetically modified to express neurotrophins promote spiral ganglion neuron survival in vitro

    OpenAIRE

    Pettingill, Lisa N.; Minter, Ricki L.; Shepherd, Robert K.

    2008-01-01

    The intracochlear infusion of neurotrophic factors via a mini-osmotic pump has been shown to prevent deafness-induced spiral ganglion neuron (SGN) degeneration; however, the use of pumps may increase the incidence of infection within the cochlea, making this technique unsuitable for neurotrophin administration in a clinical setting. Cell- and gene-based therapies are potential therapeutic options. This study investigated whether Schwann cells which were genetically modified to over-express th...

  9. Generation and Expansion of highly-pure Motor Neuron Progenitors from Human Pluripotent Stem Cells

    OpenAIRE

    Du, Zhong-Wei; Chen, Hong; Liu, Huisheng; Lu, Jianfeng; Qian, Kun; Huang, Cindy Tzu-Ling.; Zhong, Xiaofen; Fan, Frank; Zhang, Su-Chun

    2015-01-01

    SUMMARY Human pluripotent stem cells (hPSCs) have opened new opportunities for understanding human development, modeling disease processes and developing new therapeutics. However, these applications are hindered by low-efficiency and heterogeneity of target cell types differentiated from hPSCs, such as motor neurons (MNs), as well as our inability to maintain the potency of lineage committed progenitors. Here, by using a combination of small molecules that regulate multiple signaling pathway...

  10. Direct conversion of C. elegans germ cells into specific neuron types

    OpenAIRE

    Tursun, Baris; Patel, Tulsi; Kratsios, Paschalis; Hobert, Oliver

    2010-01-01

    The ability of transcription factors to directly reprogram the identity of cell types is usually restricted and is defined by cellular context. We show here that through ectopic expression of single C. elegans transcription factors, the identity of mitotic germ cells can be directly converted into that of specific neuron types (glutamatergic, cholinergic or GABAergic). This reprogramming event requires the removal of the histone chaperone LIN-53/RbAp48, a component of several histone remodeli...

  11. Neuronal Cell Fate Specification by the Convergence of Different Spatiotemporal Cues on a Common Terminal Selector Cascade.

    Directory of Open Access Journals (Sweden)

    Hugo Gabilondo

    2016-05-01

    Full Text Available Specification of the myriad of unique neuronal subtypes found in the nervous system depends upon spatiotemporal cues and terminal selector gene cascades, often acting in sequential combinatorial codes to determine final cell fate. However, a specific neuronal cell subtype can often be generated in different parts of the nervous system and at different stages, indicating that different spatiotemporal cues can converge on the same terminal selectors to thereby generate a similar cell fate. However, the regulatory mechanisms underlying such convergence are poorly understood. The Nplp1 neuropeptide neurons in the Drosophila ventral nerve cord can be subdivided into the thoracic-ventral Tv1 neurons and the dorsal-medial dAp neurons. The activation of Nplp1 in Tv1 and dAp neurons depends upon the same terminal selector cascade: col>ap/eya>dimm>Nplp1. However, Tv1 and dAp neurons are generated by different neural progenitors (neuroblasts with different spatiotemporal appearance. Here, we find that the same terminal selector cascade is triggered by Kr/pdm>grn in dAp neurons, but by Antp/hth/exd/lbe/cas in Tv1 neurons. Hence, two different spatiotemporal combinations can funnel into a common downstream terminal selector cascade to determine a highly related cell fate.

  12. From stem cells to dopamine neurons: developmental biology meets neurodegeneration.

    Science.gov (United States)

    Tailor, Jignesh; Andreska, Thomas; Kittappa, Raja

    2012-11-01

    Neurodegenerative disease affects tens of millions of people, worldwide, and comes at a cost to the public of billions of dollars. Stem cell therapy, in recent years, has generated a lot of enthusiasm as a novel treatment for neurodegenerative disease. In particular, Parkinson's disease has been identified as the ideal neurodegenerative disease to be treated using stem cells. Despite years of setbacks, recent experimental results have renewed optimism in the validity of stem cell therapy for the treatment of Parkinson's disease. In this review, we discuss advances in our understanding of the embryonic development of the dopamine system and the importance of these discoveries in the continued efforts towards stem cell therapy for Parkinson's disease.

  13. Estrogen receptor beta-selective agonists stimulate calcium oscillations in human and mouse embryonic stem cell-derived neurons.

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    Lili Zhang

    Full Text Available Estrogens are used extensively to treat hot flashes in menopausal women. Some of the beneficial effects of estrogens in hormone therapy on the brain might be due to nongenomic effects in neurons such as the rapid stimulation of calcium oscillations. Most studies have examined the nongenomic effects of estrogen receptors (ER in primary neurons or brain slices from the rodent brain. However, these cells can not be maintained continuously in culture because neurons are post-mitotic. Neurons derived from embryonic stem cells could be a potential continuous, cell-based model to study nongenomic actions of estrogens in neurons if they are responsive to estrogens after differentiation. In this study ER-subtype specific estrogens were used to examine the role of ERalpha and ERbeta on calcium oscillations in neurons derived from human (hES and mouse embryonic stem cells. Unlike the undifferentiated hES cells the differentiated cells expressed neuronal markers, ERbeta, but not ERalpha. The non-selective ER agonist 17beta-estradiol (E(2 rapidly increased [Ca2+]i oscillations and synchronizations within a few minutes. No change in calcium oscillations was observed with the selective ERalpha agonist 4,4',4''-(4-Propyl-[1H]-pyrazole-1,3,5-triyltrisphenol (PPT. In contrast, the selective ERbeta agonists, 2,3-bis(4-Hydroxyphenyl-propionitrile (DPN, MF101, and 2-(3-fluoro-4-hydroxyphenyl-7-vinyl-1,3 benzoxazol-5-ol (ERB-041; WAY-202041 stimulated calcium oscillations similar to E(2. The ERbeta agonists also increased calcium oscillations and phosphorylated PKC, AKT and ERK1/2 in neurons derived from mouse ES cells, which was inhibited by nifedipine demonstrating that ERbeta activates L-type voltage gated calcium channels to regulate neuronal activity. Our results demonstrate that ERbeta signaling regulates nongenomic pathways in neurons derived from ES cells, and suggest that these cells might be useful to study the nongenomic mechanisms of estrogenic compounds.

  14. The roadmap for estimation of cell-type-specific neuronal activity from non-invasive measurements.

    Science.gov (United States)

    Uhlirova, Hana; Kılıç, Kıvılcım; Tian, Peifang; Sakadžić, Sava; Gagnon, Louis; Thunemann, Martin; Desjardins, Michèle; Saisan, Payam A; Nizar, Krystal; Yaseen, Mohammad A; Hagler, Donald J; Vandenberghe, Matthieu; Djurovic, Srdjan; Andreassen, Ole A; Silva, Gabriel A; Masliah, Eliezer; Kleinfeld, David; Vinogradov, Sergei; Buxton, Richard B; Einevoll, Gaute T; Boas, David A; Dale, Anders M; Devor, Anna

    2016-10-01

    The computational properties of the human brain arise from an intricate interplay between billions of neurons connected in complex networks. However, our ability to study these networks in healthy human brain is limited by the necessity to use non-invasive technologies. This is in contrast to animal models where a rich, detailed view of cellular-level brain function with cell-type-specific molecular identity has become available due to recent advances in microscopic optical imaging and genetics. Thus, a central challenge facing neuroscience today is leveraging these mechanistic insights from animal studies to accurately draw physiological inferences from non-invasive signals in humans. On the essential path towards this goal is the development of a detailed 'bottom-up' forward model bridging neuronal activity at the level of cell-type-specific populations to non-invasive imaging signals. The general idea is that specific neuronal cell types have identifiable signatures in the way they drive changes in cerebral blood flow, cerebral metabolic rate of O2 (measurable with quantitative functional Magnetic Resonance Imaging), and electrical currents/potentials (measurable with magneto/electroencephalography). This forward model would then provide the 'ground truth' for the development of new tools for tackling the inverse problem-estimation of neuronal activity from multimodal non-invasive imaging data.This article is part of the themed issue 'Interpreting BOLD: a dialogue between cognitive and cellular neuroscience'. PMID:27574309

  15. Denervated hippocampus provides a favorable microenvironment for neuronal differentiation of endogenous neural stem cells

    Institute of Scientific and Technical Information of China (English)

    Lei Zhang; Xiao Han; Xiang Cheng; Xue-feng Tan; He-yan Zhao; Xin-hua Zhang

    2016-01-01

    Fimbria-fornix transection induces both exogenous and endogenous neural stem cells to differentiate into neurons in the hippocampus. This indicates that the denervated hippocampus provides an environment for neuronal differentiation of neural stem cells. However, the pathways and mechanisms in this process are still unclear. Seven days after ifmbria fornix transection, our reverse transcription polymerase chain reaction, western blot assay, and enzyme linked immunosorbent assay results show a signiifcant increase in ciliary neurotrophic factor mRNA and protein expression in the denervated hippocampus. Moreover, neural stem cells derived from hippocampi of fetal (em-bryonic day 17) Sprague-Dawley rats were treated with ciliary neurotrophic factor for 7 days, with an increased number of microtubule associated protein-2-positive cells and decreased number of glial ifbrillary acidic protein-positive cells detected. Our results show that cili-ary neurotrophic factor expression is up-regulated in the denervated hippocampus, which may promote neuronal differentiation of neural stem cells in the denervated hippocampus.

  16. Pyramidal cells in prefrontal cortex: comparative observations reveal unparalleled specializations in neuronal structure among primate species.

    Directory of Open Access Journals (Sweden)

    Guy eElston

    2011-02-01

    Full Text Available The most ubiquitous neuron in the cerebral cortex, the pyramidal cell, is characterised by markedly different dendritic structure among different cortical areas. The complex pyramidal cell phenotype in granular prefrontal cortex (gPFC of higher primates endows specific biophysical properties and patterns of connectivity, which differ to those in other cortical regions. However, within the gPFC, data have been sampled from only a select few cortical areas. The gPFC of species such as human and macaque monkey includes more than 10 cortical areas. It remains unknown as to what degree pyramidal cell structure may vary among these cortical areas. Here we undertook a survey of pyramidal cells in the dorsolateral, medial and orbital gPFC of cercopethicid primates. We found marked heterogeneity in pyramidal cell structure within and between these regions. Moreover, trends for gradients in neuronal complexity varied among species. As neuron structure determines it’s computational abilities and memory storage capacity and connectivity, we propose that these specializations in the pyramidal cell phenotype are an important determinant of species specific executive cortical functions in primates.

  17. Enhancement of neurite outgrowth in neuron cancer stem cells by growth on 3-D collagen scaffolds

    International Nuclear Information System (INIS)

    Highlights: ► Neuron cancer stem cells (NCSCs) behave high multiply of growth on collagen scaffold. ► Enhancement of NCSCs neurite outgrowth on porous collagen scaffold. ► 3-D collagen culture of NCSCs shows an advance differentiation than 2-D culture. -- Abstract: Collagen is one component of the extracellular matrix that has been widely used for constructive remodeling to facilitate cell growth and differentiation. The 3-D distribution and growth of cells within the porous scaffold suggest a clinical significance for nerve tissue engineering. In the current study, we investigated proliferation and differentiation of neuron cancer stem cells (NCSCs) on a 3-D porous collagen scaffold that mimics the natural extracellular matrix. We first generated green fluorescence protein (GFP) expressing NCSCs using a lentiviral system to instantly monitor the transitions of morphological changes during growth on the 3-D scaffold. We found that proliferation of GFP-NCSCs increased, and a single cell mass rapidly grew with unrestricted expansion between days 3 and 9 in culture. Moreover, immunostaining with neuronal nuclei (NeuN) revealed that NCSCs grown on the 3-D collagen scaffold significantly enhanced neurite outgrowth. Our findings confirmed that the 80 μm porous collagen scaffold could enhance attachment, viability and differentiation of the cancer neural stem cells. This result could provide a new application for nerve tissue engineering and nerve regeneration.

  18. Polarized trafficking of the sorting receptor SorLA in neurons and MDCK cells.

    Science.gov (United States)

    Klinger, Stine C; Højland, Anne; Jain, Shweta; Kjolby, Mads; Madsen, Peder; Svendsen, Anna Dorst; Olivecrona, Gunilla; Bonifacino, Juan S; Nielsen, Morten S

    2016-07-01

    The sorting receptor SorLA is highly expressed in neurons and is also found in other polarized cells. The receptor has been reported to participate in the trafficking of several ligands, some of which are linked to human diseases, including the amyloid precursor protein, TrkB, and Lipoprotein Lipase (LpL). Despite this, only the trafficking in nonpolarized cells has been described so far. Due to the many differences between polarized and nonpolarized cells, we examined the localization and trafficking of SorLA in epithelial Madin-Darby canine kidney (MDCK) cells and rat hippocampal neurons. We show that SorLA is mainly found in sorting endosomes and on the basolateral surface of MDCK cells and in the somatodendritic domain of neurons. This polarized distribution of SorLA respectively depends on an acidic cluster and an extended version of this cluster and involves the cellular adaptor complex AP-1. Furthermore, we show that SorLA can mediate transcytosis across a tight cell layer. PMID:27192064

  19. Protection of neuronal cells against reactive oxygen species by carnosine and related compounds.

    Science.gov (United States)

    Boldyrev, Alexander; Bulygina, Elena; Leinsoo, Toomas; Petrushanko, Irina; Tsubone, Shiori; Abe, Hiroki

    2004-01-01

    Carnosine and related compounds were compared in terms of their abilities to decrease the levels of reactive oxygen species (ROS) in suspensions of isolated neurons activated by N-methyl-D-aspartic acid (NMDA) using both stationary fluorescence measurements and flow cytometry. Carnosine was found to suppress the fluorescent signal induced by ROS production and decreased the proportion of highly fluorescent neurons, while histidine showed opposite effects. N-Acetylated derivatives of both carnosine and histidine demonstrated weak (statistically indistinguishable) suppressive effects on the ROS signal. N-Methylated derivatives of carnosine suppressed intracellular ROS generation to the same extent as carnosine. This rank of effectiveness is distinct from that previously obtained for the anti-radical ability of CRCs (anserine>carnosine>ophidine). These differences suggest that the similar ability of carnosine and its N-methylated derivatives to protect neuronal cells against the excitotoxic effect of NMDA is not solely related to the antioxidant properties of these compounds. PMID:14698913

  20. Coculture of Primary Motor Neurons and Schwann Cells as a Model for In Vitro Myelination.

    Science.gov (United States)

    Hyung, Sujin; Yoon Lee, Bo; Park, Jong-Chul; Kim, Jinseok; Hur, Eun-Mi; Francis Suh, Jun-Kyo

    2015-10-12

    A culture system that can recapitulate myelination in vitro will not only help us better understand the mechanism of myelination and demyelination, but also find out possible therapeutic interventions for treating demyelinating diseases. Here, we introduce a simple and reproducible myelination culture system using mouse motor neurons (MNs) and Schwann cells (SCs). Dissociated motor neurons are plated on a feeder layer of SCs, which interact with and wrap around the axons of MNs as they differentiate in culture. In our MN-SC coculture system, MNs survived over 3 weeks and extended long axons. Both viability and axon growth of MNs in the coculture were markedly enhanced as compared to those of MN monoculture. Co-labeling of myelin basic proteins (MBPs) and neuronal microtubules revealed that SC formed myelin sheaths by wrapping around the axons of MNs. Furthermore, using the coculture system we found that treatment of an antioxidant substance coenzyme Q10 (Co-Q10) markedly facilitated myelination.

  1. Electrophysiological study on differentiation of rat bone marrow stromal stem cells into neuron-like cells in vitro by edaravone

    Institute of Scientific and Technical Information of China (English)

    ZENG Rong; HU Zi-bing; GUO Wei-tao; LIN Hao; SUN Xin; WEI Jin-song; WU Shao-ke

    2009-01-01

    To explore the electrophysiological proper-ties of differentiation of rat bone marrow-derived stromal stem cells (rBMSCs) to neuron-like cells in vitro by edaravone, a new type of free radical scavenger. Methods: Stromal stem cells were separated from rat bone marrow with Ficoll-Paque reagent and expanded in different culture medium in vitro, rBMSCs were induced by edaravone containing serum-free L-DMEM. Morphologic observation and Western blot analysis including the ex-pression of Nav1.6, Kv1.2, Kv1.3, Cav1.2 were performed, and whole patch-clamp technique was used. Results: Cyton contraction and long processes were shown in differentiated stromal stem cells. Nav1.6, Kv1.2, Kv1.3 and Cav1.2 were expressed in both differentiated and undifferentiated cells. However, the expression of channel proteins in differentiated cells was up-regulated. Consistently, their resting potential and outward currents were also enhanced in the differentiated cells, which was especially significant in the outward rectifier potassium current. Conclusion: In vitro, neuron-like cells derived from rBMSCs, induced by edaravone, possess electrophysiologi-cal properties of neurons.

  2. Detection and Characterization of Autoantibodies to Neuronal Cell-Surface Antigens in the Central Nervous System.

    Science.gov (United States)

    van Coevorden-Hameete, Marleen H; Titulaer, Maarten J; Schreurs, Marco W J; de Graaff, Esther; Sillevis Smitt, Peter A E; Hoogenraad, Casper C

    2016-01-01

    Autoimmune encephalitis (AIE) is a group of disorders in which autoantibodies directed at antigens located on the plasma membrane of neurons induce severe neurological symptoms. In contrast to classical paraneoplastic disorders, AIE patients respond well to immunotherapy. The detection of neuronal surface autoantibodies in patients' serum or CSF therefore has serious consequences for the patients' treatment and follow-up and requires the availability of sensitive and specific diagnostic tests. This mini-review provides a guideline for both diagnostic and research laboratories that work on the detection of known surface autoantibodies and/or the identification of novel surface antigens. We discuss the strengths and pitfalls of different techniques for anti-neuronal antibody detection: (1) Immunohistochemistry (IHC) and immunofluorescence on rat/primate brain sections; (2) Immunocytochemistry (ICC) of living cultured hippocampal neurons; and (3) Cell Based Assay (CBA). In addition, we discuss the use of immunoprecipitation and mass spectrometry analysis for the detection of novel neuronal surface antigens, which is a crucial step in further disease classification and the development of novel CBAs. PMID:27303263

  3. Detection and Characterization of Autoantibodies to Neuronal Cell-Surface Antigens in the Central Nervous System

    Directory of Open Access Journals (Sweden)

    Marleen eVan Coevorden-Hameete

    2016-05-01

    Full Text Available Autoimmune encephalitis (AIE is a group of disorders in which autoantibodies directed at antigens located on the plasma membrane of neurons induce severe neurological symptoms. In contrast to classical paraneoplastic disorders, AIE patients respond well to immunotherapy. The detection of neuronal surface autoantibodies in patients’ serum or CSF therefore has serious consequences for the patients’ treatment and follow-up and requires the availability of sensitive and specific diagnostic tests. This mini-review provides a guideline for both diagnostic and research laboratories that work on the detection of known surface autoantibodies and/or the identification of novel surface antigens. We discuss the strengths and pitfalls of different techniques for anti-neuronal antibody detection: 1 Immunohistochemistry and immunofluorescence on rat/ primate brain sections, 2 Immunocytochemistry of living cultured hippocampal neurons, 3 Cell Based Assay (CBA. In addition, we discuss the use of immunoprecipitation and mass spectrometry analysis for the detection of novel neuronal surface antigens, which is a crucial step in further disease classification and the development of novel CBAs.

  4. Methamphetamine decreases dentate gyrus stem cell self-renewal and shifts the differentiation towards neuronal fate

    OpenAIRE

    Sofia Baptista; Charlène Lasgi; Caroline Benstaali; Nuno Milhazes; Fernanda Borges; Carlos Fontes-Ribeiro; Fabienne Agasse; Ana Paula Silva

    2014-01-01

    Methamphetamine (METH) is a highly addictive psychostimulant drug of abuse that negatively interferes with neurogenesis. In fact, we have previously shown that METH triggers stem/progenitor cell death and decreases neuronal differentiation in the dentate gyrus (DG). Still, little is known regarding its effect on DG stem cell properties. Herein, we investigate the impact of METH on mice DG stem/progenitor cell self-renewal functions. METH (10 nM) decreased DG stem cell self-renewal, while 1 nM...

  5. A Neuroprotective Sericin Hydrogel As an Effective Neuronal Cell Carrier for the Repair of Ischemic Stroke.

    Science.gov (United States)

    Wang, Zheng; Wang, Jian; Jin, Yang; Luo, Zhen; Yang, Wen; Xie, Hongjian; Huang, Kai; Wang, Lin

    2015-11-11

    Ischemic stroke causes extensive cellular loss that impairs brain functions, resulting in severe disabilities. No effective treatments are currently available for brain tissue regeneration. The need to develop effective therapeutic approaches for treating stroke is compelling. A tissue engineering approach employing a hydrogel carrying both cells and neurotrophic cytokines to damaged regions is an encouraging alternative for neuronal repair. However, this approach is often challenged by low in vivo cell survival rate, and low encapsulation efficiency and loss of cytokines. To address these limitations, we propose to develop a biomaterial that can form a matrix capable of improving in vivo survival of transplanted cells and reducing in vivo loss of cytokines. Here, we report that using sericin, a natural protein from silk, we have fabricated a genipin-cross-linked sericin hydrogel (GSH) with porous structure and mild swelling ratio. The GSH supports the effective attachment and growth of neurons in vitro. Strikingly, our data reveal that sericin protein is intrinsically neurotrophic and neuroprotective, promoting axon extension and branching as well as preventing primary neurons from hypoxia-induced cell death. Notably, these functions are inherited by the GSH's degradation products, which might spare a need of incorporating costly cytokines. We further demonstrate that this neurotrophic effect is dependent on the Lkb1-Nuak1 pathway, while the neuroprotective effect is realized through regulating the Bcl-2/Bax protein ratio. Importantly, when transplanted in vivo, the GSH gives a high cell survival rate and allows the cells to continuously proliferate. Together, this work unmasks the neurotrophic and neuroprotective functions for sericin and provides strong evidence justifying the GSH's suitability as a potential neuronal cell delivery vehicle for ischemic stroke repair. PMID:26478947

  6. Three counting methods agree on cell and neuron number in chimpanzee primary visual cortex

    Directory of Open Access Journals (Sweden)

    Daniel James Miller

    2014-05-01

    Full Text Available Determining the cellular composition of specific brain regions is crucial to our understanding of the function of neurobiological systems. It is therefore useful to identify the extent to which different methods agree when estimating the same properties of brain circuitry. In this study, we estimated the number of neuronal and non-neuronal cells in the primary visual cortex (area 17 or V1 of both hemispheres from a single chimpanzee. Specifically, we processed samples distributed across V1 of the right hemisphere after cortex was flattened into a sheet using two variations of the isotropic fractionator cell and neuron counting method. We processed the left hemisphere as serial brain slices for stereological investigation. The goal of this study was to evaluate the agreement between these methods in the most direct manner possible by comparing estimates of cell density across one brain region of interest in a single individual. In our hands, these methods produced similar estimates of the total cellular population (approximately 1 billion as well as the number of neurons (approximately 675 million in chimpanzee V1, providing evidence that both techniques estimate the same parameters of interest. In addition, our results indicate the strengths of each distinct tissue preparation procedure, highlighting the importance of attention to anatomical detail. In summary, we found that the isotropic fractionator and the stereological optical fractionator produced concordant estimates of the cellular composition of V1, and that this result supports the conclusion that chimpanzees conform to the primate pattern of exceptionally high packing density in V1. Ultimately, our data suggest that investigators can optimize their experimental approach by using any of these counting methods to obtain reliable cell and neuron counts.

  7. Recapitulation of spinal motor neuron-specific disease phenotypes in a human cell model of spinal muscular atrophy

    Institute of Scientific and Technical Information of China (English)

    Zhi-Bo Wang; Xiaoqing Zhang; Xue-Jun Li

    2013-01-01

    Establishing human cell models of spinal muscular atrophy (SMA) to mimic motor neuron-specific phenotypes holds the key to understanding the pathogenesis of this devastating disease.Here,we developed a closely representative cell model of SMA by knocking down the disease-determining gene,survival motor neuron (SMN),in human embryonic stem cells (hESCs).Our study with this cell model demonstrated that knocking down of SMN does not interfere with neural induction or the initial specification of spinal motor neurons.Notably,the axonal outgrowth of spinal motor neurons was significantly impaired and these disease-mimicking neurons subsequently degenerated.Furthermore,these disease phenotypes were caused by SMN-full length (SMN-FL) but not SMN-A7 (lacking exon 7)knockdown,and were specific to spinal motor neurons.Restoring the expression of SMN-FL completely ameliorated all of the disease phenotypes,including specific axonal defects and motor neuron loss.Finally,knockdown of SMNFL led to excessive mitochondrial oxidative stress in human motor neuron progenitors.The involvement of oxidative stress in the degeneration of spinal motor neurons in the SMA cell model was further confirmed by the administration of N-acetylcysteine,a potent antioxidant,which prevented disease-related apoptosis and subsequent motor neuron death.Thus,we report here the successful establishment of an hESC-based SMA model,which exhibits disease gene isoform specificity,cell type specificity,and phenotype reversibility.Our model provides a unique paradigm for studying how motor neurons specifically degenerate and highlights the potential importance of antioxidants for the treatment of SMA.

  8. Neuronal Cell Death Induced by Mechanical Percussion Trauma in Cultured Neurons is not Preceded by Alterations in Glucose, Lactate and Glutamine Metabolism.

    Science.gov (United States)

    Jayakumar, A R; Bak, L K; Rama Rao, K V; Waagepetersen, H S; Schousboe, A; Norenberg, M D

    2016-02-01

    Traumatic brain injury (TBI) is a devastating neurological disorder that usually presents in acute and chronic forms. Brain edema and associated increased intracranial pressure in the early phase following TBI are major consequences of acute trauma. On the other hand, neuronal injury, leading to neurobehavioral and cognitive impairments, that usually develop months to years after single or repetitive episodes of head trauma, are major consequences of chronic TBI. The molecular mechanisms responsible for TBI-induced injury, however, are unclear. Recent studies have suggested that early mitochondrial dysfunction and subsequent energy failure play a role in the pathogenesis of TBI. We therefore examined whether oxidative metabolism of (13)C-labeled glucose, lactate or glutamine is altered early following in vitro mechanical percussion-induced trauma (5 atm) to neurons (4-24 h), and whether such events contribute to the development of neuronal injury. Cell viability was assayed using the release of the cytoplasmic enzyme lactate dehydrogenase (LDH), together with fluorescence-based cell staining (calcein and ethidium homodimer-1 for live and dead cells, respectively). Trauma had no effect on the LDH release in neurons from 1 to 18 h. However, a significant increase in LDH release was detected at 24 h after trauma. Similar findings were identified when traumatized neurons were stained with fluorescent markers. Additionally (13)C-labeling of glutamate showed a small, but statistically significant decrease at 14 h after trauma. However, trauma had no effect on the cycling ratio of the TCA cycle at any time-period examined. These findings indicate that trauma does not cause a disturbance in oxidative metabolism of any of the substrates used for neurons. Accordingly, such metabolic disturbance does not appear to contribute to the neuronal death in the early stages following trauma. PMID:26729365

  9. The atypical cadherin Celsr1 functions non-cell autonomously to block rostral migration of facial branchiomotor neurons in mice.

