Amyloid Fibrils Trigger the Release of Neutrophil Extracellular Traps (NETs), Causing Fibril Fragmentation by NET-associated Elastase

The accumulation of amyloid fibrils is a feature of amyloid diseases, where cell toxicity is due to soluble oligomeric species that precede fibril formation or are formed by fibril fragmentation, but the mechanism(s) of fragmentation is still unclear. Neutrophil-derived elastase and histones were found in amyloid deposits from patients with different systemic amyloidoses. Neutrophil extracellular traps (NETs) are key players in a death mechanism in which neutrophils release DNA traps decorated with proteins such as elastase and histones to entangle pathogens. Here, we asked whether NETs are triggered by amyloid fibrils, reasoning that because proteases are present in NETs, protease digestion of amyloid may generate soluble, cytotoxic species. We show that amyloid fibrils from three different sources (α-synuclein, Sup35, and transthyretin) induced NADPH oxidase-dependent NETs in vitro from human neutrophils. Surprisingly, NET-associated elastase digested amyloid fibrils into short species that were cytotoxic for BHK-21 and HepG2 cells. In tissue sections from patients with primary amyloidosis, we also observed the co-localization of NETs with amyloid deposits as well as with oligomers, which are probably derived from elastase-induced fibril degradation (amyloidolysis). These data reveal that release of NETs, so far described to be elicited by pathogens, can also be triggered by amyloid fibrils. Moreover, the involvement of NETs in amyloidoses might be crucial for the production of toxic species derived from fibril fragmentation.     

Amyloidogenic properties of the prion protein fragment PrP(185-208): comparison with Alzheimer's peptide Abeta(1-28), influence of heparin and cell toxicity

Amyloid fibrils are a hallmark of Alzheimer’s and prion diseases. In both pathologies fibrils are found associated to glycosaminoglycans, modulators of the aggregation process. Amyloid peptides and proteins with very poor sequence homologies originate very similar aggregates. This implies the possible existence of a common formation mechanism. A homologous structural motif has recently been described for the Alzheimer’s peptide Aβ(1-28) and the prion protein fragment PrP(185-208). We have studied the influence histidine residues and heparin on the aggregation process of both peptides and determined the possible amyloid characteristics of PrP(185-208), still unknown. The results show that PrP(185-208) forms amyloid aggregates in the presence of heparin. Histidines influence the aggregation kinetics, as in Aβ(1-28), although to a lesser extent. Other spectroscopic properties of the PrP(185-208) fragment are shown to be equivalent to those of other amyloid peptides and PrP(185-208) is shown to be cytotoxic using a neuroblastoma cell line.     

Understanding protein folding from globular to amyloid state: Aggregation: Darker side of protein

Folding and unfolding are crucial ways of modulating biological activity and targeting proteins to different cellular locations. In the living system, protein folding occurs in a very crowded environment, often assisted with helper proteins. In some cases this pathway can go off beam and the protein can either misfold or aggregate or form structures of elongated-unbranched morphology known as amyloid fibrils. Protein folding is not just an academic matter. Recombinant biotechnology and pharmaceutical industries are some of the fields where both theoretical and practical knowledge of protein folding is required. Misfolded protein and amyloid fibrils that escape the cellular quality control check are the basic reason of a number of increasingly widespread neurodegenerative diseases such as Alzheimer's and variant Creutzfeldt-Jakob etc. Thus, protein folding study also emerges as an interesting area in the field of biomedical research. This review deals with basic concepts related to protein folding and misfolding forming toxic aggregates and amyloid fibrils as well as disease associated with them. A more practical approach will be revealed to the early diagnosis of aggregation-prone diseases and amyloid states and their balanced therapeutics.     

Evaluating nuclei concentration in amyloid fibrillation reactions using back-calculation approach

Background

In spite of our extensive knowledge of the more than 20 proteins associated with different amyloid diseases, we do not know how amyloid toxicity occurs or how to block its action. Recent contradictory reports suggest that the fibrils and/or the oligomer precursors cause toxicity. An estimate of their temporal concentration may broaden understanding of the amyloid aggregation process.

