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Sample records for amyloid peptide aggregation

  1. Interaction between amyloid beta peptide and an aggregation blocker peptide mimicking islet amyloid polypeptide.

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    Nasrollah Rezaei-Ghaleh

    Full Text Available Assembly of amyloid-beta peptide (Aβ into cytotoxic oligomeric and fibrillar aggregates is believed to be a major pathologic event in Alzheimer's disease (AD and interfering with Aβ aggregation is an important strategy in the development of novel therapeutic approaches. Prior studies have shown that the double N-methylated analogue of islet amyloid polypeptide (IAPP IAPP-GI, which is a conformationally constrained IAPP analogue mimicking a non-amyloidogenic IAPP conformation, is capable of blocking cytotoxic self-assembly of Aβ. Here we investigate the interaction of IAPP-GI with Aβ40 and Aβ42 using NMR spectroscopy. The most pronounced NMR chemical shift changes were observed for residues 13-20, while residues 7-9, 15-16 as well as the C-terminal half of Aβ--that is both regions of the Aβ sequence that are converted into β-strands in amyloid fibrils--were less accessible to solvent in the presence of IAPP-GI. At the same time, interaction of IAPP-GI with Aβ resulted in a concentration-dependent co-aggregation of Aβ and IAPP-GI that was enhanced for the more aggregation prone Aβ42 peptide. On the basis of the reduced toxicity of the Aβ peptide in the presence of IAPP-GI, our data are consistent with the suggestion that IAPP-GI redirects Aβ into nontoxic "off-pathway" aggregates.

  2. Aggregation and toxicity of amyloid-beta peptide in relation to peptide sequence variation

    OpenAIRE

    Vandersteen, A.

    2012-01-01

    Generally, aggregation of the amyloidpeptide is considered the cause of neuronal death in Alzheimer disease. The heterogenous Aß peptide occurs in various lengths in vivo: Aß40 and Aß42 are the predominant forms while both shorter and longer peptides exist. Aß40 and shorter isoforms are less aggregation-prone and hence considered less dangerous than Aß42 and longer isoforms, which are more aggregation-prone. Up to now research mainly focussed on the predominant Aß peptides and their indivi...

  3. A comparative analysis of the aggregation behavior of amyloidpeptide variants

    NARCIS (Netherlands)

    Vandersteen, Annelies; Hubin, Ellen; Sarroukh, Rabia; De Baets, Greet; Schymkowitz, Joost; Rousseau, Frederic; Subramaniam, Vinod; Raussens, Vincent; Wenschuh, Holger; Wildemann, Dirk; Broersen, Kerensa

    2012-01-01

    Aggregated forms of the amyloidpeptide are hypothesized to act as the prime toxic agents in Alzheimer disease (AD). The in vivo amyloidpeptide pool consists of both C- and N-terminally truncated or mutated peptides, and the composition thereof significantly determines AD risk. Other

  4. Aggregation properties of a short peptide that mediates amyloid fibril ...

    Indian Academy of Sciences (India)

    Short peptides have been identified from amyloidogenic proteins that form amyloid fibrils in isolation. The ... proteins. These peptide fibrils have the conformational features of β-structure that .... water and immediately deposited on freshly cleaved surface of mica .... with the peptide via electrostatic interactions. NaCl would.

  5. Uptake of raft components into amyloid β-peptide aggregates and membrane damage.

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    Sasahara, Kenji; Morigaki, Kenichi; Mori, Yasuko

    2015-07-15

    Amyloid aggregation and deposition of amyloid β-peptide (Aβ) are pathologic characteristics of Alzheimer's disease (AD). Recent reports have shown that the association of Aβ with membranes containing ganglioside GM1 (GM1) plays a pivotal role in amyloid deposition and the pathogenesis of AD. However, the molecular interactions responsible for membrane damage associated with Aβ deposition are not fully understood. In this study, we microscopically observed amyloid aggregation of Aβ in the presence of lipid vesicles and on a substrate-supported planar membrane containing raft components and GM1. The experimental system enabled us to observe lipid-associated aggregation of Aβ, uptake of the raft components into Aβ aggregates, and relevant membrane damage. The results indicate that uptake of raft components from the membrane into Aβ deposits induces macroscopic heterogeneity of the membrane structure. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Distinct thermodynamic signatures of oligomer generation in the aggregation of the amyloidpeptide

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    Cohen, Samuel I. A.; Cukalevski, Risto; Michaels, Thomas C. T.; Šarić, Andela; Törnquist, Mattias; Vendruscolo, Michele; Dobson, Christopher M.; Buell, Alexander K.; Knowles, Tuomas P. J.; Linse, Sara

    2018-05-01

    Mapping free-energy landscapes has proved to be a powerful tool for studying reaction mechanisms. Many complex biomolecular assembly processes, however, have remained challenging to access using this approach, including the aggregation of peptides and proteins into amyloid fibrils implicated in a range of disorders. Here, we generalize the strategy used to probe free-energy landscapes in protein folding to determine the activation energies and entropies that characterize each of the molecular steps in the aggregation of the amyloidpeptide (Aβ42), which is associated with Alzheimer's disease. Our results reveal that interactions between monomeric Aβ42 and amyloid fibrils during fibril-dependent secondary nucleation fundamentally reverse the thermodynamic signature of this process relative to primary nucleation, even though both processes generate aggregates from soluble peptides. By mapping the energetic and entropic contributions along the reaction trajectories, we show that the catalytic efficiency of Aβ42 fibril surfaces results from the enthalpic stabilization of adsorbing peptides in conformations amenable to nucleation, resulting in a dramatic lowering of the activation energy for nucleation.

  7. Hydrodynamic effects on β-amyloid (16-22) peptide aggregation

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    Chiricotto, Mara; Sterpone, Fabio, E-mail: fabio.sterpone@ibpc.fr [Laboratoire de Biochimie Théorique, IBPC, CNRS UPR9080, University Paris Diderot, Sorbonne Paris Cité, 13 rue Pierre et Marie Curie, 75005 Paris (France); Melchionna, Simone [CNR-ISC, Institute for Complex System, Consiglio Nazionale delle Ricerche, Rome (Italy); Derreumaux, Philippe [Laboratoire de Biochimie Théorique, IBPC, CNRS UPR9080, University Paris Diderot, Sorbonne Paris Cité, 13 rue Pierre et Marie Curie, 75005 Paris (France); IUF, Institut Universitaire de France, Boulevard Saint Michel, 75005 Paris (France)

    2016-07-21

    Computer simulations based on simplified representations are routinely used to explore the early steps of amyloid aggregation. However, when protein models with implicit solvent are employed, these simulations miss the effect of solvent induced correlations on the aggregation kinetics and lifetimes of metastable states. In this work, we apply the multi-scale Lattice Boltzmann Molecular Dynamics technique (LBMD) to investigate the initial aggregation phases of the amyloid Aβ{sub 16−22} peptide. LBMD includes naturally hydrodynamic interactions (HIs) via a kinetic on-lattice representation of the fluid kinetics. The peptides are represented by the flexible OPEP coarse-grained force field. First, we have tuned the essential parameters that control the coupling between the molecular and fluid evolutions in order to reproduce the experimental diffusivity of elementary species. The method is then deployed to investigate the effect of HIs on the aggregation of 100 and 1000 Aβ{sub 16−22} peptides. We show that HIs clearly impact the aggregation process and the fluctuations of the oligomer sizes by favouring the fusion and exchange dynamics of oligomers between aggregates. HIs also guide the growth of the leading largest cluster. For the 100 Aβ{sub 16−22} peptide system, the simulation of ∼300 ns allowed us to observe the transition from ellipsoidal assemblies to an elongated and slightly twisted aggregate involving almost the totality of the peptides. For the 1000 Aβ{sub 16−22} peptides, a system of unprecedented size at quasi-atomistic resolution, we were able to explore a branched disordered fibril-like structure that has never been described by other computer simulations, but has been observed experimentally.

  8. Exploring the early steps of aggregation of amyloid-forming peptide KFFE

    International Nuclear Information System (INIS)

    Wei Guanghong; Mousseau, Normand; Derreumaux, Philippe

    2004-01-01

    It has been shown recently that even a tetrapeptide can form amyloid fibrils sharing all the characteristics of amyloid fibrils built from large proteins. Recent experimental studies also suggest that the toxicity observed in several neurodegenerative diseases, such as Alzheimer's disease and Creutzfeldt-Jakob disease, is not only related to the mature fibrils themselves, but also to the soluble oligomers formed early in the process of fibrillogenesis. This raises the interest in studying the early steps of the aggregation process. Although fibril formation follows the nucleation-condensation process, characterized by the presence of lag phase, the exact pathways remain to be determined. In this study, we used the activation-relaxation technique and a generic energy model to explore the process of self-assembly and the structures of the resulting aggregates of eight KFFE peptides. Our simulations show, starting from different states with a preformed antiparallel dimer, that eight chains can self-assemble to adopt, with various orientations, four possible distant oligomeric well-aligned structures of similar energy. Two of these structures show a double-layer β-sheet organization, in agreement with the structure of amyloid fibrils as observed by x-ray diffraction; another two are mixtures of dimers and trimers. Our results also suggest that octamers are likely to be below the critical size for nucleation of amyloid fibrils for small peptides

  9. Exploring the early steps of aggregation of amyloid-forming peptide KFFE

    Energy Technology Data Exchange (ETDEWEB)

    Wei Guanghong [Departement de Physique and Regroupement Quebecois sur les Materiaux de Pointe, Universite de Montreal, CP 6128, succursale centre-ville, Montreal, QC, H3C 3J7 (Canada); Mousseau, Normand [Departement de Physique and Regroupement Quebecois sur les Materiaux de Pointe, Universite de Montreal, CP 6128, succursale centre-ville, Montreal, QC, H3C 3J7 (Canada); Derreumaux, Philippe [Laboratoire de Biochimie, Theorique, UPR 9080 CNRS, IBPC, Universite Paris 7 Denis-Diderot, 13 rue Pierre et Marie Curie, 75005 Paris (France)

    2004-11-10

    It has been shown recently that even a tetrapeptide can form amyloid fibrils sharing all the characteristics of amyloid fibrils built from large proteins. Recent experimental studies also suggest that the toxicity observed in several neurodegenerative diseases, such as Alzheimer's disease and Creutzfeldt-Jakob disease, is not only related to the mature fibrils themselves, but also to the soluble oligomers formed early in the process of fibrillogenesis. This raises the interest in studying the early steps of the aggregation process. Although fibril formation follows the nucleation-condensation process, characterized by the presence of lag phase, the exact pathways remain to be determined. In this study, we used the activation-relaxation technique and a generic energy model to explore the process of self-assembly and the structures of the resulting aggregates of eight KFFE peptides. Our simulations show, starting from different states with a preformed antiparallel dimer, that eight chains can self-assemble to adopt, with various orientations, four possible distant oligomeric well-aligned structures of similar energy. Two of these structures show a double-layer {beta}-sheet organization, in agreement with the structure of amyloid fibrils as observed by x-ray diffraction; another two are mixtures of dimers and trimers. Our results also suggest that octamers are likely to be below the critical size for nucleation of amyloid fibrils for small peptides.

  10. Effect of osmolytes on the conformation and aggregation of some amyloid peptides: CD spectroscopic data

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    Mohammed Inayathullah

    2016-06-01

    Full Text Available Protein misfolding and aggregation are responsible for a large number of diseases called protein conformational diseases or disorders that include Alzheimer׳s disease, Huntington׳s diseases, Prion related encephalopathies and type-II diabetes (http://dx.doi.org/10.1038/35041139 (Kopito and Ron, 2000 [1]. A variety of studies have shown that some small organic molecules, known as osmolytes have the ability to stabilize native conformation of proteins and prevent misfolding and aggregation (http://www.la-press.com/article.php?article_id=447 (Zhao et al., 2008 [2]. It has been shown that certain short segment or fragment of respective proteins can also form amyloids, and the segments also promote the aggregation in the full-length protein (http://dx.doi.org/10.2174/0929867023369187 (Gazit, 2002 [3]. This article presents circular dichroism spectroscopic data on conformational analysis and effect of osmolytes on Aβ peptide fragments, different lengths of polyglutamine peptide and the amyloidogenic segment of islet amyloid polypeptide.

  11. Evaluation of the amyloid beta-GFP fusion protein as a model of amyloid beta peptides-mediated aggregation: A study of DNAJB6 chaperone

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    Rasha Mohamed Hussein

    2015-07-01

    Full Text Available Alzheimer’s disease is a progressive neurodegenerative disease characterized by the accumulation and aggregation of extracellular amyloid β (Aβ peptides and intracellular aggregation of hyper-phosphorylated tau protein. Recent evidence indicates that accumulation and aggregation of intracellular amyloid β peptides may also play a role in disease pathogenesis. This would suggest that intracellular Heat Shock Proteins (HSP that maintain cellular protein homeostasis might be candidates for disease amelioration. We recently found that DNAJB6, a member of DNAJ family of heat shock proteins, effectively prevented the aggregation of short aggregation-prone peptides containing large poly glutamines (associated with CAG repeat diseases both in vitro and in cells. Moreover, recent in vitro data showed that DNAJB6 can delay the aggregation of Aβ42 peptides. In this study, we investigated the ability of DNAJB6 to prevent the aggregation of extracellular and intracellular Aβ peptides using transfection of HEK293 cells with Aβ-GFP fusion construct and performing western blotting and immunofluorescence techniques. We found that DNAJB6 indeed suppresses Aβ-GFP aggregation, but not seeded aggregation initiated by extracellular Aβ peptides. Unexpectedly and unlike what we found for peptide-mediated aggregation, DNAJB6 required interaction with HSP70 to prevent the aggregation of the Aβ-GFP fusion protein and its J-domain was crucial for its anti-aggregation effect. In addition, other DNAJ proteins as well as HSPA1a overexpression also suppressed Aβ-GFP aggregation efficiently. Our findings suggest that Aβ aggregation differs from poly Q peptide induced aggregation in terms of chaperone handling and sheds doubt on the usage of Aβ-GFP fusion construct for studying Aβ peptide aggregation in cells.

  12. The Aggregation Potential of the 1-15-and 1-16-Fragments of the Amyloid beta Peptide and Their Influence on the Aggregation of A beta 40

    NARCIS (Netherlands)

    Shabestari, M.; Plug, T.; Motazacker, M. M.; Meeuwenoord, N. J.; Filippov, D. V.; Meijers, J. C. M.; Huber, M.

    2013-01-01

    The aggregation of amyloid beta (A beta) peptide is important in Alzheimer's disease. Shorter A beta fragments may reduce A beta's cytotoxicity and are used in diagnostics. The aggregation of A beta 16 is controversial; Liu et al. (J. Neurosci. Res. 75:162-171, 2004) and Liao et al. (FEBS Lett.

  13. 9,10-Anthraquinone hinders β-aggregation: How does a small molecule interfere with Aβ-peptide amyloid fibrillation?

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    Convertino, Marino; Pellarin, Riccardo; Catto, Marco; Carotti, Angelo; Caflisch, Amedeo

    2009-01-01

    Amyloid aggregation is linked to a number of neurodegenerative syndromes, the most prevalent one being Alzheimer's disease. In this pathology, the β-amyloid peptides (Aβ) aggregate into oligomers, protofibrils, and fibrils and eventually into plaques, which constitute the characteristic hallmark of Alzheimer's disease. Several low-molecular-weight compounds able to impair the Aβ aggregation process have been recently discovered; yet, a detailed description of their interactions with oligomers and fibrils is hitherto missing. Here, molecular dynamics simulations are used to investigate the influence of two relatively similar tricyclic, planar compounds, that is, 9, 10-anthraquinone (AQ) and anthracene (AC), on the early phase of the aggregation of the Aβ heptapeptide segment H14QKLVFF20, the hydrophobic stretch that promotes the Aβ self-assembly. The simulations show that AQ interferes with β-sheet formation more than AC. In particular, AQ intercalates into the β-sheet because polar interactions between the compound and the peptide backbone destabilize the interstrand hydrogen bonds, thereby favoring disorder. The thioflavin T-binding assay indicates that AQ, but not AC, sensibly reduces the amount of aggregated Aβ1–40 peptide. Taken together, the in silico and in vitro results provide evidence that structural perturbations by AQ can remarkably affect ordered oligomerization. Moreover, the simulations shed light at the atomic level on the interactions between AQ and Aβ oligomers, providing useful insights for the design of small-molecule inhibitors of aggregation with therapeutic potential in Alzheimer's disease. PMID:19309732

  14. Rationally Designed Peptides and Peptidomimetics as Inhibitors of Amyloid-β (Aβ) Aggregation: Potential Therapeutics of Alzheimer's Disease.

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    Goyal, Deepti; Shuaib, Suniba; Mann, Sukhmani; Goyal, Bhupesh

    2017-02-13

    Alzheimer's disease (AD) is a progressive neurodegenerative disease with no clinically accepted treatment to cure or halt its progression. The worldwide effort to develop peptide-based inhibitors of amyloid-β (Aβ) aggregation can be considered an unplanned combinatorial experiment. An understanding of what has been done and achieved may advance our understanding of AD pathology and the discovery of effective therapeutic agents. We review here the history of such peptide-based inhibitors, including those based on the Aβ sequence and those not derived from that sequence, containing both natural and unnatural amino acid building blocks. Peptide-based aggregation inhibitors hold significant promise for future AD therapy owing to their high selectivity, effectiveness, low toxicity, good tolerance, low accumulation in tissues, high chemical and biological diversity, possibility of rational design, and highly developed methods for analyzing their mode of action, proteolytic stability (modified peptides), and blood-brain barrier (BBB) permeability.

  15. Short-term effects of beta-amyloid25-35 peptide aggregates on transmitter release in neuromuscular synapses.

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    Garcia, Neus; Santafé, Manel M; Tomàs, Marta; Lanuza, Maria A; Tomàs, Josep

    2008-03-01

    The beta-amyloid (AB) peptide25-35 contains the functional domain of the AB precursor protein that is both required for neurotrophic effects in normal neural tissues and is involved in the neurotoxic effects in Alzheimer disease. We demonstrated the presence of the amyloid precursor protein/AB peptide in intramuscular axons, presynaptic motor nerve terminals, terminal and myelinating Schwann cells, and the postsynaptic and subsarcolemmal region in the Levator auris longus muscle of adult rats by immunocytochemistry. Using intracellular recording, we investigated possible short-term functional effects of the AB fragment (0.1-10 micromol/L) on acetylcholine release in adult and newborn motor end plates. We found no change in evoked, spontaneous transmitter release or resting membrane potential of the muscle cells. A previous block of the presynaptic muscarinic receptor subtypes and a previous block or stimulation of protein kinase C revealed no masked effect of the peptide on the regulation of transmitter release. The aggregated form of AB peptide25-35, however, interfered acutely with acetylcholine release (quantal content reduction) when synaptic activity was maintained by electric stimulation. The possible relevance of this inhibition of neurotransmission by AB peptide25-35 to the pathogenesis of Alzheimer remains to be determined.

  16. Surface Plasmon Resonance Based Biosensors for Exploring the Influence of Alkaloids on Aggregation of AmyloidPeptide

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    Hanna Radecka

    2011-04-01

    Full Text Available The main objective of the presented study was the development of a simple analytical tool for exploring the influence of naturally occurring compounds on the aggregation of amyloidpeptide (Aβ40 in order to find potential anti-neurodegenerative drugs. The gold discs used for surface plasmon resonance (SPR measurements were modified with thioaliphatic acid. The surface functionalized with carboxylic groups was used for covalent attaching of Aβ40 probe by creation of amide bonds in the presence of EDC/NHS. The modified SPR gold discs were used for exploring the Aβ40 aggregation process in the presence of selected alkaloids: arecoline hydrobromide, pseudopelletierine hydrochloride, trigonelline hydrochloride and α-lobeline hydrochloride. The obtained results were discussed with other parameters which govern the phenomenon studied such as lipophilicity/ hydrophilicy and Aβ40-alkaloid association constants.

  17. Mutation-based structural modification and dynamics study of amyloid beta peptide (1–42: An in-silico-based analysis to cognize the mechanism of aggregation

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    Pritam Kumar Panda

    2016-03-01

    Full Text Available Alzheimer's disease is the prevalent cause of premature senility, a progressive mental disorder due to degeneration in brain and deposition of amyloid β peptide (1–42, a misfolded protein in the form of aggregation that prevails for a prolonged time and obstructs every aspect of life. One of the primary hallmarks of the neuropathological disease is the accretion of amyloid β peptide in the brain that leads to Alzheimer's disease, but the mechanism is still a mystery. Several investigations have shown that mutations at specific positions have a significant impact in stability of the peptide as predicted from aggregation profiles. Here in our study, we have analyzed the mutations by substituting residues at position A22G, E22G, E22K, E22Q, D23N, L34V and molecular dynamics have been performed to check the deviation in stability and conformation of the peptide. The results validated that the mutations at specific positions lead to instability and the proline substitution at E22P and L34P stalled the aggregation of the peptide. Keywords: Amyloid β peptide, Alzheimer's disease, Aggregation, Mutational analysis, NAMD, UCSF Chimera, Discovery Studio Visualizer

  18. Using Multifunctional Peptide Conjugated Au Nanorods for Monitoring β-amyloid Aggregation and Chemo-Photothermal Treatment of Alzheimer's Disease.

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    Li, Meng; Guan, Yijia; Zhao, Andong; Ren, Jinsong; Qu, Xiaogang

    2017-01-01

    Development of sensitive detectors of Aβ aggregates and effective inhibitors of Aβ aggregation are of diagnostic importance and therapeutic implications for Alzheimer's disease (AD) treatment. Herein, a novel strategy has been presented by self-assembly of peptide conjugated Au nanorods (AuP) as multifunctional Aβ fibrillization detectors and inhibitors. Our design combines the unique high NIR absorption property of AuNRs with two known Aβ inhibitors, Aβ15-20 and polyoxometalates (POMs). The synthesized AuP can effectively inhibit Aβ aggregation and dissociate amyloid deposits with NIR irradiation both in buffer and in mice cerebrospinal fluid (CSF), and protect cells from Aβ-related toxicity upon NIR irradiation. In addition, with the shape and size-dependent optical properties, the nanorods can also act as effective diagnostic probes to sensitively detect the Aβ aggregates. This is the first report to integrate 3 segments, an Aβ-targeting element, a reporter and inhibitors, in one drug delivery system for AD treatment.

  19. Posttranslational nitro-glycative modifications of albumin in Alzheimer's disease: implications in cytotoxicity and amyloidpeptide aggregation.

    Science.gov (United States)

    Ramos-Fernández, Eva; Tajes, Marta; Palomer, Ernest; Ill-Raga, Gerard; Bosch-Morató, Mònica; Guivernau, Biuse; Román-Dégano, Irene; Eraso-Pichot, Abel; Alcolea, Daniel; Fortea, Juan; Nuñez, Laura; Paez, Antonio; Alameda, Francesc; Fernández-Busquets, Xavier; Lleó, Alberto; Elosúa, Roberto; Boada, Mercé; Valverde, Miguel A; Muñoz, Francisco J

    2014-01-01

    Glycation and nitrotyrosination are pathological posttranslational modifications that make proteins prone to losing their physiological properties. Since both modifications are increased in Alzheimer's disease (AD) due to amyloidpeptide (Aβ) accumulation, we have studied their effect on albumin, the most abundant protein in cerebrospinal fluid and blood. Brain and plasmatic levels of glycated and nitrated albumin were significantly higher in AD patients than in controls. In vitro turbidometry and electron microscopy analyses demonstrated that glycation and nitrotyrosination promote changes in albumin structure and biochemical properties. Glycated albumin was more resistant to proteolysis and less uptake by hepatoma cells occurred. Glycated albumin also reduced the osmolarity expected for a solution containing native albumin. Both glycation and nitrotyrosination turned albumin cytotoxic in a cell type-dependent manner for cerebral and vascular cells. Finally, of particular relevance to AD, these modified albumins were significantly less effective in avoiding Aβ aggregation than native albumin. In summary, nitrotyrosination and especially glycation alter albumin structural and biochemical properties, and these modifications might contribute for the progression of AD.

  20. Investigation of the inhibitory effects of TiO{sub 2} on the β-amyloid peptide aggregation

    Energy Technology Data Exchange (ETDEWEB)

    Ahmed, Mukhtar H., E-mail: ahmed-m@email.ulster.ac.uk [School of Chemical Science, National Centre for Sensor Research, Dublin City University, Dublin 9 (Ireland); Nanotechnology Integrated Bioengineering Centre, University of Ulster, Jordanstown, BT37 0QB Belfast (United Kingdom); Byrne, John A. [Nanotechnology Integrated Bioengineering Centre, University of Ulster, Jordanstown, BT37 0QB Belfast (United Kingdom); Keyes, Tia E. [School of Chemical Science, National Centre for Sensor Research, Dublin City University, Dublin 9 (Ireland)

    2014-06-01

    TiO{sub 2} thin films are of great interest as biocompatible coatings and also as photocatalytic self-cleaning and antimicrobial coatings. In this work we used β-amyloid as a model for infectious protein to investigate the attachment and photocatalytic degradation. TiO{sub 2} films were prepared on stainless steel substrates using magnetron sputtering. The films were characterised before and after exposure to β-amyloid (1–42), using XRD, Raman spectroscopy, XPS and AFM. The TiO{sub 2} film was mostly composed of the anatase phase with a relatively high surface roughness. The presence of Raman peaks at 1668 cm{sup −1} and 1263 cm{sup −1}, with the XPS spectral feature for nitrogen at 400 eV, confirmed the adsorption of amyloid on surface. Following exposure of the β-amyloid contaminated TiO{sub 2} to UV-B irradiation a slight shift of amide modes was observed. Furthermore, the amide I spectra show an overall decrease in α-helix content with presence of a minor peak around 1591 cm{sup −1}, which is related to tryptophanyl and tyrosinyl radicals, which can lead to conformational change of β-amyloid. The C1s band at 292.2 eV suggests the formation of free carboxylic acid. The loss in the crucial structure of β-amyloid leads to reduce the fibril formation, thought to be induced through a photocatalytic process. - Highlights: • TiO{sub 2} thin films synthesised and characterised • Absorption study using β-amyloid (1–42) • Investigation of peptide configuration via Raman, AFM and XPS spectroscopies • β-Amyloid was subsequently degraded by photocatalytic activity of TiO{sub 2}.

  1. Rosuvastatin ameliorates cognitive impairment in rats fed with high-salt and cholesterol diet via inhibiting acetylcholinesterase activity and amyloid beta peptide aggregation.

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    Husain, I; Akhtar, M; Abdin, M Zainul; Islamuddin, M; Shaharyar, M; Najmi, A K

    2018-04-01

    Amyloid beta (Aβ) peptide aggregation and cholinergic neurodegeneration are involved in the development of cognitive impairment. Therefore, in this article, we examined rosuvastatin (RSV), an oral hypolipidemic drug, to determine its potential as a dual inhibitor of acetylcholinesterase (AChE) and Aβ peptide aggregation for the treatment of cognitive impairment. Molecular docking study was done to examine the affinity of RSV with Aβ 1-42 and AChE in silico. We also employed neurobehavioral activity tests, biochemical estimation, and histopathology to study the anti-Aβ 1-42 aggregation capability of RSV in vivo. Molecular docking study provided evidence that RSV has the best binding conformer at its receptor site or active site of an enzyme. The cognitive impairment in female Wistar rats was induced by high-salt and cholesterol diet (HSCD) ad libitum for 8 weeks. RSV ameliorated serum cholesterol level, AChE activity, and Aβ 1-42 peptide aggregations in HSCD induced cognitive impairment. In addition, RSV-treated rats showed greater scores in the open field (locomotor activity) test. Moreover, the histopathological studies in the hippocampus and cortex of rat brain also supported that RSV markedly reduced the cognitive impairment and preserved the normal histoarchitectural pattern of the hippocampus and cortex. Taken together, these data indicate that RSV may act as a dual inhibitor of AChE and Aβ 1-42 peptide aggregation, therefore suggesting a therapeutic strategy for cognitive impairment treatment.

  2. Zn(II)- and Cu(II)-induced non-fibrillar aggregates of amyloid-beta (1-42) peptide are transformed to amyloid fibrils, both spontaneously and under the influence of metal chelators.

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    Tõugu, Vello; Karafin, Ann; Zovo, Kairit; Chung, Roger S; Howells, Claire; West, Adrian K; Palumaa, Peep

    2009-09-01

    Aggregation of amyloid-beta (Abeta) peptides is a central phenomenon in Alzheimer's disease. Zn(II) and Cu(II) have profound effects on Abeta aggregation; however, their impact on amyloidogenesis is unclear. Here we show that Zn(II) and Cu(II) inhibit Abeta(42) fibrillization and initiate formation of non-fibrillar Abeta(42) aggregates, and that the inhibitory effect of Zn(II) (IC(50) = 1.8 micromol/L) is three times stronger than that of Cu(II). Medium and high-affinity metal chelators including metallothioneins prevented metal-induced Abeta(42) aggregation. Moreover, their addition to preformed aggregates initiated fast Abeta(42) fibrillization. Upon prolonged incubation the metal-induced aggregates also transformed spontaneously into fibrils, that appear to represent the most stable state of Abeta(42). H13A and H14A mutations in Abeta(42) reduced the inhibitory effect of metal ions, whereas an H6A mutation had no significant impact. We suggest that metal binding by H13 and H14 prevents the formation of a cross-beta core structure within region 10-23 of the amyloid fibril. Cu(II)-Abeta(42) aggregates were neurotoxic to neurons in vitro only in the presence of ascorbate, whereas monomers and Zn(II)-Abeta(42) aggregates were non-toxic. Disturbed metal homeostasis in the vicinity of zinc-enriched neurons might pre-dispose formation of metal-induced Abeta aggregates, subsequent fibrillization of which can lead to amyloid formation. The molecular background underlying metal-chelating therapies for Alzheimer's disease is discussed in this light.

  3. Synthetic Curcumin Analogs as Inhibitors of β -Amyloid Peptide Aggregation: Potential Therapeutic and Diagnostic Agents for Alzheimer's Disease.

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    Bukhari, Syed Nasir Abbas; Jantan, Ibrahim

    2015-01-01

    There is a crucial need to develop new effective drugs for Alzheimer's disease (AD) as the currently available AD treatments provide only momentary and incomplete symptomatic relief. Amongst natural products, curcumin, a major constituent of turmeric, has been intensively investigated for its neuroprotective effect against β-amyloid (Aβ)-induced toxicity in cultured neuronal cells. The ability of curcumin to attach to Aβ peptide and prevent its accumulation is attributed to its three structural characteristics such as the presence of two aromatic end groups and their co-planarity, the length and rigidity of the linker region and the substitution conformation of these aromatics. However, curcumin failed to reach adequate brain levels after oral absorption in AD clinical trials due to its low water solubility and poor oral bioavailability. A number of new curcumin analogs that mimic the active site of the compound along with analogs that mimic the curcumin anti-amyloid effect combined with anticholinesterase effect have been developed to enhance the bioavailability, pharmacokinetics, water solubility, stability at physiological conditions and delivery of curcumin. In this article, we have summarized all reported synthetic analogs of curcumin showing effects on β-amyloid and discussed their potential as therapeutic and diagnostic agents for AD.

  4. Amyloid β-sheet mimics that antagonize protein aggregation and reduce amyloid toxicity

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    Cheng, Pin-Nan; Liu, Cong; Zhao, Minglei; Eisenberg, David; Nowick, James S.

    2012-11-01

    The amyloid protein aggregation associated with diseases such as Alzheimer's, Parkinson's and type II diabetes (among many others) features a bewildering variety of β-sheet-rich structures in transition from native proteins to ordered oligomers and fibres. The variation in the amino-acid sequences of the β-structures presents a challenge to developing a model system of β-sheets for the study of various amyloid aggregates. Here, we introduce a family of robust β-sheet macrocycles that can serve as a platform to display a variety of heptapeptide sequences from different amyloid proteins. We have tailored these amyloid β-sheet mimics (ABSMs) to antagonize the aggregation of various amyloid proteins, thereby reducing the toxicity of amyloid aggregates. We describe the structures and inhibitory properties of ABSMs containing amyloidogenic peptides from the amyloidpeptide associated with Alzheimer's disease, β2-microglobulin associated with dialysis-related amyloidosis, α-synuclein associated with Parkinson's disease, islet amyloid polypeptide associated with type II diabetes, human and yeast prion proteins, and Tau, which forms neurofibrillary tangles.

  5. Novel β-amyloid aggregation inhibitors possessing a turn mimic.

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    Hamada, Yoshio; Miyamoto, Naoko; Kiso, Yoshiaki

    2015-04-01

    Amyloid β peptide, the main component of senile plaques found in the brain of Alzheimer disease (AD) patients, is a molecular target for AD therapeutic intervention. A number of potential AD therapeutics have been reported, including inhibitors of β-secretase, γ-secretase, and Aβ aggregation, and anti-amyloid agents, such as neprilysin, insulin degrading enzyme (IDE), and Aβ antibodies. Recently, we reported potent small-sized β-secretase (BACE1) inhibitors, which could serve as anti-AD drugs. However AD is a progressive disorder, where dementia symptoms gradually worsen over several decades, and therefore may require many years to get cured. One possible way to achieve a greater therapeutic effect is through simultaneous administration of multiple drugs, similar to those used in Highly Active Anti-Retroviral Therapy (HAART) used to treat AIDS. In order to overcome AD, we took a drug discovery approach to evaluate, novel β-amyloid aggregation inhibitors. Previously, we reported that a tong-type compound possessing a turn mimic as the inhibitor of HIV-1 protease dimerization. Oligomerized amyloid β peptides contain a turn structure within the molecule. Here, we designed and synthesized novel β-amyloid aggregation inhibitors with a turn-mimic template, based on the turn conformer of the oligomerized amyloid β peptides. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Inhibition of Alzheimer amyloid {beta} aggregation by polyvalent trehalose

    Energy Technology Data Exchange (ETDEWEB)

    Miura, Yoshiko; You, Chouga [School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa 923-1292 (Japan); Ohnishi, Reiko [Department of Molecular Design and Engineering, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603 (Japan)], E-mail: miuray@jaist.ac.jp

    2008-04-15

    A glycopolymer carrying trehalose was found to suppress the formation of amyloid fibrils from the amyloid {beta} peptide (1-42) (A{beta}), as evaluated by thioflavin T assay and atomic force microscopy. Glycopolymers carrying sugar alcohols also changed the aggregation properties of A{beta}, and the inhibitory effect depended on the type of sugar and alkyl side chain. Neutralization activity was confirmed by in vitro assay using HeLa cells. The glycopolymer carrying trehalose strongly inhibited amyloid formation and neutralized cytotoxicity.

  7. Conformational changes of the amyloid beta-peptide (1-40) adsorbed on solid surfaces

    NARCIS (Netherlands)

    Giacomelli, CE; Norde, W

    2005-01-01

    The conformational change of the 39-43 residues of the amyloid beta-peptide (A beta) toward a beta-sheet enriched state promotes self-aggregation of the peptide molecules and constitutes the major peptide component of the amyloid plaques in Alzheimer patients. The crucial question behind the

  8. Conformational changes of the amyloid beta-peptide (1-40) adsorbed on solid surfaces

    NARCIS (Netherlands)

    Giacomelli, C.E.; Norde, W.

    2005-01-01

    The conformational change of the 39-43 residues of the amyloid beta -peptide (A beta) toward a beta -sheet enriched state promotes self-aggregation of the peptide molecules and constitutes the major peptide component of the amyloid plaques in Alzheimer patients. The crucial question behind the

  9. Curcumin Attenuates AmyloidAggregate Toxicity and Modulates AmyloidAggregation Pathway.

    Science.gov (United States)

    Thapa, Arjun; Jett, Stephen D; Chi, Eva Y

    2016-01-20

    The abnormal misfolding and aggregation of amyloid-β (Aβ) peptides into β-sheet enriched insoluble deposits initiates a cascade of events leading to pathological processes and culminating in cognitive decline in Alzheimer's disease (AD). In particular, soluble oligomeric/prefibrillar Aβ have been shown to be potent neurotoxins. The naturally occurring polyphenol curcumin has been shown to exert a neuroprotective effect against age-related neurodegenerative diseases such as AD. However, its protective mechanism remains unclear. In this study, we investigated the effects of curcumin on the aggregation of Aβ40 as well as Aβ40 aggregate induced neurotoxicity. Our results show that the curcumin does not inhibit Aβ fibril formation, but rather enriches the population of "off-pathway" soluble oligomers and prefibrillar aggregates that were nontoxic. Curcumin also exerted a nonspecific neuroprotective effect, reducing toxicities induced by a range of Aβ conformers, including monomeric, oligomeric, prefibrillar, and fibrillar Aβ. The neuroprotective effect is possibly membrane-mediated, as curcumin reduced the extent of cell membrane permeabilization induced by Aβ aggregates. Taken together, our study shows that curcumin exerts its neuroprotective effect against Aβ induced toxicity through at least two concerted pathways, modifying the Aβ aggregation pathway toward the formation of nontoxic aggregates and ameliorating Aβ-induced toxicity possibly through a nonspecific pathway.

  10. AmyloidPeptide Induces Prion Protein Amyloid Formation: Evidence for Its Widespread Amyloidogenic Effect.

    Science.gov (United States)

    Honda, Ryo

    2018-04-12

    Transmissible spongiform encephalopathy is associated with misfolding of prion protein (PrP) into an amyloid β-rich aggregate. Previous studies have indicated that PrP interacts with Alzheimer's disease amyloidpeptide (Aβ), but it remains elusive how this interaction impacts on the misfolding of PrP. This study presents the first in vitro evidence that Aβ induces PrP-amyloid formation at submicromolar concentrations. Interestingly, systematic mutagenesis of PrP revealed that Aβ requires no specific amino acid sequences in PrP, and induces the misfolding of other unrelated proteins (insulin and lysozyme) into amyloid fibrils in a manner analogous to PrP. This unanticipated nonspecific amyloidogenic effect of Aβ indicates that this peptide might be involved in widespread protein aggregation, regardless of the amino acid sequences of target proteins, and exacerbate the pathology of many neurodegenerative diseases. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Stabilization of a β-hairpin in monomeric Alzheimer's amyloidpeptide inhibits amyloid formation

    OpenAIRE

    Hoyer, Wolfgang; Grönwall, Caroline; Jonsson, Andreas; Ståhl, Stefan; Härd, Torleif

    2008-01-01

    According to the amyloid hypothesis, the pathogenesis of Alzheimer's disease is triggered by the oligomerization and aggregation of the amyloid-β (Aβ) peptide into protein plaques. Formation of the potentially toxic oligomeric and fibrillar Aβ assemblies is accompanied by a conformational change toward a high content of β-structure. Here, we report the solution structure of Aβ(1–40) in complex with the phage-display selected affibody protein ZAβ3, a binding protein of nanomolar affinity. Boun...

  12. Star Polymers Reduce Islet Amyloid Polypeptide Toxicity via Accelerated Amyloid Aggregation.

    Science.gov (United States)

    Pilkington, Emily H; Lai, May; Ge, Xinwei; Stanley, William J; Wang, Bo; Wang, Miaoyi; Kakinen, Aleksandr; Sani, Marc-Antonie; Whittaker, Michael R; Gurzov, Esteban N; Ding, Feng; Quinn, John F; Davis, Thomas P; Ke, Pu Chun

    2017-12-11

    Protein aggregation into amyloid fibrils is a ubiquitous phenomenon across the spectrum of neurodegenerative disorders and type 2 diabetes. A common strategy against amyloidogenesis is to minimize the populations of toxic oligomers and protofibrils by inhibiting protein aggregation with small molecules or nanoparticles. However, melanin synthesis in nature is realized by accelerated protein fibrillation to circumvent accumulation of toxic intermediates. Accordingly, we designed and demonstrated the use of star-shaped poly(2-hydroxyethyl acrylate) (PHEA) nanostructures for promoting aggregation while ameliorating the toxicity of human islet amyloid polypeptide (IAPP), the peptide involved in glycemic control and the pathology of type 2 diabetes. The binding of PHEA elevated the β-sheet content in IAPP aggregates while rendering a new morphology of "stelliform" amyloids originating from the polymers. Atomistic molecular dynamics simulations revealed that the PHEA arms served as rodlike scaffolds for IAPP binding and subsequently accelerated IAPP aggregation by increased local peptide concentration. The tertiary structure of the star nanoparticles was found to be essential for driving the specific interactions required to impel the accelerated IAPP aggregation. This study sheds new light on the structure-toxicity relationship of IAPP and points to the potential of exploiting star polymers as a new class of therapeutic agents against amyloidogenesis.

  13. New Cyclolignans from Origanumglandulosum Active Against b -amyloid Aggregation

    Directory of Open Access Journals (Sweden)

    Abdelkader Basli

    2014-05-01

    Full Text Available Origanum glandulosum Desf is an endemic flavoring herb widely distributed in North Africa that is commonly used in traditional medicine. This oregano species is rich in essential oils but little is known about its phenolic composition. In the present study, a crude extract of O. glandulosum was prepared in order to isolate and investigate its neuroprotective potential to inhibit β-amyloid peptide (Aβ aggregation. The three major compounds of the extract were isolated: rosmarinic acid and two cyclolignans in Origanum genus, globoidnan A and a new derivative named globoidnan B. Rosmarinic acid and globoidnan A showed significant anti-aggregative activity against β amyloid aggregation (IC50 7.0 and 12.0 µM, respectively. In contrast, globoidnan B was found to be less active.

  14. Influence of hydrophobic Teflon particles on the structure of amyloid beta-peptide

    NARCIS (Netherlands)

    Giacomelli, C.E.; Norde, W.

    2003-01-01

    The amyloid beta-protein (Abeta) constitutes the major peptide component of the amyloid plaque deposits of Alzheimer's disease in humans. The Abeta changes from a nonpathogenic to a pathogenic conformation resulting in self-aggregation and deposition of the peptide. It has been established that

  15. Cross-interactions between the Alzheimer Disease AmyloidPeptide and Other Amyloid Proteins: A Further Aspect of the Amyloid Cascade Hypothesis.

    Science.gov (United States)

    Luo, Jinghui; Wärmländer, Sebastian K T S; Gräslund, Astrid; Abrahams, Jan Pieter

    2016-08-05

    Many protein folding diseases are intimately associated with accumulation of amyloid aggregates. The amyloid materials formed by different proteins/peptides share many structural similarities, despite sometimes large amino acid sequence differences. Some amyloid diseases constitute risk factors for others, and the progression of one amyloid disease may affect the progression of another. These connections are arguably related to amyloid aggregates of one protein being able to directly nucleate amyloid formation of another, different protein: the amyloid cross-interaction. Here, we discuss such cross-interactions between the Alzheimer disease amyloid-β (Aβ) peptide and other amyloid proteins in the context of what is known from in vitro and in vivo experiments, and of what might be learned from clinical studies. The aim is to clarify potential molecular associations between different amyloid diseases. We argue that the amyloid cascade hypothesis in Alzheimer disease should be expanded to include cross-interactions between Aβ and other amyloid proteins. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Disrupting beta-amyloid aggregation for Alzheimer disease treatment.

    Science.gov (United States)

    Estrada, L D; Soto, C

    2007-01-01

    Alzheimer's disease is a devastating degenerative disorder for which there is no cure or effective treatment. Although the etiology of Alzheimer's disease is not fully understood, compelling evidence indicates that deposition of aggregates composed by a misfolded form of the amyloid beta peptide (Abeta) is the central event in the disease pathogenesis. Therefore, an attractive therapeutic strategy is to prevent or reverse Abeta misfolding and aggregation. Diverse strategies have been described to identify inhibitors of this process, including screening of libraries of small molecules chemical compounds, rational design of synthetic peptides, assessment of natural Abeta-binding proteins and stimulation of the immune system by vaccination. In this article we describe these different approaches, their principles and their potential strengths and weaknesses. Overall the available data suggest that the development of drugs to interfere with Abeta misfolding and aggregation is a feasible target that hold great promise for the treatment of Alzheimer's disease.

  17. Amyloid beta peptide immunotherapy in Alzheimer disease.

    Science.gov (United States)

    Delrieu, J; Ousset, P J; Voisin, T; Vellas, B

    2014-12-01

    Recent advances in the understanding of Alzheimer's disease pathogenesis have led to the development of numerous compounds that might modify the disease process. Amyloid β peptide represents an important molecular target for intervention in Alzheimer's disease. The main purpose of this work is to review immunotherapy studies in relation to the Alzheimer's disease. Several types of amyloid β peptide immunotherapy for Alzheimer's disease are under investigation, active immunization and passive administration with monoclonal antibodies directed against amyloid β peptide. Although immunotherapy approaches resulted in clearance of amyloid plaques in patients with Alzheimer's disease, this clearance did not show significant cognitive effect for the moment. Currently, several amyloid β peptide immunotherapy approaches are under investigation but also against tau pathology. Results from amyloid-based immunotherapy studies in clinical trials indicate that intervention appears to be more effective in early stages of amyloid accumulation in particular solanezumab with a potential impact at mild Alzheimer's disease, highlighting the importance of diagnosing Alzheimer's disease as early as possible and undertaking clinical trials at this stage. In both phase III solanezumab and bapineuzumab trials, PET imaging revealed that about a quarter of patients lacked fibrillar amyloid pathology at baseline, suggesting that they did not have Alzheimer's disease in the first place. So a new third phase 3 clinical trial for solanezumab, called Expedition 3, in patients with mild Alzheimer's disease and evidence of amyloid burden has been started. Thus, currently, amyloid intervention is realized at early stage of the Alzheimer's disease in clinical trials, at prodromal Alzheimer's disease, or at asymptomatic subjects or at risk to develop Alzheimer's disease and or at asymptomatic subjects with autosomal dominant mutation. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  18. Interaction of amyloid inhibitor proteins with amyloid beta peptides: insight from molecular dynamics simulations.

    Directory of Open Access Journals (Sweden)

    Payel Das

    Full Text Available Knowledge of the detailed mechanism by which proteins such as human αB- crystallin and human lysozyme inhibit amyloid beta (Aβ peptide aggregation is crucial for designing treatment for Alzheimer's disease. Thus, unconstrained, atomistic molecular dynamics simulations in explicit solvent have been performed to characterize the Aβ17-42 assembly in presence of the αB-crystallin core domain and of lysozyme. Simulations reveal that both inhibitor proteins compete with inter-peptide interaction by binding to the peptides during the early stage of aggregation, which is consistent with their inhibitory action reported in experiments. However, the Aβ binding dynamics appear different for each inhibitor. The binding between crystallin and the peptide monomer, dominated by electrostatics, is relatively weak and transient due to the heterogeneous amino acid distribution of the inhibitor surface. The crystallin-bound Aβ oligomers are relatively long-lived, as they form more extensive contact surface with the inhibitor protein. In contrast, a high local density of arginines from lysozyme allows strong binding with Aβ peptide monomers, resulting in stable complexes. Our findings not only illustrate, in atomic detail, how the amyloid inhibitory mechanism of human αB-crystallin, a natural chaperone, is different from that of human lysozyme, but also may aid de novo design of amyloid inhibitors.

  19. Probing the role of metal cations on the aggregation behavior of amyloid β-peptide at a single molecule level by AFM

    Energy Technology Data Exchange (ETDEWEB)

    Xie, Yang; Wang, Jianhua, E-mail: wjh@cqu.edu.cn; Liu, Chundong [Chongqing University, Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering (China)

    2016-09-15

    With the development of nanotechnology, understanding of intermolecular interactions on a single molecule level by atomic force spectroscopy (AFM) has played an important role in molecular biology and biomedical science. In recent years, some research suggested that the presence of metal cations is an important regulator in the processes of misfolding and aggregation of the amyloid β-protein (Aβ), which may be an important etiological factor of Alzheimer’s disease. However, the knowledge on the principle of interactions between Aβ and metal cations at the single molecule level is still poor understood. In this paper, the amyloid β-protein (Aβ) was fabricated on substrate of mixed thiol-modified gold nanoparticles using self-assembled monolayer method and the adhesion force in the longitudinal direction between metal cations and Aβ42 were investigated by AFM. The role of metal ions on Aβ aggregation is discussed from the perspective of single molecular force. The force results showed that the specific adhesion force F{sub i} and the nonspecific force F{sub 0} between a single Aβ–Aβ pair in control experiment were calculated as 42 ± 3 and 80 pN, respectively. However, F{sub i} between a single Aβ–Aβ pair in the presence of Cu{sup 2+}, Zn{sup 2+}, Ca{sup 2+} and Al{sup 3+} increased dramatically to 84 ± 6, 89 ± 3, 73 ± 5, 95 ± 5 pN successively, which indicated that unbinding between Aβ proteins is accelerated in the presence of metal cations. What is more, the imaging results showed that substoichiometric copper cations accelerate the formation of fibrils within 3 days. The combined atomic force spectroscopy and imaging analysis indicate that metal cations play a role in promoting the aggregating behavior of Aβ42.

  20. Probing the role of metal cations on the aggregation behavior of amyloid β-peptide at a single molecule level by AFM

    Science.gov (United States)

    Xie, Yang; Wang, Jianhua; Liu, Chundong

    2016-09-01

    With the development of nanotechnology, understanding of intermolecular interactions on a single molecule level by atomic force spectroscopy (AFM) has played an important role in molecular biology and biomedical science. In recent years, some research suggested that the presence of metal cations is an important regulator in the processes of misfolding and aggregation of the amyloid β-protein (Aβ), which may be an important etiological factor of Alzheimer's disease. However, the knowledge on the principle of interactions between Aβ and metal cations at the single molecule level is still poor understood. In this paper, the amyloid β-protein (Aβ) was fabricated on substrate of mixed thiol-modified gold nanoparticles using self-assembled monolayer method and the adhesion force in the longitudinal direction between metal cations and Aβ42 were investigated by AFM. The role of metal ions on Aβ aggregation is discussed from the perspective of single molecular force. The force results showed that the specific adhesion force F i and the nonspecific force F 0 between a single Aβ-Aβ pair in control experiment were calculated as 42 ± 3 and 80 pN, respectively. However, F i between a single Aβ-Aβ pair in the presence of Cu2+, Zn2+, Ca2+ and Al3+ increased dramatically to 84 ± 6, 89 ± 3, 73 ± 5, 95 ± 5 pN successively, which indicated that unbinding between Aβ proteins is accelerated in the presence of metal cations. What is more, the imaging results showed that substoichiometric copper cations accelerate the formation of fibrils within 3 days. The combined atomic force spectroscopy and imaging analysis indicate that metal cations play a role in promoting the aggregating behavior of Aβ42.

  1. Probing the role of metal cations on the aggregation behavior of amyloid β-peptide at a single molecule level by AFM

    International Nuclear Information System (INIS)

    Xie, Yang; Wang, Jianhua; Liu, Chundong

    2016-01-01

    With the development of nanotechnology, understanding of intermolecular interactions on a single molecule level by atomic force spectroscopy (AFM) has played an important role in molecular biology and biomedical science. In recent years, some research suggested that the presence of metal cations is an important regulator in the processes of misfolding and aggregation of the amyloid β-protein (Aβ), which may be an important etiological factor of Alzheimer’s disease. However, the knowledge on the principle of interactions between Aβ and metal cations at the single molecule level is still poor understood. In this paper, the amyloid β-protein (Aβ) was fabricated on substrate of mixed thiol-modified gold nanoparticles using self-assembled monolayer method and the adhesion force in the longitudinal direction between metal cations and Aβ42 were investigated by AFM. The role of metal ions on Aβ aggregation is discussed from the perspective of single molecular force. The force results showed that the specific adhesion force F_i and the nonspecific force F_0 between a single Aβ–Aβ pair in control experiment were calculated as 42 ± 3 and 80 pN, respectively. However, F_i between a single Aβ–Aβ pair in the presence of Cu"2"+, Zn"2"+, Ca"2"+ and Al"3"+ increased dramatically to 84 ± 6, 89 ± 3, 73 ± 5, 95 ± 5 pN successively, which indicated that unbinding between Aβ proteins is accelerated in the presence of metal cations. What is more, the imaging results showed that substoichiometric copper cations accelerate the formation of fibrils within 3 days. The combined atomic force spectroscopy and imaging analysis indicate that metal cations play a role in promoting the aggregating behavior of Aβ42.

  2. Surface Mediated Self-Assembly of Amyloid Peptides

    Science.gov (United States)

    Fakhraai, Zahra

    2015-03-01

    Amyloid fibrils have been considered as causative agents in many neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, type II diabetes and amyloidosis. Amyloid fibrils form when proteins or peptides misfold into one dimensional crystals of stacked beta-sheets. In solution, amyloid fibrils form through a nucleation and growth mechanism. The rate limiting nucleation step requires a critical concentration much larger than those measured in physiological conditions. As such the exact origins of the seeds or oligomers that result in the formation of fully mature fibrils in the body remain topic intense studies. It has been suggested that surfaces and interfaces can enhance the fibrillization rate. However, studies of the mechanism and kinetics of the surface-mediated fibrillization are technologically challenging due to the small size of the oligomer and protofibril species. Using smart sample preparation technique to dry the samples after various incubation times we are able to study the kinetics of fibril formation both in solution and in the vicinity of various surfaces using high-resolution atomic force microscopy. These studies elucidate the role of surfaces in catalyzing amyloid peptide formation through a nucleation-free process. The nucleation free self-assembly is rapid and requires much smaller concentrations of peptides or proteins. We show that this process resembles diffusion limited aggregation and is governed by the peptide adhesion rate, two -dimensional diffusion of the peptides on the surface, and preferential interactions between the peptides. These studies suggest an alternative pathway for amyloid formation may exist, which could lead to new criteria for disease prevention and alternative therapies. Research was partially supported by a seed grant from the National Institute of Aging of the National Institutes of Health (NIH) under Award Number P30AG010124 (PI: John Trojanowski) and the University of Pennsylvania.

  3. Protein Folding and Aggregation into Amyloid: The Interference by Natural Phenolic Compounds

    Directory of Open Access Journals (Sweden)

    Massimo Stefani

    2013-06-01

    Full Text Available Amyloid aggregation is a hallmark of several degenerative diseases affecting the brain or peripheral tissues, whose intermediates (oligomers, protofibrils and final mature fibrils display different toxicity. Consequently, compounds counteracting amyloid aggregation have been investigated for their ability (i to stabilize toxic amyloid precursors; (ii to prevent the growth of toxic oligomers or speed that of fibrils; (iii to inhibit fibril growth and deposition; (iv to disassemble preformed fibrils; and (v to favor amyloid clearance. Natural phenols, a wide panel of plant molecules, are one of the most actively investigated categories of potential amyloid inhibitors. They are considered responsible for the beneficial effects of several traditional diets being present in green tea, extra virgin olive oil, red wine, spices, berries and aromatic herbs. Accordingly, it has been proposed that some natural phenols could be exploited to prevent and to treat amyloid diseases, and recent studies have provided significant information on their ability to inhibit peptide/protein aggregation in various ways and to stimulate cell defenses, leading to identify shared or specific mechanisms. In the first part of this review, we will overview the significance and mechanisms of amyloid aggregation and aggregate toxicity; then, we will summarize the recent achievements on protection against amyloid diseases by many natural phenols.

  4. Real-time amyloid aggregation monitoring with a photonic crystal-based approach.

    Science.gov (United States)

    Santi, Sara; Musi, Valeria; Descrovi, Emiliano; Paeder, Vincent; Di Francesco, Joab; Hvozdara, Lubos; van der Wal, Peter; Lashuel, Hilal A; Pastore, Annalisa; Neier, Reinhard; Herzig, Hans Peter

    2013-10-21

    We propose the application of a new label-free optical technique based on photonic nanostructures to real-time monitor the amyloid-beta 1-42 (Aβ(1-42)) fibrillization, including the early stages of the aggregation process, which are related to the onset of the Alzheimer's Disease (AD). The aggregation of Aβ peptides into amyloid fibrils has commonly been associated with neuronal death, which culminates in the clinical features of the incurable degenerative AD. Recent studies revealed that cell toxicity is determined by the formation of soluble oligomeric forms of Aβ peptides in the early stages of aggregation. At this phase, classical amyloid detection techniques lack in sensitivity. Upon a chemical passivation of the sensing surface by means of polyethylene glycol, the proposed approach allows an accurate, real-time monitoring of the refractive index variation of the solution, wherein Aβ(1-42) peptides are aggregating. This measurement is directly related to the aggregation state of the peptide throughout oligomerization and subsequent fibrillization. Our findings open new perspectives in the understanding of the dynamics of amyloid formation, and validate this approach as a new and powerful method to screen aggregation at early stages. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Halogenation dictates the architecture of amyloid peptide nanostructures.

    Science.gov (United States)

    Pizzi, Andrea; Pigliacelli, Claudia; Gori, Alessandro; Nonappa; Ikkala, Olli; Demitri, Nicola; Terraneo, Giancarlo; Castelletto, Valeria; Hamley, Ian W; Baldelli Bombelli, Francesca; Metrangolo, Pierangelo

    2017-07-20

    Amyloid peptides yield a plethora of interesting nanostructures though difficult to control. Here we report that depending on the number, position, and nature of the halogen atoms introduced into either one or both phenylalanine benzene rings of the amyloid β peptide-derived core-sequence KLVFF, four different architectures were obtained in a controlled manner. Our findings demonstrate that halogenation may develop as a general strategy to engineer amyloidal peptide self-assembly and obtain new amyloidal nanostructures.

  6. Exploring new biological functions of amyloids: bacteria cell agglutination mediated by host protein aggregation.

    Directory of Open Access Journals (Sweden)

    Marc Torrent

    Full Text Available Antimicrobial proteins and peptides (AMPs are important effectors of the innate immune system that play a vital role in the prevention of infections. Recent advances have highlighted the similarity between AMPs and amyloid proteins. Using the Eosinophil Cationic Protein as a model, we have rationalized the structure-activity relationships between amyloid aggregation and antimicrobial activity. Our results show how protein aggregation can induce bacteria agglutination and cell death. Using confocal and total internal reflection fluorescence microscopy we have tracked the formation in situ of protein amyloid-like aggregates at the bacteria surface and on membrane models. In both cases, fibrillar aggregates able to bind to amyloid diagnostic dyes were detected. Additionally, a single point mutation (Ile13 to Ala can suppress the protein amyloid behavior, abolishing the agglutinating activity and impairing the antimicrobial action. The mutant is also defective in triggering both leakage and lipid vesicle aggregation. We conclude that ECP aggregation at the bacterial surface is essential for its cytotoxicity. Hence, we propose here a new prospective biological function for amyloid-like aggregates with potential biological relevance.

  7. Vitamin k3 inhibits protein aggregation: Implication in the treatment of amyloid diseases

    OpenAIRE

    Parvez Alam; Sumit Kumar Chaturvedi; Mohammad Khursheed Siddiqi; Ravi Kant Rajpoot; Mohd Rehan Ajmal; Masihuz Zaman; Rizwan Hasan Khan

    2016-01-01

    Protein misfolding and aggregation have been associated with several human diseases such as Alzheimer?s, Parkinson?s and familial amyloid polyneuropathy etc. In this study, anti-fibrillation activity of vitamin k3 and its effect on the kinetics of amyloid formation of hen egg white lysozyme (HEWL) and A?-42 peptide were investigated. Here, in combination with Thioflavin T (ThT) fluorescence assay, circular dichroism (CD), transmission electron microscopy and cell cytotoxicity assay, we demons...

  8. Simultaneous measurement of amyloid fibril formation by dynamic light scattering and fluorescence reveals complex aggregation kinetics.

    Directory of Open Access Journals (Sweden)

    Aaron M Streets

    Full Text Available An apparatus that combines dynamic light scattering and Thioflavin T fluorescence detection is used to simultaneously probe fibril formation in polyglutamine peptides, the aggregating subunit associated with Huntington's disease, in vitro. Huntington's disease is a neurodegenerative disorder in a class of human pathologies that includes Alzheimer's and Parkinson's disease. These pathologies are all related by the propensity of their associated protein or polypeptide to form insoluble, β-sheet rich, amyloid fibrils. Despite the wide range of amino acid sequence in the aggregation prone polypeptides associated with these diseases, the resulting amyloids display strikingly similar physical structure, an observation which suggests a physical basis for amyloid fibril formation. Thioflavin T fluorescence reports β-sheet fibril content while dynamic light scattering measures particle size distributions. The combined techniques allow elucidation of complex aggregation kinetics and are used to reveal multiple stages of amyloid fibril formation.

  9. Sequence dependent aggregation of peptides and fibril formation

    Science.gov (United States)

    Hung, Nguyen Ba; Le, Duy-Manh; Hoang, Trinh X.

    2017-09-01

    Deciphering the links between amino acid sequence and amyloid fibril formation is key for understanding protein misfolding diseases. Here we use Monte Carlo simulations to study the aggregation of short peptides in a coarse-grained model with hydrophobic-polar (HP) amino acid sequences and correlated side chain orientations for hydrophobic contacts. A significant heterogeneity is observed in the aggregate structures and in the thermodynamics of aggregation for systems of different HP sequences and different numbers of peptides. Fibril-like ordered aggregates are found for several sequences that contain the common HPH pattern, while other sequences may form helix bundles or disordered aggregates. A wide variation of the aggregation transition temperatures among sequences, even among those of the same hydrophobic fraction, indicates that not all sequences undergo aggregation at a presumable physiological temperature. The transition is found to be the most cooperative for sequences forming fibril-like structures. For a fibril-prone sequence, it is shown that fibril formation follows the nucleation and growth mechanism. Interestingly, a binary mixture of peptides of an aggregation-prone and a non-aggregation-prone sequence shows the association and conversion of the latter to the fibrillar structure. Our study highlights the role of a sequence in selecting fibril-like aggregates and also the impact of a structural template on fibril formation by peptides of unrelated sequences.

  10. Cooperative structural transitions in amyloid-like aggregation

    Science.gov (United States)

    Steckmann, Timothy; Bhandari, Yuba R.; Chapagain, Prem P.; Gerstman, Bernard S.

    2017-04-01

    Amyloid fibril aggregation is associated with several horrific diseases such as Alzheimer's, Creutzfeld-Jacob, diabetes, Parkinson's, and others. Although proteins that undergo aggregation vary widely in their primary structure, they all produce a cross-β motif with the proteins in β-strand conformations perpendicular to the fibril axis. The process of amyloid aggregation involves forming myriad different metastable intermediate aggregates. To better understand the molecular basis of the protein structural transitions and aggregation, we report on molecular dynamics (MD) computational studies on the formation of amyloid protofibrillar structures in the small model protein ccβ, which undergoes many of the structural transitions of the larger, naturally occurring amyloid forming proteins. Two different structural transition processes involving hydrogen bonds are observed for aggregation into fibrils: the breaking of intrachain hydrogen bonds to allow β-hairpin proteins to straighten, and the subsequent formation of interchain H-bonds during aggregation into amyloid fibrils. For our MD simulations, we found that the temperature dependence of these two different structural transition processes results in the existence of a temperature window that the ccβ protein experiences during the process of forming protofibrillar structures. This temperature dependence allows us to investigate the dynamics on a molecular level. We report on the thermodynamics and cooperativity of the transformations. The structural transitions that occurred in a specific temperature window for ccβ in our investigations may also occur in other amyloid forming proteins but with biochemical parameters controlling the dynamics rather than temperature.

  11. Entrapment of Aβ1-40 peptide in unstructured aggregates

    International Nuclear Information System (INIS)

    Corsale, C; Carrotta, R; Mangione, M R; Vilasi, S; Provenzano, A; Bulone, D; San Biagio, P L; Cavallaro, G

    2012-01-01

    Recognizing the complexity of the fibrillogenesis process provides a solid ground for the development of therapeutic strategies aimed at preventing or inhibiting protein-protein aggregation. Under this perspective, it is meaningful to identify the possible aggregation pathways and their relative products. We found that Aβ-peptide dissolved in a pH 7.4 solution at small peptide concentration and low ionic strength forms globular aggregates without typical amyloid β-conformation. ThT binding kinetics was used to monitor aggregate formation. Circular dichroism spectroscopy, AFM imaging, static and dynamic light scattering were used for structural and morphological characterization of the aggregates. They appear stable or at least metastable with respect to fiber growth, therefore appearing as an incidental product in the pathway of fibrillogenesis. (paper)

  12. Detection of Alzheimer's amyloid beta aggregation by capturing molecular trails of individual assemblies

    International Nuclear Information System (INIS)

    Vestergaard, Mun'delanji; Hamada, Tsutomu; Saito, Masato; Yajima, Yoshifumi; Kudou, Monotori; Tamiya, Eiichi; Takagi, Masahiro

    2008-01-01

    Assembly of Amyloid beta (Aβ) peptides, in particular Aβ-42 is central to the formation of the amyloid plaques associated with neuro-pathologies such as Alzheimer's disease (AD). Molecular assembly of individual Aβ-42 species was observed using a simple fluorescence microscope. From the molecular movements (aka Brownian motion) of the individual peptide assemblies, we calculated a temporal evolution of the hydrodynamic radius (R H ) of the peptide at physiological temperature and pH. The results clearly show a direct relationship between R H of Aβ-42 and incubation period, corresponding to the previously reported peptide's aggregation kinetics. The data correlates highly with in solution-based label-free electrochemical detection of the peptide's aggregation, and Aβ-42 deposited on a solid surface and analysed using atomic force microscopy (AFM). To the best of our knowledge, this is the first analysis and characterisation of Aβ aggregation based on capturing molecular trails of individual assemblies. The technique enables both real-time observation and a semi-quantitative distribution profile of the various stages of Aβ assembly, at microM peptide concentration. Our method is a promising candidate for real-time observation and analysis of the effect of other pathologically-relevant molecules such as metal ions on pathways to Aβ oligomerisation and aggregation. The method is also a promising screening tool for AD therapeutics that target Aβ assembly.

  13. Characterization of amyloid beta peptides from brain extracts of transgenic mice overexpressing the London mutant of human amyloid precursor protein.

    Science.gov (United States)

    Pype, Stefan; Moechars, Dieder; Dillen, Lieve; Mercken, Marc

    2003-02-01

    Alzheimer's disease (AD) is marked by the presence of neurofibrillary tangles and amyloid plaques in the brain of patients. To study plaque formation, we report on further quantitative and qualitative analysis of human and mouse amyloid beta peptides (Abeta) from brain extracts of transgenic mice overexpressing the London mutant of human amyloid precursor protein (APP). Using enzyme-linked immunosorbant assays (ELISAs) specific for either human or rodent Abeta, we found that the peptides from both species aggregated to form plaques. The ratios of deposited Abeta1-42/1-40 were in the order of 2-3 for human and 8-9 for mouse peptides, indicating preferential deposition of Abeta42. We also determined the identity and relative levels of other Abeta variants present in protein extracts from soluble and insoluble brain fractions. This was done by combined immunoprecipitation and mass spectrometry (IP/MS). The most prominent peptides truncated either at the carboxyl- or the amino-terminus were Abeta1-38 and Abeta11-42, respectively, and the latter was strongly enriched in the extracts of deposited peptides. Taken together, our data indicate that plaques of APP-London transgenic mice consist of aggregates of multiple human and mouse Abeta variants, and the human variants that we identified were previously detected in brain extracts of AD patients.

  14. Stable, metastable, and kinetically trapped amyloid aggregate phases.

    Science.gov (United States)

    Miti, Tatiana; Mulaj, Mentor; Schmit, Jeremy D; Muschol, Martin

    2015-01-12

    Self-assembly of proteins into amyloid fibrils plays a key role in a multitude of human disorders that range from Alzheimer's disease to type II diabetes. Compact oligomeric species, observed early during amyloid formation, are reported as the molecular entities responsible for the toxic effects of amyloid self-assembly. However, the relation between early-stage oligomeric aggregates and late-stage rigid fibrils, which are the hallmark structure of amyloid plaques, has remained unclear. We show that these different structures occupy well-defined regions in a peculiar phase diagram. Lysozyme amyloid oligomers and their curvilinear fibrils only form after they cross a salt and protein concentration-dependent threshold. We also determine a boundary for the onset of amyloid oligomer precipitation. The oligomeric aggregates are structurally distinct from rigid fibrils and are metastable against nucleation and growth of rigid fibrils. These experimentally determined boundaries match well with colloidal model predictions that account for salt-modulated charge repulsion. The model also incorporates the metastable and kinetic character of oligomer phases. Similarities and differences of amyloid oligomer assembly to metastable liquid-liquid phase separation of proteins and to surfactant aggregation are discussed.

  15. Stabilization of a β-hairpin in monomeric Alzheimer's amyloidpeptide inhibits amyloid formation

    Science.gov (United States)

    Hoyer, Wolfgang; Grönwall, Caroline; Jonsson, Andreas; Ståhl, Stefan; Härd, Torleif

    2008-01-01

    According to the amyloid hypothesis, the pathogenesis of Alzheimer's disease is triggered by the oligomerization and aggregation of the amyloid-β (Aβ) peptide into protein plaques. Formation of the potentially toxic oligomeric and fibrillar Aβ assemblies is accompanied by a conformational change toward a high content of β-structure. Here, we report the solution structure of Aβ(1–40) in complex with the phage-display selected affibody protein ZAβ3, a binding protein of nanomolar affinity. Bound Aβ(1–40) features a β-hairpin comprising residues 17–36, providing the first high-resolution structure of Aβ in β conformation. The positions of the secondary structure elements strongly resemble those observed for fibrillar Aβ. ZAβ3 stabilizes the β-sheet by extending it intermolecularly and by burying both of the mostly nonpolar faces of the Aβ hairpin within a large hydrophobic tunnel-like cavity. Consequently, ZAβ3 acts as a stoichiometric inhibitor of Aβ fibrillation. The selected Aβ conformation allows us to suggest a structural mechanism for amyloid formation based on soluble oligomeric hairpin intermediates. PMID:18375754

  16. Vitamin k3 inhibits protein aggregation: Implication in the treatment of amyloid diseases.

    Science.gov (United States)

    Alam, Parvez; Chaturvedi, Sumit Kumar; Siddiqi, Mohammad Khursheed; Rajpoot, Ravi Kant; Ajmal, Mohd Rehan; Zaman, Masihuz; Khan, Rizwan Hasan

    2016-05-27

    Protein misfolding and aggregation have been associated with several human diseases such as Alzheimer's, Parkinson's and familial amyloid polyneuropathy etc. In this study, anti-fibrillation activity of vitamin k3 and its effect on the kinetics of amyloid formation of hen egg white lysozyme (HEWL) and Aβ-42 peptide were investigated. Here, in combination with Thioflavin T (ThT) fluorescence assay, circular dichroism (CD), transmission electron microscopy and cell cytotoxicity assay, we demonstrated that vitamin k3 significantly inhibits fibril formation as well as the inhibitory effect is dose dependent manner. Our experimental studies inferred that vitamin k3 exert its neuro protective effect against amyloid induced cytotoxicity through concerted pathway, modifying the aggregation formation towards formation of nontoxic aggregates. Molecular docking demonstrated that vitamin k3 mediated inhibition of HEWL and Aβ-42 fibrillogenesis may be initiated by interacting with proteolytic resistant and aggregation prone regions respectively. This work would provide an insight into the mechanism of protein aggregation inhibition by vitamin k3; pave the way for discovery of other small molecules that may exert similar effect against amyloid formation and its associated neurodegenerative diseases.

  17. CPAD, Curated Protein Aggregation Database: A Repository of Manually Curated Experimental Data on Protein and Peptide Aggregation.

    Science.gov (United States)

    Thangakani, A Mary; Nagarajan, R; Kumar, Sandeep; Sakthivel, R; Velmurugan, D; Gromiha, M Michael

    2016-01-01

    Accurate distinction between peptide sequences that can form amyloid-fibrils or amorphous β-aggregates, identification of potential aggregation prone regions in proteins, and prediction of change in aggregation rate of a protein upon mutation(s) are critical to research on protein misfolding diseases, such as Alzheimer's and Parkinson's, as well as biotechnological production of protein based therapeutics. We have developed a Curated Protein Aggregation Database (CPAD), which has collected results from experimental studies performed by scientific community aimed at understanding protein/peptide aggregation. CPAD contains more than 2300 experimentally observed aggregation rates upon mutations in known amyloidogenic proteins. Each entry includes numerical values for the following parameters: change in rate of aggregation as measured by fluorescence intensity or turbidity, name and source of the protein, Uniprot and Protein Data Bank codes, single point as well as multiple mutations, and literature citation. The data in CPAD has been supplemented with five different types of additional information: (i) Amyloid fibril forming hexa-peptides, (ii) Amorphous β-aggregating hexa-peptides, (iii) Amyloid fibril forming peptides of different lengths, (iv) Amyloid fibril forming hexa-peptides whose crystal structures are available in the Protein Data Bank (PDB) and (v) Experimentally validated aggregation prone regions found in amyloidogenic proteins. Furthermore, CPAD is linked to other related databases and resources, such as Uniprot, Protein Data Bank, PUBMED, GAP, TANGO, WALTZ etc. We have set up a web interface with different search and display options so that users have the ability to get the data in multiple ways. CPAD is freely available at http://www.iitm.ac.in/bioinfo/CPAD/. The potential applications of CPAD have also been discussed.

  18. Design of non-aggregating variants of Aβ peptide

    Energy Technology Data Exchange (ETDEWEB)

    Caine, Joanne M., E-mail: jo.caine@csiro.au [CSIRO Materials Science and Engineering, 343 Royal Parade, Parkville, Victoria 3052 (Australia); Preventative Health Flagship, 343 Royal Parade, Parkville, Victoria 3052 (Australia); CRC for Mental Health, Level 2, 161 Barry Street, Carlton South, Victoria 3053 (Australia); Churches, Quentin; Waddington, Lynne [CSIRO Materials Science and Engineering, 343 Royal Parade, Parkville, Victoria 3052 (Australia); Preventative Health Flagship, 343 Royal Parade, Parkville, Victoria 3052 (Australia); Nigro, Julie; Breheney, Kerry [CSIRO Materials Science and Engineering, 343 Royal Parade, Parkville, Victoria 3052 (Australia); Preventative Health Flagship, 343 Royal Parade, Parkville, Victoria 3052 (Australia); CRC for Mental Health, Level 2, 161 Barry Street, Carlton South, Victoria 3053 (Australia); Masters, Colin L. [CRC for Mental Health, Level 2, 161 Barry Street, Carlton South, Victoria 3053 (Australia); Florey Institute for Neuroscience and Mental Health, 30 Royal Parade, Parkville, Victoria 3052 (Australia); Nuttall, Stewart D. [CSIRO Materials Science and Engineering, 343 Royal Parade, Parkville, Victoria 3052 (Australia); Preventative Health Flagship, 343 Royal Parade, Parkville, Victoria 3052 (Australia); CRC for Mental Health, Level 2, 161 Barry Street, Carlton South, Victoria 3053 (Australia); Streltsov, Victor A., E-mail: victor.streltsov@csiro.au [CSIRO Materials Science and Engineering, 343 Royal Parade, Parkville, Victoria 3052 (Australia); Preventative Health Flagship, 343 Royal Parade, Parkville, Victoria 3052 (Australia); CRC for Mental Health, Level 2, 161 Barry Street, Carlton South, Victoria 3053 (Australia)

    2014-10-24

    Highlights: • Non-aggregating, non-toxic variants of Aβ peptide were designed using Aβ structure. • Mutations reduce aggregation by stabilising Aβ into small non-toxic oligomers. • Identification of these residues will assist the design of future therapeutic peptides. - Abstract: Self association of the amyloid-β (Aβ{sub 42}) peptide into oligomers, high molecular weight forms, fibrils and ultimately neuritic plaques, has been correlated with progressive cognitive decline in Alzheimer’s disease. Thus, insights into the drivers of the aggregation pathway have the capacity to significantly contribute to our understanding of disease mechanism. Functional assays and a three-dimensional crystal structure of the P3 amyloidogenic region 18–41 of Aβ were used to identify residues important in self-association and to design novel non-aggregating variants of the peptide. Biophysical studies (gel filtration, SDS–PAGE, dynamic light scattering, thioflavin T assay, and electron microscopy) demonstrate that in contrast to wild type Aβ these targeted mutations lose the ability to self-associate. Loss of aggregation also correlates with reduced neuronal toxicity. Our results highlight residues and regions of the Aβ peptide important for future targeting agents aimed at the amelioration of Alzheimer’s disease.

  19. Engineered aggregation inhibitor fusion for production of highly amyloidogenic human islet amyloid polypeptide.

    Science.gov (United States)

    Mirecka, Ewa Agnieszka; Gremer, Lothar; Schiefer, Stephanie; Oesterhelt, Filipp; Stoldt, Matthias; Willbold, Dieter; Hoyer, Wolfgang

    2014-12-10

    Human islet amyloid polypeptide (IAPP) is the major component of pancreatic amyloid deposits in type 2 diabetes. The structural conversion of IAPP from a monomeric state into amyloid assemblies is the subject of intense research. Recombinant production of IAPP is, however, difficult due to its extreme aggregation propensity. Here we describe a novel strategy for expression of IAPP in Escherichia coli, based on an engineered protein tag, which sequesters IAPP monomers and prevents IAPP aggregation. The IAPP-binding protein HI18 was selected by phage display from a β-wrapin library. Fusion of HI18 to IAPP enabled the soluble expression of the construct. IAPP was cleaved from the fusion construct and purified to homogeneity with a yield of 3mg of isotopically labeled peptide per liter of culture. In the monomeric state, IAPP was largely disordered as evidenced by far-UV CD and liquid-state NMR spectroscopy but competent to form amyloid fibrils according to atomic force microscopy. These results demonstrate the ability of the engineered β-wrapin HI18 for shielding the hydrophobic sequence of IAPP during expression and purification. Fusion of aggregation-inhibiting β-wrapins is a suitable approach for the recombinant production of aggregation-prone proteins. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. A Peptide-Fc Opsonin with Pan-Amyloid Reactivity

    Directory of Open Access Journals (Sweden)

    James S. Foster

    2017-09-01

    Full Text Available There is a continuing need for therapeutic interventions for patients with the protein misfolding disorders that result in systemic amyloidosis. Recently, specific antibodies have been employed to treat AL amyloidosis by opsonizing tissue amyloid deposits thereby inducing cell-mediated dissolution and organ improvement. To develop a pan-amyloid therapeutic agent, we have produced an Fc-fusion product incorporating a peptide, p5, which binds many if not all forms of amyloid. This protein, designated Fcp5, expressed in mammalian cells, forms the desired bivalent dimer structure and retains pan-amyloid reactivity similar to the p5 peptide as measured by immunosorbent assays, immunohistochemistry, surface plasmon resonance, and pulldown assays using radioiodinated Fcp5. Additionally, Fcp5 was capable of opsonizing amyloid fibrils in vitro using a pH-sensitive fluorescence assay of phagocytosis. In mice,125 I-labeled Fcp5 exhibited an extended serum circulation time, relative to the p5 peptide. It specifically bound AA amyloid deposits in diseased mice, as evidenced by biodistribution and microautoradiographic methods, which coincided with an increase in active, Iba-1-positive macrophages in the liver at 48 h postinjection of Fcp5. In healthy mice, no specific tissue accumulation was observed. The data indicate that polybasic, pan-amyloid-targeting peptides, in the context of an Fc fusion, can yield amyloid reactive, opsonizing reagents that may serve as next-generation immunotherapeutics.

