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

Chanshuai Han; Emmanuelle Nguyen-Renou; Faiza Benaliouad; Wen Luo; Carol X-Q Chen; Aeshah Alluli; Lorenza Villegas; Lenore K Beitel; Irina Shlaifer; Wolfgang E Reintsch; Andrea I Krahn; Esther Del Cid Pellitero; Edward A Fon; Thomas M Durcan

doi:10.1016/j.ymeth.2026.06.005

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

Methods. 2026 Jun 15:254:29-41. doi: 10.1016/j.ymeth.2026.06.005. Online ahead of print.

Affiliations

¹ The Neuro's Early Drug Discovery Unit (EDDU), McGill University, 3801 University Street, Montreal, Quebec H3A 2B4, Canada.
² Neurodegenerative Disorders Research Group, Department of Neurology and Neurosurgery, Montreal Neurological Institute-Hospital (The Neuro), McGill University, 3801 University Street, Montreal, QC H3A 2B4, Canada.
³ The Neuro's Early Drug Discovery Unit (EDDU), McGill University, 3801 University Street, Montreal, Quebec H3A 2B4, Canada; Neurodegenerative Disorders Research Group, Department of Neurology and Neurosurgery, Montreal Neurological Institute-Hospital (The Neuro), McGill University, 3801 University Street, Montreal, QC H3A 2B4, Canada. Electronic address: thomas.durcan@mcgill.ca.

PMID: 42297199
DOI: 10.1016/j.ymeth.2026.06.005

Abstract

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 number variants and it offers a platform for assessing compounds and therapeutics that may impede α-syn aggregate formation.

Keywords: DA NPCs: dopaminergic neural progenitor cells; DNs: dopaminergic neurons; PD: Parkinson’s disease; PFFs: preformed fibrils; iPSCs: induced pluripotent stem cells; pS129-syn: phosphorylated alpha-synuclein at serine 129; α-syn: alpha-synuclein.