The misfolding and aggregation of proteins into amyloid fibrils characterizes many neurodegenerative disorders such as Parkinson's and Alzheimer's diseases. We report here a method, termed SAVE (single aggregate visualization by enhancement) imaging, for the ultrasensitive detection of individual amyloid fibrils and oligomers using single-molecule fluorescence microscopy. We demonstrate that this method is able to detect the presence of amyloid aggregates of α-synuclein, tau, and amyloid-β. In addition, we show that aggregates can also be identified in human cerebrospinal fluid (CSF). Significantly, we see a twofold increase in the average aggregate concentration in CSF from Parkinson's disease patients compared to age-matched controls. Taken together, we conclude that this method provides an opportunity to characterize the structural nature of amyloid aggregates in a key biofluid, and therefore has the potential to study disease progression in both animal models and humans to enhance our understanding of neurodegenerative disorders.

Amyotrophic Lateral Sclerosis is characterized by a focal onset of symptoms followed by a progressive spread of pathology that has been likened to transmission of infectious prions. Cell-to-cell transmission of SOD1 protein aggregates is dependent on fluid-phase endocytosis pathways, although the precise molecular mechanisms remain to be elucidated.

Protein aggregation is a complex process resulting in the formation of heterogeneous mixtures of aggregate populations that are closely linked to neurodegenerative conditions, such as Alzheimer's disease. Here, we find that soluble aggregates formed at different stages of the aggregation process of amyloid beta (Aβ42) induce the disruption of lipid bilayers and an inflammatory response to different extents. Further, by using gradient ultracentrifugation assay, we show that the smaller aggregates are those most potent at inducing membrane permeability and most effectively inhibited by antibodies binding to the C-terminal region of Aβ42. By contrast, we find that the larger soluble aggregates are those most effective at causing an inflammatory response in microglia cells and more effectively inhibited by antibodies targeting the N-terminal region of Aβ42. These findings suggest that different toxic mechanisms driven by different soluble aggregated species of Aβ42 may contribute to the onset and progression of Alzheimer's disease.

The aggregation of α-synuclein, a protein involved in neurotransmitter release at presynaptic terminals, is associated with a range of highly debilitating neurodegenerative conditions, most notably Parkinson's disease. Intraneuronal inclusion bodies, primarily composed of α-synuclein fibrils, are the major histopathological hallmarks of these disorders, although small oligomeric assemblies are believed to play a crucial role in neuronal impairment. We have probed the mechanism of neurotoxicity of α-synuclein oligomers isolated in vitro using antibodies targeting the N-terminal region of the protein and found that the presence of the antibody resulted in a substantial reduction of the damage induced by the aggregates when incubated with primary cortical neurons and neuroblastoma cells. We observed a similar behavior in vivo using a strain of C. elegans overexpressing α-synuclein, where the aggregation process itself is also partially inhibited as a result of incubation with the antibodies. The similar effects of the antibodies in reducing the toxicity of the aggregated species formed in vitro and in vivo provide evidence for a common origin of cellular impairment induced by α-synuclein aggregates.

As a key player of the protein quality control network of the cell, the molecular chaperone Hsp70 inhibits the aggregation of the amyloid protein tau. To date, the mechanism of this inhibition and the tau species targeted by Hsp70 remain unknown. This is partly due to the inherent difficulty of studying amyloid aggregates because of their heterogeneous and transient nature. Here, we used ensemble and single-molecule fluorescence measurements to dissect how Hsp70 counteracts the self-assembly process of the K18 ΔK280 tau variant. We found that Hsp70 blocks the early stages of tau aggregation by suppressing the formation of tau nuclei. Additionally, Hsp70 sequesters oligomers and mature tau fibrils with nanomolar affinity into a protective complex, efficiently neutralizing their ability to damage membranes and seed further tau aggregation. Our results provide novel insights into the molecular mechanisms by which the chaperone Hsp70 counteracts the formation, propagation, and toxicity of tau aggregates.

