Seed amplification and neurodegeneration marker trajectories in individuals at risk of prion disease.

Human prion diseases are remarkable for long incubation times followed typically by rapid clinical decline. Seed amplification assays and neurodegeneration biofluid biomarkers are remarkably useful in the clinical phase, but their potential to predict clinical onset in healthy people remains unclear. This is relevant not only to the design of preventive strategies in those at-risk of prion diseases, but more broadly, because prion-like mechanisms are thought to underpin many neurodegenerative disorders. Here, we report the accrual of a longitudinal biofluid resource in patients, controls and healthy people at risk of prion diseases, to which ultrasensitive techniques such as real-time quaking-induced conversion (RT-QuIC) and single molecule array (Simoa) digital immunoassays were applied for preclinical biomarker discovery. We studied 648 CSF and plasma samples, including 16 people who had samples taken when healthy but later developed inherited prion disease (IPD) ('converters'; range from 9.9 prior to, and 7.4 years after onset). Symptomatic IPD CSF samples were screened by RT-QuIC assay variations, before testing the entire collection of at-risk samples using the most sensitive assay. Glial fibrillary acidic protein (GFAP), neurofilament light (NfL), tau and UCH-L1 levels were measured in plasma and CSF. Second generation (IQ-CSF) RT-QuIC proved 100% sensitive and specific for sporadic Creutzfeldt-Jakob disease (CJD), iatrogenic and familial CJD phenotypes, and subsequently detected seeding activity in four presymptomatic CSF samples from three E200K carriers; one converted in under 2 months while two remain asymptomatic after at least 3 years' follow-up. A bespoke HuPrP P102L RT-QuIC showed partial sensitivity for P102L disease. No compatible RT-QuIC assay was discovered for classical 6-OPRI, A117V and D178N, and these at-risk samples tested negative with bank vole RT-QuIC. Plasma GFAP and NfL, and CSF NfL levels emerged as proximity markers of neurodegeneration in the typically slow IPDs (e.g. P102L), with significant differences in mean values segregating healthy control from IPD carriers (within 2 years to onset) and symptomatic IPD cohorts; plasma GFAP appears to change before NfL, and before clinical conversion. In conclusion, we show distinct biomarker trajectories in fast and slow IPDs. Specifically, we identify several years of presymptomatic seeding positivity in E200K, a new proximity marker (plasma GFAP) and sequential neurodegenerative marker evolution (plasma GFAP followed by NfL) in slow IPDs. We suggest a new preclinical staging system featuring clinical, seeding and neurodegeneration aspects, for validation with larger prion at-risk cohorts, and with potential application to other neurodegenerative proteopathies.

Desmin forms toxic, seeding-competent amyloid aggregates that persist in muscle fibers.

Desmin-associated myofibrillar myopathy (MFM) has pathologic similarities to neurodegeneration-associated protein aggregate diseases. Desmin is an abundant muscle-specific intermediate filament, and disease mutations lead to its aggregation in cells, animals, and patients. We reasoned that similar to neurodegeneration-associated proteins, desmin itself may form amyloid. Desmin peptides corresponding to putative amyloidogenic regions formed seeding-competent amyloid fibrils. Amyloid formation was increased when disease-associated mutations were made within the peptide, and this conversion was inhibited by the anti-amyloid compound epigallocatechin-gallate. Moreover, a purified desmin fragment (aa 117 to 348) containing both amyloidogenic regions formed amyloid fibrils under physiologic conditions. Desmin fragment-derived amyloid coaggregated with full-length desmin and was able to template its conversion into fibrils in vitro. Desmin amyloids were cytotoxic to myotubes and disrupted their myofibril organization compared with desmin monomer or other nondesmin amyloids. Finally, desmin fragment amyloid persisted when introduced into mouse skeletal muscle. These data suggest that desmin forms seeding-competent amyloid that is toxic to myofibers. Moreover, small molecules known to interfere with amyloid formation and propagation may have therapeutic potential in MFM.

