Dembska, J.; Mahul-Mellier, A.-L.; Tollenaere, A.; Jasiqi, Y.; Suter, D. M.

Disturbances in protein homeostasis are a defining feature of aging and many neurodegenerative conditions. However, the balance of protein synthesis and clearance (i.e. protein turnover), remains challenging to quantify and to pharmacologically interrogate in post-mitotic human neurons at screening scale. Here, we establish a fluorescent timer-based, live-cell high-content imaging platform for protein turnover perturbation in human embryonic stem cell-derived neurons. We screened for 5,897 small molecules and identified 199 compounds that enhanced protein turnover. Dose-response assay of 47 prioritized candidates revealed several robust turnover modulators spanning diverse target classes. Transcriptome profiling and label-free quantitative proteomics on neurons treated with three selected compounds revealed upregulated expression of translation-associated genes. These compounds suppressed pathogenic synuclein (pS129) in a mouse primary neuron model of Lewy body-like pathology, and for one of them in human dopaminergic neurons. Together, this work provides a scalable discovery framework for protein turnover modulation in healthy and pathological contexts.