    Science.gov (United States)

    Glasco, Derrick M; Pike, Whitney; Qu, Yibo; Reustle, Lindsay; Misra, Kamana; Di Bonito, Maria; Studer, Michele; Fritzsch, Bernd; Goffinet, André M; Tissir, Fadel; Chandrasekhar, Anand

    2016-09-01

    The caudal migration of facial branchiomotor (FBM) neurons from rhombomere (r) 4 to r6 in the hindbrain is an excellent model to study neuronal migration mechanisms. Although several Wnt/Planar Cell Polarity (PCP) components are required for FBM neuron migration, only Celsr1, an atypical cadherin, regulates the direction of migration in mice. In Celsr1 mutants, a subset of FBM neurons migrates rostrally instead of caudally. Interestingly, Celsr1 is not expressed in the migrating FBM neurons, but rather in the adjacent floor plate and adjoining ventricular zone. To evaluate the contribution of different expression domains to neuronal migration, we conditionally inactivated Celsr1 in specific cell types. Intriguingly, inactivation of Celsr1 in the ventricular zone of r3-r5, but not in the floor plate, leads to rostral migration of FBM neurons, greatly resembling the migration defect of Celsr1 mutants. Dye fill experiments indicate that the rostrally-migrated FBM neurons in Celsr1 mutants originate from the anterior margin of r4. These data suggest strongly that Celsr1 ensures that FBM neurons migrate caudally by suppressing molecular cues in the rostral hindbrain that can attract FBM neurons. PMID:27395006

  10. The atypical cadherin Celsr1 functions non-cell autonomously to block rostral migration of facial branchiomotor neurons in mice.

    Science.gov (United States)

    Glasco, Derrick M; Pike, Whitney; Qu, Yibo; Reustle, Lindsay; Misra, Kamana; Di Bonito, Maria; Studer, Michele; Fritzsch, Bernd; Goffinet, André M; Tissir, Fadel; Chandrasekhar, Anand

    2016-09-01

    The caudal migration of facial branchiomotor (FBM) neurons from rhombomere (r) 4 to r6 in the hindbrain is an excellent model to study neuronal migration mechanisms. Although several Wnt/Planar Cell Polarity (PCP) components are required for FBM neuron migration, only Celsr1, an atypical cadherin, regulates the direction of migration in mice. In Celsr1 mutants, a subset of FBM neurons migrates rostrally instead of caudally. Interestingly, Celsr1 is not expressed in the migrating FBM neurons, but rather in the adjacent floor plate and adjoining ventricular zone. To evaluate the contribution of different expression domains to neuronal migration, we conditionally inactivated Celsr1 in specific cell types. Intriguingly, inactivation of Celsr1 in the ventricular zone of r3-r5, but not in the floor plate, leads to rostral migration of FBM neurons, greatly resembling the migration defect of Celsr1 mutants. Dye fill experiments indicate that the rostrally-migrated FBM neurons in Celsr1 mutants originate from the anterior margin of r4. These data suggest strongly that Celsr1 ensures that FBM neurons migrate caudally by suppressing molecular cues in the rostral hindbrain that can attract FBM neurons.

  11. Fibroblast growth factor-20 increases the yield of midbrain dopaminergic neurons derived from human embryonic stem cells

    Directory of Open Access Journals (Sweden)

    Ana Sofia Correia

    2007-12-01

    Full Text Available In the central nervous system, fibroblast growth factor (FGF-20 has been reported to act preferentially on midbrain dopaminergic neurons. It also promotes the dopaminergic differentiation of stem cells. We have analyzed the effects of FGF-20 on human embryonic stem cells (hESCs differentiation into dopaminergic neurons. We induced neuronal differentiation of hESCs by co-culturing those with PA6 mouse stromal cells for 3 weeks. When we supplemented the culture medium with FGF-20, the number of tyrosine hydroxylase (TH- expressing neurons increased fivefold, from 3% to 15% of the hESC-derived cells. The cultured cells also expressed other midbrain dopaminergic markers (PITX3, En1, Msx1, and Aldh1, suggesting that some had differentiated into midbrain dopaminergic neurons. We observed no effect of FGF-20 on the size of the soma area or neurite length of the TH-immunopositive neurons. Regardless of whether FGF-20 had been added or not, 17% of the hESC-derived cells expressed the pan-neuronal marker b-III-Tubulin. The proportion of proliferating cells positive for Ki-67 was also not affected by FGF-20 (7% of the hESC-derived cells. By contrast, after 3 weeks in culture FGF-20 significantly reduced the proportion of cells undergoing cell death, as revealed by immunoreactivity for cleaved caspase-8, Bcl-2 associated X protein (BAX and cleaved caspase-3 (2.5% to 1.2% of cleaved caspase-3-positive cells out of the hESC-derived cells. Taken together, our results indicate that FGF-20 specifically increases the yield of dopaminergic neurons from hESCs grown on PA6 feeder cells and at least part of this effect is due to a reduction in cell death.

  12. Communication between corneal epithelial cells and trigeminal neurons is facilitated by purinergic (P2 and glutamatergic receptors.

    Directory of Open Access Journals (Sweden)

    Duane J Oswald

    Full Text Available Previously, we demonstrated that nucleotides released upon mechanical injury to corneal epithelium activate purinergic (P2 receptors resulting in mobilization of a Ca(2+ wave. However, the tissue is extensively innervated and communication between epithelium and neurons is critical and not well understood. Therefore, we developed a co-culture of primary trigeminal neurons and human corneal limbal epithelial cells. We demonstrated that trigeminal neurons expressed a repertoire of P2Yand P2X receptor transcripts and responded to P2 agonists in a concentration-dependent manner. Mechanical injuries to epithelia in the co-cultures elicited a Ca(2+ wave that mobilized to neurons and was attenuated by Apyrase, an ectonucleotidase. To elucidate the role of factors released from each cell type, epithelial and neuronal cells were cultured, injured, and the wound media from one cell type was collected and added to the other cell type. Epithelial wound media generated a rapid Ca(2+ mobilization in neuronal cells that was abrogated in the presence of Apyrase, while neuronal wound media elicited a complex response in epithelial cells. The rapid Ca(2+ mobilization was detected, which was abrogated with Apyrase, but it was followed by Ca(2+ waves that occurred in cell clusters. When neuronal wound media was preincubated with a cocktail of N-methyl-D-aspartate (NMDA receptor inhibitors, the secondary response in epithelia was diminished. Glutamate was detected in the neuronal wound media and epithelial expression of NMDA receptor subunit transcripts was demonstrated. Our results indicate that corneal epithelia and neurons communicate via purinergic and NMDA receptors that mediate the wound response in a highly orchestrated manner.

  13. MHC mismatch inhibits neurogenesis and neuron maturation in stem cell allografts.

    Directory of Open Access Journals (Sweden)

    Zhiguo Chen

    Full Text Available BACKGROUND: The role of histocompatibility and immune recognition in stem cell transplant therapy has been controversial, with many reports arguing that undifferentiated stem cells are protected from immune recognition due to the absence of major histocompatibility complex (MHC markers. This argument is even more persuasive in transplantation into the central nervous system (CNS where the graft rejection response is minimal. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we evaluate graft survival and neuron production in perfectly matched vs. strongly mismatched neural stem cells transplanted into the hippocampus in mice. Although allogeneic cells survive, we observe that MHC-mismatch decreases surviving cell numbers and strongly inhibits the differentiation and retention of graft-derived as well as endogenously produced new neurons. Immune suppression with cyclosporine-A did not improve outcome but non-steroidal anti-inflammatory drugs, indomethacin or rosiglitazone, were able to restore allogeneic neuron production, integration and retention to the level of syngeneic grafts. CONCLUSIONS/SIGNIFICANCE: These results suggest an important but unsuspected role for innate, rather than adaptive, immunity in the survival and function of MHC-mismatched cellular grafts in the CNS.

  14. Photoelectrical Stimulation of Neuronal Cells by an Organic Semiconductor-Electrolyte Interface.

    Science.gov (United States)

    Abdullaeva, Oliya S; Schulz, Matthias; Balzer, Frank; Parisi, Jürgen; Lützen, Arne; Dedek, Karin; Schiek, Manuela

    2016-08-23

    As a step toward the realization of neuroprosthetics for vision restoration, we follow an electrophysiological patch-clamp approach to study the fundamental photoelectrical stimulation mechanism of neuronal model cells by an organic semiconductor-electrolyte interface. Our photoactive layer consisting of an anilino-squaraine donor blended with a fullerene acceptor is supporting the growth of the neuronal model cell line (N2A cells) without an adhesion layer on it and is not impairing cell viability. The transient photocurrent signal upon illumination from the semiconductor-electrolyte layer is able to trigger a passive response of the neuronal cells under physiological conditions via a capacitive coupling mechanism. We study the dynamics of the capacitive transmembrane currents by patch-clamp recordings and compare them to the dynamics of the photocurrent signal and its spectral responsivity. Furthermore, we characterize the morphology of the semiconductor-electrolyte interface by atomic force microscopy and study the stability of the interface in dark and under illuminated conditions. PMID:27480642

  15. Regulation of Neuronal Cell Death by c-Abl-Hippo/MST2 Signaling Pathway

    Science.gov (United States)

    Xiao, Lei; Bai, Yujie; Qu, Aiqin; Zheng, Zheng; Yuan, Zengqiang

    2012-01-01

    Background Mammalian Ste20-like kinases (MSTs) are the mammalian homologue of Drosophila hippo and play critical roles in regulation of cell death, organ size control, proliferation and tumorigenesis. MSTs exert pro-apoptotic function through cleavage, autophosphorylation and in turn phosphorylation of downstream targets, such as Histone H2B and FOXO (Forkhead box O). Previously we reported that protein kinase c-Abl mediates oxidative stress-induced neuronal cell death through phosphorylating MST1 at Y433, which is not conserved among mammalian MST2, Drosophila Hippo and C.elegans cst-1/2. Methodology/Principal Findings Using immunoblotting, in vitro kinase and cell death assay, we demonstrate that c-Abl kinase phosphorylates MST2 at an evolutionarily conserved site, Y81, within the kinase domain. We further show that the phosphorylation of MST2 by c-Abl leads to the disruption of the interaction with Raf-1 proteins and the enhancement of homodimerization of MST2 proteins. It thereby enhances the MST2 activation and induces neuronal cell death. Conclusions/Significance The identification of the c-Abl tyrosine kinase as a novel upstream activator of MST2 suggests that the conserved c-Abl-MST signaling cascade plays an important role in oxidative stress-induced neuronal cell death. PMID:22590567

  16. Neuronal, neurohormonal, and autocrine control of Xenopus melanotrope cell activity

    NARCIS (Netherlands)

    Roubos, E.W.; Scheenen, W.J.J.M.; Jenks, B.G.

    2005-01-01

    Amphibian pituitary melanotropes are used to investigate principles of neuroendocrine translation of neural input into hormonal output. Here, the steps in this translation process are outlined for the melanotrope cell of Xenopus laevis, with attention to external stimuli, neurochemical messengers, r

  17. Postscript: About Grandmother Cells and Jennifer Aniston Neurons

    Science.gov (United States)

    Quian Quiroga, Rodrigo; Kreiman, Gabriel

    2010-01-01

    The current authors reply to a response by Bowers on a comment by the current authors on the original article. A typical problem in any discussion about grandmother cells is that there is not a general consensus about what should be called as such. Here, we discuss possible interpretations in turn and contrast them with what we find in our own…

  18. The role of alpha-synuclein in melanin synthesis in melanoma and dopaminergic neuronal cells.

    Directory of Open Access Journals (Sweden)

    Tianhong Pan

    Full Text Available The relatively high co-occurrence of Parkinson's disease (PD and melanoma has been established by a large number of epidemiological studies. However, a clear biological explanation for this finding is still lacking. Ultra-violet radiation (UVR-induced skin melanin synthesis is a defense mechanism against UVR-induced damage relevant to the initiation of melanoma, whereas, increased neuromelanin (NM, the melanin synthesized in dopaminergic neurons, may enhance the susceptibility to oxidative stress-induced neuronal injury relevant to PD. SNCA is a PD-causing gene coding for alpha-Synuclein (α-Syn that expresses not only in brain, but also in skin as well as in tumors, such as melanoma. The findings that α-Syn can interact with tyrosinase (TYR and inhibit tyrosine hydroxylase (TH, both of which are enzymes involved in the biosynthesis of melanin and dopamine (DA, led us to propose that α-Syn may participate in the regulation of melanin synthesis. In this study, by applying ultraviolet B (UVB light, a physiologically relevant stimulus of melanogenesis, we detected melanin synthesis in A375 and SK-MEL-28 melanoma cells and in SH-SY5Y and PC12 dopaminergic neuronal cells and determined effects of α-Syn on melanin synthesis. Our results showed that UVB light exposure increased melanin synthesis in all 4 cell lines. However, we found that α-Syn expression reduced UVB light-induced increase of melanin synthesis and that melanin content was lower when melanoma cells were expressed with α-Syn, indicating that α-Syn may have inhibitory effects on melanin synthesis in melanoma cells. Different from melanoma cells, the melanin content was higher in α-Syn-over-expressed dopaminergic neuronal SH-SY5Y and PC12 cells, cellular models of PD, than that in non-α-Syn-expressed control cells. We concluded that α-Syn could be one of the points responsible for the positive association between PD and melanoma via its differential roles in melanin synthesis in

  19. Higher sensitivity to cadmium induced cell death of basal forebrain cholinergic neurons: A cholinesterase dependent mechanism

    International Nuclear Information System (INIS)

    Cadmium is an environmental pollutant, which is a cause of concern because it can be greatly concentrated in the organism causing severe damage to a variety of organs including the nervous system which is one of the most affected. Cadmium has been reported to produce learning and memory dysfunctions and Alzheimer like symptoms, though the mechanism is unknown. On the other hand, cholinergic system in central nervous system (CNS) is implicated on learning and memory regulation, and it has been reported that cadmium can affect cholinergic transmission and it can also induce selective toxicity on cholinergic system at peripheral level, producing cholinergic neurons loss, which may explain cadmium effects on learning and memory processes if produced on central level. The present study is aimed at researching the selective neurotoxicity induced by cadmium on cholinergic system in CNS. For this purpose we evaluated, in basal forebrain region, the cadmium toxic effects on neuronal viability and the cholinergic mechanisms related to it on NS56 cholinergic mourine septal cell line. This study proves that cadmium induces a more pronounced, but not selective, cell death on acetylcholinesterase (AChE) on cholinergic neurons. Moreover, MTT and LDH assays showed a dose dependent decrease of cell viability in NS56 cells. The ACh treatment of SN56 cells did not revert cell viability reduction induced by cadmium, but siRNA transfection against AChE partially reduced it. Our present results provide new understanding of the mechanisms contributing to the harmful effects of cadmium on the function and viability of neurons, and the possible relevance of cadmium in the pathogenesis of neurodegenerative diseases

  20. Connectivity from OR37 expressing olfactory sensory neurons to distinct cell types in the hypothalamus

    Directory of Open Access Journals (Sweden)

    Andrea eBader

    2012-11-01

    Full Text Available Olfactory sensory neurons which express a member from the OR37 subfamily of odorant receptor genes are wired to the main olfactory bulb in a unique monoglomerular fashion; from these glomeruli an untypical connectivity into higher brain centers exists. In the present study we have investigated by DiI and transsynaptic tracing approaches how the connection pattern from these glomeruli into distinct hypothalamic nuclei is organized. The application of DiI onto the ventral domain of the bulb which harbors the OR37 glomeruli resulted in the labeling of fibers within the paraventricular and supraoptic nucleus of the hypothalamus; some of these fibers were covered with varicose-like structures. No DiI-labeled cell somata were detectable in these nuclei. The data indicate that projection neurons which originate in the OR37 region of the main olfactory bulb form direct connections into these nuclei. The cells that were labeled by the transsynaptic tracer WGA in these nuclei were further characterized. Their distribution pattern in the paraventricular nucleus was reminiscent of cells which produce distinct neuropeptides. Double labeling experiments confirmed that they contained vasopressin, but not the related neuropeptide oxytocin. Morphological analysis revealed that they comprise of magno- and parvocellular cells. A comparative investigation of the WGA-positive cells in the supraoptic nucleus demonstrated that these were vasopressin-positive, as well, whereas oxytocin-producing cells of this nucleus also contained no transsynaptic tracer. Together, the data demonstrate a connectivity from OR37 expressing sensory neurons to distinct hypothalamic neurons with the same neuropeptide content.

  1. Regional differentiation of retinoic acid-induced human pluripotent embryonic carcinoma stem cell neurons.

    Directory of Open Access Journals (Sweden)

    Dennis E Coyle

    Full Text Available The NTERA2 cl D1 (NT2 cell line, derived from human teratocarcinoma, exhibits similar properties as embryonic stem (ES cells or very early neuroepithelial progenitors. NT2 cells can be induced to become postmitotic central nervous system neurons (NT2N with retinoic acid. Although neurons derived from pluripotent cells, such as NT2N, have been characterized for their neurotransmitter phenotypes, their potential suitability as a donor source for neural transplantation also depends on their ability to respond to localized environmental cues from a specific region of the CNS. Therefore, our study aimed to characterize the regional transcription factors that define the rostocaudal and dorsoventral identity of NT2N derived from a monolayer differentiation paradigm using quantitative PCR (qPCR. Purified NT2N mainly expressed both GABAergic and glutamatergic phenotypes and were electrically active but did not form functional synapses. The presence of immature astrocytes and possible radial glial cells was noted. The NT2N expressed a regional transcription factor code consistent with forebrain, hindbrain and spinal cord neural progenitors but showed minimal expression of midbrain phenotypes. In the dorsoventral plane NT2N expressed both dorsal and ventral neural progenitors. Of major interest was that even under the influence of retinoic acid, a known caudalization factor, the NT2N population maintained a rostral phenotype subpopulation which expressed cortical regional transcription factors. It is proposed that understanding the regional differentiation bias of neurons derived from pluripotent stem cells will facilitate their successful integration into existing neuronal networks within the CNS.

  2. Down regulation of ribosomal protein mRNAs during neuronal differentiation of human NTERA2 cells.

    Science.gov (United States)

    Bévort, M; Leffers, H

    2000-10-01

    We have analysed the expression of 32 ribosomal protein (RP) mRNAs during retinoic acid induced neuronal differentiation of human NTERA2 cells. Except for a new S27 variant (S27v), all were down regulated both in selectively replated differentiated neurons and the most differentiated continuous cultures, i.e., non-replated cultures. However, the expression profiles of the individual RP mRNAs were different, most (L3, L7, L8, L10, L13, L23a, L27a, L36a, L39, P0, S2, S3, S3a, S4X, S6, S9, S12, S13, S16, S19, S20, S23, and S27a) exhibited a constant down regulation, whereas a few were either initially constant (L11, L32, S8, and S11) or up regulated (L6, L15, L17, L31, and S27y) and then down regulated. The expression of S27v remained elevated in the most differentiated continuous cultures but was down regulated in replated differentiated neurons. The down regulation of RP mRNAs was variable: the expression levels in differentiated replated neurons were between 10% (S3) and 90% (S11) of the levels in undifferentiated cells. The ratio between rRNA and RP mRNA changed during the differentiation; in differentiated neurons there were, on average, about half the number of RP mRNAs per rRNA as compared to undifferentiated cells. The expression profiles of a few translation-related proteins were also determined. EF1alpha1, EF1beta1, and EF1delta were down regulated, whereas the expression of the neuron and muscle specific EF1alpha2 increased. The reduction in the expression of RP mRNAs was coordinated with a reduction in the expression level of the proliferation marker PCNA. The expression levels of most RP mRNAs were lower in purified differentiated post-mitotic neurons than in the most differentiated continuous cultures, despite similar levels of PCNA, suggesting that both the differentiation state and the proliferative status of the cells affect the expression of RP mRNAs.

  3. Neurospheres induced from bone marrow stromal cells are multipotent for differentiation into neuron, astrocyte, and oligodendrocyte phenotypes

    International Nuclear Information System (INIS)

    Bone marrow stromal cells (MSCs) can be expanded rapidly in vitro and have the potential to be differentiated into neuronal, glial and endodermal cell types. However, induction for differentiation does not always have stable result. We present a new method for efficient induction and acquisition of neural progenitors, neuronal- and glial-like cells from MSCs. We demonstrate that rat MSCs can be induced to neurospheres and most cells are positive for nestin, which is an early marker of neuronal progenitors. In addition, we had success in proliferation of these neurospheres with undifferentiated characteristics and finally we could obtain large numbers of neuronal and glial phenotypes. Many of the cells expressed β-tubulin III when they were cultivated with our method. MSCs can become a valuable cell source as an autograft for clinical application involving regeneration of the central nervous system

  4. Tangential migration of glutamatergic neurons and cortical patterning during development: Lessons from Cajal-Retzius cells.

    Science.gov (United States)

    Barber, Melissa; Pierani, Alessandra

    2016-08-01

    Tangential migration is a mode of cell movement, which in the developing cerebral cortex, is defined by displacement parallel to the ventricular surface and orthogonal to the radial glial fibers. This mode of long-range migration is a strategy by which distinct neuronal classes generated from spatially and molecularly distinct origins can integrate to form appropriate neural circuits within the cortical plate. While it was previously believed that only GABAergic cortical interneurons migrate tangentially from their origins in the subpallial ganglionic eminences to integrate in the cortical plate, it is now known that transient populations of glutamatergic neurons also adopt this mode of migration. These include Cajal-Retzius cells (CRs), subplate neurons (SPs), and cortical plate transient neurons (CPTs), which have crucial roles in orchestrating the radial and tangential development of the embryonic cerebral cortex in a noncell-autonomous manner. While CRs have been extensively studied, it is only in the last decade that the molecular mechanisms governing their tangential migration have begun to be elucidated. To date, the mechanisms of SPs and CPTs tangential migration remain unknown. We therefore review the known signaling pathways, which regulate parameters of CRs migration including their motility, contact-redistribution and adhesion to the pial surface, and discuss this in the context of how CR migration may regulate their signaling activity in a spatial and temporal manner. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 847-881, 2016. PMID:26581033

  5. Cell-intrinsic mechanisms of temperature compensation in a grasshopper sensory receptor neuron.

    Science.gov (United States)

    Roemschied, Frederic A; Eberhard, Monika Jb; Schleimer, Jan-Hendrik; Ronacher, Bernhard; Schreiber, Susanne

    2014-01-01

    Changes in temperature affect biochemical reaction rates and, consequently, neural processing. The nervous systems of poikilothermic animals must have evolved mechanisms enabling them to retain their functionality under varying temperatures. Auditory receptor neurons of grasshoppers respond to sound in a surprisingly temperature-compensated manner: firing rates depend moderately on temperature, with average Q10 values around 1.5. Analysis of conductance-based neuron models reveals that temperature compensation of spike generation can be achieved solely relying on cell-intrinsic processes and despite a strong dependence of ion conductances on temperature. Remarkably, this type of temperature compensation need not come at an additional metabolic cost of spike generation. Firing rate-based information transfer is likely to increase with temperature and we derive predictions for an optimal temperature dependence of the tympanal transduction process fostering temperature compensation. The example of auditory receptor neurons demonstrates how neurons may exploit single-cell mechanisms to cope with multiple constraints in parallel.DOI: http://dx.doi.org/10.7554/eLife.02078.001. PMID:24843016

  6. Tangential migration of glutamatergic neurons and cortical patterning during development: Lessons from Cajal-Retzius cells.

    Science.gov (United States)

    Barber, Melissa; Pierani, Alessandra

    2016-08-01

    Tangential migration is a mode of cell movement, which in the developing cerebral cortex, is defined by displacement parallel to the ventricular surface and orthogonal to the radial glial fibers. This mode of long-range migration is a strategy by which distinct neuronal classes generated from spatially and molecularly distinct origins can integrate to form appropriate neural circuits within the cortical plate. While it was previously believed that only GABAergic cortical interneurons migrate tangentially from their origins in the subpallial ganglionic eminences to integrate in the cortical plate, it is now known that transient populations of glutamatergic neurons also adopt this mode of migration. These include Cajal-Retzius cells (CRs), subplate neurons (SPs), and cortical plate transient neurons (CPTs), which have crucial roles in orchestrating the radial and tangential development of the embryonic cerebral cortex in a noncell-autonomous manner. While CRs have been extensively studied, it is only in the last decade that the molecular mechanisms governing their tangential migration have begun to be elucidated. To date, the mechanisms of SPs and CPTs tangential migration remain unknown. We therefore review the known signaling pathways, which regulate parameters of CRs migration including their motility, contact-redistribution and adhesion to the pial surface, and discuss this in the context of how CR migration may regulate their signaling activity in a spatial and temporal manner. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 847-881, 2016.

  7. Microsphere-Incorporated Hybrid Thermogel for Neuronal Differentiation of Tonsil Derived Mesenchymal Stem Cells.

    Science.gov (United States)

    Patel, Madhumita; Moon, Hyo Jung; Jung, Bo Kyung; Jeong, Byeongmoon

    2015-07-15

    Neuronal differentiation of tonsil-derived mesenchymal stem cells (TMSCs) is investigated in a 3D hybrid system. The hybrid system is prepared by increasing the temperature of poly(ethylene glycol)-poly(l-alanine) aqueous solution to 37 °C through the heat-induced sol-to-gel transition, in which TMSCs and growth factor releasing microspheres are suspended. The in situ formed gel exhibits a modulus of 800 Pa at 37 °C, similar to that of brain tissue, and it is robust enough to hold the microspheres and cells during the 3D culture of TMSCs. The neuronal growth factors are released over 12-18 d, and the TMSCs in a spherical shape initially undergo multipolar elongation during the 3D culture. Significantly higher expressions of the neuronal biomarkers such as nuclear receptor related protein (Nurr-1), neuron specific enolase, microtubule associated protein-2, neurofilament-M, and glial fibrillary acidic protein are observed in both mRNA level and protein level in the hybrid systems than in the control experiments. This study proves the significance of a controlled drug delivery concept in tissue engineering or regenerative medicine, and a 3D hybrid system with controlled release of growth factors from microspheres in a thermogel can be a very promising tool. PMID:26033880

  8. Antiproliferative, genotoxic and oxidant activities of cyclosativene in rat neuron and neuroblastoma cell lines

    Directory of Open Access Journals (Sweden)

    Toğar Başak

    2014-01-01

    Full Text Available Cyclosativene (CSV is a tetracyclic sesquiterpene found in the essential oils of Centaurea cineraria (Asteraceae and Abies magnifica A. Murray (Pinaceae plants. To the best of our knowledge, its cytotoxic, genotoxic and oxidant effects have never been studied on any cell lines. Therefore, we aimed to investigate the in vitro antiproliferative and/or cytotoxic properties, antioxidant/oxidant activity and genotoxic damage potential of CSV in healthy neurons and N2a neuroblastoma (N2a-NB cell cultures. After treatment with 10-400 μg/ml of CSV for 24 h, cell proliferation was measured by the MTT (3,(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide assay. The antioxidant activity was assessed by the total antioxidant capacity (TAC and total oxidative stress (TOS assays. To evaluate the level of DNA damage, single cell gel alkaline electrophoresis (SCGE was used. The MTT assay showed that the application of CSV significantly reduced cell viability in both cell types. CSV treatments at higher doses led to decreases of TAC levels and increases of TOS levels in neuron and N2a-NB cells. The mean values of the total scores of cells showing DNA damage were not found to be significantly different from the control values in both cells. In conclusion, this study suggests that CSV has weak anticancer potential.

  9. Directed neuronal differentiation of mouse embryonic and induced pluripotent stem cells and their gene expression profiles.