Parkinson's disease is associated with the aberrant aggregation of α-synuclein. Although the causes of this process are still unclear, post-translational modifications of α-synuclein are likely to play a modulatory role. Since α-synuclein is constitutively N-terminally acetylated, we investigated how this post-translational modification alters the aggregation behavior of this protein. By applying a three-pronged aggregation kinetics approach, we observed that N-terminal acetylation results in a reduced rate of lipid-induced aggregation and slows down both elongation and fibril-catalyzed aggregate proliferation. An analysis of the amyloid fibrils produced by the aggregation process revealed different morphologies for the acetylated and non-acetylated forms in both lipid-induced aggregation and seed-induced aggregation assays. In addition, we found that fibrils formed by acetylated α-synuclein exhibit a lower β-sheet content. These findings indicate that N-terminal acetylation of α-synuclein alters its lipid-dependent aggregation behavior, reduces its rate of in vitro aggregation, and affects the structural properties of its fibrillar aggregates.

The human molecular chaperone protein DNAJB6 was recently found to inhibit the formation of amyloid fibrils from polyglutamine peptides associated with neurodegenerative disorders such as Huntington disease. We show in the present study that DNAJB6 also inhibits amyloid formation by an even more aggregation-prone peptide (the amyloid-beta peptide, Aβ42, implicated in Alzheimer disease) in a highly efficient manner. By monitoring fibril formation using Thioflavin T fluorescence and far-UV CD spectroscopy, we have found that the aggregation of Aβ42 is retarded by DNAJB6 in a concentration-dependent manner, extending to very low sub-stoichiometric molar ratios of chaperone to peptide. Quantitative kinetic analysis and immunochemistry studies suggest that the high inhibitory efficiency is due to the interactions of the chaperone with aggregated forms of Aβ42 rather than the monomeric form of the peptide. This interaction prevents the growth of such species to longer fibrils and inhibits the formation of new amyloid fibrils through both primary and secondary nucleation. A low dissociation rate of DNAJB6 from Aβ42 aggregates leads to its incorporation into growing fibrils and hence to its gradual depletion from solution with time. When DNAJB6 is eventually depleted, fibril proliferation takes place, but the inhibitory activity can be prolonged by introducing DNAJB6 at regular intervals during the aggregation reaction. These results reveal the highly efficacious mode of action of this molecular chaperone against protein aggregation, and demonstrate that the role of molecular chaperones can involve interactions with multiple aggregated species leading to the inhibition of both principal nucleation pathways through which aggregates are able to form.

The conversion of the β-amyloid (Aβ) peptide into pathogenic aggregates is linked to the onset and progression of Alzheimer's disease. Although this observation has prompted an extensive search for therapeutic agents to modulate the concentration of Aβ or inhibit its aggregation, all clinical trials with these objectives have so far failed, at least in part because of a lack of understanding of the molecular mechanisms underlying the process of aggregation and its inhibition. To address this problem, we describe a chemical kinetics approach for rational drug discovery, in which the effects of small molecules on the rates of specific microscopic steps in the self-assembly of Aβ42, the most aggregation-prone variant of Aβ, are analyzed quantitatively. By applying this approach, we report that bexarotene, an anticancer drug approved by the U.S. Food and Drug Administration, selectively targets the primary nucleation step in Aβ42 aggregation, delays the formation of toxic species in neuroblastoma cells, and completely suppresses Aβ42 deposition and its consequences in a Caenorhabditis elegans model of Aβ42-mediated toxicity. These results suggest that the prevention of the primary nucleation of Aβ42 by compounds such as bexarotene could potentially reduce the risk of onset of Alzheimer's disease and, more generally, that our strategy provides a general framework for the rational identification of a range of candidate drugs directed against neurodegenerative disorders.