Detection of TAR DNA-binding protein 43 (TDP-43) oligomers as initial intermediate species during aggregate formation.

Aggregates of the RNA-binding protein TDP-43 (TAR DNA-binding protein) are a hallmark of the overlapping neurodegenerative disorders amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. The process of TDP-43 aggregation remains poorly understood, and whether it includes formation of intermediate complexes is unknown. Here, we analyzed aggregates derived from purified TDP-43 under semidenaturing conditions, identifying distinct oligomeric complexes at the initial time points before the formation of large aggregates. We found that this early oligomerization stage is primarily driven by TDP-43's RNA-binding region. Specific binding to GU-rich RNA strongly inhibited both TDP-43 oligomerization and aggregation, suggesting that RNA interactions are critical for maintaining TDP-43 solubility. Moreover, we analyzed TDP-43 liquid-liquid phase separation and detected similar detergent-resistant oligomers upon maturation of liquid droplets into solid-like fibrils. These results strongly suggest that the oligomers form during the early steps of TDP-43 misfolding. Importantly, the ALS-linked TDP-43 mutations A315T and M337V significantly accelerate aggregation, rapidly decreasing the monomeric population and shortening the oligomeric phase. We also show that aggregates generated from purified TDP-43 seed intracellular aggregation detected by established TDP-43 pathology markers. Remarkably, cytoplasmic aggregate seeding was detected earlier for the A315T and M337V variants and was 50% more widespread than for WT TDP-43 aggregates. We provide evidence for an initial step of TDP-43 self-assembly into intermediate oligomeric complexes, whereby these complexes may provide a scaffold for aggregation. This process is altered by ALS-linked mutations, underscoring the role of perturbations in TDP-43 homeostasis in protein aggregation and ALS-FTD pathogenesis.

Aggregation of Full-length Immunoglobulin Light Chains from Systemic Light Chain Amyloidosis (AL) Patients Is Remodeled by Epigallocatechin-3-gallate.

Intervention into amyloid deposition with anti-amyloid agents like the polyphenol epigallocatechin-3-gallate (EGCG) is emerging as an experimental secondary treatment strategy in systemic light chain amyloidosis (AL). In both AL and multiple myeloma (MM), soluble immunoglobulin light chains (LC) are produced by clonal plasma cells, but only in AL do they form amyloid deposits in vivo We investigated the amyloid formation of patient-derived LC and their susceptibility to EGCG in vitro to probe commonalities and systematic differences in their assembly mechanisms. We isolated nine LC from the urine of AL and MM patients. We quantified their thermodynamic stabilities and monitored their aggregation under physiological conditions by thioflavin T fluorescence, light scattering, SDS stability, and atomic force microscopy. LC from all patients formed amyloid-like aggregates, albeit with individually different kinetics. LC existed as dimers, ∼50% of which were linked by disulfide bridges. Our results suggest that cleavage into LC monomers is required for efficient amyloid formation. The kinetics of AL LC displayed a transition point in concentration dependence, which MM LC lacked. The lack of concentration dependence of MM LC aggregation kinetics suggests that conformational change of the light chain is rate-limiting for these proteins. Aggregation kinetics displayed two distinct phases, which corresponded to the formation of oligomers and amyloid fibrils, respectively. EGCG specifically inhibited the second aggregation phase and induced the formation of SDS-stable, non-amyloid LC aggregates. Our data suggest that EGCG intervention does not depend on the individual LC sequence and is similar to the mechanism observed for amyloid-ß and α-synuclein.

Super-resolution Imaging of Amyloid Structures over Extended Times by Using Transient Binding of Single Thioflavin†T Molecules.

