N-Terminal Acetylation of α-Synuclein Slows down Its Aggregation Process and Alters the Morphology of the Resulting 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 ß-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.

Wild-type sTREM2 blocks Aß aggregation and neurotoxicity, but the Alzheimer's R47H mutant increases Aß aggregation.

TREM2 is a pattern recognition receptor, expressed on microglia and myeloid cells, detecting lipids and Aß and inducing an innate immune response. Missense mutations (e.g., R47H) of TREM2 increase risk of Alzheimer's disease (AD). The soluble ectodomain of wild-type TREM2 (sTREM2) has been shown to protect against AD in vivo, but the underlying mechanisms are unclear. We show that Aß oligomers bind to cellular TREM2, inducing shedding of the sTREM2 domain. Wild-type sTREM2 bound to Aß oligomers (measured by single-molecule imaging, dot blots, and Bio-Layer Interferometry) inhibited Aß oligomerization and disaggregated preformed Aß oligomers and protofibrils (measured by transmission electron microscopy, dot blots, and size-exclusion chromatography). Wild-type sTREM2 also inhibited Aß fibrillization (measured by imaging and thioflavin T fluorescence) and blocked Aß-induced neurotoxicity (measured by permeabilization of artificial membranes and by loss of neurons in primary neuronal-glial cocultures). In contrast, the R47H AD-risk variant of sTREM2 is less able to bind and disaggregate oligomeric Aß but rather promotes Aß protofibril formation and neurotoxicity. Thus, in addition to inducing an immune response, wild-type TREM2 may protect against amyloid pathology by the Aß-induced release of sTREM2, which blocks Aß aggregation and neurotoxicity. In contrast, R47H sTREM2 promotes Aß aggregation into protofibril that may be toxic to neurons. These findings may explain how wild-type sTREM2 apparently protects against AD in vivo and why a single copy of the R47H variant gene is associated with increased AD risk.

Monomeric and fibrillar α-synuclein exert opposite effects on the catalytic cycle that promotes the proliferation of Aß42 aggregates.

The coaggregation of the amyloid-ß peptide (Aß) and α-synuclein is commonly observed in a range of neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. The complex interplay between Aß and α-synuclein has led to seemingly contradictory results on whether α-synuclein promotes or inhibits Aß aggregation. Here, we show how these conflicts can be rationalized and resolved by demonstrating that different structural forms of α-synuclein exert different effects on Aß aggregation. Our results demonstrate that whereas monomeric α-synuclein blocks the autocatalytic proliferation of Aß42 (the 42-residue form of Aß) fibrils, fibrillar α-synuclein catalyses the heterogeneous nucleation of Aß42 aggregates. It is thus the specific balance between the concentrations of monomeric and fibrillar α-synuclein that determines the outcome of the Aß42 aggregation reaction.

A natural product inhibits the initiation of α-synuclein aggregation and suppresses its toxicity.

The self-assembly of α-synuclein is closely associated with Parkinson's disease and related syndromes. We show that squalamine, a natural product with known anticancer and antiviral activity, dramatically affects α-synuclein aggregation in vitro and in vivo. We elucidate the mechanism of action of squalamine by investigating its interaction with lipid vesicles, which are known to stimulate nucleation, and find that this compound displaces α-synuclein from the surfaces of such vesicles, thereby blocking the first steps in its aggregation process. We also show that squalamine almost completely suppresses the toxicity of α-synuclein oligomers in human neuroblastoma cells by inhibiting their interactions with lipid membranes. We further examine the effects of squalamine in a Caenorhabditis elegans strain overexpressing α-synuclein, observing a dramatic reduction of α-synuclein aggregation and an almost complete elimination of muscle paralysis. These findings suggest that squalamine could be a means of therapeutic intervention in Parkinson's disease and related conditions.

Mutations associated with familial Parkinson's disease alter the initiation and amplification steps of α-synuclein aggregation.