  1. Two-Step Amyloid Aggregation: Sequential Lag Phase Intermediates

    Science.gov (United States)

    Castello, Fabio; Paredes, Jose M.; Ruedas-Rama, Maria J.; Martin, Miguel; Roldan, Mar; Casares, Salvador; Orte, Angel

    2017-01-01

    The self-assembly of proteins into fibrillar structures called amyloid fibrils underlies the onset and symptoms of neurodegenerative diseases, such as Alzheimer’s and Parkinson’s. However, the molecular basis and mechanism of amyloid aggregation are not completely understood. For many amyloidogenic proteins, certain oligomeric intermediates that form in the early aggregation phase appear to be the principal cause of cellular toxicity. Recent computational studies have suggested the importance of nonspecific interactions for the initiation of the oligomerization process prior to the structural conversion steps and template seeding, particularly at low protein concentrations. Here, using advanced single-molecule fluorescence spectroscopy and imaging of a model SH3 domain, we obtained direct evidence that nonspecific aggregates are required in a two-step nucleation mechanism of amyloid aggregation. We identified three different oligomeric types according to their sizes and compactness and performed a full mechanistic study that revealed a mandatory rate-limiting conformational conversion step. We also identified the most cytotoxic species, which may be possible targets for inhibiting and preventing amyloid aggregation.

  2. Trifluoroethanol modulates α-synuclein amyloid-like aggregate formation, stability and dissolution

    DEFF Research Database (Denmark)

    Di Carlo, Maria Giovanna; Vetri, Valeria; Buscarino, Gianpiero

    2016-01-01

    The conversion of proteins into amyloid fibrils and other amyloid-like aggregates is closely connected to the onset of a series of age-related pathologies. Upon changes in environmental conditions, amyloid-like aggregates may also undergo disassembly into oligomeric aggregates, the latter being r...

  3. Modeling the Aggregation Propensity and Toxicity of Amyloid-β Variants

    DEFF Research Database (Denmark)

    Tiwari, Manish Kumar; Kepp, Kasper Planeta

    2015-01-01

    Protein aggregation is a hallmark of many neurodegenerative disorders. Alzheimer’s disease (AD) is directly linked to deposits of amyloid-β (Aβ) derived from the amyloid-β protein precursor (AβPP), and multiple experimental studies have investigated the aggregation behavior of these amyloids...

  4. Oxidative stress and the amyloid beta peptide in Alzheimer’s disease

    Directory of Open Access Journals (Sweden)

    C. Cheignon

    2018-04-01

    Full Text Available Oxidative stress is known to play an important role in the pathogenesis of a number of diseases. In particular, it is linked to the etiology of Alzheimer’s disease (AD, an age-related neurodegenerative disease and the most common cause of dementia in the elderly. Histopathological hallmarks of AD are intracellular neurofibrillary tangles and extracellular formation of senile plaques composed of the amyloid-beta peptide (Aβ in aggregated form along with metal-ions such as copper, iron or zinc. Redox active metal ions, as for example copper, can catalyze the production of Reactive Oxygen Species (ROS when bound to the amyloid-β (Aβ. The ROS thus produced, in particular the hydroxyl radical which is the most reactive one, may contribute to oxidative damage on both the Aβ peptide itself and on surrounding molecule (proteins, lipids, …. This review highlights the existing link between oxidative stress and AD, and the consequences towards the Aβ peptide and surrounding molecules in terms of oxidative damage. In addition, the implication of metal ions in AD, their interaction with the Aβ peptide and redox properties leading to ROS production are discussed, along with both in vitro and in vivo oxidation of the Aβ peptide, at the molecular level. Keywords: Oxidative stress, Amyloid beta peptide, Metal-ions, Reactive oxygen species, Oxidative damages

  5. The Nucleation of Protein Aggregates - From Crystals to Amyloid Fibrils.

    Science.gov (United States)

    Buell, Alexander K

    2017-01-01

    The condensation and aggregation of individual protein molecules into dense insoluble phases is of relevance in such diverse fields as materials science, medicine, structural biology and pharmacology. A common feature of these condensation phenomena is that they usually are nucleated processes, i.e. the first piece of the condensed phase is energetically costly to create and hence forms slowly compared to its subsequent growth. Here we give a compact overview of the differences and similarities of various protein nucleation phenomena, their theoretical description in the framework of colloid and polymer science and their experimental study. Particular emphasis is put on the nucleation of a specific type of filamentous protein aggregates, amyloid fibrils. The current experimentally derived knowledge on amyloid fibril nucleation is critically assessed, and we argue that it is less advanced than is generally believed. This is due to (I) the lack of emphasis that has been put on the distinction between homogeneous and heterogeneous nucleation in experimental studies (II) the use of oversimplifying and/or inappropriate theoretical frameworks for the analysis of kinetic data of amyloid fibril nucleation. A strategy is outlined and advocated of how our understanding of this important class of processes can be improved in the future. © 2017 Elsevier Inc. All rights reserved.

  6. Structural and Thermodynamic Properties of AmyloidPeptides: Impact of Fragment Size

    Science.gov (United States)

    Kitahara, T.; Wise-Scira, O.; Coskuner, O.

    2010-10-01

    Alzheimer's disease is a progressive neurodegenerative disease whose physiological characteristics include the accumulation of amyloid-containing deposits in the brain and consequent synapse and neuron loss. Unfortunately, most widely used drugs for the treatment can palliate the outer symptoms but cannot cure the disease itself. Hence, developing a new drug that can cure it. Most recently, the ``early aggregation and monomer'' hypothesis has become popular and a few drugs have been developed based on this hypothesis. Detailed understanding of the amyloidpeptide structure can better help us to determine more effective treatment strategies; indeed, the structure of Amyloid has been studied extensively employing experimental and theoretical tools. Nevertheless, those studies have employed different fragment sizes of Amyloid and characterized its conformational nature in different media. Thus, the structural properties might be different from each other and provide a reason for the existing debates in the literature. Here, we performed all-atom MD simulations and present the structural and thermodynamic properties of Aβ1-16, Aβ1-28, and Aβ1-42 in the gas phase and in aqueous solution. Our studies show that the overall structures, secondary structures, and the calculated thermodynamic properties change with increasing peptide size. In addition, we find that the structural properties of those peptides are different from each other in the gas phase and in aqueous solution.

  7. Advances toward multifunctional cholinesterase and β-amyloid aggregation inhibitors.

    Science.gov (United States)

    Panek, Dawid; Wichur, Tomasz; Godyń, Justyna; Pasieka, Anna; Malawska, Barbara

    2017-10-01

    The emergence of a multitarget design approach in the development of new potential anti-Alzheimer's disease agents has resulted in the discovery of many multifunctional compounds focusing on various targets. Among them the largest group comprises inhibitors of both cholinesterases, with additional anti-β-amyloid aggregation activity. This review describes recent advances in this research area and presents the most interesting compounds reported over a 2-year span (2015-2016). The majority of hybrids possess heterodimeric structures obtained by linking structurally active fragments interacting with different targets. Multipotent cholinesterase inhibitors with β-amyloid antiaggregating activity may additionally possess antioxidative, neuroprotective or metal-chelating properties or less common features such as anti-β-secretase or τ-antiaggregation activity.

  8. Structure activity relationship study of curcumin analogues toward the amyloid-beta aggregation inhibitor.

    Science.gov (United States)

    Endo, Hitoshi; Nikaido, Yuri; Nakadate, Mamiko; Ise, Satomi; Konno, Hiroyuki

    2014-12-15

    Inhibition of the amyloid β aggregation process could possibly prevent the onset of Alzheimer's disease. In this article, we report a structure-activity relationship study of curcumin analogues for anti amyloid β aggregation activity. Compound 7, the ideal amyloid β aggregation inhibitor in vitro among synthesized curcumin analogues, has not only potent anti amyloid β aggregation effects, but also water solubility more than 160 times that of curcumin. In addition, new approaches to improve water solubility of curcumin-type compounds are proposed. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. Determination of critical nucleation number for a single nucleation amyloidaggregation model.

    Science.gov (United States)

    Ghosh, Preetam; Vaidya, Ashwin; Kumar, Amit; Rangachari, Vijayaraghavan

    2016-03-01

    Aggregates of amyloid-β (Aβ) peptide are known to be the key pathological agents in Alzheimer disease (AD). Aβ aggregates to form large, insoluble fibrils that deposit as senile plaques in AD brains. The process of aggregation is nucleation-dependent in which the formation of a nucleus is the rate-limiting step, and controls the physiochemical fate of the aggregates formed. Therefore, understanding the properties of nucleus and pre-nucleation events will be significant in reducing the existing knowledge-gap in AD pathogenesis. In this report, we have determined the plausible range of critical nucleation number (n(*)), the number of monomers associated within the nucleus for a homogenous aggregation model with single unique nucleation event, by two independent methods: A reduced-order stability analysis and ordinary differential equation based numerical analysis, supported by experimental biophysics. The results establish that the most likely range of n(*) is between 7 and 14 and within, this range, n(*) = 12 closely supports the experimental data. These numbers are in agreement with those previously reported, and importantly, the report establishes a new modeling framework using two independent approaches towards a convergent solution in modeling complex aggregation reactions. Our model also suggests that the formation of large protofibrils is dependent on the nature of n(*), further supporting the idea that pre-nucleation events are significant in controlling the fate of larger aggregates formed. This report has re-opened an old problem with a new perspective and holds promise towards revealing the molecular events in amyloid pathologies in the future. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Thioflavin T templates amyloid β(1-40) conformation and aggregation pathway

    DEFF Research Database (Denmark)

    Di Carlo, Maria Giovanna; Minicozzi, Velia; Foderà, Vito

    2015-01-01

    Aβ(1-40) peptide supramolecular assembly and fibril formation processes are widely recognized to have direct implications in the progression of Alzheimer's disease. The molecular basis of this biological process is still unknown and there is a strong need of developing effective strategies...... in turn rests on the reliability of the probe/labels involved. Here we present evidences of the effect of Thioflavin T (ThT), a worldwide used fluorescent dye to monitor amyloid growth, on the Aβ(1-40) conformation, stability and aggregation. By combining experimental information and Molecular Dynamics...... simulation results, we show that the presence of ThT in solution affects peptide conformation inducing peculiar supramolecular association. In particular ThT interactions with specific Aβ(1-40) residues promote a rigid partially-folded conformation which shifts the balance between different species...

  11. Studies of the aggregation of mutant proteins in vitro provide insights into the genetics of amyloid diseases.

    Science.gov (United States)

    Chiti, Fabrizio; Calamai, Martino; Taddei, Niccolo; Stefani, Massimo; Ramponi, Giampietro; Dobson, Christopher M

    2002-12-10

    Protein aggregation and the formation of highly insoluble amyloid structures is associated with a range of debilitating human conditions, which include Alzheimer's disease, Parkinson's disease, and the Creutzfeldt-Jakob disease. Muscle acylphosphatase (AcP) has already provided significant insights into mutational changes that modulate amyloid formation. In the present paper, we have used this system to investigate the effects of mutations that modify the charge state of a protein without affecting significantly the hydrophobicity or secondary structural propensities of the polypeptide chain. A highly significant inverse correlation was found to exist between the rates of aggregation of the protein variants under denaturing conditions and their overall net charge. This result indicates that aggregation is generally favored by mutations that bring the net charge of the protein closer to neutrality. In light of this finding, we have analyzed natural mutations associated with familial forms of amyloid diseases that involve alteration of the net charge of the proteins or protein fragments associated with the diseases. Sixteen mutations have been identified for which the mechanism of action that causes the pathological condition is not yet known or fully understood. Remarkably, 14 of these 16 mutations cause the net charge of the corresponding peptide or protein that converts into amyloid deposits to be reduced. This result suggests that charge has been a key parameter in molecular evolution to ensure the avoidance of protein aggregation and identifies reduction of the net charge as an important determinant in at least some forms of protein deposition diseases.

  12. Association of Cerebral AmyloidAggregation With Cognitive Functioning in Persons Without Dementia

    DEFF Research Database (Denmark)

    Jansen, Willemijn J; Ossenkoppele, Rik; Tijms, Betty M

    2018-01-01

    Importance: Cerebral amyloidaggregation is an early event in Alzheimer disease (AD). Understanding the association between amyloid aggregation and cognitive manifestation in persons without dementia is important for a better understanding of the course of AD and for the design of prevention tr...

  13. Polymorphism of amyloid fibrils formed by a peptide from the yeast prion protein Sup35: AFM and Tip-Enhanced Raman Scattering studies

    Energy Technology Data Exchange (ETDEWEB)

    Krasnoslobodtsev, Alexey V., E-mail: akrasnos@unomaha.edu [Department of Pharmaceutical Sciences, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, NE 68198 (United States); Department of Physics, University of Nebraska Omaha, Omaha, NE 68182 (United States); Deckert-Gaudig, Tanja [IPHT-Leibniz Institute of Photonic Technology, Albert-Einstein-Str. 9, D-07745 Jena (Germany); Zhang, Yuliang [Department of Pharmaceutical Sciences, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, NE 68198 (United States); Deckert, Volker [IPHT-Leibniz Institute of Photonic Technology, Albert-Einstein-Str. 9, D-07745 Jena (Germany); Institute for Physical Chemistry and Abbe Center of Photonics, University of Jena, Helmholtzweg 4, D-07743 Jena (Germany); Lyubchenko, Yuri L., E-mail: ylyubchenko@unmc.edu [Department of Pharmaceutical Sciences, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, NE 68198 (United States)

    2016-06-15

    Aggregation of prion proteins is the cause of various prion related diseases. The infectious form of prions, amyloid aggregates, exist as multiple strains. The strains are thought to represent structurally different prion protein molecules packed into amyloid aggregates, but the knowledge on the structure of different types of aggregates is limited. Here we report on the use of AFM (Atomic Force Microscopy) and TERS (Tip-Enhanced Raman Scattering) to study morphological heterogeneity and access underlying conformational features of individual amyloid aggregates. Using AFM we identified the morphology of amyloid fibrils formed by the peptide (CGNNQQNY) from the yeast prion protein Sup35 that is critically involved in the aggregation of the full protein. TERS results demonstrate that morphologically different amyloid fibrils are composed of a distinct set of conformations. Fibrils formed at pH 5.6 are composed of a mixture of peptide conformations (β-sheets, random coil and α-helix) while fibrils formed in pH~2 solution primarily have β-sheets. Additionally, peak positions in the amide III region of the TERS spectra suggested that peptides have parallel arrangement of β-sheets for pH~2 fibrils and antiparallel arrangement for fibrils formed at pH 5.6. We also developed a methodology for detailed analysis of the peptide secondary structure by correlating intensity changes of Raman bands in different regions of TERS spectra. Such correlation established that structural composition of peptides is highly localized with large contribution of unordered secondary structures on a fibrillar surface. - Highlights: • Amyloid polymorphs were characterized by AFM and TERS. • A mixture of peptide secondary structures in fibrils were identified using TERS. • TERS recognizes packing arrangement (parallel versus antiparallel) of peptides. • TERS is a powerful tool for high resolution structural analysis of fibrils.

  14. Small static electric field strength promotes aggregation-prone structures in amyloid-β(29-42)

    Science.gov (United States)

    Lu, Yan; Shi, Xiao-Feng; Salsbury, Freddie R.; Derreumaux, Philippe

    2017-04-01

    The formation of senile plaques in central neural system resulting from the aggregation of the amyloid β (Aβ) of 40 and 42 residues is one of the two hallmarks of Alzheimer's disease. Numerous experiments and computational studies have shown that the aggregation of Aβ peptides in vitro is very complex and depends on many factors such as pH, agitation, temperature, and peptide concentration. The impact of a static electric field (EF) on amyloid peptide aggregation has been much less studied, although EFs may have some applications to treat Parkinson's disease symptoms. Here, we study the influence of an EF strength of 20 mV/nm, present in the human brains, on the conformation of the Aβ29-42 dimer. Our 7 μs non-equilibrium atomistic simulations in aqueous solution show that this field-strength promotes substantially the formation of β-hairpins, believed to be a very important intermediate state during aggregation. This work also suggests that structural biology experiments conducted under appropriate EF strengths may help reduce the conformational heterogeneity of Aβ1-40/Aβ1-42 dimers and provide significant insights into their structures that may be disease-causing.

  15. The Alzheimer Disease Protective Mutation A2T Modulates Kinetic and Thermodynamic Properties of Amyloid-β (Aβ) Aggregation*

    Science.gov (United States)

    Benilova, Iryna; Gallardo, Rodrigo; Ungureanu, Andreea-Alexandra; Castillo Cano, Virginia; Snellinx, An; Ramakers, Meine; Bartic, Carmen; Rousseau, Frederic; Schymkowitz, Joost; De Strooper, Bart

    2014-01-01

    Missense mutations in alanine 673 of the amyloid precursor protein (APP), which corresponds to the second alanine of the amyloid β (Aβ) sequence, have dramatic impact on the risk for Alzheimer disease; A2V is causative, and A2T is protective. Assuming a crucial role of amyloid-Aβ in neurodegeneration, we hypothesized that both A2V and A2T mutations cause distinct changes in Aβ properties that may at least partially explain these completely different phenotypes. Using human APP-overexpressing primary neurons, we observed significantly decreased Aβ production in the A2T mutant along with an enhanced Aβ generation in the A2V mutant confirming earlier data from non-neuronal cell lines. More importantly, thioflavin T fluorescence assays revealed that the mutations, while having little effect on Aβ42 peptide aggregation, dramatically change the properties of the Aβ40 pool with A2V accelerating and A2T delaying aggregation of the Aβ peptides. In line with the kinetic data, Aβ A2T demonstrated an increase in the solubility at equilibrium, an effect that was also observed in all mixtures of the A2T mutant with the wild type Aβ40. We propose that in addition to the reduced β-secretase cleavage of APP, the impaired propensity to aggregate may be part of the protective effect conferred by A2T substitution. The interpretation of the protective effect of this mutation is thus much more complicated than proposed previously. PMID:25253695

  16. Mechanism by which DHA inhibits the aggregation of KLVFFA peptides: A molecular dynamics study

    Science.gov (United States)

    Zhou, Hong; Liu, Shengtang; Shao, Qiwen; Ma, Dongfang; Yang, Zaixing; Zhou, Ruhong

    2018-03-01

    Docosahexaenoic acid (DHA) is one of the omega-3 polyunsaturated fatty acids, which has shown promising applications in lowering Aβ peptide neurotoxicity in vitro by preventing aggregation of Aβ peptides and relieving accumulation of Aβ fibrils. Unfortunately, the underlying molecular mechanisms of how DHA interferes with the aggregation of Aβ peptides remain largely enigmatic. Herein, aggregation behaviors of amyloid-β(Aβ)16-21 peptides (KLVFFA) with or without the presence of a DHA molecule were comparatively studied using extensive all-atom molecular dynamics simulations. We found that DHA could effectively suppress the aggregation of KLVFFA peptides by redirecting peptides to unstructured oligomers. The highly hydrophobic and flexible nature of DHA made it randomly but tightly entangled with Leu-17, Phe-19, and Phe-20 residues to form unstructured but stable complexes. These lower-ordered unstructured oligomers could eventually pass through energy barriers to form ordered β-sheet structures through large conformational fluctuations. This study depicts a microscopic picture for understanding the role and mechanism of DHA in inhibition of aggregation of Aβ peptides, which is generally believed as one of the important pathogenic mechanisms of Alzheimer's disease.

  17. Recombinant amyloid beta-peptide production by coexpression with an affibody ligand

    Directory of Open Access Journals (Sweden)

    Dobson Christopher M

    2008-10-01

    Full Text Available Abstract Background Oligomeric and fibrillar aggregates of the amyloid β-peptide (Aβ have been implicated in the pathogenesis of Alzheimer's disease (AD. The characterization of Aβ assemblies is essential for the elucidation of the mechanisms of Aβ neurotoxicity, but requires large quantities of pure peptide. Here we describe a novel approach to the recombinant production of Aβ. The method is based on the coexpression of the affibody protein ZAβ3, a selected affinity ligand derived from the Z domain three-helix bundle scaffold. ZAβ3 binds to the amyloidogenic central and C-terminal part of Aβ with nanomolar affinity and consequently inhibits aggregation. Results Coexpression of ZAβ3 affords the overexpression of both major Aβ isoforms, Aβ(1–40 and Aβ(1–42, yielding 4 or 3 mg, respectively, of pure 15N-labeled peptide per liter of culture. The method does not rely on a protein-fusion or -tag and thus does not require a cleavage reaction. The purified peptides were characterized by NMR, circular dichroism, SDS-PAGE and size exclusion chromatography, and their aggregation propensities were assessed by thioflavin T fluorescence and electron microscopy. The data coincide with those reported previously for monomeric, largely unstructured Aβ. ZAβ3 coexpression moreover permits the recombinant production of Aβ(1–42 carrying the Arctic (E22G mutation, which causes early onset familial AD. Aβ(1–42E22G is obtained in predominantly monomeric form and suitable, e.g., for NMR studies. Conclusion The coexpression of an engineered aggregation-inhibiting binding protein offers a novel route to the recombinant production of amyloidogenic Aβ peptides that can be advantageously employed to study the molecular basis of AD. The presented expression system is the first for which expression and purification of the aggregation-prone Arctic variant (E22G of Aβ(1–42 is reported.

  18. Recombinant amyloid beta-peptide production by coexpression with an affibody ligand

    Science.gov (United States)

    Macao, Bertil; Hoyer, Wolfgang; Sandberg, Anders; Brorsson, Ann-Christin; Dobson, Christopher M; Härd, Torleif

    2008-01-01

    Background Oligomeric and fibrillar aggregates of the amyloid β-peptide (Aβ) have been implicated in the pathogenesis of Alzheimer's disease (AD). The characterization of Aβ assemblies is essential for the elucidation of the mechanisms of Aβ neurotoxicity, but requires large quantities of pure peptide. Here we describe a novel approach to the recombinant production of Aβ. The method is based on the coexpression of the affibody protein ZAβ3, a selected affinity ligand derived from the Z domain three-helix bundle scaffold. ZAβ3 binds to the amyloidogenic central and C-terminal part of Aβ with nanomolar affinity and consequently inhibits aggregation. Results Coexpression of ZAβ3 affords the overexpression of both major Aβ isoforms, Aβ(1–40) and Aβ(1–42), yielding 4 or 3 mg, respectively, of pure 15N-labeled peptide per liter of culture. The method does not rely on a protein-fusion or -tag and thus does not require a cleavage reaction. The purified peptides were characterized by NMR, circular dichroism, SDS-PAGE and size exclusion chromatography, and their aggregation propensities were assessed by thioflavin T fluorescence and electron microscopy. The data coincide with those reported previously for monomeric, largely unstructured Aβ. ZAβ3 coexpression moreover permits the recombinant production of Aβ(1–42) carrying the Arctic (E22G) mutation, which causes early onset familial AD. Aβ(1–42)E22G is obtained in predominantly monomeric form and suitable, e.g., for NMR studies. Conclusion The coexpression of an engineered aggregation-inhibiting binding protein offers a novel route to the recombinant production of amyloidogenic Aβ peptides that can be advantageously employed to study the molecular basis of AD. The presented expression system is the first for which expression and purification of the aggregation-prone Arctic variant (E22G) of Aβ(1–42) is reported. PMID:18973685

  19. The molecular mass of dextran used to modify magnetite nanoparticles affects insulin amyloid aggregation

    Energy Technology Data Exchange (ETDEWEB)

    Siposova, Katarina [Department of Biophysics, Institute of Experimental Physics, Slovak Academy of Sciences, Kosice (Slovakia); Pospiskova, Kristyna [Regional Centre of Advanced Technologies and Materials, Palacky University, Olomouc (Czech Republic); Bednarikova, Zuzana [Department of Biophysics, Institute of Experimental Physics, Slovak Academy of Sciences, Kosice (Slovakia); Department of Biochemistry, Faculty of Science, Safarik University, Kosice (Slovakia); Safarik, Ivo [Regional Centre of Advanced Technologies and Materials, Palacky University, Olomouc (Czech Republic); Department of Nanobiotechnology, Biology Centre, ISB, CAS, Ceske Budejovice (Czech Republic); Safarikova, Mirka [Department of Nanobiotechnology, Biology Centre, ISB, CAS, Ceske Budejovice (Czech Republic); Kubovcikova, Martina; Kopcansky, Peter [Department of Magnetism, Institute of Experimental Physics, Slovak Academy of Sciences, Kosice (Slovakia); Gazova, Zuzana, E-mail: gazova@saske.sk [Department of Biophysics, Institute of Experimental Physics, Slovak Academy of Sciences, Kosice (Slovakia)

    2017-04-01

    Protein transformation from its soluble state into amyloid aggregates is associated with amyloid-related diseases. Amyloid deposits of insulin fibrils have been found in the sites of subcutaneous insulin application in patients with prolonged diabetes. Using atomic force microscopy and ThT fluorescence assay we have investigated the interference of insulin amyloid aggregation with superparamagnetic Fe{sub 3}O{sub 4}-based nanoparticles (SPIONs) coated with dextran (DEX); molecular mass of dextran was equal to 15–20, 40 or 70 kDa. The obtained data indicate that all three types of dextran coated nanoparticles (NP-FeDEXs) are able to inhibit insulin fibrillization and to destroy amyloid fibrils. The extent of anti-amyloid activities depends on the properties of NP-FeDEXs, mainly on the size of nanoparticles which is determined by molecular mass of dextran molecules. The most effective inhibiting activity was observed for the smallest nanoparticles coated with 15–20 kDa dextran. Contrary, the highest destroying activity was observed for the largest NP-FeDEX (70 kDa dextran). - Highlights: • Interference of dextran- magnetite nanoparticles with insulin amyloid aggregation. • Nanoparticles inhibited insulin fibrillization and depolymerized insulin amyloid fibrils. • Size of nanoparticles significantly influences their anti-amyloid activities. • The most effective inhibition of insulin amyloid fibrillization was detected for the smallest nanoparticles. • Contrary, DC{sub 50} values decreased with increasing size of nanoparticles.

  20. The molecular mass of dextran used to modify magnetite nanoparticles affects insulin amyloid aggregation

    International Nuclear Information System (INIS)

    Siposova, Katarina; Pospiskova, Kristyna; Bednarikova, Zuzana; Safarik, Ivo; Safarikova, Mirka; Kubovcikova, Martina; Kopcansky, Peter; Gazova, Zuzana

    2017-01-01

    Protein transformation from its soluble state into amyloid aggregates is associated with amyloid-related diseases. Amyloid deposits of insulin fibrils have been found in the sites of subcutaneous insulin application in patients with prolonged diabetes. Using atomic force microscopy and ThT fluorescence assay we have investigated the interference of insulin amyloid aggregation with superparamagnetic Fe 3 O 4 -based nanoparticles (SPIONs) coated with dextran (DEX); molecular mass of dextran was equal to 15–20, 40 or 70 kDa. The obtained data indicate that all three types of dextran coated nanoparticles (NP-FeDEXs) are able to inhibit insulin fibrillization and to destroy amyloid fibrils. The extent of anti-amyloid activities depends on the properties of NP-FeDEXs, mainly on the size of nanoparticles which is determined by molecular mass of dextran molecules. The most effective inhibiting activity was observed for the smallest nanoparticles coated with 15–20 kDa dextran. Contrary, the highest destroying activity was observed for the largest NP-FeDEX (70 kDa dextran). - Highlights: • Interference of dextran- magnetite nanoparticles with insulin amyloid aggregation. • Nanoparticles inhibited insulin fibrillization and depolymerized insulin amyloid fibrils. • Size of nanoparticles significantly influences their anti-amyloid activities. • The most effective inhibition of insulin amyloid fibrillization was detected for the smallest nanoparticles. • Contrary, DC 50 values decreased with increasing size of nanoparticles.

  1. Lipopolysaccharide induces amyloid formation of antimicrobial peptide HAL-2.

    Science.gov (United States)

    Wang, Jiarong; Li, Yan; Wang, Xiaoming; Chen, Wei; Sun, Hongbin; Wang, Junfeng

    2014-11-01

    Lipopolysaccharide (LPS), the important component of the outer membrane of Gram-negative bacteria, contributes to the integrity of the outer membrane and protects the cell against bactericidal agents, including antimicrobial peptides. However, the mechanisms of interaction between antimicrobial peptides and LPS are not clearly understood. Halictines-2 (HAL-2), one of the novel antimicrobial peptides, was isolated from the venom of the eusocial bee Halictus sexcinctus. HAL-2 has exhibited potent antimicrobial activity against Gram-positive and Gram-negative bacteria and even against cancer cells. Here, we studied the interactions between HAL-2 and LPS to elucidate the antibacterial mechanism of HAL-2 in vitro. Our results show that HAL-2 adopts a significant degree of β-strand structure in the presence of LPS. LPS is capable of inducing HAL-2 amyloid formation, which may play a vital role in its antimicrobial activity. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Indexing amyloid peptide diffraction from serial femtosecond crystallography: new algorithms for sparse patterns

    Energy Technology Data Exchange (ETDEWEB)

    Brewster, Aaron S. [Lawrence Berkeley National Laboratory, Berkeley, CA 94720 (United States); Sawaya, Michael R. [University of California, Los Angeles, CA 90095-1570 (United States); University of California, Los Angeles, CA 90095-1570 (United States); University of California, Los Angeles, CA 90095-1570 (United States); Rodriguez, Jose [University of California, Los Angeles, CA 90095-1570 (United States); University of California, Los Angeles, CA 90095-1570 (United States); Hattne, Johan; Echols, Nathaniel [Lawrence Berkeley National Laboratory, Berkeley, CA 94720 (United States); McFarlane, Heather T. [University of California, Los Angeles, CA 90095-1570 (United States); University of California, Los Angeles, CA 90095-1570 (United States); Cascio, Duilio [University of California, Los Angeles, CA 90095-1570 (United States); University of California, Los Angeles, CA 90095-1570 (United States); University of California, Los Angeles, CA 90095-1570 (United States); Adams, Paul D. [Lawrence Berkeley National Laboratory, Berkeley, CA 94720 (United States); University of California, Berkeley, CA 94720 (United States); Eisenberg, David S. [University of California, Los Angeles, CA 90095-1570 (United States); University of California, Los Angeles, CA 90095-1570 (United States); University of California, Los Angeles, CA 90095-1570 (United States); Sauter, Nicholas K., E-mail: nksauter@lbl.gov [Lawrence Berkeley National Laboratory, Berkeley, CA 94720 (United States)

    2015-02-01

    Special methods are required to interpret sparse diffraction patterns collected from peptide crystals at X-ray free-electron lasers. Bragg spots can be indexed from composite-image powder rings, with crystal orientations then deduced from a very limited number of spot positions. Still diffraction patterns from peptide nanocrystals with small unit cells are challenging to index using conventional methods owing to the limited number of spots and the lack of crystal orientation information for individual images. New indexing algorithms have been developed as part of the Computational Crystallography Toolbox (cctbx) to overcome these challenges. Accurate unit-cell information derived from an aggregate data set from thousands of diffraction patterns can be used to determine a crystal orientation matrix for individual images with as few as five reflections. These algorithms are potentially applicable not only to amyloid peptides but also to any set of diffraction patterns with sparse properties, such as low-resolution virus structures or high-throughput screening of still images captured by raster-scanning at synchrotron sources. As a proof of concept for this technique, successful integration of X-ray free-electron laser (XFEL) data to 2.5 Å resolution for the amyloid segment GNNQQNY from the Sup35 yeast prion is presented.

  3. In vitro fibrillization of Alzheimer’s amyloidpeptide (1-42)

    Energy Technology Data Exchange (ETDEWEB)

    Tiiman, Ann [Department of Gene Technology, Tallinn University of Technology, Tallinn 12618 (Estonia); Department of Biochemistry and Biophysics, Arrhenius Laboratories, Stockholm University, Stockholm, 106 91 (Sweden); Krishtal, Jekaterina; Palumaa, Peep; Tõugu, Vello, E-mail: vello.tougu@ttu.ee [Department of Gene Technology, Tallinn University of Technology, Tallinn 12618 (Estonia)

    2015-09-15

    The amyloid deposition in the form of extracellular fibrillar aggregates of amyloid-β (Aβ) peptide is a critical pathological event in Alzheimer’s disease. Here, we report a systematic investigation of the effects of environmental factors on the kinetics of Aβ fibrillization in vitro. The effects of Aβ42 peptide concentration, temperature, pH, added solvents and the ratio of Aβ40 and Aβ42 on the peptide fibrillization under agitated conditions was studied. The analysis show that the rate of fibril growth by monomer addition is not limited by diffusion but by rearrangement in the monomer structure, which is enhanced by low concentrations of fluorinated alcohols and characterized by the activation energy of 12 kcal/mol. Fibrillization rate decreases at pH values below 7.0 where simultaneous protonation of His 13 and 14 inhibits fibril formation. The lag period for Aβ42 was only twofold shorter and the fibril growth rate twofold faster than those of Aβ40. Lag period was shortened and the fibrillization rate was increased only at 90% content of Aβ42.

  4. Bifunctional fluorescent probes for detection of amyloid aggregates and reactive oxygen species.

    Science.gov (United States)

    Needham, Lisa-Maria; Weber, Judith; Fyfe, James W B; Kabia, Omaru M; Do, Dung T; Klimont, Ewa; Zhang, Yu; Rodrigues, Margarida; Dobson, Christopher M; Ghandi, Sonia; Bohndiek, Sarah E; Snaddon, Thomas N; Lee, Steven F

    2018-02-01

    Protein aggregation into amyloid deposits and oxidative stress are key features of many neurodegenerative disorders including Parkinson's and Alzheimer's disease. We report here the creation of four highly sensitive bifunctional fluorescent probes, capable of H 2 O 2 and/or amyloid aggregate detection. These bifunctional sensors use a benzothiazole core for amyloid localization and boronic ester oxidation to specifically detect H 2 O 2 . We characterized the optical properties of these probes using both bulk fluorescence measurements and single-aggregate fluorescence imaging, and quantify changes in their fluorescence properties upon addition of amyloid aggregates of α-synuclein and pathophysiological H 2 O 2 concentrations. Our results indicate these new probes will be useful to detect and monitor neurodegenerative disease.

  5. Bifunctional fluorescent probes for detection of amyloid aggregates and reactive oxygen species

    Science.gov (United States)

    Needham, Lisa-Maria; Weber, Judith; Fyfe, James W. B.; Kabia, Omaru M.; Do, Dung T.; Klimont, Ewa; Zhang, Yu; Rodrigues, Margarida; Dobson, Christopher M.; Ghandi, Sonia; Bohndiek, Sarah E.; Snaddon, Thomas N.; Lee, Steven F.

    2018-02-01

    Protein aggregation into amyloid deposits and oxidative stress are key features of many neurodegenerative disorders including Parkinson's and Alzheimer's disease. We report here the creation of four highly sensitive bifunctional fluorescent probes, capable of H2O2 and/or amyloid aggregate detection. These bifunctional sensors use a benzothiazole core for amyloid localization and boronic ester oxidation to specifically detect H2O2. We characterized the optical properties of these probes using both bulk fluorescence measurements and single-aggregate fluorescence imaging, and quantify changes in their fluorescence properties upon addition of amyloid aggregates of α-synuclein and pathophysiological H2O2 concentrations. Our results indicate these new probes will be useful to detect and monitor neurodegenerative disease.

  6. Inhibitory Activities of Antioxidant Flavonoids from Tamarix gallica on Amyloid Aggregation Related to Alzheimer's and Type 2 Diabetes Diseases.

    Science.gov (United States)

    Ben Hmidene, Asma; Hanaki, Mizuho; Murakami, Kazuma; Irie, Kazuhiro; Isoda, Hiroko; Shigemori, Hideyuki

    2017-01-01

    The prevention of amyloid aggregation is promising for the treatment of age-related diseases such as Alzheimer's (AD) and type 2 diabetes (T2D). Ten antioxidant flavonoids isolated from the medicinal halophyte Tamarix gallica were tested for their amyloid aggregation inhibition potential. Glucuronosylated flavonoids show relatively strong inhibitory activity of Amyloid β (Aβ) and human islet amyloid polypeptide (hIAPP) aggregation compared to their aglycone analogs. Structure-activity relationship of the flavonoids suggests that the catechol moiety is important for amyloid aggregation inhibition, while the methylation of the carboxyl group in the glucuronide moiety and of the hydroxyl group in the aglycone flavonoids decreased it.

  7. Small-molecule aggregation inhibitors reduce excess amyloid in a trisomy 16 mouse cortical cell line

    Directory of Open Access Journals (Sweden)

    ANDRÉA C PAULA LIMA

    2008-01-01

    Full Text Available We have previously characterized a number of small molecule organic compounds that prevent the aggregation of the β-amyloid peptide and its neurotoxicity in hippocampal neuronal cultures. We have now evaluated the effects of such compounds on amyloid precursor protein (APP accumulation in the CTb immortalized cell line derived from the cerebral cortex of a trisomy 16 mouse, an animal model of Down's syndrome. Compared to a non-trisomic cortical cell line (CNh, CTb cells overexpress APP and exhibit slightly elevated resting intracellular Ca2+ levéis ([Ca2+]¡. Here, we show that the compounds 2,4-dinitrophenol, 3-nitrophenol and 4-anisidine decreased intracellular accumulation of APP in CTb cells. Those compounds were non-toxic to the cells, and slightly increased the basal [Ca2+]¡. Results indícate that the compounds tested can be leads for the development of drugs to decrease intracellular vesicular accumulation of APP in trisomic cells.

  8. Novel Detox Gel Depot sequesters β-Amyloid Peptides in a mouse model of Alzheimer's Disease.

    Science.gov (United States)

    Sundaram, Ranjini K; Kasinathan, Chinnaswamy; Stein, Stanley; Sundaram, Pazhani

    2012-06-01

    Alzheimer's Disease (AD), a debilitating neurodegenerative disease is caused by aggregation and accumulation of a 39-43 amino acid peptide (amyloid β or Aβ) in brain parenchyma and cerebrovasculature. The rational approach would be to use drugs that interfere with Aβ-Aβ interaction and disrupt polymerization. Peptide ligands capable of binding to the KLVFF (amino acids 16-20) region in the Aβ molecule have been investigated as possible drug candidates. Retro-inverso (RI) peptide of this pentapeptide, ffvlk, has been shown to bind artificial fibrils made from Aβ with moderate affinity. We hypothesized that a 'detox gel', which is synthesized by covalently linking a tetrameric version of RI peptide ffvlk to poly (ethylene glycol) polymer chains will act like a 'sink' to capture Aβ peptides from the surrounding environment. We previously demonstrated that this hypothesis works in an in vitro system. The present study extended this hypothesis to an in vivo mouse model of Alzheimer's Disease and determined the therapeutic effect of our detox gel. We injected detox gel subcutaneously to AD model mice and analyzed brain levels of Aβ-42 and improvement in memory parameters. The results showed a reduction of brain amyloid burden in detox gel treated mice. Memory parameters in the treated mice improved. No undesirable immune response was observed. The data strongly suggest that our detox gel can be used as an effective therapy to deplete brain Aβ levels. Considering recent abandonment of failed antibody based therapies, our detox gel appears to have the advantage of being a non-immune based therapy.

  9. Thermodynamic description of polymorphism in Q- and N-rich peptide aggregates revealed by atomistic simulation.

    Science.gov (United States)

    Berryman, Joshua T; Radford, Sheena E; Harris, Sarah A

    2009-07-08

    Amyloid fibrils are long, helically symmetric protein aggregates that can display substantial variation (polymorphism), including alterations in twist and structure at the beta-strand and protofilament levels, even when grown under the same experimental conditions. The structural and thermodynamic origins of this behavior are not yet understood. We performed molecular-dynamics simulations to determine the thermodynamic properties of different polymorphs of the peptide GNNQQNY, modeling fibrils containing different numbers of protofilaments based on the structure of amyloid-like cross-beta crystals of this peptide. We also modeled fibrils with new orientations of the side chains, as well as a de novo designed structure based on antiparallel beta-strands. The simulations show that these polymorphs are approximately isoenergetic under a range of conditions. Structural analysis reveals a dynamic reorganization of electrostatics and hydrogen bonding in the main and side chains of the Gln and Asn residues that characterize this peptide sequence. Q/N-rich stretches are found in several amyloidogenic proteins and peptides, including the yeast prions Sup35-N and Ure2p, as well as in the human poly-Q disease proteins, including the ataxins and huntingtin. Based on our results, we propose that these residues imbue a unique structural plasticity to the amyloid fibrils that they comprise, rationalizing the ability of proteins enriched in these amino acids to form prion strains with heritable and different phenotypic traits.

  10. A 31-residue peptide induces aggregation of tau's microtubule-binding region in cells

    Science.gov (United States)

    Stöhr, Jan; Wu, Haifan; Nick, Mimi; Wu, Yibing; Bhate, Manasi; Condello, Carlo; Johnson, Noah; Rodgers, Jeffrey; Lemmin, Thomas; Acharya, Srabasti; Becker, Julia; Robinson, Kathleen; Kelly, Mark J. S.; Gai, Feng; Stubbs, Gerald; Prusiner, Stanley B.; Degrado, William F.

    2017-09-01

    The self-propagation of misfolded conformations of tau underlies neurodegenerative diseases, including Alzheimer's. There is considerable interest in discovering the minimal sequence and active conformational nucleus that defines this self-propagating event. The microtubule-binding region, spanning residues 244-372, reproduces much of the aggregation behaviour of tau in cells and animal models. Further dissection of the amyloid-forming region to a hexapeptide from the third microtubule-binding repeat resulted in a peptide that rapidly forms fibrils in vitro. We show that this peptide lacks the ability to seed aggregation of tau244-372 in cells. However, as the hexapeptide is gradually extended to 31 residues, the peptides aggregate more slowly and gain potent activity to induce aggregation of tau244-372 in cells. X-ray fibre diffraction, hydrogen-deuterium exchange and solid-state NMR studies map the beta-forming region to a 25-residue sequence. Thus, the nucleus for self-propagating aggregation of tau244-372 in cells is packaged in a remarkably small peptide.

  11. Reduction and degradation of amyloid aggregates by a pulsed radio-frequency cold atmospheric plasma jet

    International Nuclear Information System (INIS)

    Bayliss, D L; Walsh, J L; Iza, F; Kong, M G; Shama, G

    2009-01-01

    Surface-borne amyloid aggregates with mature fibrils are used as a non-infectious prion model to evaluate cold atmospheric plasmas (CAPs) as a prion inactivation strategy. Using a helium-oxygen CAP jet with pulsed radio-frequency (RF) excitation, amyloid aggregates deposited on freshly cleaved mica discs are reduced substantially leaving only a few spherical fragments of sub-micrometer sizes in areas directly treated by the CAP jet. Outside the light-emitting part of the CAP jet, plasma treatment results in a 'skeleton' of much reduced amyloid stacks with clear evidence of fibril fragmentation. Analysis of possible plasma species and the physical configuration of the jet-sample interaction suggests that the skeleton structures observed are unlikely to have arisen as a result of physical forces of detachment, but instead by progressive diffusion of oxidizing plasma species into porous amyloid aggregates. Composition of chemical bonds of this reduced amyloid sample is very different from that of intact amyloid aggregates. These suggest the possibility of on-site degradation by CAP treatment with little possibility of spreading contamination elsewhere , thus offering a new reaction chemistry route to protein infectivity control with desirable implications for the practical implementation of CAP-based sterilization systems.

  12. Reduction and degradation of amyloid aggregates by a pulsed radio-frequency cold atmospheric plasma jet

    Energy Technology Data Exchange (ETDEWEB)

    Bayliss, D L; Walsh, J L; Iza, F; Kong, M G [Department of Electronic and Electrical Engineering, Loughborough University, Leicestershire LE11 3TU (United Kingdom); Shama, G [Department of Chemical Engineering, Loughborough University, Leicestershire LE11 3TU (United Kingdom)], E-mail: m.g.kong@lboro.ac.uk

    2009-11-15

    Surface-borne amyloid aggregates with mature fibrils are used as a non-infectious prion model to evaluate cold atmospheric plasmas (CAPs) as a prion inactivation strategy. Using a helium-oxygen CAP jet with pulsed radio-frequency (RF) excitation, amyloid aggregates deposited on freshly cleaved mica discs are reduced substantially leaving only a few spherical fragments of sub-micrometer sizes in areas directly treated by the CAP jet. Outside the light-emitting part of the CAP jet, plasma treatment results in a 'skeleton' of much reduced amyloid stacks with clear evidence of fibril fragmentation. Analysis of possible plasma species and the physical configuration of the jet-sample interaction suggests that the skeleton structures observed are unlikely to have arisen as a result of physical forces of detachment, but instead by progressive diffusion of oxidizing plasma species into porous amyloid aggregates. Composition of chemical bonds of this reduced amyloid sample is very different from that of intact amyloid aggregates. These suggest the possibility of on-site degradation by CAP treatment with little possibility of spreading contamination elsewhere , thus offering a new reaction chemistry route to protein infectivity control with desirable implications for the practical implementation of CAP-based sterilization systems.

  13. Small surfactant-like peptides can drive soluble proteins into active aggregates

    Directory of Open Access Journals (Sweden)

    Zhou Bihong

    2012-01-01

    Full Text Available Abstract Background Inactive protein inclusion bodies occur commonly in Escherichia coli (E. coli cells expressing heterologous proteins. Previously several independent groups have found that active protein aggregates or pseudo inclusion bodies can be induced by a fusion partner such as a cellulose binding domain from Clostridium cellulovorans (CBDclos when expressed in E. coli. More recently we further showed that a short amphipathic helical octadecapeptide 18A (EWLKAFYEKVLEKLKELF and a short beta structure peptide ELK16 (LELELKLKLELELKLK have a similar property. Results In this work, we explored a third type of peptides, surfactant-like peptides, for performing such a "pulling-down" function. One or more of three such peptides (L6KD, L6K2, DKL6 were fused to the carboxyl termini of model proteins including Aspergillus fumigatus amadoriase II (AMA, all three peptides were used, Bacillus subtilis lipase A (LipA, only L6KD was used, hereinafter the same, Bacillus pumilus xylosidase (XynB, and green fluorescent protein (GFP, and expressed in E. coli. All fusions were found to predominantly accumulate in the insoluble fractions, with specific activities ranging from 25% to 92% of the native counterparts. Transmission electron microscopic (TEM and confocal fluorescence microscopic analyses confirmed the formation of protein aggregates in the cell. Furthermore, binding assays with amyloid-specific dyes (thioflavin T and Cong red to the AMA-L6KD aggregate and the TEM analysis of the aggregate following digestion with protease K suggested that the AMA-L6KD aggregate may contain structures reminiscent of amyloids, including a fibril-like structure core. Conclusions This study shows that the surfactant-like peptides L6KD and it derivatives can act as a pull-down handler for converting soluble proteins into active aggregates, much like 18A and ELK16. These peptide-mediated protein aggregations might have important implications for protein aggregation in

  14. Nasal administration of amyloid-beta peptide decreases cerebral amyloid burden in a mouse model of Alzheimer's disease

    DEFF Research Database (Denmark)

    Weiner, H L; Lemere, C A; Maron, R

    2000-01-01

    Progressive cerebral deposition of amyloid-beta (Abeta) peptide, an early and essential feature of Alzheimer's disease (AD), is accompanied by an inflammatory reaction marked by microgliosis, astrocytosis, and the release of proinflammatory cytokines. Mucosal administration of disease-implicated ......Progressive cerebral deposition of amyloid-beta (Abeta) peptide, an early and essential feature of Alzheimer's disease (AD), is accompanied by an inflammatory reaction marked by microgliosis, astrocytosis, and the release of proinflammatory cytokines. Mucosal administration of disease...... cerebral Abeta deposition, suggesting a novel mucosal immunological approach for the treatment and prevention of AD....

  15. Amyloid fibril formation in vitro from halophilic metal binding protein: Its high solubility and reversibility minimized formation of amorphous protein aggregations

    Science.gov (United States)

    Tokunaga, Yuhei; Matsumoto, Mitsuharu; Tokunaga, Masao; Arakawa, Tsutomu; Sugimoto, Yasushi

    2013-01-01

    Halophilic proteins are characterized by high net negative charges and relatively small fraction of hydrophobic amino acids, rendering them aggregation resistant. These properties are also shared by histidine-rich metal binding protein (HP) from moderate halophile, Chromohalobacter salexigens, used in this study. Here, we examined how halophilic proteins form amyloid fibrils in vitro. His-tagged HP, incubated at pH 2.0 and 58°C, readily formed amyloid fibrils, as observed by thioflavin fluorescence, CD spectra, and transmission or atomic force microscopies. Under these low-pH harsh conditions, however, His-HP was promptly hydrolyzed to smaller peptides most likely responsible for rapid formation of amyloid fibril. Three major acid-hydrolyzed peptides were isolated from fibrils and turned out to readily form fibrils. The synthetic peptides predicted to form fibrils in these peptide sequences by Waltz software also formed fibrils. Amyloid fibril was also readily formed from full-length His-HP when incubated with 10–20% 2,2,2-trifluoroethanol at pH 7.8 and 25°C without peptide bond cleavage. PMID:24038709

  16. Iron and aluminum interaction with amyloid-beta peptides associated with Alzheimer’s disease

    Energy Technology Data Exchange (ETDEWEB)

    Drochioiu, Gabi; Ion, Laura [Alexandru Ioan Cuza University of Iasi, 11 Carol I, Iasi 700506 (Romania); Murariu, Manuela; Habasescu, Laura [Petru Poni Institute of Macromolecular Chemistry, 41A Grigore Ghica Voda Alley, Iasi 700487 (Romania)

    2014-10-06

    An elevation in the concentration of heavy metal ions in Alzheimer’s disease (AD) brain has been demonstrated in many studies. Aβ precipitation and toxicity in AD brains seem to be caused by abnormal interactions with neocortical metal ions, especially iron, copper, zinc, and aluminum [1–3]. There is increasing evidence that iron and aluminum ions are involved in the mechanisms that underlie the neurodegenerative diseases [4,5]. However, evidence was brought to demonstrate that some Aβ fragments, at physiological pH, are not able to form binary complexes with Fe(III) ions of sufficient stability to compete with metal hydroxide precipitation [6]. On the contrary, multiple metal ions are known to interact with Aβ peptides [7]. Consequently, we investigated here the interaction of Fe(II/III) and Al(III) ions with some amyloidpeptides and fragments that results in peptide aggregation and fibrillation [8,9]. Infrared spectroscopy, atomic force microscopy, scanning electron microscopy, electrophoresis and mass spectrometry demonstrated conformational changes of peptides in the presence of such metals.

  17. Unwinding fibril formation of medin, the peptide of the most common form of human amyloid

    International Nuclear Information System (INIS)

    Larsson, Annika; Soederberg, Linda; Westermark, Gunilla T.; Sletten, Knut; Engstroem, Ulla; Tjernberg, Lars O.; Naeslund, Jan; Westermark, Per

    2007-01-01

    Medin amyloid affects the medial layer of the thoracic aorta of most people above 50 years of age. The consequences of this amyloid are not completely known but the deposits may contribute to diseases such as thoracic aortic aneurysm and dissection or to the general diminished elasticity of blood vessels seen in elderly people. We show that the 50-amino acid residue peptide medin forms amyloid-like fibrils in vitro. With the use of Congo red staining, Thioflavin T fluorescence, electron microscopy, and a solid-phase binding assay on different synthetic peptides, we identified the last 18-19 amino acid residues to constitute the amyloid-promoting region of medin. We also demonstrate that the two C-terminal phenylalanines, previously suggested to be of importance for amyloid formation, are not required for medin amyloid formation

  18. Amyloid-beta aggregates cause alterations of astrocytic metabolic phenotype: impact on neuronal viability.

    Science.gov (United States)

    Allaman, Igor; Gavillet, Mathilde; Bélanger, Mireille; Laroche, Thierry; Viertl, David; Lashuel, Hilal A; Magistretti, Pierre J

    2010-03-03

    Amyloid-beta (Abeta) peptides play a key role in the pathogenesis of Alzheimer's disease and exert various toxic effects on neurons; however, relatively little is known about their influence on glial cells. Astrocytes play a pivotal role in brain homeostasis, contributing to the regulation of local energy metabolism and oxidative stress defense, two aspects of importance for neuronal viability and function. In the present study, we explored the effects of Abeta peptides on glucose metabolism in cultured astrocytes. Following Abeta(25-35) exposure, we observed an increase in glucose uptake and its various metabolic fates, i.e., glycolysis (coupled to lactate release), tricarboxylic acid cycle, pentose phosphate pathway, and incorporation into glycogen. Abeta increased hydrogen peroxide production as well as glutathione release into the extracellular space without affecting intracellular glutathione content. A causal link between the effects of Abeta on glucose metabolism and its aggregation and internalization into astrocytes through binding to members of the class A scavenger receptor family could be demonstrated. Using astrocyte-neuron cocultures, we observed that the overall modifications of astrocyte metabolism induced by Abeta impair neuronal viability. The effects of the Abeta(25-35) fragment were reproduced by Abeta(1-42) but not by Abeta(1-40). Finally, the phosphoinositide 3-kinase (PI3-kinase) pathway appears to be crucial in these events since both the changes in glucose utilization and the decrease in neuronal viability are prevented by LY294002, a PI3-kinase inhibitor. This set of observations indicates that Abeta aggregation and internalization into astrocytes profoundly alter their metabolic phenotype with deleterious consequences for neuronal viability.

  19. Ginsenoside compound K promotes β-amyloid peptide clearance in primary astrocytes via autophagy enhancement.

    Science.gov (United States)

    Guo, Jinhui; Chang, Li; Zhang, Xin; Pei, Sujuan; Yu, Meishuang; Gao, Jianlian

    2014-10-01

    The aim of the present study was to investigate the effect of ginsenoside compound K on β-amyloid (Aβ) peptide clearance in primary astrocytes. Aβ degradation in primary astrocytes was determined using an intracellular Aβ clearance assay. Aggregated LC3 in astrocyte cells, which is a marker for the level of autophagy, was detected using laser scanning confocal microscope. The effect of compound K on the mammalian target of rapamycin (mTOR)/autophagy pathway was determined using western blot analysis, and an enzyme-linked immunosorbent assay was used for Aβ detection. The results demonstrated that compound K promoted the clearance of Aβ and enhanced autophagy in primary astrocytes. In addition, it was found that phosphorylation of mTOR was inhibited by compound K, which may have contributed to the enhanced autophagy. In conclusion, compound K promotes Aβ clearance by enhancing autophagy via the mTOR signaling pathway in primary astrocytes.

  20. Controlling amyloid-beta peptide(1-42) oligomerization and toxicity by fluorinated nanoparticles.

    Science.gov (United States)

    Saraiva, Ana M; Cardoso, Isabel; Pereira, M Carmo; Coelho, Manuel A N; Saraiva, Maria João; Möhwald, Helmuth; Brezesinski, Gerald

    2010-09-03

    The amyloid-beta peptide (Abeta) is a major fibrillar component of neuritic plaques in Alzheimer's disease brains and is related to the pathogenesis of the disease. Soluble oligomers that precede fibril formation have been proposed as the main neurotoxic species that contributes to neurodegeneration and dementia. We hypothesize that oligomerization and cytotoxicity can be repressed by nanoparticles (NPs) that induce conformational changes in Abeta42. We show here that fluorinated and hydrogenated NPs with different abilities to change Abeta42 conformation influence oligomerization as assessed by atomic force microscopy, immunoblot and SDS-PAGE. Fluorinated NPs, which promote an increase in alpha-helical content, exert an antioligomeric effect, whereas hydrogenated analogues do not and lead to aggregation. Cytotoxicity assays confirmed our hypothesis by indicating that the conformational conversion of Abeta42 into an alpha-helical-enriched secondary structure also has antiapoptotic activity, thereby increasing the viability of cells treated with oligomeric species.

  1. Interaction of the amyloid β peptide with sodium dodecyl sulfate as a membrane-mimicking detergent.

    NARCIS (Netherlands)

    Hashemi, Shabestari M.; Meeuwenoord, N.J.; Filippov, D.V.; Huber, M.I.

    2016-01-01

    The amyloid β (A β) peptide is important in the context of Alzheimer's disease, since it is one of the major components of the fibrils that constitute amyloid plaques. Agents that can influence fibril formation are important, and of those, membrane mimics are particularly relevant, because the

  2. Key aromatic/hydrophobic amino acids controlling a cross-amyloid peptide interaction versus amyloid self-assembly.

    Science.gov (United States)

    Bakou, Maria; Hille, Kathleen; Kracklauer, Michael; Spanopoulou, Anna; Frost, Christina V; Malideli, Eleni; Yan, Li-Mei; Caporale, Andrea; Zacharias, Martin; Kapurniotu, Aphrodite

    2017-09-01

    The interaction of the intrinsically disordered polypeptide islet amyloid polypeptide (IAPP), which is associated with type 2 diabetes (T2D), with the Alzheimer's disease amyloid-β (Aβ) peptide modulates their self-assembly into amyloid fibrils and may link the pathogeneses of these two cell-degenerative diseases. However, the molecular determinants of this interaction remain elusive. Using a systematic alanine scan approach, fluorescence spectroscopy, and other biophysical methods, including heterocomplex pulldown assays, far-UV CD spectroscopy, the thioflavin T binding assay, transmission EM, and molecular dynamics simulations, here we identified single aromatic/hydrophobic residues within the amyloid core IAPP region as hot spots or key residues of its cross-interaction with Aβ40(42) peptide. Importantly, we also find that none of these residues in isolation plays a key role in IAPP self-assembly, whereas simultaneous substitution of four aromatic/hydrophobic residues with Ala dramatically impairs both IAPP self-assembly and hetero-assembly with Aβ40(42). Furthermore, our experiments yielded several novel IAPP analogs, whose sequences are highly similar to that of IAPP but have distinct amyloid self- or cross-interaction potentials. The identified similarities and major differences controlling IAPP cross-peptide interaction with Aβ40(42) versus its amyloid self-assembly offer a molecular basis for understanding the underlying mechanisms. We propose that these insights will aid in designing intervention strategies and novel IAPP analogs for the management of type 2 diabetes, Alzheimer's disease, or other diseases related to IAPP dysfunction or cross-amyloid interactions. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Self-assembled lipoprotein based gold nanoparticles for detection and photothermal disaggregation of β-amyloid aggregates

    KAUST Repository

    Martins, P. A. T.; Alsaiari, Shahad K.; Julfakyan, Khachatur; Nie, Z.; Khashab, Niveen M.

    2017-01-01

    We present a reconstituted lipoprotein-based nanoparticle platform comprising a curcumin fluorescent motif and an NIR responsive gold core. This multifunctional nanosystem is successfully used for aggregation-dependent fluorescence detection and photothermal disassembly of insoluble amyloid aggregates.

  4. Self-assembled lipoprotein based gold nanoparticles for detection and photothermal disaggregation of β-amyloid aggregates

    KAUST Repository

    Martins, P. A. T.

    2017-01-10

    We present a reconstituted lipoprotein-based nanoparticle platform comprising a curcumin fluorescent motif and an NIR responsive gold core. This multifunctional nanosystem is successfully used for aggregation-dependent fluorescence detection and photothermal disassembly of insoluble amyloid aggregates.

  5. Evidence that a synthetic amyloid-ß oligomer-binding peptide (ABP) targets amyloid-ß deposits in transgenic mouse brain and human Alzheimer's disease brain.

    Science.gov (United States)

    Chakravarthy, Balu; Ito, Shingo; Atkinson, Trevor; Gaudet, Chantal; Ménard, Michel; Brown, Leslie; Whitfield, James

    2014-03-14

    The synthetic ~5 kDa ABP (amyloid-ß binding peptide) consists of a region of the 228 kDa human pericentrioloar material-1 (PCM-1) protein that selectively and avidly binds in vitro Aβ1-42 oligomers, believed to be key co-drivers of Alzheimer's disease (AD), but not monomers (Chakravarthy et al., (2013) [3]). ABP also prevents Aß1-42 from triggering the apoptotic death of cultured human SHSY5Y neuroblasts, likely by sequestering Aß oligomers, suggesting that it might be a potential AD therapeutic. Here we support this possibility by showing that ABP also recognizes and binds Aβ1-42 aggregates in sections of cortices and hippocampi from brains of AD transgenic mice and human AD patients. More importantly, ABP targets Aβ1-42 aggregates when microinjected into the hippocampi of the brains of live AD transgenic mice. Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.

  6. Inhibition of peptide aggregation by means of enzymatic phosphorylation

    Directory of Open Access Journals (Sweden)

    Kristin Folmert

    2016-11-01

    Full Text Available As is the case in numerous natural processes, enzymatic phosphorylation can be used in the laboratory to influence the conformational populations of proteins. In nature, this information is used for signal transduction or energy transfer, but has also been shown to play an important role in many diseases like tauopathies or diabetes. With the goal of determining the effect of phosphorylation on amyloid fibril formation, we designed a model peptide which combines structural characteristics of α-helical coiled-coils and β-sheets in one sequence. This peptide undergoes a conformational transition from soluble structures into insoluble amyloid fibrils over time and under physiological conditions and contains a recognition motif for PKA (cAMP-dependent protein kinase that enables enzymatic phosphorylation. We have analyzed the pathway of amyloid formation and the influence of enzymatic phosphorylation on the different states along the conformational transition from random-coil to β-sheet-rich oligomers to protofilaments and on to insoluble amyloid fibrils, and we found a remarkable directing effect from β-sheet-rich structures to unfolded structures in the initial growth phase, in which small oligomers and protofilaments prevail if the peptide is phosphorylated.

  7. The molecular mass of dextran used to modify magnetite nanoparticles affects insulin amyloid aggregation

    Science.gov (United States)

    Siposova, Katarina; Pospiskova, Kristyna; Bednarikova, Zuzana; Safarik, Ivo; Safarikova, Mirka; Kubovcikova, Martina; Kopcansky, Peter; Gazova, Zuzana

    2017-04-01

    Protein transformation from its soluble state into amyloid aggregates is associated with amyloid-related diseases. Amyloid deposits of insulin fibrils have been found in the sites of subcutaneous insulin application in patients with prolonged diabetes. Using atomic force microscopy and ThT fluorescence assay we have investigated the interference of insulin amyloid aggregation with superparamagnetic Fe3O4-based nanoparticles (SPIONs) coated with dextran (DEX); molecular mass of dextran was equal to 15-20, 40 or 70 kDa. The obtained data indicate that all three types of dextran coated nanoparticles (NP-FeDEXs) are able to inhibit insulin fibrillization and to destroy amyloid fibrils. The extent of anti-amyloid activities depends on the properties of NP-FeDEXs, mainly on the size of nanoparticles which is determined by molecular mass of dextran molecules. The most effective inhibiting activity was observed for the smallest nanoparticles coated with 15-20 kDa dextran. Contrary, the highest destroying activity was observed for the largest NP-FeDEX (70 kDa dextran).

  8. Amyloid-beta aggregates cause alterations of astrocytic metabolic phenotype: impact on neuronal viability

    OpenAIRE

    Allaman, Igor; Gavillet, Mathilde; Bélanger, Mireille; Laroche, Thierry; Viertl, David; Lashuel, Hilal A.; Magistretti, Pierre J.

    2010-01-01

    Amyloid-beta (Abeta) peptides play a key role in the pathogenesis of Alzheimer's disease and exert various toxic effects on neurons; however, relatively little is known about their influence on glial cells. Astrocytes play a pivotal role in brain homeostasis, contributing to the regulation of local energy metabolism and oxidative stress defense, two aspects of importance for neuronal viability and function. In the present study, we explored the effects of Abeta peptides on glucose metabolism ...

  9. Functional and structural effects of amyloidaggregate on Xenopus laevis oocytes.

    Science.gov (United States)

    Parodi, Jorge; Ochoa-de la Paz, Lenin; Miledi, Ricardo; Martínez-Torres, Ataúlfo

    2012-10-01

    Xenopus laevis oocytes exposed to amyloidaggregate generated oscillatory electric activity (blips) that was recorded by two-microelectrode voltage-clamp. The cells exhibited a series of "spontaneous" blips ranging in amplitude from 3.8 ± 0.9 nA at the beginning of the recordings to 6.8 ± 1.7 nA after 15 min of exposure to 1 μM aggregate. These blips were similar in amplitude to those induced by the channel-forming antimicrobial agents amphotericin B (7.8 ± 1.2 nA) and gramicidin (6.3 ± 1.1 nA). The amyloid aggregate-induced currents were abolished when extracellular Ca(2+) was removed from the bathing solution, suggesting a central role for this cation in generating the spontaneous electric activity. The amyloid aggregate also affected the Ca(2+)-dependent Cl(-) currents of oocytes, as shown by increased amplitude of the transient-outward chloride current (T(out)) and the serum-activated, oscillatory Cl(-) currents. Electron microcopy revealed that amyloid aggregate induced the dissociation of the follicular cells that surround the oocyte, thus leading to a failure in the electro-chemical communication between these cells. This was also evidenced by the suppression of the oscillatory Ca(2+)-dependent ATP-currents, which require proper coupling between oocytes and the follicular cell layer. These observations, made using the X. laevis oocytes as a versatile experimental model, may help to understand the effects of amyloid aggregate on cellular communication.

  10. Microglial responses to amyloid β peptide opsonization and indomethacin treatment

    Directory of Open Access Journals (Sweden)

    Leonard Brian

    2005-08-01

    Full Text Available Abstract Background Recent studies have suggested that passive or active immunization with anti-amyloid β peptide (Aβ antibodies may enhance microglial clearance of Aβ deposits from the brain. However, in a human clinical trial, several patients developed secondary inflammatory responses in brain that were sufficient to halt the study. Methods We have used an in vitro culture system to model the responses of microglia, derived from rapid autopsies of Alzheimer's disease patients, to Aβ deposits. Results Opsonization of the deposits with anti-Aβ IgG 6E10 enhanced microglial chemotaxis to and phagocytosis of Aβ, as well as exacerbated microglial secretion of the pro-inflammatory cytokines TNF-α and IL-6. Indomethacin, a common nonsteroidal anti-inflammatory drug (NSAID, had no effect on microglial chemotaxis or phagocytosis, but did significantly inhibit the enhanced production of IL-6 after Aβ opsonization. Conclusion These results are consistent with well known, differential NSAID actions on immune cell functions, and suggest that concurrent NSAID administration might serve as a useful adjunct to Aβ immunization, permitting unfettered clearance of Aβ while dampening secondary, inflammation-related adverse events.

  11. Influence of Aluminium and EGCG on Fibrillation and Aggregation of Human Islet Amyloid Polypeptide

    Directory of Open Access Journals (Sweden)

    Zhi-Xue Xu

    2016-01-01

    Full Text Available The abnormal fibrillation of human islet amyloid polypeptide (hIAPP has been implicated in the development of type II diabetes. Aluminum is known to trigger the structural transformation of many amyloid proteins and induce the formation of toxic aggregate species. The (−-epigallocatechin gallate (EGCG is considered capable of binding both metal ions and amyloid proteins with inhibitory effect on the fibrillation of amyloid proteins. However, the effect of Al(III/EGCG complex on hIAPP fibrillation is unclear. In the present work, we sought to view insight into the structures and properties of Al(III and EGCG complex by using spectroscopic experiments and quantum chemical calculations and also investigated the influence of Al(III and EGCG on hIAPP fibrillation and aggregation as well as their combined interference on this process. Our studies demonstrated that Al(III could promote fibrillation and aggregation of hIAPP, while EGCG could inhibit the fibrillation of hIAPP and lead to the formation of hIAPP amorphous aggregates instead of the ordered fibrils. Furthermore, we proved that the Al(III/EGCG complex in molar ratio of 1 : 1 as Al(EGCG(H2O2 could inhibit the hIAPP fibrillation more effectively than EGCG alone. The results provide the invaluable reference for the new drug development to treat type II diabetes.

  12. The molecular mass of dextran used to modify magnetite nanoparticles affects insulin amyloid aggregation

    Czech Academy of Sciences Publication Activity Database

    Sipošová, K.; Pospíšková, K.; Bednáriková, Z.; Šafařík, Ivo; Šafaříková, Miroslava; Kubovčíková, M.; Kopčanský, P.; Gázová, Z.

    2017-01-01

    Roč. 427, April (2017), s. 48-53 ISSN 0304-8853 Institutional support: RVO:60077344 Keywords : amyloid aggregation * nanoparticles * magnetic fluid * dextran * insulin Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 2.630, year: 2016

  13. Manipulating Aggregation Behavior of the Uncharged Peptide Carbetocin

    DEFF Research Database (Denmark)

    Høgstedt, Ulrich B; Østergaard, Jesper; Weiss, Torsten

    2018-01-01

    Peptides are usually administered through subcutaneous injection. For low potency drugs, this may require high concentration formulations increasing the risk of peptide aggregation, especially for compounds without any intrinsic chargeable groups. Carbetocin was used as a model to study the behav...

  14. Effect of Aggregated β-Amyloid (1-42 on Synaptic Plasticity of Hippocampal Dentate Gyrus Granule Cells in Vivo

    Directory of Open Access Journals (Sweden)

    Shirin Babri

    2012-12-01

    Full Text Available Introduction: Alzheimer’s disease (AD is a common neurodegenerative disorder in elderly people with an impairment of cognitive decline and memory loss. β-amyloid (Aβ as a potent neurotoxic peptide has a pivotal role in the pathogenesis of AD. This disease begins with impairment in synaptic functions before developing into later neuro­degeneration and neuronal loss. The aim of this study was to evaluate the synaptic plasticity and electrophysiological function of granule cells in hippocampal dentate gyrus (DG after intracerebroventricular (i.c.v. administration of aggregated Aβ (1-42 peptide in vivo. Methods: Animals were divided to control and Aβ (1-42 groups. Long-term potentia­tion (LTP in perforant path-DG synapses was assessed in order to investigate the effect of aggregated Aβ (1-42 on synaptic plasticity. Field excitatory post-synaptic potential (fEPSP slope and population spike (PS amplitude were measured. Results: Administration of Aβ (1-42 significantly decreased fEPSP slope and PS amplitude in Aβ (1-42 group comparing with the control group and had no effect on baseline activity of neurons. Conclusion: The present study indicates that administration of aggregated form of Aβ (1-42 into the lateral ventricle effectively inhibits LTP in granular cells of the DG in hippocampus in vivo.

  15. Stereoselective determination of amino acids in beta-amyloid peptides and senile plaques.

    Science.gov (United States)

    Thorsén, G; Bergquist, J; Westlind-Danielsson, A; Josefsson, B

    2001-06-01

    A novel method for the determination of the enantiomeric composition of peptides is presented. In this paper, the focus has been on beta-amyloid peptides from deceased Alzheimer's disease patients. The peptides are hydrolyzed using mineral acid. The free amino acids are derivatized with the chiral reagent (+)- or (-)-1-(9-anthryl)-2-propyl chloroformate and subsequently separated using micellar electrokinetic chromatography (MEKC) and detected using laser-induced fluorescence (LIF) detection. The high separation efficiency of the MEKC-LIF system, yielding approximately 1 million theoretical plates/m for most amino acids, facilitates the simultaneous chiral determination of nine amino acids. The samples that have been analyzed were standard 1-40 beta-amyloid peptides, in vitro precipitated beta-amyloid fibrils, and human senile plaque samples.

  16. The architecture of amyloid-like peptide fibrils revealed by X-ray scattering, diffraction and electron microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Langkilde, Annette E., E-mail: annette.langkilde@sund.ku.dk [University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen (Denmark); Morris, Kyle L.; Serpell, Louise C. [University of Sussex, Falmer, Brighton (United Kingdom); Svergun, Dmitri I. [European Molecular Biology Laboratory, Hamburg Outstation, 22607 Hamburg (Germany); Vestergaard, Bente [University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen (Denmark)

    2015-04-01

    The aggregation process and the fibril state of an amyloidogenic peptide suggest monomer addition to be the prevailing mechanism of elongation and a model of the peptide packing in the fibrils has been obtained. Structural analysis of protein fibrillation is inherently challenging. Given the crucial role of fibrils in amyloid diseases, method advancement is urgently needed. A hybrid modelling approach is presented enabling detailed analysis of a highly ordered and hierarchically organized fibril of the GNNQQNY peptide fragment of a yeast prion protein. Data from small-angle X-ray solution scattering, fibre diffraction and electron microscopy are combined with existing high-resolution X-ray crystallographic structures to investigate the fibrillation process and the hierarchical fibril structure of the peptide fragment. The elongation of these fibrils proceeds without the accumulation of any detectable amount of intermediate oligomeric species, as is otherwise reported for, for example, glucagon, insulin and α-synuclein. Ribbons constituted of linearly arranged protofilaments are formed. An additional hierarchical layer is generated via the pairing of ribbons during fibril maturation. Based on the complementary data, a quasi-atomic resolution model of the protofilament peptide arrangement is suggested. The peptide structure appears in a β-sheet arrangement reminiscent of the β-zipper structures evident from high-resolution crystal structures, with specific differences in the relative peptide orientation. The complexity of protein fibrillation and structure emphasizes the need to use multiple complementary methods.

  17. Lattice model for amyloid peptides: OPEP force field parametrization and applications to the nucleus size of Alzheimer’s peptides

    Energy Technology Data Exchange (ETDEWEB)

    Tran, Thanh Thuy; Nguyen, Phuong H., E-mail: phuong.nguyen@ibpc.fr; Derreumaux, Philippe, E-mail: philippe.derreumaux@ibpc.fr [Laboratoire de Biochimie Théorique, UPR 9080, CNRS, Université Denis Diderot, Sorbonne Paris Cité IBPC, 13 rue Pierre et Marie Curie, 75005 Paris (France)

    2016-05-28

    Coarse-grained protein lattice models approximate atomistic details and keep the essential interactions. They are, therefore, suitable for capturing generic features of protein folding and amyloid formation at low computational cost. As our aim is to study the critical nucleus sizes of two experimentally well-characterized peptide fragments Aβ{sub 16−22} and Aβ{sub 37−42} of the full length Aβ{sub 1−42} Alzheimer’s peptide, it is important that simulations with the lattice model reproduce all-atom simulations. In this study, we present a comprehensive force field parameterization based on the OPEP (Optimized Potential for Efficient protein structure Prediction) force field for an on-lattice protein model, which incorporates explicitly the formation of hydrogen bonds and directions of side-chains. Our bottom-up approach starts with the determination of the best lattice force parameters for the Aβ{sub 16−22} dimer by fitting its equilibrium parallel and anti-parallel β-sheet populations to all-atom simulation results. Surprisingly, the calibrated force field is transferable to the trimer of Aβ{sub 16−22} and the dimer and trimer of Aβ{sub 37−42}. Encouraged by this finding, we characterized the free energy landscapes of the two decamers. The dominant structure of the Aβ{sub 16−22} decamer matches the microcrystal structure. Pushing the simulations for aggregates between 4-mer and 12-mer suggests a nucleus size for fibril formation of 10 chains. In contrast, the Aβ{sub 37−42} decamer is largely disordered with mixed by parallel and antiparallel chains, suggesting that the nucleus size is >10 peptides. Our refined force field coupled to this on-lattice model should provide useful insights into the critical nucleation number associated with neurodegenerative diseases.

  18. Protein kinase C involvement in the acetylcholine release reduction induced by amyloid-beta(25-35) aggregates on neuromuscular synapses.

    Science.gov (United States)

    Tomàs, Marta; Garcia, Neus; Santafé, Manuel M; Lanuza, Maria; Tomàs, Josep

    2009-01-01

    Using intracellular recording of the diaphragm muscle of adult rats, we have investigated the short-term functional effects of amyloid-beta (Abeta(25-35) peptide aggregates on the modulation of acetylcholine (ACh) release and the involvement of protein kinase C (PKC). The non-aggregated form of this peptide does not change the evoked and spontaneous transmitter release parameters on the neuromuscular synapse. However, the aggregated form of Abeta(25-35) acutely interferes with evoked quantal ACh release (approximately 40% reduction) when synaptic activity in the ex vivo neuromuscular preparation is maintained by low frequency (1 Hz) electrical stimulation. This effect is partially dependent on the activity of PKC that may have a permissive action. The end result of Abeta(25-35) is in opposition to the PKC-dependent maintenance effect on ACh release manifested in active synapses.

