Biology subjects

Silver, N. R. G.

Publications and source records attributed to Silver, N. R. G..

4 recordsLinked to original sources

bioRxiv2026

Differently sized soluble α-synuclein species from multiple system atrophy and Lewy body disease brains display different seeding propensities

Different conformations, or strains, of -synuclein (-syn) aggregates are believed to be responsible for the distinct seeding propensities, propagation profiles, and clinical presentations in Lewy body diseases (LBD) and multiple system atrophy (MSA). While biochemical properties and strain differences of insoluble deposits have been extensively characterized, the understanding of what influence soluble -syn species may have on these processes is limited to a small number of studies focusing on complex mixtures of soluble species or on a single - synucleinopathy. Given that soluble oligomers are considered highly pathologically relevant, we isolated and characterized the biochemical, seeding, and toxicity properties of size-fractionated soluble -syn species from MSA and LBD brains, comparing them to species from control brains without known neurological disease (Ctrl). We observed that levels of differently sized oligomers phosphorylated at Ser129, as well as soluble large oligomers (>450 kDa), were increased in LBD compared to both MSA and Ctrl brains. Nevertheless, species derived from MSA brain exhibited seeding activity across the spectrum of -syn species (oligomers, monomers, and truncated forms) in the seed amplification assay, whereas only oligomeric species (>150 kDa) from LBD cases were seeding-prone. In the HEK293 -syn (A53T)-YFP biosensor line, as well as in murine primary neurons, only large oligomers (>450 kDa) from MSA cases induced seeding and aggregation of -syn. Taken together, our study suggests that soluble -syn species derived from MSA and LBD brains show different biochemical, aggregation and seeding patterns, presumably due to strain variations of the respective oligomers. Our findings provide novel insight into the pathogenesis of different -synucleinopathies, which may guide us in the development of targeted therapeutics.

neuroscience↗

bioRxiv2026

α-Synuclein strain homogeneity in multiple system atrophy clinical subtypes

Conformationally distinct strains of -synuclein aggregates are believed to contribute to the clinical and pathological diversity observed among synucleinopathies such as multiple system atrophy (MSA) and Parkinsons disease. Cases of MSA can be classified into two distinct clinical subtypes: the cerebellar variant, MSA-C, and the parkinsonian variant, MSA-P. To assess whether distinct -synuclein strains may be present in individuals with MSA-C versus MSA-P, we characterized the conformational and seeding properties of -synuclein aggregates in various brain regions from MSA-C and MSA-P patients and performed propagation studies in M83 transgenic mice. Biochemical fingerprinting of -synuclein aggregates using limited proteolysis and a conformational stability assay failed to reveal differences between MSA-C and MSA-P either before or after propagation in mice. Similarly, using brain extracts from either MSA patients or MSA-inoculated mice, MSA-C and MSA-P -synuclein aggregates exhibited indistinguishable seeding attributes in a seed amplification assay. Finally, no differences were observed in either the kinetics of disease progression or the extent of cerebral -synuclein deposition in M83 mice inoculated with either MSA-C or MSA-P, regardless of the brain region from which the injected -synuclein aggregates were derived. These results suggest that MSA clinical subtypes are unlikely to arise due to distinct -synuclein strains. Instead, our findings support a model in which the same -synuclein strain initially forms in different brain regions, leading to differences in disease manifestation.

neuroscience↗

bioRxiv2025

Stochastic Misfolding Drives the Emergence of Distinct α-Synuclein Strains

The existence of -synuclein conformational strains provides a potential explanation for the clinical and pathological differences among synucleinopathies such as Parkinsons disease and multiple system atrophy. However, how distinct -synuclein strains are formed in vivo remains unknown. Here, we examined whether unique strains of self-propagating -synuclein aggregates can arise within a consistent molecular environment. Unexpectedly, we observed conformational heterogeneity between individual preparations of -synuclein pre-formed fibrils (PFFs) generated by polymerizing recombinant wild-type or A53T-mutant human -synuclein under identical conditions. Moreover, we found that -synuclein aggregates formed spontaneously in the brains of a transgenic synucleinopathy mouse model were conformationally diverse, leading to the identification of three distinct disease subtypes. Propagation of putative PFF- and brain-derived -synuclein strains in mice initiated several distinct synucleinopathies, characterized by differences in disease onset times, cerebral -synuclein deposition patterns, and the conformational attributes of -synuclein aggregates. The conformational diversity of -synuclein aggregates across PFF preparations and between the brains of individual transgenic mice demonstrates that -synuclein can spontaneously form multiple self-propagating strains within an identical environment both in vitro and in vivo. This suggests that stochastic misfolding into distinct aggregate structures drives the emergence of -synuclein strains and implies that the intrinsic variability of common synucleinopathy research tools must be considered when designing and interpreting experiments.

neuroscience↗

bioRxiv2025

α-Synuclein purification significantly impacts seed amplification assay performance and consistency

-Synuclein seed amplification assays are a promising diagnostic tool for synucleinopathies such as Parkinsons disease and multiple system atrophy. Standardized conditions are required to ensure a high degree of inter- and intra-laboratory reproducibility when performing these assays. A significant issue that hinders the utility of seed amplification assays is the de novo aggregation propensity of the -synuclein substrate as well as inter-batch heterogeneity. While much work has focused on determining appropriate seed amplification assay buffer compositions as well as the type and amount of seed used, a robust comparison of -synuclein substrate purification methods has not been reported. We therefore compared the utility of recombinant -synuclein purified using four different methods as seed amplification assay substrates across two laboratories. Osmotic shock-purified -synuclein monomer substrate showed the lowest propensity for de novo aggregation, which translated into being the best substrate for seed amplification assay reactions seeded with -synuclein preformed fibrils or patient brain homogenates. Furthermore, osmotic shock -synuclein monomer showed the best inter-batch reproducibility compared to all other substrates tested. As -synuclein seed amplification assays continue to evolve and move towards adoption in the clinical realm, this work showcases the vital importance of standardizing the production and characterization of recombinant -synuclein substrate. We encourage the widespread adoption of osmotic shock -synuclein monomer as the universal substrate for seed amplification assays to maximize intra- and inter-laboratory reproducibility.

biochemistry↗