Parkinson's disease is a highly debilitating neurodegenerative condition whose pathological hallmark is the presence in nerve cells of proteinacious deposits, known as Lewy bodies, composed primarily of amyloid fibrils of α-synuclein. Several missense mutations in the gene encoding α-synuclein have been associated with familial variants of Parkinson's disease and have been shown to affect the kinetics of the aggregation of the protein. Using a combination of experimental and theoretical approaches, we present a systematic in vitro study of the influence of disease-associated single-point mutations on the individual processes involved in α-synuclein aggregation into amyloid fibrils. We find that lipid-induced fibril production and surface catalyzed fibril amplification are the processes most strongly affected by these mutations and show that familial mutations can induce dramatic changes in the crucial processes thought to be associated with the initiation and spreading of the aggregation of α-synuclein.

Chemical properties of lipids strongly affect the kinetics of the membrane-induced aggregation of α-synuclein.

Intracellular α-synuclein deposits, known as Lewy bodies, have been linked to a range of neurodegenerative disorders, including Parkinson's disease. α-Synuclein binds to synthetic and biological lipids, and this interaction has been shown to play a crucial role for both α-synuclein's native function, including synaptic plasticity, and the initiation of its aggregation. Here, we describe the interplay between the lipid properties and the lipid binding and aggregation propensity of α-synuclein. In particular, we have observed that the binding of α-synuclein to model membranes is much stronger when the latter is in the fluid rather than the gel phase, and that this binding induces a segregation of the lipids into protein-poor and protein-rich populations. In addition, α-synuclein was found to aggregate at detectable rates only when interacting with membranes composed of the most soluble lipids investigated here. Overall, our results show that the chemical properties of lipids determine whether or not the lipids can trigger the aggregation of α-synuclein, thus affecting the balance between functional and aberrant behavior of the protein.

Mapping Surface Hydrophobicity of α-Synuclein Oligomers at the Nanoscale.

Proteins fold into a single structural ensemble but can also misfold into many diverse structures including small aggregates and fibrils, which differ in their toxicity. The aggregate surface properties play an important role in how they interact with the plasma membrane and cellular organelles, potentially inducing cellular toxicity, however, these properties have not been measured to date due to the lack of suitable methods. Here, we used a spectrally resolved, super-resolution imaging method combined with an environmentally sensitive fluorescent dye to measure the surface hydrophobicity of individual aggregates formed by the protein α-synuclein (αS), whose aggregation is associated with Parkinson's disease. We show that the surface of soluble oligomers is more hydrophobic than fibrils and populates a diverse range of coexisting states. Overall, our data show that the conversion of oligomers to fibril-like aggregates and ultimately to fibrils results in a reduction in both hydrophobicity and the variation in hydrophobicity. This funneling characteristic of the energy landscape explains many of the observed properties of αS aggregates and may be a common feature of aggregating proteins.

Nanoscopic Characterisation of Individual Endogenous Protein Aggregates in Human Neuronal Cells.

The aberrant misfolding and subsequent conversion of monomeric protein into amyloid aggregates characterises many neurodegenerative disorders, including Parkinson's and Alzheimer's diseases. These aggregates are highly heterogeneous in structure, generally of low abundance and typically smaller than the diffraction limit of light (≈250 nm). To overcome the challenges these characteristics pose to the study of endogenous aggregates formed in cells, we have developed a method to characterise them at the nanometre scale without the need for a conjugated fluorophore. Using a combination of DNA PAINT and an amyloid-specific aptamer, we demonstrate that this technique is able to detect and super-resolve a range of aggregated species, including those formed by α-synuclein and amyloid-ß. Additionally, this method enables endogenous protein aggregates within cells to be characterised. We found that neuronal cells derived from patients with Parkinson's disease contain a larger number of protein aggregates than those from healthy controls.

Optical Structural Analysis of Individual α-Synuclein Oligomers.

