An automated workflow for quantifying the formation of synuclein aggregates in human dopaminergic neurons

作者：Chanshuai Han, Emmanuelle Nguyen-Renou, Faïza Benaliouad, Wen Luo, Carol X-Q Chen, Aeshah Alluli, Lorenza Villegas, Lenore K. Beitel, Irina Shlaifer, Wolfgang Reintsch, Andrea I. Krahn, Esther Del Cid Pellitero, Edward A. Fon, Thomas M. Durcan · 发表于：Methods · 年份：2026 · DOI：10.1016/j.ymeth.2026.06.005 · 研究领域：Parkinson's Disease Mechanisms and Treatments、Neurological disorders and treatments、Cell Image Analysis Techniques

Parkinson's disease (PD) is a neurodegenerative disorder characterized by alpha-synuclein (α-syn) aggregates termed Lewy bodies. To model PD pathology in vitro, preformed fibrils of α-syn (PFFs), which can be taken up by cells, provide a seed that drives misfolding and aggregation of endogenous α-syn, with new aggregates amplifying this process. External application of PFFs to dopaminergic neurons (DNs) increases aggregate formation, marked by α-syn phosphorylation at serine 129 (pS129-syn), a pathological PD marker. Building on this, we developed an automated synuclein seeding assay to quantify new α-syn aggregates in iPSC-derived DNs. Using pS129-syn as a readout, we show that PFFs elicit a time- and dose-dependent increase in pS129-syn aggregates. Our automated seeding assay further revealed that aggregate formation depends on endogenous α-syn levels. Treatment with PFFs produced a greater increase in pS129-syn aggregates in iPSC DNs derived from a PD patient with a triplication in the SNCA gene, which encodes the α-syn protein and which elevates total α-syn levels, relative to DNs from an isogenic iPSC line from the same individual, in which the SNCA gene mutation had been corrected by CRISPR/Cas9. In contrast, no pS129-syn signal was detected in neurons in which all copies of the SNCA gene had been knocked out (KO). This proof-of-principle automated high-content imaging workflow for synuclein seeding has been validated using isogenic cell lines with defined SNCA copy num...