  19. Peptide p5 binds both heparinase-sensitive glycosaminoglycans and fibrils in patient-derived AL amyloid extracts

    Energy Technology Data Exchange (ETDEWEB)

    Martin, Emily B.; Williams, Angela [Department of Medicine, University of Tennessee Graduate School of Medicine, 1924 Alcoa Highway, Knoxville, TN 37922 (United States); Heidel, Eric [Department of Surgery, University of Tennessee Graduate School of Medicine, 1924 Alcoa Highway, Knoxville, TN 37922 (United States); Macy, Sallie [Department of Medicine, University of Tennessee Graduate School of Medicine, 1924 Alcoa Highway, Knoxville, TN 37922 (United States); Kennel, Stephen J. [Department of Medicine, University of Tennessee Graduate School of Medicine, 1924 Alcoa Highway, Knoxville, TN 37922 (United States); Department of Radiology, University of Tennessee Graduate School of Medicine, 1924 Alcoa Highway, Knoxville, TN 37922 (United States); Wall, Jonathan S., E-mail: jwall@utmck.edu [Department of Medicine, University of Tennessee Graduate School of Medicine, 1924 Alcoa Highway, Knoxville, TN 37922 (United States); Department of Radiology, University of Tennessee Graduate School of Medicine, 1924 Alcoa Highway, Knoxville, TN 37922 (United States)

    2013-06-21

    Highlights: •Polybasic peptide p5 binds human light chain amyloid extracts. •The binding of p5 with amyloid involves both glycosaminoglycans and fibrils. •Heparinase treatment led to a correlation between p5 binding and fibril content. •p5 binding to AL amyloid requires electrostatic interactions. -- Abstract: In previously published work, we have described heparin-binding synthetic peptides that preferentially recognize amyloid deposits in a mouse model of reactive systemic (AA) amyloidosis and can be imaged by using positron and single photon emission tomographic imaging. We wanted to extend these findings to the most common form of visceral amyloidosis, namely light chain (AL); however, there are no robust experimental animal models of AL amyloidosis. To further define the binding of the lead peptide, p5, to AL amyloid, we characterized the reactivity in vitro of p5 with in situ and patient-derived AL amyloid extracts which contain both hypersulfated heparan sulfate proteoglycans as well as amyloid fibrils. Histochemical staining demonstrated that the peptide specifically localized with tissue-associated AL amyloid deposits. Although we anticipated that p5 would undergo electrostatic interactions with the amyloid-associated glycosaminoglycans expressing heparin-like side chains, no significant correlation between peptide binding and glycosaminoglycan content within amyloid extracts was observed. In contrast, following heparinase I treatment, although overall binding was reduced, a positive correlation between peptide binding and amyloid fibril content became evident. This interaction was further confirmed using synthetic light chain fibrils that contain no carbohydrates. These data suggest that p5 can bind to both the sulfated glycosaminoglycans and protein fibril components of AL amyloid. Understanding these complex electrostatic interactions will aid in the optimization of synthetic peptides for use as amyloid imaging agents and potentially as

  20. Small stress molecules inhibit aggregation and neurotoxicity of prion peptide 106-126

    International Nuclear Information System (INIS)

    Kanapathipillai, Mathumai; Ku, Sook Hee; Girigoswami, Koyeli; Park, Chan Beum

    2008-01-01

    In prion diseases, the posttranslational modification of host-encoded prion protein PrP c yields a high β-sheet content modified protein PrP sc , which further polymerizes into amyloid fibrils. PrP106-126 initiates the conformational changes leading to the conversion of PrP c to PrP sc . Molecules that can defunctionalize such peptides can serve as a potential tool in combating prion diseases. In microorganisms during stressed conditions, small stress molecules (SSMs) are formed to prevent protein denaturation and maintain protein stability and function. The effect of such SSMs on PrP106-126 amyloid formation is explored in the present study using turbidity, atomic force microscopy (AFM), and cellular toxicity assay. Turbidity and AFM studies clearly depict that the SSMs-ectoine and mannosylglyceramide (MGA) inhibit the PrP106-126 aggregation. Our study also connotes that ectoine and MGA offer strong resistance to prion peptide-induced toxicity in human neuroblastoma cells, concluding that such molecules can be potential inhibitors of prion aggregation and toxicity

  1. Active protein aggregates induced by terminally attached self-assembling peptide ELK16 in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Zhou Bihong

    2011-02-01

    Full Text Available Abstract Background In recent years, it has been gradually realized that bacterial inclusion bodies (IBs could be biologically active. In particular, several proteins including green fluorescent protein, β-galactosidase, β-lactamase, alkaline phosphatase, D-amino acid oxidase, polyphosphate kinase 3, maltodextrin phosphorylase, and sialic acid aldolase have been successfully produced as active IBs when fused to an appropriate partner such as the foot-and-mouth disease virus capsid protein VP1, or the human β-amyloid peptide Aβ42(F19D. As active IBs may have many attractive advantages in enzyme production and industrial applications, it is of considerable interest to explore them further. Results In this paper, we report that an ionic self-assembling peptide ELK16 (LELELKLK2 was able to effectively induce the formation of cytoplasmic inclusion bodies in Escherichia coli (E. coli when attached to the carboxyl termini of four model proteins including lipase A, amadoriase II, β-xylosidase, and green fluorescent protein. These aggregates had a general appearance similar to the usually reported cytoplasmic inclusion bodies (IBs under transmission electron microscopy or fluorescence confocal microscopy. Except for lipase A-ELK16 fusion, the three other fusion protein aggregates retained comparable specific activities with the native counterparts. Conformational analyses by Fourier transform infrared spectroscopy revealed the existence of newly formed antiparallel beta-sheet structures in these ELK16 peptide-induced inclusion bodies, which is consistent with the reported assembly of the ELK16 peptide. Conclusions This has been the first report where a terminally attached self-assembling β peptide ELK16 can promote the formation of active inclusion bodies or active protein aggregates in E. coli. It has the potential to render E. coli and other recombinant hosts more efficient as microbial cell factories for protein production. Our observation might

  2. Pleomorphic copper coordination by Alzheimer's disease amyloid-beta peptide.

    Science.gov (United States)

    Drew, Simon C; Noble, Christopher J; Masters, Colin L; Hanson, Graeme R; Barnham, Kevin J

    2009-01-28

    Numerous conflicting models have been proposed regarding the nature of the Cu(2+) coordination environment of the amyloid beta (Abeta) peptide, the causative agent of Alzheimer's disease. This study used multifrequency CW-EPR spectroscopy to directly resolve the superhyperfine interactions between Cu(2+) and the ligand nuclei of Abeta, thereby avoiding ambiguities associated with introducing point mutations. Using a library of Abeta16 analogues with site-specific (15)N-labeling at Asp1, His6, His13, and His14, numerical simulations of the superhyperfine resonances delineated two independent 3N1O Cu(2+) coordination modes, {N(a)(D1), O, N(epsilon)(H6), N(epsilon)(H13)} (component Ia) and {N(a)(D1), O, N(epsilon)(H6), N(epsilon)(H14)} (component Ib), between pH 6-7. A third coordination mode (component II) was identified at pH 8.0, and simulation of the superhyperfine resonances indicated a 3N1O coordination sphere involving nitrogen ligation by His6, His13, and His14. No differences were observed upon (17)O-labeling of the phenolic oxygen of Tyr10, confirming it is not a key oxygen ligand in the physiological pH range. Hyperfine sublevel correlation (HYSCORE) spectroscopy, in conjunction with site-specific (15)N-labeling, provided additional support for the common role of His6 in components Ia and Ib, and for the assignment of a {O, N(epsilon)(H6), N(epsilon)(H13), N(epsilon)(H14)} coordination sphere to component II. HYSCORE studies of a peptide analogue with selective (13)C-labeling of Asp1 revealed (13)C cross-peaks characteristic of equatorial coordination by the carboxylate oxygen of Asp1 in component Ia/b coordination. The direct resolution of Cu(2+) ligand interactions, together with the key finding that component I is composed of two distinct coordination modes, provides valuable insight into a range of conflicting ligand assignments and highlights the complexity of Cu(2+)/Abeta interactions.

  3. Liposomes bi-functionalized with phosphatidic acid and an ApoE-derived peptide affect Aβ aggregation features and cross the blood-brain-barrier: implications for therapy of Alzheimer disease

    NARCIS (Netherlands)

    Bana, Laura; Minniti, Stefania; Salvati, Elisa; Sesana, Silvia; Zambelli, Vanessa; Cagnotto, Alfredo; Orlando, Antonina; Cazzaniga, Emanuela; Zwart, Rob; Scheper, Wiep; Masserini, Massimo; Re, Francesca

    2014-01-01

    Targeting amyloidpeptide (Aβ) within the brain is a strategy actively sought for therapy of Alzheimer's disease (AD). We investigated the ability of liposomes bi-functionalized with phosphatidic acid and with a modified ApoE-derived peptide (mApoE-PA-LIP) to affect Aβ aggregation/disaggregation

  4. PEGylated nanoparticles bind to and alter amyloid-beta peptide conformation

    DEFF Research Database (Denmark)

    Brambilla, Davide; Verpillot, Romain; Le Droumaguet, Benjamin

    2012-01-01

    We have demonstrated that the polyethylene glycol (PEG) corona of long-circulating polymeric nanoparticles (NPs) favors interaction with the amyloid-beta (Aß(1-42)) peptide both in solution and in serum. The influence of PEGylation of poly(alkyl cyanoacrylate) and poly(lactic acid) NPs on the int......We have demonstrated that the polyethylene glycol (PEG) corona of long-circulating polymeric nanoparticles (NPs) favors interaction with the amyloid-beta (Aß(1-42)) peptide both in solution and in serum. The influence of PEGylation of poly(alkyl cyanoacrylate) and poly(lactic acid) NPs...

  5. Drosophila Melanogaster as a Model System for Studies of Islet Amyloid Polypeptide Aggregation

    Science.gov (United States)

    Schultz, Sebastian Wolfgang; Nilsson, K. Peter R.; Westermark, Gunilla Torstensdotter

    2011-01-01

    Background Recent research supports that aggregation of islet amyloid polypeptide (IAPP) leads to cell death and this makes islet amyloid a plausible cause for the reduction of beta cell mass, demonstrated in patients with type 2 diabetes. IAPP is produced by the beta cells as a prohormone, and proIAPP is processed into IAPP by the prohormone convertases PC1/3 and PC2 in the secretory granules. Little is known about the pathogenesis for islet amyloid and which intracellular mechanisms are involved in amyloidogenesis and induction of cell death. Methodology/Principal Findings We have established expression of human proIAPP (hproIAPP), human IAPP (hIAPP) and the non-amyloidogenic mouse IAPP (mIAPP) in Drosophila melanogaster, and compared survival of flies with the expression driven to different cell populations. Only flies expressing hproIAPP in neurons driven by the Gal4 driver elavC155,Gal4 showed a reduction in lifespan whereas neither expression of hIAPP or mIAPP influenced survival. Both hIAPP and hproIAPP expression caused formation of aggregates in CNS and fat body region, and these aggregates were both stained by the dyes Congo red and pFTAA, both known to detect amyloid. Also, the morphology of the highly organized protein granules that developed in the fat body of the head in hIAPP and hproIAPP expressing flies was characterized, and determined to consist of 15.8 nm thick pentagonal rod-like structures. Conclusions/Significance These findings point to a potential for Drosophila melanogaster to serve as a model system for studies of hproIAPP and hIAPP expression with subsequent aggregation and developed pathology. PMID:21695120

  6. β-Hairpin of Islet Amyloid Polypeptide Bound to an Aggregation Inhibitor

    Science.gov (United States)

    Mirecka, Ewa A.; Feuerstein, Sophie; Gremer, Lothar; Schröder, Gunnar F.; Stoldt, Matthias; Willbold, Dieter; Hoyer, Wolfgang

    2016-01-01

    In type 2 diabetes, the formation of islet amyloid consisting of islet amyloid polypeptide (IAPP) is associated with reduction in β-cell mass and contributes to the failure of islet cell transplantation. Rational design of inhibitors of IAPP amyloid formation has therapeutic potential, but is hampered by the lack of structural information on inhibitor complexes of the conformationally flexible, aggregation-prone IAPP. Here we characterize a β-hairpin conformation of IAPP in complex with the engineered binding protein β-wrapin HI18. The β-strands correspond to two amyloidogenic motifs, 12-LANFLVH-18 and 22-NFGAILS-28, which are connected by a turn established around Ser-20. Besides backbone hydrogen bonding, the IAPP:HI18 interaction surface is dominated by non-polar contacts involving hydrophobic side chains of the IAPP β-strands. Apart from monomers, HI18 binds oligomers and fibrils and inhibits IAPP aggregation and toxicity at low substoichiometric concentrations. The IAPP β-hairpin can serve as a molecular recognition motif enabling control of IAPP aggregation. PMID:27641459

  7. Novel Detox Gel Depot sequesters β-Amyloid Peptides in a mouse model of Alzheimer’s Disease

    Science.gov (United States)

    Sundaram, Ranjini K.; Kasinathan, Chinnaswamy; Stein, Stanley; Sundaram, Pazhani

    2012-01-01

    Alzheimer’s Disease (AD), a debilitating neurodegenerative disease is caused by aggregation and accumulation of a 39–43 amino acid peptide (amyloid β or Aβ) in brain parenchyma and cerebrovasculature. The rational approach would be to use drugs that interfere with Aβ-Aβ interaction and disrupt polymerization. Peptide ligands capable of binding to the KLVFF (amino acids 16–20) region in the Aβ molecule have been investigated as possible drug candidates. Retro-inverso (RI) peptide of this pentapeptide, ffvlk, has been shown to bind artificial fibrils made from Aβ with moderate affinity. We hypothesized that a ‘detox gel’, which is synthesized by covalently linking a tetrameric version of RI peptide ffvlk to poly (ethylene glycol) polymer chains will act like a ‘sink’ to capture Aβ peptides from the surrounding environment. We previously demonstrated that this hypothesis works in an in vitro system. The present study extended this hypothesis to an in vivo mouse model of Alzheimer’s Disease and determined the therapeutic effect of our detox gel. We injected detox gel subcutaneously to AD model mice and analyzed brain levels of Aβ-42 and improvement in memory parameters. The results showed a reduction of brain amyloid burden in detox gel treated mice. Memory parameters in the treated mice improved. No undesirable immune response was observed. The data strongly suggest that our detox gel can be used as an effective therapy to deplete brain Aβ levels. Considering recent abandonment of failed antibody based therapies, our detox gel appears to have the advantage of being a non-immune based therapy. PMID:22712003

  8. Glutamate system, amyloid β peptides and tau protein: functional interrelationships and relevance to Alzheimer disease pathology

    Science.gov (United States)

    Revett, Timothy J.; Baker, Glen B.; Jhamandas, Jack; Kar, Satyabrata

    2013-01-01

    Alzheimer disease is the most prevalent form of dementia globally and is characterized premortem by a gradual memory loss and deterioration of higher cognitive functions and postmortem by neuritic plaques containing amyloid β peptide and neurofibrillary tangles containing phospho-tau protein. Glutamate is the most abundant neurotransmitter in the brain and is essential to memory formation through processes such as long-term potentiation and so might be pivotal to Alzheimer disease progression. This review discusses how the glutamatergic system is impaired in Alzheimer disease and how interactions of amyloid β and glutamate influence synaptic function, tau phosphorylation and neurodegeneration. Interestingly, glutamate not only influences amyloid β production, but also amyloid β can alter the levels of glutamate at the synapse, indicating that small changes in the concentrations of both molecules could influence Alzheimer disease progression. Finally, we describe how the glutamate receptor antagonist, memantine, has been used in the treatment of individuals with Alzheimer disease and discuss its effectiveness. PMID:22894822

  9. Human amyloid β peptide and tau co-expression impairs behavior and causes specific gene expression changes in Caenorhabditis elegans.

    Science.gov (United States)

    Wang, Chenyin; Saar, Valeria; Leung, Ka Lai; Chen, Liang; Wong, Garry

    2018-01-01

    Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the presence of extracellular amyloid plaques consisting of Amyloidpeptide (Aβ) aggregates and neurofibrillary tangles formed by aggregation of hyperphosphorylated microtubule-associated protein tau. We generated a novel invertebrate model of AD by crossing Aβ1-42 (strain CL2355) with either pro-aggregating tau (strain BR5270) or anti-aggregating tau (strain BR5271) pan-neuronal expressing transgenic Caenorhabditis elegans. The lifespan and progeny viability of the double transgenic strains were significantly decreased compared with wild type N2 (P5E-21). RNA interference of 13 available top up-regulated genes in Aβ1-42+pro-aggregating tau animals revealed that F-box family genes and nep-4 could enhance life span deficits and chemotaxis deficits while Y39G8C.2 (TTBK2) could suppress these behaviors. Comparing the list of regulated genes from C. elegans to the top 60 genes related to human AD confirmed an overlap of 8 genes: patched homolog 1, PTCH1 (ptc-3), the Rab GTPase activating protein, TBC1D16 (tbc-16), the WD repeat and FYVE domain-containing protein 3, WDFY3 (wdfy-3), ADP-ribosylation factor guanine nucleotide exchange factor 2, ARFGEF2 (agef-1), Early B-cell Factor, EBF1 (unc-3), d-amino-acid oxidase, DAO (daao-1), glutamate receptor, metabotropic 1, GRM1 (mgl-2), prolyl 4-hydroxylase subunit alpha 2, P4HA2 (dpy-18 and phy-2). Taken together, our C. elegans double transgenic model provides insight on the fundamental neurobiologic processes underlying human AD and recapitulates selected transcriptomic changes observed in human AD brains. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Manipulation of self-assembly amyloid peptide nanotubes by dielectrophoresis (DEP)

    DEFF Research Database (Denmark)

    Castillo, Jaime; Tanzi, Simone; Dimaki, Maria

    2008-01-01

    Self-assembled amyloid peptide nanotubes (SAPNT) were manipulated and immobilized using dielectrophoresis. Micro-patterned electrodes of Au were fabricated by photolithography and lifted off on a silicon dioxide layer. SAPNT were manipulated by adjusting the amplitude and frequency of the applied...

  11. Halogenation dictates the architecture of amyloid peptide nanostructures† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7nr03263c

    Science.gov (United States)

    Pizzi, Andrea; Pigliacelli, Claudia; Gori, Alessandro; Nonappa; Ikkala, Olli; Demitri, Nicola; Terraneo, Giancarlo; Castelletto, Valeria; Hamley, Ian W.; Baldelli Bombelli, Francesca

    2017-01-01

    Amyloid peptides yield a plethora of interesting nanostructures though difficult to control. Here we report that depending on the number, position, and nature of the halogen atoms introduced into either one or both phenylalanine benzene rings of the amyloid β peptide-derived core-sequence KLVFF, four different architectures were obtained in a controlled manner. Our findings demonstrate that halogenation may develop as a general strategy to engineer amyloidal peptide self-assembly and obtain new amyloidal nanostructures. PMID:28696473

  12. New pyridine derivatives as inhibitors of acetylcholinesterase and amyloid aggregation.

    Science.gov (United States)

    Pandolfi, Fabiana; De Vita, Daniela; Bortolami, Martina; Coluccia, Antonio; Di Santo, Roberto; Costi, Roberta; Andrisano, Vincenza; Alabiso, Francesco; Bergamini, Christian; Fato, Romana; Bartolini, Manuela; Scipione, Luigi

    2017-12-01

    A new series of pyridine derivatives with carbamic or amidic function has been designed and synthesized to act as cholinesterase inhibitors. The synthesized compounds were tested toward EeAChE and hAChE and toward eqBChE and hBChE. The carbamate 8 was the most potent hAChE inhibitor (IC 50  = 0.153 ± 0.016 μM) while the carbamate 11 was the most potent inhibitor of hBChE (IC 50  = 0.828 ± 0.067 μM). A molecular docking study indicated that the carbamate 8 was able to bind AChE by interacting with both CAS and PAS, in agreement with the mixed inhibition mechanism. Furthermore, the carbamates 8, 9 and 11 were able to inhibit Aβ 42 self-aggregation and possessed quite low toxicity against human astrocytoma T67 and HeLa cell lines, being the carbamate 8 the less toxic compound on both cell lines. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  13. Insights into amyloid-like aggregation of H2 region of the C-terminal domain of nucleophosmin.

    Science.gov (United States)

    Russo, Anna; Diaferia, Carlo; La Manna, Sara; Giannini, Cinzia; Sibillano, Teresa; Accardo, Antonella; Morelli, Giancarlo; Novellino, Ettore; Marasco, Daniela

    2017-02-01

    Nucleophosmin (NPM1) is a multifunctional protein involved in a variety of biological processes including the pathogenesis of several human malignancies and is the most frequently mutated gene in Acute Myeloid Leukemia (AML). To deepen the role of protein regions in its biological activities, lately we reported on the structural behavior of dissected C-terminal domain (CTD) helical fragments. Unexpectedly the H2 (residues 264-277) and H3 AML-mutated regions showed a remarkable tendency to form amyloid-like assemblies with fibrillar morphology and β-sheet structure that resulted as toxic when exposed to human neuroblastoma cells. More recently NPM1 was found to be highly expressed and toxic in neurons of mouse models of Huntington's disease (HD). Here we investigate the role of each residue in the β-strand aggregation process of H2 region of NPM1 by performing a systematic alanine scan of its sequence and structural and kinetic analyses of aggregation of derived peptides by means of Circular Dichorism (CD) and Thioflavin T (Th-T) assay. These solution state investigations pointed out the crucial role exerted by the basic amyloidogenic stretch of H2 (264-271) and to shed light on the initial and main interactions involved in fibril formation we performed studies on fibrils deriving from the related Ala peptides through the analysis of fibrils with birefringence of polarized optical microscopy and wide-angle X-ray scattering (WAXS). This analysis suggested that the presence of branched Ile 269 conferred preferential packing patterns that, instead, appeared geometrically hampered by the aromatic side-chain of Phe 268 . Present investigations could be useful to deepen the knowledge of AML molecular mechanisms and the role of cytoplasmatic aggregates of NPM1c+. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Small heat shock protein HspB8: its distribution in Alzheimer's disease brains and its inhibition of amyloid-beta protein aggregation and cerebrovascular amyloid-beta toxicity.

    NARCIS (Netherlands)

    Wilhelmus, M.M.M.; Boelens, W.C.; Otte-Holler, I.; Kamps, B.; Kusters, B.; Maat-Schieman, M.L.; Waal, R.M.W. de; Verbeek, M.M.

    2006-01-01

    Alzheimer's disease (AD) is characterized by pathological lesions, such as senile plaques (SPs) and cerebral amyloid angiopathy (CAA), both predominantly consisting of a proteolytic cleavage product of the amyloid-beta precursor protein (APP), the amyloid-beta peptide (Abeta). CAA is also the major

  15. A binding-site barrier affects imaging efficiency of high affinity amyloid-reactive peptide radiotracers in vivo.

    Science.gov (United States)

    Wall, Jonathan S; Williams, Angela; Richey, Tina; Stuckey, Alan; Huang, Ying; Wooliver, Craig; Macy, Sallie; Heidel, Eric; Gupta, Neil; Lee, Angela; Rader, Brianna; Martin, Emily B; Kennel, Stephen J

    2013-01-01

    Amyloid is a complex pathology associated with a growing number of diseases including Alzheimer's disease, type 2 diabetes, rheumatoid arthritis, and myeloma. The distribution and extent of amyloid deposition in body organs establishes the prognosis and can define treatment options; therefore, determining the amyloid load by using non-invasive molecular imaging is clinically important. We have identified a heparin-binding peptide designated p5 that, when radioiodinated, was capable of selectively imaging systemic visceral AA amyloidosis in a murine model of the disease. The p5 peptide was posited to bind effectively to amyloid deposits, relative to similarly charged polybasic heparin-reactive peptides, because it adopted a polar α helix secondary structure. We have now synthesized a variant, p5R, in which the 8 lysine amino acids of p5 have been replaced with arginine residues predisposing the peptide toward the α helical conformation in an effort to enhance the reactivity of the peptide with the amyloid substrate. The p5R peptide had higher affinity for amyloid and visualized AA amyloid in mice by using SPECT/CT imaging; however, the microdistribution, as evidenced in micro-autoradiographs, was dramatically altered relative to the p5 peptide due to its increased affinity and a resultant "binding site barrier" effect. These data suggest that radioiodinated peptide p5R may be optimal for the in vivo detection of discreet, perivascular amyloid, as found in the brain and pancreatic vasculature, by using molecular imaging techniques; however, peptide p5, due to its increased penetration, may yield more quantitative imaging of expansive tissue amyloid deposits.

  16. A binding-site barrier affects imaging efficiency of high affinity amyloid-reactive peptide radiotracers in vivo.

    Directory of Open Access Journals (Sweden)

    Jonathan S Wall

    Full Text Available Amyloid is a complex pathology associated with a growing number of diseases including Alzheimer's disease, type 2 diabetes, rheumatoid arthritis, and myeloma. The distribution and extent of amyloid deposition in body organs establishes the prognosis and can define treatment options; therefore, determining the amyloid load by using non-invasive molecular imaging is clinically important. We have identified a heparin-binding peptide designated p5 that, when radioiodinated, was capable of selectively imaging systemic visceral AA amyloidosis in a murine model of the disease. The p5 peptide was posited to bind effectively to amyloid deposits, relative to similarly charged polybasic heparin-reactive peptides, because it adopted a polar α helix secondary structure. We have now synthesized a variant, p5R, in which the 8 lysine amino acids of p5 have been replaced with arginine residues predisposing the peptide toward the α helical conformation in an effort to enhance the reactivity of the peptide with the amyloid substrate. The p5R peptide had higher affinity for amyloid and visualized AA amyloid in mice by using SPECT/CT imaging; however, the microdistribution, as evidenced in micro-autoradiographs, was dramatically altered relative to the p5 peptide due to its increased affinity and a resultant "binding site barrier" effect. These data suggest that radioiodinated peptide p5R may be optimal for the in vivo detection of discreet, perivascular amyloid, as found in the brain and pancreatic vasculature, by using molecular imaging techniques; however, peptide p5, due to its increased penetration, may yield more quantitative imaging of expansive tissue amyloid deposits.

  17. Curcumin Decreases AmyloidPeptide Levels by Attenuating the Maturation of Amyloid-β Precursor Protein*

    Science.gov (United States)

    Zhang, Can; Browne, Andrew; Child, Daniel; Tanzi, Rudolph E.

    2010-01-01

    Alzheimer disease (AD) is a devastating neurodegenerative disease with no cure. The pathogenesis of AD is believed to be driven primarily by amyloid-β (Aβ), the principal component of senile plaques. Aβ is an ∼4-kDa peptide generated via cleavage of the amyloid-β precursor protein (APP). Curcumin is a compound in the widely used culinary spice, turmeric, which possesses potent and broad biological activities, including anti-inflammatory and antioxidant activities, chemopreventative effects, and effects on protein trafficking. Recent in vivo studies indicate that curcumin is able to reduce Aβ-related pathology in transgenic AD mouse models via unknown molecular mechanisms. Here, we investigated the effects of curcumin on Aβ levels and APP processing in various cell lines and mouse primary cortical neurons. We show for the first time that curcumin potently lowers Aβ levels by attenuating the maturation of APP in the secretory pathway. These data provide a mechanism of action for the ability of curcumin to attenuate amyloid-β pathology. PMID:20622013

  18. Curcumin decreases amyloid-beta peptide levels by attenuating the maturation of amyloid-beta precursor protein.

    Science.gov (United States)

    Zhang, Can; Browne, Andrew; Child, Daniel; Tanzi, Rudolph E

    2010-09-10

    Alzheimer disease (AD) is a devastating neurodegenerative disease with no cure. The pathogenesis of AD is believed to be driven primarily by amyloid-beta (Abeta), the principal component of senile plaques. Abeta is an approximately 4-kDa peptide generated via cleavage of the amyloid-beta precursor protein (APP). Curcumin is a compound in the widely used culinary spice, turmeric, which possesses potent and broad biological activities, including anti-inflammatory and antioxidant activities, chemopreventative effects, and effects on protein trafficking. Recent in vivo studies indicate that curcumin is able to reduce Abeta-related pathology in transgenic AD mouse models via unknown molecular mechanisms. Here, we investigated the effects of curcumin on Abeta levels and APP processing in various cell lines and mouse primary cortical neurons. We show for the first time that curcumin potently lowers Abeta levels by attenuating the maturation of APP in the secretory pathway. These data provide a mechanism of action for the ability of curcumin to attenuate amyloid-beta pathology.

  19. A Redox-Active, Compact Molecule for Cross-Linking Amyloidogenic Peptides into Nontoxic, Off-Pathway Aggregates: In Vitro and In Vivo Efficacy and Molecular Mechanisms

    Energy Technology Data Exchange (ETDEWEB)

    Derrick, Jeffrey S.; Kerr, Richard A.; Nam, Younwoo; Oh, Shin Bi; Lee, Hyuck Jin; Earnest, Kaylin G.; Suh, Nayoung; Peck, Kristy L.; Ozbil, Mehmet; Korshavn, Kyle J.; Ramamoorthy, Ayyalusamy; Prabhakar, Rajeev; Merino, Edward J.; Shearer, Jason; Lee, Joo-Yong; Ruotolo, Brandon T.; Lim, Mi Hee

    2015-11-25

    Chemical reagents targeting and controlling amyloidogenic peptides have received much attention for helping identify their roles in the pathogenesis of protein-misfolding disorders. Herein, we report a novel strategy for redirecting amyloidogenic peptides into nontoxic, off-pathway aggregates, which utilizes redox properties of a small molecule (DMPD, N,N-dimethyl-p-phenylenediamine) to trigger covalent adduct formation with the peptide. In addition, for the first time, biochemical, biophysical, and molecular dynamics simulation studies have been performed to demonstrate a mechanistic understanding for such an interaction between a small molecule (DMPD) and amyloid-β (Aβ) and its subsequent anti-amyloidogenic activity, which, upon its transformation, generates ligand–peptide adducts via primary amine-dependent intramolecular cross-linking correlated with structural compaction. Furthermore, in vivo efficacy of DMPD toward amyloid pathology and cognitive impairment was evaluated employing 5xFAD mice of Alzheimer’s disease (AD). Such a small molecule (DMPD) is indicated to noticeably reduce the overall cerebral amyloid load of soluble Aβ forms and amyloid deposits as well as significantly improve cognitive defects in the AD mouse model. Overall, our in vitro and in vivo studies of DMPD toward Aβ with the first molecular-level mechanistic investigations present the feasibility of developing new, innovative approaches that employ redox-active compounds without the structural complexity as next-generation chemical tools for amyloid management.

  20. Adsorption and Orientation of Human Islet Amyloid Polypeptide (hIAPP Monomer at Anionic Lipid Bilayers: Implications for Membrane-Mediated Aggregation

    Directory of Open Access Journals (Sweden)

    Guanghong Wei

    2013-03-01

    Full Text Available Protein misfolding and aggregation cause serious degenerative diseases, such as Alzheimer’s and type II diabetes. Human islet amyloid polypeptide (hIAPP is the major component of amyloid deposits found in the pancreas of type II diabetic patients. Increasing evidence suggests that β-cell death is related to the interaction of hIAPP with the cellular membrane, which accelerates peptide aggregation. In this study, as a first step towards understanding the membrane-mediated hIAPP aggregation, we investigate the atomic details of the initial step of hIAPP-membrane interaction, including the adsorption orientation and conformation of hIAPP monomer at an anionic POPG lipid bilayer by performing all-atom molecular dynamics simulations. We found that hIAPP monomer is quickly adsorbed to bilayer surface, and the adsorption is initiated from the N-terminal residues driven by strong electrostatic interactions of the positively-charged residues K1 and R11 with negatively-charged lipid headgroups. hIAPP binds parallel to the lipid bilayer surface as a stable helix through residues 7–22, consistent with previous experimental study. Remarkably, different simulations lead to the same binding orientation stabilized by electrostatic and H-bonding interactions, with residues R11, F15 and S19 oriented towards membrane and hydrophobic residues L12, A13, L16 and V17 exposed to solvent. Implications for membrane-mediated hIAPP aggregation are discussed.

  1. Keampferol-3-O-rhamnoside abrogates amyloid beta toxicity by modulating monomers and remodeling oligomers and fibrils to non-toxic aggregates

    Directory of Open Access Journals (Sweden)

    Sharoar Md

    2012-12-01

    Full Text Available Abstract Background Aggregation of soluble, monomeric β- amyloid (Aβ to oligomeric and then insoluble fibrillar Aβ is a key pathogenic feature in development of Alzheimer’s disease (AD. Increasing evidence suggests that toxicity is linked to diffusible Aβ oligomers, rather than to insoluble fibrils. The use of naturally occurring small molecules for inhibition of Aβ aggregation has recently attracted significant interest for development of effective therapeutic strategies against the disease. A natural polyphenolic flavone, Kaempferol-3-O-rhamnoside (K-3-rh, was utilized to investigate its effects on aggregation and cytotoxic effects of Aβ42 peptide. Several biochemical techniques were used to determine the conformational changes and cytotoxic effect of the peptide in the presence and absence of K-3-rh. Results K-3-rh showed a dose-dependent effect against Aβ42 mediated cytotoxicity. Anti-amyloidogenic properties of K-3-rh were found to be efficient in inhibiting fibrilogenesis and secondary structural transformation of the peptide. The consequence of these inhibitions was the accumulation of oligomeric structural species. The accumulated aggregates were smaller, soluble, non-β-sheet and non-toxic aggregates, compared to preformed toxic Aβ oligomers. K-3-rh was also found to have the remodeling properties of preformed soluble oligomers and fibrils. Both of these conformers were found to remodel into non-toxic aggregates. The results showed that K-3-rh interacts with different Aβ conformers, which affects fibril formation, oligomeric maturation and fibrillar stabilization. Conclusion K-3-rh is an efficient molecule to hinder the self assembly and to abrogate the cytotoxic effects of Aβ42 peptide. Hence, K-3-rh and small molecules with similar structure might be considered for therapeutic development against AD.

  2. A Binding-Site Barrier Affects Imaging Efficiency of High Affinity Amyloid-Reactive Peptide Radiotracers In Vivo

    OpenAIRE

    Wall, Jonathan S.; Williams, Angela; Richey, Tina; Stuckey, Alan; Huang, Ying; Wooliver, Craig; Macy, Sallie; Heidel, Eric; Gupta, Neil; Lee, Angela; Rader, Brianna; Martin, Emily B.; Kennel, Stephen J.

    2013-01-01

    Amyloid is a complex pathology associated with a growing number of diseases including Alzheimer's disease, type 2 diabetes, rheumatoid arthritis, and myeloma. The distribution and extent of amyloid deposition in body organs establishes the prognosis and can define treatment options; therefore, determining the amyloid load by using non-invasive molecular imaging is clinically important. We have identified a heparin-binding peptide designated p5 that, when radioiodinated, was capable of selecti...

  3. β-Amyloid-derived pentapeptide RIIGLa inhibits Aβ1-42 aggregation and toxicity

    International Nuclear Information System (INIS)

    Fueloep, Livia; Zarandi, Marta; Datki, Zsolt; Soos, Katalin; Penke, Botond

    2004-01-01

    Pr-IIGL a , a derivative of the tetrapeptide β-amyloid 31-34 (Aβ 31-34 ), exerts controversial effects: it is toxic in a neuroblastoma culture, but it protects glial cells from the cytotoxic action of Aβ 1-42 . For an understanding of this phenomenon, a new pentapeptide, RIIGL a was synthetized, and both compounds were studied by different physicochemical and biological methods. Transmission electron microscopic (TEM) studies revealed that Pr-IIGL a forms fibrillar aggregates, whereas RIIGL a does not form fibrils. Congo red binding studies furnished the same results. Aggregated Pr-IIGL a acts as a cytotoxic agent in neuroblastoma cultures, but RIIGL a does not display inherent toxicity. RIIGL a co-incubated with Aβ 1-42 inhibits the formation of mature amyloid fibres (TEM studies) and reduces the cytotoxic effect of fibrillar Aβ 1-42 . These results indicate that RIIGL a is an effective inhibitor of both the aggregation and the toxic effects of Aβ 1-42 and can serve as a lead compound for the design of novel neuroprotective peptidomimetics

  4. Structural insights into mechanisms for inhibiting amyloid β42 aggregation by non-catechol-type flavonoids.

    Science.gov (United States)

    Hanaki, Mizuho; Murakami, Kazuma; Akagi, Ken-ichi; Irie, Kazuhiro

    2016-01-15

    The prevention of 42-mer amyloid β-protein (Aβ42) aggregation is promising for the treatment of Alzheimer's disease. We previously described the site-specific inhibitory mechanism for Aβ42 aggregation by a catechol-type flavonoid, (+)-taxifolin, targeting Lys16,28 after its autoxidation. In contrast, non-catechol-type flavonoids (morin, datiscetin, and kaempferol) inhibited Aβ42 aggregation without targeting Lys16,28 with almost similar potencies to that of (+)-taxifolin. We herein provided structural insights into their mechanisms for inhibiting Aβ42 aggregation. Physicochemical analyses revealed that their inhibition did not require autoxidation. The (1)H-(15)N SOFAST-HMQC NMR of Aβ42 in the presence of morin and datiscetin revealed the significant perturbation of chemical shifts of His13,14 and Gln15, which were close to the intermolecular β-sheet region, Gln15-Ala21. His13,14 also played a role in radical formation at Tyr10, thereby inducing the oxidation of Met35, which has been implicated in Aβ42 aggregation. These results suggest the direct interaction of morin and datiscetin with the Aβ42 monomer. Although only kaempferol was oxidatively-degraded during incubation, its degradation products as well as kaempferol itself suppressed Aβ42 aggregation. However, neither kaempferol nor its decomposed products perturbed the chemical shifts of the Aβ42 monomer. Aggregation experiments using 1,1,1,3,3,3-hexafluoro-2-propanol-treated Aβ42 demonstrated that kaempferol and its degradation products inhibited the elongation rather than nucleation phase, implying that they interacted with small aggregates of Aβ42, but not with the monomer. In contrast, morin and datiscetin inhibited both phases. The position and number of hydroxyl groups on the B-ring of non-catechol-type flavonoids could be important for their inhibitory potencies and mechanisms against Aβ42 aggregation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. AFM-based force spectroscopy measurements of mature amyloid fibrils of the peptide glucagon

    DEFF Research Database (Denmark)

    Dong, M. D.; Hovgaard, M. B.; Mamdouh, W.

    2008-01-01

    We report on the mechanical characterization of individual mature amyloid fibrils by atomic force microscopy (AFM) and AFM-based single-molecule force spectroscopy (SMFS). These self-assembling materials, formed from the 29-residue amphiphatic peptide hormone glucagon, were found to display...... a reversible elastic behaviour. Based on AFM morphology and SMFS studies, we suggest that the observed elasticity is due to a force-induced conformational transition which is reversible due to the beta-helical conformation of protofibrils, allowing a high degree of extension. The elastic properties...... of such mature fibrils contribute to their high stability, suggesting that the internal hydrophobic interactions of amyloid fibrils are likely to be of fundamental importance in the assembly of amyloid fibrils and therefore for the understanding of the progression of their associated pathogenic disorders...

  6. Comparison of the amyloid pore forming properties of rat and human Alzheimer’s beta-amyloid peptide 1-42: Calcium imaging data

    Directory of Open Access Journals (Sweden)

    Coralie Di Scala

    2016-03-01

    Full Text Available The data here consists of calcium imaging of human neuroblastoma SH-SY5Y cells treated with the calcium-sensitive dye Fluo-4AM and then incubated with nanomolar concentrations of either human or rat Alzheimer’s β-amyloid peptide Aβ1-42. These data are both of a qualitative (fluorescence micrographs and semi-quantitative nature (estimation of intracellular calcium concentrations of cells probed by Aβ1-42 peptides vs. control untreated cells. Since rat Aβ1-42 differs from its human counterpart at only three amino acid positions, this comparative study is a good assessment of the specificity of the amyloid pore forming assay. The interpretation of this dataset is presented in the accompanying study “Broad neutralization of calcium-permeable amyloid pore channels with a chimeric Alzheimer/Parkinson peptide targeting brain gangliosides” [1].

  7. Dynamic PET and SPECT imaging with radioiodinated, amyloid-reactive peptide p5 in mice: a positive role for peptide dehalogenation.

    Science.gov (United States)

    Martin, Emily B; Kennel, Stephen J; Richey, Tina; Wooliver, Craig; Osborne, Dustin; Williams, Angela; Stuckey, Alan; Wall, Jonathan S

    2014-10-01

    Dynamic molecular imaging provides bio-kinetic data that is used to characterize novel radiolabeled tracers for the detection of disease. Amyloidosis is a rare protein misfolding disease that can affect many organs. It is characterized by extracellular deposits composed principally of fibrillar proteins and hypersulfated proteoglycans. We have previously described a peptide, p5, which binds preferentially to amyloid deposits in a murine model of reactive (AA) amyloidosis. We have determined the whole body distribution of amyloid by molecular imaging techniques using radioiodinated p5. The loss of radioiodide from imaging probes due to enzymatic reaction has plagued the use of radioiodinated peptides and antibodies. Therefore, we studied iodine-124-labeled p5 by using dynamic PET imaging of both amyloid-laden and healthy mice to assess the rates of amyloid binding, the relevance of dehalogenation and the fate of the radiolabeled peptide. Rates of blood pool clearance, tissue accumulation and dehalogenation of the peptide were estimated from the images. Comparisons of these properties between the amyloid-laden and healthy mice provided kinetic profiles whose differences may prove to be indicative of the disease state. Additionally, we performed longitudinal SPECT/CT imaging with iodine-125-labeled p5 up to 72h post injection to determine the stability of the radioiodinated peptide when bound to the extracellular amyloid. Our data show that amyloid-associated peptide, in contrast to the unbound peptide, is resistant to dehalogenation resulting in enhanced amyloid-specific imaging. These data further support the utility of this peptide for detecting amyloidosis and monitoring potential therapeutic strategies in patients. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. Syntheses and characterization of novel oxoisoaporphine derivatives as dual inhibitors for cholinesterases and amyloid beta aggregation.

    Science.gov (United States)

    Li, Yan-Ping; Ning, Fang-Xian; Yang, Meng-Bi; Li, Yong-Cheng; Nie, Min-Hua; Ou, Tian-Miao; Tan, Jia-Heng; Huang, Shi-Liang; Li, Ding; Gu, Lian-Quan; Huang, Zhi-Shu

    2011-05-01

    A series of 3-substituted (5c-5f, 6c-6f) and 4-substituted (10a-10g) oxoisoaporphine derivatives were synthesized. It was found that all these synthetic compounds had IC50 values at micro or nano molar range for cholinesterase inhibition, and most of them could inhibit amyloid β (Aβ) self-induced aggregation with inhibition ratio from 31.8% to 57.6%. The structure-activity relationship studies revealed that the derivatives with higher selectivity on AChE also showed better inhibition on Aβ self-induced aggregation. The results from cell toxicity study indicated that most quaternary methiodide salts had higher IC50 values than the corresponding non-quaternary compounds. This study provided potentially important information for further development of oxoisoaporphine derivatives as lead compounds for the treatment of Alzheimer's disease. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  9. Aggregation and Fibril Morphology of the Arctic Mutation of Alzheimer’s Aβ peptide by CD, TEM, STEM and in situ AFM

    Science.gov (United States)

    Norlin, Nils; Hellberg, Magnus; Filippov, Andrei; Sousa, Alioscka A.; Gröbner, Gerhard; Leapman, Richard D.; Almqvist, Nils; Antzutkin, Oleg N.

    2012-01-01

    Morphology of aggregation intermediates, polymorphism of amyloid fibrils and aggregation kinetics of the “Arctic” mutant of the Alzheimer’s amyloid β-peptide, Aβ(1-40)(E22G), in a physiologically relevant TRIS buffer (pH 7.4) were thoroughly explored in comparison with the human wild type Alzheimer’s amyloid peptide, wt-Aβ(1-40), using both in situ atomic force and electron microscopy, circular dichroism and thioflavin T fluorescence assays. For arc-Aβ(1-40) at the end of the ‘lag’-period of fibrillization an abrupt appearance of ~3 nm size ‘spherical aggregates’ with a homogeneous morphology, was identified. Then, the aggregation proceeds with a rapid growth of amyloid fibrils with a variety of morphologies, while the spherical aggregates eventually disappeared during in situ measurements. Arc-Aβ(1-40) was also shown to form fibrils at much lower concentrations than wt-Aβ(1-40): ≤2.5 μM and 12.5 μM, respectively. Moreover, at the same concentration, 50 μM, the aggregation process proceeds more rapidly for arc-Aβ(1-40): The first amyloid fibrils were observed after ca 72 hours from the onset of incubation as compared to approximately 7 days for wt-Aβ(1-40). Amyloid fibrils of arc-Aβ(1-40) exhibit a large variety of polymorphs, at least five, both coiled and non-coiled distinct fibril structures were recognized by AFM, while at least four types of arc-Aβ(1-40) fibrils were identified by TEM and STEM and their mass-per-length statistics were collected suggesting supramolecular structures with two, four and six β-sheet laminae. Our results suggest a pathway of fibrillogenesis for full-length Alzheimer’s peptides with small and structurally ordered transient spherical aggregates as on-pathway immediate precursors of amyloid fibrils. PMID:22750418

  10. Rationally designed turn promoting mutation in the amyloidpeptide sequence stabilizes oligomers in solution.

    Directory of Open Access Journals (Sweden)

    Jayakumar Rajadas

    Full Text Available Enhanced production of a 42-residue beta amyloid peptide (Aβ(42 in affected parts of the brain has been suggested to be the main causative factor for the development of Alzheimer's Disease (AD. The severity of the disease depends not only on the amount of the peptide but also its conformational transition leading to the formation of oligomeric amyloid-derived diffusible ligands (ADDLs in the brain of AD patients. Despite being significant to the understanding of AD mechanism, no atomic-resolution structures are available for these species due to the evanescent nature of ADDLs that hinders most structural biophysical investigations. Based on our molecular modeling and computational studies, we have designed Met35Nle and G37p mutations in the Aβ(42 peptide (Aβ(42Nle35p37 that appear to organize Aβ(42 into stable oligomers. 2D NMR on the Aβ(42Nle35p37 peptide revealed the occurrence of two β-turns in the V24-N27 and V36-V39 stretches that could be the possible cause for the oligomer stability. We did not observe corresponding NOEs for the V24-N27 turn in the Aβ(21-43Nle35p37 fragment suggesting the need for the longer length amyloid peptide to form the stable oligomer promoting conformation. Because of the presence of two turns in the mutant peptide which were absent in solid state NMR structures for the fibrils, we propose, fibril formation might be hindered. The biophysical information obtained in this work could aid in the development of structural models for toxic oligomer formation that could facilitate the development of therapeutic approaches to AD.

  11. Molecular basis and pharmacological implications of Alzheimer amyloid ß-peptide fibril formation,

    OpenAIRE

    Tjernberg, Lars

    1998-01-01

    Alzheimer's disease is a progressive neurodegenerative disease, mostly affectingelderly. The invariable deposition of protease-resistant fibrils of Alzheimer amyloidß-peptide (Aß) in the parenchyma and blood vessels of the brain is a centralevent. The aim of this study was to investigate whether Aß develops proteaseresistance upon polymerization and whether Aß may be generated through non specificproteolysis of a polymerized precursor, to identify Aß-Aß binding and fibrilfor...

  12. The antigen-binding fragment of human gamma immunoglobulin prevents amyloid β-peptide folding into β-sheet to form oligomers

    Science.gov (United States)

    Valls-Comamala, Victòria; Guivernau, Biuse; Bonet, Jaume; Puig, Marta; Perálvarez-Marín, Alex; Palomer, Ernest; Fernàndez-Busquets, Xavier; Altafaj, Xavier; Tajes, Marta; Puig-Pijoan, Albert; Vicente, Rubén; Oliva, Baldomero; Muñoz, Francisco J.

    2017-01-01

    The amyloid beta-peptide (Aβ) plays a leading role in Alzheimer's disease (AD) physiopathology. Even though monomeric forms of Aβ are harmless to cells, Aβ can aggregate into β-sheet oligomers and fibrils, which are both neurotoxic. Therefore, one of the main therapeutic approaches to cure or delay AD onset and progression is targeting Aβ aggregation. In the present study, we show that a pool of human gamma immunoglobulins (IgG) protected cortical neurons from the challenge with Aβ oligomers, as assayed by MTT reduction, caspase-3 activation and cytoskeleton integrity. In addition, we report the inhibitory effect of IgG on Aβ aggregation, as shown by Thioflavin T assay, size exclusion chromatography and atomic force microscopy. Similar results were obtained with Palivizumab, a human anti-sincitial virus antibody. In order to dissect the important domains, we cleaved the pool of human IgG with papain to obtain Fab and Fc fragments. Using these cleaved fragments, we functionally identified Fab as the immunoglobulin fragment inhibiting Aβ aggregation, a result that was further confirmed by an in silico structural model. Interestingly, bioinformatic tools show a highly conserved structure able to bind amyloid in the Fab region. Overall, our data strongly support the inhibitory effect of human IgG on Aβ aggregation and its neuroprotective role. PMID:28467807

  13. Loss of metal ions, disulfide reduction and mutations related to familial ALS promote formation of amyloid-like aggregates from superoxide dismutase.

    Directory of Open Access Journals (Sweden)

    Zeynep A Oztug Durer

    Full Text Available Mutations in the gene encoding Cu-Zn superoxide dismutase (SOD1 are one of the causes of familial amyotrophic lateral sclerosis (FALS. Fibrillar inclusions containing SOD1 and SOD1 inclusions that bind the amyloid-specific dye thioflavin S have been found in neurons of transgenic mice expressing mutant SOD1. Therefore, the formation of amyloid fibrils from human SOD1 was investigated. When agitated at acidic pH in the presence of low concentrations of guanidine or acetonitrile, metalated SOD1 formed fibrillar material which bound both thioflavin T and Congo red and had circular dichroism and infrared spectra characteristic of amyloid. While metalated SOD1 did not form amyloid-like aggregates at neutral pH, either removing metals from SOD1 with its intramolecular disulfide bond intact or reducing the intramolecular disulfide bond of metalated SOD1 was sufficient to promote formation of these aggregates. SOD1 formed amyloid-like aggregates both with and without intermolecular disulfide bonds, depending on the incubation conditions, and a mutant SOD1 lacking free sulfhydryl groups (AS-SOD1 formed amyloid-like aggregates at neutral pH under reducing conditions. ALS mutations enhanced the ability of disulfide-reduced SOD1 to form amyloid-like aggregates, and apo-AS-SOD1 formed amyloid-like aggregates at pH 7 only when an ALS mutation was also present. These results indicate that some mutations related to ALS promote formation of amyloid-like aggregates by facilitating the loss of metals and/or by making the intramolecular disulfide bond more susceptible to reduction, thus allowing the conversion of SOD1 to a form that aggregates to form resembling amyloid. Furthermore, the occurrence of amyloid-like aggregates per se does not depend on forming intermolecular disulfide bonds, and multiple forms of such aggregates can be produced from SOD1.

  14. An infrared spectroscopy approach to follow β-sheet formation in peptide amyloid assemblies

    Science.gov (United States)

    Seo, Jongcheol; Hoffmann, Waldemar; Warnke, Stephan; Huang, Xing; Gewinner, Sandy; Schöllkopf, Wieland; Bowers, Michael T.; von Helden, Gert; Pagel, Kevin

    2017-01-01

    Amyloidogenic peptides and proteins play a crucial role in a variety of neurodegenerative disorders such as Alzheimer's and Parkinson's disease. These proteins undergo a spontaneous transition from a soluble, often partially folded form, into insoluble amyloid fibrils that are rich in β-sheets. Increasing evidence suggests that highly dynamic, polydisperse folding intermediates, which occur during fibril formation, are the toxic species in the amyloid-related diseases. Traditional condensed-phase methods are of limited use for characterizing these states because they typically only provide ensemble averages rather than information about individual oligomers. Here we report the first direct secondary-structure analysis of individual amyloid intermediates using a combination of ion mobility spectrometry-mass spectrometry and gas-phase infrared spectroscopy. Our data reveal that oligomers of the fibril-forming peptide segments VEALYL and YVEALL, which consist of 4-9 peptide strands, can contain a significant amount of β-sheet. In addition, our data show that the more-extended variants of each oligomer generally exhibit increased β-sheet content.

  15. Role of amyloid peptides in vascular dysfunction and platelet dysregulation in Alzheimer's disease

    Directory of Open Access Journals (Sweden)

    Ilaria eCanobbio

    2015-03-01

    Full Text Available Alzheimer’s disease (AD is the most common neurodegenerative cause of dementia in the elderly. AD is accompanied by the accumulation of amyloid peptides in the brain parenchyma and in the cerebral vessels. The sporadic form of the AD accounts for about 95% of all cases. It is characterized by a late onset, typically after the age of 65, with a complex and still poorly understood aetiology. Several observations point towards a central role of cerebrovascular dysfunction in the onset of sporadic AD. According to the vascular hypothesis, AD may be initiated by vascular dysfunctions that precede and promote the neurodegenerative process. In accordance to this, AD patients show increased hemorragic or ischemic stroke risks. It is now clear that multiple bidirectional connections exist between AD and cerebrovascular disease, and in this new scenario, the effect of amyloid peptides on vascular cells and blood platelets appear to be central to AD. In this review we analyse the effect of amyloid peptides on vascular function and platelet activation and its contribution to the cerebrovascular pathology associated with AD and the progression of this disease.

  16. Electrochemical sensing of 2D condensation in amyloid peptides

    Czech Academy of Sciences Publication Activity Database

    Kurzatkowska, K.; Ostatná, Veronika; Hamley, I.W.; Doneux, T.; Paleček, Emil

    2013-01-01

    Roč. 106, SEP2013 (2013), s. 43-48 ISSN 0013-4686 R&D Projects: GA ČR(CZ) GAP301/11/2055 Institutional support: RVO:68081707 Keywords : QUARTZ-CRYSTAL-MICROBALANCE * SELF -ASSEMBLED MONOLAYERS * BETA-SHEET PEPTIDES Subject RIV: BO - Biophysics Impact factor: 4.086, year: 2013

  17. PrP aggregation can be seeded by pre-formed recombinant PrP amyloid fibrils without the replication of infectious prions.

    Science.gov (United States)

    Barron, Rona M; King, Declan; Jeffrey, Martin; McGovern, Gillian; Agarwal, Sonya; Gill, Andrew C; Piccardo, Pedro

    2016-10-01

    Mammalian prions are unusual infectious agents, as they are thought to consist solely of aggregates of misfolded prion protein (PrP). Generation of synthetic prions, composed of recombinant PrP (recPrP) refolded into fibrils, has been utilised to address whether PrP aggregates are, indeed, infectious prions. In several reports, neurological disease similar to transmissible spongiform encephalopathy (TSE) has been described following inoculation and passage of various forms of fibrils in transgenic mice and hamsters. However, in studies described here, we show that inoculation of recPrP fibrils does not cause TSE disease, but, instead, seeds the formation of PrP amyloid plaques in PrP-P101L knock-in transgenic mice (101LL). Importantly, both WT-recPrP fibrils and 101L-recPrP fibrils can seed plaque formation, indicating that the fibrillar conformation, and not the primary sequence of PrP in the inoculum, is important in initiating seeding. No replication of infectious prions or TSE disease was observed following both primary inoculation and subsequent subpassage. These data, therefore, argue against recPrP fibrils being infectious prions and, instead, indicate that these pre-formed seeds are acting to accelerate the formation of PrP amyloid plaques in 101LL Tg mice. In addition, these data reproduce a phenotype which was previously observed in 101LL mice following inoculation with brain extract containing in vivo-generated PrP amyloid fibrils, which has not been shown for other synthetic prion models. These data are reminiscent of the "prion-like" spread of aggregated forms of the beta-amyloid peptide (Aβ), α-synuclein and tau observed following inoculation of transgenic mice with pre-formed seeds of each misfolded protein. Hence, even when the protein is PrP, misfolding and aggregation do not reproduce the full clinicopathological phenotype of disease. The initiation and spread of protein aggregation in transgenic mouse lines following inoculation with pre

  18. Interleukin-3 prevents neuronal death induced by amyloid peptide

    Directory of Open Access Journals (Sweden)

    Otth Carola

    2007-10-01

    Full Text Available Abstract Background Interleukin-3 (IL-3 is an important glycoprotein involved in regulating biological responses such as cell proliferation, survival and differentiation. Its effects are mediated via interaction with cell surface receptors. Several studies have demonstrated the expression of IL-3 in neurons and astrocytes of the hippocampus and cortices in normal mouse brain, suggesting a physiological role of IL-3 in the central nervous system. Although there is evidence indicating that IL-3 is expressed in some neuronal populations, its physiological role in these cells is poorly known. Results In this study, we demonstrated the expression of IL-3 receptor in cortical neurons, and analyzed its influence on amyloid β (Aβ-treated cells. In these cells, IL-3 can activate at least three classical signalling pathways, Jak/STAT, Ras/MAP kinase and the PI 3-kinase. Viability assays indicated that IL-3 might play a neuroprotective role in cells treated with Aβ fibrils. It is of interest to note that our results suggest that cell survival induced by IL-3 required PI 3-kinase and Jak/STAT pathway activation, but not MAP kinase. In addition, IL-3 induced an increase of the anti-apoptotic protein Bcl-2. Conclusion Altogether these data strongly suggest that IL-3 neuroprotects neuronal cells against neurodegenerative agents like Aβ.

  19. Dynamic membrane interactions of antibacterial and antifungal biomolecules, and amyloid peptides, revealed by solid-state NMR spectroscopy.

    Science.gov (United States)

    Naito, Akira; Matsumori, Nobuaki; Ramamoorthy, Ayyalusamy

    2018-02-01

    A variety of biomolecules acting on the cell membrane folds into a biologically active structure in the membrane environment. It is, therefore, important to determine the structures and dynamics of such biomolecules in a membrane environment. While several biophysical techniques are used to obtain low-resolution information, solid-state NMR spectroscopy is one of the most powerful means for determining the structure and dynamics of membrane bound biomolecules such as antibacterial biomolecules and amyloidogenic proteins; unlike X-ray crystallography and solution NMR spectroscopy, applications of solid-state NMR spectroscopy are not limited by non-crystalline, non-soluble nature or molecular size of membrane-associated biomolecules. This review article focuses on the applications of solid-state NMR techniques to study a few selected antibacterial and amyloid peptides. Solid-state NMR studies revealing the membrane inserted bent α-helical structure associated with the hemolytic activity of bee venom melittin and the chemical shift oscillation analysis used to determine the transmembrane structure (with α-helix and 3 10 -helix in the N- and C-termini, respectively) of antibiotic peptide alamethicin are discussed in detail. Oligomerization of an amyloidogenic islet amyloid polypeptide (IAPP, or also known as amylin) resulting from its aggregation in a membrane environment, molecular interactions of the antifungal natural product amphotericin B with ergosterol in lipid bilayers, and the mechanism of lipid raft formation by sphingomyelin studied using solid state NMR methods are also discussed in this review article. This article is part of a Special Issue entitled "Biophysical Exploration of Dynamical Ordering of Biomolecular Systems" edited by Dr. Koichi Kato. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Amyloid fibril formation of peptides derived from the C-terminus of CETP modulated by lipids

    Energy Technology Data Exchange (ETDEWEB)

    García-González, Victor [Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, 04510 México, DF (Mexico); Mas-Oliva, Jaime, E-mail: jmas@ifc.unam.mx [Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, 04510 México, DF (Mexico); División de Investigación, Facultad de Medicina, Universidad Nacional Autónoma de México, 04510 México, DF (Mexico)

    2013-04-26

    Highlights: •The secondary structure of a C-terminal peptide derived from CETP was studied. •Lipids modulate secondary structure changes of a C-terminal peptide derived from CETP. •Lysophosphatidic acid maintains a functional α-helix and prevents fibril formation. •Transfer of lipids by CETP is related to the presence of an α-helix at its C-end. -- Abstract: Cholesteryl-ester transfer protein (CETP) is a plasmatic protein involved in neutral lipid transfer between lipoproteins. Focusing on the last 12 C-terminus residues we have previously shown that mutation D{sub 470}N promotes a conformational change towards a β-secondary structure. In turn, this modification leads to the formation of oligomers and fibrillar structures, which cause cytotoxic effects similar to the ones provoked by amyloid peptides. In this study, we evaluated the role of specific lipid arrangements on the structure of peptide helix-Z (D{sub 470}N) through the use of thioflavin T fluorescence, peptide bond absorbance, circular dichroism and electron microscopy. The results indicate that the use of micelles formed with lysophosphatidylcholine and lysophosphatidic acid (LPA) under neutral pH induce a conformational transition of peptide helix-Z containing a β-sheet conformation to a native α-helix structure, therefore avoiding the formation of amyloid fibrils. In contrast, incubation with phosphatidic acid does not change the profile for the β-sheet conformation. When the electrostatic charge at the surface of micelles or vesicles is regulated through the use of lipids such as phospholipid and LPA, minimal changes and the presence of β-structures were recorded. Mixtures with a positive net charge diminished the percentage of β-structure and the amount of amyloid fibrils. Our results suggest that the degree of solvation determined by the presence of a free hydroxyl group on lipids such as LPA is a key condition that can modulate the secondary structure and the consequent formation of

  1. Amyloid fibril formation of peptides derived from the C-terminus of CETP modulated by lipids

    International Nuclear Information System (INIS)

    García-González, Victor; Mas-Oliva, Jaime

    2013-01-01

    Highlights: •The secondary structure of a C-terminal peptide derived from CETP was studied. •Lipids modulate secondary structure changes of a C-terminal peptide derived from CETP. •Lysophosphatidic acid maintains a functional α-helix and prevents fibril formation. •Transfer of lipids by CETP is related to the presence of an α-helix at its C-end. -- Abstract: Cholesteryl-ester transfer protein (CETP) is a plasmatic protein involved in neutral lipid transfer between lipoproteins. Focusing on the last 12 C-terminus residues we have previously shown that mutation D 470 N promotes a conformational change towards a β-secondary structure. In turn, this modification leads to the formation of oligomers and fibrillar structures, which cause cytotoxic effects similar to the ones provoked by amyloid peptides. In this study, we evaluated the role of specific lipid arrangements on the structure of peptide helix-Z (D 470 N) through the use of thioflavin T fluorescence, peptide bond absorbance, circular dichroism and electron microscopy. The results indicate that the use of micelles formed with lysophosphatidylcholine and lysophosphatidic acid (LPA) under neutral pH induce a conformational transition of peptide helix-Z containing a β-sheet conformation to a native α-helix structure, therefore avoiding the formation of amyloid fibrils. In contrast, incubation with phosphatidic acid does not change the profile for the β-sheet conformation. When the electrostatic charge at the surface of micelles or vesicles is regulated through the use of lipids such as phospholipid and LPA, minimal changes and the presence of β-structures were recorded. Mixtures with a positive net charge diminished the percentage of β-structure and the amount of amyloid fibrils. Our results suggest that the degree of solvation determined by the presence of a free hydroxyl group on lipids such as LPA is a key condition that can modulate the secondary structure and the consequent formation of amyloid

  2. A Kinetic Model for β-Amyloid Adsorption at the Air/Solution Interface and Its Implication to the β-Amyloid Aggregation Process

    Science.gov (United States)

    Jiang, Dianlu; Dinh, Kim Lien; Ruthenburg, Travis; Zhang, Yi; Su, Lei; Land, Donald; Zhou, Feimeng

    2011-01-01

    The kinetics of adsorption at the air/buffer solution interface of amyloid beta peptide, Aβ(1–42), whose bulk concentration (submicromolar) is more than two orders of magnitude lower than that typically used in other in vitro aggregation studies, has been studied using a Langmuir-Blodgett trough. The pressure–time curves exhibit a lag phase, wherein the surface pressure essentially remains at zero, and a rising phase, corresponding to the Aβ adsorption at the interface. The duration of the lag phase was found to be highly dependent on both the Aβ bulk concentration and the solution temperature. A large activation energy (62.2 ± 4.1 KJ/mol) was determined and the apparent adsorption rate constant was found to be linearly dependent on the Aβ bulk concentration. Attenuated total reflection-IR spectra of the adsorbed Aβ transferred to a solid substrate and circular dichroism measurements of Aβ in the solution layer near the interface reveal that the natively unstructured Aβ in the bulk undergo a conformation change (folding) to mainly the α-helical structure. The results suggest that, prior to the adsorption step, an equilibrium between Aβ conformations is established within the subsurface. The kinetic equation derived from this model confirms that the overall Aβ adsorption is kinetically controlled and the apparent rate constant is proportional to the Aβ bulk concentration. This model also indicates that interfaces such as cell membranes and lipid bilayers may facilitate Aβ aggregation/fibrillation by providing a thin hydrophobic layer adjacent to the interface for the initial Aβ conformation change (misfolding) and accumulation. Such a preconcentration effect offers a plausible explanation of the fact that Aβ fibrillation occurs in vivo at nanomolar concentrations. Another important biological implication from our work is that Aβ misfolding may occur before its adsorption onto a cell membrane. This general kinetic model should also find

  3. A kinetic model for beta-amyloid adsorption at the air/solution interface and its implication to the beta-amyloid aggregation process.

    Science.gov (United States)

    Jiang, Dianlu; Dinh, Kim Lien; Ruthenburg, Travis C; Zhang, Yi; Su, Lei; Land, Donald P; Zhou, Feimeng

    2009-03-12

    At the air/buffer solution interface the kinetics of adsorption of amyloid beta peptide, Abeta(1-42), whose bulk concentration (submicromolar) is more than 2 orders of magnitude lower than that typically used in other in vitro aggregation studies, has been studied using a Langmuir-Blodgett trough. The pressure-time curves exhibit a lag phase, wherein the surface pressure essentially remains at zero, and a rising phase, corresponding to the Abeta adsorption at the interface. The duration of the lag phase was found to be highly dependent on both the Abeta bulk concentration and the solution temperature. A large activation energy (62.2 +/- 4.1 KJ/mol) was determined and the apparent adsorption rate constant was found to be linearly dependent on the Abeta bulk concentration. Attenuated total reflection-IR spectra of the adsorbed Abeta transferred to a solid substrate and circular dichroism measurements of Abeta in the solution layer near the interface reveal that the natively unstructured Abeta in the bulk undergo a conformation change (folding) to mainly the alpha-helical structure. The results suggest that, prior to the adsorption step, an equilibrium between Abeta conformations is established within the subsurface. The kinetic equation derived from this model confirms that the overall Abeta adsorption is kinetically controlled and the apparent rate constant is proportional to the Abeta bulk concentration. This model also indicates that interfaces such as cell membranes and lipid bilayers may facilitate Abeta aggregation/ fibrillation by providing a thin hydrophobic layer adjacent to the interface for the initial A/beta conformation change (misfolding) and accumulation. Such a preconcentration effect offers a plausible explanation of the fact that Abeta fibrillation occurs in vivo at nanomolar concentrations. Another important biological implication from our work is that Abeta misfolding may occur before its adsorption onto a cell membrane. This general kinetic model

  4. Successful adjuvant-free vaccination of BALB/c mice with mutated amyloid β peptides

    Directory of Open Access Journals (Sweden)

    Wahi Monika M

    2008-02-01

    Full Text Available Abstract Background A recent human clinical trial of an Alzheimer's disease (AD vaccine using amyloid beta (Aβ 1–42 plus QS-21 adjuvant produced some positive results, but was halted due to meningoencephalitis in some participants. The development of a vaccine with mutant Aβ peptides that avoids the use of an adjuvant may result in an effective and safer human vaccine. Results All peptides tested showed high antibody responses, were long-lasting, and demonstrated good memory response. Epitope mapping indicated that peptide mutation did not lead to epitope switching. Mutant peptides induced different inflammation responses as evidenced by cytokine profiles. Ig isotyping indicated that adjuvant-free vaccination with peptides drove an adequate Th2 response. All anti-sera from vaccinated mice cross-reacted with human Aβ in APP/PS1 transgenic mouse brain tissue. Conclusion Our study demonstrated that an adjuvant-free vaccine with different Aβ peptides can be an effective and safe vaccination approach against AD. This study represents the first report of adjuvant-free vaccines utilizing Aβ peptides carrying diverse mutations in the T-cell epitope. These largely positive results provide encouragement for the future of the development of human vaccinations for AD.

  5. Laboratory Exercise for Studying the Morphology of Heat-Denatured and Amyloid Aggregates of Lysozyme by Atomic Force Microscopy

    Science.gov (United States)

    Gokalp, Sumeyra; Horton, William; Jónsdóttir-Lewis, Elfa B.; Foster, Michelle; Török, Marianna

    2018-01-01

    To facilitate learning advanced instrumental techniques, essential tools for visualizing biomaterials, a simple and versatile laboratory exercise demonstrating the use of Atomic Force Microscopy (AFM) in biomedical applications was developed. In this experiment, the morphology of heat-denatured and amyloid-type aggregates formed from a low-cost…

  6. Fibrils from designed non-amyloid-related synthetic peptides induce AA-amyloidosis during inflammation in an animal model.

    Directory of Open Access Journals (Sweden)

    Per Westermark

    Full Text Available BACKGROUND: Mouse AA-amyloidosis is a transmissible disease by a prion-like mechanism where amyloid fibrils act by seeding. Synthetic peptides with no amyloid relationship can assemble into amyloid-like fibrils and these may have seeding capacity for amyloid proteins. PRINCIPAL FINDINGS: Several synthetic peptides, designed for nanotechnology, have been examined for their ability to produce fibrils with Congo red affinity and concomitant green birefringence, affinity for thioflavin S and to accelerate AA-amyloidosis in mice. It is shown that some amphiphilic fibril-forming peptides not only produced Congo red birefringence and showed affinity for thioflavin S, but they also shortened the lag phase for systemic AA-amyloidosis in mice when they were given intravenously at the time of inflammatory induction with silver nitride. Peptides, not forming amyloid-like fibrils, did not have such properties. CONCLUSIONS: These observations should caution researchers and those who work with synthetic peptides and their derivatives to be aware of the potential health concerns.

  7. Benchmarking of copper(II) LFMM parameters for studying amyloidpeptides.

    Science.gov (United States)

    Mutter, Shaun T; Deeth, Robert J; Turner, Matthew; Platts, James A

    2018-04-01

    Ligand field molecular mechanics (LFMM) parameters have been benchmarked for copper (II) bound to the amyloid-β 1-16 peptide fragment. Several density functional theory (DFT) optimised small test models, representative of different possible copper coordination modes, have been used to test the accuracy of the LFMM copper bond lengths and angles, resulting in errors typically less than 0.1 Å and 5°. Ligand field molecular dynamics (LFMD) simulations have been carried out on the copper bound amyloid-β 1-16 peptide and snapshots extracted from the subsequent trajectory. Snapshots have been optimised using DFT and the semi-empirical PM7 method resulting in good agreement against the LFMM calculated geometry. Analysis of substructures within snapshots shows that the larger contribution of geometrical difference, as measured by RMSD, lies within the peptide backbone, arising from differences in DFT and AMBER, and the copper coordination sphere is reproduced well by LFMM. PM7 performs excellently against LFMM with an average RMSD of 0.2 Å over 21 tested snapshots. Further analysis of the LFMD trajectory shows that copper bond lengths and angles have only small deviations from average values, with the exception of a carbonyl moiety from the N-terminus, which can act as a weakly bound fifth ligand.

  8. Substitution of proline32 by α-methylproline preorganizes β2-microglobulin for oligomerization but not for aggregation into amyloids.

    Science.gov (United States)

    Torbeev, Vladimir; Ebert, Marc-Olivier; Dolenc, Jozica; Hilvert, Donald

    2015-02-25

    Conversion of soluble folded proteins into insoluble amyloids generally proceeds in three distinct mechanistic stages: (1) initial protein misfolding into aggregation-competent conformers, (2) subsequent formation of oligomeric species and, finally, (3) self-assembly into extended amyloid fibrils. In the work reported herein, we interrogated the amyloidogenesis mechanism of human β2-microglobulin (β2m), which is thought to be triggered by a pivotal cis-trans isomerization of a proline residue at position 32 in the polypeptide, with nonstandard amino acids. Using chemical protein synthesis we prepared a β2m analogue in which Pro32 was replaced by the conformationally constrained amino acid α-methylproline (MePro). The strong propensity of MePro to adopt a trans prolyl bond led to enhanced population of a non-native [trans-MePro32]β2m protein conformer, which readily formed oligomers at neutral pH. In the presence of the antibiotic rifamycin SV, which inhibits amyloid growth of wild-type β2m, [MePro32]β2m was nearly quantitatively converted into different spherical oligomeric species. Self-assembly into amyloid fibrils was not observed in the absence of seeding, however, even at low pH (<3), where wild-type β2m spontaneously forms amyloids. Nevertheless, we found that aggregation-preorganized [MePro32]β2m can act in a prion-like fashion, templating misfolded conformations in a natively folded protein. Overall, these results provide detailed insight into the role of cis-trans isomerization of Pro32 and ensuing structural rearrangements that lead to initial β2m misfolding and aggregation. They corroborate the view that conformational protein dynamics enabled by reversible Pro32 cis-trans interconversion rather than simple population of the trans conformer is critical for both nucleation and subsequent growth of β2m amyloid structures.

  9. Impairment of context memory by β-amyloid peptide in terrestrial snail

    Directory of Open Access Journals (Sweden)

    2008-09-01

    Full Text Available We examined influence of the β-amyloid peptide (25-35 neurotoxic fragment (βAP on Helix lucorum food-aversion learning. Testing with aversively conditioned carrot showed that 2, 5, and 14 days after training the βAP-injected group responded in a significantly larger number of cases and with a significantly smaller latency than the sham-injected control group. The results demonstrate that the amyloid peptide partially impairs the learning process. In an attempt to specify what component of memory is impaired we compared responses in a context in which the snails were aversively trained, and in a neutral context. It was found that the sham-injected learned snails significantly less frequently took the aversively conditioned food in the context in which the snails were shocked, while the βAP-injected snails remembered the aversive context 2 days after associative training, but were not able to distinguish two contexts 5, and 14 days after training. In a separate series of experiments a specific context was associated with electric shock, and changes in general responsiveness were tested in two contexts several days later. It was found that the βAP-injected snails significantly increased withdrawal responses in all tested contexts, while the sham-injected control animals selectively increased responsiveness only in the context in which they were reinforced with electric shocks. These results demonstrate that the β-amyloid peptide (25-35 interferes with the learning process, and may play a significant role in behavioral plasticity and memory by selectively impairing only one

  10. Broad neutralization of calcium-permeable amyloid pore channels with a chimeric Alzheimer/Parkinson peptide targeting brain gangliosides.

    Science.gov (United States)

    Di Scala, Coralie; Yahi, Nouara; Flores, Alessandra; Boutemeur, Sonia; Kourdougli, Nazim; Chahinian, Henri; Fantini, Jacques

    2016-02-01

    Growing evidence supports a role for brain gangliosides in the pathogenesis of neurodegenerative diseases including Alzheimer's and Parkinson's. Recently we deciphered the ganglioside-recognition code controlling specific ganglioside binding to Alzheimer's β-amyloid (Aβ1-42) peptide and Parkinson's disease-associated protein α-synuclein. Cracking this code allowed us to engineer a short chimeric Aβ/α-synuclein peptide that recognizes all brain gangliosides. Here we show that ganglioside-deprived neural cells do no longer sustain the formation of zinc-sensitive amyloid pore channels induced by either Aβ1-42 or α-synuclein, as assessed by single-cell Ca(2+) fluorescence microscopy. Thus, amyloid channel formation, now considered a key step in neurodegeneration, is a ganglioside-dependent process. Nanomolar concentrations of chimeric peptide competitively inhibited amyloid pore formation induced by Aβ1-42 or α-synuclein in cultured neural cells. Moreover, this peptide abrogated the intracellular calcium increases induced by Parkinson's-associated mutant forms of α-synuclein (A30P, E46K and A53T). The chimeric peptide also prevented the deleterious effects of Aβ1-42 on synaptic vesicle trafficking and decreased the Aβ1-42-induced impairment of spontaneous activity in rat hippocampal slices. Taken together, these data show that the chimeric peptide has broad anti-amyloid pore activity, suggesting that a common therapeutic strategy based on the prevention of amyloid-ganglioside interactions is a reachable goal for both Alzheimer's and Parkinson's diseases. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. A sensitive and selective electrochemical biosensor for the determination of beta-amyloid oligomer by inhibiting the peptide-triggered in situ assembly of silver nanoparticles

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    Xing Y

    2017-04-01

    Full Text Available Yun Xing,1,2 Xiao-Zhen Feng,2 Lipeng Zhang,1 Jiating Hou,2 Guo-Cheng Han,2 Zhencheng Chen2 1Henan Province of Key Laboratory of New Optoelectronic Functional Materials, College of Chemistry and Chemical Engineering, Anyang Normal University, Anyang, 2School of Life and Environmental Sciences, Guilin University of Electronic Technology, Guilin, Guangxi, People’s Republic of China Abstract: Soluble beta-amyloid (Aβ oligomer is believed to be the most important toxic species in the brain of Alzheimer’s disease (AD patients. Thus, it is critical to develop a simple method for the selective detection of Aβ oligomer with low cost and high sensitivity. In this paper, we report an electrochemical method for the detection of Aβ oligomer with a peptide as the bioreceptor and silver nanoparticle (AgNP aggregates as the redox reporters. This strategy is based on the conversion of AgNP-based colorimetric assay into electrochemical analysis. Specifically, the peptide immobilized on the electrode surface and presented in solution triggered together the in situ formation of AgNP aggregates, which produced a well-defined electrochemical signal. However, the specific binding of Aβ oligomer to the immobilized peptide prevented the in situ assembly of AgNPs. As a result, a poor electrochemical signal was observed. The detection limit of the method was found to be 6 pM. Furthermore, the amenability of this method for the analysis of Aβ oligomer in serum and artificial cerebrospinal fluid (aCSF samples was demonstrated. Keywords: electrochemical biosensors, Alzheimer’s disease, beta-amyloid oligomer, peptide, silver nanoparticles

  12. Beta-amyloid peptides undergo regulated co-secretion with neuropeptide and catecholamine neurotransmitters.

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    Toneff, Thomas; Funkelstein, Lydiane; Mosier, Charles; Abagyan, Armen; Ziegler, Michael; Hook, Vivian

    2013-08-01

    Beta-amyloid (Aβ) peptides are secreted from neurons, resulting in extracellular accumulation of Aβ and neurodegeneration of Alzheimer's disease. Because neuronal secretion is fundamental for the release of neurotransmitters, this study assessed the hypothesis that Aβ undergoes co-release with neurotransmitters. Model neuronal-like chromaffin cells were investigated, and results illustrate regulated, co-secretion of Aβ(1-40) and Aβ(1-42) with peptide neurotransmitters (galanin, enkephalin, and NPY) and catecholamine neurotransmitters (dopamine, norepinephrine, and epinephrine). Regulated secretion from chromaffin cells was stimulated by KCl depolarization and nicotine. Forskolin, stimulating cAMP, also induced co-secretion of Aβ peptides with peptide and catecholamine neurotransmitters. These data suggested the co-localization of Aβ with neurotransmitters in dense core secretory vesicles (DCSV) that store and secrete such chemical messengers. Indeed, Aβ was demonstrated to be present in DCSV with neuropeptide and catecholamine transmitters. Furthermore, the DCSV organelle contains APP and its processing proteases, β- and γ-secretases, that are necessary for production of Aβ. Thus, Aβ can be generated in neurotransmitter-containing DCSV. Human IMR32 neuroblastoma cells also displayed regulated secretion of Aβ(1-40) and Aβ(1-42) with the galanin neurotransmitter. These findings illustrate that Aβ peptides are present in neurotransmitter-containing DCSV, and undergo co-secretion with neuropeptide and catecholamine neurotransmitters that regulate brain functions. Copyright © 2013 Elsevier Inc. All rights reserved.

  13. Benzothiazole aniline tetra(ethylene glycol) and 3-amino-1,2,4-triazole inhibit neuroprotection against amyloid peptides by catalase overexpression in vitro.

    Science.gov (United States)

    Chilumuri, Amrutha; Odell, Mark; Milton, Nathaniel G N

    2013-11-20

    Alzheimer's disease, Familial British dementia, Familial Danish dementia, Type 2 diabetes mellitus, plus Creutzfeldt-Jakob disease are associated with amyloid fibril deposition and oxidative stress. The antioxidant enzyme catalase is a neuroprotective amyloid binding protein. Herein the effects of catalase overexpression in SH-SY5Y neuronal cells on the toxicity of amyloid-β (Aβ), amyloid-Bri (ABri), amyloid-Dan (ADan), amylin (IAPP), and prion protein (PrP) peptides were determined. Results showed catalase overexpression was neuroprotective against Aβ, ABri, ADan, IAPP, and PrP peptides. The catalase inhibitor 3-amino-1,2,4-triazole (3-AT) and catalase-amyloid interaction inhibitor benzothiazole aniline tetra(ethylene glycol) (BTA-EG4) significantly enhanced neurotoxicity of amyloid peptides in catalase overexpressing neuronal cells. This suggests catalase neuroprotection involves breakdown of hydrogen peroxide (H2O2) plus a direct binding interaction between catalase and the Aβ, ABri, ADan, IAPP, and PrP peptides. Kisspeptin 45-50 had additive neuroprotective actions against the Aβ peptide in catalase overexpressing cells. The effects of 3-AT had an intracellular site of action, while catalase-amyloid interactions had an extracellular component. These results suggest that the 3-AT and BTA-EG4 compounds may be able to inhibit endogenous catalase mediated neuroprotection. Use of BTA-EG4, or compounds that inhibit catalase binding to amyloid peptides, as potential therapeutics for Neurodegenerative diseases may therefore result in unwanted effects.