    Science.gov (United States)

    Chen, Xuesong; Gu, Qi; Wang, Xiang; Ma, Qingwen; Tang, Huixiang; Yan, Xiaoshuang; Guo, Xinbing; Yan, Hao; Hao, Jie; Zeng, Fanyi

    2013-07-01

    Embryonic stem cells (ESCs) may be useful as a therapeutic source of cells for the production of healthy tissue; however, they are associated with certain challenges including immunorejection as well as ethical issues. Induced pluripotent stem cells (iPSCs) are a promising substitute since a patient's own adult cells would serve as tissue precursors. Ethical concerns prevent a full evaluation of the developmental potency of human ESCs and iPSCs, therefore, mouse iPSC models are required for protocol development and safety assessments. We used a modified culturing protocol to differentiate pluripotent cells from a mouse iPS cell line and two mouse ES cell lines into neurons. Our results indicated that all three pluripotent stem cell lines underwent nearly the same differentiation process when induced to form neurons in vitro. Genomic expression microarray profiling and single-cell RT-qPCR were used to analyze the neural lineage differentiation process, and more than one thousand differentially expressed genes involved in multiple molecular processes relevant to neural development were identified.

  10. Running rewires the neuronal network of adult-born dentate granule cells.

    Science.gov (United States)

    Vivar, Carmen; Peterson, Benjamin D; van Praag, Henriette

    2016-05-01

    Exercise improves cognition in humans and animals. Running increases neurogenesis in the dentate gyrus of the hippocampus, a brain area important for learning and memory. It is unclear how running modifies the circuitry of new dentate gyrus neurons to support their role in memory function. Here we combine retroviral labeling with rabies virus mediated trans-synaptic retrograde tracing to define and quantify new neuron afferent inputs in young adult male C57Bl/6 mice, housed with or without a running wheel for one month. Exercise resulted in a shift in new neuron networks that may promote sparse encoding and pattern separation. Neurogenesis increased in the dorsal, but not the ventral, dentate gyrus by three-fold, whereas afferent traced cell labeling doubled in number. Regional analysis indicated that running differentially affected specific inputs. Within the hippocampus the ratio of innervation from inhibitory interneurons and glutamatergic mossy cells to new neurons was reduced. Distal traced cells were located in sub-cortical and cortical regions, including perirhinal, entorhinal and sensory cortices. Innervation from entorhinal cortex (EC) was augmented, in proportion to the running-induced enhancement of adult neurogenesis. Within EC afferent input and short-term synaptic plasticity from lateral entorhinal cortex, considered to convey contextual information to the hippocampus was increased. Furthermore, running upregulated innervation from regions important for spatial memory and theta rhythm generation, including caudo-medial entorhinal cortex and subcortical medial septum, supra- and medial mammillary nuclei. Altogether, running may facilitate contextual, spatial and temporal information encoding by increasing adult hippocampal neurogenesis and by reorganization of new neuron circuitry. PMID:26589333

  11. Rodent CNS neuron development: Timing of cell birth and death

    Science.gov (United States)

    Keefe, J. R.

    1984-01-01

    Data obtained from a staged series of single paired injections of tritiated thymidine to pregnant Wistar rats or C57B16/j mice on selected embryonic days and several postnatal times are reported. All injected specimens were allowed to come to term, each litter culled to six pups and specimens were sacrificed on PN28, with fixation and embedding for paraffin and plastic embedding. The results are derived from serial paraffin sections of PN28 animals exposed to autoradiographic processing and plotted with respect to heavily labelled cell nuclei present in the selected brain stem nuclei and sensory ganglia. Counts from each time sample/structure are totalled and the percentage of cells in the total labelled population/structure represented by each injection time interval plotted.

  12. Quantitative phosphoproteomic analysis of prion-infected neuronal cells

    Directory of Open Access Journals (Sweden)

    Löwer Johannes

    2010-09-01

    Full Text Available Abstract Prion diseases or transmissible spongiform encephalopathies (TSEs are fatal diseases associated with the conversion of the cellular prion protein (PrPC to the abnormal prion protein (PrPSc. Since the molecular mechanisms in pathogenesis are widely unclear, we analyzed the global phospho-proteome and detected a differential pattern of tyrosine- and threonine phosphorylated proteins in PrPSc-replicating and pentosan polysulfate (PPS-rescued N2a cells in two-dimensional gel electrophoresis. To quantify phosphorylated proteins, we performed a SILAC (stable isotope labeling by amino acids in cell culture analysis and identified 105 proteins, which showed a regulated phosphorylation upon PrPSc infection. Among those proteins, we validated the dephosphorylation of stathmin and Cdc2 and the induced phosphorylation of cofilin in PrPSc-infected N2a cells in Western blot analyses. Our analysis showed for the first time a differentially regulated phospho-proteome in PrPSc infection, which could contribute to the establishment of novel protein markers and to the development of novel therapeutic intervention strategies in targeting prion-associated disease.

  13. Reexpression of glial fibrillary acidic protein rescues the ability of astrocytoma cells to form processes in response to neurons

    OpenAIRE

    1994-01-01

    Astroglial cells play an important role in orchestrating the migration and positioning of neurons during central nervous system development. Primary astroglia, as well as astrocytoma cells will extend long stable processes when co-cultured with granule neurons. In order to determine the function of the glial fibrillary acidic protein (GFAP), the major intermediate filament protein in astroglia and astrocytoma cells, we suppressed the expression of GFAP by stable transfection of an anti- sense...

  14. Staurosporine and extracellular matrix proteins mediate the conversion of small cell lung carcinoma cells into a neuron-like phenotype.

    Science.gov (United States)

    Murmann, Tamara; Carrillo-García, Carmen; Veit, Nadine; Courts, Cornelius; Glassmann, Alexander; Janzen, Viktor; Madea, Burkhard; Reinartz, Markus; Harzen, Anne; Nowak, Michael; Perner, Sven; Winter, Jochen; Probstmeier, Rainer

    2014-01-01

    Small cell lung carcinomas (SCLCs) represent highly aggressive tumors with an overall five-year survival rate in the range of 5 to 10%. Here, we show that four out of five SCLC cell lines reversibly develop a neuron-like phenotype on extracellular matrix constituents such as fibronectin, laminin or thrombospondin upon staurosporine treatment in an RGD/integrin-mediated manner. Neurite-like processes extend rapidly with an average speed of 10 µm per hour. Depending on the cell line, staurosporine treatment affects either cell cycle arrest in G2/M phase or induction of polyploidy. Neuron-like conversion, although not accompanied by alterations in the expression pattern of a panel of neuroendocrine genes, leads to changes in protein expression as determined by two-dimensional gel electrophoresis. It is likely that SCLC cells already harbour the complete molecular repertoire to convert into a neuron-like phenotype. More extensive studies are needed to evaluate whether the conversion potential of SCLC cells is suitable for therapeutic interventions.

  15. Staurosporine and extracellular matrix proteins mediate the conversion of small cell lung carcinoma cells into a neuron-like phenotype.

    Directory of Open Access Journals (Sweden)

    Tamara Murmann

    Full Text Available Small cell lung carcinomas (SCLCs represent highly aggressive tumors with an overall five-year survival rate in the range of 5 to 10%. Here, we show that four out of five SCLC cell lines reversibly develop a neuron-like phenotype on extracellular matrix constituents such as fibronectin, laminin or thrombospondin upon staurosporine treatment in an RGD/integrin-mediated manner. Neurite-like processes extend rapidly with an average speed of 10 µm per hour. Depending on the cell line, staurosporine treatment affects either cell cycle arrest in G2/M phase or induction of polyploidy. Neuron-like conversion, although not accompanied by alterations in the expression pattern of a panel of neuroendocrine genes, leads to changes in protein expression as determined by two-dimensional gel electrophoresis. It is likely that SCLC cells already harbour the complete molecular repertoire to convert into a neuron-like phenotype. More extensive studies are needed to evaluate whether the conversion potential of SCLC cells is suitable for therapeutic interventions.

  16. Zinc Deficiency Induces Apoptosis via Mitochondrial p53- and Caspase-Dependent Pathways in Human Neuronal Precursor Cells

    Science.gov (United States)

    Seth, Rohit; Corniola, Rikki S.; Gower-Winter, Shannon D.; Morgan, Thomas J., Jr.; Bishop, Brian; Levenson, Cathy W.

    2015-01-01

    Previous studies have shown that zinc deficiency leads to apoptosis of neuronal precursor cells in vivo and in vitro. In addition to the role of p53 as a nuclear transcription factor in zinc deficient cultured human neuronal precursors (NT-2), we have now identified the translocation of phosphorylated p53 to the mitochondria and p53-dependent…

  17. Effects of cerebrolysin on motor-neuron-like NSC-34 cells.

    Science.gov (United States)

    Keilhoff, Gerburg; Lucas, Benjamin; Pinkernelle, Josephine; Steiner, Michael; Fansa, Hisham

    2014-10-01

    Although the peripheral nervous system is capable of regeneration, this capability is limited. As a potential means of augmenting nerve regeneration, the effects of cerebrolysin (CL)--a proteolytic peptide fraction--were tested in vitro on the motor-neuron-like NSC-34 cell line and organotypic spinal cord cultures. Therefore, NSC-34 cells were subjected to mechanical stress by changing media and metabolic stress by oxygen glucose deprivation. Afterwards, cell survival/proliferation using MTT and BrdU-labeling (FACS) and neurite sprouting using ImageJ analysis were evaluated. Calpain-1, Src and α-spectrin protein expression were analyzed by Western blot. In organotypic cultures, the effect of CL on motor neuron survival and neurite sprouting was tested by immunohistochemistry. CL had a temporary anti-proliferative but initially neuroprotective effect on OGD-stressed NSC-34 cells. High-dosed or repeatedly applied CL was deleterious for cell survival. CL amplified neurite reconstruction to limited extent, affected calpain-1 protein expression and influenced calpain-mediated spectrin cleavage as a function of Src expression. In organotypic spinal cord slice cultures, CL was not able to support motor neuron survival/neurite sprouting. Moreover, it hampered astroglia and microglia activities. The data suggest that CL may have only isolated positive effects on injured spinal motor neurons. High-dosed or accumulated CL seemed to have adverse effects in treatment of spinal cord injury. Further experiments are required to optimize the conditions for a safe clinical administration of CL in spinal cord injuries. PMID:24997385

  18. pSVPoMcat modifying Schwann cell to protect injured spinal neurons in rats

    Institute of Scientific and Technical Information of China (English)

    陈礼刚; 高立达; 等

    2002-01-01

    Objective:To investigate the protective effect of pSVPoMcat(myelin basic protein microgene)modifying Schwann cell on injured spinal neurons.Methods;A model of rat spinal cord injured by hemisection was used.One hundred and twenty healthy SD rats of both sexes weighing 250-300g were divided into three groups:GroupA(n=40,treated with implantation of pSPVoMcat modifying Schwann cell),GroupB(n=40,treated with implantation of Schwann cell only)and GroupC(n=400,treated with sham operation as the control).One week after operation the rat functional recovery was observed dynamically by using combined behavioral score(CBS)and cortical somatasensory evoked potentials,the spinal cord sections were stained by Nissl,acid phosphatase enzyme histochemistry and cell apoptosis was examined by metye green,terminal deoxynucleotidyl and the dUTP Nick end labeling technique.Quantitative analysis was done by computer image analysis system.Results:In Group A the injured neurons recovered well morphologically.The imaging analysis showed a result of GroupA>GroupB>Group Cin the size of the neurons (P<0.01),The percentage of ACP(acid phosphatase) stained area and the rate of apoptosis sequence were groupsAcell implantation has protective effect on injured spinal neurons and promotes recovery of injured spinal cord function in rats.

  19. Electrospun polyurethane scaffolds for proliferation and neuronal differentiation of human embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Carlberg, Bjoern; Liu, Johan [BioNano Systems Laboratory, Department of Microtechnology and Nanoscience, Chalmers University of Technology, Goeteborg, SE-412 96 (Sweden); Axell, Mathilda Zetterstroem; Kuhn, H Georg [Center for Brain Repair and Rehabilitation, Institute of Neuroscience and Physiology, University of Gothenburg, Goeteborg, SE-413 45 (Sweden); Nannmark, Ulf, E-mail: bjorn.carlberg@chalmers.s, E-mail: mathilda.zetterstrom@neuro.gu.s, E-mail: georg.kuhn@neuro.gu.s, E-mail: ulf.nannmark@anatcell.gu.s, E-mail: jliu@chalmers.s [Department of Medical Chemistry and Cell Biology, Institute of Biomedicine, University of Gothenburg, Goeteborg, SE-405 30 (Sweden)

    2009-08-15

    Adult central nervous system (CNS) tissue has a limited capacity to recover after trauma or disease. Hence, tissue engineering scaffolds intended for CNS repair and rehabilitation have been subject to intense research effort. Electrospun porous scaffolds, mimicking the natural three-dimensional environment of the in vivo extracellular matrix (ECM) and providing physical support, have been identified as promising candidates for CNS tissue engineering. The present study demonstrates in vitro culturing and neuronal differentiation of human embryonic stem cells (hESCs) on electrospun fibrous polyurethane scaffolds. Electrospun scaffolds composed of biocompatible polyurethane resin (Desmopan 9370A, Bayer MaterialScience AG) were prepared with a vertical electrospinning setup. Resulting scaffolds, with a thickness of approximately 150{mu}m, exhibited high porosity (84%) and a bimodal pore size distribution with peaks at 5-6 and 1{mu}m. The mean fiber diameter was measured to approximately 360 nm with a standard deviation of 80 nm. The undifferentiated hESC line SA002 (Cellartis AB, Goeteborg, Sweden) was seeded and cultured on the produced scaffolds and allowed propagation and then differentiation for up to 47 days. Cultivation of hESC on electrospun fibrous scaffolds proved successful and neuronal differentiation was observed via standard immunocytochemistry. The results indicate that predominantly dopaminergic tyrosine hydroxylase (TH) positive neurons are derived in co-culture with fibrous scaffolds, in comparison to reference cultures under the same differentiation conditions displaying large proportions of GFAP positive cell types. Scanning electron micrographs confirm neurite outgrowth and connection to adjacent cells, as well as cell attachment to individual fibers of the fibrous scaffold. Consequently, electrospun polyurethane scaffolds have been proven feasible as a substrate for hESC propagation and neuronal differentiation. The physical interaction between

  20. Electrospun polyurethane scaffolds for proliferation and neuronal differentiation of human embryonic stem cells

    International Nuclear Information System (INIS)

    Adult central nervous system (CNS) tissue has a limited capacity to recover after trauma or disease. Hence, tissue engineering scaffolds intended for CNS repair and rehabilitation have been subject to intense research effort. Electrospun porous scaffolds, mimicking the natural three-dimensional environment of the in vivo extracellular matrix (ECM) and providing physical support, have been identified as promising candidates for CNS tissue engineering. The present study demonstrates in vitro culturing and neuronal differentiation of human embryonic stem cells (hESCs) on electrospun fibrous polyurethane scaffolds. Electrospun scaffolds composed of biocompatible polyurethane resin (Desmopan 9370A, Bayer MaterialScience AG) were prepared with a vertical electrospinning setup. Resulting scaffolds, with a thickness of approximately 150 μm, exhibited high porosity (84%) and a bimodal pore size distribution with peaks at 5-6 and 1 μm. The mean fiber diameter was measured to approximately 360 nm with a standard deviation of 80 nm. The undifferentiated hESC line SA002 (Cellartis AB, Goeteborg, Sweden) was seeded and cultured on the produced scaffolds and allowed propagation and then differentiation for up to 47 days. Cultivation of hESC on electrospun fibrous scaffolds proved successful and neuronal differentiation was observed via standard immunocytochemistry. The results indicate that predominantly dopaminergic tyrosine hydroxylase (TH) positive neurons are derived in co-culture with fibrous scaffolds, in comparison to reference cultures under the same differentiation conditions displaying large proportions of GFAP positive cell types. Scanning electron micrographs confirm neurite outgrowth and connection to adjacent cells, as well as cell attachment to individual fibers of the fibrous scaffold. Consequently, electrospun polyurethane scaffolds have been proven feasible as a substrate for hESC propagation and neuronal differentiation. The physical interaction between cells

  1. Effects of cerebrolysin on motor-neuron-like NSC-34 cells.

    Science.gov (United States)

    Keilhoff, Gerburg; Lucas, Benjamin; Pinkernelle, Josephine; Steiner, Michael; Fansa, Hisham

    2014-10-01

    Although the peripheral nervous system is capable of regeneration, this capability is limited. As a potential means of augmenting nerve regeneration, the effects of cerebrolysin (CL)--a proteolytic peptide fraction--were tested in vitro on the motor-neuron-like NSC-34 cell line and organotypic spinal cord cultures. Therefore, NSC-34 cells were subjected to mechanical stress by changing media and metabolic stress by oxygen glucose deprivation. Afterwards, cell survival/proliferation using MTT and BrdU-labeling (FACS) and neurite sprouting using ImageJ analysis were evaluated. Calpain-1, Src and α-spectrin protein expression were analyzed by Western blot. In organotypic cultures, the effect of CL on motor neuron survival and neurite sprouting was tested by immunohistochemistry. CL had a temporary anti-proliferative but initially neuroprotective effect on OGD-stressed NSC-34 cells. High-dosed or repeatedly applied CL was deleterious for cell survival. CL amplified neurite reconstruction to limited extent, affected calpain-1 protein expression and influenced calpain-mediated spectrin cleavage as a function of Src expression. In organotypic spinal cord slice cultures, CL was not able to support motor neuron survival/neurite sprouting. Moreover, it hampered astroglia and microglia activities. The data suggest that CL may have only isolated positive effects on injured spinal motor neurons. High-dosed or accumulated CL seemed to have adverse effects in treatment of spinal cord injury. Further experiments are required to optimize the conditions for a safe clinical administration of CL in spinal cord injuries.

  2. Ethanol Activation of PKA Mediates Single-Minded 2 Expression in Neuronal Cells.

    Science.gov (United States)

    Wang, Xiaolan; Yang, Zhihua; Sun, Yinan; Zhou, Hanjing; Chu, Guangpin; Zhang, Jing; Meng, Xianfang

    2015-12-01

    Prenatal ethanol exposure can cause extensive apoptotic neurodegeneration throughout the developing central nervous system (CNS), which results in cognitive deficits and memory decline. However, the underlying mechanisms need further study. Single-minded 2 (Sim2), a transcriptional repressor, is reportedly involved in diseases that impair learning and memory, such as Down syndrome (DS) and Alzheimer's disease. It is still unknown whether Sim2 is involved in regulating ethanol-mediated neuronal injury that might ultimately lead to neuronal dysfunction and subsequent learning and memory deficits. To study the effects of ethanol on Sim2 expression and neuronal injury, we used animal models and cell culture experiments. Our results indicated that in SH-SY5Y cells, ethanol exposure increased Sim2 expression and levels of cleaved caspase 3, which is a marker for cells undergoing apoptosis. Silencing Sim2 expression attenuated caspase 3 activation and cellular apoptosis. We also found that protein kinase A (PKA) activation induced Sim2 expression, as did ethanol. Inhibiting the PKA signaling pathway with H-89 decreased Sim2 expression and cleavage of caspase 3 that was induced by ethanol in vivo and in vitro. We further found that PKA regulated Sim2 expression at the transcriptional level. These results demonstrate that ethanol leads to increased Sim2 expression via the PKA pathway, ultimately resulting in apoptotic cell death.

  3. Mechanistic studies of the toxicity of zinc gluconate in the olfactory neuronal cell line Odora.

    Science.gov (United States)

    Hsieh, Heidi; Vignesh, Kavitha Subramanian; Deepe, George S; Choubey, Divaker; Shertzer, Howard G; Genter, Mary Beth

    2016-09-01

    Zinc is both an essential and potentially toxic metal. It is widely believed that oral zinc supplementation can reduce the effects of the common cold; however, there is strong clinical evidence that intranasal (IN) zinc gluconate (ZG) gel treatment for this purpose causes anosmia, or the loss of the sense of smell, in humans. Using the rat olfactory neuron cell line, Odora, we investigated the molecular mechanism by which zinc exposure exerts its toxic effects on olfactory neurons. Following treatment of Odora cells with 100 and 200μM ZG for 0-24h, RNA-seq and in silico analyses revealed up-regulation of pathways associated with zinc metal response, oxidative stress, and ATP production. We observed that Odora cells recovered from zinc-induced oxidative stress, but ATP depletion persisted with longer exposure to ZG. ZG exposure increased levels of NLRP3 and IL-1β protein levels in a time-dependent manner, suggesting that zinc exposure may cause an inflammasome-mediated cell death, pyroptosis, in olfactory neurons. PMID:27179668

  4. Three-dimensional scaffolding to investigate neuronal derivatives of human embryonic stem cells.

    Science.gov (United States)

    Soman, Pranav; Tobe, Brian T D; Lee, Jin Woo; Winquist, Alicia A M; Singec, Ilyas; Vecchio, Kenneth S; Snyder, Evan Y; Chen, Shaochen

    2012-10-01

    Access to unlimited numbers of live human neurons derived from stem cells offers unique opportunities for in vitro modeling of neural development, disease-related cellular phenotypes, and drug testing and discovery. However, to develop informative cellular in vitro assays, it is important to consider the relevant in vivo environment of neural tissues. Biomimetic 3D scaffolds are tools to culture human neurons under defined mechanical and physico-chemical properties providing an interconnected porous structure that may potentially enable a higher or more complex organization than traditional two-dimensional monolayer conditions. It is known that even minor variations in the internal geometry and mechanical properties of 3D scaffolds can impact cell behavior including survival, growth, and cell fate choice. In this report, we describe the design and engineering of 3D synthetic polyethylene glycol (PEG)-based and biodegradable gelatin-based scaffolds generated by a free form fabrication technique with precise internal geometry and elastic stiffnesses. We show that human neurons, derived from human embryonic stem (hESC) cells, are able to adhere to these scaffolds and form organoid structures that extend in three dimensions as demonstrated by confocal and electron microscopy. Future refinements of scaffold structure, size and surface chemistries may facilitate long term experiments and designing clinically applicable bioassays.

  5. Efficient derivation of neural precursor cells, spinal motor neurons and midbrain dopaminergic neurons from human ES cells at 3% oxygen

    OpenAIRE

    Stacpoole, SRL; Bilican, B.; Webber, DJ; Luzhynskaya, A; He, XL; COMPSTON, A.; Karadottir, R.; Franklin, RJM; Chandran, S

    2011-01-01

    This protocol has been designed to generate neural precursor cells (NPCs) from human embryonic stem cells (hESCs) using a physiological oxygen (O2) level of 3% and chemically defined conditions. The first stage involves suspension culture of hESC colonies at 3% O2, where they acquire a neuroepithelial identity over two weeks. This timescale is comparable to that at 20% O2, but survival is enhanced. Sequential application of retinoic acid (RA) and purmorphamine (PM), from day 14 to 28, directs...

  6. Functional neuromuscular junctions formed by embryonic stem cell-derived motor neurons.

    Directory of Open Access Journals (Sweden)

    Joy A Umbach

    Full Text Available A key objective of stem cell biology is to create physiologically relevant cells suitable for modeling disease pathologies in vitro. Much progress towards this goal has been made in the area of motor neuron (MN disease through the development of methods to direct spinal MN formation from both embryonic and induced pluripotent stem cells. Previous studies have characterized these neurons with respect to their molecular and intrinsic functional properties. However, the synaptic activity of stem cell-derived MNs remains less well defined. In this study, we report the development of low-density co-culture conditions that encourage the formation of active neuromuscular synapses between stem cell-derived MNs and muscle cells in vitro. Fluorescence microscopy reveals the expression of numerous synaptic proteins at these contacts, while dual patch clamp recording detects both spontaneous and multi-quantal evoked synaptic responses similar to those observed in vivo. Together, these findings demonstrate that stem cell-derived MNs innervate muscle cells in a functionally relevant manner. This dual recording approach further offers a sensitive and quantitative assay platform to probe disorders of synaptic dysfunction associated with MN disease.

  7. ZFPIP/Zfp462 is involved in P19 cell pluripotency and in their neuronal fate

    International Nuclear Information System (INIS)

    The nuclear zinc finger protein ZFPIP/Zfp462 is an important factor involved in cell division during the early embryonic development of vertebrates. In pluripotent P19 cells, ZFPIP/Zfp462 takes part in cell proliferation, likely via its role in maintaining chromatin structure. To further define the function of ZFPIP/Zfp462 in the mechanisms of pluripotency and cell differentiation, we constructed a stable P19 cell line in which ZFPIP/Zfp462 knockdown is inducible. We report that ZFPIP/Zfp462 was vital for mitosis and self-renewal in pluripotent P19 cells. Its depletion induced substantial decreases in the expression of the pluripotency genes Nanog, Oct4 and Sox2 and was associated with the transient expression of specific neuronal differentiation markers. We also demonstrated that ZFPIP/Zfp462 expression appears to be unnecessary after neuronal differentiation is induced in P19 cells. Taken together, our results strongly suggest that ZFPIP/Zfp462 is a key chromatin factor involved in maintaining P19 pluripotency and in the early mechanisms of neural differentiation but that it is dispensable in differentiated P19 cells.

  8. ZFPIP/Zfp462 is involved in P19 cell pluripotency and in their neuronal fate

    Energy Technology Data Exchange (ETDEWEB)

    Masse, Julie [CNRS UMR 6061, Institut de Genetique et Developpement de Rennes (IGDR), Rennes (France); Universite de Rennes 1, 35043 Rennes cedex (France); Piquet-Pellorce, Claire [Universite de Rennes 1, 35043 Rennes cedex, EA 4427 SeRAIC (France); Viet, Justine; Guerrier, Daniel; Pellerin, Isabelle [CNRS UMR 6061, Institut de Genetique et Developpement de Rennes (IGDR), Rennes (France); Universite de Rennes 1, 35043 Rennes cedex (France); Deschamps, Stephane, E-mail: stephane.deschamps@univ-rennes1.fr [CNRS UMR 6061, Institut de Genetique et Developpement de Rennes (IGDR), Rennes (France); Universite de Rennes 1, 35043 Rennes cedex (France)

    2011-08-01

    The nuclear zinc finger protein ZFPIP/Zfp462 is an important factor involved in cell division during the early embryonic development of vertebrates. In pluripotent P19 cells, ZFPIP/Zfp462 takes part in cell proliferation, likely via its role in maintaining chromatin structure. To further define the function of ZFPIP/Zfp462 in the mechanisms of pluripotency and cell differentiation, we constructed a stable P19 cell line in which ZFPIP/Zfp462 knockdown is inducible. We report that ZFPIP/Zfp462 was vital for mitosis and self-renewal in pluripotent P19 cells. Its depletion induced substantial decreases in the expression of the pluripotency genes Nanog, Oct4 and Sox2 and was associated with the transient expression of specific neuronal differentiation markers. We also demonstrated that ZFPIP/Zfp462 expression appears to be unnecessary after neuronal differentiation is induced in P19 cells. Taken together, our results strongly suggest that ZFPIP/Zfp462 is a key chromatin factor involved in maintaining P19 pluripotency and in the early mechanisms of neural differentiation but that it is dispensable in differentiated P19 cells.