The propensity to misfold and self-assemble into stable aggregates is increasingly being recognized as a common feature of protein molecules. Our understanding of this phenomenon and of its links with human disease has improved substantially over the past two decades. Studies thus far, however, have been almost exclusively focused on cytosolic proteins, resulting in a lack of detailed information about the misfolding and aggregation of membrane proteins. As a consequence, although such proteins make up approximately 30% of the human proteome and have high propensities to aggregate, relatively little is known about the biophysical nature of their assemblies. To shed light on this issue, we have studied as a model system an archetypical representative of the ubiquitous major facilitator superfamily, the Escherichia coli lactose permease (LacY). By using a combination of established indicators of cross-β structure and morphology, including the amyloid diagnostic dye thioflavin-T, circular dichroism spectroscopy, Fourier transform infrared spectroscopy, X-ray fiber diffraction, and transmission electron microscopy, we show that LacY can form amyloid-like fibrils under destabilizing conditions. These results indicate that transmembrane α-helical proteins, similarly to cytosolic proteins, have the ability to adopt this generic state.

Scanning probe microscopy provides a unique window into the morphology, mechanics, and structure of proteins and their complexes on the nanoscale. Such measurements require, however, deposition of samples onto substrates. This process can affect conformations and assembly states of the molecular species under investigation and can bias the molecular populations observed in heterogeneous samples through differential adsorption. Here, we show that these limitations can be overcome with a single-step microfluidic spray deposition platform. This method transfers biological solutions to substrates as microdroplets with subpicoliter volume, drying in milliseconds, a timescale that is shorter than typical diffusion times of proteins on liquid-solid interfaces, thus avoiding surface mass transport and change to the assembly state. Finally, the single-step deposition ensures the attachment of the full molecular content of the sample to the substrate, allowing quantitative measurements of different molecular populations within heterogeneous systems, including protein aggregates.

Increasing evidence indicates that peripheral immune cells play a prominent role in neurodegeneration connected to protein misfolding, which are associated with formation of aberrant aggregates, including soluble protein misfolded oligomers. The precise links, however, between the physicochemical features of diverse oligomers and their effects on the immune system, particularly on adaptive immunity, remain currently unexplored, due partly to the transient and heterogeneous nature of the oligomers themselves. To overcome these limitations, we took advantage of two stable and well-characterized types of model oligomers (A and B), formed by HypF-N bacterial protein, type B oligomers displaying lower solvent-exposed hydrophobicity. Exposure to oligomers of human peripheral blood mononuclear cells (PBMCs) revealed differential effects, with type B, but not type A, oligomers leading to a reduction in CD4+ cells. Type A oligomers promoted enhanced differentiation towards CD4+ CD25High FoxP3+ Tregs and displayed a higher suppressive effect on lymphocyte proliferation than Tregs treated with oligomers B or untreated cells. Moreover, our results reveal Th1 and Th17 lymphocyte differentiation mediated by type A oligomers and a differential balance of TGF-β, IL-6, IL-23, IFN-γ and IL-10 mediators. These results indicate that type B oligomers recapitulate some of the biological responses associated with Parkinson's disease in peripheral immunocompetent cells, while type A oligomers resemble responses associated with Alzheimer's disease. We anticipate that further studies characterizing the differential effects of protein misfolded oligomers on the peripheral immune system may lead to the development of blood-based diagnostics, which could report on the type and properties of oligomers present in patients.

In Alzheimer's disease, aggregates of Aβ and tau in amyloid plaques and neurofibrillary tangles spread progressively across brain tissues following a characteristic pattern, implying a tissue-specific vulnerability to the disease. We report a transcriptional analysis of healthy brains and identify an expression signature that predicts-at ages well before the typical onset-the tissue-specific progression of the disease. We obtain this result by finding a quantitative correlation between the histopathological staging of the disease and the expression patterns of the proteins that coaggregate in amyloid plaques and neurofibrillary tangles, together with those of the protein homeostasis components that regulate Aβ and tau. Because this expression signature is evident in healthy brains, our analysis provides an explanatory link between a tissue-specific environmental risk of protein aggregation and a corresponding vulnerability to Alzheimer's disease.

