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Sample records for nonfibrillar amyloid oligomers

  1. Conversion of non-fibrillar beta-sheet oligomers into amyloid fibrils in Alzheimer's disease amyloid peptide aggregation.

    PubMed

    Benseny-Cases, Núria; Cócera, Mercedes; Cladera, Josep

    2007-10-05

    Abeta(1-40) is one of the main components of the fibrils found in amyloid plaques, a hallmark of brains affected by Alzheimer's disease. It is known that prior to the formation of amyloid fibrils in which the peptide adopts a well-ordered intermolecular beta-sheet structure, peptide monomers associate forming low and high molecular weight oligomers. These oligomers have been previously described in electron microscopy, AFM, and exclusion chromatography studies. Their specific secondary structures however, have not yet been well established. A major problem when comparing aggregation and secondary structure determinations in concentration-dependent processes such as amyloid aggregation is the different concentration range required in each type of experiment. In the present study we used the dye Thioflavin T (ThT), Fourier-transform infrared spectroscopy, and electron microscopy in order to structurally characterize the different aggregated species which form during the Abeta(1-40) fibril formation process. A unique sample containing 90microM peptide was used. The results show that oligomeric species which form during the lag phase of the aggregation kinetics are a mixture of unordered, helical, and intermolecular non-fibrillar beta-structures. The number of oligomers and the amount of non-fibrillar beta-structures grows throughout the lag phase and during the elongation phase these non-fibrillar beta-structures are transformed into fibrillar (amyloid) beta-structures, formed by association of high molecular weight intermediates.

  2. Conversion of non-fibrillar {beta}-sheet oligomers into amyloid fibrils in Alzheimer's disease amyloid peptide aggregation

    SciTech Connect

    Benseny-Cases, Nuria; Cocera, Mercedes; Cladera, Josep

    2007-10-05

    A{beta}(1-40) is one of the main components of the fibrils found in amyloid plaques, a hallmark of brains affected by Alzheimer's disease. It is known that prior to the formation of amyloid fibrils in which the peptide adopts a well-ordered intermolecular {beta}-sheet structure, peptide monomers associate forming low and high molecular weight oligomers. These oligomers have been previously described in electron microscopy, AFM, and exclusion chromatography studies. Their specific secondary structures however, have not yet been well established. A major problem when comparing aggregation and secondary structure determinations in concentration-dependent processes such as amyloid aggregation is the different concentration range required in each type of experiment. In the present study we used the dye Thioflavin T (ThT), Fourier-transform infrared spectroscopy, and electron microscopy in order to structurally characterize the different aggregated species which form during the A{beta}(1-40) fibril formation process. A unique sample containing 90 {mu}M peptide was used. The results show that oligomeric species which form during the lag phase of the aggregation kinetics are a mixture of unordered, helical, and intermolecular non-fibrillar {beta}-structures. The number of oligomers and the amount of non-fibrillar {beta}-structures grows throughout the lag phase and during the elongation phase these non-fibrillar {beta}-structures are transformed into fibrillar (amyloid) {beta}-structures, formed by association of high molecular weight intermediates.

  3. The Conformational Stability of Nonfibrillar Amyloid-β Peptide Oligomers Critically Depends on the C-Terminal Peptide Length

    PubMed Central

    2014-01-01

    The amyloid-β (Aβ) peptide is one key molecule in the pathogenesis of Alzheimer’s disease. We investigated the conformational stability of a nonfibrillar tetrameric Aβ structure by molecular dynamics (MD) simulations revealing that the stability of the Aβ tetramer depends critically on the C-terminal length. In contrast to the Aβ17–40 tetramer, which proved to be instable, the simulations demonstrate structural integrity of the Aβ17–42 and Aβ17–43 tetramers. These differences in stability can be attributed to an extension of the middle strand of a three-stranded antiparallel β sheet through residues 41–43, only present in the longer Aβ species that aggregate faster and are more neurotoxic. Additional MD simulations demonstrate that this higher stability is also present in the monomers forming the tetramer. In conclusion, our findings suggest the existence of a nonfibrillar oligomer topology that is significantly more stable for the longer Aβ species, thus offering a structural explanation for their higher neurotoxicity. PMID:24494584

  4. Specific soluble oligomers of amyloid-β peptide undergo replication and form non-fibrillar aggregates in interfacial environments.

    PubMed

    Kumar, Amit; Paslay, Lea C; Lyons, Daniel; Morgan, Sarah E; Correia, John J; Rangachari, Vijayaraghavan

    2012-06-15

    Aggregates of amyloid-β (Aβ) peptides have been implicated in the etiology of Alzheimer disease. Among the different forms of Aβ aggregates, low molecular weight species ranging between ~2- and 50-mers, also called "soluble oligomers," have emerged as the species responsible for early synaptic dysfunction and neuronal loss. Emerging evidence suggests that the neurotoxic oligomers need not be formed along the obligatory nucleation-dependant fibril formation pathway. In our earlier work, we reported the isolation of one such "off-pathway" 12-18-mer species of Aβ42 generated from fatty acids called large fatty acid-derived oligomers (LFAOs) (Kumar, A., Bullard, R. L., Patel, P., Paslay, L. C., Singh, D., Bienkiewicz, E. A., Morgan, S. E., and Rangachari, V. (2011) PLoS One 6, e18759). Here, we report the physiochemical aspects of LFAO-monomer interactions as well as LFAO-LFAO associations in the presence of interfaces. We discovered that LFAOs are a replicating strain of oligomers that recruit Aβ42 monomers and quantitatively convert them into LFAO assemblies at the expense of fibrils, a mechanism similar to prion propagation. We also found that in the presence of hexane-buffer or chloroform-buffer interfaces LFAOs are able to associate with themselves to form larger but non-fibrillar aggregates. These results further support the hypothesis that low molecular weight oligomers can be generated via non-fibril formation pathways. Furthermore, the unique replicating property of off-pathway oligomers may hold profound significance for Alzheimer disease pathology.

  5. A Multistage Pathway for Human Prion Protein Aggregation in Vitro: From Multimeric Seeds to β-Oligomers and Nonfibrillar Structures

    PubMed Central

    Cho, Kang R.; Yu, Shuiliang; Yin, Shaoman; Plomp, Marco; Qiu, S. Roger; Lakshminarayanan, Rajamani; Moradian-Oldak, Janet; Sy, Man-Sun

    2015-01-01

    Aberrant protein aggregation causes numerous neurological diseases including Creutzfeldt—Jakob disease (CJD), but the aggregation mechanisms remain poorly understood. Here, we report AFM results on the formation pathways of β-oligomers and nonfibrillar aggregates from wild-type full-length recombinant human prion protein (WT) and an insertion mutant (10OR) with five additional octapeptide repeats linked to familial CJD. Upon partial denaturing, seeds consisting of 3–4 monomers quickly appeared. Oligomers of ∼11–12 monomers then formed through direct interaction of seeds, rather than by subsequent monomer attachment. All larger aggregates formed through association of these β-oligomers. Although both WT and 10OR exhibited identical aggregation mechanisms, the latter oligomerized faster due to lower solubility and, hence, thermodynamic stability. This novel aggregation pathway has implications for prion diseases as well as others caused by protein aggregation. PMID:21534611

  6. Non-fibrillar amyloid-{beta} peptide reduces NMDA-induced neurotoxicity, but not AMPA-induced neurotoxicity

    SciTech Connect

    Niidome, Tetsuhiro; Goto, Yasuaki; Kato, Masaru; Wang, Pi-Lin; Goh, Saori; Tanaka, Naoki; Akaike, Akinori; Kihara, Takeshi; Sugimoto, Hachiro

    2009-09-04

    Amyloid-{beta} peptide (A{beta}) is thought to be linked to the pathogenesis of Alzheimer's disease. Recent studies suggest that A{beta} has important physiological roles in addition to its pathological roles. We recently demonstrated that A{beta}42 protects hippocampal neurons from glutamate-induced neurotoxicity, but the relationship between A{beta}42 assemblies and their neuroprotective effects remains largely unknown. In this study, we prepared non-fibrillar and fibrillar A{beta}42 based on the results of the thioflavin T assay, Western blot analysis, and atomic force microscopy, and examined the effects of non-fibrillar and fibrillar A{beta}42 on glutamate-induced neurotoxicity. Non-fibrillar A{beta}42, but not fibrillar A{beta}42, protected hippocampal neurons from glutamate-induced neurotoxicity. Furthermore, non-fibrillar A{beta}42 decreased both neurotoxicity and increases in the intracellular Ca{sup 2+} concentration induced by N-methyl-D-aspartate (NMDA), but not by {alpha}-amino-3-hydrozy-5-methyl-4-isoxazole propionic acid (AMPA). Our results suggest that non-fibrillar A{beta}42 protects hippocampal neurons from glutamate-induced neurotoxicity through regulation of the NMDA receptor.

  7. Crystal structure of the amyloid-β p3 fragment provides a model for oligomer formation in Alzheimer's disease.

    PubMed

    Streltsov, Victor A; Varghese, Joseph N; Masters, Colin L; Nuttall, Stewart D

    2011-01-26

    Alzheimer's disease is a progressive neurodegenerative disorder associated with the presence of amyloid-β (Aβ) peptide fibrillar plaques in the brain. However, current evidence suggests that soluble nonfibrillaroligomers may be the major drivers of Aβ-mediated synaptic dysfunction. Structural information on these Aβ species has been very limited because of their noncrystalline and unstable nature. Here, we describe a crystal structure of amylogenic residues 18-41 of the Aβ peptide (equivalent to the p3 α/γ-secretase fragment of amyloid precursor protein) presented within the CDR3 loop region of a shark Ig new antigen receptor (IgNAR) single variable domain antibody. The predominant oligomeric species is a tightly associated Aβ dimer, with paired dimers forming a tetramer in the crystal caged within four IgNAR domains, preventing uncontrolled amyloid formation. Our structure correlates with independently observed features of small nonfibrillaroligomers and reveals conserved elements consistent with residues and motifs predicted as critical in Aβ folding and oligomerization, thus potentially providing a model system for nonfibrillar oligomer formation in Alzheimer's disease.

  8. Amyloid-β Forms Fibrils by Nucleated Conformational Conversion of Oligomers

    PubMed Central

    Lee, Jiyong; Culyba, Elizabeth K.; Powers, Evan T.; Kelly, Jeffery W.

    2011-01-01

    Aβ amyloidogenesis is reported to occur via a nucleated polymerization mechanism, if so the energetically unfavorable oligomeric nucleus should be very hard to detect. However, many laboratories have detected early non-fibrillaroligomers without observing amyloid fibrils, suggesting a mechanistic revision may be needed. Herein, we introduce Cys-Cys-Aβ1-40 that cannot bind to the latent fluorophore FlAsH as a monomer, but is capable of binding FlAsH as an non-fibrillar oligomer or as a fibril, rendering the conjugates fluorescent. FlAsH monitoring of Cys-Cys-Aβ1-40 aggregation provides compelling evidence that Aβ1-40 very rapidly and efficiently forms spherical oligomers in vitro (85% yield) that are kinetically competent to slowly convert to amyloid fibrils by a nucleated conformational conversion mechanism (seedable). Moreover, this methodology demonstrated that plasmalogen ethanolamine vesicles eliminate the proteotoxicity-associated oligomerization phase of Aβ amyloidogenesis, while allowing fibril formation, rationalizing how low plasmalogen ethanolamine levels in the brain are epidemiologically linked to Alzheimer’s disease. PMID:21804535

  9. Zinc-Amyloid Interactions on a Millisecond Time-Scale Stabilize Non-Fibrillar Alzheimer Related Species

    SciTech Connect

    Noy,D.; Solomonov, I.; Sinkevich, O.; Arad, A.; Kjaer, K.; Sagi, I.

    2008-01-01

    The role of zinc, an essential element for normal brain function, in the pathology of Alzheimer's disease (AD) is poorly understood. On one hand, physiological and genetic evidence from transgenic mouse models supports its pathogenic role in promoting the deposition of the amyloid {beta}-protein (A{beta}) in senile plaques. On the other hand, levels of extracellular ('free') zinc in the brain, as inferred by the levels of zinc in cerebrospinal fluid, were found to be too low for inducing A{beta} aggregation. Remarkably, the release of transient high local concentrations of zinc during rapid synaptic events was reported. The role of such free zinc pulses in promoting A{beta} aggregation has never been established. Using a range of time-resolved structural and spectroscopic techniques, we found that zinc, when introduced in millisecond pulses of micromolar concentrations, immediately interacts with A{beta} 1-40 and promotes its aggregation. These interactions specifically stabilize non-fibrillar pathogenic related aggregate forms and prevent the formation of A{beta} fibrils (more benign species) presumably by interfering with the self-assembly process of A{beta}. These in vitro results strongly suggest a significant role for zinc pulses in A{beta} pathology. We further propose that by interfering with A{beta} self-assembly, which leads to insoluble, non-pathological fibrillar forms, zinc stabilizes transient, harmful amyloid forms. This report argues that zinc represents a class of molecular pathogens that effectively perturb the self-assembly of benign A{beta} fibrils, and stabilize harmful non-fibrillar forms.

  10. Atomic View of a Toxic Amyloid Small Oligomer

    SciTech Connect

    Laganowsky, Arthur; Liu, Cong; Sawaya, Michael R.; Whitelegge, Julian P.; Park, Jiyong; Zhao, Minglei; Pensalfini, Anna; Soriaga, Angela B.; Landau, Meytal; Teng, Poh K.; Cascio, Duilio; Glabe, Charles; Eisenberg, David

    2012-04-30

    Amyloid diseases, including Alzheimer's, Parkinson's, and the prion conditions, are each associated with a particular protein in fibrillar form. These amyloid fibrils were long suspected to be the disease agents, but evidence suggests that smaller, often transient and polymorphic oligomers are the toxic entities. Here, we identify a segment of the amyloid-forming protein {alpha}{beta} crystallin, which forms an oligomeric complex exhibiting properties of other amyloid oligomers: {beta}-sheet-rich structure, cytotoxicity, and recognition by an oligomer-specific antibody. The x-ray-derived atomic structure of the oligomer reveals a cylindrical barrel, formed from six antiparallel protein strands, that we term a cylindrin. The cylindrin structure is compatible with a sequence segment from the {beta}-amyloid protein of Alzheimer's disease. Cylindrins offer models for the hitherto elusive structures of amyloid oligomers.

  11. Pramipexole prevents neurotoxicity induced by oligomers of beta-amyloid.

    PubMed

    Uberti, Daniela; Bianchi, Irene; Olivari, Luca; Ferrari-Toninelli, Giulia; Canonico, PierLuigi; Memo, Maurizio

    2007-08-27

    Here we demonstrate that pramipexole, an antiparkinsonian dopamine receptor agonist drug, exerts neuroprotective effects against beta-amyloid neurotoxicity. Using a specific protocol to test individually oligomers, fibrils, or unaggregated amyloid beta-peptide, we found pramipexole able to protect cells against oligomers and fibrils. Unaggregated amyloid beta-peptide was found unable to cause cell death. Fibrils and oligomers were also found to produce elevated amount of free radicals, and this effect was prevented by pramipexole. We propose pramipexole may become in the future a coadjuvant in the treatment of neuropathologies, besides Parkinson's disease, where amyloid beta-peptide-mediated oxidative injury exerts a relevant role.

  12. Non-fibrillar oligomeric species of the amyloid ABri peptide, implicated in familial British dementia, are more potent at inducing apoptotic cell death than protofibrils or mature fibrils.

    PubMed

    El-Agnaf, O M; Nagala, S; Patel, B P; Austen, B M

    2001-06-29

    Familial British dementia (FBD) is an autosomal dominant neurodegenerative disorder, with biochemical and pathological similarities to Alzheimer's disease. FBD is associated with a point mutation in the stop codon of the BRI gene. The mutation extends the length of the wild-type protein by 11 amino acids, and following proteolytic cleavage, results in the production of a cyclic peptide (ABri) 11 amino acids longer than the wild-type (WT) peptide produced from the normal gene BRI. ABri was found to be the main component of amyloid deposits in FBD brains. However, pathological examination of FBD brains has shown the presence of ABri as non-fibrillar deposits as well as amyloid fibrils. Taken together, the genetic, pathological and biochemical data support the hypothesis that ABri deposits play a central role in the pathogenesis of FBD. Here we report that ABri, but not WT peptide, can oligomerise and form amyloid-like fibrils. We show for the first time that ABri induces apoptotic cell death, whereas WT is not toxic to cells. Moreover, we report the novel findings that non-fibrillar oligomeric species of ABri are more toxic than protofibrils and mature fibrils. These findings provide evidence that non-fibrillar oligomeric species are likely to play a critical role in the pathogenesis of FBD and suggest that a similar process may also operate in other neurodegenerative diseases. Copyright 2001 Academic Press.

  13. High-resolution NMR characterization of low abundance oligomers of amyloid-β without purification.

    PubMed

    Kotler, Samuel A; Brender, Jeffrey R; Vivekanandan, Subramanian; Suzuki, Yuta; Yamamoto, Kazutoshi; Monette, Martine; Krishnamoorthy, Janarthanan; Walsh, Patrick; Cauble, Meagan; Holl, Mark M Banaszak; Marsh, E Neil G; Ramamoorthy, Ayyalusamy

    2015-07-03

    Alzheimer's disease is characterized by the misfolding and self-assembly of the amyloidogenic protein amyloid-β (Aβ). The aggregation of Aβ leads to diverse oligomeric states, each of which may be potential targets for intervention. Obtaining insight into Aβ oligomers at the atomic level has been a major challenge to most techniques. Here, we use magic angle spinning recoupling (1)H-(1)H NMR experiments to overcome many of these limitations. Using (1)H-(1)H dipolar couplings as a NMR spectral filter to remove both high and low molecular weight species, we provide atomic-level characterization of a non-fibrillar aggregation product of the Aβ1-40 peptide using non-frozen samples without isotopic labeling. Importantly, this spectral filter allows the detection of the specific oligomer signal without a separate purification procedure. In comparison to other solid-state NMR techniques, the experiment is extraordinarily selective and sensitive. A resolved 2D spectra could be acquired of a small population of oligomers (6 micrograms, 7% of the total) amongst a much larger population of monomers and fibers (93% of the total). By coupling real-time (1)H-(1)H NMR experiments with other biophysical measurements, we show that a stable, primarily disordered Aβ1-40 oligomer 5-15 nm in diameter can form and coexist in parallel with the well-known cross-β-sheet fibrils.

  14. High-resolution NMR characterization of low abundance oligomers of amyloid-β without purification

    NASA Astrophysics Data System (ADS)

    Kotler, Samuel A.; Brender, Jeffrey R.; Vivekanandan, Subramanian; Suzuki, Yuta; Yamamoto, Kazutoshi; Monette, Martine; Krishnamoorthy, Janarthanan; Walsh, Patrick; Cauble, Meagan; Holl, Mark M. Banaszak; Marsh, E. Neil. G.; Ramamoorthy, Ayyalusamy

    2015-07-01

    Alzheimer’s disease is characterized by the misfolding and self-assembly of the amyloidogenic protein amyloid-β (Aβ). The aggregation of Aβ leads to diverse oligomeric states, each of which may be potential targets for intervention. Obtaining insight into Aβ oligomers at the atomic level has been a major challenge to most techniques. Here, we use magic angle spinning recoupling 1H-1H NMR experiments to overcome many of these limitations. Using 1H-1H dipolar couplings as a NMR spectral filter to remove both high and low molecular weight species, we provide atomic-level characterization of a non-fibrillar aggregation product of the Aβ1-40 peptide using non-frozen samples without isotopic labeling. Importantly, this spectral filter allows the detection of the specific oligomer signal without a separate purification procedure. In comparison to other solid-state NMR techniques, the experiment is extraordinarily selective and sensitive. A resolved 2D spectra could be acquired of a small population of oligomers (6 micrograms, 7% of the total) amongst a much larger population of monomers and fibers (93% of the total). By coupling real-time 1H-1H NMR experiments with other biophysical measurements, we show that a stable, primarily disordered Aβ1-40 oligomer 5-15 nm in diameter can form and coexist in parallel with the well-known cross-β-sheet fibrils.

  15. High-resolution NMR characterization of low abundance oligomers of amyloid-β without purification

    PubMed Central

    Kotler, Samuel A.; Brender, Jeffrey R.; Vivekanandan, Subramanian; Suzuki, Yuta; Yamamoto, Kazutoshi; Monette, Martine; Krishnamoorthy, Janarthanan; Walsh, Patrick; Cauble, Meagan; Holl, Mark M. Banaszak; Marsh, E. Neil. G.; Ramamoorthy, Ayyalusamy

    2015-01-01

    Alzheimer’s disease is characterized by the misfolding and self-assembly of the amyloidogenic protein amyloid-β (Aβ). The aggregation of Aβ leads to diverse oligomeric states, each of which may be potential targets for intervention. Obtaining insight into Aβ oligomers at the atomic level has been a major challenge to most techniques. Here, we use magic angle spinning recoupling 1H-1H NMR experiments to overcome many of these limitations. Using 1H-1H dipolar couplings as a NMR spectral filter to remove both high and low molecular weight species, we provide atomic-level characterization of a non-fibrillar aggregation product of the Aβ1-40 peptide using non-frozen samples without isotopic labeling. Importantly, this spectral filter allows the detection of the specific oligomer signal without a separate purification procedure. In comparison to other solid-state NMR techniques, the experiment is extraordinarily selective and sensitive. A resolved 2D spectra could be acquired of a small population of oligomers (6 micrograms, 7% of the total) amongst a much larger population of monomers and fibers (93% of the total). By coupling real-time 1H-1H NMR experiments with other biophysical measurements, we show that a stable, primarily disordered Aβ1-40 oligomer 5–15 nm in diameter can form and coexist in parallel with the well-known cross-β-sheet fibrils. PMID:26138908

  16. Prion Protein-mediated Toxicity of AmyloidOligomers Requires Lipid Rafts and the Transmembrane LRP1*

    PubMed Central

    Rushworth, Jo V.; Griffiths, Heledd H.; Watt, Nicole T.; Hooper, Nigel M.

    2013-01-01

    Soluble oligomers of the amyloid-β (Aβ) peptide cause neurotoxicity, synaptic dysfunction, and memory impairments that underlie Alzheimer disease (AD). The cellular prion protein (PrPC) was recently identified as a high affinity neuronal receptor for Aβ oligomers. We report that fibrillar Aβ oligomers recognized by the OC antibody, which have been shown to correlate with the onset and severity of AD, bind preferentially to cells and neurons expressing PrPC. The binding of Aβ oligomers to cell surface PrPC, as well as their downstream activation of Fyn kinase, was dependent on the integrity of cholesterol-rich lipid rafts. In SH-SY5Y cells, fluorescence microscopy and co-localization with subcellular markers revealed that the Aβ oligomers co-internalized with PrPC, accumulated in endosomes, and subsequently trafficked to lysosomes. The cell surface binding, internalization, and downstream toxicity of Aβ oligomers was dependent on the transmembrane low density lipoprotein receptor-related protein-1 (LRP1). The binding of Aβ oligomers to cell surface PrPC impaired its ability to inhibit the activity of the β-secretase BACE1, which cleaves the amyloid precursor protein to produce Aβ. The green tea polyphenol (−)-epigallocatechin gallate and the red wine extract resveratrol both remodeled the fibrillar conformation of Aβ oligomers. The resulting nonfibrillar oligomers displayed significantly reduced binding to PrPC-expressing cells and were no longer cytotoxic. These data indicate that soluble, fibrillar Aβ oligomers bind to PrPC in a conformation-dependent manner and require the integrity of lipid rafts and the transmembrane LRP1 for their cytotoxicity, thus revealing potential targets to alleviate the neurotoxic properties of Aβ oligomers in AD. PMID:23386614

  17. Stabilization, Characterization, and Selective Removal of Cystatin C Amyloid Oligomers*

    PubMed Central

    Östner, Gustav; Lindström, Veronica; Hjort Christensen, Per; Kozak, Maciej; Abrahamson, Magnus; Grubb, Anders

    2013-01-01

    The pathophysiological process in amyloid disorders usually involves the transformation of a functional monomeric protein via potentially toxic oligomers into amyloid fibrils. The structure and properties of the intermediary oligomers have been difficult to study due to their instability and dynamic equilibrium with smaller and larger species. In hereditary cystatin C amyloid angiopathy, a cystatin C variant is deposited in arterial walls and cause brain hemorrhage in young adults. In the present investigation, we use redox experiments of monomeric cystatin C, stabilized against domain swapping by an intramolecular disulfide bond, to generate stable oligomers (dimers, trimers, tetramers, decamers, and high molecular weight oligomers). These oligomers were characterized concerning size by gel filtration, polyacrylamide gel electrophoresis, and mass spectrometry, shape by electron and atomic force microscopy, and, function by assays of their capacity to inhibit proteases. The results showed the oligomers to be highly ordered, domain-swapped assemblies of cystatin C and that the oligomers could not build larger oligomers, or fibrils, without domain swapping. The stabilized oligomers were used to induce antibody formation in rabbits. After immunosorption, using immobilized monomeric cystatin C, and elution from columns with immobilized cystatin C oligomers, oligomer-specific antibodies were obtained. These could be used to selectively remove cystatin C dimers from biological fluids containing both dimers and monomers. PMID:23629649

  18. Toxic species in amyloid disorders: Oligomers or mature fibrils

    PubMed Central

    Verma, Meenakshi; Vats, Abhishek; Taneja, Vibha

    2015-01-01

    Protein aggregation is the hallmark of several neurodegenerative disorders. These protein aggregation (fibrillization) disorders are also known as amyloid disorders. The mechanism of protein aggregation involves conformation switch of the native protein, oligomer formation leading to protofibrils and finally mature fibrils. Mature fibrils have long been considered as the cause of disease pathogenesis; however, recent evidences suggest oligomeric intermediates formed during fibrillization to be toxic. In this review, we have tried to address the ongoing debate for these toxic amyloid species. We did an extensive literature search and collated information from Pubmed (http://www.ncbi.nlm.nih.gov) and Google search using various permutations and combinations of the following keywords: Neurodegeneration, amyloid disorders, protein aggregation, fibrils, oligomers, toxicity, Alzheimer's Disease, Parkinson's Disease. We describe different instances showing the toxicity of mature fibrils as well as oligomers in Alzheimer's Disease and Parkinson's Disease. Distinct structural framework and morphology of amyloid oligomers suggests difference in toxic effect between oligomers and fibrils. We highlight the difference in structure and proposed toxicity pathways for fibrils and oligomers. We also highlight the evidences indicating that intermediary oligomeric species can act as potential diagnostic biomarker. Since the formation of these toxic species follow a common structural switch among various amyloid disorders, the protein aggregation events can be targeted for developing broad-range therapeutics. The therapeutic trials based on the understanding of different protein conformers (monomers, oligomers, protofibrils and fibrils) in amyloid cascade are also described. PMID:26019408

  19. Amyloid β-protein oligomers and Alzheimer’s disease

    PubMed Central

    2013-01-01

    The oligomer cascade hypothesis, which states that oligomers are the initiating pathologic agents in Alzheimer’s disease, has all but supplanted the amyloid cascade hypothesis, which suggested that fibers were the key etiologic agents in Alzheimer’s disease. We review here the results of in vivo, in vitro and in silico studies of amyloid β-protein oligomers, and discuss important caveats that should be considered in the evaluation of these results. This article is divided into four sections that mirror the main approaches used in the field to better understand oligomers: (1) attempts to locate and examine oligomers in vivo in situ; that is, without removing these species from their environment; (2) studies involving oligomers extracted from human or animal tissues and the subsequent characterization of their properties ex vivo; (3) studies of oligomers that have been produced synthetically and studied using a reductionist approach in relatively simple in vitro biophysical systems; and (4) computational studies of oligomers in silico. These multiple orthogonal approaches have revealed much about the molecular and cell biology of amyloid β-protein. However, as informative as these approaches have been, the amyloid β-protein oligomer system remains enigmatic. PMID:24289820

  20. Calcium Ions Promote Superoxide Dismutase 1 (SOD1) Aggregation into Non-fibrillar Amyloid

    PubMed Central

    Leal, Sónia S.; Cardoso, Isabel; Valentine, Joan S.; Gomes, Cláudio M.

    2013-01-01

    Imbalance in metal ion homeostasis is a hallmark in neurodegenerative conditions involving protein deposition, and amyotrophic lateral sclerosis (ALS) is no exception. In particular, Ca2+ dysregulation has been shown to correlate with superoxide dismutase-1 (SOD1) aggregation in a cellular model of ALS. Here we present evidence that SOD1 aggregation is enhanced and modulated by Ca2+. We show that at physiological pH, Ca2+ induces conformational changes that increase SOD1 β-sheet content, as probed by far UV CD and attenuated total reflectance-FTIR, and enhances SOD1 hydrophobicity, as probed by ANS fluorescence emission. Moreover, dynamic light scattering analysis showed that Ca2+ boosts the onset of SOD1 aggregation. In agreement, Ca2+ decreases SOD1 critical concentration and nucleation time during aggregation kinetics, as evidenced by thioflavin T fluorescence emission. Attenuated total reflectance FTIR analysis showed that Ca2+ induced aggregates consisting preferentially of antiparallel β-sheets, thus suggesting a modulation effect on the aggregation pathway. Transmission electron microscopy and analysis with conformational anti-fibril and anti-oligomer antibodies showed that oligomers and amyloidogenic aggregates constitute the prevalent morphology of Ca2+-induced aggregates, thus indicating that Ca2+ diverts SOD1 aggregation from fibrils toward amorphous aggregates. Interestingly, the same heterogeneity of conformations is found in ALS-derived protein inclusions. We thus hypothesize that transient variations and dysregulation of cellular Ca2+ levels contribute to the formation of SOD1 aggregates in ALS patients. In this scenario, Ca2+ may be considered as a pathogenic effector in the formation of ALS proteinaceous inclusions. PMID:23861388

  1. Brain amyloidoligomers in ageing and Alzheimer's disease.

    PubMed

    Lesné, Sylvain E; Sherman, Mathew A; Grant, Marianne; Kuskowski, Michael; Schneider, Julie A; Bennett, David A; Ashe, Karen H

    2013-05-01

    Alzheimer's disease begins about two decades before the onset of symptoms or neuron death, and is believed to be caused by pathogenic amyloid-β aggregates that initiate a cascade of molecular events culminating in widespread neurodegeneration. The microtubule binding protein tau may mediate the effects of amyloid-β in this cascade. Amyloid plaques comprised of insoluble, fibrillar amyloid-β aggregates are the most characteristic feature of Alzheimer's disease. However, the correspondence between the distribution of plaques and the pattern of neurodegeneration is tenuous. This discrepancy has stimulated the investigation of other amyloid-β aggregates, including soluble amyloidoligomers. Different soluble amyloidoligomers have been studied in several mouse models, but not systematically in humans. Here, we measured three amyloidoligomers previously described in mouse models-amyloid-β trimers, Aβ*56 and amyloid-β dimers-in brain tissue from 75 cognitively intact individuals, ranging from young children to the elderly, and 58 impaired subjects with mild cognitive impairment or probable Alzheimer's disease. As in mouse models, where amyloid-β trimers appear to be the fundamental amyloid-β assembly unit of Aβ*56 and are present in young mice prior to memory decline, amyloid-β trimers in humans were present in children and adolescents; their levels rose gradually with age and were significantly above baseline in subjects in their 70s. Aβ*56 levels were negligible in children and young adults, rose significantly above baseline in subjects in their 40s and increased steadily thereafter. Amyloid-β dimers were undetectable until subjects were in their 60s; their levels then increased sharply and correlated with plaque load. Remarkably, in cognitively intact individuals we found strong positive correlations between Aβ*56 and two pathological forms of soluble tau (tau-CP13 and tau-Alz50), and negative correlations between Aβ*56 and two postsynaptic

  2. Lipid raft disruption protects mature neurons against amyloid oligomer toxicity.

    PubMed

    Malchiodi-Albedi, Fiorella; Contrusciere, Valentina; Raggi, Carla; Fecchi, Katia; Rainaldi, Gabriella; Paradisi, Silvia; Matteucci, Andrea; Santini, Maria Teresa; Sargiacomo, Massimo; Frank, Claudio; Gaudiano, Maria Cristina; Diociaiuti, Marco

    2010-04-01

    A specific neuronal vulnerability to amyloid protein toxicity may account for brain susceptibility to protein misfolding diseases. To investigate this issue, we compared the effects induced by oligomers from salmon calcitonin (sCTOs), a neurotoxic amyloid protein, on cells of different histogenesis: mature and immature primary hippocampal neurons, primary astrocytes, MG63 osteoblasts and NIH-3T3 fibroblasts. In mature neurons, sCTOs increased apoptosis and induced neuritic and synaptic damages similar to those caused by amyloid beta oligomers. Immature neurons and the other cell types showed no cytotoxicity. sCTOs caused cytosolic Ca(2+) rise in mature, but not in immature neurons and the other cell types. Comparison of plasma membrane lipid composition showed that mature neurons had the highest content in lipid rafts, suggesting a key role for them in neuronal vulnerability to sCTOs. Consistently, depletion in gangliosides protected against sCTO toxicity. We hypothesize that the high content in lipid rafts makes mature neurons especially vulnerable to amyloid proteins, as compared to other cell types; this may help explain why the brain is a target organ for amyloid-related diseases.

  3. Common structure and toxic function of amyloid oligomers implies a common mechanism of pathogenesis.

    PubMed

    Glabe, Charles G; Kayed, Rakez

    2006-01-24

    Recent findings indicate that soluble amyloid oligomers may represent the primary pathologic species in degenerative diseases. These amyloid oligomers share common structural features and the ability to permeabilize membranes, suggesting that they also share a common primary mechanism of pathogenesis. Membrane permeabilization by amyloid oligomers may initiate a common group of downstream pathologic processes, including intracellular calcium dyshomeostasis, production of reactive oxygen species, altered signaling pathways, and mitochondrial dysfunction that represent key effectors of cellular dysfunction and cell death in amyloid-associated degenerative disease, such as sporadic inclusion-body myositis.

  4. Size-dependent neurotoxicity of β-amyloid oligomers

    PubMed Central

    Cizas, Paulius; Budvytyte, Rima; Morkuniene, Ramune; Moldovan, Radu; Broccio, Matteo; Lösche, Mathias; Niaura, Gediminas; Valincius, Gintaras; Borutaite, Vilmante

    2010-01-01

    The link between the size of soluble amyloid β (Aβ) oligomers and their toxicity to rat cerebellar granule cells (CGC) was investigated. Variation in conditions during in vitro oligomerization of Aβ1-42 resulted in peptide assemblies with different particle size as measured by atomic force microscopy and confirmed by the dynamic light scattering and fluorescence correlation spectroscopy. Small oligomers of Aβ1-42 with a mean particle z-height of 1-2 nm exhibited propensity to bind to the phospholipid vesicles and they were the most toxic species that induced rapid neuronal necrosis at submicromolar concentrations whereas the bigger aggregates (z-height above 4-5 nm) did not bind vesicles and did not cause detectable neuronal death. Similar neurotoxic pattern was also observed in primary cultures of cortex neurons whereas Aβ1–42 oligomers, monomers and fibrils were non-toxic to glial cells in CGC cultures or macrophage J774 cells. However, both oligomeric forms of Aβ1-42 induced reduction of neuronal cell densities in the CGC cultures. PMID:20153288

  5. Size-dependent neurotoxicity of beta-amyloid oligomers.

    PubMed

    Cizas, Paulius; Budvytyte, Rima; Morkuniene, Ramune; Moldovan, Radu; Broccio, Matteo; Lösche, Mathias; Niaura, Gediminas; Valincius, Gintaras; Borutaite, Vilmante

    2010-04-15

    The link between the size of soluble amyloid beta (Abeta) oligomers and their toxicity to rat cerebellar granule cells (CGC) was investigated. Variation in conditions during in vitro oligomerization of Abeta(1-42) resulted in peptide assemblies with different particle size as measured by atomic force microscopy and confirmed by dynamic light scattering and fluorescence correlation spectroscopy. Small oligomers of Abeta(1-42) with a mean particle z-height of 1-2 nm exhibited propensity to bind to phospholipid vesicles and they were the most toxic species that induced rapid neuronal necrosis at submicromolar concentrations whereas the bigger aggregates (z-height above 4-5 nm) did not bind vesicles and did not cause detectable neuronal death. A similar neurotoxic pattern was also observed in primary cultures of cortex neurons whereas Abeta(1-42) oligomers, monomers and fibrils were non-toxic to glial cells in CGC cultures or macrophage J774 cells. However, both oligomeric forms of Abeta(1-42) induced reduction of neuronal cell densities in the CGC cultures.

  6. The Essential Role of Soluble Aβ Oligomers in Alzheimer's Disease.

    PubMed

    Wang, Zi-Xuan; Tan, Lan; Liu, Jinyuan; Yu, Jin-Tai

    2016-04-01

    Alzheimer's disease (AD) is a common neurodegenerative disease characterized by amyloid plaque and neurofibrillary tangles (NFT). With the finding that soluble nonfibrillar Aβ levels actually correlate strongly with the severity of the disease, the initial focus on amyloid plaques shifted to the contemporary concept that AD memory failure is caused by soluble Aβ oligomers. The soluble Aβ are known to be more neurotoxicthan fibrillar Aβ species. In this paper, we summarize the essential role of soluble Aβ oligomers in AD and discuss therapeutic strategies that target soluble Aβ oligomers.

  7. Apoptosis induced by islet amyloid polypeptide soluble oligomers is neutralized by diabetes-associated specific antibodies

    PubMed Central

    Bram, Yaron; Frydman-Marom, Anat; Yanai, Inbal; Gilead, Sharon; Shaltiel-Karyo, Ronit; Amdursky, Nadav; Gazit, Ehud

    2014-01-01

    Soluble oligomeric assemblies of amyloidal proteins appear to act as major pathological agents in several degenerative disorders. Isolation and characterization of these oligomers is a pivotal step towards determination of their pathological relevance. Here we describe the isolation of Type 2 diabetes-associated islet amyloid polypeptide soluble cytotoxic oligomers; these oligomers induced apoptosis in cultured pancreatic cells, permeated model lipid vesicles and interacted with cell membranes following complete internalization. Moreover, antibodies which specifically recognized these assemblies, but not monomers or amyloid fibrils, were exclusively identified in diabetic patients and were shown to neutralize the apoptotic effect induced by these oligomers. Our findings support the notion that human IAPP peptide can form highly toxic oligomers. The presence of antibodies identified in the serum of diabetic patients confirms the pathological relevance of the oligomers. In addition, the newly identified structural epitopes may also provide new mechanistic insights and a molecular target for future therapy. PMID:24589570

  8. Apoptosis induced by islet amyloid polypeptide soluble oligomers is neutralized by diabetes-associated specific antibodies.

    PubMed

    Bram, Yaron; Frydman-Marom, Anat; Yanai, Inbal; Gilead, Sharon; Shaltiel-Karyo, Ronit; Amdursky, Nadav; Gazit, Ehud

    2014-03-04

    Soluble oligomeric assemblies of amyloidal proteins appear to act as major pathological agents in several degenerative disorders. Isolation and characterization of these oligomers is a pivotal step towards determination of their pathological relevance. Here we describe the isolation of Type 2 diabetes-associated islet amyloid polypeptide soluble cytotoxic oligomers; these oligomers induced apoptosis in cultured pancreatic cells, permeated model lipid vesicles and interacted with cell membranes following complete internalization. Moreover, antibodies which specifically recognized these assemblies, but not monomers or amyloid fibrils, were exclusively identified in diabetic patients and were shown to neutralize the apoptotic effect induced by these oligomers. Our findings support the notion that human IAPP peptide can form highly toxic oligomers. The presence of antibodies identified in the serum of diabetic patients confirms the pathological relevance of the oligomers. In addition, the newly identified structural epitopes may also provide new mechanistic insights and a molecular target for future therapy.

  9. Amyloid oligomer structure characterization from simulations: A general method

    NASA Astrophysics Data System (ADS)

    Nguyen, Phuong H.; Li, Mai Suan; Derreumaux, Philippe

    2014-03-01

    Amyloid oligomers and plaques are composed of multiple chemically identical proteins. Therefore, one of the first fundamental problems in the characterization of structures from simulations is the treatment of the degeneracy, i.e., the permutation of the molecules. Second, the intramolecular and intermolecular degrees of freedom of the various molecules must be taken into account. Currently, the well-known dihedral principal component analysis method only considers the intramolecular degrees of freedom, and other methods employing collective variables can only describe intermolecular degrees of freedom at the global level. With this in mind, we propose a general method that identifies all the structures accurately. The basis idea is that the intramolecular and intermolecular states are described in terms of combinations of single-molecule and double-molecule states, respectively, and the overall structures of oligomers are the product basis of the intramolecular and intermolecular states. This way, the degeneracy is automatically avoided. The method is illustrated on the conformational ensemble of the tetramer of the Alzheimer's peptide Aβ9-40, resulting from two atomistic molecular dynamics simulations in explicit solvent, each of 200 ns, starting from two distinct structures.

  10. Amyloid oligomer structure characterization from simulations: a general method.

    PubMed

    Nguyen, Phuong H; Li, Mai Suan; Derreumaux, Philippe

    2014-03-07

    Amyloid oligomers and plaques are composed of multiple chemically identical proteins. Therefore, one of the first fundamental problems in the characterization of structures from simulations is the treatment of the degeneracy, i.e., the permutation of the molecules. Second, the intramolecular and intermolecular degrees of freedom of the various molecules must be taken into account. Currently, the well-known dihedral principal component analysis method only considers the intramolecular degrees of freedom, and other methods employing collective variables can only describe intermolecular degrees of freedom at the global level. With this in mind, we propose a general method that identifies all the structures accurately. The basis idea is that the intramolecular and intermolecular states are described in terms of combinations of single-molecule and double-molecule states, respectively, and the overall structures of oligomers are the product basis of the intramolecular and intermolecular states. This way, the degeneracy is automatically avoided. The method is illustrated on the conformational ensemble of the tetramer of the Alzheimer's peptide Aβ9-40, resulting from two atomistic molecular dynamics simulations in explicit solvent, each of 200 ns, starting from two distinct structures.

  11. Amyloid oligomer structure characterization from simulations: A general method

    SciTech Connect

    Nguyen, Phuong H.; Li, Mai Suan

    2014-03-07

    Amyloid oligomers and plaques are composed of multiple chemically identical proteins. Therefore, one of the first fundamental problems in the characterization of structures from simulations is the treatment of the degeneracy, i.e., the permutation of the molecules. Second, the intramolecular and intermolecular degrees of freedom of the various molecules must be taken into account. Currently, the well-known dihedral principal component analysis method only considers the intramolecular degrees of freedom, and other methods employing collective variables can only describe intermolecular degrees of freedom at the global level. With this in mind, we propose a general method that identifies all the structures accurately. The basis idea is that the intramolecular and intermolecular states are described in terms of combinations of single-molecule and double-molecule states, respectively, and the overall structures of oligomers are the product basis of the intramolecular and intermolecular states. This way, the degeneracy is automatically avoided. The method is illustrated on the conformational ensemble of the tetramer of the Alzheimer's peptide Aβ{sub 9−40}, resulting from two atomistic molecular dynamics simulations in explicit solvent, each of 200 ns, starting from two distinct structures.

  12. Oligomers of the amyloid-beta protein disrupt working memory: confirmation with two behavioral procedures.

    PubMed

    Poling, Alan; Morgan-Paisley, Kineta; Panos, John J; Kim, Eun-Mee; O'Hare, Eugene; Cleary, James P; Lesné, Sylvain; Ashe, Karen H; Porritt, Matthew; Baker, Lisa E

    2008-11-21

    Converging lines of evidence suggest that oligomers of amyloid-beta play a role in the cognitive impairment characteristic of Alzheimer's disease, but only three studies have provided experimental evidence of such impairment. To provide additional information about the effects of these oligomers on memory, the present study examined the memory of groups of rats exposed to ICV injections of the culture media (CM) of Chinese Hamster Ovary cells that were (7PA2) and were not (CHO-) transfected with a human mutation of amyloid precursor protein that appears to cause early-onset Alzheimer's disease. The 7PA2 CM, which contained concentrations of soluble amyloid-beta oligomers physiologically relevant to those found in human brain, significantly disrupted working memory in rats tested in a radial-arm maze. In contrast, CHO- CM, which did not contain such oligomers, had no effect on memory. The disruptive effects of 7PA2-derived amyloid-beta oligomers, evident 2h after exposure, disappeared within a day. These findings are compared to results from 7PA2 CM tested under a complex procedure thought to measure aspects of executive function. The results confirm the disruptive effects of low-n amyloid-beta oligomers and extend them to a well-established rat model of memory.

  13. Islet Amyloid in Type 2 Diabetes, and the Toxic Oligomer Hypothesis

    PubMed Central

    Haataja, Leena; Gurlo, Tatyana; Huang, Chang J.; Butler, Peter C.

    2008-01-01

    Type 2 diabetes (T2DM) is characterized by insulin resistance, defective insulin secretion, loss of β-cell mass with increased β-cell apoptosis and islet amyloid. The islet amyloid is derived from islet amyloid polypeptide (IAPP, amylin), a protein coexpressed and cosecreted with insulin by pancreatic β-cells. In common with other amyloidogenic proteins, IAPP has the propensity to form membrane permeant toxic oligomers. Accumulating evidence suggests that these toxic oligomers, rather than the extracellular amyloid form of these proteins, are responsible for loss of neurons in neurodegenerative diseases. In this review we discuss emerging evidence to suggest that formation of intracellular IAPP oligomers may contribute to β-cell loss in T2DM. The accumulated evidence permits the amyloid hypothesis originally developed for neurodegenerative diseases to be reformulated as the toxic oligomer hypothesis. However, as in neurodegenerative diseases, it remains unclear exactly why amyloidogenic proteins form oligomers in vivo, what their exact structure is, and to what extent these oligomers play a primary or secondary role in the cytotoxicity in what are now often called unfolded protein diseases. PMID:18314421

  14. Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer's amyloid beta-peptide.

    PubMed

    Haass, Christian; Selkoe, Dennis J

    2007-02-01

    The distinct protein aggregates that are found in Alzheimer's, Parkinson's, Huntington's and prion diseases seem to cause these disorders. Small intermediates - soluble oligomers - in the aggregation process can confer synaptic dysfunction, whereas large, insoluble deposits might function as reservoirs of the bioactive oligomers. These emerging concepts are exemplified by Alzheimer's disease, in which amyloid beta-protein oligomers adversely affect synaptic structure and plasticity. Findings in other neurodegenerative diseases indicate that a broadly similar process of neuronal dysfunction is induced by diffusible oligomers of misfolded proteins.

  15. Aromatic small molecules remodel toxic soluble oligomers of amyloid beta through three independent pathways.

    PubMed

    Ladiwala, Ali Reza A; Dordick, Jonathan S; Tessier, Peter M

    2011-02-04

    In protein conformational disorders ranging from Alzheimer to Parkinson disease, proteins of unrelated sequence misfold into a similar array of aggregated conformers ranging from small oligomers to large amyloid fibrils. Substantial evidence suggests that small, prefibrillar oligomers are the most toxic species, yet to what extent they can be selectively targeted and remodeled into non-toxic conformers using small molecules is poorly understood. We have evaluated the conformational specificity and remodeling pathways of a diverse panel of aromatic small molecules against mature soluble oligomers of the Aβ42 peptide associated with Alzheimer disease. We find that small molecule antagonists can be grouped into three classes, which we herein define as Class I, II, and III molecules, based on the distinct pathways they utilize to remodel soluble oligomers into multiple conformers with reduced toxicity. Class I molecules remodel soluble oligomers into large, off-pathway aggregates that are non-toxic. Moreover, Class IA molecules also remodel amyloid fibrils into the same off-pathway structures, whereas Class IB molecules fail to remodel fibrils but accelerate aggregation of freshly disaggregated Aβ. In contrast, a Class II molecule converts soluble Aβ oligomers into fibrils, but is inactive against disaggregated and fibrillar Aβ. Class III molecules disassemble soluble oligomers (as well as fibrils) into low molecular weight species that are non-toxic. Strikingly, Aβ non-toxic oligomers (which are morphologically indistinguishable from toxic soluble oligomers) are significantly more resistant to being remodeled than Aβ soluble oligomers or amyloid fibrils. Our findings reveal that relatively subtle differences in small molecule structure encipher surprisingly large differences in the pathways they employ to remodel Aβ soluble oligomers and related aggregated conformers.

  16. Distinct Annular Oligomers Captured along the Assembly and Disassembly Pathways of Transthyretin Amyloid Protofibrils

    PubMed Central

    Pires, Ricardo H.; Karsai, Árpád; Saraiva, Maria J.; Damas, Ana M.; Kellermayer, Miklós S. Z.

    2012-01-01

    Background Defects in protein folding may lead to severe degenerative diseases characterized by the appearance of amyloid fibril deposits. Cytotoxicity in amyloidoses has been linked to poration of the cell membrane that may involve interactions with amyloid intermediates of annular shape. Although annular oligomers have been detected in many amyloidogenic systems, their universality, function and molecular mechanisms of appearance are debated. Methodology/Principal Findings We investigated with high-resolution in situ atomic force microscopy the assembly and disassembly of transthyretin (TTR) amyloid protofibrils formed of the native protein by pH shift. Annular oligomers were the first morphologically distinct intermediates observed in the TTR aggregation pathway. Morphological analysis suggests that they can assemble into a double-stack of octameric rings with a 16±2 nm diameter, and displaying the tendency to form linear structures. According to light scattering data coupled to AFM imaging, annular oligomers appeared to undergo a collapse type of structural transition into spheroid oligomers containing 8–16 monomers. Disassembly of TTR amyloid protofibrils also resulted in the rapid appearance of annular oligomers but with a morphology quite distinct from that observed in the assembly pathway. Conclusions/Significance Our observations indicate that annular oligomers are key dynamic intermediates not only in the assembly but also in the disassembly of TTR protofibrils. The balance between annular and more compact forms of aggregation could be relevant for cytotoxicity in amyloidogenic disorders. PMID:22984597

  17. Emerging structural details of transient amyloidoligomers suggest designs for effective small molecule modulators

    NASA Astrophysics Data System (ADS)

    Chandra, Bappaditya; Halder, Swagata; Adler, Juliane; Korn, Alexander; Huster, Daniel; Maiti, Sudipta

    2017-05-01

    Small oligomers are the major toxic species in many amyloid related diseases, but they are difficult to characterize and target. Here we construct tetra-peptides FXFX (X = F/K), designed to exploit cation-π, π-π and hydrophobic interactions to disrupt the critical F19-L34 contact recently found in Aβ40 oligomers. FRFR accelerates Aβ40 aggregation, and strongly inhibits its binding to lipid membranes, which is important in the context of toxicity. FKFK lacks both of these effects, which correlates with the weaker interaction of K with aromatic residues. Thus it appears possible to tune specific contacts in the oligomer and effectively change its properties.

  18. The Role of AmyloidOligomers in Toxicity, Propagation, and Immunotherapy

    PubMed Central

    Sengupta, Urmi; Nilson, Ashley N.; Kayed, Rakez

    2016-01-01

    The incidence of Alzheimer's disease (AD) is growing every day and finding an effective treatment is becoming more vital. Amyloid-β (Aβ) has been the focus of research for several decades. The recent shift in the Aβ cascade hypothesis from all Aβ to small soluble oligomeric intermediates is directing the search for therapeutics towards the toxic mediators of the disease. Targeting the most toxic oligomers may prove to be an effective treatment by preventing their spread. Specific targeting of oligomers has been shown to protect cognition in rodent models. Additionally, the heterogeneity of research on Aβ oligomers may seem contradictory until size and conformation are taken into account. In this review, we will discuss Aβ oligomers and their toxicity in relation to size and conformation as well as their influence on inflammation and the potential of Aβ oligomer immunotherapy. PMID:27211547

  19. Quantitative analysis of co-oligomer formation by amyloid-beta peptide isoforms

    NASA Astrophysics Data System (ADS)

    Iljina, Marija; Garcia, Gonzalo A.; Dear, Alexander J.; Flint, Jennie; Narayan, Priyanka; Michaels, Thomas C. T.; Dobson, Christopher M.; Frenkel, Daan; Knowles, Tuomas P. J.; Klenerman, David

    2016-06-01

    Multiple isoforms of aggregation-prone proteins are present under physiological conditions and have the propensity to assemble into co-oligomers with different properties from self-oligomers, but this process has not been quantitatively studied to date. We have investigated the amyloid-β (Aβ) peptide, associated with Alzheimer’s disease, and the aggregation of its two major isoforms, Aβ40 and Aβ42, using a statistical mechanical modelling approach in combination with in vitro single-molecule fluorescence measurements. We find that at low concentrations of Aβ, corresponding to its physiological abundance, there is little free energy penalty in forming co-oligomers, suggesting that the formation of both self-oligomers and co-oligomers is possible under these conditions. Our model is used to predict the oligomer concentration and size at physiological concentrations of Aβ and suggests the mechanisms by which the ratio of Aβ42 to Aβ40 can affect cell toxicity. An increased ratio of Aβ42 to Aβ40 raises the fraction of oligomers containing Aβ42, which can increase the hydrophobicity of the oligomers and thus promote deleterious binding to the cell membrane and increase neuronal damage. Our results suggest that co-oligomers are a common form of aggregate when Aβ isoforms are present in solution and may potentially play a significant role in Alzheimer’s disease.

  20. Direct Correlation Between Ligand-Induced α-Synuclein Oligomers and Amyloid-like Fibril Growth

    PubMed Central

    Nors Perdersen, Martin; Foderà, Vito; Horvath, Istvan; van Maarschalkerweerd, Andreas; Nørgaard Toft, Katrine; Weise, Christoph; Almqvist, Fredrik; Wolf-Watz, Magnus; Wittung-Stafshede, Pernilla; Vestergaard, Bente

    2015-01-01

    Aggregation of proteins into amyloid deposits is the hallmark of several neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease. The suggestion that intermediate oligomeric species may be cytotoxic has led to intensified investigations of pre-fibrillar oligomers, which are complicated by their transient nature and low population. Here we investigate alpha-synuclein oligomers, enriched by a 2-pyridone molecule (FN075), and the conversion of oligomers into fibrils. As probed by leakage assays, the FN075 induced oligomers potently disrupt vesicles in vitro, suggesting a potential link to disease related degenerative activity. Fibrils formed in the presence and absence of FN075 are indistinguishable on microscopic and macroscopic levels. Using small angle X-ray scattering, we reveal that FN075 induced oligomers are similar, but not identical, to oligomers previously observed during alpha-synuclein fibrillation. Since the levels of FN075 induced oligomers correlate with the amounts of fibrils among different FN075:protein ratios, the oligomers appear to be on-pathway and modeling supports an ‘oligomer stacking model’ for alpha-synuclein fibril elongation. PMID:26020724

  1. Taxifolin inhibits amyloidoligomer formation and fully restores vascular integrity and memory in cerebral amyloid angiopathy.

    PubMed

    Saito, Satoshi; Yamamoto, Yumi; Maki, Takakuni; Hattori, Yorito; Ito, Hideki; Mizuno, Katsuhiko; Harada-Shiba, Mariko; Kalaria, Raj N; Fukushima, Masanori; Takahashi, Ryosuke; Ihara, Masafumi

    2017-04-04

    Cerebral amyloid angiopathy (CAA) induces various forms of cerebral infarcts and hemorrhages from vascular amyloid-β accumulation, resulting in acceleration of cognitive impairment, which is currently untreatable. Soluble amyloid-β protein likely impairs cerebrovascular integrity as well as cognitive function in early stage Alzheimer's disease. Taxifolin, a flavonol with strong anti-oxidative and anti-glycation activities, has been reported to disassemble amyloid-β in vitro but the in vivo relevance remains unknown. Here, we investigated whether taxifolin has therapeutic potential in attenuating CAA, hypothesizing that inhibiting amyloid-β assembly may facilitate its clearance through several elimination pathways. Vehicle- or taxifolin-treated Tg-SwDI mice (commonly used to model CAA) were used in this investigation. Cognitive and cerebrovascular function, as well as the solubility and oligomerization of brain amyloid-β proteins, were investigated. Spatial reference memory was assessed by water maze test. Cerebral blood flow was measured with laser speckle flowmetry and cerebrovascular reactivity evaluated by monitoring cerebral blood flow changes in response to hypercapnia. Significantly reduced cerebrovascular pan-amyloid-β and amyloid-β1-40 accumulation was found in taxifolin-treated Tg-SwDI mice compared to vehicle-treated counterparts (n = 5). Spatial reference memory was severely impaired in vehicle-treated Tg-SwDI mice but normalized after taxifolin treatment, with scoring similar to wild type mice (n = 10-17). Furthermore, taxifolin completely restored decreased cerebral blood flow and cerebrovascular reactivity in Tg-SwDI mice (n = 4-6). An in vitro thioflavin-T assay showed taxifolin treatment resulted in efficient inhibition of amyloid-β1-40 assembly. In addition, a filter trap assay and ELISA showed Tg-SwDI mouse brain homogenates exhibited significantly reduced levels of amyloidoligomers in vivo after taxifolin treatment (n

  2. Amyloid-beta oligomers increase the localization of prion protein at the cell surface.

    PubMed

    Caetano, Fabiana A; Beraldo, Flavio H; Hajj, Glaucia N M; Guimaraes, Andre L; Jürgensen, Sofia; Wasilewska-Sampaio, Ana Paula; Hirata, Pedro H F; Souza, Ivana; Machado, Cleiton F; Wong, Daisy Y-L; De Felice, Fernanda G; Ferreira, Sergio T; Prado, Vania F; Rylett, R Jane; Martins, Vilma R; Prado, Marco A M

    2011-05-01

    In Alzheimer's disease, the amyloid-β peptide (Aβ) interacts with distinct proteins at the cell surface to interfere with synaptic communication. Recent data have implicated the prion protein (PrP(C)) as a putative receptor for Aβ. We show here that Aβ oligomers signal in cells in a PrP(C)-dependent manner, as might be expected if Aβ oligomers use PrP(C) as a receptor. Immunofluorescence, flow cytometry and cell surface protein biotinylation experiments indicated that treatment with Aβ oligomers, but not monomers, increased the localization of PrP(C) at the cell surface in cell lines. These results were reproduced in hippocampal neuronal cultures by labeling cell surface PrP(C). In order to understand possible mechanisms involved with this effect of Aβ oligomers, we used live cell confocal and total internal reflection microscopy in cell lines. Aβ oligomers inhibited the constitutive endocytosis of PrP(C), but we also found that after Aβ oligomer-treatment PrP(C) formed more clusters at the cell surface, suggesting the possibility of multiple effects of Aβ oligomers. Our experiments show for the first time that Aβ oligomers signal in a PrP(C)-dependent way and that they can affect PrP(C) trafficking, increasing its localization at the cell surface.

  3. Pre-amyloid oligomers budding:a metastatic mechanism of proteotoxicity

    NASA Astrophysics Data System (ADS)

    Bernini, Fabrizio; Malferrari, Daniele; Pignataro, Marcello; Bortolotti, Carlo Augusto; di Rocco, Giulia; Lancellotti, Lidia; Brigatti, Maria Franca; Kayed, Rakez; Borsari, Marco; Del Monte, Federica; Castellini, Elena

    2016-10-01

    The pathological hallmark of misfolded protein diseases and aging is the accumulation of proteotoxic aggregates. However, the mechanisms of proteotoxicity and the dynamic changes in fiber formation and dissemination remain unclear, preventing a cure. Here we adopted a reductionist approach and used atomic force microscopy to define the temporal and spatial changes of amyloid aggregates, their modes of dissemination and the biochemical changes that may influence their growth. We show that pre-amyloid oligomers (PAO) mature to form linear and circular protofibrils, and amyloid fibers, and those can break reforming PAO that can migrate invading neighbor structures. Simulating the effect of immunotherapy modifies the dynamics of PAO formation. Anti-fibers as well as anti-PAO antibodies fragment the amyloid fibers, however the fragmentation using anti-fibers antibodies favored the migration of PAO. In conclusion, we provide evidence for the mechanisms of misfolded protein maturation and propagation and the effects of interventions on the resolution and dissemination of amyloid pathology.

  4. Desmin modifications associate with amyloid-like oligomers deposition in heart failure.

    PubMed

    Agnetti, Giulio; Halperin, Victoria L; Kirk, Jonathan A; Chakir, Khalid; Guo, Yurong; Lund, Linda; Nicolini, Francesco; Gherli, Tiziano; Guarnieri, Carlo; Caldarera, Claudio M; Tomaselli, Gordon F; Kass, David A; Van Eyk, Jennifer E

    2014-04-01

    The ultimate cause of heart failure (HF) is not known to date. The cytoskeletal protein desmin is differentially modified and forms amyloid-like oligomers in HF. We postulated that desmin post-translational modifications (PTMs) could drive aberrant desmin aggregation in HF. Therefore, we identified these PTMs and investigated their impact on desmin amyloidogenicity in human and experimental HF. We detected increased levels of selectively phosphorylated and cleaved desmin in a canine pacing model of dyssynchronous HF (DHF) compared with either controls or animals treated with cardiac resynchronization therapy (CRT). This unique animal model combines clinically relevant features with the possibility of a partly rescued phenotype. We confirmed analogous changes in desmin modifications in human HF and identified two phosphorylation sites within a glycogen synthase kinase 3 (GSK3) consensus sequence. Desmin-positive oligomers were also increased in DHF hearts compared with controls. Their amyloid properties were decreased by treatment with CRT or an anti-amyloid small molecule. Finally, we confirmed GSK3's involvement with desmin phosphorylation using an in vitro model. Based on these findings, we postulate a new mechanism of cardiac toxicity based on the PTM-driven accumulation of desmin amyloid-like oligomers. Phosphorylation and cleavage as well as oligomers formation are reduced by treatment (CRT) indicating a relationship between the three. Finally, the decrease of desmin amyloid-like oligomers with CRT or small molecules points both to a general mechanism of HF based on desmin toxicity that is independent of protein mutations and to novel potential therapies.

  5. Stabilization of neurotoxic Alzheimer amyloidoligomers by protein engineering

    PubMed Central

    Sandberg, Anders; Luheshi, Leila M.; Söllvander, Sofia; Pereira de Barros, Teresa; Macao, Bertil; Knowles, Tuomas P. J.; Biverstål, Henrik; Lendel, Christofer; Ekholm-Petterson, Frida; Dubnovitsky, Anatoly; Lannfelt, Lars; Dobson, Christopher M.; Härd, Torleif

    2010-01-01

    Soluble oligomeric aggregates of the amyloid-β peptide (Aβ) have been implicated in the pathogenesis of Alzheimer’s disease (AD). Although the conformation adopted by Aβ within these aggregates is not known, a β-hairpin conformation is known to be accessible to monomeric Aβ. Here we show that this β-hairpin is a building block of toxic Aβ oligomers by engineering a double-cysteine mutant (called Aβcc) in which the β-hairpin is stabilized by an intramolecular disulfide bond. Aβ40cc and Aβ42cc both spontaneously form stable oligomeric species with distinct molecular weights and secondary-structure content, but both are unable to convert into amyloid fibrils. Biochemical and biophysical experiments and assays with conformation-specific antibodies used to detect Aβ aggregates in vivo indicate that the wild-type oligomer structure is preserved and stabilized in Aβcc oligomers. Stable oligomers are expected to become highly toxic and, accordingly, we find that β-sheet-containing Aβ42cc oligomers or protofibrillar species formed by these oligomers are 50 times more potent inducers of neuronal apoptosis than amyloid fibrils or samples of monomeric wild-type Aβ42, in which toxic aggregates are only transiently formed. The possibility of obtaining completely stable and physiologically relevant neurotoxic Aβ oligomer preparations will facilitate studies of their structure and role in the pathogenesis of AD. For example, here we show how kinetic partitioning into different aggregation pathways can explain why Aβ42 is more toxic than the shorter Aβ40, and why certain inherited mutations are linked to protofibril formation and early-onset AD. PMID:20713699

  6. The Anti-Prion Antibody 15B3 Detects Toxic AmyloidOligomers

    PubMed Central

    Stravalaci, Matteo; Tapella, Laura; Beeg, Marten; Rossi, Alessandro; Joshi, Pooja; Pizzi, Erika; Mazzanti, Michele; Balducci, Claudia; Forloni, Gianluigi; Biasini, Emiliano; Salmona, Mario; Diomede, Luisa; Chiesa, Roberto; Gobbi, Marco

    2016-01-01

    15B3 is a monoclonal IgM antibody that selectively detects pathological aggregates of the prion protein (PrP). We report the unexpected finding that 15B3 also recognizes oligomeric but not monomeric forms of amyloid-β (Aβ)42, an aggregating peptide implicated in the pathogenesis of Alzheimer’s disease (AD). The 15B3 antibody: i) inhibits the binding of synthetic Aβ42 oligomers to recombinant PrP and neuronal membranes; ii) prevents oligomer-induced membrane depolarization; iii) antagonizes the inhibitory effects of oligomers on the physiological pharyngeal contractions of the nematode Caenorhabditis elegans; and iv) counteracts the memory deficits induced by intracerebroventricular injection of Aβ42 oligomers in mice. Thus this antibody binds to pathologically relevant forms of Aβ, and offers a potential research, diagnostic, and therapeutic tool for AD. PMID:27392850

  7. Unmodified and pyroglutamylated amyloid β peptides form hypertoxic hetero-oligomers of unique secondary structure.

    PubMed

    Goldblatt, Greg; Cilenti, Lucia; Matos, Jason O; Lee, Briana; Ciaffone, Nicholas; Wang, Qing X; Tetard, Laurene; Teter, Ken; Tatulian, Suren A

    2017-05-01

    Amyloid β (Aβ) peptide plays a major role in Alzheimer's disease (AD) and occurs in multiple forms, including pyroglutamylated Aβ (AβpE). Identification and characterization of the most cytotoxic Aβ species is necessary for advancement in AD diagnostics and therapeutics. While in brain tissue multiple Aβ species act in combination, structure/toxicity studies and immunotherapy trials have been focused on individual forms of Aβ. As a result, the molecular composition and the structural features of "toxic Aβ oligomers" have remained unresolved. Here, we have used a novel approach, hydration from gas phase coupled with isotope-edited Fourier transform infrared (FTIR) spectroscopy, to identify the prefibrillar assemblies formed by Aβ and AβpE and to resolve the structures of both peptides in combination. The peptides form unusual β-sheet oligomers stabilized by intramolecular H-bonding as opposed to intermolecular H-bonding in the fibrils. Time-dependent morphological changes in peptide assemblies have been visualized by atomic force microscopy. Aβ/AβpE hetero-oligomers exert unsurpassed cytotoxic effect on PC12 cells as compared to oligomers of individual peptides or fibrils. These findings lead to a novel concept that Aβ/AβpE hetero-oligomers, not just Aβ or AβpE oligomers, constitute the main neurotoxic conformation. The hetero-oligomers thus present a new biomarker that may be targeted for development of more efficient diagnostic and immunotherapeutic strategies to combat AD. © 2017 Federation of European Biochemical Societies.

  8. The Role of Initial Oligomers in Amyloid Fibril Formation by Human Stefin B

    PubMed Central

    Taler-Verčič, Ajda; Kirsipuu, Tiina; Friedemann, Merlin; Noormägi, Andra; Polajnar, Mira; Smirnova, Julia; Žnidarič, Magda Tušek; Žganec, Matjaž; Škarabot, Miha; Vilfan, Andrej; Staniforth, Rosemary A.; Palumaa, Peep; Žerovnik, Eva

    2013-01-01

    Oligomers are commonly observed intermediates at the initial stages of amyloid fibril formation. They are toxic to neurons and cause decrease in neural transmission and long-term potentiation. We describe an in vitro study of the initial steps in amyloid fibril formation by human stefin B, which proved to be a good model system. Due to relative stability of the initial oligomers of stefin B, electrospray ionization mass spectrometry (ESI MS) could be applied in addition to size exclusion chromatography (SEC). These two techniques enabled us to separate and detect distinguished oligomers from the monomers: dimers, trimers, tetramers, up to decamers. The amyloid fibril formation process was followed at different pH and temperatures, including such conditions where the process was slow enough to detect the initial oligomeric species at the very beginning of the lag phase and those at the end of the lag phase. Taking into account the results of the lower-order oligomers transformations early in the process, we were able to propose an improved model for the stefin B fibril formation. PMID:24013380

  9. Amyloid Oligomers and Mature Fibrils Prepared from an Innocuous Protein Cause Diverging Cellular Death Mechanisms*

    PubMed Central

    Harte, Níal P.; Klyubin, Igor; McCarthy, Eoin K.; Min, Soyoung; Garrahy, Sarah Ann; Xie, Yongjing; Davey, Gavin P.; Boland, John J.; Rowan, Michael J.; Mok, K. Hun

    2015-01-01

    Despite significant advances, the molecular identity of the cytotoxic species populated during in vivo amyloid formation crucial for the understanding of neurodegenerative disorders is yet to be revealed. In this study lysozyme prefibrillar oligomers and fibrils in both mature and sonicated states have been isolated through an optimized ultrafiltration/ultracentrifugation method and characterized with various optical spectroscopic techniques, atomic force microscopy, and transmission electron microscopy. We examined their level and mode of toxicity on rat pheochromocytoma (PC12) cells in both differentiated and undifferentiated states. We find that oligomers and fibrils display cytotoxic capabilities toward cultured cells in vitro, with oligomers producing elevated levels of cellular injury toward undifferentiated PC12 cells (PC12undiff). Furthermore, dual flow cytometry staining experiments demonstrate that the oligomers and mature fibrils induce divergent cellular death pathways (apoptosis and secondary necrosis, respectively) in these PC12 cells. We have also shown that oligomers but not sonicated mature fibrils inhibit hippocampal long term potentiation, a form of synaptic plasticity implicated in learning and memory, in vivo. We conclude that our in vitro and in vivo findings confer a level of resistance toward amyloid fibrils, and that the PC 12-based comparative cytotoxicity assay can provide insights into toxicity differences between differently aggregated protein species. PMID:26221033

  10. Structural fingerprints and their evolution during oligomeric vs. oligomer-free amyloid fibril growth

    NASA Astrophysics Data System (ADS)

    Foley, Joseph; Hill, Shannon E.; Miti, Tatiana; Mulaj, Mentor; Ciesla, Marissa; Robeel, Rhonda; Persichilli, Christopher; Raynes, Rachel; Westerheide, Sandy; Muschol, Martin

    2013-09-01

    Deposits of fibrils formed by disease-specific proteins are the molecular hallmark of such diverse human disorders as Alzheimer's disease, type II diabetes, or rheumatoid arthritis. Amyloid fibril formation by structurally and functionally unrelated proteins exhibits many generic characteristics, most prominently the cross β-sheet structure of their mature fibrils. At the same time, amyloid formation tends to proceed along one of two separate assembly pathways yielding either stiff monomeric filaments or globular oligomers and curvilinear protofibrils. Given the focus on oligomers as major toxic species, the very existence of an oligomer-free assembly pathway is significant. Little is known, though, about the structure of the various intermediates emerging along different pathways and whether the pathways converge towards a common or distinct fibril structures. Using infrared spectroscopy we probed the structural evolution of intermediates and late-stage fibrils formed during in vitro lysozyme amyloid assembly along an oligomeric and oligomer-free pathway. Infrared spectroscopy confirmed that both pathways produced amyloid-specific β-sheet peaks, but at pathway-specific wavenumbers. We further found that the amyloid-specific dye thioflavin T responded to all intermediates along either pathway. The relative amplitudes of thioflavin T fluorescence responses displayed pathway-specific differences and could be utilized for monitoring the structural evolution of intermediates. Pathway-specific structural features obtained from infrared spectroscopy and Thioflavin T responses were identical for fibrils grown at highly acidic or at physiological pH values and showed no discernible effects of protein hydrolysis. Our results suggest that late-stage fibrils formed along either pathway are amyloidogenic in nature, but have distinguishable structural fingerprints. These pathway-specific fingerprints emerge during the earliest aggregation events and persist throughout the

  11. Novel demonstration of amyloidoligomers in sporadic inclusion-body myositis muscle fibers.

    PubMed

    Nogalska, Anna; D'Agostino, Carla; Engel, W King; Klein, William L; Askanas, Valerie

    2010-11-01

    Accumulation of amyloid-β (Aβ) within muscle fibers has been considered an upstream step in the development of the s-IBM pathologic phenotype. Aβ42, which is considered more cytotoxic than Aβ40 and has a higher propensity to oligomerize, is preferentially increased in s-IBM muscle fibers. In Alzheimer disease (AD), low-molecular weight Aβ oligomers and toxic oligomers, also referred to as "Aβ-Derived Diffusible Ligands" (ADDLs), are considered strongly cytotoxic and proposed to play an important pathogenic role. ADDLs have been shown to be increased in AD brain. We now report for the first time that in s-IBM muscle biopsies Aβ-dimer, -trimer, and -tetramer are identifiable by immunoblots. While all the s-IBM samples we studied had Aβ-oligomers, their molecular weights and intensity varied between the patient samples. None of the control muscle biopsies had Aβ oligomers. Dot-immunoblots using highly specific anti-ADDL monoclonal antibodies also showed highly increased ADDLs in all s-IBM biopsies studied, while controls were negative. By immunofluorescence, in some of the abnormal s-IBM muscle fibers ADDLs were accumulated in the form of plaque-like inclusions, and were often increased diffusely in very small fibers. Normal and disease-controls were negative. By gold-immuno-electron microscopy, ADDL-immunoreactivities were in close proximity to 6-10 nm amyloid-like fibrils, and also were immunodecorating amorphous and floccular material. In cultured human muscle fibers, we found that inhibition of autophagy led to the accumulation of Aβ oligomers. This novel demonstration of Aβ42 oligomers in s-IBM muscle biopsy provides additional evidence that intra-muscle fiber accumulation of Aβ42 oligomers in s-IBM may contribute importantly to s-IBM pathogenic cascade.

  12. Amyloid Oligomer Neurotoxicity, Calcium Dysregulation, and Lipid Rafts

    PubMed Central

    Malchiodi-Albedi, Fiorella; Paradisi, Silvia; Matteucci, Andrea; Frank, Claudio; Diociaiuti, Marco

    2011-01-01

    Amyloid proteins constitute a chemically heterogeneous group of proteins, which share some biophysical and biological characteristics, the principal of which are the high propensity to acquire an incorrect folding and the tendency to aggregate. A number of diseases are associated with misfolding and aggregation of proteins, although only in some of them—most notably Alzheimer's disease (AD) and transmissible spongiform encephalopathies (TSEs)—a pathogenetic link with misfolded proteins is now widely recognized. Lipid rafts (LRs) have been involved in the pathophysiology of diseases associated with protein misfolding at several levels, including aggregation of misfolded proteins, amyloidogenic processing, and neurotoxicity. Among the pathogenic misfolded proteins, the AD-related protein amyloid β (Aβ) is by far the most studied protein, and a large body of evidence has been gathered on the role played by LRs in Aβ pathogenicity. However, significant amount of data has also been collected for several other amyloid proteins, so that their ability to interact with LRs can be considered an additional, shared feature characterizing the amyloid protein family. In this paper, we will review the evidence on the role of LRs in the neurotoxicity of huntingtin, α-synuclein, prion protein, and calcitonin. PMID:21331330

  13. The relationship of a serum protein, C1t, to a common nonfibrillar constituent of amyloid (P component) as revealed by immunohistochemical studies.

    PubMed Central

    Katz, A.; Weicker-Thorne, J.; Painter, R. H.

    1977-01-01

    C1t, a serum protein isolated by affinity chromatography on Sepharose bears a remarkable ultrastructural and physiocochemical resemblance to P component, a common constituent of amyloid. This study provides further evidence for their similarity by the demonstration of immunologic identity and by the presence of C1t in amyloid deposits of various types using immunoperoxidase and immunofluorescent techniques. In addition, subcomponents of C1, as well as C3, C4, C5, and properdin, were demonstrated to a limited extent. The possible role of C1t/P component in amyloidogenesis is discussed in the light of recent advances in our knowledge of the nature of amyloid substance. Images Figure 2 Figure 6 Figure 3 Figure 4 Figure 5 Figure 1 PMID:329683

  14. Fibrillar seeds alleviate amyloid-β cytotoxicity by omitting formation of higher-molecular-weight oligomers.

    PubMed

    Wu, Wei-hui; Liu, Qian; Sun, Xun; Yu, Ji-sheng; Zhao, De-sheng; Yu, Ye-ping; Luo, Jun-jie; Hu, Jia; Yu, Zhi-wu; Zhao, Yu-fen; Li, Yan-mei

    2013-09-27

    Amyloid-β (Aβ) peptides can exist in distinct forms including monomers, oligomers and fibrils, consisting of increased numbers of monomeric units. Among these, Aβ oligomers are implicated as the primary toxic species as pointed by multiple lines of evidence. It has been suggested that toxicity could be rendered by the soluble higher-molecular-weight (high-n) Aβ oligomers. Yet, the most culpable form in the pathogenesis of Alzheimer's disease (AD) remains elusive. Moreover, the potential interaction among the insoluble fibrils that have been excluded from the responsible aggregates in AD development, Aβ monomers and high-n oligomers is undetermined. Here, we report that insoluble Aβ fibrillar seeds can interact with Aβ monomers at the stoichiometry of 1:2 (namely, each Aβ molecule of seed can bind to two Aβ monomers at a time) facilitating the fibrillization by omitting the otherwise mandatory formation of the toxic high-n oligomers during the fibril maturation. As a result, the addition of exogenous Aβ fibrillar seeds is seen to rescue neuronal cells from Aβ cytotoxicity presumably exerted by high-n oligomers, suggesting an unexpected protective role of Aβ fibrillar seeds. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. Soluble Prion Protein Binds Isolated Low Molecular Weight AmyloidOligomers Causing Cytotoxicity Inhibition.

    PubMed

    Williams, Thomas L; Choi, Jin-Kyu; Surewicz, Krystyna; Surewicz, Witold K

    2015-12-16

    A growing number of observations indicate that soluble amyloid-β (Aβ) oligomers play a major role in Alzheimer's disease. Recent studies strongly suggest that at least some of the neurotoxic effects of these oligomers are mediated by cellular, membrane-anchored prion protein and that Aβ neurotoxicity can be inhibited by soluble recombinant prion protein (rPrP) and its fragments. However, the mechanism by which rPrP interacts with Aβ oligomers and prevents their toxicity is largely unknown, and studies in this regard are hindered by the large structural heterogeneity of Aβ oligomers. To overcome this difficulty, here we used photoinduced cross-linking of unmodified proteins (PICUP) to isolate well-defined oligomers of Aβ42 and characterize these species with regard to their cytotoxicity and interaction with rPrP, as well the mechanism by which rPrP inhibits Aβ42 cytotoxicity. Our data shows that the addition of rPrP to the assembling Aβ42 results in a shift in oligomer size distribution, decreasing the population of toxic tetramers and higher order oligomers and increasing the population of nontoxic (and possibly neuroprotective) monomers. Isolated oligomeric species of Aβ42 are cytotoxic to primary neurons and cause permeation of model lipid bilayers. These toxic effects, which are oligomer size-dependent, can be inhibited by the addition of rPrP, and our data suggest potential mechanisms of this inhibitory action. This insight should help in current efforts to develop PrP-based therapeutics for Alzheimer's disease.