  14. Benzothiazole Aniline Tetra(ethylene glycol) and 3-Amino-1,2,4-triazole Inhibit Neuroprotection against Amyloid Peptides by Catalase Overexpression in Vitro

    Science.gov (United States)

    2013-01-01

    Alzheimer’s disease, Familial British dementia, Familial Danish dementia, Type 2 diabetes mellitus, plus Creutzfeldt-Jakob disease are associated with amyloid fibril deposition and oxidative stress. The antioxidant enzyme catalase is a neuroprotective amyloid binding protein. Herein the effects of catalase overexpression in SH-SY5Y neuronal cells on the toxicity of amyloid-β (Aβ), amyloid-Bri (ABri), amyloid-Dan (ADan), amylin (IAPP), and prion protein (PrP) peptides were determined. Results showed catalase overexpression was neuroprotective against Aβ, ABri, ADan, IAPP, and PrP peptides. The catalase inhibitor 3-amino-1,2,4-triazole (3-AT) and catalase-amyloid interaction inhibitor benzothiazole aniline tetra(ethylene glycol) (BTA-EG4) significantly enhanced neurotoxicity of amyloid peptides in catalase overexpressing neuronal cells. This suggests catalase neuroprotection involves breakdown of hydrogen peroxide (H2O2) plus a direct binding interaction between catalase and the Aβ, ABri, ADan, IAPP, and PrP peptides. Kisspeptin 45–50 had additive neuroprotective actions against the Aβ peptide in catalase overexpressing cells. The effects of 3-AT had an intracellular site of action, while catalase-amyloid interactions had an extracellular component. These results suggest that the 3-AT and BTA-EG4 compounds may be able to inhibit endogenous catalase mediated neuroprotection. Use of BTA-EG4, or compounds that inhibit catalase binding to amyloid peptides, as potential therapeutics for Neurodegenerative diseases may therefore result in unwanted effects. PMID:23968537

  15. A cocoa peptide protects Caenorhabditis elegans from oxidative stress and β-amyloid peptide toxicity.

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    Patricia Martorell

    Full Text Available BACKGROUND: Cocoa and cocoa-based products contain different compounds with beneficial properties for human health. Polyphenols are the most frequently studied, and display antioxidant properties. Moreover, protein content is a very interesting source of antioxidant bioactive peptides, which can be used therapeutically for the prevention of age-related diseases. METHODOLOGY/PRINCIPAL FINDINGS: A bioactive peptide, 13L (DNYDNSAGKWWVT, was obtained from a hydrolyzed cocoa by-product by chromatography. The in vitro inhibition of prolyl endopeptidase (PEP was used as screening method to select the suitable fraction for peptide identification. Functional analysis of 13L peptide was achieved using the transgenic Caenorhabditis elegans strain CL4176 expressing the human Aβ₁₋₄₂ peptide as a pre-clinical in vivo model for Alzheimer's disease. Among the peptides isolated, peptide 13L (1 µg/mL showed the highest antioxidant activity (P≤0.001 in the wild-type strain (N2. Furthermore, 13L produced a significant delay in body paralysis in strain CL4176, especially in the 24-47 h period after Aβ₁₋₄₂ peptide induction (P≤0.0001. This observation is in accordance with the reduction of Aβ deposits in CL4176 by western blot. Finally, transcriptomic analysis in wild-type nematodes treated with 13L revealed modulation of the proteosomal and synaptic functions as the main metabolic targets of the peptide. CONCLUSIONS/SIGNIFICANCE: These findings suggest that the cocoa 13L peptide has antioxidant activity and may reduce Aβ deposition in a C. elegans model of Alzheimer's disease; and therefore has a putative therapeutic potential for prevention of age-related diseases. Further studies in murine models and humans will be essential to analyze the effectiveness of the 13L peptide in higher animals.

  16. Hydrolysis of Whey Protein Isolate with Bacillus licheniformis Protease: Fractionation and Identification of Aggregating Peptides

    NARCIS (Netherlands)

    Creusot, N.P.; Gruppen, H.

    2007-01-01

    The objective of this work was to identify the dominant aggregating peptides from a whey protein hydrolysate (degree of hydrolysis of 6.8%) obtained with Bacillus licheniformis protease. The aggregating peptides were fractionated with preparative reversed-phase chromatography and identified with

  17. Controlling the aggregation and gelation of ß-lactoglobulin by the addition of its peptides

    NARCIS (Netherlands)

    Kosters, H.A.

    2012-01-01

    In this thesis the effects of peptides, or protein hydrolysates on the heat-induced aggregation and gelation of (concentrated) protein systems were studied. First, it was investigated if specific peptides could influence the heat-induced denaturation and aggregation of intact proteins solutions,

  18. Tuning calcium carbonate growth through physical confinement and templating with amyloid-like polypeptide aggregates

    Science.gov (United States)

    Colaco, Martin Francis

    that this methodology does not extend to three-dimensional confined systems, as the water has no method of escape. Through the addition of an insoluble hydroscopic polymer to our microreactors, amorphous calcium carbonate of controllable sizes can be grown. However, crystalline calcium carbonate cannot be grown without some type of templating. Studies of calcium carbonate templating have predominantly been performed on SAMs or in poorly characterized gels or protein films. The use of ordered protein or polypeptide aggregates for templating permits both geometry and charge surface density to be varied. We have studied the kinetics and final morphology of ordered aggregates of poly-L-glutamic acid and a copolymer of glutamic acid and alanine through experiments and simulations. Electrostatics, not structure, of the monomer appeared to be the dominating factor in the aggregation, as pH and salt concentration changes led to dramatic changes in the kinetics. Examining our experimental with existing models provided inconsistent results, so we developed a new model that yielded physically realistic rate constants, while generating better fits with longer lag phases and faster growths. However, despite the similarity of aggregation conditions, the two polypeptides yielded vastly different morphologies, with the PEA forming typical amyloid-like fibrils and PE forming larger, twisted lamellar aggregates. Templating with these aggregates also yielded dramatically different patterns. Polycrystalline rhombohedral calcite with smooth faces and edges grew on PEA fibrils, with minimal templating in evidence. However, on PE, numerous calcite crystals with triangular projections tracked the surface of the aggregate. The PE lamellae are characterized by extensive beta-sheet structure. In this conformation, the glutamic acid spacings on the surface of the aggregates can mimic the spacings of the carboxylates in the calcite lattice. In addition, the high negative charge density on the

  19. Neuroprotective Effects of Pomegranate Peel Extract after Chronic Infusion with AmyloidPeptide in Mice

    Science.gov (United States)

    Morzelle, Maressa Caldeira; Salgado, Jocelem Mastrodi; Telles, Milena; Mourelle, Danilo; Bachiega, Patricia; Buck, Hudson Sousa

    2016-01-01

    Alzheimer’s disease is a chronic and degenerative condition that had no treatment until recently. The current therapeutic strategies reduce progression of the disease but are expensive and commonly cause side effects that are uncomfortable for treated patients. Functional foods to prevent and/or treat many conditions, including neurodegenerative diseases, represent a promising field of study currently gaining attention. To this end, here we demonstrate the effects of pomegranate (Punica granatum) peel extract (PPE) regarding spatial memory, biomarkers of neuroplasticity, oxidative stress and inflammation in a mouse model of neurodegeneration. Male C57Bl/6 mice were chronically infused for 35 days with amyloidpeptide 1–42 (Aβ) or vehicle (control) using mini-osmotic pumps. Another group, also infused with Aβ, was treated with PPE (p.o.– βA+PPE, 800 mg/kg/day). Spatial memory was evaluated in the Barnes maze. Animals treated with PPE and in the control group exhibited a reduction in failure to find the escape box, a finding that was not observed in the Aβ group. The consumption of PPE reduced amyloid plaque density, increased the expression of neurotrophin BDNF and reduced the activity of acetylcholinesterase enzyme. A reduction in lipid peroxidation and in the concentration of the pro-inflammatory cytokine TNF-α was also observed in the PPE group. No hepatic lesions were observed in animals treated with PPE. In conclusion, administration of pomegranate peel extract has neuroprotective effects involving multiple mechanisms to prevent establishment and progression of the neurodegenerative process induced by infusion with amyloidpeptide in mice. PMID:27829013

  20. Differences between amyloidaggregation in solution and on the membrane: insights into elucidation of the mechanistic details of Alzheimer's disease.

    Science.gov (United States)

    Kotler, Samuel A; Walsh, Patrick; Brender, Jeffrey R; Ramamoorthy, Ayyalusamy

    2014-10-07

    The association of the amyloid-β (Aβ) peptide with cellular membranes is hypothesized to be the underlying phenomenon of neurotoxicity in Alzheimer's disease. Misfolding of proteins and peptides, as is the case with Aβ, follows a progression from a monomeric state, through intermediates, ending at long, unbranched amyloid fibers. This tutorial review offers a perspective on the association of toxic Aβ structures with membranes as well as details of membrane-associated mechanisms of toxicity.

  1. Differences between amyloidaggregation in solution and on the membrane: Insights towards elucidation of the mechanistic details of Alzheimer’s disease

    Science.gov (United States)

    Kotler, Samuel A.; Walsh, Patrick; Brender, Jeffrey R.; Ramamoorthy, Ayyalusamy

    2014-01-01

    The association of the amyloid-β (Aβ) peptide with cellular membranes is hypothesized to be the underlying phenomenon of neurotoxicity in Alzheimer’s disease. Misfolding of proteins and peptides, as is the case with Aβ, follows a progression from a monomeric state, through intermediates, ending at long, unbranched amyloid fibers. This tutorial review offers a perspective into the association of toxic Aβ structures with membrane as well as details into membrane-associated mechanisms of toxicity. PMID:24464312

  2. Stability of transmembrane amyloid β-peptide and membrane integrity tested by molecular modeling of site-specific Aβ42 mutations.

    Directory of Open Access Journals (Sweden)

    Chetan Poojari

    Full Text Available Interactions of the amyloid β-protein (Aβ with neuronal cell membranes, leading to the disruption of membrane integrity, are considered to play a key role in the development of Alzheimer's disease. Natural mutations in Aβ42, such as the Arctic mutation (E22G have been shown to increase Aβ42 aggregation and neurotoxicity, leading to the early-onset of Alzheimer's disease. A correlation between the propensity of Aβ42 to form protofibrils and its effect on neuronal dysfunction and degeneration has been established. Using rational mutagenesis of the Aβ42 peptide it was further revealed that the aggregation of different Aβ42 mutants in lipid membranes results in a variety of polymorphic aggregates in a mutation dependent manner. The mutant peptides also have a variable ability to disrupt bilayer integrity. To further test the connection between Aβ42 mutation and peptide-membrane interactions, we perform molecular dynamics simulations of membrane-inserted Aβ42 variants (wild-type and E22G, D23G, E22G/D23G, K16M/K28M and K16M/E22G/D23G/K28M mutants as β-sheet monomers and tetramers. The effects of charged residues on transmembrane Aβ42 stability and membrane integrity are analyzed at atomistic level. We observe an increased stability for the E22G Aβ42 peptide and a decreased stability for D23G compared to wild-type Aβ42, while D23G has the largest membrane-disruptive effect. These results support the experimental observation that the altered toxicity arising from mutations in Aβ is not only a result of the altered aggregation propensity, but also originates from modified Aβ interactions with neuronal membranes.

  3. Modeling the Interaction between β-Amyloid Aggregates and Choline Acetyltransferase Activity and Its Relation with Cholinergic Dysfunction through Two-Enzyme/Two-Compartment Model

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    Hedia Fgaier

    2015-01-01

    Full Text Available The effect of β-amyloid aggregates on activity of choline acetyltransferase (ChAT which is responsible for synthesizing acetylcholine (ACh in human brain is investigated through the two-enzyme/two-compartment (2E2C model where the presynaptic neuron is considered as compartment 1 while both the synaptic cleft and the postsynaptic neuron are considered as compartment 2 through suggesting three different kinetic mechanisms for the inhibition effect. It is found that the incorporation of ChAT inhibition by β-amyloid aggregates into the 2E2C model is able to yield dynamic solutions for concentrations of generated β-amyloid, ACh, choline, acetate, and pH in addition to the rates of ACh synthesis and ACh hydrolysis in compartments 1 and 2. It is observed that ChAT activity needs a high concentration of β-amyloid aggregates production rate. It is found that ChAT activity is reduced significantly when neurons are exposed to high levels of β-amyloid aggregates leading to reduction in levels of ACh which is one of the most significant physiological symptoms of AD. Furthermore, the system of ACh neurocycle is dominated by the oscillatory behavior when ChAT enzyme is completely inhibited by β-amyloid. It is observed that the direct inactivation of ChAT by β-amyloid aggregates may be a probable mechanism contributing to the development of AD.

  4. Amyloidpeptide-specific DARPins as a novel class of potential therapeutics for Alzheimer disease.

    Science.gov (United States)

    Hanenberg, Michael; McAfoose, Jordan; Kulic, Luka; Welt, Tobias; Wirth, Fabian; Parizek, Petra; Strobel, Lisa; Cattepoel, Susann; Späni, Claudia; Derungs, Rebecca; Maier, Marcel; Plückthun, Andreas; Nitsch, Roger M

    2014-09-26

    Passive immunization with anti-amyloidpeptide (Aβ) antibodies is effective in animal models of Alzheimer disease. With the advent of efficient in vitro selection technologies, the novel class of designed ankyrin repeat proteins (DARPins) presents an attractive alternative to the immunoglobulin scaffold. DARPins are small and highly stable proteins with a compact modular architecture ideal for high affinity protein-protein interactions. In this report, we describe the selection, binding profile, and epitope analysis of Aβ-specific DARPins. We further showed their ability to delay Aβ aggregation and prevent Aβ-mediated neurotoxicity in vitro. To demonstrate their therapeutic potential in vivo, mono- and trivalent Aβ-specific DARPins (D23 and 3×D23) were infused intracerebroventricularly into the brains of 11-month-old Tg2576 mice over 4 weeks. Both D23 and 3×D23 treatments were shown to result in improved cognitive performance and reduced soluble Aβ levels. These findings demonstrate the therapeutic potential of Aβ-specific DARPins for the treatment of Alzheimer disease. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Intravenous immunglobulin binds beta amyloid and modifies its aggregation, neurotoxicity and microglial phagocytosis in vitro.

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    Susann Cattepoel

    Full Text Available Intravenous Immunoglobulin (IVIG has been proposed as a potential therapeutic for Alzheimer's disease (AD and its efficacy is currently being tested in mild-to-moderate AD. Earlier studies reported the presence of anti-amyloid beta (Aβ antibodies in IVIG. These observations led to clinical studies investigating the potential role of IVIG as a therapeutic agent in AD. Also, IVIG is known to mediate beneficial effects in chronic inflammatory and autoimmune conditions by interfering with various pathological processes. Therefore, we investigated the effects of IVIG and purified polyclonal Aβ-specific antibodies (pAbs-Aβ on aggregation, toxicity and phagocytosis of Aβ in vitro, thus elucidating some of the potential mechanisms of action of IVIG in AD patients. We report that both IVIG and pAbs-Aβ specifically bound to Aβ and inhibited its aggregation in a dose-dependent manner as measured by Thioflavin T assay. Additionally, IVIG and the purified pAbs-Aβ inhibited Aβ-induced neurotoxicity in the SH-SY5Y human neuroblastoma cell line and prevented Aβ binding to rat primary cortical neurons. Interestingly, IVIG and pAbs-Aβ also increased the number of phagocytosing cells as well as the amount of phagocytosed fibrillar Aβ by BV-2 microglia. Phagocytosis of Aβ depended on receptor-mediated endocytosis and was accompanied by upregulation of CD11b expression. Importantly, we could also show that Privigen dose-dependently reversed Aβ-mediated LTP inhibition in mouse hippocampal slices. Therefore, our in vitro results suggest that IVIG may have an impact on different processes involved in AD pathogenesis, thereby promoting further understanding of the effects of IVIG observed in clinical studies.

  6. Dynamic behavior of small heat shock protein inhibition on amyloid fibrillization of a small peptide (SSTSAA) from RNase A

    International Nuclear Information System (INIS)

    Xi, Dong; Dong, Xiao; Deng, Wei; Lai, Luhua

    2011-01-01

    Highlights: ► Mechanism of small heat shock protein inhibition on fibril formation was studied. ► Peptide SSTSAA with modified ends was used for amyloid fibril formation. ► FRET signal was followed during the fibril formation. ► Mj HSP16.5 inhibits fibril formation when introduced in the lag phase. ► Mj HSP16.5 slows down fibril formation when introduced after the lag phase. -- Abstract: Small heat shock proteins, a class of molecular chaperones, are reported to inhibit amyloid fibril formation in vitro, while the mechanism of inhibition remains unknown. In the present study, we investigated the mechanism by which Mj HSP16.5 inhibits amyloid fibril formation of a small peptide (SSTSAA) from RNase A. A model peptide (dansyl-SSTSAA-W) was designed by introducing a pair of fluorescence resonance energy transfer (FRET) probes into the peptide, allowing for the monitoring of fibril formation by this experimental model. Mj HSP16.5 completely inhibited fibril formation of the model peptide at a molar ratio of 1:120. The dynamic process of fibril formation, revealed by FRET, circular dichroism, and electron microscopy, showed a lag phase of about 2 h followed by a fast growth period. The effect of Mj HSP16.5 on amyloid fibril formation was investigated by adding it into the incubation solution during different growth phases. Adding Mj HSP16.5 to the incubating peptide before or during the lag phase completely inhibited fibril formation. However, introducing Mj HSP16.5 after the lag phase only slowed down the fibril formation process by adhering to the already formed fibrils. These findings provide insight into the inhibitory roles of small heat shock proteins on amyloid fibril formation at the molecular level.

  7. Dynamic behavior of small heat shock protein inhibition on amyloid fibrillization of a small peptide (SSTSAA) from RNase A

    Energy Technology Data Exchange (ETDEWEB)

    Xi, Dong [BNLMS, State Key Laboratory of Structural Chemistry for Unstable and Stable Species, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871 (China); Center for Theoretical Biology, Peking University, Beijing 100871 (China); Dong, Xiao; Deng, Wei [BNLMS, State Key Laboratory of Structural Chemistry for Unstable and Stable Species, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871 (China); Lai, Luhua, E-mail: lhlai@pku.edu.cn [BNLMS, State Key Laboratory of Structural Chemistry for Unstable and Stable Species, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871 (China); Center for Theoretical Biology, Peking University, Beijing 100871 (China)

    2011-12-09

    Highlights: Black-Right-Pointing-Pointer Mechanism of small heat shock protein inhibition on fibril formation was studied. Black-Right-Pointing-Pointer Peptide SSTSAA with modified ends was used for amyloid fibril formation. Black-Right-Pointing-Pointer FRET signal was followed during the fibril formation. Black-Right-Pointing-Pointer Mj HSP16.5 inhibits fibril formation when introduced in the lag phase. Black-Right-Pointing-Pointer Mj HSP16.5 slows down fibril formation when introduced after the lag phase. -- Abstract: Small heat shock proteins, a class of molecular chaperones, are reported to inhibit amyloid fibril formation in vitro, while the mechanism of inhibition remains unknown. In the present study, we investigated the mechanism by which Mj HSP16.5 inhibits amyloid fibril formation of a small peptide (SSTSAA) from RNase A. A model peptide (dansyl-SSTSAA-W) was designed by introducing a pair of fluorescence resonance energy transfer (FRET) probes into the peptide, allowing for the monitoring of fibril formation by this experimental model. Mj HSP16.5 completely inhibited fibril formation of the model peptide at a molar ratio of 1:120. The dynamic process of fibril formation, revealed by FRET, circular dichroism, and electron microscopy, showed a lag phase of about 2 h followed by a fast growth period. The effect of Mj HSP16.5 on amyloid fibril formation was investigated by adding it into the incubation solution during different growth phases. Adding Mj HSP16.5 to the incubating peptide before or during the lag phase completely inhibited fibril formation. However, introducing Mj HSP16.5 after the lag phase only slowed down the fibril formation process by adhering to the already formed fibrils. These findings provide insight into the inhibitory roles of small heat shock proteins on amyloid fibril formation at the molecular level.

  8. Kinetic and structural characterization of amyloidpeptide hydrolysis by human angiotensin-1-converting enzyme.

    Science.gov (United States)

    Larmuth, Kate M; Masuyer, Geoffrey; Douglas, Ross G; Schwager, Sylva L; Acharya, K Ravi; Sturrock, Edward D

    2016-03-01

    Angiotensin-1-converting enzyme (ACE), a zinc metallopeptidase, consists of two homologous catalytic domains (N and C) with different substrate specificities. Here we report kinetic parameters of five different forms of human ACE with various amyloid beta (Aβ) substrates together with high resolution crystal structures of the N-domain in complex with Aβ fragments. For the physiological Aβ(1-16) peptide, a novel ACE cleavage site was found at His14-Gln15. Furthermore, Aβ(1-16) was preferentially cleaved by the individual N-domain; however, the presence of an inactive C-domain in full-length somatic ACE (sACE) greatly reduced enzyme activity and affected apparent selectivity. Two fluorogenic substrates, Aβ(4-10)Q and Aβ(4-10)Y, underwent endoproteolytic cleavage at the Asp7-Ser8 bond with all ACE constructs showing greater catalytic efficiency for Aβ(4-10)Y. Surprisingly, in contrast to Aβ(1-16) and Aβ(4-10)Q, sACE showed positive domain cooperativity and the double C-domain (CC-sACE) construct no cooperativity towards Aβ(4-10)Y. The structures of the Aβ peptide-ACE complexes revealed a common mode of peptide binding for both domains which principally targets the C-terminal P2' position to the S2' pocket and recognizes the main chain of the P1' peptide. It is likely that N-domain selectivity for the amyloid peptide is conferred through the N-domain specific S2' residue Thr358. Additionally, the N-domain can accommodate larger substrates through movement of the N-terminal helices, as suggested by the disorder of the hinge region in the crystal structures. Our findings are important for the design of domain selective inhibitors as the differences in domain selectivity are more pronounced with the truncated domains compared to the more physiological full-length forms. The atomic coordinates and structure factors for N-domain ACE with Aβ peptides 4-10 (5AM8), 10-16 (5AM9), 1-16 (5AMA), 35-42 (5AMB) and (4-10)Y (5AMC) complexes have been deposited in the

  9. Monomeric Amyloid Beta Peptide in Hexafluoroisopropanol Detected by Small Angle Neutron Scattering.

    Directory of Open Access Journals (Sweden)

    Bo Zhang-Haagen

    Full Text Available Small proteins like amyloid beta (Aβ monomers are related to neurodegenerative disorders by aggregation to insoluble fibrils. Small angle neutron scattering (SANS is a nondestructive method to observe the aggregation process in solution. We show that SANS is able to resolve monomers of small molecular weight like Aβ for aggregation studies. We examine Aβ monomers after prolonged storing in d-hexafluoroisopropanol (dHFIP by using SANS and dynamic light scattering (DLS. We determined the radius of gyration from SANS as 1.0±0.1 nm for Aβ1-40 and 1.6±0.1 nm for Aβ1-42 in agreement with 3D NMR structures in similar solvents suggesting a solvent surface layer with 5% increased density. After initial dissolution in dHFIP Aβ aggregates sediment with a major component of pure monomers showing a hydrodynamic radius of 1.8±0.3 nm for Aβ1-40 and 3.2±0.4 nm for Aβ1-42 including a surface layer of dHFIP solvent molecules.

  10. The interaction with gold suppresses fiber-like conformations of the amyloid β (16-22) peptide

    Science.gov (United States)

    Bellucci, Luca; Ardèvol, Albert; Parrinello, Michele; Lutz, Helmut; Lu, Hao; Weidner, Tobias; Corni, Stefano

    2016-04-01

    Inorganic surfaces and nanoparticles can accelerate or inhibit the fibrillation process of proteins and peptides, including the biomedically relevant amyloid β peptide. However, the microscopic mechanisms that determine such an effect are still poorly understood. By means of large-scale, state-of-the-art enhanced sampling molecular dynamics simulations, here we identify an interaction mechanism between the segments 16-22 of the amyloid β peptide, known to be fibrillogenic by itself, and the Au(111) surface in water that leads to the suppression of fiber-like conformations from the peptide conformational ensemble. Moreover, thanks to advanced simulation analysis techniques, we characterize the conformational selection vs. induced fit nature of the gold effect. Our results disclose an inhibition mechanism that is rooted in the details of the microscopic peptide-surface interaction rather than in general phenomena such as peptide sequestration from the solution.Inorganic surfaces and nanoparticles can accelerate or inhibit the fibrillation process of proteins and peptides, including the biomedically relevant amyloid β peptide. However, the microscopic mechanisms that determine such an effect are still poorly understood. By means of large-scale, state-of-the-art enhanced sampling molecular dynamics simulations, here we identify an interaction mechanism between the segments 16-22 of the amyloid β peptide, known to be fibrillogenic by itself, and the Au(111) surface in water that leads to the suppression of fiber-like conformations from the peptide conformational ensemble. Moreover, thanks to advanced simulation analysis techniques, we characterize the conformational selection vs. induced fit nature of the gold effect. Our results disclose an inhibition mechanism that is rooted in the details of the microscopic peptide-surface interaction rather than in general phenomena such as peptide sequestration from the solution. Electronic supplementary information (ESI

  11. Modeling of Platinum-Aryl Interaction with AmyloidPeptide.

    Science.gov (United States)

    Turner, Matthew; Platts, James A; Deeth, Robert J

    2016-03-08

    Ligand field molecular mechanics (LFMM), density functional theory (DFT), and semiempirical PM7 methods are used to study the binding of two Pt(II)-L systems to an N-terminal fragment of the amyloidpeptide, where L = 2,2-bipyridyl or 1,10-phenanthroline. Molecular dynamics simulations are used to explore the conformational freedom of the peptide using LFMM combined with AMBER molecular mechanics parameters. We establish a modeling protocol, allowing for identification and analysis of favorable platinum-binding modes and peptide conformations. Preferred binding modes are identified for each ligand investigated; metal coordination occurs via Nε in His residues for both ligands--His6ε-His13ε and His6ε-His14ε for the bipyridyl and phenanthroline ligands, respectively. The observed change in binding mode for the different ligands suggests that the binding mode of these platinum-based structures can be controlled by the choice of ligand. In the bipy systems, Boltzmann population at 310 K is dominated by a single conformer, while in the phenanthroline case, three conformations make significant contributions to the ensemble. The relative stability of these conformations is due to the inherent stability of binding platinum via Nε in addition to subtle H-bonding effects.

  12. Bloodstream Amyloid-beta (1-40) Peptide, Cognition, and Outcomes in Heart Failure.

    Science.gov (United States)

    Bayes-Genis, Antoni; Barallat, Jaume; de Antonio, Marta; Domingo, Mar; Zamora, Elisabet; Vila, Joan; Subirana, Isaac; Gastelurrutia, Paloma; Pastor, M Cruz; Januzzi, James L; Lupón, Josep

    2017-11-01

    In the brain, amyloid-beta generation participates in the pathophysiology of cognitive disorders; in the bloodstream, the role of amyloid-beta is uncertain but may be linked to sterile inflammation and senescence. We explored the relationship between blood levels of amyloid-beta 1-40 peptide (Aβ40), cognition, and mortality (all-cause, cardiovascular, and heart failure [HF]-related) in ambulatory patients with HF. Bloodstream Aβ40 was measured in 939 consecutive patients with HF. Cognition was evaluated with the Pfeiffer questionnaire (adjusted for educational level) at baseline and during follow-up. Multivariate Cox regression analyses and measurements of performance (discrimination, calibration, and reclassification) were used, with competing risk for specific causes of death. Over 5.1 ± 2.9 years, 471 patients died (all-cause): 250 from cardiovascular causes and 131 HF-related. The median Aβ40 concentration was 519.1 pg/mL [Q1-Q3: 361.8-749.9 pg/mL]. The Aβ40 concentration correlated with age, body mass index, renal dysfunction, and New York Heart Association functional class (all P < .001). There were no differences in Aβ40 in patients with and without cognitive impairment at baseline (P = .97) or during follow-up (P = .20). In multivariable analysis, including relevant clinical predictors and N-terminal pro-B-type natriuretic peptide, Aβ40 remained significantly associated with all-cause death (HR, 1.22; 95%CI, 1.10-1.35; P < .001) and cardiovascular death (HR, 1.18; 95%CI, 1.03-1.36; P = .02), but not with HF-related death (HR, 1.13; 95%CI, 0.93-1.37; P = .22). Circulating Aβ40 improved calibration and patient reclassification. Blood levels of Aβ40 are not associated with cognitive decline in HF. Circulating Aβ40 was predictive of mortality and may indicate systemic aging. Copyright © 2017 Sociedad Española de Cardiología. Published by Elsevier España, S.L.U. All rights reserved.

  13. 2A4 binds soluble and insoluble light chain aggregates from AL amyloidosis patients and promotes clearance of amyloid deposits by phagocytosis †.

    Science.gov (United States)

    Renz, Mark; Torres, Ronald; Dolan, Philip J; Tam, Stephen J; Tapia, Jose R; Li, Lauri; Salmans, Joshua R; Barbour, Robin M; Shughrue, Paul J; Nijjar, Tarlochan; Schenk, Dale; Kinney, Gene G; Zago, Wagner

    2016-09-01

    Amyloid light chain (AL) amyloidosis is characterized by misfolded light chain (LC) (amyloid) deposition in various peripheral organs, leading to progressive dysfunction and death. There are no regulatory agency-approved treatments for AL amyloidosis, and none of the available standard of care approaches directly targets the LC protein that constitutes the amyloid. NEOD001, currently in late-stage clinical trials, is a conformation-specific, anti-LC antibody designed to specifically target misfolded LC aggregates and promote phagocytic clearance of AL amyloid deposits. The present study demonstrated that the monoclonal antibody 2A4, the murine form of NEOD001, binds to patient-derived soluble and insoluble LC aggregates and induces phagocytic clearance of AL amyloid in vitro. 2A4 specifically labeled all 21 fresh-frozen organ samples studied, which were derived from 10 patients representing both κ and λ LC amyloidosis subtypes. 2A4 immunoreactivity largely overlapped with thioflavin T-positive labeling, and 2A4 bound both soluble and insoluble LC aggregates extracted from patient tissue. Finally, 2A4 induced macrophage engagement and phagocytic clearance of AL amyloid deposits in vitro. These findings provide further evidence that 2A4/NEOD001 can effectively clear and remove human AL-amyloid from tissue and further support the rationale for the evaluation of NEOD001 in patients with AL amyloidosis.

  14. 2A4 binds soluble and insoluble light chain aggregates from AL amyloidosis patients and promotes clearance of amyloid deposits by phagocytosis †

    Science.gov (United States)

    Renz, Mark; Torres, Ronald; Dolan, Philip J.; Tam, Stephen J.; Tapia, Jose R.; Li, Lauri; Salmans, Joshua R.; Barbour, Robin M.; Shughrue, Paul J.; Nijjar, Tarlochan; Schenk, Dale; Kinney, Gene G.; Zago, Wagner

    2016-01-01

    Abstract Amyloid light chain (AL) amyloidosis is characterized by misfolded light chain (LC) (amyloid) deposition in various peripheral organs, leading to progressive dysfunction and death. There are no regulatory agency–approved treatments for AL amyloidosis, and none of the available standard of care approaches directly targets the LC protein that constitutes the amyloid. NEOD001, currently in late-stage clinical trials, is a conformation-specific, anti-LC antibody designed to specifically target misfolded LC aggregates and promote phagocytic clearance of AL amyloid deposits. The present study demonstrated that the monoclonal antibody 2A4, the murine form of NEOD001, binds to patient-derived soluble and insoluble LC aggregates and induces phagocytic clearance of AL amyloid in vitro. 2A4 specifically labeled all 21 fresh-frozen organ samples studied, which were derived from 10 patients representing both κ and λ LC amyloidosis subtypes. 2A4 immunoreactivity largely overlapped with thioflavin T–positive labeling, and 2A4 bound both soluble and insoluble LC aggregates extracted from patient tissue. Finally, 2A4 induced macrophage engagement and phagocytic clearance of AL amyloid deposits in vitro. These findings provide further evidence that 2A4/NEOD001 can effectively clear and remove human AL-amyloid from tissue and further support the rationale for the evaluation of NEOD001 in patients with AL amyloidosis. PMID:27494229

  15. Inhibition of amyloid β aggregation and protective effect on SH-SY5Y cells by triterpenoid saponins from the cactus Polaskia chichipe.

    Science.gov (United States)

    Fujihara, Koji; Koike, Shin; Ogasawara, Yuki; Takahashi, Kunio; Koyama, Kiyotaka; Kinoshita, Kaoru

    2017-07-01

    Alzheimer's disease (AD) destroys brain function, especially in the hippocampus, and is a social problem worldwide. A major pathogenesis of AD is related to the accumulation of amyloid beta (Aβ) peptides, resulting in neuronal cell death in the brain. Here, we isolated four saponins (1-4) and elucidated their structures from 1D and 2D NMR and HRFABMS spectral data. The structures of 1 and 2 were determined as new saponins which have cochalic acid as the aglycon, and 3 was determined as a new saponin with oleanolic acid as the aglycon. Compound 4 was confirmed as the known saponin chikusetsusaponin V (=ginsenoside R 0 ). Isolated saponins (1-4) and six previously reported saponins (5-10) were tested for their inhibitory effects of Aβ aggregation and their protective effects on SH-SY5Y cells against Aβ-associated toxicity. As the results, compounds 3 and 4 showed inhibitory effect of Aβ aggregation and compounds 5-8 exerted the protective effects on SH-SY5Y cells against Aβ-associated toxicity. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Preparation of Amyloid Fibrils Seeded from Brain and Meninges.

    Science.gov (United States)

    Scherpelz, Kathryn P; Lu, Jun-Xia; Tycko, Robert; Meredith, Stephen C

    2016-01-01

    Seeding of amyloid fibrils into fresh solutions of the same peptide or protein in disaggregated form leads to the formation of replicate fibrils, with close structural similarity or identity to the original fibrillar seeds. Here we describe procedures for isolating fibrils composed mainly of β-amyloid (Aβ) from human brain and from leptomeninges, a source of cerebral blood vessels, for investigating Alzheimer's disease and cerebral amyloid angiopathy. We also describe methods for seeding isotopically labeled, disaggregated Aβ peptide solutions for study using solid-state NMR and other techniques. These methods should be applicable to other types of amyloid fibrils, to Aβ fibrils from mice or other species, tissues other than brain, and to some non-fibrillar aggregates. These procedures allow for the examination of authentic amyloid fibrils and other protein aggregates from biological tissues without the need for labeling the tissue.

  17. Electrochemical quantification of the Alzheimer’s disease amyloid-β (1–40 using amyloid-β fibrillization promoting peptide

    Directory of Open Access Journals (Sweden)

    Satoshi Fujii

    2015-12-01

    Full Text Available Amyloidpeptide (Aβ is believed to be an important biomarker for the early diagnosis of Alzheimer’s disease. Therefore, practical and reliable methods to assay Aβ levels have been coveted. In this study, a rapid, sensitive, and selective electrochemical method for Aβ(1–40 detection using Cu2+ redox cycling on peptide-modified gold electrodes was developed. A 19-residue peptide that can promote Aβ fibrillization (AFPP was immobilized onto a gold electrode. After incubating an Aβ solution with the modified electrode for 1 h, a Cu2+ solution was added and cyclic voltammetry measurements were conducted. The voltammetric response was found to be proportional to the Aβ(1–40 concentration in the 0.1–5 μM range, and a detection limit of 18 nM was achieved. Washing with sodium hydroxide and ethylenediaminetetraacetate solutions easily reinitialized the modified electrode. Results obtained using the reinitialized electrode showed good reproducibility. Furthermore, when another amyloidogenic and Cu2+-binding protein amylin was used as the target, no voltammetric response was observed. These results indicate that the AFPP-modified electrode provides a promising, label-free, sensitive, selective, cost-effective, and easy method for the quantification of Aβ. Keywords: Amyloid-β, Alzheimer’s disease (AD, Fibrillization, Electrochemical detection, Nanobiochip, Cu redox

  18. How cholesterol constrains glycolipid conformation for optimal recognition of Alzheimer's beta amyloid peptide (Abeta1-40).

    Science.gov (United States)

    Yahi, Nouara; Aulas, Anaïs; Fantini, Jacques

    2010-02-05

    Membrane lipids play a pivotal role in the pathogenesis of Alzheimer's disease, which is associated with conformational changes, oligomerization and/or aggregation of Alzheimer's beta-amyloid (Abeta) peptides. Yet conflicting data have been reported on the respective effect of cholesterol and glycosphingolipids (GSLs) on the supramolecular assembly of Abeta peptides. The aim of the present study was to unravel the molecular mechanisms by which cholesterol modulates the interaction between Abeta(1-40) and chemically defined GSLs (GalCer, LacCer, GM1, GM3). Using the Langmuir monolayer technique, we show that Abeta(1-40) selectively binds to GSLs containing a 2-OH group in the acyl chain of the ceramide backbone (HFA-GSLs). In contrast, Abeta(1-40) did not interact with GSLs containing a nonhydroxylated fatty acid (NFA-GSLs). Cholesterol inhibited the interaction of Abeta(1-40) with HFA-GSLs, through dilution of the GSL in the monolayer, but rendered the initially inactive NFA-GSLs competent for Abeta(1-40) binding. Both crystallographic data and molecular dynamics simulations suggested that the active conformation of HFA-GSL involves a H-bond network that restricts the orientation of the sugar group of GSLs in a parallel orientation with respect to the membrane. This particular conformation is stabilized by the 2-OH group of the GSL. Correspondingly, the interaction of Abeta(1-40) with HFA-GSLs is strongly inhibited by NaF, an efficient competitor of H-bond formation. For NFA-GSLs, this is the OH group of cholesterol that constrains the glycolipid to adopt the active L-shape conformation compatible with sugar-aromatic CH-pi stacking interactions involving residue Y10 of Abeta(1-40). We conclude that cholesterol can either inhibit or facilitate membrane-Abeta interactions through fine tuning of glycosphingolipid conformation. These data shed some light on the complex molecular interplay between cell surface GSLs, cholesterol and Abeta peptides, and on the influence

  19. How cholesterol constrains glycolipid conformation for optimal recognition of Alzheimer's beta amyloid peptide (Abeta1-40.

    Directory of Open Access Journals (Sweden)

    Nouara Yahi

    Full Text Available Membrane lipids play a pivotal role in the pathogenesis of Alzheimer's disease, which is associated with conformational changes, oligomerization and/or aggregation of Alzheimer's beta-amyloid (Abeta peptides. Yet conflicting data have been reported on the respective effect of cholesterol and glycosphingolipids (GSLs on the supramolecular assembly of Abeta peptides. The aim of the present study was to unravel the molecular mechanisms by which cholesterol modulates the interaction between Abeta(1-40 and chemically defined GSLs (GalCer, LacCer, GM1, GM3. Using the Langmuir monolayer technique, we show that Abeta(1-40 selectively binds to GSLs containing a 2-OH group in the acyl chain of the ceramide backbone (HFA-GSLs. In contrast, Abeta(1-40 did not interact with GSLs containing a nonhydroxylated fatty acid (NFA-GSLs. Cholesterol inhibited the interaction of Abeta(1-40 with HFA-GSLs, through dilution of the GSL in the monolayer, but rendered the initially inactive NFA-GSLs competent for Abeta(1-40 binding. Both crystallographic data and molecular dynamics simulations suggested that the active conformation of HFA-GSL involves a H-bond network that restricts the orientation of the sugar group of GSLs in a parallel orientation with respect to the membrane. This particular conformation is stabilized by the 2-OH group of the GSL. Correspondingly, the interaction of Abeta(1-40 with HFA-GSLs is strongly inhibited by NaF, an efficient competitor of H-bond formation. For NFA-GSLs, this is the OH group of cholesterol that constrains the glycolipid to adopt the active L-shape conformation compatible with sugar-aromatic CH-pi stacking interactions involving residue Y10 of Abeta(1-40. We conclude that cholesterol can either inhibit or facilitate membrane-Abeta interactions through fine tuning of glycosphingolipid conformation. These data shed some light on the complex molecular interplay between cell surface GSLs, cholesterol and Abeta peptides, and on the

  20. Towards Alzheimer's beta-amyloid vaccination.

    Science.gov (United States)

    Frenkel, D; Solomon, B

    2001-01-01

    Beta-amyloid pathology, the main hallmark of Alzheimer's disease (AD), has been linked to its conformational status and aggregation. We recently showed that site-directed monoclonal antibodies (mAbs) towards the N-terminal region of the human beta-amyloid peptide bind to preformed beta-amyloid fibrils (Abeta), leading to disaggregation and inhibition of their neurotoxic effect. Here we report the development of a novel immunization procedure to raise effective anti-aggregating amyloid beta-protein (AbetaP) antibodies, using as antigen filamentous phages displaying the only EFRH peptide found to be the epitope of these antibodies. Due to the high antigenicity of the phage no adjuvant is required to obtain high affinity anti-aggregating IgG antibodies in animals model, that exhibit identity to human AbetaP. Such antibodies are able to sequester peripheral AbetaP, thus avoiding passage through the blood brain barrier (BBB) and, as recently shown in a transgenic mouse model, to cross the BBB and dissolve already formed beta-amyloid plaques. To our knowledge, this is the first attempt to use as a vaccine a self-anti-aggregating epitope displayed on a phage, and this may pave the way to treat abnormal accumulation-peptide diseases, such as Alzheimer's disease or other amyloidogenic diseases. Copyright 2001 The International Association for Biologicals.

  1. Peptides of presenilin-1 bind the amyloid precursor protein ectodomain and offer a novel and specific therapeutic approach to reduce ß-amyloid in Alzheimer's disease.

    Science.gov (United States)

    Dewji, Nazneen N; Singer, S Jonathan; Masliah, Eliezer; Rockenstein, Edward; Kim, Mihyun; Harber, Martha; Horwood, Taylor

    2015-01-01

    β-Amyloid (Aβ) accumulation in the brain is widely accepted to be critical to the development of Alzheimer's disease (AD). Current efforts at reducing toxic Aβ40 or 42 have largely focused on modulating γ-secretase activity to produce shorter, less toxic Aβ, while attempting to spare other secretase functions. In this paper we provide data that offer the potential for a new approach for the treatment of AD. The method is based on our previous findings that the production of Aβ from the interaction between the β-amyloid precursor protein (APP) and Presenilin (PS), as part of the γ-secretase complex, in cell culture is largely inhibited if the entire water-soluble NH2-terminal domain of PS is first added to the culture. Here we demonstrate that two small, non-overlapping water-soluble peptides from the PS-1 NH2-terminal domain can substantially and specifically inhibit the production of total Aβ as well as Aβ40 and 42 in vitro and in vivo in the brains of APP transgenic mice. These results suggest that the inhibitory activity of the entire amino terminal domain of PS-1 on Aβ production is largely focused in a few smaller sequences within that domain. Using biolayer interferometry and confocal microscopy we provide evidence that peptides effective in reducing Aβ give a strong, specific and biologically relevant binding with the purified ectodomain of APP 695. Finally, we demonstrate that the reduction of Aβ by the peptides does not affect the catalytic activities of β- or γ-secretase, or the level of APP. P4 and P8 are the first reported protein site-specific small peptides to reduce Aβ production in model systems of AD. These peptides and their derivatives offer new potential drug candidates for the treatment of AD.

  2. Peptides of presenilin-1 bind the amyloid precursor protein ectodomain and offer a novel and specific therapeutic approach to reduce ß-amyloid in Alzheimer's disease.

    Directory of Open Access Journals (Sweden)

    Nazneen N Dewji

    Full Text Available β-Amyloid (Aβ accumulation in the brain is widely accepted to be critical to the development of Alzheimer's disease (AD. Current efforts at reducing toxic Aβ40 or 42 have largely focused on modulating γ-secretase activity to produce shorter, less toxic Aβ, while attempting to spare other secretase functions. In this paper we provide data that offer the potential for a new approach for the treatment of AD. The method is based on our previous findings that the production of Aβ from the interaction between the β-amyloid precursor protein (APP and Presenilin (PS, as part of the γ-secretase complex, in cell culture is largely inhibited if the entire water-soluble NH2-terminal domain of PS is first added to the culture. Here we demonstrate that two small, non-overlapping water-soluble peptides from the PS-1 NH2-terminal domain can substantially and specifically inhibit the production of total Aβ as well as Aβ40 and 42 in vitro and in vivo in the brains of APP transgenic mice. These results suggest that the inhibitory activity of the entire amino terminal domain of PS-1 on Aβ production is largely focused in a few smaller sequences within that domain. Using biolayer interferometry and confocal microscopy we provide evidence that peptides effective in reducing Aβ give a strong, specific and biologically relevant binding with the purified ectodomain of APP 695. Finally, we demonstrate that the reduction of Aβ by the peptides does not affect the catalytic activities of β- or γ-secretase, or the level of APP. P4 and P8 are the first reported protein site-specific small peptides to reduce Aβ production in model systems of AD. These peptides and their derivatives offer new potential drug candidates for the treatment of AD.

  3. A microliter-scale high-throughput screening system with quantum-dot nanoprobes for amyloidaggregation inhibitors.

    Directory of Open Access Journals (Sweden)

    Yukako Ishigaki

    Full Text Available The aggregation of amyloid β protein (Aβ is a key step in the pathogenesis of Alzheimer's disease (AD, and therefore inhibitory substances for Aβ aggregation may have preventive and/or therapeutic potential for AD. Here we report a novel microliter-scale high-throughput screening system for Aβ aggregation inhibitors based on fluorescence microscopy-imaging technology with quantum-dot Nanoprobes. This screening system could be analyzed with a 5-µl sample volume when a 1536-well plate was used, and the inhibitory activity could be estimated as half-maximal effective concentrations (EC50. We attempted to comprehensively screen Aβ aggregation inhibitors from 52 spices using this system to assess whether this novel screening system is actually useful for screening inhibitors. Screening results indicate that approximately 90% of the ethanolic extracts from the spices showed inhibitory activity for Aβ aggregation. Interestingly, spices belonging to the Lamiaceae, the mint family, showed significantly higher activity than the average of tested spices. Furthermore, we tried to isolate the main inhibitory compound from Saturejahortensis, summer savory, a member of the Lamiaceae, using this system, and revealed that the main active compound was rosmarinic acid. These results demonstrate that this novel microliter-scale high-throughput screening system could be applied to the actual screening of Aβ aggregation inhibitors. Since this system can analyze at a microscopic scale, it is likely that further minimization of the system would easily be possible such as protein microarray technology.

  4. Amyloidogenic peptides for design of inhibitors of peptide aggregation and as templates for new bionanomaterials

    NARCIS (Netherlands)

    Elgersma, R.C.

    2008-01-01

    Misfolding of proteins from their soluble form into highly insoluble fibrillar deposits can lead to (non-)neurodegenerative disorders or systemic amyloidosis. This class of diseases (for which no therapy is available yet) is called amyloid diseases. Amyloid refers to the extracellular proteinaceous

  5. Chronic cladribine administration increases amyloid beta peptide generation and plaque burden in mice.

    Directory of Open Access Journals (Sweden)

    Crystal D Hayes

    Full Text Available The clinical uses of 2-chloro-2'-deoxyadenosine (2-CDA or cladribine which was initially prescribed to patients with hematological and lymphoid cancers is now extended to treat patients with multiple sclerosis (MS. Previous data has shown that 2-CDA has high affinity to the brain and readily passes through the blood brain barrier reaching CSF concentrations 25% of that found in plasma. However, whether long-term administration of 2-CDA can lead to any adverse effects in patients or animal models is not yet clearly known.Here we show that exposure of 2-CDA to CHO cells stably expressing wild-type APP751 increased generation and secretion of amyloid β peptide (Aβ in to the conditioned medium. Interestingly, increased Aβ levels were noticed even at non-toxic concentrations of 2-CDA. Remarkably, chronic treatment of APdE9 mice, a model of Alzheimer's disease with 2-CDA for 60 days increased amyloid plaque burden by more than 1-fold. Increased Aβ generation appears to result from increased turnover of APP as revealed by cycloheximide-chase experiments. Additionally, surface labeling of APP with biotin and immunoprecipitation of surface labeled proteins with anti-biotin antibody also indicated increased APP at the cell surface in 2-CDA treated cells compared to controls. Increased turnover of APP by 2-CDA in turn might be a consequence of decreased protein levels of PIN 1, which is known to regulate cis-trans isomerization and phosphorylation of APP. Most importantly, like many other oncology drugs, 2-CDA administration led to significant delay in acquiring a reward-based learning task in a T maze paradigm.Taken together, these data provide compelling evidence for the first time that chronic 2-CDA administration can increase amyloidogenic processing of APP leading to robustly increased plaque burden which may be responsible for the observed deficits in learning skills. Thus chronic treatment of mice with 2-CDA can have deleterious effects in vivo.

  6. Mechanisms of plastein formation, and prospective food and nutraceutical applications of the peptide aggregates

    Directory of Open Access Journals (Sweden)

    Min Gong

    2015-03-01

    Full Text Available Plastein is a protease-induced peptide aggregate with prospective application in enhancing the nutritional quality of proteins and debittering protein hydrolysates. These properties are yet to be applied in product development possibly due to economic considerations (production cost vs. product yields. This paper reviews currently proposed mechanisms of plastein formation including condensation, transpeptidation and physical interaction of aggregating peptides. Emerging findings indicate that plastein possesses bioactivities, thereby expanding its prospective application. The role of proteases in inducing peptide interaction in plastein remains unclear. Understanding the protease function will facilitate the development of efficient proteases and scalable industrial processes for plastein production.

  7. Hypocretin and brain β-amyloid peptide interactions in cognitive disorders and narcolepsy

    Directory of Open Access Journals (Sweden)

    Yves A Dauvilliers

    2014-06-01

    Full Text Available Objective: To examine relationships between cerebrospinal fluid (CSF Alzheimer’ disease (AD biomarkers and hypocretin-1 levels in patients with cognitive abnormalities and hypocretin-deficient narcolepsy-cataplexy (NC, estimate diagnostic accuracy, and determine correlations with sleep disturbances. Background: Sleep disturbances are frequent in AD. Interactions between brain β-amyloid (Aβ aggregation and a wake-related neurotransmitter hypocretin have been reported in a mouse model of AD. Methods: Ninety-one cognitive patients (37 AD, 16 mild cognitive impairment – MCI that converts to AD, 38 other dementias and 15 elderly patients with NC were recruited. Patients were diagnosed blind to CSF results. CSF A42, total tau, ptau181, and hypocretin-1 were measured. Sleep disturbances were assessed with questionnaires in 32 cognitive patients. Results: Lower CSF Aβ42 but higher tau and P-tau levels were found in AD and MCI compared to other dementias. CSF hypocretin-1 levels were higher in patients with MCI due to AD compared to other dementias, with a similar tendency for patients with advanced AD. CSF hypocretin-1 was significantly and independently associated with AD/MCI due to AD, with an OR of 2.70 after full adjustment, exceeding that for Aβ42. Aβ42 correlated positively with hypocretin-1 levels in advanced stage AD. No association was found between sleep disturbances and CSF biomarkers. No patients with NC achieved pathological cutoffs for Aβ42, with respectively one and four patients with NC above tau and P-tau cutoffs and no correlations between hypocretin-1 and other biomarkers. Conclusions: Our results suggest a pathophysiological relationship between Aβ42 and hypocretin-1 in the AD process, with higher CSF hypocretin-1 levels in early disease stages. Further longitudinal studies are needed to validate these biomarker interactions and to determine the cause-effect relationship and the role of wake/sleep behavior in amyloid

  8. Neurofibrillary tangles and the deposition of a beta amyloid peptide with a novel N-terminal epitope in the brains of wild Tsushima leopard cats.

    Directory of Open Access Journals (Sweden)

    James K Chambers

    Full Text Available Beta amyloid (Aβ deposits are seen in aged individuals in many of the mammalian species that possess the same Aβ amino acid sequence as humans. Conversely, neurofibrillary tangles (NFT, the other hallmark lesion of Alzheimer's disease (AD, are extremely rare in these animals. We detected Aβ deposits in the brains of Tsushima leopard cats (Prionailurus bengalensis euptilurus that live exclusively on Tsushima Island, Japan. Aβ42 was deposited in a granular pattern in the neuropil of the pyramidal cell layer, but did not form argyrophilic senile plaques. These Aβ deposits were not immunolabeled with antibodies to the N-terminal of human Aβ. Sequence analysis of the amyloid precursor protein revealed an amino acid substitution at the 7th residue of the Aβ peptide. In a comparison with other mammalian animals that do develop argyrophilic senile plaques, we concluded that the alternative Aβ amino acid sequence displayed by leopard cats is likely to be related to its distinctive deposition pattern. Interestingly, most of the animals with these Aβ deposits also developed NFTs. The distributions of hyperphosphorylated tau-positive cells and the two major isoforms of aggregated tau proteins were quite similar to those seen in Alzheimer's disease. In addition, the unphosphorylated form of GSK-3β colocalized with hyperphosphorylated tau within the affected neurons. In conclusion, this animal species develops AD-type NFTs without argyrophilic senile plaques.

  9. Genetic Mechanisms of Coffee Extract Protection in a Caenorhabditis elegans Model of β-Amyloid Peptide Toxicity

    OpenAIRE

    Dostal, Vishantie; Roberts, Christine M.; Link, Christopher D.

    2010-01-01

    Epidemiological studies have reported that coffee and/or caffeine consumption may reduce Alzheimer's disease (AD) risk. We found that coffee extracts can similarly protect against β-amyloid peptide (Aβ) toxicity in a transgenic Caenorhabditis elegans Alzheimer's disease model. The primary protective component(s) in this model is not caffeine, although caffeine by itself can show moderate protection. Coffee exposure did not decrease Aβ transgene expression and did not need to be present during...

  10. Novel hybrids of oxoisoaporphine-tryptamine as acetylcholinesterase-induced β-amyloid aggregation inhibitors with improved antioxidant properties

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    Zhao Hai-Tao

    2015-01-01

    Full Text Available A series of dual binding site acetylcholinesterase (AChE inhibitors have been designed, synthesized, and tested for their antioxidant ability and inhibitory potency on AChE and AChE-induced b-amyloid (Ab aggregation. The new hybrids consist of a unit of 1-azabenzanthrone and a tryptamine or its derivative, connected through a a,w - alkyldiamide bridge. These hybrids exhibit moderate AChE inhibitory activity with IC50 values in the micromolar range and significant in vitro inhibitory activity toward the AChE-induced Ab aggregation. Moreover, six out of the nine hybrids of this series exhibit a higher oxygen radical absorbance capacity than trolox, which makes them promising anti-Alzheimer drug candidates.

  11. Identification of Small Peptides in Human Cerebrospinal Fluid upon Amyloid-β Degradation.

    Science.gov (United States)

    Mizuta, Naoki; Yanagida, Kanta; Kodama, Takashi; Tomonaga, Takeshi; Takami, Mako; Oyama, Hiroshi; Kudo, Takashi; Ikeda, Manabu; Takeda, Masatoshi; Tagami, Shinji; Okochi, Masayasu

    2017-01-01

    Amyloid-β (Aβ) degradation in brains of Alzheimer disease patients is a crucial focus for the clarification of disease pathogenesis. Nevertheless, the mechanisms underlying Aβ degradation in the human brain remain unclear. This study aimed to quantify the levels of small C-terminal Aβ fragments generated upon Aβ degradation in human cerebrospinal fluid (CSF). A fraction containing small peptides was isolated and purified from human CSF by high-pressure liquid chromatography. Degradation products of Aβ C termini were identified and measured by liquid chromatography-tandem mass spectrometry. The C-terminal fragments of Aβ in the conditioned medium of cultured cells transfected with the Swedish variant of βAPP (sw βAPP) were analyzed. These fragments in brains of PS1 I213T knock-in transgenic mice, overexpressing sw βAPP, were also analyzed. The peptide fragments GGVV and GVV, produced by the cleavage of Aβ40, were identified in human CSF as well as in the brains of the transgenic mice and in the conditioned medium of the cultured cells. Relative to Aβ40 levels, GGVV and GVV levels were 7.6 ± 0.81 and 1.5 ± 0.18%, respectively, in human CSF. Levels of the GGVV fragment did not increase by the introduction of genes encoding neprilysin and insulin-degrading enzyme to the cultured cells. Our results indicate that a substantial amount of Aβ40 in human brains is degraded via a neprilysin- or insulin-degrading enzyme-independent pathway. © 2017 S. Karger AG, Basel.

  12. The Alzheimer's Disease-Associated Amyloid β-Protein Is an Antimicrobial Peptide

    Science.gov (United States)

    Soscia, Stephanie J.; Kirby, James E.; Washicosky, Kevin J.; Tucker, Stephanie M.; Ingelsson, Martin; Hyman, Bradley; Burton, Mark A.; Goldstein, Lee E.; Duong, Scott; Tanzi, Rudolph E.; Moir, Robert D.

    2010-01-01

    Background The amyloid β-protein (Aβ) is believed to be the key mediator of Alzheimer's disease (AD) pathology. Aβ is most often characterized as an incidental catabolic byproduct that lacks a normal physiological role. However, Aβ has been shown to be a specific ligand for a number of different receptors and other molecules, transported by complex trafficking pathways, modulated in response to a variety of environmental stressors, and able to induce pro-inflammatory activities. Methodology/Principal Findings Here, we provide data supporting an in vivo function for Aβ as an antimicrobial peptide (AMP). Experiments used established in vitro assays to compare antimicrobial activities of Aβ and LL-37, an archetypical human AMP. Findings reveal that Aβ exerts antimicrobial activity against eight common and clinically relevant microorganisms with a potency equivalent to, and in some cases greater than, LL-37. Furthermore, we show that AD whole brain homogenates have significantly higher antimicrobial activity than aged matched non-AD samples and that AMP action correlates with tissue Aβ levels. Consistent with Aβ-mediated activity, the increased antimicrobial action was ablated by immunodepletion of AD brain homogenates with anti-Aβ antibodies. Conclusions/Significance Our findings suggest Aβ is a hitherto unrecognized AMP that may normally function in the innate immune system. This finding stands in stark contrast to current models of Aβ-mediated pathology and has important implications for ongoing and future AD treatment strategies. PMID:20209079

  13. The Alzheimer's disease-associated amyloid beta-protein is an antimicrobial peptide.

    Directory of Open Access Journals (Sweden)

    Stephanie J Soscia

    2010-03-01

    Full Text Available The amyloid beta-protein (Abeta is believed to be the key mediator of Alzheimer's disease (AD pathology. Abeta is most often characterized as an incidental catabolic byproduct that lacks a normal physiological role. However, Abeta has been shown to be a specific ligand for a number of different receptors and other molecules, transported by complex trafficking pathways, modulated in response to a variety of environmental stressors, and able to induce pro-inflammatory activities.Here, we provide data supporting an in vivo function for Abeta as an antimicrobial peptide (AMP. Experiments used established in vitro assays to compare antimicrobial activities of Abeta and LL-37, an archetypical human AMP. Findings reveal that Abeta exerts antimicrobial activity against eight common and clinically relevant microorganisms with a potency equivalent to, and in some cases greater than, LL-37. Furthermore, we show that AD whole brain homogenates have significantly higher antimicrobial activity than aged matched non-AD samples and that AMP action correlates with tissue Abeta levels. Consistent with Abeta-mediated activity, the increased antimicrobial action was ablated by immunodepletion of AD brain homogenates with anti-Abeta antibodies.Our findings suggest Abeta is a hitherto unrecognized AMP that may normally function in the innate immune system. This finding stands in stark contrast to current models of Abeta-mediated pathology and has important implications for ongoing and future AD treatment strategies.

  14. The Physiological Roles of AmyloidPeptide Hint at New Ways to Treat Alzheimer's Disease

    Directory of Open Access Journals (Sweden)

    Holly M. Brothers

    2018-04-01

    Full Text Available Amyloid-ß (Aß is best known as the misfolded peptide that is involved in the pathogenesis of Alzheimer's disease (AD, and it is currently the primary therapeutic target in attempts to arrest the course of this disease. This notoriety has overshadowed evidence that Aß serves several important physiological functions. Aß is present throughout the lifespan, it has been found in all vertebrates examined thus far, and its molecular sequence shows a high degree of conservation. These features are typical of a factor that contributes significantly to biological fitness, and this suggestion has been supported by evidence of functions that are beneficial for the brain. The putative roles of Aß include protecting the body from infections, repairing leaks in the blood-brain barrier, promoting recovery from injury, and regulating synaptic function. Evidence for these beneficial roles comes from in vitro and in vivo studies, which have shown that the cellular production of Aß rapidly increases in response to a physiological challenge and often diminishes upon recovery. These roles are further supported by the adverse outcomes of clinical trials that have attempted to deplete Aß in order to treat AD. We suggest that anti-Aß therapies will produce fewer adverse effects if the known triggers of Aß deposition (e.g., pathogens, hypertension, and diabetes are addressed first.

  15. Viscoelastic response of neural cells governed by the deposition of amyloidpeptides (Aβ)

    Science.gov (United States)

    Gong, Ze; You, Ran; Chang, Raymond Chuen-Chung; Lin, Yuan

    2016-06-01

    Because of its intimate relation with Alzheimer's disease (AD), the question of how amyloidpeptide (Aβ) deposition alters the membrane and cytoskeltal structure of neural cells and eventually their mechanical response has received great attention. In this study, the viscoelastic properties of primary neurons subjected to various Aβ treatments were systematically characterized using atomic force microrheology. It was found that both the storage ( G ') and loss ( G ″) moduli of neural cells are rate-dependent and grow by orders of magnitude as the driving frequency ω varies from 1 to 100 Hz. However, a much stronger frequency dependence was observed in the loss moduli (with a scaling exponent of ˜0.96) than that in G ' ( ˜ ω 0.2 ). Furthermore, both cell moduli increase gradually within the first 6 h of Aβ treatment before steady-state values are reached, with a higher dosage of Aβ leading to larger changes in cell properties. Interestingly, we showed that the measured neuron response can be well-explained by a power law structural damping model. Findings here establish a quantitative link between Aβ accumulation and the physical characteristics of neural cells and hence could provide new insights into how disorders like AD affect the progression of different neurological processes from a mechanics point of view.

  16. peptide

    Indian Academy of Sciences (India)

    Prakash

    effects can be observed under certain conditions but these are not always .... of proteins with amyloid characteristics in muscle (Jayaraman et al. 2008) ... not enhance the growth of dangerous fibrils generated at pH. 7.4. ..... The lower chart shows Aβ(25-35) aggregation kinetics during the first 4 min of monitoring. Results are ...

  17. Effect of electrostatics on aggregation of prion protein Sup35 peptide

    International Nuclear Information System (INIS)

    Portillo, Alexander M; Krasnoslobodtsev, Alexey V; Lyubchenko, Yuri L

    2012-01-01

    Self-assembly of misfolded proteins into ordered fibrillar structures is a fundamental property of a wide range of proteins and peptides. This property is also linked with the development of various neurodegenerative diseases such as Alzheimer’s and Parkinson’s. Environmental conditions modulate the misfolding and aggregation processes. We used a peptide, CGNNQQNY, from yeast prion protein Sup35, as a model system to address effects of environmental conditions on aggregate formation. The GNNQQNY peptide self-assembles in fibrils with structural features that are similar to amyloidogenic proteins. Atomic force microscopy (AFM) and thioflavin T (ThT) fluorescence assay were employed to follow the aggregation process at various pHs and ionic strengths. We also used single molecule AFM force spectroscopy to probe interactions between the peptides under various conditions. The ThT fluorescence data showed that the peptide aggregates fast at pH values approaching the peptide isoelectric point (pI = 5.3) and the kinetics is 10 times slower at acidic pH (pH 2.0), suggesting that electrostatic interactions contribute to the peptide self-assembly into aggregates. This hypothesis was tested by experiments performed at low (11 mM) and high (150 mM) ionic strengths. Indeed, the aggregation lag time measured at pH 2 at low ionic strength (11 mM) is 195 h, whereas the lag time decreases ∼5 times when the ionic strength is increased to 150 mM. At conditions close to the pI value, pH 5.6, the aggregation lag time is 12 ± 6 h under low ionic strength, and there is minimal change to the lag time at 150 mM NaCl. The ionic strength also influences the morphology of aggregates visualized with AFM. In pH 2.0 and at high ionic strength, the aggregates are twofold taller than those formed at low ionic strength. In parallel, AFM force spectroscopy studies revealed minimal contribution of electrostatics to dissociation of transient peptide dimers. (paper)

  18. Proposal for novel curcumin derivatives as potent inhibitors against Alzheimer's disease: Ab initio molecular simulations on the specific interactions between amyloid-beta peptide and curcumin

    Science.gov (United States)

    Ota, Shintaro; Fujimori, Mitsuki; Ishimura, Hiromi; Shulga, Sergiy; Kurita, Noriyuki

    2017-10-01

    Accumulation of amyloid-β (Aβ) peptides in a brain is closely related with the pathogenesis of Alzheimer's disease. To suppress the production of Aβ peptides, we propose novel curcumin derivatives and investigate their binding properties with the amyloid precursor protein (APP), using protein-ligand docking as well as ab initio molecular simulations. Our proposed derivative (curcumin XIV) is found to have a large binding energy with APP and interacts strongly with the cleavage site Ala19 by secretase. It is thus expected that curcumin XIV can protect APP from the secretase attack and be a potent inhibitor against the production of Aβ peptides.

  19. A carboxylated Zn-phthalocyanine inhibits fibril formation of Alzheimer's amyloid β peptide.

    Science.gov (United States)

    Tabassum, Shatera; Sheikh, Abdullah M; Yano, Shozo; Ikeue, Takafumi; Handa, Makoto; Nagai, Atsushi

    2015-02-01

    Amyloid β (Aβ), a 39-42 amino acid peptide derived from amyloid precursor protein, is deposited as fibrils in Alzheimer's disease brains, and is considered to play a major role in the pathogenesis of the disease. We have investigated the effects of a water-soluble Zn-phthalocyanine, ZnPc(COONa)₈, a macrocyclic compound with near-infrared optical properties, on Aβ fibril formation in vitro. A thioflavin T fluorescence assay showed that ZnPc(COONa)₈ significantly inhibited Aβ fibril formation, increasing the lag time and dose-dependently decreasing the plateau level of fibril formation. Moreover, it destabilized pre-formed Aβ fibrils, resulting in an increase in low-molecular-weight species. After fibril formation in the presence of ZnPc(COONa)₈, immunoprecipitation of Aβ₁₋₄₂ using Aβ-specific antibody followed by near-infrared scanning demonstrated binding of ZnPc(COONa)₈ to Aβ₁₋₄₂. A study using the hydrophobic fluorescent probe 8-anilino-1-naphthalenesulfonic acid showed that ZnPc(COONa)8 decreased the hydrophobicity during Aβ₁₋₄₂ fibril formation. CD spectroscopy showed an increase in the α helix structure and a decrease in the β sheet structure of Aβ₁₋₄₀ in fibril-forming buffer containing ZnPc(COONa)₈. SDS/PAGE and a dot-blot immunoassay showed that ZnPc(COONa)₈ delayed the disappearance of low-molecular-weight species and the appearance of higher-molecular-weight oligomeric species of Aβ₁₋₄₂. A cell viability assay showed that ZnPc(COONa)₈ was not toxic to a neuronal cell line (A1), but instead protected A1 cells against Aβ₁₋₄₂-induced toxicity. Overall, our results indicate that ZnPc(COONa)₈ binds to Aβ and decreases the hydrophobicity, and this change is unfavorable for Aβ oligomerization and fibril formation. © 2014 FEBS.

  20. Importance of lipopolysaccharide aggregate disruption for the anti-endotoxic effects of heparin cofactor II peptides.

    Science.gov (United States)

    Singh, Shalini; Papareddy, Praveen; Kalle, Martina; Schmidtchen, Artur; Malmsten, Martin

    2013-11-01

    Lipid membrane and lipopolysaccharide (LPS) interactions were investigated for a series of amphiphilic and cationic peptides derived from human heparin cofactor II (HCII), using dual polarization interferometry, ellipsometry, circular dichroism (CD), cryoTEM, and z-potential measurements. Antimicrobial effects of these peptides were compared to their ability to disorder bacterial lipid membranes, while their capacity to block endotoxic effects of LPS was correlated to the binding of these peptides to LPS and its lipid A moiety, and to charge, secondary structure, and morphology of peptide/LPS complexes. While the peptide KYE28 (KYEITTIHNLFRKLTHRLFRRNFGYTLR) displayed potent antimicrobial and anti-endotoxic effects, its truncated variants KYE21 (KYEITTIHNLFRKLTHRLFRR) and NLF20 (NLFRKLTHRLFRRNFGYTLR) provide some clues on structure-activity relations, since KYE21 retains both the antimicrobial and anti-endotoxic effects of KYE28 (although both attenuated), while NLF20 retains the antimicrobial but only a fraction of the anti-endotoxic effect, hence locating the anti-endotoxic effects of KYE28 to its N-terminus. The antimicrobial effect, on the other hand, is primarily located at the C-terminus of KYE28. While displaying quite different endotoxic effects, these peptides bind to a similar extent to both LPS and lipid A, and also induce comparable LPS scavenging on model eukaryotic membranes. In contrast, fragmentation and densification of LPS aggregates, in turn dependent on the secondary structure in the peptide/LPS aggregates, correlate to the anti-endotoxic effect of these peptides, thus identifying peptide-induced packing transitions in LPS aggregates as key for anti-endotoxic functionality. This aspect therefore needs to be taken into account in the development of novel anti-endotoxic peptide therapeutics. Copyright © 2013. Published by Elsevier B.V.

  1. COPS5 (Jab1) protein increases β site processing of amyloid precursor protein and amyloid β peptide generation by stabilizing RanBP9 protein levels.

    Science.gov (United States)

    Wang, Hongjie; Dey, Debleena; Carrera, Ivan; Minond, Dmitriy; Bianchi, Elisabetta; Xu, Shaohua; Lakshmana, Madepalli K

    2013-09-13

    Increased processing of amyloid precursor protein (APP) and accumulation of neurotoxic amyloid β peptide (Aβ) in the brain is central to the pathogenesis of Alzheimer's disease (AD). Therefore, the identification of molecules that regulate Aβ generation is crucial for future therapeutic approaches for AD. We demonstrated previously that RanBP9 regulates Aβ generation in a number of cell lines and primary neuronal cultures by forming tripartite protein complexes with APP, low-density lipoprotein-related protein, and BACE1, consequently leading to increased amyloid plaque burden in the brain. RanBP9 is a scaffold protein that exists and functions in multiprotein complexes. To identify other proteins that may bind RanBP9 and regulate Aβ levels, we used a two-hybrid analysis against a human brain cDNA library and identified COPS5 as a novel RanBP9-interacting protein. This interaction was confirmed by coimmunoprecipitation experiments in both neuronal and non-neuronal cells and mouse brain. Colocalization of COPS5 and RanBP9 in the same subcellular compartments further supported the interaction of both proteins. Furthermore, like RanBP9, COPS5 robustly increased Aβ generation, followed by increased soluble APP-β (sAPP-β) and decreased soluble-APP-α (sAPP-α) levels. Most importantly, down-regulation of COPS5 by siRNAs reduced Aβ generation, implying that endogenous COPS5 regulates Aβ generation. Finally, COPS5 levels were increased significantly in AD brains and APΔE9 transgenic mice, and overexpression of COPS5 strongly increased RanBP9 protein levels by increasing its half-life. Taken together, these results suggest that COPS5 increases Aβ generation by increasing RanBP9 levels. Thus, COPS5 is a novel RanBP9-binding protein that increases APP processing and Aβ generation by stabilizing RanBP9 protein levels.

  2. pH-dependent kinetics of copper ions binding to amyloidpeptide.

    Science.gov (United States)

    Bin, Yannan; Chen, Shu; Xiang, Juan

    2013-02-01

    Interactions of amyloidpeptide (Aβ) with Cu(2+) are known to be pH-dependent and believed to play a crucial role in the neurotoxicity of Alzheimer's disease (AD). Some research has revealed that injured brains with lowered pH have higher risks of developing AD. However, reported experiments were performed under neutral or mildly acidic conditions, and no reports about the affinity of Aβ-Cu(2+) below pH6.0. In this study, surface plasmon resonance (SPR) sensor with immobilized Aβ was used to investigate the formation of Aβ-Cu(2+) complexes under acidic pH conditions. Dissociation constants were calculated and shown to be pH-dependent, ranging from 3.5×10(-8)M to 8.7×10(-3)M in the pH range from 7.0 to 4.0. The physiological significance of K(d) was preliminarily investigated by monitoring the generation of OH() in aerobic solutions containing Aβ-Cu(2+) and Cu(2+). The results imply that acidic conditions could aggravate the oxidative stress in the presence of Cu(2+), and the weak affinities of Aβ-Cu(2+) under mildly acidic pH of 5.0-6.0 could further enhance the oxidative damage. However, the oxidative stress effect of Aβ is negligible due to the suppressed formation of Aβ-Cu(2+) below pH5.0. This work is useful for the in-depth understanding of the role of Aβ-Cu(2+) in AD neuropathology. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

  3. Apolipoproteins E and J interfere with amyloid-beta uptake by primary human astrocytes and microglia in vitro

    NARCIS (Netherlands)

    Mulder, S.D.; Nielsen, H.M.; Blankenstein, M.A.; Eikelenboom, P.; Veerhuis, R.

    2014-01-01

    Defective clearance of the amyloidpeptide (Aβ) from the brain is considered a strong promoter in Alzheimer's disease (AD) pathogenesis. Astrocytes and microglia are important mediators of Aβ clearance and Aβ aggregation state and the presence of amyloid associated proteins (AAPs), such as

  4. Cannabinoid effects on β amyloid fibril and aggregate formation, neuronal and microglial-activated neurotoxicity in vitro.

    Science.gov (United States)

    Janefjord, Emelie; Mååg, Jesper L V; Harvey, Benjamin S; Smid, Scott D

    2014-01-01

    Cannabinoid (CB) ligands have demonstrated neuroprotective properties. In this study we compared the effects of a diverse set of CB ligands against β amyloid-mediated neuronal toxicity and activated microglial-conditioned media-based neurotoxicity in vitro, and compared this with a capacity to directly alter β amyloid (Aβ) fibril or aggregate formation. Neuroblastoma (SH-SY5Y) cells were exposed to Aβ1-42 directly or microglial (BV-2 cells) conditioned media activated with lipopolysaccharide (LPS) in the presence of the CB1 receptor-selective agonist ACEA, CB2 receptor-selective agonist JWH-015, phytocannabinoids Δ(9)-THC and cannabidiol (CBD), the endocannabinoids 2-arachidonoyl glycerol (2-AG) and anandamide or putative GPR18/GPR55 ligands O-1602 and abnormal-cannabidiol (Abn-CBD). TNF-α and nitrite production was measured in BV-2 cells to compare activation via LPS or albumin with Aβ1-42. Aβ1-42 evoked a concentration-dependent loss of cell viability in SH-SY5Y cells but negligible TNF-α and nitrite production in BV-2 cells compared to albumin or LPS. Both albumin and LPS-activated BV-2 conditioned media significantly reduced neuronal cell viability but were directly innocuous to SH-SY5Y cells. Of those CB ligands tested, only 2-AG and CBD were directly protective against Aβ-evoked SH-SY5Y cell viability, whereas JWH-015, THC, CBD, Abn-CBD and O-1602 all protected SH-SY5Y cells from BV-2 conditioned media activated via LPS. While CB ligands variably altered the morphology of Aβ fibrils and aggregates, there was no clear correlation between effects on Aβ morphology and neuroprotective actions. These findings indicate a neuroprotective action of CB ligands via actions at microglial and neuronal cells.

  5. Einfluß einer In-vitro- und In-vivo-Cholesterol-Modulation in Hirnmembranen auf die zellulären Effekte von Amyloid-beta-Peptid

    OpenAIRE

    Kirsch, Christopher

    2003-01-01

    Die exzessive Bildung und Ablagerung von aggregiertem Amyloid beta-Peptid im Gehirn von Alzheimer Patienten wird allgemein als zentrales Ereignis im Rahmen des Neurodegenerationsprozesses der Alzheimer Demenz betrachtet. Der Amyloid-Stoffwechsel ist dabei in sehr vielfältiger Weise mit dem zellulären Cholesterol-Stoffwechsel verknüpft. Hohe Cholesterolspiegel in spezifischen Membrandomänen wie Lipid-Rafts forcieren sehr wahrscheinlich die zelluläre Produktion als auch die Fibrillogenese von A...

  6. A Peptide Derived from the HIV-1 gp120 Coreceptor-Binding Region Promotes Formation of PAP248-286 Amyloid Fibrils to Enhance HIV-1 Infection.

    Directory of Open Access Journals (Sweden)

    Jinquan Chen

    Full Text Available Semen is a major vehicle for HIV transmission. Prostatic acid phosphatase (PAP fragments, such as PAP248-286, in human semen can form amyloid fibrils to enhance HIV infection. Other endogenous or exogenous factors present during sexual intercourse have also been reported to promote the formation of seminal amyloid fibrils.Here, we demonstrated that a synthetic 15-residue peptide derived from the HIV-1 gp120 coreceptor-binding region, designated enhancing peptide 2 (EP2, can rapidly self-assemble into nanofibers. These EP2-derivated nanofibers promptly accelerated the formation of semen amyloid fibrils by PAP248-286, as shown by Thioflavin T (ThT and Congo red assays. The amyloid fibrils presented similar morphology, assessed via transmission electron microscopy (TEM, in the presence or absence of EP2. Circular dichroism (CD spectroscopy revealed that EP2 accelerates PAP248-286 amyloid fibril formation by promoting the structural transition of PAP248-286 from a random coil into a cross-β-sheet. Newly formed semen amyloid fibrils effectively enhanced HIV-1 infection in TZM-bl cells and U87 cells by promoting the binding of HIV-1 virions to target cells.Nanofibers composed of EP2 promote the formation of PAP248-286 amyloid fibrils and enhance HIV-1 infection.

  7. The role of Metals in Amyloid Aggregation: A Test Case for ab initio Simulations

    International Nuclear Information System (INIS)

    Minicozzi, V.; Rossi, G. C.; Stellato, F.; Morante, S.

    2007-01-01

    First principle ab initio molecular dynamics simulations of the Car-Parrinello type have proved to be of invaluable help in understanding the microscopic mechanisms of chemical bonding both in solid state physics and in structural biophysics. In this work we present as test cases the study of the Cu coordination mode in two especially important examples: Prion protein and β-amyloids. Using medium size PC-clusters as well as larger parallel platforms, we are able to deal with systems comprising 300 to 500 atoms and 1000 to 1500 electrons for as long as 2-3 ps. We present structural results which confirm indications coming from NMR and XAS data

  8. Probing amyloid protein aggregation with optical superresolution methods: from the test tube to models of disease.

    Science.gov (United States)

    Kaminski, Clemens F; Kaminski Schierle, Gabriele S

    2016-10-01

    The misfolding and self-assembly of intrinsically disordered proteins into insoluble amyloid structures are central to many neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Optical imaging of this self-assembly process in vitro and in cells is revolutionizing our understanding of the molecular mechanisms behind these devastating conditions. In contrast to conventional biophysical methods, optical imaging and, in particular, optical superresolution imaging, permits the dynamic investigation of the molecular self-assembly process in vitro and in cells, at molecular-level resolution. In this article, current state-of-the-art imaging methods are reviewed and discussed in the context of research into neurodegeneration.