Oligomeric amyloid structures are crucial therapeutic targets in Alzheimer's and other amyloid diseases. However, these oligomers are too small to be resolved by standard light microscopy. We have developed a simple and versatile tool to image amyloid structures by using thioflavin T without the need for covalent labeling or immunostaining. The dynamic binding of single dye molecules generates photon bursts that are used for fluorophore localization on a nanometer scale. Thus, photobleaching cannot degrade image quality, allowing for extended observation times. Super-resolution transient amyloid binding microscopy promises to directly image native amyloid by using standard probes and record amyloid dynamics over minutes to days. We imaged amyloid fibrils from multiple polypeptides, oligomeric, and fibrillar structures formed during different stages of amyloid-ß aggregation, as well as the structural remodeling of amyloid-ß fibrils by the compound epi-gallocatechin gallate.

Amyloid-ß(1-42) Aggregation Initiates Its Cellular Uptake and Cytotoxicity.

The accumulation of amyloid ß peptide(1-42) (Aß(1-42)) in extracellular plaques is one of the pathological hallmarks of Alzheimer disease (AD). Several studies have suggested that cellular reuptake of Aß(1-42) may be a crucial step in its cytotoxicity, but the uptake mechanism is not yet understood. Aß may be present in an aggregated form prior to cellular uptake. Alternatively, monomeric peptide may enter the endocytic pathway and conditions in the endocytic compartments may induce the aggregation process. Our study aims to answer the question whether aggregate formation is a prerequisite or a consequence of Aß endocytosis. We visualized aggregate formation of fluorescently labeled Aß(1-42) and tracked its internalization by human neuroblastoma cells and neurons. ß-Sheet-rich Aß(1-42) aggregates entered the cells at low nanomolar concentration of Aß(1-42). In contrast, monomer uptake faced a concentration threshold and occurred only at concentrations and time scales that allowed Aß(1-42) aggregates to form. By uncoupling membrane binding from internalization, we found that Aß(1-42) monomers bound rapidly to the plasma membrane and formed aggregates there. These structures were subsequently taken up and accumulated in endocytic vesicles. This process correlated with metabolic inhibition. Our data therefore imply that the formation of ß-sheet-rich aggregates is a prerequisite for Aß(1-42) uptake and cytotoxicity.

Glucose directs amyloid-beta into membrane-active oligomers.

Oligomeric amyloid-ß 1-42 (Aß-42) peptides are considered to be the most toxic species connected to the occurrence of Alzheimer's disease. However, not all aggregation conditions promote oligomer formation in vitro, raising the question whether oligomer formation in vivo also requires a specific suitable cellular environment. We recently found that interaction with neuronal membranes initiates aggregation of Aß-42 and neuronal uptake. Our data suggest that small molecules in the extracellular space can facilitate the formation of membrane-active Aß-42 oligomers. We analyzed the early stage of Aß-42 aggregation in the presence of glucose and sucrose and found that these sugars strongly favor Aß-42 oligomer formation. We characterized oligomers by dynamic light scattering, atomic force microscopy, immuno-transmission electron microscopy and fluorescence cross correlation spectroscopy. We found that Aß-42 spontaneously and rapidly forms low molecular weight oligomers in the presence of sugars. Slightly acidic pH (6.7-7) greatly favors oligomer formation when compared to the extracellular physiological pH (7.4). Circular dichroism demonstrated that these Aß-42 oligomers did not adopt a ß-sheet structure. Unstructured oligomeric Aß-42 interacted with membrane bilayers of giant unilamellar vesicles (GUV) and neuronal model cells, facilitated cellular uptake of Aß-42, and inhibition of mitochondrial activity. Our data therefore suggest that elevated concentrations of glucose within the range observed in diabetic individuals (10 mM) facilitate the formation of membrane-active Aß-42 oligomers.

Stabilization of α-Synuclein Fibril Clusters Prevents Fragmentation and Reduces Seeding Activity and Toxicity.