  9. Pathways to ischemic neuronal cell death: are sex differences relevant?

    Directory of Open Access Journals (Sweden)

    McCullough Louise D

    2008-06-01

    Full Text Available Abstract We have known for some time that the epidemiology of human stroke is sexually dimorphic until late in life, well beyond the years of reproductive senescence and menopause. Now, a new concept is emerging: the mechanisms and outcome of cerebral ischemic injury are influenced strongly by biological sex as well as the availability of sex steroids to the brain. The principal mammalian estrogen (17 β estradiol or E2 is neuroprotective in many types of brain injury and has been the major focus of investigation over the past several decades. However, it is becoming increasingly clear that although hormones are a major contributor to sex-specific outcomes, they do not fully account for sex-specific responses to cerebral ischemia. The purpose of this review is to highlight recent studies in cell culture and animal models that suggest that genetic sex determines experimental stroke outcome and that divergent cell death pathways are activated after an ischemic insult. These sex differences need to be identified if we are to develop efficacious neuroprotective agents for use in stroke patients.

  10. Carnosine decreased neuronal cell death through targeting glutamate system and astrocyte mitochondrial bioenergetics in cultured neuron/astrocyte exposed to OGD/recovery.

    Science.gov (United States)

    Ouyang, Li; Tian, Yueyang; Bao, Yun; Xu, Huijuan; Cheng, Jiaoyan; Wang, Bingyu; Shen, Yao; Chen, Zhong; Lyu, Jianxin

    2016-06-01

    Previously, we showed that carnosine upregulated the expression level of glutamate transporter 1 (GLT-1), which has been recognized as an important participant in the astrocyte-neuron lactate shuttle (ANLS), with ischemic model in vitro and in vivo. This study was designed to investigate the protective effect of carnosine on neuron/astrocyte co-cultures exposed to OGD/recovery, and to explore whether the ANLS or any other mechanism contributes to carnosine-induced neuroprotection on neuron/astrocyte. Co-cultures were treated with carnosine and exposed to OGD/recovery. Cell death and the extracellular levels of glutamate and GABA were measured. The mitochondrial respiration and glycolysis were detected by Seahorse Bioscience XF96 Extracellular Flux Analyzer. Results showed that carnosine decreased neuronal cell death, increased extracellular GABA level, and abolished the increase in extracellular glutamate and reversed the mitochondrial energy metabolism disorder induced by OGD/recovery. Carnosine also upregulated the mRNA level of neuronal glutamate transporter EAAC1 at 2h after OGD. Dihydrokainate, a specific inhibitor of GLT-1, decreased glycolysis but it did not affect mitochondrial respiration of the cells, and it could not reverse the increase in mitochondrial OXPHOS induced by carnosine in the co-cultures. The levels of mRNAs for monocarboxylate transporter1, 4 (MCT1, 4), which were expressed in astrocytes, and MCT2, the main neuronal MCT, were significantly increased at the early stage of recovery. Carnosine only partly reversed the increased expression of astrocytic MCT1 and MCT4. These results suggest that regulating astrocytic energy metabolism and extracellular glutamate and GABA levels but not the ANLS are involved in the carnosine-induced neuroprotection. PMID:27040711

  11. Sexual dimorphism in the human olfactory bulb: females have more neurons and glial cells than males.

    Directory of Open Access Journals (Sweden)

    Ana V Oliveira-Pinto

    Full Text Available Sex differences in the human olfactory function reportedly exist for olfactory sensitivity, odorant identification and memory, and tasks in which odors are rated based on psychological features such as familiarity, intensity, pleasantness, and others. Which might be the neural bases for these behavioral differences? The number of cells in olfactory regions, and especially the number of neurons, may represent a more accurate indicator of the neural machinery than volume or weight, but besides gross volume measures of the human olfactory bulb, no systematic study of sex differences in the absolute number of cells has yet been undertaken. In this work, we investigate a possible sexual dimorphism in the olfactory bulb, by quantifying postmortem material from 7 men and 11 women (ages 55-94 years with the isotropic fractionator, an unbiased and accurate method to estimate absolute cell numbers in brain regions. Female bulbs weighed 0.132 g in average, while male bulbs weighed 0.137 g, a non-significant difference; however, the total number of cells was 16.2 million in females, and 9.2 million in males, a significant difference of 43.2%. The number of neurons in females reached 6.9 million, being no more than 3.5 million in males, a difference of 49.3%. The number of non-neuronal cells also proved higher in women than in men: 9.3 million and 5.7 million, respectively, a significant difference of 38.7%. The same differences remained when corrected for mass. Results demonstrate a sex-related difference in the absolute number of total, neuronal and non-neuronal cells, favoring women by 40-50%. It is conceivable that these differences in quantitative cellularity may have functional impact, albeit difficult to infer how exactly this would be, without knowing the specific circuits cells make. However, the reported advantage of women as compared to men may stimulate future work on sex dimorphism of synaptic microcircuitry in the olfactory bulb.

  12. Role of gangliosides in the differentiation of human mesenchymal-derived stem cells into osteoblasts and neuronal cells.

    Science.gov (United States)

    Moussavou, Ghislain; Kwak, Dong Hoon; Lim, Malg-Um; Kim, Ji-Su; Kim, Sun-Uk; Chang, Kyu-Tae; Choo, Young-Kug

    2013-11-01

    Gangliosides are complex glycosphingolipids that are the major component of cytoplasmic cell membranes, and play a role in the control of biological processes. Human mesenchymal stem cells (hMSCs) have received considerable attention as alternative sources of adult stem cells because of their potential to differentiate into multiple cell lineages. In this study, we focus on various functional roles of gangliosides in the differentiation of hMSCs into osteoblasts or neuronal cells. A relationship between gangliosides and epidermal growth factor receptor (EGFR) activation during osteoblastic differentiation of hMSCs was observed, and the gangliosides may play a major role in the regulation of the differentiation. The roles of gangliosides in osteoblast differentiation are dependent on the origin of hMSCs. The reduction of ganglioside biosynthesis inhibited the neuronal differentiation of hMSCs during an early stage of the differentiation process, and the ganglioside expression can be used as a marker for the identification of neuronal differentiation from hMSCs.

  13. [NEURONAL DIFFERENTIATION OF PC12 CELL LINE AND MURINE NEURAL STEM CELLS ON THE CARBON NANOTUBES FILMS].

    Science.gov (United States)

    Posypanova, G A; Gaiduchenko, A I; Moskaleva, E Yu; Fedorov, G E

    2016-01-01

    The study of the interaction of nerve cells with specially designed substrates (scaffolds) with different surface characteristics at the nanoscale is a necessary step in the development of methods of stimulation of regeneration of nervous tissues, as well as to create next generation of bioelectronic devices. A promising material for such scaffolds may be carbon nanotubes (CNT) that are flexible films of graphene rolled into nano-sized cylindrical tubes. CNT were produced by chemical deposition from the gas phase. The analysis of the PC12 cells cultivated on quartz glass coated by carbon nanotubes films using electron and light microscopy has shown that CNT stimulate the proliferation and do not inhibit neuronal differentiation of PC12 cells. We have found that it is possible to obtain differentiated neurons from murine neural stem cells on the quartz glasses covered with CNT films. The data obtained indicate that the CNT films produced by chemical deposition from the gas phase onto quartz glass may be used as the electro conductive scaffold to obtain and study the functions of neural cells and possibly of mature neurons. PMID:27228654

  14. Chandelier cells control excessive cortical excitation: characteristics of whisker-evoked synaptic responses of layer 2/3 nonpyramidal and pyramidal neurons.

    Science.gov (United States)

    Zhu, Yinghua; Stornetta, Ruth L; Zhu, J Julius

    2004-06-01

    Chandelier cells form inhibitory axo-axonic synapses on pyramidal neurons with their characteristic candlestick-like axonal terminals. The functional role of chandelier cells is still unclear, although the preferential loss of this cell type at epileptic loci suggests a role in epilepsy. Here we report an examination of whisker- and spontaneous activity-evoked responses in chandelier cells and other fast-spiking nonpyramidal neurons and regular-spiking pyramidal neurons in layer 2/3 of the barrel cortex. Fast-spiking nonpyramidal neurons, including chandelier cells, basket cells, neurogliaform cells, double bouquet cells, net basket cells, bitufted cells, and regular-spiking pyramidal neurons all respond to stimulation of multiple whiskers on the contralateral face. Whisker stimulation, however, evokes small, delayed EPSPs preceded by an earlier IPSP and no action potentials in chandelier cells, different from other nonpyramidal and pyramidal neurons. In addition, chandelier cells display a larger receptive field with lower acuity than other fast-spiking nonpyramidal neurons and pyramidal neurons. Notably, simultaneous dual whole-cell in vivo recordings show that chandelier cells, which rarely fire action potentials spontaneously, fire more robustly than other types of cortical neurons when the overall cortical excitation increases. Thus, chandelier cells may not process fast ascending sensory information but instead may be reserved to prevent excessive excitatory activity in neuronal networks. PMID:15175379

  15. FolR1: a novel cell surface marker for isolating midbrain dopamine neural progenitors and nascent dopamine neurons

    Science.gov (United States)

    Gennet, Nicole; Tamburini, Claudia; Nan, Xinsheng; Li, Meng

    2016-01-01

    Cell type-specific surface markers offer a powerful tool for purifying defined cell types for restorative therapies and drug screenings. Midbrain dopaminergic neurons (mesDA) are the nerve cells preferentially lost in the brains of Parkinson’s disease patients. Clinical trials of transplantation of fetal neural precursors suggest that cell therapy may offer a cure for this devastating neurological disease. Many lines of preclinical studies demonstrate that neural progenitors committed to dopaminergic fate survive and integrate better than postmitotic DA neurons. We show that the folate-receptor 1 (FolR1), a GPI-anchored cell surface molecule, specifically marks mesDA neural progenitors and immature mesDA neurons. FolR1 expression superimposes with Lmx1a, a bona-fide mesDA lineage marker, during the active phase of mesDA neurogenesis from E9.5 to E14.5 during mouse development, as well as in ESC-derived mesDA lineage. FolR1+ neural progenitors can be isolated by FACS or magnetic sorting (MAC) which give rise to dopamine neurons expressing TH and Pitx3, whilst FolR1 negative cells generate non-dopaminergic neurons and glia cells. This study identifies FolR1 as a new cell surface marker selectively expressed in mesDA progenitors in vivo and in vitro and that can be used to enrich in vitro differentiated TH neurons. PMID:27580818

  16. FolR1: a novel cell surface marker for isolating midbrain dopamine neural progenitors and nascent dopamine neurons.

    Science.gov (United States)

    Gennet, Nicole; Tamburini, Claudia; Nan, Xinsheng; Li, Meng

    2016-01-01

    Cell type-specific surface markers offer a powerful tool for purifying defined cell types for restorative therapies and drug screenings. Midbrain dopaminergic neurons (mesDA) are the nerve cells preferentially lost in the brains of Parkinson's disease patients. Clinical trials of transplantation of fetal neural precursors suggest that cell therapy may offer a cure for this devastating neurological disease. Many lines of preclinical studies demonstrate that neural progenitors committed to dopaminergic fate survive and integrate better than postmitotic DA neurons. We show that the folate-receptor 1 (FolR1), a GPI-anchored cell surface molecule, specifically marks mesDA neural progenitors and immature mesDA neurons. FolR1 expression superimposes with Lmx1a, a bona-fide mesDA lineage marker, during the active phase of mesDA neurogenesis from E9.5 to E14.5 during mouse development, as well as in ESC-derived mesDA lineage. FolR1(+) neural progenitors can be isolated by FACS or magnetic sorting (MAC) which give rise to dopamine neurons expressing TH and Pitx3, whilst FolR1 negative cells generate non-dopaminergic neurons and glia cells. This study identifies FolR1 as a new cell surface marker selectively expressed in mesDA progenitors in vivo and in vitro and that can be used to enrich in vitro differentiated TH neurons. PMID:27580818

  17. Enriched population of PNS neurons derived from human embryonic stem cells as a platform for studying peripheral neuropathies.

    Directory of Open Access Journals (Sweden)

    Moran Valensi-Kurtz

    Full Text Available BACKGROUND: The absence of a suitable cellular model is a major obstacle for the study of peripheral neuropathies. Human embryonic stem cells hold the potential to be differentiated into peripheral neurons which makes them a suitable candidate for this purpose. However, so far the potential of hESC to differentiate into derivatives of the peripheral nervous system (PNS was not investigated enough and in particular, the few trials conducted resulted in low yields of PNS neurons. Here we describe a novel hESC differentiation method to produce enriched populations of PNS mature neurons. By plating 8 weeks hESC derived neural progenitors (hESC-NPs on laminin for two weeks in a defined medium, we demonstrate that over 70% of the resulting neurons express PNS markers and 30% of these cells are sensory neurons. METHODS/FINDINGS: Our method shows that the hNPs express neuronal crest lineage markers in a temporal manner, and by plating 8 weeks hESC-NPs into laminin coated dishes these hNPs were promoted to differentiate and give rise to homogeneous PNS neuronal populations, expressing several PNS lineage-specific markers. Importantly, these cultures produced functional neurons with electrophysiological activities typical of mature neurons. Moreover, supporting this physiological capacity implantation of 8 weeks old hESC-NPs into the neural tube of chick embryos also produced human neurons expressing specific PNS markers in vivo in just a few days. Having the enriched PNS differentiation system in hand, we show for the first time in human PNS neurons the expression of IKAP/hELP1 protein, where a splicing mutation on the gene encoding this protein causes the peripheral neuropathy Familial Dysautonomia. CONCLUSIONS/SIGNIFICANCE: We conclude that this differentiation system to produce high numbers of human PNS neurons will be useful for studying PNS related neuropathies and for developing future drug screening applications for these diseases.

  18. Possible involvement of DNA methylation in 5-azacytidine-induced neuronal cell apoptosis

    OpenAIRE

    Nakayama, Hiroyuki; Kajikawa, S.; Shinozuka, J.; Su, W. P.; Doi, K

    1999-01-01

    Eight chemicals that are cytidine analogues or nucleosides (5-azacytidine (SAzC), 5-azadeoxycytidine, 6-azacytidine, 5-azacytosin, cytidine, 3-deazaadenine, 3-deazauridine and 6-azauridine) were examined for the ability to induce neuronal apoptosis. 5AzC and 5-azadeoxycytidine induced apoptosis in the brain and spinal cord of the fetuses at 24 hr after the injection to dams, while the other chemicals tested failed to induce apoptosis. In the system of PC12 cell...

  19. Natural apoptosis in developing mice dopamine midbrain neurons and vermal Purkinje cells.

    Science.gov (United States)

    Martí-Clúa, J

    2016-01-01

    Natural cell death by apoptosis was studied in two neuronal populations of BALB/c, C57BL/6 and B6CBA-Aw-j/A hybrid stock mice: (I) dopaminergic (DA) neurons in choosing coronal levels throughout the anteroposterior extent of the substantia nigra pars compacta (SNc), and (II) Purkinje cells (PCs) in each vermal lobe of the cerebellar cortex. Mice were collected at postnatal day (P) 2 and P14 for the midbrain study, and at P4 and P7 for the analysis of the cerebellum. No DA cells with morphologic criteria for apoptosis were found. Moreover, when the combination of tyrosine hydroxylase and TUNEL or tyrosine hydroxylase and active caspase-3 immunohistochemistry were performed in the same tissue section, no DA cells TUNEL positives or active caspase-3-stained DA neurons were seen. On the other hand, when PCs were considered, data analysis revealed that more dying PCs were observed at P4 than at P7. Values of neuron death were highest in the central lobe; this was followed by the posterior and anterior lobes and then by the inferior lobe. To determine if apoptotic death of PCs is linked to their time-of-origin profiles, pregnant dams were administered with [3H]TdR on embryonic days 11-12, 12-13, 13-14 and 14-15. When TUNEL and [3H]TdR autoradiography or active caspase-3 immunohistochemistry and [3H]TdR autoradiography were combined in the same tissue section, results reveal that the naturally occurring PC death is not related to its time of origin but, rather, is random across age. PMID:27543775

  20. Coupled electrophysiological recording and single cell transcriptome analyses revealed molecular mechanisms underlying neuronal maturation

    OpenAIRE

    Chen, Xiaoying; Zhang, Kunshan; Zhou, Liqiang; Gao, Xinpei; Wang, Junbang; Yao, Yinan; He, Fei; Luo, Yuping; Yu, Yongchun; Li, Siguang; Cheng, Liming; Sun, Yi E.

    2016-01-01

    The mammalian brain is heterogeneous, containing billions of neurons and trillions of synapses forming various neural circuitries, through which sense, movement, thought, and emotion arise. The cellular heterogeneity of the brain has made it difficult to study the molecular logic of neural circuitry wiring, pruning, activation, and plasticity, until recently, transcriptome analyses with single cell resolution makes decoding of gene regulatory networks underlying aforementioned circuitry prope...

  1. Nitro-Oxidative Stress after Neuronal Ischemia Induces Protein Nitrotyrosination and Cell Death

    Directory of Open Access Journals (Sweden)

    Marta Tajes

    2013-01-01

    Full Text Available Ischemic stroke is an acute vascular event that obstructs blood supply to the brain, producing irreversible damage that affects neurons but also glial and brain vessel cells. Immediately after the stroke, the ischemic tissue produces nitric oxide (NO to recover blood perfusion but also produces superoxide anion. These compounds interact, producing peroxynitrite, which irreversibly nitrates protein tyrosines. The present study measured NO production in a human neuroblastoma (SH-SY5Y, a murine glial (BV2, a human endothelial cell line (HUVEC, and in primary cultures of human cerebral myocytes (HC-VSMCs after experimental ischemia in vitro. Neuronal, endothelial, and inducible NO synthase (NOS expression was also studied up to 24 h after ischemia, showing a different time course depending on the NOS type and the cells studied. Finally, we carried out cell viability experiments on SH-SY5Y cells with H2O2, a prooxidant agent, and with a NO donor to mimic ischemic conditions. We found that both compounds were highly toxic when they interacted, producing peroxynitrite. We obtained similar results when all cells were challenged with peroxynitrite. Our data suggest that peroxynitrite induces cell death and is a very harmful agent in brain ischemia.

  2. MicroRNA-9 promotes the neuronal differentiation of rat bone marrow mesenchymal stem cells by activating autophagy

    Directory of Open Access Journals (Sweden)

    Guang-yu Zhang

    2015-01-01

    Full Text Available MicroRNA-9 (miR-9 has been shown to promote the differentiation of bone marrow mesenchymal stem cells into neuronal cells, but the precise mechanism is unclear. Our previous study confirmed that increased autophagic activity improved the efficiency of neuronal differentiation in bone marrow mesenchymal stem cells. Accumulating evidence reveals that miRNAs adjust the autophagic pathways. This study used miR-9-1 lentiviral vector and miR-9-1 inhibitor to modulate the expression level of miR-9. Autophagic activity and neuronal differentiation were measured by the number of light chain-3 (LC3-positive dots, the ratio of LC3-II/LC3, and the expression levels of the neuronal markers enolase and microtubule-associated protein 2. Results showed that LC3-positive dots, the ratio of LC3-II/LC3, and expression of neuron specific enolase and microtubule-associated protein 2 increased in the miR-9 + group. The above results suggest that autophagic activity increased and bone marrow mesenchymal stem cells were prone to differentiate into neuronal cells when miR-9 was overexpressed, demonstrating that miR-9 can promote neuronal differentiation by increasing autophagic activity.

  3. MicroRNA-9 promotes the neuronal differentiation of rat bone marrow mesenchymal stem cells by activating autophagy

    Institute of Scientific and Technical Information of China (English)

    Guang-yu Zhang; Jun Wang; Yan-jie Jia; Rui Han; Ping Li; Deng-na Zhu

    2015-01-01

    MicroRNA-9 (miR-9) has been shown to promote the differentiation of bone marrow mesen-chymal stem cells into neuronal cells, but the precise mechanism is unclear. Our previous study conifrmed that increased autophagic activity improved the efifciency of neuronal differentiation in bone marrow mesenchymal stem cells. Accumulating evidence reveals that miRNAs adjust the autophagic pathways. This study used miR-9-1 lentiviral vector and miR-9-1 inhibitor to modulate the expression level of miR-9. Autophagic activity and neuronal differentiation were measured by the number of light chain-3 (LC3)-positive dots, the ratio of LC3-II/LC3, and the expression levels of the neuronal markers enolase and microtubule-associated protein 2. Re-sults showed that LC3-positive dots, the ratio of LC3-II/LC3, and expression of neuron speciifc enolase and microtubule-associated protein 2 increased in the miR-9+ group. The above results suggest that autophagic activity increased and bone marrow mesenchymal stem cells were prone to differentiate into neuronal cells when miR-9 was overexpressed, demonstrating that miR-9 can promote neuronal differentiation by increasing autophagic activity.

  4. Stretch Injury of Human Induced Pluripotent Stem Cell Derived Neurons in a 96 Well Format

    Science.gov (United States)

    Sherman, Sydney A.; Phillips, Jack K.; Costa, J. Tighe; Cho, Frances S.; Oungoulian, Sevan R.; Finan, John D.

    2016-01-01

    Traumatic brain injury (TBI) is a major cause of mortality and morbidity with limited therapeutic options. Traumatic axonal injury (TAI) is an important component of TBI pathology. It is difficult to reproduce TAI in animal models of closed head injury, but in vitro stretch injury models reproduce clinical TAI pathology. Existing in vitro models employ primary rodent neurons or human cancer cell line cells in low throughput formats. This in vitro neuronal stretch injury model employs human induced pluripotent stem cell-derived neurons (hiPSCNs) in a 96 well format. Silicone membranes were attached to 96 well plate tops to create stretchable, culture substrates. A custom-built device was designed and validated to apply repeatable, biofidelic strains and strain rates to these plates. A high content approach was used to measure injury in a hypothesis-free manner. These measurements are shown to provide a sensitive, dose-dependent, multi-modal description of the response to mechanical insult. hiPSCNs transition from healthy to injured phenotype at approximately 35% Lagrangian strain. Continued development of this model may create novel opportunities for drug discovery and exploration of the role of human genotype in TAI pathology. PMID:27671211

  5. The role of mitochondrial function in glutamate-dependent metabolism in neuronal cells.

    Science.gov (United States)

    Smaili, S S; Ureshino, R P; Rodrigues, L; Rocha, K K; Carvalho, J T; Oseki, K T; Bincoletto, C; Lopes, G S; Hirata, H

    2011-12-01

    Glutamate is an important neurotransmitter in neurons and glial cells and it is one of the keys to the neuron-glial interaction in the brain. Glutamate transmission is strongly dependent on calcium homeostasis and on mitochondrial function. In the present work we presented several aspects related to the role of mitochondria in glutamate signaling and in brain diseases. We focused on glutamateinduced calcium signaling and its relation to the organelle dysfunction with cell death processes. In addition, we have discussed how alterations in this pathway may lead or aggravate a variety of neurodegenerative diseases. We compiled information on how mitochondria can influence cell fate during glutamate stimulation and calcium signaling. These organelles play a pivotal role in neuron and glial exchange, in synaptic plasticity and several pathological conditions related to Aging, Alzheimer's, Parkinson's and Huntington's diseases. We have also presented autophagy as a mechanism activated during mitochondrial dysfunction which may function as a protective mechanism during injury. Furthermore, some new perspectives and approaches to treat these neurodegenerative diseases are offered and evaluated.

  6. ENA/VASP downregulation triggers cell death by impairing axonal maintenance in hippocampal neurons.

    Science.gov (United States)

    Franco, D Lorena; Rezával, Carolina; Cáceres, Alfredo; Schinder, Alejandro F; Ceriani, M Fernanda

    2010-06-01

    Neurodegenerative diseases encompass a broad variety of motor and cognitive disorders that are accompanied by death of specific neuronal populations or brain regions. Cellular and molecular mechanisms underlying these complex disorders remain largely unknown. In a previous work we searched for novel Drosophila genes relevant for neurodegeneration and singled out enabled (ena), which encodes a protein involved in cytoskeleton remodeling. To extend our understanding on the mechanisms of ENA-triggered degeneration we now investigated the effect of silencing ena ortholog genes in mouse hippocampal neurons. We found that ENA/VASP downregulation led to neurite retraction and concomitant neuronal cell death through an apoptotic pathway. Remarkably, this retraction initially affected the axonal structure, showing no effect on dendrites. Reduction in ENA/VASP levels blocked the neuritogenic effect of a specific RhoA kinase (ROCK) inhibitor, thus suggesting that these proteins could participate in the Rho-signaling pathway. Altogether these observations demonstrate that ENA/VASP proteins are implicated in the establishment and maintenance of the axonal structure and that a change on their expression levels triggers neuronal degeneration.

  7. Various tolerances to arsenic trioxide between human cortical neurons and leukemic cells

    Institute of Scientific and Technical Information of China (English)

    ZHOU Jin; MENG Ran; SUI Xinhua; LI Wenbin; YANG Baofeng

    2006-01-01

    Arsenic trioxide (As2O3) is very effective for treatment of acute promyelocytic leukaemia (APL) but little can pass through the blood-brain-barrier (BBB),which limits its use in the prevention and treatment of central nervous system leukaemia (CNSL). Before creating a non-invasive method to help As2O3 's access, the safe and effective therapeutic concentration of As2O3 in the CNS ought to be known. The changes of apoptosis biomarkers, [Ca2+]i and PKC activity of both leukaemia cells and human cortical neurons, were monitored before and after being treated with As2O3 in vitro with laser confocal microscopy and Western blot. NSE concentration, the neuron invasive biomarker, was monitored by enzyme immunoassay (NSE-EIA). This study revealed that cortical neuron was more tolerable to As2O3 compared to NB4. 1.0 μmol / L As2O3 showed little influence on cortical neuron but effectively promoted apoptosis and induced differentiation of NB4.

  8. In vitro induction and differentiation of umbilical cord mesenchymal stem cells into neuron-like cells by alltrans retinoic acid

    Institute of Scientific and Technical Information of China (English)

    Wei; Jin; Yao-Peng; Xu; An-Huai; Yang; Yi-Qiao; Xing

    2015-01-01

    AIM: To determine the optimal concentration for inducing the differentiation of human umbilical cord-derived mesenchymal stem cells(h UC-MSCs) into neuron-like cells, although it is understood that all-trans retinoic acid(ATRA) regulates cell proliferation in the nervous system by modulating the balance between mitosis and apoptosis.METHODS: The abilities of ATRA to promote apoptosis as well as neural differentiation were assessed in cultured h UC-MSCs by morphological observation, MTT assay, annexin V-FITC/PI flow cytometry and immunocytochemistry.RESULTS: The data showed that low concentrations of ATRA(0.5 μmol, 0.25 μmol) had no effect on the number of cells. However, treatment with 1.0 μmol or 2.0 μmol ATRA induced a 24.16% and 52.67% reduction in cell number, respectively, compared with vehicle-treated cultures. Further, 4.0 μmol ATRA had a potent effect on cell number, with almost no adherent cells recovered after 24 h. We further showed that 0.5 μmol ATRA caused these cells to express characteristic markers of neuronal progenitor cells.CONCLUSION: Taken together, we conclude that ATRA has a dose-dependent influence on the neural differentiation and apoptosis of h UC-MSCs. These findings have implications on the use of ATRA-differentiated h UC-MSCs for the study of neural degeneration diseases.

