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.     

Sedimentation velocity analysis of amyloid oligomers and fibrils using fluorescence detection

The assembly of proteins into large fibrillar aggregates, known as amyloid fibrils, is associated with a number of common and debilitating diseases. In some cases, proteins deposit extracellularly, while in others the aggregation is intracellular. A common feature of these diseases is the presence of aggregates of different sizes, including mature fibrils, small oligomeric precursors, and other less well understood structural forms such as amorphous aggregates. These various species possess distinct biochemical, biophysical, and pathological properties. Here, we detail a number of techniques that can be employed to examine amyloid fibrils and oligomers using a fluorescence-detection system (FDS) coupled with the analytical ultracentrifuge. Sedimentation velocity analysis using fluorescence detection is a particularly useful method for resolving the complex heterogeneity present in amyloid systems and can be used to characterize aggregation in exceptional detail. Furthermore, the fluorescence detection module provides a number of particularly attractive features for the analysis of aggregating proteins. It expands the practical range of concentrations of aggregating proteins under study, which is useful for greater insight into the aggregation process. It also enables the assessment of aggregation behavior in complex biological solutions, such as cell lysates, and the assessment of processes that regulate in-cell or extracellular aggregation kinetics. Four methods of fluorescent detection that are compatible with the current generation of FDS instrumentation are described: (1) Detection of soluble amyloid fibrils using a covalently bound fluorophore. (2) Detection of amyloid fibrils using an extrinsic dye that emits fluorescence when bound to fibrils. (3) Detection of fluorescently-labeled lipids and their interaction with oligomeric amyloid intermediates. (4) Detection of green fluorescence protein (GFP) constructs and their interactions within mammalian cell lysates.     

Heat shock protein 70 prevents both tau aggregation and the inhibitory effects of preexisting tau aggregates on fast axonal transport

Aggregation and accumulation of the microtubule-associated protein tau are associated with cognitive decline and neuronal degeneration in Alzheimer's disease and other tauopathies. Thus, preventing the transition of tau from a soluble state to insoluble aggregates and/or reversing the toxicity of existing aggregates would represent a reasonable therapeutic strategy for treating these neurodegenerative diseases. Here we demonstrate that molecular chaperones of the heat shock protein 70 (Hsp70) family are potent inhibitors of tau aggregation in vitro, preventing the formation of both mature fibrils and oligomeric intermediates. Remarkably, addition of Hsp70 to a mixture of oligomeric and fibrillar tau aggregates prevents the toxic effect of these tau species on fast axonal transport, a critical process for neuronal function. When incubated with preformed tau aggregates, Hsp70 preferentially associated with oligomeric over fibrillar tau, suggesting that prefibrillar oligomeric tau aggregates play a prominent role in tau toxicity. Taken together, our data provide a novel molecular basis for the protective effect of Hsp70 in tauopathies.     

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

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.     

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.     

Polymorphism in the intermediates and products of amyloid assembly

Amyloid formation reactions exhibit two classes of polymorphisms: the metastable intermediates commonly observed during amyloid formation and the range of conformationally distinct mature fibrils often seen at the reaction endpoint. Although recent data suggest that spherical oligomers and protofibrils in most cases are not obligate intermediates of amyloid assembly, oligomeric states might sometimes serve as on-pathway intermediates. Mature amyloid polymorphs self-propagate as a result of the normally very high fidelity of amyloid elongation, giving rise to strain behavior and species barriers in prion phenomena. Oligomers, protofibrils and various polymorphic forms of mature amyloid fibrils seem to be distinguished by differences in atomic structure that give rise to differences in observed morphologies.     

Dense shell glycodendrimers as potential nontoxic anti-amyloidogenic agents in Alzheimer's disease. Amyloid-dendrimer aggregates morphology and cell toxicity

Dendrimers have been proved to interact with amyloids, although most of dendrimers assayed in amyloidogenic systems are toxic to cells. The development of glycodendrimers, poly(propyleneimine) (PPI) dendrimers decorated with maltose (Mal), represents the possibility of using dendrimers with a low intrinsic toxicity. In the present paper we show that fourth (PPI-G4-Mal) and fifth (PPI-G5-Mal) generation glycodendrimers have the capacity to interfere with Alzheimer's amyloid peptide Aβ(1-40) fibrilization. The interaction is generation dependent: PPI-G5-Mal blocks amyloid fibril formation generating granular nonfibrillar amorphous aggregates, whereas PPI-G4-Mal generates clumped fibrils at low dendrimer-peptide ratios and amorphous aggregates at high ratios. Both PPI-G4-Mal and PPI-G5-Mal are nontoxic to PC12 and SH-SY5Y cells. PPI-G4-Mal reduces amyloid toxicity by clumping fibrils together, whereas amorphous aggregates are toxic to PC12 cells. The results show that glycodendrimers are promising nontoxic agents in the search for anti-amyloidogenic compounds. Fibril clumping may be an anti-amyloid toxicity strategy.     