Methodology/Principal Findings

Assuming that conversion of folded protein to fibril is initiated by a nucleation event, we back-calculate the distribution of nuclei concentration. The temporal in vitro concentration of nuclei for the model hormone, recombinant human insulin, is estimated to be in the picomolar range. This is a conservative estimate since the back-calculation method is likely to overestimate the nuclei concentration because it does not take into consideration fibril fragmentation, which would lower the amount of nuclei

Conclusions

Because of their propensity to form aggregates (non-ordered) and fibrils (ordered), this very low concentration could explain the difficulty in isolating and blocking oligomers or nuclei toxicity and the long onset time for amyloid diseases.     

Stepwise Organization of the β-Structure Identifies Key Regions Essential for the Propagation and Cytotoxicity of Insulin Amyloid Fibrils

Amyloid fibrils are supramolecular assemblies, the deposition of which is associated with many serious diseases including Alzheimer, prion, and Huntington diseases. Several smaller aggregates such as oligomers and protofibrils have been proposed to play a role in early stages of the fibrillation process; however, little is known about how these species contribute to the formation of mature amyloid fibrils with a rigid cross-β structure. Here, we identified a new pathway for the formation of insulin amyloid fibrils at a high concentration of salt in which mature fibrils were formed in a stepwise manner via a prefibrillar intermediate: minute prefibrillar species initially accumulated, followed by the subsequent formation of thicker amyloid fibrils. Fourier transform infrared spectra suggested the sequential formation of two types of β-sheets with different strength hydrogen bonds, one of which was developed concomitantly with the mutual assembly of the prefibrillar intermediate to form mature fibrils. Interestingly, fibril propagation and cellular toxicity appeared only after the later step of structural organization, and a comparison of β-sheet regions between the prefibrillar intermediate and mature fibrils using proteolysis led to the proposal of specific regions essential for manifestation of these properties.     

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 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.     

Vortex-Induced Formation of Insulin Amyloid Superstructures Probed by Time-Lapse Atomic Force Microscopy and Circular Dichroism Spectroscopy

Misfolding and aggregation of proteins are multipathway processes that result in polymorphism of amyloid fibrils. While agitation is one of the most common means of inducing structural variants of fibrils (the so-called ‘amyloid strains’), there is as yet no mechanistic explanation for this effect. In this study, time-lapse atomic force microscopy has been employed to probe insulin fibrillation upon intensive agitation. At 60 °C, the initial stages of aggregation in agitated samples are similar to those in quiescent solutions; however, in vortexed samples, an abrupt and highly cooperative collapse of early filaments occurs, yielding twisted and laterally aligned aggregates with defined chiroptical properties. In the absence of any detectable birefringence and linear dichroism, the observed strong Cotton effect is attributed to twisted chiral amyloid superstructures. Early fibrils formed in agitated samples, but transferred to quiescent conditions before the collapse event, do not form the superstructures. On the other hand, mature insulin fibrils grown in quiescent samples and later subjected to rapid vortexing transform into clumps of finely broken fibers lacking the superstructural chirality and chiroptical properties of the continuously agitated samples. A generalized mechanism of inducing structural variants of amyloid fibrils by hydrodynamic forces favoring secondary nucleation events over elongation of fibrils is put forward. We propose that competition between low-aspect-ratio and high-aspect-ratio amyloidogenic pathways driven by fluid dynamics may play an important role in promoting distinct amyloid strains.     

Nucleation of β-rich oligomers and β-barrels in the early aggregation of human islet amyloid polypeptide

The self-assembly of human islet amyloid polypeptide (hIAPP) into β-sheet rich amyloid aggregates is associated with pancreatic β-cell death in type 2 diabetes (T2D). Prior experimental studies of hIAPP aggregation reported the early accumulation of α-helical intermediates before the rapid conversion into β-sheet rich amyloid fibrils, as also corroborated by our experimental characterizations with transmission electron microscopy and Fourier transform infrared spectroscopy. Although increasing evidence suggests that small oligomers populating early hIAPP aggregation play crucial roles in cytotoxicity, structures of these oligomer intermediates and their conformational conversions remain unknown, hindering our understanding of T2D disease mechanism and therapeutic design targeting these early aggregation species. We further applied large-scale discrete molecule dynamics simulations to investigate the oligomerization of full-length hIAPP, employing multiple molecular systems of increasing number of peptides. We found that the oligomerization process was dynamic, involving frequent inter-oligomeric exchanges. On average, oligomers had more α-helices than β-sheets, consistent with ensemble-based experimental measurements. However, in ~4–6% independent simulations, β-rich oligomers expected as the fibrillization intermediates were observed, especially in the pentamer and hexamer simulations. These β-rich oligomers could adopt β-barrel conformations, recently postulated to be the toxic oligomer species but only observed computationally in the aggregates of short amyloid protein fragments. Free-energy analysis revealed high energies of these β-rich oligomers, supporting the nucleated conformational changes of oligomers in amyloid aggregation. β-barrel oligomers of full-length hIAPP with well-defined three-dimensional structures may play an important pathological role in T2D etiology and may be a therapeutic target for the disease.     