  9. Magnolia polyphenols attenuate oxidative and inflammatory responses in neurons and microglial cells

    Directory of Open Access Journals (Sweden)

    Chuang Dennis Y

    2013-01-01

    Full Text Available Abstract Background The bark of magnolia has been used in Oriental medicine to treat a variety of remedies, including some neurological disorders. Magnolol (Mag and honokiol (Hon are isomers of polyphenolic compounds from the bark of Magnolia officinalis, and have been identified as major active components exhibiting anti-oxidative, anti-inflammatory, and neuroprotective effects. In this study, we investigate the ability of these isomers to suppress oxidative stress in neurons stimulated by the ionotropic glutamate receptor agonist N-methyl-D-aspartate (NMDA and oxidative and inflammatory responses in microglial cells activated by interferon-γ (IFNγ and lipopolysaccharide (LPS. We also attempt to elucidate the mechanism and signaling pathways involved in cytokine-induced production of reactive oxygen species (ROS in microglial cells. Methods Dihydroethidium (DHE was used to assay superoxide production in neurons, while CM-H2DCF-DA was used to test for ROS production in murine (BV-2 and rat (HAPI immortalized microglial cells. NADPH oxidase inhibitors (for example, diphenyleneiodonium (DPI, AEBSF, and apocynin and immunocytochemistry targeting p47phox and gp91phox were used to assess the involvement of NADPH oxidase. Western blotting was used to assess iNOS and ERK1/2 expression, and the Griess reaction protocol was employed to determine nitric oxide (NO concentration. Results Exposure of Hon and Mag (1–10 μM to neurons for 24 h did not alter neuronal viability, but both compounds (10 μM inhibited NMDA-stimulated superoxide production, a pathway known to involve NADPH oxidase. In microglial cells, Hon and Mag inhibited IFNγ±LPS-induced iNOS expression, NO, and ROS production. Studies with inhibitors and immunocytochemical assay further demonstrated the important role of IFNγ activating the NADPH oxidase through the p-ERK-dependent pathway. Hon and, to a lesser extent, Mag inhibited IFNγ-induced p-ERK1/2 and its downstream pathway for

  10. Regulation of Neuronal Stem Cell Proliferation in the Hippocampus by Endothelial Ceramide

    Directory of Open Access Journals (Sweden)

    Anne Gulbins

    2016-08-01

    Full Text Available Background/Aims: Major depressive disorder is one of the most common diseases in western countries. The disease is mainly defined by its psychiatric symptoms. However, the disease has also many symptoms outside the central nervous system, in particular cardiovascular symptoms. Recent studies demonstrated that the acid sphingomyelinase/ceramide system plays an important role in the development of major depressive disorder and functions as a target of antidepressants. Methods: Here, we investigated (i whether ceramide accumulates in endothelial cells in the neurogenetic zone of the hippocampus after glucocorticosterone-mediated stress, (ii whether ceramide is released into the extracellular space of the hippocampus and (iii whether extracellular ceramide inhibits neuronal proliferation. Ceramide was determined in endothelial cell culture supernatants or extracellular hippocampus extracts by a kinase assay. Endothelial ceramide in the hippocampus was analyzed by confocal microscopy of brain sections stained with Cy3-labelled anti-ceramide antibodies and FITC-Isolectin B4. Neuronal proliferation was measured by incubation of pheochromocytoma neuronal cells with culture supernatants and extracellular hippocampus extracts. Results: Treatment of cultured endothelial cells with glucocorticosterone induces a release of ceramide into the supernatant. Likewise, treatment of mice with glucocorticosterone triggers a release of ceramide into the extracellular space of the hippocampus. The release of ceramide is inhibited by concomitant treatment with the antidepressant amitriptyline, which also inhibits the activity of the acid sphingomyelinase. Studies employing confocal microscopy revealed that ceramide is formed and accumulates exclusively in endothelial cells in the hippocampus of stressed mice, a process that was again prevented by co-application of amitriptyline. Ceramide released in the culture supernatant or into the extracellular space of the

  11. Immortalized mesenchymal stem cells: an alternative to primary mesenchymal stem cells in neuronal differentiation and neuroregeneration associated studies

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    Gong Min

    2011-11-01

    Full Text Available Abstract Background Mesenchymal stem cells (MSCs can be induced to differentiate into neuronal cells under appropriate cellular conditions and transplanted in brain injury and neurodegenerative diseases animal models for neuroregeneration studies. In contrast to the embryonic stem cells (ESCs, MSCs are easily subject to aging and senescence because of their finite ability of self-renewal. MSCs senescence seriously affected theirs application prospects as a promising tool for cell-based regenerative medicine and tissue engineering. In the present study, we established a reversible immortalized mesenchymal stem cells (IMSCs line by using SSR#69 retrovirus expressing simian virus 40 large T (SV40T antigen as an alternative to primary MSCs. Methods The retroviral vector SSR#69 expressing simian virus 40 large T (SV40T antigen was used to construct IMSCs. IMSCs were identified by flow cytometry to detect cell surface makers. To investigate proliferation and differentiation potential of IMSCs, cell growth curve determination and mesodermal trilineage differentiation tests were performed. Neuronal differentiation characteristics of IMSCs were detected in vitro. Before IMSCs transplantation, we excluded its tumorigenicity in nude mice firstly. The Morris water maze tests and shuttle box tests were performed five weeks after HIBD models received cells transplantation therapy. Results In this study, reversible IMSCs were constructed successfully and had the similar morphology and cell surface makers as primary MSCs. IMSCs possessed better ability of proliferation and anti-senescence compared with primary MSCs, while maintained multilineage differentiation capacity. Neural-like cells derived from IMSCs had similar expressions of neural-specific genes, protein expression patterns and resting membrane potential (RMP compared with their counterparts derived from primary MSCs. There was no bump formation in nude mice subcutaneously injected with IMSCs. IMSCs

  12. Quantitative assessment of neurite outgrowth in human embryonic stem-cell derived neurons using automated high-content image analysis

    Science.gov (United States)

    During development neurons undergo a number of morphological changes including neurite outgrowth from the cell body. Exposure to neurotoxicants that interfere with this process may cause in permanent deficits in nervous system function. While many studies have used rodent primary...

  13. Combined exposure to simulated microgravity and acute or chronic radiation reduces neuronal network integrity and cell survival

    Science.gov (United States)

    Benotmane, Rafi

    During orbital or interplanetary space flights, astronauts are exposed to cosmic radiations and microgravity. This study aimed at assessing the effect of these combined conditions on neuronal network density, cell morphology and survival, using well-connected mouse cortical neuron cultures. To this end, neurons were exposed to acute low and high doses of low LET (X-rays) radiation or to chronic low dose-rate of high LET neutron irradiation (Californium-252), under the simulated microgravity generated by the Random Positioning Machine (RPM, Dutch space). High content image analysis of cortical neurons positive for the neuronal marker βIII-tubulin unveiled a reduced neuronal network integrity and connectivity, and an altered cell morphology after exposure to acute/chronic radiation or to simulated microgravity. Additionally, in both conditions, a defect in DNA-repair efficiency was revealed by an increased number of γH2AX-positive foci, as well as an increased number of Annexin V-positive apoptotic neurons. Of interest, when combining both simulated space conditions, we noted a synergistic effect on neuronal network density, neuronal morphology, cell survival and DNA repair. Furthermore, these observations are in agreement with preliminary gene expression data, revealing modulations in cytoskeletal and apoptosis-related genes after exposure to simulated microgravity. In conclusion, the observed in vitro changes in neuronal network integrity and cell survival induced by space simulated conditions provide us with mechanistic understanding to evaluate health risks and the development of countermeasures to prevent neurological disorders in astronauts over long-term space travels. Acknowledgements: This work is supported partly by the EU-FP7 projects CEREBRAD (n° 295552)

  14. Effects of Bee Venom on Glutamate-Induced Toxicity in Neuronal and Glial Cells

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    Sang Min Lee

    2012-01-01

    Full Text Available Bee venom (BV, which is extracted from honeybees, is used in traditional Korean medical therapy. Several groups have demonstrated the anti-inflammatory effects of BV in osteoarthritis both in vivo and in vitro. Glutamate is the predominant excitatory neurotransmitter in the central nervous system (CNS. Changes in glutamate release and uptake due to alterations in the activity of glutamate transporters have been reported in many neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis. To assess if BV can prevent glutamate-mediated neurotoxicity, we examined cell viability and signal transduction in glutamate-treated neuronal and microglial cells in the presence and absence of BV. We induced glutamatergic toxicity in neuronal cells and microglial cells and found that BV protected against cell death. Furthermore, BV significantly inhibited the cellular toxicity of glutamate, and pretreatment with BV altered MAP kinase activation (e.g., JNK, ERK, and p38 following exposure to glutamate. These findings suggest that treatment with BV may be helpful in reducing glutamatergic cell toxicity in neurodegenerative diseases.

  15. A transient expression of Prospero promotes cell cycle exit of Drosophila postembryonic neurons through the regulation of Dacapo.

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    Jordi Colonques

    Full Text Available Cell proliferation, specification and terminal differentiation must be precisely coordinated during brain development to ensure the correct production of different neuronal populations. Most Drosophila neuroblasts (NBs divide asymmetrically to generate a new NB and an intermediate progenitor called ganglion mother cell (GMC which divides only once to generate two postmitotic cells called ganglion cells (GCs that subsequently differentiate into neurons. During the asymmetric division of NBs, the homeodomain transcription factor PROSPERO is segregated into the GMC where it plays a key role as cell fate determinant. Previous work on embryonic neurogenesis has shown that PROSPERO is not expressed in postmitotic neuronal progeny. Thus, PROSPERO is thought to function in the GMC by repressing genes required for cell-cycle progression and activating genes involved in terminal differentiation. Here we focus on postembryonic neurogenesis and show that the expression of PROSPERO is transiently upregulated in the newly born neuronal progeny generated by most of the larval NBs of the OL and CB. Moreover, we provide evidence that this expression of PROSPERO in GCs inhibits their cell cycle progression by activating the expression of the cyclin-dependent kinase inhibitor (CKI DACAPO. These findings imply that PROSPERO, in addition to its known role as cell fate determinant in GMCs, provides a transient signal to ensure a precise timing for cell cycle exit of prospective neurons, and hence may link the mechanisms that regulate neurogenesis and those that control cell cycle progression in postembryonic brain development.

  16. Cell Signaling Mechanisms by which Geniposide Regulates Insulin- Degrading Enzyme Expression in Primary Cortical Neurons.

    Science.gov (United States)

    Zhang, Yonglan; Xia, Zhining; Liu, Jianhui; Yin, Fei

    2015-01-01

    An increasing number of studies have demonstrated that insulin-degrading enzyme (IDE) plays an essential role in both the degradation and its activity of β-amyloid (Aβ). Therefore, the regulation of IDE expression and/or modification of IDE-dependent actions are two emerging strategies for the treatment of Alzheimer's disease (AD). We previously observed that geniposide, a novel agonist of glucagon-like peptide 1 receptor (GLP-1R), could attenuate Aβ-induced neurotoxicity by regulating the expression of IDE in primary cortical neurons. However, the signal transduction mechanisms underlying this effect were not elucidated. The present study, therefore examined and explored the cell signaling transduction and molecular mechanisms by which geniposide induces the expression of IDE in primary cortical neurons. The current study revealed that LY294002 (an inhibitor for phosphatidyl inositol 3-kinase, PI3K), PP1 (inhibitor for c-Src), GW9662 (antagonist for peroxisome proliferator-activated receptor γ, PPARγ), H89 (an inhibitor for protein kinase A, PKA) and AG1478 (an antagonist for epidermal growth factor receptor, EGFR) prohibited the up-regulation of IDE induced by geniposide in primary cortical neurons. Further, geniposide also enhanced the phosphorylation of PPARγ and accelerated the release of phosphorylated FoxO1 (forkhead box O1) from nuclear fraction to the cytosol. Moreover, geniposide directly activated the activity of IDE promoter in PC12 cells, which confirmed the presence of the GLP-1 receptor. Taken together, our findings reveal for the first time the cell signaling transduction pathway of geniposide regulating the expression of IDE in neurons.

  17. Neurons and Glial Cells Are Added to the Female Rat Anteroventral Periventricular Nucleus During Puberty.

    Science.gov (United States)

    Mohr, Margaret A; Garcia, Francisca L; DonCarlos, Lydia L; Sisk, Cheryl L

    2016-06-01

    The anteroventral periventricular nucleus (AVPV) orchestrates the neuroendocrine-positive feedback response that triggers ovulation in female rodents. The AVPV is larger and more cell-dense in females than in males, and during puberty, only females develop the capacity to show a positive feedback response. We previously reported a potential new mechanism to explain this female-specific gain of function during puberty, namely a female-biased sex difference in the pubertal addition of new cells to the rat AVPV. Here we first asked whether this sex difference is due to greater cell proliferation and/or survival in females. Female and male rats received the cell birthdate marker 5-bromo-2'-deoxyuridine (BrdU; 200 mg/kg, ip) on postnatal day (P) 30; brains were collected at short and long intervals after BrdU administration to assess cell proliferation and survival, respectively. Overall, females had more BrdU-immunoreactive cells in the AVPV than did males, with no sex differences in the rate of cell attrition over time. Thus, the sex difference in pubertal addition of AVPV cells appears to be due to greater cell proliferation in females. Next, to determine the phenotype of pubertally born AVPV cells, daily BrdU injections were given to female rats on P28-56, and tissue was collected on P77 to assess colocalization of BrdU and markers for mature neurons or glia. Of the pubertally born AVPV cells, approximately 15% differentiated into neurons, approximately 19% into astrocytes, and approximately 23% into microglia. Thus, both neuro- and gliogenesis occur in the pubertal female rat AVPV and potentially contribute to maturation of female reproductive function. PMID:27145006

  18. Ctip2-, Satb2-, Prox1-, and GAD65-Expressing Neurons in Rat Cultures: Preponderance of Single- and Double-Positive Cells, and Cell Type-Specific Expression of Neuron-Specific Gene Family Members, Nsg-1 (NEEP21) and Nsg-2 (P19).

    Science.gov (United States)

    Digilio, Laura; Yap, Chan Choo; Winckler, Bettina

    2015-01-01

    The brain consists of many distinct neuronal cell types, but which cell types are present in widely used primary cultures of embryonic rodent brain is often not known. We characterized how abundantly four cell type markers (Ctip2, Satb2, Prox1, GAD65) were represented in cultured rat neurons, how easily neurons expressing different markers can be transfected with commonly used plasmids, and whether neuronal-enriched endosomal proteins Nsg-1 (NEEP21) and Nsg-2 (P19) are ubiquitously expressed in all types of cultured neurons. We found that cultured neurons stably maintain cell type identities that are reflective of cell types in vivo. This includes neurons maintaining simultaneous expression of two transcription factors, such as Ctip2+/Satb2+ or Prox1+/Ctip2+ double-positive cells, which have also been described in vivo. Secondly, we established the superior efficiency of CAG promoters for both Lipofectamine-mediated transfection as well as for electroporation. Thirdly, we discovered that Nsg-1 and Nsg-2 were not expressed equally in all neurons: whereas high levels of both Nsg-1 and Nsg-2 were found in Satb2-, Ctip2-, and GAD65-positive neurons, Prox1-positive neurons in hippocampal cultures expressed low levels of both. Our findings thus highlight the importance of identifying neuronal cell types for doing cell biology in cultured neurons: Keeping track of neuronal cell type might uncover effects in assays that might otherwise be masked by the mixture of responsive and non-responsive neurons in the dish.

  19. Efficient Generation of Corticofugal Projection Neurons from Human Embryonic Stem Cells

    OpenAIRE

    Xiaoqing Zhu; Zongyong Ai; Xintian Hu; Tianqing Li

    2016-01-01

    Efforts to study development and function of corticofugal projection neurons (CfuPNs) in the human cerebral cortex for health and disease have been limited by the unavailability of highly enriched CfuPNs. Here, we develop a robust, two-step process for generating CfuPNs from human embryonic stem cells (hESCs): directed induction of neuroepithelial stem cells (NESCs) from hESCs and efficient differentiation of NESCs to about 80% of CfuPNs. NESCs or a NESC faithfully maintain unlimitedly self-r...

  20. Compartmentalization of GABA synthesis by GAD67 differs between pancreatic beta cells and neurons

    DEFF Research Database (Denmark)

    Kanaani, Jamil; Cianciaruso, Chiara; Phelps, Edward A;

    2015-01-01

    The inhibitory neurotransmitter GABA is synthesized by the enzyme glutamic acid decarboxylase (GAD) in neurons and in pancreatic β-cells in islets of Langerhans where it functions as a paracrine and autocrine signaling molecule regulating the function of islet endocrine cells. The localization...... of the two non-allelic isoforms GAD65 and GAD67 to vesicular membranes is important for rapid delivery and accumulation of GABA for regulated secretion. While the membrane anchoring and trafficking of GAD65 are mediated by intrinsic hydrophobic modifications, GAD67 remains hydrophilic, and yet is targeted...... accumulation of newly synthesized GABA for regulated secretion and fine tuning of GABA-signaling in islets of Langerhans....

  1. Cell-attached recordings of responses evoked by photorelease of GABA in the immature cortical neurons

    OpenAIRE

    Marat eMinlebaev; Guzel eValeeva; Vadim eTcheremiskine; Gaelle eCoustillier; Rustem eKhazipov

    2013-01-01

    We present a novel non-invasive technique to measure the polarity of GABAergic responses based on cell-attached recordings of currents activated by laser-uncaging of GABA. For these recordings, a patch pipette was filled with a solution containing RuBi-GABA, and GABA was released from this complex by a laser beam conducted to the tip of the patch pipette via an optic fiber. In cell-attached recordings from neocortical and hippocampal neurons in postnatal days P2-5 rat brain slices in vitro, w...

  2. Accelerated neuronal cell recovery from Botulinum neurotoxin intoxication by targeted ubiquitination.

    Directory of Open Access Journals (Sweden)

    Chueh-Ling Kuo

    Full Text Available Botulinum neurotoxin (BoNT, a Category A biodefense agent, delivers a protease to motor neuron cytosol that cleaves one or more soluble NSF attachment protein receptors (SNARE proteins involved in neurotransmission to cause a flaccid paralysis. No antidotes exist to reverse symptoms of BoNT intoxication so severely affected patients require artificial respiration with prolonged intensive care. Time to recovery depends on toxin serotype because the intraneuronal persistence of the seven known BoNT serotypes varies widely from days to many months. Our therapeutic antidote strategy is to develop 'targeted F-box' (TFB agents that target the different intraneuronal BoNT proteases for accelerated degradation by the ubiquitin proteasome system (UPS, thus promoting rapid recovery from all serotypes. These agents consist of a camelid heavy chain-only V(H (VHH domain specific for a BoNT protease fused to an F-box domain recognized by an intraneuronal E3-ligase. A fusion protein containing the 14 kDa anti-BoNT/A protease VHH, ALcB8, joined to a 15 kDa F-box domain region of TrCP (D5 was sufficient to cause increased ubiquitination and accelerate turnover of the targeted BoNT/A protease within neurons. Neuronal cells expressing this TFB, called D5-B8, were also substantially resistant to BoNT/A intoxication and recovered from intoxication at least 2.5 fold quicker than control neurons. Fusion of D5 to a VHH specific for BoNT/B protease (BLcB10 led to accelerated turnover of the targeted protease within neurons, thus demonstrating the modular nature of these therapeutic agents and suggesting that development of similar therapeutic agents specific to all botulinum serotypes should be readily achievable.

  3. Accelerated neuronal cell recovery from Botulinum neurotoxin intoxication by targeted ubiquitination.

    Science.gov (United States)

    Kuo, Chueh-Ling; Oyler, George A; Shoemaker, Charles B

    2011-01-01

    Botulinum neurotoxin (BoNT), a Category A biodefense agent, delivers a protease to motor neuron cytosol that cleaves one or more soluble NSF attachment protein receptors (SNARE) proteins involved in neurotransmission to cause a flaccid paralysis. No antidotes exist to reverse symptoms of BoNT intoxication so severely affected patients require artificial respiration with prolonged intensive care. Time to recovery depends on toxin serotype because the intraneuronal persistence of the seven known BoNT serotypes varies widely from days to many months. Our therapeutic antidote strategy is to develop 'targeted F-box' (TFB) agents that target the different intraneuronal BoNT proteases for accelerated degradation by the ubiquitin proteasome system (UPS), thus promoting rapid recovery from all serotypes. These agents consist of a camelid heavy chain-only V(H) (VHH) domain specific for a BoNT protease fused to an F-box domain recognized by an intraneuronal E3-ligase. A fusion protein containing the 14 kDa anti-BoNT/A protease VHH, ALcB8, joined to a 15 kDa F-box domain region of TrCP (D5) was sufficient to cause increased ubiquitination and accelerate turnover of the targeted BoNT/A protease within neurons. Neuronal cells expressing this TFB, called D5-B8, were also substantially resistant to BoNT/A intoxication and recovered from intoxication at least 2.5 fold quicker than control neurons. Fusion of D5 to a VHH specific for BoNT/B protease (BLcB10) led to accelerated turnover of the targeted protease within neurons, thus demonstrating the modular nature of these therapeutic agents and suggesting that development of similar therapeutic agents specific to all botulinum serotypes should be readily achievable. PMID:21629663

  4. Safrole oxide induced neuronal differentiation of rat bone-marrow mesenchymal stem cells by elevating Hsp70.

    Science.gov (United States)

    Zhao, YanChun; Xin, Jie; Sun, ChunHui; Zhao, BaoXiang; Zhao, Jing; Su, Le

    2012-11-01

    In a previous study, we found that at low concentrations, safrole oxide (SFO) could induce vascular endothelial cell (VEC) transdifferentiation into neuron-like cells; however, whether SFO could induce bone-marrow mesenchymal stem cell (BMSC) neural differentiation was unknown. Here, we found that SFO could effectively induce BMSC neural differentiation in the presence of serum and fibroblast growth factor 2 and did not affect cell viability at low concentrations. The levels of neuron-specific enolase and neurofilament-L were increased greatly, but that of glial fibrillary acidic protein was absent with SFO treatment for 48h. Furthermore, SFO could increase the level of heat shock protein 70 (Hsp70), an important factor in neuronal differentiation. Knockdown of Hsp70 by its small interfering RNA blocked SFO-induced BMSC differentiation. Thus, SFO is a novel inducer of BMSC differentiation to neuron-like cells and Hsp70 is implicated in the differentiation process. We provide a new tool for obtaining neuron-like cells from BMSCs and for further investigating the new effect of Hsp70 on BMSC neuronal differentiation.

  5. Methamphetamine decreases dentate gyrus stem cell self-renewal and shifts the differentiation towards neuronal fate

    Directory of Open Access Journals (Sweden)

    Sofia Baptista

    2014-09-01

    Full Text Available Methamphetamine (METH is a highly addictive psychostimulant drug of abuse that negatively interferes with neurogenesis. In fact, we have previously shown that METH triggers stem/progenitor cell death and decreases neuronal differentiation in the dentate gyrus (DG. Still, little is known regarding its effect on DG stem cell properties. Herein, we investigate the impact of METH on mice DG stem/progenitor cell self-renewal functions. METH (10 nM decreased DG stem cell self-renewal, while 1 nM delayed cell cycle in the G0/G1-to-S phase transition and increased the number of quiescent cells (G0 phase, which correlated with a decrease in cyclin E, pEGFR and pERK1/2 protein levels. Importantly, both drug concentrations (1 or 10 nM did not induce cell death. In accordance with the impairment of self-renewal capacity, METH (10 nM decreased Sox2+/Sox2+ while increased Sox2−/Sox2− pairs of daughter cells. This effect relied on N-methyl-d-aspartate (NMDA signaling, which was prevented by the NMDA receptor antagonist, MK-801 (10 μM. Moreover, METH (10 nM increased doublecortin (DCX protein levels consistent with neuronal differentiation. In conclusion, METH alters DG stem cell properties by delaying cell cycle and decreasing self-renewal capacities, mechanisms that may contribute to DG neurogenesis impairment followed by cognitive deficits verified in METH consumers.

  6. CD8 T cell-mediated killing of orexinergic neurons induces a narcolepsy-like phenotype in mice.

    Science.gov (United States)

    Bernard-Valnet, Raphaël; Yshii, Lidia; Quériault, Clémence; Nguyen, Xuan-Hung; Arthaud, Sébastien; Rodrigues, Magda; Canivet, Astrid; Morel, Anne-Laure; Matthys, Arthur; Bauer, Jan; Pignolet, Béatrice; Dauvilliers, Yves; Peyron, Christelle; Liblau, Roland S

    2016-09-27

    Narcolepsy with cataplexy is a rare and severe sleep disorder caused by the destruction of orexinergic neurons in the lateral hypothalamus. The genetic and environmental factors associated with narcolepsy, together with serologic data, collectively point to an autoimmune origin. The current animal models of narcolepsy, based on either disruption of the orexinergic neurotransmission or neurons, do not allow study of the potential autoimmune etiology. Here, we sought to generate a mouse model that allows deciphering of the immune mechanisms leading to orexin(+) neuron loss and narcolepsy development. We generated mice expressing the hemagglutinin (HA) as a "neo-self-antigen" specifically in hypothalamic orexin(+) neurons (called Orex-HA), which were transferred with effector neo-self-antigen-specific T cells to assess whether an autoimmune process could be at play in narcolepsy. Given the tight association of narcolepsy with the human leukocyte antigen (HLA) HLA-DQB1*06:02 allele, we first tested the pathogenic contribution of CD4 Th1 cells. Although these T cells readily infiltrated the hypothalamus and triggered local inflammation, they did not elicit the loss of orexin(+) neurons or clinical manifestations of narcolepsy. In contrast, the transfer of cytotoxic CD8 T cells (CTLs) led to both T-cell infiltration and specific destruction of orexin(+) neurons. This phenotype was further aggravated upon repeated injections of CTLs. In situ, CTLs interacted directly with MHC class I-expressing orexin(+) neurons, resulting in cytolytic granule polarization toward neurons. Finally, drastic neuronal loss caused manifestations mimicking human narcolepsy, such as cataplexy and sleep attacks. This work demonstrates the potential role of CTLs as final effectors of the immunopathological process in narcolepsy.

  7. Sumoylation regulates nuclear localization of lipin-1alpha in neuronal cells.

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    Guang-Hui Liu

    Full Text Available Lipin-1 is a protein that has dual functions as a phosphatidic acid phosphohydrolase (PAP and a nuclear transcriptional coactivator. It remains unknown how the nuclear localization and coactivator functions of lipin-1 are regulated. Here, we show that lipin-1 (including both the alpha and beta isoforms is modified by sumoylation at two consensus sumoylation sites. We are unable to detect sumoylation of the related proteins lipin-2 and lipin-3. Lipin-1 is sumoylated at relatively high levels in brain, where lipin-1alpha is the predominant form. In cultured embryonic cortical neurons and SH-SY5Y neuronal cells, ectopically expressed lipin-1alpha is localized in both the nucleus and the cytoplasm, and the nuclear localization is abrogated by mutating the consensus sumyolation motifs. The sumoylation site mutant of lipin-1alpha loses the capacity to coactivate the transcriptional (co- activators PGC-1alpha and MEF2, consistent with its nuclear exclusion. Thus, these results show that sumoylation facilitates the nuclear localization and transcriptional coactivator behavior of lipin-1alpha that we observe in cultured neuronal cells, and suggest that lipin-1alpha may act as a sumoylation-regulated transcriptional coactivator in brain.

  8. Nogo receptor 1 is expressed in both primary cultured glial cells and neurons

    Science.gov (United States)

    Ukai, Junichi; Imagama, Shiro; Ohgomori, Tomohiro; Ito, Zenya; Ando, Kei; Ishiguro, Naoki; Kadomatsu, Kenji

    2016-01-01

    ABSTRACT Nogo receptor (NgR) is common in myelin-derived molecules, i.e., Nogo, MAG, and OMgp, and plays important roles in both axon fasciculation and the inhibition of axonal regeneration. In contrast to NgR’s roles in neurons, its roles in glial cells have been poorly explored. Here, we found a dynamic regulation of NgR1 expression during development and neuronal injury. NgR1 mRNA was consistently expressed in the brain from embryonic day 18 to postnatal day 25. In contrast, its expression significantly decreased in the spinal cord during development. Primary cultured neurons, microglia, and astrocytes expressed NgR1. Interestingly, a contusion injury in the spinal cord led to elevated NgR1 mRNA expression at the injury site, but not in the motor cortex, 14 days after injury. Consistent with this, astrocyte activation by TGFβ1 increased NgR1 expression, while microglia activation rather decreased NgR1 expression. These results collectively suggest that NgR1 expression is enhanced in a milieu of neural injury. Our findings may provide insight into the roles of NgR1 in glial cells.