  16. Amyloidoligomers are sequestered by both intracellular and extracellular chaperones

    PubMed Central

    Narayan, Priyanka; Meehan, Sarah; Carver, John A.; Wilson, Mark R.; Dobson, Christopher M.; Klenerman, David

    2016-01-01

    The aberrant aggregation of the amyloid-β peptide into β-sheet rich, fibrillar structures proceeds via a heterogeneous ensemble of oligomeric intermediates that have been associated with neurotoxicity in Alzheimer’s disease (AD). Of particular interest in this context are the mechanisms by which molecular chaperones, part of the primary biological defenses against protein misfolding, influence Aβ aggregation. We have used single-molecule fluorescence techniques to compare the interactions between distinct aggregation states (monomers, oligomers, amyloid fibrils) of the AD-associated amyloid-β(1-40) peptide, and two molecular chaperones, both of which are upregulated in the brains of patients with AD and have been found colocalized with Aβ in senile plaques. One of the chaperones, αB-crystallin, is primarily found inside cells while the other, clusterin, is predominantly located in the extracellular environment. We find that both chaperones bind to misfolded oligomeric species and form long-lived complexes thereby preventing both their further growth into fibrils and their dissociation. From these studies, we conclude that these chaperones have a common mechanism of action based on sequestering Aβ oligomers. This conclusion suggests that these chaperones, both of which are ATP-independent, are able to inhibit potentially pathogenic Aβ oligomer-associated processes whether they occur in the extracellular or intracellular environment. PMID:23106396

  17. Does Thioflavin-T Detect Oligomers Formed During Amyloid Fibril Assembly

    NASA Astrophysics Data System (ADS)

    Persichilli, Christopher; Hill, Shannon E.; Mast, Jason; Muschol, Martin

    2011-03-01

    Recent results have shown that oligomeric intermediates of amyloid fibril assembly represent the main toxic species in disorders such as Alzheimer's disease and type II diabetes. Thioflavin-T (ThT) is among the most commonly used indicator dyes for mature amyloid fibrils in vitro. We used ThT to monitor amyloid fibril formation of lysozyme (HEWL), and correlated ThT fluorescence to concurrent dynamic light scattering and atomic force microscopy measurements. Specifically, we tested the ability of ThT to discern among oligomer-free vs. oligomeric fibril assembly pathways. We found that ThT fluorescence did not detect oligomer growth; however, fluorescence increases did coincide with the formation of monomeric filaments in the oligomer-free assembly pathway. This implies that ThT fluorescence is not generally suitable for the detection of oligomeric intermediates. The results further suggest different internal structures for oligomeric vs. monomeric filaments. This research was supported, in part, by funding through the Byrd Alzheimer's Institute (ARG-2007-22) and the BITT-Florida Center of Excellence for M.M., an NSF-REU grant (DMR-1004873) for C. P. and an NSF-IGERT fellowship for S.H.

  18. A strategy for designing a peptide probe for detection of β-amyloid oligomers.

    PubMed

    Hu, Yang; Su, Baihao; Kim, Chung-Sei; Hernandez, Michael; Rostagno, Agueda; Ghiso, Jorge; Kim, Jin Ryoun

    2010-11-22

    Aggregation of β-amyloid (Aβ) is implicated in the pathology of Alzheimer's disease. Development of a robust strategy to detect Aβ oligomeric intermediates, which have been identified as significant toxic agents, would be highly beneficial in the screening of drug candidates as well as enhancing our understanding of Aβ oligomerization. Rapid, specific and quantitative detection, currently unavailable, would be highly preferred for accurate and reliable probing of transient Aβ oligomers. Here, we report the development of a novel peptide probe, PG46, based on the nature of Aβ self-assembly and the conformation-sensitive fluorescence of the biarsenical dye, FlAsH. PG46 was found to bind to Aβ oligomers and displayed an increase in FlAsH fluorescence upon binding. No such event was observed when PG46 was co-incubated with Aβ low-molecular-weight species or Aβ fibrils. Aβ oligomer detection was fast, and occurred within one hour without any additional sample incubation or preparation. We anticipate that the development of a strategy for detection of amyloid oligomers described in this study will be directly relevant to a host of other amyloidogenic proteins.

  19. Structural Insights into Amyloid Oligomers of the Parkinson Disease-related Protein α-Synuclein*

    PubMed Central

    Gallea, J. Ignacio; Celej, M. Soledad

    2014-01-01

    The presence of intraneuronal deposits mainly formed by amyloid fibrils of the presynaptic protein α-synuclein (AS) is a hallmark of Parkinson disease. Currently, neurotoxicity is attributed to prefibrillar oligomeric species rather than the insoluble aggregates, although their mechanisms of toxicity remain elusive. Structural details of the supramolecular organization of AS oligomers are critically needed to decipher the structure-toxicity relationship underlying their pathogenicity. In this study, we employed site-specific fluorescence to get a deeper insight into the internal architecture of AS oligomeric intermediates. We demonstrate that AS oligomers are ordered assemblies possessing a well defined pattern of intermolecular contacts. Some of these contacts involve regions that form the β-sheet core in the fibrillar state, although their spatial arrangement may differ in the two aggregated forms. However, even though the two termini are excluded from the fibrillar core, they are engaged in a number of intermolecular interactions within the oligomer. Therefore, substantial structural remodeling of early oligomeric interactions is essential for fibril growth. The intermolecular contacts identified in AS oligomers can serve as targets for the rational design of anti-amyloid compounds directed at preventing oligomeric interactions/reorganizations. PMID:25143382

  20. Nature of the Amyloid-β Monomer and the Monomer-Oligomer Equilibrium

    PubMed Central

    Nag, Suman; Sarkar, Bidyut; Bandyopadhyay, Arkarup; Sahoo, Bankanidhi; Sreenivasan, Varun K. A.; Kombrabail, Mamata; Muralidharan, Chandrakesan; Maiti, Sudipta

    2011-01-01

    The monomer to oligomer transition initiates the aggregation and pathogenic transformation of Alzheimer amyloid-β (Aβ) peptide. However, the monomeric state of this aggregation-prone peptide has remained beyond the reach of most experimental techniques, and a quantitative understanding of this transition is yet to emerge. Here, we employ single-molecule level fluorescence tools to characterize the monomeric state and the monomer-oligomer transition at physiological concentrations in buffers mimicking the cerebrospinal fluid (CSF). Our measurements show that the monomer has a hydrodynamic radius of 0.9 ± 0.1 nm, which confirms the prediction made by some of the in silico studies. Surprisingly, at equilibrium, both Aβ40 and Aβ42 remain predominantly monomeric up to 3 μm, above which it forms large aggregates. This concentration is much higher than the estimated concentrations in the CSF of either normal or diseased brains. If Aβ oligomers are present in the CSF and are the key agents in Alzheimer pathology, as is generally believed, then these must be released in the CSF as preformed entities. Although the oligomers are thermodynamically unstable, we find that a large kinetic barrier, which is mostly entropic in origin, strongly impedes their dissociation. Thermodynamic principles therefore allow the development of a pharmacological agent that can catalytically convert metastable oligomers into nontoxic monomers. PMID:21349839

  1. Synaptotoxic amyloidoligomers: a molecular basis for the cause, diagnosis, and treatment of Alzheimer's disease?

    PubMed

    Klein, William L

    2013-01-01

    The oligomer hypothesis for Alzheimer's disease (AD)was introduced in 1998. It was based on evidence that oligomers could exist free of amyloid fibrils, that fibril-free oligomer solutions rapidly inhibited long term potentiation, and that oligomers ultimately caused a highly selective nerve cell death. Fibrils no longer were the only toxins made by amyloid-β (Aβ), and likely not the most important ones. Oligomers provided a new basis for instigating AD. Since introduction of the hypothesis, more than 1,500 articles on oligomers have been published. Articles for this review were selected for contributions to oligomer theory at three different levels. The first set demonstrated new aspects of oligomer pathobiology in cell models, showing that exposure of neurons to oligomers is sufficient to cause key features of AD neuropathology. The second set confirmed the relationship between oligomers and salient AD neuropathology in animal models, consistent with other in vivo studies that overall have substantiated cell-based discoveries. The third set developed strategies for therapeutic targeting of oligomers, introducing both small molecule and antibody-based approaches. These and related findings from many groups have helped establish oligomers as central to the mechanism of AD pathogenesis. Comprising a ligand-based attack on specific synapses, the action of toxic oligomers gives a molecular basis to account for key features of AD neuropathology and to explain why early disease targets memory. Although there still is no effective treatment for AD, insights over the past five years raise hopes that new approaches targeting Aβ oligomers could finally bring disease-modifying therapeutics.

  2. Discrete Molecular Dynamics Study of Oligomer Formation by N-Terminally Truncated Amyloid β-Protein

    PubMed Central

    Meral, Derya; Urbanc, Brigita

    2013-01-01

    In Alzheimer’s disease (AD), amyloid β-protein (Aβ) self–assembles into toxic oligomers. Of the two predominant Aβ alloforms, Aβ1–40 and Aβ1–42, the latter is particularly strongly linked to AD. N-terminally truncated and pyroglutamated Aβ peptides were recently shown to seed Aβ aggregation and contribute significantly to Aβ–mediated toxicity, yet their folding and assembly were not explored computationally. Discrete molecular dynamics (DMD) approach previously captured in vitro–derived distinct Aβ1–40 and Aβ1–42 oligomer size distributions and predicted that the more toxic Aβ1–42 oligomers had more flexible and solvent exposed N-termini than Aβ1–40 oligomers. Here, we examined oligomer formation of Aβ3–40, Aβ3–42, Aβ11–40, and Aβ11–42 by the DMD approach. The four N-terminally truncated peptides showed increased oligomerization propensity relative to the full–length peptides, consistent with in vitro findings. Conformations formed by Aβ3–40/42 had significantly more flexible and solvent–exposed N-termini than Aβ1–40/42 conformations. In contrast, in Aβ11–40/42 conformations the N-termini formed more contacts and were less accessible to the solvent. The compactness of the Aβ11–40/42 conformations was in part facilitated by Val12. Two single amino acid substitutions that reduced and abolished hydrophobicity at position 12, respectively, resulted in a proportionally increased structural variability. Our results suggest that Aβ11–40 and Aβ11–42 oligomers might be less toxic than Aβ1–40 and Aβ1–42 oligomers and offer a plausible explanation for the experimentally–observed increased toxicity of Aβ3–40 and Aβ3–42 and their pyroglutamated forms. PMID:23500806

  3. Humanin Specifically Interacts with AmyloidOligomers and Counteracts Their in vivo Toxicity.

    PubMed

    Romeo, Margherita; Stravalaci, Matteo; Beeg, Marten; Rossi, Alessandro; Fiordaliso, Fabio; Corbelli, Alessandro; Salmona, Mario; Gobbi, Marco; Cagnotto, Alfredo; Diomede, Luisa

    2017-03-06

    The 24-residue peptide humanin (HN) has been proposed as peptide-based inhibitors able to interact directly with amyloid-β (Aβ) oligomers and interfere with the formation and/or biological properties of toxic Aβ species. When administered exogenously HN, or its synthetic S14G-derivative (HNG), exerted multiple cytoprotective effects, counteracting the Aβ-induced toxicity. Whether these peptides interact directly with Aβ, particularly with the soluble oligomeric assemblies, remains largely unknown. We here investigated the ability of HN and HNG to interact directly with highly aggregating Aβ42, and interfere with the formation and toxicity of its oligomers. Experiments were run in cell-free conditions and in vivo in a transgenic C. elegans strain in which the Aβ toxicity was specifically due to oligomeric species. Thioflavin-T assay indicated that both HN and HNG delay the formation and reduce the final amount of Aβ42 fibrils. In vitro surface plasmon resonance studies indicated that they interact with Aβ42 oligomers favoring the formation of amorphous larger assemblies, observed with turbidity and electron microscopy. In vivo studies indicated that both HN and HNG decrease the relative abundance of A11-positive prefibrillar oligomers as well as OC-positive fibrillar oligomers and had similar protective effects. However, while HN possibly decreased the oligomers by promoting their assembly into larger aggregates, the reduction of oligomers caused by HNG can be ascribed to a marked decrease of the total Aβ levels, likely the consequence of the HNG-induced overexpression of the Aβ-degrading enzyme neprilysin. These findings provide information on the mechanisms underlying the anti-oligomeric effects of HN and HNG and illustrate the role of S14G substitution in regulating the in vivo mechanism of action.

  4. Natural Amyloid-Beta Oligomers Acutely Impair the Formation of a Contextual Fear Memory in Mice

    PubMed Central

    Kittelberger, Kara A.; Piazza, Fabrizio; Tesco, Giuseppina; Reijmers, Leon G.

    2012-01-01

    Memory loss is one of the hallmark symptoms of Alzheimer's disease (AD). It has been proposed that soluble amyloid-beta (Abeta) oligomers acutely impair neuronal function and thereby memory. We here report that natural Abeta oligomers acutely impair contextual fear memory in mice. A natural Abeta oligomer solution containing Abeta monomers, dimers, trimers, and tetramers was derived from the conditioned medium of 7PA2 cells, a cell line that expresses human amyloid precursor protein containing the Val717Phe familial AD mutation. As a control we used 7PA2 conditioned medium from which Abeta oligomers were removed through immunodepletion. Separate groups of mice were injected with Abeta and control solutions through a cannula into the lateral brain ventricle, and subjected to fear conditioning using two tone-shock pairings. One day after fear conditioning, mice were tested for contextual fear memory and tone fear memory in separate retrieval trials. Three experiments were performed. For experiment 1, mice were injected three times: 1 hour before and 3 hours after fear conditioning, and 1 hour before context retrieval. For experiments 2 and 3, mice were injected a single time at 1 hour and 2 hours before fear conditioning respectively. In all three experiments there was no effect on tone fear memory. Injection of Abeta 1 hour before fear conditioning, but not 2 hours before fear conditioning, impaired the formation of a contextual fear memory. In future studies, the acute effect of natural Abeta oligomers on contextual fear memory can be used to identify potential mechanisms and treatments of AD associated memory loss. PMID:22238679

  5. Amyloid beta oligomers induce impairment of neuronal insulin receptors.

    PubMed

    Zhao, Wei-Qin; De Felice, Fernanda G; Fernandez, Sara; Chen, Hui; Lambert, Mary P; Quon, Michael J; Krafft, Grant A; Klein, William L

    2008-01-01

    Recent studies have indicated an association between Alzheimer's disease (AD) and central nervous system (CNS) insulin resistance. However, the cellular mechanisms underlying the link between these two pathologies have not been elucidated. Here we show that signal transduction by neuronal insulin receptors (IR) is strikingly sensitive to disruption by soluble Abeta oligomers (also known as ADDLs). ADDLs are known to accumulate in AD brain and have recently been implicated as primary candidates for initiating deterioration of synapse function, composition, and structure. Using mature cultures of hippocampal neurons, a preferred model for studies of synaptic cell biology, we found that ADDLs caused a rapid and substantial loss of neuronal surface IRs specifically on dendrites bound by ADDLs. Removal of dendritic IRs was associated with increased receptor immunoreactivity in the cell body, indicating redistribution of the receptors. The neuronal response to insulin, measured by evoked IR tyrosine autophosphorylation, was greatly inhibited by ADDLs. Inhibition also was seen with added glutamate or potassium-induced depolarization. The effects on IR function were completely blocked by NMDA receptor antagonists, tetrodotoxin, and calcium chelator BAPTA-AM. Downstream from the IR, ADDLs induced a phosphorylation of Akt at serine473, a modification associated with neurodegenerative and insulin resistance diseases. These results identify novel factors that affect neuronal IR signaling and suggest that insulin resistance in AD brain is a response to ADDLs, which disrupt insulin signaling and may cause a brain-specific form of diabetes as part of an overall pathogenic impact on CNS synapses.

  6. Large Soluble Oligomers of Amyloid β-Protein from Alzheimer Brain Are Far Less Neuroactive Than the Smaller Oligomers to Which They Dissociate.

    PubMed

    Yang, Ting; Li, Shaomin; Xu, Huixin; Walsh, Dominic M; Selkoe, Dennis J

    2017-01-04

    Soluble oligomers of amyloid β-protein (oAβ) isolated from the brains of Alzheimer's disease (AD) patients have been shown experimentally (in the absence of amyloid plaques) to impair hippocampal synaptic plasticity, decrease synapses, induce tau hyperphosphorylation and neuritic dystrophy, activate microglial inflammation, and impair memory in normal adult rodents. Nevertheless, there has been controversy about what types of oligomers actually confer these AD-like phenotypes. Here, we show that the vast majority of soluble Aβ species obtained from brains of humans who died with confirmed AD elute at high molecular weight (HMW) on nondenaturing size-exclusion chromatography. These species have little or no cytotoxic activity in several bioassays. However, incubation of HMW oAβ in mildly alkaline buffer led to their quantitative dissociation into low molecular weight oligomers (∼8-70 kDa), and these were now far more bioactive: they impaired hippocampal LTP, decreased neuronal levels of β2-adrenergic receptors, and activated microglia in wt mice in vivo Thus, most soluble Aβ assemblies in AD cortex are large and inactive but under certain circumstances can dissociate into smaller, highly bioactive species. Insoluble amyloid plaques likely sequester soluble HMW oligomers, limiting their potential to dissociate. We conclude that conditions that destabilize HMW oligomers or retard the sequestration of their smaller, more bioactive components are important drivers of Aβ toxicity. Selectively targeting these small, cytotoxic forms should be therapeutically beneficial.

  7. Soluble amyloidoligomers as synaptotoxins leading to cognitive impairment in Alzheimer’s disease

    PubMed Central

    Ferreira, Sergio T.; Lourenco, Mychael V.; Oliveira, Mauricio M.; De Felice, Fernanda G.

    2015-01-01

    Alzheimer’s disease (AD) is the most common form of dementia in the elderly, and affects millions of people worldwide. As the number of AD cases continues to increase in both developed and developing countries, finding therapies that effectively halt or reverse disease progression constitutes a major research and public health challenge. Since the identification of the amyloid-β peptide (Aβ) as the major component of the amyloid plaques that are characteristically found in AD brains, a major effort has aimed to determine whether and how Aβ leads to memory loss and cognitive impairment. A large body of evidence accumulated in the past 15 years supports a pivotal role of soluble Aβ oligomers (AβOs) in synapse failure and neuronal dysfunction in AD. Nonetheless, a number of basic questions, including the exact molecular composition of the synaptotoxic oligomers, the identity of the receptor(s) to which they bind, and the signaling pathways that ultimately lead to synapse failure, remain to be definitively answered. Here, we discuss recent advances that have illuminated our understanding of the chemical nature of the toxic species and the deleterious impact they have on synapses, and have culminated in the proposal of an Aβ oligomer hypothesis for Alzheimer’s pathogenesis. We also highlight outstanding questions and challenges in AD research that should be addressed to allow translation of research findings into effective AD therapies. PMID:26074767

  8. Synaptic AmyloidOligomers Precede p-Tau and Differentiate High Pathology Control Cases

    PubMed Central

    Bilousova, Tina; Miller, Carol A.; Poon, Wayne W.; Vinters, Harry V.; Corrada, Maria; Kawas, Claudia; Hayden, Eric Y.; Teplow, David B.; Glabe, Charles; Albay, Ricardo; Cole, Gregory M.; Teng, Edmond; Gylys, Karen H.

    2017-01-01

    Amyloid-β (Aβ) and hyperphosphorylated tau (p-tau) aggregates form the two discrete pathologies of Alzheimer disease (AD), and oligomeric assemblies of each protein are localized to synapses. To determine the sequence by which pathology appears in synapses, Aβ and p-tau were quantified across AD disease stages in parietal cortex. Nondemented cases with high levels of AD-related pathology were included to determine factors that confer protection from clinical symptoms. Flow cytometric analysis of synaptosome preparations was used to quantify Aβ and p-tau in large populations of individual synaptic terminals. Soluble Aβ oligomers were assayed by a single antibody sandwich enzyme-linked immunosorbent assay. Total in situ Aβ was elevated in patients with early- and late-stage AD dementia, but not in high pathology nondemented controls compared with age-matched normal controls. However, soluble Aβ oligomers were highest in early AD synapses, and this assay distinguished early AD cases from high pathology controls. Overall, synapse-associated p-tau did not increase until late-stage disease in human and transgenic rat cortex, and p-tau was elevated in individual Aβ-positive synaptosomes in early AD. These results suggest that soluble oligomers in surviving neocortical synaptic terminals are associated with dementia onset and suggest an amyloid cascade hypothesis in which oligomeric Aβ drives phosphorylated tau accumulation and synaptic spread. These results indicate that antiamyloid therapies will be less effective once p-tau pathology is developed. PMID:26718979

  9. An Account of Amyloid Oligomers: Facts and Figures Obtained from Experiments and Simulations.

    PubMed

    Nagel-Steger, Luitgard; Owen, Michael C; Strodel, Birgit

    2016-04-15

    The deposition of amyloid in brain tissue in the context of neurodegenerative diseases involves the formation of intermediate species-termed oligomers-of lower molecular mass and with structures that deviate from those of mature amyloid fibrils. Because these oligomers are thought to be primarily responsible for the subsequent disease pathogenesis, the elucidation of their structure is of enormous interest. Nevertheless, because of the high aggregation propensity and the polydispersity of oligomeric species formed by the proteins or peptides in question, the preparation of appropriate samples for high-resolution structural methods has proven to be rather difficult. This is why theoretical approaches have been of particular importance in gaining insights into possible oligomeric structures for some time. Only recently has it been possible to achieve some progress with regard to the experimentally based structural characterization of defined oligomeric species. Here we discuss how theory and experiment are used to determine oligomer structures and what can be done to improve the integration of the two disciplines. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Pre-amyloid oligomers budding:a metastatic mechanism of proteotoxicity

    PubMed Central

    Bernini, Fabrizio; Malferrari, Daniele; Pignataro, Marcello; Bortolotti, Carlo Augusto; Di Rocco, Giulia; Lancellotti, Lidia; Brigatti, Maria Franca; Kayed, Rakez; Borsari, Marco; del Monte, Federica; Castellini, Elena

    2016-01-01

    The pathological hallmark of misfolded protein diseases and aging is the accumulation of proteotoxic aggregates. However, the mechanisms of proteotoxicity and the dynamic changes in fiber formation and dissemination remain unclear, preventing a cure. Here we adopted a reductionist approach and used atomic force microscopy to define the temporal and spatial changes of amyloid aggregates, their modes of dissemination and the biochemical changes that may influence their growth. We show that pre-amyloid oligomers (PAO) mature to form linear and circular protofibrils, and amyloid fibers, and those can break reforming PAO that can migrate invading neighbor structures. Simulating the effect of immunotherapy modifies the dynamics of PAO formation. Anti-fibers as well as anti-PAO antibodies fragment the amyloid fibers, however the fragmentation using anti-fibers antibodies favored the migration of PAO. In conclusion, we provide evidence for the mechanisms of misfolded protein maturation and propagation and the effects of interventions on the resolution and dissemination of amyloid pathology. PMID:27775057

  11. Alzheimer's therapeutics targeting amyloid beta 1-42 oligomers II: Sigma-2/PGRMC1 receptors mediate Abeta 42 oligomer binding and synaptotoxicity.

    PubMed

    Izzo, Nicholas J; Xu, Jinbin; Zeng, Chenbo; Kirk, Molly J; Mozzoni, Kelsie; Silky, Colleen; Rehak, Courtney; Yurko, Raymond; Look, Gary; Rishton, Gilbert; Safferstein, Hank; Cruchaga, Carlos; Goate, Alison; Cahill, Michael A; Arancio, Ottavio; Mach, Robert H; Craven, Rolf; Head, Elizabeth; LeVine, Harry; Spires-Jones, Tara L; Catalano, Susan M

    2014-01-01

    Amyloid beta (Abeta) 1-42 oligomers accumulate in brains of patients with Mild Cognitive Impairment (MCI) and disrupt synaptic plasticity processes that underlie memory formation. Synaptic binding of Abeta oligomers to several putative receptor proteins is reported to inhibit long-term potentiation, affect membrane trafficking and induce reversible spine loss in neurons, leading to impaired cognitive performance and ultimately to anterograde amnesia in the early stages of Alzheimer's disease (AD). We have identified a receptor not previously associated with AD that mediates the binding of Abeta oligomers to neurons, and describe novel therapeutic antagonists of this receptor capable of blocking Abeta toxic effects on synapses in vitro and cognitive deficits in vivo. Knockdown of sigma-2/PGRMC1 (progesterone receptor membrane component 1) protein expression in vitro using siRNA results in a highly correlated reduction in binding of exogenous Abeta oligomers to neurons of more than 90%. Expression of sigma-2/PGRMC1 is upregulated in vitro by treatment with Abeta oligomers, and is dysregulated in Alzheimer's disease patients' brain compared to age-matched, normal individuals. Specific, high affinity small molecule receptor antagonists and antibodies raised against specific regions on this receptor can displace synthetic Abeta oligomer binding to synaptic puncta in vitro and displace endogenous human AD patient oligomers from brain tissue sections in a dose-dependent manner. These receptor antagonists prevent and reverse the effects of Abeta oligomers on membrane trafficking and synapse loss in vitro and cognitive deficits in AD mouse models. These findings suggest sigma-2/PGRMC1 receptors mediate saturable oligomer binding to synaptic puncta on neurons and that brain penetrant, small molecules can displace endogenous and synthetic oligomers and improve cognitive deficits in AD models. We propose that sigma-2/PGRMC1 is a key mediator of the pathological effects of

  12. Alzheimer's Therapeutics Targeting Amyloid Beta 1–42 Oligomers II: Sigma-2/PGRMC1 Receptors Mediate Abeta 42 Oligomer Binding and Synaptotoxicity

    PubMed Central

    Izzo, Nicholas J.; Xu, Jinbin; Zeng, Chenbo; Kirk, Molly J.; Mozzoni, Kelsie; Silky, Colleen; Rehak, Courtney; Yurko, Raymond; Look, Gary; Rishton, Gilbert; Safferstein, Hank; Cruchaga, Carlos; Goate, Alison; Cahill, Michael A.; Arancio, Ottavio; Mach, Robert H.; Craven, Rolf; Head, Elizabeth; LeVine, Harry; Spires-Jones, Tara L.; Catalano, Susan M.

    2014-01-01

    Amyloid beta (Abeta) 1–42 oligomers accumulate in brains of patients with Mild Cognitive Impairment (MCI) and disrupt synaptic plasticity processes that underlie memory formation. Synaptic binding of Abeta oligomers to several putative receptor proteins is reported to inhibit long-term potentiation, affect membrane trafficking and induce reversible spine loss in neurons, leading to impaired cognitive performance and ultimately to anterograde amnesia in the early stages of Alzheimer's disease (AD). We have identified a receptor not previously associated with AD that mediates the binding of Abeta oligomers to neurons, and describe novel therapeutic antagonists of this receptor capable of blocking Abeta toxic effects on synapses in vitro and cognitive deficits in vivo. Knockdown of sigma-2/PGRMC1 (progesterone receptor membrane component 1) protein expression in vitro using siRNA results in a highly correlated reduction in binding of exogenous Abeta oligomers to neurons of more than 90%. Expression of sigma-2/PGRMC1 is upregulated in vitro by treatment with Abeta oligomers, and is dysregulated in Alzheimer's disease patients' brain compared to age-matched, normal individuals. Specific, high affinity small molecule receptor antagonists and antibodies raised against specific regions on this receptor can displace synthetic Abeta oligomer binding to synaptic puncta in vitro and displace endogenous human AD patient oligomers from brain tissue sections in a dose-dependent manner. These receptor antagonists prevent and reverse the effects of Abeta oligomers on membrane trafficking and synapse loss in vitro and cognitive deficits in AD mouse models. These findings suggest sigma-2/PGRMC1 receptors mediate saturable oligomer binding to synaptic puncta on neurons and that brain penetrant, small molecules can displace endogenous and synthetic oligomers and improve cognitive deficits in AD models. We propose that sigma-2/PGRMC1 is a key mediator of the pathological effects of

  13. Tetracycline and its analogues protect Caenorhabditis elegans from β amyloid-induced toxicity by targeting oligomers.

    PubMed

    Diomede, Luisa; Cassata, Giuseppe; Fiordaliso, Fabio; Salio, Monica; Ami, Diletta; Natalello, Antonino; Doglia, Silvia Maria; De Luigi, Ada; Salmona, Mario

    2010-11-01

    The accumulation and deposition of amyloid beta (Aβ) peptide in extracellular dense plaques in the brain is a key phase in Alzheimer's disease (AD). Small oligomeric forms of Aβ are responsible for the toxicity and the early cognitive impairment observed in patients before the amyloid plaque deposits appear. It is essential for the development of an efficient cure for AD to identify compounds that interfere with Aβ aggregation, counteracting the molecular mechanisms involved in conversion of the monomeric amyloid protein into oligomeric and fibrillar forms. Tetracyclines have been proposed for AD therapy, although their effects on the aggregation of Aβ protein, particularly their ability to interact in vivo with the Aβ oligomers and/or aggregates, remain to be understood. Using transgenic Caenorhabditis elegans as a simplified invertebrate model of AD, we evaluated the ability of tetracyclines to interfere with the sequence of events leading to Aβ proteotoxicity. The drugs directly interact with the Aβ assemblies in vivo and reduce Aβ oligomer deposition, protecting C. elegans from oxidative stress and the onset of the paralysis phenotype. These effects were specific, dose-related and not linked to any antibiotic activity, suggesting that the drugs might offer an effective therapeutic strategy to target soluble Aβ aggregates.

  14. Folic acid attenuates the effects of amyloid β oligomers on DNA methylation in neuronal cells.

    PubMed

    Liu, Huan; Li, Wen; Zhao, Shijing; Zhang, Xumei; Zhang, Meilin; Xiao, Yanyu; Wilson, John X; Huang, Guowei

    2016-08-01

    Alzheimer's disease (AD) is a highly prevalent type of dementia. The epigenetic mechanism of gene methylation provides a putative link between nutrition, one-carbon metabolism, and disease progression because folate deficiency may cause hypomethylation of promoter regions in AD-relevant genes. We hypothesized that folic acid supplementation may protect neuron cells from amyloid β (Aβ) oligomer-induced toxicity by modulating DNA methylation of APP and PS1 in AD models. Primary hippocampal neuronal cells and hippocampal HT-22 cells were incubated for 24 h with a combination of folic acid and either Aβ oligomers or vehicle and were then incubated for 72 h with various concentrations of folic acid. AD transgenic mice were fed either folate-deficient or control diets and gavaged daily with various doses of folic acid (0 or 600 μg/kg). DNA methyltransferase (DNMT) activity, cell viability, methylation potential of cells, APP and PS1 expression, and the methylation of the respective promoters were determined. Aβ oligomers lowered DNMT activity, increased PS1 and APP expression, and decreased cell viability. Folic acid dose-dependently stimulated methylation potential and DNMT activity, altered PS1 and APP promoter methylation, decreased PS1 and APP expression, and partially preserved cell viability. Folic acid increased PS1 and APP promoter methylation in AD transgenic mice. These results suggest a mechanism by which folic acid may prevent Aβ oligomer-induced neuronal toxicity.

  15. Targeting β-amyloid plaques and oligomers: development of near-IR fluorescence imaging probes.

    PubMed

    Liu, Hongwu; Yang, Jian; Wang, Letian; Xu, Yungen; Zhang, Siyuan; Lv, Jie; Ran, Chongzhao; Li, Yuyan

    2017-02-01

    Evidence indicated that shifting treatment to a presymptomatic stage may produce significant benefits to prevent/alleviate the progression of Alzheimer's disease (AD); in particular, early incorporation of noninvasive imaging and biomarker testing will be significantly beneficial for AD drug development. Based on amyloid cascade hypothesis and its revised version, both β-amyloid deposition and soluble oligomeric species could be good diagnostic biomarkers for AD. Near-IR fluorescence (NIRF) imaging, which so far is limited to animal studies, is a promising method for its incomparable advantages such as low cost, high-throughput and easy operation. This review focuses on recent reported NIRF probes that showed excellent binding to plaques and oligomers. We hope that this review will shed light on the future of NIRF probes' discovery.

  16. Understanding amyloid fibril nucleation and aβ oligomer/drug interactions from computer simulations.

    PubMed

    Nguyen, Phuong; Derreumaux, Philippe

    2014-02-18

    Evolution has fine-tuned proteins to accomplish a variety of tasks. Yet, with aging, some proteins assemble into harmful amyloid aggregates associated with neurodegenerative diseases, such as Alzheimer's disease (AD), which presents a complex and costly challenge to our society. Thus, far, drug after drug has failed to slow the progression of AD, characterized by the self-assembly of the 39-43 amino acid β-amyloid (Aβ) protein into extracellular senile plaques that form a cross-β structure. While there is experimental evidence that the Aβ small oligomers are the primary toxic species, standard tools of biology have failed to provide structures of these transient, inhomogeneous assemblies. Despite extensive experimental studies, researchers have not successfully characterized the nucleus ensemble, the starting point for rapid fibril formation. Similarly scientists do not have atomic data to show how the compounds that reduce both fibril formation and toxicity in cells bind to Aβ42 oligomers. In this context, computer simulations are important tools for gaining insights into the self-assembly of amyloid peptides and the molecular mechanism of inhibitors. This Account reviews what analytical models and simulations at different time and length scales tell us about the dynamics, kinetics, and thermodynamics of amyloid fibril formation and, notably, the nucleation process. Though coarse-grained and mesoscopic protein models approximate atomistic details by averaging out unimportant degrees of freedom, they provide generic features of amyloid formation and insights into mechanistic details of the self-assembly process. The thermodynamics and kinetics vary from linear peptides adopting straight β-strands in fibrils to longer peptides adopting in parallel U shaped conformations in fibrils. In addition, these properties change with the balance between electrostatic and hydrophobic interactions and the intrinsic disorder of the system. However, simulations suggest that

  17. Postsynaptic Receptors for AmyloidOligomers as Mediators of Neuronal Damage in Alzheimer's Disease.

    PubMed

    Dinamarca, Margarita C; Ríos, Juvenal A; Inestrosa, Nibaldo C

    2012-01-01

    The neurotoxic effect of amyloid-β peptide (Aβ) over the central synapses has been described and is reflected in the decrease of some postsynaptic excitatory proteins, the alteration in the number and morphology of the dendritic spines, and a decrease in long-term potentiation. Many studies has been carried out to identify the putative Aβ receptors in neurons, and is still no clear why the Aβ oligomers only affect the excitatory synapses. Aβ oligomers bind to neurite and preferentially to the postsynaptic region, where the postsynaptic protein-95 (PSD-95) is present in the glutamatergic synapse, and interacts directly with the N-methyl-D-aspartate receptor (NMDAR) and neuroligin (NL). NL is a postsynaptic protein which binds to the presynaptic protein, neurexin to form a heterophilic adhesion complex, the disruption of this interaction affects the integrity of the synaptic contact. Structurally, NL has an extracellular domain homolog to acetylcholinesterase, the first synaptic protein that was found to interact with Aβ. In the present review we will document the interaction between Aβ and the extracellular domain of NL-1 at the excitatory synapse, as well as the interaction with other postsynaptic components, including the glutamatergic receptors (NMDA and mGluR5), the prion protein, the neurotrophin receptor, and the α7-nicotinic acetylcholine receptor. We conclude that several Aβ oligomers receptors exist at the excitatory synapse, which could be the responsible for the neurotoxic effect described for the Aβ oligomers. The characterization of the interaction between Aβ receptors and Aβ oligomers could help to understand the source of the neurologic damage observed in the brain of the Alzheimer's disease patients.

  18. Salt anions promote the conversion of HypF-N into amyloid-like oligomers and modulate the structure of the oligomers and the monomeric precursor state.

    PubMed

    Campioni, Silvia; Mannini, Benedetta; López-Alonso, Jorge P; Shalova, Irina N; Penco, Amanda; Mulvihill, Estefania; Laurents, Douglas V; Relini, Annalisa; Chiti, Fabrizio

    2012-12-07

    An understanding of the solution factors contributing to the rate of aggregation of a protein into amyloid oligomers, to the modulation of the conformational state populated prior to aggregation and to the structure/morphology of the resulting oligomers is one of the goals of present research in this field. We have studied the influence of six different salts on the conversion of the N-terminal domain of Escherichiacoli HypF (HypF-N) into amyloid-like oligomers under conditions of acidic pH. Our results show that salts having different anions (NaCl, NaClO(4), NaI, Na(2)SO(4)) accelerate oligomerization with an efficacy that follows the electroselectivity series of the anions (SO(4)(2-)≥ ClO(4)(-)>I(-)>Cl(-)). By contrast, salts with different cations (NaCl, LiCl, KCl) have similar effects. We also investigated the effect of salts on the structure of the final and initial states of HypF-N aggregation. The electroselectivity series does not apply to the effect of anions on the structure of the oligomers. By contrast, it applies to their effect on the content of secondary structure and on the exposure of hydrophobic clusters of the monomeric precursor state. The results therefore indicate that the binding of anions to the positively charged residues of HypF-N at low pH is the mechanism by which salts modulate the rate of oligomerization and the structure of the monomeric precursor state but not the structure of the resulting oligomers. Overall, the data contribute to rationalize the effect of salts on amyloid-like oligomer formation and to explain the role of charged biological macromolecules in protein aggregation processes.

  19. Real-time investigation of cytochrome c release profiles in living neuronal cells undergoing amyloid beta oligomer-induced apoptosis

    NASA Astrophysics Data System (ADS)

    Lee, Jae Young; Park, Younggeun; Pun, San; Lee, Sung Sik; Lo, Joe F.; Lee, Luke P.

    2015-06-01

    Intracellular Cyt c release profiles in living human neuroblastoma undergoing amyloid β oligomer (AβO)-induced apoptosis, as a model Alzheimer's disease-associated pathogenic molecule, were analysed in a real-time manner using plasmon resonance energy transfer (PRET)-based spectroscopy.Intracellular Cyt c release profiles in living human neuroblastoma undergoing amyloid β oligomer (AβO)-induced apoptosis, as a model Alzheimer's disease-associated pathogenic molecule, were analysed in a real-time manner using plasmon resonance energy transfer (PRET)-based spectroscopy. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr02390d

  20. Large Soluble Oligomers of Amyloid β-Protein from Alzheimer Brain Are Far Less Neuroactive Than the Smaller Oligomers to Which They Dissociate

    PubMed Central

    Yang, Ting; Li, Shaomin; Xu, Huixin

    2017-01-01

    Soluble oligomers of amyloid β-protein (oAβ) isolated from the brains of Alzheimer's disease (AD) patients have been shown experimentally (in the absence of amyloid plaques) to impair hippocampal synaptic plasticity, decrease synapses, induce tau hyperphosphorylation and neuritic dystrophy, activate microglial inflammation, and impair memory in normal adult rodents. Nevertheless, there has been controversy about what types of oligomers actually confer these AD-like phenotypes. Here, we show that the vast majority of soluble Aβ species obtained from brains of humans who died with confirmed AD elute at high molecular weight (HMW) on nondenaturing size-exclusion chromatography. These species have little or no cytotoxic activity in several bioassays. However, incubation of HMW oAβ in mildly alkaline buffer led to their quantitative dissociation into low molecular weight oligomers (∼8–70 kDa), and these were now far more bioactive: they impaired hippocampal LTP, decreased neuronal levels of β2-adrenergic receptors, and activated microglia in wt mice in vivo. Thus, most soluble Aβ assemblies in AD cortex are large and inactive but under certain circumstances can dissociate into smaller, highly bioactive species. Insoluble amyloid plaques likely sequester soluble HMW oligomers, limiting their potential to dissociate. We conclude that conditions that destabilize HMW oligomers or retard the sequestration of their smaller, more bioactive components are important drivers of Aβ toxicity. Selectively targeting these small, cytotoxic forms should be therapeutically beneficial. SIGNIFICANCE STATEMENT Oligomers of amyloid β-protein (oAβ) are tought to play an important role in Alzheimer's disease (AD), but there is confusion and controversy about what types and sizes of oligomers have disease-relevant activity. Using size-exclusion chromatography and three distinct measures of bioactivity, we show that the predominant forms of Aβ in aqueous extracts of AD brain are

  1. Prevention of amyloid-beta oligomer-induced neuronal death by EGTA, estradiol, and endocytosis inhibitor.

    PubMed

    Cižas, Paulius; Jekabsonė, Aistė; Borutaitė, Vilmantė; Morkūnienė, Ramunė

    2011-01-01

    BACKGROUND AND OBJECTIVE. Alzheimer's disease is a progressive neurodegenerative disease that is biochemically characterized by the accumulation of amyloid beta (Aβ) peptides in the brain. The current hypothesis suggests that Aβ oligomers rather than fibrillar aggregates are the most toxic species of Aβ though the mechanisms of their neurotoxicity are unclear. The authors have previously shown that small Aβ(1-42) oligomers at around 1 µM concentration caused rapid (in 24 h) neuronal death in cerebellar granule cell (CGC) cultures. In this study, we aimed to investigate whether protracted (up to 7 days) incubation of CGC cultures with lower submicromolar concentration of various aggregates of Aβ(1-42) had an effect on viability of neurons. In order to get some insight into the mechanism of Aβ-induced cell death, we also sought to determine whether extracellular Ca(2+) and process of endocytosis contributed to Aβ oligomer-induced neurotoxicity and whether pharmacological interventions into these processes would prevent Aβ oligomer-induced cell death. MATERIAL AND METHODS. Primary cultures of CGC were treated with various aggregate forms of Aβ(1-42). Cell viability was assessed by fluorescent microscopy using propidium iodide and Hoechst 33342 staining. RESULTS. Exposure of neurons to 500 nM Aβ(1-42) oligomers for 72-168 h caused extensive neuronal necrosis. Lower concentrations (100-250 nM) were not toxic to cells during 7 days of incubation. Aβ(1-42) monomers and fibrils had no effect on neuronal viability even after 7 days of incubation. Treatment of neurons with EGTA, steroid hormone 17β-estradiol, and methyl-β-cyclodextrin significantly reduced Aβ(1-42) oligomers-induced neuronal death. CONCLUSIONS. The results show that submicromolar concentrations of Aβ(1-42) oligomers were highly toxic to neurons during protracted incubation inducing neuronal necrosis that can be prevented by chelating extracellular Ca(2+) with EGTA, inhibiting endocytosis with

  2. CD45 Deficiency Drives Amyloid-β Peptide Oligomers and Neuronal Loss in Alzheimer's Disease Mice

    PubMed Central

    Zhu, Yuyan; Hou, Huayan; Rezai-Zadeh, Kavon; Giunta, Brian; Ruscin, Amanda; Gemma, Carmelina; Jin, JingJi; Dragicevic, Natasa; Bradshaw, Patrick; Rasool, Suhail; Glabe, Charles G.; Ehrhart, Jared; Bickford, Paula; Mori, Takashi; Obregon, Demian; Town, Terrence; Tan, Jun

    2011-01-01

    Converging lines of evidence indicate dysregulation of the key immunoregulatory molecule CD45 (also known as leukocyte common antigen) in Alzheimer's disease (AD). We report that transgenic mice overproducing amyloid-β peptide (Aβ) but deficient in CD45 (PSAPP/CD45–/– mice) faithfully recapitulate AD neuropathology. Specifically, we find increased abundance of cerebral intracellular and extracellular soluble oligomeric and insoluble Aβ, decreased plasma soluble Aβ, increased abundance of microglial neurotoxic cytokines tumor necrosis factor-α and interleukin-1β, and neuronal loss in PSAPP/CD45–/– mice compared with CD45-sufficient PSAPP littermates (bearing mutant human amyloid precursor protein and mutant human presenilin-1 transgenes). After CD45 ablation, in vitro and in vivo studies demonstrate an anti-Aβ phagocytic but proinflammatory microglial phenotype. This form of microglial activation occurs with elevated Aβ oligomers and neural injury and loss as determined by decreased ratio of anti-apoptotic Bcl-xL to proapoptotic Bax, increased activated caspase-3, mitochondrial dysfunction, and loss of cortical neurons in PSAPP/CD45–/– mice. These data show that deficiency in CD45 activity leads to brain accumulation of neurotoxic Aβ oligomers and validate CD45-mediated microglial clearance of oligomeric Aβ as a novel AD therapeutic target. PMID:21273420

  3. Heat shock treatment reduces beta amyloid toxicity in vivo by diminishing oligomers.

    PubMed

    Wu, Yanjue; Cao, Zhiming; Klein, William L; Luo, Yuan

    2010-06-01

    Heat shock response, mediated by heat shock proteins, is a highly conserved physiological process in multicellular organisms for reestablishment of cellular homeostasis. Expression of heat shock factors and subsequent heat shock protein plays a role in protection against proteotoxicity in invertebrate and vertebrate models. Proteotoxicity due to beta-amyloid peptide (Abeta) oligomerization has been linked to the pathogenesis of Alzheimer's disease. Previously, we demonstrated that progressive paralysis induced by expression of human Abeta(1-42) in transgenic Caenorhabditis elegans was alleviated by Abeta oligomer inhibitors Ginkgo biloba extract and its constituents [Wu, Y., Wu, Z., Butko, P., Christen, Y., Lambert, M.P., Klein, W.L., Link, C.D., Luo, Y., 2006. Amyloid-beta-induced pathological behaviors are suppressed by Ginkgo biloba extract EGb 761 and ginkgolides in transgenic Caenorhabditis elegans. J. Neurosci. 26(50): 13102-13113]. In this study, we apply a protective heat shock to the transgenic C. elegans and demonstrate: (1) a delay in paralysis, (2) increased expression of small heat shock protein HSP16.2, and (3) significant reduction of Abeta oligomers in a heat shock time-dependent manner. These results suggest that transient heat shock lessens Abeta toxicity by diminishing Abeta oligomerization, which provides a link between up regulation of endogenous chaperone proteins and protection against Abeta proteotoxicity in vivo.

  4. Probing the Nucleus Model for Oligomer Formation during Insulin Amyloid Fibrillogenesis

    PubMed Central

    Pease, Leonard F.; Sorci, Mirco; Guha, Suvajyoti; Tsai, De-Hao; Zachariah, Michael R.; Tarlov, Michael J.; Belfort, Georges

    2010-01-01

    We find evidence for a direct transition of insulin monomers into amyloid fibrils without measurable concentrations of oligomers or protofibrils, suggesting that fibrillogenesis may occur directly from assembly of denaturing insulin monomers rather than by successive transitions through protofibril nuclei. To support our finding, we obtain size distributions using electrospray differential mobility analysis (ES-DMA), which provides excellent resolution to clearly distinguish among small oligomers and rapidly generates statistically significant size distributions. The distributions detect an absence of significant peaks between 6 nm and 17 nm as the monomer reacts into fibers—exactly the size range observed by others for small-angle-neutron-scattering-measured intermediates and for circular supramolecular structures. They report concentrations in the nanomolar range, whereas our limit of detection remains three-orders-of-magnitude lower (<5 pmol/L). This finding, along with the lack of significant increases in the β-sheet content of monomers using circular dichroism, suggests monomers do not first structurally rearrange and accumulate in a β-rich state but react and reorganize at the growing fiber's tip. These results quantitatively inform reaction-based theories of amyloid fiber formation and have implications for neurodegenerative, protein conformation ailments including Alzheimer's disease and bovine spongiform encephalopathy. PMID:21156140

  5. Energetic contributions of residues to the formation of early amyloidoligomers.

    PubMed

    Pouplana, R; Campanera, J M

    2015-01-28

    Low-weight amyloid-β (Aβ) oligomers formed at early stages of oligomerization rather than fibril assemblies seem to be the toxic components that drive neurodegeneration in Alzheimer's disease. Unfortunately, detailed knowledge of the structure of these early oligomers at the residue level is not yet available. In this study, we performed all-atom explicit solvent molecular dynamics simulations to examine the oligomerization process of Aβ10-35 monomers when forming dimers, trimers, tetramers and octamers, with four independent simulations of a total simulated time of 3 μs for each oligomer system. The decomposition of the stability free energy by MM-GBSA methodology allowed us to unravel the network of energetic interactions that stabilize such oligomers. The contribution of the intermonomeric van der Waals term is the most significant energy feature of the oligomerization process, consistent with the so-called hydrophobic effect. Furthermore, the decomposition of the stability free energy into residues and residue-pairwise terms revealed that it is mainly apolar interactions between the three specific hydrophobic fragments 31-35 (C-terminal region), 17-20 (central hydrophobic core) and 12-14 (N-terminal region) that are responsible for such a favourable effect. The conformation in which the hydrophobic cthr-chc interaction is oriented perpendicularly is particularly important. We propose three other model substructures that favour the oligomerization process and can thus be considered as molecular targets for future inhibitors. Understanding Aβ oligomerization at the residue level could lead to more efficient design of inhibitors of this process.

  6. Probing and trapping a sensitive conformation: amyloid-β fibrils, oligomers, and dimers.

    PubMed

    Fawver, Janelle N; Duong, Karen T; Wise-Scira, Olivia; Petrofes Chapa, Rachel; Schall, Hayley E; Coskuner, Orkid; Zhu, Xiongwei; Colom, Luis V; Murray, Ian V J

    2012-01-01

    Alzheimer's disease (AD) is a devastating neurodegenerative disease with pathological misfolding of amyloid-β protein (Aβ). The recent interest in Aβ misfolding intermediates necessitates development of novel detection methods and ability to trap these intermediates. We speculated that two regions of Aβ may allow for detection of specific Aβ species: the N-terminal and 22-35, both likely important in oligomer interaction and formation. We determined via epitomics, proteomic assays, and electron microscopy that the Aβ(42) species (wild type, ΔE22, and MetOx) predominantly formed fibrils, oligomers, or dimers, respectively. The 2H4 antibody to the N-terminal of Aβ, in the presence of 2% SDS, primarily detected fibrils, and an antibody to the 22-35 region detected low molecular weight Aβ species. Simulated molecular modeling provided insight into these SDS-induced structural changes. We next determined if these methods could be used to screen anti-Aβ drugs as well as identify compounds that trap Aβ in various conformations. Immunoblot assays determined that taurine, homotaurine (Tramiprosate), myoinositol, methylene blue, and curcumin did not prevent Aβ aggregation. However, calmidazolium chloride trapped Aβ at oligomers, and berberine reduced oligomer formation. Finally, pretreatment of AD brain tissues with SDS enhanced 2H4 antibody immunostaining of fibrillar Aβ. Thus we identified and characterized Aβs that adopt specific predominant conformations (modified Aβ or via interactions with compounds), developed a novel assay for aggregated Aβ, and applied it to drug screening and immunohistochemistry. In summary, our novel approach facilitates drug screening, increases the probability of success of antibody therapeutics, and improves antibody-based detection and identification of different conformations of Aβ.

  7. A Foldamer-Dendrimer Conjugate Neutralizes Synaptotoxic β-Amyloid Oligomers

    PubMed Central

    Fülöp, Lívia; Mándity, István M.; Juhász, Gábor; Szegedi, Viktor; Hetényi, Anasztázia; Wéber, Edit; Bozsó, Zsolt; Simon, Dóra; Benkő, Mária; Király, Zoltán; Martinek, Tamás A.

    2012-01-01

    Background and Aims Unnatural self-organizing biomimetic polymers (foldamers) emerged as promising materials for biomolecule recognition and inhibition. Our goal was to construct multivalent foldamer-dendrimer conjugates which wrap the synaptotoxic β-amyloid (Aβ) oligomers with high affinity through their helical foldamer tentacles. Oligomeric Aβ species play pivotal role in Alzheimer's disease, therefore recognition and direct inhibition of this undruggable target is a great current challenge. Methods and Results Short helical β-peptide foldamers with designed secondary structures and side chain chemistry patterns were applied as potential recognition segments and their binding to the target was tested with NMR methods (saturation transfer difference and transferred-nuclear Overhauser effect). Helices exhibiting binding in the µM region were coupled to a tetravalent G0-PAMAM dendrimer. In vitro biophysical (isothermal titration calorimetry, dynamic light scattering, transmission electron microscopy and size-exclusion chromatography) and biochemical tests (ELISA and dot blot) indicated the tight binding between the foldamer conjugates and the Aβ oligomers. Moreover, a selective low nM interaction with the low molecular weight fraction of the Aβ oligomers was found. Ex vivo electrophysiological experiments revealed that the new material rescues the long-term potentiation from the toxic Aβ oligomers in mouse hippocampal slices at submicromolar concentration. Conclusions The combination of the foldamer methodology, the fragment-based approach and the multivalent design offers a pathway to unnatural protein mimetics that are capable of specific molecular recognition, and has already resulted in an inhibitor for an extremely difficult target. PMID:22859942

  8. Alzheimer's Therapeutics Targeting Amyloid Beta 1–42 Oligomers I: Abeta 42 Oligomer Binding to Specific Neuronal Receptors Is Displaced by Drug Candidates That Improve Cognitive Deficits

    PubMed Central

    Izzo, Nicholas J.; Staniszewski, Agnes; To, Lillian; Fa, Mauro; Teich, Andrew F.; Saeed, Faisal; Wostein, Harrison; Walko, Thomas; Vaswani, Anisha; Wardius, Meghan; Syed, Zanobia; Ravenscroft, Jessica; Mozzoni, Kelsie; Silky, Colleen; Rehak, Courtney; Yurko, Raymond; Finn, Patricia; Look, Gary; Rishton, Gilbert; Safferstein, Hank; Miller, Miles; Johanson, Conrad; Stopa, Edward; Windisch, Manfred; Hutter-Paier, Birgit; Shamloo, Mehrdad; Arancio, Ottavio; LeVine, Harry; Catalano, Susan M.

    2014-01-01

    Synaptic dysfunction and loss caused by age-dependent accumulation of synaptotoxic beta amyloid (Abeta) 1–42 oligomers is proposed to underlie cognitive decline in Alzheimer's disease (AD). Alterations in membrane trafficking induced by Abeta oligomers mediates reduction in neuronal surface receptor expression that is the basis for inhibition of electrophysiological measures of synaptic plasticity and thus learning and memory. We have utilized phenotypic screens in mature, in vitro cultures of rat brain cells to identify small molecules which block or prevent the binding and effects of Abeta oligomers. Synthetic Abeta oligomers bind saturably to a single site on neuronal synapses and induce deficits in membrane trafficking in neuronal cultures with an EC50 that corresponds to its binding affinity. The therapeutic lead compounds we have found are pharmacological antagonists of Abeta oligomers, reducing the binding of Abeta oligomers to neurons in vitro, preventing spine loss in neurons and preventing and treating oligomer-induced deficits in membrane trafficking. These molecules are highly brain penetrant and prevent and restore cognitive deficits in mouse models of Alzheimer's disease. Counter-screening these compounds against a broad panel of potential CNS targets revealed they are highly potent and specific ligands of the sigma-2/PGRMC1 receptor. Brain concentrations of the compounds corresponding to greater than 80% receptor occupancy at the sigma-2/PGRMC1 receptor restore cognitive function in transgenic hAPP Swe/Ldn mice. These studies demonstrate that synthetic and human-derived Abeta oligomers act as pharmacologically-behaved ligands at neuronal receptors - i.e. they exhibit saturable binding to a target, they exert a functional effect related to their binding and their displacement by small molecule antagonists blocks their functional effect. The first-in-class small molecule receptor antagonists described here restore memory to normal in multiple AD

  9. Alzheimer's therapeutics targeting amyloid beta 1-42 oligomers I: Abeta 42 oligomer binding to specific neuronal receptors is displaced by drug candidates that improve cognitive deficits.

    PubMed

    Izzo, Nicholas J; Staniszewski, Agnes; To, Lillian; Fa, Mauro; Teich, Andrew F; Saeed, Faisal; Wostein, Harrison; Walko, Thomas; Vaswani, Anisha; Wardius, Meghan; Syed, Zanobia; Ravenscroft, Jessica; Mozzoni, Kelsie; Silky, Colleen; Rehak, Courtney; Yurko, Raymond; Finn, Patricia; Look, Gary; Rishton, Gilbert; Safferstein, Hank; Miller, Miles; Johanson, Conrad; Stopa, Edward; Windisch, Manfred; Hutter-Paier, Birgit; Shamloo, Mehrdad; Arancio, Ottavio; LeVine, Harry; Catalano, Susan M

    2014-01-01

    Synaptic dysfunction and loss caused by age-dependent accumulation of synaptotoxic beta amyloid (Abeta) 1-42 oligomers is proposed to underlie cognitive decline in Alzheimer's disease (AD). Alterations in membrane trafficking induced by Abeta oligomers mediates reduction in neuronal surface receptor expression that is the basis for inhibition of electrophysiological measures of synaptic plasticity and thus learning and memory. We have utilized phenotypic screens in mature, in vitro cultures of rat brain cells to identify small molecules which block or prevent the binding and effects of Abeta oligomers. Synthetic Abeta oligomers bind saturably to a single site on neuronal synapses and induce deficits in membrane trafficking in neuronal cultures with an EC50 that corresponds to its binding affinity. The therapeutic lead compounds we have found are pharmacological antagonists of Abeta oligomers, reducing the binding of Abeta oligomers to neurons in vitro, preventing spine loss in neurons and preventing and treating oligomer-induced deficits in membrane trafficking. These molecules are highly brain penetrant and prevent and restore cognitive deficits in mouse models of Alzheimer's disease. Counter-screening these compounds against a broad panel of potential CNS targets revealed they are highly potent and specific ligands of the sigma-2/PGRMC1 receptor. Brain concentrations of the compounds corresponding to greater than 80% receptor occupancy at the sigma-2/PGRMC1 receptor restore cognitive function in transgenic hAPP Swe/Ldn mice. These studies demonstrate that synthetic and human-derived Abeta oligomers act as pharmacologically-behaved ligands at neuronal receptors--i.e. they exhibit saturable binding to a target, they exert a functional effect related to their binding and their displacement by small molecule antagonists blocks their functional effect. The first-in-class small molecule receptor antagonists described here restore memory to normal in multiple AD models

  10. The slowly aggregating salmon Calcitonin: a useful tool for the study of the amyloid oligomers structure and activity.

    PubMed

    Diociaiuti, Marco; Gaudiano, Maria Cristina; Malchiodi-Albedi, Fiorella

    2011-01-01

    Amyloid proteins of different aminoacidic composition share the tendency to misfold and aggregate in a similar way, following common aggregation steps. The process includes the formation of dimers, trimers, and low molecular weight prefibrillar oligomers, characterized by the typical morphology of globules less than 10 nm diameter. The globules spontaneously form linear or annular structures and, eventually, mature fibers. The rate of this process depends on characteristics intrinsic to the different proteins and to environmental conditions (i.e., pH, ionic strength, solvent composition, temperature). In the case of neurodegenerative diseases, it is now generally agreed that the pathogenic aggregates are not the mature fibrils, but the intermediate, soluble oligomers. However, the molecular mechanism by which these oligomers trigger neuronal damage is still unclear. In particular, it is not clear if there is a peculiar structure at the basis of the neurotoxic effect and how this structure interacts with neurons. This review will focus on the results we obtained using salmon Calcitonin, an amyloid protein characterized by a very slow aggregation rate, which allowed us to closely monitor the aggregation process. We used it as a tool to investigate the characteristics of amyloid oligomers formation and their interactions with neuronal cells. Our results indicate that small globules of about 6 nm could be the responsible for the neurotoxic effects. Moreover, our data suggest that the rich content in lipid rafts of neuronal cell plasma membrane may render neurons particularly vulnerable to the amyloid protein toxic effect.

  11. The load of amyloidoligomers is decreased in the cerebrospinal fluid of Alzheimer's disease patients.

    PubMed

    Sancesario, Giulia M; Cencioni, Maria T; Esposito, Zaira; Borsellino, Giovanna; Nuccetelli, Marzia; Martorana, Alessandro; Battistini, Luca; Sorge, Roberto; Spalletta, Gianfranco; Ferrazzoli, Davide; Bernardi, Giorgio; Bernardini, Sergio; Sancesario, Giuseppe

    2012-01-01

    Amyloid-β (Aβ) oligomers are heterogeneous and instable compounds of variable molecular weight. Flow cytometry and fluorescence resonance energy transfer (FRET)-based methods allow the simultaneous detection of Aβ oligomers with low and high molecular weight in their native form. We evaluated whether an estimate of different species of Aβ oligomers in the cerebrospinal fluid (CSF) with or without dilution with RIPA buffer could be more useful in the diagnosis of Alzheimer's disease (AD) than the measurement of Aβ42 monomers, total tau (t-tau), and phosphorylated tau (p-tau). Increased t-tau (p < 0.01) and p-tau (p < 0.01), and decreased Aβ42 (p < 0.01), were detected in the CSF of patients with AD (n = 46), compared to patients with other dementia (OD) (n = 35) or with other neurological disorders (OND) (n = 56). In native CSF (n = 137), the levels of Aβ oligomers were lower (p < 0.05) in AD than in OD and OND patients; in addition, the ratio Aβ oligomers/p-tau was lower in AD than in OD (p < 0.01) and OND (p < 0.05) patients, yielding a sensitivity of 75% and a specificity of 64%. However, in CSF diluted with RIPA (n = 30), Aβ oligomers appeared higher (p < 0.05) in AD than in OND patients, suggesting they become partially disaggregated and more easily detectable after RIPA. In conclusion, FRET analysis in native CSF is essential to correctly determine the composition of Aβ oligomers. In this experimental setting, the simultaneous estimate of low and high molecular weight Aβ oligomers is as useful as the other biomarkers in the diagnosis of AD. The low amount of Aβ oligomers detected in native CSF of AD may be inversely related to their levels in the brain, as occurs for Aβ monomers, representing a biomarker for the amyloid pathogenic cascade.

  12. Oligomer stability of Amyloid- β (A β) 25-35: A Dissipative Particle Dynamics study

    NASA Astrophysics Data System (ADS)

    Pivkin, Igor; Peter, Emanuel

    Alzheimer's disease is strongly associated with an accumulation of Amyloid- β (A β) peptide plaques in the human brain. A β is a 43 residues long intrinsically disordered peptide and has a strong tendency to form aggregates. Evidence accumulates that A β acts toxic to the neurons in the brain through the formation of small soluble oligomers. A β 25-35 is the smallest fragment of A β which still retains its toxicity and its ability to form extended fibrils. In this talk we will present the results from simulations of aggregation of up to 100 A β 25-35 peptides using a novel polarizable coarse-grained protein model in combination with Dissipative Particle Dynamics.

  13. Conformation-specific antibodies to target amyloid β oligomers and their application to immunotherapy for Alzheimer's disease.

    PubMed

    Murakami, Kazuma

    2014-01-01

    Amyloid β-protein (Aβ) oligomers, intermediates of Aβ aggregation, cause cognitive impairment and synaptotoxicity in the pathogenesis of Alzheimer's disease (AD). Immunotherapy using anti-Aβ antibody is one of the most promising approaches for AD treatment. However, most clinical trials using conventional sequence-specific antibodies have proceeded with difficulty. This is probably due to the unintended removal of the non-pathological monomer and fibrils of Aβ as well as the pathological oligomers by these antibodies that recognize Aβ sequence, which is not involved in synaptotoxicity. Several efforts have been made recently to develop conformation-specific antibodies that target the tertiary structure of Aβ oligomers. Here, we review the recent findings of Aβ oligomers and anti-Aβ antibodies including our own, and discuss their potential as therapeutic and diagnostic tools.

  14. Transthyretin as both a sensor and a scavenger of β-amyloid oligomers.

    PubMed

    Yang, Dennis T; Joshi, Gururaj; Cho, Patricia Y; Johnson, Jeffrey A; Murphy, Regina M

    2013-04-30

    Transthyretin (TTR) is a homotetrameric transport protein, assembled from monomers that each contain two four-stranded β-sheets and a short α-helix and loop. In the tetramer, the "inner" β-sheet forms a hydrophobic pocket while the helix and loop are solvent-exposed. β-Amyloid (Aβ) aggregates bind to TTR, and the level of binding is significantly reduced in mutants L82A (on the loop) and L110A (on the inner β-sheet). Protection against Aβ toxicity was demonstrated for wild-type TTR but not L82A or L110A, providing a direct link between TTR-Aβ binding and TTR-mediated cytoprotection. Protection is afforded at substoichiometric (1:100) TTR:Aβ molar ratios, and the level of binding of Aβ to TTR is highest for partially aggregated materials and decreased for freshly prepared or heavily aggregated Aβ, suggesting that TTR binds selectively to soluble toxic Aβ aggregates. A novel technique, nanoparticle tracking, is used to show that TTR arrests Aβ aggregation by both preventing formation of new aggregates and inhibiting growth of existing aggregates. TTR tetramers are normally quite stable; tetrameric structure is necessary for the protein's transport functions, and mutations that decrease tetramer stability have been linked to TTR amyloid diseases. However, TTR monomers bind more Aβ than do tetramers, presumably because the hydrophobic inner sheet is solvent-exposed upon tetramer disassembly. Wild-type and L110A tetramers, but not L82A, were destabilized upon being co-incubated with Aβ, suggesting that binding of Aβ to L82 triggers tetramer dissociation. Taken together, these results suggest a novel mechanism of action for TTR: the EF helix/loop "senses" the presence of soluble toxic Aβ oligomers, triggering destabilization of TTR tetramers and exposure of the hydrophobic inner sheet, which then "scavenges" these toxic oligomers and prevents them from causing cell death.

  15. AmyloidOligomers Induce Differential Gene Expression in Adult Human Brain Slices*

    PubMed Central

    Sebollela, Adriano; Freitas-Correa, Leo; Oliveira, Fabio F.; Paula-Lima, Andrea C.; Saraiva, Leonardo M.; Martins, Samantha M.; Mota, Louise D.; Torres, Cesar; Alves-Leon, Soniza; de Souza, Jorge M.; Carraro, Dirce M.; Brentani, Helena; De Felice, Fernanda G.; Ferreira, Sergio T.

    2012-01-01

    Cognitive decline in Alzheimer disease (AD) is increasingly attributed to the neuronal impact of soluble oligomers of the amyloid-β peptide (AβOs). Current knowledge on the molecular and cellular mechanisms underlying the toxicity of AβOs stems largely from rodent-derived cell/tissue culture experiments or from transgenic models of AD, which do not necessarily recapitulate the complexity of the human disease. Here, we used DNA microarray and RT-PCR to investigate changes in transcription in adult human cortical slices exposed to sublethal doses of AβOs. The results revealed a set of 27 genes that showed consistent differential expression upon exposure of slices from three different donors to AβOs. Functional classification of differentially expressed genes revealed that AβOs impact pathways important for neuronal physiology and known to be dysregulated in AD, including vesicle trafficking, cell adhesion, actin cytoskeleton dynamics, and insulin signaling. Most genes (70%) were down-regulated by AβO treatment, suggesting a predominantly inhibitory effect on the corresponding pathways. Significantly, AβOs induced down-regulation of synaptophysin, a presynaptic vesicle membrane protein, suggesting a mechanism by which oligomers cause synapse failure. The results provide insight into early mechanisms of pathogenesis of AD and suggest that the neuronal pathways affected by AβOs may be targets for the development of novel diagnostic or therapeutic approaches. PMID:22235132

  16. Environmental enrichment strengthens corticocortical interactions and reduces amyloidoligomers in aged mice

    PubMed Central

    Mainardi, Marco; Di Garbo, Angelo; Caleo, Matteo; Berardi, Nicoletta; Sale, Alessandro; Maffei, Lamberto

    2013-01-01

    Brain aging is characterized by global changes which are thought to underlie age-related cognitive decline. These include variations in brain activity and the progressive increase in the concentration of soluble amyloid-β (Aβ) oligomers, directly impairing synaptic function and plasticity even in the absence of any neurodegenerative disorder. Considering the high social impact of the decline in brain performance associated to aging, there is an urgent need to better understand how it can be prevented or contrasted. Lifestyle components, such as social interaction, motor exercise and cognitive activity, are thought to modulate brain physiology and its susceptibility to age-related pathologies. However, the precise functional and molecular factors that respond to environmental stimuli and might mediate their protective action again pathological aging still need to be clearly identified. To address this issue, we exploited environmental enrichment (EE), a reliable model for studying the effect of experience on the brain based on the enhancement of cognitive, social and motor experience, in aged wild-type mice. We analyzed the functional consequences of EE on aged brain physiology by performing in vivo local field potential (LFP) recordings with chronic implants. In addition, we also investigated changes induced by EE on molecular markers of neural plasticity and on the levels of soluble Aβ oligomers. We report that EE induced profound changes in the activity of the primary visual and auditory cortices and in their functional interaction. At the molecular level, EE enhanced plasticity by an upward shift of the cortical excitation/inhibition balance. In addition, EE reduced brain Aβ oligomers and increased synthesis of the Aβ-degrading enzyme neprilysin. Our findings strengthen the potential of EE procedures as a non-invasive paradigm for counteracting brain aging processes. PMID:24478697

  17. AMYLOID BETA OLIGOMERS IMPAIR FEAR CONDITIONED MEMORY IN A CALCINEURIN-DEPENDENT FASHION IN MICE

    PubMed Central

    Dineley, Kelly T.; Kayed, Rakez; Neugebauer, Volker; Fu, Yu; Zhang, Wenru; Reese, Lindsay C.; Taglialatela, Giulio

    2010-01-01

    Soluble oligomeric aggregates of the amyloid beta (Aβ) peptide are believed to be the most neurotoxic Aβ species affecting the brain in Alzheimer Disease (AD), a terminal neurodegenerative disorder involving severe cognitive decline underlined by initial synaptic dysfunction and later extensive neuronal death in the CNS. Recent evidence indicates that Aβ oligomers are recruited at the synapse, oppose expression of long term potentiation (LTP), perturb intracellular calcium balance, disrupt dendritic spines and induce memory deficits. However, the molecular mechanisms behind these outcomes are only partially understood; achieving such insight is necessary for the comprehension of Aβ-mediated neuronal dysfunction. We have investigated the role of the phosphatase calcineurin (CaN) in the pathological processes of AD. CaN is especially abundant in the CNS where it is involved in synaptic activity, LTP and memory function. Here, we describe how oligomeric Aβ treatment causes memory deficits and depresses LTP expression in a CaN-dependent fashion. Mice given a single intracerebroventricular injection of Aβ oligomers exhibited increased CaN activity and decreased pCREB, a transcription factor involved in proper synaptic function, accompanied by decreased memory in a fear conditioning task. These effects were reversed by treatment with the CaN inhibitor FK506. We further found that expression of hippocampal LTP in acutely cultured rodent brain slices was opposed by Aβ oligomers and that this effect was also reversed by FK506. Collectively, these results indicate that CaN activation may play a central role in mediating synaptic and memory disrupting effect induced by acute oligomeric Aβ treatment in mice. PMID:20544830

  18. Amyloid-beta oligomers impair fear conditioned memory in a calcineurin-dependent fashion in mice.

    PubMed

    Dineley, Kelly T; Kayed, Rakez; Neugebauer, Volker; Fu, Yu; Zhang, Wenru; Reese, Lindsay C; Taglialatela, Giulio

    2010-10-01

    Soluble oligomeric aggregates of the amyloid-beta (A beta) peptide are believed to be the most neurotoxic A beta species affecting the brain in Alzheimer disease (AD), a terminal neurodegenerative disorder involving severe cognitive decline underscored by initial synaptic dysfunction and later extensive neuronal death in the CNS. Recent evidence indicates that A beta oligomers are recruited at the synapse, oppose expression of long-term potentiation (LTP), perturb intracellular calcium balance, disrupt dendritic spines, and induce memory deficits. However, the molecular mechanisms behind these outcomes are only partially understood; achieving such insight is necessary for the comprehension of A beta-mediated neuronal dysfunction. We have investigated the role of the phosphatase calcineurin (CaN) in these pathological processes of AD. CaN is especially abundant in the CNS, where it is involved in synaptic activity, LTP, and memory function. Here, we describe how oligomeric A beta treatment causes memory deficits and depresses LTP expression in a CaN-dependent fashion. Mice given a single intracerebroventricular injection of A beta oligomers exhibited increased CaN activity and decreased pCREB, a transcription factor involved in proper synaptic function, accompanied by decreased memory in a fear conditioning task. These effects were reversed by treatment with the CaN inhibitor FK506. We further found that expression of hippocampal LTP in acutely cultured rodent brain slices was opposed by A beta oligomers and that this effect was also reversed by FK506. Collectively, these results indicate that CaN activation may play a central role in mediating synaptic and memory disruption induced by acute oligomeric A beta treatment in mice. (c) 2010 Wiley-Liss, Inc.

  19. Direct observation of single amyloid-β(1-40) oligomers on live cells: binding and growth at physiological concentrations.

    PubMed

    Johnson, Robin D; Schauerte, Joseph A; Wisser, Kathleen C; Gafni, Ari; Steel, Duncan G

    2011-01-01

    Understanding how amyloid-β peptide interacts with living cells on a molecular level is critical to development of targeted treatments for Alzheimer's disease. Evidence that oligomeric Aβ interacts with neuronal cell membranes has been provided, but the mechanism by which membrane binding occurs and the exact stoichiometry of the neurotoxic aggregates remain elusive. Physiologically relevant experimentation is hindered by the high Aβ concentrations required for most biochemical analyses, the metastable nature of Aβ aggregates, and the complex variety of Aβ species present under physiological conditions. Here we use single molecule microscopy to overcome these challenges, presenting direct optical evidence that small Aβ(1-40) oligomers bind to living neuroblastoma cells at physiological Aβ concentrations. Single particle fluorescence intensity measurements indicate that cell-bound Aβ species range in size from monomers to hexamers and greater, with the majority of bound oligomers falling in the dimer-to-tetramer range. Furthermore, while low-molecular weight oligomeric species do form in solution, the membrane-bound oligomer size distribution is shifted towards larger aggregates, indicating either that bound Aβ oligomers can rapidly increase in size or that these oligomers cluster at specific sites on the membrane. Calcium indicator studies demonstrate that small oligomer binding at physiological concentrations induces only mild, sporadic calcium leakage. These findings support the hypothesis that small oligomers are the primary Aβ species that interact with neurons at physiological concentrations.

  20. Protective spin-labeled fluorenes maintain amyloid beta peptide in small oligomers and limit transitions in secondary structure

    SciTech Connect

    Altman, Robin; Ly, Sonny; Hilt, Silvia; Petrlova, Jitka; Maezawa, Izumi; Kálai, Tamás; Hideg, Kálmán; Jin, Lee-Way; Laurence, Ted A.; Voss, John C.

    2015-12-01

    Alzheimer’s disease is characterized by the presence of extracellular plaques comprised of amyloid beta (Aβ) peptides. Soluble oligomers of the Aβ peptide underlie a cascade of neuronal loss and dysfunction associated with Alzheimer's disease. Single particle analyses of Aβ oligomers in solution by fluorescence correlation spectroscopy (FCS) were used to provide real-time descriptions of how spin-labeled fluorenes (SLFs; bi-functional small molecules that block the toxicity of Aβ) prevent and disrupt oligomeric assemblies of Aβ in solution. The FCS results, combined with electron paramagnetic resonance spectroscopy and circular dichroism spectroscopy, demonstrate SLFs can inhibit the growth of Aβ oligomers and disrupt existing oligomers while retaining Aβ in a largely disordered state. Furthermore, while the ability of SLF to block Aβ toxicity correlates with a reduction in oligomer size, our results suggest the conformation of Aβ within the oligomer determines the toxicity of the species. Attenuation of Aβ toxicity, which has been associated primarily with the soluble oligomeric form, can be achieved through redistribution of the peptides into smaller oligomers and arrest of the fractional increase in beta secondary structure.

  1. Protective spin-labeled fluorenes maintain amyloid beta peptide in small oligomers and limit transitions in secondary structure

    PubMed Central

    Altman, Robin; Ly, Sonny; Hilt, Silvia; Petrlova, Jitka; Maezawa, Izumi; Kálai, Tamás; Hideg, Kálmán; Jin, Lee-Way; Laurence, Ted A.; Voss, John C.

    2015-01-01

    Alzheimer’s disease is characterized by the presence of extracellular plaques comprised of amyloid beta (Aβ) peptides. Soluble oligomers of the Aβ peptide underlie a cascade of neuronal loss and dysfunction associated with Alzheimer’s disease. Single particle analyses of Aβ oligomers in solution by fluorescence correlation spectroscopy (FCS) were used to provide real-time descriptions of how spin-labeled fluorenes (SLFs; bi-functional small molecules that block the toxicity of Aβ) prevent and disrupt oligomeric assemblies of Aβ in solution. Furthermore, the circular dichroism (CD) spectrum of untreated Aβ shows a continuous, progressive change over a 24-hour period, while the spectrum of Aβ treated with SLF remains relatively constant following initial incubation. These findings suggest the conformation of Aβ within the oligomer provides a complementary determinant of Aβ toxicity in addition to oligomer growth and size. Although SLF does not produce a dominant state of secondary structure in Aβ, it does induce a net reduction in beta secondary content compared to untreated samples of Aβ. The FCS results, combined with electron paramagnetic resonance spectroscopy and CD spectroscopy, demonstrate SLFs can inhibit the growth of Aβ oligomers and disrupt existing oligomers, while retaining Aβ as a population of smaller, yet largely disordered oligomers. PMID:26374940

  2. Acute amnestic encephalopathy in amyloidoligomer-injected mice is due to their widespread diffusion in vivo.

    PubMed

    Epelbaum, Stéphane; Youssef, Ihsen; Lacor, Pascale N; Chaurand, Pierre; Duplus, Eric; Brugg, Bernard; Duyckaerts, Charles; Delatour, Benoît

    2015-06-01

    Amyloid-β (Aβ) oligomers are the suspected culprit as initiators of Alzheimer's disease (AD). However, their diffusion in the brain remains unknown. Here, we studied Aβ oligomers' dissemination and evaluated their in vivo toxicity. Wild-type mice were injected with 50 pmol of synthetic Aβ oligomers (of different size) in the hippocampus. Oligomers diffused largely in the brain as soon as 1 hour and up to 7 days after injection. A transient encephalopathy with memory impairment was induced by this unique injection. The immunoreactivity of the postsynaptic marker PSD95 was diffusely decreased. Similar results (both on memory and PSD95 immunoreactivity) were obtained with delipidated and high molecular weight oligomers (>50 kDa) but not with smaller assemblies. Tau hyperphosphorylation was observed in the oligomer-injected brains. Finally, fos immunostaining was increased in Aβ-derived diffusible ligands-injected mice, suggesting neuronal hyperactivity. Rapid and widespread diffusion of Aβ oligomers was demonstrated in vivo and associated with decreased synaptic markers and memory deficits which gives new insight to the pathogenicity of Aβ.

  3. Effects of secreted oligomers of amyloid β-protein on hippocampal synaptic plasticity: a potent role for trimers

    PubMed Central

    Townsend, Matthew; Shankar, Ganesh M; Mehta, Tapan; Walsh, Dominic M; Selkoe, Dennis J

    2006-01-01

    The accumulation of amyloid β-protein (Aβ) in brain regions serving memory and cognition is a central pathogenic feature of Alzheimer's disease (AD). We have shown that small soluble oligomers of human Aβ that are naturally secreted by cultured cells inhibit hippocampal long-term potentiation (LTP) in vitro and in vivo and transiently impair the recall of a complex learned behaviour in rats. These results support the hypothesis that diffusible oligomers of Aβ initiate a synaptic dysfunction that may be an early event in AD. We now report detailed electrophysiological analyses that define conditions under which acute application of soluble Aβ inhibits hippocampal synaptic plasticity in wild-type mice. To ascertain which Aβ assemblies contribute to the impairment of LTP, we fractionated oligomers by size-exclusion chromatography and found that Aβ trimers fully inhibit LTP, whereas dimers and tetramers have an intermediate potency. Natural Aβ oligomers are sensitive to heat denaturation, primarily inhibit the induction phase of LTP, and cause a sustained impairment of LTP even after extensive washout. We observed no effects of Aβ oligomers on presynaptic vesicle release. LTP in juvenile mice is resistant to the effects of Aβ oligomers, as is brain-derived-neurotrophic-factor-induced LTP in adult hippocampus. We conclude that specific assemblies, particularly timers, of naturally secreted Aβ oligomers are potent and selective inhibitors of certain forms of hippocampal LTP. PMID:16469784

  4. Dynamic assessment of Amyloid oligomers - cell membrane interaction by advanced impedance spectroscopy

    NASA Astrophysics Data System (ADS)

    Gheorghiu, M.; David, S.; Polonschii, C.; Bratu, D.; Gheorghiu, E.

    2013-04-01

    The amyloid β (Aβ) peptides are believed to be pivotal in Alzheimer's disease (AD) pathogenesis and onset of vascular dysfunction. Recent studies indicate that Aβ1-42 treatment influences the expression of tight junction protein complexes, stress fibre formation, disruption and aggregation of actin filaments and cellular gap formation. Aiming for functional characterization of model cells upon Aβ1-42 treatment, we deployed an advanced Electric Cell-substrate Impedance Sensing for monitoring cell evolution. A precision Impedance Analyzer with a multiplexing module developed in house was used for recording individual electrode sets in the 40 Hz - 100 KHz frequency range. In a step forward from the classical ECIS assays, we report on a novel data analysis algorithm that enables access to cellular and paracellular electrical parameters and cell surface interaction with fully developed cell monolayers. The evolution of the impedance at selected frequencies provides evidence for a dual effect of Aβ42 exposure, at both paracellular permeability and cell adherence level, with intricate dynamics that open up new perspectives on Aβ1-42 oligomers - cell membrane interaction. Validation of electrical impedance assays of the amyloid fibrils effect on cell membrane structure is achieved by both AFM analysis and Surface Plasmon Resonance studies. The capabilities of this noninvasive, real time platform for cell analysis in a wider applicative context are outlined.

