Small soluble α-synuclein aggregates are the toxic species in Parkinson's disease

Parkinson's disease (PD), the second most common neurodegenerative disorder, is characterized by the irreversible loss of dopaminergic neurons in the substantia nigra, leading to motor symptoms. The presence of Lewy bodies, containing aggregated α-synuclein, is a hallmark of PD, but the specific toxic species among the heterogeneous α-synuclein aggregates remains unclear. While some studies suggest small soluble oligomers are most cytotoxic, others implicate mature fibrils. The challenge lies in the co-existence of various α-synuclein species, hindering the isolation and study of their individual toxic properties. This research aims to determine the toxicity of isolated α-synuclein aggregates as a function of size, bridging the gap between in vitro studies and the complex reality of human brain tissue.

Previous research has linked soluble α-synuclein aggregates, particularly oligomers, to neuronal death. However, there's no consensus on the precise definition or characterization of oligomers, which range in size between monomers and insoluble fibrils. Several mechanisms of α-synuclein-induced toxicity have been proposed, including membrane disruption, synaptic loss, mitochondrial and endoplasmic reticulum dysfunction, seeding capacity, and neuroinflammation. While some studies suggest that small prefibrillar oligomers are the most toxic, others show that fibrils, capable of seeding further aggregation, are more harmful. Many of these studies used acute doses, so their relevance at physiological aggregate concentrations remains questionable. The main challenge is to determine the toxicity of various aggregate sizes in a way that directly relates to the aggregates found in human brains. Prior studies often used trapped oligomers, stabilizers, high concentrations, or mutated α-synuclein, leading to uncertainties about the relevance to naturally occurring aggregates.

This study employed a size-exclusion centrifugation method using a sucrose density gradient to separate α-synuclein aggregates by size. In vitro α-synuclein aggregates were generated and fractionated into different sucrose concentrations (10%, 20%, 30%, 40%, and 50%). The structure of the aggregates in each fraction was characterized using various techniques: transmission electron microscopy (TEM), total internal reflection fluorescence (TIRF) microscopy with thioflavin T (ThT) and thioflavin X (ThX) dyes, atomic force microscopy (AFM), and single-molecule pulldown (SIMPull) assay with conformation-specific and sequence-specific antibodies. Toxicity was assessed using two assays: a membrane disruption assay measuring calcium influx into liposomes, and an inflammatory response assay measuring TNFα release from BV2 microglial cells. Soluble aggregates were also extracted from post-mortem human brain tissue (amygdala) from Parkinson's disease patients and controls. The size and inflammatory capacity of these brain-derived aggregates were analyzed using AD-PAINT super-resolution microscopy and AFM. Statistical analyses included t-tests, ANOVA, Kolmogorov-Smirnov tests, and Mann–Whitney U tests.

Structural characterization revealed that aggregate size increased with sucrose concentration. Small, predominantly spherical aggregates were found in the 20% fraction, transitioning to elongated structures in the 30% fraction and fibrils in the 40% and 50% fractions. AFM confirmed an increase in height and diameter with increasing sucrose concentration, with the 20% and 30% fractions containing protofilaments and the 40% and 50% fractions containing larger aggregates. Antibody-based imaging showed that the C-terminus of α-synuclein was more accessible in smaller aggregates. Toxicity assays demonstrated that the smaller aggregates (10% and 20% fractions, <200 nm) were significantly more toxic than larger aggregates in terms of membrane disruption and inflammatory response (TNFα release). Analysis of post-mortem human brain samples revealed that soluble α-synuclein aggregates in Parkinson's disease brains were significantly smaller (<100 nm) and more inflammatory than those in control brains. These smaller aggregates in PD brains also induced a greater inflammatory response in BV2 cells over a 96-hour period. The difference in inflammatory capacity correlated with size, not total aggregate amount.

This study demonstrates a clear link between α-synuclein aggregate size and toxicity. Smaller, soluble aggregates, rather than larger fibrils, are the primary drivers of membrane disruption and neuroinflammation. The findings in human brain samples strongly support this conclusion, showing that smaller, more inflammatory aggregates are characteristic of Parkinson's disease. These results highlight the importance of focusing on the small soluble aggregates as therapeutic targets for Parkinson's disease. The methods developed in this study provide a robust approach for characterizing the heterogeneity of α-synuclein aggregates and assessing their toxicity.

This research conclusively demonstrates that small soluble, non-fibrillar α-synuclein aggregates are the most toxic species in Parkinson's disease, driving neuroinflammation and potentially disease progression. These findings suggest that therapeutic strategies targeting these specific aggregates could be highly effective. Further research should focus on understanding the specific mechanisms by which these small aggregates cause toxicity and on developing targeted therapies to remove or neutralize them.

The study primarily focused on the amygdala region of the brain. While this region is relevant to PD dementia, further investigation in other brain regions affected by PD is needed. The sample size for the human brain tissue analysis was relatively small (three PD cases and three controls), limiting the generalizability of the findings. The membrane permeabilization assay lacked sensitivity for brain-derived samples, potentially due to lower aggregate concentration compared to recombinant preparations.