An evaluation of the self-assembly enhancing properties of cell-derived hexameric amyloid-β.
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ABSTRACT:
SUBMITTER: Vadukul DM
PROVIDER: S-EPMC8172837 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Epidemiological evidence suggests that moderate consumption of red wine reduces the incidence of Alzheimer disease (AD). To study the protective effects of red wine, experiments recently were executed in the Tg2576 mouse model of AD. These studies showed that a commercially available grape seed polyphenolic extract, MegaNatural-AZ (MN), significantly attenuated AD-type cognitive deterioration and reduced cerebral amyloid deposition (Wang, J., Ho, L., Zhao, W., Ono, K., Rosensweig, C., Chen, L., Humala, N., Teplow, D. B., and Pasinetti, G. M. (2008) J. Neurosci. 28, 6388-6392). To elucidate the mechanistic bases for these observations, here we used CD spectroscopy, photo-induced cross-linking of unmodified proteins, thioflavin T fluorescence, size exclusion chromatography, and electron microscopy to examine the effects of MN on the assembly of the two predominant disease-related amyloid beta-protein alloforms, Abeta40 and Abeta42. We also examined the effects of MN on Abeta-induced cytotoxicity by assaying 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide metabolism and lactate dehydrogenase activity in Abeta-treated, differentiated pheochromocytoma (PC12) cells. Initial studies revealed that MN blocked Abeta fibril formation. Subsequent evaluation of the assembly stage specificity of the effect showed that MN was able to inhibit protofibril formation, pre-protofibrillar oligomerization, and initial coil --> alpha-helix/beta-sheet secondary structure transitions. Importantly, MN had protective effects in assays of cytotoxicity in which MN was mixed with Abeta prior to peptide assembly or following assembly and just prior to peptide addition to cells. These data suggest that MN is worthy of consideration as a therapeutic agent for AD.
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Project description:The self-assembly of two derivatives of KLVFF, a fragment Aβ(16-20) of the amyloid beta (Aβ) peptide, is investigated and recovery of viability of neuroblastoma cells exposed to Aβ (1-42) is observed at sub-stoichiometric peptide concentrations. Fluorescence assays show that NH2-KLVFF-CONH2 undergoes hydrophobic collapse and amyloid formation at the same critical aggregation concentration (cac). In contrast, NH2-K(Boc)LVFF-CONH2 undergoes hydrophobic collapse at a low concentration, followed by amyloid formation at a higher cac. These findings are supported by the β-sheet features observed by FTIR. Electrospray ionization mass spectrometry indicates that NH2-K(Boc)LVFF-CONH2 forms a significant population of oligomeric species above the cac. Cryo-TEM, used together with SAXS to determine fibril dimensions, shows that the length and degree of twisting of peptide fibrils seem to be influenced by the net peptide charge. Grazing incidence X-ray scattering from thin peptide films shows features of β-sheet ordering for both peptides, along with evidence for lamellar ordering of NH2-KLVFF-CONH2. This work provides a comprehensive picture of the aggregation properties of these two KLVFF derivatives and shows their utility, in unaggregated form, in restoring the viability of neuroblastoma cells against Aβ-induced toxicity.
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Project description:Alzheimer's disease (AD) is a fatal neurodegenerative disorder associated with severe dementia, progressive cognitive decline, and irreversible memory loss. Although its etiopathogenesis is still unclear, the aggregation of amyloid-β (Aβ) peptides into supramolecular structures and their accumulation in the central nervous system play a critical role in the onset and progression of the disease. On such a premise, the inhibition of the early stages of Aβ aggregation is a potential prevention strategy for the treatment of AD. Since several natural occurring compounds, as well as metal-based molecules, showed promising inhibitory activities toward Aβ aggregation, we herein characterized the interaction of an organoruthenium derivative of curcumin with Aβ(1-40) and Aβ(1-42) peptides, and we evaluated its ability to inhibit the oligomerization/fibrillogenesis processes by combining in silico and in vitro methods. In general, besides being less toxic to neuronal cells, the derivative preserved the amyloid binding ability of the parent compound in terms of equilibrium dissociation constants but (most notably) was more effective both in retarding the formation and limiting the size of amyloid aggregates by virtue of a higher hindering effect on the amyloid-amyloid elongation surface. Additionally, the complex protected neuronal cells from amyloid toxicity.