  5. Amyloid β oligomers in Alzheimer’s disease pathogenesis, treatment, and diagnosis

    PubMed Central

    Viola, Kirsten L.; Klein, William L.

    2015-01-01

    Protein aggregation is common to dozens of diseases including prionoses, diabetes, Parkinson’s and Alzheimer’s. Over the past 15 years, there has been a paradigm shift in understanding the structural basis for these proteinopathies. Precedent for this shift has come from investigation of soluble Aβ oligomers (AβOs), toxins now widely regarded as instigating neuron damage leading to Alzheimer’s dementia. Toxic AβOs accumulate in AD brain and constitute long-lived alternatives to the disease-defining Aβ fibrils deposited in amyloid plaques. Key experiments using fibril-free AβO solutions demonstrated that while Aβ is essential for memory loss, the fibrillar Aβ in amyloid deposits is not the agent. The AD-like cellular pathologies induced by AβOs suggest their impact provides a unifying mechanism for AD pathogenesis, explaining why early stage disease is specific for memory and accounting for major facets of AD neuropathology. Alternative ideas for triggering mechanisms are being actively investigated. Some research favors insertion of AβOs into membrane, while other evidence supports ligand-like accumulation at particular synapses. Over a dozen candidate toxin receptors have been proposed. AβO binding triggers a redistribution of critical synaptic proteins and induces hyperactivity in metabotropic and ionotropic glutamate receptors. This leads to Ca2+ overload and instigates major facets of AD neuropathology, including tau hyperphosphorylation, insulin resistance, oxidative stress, and synapse loss. Because different species of AβOs have been identified, a remaining question is which oligomer is the major pathogenic culprit. The possibility has been raised that more than one species plays a role. Despite some key unknowns, the clinical relevance of AβOs has been established, and new studies are beginning to point to co-morbidities such as diabetes and hypercholesterolemia as etiological factors. Because pathogenic AβOs appear early in the disease

  6. Structural evolution and membrane interactions of Alzheimer's amyloid-beta peptide oligomers: new knowledge from single-molecule fluorescence studies.

    PubMed

    Johnson, Robin D; Steel, Duncan G; Gafni, Ari

    2014-07-01

    Amyloid-β peptide (Aβ) oligomers may represent the proximal neurotoxin in Alzheimer's disease. Single-molecule microscopy (SMM) techniques have recently emerged as a method for overcoming the innate difficulties of working with amyloid-β, including the peptide's low endogenous concentrations, the dynamic nature of its oligomeric states, and its heterogeneous and complex membrane interactions. SMM techniques have revealed that small oligomers of the peptide bind to model membranes and cells at low nanomolar-to-picomolar concentrations and diffuse at rates dependent on the membrane characteristics. These methods have also shown that oligomers grow or dissociate based on the presence of specific inhibitors or promoters and on the ratio of Aβ40 to Aβ42. Here, we discuss several types of single-molecule imaging that have been applied to the study of Aβ oligomers and their membrane interactions. We also summarize some of the recent insights SMM has provided into oligomer behavior in solution, on planar lipid membranes, and on living cell membranes. A brief overview of the current limitations of the technique, including the lack of sensitive assays for Aβ-induced toxicity, is included in hopes of inspiring future development in this area of research. © 2014 The Protein Society.

  7. Structural evolution and membrane interactions of Alzheimer's amyloid-beta peptide oligomers: New knowledge from single-molecule fluorescence studies

    PubMed Central

    Johnson, Robin D; Steel, Duncan G; Gafni, Ari

    2014-01-01

    Amyloid-β peptide (Aβ) oligomers may represent the proximal neurotoxin in Alzheimer's disease. Single-molecule microscopy (SMM) techniques have recently emerged as a method for overcoming the innate difficulties of working with amyloid-β, including the peptide's low endogenous concentrations, the dynamic nature of its oligomeric states, and its heterogeneous and complex membrane interactions. SMM techniques have revealed that small oligomers of the peptide bind to model membranes and cells at low nanomolar-to-picomolar concentrations and diffuse at rates dependent on the membrane characteristics. These methods have also shown that oligomers grow or dissociate based on the presence of specific inhibitors or promoters and on the ratio of Aβ40 to Aβ42. Here, we discuss several types of single-molecule imaging that have been applied to the study of Aβ oligomers and their membrane interactions. We also summarize some of the recent insights SMM has provided into oligomer behavior in solution, on planar lipid membranes, and on living cell membranes. A brief overview of the current limitations of the technique, including the lack of sensitive assays for Aβ-induced toxicity, is included in hopes of inspiring future development in this area of research. PMID:24753305

  8. Amyloid-β Protein Oligomer at Low Nanomolar Concentrations Activates Microglia and Induces Microglial Neurotoxicity*

    PubMed Central

    Maezawa, Izumi; Zimin, Pavel I.; Wulff, Heike; Jin, Lee-Way

    2011-01-01

    Neuroinflammation and associated neuronal dysfunction mediated by activated microglia play an important role in the pathogenesis of Alzheimer disease (AD). Microglia are activated by aggregated forms of amyloid-β protein (Aβ), usually demonstrated in vitro by stimulating microglia with micromolar concentrations of fibrillar Aβ, a major component of amyloid plaques in AD brains. Here we report that amyloidoligomer (AβO), at 5–50 nm, induces a unique pattern of microglia activation that requires the activity of the scavenger receptor A and the Ca2+-activated potassium channel KCa3.1. AβO treatment induced an activated morphological and biochemical profile of microglia, including activation of p38 MAPK and nuclear factor κB. Interestingly, although increasing nitric oxide (NO) production, AβO did not increase several proinflammatory mediators commonly induced by lipopolyliposacharides or fibrillar Aβ, suggesting that AβO stimulates both common and divergent pathways of microglia activation. AβO at low nanomolar concentrations, although not neurotoxic, induced indirect, microglia-mediated damage to neurons in dissociated cultures and in organotypic hippocampal slices. The indirect neurotoxicity was prevented by (i) doxycycline, an inhibitor of microglia activation; (ii) TRAM-34, a selective KCa3.1 blocker; and (iii) two inhibitors of inducible NO synthase, indicating that KCa3.1 activity and excessive NO release are required for AβO-induced microglial neurotoxicity. Our results suggest that AβO, generally considered a neurotoxin, may more potently cause neuronal damage indirectly by activating microglia in AD. PMID:20971854

  9. Pre-amyloid oligomers of the proteotoxic RepA-WH1 prionoid assemble at the bacterial nucleoid

    PubMed Central

    Moreno-del Álamo, María; de la Espina, Susana Moreno-Díaz; Fernández-Tresguerres, M. Elena; Giraldo, Rafael

    2015-01-01

    Upon binding to short specific dsDNA sequences in vitro, the N-terminal WH1 domain of the plasmid DNA replication initiator RepA assembles as amyloid fibres. These are bundles of single or double twisted tubular filaments in which distorted RepA-WH1 monomers are the building blocks. When expressed in Escherichia coli, RepA-WH1 triggers the first synthetic amyloid proteinopathy in bacteria, recapitulating some of the features of mammalian prion diseases: it is vertically transmissible, albeit non-infectious, showing up in at least two phenotypically distinct and interconvertible strains. Here we report B3h7, a monoclonal antibody specific for oligomers of RepA-WH1, but which does not recognize the mature amyloid fibres. Unlike a control polyclonal antibody generated against the soluble protein, B3h7 interferes in vitro with DNA-promoted or amyloid-seeded assembly of RepA-WH1 fibres, thus the targeted oligomers are on-pathway amyloidogenic intermediates. Immuno-electron microscopy with B3h7 on thin sections of E. coli cells expressing RepA-WH1 consistently labels the bacterial nucleoid, but not the large cytoplasmic aggregates of the protein. This observation points to the nucleoid as the place where oligomeric amyloid precursors of RepA-WH1 are generated, and suggests that, once nucleated by DNA, further growth must continue in the cytoplasm due to entropic exclusion. PMID:26423724

  10. Fabrication of an antibody-aptamer sandwich assay for electrochemical evaluation of levels of β-amyloid oligomers

    PubMed Central

    Zhou, Yanli; Zhang, Huanqing; Liu, Lantao; Li, Congming; Chang, Zhu; Zhu, Xu; Ye, Baoxian; Xu, Maotian

    2016-01-01

    Amyloid β-peptide (Aβ) in its oligomeric form is often considered as the most toxic species in Alzheimer’s disease (AD), and thus Aβ oligomer is a potentially promising candidate biomarker for AD diagnosis. The development of a sensitive and reliable method for monitoring the Aβ oligomer levels in body fluids is an urgent requirement in order to predict the severity and progression at early or preclinical stages of AD. Here, we show a proof of concept for a sensitive and specific detection of Aβ oligomers by an antibody-aptamer sandwich assay. The antibodies of Aβ oligomers and a nanocomposite of gold nanoparticles with aptamer and thionine (aptamer-Au-Th) were used as the recognition element and the detection probe for specifically binding to Aβ oligomers, respectively. The electrochemical signal of Th reduction could provide measurable electrochemical signals, and a low limit of detection (100 pM) was achieved due to the signal amplification by high loading of Th on the gold nanoparticles. The feasibility of the assay was verified by test of Aβ oligomers in artificial cerebrospinal fluid. The proposed strategy presents valuable information related to early diagnosis of AD process. PMID:27725775

  11. Aβ42-oligomer Interacting Peptide (AIP) neutralizes toxic amyloid-β42 species and protects synaptic structure and function

    NASA Astrophysics Data System (ADS)

    Barucker, Christian; Bittner, Heiko J.; Chang, Philip K.-Y.; Cameron, Scott; Hancock, Mark A.; Liebsch, Filip; Hossain, Shireen; Harmeier, Anja; Shaw, Hunter; Charron, François M.; Gensler, Manuel; Dembny, Paul; Zhuang, Wei; Schmitz, Dietmar; Rabe, Jürgen P.; Rao, Yong; Lurz, Rudi; Hildebrand, Peter W.; McKinney, R. Anne; Multhaup, Gerhard

    2015-10-01

    The amyloid-β42 (Aβ42) peptide is believed to be the main culprit in the pathogenesis of Alzheimer disease (AD), impairing synaptic function and initiating neuronal degeneration. Soluble Aβ42 oligomers are highly toxic and contribute to progressive neuronal dysfunction, loss of synaptic spine density, and affect long-term potentiation (LTP). We have characterized a short, L-amino acid Aβ-oligomer Interacting Peptide (AIP) that targets a relatively well-defined population of low-n Aβ42 oligomers, rather than simply inhibiting the aggregation of Aβ monomers into oligomers. Our data show that AIP diminishes the loss of Aβ42-induced synaptic spine density and rescues LTP in organotypic hippocampal slice cultures. Notably, the AIP enantiomer (comprised of D-amino acids) attenuated the rough-eye phenotype in a transgenic Aβ42 fly model and significantly improved the function of photoreceptors of these flies in electroretinography tests. Overall, our results indicate that specifically “trapping” low-n oligomers provides a novel strategy for toxic Aβ42-oligomer recognition and removal.

  12. Aβ42-oligomer Interacting Peptide (AIP) neutralizes toxic amyloid-β42 species and protects synaptic structure and function.

    PubMed

    Barucker, Christian; Bittner, Heiko J; Chang, Philip K-Y; Cameron, Scott; Hancock, Mark A; Liebsch, Filip; Hossain, Shireen; Harmeier, Anja; Shaw, Hunter; Charron, François M; Gensler, Manuel; Dembny, Paul; Zhuang, Wei; Schmitz, Dietmar; Rabe, Jürgen P; Rao, Yong; Lurz, Rudi; Hildebrand, Peter W; McKinney, R Anne; Multhaup, Gerhard

    2015-10-29

    The amyloid-β42 (Aβ42) peptide is believed to be the main culprit in the pathogenesis of Alzheimer disease (AD), impairing synaptic function and initiating neuronal degeneration. Soluble Aβ42 oligomers are highly toxic and contribute to progressive neuronal dysfunction, loss of synaptic spine density, and affect long-term potentiation (LTP). We have characterized a short, L-amino acid Aβ-oligomer Interacting Peptide (AIP) that targets a relatively well-defined population of low-n Aβ42 oligomers, rather than simply inhibiting the aggregation of Aβ monomers into oligomers. Our data show that AIP diminishes the loss of Aβ42-induced synaptic spine density and rescues LTP in organotypic hippocampal slice cultures. Notably, the AIP enantiomer (comprised of D-amino acids) attenuated the rough-eye phenotype in a transgenic Aβ42 fly model and significantly improved the function of photoreceptors of these flies in electroretinography tests. Overall, our results indicate that specifically "trapping" low-n oligomers provides a novel strategy for toxic Aβ42-oligomer recognition and removal.

  13. Aβ42-oligomer Interacting Peptide (AIP) neutralizes toxic amyloid-β42 species and protects synaptic structure and function

    PubMed Central

    Barucker, Christian; Bittner, Heiko J.; Chang, Philip K.-Y.; Cameron, Scott; Hancock, Mark A.; Liebsch, Filip; Hossain, Shireen; Harmeier, Anja; Shaw, Hunter; Charron, François M.; Gensler, Manuel; Dembny, Paul; Zhuang, Wei; Schmitz, Dietmar; Rabe, Jürgen P.; Rao, Yong; Lurz, Rudi; Hildebrand, Peter W.; McKinney, R. Anne; Multhaup, Gerhard

    2015-01-01

    The amyloid-β42 (Aβ42) peptide is believed to be the main culprit in the pathogenesis of Alzheimer disease (AD), impairing synaptic function and initiating neuronal degeneration. Soluble Aβ42 oligomers are highly toxic and contribute to progressive neuronal dysfunction, loss of synaptic spine density, and affect long-term potentiation (LTP). We have characterized a short, L-amino acid Aβ-oligomer Interacting Peptide (AIP) that targets a relatively well-defined population of low-n Aβ42 oligomers, rather than simply inhibiting the aggregation of Aβ monomers into oligomers. Our data show that AIP diminishes the loss of Aβ42-induced synaptic spine density and rescues LTP in organotypic hippocampal slice cultures. Notably, the AIP enantiomer (comprised of D-amino acids) attenuated the rough-eye phenotype in a transgenic Aβ42 fly model and significantly improved the function of photoreceptors of these flies in electroretinography tests. Overall, our results indicate that specifically “trapping” low-n oligomers provides a novel strategy for toxic Aβ42-oligomer recognition and removal. PMID:26510576

  14. Beta-amyloid oligomers induce early loss of presynaptic proteins in primary neurons by caspase-dependent and proteasome-dependent mechanisms.

    PubMed

    Jang, Bong Geum; In, Sua; Choi, Boyoung; Kim, Min-Ju

    2014-11-12

    Beta-amyloid is a major pathogenic molecule for Alzheimer's disease (AD) and can be aggregated into a soluble oligomer, which is a toxic intermediate, before amyloid fibril formation. Beta-amyloid oligomers are associated closely with early synaptic loss in AD. However, it is still unknown which synaptic proteins are involved in the synaptotoxicity, and a direct comparison among the synaptic proteins should also be addressed. Here, we investigated changes in the expression of several presynaptic and postsynaptic proteins in primary neurons after treatment with a low-molecular weight and a high-molecular weight beta-amyloid oligomer. Both oligomers induced early neuronal dysfunction after 4 h and significantly reduced presynaptic protein (synaptophysin, syntaxin, synapsin, and synaptotagmin) expression. However, the expression of postsynaptic proteins (PSD95, NMDAR2A/B, and GluR2/3), except NMDAR1 was not reduced, and some protein expression levels were increased. Glutamate treatment, which is correlated with postsynaptic activation, showed more postsynaptic-specific protein loss compared with beta-amyloid oligomer treatment. Finally, the caspase inhibitor zVAD and the proteasomal inhibitor MG132 attenuated presynaptic protein loss. Thus, our data showed changes in synaptic proteins by beta-amyloid oligomers, which provides an understanding of early synaptotoxicity and suggests new approaches for AD treatment.

  15. Structural polymorphism of human islet amyloid polypeptide (hIAPP) oligomers highlights the importance of interfacial residue interactions.

    PubMed

    Zhao, Jun; Yu, Xiang; Liang, Guizhao; Zheng, Jie

    2011-01-10

    A 37-residue of human islet amyloid polypeptide (hIAPP or amylin) is a main component of amyloid plaques found in the pancreas of ∼90% of type II diabetes patients. It is reported that hIAPP oligomers, rather than mature fibrils, are major toxic species responsible for pancreatic islet β-cell dysfunction and even cell death, but molecular structures of these oligomers remain elusive. In this work, on the basis of recent solid-state NMR and mass-per-length (MPL) data, we model a series of hIAPP oligomers with different β-layers (one, two, and three layers), symmetries (symmetry and asymmetry), and associated interfaces using molecular dynamics simulations. Three distinct interfaces formed by C-terminal β-sheet and C-terminal β-sheet (CC), N-terminal β-sheet and N-terminal β-sheet (NN), and C-terminal β-sheet and N-terminal β-sheet (CN) are identified to drive multiple cross-β-layers laterally associated together to form different amyloid organizations via different intermolecular interactions, in which the CC interface is dominated by polar interactions, the NN interface is dominated by hydrophobic interactions, and the CN interface is dominated by mixed polar and hydrophobic interactions. Overall, the structural stability of the proposed hIAPP oligomers is a result of delicate balance between maximization of favorable peptide-peptide interactions at the interfaces and optimization of solvation energy with globular structure. Different hIAPP oligomeric models indicate a general and intrinsic nature of amyloid polymorphism, driven by different interfacial side-chain interactions. The proposed models are compatible with recent experimental data in overall size, cross-section area, and molecular weight. A general hIAPP aggregation mechanism is proposed on the basis of our simulated models and experimental data.

  16. AmyloidOligomers Interact with Neurexin and Diminish Neurexin-mediated Excitatory Presynaptic Organization

    PubMed Central

    Naito, Yusuke; Tanabe, Yuko; Lee, Alfred Kihoon; Hamel, Edith; Takahashi, Hideto

    2017-01-01

    Alzheimer’s disease (AD) is characterized by excessive production and deposition of amyloid-beta (Aβ) proteins as well as synapse dysfunction and loss. While soluble Aβ oligomers (AβOs) have deleterious effects on synapse function and reduce synapse number, the underlying molecular mechanisms are not well understood. Here we screened synaptic organizer proteins for cell-surface interaction with AβOs and identified a novel interaction between neurexins (NRXs) and AβOs. AβOs bind to NRXs via the N-terminal histidine-rich domain (HRD) of β-NRX1/2/3 and alternatively-spliced inserts at splicing site 4 of NRX1/2. In artificial synapse-formation assays, AβOs diminish excitatory presynaptic differentiation induced by NRX-interacting proteins including neuroligin1/2 (NLG1/2) and the leucine-rich repeat transmembrane protein LRRTM2. Although AβOs do not interfere with the binding of NRX1β to NLG1 or LRRTM2, time-lapse imaging revealed that AβO treatment reduces surface expression of NRX1β on axons and that this reduction depends on the NRX1β HRD. In transgenic mice expressing mutated human amyloid precursor protein, synaptic expression of β-NRXs, but not α-NRXs, decreases. Thus our data indicate that AβOs interact with NRXs and that this interaction inhibits NRX-mediated presynaptic differentiation by reducing surface expression of axonal β-NRXs, providing molecular and mechanistic insights into how AβOs lead to synaptic pathology in AD. PMID:28211900

  17. Non-fibrillar beta-amyloid abates spike-timing-dependent synaptic potentiation at excitatory synapses in layer 2/3 of the neocortex by targeting postsynaptic AMPA receptors.

    PubMed

    Shemer, Isaac; Holmgren, Carl; Min, Rogier; Fülöp, Livia; Zilberter, Misha; Sousa, Kyle M; Farkas, Tamás; Härtig, Wolfgang; Penke, Botond; Burnashev, Nail; Tanila, Heikki; Zilberter, Yuri; Harkany, Tibor

    2006-04-01

    Cognitive decline in Alzheimer's disease (AD) stems from the progressive dysfunction of synaptic connections within cortical neuronal microcircuits. Recently, soluble amyloid beta protein oligomers (Abeta(ol)s) have been identified as critical triggers for early synaptic disorganization. However, it remains unknown whether a deficit of Hebbian-related synaptic plasticity occurs during the early phase of AD. Therefore, we studied whether age-dependent Abeta accumulation affects the induction of spike-timing-dependent synaptic potentiation at excitatory synapses on neocortical layer 2/3 (L2/3) pyramidal cells in the APPswe/PS1dE9 transgenic mouse model of AD. Synaptic potentiation at excitatory synapses onto L2/3 pyramidal cells was significantly reduced at the onset of Abeta pathology and was virtually absent in mice with advanced Abeta burden. A decreased alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)/N-methyl-D-aspartate (NMDA) receptor-mediated current ratio implicated postsynaptic mechanisms underlying Abeta synaptotoxicity. The integral role of Abeta(ol)s in these processes was verified by showing that pretreatment of cortical slices with Abeta((25-35)ol)s disrupted spike-timing-dependent synaptic potentiation at unitary connections between L2/3 pyramidal cells, and reduced the amplitude of miniature excitatory postsynaptic currents therein. A robust decrement of AMPA, but not NMDA, receptor-mediated currents in nucleated patches from L2/3 pyramidal cells confirmed that Abeta(ol)s perturb basal glutamatergic synaptic transmission by affecting postsynaptic AMPA receptors. Inhibition of AMPA receptor desensitization by cyclothiazide significantly increased the amplitude of excitatory postsynaptic potentials evoked by afferent stimulation, and rescued synaptic plasticity even in mice with pronounced Abeta pathology. We propose that soluble Abeta(ol)s trigger the diminution of synaptic plasticity in neocortical pyramidal cell networks during early

  18. Assessing the Effects of Acute Amyloid β Oligomer Exposure in the Rat

    PubMed Central

    Wong, Ryan S.; Cechetto, David F.; Whitehead, Shawn N.

    2016-01-01

    Alzheimer’s disease (AD) is the most common form of dementia, yet there are no therapeutic treatments that can either cure or delay its onset. Currently, the pathogenesis of AD is still uncertain, especially with respect to how the disease develops from a normal healthy brain. Amyloid β oligomers (AβO) are highly neurotoxic proteins and are considered potential initiators to the pathogenesis of AD. Rat brains were exposed to AβO via bilateral intracerebroventricular injections. Rats were then euthanized at either 1, 3, 7 or 21-days post surgery. Rat behavioural testing was performed using the Morris water maze and open field tests. Post-mortem brain tissue was immunolabelled for Aβ, microglia, and cholinergic neurons. Rats exposed to AβO showed deficits in spatial learning and anxiety-like behaviour. Acute positive staining for Aβ was only observed in the corpus callosum surrounding the lateral ventricles. AβO exposed rat brains also showed a delayed increase in activated microglia within the corpus callosum and a decreased number of cholinergic neurons within the basal forebrain. Acute exposure to AβO resulted in mild learning and memory impairments with co-concomitant white matter pathology within the corpus callosum and cholinergic cell loss within the basal forebrain. Results suggest that acute exposure to AβO in the rat may be a useful tool in assessing the early phases for the pathogenesis of AD. PMID:27563885

  19. Oleocanthal ameliorates amyloidoligomers' toxicity on astrocytes and neuronal cells: In vitro studies.

    PubMed

    Batarseh, Yazan S; Mohamed, Loqman A; Al Rihani, Sweilem B; Mousa, Youssef M; Siddique, Abu Bakar; El Sayed, Khalid A; Kaddoumi, Amal

    2017-04-07

    Extra-virgin olive oil (EVOO) has several health promoting effects. Evidence have shown that EVOO attenuates the pathology of amyloid-β (Aβ) and improves cognitive function in experimental animal models, suggesting it's potential to protect and reduce the risk of developing Alzheimer's disease (AD). Available studies have linked this beneficial effect to oleocanthal, one of the active components in EVOO. The effect of oleocanthal against AD pathology has been linked to its ability to attenuate Aβ and tau aggregation in vitro, and enhance Aβ clearance from the brains of wild-type and AD transgenic mice in vivo. However, the ability of oleocanthal to alter the toxic effect of Aβ on brain parenchymal cells is unknown. In the current study, we investigated oleocanthal effect on modulating Aβ oligomers (Aβo) pathological events in neurons and astrocytes. Our findings demonstrated oleocanthal prevented Aβo-induced synaptic proteins, SNAP-25 and PSD-95, down-regulation in neurons, and attenuated Aβo-induced inflammation, glutamine transporter (GLT1) and glucose transporter (GLUT1) down-regulation in astrocytes. Aβo-induced inflammation was characterized by interleukin-6 (IL-6) increase and glial fibrillary acidic protein (GFAP) upregulation that were reduced by oleocanthal. In conclusion, this study provides further evidence to support the protective effect of EVOO-derived phenolic secoiridoid oleocanthal against AD pathology.

  20. Selective amyloid β oligomer assay based on abasic site-containing molecular beacon and enzyme-free amplification.

    PubMed

    Zhu, Linling; Zhang, Junying; Wang, Fengyang; Wang, Ya; Lu, Linlin; Feng, Chongchong; Xu, Zhiai; Zhang, Wen

    2016-04-15

    Amyloid-beta (Aβ) oligomers are highly toxic species in the process of Aβ aggregation and are regarded as potent therapeutic targets and diagnostic markers for Alzheimer's disease (AD). Herein, a label-free molecular beacon (MB) system integrated with enzyme-free amplification strategy was developed for simple and highly selective assay of Aβ oligomers. The MB system was constructed with abasic site (AP site)-containing stem-loop DNA and a fluorescent ligand 2-amino-5,6,7-trimethyl-1,8-naphyridine (ATMND), of which the fluorescence was quenched upon binding to the AP site in DNA stem. Enzyme-free amplification was realized by target-triggered continuous opening of two delicately designed MBs (MB1 and MB2). Target DNA hybridization with MB1 and then MB2 resulted in the release of two ATMND molecules in one binding event. Subsequent target recycling could greatly amplify the detection sensitivity due to the greatly enhanced turn-on emission of ATMND fluorescence. Combining with Aβ oligomers aptamers, the strategy was applied to analyze Aβ oligomers and the results showed that it could quantify Aβ oligomers with high selectivity and monitor the Aβ aggregation process. This novel method may be conducive to improve the diagnosis and pathogenic study of Alzheimer's disease. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Amyloid β oligomers induce interleukin-1β production in primary microglia in a cathepsin B- and reactive oxygen species-dependent manner

    SciTech Connect

    Taneo, Jun; Adachi, Takumi; Yoshida, Aiko; Takayasu, Kunio; Takahara, Kazuhiko; Inaba, Kayo

    2015-03-13

    Amyloid β (Aβ) peptide, a causative agent of Alzheimer's disease, forms two types of aggregates: oligomers and fibrils. These aggregates induce inflammatory responses, such as interleukin-1β (IL-1β) production by microglia, which are macrophage-like cells located in the brain. In this study, we examined the effect of the two forms of Aβ aggregates on IL-1β production in mouse primary microglia. We prepared Aβ oligomer and fibril from Aβ (1–42) peptide in vitro. We analyzed the characteristics of these oligomers and fibrils by electrophoresis and atomic force microscopy. Interestingly, Aβ oligomers but not Aβ monomers or fibrils induced robust IL-1β production in the presence of lipopolysaccharide. Moreover, Aβ oligomers induced endo/phagolysosome rupture, which released cathepsin B into the cytoplasm. Aβ oligomer-induced IL-1β production was inhibited not only by the cathepsin B inhibitor CA-074-Me but also by the reactive oxygen species (ROS) inhibitor N-acetylcysteine. Random chemical crosslinking abolished the ability of the oligomers to induce IL-1β. Thus, multimerization and fibrillization causes Aβ oligomers to lose the ability to induce IL-1β. These results indicate that Aβ oligomers, but not fibrils, induce IL-1β production in primary microglia in a cathepsin B- and ROS-dependent manner. - Highlights: • We prepared amyloid β (Aβ) fibrils with minimum contamination of Aβ oligomers. • Primary microglia (MG) produced IL-1β in response to Aβ oligomers, but not fibrils. • Only Aβ oligomers induced leakage of cathepsin B from endo/phagolysosomes. • IL-1β production in response to Aβ oligomers depended on both cathepsin B and ROS. • Crosslinking reduced the ability of the Aβ oligomers to induce IL-1β from MG.

  2. Dispersible amyloid β-protein oligomers, protofibrils, and fibrils represent diffusible but not soluble aggregates: their role in neurodegeneration in amyloid precursor protein (APP) transgenic mice.

    PubMed

    Rijal Upadhaya, Ajeet; Capetillo-Zarate, Estibaliz; Kosterin, Irina; Abramowski, Dorothee; Kumar, Sathish; Yamaguchi, Haruyasu; Walter, Jochen; Fändrich, Marcus; Staufenbiel, Matthias; Thal, Dietmar Rudolf

    2012-11-01

    Soluble amyloid β-protein (Aβ) aggregates have been identified in the Alzheimer's disease (AD) brain. Dispersed Aβ aggregates in the brain parenchyma are different from soluble, membrane-associated and plaque-associated solid aggregates. They are in mixture with the extra- or intracellular fluid but can be separated from soluble proteins by ultracentrifugation. To clarify the role of dispersible Aβ aggregates for neurodegeneration we analyzed 2 different amyloid precursor protein (APP)-transgenic mouse models. APP23 mice overexpress human mutant APP with the Swedish mutation. APP51/16 mice express high levels of human wild type APP. Both mice develop Aβ-plaques. Dendritic degeneration, neuron loss, and loss of asymmetric synapses were seen in APP23 but not in APP51/16 mice. The soluble and dispersible fractions not separated from one another were received as supernatant after centrifugation of native forebrain homogenates at 14,000 × g. Subsequent ultracentrifugation separated the soluble, i.e., the supernatant, from the dispersible fraction, i.e., the resuspended pellet. The major biochemical difference between APP23 and APP51/16 mice was that APP23 mice exhibited higher levels of dispersible Aβ oligomers, protofibrils and fibrils precipitated with oligomer (A11) and protofibril/fibril (B10AP) specific antibodies than APP51/16 mice. These differences, rather than soluble Aβ and Aβ plaque pathology were associated with dendritic degeneration, neuron, and synapse loss in APP23 mice in comparison with APP51/16 mice. Immunoprecipitation of dispersible Aβ oligomers, protofibrils, and fibrils revealed that they were associated with APP C-terminal fragments (APP-CTFs). These results indicate that dispersible Aβ oligomers, protofibrils, and fibrils represent an important pool of Aβ aggregates in the brain that critically interact with membrane-associated APP C-terminal fragments. The concentration of dispersible Aβ aggregates, thereby, presumably determines

  3. Reduction of Blood AmyloidOligomers in Alzheimer's Disease Transgenic Mice by c-Abl Kinase Inhibition.

    PubMed

    Estrada, Lisbell D; Chamorro, David; Yañez, María José; Gonzalez, Marcelo; Leal, Nancy; von Bernhardi, Rommy; Dulcey, Andrés E; Marugan, Juan; Ferrer, Marc; Soto, Claudio; Zanlungo, Silvana; Inestrosa, Nibaldo C; Alvarez, Alejandra R

    2016-10-04

    One of the pathological hallmarks of Alzheimer's disease (AD) is the presence of amyloid plaques, which are deposits of misfolded and aggregated amyloid-beta peptide (Aβ). The role of the c-Abl tyrosine kinase in Aβ-mediated neurodegeneration has been previously reported. Here, we investigated the therapeutic potential of inhibiting c-Abl using imatinib. We developed a novel method, based on a technique used to detect prions (PMCA), to measure minute amounts of misfolded-Aβ in the blood of AD transgenic mice. We found that imatinib reduces Aβ-oligomers in plasma, which correlates with a reduction of AD brain features such as plaques and oligomers accumulation, neuroinflammation, and cognitive deficits. Cells exposed to imatinib and c-Abl KO mice display decreased levels of β-CTF fragments, suggesting that an altered processing of the amyloid-beta protein precursor is the most probable mechanism behind imatinib effects. Our findings support the role of c-Abl in Aβ accumulation and AD, and propose AD-PMCA as a new tool to evaluate AD progression and screening for drug candidates.

  4. Role of the Fast Kinetics of Pyroglutamate-Modified AmyloidOligomers in Membrane Binding and Membrane Permeability

    PubMed Central

    2015-01-01

    Membrane permeability to ions and small molecules is believed to be a critical step in the pathology of Alzheimer’s disease (AD). Interactions of oligomers formed by amyloid-β (Aβ) peptides with the plasma cell membrane are believed to play a fundamental role in the processes leading to membrane permeability. Among the family of Aβs, pyroglutamate (pE)-modified Aβ peptides constitute the most abundant oligomeric species in the brains of AD patients. Although membrane permeability mechanisms have been studied for full-length Aβ1–40/42 peptides, these have not been sufficiently characterized for the more abundant AβpE3–42 fragment. Here we have compared the adsorbed and membrane-inserted oligomeric species of AβpE3–42 and Aβ1–42 peptides. We find lower concentrations and larger dimensions for both species of membrane-associated AβpE3–42 oligomers. The larger dimensions are attributed to the faster self-assembly kinetics of AβpE3–42, and the lower concentrations are attributed to weaker interactions with zwitterionic lipid headgroups. While adsorbed oligomers produced little or no significant membrane structural damage, increased membrane permeabilization to ionic species is understood in terms of enlarged membrane-inserted oligomers. Membrane-inserted AβpE3–42 oligomers were also found to modify the mechanical properties of the membrane. Taken together, our results suggest that membrane-inserted oligomers are the primary species responsible for membrane permeability. PMID:24950761

  5. Domains of STIP1 responsible for regulating PrPC-dependent amyloidoligomer toxicity.

    PubMed

    Maciejewski, Andrzej; Ostapchenko, Valeriy G; Beraldo, Flavio H; Prado, Vania F; Prado, Marco A M; Choy, Wing-Yiu

    2016-07-15

    Soluble oligomers of amyloid-beta peptide (AβO) transmit neurotoxic signals through the cellular prion protein (PrP(C)) in Alzheimer's disease (AD). Secreted stress-inducible phosphoprotein 1 (STIP1), an Hsp70 and Hsp90 cochaperone, inhibits AβO binding to PrP(C) and protects neurons from AβO-induced cell death. Here, we investigated the molecular interactions between AβO and STIP1 binding to PrP(C) and their effect on neuronal cell death. We showed that residues located in a short region of PrP (90-110) mediate AβO binding and we narrowed the major interaction in this site to amino acids 91-100. In contrast, multiple binding sites on STIP1 (DP1, TPR1 and TPR2A) contribute to PrP binding. DP1 bound the N-terminal of PrP (residues 23-95), whereas TPR1 and TPR2A showed binding to the C-terminal of PrP (residues 90-231). Importantly, only TPR1 and TPR2A directly inhibit both AβO binding to PrP and cell death. Furthermore, our structural studies reveal that TPR1 and TPR2A bind to PrP through distinct regions. The TPR2A interface was shown to be much more extensive and to partially overlap with the Hsp90 binding site. Our data show the possibility of a PrP, STIP1 and Hsp90 ternary complex, which may influence AβO-mediated cell death. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

  6. Binding affinity of amyloid oligomers to cellular membranes is a generic indicator of cellular dysfunction in protein misfolding diseases

    PubMed Central

    Evangelisti, Elisa; Cascella, Roberta; Becatti, Matteo; Marrazza, Giovanna; Dobson, Christopher M.; Chiti, Fabrizio; Stefani, Massimo; Cecchi, Cristina

    2016-01-01

    The conversion of peptides or proteins from their soluble native states into intractable amyloid deposits is associated with a wide range of human disorders. Misfolded protein oligomers formed during the process of aggregation have been identified as the primary pathogenic agents in many such conditions. Here, we show the existence of a quantitative relationship between the degree of binding to neuronal cells of different types of oligomers formed from a model protein, HypF-N, and the GM1 content of the plasma membranes. In addition, remarkably similar behavior is observed for oligomers of the Aβ42 peptide associated with Alzheimer’s disease. Further analysis has revealed the existence of a linear correlation between the level of the influx of Ca2+ across neuronal membranes that triggers cellular damage, and the fraction of oligomeric species bound to the membrane. Our findings indicate that the susceptibility of neuronal cells to different types of misfolded oligomeric assemblies is directly related to the extent of binding of such oligomers to the cellular membrane. PMID:27619987

  7. Binding affinity of amyloid oligomers to cellular membranes is a generic indicator of cellular dysfunction in protein misfolding diseases.

    PubMed

    Evangelisti, Elisa; Cascella, Roberta; Becatti, Matteo; Marrazza, Giovanna; Dobson, Christopher M; Chiti, Fabrizio; Stefani, Massimo; Cecchi, Cristina

    2016-09-13

    The conversion of peptides or proteins from their soluble native states into intractable amyloid deposits is associated with a wide range of human disorders. Misfolded protein oligomers formed during the process of aggregation have been identified as the primary pathogenic agents in many such conditions. Here, we show the existence of a quantitative relationship between the degree of binding to neuronal cells of different types of oligomers formed from a model protein, HypF-N, and the GM1 content of the plasma membranes. In addition, remarkably similar behavior is observed for oligomers of the Aβ42 peptide associated with Alzheimer's disease. Further analysis has revealed the existence of a linear correlation between the level of the influx of Ca(2+) across neuronal membranes that triggers cellular damage, and the fraction of oligomeric species bound to the membrane. Our findings indicate that the susceptibility of neuronal cells to different types of misfolded oligomeric assemblies is directly related to the extent of binding of such oligomers to the cellular membrane.

  8. Cu K-edge X-ray Absorption Spectroscopy Reveals Differential Copper Coordimation Within Amyloid-beta Oligomers Compared to Amyloid-beta Monomers

    SciTech Connect

    J Shearer; P Callan; T Tran; V Szalai

    2011-12-31

    The fatal neurodegenerative disorder Alzheimer's disease (AD) has been linked to the formation of soluble neurotoxic oligomers of amyloid-{beta} (A{beta}) peptides. These peptides have high affinities for copper cations. Despite their potential importance in AD neurodegeneration few studies have focused on probing the Cu{sup 2+/1+} coordination environment within A{beta} oligomers. Herein we present a Cu K-edge X-ray absorption spectroscopic study probing the copper-coordination environment within oligomers of A{beta}(42) (sequence: DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA). We find that the Cu{sup 2+} cation is contained within a square planar mixed N/O ligand environment within A{beta}(42) oligomers, which is similar to the copper coordination environment of the monomeric forms of {l_brace}Cu{sup II}A{beta}(40){r_brace} and {l_brace}Cu{sup II}A{beta}(16){r_brace}. Reduction of the Cu{sup 2+} cation within the A{beta}(42) oligomers to Cu{sup 1+} yields a highly dioxygen sensitive copper-species that contains Cu{sup 1+} in a tetrahedral coordination geometry. This can be contrasted with monomers of {l_brace}Cu{sup I}A{beta}(40){r_brace} and {l_brace}Cu{sup I}A{beta}(16){r_brace}, which contain copper in a dioxygen inert linear bis-histidine ligand environment [Shearer and Szalai, J. Am. Chem. Soc., 2008, 130, 17826]. The biological implications of these findings are discussed.

  9. Soluble Amyloid β-Oligomers Affect Dielectric Membrane Properties by Bilayer Insertion and Domain Formation: Implications for Cell Toxicity

    PubMed Central

    Valincius, Gintaras; Heinrich, Frank; Budvytyte, Rima; Vanderah, David J.; McGillivray, Duncan J.; Sokolov, Yuri; Hall, James E.; Lösche, Mathias

    2008-01-01

    It is well established that Alzheimer's amyloid β-peptides reduce the membrane barrier to ion transport. The prevailing model ascribes the resulting interference with ion homeostasis to the formation of peptide pores across the bilayer. In this work, we examine the interaction of soluble prefibrillar amyloid β (Aβ1–42)-oligomers with bilayer models, observing also dramatic increases in ion current at micromolar peptide concentrations. We demonstrate that the Aβ-induced ion conductances across free-standing membranes and across substrate-supported “tethered” bilayers are quantitatively similar and depend on membrane composition. However, characteristic signatures of the molecular transport mechanism were distinctly different from ion transfer through water-filled pores, as shown by a quantitative comparison of the membrane response to Aβ-oligomers and to the bacterial toxin α-hemolysin. Neutron reflection from tethered membranes showed that Aβ-oligomers insert into the bilayer, affecting both membrane leaflets. By measuring the capacitance of peptide-free membranes, as well as their geometrical thicknesses, the dielectric constants in the aliphatic cores of 1,2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-diphytanoyl-sn-glycero-3-phosphocholine bilayers were determined to be ɛ = 2.8 and 2.2, respectively. The magnitude of the Aβ-induced increase in ɛ indicates that Aβ-oligomers affect membranes by inducing lateral heterogeneity in the bilayers, but an increase in the water content of the bilayers was not observed. The activation energy for Aβ-induced ion transport across the membrane is at least three times higher than that measured for membranes reconstituted with α-hemolysin pores, Ea = 36.8 vs. 9.9 kJ/mol, indicating that the molecular mechanisms underlying both transport processes are fundamentally different. The Aβ-induced membrane conductance shows a nonlinear dependence on the peptide concentration in the membrane. Moreover, Ea depends on

  10. Postsynaptic Receptors for AmyloidOligomers as Mediators of Neuronal Damage in Alzheimer’s Disease

    PubMed Central

    Dinamarca, Margarita C.; Ríos, Juvenal A.; Inestrosa, Nibaldo C.

    2012-01-01

    The neurotoxic effect of amyloid-β peptide (Aβ) over the central synapses has been described and is reflected in the decrease of some postsynaptic excitatory proteins, the alteration in the number and morphology of the dendritic spines, and a decrease in long-term potentiation. Many studies has been carried out to identify the putative Aβ receptors in neurons, and is still no clear why the Aβ oligomers only affect the excitatory synapses. Aβ oligomers bind to neurite and preferentially to the postsynaptic region, where the postsynaptic protein-95 (PSD-95) is present in the glutamatergic synapse, and interacts directly with the N-methyl-D-aspartate receptor (NMDAR) and neuroligin (NL). NL is a postsynaptic protein which binds to the presynaptic protein, neurexin to form a heterophilic adhesion complex, the disruption of this interaction affects the integrity of the synaptic contact. Structurally, NL has an extracellular domain homolog to acetylcholinesterase, the first synaptic protein that was found to interact with Aβ. In the present review we will document the interaction between Aβ and the extracellular domain of NL-1 at the excitatory synapse, as well as the interaction with other postsynaptic components, including the glutamatergic receptors (NMDA and mGluR5), the prion protein, the neurotrophin receptor, and the α7-nicotinic acetylcholine receptor. We conclude that several Aβ oligomers receptors exist at the excitatory synapse, which could be the responsible for the neurotoxic effect described for the Aβ oligomers. The characterization of the interaction between Aβ receptors and Aβ oligomers could help to understand the source of the neurologic damage observed in the brain of the Alzheimer’s disease patients. PMID:23267328

  11. An N-terminal antibody promotes the transformation of amyloid fibrils into oligomers and enhances the neurotoxicity of amyloid-beta: the dust-raising effect.

    PubMed

    Liu, Yu-Hui; Bu, Xian-Le; Liang, Chun-Rong; Wang, Ye-Ran; Zhang, Tao; Jiao, Shu-Sheng; Zeng, Fan; Yao, Xiu-Qing; Zhou, Hua-Dong; Deng, Juan; Wang, Yan-Jiang

    2015-08-28

    Senile plaques consisting of amyloid-beta (Aβ) are the major pathological hallmark of Alzheimer's disease (AD) and have been the primary therapeutic target. Immunotherapies, which are designed to remove brain Aβ deposits, increased levels of soluble Aβ and accelerated brain atrophy in some clinical trials, suggesting that the solubilization of Aβ deposition might facilitate the formation of more toxic Aβ oligomers and enhance neurotoxicity. The capacity of antibodies against different epitopes of Aβ to disaggregate preformed Aβ fibrils was investigated. The co-incubation of antibodies and Aβ fibrils was then tested for neurotoxicity both in vitro and in vivo. After the incubation of preformed Aβ fibrils with the N-terminal antibody 6E10, the fibrils were decreased, while the oligomers, mostly dimers and trimers, were significantly increased. However, no such effects were observed for antibodies targeting the middle domain (4G8) and C-terminus of Aβ (8G7). The co-incubates of preformed Aβ fibrils with 6E10 were more neurotoxic, both in vitro and in vivo, than the co-incubates with 4G8 and 8G7. Our results indicate that the antibody targeting the N-terminus of Aβ promoted the transformation of Aβ from fibrils into oligomers and increased neurotoxicity. Immunotherapies should take into consideration the enhanced neurotoxicity associated with the solubilization of Aβ deposits by antibodies against the Nterminus of Aβ.

  12. A glycine zipper motif mediates the formation of toxic β-amyloid oligomers in vitro and in vivo.

    PubMed

    Fonte, Virginia; Dostal, Vishantie; Roberts, Christine M; Gonzales, Patrick; Lacor, Pascale N; Lacor, Pascale; Velasco, Pauline T; Magrane, Jordi; Dingwell, Natalie; Fan, Emily Y; Silverman, Michael A; Stein, Gretchen H; Link, Christopher D

    2011-08-23

    The β-amyloid peptide (Aβ) contains a Gly-XXX-Gly-XXX-Gly motif in its C-terminal region that has been proposed to form a "glycine zipper" that drives the formation of toxic Aβ oligomers. We have tested this hypothesis by examining the toxicity of Aβ variants containing substitutions in this motif using a neuronal cell line, primary neurons, and a transgenic C. elegans model. We found that a Gly37Leu substitution dramatically reduced Aβ toxicity in all models tested, as measured by cell dysfunction, cell death, synaptic alteration, or tau phosphorylation. We also demonstrated in multiple models that Aβ Gly37Leu is actually anti-toxic, thereby supporting the hypothesis that interference with glycine zipper formation blocks assembly of toxic Aβ oligomers. To test this model rigorously, we engineered second site substitutions in Aβ predicted by the glycine zipper model to compensate for the Gly37Leu substitution and expressed these in C. elegans. We show that these second site substitutions restore in vivo Aβtoxicity, further supporting the glycine zipper model. Our structure/function studies support the view that the glycine zipper motif present in the C-terminal portion of Aβ plays an important role in the formation of toxic Aβ oligomers. Compounds designed to interfere specifically with formation of the glycine zipper could have therapeutic potential.

  13. Concentrations of antibodies against β-amyloid 40/42 monomer and oligomers in Chinese intravenous immunoglobulins.

    PubMed

    Ye, Shengliang; Zeng, Renyong; Jiang, Peng; Hou, Mingxia; Liu, Fengjuan; Wang, Zongkui; Du, Xi; Yuan, Jing; Chen, Yunhua; Cao, Haijun; Ma, Li; Li, Changqing

    2017-02-17

    Intravenous immunoglobulin (IVIg) preparations are being investigated as a potential agent for treatment or prevention of Alzheimer's disease (AD). Antibodies towards soluble β-amyloid (Aβ) contained in IVIg were considered to be the major component contributing to the beneficial effect of the preparations in pilot studies. This study compared the antibody concentrations against Aβ in Octagam(®) IVIg (Octapharma) and 9 IVIg preparations from different Chinese manufacturers by ELISA, using Aβ40 monomer, Aβ40 soluble oligomers, Aβ42 monomer and Aβ42 soluble oligomers as the antigens. The results showed that each preparation contained different antibody levels against the four Aβ forms. The median values of the four antibody concentrations in Chinese IVIg preparations were 16.53, 8.47, 24.36 and 33.25μg/mL, which were remarkably higher than that in Octagam(®) IVIg (1.66, 2.07, 4.61 and 4.64μg/mL). Moreover, the anti-Aβ42 oligomer antibody levels in almost all IVIg preparations were higher than the anti-Aβ42 monomer antibody, and the concentrations of anti-Aβ42 antibodies in most of the IVIg preparations were significantly higher than that of anti-Aβ40 antibodies. These findings will contribute to an increased understanding of the uniqueness of Chinese IVIg preparations, and could provide support for a trial of a Chinese IVIg product in AD patients.

  14. Surface-enhanced Raman spectroscopy for detection of toxic amyloid β oligomers adsorbed on self-assembled monolayers

    NASA Astrophysics Data System (ADS)

    Voiciuk, Vladislava; Valincius, Gintaras; Budvytytė, Rima; Matijoška, Algirdas; Matulaitienė, Ieva; Niaura, Gediminas

    Surface-enhanced Raman spectroscopy (SERS) was used to detect different spectral features of small (1-2 nm) and large (5-10 nm) synthetic amyloid Aβ-42 oligomers, exhibiting high and no detectable neurotoxicities, respectively. Adsorption of peptides at self-assembled monolayers (SAM) terminated by methyl and pyridinium groups was employed to differentiate toxic and non-toxic oligomers. Three SAMs were analyzed: hydrophobic heptanethiol (HT) and octadecanethiol (ODT) as well as positively charged N-(6-mercapto)hexylpyridinium (MHP) chloride. SERS study revealed twofold adsorption effect, changes in the monolayer structure and appearance of new bands associated with the adsorbed peptides. A band at 1387 cm-1, observed as a result of the SAM and Aβ-42 interaction, is tentatively assigned to the peptide symmetric stretching vibration of carboxylate groups, and appears to be the most prominent spectral feature distinguishing toxic oligomers from the non-toxic Aβ-42 forms. This band was identified in the spectra of Aβ-42 adsorption on heptanethiol and MHP monolayers, while no clear perturbations were observed in the case of ODT monolayer.

  15. Beta-amyloid 1-42 monomers, but not oligomers, produce PHF-like conformation of Tau protein.

    PubMed

    Manassero, Giusi; Guglielmotto, Michela; Zamfir, Raluca; Borghi, Roberta; Colombo, Laura; Salmona, Mario; Perry, George; Odetti, Patrizio; Arancio, Ottavio; Tamagno, Elena; Tabaton, Massimo

    2016-10-01

    The mechanistic relationship between amyloid β1-42 (Aβ1-42) and the alteration of Tau protein are debated. We investigated the effect of Aβ1-42 monomers and oligomers on Tau, using mice expressing wild-type human Tau that do not spontaneously develop Tau pathology. After intraventricular injection of Aβ1-42, mice were sacrificed after 3 h or 4 days. The short-lasting treatment with Aβ monomers, but not oligomers, showed a conformational PHF-like change of Tau, together with hyperphosphorylation. The same treatment induced increase in concentration of GSK3 and MAP kinases. The inhibition of the kinases rescued the Tau changes. Aβ monomers increased the levels of total Tau, through the inhibition of proteasomal degradation. Aβ oligomers reproduced all the aforementioned alterations only after 4 days of treatment. It is known that Aβ1-42 monomers foster synaptic activity. Our results suggest that Aβ monomers physiologically favor Tau activity and dendritic sprouting, whereas their excess causes Tau pathology. Moreover, our study indicates that anti-Aβ therapies should be targeted to Aβ1-42 monomers too.

  16. Computational insights into the inhibition and destabilization of morin on the oligomer of full-length human islet amyloid polypeptide.

    PubMed

    Wang, Qianqian; Zhou, Shuangyan; Wei, Wei; Yao, Xiaojun; Liu, Huanxiang; Hu, Zhide

    2015-11-21

    The aggregation of human islet amyloid polypeptide (hIAPP) is closely related with the occurrence of type 2 diabetes (T2D). Natural flavonoid morin was confirmed to not only inhibit the amyloid formation of hIAPP, but disaggregate its preformed amyloid fibrils. In this study, with the goal of elucidating the molecular mechanism of inhibition and destabilization of morin on the full-length hIAPP(1-37) oligomer, molecular dynamics simulations were performed for hIAPP(1-37) pentamer in the presence and absence of morin. The obtained results show that during the protein-inhibitor interaction, morin can notably alter the structural properties of hIAPP(1-37) pentamer, such as morphology, solvent accessible surface area and secondary structure. Moreover, we identified three possible binding sites of morin on hIAPP, all of which located near the amyloidogenic region of this protein. From the binding free energy calculations, we found that Site II was the most possible one. Further conformational analysis together with energy decomposition showed that the residues His18, Phe23 and Ile26 play a key role in the binding with morin by hydrogen bond, π-π and hydrophobic interactions. The proposal of the theoretical mechanism of morin against hIAPP aggregation will provide valuable information for the development of new drugs to inhibit hIAPP aggregation.

  17. Small angle X-ray scattering analysis of Cu(2+)-induced oligomers of the Alzheimer's amyloid β peptide.

    PubMed

    Ryan, Timothy M; Kirby, Nigel; Mertens, Haydyn D T; Roberts, Blaine; Barnham, Kevin J; Cappai, Roberto; Pham, Chi Le Lan; Masters, Colin L; Curtain, Cyril C

    2015-03-01

    Research into causes of Alzheimer's disease and its treatment has produced a tantalising array of hypotheses about the role of transition metal dyshomeostasis, many of them on the interaction of these metals with the neurotoxic amyloid-β peptide (Aβ). Here, we have used small angle X-ray scattering (SAXS) to study the effect of the molar ratio, Cu(2+)/Aβ, on the early three-dimensional structures of the Aβ1-40 and Cu(2+)/Aβ1-42 peptides in solution. We found that at molar ratios of 0.5 copper to peptide Aβ1-40 aggregated, while Aβ1-42 adopted a relatively monodisperse cylindrical shape, and at a ratio of 1.5 copper to peptide Aβ1-40 adopted a monodisperse cylindrical shape, while Aβ1-42 adopted the shape of an ellipsoid of rotation. We also found, via in-line rapid mixing SAXS analysis, that both peptides in the absence of copper were monodisperse at very short timeframes (<2 s). Kratky plots of these scattering profiles indicated that immediately after mixing both were intrinsically disordered. Ensemble optimisation modelling reflected this, indicating a wide range of structural conformers. These data reflect the ensembles from which the Cu(2+)-promoted oligomers were derived. Our results are discussed in the light of other studies that have shown that the Cu(2+)/Aβ has a marked effect on fibril and oligomer formation by this peptide, with a higher ratio favouring the formation of cytotoxic non-amyloid oligomers. Our results are relatively consistent with previous two-dimensional studies of the conformations of these Cu(2+)-induced entities, made on a much longer time-scale than SAXS, by transmission electron microscopy and atomic force microscopy, which showed that a range of oligomeric species are formed. We propose that SAXS carried out on a modern synchrotron beamline enables studies on initial events in disordered protein folding on physiologically-relevant time-scales, and will likely provide great insight into the initiating processes of the A

  18. A mechanistic model of tau amyloid aggregation based on direct observation of oligomers

    NASA Astrophysics Data System (ADS)

    Shammas, Sarah L.; Garcia, Gonzalo A.; Kumar, Satish; Kjaergaard, Magnus; Horrocks, Mathew H.; Shivji, Nadia; Mandelkow, Eva; Knowles, Tuomas P. J.; Mandelkow, Eckhard; Klenerman, David

    2015-04-01

    Protein aggregation plays a key role in neurodegenerative disease, giving rise to small oligomers that may become cytotoxic to cells. The fundamental microscopic reactions taking place during aggregation, and their rate constants, have been difficult to determine due to lack of suitable methods to identify and follow the low concentration of oligomers over time. Here we use single-molecule fluorescence to study the aggregation of the repeat domain of tau (K18), and two mutant forms linked with familial frontotemporal dementia, the deletion mutant ΔK280 and the point mutant P301L. Our kinetic analysis reveals that aggregation proceeds via monomeric assembly into small oligomers, and a subsequent slow structural conversion step before fibril formation. Using this approach, we have been able to quantitatively determine how these mutations alter the aggregation energy landscape.

  19. The Familial British Dementia Mutation Promotes Formation of Neurotoxic Cystine Cross-linked Amyloid Bri (ABri) Oligomers.

    PubMed

    Cantlon, Adam; Frigerio, Carlo Sala; Freir, Darragh B; Boland, Barry; Jin, Ming; Walsh, Dominic M

    2015-07-03

    Familial British dementia (FBD) is an inherited neurodegenerative disease believed to result from a mutation in the BRI2 gene. Post-translational processing of wild type BRI2 and FBD-BRI2 result in the production of a 23-residue long Bri peptide and a 34-amino acid long ABri peptide, respectively, and ABri is found deposited in the brains of individuals with FBD. Similarities in the neuropathology and clinical presentation shared by FBD and Alzheimer disease (AD) have led some to suggest that ABri and the AD-associated amyloid β-protein (Aβ) are molecular equivalents that trigger analogous pathogenic cascades. But the sequences and innate properties of ABri and Aβ are quite different, notably ABri contains two cysteine residues that can form disulfide bonds. Thus we sought to determine whether ABri was neurotoxic and if this activity was regulated by oxidation and/or aggregation. Crucially, the type of oxidative cross-linking dramatically influenced both ABri aggregation and toxicity. Cyclization of Bri and ABri resulted in production of biologically inert monomers that showed no propensity to assemble, whereas reduced ABri and reduced Bri aggregated forming thioflavin T-positive amyloid fibrils that lacked significant toxic activity. ABri was more prone to form inter-molecular disulfide bonds than Bri and the formation of covalently stabilized ABri oligomers was associated with toxicity. These results suggest that extension of the C-terminal of Bri causes a shift in the type of disulfide bonds formed and that structures built from covalently cross-linked oligomers can interact with neurons and compromise their function and viability. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. The Familial British Dementia Mutation Promotes Formation of Neurotoxic Cystine Cross-linked Amyloid Bri (ABri) Oligomers*

    PubMed Central

    Cantlon, Adam; Frigerio, Carlo Sala; Freir, Darragh B.; Boland, Barry; Jin, Ming; Walsh, Dominic M.

    2015-01-01

    Familial British dementia (FBD) is an inherited neurodegenerative disease believed to result from a mutation in the BRI2 gene. Post-translational processing of wild type BRI2 and FBD-BRI2 result in the production of a 23-residue long Bri peptide and a 34-amino acid long ABri peptide, respectively, and ABri is found deposited in the brains of individuals with FBD. Similarities in the neuropathology and clinical presentation shared by FBD and Alzheimer disease (AD) have led some to suggest that ABri and the AD-associated amyloid β-protein (Aβ) are molecular equivalents that trigger analogous pathogenic cascades. But the sequences and innate properties of ABri and Aβ are quite different, notably ABri contains two cysteine residues that can form disulfide bonds. Thus we sought to determine whether ABri was neurotoxic and if this activity was regulated by oxidation and/or aggregation. Crucially, the type of oxidative cross-linking dramatically influenced both ABri aggregation and toxicity. Cyclization of Bri and ABri resulted in production of biologically inert monomers that showed no propensity to assemble, whereas reduced ABri and reduced Bri aggregated forming thioflavin T-positive amyloid fibrils that lacked significant toxic activity. ABri was more prone to form inter-molecular disulfide bonds than Bri and the formation of covalently stabilized ABri oligomers was associated with toxicity. These results suggest that extension of the C-terminal of Bri causes a shift in the type of disulfide bonds formed and that structures built from covalently cross-linked oligomers can interact with neurons and compromise their function and viability. PMID:25957407

  1. Pharmacokinetic Properties of a Novel D-Peptide Developed to be Therapeutically Active Against Toxic β-Amyloid Oligomers.

    PubMed

    Leithold, Leonie H E; Jiang, Nan; Post, Julia; Ziehm, Tamar; Schartmann, Elena; Kutzsche, Janine; Shah, N Jon; Breitkreutz, Jörg; Langen, Karl-Josef; Willuweit, Antje; Willbold, Dieter

    2016-02-01

    It has been shown that amyloid β (Aβ) oligomers play an important role in the pathology of Alzheimer's disease (AD). D3, a peptide consisting solely of D-enantiomeric amino acid residues, was developed to specifically eliminate Aβ oligomers and is therapeutically active in transgenic AD mice. D-peptides have several advantages over L-peptides, but little is known about their pharmacokinetic potential in vivo. Here, we analysed the pharmacokinetic properties of RD2, a rationally designed and potent D3 derivative. The pharmacokinetic analysis was performed using (3)H-RD2 after administration via several routes in mice. The time dependent amount of radiolabelled RD2 was measured in plasma and several organ homogenates by liquid scintillation counting. Furthermore, binding to plasma proteins was estimated. RD2 penetrates into the brain, where it is thought to implement its therapeutic function. All administration routes result in a maximal brain concentration per dose (Cmax/D) of 0.06 (μg/g)/(mg/kg) with brain/plasma ratios ranging between 0.7 and 1.0. RD2 shows a small elimination constant and a long terminal half-life in plasma of more than 2 days. It also exhibits high bioavailability after i.p., s.c. or p.o. administration. These excellent pharmacokinetic properties confirm that RD2 is a very promising drug candidate for AD.

  2. Alzheimer AmyloidOligomer Bound to Post-Synaptic Prion Protein Activates Fyn to Impair Neurons

    PubMed Central

    Um, Ji Won; Nygaard, Haakon B.; Heiss, Jacqueline K.; Kostylev, Mikhail A.; Stagi, Massimiliano; Vortmeyer, Alexander; Wisniewski, Thomas; Gunther, Erik C.; Strittmatter, Stephen M.

    2012-01-01

    SUMMARY Amyloid-beta (Aβ) oligomers are thought to trigger Alzheimer’s disease (AD) pathophysiology. Cellular Prion Protein (PrPC) selectively binds oligomeric Aβ and can mediate AD-related phenotypes. Here, we examined the specificity, distribution and signaling from Aβ/PrP complexes, seeking to explain how they might alter the function of NMDA receptors in neurons. PrPC is enriched in post-synaptic densities, and Aβ/PrPC interaction leads to Fyn kinase activation. Soluble Aβ assemblies derived from human AD brain interact with PrPC to activate Fyn. Aβ engagement of PrPC/Fyn signaling yields phosphorylation of the NR2B subunit of NMDA-receptors, which is coupled to an initial increase and then loss of surface NMDA-receptors. Aβ-induced LDH release and dendritic spine loss require both PrPC and Fyn, and human familial AD transgene-induced convulsive seizures do not occur in mice lacking PrPC. These results delineate an Aβ oligomer signal transduction pathway requiring PrPC and Fyn to alter synaptic function with relevance to AD. PMID:22820466

  3. Astrocytic Expression of the Immunoreceptor CD300f Protects Hippocampal Neurons from AmyloidOligomer Toxicity In Vitro.

    PubMed

    Lima, Thiago Zaqueu; Sardinha, Luis Roberto; Sayos, Joan; Mello, Luiz Eugenio; Peluffo, Hugo

    2017-01-01

    Astrocytes contribute to neuroinflammation that accompanies neurodegenerative disorders such as Alzheimer's disease (AD). In this sense, the toxicity of these diseases might be attenuated through the modulation of astrocytic inflammatory responses. Recently, the CD300f immunoreceptor was described as a new member of the CD300 immunoreceptor family, showing promising modulatory properties. Here, we investigated whether overexpression of hCD300f (the human isoform of CD300f) in astrocytes protects hippocampal neurons against the degeneration induced by amyloid-beta (Aβ) oligomer. Astrocyte monolayers were transfected with hCD300f before seeding the hippocampal neurons, and then the co-culture was exposed to Aβ1-42 oligomers (5 μM, 48h). hCD300f expression significantly abrogated the neuronal loss elicited by Aβ. This effect was dependent on neuron-astrocyte cell-cell interactions since no protection was observed using conditioned media from transfected astrocytes. Astrocyte modulation was dependent on the cytoplasmic signaling tail of hCD300f. Furthermore hCD300f expression did not affect the ability of astrocytes to uptake Aβ1- 42 oligomers by endocytosis, which discards the possibility that increased Aβ1-42 clearance could mediate neuroprotection by hCD300f. These results suggest that the astrocyte-directed expression of the hCD300f immune receptor can be a neuroprotective strategy in AD disease. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  4. Enhancement of β-amyloid oligomer accumulation after intracerebroventricular injection of streptozotocin, which involves central insulin signaling in a transgenic mouse model.

    PubMed

    Lin, Fangju; Jia, Jianping; Qin, Wei

    2014-11-12

    The β-amyloid (Aβ) oligomer rather than fibrillar Aβ has become the important focus of recent studies on the pathogenesis of Alzheimer's disease (AD). Insulin signaling plays important roles in cognitive disease, such as AD. However, in-vivo evidence for the link between central insulin signaling and the Aβ oligomer are lacking, and the mechanisms underlying the effect of central insulin signaling on AD are still elusive. Our team has established the Presenilin-1 Val97Leu mutant transgenic (PS1V97L) AD mouse model with the intraneuronal Aβ oligomer as the potential initiator for other pathologies, but without extracellular amyloid plaque formation. Using this model, we investigated the roles of disturbed central insulin signaling induced by intracerebroventricular injection of streptozotocin (STZ) in the progression of AD. We observed that PS1V97L mice after intracerebroventricular injection of STZ showed increased Aβ oligomer accumulation and aggravated spatial learning and memory deficit in the absence of diabetes symptoms. Furthermore, STZ administration inhibited the activation of the insulin receptor and enhanced the activation of c-Jun NH2-terminal kinase, which was accompanied by increased production of carboxy-terminal fragments from the amyloid precursor protein, in the brain of PS1V97L mice. Overall, our study provided in-vivo evidence for a role of central insulin signaling in AD progression.

  5. Lithium and memantine improve spatial memory impairment and neuroinflammation induced by β-amyloid 1-42 oligomers in rats.

    PubMed

    Budni, J; Feijó, D P; Batista-Silva, H; Garcez, M L; Mina, F; Belletini-Santos, T; Krasilchik, L R; Luz, A P; Schiavo, G L; Quevedo, J

    2017-05-01

    Alzheimer's disease (AD) is the most common cause of dementia in the elderly. The main hallmarks of this disease include progressive cognitive dysfunction and an accumulation of soluble oligomers of β-amyloid (Aβ) 1-42 peptide. In this research, we show the effects of lithium and memantine on spatial memory and neuroinflammation in an Aβ1-42 oligomers-induced animal model of dementia in rats. Aβ 1-42 oligomers were administered intrahippocampally to male wistar rats to induce dementia. Oral treatments with memantine (5mg/kg), lithium (5mg/kg), or both drugs in combination were performed over a period of 17days. 14days after the administration of the Aβ1-42 oligomers, the radial arm-maze task was performed. At the end of the test period, the animals were euthanized, and the frontal cortex and hippocampus were removed for use in our analysis. Our results showed that alone treatments with lithium or memantine ameliorate the spatial memory damage caused by Aβ1-42. The animals that received combined doses of lithium and memantine showed better cognitive performance in their latency time and total errors to find food when compared to the results from alone treatments. Moreover, in our study, lithium and/or memantine were able to reverse the decreases observed in the levels of interleukin (IL)-4 that were induced by Aβ1-42 in the frontal cortex. In the hippocampus, only memantine and the association of memantine and lithium were able to reverse this effect. Alone doses of lithium and memantine or the association of lithium and memantine caused reductions in the levels of IL-1β in the frontal cortex and hippocampus, and decreased the levels of TNF-α in the hippocampus. Taken together, these data suggest that lithium and memantine might be a potential therapy against cognitive impairment and neuroinflammation induced by Aβ1-42, and their association may be a promising alternative to be investigated in the treatment of AD-like dementia. Copyright © 2017 Elsevier

  6. SERS Detection of Amyloid Oligomers on Metallorganic-Decorated Plasmonic Beads.

    PubMed

    Guerrini, Luca; Arenal, Raul; Mannini, Benedetta; Chiti, Fabrizio; Pini, Roberto; Matteini, Paolo; Alvarez-Puebla, Ramon A

    2015-05-13

    Protein misfolded proteins are among the most toxic endogenous species of macromolecules. These chemical entities are responsible for neurodegenerative disorders such as Alzheimer's, Parkinson's, Creutzfeldt-Jakob's and different non-neurophatic amyloidosis. Notably, these oligomers show a combination of marked heterogeneity and low abundance in body fluids, which have prevented a reliable detection by immunological methods so far. Herein we exploit the selectivity of proteins to react with metallic ions and the sensitivity of surface-enhanced Raman spectroscopy (SERS) toward small electronic changes in coordination compounds to design and engineer a reliable optical sensor for protein misfolded oligomers. Our strategy relies on the functionalization of Au nanoparticle-decorated polystyrene beads with an effective metallorganic Raman chemoreceptor, composed by Al(3+) ions coordinated to 4-mercaptobenzoic acid (MBA) with high Raman cross-section, that selectively binds aberrant protein oligomers. The mechanical deformations of the MBA phenyl ring upon complexation with the oligomeric species are registered in its SERS spectrum and can be quantitatively correlated with the concentration of the target biomolecule. The SERS platform used here appears promising for future implementation of diagnostic tools of aberrant species associated with protein deposition diseases, including those with a strong social and economic impact, such as Alzheimer's and Parkinson's diseases.

  7. Getting a grip on prions: oligomers, amyloids and pathological membrane interactions

    PubMed Central

    Caughey, Byron; Baron, Gerald S.; Chesebro, Bruce; Jeffrey, Martin

    2009-01-01

    The prion (infectious protein) concept has evolved with the discovery of new self-propagating protein states in organisms as diverse as mammals and fungi. The infectious agent of the mammalian transmissible spongiform encephalopathies (TSE) has long been considered to be the prototypical prion, and recent cell-free propagation and biophysical analyses of TSE infectivity have now firmly established its prion credentials. Other disease-associated protein aggregates, such as some amyloids, can also have prion-like characteristics under certain experimental conditions. However, most amyloids appear to lack the natural transmissibility of TSE prions. One feature that distinguishes the latter from the former is the glycophosphatidylinositol membrane anchor on prion protein, the molecule that is corrupted in TSE diseases. The presence of this anchor profoundly affects TSE pathogenesis, which involves major membrane distortions in the brain, and may be a key reason for the greater neurovirulence of TSE prions relative to many other autocatalytic protein aggregates. PMID:19231987

  8. Low molecular weight oligomers of amyloid peptides display β-barrel conformations: A replica exchange molecular dynamics study in explicit solvent

    NASA Astrophysics Data System (ADS)

    De Simone, Alfonso; Derreumaux, Philippe

    2010-04-01

    The self-assembly of proteins and peptides into amyloid fibrils is connected to over 40 pathological conditions including neurodegenerative diseases and systemic amyloidosis. Diffusible, low molecular weight protein and peptide oligomers that form in the early steps of aggregation appear to be the harmful cytotoxic species in the molecular etiology of these diseases. So far, the structural characterization of these oligomers has remained elusive owing to their transient and dynamic features. We here address, by means of full atomistic replica exchange molecular dynamics simulations, the energy landscape of heptamers of the amyloidogenic peptide NHVTLSQ from the beta-2 microglobulin protein. The simulations totaling 5 μs show that low molecular weight oligomers in explicit solvent consist of β-barrels in equilibrium with amorphous states and fibril-like assemblies. The results, also accounting for the influence of the pH on the conformational properties, provide a strong evidence of the formation of transient β-barrel assemblies in the early aggregation steps of amyloid-forming systems. Our findings are discussed in terms of oligomers cytotoxicity.

  9. Low molecular weight oligomers of amyloid peptides display beta-barrel conformations: a replica exchange molecular dynamics study in explicit solvent.

    PubMed

    De Simone, Alfonso; Derreumaux, Philippe

    2010-04-28

    The self-assembly of proteins and peptides into amyloid fibrils is connected to over 40 pathological conditions including neurodegenerative diseases and systemic amyloidosis. Diffusible, low molecular weight protein and peptide oligomers that form in the early steps of aggregation appear to be the harmful cytotoxic species in the molecular etiology of these diseases. So far, the structural characterization of these oligomers has remained elusive owing to their transient and dynamic features. We here address, by means of full atomistic replica exchange molecular dynamics simulations, the energy landscape of heptamers of the amyloidogenic peptide NHVTLSQ from the beta-2 microglobulin protein. The simulations totaling 5 micros show that low molecular weight oligomers in explicit solvent consist of beta-barrels in equilibrium with amorphous states and fibril-like assemblies. The results, also accounting for the influence of the pH on the conformational properties, provide a strong evidence of the formation of transient beta-barrel assemblies in the early aggregation steps of amyloid-forming systems. Our findings are discussed in terms of oligomers cytotoxicity.

  10. Monomer adds to preformed structured oligomers of Abeta-peptides by a two-stage dock-lock mechanism.

    PubMed

    Nguyen, Phuong H; Li, Mai Suan; Stock, Gerhard; Straub, John E; Thirumalai, D

    2007-01-02

    Nonfibrillar soluble oligomers, which are intermediates in the transition from monomers to amyloid fibrils, may be the toxic species in Alzheimer's disease. To monitor the early events that direct assembly of amyloidogenic peptides we probe the dynamics of formation of (Abeta(16-22))(n) by adding a monomer to a preformed (Abeta(16-22))(n-1) (n = 4-6) oligomer in which the peptides are arranged in an antiparallel beta-sheet conformation. All atom molecular dynamics simulations in water and multiple long trajectories, for a cumulative time of 6.9 mus, show that the oligomer grows by a two-stage dock-lock mechanism. The largest conformational change in the added disordered monomer occurs during the rapid ( approximately 50 ns) first dock stage in which the beta-strand content of the monomer increases substantially from a low initial value. In the second slow-lock phase, the monomer rearranges to form in register antiparallel structures. Surprisingly, the mobile structured oligomers undergo large conformational changes in order to accommodate the added monomer. The time needed to incorporate the monomer into the fluid-like oligomer grows even when n = 6, which suggests that the critical nucleus size must exceed six. Stable antiparallel structure formation exceeds hundreds of nanoseconds even though frequent interpeptide collisions occur at elevated monomer concentrations used in the simulations. The dock-lock mechanism should be a generic mechanism for growth of oligomers of amyloidogenic peptides.

  11. Monomer adds to preformed structured oligomers of Aβ-peptides by a two-stage dock–lock mechanism

    PubMed Central

    Nguyen, Phuong H.; Li, Mai Suan; Stock, Gerhard; Straub, John E.; Thirumalai, D.

    2007-01-01

    Nonfibrillar soluble oligomers, which are intermediates in the transition from monomers to amyloid fibrils, may be the toxic species in Alzheimer's disease. To monitor the early events that direct assembly of amyloidogenic peptides we probe the dynamics of formation of (Aβ16–22)n by adding a monomer to a preformed (Aβ16–22)n−1 (n = 4–6) oligomer in which the peptides are arranged in an antiparallel β-sheet conformation. All atom molecular dynamics simulations in water and multiple long trajectories, for a cumulative time of 6.9 μs, show that the oligomer grows by a two-stage dock–lock mechanism. The largest conformational change in the added disordered monomer occurs during the rapid (≈50 ns) first dock stage in which the β-strand content of the monomer increases substantially from a low initial value. In the second slow-lock phase, the monomer rearranges to form in register antiparallel structures. Surprisingly, the mobile structured oligomers undergo large conformational changes in order to accommodate the added monomer. The time needed to incorporate the monomer into the fluid-like oligomer grows even when n = 6, which suggests that the critical nucleus size must exceed six. Stable antiparallel structure formation exceeds hundreds of nanoseconds even though frequent interpeptide collisions occur at elevated monomer concentrations used in the simulations. The dock–lock mechanism should be a generic mechanism for growth of oligomers of amyloidogenic peptides. PMID:17190811

  12. Stabilization of native amyloid β-protein oligomers by Copper and Hydrogen peroxide Induced Cross-linking of Unmodified Proteins (CHICUP).

    PubMed

    Williams, Thomas L; Serpell, Louise C; Urbanc, Brigita

    2016-03-01

    Oligomeric assemblies are postulated to be proximate neurotoxic species in human diseases associated with aberrant protein aggregation. Their heterogeneous and transient nature makes their structural characterization difficult. Size distributions of oligomers of several amyloidogenic proteins, including amyloid β-protein (Aβ) relevant to Alzheimer's disease (AD), have been previously characterized in vitro by photo-induced cross-linking of unmodified proteins (PICUP) followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Due to non-physiological conditions associated with the PICUP chemistry, Aβ oligomers cross-linked by PICUP may not be representative of in vivo conditions. Here, we examine an alternative Copper and Hydrogen peroxide Induced Cross-linking of Unmodified Proteins (CHICUP), which utilizes naturally occurring divalent copper ions and hydrogen peroxide and does not require photo activation. Our results demonstrate that CHICUP and PICUP applied to the two predominant Aβ alloforms, Aβ40 and Aβ42, result in similar oligomer size distributions. Thioflavin T fluorescence data and atomic force microscopy images demonstrate that both CHICUP and PICUP stabilize Aβ oligomers and attenuate fibril formation. Relative to noncross-linked peptides, CHICUP-treated Aβ40 and Aβ42 cause prolonged disruption to biomimetic lipid vesicles. CHICUP-stabilized Aβ oligomers link the amyloid cascade, metal, and oxidative stress hypotheses of AD into a more comprehensive understanding of the molecular basis of AD pathology. Because copper and hydrogen peroxide are elevated in the AD brain, CHICUP-stabilized Aβ oligomers are biologically relevant and should be further explored as a new therapeutic target.

  13. Electrochemical Detection of AmyloidOligomers Based on the Signal Amplification of a Network of Silver Nanoparticles.

    PubMed

    Xia, Ning; Wang, Xin; Zhou, Binbin; Wu, Yangyang; Mao, Wenhui; Liu, Lin

    2016-08-03

    Amyloidoligomers (AβOs) are the most important toxic species in the brain of Alzheimer's disease (AD) patient. AβOs, therefore, are considered reliable molecular biomarkers for the diagnosis of AD. Herein, we reported a simple and sensitive electrochemical method for the selective detection of AβOs using silver nanoparticles (AgNPs) as the redox reporters and PrP(95-110), an AβOs-specific binding peptide, as the receptor. Specifically, adamantine (Ad)-labeled PrP(95-110), denoted as Ad-PrP(95-110), induced the aggregation and color change of AgNPs and the follow-up formation of a network of Ad-PrP(95-110)-AgNPs. Then, Ad-PrP(95-110)-AgNPs were anchored onto a β-cyclodextrin (β-CD)-covered electrode surface through the host-guest interaction between Ad and β-CD, thus producing an amplified electrochemical signal through the solid-state Ag/AgCl reaction by the AgNPs. In the presence of AβOs, Ad-PrP(95-110) interacted specifically with the AβOs, thus losing the capability to bind AgNPs and to induce the formation of an AgNPs-based network on the electrode surface. Consequently, the electrochemical signal decreased with an increase in the concentration of AβOs in the range of 20 pM to 100 nM. The biosensor had a detection limit of 8 pM and showed no response to amyloid-β monomers (AβMs) and fibrils (AβFs). On the basis of the well-defined and amplified electrochemical signal of the AgNPs-based network architecture, these results should be valuable for the design of novel electrochemical biosensors by marrying specific receptors.

  14. Association thermodynamics and conformational stability of beta-sheet amyloid beta(17-42) oligomers: effects of E22Q (Dutch) mutation and charge neutralization.