Protein misfolding results in the accumulation of aggregated ß-sheet-rich structures in Parkinson's disease (PD) and Alzheimer's disease. The toxic oligomer hypothesis stipulates that prefibrillar assemblies, such as soluble oligomers or protofibrils, are responsible for the poor prognosis of these diseases. Previous studies demonstrated that a small molecule related to the natural compound orcein, O4, directly binds to amyloid-ß fibrils and stabilizes them, accelerating the formation of end-stage mature fibrils. Here we demonstrate a similar phenomenon during O4 treatment of α-synuclein (αsyn) aggregates, the protein responsible for PD pathology. While the drug did not change the kinetics of aggregate formation as measured by the amyloidophilic dye thioflavin T, O4 depleted αsyn oligomers and promoted the formation of sodium dodecyl sulfate and proteinase K resistant aggregates consisting of large fibril clusters. These fibril clusters exhibited reduced toxicity to human neuronal model cells and reduced seeding activity in vitro. The effectiveness of O4 decreased when it was added at later points in the αsyn aggregation pathway, which suggests that the incorporation of O4 into fibril assemblies stabilizes them against chemical, enzymatic, and mechanic degradation. These findings suggest that small molecules, which stabilize amyloid fibrils, can prevent fibril fragmentation and seeding and consequently prevent prion-like replication of misfolded αsyn. Inhibiting prion replication by fibril stabilization could thus be a therapeutic strategy for PD.

Tau Trimers Are the Minimal Propagation Unit Spontaneously Internalized to Seed Intracellular Aggregation.

Tau amyloid assemblies propagate aggregation from the outside to the inside of a cell, which may mediate progression of the tauopathies. The critical size of Tau assemblies, or "seeds," responsible for this activity is currently unknown, but this could be important for the design of effective therapies. We studied recombinant Tau repeat domain (RD) and Tau assemblies purified from Alzheimer disease (AD) brain composed largely of full-length Tau. Large RD fibrils were first sonicated to create a range of assembly sizes. We confirmed our ability to resolve stable assemblies ranging from n = 1 to >100 units of Tau using size exclusion chromatography, fluorescence correlation spectroscopy, cross-linking followed by Western blot, and mass spectrometry. All recombinant Tau assemblies bound heparan sulfate proteoglycans on the cell surface, which are required for Tau uptake and seeding, because they were equivalently sensitive to inhibition by heparin and chlorate. However, cells only internalized RD assemblies of n ≥ 3 units. We next analyzed Tau assemblies from AD or control brains. AD brains contained aggregated species, whereas normal brains had predominantly monomer, and no evidence of large assemblies. HEK293 cells and primary neurons spontaneously internalized Tau of n ≥ 3 units from AD brain in a heparin- and chlorate-sensitive manner. Only n ≥ 3-unit assemblies from AD brain spontaneously seeded intracellular Tau aggregation in HEK293 cells. These results indicate that a clear minimum size (n = 3) of Tau seed exists for spontaneous propagation of Tau aggregation from the outside to the inside of a cell, whereas many larger sizes of soluble aggregates trigger uptake and seeding.

The Effect of (-)-Epigallo-catechin-(3)-gallate on Amyloidogenic Proteins Suggests a Common Mechanism.

Studies on the interaction of the green tea polyphenol (-)-Epigallocatechin-3-gallate (EGCG) with fourteen disease-related amyloid polypeptides and prions Huntingtin, Amyloid-beta, alpha-Synuclein, islet amyloid polypeptide (IAPP), Sup35, NM25 and NM4, tau, MSP2, semen-derived enhancer of virus infection (SEVI), immunoglobulin light chains, beta-microglobulin, prion protein (PrP) and Insulin, have yielded a variety of experimental observations. Here, we analyze whether these observations could be explained by a common mechanism and give a broad overview of the published experimental data on the actions of EGCG. Firstly, we look at the influence of EGCG on aggregate toxicity, morphology, seeding competence, stability and conformational changes. Secondly, we screened publications elucidating the biochemical mechanism of EGCG intervention, notably the effect of EGCG on aggregation kinetics, oligomeric aggregation intermediates, and its binding mode to polypeptides. We hypothesize that the experimental results may be reconciled in a common mechanism, in which EGCG binds to cross-beta sheet aggregation intermediates. The relative position of these species in the energy profile of the amyloid cascade would determine the net effect of EGCG on aggregation and disaggregation of amyloid fibrils.