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Project description:Photo-oxygenation of β-amyloid (Aβ) has been considered an efficient way to inhibit Aβ aggregation in Alzheimer's disease (AD). However, current photosensitizers cannot simultaneously achieve enhanced blood-brain barrier (BBB) permeability and selective photooxygenation of Aβ, leading to poor therapeutic efficacy, severe off-target toxicity, and substandard bioavailability. Herein, an Aβ target-driven supramolecular self-assembly (PKNPs) with enhanced BBB penetrability and switchable photoactivity is designed and demonstrated to be effective in preventing Aβ aggregation in vivo. PKNPs are prepared by the self-assembly of the Aβ-targeting peptide KLVFF and an FDA-approved porphyrin derivative (5-(4-carboxyphenyl)-10,15,20-triphenylporphyrin). Due to the photothermal effect of PKNPs, the BBB permeability of PKNPs under irradiation is 8.5-fold higher than that of porphyrin alone. Moreover, upon selective interaction with Aβ, PKNPs undergo morphological change from the spherical to the amorphous form, resulting in a smart transformation from photothermal activity to photodynamic activity. Consequently, the disassembled PKNPs can selectively oxygenate Aβ without affecting off-target proteins (insulin, bovine serum albumin, and human serum albumin). The well-designed PKNPs exhibit not only improved BBB permeability but also highly selective Aβ photooxygenation. Furthermore, in vivo experiments demonstrate that PKNPs can alleviate Aβ-induced neurotoxicity and prolong the life span of the commonly used AD transgenic Caenorhabditis elegans CL2006. Our work may open a new path for using supramolecular self-assemblies as switchable phototheranostics for the selective and effective prevention of Aβ aggregation and related neurotoxicity in AD.
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Project description:Amyloid-β (Aβ) self-assembly into cross-β amyloid fibrils is implicated in a causative role in Alzheimer's disease pathology. Uncertainties persist regarding the mechanisms of amyloid self-assembly and the role of metastable prefibrillar aggregates. Aβ fibrils feature a sheet-turn-sheet motif in the constituent β-strands; as such, turn nucleation has been proposed as a rate-limiting step in the self-assembly pathway. Herein, we report the use of an azobenzene β-hairpin mimetic to study the role turn nucleation plays on Aβ self-assembly. [3-(3-Aminomethyl)phenylazo]phenylacetic acid (AMPP) was incorporated into the putative turn region of Aβ42 to elicit temporal control over Aβ42 turn nucleation; it was hypothesized that self-assembly would be favored in the cis-AMPP conformation if β-hairpin formation occurs during Aβ self-assembly and that the trans-AMPP conformer would display attenuated fibrillization propensity. It was unexpectedly observed that the trans-AMPP Aβ42 conformer forms fibrillar constructs that are similar in almost all characteristics, including cytotoxicity, to wild-type Aβ42. Conversely, the cis-AMPP Aβ42 congeners formed nonfibrillar, amorphous aggregates that exhibited no cytotoxicity. Additionally, cis-trans photoisomerization resulted in rapid formation of native-like amyloid fibrils and trans-cis conversion in the fibril state reduced the population of native-like fibrils. Thus, temporal photocontrol over Aβ turn conformation provides significant insight into Aβ self-assembly. Specifically, Aβ mutants that adopt stable β-turns form aggregate structures that are unable to enter folding pathways leading to cross-β fibrils and cytotoxic prefibrillar intermediates.
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Project description:The self-assembly of peptides and proteins into amyloid fibrils and other aggregates are linked to several diseases. One of the most studied cases is the peptide amyloid-β (Aβ), found self-assembled in Alzheimer's disease patients' brains. In test tubes, assays with chemically synthesized or recombinant Aβ are widely investigated to understand the aggregation process and to find modulators, which could be of therapeutic interest. Experience over more than a decade in our laboratory through discussions with colleagues, expertly studying the literature, and as reviewers revealed to us the widely encountered difficulty to control the aggregation and obtain reproducible results in the test tube. However, this issue is scarcely reported and discussed in the publications, which we think hampers strongly the progress in this field and can deceive newcomers. Here, we describe the difficulty and potential reasons to obtain reproducible aggregation data and propose some guidelines for working with it.
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Project description:Amyloid formation is associated with devastating diseases such as Alzheimer's, Parkinson's and Type-2 diabetes. The large amyloid deposits found in patients suffering from these diseases have remained difficult to probe by structural means. Recent NMR models also predict heterotypic interactions from distinct peptide fragments but limited evidence of heterotypic packed sheets is observed in solution. Here we characterize two segments of the protein amyloid β (Aβ) known to form fibrils in Alzheimer's disease patients. We designed two variants of Aβ(19-24) and Aβ(27-32), IFAEDV (I6V) and NKGAIF (N6F) to lower the aggregation propensity of individual peptides while maintaining the similar interactions between the two segments in their native forms. We found that the variants do not form significant amyloid fibrils individually but a 1:1 mixture forms abundant fibrils. Using ion mobility-mass spectrometry (IM-MS), hetero-oligomers up to decamers were found in the mixture while the individual peptides formed primarily dimers and some tetramers consistent with a strong heterotypic interaction between the two segments. We showed by X-ray crystallography that I6V formed a Class 7 zipper with a weakly packed pair of β-sheets and no segregated dry interface, while N6F formed a more stable Class 1 zipper. In a mixture of equimolar N6F:I6V, I6V forms a more stable zipper than in I6V alone while no N6F or hetero-typic zippers are observed. These data are consistent with a mechanism where N6F catalyzes assembly of I6V into a stable zipper and perhaps into stable, pure I6V fibrils that are observed in AFM measurements.