    PubMed

    Blinov, Nikolay; Dorosh, Lyudmyla; Wishart, David; Kovalenko, Andriy

    2010-01-20

    Amyloid fibrils are associated with many neurodegenerative diseases. It was found that amyloidogenic oligomers, not mature fibrils, are neurotoxic agents related to these diseases. Molecular mechanisms of infectivity, pathways of aggregation, and molecular structure of these oligomers remain elusive. Here, we use all-atom molecular dynamics, molecular mechanics combined with solvation analysis by statistical-mechanical, three-dimensional molecular theory of solvation (also known as 3D-RISM-KH) in a new MM-3D-RISM-KH method to study conformational stability, and association thermodynamics of small wild-type Abeta(17-42) oligomers with different protonation states of Glu(22), as well the E22Q (Dutch) mutants. The association free energy of small beta-sheet oligomers shows near-linear trend with the dimers being thermodynamically more stable relative to the larger constructs. The linear (within statistical uncertainty) dependence of the association free energy on complex size is a consequence of the unilateral stacking of monomers in the beta-sheet oligomers. The charge reduction of the wild-type Abeta(17-42) oligomers upon protonation of the solvent-exposed Glu(22) at acidic conditions results in lowering the association free energy compared to the wild-type oligomers at neutral pH and the E22Q mutants. The neutralization of the peptides because of the E22Q mutation only marginally affects the association free energy, with the reduction of the direct electrostatic interactions mostly compensated by the unfavorable electrostatic solvation effects. For the wild-type oligomers at acidic conditions such compensation is not complete, and the electrostatic interactions, along with the gas-phase nonpolar energetic and the overall entropic effects, contribute to the lowering of the association free energy. The differences in the association thermodynamics between the wild-type Abeta(17-42) oligomers at neutral pH and the Dutch mutants, on the one hand, and the Abeta(17

  15. Protective effects of glycycoumarin and procyanidin B1, active components of traditional Japanese medicine yokukansan, on amyloid β oligomer-induced neuronal death.

    PubMed

    Kanno, Hitomi; Kawakami, Zenji; Tabuchi, Masahiro; Mizoguchi, Kazushige; Ikarashi, Yasushi; Kase, Yoshio

    2015-01-15

    Yokukansan, a traditional Japanese (Kampo) medicine, is composed of seven medicinal herbs, and has been traditionally used to treat neurosis, insomnia, and night crying and irritability in children. Yokukansan and its constituent herbs, Glycyrrhiza and Uncaria Hook, have recently been shown to have protective effects against amyloid β (Aβ) oligomer-induced apoptosis by suppressing the activation of caspase-3 in primary cultured neurons. The aim of the present study was to identify the effective components of Glycyrrhiza and Uncaria Hook against Aβ oligomer-induced neurotoxicity. We also attempted to clarify the mechanisms by which yokukansan and these herbs, as well as their components, suppressed the activation of caspase-3 in Aβ oligomer-treated neurons. Rat primary cultured cortical neurons were treated with Aβ oligomer (3 μM). The protective effects of 16 components derived from Glycyrrhiza or Uncaria Hook against Aβ oligomer-induced neurotoxicity were determined using the MTT reduction assay 48 h after the treatment. The suppressive effects of the test substances, i.e., yokukansan, Glycyrrhiza, Uncaria Hook, and screened components, on the Aβ oligomer-induced activation of caspase-3(/7) were evaluated using the caspase-Glo assay 48 h after the Aβ oligomer treatment. The suppressive effects of the test substances on the activation of caspase-8 and -9, both of which are located upstream of caspase-3, were also examined 24h after the Aβ oligomer treatment. Two of the 16 components tested, glycycoumarin derived from Glycyrrhiza and procyanidin B1 derived from Uncaria Hook, significantly inhibited Aβ oligomer-induced neuronal death in a dose-dependent manner. Glycyrrhiza, Uncaria Hook, and yokukansan significantly suppressed the Aβ oligomer-induced activation of caspase-3 as well as caspase-8 and -9. Glycycoumarin also suppressed the activation of caspase-3, but not caspase-8 and -9. Procyanidin B1 suppressed the activation of caspase-3, -8, and -9. Our

  16. Amyloidoligomers induce tau-independent disruption of BDNF axonal transport via calcineurin activation in cultured hippocampal neurons

    PubMed Central

    Ramser, Elisa M.; Gan, Kathlyn J.; Decker, Helena; Fan, Emily Y.; Suzuki, Matthew M.; Ferreira, Sergio T.; Silverman, Michael A.

    2013-01-01

    Disruption of fast axonal transport (FAT) is an early pathological event in Alzheimer's disease (AD). Soluble amyloidoligomers (AβOs), increasingly recognized as proximal neurotoxins in AD, impair organelle transport in cultured neurons and transgenic mouse models. AβOs also stimulate hyperphosphorylation of the axonal microtubule-associated protein, tau. However, the role of tau in FAT disruption is controversial. Here we show that AβOs reduce vesicular transport of brain-derived neurotrophic factor (BDNF) in hippocampal neurons from both wild-type and tau-knockout mice, indicating that tau is not required for transport disruption. FAT inhibition is not accompanied by microtubule destabilization or neuronal death. Significantly, inhibition of calcineurin (CaN), a calcium-dependent phosphatase implicated in AD pathogenesis, rescues BDNF transport. Moreover, inhibition of protein phosphatase 1 and glycogen synthase kinase 3β, downstream targets of CaN, prevents BDNF transport defects induced by AβOs. We further show that AβOs induce CaN activation through nonexcitotoxic calcium signaling. Results implicate CaN in FAT regulation and demonstrate that tau is not required for AβO-induced BDNF transport disruption. PMID:23783030

  17. Distribution of Amyloid-Like and Oligomeric Species from Protein Aggregation Kinetics.

    PubMed

    Silva, Alexandra; Almeida, Bruno; Fraga, Joana S; Taboada, Pablo; Martins, Pedro M; Macedo-Ribeiro, Sandra

    2017-09-14

    Amyloid fibrils and soluble oligomers are two types of protein aggregates associated with neurodegeneration. Classic therapeutic strategies try to prevent the nucleation and spread of amyloid fibrils, whilst diffusible oligomers have emerged as promising drug targets affecting downstream pathogenic processes. We developed a generic protein aggregation model and validate it against measured compositions of fibrillar and non-fibrillar assemblies of ataxin-3, a protein implicated in Machado-Joseph disease. The derived analytic rate-law equations can be used to 1) identify the presence of parallel aggregation pathways and 2) estimate the critical sizes of amyloid fibrils. The discretized population balance supporting our model is the first to quantitatively fit time-resolved measurements of size and composition of both amyloid-like and oligomeric species. The new theoretical framework can be used to screen a new class of drugs specifically targeting toxic oligomers. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Valproic acid and its congener propylisopropylacetic acid reduced the amount of soluble amyloidoligomers released from 7PA2 cells.

    PubMed

    Williams, Robin S B; Bate, Clive

    2017-09-22

    The amyloid hypothesis of Alzheimer's disease suggests that synaptic degeneration and pathology is caused by the accumulation of amyloid-β (Aβ) peptides derived from the amyloid precursor protein (APP). Subsequently, soluble Aβ oligomers cause the loss of synaptic proteins from neurons, a histopathological feature of Alzheimer's disease that correlates with the degree of dementia. In this study, the production of toxic forms of Aβ was examined in vitro using 7PA2 cells stably transfected with human APP. We show that conditioned media from 7PA2 cells containing Aβ oligomers caused synapse degeneration as measured by the loss of synaptic proteins, including synaptophysin and cysteine-string protein, from cultured neurons. Critically, conditioned media from 7PA2 cells treated with valproic acid (2-propylpentanoic acid (VPA)) or propylisopropylacetic acid (PIA) did not cause synapse damage. Treatment with VPA or PIA did not significantly affect total Aβ42 concentrations; rather these drugs selectively reduced the concentrations of Aβ42 oligomers in conditioned media. In contrast, treatment significantly increased the concentrations of Aβ42 monomers in conditioned media. VPA or PIA treatment reduced the concentrations of APP within lipid rafts, membrane compartments associated with Aβ production. These effects of VPA and PIA were reversed by the addition of platelet-activating factor, a bioactive phospholipid produced following activation of phospholipase A2, an enzyme sensitive to VPA and PIA. Collectively these data suggest that VPA and PIA reduce Aβ oligomers through inhibition of phospholipase A2 and suggest a novel therapeutic approach to Alzheimer's treatment. Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.

  19. Environmental Enrichment Potently Prevents Microglia-Mediated Neuroinflammation by Human Amyloid β-Protein Oligomers

    PubMed Central

    Xu, Huixin; Gelyana, Eilrayna; Rajsombath, Molly; Yang, Ting; Li, Shaomin

    2016-01-01

    Microglial dysfunction is increasingly recognized as a key contributor to the pathogenesis of Alzheimer's disease (AD). Environmental enrichment (EE) is well documented to enhance neuronal form and function, but almost nothing is known about whether and how it alters the brain's innate immune system. Here we found that prolonged exposure of naive wild-type mice to EE significantly altered microglial density and branching complexity in the dentate gyrus of hippocampus. In wild-type mice injected intraventricularly with soluble Aβ oligomers (oAβ) from hAPP-expressing cultured cells, EE prevented several morphological features of microglial inflammation and consistently prevented oAβ-mediated mRNA changes in multiple inflammatory genes both in vivo and in primary microglia cultured from the mice. Microdialysis in behaving mice confirmed that EE normalized increases in the extracellular levels of the key cytokines (CCL3, CCL4, TNFα) identified by the mRNA analysis. Moreover, EE prevented the changes in microglial gene expression caused by ventricular injection of oAβ extracted directly from AD cerebral cortex. We conclude that EE potently alters the form and function of microglia in a way that prevents their inflammatory response to human oAβ, suggesting that prolonged environmental enrichment could protect against AD by modulating the brain's innate immune system. SIGNIFICANCE STATEMENT Environmental enrichment (EE) is a potential therapy to delay Alzheimer's disease (AD). Microglial inflammation is associated with the progression of AD, but the influence of EE on microglial inflammation is unclear. Here we systematically applied in vivo methods to show that EE alters microglia in the dentate gyrus under physiological conditions and robustly prevents microglial inflammation induced by human Aβ oligomers, as shown by neutralized microglial inflammatory morphology, mRNA changes, and brain interstitial fluid cytokine levels. Our findings suggest that EE alters the

  20. Disruption of Amyloid Plaques Integrity Affects the Soluble Oligomers Content from Alzheimer Disease Brains

    PubMed Central

    Moyano, Javier; Sanchez-Mico, María; Torres, Manuel; Davila, Jose Carlos; Vizuete, Marisa; Gutierrez, Antonia; Vitorica, Javier

    2014-01-01

    The implication of soluble Abeta in the Alzheimer’s disease (AD) pathology is currently accepted. In fact, the content of soluble extracellular Abeta species, such as monomeric and/or oligomeric Abeta, seems to correlate with the clinico-pathological dysfunction observed in AD patients. However, the nature (monomeric, dimeric or other oligomers), the relative abundance, and the origin (extra-/intraneuronal or plaque-associated), of these soluble species are actually under debate. In this work we have characterized the soluble (defined as soluble in Tris-buffered saline after ultracentrifugation) Abeta, obtained from hippocampal samples of Braak II, Braak III–IV and Braak V–VI patients. Although the content of both Abeta40 and Abeta42 peptides displayed significant increase with pathology progression, our results demonstrated the presence of low, pg/µg protein, amount of both peptides. This low content could explain the absence (or below detection limits) of soluble Abeta peptides detected by western blots or by immunoprecipitation-western blot analysis. These data were in clear contrast to those published recently by different groups. Aiming to explain the reasons that determine these substantial differences, we also investigated whether the initial homogenization could mobilize Abeta from plaques, using 12-month-old PS1xAPP cortical samples. Our data demonstrated that manual homogenization (using Dounce) preserved the integrity of Abeta plaques whereas strong homogenization procedures (such as sonication) produced a vast redistribution of the Abeta species in all soluble and insoluble fractions. This artifact could explain the dissimilar and somehow controversial data between different groups analyzing human AD samples. PMID:25485545

  1. Kinetically driven refolding of the hyperstable EBNA1 origin DNA-binding dimeric beta-barrel domain into amyloid-like spherical oligomers.

    PubMed

    Freire, Eleonora; Oddo, Cristian; Frappier, Lori; de Prat-Gay, Gonzalo

    2008-02-01

    -translational and directional folding of EBNA1 in vivo, possibly assisted by folding accessory proteins. Finally, the oligomers bind Congo red and thioflavin-T, both characteristic of repetitive beta-sheet elements of structure found in amyloids and their soluble precursors. The stable nature of the "kinetically trapped" oligomers suggest their value as models for understanding amyloid intermediates, their toxic nature, and the progress to amyloid fibers in misfolding diseases. The possible role of the EBNA1 spherical oligomers in the virus biology is discussed. (c) 2007 Wiley-Liss, Inc.

  2. Structure of ring-shaped Aβ42 oligomers determined by conformational selection

    NASA Astrophysics Data System (ADS)

    Tran, Linh; Basdevant, Nathalie; Prévost, Chantal; Ha-Duong, Tâp

    2016-02-01

    The oligomerization of amyloid beta (Aβ) peptides into soluble non-fibrillar species plays a critical role in the pathogenesis of Alzheimer’s disease. However, it has been challenging to characterize the tertiary and quaternary structures of Aβ peptides due to their disordered nature and high aggregation propensity. In this work, replica exchange molecular dynamics simulations were used to explore the conformational space of Aβ42 monomer. Among the most populated transient states, we identified a particular conformation which was able to generate ring-shaped pentamers and hexamers, when docked onto itself. The structures of these aggregates were stable during microsecond all-atom MD simulations in explicit solvent. In addition to high resolution models of these oligomers, this study provides support for the conformational selection mechanism of Aβ peptide self-assembly.

  3. Molecular Structure of Aggregated Amyloid-β: Insights from Solid State Nuclear Magnetic Resonance

    PubMed Central

    Tycko, Robert

    2016-01-01

    Amyloid-β (Aβ) peptides aggregate to form polymorphic amyloid fibrils and a variety of intermediate assemblies, including oligomers and protofibrils, both in vitro and in human brain tissue. Since the beginning of the 21st century, considerable progress has been made on characterization of the molecular structures of Aβ aggregates. Full molecular structural models that are based primarily on data from solid state nuclear magnetic resonance measurements have been developed for several in vitro Aβ fibrils and one metastable protofibril. Partial structural characterization of other aggregation intermediates has been achieved. One full structural model for fibrils derived from brain tissue has also been reported. Future work is likely to focus on additional structures from brain tissue and on further clarification of nonfibrillar Aβ aggregates. PMID:27481836

  4. An Oral Aβ Vaccine Using a Recombinant Adeno-Associated Virus Vector in Aged Monkeys: Reduction in Plaque Amyloid and Increase in Aβ Oligomers.

    PubMed

    Hara, Hideo; Ono, Fumiko; Nakamura, Shinichiro; Matsumoto, Shin-Ei; Jin, Haifeng; Hattori, Nobutaka; Tabira, Takeshi

    2016-10-04

    With the objective to improve the amyloid-β (Aβ) targeting immunotherapy, we investigated the safety and efficacy of an oral vaccine with recombinant adeno-associated virus vector carrying a signal sequence and Aβ1-43 cDNA (rAAV/Aβ) in old non-human primates, 12 African green and 10 cynomolgus monkeys. The enteric-dissolving coated capsules containing rAAV/Aβ were orally administered once or twice, then monkeys' conditions were carefully observed with complete blood count and laboratory examinations of the sera. General conditions, food intake, water intake, stool conditions, body weight changes, and menstruation cycles were not significantly altered, and laboratory tests and pathological examinations of the systemic organs were unremarkable. Pathological examinations of the brain showed significant reduction of the amyloid plaque burden and intracellular Aβ without inflammatory or hemorrhagic changes in the brain. However, soluble Aβ and some Aβ oligomers were increased in rAAV-treated monkey brains without changes of the neuronal density and vascular amyloidosis. Thus, this vaccine seems to be safe in general, but we must be cautious about the increase of Aβ oligomers after vaccination. This vaccine may be recommended at a very early stage of Alzheimer's disease when little amyloid is deposited.

  5. Molecular insight into amyloid oligomer destabilizing mechanism of flavonoid derivative 2-(4' benzyloxyphenyl)-3-hydroxy-chromen-4-one through docking and molecular dynamics simulations.

    PubMed

    Kumar, Akhil; Srivastava, Swati; Tripathi, Shubhandra; Singh, Sandeep Kumar; Srikrishna, Saripella; Sharma, Ashok

    2016-06-01

    Aggregation of amyloid peptide (Aβ) has been shown to be directly related to progression of Alzheimer's disease (AD). Aβ is neurotoxic and its deposition and aggregation ultimately lead to cell death. In our previous work, we reported flavonoid derivative (compound 1) showing promising result in transgenic AD model of Drosophila. Compound 1 showed prevention of Aβ-induced neurotoxicity and neuroprotective efficacy in Drosophila system. However, mechanism of action of compound 1 and its effect on the amyloid is not known. We therefore performed molecular docking and atomistic, explicit-solvent molecular dynamics simulations to investigate the process of Aβ interaction, inhibition, and destabilizing mechanism. Results showed different preferred binding sites of compound 1 and good affinity toward the target. Through the course of 35 ns molecular dynamics simulation, conformations_5 of compound 1 intercalates into the hydrophobic core near the salt bridge and showed major structural changes as compared to other conformations. Compound 1 showed interference with the salt bridge and thus reducing the inter strand hydrogen bound network. This minimizes the side chain interaction between the chains A-B leading to disorder in oligomer. Contact map analysis of amino acid residues between chains A and B also showed lesser interaction with adjacent amino acids in the presence of compound 1 (conformations_5). The study provides an insight into how compound 1 interferes and disorders the Aβ peptide. These findings will further help to design better inhibitors for aggregation of the amyloid oligomer.

  6. Activation of D1/D5 Dopamine Receptors Protects Neurons from Synapse Dysfunction Induced by AmyloidOligomers*

    PubMed Central

    Jürgensen, Sofia; Antonio, Leandro L.; Mussi, Gabriela E. A.; Brito-Moreira, Jordano; Bomfim, Theresa R.; De Felice, Fernanda G.; Garrido-Sanabria, Emilio R.; Cavalheiro, Ésper A.; Ferreira, Sergio T.

    2011-01-01

    Soluble oligomers of the amyloid-β peptide (AβOs) accumulate in the brains of Alzheimer disease (AD) patients and are implicated in synapse failure and early memory loss in AD. AβOs have been shown to impact synapse function by inhibiting long term potentiation, facilitating the induction of long term depression and inducing internalization of both AMPA and NMDA glutamate receptors, critical players in plasticity mechanisms. Because activation of dopamine D1/D5 receptors plays important roles in memory circuits by increasing the insertion of AMPA and NMDA receptors at synapses, we hypothesized that selective activation of D1/D5 receptors could protect synapses from the deleterious action of AβOs. We show that SKF81297, a selective D1/D5 receptor agonist, prevented the reduction in surface levels of AMPA and NMDA receptors induced by AβOs in hippocampal neurons in culture. Protection by SKF81297 was abrogated by the specific D1/D5 antagonist, SCH23390. Levels of AMPA receptor subunit GluR1 phosphorylated at Ser845, which regulates AMPA receptor association with the plasma membrane, were reduced in a calcineurin-dependent manner in the presence of AβOs, and treatment with SKF81297 prevented this reduction. Establishing the functional relevance of these findings, SKF81297 blocked the impairment of long term potentiation induced by AβOs in hippocampal slices. Results suggest that D1/D5 receptors may be relevant targets for development of novel pharmacological approaches to prevent synapse failure in AD. PMID:21115476

  7. Activation of D1/D5 dopamine receptors protects neurons from synapse dysfunction induced by amyloid-beta oligomers.

    PubMed

    Jürgensen, Sofia; Antonio, Leandro L; Mussi, Gabriela E A; Brito-Moreira, Jordano; Bomfim, Theresa R; De Felice, Fernanda G; Garrido-Sanabria, Emilio R; Cavalheiro, Ésper A; Ferreira, Sergio T

    2011-02-04

    Soluble oligomers of the amyloid-β peptide (AβOs) accumulate in the brains of Alzheimer disease (AD) patients and are implicated in synapse failure and early memory loss in AD. AβOs have been shown to impact synapse function by inhibiting long term potentiation, facilitating the induction of long term depression and inducing internalization of both AMPA and NMDA glutamate receptors, critical players in plasticity mechanisms. Because activation of dopamine D1/D5 receptors plays important roles in memory circuits by increasing the insertion of AMPA and NMDA receptors at synapses, we hypothesized that selective activation of D1/D5 receptors could protect synapses from the deleterious action of AβOs. We show that SKF81297, a selective D1/D5 receptor agonist, prevented the reduction in surface levels of AMPA and NMDA receptors induced by AβOs in hippocampal neurons in culture. Protection by SKF81297 was abrogated by the specific D1/D5 antagonist, SCH23390. Levels of AMPA receptor subunit GluR1 phosphorylated at Ser(845), which regulates AMPA receptor association with the plasma membrane, were reduced in a calcineurin-dependent manner in the presence of AβOs, and treatment with SKF81297 prevented this reduction. Establishing the functional relevance of these findings, SKF81297 blocked the impairment of long term potentiation induced by AβOs in hippocampal slices. Results suggest that D1/D5 receptors may be relevant targets for development of novel pharmacological approaches to prevent synapse failure in AD.

  8. Alzheimer’s Toxic Amyloid Beta Oligomers: Unwelcome Visitors to the Na/K ATPase alpha3 Docking Station

    PubMed Central

    DiChiara, Thomas; DiNunno, Nadia; Clark, Jeffrey; Bu, Riana Lo; Cline, Erika N.; Rollins, Madeline G.; Gong, Yuesong; Brody, David L.; Sligar, Stephen G.; Velasco, Pauline T.; Viola, Kirsten L.; Klein, William L.

    2017-01-01

    Toxic amyloid beta oligomers (AβOs) are known to accumulate in Alzheimer’s disease (AD) and in animal models of AD. Their structure is heterogeneous, and they are found in both intracellular and extracellular milieu. When given to CNS cultures or injected ICV into non-human primates and other non-transgenic animals, AβOs have been found to cause impaired synaptic plasticity, loss of memory function, tau hyperphosphorylation and tangle formation, synapse elimination, oxidative and ER stress, inflammatory microglial activation, and selective nerve cell death. Memory loss and pathology in transgenic models are prevented by AβO antibodies, while Aducanumab, an antibody that targets AβOs as well as fibrillar Aβ, has provided cognitive benefit to humans in early clinical trials. AβOs have now been investigated in more than 3000 studies and are widely thought to be the major toxic form of Aβ. Although much has been learned about the downstream mechanisms of AβO action, a major gap concerns the earliest steps: How do AβOs initially interact with surface membranes to generate neuron-damaging transmembrane events? Findings from Ohnishi et al (PNAS 2005) combined with new results presented here are consistent with the hypothesis that AβOs act as neurotoxins because they attach to particular membrane protein docks containing Na/K ATPase-α3, where they inhibit ATPase activity and pathologically restructure dock composition and topology in a manner leading to excessive Ca++ build-up. Better understanding of the mechanism that makes attachment of AβOs to vulnerable neurons a neurotoxic phenomenon should open the door to therapeutics and diagnostics targeting the first step of a complex pathway that leads to neural damage and dementia. PMID:28356893

  9. Characterization of Oligomers of Heterogeneous Size as Precursors of Amyloid Fibril Nucleation of an SH3 Domain: An Experimental Kinetics Study

    PubMed Central

    Ruzafa, David; Morel, Bertrand; Varela, Lorena; Azuaga, Ana I.; Conejero-Lara, Francisco

    2012-01-01

    Understanding the earliest molecular events during nucleation of the amyloid aggregation cascade is of fundamental significance to prevent amyloid related disorders. We report here an experimental kinetic analysis of the amyloid aggregation of the N47A mutant of the α-spectrin SH3 domain (N47A Spc-SH3) under mild acid conditions, where it is governed by rapid formation of amyloid nuclei. The initial rates of formation of amyloid structures, monitored by thioflavine T fluorescence at different protein concentrations, agree quantitatively with high-order kinetics, suggesting an oligomerization pre-equilibrium preceding the rate-limiting step of amyloid nucleation. The curves of native state depletion also follow high-order irreversible kinetics. The analysis is consistent with the existence of low-populated and heterogeneous oligomeric precursors of fibrillation that form by association of partially unfolded protein monomers. An increase in NaCl concentration accelerates fibrillation but reduces the apparent order of the nucleation kinetics; and a double mutant (K43A, N47A) Spc-SH3 domain, largely unfolded under native conditions and prone to oligomerize, fibrillates with apparent first order kinetics. On the light of these observations, we propose a simple kinetic model for the nucleation event, in which the monomer conformational unfolding and the oligomerization of an amyloidogenic intermediate are rapidly pre-equilibrated. A conformational change of the polypeptide chains within any of the oligomers, irrespective of their size, is the rate-limiting step leading to the amyloid nuclei. This model is able to explain quantitatively the initial rates of aggregation and the observed variations in the apparent order of the kinetics and, more importantly, provides crucial thermodynamic magnitudes of the processes preceding the nucleation. This kinetic approach is simple to use and may be of general applicability to characterize the amyloidogenic intermediates and

  10. Single-Molecule Imaging Reveals that Small Amyloid-β1–42 Oligomers Interact with the Cellular Prion Protein (PrPC)

    PubMed Central

    Ganzinger, Kristina A; Narayan, Priyanka; Qamar, Seema S; Weimann, Laura; Ranasinghe, Rohan T; Aguzzi, Adriano; Dobson, Christopher M; McColl, James; St George-Hyslop, Peter; Klenerman, David

    2014-01-01

    Oligomers of the amyloid-β peptide (Aβ) play a central role in the pathogenesis of Alzheimer’s disease and have been suggested to induce neurotoxicity by binding to a plethora of cell-surface receptors. However, the heterogeneous mixtures of oligomers of varying sizes and conformations formed by Aβ42 have obscured the nature of the oligomeric species that bind to a given receptor. Here, we have used single-molecule imaging to characterize Aβ42 oligomers (oAβ42) and to confirm the controversial interaction of oAβ42 with the cellular prion protein (PrPC) on live neuronal cells. Our results show that, at nanomolar concentrations, oAβ42 interacts with PrPC and that the species bound to PrPC are predominantly small oligomers (dimers and trimers). Single-molecule biophysical studies can thus aid in deciphering the mechanisms that underlie receptor-mediated oAβ-induced neurotoxicity, and ultimately facilitate the discovery of novel inhibitors of these pathways. PMID:25294384

  11. Peripherally administered sera antibodies recognizing amyloidoligomers mitigate Alzheimer's disease-like pathology and cognitive decline in aged 3× Tg-AD mice.

    PubMed

    Wang, Hai-Chao; Yu, Yun-Zhou; Liu, Si; Zhao, Meng; Xu, Qing

    2016-04-04

    Active and passive immunotherapy targeting amyloid-β (Aβ) may be the most promising strategy to prevent or treat Alzheimer's disease (AD). Previously, immunization with the recombinant 6Aβ15-T antigen generated robust anti-Aβ serum antibodies that strongly recognized Aβ42 oligomers in different mice, markedly reduced the amyloid burden, and improved behavioral performance of immunized older AD mice. Here, we further determined that these anti-6Aβ15-T serum antibodies from different strains of mice displayed anti-Aβ antibody responses against the same epitopes in the Aβ1-15 region. Peripheral administration of anti-6Aβ15-T serum antibodies was also effective to mitigate AD-like pathology and cognitive decline in aged 3× Tg-AD mice. Specifically, the levels of Aβ and tau in the brains of 3× Tg-AD mice were significantly reduced after passive immunotherapy, which seemed necessary or beneficial to ameliorate memory impairment. In addition, our results showed that this immunotherapy also prevented presynaptic dynamin 1 degradation, which might help to further protect synaptic functions and allow functional recovery of cognition. Moreover, immunization with 6Aβ15-T in rabbits induced a similar antibody response as that in mice, and the rabbit serum antibodies reacted strongly with Aβ42 oligomers and inhibited oligomer-mediated neurotoxicity. We concluded that passive immunization with Aβ42 oligomer conformation-sensitive anti-6Aβ15-T serum antibodies is effective in providing potentially therapeutic effects in aged 3× Tg-AD mice by reducing Aβ and tau.

  12. Ac-LPFFD-Th: A Trehalose-Conjugated Peptidomimetic as a Strong Suppressor of AmyloidOligomer Formation and Cytotoxicity.

    PubMed

    Sinopoli, Alessandro; Giuffrida, Alessandro; Tomasello, Marianna Flora; Giuffrida, Maria Laura; Leone, Marilisa; Attanasio, Francesco; Caraci, Filippo; De Bona, Paolo; Naletova, Irina; Saviano, Michele; Copani, Agata; Pappalardo, Giuseppe; Rizzarelli, Enrico

    2016-08-17

    The inhibition of amyloid formation is a promising therapeutic approach for the treatment of neurodegenerative diseases. Peptide-based inhibitors, which have been widely investigated, are generally derived from original amyloid sequences. Most interestingly, trehalose, a nonreducing disaccharide of α-glucose, is effective in preventing the aggregation of numerous proteins. We have determined that the development of hybrid compounds could provide new molecules with improved properties that might synergically increase the potency of their single moieties. In this work, the ability of Ac-LPFFD-Th, a C-terminally trehalose-conjugated derivative, to slow down the Aβ aggregation process was investigated by means of different biophysical techniques, including thioflavin T fluorescence, dynamic light scattering, ESI-MS, and NMR spectroscopy. Moreover, we demonstrate that Ac-LPFFD-Th modifies the aggregation features of Aβ and protects neurons from Aβ oligomers' toxic insult.

  13. Two distinct amyloid beta-protein (Abeta) assembly pathways leading to oligomers and fibrils identified by combined fluorescence correlation spectroscopy, morphology, and toxicity analyses.

    PubMed

    Matsumura, Satoko; Shinoda, Keiko; Yamada, Mayumi; Yokojima, Satoshi; Inoue, Masafumi; Ohnishi, Takayuki; Shimada, Tetsuya; Kikuchi, Kazuya; Masui, Dai; Hashimoto, Shigeki; Sato, Michio; Ito, Akane; Akioka, Manami; Takagi, Shinsuke; Nakamura, Yoshihiro; Nemoto, Kiyokazu; Hasegawa, Yutaka; Takamoto, Hisayoshi; Inoue, Haruo; Nakamura, Shinichiro; Nabeshima, Yo-ichi; Teplow, David B; Kinjo, Masataka; Hoshi, Minako

    2011-04-01

    Nonfibrillar assemblies of amyloid β-protein (Aβ) are considered to play primary roles in Alzheimer disease (AD). Elucidating the assembly pathways of these specific aggregates is essential for understanding disease pathogenesis and developing knowledge-based therapies. However, these assemblies cannot be monitored in vivo, and there has been no reliable in vitro monitoring method at low protein concentration. We have developed a highly sensitive in vitro monitoring method using fluorescence correlation spectroscopy (FCS) combined with transmission electron microscopy (TEM) and toxicity assays. Using Aβ labeled at the N terminus or Lys(16), we uncovered two distinct assembly pathways. One leads to highly toxic 10-15-nm spherical Aβ assemblies, termed amylospheroids (ASPDs). The other leads to fibrils. The first step in ASPD formation is trimerization. ASPDs of ∼330 kDa in mass form from these trimers after 5 h of slow rotation. Up to at least 24 h, ASPDs remain the dominant structures in assembly reactions. Neurotoxicity studies reveal that the most toxic ASPDs are ∼128 kDa (∼32-mers). In contrast, fibrillogenesis begins with dimer formation and then proceeds to formation of 15-40-nm spherical intermediates, from which fibrils originate after 15 h. Unlike ASPD formation, the Lys(16)-labeled peptide disturbed fibril formation because the Aβ(16-20) region is critical for this final step. These differences in the assembly pathways clearly indicated that ASPDs are not fibril precursors. The method we have developed should facilitate identifying Aβ assembly steps at which inhibition may be beneficial.

  14. Regulation of Amyloid β Oligomer Binding to Neurons and Neurotoxicity by the Prion Protein-mGluR5 Complex.

    PubMed

    Beraldo, Flavio H; Ostapchenko, Valeriy G; Caetano, Fabiana A; Guimaraes, Andre L S; Ferretti, Giulia D S; Daude, Nathalie; Bertram, Lisa; Nogueira, Katiane O P C; Silva, Jerson L; Westaway, David; Cashman, Neil R; Martins, Vilma R; Prado, Vania F; Prado, Marco A M

    2016-10-14

    The prion protein (PrP(C)) has been suggested to operate as a scaffold/receptor protein in neurons, participating in both physiological and pathological associated events. PrP(C), laminin, and metabotropic glutamate receptor 5 (mGluR5) form a protein complex on the plasma membrane that can trigger signaling pathways involved in neuronal differentiation. PrP(C) and mGluR5 are co-receptors also for β-amyloid oligomers (AβOs) and have been shown to modulate toxicity and neuronal death in Alzheimer's disease. In the present work, we addressed the potential crosstalk between these two signaling pathways, laminin-PrP(C)-mGluR5 or AβO-PrP(C)-mGluR5, as well as their interplay. Herein, we demonstrated that an existing complex containing PrP(C)-mGluR5 has an important role in AβO binding and activity in neurons. A peptide mimicking the binding site of laminin onto PrP(C) (Ln-γ1) binds to PrP(C) and induces intracellular Ca(2+) increase in neurons via the complex PrP(C)-mGluR5. Ln-γ1 promotes internalization of PrP(C) and mGluR5 and transiently decreases AβO biding to neurons; however, the peptide does not impact AβO toxicity. Given that mGluR5 is critical for toxic signaling by AβOs and in prion diseases, we tested whether mGlur5 knock-out mice would be susceptible to prion infection. Our results show mild, but significant, effects on disease progression, without affecting survival of mice after infection. These results suggest that PrP(C)-mGluR5 form a functional response unit by which multiple ligands can trigger signaling. We propose that trafficking of PrP(C)-mGluR5 may modulate signaling intensity by different PrP(C) ligands. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. The Transient Receptor Potential Melastatin 2 (TRPM2) Channel Contributes to β-Amyloid Oligomer-Related Neurotoxicity and Memory Impairment.

    PubMed

    Ostapchenko, Valeriy G; Chen, Megan; Guzman, Monica S; Xie, Yu-Feng; Lavine, Natalie; Fan, Jue; Beraldo, Flavio H; Martyn, Amanda C; Belrose, Jillian C; Mori, Yasuo; MacDonald, John F; Prado, Vania F; Prado, Marco A M; Jackson, Michael F

    2015-11-11

    In Alzheimer's disease, accumulation of soluble oligomers of β-amyloid peptide is known to be highly toxic, causing disturbances in synaptic activity and neuronal death. Multiple studies relate these effects to increased oxidative stress and aberrant activity of calcium-permeable cation channels leading to calcium imbalance. The transient receptor potential melastatin 2 (TRPM2) channel, a Ca(2+)-permeable nonselective cation channel activated by oxidative stress, has been implicated in neurodegenerative diseases, and more recently in amyloid-induced toxicity. Here we show that the function of TRPM2 is augmented by treatment of cultured neurons with β-amyloid oligomers. Aged APP/PS1 Alzheimer's mouse model showed increased levels of endoplasmic reticulum stress markers, protein disulfide isomerase and phosphorylated eukaryotic initiation factor 2α, as well as decreased levels of the presynaptic marker synaptophysin. Elimination of TRPM2 in APP/PS1 mice corrected these abnormal responses without affecting plaque burden. These effects of TRPM2 seem to be selective for β-amyloid toxicity, as ER stress responses to thapsigargin or tunicamycin in TRPM2(-/-) neurons was identical to that of wild-type neurons. Moreover, reduced microglial activation was observed in TRPM2(-/-)/APP/PS1 hippocampus compared with APP/PS1 mice. In addition, age-dependent spatial memory deficits in APP/PS1 mice were reversed in TRPM2(-/-)/APP/PS1 mice. These results reveal the importance of TRPM2 for β-amyloid neuronal toxicity, suggesting that TRPM2 activity could be potentially targeted to improve outcomes in Alzheimer's disease. Transient receptor potential melastatin 2 (TRPM2) is an oxidative stress sensing calcium-permeable channel that is thought to contribute to calcium dysregulation associated with neurodegenerative diseases, including Alzheimer's disease. Here we show that oligomeric β-amyloid, the toxic peptide in Alzheimer's disease, facilitates TRPM2 channel activation. In mice

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

    PubMed Central

    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

  17. Neurotoxicity and memory deficits induced by soluble low-molecular-weight amyloid-β1-42 oligomers are revealed in vivo by using a novel animal model.

    PubMed

    Brouillette, Jonathan; Caillierez, Raphaëlle; Zommer, Nadège; Alves-Pires, Claire; Benilova, Iryna; Blum, David; De Strooper, Bart; Buée, Luc

    2012-06-06

    Neuronal and synaptic degeneration are the best pathological correlates for memory decline in Alzheimer's disease (AD). Although the accumulation of soluble low-molecular-weight amyloid-β (Aβ) oligomers has been suggested to trigger neurodegeneration in AD, animal models overexpressing or infused with Aβ lack neuronal loss at the onset of memory deficits. Using a novel in vivo approach, we found that repeated hippocampal injections of small soluble Aβ(1-42) oligomers in awake, freely moving mice were able to induce marked neuronal loss, tau hyperphosphorylation, and deficits in hippocampus-dependent memory. The neurotoxicity of small Aβ(1-42) species was observed in vivo as well as in vitro in association with increased caspase-3 activity and reduced levels of the NMDA receptor subunit NR2B. We found that the sequestering agent transthyretin is able to bind the toxic Aβ(1-42) species and attenuated the loss of neurons and memory deficits. Our novel mouse model provides evidence that small, soluble Aβ(1-42) oligomers are able to induce extensive neuronal loss in vivo and initiate a cascade of events that mimic the key neuropathological hallmarks of AD.

  18. Validation and Characterization of a Novel Peptide That Binds Monomeric and Aggregated β-Amyloid and Inhibits the Formation of Neurotoxic Oligomers*

    PubMed Central

    Barr, Renae K.; Verdile, Giuseppe; Wijaya, Linda K.; Morici, Michael; Taddei, Kevin; Gupta, Veer B.; Pedrini, Steve; Jin, Liang; Nicolazzo, Joseph A.; Knock, Erin; Fraser, Paul E.; Martins, Ralph N.

    2016-01-01

    Although the formation of β-amyloid (Aβ) deposits in the brain is a hallmark of Alzheimer disease (AD), the soluble oligomers rather than the mature amyloid fibrils most likely contribute to Aβ toxicity and neurodegeneration. Thus, the discovery of agents targeting soluble Aβ oligomers is highly desirable for early diagnosis prior to the manifestation of a clinical AD phenotype and also more effective therapies. We have previously reported that a novel 15-amino acid peptide (15-mer), isolated via phage display screening, targeted Aβ and attenuated its neurotoxicity (Taddei, K., Laws, S. M., Verdile, G., Munns, S., D'Costa, K., Harvey, A. R., Martins, I. J., Hill, F., Levy, E., Shaw, J. E., and Martins, R. N. (2010) Neurobiol. Aging 31, 203–214). The aim of the current study was to generate and biochemically characterize analogues of this peptide with improved stability and therapeutic potential. We demonstrated that a stable analogue of the 15-amino acid peptide (15M S.A.) retained the activity and potency of the parent peptide and demonstrated improved proteolytic resistance in vitro (stable to t = 300 min, c.f. t = 30 min for the parent peptide). This candidate reduced the formation of soluble Aβ42 oligomers, with the concurrent generation of non-toxic, insoluble aggregates measuring up to 25–30 nm diameter as determined by atomic force microscopy. The 15M S.A. candidate directly interacted with oligomeric Aβ42, as shown by coimmunoprecipitation and surface plasmon resonance/Biacore analysis, with an affinity in the low micromolar range. Furthermore, this peptide bound fibrillar Aβ42 and also stained plaques ex vivo in brain tissue from AD model mice. Given its multifaceted ability to target monomeric and aggregated Aβ42 species, this candidate holds promise for novel preclinical AD imaging and therapeutic strategies. PMID:26538562

  19. Probing the sources of the apparent irreproducibility of amyloid formation: drastic changes in kinetics and a switch in mechanism due to micellelike oligomer formation at critical concentrations of IAPP.

    PubMed

    Brender, Jeffrey R; Krishnamoorthy, Janarthanan; Sciacca, Michele F M; Vivekanandan, Subramanian; D'Urso, Luisa; Chen, Jennifer; La Rosa, Carmelo; Ramamoorthy, Ayyalusamy

    2015-02-19

    The aggregation of amyloidogenic proteins is infamous for being highly chaotic, with small variations in conditions sometimes leading to large changes in aggregation rates. Using the amyloidogenic protein IAPP (islet amyloid polypeptide protein, also known as amylin) as an example, we show that a part of this phenomenon may be related to the formation of micellelike oligomers at specific critical concentrations and temperatures. We show that pyrene fluorescence can sensitively detect micellelike oligomer formation by IAPP and discriminate between micellelike oligomers from fibers and monomers, making pyrene one of the few chemical probes specific to a prefibrillar oligomer. We further show that oligomers of this type reversibly form at critical concentrations in the low micromolar range and at specific critical temperatures. Micellelike oligomer formation has several consequences for amyloid formation by IAPP. First, the kinetics of fiber formation increase substantially as the critical concentration is approached but are nearly independent of concentration below it, suggesting a direct role for the oligomers in fiber formation. Second, the critical concentration is strongly correlated with the propensity to form amyloid: higher critical concentrations are observed for both IAPP variants with lower amyloidogenicity and for native IAPP at acidic pH in which aggregation is greatly slowed. Furthermore, using the DEST NMR technique, we show that the pathway of amyloid formation switches as the critical point is approached, with self-interactions primarily near the N-terminus below the critical temperature and near the central region above the critical temperature, reconciling two apparently conflicting views of the initiation of IAPP aggregation.

  20. Early accumulation of intracellular fibrillar oligomers and late congophilic amyloid angiopathy in mice expressing the Osaka intra-Aβ APP mutation.

    PubMed

    Kulic, L; McAfoose, J; Welt, T; Tackenberg, C; Späni, C; Wirth, F; Finder, V; Konietzko, U; Giese, M; Eckert, A; Noriaki, K; Shimizu, T; Murakami, K; Irie, K; Rasool, S; Glabe, C; Hock, C; Nitsch, R M

    2012-11-13

    Pathogenic amyloid-β peptide precursor (APP) mutations clustered around position 693 of APP-position 22 of the Aβ sequence--are commonly associated with congophilic amyloid angiopathy (CAA) and intracerebral hemorrhages. In contrast, the Osaka (E693Δ) intra-Aβ APP mutation shows a recessive pattern of inheritance that leads to AD-like dementia despite low brain amyloid on in vivo positron emission tomography imaging. Here, we investigated the effects of the Osaka APP mutation on Aβ accumulation and deposition in vivo using a newly generated APP transgenic mouse model (E22ΔAβ) expressing the Osaka mutation together with the Swedish (K670N/M671L) double mutation. E22ΔAβ mice exhibited reduced α-processing of APP and early accumulation of intraneuronal fibrillar Aβ oligomers associated with cognitive deficits. In line with our in vitro findings that recombinant E22Δ-mutated Aβ peptides form amyloid fibrils, aged E22ΔAβ mice showed extracellular CAA deposits in leptomeningeal cerebellar and cortical vessels. In vitro results from thioflavin T aggregation assays with recombinant Aβ peptides revealed a yet unknown antiamyloidogenic property of the E693Δ mutation in the heterozygous state and an inhibitory effect of E22Δ Aβ42 on E22Δ Aβ40 fibrillogenesis. Moreover, E22Δ Aβ42 showed a unique aggregation kinetics lacking exponential fibril growth and poor seeding effects on wild-type Aβ aggregation. These results provide a possible explanation for the recessive trait of inheritance of the Osaka APP mutation and the apparent lack of amyloid deposition in E693Δ mutation carriers.

  1. Biophysical analyses of synthetic amyloid-beta(1-42) aggregates before and after covalent cross-linking. Implications for deducing the structure of endogenous amyloid-beta oligomers.

    PubMed

    Moore, Brenda D; Rangachari, Vijayaraghavan; Tay, William M; Milkovic, Nicole M; Rosenberry, Terrone L

    2009-12-15

    A neuropathological hallmark of Alzheimer's disease (AD) is the presence of large numbers of senile plaques in the brain. These deposits are rich in fibrils that are composed of 40- and 42-residue amyloid-beta (Abeta) peptides. Several lines of evidence indicate that soluble Abeta aggregates as well as fibrils are important in the etiology of AD. Low levels of endogenous soluble Abeta aggregates make them difficult to characterize, but several species in extracts of AD brains have been detected by gel electrophoresis in sodium dodecyl sulfate (SDS) and immunoblotting. Individual Abeta oligomers ranging in size from dimers through dodecamers of 4 kDa monomeric Abeta have been resolved in other laboratories as discrete species by size exclusion chromatography (SEC). In an effort to reconstitute soluble Abeta aggregates in vitro that resemble the endogenous soluble Abeta aggregates, we previously found that monomeric Abeta(1-42) rapidly forms soluble oligomers in the presence of dilute SDS micelles. Here we extend this work in two directions. First, we contrast the size and secondary structure of these oligomers with those of synthetic Abeta(1-42) fibrils. SEC and multiangle light scattering were used to obtain a molecular mass of 150 kDa for the isolated oligomers. The oligomers partially dissociated to monomers through nonamers when incubated with SDS, but in contrast to endogenous oligomers, we saw no evidence of these discrete species prior to SDS treatment. One hypothesis to explain this difference is that endogenous oligomers are stabilized by covalent cross-linking induced by unknown cellular agents. To explore this hypothesis, optimal mass spectrometry (MS) analysis procedures need to be developed for Abeta cross-linked in vitro. In our second series of studies, we began this process by treating monomeric and aggregated Abeta(1-42) with three cross-linking agents: transglutaminase, glutaraldehyde, and Cu(II) with peroxide. We compared the efficiency of

  2. Copper ligation to soluble oligomers of the English mutant of the amyloid-β peptide yields a linear Cu(I) site that is resistant to O2 oxidation.

    PubMed

    Peck, Kristy L; Clewett, Heather S; Schmitt, Jennifer C; Shearer, Jason

    2013-05-25

    Copper coordination to soluble oligomers of the English (AβH(6)R) mutant of the amyloid-β peptide is probed. Cu(II) coordination yields a square planar (N/O)4 coordination environment, while reduction yields an O2 inert linear bis-His Cu(I) centre.

  3. A synthetic peptide corresponding to a region of the human pericentriolar material 1 (PCM-1) protein binds β-amyloid (Aβ1-42 ) oligomers.

    PubMed

    Chakravarthy, Balu; Ménard, Michel; Brown, Leslie; Hewitt, Melissa; Atkinson, Trevor; Whitfield, James

    2013-08-01

    We have recently reported that a ~19-kDa polypeptide, rPK-4, is a protein kinase Cs inhibitor that is 89% homologous to the 1171-1323 amino acid region of the 228-kDa human pericentriolar material-1 (PCM-1) protein (Chakravarthy et al. 2012). We have now discovered that rPK-4 binds oligomeric amyloid-β peptide (Aβ)1-42 with high affinity. Most importantly, a PCM-1-selective antibody co-precipitated Aβ and amyloid β precursor protein (AβPP) from cerebral cortices and hippocampi from AD (Alzheimer's disease) transgenic mice that produce human AβPP and Aβ1-42 , suggesting that PCM-1 may interact with amyloid precursor protein/Aβ in vivo. We have identified rPK-4's Aβ-binding domain using a set of overlapping synthetic peptides. We have found with ELISA, dot-blot, and polyacrylamide gel electrophoresis techniques that a ~ 5 kDa synthetic peptide, amyloid binding peptide (ABP)-p4-5 binds Aβ1-42 at nM levels. Most importantly, ABP-p4-5, like rPK-4, appears to preferentially bind Aβ1-42 oligomers, believed to be the toxic AD-drivers. As expected from these observations, ABP-p4-5 prevented Aβ1-42 from killing human SH-SY5Y neuroblastoma cells via apoptosis. These findings indicate that ABP-p4-5 is a possible candidate therapeutic for AD. © Her Majesty the Queen in Right of Canada 2013. Reproduced with the permission of the Minister of Industry.

  4. Aging and amyloid β oligomers enhance TLR4 expression, LPS-induced Ca(2+) responses, and neuron cell death in cultured rat hippocampal neurons.

    PubMed

    Calvo-Rodríguez, María; de la Fuente, Carmen; García-Durillo, Mónica; García-Rodríguez, Carmen; Villalobos, Carlos; Núñez, Lucía

    2017-01-31

    Toll-like receptors (TLRs) are transmembrane pattern-recognition receptors of the innate immune system recognizing diverse pathogen-derived and tissue damage-related ligands. It has been suggested that TLR signaling contributes to the pathogenesis of age-related, neurodegenerative diseases, including Alzheimer's disease (AD). AD is associated to oligomers of the amyloid β peptide (Aβo) that cause intracellular Ca(2+) dishomeostasis and neuron cell death in rat hippocampal neurons. Here we assessed the interplay between inflammation and Aβo in long-term cultures of rat hippocampal neurons, an in vitro model of neuron aging and/or senescence. Ca(2+) imaging and immunofluorescence against annexin V and TLR4 were applied in short- and long-term cultures of rat hippocampal neurons to test the effects of TLR4-agonist LPS and Aβo on cytosolic [Ca(2+)] and on apoptosis as well as on expression of TLR4. LPS increases cytosolic [Ca(2+)] and promotes apoptosis in rat hippocampal neurons in long-term culture considered aged and/or senescent neurons, but not in short-term cultured neurons considered young neurons. TLR4 antagonist CAY10614 prevents both effects. TLR4 expression in rat hippocampal neurons is significantly larger in aged hippocampal cultures. Treatment of aged hippocampal cultures with Aβo increases TLR4 expression and enhances LPS-induced Ca(2+) responses and neuron cell death. Aging and amyloid β oligomers, the neurotoxin involved in Alzheimer's disease, enhance TLR4 expression as well as LPS-induced Ca(2+) responses and neuron cell death in rat hippocampal neurons aged in vitro.

  5. Involvement of insulin-like growth factor 1 receptor signaling in the amyloid-β peptide oligomers-induced p75 neurotrophin receptor protein expression in mouse hippocampus.

    PubMed

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

    2012-01-01

    The p75 neurotrophin receptor (p75NTR) has been thought to play a critical role in amyloid-β peptide (Aβ)-mediated neurodegeneration and Aβ metabolism in Alzheimer's disease (AD) brains. Our previous report showed that membrane-associated p75NTR protein expression was significantly increased in the hippocampi of two different strains of transgenic AD mice and was associated with the age-dependent elevation of Aβ1-42 levels. Here, we provide evidence that the Aβ1-42 oligomers known as ADDLs (Aβ-derived diffusible ligands) induce p75NTR protein expression through insulin-like growth factor 1 receptor (IGF-1R) phosphorylation in SH-SY5Y human neuroblastoma cells. An in vivo microinjection study demonstrated that microinjected ADDLs increased the p75NTR protein expression by 1.4-fold in the ipsilateral hippocampus compared to the contralateral hippocampus. In addition, ADDLs microinjected into mouse hippocampi facilitated IGF-1R phosphorylation within 30 min and the co-administration of picropodophyllin, an IGF-1R kinase inhibitor, blocked ADDLs-induced p75NTR expression. We examined the possible involvement of IGF-1R in the increased p75NTR protein expression in the hippocampi of 6-month-old AβPPswe/PS1dE9 AD model mice that had accumulated significant amounts of Aβ1-42 and showed significantly higher p75NTR expression than age-matched wild-type mice. We found that IGF-1R phosphorylation in these transgenic mice was higher than that in the wild-type mice. These findings indicate that Aβ1-42 oligomers stimulate the p75NTR protein expression in the hippocampus through IGF-1R signaling. Thus, Aβ1-42 oligomers-mediated IGF-1R activation may trigger an increase in p75NTR protein expression in the hippocampus of AD brain during the early stages of disease development.

  6. Amyloidogenic peptide oligomer accumulation in autophagy-deficient β cells induces diabetes

    PubMed Central

    Kim, Jinyoung; Cheon, Hwanju; Jeong, Yeon Taek; Quan, Wenying; Kim, Kook Hwan; Cho, Jae Min; Lim, Yu-Mi; Oh, Seung Hoon; Jin, Sang-Man; Kim, Jae Hyeon; Lee, Moon-Kyu; Kim, Sunshin; Komatsu, Masaaki; Kang, Sang-Wook; Lee, Myung-Shik

    2014-01-01

    Islet amyloid accumulation is a hallmark of human type 2 diabetes (T2D). In contrast to human islet amyloid polypeptide (hIAPP), murine islet amyloid polypeptide (mIAPP) does not exhibit amyloidogenic propensity. Because autophagy is important in the clearance of amyloid-like proteins, we studied transgenic mice with β cell–specific expression of hIAPP to evaluate the contribution of autophagy in T2D-associated accumulation of hIAPP. In mice with β cell–specific expression of hIAPP, a deficiency in autophagy resulted in development of overt diabetes, which was not observed in mice expressing hIAPP alone or lacking autophagy alone. Furthermore, lack of autophagy in hIAPP-expressing animals resulted in hIAPP oligomer and amyloid accumulation in pancreatic islets, leading to increased death and decreased mass of β cells. Expression of hIAPP in purified monkey islet cells or a murine β cell line resulted in pro-hIAPP dimer formation, while dimer formation was absent or reduced dramatically in cells expressing either nonamyloidogenic mIAPP or nonfibrillar mutant hIAPP. In autophagy-deficient cells, accumulation of pro-hIAPP dimers increased markedly, and pro-hIAPP trimers were detected in the detergent-insoluble fraction. Enhancement of autophagy improved the metabolic profile of hIAPP-expressing mice fed a high-fat diet. These results suggest that autophagy promotes clearance of amyloidogenic hIAPP, autophagy deficiency exacerbates pathogenesis of human T2D, and autophagy enhancers have therapeutic potential for islet amyloid accumulation-associated human T2D. PMID:25036705

  7. Inhibition of Alzheimer's amyloid toxicity with a tricyclic pyrone molecule in vitro and in vivo.

    PubMed

    Hong, Hyun-Seok; Rana, Sandeep; Barrigan, Lydia; Shi, Aibin; Zhang, Yi; Zhou, Feimeng; Jin, Lee-Way; Hua, Duy H

    2009-02-01

    Small beta-amyloid (Abeta) 1-42 aggregates are toxic to neurons and may be the primary toxic species in Alzheimer's disease (AD). Methods to reduce the level of Abeta, prevent Abeta aggregation, and eliminate existing Abeta aggregates have been proposed for treatment of AD. A tricyclic pyrone named CP2 is found to prevent cell death associated with Abeta oligomers. We studied the possible mechanisms of neuroprotection by CP2. Surface plasmon resonance spectroscopy shows a direct binding of CP2 with Abeta42 oligomer. Circular dichroism spectroscopy reveals monomeric Abeta42 peptide remains as a random coil/alpha-helix structure in the presence of CP2 over 48 h. Atomic force microscopy studies show CP2 exhibits similar ability to inhibit Abeta42 aggregation as that of Congo red and curcumin. Atomic force microscopy closed-fluid cell study demonstrates that CP2 disaggregates Abeta42 oligomers and protofibrils. CP2 also blocks Abeta fibrillations using a protein quantification method. Treatment of 5x familial Alzheimer's disease mice, a robust Abeta42-producing animal model of AD, with a 2-week course of CP2 resulted in 40% and 50% decreases in non-fibrillar and fibrillar Abeta species, respectively. Our results suggest that CP2 might be beneficial to AD patients by preventing Abeta aggregation and disaggregating existing Abeta oligomers and protofibrils.

  8. Detection of Soluble AmyloidOligomers and Insoluble High-Molecular-Weight Particles in CSF: Development of Methods with Potential for Diagnosis and Therapy Monitoring of Alzheimer's Disease.

    PubMed

    Funke, Susanne Aileen

    2011-01-01

    The diagnosis of probable Alzheimer's disease (AD) can be established premortem based on clinical criteria like neuropsychological tests. Post mortem, specific neuropathological changes like amyloid plaques define AD. However, the standard criteria based on medical history and mental status examinations do not take into account the long preclinical features of the disease, and a biomarker for improved diagnosis of AD is urgently needed. In a large number of studies, amyloid-β (Aβ) monomer concentrations in CSF of AD patients are consistently and significantly reduced when compared to healthy controls. Therefore, monomeric Aβ in CSF was suggested to be a helpful biomarker for the diagnosis of preclinical AD. However, not the monomeric form, but Aβ oligomers have been shown to be the toxic species in AD pathology, and their quantification and characterization could facilitate AD diagnosis and therapy monitoring. Here, we review the current status of assay development to reliably and routinely detect Aβ oligomers and high-molecular-weight particles in CSF.

  9. Extracellular Tau Oligomers Produce An Immediate Impairment of LTP and Memory.

    PubMed

    Fá, M; Puzzo, D; Piacentini, R; Staniszewski, A; Zhang, H; Baltrons, M A; Li Puma, D D; Chatterjee, I; Li, J; Saeed, F; Berman, H L; Ripoli, C; Gulisano, W; Gonzalez, J; Tian, H; Costa, J A; Lopez, P; Davidowitz, E; Yu, W H; Haroutunian, V; Brown, L M; Palmeri, A; Sigurdsson, E M; Duff, K E; Teich, A F; Honig, L S; Sierks, M; Moe, J G; D'Adamio, L; Grassi, C; Kanaan, N M; Fraser, P E; Arancio, O

    2016-01-20

    Non-fibrillar soluble oligomeric forms of amyloid-β peptide (oAβ) and tau proteins are likely to play a major role in Alzheimer's disease (AD). The prevailing hypothesis on the disease etiopathogenesis is that oAβ initiates tau pathology that slowly spreads throughout the medial temporal cortex and neocortices independently of Aβ, eventually leading to memory loss. Here we show that a brief exposure to extracellular recombinant human tau oligomers (oTau), but not monomers, produces an impairment of long-term potentiation (LTP) and memory, independent of the presence of high oAβ levels. The impairment is immediate as it raises as soon as 20 min after exposure to the oligomers. These effects are reproduced either by oTau extracted from AD human specimens, or naturally produced in mice overexpressing human tau. Finally, we found that oTau could also act in combination with oAβ to produce these effects, as sub-toxic doses of the two peptides combined lead to LTP and memory impairment. These findings provide a novel view of the effects of tau and Aβ on memory loss, offering new therapeutic opportunities in the therapy of AD and other neurodegenerative diseases associated with Aβ and tau pathology.

  10. Non-esterified fatty acids generate distinct low-molecular weight amyloid-β (Aβ42) oligomers along pathway different from fibril formation.

    PubMed

    Kumar, Amit; Bullard, Rebekah L; Patel, Pritesh; Paslay, Lea C; Singh, Dipti; Bienkiewicz, Ewa A; Morgan, Sarah E; Rangachari, Vijayaraghavan

    2011-04-19

    Amyloid-β (Aβ) peptide aggregation is known to play a central role in the etiology of Alzheimer's disease (AD). Among various aggregates, low-molecular weight soluble oligomers of Aβ are increasingly believed to be the primary neurotoxic agents responsible for memory impairment. Anionic interfaces are known to influence the Aβ aggregation process significantly. Here, we report the effects of interfaces formed by medium-chain (C9-C12), saturated non-esterified fatty acids (NEFAs) on Aβ42 aggregation. NEFAs uniquely affected Aβ42 aggregation rates that depended on both the ratio of Aβ:NEFA as well the critical micelle concentration (CMC) of the NEFAs. More importantly, irrespective of the kind of NEFA used, we observed that two distinct oligomers, 12-18 mers and 4-5 mers were formed via different pathway of aggregation under specific experimental conditions: (i) 12-18 mers were generated near the CMC in which NEFAs augment the rate of Aβ42 aggregation towards fibril formation, and, (ii) 4-5 mers were formed above the CMC, where NEFAs inhibit fibril formation. The data indicated that both 12-18 mers and 4-5 mers are formed along an alternate pathway called 'off-pathway' that did not result in fibril formation and yet have subtle structural and morphological differences that distinguish their bulk molecular behavior. These observations, (i) reflect the possible mechanism of Aβ aggregation in physiological lipid-rich environments, and (ii) reiterate the fact that all oligomeric forms of Aβ need not be obligatory intermediates of the fibril formation pathway.

  11. Droplet-based magnetic bead immunoassay using microchannel-connected multiwell plates (μCHAMPs) for the detection of amyloid beta oligomers.

    PubMed

    Park, Min Cheol; Kim, Moojong; Lim, Gun Taek; Kang, Sung Min; An, Seong Soo A; Kim, Tae Song; Kang, Ji Yoon

    2016-06-21

    Multiwell plates are regularly used in analytical research and clinical diagnosis but often require laborious washing steps and large sample or reagent volumes (typically, 100 μL per well). To overcome such drawbacks in the conventional multiwell plate, we present a novel microchannel-connected multiwell plate (μCHAMP) that can be used for automated disease biomarker detection in a small sample volume by performing droplet-based magnetic bead immunoassay inside the plate. In this μCHAMP-based immunoassay platform, small volumes (30-50 μL) of aqueous-phase working droplets are stably confined within each well by the simple microchannel structure (200-300 μm in height and 0.5-1 mm in width), and magnetic beads are exclusively transported into an adjacent droplet through the oil-filled microchannels assisted by a magnet array aligned beneath and controlled by a XY-motorized stage. Using this μCHAMP-based platform, we were able to perform parallel detection of synthetic amyloid beta (Aβ) oligomers as a model analyte for the early diagnosis of Alzheimer's disease (AD). This platform easily simplified the laborious and consumptive immunoassay procedure by achieving automated parallel immunoassay (32 assays per operation in 3-well connected 96-well plate) within 1 hour and at low sample consumption (less than 10 μL per assay) with no cumbersome manual washing step. Moreover, it could detect synthetic Aβ oligomers even below 10 pg mL(-1) concentration with a calculated detection limit of ∼3 pg mL(-1). Therefore, the μCHAMP and droplet-based magnetic bead immunoassay, with the combination of XY-motorized magnet array, would be a useful platform in the diagnosis of human disease, including AD, which requires low consumption of the patient's body fluid sample and automation of the entire immunoassay procedure for high processing capacity.

  12. Amyloid β oligomer-induced ERK1/2-dependent serine 636/639 phosphorylation of insulin receptor substrate-1 impairs insulin signaling and glycogen storage in human astrocytes.

    PubMed

    Zhang, Qinghua; Guo, Shougang; Zhang, Xiao; Tang, Shi; Wang, Lu; Han, Xiaojuan; Shao, Wen; Cong, Lin; Du, Yifeng

    2015-04-25

    This study is to investigate the effect of amyloid β1-42 oligomers on insulin signaling in astrocytes. Synthetic Aβ1-42 oligomers were prepared and the oligomeric form of Aβ1-42 was verified by an electron microscope. Normal human astrocytes were cultured in Dulbecco's Modified Eagle Medium. Western blotting was employed to measure the amount of lysate proteins. Immunofluorescence was performed to detect the distribution of phosphorylated insulin receptor substrate-1 and expression of P-GSK3β in astrocytes under confocal microscopy and fluorescent microscopy, respectively. Periodic Acid-Schiff staining was used to detect glycogen, the content of which was measured using glycogen assay. Our data showed that Aβ1-42 oligomers inhibited insulin-induced serine phosphorylation of Akt at 473 and GSK3β at serine 9, as well as glycogen storage. However, the levels of phosphorylated GSK3β at tyrosine 216 were significantly increased in the presence of Aβ1-42 oligomers. In addition, the levels of phosphorylated ERK1/2 and insulin receptor substrate-1 at serine 636/639 were significantly increased in response to treatment with Aβ1-42 oligomers. Of note, the responses and inhibitory effects of Aβ1-42 oligomers on insulin signaling were partially reversed by ERK1/2 upstream inhibitor PD98059. Our results demonstrated that Aβ1-42 oligomers impaired insulin signaling and suppressed insulin-induced glycogen storage in human astrocytes, probably due to ERK1/2-dependent serine phosphorylation of insulin receptor substrate-1 at 636/639 induced by Aβ1-42 oligomers. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Brain transit and ameliorative effects of intranasally delivered anti-amyloidoligomer antibody in 5XFAD mice.

    PubMed

    Xiao, Chun; Davis, Francesca J; Chauhan, Balwantsinh C; Viola, Kirsten L; Lacor, Pascale N; Velasco, Pauline T; Klein, William L; Chauhan, Neelima B

    2013-01-01

    Alzheimer's disease (AD) is a global health crisis with limited treatment options. Despite major advances in neurotherapeutics, poor brain penetration due to the blood-brain barrier continues to pose a big challenge in overcoming the access of therapeutics to the central nervous system. In that regard, the non-invasive intranasal route of brain targeting is gaining considerable attention. The nasal mucosa offers a large surface area, rapid absorption, and avoidance of first-pass metabolism increasing drug bioavailability with less systemic side effects. Intranasal delivery is known to utilize olfactory, rostral migratory stream, and trigeminal routes to reach the brain. This investigation confirmed that intranasal delivery of oligomeric amyloid-β antibody (NU4) utilized all three routes to enter the brain with a resident time of 96 hours post single bolus intranasal administration, and showed evidence of perikaryal and parenchymal uptake of NU4 in 5XFAD mouse brain, confirming the intranasal route as a non-invasive and efficient way of delivering therapeutics to the brain. In addition, this study demonstrated that intranasal delivery of NU4 antibody lowered cerebral amyloid-β and improved spatial learning in 5XFAD mice.

  14. Amyloid beta oligomers induce neuronal elasticity changes in age-dependent manner: a force spectroscopy study on living hippocampal neurons

    PubMed Central

    Ungureanu, Andreea-Alexandra; Benilova, Iryna; Krylychkina, Olga; Braeken, Dries; De Strooper, Bart; Van Haesendonck, Chris; Dotti, Carlos G.; Bartic, Carmen

    2016-01-01

    Small soluble species of amyloid-beta (Aβ) formed during early peptide aggregation stages are responsible for several neurotoxic mechanisms relevant to the pathology of Alzheimer’s disease (AD), although their interaction with the neuronal membrane is not completely understood. This study quantifies the changes in the neuronal membrane elasticity induced by treatment with the two most common Aβ isoforms found in AD brains: Aβ40 and Aβ42. Using quantitative atomic force microscopy (AFM), we measured for the first time the static elastic modulus of living primary hippocampal neurons treated with pre-aggregated Aβ40 and Aβ42 soluble species. Our AFM results demonstrate changes in the elasticity of young, mature and aged neurons treated for a short time with the two Aβ species pre-aggregated for 2 hours. Neurons aging under stress conditions, showing aging hallmarks, are the most susceptible to amyloid binding and show the largest decrease in membrane stiffness upon Aβ treatment. Membrane stiffness defines the way in which cells respond to mechanical forces in their environment and has been shown to be important for processes such as gene expression, ion-channel gating and neurotransmitter vesicle transport. Thus, one can expect that changes in neuronal membrane elasticity might directly induce functional changes related to neurodegeneration. PMID:27173984

  15. Prion-Protein-interacting AmyloidOligomers of High Molecular Weight Are Tightly Correlated with Memory Impairment in Multiple Alzheimer Mouse Models.

    PubMed

    Kostylev, Mikhail A; Kaufman, Adam C; Nygaard, Haakon B; Patel, Pujan; Haas, Laura T; Gunther, Erik C; Vortmeyer, Alexander; Strittmatter, Stephen M

    2015-07-10

    Alzheimer disease (AD) is characterized by amyloid-β accumulation, with soluble oligomers (Aβo) being the most synaptotoxic. However, the multivalent and unstable nature of Aβo limits molecular characterization and hinders research reproducibility. Here, we characterized multiple Aβo forms throughout the life span of various AD mice and in post-mortem human brain. Aβo exists in several populations, where prion protein (PrP(C))-interacting Aβo is a high molecular weight Aβ assembly present in multiple mice and humans with AD. Levels of PrP(C)-interacting Aβo match closely with mouse memory and are equal or superior to other Aβ measures in predicting behavioral impairment. However, Aβo metrics vary considerably between mouse strains. Deleting PrP(C) expression in mice with relatively low PrP(C)-interacting Aβo (Tg2576) results in partial rescue of cognitive performance as opposed to complete recovery in animals with a high percentage of PrP(C)-interacting Aβo (APP/PSEN1). These findings highlight the relative contributions and interplay of Aβo forms in AD.

  16. Soluble amyloid beta oligomers block the learning-induced increase in hippocampal sharp wave-ripple rate and impair spatial memory formation.

    PubMed

    Nicole, Olivier; Hadzibegovic, Senka; Gajda, Judyta; Bontempi, Bruno; Bem, Tiaza; Meyrand, Pierre

    2016-03-07

    Post-learning hippocampal sharp wave-ripples (SWRs) generated during slow wave sleep are thought to play a crucial role in memory formation. While in Alzheimer's disease, abnormal hippocampal oscillations have been reported, the functional contribution of SWRs to the typically observed spatial memory impairments remains unclear. These impairments have been related to degenerative synaptic changes produced by soluble amyloid beta oligomers (Aβos) which, surprisingly, seem to spare the SWR dynamics during routine behavior. To unravel a potential effect of Aβos on SWRs in cognitively-challenged animals, we submitted vehicle- and Aβo-injected mice to spatial recognition memory testing. While capable of forming short-term recognition memory, Aβ mice exhibited faster forgetting, suggesting successful encoding but an inability to adequately stabilize and/or retrieve previously acquired information. Without prior cognitive requirements, similar properties of SWRs were observed in both groups. In contrast, when cognitively challenged, the post-encoding and -recognition peaks in SWR occurrence observed in controls were abolished in Aβ mice, indicating impaired hippocampal processing of spatial information. These results point to a crucial involvement of SWRs in spatial memory formation and identify the Aβ-induced impairment in SWRs dynamics as a disruptive mechanism responsible for the spatial memory deficits associated with Alzheimer's disease.

  17. Prion-Protein-interacting AmyloidOligomers of High Molecular Weight Are Tightly Correlated with Memory Impairment in Multiple Alzheimer Mouse Models*

    PubMed Central

    Kostylev, Mikhail A.; Kaufman, Adam C.; Nygaard, Haakon B.; Patel, Pujan; Haas, Laura T.; Gunther, Erik C.; Vortmeyer, Alexander; Strittmatter, Stephen M.

    2015-01-01

    Alzheimer disease (AD) is characterized by amyloid-β accumulation, with soluble oligomers (Aβo) being the most synaptotoxic. However, the multivalent and unstable nature of Aβo limits molecular characterization and hinders research reproducibility. Here, we characterized multiple Aβo forms throughout the life span of various AD mice and in post-mortem human brain. Aβo exists in several populations, where prion protein (PrPC)-interacting Aβo is a high molecular weight Aβ assembly present in multiple mice and humans with AD. Levels of PrPC-interacting Aβo match closely with mouse memory and are equal or superior to other Aβ measures in predicting behavioral impairment. However, Aβo metrics vary considerably between mouse strains. Deleting PrPC expression in mice with relatively low PrPC-interacting Aβo (Tg2576) results in partial rescue of cognitive performance as opposed to complete recovery in animals with a high percentage of PrPC-interacting Aβo (APP/PSEN1). These findings highlight the relative contributions and interplay of Aβo forms in AD. PMID:26018073

  18. Soluble amyloid beta oligomers block the learning-induced increase in hippocampal sharp wave-ripple rate and impair spatial memory formation

    PubMed Central

    Nicole, Olivier; Hadzibegovic, Senka; Gajda, Judyta; Bontempi, Bruno; Bem, Tiaza; Meyrand, Pierre

    2016-01-01

    Post-learning hippocampal sharp wave-ripples (SWRs) generated during slow wave sleep are thought to play a crucial role in memory formation. While in Alzheimer’s disease, abnormal hippocampal oscillations have been reported, the functional contribution of SWRs to the typically observed spatial memory impairments remains unclear. These impairments have been related to degenerative synaptic changes produced by soluble amyloid beta oligomers (Aβos) which, surprisingly, seem to spare the SWR dynamics during routine behavior. To unravel a potential effect of Aβos on SWRs in cognitively-challenged animals, we submitted vehicle- and Aβo-injected mice to spatial recognition memory testing. While capable of forming short-term recognition memory, Aβ mice exhibited faster forgetting, suggesting successful encoding but an inability to adequately stabilize and/or retrieve previously acquired information. Without prior cognitive requirements, similar properties of SWRs were observed in both groups. In contrast, when cognitively challenged, the post-encoding and -recognition peaks in SWR occurrence observed in controls were abolished in Aβ mice, indicating impaired hippocampal processing of spatial information. These results point to a crucial involvement of SWRs in spatial memory formation and identify the Aβ-induced impairment in SWRs dynamics as a disruptive mechanism responsible for the spatial memory deficits associated with Alzheimer’s disease. PMID:26947247

  19. Mixed oligomers and monomeric amyloid-β disrupts endothelial cells integrity and reduces monomeric amyloid-β transport across hCMEC/D3 cell line as an in vitro blood-brain barrier model.

    PubMed

    Qosa, Hisham; LeVine, Harry; Keller, Jeffrey N; Kaddoumi, Amal

    2014-09-01

    Senile amyloid plaques are one of the diagnostic hallmarks of Alzheimer's disease (AD). However, the severity of clinical symptoms of AD is weakly correlated with the plaque load. AD symptoms severity is reported to be more strongly correlated with the level of soluble amyloid-β (Aβ) assemblies. Formation of soluble Aβ assemblies is stimulated by monomeric Aβ accumulation in the brain, which has been related to its faulty cerebral clearance. Studies tend to focus on the neurotoxicity of specific Aβ species. There are relatively few studies investigating toxic effects of Aβ on the endothelial cells of the blood-brain barrier (BBB). We hypothesized that a soluble Aβ pool more closely resembling the in vivo situation composed of a mixture of Aβ40 monomer and Aβ42 oligomer would exert higher toxicity against hCMEC/D3 cells as an in vitro BBB model than either component alone. We observed that, in addition to a disruptive effect on the endothelial cells integrity due to enhancement of the paracellular permeability of the hCMEC/D3 monolayer, the Aβ mixture significantly decreased monomeric Aβ transport across the cell culture model. Consistent with its effect on Aβ transport, Aβ mixture treatment for 24h resulted in LRP1 down-regulation and RAGE up-regulation in hCMEC/D3 cells. The individual Aβ species separately failed to alter Aβ clearance or the cell-based BBB model integrity. Our study offers, for the first time, evidence that a mixture of soluble Aβ species, at nanomolar concentrations, disrupts endothelial cells integrity and its own transport across an in vitro model of the BBB.

  20. Synaptic transmission is impaired prior to plaque formation in amyloid precursor protein-overexpressing mice without altering behaviorally-correlated sharp wave-ripple complexes.

    PubMed

    Hermann, D; Both, M; Ebert, U; Gross, G; Schoemaker, H; Draguhn, A; Wicke, K; Nimmrich, V

    2009-09-15

    One of the hallmarks of Alzheimer's disease is the accumulation of amyloid plaques in brains of affected patients. Several recent studies provided evidence that soluble oligomer forms of amyloid-beta (Abeta) rather than plaques determine cognitive decline. In vitro studies using artificial Abeta oligomer preparations suggest that such pathophysiology is caused by a specific impairment of synaptic function. We examined whether synaptic deficits occur before deposition of insoluble fibrillar Abeta by analyzing brain slices taken from young Tg2576 mice overexpressing mutant amyloid precursor protein. Excitatory synaptic transmission in the hippocampal CA1 region was strongly impaired before plaque development, suggesting a dissociation of an early synaptic impairment, probably caused by soluble oligomeric amyloid-beta, from subsequent plaque formation. At higher age neurotransmission was also decreased in wild type mice, paralleling a cognitive decline of normal aged animals. Memory formation in rats is accompanied by distinct hippocampal network oscillations. It has recently been shown that hippocampal gamma oscillations, a network correlate of exploratory behavior, are impaired in amyloid precursor protein (APP)-overexpressing mice. We determined whether sharp wave-ripple complexes, which contribute to memory consolidation during slow wave-sleep, are modified in Tg2576 mice. Interestingly, neither sharp waves nor superimposed ripples were changed at pre-plaque or plaque stages. During aging, however, there was a strong reduction of sharp wave frequency and ripple energy in wild type and APP-overexpressing animals. This indicates that the reported changes in network oscillations following APP-overexpression are specific for gamma oscillations, whereas aging has a more general effect on network properties. Taken together our data suggest that non-fibrillar forms of Abeta--possibly Abeta oligomers--specifically interfere with synaptic function in Tg2576, but do not

  1. The antineoplastic drug flavopiridol reverses memory impairment induced by Amyloid-ß1-42 oligomers in mice.

    PubMed

    Leggio, Gian Marco; Catania, Maria Vincenza; Puzzo, Daniela; Spatuzza, Michela; Pellitteri, Rosalia; Gulisano, Walter; Torrisi, Sebastiano Alfio; Giurdanella, Giovanni; Piazza, Cateno; Impellizzeri, Agata Rita; Gozzo, Lucia; Navarria, Andrea; Bucolo, Claudio; Nicoletti, Ferdinando; Palmeri, Agostino; Salomone, Salvatore; Copani, Agata; Caraci, Filippo; Drago, Filippo

    2016-04-01

    The ectopic re-activation of cell cycle in neurons is an early event in the pathogenesis of Alzheimer's disease (AD), which could lead to synaptic failure and ensuing cognitive deficits before frank neuronal death. Cytostatic drugs that act as cyclin-dependent kinase (CDK) inhibitors have been poorly investigated in animal models of AD. In the present study, we examined the effects of flavopiridol, an inhibitor of CDKs currently used as antineoplastic drug, against cell cycle reactivation and memory loss induced by intracerebroventricular injection of Aß1-42 oligomers in CD1 mice. Cycling neurons, scored as NeuN-positive cells expressing cyclin A, were found both in the frontal cortex and in the hippocampus of Aβ-injected mice, paralleling memory deficits. Starting from three days after Aβ injection, flavopiridol (0.5, 1 and 3mg/kg) was intraperitoneally injected daily, for eleven days. Here we show that a treatment with flavopiridol (0.5 and 1mg/kg) was able to rescue the loss of memory induced by Aβ1-42, and to prevent the occurrence of ectopic cell-cycle events in the mouse frontal cortex and hippocampus. This is the first evidence that a cytostatic drug can prevent cognitive deficits in a non-transgenic animal model of AD.

  2. AmyloidOligomers Transiently Inhibit AMP-activated kinase and Cause Metabolic Defects in Hippocampal Neurons.

    PubMed

    Seixas da Silva, Gisele S; Melo, Helen M; Lourenco, Mychael V; Lyra E Silva, Natalia de M; de Carvalho, Marcelo B; Alves-Leon, Soniza; de Souza, Jorge M; Klein, William L; da-Silva, Wagner S; Ferreira, Sergio T; De Felice, Fernanda G

    2017-03-16

    AMP-activated kinase (AMPK) is a key player in energy sensing and metabolic reprogramming under cellular energy restriction. Several studies have linked impaired AMPK function to peripheral metabolic diseases such as diabetes. However, the impact of neurological disorders, such as Alzheimer disease (AD), on AMPK function and downstream effects of altered AMPK activity on neuronal metabolism have been investigated only recently. Here, we report the impact of A β oligomers (AβOs), synaptotoxins that accumulate in AD brains, on neuronal AMPK activity. Short-term exposure of cultured rat hippocampal neurons or ex vivo human cortical slices to AβOs transiently decreased intracellular ATP levels and AMPK activity, as evaluated by its phosphorylation at threonine residue 172 (AMPKpThr172). The AβO-dependent reduction in AMPKpThr172 levels was mediated by glutamate receptors of the N-methyl-D-aspartate (NMDA) subtype, and resulted in removal of glucose transporters (GLUTs) from the surfaces of dendritic processes in hippocampal neurons. Importantly, insulin prevented the AβO-induced inhibition of AMPK. Our results establish a novel toxic impact of A βOs on neuronal metabolism and suggest that AβO-induced, NMDA receptor-mediated AMPK inhibition may play a key role in early brain metabolic defects in AD.

  3. Effect of the disulfide bridge and the C-terminal extension on the oligomerization of the amyloid peptide ABri implicated in familial British dementia.

