Liquid-liquid phase separation of α-synuclein is highly sensitive to sequence complexity.

The Parkinson's-associated protein α-synuclein (α-syn) can undergo liquid-liquid phase separation (LLPS), which typically leads to the formation of amyloid fibrils. The coincidence of LLPS and amyloid formation has complicated the identification of the molecular determinants unique to LLPS of α-syn. Moreover, the lack of strategies to selectively perturb LLPS makes it difficult to dissect the biological roles specific to α-syn LLPS, independent of fibrillation. Herein, using a combination of subtle missense mutations, we show that LLPS of α-syn is highly sensitive to its sequence complexity. In fact, we find that even a highly conservative mutation (V16I) that increases sequence complexity without perturbing physicochemical and structural properties, is sufficient to reduce LLPS by 75%; this effect can be reversed by an adjacent V-to-I mutation (V15I) that restores the original sequence complexity. A18T, a complexity-enhancing PD-associated mutation, was likewise found to reduce LLPS, implicating sequence complexity in α-syn pathogenicity. Furthermore, leveraging the differences in LLPS propensities among different α-syn variants, we demonstrate that fibrillation of α-syn does not necessarily correlate with its LLPS. In fact, we identify mutations that selectively perturb LLPS or fibrillation of α-syn, unlike previously studied mutations. The variants and design principles reported herein should therefore empower future studies to disentangle these two phenomena and distinguish their (patho)biological roles.

Liquid-liquid phase separation of α-synuclein is highly sensitive to sequence complexity.

The Parkinson's-associated protein α-synuclein (α-syn) can undergo liquid-liquid phase separation (LLPS), which typically leads to the formation of amyloid fibrils. The coincidence of LLPS and amyloid formation has complicated the identification of the molecular determinants unique to LLPS of α-syn. Moreover, the lack of strategies to selectively perturb LLPS makes it difficult to dissect the biological roles specific to α-syn LLPS, independent of fibrillation. Herein, using a combination of subtle missense mutations, we show that LLPS of α-syn is highly sensitive to its sequence complexity. In fact, we find that even a highly conservative mutation (V16I) that increases sequence complexity without perturbing physicochemical and structural properties, is sufficient to reduce LLPS by 75%; this effect can be reversed by an adjacent V-to-I mutation (V15I) that restores the original sequence complexity. A18T, a complexity-enhancing PD-associated mutation, was likewise found to reduce LLPS, implicating sequence complexity in α-syn pathogenicity. Furthermore, leveraging the differences in LLPS propensities among different α-syn variants, we demonstrate that fibrillation of α-syn does not necessarily correlate with its LLPS. In fact, we identify mutations that selectively perturb LLPS or fibrillation of α-syn, unlike previously studied mutations. The variants and design principles reported herein should therefore empower future studies to disentangle these two phenomena and distinguish their (patho)biological roles.

Cholesterol in Synaptic Vesicle Membranes Regulates the Vesicle-Binding, Function, and Aggregation of α-Synuclein.

Loss of function and aggregation of the neuronal protein α-Synuclein (A-Syn) underlies the pathogenesis of Parkinson's disease (PD), and both the function and aggregation of this protein happen to be mediated via its binding to the synaptic vesicles (SVs) at the presynaptic termini. An essential constituent of SV membranes is cholesterol, with which A-Syn directly interacts while binding to membranes. Thus, cholesterol content in SV membranes is likely to affect the binding of A-Syn to these vesicles and consequently its functional and pathogenic behaviors. Interestingly, the dyshomeostasis of cholesterol has often been associated with PD, with reports linking both high and low cholesterol levels to an increased risk of neurodegeneration. Herein, using SV-mimicking liposomes containing increasing percentages of membrane cholesterol, we show (with mathematical interpretation) that the binding of A-Syn to synaptic-like vesicles is strongest in the presence of an optimum cholesterol content, which correlates to its maximum function and minimum aggregation. This implicates a minimum risk of neurodegeneration at optimum cholesterol levels and rationalizes the existing controversial relationship between cholesterol levels and PD. Increased membrane cholesterol was, however, found to protect against damage caused by aggregated A-Syn, complementing previous reports and portraying one advantage of high cholesterol over low.

Modulation of α-Synuclein Fibrillation by Ultrasmall and Biocompatible Gold Nanoclusters.

Parkinson's disease (PD) is the second most common neurodegenerative disorder, the pathogenesis of which is closely linked to the misfolding and aggregation of the neuronal protein α-Synuclein (A-Syn). Numerous molecules that inhibit/modulate the pathogenic aggregation of A-Syn in an effort to tackle PD pathogenesis have been reported, but none so far have been successful in treating the disease at the clinic. One major reason for this is the poor blood-brain barrier (BBB) permeability of most of the molecules being used. Therefore, using BBB-permeable (and biocompatible) nanomaterials as fibrillation modulators is gaining importance. In the present work, we show how nontoxic and ultrasmall gold nanoclusters (AuNCs) can systematically modulate the pathogenic fibrillation of A-Syn in vitro, based on the chemical nature of their capping agents, using two reported easily synthesizable AuNCs as models. In addition, we detect the BBB permeability in mice of one of these AuNCs solely by making use of its intrinsic fluorescence. Thus, our work exemplifies how AuNCs can be potential therapeutics against PD; while also acting as fluorescent probes for their own BBB permeability.

An aminoglycoside antibiotic inhibits both lipid-induced and solution-phase fibrillation of α-synuclein in vitro.

Parkinson's disease (PD), closely associated with the misfolding and aggregation of the neuronal protein α-synuclein (A-Syn), is a neurodegenerative disorder with no cure to date. Here, we show that the commercially available, inexpensive, aminoglycoside antibiotic kanamycin effectively inhibits both lipid-induced and solution-phase aggregation of A-Syn in vitro, pointing towards the prospective repurposing of kanamycin as a potential anti-PD drug.

Modulation of α-Synuclein Aggregation by Cytochrome c Binding and Hetero-dityrosine Adduct Formation.

The aggregation of α-synuclein (α-Syn) has been implicated strongly in Parkinson's disease (PD). The intrinsically disordered nature of α-Syn makes this protein prone to self-association or heteroassociation with another protein or lipid. While conformational fluctuation and free radical chemistry have been shown to play important roles in its ability toward self- and heteroassociation, any systematic understanding of their contributions is missing. Here, we report an in vitro investigation of the interaction between α-Syn and cytochrome c in the oxidized (cyt c III) and reduced forms (cyt c II), in which cyt c III was found to induce a large compaction of α-Syn and inhibit the aggregation by favoring a hetero-dityrosine bond formation. In contrast, the presence of cyt c II did not result in any compaction and its presence was found to facilitate α-Syn aggregation. The variation in the charge distribution of the surface residues of cyt c III and cyt c II is expected to play a decisive role in their interaction with α-Syn.