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

Several lines of evidence indicate that prefibrillar assemblies of amyloid-ß (Aß) 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 Aß fibrillogenesis through the action of the orcein-related small molecule O4, which directly binds to hydrophobic amino acid residues in Aß peptides and stabilizes the self-assembly of seeding-competent, ß-sheet-rich protofibrils and fibrils. Notably, the O4-mediated acceleration of amyloid fibril formation efficiently decreases the concentration of small, toxic Aß oligomers in complex, heterogeneous aggregation reactions. In addition, O4 treatment suppresses inhibition of long-term potentiation by Aß 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.

EGCG remodels mature alpha-synuclein and amyloid-beta fibrils and reduces cellular toxicity.

Protein misfolding and formation of beta-sheet-rich amyloid fibrils or aggregates is related to cellular toxicity and decay in various human disorders including Alzheimer's and Parkinson's disease. Recently, we demonstrated that the polyphenol (-)-epi-gallocatechine gallate (EGCG) inhibits alpha-synuclein and amyloid-beta fibrillogenesis. It associates with natively unfolded polypeptides and promotes the self-assembly of unstructured oligomers of a new type. Whether EGCG disassembles preformed amyloid fibrils, however, remained unclear. Here, we show that EGCG has the ability to convert large, mature alpha-synuclein and amyloid-beta fibrils into smaller, amorphous protein aggregates that are nontoxic to mammalian cells. Mechanistic studies revealed that the compound directly binds to beta-sheet-rich aggregates and mediates the conformational change without their disassembly into monomers or small diffusible oligomers. These findings suggest that EGCG is a potent remodeling agent of mature amyloid fibrils.

Direct Observation of Competing Prion Protein Fibril Populations with Distinct Structures and Kinetics.

In prion diseases, fibrillar assemblies of misfolded prion protein (PrP) self-propagate by incorporating PrP monomers. These assemblies can evolve to adapt to changing environments and hosts, but the mechanism of prion evolution is poorly understood. We show that PrP fibrils exist as a population of competing conformers, which are selectively amplified under different conditions and can "mutate" during elongation. Prion replication therefore possesses the steps necessary for molecular evolution analogous to the quasispecies concept of genetic organisms. We monitored structure and growth of single PrP fibrils by total internal reflection and transient amyloid binding super-resolution microscopy and detected at least two main fibril populations, which emerged from seemingly homogeneous PrP seeds. All PrP fibrils elongated in a preferred direction by an intermittent "stop-and-go" mechanism, but each population possessed distinct elongation mechanisms that incorporated either unfolded or partially folded monomers. Elongation of RML and ME7 prion rods likewise exhibited distinct kinetic features. The discovery of polymorphic fibril populations growing in competition, which were previously hidden in ensemble measurements, suggests that prions and other amyloid replicating by prion-like mechanisms may represent quasispecies of structural isomorphs that can evolve to adapt to new hosts and conceivably could evade therapeutic intervention.

Loss of Residues 119-136, Including the First ß-strand of Human Prion Protein, Generates an Aggregation-competent Partially "Open" Form.

In prion replication, the cellular form of prion protein (PrPC) must undergo a full conformational transition to its disease-associated fibrillar form. Transmembrane forms of PrP have been implicated in this structural conversion. The cooperative unfolding of a structural core in PrPC presents a substantial energy barrier to prion formation, with membrane insertion and detachment of parts of PrP presenting a plausible route to its reduction. Here, we examined the removal of residues 119-136 of PrP, a region which includes the first ß-strand and a substantial portion of the conserved hydrophobic region of PrP, a region which associates with the ER membrane, on the structure, stability and self-association of the folded domain of PrPC. We see an "open" native-like conformer with increased solvent exposure which fibrilises more readily than the native state. These data suggest a stepwise folding transition, which is initiated by the conformational switch to this "open" form of PrPC.

Essential Components of Synthetic Infectious Prion Formation De Novo.