    PubMed

    El-Agnaf, O M; Sheridan, J M; Sidera, C; Siligardi, G; Hussain, R; Haris, P I; Austen, B M

    2001-03-27

    Familial British dementia (FBD) is a rare neurodegenerative disorder and shares features with Alzheimer's disease, including amyloid plaque deposits, neurofibrillary tangles, neuronal loss, and progressive dementia. Immunohistochemical and biochemical analysis of plaques and vascular amyloid of FBD brains revealed that a 4 kDa peptide named ABri is the main component of the highly insoluble amyloid deposits. In FBD patients, the ABri peptide is produced as a result of a point mutation in the usual stop codon of the BRI gene. This mutation produces a BRI precursor protein 11 amino acids longer than the wild-type protein. Mutant and wild-type precursor proteins both undergo furin cleavage between residues 243 and 244, producing a peptide of 34 amino acids in the case of ABri and 23 amino acids in the case of the wild-type (WT) peptide. Here we demonstrate that the intramolecular disulfide bond in ABri and the C-terminal extension are required to elongate initially formed dimers to oligomers and fibrils. In contrast, the shorter WT peptide did not aggregate under the same conditions. Conformational analyses indicate that the disulfide bond and the C-terminal extension of ABri are required for the formation of beta-sheet structure. Soluble nonfibrillar ABri oligomers were observed prior to the appearance of mature fibrils. A molecular model of ABri containing three beta-strands, and two beta-hairpins annealed by a disulfide bond, has been constructed, and predicts a hydrophobic surface which is instrumental in promoting oligomerization.

  4. Magnetic bead droplet immunoassay of oligomer amyloid β for the diagnosis of Alzheimer's disease using micro-pillars to enhance the stability of the oil-water interface.

    PubMed

    Kim, Jeong Ah; Kim, Moojong; Kang, Sung Min; Lim, Kun Taek; Kim, Tae Song; Kang, Ji Yoon

    2015-05-15

    Despite scientific progress in the study of Alzheimer's disease (AD), it is still challenging to develop a robust and sensitive methodology for the early diagnosis of AD due to the lack of a decisive biomarker in blood. Recent reports on the oligomer amyloid β (Aβ) as a biomarker demonstrated its possibility for identifying early onset of AD in patients, but its low concentration in blood requires highly reliable detection techniques. To overcome the low reliability and labor-intensive procedures of conventional enzyme-linked immunosorbent assay (ELISA), we present a magnetic bead-droplet immunoassay platform for simple and highly sensitive detection of oligomer Aβ for the diagnosis of AD. This microchip consists of chambers that contain water-based reagents or oil for consecutive assay procedures, and there are arrays of micro-pillars fabricated between the two adjacent chambers to form robust water-oil interfaces. With the aid of these micro-pillars, magnetic beads can stably pass through each chamber by linearly actuating a magnet along the microchip. The robust water-oil interface and simple procedures of the assay make it possible to obtain reliable results from this microchip. The intensity of the fluorescence at the read-out chamber increased quantitatively and linearly, depending on the amount of serially-diluted standard Aβ solution. The results of the assay indicated that the limit of detection was about 10 pg/mL even though it was done with manual manipulation of the magnet. This platform simplified the complicated ELISA procedure and achieved high sensitivity that was no lower than that of the conventional magnetic bead immunoassay. The magnetic bead-droplet platform reduced the assay time to 45 min, and it also reduced the amount of antibody usage in a single diagnosis significantly (10-30 ng of antibody per single assay). Consequently, this microfluidic chip has strong potential as a feasible system for use in the diagnosis of AD with a fast and

  5. Protective effect of leptin and ghrelin against toxicity induced by amyloidoligomers in a hypothalamic cell line.

    PubMed

    Gomes, S; Martins, I; Fonseca, A C R G; Oliveira, C R; Resende, R; Pereira, C M F

    2014-03-01

    In addition to cognitive decline, Alzheimer's disease (AD) patients also exhibit an unexplained weight loss that correlates with disease progression. In young and middle-aged AD patients, large amounts of amyloid-β (Aβ) deposits were observed in the hypothalamus, a brain region involved in the control of feeding and body weight through the action of peripheral metabolic peptides, which have recently been shown to have neuroprotective effects. Moreover, levels of peripheral metabolic peptides, such as leptin and ghrelin, are changed in AD patients. The present study aimed to investigate the role of Aβ peptide in the survival of hypothalamic cells and to explore the receptor-mediated protective effect of leptin and ghrelin against Aβ-induced toxicity in these cells. Using the mHypoE-N42 cell line, we demonstrated for the first time that oligomeric Aβ is toxic to hypothalamic cells, leading to cell death. It was also demonstrated that leptin and ghrelin protect these cells against AβO-induced cell death through the activation of the leptin and ghrelin receptors, respectively. Furthermore, ghrelin and leptin prevented superoxide production, calcium rise and mitochondrial dysfunction triggered by AβO. Taken together, these results suggest that peripheral metabolic peptides, in particular leptin and ghrelin, might be considered as preventive strategies for ameliorating hypothalamic alterations in AD.

  6. EphA4 Activation of c-Abl Mediates Synaptic Loss and LTP Blockade Caused by AmyloidOligomers

    PubMed Central

    M. Vargas, Lina; Leal, Nancy; Estrada, Lisbell D.; González, Adrian; Serrano, Felipe; Araya, Katherine; Gysling, Katia; Inestrosa, Nibaldo C.; Pasquale, Elena B.; Alvarez, Alejandra R.

    2014-01-01

    The early stages of Alzheimer's disease are characterised by impaired synaptic plasticity and synapse loss. Here, we show that amyloidoligomers (AβOs) activate the c-Abl kinase in dendritic spines of cultured hippocampal neurons and that c-Abl kinase activity is required for AβOs-induced synaptic loss. We also show that the EphA4 receptor tyrosine kinase is upstream of c-Abl activation by AβOs. EphA4 tyrosine phosphorylation (activation) is increased in cultured neurons and synaptoneurosomes exposed to AβOs, and in Alzheimer-transgenic mice brain. We do not detect c-Abl activation in EphA4-knockout neurons exposed to AβOs. More interestingly, we demonstrate EphA4/c-Abl activation is a key-signalling event that mediates the synaptic damage induced by AβOs. According to this results, the EphA4 antagonistic peptide KYL and c-Abl inhibitor STI prevented i) dendritic spine reduction, ii) the blocking of LTP induction and iii) neuronal apoptosis caused by AβOs. Moreover, EphA4-/- neurons or sh-EphA4-transfected neurons showed reduced synaptotoxicity by AβOs. Our results are consistent with EphA4 being a novel receptor that mediates synaptic damage induced by AβOs. EphA4/c-Abl signalling could be a relevant pathway involved in the early cognitive decline observed in Alzheimer's disease patients. PMID:24658113

  7. EphA4 activation of c-Abl mediates synaptic loss and LTP blockade caused by amyloidoligomers.

    PubMed

    Vargas, Lina M; Leal, Nancy; Estrada, Lisbell D; González, Adrian; Serrano, Felipe; Araya, Katherine; Gysling, Katia; Inestrosa, Nibaldo C; Pasquale, Elena B; Alvarez, Alejandra R

    2014-01-01

    The early stages of Alzheimer's disease are characterised by impaired synaptic plasticity and synapse loss. Here, we show that amyloidoligomers (AβOs) activate the c-Abl kinase in dendritic spines of cultured hippocampal neurons and that c-Abl kinase activity is required for AβOs-induced synaptic loss. We also show that the EphA4 receptor tyrosine kinase is upstream of c-Abl activation by AβOs. EphA4 tyrosine phosphorylation (activation) is increased in cultured neurons and synaptoneurosomes exposed to AβOs, and in Alzheimer-transgenic mice brain. We do not detect c-Abl activation in EphA4-knockout neurons exposed to AβOs. More interestingly, we demonstrate EphA4/c-Abl activation is a key-signalling event that mediates the synaptic damage induced by AβOs. According to this results, the EphA4 antagonistic peptide KYL and c-Abl inhibitor STI prevented i) dendritic spine reduction, ii) the blocking of LTP induction and iii) neuronal apoptosis caused by AβOs. Moreover, EphA4-/- neurons or sh-EphA4-transfected neurons showed reduced synaptotoxicity by AβOs. Our results are consistent with EphA4 being a novel receptor that mediates synaptic damage induced by AβOs. EphA4/c-Abl signalling could be a relevant pathway involved in the early cognitive decline observed in Alzheimer's disease patients.

  8. Oligomers of Amyloid β Prevent Physiological Activation of the Cellular Prion Protein-Metabotropic Glutamate Receptor 5 Complex by Glutamate in Alzheimer Disease*

    PubMed Central

    Haas, Laura T.

    2016-01-01

    The dysfunction and loss of synapses in Alzheimer disease are central to dementia symptoms. We have recently demonstrated that pathological Amyloid β oligomer (Aβo) regulates the association between intracellular protein mediators and the synaptic receptor complex composed of cellular prion protein (PrPC) and metabotropic glutamate receptor 5 (mGluR5). Here we sought to determine whether Aβo alters the physiological signaling of the PrPC-mGluR5 complex upon glutamate activation. We provide evidence that acute exposure to Aβo as well as chronic expression of familial Alzheimer disease mutant transgenes in model mice prevents protein-protein interaction changes of the complex induced by the glutamate analog 3,5-dihydroxyphenylglycine. We further show that 3,5-dihydroxyphenylglycine triggers the phosphorylation and activation of protein-tyrosine kinase 2-β (PTK2B, also referred to as Pyk2) and of calcium/calmodulin-dependent protein kinase II in wild-type brain slices but not in Alzheimer disease transgenic brain slices or wild-type slices incubated with Aβo. This study further distinguishes two separate Aβo-dependent signaling cascades, one dependent on extracellular Ca2+ and Fyn kinase activation and the other dependent on the release of Ca2+ from intracellular stores. Thus, Aβo triggers multiple distinct PrPC-mGluR5-dependent events implicated in neurodegeneration and dementia. We propose that targeting the PrPC-mGluR5 complex will reverse aberrant Aβo-triggered states of the complex to allow physiological fluctuations of glutamate signaling. PMID:27325698

  9. Peroxiredoxin 5 prevents amyloid-beta oligomer-induced neuronal cell death by inhibiting ERK-Drp1-mediated mitochondrial fragmentation.

    PubMed

    Kim, Bokyung; Park, Junghyung; Chang, Kyu-Tae; Lee, Dong-Seok

    2016-01-01

    Alzheimer's disease (AD), a neurodegenerative disorder, is caused by amyloid-beta oligomers (AβOs). AβOs induce cell death by triggering oxidative stress and mitochondrial dysfunction. A recent study showed that AβO-induced oxidative stress is associated with extracellular signal-regulated kinase (ERK)-dynamin related protein 1 (Drp1)-mediated mitochondrial fission. Reactive oxygen species (ROS) are regulated by antioxidant enzymes, especially peroxiredoxins (Prxs) that scavenge H2O2. These enzymes inhibit neuronal cell death induced by various neurotoxic reagents. However, it is unclear whether Prx5, which is specifically expressed in neuronal cells, protects these cells from AβO-induced damage. In this study, we found that Prx5 expression was upregulated by AβO-induced oxidative stress and that Prx5 decreased ERK-Drp1-mediated mitochondrial fragmentation and apoptosis of HT-22 neuronal cells. Prx5 expression was affected by AβO, and amelioration of oxidative stress by N-acetyl-L-cysteine decreased AβO-induced Prx5 expression. Prx5 overexpression reduced ROS as well as RNS and apoptotic cell death but Prx5 knockdown did not. In addition, Prx5 overexpression ameliorated ERK-Drp1-mediated mitochondrial fragmentation but Prx5 knockdown did not. These results indicated that inducible Prx5 expression by AβO plays a key role in inhibiting both ERK-Drp1-induced mitochondrial fragmentation and neuronal cell death by regulating oxidative stress. Thus, Prx5 may be a new therapeutic agent for treating AD.

  10. Neurodegeneration in an Animal Model of Chronic Amyloid-beta Oligomer Infusion Is Counteracted by Antibody Treatment Infused with Osmotic Pumps.

    PubMed

    Sajadi, Ahmadali; Provost, Chloé; Pham, Brendon; Brouillette, Jonathan

    2016-08-14

    Decline in hippocampal-dependent explicit memory (memory for facts and events) is one of the earliest clinical symptom of Alzheimer's disease (AD). It is well established that synapse loss and ensuing neurodegeneration are the best predictors for memory impairments in AD. Latest studies have emphasized the neurotoxic role of soluble amyloid-beta oligomers (Aβo) that begin to accumulate in the human brain approximately 10 to 15 yr before the clinical symptoms become apparent. Many reports indicate that soluble Aβo correlate with memory deficits in AD models and humans. The Aβo-induced neurodegeneration observed in neuronal and brain slice cultures has been more challenging to reproduce in many animal models. The model of repeated Aβo infusions shown here overcome this issue and allow addressing two key domains for developing new disease modifying therapies: identify biological markers to diagnose early AD, and determine the molecular mechanisms underpinning Aβo-induced memory deficits at the onset of AD. Since soluble Aβo aggregate relatively fast into insoluble Aβ fibrils that correlate poorly with the clinical state of patients, soluble Aβo are prepared freshly and injected once per day during six days to produce marked cell death in the hippocampus. We used cannula specially design for simultaneous infusions of Aβo and continuous infusion of Aβo antibody (6E10) in the hippocampus using osmotic pumps. This innovative in vivo method can now be used in preclinical studies to validate the efficiency of new AD therapies that might prevent the deposition and neurotoxicity of Aβo in pre-dementia patients.

  11. Neurodegeneration in an Animal Model of Chronic Amyloid-beta Oligomer Infusion Is Counteracted by Antibody Treatment Infused with Osmotic Pumps

    PubMed Central

    Sajadi, Ahmadali; Provost, Chloé; Pham, Brendon; Brouillette, Jonathan

    2016-01-01

    Decline in hippocampal-dependent explicit memory (memory for facts and events) is one of the earliest clinical symptom of Alzheimer's disease (AD). It is well established that synapse loss and ensuing neurodegeneration are the best predictors for memory impairments in AD. Latest studies have emphasized the neurotoxic role of soluble amyloid-beta oligomers (Aβo) that begin to accumulate in the human brain approximately 10 to 15 yr before the clinical symptoms become apparent. Many reports indicate that soluble Aβo correlate with memory deficits in AD models and humans. The Aβo-induced neurodegeneration observed in neuronal and brain slice cultures has been more challenging to reproduce in many animal models. The model of repeated Aβo infusions shown here overcome this issue and allow addressing two key domains for developing new disease modifying therapies: identify biological markers to diagnose early AD, and determine the molecular mechanisms underpinning Aβo-induced memory deficits at the onset of AD. Since soluble Aβo aggregate relatively fast into insoluble Aβ fibrils that correlate poorly with the clinical state of patients, soluble Aβo are prepared freshly and injected once per day during six days to produce marked cell death in the hippocampus. We used cannula specially design for simultaneous infusions of Aβo and continuous infusion of Aβo antibody (6E10) in the hippocampus using osmotic pumps. This innovative in vivo method can now be used in preclinical studies to validate the efficiency of new AD therapies that might prevent the deposition and neurotoxicity of Aβo in pre-dementia patients. PMID:27585306

  12. Soluble oligomers of amyloid-β cause changes in redox state, DNA methylation, and gene transcription by inhibiting EAAT3 mediated cysteine uptake.

    PubMed

    Hodgson, Nathaniel; Trivedi, Malav; Muratore, Christina; Li, Shaomin; Deth, Richard

    2013-01-01

    Oxidative stress, hyperhomocysteinemia, altered DNA methylation, and insulin resistance in the brain are associated with Alzheimer's disease (AD), but the role of amyloid-β (Aβ) in these events remains unclear. Intracellular cysteine is rate-limiting for synthesis of the antioxidant glutathione (GSH), and factors regulating cysteine uptake exert a powerful influence over cellular redox status, especially in mature neurons where cysteine synthesis via transsulfuration of homocysteine (HCY) is restricted. We investigated the effect of soluble Aβ oligomers (oAβ) on basal and insulin-like growth factor-1 (IGF-1)-induced cysteine uptake mediated by the excitatory amino acid transporter 3 (EAAT3) in cultured human neuronal cells. We also examined the effect of oAβ on intracellular thiol metabolite levels, DNA methylation, and the transcription status of redox and methylation-associated genes. oAβ inhibited EAAT3-mediated cysteine uptake, causing a decrease in intracellular cysteine and GSH levels. The ratio of the methyl donor S-adenosylmethionine to the methylation inhibitor S-adenosylhomocysteine was decreased, in association with an increase in HCY and a global decrease in DNA methylation, indicative of decreased activity of the redox-sensitive enzyme methionine synthase. These metabolic effects of oAβ coincided with changes in the expression of redox and methylation pathway genes. The ability of oAβ to modulate gene expression via their redox and methylation-dependent epigenetic effects may contribute to the pathology of AD and recognition of this mechanism may lead to novel treatment approaches. We describe a role of IGF-1 signaling in regulating redox and methylation homeostasis, and propose this to be a pathogenic target of oAβ.

  13. Interaction of amyloid-β (Aβ) oligomers with neurexin 2α and neuroligin 1 mediates synapse damage and memory loss in mice.

    PubMed

    Brito-Moreira, Jordano; Lourenco, Mychael V; Oliveira, Mauricio M; Ribeiro, Felipe C; Ledo, José Henrique; Diniz, Luan P; Vital, Juliana F S; Magdesian, Margaret H; Melo, Helen M; Barros-Aragão, Fernanda; de Souza, Jorge M; Alves-Leon, Soniza V; Gomes, Flavia C A; Clarke, Julia R; Figueiredo, Cláudia P; De Felice, Fernanda G; Ferreira, Sergio T

    2017-05-05

    Brain accumulation of the amyloid-β protein (Aβ) and synapse loss are neuropathological hallmarks of Alzheimer disease (AD). Aβ oligomers (AβOs) are synaptotoxins that build up in the brains of patients and are thought to contribute to memory impairment in AD. Thus, identification of novel synaptic components that are targeted by AβOs may contribute to the elucidation of disease-relevant mechanisms. Trans-synaptic interactions between neurexins (Nrxs) and neuroligins (NLs) are essential for synapse structure, stability, and function, and reduced NL levels have been associated recently with AD. Here we investigated whether the interaction of AβOs with Nrxs or NLs mediates synapse damage and cognitive impairment in AD models. We found that AβOs interact with different isoforms of Nrx and NL, including Nrx2α and NL1. Anti-Nrx2α and anti-NL1 antibodies reduced AβO binding to hippocampal neurons and prevented AβO-induced neuronal oxidative stress and synapse loss. Anti-Nrx2α and anti-NL1 antibodies further blocked memory impairment induced by AβOs in mice. The results indicate that Nrx2α and NL1 are targets of AβOs and that prevention of this interaction reduces the deleterious impact of AβOs on synapses and cognition. Identification of Nrx2α and NL1 as synaptic components that interact with AβOs may pave the way for development of novel approaches aimed at halting synapse failure and cognitive loss in AD. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Oligomers of Amyloid β Prevent Physiological Activation of the Cellular Prion Protein-Metabotropic Glutamate Receptor 5 Complex by Glutamate in Alzheimer Disease.

    PubMed

    Haas, Laura T; Strittmatter, Stephen M

    2016-08-12

    The dysfunction and loss of synapses in Alzheimer disease are central to dementia symptoms. We have recently demonstrated that pathological Amyloid β oligomer (Aβo) regulates the association between intracellular protein mediators and the synaptic receptor complex composed of cellular prion protein (PrP(C)) and metabotropic glutamate receptor 5 (mGluR5). Here we sought to determine whether Aβo alters the physiological signaling of the PrP(C)-mGluR5 complex upon glutamate activation. We provide evidence that acute exposure to Aβo as well as chronic expression of familial Alzheimer disease mutant transgenes in model mice prevents protein-protein interaction changes of the complex induced by the glutamate analog 3,5-dihydroxyphenylglycine. We further show that 3,5-dihydroxyphenylglycine triggers the phosphorylation and activation of protein-tyrosine kinase 2-β (PTK2B, also referred to as Pyk2) and of calcium/calmodulin-dependent protein kinase II in wild-type brain slices but not in Alzheimer disease transgenic brain slices or wild-type slices incubated with Aβo. This study further distinguishes two separate Aβo-dependent signaling cascades, one dependent on extracellular Ca(2+) and Fyn kinase activation and the other dependent on the release of Ca(2+) from intracellular stores. Thus, Aβo triggers multiple distinct PrP(C)-mGluR5-dependent events implicated in neurodegeneration and dementia. We propose that targeting the PrP(C)-mGluR5 complex will reverse aberrant Aβo-triggered states of the complex to allow physiological fluctuations of glutamate signaling.

  15. Aβ levels in the jugular vein and high molecular weight Aβ oligomer levels in CSF can be used as biomarkers to indicate the anti-amyloid effect of IVIg for Alzheimer's disease.

    PubMed

    Kasai, Takashi; Kondo, Masaki; Ishii, Ryotaro; Tanaka, Akihiro; Ataka, Suzuka; Shimada, Hiroyuki; Tomiyama, Takami; Mori, Hiroshi; Taylor, Mark; Allsop, David; Nakagawa, Masanori; Mizuno, Toshiki; Tokuda, Takahiko

    2017-01-01

    Intravenous immunoglobulin (IVIg) has been a candidate as a potential anti-amyloid immunotherapy for Alzheimer disease (AD) because it contains anti-amyloid β (Aβ) antibodies. Although several studies with IVIg in AD have been published, changing levels of Aβ efflux from the brain, or disaggregation of Aβ species induced by immunotherapy, have not been properly investigated. Here, we carried out an open label study of therapy with IVIg in five patients with AD. We collected plasma from a peripheral vein (peripheral-plasma) and from the internal jugular vein (jugular-plasma) to estimate directly the efflux of soluble Aβ from the brain. We also measured high molecular weight (HMW) Aβ oligomers in CSF as a marker to detect disaggregated Aβ. IVIg infusions were well tolerated in the majority of cases. However, one study subject had epileptic seizures after IVIg. Levels of HMW CSF Aβ oligomers in all participants were significantly increased after IVIg. Aβ40 and Aβ42 levels in jugular-plasma were continuously or temporarily elevated after treatment in three of five patients who showed preserved cognitive function, whereas levels of those in peripheral-plasma did not correlate with reactivity to the treatment. Other conventional biomarkers including 11C-Pittsburgh compound B retention were not altered after the treatment. These findings imply that HMW Aβ oligomer levels could be a better biomarker to reflect the anti-amyloid effects of IVIg than conventional Aβ species; moreover, Aβ in jugular-plasma seems to be a more direct and precise biomarker to estimate clearance of Aβ from the brain rather than Aβ in peripheral-plasma. UMIN000022319.

  16. RyR2-Mediated Ca(2+) Release and Mitochondrial ROS Generation Partake in the Synaptic Dysfunction Caused by Amyloid β Peptide Oligomers.

    PubMed

    SanMartín, Carol D; Veloso, Pablo; Adasme, Tatiana; Lobos, Pedro; Bruna, Barbara; Galaz, Jose; García, Alejandra; Hartel, Steffen; Hidalgo, Cecilia; Paula-Lima, Andrea C

    2017-01-01

    Amyloid β peptide oligomers (AβOs), toxic aggregates with pivotal roles in Alzheimer's disease, trigger persistent and low magnitude Ca(2+) signals in neurons. We reported previously that these Ca(2+) signals, which arise from Ca(2+) entry and subsequent amplification by Ca(2+) release through ryanodine receptor (RyR) channels, promote mitochondrial network fragmentation and reduce RyR2 expression. Here, we examined if AβOs, by inducing redox sensitive RyR-mediated Ca(2+) release, stimulate mitochondrial Ca(2+)-uptake, ROS generation and mitochondrial fragmentation, and also investigated the effects of the antioxidant N-acetyl cysteine (NAC) and the mitochondrial antioxidant EUK-134 on AβOs-induced mitochondrial dysfunction. In addition, we studied the contribution of the RyR2 isoform to AβOs-induced Ca(2+) release, mitochondrial Ca(2+) uptake and fragmentation. We show here that inhibition of NADPH oxidase type-2 prevented the emergence of RyR-mediated cytoplasmic Ca(2+) signals induced by AβOs in primary hippocampal neurons. Treatment with AβOs promoted mitochondrial Ca(2+) uptake and increased mitochondrial superoxide and hydrogen peroxide levels; ryanodine, at concentrations that suppress RyR activity, prevented these responses. The antioxidants NAC and EUK-134 impeded the mitochondrial ROS increase induced by AβOs. Additionally, EUK-134 prevented the mitochondrial fragmentation induced by AβOs, as previously reported for NAC and ryanodine. These findings show that both antioxidants, NAC and EUK-134, prevented the Ca(2+)-mediated noxious effects of AβOs on mitochondrial function. Our results also indicate that Ca(2+) release mediated by the RyR2 isoform causes the deleterious effects of AβOs on mitochondrial function. Knockdown of RyR2 with antisense oligonucleotides reduced by about 50% RyR2 mRNA and protein levels in primary hippocampal neurons, decreased by 40% Ca(2+) release induced by the RyR agonist 4-chloro-m-cresol, and significantly reduced

  17. The second-generation active Aβ immunotherapy CAD106 reduces amyloid accumulation in APP transgenic mice while minimizing potential side effects.

    PubMed

    Wiessner, Christoph; Wiederhold, Karl-Heinz; Tissot, Alain C; Frey, Peter; Danner, Simone; Jacobson, Laura H; Jennings, Gary T; Lüönd, Rainer; Ortmann, Rainer; Reichwald, Julia; Zurini, Mauro; Mir, Anis; Bachmann, Martin F; Staufenbiel, Matthias

    2011-06-22

    Immunization against amyloid-β (Aβ) can reduce amyloid accumulation in vivo and is considered a potential therapeutic approach for Alzheimer's disease. However, it has been associated with meningoencephalitis thought to be mediated by inflammatory T-cells. With the aim of producing an immunogenic vaccine without this side effect, we designed CAD106 comprising Aβ1-6 coupled to the virus-like particle Qβ. Immunization with this vaccine did not activate Aβ-specific T-cells. In APP transgenic mice, CAD106 induced efficacious Aβ antibody titers of different IgG subclasses mainly recognizing the Aβ3-6 epitope. CAD106 reduced brain amyloid accumulation in two APP transgenic mouse lines. Plaque number was a more sensitive readout than plaque area, followed by Aβ42 and Aβ40 levels. Studies with very strong overall amyloid reduction showed an increase in vascular Aβ, which atypically was nonfibrillar. The efficacy of Aβ immunotherapy depended on the Aβ levels and thus differed between animal models, brain regions, and stage of amyloid deposition. Therefore, animal studies may not quantitatively predict the effect in human Alzheimer's disease. Our studies provided no evidence for increased microhemorrhages or inflammatory reactions in amyloid-containing brain. In rhesus monkeys, CAD106 induced a similar antibody response as in mice. The antibodies stained amyloid deposits on tissue sections of mouse and human brain but did not label cellular structures containing APP. They reacted with Aβ monomers and oligomers and blocked Aβ toxicity in cell culture. We conclude that CAD106 immunization is suited to interfere with Aβ aggregation and its downstream detrimental effects.

  18. TANGO-Inspired Design of Anti-Amyloid Cyclic Peptides.

    PubMed

    Lu, Xiaomeng; Brickson, Claire R; Murphy, Regina M

    2016-09-21

    β-Amyloid peptide (Aβ) self-associates into oligomers and fibrils, in a process that is believed to directly lead to neuronal death in Alzheimer's disease. Compounds that bind to Aβ, and inhibit fibrillogenesis and neurotoxicity, are of interest as an anti-Alzheimer therapeutic strategy. Peptides are particularly attractive for this purpose, because they have advantages over small molecules in their ability to disrupt protein-protein interactions, yet they are amenable to tuning of their properties through chemical means, unlike antibodies. Self-complementation and peptide library screening are two strategies that have been employed in the search for peptides that bind to Aβ. We have taken a different approach, by designing Aβ-binding peptides using transthyretin (TTR) as a template. Previously, we demonstrated that a cyclic peptide, with sequence derived from the known Aβ-binding site on TTR, suppressed Aβ aggregation into fibrils and protected neurons against Aβ toxicity. Here, we searched for cyclic peptides with improved efficacy, by employing the algorithm TANGO, designed originally to identify amyloidogenic sequences in proteins. By using TANGO as a guide to predict the effect of sequence modifications on conformation and aggregation, we synthesized a significantly improved cyclic peptide. We demonstrate that the peptide, in binding to Aβ, redirects Aβ toward protease-sensitive, nonfibrillar aggregates. Cyclic peptides designed using this strategy have attractive solubility, specificity, and stability characteristics.

  19. β-Amyloid Oligomers Induce Phosphorylation of Tau and Inactivation of Insulin Receptor Substrate via c-Jun N-Terminal Kinase Signaling: Suppression by Omega-3 Fatty Acids and Curcumin

    PubMed Central

    Ma, Qiu-Lan; Yang, Fusheng; Rosario, Emily R.; Ubeda, Oliver J.; Beech, Walter; Gant, Dana J.; Chen, Ping Ping; Hudspeth, Beverly; Chen, Cory; Zhao, Yongle; Vinters, Harry V.; Frautschy, Sally A.

    2009-01-01

    Both insulin resistance (type II diabetes) and β-amyloid (Aβ) oligomers are implicated in Alzheimer's disease (AD). Here, we investigate the role of Aβ oligomer-induced c-Jun N-terminal kinase (JNK) activation leading to phosphorylation and degradation of the adaptor protein insulin receptor substrate-1 (IRS-1). IRS-1 couples insulin and other trophic factor receptors to downstream kinases and neuroprotective signaling. Increased phospho-IRS-1 is found in AD brain and insulin-resistant tissues from diabetics. Here, we report Aβ oligomers significantly increased active JNK and phosphorylation of IRS-1 (Ser616) and tau (Ser422) in cultured hippocampal neurons, whereas JNK inhibition blocked these responses. The omega-3 fatty acid docosahexaenoic acid (DHA) similarly inhibited JNK and the phosphorylation of IRS-1 and tau in cultured hippocampal neurons. Feeding 3xTg-AD transgenic mice a diet high in saturated and omega-6 fat increased active JNK and phosphorylated IRS-1 and tau. Treatment of the 3xTg-AD mice on high-fat diet with fish oil or curcumin or a combination of both for 4 months reduced phosphorylated JNK, IRS-1, and tau and prevented the degradation of total IRS-1. This was accompanied by improvement in Y-maze performance. Mice fed with fish oil and curcumin for 1 month had more significant effects on Y-maze, and the combination showed more significant inhibition of JNK, IRS-1, and tau phosphorylation. These data indicate JNK mediates Aβ oligomer inactivation of IRS-1 and phospho-tau pathology and that dietary treatment with fish oil/DHA, curcumin, or a combination of both has the potential to improve insulin/trophic signaling and cognitive deficits in AD. PMID:19605645

  20. Dendritic spine density, morphology, and fibrillar actin content surrounding amyloid-β plaques in a mouse model of amyloid-β deposition.

    PubMed

    Kirkwood, Caitlin M; Ciuchta, Jennifer; Ikonomovic, Milos D; Fish, Kenneth N; Abrahamson, Eric E; Murray, Patrick S; Klunk, William E; Sweet, Robert A

    2013-08-01

    Dendritic spines are the site of most excitatory synapses, the loss of which correlates with cognitive impairment in patients with Alzheimer disease. Substantial evidence indicates that amyloid-β (Aβ) peptide, either insoluble fibrillar Aβ deposited into plaques or soluble nonfibrillar Aβ species, can cause spine loss but the concurrent contributions of fibrillar Aβ and nonfibrillar Aβ to spine loss has not been previously assessed. We used multiple-label immunohistochemistry to measure spine density, size, and F-actin content surrounding plaques in the cerebral cortex in the PSAPP mouse model of Aβ deposition. Our approach allowed us to measure fibrillar Aβ plaque content and an index of nonfibrillar Aβ species concurrently. We found that spine density was reduced within 6 μm of the plaque perimeter, remaining spines were more compact, and F-actin content per spine was increased. Measures of fibrillar Aβ plaque content were associated with reduced spine density near plaques, whereas measures of nonfibrillar Aβ species were associated with reduced spine density and size but not altered F-actin content. These findings suggest that strategies to preserve dendritic spines in AD patients may need to address both nonfibrillar and fibrillar forms of Aβ and that nonfibrillar Aβ may exert spine toxicity through pathways not mediated by depolymerization of F-actin.

  1. Dietary arachidonic acid increases deleterious effects of amyloidoligomers on learning abilities and expression of AMPA receptors: putative role of the ACSL4-cPLA2 balance.

    PubMed

    Thomas, Mélanie H; Paris, Cédric; Magnien, Mylène; Colin, Julie; Pelleïeux, Sandra; Coste, Florence; Escanyé, Marie-Christine; Pillot, Thierry; Olivier, Jean-Luc

    2017-08-29

    Polyunsaturated fatty acids play a crucial role in neuronal function, and the modification of these compounds in the brain could have an impact on neurodegenerative diseases such as Alzheimer's disease. Despite the fact that arachidonic acid is the second foremost polyunsaturated fatty acid besides docosahexaenoic acid, its role and the regulation of its transfer and mobilization in the brain are poorly known. Two groups of 39 adult male BALB/c mice were fed with an arachidonic acid-enriched diet or an oleic acid-enriched diet, respectively, for 12 weeks. After 10 weeks on the diet, mice received intracerebroventricular injections of either NaCl solution or amyloid-β peptide (Aβ) oligomers. Y-maze and Morris water maze tests were used to evaluate short- and long-term memory. At 12 weeks on the diet, mice were killed, and blood, liver, and brain samples were collected for lipid and protein analyses. We found that the administration of an arachidonic acid-enriched diet for 12 weeks induced short-term memory impairment and increased deleterious effects of Aβ oligomers on learning abilities. These cognitive alterations were associated with modifications of expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, postsynaptic density protein 95, and glial fibrillary acidic protein in mouse cortex or hippocampus by the arachidonic acid-enriched diet and Aβ oligomer administration. This diet also led to an imbalance between the main ω-6 fatty acids and the ω-3 fatty acids in favor of the first one in erythrocytes and the liver as well as in the hippocampal and cortical brain structures. In the cortex, the dietary arachidonic acid also induced an increase of arachidonic acid-containing phospholipid species in phosphatidylserine class, whereas intracerebroventricular injections modified several arachidonic acid- and docosahexaenoic acid-containing species in the four phospholipid classes. Finally, we observed that dietary arachidonic acid

  2. Single Particle Characterization of Aβ Oligomers in Solution

    PubMed Central

    Yusko, Erik C.; Prangkio, Panchika; Sept, David; Rollings, Ryan C.; Li, Jiali; Mayer, Michael

    2012-01-01

    Determining the pathological role of amyloids in amyloid-associated diseases will require a method for determining the dynamic distributions in size and shape of amyloid oligomers with high resolution. Here, we explored the potential of resistive-pulse sensing through lipid bilayer-coated nanopores to measure the size of individual amyloidoligomers directly in solution and without chemical modification. This method classified individual amyloid-β aggregates as spherical oligomers, protofibrils, or mature fibers and made it possible to account for the large heterogeneity of amyloid-β aggregate sizes. The approach revealed the distribution of protofibrillar lengths as well as the average cross-sectional area of protofibrils and fibers. PMID:22686709

  3. High-mobility group box-1 protein and β-amyloid oligomers promote neuronal differentiation of adult hippocampal neural progenitors via receptor for advanced glycation end products/nuclear factor-κB axis: relevance for Alzheimer's disease.

    PubMed

    Meneghini, Vasco; Bortolotto, Valeria; Francese, Maria Teresa; Dellarole, Anna; Carraro, Lorenzo; Terzieva, Slavica; Grilli, Mariagrazia

    2013-04-03

    Dysregulated hippocampal neurogenesis has been associated with neurodegenerative disorders, including Alzheimer's disease (AD), in which it may potentially represent an auto-reparatory mechanism that could counteract neuronal loss and cognitive impairment. We evaluated hippocampal neurogenesis in TgCRND8 mice and reported that, at 32 weeks of age, corresponding to an advanced AD-like neuropathology stage, increased numbers of proliferating cells, doublecortin-expressing progenitors/neuroblasts, and early postmitotic calretinin-expressing neurons were present compared with wild-type (WT) littermates. When hippocampal neural progenitor cells (NPCs) were isolated from TgCRND8 mice, we demonstrated that (1) their neurogenic potential was higher compared with WT NPCs; (2) medium conditioned by TgCRND8 NPC promoted neuronal differentiation of WT NPCs; and (3) the proneurogenic effect of TgCRND8-conditioned medium was counteracted by blockade of the receptor for advanced glycation end products (RAGE)/nuclear factor-κB (NF-κB) axis. Furthermore, we showed that β-amyloid 1-42 (Aβ(1-42)) oligomers, but not monomers and fibrils, and the alarmin high-mobility group box-1 protein (HMGB-1) could promote neuronal differentiation of NPCs via activation of the RAGE/NF-κB axis. Altogether, these data suggest that, in AD brain, an endogenous proneurogenic response could be potentially triggered and involve signals (Aβ(1-42) oligomers and HMGB-1) and pathways (RAGE/NF-κB activation) that also contribute to neuroinflammation/neurotoxicity. A more detailed analysis confirmed no significant increase of new mature neurons in hippocampi of TgCRND8 compared with WT mice, suggesting reduced survival and/or integration of newborn neurons. Therapeutic strategies in AD should ideally combine the ability of sustaining hippocampal neurogenesis as well as of counteracting an hostile brain microenvironment so to promote survival of vulnerable cell populations, including adult generated

  4. New Altered Non-Fibrillar Collagens in Human Dilated Cardiomyopathy: Role in the Remodeling Process

    PubMed Central

    Ortega, Ana; Tarazón, Estefanía; Triviño, Juan Carlos; Martínez-Dolz, Luis; González-Juanatey, José Ramón; Lago, Francisca; Portolés, Manuel; Rivera, Miguel

    2016-01-01

    Background In dilated cardiomyopathy (DCM), cardiac failure is accompanied by profound alterations of extracellular matrix associated with the progression of cardiac dilation and left ventricular (LV) dysfunction. Recently, we reported alterations of non-fibrillar collagen expression in ischemic cardiomyopathy linked to fibrosis and cardiac remodeling. We suspect that expression changes in genes coding for non-fibrillar collagens may have a potential role in DCM development. Objectives This study sought to analyze changes in the expression profile of non-fibrillar collagen genes in patients with DCM and to examine relationships between cardiac remodeling parameters and the expression levels of these genes. Methods and Results Twenty-three human left ventricle tissue samples were obtained from DCM patients (n = 13) undergoing heart transplantation and control donors (n = 10) for RNA sequencing analysis. We found increased mRNA levels of six non-fibrillar collagen genes, such as COL4A5, COL9A1, COL21A1, and COL23A1 (P < 0.05 for all), not previously described in DCM. Protein levels of COL8A1 and COL16A1 (P < 0.05 for both), were correspondingly increased. We also identified TGF-β1 significantly upregulated and related to both COL8A1 and COL16A1. Interestingly, we found a significant relationship between LV mass index and the gene expression level of COL8A1 (r = 0.653, P < 0.05). Conclusions In our research, we identified new non-fibrillar collagens with altered expression in DCM, being COL8A1 overexpression directly related to LV mass index, suggesting that they may be involved in the progression of cardiac dilation and remodeling. PMID:27936202

  5. Amyloid-β nanotubes are associated with prion protein-dependent synaptotoxicity

    PubMed Central

    Nicoll, Andrew J.; Panico, Silvia; Freir, Darragh B.; Wright, Daniel; Terry, Cassandra; Risse, Emmanuel; Herron, Caroline E.; O’Malley, Tiernan; Wadsworth, Jonathan D. F.; Farrow, Mark A.; Walsh, Dominic M.; Saibil, Helen R.; Collinge, John

    2013-01-01

    Growing evidence suggests water-soluble, non-fibrillar forms of amyloid-β protein (Aβ) have important roles in Alzheimer’s disease with toxicities mimicked by synthetic Aβ1–42. However, no defined toxic structures acting via specific receptors have been identified and roles of proposed receptors, such as prion protein (PrP), remain controversial. Here we quantify binding to PrP of Aβ1–42 after different durations of aggregation. We show PrP-binding and PrP-dependent inhibition of long-term potentiation (LTP) correlate with the presence of protofibrils. Globular oligomers bind less avidly to PrP and do not inhibit LTP, whereas fibrils inhibit LTP in a PrP-independent manner. That only certain transient Aβ assemblies cause PrP-dependent toxicity explains conflicting reports regarding the involvement of PrP in Aβ-induced impairments. We show that these protofibrils contain a defined nanotubular structure with a previously unidentified triple helical conformation. Blocking the formation of Aβ nanotubes or their interaction with PrP might have a role in treatment of Alzheimer’s disease. PMID:24022506

  6. Amyloid-β nanotubes are associated with prion protein-dependent synaptotoxicity.

    PubMed

    Nicoll, Andrew J; Panico, Silvia; Freir, Darragh B; Wright, Daniel; Terry, Cassandra; Risse, Emmanuel; Herron, Caroline E; O'Malley, Tiernan; Wadsworth, Jonathan D F; Farrow, Mark A; Walsh, Dominic M; Saibil, Helen R; Collinge, John

    2013-01-01

    Growing evidence suggests water-soluble, non-fibrillar forms of amyloid-β protein (Aβ) have important roles in Alzheimer's disease with toxicities mimicked by synthetic Aβ(1-42). However, no defined toxic structures acting via specific receptors have been identified and roles of proposed receptors, such as prion protein (PrP), remain controversial. Here we quantify binding to PrP of Aβ(1-42) after different durations of aggregation. We show PrP-binding and PrP-dependent inhibition of long-term potentiation (LTP) correlate with the presence of protofibrils. Globular oligomers bind less avidly to PrP and do not inhibit LTP, whereas fibrils inhibit LTP in a PrP-independent manner. That only certain transient Aβ assemblies cause PrP-dependent toxicity explains conflicting reports regarding the involvement of PrP in Aβ-induced impairments. We show that these protofibrils contain a defined nanotubular structure with a previously unidentified triple helical conformation. Blocking the formation of Aβ nanotubes or their interaction with PrP might have a role in treatment of Alzheimer's disease.

  7. Sedimentation studies on human amylin fail to detect low-molecular-weight oligomers.

    PubMed

    Vaiana, Sara M; Ghirlando, Rodolfo; Yau, Wai-Ming; Eaton, William A; Hofrichter, James

    2008-04-01

    Sedimentation velocity experiments show that only monomers coexist with amyloid fibrils of human islet amyloid-polypeptide. No oligomers containing <100 monomers could be detected, suggesting that the putative toxic oligomers are much larger than those found for the Alzheimer's peptide, Abeta(1-42).

  8. Induced β-Barrel Formation of the Alzheimer's Aβ25–35 Oligomers on Carbon Nanotube Surfaces: Implication for Amyloid Fibril Inhibition

    PubMed Central

    Fu, Zhaoming; Luo, Yin; Derreumaux, Philippe; Wei, Guanghong

    2009-01-01

    Recent experimental studies show that carbon nanotubes impact the aggregation process of proteins associated with neurodegenerative diseases. However, the details of molecular interactions between proteins and carbon nanotubes are still not well understood. In this study, we investigate the initial adsorption features and dynamics of the Alzheimer's amyloid-β peptide spanning residues 25–35 (Aβ25–35) on a single-walled carbon nanotube (SWNT) surface using fully atomic molecular dynamics simulations (MD) in explicit solvent. The initial configurations of the Aβ25–35 peptides consist of two preformed bilayer β-sheets, each with four or five β-strands in parallel or mixed antiparallel-parallel orientations. Our simulations show, for what we believe is the first time, that two disjointed Aβ25–35 β-sheets with mixed antiparallel-parallel strands can assemble into β-barrels wrapping the SWNT. In contrast, both simulations of Aβ25–35 without SWNT, and simulations of SWNT−Aβ25–35 with purely parallel β-strands, lead to disordered aggregates. We find that Aβ25–35 β-barrel formation involves at least two steps: i), curving of the Aβ25–35 β-sheets as a result of strong hydrophobic interactions with carbon nanotube concomitantly with dehydration of the SWNT-peptide interface; and ii), intersheet backbone hydrogen bond formation with fluctuating intrasheet hydrogen bonds. Detailed analysis of the conversion shows that β-barrel formation on SWNT surface results from the interplay of dehydration and peptide-SWNT/peptide-peptide interactions. Implications of our results on amyloid fibril inhibition are discussed. PMID:19751686

  9. β-Amyloid (Aβ) Oligomers Impair Brain-derived Neurotrophic Factor Retrograde Trafficking by Down-regulating Ubiquitin C-terminal Hydrolase, UCH-L1*

    PubMed Central

    Poon, Wayne W.; Carlos, Anthony J.; Aguilar, Brittany L.; Berchtold, Nicole C.; Kawano, Crystal K.; Zograbyan, Vahe; Yaopruke, Tim; Shelanski, Michael; Cotman, Carl W.

    2013-01-01

    We previously found that BDNF-dependent retrograde trafficking is impaired in AD transgenic mouse neurons. Utilizing a novel microfluidic culture chamber, we demonstrate that Aβ oligomers compromise BDNF-mediated retrograde transport by impairing endosomal vesicle velocities, resulting in impaired downstream signaling driven by BDNF/TrkB, including ERK5 activation, and CREB-dependent gene regulation. Our data suggest that a key mechanism mediating the deficit involves ubiquitin C-terminal hydrolase L1 (UCH-L1), a deubiquitinating enzyme that functions to regulate cellular ubiquitin. Aβ-induced deficits in BDNF trafficking and signaling are mimicked by LDN (an inhibitor of UCH-L1) and can be reversed by increasing cellular UCH-L1 levels, demonstrated here using a transducible TAT-UCH-L1 strategy. Finally, our data reveal that UCH-L1 mRNA levels are decreased in the hippocampi of AD brains. Taken together, our data implicate that UCH-L1 is important for regulating neurotrophin receptor sorting to signaling endosomes and supporting retrograde transport. Further, our results support the idea that in AD, Aβ may down-regulate UCH-L1 in the AD brain, which in turn impairs BDNF/TrkB-mediated retrograde signaling, compromising synaptic plasticity and neuronal survival. PMID:23599427

  10. Cross Talk Between Brain Innate Immunity and Serotonin Signaling Underlies Depressive-Like Behavior Induced by Alzheimer's AmyloidOligomers in Mice.

    PubMed

    Ledo, Jose Henrique; Azevedo, Estefania P; Beckman, Danielle; Ribeiro, Felipe C; Santos, Luis E; Razolli, Daniela S; Kincheski, Grasielle C; Melo, Helen M; Bellio, Maria; Teixeira, Antonio L; Velloso, Licio A; Foguel, Debora; De Felice, Fernanda G; Ferreira, Sergio T

    2016-11-30

    Considerable clinical and epidemiological evidence links Alzheimer's disease (AD) and depression. However, the molecular mechanisms underlying this connection are largely unknown. We reported recently that soluble Aβ oligomers (AβOs), toxins that accumulate in AD brains and are thought to instigate synapse damage and memory loss, induce depressive-like behavior in mice. Here, we report that the mechanism underlying this action involves AβO-induced microglial activation, aberrant TNF-α signaling, and decreased brain serotonin levels. Inactivation or ablation of microglia blocked the increase in brain TNF-α and abolished depressive-like behavior induced by AβOs. Significantly, we identified serotonin as a negative regulator of microglial activation. Finally, AβOs failed to induce depressive-like behavior in Toll-like receptor 4-deficient mice and in mice harboring a nonfunctional TLR4 variant in myeloid cells. Results establish that AβOs trigger depressive-like behavior via a double impact on brain serotonin levels and microglial activation, unveiling a cross talk between brain innate immunity and serotonergic signaling as a key player in mood alterations in AD.

  11. β-Amyloid (Aβ) oligomers impair brain-derived neurotrophic factor retrograde trafficking by down-regulating ubiquitin C-terminal hydrolase, UCH-L1.

    PubMed

    Poon, Wayne W; Carlos, Anthony J; Aguilar, Brittany L; Berchtold, Nicole C; Kawano, Crystal K; Zograbyan, Vahe; Yaopruke, Tim; Shelanski, Michael; Cotman, Carl W

    2013-06-07

    We previously found that BDNF-dependent retrograde trafficking is impaired in AD transgenic mouse neurons. Utilizing a novel microfluidic culture chamber, we demonstrate that Aβ oligomers compromise BDNF-mediated retrograde transport by impairing endosomal vesicle velocities, resulting in impaired downstream signaling driven by BDNF/TrkB, including ERK5 activation, and CREB-dependent gene regulation. Our data suggest that a key mechanism mediating the deficit involves ubiquitin C-terminal hydrolase L1 (UCH-L1), a deubiquitinating enzyme that functions to regulate cellular ubiquitin. Aβ-induced deficits in BDNF trafficking and signaling are mimicked by LDN (an inhibitor of UCH-L1) and can be reversed by increasing cellular UCH-L1 levels, demonstrated here using a transducible TAT-UCH-L1 strategy. Finally, our data reveal that UCH-L1 mRNA levels are decreased in the hippocampi of AD brains. Taken together, our data implicate that UCH-L1 is important for regulating neurotrophin receptor sorting to signaling endosomes and supporting retrograde transport. Further, our results support the idea that in AD, Aβ may down-regulate UCH-L1 in the AD brain, which in turn impairs BDNF/TrkB-mediated retrograde signaling, compromising synaptic plasticity and neuronal survival.

  12. MR microscopy of human amyloid-β deposits: characterization of parenchymal amyloid, diffuse plaques, and vascular amyloid.

    PubMed

    Nabuurs, Rob J A; Natté, Remco; de Ronde, Fenna M; Hegeman-Kleinn, Ingrid; Dijkstra, Jouke; van Duinen, Sjoerd G; Webb, Andrew G; Rozemuller, Annemieke J; van Buchem, Mark A; van der Weerd, Louise

    2013-01-01

    Cerebral deposits of amyloid-β peptides (Aβ) form the neuropathological hallmarks of Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA). In the brain, Aβ can aggregate as insoluble fibrils present in amyloid plaques and vascular amyloid, or as diffuse plaques consisting of mainly non-fibrillar Aβ. Previously, magnetic resonance imaging (MRI) has been shown to be capable of detecting individual amyloid plaques, not only via the associated iron, but also Aβ itself has been suggested to be responsible for a decrease in the image intensity. In this current study we aim to investigate the MRI properties of the different cerebral Aβ deposits including diffuse plaques and vascular amyloid. Postmortem 60-μm-thick brain sections of AD, CAA, and Down's syndrome patients, known to contain Aβ, were studied. High resolution T2*- and T2-weighted MRI scans and quantitative relaxation maps were acquired using a microcoil on a Bruker 9.4T MRI system. Specific MRI characteristics of each type of Aβ deposit were examined by co-registration of the MRI with Congo Red and Aβ-immunostainings of the same sections. Our results show that only fibrillar Aβ, present in both vascular and parenchymal amyloid, induced a significant change in T2* and T2 values. However, signal changes were not as consistent for all of the vessels affected by CAA, irrespective of possible dyshoric changes. In contrast, the non-fibrillar diffuse plaques did not create any detectable MRI signal changes. These findings are relevant for the interpretation and further development of (quantitative) MRI methods for the detection and follow-up of AD and CAA.

  13. Regional distribution of amyloid-Bri deposition and its association with neurofibrillary degeneration in familial British dementia.

    PubMed

    Holton, J L; Ghiso, J; Lashley, T; Rostagno, A; Guerin, C J; Gibb, G; Houlden, H; Ayling, H; Martinian, L; Anderton, B H; Wood, N W; Vidal, R; Plant, G; Frangione, B; Revesz, T

    2001-02-01

    Familial British dementia (FBD), pathologically characterized by cerebral amyloid angiopathy (CAA), amyloid plaques, and neurofibrillary degeneration, is associated with a stop codon mutation in the BRI gene resulting in the production of an amyloidogenic fragment, amyloid-Bri (ABri). The aim of this study was to assess the distribution of ABri fibrillar and nonfibrillar lesions and their relationship to neurofibrillary pathology, astroglial and microglial response using immunohistochemistry, confocal microscopy, and immunoelectron microscopy in five cases of FBD. Abnormal tau was studied with immunoblotting. We present evidence that ABri is deposited throughout the central nervous system in blood vessels and parenchyma where both amyloid (fibrillar) and pre-amyloid (nonfibrillar) lesions are formed. Ultrastructurally amyloid lesions appear as bundles of fibrils recognized by an antibody raised against ABri, whereas Thioflavin S-negative diffuse deposits consist of amorphous electron-dense material with sparse, dispersed fibrils. In contrast to nonfibrillar lesions, fibrillar ABri is associated with a marked astrocytic and microglial response. Neurofibrillary tangles and neuropil threads occurring mainly in limbic structures, are found in areas affected by all types of ABri lesions whereas abnormal neurites are present around amyloid lesions. Immunoblotting for tau revealed a triplet electrophoretic migration pattern. Our observations confirm a close link between ABri deposition and neurodegeneration in FBD.

  14. Carbon Nanotube Inhibits the Formation of β-Sheet-Rich Oligomers of the Alzheimer's Amyloid-β(16-22) Peptide

    PubMed Central

    Li, Huiyu; Luo, Yin; Derreumaux, Philippe; Wei, Guanghong

    2011-01-01

    Alzheimer's disease is associated with the abnormal self-assembly of the amyloid-β (Aβ) peptide into toxic β-rich aggregates. Experimental studies have shown that hydrophobic nanoparticles retard Aβ fibrillation by slowing down the nucleation process; however, the effects of nanoparticles on Aβ oligomeric structures remain elusive. In this study, we investigate the conformations of Aβ(16-22) octamers in the absence and presence of a single-walled carbon nanotube (SWCNT) by performing extensive all-atom replica exchange molecular-dynamics simulations in explicit solvent. Our simulations starting from eight random chains demonstrate that the addition of SWCNT into Aβ(16-22) solution prevents β-sheet formation. Simulation starting from a prefibrillar β-sheet octamer shows that SWCNT destabilizes the β-sheet structure. A detailed analysis of the Aβ(16-22)/SWCNT/water interactions reveals that both the inhibition of β-sheet formation and the destabilization of prefibrillar β-sheets by SWCNT result from the same physical forces: hydrophobic and π-stacking interactions (with the latter playing a more important role). By analyzing the stacking patterns between the Phe aromatic rings and the SWCNT carbon rings, we find that short ring–centroid distances mostly favor parallel orientation, whereas large distances allow all other orientations to be populated. Overall, our computational study provides evidence that SWCNT is likely to inhibit Aβ(16-22) and full-length Aβ fibrillation. PMID:22067167

  15. Pathogenesis of Abeta oligomers in synaptic failure.

    PubMed

    Sivanesan, Senthilkumar; Tan, Aaron; Rajadas, Jayakumar

    2013-03-01

    The soluble Abeta oligomers in brain are highly correlated with memory related synaptic dysfunctions in Alzheimer's disease (AD). However, more recent studies implicate the involvement of Abeta dimers and trimers in memory related AD pathology. Apparently, Abeta oligomers can bind with cellular prion protein at the membrane receptors, forming annular amyloid pores and membrane ion channels to induce aberrant spine cytoskeletal changes. Hence synapse targeting of Abeta oligomers involves activation of many receptors such as N-Methyl-D-aspartate (NMDA), alpha-amino-3- hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), nicotinic acetylcholine (nAChRs), p75 neurotrophin (p75NTR) following aberrant clustering of metabotropic glutamate receptors (mGluR5) leading to neuronal loss and LTP failure. In particular, NMDA and AMPA receptor activation by soluble amyloid oligomers involves calcium mediated mitochondrial dysfunction, decreased Ca((2+))/calmodulin-dependent protein kinase II (CaMKII) levels at the synapses accompanying dramatic loss of synaptic proteins such as postsynaptic density-95 (PSD-95), dynamin-1 and synaptophysin. This kind of receptor-Abeta oligomer interaction might eventually affect the neuronal membrane integrity by altering dielectric barrier, various synaptic proteins, spine morphology and density and P/Q calcium currents that might provoke a cascade of events leading to neuronal loss and memory failure. In this review, we try to explain in detail the various possible mechanisms that connect Abeta oligomers with synapse damage and memory failure.

  16. Neurodegeneration in familial amyloid polyneuropathy: from pathology to molecular signaling.

    PubMed

    Sousa, Mónica Mendes; Saraiva, Maria João

    2003-12-01

    Familial amyloid polyneuropathy (FAP) is an autosomal dominant neurodegenerative disorder related to the systemic deposition of mutated transthyretin (TTR) amyloid fibrils, particularly in peripheral nervous system (PNS). TTR fibrils are diffusely distributed in the PNS of FAP patients, involving nerve trunks, plexuses and ganglia. In peripheral nerves, amyloid deposits are prominent in the endoneurium, near blood vessels, Schwann cells and collagen fibrils. Fiber degeneration is axonal, beginning in the unmyelinated and low diameter myelinated fibers. Several hypotheses have been raised to explain axonal and neuronal loss: (i) compression of the nervous tissue by amyloid; however, a cause-effect relationship between amyloid deposition, structural nerve changes and degeneration was never clearly made; (ii) role of nerve ischemia secondary to lesions caused by perivascular amyloid, which is also doubtful as compromised blood flow was never demonstrated; (iii) lesions in the dorsal root ganglia neurons or Schwann cells. Recently, evidence for the presence of toxic non-fibrillar TTR aggregates early in FAP nerves constituted a first step to unravel molecular signaling related to neurodegeneration in FAP. The toxic nature of TTR non-fibrillar aggregates, and not mature TTR fibrils, was evidenced by their ability to induce the expression of oxidative stress and inflammation-related molecules in neuronal cells, driving them into apoptotic pathways. How these TTR aggregates exert their effects is debatable; interaction with cellular receptors, namely, the receptor for advanced glycation endproducts (RAGE), is a probable candidate mechanism. The pathology and the yet unknown molecular signaling mechanisms responsible for neurodegeneration in FAP are discussed.

  17. Pathogenic mechanisms of prion protein, amyloid-β and α-synuclein misfolding: the prion concept and neurotoxicity of protein oligomers.

    PubMed

    Ugalde, Cathryn L; Finkelstein, David I; Lawson, Victoria A; Hill, Andrew F

    2016-10-01

    Proteinopathies represent a group of diseases characterized by the unregulated misfolding and aggregation of proteins. Accumulation of misfolded protein in the central nervous system (CNS) is associated with neurodegenerative diseases, such as the transmissible spongiform encephalopathies (or prion diseases), Alzheimer's disease, and the synucleinopathies (the most common of which is Parkinson's disease). Of these, the pathogenic mechanisms of prion diseases are particularly striking where the transmissible, causative agent of disease is the prion, or proteinaceous infectious particle. Prions are composed almost exclusively of PrP(Sc) ; a misfolded isoform of the normal cellular protein, PrP(C) , which is found accumulated in the CNS in disease. Today, mounting evidence suggests other aggregating proteins, such as amyloid-β (Aβ) and α-synuclein (α-syn), proteins associated with Alzheimer's disease and synucleinopathies, respectively, share similar biophysical and biochemical properties with PrP(Sc) that influences how they misfold, aggregate, and propagate in disease. In this regard, the definition of a 'prion' may ultimately expand to include other pathogenic proteins. Unifying knowledge of folded proteins may also reveal common mechanisms associated with other features of disease that are less understood, such as neurotoxicity. This review discusses the common features Aβ and α-syn share with PrP and neurotoxic mechanisms associated with these misfolded proteins. Several proteins are known to misfold and accumulate in the central nervous system causing a range of neurodegenerative diseases, such as Alzheimer's, Parkinson's, and the prion diseases. Prions are transmissible misfolded conformers of the prion protein, PrP, which seed further generation of infectious proteins. Similar effects have recently been observed in proteins associated with Alzheimer's disease and the synucleinopathies, leading to the proposition that the definition of a 'prion' may

  18. Age-dependent accumulation of soluble amyloid beta (Abeta) oligomers reverses the neuroprotective effect of soluble amyloid precursor protein-alpha (sAPP(alpha)) by modulating phosphatidylinositol 3-kinase (PI3K)/Akt-GSK-3beta pathway in Alzheimer mouse model.

    PubMed

    Jimenez, Sebastian; Torres, Manuel; Vizuete, Marisa; Sanchez-Varo, Raquel; Sanchez-Mejias, Elisabeth; Trujillo-Estrada, Laura; Carmona-Cuenca, Irene; Caballero, Cristina; Ruano, Diego; Gutierrez, Antonia; Vitorica, Javier

    2011-05-27

    Neurotrophins, activating the PI3K/Akt signaling pathway, control neuronal survival and plasticity. Alterations in NGF, BDNF, IGF-1, or insulin signaling are implicated in the pathogenesis of Alzheimer disease. We have previously characterized a bigenic PS1×APP transgenic mouse displaying early hippocampal Aβ deposition (3 to 4 months) but late (17 to 18 months) neurodegeneration of pyramidal cells, paralleled to the accumulation of soluble Aβ oligomers. We hypothesized that PI3K/Akt/GSK-3β signaling pathway could be involved in this apparent age-dependent neuroprotective/neurodegenerative status. In fact, our data demonstrated that, as compared with age-matched nontransgenic controls, the Ser-9 phosphorylation of GSK-3β was increased in the 6-month PS1×APP hippocampus, whereas in aged PS1×APP animals (18 months), GSK-3β phosphorylation levels displayed a marked decrease. Using N2a and primary neuronal cell cultures, we demonstrated that soluble amyloid precursor protein-α (sAPPα), the predominant APP-derived fragment in young PS1×APP mice, acting through IGF-1 and/or insulin receptors, activated the PI3K/Akt pathway, phosphorylated the GSK-3β activity, and in consequence, exerted a neuroprotective action. On the contrary, several oligomeric Aβ forms, present in the soluble fractions of aged PS1×APP mice, inhibited the induced phosphorylation of Akt/GSK-3β and decreased the neuronal survival. Furthermore, synthetic Aβ oligomers blocked the effect mediated by different neurotrophins (NGF, BDNF, insulin, and IGF-1) and sAPPα, displaying high selectivity for NGF. In conclusion, the age-dependent appearance of APP-derived soluble factors modulated the PI3K/Akt/GSK-3β signaling pathway through the major neurotrophin receptors. sAPPα stimulated and Aβ oligomers blocked the prosurvival signaling. Our data might provide insights into the selective vulnerability of specific neuronal groups in Alzheimer disease.

  19. Capping of Aβ42 Oligomers by Small Molecule Inhibitors

    PubMed Central

    2015-01-01

    Aβ42 peptides associate into soluble oligomers and protofibrils in the process of forming the amyloid fibrils associated with Alzheimer’s disease. The oligomers have been reported to be more toxic to neurons than fibrils, and have been targeted by a wide range of small molecule and peptide inhibitors. With single touch atomic force microscopy (AFM), we show that monomeric Aβ42 forms two distinct types of oligomers, low molecular weight (MW) oligomers with heights of 1–2 nm and high MW oligomers with heights of 3–5 nm. In both cases, the oligomers are disc-shaped with diameters of ∼10–15 nm. The similar diameters suggest that the low MW species stack to form the high MW oligomers. The ability of Aβ42 inhibitors to interact with these oligomers is probed using atomic force microscopy and NMR spectroscopy. We show that curcumin and resveratrol bind to the N-terminus (residues 5–20) of Aβ42 monomers and cap the height of the oligomers that are formed at 1–2 nm. A second class of inhibitors, which includes sulindac sulfide and indomethacin, exhibit very weak interactions across the Aβ42 sequence and do not block the formation of the high MW oligomers. The correlation between N-terminal interactions and capping of the height of the Aβ oligomers provides insights into the mechanism of inhibition and the pathway of Aβ aggregation. PMID:25422864

  20. Gamma-cross-linked nonfibrillar collagen gel as a scaffold for osteogenic differentiation of mesenchymal stem cells.

    PubMed

    Takitoh, Takako; Bessho, Masahiko; Hirose, Motohiro; Ohgushi, Hajime; Mori, Hideki; Hara, Masayuki

    2015-02-01

    We fabricated a transparent nonfibrillar collagen gel using gamma irradiation (5 kGy) and cultured rat mesenchymal stem cells (MSCs) on both the gamma-irradiated collagen gel and on unirradiated fibrillar collagen gel. Cells attached well and proliferated with high viability on the surface of both gels. The cells cultured on the gamma-irradiated nonfibrillar gel had a unique elongated shape and adhered to each other in culture. After 21 days of culture in dexamethasone-containing culture medium, the contents of bone-specific osteocalcin and calcium on the gamma-irradiated nonfibrillar gel were 1.4 and 1.9 times higher than those on fibrillar collagen gel, respectively. These data show that osteogenic differentiation of MSCs was promoted more efficiently on the gamma-cross-linked nonfibrillar gel than on the fibrillar gel and demonstrate the potential of the gamma-irradiated collagen gel for use in bone tissue engineering. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  1. Phenylethynyl terminated imide oligomers

    NASA Technical Reports Server (NTRS)

    Hergenrother, Paul M. (Inventor); Bryant, Robert G. (Inventor); Jensen, Brian J. (Inventor); Havens, Stephen J. (Inventor)

    1995-01-01

    Four phenylethynyl amine compounds - 3 and 4-aminophenoxy-4'-phenylethynylbenzophenone, and 3 and 4-amino-4'-phenylethynylbenzophenone - were readily prepared and were used to endcap imide oligomers. Phenylethynyl-terminated amide acid oligomers and phenylethynyl-terminated imide oligomers with various molecular weights and compositions were prepared and characterized. These oligomers were cured at 300 to 400 C to provide crosslinked polyimides with excellent solvent resistance, high strength and modulus, and good high temperature properties. Adhesive panels, composites, films, and moldings from these phenylethynyl terminated imide oligomers gave excellent mechanical performance.

  2. Mechanical and structural contribution of non-fibrillar matrix in uniaxial tension: a collagen-agarose co-gel model.

    PubMed

    Lake, Spencer P; Barocas, Victor H

    2011-07-01

    The mechanical role of non-fibrillar matrix and the nature of its interaction with the collagen network in soft tissues remain poorly understood, in part because of the lack of a simple experimental model system to quantify these interactions. This study's objective was to examine mechanical and structural properties of collagen-agarose co-gels, utilized as a simplified model system, to understand better the relationships between the collagen network and non-fibrillar matrix. We hypothesized that the presence of agarose would have a pronounced effect on microstructural reorganization and mechanical behavior. Samples fabricated from gel solutions containing 1.0 mg/mL collagen and 0, 0.125, or 0.25% w/v agarose were evaluated via scanning electron microscopy, incremental tensile stress-relaxation tests, and polarized light imaging. While the incorporation of agarose did not dramatically alter collagen network morphology, agarose led to concentration-dependent changes in mechanical and structural properties. Specifically, resistance of co-gels to volume change corresponded with differences in fiber reorientation and elastic/viscoelastic mechanics. Results demonstrate strong relationships between tissue properties and offer insight into behavior of tissues of varying Poisson's ratio and fiber kinematics. Results also suggest that non-fibrillar material may have significant effects on properties of artificial and native tissues even in tension, which is generally assumed to be collagen dominated.

  3. Targeting the proper amyloid-beta neuronal toxins: a path forward for Alzheimer’s disease immunotherapeutics

    PubMed Central

    2014-01-01

    Levels of amyloid-beta monomer and deposited amyloid-beta in the Alzheimer’s disease brain are orders of magnitude greater than soluble amyloid-beta oligomer levels. Monomeric amyloid-beta has no known direct toxicity. Insoluble fibrillar amyloid-beta has been proposed to be an in vivo mechanism for removal of soluble amyloid-beta and exhibits relatively low toxicity. In contrast, soluble amyloid-beta oligomers are widely reported to be the most toxic amyloid-beta form, both causing acute synaptotoxicity and inducing neurodegenerative processes. None of the amyloid-beta immunotherapies currently in clinical development selectively target soluble amyloid-beta oligomers, and their lack of efficacy is not unexpected considering their selectivity for monomeric or fibrillar amyloid-beta (or both) rather than soluble amyloid-beta oligomers. Because they exhibit acute, memory-compromising synaptic toxicity and induce chronic neurodegenerative toxicity and because they exist at very low in vivo levels in the Alzheimer’s disease brain, soluble amyloid-beta oligomers constitute an optimal immunotherapeutic target that should be pursued more aggressively. PMID:25045405

  4. Pyrroloquinoline quinone inhibits the fibrillation of amyloid proteins

    PubMed Central

    Kim, Jihoon; Kobayashi, Masaki; Fukuda, Makoto; Ogasawara, Daisuke; Kobayashi, Natsuki; Han, Sungwoong; Nakamura, Chikashi; Inada, Masaki; Miyaura, Chisato; Ikebukuro, Kazunori

    2010-01-01

    Several neurodegenerative diseases involve the selective damage of neuron cells resulting from the accumulation of amyloid fibril formation. Considering that the formation of amyloid fibrils as well as their precursor oligomers is cytotoxic, the agents that prevent the formation of oligomers and/or fibrils might allow the development of a novel therapeutic approach to neurodegenerative diseases. Here, we show pyrroloquinoline quinone (PQQ) inhibits the amyloid fibril formation of the amyloid proteins, amyloid β (1–42) and mouse prion protein. The fibril formation of mouse prion protein in the presence of PQQ was dramatically prevented. Similarly, the fibril formation of amyloid β (1–42) also decreased. With further advanced pharmacological approaches, PQQ may become a leading anti-neurodegenerative compound in the treatment of neurodegenerative diseases. PMID:20083898

  5. A Generic Method for Design of Oligomer-Specific Antibodies

    PubMed Central

    Brännström, Kristoffer; Lindhagen-Persson, Malin; Gharibyan, Anna L.; Iakovleva, Irina; Vestling, Monika; Sellin, Mikael E.; Brännström, Thomas; Morozova-Roche, Ludmilla; Forsgren, Lars; Olofsson, Anders

    2014-01-01

    Antibodies that preferentially and specifically target pathological oligomeric protein and peptide assemblies, as opposed to their monomeric and amyloid counterparts, provide therapeutic and diagnostic opportunities for protein misfolding diseases. Unfortunately, the molecular properties associated with oligomer-specific antibodies are not well understood, and this limits targeted design and development. We present here a generic method that enables the design and optimisation of oligomer-specific antibodies. The method takes a two-step approach where discrimination between oligomers and fibrils is first accomplished through identification of cryptic epitopes exclusively buried within the structure of the fibrillar form. The second step discriminates between monomers and oligomers based on differences in avidity. We show here that a simple divalent mode of interaction, as within e.g. the IgG isotype, can increase the binding strength of the antibody up to 1500 times compared to its monovalent counterpart. We expose how the ability to bind oligomers is affected by the monovalent affinity and the turnover rate of the binding and, importantly, also how oligomer specificity is only valid within a specific concentration range. We provide an example of the method by creating and characterising a spectrum of different monoclonal antibodies against both the Aβ peptide and α-synuclein that are associated with Alzheimer's and Parkinson's diseases, respectively. The approach is however generic, does not require identification of oligomer-specific architectures, and is, in essence, applicable to all polypeptides that form oligomeric and fibrillar assemblies. PMID:24618582

  6. Elucidating molecular mass and shape of a neurotoxic Aβ oligomer.

    PubMed

    Sebollela, Adriano; Mustata, Gina-Mirela; Luo, Kevin; Velasco, Pauline T; Viola, Kirsten L; Cline, Erika N; Shekhawat, Gajendra S; Wilcox, Kyle C; Dravid, Vinayak P; Klein, William L

    2014-12-17

    Alzheimer's disease (AD), the most prevalent type of dementia, has been associated with the accumulation of amyloid β oligomers (AβOs) in the central nervous system. AβOs vary widely in size, ranging from dimers to larger than 100 kDa. Evidence indicates that not all oligomers are toxic, and there is yet no consensus on the size of the actual toxic oligomer. Here we used NU4, a conformation-dependent anti-AβO monoclonal antibody, to investigate size and shape of a toxic AβO assembly. By using size-exclusion chromatography and immuno-based detection, we isolated an AβO-NU4 complex amenable for biochemical and morphological studies. The apparent molecular mass of the NU4-targeted oligomer was 80 kDa. Atomic force microscopy imaging of the AβO-NU4 complex showed a size distribution centered at 5.37 nm, an increment of 1.5 nm compared to the size of AβOs (3.85 nm). This increment was compatible with the size of NU4 (1.3 nm), suggesting a 1:1 oligomer to NU4 ratio. NU4-reactive oligomers extracted from AD human brain concentrated in a molecular mass range similar to that found for in vitro prepared oligomers, supporting the relevance of the species herein studied. These results represent an important step toward understanding the connection between AβO size and toxicity.

  7. Towards a Pharmacophore for Amyloid

    SciTech Connect

    Landau, Meytal; Sawaya, Michael R.; Faull, Kym F.; Laganowsky, Arthur; Jiang, Lin; Sievers, Stuart A.; Liu, Jie; Barrio, Jorge R.; Eisenberg, David

    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 cocktail of compounds

  8. Memantine rescues transient cognitive impairment caused by high-molecular-weight aβ oligomers but not the persistent impairment induced by low-molecular-weight oligomers.

    PubMed

    Figueiredo, Cláudia P; Clarke, Julia R; Ledo, José Henrique; Ribeiro, Felipe C; Costa, Carine V; Melo, Helen M; Mota-Sales, Axa P; Saraiva, Leonardo M; Klein, William L; Sebollela, Adriano; De Felice, Fernanda G; Ferreira, Sergio T

    2013-06-05

    Brain accumulation of soluble amyloidoligomers (AβOs) has been implicated in synapse failure and cognitive impairment in Alzheimer's disease (AD). However, whether and how oligomers of different sizes induce synapse dysfunction is a matter of controversy. Here, we report that low-molecular-weight (LMW) and high-molecular-weight (HMW) Aβ oligomers differentially impact synapses and memory. A single intracerebroventricular injection of LMW AβOs (10 pmol) induced rapid and persistent cognitive impairment in mice. On the other hand, memory deficit induced by HMW AβOs (10 pmol) was found to be reversible. While memory impairment in LMW oligomer-injected mice was associated with decreased hippocampal synaptophysin and GluN2B immunoreactivities, synaptic pathology was not detected in the hippocampi of HMW oligomer-injected mice. On the other hand, HMW oligomers, but not LMW oligomers, induced oxidative stress in hippocampal neurons. Memantine rescued both neuronal oxidative stress and the transient memory impairment caused by HMW oligomers, but did not prevent the persistent cognitive deficit induced by LMW oligomers. Results establish that different Aβ oligomer assemblies act in an orchestrated manner, inducing different pathologies and leading to synapse dysfunction. Furthermore, results suggest a mechanistic explanation for the limited efficacy of memantine in preventing memory loss in AD.

  9. Soluble Aβ oligomer production and toxicity

    PubMed Central

    Larson, Megan E.; Lesné, Sylvain E.

    2011-01-01

    For nearly 100 years following the first description of this neurological disorder by Dr. Alois Alzheimer, amyloid plaques and neurofibrillary tangles have been hypothesized to cause neuronal loss. With evidence that the extent of insoluble, deposited amyloid poorly correlated with cognitive impairment, research efforts focused on soluble forms of Aβ, also referred as Aβ oligomers. Following a decade of studies, soluble oligomeric forms of Aβ are now believed to induce the deleterious cascade(s) involved in the pathophysiology of Alzheimer’s disease. In this review, we will discuss our current understanding about endogenous oligomeric Aβ production, their relative toxicity in vivo and in vitro, and explore the potential future directions needed for the field. PMID:22121920

  10. Capping of aβ42 oligomers by small molecule inhibitors.

    PubMed

    Fu, Ziao; Aucoin, Darryl; Ahmed, Mahiuddin; Ziliox, Martine; Van Nostrand, William E; Smith, Steven O

    2014-12-23

    Aβ42 peptides associate into soluble oligomers and protofibrils in the process of forming the amyloid fibrils associated with Alzheimer's disease. The oligomers have been reported to be more toxic to neurons than fibrils, and have been targeted by a wide range of small molecule and peptide inhibitors. With single touch atomic force microscopy (AFM), we show that monomeric Aβ42 forms two distinct types of oligomers, low molecular weight (MW) oligomers with heights of 1-2 nm and high MW oligomers with heights of 3-5 nm. In both cases, the oligomers are disc-shaped with diameters of ~10-15 nm. The similar diameters suggest that the low MW species stack to form the high MW oligomers. The ability of Aβ42 inhibitors to interact with these oligomers is probed using atomic force microscopy and NMR spectroscopy. We show that curcumin and resveratrol bind to the N-terminus (residues 5-20) of Aβ42 monomers and cap the height of the oligomers that are formed at 1-2 nm. A second class of inhibitors, which includes sulindac sulfide and indomethacin, exhibit very weak interactions across the Aβ42 sequence and do not block the formation of the high MW oligomers. The correlation between N-terminal interactions and capping of the height of the Aβ oligomers provides insights into the mechanism of inhibition and the pathway of Aβ aggregation.

  11. Interruptions between the triple helix peptides can promote the formation of amyloid-like fibrils

    NASA Astrophysics Data System (ADS)

    Parmar, Avanish; Hwang, Eileen; Brodsky, Barbara

    2010-03-01

    It has been reported that collagen can initiate or accelerate the formation of amyloid fibrils. Non-fibrillar collagen types have sites where the repeating (Gly-Xaa-Yaa)n sequences are interrupted by non- Gly-Xaa-Yaa sequences, and we are investigating the hypothesis that some of these interruptions can promote amyloid formation. Our experimental data show that model peptides containing an 8 or 9 residue interruption sequence between (Gly-Pro-Hyp)n domains have a strong propensity for self association to form fibrous structures. A peptide containing only the 9-residue interruption sequence forms amyloid like fibrils with anti-parallel β sheet. Computational analysis predicts that 33 out of 374 naturally occurring human non-fibrillar collagen sequences within or between triple-helical sequences have significant cross-β aggregation potential, including the 8 and 9 residue sequences studied in peptides. Further studies are in progress to investigate whether a triple-helix peptide promotes amyloidogenesis and whether amyloid interferes with collagen fibrillogenesis.

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

    PubMed Central

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

    2016-01-01

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

  13. A mimotope of Aβ oligomers may also behave as a β-sheet inhibitor.

    PubMed

    Zhang, Yang-Xin; Wang, Shao-Wei; Lu, Shuai; Zhang, Ling-Xiao; Liu, Dong-Qun; Ji, Mei; Wang, Wei-Yun; Liu, Rui-Tian

    2017-10-04

    Beta-amyloid (Aβ) oligomers are strongly associated with the cascade of harmful events leading to neurodegeneration in Alzheimer's disease (AD). Elimination of Aβ oligomers or inhibition of Aβ assembly is a valuable therapeutic approach for the treatment of AD. Here, we obtained a mimotope of Aβ oligomers, AOEP2, by screening a peptide library using oligomer-specific antibodies. The antibodies induced by AOEP2 specifically recognize Aβ oligomers rather than monomers and fibrils. Interestingly, the AOEP2 peptide binds to Aβ monomers and inhibits the formation of Aβ oligomers and β-sheet structure, reduces Aβ42-induced neurotoxicity and decreases the release of proinflammatory cytokines. Taken together, AOEP2, a novel multifunctional peptide directly or indirectly targeting Aβ, has promising therapeutic potential for AD. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  14. A Kinetic Model for Cell Damage Caused by Oligomer Formation.

    PubMed

    Hong, Liu; Huang, Ya-Jing; Yong, Wen-An

    2015-10-06

    It is well known that the formation of amyloid fiber may cause invertible damage to cells, although the underlying mechanism has not been fully understood. In this article, a microscopic model considering the detailed processes of amyloid formation and cell damage is constructed based on four simple assumptions, one of which is that cell damage is raised by oligomers rather than mature fibrils. By taking the maximum entropy principle, this microscopic model in the form of infinite mass-action equations together with two reaction-convection partial differential equations (PDEs) has been greatly coarse-grained into a macroscopic system consisting of only five ordinary differential equations (ODEs). With this simple model, the effects of primary nucleation, elongation, fragmentation, and protein and seeds concentration on amyloid formation and cell damage have been extensively explored and compared with experiments. We hope that our results will provide new insights into the quantitative linkage between amyloid formation and cell damage.

