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bioRxiv · 10.64898/2025.12.19.695578

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

Han, C.·Nguyen-Renou, E.·Benaliouad, F.·Luo, W.·Chen, C. X.-Q.·Alluli, A.·Villegas, L.·Beitel, L. K.·Shlaifer, I.·Reintsch, W. E.·Krahn, A. I.·Del Cid Pellitero, E.·Fon, E. A.·Durcan, T. M.
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Abstract

Parkinsons disease (PD) is a neurodegenerative disorder characterized by alpha-synuclein (-syn) aggregates termed Lewy bodies. To model PD pathology in vitro, preformed fibrils of -syn (PFFs), which can be taken up by cells, provide a seed that drives misfolding and aggregation of endogenous -syn, with new aggregates amplifying this process. External application of PFFs to dopaminergic neurons (DNs) increases aggregate formation, marked by -syn phosphorylation at serine 129 (pS129-syn), a pathological PD marker. Building on this, we developed an automated synuclein seeding assay to quantify new -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 high-throughput assay further revealed that aggregate formation depends on endogenous -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 -syn protein and which elevates total -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 high-content imaging assay for synuclein seeding offers a platform for assessing compounds and therapeutics that may impede -syn aggregate formation.

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Han, C., Nguyen-Renou, E., Benaliouad, F., Luo, W., Chen, C. X.-Q., Alluli, A., Villegas, L., Beitel, L. K., Shlaifer, I., Reintsch, W. E., Krahn, A. I., Del Cid Pellitero, E., Fon, E. A., Durcan, T. M.. 2025-12-23. An automated workflow for quantifying the formation of synuclein aggregates in human dopaminergic neurons. https://doi.org/10.64898/2025.12.19.695578

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