Protein aggregation inhibitors induce divergent transcriptional responses in a cellular model of ɑ-synuclein seeded aggregation

Abstract Parkinson’s disease (PD), dementia with Lewy Bodies (DLB) and multiple system atrophy (MSA) are progressive neurodegenerative disorders marked by the pathological aggregation of alpha-synuclein (ɑSyn). Despite significant research efforts, effective therapeutic interventions remain elusive due to limited understanding of the cellular effects of ɑSyn aggregation and propagation. This study presents the development of a scalable cellular seeding assay for screening small molecules targeting cellular ɑSyn seeded aggregation. By leveraging a fluorescent reporter of ɑSyn and phenotypic screening, the assay enables high-throughput evaluation of potential inhibitors in a cellular environment mimicking disease pathology. We evaluated three different αSyn aggregation inhibitors tested in clinical trials for PD: Minzasolmin, Emrusolmin and EGCG and profiled gene expression using multiplexed single cell RNA sequencing in order to examine their distinct effects on cellular pathways associated with ɑSyn overexpression or seeded aggregation. Two cellular activities were prominently affected: lipid metabolism and rRNA processing. Notably, while EGCG effects were confined to cells with aggregated αSyn, Minzasolmin and Emrusolmin also produced transcriptional changes in cells without aggregated αSyn. Each of the compounds tested induced a partial reversal of transcriptional effects resulting from αSyn seeded aggregation. We identified 391 genes that were no longer significantly differentially expressed upon addition of compound, relative to cells with seeded aggregation. This platform bridges phenotypic screening and molecular pathway analysis, providing insights into druggable pathways for synucleinopathies. The molecular signatures identified here can assist in testing and benchmarking future drug discovery leads.