  15. Goodpasture Antigen-binding Protein/Ceramide Transporter Binds to Human Serum Amyloid P-Component and Is Present in Brain Amyloid Plaques*

    PubMed Central

    Mencarelli, Chiara; Bode, Gerard H.; Losen, Mario; Kulharia, Mahesh; Molenaar, Peter C.; Veerhuis, Robert; Steinbusch, Harry W. M.; De Baets, Marc H.; Nicolaes, Gerry A. F.; Martinez-Martinez, Pilar

    2012-01-01

    Serum amyloid P component (SAP) is a non-fibrillar glycoprotein belonging to the pentraxin family of the innate immune system. SAP is present in plasma, basement membranes, and amyloid deposits. This study demonstrates, for the first time, that the Goodpasture antigen-binding protein (GPBP) binds to human SAP. GPBP is a nonconventional Ser/Thr kinase for basement membrane type IV collagen. Also GPBP is found in plasma and in the extracellular matrix. In the present study, we demonstrate that GPBP specifically binds SAP in its physiological conformations, pentamers and decamers. The START domain in GPBP is important for this interaction. SAP and GPBP form complexes in blood and partly colocalize in amyloid plaques from Alzheimer disease patients. These data suggest the existence of complexes of SAP and GPBP under physiological and pathological conditions. These complexes are important for understanding basement membrane, blood physiology, and plaque formation in Alzheimer disease. PMID:22396542

  16. Amyloid fibrils compared to peptide nanotubes.

    PubMed

    Zganec, Matjaž; Zerovnik, Eva

    2014-09-01

    Prefibrillar oligomeric states and amyloid fibrils of amyloid-forming proteins qualify as nanoparticles. We aim to predict what biophysical and biochemical properties they could share in common with better researched peptide nanotubes. We first describe what is known of amyloid fibrils and prefibrillar aggregates (oligomers and protofibrils): their structure, mechanisms of formation and putative mechanism of cytotoxicity. In distinction from other neuronal fibrillar constituents, amyloid fibrils are believed to cause pathology, however, some can also be functional. Second, we give a review of known biophysical properties of peptide nanotubes. Finally, we compare properties of these two macromolecular states side by side and discuss which measurements that have already been done with peptide nanotubes could be done with amyloid fibrils as well.

  17. Exploring the assembly mechanism of tetrapeptide oligomers using the Activation-Relaxation Technique

    NASA Astrophysics Data System (ADS)

    Wei, Guanghong; Mousseau, Normand; Derreumaux, Philippe

    2004-03-01

    Alzheimer's disease and Parkinson's disease are associated with formation of amyloid fibrils. All amyloid fibrils seem to share a common cross β-sheet structure. Experimental studies have shown that peptides as short as 4 amino acids can form amyloid fibrils. It has also been shown that the oligomers that form early in the aggregation process of even non-disease-related proteins may be cytotoxic. We report a detailed study of the assembly mechanisms of the tetrapeptides into different size oligomers: trimers, hexamers and more. The assembly of the oligomers, in which the peptides form β-sheets through interpeptide interactions, are studied using the activation-relaxation technique (ART) in combination with a reduced off-lattice energy model (OPEP). We also describe the multiple pathways of oligomerization as well as categorize the various oligomeric intermediates, providing information of the early events of β-sheet formation.

  18. Preferential association of serum amyloid P component with fibrillar deposits in familial British and Danish dementias: similarities with Alzheimer's disease.

    PubMed

    Rostagno, Agueda; Lashley, Tammaryn; Ng, Douglas; Meyerson, Jordana; Braendgaard, Hans; Plant, Gordon; Bojsen-Møller, Marie; Holton, Janice; Frangione, Blas; Revesz, Tamas; Ghiso, Jorge

    2007-06-15

    Two hereditary forms of cerebrovascular amyloidosis, familial British and Danish dementias (FBD and FDD), share striking similarities with Alzheimer's disease (AD) despite structural differences among their amyloid subunits (ABri in FBD, ADan in FDD, and Abeta in AD). Neuropathological lesions in these disorders include neurofibrillary tangles, parenchymal amyloid and pre-amyloid deposits and overwhelming cerebral amyloid angiopathy co-localizing with reactive microglia and multiple amyloid associated proteins including activation products of the complement cascade. Immunohistochemical analysis of FBD and FDD brain lesions unveiled the presence of serum amyloid P-component (SAP) primarily associated with thioflavin positive amyloid deposits in spite of the significant pre-amyloid burden existing in both disorders. Using affinity chromatography and ELISA binding assays we demonstrated specific, calcium-dependent, saturable, high affinity binding interactions between SAP and ABri/ADan peptides, with dissociation constant values in the sub-nanomolar range and within the same order of magnitude as those resulting from the interaction of SAP with Alzheimer's Abeta1-40 and Abeta1-42. The preferential association of SAP with fibrillar amyloid lesions and not with non-fibrillar pre-amyloid deposits is puzzling, suggesting that SAP modulates the assembly and stability of the final fibril rather than participating in the early steps of protein misfolding and oligomerization.

  19. Single-Molecule Imaging Reveals Aβ42:Aβ40 Ratio-Dependent Oligomer Growth on Neuronal Processes

    PubMed Central

    Johnson, Robin D.; Schauerte, Joseph A.; Chang, Chun-Chieh; Wisser, Kathleen C.; Althaus, John Christian; Carruthers, Cynthia J.L.; Sutton, Michael A.; Steel, Duncan G.; Gafni, Ari

    2013-01-01

    Soluble oligomers of the amyloid-β peptide have been implicated as proximal neurotoxins in Alzheimer’s disease. However, the identity of the neurotoxic aggregate(s) and the mechanisms by which these species induce neuronal dysfunction remain uncertain. Physiologically relevant experimentation is hindered by the low endogenous concentrations of the peptide, the metastability of Aβ oligomers, and the wide range of observed interactions between Aβ and biological membranes. Single-molecule microscopy represents one avenue for overcoming these challenges. Using this technique, we find that Aβ binds to primary rat hippocampal neurons at physiological concentrations. Although amyloid-β(1–40) as well as amyloid-β(1–42) initially form larger oligomers on neurites than on glass slides, a 1:1 mix of the two peptides result in smaller neurite-bound oligomers than those detected on-slide or for either peptide alone. With 1 nM peptide in solution, Aβ40 oligomers do not grow over the course of 48 h, Aβ42 oligomers grow slightly, and oligomers of a 1:1 mix grow substantially. Evidently, small Aβ oligomers are capable of binding to neurons at physiological concentrations and grow at rates dependent on local Aβ42:Aβ40 ratios. These results are intriguing in light of the increased Aβ42:Aβ40 ratios shown to correlate with familial Alzheimer’s disease mutations. PMID:23442968

  20. Natural polyphenols binding to amyloid: a broad class of compounds to treat different human amyloid diseases.

    PubMed

    Ngoungoure, Viviane L Ndam; Schluesener, Jan; Moundipa, Paul F; Schluesener, Hermann

    2015-01-01

    Polyphenols are a large group of phytonutrients found in herbal beverages and foods. They have manifold biological activities, including antioxidative, antimicrobial, and anti-inflammatory properties. Interestingly, some polyphenols bind to amyloid and substantially ameliorate amyloid diseases. Misfolding, aggregation, and accumulation of amyloid fibrils in tissues or organs leads to a group of disorders, called amyloidoses. Prominent diseases are Alzheimer's, Parkinson's, and Huntington's disease, but there are other, less well-known diseases wherein accumulation of misfolded protein is a prominent feature. Amyloidoses are a major burden to public health. In particular, Alzheimer's disease shows a strong increase in patient numbers. Accelerated development of effective therapies for amyloidoses is a necessity. A viable strategy can be the prevention or reduction of protein misfolding, thus reducing amyloid build-up by restoring the cellular aggretome. Amyloid-binding polyphenols affect amyloid formation on various levels, e.g. by inhibiting fibril formation or steering oligomer formation into unstructured, nontoxic pathways. Consequently, preclinical studies demonstrate reduction of amyloid-formation by polyphenols. Amyloid-binding polyphenols might be suitable lead structures for development of imaging agents for early detection of disease and monitoring amyloid deposition. Intake of dietary polyphenols might be relevant to the prevention of amyloidoses. Nutraceutical strategies might be a way to reduce amyloid diseases.

  1. Difference in aggregation between functional and toxic amyloids studied by atomistic simulations

    NASA Astrophysics Data System (ADS)

    Carballo Pacheco, Martin; Ismail, Ahmed E.; Strodel, Birgit

    Amyloids are highly structured protein aggregates, normally associated with neurodegenerative diseases such as Alzheimer's disease. In recent years, a number of nontoxic amyloids with physiologically normal functions, called functional amyloids, have been found. It is known that soluble small oligomers are more toxic than large fibrils. Thus, we study with atomistic explicit-solvent molecular dynamics simulations the oligomer formation of the amyloid- β peptide Aβ25 - 35, associated with Alzheimer's disease, and two functional amyloid-forming tachykinin peptides: kassinin and neuromedin K. Our simulations show that monomeric peptides in extended conformations aggregate faster than those in collapsed hairpin-like conformations. In addition, we observe faster aggregation by functional amyloids than toxic amyloids, which could explain their lack of toxicity.

  2. Monofunctional hyperbranched ethylene oligomers.

    PubMed

    Wiedemann, Thomas; Voit, Gregor; Tchernook, Alexandra; Roesle, Philipp; Göttker-Schnetmann, Inigo; Mecking, Stefan

    2014-02-05

    The neutral κ(2)N,O-salicylaldiminato Ni(II) complexes [κ(2)N,O-{(2,6-(3',5'-R2C6H3)2C6H3-N═C(H)-(3,5-I2-2-O-C6H2)}]NiCH3(pyridine)] (1a-pyr, R = Me; 1b-pyr, R = Et; 1c-pyr, R = iPr) convert ethylene to hyperbranched low-molecular-weight oligomers (Mn ca. 1000 g mol(-1)) with high productivities. While all three catalysts are capable of generating hyperbranched structures, branching densities decrease significantly with the nature of the remote substituent along Me > Et > iPr and oligomer molecular weights increase. Consequently, only 1a-pyr forms hyperbranched structures over a wide range of reaction conditions (ethylene pressure 5-30 atm and 20-70 °C). An in situ catalyst system achieves similar activities and identical highly branched oligomer microstructures, eliminating the bottleneck given by the preparation and isolation of Ni-Me catalyst precursor species. Selective introduction of one primary carboxylic acid ester functional group per highly branched oligoethylene molecule was achieved by isomerizing ethoxycarbonylation and alternatively cross metathesis with ethyl acrylate followed by hydrogenation. The latter approach results in complete functionalization and no essential loss of branched oligomer material and molecular weight, as the reacting double bonds are close to a chain end. Reduction yielded a monoalcohol-functionalized oligomer. Introduction of one reactive epoxide group per branched oligomer occurs completely and selectively under mild conditions. All reaction steps involved in oligomerization and monofunctionalization are efficient and readily scalable.

  3. Optimized Ultrasonic Irradiation Finds Out Ultrastable Aβ1-40 Oligomers.

    PubMed

    Nakajima, Kichitaro; So, Masatomo; Takahashi, Kazuma; Tagawa, Yoh-Ichi; Hirao, Masahiko; Goto, Yuji; Ogi, Hirotsugu

    2017-03-30

    Oligomer species of amyloid β (Aβ) peptides are intensively investigated because of their relevance to Alzheimer's disease (AD), and a stable oligomer will be a cause of AD. In this article, we investigate the structural stability of two representative Aβ1-40 oligomers, which are with and without the β-sheet structure, denoted by β and non-β oligomers, respectively, using optimized ultrasonic irradiation (OUI). Recent studies reveal that OUI significantly accelerates the fibril formation in Aβ1-40 monomers; it is capable of transforming any unstable oligomers into fibrils (the dead-end products) in a short time. First, we find that β oligomers can be produced under high-speed stirring agitation; their β-sheet structures are evaluated by the circular-dichroism spectrum measurement, by the immunoassay using the fibril-specific OC antibody, and by the seeding experiment, showing identical characteristics to those formed in previous reports. Second, we form non-β oligomers in a high-concentration NaCl solution and confirm that they include no β-sheet structure, and they are recognized by the oligomer-specific A11 antibody. Furthermore, we confirm the neurotoxicity of the two types of oligomers using the neural tissue derived from mouse embryonic stem cells. We apply the OUI agitation to the β and non-β oligomers. The non-β oligomers are transformed into the fibrils, indicating that they are intermediate species in the fibrillation pathway. However, the β oligomers are surprisingly unaffected by OUI, indicating their high thermodynamic stability. We conclude that the β oligomers should be the independent dead-end products of another pathway, different from the fibrillation pathway.

  4. Resting microglia react to Aβ42 fibrils but do not detect oligomers or oligomer-induced neuronal damage.

    PubMed

    Ferrera, Denise; Mazzaro, Nadia; Canale, Claudio; Gasparini, Laura

    2014-11-01

    In Alzheimer's disease (AD), amyloid-β (Aβ) deposits accumulate in the brain parenchyma and contain fibrils of aggregated heterogeneous Aβ peptides. In addition to fibrils, Aβ aggregates into stable soluble species (termed Aβ oligomers), which are increasingly viewed as the key drivers of early neurodegenerative events in AD. Aβ aggregates stimulate microglia recruitment and activation. In the AD brain, microglia surround Aβ deposits, activate, and abnormally produce inflammatory mediators, contributing to AD pathogenesis. However, it remains unclear to which of the conformationally diverse Aβ species microglia specifically react. Here, we explore the "sensor" capability of murine microglia. We examine whether they can detect and discriminate the toxic Aβ oligomers, Aβ fibrils, and Aβ-induced neuronal damage and investigate whether they are activated by diverse human Aβ species cell autonomously or through neuron-derived factors. We find that, on aggregation in vitro, Aβ42 peptides form stable oligomers and fibrils, which are neurotoxic and trigger dendritic spine loss in mature primary mouse hippocampal neurons. Further, in resting primary murine microglia, Aβ42 fibrils induce a pattern of expression of inflammatory genes typical of the classical inflammatory response induced by infectious agents (e.g., the bacterial toxin lipopolysaccharide). Conversely, Aβ42 oligomers never elicit a microglia inflammatory response, whether applied alone, in combination with neuron-derived secreted factors, or in contact with neurons. Thus, microglia strongly react to Aβ42 fibrils, but do not sense Aβ oligomers or oligomer-induced neuronal damage. This suggests that early neurotoxic species can escape detection by microglia, leading to the chronic unfolding of amyloid pathology in AD. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. Alzheimer's β-amyloid peptides can activate the early components of complement classical pathway in a C1q-independent manner

    PubMed Central

    Bergamaschini, L; Canziani, S; Bottasso, B; Cugno, M; Braidotti, P; Agostoni, A

    1999-01-01

    β-Amyloid (β-A) accumulates in the brain of patients with Alzheimer's disease (AD) and is presumably involved in the pathogenesis of this disease, on account of its neurotoxicity and complement-activating ability. Although assembly of β-A in particular aggregates seems to be crucial, soluble non-fibrillar β-A may also be involved. Non-fibrillar β-A does not bind C1q, so we investigated alternative mechanisms of β-A-dependent complement activation in vitro. On incubation with normal human plasma, non-fibrillar β-A 1-42, and truncated peptide 1–28, induced dose-dependent activation of C1s and C4, sparing C3, as assessed by densitometric analysis of immunostained membrane after SDS–PAGE and Western blotting. The mechanism of C4 activation was not dependent on C1q, because non-fibrillar β-A can still activate C1s and C4 in plasma genetically deficient in C1q (C1qd). In Factor XII-deficient plasma (F.XIId) the amount of cleaved C4 was about 5–10% less that in C1qd and in normal EDTA plasma; the reconstitution of F.XIId plasma with physiologic concentrations of F.XII resulted in an increased (8–15%) β-A-dependent cleavage of C4. Thus our results indicate that the C1q-independent activation of C1 and C4 can be partially mediated by the activation products of contact system. Since the activation of contact system and of C4 leads to generation of several humoral inflammatory peptides, non-fibrillar β-A might play a role in initiating the early inflammatory reactions leading to a multistep cascade contributing to neuronal and clinical dysfunction of AD brain. PMID:10193429

  6. Alzheimer's beta-amyloid peptides can activate the early components of complement classical pathway in a C1q-independent manner.

    PubMed

    Bergamaschini, L; Canziani, S; Bottasso, B; Cugno, M; Braidotti, P; Agostoni, A

    1999-03-01

    beta-Amyloid (beta-A) accumulates in the brain of patients with Alzheimer's disease (AD) and is presumably involved in the pathogenesis of this disease, on account of its neurotoxicity and complement-activating ability. Although assembly of beta-A in particular aggregates seems to be crucial, soluble non-fibrillar beta-A may also be involved. Non-fibrillar beta-A does not bind C1q, so we investigated alternative mechanisms of beta-A-dependent complement activation in vitro. On incubation with normal human plasma, non-fibrillar beta-A 1-42, and truncated peptide 1-28, induced dose-dependent activation of C1s and C4, sparing C3, as assessed by densitometric analysis of immunostained membrane after SDS-PAGE and Western blotting. The mechanism of C4 activation was not dependent on C1q, because non-fibrillar beta-A can still activate C1s and C4 in plasma genetically deficient in C1q (C1qd). In Factor XII-deficient plasma (F.XIId) the amount of cleaved C4 was about 5-10% less that in C1qd and in normal EDTA plasma; the reconstitution of F.XIId plasma with physiologic concentrations of F.XII resulted in an increased (8-15%) beta-A-dependent cleavage of C4. Thus our results indicate that the C1q-independent activation of C1 and C4 can be partially mediated by the activation products of contact system. Since the activation of contact system and of C4 leads to generation of several humoral inflammatory peptides, non-fibrillar beta-A might play a role in initiating the early inflammatory reactions leading to a multistep cascade contributing to neuronal and clinical dysfunction of AD brain.

  7. Destroying activity of magnetoferritin on lysozyme amyloid fibrils

    NASA Astrophysics Data System (ADS)

    Kopcansky, Peter; Siposova, Katarina; Melnikova, Lucia; Bednarikova, Zuzana; Timko, Milan; Mitroova, Zuzana; Antosova, Andrea; Garamus, Vasil M.; Petrenko, Viktor I.; Avdeev, Mikhail V.; Gazova, Zuzana

    2015-03-01

    Presence of protein amyloid aggregates (oligomers, protofilaments, fibrils) is associated with many diseases as diabetes mellitus or Alzheimer's disease. The interaction between lysozyme amyloid fibrils and magnetoferritin loaded with different amount of iron atoms (168 or 532 atoms) has been investigated by small-angle X-rays scattering and thioflavin T fluorescence measurements. Results suggest that magnetoferritin caused an iron atom-concentration dependent reduction of lysozyme fibril size.

  8. Phenylethynyl terminated reactive oligomer

    NASA Technical Reports Server (NTRS)

    Bryant, Robert G. (Inventor); Jensen, Brian J. (Inventor); Hergenrother, Paul M. (Inventor)

    1995-01-01

    A composition of matter having the general structure: ##STR1## (wherein X is F, Cl, or NO.sub.2, and Y is CO, SO.sub.2 or C(CF.sub.3).sub.2) is employed to terminate a nucleophilic reagent, resulting in the exclusive production of phenylethynyl terminated reactive oligomers which display unique thermal characteristics. A reactive diluent having the general structure: ##STR2## (wherein R is any aliphatic or aromatic moiety) is employed to decrease the melt viscosity of a phenylethynyl terminated reactive oligomer and to subsequently react therewith to provide a thermosetting material of enhanced density. These materials have features which make them attractive candidates for use as composite matrices and adhesives.

  9. Amyloid neuropathies.

    PubMed

    Adams, David; Lozeron, Pierre; Lacroix, Catherine

    2012-10-01

    As amyloid neuropathies have benefited from recent major progress, this review is timely and relevant. The main recent articles on amyloid neuropathy cover its description, methods for diagnosis and therapies. Varied clinical presentations are described in transthyretin (TTR)-familial amyloidosis with polyneuropathy (FAP) and light chain amyloid neuropathy. Mass spectrometry is able to identify the biochemical nature of amyloidogenic protein in nerve biopsy and skin biopsy samples for diagnosis of small fiber polyneuropathy. Both nerve biopsy and TTR gene sequencing are important to identify sporadic cases of amyloid neuropathy. Nerve biopsy is useful in demonstrating the amyloid origin of neuropathies developing after domino liver transplant recipients. Liver transplantation improves long-term survival in Met30 TTR-FAP. Factors recognized as leading to cardiomyopathy progression or heart involvement after liver transplantation are late disease onset and fibril composition. Combined heart and liver transplantation is recommended in severe restrictive cardiomyopathy. Antiamyloid drugs are emerging: tafamidis, a TTR stabilizer, showed in a phase III controlled study its ability to slow stage 1 FAP progression. Other strategies are emerging for TTR-FAP (combination doxycycline-tauroursodeoxycholic acid, small interfering RNA, antisense oligonucleotide, monoclonal antibody antiserum amyloid P component). For light chain neuropathy, intensive chemotherapy may be helpful. There is better recognition of amyloid neuropathies, and hope for enrolling patients with FAP in future clinical trials testing new antiamyloid drugs.

  10. Therapeutic approaches against common structural features of toxic oligomers shared by multiple amyloidogenic proteins.

    PubMed

    Guerrero-Muñoz, Marcos J; Castillo-Carranza, Diana L; Kayed, Rakez

    2014-04-15

    Impaired proteostasis is one of the main features of all amyloid diseases, which are associated with the formation of insoluble aggregates from amyloidogenic proteins. The aggregation process can be caused by overproduction or poor clearance of these proteins. However, numerous reports suggest that amyloid oligomers are the most toxic species, rather than insoluble fibrillar material, in Alzheimer's, Parkinson's, and Prion diseases, among others. Although the exact protein that aggregates varies between amyloid disorders, they all share common structural features that can be used as therapeutic targets. In this review, we focus on therapeutic approaches against shared features of toxic oligomeric structures and future directions. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. Islet amyloid polypeptide (IAPP): a second amyloid in Alzheimer's disease.

    PubMed

    Fawver, Janelle N; Ghiwot, Yonatan; Koola, Catherine; Carrera, Wesley; Rodriguez-Rivera, Jennifer; Hernandez, Caterina; Dineley, Kelly T; Kong, Yu; Li, Jianrong; Jhamandas, Jack; Perry, George; Murray, Ian V J

    2014-01-01

    Amyloid formation is the pathological hallmark of type 2 diabetes (T2D) and Alzheimer's disease (AD). These diseases are marked by extracellular amyloid deposits of islet amyloid polypeptide (IAPP) in the pancreas and amyloid β (Aβ) in the brain. Since IAPP may enter the brain and disparate amyloids can cross-seed each other to augment amyloid formation, we hypothesized that pancreatic derived IAPP may enter the brain to augment misfolding of Aβ in AD. The corollaries for validity of this hypothesis are that IAPP [1] enters the brain, [2] augments Aβ misfolding, [3] associates with Aβ plaques, and most importantly [4] plasma levels correlate with AD diagnosis. We demonstrate the first 3 corollaries that: (1) IAPP is present in the brain in human cerebrospinal fluid (CSF), (2) synthetic IAPP promoted oligomerization of Aβ in vitro, and (3) endogenous IAPP localized to Aβ oligomers and plaques. For the 4th corollary, we did not observe correlation of peripheral IAPP levels with AD pathology in either an African American cohort or AD transgenic mice. In the African American cohort, with increased risk for both T2D and AD, peripheral IAPP levels were not significantly different in samples with no disease, T2D, AD, or both T2D and AD. In the Tg2576 AD mouse model, IAPP plasma levels were not significantly elevated at an age where the mice exhibit the glucose intolerance of pre-diabetes. Based on this negative data, it appears unlikely that peripheral IAPP cross-seeds or "infects" Aβ pathology in AD brain. However, we provide novel and additional data which demonstrate that IAPP protein is present in astrocytes in murine brain and secreted from primary cultured astrocytes. This preliminary report suggests a potential and novel association between brain derived IAPP and AD, however whether astrocytic derived IAPP cross-seeds Aβ in the brain requires further research.

  12. Wnt-5a occludes Abeta oligomer-induced depression of glutamatergic transmission in hippocampal neurons.

    PubMed

    Cerpa, Waldo; Farías, Ginny G; Godoy, Juan A; Fuenzalida, Marco; Bonansco, Christian; Inestrosa, Nibaldo C

    2010-01-18

    Soluble amyloid-beta (Abeta;) oligomers have been recognized to be early and key intermediates in Alzheimer's disease (AD)-related synaptic dysfunction. Abeta oligomers block hippocampal long-term potentiation (LTP) and impair rodent spatial memory. Wnt signaling plays an important role in neural development, including synaptic differentiation. We report here that the Wnt signaling activation prevents the synaptic damage triggered by Abeta oligomers. Electrophysiological analysis of Schaffer collaterals-CA1 glutamatergic synaptic transmission in hippocampal slices indicates that Wnt-5a increases the amplitude of field excitatory postsynaptic potentials (fEPSP) and both AMPA and NMDA components of the excitatory postsynaptic currents (EPSCs), without modifying the paired pulse facilitation (PPF). Conversely, in the presence of Abeta oligomers the fEPSP and EPSCs amplitude decreased without modification of the PPF, while the postsynaptic scaffold protein (PSD-95) decreased as well. Co-perfusion of hippocampal slices with Wnt-5a and Abeta oligomers occludes against the synaptic depression of EPSCs as well as the reduction of PSD-95 clusters induced by Abeta oligomers in neuronal cultures. Taken together these results indicate that Wnt-5a and Abeta oligomers inversely modulate postsynaptic components. These results indicate that post-synaptic damage induced by Abeta oligomers in hippocampal neurons is prevented by non-canonical Wnt pathway activation.

  13. Wnt-5a occludes Aβ oligomer-induced depression of glutamatergic transmission in hippocampal neurons

    PubMed Central

    2010-01-01

    Background Soluble amyloid-β (Aβ;) oligomers have been recognized to be early and key intermediates in Alzheimer's disease (AD)-related synaptic dysfunction. Aβ oligomers block hippocampal long-term potentiation (LTP) and impair rodent spatial memory. Wnt signaling plays an important role in neural development, including synaptic differentiation. Results We report here that the Wnt signaling activation prevents the synaptic damage triggered by Aβ oligomers. Electrophysiological analysis of Schaffer collaterals-CA1 glutamatergic synaptic transmission in hippocampal slices indicates that Wnt-5a increases the amplitude of field excitatory postsynaptic potentials (fEPSP) and both AMPA and NMDA components of the excitatory postsynaptic currents (EPSCs), without modifying the paired pulse facilitation (PPF). Conversely, in the presence of Aβ oligomers the fEPSP and EPSCs amplitude decreased without modification of the PPF, while the postsynaptic scaffold protein (PSD-95) decreased as well. Co-perfusion of hippocampal slices with Wnt-5a and Aβ oligomers occludes against the synaptic depression of EPSCs as well as the reduction of PSD-95 clusters induced by Aβ oligomers in neuronal cultures. Taken together these results indicate that Wnt-5a and Aβ oligomers inversely modulate postsynaptic components. Conclusion These results indicate that post-synaptic damage induced by Aβ oligomers in hippocampal neurons is prevented by non-canonical Wnt pathway activation. PMID:20205789

  14. Polyphenols protect mitochondrial membrane against permeabilization induced by HEWL oligomers: Possible mechanism of action.

    PubMed

    Roqanian, Shaqayeq; Meratan, Ali Akbar; Ahmadian, Shahin; Shafizadeh, Mahshid; Ghasemi, Atiyeh; Karami, Leila

    2017-10-01

    Increasing body of evidence suggests that polyphenols frequently interacting with amyloid aggregates and/or interfering with aggregate species to bind biomembranes may serve as a therapeutic approach for the treatment of amyloid-related diseases. Hence, in the present study, the possible effects of three naturally occurring polyphenols including Curcumin, Quercetin, and Resveratrol on mitochondrial membrane permeabilization induced by Hen Egg White Lysozyme (HEWL) oligomers were investigated. Our results indicated that pre-incubation of mitochondrial homogenate with polyphenols considerably inhibit membrane permeabilization in a concentration dependent manner. In parallel, HEWL oligomers, which were co-incubated with the polyphenols, showed less effectiveness on membrane permeabilization, suggesting that toxicity of oligomers was hindered. Using a range of techniques including fluorescence quenching, Nile red binding assay, zeta potential and size measurements, CD (far- and near-UV) spectroscopy, and molecular docking, we found that the polyphenols, structure-dependently, interact with and induce conformational changes in HEWL oligomers, thereby inhibit their toxicity. We proposed a mechanism by which selected polyphenols induce their protective effects through binding to mitochondria and interfering with HEWL oligomer-membrane interactions and/or by direct interaction with HEWL oligomers, induction of conformational changes, and generating far less toxic species. However, additional studies are needed to elucidate the detailed mechanisms involved. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    NASA Astrophysics Data System (ADS)

    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 amyloid-β peptide 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.

  16. Amyloid-β induced signaling by cellular prion protein and Fyn kinase in Alzheimer disease.

    PubMed

    Um, Ji Won; Strittmatter, Stephen M

    2013-01-01

    Alzheimer disease (AD) is the most prevalent cause of dementia. Amyloid-β (Aβ) oligomers are potent synaptotoxins thought to mediate AD-related phenotypes. Cellular prion protein (PrP(C)) has been identified as a high-affinity receptor for Aβ oligomers. Herein, we review the functional consequences of Aβ oligomer binding to PrP(C) on the neuronal surface. We highlight recent evidence that Fyn kinase mediates signal transduction downstream of the PrP(C)-Aβ oligomer complex. These studies suggest that PrP(C) has a central role in AD pathogenesis and may provide a target for therapeutic intervention in AD.

  17. Metastable Amyloid Phases and their Conversion to Mature Fibrils

    NASA Astrophysics Data System (ADS)

    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.

  18. Inhibition of Toxic IAPP Amyloid by Extracts of Common Fruits.

    PubMed

    Kao, Pei-Yu; Green, Evangeline; Pereira, Catalina; Ekimura, Shauna; Juarez, Dennis; Whyte, Travis; Arhar, Taylor; Malaspina, Bianca; Nogaj, Luiza A; Moffet, David A

    2015-01-01

    The aggregation of the 37-amino acid polypeptide islet amyloid polypeptide (IAPP, amylin), as either insoluble amyloid or as small oligomers, appears to play a direct role in the death of pancreatic β-islet cells in type 2 diabetes. It is believed that inhibiting the aggregation of IAPP may slow down, if not prevent entirely, the progression of this disease. Extracts of thirteen different common fruits were analyzed for their ability to prevent the aggregation of amyloidogenic IAPP. Thioflavin T binding, immuno-detection and circular dichroism assays were performed to test the in vitro inhibitory potential of each extract. Atomic force microscopy was used to visualize the formation of amyloid fibrils with and without each fruit extract. Finally, extracts were tested for their ability to protect living mammalian cells from the toxic effects of amyloid IAPP. Several fruits showed substantial ability to inhibit IAPP aggregation and protect living cells from toxic IAPP amyloid.

  19. Label-free imaging of amyloids using their intrinsic linear and nonlinear optical properties

    PubMed Central

    Johansson, Patrik K.; Koelsch, Patrick

    2017-01-01

    The optical properties of amyloid fibers are often distinct from those of the source protein in its non-fibrillar form. These differences can be utilized for label-free imaging or characterization of such structures, which is particularly important for understanding amyloid fiber related diseases such as Alzheimer’s and Parkinson’s disease. We demonstrate that two amyloid forming proteins, insulin and β-lactoglobulin (β-LG), show intrinsic fluorescence with emission spectra that are dependent on the excitation wavelength. Additionally, a new fluorescence peak at about 430 nm emerges for β-LG in its amyloid state. The shift in emission wavelength is related to the red edge excitation shift (REES), whereas the additional fluorescence peak is likely associated with charge delocalization along the fiber backbone. Furthermore, the spherulitic amyloid plaque-like superstructures formed from the respective proteins were imaged label-free with confocal fluorescence, multiphoton excitation fluorescence (MPEF), and second-harmonic generation (SHG) microscopy. The latter two techniques in particular yield images with a high contrast between the amyloid fiber regions and the core of amorphously structured protein. Strong multiphoton absorption (MPA) for the amyloid fibers is a likely contributor to the observed contrast in the MPEF images. The crystalline fibrillar region provides even higher contrast in the SHG images, due to the inherently ordered non-centrosymmetric structure of the fibers together with their non-isotropic arrangement. Finally, we show that MPEF from the insulin spherulites exhibits a spectral dependence on the excitation wavelength. This behavior is consistent with the REES phenomenon, which we hypothesize is the origin of this observation. The presented results suggest that amyloid deposits can be identified and structurally characterized based on their intrinsic optical properties, which is important for probe-less and label-free identification

  20. Structural characterization of toxic oligomers that are kinetically trapped during α-synuclein fibril formation

    PubMed Central

    Chen, Serene W.; Drakulic, Srdja; Deas, Emma; Ouberai, Myriam; Aprile, Francesco A.; Arranz, Rocío; Ness, Samuel; Roodveldt, Cintia; Guilliams, Tim; De-Genst, Erwin J.; Klenerman, David; Wood, Nicholas W.; Knowles, Tuomas P.J.; Alfonso, Carlos; Rivas, Germán; Abramov, Andrey Y.; Valpuesta, José María; Dobson, Christopher M.; Cremades, Nunilo

    2015-01-01

    We describe the isolation and detailed structural characterization of stable toxic oligomers of α-synuclein that have accumulated during the process of amyloid formation. Our approach has allowed us to identify distinct subgroups of oligomers and to probe their molecular architectures by using cryo-electron microscopy (cryoEM) image reconstruction techniques. Although the oligomers exist in a range of sizes, with different extents and nature of β-sheet content and exposed hydrophobicity, they all possess a hollow cylindrical architecture with similarities to certain types of amyloid fibril, suggesting that the accumulation of at least some forms of amyloid oligomers is likely to be a consequence of very slow rates of rearrangement of their β-sheet structures. Our findings reveal the inherent multiplicity of the process of protein misfolding and the key role the β-sheet geometry acquired in the early stages of the self-assembly process plays in dictating the kinetic stability and the pathological nature of individual oligomeric species. PMID:25855634

  1. Structural characterization of toxic oligomers that are kinetically trapped during α-synuclein fibril formation.

    PubMed

    Chen, Serene W; Drakulic, Srdja; Deas, Emma; Ouberai, Myriam; Aprile, Francesco A; Arranz, Rocío; Ness, Samuel; Roodveldt, Cintia; Guilliams, Tim; De-Genst, Erwin J; Klenerman, David; Wood, Nicholas W; Knowles, Tuomas P J; Alfonso, Carlos; Rivas, Germán; Abramov, Andrey Y; Valpuesta, José María; Dobson, Christopher M; Cremades, Nunilo

    2015-04-21

    We describe the isolation and detailed structural characterization of stable toxic oligomers of α-synuclein that have accumulated during the process of amyloid formation. Our approach has allowed us to identify distinct subgroups of oligomers and to probe their molecular architectures by using cryo-electron microscopy (cryoEM) image reconstruction techniques. Although the oligomers exist in a range of sizes, with different extents and nature of β-sheet content and exposed hydrophobicity, they all possess a hollow cylindrical architecture with similarities to certain types of amyloid fibril, suggesting that the accumulation of at least some forms of amyloid oligomers is likely to be a consequence of very slow rates of rearrangement of their β-sheet structures. Our findings reveal the inherent multiplicity of the process of protein misfolding and the key role the β-sheet geometry acquired in the early stages of the self-assembly process plays in dictating the kinetic stability and the pathological nature of individual oligomeric species.

  2. Insights into the mechanism of Alzheimer's β-amyloid aggregation as a function of concentration by using atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Mustata, Gina-Mirela; Shekhawat, Gajendra S.; Lambert, Mary P.; Viola, Kirsten L.; Velasco, Pauline T.; Klein, William L.; Dravid, Vinayak P.

    2012-03-01

    The size and shape of Alzheimer's β-amyloid structures, as well as the kinetics of their self-assembly, exhibits a very pronounced dependence on concentration and environment. In the present study, we are reporting the direct observation of Aβ oligomers and fibrils assemblies using atomic force microscopy imaging in fluid environment. These results demonstrate that in the Aβ preparations at lower concentrations, predominant are the globular, smaller oligomers, while for higher concentrations, globular oligomers co-exist with higher molecular weight fibrillar structures. At higher concentrations, the fibril formation is a dynamic and continuous process, yielding amyloid fibrils with multiple structures and diameters.

  3. Alzheimer's disease amyloid peptides interact with DNA, as proved by surface plasmon resonance.

    PubMed

    Barrantes, Alejandro; Camero, Sergio; Garcia-Lucas, Angel; Navarro, Pedro J; Benitez, María J; Jiménez, Juan S

    2012-10-01

    According to the amyloid hypothesis, abnormal processing of the β-amyloid precursor protein in Alzheimer's disease patients increases the production of β-amyloid toxic peptides, which, after forming highly aggregated fibrillar structures, lead to extracellular plaques formation, neuronal loss and dementia. However, a great deal of evidence has point to intracellular small oligomers of amyloid peptides, probably transient intermediates in the process of fibrillar structures formation, as the most toxic species. In order to study the amyloid-DNA interaction, we have selected here three different forms of the amyloid peptide: Aβ1-40, Aβ25-35 and a scrambled form of Aβ25-35. Surface Plasmon Resonance was used together with UV-visible spectroscopy, Electrophoresis and Electronic Microscopy to carry out this study. Our results prove that, similarly to the full length Aβ1-42, all conformations of toxic amyloid peptides, Aβ1-40 and Aβ25-35, may bind DNA. In contrast, the scrambled form of Aβ25-35, a non-aggregating and nontoxic form of this peptide, could not bind DNA. We conclude that although the amyloid-DNA interaction is closely related to the amyloid aggregation proneness, this cannot be the only factor which determines the interaction, since small oligomers of amyloid peptides may also bind DNA if their predominant negatively charged amino acid residues are previously neutralized.

  4. Prefibrillar transthyretin oligomers and cold stored native tetrameric transthyretin are cytotoxic in cell culture

    SciTech Connect

    Soergjerd, Karin; Klingstedt, Therese; Lindgren, Mikael; Kagedal, Katarina; Hammarstroem, Per

    2008-12-26

    Recent studies suggest that soluble, oligomeric species, which are intermediates in the fibril formation process in amyloid disease, might be the key species in amyloid pathogenesis. Soluble oligomers of human wild type transthyretin (TTR) were produced to elucidate oligomer properties. Employing ThT fluorescence, time-resolved fluorescence anisotropy of pyrene-labeled TTR, chemical cross-linking, and electron microscopy we demonstrated that early formed soluble oligomers (within minutes) from A-state TTR comprised on the average 20-30 TTR monomers. When administered to neuroblastoma cells these early oligomers proved highly cytotoxic and induced apoptosis after 48 h of incubation. More mature fibrils (>24 h of fibrillation) were non-toxic. Surprisingly, we also found that native tetrameric TTR, when purified and stored under cold conditions (4 deg. C) was highly cytotoxic. The effect could be partially restored by increasing the temperature of the protein. The cytotoxic effects of native tetrameric TTR likely stems from a hitherto unexplored low temperature induced rearrangement of the tetramer conformation that possibly is related to the conformation of misfolded TTR in amyloigogenic oligomers.

  5. Crystal structure of a human prion protein fragment reveals a motif for oligomer formation.

    PubMed

    Apostol, Marcin I; Perry, Kay; Surewicz, Witold K

    2013-07-17

    The structural transition of the prion protein from α-helical- to β-sheet-rich underlies its conversion into infectious and disease-associated isoforms. Here we describe the crystal structure of a fragment from human prion protein consisting of the disulfide-bond-linked portions of helices 2 and 3. Instead of forming a pair-of-sheets steric zipper structure characteristic of amyloid fibers, this fragment crystallized into a β-sheet-rich assembly of hexameric oligomers. This study reveals a never before observed structural motif for ordered protein aggregates and suggests a possible mechanism for self-propagation of misfolded conformations by such nonamyloid oligomers.

  6. Self-propagative replication of Aβ oligomers suggests potential transmissibility in Alzheimer disease.

    PubMed

    Kumar, Amit; Pate, Kayla M; Moss, Melissa A; Dean, Dexter N; Rangachari, Vijayaraghavan

    2014-01-01

    The aggregation of amyloid-β (Aβ) peptide and its deposition in parts of the brain form the central processes in the etiology of Alzheimer disease (AD). The low-molecular weight oligomers of Aβ aggregates (2 to 30 mers) are known to be the primary neurotoxic agents whose mechanisms of cellular toxicity and synaptic dysfunction have received substantial attention in the recent years. However, how these toxic agents proliferate and induce widespread amyloid deposition throughout the brain, and what mechanism is involved in the amplification and propagation of toxic oligomer species, are far from clear. Emerging evidence based on transgenic mice models indicates a transmissible nature of Aβ aggregates and implicates a prion-like mechanism of oligomer propagation, which manifests as the dissemination and proliferation of Aβ toxicity. Despite accumulating evidence in support of a transmissible nature of Aβ aggregates, a clear, molecular-level understanding of this intriguing mechanism is lacking. Recently, we reported the characterization of unique replicating oligomers of Aβ42 (12-24 mers) in vitro called Large Fatty Acid-derived Oligomers (LFAOs) (Kumar et al., 2012, J. Biol. Chem). In the current report, we establish that LFAOs possess physiological activity by activating NF-κB in human neuroblastoma cells, and determine the experimental parameters that control the efficiency of LFAO replication by self-propagation. These findings constitute the first detailed report on monomer - oligomer lateral propagation reactions that may constitute potential mechanism governing transmissibility among Aβ oligomers. These data support the previous reports on transmissible mechanisms observed in transgenic animal models.

  7. Common molecular mechanism of amyloid pore formation by Alzheimer's β-amyloid peptide and α-synuclein.

    PubMed

    Di Scala, Coralie; Yahi, Nouara; Boutemeur, Sonia; Flores, Alessandra; Rodriguez, Léa; Chahinian, Henri; Fantini, Jacques

    2016-06-29

    Calcium-permeable pores formed by small oligomers of amyloid proteins are the primary pathologic species in Alzheimer's and Parkinson's diseases. However, the molecular mechanisms underlying the assembly of these toxic oligomers in the plasma membrane of brain cells remain unclear. Here we have analyzed and compared the pore-forming capability of a large panel of amyloid proteins including wild-type, variant and truncated forms, as well as synthetic peptides derived from specific domains of Aβ1-42 and α-synuclein. We show that amyloid pore formation involves two membrane lipids, ganglioside and cholesterol, that physically interact with amyloid proteins through specific structural motifs. Mutation or deletion of these motifs abolished pore formation. Moreover, α-synuclein (Parkinson) and Aβ peptide (Alzheimer) did no longer form Ca(2+)-permeable pores in presence of drugs that target either cholesterol or ganglioside or both membrane lipids. These results indicate that gangliosides and cholesterol cooperate to favor the formation of amyloid pores through a common molecular mechanism that can be jammed at two different steps, suggesting the possibility of a universal therapeutic approach for neurodegenerative diseases. Finally we present the first successful evaluation of such a new therapeutic approach (coined "membrane therapy") targeting amyloid pores formed by Aβ1-42 and α-synuclein.

  8. Common molecular mechanism of amyloid pore formation by Alzheimer’s β-amyloid peptide and α-synuclein

    PubMed Central

    Di Scala, Coralie; Yahi, Nouara; Boutemeur, Sonia; Flores, Alessandra; Rodriguez, Léa; Chahinian, Henri; Fantini, Jacques

    2016-01-01

    Calcium-permeable pores formed by small oligomers of amyloid proteins are the primary pathologic species in Alzheimer’s and Parkinson’s diseases. However, the molecular mechanisms underlying the assembly of these toxic oligomers in the plasma membrane of brain cells remain unclear. Here we have analyzed and compared the pore-forming capability of a large panel of amyloid proteins including wild-type, variant and truncated forms, as well as synthetic peptides derived from specific domains of Aβ1-42 and α-synuclein. We show that amyloid pore formation involves two membrane lipids, ganglioside and cholesterol, that physically interact with amyloid proteins through specific structural motifs. Mutation or deletion of these motifs abolished pore formation. Moreover, α-synuclein (Parkinson) and Aβ peptide (Alzheimer) did no longer form Ca2+-permeable pores in presence of drugs that target either cholesterol or ganglioside or both membrane lipids. These results indicate that gangliosides and cholesterol cooperate to favor the formation of amyloid pores through a common molecular mechanism that can be jammed at two different steps, suggesting the possibility of a universal therapeutic approach for neurodegenerative diseases. Finally we present the first successful evaluation of such a new therapeutic approach (coined “membrane therapy”) targeting amyloid pores formed by Aβ1-42 and α-synuclein. PMID:27352802

  9. Tg-SwDI Transgenic Mice Exhibit Novel Alterations in AβPP Processing, Aβ Degradation, and Resilient Amyloid Angiopathy

    PubMed Central

    Van Vickle, Gregory D.; Esh, Chera L.; Daugs, Ian D.; Kokjohn, Tyler A.; Kalback, Walter M.; Patton, R. Lyle; Luehrs, Dean C.; Walker, Douglas G.; Lue, Lih-Fen; Beach, Thomas G.; Davis, Judianne; Van Nostrand, William E.; Castaño, Eduardo M.; Roher, Alex E.

    2008-01-01

    Alzheimer’s disease (AD) is characterized by the accumulation of extracellular insoluble amyloid, primarily derived from polymerized amyloid-β (Aβ) peptides. We characterized the chemical composition of the Aβ peptides deposited in the brain parenchyma and cerebrovascular walls of triple transgenic Tg-SwDI mice that produce a rapid and profuse Aβ accumulation. The processing of the N- and C-terminal regions of mutant AβPP differs substantially from humans because the brain parenchyma accumulates numerous, diffuse, nonfibrillar plaques, whereas the thalamic microvessels harbor overwhelming amounts of compact, fibrillar, thioflavine-S- and apolipoprotein E-positive amyloid deposits. The abundant accretion of vascular amyloid, despite low AβPP transgene expression levels, suggests that inefficient Aβ proteolysis because of conformational changes and dimerization may be key pathogenic factors in this animal model. The disruption of amyloid plaque cores by immunotherapy is accompanied by increased perivascular deposition in both humans and transgenic mice. This analogous susceptibility and response to the disruption of amyloid deposits suggests that Tg-SwDI mice provide an excellent model in which to study the functional aftermath of immunotherapeutic interventions. These mice might also reveal new avenues to promote amyloidogenic AβPP processing and fundamental insights into the faulty degradation and clearance of Aβ in AD, pivotal issues in understanding AD pathophysiology and the assessment of new therapeutic agents. PMID:18599612

  10. [Amyloid goiter].

    PubMed

    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.

  11. Biochemistry of Amyloid β-Protein and Amyloid Deposits in Alzheimer Disease

    PubMed Central

    Masters, Colin L.; Selkoe, Dennis J.

    2012-01-01

    Progressive cerebral deposition of the amyloid β-protein (Aβ) in brain regions serving memory and cognition is an invariant and defining feature of Alzheimer disease. A highly similar but less robust process accompanies brain aging in many nondemented humans, lower primates, and some other mammals. The discovery of Aβ as the subunit of the amyloid fibrils in meningocerebral blood vessels and parenchymal plaques has led to innumerable studies of its biochemistry and potential cytotoxic properties. Here we will review the discovery of Aβ, numerous aspects of its complex biochemistry, and current attempts to understand how a range of Aβ assemblies, including soluble oligomers and insoluble fibrils, may precipitate and promote neuronal and glial alterations that underlie the development of dementia. Although the role of Aβ as a key molecular factor in the etiology of Alzheimer disease remains controversial, clinical trials of amyloid-lowering agents, reviewed elsewhere in this book, are poised to resolve the question of its pathogenic primacy. PMID:22675658

  12. Oligomer-oligomer versus oligomer-monomer C(2)-C(2)' coupling reactions in polypyrrole growth.

    PubMed

    Lacroix, J C; Maurel, F; Lacaze, P C

    2001-03-07

    The C(2)-C(2)' coupling reactions of oligopyrrole radical-cations of increasing length generated by electrochemical oxidation have been modeled by transition state calculations. The modeling approach takes into account solvent effects and (i) shows that the coupling distance in the transition state decreases with oligomer length, (ii) demonstrates that dimerization rates in the gas phase decrease with oligomer length but increase in water, (iii) suggests that in a less solvating medium the dimerization rates could be equivalent, (iv) indicates that in all solvents quaterpyrrole and sexipyrrole formation is faster through a coupling reaction between oligomer and monomer radical-cations than two oligomer radical-cations, and (v) suggests that for the formation of a long oligopyrrole from oligopyrrole-pyrrole reactions the mechanism might involve the coupling of the oligopyrrole dication with a non-oxidized pyrrole unit instead of the coupling of two radical-cations or that of the oligopyrrole dication with a pyrrole radical-cation.

  13. Interaction of toxic and non-toxic HypF-N oligomers with lipid bilayers investigated at high resolution with atomic force microscopy

    PubMed Central

    Oropesa-Nuñez, Reinier; Seghezza, Silvia; Dante, Silvia; Diaspro, Alberto; Cascella, Roberta; Cecchi, Cristina; Stefani, Massimo; Chiti, Fabrizio; Canale, Claudio

    2016-01-01

    Protein misfolded oligomers are considered the most toxic species amongst those formed in the process of amyloid formation and the molecular basis of their toxicity, although not completely understood, is thought to originate from the interaction with the cellular membrane. Here, we sought to highlight the molecular determinants of oligomer-membrane interaction by atomic force microscopy. We monitored the interaction between multiphase supported lipid bilayers and two types of HypF-N oligomers displaying different structural features and cytotoxicities. By our approach we imaged with unprecedented resolution the ordered and disordered lipid phases of the bilayer and different oligomer structures interacting with either phase. We identified the oligomers and lipids responsible for toxicity and, more generally, we established the importance of the membrane lipid component in mediating oligomer toxicity. Our findings support the importance of GM1 ganglioside in mediating the oligomer-bilayer interaction and support a mechanism of oligomer cytotoxicity involving bilayer destabilization by globular oligomers within GM1-rich ordered raft regions rather than by annular oligomers in the surrounding disordered membrane domains. PMID:27391440

  14. Effect of Zn(2+) ions on the assembly of amylin oligomers: insight into the molecular mechanisms.

    PubMed

    Wineman-Fisher, Vered; Miller, Yifat

    2016-08-03

    Amylin is an endocrine hormone and is a member of the family of amyloid peptides and proteins that emerge as potential scaffolds by self-assembly processes. Zn(2+) ions can bind to amylin peptides to form self-assembled Zn(2+)-amylin oligomers. In the current work the binding sites of Zn(2+) ions in the self-assembled amylin oligomers at various concentrations of zinc have been investigated. Our results yield two conclusions. First, in the absence of Zn(2+) ions polymorphic states (i.e. various classes of amylin oligomers) are obtained, but when Zn(2+) ions bind to amylin peptides to form Zn(2+)-amylin oligomers, the polymorphism is decreased, i.e. Zn(2+) ions bind only to specific classes of amylin. At low concentrations of Zn(2+) ions the polymorphism is smaller than at high concentrations. Second, the structural features of the self-assembled amylin oligomers are not affected by the presence of Zn(2+) ions. This study proposes new molecular mechanisms of the self-assembly of Zn(2+)-amylin oligomers.

  15. Aβ42 assembles into specific β-barrel pore-forming oligomers in membrane-mimicking environments

    PubMed Central

    Serra-Batiste, Montserrat; Ninot-Pedrosa, Martí; Bayoumi, Mariam; Gairí, Margarida; Maglia, Giovanni; Carulla, Natàlia

    2016-01-01

    The formation of amyloid-β peptide (Aβ) oligomers at the cellular membrane is considered to be a crucial process underlying neurotoxicity in Alzheimer’s disease (AD). Therefore, it is critical to characterize the oligomers that form within a membrane environment. To contribute to this characterization, we have applied strategies widely used to examine the structure of membrane proteins to study the two major Aβ variants, Aβ40 and Aβ42. Accordingly, various types of detergent micelles were extensively screened to identify one that preserved the properties of Aβ in lipid environments—namely the formation of oligomers that function as pores. Remarkably, under the optimized detergent micelle conditions, Aβ40 and Aβ42 showed different behavior. Aβ40 aggregated into amyloid fibrils, whereas Aβ42 assembled into oligomers that inserted into lipid bilayers as well-defined pores and adopted a specific structure with characteristics of a β-barrel arrangement that we named β-barrel pore-forming Aβ42 oligomers (βPFOsAβ42). Because Aβ42, relative to Aβ40, has a more prominent role in AD, the higher propensity of Aβ42 to form βPFOs constitutes an indication of their relevance in AD. Moreover, because βPFOsAβ42 adopt a specific structure, this property offers an unprecedented opportunity for testing a hypothesis regarding the involvement of βPFOs and, more generally, membrane-associated Aβ oligomers in AD. PMID:27621459

  16. Polymorphism of Alzheimer's Abeta17-42 (p3) oligomers: the importance of the turn location and its conformation.

    PubMed

    Miller, Yifat; Ma, Buyong; Nussinov, Ruth

    2009-08-19

    Abeta(17-42) (so-called p3) amyloid is detected in vivo in the brains of individuals with Alzheimer's disease or Down's syndrome. We investigated the polymorphism of Abeta(17-42) oligomers based on experimental data from steady-state NMR measurements, electron microscopy, two-dimensional hydrogen exchange, and mutational studies, using all-atom molecular-dynamics simulation with explicit solvent. We assessed the structural stability and the populations. Our results suggest that conformational differences in the U-turn of Abeta(17-42) lead to polymorphism in beta-sheet registration and retention of an ordered beta-strand organization at the termini. Further, although the parallel Abeta(17-42) oligomer organization is the most stable of the conformers investigated here, different antiparallel Abeta(17-42) organizations are also stable and compete with the parallel architectures, presenting a polymorphic population. In this study we propose that 1), the U-turn conformation is the primary factor leading to polymorphism in the assembly of Abeta(17-42) oligomers, and is also coupled to oligomer growth; and 2), both parallel Abeta(17-42) oligomers and an assembly of Abeta(17-42) oligomers that includes both parallel and antiparallel organizations contribute to amyloid fibril formation. Finally, since a U-turn motif generally appears in amyloids formed by full proteins or long fragments, and since to date these have been shown to exist only in parallel architectures, our results apply to a broad range of oligomers and fibrils.

  17. Counterion condensation on ionic oligomers

    NASA Astrophysics Data System (ADS)

    Manning, Gerald S.; Mohanty, Udayan

    1997-02-01

    The Ramanathan-Woodbury formulas representing the charge density critical for the onset of counterion condensation on finite-length polymers are derived by three alternate methods, an extension of Debye-Huckel theory, a theory of end effects, and by density functional theory. For charged oligomers with length of the same order as the Debye length, the threshold for condensation is the same as for polymers of length much greater than the Debye lenght. However, the threshold depends both on length and salt concentration if the oligomer is shorter than the Debye length, in such a way as to recede to infinity as the ratio of oligomer length to Debye length tends to zero (i.e., condensation vanishes in this limit). The extended Debye-Huckel theory additionally provides a new result for the partition function of the condensed layer, showing that the free energy of the condensed counterions is different on an oligomer and a polymer, even when the fractional extent of condensation is the same. The end effect theory discloses a hitherto unnoticed connection between the number of counterions condensed at the ends of a long polymer and the number condensed on a short oligomer.

  18. The Aβ oligomer hypothesis for synapse failure and memory loss in Alzheimer's diseas

    PubMed Central

    Ferreira, Sergio T; Klein, William L

    2015-01-01

    Alzheimer's disease is the 3rd most costly disease and is estimated to be the 6th leading cause of death. Alzheimer's disease (AD) is fatal and affected individuals can sometimes linger many years. Current treatments are palliative and transient, not disease modifying. This article reviews progress in the search to identify the primary AD-causing toxins. We summarize the shift from an initial focus on amyloid plaques to the contemporary concept that AD memory failure is caused by small soluble oligomers of the Aβ peptide, toxins that target and disrupt particular synapses. Evidence is presented that links Aβ oligomers to pathogenesis in animal models and humans, with reference to seminal discoveries from cell biology and new ideas concerning pathogenic mechanisms. These findings have established the oligomer hypothesis as a new molecular basis for the cause, diagnosis, and treatment of AD. PMID:21914486

  19. Amyloid cascade in Alzheimer's disease: Recent advances in medicinal chemistry.

    PubMed

    Mohamed, Tarek; Shakeri, Arash; Rao, Praveen P N

    2016-05-04

    Alzheimer's disease is of major concern all over the world due to a number of factors including (i) an aging population (ii) increasing life span and (iii) lack of effective pharmacotherapy options. The past decade has seen intense research in discovering disease-modifying multitargeting small molecules as therapeutic options. The pathophysiology of Alzheimer's disease is attributed to a number of factors such as the cholinergic dysfunction, amyloid/tau toxicity and oxidative stress/mitochondrial dysfunction. In recent years, targeting the amyloid cascade has emerged as an attractive strategy to discover novel neurotherapeutics. Formation of beta-amyloid species, with different degrees of solubility and neurotoxicity is associated with the gradual decline in cognition leading to dementia. The two commonly used approaches to prevent beta-amyloid accumulation in the brain include (i) development of beta-secretase inhibitors and (ii) designing direct inhibitors of beta-amyloid (self-induced) aggregation. This review highlights the amyloid cascade hypothesis and the key chemical features required to design small molecules that inhibit lower and higher order beta-amyloid aggregates. Several recent examples of small synthetic molecules with disease-modifying properties were considered and their molecular docking studies were conducted using either a dimer or steric-zipper assembly of beta-amyloid. These investigations provide a mechanistic understanding on the structural requirements needed to design novel small molecules with anti-amyloid aggregation properties. Significantly, this work also demonstrates that the structural requirements to prevent aggregation of various amyloid species differs considerably, which explains the fact that many small molecules do not exhibit similar inhibition profile toward diverse amyloid species such as dimers, trimers, tetramers, oligomers, protofibrils and fibrils. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  20. Stabilizing Off-pathway Oligomers by Polyphenol Nanoassemblies for IAPP Aggregation Inhibition

    NASA Astrophysics Data System (ADS)

    Nedumpully-Govindan, Praveen; Kakinen, Aleksandr; Pilkington, Emily H.; Davis, Thomas P.; Chun Ke, Pu; Ding, Feng

    2016-01-01

    Experimental studies have shown that many naturally occurring polyphenols have inhibitory effect on the aggregation of several proteins. Here, we use discrete molecular dynamics (DMD) simulations and high-throughput dynamic light scattering (DLS) experiments to study the anti-aggregation effects of two polyphenols, curcumin and resveratrol, on the aggregation of islet amyloid polypeptide (IAPP or amylin). Our DMD simulations suggest that the aggregation inhibition is caused by stabilization of small molecular weight IAPP off-pathway oligomers by the polyphenols. Our analysis indicates that IAPP-polyphenol hydrogen bonds and π-π stacking combined with hydrophobic interactions are responsible for the stabilization of oligomers. The presence of small oligomers is confirmed with DLS measurements in which nanometer-sized oligomers are found to be stable for up to 7.5 hours, the time frame within which IAPP aggregates in the absence of polyphenols. Our study offers a general anti-aggregation mechanism for polyphenols, and further provides a computational framework for the future design of anti-amyloid aggregation therapeutics.

  1. Stabilizing Off-pathway Oligomers by Polyphenol Nanoassemblies for IAPP Aggregation Inhibition

    PubMed Central

    Nedumpully-Govindan, Praveen; Kakinen, Aleksandr; Pilkington, Emily H.; Davis, Thomas P.; Chun Ke, Pu; Ding, Feng

    2016-01-01

    Experimental studies have shown that many naturally occurring polyphenols have inhibitory effect on the aggregation of several proteins. Here, we use discrete molecular dynamics (DMD) simulations and high-throughput dynamic light scattering (DLS) experiments to study the anti-aggregation effects of two polyphenols, curcumin and resveratrol, on the aggregation of islet amyloid polypeptide (IAPP or amylin). Our DMD simulations suggest that the aggregation inhibition is caused by stabilization of small molecular weight IAPP off-pathway oligomers by the polyphenols. Our analysis indicates that IAPP-polyphenol hydrogen bonds and π-π stacking combined with hydrophobic interactions are responsible for the stabilization of oligomers. The presence of small oligomers is confirmed with DLS measurements in which nanometer-sized oligomers are found to be stable for up to 7.5 hours, the time frame within which IAPP aggregates in the absence of polyphenols. Our study offers a general anti-aggregation mechanism for polyphenols, and further provides a computational framework for the future design of anti-amyloid aggregation therapeutics. PMID:26763863

  2. Molecular Mechanism for the (-)-Epigallocatechin Gallate-Induced Toxic to Nontoxic Remodeling of Aβ Oligomers.

    PubMed

    Ahmed, Rashik; VanSchouwen, Bryan; Jafari, Naeimeh; Ni, Xiaodan; Ortega, Joaquin; Melacini, Giuseppe

    2017-10-04

    (-)-Epigallocatechin gallate (EGCG) effectively reduces the cytotoxicity of the Alzheimer's disease β-amyloid peptide (Aβ) by remodeling seeding-competent Aβ oligomers into off-pathway seeding-incompetent Aβ assemblies. However, the mechanism of EGCG-induced remodeling is not fully understood. Here we combine (15)N and (1)H dark-state exchange saturation transfer (DEST), relaxation, and chemical shift projection NMR analyses with fluorescence, dynamic light scattering, and electron microscopy to elucidate how EGCG remodels Aβ oligomers. We show that the remodeling adheres to a Hill-Scatchard model whereby the Aβ(1-40) self-association occurs cooperatively and generates Aβ(1-40) oligomers with multiple independent binding sites for EGCG with a Kd ∼10-fold lower than that for the Aβ(1-40) monomers. Upon binding to EGCG, the Aβ(1-40) oligomers become less solvent exposed, and the β-regions, which are involved in direct monomer-protofibril contacts in the absence of EGCG, undergo a direct-to-tethered contact shift. This switch toward less engaged monomer-protofibril contacts explains the seeding incompetency observed upon EGCG remodeling and suggests that EGCG interferes with secondary nucleation events known to generate toxic Aβ assemblies. Unexpectedly, the N-terminal residues experience an opposite EGCG-induced shift from tethered to direct contacts, explaining why EGCG remodeling occurs without release of Aβ(1-40) monomers. We also show that upon binding Aβ(1-40) oligomers the relative positions of the EGCG B and D rings change with respect to that of ring A. These distinct structural changes occurring in both Aβ(1-40) oligomers and EGCG during remodeling offer a foundation for understanding the molecular mechanism of EGCG as a neurotoxicity inhibitor. Furthermore, the results reported here illustrate the effectiveness of DEST-based NMR approaches in investigating the mechanism of low-molecular-weight amyloid inhibitors.

  3. Monitoring and targeting the initial dimerization stage of amyloid self-assembly.

    PubMed

    Bram, Yaron; Lampel, Ayala; Shaltiel-Karyo, Ronit; Ezer, Anat; Scherzer-Attali, Roni; Segal, Daniel; Gazit, Ehud

    2015-02-09

    Amyloid deposits are pathological hallmark of a large group of human degenerative disorders of unrelated etiologies. While accumulating evidence suggests that early oligomers may account for tissue degeneration, most detection tools do not allow the monitoring of early association events. Here we exploit bimolecular fluorescence complementation analysis to detect and quantify the dimerization of three major amyloidogenic polypeptides; islet amyloid polypeptide, β-amyloid and α-synuclein. The constructed systems provided direct visualization of protein-protein interactions in which only assembled dimers display strong fluorescent signal. Potential inhibitors that interfere with the initial intermolecular interactions of islet amyloid polypeptide were further identified using this system. Moreover, the identified compounds were able to inhibit the aggregation and cytotoxicity of islet amyloid polypeptide, demonstrating the importance of targeting amyloid dimer formation for future drug development. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. AMYLOID NEUROPATHIES

    PubMed Central

    Shin, Susan C.; Robinson-Papp, Jessica

    2012-01-01

    Peripheral neuropathy is a common complication of many of the systemic amyloidoses. Although the cause of neuropathy is not entirely clear, it is likely related to amyloid deposition within the nerve. This may lead to focal, multifocal, or diffuse neuropathies involving sensory, motor and/or autonomic fibers. The presenting symptoms depend on the distribution of nerves affected. One of the most common phenotypes is sensorimotor polyneuropathy, which is characterized by symptoms of neuropathic pain, numbness, and in advanced cases weakness. Symptoms begin in the feet and ultimately progress to the proximal legs and hands. The most common focal neuropathy is a median neuropathy at the wrist, or clinically known as carpal tunnel syndrome. Carpal tunnel symptoms may include pain and sensory disturbances in the lateral palm and fingers; hand weakness may ensue if the focal neuropathy is severe. Autonomic neuropathy may affect a variety of organ systems such as the cardiovascular, gastrointestinal, and genitourinary systems. Symptoms may be non-specific making the diagnosis of autonomic neuropathy more difficult to identify. However, it is important to recognize and distinguish autonomic neuropathy from diseases of the end-organs themselves. This chapter reviews the inherited and acquired amyloidoses that affect the peripheral nervous system including familial amyloid polyneuropathy, and primary, secondary and senile amyloidosis. We emphasize the clinical presentation of the neurologic aspects of these diseases, physical examination findings, appropriate diagnostic evaluation, treatment and prognosis. PMID:23239211

  5. Amyloid neuropathies.

    PubMed

    Shin, Susan C; Robinson-Papp, Jessica

    2012-01-01

    Peripheral neuropathy is a common complication of many of the systemic amyloidoses. Although the cause of neuropathy is not entirely clear, it is likely related to amyloid deposition within the nerve. This may lead to focal, multifocal, or diffuse neuropathies involving sensory, motor and/or autonomic fibers. The presenting symptoms depend on the distribution of nerves affected. One of the most common phenotypes is sensorimotor polyneuropathy, which is characterized by symptoms of neuropathic pain, numbness, and in advanced cases weakness. Symptoms begin in the feet and ultimately progress to the proximal legs and hands. The most common focal neuropathy is a median neuropathy at the wrist, clinically known as carpal tunnel syndrome. Carpal tunnel symptoms may include pain and sensory disturbances in the lateral palm and fingers; hand weakness may ensue if the focal neuropathy is severe. Autonomic neuropathy may affect a variety of organ systems such as the cardiovascular, gastrointestinal, and genitourinary systems. Symptoms may be non-specific making the diagnosis of autonomic neuropathy more difficult to identify. However, it is important to recognize and distinguish autonomic neuropathy from diseases of the end-organs themselves. This article reviews the inherited and acquired amyloidoses that affect the peripheral nervous system including familial amyloid polyneuropathy, and primary, secondary and senile amyloidosis. We emphasize the clinical presentation of the neurologic aspects of these diseases, physical examination findings, appropriate diagnostic evaluation, treatment and prognosis. © 2012 Mount Sinai School of Medicine.

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

    PubMed Central

    Stefani, Massimo; Rigacci, Stefania

    2013-01-01

    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. PMID:23765219

  7. ABCA7 Mediates Phagocytic Clearance of Amyloid-β in the Brain.

    PubMed

    Fu, YuHong; Hsiao, Jen-Hsiang T; Paxinos, George; Halliday, Glenda M; Kim, Woojin Scott

    2016-09-06

    Alzheimer's disease (AD) is a neurodegenerative disorder characterized by dementia and abnormal deposits of aggregated amyloid-β in the brain. Recent genome-wide association studies have revealed that ABCA7 is strongly associated with AD. In vitro evidence suggests that the role of ABCA7 is related to phagocytic activity. Deletion of ABCA7 in a mouse model of AD exacerbates cerebral amyloid-β plaque load. However, the biological role of ABCA7 in AD brain pathogenesis is unknown. We show that ABCA7 is highly expressed in microglia and when monocytes are differentiated into macrophages. We hypothesized that ABCA7 plays a protective role in the brain that is related to phagocytic clearance of amyloid-β. We isolated microglia and macrophages from Abca7-/- and wild type mice and tested them for their capacity to phagocytose amyloidoligomers. We found that the phagocytic clearance of amyloid-β was substantially reduced in both microglia and macrophages from Abca7-/- mice compared to wild type mice. Consistent with these results, in vivo phagocytic clearance of amyloidoligomers in the hippocampus was reduced in Abca7-/- mice. Furthermore, ABCA7 transcription was upregulated in AD brains and in amyloidogenic mouse brains specifically in the hippocampus as a response to the amyloid-β pathogenic state. Together these results indicate that ABCA7 mediates phagocytic clearance of amyloid-β in the brain, and reveal a mechanism by which loss of function of ABCA7 increases the susceptibility to AD.

  8. Protection against β-amyloid-induced synaptic and memory impairments via altering β-amyloid assembly by bis(heptyl)-cognitin

    PubMed Central

    Chang, Lan; Cui, Wei; Yang, Yong; Xu, Shujun; Zhou, Wenhua; Fu, Hongjun; Hu, Shengquan; Mak, Shinghung; Hu, Juwei; Wang, Qin; Pui-Yan Ma, Victor; Chung-lit Choi, Tony; Dik-lung Ma, Edmond; Tao, Liang; Pang, Yuanping; Rowan, Michael J.; Anwyl, Roger; Han, Yifan; Wang, Qinwen

    2015-01-01

    β-amyloid (Aβ) oligomers have been closely implicated in the pathogenesis of Alzheimer’s disease (AD). We found, for the first time, that bis(heptyl)-cognitin, a novel dimeric acetylcholinesterase (AChE) inhibitor derived from tacrine, prevented Aβ oligomers-induced inhibition of long-term potentiation (LTP) at concentrations that did not interfere with normal LTP. Bis(heptyl)-cognitin also prevented Aβ oligomers-induced synaptotoxicity in primary hippocampal neurons. In contrast, tacrine and donepezil, typical AChE inhibitors, could not prevent synaptic impairments in these models, indicating that the modification of Aβ oligomers toxicity by bis(heptyl)-cognitin might be attributed to a mechanism other than AChE inhibition. Studies by using dot blotting, immunoblotting, circular dichroism spectroscopy, and transmission electron microscopy have shown that bis(heptyl)-cognitin altered Aβ assembly via directly inhibiting Aβ oligomers formation and reducing the amount of preformed Aβ oligomers. Molecular docking analysis further suggested that bis(heptyl)-cognitin presumably interacted with the hydrophobic pockets of Aβ, which confers stabilizing powers and assembly alteration effects on Aβ. Most importantly, bis(heptyl)-cognitin significantly reduced cognitive impairments induced by intra-hippocampal infusion of Aβ oligomers in mice. These results clearly demonstrated how dimeric agents prevent Aβ oligomers-induced synaptic and memory impairments, and offered a strong support for the beneficial therapeutic effects of bis(heptyl)-cognitin in the treatment of AD. PMID:26194093

  9. Amyloid β Oligomeric Species Present in the Lag Phase of Amyloid Formation

    PubMed Central

    Wolff, Martin; Unuchek, Dmitry; Zhang, Bo; Gordeliy, Valentin; Willbold, Dieter; Nagel-Steger, Luitgard

    2015-01-01

    Alzheimer’s disease (AD)-associated amyloid β peptide (Aβ) is one of the main actors in AD pathogenesis. Aβ is characterized by its high tendency to self-associate, leading to the generation of oligomers and amyloid fibrils. The elucidation of pathways and intermediates is crucial for the understanding of protein assembly mechanisms in general and in conjunction with neurodegenerative diseases, e.g., for the identification of new therapeutic targets. Our study focused on Aβ42 and its oligomeric assemblies in the lag phase of amyloid formation, as studied by sedimentation velocity (SV) centrifugation. The assembly state of Aβ during the lag phase, the time required by an Aβ solution to reach the exponential growth phase of aggregation, was characterized by a dominant monomer fraction below 1 S and a population of oligomeric species between 4 and 16 S. From the oligomer population, two major species close to a 12-mer and an 18-mer with a globular shape were identified. The recurrence of these two species at different initial concentrations and experimental conditions as the smallest assemblies present in solution supports the existence of distinct, energetically favored assemblies in solution. The sizes of the two species suggest an Aβ42 aggregation pathway that is based on a basic hexameric building block. The study demonstrates the potential of SV analysis for the evaluation of protein aggregation pathways. PMID:26024352

  10. Different effects of Alzheimer's peptide Aβ(1-40) oligomers and fibrils on supported lipid membranes.

    PubMed

    Canale, Claudio; Seghezza, Silvia; Vilasi, Silvia; Carrotta, Rita; Bulone, Donatella; Diaspro, Alberto; San Biagio, Pier Luigi; Dante, Silvia

    2013-12-01

    Beta-amyloid (1-40) is one of the two most abundant species of amyloid-beta peptides present as fibrils in the extracellular senile plaques in the brain of Alzheimer's patients. Recently, the molecular aggregates constituting the early stage of fibril formation, i.e., oligomers and protofibrils, have been investigated as the main responsible for amyloid-beta cytotoxic effect. The molecular mechanism leading to neurodegeneration is still under debate, and it is common opinion that it may reside in the interaction between amyloid species and the neural membrane. In this investigation Atomic Force Microscopy and spectroscopy have been used to understand how structural (and mechanical) properties of POPC/POPS lipid bilayers, simulating the phospholipid composition and negative net charge of neuritic cell membranes, are influenced by the interaction with Aβ(1-40), in different stages of the peptide aggregation. Substantial differences in the damage caused to the lipid bilayers have been observed, confirming the toxic effect exerted especially by Aβ(1-40) prefibrillar oligomers. © 2013 Elsevier B.V. All rights reserved.

  11. Study of neurotoxic intracellular calcium signalling triggered by amyloids.

    PubMed

    Villalobos, Carlos; Caballero, Erica; Sanz-Blasco, Sara; Núñez, Lucía

    2012-01-01

    Neurotoxicity in Alzheimer's disease (AD) is associated to dishomeostasis of intracellular Ca(2+) induced by amyloid β peptide (Aβ) species. Understanding of the effects of Aβ on intracellular Ca(2+) homeostasis requires preparation of the different Aβ assemblies including oligomers and fibrils and the testing of their effects on cytosolic and mitochondrial Ca(2+) in neurons. Procedures for cerebellar granule cell culture, preparation of Aβ species as well as fluorescence and bioluminescence imaging of cytosolic and mitochondrial Ca(2+) in neurons are described.

  12. Regional brain hypometabolism is unrelated to regional amyloid plaque burden

    PubMed Central

    Altmann, Andre; Ng, Bernard; Landau, Susan M.; Jagust, William J.

    2015-01-01

    See Sorg and Grothe (doi:10.1093/brain/awv302) for a scientific commentary on this article. In its original form, the amyloid cascade hypothesis of Alzheimer’s disease holds that fibrillar deposits of amyloid are an early, driving force in pathological events leading ultimately to neuronal death. Early clinicopathological investigations highlighted a number of inconsistencies leading to an updated hypothesis in which amyloid plaques give way to amyloid oligomers as the driving force in pathogenesis. Rather than focusing on the inconsistencies, amyloid imaging studies have tended to highlight the overlap between regions that show early amyloid plaque signal on positron emission tomography and that also happen to be affected early in Alzheimer’s disease. Recent imaging studies investigating the regional dependency between metabolism and amyloid plaque deposition have arrived at conflicting results, with some showing regional associations and other not. We extracted multimodal neuroimaging data from the Alzheimer’s disease neuroimaging database for 227 healthy controls and 434 subjects with mild cognitive impairment. We analysed regional patterns of amyloid deposition, regional glucose metabolism and regional atrophy using florbetapir (18F) positron emission tomography, 18F-fluordeoxyglucose positron emission tomography and T1-weighted magnetic resonance imaging, respectively. Specifically, we derived grey matter density and standardized uptake value ratios for both positron emission tomography tracers in 404 functionally defined regions of interest. We examined the relation between regional glucose metabolism and amyloid plaques using linear models. For each region of interest, correcting for regional grey matter density, age, education and disease status, we tested the association of regional glucose metabolism with (i) cortex-wide florbetapir uptake; (ii) regional (i.e. in the same region of interest) florbetapir uptake; and (iii) regional florbetapir uptake

  13. Regional brain hypometabolism is unrelated to regional amyloid plaque burden.

    PubMed

    Altmann, Andre; Ng, Bernard; Landau, Susan M; Jagust, William J; Greicius, Michael D

    2015-12-01

    In its original form, the amyloid cascade hypothesis of Alzheimer's disease holds that fibrillar deposits of amyloid are an early, driving force in pathological events leading ultimately to neuronal death. Early clinicopathological investigations highlighted a number of inconsistencies leading to an updated hypothesis in which amyloid plaques give way to amyloid oligomers as the driving force in pathogenesis. Rather than focusing on the inconsistencies, amyloid imaging studies have tended to highlight the overlap between regions that show early amyloid plaque signal on positron emission tomography and that also happen to be affected early in Alzheimer's disease. Recent imaging studies investigating the regional dependency between metabolism and amyloid plaque deposition have arrived at conflicting results, with some showing regional associations and other not. We extracted multimodal neuroimaging data from the Alzheimer's disease neuroimaging database for 227 healthy controls and 434 subjects with mild cognitive impairment. We analysed regional patterns of amyloid deposition, regional glucose metabolism and regional atrophy using florbetapir ((18)F) positron emission tomography, (18)F-fluordeoxyglucose positron emission tomography and T1-weighted magnetic resonance imaging, respectively. Specifically, we derived grey matter density and standardized uptake value ratios for both positron emission tomography tracers in 404 functionally defined regions of interest. We examined the relation between regional glucose metabolism and amyloid plaques using linear models. For each region of interest, correcting for regional grey matter density, age, education and disease status, we tested the association of regional glucose metabolism with (i) cortex-wide florbetapir uptake; (ii) regional (i.e. in the same region of interest) florbetapir uptake; and (iii) regional florbetapir uptake while correcting in addition for cortex-wide florbetapir uptake. P-values for each setting

  14. Targeting Cancer with Antisense Oligomers

    SciTech Connect

    Hnatowich, DJ

    2008-10-28

    With financial assistance from the Department of Energy, we have shown definitively that radiolabeled antisense DNAs and other oligomers will accumulate in target cancer cells in vitro and in vivo by an antisense mechanism. We have also shown that the number of mRNA targets for our antisense oligomers in the cancer cell types that we have investigated so far is sufficient to provide and antisense image and/or radiotherapy of cancer in mice. These studies have been reported in about 10 publications. However our observation over the past several years has shown that radiolabeled antisense oligomers administered intravenously in their native and naked form will accumulate and be retained in target xenografts by an antisense mechanism but will also accumulate at high levels in normal organs such as liver, spleen and kidneys. We have investigated unsuccessfully several commercially available vectors. Thus the use of radiolabeled antisense oligomers for the imaging of cancer must await novel approaches to delivery. This laboratory has therefore pursued two new paths, optical imaging of tumor and Auger radiotherapy. We are developing a novel method of optical imaging tumor using antisense oligomers with a fluorophore is administered while hybridized with a shorter complementary oligomer with an inhibitor. In culture and in tumored mice that the duplex remains intact and thus nonfluorescent until it encounters its target mRNA at which time it dissociates and the antisense oligomer binds along with its fluorophore to the target. Simultaneous with the above, we have also observed, as have others, that antisense oligomers migrate rapidly and quantitatively to the nucleus upon crossing cell membranes. The Auger electron radiotherapy path results from this observation since the nuclear migration properties could be used effectively to bring and to retain in the nucleus an Auger emitting radionuclide such as 111In or 125I bound to the antisense oligomer. Since the object becomes

  15. Alzheimer's-associated Abeta oligomers show altered structure, immunoreactivity and synaptotoxicity with low doses of oleocanthal.