Removing or preventing the formation of [Formula: see text]-synuclein aggregates is a plausible strategy against Parkinson's disease. To this end, we have engineered the [Formula: see text]-wrapin AS69 to bind monomeric [Formula: see text]-synuclein with high affinity. In cultured cells, AS69 reduced the self-interaction of [Formula: see text]-synuclein and formation of visible [Formula: see text]-synuclein aggregates. In flies, AS69 reduced [Formula: see text]-synuclein aggregates and the locomotor deficit resulting from [Formula: see text]-synuclein expression in neuronal cells. In biophysical experiments in vitro, AS69 highly sub-stoichiometrically inhibited both primary and autocatalytic secondary nucleation processes, even in the presence of a large excess of monomer. We present evidence that the AS69-[Formula: see text]-synuclein complex, rather than the free AS69, is the inhibitory species responsible for sub-stoichiometric inhibition of secondary nucleation. These results represent a new paradigm that high affinity monomer binders can lead to strongly sub-stoichiometric inhibition of nucleation processes.

The onset and progression of numerous protein misfolding diseases are associated with the presence of oligomers formed during the aberrant aggregation of several different proteins, including amyloid-β (Aβ) in Alzheimer's disease and α-synuclein (αS) in Parkinson's disease. These small, soluble aggregates are currently major targets for drug discovery. In this study, we show that trodusquemine, a naturally-occurring aminosterol, markedly reduces the cytotoxicity of αS, Aβ and HypF-N oligomers to human neuroblastoma cells by displacing the oligomers from cell membranes in the absence of any substantial morphological and structural changes to the oligomers. These results indicate that the reduced toxicity results from a mechanism that is common to oligomers from different proteins, shed light on the origin of the toxicity of the most deleterious species associated with protein aggregation and suggest that aminosterols have the therapeutically-relevant potential to protect cells from the oligomer-induced cytotoxicity associated with numerous protein misfolding diseases.

The conversion of peptides or proteins from their soluble native states into intractable amyloid deposits is associated with a wide range of human disorders. Misfolded protein oligomers formed during the process of aggregation have been identified as the primary pathogenic agents in many such conditions. Here, we show the existence of a quantitative relationship between the degree of binding to neuronal cells of different types of oligomers formed from a model protein, HypF-N, and the GM1 content of the plasma membranes. In addition, remarkably similar behavior is observed for oligomers of the Aβ42 peptide associated with Alzheimer's disease. Further analysis has revealed the existence of a linear correlation between the level of the influx of Ca(2+) across neuronal membranes that triggers cellular damage, and the fraction of oligomeric species bound to the membrane. Our findings indicate that the susceptibility of neuronal cells to different types of misfolded oligomeric assemblies is directly related to the extent of binding of such oligomers to the cellular membrane.

The self-replicating properties of proteins into amyloid fibrils is a common phenomenon and underlies a variety of neurodegenerative diseases. Because propagation-active fibrils are chemically indistinguishable from innocuous aggregates and monomeric precursors, their detection requires measurements of their replicative capacity. Here we present a digital amyloid quantitative assay (d-AQuA) with insulin as model protein for the absolute quantification of single replicative units, propagons. D-AQuA is a microfluidics-based technology that performs miniaturized simultaneous propagon-induced amplification chain reactions within hundreds to thousands of picoliter-sized droplets. At limiting dilutions, the d-AQuA reactions follow a stochastic regime indicative of the detection of single propagons. D-AQuA thus enables absolute quantification of single propagons present in a given sample at very low concentrations. The number of propagons quantified by d-AQuA was similar to that of fibrillar insulin aggregates detected by atomic-force microscopy and to an equivalent microplate-based assay, providing independent evidence for the identity of insulin propagons with a subset of morphologically defined protein aggregates. The sensitivity, precision, and accuracy of d-AQuA enable it to be suitable for multiple biotechnological and medical applications.

Neurodegenerative diseases, such as Alzheimer's disease (AD), are associated with protein misfolding and aggregation into amyloid fibrils. Increasing evidence suggests that soluble, low-molecular-weight aggregates play a key role in disease-associated toxicity. Within this population of aggregates, closed-loop pore-like structures have been observed for a variety of amyloid systems, and their presence in brain tissues is associated with high levels of neuropathology. However, their mechanism of formation and relationship with mature fibrils have largely remained challenging to elucidate. Here, we use atomic force microscopy and statistical theory of biopolymers to characterize amyloid ring structures derived from the brains of AD patients. We analyze the bending fluctuations of protofibrils and show that the process of loop formation is governed by the mechanical properties of their chains. We conclude that ex vivo protofibril chains possess greater flexibility than that imparted by hydrogen-bonded networks characteristic of mature amyloid fibrils, such that they are able to form end-to-end connections. These results explain the diversity in the structures formed from protein aggregation and shed light on the links between early forms of flexible ring-forming aggregates and their role in disease.