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Project description:Epidemiological evidence that red wine consumption negatively correlates with risk of Alzheimer's disease has led to experimental studies demonstrating that grape seed extracts inhibit the aggregation and oligomerization of Aβ in vitro and ameliorate neuropathology and behavioral deficits in a mouse model of Alzheimer's disease. The active agent in the extracts is a mixed population of polyphenolic compounds. To evaluate the relative potency of each of these compounds, HPLC was used to fractionate the mixture into monomers, dimers, and oligomers. Each fraction was analyzed for its effect on Aβ conformational dynamics (circular dichroism), oligomerization (zero-length photochemical cross-linking), aggregation kinetics (Thioflavin T fluorescence), and morphology (electron microscopy). The relative activities of each fraction were determined on the basis of molar concentration (mol/L) or mass concentration (g/L). When molar concentration, the number concentration of each polyphenolic compound, was considered, the oligomer fraction was the most potent inhibitor of Aβ oligomerization and aggregation. However, when mass concentration, the number concentration of phenolic groups, was considered, monomers were the most potent inhibitors. To understand these ostensibly contradictory results, a model of polyphenol:Aβ complexation was developed. This model, which was found to be consistent with published X-ray crystallographic studies, offers an explanation for the effects of functional group polyvalency on inhibitor activity. Our data emphasize the importance of an in-depth understanding of the mechanism(s) underlying 'concentration dependence' in inhibitor systems involving polyfunctional agents.
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Project description:Determining the structural origins of amyloid fibrillation is essential for understanding both the pathology of amyloidosis and the rational design of inhibitors to prevent or reverse amyloid formation. In this work, the decisive roles of peptide structures on amyloid self-assembly and morphological diversity were investigated by the design of eight amyloidogenic peptides derived from islet amyloid polypeptide. Among the segments, two distinct morphologies were highlighted in the form of twisted and planar (untwisted) ribbons with varied diameters, thicknesses, and lengths. In particular, transformation of amyloid fibrils from twisted ribbons into untwisted structures was triggered by substitution of the C-terminal serine with threonine, where the side chain methyl group was responsible for the distinct morphological change. This effect was confirmed following serine substitution with alanine and valine and was ascribed to the restriction of intersheet torsional strain through the increased hydrophobic interactions and hydrogen bonding. We also studied the variation of fibril morphology (i.e., association and helicity) and peptide aggregation propensity by increasing the hydrophobicity of the peptide side group, capping the N-terminus, and extending sequence length. We anticipate that our insights into sequence-dependent fibrillation and morphological diversity will shed light on the structural interpretation of amyloidogenesis and development of structure-specific imaging agents and aggregation inhibitors.
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Project description:The β-amyloid peptide (Aβ) is found as amyloid fibrils in senile plaques, a typical hallmark of Alzheimer's disease (AD). However, intermediate soluble oligomers of Aβ are now recognized as initiators of the pathogenic cascade leading to AD. Studies using recombinant Aβ have shown that hexameric Aβ in particular acts as a critical nucleus for Aβ self-assembly. We recently isolated hexameric Aβ assemblies from a cellular model, and demonstrated their ability to enhance Aβ aggregation in vitro. Here, we report the presence of similar hexameric-like Aβ assemblies across several cellular models, including neuronal-like cell lines. In order to better understand how they are produced in a cellular context, we investigated the role of presenilin-1 (PS1) and presenilin-2 (PS2) in their formation. PS1 and PS2 are the catalytic subunits of the γ-secretase complex that generates Aβ. Using CRISPR-Cas9 to knockdown each of the two presenilins in neuronal-like cell lines, we observed a direct link between the PS2-dependent processing pathway and the release of hexameric-like Aβ assemblies in extracellular vesicles. Further, we assessed the contribution of hexameric Aβ to the development of amyloid pathology. We report the early presence of hexameric-like Aβ assemblies in both transgenic mice brains exhibiting human Aβ pathology and in the cerebrospinal fluid of AD patients, suggesting hexameric Aβ as a potential early AD biomarker. Finally, cell-derived hexameric Aβ was found to seed other human Aβ forms, resulting in the aggravation of amyloid deposition in vivo and neuronal toxicity in vitro.
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