    PubMed

    Pitt, Jason; Roth, William; Lacor, Pascale; Smith, Amos B; Blankenship, Matthew; Velasco, Pauline; De Felice, Fernanda; Breslin, Paul; Klein, William L

    2009-10-15

    It now appears likely that soluble oligomers of amyloid-beta1-42 peptide, rather than insoluble fibrils, act as the primary neurotoxin in Alzheimer's disease (AD). Consequently, compounds capable of altering the assembly state of these oligomers (referred to as ADDLs) may have potential for AD therapeutics. Phenolic compounds are of particular interest for their ability to disrupt Abeta oligomerization and reduce pathogenicity. This study has focused on oleocanthal (OC), a naturally-occurring phenolic compound found in extra-virgin olive oil. OC increased the immunoreactivity of soluble Abeta species, when assayed with both sequence- and conformation-specific Abeta antibodies, indicating changes in oligomer structure. Analysis of oligomers in the presence of OC showed an upward shift in MW and a ladder-like distribution of SDS-stable ADDL subspecies. In comparison with control ADDLs, oligomers formed in the presence of OC (Abeta-OC) showed equivalent colocalization at synapses but exhibited greater immunofluorescence as a result of increased antibody recognition. The enhanced signal at synapses was not due to increased synaptic binding, as direct detection of fluorescently-labeled ADDLs showed an overall reduction in ADDL signal in the presence of OC. Decreased binding to synapses was accompanied by significantly less synaptic deterioration assayed by drebrin loss. Additionally, treatment with OC improved antibody clearance of ADDLs. These results indicate oleocanthal is capable of altering the oligomerization state of ADDLs while protecting neurons from the synaptopathological effects of ADDLs and suggest OC as a lead compound for development in AD therapeutics.

  16. Mitochondrial Ca2+ Overload Underlies Aβ Oligomers Neurotoxicity Providing an Unexpected Mechanism of Neuroprotection by NSAIDs

    PubMed Central

    Sanz-Blasco, Sara; Valero, Ruth A.; Rodríguez-Crespo, Ignacio; Villalobos, Carlos; Núñez, Lucía

    2008-01-01

    Dysregulation of intracellular Ca2+ homeostasis may underlie amyloid β peptide (Aβ) toxicity in Alzheimer's Disease (AD) but the mechanism is unknown. In search for this mechanism we found that Aβ1–42 oligomers, the assembly state correlating best with cognitive decline in AD, but not Aβ fibrils, induce a massive entry of Ca2+ in neurons and promote mitochondrial Ca2+ overload as shown by bioluminescence imaging of targeted aequorin in individual neurons. Aβ oligomers induce also mitochondrial permeability transition, cytochrome c release, apoptosis and cell death. Mitochondrial depolarization prevents mitochondrial Ca2+ overload, cytochrome c release and cell death. In addition, we found that a series of non-steroidal anti-inflammatory drugs (NSAIDs) including salicylate, sulindac sulfide, indomethacin, ibuprofen and R-flurbiprofen depolarize mitochondria and inhibit mitochondrial Ca2+ overload, cytochrome c release and cell death induced by Aβ oligomers. Our results indicate that i) mitochondrial Ca2+ overload underlies the neurotoxicity induced by Aβ oligomers and ii) inhibition of mitochondrial Ca2+ overload provides a novel mechanism of neuroprotection by NSAIDs against Aβ oligomers and AD. PMID:18648507

  17. Diaryl hydrazones as multifunctional inhibitors of amyloid self-assembly.

    PubMed

    Török, Béla; Sood, Abha; Bag, Seema; Tulsan, Rekha; Ghosh, Sanjukta; Borkin, Dmitry; Kennedy, Arleen R; Melanson, Michelle; Madden, Richard; Zhou, Weihong; Levine, Harry; Török, Marianna

    2013-02-19

    The design and application of an effective, new class of multifunctional small molecule inhibitors of amyloid self-assembly are described. Several compounds based on the diaryl hydrazone scaffold were designed. Forty-four substituted derivatives of this core structure were synthesized using a variety of benzaldehydes and phenylhydrazines and characterized. The inhibitor candidates were evaluated in multiple assays, including the inhibition of amyloid β (Aβ) fibrillogenesis and oligomer formation and the reverse processes, the disassembly of preformed fibrils and oligomers. Because the structure of the hydrazone-based inhibitors mimics the redox features of the antioxidant resveratrol, the radical scavenging effect of the compounds was evaluated by colorimetric assays against 2,2-diphenyl-1-picrylhydrazyl and superoxide radicals. The hydrazone scaffold was active in all of the different assays. The structure-activity relationship revealed that the substituents on the aromatic rings had a considerable effect on the overall activity of the compounds. The inhibitors showed strong activity in fibrillogenesis inhibition and disassembly, and even greater potency in the inhibition of oligomer formation and oligomer disassembly. Supporting the quantitative fluorometric and colorimetric assays, size exclusion chromatographic studies indicated that the best compounds practically eliminated or substantially inhibited the formation of soluble, aggregated Aβ species, as well. Atomic force microscopy was also applied to monitor the morphology of Aβ deposits. The compounds also possessed the predicted antioxidant properties; approximately 30% of the synthesized compounds showed a radical scavenging effect equal to or better than that of resveratrol or ascorbic acid.

  18. Ion mobility-mass spectrometry and orthogonal gas-phase techniques to study amyloid formation and inhibition.

    PubMed

    Hoffmann, Waldemar; von Helden, Gert; Pagel, Kevin

    2017-03-23

    Amyloidogenic peptide oligomers are responsible for a variety of neurodegenerative disorders such as Alzheimer's and Parkinson's disease. Due to their dynamic, polydisperse, and polymorphic nature, these oligomers are very challenging to characterize using traditional condensed-phase methods. In the last decade, ion mobility-mass spectrometry (IM-MS) and related gas-phase techniques have emerged as a powerful alternative to disentangle the structure and assembly characteristics of amyloid forming systems. This review highlights recent advances in which IM-MS was used to characterize amyloid oligomers and their underlying assembly pathway. In addition, we summarize recent studies in which IM-MS was used to size- and mass-select species for a further spectroscopic investigation and outline the potential of IM-MS as a tool for the screening of amyloid inhibitors.

  19. Adeno-associated Virus Gene Therapy With Cholesterol 24-Hydroxylase Reduces the Amyloid Pathology Before or After the Onset of Amyloid Plaques in Mouse Models of Alzheimer's Disease

    PubMed Central

    Hudry, Eloise; Van Dam, Debby; Kulik, Wim; De Deyn, Peter P; Stet, Femke S; Ahouansou, Ornella; Benraiss, Abdellatif; Delacourte, André; Bougnères, Pierre; Aubourg, Patrick; Cartier, Nathalie

    2009-01-01

    The development of Alzheimer's disease (AD) is closely connected with cholesterol metabolism. Cholesterol increases the production and deposition of amyloid-β (Aβ) peptides that result in the formation of amyloid plaques, a hallmark of the pathology. In the brain, cholesterol is synthesized in situ but cannot be degraded nor cross the blood–brain barrier. The major exportable form of brain cholesterol is 24S-hydroxycholesterol, an oxysterol generated by the neuronal cholesterol 24-hydroxylase encoded by the CYP46A1 gene. We report that the injection of adeno-associated vector (AAV) encoding CYP46A1 in the cortex and hippocampus of APP23 mice before the onset of amyloid deposits markedly reduces Aβ peptides, amyloid deposits and trimeric oligomers at 12 months of age. The Morris water maze (MWM) procedure also demonstrated improvement of spatial memory at 6 months, before the onset of amyloid deposits. AAV5-wtCYP46A1 vector injection in the cortex and hippocampus of amyloid precursor protein/presenilin 1 (APP/PS) mice after the onset of amyloid deposits also reduced markedly the number of amyloid plaques in the hippocampus, and to a less extent in the cortex, 3 months after the injection. Our data demonstrate that neuronal overexpression of CYP46A1 before or after the onset of amyloid plaques significantly reduces Aβ pathology in mouse models of AD. PMID:19654569

  20. Intracellular soluble α‐synuclein oligomers reduce pyramidal cell excitability

    PubMed Central

    Kaufmann, Timothy J.; Harrison, Paul M.; Richardson, Magnus J. E.; Pinheiro, Teresa J. T.

    2016-01-01

    Key points The presynaptic protein α‐synuclein forms aggregates during Parkinson's disease.Accumulating evidence suggests that the small soluble oligomers of α‐synuclein are more toxic than the larger aggregates appearing later in the disease.The link between oligomer toxicity and structure still remains unclear.In the present study, we have produced two structurally‐defined oligomers that have a similar morphology but differ in secondary structure.These oligomers were introduced into neocortical pyramidal cells during whole‐cell recording and, using a combination of experimentation and modelling, electrophysiological parameters were extracted.Both oligomeric species had similar effects on neuronal properties reducing input resistance, time constant and increasing capacitance. The net effect was a marked reduction in neuronal excitability that could impact on network activity. Abstract The presynaptic protein α‐synuclein (αSyn) aggregates during Parkinson's disease (PD) to form large proteinaceous amyloid plaques, the spread of which throughout the brain clinically defines the severity of the disease. During early stages of aggregation, αSyn forms soluble annular oligomers that show greater toxicity than much larger fibrils. These oligomers produce toxicity via a number of possible mechanisms, including the production of pore‐forming complexes that permeabilize membranes. In the present study, two well‐defined species of soluble αSyn oligomers were produced by different protocols: by polymerization of monomer and by sonication of fibrils. The two oligomeric species produced were morphologically similar, with both having an annular structure and consisting of approximately the same number of monomer subunits, although they differed in their secondary structure. Oligomeric and monomeric αSyn were injected directly into the soma of pyramidal neurons in mouse neocortical brain slices during whole‐cell patch clamp recording. Using a combined

  1. Out-of-register β-sheets suggest a pathway to toxic amyloid aggregates.

    PubMed

    Liu, Cong; Zhao, Minglei; Jiang, Lin; Cheng, Pin-Nan; Park, Jiyong; Sawaya, Michael R; Pensalfini, Anna; Gou, Dawei; Berk, Arnold J; Glabe, Charles G; Nowick, James; Eisenberg, David

    2012-12-18

    Although aberrant protein aggregation has been conclusively linked to dozens of devastating amyloid diseases, scientists remain puzzled about the molecular features that render amyloid fibrils or small oligomers toxic. Here, we report a previously unobserved type of amyloid fibril that tests as cytotoxic: one in which the strands of the contributing β-sheets are out of register. In all amyloid fibrils previously characterized at the molecular level, only in-register β-sheets have been observed, in which each strand makes its full complement of hydrogen bonds with the strands above and below it in the fibril. In out-of-register sheets, strands are sheared relative to one another, leaving dangling hydrogen bonds. Based on this finding, we designed out-of-register β-sheet amyloid mimics, which form both cylindrin-like oligomers and fibrils, and these mimics are cytotoxic. Structural and energetic considerations suggest that out-of-register fibrils can readily convert to toxic cylindrins. We propose that out-of-register β-sheets and their related cylindrins are part of a toxic amyloid pathway, which is distinct from the more energetically favored in-register amyloid pathway.

  2. Detection of β-Amyloid Peptide Dimer in Solution by Fluorescence Resonance Energy Transfer

    NASA Astrophysics Data System (ADS)

    Han, Jun; Mei, Erwen; Kung, Mei-Ping; Kung, Hank; Dai, Hai-Lung

    2006-03-01

    Studies have suggested that there is a connection between ß-amyloid-derived diffusible ligands (ADDLs), small oligomers formed from clustering of peptides with 39-42 amino acid units, and pathogenicity of Alzheimer's disease. It is believed that the soluble ADDL oligomers eventually coagulate and precipitate into fibrils that cause neurotoxicity. Although there have been studies characterizing the fibrils structure and the large coagulate formation kinetics, little experimental information exists for the oligomers in the solution phase. We report here the use of fluorescence resonance energy transfer detected through a confocal microscope under single molecule conditions for the detection of the β-amyloid (1-40) peptide dimer in solution. The structure of the dimer is characterized in terms of the distance of the two N-terminals.

  3. Accumulation of murine amyloid-β mimics early Alzheimer's disease.

    PubMed

    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 amyloid-β peptides mimics only inherited (familiar) Alzheimer's disease, which accounts for <1% of all 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 amyloidoligomers. Thus, neprilysin × ABCC1 double-deficient mice present a new model for early effects of amyloid-β-related mild cognitive impairment that allows

  4. Single-Molecule Imaging of Individual Amyloid Protein Aggregates in Human Biofluids.

    PubMed

    Horrocks, Mathew H; Lee, Steven F; Gandhi, Sonia; Magdalinou, Nadia K; Chen, Serene W; Devine, Michael J; Tosatto, Laura; Kjaergaard, Magnus; Beckwith, Joseph S; Zetterberg, Henrik; Iljina, Marija; Cremades, Nunilo; Dobson, Christopher M; Wood, Nicholas W; Klenerman, David

    2016-03-16

    The misfolding and aggregation of proteins into amyloid fibrils characterizes many neurodegenerative disorders such as Parkinson's and Alzheimer's diseases. We report here a method, termed SAVE (single aggregate visualization by enhancement) imaging, for the ultrasensitive detection of individual amyloid fibrils and oligomers using single-molecule fluorescence microscopy. We demonstrate that this method is able to detect the presence of amyloid aggregates of α-synuclein, tau, and amyloid-β. In addition, we show that aggregates can also be identified in human cerebrospinal fluid (CSF). Significantly, we see a twofold increase in the average aggregate concentration in CSF from Parkinson's disease patients compared to age-matched controls. Taken together, we conclude that this method provides an opportunity to characterize the structural nature of amyloid aggregates in a key biofluid, and therefore has the potential to study disease progression in both animal models and humans to enhance our understanding of neurodegenerative disorders.

  5. Single-Molecule Imaging of Individual Amyloid Protein Aggregates in Human Biofluids

    PubMed Central

    2016-01-01

    The misfolding and aggregation of proteins into amyloid fibrils characterizes many neurodegenerative disorders such as Parkinson’s and Alzheimer’s diseases. We report here a method, termed SAVE (single aggregate visualization by enhancement) imaging, for the ultrasensitive detection of individual amyloid fibrils and oligomers using single-molecule fluorescence microscopy. We demonstrate that this method is able to detect the presence of amyloid aggregates of α-synuclein, tau, and amyloid-β. In addition, we show that aggregates can also be identified in human cerebrospinal fluid (CSF). Significantly, we see a twofold increase in the average aggregate concentration in CSF from Parkinson’s disease patients compared to age-matched controls. Taken together, we conclude that this method provides an opportunity to characterize the structural nature of amyloid aggregates in a key biofluid, and therefore has the potential to study disease progression in both animal models and humans to enhance our understanding of neurodegenerative disorders. PMID:26800462

  6. Current and future treatment of amyloid diseases

    PubMed Central

    Ankarcrona, M.; Winblad, B.; Monteiro, C.; Fearns, C.; Powers, E. T.; Johansson, J.; Westermark, G. T.; Presto, J.; Ericzon, B-G.; Kelly, J. W.

    2016-01-01

    There are more than 30 human proteins whose aggregation appears to cause degenerative maladies referred to as amyloid diseases or amyloidoses. These disorders are named after the characteristic cross-β-sheet amyloid fibrils that accumulate systemically or are localized to specific organs. In most cases current treatment is limited to symptomatic approaches and thus disease-modifying therapies are needed. Alzheimer’s disease is a neurodegenerative disorder with amyloid β-peptide (Aβ) plaques and tau neurofibrillary tangles as pathological hallmarks. Numerous clinical trials have been conducted with vaccines and small molecules to target Aβ formation and aggregation and also enhance Aβ clearance; so far such clinical trials have been unsuccessful. Novel strategies are therefore required and here we will discuss the possibility of utilizing the chaperone BRICHOS to prevent Aβ aggregation and toxicity. Type 2 diabetes mellitus is symptomatically treated with insulin. However the disease is linked to the aggregation and progressive accumulation of islet amyloid polypeptide and oligomers of this peptide are cytotoxic. Several compounds have been shown to inhibit islet amyloid aggregation and cytotoxicity in vitro. Future animal studies and clinical trials have to be be conducted to determine their efficacy in vivo. Transthyretin (TTR) amyloidoses are a group of systemic degenerative diseases involving multiple organ systems and caused by TTR aggregation. Liver transplantation decreases the generation of misfolded TTR and improves the quality of life for a subgroup of this patient population. Compounds that stabilize the natively folded, non-amyloidogenic, tetrameric conformation of TTR have been developed and the drug tafamidis is available as treatment. PMID:27165517

  7. Current and future treatment of amyloid diseases.

    PubMed

    Ankarcrona, M; Winblad, B; Monteiro, C; Fearns, C; Powers, E T; Johansson, J; Westermark, G T; Presto, J; Ericzon, B-G; Kelly, J W

    2016-08-01

    There are more than 30 human proteins whose aggregation appears to cause degenerative maladies referred to as amyloid diseases or amyloidoses. These disorders are named after the characteristic cross-β-sheet amyloid fibrils that accumulate systemically or are localized to specific organs. In most cases, current treatment is limited to symptomatic approaches and thus disease-modifying therapies are needed. Alzheimer's disease is a neurodegenerative disorder with extracellular amyloid β-peptide (Aβ) fibrils and intracellular tau neurofibrillary tangles as pathological hallmarks. Numerous clinical trials have been conducted with passive and active immunotherapy, and small molecules to inhibit Aβ formation and aggregation or to enhance Aβ clearance; so far such clinical trials have been unsuccessful. Novel strategies are therefore required and here we will discuss the possibility of utilizing the chaperone BRICHOS to prevent Aβ aggregation and toxicity. Type 2 diabetes mellitus is symptomatically treated with insulin. However, the underlying pathology is linked to the aggregation and progressive accumulation of islet amyloid polypeptide as fibrils and oligomers, which are cytotoxic. Several compounds have been shown to inhibit islet amyloid aggregation and cytotoxicity in vitro. Future animal studies and clinical trials have to be conducted to determine their efficacy in vivo. The transthyretin (TTR) amyloidoses are a group of systemic degenerative diseases compromising multiple organ systems, caused by TTR aggregation. Liver transplantation decreases the generation of misfolded TTR and improves the quality of life for a subgroup of this patient population. Compounds that stabilize the natively folded, nonamyloidogenic, tetrameric conformation of TTR have been developed and the drug tafamidis is available as a promising treatment.

  8. Organic solvent mediated self-association of an amyloid forming peptide from beta2-microglobulin: an atomic force microscopy study.

    PubMed

    Chaudhary, Nitin; Singh, Shashi; Nagaraj, Ramakrishnan

    2008-01-01

    Human beta(2)-microglobulin (beta(2)m) forms amyloid fibrils in hemodialysis related amyloidosis. Peptides spanning the beta strands of beta(2)m have been shown to form amyloid fibrils in isolation. We have studied the self-association of a 13-residue peptide Ac-DWSFYLLYYTEFT-am (Pbeta(2)m) spanning one of the beta-strands of human beta(2)-microglobulin when dissolved in various organic solvents such as methanol (MeOH), trifluoroethanol (TFE), hexafluoroisopropanol (HFIP), and dimethylsulfoxide. We have observed that Pbeta(2)m forms amyloid fibrils when diluted from organic solvents into aqueous buffer at pH 7.0 as judged by increase in thioflavin T fluorescence. Fibril formation was observed to depend on the solvents in which peptide stock solutions were prepared. Circular dichroism spectra indicated propensity for helical conformation in MeOH, TFE, and HFIP. In buffer, beta-structure was observed irrespective of the solvent in which the peptide stock solutions were prepared. Atomic force microscopy images obtained by drying the peptide on mica from organic solvents indicated the ability of Pbeta(2)m to self-associate to form nonfibrillar structures. Morphology of the structures was dependent on the solvent in which the peptide was dissolved. Peptides that have the ability to self-associate such as amyloid-forming peptides would be attractive candidates for the generation of self-assembled structures with varying morphologies by appropriate choice of surfaces and solvents for dissolution. Copyright 2008 Wiley Periodicals, Inc.

  9. AD synapses contain abundant Aβ monomer and multiple soluble oligomers, including a 56 kDa assembly

    PubMed Central

    Sokolow, Sophie; Henkins, Kristen M.; Bilousova, Tina; Miller, Carol A.; Vinters, Harry V.; Poon, Wayne; Cole, Gregory M.; Gylys, Karen Hoppens

    2011-01-01

    Much evidence indicates that soluble amyloid beta (Aβ) oligomers are key mediators of early cognitive loss, but the localization and key peptide species remain unclear. We have used flow cytometry analysis to demonstrate that surviving Alzheimer's disease (AD) synapses accumulate both Aβ and p-tau. The present experiments use peptide-specific xMAP assays and Western blotting to identify the Aβ peptide species in synaptosome-enriched samples from normal human subjects, neurologic controls, and AD cases. Aβ40 peptide levels did not vary, but both Aβ42 and Aβ oligomers were increased in soluble AD extracts, with oligomer levels 20-fold higher in aqueous compared to detergent extracts. In Western blots, a ladder of SDS-stable oligomers was observed in AD cases, varying in size from monomer, the major peptide observed, to larger assemblies up to about 200 kD and larger. Multiple oligomers, including monomer, small oligomers, a 56 kD assembly, and APP were correlated with the Aβ level measured in flow cytomety-purified synaptosomes. These results suggest that multiple APP processing pathways are active in AD synapses and multiple soluble oligomeric assemblies may contribute to synaptic dysfunction. PMID:21741125

  10. Role of water in Protein Aggregation and Amyloid Polymorphism

    PubMed Central

    Thirumalai, D.; Reddy, Govardhan; Straub, John E.

    2011-01-01

    Conspectus The link between oligomers and amyloid fibrils and a variety of neurodegenerative diseases raises the need to decipher the principles governing protein aggregation. Mechanisms of in vivo amyloid formation involve a number of coconspirators and complex interactions with membranes. Nevertheless, it is believed that understanding the biophysical basis of in vitro amyloid formation in well-defined systems is important in discovering ligands that preferentially bind to regions that harbor amyloidogenic tendencies. Determination of structures of fibrils of a variety of peptides has set the stage for probing the dynamics of oligomer formation and amyloid growth using computer simulations. Most experimental and simulation studies have been interpreted largely from the perspective of proteins without much consideration of the role of solvent in enabling or inhibiting oligomer formation and assembly to protofilaments and amyloid fibrils. Here, we provide a perspective on how interactions with water affect folding landscapes of Aβ monomers, oligomer formation in Aβ16–22 fragment, protofilament formation in a peptide from yeast prion Sup35. Explicit molecular dynamics simulations of these systems illustrate how water controls the self-assembly of higher order structures and provide a structural basis for understanding the kinetics of oligomer and fibril growth. Simulations show that monomers of Aβ-peptides sample a number of compact conformations. Population of aggregation-prone structures (N*) with salt-bridge, which bear a striking similarity to the peptide structure in the fibril, requires overcoming a high desolvation barrier. In general, sequences for which N* structures are not significantly populated are unlikely to aggregate. Generically oligomers and fibrils form in two steps. In the first stage water is expelled from the region between peptides rich in hydrophobic residues (for example Aβ16–22) resulting in the disordered oligomers. In the second

  11. Human cyclophilin 40 unravels neurotoxic amyloids.

    PubMed

    Baker, Jeremy D; Shelton, Lindsey B; Zheng, Dali; Favretto, Filippo; Nordhues, Bryce A; Darling, April; Sullivan, Leia E; Sun, Zheying; Solanki, Parth K; Martin, Mackenzie D; Suntharalingam, Amirthaa; Sabbagh, Jonathan J; Becker, Stefan; Mandelkow, Eckhard; Uversky, Vladimir N; Zweckstetter, Markus; Dickey, Chad A; Koren, John; Blair, Laura J

    2017-06-01

    The accumulation of amyloidogenic proteins is a pathological hallmark of neurodegenerative disorders. The aberrant accumulation of the microtubule associating protein tau (MAPT, tau) into toxic oligomers and amyloid deposits is a primary pathology in tauopathies, the most common of which is Alzheimer's disease (AD). Intrinsically disordered proteins, like tau, are enriched with proline residues that regulate both secondary structure and aggregation propensity. The orientation of proline residues is regulated by cis/trans peptidyl-prolyl isomerases (PPIases). Here we show that cyclophilin 40 (CyP40), a PPIase, dissolves tau amyloids in vitro. Additionally, CyP40 ameliorated silver-positive and oligomeric tau species in a mouse model of tau accumulation, preserving neuronal health and cognition. Nuclear magnetic resonance (NMR) revealed that CyP40 interacts with tau at sites rich in proline residues. CyP40 was also able to interact with and disaggregate other aggregating proteins that contain prolines. Moreover, CyP40 lacking PPIase activity prevented its capacity for disaggregation in vitro. Finally, we describe a unique structural property of CyP40 that may permit disaggregation to occur in an energy-independent manner. This study identifies a novel human protein disaggregase and, for the first time, demonstrates its capacity to dissolve intracellular amyloids.

  12. Human cyclophilin 40 unravels neurotoxic amyloids

    PubMed Central

    Favretto, Filippo; Nordhues, Bryce A.; Darling, April; Sullivan, Leia E.; Sun, Zheying; Solanki, Parth K.; Martin, Mackenzie D.; Suntharalingam, Amirthaa; Sabbagh, Jonathan J.; Becker, Stefan; Mandelkow, Eckhard; Uversky, Vladimir N.; Zweckstetter, Markus; Dickey, Chad A.; Koren, John; Blair, Laura J.

    2017-01-01

    The accumulation of amyloidogenic proteins is a pathological hallmark of neurodegenerative disorders. The aberrant accumulation of the microtubule associating protein tau (MAPT, tau) into toxic oligomers and amyloid deposits is a primary pathology in tauopathies, the most common of which is Alzheimer’s disease (AD). Intrinsically disordered proteins, like tau, are enriched with proline residues that regulate both secondary structure and aggregation propensity. The orientation of proline residues is regulated by cis/trans peptidyl-prolyl isomerases (PPIases). Here we show that cyclophilin 40 (CyP40), a PPIase, dissolves tau amyloids in vitro. Additionally, CyP40 ameliorated silver-positive and oligomeric tau species in a mouse model of tau accumulation, preserving neuronal health and cognition. Nuclear magnetic resonance (NMR) revealed that CyP40 interacts with tau at sites rich in proline residues. CyP40 was also able to interact with and disaggregate other aggregating proteins that contain prolines. Moreover, CyP40 lacking PPIase activity prevented its capacity for disaggregation in vitro. Finally, we describe a unique structural property of CyP40 that may permit disaggregation to occur in an energy-independent manner. This study identifies a novel human protein disaggregase and, for the first time, demonstrates its capacity to dissolve intracellular amyloids. PMID:28654636

  13. The effects of soluble Aβ oligomers on neurodegeneration in Alzheimer's disease.

    PubMed

    Brouillette, Jonathan

    2014-01-01

    The neurodegenerative process that defines Alzheimer''s disease (AD) is initially characterized by synaptic alterations followed by synapse loss and ultimately cell death. Decreased synaptic density that precedes neuronal death is the strongest pathological correlate of cognitive deficits observed in AD. Substantial synapse and neuron loss occur early in disease progression in the entorhinal cortex (EC) and the CA1 region of the hippocampus, when memory deficits become clinically detectable. Mounting evidence suggests that soluble amyloid-β (Aβ) oligomers trigger synapse dysfunction both in vitro and in vivo. However, the neurodegenerative effect of Aβ species observed on neuronal culture or organotypic brain slice culture has been more challenging to mimic in animal models. While most of the transgenic mice that overexpress Aβ show abundant amyloid plaque pathology and early synaptic alterations, these models have been less successful in recapitulating the spatiotemporal pattern of cell loss observed in AD. Recently we developed a novel animal model that revealed the neurodegenerative effect of soluble low-molecular-weight Aβ oligomers in vivo. This new approach may now serve to determine the molecular and cellular mechanisms linking soluble Aβ species to neurodegeneration in animals. In light of the low efficiency of AD therapies based on the amyloid cascade hypothesis, a novel framework, the aging factor cascade hypothesis, is proposed in an attempt to integrate the new data and concepts that emerged from recent research to develop disease modifying therapies.

  14. Hyperphosphorylation-induced tau oligomers.

    PubMed

    Iqbal, Khalid; Gong, Cheng-Xin; Liu, Fei

    2013-01-01

    In normal adult brain the microtubule associated protein (MAP) tau contains 2-3 phosphates per mol of the protein and at this level of phosphorylation it is a soluble cytosolic protein. The normal brain tau interacts with tubulin and promotes its assembly into microtubules and stabilizes these fibrils. In Alzheimer disease (AD) brain tau is three to fourfold hyperphosphorylated. The abnormally hyperphosphorylated tau binds to normal tau instead of the tubulin and this binding leads to the formation of tau oligomers. The tau oligomers can be sedimented at 200,000 × g whereas the normal tau under these conditions remains in the supernatant. The abnormally hyperphosphorylated tau is capable of sequestering not only normal tau but also MAP MAP1 and MAP2 and causing disruption of the microtubule network promoted by these proteins. Unlike Aβ and prion protein (PrP) oligomers, tau oligomerization in AD and related tauopathies is hyperphosphorylation-dependent; in vitro dephosphorylation of AD P-tau with protein phosphatase 2A (PP2A) inhibits and rehyperphosphorylation of the PP2A-AD P-tau with more than one combination of tau protein kinases promotes its oligomerization. In physiological assembly conditions the AD P-tau readily self-assembles into paired helical filaments. Missense tau mutations found in frontotemporal dementia apparently lead to tau oligomerization and neurofibrillary pathology by promoting its abnormal hyperphosphorylation. Dysregulation of the alternative splicing of tau that alters the 1:1 ratio of the 3-repeat: 4-repeat taus such as in Down syndrome, Pick disease, and progressive supranuclear palsy leads to the abnormal hyperphosphorylation of tau.

  15. Counterion condensation on heparin oligomers.

    PubMed

    Minsky, Burcu Baykal; Atmuri, Anand; Kaltashov, Igor A; Dubin, Paul L

    2013-04-08

    The electropherogram of native heparin shows a broad distribution of mobilities μ, which truncates abruptly at a notably high μ = 4.7 × 10(-4) cm(2) V(-1) s(-1). This highly skewed mobility distribution is also found for the 20-saccharide chain, which shows from mass spectrometry a more uniform (symmetrical) with respect to sulfation level. Since a partially degraded heparin exhibits oligomer peaks with μ> 5 × 10(-4) cm(2) V(-1) s(-1) (appearing to escape the limitation of the mobility value for native heparin), we examined the electrophoretic behavior of chain-length monodisperse heparin oligomers. Their mobilities varied inversely with the logarithm of the contour length, L, for L from 3 to 10 nm and reached an asymptotic limit for L > 20 nm. The generality of this effect was indicated by similar behavior for oligomers of poly(styrene sulfonate). A recent theory of polyelectrolyte end effects (Manning, G. S. Macromolecules2008, 41, 6217-6227), in which chain termini exhibit reduced counterion condensation was found to quantitatively account for these results. A qualitative explanation for the anomalously high value of μ of native heparin, 10-20% higher than those seen for synthetic polyelectrolytes of higher linear charge density, is suggested on the basis of similar junction effects (Manning, G. S. Macromolecules2008, 41, 6217-6227), which reduce counterion condensation at the interfaces of regions of high and low sulfation. We suggest that these effects should be considered in models for the biofunctionality of the regulated high and low sulfation (NS/NA) domains of heparan sulfate.

  16. Isolating Toxic Insulin Amyloid Reactive Species that Lack β-Sheets and Have Wide pH Stability

    PubMed Central

    Heldt, Caryn L.; Kurouski, Dmitry; Sorci, Mirco; Grafeld, Elizabeth; Lednev, Igor K.; Belfort, Georges

    2011-01-01

    Amyloid diseases, including Alzheimer's disease, are characterized by aggregation of normally functioning proteins or peptides into ordered, β-sheet rich fibrils. Most of the theories on amyloid toxicity focus on the nuclei or oligomers in the fibril formation process. The nuclei and oligomers are transient species, making their full characterization difficult. We have isolated toxic protein species that act like an oligomer and may provide the first evidence of a stable reactive species created by disaggregation of amyloid fibrils. This reactive species was isolated by dissolving amyloid fibrils at high pH and it has a mass >100 kDa and a diameter of 48 ± 15 nm. It seeds the formation of fibrils in a dose dependent manner, but using circular dichroism and deep ultraviolet resonance Raman spectroscopy, the reactive species was found to not have a β-sheet rich structure. We hypothesize that the reactive species does not decompose at high pH and maintains its structure in solution. The remaining disaggregated insulin, excluding the toxic reactive species that elongated the fibrils, returned to native structured insulin. This is the first time, to our knowledge, that a stable reactive species of an amyloid reaction has been separated and characterized by disaggregation of amyloid fibrils. PMID:21641325

  17. Functional amyloids in bacteria.

    PubMed

    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.

  18. Stabilization of nontoxic Aβ-oligomers: insights into the mechanism of action of hydroxyquinolines in Alzheimer's disease.

    PubMed

    Ryan, Timothy M; Roberts, Blaine R; McColl, Gawain; Hare, Dominic J; Doble, Philip A; Li, Qiao-Xin; Lind, Monica; Roberts, Anne M; Mertens, Haydyn D T; Kirby, Nigel; Pham, Chi L L; Hinds, Mark G; Adlard, Paul A; Barnham, Kevin J; Curtain, Cyril C; Masters, Colin L

    2015-02-18

    The extracellular accumulation of amyloid β (Aβ) peptides is characteristic of Alzheimer's disease (AD). However, formation of diffusible, oligomeric forms of Aβ, both on and off pathways to amyloid fibrils, is thought to include neurotoxic species responsible for synaptic loss and neurodegeneration, rather than polymeric amyloid aggregates. The 8-hydroxyquinolines (8-HQ) clioquinol (CQ) and PBT2 were developed for their ability to inhibit metal-mediated generation of reactive oxygen species from Aβ:Cu complexes and have both undergone preclinical and Phase II clinical development for the treatment of AD. Their respective modes of action are not fully understood and may include both inhibition of Aβ fibrillar polymerization and direct depolymerization of existing Aβ fibrils. In the present study, we find that CQ and PBT2 can interact directly with Aβ and affect its propensity to aggregate. Using a combination of biophysical techniques, we demonstrate that, in the presence of these 8-HQs and in the absence of metal ions, Aβ associates with two 8-HQ molecules and forms a dimer. Furthermore, 8-HQ bind Aβ with an affinity of 1-10 μm and suppress the formation of large (>30 kDa) oligomers. The stabilized low molecular weight species are nontoxic. Treatment with 8-HQs also reduces the levels of in vivo soluble oligomers in a Caenorhabditis elegans model of Aβ toxicity. We propose that 8-HQs possess an additional mechanism of action that neutralizes neurotoxic Aβ oligomer formation through stabilization of small (dimeric) nontoxic Aβ conformers.

  19. Optimization of the All-D Peptide D3 for Aβ Oligomer Elimination.

    PubMed

    Klein, Antonia Nicole; Ziehm, Tamar; Tusche, Markus; Buitenhuis, Johan; Bartnik, Dirk; Boeddrich, Annett; Wiglenda, Thomas; Wanker, Erich; Funke, Susanne Aileen; Brener, Oleksandr; Gremer, Lothar; Kutzsche, Janine; Willbold, Dieter

    2016-01-01

    The aggregation of amyloid-β (Aβ) is postulated to be the crucial event in Alzheimer's disease (AD). In particular, small neurotoxic Aβ oligomers are considered to be responsible for the development and progression of AD. Therefore, elimination of thesis oligomers represents a potential causal therapy of AD. Starting from the well-characterized d-enantiomeric peptide D3, we identified D3 derivatives that bind monomeric Aβ. The underlying hypothesis is that ligands bind monomeric Aβ and stabilize these species within the various equilibria with Aβ assemblies, leading ultimately to the elimination of Aβ oligomers. One of the hereby identified d-peptides, DB3, and a head-to-tail tandem of DB3, DB3DB3, were studied in detail. Both peptides were found to: (i) inhibit the formation of Thioflavin T-positive fibrils; (ii) bind to Aβ monomers with micromolar affinities; (iii) eliminate Aβ oligomers; (iv) reduce Aβ-induced cytotoxicity; and (v) disassemble preformed Aβ aggregates. The beneficial effects of DB3 were improved by DB3DB3, which showed highly enhanced efficacy. Our approach yielded Aβ monomer-stabilizing ligands that can be investigated as a suitable therapeutic strategy against AD.

  20. WASP-1, a canonical Wnt signaling potentiator, rescues hippocampal synaptic impairments induced by Aβ oligomers.

    PubMed

    Vargas, Jessica Y; Ahumada, Juan; Arrázola, Macarena S; Fuenzalida, Marco; Inestrosa, Nibaldo C

    2015-02-01

    Amyloid-β (Aβ) oligomers are a key factor in Alzheimer's disease (AD)-associated synaptic dysfunction. Aβ oligomers block the induction of hippocampal long-term potentiation (LTP) in rodents. The activation of Wnt signaling prevents Aβ oligomer-induced neurotoxic effects. The compound WASP-1 (Wnt-activating small molecule potentiator-1), has been described as a synergist of the ligand Wnt-3a, enhancing the activation of Wnt/β-catenin signaling. Herein, we report that WASP-1 administration successfully rescued Aβ-induced synaptic impairments both in vitro and in vivo. The activation of canonical Wnt/β-catenin signaling by WASP-1 increased synaptic transmission and rescued hippocampal LTP impairments induced by Aβ oligomers. Additionally, intra-hippocampal administration of WASP-1 to the double transgenic APPswe/PS1dE9 mouse model of AD prevented synaptic protein loss and reduced tau phosphorylation levels. Moreover, we found that WASP-1 blocked Aβ aggregation in vitro and reduced pathological tau phosphorylation in vivo. These results indicate that targeting canonical Wnt signaling with WASP-1 could have value for treating AD.

  1. Cytotoxic Helix-Rich Oligomer Formation by Melittin and Pancreatic Polypeptide

    PubMed Central

    Singh, Pradeep K.; Ghosh, Dhiman; Tewari, Debanjan; Mohite, Ganesh M.; Carvalho, Edmund; Jha, Narendra Nath; Jacob, Reeba S.; Sahay, Shruti; Banerjee, Rinti; Bera, Amal K.; Maji, Samir K.

    2015-01-01

    Conversion of amyloid fibrils by many peptides/proteins involves cytotoxic helix-rich oligomers. However, their toxicity and biophysical studies remain largely unknown due to their highly dynamic nature. To address this, we chose two helical peptides (melittin, Mel and pancreatic polypeptide, PP) and studied their aggregation and toxicity. Mel converted its random coil structure to oligomeric helical structure upon binding to heparin; however, PP remained as helix after oligomerization. Interestingly, similar to Parkinson’s associated α-synuclein (AS) oligomers, Mel and PP also showed tinctorial properties, higher hydrophobic surface exposure, cellular toxicity and membrane pore formation after oligomerization in the presence of heparin. We suggest that helix-rich oligomers with exposed hydrophobic surface are highly cytotoxic to cells irrespective of their disease association. Moreover as Mel and PP (in the presence of heparin) instantly self-assemble into stable helix-rich amyloidogenic oligomers; they could be represented as models for understanding the biophysical and cytotoxic properties of helix-rich intermediates in detail. PMID:25803428

  2. Optimization of the All-D Peptide D3 for Aβ Oligomer Elimination

    PubMed Central

    Klein, Antonia Nicole; Ziehm, Tamar; Tusche, Markus; Buitenhuis, Johan; Bartnik, Dirk; Boeddrich, Annett; Wiglenda, Thomas; Wanker, Erich; Funke, Susanne Aileen; Brener, Oleksandr; Gremer, Lothar; Kutzsche, Janine; Willbold, Dieter

    2016-01-01

    The aggregation of amyloid-β (Aβ) is postulated to be the crucial event in Alzheimer’s disease (AD). In particular, small neurotoxic Aβ oligomers are considered to be responsible for the development and progression of AD. Therefore, elimination of thesis oligomers represents a potential causal therapy of AD. Starting from the well-characterized d-enantiomeric peptide D3, we identified D3 derivatives that bind monomeric Aβ. The underlying hypothesis is that ligands bind monomeric Aβ and stabilize these species within the various equilibria with Aβ assemblies, leading ultimately to the elimination of Aβ oligomers. One of the hereby identified d-peptides, DB3, and a head-to-tail tandem of DB3, DB3DB3, were studied in detail. Both peptides were found to: (i) inhibit the formation of Thioflavin T-positive fibrils; (ii) bind to Aβ monomers with micromolar affinities; (iii) eliminate Aβ oligomers; (iv) reduce Aβ-induced cytotoxicity; and (v) disassemble preformed Aβ aggregates. The beneficial effects of DB3 were improved by DB3DB3, which showed highly enhanced efficacy. Our approach yielded Aβ monomer-stabilizing ligands that can be investigated as a suitable therapeutic strategy against AD. PMID:27105346

  3. Oligomer-targeting with a conformational antibody fragment promotes toxicity in Aβ-expressing flies

    PubMed Central

    2014-01-01

    Introduction The self-assembly of Aβ peptides into a range of conformationally heterogeneous amyloid states represents a fundamental event in Alzheimer’s disease. Within these structures oligomeric intermediates are considered to be particularly pathogenic. To test this hypothesis we have used a conformational targeting approach where particular conformational states, such as oligomers or fibrils, are recognized in vivo by state-specific antibody fragments. Results We show that oligomer targeting with the KW1 antibody fragment, but not fibril targeting with the B10 antibody fragment, affects toxicity in Aβ-expressing Drosophila melanogaster. The effect of KW1 is observed to occur selectively with flies expressing Aβ(1–40) and not with those expressing Aβ(1–42) or the arctic variant of Aβ(1–42) This finding is consistent with the binding preference of KW1 for Aβ(1–40) oligomers that has been established in vitro. Strikingly, and in contrast to the previously demonstrated in vitro ability of this antibody fragment to block oligomeric toxicity in long-term potentiation measurements, KW1 promotes toxicity in the flies rather than preventing it. This result shows the crucial importance of the environment in determining the influence of antibody binding on the nature and consequences of the protein misfolding and aggregation. Conclusions While our data support to the pathological relevance of oligomers, they highlight the issues to be addressed when developing inhibitory strategies that aim to neutralize these states by means of antagonistic binding agents. PMID:24725347

  4. Oligovalent Amyloid-Binding Agents Reduce SEVI-Mediated Enhancement of HIV-1 Infection

    PubMed Central

    Capule, Christina C.; Brown, Caitlin; Olsen, Joanna S.; Dewhurst, Stephen; Yang, Jerry

    2012-01-01

    This paper evaluates the use of oligovalent amyloid-binding molecules as potential agents that can reduce the enhancement of HIV-1 infection in cells by SEVI fibrils. These naturally occurring amyloid fibrils found in semen have been implicated as mediators that can facilitate the attachment and internalization of HIV-1 virions to immune cells. Molecules that are capable of reducing the role of SEVI in HIV-1 infection may, therefore, represent a novel strategy to reduce the rate of sexual transmission of HIV-1 in humans. Here, we evaluated a set of synthetic, oligovalent derivatives of BTA (a known amyloid-binding molecule) for their capability to bind cooperatively to aggregated amyloid peptides and to neutralize the effects of SEVI in HIV-1 infection. We demonstrate that these BTA derivatives exhibit a general trend of increased binding to aggregated amyloids as a function of increasing valence number of the oligomer. Importantly, we find that oligomers of BTA show improved capability to reduce SEVI-mediated infection of HIV-1 in cells compared to a BTA monomer, with the pentamer exhibiting a 65-fold improvement in efficacy compared to a previously reported monomeric BTA derivative. These results, thus, support the use of amyloid-targeting molecules as potential supplements for microbicides to curb the spread of HIV-1 through sexual contact. PMID:22239120

  5. The Aβ peptide forms non-amyloid fibrils in the presence of carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Luo, Jinghui; Wärmländer, Sebastian K. T. S.; Yu, Chien-Hung; Muhammad, Kamran; Gräslund, Astrid; Pieter Abrahams, Jan

    2014-05-01

    Carbon nanotubes have specific properties that make them potentially useful in biomedicine and biotechnology. However, carbon nanotubes may themselves be toxic, making it imperative to understand how carbon nanotubes interact with biomolecules such as proteins. Here, we used NMR, CD, and ThT/fluorescence spectroscopy together with AFM imaging to study pH-dependent molecular interactions between single walled carbon nanotubes (SWNTs) and the amyloid-beta (Aβ) peptide. The aggregation of the Aβ peptide, first into oligomers and later into amyloid fibrils, is considered to be the toxic mechanism behind Alzheimer's disease. We found that SWNTs direct the Aβ peptides to form a new class of β-sheet-rich yet non-amyloid fibrils.Carbon nanotubes have specific properties that make them potentially useful in biomedicine and biotechnology. However, carbon nanotubes may themselves be toxic, making it imperative to understand how carbon nanotubes interact with biomolecules such as proteins. Here, we used NMR, CD, and ThT/fluorescence spectroscopy together with AFM imaging to study pH-dependent molecular interactions between single walled carbon nanotubes (SWNTs) and the amyloid-beta (Aβ) peptide. The aggregation of the Aβ peptide, first into oligomers and later into amyloid fibrils, is considered to be the toxic mechanism behind Alzheimer's disease. We found that SWNTs direct the Aβ peptides to form a new class of β-sheet-rich yet non-amyloid fibrils. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr00291a

  6. QIAD assay for quantitating a compound’s efficacy in elimination of toxic Aβ oligomers

    PubMed Central

    Brener, Oleksandr; Dunkelmann, Tina; Gremer, Lothar; van Groen, Thomas; Mirecka, Ewa A.; Kadish, Inga; Willuweit, Antje; Kutzsche, Janine; Jürgens, Dagmar; Rudolph, Stephan; Tusche, Markus; Bongen, Patrick; Pietruszka, Jörg; Oesterhelt, Filipp; Langen, Karl-Josef; Demuth, Hans-Ulrich; Janssen, Arnold; Hoyer, Wolfgang; Funke, Susanne A.; Nagel-Steger, Luitgard; Willbold, Dieter

    2015-01-01

    Strong evidence exists for a central role of amyloid β-protein (Aβ) oligomers in the pathogenesis of Alzheimer’s disease. We have developed a fast, reliable and robust in vitro assay, termed QIAD, to quantify the effect of any compound on the Aβ aggregate size distribution. Applying QIAD, we studied the effect of homotaurine, scyllo-inositol, EGCG, the benzofuran derivative KMS88009, ZAβ3W, the D-enantiomeric peptide D3 and its tandem version D3D3 on Aβ aggregation. The predictive power of the assay for in vivo efficacy is demonstrated by comparing the oligomer elimination efficiency of D3 and D3D3 with their treatment effects in animal models of Alzheimer´s disease. PMID:26394756

  7. Mechanics of a fiber network within a non-fibrillar matrix: model and comparison with collagen-agarose co-gels

    PubMed Central

    Lake, Spencer P.; Hadi, Mohammad F.; Lai, Victor K.; Barocas, Victor H.

    2013-01-01

    While collagen is recognized as the predominant mechanical component of soft connective tissues, the role of the non-fibrillar matrix (NFM) is less well understood. Even model systems, such as the collagen-agarose co-gel, can exhibit complex behavior, making it difficult to identify relative contributions of specific tissue constituents. In the present study, we developed a two-component microscale model of collagen-agarose tissue analogs and used it to elucidate the interaction between collagen and NFM in uniaxial tension. Collagen fibers were represented with Voronoi networks, and the NFM was modeled as a neo-Hookean solid. Model predictions of total normal stress and Poisson’s ratio matched experimental observations well (including high Poisson’s values of ~3), and the addition of NFM led to composition-dependent decreases in volume change and increases in fiber stretch. Because the NFM was more resistant to volume change than the fiber network, extension of the composite led to pressurization of the NFM. Within a specific range of parameter values (low shear modulus and moderate Poisson’s ratio), the magnitude of the reaction force decreased relative to this pressurization component resulting in a negative (compressive) NFM stress in the loading direction, even though the composite tissue was in tension. PMID:22565816

  8. Interactions between Amyloid-β and Tau Fragments Promote Aberrant Aggregates: Implications for Amyloid Toxicity

    PubMed Central

    2015-01-01

    We have investigated at the oligomeric level interactions between Aβ(25–35) and Tau(273–284), two important fragments of the amyloid-β and Tau proteins, implicated in Alzheimer’s disease. We are able to directly observe the coaggregation of these two peptides by probing the conformations of early heteroligomers and the macroscopic morphologies of the aggregates. Ion-mobility experiment and theoretical modeling indicate that the interactions of the two fragments affect the self-assembly processes of both peptides. Tau(273–284) shows a high affinity to form heteroligomers with existing Aβ(25–35) monomer and oligomers in solution. The configurations and characteristics of the heteroligomers are determined by whether the population of Aβ(25–35) or Tau(273–284) is dominant. As a result, two types of aggregates are observed in the mixture with distinct morphologies and dimensions from those of pure Aβ(25–35) fibrils. The incorporation of some Tau into β-rich Aβ(25–35) oligomers reduces the aggregation propensity of Aβ(25–35) but does not fully abolish fibril formation. On the other hand, by forming complexes with Aβ(25–35), Tau monomers and dimers can advance to larger oligomers and form granular aggregates. These heteroligomers may contribute to toxicity through loss of normal function of Tau or inherent toxicity of the aggregates themselves. PMID:25153942

  9. Interactions between amyloid-β and Tau fragments promote aberrant aggregates: implications for amyloid toxicity.

    PubMed

    Do, Thanh D; Economou, Nicholas J; Chamas, Ali; Buratto, Steven K; Shea, Joan-Emma; Bowers, Michael T

    2014-09-25

    We have investigated at the oligomeric level interactions between Aβ(25-35) and Tau(273-284), two important fragments of the amyloid-β and Tau proteins, implicated in Alzheimer's disease. We are able to directly observe the coaggregation of these two peptides by probing the conformations of early heteroligomers and the macroscopic morphologies of the aggregates. Ion-mobility experiment and theoretical modeling indicate that the interactions of the two fragments affect the self-assembly processes of both peptides. Tau(273-284) shows a high affinity to form heteroligomers with existing Aβ(25-35) monomer and oligomers in solution. The configurations and characteristics of the heteroligomers are determined by whether the population of Aβ(25-35) or Tau(273-284) is dominant. As a result, two types of aggregates are observed in the mixture with distinct morphologies and dimensions from those of pure Aβ(25-35) fibrils. The incorporation of some Tau into β-rich Aβ(25-35) oligomers reduces the aggregation propensity of Aβ(25-35) but does not fully abolish fibril formation. On the other hand, by forming complexes with Aβ(25-35), Tau monomers and dimers can advance to larger oligomers and form granular aggregates. These heteroligomers may contribute to toxicity through loss of normal function of Tau or inherent toxicity of the aggregates themselves.

  10. Microspectroscopy (μFTIR) reveals co-localization of lipid oxidation and amyloid plaques in human Alzheimer disease brains.

    PubMed

    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.

  11. Key Points Concerning Amyloid Infectivity and Prion-Like Neuronal Invasion

    PubMed Central

    Espargaró, Alba; Busquets, Maria Antònia; Estelrich, Joan; Sabate, Raimon

    2016-01-01

    Amyloid aggregation has been related to an increasing number of human illnesses, from Alzheimer’s and Parkinson’s diseases (AD/PD) to Creutzfeldt-Jakob disease. Commonly, only prions have been considered as infectious agents with a high capacity of propagation. However, recent publications have shown that many amyloid proteins, including amyloid β-peptide, α-synuclein (α-syn) and tau protein, also propagate in a “prion-like” manner. Meanwhile, no link between propagation of pathological proteins and neurotoxicity has been demonstrated. The extremely low infectivity under natural conditions of most non-prion amyloids is far below the capacity to spread exhibited by prions. Nonetheless, it is important to elucidate the key factors that cause non-prion amyloids to become infectious agents. In recent years, important advances in our understanding of the amyloid processes of amyloid-like proteins and unrelated prions (i.e., yeast and fungal prions) have yielded essential information that can shed light on the prion phenomenon in mammals and humans. As shown in this review, recent evidence suggests 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 all be key factors affecting their spread. In short, those factors that favor the high concentration of extracellular nuclei or oligomers, characterized by small size, with a low toxicity could dramatically increase prion propensity; whereas low concentrations of highly toxic intracellular amyloids, with a large size, would effectively prevent infectivity. PMID:27147962

  12. Amyloid β-Protein C-Terminal Fragments: Formation of Cylindrins and β-Barrels.

    PubMed

    Do, Thanh D; LaPointe, Nichole E; Nelson, Rebecca; Krotee, Pascal; Hayden, Eric Y; Ulrich, Brittany; Quan, Sarah; Feinstein, Stuart C; Teplow, David B; Eisenberg, David; Shea, Joan-Emma; Bowers, Michael T

    2016-01-20

    In order to evaluate potential therapeutic targets for treatment of amyloidoses such as Alzheimer's disease (AD), it is essential to determine the structures of toxic amyloid oligomers. However, for the amyloid β-protein peptide (Aβ), thought to be the seminal neuropathogenetic agent in AD, its fast aggregation kinetics and the rapid equilibrium dynamics among oligomers of different size pose significant experimental challenges. Here we use ion-mobility mass spectrometry, in combination with electron microscopy, atomic force microscopy, and computational modeling, to test the hypothesis that Aβ peptides can form oligomeric structures resembling cylindrins and β-barrels. These structures are hypothesized to cause neuronal injury and death through perturbation of plasma membrane integrity. We show that hexamers of C-terminal Aβ fragments, including Aβ(24-34), Aβ(25-35) and Aβ(26-36), have collision cross sections similar to those of cylindrins. We also show that linking two identical fragments head-to-tail using diglycine increases the proportion of cylindrin-sized oligomers. In addition, we find that larger oligomers of these fragments may adopt β-barrel structures and that β-barrels can be formed by folding an out-of-register β-sheet, a common type of structure found in amyloid proteins.

  13. Amyloid β-Protein C-terminal Fragments: Formation of Cylindrins and β-barrels

    PubMed Central

    Do, Thanh D.; LaPointe, Nichole E.; Nelson, Rebecca; Krotee, Pascal; Hayden, Eric Y.; Ulrich, Brittany; Quan, Sarah; Feinstein, Stuart C.; Teplow, David B.; Eisenberg, David; Shea, Joan-Emma; Bowers, Michael T.

    2016-01-01

    In order to evaluate potential therapeutic targets for treatment of amyloidoses such as Alzheimer’s disease (AD), it is essential to determine the structures of toxic amyloid oligomers. However, for the amyloid β-protein peptide (Aβ), thought to be the seminal neuropathogenetic agent in AD, its fast aggregation kinetics and the rapid equilibrium dynamics among oligomers of different size pose significant experimental challenges. Here we use ion-mobility mass spectrometry, in combination with electron microscopy, atomic force microscopy, and computational modeling, to test the hypothesis that Aβ peptides can form oligomeric structures resembling cylindrins and β-barrels. These structures are hypothesized to cause neuronal injury and death through perturbation of plasma membrane integrity. We show that hexamers of C-terminal Aβ fragments, including Aβ(24-34), Aβ(25-35) and Aβ(26-36), have collision cross-sections similar to those of cylindrins. We also show that linking two identical fragments head-to-tail using di-glycine increases the proportion of cylindrin-sized oligomers. In addition, we find that larger oligomers of these fragments may adopt β-barrel structures and that β-barrels can be formed by folding an out-of-register β-sheet, a common type of structure found in amyloid proteins. PMID:26700445

  14. Lessons from a Rare Familial Dementia: Amyloid and Beyond.

    PubMed

    Cantlon, Adam; Frigerio, Carlo Sala; Walsh, Dominic M

    2015-08-01

    Here we review the similarities between a rare inherited disorder, familial British dementia (FBD), and the most common of all late-life neurological conditions, Alzheimer's diseases (AD). We describe the symptoms, pathology and genetics of FBD, the biology of the BRI2 protein and mouse models of FBD and familial Danish dementia. In particular, we focus on the evolving recognition of the importance of protein oligomers and aberrant processing of the amyloid β-protein precursor (APP) - themes that are common to both FBD and AD. The initial discovery that FBD is phenotypically similar to AD, but associated with the deposition of an amyloid peptide (ABri) distinct from the amyloid β-protein (Aβ) led many to assume that amyloid production alone is sufficient to initiate disease and that ABri is the molecular equivalent of Aβ. Parallel with work on Aβ, studies of ABri producing animal models and in vitro ABri toxicity experiments caused a revision of the amyloid hypothesis and a focus on soluble oligomers of Aβ and ABri. Contemporaneous other studies suggested that loss of the ABri precursor protein (BRI2) may underlie the cognitive deficits in FBD. In this regard it is important to note that BRI2 has been shown to interact with and regulate the processing of APP, and that mutant BRI2 leads to altered cleavage of APP. A synthesis of these results suggests that a "two-hit mechanism" better explains FBD than earlier toxic gain of function and toxic loss of function models. The lessons learned from the study of FBD imply that the molecular pathology of AD is also likely to involve both aberrant aggregation (in AD, Aβ) and altered APP processing. With regard to FBD, we propose that the C-terminal 11 amino acid of FBD-BRI2 interfere with both the normal function of BRI2 and promotes the production of cystine cross-linked toxic ABri oligomers. In this scenario, loss of BRI2 function leads to altered APP processing in as yet underappreciated ways. Given the similarities

  15. Lessons from a Rare Familial Dementia: Amyloid and Beyond

    PubMed Central

    Cantlon, Adam; Frigerio, Carlo Sala; Walsh, Dominic M.

    2015-01-01

    Here we review the similarities between a rare inherited disorder, familial British dementia (FBD), and the most common of all late-life neurological conditions, Alzheimer's diseases (AD). We describe the symptoms, pathology and genetics of FBD, the biology of the BRI2 protein and mouse models of FBD and familial Danish dementia. In particular, we focus on the evolving recognition of the importance of protein oligomers and aberrant processing of the amyloid β-protein precursor (APP) - themes that are common to both FBD and AD. The initial discovery that FBD is phenotypically similar to AD, but associated with the deposition of an amyloid peptide (ABri) distinct from the amyloid β-protein (Aβ) led many to assume that amyloid production alone is sufficient to initiate disease and that ABri is the molecular equivalent of Aβ. Parallel with work on Aβ, studies of ABri producing animal models and in vitro ABri toxicity experiments caused a revision of the amyloid hypothesis and a focus on soluble oligomers of Aβ and ABri. Contemporaneous other studies suggested that loss of the ABri precursor protein (BRI2) may underlie the cognitive deficits in FBD. In this regard it is important to note that BRI2 has been shown to interact with and regulate the processing of APP, and that mutant BRI2 leads to altered cleavage of APP. A synthesis of these results suggests that a “two-hit mechanism” better explains FBD than earlier toxic gain of function and toxic loss of function models. The lessons learned from the study of FBD imply that the molecular pathology of AD is also likely to involve both aberrant aggregation (in AD, Aβ) and altered APP processing. With regard to FBD, we propose that the C-terminal 11 amino acid of FBD-BRI2 interfere with both the normal function of BRI2 and promotes the production of cystine cross-linked toxic ABri oligomers. In this scenario, loss of BRI2 function leads to altered APP processing in as yet underappreciated ways. Given the

  16. Stereoisomers Probe Steric Zippers in Amyloid-β.

    PubMed

    Chandra, Bappaditya; Korn, Alexander; Maity, Barun Kumar; Adler, Juliane; Rawat, Anoop; Krueger, Martin; Huster, Daniel; Maiti, Sudipta

    2017-02-14

    Shape complementarity between close-packed residues plays a critical role in the amyloid aggregation process. Here, we probe such "steric zipper" interactions in amyloid-β (Aβ40), whose aggregation is linked to Alzheimer's disease, by replacing natural residues by their stereoisomers. Such mutations are expected to specifically destabilize the shape sensitive "packing" interactions, which may potentially increase their solubility and change other properties. We study the stereomutants DF19 and DL34 and also the DA2/DF4/DH6/DS8 mutant of Aβ40. F19-L34 is a critical contact in a tightly packed region of Aβ, while residues 1-9 are known to be disordered. While both DF19 and DL34 slow down the kinetics of aggregation and form amyloid fibrils efficiently, only DL34 increases the final solubility. DF19 gives rise to additional off-pathway aggregation which results in large, kinetically stable aggregates, and has lower net solubility. DA2/DF4/DH6/DS8 does not have an effect on the kinetics or the solubility. Notably, both DF19 and DL34 oligomers have a significantly lower level of interactions with lipid vesicles and live cells. We conclude that stereoisomers can cause complex site dependent changes in amyloid properties, and provide an effective tool to determine the role of individual residues in shaping the packed interiors of amyloid aggregates.

  17. Amyloid Structure and Assembly: Insights from Scanning Transmission Electron Microscopy

    SciTech Connect

    Goldsbury, C.; Wall, J.; Baxa, U.; Simon, M. N.; Steven, A. C.; Engel, A.; Aebi, U.; Muller, S. A.

    2011-01-01

    Amyloid fibrils are filamentous protein aggregates implicated in several common diseases such as Alzheimer's disease and type II diabetes. Similar structures are also the molecular principle of the infectious spongiform encephalopathies such as Creutzfeldt-Jakob disease in humans, scrapie in sheep, and of the so-called yeast prions, inherited non-chromosomal elements found in yeast and fungi. Scanning transmission electron microscopy (STEM) is often used to delineate the assembly mechanism and structural properties of amyloid aggregates. In this review we consider specifically contributions and limitations of STEM for the investigation of amyloid assembly pathways, fibril polymorphisms and structural models of amyloid fibrils. This type of microscopy provides the only method to directly measure the mass-per-length (MPL) of individual filaments. Made on both in vitro assembled and ex vivo samples, STEM mass measurements have illuminated the hierarchical relationships between amyloid fibrils and revealed that polymorphic fibrils and various globular oligomers can assemble simultaneously from a single polypeptide. The MPLs also impose strong constraints on possible packing schemes, assisting in molecular model building when combined with high-resolution methods like solid-state nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR).

  18. The effect of tachykinin neuropeptides on amyloid {beta} aggregation

    SciTech Connect

    Flashner, Efrat; Raviv, Uri; Friedler, Assaf

    2011-04-01

    Research highlights: {yields} Mechanistic explanation of how tachykinin neuropeptides reduce A{beta}-induced neurotoxicity. {yields} Biophysical studies suggest that tachykinins do not modulate the distribution of A{beta} oligomeric states, but rather may incorporate into the fibrils. {yields} A possible strategy to inhibit toxicity of amyloid fibrils. -- Abstract: A hallmark of Alzheimer's disease is production of amyloid {beta} peptides resulting from aberrant cleavage of the amyloid precursor protein. Amyloid {beta} assembles into fibrils under physiological conditions, through formation of neurotoxic intermediate oligomers. Tachykinin peptides are known to affect amyloid {beta} neurotoxicity in cells. To understand the mechanism of this effect, we studied how tachykinins affect A{beta}(1-40) aggregation in vitro. Fibrils grown in the presence of tachykinins exhibited reduced thioflavin T (ThT) fluorescence, while their morphology, observed in transmission electron microscopy (TEM), did not alter. Cross linking studies revealed that the distribution of low molecular weight species was not affected by tachykinins. Our results suggest that there may be a specific interaction between tachykinins and A{beta}(1-40) that allows them to co-assemble. This effect may explain the reduction of A{beta}(1-40) neurotoxicity in cells treated with tachykinins.

  19. Amyloid Structure and Assembly: Insights from Scanning Transmission Electron Microscopy

    PubMed Central

    Goldsbury, Claire; Baxa, Ulrich; Simon, Martha N.; Steven, Alasdair C.; Engel, Andreas; Wall, Joseph S.; Aebi, Ueli; Müller, Shirley A.

    2010-01-01

    Amyloid fibrils are filamentous protein aggregates implicated in several common diseases like Alzheimer’s disease and type II diabetes. Similar structures are also the molecular principle of the infectious spongiform encephalopathies like Creutzfeldt-Jakob disease in humans, scrapie in sheep, and of the so-called yeast prions, inherited non-chromosomal elements found in yeast and fungi. Scanning transmission electron microscopy (STEM) is often used to delineate the assembly mechanism and structural properties of amyloid aggregates. In this review we consider specifically contributions and limitations of STEM for the investigation of amyloid assembly pathways, fibril polymorphisms and structural models of amyloid fibrils. This type of microscopy provides the only method to directly measure the mass-per-length (MPL) of individual filaments. Made on both in vitro assembled and ex vivo samples, STEM mass measurements have illuminated the hierarchical relationships between amyloid fibrils and revealed that polymorphic fibrils and various globular oligomers can assemble simultaneously from a single polypeptide. The MPLs also impose strong constraints on possible packing schemes, assisting in molecular model building when combined with high-resolution methods like solid-state nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR). PMID:20868754

  20. Amyloid structure and assembly: insights from scanning transmission electron microscopy.

    PubMed

    Goldsbury, Claire; Baxa, Ulrich; Simon, Martha N; Steven, Alasdair C; Engel, Andreas; Wall, Joseph S; Aebi, Ueli; Müller, Shirley A

    2011-01-01

    Amyloid fibrils are filamentous protein aggregates implicated in several common diseases such as Alzheimer's disease and type II diabetes. Similar structures are also the molecular principle of the infectious spongiform encephalopathies such as Creutzfeldt-Jakob disease in humans, scrapie in sheep, and of the so-called yeast prions, inherited non-chromosomal elements found in yeast and fungi. Scanning transmission electron microscopy (STEM) is often used to delineate the assembly mechanism and structural properties of amyloid aggregates. In this review we consider specifically contributions and limitations of STEM for the investigation of amyloid assembly pathways, fibril polymorphisms and structural models of amyloid fibrils. This type of microscopy provides the only method to directly measure the mass-per-length (MPL) of individual filaments. Made on both in vitro assembled and ex vivo samples, STEM mass measurements have illuminated the hierarchical relationships between amyloid fibrils and revealed that polymorphic fibrils and various globular oligomers can assemble simultaneously from a single polypeptide. The MPLs also impose strong constraints on possible packing schemes, assisting in molecular model building when combined with high-resolution methods like solid-state nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR). Copyright © 2010 Elsevier Inc. All rights reserved.

  1. Surface Mediated Self-Assembly of Amyloid Peptides

    NASA Astrophysics Data System (ADS)

    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.

  2. Brazilin inhibits amyloid β-protein fibrillogenesis, remodels amyloid fibrils and reduces amyloid cytotoxicity

    NASA Astrophysics Data System (ADS)

    Du, Wen-Jie; Guo, Jing-Jing; Gao, Ming-Tao; Hu, Sheng-Quan; Dong, Xiao-Yan; Han, Yi-Fan; Liu, Fu-Feng; Jiang, Shaoyi; Sun, Yan

    2015-01-01

    Soluble amyloid β-protein (Aβ) oligomers, the main neurotoxic species, are predominantly formed from monomers through a fibril-catalyzed secondary nucleation. Herein, we virtually screened an in-house library of natural compounds and discovered brazilin as a dual functional compound in both Aβ42 fibrillogenesis inhibition and mature fibril remodeling, leading to significant reduction in Aβ42 cytotoxicity. The potent inhibitory effect of brazilin was proven by an IC50 of 1.5 +/- 0.3 μM, which was smaller than that of (-)-epigallocatechin gallate in Phase III clinical trials and about one order of magnitude smaller than those of curcumin and resveratrol. Most importantly, it was found that brazilin redirected Aβ42 monomers and its mature fibrils into unstructured Aβ aggregates with some β-sheet structures, which could prevent both the primary nucleation and the fibril-catalyzed secondary nucleation. Molecular simulations demonstrated that brazilin inhibited Aβ42 fibrillogenesis by directly binding to Aβ42 species via hydrophobic interactions and hydrogen bonding and remodeled mature fibrils by disrupting the intermolecular salt bridge Asp23-Lys28 via hydrogen bonding. Both experimental and computational studies revealed a different working mechanism of brazilin from that of known inhibitors. These findings indicate that brazilin is of great potential as a neuroprotective and therapeutic agent for Alzheimer's disease.

  3. Proliferation of amyloid-β42 aggregates occurs through a secondary nucleation mechanism

    PubMed Central

    Cohen, Samuel I. A.; Linse, Sara; Luheshi, Leila M.; Hellstrand, Erik; White, Duncan A.; Rajah, Luke; Otzen, Daniel E.; Vendruscolo, Michele; Dobson, Christopher M.; Knowles, Tuomas P. J.

    2013-01-01

    The generation of toxic oligomers during the aggregation of the amyloid-β (Aβ) peptide Aβ42 into amyloid fibrils and plaques has emerged as a central feature of the onset and progression of Alzheimer’s disease, but the molecular pathways that control pathological aggregation have proved challenging to identify. Here, we use a combination of kinetic studies, selective radiolabeling experiments, and cell viability assays to detect directly the rates of formation of both fibrils and oligomers and the resulting cytotoxic effects. Our results show that once a small but critical concentration of amyloid fibrils has accumulated, the toxic oligomeric species are predominantly formed from monomeric peptide molecules through a fibril-catalyzed secondary nucleation reaction, rather than through a classical mechanism of homogeneous primary nucleation. This catalytic mechanism couples together the growth of insoluble amyloid fibrils and the generation of diffusible oligomeric aggregates that are implicated as neurotoxic agents in Alzheimer’s disease. These results reveal that the aggregation of Aβ42 is promoted by a positive feedback loop that originates from the interactions between the monomeric and fibrillar forms of this peptide. Our findings bring together the main molecular species implicated in the Aβ aggregation cascade and suggest that perturbation of the secondary nucleation pathway identified in this study could be an effective strategy to control the proliferation of neurotoxic Aβ42 oligomers. PMID:23703910

  4. [Physico-chemical methods for studing β-amyloid aggregation].

    PubMed

    Radko, S P; Khmeleva, S A; Suprun, E V; Kozin, S A; Bodoev, N V; Makarov, A A; Archakov, A I; Shumyantseva, V V

    2015-01-01

    Alzheimer's disease is the most prevalent neurodegenerative pathology. According to the amyloid cascade hypothesis, a key event of the Alzheimer's disease pathogenesis is a transition of the β-amyloid peptide (Аβ) from the monomeric form to the aggregated state. The mechanism of Аβ aggregation is intensively studied in vitro, by means of synthetic peptides and various physico-chemical methods allowing evaluation of size, molecular structure, and morphology of the formed aggregates. The paper reviews both the well-known and recently introduced physico-chemical methods for analysis of Аβ aggregation, including microscopу, optical and fluorescent methods, method of electron paramagnetic resonance, electrochemical and electrophoretic methods, gel-filtration, and mass spectrometric methods. Merits and drawbacks of the methods are discussed. The unique possibility to simultaneously observe Аβ monomers as well oligomers and large aggregates by means of atomic force microscopy or fluorescence correlation spectroscopy is emphasized. The high detection sensitivity of the latter method, monitoring the aggregation process in Аβ solutions at low peptide concentrations is underlined. Among mass spectrometric methods, the ion mobility mass spectrometry is marked out as a method enabling to obtain information about both the spectrum of Аβ oligomers and their structure. It is pointed out that the use of several methods giving the complementary data about Аβ aggregates is the best experimental approach to studying the process of b-amyloid peptide aggregation in vitro.

  5. Amyloid-like aggregates formation by bovine apo-carbonic anhydrase in various alcohols: A comparative study.

    PubMed

    Es-Haghi, Ali; Ebrahim-Habibi, Azadeh; Sabbaghian, Marjan; Nemat-Gorgani, Mohsen

    2016-11-01

    Peptides and proteins convert from their native states to amyloid fibrillar aggregates in a number of pathological conditions. Characterizing these species could provide useful information on their pathogenicity and the key factors involved in their generation. In this study, we have observed the ability of the model protein apo-bovine carbonic anhydrase (apo-BCA) to form amyloid-like aggregates in the presence of halogenated and non-halogenated alcohols. Far-UV circular dichroism, ThT fluorescence, atomic force microscopy and dynamic light scattering were used to characterize these structures. The concentration required for effective protein aggregation varied between the solvents, with non-halogenated alcohols acting in a wider range. These aggregates show amyloid-like structures as determined by specific techniques used for characterizing amyloid structures. Oligomers were obtained with various size distributions, but fibrillar structures were not observed. Use of halogenated alcohols resulted into smaller hydrodynamic radii, and most stable oligomers were formed in hexafluoropropan-2-ol (HFIP). At optimal concentrations used to generate these structures, the non-halogenated alcohols showed higher hydrophobicity, which may be related to the lower stability of the generated oligomers. These oligomers have the potential to be used as models in the search for effective treatments in proteinopathies.

  6. Ballistic Energy Transport in Oligomers.

    PubMed

    Rubtsova, Natalia I; Qasim, Layla N; Kurnosov, Arkady A; Burin, Alexander L; Rubtsov, Igor V

    2015-09-15

    The development of nanocomposite materials with desired heat management properties, including nanowires, layered semiconductor structures, and self-assembled monolayer (SAM) junctions, attracts broad interest. Such materials often involve polymeric/oligomeric components and can feature high or low thermal conductivity, depending on their design. For example, in SAM junctions made of alkane chains sandwiched between metal layers, the thermal conductivity can be very low, whereas the fibers of ordered polyethylene chains feature high thermal conductivity, exceeding that of many pure metals. The thermal conductivity of nanostructured materials is determined by the energy transport between and within each component of the material, which all need to be understood for optimizing the properties. For example, in the SAM junctions, the energy transport across the metal-chain interface as well as the transport through the chains both determine the overall heat conductivity, however, to separate these contributions is difficult. Recently developed relaxation-assisted two-dimensional infrared (RA 2DIR) spectroscopy is capable of studying energy transport in individual molecules in the time domain. The transport in a molecule is initiated by exciting an IR-active group (a tag); the method records the influence of the excess energy on another mode in the molecule (a reporter). The energy transport time can be measured for different reporters, and the transport speed through the molecule is evaluated. Various molecules were interrogated by RA 2DIR: in molecules without repeating units (disordered), the transport mechanism was expected and found to be diffusive. The transport via an oligomer backbone can potentially be ballistic, as the chain offers delocalized vibrational states. Indeed, the transport regime via three tested types of oligomers, alkanes, polyethyleneglycols, and perfluoroalkanes was found to be ballistic, whereas the transport within the end groups was diffusive

  7. Hologram recording in azobenzene oligomers

    NASA Astrophysics Data System (ADS)

    Ozols, Andris O.; Kampars, Valdis; Reinfelde, Mara; Kokars, Valdis

    2003-08-01

    Elementary hologram (holographic grating) recording and their coherent optical erasure have been experimentally studied in azobenzene oligomer (ABO) layers differing by their chemical composition, matrices and by the connection type of azobenzene chromophores to the matrix (dispersed or covalently bound). The best holographic parameters (7.9% diffraction efficiency and 86 J/cm2 specific recording energy) were achieved in the samples with covalent bonding to the matrix. Vector recording is also possible. Recording is unstable and reversible. The coherent optical erasure studies have shown its efficiency dependencies on the initial diffraction efficiency, erasing beam intensity and grating period which are different for three groups of ABO samples. The conclusion is made that recording is due to the photoinduced alignment of the azobenzene chromophores followed by refractive index changes. These are the first results and further studies are in progress.

  8. Optical Spectra of Silicon Oligomers

    NASA Astrophysics Data System (ADS)

    Kishida, Hideo; Tachibana, Hiroaki; Sakurai, Kouhei; Matsumoto, Mutsuyoshi; Abe, Shuji; Tokura, Yoshinori

    1996-06-01

    Optical absorption spectra have been measured for finite-chain analogs of linear polysilane, silicon oligomers CH3[Si(CH3)2]nCH3, with controlled chain length n(=2 to 16).The intense lowest electronic absorption peak and its higher-lying side bands,which correspond to the one-dimensional exciton series in the infinite chain,shift to higher energy with decrease of the chain length because of the confinement of the excited states.The oscillator strength of the main absorption peak increases with the chain length, while the linewidth of the main peak drastically decreases, especially in the region n=2 to 6.These finite size effects of the electronic (excitonic) absorptionare argued in terms of spatial extension of the excited states, motional narrowing and electron correlation effect.

  9. Optimization of d-Peptides for Aβ Monomer Binding Specificity Enhances Their Potential to Eliminate Toxic Aβ Oligomers.

    PubMed

    Klein, Antonia Nicole; Ziehm, Tamar; van Groen, Thomas; Kadish, Inga; Elfgen, Anne; Tusche, Markus; Thomaier, Maren; Reiss, Kerstin; Brener, Oleksandr; Gremer, Lothar; Kutzsche, Janine; Willbold, Dieter

    2017-09-20

    Amyloid-beta (Aβ) oligomers are thought to be causative for the development and progression of Alzheimer's disease (AD). Starting from the Aβ oligomer eliminating d-enantiomeric peptide D3, we developed and applied a two-step procedure based on peptide microarrays to identify D3 derivatives with increased binding affinity and specificity for monomeric Aβ(1-42) to further enhance the Aβ oligomer elimination efficacy. Out of more than 1000 D3 derivatives, we selected seven novel d-peptides, named ANK1 to ANK7, and characterized them in more detail in vitro. All ANK peptides bound to monomeric Aβ(1-42), eliminated Aβ(1-42) oligomers, inhibited Aβ(1-42) fibril formation, and reduced Aβ(1-42)-induced cytotoxicity more efficiently than D3. Additionally, ANK6 completely inhibited the prion-like propagation of preformed Aβ(1-42) seeds and showed a nonsignificant tendency for improving memory performance of tg-APPSwDI mice after i.p. application for 4 weeks. This supports the hypothesis that stabilization of Aβ monomers and thereby induced elimination of Aβ oligomers is a suitable therapeutic strategy.

  10. Chronic stress exposure following photothrombotic stroke is associated with increased levels of Amyloid beta accumulation and altered oligomerisation at sites of thalamic secondary neurodegeneration in mice.

    PubMed

    Ong, Lin Kooi; Zhao, Zidan; Kluge, Murielle; Walker, Frederick R; Nilsson, Michael

    2017-04-01

    Exposure to severe stress following stroke is recognised to complicate the recovery process. We have identified that stress can exacerbate the severity of post-stroke secondary neurodegeneration in the thalamus. In this study, we investigated whether exposure to stress could influence the accumulation of the neurotoxic protein Amyloid-β. Using an experimental model of focal cortical ischemia in adult mice combined with exposure to chronic restraint stress, we examined changes within the contra- and ipsilateral thalamus at six weeks post-stroke using Western blotting and immunohistochemical approaches. Western blotting analysis indicated that stroke was associated with a significant enhancement of the 25 and 50 kDa oligomers within the ipsilateral hemisphere and the 20 kDa oligomer within the contralateral hemisphere. Stroked animals exposed to stress exhibited an additional increase in multiple forms of Amyloid-beta oligomers. Immunohistochemistry analysis confirmed that stroke was associated with a significant accumulation of Amyloid-beta within the thalami of both hemispheres, an effect that was exacerbated in stroke animals exposed to stress. Given that Amyloid-beta oligomers, most notably the 30-40 and 50 kDa oligomers, are recognised to correlate with accelerated cognitive decline, our results suggest that monitoring stress levels in patients recovering from stroke may merit consideration in the future.

  11. Diaryl Hydrazones as Multifunctional Inhibitors of Amyloid Self-Assembly†

    PubMed Central

    Török, Béla; Sood, Abha; Bag, Seema; Tulsan, Rekha; Ghosh, Sanjukta; Borkin, Dmitry; Kennedy, Arleen R.; Melanson, Michelle; Madden, Richard; Zhou, Weihong; LeVine, Harry; Török, Marianna

    2013-01-01

    The design and application of an effective, new class of multifunctional small molecule inhibitors of amyloid self-assembly are described. Several compounds, based on the diaryl hydrazone scaffold were designed. Forty-four substituted derivatives of this core structure were synthesized using a variety of benzaldehydes and phenylhydrazines and were characterized. The inhibitor candidates were evaluated in multiple assays, including the inhibition of Aβ fibrillogenesis and oligomer formation and the reverse processes, the disassembly of preformed fibrils and oligomers. Since the structure of the hydrazone-based inhibitors mimic the redox features of the antioxidant resveratrol the radical scavenging effect of the compounds was evaluated by colorimetric assays against 2,2-diphenyl-lpicrylhydrazyl (DPPH) and superoxide radicals. The hydrazone scaffold was active in all of the different assays. The structure-activity relationship revealed that the substituents on the aromatic rings had considerable effect on the overall activity of the compounds. The inhibitors showed strong activity in the fibrillogenesis inhibition and disassembly, and even greater potency in the inhibition of oligomer formation and oligomer disassembly. Supporting the quantitative fluorometric and colorimetric assays, size exclusion chromatographic studies indicated that the best compounds practically eliminated or substantially inhibited the formation of soluble, aggregated Aβ species, as well. Atomic Force Microscopy was also applied to monitor the morphology of Aβ deposits. The compounds also possessed the predicted antioxidant properties; approximately 30% of the synthesized compounds showed equal or better radical scavenging effect than resveratrol or ascorbic acid. PMID:23346953

  12. [Twenty-five years of the amyloid hypothesis of alzheimer disease: advances, failures and new perspectives].