  9. Novel role of KCNQ2/3 channels in regulating neuronal cell viability.

    Science.gov (United States)

    Zhou, X; Wei, J; Song, M; Francis, K; Yu, S P

    2011-03-01

    Overactivation of certain K(+) channels can mediate excessive K(+) efflux and intracellular K(+) depletion, which are early ionic events in apoptotic cascade. The present investigation examined a possible role of the KCNQ2/3 channel or M-channel (also named Kv7.2/7.3 channels) in the pro-apoptotic process. Whole-cell recordings detected much larger M-currents (212 ± 31 pA or 10.5 ± 1.5 pA/pF) in cultured hippocampal neurons than that in cultured cortical neurons (47 ± 21 pA or 2.4 ± 0.8 pA/pF). KCNQ2/3 channel openers N-ethylmaleimide (NEM) and flupirtine caused dose-dependent K(+) efflux, intracellular K(+) depletion, and cell death in hippocampal cultures, whereas little cell death was induced by NEM in cortical cultures. The NEM-induced cell death was antagonized by co-applied KCNQ channel inhibitor XE991 (10 μM), or by elevated extracellular K(+) concentration. Supporting a mediating role of KCNQ2/3 channels in apoptosis, expression of KCNQ2 or KCNQ2/3 channels in Chinese hamster ovary (CHO) cells initiated caspase-3 activation. Consistently, application of NEM (20 μM, 8 h) in hippocampal cultures similarly caused caspase-3 activation assessed by immunocytochemical staining and western blotting. NEM increased the expression of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), induced mitochondria membrane depolarization, cytochrome c release, formation of apoptosome complex, and apoptosis-inducing factor (AIF) translocation into nuclear. All these events were attenuated by blocking KCNQ2/3 channels. These findings provide novel evidence that KCNQ2/3 channels could be an important regulator in neuronal apoptosis. PMID:20885443

  10. The Cell Death Pathway Regulates Synapse Elimination through Cleavage of Gelsolin in Caenorhabditis elegans Neurons

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    Lingfeng Meng

    2015-06-01

    Full Text Available Synapse elimination occurs in development, plasticity, and disease. Although the importance of synapse elimination has been documented in many studies, the molecular mechanisms underlying this process are unclear. Here, using the development of C. elegans RME neurons as a model, we have uncovered a function for the apoptosis pathway in synapse elimination. We find that the conserved apoptotic cell death (CED pathway and axonal mitochondria are required for the elimination of transiently formed clusters of presynaptic components in RME neurons. This function of the CED pathway involves the activation of the actin-filament-severing protein, GSNL-1. Furthermore, we show that caspase CED-3 cleaves GSNL-1 at a conserved C-terminal region and that the cleaved active form of GSNL-1 promotes its actin-severing ability. Our data suggest that activation of the CED pathway contributes to selective elimination of synapses through disassembly of the actin filament network.

  11. Novel role of KCNQ2/3 channels in regulating neuronal cell viability

    OpenAIRE

    Zhou, X.; Wei, J; Song, M; Francis, K.; Yu, S. P.

    2010-01-01

    Overactivation of certain K+ channels can mediate excessive K+ efflux and intracellular K+ depletion, which are early ionic events in apoptotic cascade. The present investigation examined a possible role of the KCNQ2/3 channel or M-channel (also named Kv7.2/7.3 channels) in the pro-apoptotic process. Whole-cell recordings detected much larger M-currents (212±31 pA or 10.5±1.5 pA/pF) in cultured hippocampal neurons than that in cultured cortical neurons (47±21 pA or 2.4±0.8 pA/pF). KCNQ2/3 cha...

  12. StearoylCoA desaturase-5: a novel regulator of neuronal cell proliferation and differentiation.

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    Debora I Sinner

    Full Text Available Recent studies have demonstrated that human stearoylCoA desaturase-1 (SCD1, a Δ9-desaturase that converts saturated fatty acids (SFA into monounsaturated fatty acids, controls the rate of lipogenesis, cell proliferation and tumorigenic capacity in cancer cells. However, the biological function of stearoylCoA desaturase-5 (SCD5, a second isoform of human SCD that is highly expressed in brain, as well as its potential role in human disease, remains unknown. In this study we report that the constitutive overexpression of human SCD5 in mouse Neuro2a cells, a widely used cell model of neuronal growth and differentiation, displayed a greater n-7 MUFA-to-SFA ratio in cell lipids compared to empty-vector transfected cells (controls. De novo synthesis of phosphatidylcholine and cholesterolesters was increased whereas phosphatidylethanolamine and triacylglycerol formation was reduced in SCD5-expressing cells with respect to their controls, suggesting a differential use of SCD5 products for lipogenic reactions. We also observed that SCD5 expression markedly accelerated the rate of cell proliferation and suppressed the induction of neurite outgrowth, a typical marker of neuronal differentiation, by retinoic acid indicating that the desaturase plays a key role in the mechanisms of cell division and differentiation. Critical signal transduction pathways that are known to modulate these processes, such epidermal growth factor receptor (EGFRAkt/ERK and Wnt, were affected by SCD5 expression. Epidermal growth factor-induced phosphorylation of EGFR, Akt and ERK was markedly blunted in SCD5-expressing cells. Furthermore, the activity of canonical Wnt was reduced whereas the non-canonical Wnt was increased by the presence of SCD5 activity. Finally, SCD5 expression increased the secretion of recombinant Wnt5a, a non-canonical Wnt, whereas it reduced the cellular and secreted levels of canonical Wnt7b. Our data suggest that, by a coordinated modulation of key

  13. Investigation of Neuronal Cell Type-Specific Gene Expression of Ca2+/Calmodulin-dependent Protein Kinase II.

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    Mima Kazuko

    2002-01-01

    Full Text Available The promoter activity of the rat Ca2+/calmodulin-dependent protein kinase II gene was analyzed using the luciferase reporter gene in neuronal and non-neuronal cell lines. Neuronal cell type-specific promoter activity was found in the 5'-flanking region of &agr; and &bgr; isoform genes of the kinase. Silencer elements were also found further upstream of promoter regions. A brain-specific protein bound to the DNA sequence of the 5'-flanking region of the gene was found by gel mobility shift analysis in the nuclear extract of the rat brain, including the cerebellum, forebrain, and brainstem, but not in that of non-neuronal tissues, including liver, kidney and spleen. The luciferase expression system and gel shift analysis can be used as an additional and better index by which to monitor gene expression in most cell types.

  14. The tumorigenicity of mouse embryonic stem cells and in vitro differentiated neuronal cells is controlled by the recipients' immune response.

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    Ralf Dressel

    Full Text Available Embryonic stem (ES cells have the potential to differentiate into all cell types and are considered as a valuable source of cells for transplantation therapies. A critical issue, however, is the risk of teratoma formation after transplantation. The effect of the immune response on the tumorigenicity of transplanted cells is poorly understood. We have systematically compared the tumorigenicity of mouse ES cells and in vitro differentiated neuronal cells in various recipients. Subcutaneous injection of 1x10(6 ES or differentiated cells into syngeneic or allogeneic immunodeficient mice resulted in teratomas in about 95% of the recipients. Both cell types did not give rise to tumors in immunocompetent allogeneic mice or xenogeneic rats. However, in 61% of cyclosporine A-treated rats teratomas developed after injection of differentiated cells. Undifferentiated ES cells did not give rise to tumors in these rats. ES cells turned out to be highly susceptible to killing by rat natural killer (NK cells due to the expression of ligands of the activating NK receptor NKG2D on ES cells. These ligands were down-regulated on differentiated cells. The activity of NK cells which is not suppressed by cyclosporine A might contribute to the prevention of teratomas after injection of ES cells but not after inoculation of differentiated cells. These findings clearly point to the importance of the immune response in this process. Interestingly, the differentiated cells must contain a tumorigenic cell population that is not present among ES cells and which might be resistant to NK cell-mediated killing.

  15. Genetic cell targeting uncovers specific neuronal types and distinct subregions in the bed nucleus of the stria terminalis.

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    Nguyen, Amanda Q; Dela Cruz, Julie A D; Sun, Yanjun; Holmes, Todd C; Xu, Xiangmin

    2016-08-15

    The bed nucleus of the stria terminalis (BNST) plays an important role in fear, stress, and anxiety. It contains a collection of subnuclei delineated by gross cytoarchitecture features; however, there has yet to be a systematic examination of specific BNST neuronal types and their associated neurochemical makeup. The present study focuses on improved characterization of the anterior BNST based on differing molecular and chemical expression aided by mouse genetics. Specific Cre driver lines crossed with a fluorescent reporter line were used for genetic cell targeting and immunochemical staining. Using this new approach, we were able to robustly identify specific excitatory and inhibitory cell types in the BNST. The presence and distribution of excitatory neurons were firmly established; glutamatergic neurons in the anterior BNST accounted for about 14% and 31% of dorsal and ventral BNST cells, respectively. GABAergic neurons expressing different isoforms of glutamic acid decarboxylase were found to have differential subregional distributions. Almost no parvalbumin-expressing cells were found in the BNST, while somatostatin-expressing cells and calretinin-expressing cells account for modest proportions of BNST cells. In addition, vasoactive intestinal peptide-expressing axonal plexuses were prominent in the oval and juxtacapsular subregions. In addition, we discovered that corticotropin-releasing hormone-expressing cells contain GABAergic and glutamatergic subpopulations. Together, this study reveals new information on excitatory and inhibitory neurons in the BNST, which will facilitate genetic dissection and functional studies of BNST subregions. J. Comp. Neurol. 524:2379-2399, 2016. © 2016 Wiley Periodicals, Inc. PMID:26718312

  16. Progesterone promotes neuronal differentiation of human umbilical cord mesenchymal stem cells in culture conditions that mimic the brain microenvironment

    Institute of Scientific and Technical Information of China (English)

    Xianying Wang; Honghai Wu; Gai Xue; Yanning Hou

    2012-01-01

    In this study, human umbilical cord mesenchymal stem cells from full-term neonates born by vaginal delivery were cultured in medium containing 150 mg/mL of brain tissue extracts from Sprague-Dawley rats (to mimic the brain microenvironment). Immunocytochemical analysis demonstrated that the cells differentiated into neuron-like cells. To evaluate the effects of progesterone as a neurosteroid on the neuronal differentiation of human umbilical cord mesenchymal stem cells, we cultured the cells in medium containing progesterone (0.1, 1, 10 μM) in addition to brain tissue extracts. Reverse transcription-PCR and flow cytometric analysis of neuron specific enolase-positive cells revealed that the percentages of these cells increased significantly following progesterone treatment, with the optimal progesterone concentration for neuron-like differentiation being 1 μM. These results suggest that progesterone can enhance the neuronal differentiation of human umbilical cord mesenchymal stem cells in culture medium containing brain tissue extracts to mimic the brain microenvironment.

  17. An Improved Method for Directional Differentiation and Efficient Production of Neurons from Embryonic Stem Cells in vitro

    Institute of Scientific and Technical Information of China (English)

    ZHOU Yufeng; FANG Feng; DONG Yongsui; LI Ge; ZHEN Hong; YI Wenlong; XIANG Zhidan

    2005-01-01

    To establish a method of directional differentiation and efficient production of neurons from embryonic stem cells (ES cells) in vitro, based on the 4-/4+ protocol described by Bain, a new method was established to induce ES cells differentiating into neurons by means of three-step differentiation using all-trans retinoic acid (ATRA) combined with astrocyte-conditioned medium (ACM) in Vitro. The totipotency of ES cells was identified by observation of cells' morphology and formations of teratoma in immunocompromised mice. The cells' differentiation was evaluated continuously by the detection of the specific cellular markers of neural stem ceils, neurons and astrocytes,including nestin, NSE and GFAP using immunohistochemistry assay. The NSE positive cells' ratio of the differentiated cells was determined by flow cytometry. It was found that the transparent circular clusters surrounding embryoid bodies induced with combining induction protocol formed just after 24 h and gradually enlarged later. This phenomenon could not be observed in EBs induced only by ATRA. The NSE positive cells' ratio irthe cells induced with ATRA and ACM was higher than that of the cells induced by ATRA at different time points of differentiation, and finally reached up to 73.5 % among the total differentiated population. It was concluded that ES cells could be induced into neurons with high purity and yield by means of inducing method combining with ATRA and ACM.

  18. MicroRNA-125b promotes neuronal differentiation in human cells by repressing multiple targets.

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    Le, Minh T N; Xie, Huangming; Zhou, Beiyan; Chia, Poh Hui; Rizk, Pamela; Um, Moonkyoung; Udolph, Gerald; Yang, Henry; Lim, Bing; Lodish, Harvey F

    2009-10-01

    MicroRNAs (miRNAs) are a class of small noncoding RNAs that regulate gene expression at the posttranscriptional level. Research on miRNAs has highlighted their importance in neural development, but the specific functions of neurally enriched miRNAs remain poorly understood. We report here the expression profile of miRNAs during neuronal differentiation in the human neuroblastoma cell line SH-SY5Y. Six miRNAs were significantly upregulated during differentiation induced by all-trans-retinoic acid and brain-derived neurotrophic factor. We demonstrated that the ectopic expression of either miR-124a or miR-125b increases the percentage of differentiated SH-SY5Y cells with neurite outgrowth. Subsequently, we focused our functional analysis on miR-125b and demonstrated the important role of this miRNA in both the spontaneous and induced differentiations of SH-SH5Y cells. miR-125b is also upregulated during the differentiation of human neural progenitor ReNcell VM cells, and miR-125b ectopic expression significantly promotes the neurite outgrowth of these cells. To identify the targets of miR-125b regulation, we profiled the global changes in gene expression following miR-125b ectopic expression in SH-SY5Y cells. miR-125b represses 164 genes that contain the seed match sequence of the miRNA and/or that are predicted to be direct targets of miR-125b by conventional methods. Pathway analysis suggests that a subset of miR-125b-repressed targets antagonizes neuronal genes in several neurogenic pathways, thereby mediating the positive effect of miR-125b on neuronal differentiation. We have further validated the binding of miR-125b to the miRNA response elements of 10 selected mRNA targets. Together, we report here for the first time the important role of miR-125b in human neuronal differentiation.

  19. A new glucocerebrosidase-deficient neuronal cell model provides a tool to probe pathophysiology and therapeutics for Gaucher disease

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    Wendy Westbroek

    2016-07-01

    Full Text Available Glucocerebrosidase is a lysosomal hydrolase involved in the breakdown of glucosylceramide. Gaucher disease, a recessive lysosomal storage disorder, is caused by mutations in the gene GBA1. Dysfunctional glucocerebrosidase leads to accumulation of glucosylceramide and glycosylsphingosine in various cell types and organs. Mutations in GBA1 are also a common genetic risk factor for Parkinson disease and related synucleinopathies. In recent years, research on the pathophysiology of Gaucher disease, the molecular link between Gaucher and Parkinson disease, and novel therapeutics, have accelerated the need for relevant cell models with GBA1 mutations. Although induced pluripotent stem cells, primary rodent neurons, and transfected neuroblastoma cell lines have been used to study the effect of glucocerebrosidase deficiency on neuronal function, these models have limitations because of challenges in culturing and propagating the cells, low yield, and the introduction of exogenous mutant GBA1. To address some of these difficulties, we established a high yield, easy-to-culture mouse neuronal cell model with nearly complete glucocerebrosidase deficiency representative of Gaucher disease. We successfully immortalized cortical neurons from embryonic null allele gba−/− mice and the control littermate (gba+/+ by infecting differentiated primary cortical neurons in culture with an EF1α-SV40T lentivirus. Immortalized gba−/− neurons lack glucocerebrosidase protein and enzyme activity, and exhibit a dramatic increase in glucosylceramide and glucosylsphingosine accumulation, enlarged lysosomes, and an impaired ATP-dependent calcium-influx response; these phenotypical characteristics were absent in gba+/+ neurons. This null allele gba−/− mouse neuronal model provides a much-needed tool to study the pathophysiology of Gaucher disease and to evaluate new therapies.

  20. Protein carbonylation, protein aggregation and neuronal cell death in a murine model of multiple sclerosis

    Science.gov (United States)

    Dasgupta, Anushka

    Many studies have suggested that oxidative stress plays an important role in the pathophysiology of both multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). Yet, the mechanism by which oxidative stress leads to tissue damage in these disorders is unclear. Recent work from our laboratory has revealed that protein carbonylation, a major oxidative modification caused by severe and/or chronic oxidative stress conditions, is elevated in MS and EAE. Furthermore, protein carbonylation has been shown to alter protein structure leading to misfolding/aggregation. These findings prompted me to hypothesize that carbonylated proteins, formed as a consequence of oxidative stress and/or decreased proteasomal activity, promote protein aggregation to mediate neuronal apoptosis in vitro and in EAE. To test this novel hypothesis, I first characterized protein carbonylation, protein aggregation and apoptosis along the spinal cord during the course of myelin-oligodendrocyte glycoprotein (MOG)35-55 peptide-induced EAE in C57BL/6 mice [Chapter 2]. The results show that carbonylated proteins accumulate throughout the course of the disease, albeit by different mechanisms: increased oxidative stress in acute EAE and decreased proteasomal activity in chronic EAE. I discovered not only that there is a temporal correlation between protein carbonylation and apoptosis but also that carbonyl levels are significantly higher in apoptotic cells. A high number of juxta-nuclear and cytoplasmic protein aggregates containing the majority of the oxidized proteins are also present during the course of EAE, which seems to be due to reduced autophagy. In chapter 3, I show that when gluthathione levels are reduced to those in EAE spinal cord, both neuron-like PC12 (nPC12) cells and primary neuronal cultures accumulate carbonylated proteins and undergo cell death (both by necrosis and apoptosis). Immunocytochemical and biochemical studies also revealed a temporal

  1. Extracellular vesicles from a muscle cell line (C2C12 enhance cell survival and neurite outgrowth of a motor neuron cell line (NSC-34

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    Roger D. Madison

    2014-02-01

    Full Text Available Introduction: There is renewed interest in extracellular vesicles over the past decade or 2 after initially being thought of as simple cellular garbage cans to rid cells of unwanted components. Although there has been intense research into the role of extracellular vesicles in the fields of tumour and stem cell biology, the possible role of extracellular vesicles in nerve regeneration is just in its infancy. Background: When a peripheral nerve is damaged, the communication between spinal cord motor neurons and their target muscles is disrupted and the result can be the loss of coordinated muscle movement. Despite state-of-the-art surgical procedures only approximately 10% of adults will recover full function after peripheral nerve repair. To improve upon such results will require a better understanding of the basic mechanisms that influence axon outgrowth and the interplay between the parent motor neuron and the distal end organ of muscle. It has previously been shown that extracellular vesicles are immunologically tolerated, display targeting ligands on their surface, and can be delivered in vivo to selected cell populations. All of these characteristics suggest that extracellular vesicles could play a significant role in nerve regeneration. Methods: We have carried out studies using 2 very well characterized cell lines, the C2C12 muscle cell line and the motor neuron cell line NSC-34 to ask the question: Do extracellular vesicles from muscle influence cell survival and/or neurite outgrowth of motor neurons? Conclusion: Our results show striking effects of extracellular vesicles derived from the muscle cell line on the motor neuron cell line in terms of neurite outgrowth and survival.

  2. Induction of cell stress in neurons from transgenic mice expressing yellow fluorescent protein: implications for neurodegeneration research.

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    Laura H Comley

    Full Text Available BACKGROUND: Mice expressing fluorescent proteins in neurons are one of the most powerful tools in modern neuroscience research and are increasingly being used for in vivo studies of neurodegeneration. However, these mice are often used under the assumption that the fluorescent proteins present are biologically inert. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that thy1-driven expression of yellow fluorescent protein (YFP in neurons triggers multiple cell stress responses at both the mRNA and protein levels in vivo. The presence of YFP in neurons also subtly altered neuronal morphology and modified the time-course of dying-back neurodegeneration in experimental axonopathy, but not in Wallerian degeneration triggered by nerve injury. CONCLUSIONS/SIGNIFICANCE: We conclude that fluorescent protein expressed in thy1-YFP mice is not biologically inert, modifies molecular and cellular characteristics of neurons in vivo, and has diverse and unpredictable effects on neurodegeneration pathways.

  3. Cell-autonomous requirement of the USP/EcR-B ecdysone receptor for mushroom body neuronal remodeling in Drosophila.

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    Lee, T; Marticke, S; Sung, C; Robinow, S; Luo, L

    2000-12-01

    Neuronal process remodeling occurs widely in the construction of both invertebrate and vertebrate nervous systems. During Drosophila metamorphosis, gamma neurons of the mushroom bodies (MBs), the center for olfactory learning in insects, undergo pruning of larval-specific dendrites and axons followed by outgrowth of adult-specific processes. To elucidate the underlying molecular mechanisms, we conducted a genetic mosaic screen and identified one ultraspiracle (usp) allele defective in larval process pruning. Consistent with the notion that USP forms a heterodimer with the ecdysone receptor (EcR), we found that the EcR-B1 isoform is specifically expressed in the MB gamma neurons, and is required for the pruning of larval processes. Surprisingly, most identified primary EcR/USP targets are dispensable for MB neuronal remodeling. Our study demonstrates cell-autonomous roles for EcR/USP in controlling neuronal remodeling, potentially through novel downstream targets. PMID:11163268

  4. Mushroom extract protects against hydrogen peroxide-induced toxicity in hepatic and neuronal human cultured cells.

    Science.gov (United States)

    Guizani, Nejib; Waly, Mostafa I

    2012-11-15

    Hydrogen peroxide is an oxidative stress agent that is associated with depletion of intracellular glutathione and inhibition of antioxidant enzymes in different cell lines. Consumption of antioxidant-rich foods reduces cellular oxidative stress and its related health problems. This study aimed to assess the antioxidant properties of mushroom, Agaricus bisporous cultivar extract, against hydrogen peroxide induced oxidative stress in cultured human hepatic (HepG2) and neuronal (SH-SY5Y) cells. In this study, hydrogen peroxide caused significant oxidative stress in HepG2 and SH-SY5Y cells as demonstrated by glutathione depletion, impairment of total antioxidant capacity and inhibition of antioxidant enzymes (glutathione peroxidase, catalase and superoxide dismutase). Agaricusbisporous extract ameliorated the observed hydrogen peroxide-induced oxidative cellular insult as indicated by restoring the activity of glutathione and the assayed antioxidant enzymes to control levels. The results suggest that mushroom extract as antioxidant properties and protects against the oxidative stress induced by hydrogen peroxide-in cultured human hepatic and neuronal cells. PMID:24261122

  5. Neuroprotective effects of bovine colostrum on intracerebral hemorrhage-induced apoptotic neuronal cell death in rats.

    Science.gov (United States)

    Kim, Sung Eun; Ko, Il Gyu; Shin, Mal Soon; Kim, Chang Ju; Ko, Young Gwan; Cho, Hanjin

    2012-08-01

    Brain cell death after intracerebral hemorrhage may be mediated in part by an apoptotic mechanism. Colostrum is the first milk produced by mammals for their young. It plays an important role in protection and development by providing various antibodies, growth factors and nutrients, and has been used for various diseases in many countries. In the present study, we investigated the anti-apoptotic effects of bovine colostrum using organotypic hippocampal slice cultures and an intracerebral hemorrhage animal model. We performed densitometric measurements of propidium iodide uptake, a step-down avoidance task, Nissl staining, and caspase-3 immunohistochemistry. The present results revealed that colostrum treatment significantly suppressed N-methyl-D-aspartic acid-induced neuronal cell death in the rat hippocampus. Moreover, colostrum treatment improved short-term memory by suppressing hemorrhage-induced apoptotic neuronal cell death and decreasing the volume of the lesion induced by intracerebral hemorrhage in the rat hippocampus. These results suggest that colostrum may have a beneficial role in recovering brain function following hemorrhagic stroke by suppressing apoptotic cell death. PMID:25624793

  6. Neuroprotective effects of bovine colostrum on intracerebral hemorrhage-induced apoptotic neuronal cell death in rats☆

    Science.gov (United States)

    Kim, Sung Eun; Ko, Il Gyu; Shin, Mal Soon; Kim, Chang Ju; Ko, Young Gwan; Cho, Hanjin

    2012-01-01

    Brain cell death after intracerebral hemorrhage may be mediated in part by an apoptotic mechanism. Colostrum is the first milk produced by mammals for their young. It plays an important role in protection and development by providing various antibodies, growth factors and nutrients, and has been used for various diseases in many countries. In the present study, we investigated the anti-apoptotic effects of bovine colostrum using organotypic hippocampal slice cultures and an intracerebral hemorrhage animal model. We performed densitometric measurements of propidium iodide uptake, a step-down avoidance task, Nissl staining, and caspase-3 immunohistochemistry. The present results revealed that colostrum treatment significantly suppressed N-methyl-D-aspartic acid-induced neuronal cell death in the rat hippocampus. Moreover, colostrum treatment improved short-term memory by suppressing hemorrhage-induced apoptotic neuronal cell death and decreasing the volume of the lesion induced by intracerebral hemorrhage in the rat hippocampus. These results suggest that colostrum may have a beneficial role in recovering brain function following hemorrhagic stroke by suppressing apoptotic cell death. PMID:25624793

  7. Natural cerebrolysin induces neuronal differentiation in bone marrow mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    Zhengzhi Wu; Yinghong Li; Andrew C. J. Huang O; Ming Li; Min Yang; Manyin Chen

    2009-01-01

    BACKGROUND: Bone marrow mesenchymal stem cells (MSCs) have been shown to differentiate into neuronal-like cells through the use of several factors, such as 2-mercaptoethanol, dimethyl sulfoxide, or monothioglycero However, these factors are not suitable for human use due to toxicity. Theoretically speaking, traditional Chinese medicine could be used as potential and safe factors.OBJECTIVE: To investigate the effect of natural cerebrolysin on neuronal-like differentiation of MSCs, based on protein and mRNA analyses.DESIGN, TIME AND SETTING: A parallel controlled, in vitro experiment was performed at the Institute of Integrated Chinese and Western Medicine, Shenzhen Hospital, Southern Medical University, between June 2006 and April 2008.MATERIALS: Natural cerebrolysin was provided by Shenzhen Institute of Integrated Chinese and Western Medicine, China. it primarily consisted of Renshen (Radix Ginseng), Tianma (Rhizoma Gastrodiae), and Yinxingye (Ginkgo Leaf) at a proportion of 1:2:2. Natural cerebrolysin extract (1:20) was prepared using conventional water extraction methodology. Each gram of extract equaled 20 grams of the crude drug. Twelve adult, male, New Zealand rabbits were included, six of which underwent intragastric administration of natural cerebrolysin extract (0.976 g/kg per day)for 1 month for natural cerebrolysin-containing serum. The remaining six rabbits received intragastric administration of equal volumes of physiological saline for normal blank serum.METHODS: Sprague Dawley male rats, 6-8 weeks old, were used to harvest tibial and femoral bone marrow. Isolation and purification of MSCs were established from the whole bone marrow by removing the non-adherent cells in primary and passage cultures. For cellular identification, MSCs from four to five passages were co-cultured with LG-DMEM media containing 10% natural cerebrolysin. Simultaneously, MSCs cultured in LG-DMEM media containing 10% blank rabbit serum served as the control group.MAIN OUTCOME

  8. Maintenance and neuronal cell differentiation of neural stem cells C17.2 correlated to medium availability sets design criteria in microfluidic systems.

