Seed amplification of released alpha-synuclein strains reveals genotype-specific differences in Parkinson’s disease neurons

Abstract

Parkinson’s disease is one of a group of diseases collectively termed α-synucleinopathies, all of which are characterized by abnormal accumulation of toxic α-synuclein (α-syn) assemblies which are thought to follow a prion-like propagation model and drive neuronal death. Both intrinsic strain properties and the genetic background of affected individuals are hypothesized to influence α-syn aggregate accumulation, propagation, and extracellular release. As such, the aim of this study is to characterize how different genetic backgrounds modulate α-syn aggregate accumulation and release in response to treatment with different α-syn strains. iPSC-derived neurons from five different groups were employed (healthy non-PD controls (NPC), idiopathic PD (iPD), GBA1 N370S, LRRK2 G2019S and SNCA A53T) ( n = 15 cell lines, three lines per group). Individual aggregates in conditioned medium (CM) were measured using a single-particle detection system to quantify released α-syn species and assess strain persistence across genotype groups. Seed amplification assay (SAA) was then applied and linear discriminant analysis (LDA) of single particle properties from CM of α-syn fibril-treated neurons was used to discriminate between groups. α-Syn released into CM showed strain persistence in LRRK2 G2019S and GBA1 N370S genotype groups, while SNCA A53T neurons exhibited evidence of strain remodeling. Interestingly, amplification-response Δ-feature analysis of single particle properties showed group discrimination beyond either pre- or post- amplification measurements independently, reaching a maximum LOO-CV accuracy of 66.7% (3.3 × chance level) at day 10 post-treatment in CM from fibrils-treated neurons. These findings indicate that genotype-specific cellular responses differentially shape the structural properties of released α-syn in a manner that determines amplification responses, enabling inter-group discrimination at the single-molecule level.