Oligomeric forms of insulin amyloid aggregation disrupt outgrowth and complexity of neuron-like PC12 cells

Formation of protein amyloid fibrils consists of a series of intermediates including oligomeric aggregates, proto-fibrillar structures, and finally mature fibrils. Recent studies show higher toxicity for oligomeric and proto-fibrillar intermediates of protein relative to their mature fibrils. Here the kinetic of the insulin amyloid fibrillation was evaluated using a variety of techniques including ThT fluorescence, Congo red absorbance, circular dichroism, and atomic force microscopy (AFM). The solution surface tension changes were attributed to hydrophobic changes in insulin structure and were detected by Du Noüy Ring method. Determination of the surface tension of insulin oligomeric, proto-fibrillar and fibrillar forms indicated that the hydrophobicity of solution is enhanced by the formation of the oligomeric forms of insulin compared to other forms. In order to investigate the toxicity of the different forms of insulin we monitored morphological alterations of the differentiated neuron-like PC12 cells following incubation with native, oligomeric aggregates, proto-fibrillar, and fibrillar forms of insulin. The cell body area, average neurite length, neurite width, number of primary neurites, and percent of bipolar cells and node/primary neurite ratios were used to assess the growth and complexity of PC12 cells exposed to different forms of insulin. We observed that the oligomeric form of insulin impaired the growth and complexity of PC12 cells compared to other forms. Together our data suggest that the lower surface tension of oligomers and their perturbation affects the morphology of PC12 cells, mainly due to their enhanced hydrophobicity and detergent-like structures.     

Inhibition of toxicity in the beta-amyloid peptide fragment beta -(25-35) using N-methylated derivatives: a general strategy to prevent amyloid formation

beta-(25-35) is a synthetic derivative of beta-amyloid, the peptide that is believed to cause Alzheimer's disease. As it is highly toxic and forms fibrillar aggregates typical of beta-amyloid, it is suitable as a model for testing inhibitors of aggregation and toxicity. We demonstrate that N-methylated derivatives of beta-(25-35), which in isolation are soluble and non-toxic, can prevent the aggregation and inhibit the resulting toxicity of the wild type peptide. N-Methylation can block hydrogen bonding on the outer edge of the assembling amyloid. The peptides are assayed by Congo red and thioflavin T binding, electron microscopy, and a 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) toxicity assay on PC12 cells. One peptide (Gly(25) N-methylated) has properties similar to the wild type, whereas five have varying effects on prefolded fibrils and fibril assembly. In particular, beta-(25-35) with Gly(33) N-methylated is able to completely prevent fibril assembly and to reduce the toxicity of prefolded amyloid. With Leu(34) N-methylated, the fibril morphology is altered and the toxicity reduced. We suggest that the use of N-methylated derivatives of amyloidogenic peptides and proteins could provide a general solution to the problem of amyloid deposition and toxicity.     

Formation of toxic Abeta(1-40) fibrils on GM1 ganglioside-containing membranes mimicking lipid rafts: polymorphisms in Abeta(1-40) fibrils

The abnormal aggregation and deposition of amyloid β protein (Aβ) on neuronal cells are critical to the onset of Alzheimer's disease. The entity (oligomers or fibrils) of toxic Aβ species responsible for the pathogenesis of the disease has been controversial. We have reported that the Aβ aggregates on ganglioside-rich domains of neuronal PC12 cells as well as in raft-like model membranes. Here, we identified toxic Aβ(1-40) aggregates formed with GM1-ganglioside-containing membranes. Aβ(1-40) was incubated with raft-like liposomes composed of GM1/cholesterol/sphingomyelin at 1:2:2 and 37 °C. After a lag period, toxic amyloid fibrils with a width of 12 nm were formed and subsequently laterally assembled with slight changes in their secondary structure as confirmed by viability assay, thioflavin-T fluorescence, circular dichroism, and transmission electron microscopy. In striking contrast, Aβ fibrils formed without membranes were thinner (6.7 nm) and much less toxic because of weaker binding to cell membranes and a smaller surface hydrophobicity. This study suggests that toxic Aβ(1-40) species formed on membranes are not soluble oligomers but amyloid fibrils and that Aβ(1-40) fibrils exhibit polymorphisms.     