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    Bu Wang

    Full Text Available BACKGROUND: Neural stem cells (NSCs play an important role in developing potential cell-based therapeutics for neurodegenerative disease. Microfluidics has proven a powerful tool in mechanistic studies of NSC differentiation. However, NSCs are prone to differentiate when the nutrients are limited, which occurs unfavorable by fast medium consumption in miniaturized culture environment. For mechanistic studies of NSCs in microfluidics, it is vital that neuronal cell differentiation is triggered by controlled factors only. Thus, we studied the correlation between available cell medium and spontaneous neuronal cell differentiation of C17.2 NSCs in standard culture medium, and proposed the necessary microfluidic design criteria to prevent undesirable cell phenotype changes. METHODOLOGY/PRINCIPAL FINDINGS: A series of microchannels with specific geometric parameters were designed to provide different amount of medium to the cells over time. A medium factor (MF, defined as the volume of stem cell culture medium divided by total number of cells at seeding and number of hours between medium replacement successfully correlated the amount of medium available to each cell averaged over time to neuronal cell differentiation. MF smaller than 8.3×10(4 µm3/cell⋅hour produced significant neuronal cell differentiation marked by cell morphological change and significantly more cells with positive β-tubulin-III and MAP2 staining than the control. When MF was equal or greater than 8.3×10(4 µm3/cell⋅hour, minimal spontaneous neuronal cell differentiation happened relative to the control. MF had minimal relation with the average neurite length. SIGNIFICANCE: MFs can be controlled easily to maintain the stem cell status of C17.2 NSCs or to induce spontaneous neuronal cell differentiation in standard stem cell culture medium. This finding is useful in designing microfluidic culture platforms for controllable NSC maintenance and differentiation. This study also

  9. Ectopic expression of neurogenin 2 alone is sufficient to induce differentiation of embryonic stem cells into mature neurons.

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    Eva C Thoma

    Full Text Available Recent studies show that combinations of defined key developmental transcription factors (TFs can reprogram somatic cells to pluripotency or induce cell conversion of one somatic cell type to another. However, it is not clear if single genes can define a cell̀s identity and if the cell fate defining potential of TFs is also operative in pluripotent stem cells in vitro. Here, we show that ectopic expression of the neural TF Neurogenin2 (Ngn2 is sufficient to induce rapid and efficient differentiation of embryonic stem cells (ESCs into mature glutamatergic neurons. Ngn2-induced neuronal differentiation did not require any additional external or internal factors and occurred even under pluripotency-promoting conditions. Differentiated cells displayed neuron-specific morphology, protein expression, and functional features, most importantly the generation of action potentials and contacts with hippocampal neurons. Gene expression analyses revealed that Ngn2-induced in vitro differentiation partially resembled neurogenesis in vivo, as it included specific activation of Ngn2 target genes and interaction partners. These findings demonstrate that a single gene is sufficient to determine cell fate decisions of uncommitted stem cells thus giving insights into the role of key developmental genes during lineage commitment. Furthermore, we present a promising tool to improve directed differentiation strategies for applications in both stem cell research and regenerative medicine.

  10. Dynamic changes in hair cell ribbon synapse induced by loss of spiral ganglion neurons in mice

    Institute of Scientific and Technical Information of China (English)

    Yuan Yasheng; Chi Fanglu

    2014-01-01

    Background Previous studies have suggested that primary degeneration of hair cells causes secondary degeneration of spiral ganglion neurons (SGNs),but the effect of SGN degeneration on hair cells has not been studied.In the adult mouse inner ear ouabain can selectively and permanently induce the degeneration of type 1 SGNs while leaving type 2 SGNs,efferent fibers,and sensory hair cells relatively intact.This study aimed to investigate the dynamic changes in hair cell ribbon synapse induced by loss of SGNs using ouabain application to the round window niche of adult mice.Methods In the analysis,24 CBA/CAJ mice aged 8-10 weeks,were used,of which 6 normal mice were used as the control group.After ouabain application in the round window niche 6 times in an hour,ABR threshold shifts at least 30 dB in the three experimental groups which had six mice for 1-week group,six for 1-month group,and six for 3-month group.All 24 animals underwent function test at 1 week and then immunostaining at 1 week,1 month,and 3 months.Results The loss of neurons was followed by degeneration of postsynaptic specializations at the afferent synapse with hair cells.One week after ouabain treatment,the nerve endings of type 1 SGNs and postsynaptic densities,as measured by Na/K ATPase and PSD-95,were affected but not entirely missing,but their partial loss had consequences for synaptic ribbons that form the presynaptic specialization at the synapse between hair cells and primary afferent neurons.Ribbon numbers in inner hair cells decreased (some of them broken and the ribbon number much decreased),and the arrangement of the synaptic ribbons had undergone a dynamic reorganization:ribbons with or without associated postsynaptic densities moved from their normal location in the basal membrane of the cell to a more apical location and the neural endings alone were also found at more apical locations without associated ribbons.After 1 month,when the neural postsynaptic densities had completed their

  11. Prion infection impairs lysosomal degradation capacity by interfering with rab7 membrane attachment in neuronal cells.

    Science.gov (United States)

    Shim, Su Yeon; Karri, Srinivasarao; Law, Sampson; Schatzl, Hermann M; Gilch, Sabine

    2016-01-01

    Prions are proteinaceous infectious particles which cause fatal neurodegenerative disorders in humans and animals. They consist of a mostly β-sheeted aggregated isoform (PrP(Sc)) of the cellular prion protein (PrP(c)). Prions replicate autocatalytically in neurons and other cell types by inducing conformational conversion of PrP(c) into PrP(Sc). Within neurons, PrP(Sc) accumulates at the plasma membrane and in vesicles of the endocytic pathway. To better understand the mechanisms underlying neuronal dysfunction and death it is critical to know the impact of PrP(Sc) accumulation on cellular pathways. We have investigated the effects of prion infection on endo-lysosomal transport. Our study demonstrates that prion infection interferes with rab7 membrane association. Consequently, lysosomal maturation and degradation are impaired. Our findings indicate a mechanism induced by prion infection that supports stable prion replication. We suggest modulation of endo-lysosomal vesicle trafficking and enhancement of lysosomal maturation as novel targets for the treatment of prion diseases. PMID:26865414

  12. SpineLab: tool for three-dimensional reconstruction of neuronal cell morphology

    Science.gov (United States)

    Jungblut, Daniel; Vlachos, Andreas; Schuldt, Gerlind; Zahn, Nadine; Deller, Thomas; Wittum, Gabriel

    2012-07-01

    SpineLab is a software tool developed for reconstructing neuronal feature skeletons from three-dimensional single- or multi-photon image stacks. These images often suffer from limited resolution and a low signal-to-noise ratio, making the extraction of morphometric information difficult. To overcome this limitation, we have developed a software tool that offers the possibility to create feature skeletons in various modes--automatically as well as with manual interaction. We have named this novel tool SpineLab. In a first step, an investigator adjusts a set of parameters for automatic analysis in an interactive manner, i.e., with online visual feedback, followed by a second step, in which the neuronal feature skeleton can be modified by hand. We validate the ability of SpineLab to reconstruct the entire dendritic tree of identified GFP-expressing neurons and evaluate the accuracy of dendritic spine detection. We report that SpineLab is capable of significantly facilitating the reconstruction of dendrites and spines. Moreover, the automatic approach appears sufficient to detect spine density changes in time-lapse imaging experiments. Taken together, we conclude that SpineLab is an ideal software tool for partially automatic reconstruction of neural cell morphology.

  13. Multidendritic sensory neurons in the adult Drosophila abdomen: origins, dendritic morphology, and segment- and age-dependent programmed cell death

    Directory of Open Access Journals (Sweden)

    Sugimura Kaoru

    2009-10-01

    Full Text Available Abstract Background For the establishment of functional neural circuits that support a wide range of animal behaviors, initial circuits formed in early development have to be reorganized. One way to achieve this is local remodeling of the circuitry hardwiring. To genetically investigate the underlying mechanisms of this remodeling, one model system employs a major group of Drosophila multidendritic sensory neurons - the dendritic arborization (da neurons - which exhibit dramatic dendritic pruning and subsequent growth during metamorphosis. The 15 da neurons are identified in each larval abdominal hemisegment and are classified into four categories - classes I to IV - in order of increasing size of their receptive fields and/or arbor complexity at the mature larval stage. Our knowledge regarding the anatomy and developmental basis of adult da neurons is still fragmentary. Results We identified multidendritic neurons in the adult Drosophila abdomen, visualized the dendritic arbors of the individual neurons, and traced the origins of those cells back to the larval stage. There were six da neurons in abdominal hemisegment 3 or 4 (A3/4 of the pharate adult and the adult just after eclosion, five of which were persistent larval da neurons. We quantitatively analyzed dendritic arbors of three of the six adult neurons and examined expression in the pharate adult of key transcription factors that result in the larval class-selective dendritic morphologies. The 'baseline design' of A3/4 in the adult was further modified in a segment-dependent and age-dependent manner. One of our notable findings is that a larval class I neuron, ddaE, completed dendritic remodeling in A2 to A4 and then underwent caspase-dependent cell death within 1 week after eclosion, while homologous neurons in A5 and in more posterior segments degenerated at pupal stages. Another finding is that the dendritic arbor of a class IV neuron, v'ada, was immediately reshaped during post

  14. Inducible transcript expressed by reactive epithelial cells at sites of olfactory sensory neuron proliferation.

    Science.gov (United States)

    Stoss, Thomas D; Nickell, Melissa D; Hardin, Debra; Derby, Charles D; McClintock, Timothy S

    2004-02-15

    The continuous replacement of cells in the spiny lobster olfactory organ depends on proliferation of new cells at a specific site, the proximal proliferation zone (PPZ). Using representational difference analysis of cDNA, we identified transcripts enriched in the PPZ compared to the mature zone (MZ) of the organ. The 12 clones identified included four novel sequences, three exoskeletal proteins, a serine protease, two protease inhibitors, a putative growth factor, and a sequence named PET-15 that has similarity to antimicrobial proteins of the crustin type. PET-15 mRNA was only detected in epithelial cells. It was abundant in all epithelial cells of the PPZ, but was only detected in the MZ at sites of damage to the olfactory organ. PET-15 mRNA was increased by types of damage that are known to induce proliferation of new olfactory sensory neurons in the olfactory organ. It increased in the PPZ after partial ablation of the olfactory organ and in the MZ after shaving of aesthetasc sensilla. These ipsilateral effects were mirrored by smaller increases in the undamaged contralateral olfactory organ. These contralateral effects are most parsimoniously explained by the action of a diffusible signal. Because epithelial cells are the source of proliferating progenitors in the olfactory organ, the same diffusible signal may stimulate increases in both cellular proliferation and PET-15 mRNA. The uniformity of expression of PET-15 in the PPZ epithelium suggests that the epithelial cells that give rise to new olfactory sensory neurons are a subset of cells that express PET-15.

  15. Diabetes Accelerates Retinal Neuronal Cell Death In A Mouse Model of Endogenous Hyperhomocysteinemia

    Directory of Open Access Journals (Sweden)

    Preethi S. Ganapathy

    2009-07-01

    Full Text Available Hyperhomocysteinemia has been implicated in visual dysfunction. We reported recently that mice with endogenous hyperhomocysteinemia, due to mutation of the cystathionine-β-synthase (cbs gene, demonstrate loss of neurons in the retinal ganglion cell (RGC layer and other retinal layers as homocysteine levels increase. Some clinical studies implicate hyperhomocysteinemia in the pathogenesis of diabetic retinopathy, which is also characterized by RGC loss. The present study used cbs+/- mice to determine whether modest elevation of plasma homocysteine, in the presence of diabetes, accelerates neuronal cell loss. Diabetes (DB was induced in 3 wk old cbs+/- and wildtype mice using streptozotocin; four groups of mice were studied: DB cbs+/-; non-DB cbs+/-; DB cbs+/+; non-DB cbs+/+. One group of diabetic cbs+/- mice was maintained on a high methionine diet (HMD, 0.5% methionine drinking water to increase plasma homocysteine slightly. Eyes were harvested at 5, 10 and 15 weeks post-onset of diabetes; retinal cryosections were examined by light microscopy and subjected to systematic morphometric analysis. Diabetic cbs+/- had significantly fewer RGCs at 5 weeks compared to age-matched, non-diabetic cbs+/- and wildtype controls (10.0 ± 0.5 versus 14.9 ± 0.5 and 15.8 ± 0.6 cells/100 µm retina length, respectively. Significant differences in retinas of DB/high homocysteine versus controls were obtained 15 wks post-onset of diabetes including fewer RGCS and decreased thickness of inner nuclear and plexiform layers. Moderate increases in plasma homocysteine coupled with diabetes cause a more dramatic alteration of retinal phenotype than elevated homocysteine or diabetes alone and suggest that diabetes accelerates the retinal neuronal death in hyperhomocysteinemic mice.

  16. HMGB4 is expressed by neuronal cells and affects the expression of genes involved in neural differentiation

    Science.gov (United States)

    Rouhiainen, Ari; Zhao, Xiang; Vanttola, Päivi; Qian, Kui; Kulesskiy, Evgeny; Kuja-Panula, Juha; Gransalke, Kathleen; Grönholm, Mikaela; Unni, Emmanual; Meistrich, Marvin; Tian, Li; Auvinen, Petri; Rauvala, Heikki

    2016-09-01

    HMGB4 is a new member in the family of HMGB proteins that has been characterized in sperm cells, but little is known about its functions in somatic cells. Here we show that HMGB4 and the highly similar rat Transition Protein 4 (HMGB4L1) are expressed in neuronal cells. Both proteins had slow mobility in nucleus of living NIH-3T3 cells. They interacted with histones and their differential expression in transformed cells of the nervous system altered the post-translational modification statuses of histones in vitro. Overexpression of HMGB4 in HEK 293T cells made cells more susceptible to cell death induced by topoisomerase inhibitors in an oncology drug screening array and altered variant composition of histone H3. HMGB4 regulated over 800 genes in HEK 293T cells with a p-value ≤0.013 (n = 3) in a microarray analysis and displayed strongest association with adhesion and histone H2A -processes. In neuronal and transformed cells HMGB4 regulated the expression of an oligodendrocyte marker gene PPP1R14a and other neuronal differentiation marker genes. In conclusion, our data suggests that HMGB4 is a factor that regulates chromatin and expression of neuronal differentiation markers.

  17. Cholecystokinin-2 receptor mediated gene expression in neuronal PC12 cells

    DEFF Research Database (Denmark)

    Hansen, Thomas v O; Borup, Rehannah; Marstrand, Troels;

    2007-01-01

    Cholecystokinin (CCK) is abundantly expressed in the CNS, in which it regulates feeding behavior and long-term memory. Moreover, CCK has been implicated in mental disorders, such as anxiety and schizophrenia. Despite its manifest physiological and pathophysiological role, the molecular targets...... could be identified. Comparison with forskolin- and nerve growth factor (NGF)-treated PC12 cells showed that CCK induced a separate set of target genes. Taken together, we propose that neuronal CCK may have a role in the regulation of the circadian rhythm, the metabolism of cerebral cholesterol...

  18. Mouse ES cells overexpressing DNMT1 produce abnormal neurons with upregulated NR1

    OpenAIRE

    D'Aiuto, Leonardo; Di Maio, Roberto; Mohan, K. Naga; Minervini, Crescenzio; Saporiti, Federica; Soreca, Isabella; Greenamyre, J. Timothy; Chaillet, J. Richard

    2011-01-01

    High levels of DNA methyltransferase 1 (DNMT1), hypermethylation, and downregulation of GAD67 and reelin have been described in GABAergic interneurons of patients with schizophrenia (SZ) and bipolar (BP) disorders. However, overexpression of DNMT1 is lethal, making it difficult to assess the direct effect of high levels of DNMT1 on neuronal development in vivo. We therefore used Dnmt1tet/tet mouse ES cells that overexpress DNMT1 as an in vitro model to investigate the impact of high levels of...

  19. The clinical spectrum of mutations in L1, a neuronal cell adhesion molecule

    Energy Technology Data Exchange (ETDEWEB)

    Fransen, E.; Vits, L.; Van Camp, G.; Willems, P.J. [Univ. of Antwerp (Belgium)

    1996-07-12

    Mutations in the gene encoding the neuronal cell adhesion molecule L1 are responsible for several syndromes with clinical overlap, including X-linked hydrocephalus (XLH, HSAS), MASA (mental retardation, aphasia, shuffling gait, adducted thumbs) syndrome, complicated X-linked spastic paraplegia (SP 1), X-linked mental retardation-clasped thumb (MR-CT) syndrome, and some forms of X-linked agenesis of the corpus callosum (ACC). We review 34 L1 mutations in patients with these phenotypes. 22 refs., 3 figs., 4 tabs.

  20. Efficient Generation of Corticofugal Projection Neurons from Human Embryonic Stem Cells.

    Science.gov (United States)

    Zhu, Xiaoqing; Ai, Zongyong; Hu, Xintian; Li, Tianqing

    2016-06-27

    Efforts to study development and function of corticofugal projection neurons (CfuPNs) in the human cerebral cortex for health and disease have been limited by the unavailability of highly enriched CfuPNs. Here, we develop a robust, two-step process for generating CfuPNs from human embryonic stem cells (hESCs): directed induction of neuroepithelial stem cells (NESCs) from hESCs and efficient differentiation of NESCs to about 80% of CfuPNs. NESCs or a NESC faithfully maintain unlimitedly self-renewal and self-organized abilities to develop into miniature neural tube-like structures. NESCs retain a stable propensity toward neuronal differentiation over culture as fate-restricted progenitors of CfuPNs and interneurons. When grafted into mouse brains, NESCs successfully integrate into the host brains, differentiate into CfuPNs and effectively reestablish specific patterns of subcortical projections and synapse structures. Efficient generation of CfuPNs in vitro and in vivo will facilitate human cortex development and offer sufficient CfuPNs for cell therapy.

  1. Process Extension from Embryonic Stem Cell-Derived Motor Neurons through Synthetic Extracellular Matrix Mimics

    Science.gov (United States)

    McKinnon, Daniel Devaud

    This thesis focuses on studying the extension of motor axons through synthetic poly(ethylene glycol) PEG hydrogels that have been modified with biochemical functionalities to render them more biologically relevant. Specifically, the research strategy is to encapsulate embryonic stem cell-derived motor neurons (ESMNs) in synthetic PEG hydrogels crosslinked through three different chemistries providing three mechanisms for dynamically tuning material properties. First, a covalently crosslinked, enzymatically degradable hydrogel is developed and exploited to study the biophysical dynamics of axon extension and matrix remodeling. It is demonstrated that dispersed motor neurons require a battery of adhesive peptides and growth factors to maintain viability and extend axons while those in contact with supportive neuroglial cells do not. Additionally, cell-degradable crosslinker peptides and a soft modulus mimicking that of the spinal cord are requirements for axon extension. However, because local degradation of the hydrogel results in a cellular environment significantly different than that of the bulk, enzymatically degradable peptide crosslinkers were replaced with reversible covalent hydrazone bonds to study the effect of hydrogel modulus on axon extension. This material is characterized in detail and used to measure forces involved in axon extension. Finally, a hydrogel with photocleavable linkers incorporated into the network structure is exploited to explore motor axon response to physical channels. This system is used to direct the growth of motor axons towards co-cultured myotubes, resulting in the formation of an in vitro neural circuit.

  2. Translation dynamics of single mRNAs in live cells and neurons.

    Science.gov (United States)

    Wu, Bin; Eliscovich, Carolina; Yoon, Young J; Singer, Robert H

    2016-06-17

    Translation is the fundamental biological process converting mRNA information into proteins. Single-molecule imaging in live cells has illuminated the dynamics of RNA transcription; however, it is not yet applicable to translation. Here, we report single-molecule imaging of nascent peptides (SINAPS) to assess translation in live cells. The approach provides direct readout of initiation, elongation, and location of translation. We show that mRNAs coding for endoplasmic reticulum (ER) proteins are translated when they encounter the ER membrane. Single-molecule fluorescence recovery after photobleaching provides direct measurement of elongation speed (5 amino acids per second). In primary neurons, mRNAs are translated in proximal dendrites but repressed in distal dendrites and display "bursting" translation. This technology provides a tool with which to address the spatiotemporal translation mechanism of single mRNAs in living cells. PMID:27313041

  3. Nanosensors for label-free measurement of sodium ion fluxes of neuronal cells

    Energy Technology Data Exchange (ETDEWEB)

    Gebinoga, Michael, E-mail: michael.gebinoga@tu-ilmenau.de [ZIK MacroNano Microfluidics and Biosensors, Technical University Ilmenau, P.O. Box 100565, D-98684 Ilmenau (Germany); Silveira, Liele; Cimalla, Irina [ZIK MacroNano Microfluidics and Biosensors, Technical University Ilmenau, P.O. Box 100565, D-98684 Ilmenau (Germany); Dumitrescu, Andreea [University of Pennsylvania - School of Engineering and Applied Science, Philadelphia, PA 19104-6391 (United States); Kittler, Mario; Luebbers, Benedikt; Becker, Annette [ZIK MacroNano Microfluidics and Biosensors, Technical University Ilmenau, P.O. Box 100565, D-98684 Ilmenau (Germany); Lebedev, Vadim [Fraunhofer Institute for Solid State Physics, Tullastr. 7, D-79108 Freiburg (Germany); Schober, Andreas [ZIK MacroNano Microfluidics and Biosensors, Technical University Ilmenau, P.O. Box 100565, D-98684 Ilmenau (Germany)

    2010-05-25

    Novel nanosensors based on aluminium gallium nitrides (AlGaN/GaN) high electron mobility transistors have been of high interest during the last years, especially for their electrical characteristics as open gate field effect transistors. These nanosensors provide a valuable tool for high content screening in drug discovery, cell monitoring and liquid analyses focusing on applications of electrochemical detection technology. Our own measurements with these sensors confirm their pH sensitivity and in addition the possibility of detection of other ions in aqueous media. These measurements deal with the reactions of NG 108-15 (mouse neuroblastoma x rat glioma hybrid) neuronal cells in response to different acetylcholinesterase inhibitors. Our experimental approach shows some advantages. The first advantage is the label-free measurement of ion fluxes, and another advantage is the possibility non-destructively to estimate cell signals.

  4. The mast cell stabilizer sodium cromoglycate reduces histamine release and status epilepticus-induced neuronal damage in the rat hippocampus.

    Science.gov (United States)

    Valle-Dorado, María Guadalupe; Santana-Gómez, César Emmanuel; Orozco-Suárez, Sandra Adela; Rocha, Luisa

    2015-05-01

    Experiments were designed to evaluate changes in the histamine release, mast cell number and neuronal damage in hippocampus induced by status epilepticus. We also evaluated if sodium cromoglycate, a stabilizer of mast cells with a possible stabilizing effect on the membrane of neurons, was able to prevent the release of histamine, γ-aminobutyric acid (GABA) and glutamate during the status epilepticus. During microdialysis experiments, rats were treated with saline (SS-SE) or sodium cromoglycate (CG-SE) and 30 min later received the administration of pilocarpine to induce status epilepticus. Twenty-four hours after the status epilepticus, the brains were used to determine the neuronal damage and the number of mast cells in hippocampus. During the status epilepticus, SS-SE group showed an enhanced release of histamine (138.5%, p = 0.005), GABA (331 ± 91%, p ≤ 0.001) and glutamate (467%, p ≤ 0.001), even after diazepam administration. One day after the status epilepticus, SS-SE group demonstrated increased number of mast cells in Stratum pyramidale of CA1 (88%, p status epilepticus (p = 0.048), absence of wet-dog shakes, reduced histamine (but not GABA and glutamate) release, lower number of mast cells (p = 0.008) and reduced neuronal damage in hippocampus. Our data revealed that histamine, possibly from mast cells, is released in hippocampus during the status epilepticus. This effect may be involved in the subsequent neuronal damage and is diminished with sodium cromoglycate pretreatment.

  5. A Flavonoid Compound Promotes Neuronal Differentiation of Embryonic Stem Cells via PPAR-β Modulating Mitochondrial Energy Metabolism

    Science.gov (United States)

    Mei, Yu-qin; Pan, Zong-fu; Chen, Wen-teng; Xu, Min-hua; Zhu, Dan-yan; Yu, Yong-ping; Lou, Yi-jia

    2016-01-01

    Relatively little is known regarding mitochondrial metabolism in neuronal differentiation of embryonic stem (ES) cells. By using a small molecule, present research has investigated the pattern of cellular energy metabolism in neural progenitor cells derived from mouse ES cells. Flavonoid compound 4a faithfully facilitated ES cells to differentiate into neurons morphologically and functionally. The expression and localization of peroxisome proliferator-activated receptors (PPARs) were examined in neural progenitor cells. PPAR-β expression showed robust upregulation compared to solvent control. Treatment with PPAR-β agonist L165041 alone or together with compound 4a significantly promoted neuronal differentiation, while antagonist GSK0660 blocked the neurogenesis-promoting effect of compound 4a. Consistently, knockdown of PPAR-β in ES cells abolished compound 4a-induced neuronal differentiation. Interestingly, we found that mitochondrial fusion protein Mfn2 was also abolished by sh-PPAR-β, resulting in abnormal mitochondrial Ca2+ ([Ca2+]M) transients as well as impaired mitochondrial bioenergetics. In conclusion, we demonstrated that by modulating mitochondrial energy metabolism through Mfn2 and mitochondrial Ca2+, PPAR-β took an important role in neuronal differentiation induced by flavonoid compound 4a. PMID:27315062

  6. Neurogliaform and Ivy cells: a major class of nNOS expressing GABAergic neurons

    Directory of Open Access Journals (Sweden)

    Caren eArmstrong

    2012-05-01

    Full Text Available Neurogliaform and Ivy cells are members of an abundant class of neuronal nitric oxide synthase (nNOS expressing GABAergic interneurons found in diverse brain regions. These cells have a defining dense local axonal plexus, and display unique synaptic properties, including a biphasic postsynaptic response with both a slow GABAA component, and a GABAB component, following even a single action potential. The type of transmission displayed by these cells has been termed ‘volume transmission,’ distinct from both tonic and classical synaptic transmission. Electrical connections are also notable in that, unlike other classes, neurogliaform family cells will form gap junctions not only with other neurogliaform cells, but also with members of other GABAergic cell classes. In this review we focus on neurogliaform and Ivy cells throughout the hippocampal formation, where recent studies highlight their role in feedforward inhibition, uncover their ability to display a phenomenon called persistent firing, and reveal their modulation by opioids. The unique properties of this class of cells, their abundance, rich connectivity, and modulation by clinically relevant drugs make them an attractive target for future studies in vivo during different behavioral and pharmacological conditions.

  7. Differentiation of Embryonic Stem Cells into Neurons and Retina—like Structure in Nude Mice

    Institute of Scientific and Technical Information of China (English)

    LiYP; GeJ

    1999-01-01

    Purpose:To investigate the intraocular growth and biological characteristics of mice embryonic stem cells in nude mice.Methods:Murine embryonic stem cells(D3 cell line)were cultured and maintained in an undifferentiated state in vitro,then transplanted into the anterior chamber of nude mice.Mophological and immunohistochemical examinations were implemented.Results:Two to three days after transplantation,yellow-white floating granules,sheets and masses were seen inside the anterior chamber and vitreous cavity,and enlarged gradually,14-20days later,the mice were executed.Morphological examination showed that there were undifferentiated cells and some round or polygonal differentiated cells in anterior chamber and vitreous cavity.The morphology of these differentiated cells were similar to that of the retina.The cells were highly positive in NSE staining.Conclusion:The tranplanted embryonic stem cells cold grow in the eyes of nude mice with tendency to differentiate into neurons and retina-like structure.