Small aggregates of misfolded proteins play a key role in neurodegenerative disorders. Such species have proved difficult to study due to the lack of suitable methods capable of resolving these heterogeneous aggregates, which are smaller than the optical diffraction limit. We demonstrate here an all-optical fluorescence microscopy method to characterise the structure of individual protein aggregates based on the fluorescence anisotropy of dyes such as thioflavin-T, and show that this technology is capable of studying oligomers in human biofluids such as cerebrospinal fluid. We first investigated in vitro the structural changes in individual oligomers formed during the aggregation of recombinant α-synuclein. By studying the diffraction-limited aggregates we directly evaluated their structural conversion and correlated this with the potential of aggregates to disrupt lipid bilayers. We finally characterised the structural features of aggregates present in cerebrospinal fluid of Parkinson's disease patients and age-matched healthy controls.

Ultrasensitive Measurement of Ca2+ Influx into Lipid Vesicles Induced by Protein Aggregates.

To quantify and characterize the potentially toxic protein aggregates associated with neurodegenerative diseases, a high-throughput assay based on measuring the extent of aggregate-induced Ca2+ entry into individual lipid vesicles has been developed. This approach was implemented by tethering vesicles containing a Ca2+ sensitive fluorescent dye to a passivated surface and measuring changes in the fluorescence as a result of membrane disruption using total internal reflection microscopy. Picomolar concentrations of Aß42 oligomers could be observed to induce Ca2+ influx, which could be inhibited by the addition of a naturally occurring chaperone and a nanobody designed to bind to the Aß peptide. We show that the assay can be used to study aggregates from other proteins, such as α-synuclein, and to probe the effects of complex biofluids, such as cerebrospinal fluid, and thus has wide applicability.

Elongation rate and average length of amyloid fibrils in solution using isotope-labelled small-angle neutron scattering.

We demonstrate a solution method that allows both elongation rate and average fibril length of assembling amyloid fibrils to be estimated. The approach involves acquisition of real-time neutron scattering data during the initial stages of seeded growth, using contrast matched buffer to make the seeds effectively invisible to neutrons. As deuterated monomers add on to the seeds, the labelled growing ends give rise to scattering patterns that we model as cylinders whose increase in length with time gives an elongation rate. In addition, the absolute intensity of the signal can be used to determine the number of growing ends per unit volume, which in turn provides an estimate of seed length. The number of ends did not change significantly during elongation, demonstrating that any spontaneous or secondary nucleation was not significant compared with growth on the ends of pre-existing fibrils, and in addition providing a method of internal validation for the technique. Our experiments on initial growth of alpha synuclein fibrils using 1.2 mg ml-1 seeds in 2.5 mg ml-1 deuterated monomer at room temperature gave an elongation rate of 6.3 ± 0.5 Å min-1, and an average seed length estimate of 4.2 ± 1.3 µm.

Comparative Studies in the A30P and A53T α-Synuclein C. elegans Strains to Investigate the Molecular Origins of Parkinson's Disease.

The aggregation of α-synuclein is a hallmark of Parkinson's disease (PD) and a variety of related neurological disorders. A number of mutations in this protein, including A30P and A53T, are associated with familial forms of the disease. Patients carrying the A30P mutation typically exhibit a similar age of onset and symptoms as sporadic PD, while those carrying the A53T mutation generally have an earlier age of onset and an accelerated progression. We report two C. elegans models of PD (PDA30P and PDA53T), which express these mutational variants in the muscle cells, and probed their behavior relative to animals expressing the wild-type protein (PDWT). PDA30P worms showed a reduced speed of movement and an increased paralysis rate, control worms, but no change in the frequency of body bends. By contrast, in PDA53T worms both speed and frequency of body bends were significantly decreased, and paralysis rate was increased. α-Synuclein was also observed to be less well localized into aggregates in PDA30P worms compared to PDA53T and PDWT worms, and amyloid-like features were evident later in the life of the animals, despite comparable levels of expression of α-synuclein. Furthermore, squalamine, a natural product currently in clinical trials for treating symptomatic aspects of PD, was found to reduce significantly the aggregation of α-synuclein and its associated toxicity in PDA53T and PDWT worms, but had less marked effects in PDA30P. In addition, using an antibody that targets the N-terminal region of α-synuclein, we observed a suppression of toxicity in PDA30P, PDA53T and PDWT worms. These results illustrate the use of these two C. elegans models in fundamental and applied PD research.