  9. Lithium chloride increases the production of amyloid-beta peptide independently from its inhibition of glycogen synthase kinase 3.

    Science.gov (United States)

    Feyt, Christine; Kienlen-Campard, Pascal; Leroy, Karelle; N'Kuli, Francisca; Courtoy, Pierre J; Brion, Jean-Pierre; Octave, Jean-Noël

    2005-09-30

    Glycogen synthase kinase 3 (GSK3) is able to phosphorylate tau at many sites that are found to be phosphorylated in paired helical filaments in Alzheimer disease. Lithium chloride (LiCl) efficiently inhibits GSK3 and was recently reported to also decrease the production of amyloid-beta peptide (Abeta) from its precursor, the amyloid precursor protein. Therefore, lithium has been proposed as a combined therapeutic agent, inhibiting both the hyperphosphorylation of tau and the production of Abeta. Here, we demonstrate that the inhibition of GSK3 by LiCl induced the nuclear translocation of beta-catenin in Chinese hamster ovary cells and rat cultured neurons, in which a decrease in tau phosphorylation was observed. In both cellular models, a nontoxic concentration of LiCl increased the production of Abeta by increasing the beta-cleavage of amyloid precursor protein, generating more substrate for an unmodified gamma-secretase activity. SB415286, another GSK3 inhibitor, induced the nuclear translocation of beta-catenin and slightly decreased Abeta production. It is concluded that the LiCl-mediated increase in Abeta production is not related to GSK3 inhibition.

  10. Distinguishing d- and l-aspartic and isoaspartic acids in amyloid β peptides with ultrahigh resolution ion mobility spectrometry.

    Science.gov (United States)

    Zheng, Xueyun; Deng, Liulin; Baker, Erin S; Ibrahim, Yehia M; Petyuk, Vladislav A; Smith, Richard D

    2017-07-11

    While α-linked amino acids in the l-form are exclusively utilized in mammalian protein building, β-linked and d-form amino acids also have important biological roles. Unfortunately, the structural elucidation and separation of these different amino acid types in peptides has been analytically challenging to date due to the numerous isomers present, limiting our knowledge about their existence and biological roles. Here, we utilized an ultrahigh resolution ion mobility spectrometry platform coupled with mass spectrometry (IMS-MS) to separate amyloid β (Aβ) peptides containing l-aspartic acid, d-aspartic acid, l-isoaspartic acid, and d-isoaspartic acid residues which span α- and β-linked amino acids in both d- and l-forms. The results illustrate how IMS-MS could be used to better understand age-related diseases or protein folding disorders resulting from amino acid modifications.

  11. Cross-linking of cell surface amyloid precursor protein leads to increased β-amyloid peptide production in hippocampal neurons: implications for Alzheimer's disease.

    Science.gov (United States)

    Lefort, Roger; Pozueta, Julio; Shelanski, Michael

    2012-08-01

    The accumulation of the β-amyloid peptide (Aβ) in Alzheimer's disease (AD) is thought to play a causative role in triggering synaptic dysfunction in neurons, leading to their eventual demise through apoptosis. Aβ is produced and secreted upon sequential cleavage of the amyloid precursor protein (APP) by β-secretases and γ-secretases. However, while Aβ levels have been shown to be increased in the brains of AD patients, little is known about how the cleavage of APP and the subsequent generation of Aβ is influenced, or whether the cleavage process changes over time. It has been proposed that Aβ can bind APP and promote amyloidogenic processing of APP, further enhancing Aβ production. Proof of this idea has remained elusive because a clear mechanism has not been identified, and the promiscuous nature of Aβ binding complicates the task of demonstrating the idea. To work around these problems, we used an antibody-mediated approach to bind and cross-link cell-surface APP in cultured rat primary hippocampal neurons. Here we show that cross-linking of APP is sufficient to raise the levels of Aβ in viable neurons with a concomitant increase in the levels of the β-secretase BACE1. This appears to occur as a result of a sorting defect that stems from the caspase-3-mediated inactivation of a key sorting adaptor protein, namely GGA3, which prevents the lysosomal degradation of BACE1. Together, our data suggest the occurrence of a positive pathogenic feedback loop involving Aβ and APP in affected neurons possibly allowing Aβ to spread to nearby healthy neurons.

  12. Possible Function of Molecular Chaperones in Diseases Caused by Propagating Amyloid Aggregates

    Directory of Open Access Journals (Sweden)

    Vladimir F. Lazarev

    2017-05-01

    Full Text Available The vast majority of neurodegenerative pathologies stem from the formation of toxic oligomers and aggregates composed of wrongly folded proteins. These protein complexes can be released from pathogenic cells and enthralled by other cells, causing the formation of new aggregates in a prion-like manner. By this mechanism, migrating complexes can transmit a disorder to distant regions of the brain and promote gradually transmitting degenerative processes. Molecular chaperones can counteract the toxicity of misfolded proteins. In this review, we discuss recent data on the possible cytoprotective functions of chaperones in horizontally transmitting neurological disorders.

  13. Protective Effects Induced by Microwave-Assisted Aqueous Harpagophytum Extract on Rat Cortex Synaptosomes Challenged with Amyloid β-Peptide.

    Science.gov (United States)

    Ferrante, Claudio; Recinella, Lucia; Locatelli, Marcello; Guglielmi, Paolo; Secci, Daniela; Leporini, Lidia; Chiavaroli, Annalisa; Leone, Sheila; Martinotti, Sara; Brunetti, Luigi; Vacca, Michele; Menghini, Luigi; Orlando, Giustino

    2017-08-01

    Harpagophytum procumbens is a plant species that displays anti-inflammatory properties in multiple tissues. The iridoid glycosides arpagoside, harpagide, and procumbide appear to be the most therapeutically important constituents. In addition, harpagoside treatment exerted neuroprotective effects both in vitro and in vivo. Considering these findings, the aim of the present work is to explore the possible protective role of the previously described microwave-assisted aqueous extract of H. procumbens on rat hypothalamic (Hypo-E22) cells, and in rat cortex challenged with amyloid β-peptide (1-40). In this context, we assayed the protective effects induced by H. procumbens by measuring the levels of malondialdehyde, 3-hydroxykynurenine (3-HK), brain-derived neurotrophic factor, and tumor necrosis factor-α, 3-HK. Finally, we evaluated the effects of H. procumbens treatment on cortex levels of dopamine, norepinephrine, and serotonin. H. procumbens extract was well tolerated by Hypo-E22 cells and upregulated brain-derived neurotrophic factor gene expression but down-regulated tumor necrosis factor-α gene expression. In addition, the extract reduced amyloid β-peptide stimulation of malondialdehyde and 3-HK and blunted the decrease of dopamine, norepinephrine, and serotonin, in the cortex. In this context, our work supports further studies for the evaluation and confirmation of Harpagophytum in the management of the clinical symptoms related to Alzheimer's disease. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  14. DNAJB6 is a peptide-binding chaperone which can suppress amyloid fibrillation of polyglutamine peptides at substoichiometric molar ratios

    NARCIS (Netherlands)

    Mansson, Cecilia; Kakkar, Vaishali; Monsellier, Elodie; Sourigues, Yannick; Harmark, Johan; Kampinga, Harm H.; Melki, Ronald; Emanuelsson, Cecilia

    Expanded polyglutamine (polyQ) stretches lead to protein aggregation and severe neurodegenerative diseases. A highly efficient suppressor of polyQ aggregation was identified, the DNAJB6, when molecular chaperones from the HSPH, HSPA, and DNAJ families were screened for huntingtin exon 1 aggregation

  15. AmyloidPeptide Is Needed for cGMP-Induced Long-Term Potentiation and Memory.

    Science.gov (United States)

    Palmeri, Agostino; Ricciarelli, Roberta; Gulisano, Walter; Rivera, Daniela; Rebosio, Claudia; Calcagno, Elisa; Tropea, Maria Rosaria; Conti, Silvia; Das, Utpal; Roy, Subhojit; Pronzato, Maria Adelaide; Arancio, Ottavio; Fedele, Ernesto; Puzzo, Daniela

    2017-07-19

    High levels of amyloidpeptide (Aβ) have been related to Alzheimer's disease pathogenesis. However, in the healthy brain, low physiologically relevant concentrations of Aβ are necessary for long-term potentiation (LTP) and memory. Because cGMP plays a key role in these processes, here we investigated whether the cyclic nucleotide cGMP influences Aβ levels and function during LTP and memory. We demonstrate that the increase of cGMP levels by the phosphodiesterase-5 inhibitors sildenafil and vardenafil induces a parallel release of Aβ due to a change in the approximation of amyloid precursor protein (APP) and the β-site APP cleaving enzyme 1. Moreover, electrophysiological and behavioral studies performed on animals of both sexes showed that blocking Aβ function, by using anti-murine Aβ antibodies or APP knock-out mice, prevents the cGMP-dependent enhancement of LTP and memory. Our data suggest that cGMP positively regulates Aβ levels in the healthy brain which, in turn, boosts synaptic plasticity and memory. SIGNIFICANCE STATEMENT Amyloid-β (Aβ) is a key pathogenetic factor in Alzheimer's disease. However, low concentrations of endogenous Aβ, mimicking levels of the peptide in the healthy brain, enhance hippocampal long-term potentiation (LTP) and memory. Because the second messenger cGMP exerts a central role in LTP mechanisms, here we studied whether cGMP affects Aβ levels and function during LTP. We show that cGMP enhances Aβ production by increasing the APP/BACE-1 convergence in endolysosomal compartments. Moreover, the cGMP-induced enhancement of LTP and memory was disrupted by blockade of Aβ, suggesting that the physiological effect of the cyclic nucleotide on LTP and memory is dependent upon Aβ. Copyright © 2017 the authors 0270-6474/17/376926-12$15.00/0.

  16. Multitarget Therapeutic Leads for Alzheimer's Disease: Quinolizidinyl Derivatives of Bi- and Tricyclic Systems as Dual Inhibitors of Cholinesterases and β-Amyloid (Aβ) Aggregation.

    Science.gov (United States)

    Tonelli, Michele; Catto, Marco; Tasso, Bruno; Novelli, Federica; Canu, Caterina; Iusco, Giovanna; Pisani, Leonardo; Stradis, Angelo De; Denora, Nunzio; Sparatore, Anna; Boido, Vito; Carotti, Angelo; Sparatore, Fabio

    2015-06-01

    Multitarget therapeutic leads for Alzheimer's disease were designed on the models of compounds capable of maintaining or restoring cell protein homeostasis and of inhibiting β-amyloid (Aβ) oligomerization. Thirty-seven thioxanthen-9-one, xanthen-9-one, naphto- and anthraquinone derivatives were tested for the direct inhibition of Aβ(1-40) aggregation and for the inhibition of electric eel acetylcholinesterase (eeAChE) and horse serum butyrylcholinesterase (hsBChE). These compounds are characterized by basic side chains, mainly quinolizidinylalkyl moieties, linked to various bi- and tri-cyclic (hetero)aromatic systems. With very few exceptions, these compounds displayed inhibitory activity on both AChE and BChE and on the spontaneous aggregation of β-amyloid. In most cases, IC50 values were in the low micromolar and sub-micromolar range, but some compounds even reached nanomolar potency. The time course of amyloid aggregation in the presence of the most active derivative (IC50 =0.84 μM) revealed that these compounds might act as destabilizers of mature fibrils rather than mere inhibitors of fibrillization. Many compounds inhibited one or both cholinesterases and Aβ aggregation with similar potency, a fundamental requisite for the possible development of therapeutics exhibiting a multitarget mechanism of action. The described compounds thus represent interesting leads for the development of multitarget AD therapeutics. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Effects of Amyloid Precursor Protein 17 Peptide on the Protection of Diabetic Encephalopathy and Improvement of Glycol Metabolism in the Diabetic Rat

    Directory of Open Access Journals (Sweden)

    Heng Meng

    2013-01-01

    Full Text Available Researchers have proposed that amyloid precursor protein 17 peptide (APP17 peptide, an active fragment of amyloid precursor protein (APP in the nervous system, has therapeutic effects on neurodegeneration. Diabetic encephalopathy (DE is a neurological disease caused by diabetes. Here we use multiple experimental approaches to investigate the effect of APP17 peptide on changes in learning behavior and glycol metabolism in rats. It was found that rats with DE treated by APP17 peptide showed reversed behavioral alternation. The [18F]-FDG-PET images and other results all showed that the APP17 peptide could promote glucose metabolism in the brain of the DE rat model. Meanwhile, the insulin signaling was markedly increased as shown by increased phosphorylation of Akt and enhanced GLUT4 activation. Compared with the DE group, the activities of SOD, GSH-Px, and CAT in the rat hippocampal gyrus were increased, while MDA decreased markedly in the DE + APP17 peptide group. No amyloid plaques in the cortex and the hippocampus were detected in either group, indicating that the experimental animals in the current study were not suffering from Alzheimer’s disease. These results indicate that APP17 peptide could be used to treat DE effectively.

  18. Human stefin B normal and patho-physiological role: molecular and cellular aspects of amyloid-type aggregation of certain EPM1 mutants.

    Directory of Open Access Journals (Sweden)

    Mira ePolajnar

    2012-08-01

    Full Text Available Epilepsies are characterised by abnormal electrophysiological activity of the brain. Among various types of inherited epilepsies different epilepsy syndromes, among them progressive myoclonus epilepsies with features of ataxia and neurodegeneration, are counted. The progressive myoclonus epilepsy of type 1 (EPM1, also known as Unverricht-Lundborg disease presents with features of cerebellar atrophy and increased oxidative stress. It has been found that EPM1 is caused by mutations in human cystatin B gene (human stefin B. We first describe the role of protein aggregation in other neurodegenerative conditions. Protein aggregates appear intraneurally but are also excreted, such as is the case with senile plaques of amyloid- β (Aβ that accumulate in the brain parenchyma and vessel walls. A common characteristic of such diseases is the change of the protein conformation towards β secondary structure that accounts for the strong tendency of such proteins to aggregate and form amyloid fibrils. Second, we describe the patho-physiology of EPM1 and the normal and aberrant roles of stefin B in a mouse model of the disease. Furthermore, we discuss how the increased protein aggregation observed with some of the mutants of human stefin B may relate to the neurodegeneration that occurs in rare EPM1 patients. Our hypothesis (Ceru et al., 2005 states that some of the EPM1 mutants of human stefin B may undergo aggregation in neural cells, thus gaining additional toxic function (apart from loss of normal function. Our in vitro experiments thus far have confirmed that 4 mutants undergo increased aggregation relative to the wild-type protein. It has been shown that the R68X mutant forms amyloid-fibrils very rapidly, even at neutral pH and forms perinuclear inclusions, whereas the G4R mutant exhibits a prolonged lag phase, during which the toxic prefibrillar aggregates accumulate and are scattered more diffusely over the cytoplasm. Initial experiments on the G50E

  19. Resveratrol and Amyloid-Beta: Mechanistic Insights

    Directory of Open Access Journals (Sweden)

    Yongming Jia

    2017-10-01

    Full Text Available The amyloid-beta (Aβ hypothesis that dyshomeostasis between Aβ production and clearance is a very early, key molecular factor in the etiology of Alzheimer’s disease (AD has been proposed and examined in the AD research field. Scientists have focused on seeking natural products or drugs to influence the dynamic equilibrium of Aβ, targeting production and clearance of Aβ. There is emerging evidence that resveratrol (Res, a naturally occurring polyphenol mainly found in grapes and red wine, acts on AD in numerous in vivo and in vitro models. Res decreases the amyloidogenic cleavage of the amyloid precursor protein (APP, enhances clearance of amyloid beta-peptides, and reduces Aβ aggregation. Moreover, Res also protects neuronal functions through its antioxidant properties. This review discusses the action of Res on Aβ production, clearance and aggregation and multiple potential mechanisms, providing evidence of the useful of Res for AD treatment.

  20. New Method Based on Capillary Electrophoresis with Laser-Induced Fluorescence Detection (CE-LIF) to Monitor Interaction between Nanoparticles and the AmyloidPeptide

    NARCIS (Netherlands)

    Brambilla, Davide; Verpillot, Romain; Taverna, Myriam; de Kimpe, Line; Le Droumaguet, Benjamin; Nicolas, Julien; Canovi, Mara; Gobbi, Marco; Mantegazza, Francesco; Salmona, Mario; Nicolas, Valérie; Scheper, Wiep; Couvreur, Patrick; Andrieux, Karine

    2010-01-01

    A novel application of capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was proposed to efficiently detect and monitor the interaction between polymeric nanoparticles and the β-Amyloid peptide (Aβ(1-42)), a biomarker for Alzheimer's Disease (AD), at concentrations close

  1. Chronic exposure of NG108-15 cells to amyloid beta peptide (A beta(1-42)) abolishes calcium influx via N-type calcium channels

    Czech Academy of Sciences Publication Activity Database

    Kašparová, Jana; Lisá, Věra; Tuček, Stanislav; Doležal, Vladimír

    2001-01-01

    Roč. 26, 8-9 (2001), s. 1079-1084 ISSN 0364-3190 R&D Projects: GA MZd NF5183 Institutional research plan: CEZ:AV0Z5011922 Keywords : amyloid beta peptide * Alzheimer's disease * calcium Subject RIV: FH - Neurology Impact factor: 1.638, year: 2001

  2. Monodisperse carboxyl-functionalized poly(ethylene glycol)-coated magnetic poly(glycidyl methacrylate) microspheres: application to the immunocapture of .beta.-amyloid peptides

    Czech Academy of Sciences Publication Activity Database

    Horák, Daniel; Hlídková, Helena; Hiraoui, M.; Taverna, M.; Proks, Vladimír; Mázl Chánová, Eliška; Smadja, C.; Kučerová, Z.

    2014-01-01

    Roč. 14, č. 11 (2014), s. 1590-1599 ISSN 1616-5187 R&D Projects: GA MŠk 7E12053 EU Projects: European Commission(XE) 246513 - NADINE Institutional support: RVO:61389013 Keywords : β-amyloid peptides * CE-LIF detection * functionalization Subject RIV: CD - Macromolecular Chemistry Impact factor: 3.851, year: 2014

  3. Functional Amyloids in Reproduction.

    Science.gov (United States)

    Hewetson, Aveline; Do, Hoa Quynh; Myers, Caitlyn; Muthusubramanian, Archana; Sutton, Roger Bryan; Wylie, Benjamin J; Cornwall, Gail A

    2017-06-29

    Amyloids are traditionally considered pathological protein aggregates that play causative roles in neurodegenerative disease, diabetes and prionopathies. However, increasing evidence indicates that in many biological systems nonpathological amyloids are formed for functional purposes. In this review, we will specifically describe amyloids that carry out biological roles in sexual reproduction including the processes of gametogenesis, germline specification, sperm maturation and fertilization. Several of these functional amyloids are evolutionarily conserved across several taxa, including human, emphasizing the critical role amyloids perform in reproduction. Evidence will also be presented suggesting that, if altered, some functional amyloids may become pathological.

  4. Simultaneous membrane interaction of amphipathic peptide monomers, self-aggregates and cargo complexes detected by fluorescence correlation spectroscopy.

    Science.gov (United States)

    Vasconcelos, Luís; Lehto, Tõnis; Madani, Fatemeh; Radoi, Vlad; Hällbrink, Mattias; Vukojević, Vladana; Langel, Ülo

    2018-02-01

    Peptides able to translocate cell membranes while carrying macromolecular cargo, as cell-penetrating peptides (CPPs), can contribute to the field of drug delivery by enabling the transport of otherwise membrane impermeable molecules. Formation of non-covalent complexes between amphipathic peptides and oligonucleotides is driven by electrostatic and hydrophobic interactions. Here we investigate and quantify the coexistence of distinct molecular species in multiple equilibria, namely peptide monomer, peptide self-aggregates and peptide/oligonucleotide complexes. As a model for the complexes, we used a stearylated peptide from the PepFect family, PF14 and siRNA. PF14 has a cationic part and a lipid part, resembling some characteristics of cationic lipids. Fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) were used to detect distinct molecular entities in solution and at the plasma membrane of live cells. For that, we labeled the peptide with carboxyrhodamine 6G and the siRNA with Cyanine 5. We were able to detect fluorescent entities with diffusional properties characteristic of the peptide monomer as well as of peptide aggregates and peptide/oligonucleotide complexes. Strategies to avoid peptide adsorption to solid surfaces and self-aggregation were developed and allowed successful FCS measurements in solution and at the plasma membrane. The ratio between the detected molecular species was found to vary with pH, peptide concentration and the proximity to the plasma membrane. The present results suggest that the diverse cellular uptake mechanisms, often reported for amphipathic CPPs, might result from the synergistic effect of peptide monomers, self-aggregates and cargo complexes, distributed unevenly at the plasma membrane. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Cysteine Cathepsins in the secretory vesicle produce active peptides: Cathepsin L generates peptide neurotransmitters and cathepsin B produces beta-amyloid of Alzheimer's disease.

    Science.gov (United States)

    Hook, Vivian; Funkelstein, Lydiane; Wegrzyn, Jill; Bark, Steven; Kindy, Mark; Hook, Gregory

    2012-01-01

    Recent new findings indicate significant biological roles of cysteine cathepsin proteases in secretory vesicles for production of biologically active peptides. Notably, cathepsin L in secretory vesicles functions as a key protease for proteolytic processing of proneuropeptides (and prohormones) into active neuropeptides that are released to mediate cell-cell communication in the nervous system for neurotransmission. Moreover, cathepsin B in secretory vesicles has been recently identified as a β-secretase for production of neurotoxic β- amyloid (Aβ) peptides that accumulate in Alzheimer's disease (AD), participating as a notable factor in the severe memory loss in AD. These secretory vesicle functions of cathepsins L and B for production of biologically active peptides contrast with the well-known role of cathepsin proteases in lysosomes for the degradation of proteins to result in their inactivation. The unique secretory vesicle proteome indicates proteins of distinct functional categories that provide the intravesicular environment for support of cysteine cathepsin functions. Features of the secretory vesicle protein systems insure optimized intravesicular conditions that support the proteolytic activity of cathepsins. These new findings of recently discovered biological roles of cathepsins L and B indicate their significance in human health and disease. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome. Copyright © 2011 Elsevier B.V. All rights reserved.

  6. Microfluidic Isoelectric Focusing of Amyloid Beta Peptides Followed by Micropillar-Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry.

    Science.gov (United States)

    Mikkonen, Saara; Jacksén, Johan; Roeraade, Johan; Thormann, Wolfgang; Emmer, Åsa

    2016-10-18

    A novel method for preconcentration and purification of the Alzheimer's disease related amyloid beta (Aβ) peptides by isoelectric focusing (IEF) in 75 nL microchannels combined with their analysis by micropillar-matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) is presented. A semiopen chip-based setup, consisting of open microchannels covered by a lid of a liquid fluorocarbon, was used. IEF was performed in a mixture of four small and chemically well-defined amphoteric carriers, glutamic acid, aspartyl-histidine (Asp-His), cycloserine (cSer), and arginine, which provided a stepwise pH gradient tailored for focusing of the C-terminal Aβ peptides with a pI of 5.3 in the boundary between cSer and Asp-His. Information about the focusing dynamics and location of the foci of Aβ peptides and other compounds was obtained using computer simulation and by performing MALDI-MS analysis directly from the open microchannel. With the established configuration, detection was performed by direct sampling of a nanoliter volume containing the focused Aβ peptides from the microchannel, followed by deposition of this volume onto a chip with micropillar MALDI targets. In addition to purification, IEF preconcentration provides at least a 10-fold increase of the MALDI-MS-signal. After immunoprecipitation and concentration of the eluate in the microchannel, IEF-micropillar-MALDI-MS is demonstrated to be a suitable platform for detection of Aβ peptides in human cerebrospinal fluid as well as in blood plasma.

  7. Chirality and chiroptical properties of amyloid fibrils.

    Science.gov (United States)

    Dzwolak, Wojciech

    2014-09-01

    Chirality of amyloid fibrils-linear beta-sheet-rich aggregates of misfolded protein chains-often manifests in morphological traits such as helical twist visible in atomic force microscopy and in chiroptical properties accessible to vibrational circular dichroism (VCD). According to recent studies the relationship between molecular chirality of polypeptide building blocks and superstructural chirality of amyloid fibrils may be more intricate and less deterministic than previously assumed. Several puzzling experimental findings have put into question earlier intuitive ideas on: 1) the bottom-up chirality transfer upon amyloidogenic self-assembly, and 2) the structural origins of chiroptical properties of protein aggregates. For example, removal of a single amino acid residue from an amyloidogenic all-L peptide was shown to reverse handedness of fibrils. On the other hand, certain types of amyloid aggregates revealed surprisingly strong VCD spectra with the sign and shape dependent on the conditions of fibrillation. Hence, microscopic and chiroptical studies have highlighted chirality as one more aspect of polymorphism of amyloid fibrils. This brief review is intended to outline the current state of research on amyloid-like fibrils from the perspective of their structural and superstructural chirality and chiroptical properties. © 2014 Wiley Periodicals, Inc.

  8. Assembly of Triblock Amphiphilic Peptides into One-Dimensional Aggregates and Network Formation.

    Science.gov (United States)

    Ozgur, Beytullah; Sayar, Mehmet

    2016-10-06

    Peptide assembly plays a key role in both neurological diseases and development of novel biomaterials with well-defined nanostructures. Synthetic model peptides provide a unique platform to explore the role of intermolecular interactions in the assembly process. A triblock peptide architecture designed by the Hartgerink group is a versatile system which relies on Coulomb interactions, hydrogen bonding, and hydrophobicity to guide these peptides' assembly at three different length scales: β-sheets, double-wall ribbon-like aggregates, and finally a highly porous network structure which can support gels with ≤1% by weight peptide concentration. In this study, by using molecular dynamics simulations of a structure based implicit solvent coarse grained model, we analyzed this hierarchical assembly process. Parametrization of our CG model is based on multiple-state points from atomistic simulations, which enables this model to represent the conformational adaptability of the triblock peptide molecule based on the surrounding medium. Our results indicate that emergence of the double-wall β-sheet packing mechanism, proposed in light of the experimental evidence, strongly depends on the subtle balance of the intermolecular forces. We demonstrate that, even though backbone hydrogen bonding dominates the early nucleation stages, depending on the strength of the hydrophobic and Coulomb forces, alternative structures such as zero-dimensional aggregates with two β-sheets oriented orthogonally (which we refer to as a cross-packed structure) and β-sheets with misoriented hydrophobic side chains are also feasible. We discuss the implications of these competing structures for the three different length scales of assembly by systematically investigating the influence of density, counterion valency, and hydrophobicity.

  9. Anti-amyloid aggregation activity of novel carotenoids: implications for Alzheimer’s drug discovery

    Directory of Open Access Journals (Sweden)

    Lakey-Beitia J

    2017-05-01

    Full Text Available Johant Lakey-Beitia,1,2 Deborah Doens,2,3 D Jagadeesh Kumar,4 Enrique Murillo,5 Patricia L Fernandez,3 KS Rao,6 Armando A Durant-Archibold1,5 1Center for Biodiversity and Drug Discovery, Instituto de Investigaciones Científicas y Servicios de Alta Tecnología (INDICASAT AIP, Panama, Republic of Panama; 2Department of Biotechnology, Acharya Nagarjuna University, Guntur, India; 3Center for Molecular and Cellular Biology of Diseases, Instituto de Investigaciones Científicas y Servicios de Alta Tecnología (INDICASAT AIP, Panama, Republic of Panama; 4Department of Biotechnology, Sir M Visvesvaraya Institute of Technology, Bangalore, India; 5Department of Biochemistry, College of Natural, Exact Sciences and Technology, University of Panama, Panama, Republic of Panama; 6Center for Neuroscience, Instituto de Investigaciones Científicas y Servicios de Alta Tecnología (INDICASAT AIP, Panama, Republic of Panama Abstract: Alzheimer’s disease (AD is the leading cause of dementia, affecting approximately 33.5 million people worldwide. Aging is the main risk factor associated with AD. Drug discovery based on nutraceutical molecules for prevention and treatment of AD is a growing topic. In this sense, carotenoids are phytochemicals present mainly in fruits and vegetables with reported benefits for human health. In this research, the anti-amyloidogenic activity of three carotenoids, cryptocapsin, cryptocapsin-5,6-epoxide, and zeaxanthin, was assessed. Cryptocapsin showed the highest bioactivity, while cryptocapsin-5,6-epoxide and zeaxanthin exhibited similar activity on anti-aggregation assays. Molecular modeling analysis revealed that the evaluated carotenoids might follow two mechanisms for inhibiting Aβ aggregation: by preventing the formation of the fibril and through disruption of the Aβ aggregates. Our studies provided evidence that cryptocapsin, cryptocapsin-5,6-epoxide, and zeaxanthin have anti-amyloidogenic potential and could be used for

  10. The metabolic enhancer piracetam ameliorates the impairment of mitochondrial function and neurite outgrowth induced by beta-amyloid peptide.

    Science.gov (United States)

    Kurz, C; Ungerer, I; Lipka, U; Kirr, S; Schütt, T; Eckert, A; Leuner, K; Müller, W E

    2010-05-01

    beta-Amyloid peptide (Abeta) is implicated in the pathogenesis of Alzheimer's disease by initiating a cascade of events from mitochondrial dysfunction to neuronal death. The metabolic enhancer piracetam has been shown to improve mitochondrial dysfunction following brain aging and experimentally induced oxidative stress. We used cell lines (PC12 and HEK cells) and murine dissociated brain cells. The protective effects of piracetam in vitro and ex vivo on Abeta-induced impairment of mitochondrial function (as mitochondrial membrane potential and ATP production), on secretion of soluble Abeta and on neurite outgrowth in PC12 cells were investigated. Piracetam improves mitochondrial function of PC12 cells and acutely dissociated brain cells from young NMRI mice following exposure to extracellular Abeta(1-42). Similar protective effects against Abeta(1-42) were observed in dissociated brain cells from aged NMRI mice, or mice transgenic for mutant human amyloid precursor protein (APP) treated with piracetam for 14 days. Soluble Abeta load was markedly diminished in the brain of those animals after treatment with piracetam. Abeta production by HEK cells stably transfected with mutant human APP was elevated by oxidative stress and this was reduced by piracetam. Impairment of neuritogenesis is an important consequence of Abeta-induced mitochondrial dysfunction and Abeta-induced reduction of neurite growth in PC12 cells was substantially improved by piracetam. Our findings strongly support the concept of improving mitochondrial function as an approach to ameliorate the detrimental effects of Abeta on brain function.

  11. The pathogenic implication of abnormal interaction between apolipoprotein E isoforms, amyloid-beta peptides, and sulfatides in Alzheimer's disease.

    Science.gov (United States)

    Han, Xianlin

    2010-06-01

    Alzheimer's disease (AD) is the most common cause of dementia in the aging population. Prior work has shown that the epsilon4 allele of apolipoprotein E (apoE4) is a major risk factor for "sporadic" AD, which accounts for >99% of AD cases without a defined underlying mechanism. Recently, we have demonstrated that sulfatides are substantially and specifically depleted at the very early stage of AD. To identify the mechanism(s) of sulfatide loss concurrent with AD onset, we have found that: (1) sulfatides are specifically associated with apoE-associated particles in cerebrospinal fluid (CSF); (2) apoE modulates cellular sulfatide levels; and (3) the modulation of sulfatide content is apoE isoform dependent. These findings not only lead to identification of the potential mechanisms underlying sulfatide depletion at the earliest stages of AD but also serve as mechanistic links to explain the genetic association of apoE4 with AD. Moreover, our recent studies further demonstrated that (1) apoE mediates sulfatide depletion in amyloid-beta precursor protein transgenic mice; (2) sulfatides enhance amyloid beta (Abeta) peptides binding to apoE-associated particles; (3) Abeta42 content notably correlates with sulfatide content in CSF; (4) sulfatides markedly enhance the uptake of Abeta peptides; and (5) abnormal sulfatide-facilitated Abeta uptake results in the accumulation of Abeta in lysosomes. Collectively, our studies clearly provide a link between apoE, Abeta, and sulfatides in AD and establish a foundation for the development of effective therapeutic interventions for AD.

  12. Intracellular amyloid formation in muscle cells of Aβ-transgenic Caenorhabditis elegans: determinants and physiological role in copper detoxification

    Directory of Open Access Journals (Sweden)

    Bush Ashley I

    2009-01-01

    Full Text Available Abstract Background The amyloid β-peptide is a ubiquitous peptide, which is prone to aggregate forming soluble toxic oligomers and insoluble less-toxic aggregates. The intrinsic and external/environmental factors that determine Aβ aggregation in vivo are poorly understood, as well as the cellular meaning of this process itself. Genetic data as well as cell biological and biochemical evidence strongly support the hypothesis that Aβ is a major player in the onset and development of Alzheimer's disease. In addition, it is also known that Aβ is involved in Inclusion Body Myositis, a common myopathy of the elderly in which the peptide accumulates intracellularly. Results In the present work, we found that intracellular Aβ aggregation in muscle cells of Caenorhabditis elegans overexpressing Aβ peptide is affected by two single amino acid substitutions, E22G (Arctic and V18A (NIC. Both variations show decrease intracellular amyloidogenesis compared to wild type Aβ. We show that intracellular amyloid aggregation of wild type Aβ is accelerated by Cu2+ and diminished by copper chelators. Moreover, we demonstrate through toxicity and behavioral assays that Aβ-transgenic worms display a higher tolerance to Cu2+ toxic effects and that this resistance may be linked to the formation of amyloid aggregates. Conclusion Our data show that intracellular Aβ amyloid aggregates may trap excess of free Cu2+ buffering its cytotoxic effects and that accelerated intracellular Aβ aggregation may be part of a cell protective mechanism.

  13. In situ analysis of Bacillus licheniformis biofilms: amyloid-like polymers and eDNA are involved in the adherence and aggregation of the extracellular matrix.

    Science.gov (United States)

    Randrianjatovo-Gbalou, I; Rouquette, P; Lefebvre, D; Girbal-Neuhauser, E; Marcato-Romain, C-E

    2017-05-01

    This study attempts to determine which of the exopolymeric substances are involved in the adherence and aggregation of a Bacillus licheniformis biofilm. The involvement of extracellular proteins and eDNA were particularly investigated using DNase and proteinase K treatment. The permeability of the biofilms increased fivefold after DNase I treatment. The quantification of the matrix components showed that, irrespective to the enzyme tested, eDNA and amyloid-like polymers were removed simultaneously. Size-exclusion chromatography analyses supported these observations and revealed the presence of associated nucleic acid and protein complexes in the biofilm lysates. These data suggest that some extracellular DNA and amyloid-like proteins were closely interlaced within the matrix. Finally, confocal laser scanning microscopy imaging gave supplementary clues about the 3D organization of the biofilms, confirming that eDNA and exoproteins were essentially layered under and around the bacterial cells, whereas the amyloid-like fractions were homogeneously distributed within the matrix. These results confirm that some DNA-amyloid complexes play a key role in the modulation of the mechanical resistance of B. licheniformis biofilms. The study highlights the need to consider the whole structure of biofilms and to target the interactions between matrix components. A better understanding of B. licheniformis biofilm physiology and the structural organization of the matrix will strengthen strategies of biofilm control. © 2017 The Society for Applied Microbiology.

  14. Involvement of insulin-degrading enzyme in insulin- and atrial natriuretic peptide-sensitive internalization of amyloidpeptide in mouse brain capillary endothelial cells.

    Science.gov (United States)

    Ito, Shingo; Ohtsuki, Sumio; Murata, Sho; Katsukura, Yuki; Suzuki, Hiroya; Funaki, Miho; Tachikawa, Masanori; Terasaki, Tetsuya

    2014-01-01

    Cerebral clearance of amyloidpeptide (Aβ), which is implicated in Alzheimer's disease, involves elimination across the blood-brain barrier (BBB), and we previously showed that an insulin-sensitive process is involved in the case of Aβ1-40. The purpose of this study was to clarify the molecular mechanism of the insulin-sensitive Aβ1-40 elimination across mouse BBB. An in vivo cerebral microinjection study demonstrated that [125I]hAβ1-40 elimination from mouse brain was inhibited by human natriuretic peptide (hANP), and [125I]hANP elimination was inhibited by hAβ1-40, suggesting that hAβ1-40 and hANP share a common elimination process. Internalization of [125I]hAβ1-40 into cultured mouse brain capillary endothelial cells (TM-BBB4) was significantly inhibited by either insulin, hANP, other natriuretic peptides or insulin-degrading enzyme (IDE) inhibitors, but was not inhibited by phosphoramidon or thiorphan. Although we have reported the involvement of natriuretic peptide receptor C (Npr-C) in hANP internalization, cells stably expressing Npr-C internalized [125I]hANP but not [125I]hAβ1-40, suggesting that there is no direct interaction between Npr-C and hAβ1-40. IDE was detected in plasma membrane of TM-BBB4 cells, and internalization of [125I]hAβ1-40 by TM-BBB4 cells was reduced by IDE-targeted siRNAs. We conclude that elimination of hAβ1-40 from mouse brain across the BBB involves an insulin- and ANP-sensitive process, mediated by IDE expressed in brain capillary endothelial cells.

  15. Hyperforin prevents beta-amyloid neurotoxicity and spatial memory impairments by disaggregation of Alzheimer's amyloid-beta-deposits.

    Science.gov (United States)

    Dinamarca, M C; Cerpa, W; Garrido, J; Hancke, J L; Inestrosa, N C

    2006-11-01

    The major protein constituent of amyloid deposits in Alzheimer's disease (AD) is the amyloid beta-peptide (Abeta). In the present work, we have determined the effect of hyperforin an acylphloroglucinol compound isolated from Hypericum perforatum (St John's Wort), on Abeta-induced spatial memory impairments and on Abeta neurotoxicity. We report here that hyperforin: (1) decreases amyloid deposit formation in rats injected with amyloid fibrils in the hippocampus; (2) decreases the neuropathological changes and behavioral impairments in a rat model of amyloidosis; (3) prevents Abeta-induced neurotoxicity in hippocampal neurons both from amyloid fibrils and Abeta oligomers, avoiding the increase in reactive oxidative species associated with amyloid toxicity. Both effects could be explained by the capacity of hyperforin to disaggregate amyloid deposits in a dose and time-dependent manner and to decrease Abeta aggregation and amyloid formation. Altogether these evidences suggest that hyperforin may be useful to decrease amyloid burden and toxicity in AD patients, and may be a putative therapeutic agent to fight the disease.

  16. Aggregation, impaired degradation and immunization targeting of amyloid-beta dimers in Alzheimer’s disease: a stochastic modelling approach

    Directory of Open Access Journals (Sweden)

    Proctor Carole J

    2012-07-01

    Full Text Available Abstract Background Alzheimer’s disease (AD is the most frequently diagnosed neurodegenerative disorder affecting humans, with advanced age being the most prominent risk factor for developing AD. Despite intense research efforts aimed at elucidating the precise molecular underpinnings of AD, a definitive answer is still lacking. In recent years, consensus has grown that dimerisation of the polypeptide amyloid-beta (Aß, particularly Aß42, plays a crucial role in the neuropathology that characterise AD-affected post-mortem brains, including the large-scale accumulation of fibrils, also referred to as senile plaques. This has led to the realistic hope that targeting Aß42 immunotherapeutically could drastically reduce plaque burden in the ageing brain, thus delaying AD onset or symptom progression. Stochastic modelling is a useful tool for increasing understanding of the processes underlying complex systems-affecting disorders such as AD, providing a rapid and inexpensive strategy for testing putative new therapies. In light of the tool’s utility, we developed computer simulation models to examine Aß42 turnover and its aggregation in detail and to test the effect of immunization against Aß dimers. Results Our model demonstrates for the first time that even a slight decrease in the clearance rate of Aß42 monomers is sufficient to increase the chance of dimers forming, which could act as instigators of protofibril and fibril formation, resulting in increased plaque levels. As the process is slow and levels of Aβ are normally low, stochastic effects are important. Our model predicts that reducing the rate of dimerisation leads to a significant reduction in plaque levels and delays onset of plaque formation. The model was used to test the effect of an antibody mediated immunological response. Our results showed that plaque levels were reduced compared to conditions where antibodies are not present. Conclusion Our model supports the current

  17. Development and validation of dried matrix spot sampling for the quantitative determination of amyloid β peptides in cerebrospinal fluid.

    Science.gov (United States)

    Delaby, Constance; Gabelle, Audrey; Meynier, Philippe; Loubiere, Vincent; Vialaret, Jérôme; Tiers, Laurent; Ducos, Jacques; Hirtz, Christophe; Lehmann, Sylvain

    2014-05-01

    The use of dried blood spots on filter paper is well documented as an affordable and practical alternative to classical venous sampling for various clinical needs. This technique has indeed many advantages in terms of collection, biological safety, storage, and shipment. Amyloid β (Aβ) peptides are useful cerebrospinal fluid (CSF) biomarkers for Alzheimer disease diagnosis. However, Aβ determination is hindered by preanalytical difficulties in terms of sample collection and stability in tubes. We compared the quantification of Aβ peptides (1-40, 1-42, and 1-38) by simplex and multiplex ELISA, following either a standard operator method (liquid direct quantification) or after spotting CSF onto dried matrix paper card. The use of dried matrix spot (DMS) overcame preanalytical problems and allowed the determination of Aβ concentrations that were highly commutable (Bland-Altman) with those obtained using CSF in classical tubes. Moreover, we found a positive and significant correlation (r2=0.83, Pearson coefficient p=0.0329) between the two approaches. This new DMS method for CSF represents an interesting alternative that increases the quality and efficiency in preanalytics. This should enable the better exploitation of Aβ analytes for Alzheimer's diagnosis.

  18. High frequency NcoI RFLP detected in the Alzheimer amyloid peptide gene

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Y; Dobkin, C; Devine-Gage, E; Ramakrishna, N; Brown, W T; Wisniewski, H M; Robakis, N K

    1988-02-25

    Probe pAMB2.3 contains a 778bp cDNA fragment encoding the amyloid polypeptide of Alzheimer disease and Down Syndrome (beta protein) inserted into pUC18. The probe was sublocalized to chromosome 21q21. NcoI identifies a two allele polymorphism of a band at either 15 kb or 8.5 kb. Autosomal codominant inheritance was shown in five informative families. Two non polymorphic bands, 7.0kb and 6.0kb, are seen only occasionally. Their presence seems to depend on the particular DNA preparation.

  19. The Peptide Vaccine Combined with Prior Immunization of a Conventional Diphtheria-Tetanus Toxoid Vaccine Induced Amyloid β Binding Antibodies on Cynomolgus Monkeys and Guinea Pigs

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    Akira Yano

    2015-01-01

    Full Text Available The reduction of brain amyloid beta (Aβ peptides by anti-Aβ antibodies is one of the possible therapies for Alzheimer’s disease. We previously reported that the Aβ peptide vaccine including the T-cell epitope of diphtheria-tetanus combined toxoid (DT induced anti-Aβ antibodies, and the prior immunization with conventional DT vaccine enhanced the immunogenicity of the peptide. Cynomolgus monkeys were given the peptide vaccine subcutaneously in combination with the prior DT vaccination. Vaccination with a similar regimen was also performed on guinea pigs. The peptide vaccine induced anti-Aβ antibodies in cynomolgus monkeys and guinea pigs without chemical adjuvants, and excessive immune responses were not observed. Those antibodies could preferentially recognize Aβ40, and Aβ42 compared to Aβ fibrils. The levels of serum anti-Aβ antibodies and plasma Aβ peptides increased in both animals and decreased the brain Aβ40 level of guinea pigs. The peptide vaccine could induce a similar binding profile of anti-Aβ antibodies in cynomolgus monkeys and guinea pigs. The peptide vaccination could be expected to reduce the brain Aβ peptides and their toxic effects via clearance of Aβ peptides by generated antibodies.

  20. A novel antagonistic role of natural compound icariin on neurotoxicity of amyloid β peptide

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    Jianhui Liu

    2015-01-01

    Interpretation & conclusions: The results indicated a novel antagonistic role of icariin in the neurotoxicity of Aβ1-42 via inhibiting its aggregation, suggesting that icariin might have potential therapeutic benefits to delay or modify the progression of AD.

  1. Gold Nanoparticles and Microwave Irradiation Inhibit Beta-Amyloid Amyloidogenesis

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    Bastus Neus

    2008-01-01

    Full Text Available Abstract Peptide-Gold nanoparticles selectively attached to β-amyloid protein (Aβ amyloidogenic aggregates were irradiated with microwave. This treatment produces dramatic effects on the Aβ aggregates, inhibiting both the amyloidogenesis and the restoration of the amyloidogenic potential. This novel approach offers a new strategy to inhibit, locally and remotely, the amyloidogenic process, which could have application in Alzheimer’s disease therapy. We have studied the irradiation effect on the amyloidogenic process in the presence of conjugates peptide-nanoparticle by transmission electronic microscopy observations and by Thioflavine T assays to quantify the amount of fibrils in suspension. The amyloidogenic aggregates rather than the amyloid fibrils seem to be better targets for the treatment of the disease. Our results could contribute to the development of a new therapeutic strategy to inhibit the amyloidogenic process in Alzheimer’s disease.

  2. Conformation and Aggregation of LKα14 Peptide in Bulk Water and at the Air/Water Interface.

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    Dalgicdir, Cahit; Sayar, Mehmet

    2015-12-10

    Historically, the protein folding problem has mainly been associated with understanding the relationship between amino acid sequence and structure. However, it is known that both the conformation of individual molecules and their aggregation strongly depend on the environmental conditions. Here, we study the aggregation behavior of the model peptide LKα14 (with amino acid sequence LKKLLKLLKKLLKL) in bulk water and at the air/water interface. We start by a quantitative analysis of the conformational space of a single LKα14 in bulk water. Next, in order to analyze the aggregation tendency of LKα14, by using the umbrella sampling technique we calculate the potential of mean force for pulling a single peptide from an n-molecule aggregate. In agreement with the experimental results, our calculations yield the optimal aggregate size as four. This equilibrium state is achieved by two opposing forces: Coulomb repulsion between the lysine side chains and the reduction of solvent accessible hydrophobic surface area upon aggregation. At the vacuum/water interface, however, even dimers of LKα14 become marginally stable, and any larger aggregate falls apart instantaneously. Our results indicate that even though the interface is highly influential in stabilizing the α-helix conformation for a single molecule, it significantly reduces the attraction between two LKα14 peptides, along with their aggregation tendency.

  3. Dodecylphosphocholine Micelles Induce Amyloid Formation of the PrP(110-136 Peptide via an α-Helical Metastable Conformation.

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    Simon Sauvé

    Full Text Available A peptide encompassing the conserved hydrophobic region and the first β-strand of the prion protein (PrP(110-136 shown to interact with the surface of dodecylphosphocholine micelles adopts an α-helical conformation that is localized below the head-group layer. This surface-bound peptide has a half-life of one day, and readily initiates the formation of amyloid fibrils. The presence of the latter was confirmed using birefringence microscopy upon Congo red binding and thioflavin T-binding induced fluorescence. The observation of this metastable α-helical conformer provides a unique snapshot of the early steps of the inter-conversion pathway. These findings together with the body of evidence from the prion literature allowed us to propose a mechanism for the conversion of PrPC to amyloid material.

  4. Influence of dendrimer's structure on its activity against amyloid fibril formation

    International Nuclear Information System (INIS)

    Klajnert, B.; Cortijo-Arellano, M.; Cladera, J.; Bryszewska, M.

    2006-01-01

    Inhibition of fibril assembly is a potential therapeutic strategy in neurodegenerative disorders such as prion and Alzheimer's diseases. Highly branched, globular polymers-dendrimers-are novel promising inhibitors of fibril formation. In this study, the effect of polyamidoamine (PAMAM) dendrimers (generations 3rd, 4th, and 5th) on amyloid aggregation of the prion peptide PrP 185-208 and the Alzheimer's peptide Aβ 1-28 was examined. Amyloid fibrils were produced in vitro and their formation was monitored using the dye thioflavin T (ThT). Fluorescence studies were complemented with electron microscopy. The results show that the higher the dendrimer generation, the larger the degree of inhibition of the amyloid aggregation process and the more effective are dendrimers in disrupting the already existing fibrils. A hypothesis on dendrimer-peptide interaction mechanism is presented based on the dendrimers' molecular structure

  5. An UHPLC-MS/MS method for simultaneous quantification of human amyloid beta peptides Aβ1-38, Aβ1-40 and Aβ1-42 in cerebrospinal fluid using micro-elution solid phase extraction.

    Science.gov (United States)

    Lin, Ping-Ping; Chen, Wei-Li; Yuan, Fei; Sheng, Lei; Wu, Yu-Jia; Zhang, Wei-Wei; Li, Guo-Qing; Xu, Hong-Rong; Li, Xue-Ning

    2017-12-01

    Amyloid beta (Aβ) peptides in cerebrospinal fluid are extensively estimated for identification of Alzheimer's disease (AD) as diagnostic biomarkers. Unfortunately, their pervasive application is hampered by interference from Aβ propensity of self-aggregation, nonspecifically bind to surfaces and matrix proteins, and by lack of quantitive standardization. Here we report on an alternative Ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for simultaneous measurement of human amyloid beta peptides Aβ1-38, Aβ1-40 and Aβ1-42 in cerebrospinal fluid (CSF) using micro-elution solid phase extraction (SPE). Samples were pre-processing by the mixed-mode micro-elution solid phase extraction and quantification was performed in the positive ion multiple reaction monitoring (MRM) mode using electrospray ionization. The stable-isotope labeled Aβ peptides 15 N 51 - Aβ1-38, 15 N 53 - Aβ1-40 and 15 N 55 - Aβ1-42 peptides were used as internal standards. And the artificial cerebrospinal fluid (ACSF) containing 5% rat plasma was used as a surrogate matrix for calibration curves. The quality control (QC) samples at 0.25, 2 and 15ng/mL were prepared. A "linear" regression (1/x 2 weighting): y=ax+b was used to fit the calibration curves over the concentration range of 0.1-20ng/mL for all three peptides. Coefficient of variation (CV) of intra-batch and inter-batch assays were all less than 6.44% for Aβ1-38, 6.75% for Aβ1-40 and 10.74% for Aβ1-42. The precision values for all QC samples of three analytes met the acceptance criteria. Extract recoveries of Aβ1-38, Aβ1-40 and Aβ1-42 were all greater than 70.78%, both in low and high QC samples. The stability assessments showed that QC samples at both low and high levels could be stable for at least 24h at 4°C, 4h at room temperature and through three freeze-thaw cycles without sacrificing accuracy or precision. And no significant carryover effect was observed. This validated UHPLC

  6. Hsp72 (HSPA1A Prevents Human Islet Amyloid Polypeptide Aggregation and Toxicity: A New Approach for Type 2 Diabetes Treatment.

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    Paola C Rosas

    Full Text Available Type 2 diabetes is a growing public health concern and accounts for approximately 90% of all the cases of diabetes. Besides insulin resistance, type 2 diabetes is characterized by a deficit in β-cell mass as a result of misfolded human islet amyloid polypeptide (h-IAPP which forms toxic aggregates that destroy pancreatic β-cells. Heat shock proteins (HSP play an important role in combating the unwanted self-association of unfolded proteins. We hypothesized that Hsp72 (HSPA1A prevents h-IAPP aggregation and toxicity. In this study, we demonstrated that thermal stress significantly up-regulates the intracellular expression of Hsp72, and prevents h-IAPP toxicity against pancreatic β-cells. Moreover, Hsp72 (HSPA1A overexpression in pancreatic β-cells ameliorates h-IAPP toxicity. To test the hypothesis that Hsp72 (HSPA1A prevents aggregation and fibril formation, we established a novel C. elegans model that expresses the highly amyloidogenic human pro-IAPP (h-proIAPP that is implicated in amyloid formation and β-cell toxicity. We demonstrated that h-proIAPP expression in body-wall muscles, pharynx and neurons adversely affects C. elegans development. In addition, we demonstrated that h-proIAPP forms insoluble aggregates and that the co-expression of h-Hsp72 in our h-proIAPP C. elegans model, increases h-proIAPP solubility. Furthermore, treatment of transgenic h-proIAPP C. elegans with ADAPT-232, known to induce the expression and release of Hsp72 (HSPA1A, significantly improved the growth retardation phenotype of transgenic worms. Taken together, this study identifies Hsp72 (HSPA1A as a potential treatment to prevent β-cell mass decline in type 2 diabetic patients and establishes for the first time a novel in vivo model that can be used to select compounds that attenuate h-proIAPP aggregation and toxicity.

  7. Glyceraldehyde-3-phosphate dehydrogenase aggregation inhibitor peptide: A potential therapeutic strategy against oxidative stress-induced cell death.

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    Itakura, Masanori; Nakajima, Hidemitsu; Semi, Yuko; Higashida, Shusaku; Azuma, Yasu-Taka; Takeuchi, Tadayoshi

    2015-11-13

    The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has multiple functions, including mediating oxidative stress-induced neuronal cell death. This process is associated with disulfide-bonded GAPDH aggregation. Some reports suggest a link between GAPDH and the pathogenesis of several oxidative stress-related diseases. However, the pathological significance of GAPDH aggregation in disease pathogenesis remains unclear due to the lack of an effective GAPDH aggregation inhibitor. In this study, we identified a GAPDH aggregation inhibitor (GAI) peptide and evaluated its biological profile. The decapeptide GAI specifically inhibited GAPDH aggregation in a concentration-dependent manner. Additionally, the GAI peptide did not affect GAPDH glycolytic activity or cell viability. The GAI peptide also exerted a protective effect against oxidative stress-induced cell death in SH-SY5Y cells. This peptide could potentially serve as a tool to investigate GAPDH aggregation-related neurodegenerative and neuropsychiatric disorders and as a possible therapy for diseases associated with oxidative stress-induced cell death. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Association between amylin and amyloidpeptides in plasma in the context of apolipoprotein E4 allele.

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    Wei Qiao Qiu

    Full Text Available Amylin, a pancreatic peptide that readily crosses the blood brain barrier (BBB, and amyloid-beta peptide (Aβ, the main component of amyloid plaques and a major component of Alzheimer's disease (AD pathology in the brain, share several features. These include having similar β-sheet secondary structures, binding to the same receptor, and being degraded by the same protease. Thus, amylin may be associated with Aβ, but the nature of their relationship remains unclear. In this study, we used human samples to study the relationship between plasma amylin and Aβ in the context of the apolipoprotein E alleles (ApoE. We found that concentrations of Aβ1-42 (P<0.0001 and Aβ1-40 (P<0.0001 increased with each quartile increase of amylin. Using multivariate regression analysis, the study sample showed that plasma amylin was associated with Aβ1-42 (β = +0.149, SE = 0.025, P<0.0001 and Aβ1-40 (β = +0.034, SE = 0.016, P = 0.04 as an outcome after adjusting for age, gender, ethnicity, ApoE4, BMI, diabetes, stroke, kidney function and lipid profile. This positive association between amylin and Aβ1-42 in plasma was found regardless of the ApoE genotype. In contrast, the relationship between amylin and Aβ1-40 in plasma seen in ApoE4 non-carriers disappeared in the presence of ApoE4. Using AD mouse models, our recent study demonstrates that intraperitoneal (i.p. injection of synthetic amylin enhances the removal of Aβ from the brain into blood, thus resulting in increased blood levels of both amylin and Aβ. The positive association between amylin and Aβ, especially Aβ1-42, in human blood samples is probably relevant to the findings in the AD mouse models. The presence of ApoE4 may attenuate amylin's capacity to remove Aβ, especially Aβ1-40, from the AD brain.

  9. Collapsed state of polyglutamic acid results in amyloid spherulite formation.

    Science.gov (United States)

    Stehli, Daniel; Mulaj, Mentor; Miti, Tatiana; Traina, Joshua; Foley, Joseph; Muschol, Martin

    2015-01-01

    Self-assembly of proteins and peptides into amyloid fibrils involves multiple distinct intermediates and late-stage fibrillar polymorphs. Understanding the conditions and mechanisms that promote the formation of one type of intermediate and polymorph over the other represents a fundamental challenge. Answers to this question are also of immediate biomedical relevance since different amyloid aggregate species have been shown to have distinct pathogenic potencies. One amyloid polymorph that has received comparatively little attention are amyloid spherulites. Here we report that self-assembly of the intrinsically disordered polymer poly(L-glutamic) acid (PLE) can generate amyloid spherulites. We characterize spherulite growth kinetics, as well as the morphological, optical and tinctorial features of this amyloid polymorph previously unreported for PLE. We find that PLE spherulites share both tinctorial and structural characteristics with their amyloid fibril counterparts. Differences in PLE's molecular weight, polydispersity or chemistry could not explain the selective propensity toward either fibril or spherulite formation. Instead, we provide evidence that PLE polymers can exist in either a collapsed globule or an extended random coil conformation. The collapsed globule consistently produces spherulites while the extended coil assembles into disordered fibril bundles. This results suggests that these 2 PLE conformers directly affect the morphology of the resulting macroscopic amyloid assembly.

  10. Collapsed state of polyglutamic acid results in amyloid spherulite formation

    Science.gov (United States)

    Stehli, Daniel; Mulaj, Mentor; Miti, Tatiana; Traina, Joshua; Foley, Joseph; Muschol, Martin

    2015-01-01

    Self-assembly of proteins and peptides into amyloid fibrils involves multiple distinct intermediates and late-stage fibrillar polymorphs. Understanding the conditions and mechanisms that promote the formation of one type of intermediate and polymorph over the other represents a fundamental challenge. Answers to this question are also of immediate biomedical relevance since different amyloid aggregate species have been shown to have distinct pathogenic potencies. One amyloid polymorph that has received comparatively little attention are amyloid spherulites. Here we report that self-assembly of the intrinsically disordered polymer poly(L-glutamic) acid (PLE) can generate amyloid spherulites. We characterize spherulite growth kinetics, as well as the morphological, optical and tinctorial features of this amyloid polymorph previously unreported for PLE. We find that PLE spherulites share both tinctorial and structural characteristics with their amyloid fibril counterparts. Differences in PLE's molecular weight, polydispersity or chemistry could not explain the selective propensity toward either fibril or spherulite formation. Instead, we provide evidence that PLE polymers can exist in either a collapsed globule or an extended random coil conformation. The collapsed globule consistently produces spherulites while the extended coil assembles into disordered fibril bundles. This results suggests that these 2 PLE conformers directly affect the morphology of the resulting macroscopic amyloid assembly. PMID:28232889

  11. Membrane aggregation and perturbation induced by antimicrobial peptide of S-thanatin

    International Nuclear Information System (INIS)

    Wu, Guoqiu; Wu, Hongbin; Li, Linxian; Fan, Xiaobo; Ding, Jiaxuan; Li, Xiaofang; Xi, Tao; Shen, Zilong

    2010-01-01

    Thanatin, a 21-residue peptide, is an inducible insect peptide. In our previous study, we have identified a novel thanatin analog of S-thanatin, which exhibited a broad antimicrobial activity against bacteria and fungi with low hemolytic activity. This study was aimed to delineate the antimicrobial mechanism of S-thanatin and identify its interaction with bacterial membranes. In this study, membrane phospholipid was found to be the target for S-thanatin. In the presence of vesicles, S-thanatin interestingly led to the aggregation of anionic vesicles and sonicated bacteria. Adding S-thanatin to Escherichia coli suspension would result in the collapse of membrane and kill bacteria. The sensitivity assay of protoplast elucidated the importance of outer membrane (OM) for S-thanatin's antimicrobial activity. Compared with other antimicrobial peptide, S-thanatin produced chaotic membrane morphology and cell debris in electron microscopic appearance. These results supported our hypothesis that S-thanatin bound to negatively charged LPS and anionic lipid, impeded membrane respiration, exhausted the intracellular potential, and released periplasmic material, which led to cell death.

  12. Proteomic analysis of highly prevalent amyloid A amyloidosis endemic to endangered island foxes.

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    Patricia M Gaffney

    Full Text Available Amyloid A (AA amyloidosis is a debilitating, often fatal, systemic amyloid disease associated with chronic inflammation and persistently elevated serum amyloid A (SAA. Elevated SAA is necessary but not sufficient to cause disease and the risk factors for AA amyloidosis remain poorly understood. Here we identify an extraordinarily high prevalence of AA amyloidosis (34% in a genetically isolated population of island foxes (Urocyon littoralis with concurrent chronic inflammatory diseases. Amyloid deposits were most common in kidney (76%, spleen (58%, oral cavity (45%, and vasculature (44% and were composed of unbranching, 10 nm in diameter fibrils. Peptide sequencing by mass spectrometry revealed that SAA peptides were dominant in amyloid-laden kidney, together with high levels of apolipoprotein E, apolipoprotein A-IV, fibrinogen-α chain, and complement C3 and C4 (false discovery rate ≤ 0.05. Reassembled peptide sequences showed island fox SAA as an 111 amino acid protein, most similar to dog and artic fox, with 5 unique amino acid variants among carnivores. SAA peptides extended to the last two C-terminal amino acids in 5 of 9 samples, indicating that near full length SAA was often present in amyloid aggregates. These studies define a remarkably prevalent AA amyloidosis in island foxes with widespread systemic amyloid deposition, a unique SAA sequence, and the co-occurrence of AA with apolipoproteins.

  13. Amyloidpeptides and tau protein as biomarkers in cerebrospinal and interstitial fluid following traumatic brain injury: A review of experimental and clinical studies

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    Parmenion P. Tsitsopoulos

    2013-06-01

    Full Text Available Traumatic brain injury (TBI survivors frequently suffer from life-long deficits in cognitive functions and a reduced quality of life. Axonal injury, observed in most severe TBI patients, results in accumulation of amyloid precursor protein (APP. Post-injury enzymatic cleavage of APP can generate amyloid-β (Aβ peptides, a hallmark finding in Alzheimer’s disease (AD. At autopsy, brains of AD and a subset of TBI victims display some similarities including accumulation of Aβ peptides and neurofibrillary tangles of hyperphosphorylated tau proteins. Most epidemiological evidence suggests a link between TBI and AD, implying that TBI has neurodegenerative sequelae. Aβ peptides and tau may be used as biomarkers in interstitial fluid (ISF using cerebral microdialysis and/or cerebrospinal fluid (CSF following clinical TBI. In the present review, the available clinical and experimental literature on Aβ peptides and tau as potential biomarkers following TBI is comprehensively analyzed. Elevated CSF and ISF tau protein levels have been observed following severe TBI and suggested to correlate with clinical outcome. Although Aβ peptides are produced by normal neuronal metabolism, high levels of long and/or fibrillary Aβ peptides may be neurotoxic. Increased CSF and/or ISF Aβ levels post-injury may be related to neuronal activity and/or the presence of axonal injury. The heterogeneity of animal models, clinical cohorts, analytical techniques and the complexity of TBI in available studies make the clinical value of tau and Aβ as biomarkers uncertain at present. Additionally, the link between early post-injury changes in tau and Aβ peptides and the future risk of developing AD remains unclear. Future studies using e.g. rapid biomarker sampling combined with enhanced analytical techniques and/or novel pharmacological tools could provide additional information on the importance of Aβ peptides and tau protein in both the acute pathophysiology and long

  14. O-Hydroxyl- or o-amino benzylamine-tacrine hybrids: multifunctional biometals chelators, antioxidants, and inhibitors of cholinesterase activity and amyloidaggregation.

    Science.gov (United States)

    Mao, Fei; Huang, Ling; Luo, Zonghua; Liu, Anqiu; Lu, Chuanjun; Xie, Zhiyong; Li, Xingshu

    2012-10-01

    In an effort to identify novel multifunctional drug candidates for the treatment of Alzheimer's disease (AD), a series of hybrid molecules were synthesised by reacting N-(aminoalkyl)tacrine with salicylic aldehyde or derivatives of 2-aminobenzaldehyde. These compounds were then evaluated as multifunctional anti-Alzheimer's disease agents. All of the hybrids are potential biometal chelators, and in addition, most of them were better antioxidants and inhibitors of cholinesterases and amyloid-β (Aβ) aggregation than the lead compound tacrine. Compound 7c has the potential to be a candidate for AD therapy: it is a much better inhibitor of acetylcholinesterase (AChE) than tacrine (IC(50): 0.55 nM vs 109 nM), has good biometal chelation ability, is able to inhibit Aβ aggregation and has moderate antioxidant activity (1.22 Trolox equivalents). Copyright © 2012 Elsevier Ltd. All rights reserved.

  15. (-)-Meptazinol-melatonin hybrids as novel dual inhibitors of cholinesterases and amyloidaggregation with high antioxidant potency for Alzheimer's therapy.

    Science.gov (United States)

    Cheng, Shaobing; Zheng, Wei; Gong, Ping; Zhou, Qiang; Xie, Qiong; Yu, Lining; Zhang, Peiyi; Chen, Liangkang; Li, Juan; Chen, Jianxing; Chen, Hailin; Chen, Hongzhuan

    2015-07-01

    The multifactorial pathogenesis of Alzheimer's disease (AD) implicates that multi-target-directed ligands (MTDLs) intervention may represent a promising therapy for AD. Amyloid-β (Aβ) aggregation and oxidative stress, two prominent neuropathological hallmarks in patients, play crucial roles in the neurotoxic cascade of this disease. In the present study, a series of novel (-)-meptazinol-melatonin hybrids were designed, synthesized and biologically characterized as potential MTDLs against AD. Among them, hybrids 7-7c displayed higher dual inhibitory potency toward cholinesterases (ChEs) and better oxygen radical absorbance capacity (ORAC) than the parental drugs. Furthermore, compound 7c could effectively inhibit Aβ self-aggregation, showed favorable safety and the blood-brain barrier (BBB) permeability. Therefore, 7c may serve as a valuable candidate that is worthy of further investigations in the treatment of AD. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Intrinsic excitability changes induced by acute treatment of hippocampal CA1 pyramidal neurons with exogenous amyloid β peptide

    Science.gov (United States)

    Scullion, Sarah; Brown, Jon T.; Randall, Andrew D.

    2015-01-01

    ABSTRACT Accumulation of beta‐amyloid (Aβ) peptides in the human brain is a canonical pathological hallmark of Alzheimer's disease (AD). Recent work in Aβ‐overexpressing transgenic mice indicates that increased brain Aβ levels can be associated with aberrant epileptiform activity. In line with this, such mice can also exhibit altered intrinsic excitability (IE) of cortical and hippocampal neurons: these observations may relate to the increased prevalence of seizures in AD patients. In this study, we examined what changes in IE are produced in hippocampal CA1 pyramidal cells after 2–5 h treatment with an oligomeric preparation of synthetic human Aβ 1–42 peptide. Whole cell current clamp recordings were compared between Aβ‐(500 nM) and vehicle‐(DMSO 0.05%) treated hippocampal slices obtained from mice. The soluble Aβ treatment did not produce alterations in sub‐threshold intrinsic properties, including membrane potential, input resistance, and hyperpolarization activated “sag”. Similarly, no changes were noted in the firing profile evoked by 500 ms square current supra‐threshold stimuli. However, Aβ 500 nM treatment resulted in the hyperpolarization of the action potential (AP) threshold. In addition, treatment with Aβ at 500 nM depressed the after‐hyperpolarization that followed both a single AP or 50 Hz trains of a number of APs between 5 and 25. These data suggest that acute exposure to soluble Aβ oligomers affects IE properties of CA1 pyramidal neurons differently from outcomes seen in transgenic models of amyloidopathy. However, in both chronic and acute models, the IE changes are toward hyperexcitability, reinforcing the idea that amyloidopathy and increased incidence in seizures might be causally related in AD patients. © 2014 The Authors Hippocampus Published by Wiley Periodicals, Inc. PMID:25515596

  17. A synthetic peptide with the putative iron binding motif of amyloid precursor protein (APP does not catalytically oxidize iron.

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    Kourosh Honarmand Ebrahimi

    Full Text Available The β-amyloid precursor protein (APP, which is a key player in Alzheimer's disease, was recently reported to possess an Fe(II binding site within its E2 domain which exhibits ferroxidase activity [Duce et al. 2010, Cell 142: 857]. The putative ligands of this site were compared to those in the ferroxidase site of ferritin. The activity was indirectly measured using transferrin, which scavenges the Fe(III product of the reaction. A 22-residue synthetic peptide, named FD1, with the putative ferroxidase site of APP, and the E2 domain of APP were each reported to exhibit 40% of the ferroxidase activity of APP and of ceruloplasmin. It was also claimed that the ferroxidase activity of APP is inhibited by Zn(II just as in ferritin. We measured the ferroxidase activity indirectly (i by the incorporation of the Fe(III product of the ferroxidase reaction into transferrin and directly (ii by monitoring consumption of the substrate molecular oxygen. The results with the FD1 peptide were compared to the established ferroxidase activities of human H-chain ferritin and of ceruloplasmin. For FD1 we observed no activity above the background of non-enzymatic Fe(II oxidation by molecular oxygen. Zn(II binds to transferrin and diminishes its Fe(III incorporation capacity and rate but it does not specifically bind to a putative ferroxidase site of FD1. Based on these results, and on comparison of the putative ligands of the ferroxidase site of APP with those of ferritin, we conclude that the previously reported results for ferroxidase activity of FD1 and - by implication - of APP should be re-evaluated.

  18. Sildenafil protects neuronal cells from mitochondrial toxicity induced by β-amyloid peptide via ATP-sensitive K+ channels.

    Science.gov (United States)

    Son, Yonghae; Kim, Koanhoi; Cho, Hyok-Rae

    2018-06-02

    To understand the molecular mechanisms underlying the beneficial effects of sildenafil in animal models of neurological disorders, we investigated the effects of sildenafil on the mitochondrial toxicity induced by β-amyloid (Aβ) peptide. Treatment of HT-22 hippocampal neuronal cells with Aβ 25∼35 results in increased mitochondrial Ca 2+ load, which is subsequently suppressed by sildenafil as well as by diazoxide, a selective opener of the ATP-sensitive K + channels (K ATP ). However, the suppressive effects of sildenafil and diazoxide are significantly attenuated by 5-hydroxydecanoic acid (5-HD), a K ATP inhibitor. The increased mitochondrial Ca 2+ overload is accompanied by decrease in the intracellular ATP concentration, increase in intracellular ROS generation, occurrence of mitochondrial permeability transition, and activation of caspase-9 and cell death. Exposure to sildenafil inhibited the mitochondria-associated changes and cell death induced by Aβ. However, the inhibitory effects of sildenafil are abolished or weakened in the presence of 5-HD, suggesting that opening of the mitochondrial K ATP is required for sildenafil to exert these effects. Taken together, these results indicate that at the mitochondrial levels, sildenafil plays a protective role towards neuronal cell in an environment rich in Aβ, and exerts its effects via the mitochondrial K ATP channels-dependent mechanisms. Copyright © 2018 Elsevier Inc. All rights reserved.

  19. Antagonizing beta-amyloid peptide neurotoxicity of the anti-aging fungus Ganoderma lucidum.

    Science.gov (United States)

    Lai, Cora Sau-Wan; Yu, Man-Shan; Yuen, Wai-Hung; So, Kwok-Fai; Zee, Sze-Yong; Chang, Raymond Chuen-Chung

    2008-01-23

    Ganoderma lucidum (Leyss. ex Fr.) Karst. (Lingzhi) is a medicinal fungus used clinically in many Asian countries to promote health and longevity. Synaptic degeneration is another key mode of neurodegeneration in Alzheimer's disease (AD). Recent studies have shown the loss of synaptic density proteins in each individual neuron during the progression of AD. It was recently reported that beta-amyloid (Abeta) could cause synaptic dysfunction and contribute to AD pathology. In this study, we reported that aqueous extract of G. lucidum significantly attenuated Abeta-induced synaptotoxicity by preserving the synaptic density protein, synaptophysin. In addition, G. lucidum aqueous extract antagonized Abeta-triggered DEVD cleavage activities in a dose-dependent manner. Further studies elucidated that phosphorylation of c-Jun N-terminal kinase, c-Jun, and p38 MAP kinase was attenuated by G. lucidum in Abeta-stressed neurons. Taken together, the results prove a hypothesis that anti-aging G. lucidum can prevent harmful effects of the exterminating toxin Abeta in AD.

  20. Accumulation of Exogenous Amyloid-Beta Peptide in Hippocampal Mitochondria Causes Their Dysfunction: A Protective Role for Melatonin

    Directory of Open Access Journals (Sweden)

    Sergio Rosales-Corral

    2012-01-01

    Full Text Available Amyloid-beta (Aβ pathology is related to mitochondrial dysfunction accompanied by energy reduction and an elevated production of reactive oxygen species (ROS. Monomers and oligomers of Aβ have been found inside mitochondria where they accumulate in a time-dependent manner as demonstrated in transgenic mice and in Alzheimer’s disease (AD brain. We hypothesize that the internalization of extracellular Aβ aggregates is the major cause of mitochondrial damage and here we report that following the injection of fibrillar Aβ into the hippocampus, there is severe axonal damage which is accompanied by the entrance of Aβ into the cell. Thereafter, Aβ appears in mitochondria where it is linked to alterations in the ionic gradient across the inner mitochondrial membrane. This effect is accompanied by disruption of subcellular structure, oxidative stress, and a significant reduction in both the respiratory control ratio and in the hydrolytic activity of ATPase. Orally administrated melatonin reduced oxidative stress, improved the mitochondrial respiratory control ratio, and ameliorated the energy imbalance.

  1. Investigation of Amyloid Structures at Nanoscale via AFM based Dynamic Nanomechncial Microscopy

    DEFF Research Database (Denmark)

    Zhang, Shuai

    2014-01-01

    Amyloid structures are one important kind of protein aggregations. They are a group of stable misfolded species, other than native states, which have been found to accumulate as plaques on neuron cells. This behavior is considered to associate with tens of human neurodegenerative diseases...... summarized the main methodologies of DNM. I also utilized DNM to explore the path way of amyloid self-assembly, and the substrate effect to the conformation of amyloid structures. Furthermore, 2D peptide based material has also been characterized by DNM....

  2. Serum β-amyloid peptide levels spike in the early stage of Alzheimer-like plaque pathology in an APP/PS1 double transgenic mouse model.

    Science.gov (United States)

    He, Jue; Qiao, Jin-Ping; Zhu, Shenghua; Xue, Mengzhou; Chen, Wenwu; Wang, Xinchun; Tempier, Adrien; Huang, Qingjun; Kong, Jiming; Li, Xin-Min

    2013-11-01

    Serum levels of β-amyloid (Aβ) peptides may represent an early biomarker in the diagnosis of Alzheimer's disease (AD). In the present study, we investigated the temporal kinetic changes in the levels of serum Aβ 1-42 and 40 in an amyloid precursor protein (APP)/presenilin (PS)1 double transgenic mouse model of AD. Serum Aβ peptide levels in 2-, 3-, 6-, 9- and 18-month old, and liver Aβ 1-40 level in 6-month old mice were measured using enzyme-linked immunosorbent assay (ELISA) kits. Results revealed that serum Aβ levels peaked in 3-month old transgenic mice, and the Aβ level in non-transgenic and transgenic mice is comparable in liver. Compared to the 6-month old transgenic mice, Congo red staining showed that the 3-month old transgenic mice had minimum brain Aβ plaques, corresponding to the early stage of Alzheimer-like plaque pathology, and confocal microscope images showed that the deposition of Aβ in their cerebral vessels was minimal. Furthermore, results of the water maze test, showed that memory was normal for the 3- month old transgenic mice when compared to age-matched non-transgenic mice. These results suggest that serum Aβ peptide levels may be peaked during the early stage of AD. Monitoring serum Aβ peptide levels in the potential AD population may provide an early diagnosis of AD prior to the appearance of clinical symptoms.

  3. Semisynthesis and Structure-Activity Studies of Uncarinic Acid C Isolated from Uncaria rhynchophylla as a Specific Inhibitor of the Nucleation Phase in Amyloid β42 Aggregation.

    Science.gov (United States)

    Yoshioka, Takuya; Murakami, Kazuma; Ido, Kyohei; Hanaki, Mizuho; Yamaguchi, Kanoko; Midorikawa, Satohiro; Taniwaki, Shinji; Gunji, Hiroki; Irie, Kazuhiro

    2016-10-28

    Oligomers of the 42-mer amyloid-β protein (Aβ42), rather than fibrils, cause synaptic dysfunction in the pathology of Alzheimer's disease (AD). The nucleation phase in a nucleation-dependent aggregation model of Aβ42 is related to the formation of oligomers. Uncaria rhynchophylla is one component of "Yokukansan", a Kampo medicine, which is widely used for treating AD symptoms. Previously, an extract of U. rhynchophylla was found to reduce the aggregation of Aβ42, but its active principles have yet to be identified. In the present work, uncarinic acid C (3) was identified as an inhibitor of Aβ42 aggregation that is present in U. rhynchophylla. Moreover, compound 3 acted as a specific inhibitor of the nucleation phase of Aβ42 aggregation. Compound 3 was synthesized from saponin A (10), an abundant byproduct of rutin purified from Uncaria elliptica. Comprehensive structure-activity studies on 3 suggest that both a C-27 ferulate and a C-28 carboxylic acid group are required for its inhibitory activity. These findings may aid the development of oligomer-specific inhibitors for AD therapy.

  4. The metabolic enhancer piracetam ameliorates the impairment of mitochondrial function and neurite outgrowth induced by ß-amyloid peptide

    Science.gov (United States)

    Kurz, C; Ungerer, I; Lipka, U; Kirr, S; Schütt, T; Eckert, A; Leuner, K; Müller, WE

    2010-01-01

    Background and purpose: β-Amyloid peptide (Aβ) is implicated in the pathogenesis of Alzheimer's disease by initiating a cascade of events from mitochondrial dysfunction to neuronal death. The metabolic enhancer piracetam has been shown to improve mitochondrial dysfunction following brain aging and experimentally induced oxidative stress. Experimental approach: We used cell lines (PC12 and HEK cells) and murine dissociated brain cells. The protective effects of piracetam in vitro and ex vivo on Aβ-induced impairment of mitochondrial function (as mitochondrial membrane potential and ATP production), on secretion of soluble Aβ and on neurite outgrowth in PC12 cells were investigated. Key results: Piracetam improves mitochondrial function of PC12 cells and acutely dissociated brain cells from young NMRI mice following exposure to extracellular Aβ1-42. Similar protective effects against Aβ1-42 were observed in dissociated brain cells from aged NMRI mice, or mice transgenic for mutant human amyloid precursor protein (APP) treated with piracetam for 14 days. Soluble Aβ load was markedly diminished in the brain of those animals after treatment with piracetam. Aβ production by HEK cells stably transfected with mutant human APP was elevated by oxidative stress and this was reduced by piracetam. Impairment of neuritogenesis is an important consequence of Aβ-induced mitochondrial dysfunction and Aβ-induced reduction of neurite growth in PC12 cells was substantially improved by piracetam. Conclusion and implications: Our findings strongly support the concept of improving mitochondrial function as an approach to ameliorate the detrimental effects of Aβ on brain function. This article is commented on by Moncada, pp. 217–219 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2010.00706.x and to view related papers by Pravdic et al. and Puerta et al. visit http://dx.doi.org/10.1111/j.1476-5381.2010.00698.x and http://dx.doi.org/10.1111/j

  5. Weak glycolipid binding of a microdomain-tracer peptide correlates with aggregation and slow diffusion on cell membranes.