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

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 (PC12^undiff). 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.     

Antifibrillizing agents catalyze the formation of unstable intermediate aggregates of beta‐amyloid

Although Alzheimer's disease (AD) is characterized by the extracellular deposition of fibrillar aggregates of beta‐amyloid (Aβ), transient oligomeric species of Aβ are increasingly implicated in the pathogenesis of AD. Natively unfolded monomeric Aβ can misfold and progressively assemble into fibrillar aggregates, following a well‐established “on pathway” seeded‐nucleation mechanism. Here, we show that three simple saccharides, mannose, sucrose, and raffinose, alter Aβ aggregation kinetics and morphology. The saccharides inhibit formation of Aβ fibrils but promote formation of various oligomeric aggregate species through different “off pathway” aggregation mechanisms at 37°C but not at 60°C. The various oligomeric Aβ aggregates formed when coincubated with the different saccharides are morphologically distinct but all are toxic toward SH‐SY5Y human neuroblastoma cells, increasing the level of toxicity and greatly prolonging toxicity compared with Aβ alone. As a wide variety of anti‐Aβ aggregation strategies are being actively pursued as potential therapeutics for AD, these studies suggest that care must be taken to ensure that the therapeutic agents also block toxic oligomeric Aβ assembly as well as inhibit fibril formation. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Amyloid toxicity in skeletal myoblasts: Implications for inclusion-body myositis

Skeletal muscle disorder, inclusion-body myositis (IBM) has been known for accumulation of amyloid characteristic proteins in muscle. To understand the biophysical basis of IBM, the interaction of amyloid fibrils with skeletal myoblast cells (SMC) has been studied in vitro. Synthetic insulin fibrils and Aβ_25-35 fibrils were used for this investigation. From the saturation binding analysis, the calculated dissociation constant (K_d) for insulin fibril and Aβ_25-35 fibrils were 69.37 ± 11.17 nM and 115.60 ± 12.17 nM, respectively. The fibrillar insulin comparatively has higher affinity binding to SMC than Aβ fibrils. The competitive binding studies with native insulin showed that the amount of bound insulin fibril was significantly decreased due to displacement of native insulin. However, the presence of native insulin is not altered the binding of β-amyloid fibril. The cytotoxicity of insulin amyloid intermediates was measured. The pre-fibrillar intermediates of insulin showed significant toxicity (35%) as compared to matured fibrils. Myoblast treated with β-amyloid fibrils showed more oxidative damage than the insulin fibril. Cell differentiating action of amyloidic insulin was assayed by creatine kinase activity. The insulin fibril treated cells differentiated more slowly compared to native insulin. However, β-amyloid fibrils do not show cell differentiation property. These findings reinforce the hypothesis that accumulation of amyloid related proteins is significant for the pathological events that could lead to muscle degeneration and weakness in IBM.     

All or none fibrillogenesis of a prion peptide.

Amyloid proteins and peptides comprise a diverse group of molecules that vary both in size and amino-acid sequence, yet assemble into amyloid fibrils that have a common core structure. Kinetic studies of amyloid fibrillogenesis have revealed that certain amyloid proteins form oligomeric intermediates prior to fibril formation. We have investigated fibril formation with a peptide corresponding to residues 195-213 of the human prion protein. Through a combination of kinetic and equilibrium studies, we have found that the fibrillogenesis of this peptide proceeds as an all-or-none reaction where oligomeric intermediates are not stably populated. This variation in whether oligomeric intermediates are stably populated during fibril formation indicates that amyloid proteins assemble into a common fibrillar structure; however, they do so through different pathways.     

Huntingtin spheroids and protofibrils as precursors in polyglutamine fibrilization

The pathology of Huntington's disease is characterized by neuronal degeneration and inclusions containing N-terminal fragments of mutant huntingtin (htt). To study htt aggregation, we examined purified htt fragments in vitro, finding globular and protofibrillar intermediates participating in the genesis of mature fibrils. These intermediates were high in beta-structure. Furthermore, Congo Red, a dye that stains amyloid fibrils, prevented the assembly of mutant htt into mature fibrils, but not the formation of protofibrils. Other proteins capable of forming ordered aggregates, such as amyloid beta and alpha-synuclein, form similar intermediates, suggesting that the mechanisms of mutant htt aggregation and possibly htt toxicity may overlap with other neurodegenerative disorders.     

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.     

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.     