Black tea theaflavins inhibit formation of toxic amyloid-β and α-synuclein fibrils

Causal therapeutic approaches for amyloid diseases such as Alzheimer's and Parkinson's disease targeting toxic amyloid oligomers or fibrils are still emerging. Here, we show that theaflavins (TF1, TF2a, TF2b, and TF3), the main polyphenolic components found in fermented black tea, are potent inhibitors of amyloid-β (Aβ) and α-synuclein (αS) fibrillogenesis. Their mechanism of action was compared to that of two established inhibitors of amyloid formation, (-)-epigallocatechin gallate (EGCG) and congo red (CR). All three compounds reduce the fluorescence of the amyloid indicator dye thioflavin T. Mapping the binding regions of TF3, EGCG, and CR revealed that all three bind to two regions of the Aβ peptide, amino acids 12-23 and 24-36, albeit with different specificities. However, their mechanisms of amyloid inhibition differ. Like EGCG but unlike congo red, theaflavins stimulate the assembly of Aβ and αS into nontoxic, spherical aggregates that are incompetent in seeding amyloid formation and remodel Aβ fibrils into nontoxic aggregates. When compared to EGCG, TF3 was less susceptible to air oxidation and had an increased efficacy under oxidizing conditions. These findings suggest that theaflavins might be used to remove toxic amyloid deposits.     

A model of amyloid's role in disease based on fibril fracture

Although the correlative evidence relating the presence of amyloid fibrils and certain disease states (e.g. Alzheimer's disease and Type 2 Diabetes) is overwhelming, a direct causative role for amyloid has proved harder to establish. Current thinking links a narrow region of the aggregate protein size distribution, the so called ‘early aggregate’ domain to cellular toxicity. A troubling feature of this theory however is that the nucleated reaction mechanism by which amyloid formation is believed to occur results in a very low number concentration of early aggregates which are rapidly extended to form amyloid fibrils. This situation means that the concentration of early aggregates is very low at the time when they are supposedly at their most toxic. Here we adopt a novel explicit simulation strategy to examine a kinetic regime involving nucleated growth combined with fibril fragmentation under which this situation can be reversed so as to produce a high number concentration of small on-pathway toxic aggregates. Dependent upon the rate of fragmentation, the time scale for generation of toxic early aggregates may be coupled, uncoupled or disassociated from the time scale for the appearance of amyloid fibrils. Furthermore the model presents itself as a biochemical ‘switch’ transitioning between modes of amyloid induced cell death dependent upon either specific amyloid toxicity or non-specific solid mass induced tissue damage.     

Synthetic Lipid Vesicles Recruit Native-Like Aggregates and Affect the Aggregation Process of the Prion Ure2p: Insights on Vesicle Permeabilization and Charge Selectivity

The yeast prion Ure2p polymerizes into native-like fibrils, retaining the overall structure and binding properties of the soluble protein. Recently we have shown that, similar to amyloid oligomers, the native-like Ure2p fibrils and their precursor oligomers are highly toxic to cultured mammalian cells when added to the culture medium, whereas Ure2p amyloid fibrils generated by heating the native-like fibrils are substantially harmless. We show here that, contrary to the nontoxic amyloid fibrils, the toxic, native-like Ure2p assemblies induce a significant calcein release from negatively charged phosphatidylserine vesicles. A minor and less-specific effect was observed with zwitterionic phosphatidylcholine vesicles, suggesting that the toxic aggregates preferentially bind to negatively charged sites on lipid membranes. We also found that cholesterol-enriched phospholipid membranes are protected against permeabilization by native-like Ure2p assemblies. Moreover, vesicle permeabilization appears charge-selective, allowing calcium, but not chloride, influx to be monitored. Finally, we found that the interaction with phosphatidylserine membranes speeds up Ure2p polymerization into oligomers and fibrils structurally and morphologically similar to the native-like Ure2p assemblies arising in free solution, although less cytotoxic. These data suggest that soluble Ure2p oligomers and native-like fibrils, but not amyloid fibrils, interact intimately with negatively charged lipid membranes, where they allow selective cation influx.     

