Screening for Protein Turnover Modulators in Human Neurons

Disturbed protein homeostasis is a hallmark of aging and many neurodegenerative disorders, reflecting imbalances in protein synthesis and clearance. The interplay of these processes defines protein turnover and maintains a functional proteome. Protein turnover regulation remains challenging to quantify, particularly in post-mitotic human neurons and at a scale compatible with small molecule or genetic screening. In this PhD thesis, we established a live-cell imaging platform based on a fluorescent timer to perturb and quantify protein turnover in stem cells-derived human neurons and to identify pharmacological modulators in a disease-relevant context. We generated human induced neurons expressing the fluorescent timer which allows quantitative determination of protein turnover. We then implemented the timer in the context of a high-content imaging workflow suitable for phenotypic screening. A primary screen of 6,000 small molecules identified compounds that enhanced protein turnover. The dose-response profiling of the prioritized candidates yielded a set of robust modulators spanning diverse target classes. From this group, we selected three hits: CI-994, CGP-52411 and AS-252424. We performed RNA-seq and LFQ proteomics of drug-treated neurons, which converged on translation- and ribosome-associated signatures, whereas the abundance of proteasome and canonical autophagy components remained largely unchanged, suggesting that turnover enhancement can arise from functional regulation of existing protein quality control machinery rather than increased abundance. To relate turnover modulation to proteotoxic stress, selected compounds were tested in an alpha-synuclein (aSyn) preformed-fibril seeding model in mouse primary neurons and human dopaminergic neurons. Pathology was quantified by high-content imaging of phosphorylated aSyn (pS129) and confirmed by immunoblotting. All three compounds suppressed the pS129 pathology in a concentration-dependent manner without altering fibril uptake, consistent with modulation of early aggregation and/or neuronal stress responses. In summary, this work describes a scalable single-cell imaging framework for protein turnover- focused screening in human neurons, provides candidate small-molecule modulators, and establishes an experimental basis to dissect how neuronal protein turnover can be tuned under basal and proteotoxic conditions. While the causal links between protein turnover modulation, long-term neuronal health, and neuroprotection require further study, the platform generalizes beyond Parkinson's disease and enables systematic exploration of proteostasis-targeting strategies across diverse conditions.