Squalamine and Its Derivatives Modulate the Aggregation of Amyloid-ß and α-Synuclein and Suppress the Toxicity of Their Oligomers.

The aberrant aggregation of proteins is a key molecular event in the development and progression of a wide range of neurodegenerative disorders. We have shown previously that squalamine and trodusquemine, two natural products in the aminosterol class, can modulate the aggregation of the amyloid-ß peptide (Aß) and of α-synuclein (αS), which are associated with Alzheimer's and Parkinson's diseases. In this work, we expand our previous analyses to two squalamine derivatives, des-squalamine and α-squalamine, obtaining further insights into the mechanism by which aminosterols modulate Aß and αS aggregation. We then characterize the ability of these small molecules to alter the physicochemical properties of stabilized oligomeric species in vitro and to suppress the toxicity of these aggregates to varying degrees toward human neuroblastoma cells. We found that, despite the fact that these aminosterols exert opposing effects on Aß and αS aggregation under the conditions that we tested, the modifications that they induced to the toxicity of oligomers were similar. Our results indicate that the suppression of toxicity is mediated by the displacement of toxic oligomeric species from cellular membranes by the aminosterols. This study, thus, provides evidence that aminosterols could be rationally optimized in drug discovery programs to target oligomer toxicity in Alzheimer's and Parkinson's diseases.

Direct measurement of lipid membrane disruption connects kinetics and toxicity of Aß42 aggregation.

The formation of amyloid deposits in human tissues is a defining feature of more than 50 medical disorders, including Alzheimer's disease. Strong genetic and histological evidence links these conditions to the process of protein aggregation, yet it has remained challenging to identify a definitive connection between aggregation and pathogenicity. Using time-resolved fluorescence microscopy of individual synthetic vesicles, we show for the Aß42 peptide implicated in Alzheimer's disease that the disruption of lipid bilayers correlates linearly with the time course of the levels of transient oligomers generated through secondary nucleation. These findings indicate a specific role of oligomers generated through the catalytic action of fibrillar species during the protein aggregation process in driving deleterious biological function and establish a direct causative connection between amyloid formation and its pathological effects.

Screening of small molecules using the inhibition of oligomer formation in α-synuclein aggregation as a selection parameter.

The aggregation of α-synuclein is a central event in Parkinsons's disease and related synucleinopathies. Since pharmacologically targeting this process, however, has not yet resulted in approved disease-modifying treatments, there is an unmet need of developing novel methods of drug discovery. In this context, the use of chemical kinetics has recently enabled accurate quantifications of the microscopic steps leading to the proliferation of protein misfolded oligomers. As these species are highly neurotoxic, effective therapeutic strategies may be aimed at reducing their numbers. Here, we exploit this quantitative approach to develop a screening strategy that uses the reactive flux toward α-synuclein oligomers as a selection parameter. Using this approach, we evaluate the efficacy of a library of flavone derivatives, identifying apigenin as a compound that simultaneously delays and reduces the formation of α-synuclein oligomers. These results demonstrate a compound selection strategy based on the inhibition of the formation of α-synuclein oligomers, which may be key in identifying small molecules in drug discovery pipelines for diseases associated with α-synuclein aggregation.

The Influence of Pathogenic Mutations in α-Synuclein on Biophysical and Structural Characteristics of Amyloid Fibrils.