    PubMed

    Levin, O S; Vasenina, E E

    2016-01-01

    Amyloid hypothesis of Alzheimer's disease (AD) has been long the primary one. During the 25-year history the concept has been dramatically changed. Accumulation of β-amyloid is associated not only with the disruption of its synthesis (as it seemed after the discovery of genetic mechanisms of some familial cases of AD) but rather with the disruption of its clearance and elimination from the brain tissue via the microcirculatory system. It has been recognized that soluble oligomers of β-amyloid, but not senile plaques that consisted of insoluble conjugates described by A. Alzheimer 100 years ago, play a key pathogenic role in the brain. Interrelation of vascular and degenerative processes is confirmed by common risk factors, clinical, neuroimaging, pathomorphological and experimental data. Insulin-resistance is also one of the links between AD degenerative and vascular processes. Based on the current state of the amyloid hypothesis, perspectives of new multimodal treatment strategies are discussed.

  13. [Twenty-five years of the amyloid hypothesis of alzheimer disease: advances, failures and new perspectives].

    PubMed

    Levin, O S; Vasenina, E E

    Amyloid hypothesis of Alzheimer's disease (AD) has been long the primary one. During the 25-year history the concept has been dramatically changed. Accumulation of β-amyloid is associated not only with the disruption of its synthesis (as it seemed after the discovery of genetic mechanisms of some familial cases of AD) but rather with the disruption of its clearance and elimination from the brain tissue via the microcirculatory system. It has been recognized that soluble oligomers of β-amyloid, but not senile plaques that consisted of insoluble conjugates described by A. Alzheimer 100 years ago, play a key pathogenic role in the brain. Interrelation of vascular and degenerative processes is confirmed by common risk factors, clinical, neuroimaging, pathomorphological and experimental data. Insulin-resistance is also one of the links between AD degenerative and vascular processes. Based on the current state of the amyloid hypothesis, perspectives of new multimodal treatment strategies are discussed.

  14. An amyloid lung

    PubMed Central

    Zundel, W. E.; Prior, A. P.

    1971-01-01

    A 55-year-old housewife died from an illness characterized by progressive respiratory incapacity. Changes were confined to the lungs and consisted of a diffuse infiltration by amyloid. No adequate cause was found for this amyloid, and we suggest that this is a case of primary alveolar septal amyloidosis. Images PMID:5559913

  15. Ultrafast propagation of β-amyloid fibrils in oligomeric cloud

    PubMed Central

    Ogi, Hirotsugu; Fukukshima, Masahiko; Hamada, Hiroki; Noi, Kentaro; Hirao, Masahiko; Yagi, Hisashi; Goto, Yuji

    2014-01-01

    Interaction between monomer peptides and seeds is essential for clarifying the fibrillation mechanism of amyloid β (Aβ) peptides. We monitored the deposition reaction of Aβ1–40 peptides on immobilized seeds grown from Aβ1–42, which caused formation of oligomers in the early stage. The deposition reaction and fibrillation procedure were monitored throughout by novel total-internal-reflection-fluorescence microscopy with a quartz-crystal microbalance (TIRFM-QCM) system. This system allows simultaneous evaluation of the amount of deposited peptides on the surface seeds by QCM and fibril nucleation and elongation by TIRFM. Most fibrils reached other nuclei, forming the fibril network across the nucleus hubs in a short time. We found a fibril-elongation rate two-orders-of-magnitude higher in an oligomeric cloud than reported values, indicating ultrafast transition of oligomers into fibrils. PMID:25376301

  16. Structures for amyloid fibrils.

    PubMed

    Makin, O Sumner; Serpell, Louise C

    2005-12-01

    Alzheimer's disease and Creutzfeldt-Jakob disease are the best-known examples of a group of diseases known as the amyloidoses. They are characterized by the extracellular deposition of toxic, insoluble amyloid fibrils. Knowledge of the structure of these fibrils is essential for understanding the process of pathology of the amyloidoses and for the rational design of drugs to inhibit or reverse amyloid formation. Structural models have been built using information from a wide variety of techniques, including X-ray diffraction, electron microscopy, solid state NMR and EPR. Recent advances have been made in understanding the architecture of the amyloid fibril. Here, we describe and compare postulated structural models for the mature amyloid fibril and discuss how the ordered structure of amyloid contributes to its stability.

  17. Aβ(1-42) aggregates into non-toxic amyloid assemblies in the presence of the natural polyphenol oleuropein aglycon.

    PubMed

    Rigacci, Stefania; Guidotti, Valentina; Bucciantini, Monica; Nichino, Daniela; Relini, Annalisa; Berti, Andrea; Stefani, Massimo

    2011-12-01

    Amyloid aggregation starts with the initial misfolding of peptide/protein precursors, with subsequent structural rearrangement into oligomers and protofibrils; the latter eventually organize into fibrils with shared basic structural features, found deposited in amyloid diseases. Mounting evidence indicates early oligomers as the most toxic amyloid species; accordingly, the search of inhibitors of their growth is considered a promising target to prevent amyloid toxicity. We recently showed that oleuropein aglycon, a polyphenol abundant in the extra virgin olive oil, interferes with the aggregation of amylin (involved in type-2 diabetes), eliminating its cytotoxicity. Here we report that oleuropein aglycon also hinders amyloid aggregation of Aβ(1-42) and its cytotoxicity, suggesting a general effect of such polyphenol. In particular, by using a wide panel of different spectroscopic, immunologic, cell viability and imaging techniques we provide a more detailed description of Aβ(1-42) structural modifications arising in the presence of the inhibitor and the resulting cytotoxicity. We here report that the polyphenol eliminates the appearance of early toxic oligomers favouring the formation of stable harmless protofibrils, structurally different from the typical Aβ(1-42) fibrils. We also show that oleuropein aglycon is maximally effective when is present at the beginning of the aggregation process; furthermore, when added to preformed fibrils, it does not induce the release of toxic oligomers but, rather, neutralizes any residual toxicity possibly arising from the residual presence of traces of soluble oligomers and other toxic aggregates. The possible use of this polyphenol as anti-aggregation molecule is discussed in the light of these data.

  18. The synaptic protein neuroligin-1 interacts with the amyloid β-peptide. Is there a role in Alzheimer's disease?

    PubMed

    Dinamarca, Margarita C; Weinstein, David; Monasterio, Octavio; Inestrosa, Nibaldo C

    2011-09-27

    Amyloid β-peptide (Aβ) is the main component of the amyloid plaques associated with Alzheimer's disease (AD). In the early steps of the disease soluble Aβ oligomers are produced. According to the current "amyloid hypothesis" these oligomers can accumulate over time, leading progressively to the loss of synaptic function and the cognitive failure characteristic of AD. To understand the role of oligomeric Aβ species in AD pathology, it is important to understand the mechanism by which Aβ oligomers are targeted to synaptic junction. We report here the interaction between Aβ with neuroligin-1 (NL-1), a postsynaptic cell-adhesion protein specific for excitatory synapses, which shares a high degree of similarity with acetylcholinesterase, the first synaptic protein described to interact with Aβ. Using intrinsic fluorescence and surface plasmon resonance, we found that Aβ binds to the extracellular domain of NL-1 with a K(d) in the nanomolar range. In the case of NL-2, a postsynaptic cell-adhesion protein specific for inhibitory synapses, just a very weak interaction with Aβ was observed. Aβ polymerization analysis-studied by thioflavin-T assay and electron microscopy-indicated that NL-1 stabilized Aβ aggregates in vitro. Moreover, NL-1 acts as a nucleating factor during the Aβ aggregation process, stimulating the formation of Aβ oligomers. Besides, immunoprecipitation assays confirm that Aβ oligomers interact with NL-1 but not with NL-2. In conclusion, our results show that NL-1 interacts with Aβ increasing the formation of Aβ oligomers, suggesting that this interaction could triggers the targeting of Aβ oligomer to the postsynaptic regions of excitatory synapses.

  19. What does make an amyloid toxic: morphology, structure or interaction with membrane?

    PubMed

    Berthelot, Karine; Cullin, Christophe; Lecomte, Sophie

    2013-01-01

    The toxicity of amyloids is a subject under intense scrutiny. Many studies link this toxicity to the existence of various intermediate structures prior to the fiber formation and/or their specific interaction with membranes. Membranes can also be a catalyst of amyloidogenesis and the composition or the charge of membrane lipids may be of particular importance. Despite intensive research in the field, such intermediates are not yet fully characterized probably because of the lack of adapted methods for their analyses, and the mechanisms of interaction with the membrane are far to be understood. The purpose of this mini-review is to highlight some in vitro characteristics that seem to be convergent to explain the toxicity observed for some amyloids. Based on a comparison between the behavior of a model non-toxic amyloid (the Prion Forming Domain of HET-s) and its toxic mutant (M8), we could establish that short oligomers and/or fibers assembled in antiparallel β-sheets strongly interact with membrane leading to its disruption. Many recent evidences are in favor of the formation of antiparallel toxic oligomers assembled in β-helices able to form pores. We may also propose a new model of amyloid interaction with membranes by a "raft-like" mode of insertion that could explain important destabilization of membranes and thus amyloid toxicity.

  20. Inhibition of beta-amyloid aggregation by fluorescent dye labels

    SciTech Connect

    Amaro, Mariana; Wellbrock, Thorben; Birch, David J. S.; Rolinski, Olaf J.

    2014-02-10

    The fluorescence decay of beta-amyloid's (Aβ) intrinsic fluorophore tyrosine has been used for sensing the oligomer formation of dye-labelled Aβ monomers and the results compared with previously studied oligomerization of the non-labelled Aβ peptides. It has been demonstrated that two different sized, covalently bound probes 7-diethylaminocoumarin-3-carbonyl and Hilyte Fluor 488 (HLF), alter the rate and character of oligomerization to different extents. The ability of HLF to inhibit formation of highly ordered structures containing beta-sheets was also shown. The implications of our findings for using fluorescence methods in amyloidosis research are discussed and the advantages of this auto-fluorescence approach highlighted.

  1. Inhibition of beta-amyloid aggregation by fluorescent dye labels

    NASA Astrophysics Data System (ADS)

    Amaro, Mariana; Wellbrock, Thorben; Birch, David J. S.; Rolinski, Olaf J.

    2014-02-01

    The fluorescence decay of beta-amyloid's (Aβ) intrinsic fluorophore tyrosine has been used for sensing the oligomer formation of dye-labelled Aβ monomers and the results compared with previously studied oligomerization of the non-labelled Aβ peptides. It has been demonstrated that two different sized, covalently bound probes 7-diethylaminocoumarin-3-carbonyl and Hilyte Fluor 488 (HLF), alter the rate and character of oligomerization to different extents. The ability of HLF to inhibit formation of highly ordered structures containing beta-sheets was also shown. The implications of our findings for using fluorescence methods in amyloidosis research are discussed and the advantages of this auto-fluorescence approach highlighted.

  2. A vaccine with Aβ oligomer-specific mimotope attenuates cognitive deficits and brain pathologies in transgenic mice with Alzheimer's disease.

    PubMed

    Wang, Shao-Wei; Liu, Dong-Qun; Zhang, Ling-Xiao; Ji, Mei; Zhang, Yang-Xin; Dong, Quan-Xiu; Liu, Shu-Ying; Xie, Xi-Xiu; Liu, Rui-Tian

    2017-06-07

    β-Amyloid peptide (Aβ) oligomers are initial factors used to induce Alzheimer's disease (AD) development, and Aβ monomers have normal physiological function. The antibodies or vaccines against Aβ monomers have serious problems, such as side effects and low curative effects. Therefore, it is essential to specifically target Aβ oligomers rather than monomers for the treatment of AD. The mimotopes of Aβ oligomers were obtained by panning the phage-displayed random peptide libraries using oligomer-specific antibodies as targets and expressed on the surface of EBY100 Saccharomyces cerevisiae to generate yeast cell base vaccines. One vaccine (AOE1) induced antibodies specifically against Aβ oligomers and was selected for further study. The APP/PS1 mice were subcutaneously immunized with AOE1 eight times. The levels and characteristics of antibodies induced by AOE1 were determined by enzyme-linked immunosorbent assay. The effect of AOE1 on the cognitive deficits of AD mice was tested by novel object recognition (NOR) and Y-maze. Dot blot analysis, Western blot analysis, and immunohistochemistry were applied to measure the effects of AOE1 on Aβ pathologies, neuroinflammation, and microhemorrhages in the brains of AD mice. Eight mimotope candidates of Aβ oligomers were selected and expressed on EBY100 S. cerevisiae. Only AOE1 vaccine containing mimotope L2 induced antibodies that specifically recognized Aβ42 oligomers rather than monomers. AOE1 immunization significantly increased the AD mice's exploration times for the novel object in the NOR test and the choices for new arms in the Y-maze test, and it reduced levels of Aβ oligomers and glial activation in the AD mouse brains. No activation of Aβ-specific T cells and microhemorrhages was observed in their brains following AOE1 vaccination. AOE1 is the first vaccine applying the oligomer-specific mimotope as an immunogen, which could induce antibodies with high specificity to Aβ oligomers. AOE1 immunization

  3. Bis(sulfosuccinimidyl) suberate (BS3) crosslinking analysis of the behavior of amyloid-β peptide in solution and in phospholipid membranes

    PubMed Central

    Pei, Jie; Liu, En-Qi; Zhang, Lin

    2017-01-01

    The structure and state of amyloid-β peptide (Aβ) oligomers often need to be checked by reliable experimental methods. Electrophoresis is a commonly applied measurement method. However, due to the presence of detergents, oligomers are easily broken during electrophoresis, which makes it very hard to accurately assess Aβ aggregate states. In the current study, bis(sulfosuccinimidyl) suberate (BS3) was used to cross-link Aβ1–42 oligomers prior to electrophoresis. When compared to a previously reported Aβ cross-linking agent, glutaraldehyde, it was quite apparent that BS3 is more suitable for detecting intra-membrane Aβ oligomers and extra-membrane Aβ oligomers states. As such, our findings provide an efficient method for analyzing Aβ proteins or other proteins that are easily aggregated in solution and in phospholipid membranes. PMID:28323849

  4. Nanophotonics of protein amyloids

    NASA Astrophysics Data System (ADS)

    Bhattacharya, Mily; Mukhopadhyay, Samrat

    2014-04-01

    Technological breakthroughs in the super-resolution optical imaging techniques have enriched our current understanding of a range of biological systems and biomolecular processes at the nanoscopic spatial resolution. Protein amyloids are an important class of ordered protein assemblies consisting of misfolded proteins that are implicated in a wide range of devastating human diseases. In order to decipher the structural basis of the supramolecular protein assembly in amyloids and their detrimental interactions with the cell membranes, it is important to employ high-resolution optical imaging techniques. Additionally, amyloids could serve as novel biological nanomaterials for a variety of potential applications. In this review, we summarize a few examples of the utility of near-field scanning optical imaging methodologies to obtain a wealth of structural information into the nanoscale amyloid assembly. Although the near-field technologies were developed several decades ago, it is only recently that these methodologies are being applied and adapted for amyloid research to yield novel information pertaining to the exciting nanoscopic world of protein aggregates. We believe that the account on the nanophotonics of amyloids described in this review will be useful for the future studies on the biophysics of amyloids.

  5. Amyloid Fibrils from Hemoglobin

    PubMed Central

    Jayawardena, Nadishka; Kaur, Manmeet; Nair, Smitha; Malmstrom, Jenny; Goldstone, David; Negron, Leonardo; Gerrard, Juliet A.; Domigan, Laura J.

    2017-01-01

    Amyloid fibrils are a class of insoluble protein nanofibers that are formed via the self-assembly of a wide range of peptides and proteins. They are increasingly exploited for a broad range of applications in bionanotechnology, such as biosensing and drug delivery, as nanowires, hydrogels, and thin films. Amyloid fibrils have been prepared from many proteins, but there has been no definitive characterization of amyloid fibrils from hemoglobin to date. Here, nanofiber formation was carried out under denaturing conditions using solutions of apo-hemoglobin extracted from bovine waste blood. A characteristic amyloid fibril morphology was confirmed by transmission electron microscopy (TEM) and atomic force microscopy (AFM), with mean fibril dimensions of approximately 5 nm diameter and up to several microns in length. The thioflavin T assay confirmed the presence of β-sheet structures in apo-hemoglobin fibrils, and X-ray fiber diffraction showed the characteristic amyloid cross-β quaternary structure. Apo-hemoglobin nanofibers demonstrated high stability over a range of temperatures (−20 to 80 °C) and pHs (2–10), and were stable in the presence of organic solvents and trypsin, confirming their potential as nanomaterials with versatile applications. This study conclusively demonstrates the formation of amyloid fibrils from hemoglobin for the first time, and also introduces a cost-effective method for amyloid fibril manufacture using meat industry by-products. PMID:28398221

  6. Amyloid-like fibril elongation follows michaelis-menten kinetics.

    PubMed

    Milto, Katazyna; Botyriute, Akvile; Smirnovas, Vytautas

    2013-01-01

    A number of proteins can aggregate into amyloid-like fibrils. It was noted that fibril elongation has similarities to an enzymatic reaction, where monomers or oligomers would play a role of substrate and nuclei/fibrils would play a role of enzyme. The question is how similar these processes really are. We obtained experimental data on insulin amyloid-like fibril elongation at the conditions where other processes which may impact kinetics of fibril formation are minor and fitted it using Michaelis-Menten equation. The correlation of the fit is very good and repeatable. It speaks in favour of enzyme-like model of fibril elongation. In addition, obtained [Formula: see text] and [Formula: see text] values at different conditions may help in better understanding influence of environmental factors on the process of fibril elongation.

  7. Intracellular Aβ-oligomers and early inflammation in a model of Alzheimer's disease.

    PubMed

    Ferretti, Maria Teresa; Bruno, Martin A; Ducatenzeiler, Adriana; Klein, William L; Cuello, A Claudio

    2012-07-01

    Lifelong use of nonsteroidal anti-inflammatory drugs (NSAIDs) has been shown to diminish the incidence of Alzheimer's disease (AD), suggesting a key role of inflammation in early stages of the pathology. While amyloid plaque-associated inflammation has been extensively studied in human and animal models, little is known about the inflammatory process prior to plaque deposition, i.e., in preclinical stages of AD. In this study we investigated microglial and neuronal inflammatory markers in preplaque transgenic McGill-Thy1-APP mice. We found evidence that prior to plaque deposition classical markers of microglial activation such as major histocompatibility complex class II (MHC-II), inducible nitric oxide synthase (i-NOS), and CD40 are already upregulated in the hippocampus of transgenic mice. Microglial cells from transgenic mice in the preplaque stage displayed intermediately activated morphology and appeared to be recruited toward intracellular amyloid-β peptide (Aβ)-oligomer burdened neurons. The inducible, neuron-specific cyclooxygenase 2 (COX-2) enzyme was found to be upregulated and specifically expressed by neurons in close relationship with Aβ-bearing cells, at this early stage of the AD-like pathology. Our study suggests that neuroinflammation might be one of the earliest pathological responses to intracellular accumulation of Aβ-oligomers. Copyright © 2012 Elsevier Inc. All rights reserved.

  8. Peptide Amyloid Surface Display

    PubMed Central

    2015-01-01

    Homomeric self-assembly of peptides into amyloid fibers is a feature of many diseases. A central role has been suggested for the lateral fiber surface affecting gains of toxic function. To investigate this, a protein scaffold that presents a discrete, parallel β-sheet surface for amyloid subdomains up to eight residues in length has been designed. Scaffolds that present the fiber surface of islet amyloid polypeptide (IAPP) were prepared. The designs show sequence-specific surface effects apparent in that they gain the capacity to attenuate rates of IAPP self-assembly in solution and affect IAPP-induced toxicity in insulin-secreting cells. PMID:25541905

  9. Competitive Mirror Image Phage Display Derived Peptide Modulates Amyloid Beta Aggregation and Toxicity

    PubMed Central

    Rudolph, Stephan; Klein, Antonia Nicole; Tusche, Markus; Schlosser, Christine; Elfgen, Anne; Brener, Oleksandr; Teunissen, Charlotte; Gremer, Lothar; Funke, Susanne Aileen; Kutzsche, Janine; Willbold, Dieter

    2016-01-01

    Alzheimer´s disease is the most prominent type of dementia and currently no causative treatment is available. According to recent studies, oligomeric species of the amyloid beta (Aβ) peptide appear to be the most toxic Aβ assemblies. Aβ monomers, however, may be not toxic per se and may even have a neuroprotective role. Here we describe a competitive mirror image phage display procedure that allowed us to identify preferentially Aβ1–42 monomer binding and thereby stabilizing peptides, which destabilize and thereby eliminate toxic oligomer species. One of the peptides, called Mosd1 (monomer specific d-peptide 1), was characterized in more detail. Mosd1 abolished oligomers from a mixture of Aβ1–42 species, reduced Aβ1–42 toxicity in cell culture, and restored the physiological phenotype in neuronal cells stably transfected with the gene coding for human amyloid precursor protein. PMID:26840229

  10. Early oligomerization stages for the non-amyloid component of α-synuclein amyloid

    NASA Astrophysics Data System (ADS)

    Eugene, Cindie; Laghaei, Rozita; Mousseau, Normand

    2014-10-01

    In recent years, much effort has focused on the early stages of aggregation and the formation of amyloid oligomers. Aggregation processes for these proteins are complex and their non-equilibrium nature makes any experimental study very difficult. Under these conditions, simulations provide a useful alternative for understanding the dynamics of the early stages of oligomerization. Here, we focus on the non-Aβ amyloid component (NAC) of the monomer, dimer, and trimer of α-synuclein, an important 35-residue sequence involved in the aggregation and fibrillation of this protein associated with Parkinson's disease. Using Hamiltonian and temperature replica exchange molecular dynamics simulations combined with the coarse grained Optimized Potential for Efficient peptide structure Prediction potential, we identify the role of the various regions and the secondary structures for the onset of oligomerization. For this sequence, we clearly observe the passage from α-helix to β-sheet, a characteristic transition of amyloid proteins. More precisely, we find that the NAC monomer is highly structured with two α-helical regions, between residues 2-13 and 19-25. As the dimer and trimer form, β-sheet structures between residues 2-14 and 26-34 appear and rapidly structure the system. The resulting conformations are much more structured than similar dimers and trimers of β-amyloid and amylin proteins and yet display a strong polymorphism at these early stages of aggregation. In addition to its inherent experimental interest, comparison with other sequences shows that NAC could be a very useful numerical model for understanding the onset of aggregation.

  11. SDS-PAGE analysis of Aβ oligomers is disserving research into Alzheimer´s disease: appealing for ESI-IM-MS

    PubMed Central

    Pujol-Pina, Rosa; Vilaprinyó-Pascual, Sílvia; Mazzucato, Roberta; Arcella, Annalisa; Vilaseca, Marta; Orozco, Modesto; Carulla, Natàlia

    2015-01-01

    The characterization of amyloid-beta peptide (Aβ) oligomer forms and structures is crucial to the advancement in the field of Alzheimer´s disease (AD). Here we report a critical evaluation of two methods used for this purpose, namely sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), extensively used in the field, and ion mobility coupled to electrospray ionization mass spectrometry (ESI-IM-MS), an emerging technique with great potential for oligomer characterization. To evaluate their performance, we first obtained pure cross-linked Aβ40 and Aβ42 oligomers of well-defined order. Analysis of these samples by SDS-PAGE revealed that SDS affects the oligomerization state of Aβ42 oligomers, thus providing flawed information on their order and distribution. In contrast, ESI-IM-MS provided accurate information, while also reported on the chemical nature and on the structure of the oligomers. Our findings have important implications as they challenge scientific paradigms in the AD field built upon SDS-PAGE characterization of Aβ oligomer samples. PMID:26450154

  12. SDS-PAGE analysis of Aβ oligomers is disserving research into Alzheimer´s disease: appealing for ESI-IM-MS.

    PubMed

    Pujol-Pina, Rosa; Vilaprinyó-Pascual, Sílvia; Mazzucato, Roberta; Arcella, Annalisa; Vilaseca, Marta; Orozco, Modesto; Carulla, Natàlia

    2015-10-09

    The characterization of amyloid-beta peptide (Aβ) oligomer forms and structures is crucial to the advancement in the field of Alzheimer´s disease (AD). Here we report a critical evaluation of two methods used for this purpose, namely sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), extensively used in the field, and ion mobility coupled to electrospray ionization mass spectrometry (ESI-IM-MS), an emerging technique with great potential for oligomer characterization. To evaluate their performance, we first obtained pure cross-linked Aβ40 and Aβ42 oligomers of well-defined order. Analysis of these samples by SDS-PAGE revealed that SDS affects the oligomerization state of Aβ42 oligomers, thus providing flawed information on their order and distribution. In contrast, ESI-IM-MS provided accurate information, while also reported on the chemical nature and on the structure of the oligomers. Our findings have important implications as they challenge scientific paradigms in the AD field built upon SDS-PAGE characterization of Aβ oligomer samples.

  13. SDS-PAGE analysis of Aβ oligomers is disserving research into Alzheimer´s disease: appealing for ESI-IM-MS

    NASA Astrophysics Data System (ADS)

    Pujol-Pina, Rosa; Vilaprinyó-Pascual, Sílvia; Mazzucato, Roberta; Arcella, Annalisa; Vilaseca, Marta; Orozco, Modesto; Carulla, Natàlia

    2015-10-01

    The characterization of amyloid-beta peptide (Aβ) oligomer forms and structures is crucial to the advancement in the field of Alzheimer´s disease (AD). Here we report a critical evaluation of two methods used for this purpose, namely sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), extensively used in the field, and ion mobility coupled to electrospray ionization mass spectrometry (ESI-IM-MS), an emerging technique with great potential for oligomer characterization. To evaluate their performance, we first obtained pure cross-linked Aβ40 and Aβ42 oligomers of well-defined order. Analysis of these samples by SDS-PAGE revealed that SDS affects the oligomerization state of Aβ42 oligomers, thus providing flawed information on their order and distribution. In contrast, ESI-IM-MS provided accurate information, while also reported on the chemical nature and on the structure of the oligomers. Our findings have important implications as they challenge scientific paradigms in the AD field built upon SDS-PAGE characterization of Aβ oligomer samples.

  14. Inhibition of Wnt signaling induces amyloidogenic processing of amyloid precursor protein and the production and aggregation of Amyloid-β (Aβ)42 peptides.

    PubMed

    Tapia-Rojas, Cheril; Burgos, Patricia V; Inestrosa, Nibaldo C

    2016-12-01

    Alzheimer's disease (AD) is the most common neurodegenerative disorder and the most frequent cause of dementia in the aged population. According to the amyloid hypothesis, the amyloid-β (Aβ) peptide plays a key role in the pathogenesis of AD. Aβ is generated from the amyloidogenic processing of amyloid precursor protein and can aggregate to form oligomers, which have been described as a major synaptotoxic agent in neurons. Dysfunction of Wnt signaling has been linked to increased Aβ formation; however, several other studies have argued against this possibility. Herein, we use multiple experimental approaches to confirm that the inhibition of Wnt signaling promoted the amyloidogenic proteolytic processing of amyloid precursor protein. We also demonstrate that inhibiting Wnt signaling increases the production of the Aβ42 peptide, the Aβ42 /Aβ40 ratio, and the levels of Aβ oligomers such as trimers and tetramers. Moreover, we show that activating Wnt signaling reduces the levels of Aβ42 and its aggregates, increases Aβ40 levels, and reduces the Aβ42 /Aβ40 ratio. Finally, we show that the protective effects observed in response to activation of the Wnt pathway rely on β-catenin-dependent transcription, which is demonstrated experimentally via the expression of various 'mutant forms of β-catenin'. Together, our findings indicate that loss of the Wnt signaling pathway may contribute to the pathogenesis of AD.

  15. Aβ40 Oligomers Identified as a Potential Biomarker for the Diagnosis of Alzheimer's Disease

    PubMed Central

    Wang, Xuemei; Magdangal, Erika; Salisbury, Cleo; Peretz, David; Zuckermann, Ronald N.; Connolly, Michael D.; Hansson, Oskar; Minthon, Lennart; Zetterberg, Henrik; Blennow, Kaj; Fedynyshyn, Joseph P.; Allauzen, Sophie

    2010-01-01

    Alzheimer's Disease (AD) is the most prevalent form of dementia worldwide, yet the development of therapeutics has been hampered by the absence of suitable biomarkers to diagnose the disease in its early stages prior to the formation of amyloid plaques and the occurrence of irreversible neuronal damage. Since oligomeric Aβ species have been implicated in the pathophysiology of AD, we reasoned that they may correlate with the onset of disease. As such, we have developed a novel misfolded protein assay for the detection of soluble oligomers composed of Aβ x-40 and x-42 peptide (hereafter Aβ40 and Aβ42) from cerebrospinal fluid (CSF). Preliminary validation of this assay with 36 clinical samples demonstrated the presence of aggregated Aβ40 in the CSF of AD patients. Together with measurements of total Aβ42, diagnostic sensitivity and specificity greater than 95% and 90%, respectively, were achieved. Although larger sample populations will be needed to confirm this diagnostic sensitivity, our studies demonstrate a sensitive method of detecting circulating Aβ40 oligomers from AD CSF and suggest that these oligomers could be a powerful new biomarker for the early detection of AD. PMID:21209907

  16. Proneurogenic Group II mGluR antagonist improves learning and reduces anxiety in Alzheimer Aβ oligomer mouse.

    PubMed

    Kim, S H; Steele, J W; Lee, S W; Clemenson, G D; Carter, T A; Treuner, K; Gadient, R; Wedel, P; Glabe, C; Barlow, C; Ehrlich, M E; Gage, F H; Gandy, S

    2014-11-01

    Proneurogenic compounds have recently shown promise in some mouse models of Alzheimer's pathology. Antagonists at Group II metabotropic glutamate receptors (Group II mGluR: mGlu2, mGlu3) are reported to stimulate neurogenesis. Agonists at those receptors trigger γ-secretase-inhibitor-sensitive biogenesis of Aβ42 peptides from isolated synaptic terminals, which is selectively suppressed by antagonist pretreatment. We have assessed the therapeutic potential of chronic pharmacological inhibition of Group II mGluR in Dutch APP (Alzheimer's amyloid precursor protein E693Q) transgenic mice that accumulate Dutch amyloid-β (Aβ) oligomers but never develop Aβ plaques. BCI-838 is a clinically well-tolerated, orally bioavailable, investigational prodrug that delivers to the brain BCI-632, the active Group II mGluR antagonist metabolite. Dutch Aβ-oligomer-forming APP transgenic mice (APP E693Q) were dosed with BCI-838 for 3 months. Chronic treatment with BCI-838 was associated with reversal of transgene-related amnestic behavior, reduction in anxiety, reduction in levels of brain Aβ monomers and oligomers, and stimulation of hippocampal neurogenesis. Group II mGluR inhibition may offer a unique package of relevant properties as an Alzheimer's disease therapeutic or prophylactic by providing both attenuation of neuropathology and stimulation of repair.

  17. When amyloids become prions.

    PubMed

    Sabate, Raimon

    2014-01-01

    The conformational diseases, linked to protein aggregation into amyloid conformations, range from non-infectious neurodegenerative disorders, such as Alzheimer disease (AD), to highly infectious ones, such as human transmissible spongiform encephalopathies (TSEs). They are commonly known as prion diseases. However, since all amyloids could be considered prions (from those involved in cell-to-cell transmission to those responsible for real neuronal invasion), it is necessary to find an underlying cause of the different capacity to infect that each of the proteins prone to form amyloids has. As proposed here, both the intrinsic cytotoxicity and the number of nuclei of aggregation per cell could be key factors in this transmission capacity of each amyloid.

  18. When amyloids become prions

    PubMed Central

    Sabate, Raimon

    2014-01-01

    The conformational diseases, linked to protein aggregation into amyloid conformations, range from non-infectious neurodegenerative disorders, such as Alzheimer's disease (AD), to highly infectious ones, such as human transmissible spongiform encephalopathies (TSEs). They are commonly known as prion diseases. However, since all amyloids could be considered prions (from those involved in cell-to-cell transmission to those responsible for real neuronal invasion), it is necessary to find an underlying cause of the different capacity to infect that each of the proteins prone to form amyloids has. As proposed here, both the intrinsic cytotoxicity and the number of nuclei of aggregation per cell could be key factors in this transmission capacity of each amyloid. PMID:24831240

  19. Interactions between Aβ oligomers and presynaptic cholinergic signaling: age-dependent effects on attentional capacities

    PubMed Central

    Parikh, Vinay; Bernard, Carcha S.; Naughton, Sean X.; Yegla, Brittney

    2014-01-01

    Substantial evidence suggests that cerebral deposition of the neurotoxic fibrillar form of amyloid precursor protein, β-amyloid (Aβ), plays a critical role in the pathogenesis of Alzheimer's disease (AD). Yet, many aspects of AD pathology including the cognitive symptoms and selective vulnerability of cortically-projecting basal forebrain (BF) cholinergic neurons are not well explained by this hypothesis. Specifically, it is not clear why cognitive decline appears early when the loss of BF cholinergic neurons and plaque deposition are manifested late in AD. Soluble oligomeric forms of Aβ are proposed to appear early in the pathology and to be better predictors of synaptic loss and cognitive deficits. The present study was designed to examine the impact of Aβ oligomers on attentional functions and presynaptic cholinergic transmission in young and aged rats. Chronic intracranial infusions of Aβ oligomers produced subtle decrements in the ability of rats to sustain attentional performance with time on task, irrespective of the age of the animals. However, Aβ oligomers produced robust detrimental effects on performance under conditions of enhanced attentional load in aged animals. In vivo electrochemical recordings show reduced depolarization-evoked cholinergic signals in Aβ-infused aged rats. Moreover, soluble Aβ disrupted the capacity of cholinergic synapses to clear exogenous choline from the extracellular space in both young and aged rats, reflecting impairments in the choline transport process that is critical for acetylcholine (ACh) synthesis and release. Although aging per se reduced the cross-sectional area of BF cholinergic neurons and presynaptic cholinergic proteins in the cortex, attentional performance and ACh release remained unaffected in aged rats infused with the control peptide. Taken together, these data suggest that soluble Aβ may marginally influence attentional functions at young ages primarily by interfering with the choline uptake

  20. Common interruptions in the repeating tripeptide sequence of non-fibrillar collagens: sequence analysis and structural studies on triple-helix peptide models.

    PubMed

    Thiagarajan, Geetha; Li, Yingjie; Mohs, Angela; Strafaci, Christopher; Popiel, Magdalena; Baum, Jean; Brodsky, Barbara

    2008-02-22

    Interruptions in the repeating (Gly-X1-X2)(n) amino acid sequence pattern are found in the triple-helix domains of all non-fibrillar collagens, and perturbations to the triple-helix at such sites are likely to play a role in collagen higher-order structure and function. This study defines the sequence features and structural consequences of the most common interruption, where one residue is missing from the tripeptide pattern, Gly-X1-X2-Gly-AA(1)-Gly-X1-X2, designated G1G interruptions. Residues found within G1G interruptions are predominantly hydrophobic (70%), followed by a significant amount of charged residues (16%), and the Gly-X1-X2 triplets flanking the interruption are atypical. Studies on peptide models indicate the degree of destabilization is much greater when Pro is in the interruption, GP, than when hydrophobic residues (GF, GY) are present, and a rigid Gly-Pro-Hyp tripeptide adjacent to the interruption leads to greater destabilization than a flexible Gly-Ala-Ala sequence. Modeling based on NMR data indicates the Phe residue within a GF interruption is located on the outside of the triple helix. The G1G interruptions resemble a previously studied collagen interruption GPOGAAVMGPO, designated G4G-type, in that both are destabilizing, but allow continuation of rod-like triple helices and maintenance of the single residue stagger throughout the imperfection, with a loss of axial register of the superhelix on both sides. Both kinds of interruptions result in a highly localized perturbation in hydrogen bonding and dihedral angles, but the hydrophobic residue of a G4G interruption packs near the central axis of the superhelix, while the hydrophobic residue of a G1G interruption is located on the triple-helix surface. The different structural consequences of G1G and G4G interruptions in the repeating tripeptide sequence pattern suggest a physical basis for their differential susceptibility to matrix metalloproteinases in type X collagen.

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

    NASA Astrophysics Data System (ADS)

    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.

  2. The cellular prion protein traps Alzheimer's Aβ in an oligomeric form and disassembles amyloid fibers

    PubMed Central

    Younan, Nadine D.; Sarell, Claire J.; Davies, Paul; Brown, David R.; Viles, John H.

    2013-01-01

    There is now strong evidence to show that the presence of the cellular prion protein (PrPC) mediates amyloid-β (Aβ) neurotoxicity in Alzheimer's disease (AD). Here, we probe the molecular details of the interaction between PrPC and Aβ and discover that substoichiometric amounts of PrPC, as little as 1/20, relative to Aβ will strongly inhibit amyloid fibril formation. This effect is specific to the unstructured N-terminal domain of PrPC. Electron microscopy indicates PrPC is able to trap Aβ in an oligomeric form. Unlike fibers, this oligomeric Aβ contains antiparallel β sheet and binds to a oligomer specific conformational antibody. Our NMR studies show that a specific region of PrPC, notably residues 95–113, binds to Aβ oligomers, but only once Aβ misfolds. The ability of PrPC to trap and concentrate Aβ in an oligomeric form and disassemble mature fibers suggests a mechanism by which PrPC might confer Aβ toxicity in AD, as oligomers are thought to be the toxic form of Aβ. Identification of a specific recognition site on PrPC that traps Aβ in an oligomeric form is potentially a therapeutic target for the treatment of Alzheimer's disease.—Younan, N. D., Sarell, C. J., Davies, P., Brown, D. R., Viles, J. H. The cellular prion protein traps Alzheimer's Aβ in an oligomeric form and disassembles amyloid fibers. PMID:23335053

  3. Amyloidβ Peptides in interaction with raft-mime model membranes: a neutron reflectivity insight

    PubMed Central

    Rondelli, Valeria; Brocca, Paola; Motta, Simona; Messa, Massimo; Colombo, Laura; Salmona, Mario; Fragneto, Giovanna; Cantù, Laura; Del Favero, Elena

    2016-01-01

    The role of first-stage β–amyloid aggregation in the development of the Alzheimer disease, is widely accepted but still unclear. Intimate interaction with the cell membrane is invoked. We designed Neutron Reflectometry experiments to reveal the existence and extent of the interaction between β–amyloid (Aβ) peptides and a lone customized biomimetic membrane, and their dependence on the aggregation state of the peptide. The membrane, asymmetrically containing phospholipids, GM1 and cholesterol in biosimilar proportion, is a model for a raft, a putative site for amyloid-cell membrane interaction. We found that the structured-oligomer of Aβ(1-42), its most acknowledged membrane-active state, is embedded as such into the external leaflet of the membrane. Conversely, the Aβ(1-42) unstructured early-oligomers deeply penetrate the membrane, likely mimicking the interaction at neuronal cell surfaces, when the Aβ(1-42) is cleaved from APP protein and the membrane constitutes a template for its further structural evolution. Moreover, the smaller Aβ(1-6) fragment, the N-terminal portion of Aβ, was also used. Aβ N-terminal is usually considered as involved in oligomer stabilization but not in the peptide-membrane interaction. Instead, it was seen to remove lipids from the bilayer, thus suggesting its role, once in the whole peptide, in membrane leakage, favouring peptide recruitment. PMID:26880066

  4. Guanidine hydrochloride denaturation of dopamine-induced α-synuclein oligomers: a small-angle X-ray scattering study.

    PubMed

    Pham, Chi L L; Kirby, Nigel; Wood, Kathleen; Ryan, Timothy; Roberts, Blaine; Sokolova, Anna; Barnham, Kevin J; Masters, Colin L; Knott, Robert B; Cappai, Roberto; Curtain, Cyril C; Rekas, Agata

    2014-01-01

    Alpha-synuclein (α-syn) forms the amyloid-containing Lewy bodies found in the brain in Parkinson's disease. The neurotransmitter dopamine (DA) reacts with α-syn to form SDS-resistant soluble, non-amyloid, and melanin-containing oligomers. Their toxicity is debated, as is the nature of their structure and their relation to amyloid-forming conformers of α-syn. The small-angle X-ray scattering technique in combination with modeling by the ensemble optimization method showed that the un-reacted native protein populated three broad classes of conformer, while reaction with DA gave a restricted ensemble range suggesting that the rigid melanin molecule played an important part in their structure. We found that 6 M guanidine hydrochloride did not dissociate α-syn DA-reacted dimers and trimers, suggesting covalent linkages. The pathological significance of covalent association is that if they are non-toxic, the oligomers would act as a sink for toxic excess DA and α-syn; if toxic, their stability could enhance their toxicity. We argue it is essential, therefore, to resolve the question of whether they are toxic or not.

  5. Effect of Synthetic Aβ Peptide Oligomers and Fluorinated Solvents on Kv1.3 Channel Properties and Membrane Conductance

    PubMed Central

    Lioudyno, Maria I.; Broccio, Matteo; Sokolov, Yuri; Rasool, Suhail; Wu, Jessica; Alkire, Michael T.; Liu, Virginia; Kozak, J. Ashot; Dennison, Philip R.; Glabe, Charles G.; Lösche, Mathias; Hall, James E.

    2012-01-01

    The impact of synthetic amyloid β (1–42) (Aβ1–42) oligomers on biophysical properties of voltage-gated potassium channels Kv 1.3 and lipid bilayer membranes (BLMs) was quantified for protocols using hexafluoroisopropanol (HFIP) or sodium hydroxide (NaOH) as solvents prior to initiating the oligomer formation. Regardless of the solvent used Aβ1–42 samples contained oligomers that reacted with the conformation-specific antibodies A11 and OC and had similar size distributions as determined by dynamic light scattering. Patch-clamp recordings of the potassium currents showed that synthetic Aβ1–42 oligomers accelerate the activation and inactivation kinetics of Kv 1.3 current with no significant effect on current amplitude. In contrast to oligomeric samples, freshly prepared, presumably monomeric, Aβ1–42 solutions had no effect on Kv 1.3 channel properties. Aβ1–42 oligomers had no effect on the steady-state current (at −80 mV) recorded from Kv 1.3-expressing cells but increased the conductance of artificial BLMs in a dose-dependent fashion. Formation of amyloid channels, however, was not observed due to conditions of the experiments. To exclude the effects of HFIP (used to dissolve lyophilized Aβ1–42 peptide), and trifluoroacetic acid (TFA) (used during Aβ1–42 synthesis), we determined concentrations of these fluorinated compounds in the stock Aβ1–42 solutions by 19F NMR. After extensive evaporation, the concentration of HFIP in the 100× stock Aβ1–42 solutions was ∼1.7 μM. The concentration of residual TFA in the 70× stock Aβ1–42 solutions was ∼20 μM. Even at the stock concentrations neither HFIP nor TFA alone had any effect on potassium currents or BLMs. The Aβ1–42 oligomers prepared with HFIP as solvent, however, were more potent in the electrophysiological tests, suggesting that fluorinated compounds, such as HFIP or structurally-related inhalational anesthetics, may affect Aβ1–42 aggregation and potentially enhance

  6. Alpha-Synuclein Oligomers Interact with Metal Ions to Induce Oxidative Stress and Neuronal Death in Parkinson's Disease

    PubMed Central

    Deas, Emma; Cremades, Nunilo; Angelova, Plamena R.; Ludtmann, Marthe H.R.; Yao, Zhi; Chen, Serene; Horrocks, Mathew H.; Banushi, Blerida; Little, Daniel; Devine, Michael J.; Gissen, Paul; Klenerman, David; Dobson, Christopher M.; Wood, Nicholas W.

    2016-01-01

    Abstract Aims: Protein aggregation and oxidative stress are both key pathogenic processes in Parkinson's disease, although the mechanism by which misfolded proteins induce oxidative stress and neuronal death remains unknown. In this study, we describe how aggregation of alpha-synuclein (α-S) from its monomeric form to its soluble oligomeric state results in aberrant free radical production and neuronal toxicity. Results: We first demonstrate excessive free radical production in a human induced pluripotent stem-derived α-S triplication model at basal levels and on application of picomolar doses of β-sheet-rich α-S oligomers. We probed the effects of different structural species of α-S in wild-type rat neuronal cultures and show that both oligomeric and fibrillar forms of α-S are capable of generating free radical production, but that only the oligomeric form results in reduction of endogenous glutathione and subsequent neuronal toxicity. We dissected the mechanism of oligomer-induced free radical production and found that it was interestingly independent of several known cellular enzymatic sources. Innovation: The oligomer-induced reactive oxygen species (ROS) production was entirely dependent on the presence of free metal ions as addition of metal chelators was able to block oligomer-induced ROS production and prevent oligomer-induced neuronal death. Conclusion: Our findings further support the causative role of soluble amyloid oligomers in triggering neurodegeneration and shed light into the mechanisms by which these species cause neuronal damage, which, we show here, can be amenable to modulation through the use of metal chelation. Antioxid. Redox Signal. 24, 376–391. PMID:26564470

  7. Early and progressive deficit of neuronal activity patterns in a model of local amyloid pathology in mouse prefrontal cortex.

    PubMed

    Koukouli, Fani; Rooy, Marie; Maskos, Uwe

    2016-12-20

    Alzheimer's Disease (AD) is the most common form of dementia. The condition predominantly affects the cerebral cortex and hippocampus and is characterized by the spread of amyloid plaques and neurofibrillary tangles (NFTs). But soluble amyloid-β (Aβ) oligomers have also been identified to accumulate in the brains of AD patients and correlate with cognitive dysfunction more than the extent of plaque deposition. Here, we developed an adeno-associated viral vector expressing the human mutated amyloid precursor protein (AAV-hAPP). Intracranial injection of the AAV into the prefrontal cortex (PFC) allowed the induction of AD-like deficits in adult mice, thereby modelling human pathology. AAV-hAPP expression caused accumulation of Aβ oligomers, microglial activation, astrocytosis and the gradual formation of amyloid plaques and NFTs. In vivo two-photon imaging revealed an increase in neuronal activity, a dysfunction characteristic of the pathology, already during the accumulation of soluble oligomers. Importantly, we found that Aβ disrupts the synchronous spontaneous activity of neurons in PFC that, as in humans, is characterized by ultraslow fluctuation patterns. Our work allowed us to track brain activity changes during disease progression and provides new insight into the early deficits of synchronous ongoing brain activity, the "default network", in the presence of Aβ peptide.

  8. Early and progressive deficit of neuronal activity patterns in a model of local amyloid pathology in mouse prefrontal cortex

    PubMed Central

    Koukouli, Fani; Rooy, Marie; Maskos, Uwe

    2016-01-01

    Alzheimer's Disease (AD) is the most common form of dementia. The condition predominantly affects the cerebral cortex and hippocampus and is characterized by the spread of amyloid plaques and neurofibrillary tangles (NFTs). But soluble amyloid-β (Aβ) oligomers have also been identified to accumulate in the brains of AD patients and correlate with cognitive dysfunction more than the extent of plaque deposition. Here, we developed an adeno-associated viral vector expressing the human mutated amyloid precursor protein (AAV-hAPP). Intracranial injection of the AAV into the prefrontal cortex (PFC) allowed the induction of AD-like deficits in adult mice, thereby modelling human pathology. AAV-hAPP expression caused accumulation of Aβ oligomers, microglial activation, astrocytosis and the gradual formation of amyloid plaques and NFTs. In vivo two-photon imaging revealed an increase in neuronal activity, a dysfunction characteristic of the pathology, already during the accumulation of soluble oligomers. Importantly, we found that Aβ disrupts the synchronous spontaneous activity of neurons in PFC that, as in humans, is characterized by ultraslow fluctuation patterns. Our work allowed us to track brain activity changes during disease progression and provides new insight into the early deficits of synchronous ongoing brain activity, the “default network”, in the presence of Aβ peptide. PMID:27999185

  9. Structural differences between Abeta(1-40) intermediate oligomers and fibrils elucidated by proteolytic fragmentation and hydrogen/deuterium exchange.

    PubMed

    Zhang, Aming; Qi, Wei; Good, Theresa A; Fernandez, Erik J

    2009-02-01

    The aggregation of amyloid-beta protein (Abeta) in vivo is a critical pathological event in Alzheimer's disease. Although more and more evidence shows that the intermediate oligomers are the primary neurotoxic species in Alzheimer's disease, the particular structural features responsible for the toxicity of these intermediates are poorly understood. We measured the peptide level solvent accessibility of multiple Abeta(1-40) aggregated states using hydrogen exchange detected by mass spectrometry. A gradual reduction in solvent accessibility, spreading from the C-terminal region to the N-terminal region was observed with ever more aggregated states of Abeta peptide. The observed hydrogen exchange protection begins with reporter peptides 20-34 and 35-40 in low molecular weight oligomers found in fresh samples and culminates with increasing solvent protection of reporter peptide 1-16 in long time aged fibrillar species. The more solvent exposed structure of intermediate oligomers in the N-termini relative to well-developed fibrils provides a novel explanation for the structure-dependent neurotoxicity of soluble oligomers reported previously.

  10. Detection of Mutant Huntingtin Aggregation Conformers and Modulation of SDS-Soluble Fibrillar Oligomers by Small Molecules

    PubMed Central

    Sontag, Emily Mitchell; Lotz, Gregor P.; Yang, Guocheng; Sontag, Christopher J.; Cummings, Brian J.; Glabe, Charles G.; Muchowski, Paul J.; Thompson, Leslie Michels

    2012-01-01

    The Huntington’s disease (HD) mutation leads to a complex process of Huntingtin (Htt) aggregation into multimeric species that eventually form visible inclusions in cytoplasm, nuclei and neuronal processes. One hypothesis is that smaller, soluble forms of amyloid proteins confer toxic effects and contribute to early cell dysfunction. However, analysis of mutant Htt aggregation intermediates to identify conformers that may represent toxic forms of the protein and represent potential drug targets remains difficult. We performed a detailed analysis of aggregation conformers in multiple in vitro, cell and ex vivo models of HD. Conformation-specific antibodies were used to identify and characterize aggregation species, allowing assessment of multiple conformers present during the aggregation process. Using a series of assays together with these antibodies, several forms could be identified. Fibrillar oligomers, defined as having a β-sheet rich conformation, are observed in vitro using recombinant protein and in protein extracts from cells in culture or mouse brain and shown to be globular, soluble and non-sedimentable structures. Compounds previously described to modulate visible inclusion body formation and reduce toxicity in HD models were also tested and consistently found to alter the formation of fibrillar oligomers. Interestingly, these compounds did not alter the rate of visible inclusion formation, indicating that fibrillar oligomers are not necessarily the rate limiting step of inclusion body formation. Taken together, we provide insights into the structure and formation of mutant Htt fibrillar oligomers that are modulated by small molecules with protective potential in HD models. PMID:24086178

  11. Structural Characterization of Monomers and Oligomers of D-Amino Acid-Containing Peptides Using T-Wave Ion Mobility Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Pang, Xueqin; Jia, Chenxi; Chen, Zhengwei; Li, Lingjun

    2017-01-01

    The D-residues are crucial to biological function of D-amino acid containing peptides (DAACPs). Previous ion mobility mass spectrometry (IM-MS) studies revealing oligomerization patterns of amyloid cascade demonstrated conversion from native soluble unstructured assembly to fibril ß-sheet oligomers, which has been implicated in amyloid diseases, such as Alzheimer's disease and type 2 diabetes. Although neuropeptides are typically present at very low concentrations in circulation, their local concentrations could be much higher in large dense core vesicles, forming dimers or oligomers. We studied the oligomerization of protonated and metal-adducted achatin I and dermorphin peptide isomers with IM-MS. Our results suggested that dimerization, oligomerization, and metal adduction augment the structural differences between D/L peptide isomers compared to protonated monomers. Dimers and oligomers enhanced the structural differences between D/L peptide isomers in both aqueous and organic solvent system. Furthermore, some oligomer forms were only observed for either D- or L-isomers, indicating the importance of chiral center in oligomerization process. The oligomerization patterns of D/L isomers appear to be similar. Potassium adducts were detected to enlarge the structural differences between D/L isomers.

  12. Lactic acid oligomers (OLAs) as prodrug moieties.

    PubMed

    Kruse, J; Lachmann, B; Lauer, R; Eppacher, S; Noe, C R

    2013-02-01

    In this paper we propose the use of lactic acid oligomers (OLAs) as prodrug moieties. Two synthetic approaches are presented, on the one hand a non selective oligomerisation of lactic acid and on the other hand a block synthesis to tetramers of lactic acid. Dimers of lactic acid were investigated with respect to their plasma stability and their adsorption to albumine. Ibuprofen was chosen as the first drug for OLAylation. The ester 19 of LA(1)-ibuprofen was evaluated with respect to the degradation to human plasma and the adsorption to albumine. All results indicate that lactic acid oligomers are promising prodrug moieties.

  13. Bacterial Chaperones CsgE and CsgC Differentially Modulate Human α-Synuclein Amyloid Formation via Transient Contacts

    PubMed Central

    Evans, Margery L.; Jain, Neha; Götheson, Anna; Åden, Jörgen; Chapman, Matthew R.; Almqvist, Fredrik; Wittung-Stafshede, Pernilla

    2015-01-01

    Amyloid formation is historically associated with cytotoxicity, but many organisms produce functional amyloid fibers (e.g., curli) as a normal part of cell biology. Two E. coli genes in the curli operon encode the chaperone-like proteins CsgC and CsgE that both can reduce in vitro amyloid formation by CsgA. CsgC was also found to arrest amyloid formation of the human amyloidogenic protein α-synuclein, which is involved in Parkinson’s disease. Here, we report that the inhibitory effects of CsgC arise due to transient interactions that promote the formation of spherical α-synuclein oligomers. We find that CsgE also modulates α-synuclein amyloid formation through transient contacts but, in contrast to CsgC, CsgE accelerates α-synuclein amyloid formation. Our results demonstrate the significance of transient protein interactions in amyloid regulation and emphasize that the same protein may inhibit one type of amyloid while accelerating another. PMID:26465894

  14. [Cerebral amyloid angiopathy].

    PubMed

    Sakai, Kenji; Yamada, Masahito

    2014-07-01

    Cerebral amyloid angiopathy (CAA) is a disorder characterized by the accumulation of amyloid proteins in the small and medium-sized blood vessels of the leptomeninges and central nervous system. Amyloid β protein (Aβ), immunoglobulin light chains, cystatin C, prion protein (PrP), ABri/ADan, transthyretin, and gelsoline, are all associated with CAA. While most CAA patients demonstrated sporadic Aβ-type amyloid deposition, a small number of patients present with familial forms, e.g. Dutch-type hereditary cerebral hemorrhage with amyloidosis (HCHWA-D), Icelandic-type HCHWA (HCHWA-I), familial British dementia (FBD), familial Danish dementia (FDD), and PrP-CAA. Deposited amyloid proteins damage smooth muscle cells in blood vessel walls leading to pathological appearances calling 'double-barreled' changes, fibrinoid necrosis, and microaneurysms. These structural abnormalities result in microinfarcts and hemorrhages in the central nervous system. Recurrent hemorrhage is a common clinical manifestation in patients with CAA; however, small multiple infarctions, progressive dementia, transient neurological symptoms, and CAA-related inflammation can also occur. The pathomechanisms of CAA remain unknown. Although improvements in imaging techniques have allowed us to identify patients with CAA more readily, pathological examination is still essential for a definite diagnosis. There have been no curative treatments for CAA so far.

  15. Neuroprotective and nootropic drug noopept rescues α-synuclein amyloid cytotoxicity.

    PubMed

    Jia, Xueen; Gharibyan, Anna L; Öhman, Anders; Liu, Yonggang; Olofsson, Anders; Morozova-Roche, Ludmilla A

    2011-12-16

    Parkinson's disease is a common neurodegenerative disorder characterized by α-synuclein (α-Syn)-containing Lewy body formation and selective loss of dopaminergic neurons in the substantia nigra. We have demonstrated the modulating effect of noopept, a novel proline-containing dipeptide drug with nootropic and neuroprotective properties, on α-Syn oligomerization and fibrillation by using thioflavin T fluorescence, far-UV CD, and atomic force microscopy techniques. Noopept does not bind to a sterically specific site in the α-Syn molecule as revealed by heteronuclear two-dimensional NMR analysis, but due to hydrophobic interactions with toxic amyloid oligomers, it prompts their rapid sequestration into larger fibrillar amyloid aggregates. Consequently, this process rescues the cytotoxic effect of amyloid oligomers on neuroblastoma SH-SY5Y cells as demonstrated by using cell viability assays and fluorescent staining of apoptotic and necrotic cells and by assessing the level of intracellular oxidative stress. The mitigating effect of noopept against amyloid oligomeric cytotoxicity may offer additional benefits to the already well-established therapeutic functions of this new pharmaceutical.

  16. Amyloid beta-induced nerve growth factor dysmetabolism in Alzheimer disease.

    PubMed

    Bruno, Martin A; Leon, Wanda C; Fragoso, Gabriela; Mushynski, Walter E; Almazan, Guillermina; Cuello, A Claudio

    2009-08-01

    We previously reported that the precursor form of nerve growth factor (pro-NGF) and not mature NGF is liberated in the CNS in an activity-dependent manner, and that its maturation and degradation occur in the extracellular space by the coordinated action of proteases.Here, we present evidence of diminished conversion of pro-NGF to its mature form and of greater NGF degradation in Alzheimer disease (AD) brain samples compared with controls. These alterations of the NGF metabolic pathway likely resulted in the increased pro-NGF levels. The pro-NGF was largely in a peroxynitrited form in the AD samples. Intrahippocampal injection of amyloid-beta oligomers provoked similar upregulation of pro-NGF in naive rats that was accompanied by evidence of microglial activation (CD40), increased levels of inducible nitric oxide synthase, and increased activity of the NGF-degrading enzyme matrix metalloproteinase 9. The elevated inducible nitric oxide synthase provoked the generation of biologically inactive, peroxynitrite-modified pro-NGF in amyloid-beta oligomer-injected rats. These parameters were corrected by minocycline treatment. Minocycline also diminished altered matrix metalloproteinase 9, inducible nitric oxide synthase, and microglial activation (CD40); improved cognitive behavior; and normalized pro-NGF levels in a transgenic mouse AD model. The effects of amyloid-beta amyloid CNS burden on NGF metabolism may explain the paradoxical upregulation of pro-NGF in AD accompanied by atrophy of forebrain cholinergic neurons.

  17. Glycines from the APP GXXXG/GXXXA Transmembrane Motifs Promote Formation of Pathogenic Aβ Oligomers in Cells

    PubMed Central

    Decock, Marie; Stanga, Serena; Octave, Jean-Noël; Dewachter, Ilse; Smith, Steven O.; Constantinescu, Stefan N.; Kienlen-Campard, Pascal

    2016-01-01

    Alzheimer’s disease (AD) is the most common neurodegenerative disorder characterized by progressive cognitive decline leading to dementia. The amyloid precursor protein (APP) is a ubiquitous type I transmembrane (TM) protein sequentially processed to generate the β-amyloid peptide (Aβ), the major constituent of senile plaques that are typical AD lesions. There is a growing body of evidence that soluble Aβ oligomers correlate with clinical symptoms associated with the disease. The Aβ sequence begins in the extracellular juxtamembrane region of APP and includes roughly half of the TM domain. This region contains GXXXG and GXXXA motifs, which are critical for both TM protein interactions and fibrillogenic properties of peptides derived from TM α-helices. Glycine-to-leucine mutations of these motifs were previously shown to affect APP processing and Aβ production in cells. However, the detailed contribution of these motifs to APP dimerization, their relation to processing, and the conformational changes they can induce within Aβ species remains undefined. Here, we describe highly resistant Aβ42 oligomers that are produced in cellular membrane compartments. They are formed in cells by processing of the APP amyloidogenic C-terminal fragment (C99), or by direct expression of a peptide corresponding to Aβ42, but not to Aβ40. By a point-mutation approach, we demonstrate that glycine-to-leucine mutations in the G29XXXG33 and G38XXXA42 motifs dramatically affect the Aβ oligomerization process. G33 and G38 in these motifs are specifically involved in Aβ oligomerization; the G33L mutation strongly promotes oligomerization, while G38L blocks it with a dominant effect on G33 residue modification. Finally, we report that the secreted Aβ42 oligomers display pathological properties consistent with their suggested role in AD, but do not induce toxicity in survival assays with neuronal cells. Exposure of neurons to these Aβ42 oligomers dramatically affects neuronal

  18. Amyloid Beta Mediates Memory Formation

    ERIC Educational Resources Information Center

    Garcia-Osta, Ana; Alberini, Cristina M.

    2009-01-01

    The amyloid precursor protein (APP) undergoes sequential cleavages to generate various polypeptides, including the amyloid [beta] (1-42) peptide (A[beta][1-42]), which is believed to play a major role in amyloid plaque formation in Alzheimer's disease (AD). Here we provide evidence that, in contrast with its pathological role when accumulated,…

  19. Amyloid Beta Mediates Memory Formation

    ERIC Educational Resources Information Center

    Garcia-Osta, Ana; Alberini, Cristina M.

    2009-01-01

    The amyloid precursor protein (APP) undergoes sequential cleavages to generate various polypeptides, including the amyloid [beta] (1-42) peptide (A[beta][1-42]), which is believed to play a major role in amyloid plaque formation in Alzheimer's disease (AD). Here we provide evidence that, in contrast with its pathological role when accumulated,…

  20. Characterization of amyloid in equine recurrent uveitis as AA amyloid.

    PubMed

    Ostevik, L; de Souza, G A; Wien, T N; Gunnes, G; Sørby, R

    2014-01-01

    Two horses with chronic uveitis and histological lesions consistent with equine recurrent uveitis (ERU) were examined. Microscopical findings in the ciliary body included deposits of amyloid lining the non-pigmented epithelium, intracytoplasmic, rod-shaped, eosinophilic inclusions and intraepithelial infiltration of T lymphocytes. Ultrastructural examination of the ciliary body of one horse confirmed the presence of abundant extracellular deposits of non-branching fibrils (9-11 nm in diameter) consistent with amyloid. Immunohistochemistry revealed strong positive labelling for AA amyloid and mass spectrometry showed the amyloid to consist primarily of serum amyloid A1 in both cases. The findings suggest that localized, intraocular AA amyloidosis may occur in horses with ERU.

  1. Amyloid Fibril Solubility.

    PubMed

    Rizzi, L G; Auer, S

    2015-11-19

    It is well established that amyloid fibril solubility is protein specific, but how solubility depends on the interactions between the fibril building blocks is not clear. Here we use a simple protein model and perform Monte Carlo simulations to directly measure the solubility of amyloid fibrils as a function of the interaction between the fibril building blocks. Our simulations confirms that the fibril solubility depends on the fibril thickness and that the relationship between the interactions and the solubility can be described by a simple analytical formula. The results presented in this study reveal general rules how side-chain-side-chain interactions, backbone hydrogen bonding, and temperature affect amyloid fibril solubility, which might prove to be a powerful tool to design protein fibrils with desired solubility and aggregation properties in general.

  2. Epigallocatechin Gallate Remodels Overexpressed Functional Amyloids in Pseudomonas aeruginosa and Increases Biofilm Susceptibility to Antibiotic Treatment.

    PubMed

    Stenvang, Marcel; Dueholm, Morten S; Vad, Brian S; Seviour, Thomas; Zeng, Guanghong; Geifman-Shochat, Susana; Søndergaard, Mads T; Christiansen, Gunna; Meyer, Rikke Louise; Kjelleberg, Staffan; Nielsen, Per Halkjær; Otzen, Daniel E

    2016-12-16

    Epigallocatechin-3-gallate (EGCG) is the major polyphenol in green tea. It has antimicrobial properties and disrupts the ordered structure of amyloid fibrils involved in human disease. The antimicrobial effect of EGCG against the opportunistic pathogen Pseudomonas aeruginosa has been shown to involve disruption of quorum sensing (QS). Functional amyloid fibrils in P. aeruginosa (Fap) are able to bind and retain quorum-sensing molecules, suggesting that EGCG interferes with QS through structural remodeling of amyloid fibrils. Here we show that EGCG inhibits the ability of Fap to form fibrils; instead, EGCG stabilizes protein oligomers. Existing fibrils are remodeled by EGCG into non-amyloid aggregates. This fibril remodeling increases the binding of pyocyanin, demonstrating a mechanism by which EGCG can affect the QS function of functional amyloid. EGCG reduced the amyloid-specific fluorescent thioflavin T signal in P. aeruginosa biofilms at concentrations known to exert an antimicrobial effect. Nanoindentation studies showed that EGCG reduced the stiffness of biofilm containing Fap fibrils but not in biofilm with little Fap. In a combination treatment with EGCG and tobramycin, EGCG had a moderate effect on the minimum bactericidal eradication concentration against wild-type P. aeruginosa biofilms, whereas EGCG had a more pronounced effect when Fap was overexpressed. Our results provide a direct molecular explanation for the ability of EGCG to disrupt P. aeruginosa QS and modify its biofilm and strengthens the case for EGCG as a candidate in multidrug treatment of persistent biofilm infections.

  3. The formation, function and regulation of amyloids: insights from structural biology.

    PubMed

    Landreh, M; Sawaya, M R; Hipp, M S; Eisenberg, D S; Wüthrich, K; Hartl, F U

    2016-08-01

    Amyloid diseases are characterized by the accumulation of insoluble, β-strand-rich aggregates. The underlying structural conversions are closely associated with cellular toxicity, but can also drive the formation of functional protein assemblies. In recent years, studies in the field of structural studies have revealed astonishing insights into the origins, mechanisms and implications of amyloid formation. Notably, high-resolution crystal structures of peptides in amyloid-like fibrils and prefibrillar oligomers have become available despite their challenging chemical nature. Nuclear magnetic resonance spectroscopy has revealed that dynamic local polymorphisms in the benign form of the prion protein affect the transformation into amyloid fibrils and the transmissibility of prion diseases. Studies of the structures and interactions of chaperone proteins help us to understand how the cellular proteostasis network is able to recognize different stages of aberrant protein folding and prevent aggregation. In this review, we will focus on recent developments that connect the different aspects of amyloid biology and discuss how understanding the process of amyloid formation and the associated defence mechanisms can reveal targets for pharmacological intervention that may become the first steps towards clinically viable treatment strategies. © 2016 The Association for the Publication of the Journal of Internal Medicine.

  4. Opposing Effects of Cucurbit[7]uril and 1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose on Amyloid β25–35 Assembly

    PubMed Central

    de Almeida, Natália E. C.; Do, Thanh D.; Tro, Michael; LaPointe, Nichole E.; Feinstein, Stuart C.; Shea, Joan-Emma; Bowers, Michael T.

    2015-01-01

    Alzheimer’s disease (AD) is a neurodegenerative disease characterized by extracellular deposits of amyloid β-protein (Aβ) in the brain. The conversion of soluble monomers to amyloid Aβ fibrils is a complicated process and involves several transient oligomeric species, which are widely believed to be highly toxic and play a crucial role in the etiology of AD. The development of inhibitors to prevent formation of small and mid-sized oligomers is a promising strategy for AD treatment. In this work, we employ ion mobility spectrometry (IMS), transmission electron microscopy (TEM) and molecular dynamics (MD) simulations to elucidate the structural modulation promoted by two potential inhibitors of Aβ oligomerization, cucurbit[7]uril (CB[7]) and 1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose (PGG), on early oligomer and fibril formation of the Aβ25–35 fragment. One and two CB[7] molecules bind to Aβ25–35 monomers and dimers, respectively, and suppress aggregation by remodeling early oligomer structures and inhibiting the formation of higher-order oligomers. On the other hand, non-selective binding was observed between PGG and Aβ25–35. The interactions between PGG and Aβ25–35, surprisingly, enhanced the formation of Aβ aggregates by promoting extended Aβ25–35 conformations in both homo- and hetero-oligomers. When both ligands were present, the inhibitory effect of CB[7] overrode the stimulatory effect of PGG on Aβ25–35 aggregation, suppressing the formation of large amyloid oligomers and eliminating the structural conversion from isotropic to β-rich topologies induced by PGG. Our results provide mechanistic insights into CB[7] and PGG action on Aβ oligomerization. They also demonstrate the power of the IMS technique to investigate mechanisms of multiple small-molecule agents on the amyloid formation process. PMID:26629788

  5. Opposing Effects of Cucurbit[7]uril and 1,2,3,4,6-Penta-O-galloyl-β-d-glucopyranose on Amyloid β25-35 Assembly.

    PubMed

    de Almeida, Natália E C; Do, Thanh D; Tro, Michael; LaPointe, Nichole E; Feinstein, Stuart C; Shea, Joan-Emma; Bowers, Michael T

    2016-02-17

    Alzheimer's disease (AD) is a neurodegenerative disease characterized by extracellular deposits of amyloid β protein (Aβ) in the brain. The conversion of soluble monomers to amyloid Aβ fibrils is a complicated process and involves several transient oligomeric species, which are widely believed to be highly toxic and play a crucial role in the etiology of AD. The development of inhibitors to prevent formation of small and midsized oligomers is a promising strategy for AD treatment. In this work, we employ ion mobility spectrometry (IMS), transmission electron microscopy (TEM), and molecular dynamics (MD) simulations to elucidate the structural modulation promoted by two potential inhibitors of Aβ oligomerization, cucurbit[7]uril (CB[7]) and 1,2,3,4,6-penta-O-galloyl-β-d-glucopyranose (PGG), on early oligomer and fibril formation of the Aβ25-35 fragment. One and two CB[7] molecules bind to Aβ25-35 monomers and dimers, respectively, and suppress aggregation by remodeling early oligomer structures and inhibiting the formation of higher-order oligomers. On the other hand, nonselective binding was observed between PGG and Aβ25-35. The interactions between PGG and Aβ25-35, surprisingly, enhanced the formation of Aβ aggregates by promoting extended Aβ25-35 conformations in both homo- and hetero-oligomers. When both ligands were present, the inhibitory effect of CB[7] overrode the stimulatory effect of PGG on Aβ25-35 aggregation, suppressing the formation of large amyloid oligomers and eliminating the structural conversion from isotropic to β-rich topologies induced by PGG. Our results provide mechanistic insights into CB[7] and PGG action on Aβ oligomerization. They also demonstrate the power of the IMS technique to investigate mechanisms of multiple small-molecule agents on the amyloid formation process.

  6. Designing Surface-Confined Coordination Oligomers

    SciTech Connect

    Altman, M.; Rachamim, M; Ichiki, T; Iron, M; Evmenenko, G; Dutta, P; van der Boom, M

    2010-01-01

    HOMO-LUMO engineering of coordination-based oligomers covalently bound to silicon or glass has been achieved by the use of a partially fluorinated chromophore (see graphic). The experimental and computationally derived physical chemical properties of these assemblies are compared to their non-fluorinated analogues.

  7. Glucosamine oligomers: 1. Preparation and characterization.

    PubMed

    Domard, A; Cartier, N

    1989-10-01

    Hydrolysis of chitosan in hot concentrated HCl led to chito-oligosaccharides [beta-(1----4) linked 2-amino-2-deoxy-D-glucose]. The time dependence of the distribution was studied. A convenient choice of the conditions for steric exclusion chromatography of these hydrolysates allowed the separation of the first 15 oligomers and of fractions up to DP = 40.

  8. Neuronal amyloid-β accumulation within cholinergic basal forebrain in ageing and Alzheimer's disease.

    PubMed

    Baker-Nigh, Alaina; Vahedi, Shahrooz; Davis, Elena Goetz; Weintraub, Sandra; Bigio, Eileen H; Klein, William L; Geula, Changiz

    2015-06-01

    The mechanisms that contribute to selective vulnerability of the magnocellular basal forebrain cholinergic neurons in neurodegenerative diseases, such as Alzheimer's disease, are not fully understood. Because age is the primary risk factor for Alzheimer's disease, mechanisms of interest must include age-related alterations in protein expression, cell type-specific markers and pathology. The present study explored the extent and characteristics of intraneuronal amyloid-β accumulation, particularly of the fibrillogenic 42-amino acid isoform, within basal forebrain cholinergic neurons in normal young, normal aged and Alzheimer's disease brains as a potential contributor to the selective vulnerability of these neurons using immunohistochemistry and western blot analysis. Amyloid-β1-42 immunoreactivity was observed in the entire cholinergic neuronal population regardless of age or Alzheimer's disease diagnosis. The magnitude of this accumulation as revealed by optical density measures was significantly greater than that in cortical pyramidal neurons, and magnocellular neurons in the globus pallidus did not demonstrate a similar extent of amyloid immunoreactivity. Immunoblot analysis with a panel of amyloid-β antibodies confirmed accumulation of high concentration of amyloid-β in basal forebrain early in adult life. There was no age- or Alzheimer-related alteration in total amyloid-β content within this region. In contrast, an increase in the large molecular weight soluble oligomer species was observed with a highly oligomer-specific antibody in aged and Alzheimer brains when compared with the young. Similarly, intermediate molecular weight oligomeric species displayed an increase in aged and Alzheimer brains when compared with the young using two amyloid-β42 antibodies. Compared to cortical homogenates, small molecular weight oligomeric species were lower and intermediate species were enriched in basal forebrain in ageing and Alzheimer's disease. Regional and age

  9. Exploring the aggregation free energy landscape of the amyloid-β protein (1-40).

    PubMed

    Zheng, Weihua; Tsai, Min-Yeh; Chen, Mingchen; Wolynes, Peter G

    2016-10-18

    A predictive coarse-grained protein force field [associative memory, water-mediated, structure, and energy model for molecular dynamics (AWSEM)-MD] is used to study the energy landscapes and relative stabilities of amyloid-β protein (1-40) in the monomer and all of its oligomeric forms up to an octamer. We find that an isolated monomer is mainly disordered with a short α-helix formed at the central hydrophobic core region (L17-D23). A less stable hairpin structure, however, becomes increasingly more stable in oligomers, where hydrogen bonds can form between neighboring monomers. We explore the structure and stability of both prefibrillar oligomers that consist of mainly antiparallel β-sheets and fibrillar oligomers with only parallel β-sheets. Prefibrillar oligomers are polymorphic but typically take on a cylindrin-like shape composed of mostly antiparallel β-strands. At the concentration of the simulation, the aggregation free energy landscape is nearly downhill. We use umbrella sampling along a structural progress coordinate for interconversion between prefibrillar and fibrillar forms to identify a conversion pathway between these forms. The fibrillar oligomer only becomes favored over its prefibrillar counterpart in the pentamer where an interconversion bottleneck appears. The structural characterization of the pathway along with statistical mechanical perturbation theory allow us to evaluate the effects of concentration on the free energy landscape of aggregation as well as the effects of the Dutch and Arctic mutations associated with early onset of Alzheimer's disease.

  10. Brain propagation of transduced α-synuclein involves non-fibrillar protein species and is enhanced in α-synuclein null mice.

    PubMed

    Helwig, Michael; Klinkenberg, Michael; Rusconi, Raffaella; Musgrove, Ruth E; Majbour, Nour K; El-Agnaf, Omar M A; Ulusoy, Ayse; Di Monte, Donato A

    2016-03-01

    oligomeric, fibrillar and/or total (monomeric and aggregated) α-synuclein. Following viral vector transduction, monomeric, oligomeric and fibrillar protein was detected within donor neurons in the medulla oblongata. In contrast, recipient axons in the pons were devoid of immunoreactivity for fibrillar α-synuclein, indicating that non-fibrillar forms of α-synuclein were primarily transferred from one neuron to the other, diffused within the brain and led to initial neuronal injury. This study elucidates a paradigm of α-synuclein propagation that may play a particularly important role under pathophysiological conditions associated with enhanced α-synuclein expression. Rapid long-distance diffusion and accumulation of monomeric and oligomeric α-synuclein does not necessarily involve pathological seeding but could still result in a significant neuronal burden during the pathogenesis of neurodegenerative diseases. © The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  11. Structural differences of amyloid-β fibrils revealed by antibodies from phage display.

    PubMed

    Droste, Patrick; Frenzel, André; Steinwand, Miriam; Pelat, Thibaut; Thullier, Philippe; Hust, Michael; Lashuel, Hilal; Dübel, Stefan

    2015-06-18

    Beside neurofibrillary tangles, amyloid plaques are the major histological hallmarks of Alzheimer's disease (AD) being composed of aggregated fibrils of β-amyloid (Aβ). During the underlying fibrillogenic pathway, starting from a surplus of soluble Aβ and leading to mature fibrils, multiple conformations of this peptide appear, including oligomers of various shapes and sizes. To further investigate the fibrillization of β-amyloid and to have tools at hand to monitor the distribution of aggregates in the brain or even act as disease modulators, it is essential to develop highly sensitive antibodies that can discriminate between diverse aggregates of Aβ. Here we report the generation and characterization of a variety of amyloid-β specific human and human-like antibodies. Distinct fractions of monomers and oligomers of various sizes were separated by size exclusion chromatography (SEC) from Aβ42 peptides. These antigens were used for the generation of two Aβ42 specific immune scFv phage display libraries from macaque (Macaca fascicularis). Screening of these libraries as well as two naïve human phage display libraries resulted in multiple unique binders specific for amyloid-β. Three of the obtained antibodies target the N-terminal part of Aβ42 although with varying epitopes, while another scFv binds to the α-helical central region of the peptide. The affinities of the antibodies to various Aβ42 aggregates as well as their ability to interfere with fibril formation and disaggregation of preformed fibrils were determined. Most significantly, one of the scFv is fibril-specific and can discriminate between two different fibril forms resulting from variations in the acidity of the milieu during fibrillogenesis. We demonstrated that the approach of animal immunization and subsequent phage display based antibody selection is applicable to generate highly specific anti β-amyloid scFvs that are capable of accurately discriminating between minute conformational

  12. Nanomaterials for reducing amyloid cytotoxicity.

    PubMed

    Zhang, Min; Mao, Xiaobo; Yu, Yue; Wang, Chen-Xuan; Yang, Yan-Lian; Wang, Chen

    2013-07-26

    This review is intended to reflect the recent progress on therapeutic applications of nanomaterials in amyloid diseases. The progress on anti-amyloid functions of various nanomaterials including inorganic nanoparticles, polymeric nanoparticles, carbon nanomaterials and biomolecular aggregates, is reviewed and discussed. The main functionalization strategies for general nanoparticle modifications are reviewed for potential applications of targeted therapeutics. The interaction mechanisms between amyloid peptides and nanomaterials are discussed from the perspectives of dominant interactions and kinetics. The encapsulation of anti-amyloid drugs, targeted drug delivery, controlled drug release and drug delivery crossing blood brain barrier by application of nanomaterials would also improve the therapeutics of amyloid diseases.

  13. Insulin-degrading enzyme degrades amyloid peptides associated with British and Danish familial dementia.

    PubMed

    Morelli, Laura; Llovera, Ramiro E; Alonso, Leonardo G; Frangione, Blas; de Prat-Gay, Gonzalo; Ghiso, Jorge; Castaño, Eduardo M

    2005-07-08

    Familial British dementia (FBD) and familial Danish dementia (FDD) are autosomal dominant disorders characterized by cerebrovascular and parenchymal amyloid deposition and neurofibrillary degeneration. In both conditions, the genetic defects cause the loss of the normal stop codon in the precursor BRI, generating novel 34-residue peptides named ABri and ADan in FBD and FDD, respectively. ABri and ADan show a strong tendency to aggregate into