    Directory of Open Access Journals (Sweden)

    Tim Lauterbach

    Full Text Available Organized assembly or aggregation of sphingolipid-binding ligands, such as certain toxins and pathogens, has been suggested to increase binding affinity of the ligand to the cell membrane and cause membrane reorganization or distortion. Here we show that the diffusion behavior of the fluorescently tagged sphingolipid-interacting peptide probe SBD (Sphingolipid Binding Domain is altered by modifications in the construction of the peptide sequence that both result in a reduction in binding to ganglioside-containing supported lipid membranes, and at the same time increase aggregation on the cell plasma membrane, but that do not change relative amounts of secondary structural features. We tested the effects of modifying the overall charge and construction of the SBD probe on its binding and diffusion behavior, by Surface Plasmon Resonance (SPR; Biacore analysis on lipid surfaces, and by Fluorescence Correlation Spectroscopy (FCS on live cells, respectively. SBD binds preferentially to membranes containing the highly sialylated gangliosides GT1b and GD1a. However, simple charge interactions of the peptide with the negative ganglioside do not appear to be a critical determinant of binding. Rather, an aggregation-suppressing amino acid composition and linker between the fluorophore and the peptide are required for optimum binding of the SBD to ganglioside-containing supported lipid bilayer surfaces, as well as for interaction with the membrane. Interestingly, the strength of interactions with ganglioside-containing artificial membranes is mirrored in the diffusion behavior by FCS on cell membranes, with stronger binders displaying similar characteristic diffusion profiles. Our findings indicate that for aggregation-prone peptides, aggregation occurs upon contact with the cell membrane, and rather than giving a stronger interaction with the membrane, aggregation is accompanied by weaker binding and complex diffusion profiles indicative of heterogeneous

  6. Metabolic changes precede proteostatic dysfunction in a Drosophila model of Abeta peptide toxicity

    DEFF Research Database (Denmark)

    Ott, Stanislav; Vishnivetskaya, Anastasia; Malmendal, Anders

    2016-01-01

    Amyloid beta (Aβ) peptide aggregation is linked to the initiation of Alzheimer's disease; accordingly, aggregation-prone isoforms of Aβ, expressed in the brain, shorten the lifespan of Drosophila melanogaster. However, the lethal effects of Aβ are not apparent until after day 15. We used shibireT...

  7. Imaging β-amyloid fibrils in Alzheimer's disease: a critical analysis through simulation of amyloid fibril polymerization

    International Nuclear Information System (INIS)

    Shoghi-Jadid, Kooresh; Barrio, Jorge R.; Kepe, Vladimir; Wu, H.-M.; Small, Gary W.; Phelps, Michael E.; Huang, S.-C.

    2005-01-01

    The polymerization of β-amyloid (Aβ) peptides into fibrillary plaques is implicated, in part, in the pathogenesis of Alzheimer's disease. Aβ molecular imaging probes (Aβ-MIPs) have been introduced in an effort to quantify amyloid burden or load, in subjects afflicted with AD by invoking the classic PET receptor model for the quantitation of neuronal receptor density. In this communication, we explore conceptual differences between imaging the density of amyloid fibril polymers and neuronal receptors. We formulate a mathematical model for the polymerization of Aβ with parameters that are mapped to biological modulators of fibrillogenesis and introduce a universal measure for amyloid load to accommodate various interactions of Aβ-MIPs with fibrils. Subsequently, we hypothesize four Aβ-MIPs and utilize the fibrillogenesis model to simulate PET tissue time activity curves (TACs). Given the unique nature of polymer growth and resulting PET TAC, the four probes report differing amyloid burdens for a given brain pathology, thus complicating the interpretation of PET images. In addition, we introduce the notion of an MIP's resolution, apparent maximal binding site concentration, optimal kinetic topology and its resolving power in characterizing the pathological progression of AD and the effectiveness of drug therapy. The concepts introduced in this work call for a new paradigm that goes beyond the classic parameters B max and K D to include binding characteristics to polymeric peptide aggregates such as amyloid fibrils, neurofibrillary tangles and prions

  8. Nanoparticles and amyloid systems: A fatal encounter?

    Energy Technology Data Exchange (ETDEWEB)

    Abel, Bernd [Leibniz Institute of Surface Modification, Chemical Department, Permoserstr. 15, D-04318 Leipzig, Germany and Wilhelm-Ostwald-Institute for Physical and Theoretical Chemistry, Linnéstr. 3, D-04103 Leipzig (Germany)

    2014-10-06

    Nanoparticles (NPs) are used in many products of our daily life, however, there has been concern that they may also be harmful to human health. Recently NPs have been found to accelerate the fibrillation kinetics of amyloid systems. In the past this has been preliminarily attributed to a nucleation effect. Nanoparticle surfaces and interfaces appear to limit the degrees of freedom of amyloid systems (i.e., peptides and proteins) due to a phase space constraint such that rapid cross-beta structures are formed faster than without interface interactions and in turn fibril formation is enhanced significantly. Here we explore if lipid bilayers in the form of liposomes (140nm) also accelerate fibril formation for amyloid systems. We have investigated a fragment NNFGAIL of the Human islet amyloid polypeptide (hIAPP) in contact with 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) liposomes in aqueous solution. We found that the lipid bilayer vesicles do accelerate fibril formation in time-resolved off-line detected atomic force microscopy experiments. Characteristic Thioflavine-T fluorescence on the same structures verify that the structures consist of aggregated peptides in a typical cross-β-structure arrangement.

  9. Neurine, an acetylcholine autolysis product, elevates secreted amyloid-beta protein precursor and amyloid-beta peptide levels, and lowers neuronal cell viability in culture: a role in Alzheimer's disease?

    Science.gov (United States)

    Tweedie, David; Brossi, Arnold; Chen, DeMoa; Ge, Yuan-Wen; Bailey, Jason; Yu, Qian-Sheng; Kamal, Mohammad A; Sambamurti, Kumar; Lahiri, Debomoy K; Greig, Nigel H

    2006-09-01

    Classical hallmarks of Alzheimer's disease (AD) are a synaptic loss, cholinergic neuron death, and abnormal protein deposition, particularly of toxic amyloid-beta peptide (Abeta) that is derived from amyloid-beta protein precursor (AbetaPP) by the action of beta- and gamma-secretases. The trigger(s) initiating the biochemical cascades that underpin these hallmarks have yet to be fully elucidated. The typical forebrain cholinergic cell demise associated with AD brain results in a loss of presynaptic cholinergic markers and acetylcholine (ACh). Neurine (vinyl-trimethyl-ammonium hydroxide) is a breakdown product of ACh, consequent to autolysis and is an organic poison found in cadavre brain. The time- and concentration-dependent actions of neurine were assessed in human neuroblastoma (NB, SK-N-SH) cells in culture by quantifying cell viability by lactate dehydrogenase (LDH) and MTS assay, and AbetaPP and Abeta levels by Western blot and ELISA. NB cells displayed evidence of toxicity to neurine at > or = 3 mg/ml, as demonstrated by elevated LDH levels in the culture media and a reduced cell viability shown by the MTS assay. Using subtoxic concentrations of neurine, elevations in AbetaPP and Abeta1-40 peptide levels were detected in conditioned media samples.

  10. Exendin-4, a glucagon-like peptide 1 receptor agonist, protects against amyloidpeptide-induced impairment of spatial learning and memory in rats.

    Science.gov (United States)

    Jia, Xiao-Tao; Ye-Tian; Yuan-Li; Zhang, Ge-Juan; Liu, Zhi-Qin; Di, Zheng-Li; Ying, Xiao-Ping; Fang, Yan; Song, Er-Fei; Qi, Jin-Shun; Pan, Yan-Fang

    2016-05-15

    Type 2 diabetes mellitus (T2DM) and Alzheimer's disease (AD) share specific molecular mechanisms, and agents with proven efficacy in one may be useful against the other. The glucagon-like peptide-1 (GLP-1) receptor agonist exendin-4 has similar properties to GLP-1 and is currently in clinical use for T2DM treatment. Thus, this study was designed to characterize the effects of exendin-4 on the impairment of learning and memory induced by amyloid protein (Aβ) and its probable molecular underlying mechanisms. The results showed that (1) intracerebroventricular (i.c.v.) injection of Aβ1-42 resulted in a significant decline of spatial learning and memory of rats in water maze tests; (2) pretreatment with exendin-4 effectively and dose-dependently protected against the Aβ1-42-induced impairment of spatial learning and memory; (3) exendin-4 treatment significantly decreased the expression of Bax and cleaved caspase-3 and increased the expression of Bcl2 in Aβ1-42-induced Alzheimer's rats. The vision and swimming speed of the rats among all groups in the visible platform tests did not show any difference. These findings indicate that systemic pretreatment with exendin-4 can effectively prevent the behavioral impairment induced by neurotoxic Aβ1-42, and the underlying protective mechanism of exendin-4 may be involved in the Bcl2, Bax and caspase-3 pathways. Thus, the application of exendin-4 or the activation of its signaling pathways may be a promising strategy to ameliorate the degenerative processes observed in AD. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Generation and Partial Characterization of Rabbit Monoclonal Antibody to AmyloidPeptide 1-37 (Aβ37).

    Science.gov (United States)

    Mehta, Pankaj D; Blain, Jean-Francois; Freeman, Emily A; Patrick, Bruce A; Barshatzky, Marc; Hrdlicka, Lori A; Mehta, Sangita P; Frackowiak, Janusz; Mazur-Kolecka, Bozena; Wegiel, Jerzy; Patzke, Holger; Miller, David L

    2017-01-01

    Secreted soluble amyloid-β 1-37 (Aβ37) peptide is one of the prominent Aβ forms next to Aβ40, and is found in cerebrospinal fluid (CSF) and blood. Recent studies have shown the importance of quantitation of CSF Aβ37 levels in combination with Aβ38, Aβ40, and Aβ42 to support the diagnosis of patients with probable Alzheimer's disease (AD), and the value of antibody to Aβ37 to facilitate drug discovery studies. However, the availability of reliable and specific monoclonal antibody to Aβ37 is very limited. Our aims were: 1) to generate and partially characterize rabbit monoclonal antibody (RabmAb) to Aβ37, and 2) to determine whether the antibody detects changes in Aβ37 levels produced by a γ-secretase modulator (GSM). Our generated RabmAb to Aβ37 was found to be specific to Aβ37, since it did not react with Aβ36, Aβ38, Aβ39, Aβ40, and Aβ42 in an ELISA or immunoblotting. The epitope of the antibody was contained in the seven C-terminal residues of Aβ37. The antibody was sensitive enough to measure CSF and plasma Aβ37 levels in ELISA. Immunohistological studies showed the presence of Aβ37-positive deposits in the brain of AD, and Down syndrome persons diagnosed with AD. Our studies also showed that the antibody detected Aβ37 increases in CSF and brains of rodents following treatment with a GSM. Thus, our antibody can be widely applied to AD research, and in a panel based approach it may have potential to support the diagnosis of probable AD, and in testing the effect of GSMs to target AD.

  12. Activation of the endoplasmic reticulum stress response by the amyloid-beta 1-40 peptide in brain endothelial cells.

    Science.gov (United States)

    Fonseca, Ana Catarina R G; Ferreiro, Elisabete; Oliveira, Catarina R; Cardoso, Sandra M; Pereira, Cláudia F

    2013-12-01

    Neurovascular dysfunction arising from endothelial cell damage is an early pathogenic event that contributes to the neurodegenerative process occurring in Alzheimer's disease (AD). Since the mechanisms underlying endothelial dysfunction are not fully elucidated, this study was aimed to explore the hypothesis that brain endothelial cell death is induced upon the sustained activation of the endoplasmic reticulum (ER) stress response by amyloid-beta (Aβ) peptide, which deposits in the cerebral vessels in many AD patients and transgenic mice. Incubation of rat brain endothelial cells (RBE4 cell line) with Aβ1-40 increased the levels of several markers of ER stress-induced unfolded protein response (UPR), in a time-dependent manner, and affected the Ca(2+) homeostasis due to the release of Ca(2+) from this intracellular store. Finally, Aβ1-40 was shown to activate both mitochondria-dependent and -independent apoptotic cell death pathways. Enhanced release of cytochrome c from mitochondria and activation of the downstream caspase-9 were observed in cells treated with Aβ1-40 concomitantly with caspase-12 activation. Furthermore, Aβ1-40 activated the apoptosis effectors' caspase-3 and promoted the translocation of apoptosis-inducing factor (AIF) to the nucleus demonstrating the involvement of caspase-dependent and -independent mechanisms during Aβ-induced endothelial cell death. In conclusion, our data demonstrate that ER stress plays a significant role in Aβ1-40-induced apoptotic cell death in brain endothelial cells suggesting that ER stress-targeted therapeutic strategies might be useful in AD to counteract vascular defects and ultimately neurodegeneration. © 2013.

  13. Isobaric Quantification of Cerebrospinal Fluid AmyloidPeptides in Alzheimer's Disease: C-Terminal Truncation Relates to Early Measures of Neurodegeneration.

    Science.gov (United States)

    Rogeberg, Magnus; Almdahl, Ina Selseth; Wettergreen, Marianne; Nilsson, Lars N G; Fladby, Tormod

    2015-11-06

    The amyloid beta (Aβ) peptide is the main constituent of the plaques characteristic of Alzheimer's disease (AD). Measurement of Aβ1-42 in cerebrospinal fluid (CSF) is a valuable marker in AD research, where low levels indicate AD. Although the use of immunoassays measuring Aβ1-38 and Aβ1-40 in addition to Aβ1-42 has increased, quantitative assays of other Aβ peptides remain rarely explored. We recently discovered novel Aβ peptides in CSF using antibodies recognizing the Aβ mid-domain region. Here we have developed a method using both Aβ N-terminal and mid-domain antibodies for immunoprecipitation in combination with isobaric labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for relative quantification of endogenous Aβ peptides in CSF. The developed method was used in a pilot study to produce Aβ peptide profiles from 38 CSF samples. Statistical comparison between CSF samples from 19 AD patients and 19 cognitively healthy controls revealed no significant differences at group level. A significant correlation was found between several larger C-terminally truncated Aβ peptides and protein biomarkers for neuronal damage, particularly prominent in the control group. Comparison of the isobaric quantification with immunoassays measuring Aβ1-38 or Aβ1-40 showed good correlation (r(2) = 0.84 and 0.85, respectively) between the two analysis methods. The developed method could be used to assess disease-modifying therapies directed at Aβ production or degradation.

  14. Aggregation process of Aβ1-40 with non-Aβ amyloid component of α-synuclein

    International Nuclear Information System (INIS)

    Eugene, Cindie; Mousseau, Normand

    2015-01-01

    Many neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases, are characterized by the presence of amyloid fibers. Recently, attention has turned from the fibers to the early stages of oligomerization where toxicity could be highest. Here, we focus on the interactions between non-Aβ amyloid component of a-synuclein (NAC) and Aβ 1-40 , two proteins found in amyloid fibrils associated with Alzheimer's disease. We combine the coarse-grained OPEP potential with a Hamiltonian and temperature replica exchange molecular dynamics simulation (HT-REMD) to identify mechanisms leading to the formation of secondary structures promoting fibrillation. We observe that the propensity to form beta-sheet remains the same for Aβ 1-40 whereas is decreases significantly for NAC. In particular, the 25-35 region of Aβ 1-40 is a significant area of secondary structure stabilization with NAC. The ionic interactions between salt-bridge D23 and K28 in Aβ 1-40 and K20 and E23 in NAC of the heterogeneous dimer are consistent with the salt-bridges found in NAC and Aβ 1-40 homogenous dimers and allow us to see that these interactions don't necessarily dominate the interchain stabilizations. Our numerical simulation also show the formation of interaction between the early oligomer of NAC and Aβ 1-40 . (paper)

  15. The influence of pathological mutations and proline substitutions in TDP-43 glycine-rich peptides on its amyloid properties and cellular toxicity.

    Directory of Open Access Journals (Sweden)

    Chia-Sui Sun

    Full Text Available TAR DNA-binding protein (TDP-43 was identified as the major ubiquitinated component deposited in the inclusion bodies in amyotrophic lateral sclerosis (ALS and frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U in 2006. Later on, numerous ALS-related mutations were found in either the glycine or glutamine/asparagine-rich region on the TDP-43 C-terminus, which hinted on the importance of mutations on the disease pathogenesis. However, how the structural conversion was influenced by the mutations and the biological significance of these peptides remains unclear. In this work, various peptides bearing pathogenic or de novo designed mutations were synthesized and displayed their ability to form twisted amyloid fibers, cause liposome leakage, and mediate cellular toxicity as confirmed by transmission electron microscopy (TEM, circular dichroism (CD, Thioflavin T (ThT assay, Raman spectroscopy, calcein leakage assay, and cell viability assay. We have also shown that replacing glycines with prolines, known to obstruct β-sheet formation, at the different positions in these peptides may influence the amyloidogenesis process and neurotoxicity. In these cases, GGG308PPP mutant was not able to form beta-amyloid, cause liposome leakage, nor jeopardized cell survival, which hinted on the importance of the glycines (308-310 during amyloidogenesis.

  16. pH-dependence of the specific binding of Cu(II) and Zn(II) ions to the amyloidpeptide

    International Nuclear Information System (INIS)

    Ghalebani, Leila; Wahlström, Anna; Danielsson, Jens; Wärmländer, Sebastian K.T.S.; Gräslund, Astrid

    2012-01-01

    Highlights: ► Cu(II) and Zn(II) display pH-dependent binding to the Aβ(1–40) peptide. ► At pH 7.4 both metal ions display residue-specific binding to the Aβ peptide. ► At pH 5.5 the binding specificity is lost for Zn(II). ► Differential Cu(II) and Zn(II) binding may help explain metal-induced AD toxicity. -- Abstract: Metal ions like Cu(II) and Zn(II) are accumulated in Alzheimer’s disease amyloid plaques. The amyloid-β (Aβ) peptide involved in the disease interacts with these metal ions at neutral pH via ligands provided by the N-terminal histidines and the N-terminus. The present study uses high-resolution NMR spectroscopy to monitor the residue-specific interactions of Cu(II) and Zn(II) with 15 N- and 13 C, 15 N-labeled Aβ(1–40) peptides at varying pH levels. At pH 7.4 both ions bind to the specific ligands, competing with one another. At pH 5.5 Cu(II) retains its specific histidine ligands, while Zn(II) seems to lack residue-specific interactions. The low pH mimics acidosis which is linked to inflammatory processes in vivo. The results suggest that the cell toxic effects of redox active Cu(II) binding to Aβ may be reversed by the protective activity of non-redox active Zn(II) binding to the same major binding site under non-acidic conditions. Under acidic conditions, the protective effect of Zn(II) may be decreased or changed, since Zn(II) is less able to compete with Cu(II) for the specific binding site on the Aβ peptide under these conditions.

  17. Calcium binding to beta-2-microglobulin at physiological pH drives the occurrence of conformational changes which cause the protein to precipitate into amorphous forms that subsequently transform into amyloid aggregates.

    Directory of Open Access Journals (Sweden)

    Sukhdeep Kumar

    Full Text Available Using spectroscopic, calorimetric and microscopic methods, we demonstrate that calcium binds to beta-2-microglobulin (β2m under physiological conditions of pH and ionic strength, in biological buffers, causing a conformational change associated with the binding of up to four calcium atoms per β2m molecule, with a marked transformation of some random coil structure into beta sheet structure, and culminating in the aggregation of the protein at physiological (serum concentrations of calcium and β2m. We draw attention to the fact that the sequence of β2m contains several potential calcium-binding motifs of the DXD and DXDXD (or DXEXD varieties. We establish (a that the microscopic aggregation seen at physiological concentrations of β2m and calcium turns into actual turbidity and visible precipitation at higher concentrations of protein and β2m, (b that this initial aggregation/precipitation leads to the formation of amorphous aggregates, (c that the formation of the amorphous aggregates can be partially reversed through the addition of the divalent ion chelating agent, EDTA, and (d that upon incubation for a few weeks, the amorphous aggregates appear to support the formation of amyloid aggregates that bind to the dye, thioflavin T (ThT, resulting in increase in the dye's fluorescence. We speculate that β2m exists in the form of microscopic aggregates in vivo and that these don't progress to form larger amyloid aggregates because protein concentrations remain low under normal conditions of kidney function and β2m degradation. However, when kidney function is compromised and especially when dialysis is performed, β2m concentrations probably transiently rise to yield large aggregates that deposit in bone joints and transform into amyloids during dialysis related amyloidosis.

  18. Soluble γ-secretase modulators selectively inhibit the production of the 42-amino acid amyloid β peptide variant and augment the production of multiple carboxy-truncated amyloid β species.

    Science.gov (United States)

    Wagner, Steven L; Zhang, Can; Cheng, Soan; Nguyen, Phuong; Zhang, Xulun; Rynearson, Kevin D; Wang, Rong; Li, Yueming; Sisodia, Sangram S; Mobley, William C; Tanzi, Rudolph E

    2014-02-04

    Alzheimer's disease (AD) is characterized pathologically by an abundance of extracellular neuritic plaques composed primarily of the 42-amino acid amyloid β peptide variant (Aβ42). In the majority of familial AD (FAD) cases, e.g., those harboring mutations in presenilin 1 (PS1), there is a relative increase in the levels of Aβ42 compared to the levels of Aβ40. We previously reported the characterization of a series of aminothiazole-bridged aromates termed aryl aminothiazole γ-secretase modulators or AGSMs [Kounnas, M. Z., et al. (2010) Neuron 67, 769-780] and showed their potential for use in the treatment of FAD [Wagner, S. L., et al. (2012) Arch. Neurol. 69, 1255-1258]. Here we describe a series of GSMs with physicochemical properties improved compared to those of AGSMs. Specific heterocycle replacements of the phenyl rings in AGSMs provided potent molecules with improved aqueous solubilities. A number of these soluble γ-secretase modulators (SGSMs) potently lowered Aβ42 levels without inhibiting proteolysis of Notch or causing accumulation of amyloid precursor protein carboxy-terminal fragments, even at concentrations approximately 1000-fold greater than their IC50 values for reducing Aβ42 levels. The effects of one potent SGSM on Aβ peptide production were verified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, showing enhanced production of a number of carboxy-truncated Aβ species. This SGSM also inhibited Aβ42 peptide production in a highly purified reconstituted γ-secretase in vitro assay system and retained the ability to modulate γ-secretase-mediated proteolysis in a stably transfected cell culture model overexpressing a human PS1 mutation validating the potential for use in FAD.

  19. Functional amyloids in bacteria.

    Science.gov (United States)

    Romero, Diego; Kolter, Roberto

    2014-06-01

    The term amyloidosis is used to refer to a family of pathologies altering the homeostasis of human organs. Despite having a name that alludes to starch content, the amyloid accumulations are made up of proteins that polymerize as long and rigid fibers. Amyloid proteins vary widely with respect to their amino acid sequences but they share similarities in their quaternary structure; the amyloid fibers are enriched in β-sheets arranged perpendicular to the axis of the fiber. This structural feature provides great robustness, remarkable stability, and insolubility. In addition, amyloid proteins specifically stain with certain dyes such as Congo red and thioflavin-T. The aggregation into amyloid fibers, however, it is not restricted to pathogenic processes, rather it seems to be widely distributed among proteins and polypeptides. Amyloid fibers are present in insects, fungi and bacteria, and they are important in maintaining the homeostasis of the organism. Such findings have motivated the use of the term "functional amyloid" to differentiate these amyloid proteins from their toxic siblings. This review focuses on systems that have evolved in bacteria that control the expression and assembly of amyloid proteins on cell surfaces, such that the robustness of amyloid proteins are used towards a beneficial end. Copyright© by the Spanish Society for Microbiology and Institute for Catalan Studies.

  20. CD147 is a regulatory subunit of the gamma-secretase complex inAlzheimer's disease amyloid beta-peptide production

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Shuxia; Zhou, Hua; Walian, Peter J.; Jap, Bing K.

    2005-04-06

    {gamma}-secretase is a membrane protein complex that cleaves the {beta}-amyloid precursor protein (APP) within the transmembrane region, following prior processing by {beta}-secretase, producing amyloid {beta}-peptides (A{beta}{sub 40} and A{beta}{sub 42}). Errant production of A{beta}-peptides that substantially increases A{beta}{sub 42} production has been associated with the formation of amyloid plaques in Alzheimer's disease patients. Biophysical and genetic studies indicate that presenilin-1 (Psn-1), which contains the proteolytic active site, and three other membrane proteins, nicastrin (Nct), APH-1, and PEN-2 are required to form the core of the active {gamma}-secretase complex. Here, we report the purification of the native {gamma}-secretase complexes from HeLa cell membranes and the identification of an additional {gamma}-secretase complex subunit, CD147, a transmembrane glycoprotein with two immunoglobulin-like domains. The presence of this subunit as an integral part of the complex itself was confirmed through co-immunoprecipitation studies of the purified protein from HeLa cells and solubilized complexes from other cell lines such as neural cell HCN-1A and HEK293. Depletion of CD147 by RNA interference was found to increase the production of A{beta} peptides without changing the expression level of the other {gamma}-secretase components or APP substrates while CD147 overexpression had no statistically significant effect on amyloid {beta}-peptide production, other {gamma}-secretase components or APP substrates, indicating that the presence of the CD147 subunit within the {gamma}-secretase complex directly down-modulates the production of A{beta}-peptides. {gamma}-secretase was first recognized through its role in the production of the A{beta} peptides that are pathogenic in Alzheimer's disease (AD) (1). {gamma}-secretase is a membrane protein complex with unusual aspartyl protease activity that cleaves a variety of type I membrane proteins

  1. Oxidation of the tryptophan 32 residue of human superoxide dismutase 1 caused by its bicarbonate-dependent peroxidase activity triggers the non-amyloid aggregation of the enzyme.

    Science.gov (United States)

    Coelho, Fernando R; Iqbal, Asif; Linares, Edlaine; Silva, Daniel F; Lima, Filipe S; Cuccovia, Iolanda M; Augusto, Ohara

    2014-10-31

    The role of oxidative post-translational modifications of human superoxide dismutase 1 (hSOD1) in the amyotrophic lateral sclerosis (ALS) pathology is an attractive hypothesis to explore based on several lines of evidence. Among them, the remarkable stability of hSOD1(WT) and several of its ALS-associated mutants suggests that hSOD1 oxidation may precede its conversion to the unfolded and aggregated forms found in ALS patients. The bicarbonate-dependent peroxidase activity of hSOD1 causes oxidation of its own solvent-exposed Trp(32) residue. The resulting products are apparently different from those produced in the absence of bicarbonate and are most likely specific for simian SOD1s, which contain the Trp(32) residue. The aims of this work were to examine whether the bicarbonate-dependent peroxidase activity of hSOD1 (hSOD1(WT) and hSOD1(G93A) mutant) triggers aggregation of the enzyme and to comprehend the role of the Trp(32) residue in the process. The results showed that Trp(32) residues of both enzymes are oxidized to a similar extent to hSOD1-derived tryptophanyl radicals. These radicals decayed to hSOD1-N-formylkynurenine and hSOD1-kynurenine or to a hSOD1 covalent dimer cross-linked by a ditryptophan bond, causing hSOD1 unfolding, oligomerization, and non-amyloid aggregation. The latter process was inhibited by tempol, which recombines with the hSOD1-derived tryptophanyl radical, and did not occur in the absence of bicarbonate or with enzymes that lack the Trp(32) residue (bovine SOD1 and hSOD1(W32F) mutant). The results support a role for the oxidation products of the hSOD1-Trp(32) residue, particularly the covalent dimer, in triggering the non-amyloid aggregation of hSOD1. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Does aluminium bind to histidine? An NMR investigation of amyloid β12 and amyloid β16 fragments.

    Science.gov (United States)

    Narayan, Priya; Krishnarjuna, Bankala; Vishwanathan, Vinaya; Jagadeesh Kumar, Dasappa; Babu, Sudhir; Ramanathan, Krishna Venkatachala; Easwaran, Kalpathy Ramaier Katchap; Nagendra, Holenarasipur Gundurao; Raghothama, Srinivasarao

    2013-07-01

    Aluminium and zinc are known to be the major triggering agents for aggregation of amyloid peptides leading to plaque formation in Alzheimer's disease. While zinc binding to histidine in Aβ (amyloid β) fragments has been implicated as responsible for aggregation, not much information is available on the interaction of aluminium with histidine. In the NMR study of the N-terminal Aβ fragments, DAEFRHDSGYEV (Aβ12) and DAEFRHDSGYEVHHQK (Aβ16) presented here, the interactions of the fragments with aluminium have been investigated. Significant chemical shifts were observed for few residues near the C-terminus when aluminium chloride was titrated with Aβ12 and Aβ16 peptides. Surprisingly, it is nonhistidine residues which seem to be involved in aluminium binding. Based on NMR constrained structure obtained by molecular modelling, aluminium-binding pockets in Aβ12 were around charged residues such as Asp, Glu. The results are discussed in terms of native structure propagation, and the relevance of histidine residues in the sequences for metal-binding interactions. We expect that the study of such short amyloid peptide fragments will not only provide clues for plaque formation in aggregated conditions but also facilitate design of potential drugs for these targets. © 2013 John Wiley & Sons A/S.

  3. The architecture of amyloid-like peptide fibrils revealed by X-ray scattering, diffraction and electron microscopy

    DEFF Research Database (Denmark)

    Langkilde, Annette Eva; Morris, Kyle L; Serpell, Louise C

    2015-01-01

    Structural analysis of protein fibrillation is inherently challenging. Given the crucial role of fibrils in amyloid diseases, method advancement is urgently needed. A hybrid modelling approach is presented enabling detailed analysis of a highly ordered and hierarchically organized fibril of the G......Structural analysis of protein fibrillation is inherently challenging. Given the crucial role of fibrils in amyloid diseases, method advancement is urgently needed. A hybrid modelling approach is presented enabling detailed analysis of a highly ordered and hierarchically organized fibril...

  4. A systematic review of amyloid-beta peptides as putative mediators of the association between affective disorders and Alzheimer's disease

    DEFF Research Database (Denmark)

    Abbasowa, Leda; Heegaard, N. H. H.

    2014-01-01

    Background: Affective disorders are associated with an increased occurrence of cognitive deficits and have been linked to cognitive impairment and Alzheimer's disease. The putative molecular mechanisms involved in these associations are however not clear. The aim of this systematic review...... were limited by very low sample numbers. Finally, different assays for amyloid-beta were utilized in the different studies, thus hampering comparisons. Conclusion: To unravel possible risk relations and causalities between affective disorder and Alzheimer's disease and to determine how amyloid...

  5. An anticancer drug suppresses the primary nucleation reaction that initiates the production of the toxic Aβ42 aggregates linked with Alzheimer's disease

    NARCIS (Netherlands)

    Habchi, Johnny; Arosio, Paolo; Perni, Michele; Costa, Ana Rita; Yagi-Utsumi, Maho; Joshi, Priyanka; Chia, Sean; Cohen, Samuel I A; Müller, Martin B D; Linse, Sara; Nollen, Ellen A A; Dobson, Christopher M; Knowles, Tuomas P J; Vendruscolo, Michele

    2016-01-01

    The conversion of the β-amyloid (Aβ) peptide into pathogenic aggregates is linked to the onset and progression of Alzheimer's disease. Although this observation has prompted an extensive search for therapeutic agents to modulate the concentration of Aβ or inhibit its aggregation, all clinical trials

  6. Effect of nanoparticles binding ß-amyloid peptide on nitric oxide production by cultured endothelial cells and macrophages

    Directory of Open Access Journals (Sweden)

    Orlando A

    2013-04-01

    Full Text Available Antonina Orlando,1 Francesca Re,1 Silvia Sesana,1 Ilaria Rivolta,1 Alice Panariti,1 Davide Brambilla,2 Julien Nicolas,2 Patrick Couvreur,2 Karine Andrieux,2 Massimo Masserini,1 Emanuela Cazzaniga1 1Department of Health Sciences, University of Milano-Bicocca, Monza, Italy; 2Institut Galien Paris Sud, University Paris-Sud, Châtenay-Malabry, France Background: As part of a project designing nanoparticles for the treatment of Alzheimer’s disease, we have synthesized and characterized a small library of nanoparticles binding with high affinity to the β-amyloid peptide and showing features of biocompatibility in vitro, which are important properties for administration in vivo. In this study, we focused on biocompatibility issues, evaluating production of nitric oxide by cultured human umbilical vein endothelial cells and macrophages, used as models of cells which would be exposed to nanoparticles after systemic administration. Methods: The nanoparticles tested were liposomes and solid lipid nanoparticles carrying phosphatidic acid or cardiolipin, and PEGylated poly(alkyl cyanoacrylate nanoparticles (PEG-PACA. We measured nitric oxide production using the Griess method as well as phosphorylation of endothelial nitric oxide synthase and intracellular free calcium, which are biochemically related to nitric oxide production. MTT viability tests and caspase-3 detection were also undertaken. Results: Exposure to liposomes did not affect the viability of endothelial cells at any concentration tested. Increased production of nitric oxide was detected only with liposomes carrying phosphatidic acid or cardiolipin at the highest concentration (120 µg/mL, together with increased synthase phosphorylation and intracellular calcium levels. Macrophages exposed to liposomes showed a slightly dose-dependent decrease in viability, with no increase in production of nitric oxide. Exposure to solid lipid nanoparticles carrying phosphatidic acid decreased viability in

  7. Effects of Yizhi Capsule (益智胶囊) on Learning and Memory Disorder and β-amyloid Peptide Induced Neurotoxicity in Rats

    Institute of Scientific and Technical Information of China (English)

    WU Hang-yu; XU Jiang-ping; LI Lin; ZHU Bai-hua

    2006-01-01

    Objective: To explore the effects of Yizhi Capsule (益智胶囊, YZC) on learning and memory disorder and β-amyloid peptide induced neurotoxicity in rats. Methods: Various doses of YZC were administered to Sprague-Dawley (SD) rats for 8 consecutive days, twice a day. On the 8th day of the experiment,scopolamine hydrobromide was intraperitoneally injected to every rat and Morris water maze test and shuttle dark avoidance test were carried out respectively to explore the changes of learning and memory capacities in the rats. Besides, after the cerebral cortical neurons of newborn SD rats aged within 3 days were cultured in vitro for 7 days, drug serum containing YZC was added to the cultured neurons before or after β amyloid peptide25-35 (Aβ25-35) intoxication to observe the protective effect of YZC on neurotoxicity by MTT assay and to determine the LDH content in the supernatant. Results: Compared with those untreated with YZC, the rats having received YZC treatment got superiority in shorter time of platform seeking in Morris water maze test,as well as elongated latent period and less times of error in shuttle dark avoidance test. On the cultured neurons, YZC drug serum could effectively increase the survival rate of Aβ25-35 intoxicated neurons and reduce the LDH contents in cultured supernatant. Conclusion: YZC has an action of improving learning and memory disorder, and good protective effect on Aβ25-35 induced neurotoxicity in SD rats.

  8. DL-3-n-butylphthalide-Edaravone hybrids as novel dual inhibitors of amyloidaggregation and monoamine oxidases with high antioxidant potency for Alzheimer's therapy.

    Science.gov (United States)

    Qiang, Xiaoming; Li, Yan; Yang, Xia; Luo, Li; Xu, Rui; Zheng, Yunxiaozhu; Cao, Zhongcheng; Tan, Zhenghuai; Deng, Yong

    2017-02-15

    Considering the complex etiology of Alzheimer's disease (AD), multifunctional agents may be beneficial for the treatment of this disease. A series of DL-3-n-butylphthalide-Edaravone hybrids were designed, synthesized and evaluated as novel dual inhibitors of amyloidaggregation and monoamine oxidases. Among them, compounds 9a-d exhibited good inhibition of self-induced Aβ 1-42 aggregation with inhibition ratio 57.7-71.5%. For MAO, these new hybrids exhibited good balance of inhibition for MAO-A and MAO-B. In addition, all target compounds retained the antioxidant activity of edaravone, showed equal or better antioxidant activity than edaravone. The results of the parallel artificial membrane permeability assay for blood-brain barrier indicated that compounds 9a-d would be able to cross the blood-brain barrier and reach their biological targets in the central nervous system. The promising results in all assays demonstrated that the strategy behind the designing of compounds was rational and favourable. Taken together, these preliminary findings suggested that the compounds with the strongest bioactivity deserves further investigated for pharmacological development in AD therapy. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Development of multifunctional, heterodimeric isoindoline-1,3-dione derivatives as cholinesterase and β-amyloid aggregation inhibitors with neuroprotective properties.

    Science.gov (United States)

    Guzior, Natalia; Bajda, Marek; Skrok, Mirosław; Kurpiewska, Katarzyna; Lewiński, Krzysztof; Brus, Boris; Pišlar, Anja; Kos, Janko; Gobec, Stanislav; Malawska, Barbara

    2015-03-06

    The presented study describes the synthesis, pharmacological evaluation (AChE and BuChE inhibition, beta amyloid anti-aggregation effect and neuroprotective effect), molecular modeling and crystallographic studies of a novel series of isoindoline-1,3-dione derivatives. The target compounds were designed as dual binding site acetylcholinesterase inhibitors with an arylalkylamine moiety binding at the catalytic site of the enzyme and connected via an alkyl chain to a heterocyclic fragment, capable of binding at the peripheral anionic site of AChE. Among these molecules, compound 15b was found to be the most potent and selective AChE inhibitor (IC50EeAChE = 0.034 μM). Moreover, compound 13b in addition to AChE inhibition (IC50 EeAChE = 0.219 μM) possesses additional properties, such as the ability to inhibit Aβ aggregation (65.96% at 10 μM) and a neuroprotective effect against Aβ toxicity at 1 and 3 μM. Compound 13b emerges as a promising multi-target ligand for the further development of the therapy for age-related neurodegenerative disorders. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  10. Generation of the beta-amyloid peptide and the amyloid precursor protein C-terminal fragment gamma are potentiated by FE65L1.

    Science.gov (United States)

    Chang, Yang; Tesco, Giuseppina; Jeong, William J; Lindsley, Loren; Eckman, Elizabeth A; Eckman, Christopher B; Tanzi, Rudolph E; Guénette, Suzanne Y

    2003-12-19

    Members of the FE65 family of adaptor proteins, FE65, FE65L1, and FE65L2, bind the C-terminal region of the amyloid precursor protein (APP). Overexpression of FE65 and FE65L1 was previously reported to increase the levels of alpha-secretase-derived APP (APPs alpha). Increased beta-amyloid (A beta) generation was also observed in cells showing the FE65-dependent increase in APPs alpha. To understand the mechanism for the observed increase in both A beta and APPs alpha given that alpha-secretase cleavage of a single APP molecule precludes A beta generation, we examined the effects of FE65L1 overexpression on APP C-terminal fragments (APP CTFs). Our data show that FE65L1 potentiates gamma-secretase processing of APP CTFs, including the amyloidogenic CTF C99, accounting for the ability of FE65L1 to increase generation of APP C-terminal domain and A beta 40. The FE65L1 modulation of these processing events requires binding of FE65L1 to APP and APP CTFs and is not because of a direct effect on gamma-secretase activity, because Notch intracellular domain generation is not altered by FE65L1. Furthermore, enhanced APP CTF processing can be detected in early endosome vesicles but not in endoplasmic reticulum or Golgi membranes, suggesting that the effects of FE65L1 occur at or near the plasma membrane. Finally, although FE65L1 increases APP C-terminal domain production, it does not mediate the APP-dependent transcriptional activation observed with FE65.

  11. Sequence-Dependent Self-Assembly and Structural Diversity of Islet Amyloid Polypeptide-Derived β-Sheet Fibrils

    International Nuclear Information System (INIS)

    Wang, Shih-Ting; Lin, Yiyang; Spencer, Ryan K.; Thomas, Michael R.; Nguyen, Andy I.

    2017-01-01

    Determining the structural origins of amyloid fibrillation is essential for understanding both the pathology of amyloidosis and the rational design of inhibitors to prevent or reverse amyloid formation. In this work, the decisive roles of peptide structures on amyloid self-assembly and morphological diversity were investigated by the design of eight amyloidogenic peptides derived from islet amyloid polypeptide. Among the segments, two distinct morphologies were highlighted in the form of twisted and planar (untwisted) ribbons with varied diameters, thicknesses, and lengths. In particular, transformation of amyloid fibrils from twisted ribbons into untwisted structures was triggered by substitution of the C-terminal serine with threonine, where the side chain methyl group was responsible for the distinct morphological change. This effect was confirmed following serine substitution with alanine and valine and was ascribed to the restriction of intersheet torsional strain through the increased hydrophobic interactions and hydrogen bonding. We also studied the variation of fibril morphology (i.e., association and helicity) and peptide aggregation propensity by increasing the hydrophobicity of the peptide side group, capping the N-terminus, and extending sequence length. Lastly, we anticipate that our insights into sequence-dependent fibrillation and morphological diversity will shed light on the structural interpretation of amyloidogenesis and development of structure-specific imaging agents and aggregation inhibitors.

  12. Human Islet Amyloid Polypeptide

    DEFF Research Database (Denmark)

    Kosicka, Iga

    2014-01-01

    Diabetes mellitus type II is a metabolic disease affecting millions of people worldwide. The disease is associated with occurence of insoluble, fibrillar, protein aggregates in islets of Langerhans in the pancreas - islet amyloid. The main constituent of these protein fibers is the human islet...... of diabetes type II, while revealing the structure(s) of islet amyloid fibrils is necessary for potential design of therapeutic agents....

  13. Imaging of Cerebral Amyloid Angiopathy with Bivalent (99m)Tc-Hydroxamamide Complexes.

    Science.gov (United States)

    Iikuni, Shimpei; Ono, Masahiro; Watanabe, Hiroyuki; Matsumura, Kenji; Yoshimura, Masashi; Kimura, Hiroyuki; Ishibashi-Ueda, Hatsue; Okamoto, Yoko; Ihara, Masafumi; Saji, Hideo

    2016-05-16

    Cerebral amyloid angiopathy (CAA), characterized by the deposition of amyloid aggregates in the walls of cerebral vasculature, is a major factor in intracerebral hemorrhage and vascular cognitive impairment and is also associated closely with Alzheimer's disease (AD). We previously reported (99m)Tc-hydroxamamide ((99m)Tc-Ham) complexes with a bivalent amyloid ligand showing high binding affinity for β-amyloid peptide (Aβ(1-42)) aggregates present frequently in the form in AD. In this article, we applied them to CAA-specific imaging probes, and evaluated their utility for CAA-specific imaging. In vitro inhibition assay using Aβ(1-40) aggregates deposited mainly in CAA and a brain uptake study were performed for (99m)Tc-Ham complexes, and all (99m)Tc-Ham complexes with an amyloid ligand showed binding affinity for Aβ(1-40) aggregates and very low brain uptake. In vitro autoradiography of human CAA brain sections and ex vivo autoradiography of Tg2576 mice were carried out for bivalent (99m)Tc-Ham complexes ([(99m)Tc]SB2A and [(99m)Tc]BT2B), and they displayed excellent labeling of Aβ depositions in human CAA brain sections and high affinity and selectivity to CAA in transgenic mice. These results may offer new possibilities for the development of clinically useful CAA-specific imaging probes based on the (99m)Tc-Ham complex.

  14. Effects of Capsule Yi -Zhi on learning and memory disorder and beta-amyloid peptide induced neurotoxicity in rats

    Institute of Scientific and Technical Information of China (English)

    XUJiang-Ping; WUHang-Yu; LILin

    2004-01-01

    AIM To investigate the effects of Capsule Yi-Zhi (CYZ) on learning and memory disorder and beta-amyloid protein induced neurotoxieity in rats. Methods Various doses of CYZ were administered to Sprague-Dawley (SD) rats for 8 days, twice a day. Then scopolamine hydrobromide (Sco) intraperitoneal injection was performed on each rat and the

  15. Chemical Kinetics for Bridging Molecular Mechanisms and Macroscopic Measurements of Amyloid Fibril Formation.

    Science.gov (United States)

    Michaels, Thomas C T; Šarić, Anđela; Habchi, Johnny; Chia, Sean; Meisl, Georg; Vendruscolo, Michele; Dobson, Christopher M; Knowles, Tuomas P J

    2018-04-20

    Understanding how normally soluble peptides and proteins aggregate to form amyloid fibrils is central to many areas of modern biomolecular science, ranging from the development of functional biomaterials to the design of rational therapeutic strategies against increasingly prevalent medical conditions such as Alzheimer's and Parkinson's diseases. As such, there is a great need to develop models to mechanistically describe how amyloid fibrils are formed from precursor peptides and proteins. Here we review and discuss how ideas and concepts from chemical reaction kinetics can help to achieve this objective. In particular, we show how a combination of theory, experiments, and computer simulations, based on chemical kinetics, provides a general formalism for uncovering, at the molecular level, the mechanistic steps that underlie the phenomenon of amyloid fibril formation.

  16. Chemical Kinetics for Bridging Molecular Mechanisms and Macroscopic Measurements of Amyloid Fibril Formation

    Science.gov (United States)

    Michaels, Thomas C. T.; Šarić, Anđela; Habchi, Johnny; Chia, Sean; Meisl, Georg; Vendruscolo, Michele; Dobson, Christopher M.; Knowles, Tuomas P. J.

    2018-04-01

    Understanding how normally soluble peptides and proteins aggregate to form amyloid fibrils is central to many areas of modern biomolecular science, ranging from the development of functional biomaterials to the design of rational therapeutic strategies against increasingly prevalent medical conditions such as Alzheimer's and Parkinson's diseases. As such, there is a great need to develop models to mechanistically describe how amyloid fibrils are formed from precursor peptides and proteins. Here we review and discuss how ideas and concepts from chemical reaction kinetics can help to achieve this objective. In particular, we show how a combination of theory, experiments, and computer simulations, based on chemical kinetics, provides a general formalism for uncovering, at the molecular level, the mechanistic steps that underlie the phenomenon of amyloid fibril formation.

  17. Effects of super-hard rice bread blended with black rice bran on amyloid β peptide production and abrupt increase in postprandial blood glucose levels in mice.

    Science.gov (United States)

    Nakamura, Sumiko; Hara, Takashi; Joh, Toshio; Kobayashi, Atsushi; Yamazaki, Akira; Kasuga, Kensaku; Ikeuchi, Takeshi; Ohtsubo, Ken'ichi

    2017-02-01

    Alzheimer's disease and type 2 diabetes are very serious diseases with the latter having been suggested to cause the former. We prepared super-hard rice bread blended with black rice bran (SRBBB), which contained a high amount of resistant starch that showed strong inhibitory activities against β-secretase and acetylcholinesterase even after heating. Black rice bran showed greater β-secretase inhibitory activity (3.6-fold) than Koshihikari rice. The bran contained more oleic acid and anthocyanin, meaning that it is potentially a biofunctional food with a high antioxidant capacity. Furthermore, aged mice, which were fed a SRBBB diet for four weeks, showed lower amyloid β 40 peptide in the blood than mice fed a commercial diet (p < 0.01). Additionally, their initial blood glucose levels (BGLs) after 12 weeks of being fed SRBBB were significantly lower than those in the control group. Taken together, our results indicate SRBBB shows promise for inhibiting not only amyloid β production, but also abrupt increases in postprandial BGLs.

  18. Disease-modifying anti-Alzheimer's drugs: inhibitors of human cholinesterases interfering with β-amyloid aggregation.

    Science.gov (United States)

    Brogi, Simone; Butini, Stefania; Maramai, Samuele; Colombo, Raffaella; Verga, Laura; Lanni, Cristina; De Lorenzi, Ersilia; Lamponi, Stefania; Andreassi, Marco; Bartolini, Manuela; Andrisano, Vincenza; Novellino, Ettore; Campiani, Giuseppe; Brindisi, Margherita; Gemma, Sandra

    2014-07-01

    We recently described multifunctional tools (2a-c) as potent inhibitors of human Cholinesterases (ChEs) also able to modulate events correlated with Aβ aggregation. We herein propose a thorough biological and computational analysis aiming at understanding their mechanism of action at the molecular level. We determined the inhibitory potency of 2a-c on Aβ1-42 self-aggregation, the interference of 2a with the toxic Aβ oligomeric species and with the postaggregation states by capillary electrophoresis analysis and transmission electron microscopy. The modulation of Aβ toxicity was assessed for 2a and 2b on human neuroblastoma cells. The key interactions of 2a with Aβ and with the Aβ-preformed fibrils were computationally analyzed. 2a-c toxicity profile was also assessed (human hepatocytes and mouse fibroblasts). Our prototypical pluripotent analogue 2a interferes with Aβ oligomerization process thus reducing Aβ oligomers-mediated toxicity in human neuroblastoma cells. 2a also disrupts preformed fibrils. Computational studies highlighted the bases governing the diversified activities of 2a. Converging analytical, biological, and in silico data explained the mechanism of action of 2a on Aβ1-42 oligomers formation and against Aβ-preformed fibrils. This evidence, combined with toxicity data, will orient the future design of safer analogues. © 2014 John Wiley & Sons Ltd.

  19. Two distinct β-sheet structures in Italian-mutant amyloid-beta fibrils : a potential link to different clinical phenotypes

    NARCIS (Netherlands)

    Hubin, Ellen; Deroo, Stéphanie; Schierle, Gabriele Kaminksi; Kaminski, Clemens; Serpell, Louise; Subramaniam, Vinod; van Nuland, Nico; Broersen, Kerensa; Raussens, Vincent; Sarroukh, Rabia

    2015-01-01

    Most Alzheimer's disease (AD) cases are late-onset and characterized by the aggregation and deposition of the amyloid-beta (Aβ) peptide in extracellular plaques in the brain. However, a few rare and hereditary Aβ mutations, such as the Italian Glu22-to-Lys (E22K) mutation, guarantee the development

  20. Cholesterol catalyses Aβ42 aggregation through a heterogeneous nucleation pathway in the presence of lipid membranes

    Science.gov (United States)

    Habchi, Johnny; Chia, Sean; Galvagnion, Céline; Michaels, Thomas C. T.; Bellaiche, Mathias M. J.; Ruggeri, Francesco Simone; Sanguanini, Michele; Idini, Ilaria; Kumita, Janet R.; Sparr, Emma; Linse, Sara; Dobson, Christopher M.; Knowles, Tuomas P. J.; Vendruscolo, Michele

    2018-06-01

    Alzheimer's disease is a neurodegenerative disorder associated with the aberrant aggregation of the amyloidpeptide. Although increasing evidence implicates cholesterol in the pathogenesis of Alzheimer's disease, the detailed mechanistic link between this lipid molecule and the disease process remains to be fully established. To address this problem, we adopt a kinetics-based strategy that reveals a specific catalytic role of cholesterol in the aggregation of Aβ42 (the 42-residue form of the amyloidpeptide). More specifically, we demonstrate that lipid membranes containing cholesterol promote Aβ42 aggregation by enhancing its primary nucleation rate by up to 20-fold through a heterogeneous nucleation pathway. We further show that this process occurs as a result of cooperativity in the interaction of multiple cholesterol molecules with Aβ42. These results identify a specific microscopic pathway by which cholesterol dramatically enhances the onset of Aβ42 aggregation, thereby helping rationalize the link between Alzheimer's disease and the impairment of cholesterol homeostasis.

  1. Role of αA-crystallin-derived αA66-80 peptide in guinea pig lens crystallin aggregation and insolubilization.

    Science.gov (United States)

    Raju, Murugesan; Mooney, Brian P; Thakkar, Kavi M; Giblin, Frank J; Schey, Kevin L; Sharma, K Krishna

    2015-03-01

    Earlier we reported that low molecular weight (LMW) peptides accumulate in aging human lens tissue and that among the LMW peptides, the chaperone inhibitor peptide αA66-80, derived from α-crystallin protein, is one of the predominant peptides. We showed that in vitro αA66-80 induces protein aggregation. The current study was undertaken to determine whether LMW peptides are also present in guinea pig lens tissue subjected to hyperbaric oxygen (HBO) in vivo. The nuclear opacity induced by HBO in guinea pig lens is the closest animal model for studying age-related cataract formation in humans. A LMW peptide profile by mass spectrometry showed the presence of an increased amount of LMW peptides in HBO-treated guinea pig lenses compared to age-matched controls. Interestingly, the mass spectrometric data also showed that the chaperone inhibitor peptide αA66-80 accumulates in HBO-treated guinea pig lens. Following incubation of synthetic chaperone inhibitor peptide αA66-80 with α-crystallin from guinea pig lens extracts, we observed a decreased ability of α-crystallin to inhibit the amorphous aggregation of the target protein alcohol dehydrogenase and the formation of large light scattering aggregates, similar to those we have observed with human α-crystallin and αA66-80 peptide. Further, time-lapse recordings showed that a preformed complex of α-crystallin and αA66-80 attracted additional crystallin molecules to form even larger aggregates. These results demonstrate that LMW peptide-mediated cataract development in aged human lens and in HBO-induced lens opacity in the guinea pig may have common molecular pathways. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Effects of an Aβ-antibody fragment on Aβ aggregation and astrocytic uptake are modulated by apolipoprotein E and J mimetic peptides.

    Directory of Open Access Journals (Sweden)

    Laia Montoliu-Gaya

    Full Text Available Aβ-Immunotherapy has long been studied in the treatment of Alzheimer's disease (AD, but not how other molecules involved in the disease can affect antibody performance. We previously designed an antibody fragment, scFv-h3D6, and showed that it precludes Aβ-induced cytotoxicity by withdrawing Aβ oligomers from the amyloid pathway towards a non-toxic, worm-like pathway. ScFv-h3D6 was effective at the behavioral, cellular, and molecular levels in the 3xTg-AD mouse model. Because scFv-h3D6 treatment restored apolipoprotein E (apoE and J (apoJ concentrations to non-pathological values, and Aβ internalization by glial cells was found to be decreased in the presence of these apolipoproteins, we now aimed to test the influence of scFv-h3D6 on Aβ aggregation and cellular uptake by primary human astrocytes in the presence of therapeutic apoE and apoJ mimetic peptides (MPs. Firstly, we demonstrated by CD and FTIR that the molecules used in this work were well folded. Next, interactions between apoE or apoJ-MP, scFv-h3D6 and Aβ were studied by CD. The conformational change induced by the interaction of Aβ with apoE-MP was much bigger than the induced with apoJ-MP, in line with the observed formation of protective worm-like fibrils by the scFv-h3D6/Aβ complex in the presence of apoJ-MP, but not of apoE-MP. ScFv-h3D6, apoJ-MP, and apoE-MP to a different extent reduced Aβ uptake by astrocytes, and apoE-MP partially interfered with the dramatic reduction by scFv-h3D6 while apoJ-MP had no effect on scFv-h3D6 action. As sustained Aβ uptake by astrocytes may impair their normal functions, and ultimately neuronal viability, this work shows another beneficence of scFv-h3D6 treatment, which is not further improved by the use of apoE or apoJ mimetic peptides.

  3. Why are Functional Amyloids Non-Toxic in Humans?

    Directory of Open Access Journals (Sweden)

    Matthew P. Jackson

    2017-09-01

    Full Text Available Amyloids were first identified in association with amyloidoses, human diseases in which proteins and peptides misfold into amyloid fibrils. Subsequent studies have identified an array of functional amyloid fibrils that perform physiological roles in humans. Given the potential for the production of toxic species in amyloid assembly reactions, it is remarkable that cells can produce these functional amyloids without suffering any obvious ill effect. Although the precise mechanisms are unclear, there are a number of ways in which amyloid toxicity may be prevented. These include regulating the level of the amyloidogenic peptides and proteins, minimising the production of prefibrillar oligomers in amyloid assembly reactions, sequestrating amyloids within membrane bound organelles, controlling amyloid assembly by other molecules, and disassembling the fibrils under physiological conditions. Crucially, a better understanding of how toxicity is avoided in the production of functional amyloids may provide insights into the prevention of amyloid toxicity in amyloidoses.

  4. [Design, synthesis and evaluation of bis-nicotine derivatives as inhibitors of cholinesterases and beta-amyloid aggregation].

    Science.gov (United States)

    Luo, Wen; Zhao, Yong-mei; Tian, Run-guo; Su, Ya-bin; Hong, Chen

    2013-11-01

    A novel series of bis-nicotine derivatives (3a-3i) were designed, synthesized and evaluated as bivalent anti-Alzheimer's disease agents. The pharmacological results indicated that compounds 3e-3i inhibited both acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in the micromolar range (IC50, 2.28-117.86 micromol x L(-1) for AChE and 1.67-125 micromol x L(-1) for BChE), which was at the same potency as rivastigmine. A Lineweaver-Burk plot and molecular modeling study showed that these derivatives targeted both the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE. Besides, these compounds could significantly inhibit the self-induced Abeta aggregation with inhibition activity (11.85%-62.14%) at the concentration of 20 micromol x L(-1).

  5. Spectroscopic study of Alzheimer's amyloid fibrils using terahertz time domain spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Jung, Euna; Kim, Jeonghoi; Han, Younho; Moon, Kiwon; Lim, Meehyun; Han, Haewook; Park, Joonhyuck; Kim, Sungjee [POSTECH, Pohang (Korea, Republic of)

    2008-11-15

    Alzheimer's disease, one of the most common neurodegenerative diseases, is characterized by extensive amyloid deposition. Amyloid deposits contain the abundant fibrils formed by amyloid β protein (Aβ). Because amyloid fibrils are associated with amyloid diseases, including Alzheimer's disease, type 2 diabetes, prion disease, Parkinson's disease, senile systemic amyloidosis and Huntington's disease, there has been considerable interest within the biomedical and biochemical research communities. In transmission electron microscopic (TEM)images, amyloid firils are 0.1∼10μm long and approximately 10nm wide. Amyloid fibrils commonly exhibit self assembled filaments, often described as twisted or parallel assemblies of finer protofilaments. They are formed by the spontaneous aggregation of a wide variety of peptides and proteins. Structural studies of amyloid fibrils have revealed that the common structural motif of virtually all amyloid fibrils consists of cross β sheets in which the peptide strands are arranged perpendicular to the long axis of the fiber. But little was known until recently about the molecular level structures of amyloid fibils. Therefore, spectroscopic investigation of both amyloid fibrils and Aβ at the molecular level can provide the significant evidence for the molecular understanding of amyloidogenesis and for the development of innovative therapeutic and diagnostic approaches. We used terahertz time domain spectroscopy (THz TDS)to investigate both Aβ and amyloid fibril. THz TDS, developed over the last two decades, is a powerful tool to extract the properties of biomaterials and provides unique spectral signatures of biomolecules within 0.1∼10THz, which exists between microwave and infrared frequency range. Current interest in THz radiation arises from its capability of probing the delocalized collective vibrational modes in proteins. Studying the collective modes of proteins in THz frequency range can play an

  6. Spectroscopic study of Alzheimer's amyloid fibrils using terahertz time domain spectroscopy

    International Nuclear Information System (INIS)

    Jung, Euna; Kim, Jeonghoi; Han, Younho; Moon, Kiwon; Lim, Meehyun; Han, Haewook; Park, Joonhyuck; Kim, Sungjee

    2008-01-01

    Alzheimer's disease, one of the most common neurodegenerative diseases, is characterized by extensive amyloid deposition. Amyloid deposits contain the abundant fibrils formed by amyloid β protein (Aβ). Because amyloid fibrils are associated with amyloid diseases, including Alzheimer's disease, type 2 diabetes, prion disease, Parkinson's disease, senile systemic amyloidosis and Huntington's disease, there has been considerable interest within the biomedical and biochemical research communities. In transmission electron microscopic (TEM)images, amyloid firils are 0.1∼10μm long and approximately 10nm wide. Amyloid fibrils commonly exhibit self assembled filaments, often described as twisted or parallel assemblies of finer protofilaments. They are formed by the spontaneous aggregation of a wide variety of peptides and proteins. Structural studies of amyloid fibrils have revealed that the common structural motif of virtually all amyloid fibrils consists of cross β sheets in which the peptide strands are arranged perpendicular to the long axis of the fiber. But little was known until recently about the molecular level structures of amyloid fibils. Therefore, spectroscopic investigation of both amyloid fibrils and Aβ at the molecular level can provide the significant evidence for the molecular understanding of amyloidogenesis and for the development of innovative therapeutic and diagnostic approaches. We used terahertz time domain spectroscopy (THz TDS)to investigate both Aβ and amyloid fibril. THz TDS, developed over the last two decades, is a powerful tool to extract the properties of biomaterials and provides unique spectral signatures of biomolecules within 0.1∼10THz, which exists between microwave and infrared frequency range. Current interest in THz radiation arises from its capability of probing the delocalized collective vibrational modes in proteins. Studying the collective modes of proteins in THz frequency range can play an important role in

  7. Neuronal Cx3cr1 Deficiency Protects against Amyloid β-Induced Neurotoxicity

    Science.gov (United States)

    Dworzak, Jenny; Renvoisé, Benoît; Habchi, Johnny; Yates, Emma V.; Combadière, Christophe; Knowles, Tuomas P.; Dobson, Christopher M.; Blackstone, Craig; Paulsen, Ole; Murphy, Philip M.

    2015-01-01

    Cx3cr1, the receptor for the chemokine Cx3cl1 (fractalkine), has been implicated in the progression and severity of Alzheimer’s disease-like pathology in mice, but the underlying mechanisms remain unclear. A complicating factor is that Cx3cr1 has been demonstrated in both neurons and microglia. Here, we have dissected the differences between neuronal and microglial Cx3cr1, specifically by comparing direct amyloid-β-induced toxicity in cultured, mature, microglia-depleted hippocampal neurons from wild-type and Cx3cr1-/- mice. Wild-type neurons expressed both Cx3cl1 and Cx3cr1 and released Cx3cl1 in response to amyloid-β. Knockout of neuronal Cx3cr1 abated amyloid-β-induced lactate dehydrogenase release. Furthermore, amyloid-β differentially induced depression of pre- and postsynaptic components of miniature excitatory postsynaptic currents, in a peptide conformation-dependent manner. Knockout of neuronal Cx3cr1 abated effects of both amyloid-β conformational states, which were differentiable by aggregation kinetics and peptide morphology. We obtained similar results after both acute and chronic treatment of cultured neurons with the Cx3cr1 antagonist F1. Thus, neuronal Cx3cr1 may impact Alzheimer’s disease-like pathology by modulating conformational state-dependent amyloid-β-induced synaptotoxicity. PMID:26038823

  8. Neuronal Cx3cr1 Deficiency Protects against Amyloid β-Induced Neurotoxicity.

    Directory of Open Access Journals (Sweden)

    Jenny Dworzak

    Full Text Available Cx3cr1, the receptor for the chemokine Cx3cl1 (fractalkine, has been implicated in the progression and severity of Alzheimer's disease-like pathology in mice, but the underlying mechanisms remain unclear. A complicating factor is that Cx3cr1 has been demonstrated in both neurons and microglia. Here, we have dissected the differences between neuronal and microglial Cx3cr1, specifically by comparing direct amyloid-β-induced toxicity in cultured, mature, microglia-depleted hippocampal neurons from wild-type and Cx3cr1-/- mice. Wild-type neurons expressed both Cx3cl1 and Cx3cr1 and released Cx3cl1 in response to amyloid-β. Knockout of neuronal Cx3cr1 abated amyloid-β-induced lactate dehydrogenase release. Furthermore, amyloid-β differentially induced depression of pre- and postsynaptic components of miniature excitatory postsynaptic currents, in a peptide conformation-dependent manner. Knockout of neuronal Cx3cr1 abated effects of both amyloid-β conformational states, which were differentiable by aggregation kinetics and peptide morphology. We obtained similar results after both acute and chronic treatment of cultured neurons with the Cx3cr1 antagonist F1. Thus, neuronal Cx3cr1 may impact Alzheimer's disease-like pathology by modulating conformational state-dependent amyloid-β-induced synaptotoxicity.

  9. Key points concerning amyloid infectivity and prion-like neuronal invasion

    Directory of Open Access Journals (Sweden)

    Alba eEspargaró

    2016-04-01

    Full Text Available Amyloid aggregation has been related to an increasing number of human illnesses, from Alzheimer and Parkinson’s diseases to Creutzfeldt-Jakob disease. Traditionally only prions have been considered as infectious agents with a high capacity of propagation. Although recent publications have showed that many amyloid proteins, including amyloid β-peptide, α-synuclein and tau protein, also propagate in a prion-like manner, the link between propagation of pathological proteins and neurotoxicity has not been evidenced. The extremely low infectivity in natural conditions of the most of non-prion amyloids is far from the spreading capacity displayed by the prions. However, it is important to elucidate the key factors that cause non-prion amyloids become infectious agents. In recent years, important advances in the understanding of the amyloid processes of amyloid-like proteins and unrelated prions (i.e., yeast and fungal prions have yielded essential information that can be applied to shed light on the prion phenomenon in mammals and humans. As shown in this review, recent evidences suggest that there are key factors that could dramatically modulate the prion capacity of proteins in the amyloid conformation. The concentration of nuclei, the presence of oligomers, and the toxicity, resistance and localization of these aggregates could be key factors affecting their spreading. In short, those factors that favor the high concentration of extracellular nuclei or oligomers, characterized by a small size, with a low toxicity could dramatically increase prion propensity; whereas low concentrations of highly toxic intracellular amyloids, with a large size, would prevent infectivity.

  10. The recombinant C-terminal fragment of tetanus toxin protects against cholinotoxicity by intraseptal injection of β-amyloid peptide (25-35) in rats.

    Science.gov (United States)

    Patricio-Martínez, A; Mendieta, L; Martínez, I; Aguilera, J; Limón, I D

    2016-02-19

    The recombinant C-terminal domain of tetanus toxin (Hc-TeTx) is a new non-toxic peptide of the tetanus toxin that exerts a protective action against glutamate excitotoxicity in motoneurons. Moreover, its efficacy as a neuroprotective agent has been demonstrated in several animal models of neurodegeneration. The eleven amino acids in the β amyloid peptide (Aβ25-35) mimic the toxic effects of the full β amyloid peptide (Aβ1-42), causing the impairment of the cholinergic system in the medial septum (MS) which, in turn, alters the septo-hippocampal pathway and leads to learning and memory impairments. The aim of this study was to examine the neuroprotective effects of the Hc-TeTx fragment against cholinotoxicity. The Hc-TeTx fragment (100 ng) was injected into the rats intercranially, with the Aβ(25-35) (2 μg) then injected into their MS. The animals were tested for spatial learning and memory in the eight-arm radial maze. The brains were removed to assess cholinergic markers, such as choline acetyltransferase (ChAT) and acetylcholinesterase (AChE), and to explore neurodegeneration in the MS and hippocampus, using amino-cupric silver and H&E staining. Finally, capase-3, a marker of apoptosis, was examined in the MS. Our results clearly demonstrate that the application of Hc-TeTx prevents the loss of cholinergic markers (ChAT and AChE), the activation of capase-3, and neurodegeneration in the MS and the CA1 and CA3 subfields of the hippocampus. All these improvements were reflected in spatial learning and memory performance, and were significantly higher compared with animals treated with Aβ(25-35). Interestingly, the single administration of Hc-TeTx into the MS modified the ChAT and AChE expression that affect cognitive processes, without inducing neurodegeneration or an increase in capase-3 expression in the MS and hippocampus. In summary, our findings suggest that the recombinant Hc-TeTx fragment offers effective protection for the septo-hippocampal pathway

  11. The amyloid beta-peptide is imported into mitochondria via the TOM import machinery and localized to mitochondrial cristae

    DEFF Research Database (Denmark)

    Hansson Petersen, Camilla A; Alikhani, Nyosha; Behbahani, Homira

    2008-01-01

    that extracellulary applied Abeta can be internalized by human neuroblastoma cells and can colocalize with mitochondrial markers. Together, these results provide further insight into the mitochondrial uptake of Abeta, a peptide considered to be of major significance in Alzheimer's disease....

  12. Effects of a Standardized Phenolic-Enriched Maple Syrup Extract on β-Amyloid Aggregation, Neuroinflammation in Microglial and Neuronal Cells, and β-Amyloid Induced Neurotoxicity in Caenorhabditis elegans.

    Science.gov (United States)

    Ma, Hang; DaSilva, Nicholas A; Liu, Weixi; Nahar, Pragati P; Wei, Zhengxi; Liu, Yongqiang; Pham, Priscilla T; Crews, Rebecca; Vattem, Dhiraj A; Slitt, Angela L; Shaikh, Zahir A; Seeram, Navindra P

    2016-11-01

    Published data supports the neuroprotective effects of several phenolic-containing natural products, including certain fruit, berries, spices, nuts, green tea, and olive oil. However, limited data are available for phenolic-containing plant-derived natural sweeteners including maple syrup. Herein, we investigated the neuroprotective effects of a chemically standardized phenolic-enriched maple syrup extract (MSX) using a combination of biophysical, in vitro, and in vivo studies. Based on biophysical data (Thioflavin T assay, transmission electron microscopy, circular dichroism, dynamic light scattering, and zeta potential), MSX reduced amyloid β 1-42 peptide (Aβ 1-42 ) fibrillation in a concentration-dependent manner (50-500 μg/mL) with similar effects as the neuroprotective polyphenol, resveratrol, at its highest test concentration (63.5 % at 500 μg/mL vs. 77.3 % at 50 μg/mL, respectively). MSX (100 μg/mL) decreased H 2 O 2 -induced oxidative stress (16.1 % decrease in ROS levels compared to control), and down-regulated the production of lipopolysaccharide (LPS)-stimulated inflammatory markers (22.1, 19.9, 74.8, and 87.6 % decrease in NOS, IL-6, PGE 2 , and TNFα levels, respectively, compared to control) in murine BV-2 microglial cells. Moreover, in a non-contact co-culture cell model, differentiated human SH-SY5Y neuronal cells were exposed to conditioned media from BV-2 cells treated with MSX (100 μg/mL) and LPS or LPS alone. MSX-BV-2 media increased SH-SY5Y cell viability by 13.8 % compared to media collected from LPS-BV-2 treated cells. Also, MSX (10 μg/mL) showed protective effects against Aβ 1-42 induced neurotoxicity and paralysis in Caenorhabditis elegans in vivo. These data support the potential neuroprotective effects of MSX warranting further studies on this natural product.

  13. Biomimetic and Aggregation-Driven Crystallization Route for Room-Temperature Material Synthesis: Growth of β-Ga2O3 Nanoparticles Using Peptide Assemblies as Nanoreactors

    Science.gov (United States)

    Lee, Sang-Yup; Gao, Xueyun; Matsui, Hiroshi

    2008-01-01

    The room temperature synthesis of β-Ga2O3 nanocrystal was examined by coupling two biomimetic crystallization techniques, the enzymatic peptide nano-assembly templating and the aggregation-driven crystallization. The catalytic template of peptide assembly nucleated and mineralized primary β-Ga2O3 crystals, and then fused them to grow single-crystalline and monodisperse nanoparticles in the cavity of the peptide assembly at room temperature. In this work, the peptide assembly was exploited as a nano-reactor with an enzymatic functionality catalyzing the hydrolysis of gallium precursors. In addition, the characteristic ring-structure of peptide assembly is expected to provide an efficient dehydration pathway and the crystallization control over the surface tension, which are advantageous for the β-Ga2O3 crystal growth. This multifunctional peptide assembly could be applied for syntheses of a variety of nanomaterials that are kinetically difficult to grow at room temperature. PMID:17302413

  14. The Na+/K+-ATPase and the amyloid-beta peptide aβ1-40 control the cellular distribution, abundance and activity of TRPC6 channels.

    Science.gov (United States)

    Chauvet, Sylvain; Boonen, Marielle; Chevallet, Mireille; Jarvis, Louis; Abebe, Addis; Benharouga, Mohamed; Faller, Peter; Jadot, Michel; Bouron, Alexandre

    2015-11-01

    The Na(+)/K(+)-ATPase interacts with the non-selective cation channels TRPC6 but the functional consequences of this association are unknown. Experiments performed with HEK cells over-expressing TRPC6 channels showed that inhibiting the activity of the Na(+)/K(+)-ATPase with ouabain reduced the amount of TRPC6 proteins and depressed Ca(2+) entry through TRPC6. This effect, not mimicked by membrane depolarization with KCl, was abolished by sucrose and bafilomycin-A, and was partially sensitive to the intracellular Ca(2+) chelator BAPTA/AM. Biotinylation and subcellular fractionation experiments showed that ouabain caused a multifaceted redistribution of TRPC6 to the plasma membrane and to an endo/lysosomal compartment where they were degraded. The amyloid beta peptide Aβ(1-40), another inhibitor of the Na(+)/K(+)-ATPase, but not the shorter peptide Aβ1-16, reduced TRPC6 protein levels and depressed TRPC6-mediated responses. In cortical neurons from embryonic mice, ouabain, veratridine (an opener of voltage-gated Na(+) channel), and Aβ(1-40) reduced TRPC6-mediated Ca(2+) responses whereas Aβ(1-16) was ineffective. Furthermore, when Aβ(1-40) was co-added together with zinc acetate it could no longer control TRPC6 activity. Altogether, this work shows the existence of a functional coupling between the Na(+)/K(+)-ATPase and TRPC6. It also suggests that the abundance, distribution and activity of TRPC6 can be regulated by cardiotonic steroids like ouabain and the naturally occurring peptide Aβ(1-40) which underlines the pathophysiological significance of these processes. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Curcumin Improves Amyloid β-Peptide (1-42 Induced Spatial Memory Deficits through BDNF-ERK Signaling Pathway.

    Directory of Open Access Journals (Sweden)

    Lu Zhang

    Full Text Available Curcumin, the most active component of turmeric, has various beneficial properties, such as antioxidant, anti-inflammatory, and antitumor effects. Previous studies have suggested that curcumin reduces the levels of amyloid and oxidized proteins and prevents memory deficits and thus is beneficial to patients with Alzheimer's disease (AD. However, the molecular mechanisms underlying curcumin's effect on cognitive functions are not well-understood. In the present study, we examined the working memory and spatial reference memory in rats that received a ventricular injection of amyloid-β1-42 (Aβ1-42, representing a rodent model of Alzheimer's disease (AD. The rats treated with Aβ1-42 exhibited obvious cognitive deficits in behavioral tasks. Chronic (seven consecutive days, once per day but not acute (once a day curcumin treatments (50, 100, and 200 mg/kg improved the cognitive functions in a dose-dependent manner. In addition, the beneficial effect of curcumin is accompanied by increased BDNF levels and elevated levels of phosphorylated ERK in the hippocampus. Furthermore, the cognition enhancement effect of curcumin could be mimicked by the overexpression of BDNF in the hippocampus and blocked by either bilateral hippocampal injections with lentiviruses that express BDNF shRNA or a microinjection of ERK inhibitor. These findings suggest that chronic curcumin ameliorates AD-related cognitive deficits and that upregulated BDNF-ERK signaling in the hippocampus may underlie the cognitive improvement produced by curcumin.

  16. Proinsulin C-peptide interferes with insulin fibril formation

    International Nuclear Information System (INIS)

    Landreh, Michael; Stukenborg, Jan-Bernd; Willander, Hanna; Söder, Olle; Johansson, Jan; Jörnvall, Hans

    2012-01-01

    Highlights: ► Insulin and C-peptide can interact under insulin fibril forming conditions. ► C-peptide is incorporated into insulin aggregates and alters aggregation lag time. ► C-peptide changes insulin fibril morphology and affects backbone accessibility. ► C-peptide may be a regulator of fibril formation by β-cell granule proteins. -- Abstract: Insulin aggregation can prevent rapid insulin uptake and cause localized amyloidosis in the treatment of type-1 diabetes. In this study, we investigated the effect of C-peptide, the 31-residue peptide cleaved from proinsulin, on insulin fibrillation at optimal conditions for fibrillation. This is at low pH and high concentration, when the fibrils formed are regular and extended. We report that C-peptide then modulates the insulin aggregation lag time and profoundly changes the fibril appearance, to rounded clumps of short fibrils, which, however, still are Thioflavine T-positive. Electrospray ionization mass spectrometry also indicates that C-peptide interacts with aggregating insulin and is incorporated into the aggregates. Hydrogen/deuterium exchange mass spectrometry further reveals reduced backbone accessibility in insulin aggregates formed in the presence of C-peptide. Combined, these effects are similar to those of C-peptide on islet amyloid polypeptide fibrillation and suggest that C-peptide has a general ability to interact with amyloidogenic proteins from pancreatic β-cell granules. Considering the concentrations, these peptide interactions should be relevant also during physiological secretion, and even so at special sites post-secretory or under insulin treatment conditions in vivo.

  17. Proinsulin C-peptide interferes with insulin fibril formation

    Energy Technology Data Exchange (ETDEWEB)

    Landreh, Michael [Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm (Sweden); Stukenborg, Jan-Bernd [Department of Women' s and Children' s Health, Astrid Lindgren Children' s Hospital, Pediatric Endocrinology Unit, Karolinska Institutet and University Hospital, S-17176 Stockholm (Sweden); Willander, Hanna [KI-Alzheimer' s Disease Research Center, NVS Department, Karolinska Institutet, S-141 86 Stockholm (Sweden); Soeder, Olle [Department of Women' s and Children' s Health, Astrid Lindgren Children' s Hospital, Pediatric Endocrinology Unit, Karolinska Institutet and University Hospital, S-17176 Stockholm (Sweden); Johansson, Jan [KI-Alzheimer' s Disease Research Center, NVS Department, Karolinska Institutet, S-141 86 Stockholm (Sweden); Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, S-751 23 Uppsala (Sweden); Joernvall, Hans, E-mail: Hans.Jornvall@ki.se [Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm (Sweden)

    2012-02-17

    Highlights: Black-Right-Pointing-Pointer Insulin and C-peptide can interact under insulin fibril forming conditions. Black-Right-Pointing-Pointer C-peptide is incorporated into insulin aggregates and alters aggregation lag time. Black-Right-Pointing-Pointer C-peptide changes insulin fibril morphology and affects backbone accessibility. Black-Right-Pointing-Pointer C-peptide may be a regulator of fibril formation by {beta}-cell granule proteins. -- Abstract: Insulin aggregation can prevent rapid insulin uptake and cause localized amyloidosis in the treatment of type-1 diabetes. In this study, we investigated the effect of C-peptide, the 31-residue peptide cleaved from proinsulin, on insulin fibrillation at optimal conditions for fibrillation. This is at low pH and high concentration, when the fibrils formed are regular and extended. We report that C-peptide then modulates the insulin aggregation lag time and profoundly changes the fibril appearance, to rounded clumps of short fibrils, which, however, still are Thioflavine T-positive. Electrospray ionization mass spectrometry also indicates that C-peptide interacts with aggregating insulin and is incorporated into the aggregates. Hydrogen/deuterium exchange mass spectrometry further reveals reduced backbone accessibility in insulin aggregates formed in the presence of C-peptide. Combined, these effects are similar to those of C-peptide on islet amyloid polypeptide fibrillation and suggest that C-peptide has a general ability to interact with amyloidogenic proteins from pancreatic {beta}-cell granules. Considering the concentrations, these peptide interactions should be relevant also during physiological secretion, and even so at special sites post-secretory or under insulin treatment conditions in vivo.