Proteinaceous deposits of α-synuclein amyloid fibrils are a hallmark of human disorders including Parkinson's disease. The onset of this disease is also associated with five familial mutations of the gene encoding the protein. However, the mechanistic link between single point mutations and the kinetics of aggregation, biophysical properties of the resulting amyloid fibrils, and an increased risk of disease is still elusive. Here, we demonstrate that the disease-associated mutations of α-synuclein generate different amyloid fibril polymorphs compared to the wild type protein. Remarkably, the α-synuclein variants forming amyloid fibrils of a comparable structure, morphology, and heterogeneity show similar microscopic steps defining the aggregation kinetics. These results demonstrate that a single point mutation can significantly alter the distribution of fibrillar polymorphs in α-synuclein, suggesting that differences in the clinical phenotypes of familial Parkinson's disease could be associated with differences in the mechanism of formation and the structural characteristics of the aggregates.

Increased Secondary Nucleation Underlies Accelerated Aggregation of the Four-Residue N-Terminally Truncated Aß42 Species Aß5-42.

Aggregation of the amyloid-ß (Aß) peptide into plaques is believed to play a crucial role in Alzheimer's disease. Amyloid plaques consist of fibrils of full length Aß peptides as well as N-terminally truncated species. ß-Site amyloid precursor protein-cleaving enzyme (BACE1) cleaves amyloid precursor protein in the first step in Aß peptide production and is an attractive therapeutic target to limit Aß generation. Inhibition of BACE1, however, induces a unique pattern of Aß peptides with increased levels of N-terminally truncated Aß peptides starting at position 5 (Aß5-X), indicating that these peptides are generated through a BACE1-independent pathway. Here we elucidate the aggregation mechanism of Aß5-42 and its influence on full-length Aß42. We find that, compared to Aß42, Aß5-42 is more aggregation prone and displays enhanced nucleation rates. Aß5-42 oligomers cause nonspecific membrane disruption to similar extent as Aß42 but appear at earlier time points in the aggregation reaction. Noteworthy, this implies similar toxicity of Aß42 and Aß5-42 and the toxic species are generated faster by Aß5-42. The increased rate of secondary nucleation on the surface of existing fibrils originates from a higher affinity of Aß5-42 monomers for fibrils, as compared to Aß42: an effect that may be related to the reduced net charge of Aß5-42. Moreover, Aß5-42 and Aß42 peptides coaggregate into heteromolecular fibrils and either species can elongate existing Aß42 or Aß5-42 fibrils but Aß42 fibrils are more catalytic than Aß5-42 fibrils. Our findings highlight the importance of the N-terminus for surface-catalyzed nucleation and thus the production of toxic oligomers.

An engineered monomer binding-protein for α-synuclein efficiently inhibits the proliferation of amyloid fibrils.

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.

Different soluble aggregates of Aß42 can give rise to cellular toxicity through different mechanisms.

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 (Aß42) 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 Aß42. 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 Aß42. These findings suggest that different toxic mechanisms driven by different soluble aggregated species of Aß42 may contribute to the onset and progression of Alzheimer's disease.

Single-Molecule Characterization of the Interactions between Extracellular Chaperones and Toxic α-Synuclein Oligomers.

The aberrant aggregation of α-synuclein is associated with several human diseases, collectively termed the α-synucleinopathies, which includes Parkinson's disease. The progression of these diseases is, in part, mediated by extracellular α-synuclein oligomers that may exert effects through several mechanisms, including prion-like transfer, direct cytotoxicity, and pro-inflammatory actions. In this study, we show that two abundant extracellular chaperones, clusterin and α2-macroglobulin, directly bind to exposed hydrophobic regions on the surface of α-synuclein oligomers. Using single-molecule fluorescence techniques, we found that clusterin, unlike α2-macroglobulin, exhibits differential binding to α-synuclein oligomers that may be related to structural differences between two previously described forms of αS oligomers. The binding of both chaperones reduces the ability of the oligomers to permeabilize lipid membranes and prevents an oligomer-induced increase in ROS production in cultured neuronal cells. Taken together, these data suggest a neuroprotective role for extracellular chaperones in suppressing the toxicity associated with α-synuclein oligomers.