Prion diseases are a class of neurodegenerative diseases that are uniquely infectious. Whilst their general replication mechanism is well understood, the components required for the formation and propagation of highly infectious prions are poorly characterized. The protein-only hypothesis posits that the prion protein (PrP) is the only component of the prion; however, additional co-factors are required for its assembly into infectious prions. These can be provided by brain homogenate, but synthetic lipids and non-coding RNA have also been used in vitro. Here, we review a range of experimental approaches, which generate PrP amyloid assemblies de novo. These synthetic PrP assemblies share some, but not necessarily all, properties of genuine infectious prions. We will discuss the different experimental approaches, how a prion is defined, the non-protein requirements of a prion, and provide an overview of the current state of prion amplification and generation in vitro.

VCP suppresses proteopathic seeding in neurons.

BACKGROUND: Neuronal uptake and subsequent spread of proteopathic seeds, such as αS (alpha-synuclein), Tau, and TDP-43, contribute to neurodegeneration. The cellular machinery participating in this process is poorly understood. One proteinopathy called multisystem proteinopathy (MSP) is associated with dominant mutations in Valosin Containing Protein (VCP). MSP patients have muscle and neuronal degeneration characterized by aggregate pathology that can include αS, Tau and TDP-43. METHODS: We performed a fluorescent cell sorting based genome-wide CRISPR-Cas9 screen in αS biosensors. αS and TDP-43 seeding activity under varied conditions was assessed using FRET/Flow biosensor cells or immunofluorescence for phosphorylated αS or TDP-43 in primary cultured neurons. We analyzed in vivo seeding activity by immunostaining for phosphorylated αS following intrastriatal injection of αS seeds in control or VCP disease mutation carrying mice. RESULTS: One hundred fifty-four genes were identified as suppressors of αS seeding. One suppressor, VCP when chemically or genetically inhibited increased αS seeding in cells and neurons. This was not due to an increase in αS uptake or αS protein levels. MSP-VCP mutation expression increased αS seeding in cells and neurons. Intrastriatal injection of αS preformed fibrils (PFF) into VCP-MSP mutation carrying mice increased phospho αS expression as compared to control mice. Cells stably expressing fluorescently tagged TDP-43 C-terminal fragment FRET pairs (TDP-43 biosensors) generate FRET when seeded with TDP-43 PFF but not monomeric TDP-43. VCP inhibition or MSP-VCP mutant expression increases TDP-43 seeding in TDP-43 biosensors. Similarly, treatment of neurons with TDP-43 PFFs generates high molecular weight insoluble phosphorylated TDP-43 after 5 days. This TDP-43 seed dependent increase in phosphorlyated TDP-43 is further augmented in MSP-VCP mutant expressing neurons. CONCLUSION: Using an unbiased screen, we identified the multifunctional AAA ATPase VCP as a suppressor of αS and TDP-43 aggregate seeding in cells and neurons. VCP facilitates the clearance of damaged lysosomes via lysophagy. We propose that VCP's surveillance of permeabilized endosomes may protect against the proteopathic spread of pathogenic protein aggregates. The spread of distinct aggregate species may dictate the pleiotropic phenotypes and pathologies in VCP associated MSP.

Black tea theaflavins inhibit formation of toxic amyloid-ß 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-ß (Aß) 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 Aß 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 Aß and αS into nontoxic, spherical aggregates that are incompetent in seeding amyloid formation and remodel Aß 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.

Bacterial inclusion bodies of Alzheimer's disease ß-amyloid peptides can be employed to study native-like aggregation intermediate states.