  18. Contact between the β1 and β2 Segments of α-Synuclein that Inhibits Amyloid Formation.

    Science.gov (United States)

    Shaykhalishahi, Hamed; Gauhar, Aziz; Wördehoff, Michael M; Grüning, Clara S R; Klein, Antonia N; Bannach, Oliver; Stoldt, Matthias; Willbold, Dieter; Härd, Torleif; Hoyer, Wolfgang

    2015-07-20

    Conversion of the intrinsically disordered protein α-synuclein (α-syn) into amyloid aggregates is a key process in Parkinson's disease. The sequence region 35-59 contains β-strand segments β1 and β2 of α-syn amyloid fibril models and most disease-related mutations. β1 and β2 frequently engage in transient interactions in monomeric α-syn. The consequences of β1-β2 contacts are evaluated by disulfide engineering, biophysical techniques, and cell viability assays. The double-cysteine mutant α-synCC, with a disulfide linking β1 and β2, is aggregation-incompetent and inhibits aggregation and toxicity of wild-type α-syn. We show that α-syn delays the aggregation of amyloidpeptide and islet amyloid polypeptide involved in Alzheimer's disease and type 2 diabetes, an effect enhanced in the α-synCC mutant. Tertiary interactions in the β1-β2 region of α-syn interfere with the nucleation of amyloid formation, suggesting promotion of such interactions as a potential therapeutic approach. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. {beta} - amyloid imaging probes

    Energy Technology Data Exchange (ETDEWEB)

    Jeong, Jae Min [Seoul National University College of Medicine, Seoul (Korea, Republic of)

    2007-04-15

    Imaging distribution of {beta} - amyloid plaques in Alzheimer's disease is very important for early and accurate diagnosis. Early trial of the {beta} -amyloid plaques includes using radiolabeled peptides which can be only applied for peripheral {beta} - amyloid plaques due to limited penetration through the blood brain barrier (BBB). Congo red or Chrysamine G derivatives were labeled with Tc-99m for imaging {beta} - amyloid plaques of Alzheimer patient's brain without success due to problem with BBB penetration. Thioflavin T derivatives gave breakthrough for {beta} - amyloid imaging in vivo, and a benzothiazole derivative [C-11]6-OH-BTA-1 brought a great success. Many other benzothiazole, benzoxazole, benzofuran, imidazopyridine, and styrylbenzene derivatives have been labeled with F-18 and I-123 to improve the imaging quality. However, [C-11]6-OH-BTA-1 still remains as the best. However, short half-life of C-11 is a limitation of wide distribution of this agent. So, it is still required to develop an Tc-99m, F-18 or I-123 labeled agent for {beta} - amyloid imaging agent.

  20. β - amyloid imaging probes

    International Nuclear Information System (INIS)

    Jeong, Jae Min

    2007-01-01

    Imaging distribution of β - amyloid plaques in Alzheimer's disease is very important for early and accurate diagnosis. Early trial of the β -amyloid plaques includes using radiolabeled peptides which can be only applied for peripheral β - amyloid plaques due to limited penetration through the blood brain barrier (BBB). Congo red or Chrysamine G derivatives were labeled with Tc-99m for imaging β - amyloid plaques of Alzheimer patient's brain without success due to problem with BBB penetration. Thioflavin T derivatives gave breakthrough for β - amyloid imaging in vivo, and a benzothiazole derivative [C-11]6-OH-BTA-1 brought a great success. Many other benzothiazole, benzoxazole, benzofuran, imidazopyridine, and styrylbenzene derivatives have been labeled with F-18 and I-123 to improve the imaging quality. However, [C-11]6-OH-BTA-1 still remains as the best. However, short half-life of C-11 is a limitation of wide distribution of this agent. So, it is still required to develop an Tc-99m, F-18 or I-123 labeled agent for β - amyloid imaging agent

  1. Accumulation of murine amyloid-β mimics early Alzheimer's disease.

    Science.gov (United States)

    Krohn, Markus; Bracke, Alexander; Avchalumov, Yosef; Schumacher, Toni; Hofrichter, Jacqueline; Paarmann, Kristin; Fröhlich, Christina; Lange, Cathleen; Brüning, Thomas; von Bohlen Und Halbach, Oliver; Pahnke, Jens

    2015-08-01

    Amyloidosis mouse models of Alzheimer's disease are generally established by transgenic approaches leading to an overexpression of mutated human genes that are known to be involved in the generation of amyloid-β in Alzheimer's families. Although these models made substantial contributions to the current knowledge about the 'amyloid hypothesis' of Alzheimer's disease, the overproduction of amyloidpeptides mimics only inherited (familiar) Alzheimer's disease, which accounts for patients with Alzheimer's disease. The inherited form is even regarded a 'rare' disease according to the regulations for funding of the European Union (www.erare.eu). Here, we show that mice that are double-deficient for neprilysin (encoded by Mme), one major amyloid-β-degrading enzyme, and the ABC transporter ABCC1, a major contributor to amyloid-β clearance from the brain, develop various aspects of sporadic Alzheimer's disease mimicking the clinical stage of mild cognitive impairment. Using behavioural tests, electrophysiology and morphological analyses, we compared different ABC transporter-deficient animals and found that alterations are most prominent in neprilysin × ABCC1 double-deficient mice. We show that these mice have a reduced probability to survive, show increased anxiety in new environments, and have a reduced working memory performance. Furthermore, we detected morphological changes in the hippocampus and amygdala, e.g. astrogliosis and reduced numbers of synapses, leading to defective long-term potentiation in functional measurements. Compared to human, murine amyloid-β is poorly aggregating, due to changes in three amino acids at N-terminal positions 5, 10, and 13. Interestingly, our findings account for the action of early occurring amyloid-β species/aggregates, i.e. monomers and small amyloid-β oligomers. Thus, neprilysin × ABCC1 double-deficient mice present a new model for early effects of amyloid-β-related mild cognitive impairment that allows investigations

  2. Role of sequence and structural polymorphism on the mechanical properties of amyloid fibrils.

    Directory of Open Access Journals (Sweden)

    Gwonchan Yoon

    Full Text Available Amyloid fibrils playing a critical role in disease expression, have recently been found to exhibit the excellent mechanical properties such as elastic modulus in the order of 10 GPa, which is comparable to that of other mechanical proteins such as microtubule, actin filament, and spider silk. These remarkable mechanical properties of amyloid fibrils are correlated with their functional role in disease expression. This suggests the importance in understanding how these excellent mechanical properties are originated through self-assembly process that may depend on the amino acid sequence. However, the sequence-structure-property relationship of amyloid fibrils has not been fully understood yet. In this work, we characterize the mechanical properties of human islet amyloid polypeptide (hIAPP fibrils with respect to their molecular structures as well as their amino acid sequence by using all-atom explicit water molecular dynamics (MD simulation. The simulation result suggests that the remarkable bending rigidity of amyloid fibrils can be achieved through a specific self-aggregation pattern such as antiparallel stacking of β strands (peptide chain. Moreover, we have shown that a single point mutation of hIAPP chain constituting a hIAPP fibril significantly affects the thermodynamic stability of hIAPP fibril formed by parallel stacking of peptide chain, and that a single point mutation results in a significant change in the bending rigidity of hIAPP fibrils formed by antiparallel stacking of β strands. This clearly elucidates the role of amino acid sequence on not only the equilibrium conformations of amyloid fibrils but also their mechanical properties. Our study sheds light on sequence-structure-property relationships of amyloid fibrils, which suggests that the mechanical properties of amyloid fibrils are encoded in their sequence-dependent molecular architecture.

  3. Activation of phospholipase A2 by temporin B: Formation of antimicrobial peptide-enzyme amyloid-type cofibrils

    NARCIS (Netherlands)

    Code, Christian; Domanov, Y.A.; Killian, J.A.; Kinnunen, P.K.J.

    2009-01-01

    Phospholipases A2 have been shown to be activated in a concentration dependent manner by a number of antimicrobial peptides, including melittin, magainin 2, indolicidin, and temporins B and L. Here we used fluorescently labelled bee venom PLA2 (PLA2D) and the saturated phospholipid substrate

  4. Microspectroscopy (μFTIR) reveals co-localization of lipid oxidation and amyloid plaques in human Alzheimer disease brains.

    Science.gov (United States)

    Benseny-Cases, Núria; Klementieva, Oxana; Cotte, Marine; Ferrer, Isidre; Cladera, Josep

    2014-12-16

    Amyloid peptides are the main component of one of the characteristic pathological hallmarks of Alzheimer's disease (AD): senile plaques. According to the amyloid cascade hypothesis, amyloid peptides may play a central role in the sequence of events that leads to neurodegeneration. However, there are other factors, such as oxidative stress, that may be crucial for the development of the disease. In the present paper, we show that it is possible, by using Fourier tranform infrared (FTIR) microscopy, to co-localize amyloid deposits and lipid peroxidation in tissue slides from patients affected by Alzheimer's disease. Plaques and lipids can be analyzed in the same sample, making use of the characteristic infrared bands for peptide aggregation and lipid oxidation. The results show that, in samples from patients diagnosed with AD, the plaques and their immediate surroundings are always characterized by the presence of oxidized lipids. As for samples from non-AD individuals, those without amyloid plaques show a lower level of lipid oxidation than AD individuals. However, it is known that plaques can be detected in the brains of some non-AD individuals. Our results show that, in such cases, the lipid in the plaques and their surroundings display oxidation levels that are similar to those of tissues with no plaques. These results point to lipid oxidation as a possible key factor in the path that goes from showing the typical neurophatological hallmarks to suffering from dementia. In this process, the oxidative power of the amyloid peptide, possibly in the form of nonfibrillar aggregates, could play a central role.

  5. Some commonly used brominated flame retardants cause Ca2+-ATPase inhibition, beta-amyloid peptide release and apoptosis in SH-SY5Y neuronal cells.

    Directory of Open Access Journals (Sweden)

    Fawaz Al-Mousa

    Full Text Available Brominated flame retardants (BFRs are chemicals commonly used to reduce the flammability of consumer products and are considered pollutants since they have become widely dispersed throughout the environment and have also been shown to bio-accumulate within animals and man. This study investigated the cytotoxicity of some of the most commonly used groups of BFRs on SH-SY5Y human neuroblastoma cells. The results showed that of the BFRs tested, hexabromocyclododecane (HBCD, tetrabromobisphenol-A (TBBPA and decabromodiphenyl ether (DBPE, all are cytotoxic at low micromolar concentrations (LC(50 being 2.7 ± 0.7 µM, 15 ± 4 µM and 28 ± 7 µM, respectively. They induced cell death, at least in part, by apoptosis through activation of caspases. They also increased intracellular [Ca(2+] levels and reactive-oxygen-species within these neuronal cells. Furthermore, these BFRs also caused rapid depolarization of the mitochondria and cytochrome c release in these neuronal cells. Elevated intracellular [Ca(2+] levels appear to occur through a mechanism involving microsomal Ca(2+-ATPase inhibition and this maybe responsible for Ca(2+-induced mitochondrial dysfunction. In addition, µM levels of these BFRs caused β-amyloid peptide (Aβ-42 processing and release from these cells with a few hours of exposure. These results therefore shows that these pollutants are both neurotoxic and amyloidogenic in-vitro.

  6. Covalent modifications of the amyloid beta peptide by hydroxynonenal: Effects on metal ion binding by monomers and insights into the fibril topology.

    Science.gov (United States)

    Grasso, G; Komatsu, H; Axelsen, P H

    2017-09-01

    Amyloid β peptides (Aβ) and metal ions are associated with oxidative stress in Alzheimer's disease (AD). Oxidative stress, acting on ω-6 polyunsaturated fatty acyl chains, produces diverse products, including 4-hydroxy-2-nonenal (HNE), which can covalently modify the Aβ that helped to produce it. To examine possible feedback mechanisms involving Aβ, metal ions and HNE production, the effects of HNE modification and fibril formation on metal ion binding was investigated. Results indicate that copper(II) generally inhibits the modification of His side chains in Aβ by HNE, but that once modified, copper(II) still binds to Aβ with high affinity. Fibril formation protects only one of the three His residues in Aβ from HNE modification, and this protection is consistent with proposed models of fibril structure. These results provide insight into a network of biochemical reactions that may be operating as a consequence of oxidative stress in AD, or as part of the pathogenic process. Copyright © 2016. Published by Elsevier Inc.

  7. Other Species in the Aqueous Environment of a Peptide Can Invert its Intrinsic Solvated Polyproline II/Beta Propensity: Implications for Amyloid Formation.

    Science.gov (United States)

    Mirkin, Noemi G; Krimm, Samuel

    2016-02-02

    As we have previously shown, the predominance of the polyproline II conformation in the circular dichroism spectra of aqueous polypeptides is related to its lower energy than that of the beta conformation. In order to test whether this is still the case in the presence of additional components in the medium, we have calculated the energy difference between these two conformations in an alanine-dipeptide/twelve-water system without and with the addition of an HCl molecule. We find in the latter case that the beta conformer is of lower energy than the polyproline II. Energy profiles near the minima in both cases also permit conclusions about the relative entropies of these structures. These results emphasize the importance of considering the peptide-plus-medium state as the relevant entity in determining the structural properties of such systems. Such an inversion could be relevant to the formation of amyloid and could thus lead to new strategies for studying its role in the development of neurodegenerative diseases. This article is protected by copyright. All rights reserved. © 2016 Wiley Periodicals, Inc.

  8. Serum amyloid A stimulates matrix-metalloproteinase-9 upregulation via formyl peptide receptor like-1-mediated signaling in human monocytic cells

    International Nuclear Information System (INIS)

    Lee, Ha Young; Kim, Mi-Kyoung; Park, Kyoung Sun; Bae, Yun Hee; Yun, Jeanho; Park, Joo-In; Kwak, Jong-Young; Bae, Yoe-Sik

    2005-01-01

    In the present study, we found that serum amyloid A (SAA) stimulated matrix-metalloproteinase-9 (MMP-9) upregulation at the transcription and translational levels in THP-1 cells. SAA stimulated the activation of nuclear factor κB (NF-κB), which was required for the MMP-9 upregulation by SAA. The signaling events induced by SAA included the activation of ERK and intracellular calcium rise, which were found to be required for MMP-9 upregulation. Formyl peptide receptor like 1 (FPRL1) was found to be involved in the upregulation of MMP-9 by SAA. Among several FPRL1 agonists, including Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm), SAA selectively stimulated MMP-9 upregulation. With respect to the molecular mechanisms involved in the differential action of SAA and WKYMVm, we found that SAA could not competitively inhibit the binding of 125 I-labeled WKYMVm to FPRL1. Taken together, we suggest that SAA plays a role in the modulation of inflammatory and immune responses via FPRL1, by inducing MMP-9 upregulation in human monocytic cells

  9. Amyloid-beta induced CA1 pyramidal cell loss in young adult rats is alleviated by systemic treatment with FGL, a neural cell adhesion molecule-derived mimetic peptide.

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    Nicola J Corbett

    Full Text Available Increased levels of neurotoxic amyloid-beta in the brain are a prominent feature of Alzheimer's disease. FG-Loop (FGL, a neural cell adhesion molecule-derived peptide that corresponds to its second fibronectin type III module, has been shown to provide neuroprotection against a range of cellular insults. In the present study impairments in social recognition memory were seen 24 days after a 5 mg/15 µl amyloid-beta(25-35 injection into the right lateral ventricle of the young adult rat brain. This impairment was prevented if the animal was given a systemic treatment of FGL. Unbiased stereology was used to investigate the ability of FGL to alleviate the deleterious effects on CA1 pyramidal cells of the amyloid-beta(25-35 injection. NeuN, a neuronal marker (for nuclear staining was used to identify pyramidal cells, and immunocytochemistry was also used to identify inactive glycogen synthase kinase 3beta (GSK3β and to determine the effects of amyloid-beta(25-35 and FGL on the activation state of GSK3β, since active GSK3β has been shown to cause a range of AD pathologies. The cognitive deficits were not due to hippocampal atrophy as volume estimations of the entire hippocampus and its regions showed no significant loss, but amyloid-beta caused a 40% loss of pyramidal cells in the dorsal CA1 which was alleviated partially by FGL. However, FGL treatment without amyloid-beta was also found to cause a 40% decrease in CA1 pyramidal cells. The action of FGL may be due to inactivation of GSK3β, as an increased proportion of CA1 pyramidal neurons contained inactive GSK3β after FGL treatment. These data suggest that FGL, although potentially disruptive in non-pathological conditions, can be neuroprotective in disease-like conditions.

  10. Protective Effects of Testosterone on Presynaptic Terminals against Oligomeric β-Amyloid Peptide in Primary Culture of Hippocampal Neurons

    Directory of Open Access Journals (Sweden)

    Chi-Fai Lau

    2014-01-01

    Full Text Available Increasing lines of evidence support that testosterone may have neuroprotective effects. While observational studies reported an association between higher bioavailable testosterone or brain testosterone levels and reduced risk of Alzheimer’s disease (AD, there is limited understanding of the underlying neuroprotective mechanisms. Previous studies demonstrated that testosterone could alleviate neurotoxicity induced by β-amyloid (Aβ, but these findings mainly focused on neuronal apoptosis. Since synaptic dysfunction and degeneration are early events during the pathogenesis of AD, we aim to investigate the effects of testosterone on oligomeric Aβ-induced synaptic changes. Our data suggested that exposure of primary cultured hippocampal neurons to oligomeric Aβ could reduce the length of neurites and decrease the expression of presynaptic proteins including synaptophysin, synaptotagmin, and synapsin-1. Aβ also disrupted synaptic vesicle recycling and protein folding machinery. Testosterone preserved the integrity of neurites and the expression of presynaptic proteins. It also attenuated Aβ-induced impairment of synaptic exocytosis. By using letrozole as an aromatase antagonist, we further demonstrated that the effects of testosterone on exocytosis were unlikely to be mediated through the estrogen receptor pathway. Furthermore, we showed that testosterone could attenuate Aβ-induced reduction of HSP70, which suggests a novel mechanism that links testosterone and its protective function on Aβ-induced synaptic damage. Taken together, our data provide further evidence on the beneficial effects of testosterone, which may be useful for future drug development for AD.

  11. Two-dimensional sum-frequency generation (2D SFG) spectroscopy: summary of principles and its application to amyloid fiber monolayers.

    Science.gov (United States)

    Ghosh, Ayanjeet; Ho, Jia-Jung; Serrano, Arnaldo L; Skoff, David R; Zhang, Tianqi; Zanni, Martin T

    2015-01-01

    By adding a mid-infrared pulse shaper to a sum-frequency generation (SFG) spectrometer, we have built a 2D SFG spectrometer capable of measuring spectra analogous to 2D IR spectra but with monolayer sensitivity and SFG selection rules. In this paper, we describe the experimental apparatus and provide an introduction to 2D SFG spectroscopy to help the reader interpret 2D SFG spectra. The main aim of this manuscript is to report 2D SFG spectra of the amyloid forming peptide FGAIL. FGAIL is a critical segment of the human islet amyloid polypeptide (hIAPP or amylin) that aggregates in people with type 2 diabetes. FGAIL is catalyzed into amyloid fibers by many types of surfaces. Here, we study the structure of FGAIL upon deposition onto a gold surface covered with a self-assembled monolayer of methyl-4-mercaptobenzoate (MMB) that produces an ester coating. FGAIL deposited on bare gold does not form ordered layers. The measured 2D SFG spectrum is consistent with amyloid fiber formation, exhibiting both the parallel (a+) and perpendicular (a-) symmetry modes associated with amyloid β-sheets. Cross peaks are observed between the ester stretches of the coating and the FGAIL peptides. Simulations are presented for two possible structures of FGAIL amyloid β-sheets that illustrate the sensitivity of the 2D SFG spectra to structure and orientation. These results provide some of the first molecular insights into surface catalyzed amyloid fiber structure.

  12. The Mechanisms of Aberrant Protein Aggregation

    Science.gov (United States)

    Cohen, Samuel; Vendruscolo, Michele; Dobson, Chris; Knowles, Tuomas

    2012-02-01

    We discuss the development of a kinetic theory for understanding the aberrant loss of solubility of proteins. The failure to maintain protein solubility results often in the assembly of organized linear structures, commonly known as amyloid fibrils, the formation of which is associated with over 50 clinical disorders including Alzheimer's and Parkinson's diseases. A true microscopic understanding of the mechanisms that drive these aggregation processes has proved difficult to achieve. To address this challenge, we apply the methodologies of chemical kinetics to the biomolecular self-assembly pathways related to protein aggregation. We discuss the relevant master equation and analytical approaches to studying it. In particular, we derive the underlying rate laws in closed-form using a self-consistent solution scheme; the solutions that we obtain reveal scaling behaviors that are very generally present in systems of growing linear aggregates, and, moreover, provide a general route through which to relate experimental measurements to mechanistic information. We conclude by outlining a study of the aggregation of the Alzheimer's amyloid-beta peptide. The study identifies the dominant microscopic mechanism of aggregation and reveals previously unidentified therapeutic strategies.

  13. Differential regulation of amyloid precursor protein sorting with pathological mutations results in a distinct effect on amyloid-β production.

    Science.gov (United States)

    Lin, Yen-Chen; Wang, Jia-Yi; Wang, Kai-Chen; Liao, Jhih-Ying; Cheng, Irene H

    2014-11-01

    The deposition of amyloid-β (Aβ) peptide, which is generated from amyloid precursor protein (APP), is the pathological hallmark of Alzheimer's disease (AD). Three APP familial AD mutations (D678H, D678N, and H677R) located at the sixth and seventh amino acid of Aβ have distinct effect on Aβ aggregation, but their influence on the physiological and pathological roles of APP remain unclear. We found that the D678H mutation strongly enhances amyloidogenic cleavage of APP, thus increasing the production of Aβ. This enhancement of amyloidogenic cleavage is likely because of the acceleration of APPD678H sorting into the endosomal-lysosomal pathway. In contrast, the APPD678N and APPH677R mutants do not cause the same effects. Therefore, this study indicates a regulatory role of D678H in APP sorting and processing, and provides genetic evidence for the importance of APP sorting in AD pathogenesis. The internalization of amyloid precursor protein (APP) increases its opportunity to be processed by β-secretase and to produce Amyloid-β (Aβ) that causes Alzheimer's disease (AD). We report a pathogenic APPD678H mutant that enhances APP internalization into the endosomal-lysosomal pathway and thus promotes the β-secretase cleavage and Aβ production. This study provides genetic evidence for the importance of APP sorting in AD pathogenesis. © 2014 International Society for Neurochemistry.

  14. secHsp70 as a tool to approach amyloid-β42 and other extracellular amyloids.

    Science.gov (United States)

    De Mena, Lorena; Chhangani, Deepak; Fernandez-Funez, Pedro; Rincon-Limas, Diego E

    2017-07-03

    Self-association of amyloidogenic proteins is the main pathological trigger in a wide variety of neurodegenerative disorders. These aggregates are deposited inside or outside the cell due to hereditary mutations, environmental exposures or even normal aging. Cumulative evidence indicates that the heat shock chaperone Hsp70 possesses robust neuroprotection against various intracellular amyloids in Drosophila and mouse models. However, its protective role against extracellular amyloids was largely unknown as its presence outside the cells is very limited. Our recent manuscript in PNAS revealed that an engineered form of secreted Hsp70 (secHsp70) is highly protective against toxicity induced by extracellular deposition of the amyloid-β42 (Aβ42) peptide. In this Extra View article, we extend our analysis to other members of the heat shock protein family. We created PhiC31-based transgenic lines for human Hsp27, Hsp40, Hsp60 and Hsp70 and compared their activities in parallel against extracellular Aβ42. Strikingly, only secreted Hsp70 exhibits robust protection against Aβ42-triggered toxicity in the extracellular milieu. These observations indicate that the ability of secHsp70 to suppress Aβ42 insults is quite unique and suggest that targeted secretion of Hsp70 may represent a new therapeutic approach against Aβ42 and other extracellular amyloids. The potential applications of this engineered chaperone are discussed.

  15. Modulation of Amyloid-β Conformation by Charge State of N-Terminal Disordered Region

    International Nuclear Information System (INIS)

    Xi Wen-Hui; Li Wen-Fei; Wang Wei

    2012-01-01

    Based on molecular dynamics simulations, we show that variations of the charge states of the histidines, which are the main effects of pH-value change and metal binding, can lead to a drastic change of the intra-peptide interactions of the segment 17–42 and the conformational distribution of the monomeric amyloid-β (Aβ). Since we already knew that the conformational distribution of monomeric Aβ can largely affect Aβ fibrillar aggregation, our results suggest that the pH value change and metal binding can affect the Aβ aggregation by much more complex mechanism than just affecting the inter-peptide interactions. To fully understand the mechanism of metal binding and pH-value induced Aβ aggregation, we also need to consider their effects on the conformational distribution of monomeric Aβ. (cross-disciplinary physics and related areas of science and technology)

  16. Breakout character of islet amyloid polypeptide hydrophobic mutations at the onset of type-2 diabetes

    Science.gov (United States)

    Frigori, Rafael B.

    2014-11-01

    Toxic fibrillar aggregates of islet amyloid polypeptide (IAPP) appear as the physical outcome of a peptidic phase transition signaling the onset of type-2 diabetes mellitus in different mammalian species. In particular, experimentally verified mutations on the amyloidogenic segment 20-29 in humans, cats, and rats are highly correlated with the molecular aggregation propensities. Through a microcanonical analysis of the aggregation of IAPP20 -29 isoforms, we show that a minimalist one-bead hydrophobic-polar continuum model for protein interactions properly quantifies those propensities from free-energy barriers. Our results highlight the central role of sequence-dependent hydrophobic mutations on hot spots for stabilization, and thus for the engineering, of such biological peptides.

  17. Antiplatelet Aggregation and Antithrombosis Efficiency of Peptides in the Snake Venom of Deinagkistrodon acutus: Isolation, Identification, and Evaluation

    Directory of Open Access Journals (Sweden)

    Bin Ding

    2015-01-01

    Full Text Available Two peptides of Pt-A (Glu-Asn-Trp 429 Da and Pt-B (Glu-Gln-Trp 443 Da were isolated from venom liquor of Deinagkistrodon acutus. Their antiplatelet aggregation effects were evaluated with platelet-rich human plasma in vitro; the respective IC50 of Pt-A and Pt-B was 66 μM and 203 μM. Both peptides exhibited protection effects on ADP-induced paralysis in mice. After ADP administration, the paralysis time of different concentration of Pt-A and Pt-B lasted as the following: 80 mg/kg Pt-B (152.8 ± 57.8 s < 40 mg/kg Pt-A (163.5 ± 59.8 s < 20 mg/kg Pt-A (253.5 ± 74.5 s < 4 mg/kg clopidogrel (a positive control, 254.5 ± 41.97 s < 40 mg/kg Pt-B (400.8 ± 35.9 s < 10 mg/kg Pt-A (422.8 ± 55.4 s, all of which were statistically shorter than the saline treatment (666 ± 28 s. Pulmonary tissue biopsy confirmed that Pt-A and Pt-B prevented the formation of thrombi in the lung. Unlike ADP injection alone, which caused significant reduction of peripheral platelet count, Pt-A treatment prevented the drop of peripheral platelet counts; interestingly, Pt-B could not, even though the same amount of Pt-B also showed protection effects on ADP-induced paralysis and thrombosis. More importantly, intravenous injection of Pt-A and Pt-B did not significantly increase the hemorrhage risks as clopidogrel.

  18. Sequestration of the Abeta peptide prevents toxicity and promotes degradation in vivo.

    Directory of Open Access Journals (Sweden)

    Leila M Luheshi

    2010-03-01

    Full Text Available Protein aggregation, arising from the failure of the cell to regulate the synthesis or degradation of aggregation-prone proteins, underlies many neurodegenerative disorders. However, the balance between the synthesis, clearance, and assembly of misfolded proteins into neurotoxic aggregates remains poorly understood. Here we study the effects of modulating this balance for the amyloid-beta (Abeta peptide by using a small engineered binding protein (Z(Abeta3 that binds with nanomolar affinity to Abeta, completely sequestering the aggregation-prone regions of the peptide and preventing its aggregation. Co-expression of Z(Abeta3 in the brains of Drosophila melanogaster expressing either Abeta(42 or the aggressive familial associated E22G variant of Abeta(42 abolishes their neurotoxic effects. Biochemical analysis indicates that monomer Abeta binding results in degradation of the peptide in vivo. Complementary biophysical studies emphasize the dynamic nature of Abeta aggregation and reveal that Z(Abeta3 not only inhibits the initial association of Abeta monomers into oligomers or fibrils, but also dissociates pre-formed oligomeric aggregates and, although very slowly, amyloid fibrils. Toxic effects of peptide aggregation in vivo can therefore be eliminated by sequestration of hydrophobic regions in monomeric peptides, even when these are extremely aggregation prone. Our studies also underline how a combination of in vivo and in vitro experiments provide mechanistic insight with regard to the relationship between protein aggregation and clearance and show that engineered binding proteins may provide powerful tools with which to address the physiological and pathological consequences of protein aggregation.

  19. The prion protein as a receptor for amyloid-beta

    NARCIS (Netherlands)

    Kessels, Helmut W.; Nguyen, Louis N.; Nabavi, Sadegh; Malinow, Roberto

    2010-01-01

    Increased levels of brain amyloid-beta, a secreted peptide cleavage product of amyloid precursor protein (APP), is believed to be critical in the aetiology of Alzheimer's disease. Increased amyloid-beta can cause synaptic depression, reduce the number of spine protrusions (that is, sites of synaptic

  20. Molecular Mechanism of the Early Stage of Amyloidogenic Hexapeptides (NFGAIL) Aggregation

    International Nuclear Information System (INIS)

    Shi Bi-Yun; Zhou Bo; Cai Zhuo-Wei; Yang Zai-Xing; Xiu Peng

    2013-01-01

    Peptides/proteins aggregation can give rise to pathological conditions of many human diseases. Small partially ordered oligomers formed in the early stage of aggregation, rather than mature fibrils, are thought to be the main toxicity agent for the living cell. Thus, understanding the pathway and the underlying physical mechanism in the early stage of aggregation is very important for prevention and treatment of these protein functional diseases. Herein we use all-atom molecular dynamics simulations to study the aggregation of four NFGAIL hexapeptides (NFGAIL peptide is a core segment of human islet amyloid polypeptide and exhibits similar aggregation kinetics as the full-length polypeptide). We observe that the peptide monomers in water mainly adopt non-structural coil configurations; the four peptides which are randomly placed in water aggregate spontaneously to partially ordered oligomer (β-sheets) through dimerization or trimerization, with the dimerization predominated. Both parallel and anti-parallel β-sheets are observed. The hydrophobic interactions drive the initial peptides associations, and the subsequent conformational fluctuations promote the formation of more hydrogen bonds between the dangling hydrogen sites in the main chains of peptides. (interdisciplinary physics and related areas of science and technology)

  1. Design, synthesis and biological evaluation of benzo[e][1,2,4]triazin-7(1H)-one and [1,2,4]-triazino[5,6,1-jk]carbazol-6-one derivatives as dual inhibitors of beta-amyloid aggregation and acetyl/butyryl cholinesterase.

    Science.gov (United States)

    Catto, Marco; Berezin, Andrey A; Lo Re, Daniele; Loizou, Georgia; Demetriades, Marina; De Stradis, Angelo; Campagna, Francesco; Koutentis, Panayiotis A; Carotti, Angelo

    2012-12-01

    Alzheimer's disease (AD) onset and progression are associated with the dysregulation of multiple and complex physiological processes and a successful therapeutic approach should therefore address more than one target. Two new chemical entities, the easily accessible heterocyclic scaffolds 1,3-diphenylbenzo[e][1,2,4]triazin-7(1H)-one (benzotriazinone I) and 2-phenyl-6H-[1,2,4]triazino[5,6,1-jk]carbazol-6-one (triazafluoranthenone II), were explored for their multitarget-directed inhibition of beta-amyloid (Aβ) fibrillization and acetyl- (AChE) and/or butyryl- (BChE) cholinesterase, three valuable targets for AD therapy. Introduction of appropriate amine substituents at positions 6 and 5 on scaffold I and II, respectively, allowed the preparation of a series of compounds that were tested as Aβ(1-40) aggregation and cholinesterase inhibitors. Potent inhibitors of Aβ self-aggregation were discovered and among them benzotriazinone 7 exhibited an outstanding IC(50) equal to 0.37 μM. Compounds bearing a basic amine linked to the heterocyclic scaffold through a linear alkyl chain of varying length also afforded good ChE inhibitors. In particular, benzotriazinone 24 and triazafluoranthenone 38 were endowed with an interesting multiple activity, the former displaying IC(50) values of 1.4, 1.5 and 1.9 μM on Aβ aggregation and AChE and BChE inhibition, respectively, and the latter showing IC(50) values of 1.4 and an outstanding 0.025 μM in the Aβ aggregation and BChE inhibition, respectively. Benzotriazinone 24 and triazafluoranthenone 29, selected owing to their suitable aqueous solubility and Aβ aggregation inhibition, were submitted to a time course kinetic assay followed with thioflavin T (ThT) spectrofluorimetry, circular dichroism (CD) and transmission electron microscopy (TEM). Experimental data indicated that 24 acted at a low concentration ratio (10 μM 24 vs. 50 μM Aβ), stabilizing the unstructured Aβ peptide and inhibiting fibrillogenesis, and that 29

  2. ALPHA-SYNUCLEIN STRUCTURE, AGGREGATION AND MODULATORS

    Directory of Open Access Journals (Sweden)

    Pinakin K. Makwana

    2016-06-01

    Full Text Available Alpha-synuclein is an intrinsically unstructured protein, involved in various neurodegenerative disorders. In vitro/in vivo experiments, as well as genetic mutation studies establish a direct link between alphasynuclein and synucleinopathies. Due to its natively unfolded state, alpha synuclein can adopt numerous conformations upon interaction with its partners and cellular factors, offering explanation for its diverse interactions. Aggregated form of alpha-synuclein has been observed in the brain of patients with synucleinopathies, a hallmark of neurodegeneration, and cell death has been attributed to aggregation induced toxicity. The process of aggregation involves nucleation, followed by intermediate oligomeric states, and finally the fibrillar amyloids. Of the various conformations/species that alpha-synuclein assumes before it transforms into mature amyloid fibrils, the oligomeric species is the most toxic. Thus, an effective way to limit disease progression is by modifying/slowing down protein aggregation/deposition in the brain. Various small natural products, synthetic chemicals, peptides and antibodies specific to alpha-synuclein have been designed/identified to reduce its rate of aggregation. Unfortunately, not even a handful of the molecules have cleared the clinical trials. Even today, medications available for Parkinson’s patients are mostly the drugs that adjust for loss of dopamine in the brain, and hence do not stop the progression of the disease or cure the symptoms. Thus, more molecular level studies are warranted to fully elucidate the process of alpha-synuclein aggregation, which in turn could help in identifying novel therapeutics and preventives. The present review summarizes the insights gained into the structure, in vitro aggregation and inhibitors/modulators of alpha-synuclein aggregation, that can be used to design better and effective inhibitors against the diseases.

  3. Degradation and aggregation of delta sleep-inducing peptide (DSIP) and two analogs in plasma and serum

    International Nuclear Information System (INIS)

    Graf, M.V.; Saegesser, B.; Schoenenberger, G.A.

    1987-01-01

    The biostability of DSIP (delta sleep-inducing peptide) and two analogs in blood was investigated in order to determine if rates of inactivation contribute to variable effects in vivo. Incubation of DSIP in human or rat blood led to release of products having retention times on a gel filtration column equivalent to Trp. Formation of products was dependent on temperature, time, and species. Incubation of 125 I-N-Tyr-DSIP and 125 I-N-Tyr-P-DSIP, a phosphorylated analog, revealed slower degradation and, in contrast to DSIP, produced complex formation. An excess of unlabeled material did not displace the radioactivity supporting the assumption of non-specific binding/aggregation. It was concluded that the rapid disappearance of injected DSIP in blood was due to degradation, whereas complex formation together with slower degradation resulted in longer persistence of apparently intact analogs. Whether this could explain the sometimes stronger and more consistent effects of DSIP-analogs remains to be examined

  4. Haematoxylin and eosin staining identifies medium to large bacterial aggregates with a reliable specificity: A comparative analysis of follicular bacterial aggregates in axillary biopsies using peptide nucleic acid-fluorescence in situ hybridization and haematoxylin and eosin staining.

    Science.gov (United States)

    Ring, Hans Christian; Theut Riis, Peter; Bay, Lene; Kallenbach, Klaus; Bjarnsholt, Thomas; Jemec, Gregor B E

    2017-10-01

    Although peptide nucleic acid (PNA), fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM) are the reference tools in the study of bacterial aggregates/biofilms, it may also be rather time-consuming. This study aimed to investigate the sensitivity and specificity between bacterial aggregates identified by haematoxylin and eosin (HE) staining vs bacterial aggregates in corresponding PNA-FISH samples. Axillary biopsies were obtained in 24 healthy controls. HE-stained and PNA-FISH samples were investigated using traditional light microscopy and CLSM, respectively. The data demonstrate that HE staining identifies large bacterial aggregates (>10 μm) with a sensitivity of 0.43 and specificity of 1. The methods, however, are not equivalent as demonstrated by a McNemar's test (P=.04). Where bacterial aggregates >10 μm in diameter, HE staining may offer a rapid and practical low-cost tool to evaluate bacterial aggregates. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  5. Chiral recognition in amyloid fiber growth.

    Science.gov (United States)

    Torbeev, Vladimir; Grogg, Marcel; Ruiz, Jérémy; Boehringer, Régis; Schirer, Alicia; Hellwig, Petra; Jeschke, Gunnar; Hilvert, Donald

    2016-05-01

    Insoluble amyloid fibers represent a pathological signature of many human diseases. To treat such diseases, inhibition of amyloid formation has been proposed as a possible therapeutic strategy. d-Peptides, which possess high proteolytic stability and lessened immunogenicity, are attractive candidates in this context. However, a molecular understanding of chiral recognition phenomena for d-peptides and l-amyloids is currently incomplete. Here we report experiments on amyloid growth of individual enantiomers and their mixtures for two distinct polypeptide systems of different length and structural organization: a 44-residue covalently-linked dimer derived from a peptide corresponding to the [20-41]-fragment of human β2-microglobulin (β2m) and the 99-residue full-length protein. For the dimeric [20-41]β2m construct, a combination of electron paramagnetic resonance of nitroxide-labeled constructs and (13) C-isotope edited FT-IR spectroscopy of (13) C-labeled preparations was used to show that racemic mixtures precipitate as intact homochiral fibers, i.e. undergo spontaneous Pasteur-like resolution into a mixture of left- and right-handed amyloids. In the case of full-length β2m, the presence of the mirror-image d-protein affords morphologically distinct amyloids that are composed largely of enantiopure domains. Removal of the l-component from hybrid amyloids by proteolytic digestion results in their rapid transformation into characteristic long straight d-β2m amyloids. Furthermore, the full-length d-enantiomer of β2m was found to be an efficient inhibitor of l-β2m amyloid growth. This observation highlights the potential of longer d-polypeptides for future development into inhibitors of amyloid propagation. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

  6. Contribution of simple saccharides to the stabilization of amyloid structure

    International Nuclear Information System (INIS)

    Fung, Justin; Darabie, Audrey A.; McLaurin, JoAnne

    2005-01-01

    The use of osmolytes or chaperones to stabilize proteins/peptides that misfold in neurodegenerative diseases is an attractive concept for drug development. We have investigated the role of a series of small carbohydrates for protection of the natively structured Alzheimer's amyloidpeptides (Aβ). Using circular dichroism spectroscopy to follow the β-structural transitions and electron microscopy to examine tertiary structural characteristics, we demonstrate that the hydrogen bonding capacity of the carbohydrate determines the inhibition or promotion of fibrillogenesis. Three sugar molecules that vary only in their distribution of potential H-bonding partners promote various structural changes in Aβ. Two of these sugar molecules are excluded from Aβ during aggregation and promote mature fibre growth, while the other binds Aβ promoting nucleation and the accumulation of protofibrils. Our studies suggest that utilization of a combinatorial strategy to alter H-bonding capacity across a simple carbohydrate molecule may represent a novel drug design strategy

  7. Functional Amyloid Formation within Mammalian Tissue.

    Directory of Open Access Journals (Sweden)

    2005-11-01

    Full Text Available Amyloid is a generally insoluble, fibrous cross-beta sheet protein aggregate. The process of amyloidogenesis is associated with a variety of neurodegenerative diseases including Alzheimer, Parkinson, and Huntington disease. We report the discovery of an unprecedented functional mammalian amyloid structure generated by the protein Pmel17. This discovery demonstrates that amyloid is a fundamental nonpathological protein fold utilized by organisms from bacteria to humans. We have found that Pmel17 amyloid templates and accelerates the covalent polymerization of reactive small molecules into melanin-a critically important biopolymer that protects against a broad range of cytotoxic insults including UV and oxidative damage. Pmel17 amyloid also appears to play a role in mitigating the toxicity associated with melanin formation by sequestering and minimizing diffusion of highly reactive, toxic melanin precursors out of the melanosome. Intracellular Pmel17 amyloidogenesis is carefully orchestrated by the secretory pathway, utilizing membrane sequestration and proteolytic steps to protect the cell from amyloid and amyloidogenic intermediates that can be toxic. While functional and pathological amyloid share similar structural features, critical differences in packaging and kinetics of assembly enable the usage of Pmel17 amyloid for normal function. The discovery of native Pmel17 amyloid in mammals provides key insight into the molecular basis of both melanin formation and amyloid pathology, and demonstrates that native amyloid (amyloidin may be an ancient, evolutionarily conserved protein quaternary structure underpinning diverse pathways contributing to normal cell and tissue physiology.

  8. Functional amyloid formation within mammalian tissue.

    Directory of Open Access Journals (Sweden)

    Douglas M Fowler

    2006-01-01

    Full Text Available Amyloid is a generally insoluble, fibrous cross-beta sheet protein aggregate. The process of amyloidogenesis is associated with a variety of neurodegenerative diseases including Alzheimer, Parkinson, and Huntington disease. We report the discovery of an unprecedented functional mammalian amyloid structure generated by the protein Pmel17. This discovery demonstrates that amyloid is a fundamental nonpathological protein fold utilized by organisms from bacteria to humans. We have found that Pmel17 amyloid templates and accelerates the covalent polymerization of reactive small molecules into melanin-a critically important biopolymer that protects against a broad range of cytotoxic insults including UV and oxidative damage. Pmel17 amyloid also appears to play a role in mitigating the toxicity associated with melanin formation by sequestering and minimizing diffusion of highly reactive, toxic melanin precursors out of the melanosome. Intracellular Pmel17 amyloidogenesis is carefully orchestrated by the secretory pathway, utilizing membrane sequestration and proteolytic steps to protect the cell from amyloid and amyloidogenic intermediates that can be toxic. While functional and pathological amyloid share similar structural features, critical differences in packaging and kinetics of assembly enable the usage of Pmel17 amyloid for normal function. The discovery of native Pmel17 amyloid in mammals provides key insight into the molecular basis of both melanin formation and amyloid pathology, and demonstrates that native amyloid (amyloidin may be an ancient, evolutionarily conserved protein quaternary structure underpinning diverse pathways contributing to normal cell and tissue physiology.

  9. Anti-amyloid treatments in Alzheimer's disease.

    Science.gov (United States)

    Sapra, Mamta; Kim, Kye Y

    2009-06-01

    Alzheimer's disease is one of the most challenging threats to the healthcare system in society. One of the main characteristic of Alzheimer's disease (AD) pathology is formation of amyloid plaques from accumulation of amyloid beta peptide. The therapeutic agents that are currently available for AD including acetylcholinesterase inhibitors (AchEIs) and the N-methyl-D-aspartate (NMDA) antagonist are focused on improving the symptoms and do not revert the progression of the disease. This limitation coupled with the burgeoning increase in the prevalence of AD and resultant impact on healthcare economics calls for more substantial treatments for AD. According to the leading amyloid hypothesis, cleavage of amyloid precursor protein to release amyloid beta peptide is the critical event in pathogenesis of Alzheimer's disease. Recently treatment strategies have been focused on modifying the formation, clearance and accumulation of neurotoxic amyloid beta peptide. This article reviews different therapeutic approaches that have been investigated to target amyloid beta ranging from secretase modulators, antiaggregation agents to amyloid immunotherapy. Authors review the different novel drugs which are in clinical trials.

  10. Data on correlation between Aβ42 structural aggregation propensity and toxicity in bacteria

    Directory of Open Access Journals (Sweden)

    Anita Carija

    2016-06-01

    Full Text Available Protein aggregation and amyloid formation is a hallmark of an increasing number of human disorders. Because protein aggregation is deleterious for the cell physiology and results in a decrease in overall cell fitness, it is thought that natural selection acts to purify aggregating proteins during evolution. This data article contains complementary figures and results related to the research article entitled “Selection against toxic aggregation-prone protein sequences in bacteria” (Navarro et al., 2014 [1]. Here, we used the AGGRESCAN3D (A3D server, a novel in house predictor that forecasts protein aggregation properties in protein structures to illustrate a striking correlation between the structure-based predictions of aggregation propensities for Alzheimer’s Aβ42 peptide variants and their previously reported deleterious effects in bacteria.

  11. Iron Biochemistry is Correlated with Amyloid Plaque Morphology in an Established Mouse Model of Alzheimer's Disease.

    Science.gov (United States)

    Telling, Neil D; Everett, James; Collingwood, Joanna F; Dobson, Jon; van der Laan, Gerrit; Gallagher, Joseph J; Wang, Jian; Hitchcock, Adam P

    2017-10-19

    A signature characteristic of Alzheimer's disease (AD) is aggregation of amyloid-beta (Aβ) fibrils in the brain. Nevertheless, the links between Aβ and AD pathology remain incompletely understood. It has been proposed that neurotoxicity arising from aggregation of the Aβ 1-42 peptide can in part be explained by metal ion binding interactions. Using advanced X-ray microscopy techniques at sub-micron resolution, we investigated relationships between iron biochemistry and AD pathology in intact cortex from an established mouse model over-producing Aβ. We found a direct correlation of amyloid plaque morphology with iron, and evidence for the formation of an iron-amyloid complex. We also show that iron biomineral deposits in the cortical tissue contain the mineral magnetite, and provide evidence that Aβ-induced chemical reduction of iron could occur in vivo. Our observations point to the specific role of iron in amyloid deposition and AD pathology, and may impact development of iron-modifying therapeutics for AD. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  12. High plasma levels of islet amyloid polypeptide in young with new-onset of type 1 diabetes mellitus.

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    Johan F Paulsson

    Full Text Available AIMS/HYPOTHESIS: Islet amyloid polypeptide (IAPP is a beta cell hormone secreted together with insulin upon glucose stimulation. IAPP participates in normal glucose regulation, but IAPP is also known for its ability to misfold and form islet amyloid. Amyloid fibrils form through smaller cell toxic intermediates and deposited amyloid disrupts normal islet architecture. Even though IAPP and amyloid formation are much discussed in type 2 diabetes, our aim was to study the significance of IAPP in type 1 diabetes. RESULTS: Plasma IAPP levels in children and adolescents with newly diagnosed type 1 diabetes (n = 224 were analysed and concentrations exceeding 100 pmol/L (127.2-888.7 pmol/L were found in 11% (25/224. The IAPP increase did not correlate with C-peptide levels. CONCLUSIONS/INTERPRETATION: Plasma levels of IAPP and insulin deviate in a subpopulation of young with newly-diagnosed type 1 diabetes. The determined elevated levels of IAPP might increase the risk for IAPP misfolding and formation of cell toxic amyloid in beta cells. This finding add IAPP-aggregation to the list over putative pathological factors causing type 1 diabetes.

  13. Alzheimer's disease amyloid-beta links lens and brain pathology in Down syndrome.

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    Juliet A Moncaster

    2010-05-01

    Full Text Available Down syndrome (DS, trisomy 21 is the most common chromosomal disorder and the leading genetic cause of intellectual disability in humans. In DS, triplication of chromosome 21 invariably includes the APP gene (21q21 encoding the Alzheimer's disease (AD amyloid precursor protein (APP. Triplication of the APP gene accelerates APP expression leading to cerebral accumulation of APP-derived amyloid-beta peptides (Abeta, early-onset AD neuropathology, and age-dependent cognitive sequelae. The DS phenotype complex also includes distinctive early-onset cerulean cataracts of unknown etiology. Previously, we reported increased Abeta accumulation, co-localizing amyloid pathology, and disease-linked supranuclear cataracts in the ocular lenses of subjects with AD. Here, we investigate the hypothesis that related AD-linked Abeta pathology underlies the distinctive lens phenotype associated with DS. Ophthalmological examinations of DS subjects were correlated with phenotypic, histochemical, and biochemical analyses of lenses obtained from DS, AD, and normal control subjects. Evaluation of DS lenses revealed a characteristic pattern of supranuclear opacification accompanied by accelerated supranuclear Abeta accumulation, co-localizing amyloid pathology, and fiber cell cytoplasmic Abeta aggregates (approximately 5 to 50 nm identical to the lens pathology identified in AD. Peptide sequencing, immunoblot analysis, and ELISA confirmed the identity and increased accumulation of Abeta in DS lenses. Incubation of synthetic Abeta with human lens protein promoted protein aggregation, amyloid formation, and light scattering that recapitulated the molecular pathology and clinical features observed in DS lenses. These results establish the genetic etiology of the distinctive lens phenotype in DS and identify the molecular origin and pathogenic mechanism by which lens pathology is expressed in this common chromosomal disorder. Moreover, these findings confirm increased Abeta

  14. Proteomic screening for amyloid proteins.

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    Anton A Nizhnikov

    Full Text Available Despite extensive study, progress in elucidation of biological functions of amyloids and their role in pathology is largely restrained due to the lack of universal and reliable biochemical methods for their discovery. All biochemical methods developed so far allowed only identification of glutamine/asparagine-rich amyloid-forming proteins or proteins comprising amyloids that form large deposits. In this article we present a proteomic approach which may enable identification of a broad range of amyloid-forming proteins independently of specific features of their sequences or levels of expression. This approach is based on the isolation of protein fractions enriched with amyloid aggregates via sedimentation by ultracentrifugation in the presence of strong ionic detergents, such as sarkosyl or SDS. Sedimented proteins are then separated either by 2D difference gel electrophoresis or by SDS-PAGE, if they are insoluble in the buffer used for 2D difference gel electrophoresis, after which they are identified by mass-spectrometry. We validated this approach by detection of known yeast prions and mammalian proteins with established capacity for amyloid formation and also revealed yeast proteins forming detergent-insoluble aggregates in the presence of human huntingtin with expanded polyglutamine domain. Notably, with one exception, all these proteins contained glutamine/asparagine-rich stretches suggesting that their aggregates arose due to polymerization cross-seeding by human huntingtin. Importantly, though the approach was developed in a yeast model, it can easily be applied to any organism thus representing an efficient and universal tool for screening for amyloid proteins.

  15. Monoamine oxidase B is elevated in Alzheimer disease neurons, is associated with γ-secretase and regulates neuronal amyloid β-peptide levels.

    Science.gov (United States)

    Schedin-Weiss, Sophia; Inoue, Mitsuhiro; Hromadkova, Lenka; Teranishi, Yasuhiro; Yamamoto, Natsuko Goto; Wiehager, Birgitta; Bogdanovic, Nenad; Winblad, Bengt; Sandebring-Matton, Anna; Frykman, Susanne; Tjernberg, Lars O

    2017-08-01

    Increased levels of the pathogenic amyloid β-peptide (Aβ), released from its precursor by the transmembrane protease γ-secretase, are found in Alzheimer disease (AD) brains. Interestingly, monoamine oxidase B (MAO-B) activity is also increased in AD brain, but its role in AD pathogenesis is not known. Recent neuroimaging studies have shown that the increased MAO-B expression in AD brain starts several years before the onset of the disease. Here, we show a potential connection between MAO-B, γ-secretase and Aβ in neurons. MAO-B immunohistochemistry was performed on postmortem human brain. Affinity purification of γ-secretase followed by mass spectrometry was used for unbiased identification of γ-secretase-associated proteins. The association of MAO-B with γ-secretase was studied by coimmunoprecipitation from brain homogenate, and by in-situ proximity ligation assay (PLA) in neurons as well as mouse and human brain sections. The effect of MAO-B on Aβ production and Notch processing in cell cultures was analyzed by siRNA silencing or overexpression experiments followed by ELISA, western blot or FRET analysis. Methodology for measuring relative intraneuronal MAO-B and Aβ42 levels in single cells was developed by combining immunocytochemistry and confocal microscopy with quantitative image analysis. Immunohistochemistry revealed MAO-B staining in neurons in the frontal cortex, hippocampus CA1 and entorhinal cortex in postmortem human brain. Interestingly, the neuronal staining intensity was higher in AD brain than in control brain in these regions. Mass spectrometric data from affinity purified γ-secretase suggested that MAO-B is a γ-secretase-associated protein, which was confirmed by immunoprecipitation and PLA, and a neuronal location of the interaction was shown. Strikingly, intraneuronal Aβ42 levels correlated with MAO-B levels, and siRNA silencing of MAO-B resulted in significantly reduced levels of intraneuronal Aβ42. Furthermore, overexpression of

  16. Causative factors for formation of toxic islet amyloid polypeptide oligomer in type 2 diabetes mellitus

    Directory of Open Access Journals (Sweden)

    Jeong HR

    2015-11-01

    Full Text Available Hye Rin Jeong, Seong Soo A AnDepartment of Bionano Technology, Gachon Medical Research Institute, Gachon University, Gyeonggi-do, Republic of KoreaAbstract: Human islet amyloid polypeptide (h-IAPP is a peptide hormone that is synthesized and cosecreted with insulin from insulin-secreting pancreatic β-cells. Recently, h-IAPP was proposed to be the main component responsible for the cytotoxic pancreatic amyloid deposits in patients with type 2 diabetes mellitus (T2DM. Since the causative factors of IAPP (or amylin oligomer aggregation are not fully understood, this review will discuss the various forms of h-IAPP aggregation. Not all forms of IAPP aggregates trigger the destruction of β-cell function and loss of β-cell mass; however, toxic oligomers do trigger these events. Once these toxic oligomers form under abnormal metabolic conditions in T2DM, they can lead to cell disruption by inducing cell membrane destabilization. In this review, the various factors that have been shown to induce toxic IAPP oligomer formation will be presented, as well as the potential mechanism of oligomer and fibril formation from pro-IAPPs. Initially, pro-IAPPs undergo enzymatic reactions to produce the IAPP monomers, which can then develop into oligomers and fibrils. By this mechanism, toxic oligomers could be generated by diverse pathway components. Thus, the interconnections between factors that influence amyloid aggregation (eg, absence of PC2 enzyme, deamidation, reduction of disulfide bonds, environmental factors in the cell, genetic mutations, copper metal ions, and heparin will be presented. Hence, this review will aid in understanding the fundamental causative factors contributing to IAPP oligomer formation and support studies for investigating novel T2DM therapeutic approaches, such as the development of inhibitory agents for preventing oligomerization at the early stages of diabetic pathology.Keywords: amyloid aggregation, causative factor, IAPP, islet

  17. Overproduction, purification, crystallization and preliminary X-ray analysis of human Fe65-PTB2 in complex with the amyloid precursor protein intracellular domain

    Energy Technology Data Exchange (ETDEWEB)

    Radzimanowski, Jens [Heidelberg University Biochemistry Center, INF328, D-69120 Heidelberg (Germany); Beyreuther, Konrad [Center for Molecular Biology, University Heidelberg, INF282, D-69120 Heidelberg (Germany); Sinning, Irmgard; Wild, Klemens, E-mail: klemens.wild@bzh.uni-heidelberg.de [Heidelberg University Biochemistry Center, INF328, D-69120 Heidelberg (Germany)

    2008-05-01

    Alzheimer’s disease is characterized by proteolytic processing of the amyloid precursor protein (APP), which releases the aggregation-prone amyloid-β (Aβ) peptide and liberates the intracellular domain (AICD) that interacts with various adaptor proteins. The crystallized AICD–Fe65-PTB2 complex is of central importance for APP translocation, nuclear signalling, processing and Aβ generation. Alzheimer’s disease is associated with typical brain deposits (senile plaques) that mainly contain the neurotoxic amyloid β peptide. This peptide results from proteolytic processing of the type I transmembrane protein amyloid precursor protein (APP). During this proteolytic pathway the APP intracellular domain (AICD) is released into the cytosol, where it associates with various adaptor proteins. The interaction of the AICD with the C-terminal phosphotyrosine-binding domain of Fe65 (Fe65-PTB2) regulates APP translocation, signalling and processing. Human AICD and Fe65-PTB2 have been cloned, overproduced and purified in large amounts in Escherichia coli. A complex of Fe65-PTB2 with the C-terminal 32 amino acids of the AICD gave well diffracting hexagonal crystals and data have been collected to 2.1 Å resolution. Initial phases obtained by the molecular-replacement method are of good quality and revealed well defined electron density for the substrate peptide.

  18. Role of Prion Protein Aggregation in Neurotoxicity

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    Tullio Florio

    2012-07-01

    Full Text Available In several neurodegenerative diseases, such as Parkinson, Alzheimer’s, Huntington, and prion diseases, the deposition of aggregated misfolded proteins is believed to be responsible for the neurotoxicity that characterizes these diseases. Prion protein (PrP, the protein responsible of prion diseases, has been deeply studied for the peculiar feature of its misfolded oligomers that are able to propagate within affected brains, inducing the conversion of the natively folded PrP into the pathological conformation. In this review, we summarize the available experimental evidence concerning the relationship between aggregation status of misfolded PrP and neuronal death in the course of prion diseases. In particular, we describe the main findings resulting from the use of different synthetic (mainly PrP106-126 and recombinant PrP-derived peptides, as far as mechanisms of aggregation and amyloid formation, and how these different spatial conformations can affect neuronal death. In particular, most data support the involvement of non-fibrillar oligomers rather than actual amyloid fibers as the determinant of neuronal death.

  19. NMR WaterLOGSY Reveals Weak Binding of Bisphenol A with Amyloid Fibers of a Conserved 11 Residue Peptide from Androgen Receptor.

    Directory of Open Access Journals (Sweden)

    Julia Asencio-Hernández

    Full Text Available There is growing evidence that bisphenol A (BPA, a molecule largely released in the environment, has detrimental effects on ecosystems and on human health. It acts as an endocrine disruptor targeting steroid hormone receptors, such as the estrogen receptor (ER, estrogen-related receptor (ERR and androgen receptor (AR. BPA-derived molecules have recently been shown to interact with the AR N-terminal domain (AR-NTD, which is known to be largely intrinsically disordered. This N-terminal domain contains an 11 residue conserved domain that forms amyloid fibers upon oxidative dimerisation through its strictly conserved Cys240 residue. We investigate here the interaction of BPA, and other potential endocrine disruptors, with AR-NTD amyloid fibers using the WaterLOGSY NMR experiment. We observed a selective binding of these compounds to the amyloid fibers formed by the AR-NTD conserved region and glutamine homopolymers. This observation suggests that the high potency of endocrine disruptors may result, in part, from their ability to bind amyloid forms of nuclear receptors in addition to their cognate binding sites. This property may be exploited to design future therapeutic strategies targeting AR related diseases such as the spinal bulbar muscular atrophy or prostate cancer. The ability of NMR WaterLOGSY experiments to detect weak interactions between small ligands and amyloid fibers may prove to be of particular interest for identifying promising hit molecules.

  20. Calcium signaling and amyloid toxicity in Alzheimer disease.

    Science.gov (United States)

    Demuro, Angelo; Parker, Ian; Stutzmann, Grace E

    2010-04-23

    Intracellular Ca(2+) signaling is fundamental to neuronal physiology and viability. Because of its ubiquitous roles, disruptions in Ca(2+) homeostasis are implicated in diverse disease processes and have become a major focus of study in multifactorial neurodegenerative diseases such as Alzheimer disease (AD). A hallmark of AD is the excessive production of beta-amyloid (Abeta) and its massive accumulation in amyloid plaques. In this minireview, we highlight the pathogenic interactions between altered cellular Ca(2+) signaling and Abeta in its different aggregation states and how these elements coalesce to alter the course of the neurodegenerative disease. Ca(2+) and Abeta intersect at several functional levels and temporal stages of AD, thereby altering neurotransmitter receptor properties, disrupting membrane integrity, and initiating apoptotic signaling cascades. Notably, there are reciprocal interactions between Ca(2+) pathways and amyloid pathology; altered Ca(2+) signaling accelerates Abeta formation, whereas Abeta peptides, particularly in soluble oligomeric forms, induce Ca(2+) disruptions. A degenerative feed-forward cycle of toxic Abeta generation and Ca(2+) perturbations results, which in turn can spin off to accelerate more global neuropathological cascades, ultimately leading to synaptic breakdown, cell death, and devastating memory loss. Although no cause or cure is currently known, targeting Ca(2+) dyshomeostasis as an underlying and integral component of AD pathology may result in novel and effective treatments for AD.

  1. Acetylcholinesterase triggers the aggregation of PrP 106-126

    International Nuclear Information System (INIS)

    Pera, M.; Roman, S.; Ratia, M.; Camps, P.; Munoz-Torrero, D.; Colombo, L.; Manzoni, C.; Salmona, M.; Badia, A.; Clos, M.V.

    2006-01-01

    Acetylcholinesterase (AChE), a senile plaque component, promotes amyloid-β-protein (Aβ) fibril formation in vitro. The presence of prion protein (PrP) in Alzheimer's disease (AD) senile plaques prompted us to assess if AChE could trigger the PrP peptides aggregation as well. Consequently, the efficacy of AChE on the PrP peptide spanning-residues 106-126 aggregation containing a coumarin fluorescence probe (coumarin-PrP 106-126) was studied. Kinetics of coumarin-PrP 106-126 aggregation showed a significant increase of maximum size of aggregates (MSA), which was dependent on AChE concentration. AChE-PrP 106-126 aggregates showed the tinctorial and optical amyloid properties as determined by polarized light and electronic microscopy analysis. A remarkable inhibition of MSA was obtained with propidium iodide, suggesting that AChE triggers PrP 106-126 and Aβ aggregation through a similar mechanism. Huprines (AChE inhibitors) also significantly decreased MSA induced by AChE as well, unveiling the potential interest for some AChE inhibitors as a novel class of potential anti-prion drugs

  2. Diabetes Drug Discovery: hIAPP1–37 Polymorphic Amyloid Structures as Novel Therapeutic Targets

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    Isaac Fernández-Gómez

    2018-03-01

    Full Text Available Human islet amyloid peptide (hIAPP1–37 aggregation is an early step in Diabetes Mellitus. We aimed to evaluate a family of pharmaco-chaperones to act as modulators that provide dynamic interventions and the multi-target capacity (native state, cytotoxic oligomers, protofilaments and fibrils of hIAPP1–37 required to meet the treatment challenges of diabetes. We used a cross-functional approach that combines in silico and in vitro biochemical and biophysical methods to study the hIAPP1–37 aggregation-oligomerization process as to reveal novel potential anti-diabetic drugs. The family of pharmaco-chaperones are modulators of the oligomerization and fibre formation of hIAPP1–37. When they interact with the amino acid in the amyloid-like steric zipper zone, they inhibit and/or delay the aggregation-oligomerization pathway by binding and stabilizing several amyloid structures of hIAPP1–37. Moreover, they can protect cerebellar granule cells (CGC from the cytotoxicity produced by the hIAPP1–37 oligomers. The modulation of proteostasis by the family of pharmaco-chaperones A–F is a promising potential approach to limit the onset and progression of diabetes and its comorbidities.

  3. Diabetes Drug Discovery: hIAPP1-37 Polymorphic Amyloid Structures as Novel Therapeutic Targets.

    Science.gov (United States)

    Fernández-Gómez, Isaac; Sablón-Carrazana, Marquiza; Bencomo-Martínez, Alberto; Domínguez, Guadalupe; Lara-Martínez, Reyna; Altamirano-Bustamante, Nelly F; Jiménez-García, Luis Felipe; Pasten-Hidalgo, Karina; Castillo-Rodríguez, Rosa Angélica; Altamirano, Perla; Marrero, Suchitil Rivera; Revilla-Monsalve, Cristina; Valdés-Sosa, Peter; Salamanca-Gómez, Fabio; Garrido-Magaña, Eulalia; Rodríguez-Tanty, Chryslaine; Altamirano-Bustamante, Myriam M

    2018-03-19

    Human islet amyloid peptide (hIAPP 1-37 ) aggregation is an early step in Diabetes Mellitus. We aimed to evaluate a family of pharmaco-chaperones to act as modulators that provide dynamic interventions and the multi-target capacity (native state, cytotoxic oligomers, protofilaments and fibrils of hIAPP 1-37 ) required to meet the treatment challenges of diabetes. We used a cross-functional approach that combines in silico and in vitro biochemical and biophysical methods to study the hIAPP 1-37 aggregation-oligomerization process as to reveal novel potential anti-diabetic drugs. The family of pharmaco-chaperones are modulators of the oligomerization and fibre formation of hIAPP 1-37 . When they interact with the amino acid in the amyloid-like steric zipper zone, they inhibit and/or delay the aggregation-oligomerization pathway by binding and stabilizing several amyloid structures of hIAPP 1-37 . Moreover, they can protect cerebellar granule cells (CGC) from the cytotoxicity produced by the hIAPP 1-37 oligomers. The modulation of proteostasis by the family of pharmaco-chaperones A - F is a promising potential approach to limit the onset and progression of diabetes and its comorbidities.

  4. Atomic Force Microscopy and MD Simulations Reveal Pore-Like Structures of All-D-Enantiomer of Alzheimer’s β-Amyloid Peptide: Relevance to the Ion Channel Mechanism of AD Pathology

    Science.gov (United States)

    Connelly, Laura; Arce, Fernando Teran; Jang, Hyunbum; Capone, Ricardo; Kotler, Samuel A.; Ramachandran, Srinivasan; Kagan, Bruce L.; Nussinov, Ruth; Lal, Ratnesh

    2012-01-01

    Alzheimer’s disease (AD) is a protein misfolding disease characterized by a build-up of β-amyloid (Aβ) peptide as senile plaques, uncontrolled neurodegeneration, and memory loss. AD pathology is linked to the destabilization of cellular ionic homeostasis and involves Aβ peptide-plasma membrane interactions. In principle, there are two possible ways through which disturbance of the ionic homeostasis can take place: directly, where the Aβ peptide either inserts into the membrane and creates ion-conductive pores or destabilizes the membrane organization; or, indirectly, where the Aβ peptide interacts with existing cell membrane receptors. To distinguish between these two possible types of Aβ-membrane interactions, we took advantage of the biochemical tenet that ligand-receptor interactions are stereospecific; L-amino acid peptides, but not their D-counterparts, bind to cell membrane receptors. However, with respect to the ion channel-mediated mechanism, like L-amino acids, D-amino acid peptides will also form ion channel-like structures. Using atomic force microscopy (AFM) we imaged the structures of both D- and L-enantiomers of the full length Aβ1-42 when reconstituted in lipid bilayers. AFM imaging shows that both L- and D-Aβ isomers form similar channel-like structures. Molecular dynamics (MD) simulations support the AFM imaged 3D structures. Earlier we have shown that D-Aβ1-42 channels conduct ions similarly to their L-counter parts. Taken together, our results support the direct mechanism of Aβ ion channel-mediated destabilization of ionic homeostasis rather than the indirect mechanism through Aβ interaction with membrane receptors. PMID:22217000

  5. Understanding curcumin-induced modulation of protein aggregation.

    Science.gov (United States)

    Ahmad, Basir; Borana, Mohanish S; Chaudhary, Ankur P

    2017-07-01

    Curcumin, a diarylheptanoid compound, found in spice turmeric is known to alter the aggregation of proteins and reduce the toxicity of the aggregates. This review looks at the molecular basis of modulating protein aggregation and toxicity of the aggregates. Foremost, we identify the interaction of curcumin and its derivatives with proteins/peptides and the effect of their interaction on the conformational stability and unfolding/folding pathway(s). The unfolding/folding processes generate partially folded/unfolded intermediate, which serve as aggregation precursor state. Secondly, we discuss the effect of curcumin binding on the kinetics parameters of the aggregation process, which give information about the mechanism of the aggregation inhibition. We describe, in addition, that curcumin can accelerate/promote fibril formation by binding to oligomeric intermediate(s) accumulated in the aggregation pathway. Finally, we discuss the correlation of curcumin-induced monomeric and/or oligomeric precursor states with aggregate structure and toxicity. On the basis of these discussions, we propose a model describing curcumin-induced inhibition/promotion of formation of amyloid-like fibrils. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Nonequilibrium and generalized-ensemble molecular dynamics simulations for amyloid fibril

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    Okumura, Hisashi [Research Center for Computational Science, Institute for Molecular Science, Okazaki, Aichi 444-8585 (Japan); Department of Structural Molecular Science, The Graduate University for Advanced Studies, Okazaki, Aichi 444-8585 (Japan)

    2015-12-31

    Amyloids are insoluble and misfolded fibrous protein aggregates and associated with more than 20 serious human diseases. We perform all-atom molecular dynamics simulations of amyloid fibril assembly and disassembly.

  7. Amyloid-β production via cleavage of amyloid-β protein precursor is modulated by cell density.

    Science.gov (United States)

    Zhang, Can; Browne, Andrew; Divito, Jason R; Stevenson, Jesse A; Romano, Donna; Dong, Yuanlin; Xie, Zhongcong; Tanzi, Rudolph E

    2010-01-01

    Mounting evidence suggests that Alzheimer's disease (AD) is caused by the accumulation of the small peptide, amyloid-β (Aβ), a proteolytic cleavage product of amyloid-β protein precursor (AβPP). Aβ is generated through a serial cleavage of AβPP by β- and γ-secretase. Aβ40 and Aβ42 are the two main components of amyloid plaques in AD brains, with Aβ42 being more prone to aggregation. AβPP can also be processed by α-secretase, which cleaves AβPP within the Aβ sequence, thereby preventing the generation of Aβ. Little is currently known regarding the effects of cell density on AβPP processing and Aβ generation. Here we assessed the effects of cell density on AβPP processing in neuronal and non-neuronal cell lines, as well as mouse primary cortical neurons. We found that decreased cell density significantly increases levels of Aβ40, Aβ42, total Aβ, and the ratio of Aβ42: Aβ40. These results also indicate that cell density is a significant modulator of AβPP processing. Overall, these findings carry profound implications for both previous and forthcoming studies aiming to assess the effects of various conditions and genetic/chemical factors, e.g., novel drugs on AβPP processing and Aβ generation in cell-based systems. Moreover, it is interesting to speculate whether cell density changes in vivo may also affect AβPP processing and Aβ levels in the AD brain.

  8. Amyloid-degrading ability of nattokinase from Bacillus subtilis natto.

    Science.gov (United States)

    Hsu, Ruei-Lin; Lee, Kung-Ta; Wang, Jung-Hao; Lee, Lily Y-L; Chen, Rita P-Y

    2009-01-28

    More than 20 unrelated proteins can form amyloid fibrils in vivo which are related to various diseases, such as Alzheimer's disease, prion disease, and systematic amyloidosis. Amyloid fibrils are an ordered protein aggregate with a lamellar cross-beta structure. Enhancing amyloid clearance is one of the targets of the therapy of these amyloid-related diseases. Although there is debate on whether the toxicity is due to amyloids or their precursors, research on the degradation of amyloids may help prevent or alleviate these diseases. In this study, we explored the amyloid-degrading ability of nattokinase, a fibrinolytic subtilisin-like serine protease, and determined the optimal conditions for amyloid hydrolysis. This ability is shared by proteinase K and subtilisin Carlsberg, but not by trypsin or plasmin.

  9. Peripherally applied synthetic peptide isoAsp7-Aβ(1-42) triggers cerebral β-amyloidosis.

    Science.gov (United States)

    Kozin, S A; Cheglakov, I B; Ovsepyan, A A; Telegin, G B; Tsvetkov, P O; Lisitsa, A V; Makarov, A A

    2013-10-01

    Intracerebral and intraperitoneal inoculation with β-amyloid-rich brain extracts originating from patients with Alzheimer's disease as well as intracerebral injection of aggregates composed of synthetic Aβ can induce cerebral β-amyloidosis, and associated cognitive dysfunctions in susceptible animal hosts. We have found that repetitive intravenous administration of 100 μg of synthetic peptide corresponding to isoAsp7-containing Aβ(1-42), an abundant age-dependent Aβ isoform present both in the pathological brain and in synthetic Aβ preparations, robustly accelerates formation of classic dense-core congophilic amyloid plaques in the brain of β-amyloid precursor protein transgenic mice. Our findings indicate this peptide as an inductive agent of cerebral β-amyloidosis in vivo.

  10. Active immunization with the peptide epitope vaccine Aβ3-10-KLH induces a Th2-polarized anti-Aβ antibody response and decreases amyloid plaques in APP/PS1 transgenic mice.

    Science.gov (United States)

    Ding, Li; Meng, Yuan; Zhang, Hui-Yi; Yin, Wen-Chao; Yan, Yi; Cao, Yun-Peng

    2016-11-10

    Active amyloid-β (Aβ) immunotherapy is effective in preventing Aβ deposition, facilitating plaque clearance, and improving cognitive functions in mouse models of Alzheimer's disease (AD). Developing a safe and effective AD vaccine requires a delicate balance between inducing adequate humoral immune responses and avoiding T cell-mediated autoimmune responses. In this study, we designed 2 peptide epitope vaccines, Aβ3-10-KLH and 5Aβ3-10, prepared respectively by coupling Aβ3-10 to the immunogenic carrier protein keyhole limpet hemocyanin (KLH) or by joining 5 Aβ3-10 epitopes linearly in tandem. Young APP/PS1 mice were immunized subcutaneously with Aβ3-10-KLH or 5Aβ3-10 mixed with Freund's adjuvant, and the immunopotencies of these Aβ3-10 peptide vaccines were tested. Aβ3-10-KLH elicited a robust Th2-polarized anti-Aβ antibody response and inhibited Aβ deposition in APP/PS1 mice. However, 5Aβ3-10 did not induce an effective humoral immune response. These results indicated that Aβ3-10-KLH may be a safe and efficient vaccine for AD and that conjugating the antigen to a carrier protein may be more effective than linking multiple peptide antigens in tandem in applications for antibody production and vaccine preparation. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  11. Design of a dual-function peptide probe as a binder of angiotensin II and an inducer of silver nanoparticle aggregation for use in label-free colorimetric assays.

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    Okochi, Mina; Kuboyama, Masashi; Tanaka, Masayoshi; Honda, Hiroyuki

    2015-09-01

    Label-free colorimetric assays using metallic nanoparticles have received much recent attention, for their application in simple and sensitive methods for detection of biomolecules. Short peptide probes that can bind to analyte biomolecules are attractive ligands in molecular nanotechnology; however, identification of biological recognition motifs is usually based on trial-and-error experiments. Herein, a peptide probe was screened for colorimetric detection of angiotensin II (Ang II) using a mechanism for non-crosslinking aggregation of silver nanoparticles (AgNPs). The dual-function peptides, which bind to the analyte and induce AgNP aggregation, were identified using a two-step strategy: (1) screening of an Ang II-binding peptide from an Ang II receptor sequence library, using SPOT technology, which enable peptides synthesis on cellulose membranes via an Fmoc method and (2) selection of peptide probes that effectively induce aggregation of AgNPs using a photolinker modified peptide array. Using the identified peptide probe, KGKNKRRR, aggregation of AgNPs was detected by observation of a pink color in the absence of Ang II, whereas AgNPs remained dispersed in the presence of Ang II (yellow). The color changes were not observed in the presence of other hormone molecules. Ang II could be detected within 15 min, with a detection limit of 10 µM, by measuring the ratio of absorbance at 400 nm and 568 nm; the signal could also be observed with the naked eye. These data suggest that the peptide identified here could be used as a probe for simple and rapid colorimetric detection of Ang II. This strategy for the identification of functional peptides shows promise for the development of colorimetric detection of various diagnostically important biomolecules. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. beta. -Amyloid gene dosage in Alzheimer's disease

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    Murdoch, G H; Manuelidis, L; Kim, J H; Manuelidis, E E

    1988-01-11

    The 4-5 kd amyloid ..beta..-peptide is a major constituent of the characteristic amyloid plaque of Alzheimer's disease. It has been reported that some cases of sporatic Alzheimer's disease are associated with at least a partial duplication of chromosome 21 containing the gene corresponding to the 695 residue precursor of this peptide. To contribute to an understanding of the frequency to such a duplication event in the overall Alzheimer's population, the authors have determined the gene dosage of the ..beta..-amyloid gene in this collection of cases. All cases had a clinical diagnosis of Alzheimer's confirmed neuropathologically. Each Alzheimer's case had an apparent normal diploid ..beta..-amyloid gene dosage, while control Down's cases had the expected triploid dosage. Thus partial duplication of chromosome 21 may be a rare finding in Alzheimer's disease. Similar conclusions were just reported in several studies of the Harvard Alzheimer collection.

  13. Pterostilbene-O-acetamidoalkylbenzylamines derivatives as novel dual inhibitors of cholinesterase with anti-β-amyloid aggregation and antioxidant properties for the treatment of Alzheimer's disease.

    Science.gov (United States)

    Li, Yuxing; Qiang, Xiaoming; Li, Yan; Yang, Xia; Luo, Li; Xiao, Ganyuan; Cao, Zhongcheng; Tan, Zhenghuai; Deng, Yong

    2016-04-15

    A series of pterostilbene-O-acetamidoalkylbenzylamines were designed, synthesized and evaluated as dual inhibitors of AChE and BuChE. To further explore the multifunctional properties of the new derivatives, their antioxidant activities and inhibitory effects on self-induced Aβ1-42 aggregation and HuAChE-induced Aβ1-40 aggregation were also tested. The results showed that most of these compounds could effectively inhibit AChE and BuChE. Particularly, compound 21d exhibited the best AChE inhibitory activity (IC50=0.06 μM) and good inhibition of BuChE (IC50=28.04 μM). Both the inhibition kinetic analysis and molecular modeling study revealed that these compounds showed mixed-type inhibition, binding simultaneously to the CAS and PAS of AChE. In addition to cholinesterase inhibitory activities, these compounds showed different levels of antioxidant activity. However, the inhibitory activities against self-induced and HuAChE-induced Aβ aggregation of these new derivatives were unsatisfied. Taking into account the results of the biological evaluation, further modifications will be designed in order to increase the potency on the different targets. The results displayed in this Letter can be a new starting point for further development of multifunctional agents for Alzheimer's disease. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Cerebral amyloid angiopathy, blood-brain barrier disruption and amyloid accumulation in SAMP8 mice.