Protein aggregation, arising from the failure of the cell to regulate the synthesis or degradation of aggregation-prone proteins, underlies many neurodegenerative disorders. However, the balance between the synthesis, clearance, and assembly of misfolded proteins into neurotoxic aggregates remains poorly understood. Here we study the effects of modulating this balance for the amyloid-beta (Abeta) peptide by using a small engineered binding protein (Z(Abeta3)) that binds with nanomolar affinity to Abeta, completely sequestering the aggregation-prone regions of the peptide and preventing its aggregation. Co-expression of Z(Abeta3) in the brains of Drosophila melanogaster expressing either Abeta(42) or the aggressive familial associated E22G variant of Abeta(42) abolishes their neurotoxic effects. Biochemical analysis indicates that monomer Abeta binding results in degradation of the peptide in vivo. Complementary biophysical studies emphasize the dynamic nature of Abeta aggregation and reveal that Z(Abeta3) not only inhibits the initial association of Abeta monomers into oligomers or fibrils, but also dissociates pre-formed oligomeric aggregates and, although very slowly, amyloid fibrils. Toxic effects of peptide aggregation in vivo can therefore be eliminated by sequestration of hydrophobic regions in monomeric peptides, even when these are extremely aggregation prone. Our studies also underline how a combination of in vivo and in vitro experiments provide mechanistic insight with regard to the relationship between protein aggregation and clearance and show that engineered binding proteins may provide powerful tools with which to address the physiological and pathological consequences of protein aggregation.

α-Synuclein is an intrinsically disordered protein that is associated with the pathogenesis of Parkinson's disease through the processes involved in the formation of amyloid fibrils. α and β-synuclein are homologous proteins found at comparable levels in presynaptic terminals but β-synuclein has a greatly reduced propensity to aggregate and indeed has been found to inhibit α-synuclein aggregation. In this paper, we describe how sequence differences between α- and β-synuclein affect individual microscopic processes in amyloid formation. In particular, we show that β-synuclein strongly suppresses both lipid-induced aggregation and secondary nucleation of α-synuclein by competing for binding sites at the surfaces of lipid vesicles and fibrils, respectively. These results suggest that β-synuclein can act as a natural inhibitor of α-synuclein aggregation by reducing both the initiation of its self-assembly and the proliferation of its aggregates.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease involving the formation of cytoplasmic aggregates by proteins including TDP-43 and SOD1, in affected cells in the central nervous system (CNS). Pathology spreads from an initial site of onset to contiguous anatomical regions. There is evidence that for disease-associated proteins, including TDP-43 and SOD1, non-native protein conformers can promote misfolding of the natively folded counterparts, and cell-to-cell transfer of pathological aggregates may underlie the spread of the disease throughout the CNS. A variety of studies have demonstrated that SOD1 is released by neuron-like cells into the surrounding culture medium, either in their free state or encapsulated in extracellular vesicles such as exosomes. Extracellular SOD1 can then be internalised by naïve cells incubated in this conditioned medium, leading to the misfolding and aggregation of endogenous intracellular SOD1; an effect that propagates over serial passages. A similar phenomenon has also been observed with other proteins associated with protein misfolding and progressive neurological disorders, including tau, α-synuclein and both mammalian and yeast prions. Conditioned media experiments using TDP-43 have been less conclusive, with evidence for this protein undergoing intercellular transfer being less straightforward. In this review, we describe the properties of TDP-43 and SOD1 and look at the evidence for their respective abilities to participate in cell-to-cell transfer via conditioned medium, and discuss how variations in the nature of cell-to-cell transfer suggests that a number of different mechanisms are involved in the spreading of pathology in ALS.