The yeast prion Ure2p native-like assemblies are toxic to mammalian cells regardless of their aggregation state

The yeast prion Ure2p assembles in vitro into oligomers and fibrils retaining the alpha-helix content and binding properties of the soluble protein. Here we show that the different forms of Ure2p native-like assemblies (dimers, oligomers, and fibrils) are similarly toxic to murine H-END cells when added to the culture medium. Interestingly, the amyloid fibrils obtained by heat treatment of the toxic native-like fibrils appear harmless. Moreover, the Ure2p C-terminal domain, lacking the N-terminal segment necessary for aggregation but containing the glutathione binding site, is not cytotoxic. This finding strongly supports the idea that Ure2p toxicity depends on the structural properties of the flexible N-terminal prion domain and can therefore be considered as an inherent feature of the protein, unrelated to its aggregation state but rather associated with a basic toxic fold shared by all of the Ure2p native-like assemblies. Indeed, the latter are able to interact with the cell surface, leading to alteration of calcium homeostasis, membrane permeabilization, and oxidative stress, whereas the heat-treated amyloid fibrils do not. Our results support the idea of a general mechanism of toxicity of any protein/peptide aggregate endowed with structural features, making it able to interact with cell membranes and to destabilize them. This evidence extends the widely accepted view that the toxicity by protein aggregates is restricted to amyloid prefibrillar aggregates and provides new insights into the mechanism by which native-like oligomers compromise cell viability.     

Amyloid toxicity is independent of polypeptide sequence, length and chirality

By using an amyloid sequence pattern, here we have identified putative six-residue amyloidogenic stretches in several relevant amyloid proteins. Hexapeptides synthesized on the bases of the sequence stretches matching the pattern have been shown to form amyloid fibrils in vitro. As larger pathological peptides such as Aβ_1-42 do, these short amyloid peptides form heterogeneous mixtures of small aggregates that induce cell death in PC12 cells and primary hippocampal neurons. Toxic mixtures of small aggregates from these hexapeptides bind to cell membranes and can be further internalized, as also observed for natural amyloid proteins. In neurons, toxic aggregates obtained from the full length Aβ_1-42 amyloid peptide or their amyloid stretch Aβ_16-21 peptide preferentially localize in synapses, leading to the re-organization of the underlying actin cytoskeleton. This process does not involve stereospecific interactions between membrane and toxic species as D-sequences are as toxic as L ones, suggesting that is not receptor mediated. Based on these results, we propose here that regardless of polypeptide sequence, length and amino acid chirality, amyloid prefibrillar aggregates exert their cytotoxic effect through a common cell death mechanism related to a particular quaternary structure. The degree of toxicity of these species seems to depend, however, on cell membrane composition.     

Neurotoxicity of acetylcholinesterase amyloid beta-peptide aggregates is dependent on the type of Abeta peptide and the AChE concentration present in the complexes.

Alzheimer's disease (AD) is a neurodegenerative disorder whose hallmark is the presence of senile plaques and neurofibrillary tangles. Senile plaques are mainly composed of amyloid beta-peptide (Abeta) fibrils and several proteins including acetylcholinesterase (AChE). AChE has been previously shown to stimulate the aggregation of Abeta1-40 into amyloid fibrils. In the present work, the neurotoxicity of different amyloid aggregates formed in the absence or presence of AChE was evaluated in rat pheochromocytoma PC12 cells. Stable AChE-Abeta complexes were found to be more toxic than those formed without the enzyme, for Abeta1-40 and Abeta1-42, but not for amyloid fibrils formed with AbetaVal18-Ala, a synthetic variant of the Abeta1-40 peptide. Of all the AChE-Abeta complexes tested the one containing the Abeta1-40 peptide was the most toxic. When increasing concentrations of AChE were used to aggregate the Abeta1-40 peptide, the neurotoxicity of the complexes increased as a function of the amount of enzyme bound to each complex. Our results show that AChE-Abeta1-40 aggregates are more toxic than those of AChE-Abeta1-42 and that the neurotoxicity depends on the amount of AChE bound to the complexes, suggesting that AChE may play a key role in the neurodegeneration observed in Alzheimer brain.     