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    del Valle, Jaume; Duran-Vilaregut, Joaquim; Manich, Gemma; Pallàs, Mercè; Camins, Antoni; Vilaplana, Jordi; Pelegrí, Carme

    2011-01-01

    Cerebrovascular dysfunction and β-amyloid peptide deposition on the walls of cerebral blood vessels might be an early event in the development of Alzheimer's disease. Here we studied the time course of amyloid deposition in blood vessels and blood-brain barrier (BBB) disruption in the CA1 subzone of the hippocampus of SAMP8 mice and the association between these two variables. We also studied the association between the amyloid deposition in blood vessels and the recently described amyloid clusters in the parenchyma, as well as the association of these clusters with vessels in which the BBB is disrupted. SAMP8 mice showed greater amyloid deposition in blood vessels than age-matched ICR-CD1 control mice. Moreover, at 12 months of age the number of vessels with a disrupted BBB had increased in both strains, especially SAMP8 animals. At this age, all the vessels with amyloid deposition showed BBB disruption, but several capillaries with an altered BBB showed no amyloid on their walls. Moreover, amyloid clusters showed no spatial association with vessels with amyloid deposition, nor with vessels in which the BBB had been disrupted. Finally, we can conclude that vascular amyloid deposition seems to induce BBB alterations, but BBB disruption may also be due to other factors. Copyright © 2011 S. Karger AG, Basel.

  15. Pro-domain removal in ASP-2 and the cleavage of the amyloid precursor are influenced by pH

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    Austen Brian

    2002-08-01

    Full Text Available Abstract Background One of the signatures of Alzheimer's disease is the accumulation of aggregated amyloid protein, Aβ, in the brain. Aβ arises from cleavage of the Amyloid Precursor protein by β and γ secretases, which present attractive candidates for therapeutic targeting. Two β-secretase candidates, ASP-1 and ASP-2, were identified as aspartic proteases, both of which cleave the amyloid precursor at the β-site. These are produced as immature transmembrane proteins containing a pro-segment. Results ASP-2 expressed in HEK293-cells cleaved the Swedish mutant amyloid precursor at different β-sites at different pHs in vitro. Recent reports show that furin cleaves the pro-peptide of ASP-2, whereas ASP-1 undergoes auto-catalysis. We show that purified recombinant ASP-2 cleaves its own pro-peptide at ph 5 but not pH 8.5 as seen by mass spectrometry, electrophoresis and N-terminal sequencing. Conclusion We suggest that ASP-2 processing as well as activity are influenced by pH, and hence the cellular localisation of the protein may have profound effects on the production of Aβ. These factors should be taken into consideration in the design of potential inhibitors for these enzymes.

  16. Systematic examination of polymorphism in amyloid fibrils by molecular-dynamics simulation.

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    Berryman, Joshua T; Radford, Sheena E; Harris, Sarah A

    2011-05-04

    Amyloid fibrils often exhibit polymorphism. Polymorphs are formed when proteins or peptides with identical sequences self-assemble into fibrils containing substantially different arrangements of the β-strands. We used atomistic molecular-dynamics simulation to examine the thermodynamic stability of a amyloid fibrils in different polymorphic forms by performing a systematic investigation of sequence and symmetry space for a series of peptides with a range of physicochemical properties. We show that the stability of fibrils depends on both sequence and the symmetry because these factors determine the availability of favorable interactions between the peptide strands within a sheet and in intersheet packing. By performing a detailed analysis of these interactions as a function of symmetry, we obtained a series of simple design rules that can be used to determine which polymorphs of a given sequence are most likely to form thermodynamically stable fibrils. These rules can potentially be employed to design peptide sequences that aggregate into a preferred polymorphic form for nanotechnological purposes. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  17. Evaluation of Polyphenol Anthocyanin-Enriched Extracts of Blackberry, Black Raspberry, Blueberry, Cranberry, Red Raspberry, and Strawberry for Free Radical Scavenging, Reactive Carbonyl Species Trapping, Anti-Glycation, Anti-β-Amyloid Aggregation, and Microglial Neuroprotective Effects

    Directory of Open Access Journals (Sweden)

    Hang Ma

    2018-02-01

    Full Text Available Glycation is associated with several neurodegenerative disorders, including Alzheimer’s disease (AD, where it potentiates the aggregation and toxicity of proteins such as β-amyloid (Aβ. Published studies support the anti-glycation and neuroprotective effects of several polyphenol-rich fruits, including berries, which are rich in anthocyanins. Herein, blackberry, black raspberry, blueberry, cranberry, red raspberry, and strawberry extracts were evaluated for: (1 total phenolic and anthocyanins contents, (2 free radical (DPPH scavenging and reactive carbonyl species (methylglyoxal; MGO trapping, (3 anti-glycation (using BSA-fructose and BSA-MGO models, (4 anti-Aβ aggregation (using thermal- and MGO-induced fibrillation models, and, (5 murine microglia (BV-2 neuroprotective properties. Berry crude extracts (CE were fractionated to yield anthocyanins-free (ACF and anthocyanins-enriched (ACE extracts. The berry ACEs (at 100 μg/mL showed superior free radical scavenging, reactive carbonyl species trapping, and anti-glycation effects compared to their respective ACFs. The berry ACEs (at 100 μg/mL inhibited both thermal- and MGO-induced Aβ fibrillation. In addition, the berry ACEs (at 20 μg/mL reduced H2O2-induced reactive oxygen species production, and lipopolysaccharide-induced nitric oxide species in BV-2 microglia as well as decreased H2O2-induced cytotoxicity and caspase-3/7 activity in BV-2 microglia. The free radical scavenging, reactive carbonyl trapping, anti-glycation, anti-Aβ fibrillation, and microglial neuroprotective effects of these berry extracts warrant further in vivo studies to evaluate their potential neuroprotective effects against AD.

  18. Lactic acid induces aberrant amyloid precursor protein processing by promoting its interaction with endoplasmic reticulum chaperone proteins.

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    Yiwen Xiang

    Full Text Available BACKGROUND: Lactic acid, a natural by-product of glycolysis, is produced at excess levels in response to impaired mitochondrial function, high-energy demand, and low oxygen availability. The enzyme involved in the production of β-amyloid peptide (Aβ of Alzheimer's disease, BACE1, functions optimally at lower pH, which led us to investigate a potential role of lactic acid in the processing of amyloid precursor protein (APP. METHODOLOGY/PRINCIPAL FINDINGS: Lactic acid increased levels of Aβ40 and 42, as measured by ELISA, in culture medium of human neuroblastoma cells (SH-SY5Y, whereas it decreased APP metabolites, such as sAPPα. In cell lysates, APP levels were increased and APP was found to interact with ER-chaperones in a perinuclear region, as determined by co-immunoprecipitation and fluorescence microscopy studies. Lactic acid had only a very modest effect on cellular pH, did increase the levels of ER chaperones Grp78 and Grp94 and led to APP aggregate formation reminiscent of aggresomes. CONCLUSIONS/SIGNIFICANCE: These findings suggest that sustained elevations in lactic acid levels could be a risk factor in amyloidogenesis related to Alzheimer's disease through enhanced APP interaction with ER chaperone proteins and aberrant APP processing leading to increased generation of amyloid peptides and APP aggregates.

  19. Coconut (Cocos nucifera) Ethanolic Leaf Extract Reduces Amyloid-β (1-42) Aggregation and Paralysis Prevalence in Transgenic Caenorhabditis elegans Independently of Free Radical Scavenging and Acetylcholinesterase Inhibition.

    Science.gov (United States)

    Manalo, Rafael Vincent; Silvestre, Maries Ann; Barbosa, Aza Lea Anne; Medina, Paul Mark

    2017-04-21

    Virgin coconut oil (VCO) has been the subject of several studies which have aimed to alleviate Alzheimer's disease (AD) pathology, focusing on in vitro antioxidant and acetylcholinesterase (AChE) inhibitory activities. Here, we studied an underutilized and lesser-valued part of the coconut tree, specifically the leaves, using in vitro and in vivo approaches. Coconut leaf extract (CLE) was screened for antioxidant and AChE inhibitory properties in vitro and therapeutic effects in two strains of transgenic Caenorhabditis elegans expressing amyloid-β 1-42 (Aβ 1-42 ) in muscle cells. CLE demonstrated free radical scavenging activity with an EC 50 that is 79-fold less compared to ascorbic acid, and an AChE inhibitory activity that is 131-fold less compared to Rivastigmine. Surprisingly, in spite of its low antioxidant activity and AChE inhibition, CLE reduced Aβ deposits by 30.31% in CL2006 in a dose-independent manner, and reduced the percentage of paralyzed nematodes at the lowest concentration of CLE (159.38 μg/mL), compared to dH₂O/vehicle (control). Phytochemical analysis detected glycosides, anthocyanins, and hydrolyzable tannins in CLE, some of which are known to be anti-amyloidogenic. Taken together, these findings suggest that CLE metabolites alternatively decrease AB 1-42 aggregation and paralysis prevalence independently of free radical scavenging and AChE inhibition, and this warrants further investigation on the bioactive compounds of CLE.

  20. Connecting macroscopic observables and microscopic assembly events in amyloid formation using coarse grained simulations.

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    Noah S Bieler

    Full Text Available The pre-fibrillar stages of amyloid formation have been implicated in cellular toxicity, but have proved to be challenging to study directly in experiments and simulations. Rational strategies to suppress the formation of toxic amyloid oligomers require a better understanding of the mechanisms by which they are generated. We report Dynamical Monte Carlo simulations that allow us to study the early stages of amyloid formation. We use a generic, coarse-grained model of an amyloidogenic peptide that has two internal states: the first one representing the soluble random coil structure and the second one the [Formula: see text]-sheet conformation. We find that this system exhibits a propensity towards fibrillar self-assembly following the formation of a critical nucleus. Our calculations establish connections between the early nucleation events and the kinetic information available in the later stages of the aggregation process that are commonly probed in experiments. We analyze the kinetic behaviour in our simulations within the framework of the theory of classical nucleated polymerisation, and are able to connect the structural events at the early stages in amyloid growth with the resulting macroscopic observables such as the effective nucleus size. Furthermore, the free-energy landscapes that emerge from these simulations allow us to identify pertinent properties of the monomeric state that could be targeted to suppress oligomer formation.

  1. Tackling amyloidogenesis in Alzheimer's disease with A2V variants of Amyloid-β.

    Science.gov (United States)

    Di Fede, Giuseppe; Catania, Marcella; Maderna, Emanuela; Morbin, Michela; Moda, Fabio; Colombo, Laura; Rossi, Alessandro; Cagnotto, Alfredo; Virgilio, Tommaso; Palamara, Luisa; Ruggerone, Margherita; Giaccone, Giorgio; Campagnani, Ilaria; Costanza, Massimo; Pedotti, Rosetta; Salvalaglio, Matteo; Salmona, Mario; Tagliavini, Fabrizio

    2016-02-11

    We developed a novel therapeutic strategy for Alzheimer's disease (AD) exploiting the properties of a natural variant of Amyloid-β (Aβ) carrying the A2V substitution, which protects heterozygous carriers from AD by its ability to interact with wild-type Aβ, hindering conformational changes and assembly thereof. As prototypic compound we designed a six-mer mutated peptide (Aβ1-6A2V), linked to the HIV-related TAT protein, which is widely used for brain delivery and cell membrane penetration of drugs. The resulting molecule [Aβ1-6A2VTAT(D)] revealed strong anti-amyloidogenic effects in vitro and protected human neuroblastoma cells from Aβ toxicity. Preclinical studies in AD mouse models showed that short-term treatment with Aβ1-6A2VTAT(D) inhibits Aβ aggregation and cerebral amyloid deposition, but a long treatment schedule unexpectedly increases amyloid burden, although preventing cognitive deterioration. Our data support the view that the AβA2V-based strategy can be successfully used for the development of treatments for AD, as suggested by the natural protection against the disease in human A2V heterozygous carriers. The undesirable outcome of the prolonged treatment with Aβ1-6A2VTAT(D) was likely due to the TAT intrinsic attitude to increase Aβ production, avidly bind amyloid and boost its seeding activity, warning against the use of the TAT carrier in the design of AD therapeutics.

  2. Tackling amyloidogenesis in Alzheimer’s disease with A2V variants of Amyloid

    Science.gov (United States)

    Di Fede, Giuseppe; Catania, Marcella; Maderna, Emanuela; Morbin, Michela; Moda, Fabio; Colombo, Laura; Rossi, Alessandro; Cagnotto, Alfredo; Virgilio, Tommaso; Palamara, Luisa; Ruggerone, Margherita; Giaccone, Giorgio; Campagnani, Ilaria; Costanza, Massimo; Pedotti, Rosetta; Salvalaglio, Matteo; Salmona, Mario; Tagliavini, Fabrizio

    2016-01-01

    We developed a novel therapeutic strategy for Alzheimer’s disease (AD) exploiting the properties of a natural variant of Amyloid-β (Aβ) carrying the A2V substitution, which protects heterozygous carriers from AD by its ability to interact with wild-type Aβ, hindering conformational changes and assembly thereof. As prototypic compound we designed a six-mer mutated peptide (Aβ1-6A2V), linked to the HIV-related TAT protein, which is widely used for brain delivery and cell membrane penetration of drugs. The resulting molecule [Aβ1-6A2VTAT(D)] revealed strong anti-amyloidogenic effects in vitro and protected human neuroblastoma cells from Aβ toxicity. Preclinical studies in AD mouse models showed that short-term treatment with Aβ1-6A2VTAT(D) inhibits Aβ aggregation and cerebral amyloid deposition, but a long treatment schedule unexpectedly increases amyloid burden, although preventing cognitive deterioration. Our data support the view that the AβA2V-based strategy can be successfully used for the development of treatments for AD, as suggested by the natural protection against the disease in human A2V heterozygous carriers. The undesirable outcome of the prolonged treatment with Aβ1-6A2VTAT(D) was likely due to the TAT intrinsic attitude to increase Aβ production, avidly bind amyloid and boost its seeding activity, warning against the use of the TAT carrier in the design of AD therapeutics. PMID:26864599

  3. General amyloid inhibitors? A critical examination of the inhibition of IAPP amyloid formation by inositol stereoisomers.

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    Hui Wang

    Full Text Available Islet amyloid polypeptide (IAPP or amylin forms amyloid deposits in the islets of Langerhans; a process that is believed to contribute to the progression of type 2 diabetes and to the failure of islet transplants. An emerging theme in amyloid research is the hypothesis that the toxic species produced during amyloid formation by different polypeptides share common features and exert their effects by common mechanisms. If correct, this suggests that inhibitors of amyloid formation by one polypeptide might be effective against other amyloidogenic sequences. IAPP and Aβ, the peptide responsible for amyloid formation in Alzheimer's disease, are particularly interesting in this regard as they are both natively unfolded in their monomeric states and share some common characteristics. Comparatively little effort has been expended on the design of IAPP amyloid inhibitors, thus it is natural to inquire if Aβ inhibitors are effective against IAPP, especially since no IAPP inhibitors have been clinically approved. A range of compounds inhibit Aβ amyloid formation, including various stereoisomers of inositol. Myo-, scyllo-, and epi-inositol have been shown to induce conformational changes in Aβ and prevent Aβ amyloid fibril formation by stabilizing non-fibrillar β-sheet structures. We investigate the ability of inositol stereoisomers to inhibit amyloid formation by IAPP. The compounds do not induce a conformational change in IAPP and are ineffective inhibitors of IAPP amyloid formation, although some do lead to modest apparent changes in IAPP amyloid fibril morphology. Thus not all classes of Aβ inhibitors are effective against IAPP. This work provides a basis of comparison to work on polyphenol based inhibitors of IAPP amyloid formation and helps provide clues as to the features which render them effective. The study also helps provide information for further efforts in rational inhibitor design.

  4. Towards a Pharmacophore for Amyloid

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    Landau, Meytal; Sawaya, Michael R.; Faull, Kym F.; Laganowsky, Arthur; Jiang, Lin; Sievers, Stuart A.; Liu, Jie; Barrio, Jorge R.; Eisenberg, David (UCLA)

    2011-09-16

    Diagnosing and treating Alzheimer's and other diseases associated with amyloid fibers remains a great challenge despite intensive research. To aid in this effort, we present atomic structures of fiber-forming segments of proteins involved in Alzheimer's disease in complex with small molecule binders, determined by X-ray microcrystallography. The fiber-like complexes consist of pairs of {beta}-sheets, with small molecules binding between the sheets, roughly parallel to the fiber axis. The structures suggest that apolar molecules drift along the fiber, consistent with the observation of nonspecific binding to a variety of amyloid proteins. In contrast, negatively charged orange-G binds specifically to lysine side chains of adjacent sheets. These structures provide molecular frameworks for the design of diagnostics and drugs for protein aggregation diseases. The devastating and incurable dementia known as Alzheimer's disease affects the thinking, memory, and behavior of dozens of millions of people worldwide. Although amyloid fibers and oligomers of two proteins, tau and amyloid-{beta}, have been identified in association with this disease, the development of diagnostics and therapeutics has proceeded to date in a near vacuum of information about their structures. Here we report the first atomic structures of small molecules bound to amyloid. These are of the dye orange-G, the natural compound curcumin, and the Alzheimer's diagnostic compound DDNP bound to amyloid-like segments of tau and amyloid-{beta}. The structures reveal the molecular framework of small-molecule binding, within cylindrical cavities running along the {beta}-spines of the fibers. Negatively charged orange-G wedges into a specific binding site between two sheets of the fiber, combining apolar binding with electrostatic interactions, whereas uncharged compounds slide along the cavity. We observed that different amyloid polymorphs bind different small molecules, revealing that a

  5. Inhibition of amyloid oligomerization into different supramolecular architectures by small molecules: mechanistic insights and design rules.

    Science.gov (United States)

    Brahmachari, Sayanti; Paul, Ashim; Segal, Daniel; Gazit, Ehud

    2017-05-01

    Protein misfolding and aggregation have been associated with several human disorders, including Alzheimer's, Parkinson's and Huntington's diseases, as well as senile systemic amyloidosis and Type II diabetes. However, there is no current disease-modifying therapy available for the treatment of these disorders. In spite of extensive academic, pharmaceutical, medicinal and clinical research, a complete mechanistic model for this family of diseases is still lacking. In this review, we primarily discuss the different types of small molecular entities which have been used for the inhibition of the aggregation process of different amyloidogenic proteins under diseased conditions. These include small peptides, polyphenols, inositols, quinones and their derivatives, and metal chelator molecules. In recent years, these groups of molecules have been extensively studied using in vitro, in vivo and computational models to understand their mechanism of action and common structural features underlying the process of inhibition. A salient feature found to be instrumental in the process of inhibition is the balance between the aromatic unit that functions as the amyloid recognition unit and the hydrophilic amyloid breaker unit. The establishment of structure-function relationship for amyloid-modifying therapies by the various functional entities should serve as an important step toward the development of efficient therapeutics.

  6. [Amyloid goiter].

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    Hrívó, A; Péter, I; Bánkúti, B; Péley, G; Baska, F; Besznyák, I

    1999-03-21

    Amyloid goitre is at an extremely rare occurrence. Authors review the origin of disease and its symptoms, diagnostic and therapeutic tools. The disease may be due to either primary or secondary systemic or local amyloidosis. Diagnosis may be made even before surgery on anamnestic data, on very rapid growth of thyroid glands, on diffuse appearance, on other symptoms of systemic amyloidosis, on findings of iconographic procedures and on detection of amyloid in aspirates. Final diagnosis is based on histology. Surgical therapy is aiming at avoidance of the existing and the threatening consequences of expanding mass. The outcome is independent from thyroid surgery, it is related to other manifestations of amyloidosis. Concerning with the present case the chronic superior vena cava syndrome and chylous pleural effusion as first described symptoms and asymptomatic hyperthyroxinaemia is emphasised. Neither other organ involvement, nor primary amyloidogenous molecula was found during the 18 months follow up, so patient has secondary and localised amyloidosis.

  7. Mechanical properties of amyloid-like fibrils defined by secondary structures

    Science.gov (United States)

    Bortolini, C.; Jones, N. C.; Hoffmann, S. V.; Wang, C.; Besenbacher, F.; Dong, M.

    2015-04-01

    Amyloid and amyloid-like fibrils represent a generic class of highly ordered nanostructures that are implicated in some of the most fatal neurodegenerative diseases. On the other hand, amyloids, by possessing outstanding mechanical robustness, have also been successfully employed as functional biomaterials. For these reasons, physical and chemical factors driving fibril self-assembly and morphology are extensively studied - among these parameters, the secondary structures and the pH have been revealed to be crucial, since a variation in pH changes the fibril morphology and net chirality during protein aggregation. It is important to quantify the mechanical properties of these fibrils in order to help the design of effective strategies for treating diseases related to the presence of amyloid fibrils. In this work, we show that by changing pH the mechanical properties of amyloid-like fibrils vary as well. In particular, we reveal that these mechanical properties are strongly related to the content of secondary structures. We analysed and estimated the Young's modulus (E) by comparing the persistence length (Lp) - measured from the observation of TEM images by using statistical mechanics arguments - with the mechanical information provided by peak force quantitative nanomechanical property mapping (PF-QNM). The secondary structure content and the chirality are investigated by means of synchrotron radiation circular dichroism (SR-CD). Results arising from this study could be fruitfully used as a protocol to investigate other medical or engineering relevant peptide fibrils.Amyloid and amyloid-like fibrils represent a generic class of highly ordered nanostructures that are implicated in some of the most fatal neurodegenerative diseases. On the other hand, amyloids, by possessing outstanding mechanical robustness, have also been successfully employed as functional biomaterials. For these reasons, physical and chemical factors driving fibril self-assembly and morphology

  8. Formation of amyloid fibers by monomeric light chain variable domains.

    Science.gov (United States)

    Brumshtein, Boris; Esswein, Shannon R; Landau, Meytal; Ryan, Christopher M; Whitelegge, Julian P; Phillips, Martin L; Cascio, Duilio; Sawaya, Michael R; Eisenberg, David S

    2014-10-03

    Systemic light chain amyloidosis is a lethal disease characterized by excess immunoglobulin light chains and light chain fragments composed of variable domains, which aggregate into amyloid fibers. These fibers accumulate and damage organs. Some light chains induce formation of amyloid fibers, whereas others do not, making it unclear what distinguishes amyloid formers from non-formers. One mechanism by which sequence variation may reduce propensity to form amyloid fibers is by shifting the equilibrium toward an amyloid-resistant quaternary structure. Here we identify the monomeric form of the Mcg immunoglobulin light chain variable domain as the quaternary unit required for amyloid fiber assembly. Dimers of Mcg variable domains remain stable and soluble, yet become prone to assemble into amyloid fibers upon disassociation into monomers. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Assessment of the nature interactions of β-amyloid protein by a nanoprobe method.

    Science.gov (United States)

    Caballero, Leonardo; Mena, Juan; Morales-Alvarez, Aurora; Kogan, Marcelo J; Melo, Francisco

    2015-01-01

    We present a method based on atomic force microscopy (AFM) to assess the work of adhesion between the interfaces of gold AFM tips functionalized with three peptides derived from β-sheet breaker LPFFD [CLPFFD-NH2 (i0) and their isomers CDLPFF-NH2 (i1) and CLPDFF-NH2 (i2)], and the beta-amyloid protein (Aβ1-42). β-Amyloid protein was deposited onto a highly oriented graphite (HOPG) surface as protofibrils and fibrils. The presence of the residues Leu (L), Phe (F), and Phe (F), which are also present in the native sequence, confirm that the peptides are able to bind to the aggregates of Aβ1-42 fibrils and protofibrils. Force of adhesion data were directly obtained from the maximum force on retraction, and the work of adhesion was calculated from the Jhonson-Kendall-Roberts model (JKR-Model). Both the polar and dispersive contributions to the surface energy of the peptides i0, i1, and i2, as well as Aβ1-42 fibrils and protofibrils, were determined by means of measuring the contact angle and using the two-fluid method. The macroscopic energies of the functionalized gold surfaces do not differ significantly between isomers, which confirms the similar nature of the peptides i0, i1, and i2 but suggests that the macroscopic measurements are not able to distinguish specific sequences. The nanoprobe reveals a typical adhesion work value associated with the interaction of protofibrils with i0 and i2; this value is three times higher than that of i1. The difference is attributed to the hydrophobic nature of protofibrils, the predominant exposition of hydrophobic residues of the peptides i0 and i2, with respect to i1, and the degree of functionalization. i0 and i2 presented a slight adhesion with Aβ fibrils, which is associated with the exposed hydrophilic groups of these fibrils (onto HOPG) compared to the protofibrils. However, i1 showed interaction with both Aβ fibrils and protofibrils. For this, we propose an explanation based on the fact that the peptide i1 locates

  10. Amyloid Imaging in Aging and Dementia: Testing the Amyloid Hypothesis In Vivo

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    G. D. Rabinovici

    2009-01-01

    Full Text Available Amyloid imaging represents a major advance in neuroscience, enabling the detection and quantification of pathologic protein aggregations in the brain. In this review we survey current amyloid imaging techniques, focusing on positron emission tomography (PET with ^{11}carbon-labelled Pittsburgh Compound-B (11C-PIB, the most extensively studied and best validated tracer. PIB binds specifically to fibrillar beta-amyloid (Aβ deposits, and is a sensitive marker for Aβ pathology in cognitively normal older individuals and patients with mild cognitive impairment (MCI and Alzheimer’s disease (AD. PIB-PET provides us with a powerful tool to examine in vivo the relationship between amyloid deposition, clinical symptoms, and structural and functional brain changes in the continuum between normal aging and AD. Amyloid imaging studies support a model in which amyloid deposition is an early event on the path to dementia, beginning insidiously in cognitively normal individuals, and accompanied by subtle cognitive decline and functional and structural brain changes suggestive of incipient AD. As patients progress to dementia, clinical decline and neurodegeneration accelerate and proceed independently of amyloid accumulation. In the future, amyloid imaging is likely to supplement clinical evaluation in selecting patients for anti-amyloid therapies, while MRI and FDG-PET may be more appropriate markers of clinical progression.

  11. Heterologous amyloid seeding: revisiting the role of acetylcholinesterase in Alzheimer's disease.

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    Létitia Jean

    2007-07-01

    Full Text Available Neurodegenerative diseases associated with abnormal protein folding and ordered aggregation require an initial trigger which may be infectious, inherited, post-inflammatory or idiopathic. Proteolytic cleavage to generate vulnerable precursors, such as amyloid-beta peptide (Abeta production via beta and gamma secretases in Alzheimer's Disease (AD, is one such trigger, but the proteolytic removal of these fragments is also aetiologically important. The levels of Abeta in the central nervous system are regulated by several catabolic proteases, including insulysin (IDE and neprilysin (NEP. The known association of human acetylcholinesterase (hAChE with pathological aggregates in AD together with its ability to increase Abeta fibrilization prompted us to search for proteolytic triggers that could enhance this process. The hAChE C-terminal domain (T40, AChE(575-614 is an exposed amphiphilic alpha-helix involved in enzyme oligomerisation, but it also contains a conformational switch region (CSR with high propensity for conversion to non-native (hidden beta-strand, a property associated with amyloidogenicity. A synthetic peptide (AChE(586-599 encompassing the CSR region shares homology with Abeta and forms beta-sheet amyloid fibrils. We investigated the influence of IDE and NEP proteolysis on the formation and degradation of relevant hAChE beta-sheet species. By combining reverse-phase HPLC and mass spectrometry, we established that the enzyme digestion profiles on T40 versus AChE(586-599, or versus Abeta, differed. Moreover, IDE digestion of T40 triggered the conformational switch from alpha- to beta-structures, resulting in surfactant CSR species that self-assembled into amyloid fibril precursors (oligomers. Crucially, these CSR species significantly increased Abeta fibril formation both by seeding the energetically unfavorable formation of amyloid nuclei and by enhancing the rate of amyloid elongation. Hence, these results may offer an explanation

  12. Prevalence of cerebral amyloid pathology in persons without dementia

    DEFF Research Database (Denmark)

    Jansen, Willemijn J; Ossenkoppele, Rik; Knol, Dirk L

    2015-01-01

    IMPORTANCE: Cerebral amyloidaggregation is an early pathological event in Alzheimer disease (AD), starting decades before dementia onset. Estimates of the prevalence of amyloid pathology in persons without dementia are needed to understand the development of AD and to design prevention studies...

  13. Quantitation of amyloid beta peptides Aβ(1-38), Aβ(1-40), and Aβ(1-42) in human cerebrospinal fluid by ultra-performance liquid chromatography-tandem mass spectrometry.

    Science.gov (United States)

    Lame, Mary E; Chambers, Erin E; Blatnik, Matthew

    2011-12-15

    Critical events in Alzheimer's disease (AD) involve an imbalance between the production and clearance of amyloid beta (Aβ) peptides from the brain. Current methods for Aβ quantitation rely heavily on immuno-based techniques. However, these assays require highly specific antibodies and reagents that are time-consuming and expensive to develop. Immuno-based assays are also characterized by poor dynamic ranges, cross-reactivity, matrix interferences, and dilution linearity problems. In particular, noncommercial immunoassays are especially subject to high intra- and interassay variability because they are not subject to more stringent manufacturing controls. Combinations of these factors make immunoassays more labor-intensive and often challenging to validate in support of clinical studies. Here we describe a mixed-mode solid-phase extraction method and an ultra-performance liquid chromatography tandem mass spectrometry (SPE UPLC-MS/MS) assay for the simultaneous quantitation of Aβ(1-38), Aβ(1-40), and Aβ(1-42) from human cerebrospinal fluid (CSF). Negative ion versus positive ion species were compared using their corresponding multiple reaction monitoring (MRM) transitions, and negative ions were approximately 1.6-fold greater in intensity but lacked selectivity in matrix. The positive ion MRM assay was more than sufficient to quantify endogenous Aβ peptides. Aβ standards were prepared in artificial CSF containing 5% rat plasma, and quality control samples were prepared in three pooled CSF sources. Extraction efficiency was greater than 80% for all three peptides, and the coefficient of variation during analysis was less than 15% for all species. Mean basal levels of Aβ species from three CSF pools were 1.64, 2.17, and 1.26 ng/ml for Aβ(1-38); 3.24, 3.63, and 2.55 ng/ml for Aβ(1-40); and 0.50, 0.63, and 0.46 ng/ml for Aβ(1-42). Copyright © 2011 Elsevier Inc. All rights reserved.

  14. Reducing the Levels of Akt Activation by PDK1 Knock-in Mutation Protects Neuronal Cultures against Synthetic Amyloid-Beta Peptides

    Directory of Open Access Journals (Sweden)

    Shaobin Yang

    2018-01-01

    Full Text Available The Akt kinase has been widely assumed for years as a key downstream effector of the PI3K signaling pathway in promoting neuronal survival. This notion was however challenged by the finding that neuronal survival responses were still preserved in mice with reduced Akt activity. Moreover, here we show that the Akt signaling is elevated in the aged brain of two different mice models of Alzheimer Disease. We manipulate the rate of Akt stimulation by employing knock-in mice expressing a mutant form of PDK1 (phosphoinositide-dependent protein kinase 1 with reduced, but not abolished, ability to activate Akt. We found increased membrane localization and activity of the TACE/ADAM17 α-secretase in the brain of the PDK1 mutant mice with concomitant TNFR1 processing, which provided neurons with resistance against TNFα-induced neurotoxicity. Opposite to the Alzheimer Disease transgenic mice, the PDK1 knock-in mice exhibited an age-dependent attenuation of the unfolding protein response, which protected the mutant neurons against endoplasmic reticulum stressors. Moreover, these two mechanisms cooperatively provide the mutant neurons with resistance against amyloid-beta oligomers, and might singularly also contribute to protect these mice against amyloid-beta pathology.

  15. Cerebral Amyloid Angiopathy

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    Mahmut Edip Gürol

    2009-03-01

    Full Text Available Cerebral amyloid angiopathy (CAA is characterized by the accumulation of amyloid beta-peptides (Ab in the walls of leptomeningeal arteries, arterioles, and veins. Despite the fact that these pathological changes were first described in 1909, major advancement in our understanding of the clinicoradiological manifestations, neurobiology, and course of CAA has occurred only during the last 30 years. No significant associations have been shown between CAA and other systemic/visceral amyloidoses or vascular risk factors, including hypertension. CAA is well known as the most common cause of spontaneous and anticoagulant-related lobar parenchymal ICH in the elderly. It also causes lobar cerebral microbleeds (CMBs, small dot-like dark susceptibility artifacts visible with gradient recalled echo (GRE-magnetic resonance imaging (MRI. CMBs are important markers of disease severity and predictors of CAA progression. Amyloid angiopathy is also a common cause of ischemic microvascular white matter disease (WMD and deep cerebral infarctions. Such WMD is defined as subcortical and periventricular white matter changes without obvious infarction, as well as a dark appearance on computerized tomography (CT and a bright appearance on fluid attenuated inversion recovery (FLAIR-MRI. CAA-related vascular dysfunction, with its hemorrhagic and ischemic complications, is a recognized contributor to vascular cognitive impairment in the elderly, an independent effect that is synergistically increased by Alzheimer pathologies, such as plaques and tangles. A set of clinicoradiological criteria was established for the accurate diagnosis of CAA. According to the Boston Criteria, patients aged 55 years and older with multiple hemorrhages (on CT or GRE-MRI restricted to the lobar, cortical, or corticosubcortical regions (cerebellar hemorrhage allowed are diagnosed as probable CAA when no other etiology is found; a single hemorrhage in the same region is classified as possible

  16. Dendrimer effects on peptide and protein fibrillation

    DEFF Research Database (Denmark)

    Heegaard, Peter M. H.; Boas, Ulrik; Otzen, Daniel E.

    2007-01-01

    involved in a range of serious and irreversibly progressive pathological conditions (protein-misfolding diseases). Interesting as this may be, the interaction of dendrimers with such generic peptidic aggregates also offers a new perspective on the molecular mechanisms governing assembly and disassembly......, they offer numerous possibilities for interactions with and responses to biological macromolecules and biostructures including cell membranes and proteins. By way of their multiple functional surface groups, they allow the design of surfaces carrying a multitude of biological motifs and/or charges giving...... rise to quite significant biological and physico-chemical effects. Here we describe the surprising ability of dendrimers to interact with and perturb polypeptide conformations, particularly efficiently towards amyloid structures; that is, the structures of highly insoluble polypeptide aggregates...

  17. Towards Prebiotic Catalytic Amyloids Using High Throughput Screening.

    Directory of Open Access Journals (Sweden)

    Michael P Friedmann

    Full Text Available Enzymes are capable of directing complex stereospecific transformations and of accelerating reaction rates many orders of magnitude. As even the simplest known enzymes comprise thousands of atoms, the question arises as to how such exquisite catalysts evolved. A logical predecessor would be shorter peptides, but they lack the defined structure and size that are apparently necessary for enzyme functions. However, some very short peptides are able to assemble into amyloids, thereby forming a well-defined tertiary structure called the cross-β-sheet, which bestows unique properties upon the peptides. We have hypothesized that amyloids could have been the catalytically active precursor to modern enzymes. To test this hypothesis, we designed an amyloid peptide library that could be screened for catalytic activity. Our approach, amenable to high-throughput methodologies, allowed us to find several peptides and peptide mixtures that form amyloids with esterase activity. These results indicate that amyloids, with their stability in a wide range of conditions and their potential as catalysts with low sequence specificity, would indeed be fitting precursors to modern enzymes. Furthermore, our approach can be efficiently expanded upon in library size, screening conditions, and target activity to yield novel amyloid catalysts with potential applications in aqueous-organic mixtures, at high temperature and in other extreme conditions that could be advantageous for industrial applications.

  18. Sorting of a HaloTag protein that has only a signal peptide sequence into exocrine secretory granules without protein aggregation.

    Science.gov (United States)

    Fujita-Yoshigaki, Junko; Matsuki-Fukushima, Miwako; Yokoyama, Megumi; Katsumata-Kato, Osamu

    2013-11-15

    The mechanism involved in the sorting and accumulation of secretory cargo proteins, such as amylase, into secretory granules of exocrine cells remains to be solved. To clarify that sorting mechanism, we expressed a reporter protein HaloTag fused with partial sequences of salivary amylase protein in primary cultured parotid acinar cells. We found that a HaloTag protein fused with only the signal peptide sequence (Met(1)-Ala(25)) of amylase, termed SS25H, colocalized well with endogenous amylase, which was confirmed by immunofluorescence microscopy. Percoll-density gradient centrifugation of secretory granule fractions shows that the distributions of amylase and SS25H were similar. These results suggest that SS25H is transported to secretory granules and is not discriminated from endogenous amylase by the machinery that functions to remove proteins other than granule cargo from immature granules. Another reporter protein, DsRed2, that has the same signal peptide sequence also colocalized with amylase, suggesting that the sorting to secretory granules is not dependent on a characteristic of the HaloTag protein. Whereas Blue Native PAGE demonstrates that endogenous amylase forms a high-molecular-weight complex, SS25H does not participate in the complex and does not form self-aggregates. Nevertheless, SS25H was released from cells by the addition of a β-adrenergic agonist, isoproterenol, which also induces amylase secretion. These results indicate that addition of the signal peptide sequence, which is necessary for the translocation in the endoplasmic reticulum, is sufficient for the transportation and storage of cargo proteins in secretory granules of exocrine cells.

  19. Liquid Crystal Enabled Early Stage Detection of Beta Amyloid Formation on Lipid Monolayers

    Energy Technology Data Exchange (ETDEWEB)

    Sadati, Monirosadat [Institute for Molecular Engineering, University of Chicago, Chicago IL 60637 USA; Apik, Aslin Izmitli [Chemical and Biological Engineering, University of Wisconsin, Madison WI 53706 USA; Armas-Perez, Julio C. [Institute for Molecular Engineering, University of Chicago, Chicago IL 60637 USA; Martinez-Gonzalez, Jose [Institute for Molecular Engineering, University of Chicago, Chicago IL 60637 USA; Hernandez-Ortiz, Juan P. [Institute for Molecular Engineering, University of Chicago, Chicago IL 60637 USA; Departamento de Materiales y Minerales, Facultad de Minas, Universidad Nacional de Colombia, Sede Medellín, Calle 75 # 79A-51, Bloque M17 Medellín Colombia; Abbott, Nicholas L. [Chemical and Biological Engineering, University of Wisconsin, Madison WI 53706 USA; de Pablo, Juan J. [Institute for Molecular Engineering, University of Chicago, Chicago IL 60637 USA; Argonne National Laboratory, Argonne IL 60439 USA

    2015-09-09

    Liquid crystals (LCs) can serve as sensitive reporters of interfacial events, and this property has been used for sensing of synthetic or biological toxins. Here it is demonstrated that LCs can distinguish distinct molecular motifs and exhibit a specific response to beta-sheet structures. That property is used to detect the formation of highly toxic protofibrils involved in neurodegenerative diseases, where it is crucial to develop methods that probe the early-stage aggregation of amyloidogenic peptides in the vicinity of biological membranes. In the proposed method, the amyloid fibrils formed at the lipid-decorated LC interface can change the orientation of LCs and form elongated and branched structures that are amplified by the mesogenic medium; however, nonamyloidogenic peptides form ellipsoidal domains of tilted LCs. Moreover, a theoretical and computational analysis is used to reveal the underlying structure of the LC, thereby providing a detailed molecular-level view of the interactions and mechanisms responsible for such motifs. The corresponding signatures can be detected at nanomolar concentrations of peptide by polarized light microscopy and much earlier than the ones that can be identified by fluorescence-based techniques. As such, it offers the potential for early diagnoses of neurodegenerative diseases and for facile testing of inhibitors of amyloid formation.

  20. Islet Amyloid Polypeptide: Structure, Function, and Pathophysiology

    Directory of Open Access Journals (Sweden)

    Rehana Akter

    2016-01-01

    Full Text Available The hormone islet amyloid polypeptide (IAPP, or amylin plays a role in glucose homeostasis but aggregates to form islet amyloid in type-2 diabetes. Islet amyloid formation contributes to β-cell dysfunction and death in the disease and to the failure of islet transplants. Recent work suggests a role for IAPP aggregation in cardiovascular complications of type-2 diabetes and hints at a possible role in type-1 diabetes. The mechanisms of IAPP amyloid formation in vivo or in vitro are not understood and the mechanisms of IAPP induced β-cell death are not fully defined. Activation of the inflammasome, defects in autophagy, ER stress, generation of reactive oxygen species, membrane disruption, and receptor mediated mechanisms have all been proposed to play a role. Open questions in the field include the relative importance of the various mechanisms of β-cell death, the relevance of reductionist biophysical studies to the situation in vivo, the molecular mechanism of amyloid formation in vitro and in vivo, the factors which trigger amyloid formation in type-2 diabetes, the potential role of IAPP in type-1 diabetes, the development of clinically relevant inhibitors of islet amyloidosis toxicity, and the design of soluble, bioactive variants of IAPP for use as adjuncts to insulin therapy.

  1. The role of beta amyloid in Alzheimer's disease: still a cause of everything or the only one who got caught?

    Science.gov (United States)

    Verdile, Giuseppe; Fuller, Stephanie; Atwood, Craig S; Laws, Simon M; Gandy, Samuel E; Martins, Ralph N

    2004-10-01

    The beta amyloid (A beta) protein is a key molecule in the pathogenesis of Alzheimer's disease (AD). The tendency of the A beta peptide to aggregate, its reported neurotoxicity, and genetic linkage studies, have led to a hypothesis of AD pathogenesis that many AD researchers term the amyloid cascade hypothesis. In this hypothesis, an increased production of A beta results in neurodegeneration and ultimately dementia through a cascade of events. In the past 15 years, debate amongst AD researchers has arisen as to whether A beta is a cause or an effect of the pathogenic process. Recent in vitro and in vivo research has consolidated the theory that A beta is the primary cause, initiating secondary events, culminating in the neuropathological hallmarks associated with AD. This research has led to the development of therapeutic agents, currently in human clinical trials, which target A beta.

  2. Evaluation of peptide adsorption-controlled liquid chromatography-tandem mass spectrometric (PAC-LC-MS/MS) method for simple and simultaneous quantitation of amyloid β 1-38, 1-40, 1-42 and 1-43 peptides in dog cerebrospinal fluid.

    Science.gov (United States)

    Goda, Ryoya; Kobayashi, Nobuhiro

    2012-05-01

    To evaluate the usefulness of the peptide adsorption-controlled liquid chromatography-tandem mass spectrometry (PAC-LC-MS/MS) for reproducible measurement of peptides in biological fluids, simultaneous quantitation of amyloid β 1-38, 1-40, 1-42 and 1-43 peptides (Aβ38, Aβ40, Aβ42 and Aβ43) in dog cerebrospinal fluid (CSF) was tried. Each stable isotope labeled Aβ was used as the internal standard to minimize the influence of CSF matrix on the reproducible Aβ quantitation. To reduce a loss of Aβ during the pretreatment procedures, the dog CSF diluted by water-acetic acid-methanol (2:6:1, v/v/v) was loaded on PAC-LC-MS/MS directly. Quantification of the Aβ in the diluted dog CSF was carried out using multiple reaction monitoring (MRM) mode. The [M+5H(5+)] and b(5+) ion fragment of each peptide were chosen as the precursor and product ions for MRM transitions of each peptide. The calibration curves were drawn from Aβ standard calibration solutions using PAC-LC-MS/MS. Analysis of dog CSF samples suggests that the basal concentration of Aβ38, Aβ40, Aβ42 and Aβ43 in dog CSF is approximately 300, 900, 200 and 30 pM, respectively. This is the first time Aβ concentrations in dog CSF have been reported. Additionally, the evaluation of intra- and inter-day reproducibility of analysis of Aβ standard solution, the freeze-thaw stability and the room temperature stability of Aβ standard solution suggest that the PAC-LC-MS/MS method enables reproducible Aβ quantitation. Copyright © 2012 Elsevier B.V. All rights reserved.

  3. Long-Term Treatment with Liraglutide, a Glucagon-Like Peptide-1 (GLP-1 Receptor Agonist, Has No Effect on β-Amyloid Plaque Load in Two Transgenic APP/PS1 Mouse Models of Alzheimer's Disease.

    Directory of Open Access Journals (Sweden)

    Henrik H Hansen

    Full Text Available One of the major histopathological hallmarks of Alzheimer's disease (AD is cerebral deposits of extracellular β-amyloid peptides. Preclinical studies have pointed to glucagon-like peptide 1 (GLP-1 receptors as a potential novel target in the treatment of AD. GLP-1 receptor agonists, including exendin-4 and liraglutide, have been shown to promote plaque-lowering and mnemonic effects of in a number of experimental models of AD. Transgenic mouse models carrying genetic mutations of amyloid protein precursor (APP and presenilin-1 (PS1 are commonly used to assess the pharmacodynamics of potential amyloidosis-lowering and pro-cognitive compounds. In this study, effects of long-term liraglutide treatment were therefore determined in two double APP/PS1 transgenic mouse models of Alzheimer's disease carrying different clinical APP/PS1 mutations, i.e. the 'London' (hAPPLon/PS1A246E and 'Swedish' mutation variant (hAPPSwe/PS1ΔE9 of APP, with co-expression of distinct PS1 variants. Liraglutide was administered in 5 month-old hAPPLon/PS1A246E mice for 3 months (100 or 500 ng/kg/day, s.c., or 7 month-old hAPPSwe/PS1ΔE9 mice for 5 months (500 ng/kg/day, s.c.. In both models, regional plaque load was quantified throughout the brain using stereological methods. Vehicle-dosed hAPPSwe/PS1ΔE9 mice exhibited considerably higher cerebral plaque load than hAPPLon/PS1A246E control mice. Compared to vehicle-dosed transgenic controls, liraglutide treatment had no effect on the plaque levels in hAPPLon/PS1A246E and hAPPSwe/PS1ΔE9 mice. In conclusion, long-term liraglutide treatment exhibited no effect on cerebral plaque load in two transgenic mouse models of low- and high-grade amyloidosis, which suggests differential sensitivity to long-term liraglutide treatment in various transgenic mouse models mimicking distinct pathological hallmarks of AD.

  4. Protein Polymers and Amyloids

    DEFF Research Database (Denmark)

    Risør, Michael Wulff

    2014-01-01

    Several human disorders are caused by a common general disease mechanism arising from abnormal folding and aggregation of the underlying protein. These include the prevalent dementias like Alzheimer’s and Parkinson’s, where accumulation of protein fibrillar structures, known as amyloid fibrils......, is a general hallmark. They also include the α1-antitrypsin deficiency, where disease-causing mutations in the serine protease inhibitor, α1-antitrypsin (α1AT), leads to accumulation of the aberrant protein in the liver of these patients. The native metastable structure of α1AT constitutes a molecular trap...... that inhibits its target protease through a large conformational change but mutations compromise this function and cause premature structural collapse into hyperstable polymers. Understanding the conformational disorders at a molecular level is not only important for our general knowledge on protein folding...

  5. Alzheimer's disease related markers, cellular toxicity and behavioral deficits induced six weeks after oligomeric amyloidpeptide injection in rats.

    Directory of Open Access Journals (Sweden)

    Charleine Zussy

    Full Text Available Alzheimer's disease (AD is a neurodegenerative pathology associated with aging characterized by the presence of senile plaques and neurofibrillary tangles that finally result in synaptic and neuronal loss. The major component of senile plaques is an amyloid-β protein (Aβ. Recently, we characterized the effects of a single intracerebroventricular (icv injection of Aβ fragment (25-35 oligomers (oAβ(25-35 for up to 3 weeks in rats and established a clear parallel with numerous relevant signs of AD. To clarify the long-term effects of oAβ(25-35 and its potential role in the pathogenesis of AD, we determined its physiological, behavioral, biochemical and morphological impacts 6 weeks after injection in rats. oAβ(25-35 was still present in the brain after 6 weeks. oAβ(25-35 injection did not affect general activity and temperature rhythms after 6 weeks, but decreased body weight, induced short- and long-term memory impairments, increased corticosterone plasma levels, brain oxidative (lipid peroxidation, mitochondrial (caspase-9 levels and reticulum stress (caspase-12 levels, astroglial and microglial activation. It provoked cholinergic neuron loss and decreased brain-derived neurotrophic factor levels. It induced cell loss in the hippocampic CA subdivisions and decreased hippocampic neurogenesis. Moreover, oAβ(25-35 injection resulted in increased APP expression, Aβ(1-42 generation, and increased Tau phosphorylation. In conclusion, this in vivo study evidenced that the soluble oligomeric forms of short fragments of Aβ, endogenously identified in AD patient brains, not only provoked long-lasting pathological alterations comparable to the human disease, but may also directly contribute to the progressive increase in amyloid load and Tau pathology, involved in the AD physiopathology.

  6. Computational Selection of Inhibitors of A-beta Aggregation and Neuronal Toxicity

    Science.gov (United States)

    Chen, Deliang; Martin, Zane S.; Soto, Claudio; Schein, Catherine H.

    2009-01-01

    Alzheimer’s Disease (AD) is characterized by the cerebral accumulation of misfolded and aggregated amyloid-β protein (Aβ). Disease symptoms can be alleviated, in vitro and in vivo, by “β-sheet breaker” pentapeptides that reduce plaque volume. However the peptide nature of these compounds, made them biologically unstable and unable to penetrate membranes with high efficiency. The main goal of this study was to use computational methods to identify small molecule mimetics with better drug-like properties. For this purpose, the docked conformations of the active peptides were used to identify compounds with similar activities. A series of related β-sheet breaker peptides were docked to solid state NMR structures of a fibrillar form of Aβ. The lowest energy conformations of the active peptides were used to design three dimensional (3D)-pharmacophores, suitable for screening the NCI database with Unity. Small molecular weight compounds with physicochemical features in a conformation similar to the active peptides were selected, ranked by docking solubility parameters. Of 16 diverse compounds selected for experimental screening, 2 prevented and reversed Aβ aggregation at 2–3 μM concentration, as measured by Thioflavin T (ThT) fluorescence and ELISA assays. They also prevented the toxic effects of aggregated Aβ on neuroblastoma cells. Their low molecular weight and aqueous solubility makes them promising lead compounds for treating AD. PMID:19540126

  7. Is Supramolecular Filament Chirality the Underlying Cause of Major Morphology Differences in Amyloid Fibrils?

    Science.gov (United States)

    2015-01-01

    The unique enhanced sensitivity of vibrational circular dichroism (VCD) to the formation and development of amyloid fibrils in solution is extended to four additional fibril-forming proteins or peptides where it is shown that the sign of the fibril VCD pattern correlates with the sense of supramolecular filament chirality and, without exception, to the dominant fibril morphology as observed in AFM or SEM images. Previously for insulin, it has been demonstrated that the sign of the VCD band pattern from filament chirality can be controlled by adjusting the pH of the incubating solution, above pH 2 for “normal” left-hand-helical filaments and below pH 2 for “reversed” right-hand-helical filaments. From AFM or SEM images, left-helical filaments form multifilament braids of left-twisted fibrils while the right-helical filaments form parallel filament rows of fibrils with a flat tape-like morphology, the two major classes of fibril morphology that from deep UV resonance Raman scattering exhibit the same cross-β-core secondary structure. Here we investigate whether fibril supramolecular chirality is the underlying cause of the major morphology differences in all amyloid fibrils by showing that the morphology (twisted versus flat) of fibrils of lysozyme, apo-α-lactalbumin, HET-s (218–289) prion, and a short polypeptide fragment of transthyretin, TTR (105–115), directly correlates to their supramolecular chirality as revealed by VCD. The result is strong evidence that the chiral supramolecular organization of filaments is the principal underlying cause of the morphological heterogeneity of amyloid fibrils. Because fibril morphology is linked to cell toxicity, the chirality of amyloid aggregates should be explored in the widely used in vitro models of amyloid-associated diseases. PMID:24484302

  8. Development and characterization of a TAPIR-like mouse monoclonal antibody to amyloid-beta.

    Science.gov (United States)

    Wang, Jun; Hara, Hideo; Makifuchi, Takao; Tabira, Takeshi

    2008-06-01

    Tissue amyloid plaque immuno-reactive (TAPIR) antibody was better related to the effect of immunotherapy in Alzheimer's disease (AD) than ELISA antibody. Here we used a hybridoma technique to develop a TAPIR-like anti-human amyloid-beta (Abeta) mouse monoclonal antibody. The obtained monoclonal antibody, 3.4A10, was an IgG2b isotype and recognized N-terminal portion of Abeta1-42 without binding denatured or native amyloid-beta protein precursor. It had higher affinity to Abeta1-42 than to Abeta1-40 by Biacore affinity analysis and stained preferably the peripheral part of senile plaques and recognized the plaque core less than 4G8. It inhibited the Abeta1-42 fibril formation as well as degraded pre-aggregated Abeta1-42 peptide in a thioflavin T fluorescence spectrophotometry assay. The in vivo studies showed that 3.4A10 treatment decreased amyloid burden compared to the control group and significantly reduced Abeta42 levels rather than Abeta40 levels in brain lysates as well as the Abeta*56 oligomer (12mer) in TBS fraction of the brain lysates. 3.4A10 entered brain and decorated some plaques, which is surrounded by more Iba1-positive microglia. 3.4A10 therapy did not induce lymphocytic infiltration and obvious increase in microhemorrhage. We conclude that 3.4A10 is a TAPIR-like anti-human amyloid monoclonal antibody, and has a potential of therapeutic application for AD.

  9. Is supramolecular filament chirality the underlying cause of major morphology differences in amyloid fibrils?

    Science.gov (United States)

    Kurouski, Dmitry; Lu, Xuefang; Popova, Ludmila; Wan, William; Shanmugasundaram, Maruda; Stubbs, Gerald; Dukor, Rina K; Lednev, Igor K; Nafie, Laurence A

    2014-02-12

    The unique enhanced sensitivity of vibrational circular dichroism (VCD) to the formation and development of amyloid fibrils in solution is extended to four additional fibril-forming proteins or peptides where it is shown that the sign of the fibril VCD pattern correlates with the sense of supramolecular filament chirality and, without exception, to the dominant fibril morphology as observed in AFM or SEM images. Previously for insulin, it has been demonstrated that the sign of the VCD band pattern from filament chirality can be controlled by adjusting the pH of the incubating solution, above pH 2 for "normal" left-hand-helical filaments and below pH 2 for "reversed" right-hand-helical filaments. From AFM or SEM images, left-helical filaments form multifilament braids of left-twisted fibrils while the right-helical filaments form parallel filament rows of fibrils with a flat tape-like morphology, the two major classes of fibril morphology that from deep UV resonance Raman scattering exhibit the same cross-β-core secondary structure. Here we investigate whether fibril supramolecular chirality is the underlying cause of the major morphology differences in all amyloid fibrils by showing that the morphology (twisted versus flat) of fibrils of lysozyme, apo-α-lactalbumin, HET-s (218-289) prion, and a short polypeptide fragment of transthyretin, TTR (105-115), directly correlates to their supramolecular chirality as revealed by VCD. The result is strong evidence that the chiral supramolecular organization of filaments is the principal underlying cause of the morphological heterogeneity of amyloid fibrils. Because fibril morphology is linked to cell toxicity, the chirality of amyloid aggregates should be explored in the widely used in vitro models of amyloid-associated diseases.

  10. Protein aggregation in bacteria: the thin boundary between functionality and toxicity.

    Science.gov (United States)

    Bednarska, Natalia G; Schymkowitz, Joost; Rousseau, Frederic; Van Eldere, Johan

    2013-09-01

    Misfolding and aggregation of proteins have a negative impact on all living organisms. In recent years, aggregation has been studied in detail due to its involvement in neurodegenerative diseases, including Alzheimer's, Parkinson's and Huntington's diseases, and type II diabetes--all associated with accumulation of amyloid fibrils. This research highlighted the central importance of protein homeostasis, or proteostasis for short, defined as the cellular state in which the proteome is both stable and functional. It implicates an equilibrium between synthesis, folding, trafficking, aggregation, disaggregation and degradation. In accordance with the eukaryotic systems, it has been documented that protein aggregation also reduces fitness of bacterial cells, but although our understanding of the cellular protein quality control systems is perhaps most detailed in bacteria, the use of bacterial proteostasis as a drug target remains little explored. Here we describe protein aggregation as a normal physiological process and its role in bacterial virulence and we shed light on how bacteria defend themselves against the toxic threat of aggregates. We review the impact of aggregates on bacterial viability and look at the ways that bacteria use to maintain a balance between aggregation and functionality. The proteostasis in bacteria can be interrupted via overexpression of proteins, certain antibiotics such as aminoglycosides, as well as antimicrobial peptides--all leading to loss of cell viability. Therefore intracellular protein aggregation and disruption of proteostatic balance in bacteria open up another strategy that should be explored towards the discovery of new antimicrobials.

  11. Fibrillar dimer formation of islet amyloid polypeptides

    Energy Technology Data Exchange (ETDEWEB)

    Chiu, Chi-cheng [Univ. of Chicago, IL (United States); Argonne National Lab. (ANL), Argonne, IL (United States); de Pablo, Juan J. [Univ. of Chicago, IL (United States); Argonne National Lab. (ANL), Argonne, IL (United States)

    2015-05-08

    Amyloid deposits of human islet amyloid polypeptide (hIAPP), a 37-residue hormone co-produced with insulin, have been implicated in the development of type 2 diabetes. Residues 20 – 29 of hIAPP have been proposed to constitute the amyloidogenic core for the aggregation process, yet the segment is mostly unstructured in the mature fibril, according to solid-state NMR data. Here we use molecular simulations combined with bias-exchange metadynamics to characterize the conformational free energies of hIAPP fibrillar dimer and its derivative, pramlintide. We show that residues 20 – 29 are involved in an intermediate that exhibits transient β-sheets, consistent with recent experimental and simulation results. By comparing the aggregation of hIAPP and pramlintide, we illustrate the effects of proline residues on inhibition of the dimerization of IAPP. The mechanistic insights presented here could be useful for development of therapeutic inhibitors of hIAPP amyloid formation.

  12. Dysfunction of different cellular degradation pathways contributes to specific β-amyloid42-induced pathologies.

    Science.gov (United States)

    Ji, Xuan-Ru; Cheng, Kuan-Chung; Chen, Yu-Ru; Lin, Tzu-Yu; Cheung, Chun Hei Antonio; Wu, Chia-Lin; Chiang, Hsueh-Cheng

    2018-03-01

    The endosomal-lysosomal system (ELS), autophagy, and ubiquitin-proteasome system (UPS) are cellular degradation pathways that each play a critical role in the removal of misfolded proteins and the prevention of the accumulation of abnormal proteins. Recent studies on Alzheimer's disease (AD) pathogenesis have suggested that accumulation of aggregated β-amyloid (Aβ) peptides in the AD brain results from a dysfunction in these cellular clearance systems. However, the specific roles of these pathways in the removal of Aβ peptides and the pathogenesis underlying AD are unclear. Our in vitro and in vivo genetic approaches revealed that ELS mainly removed monomeric β-amyloid42 (Aβ42), while autophagy and UPS clear oligomeric Aβ42. Although overproduction of phosphatidylinositol 4-phosphate-5 increased Aβ42 clearance, it reduced the life span of Aβ42 transgenic flies. Our behavioral studies further demonstrated impaired autophagy and UPS-enhanced Aβ42-induced learning and memory deficits, but there was no effect on Aβ42-induced reduction in life span. Results from genetic fluorescence imaging showed that these pathways were damaged in the following order: UPS, autophagy, and finally ELS. The results of our study demonstrate that different degradation pathways play distinct roles in the removal of Aβ42 aggregates and in disease progression. These findings also suggest that pharmacologic treatments that are designed to stimulate cellular degradation pathways in patients with AD should be used with caution.-Ji, X.-R., Cheng, K.-C., Chen, Y.-R., Lin, T.-Y., Cheung, C. H. A., Wu, C.-L., Chiang, H.-C. Dysfunction of different cellular degradation pathways contributes to specific β-amyloid42-induced pathologies.

  13. Metastable Amyloid Phases and their Conversion to Mature Fibrils

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    Muschol, Martin; Miti, Tatiana; Mulaj, Mentor; Schmit, Jeremy

    Self-assembly of proteins into amyloid fibrils plays a key role in both functional biological responses and pathogenic disorders which include Alzheimer's disease and type II diabetes. Amyloid fibril assembly frequently generates compact oligomeric and curvilinear polymeric intermediates which are implicated to be toxic to cells. Yet, the relation between these early-stage oligomeric aggregates and late-stage rigid fibrils, which are the hallmark structure of amyloid plaques, has remained unclear. Our measurements indicate that lysozyme amyloid oligomers and their curvilinear fibrils only form after crossing a salt and protein concentration dependent threshold. These oligomeric aggregates are structurally distinct from rigid fibrils and are metastable against nucleation and growth of rigid fibrils. Our experimental transition boundaries match well with colloidal model predictions accounting for salt-modulated charge repulsion. We also report our preliminary findings on the mechanism by which these metastable oligomeric phases are converted into stable amyloid fibrils.

  14. Immunotherapy of Alzheimer's disease (AD): from murine models to anti-amyloid beta (Abeta) human monoclonal antibodies.

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    Geylis, Valeria; Steinitz, Michael

    2006-01-01

    The deposition of amyloid beta (Abeta) protein is a key pathological feature in Alzheimer's disease (AD). In murine models of AD, both active and passive immunization against Abeta induce a marked reduction in amyloid brain burden and an improvement in cognitive functions. Preliminary results of a prematurely terminated clinical trial where AD patients were actively vaccinated with aggregated Abeta bear resemblance to those documented in murine models. Passive immunization of AD patients with anti-Abeta antibodies, in particular human antibodies, is a strategy that provides a more cautious management and control of any undesired side effects. Sera of all healthy adults contain anti-Abeta IgG autoimmune antibodies. Hence antigen-committed human B-cells are easily immortalized by Epstein-Barr virus (EBV) into anti-Abeta secreting cell lines. Two anti-Abeta human monoclonal antibodies which we recently prepared bind to the N-terminus of Abeta peptide and were shown to stain amyloid plaques in non-fixed brain sections from an AD patient. It is anticipated that specifically selected anti-Abeta human monoclonal antibodies could reduce and inhibit deposits of amyloid in brain while avoiding the cognitive decline that characterizes AD. In the future, this type of antibody may prove to be a promising immune therapy for the disease.

  15. Serum amyloid beta peptides in patients with dementia and age-matched non-demented controls as detected by surface-enhanced laser desorption ionisation-time of flight mass spectrometry (SELDI-TOF MS).

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    Frankfort, Suzanne V; van Campen, Jos P C M; Tulner, Linda R; Beijnen, Jos H

    2008-09-01

    By using surface enhanced laser desorption/ionisation- time of flight mass spectrometry (SELDI-TOF MS) an amyloid beta (Abeta) profile was shown in cerebrospinal fluid (CSF) of patients with dementia. To investigate the Abeta-profile in serum with SELDI-TOF MS, to evaluate if this profile resembles CSF profiles and to investigate the correlation between intensity of Abeta-peptide-peaks in serum and clinical, demographical and genetic variables. Duplicate profiling of Abeta by an SELDI-TOF MS immunocapture assay was performed in 106 patients, suffering from Alzheimer's Disease or Vascular Dementia and age-matched non-demented control patients. Linear regression analyses were performed to investigate the intensities of four selected Abeta peaks as dependent variables in relation to the independent clinical, demographic or genetic variables. Abeta37, Abeta38 and Abeta40 were found among additional unidentified Abeta peptides, with the most pronounced Abeta peak at a molecular mass of 7752. This profile partly resembled the CSF profile. The clinical diagnosis was not a predictive independent variable, however ABCB1 genotypes C1236T, G2677T/A, age and creatinine level showed to be related to Abeta peak intensities in multivariate analyses. We found an Abeta profile in serum that partly resembled the CSF profile in demented patients. Age, creatinine levels, presence of the APOE epsilon4 allele and ABCB1 genotypes (C1236T and G2677T/A) were correlated with the Abeta serum profile. The role of P-gp as an Abeta transporter and the role of ABCB1 genotypes deserves further research. The investigated serum Abeta profile is probably not useful in the diagnosis of dementia.

  16. Amyloid β42 peptide is toxic to non-neural cells in Drosophila yielding a characteristic metabolite profile and the effect can be suppressed by PI3K

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    Mercedes Arnés

    2017-11-01

    Full Text Available The human Aβ42 peptide is associated with Alzheimer's disease through its deleterious effects in neurons. Expressing the human peptide in adult Drosophila in a tissue- and time-controlled manner, we show that Aβ42 is also toxic in non-neural cells, neurosecretory and epithelial cell types in particular. This form of toxicity includes the aberrant signaling by Wingless morphogen leading to the eventual activation of Caspase 3. Preventing Caspase 3 activation by means of p53 keeps epithelial cells from elimination but maintains the Aβ42 toxicity yielding more severe deleterious effects to the organism. Metabolic profiling by nuclear magnetic resonance (NMR of adult flies at selected ages post Aβ42 expression onset reveals characteristic changes in metabolites as early markers of the pathological process. All morphological and most metabolic features of Aβ42 toxicity can be suppressed by the joint overexpression of PI3K.

  17. Distinguishing d - and l -aspartic and isoaspartic acids in amyloid β peptides with ultrahigh resolution ion mobility spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Xueyun; Deng, Liulin; Baker, Erin M.; Ibrahim, Yehia M.; Petyuk, Vladislav A.; Smith, Richard D.

    2017-01-01

    Ion mobility spectrometry (IMS) was utilized to separate Aβ peptide variants containing isomeric asparic and isoaspartic acid residues with either al- ord-form. The abundance of each variant is of great interest in Alzheimer's disease studies and also to evaluate how often these modifications are occurring in other environmental and biological samples.

  18. A method for probing the mutational landscape of amyloid structure.

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    O'Donnell, Charles W; Waldispühl, Jérôme; Lis, Mieszko; Halfmann, Randal; Devadas, Srinivas; Lindquist, Susan; Berger, Bonnie

    2011-07-01

    Proteins of all kinds can self-assemble into highly ordered β-sheet aggregates known as amyloid fibrils, important both biologically and clinically. However, the specific molecular structure of a fibril can vary dramatically depending on sequence and environmental conditions, and mutations can drastically alter amyloid function and pathogenicity. Experimental structure determination has proven extremely difficult with only a handful of NMR-based models proposed, suggesting a need for computational methods. We present AmyloidMutants, a statistical mechanics approach for de novo prediction and analysis of wild-type and mutant amyloid structures. Based on the premise of protein mutational landscapes, AmyloidMutants energetically quantifies the effects of sequence mutation on fibril conformation and stability. Tested on non-mutant, full-length amyloid structures with known chemical shift data, AmyloidMutants offers roughly 2-fold improvement in prediction accuracy over existing tools. Moreover, AmyloidMutants is the only method to predict complete super-secondary structures, enabling accurate discrimination of topologically dissimilar amyloid conformations that correspond to the same sequence locations. Applied to mutant prediction, AmyloidMutants identifies a global conformational switch between Aβ and its highly-toxic 'Iowa' mutant in agreement with a recent experimental model based on partial chemical shift data. Predictions on mutant, yeast-toxic strains of HET-s suggest similar alternate folds. When applied to HET-s and a HET-s mutant with core asparagines replaced by glutamines (both highly amyloidogenic chemically similar residues abundant in many amyloids), AmyloidMutants surprisingly predicts a greatly reduced capacity of the glutamine mutant to form amyloid. We confirm this finding by conducting mutagenesis experiments. Our tool is publically available on the web at http://amyloid.csail.mit.edu/. lindquist_admin@wi.mit.edu; bab@csail.mit.edu.

  19. Formation of soluble amyloid oligomers and amyloid fibrils by the multifunctional protein vitronectin

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    Langen Ralf

    2008-10-01

    Full Text Available Abstract Background The multifunctional protein vitronectin is present within the deposits associated with Alzheimer disease (AD, age-related macular degeneration (AMD, atherosclerosis, systemic amyloidoses, and glomerulonephritis. The extent to which vitronectin contributes to amyloid formation within these plaques, which contain misfolded, amyloidogenic proteins, and the role of vitronectin in the pathophysiology of the aforementioned diseases is currently unknown. The investigation of vitronectin aggregation is significant since the formation of oligomeric and fibrillar structures are common features of amyloid proteins. Results We observed vitronectin immunoreactivity in senile plaques of AD brain, which exhibited overlap with the amyloid fibril-specific OC antibody, suggesting that vitronectin is deposited at sites of amyloid formation. Of particular interest is the growing body of evidence indicating that soluble nonfibrillar oligomers may be responsible for the development and progression of amyloid diseases. In this study we demonstrate that both plasma-purified and recombinant human vitronectin readily form spherical oligomers and typical amyloid fibrils. Vitronectin oligomers are toxic to cultured neuroblastoma and retinal pigment epithelium (RPE cells, possibly via a membrane-dependent mechanism, as they cause leakage of synthetic vesicles. Oligomer toxicity was attenuated in RPE cells by the anti-oligomer A11 antibody. Vitronectin fibrils contain a C-terminal protease-resistant fragment, which may approximate the core region of residues essential to amyloid formation. Conclusion These data reveal the propensity of vitronectin to behave as an amyloid protein and put forth the possibilities that accumulation of misfolded vitronectin may contribute to aggregate formation seen in age-related amyloid diseases.

  20. Nearly reversible conformational change of amyloid fibrils as revealed by pH-jump experiments.

    Science.gov (United States)

    Yamaguchi, Kei-ichi; Kamatari, Yuji O; Fukuoka, Mayuko; Miyaji, Reiji; Kuwata, Kazuo

    2013-10-01

    pH-jump induced conformational transitions between substates of preformed amyloid fibrils made by a fragmented peptide of helix 2 (H2 peptide) of MoPrP were detected, and their kinetics were analyzed using a novel pH-jump apparatus specially designed for observing amyloids. Previously, we reported that H2 peptide formed ordered fibrils with a minimum at 207 nm on CD spectra at pH 2.9 (named pH 2.9 fibrils), but formed aggregate-like fibrils with a minimum at 220 nm at pH 7.5 (named pH 7.5 fibrils). When pH-jump from 2.9 to 7.5 was performed, the CD spectrum changed instantly, but the finally observed ellipticities were clearly distinct from those of pH 7.5 fibrils. Thus, the finally observed state is termed 'pH 7.5-like fibrils'. However, pH 7.5-like fibrils reverted to the conformation very similar to that of the pH 2.9 fibrils when the pH of the solution was restored to 2.9. Then, we examined the kinetics of the nearly reversible conformational changes between pH 2.9 fibrils and pH 7.5-like fibrils using ANS fluorescence stopped-flow, and we observed relatively fast phases (0.7-18 s(-1)). In contrast, the conversion between pH 7.5-like fibrils and pH 7.5 fibrils never occurred (<0.2 day(-1)). Thus, H2 fibrils can be switched readily between distinct conformations separated by a low energy barrier, while a large energy barrier clearly separated the different conformations. These conformational varieties of amyloid fibrils may explain the physical basis of the diversity in prion.

  1. Systematic development of small molecules to inhibit specific microscopic steps of Aβ42 aggregation in Alzheimer's disease.

    Science.gov (United States)

    Habchi, Johnny; Chia, Sean; Limbocker, Ryan; Mannini, Benedetta; Ahn, Minkoo; Perni, Michele; Hansson, Oskar; Arosio, Paolo; Kumita, Janet R; Challa, Pavan Kumar; Cohen, Samuel I A; Linse, Sara; Dobson, Christopher M; Knowles, Tuomas P J; Vendruscolo, Michele

    2017-01-10

    The aggregation of the 42-residue form of the amyloidpeptide (Aβ42) is a pivotal event in Alzheimer's disease (AD). The use of chemical kinetics has recently enabled highly accurate quantifications of the effects of small molecules on specific microscopic steps in Aβ42 aggregation. Here, we exploit this approach to develop a rational drug discovery strategy against Aβ42 aggregation that uses as a read-out the changes in the nucleation and elongation rate constants caused by candidate small molecules. We thus identify a pool of compounds that target specific microscopic steps in Aβ42 aggregation. We then test further these small molecules in human cerebrospinal fluid and in a Caenorhabditis elegans model of AD. Our results show that this strategy represents a powerful approach to identify systematically small molecule lead compounds, thus offering an appealing opportunity to reduce the attrition problem in drug discovery.

  2. Doubly Phosphorylated Peptide Vaccines to Protect Transgenic P301S Mice against Alzheimer’s Disease Like Tau Aggregation

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    Monique Richter

    2014-07-01

    Full Text Available Intracellular neurofibrillary tangles and extracellular senile plaques are potential targets for active and passive immunotherapies. In this study we used the transgenic mouse model P301S for active immunizations with peptide vaccines composed of a double phosphorylated tau neoepitope (pSer202/pThr205, pThr212/pSer214, pThr231/pSer235 and an immunomodulatory T cell epitope from the tetanus toxin or tuberculosis antigen Ag85B. Importantly, the designed vaccine combining Alzheimer’s disease (AD specific B cell epitopes with foreign (bacterial T cell epitopes induced fast immune responses with high IgG1 titers after prophylactic immunization that subsequently decreased over the observation period. The effectiveness of the immunization was surveyed by evaluating the animal behavior, as well as the pathology in the brain by biochemical and histochemical techniques. Immunized mice clearly lived longer with reduced paralysis than placebo-treated mice. Additionally, they performed significantly better in rotarod and beam walk tests at the age of 20 weeks, indicating that the disease development was slowed down. Forty-eight weeks old vaccinated mice passed the beam walk test significantly better than control animals, which together with the increased survival rates undoubtedly prove the treatment effect. In conclusion, the data provide strong evidence that active immune therapies can reduce toxic effects of deposits formed in AD.

  3. Ionic Strength Modulation of the Free Energy Landscape of Aβ40 Peptide Fibril Formation.

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    Abelein, Axel; Jarvet, Jüri; Barth, Andreas; Gräslund, Astrid; Danielsson, Jens

    2016-06-01

    Protein misfolding and formation of cross-β structured amyloid fibrils are linked to many neurodegenerative disorders. Although recently developed quantitative approaches have started to reveal the molecular nature of self-assembly and fibril formation of proteins and peptides, it is yet unclear how these self-organization events are precisely modulated by microenvironmental factors, which are known to strongly affect the macroscopic aggregation properties. Here, we characterize the explicit effect of ionic strength on the microscopic aggregation rates of amyloid β peptide (Aβ40) self-association, implicated in Alzheimer's disease. We found that physiological ionic strength accelerates Aβ40 aggregation kinetics by promoting surface-catalyzed secondary nucleation reactions. This promoted catalytic effect can be assigned to shielding of electrostatic repulsion between monomers on the fibril surface or between the fibril surface itself and monomeric peptides. Furthermore, we observe the formation of two different β-structured states with similar but distinct spectroscopic features, which can be assigned to an off-pathway immature state (Fβ*) and a mature stable state (Fβ), where salt favors formation of the Fβ fibril morphology. Addition of salt to preformed Fβ* accelerates transition to Fβ, underlining the dynamic nature of Aβ40 fibrils in solution. On the basis of these results we suggest a model where salt decreases the free-energy barrier for Aβ40 folding to the Fβ state, favoring the buildup of the mature fibril morphology while omitting competing, energetically less favorable structural states.

  4. Functional α7β2 nicotinic acetylcholine receptors expressed in hippocampal interneurons exhibit high sensitivity to pathological level of amyloid β peptides

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    Liu Qiang

    2012-12-01

    Full Text Available Abstract Background β-amyloid (Aβ accumulation is described as a hallmark of Alzheimer’s disease (AD. Aβ perturbs a number of synaptic components including nicotinic acetylcholine receptors containing α7 subunits (α7-nAChRs, which are abundantly expressed in the hippocampus and found on GABAergic interneurons. We have previously demonstrated the existence of a novel, heteromeric α7β2-nAChR in basal forebrain cholinergic neurons that exhibits high sensitivity to acute Aβ exposure. To extend our previous work, we evaluated the expression and pharmacology of α7β2-nAChRs in hippocampal interneurons and their sensitivity to Aβ. Results GABAergic interneurons in the CA1 subregion of the hippocampus expressed functional α7β2-nAChRs, which were characterized by relatively slow whole-cell current kinetics, pharmacological sensitivity to dihydro-β-erythroidine (DHβE, a nAChR β2* subunit selective blocker, and α7 and β2 subunit interaction using immunoprecipitation assay. In addition, α7β2-nAChRs were sensitive to 1 nM oligomeric Aβ. Similar effects were observed in identified hippocampal interneurons prepared from GFP-GAD mice. Conclusion These findings suggest that Aβ modulation of cholinergic signaling in hippocampal GABAergic interneurons via α7β2-nAChRs could be an early and critical event in Aβ-induced functional abnormalities of hippocampal function, which may be relevant to learning and memory deficits in AD.

  5. Protective effects of compound FLZ on β-amyloid peptide-(25-35)-induced mouse hippocampal injury and learning and memory impairment

    Institute of Scientific and Technical Information of China (English)

    Fang FANG; Geng-tao LIU

    2006-01-01

    Aim: To study the protective effects of compound FLZ, a novel synthetic analogue of natural squamosamide, on learning and memory impairment and lesions of the hippocampus caused by icv injection of β-amyloid25-35 (Aβ25-35) in mice. Methods: Mice were icv injected with the Aβ25-35 (15 nmol/mouse), and then treated with oral administration of 75 mg/kg or 150 mg/kg of FLZ once daily for 16 consecutive days. The impairment of learning and memory in mice were tested using step-down test and Morris water maze test. The content of malondialdehyde (MDA) and the expressions of acetylcholinesterase (AChE), Bax, and Bcl-2 in the CA1 region of the mouse hippocampus were measured by biochemical and immu-nohistochemical analysis, respectively. The pathological damages of hippocampus were observed using a microscope. Results: FLZ (75 mg/kg, 150 mg/kg) significantly attenuated Aβ25-35-induced impairment of learning and memory in the step-down test and Morris water maze test. FLZ also reduced pathological damages to the hippocampus induced by Aβ25-35 Furthermore, FLZ prevented the increase of AChE and Bax, and the decrease of Bcl-2 immunoreactive cells in the CA1 region of the hippocampus, and reduced the increase of MDA content in the hippocampus in mice injected with Aβ25-35. Conclusion: FLZ has protective action against the impairment of learning and memory and pathological damage to the hippocampus induced by icv injection of Aβ25-35 in mice.

  6. Intrahippocampal Pathways Involved in Learning/Memory Mechanisms are Affected by Intracerebral Infusions of Amyloid-β25-35 Peptide and Hydrated Fullerene C60 in Rats.

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    Gordon, Rita; Podolski, Igor; Makarova, Ekaterina; Deev, Alexander; Mugantseva, Ekaterina; Khutsyan, Sergey; Sengpiel, Frank; Murashev, Arkady; Vorobyov, Vasily

    2017-01-01

    Primary memory impairments associated with increased level of amyloid-β (Aβ) in the brain have been shown to be linked, partially, with early pathological changes in the entorhinal cortex (EC) which spre