The structures of oligomeric intermediate states in the aggregation process of Alzheimer's disease ß-amyloid peptides have been the subject of debate for many years. Bacterial inclusion bodies contain large amounts of small heat shock proteins (sHSPs), which are highly homologous to those found in the plaques of the brains of Alzheimer's disease patients. sHSPs break down amyloid fibril structure in vitro and induce oligomeric assemblies. Prokaryotic protein overexpression thus mimics the conditions encountered in the cell under stress and allows the structures of Aß aggregation intermediate states to be investigated under native-like conditions, which is not otherwise technically possible. We show that IB40/IB42 fulfil all the requirements to be classified as amyloids: they seed fibril growth, are Congo red positive and show characteristic ß-sheet-rich CD spectra. However, IB40 and IB42 are much less stable than fibrils formed in vitro and contain significant amounts of non-ß-sheet regions, as seen from FTIR studies. Quantitative analyses of solution-state NMR H/D exchange rates show that the hydrophobic cores involving residues V18-F19-F20 adopt ß-sheet conformations, whereas the C termini adopt α-helical coiled-coil structures. In the past, an α-helical intermediate-state structure has been postulated, but could not be verified experimentally. In agreement with the current literature, in which Aß oligomers are described as the most toxic state of the peptides, we find that IB42 contains SDS-resistant oligomers that are more neurotoxic than Aß42 fibrils. E. coli inclusion bodies formed by the Alzheimer's disease ß-amyloid peptides Aß40 and Aß42 thus behave structurally like amyloid aggregation intermediate states and open the possibility of studying amyloids in a native-like, cellular environment.

Brazilin Removes Toxic Alpha-Synuclein and Seeding Competent Assemblies from Parkinson Brain by Altering Conformational Equilibrium.

Alpha-synuclein (α-syn) fibrils, a major constituent of the neurotoxic Lewy Bodies in Parkinson's disease, form via nucleation dependent polymerization and can replicate by a seeding mechanism. Brazilin, a small molecule derived from red cedarwood trees in Brazil, has been shown to inhibit the fibrillogenesis of amyloid-beta (Aß) and α-syn as well as remodel mature fibrils and reduce cytotoxicity. Here we test the effects of Brazilin on both seeded and unseeded α-syn fibril formation and show that the natural polyphenol inhibits fibrillogenesis of α-syn by a unique mechanism that alters conformational equilibria in two separate points of the assembly mechanism: Brazilin preserves the natively unfolded state of α-syn by specifically binding to the compact conformation of the α-syn monomer. Brazilin also eliminates seeding competence of α-syn assemblies from Parkinson's disease patient brain tissue, and reduces toxicity of pre-formed assemblies in primary neurons by inducing the formation of large fibril clusters. Molecular docking of Brazilin shows the molecule to interact both with unfolded α-syn monomers and with the cross-ß sheet structure of α-syn fibrils. Our findings suggest that Brazilin has substantial potential as a neuroprotective and therapeutic agent for Parkinson's disease.

Megadalton-sized Dityrosine Aggregates of α-Synuclein Retain High Degrees of Structural Disorder and Internal Dynamics.

Heterogeneous aggregates of the human protein α-synuclein (αSyn) are abundantly found in Lewy body inclusions of Parkinson's disease patients. While structural information on classical αSyn amyloid fibrils is available, little is known about the conformational properties of disease-relevant, non-canonical aggregates. Here, we analyze the structural and dynamic properties of megadalton-sized dityrosine adducts of αSyn that form in the presence of reactive oxygen species and cytochrome c, a proapoptotic peroxidase that is released from mitochondria during sustained oxidative stress. In contrast to canonical cross-ß amyloids, these aggregates retain high degrees of internal dynamics, which enables their characterization by solution-state NMR spectroscopy. We find that intermolecular dityrosine crosslinks restrict αSyn motions only locally whereas large segments of concatenated molecules remain flexible and disordered. Indistinguishable aggregates form in crowded in vitro solutions and in complex environments of mammalian cell lysates, where relative amounts of free reactive oxygen species, rather than cytochrome c, are rate limiting. We further establish that dityrosine adducts inhibit classical amyloid formation by maintaining αSyn in its monomeric form and that they are non-cytotoxic despite retaining basic membrane-binding properties. Our results suggest that oxidative αSyn aggregation scavenges cytochrome c's activity into the formation of amorphous, high molecular-weight structures that may contribute to the structural diversity of Lewy body deposits.