d-Polyglutamine Amyloid Recruits l-Polyglutamine Monomers and Kills Cells

Polyglutamine (polyQ) amyloid fibrils are observed in disease tissue and have been implicated as toxic agents responsible for neurodegeneration in expanded CAG repeat diseases such as Huntington's disease. Despite intensive efforts, the mechanism of amyloid toxicity remains unknown. As a novel approach to probing polyQ toxicity, we investigate here how some cellular and physical properties of polyQ amyloid vary with the chirality of the glutamine residues in the polyQ. We challenged PC12 cells with small amyloid fibrils composed of either l- or d-polyQ peptides and found that d-fibrils are as cytotoxic as l-fibrils. We also found using fluorescence microscopy that both aggregates effectively seed the aggregation of cell-produced l-polyQ proteins, suggesting a surprising lack of stereochemical restriction in seeded elongation of polyQ amyloid. To investigate this effect further, we studied chemically synthesized d- and l-polyQ in vitro. We found that, as expected, d-polyQ monomers are not recognized by proteins that recognize l-polyQ monomers. However, amyloid fibrils prepared from d-polyQ peptides can efficiently seed the aggregation of l-polyQ monomers in vitro, and vice versa. This result is consistent with our cell results on polyQ recruitment but is inconsistent with previous literature reports on the chiral specificity of amyloid seeding. This chiral cross-seeding can be rationalized by a model for seeded elongation featuring a “rippled β-sheet” interface between seed fibril and docked monomers of opposite chirality. The lack of chiral discrimination in polyQ amyloid cytotoxicity is consistent with several toxicity mechanisms, including recruitment of cellular polyQ proteins.     

Amyloid fibrils of mammalian prion protein are highly toxic to cultured cells and primary neurons

A growing body of evidence indicates that small, soluble oligomeric species generated from a variety of proteins and peptides rather than mature amyloid fibrils are inherently highly cytotoxic. Here, we show for the first time that mature amyloid fibrils produced from full-length recombinant mammalian prion protein (rPrP) were highly toxic to cultured cells and primary hippocampal and cerebella neurons. Fibrils induced apoptotic cell death in a time- and dose-dependent manner. The toxic effect of fibrils was comparable with that exhibited by soluble small beta-oligomers generated from the same protein. Fibrils prepared from insulin were not toxic, suggesting that the toxic effect was not solely due to the highly polymeric nature of the fibrillar form. The cell death caused by rPrP fibrils or beta-oligomers was substantially reduced when expression of endogenous PrP(C) was down-regulated by small interfering RNAs. In opposition to the beta-oligomer and amyloid fibrils of rPrP, the monomeric alpha-helical form of rPrP stimulated neurite out-growth and survival of neurons. These studies illustrated that both soluble beta-oligomer and amyloid fibrils of the prion protein are intrinsically toxic and confirmed that endogenously expressed PrP(C) is required for mediating the toxicity of abnormally folded external PrP aggregates.     

Hexafluoroisopropanol induces amyloid fibrils of islet amyloid polypeptide by enhancing both hydrophobic and electrostatic interactions

Although amyloid fibrils deposit with various proteins, the comprehensive mechanism by which they form remains unclear. We studied the formation of fibrils of human islet amyloid polypeptide associated with type II diabetes in the presence of various concentrations of 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) under acidic and neutral pH conditions using CD, amyloid-specific thioflavin T fluorescence, fluorescence imaging with thioflavin T, and atomic force microscopy. At low pH, the formation of fibrils was promoted by HFIP with an optimum at 5% (v/v). At neutral pH in the absence of HFIP, significant amounts of amorphous aggregates formed in addition to the fibrils. The addition of HFIP suppressed the formation of amorphous aggregates, leading to a predominance of fibrils with an optimum effect at 25% (v/v). Under both conditions, higher concentrations of HFIP dissolved the fibrils and stabilized the α-helical structure. The results indicate that fibrils and amorphous aggregates are different types of precipitates formed by exclusion from water-HFIP mixtures. The exclusion occurs through the combined effects of hydrophobic interactions and electrostatic interactions, both of which are strengthened by low concentrations of HFIP, and a subtle balance between the two types of interactions determines whether the fibrils or amorphous aggregates dominate. We suggest a general view of how the structure of precipitates varies dramatically from single crystals to amyloid fibrils and amorphous aggregates.     

