Search

A wide variety of oligomeric structures are formed during the aggregation of proteins associated with neurodegenerative diseases. Such soluble oligomers are believed to be key toxic species in the related disorders; therefore, identification of the structural determinants of toxicity is of upmost importance. Here, we analysed toxic oligomers of α-synuclein and its pathological variants in order to identify structural features that could be related to toxicity and found a novel structural polymorphism within G51D oligomers. These G51D oligomers can adopt a variety of β-sheet-rich structures with differing degrees of α-helical content, and the helical structural content of these oligomers correlates with the level of induced cellular dysfunction in SH-SY5Y cells. This structure–function relationship observed in α-synuclein oligomers thus presents the α-helical structure as another potential structural determinant that may be linked with cellular toxicity in amyloid-related proteins.

Significance The formation of amyloid fibrils and toxic oligomeric species has been shown to be inhibited by their interactions with molecular chaperones, thus modulating monomer sequestration and toxicity in the context of neurodegenerative diseases. Understanding the physical and chemical properties underlying chaperone binding processes is essential to explore new therapeutic strategies to target toxic amyloid species. Here, we determine that the binding of the small heat-shock protein αB-crystallin to α-synculein fibrils, a protein which is related to the progression of Parkinson’s disease, is driven by entropic forces. By applying a microfluidic platform, we accurately quantified the thermodynamics and the kinetics of this intermolecular interaction in the condensed phase and hypothesize that αB-crystallin oligomers work as an entropic buffer system., Molecular chaperones are key components of the cellular proteostasis network whose role includes the suppression of the formation and proliferation of pathogenic aggregates associated with neurodegenerative diseases. The molecular principles that allow chaperones to recognize misfolded and aggregated proteins remain, however, incompletely understood. To address this challenge, here we probe the thermodynamics and kinetics of the interactions between chaperones and protein aggregates under native solution conditions using a microfluidic platform. We focus on the binding between amyloid fibrils of α-synuclein, associated with Parkinson’s disease, to the small heat-shock protein αB-crystallin, a chaperone widely involved in the cellular stress response. We find that αB-crystallin binds to α-synuclein fibrils with high nanomolar affinity and that the binding is driven by entropy rather than enthalpy. Measurements of the change in heat capacity indicate significant entropic gain originates from the disassembly of the oligomeric chaperones that function as an entropic buffer system. These results shed light on the functional roles of chaperone oligomerization and show that chaperones are stored as inactive complexes which are capable of releasing active subunits to target aberrant misfolded species.

Aggregation of the 140-residue protein α-synuclein (αSN) is a key factor in the etiology of Parkinson’s disease. Although the intensely anionic C-terminal domain (CTD) of αSN does not form part of the amyloid core region or affect membrane binding ability, truncation or reduction of charges in the CTD promotes fibrillation through as yet unknown mechanisms. Here, we study stepwise truncated CTDs and identify a threshold region around residue 121; constructs shorter than this dramatically increase their fibrillation tendency. Remarkably, these effects persist even when as little as 10% of the truncated variant is mixed with the full-length protein. Increased fibrillation can be explained by a substantial increase in self-replication, most likely via fragmentation. Paradoxically, truncation also suppresses toxic oligomer formation, and oligomers that can be formed by chemical modification show reduced membrane affinity and cytotoxicity. These remarkable changes correlate to the loss of negative electrostatic potential in the CTD and highlight a double-edged electrostatic safety guard.

Funder: Royal Society; doi: https://doi.org/10.13039/501100000288, Funder: UK Dementia Research Institute, Aggregation of α-synuclein (α-syn) is closely linked to Parkinson’s disease (PD) and the related synucleinopathies. Aggregates spread through the brain during the progression of PD, but the mechanism by which this occurs is still not known. One possibility is a self-propagating, templated-seeding mechanism, but this cannot be established without quantitative information about the efficiencies and rates of the key steps in the cellular process. To address this issue, we imaged the uptake and seeding of unlabeled exogenous α-syn fibrils by SH-SY5Y cells and the resulting secreted aggregates, using super-resolution microscopy. Externally-applied fibrils very inefficiently induced self-assembly of endogenous α-syn in a process accelerated by the proteasome. Seeding resulted in the increased secretion of nanoscopic aggregates (mean 35 nm diameter), of both α-syn and Aβ. Our results suggest that cells respond to seed-induced disruption of protein homeostasis predominantly by secreting nanoscopic aggregates; this mechanism may therefore be an important protective response by cells to protein aggregation.

The aggregation of α‐synuclein into small soluble aggregates and then fibrils is important in the development and spreading of aggregates through the brain in Parkinson's disease. Fibrillar aggregates can grow by monomer addition and then break into fragments that could spread into neighboring cells. The rate constants for fibril elongation and fragmentation have been measured but it is not known how large an aggregate needs to be before fibril formation is thermodynamically favorable. This critical size is an important parameter controlling at what stage in an aggregation reaction fibrils can form and replicate. We determined this value to be approximately 70 monomers using super‐resolution and atomic force microscopy imaging of individual α‐synuclein aggregates formed in solution over long time periods. This represents the minimum size for a stable α‐synuclein fibril and we hypothesis the formation of aggregates of this size in a cell represents a tipping point at which rapid replication occurs., The critical size for α‐synuclein to form a stable fibrils, which can then replicate by monomer addition and fragmentation, has been found to be 70 monomers. Preventing aggregates from reaching this critical size would be a key cellular protective mechanism.

Parkinson’s disease (PD) is characterized by proteinaceous aggregates named Lewy Bodies and Lewy Neurites containing $\alpha$-synuclein fibrils. The underlying aggregation mechanism of this protein is dominated by a secondary process at mildly acidic pH, as in endosomes and other organelles. This effect manifests as a strong acceleration of the aggregation in the presence of seeds and a weak dependence of the aggregation rate on monomer concentration. The molecular mechanism underlying this process could be nucleation of monomers on fibril surfaces or fibril fragmentation. Here, we aim to distinguish between these mechanisms. The nature of the secondary processes was investigated using differential sedimentation analysis, trap and seed experiments, quartz crystal microbalance experiments and super-resolution microscopy. The results identify secondary nucleation of monomers on the fibril surface as the dominant secondary process leading to rapid generation of new aggregates, while no significant contribution from fragmentation was found. The newly generated oligomeric species quickly elongate to further serve as templates for secondary nucleation and this may have important implications in the spreading of PD.

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.

α-Synuclein (α-syn) is a 140-residue intrinsically disordered protein that is involved in neuronal and synaptic vesicle plasticity, but its aggregation to form amyloid fibrils is the hallmark of Parkinson's disease (PD). The interaction between α-syn and lipid surfaces is believed to be a key feature for mediation of its normal function, but under other circumstances it is able to modulate amyloid fibril formation. Using a combination of experimental and theoretical approaches, we identify the mechanism through which facile aggregation of α-syn is induced under conditions where it binds a lipid bilayer, and we show that the rate of primary nucleation can be enhanced by three orders of magnitude or more under such conditions. These results reveal the key role that membrane interactions can have in triggering conversion of α-syn from its soluble state to the aggregated state that is associated with neurodegeneration and to its associated disease states.