Only amyloidogenic intermediates of transthyretin induce apoptosis

In diseases like Alzheimer's disease and familial amyloidotic polyneuropathy (FAP) amyloid deposits co-localize with areas of neurodegeneration. FAP is associated with mutations of the plasma protein transthyretin (TTR). We can here show an apoptotic effect of amyloidogenic mutants of TTR on a human neuroblastoma cell line. Toxicity could be blocked by catalase indicating a free oxygen radical dependent mechanism. The toxic effect was dependent on the state of aggregation and unexpectedly mature fibrils from FAP-patients who failed to exert an apoptotic response. Morphological studies revealed a correlation between toxicity and the presence of immature amyloid. Thus, we can show that toxicity is associated with early stages of fibril formation and propose that mature full-length fibrils represent an inert end stage, which might serve as a rescue mechanism.     

Can size alone explain some of the differences in toxicity between β‐amyloid oligomers and fibrils?

β‐Amyloid (Aβ) peptide is believed to play a key role in the mechanism of Alzheimer's disease (AD). Aβ tends to aggregate to form amyloid fibrils. A variety of evidence indicates that Aβ aggregates are toxic in vitro and in vivo. An early “Aβ hypothesis” postulated that AD was the consequence of neuron death induced by insoluble deposits of large Aβ fibrils. Newer findings indicate that small soluble Aβ oligomers are the neurotoxic species, yet their structure is still unknown. Many researchers have tried to probe the differences in molecular structure between Aβ oligomers, protofibrils, and fibrils that give rise to their unique toxicities, but with limited success. In this report, we examine the hypothesis that differences in the toxicity of different aggregated Aβ species are the result of differences in species concentration and diffusivity. Using a simple mathematical analysis based on the assumption of a diffusion‐limited reaction, we demonstrate that near 10‐fold differences in toxicity between spherical oligomers and fibrils can be explained from size and concentration arguments. While this work does not suggest that Aβ oligomers and fibrils have identical molecular structures, it highlights the possibility that simple physical phenomena may contribute to the biological processes induced by Aβ. Biotechnol. Bioeng. 2010;106: 333–337. © 2010 Wiley Periodicals, Inc.     

Prion-like propagation of cytosolic protein aggregates

Amyloid formation is a hallmark of several systemic and neurodegenerative diseases. Extracellular amyloid deposits or intracellular inclusions arise from the conformational transition of normally soluble proteins into highly ordered fibrillar aggregates. Amyloid fibrils are formed by nucleated polymerization, a process also shared by prions, proteinaceous infectious agents identified in mammals and fungi. Unlike so called non-infectious amyloids, the aggregation phenotype of prion proteins can be efficiently transmitted between cells and organisms. Recent discoveries in vivo now implicate that even disease-associated intracellular protein aggregates consisting of α-synuclein or Tau have the capacity to seed aggregation of homotypic native proteins and might propagate their amyloid states in a prion-like manner. Studies in tissue culture demonstrate that aggregation of diverse intracellular amyloidogenic proteins can be induced by exogenous fibrillar seeds. Still, a prerequisite for prion-like propagation is the fragmentation of proteinaceous aggregates into smaller seeds that can be transmitted to daughter cells. So far efficient propagation of the aggregation phenotype in the absence of exogenous seeds was only observed for a yeast prion domain expressed in tissue culture. Intrinsic properties of amyloidogenic protein aggregates and a suitable host environment likely determine if a protein polymer can propagate in a prion-like manner in the mammalian cytosol.     