Small-molecule conversion of toxic oligomers to nontoxic β-sheet-rich amyloid fibrils

Several lines of evidence indicate that prefibrillar assemblies of amyloid-β (Aβ) polypeptides, such as soluble oligomers or protofibrils, rather than mature, end-stage amyloid fibrils cause neuronal dysfunction and memory impairment in Alzheimer's disease. These findings suggest that reducing the prevalence of transient intermediates by small molecule-mediated stimulation of amyloid polymerization might decrease toxicity. Here we demonstrate the acceleration of Aβ fibrillogenesis through the action of the orcein-related small molecule O4, which directly binds to hydrophobic amino acid residues in Aβ peptides and stabilizes the self-assembly of seeding-competent, β-sheet-rich protofibrils and fibrils. Notably, the O4-mediated acceleration of amyloid fibril formation efficiently decreases the concentration of small, toxic Aβ oligomers in complex, heterogeneous aggregation reactions. In addition, O4 treatment suppresses inhibition of long-term potentiation by Aβ oligomers in hippocampal brain slices. These results support the hypothesis that small, diffusible prefibrillar amyloid species rather than mature fibrillar aggregates are toxic for mammalian cells.     

Structural studies reveal that the diverse morphology of beta(2)-microglobulin aggregates is a reflection of different molecular architectures

Amyloid deposition accompanies over 20 degenerative diseases in human, including Alzheimer's, Parkinson's, and prion diseases. Recent studies revealed the importance of other type of protein aggregates, e.g., non-specific aggregates, protofibrils, and small oligomers in the development of such diseases and proved their increased toxicity for living cells in comparison with mature amyloid fibrils. We carried out a comparative structural analysis of different monomeric and aggregated states of beta(2)-microglobulin, a protein responsible for hemodialysis-related amyloidosis. We investigated the structure of the native and acid-denatured states, as well as that of mature fibrils, immature fibrils, amorphous aggregates, and heat-induced filaments, prepared under various in vitro conditions. Infrared spectroscopy demonstrated that the beta-sheet compositions of immature fibrils, heat-induced filaments and amorphous aggregates are characteristic of antiparallel intermolecular beta-sheet structure while mature fibrils are different from all others suggesting a unique overall structure and assembly. Filamentous aggregates prepared by heat treatment are of importance in understanding the in vivo disease because of their stability under physiological conditions, where amyloid fibrils and protofibrils formed at acidic pH depolymerize. Atomic force microscopy of heat-induced filaments represented a morphology similar to that of the low pH immature fibrils. At a pH close to the pI of the protein, amorphous aggregates were formed readily with association of the molecules in native-like conformation, followed by formation of intermolecular beta-sheet structure in a longer time-scale. Extent of the core buried from the solvent in the various states was investigated by H/D exchange of the amide protons.     

Toxicity of non-abeta component of Alzheimer's disease amyloid, and N-terminal fragments thereof, correlates to formation of beta-sheet structure and fibrils.

The non-Abeta component of Alzheimer's disease amyloid (NAC) and its precursor alpha-synuclein have been linked to amyloidogenesis in Alzheimer's disease (AD), Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Previously we have shown that NAC forms beta-sheet structures and fibrils [El-Agnaf, O.M.A., Bodles, A.M., Guthrie, D.J.S., Harriott, P. & Irvine, G.B. (1998) Eur. J. Biochem. 258, 157-163]. As a measure of their neurotoxic potential we have examined the ability of fresh and aged NAC and fragments thereof to inhibit the reduction of the redox dye 3-(4, 5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide by rat pheochromocytoma PC12 cells. Micromolar concentrations of NAC and fragments thereof display varying degrees of toxicity. On immediate dissolution and after an incubation period for 3 days at 37 degrees C the full-length peptide and fragments NAC(3-18) and NAC(1-18) scrambled sequence [NAC(1-18 s)] were toxic, whereas fragments NAC(1-13) and NAC(6-14) were not. CD indicates that NAC(3-18) and NAC(1-18 s) exhibit beta-sheet secondary structure in aqueous solution, whereas NAC(1-13) and NAC(6-14) do not. NAC(3-18) aggregates, as indicated by concentration of peptide remaining in solution after 3 days measured by an HPLC assay, and forms fibrils, as determined by electron microscopy. However, although some fibrils were detected for NAC(1-18 s) it does not come out of solution to a significant degree. Fragments NAC(1-13) and NAC(6-14) form few fibrils and remain in solution. These findings indicate that the ability of the central region of NAC to form beta-sheet secondary structures is important for determining the toxicity of the peptide. This contrasts with what has been reported previously for most Abeta peptides as their toxicity appears to require the peptide to have formed fibrillary aggregates as well as displaying beta-sheet. These results suggest that an intermediate, which exhibits beta-sheet structure, may be responsible for the toxic properties of NAC and provides further evidence for the role of NAC in the pathogenesis of AD, PD and DLB.     

