A high-content imaging workflow to screen for molecules that reduce cellular uptake of α-synuclein preformed fibrils

• Accumulation of a-synuclein aggregates in cells is a hallmark of Parkinson disease. • Stem cell-derived neurons provide a human model to investigate aggregate uptake. • High content imaging and analysis allow to measure uptake on the cellular level. • Providing a scalable tool to screen for inhibitors of a-synuclein aggregate uptake. • Screening a compound library revealed several inhibitors of uptake. A classical pathological hallmark of many neurodegenerative diseases is the formation of protein-rich aggregates and inclusions. In Parkinson’s disease (PD), α-synuclein (α-syn) constitutes a major protein component of pathological inclusions, termed Lewy bodies. These α-syn aggregates are hypothesized to spread throughout the nervous system by cell-to-cell transmission acting as templates to amplify aggregate formation. In vitro generated α-syn aggregates, commonly called preformed fibrils (PFFs), have been used to investigate a number of aspects related to α-syn mediated pathology across different model systems. Here we describe a semi-automated assay to screen for small molecules that interfere with the cellular uptake and accumulation of PFFs. The assay uses dopaminergic progenitor cells (DPCs), derived from human induced pluripotent stem cells (hiPSCs). In an initial screen, we tested 1520 small molecules and identified several molecules that strongly reduce intracellular PFF load in DPCs. From these hits, candidate compounds were validated in dopaminergic neurons (DNs) to demonstrate the utility of the assay. This assay provides a robust, scalable and adaptable tool to screen for molecules that affect PFF uptake in hiPSC-derived cell models. Within the scope of this screen, it led to the identification of a set of compounds with diverse annotated targets that effectively reduce the accumulation of α-syn aggregates in DPCs and DNs.