Microcin amyloid fibrils A are reservoir of toxic oligomeric species

Microcin E492 (Mcc), a low molecular weight bacteriocin produced by Klebsiella pneumoniae RYC492, has been shown to exist in two forms: soluble forms that are believed to be toxic to the bacterial cell by forming pores and non-toxic fibrillar forms that share similar biochemical and biophysical properties with amyloids associated with several human diseases. Here we report that fibrils polymerized in vitro from soluble forms sequester toxic species that can be released upon changing environmental conditions such as pH, ionic strength, and upon dilution. Our results indicate that basic pH (≥8.5), low NaCl concentrations (≤50 mm), and dilution (>10-fold) destabilize Mcc fibrils into more soluble species that are found to be toxic to the target cells. Additionally, we also found a similar conversion of non-toxic fibrils into highly toxic oligomers using Mcc aggregates produced in vivo. Moreover, the soluble protein released from fibrils is able to rapidly polymerize into amyloid fibrils under fibril-forming conditions and to efficiently seed aggregation of monomeric Mcc. Our findings indicate that fibrillar forms of Mcc constitute a reservoir of toxic oligomeric species that is released into the medium upon changing the environmental conditions. These findings may have substantial implications to understand the dynamic process of interconversion between toxic and non-toxic aggregated species implicated in protein misfolding diseases.     

Amyloid fibrils compared to peptide nanotubes

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.     

Soluble oligomers are sufficient for transmission of a yeast prion but do not confer phenotype

Amyloidogenic proteins aggregate through a self-templating mechanism that likely involves oligomeric or prefibrillar intermediates. For disease-associated amyloidogenic proteins, such intermediates have been suggested to be the primary cause of cellular toxicity. However, isolation and characterization of these oligomeric intermediates has proven difficult, sparking controversy over their biological relevance in disease pathology. Here, we describe an oligomeric species of a yeast prion protein in cells that is sufficient for prion transmission and infectivity. These oligomers differ from the classic prion aggregates in that they are soluble and less resistant to SDS. We found that large, SDS-resistant aggregates were required for the prion phenotype but that soluble, more SDS-sensitive oligomers contained all the information necessary to transmit the prion conformation. Thus, we identified distinct functional requirements of two types of prion species for this endogenous epigenetic element. Furthermore, the nontoxic, self-replicating amyloid conformers of yeast prion proteins have again provided valuable insight into the mechanisms of amyloid formation and propagation in cells.     

Oligomeric assembly of native-like precursors precedes amyloid formation by beta-2 microglobulin

The deposition of beta-2-microglobulin (beta2m) as amyloid fibers results in debilitating complications for renal failure patients who are treated by hemodialysis. In vitro, wild-type beta2m can be converted to amyloid under physiological conditions by exposure to biomedically relevant concentrations of Cu(2+). In this work, we have made comparative measurements of the structural and oligomeric changes in beta2m at time points preceding fibrillogenesis. Our results show Cu(2+) mediates the formation of a monomeric, activated state followed by the formation of a discrete dimeric intermediate. The dimeric intermediates then assemble into tetra- and hexameric forms which display little additional oligomerization on the time scales of their own formation (<1 h). Amyloid fiber formation progresses from these intermediate states but on much longer time scales (>1 week). Although Cu(2+) is necessary for the generation and stabilization of these intermediates, it is not required for the stability of mature amyloid fibers. This suggests that Cu(2+) acts as an initiating factor of amyloidosis by inducing oligomer formation. (1)H NMR and near-UV circular dichroism are used to establish that oligomeric intermediates are native-like in structure. The native-like structure and discrete oligomeric size of beta2m amyloid intermediates suggest that this protein forms fibrils by structural domain swapping.     

Early mechanisms of amyloid fibril nucleation in model and disease-related proteins

Protein amyloid aggregation is a hallmark in neuropathologies and other diseases of tremendous impact such as Alzheimer’s or Parkinson’s diseases. During the last decade, it has become increasingly evident that neuronal death is mainly induced by proteinaceous oligomers rather than the mature amyloid fibrils. Therefore, the earliest molecular events occurring during the amyloid aggregation cascade represent a growing interest of study.Important breakthroughs have been achieved using experimental data from different proteins, used as models, as well as systems related to diseases. Here, we summarize the structural properties of amyloid oligomeric and fibrillar aggregates and review the recent advances on how biophysical techniques can be combined with quantitative kinetic analysis and theoretical models to study the detailed mechanism of oligomer formation and nucleation of fibrils.These insights into the mechanism of early oligomerization and amyloid nucleation are of relevant interest in drug discovery and in the design of preventive strategies against neurodegenerative diseases.     

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.