Docosahexaenoic acid stabilizes soluble amyloid-beta protofibrils and sustains amyloid-beta-induced neurotoxicity in vitro

Enrichment of diet and culture media with the polyunsaturated fatty acid docosahexaenoic acid has been found to reduce the amyloid burden in mice and lower amyloid-beta (Abeta) levels in both mice and cultured cells. However, the direct interaction of polyunsaturated fatty acids, such as docosahexaenoic acid, with Abeta, and their effect on Abeta aggregation has not been explored in detail. Therefore, we have investigated the effect of docosahexaenoic acid, arachidonic acid and the saturated fatty acid arachidic acid on monomer oligomerization into protofibrils and protofibril fibrillization into fibrils in vitro, using size exclusion chromatography. The polyunsaturated fatty acids docosahexaenoic acid and arachidonic acid at micellar concentrations stabilized soluble Abeta42 wild-type protofibrils, thereby hindering their conversion to insoluble fibrils. As a consequence, docosahexaenoic acid sustained amyloid-beta-induced toxicity in PC12 cells over time, whereas Abeta without docosahexaenoic acid stabilization resulted in reduced toxicity, as Abeta formed fibrils. Arachidic acid had no effect on Abeta aggregation, and neither of the fatty acids had any protofibril-stabilizing effect on Abeta42 harboring the Arctic mutation (AbetaE22G). Consequently, AbetaArctic-induced toxicity could not be sustained using docosahexaenoic acid. These results provide new insights into the toxicity of different Abeta aggregates and how endogenous lipids can affect Abeta aggregation.     

Synthesis of novel, multivalent glycodendrimers as ligands for HIV-1 gp120

Multivalent neoglycoconjugates are valuable tools for studying carbohydrate-protein interactions. To study the interaction of HIV-1 gp120 with its reported alternate glycolipid receptors, galactosyl ceramide (GalCer) and sulfatide, galactose- and sulfated galactose-derivatized dendrimers were synthesized, analyzed as ligands for rgp120 by surface plasmon resonance, and tested for their ability to inhibit HIV-1 infection of CXCR4- and CCR5-expressing indicator cells. Four different series of glycodendrimers were made by amine coupling spacer-arm derivatized galactose residues, either sulfated or nonsulfated, to poly(propylenimine) dendrimers, generations 1-5. One series of glycodendrimers was prepared from the ceramide saccharide derivative of purified natural GalCer, and another was from chemically synthesized 3-(beta-D-galactopyranosylthio)propionic acid. Synthesis of 3-sulfogalactopyranosyl-derivatized dendrimers was accomplished using the novel compound, 3-(beta-D-3-sulfogalactopyranosylthio)propionic acid. The fourth series was made by random sulfation of the 3-(beta-D-galactopyranosylthio)propionic acid functionalized dendrimers. Structures of the carbohydrate moieties were confirmed by NMR, and the average molecular weights and polydispersities of the different glycodendrimers were determined using MALDI-TOF MS. Surface plasmon resonance studies found that rgp120 IIIB bound to the derivatized dendrimers tested with nanomolar affinity, and to dextran sulfate with picomolar affinity. In vitro studies of the effectiveness of these compounds at inhibiting infection of U373-MAGI-CCR5 cells by HIV-1 Ba-L indicated that the sulfated glycodendrimers were better inhibitors than the nonsulfated glycodendrimers, but not as effective as dextran sulfate.     