  8. Respiratory Syncytial Virus (RSV) Infects Neuronal Cells and Processes That Innervate the Lung by a Process Involving RSV G Protein

    OpenAIRE

    Li, Xia-qing; Fu, Zhen F.; Alvarez, Rene; Henderson, Christine; Ralph A. Tripp

    2006-01-01

    Respiratory syncytial virus (RSV) is a primary cause of morbidity and life-threatening lower respiratory tract disease in infants and young children. Children with acute RSV bronchiolitis often develop respiratory sequelae, but the disease mechanisms are poorly understood. Mounting evidence suggests that RSV may mediate persistent infection. Using immunohistochemistry to identify RSV and RSV-infected cell types, we show that RSV infects primary neurons and neuronal processes that innervate th...

  9. Differences in DNA methylation between human neuronal and glial cells are concentrated in enhancers and non-CpG sites

    OpenAIRE

    Kozlenkov, Alexey; Roussos, Panos; Timashpolsky, Alisa; Barbu, Mihaela; Rudchenko, Sergei; Bibikova, Marina; Klotzle, Brandy; Byne, William; Lyddon, Rebecca; Di Narzo, Antonio Fabio; Hurd, Yasmin L.; Eugene V Koonin; Dracheva, Stella

    2013-01-01

    We applied Illumina Human Methylation450K array to perform a genomic-scale single-site resolution DNA methylation analysis in neuronal and nonneuronal (primarily glial) nuclei separated from the orbitofrontal cortex of postmortem human brain. The findings were validated using enhanced reduced representation bisulfite sequencing. We identified thousands of sites differentially methylated (DM) between neuronal and nonneuronal cells. The DM sites were depleted within CpG-island–containing promot...

  10. Overexpression of microRNA-124 promotes the neuronal differentiation of bone marrow-derived mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    Defeng Zou; Yi Chen; Yaxin Han; Chen Lv; Guanjun Tu

    2014-01-01

    microRNAs (miRNAs) play an important regulatory role in the self-renewal and differentiation of stem cells. In this study, we examined the effects of miRNA-124 (miR-124) overexpression in bone marrow-derived mesenchymal stem cells. In particular, we focused on the effect of overexpression on the differentiation of bone marrow-derived mesenchymal stem cells into neurons. First, we used GeneChip technology to analyze the expression of miRNAs inbone marrow-derived mesen-chymal stem cells, neural stem cells and neurons. miR-124 expression was substantially reduced inbone marrow-derived mesenchymal stem cells compared with the other cell types. We con-structed a lentiviral vector overexpressing miR-124 and transfected it intobone marrow-derived mesenchymal stem cells. Intracellular expression levels of the neuronal early markersβ-III tu-bulin and microtubule-associated protein-2 were signiifcantly increased, and apoptosis induced by oxygen and glucose deprivation was reduced in transfected cells. After miR-124-transfected bone marrow-derived mesenchymal stem cells were transplanted into the injured rat spinal cord, a large number of cells positive for the neuronal marker neurofilament-200 were observed in the transplanted region. The Basso-Beattie-Bresnahan locomotion scores showed that the motor function of the hind limb of rats with spinal cord injury was substantially improved. These re-sults suggest that miR-124 plays an important role in the differentiation ofbone marrow-derived mesenchymal stem cells into neurons. Our ifndings should facilitate the development of novel strategies for enhancing the therapeutic efifcacy ofbone marrow-derived mesenchymal stem cell transplantation for spinal cord injury.

  11. Transplantation of human adipose tissue-derived stem cells for repair of injured spiral ganglion neurons in deaf guinea pigs.

    Science.gov (United States)

    Jang, Sujeong; Cho, Hyong-Ho; Kim, Song-Hee; Lee, Kyung-Hwa; Cho, Yong-Bum; Park, Jong-Seong; Jeong, Han-Seong

    2016-06-01

    Excessive noise, ototoxic drugs, infections, autoimmune diseases, and aging can cause loss of spiral ganglion neurons, leading to permanent sensorineural hearing loss in mammals. Stem cells have been confirmed to be able to differentiate into spiral ganglion neurons. Little has been reported on adipose tissue-derived stem cells (ADSCs) for repair of injured spiral ganglion neurons. In this study, we hypothesized that transplantation of neural induced-human ADSCs (NI-hADSCs) can repair the injured spiral ganglion neurons in guinea pigs with neomycin-induced sensorineural hearing loss. NI-hADSCs were induced with culture medium containing basic fibroblast growth factor and forskolin and then injected to the injured cochleae. Guinea pigs that received injection of Hanks' balanced salt solution into the cochleae were used as controls. Hematoxylin-eosin staining showed that at 8 weeks after cell transplantation, the number of surviving spiral ganglion neurons in the cell transplantation group was significantly increased than that in the control group. Also at 8 weeks after cell transplantation, immunohistochemical staining showed that a greater number of NI-hADSCs in the spiral ganglions were detected in the cell transplantation group than in the control group, and these NI-hADSCs expressed neuronal markers neurofilament protein and microtubule-associated protein 2. Within 8 weeks after cell transplantation, the guinea pigs in the cell transplantation group had a gradually decreased auditory brainstem response threshold, while those in the control group had almost no response to 80 dB of clicks or pure tone burst. These findings suggest that a large amount of NI-hADSCs migrated to the spiral ganglions, survived for a period of time, repaired the injured spiral ganglion cells, and thereby contributed to the recovery of sensorineural hearing loss in guinea pigs. PMID:27482231

  12. Transplantation of human adipose tissue-derived stem cells for repair of injured spiral ganglion neurons in deaf guinea pigs

    Science.gov (United States)

    Jang, Sujeong; Cho, Hyong-Ho; Kim, Song-Hee; Lee, Kyung-Hwa; Cho, Yong-Bum; Park, Jong-Seong; Jeong, Han-Seong

    2016-01-01

    Excessive noise, ototoxic drugs, infections, autoimmune diseases, and aging can cause loss of spiral ganglion neurons, leading to permanent sensorineural hearing loss in mammals. Stem cells have been confirmed to be able to differentiate into spiral ganglion neurons. Little has been reported on adipose tissue-derived stem cells (ADSCs) for repair of injured spiral ganglion neurons. In this study, we hypothesized that transplantation of neural induced-human ADSCs (NI-hADSCs) can repair the injured spiral ganglion neurons in guinea pigs with neomycin-induced sensorineural hearing loss. NI-hADSCs were induced with culture medium containing basic fibroblast growth factor and forskolin and then injected to the injured cochleae. Guinea pigs that received injection of Hanks’ balanced salt solution into the cochleae were used as controls. Hematoxylin-eosin staining showed that at 8 weeks after cell transplantation, the number of surviving spiral ganglion neurons in the cell transplantation group was significantly increased than that in the control group. Also at 8 weeks after cell transplantation, immunohistochemical staining showed that a greater number of NI-hADSCs in the spiral ganglions were detected in the cell transplantation group than in the control group, and these NI-hADSCs expressed neuronal markers neurofilament protein and microtubule-associated protein 2. Within 8 weeks after cell transplantation, the guinea pigs in the cell transplantation group had a gradually decreased auditory brainstem response threshold, while those in the control group had almost no response to 80 dB of clicks or pure tone burst. These findings suggest that a large amount of NI-hADSCs migrated to the spiral ganglions, survived for a period of time, repaired the injured spiral ganglion cells, and thereby contributed to the recovery of sensorineural hearing loss in guinea pigs. PMID:27482231

  13. Effects of nerve growth factor and heart cell conditioned medium on neurite regeneration of aged sympathetic neurons in culture.

    Science.gov (United States)

    Uchida, Y; Tomonaga, M

    1985-11-25

    The effects of nerve growth factor (NGF) and heart-cell-conditioned medium (HCM) on the neurite regeneration of aged sympathetic neurons were investigated in culture. Investigation of HCM was carried out by two different methods: one was the use of whole HCM on collagen substratum, which reflected component(s) effective in solution (HCM-S); the other was the use of polyornithine (PORN)-binding component(s) (P-HCM). Superior cervical ganglion neurons prepared from male mice from 6 to 30 months of age were cultured in MEM-10% FCS on collagen or gelatin-PORN substratum for 3 days. The number of neurons with neurites and the length of neurites were quantified as neurite production and elongation, respectively. Neuronal survival was not affected by addition of NGF, HCM-S or P-HCM. Neurite production of early adult neurons was enhanced by NGF, HCM-S or P-HCM. In contrast, neurite production of aged neurons was enhanced by only HCM-S, but not NGF or P-HCM. HCM-S did not promote neurite elongation in neurons at any age. Neurite elongation of early adult neurons was enhanced by NGF or P-HCM. Neurite elongation of aged neurons was enhanced by P-HCM. However, responsiveness of NGF for neurite elongation varied according to substrata. No age-related difference was found in neurite production and elongation in the absence of NGF, HCM-S or P-HCM. These results indicate that responsiveness of aged sympathetic neurons is various in different growth factors. PMID:3840716

  14. Is integration and survival of newborn neurons the bottleneck for effective neural repair by endogenous neural precursor cells?

    Directory of Open Access Journals (Sweden)

    Ann eTurnley

    2014-02-01

    Full Text Available After two decades of research the existence of adult neural precursor cells and the phenomenon of adult neurogenesis is well established. However, there has been little or no effective harnessing of these endogenous cells to promote functional neuronal replacement following neural injury or disease. Neural precursor cells can respond to neural damage by proliferating, migrating to the site of injury and differentiating into neuronal or glial lineages. However, after a month or so, very few or no newborn neurons can be detected, suggesting that even though neuroblasts are generated, they generally fail to survive as mature neurons and contribute to the local circuitry. Is this lack of survival and integration one of the major bottlenecks that inhibits effective neuronal replacement and subsequent repair of the nervous system following injury or disease? In this perspective article the possibility that this bottleneck can be targeted to enhance the integration and subsequent survival of newborn neurons will be explored and will suggest some possible mechanisms that may need to be modulated for this to occur.

  15. Bispyridinium Compounds Inhibit Both Muscle and Neuronal Nicotinic Acetylcholine Receptors in Human Cell Lines.

    Science.gov (United States)

    Ring, Avi; Strom, Bjorn Oddvar; Turner, Simon R; Timperley, Christopher M; Bird, Michael; Green, A Christopher; Chad, John E; Worek, Franz; Tattersall, John E H

    2015-01-01

    Standard treatment of poisoning by organophosphorus anticholinesterases uses atropine to reduce the muscarinic effects of acetylcholine accumulation and oximes to reactivate acetylcholinesterase (the effectiveness of which depends on the specific anticholinesterase), but does not directly address the nicotinic effects of poisoning. Bispyridinium molecules which act as noncompetitive antagonists at nicotinic acetylcholine receptors have been identified as promising compounds and one has been shown to improve survival following organophosphorus poisoning in guinea-pigs. Here, we have investigated the structural requirements for antagonism and compared inhibitory potency of these compounds at muscle and neuronal nicotinic receptors and acetylcholinesterase. A series of compounds was synthesised, in which the length of the polymethylene linker between the two pyridinium moieties was increased sequentially from one to ten carbon atoms. Their effects on nicotinic receptor-mediated calcium responses were tested in muscle-derived (CN21) and neuronal (SH-SY5Y) cells. Their ability to inhibit acetylcholinesterase activity was tested using human erythrocyte ghosts. In both cell lines, the nicotinic response was inhibited in a dose-dependent manner and the inhibitory potency of the compounds increased with greater linker length between the two pyridinium moieties, as did their inhibitory potency for human acetylcholinesterase activity in vitro. These results demonstrate that bispyridinium compounds inhibit both neuronal and muscle nicotinic receptors and that their potency depends on the length of the hydrocarbon chain linking the two pyridinium moieties. Knowledge of structure-activity relationships will aid the optimisation of molecular structures for therapeutic use against the nicotinic effects of organophosphorus poisoning.

  16. Bispyridinium Compounds Inhibit Both Muscle and Neuronal Nicotinic Acetylcholine Receptors in Human Cell Lines.

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    Avi Ring

    Full Text Available Standard treatment of poisoning by organophosphorus anticholinesterases uses atropine to reduce the muscarinic effects of acetylcholine accumulation and oximes to reactivate acetylcholinesterase (the effectiveness of which depends on the specific anticholinesterase, but does not directly address the nicotinic effects of poisoning. Bispyridinium molecules which act as noncompetitive antagonists at nicotinic acetylcholine receptors have been identified as promising compounds and one has been shown to improve survival following organophosphorus poisoning in guinea-pigs. Here, we have investigated the structural requirements for antagonism and compared inhibitory potency of these compounds at muscle and neuronal nicotinic receptors and acetylcholinesterase. A series of compounds was synthesised, in which the length of the polymethylene linker between the two pyridinium moieties was increased sequentially from one to ten carbon atoms. Their effects on nicotinic receptor-mediated calcium responses were tested in muscle-derived (CN21 and neuronal (SH-SY5Y cells. Their ability to inhibit acetylcholinesterase activity was tested using human erythrocyte ghosts. In both cell lines, the nicotinic response was inhibited in a dose-dependent manner and the inhibitory potency of the compounds increased with greater linker length between the two pyridinium moieties, as did their inhibitory potency for human acetylcholinesterase activity in vitro. These results demonstrate that bispyridinium compounds inhibit both neuronal and muscle nicotinic receptors and that their potency depends on the length of the hydrocarbon chain linking the two pyridinium moieties. Knowledge of structure-activity relationships will aid the optimisation of molecular structures for therapeutic use against the nicotinic effects of organophosphorus poisoning.

  17. Glycans in sera of amyotrophic lateral sclerosis patients and their role in killing neuronal cells.

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    Meital Edri-Brami

    Full Text Available Amyotrophic lateral sclerosis (ALS is a fatal neurodegenerative disease caused by degeneration of upper and lower motor neurons. To date, glycosylation patterns of glycoproteins in fluids of ALS patients have not been described. Moreover, the aberrant glycosylation related to the pathogenesis of other neurodegenerative diseases encouraged us to explore the glycome of ALS patient sera. We found high levels of sialylated glycans and low levels of core fucosylated glycans in serum-derived N-glycans of patients with ALS, compared to healthy volunteer sera. Based on these results, we analyzed the IgG Fc N(297-glycans, as IgG are major serum glycoproteins affected by sialylation or core fucosylation and are found in the motor cortex of ALS patients. The analyses revealed a distinct glycan, A2BG2, in IgG derived from ALS patient sera (ALS-IgG. This glycan increases the affinity of IgG to CD16 on effector cells, consequently enhancing Antibody-Dependent Cellular Cytotoxicity (ADCC. Therefore, we explore whether the Fc-N(297-glycans of IgG may be involved in ALS disease. Immunostaining of brain and spinal cord tissues revealed over-expression of CD16 and co-localization of intact ALS-IgG with CD16 and in brain with activated microglia of G93A-SOD1 mice. Intact ALS-IgG enhanced effector cell activation and ADCC reaction in comparison to sugar-depleted or control IgG. ALS-IgG were localized in the synapse between brain microglia and neurons of G93A-SOD1 mice, manifesting a promising in vivo ADCC reaction. Therefore, glycans of ALS-IgG may serve as a biomarker for the disease and may be involved in neuronal damage.

  18. NSC-34 Motor Neuron-Like Cells Are Unsuitable as Experimental Model for Glutamate-Mediated Excitotoxicity

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    Madji Hounoum, Blandine; Vourc’h, Patrick; Felix, Romain; Corcia, Philippe; Patin, Franck; Guéguinou, Maxime; Potier-Cartereau, Marie; Vandier, Christophe; Raoul, Cédric; Andres, Christian R.; Mavel, Sylvie; Blasco, Hélène

    2016-01-01

    Glutamate-induced excitotoxicity is a major contributor to motor neuron degeneration in the pathogenesis of amyotrophic lateral sclerosis (ALS). The spinal cord × Neuroblastoma hybrid cell line (NSC-34) is often used as a bona fide cellular model to investigate the physiopathological mechanisms of ALS. However, the physiological response of NSC-34 to glutamate remains insufficiently described. In this study, we evaluated the relevance of differentiated NSC-34 (NSC-34D) as an in vitro model for glutamate excitotoxicity studies. NSC-34D showed morphological and physiological properties of motor neuron-like cells and expressed glutamate receptor subunits GluA1–4, GluN1 and GluN2A/D. Despite these diverse characteristics, no specific effect of glutamate was observed on cultured NSC-34D survival and morphology, in contrast to what has been described in primary culture of motor neurons (MN). Moreover, a small non sustained increase in the concentration of intracellular calcium was observed in NSC-34D after exposure to glutamate compared to primary MN. Our findings, together with the inability to obtain cultures containing only differentiated cells, suggest that the motor neuron-like NSC-34 cell line is not a suitable in vitro model to study glutamate-induced excitotoxicity. We suggest that the use of primary cultures of MN is more suitable than NSC-34 cell line to explore the pathogenesis of glutamate-mediated excitotoxicity at the cellular level in ALS and other motor neuron diseases. PMID:27242431

  19. Label-free detection of neuronal differentiation in cell populations using high-throughput live-cell imaging of PC12 cells.

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    Sebastian Weber

    Full Text Available Detection of neuronal cell differentiation is essential to study cell fate decisions under various stimuli and/or environmental conditions. Many tools exist that quantify differentiation by neurite length measurements of single cells. However, quantification of differentiation in whole cell populations remains elusive so far. Because such populations can consist of both proliferating and differentiating cells, the task to assess the overall differentiation status is not trivial and requires a high-throughput, fully automated approach to analyze sufficient data for a statistically significant discrimination to determine cell differentiation. We address the problem of detecting differentiation in a mixed population of proliferating and differentiating cells over time by supervised classification. Using nerve growth factor induced differentiation of PC12 cells, we monitor the changes in cell morphology over 6 days by phase-contrast live-cell imaging. For general applicability, the classification procedure starts out with many features to identify those that maximize discrimination of differentiated and undifferentiated cells and to eliminate features sensitive to systematic measurement artifacts. The resulting image analysis determines the optimal post treatment day for training and achieves a near perfect classification of differentiation, which we confirmed in technically and biologically independent as well as differently designed experiments. Our approach allows to monitor neuronal cell populations repeatedly over days without any interference. It requires only an initial calibration and training step and is thereafter capable to discriminate further experiments. In conclusion, this enables long-term, large-scale studies of cell populations with minimized costs and efforts for detecting effects of external manipulation of neuronal cell differentiation.

  20. Β-amyloid 1-42 oligomers impair function of human embryonic stem cell-derived forebrain cholinergic neurons.

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    Linn Wicklund

    Full Text Available Cognitive impairment in Alzheimer's disease (AD patients is associated with a decline in the levels of growth factors, impairment of axonal transport and marked degeneration of basal forebrain cholinergic neurons (BFCNs. Neurogenesis persists in the adult human brain, and the stimulation of regenerative processes in the CNS is an attractive prospect for neuroreplacement therapy in neurodegenerative diseases such as AD. Currently, it is still not clear how the pathophysiological environment in the AD brain affects stem cell biology. Previous studies investigating the effects of the β-amyloid (Aβ peptide on neurogenesis have been inconclusive, since both neurogenic and neurotoxic effects on progenitor cell populations have been reported. In this study, we treated pluripotent human embryonic stem (hES cells with nerve growth factor (NGF as well as with fibrillar and oligomeric Aβ1-40 and Aβ1-42 (nM-µM concentrations and thereafter studied the differentiation in vitro during 28-35 days. The process applied real time quantitative PCR, immunocytochemistry as well as functional studies of intracellular calcium signaling. Treatment with NGF promoted the differentiation into functionally mature BFCNs. In comparison to untreated cells, oligomeric Aβ1-40 increased the number of functional neurons, whereas oligomeric Aβ1-42 suppressed the number of functional neurons. Interestingly, oligomeric Aβ exposure did not influence the number of hES cell-derived neurons compared with untreated cells, while in contrast fibrillar Aβ1-40 and Aβ1-42 induced gliogenesis. These findings indicate that Aβ1-42 oligomers may impair the function of stem cell-derived neurons. We propose that it may be possible for future AD therapies to promote the maturation of functional stem cell-derived neurons by altering the brain microenvironment with trophic support and by targeting different aggregation forms of Aβ.

  1. A novel combination of factors, termed SPIE, which promotes dopaminergic neuron differentiation from human embryonic stem cells.

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    Tandis Vazin

    Full Text Available BACKGROUND: Stromal-Derived Inducing Activity (SDIA is one of the most efficient methods of generating dopaminergic (DA neurons from embryonic stem cells (ESC. DA neuron induction can be achieved by co-culturing ESC with the mouse stromal cell lines PA6 or MS5. The molecular nature of this effect, which has been termed "SDIA" is so far unknown. Recently, we found that factors secreted by PA6 cells provided lineage-specific instructions to induce DA differentiation of human ESC (hESC. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we compared PA6 cells to various cell lines lacking the SDIA effect, and employed genome expression analysis to identify differentially-expressed signaling molecules. Among the factors highly expressed by PA6 cells, and known to be associated with CNS development, were stromal cell-derived factor 1 (SDF-1/CXCL12, pleiotrophin (PTN, insulin-like growth factor 2 (IGF2, and ephrin B1 (EFNB1. When these four factors, the combination of which was termed SPIE, were applied to hESC, they induced differentiation to TH-positive neurons in vitro. RT-PCR and western blot analysis confirmed the expression of midbrain specific markers, including engrailed 1, Nurr1, Pitx3, and dopamine transporter (DAT in cultures influenced by these four molecules. Electrophysiological recordings showed that treatment of hESC with SPIE induced differentiation of neurons that were capable of generating action potentials and forming functional synaptic connections. CONCLUSIONS/SIGNIFICANCE: The combination of SDF-1, PTN, IGF2, and EFNB1 mimics the DA phenotype-inducing property of SDIA and was sufficient to promote differentiation of hESC to functional midbrain DA neurons. These findings provide a method for differentiating hESC to form DA neurons, without a requirement for the use of animal-derived cell lines or products.

  2. Acetylcholine secretion by motor neuron-like cells from umbilical cord mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    Xueyuan Liu; Dehua Li; Dong Jiang; Yan Fang

    2013-01-01

    Umbilical cord mesenchymal stem cel s were isolated by a double enzyme digestion method. The third passage of umbilical cord mesenchymal stem cel s was induced with heparin and/or basic fi-broblast growth factor. Results confirmed that cel morphology did not change after induction with basic fibroblast growth factor alone. However, neuronal morphology was visible, and microtu-bule-associated protein-2 expression and acetylcholine levels increased fol owing induction with heparin alone or heparin combined with basic fibroblast growth factor. Hb9 and choline acetyl-transferase expression was high fol owing inductive with heparin combined with basic fibroblast growth factor. Results indicate that the inductive effect of basic fibroblast growth factor alone was not obvious. Heparin combined with basic fibroblast growth factor noticeably promoted the differen-tiation of umbilical cord mesenchymal stem cel s into motor neuron-like cel s. Simultaneously, um-bilical cord mesenchymal stem cel s could secrete acetylcholine.

  3. Input dependent cell assembly dynamics in a model of the striatal medium spiny neuron network.

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    Ponzi, Adam; Wickens, Jeff

    2012-01-01

    The striatal medium spiny neuron (MSN) network is sparsely connected with fairly weak GABAergic collaterals receiving an excitatory glutamatergic cortical projection. Peri-stimulus time histograms (PSTH) of MSN population response investigated in various experimental studies display strong firing rate modulations distributed throughout behavioral task epochs. In previous work we have shown by numerical simulation that sparse random networks of inhibitory spiking neurons with characteristics appropriate for UP state MSNs form cell assemblies which fire together coherently in sequences on long behaviorally relevant timescales when the network receives a fixed pattern of constant input excitation. Here we first extend that model to the case where cortical excitation is composed of many independent noisy Poisson processes and demonstrate that cell assembly dynamics is still observed when the input is sufficiently weak. However if cortical excitation strength is increased more regularly firing and completely quiescent cells are found, which depend on the cortical stimulation. Subsequently we further extend previous work to consider what happens when the excitatory input varies as it would when the animal is engaged in behavior. We investigate how sudden switches in excitation interact with network generated patterned activity. We show that sequences of cell assembly activations can be locked to the excitatory input sequence and outline the range of parameters where this behavior is shown. Model cell population PSTH display both stimulus and temporal specificity, with large population firing rate modulations locked to elapsed time from task events. Thus the random network can generate a large diversity of temporally evolving stimulus dependent responses even though the input is fixed between switches. We suggest the MSN network is well suited to the generation of such slow coherent task dependent response which could be utilized by the animal in behavior.

  4. Input dependent cell assembly dynamics in a model of the striatal medium spiny neuron network

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    Adam ePonzi

    2012-03-01

    Full Text Available The striatal medium spiny neuron (MSNs network is sparsely connected with fairly weak GABAergic collaterals receiving an excitatory glutamatergic cortical projection. Peri stimulus time histograms (PSTH of MSN population response investigated in various experimental studies display strong firing rate modulations distributed throughout behavioural task epochs. In previous work we have shown by numerical simulation that sparse random networks of inhibitory spiking neurons with characteristics appropriate for UP state MSNs form cell assemblies which fire together coherently in sequences on long behaviourally relevant timescales when the network receives a fixed pattern of constant input excitation. Here we first extend that model to the case where cortical excitation is composed of many independent noisy Poisson processes and demonstrate that cell assembly dynamics is still observed when the input is sufficiently weak. However if cortical excitation strength is increased more regularly firing and completely quiescent cells are found, which depend on the cortical stimulation. Subsequently we further extend previous work to consider what happens when the excitatory input varies as it would in when the animal is engaged in behavior. We investigate how sudden switches in excitation interact with network generated patterned activity. We show that sequences of cell assembly activations can be locked to the excitatory input sequence and delineate the range of parameters where this behaviour is shown. Model cell population PSTH display both stimulus and temporal specificity, with large population firing rate modulations locked to elapsed time from task events. Thus the random network can generate a large diversity of temporally evolving stimulus dependent responses even though the input is fixed between switches. We suggest the MSN network is well suited to the generation of such slow coherent task dependent response

  5. Inhibiting Matrix Metalloproteinase 3 Ameliorates Neuronal Loss in the Ganglion Cell Layer of Rats in Retinal Ischemia/Reperfusion.

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    Hu, Tu; You, Qiuting; Chen, Dan; Tong, Jianbin; Shang, Lei; Luo, Jia; Qiu, Yi; Yu, Huimin; Zeng, Leping; Huang, Jufang

    2016-05-01

    It has been demonstrated that matrix metalloproteinase 3 (MMP3) is integrally involved in the neuronal degeneration of the central nervous system by promoting glial activation, neuronal apoptosis and damage to the brain-blood barrier. However, whether MMP3 also contributes to the neuronal degeneration induced by retinal ischemia/reperfusion is still uncertain. In the present study, we detected the cellular localization of MMP3 in adult rat retinae and explored the relationship of its expression with neuronal loss in the ganglion cell layer (GCL) in retinal ischemia/reperfusion. We found that MMP3 was widely expressed in many cells throughout the layers of the rat retinae, including Vertebrate neuron