Amyloid Protofibrils of Lysozyme Nucleate and Grow Via Oligomer Fusion

The mechanisms linking deposits of insoluble amyloid fibrils to the debilitating neuronal cell death characteristic of neurodegenerative diseases remain enigmatic. Recent findings implicate transiently formed intermediates of mature amyloid fibrils as the principal toxic agent. Hence, determining which intermediate aggregates represent on-pathway precursors or off-pathway side branches is critical for understanding amyloid self-assembly, and for devising therapeutic approaches targeting relevant toxic species. We examined amyloid fibril self-assembly in acidic solutions, using the model protein hen egg-white lysozyme. Combining in situ dynamic light scattering with calibrated atomic-force microscopy, we monitored the nucleation and growth kinetics of multiple transient aggregate species, and characterized both their morphologies and physical dimensions. Upon incubation at elevated temperatures, uniformly sized oligomers formed at a constant rate. After a lag period of several hours, protofibrils spontaneously nucleated. The nucleation kinetics of protofibrils and the tight match of their widths and heights with those of oligomers imply that protofibrils both nucleated and grew via oligomer fusion. After reaching several hundred nanometers in length, protofibrils assembled into mature fibrils. Overall, the amyloid fibril assembly of lysozyme followed a strict hierarchical aggregation pathway, with amyloid monomers, oligomers, and protofibrils forming on-pathway intermediates for assembly into successively more complex structures.     

Prion-like propagation of cytosolic protein aggregates: Insights from cell culture models

Amyloid formation is a hallmark of several systemic and neurodegenerative diseases. Extracellular amyloid deposits or intracellular inclusions arise from the conformational transition of normally soluble proteins into highly ordered fibrillar aggregates. Amyloid fibrils are formed by nucleated polymerization, a process also shared by prions, proteinaceous infectious agents identified in mammals and fungi. Unlike so called non-infectious amyloids, the aggregation phenotype of prion proteins can be efficiently transmitted between cells and organisms. Recent discoveries in vivo now implicate that even disease-associated intracellular protein aggregates consisting of α-synuclein or Tau have the capacity to seed aggregation of homotypic native proteins and might propagate their amyloid states in a prion-like manner. Studies in tissue culture demonstrate that aggregation of diverse intracellular amyloidogenic proteins can be induced by exogenous fibrillar seeds. Still, a prerequisite for prion-like propagation is the fragmentation of proteinaceous aggregates into smaller seeds that can be transmitted to daughter cells. So far efficient propagation of the aggregation phenotype in the absence of exogenous seeds was only observed for a yeast prion domain expressed in tissue culture. Intrinsic properties of amyloidogenic protein aggregates and a suitable host environment likely determine if a protein polymer can propagate in a prion-like manner in the mammalian cytosol.Key words: prion, Sup35, huntingtin, polyglutamine, Tau, co-aggregation, amyloid, α-synuclein     

Soluble oligomers from a non-disease related protein mimic Abeta-induced tau hyperphosphorylation and neurodegeneration

Protein aggregation and amyloid accumulation in different tissues are associated with cellular dysfunction and toxicity in important human pathologies, including Alzheimer's disease and various forms of systemic amyloidosis. Soluble oligomers formed at the early stages of protein aggregation have been increasingly recognized as the main toxic species in amyloid diseases. To gain insight into the mechanisms of toxicity instigated by soluble protein oligomers, we have investigated the aggregation of hen egg white lysozyme (HEWL), a normally harmless protein. HEWL initially aggregates into beta-sheet rich, roughly spherical oligomers which appear to convert with time into protofibrils and mature amyloid fibrils. HEWL oligomers are potently neurotoxic to rat cortical neurons in culture, while mature amyloid fibrils are little or non-toxic. Interestingly, when added to cortical neuronal cultures HEWL oligomers induce tau hyperphosphorylation at epitopes that are characteristically phosphorylated in neurons exposed to soluble oligomers of the amyloid-beta peptide. Furthermore, injection of HEWL oligomers in the cerebral cortices of adult rats induces extensive neurodegeneration in different brain areas. These results show that soluble oligomers from a non-disease related protein can mimic specific neuronal pathologies thought to be induced by soluble amyloid-beta peptide oligomers in Alzheimer's disease and support the notion that amyloid oligomers from different proteins may share common structural determinants that would explain their generic cytotoxicities.     