Structural studies reveal that the diverse morphology of β2-microglobulin aggregates is a reflection of different molecular architectures

Amyloid deposition accompanies over 20 degenerative diseases in human, including Alzheimer's, Parkinson's, and prion diseases. Recent studies revealed the importance of other type of protein aggregates, e.g., non-specific aggregates, protofibrils, and small oligomers in the development of such diseases and proved their increased toxicity for living cells in comparison with mature amyloid fibrils. We carried out a comparative structural analysis of different monomeric and aggregated states of β₂-microglobulin, a protein responsible for hemodialysis-related amyloidosis. We investigated the structure of the native and acid-denatured states, as well as that of mature fibrils, immature fibrils, amorphous aggregates, and heat-induced filaments, prepared under various in vitro conditions. Infrared spectroscopy demonstrated that the β-sheet compositions of immature fibrils, heat-induced filaments and amorphous aggregates are characteristic of antiparallel intermolecular β-sheet structure while mature fibrils are different from all others suggesting a unique overall structure and assembly. Filamentous aggregates prepared by heat treatment are of importance in understanding the in vivo disease because of their stability under physiological conditions, where amyloid fibrils and protofibrils formed at acidic pH depolymerize. Atomic force microscopy of heat-induced filaments represented a morphology similar to that of the low pH immature fibrils. At a pH close to the pI of the protein, amorphous aggregates were formed readily with association of the molecules in native-like conformation, followed by formation of intermolecular β-sheet structure in a longer time-scale. Extent of the core buried from the solvent in the various states was investigated by H/D exchange of the amide protons.     

An unusual soluble beta-turn-rich conformation of prion is involved in fibril formation and toxic to neuronal cells

A key molecular event in prion diseases is the conversion of the prion protein (PrP) from its normal cellular form (PrPC) to the disease-specific form (PrPSc). The transition from PrPC to PrPSc involves a major conformational change, resulting in amorphous protein aggregates and fibrillar amyloid deposits with increased beta-sheet structure. Using recombinant PrP refolded into a beta-sheet-rich form (beta-PrP) we have studied the fibrillization of beta-PrP both in solution and in association with raft membranes. In low ionic strength thick dense fibrils form large networks, which coexist with amorphous aggregates. High ionic strength results in less compact fibrils, that assemble in large sheets packed with globular PrP particles, resembling diffuse aggregates found in ex vivo preparations of PrPSc. Here we report on the finding of a beta-turn-rich conformation involved in prion fibrillization that is toxic to neuronal cells in culture. This is the first account of an intermediate in prion fibril formation that is toxic to neuronal cells. We propose that this unusual beta-turn-rich form of PrP may be a precursor of PrPSc and a candidate for the neurotoxic molecule in prion pathogenesis.     

Surface structure of amyloid-beta fibrils contributes to cytotoxicity

Amyloid beta (Abeta) toxicity has been hypothesized to initiate the pathogenesis of Alzheimer's disease (AD). The characteristic fibrillar morphology of Abeta-aggregates, that constitute the main components of senile plaque, has long been considered to account for the neurotoxicity. But recent reports argue against a primary role for mature fibrils in AD pathogenesis because of the lack of a robust correlation between the severity of neurological impairment and the extent of amyloid deposition. Toxicity from the soluble prefibrillar intermediate entity of aggregates often called oligomer has recently proposed a plausible explanation for this inconsistency. An alternative explanation is based on the observation that certain amyloid fibril morphologies are more toxic than others, indicating that not all amyloid fibrils are equally toxic. Here, we report that it is not only the beta-sheeted fibrillar structure but also the surface physicochemical composition that affects the toxicity of Abeta fibrils. For the first time, colloidal gold was used to visualize by electron microscopy positive-charge clusters on Abeta fibrils. Chemical modifications as well as point-mutated Abeta synthesis techniques were applied to change the surface structures of Abeta and to show how local structure affects surface properties that are responsible for electrostatic and hydrophobic interactions with cells. We also report that covering the surface of Abeta fibers with myelin basic protein, which has surface properties contrary to those of Abeta, suppresses Abeta toxicity. On the basis of these results, we propose that the surface structure of Abeta fibrils plays an important role in Abeta toxicity.