Converse modulation of toxic α-synuclein oligomers in living cells by N′-benzylidene-benzohydrazide derivates and ferric iron

Intracellular α-synuclein (α-syn) aggregates are the pathological hallmark in several neurodegenerative diseases including Parkinson’s disease, dementia with Lewy bodies and multiple system atrophy. Recent evidence suggests that small oligomeric aggregates rather than large amyloid fibrils represent the main toxic particle species in these diseases. We recently characterized iron-dependent toxic α-syn oligomer species by confocal single molecule fluorescence techniques and used this aggregation model to identify several N′-benzylidene-benzohydrazide (NBB) derivatives inhibiting oligomer formation in vitro. In our current work, we used the bioluminescent protein-fragment complementation assay (BPCA) to directly analyze the formation of toxic α-syn oligomers in cell culture and to investigate the effect of iron and potential drug-like compounds in living cells. Similar to our previous findings in vitro, we found a converse modulation of toxic α-syn oligomers by NBB derivates and ferric iron, which was characterized by an increase in aggregate formation by iron and an inhibitory effect of certain NBB compounds. Inhibition of α-syn oligomer formation by the NBB compound 293G02 was paralleled by a reduction in cytotoxicity indicating that toxic α-syn oligomers are present in the BPCA cell culture model and that pharmacological inhibition of oligomer formation can reduce toxicity. Thus, this approach provides a suitable model system for the development of new disease-modifying drugs targeting toxic oligomer species. Moreover, NBB compounds such as 293G02 may provide useful tool compounds to dissect the functional role of toxic oligomer species in cell culture models and in vivo.     

Steroid Hormones Block Amyloid Fibril-Induced 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) Formazan Exocytosis: Relationship to Neurotoxicity

Abstract: Perhaps the most reproducible early event induced by the interaction of amyloid β peptide (Aβ) with the cell is the inhibition of cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction. We recently demonstrated that cytotoxic amyloid peptides such as Aβ and human amylin inhibit cellular MTT reduction by dramatically enhancing MTT formazan exocytosis. We now show the following: (a) Insulin and glucagon, when converted to fibrils with β-pleated sheet structure, induce MTT formazan exocytosis that is indistinguishable from that induced by Aβ. NAC35, an amyloidogenic fragment of α-synuclein (or NACP), also induces MTT formazan exocytosis. (b) All protein fibrils with the β-pleated sheet structure examined are toxic to rat hippocampal neurons. (c) Many sterol sex hormones (e.g., estradiol and progesterone) block amyloid fibril-enhanced MTT formazan exocytosis as well as MTT formazan exocytosis in control cells by acting at a common late step in the exocytic pathway. Steroids fail, however, to protect hippocampal neurons from acute amyloid fibril toxicity. These findings suggest that the ability to enhance MTT formazan exocytosis and to induce neurotoxicity are common biological activities of protein fibrils with β-pleated sheet structure but that enhanced MTT formazan exocytosis is not sufficient for acute Aβ neurotoxicity.     

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

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.     

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.     

Analysis of the length distribution of amyloid fibrils by centrifugal sedimentation

The aggregation of normally soluble peptides and proteins into amyloid fibrils is a process associated with a wide range of pathological conditions, including Alzheimer's and Parkinson's diseases. It has become apparent that aggregates of different sizes possess markedly different biological effects, with aggregates of lower relative molecular weight being associated with stronger neurotoxicity. Yet, although many approaches exist to measure the total mass concentration of aggregates, the ability to probe the length distribution of growing aggregates in solution has remained more elusive. In this work, we applied a differential centrifugation technique to measure the sedimentation coefficients of amyloid fibrils produced during the aggregation process of the amyloid β (M1–42) peptide (Aβ42). The centrifugal method has the advantage of providing structural information on the fibril distribution directly in solution and affording a short analysis time with respect to alternative imaging and analytical centrifugation approaches. We show that under quiescent conditions interactions between Aβ42 fibrils lead to lateral association and to the formation of entangled clusters. By contrast, aggregation under shaking generates a population of filaments characterized by shorter lengths. The results, which have been validated by cryogenic transmission electron microscopy (cryo-TEM) analysis, highlight the important role that fibril–fibril assembly can play in the deposition of aggregation-prone peptides.