PRO-FitS: a novel phenotypic assay to identify enhancers of proteostasis in C. elegans

Abstract The prevalence of neurodegenerative diseases (NDs) continues to rise with the aging of populations worldwide, representing a pressing need for the establishment of therapeutic strategies. Maintaining proteostasis is crucial for healthy aging, as the accumulation of misfolded and aggregated proteins is a key contributor to age-related cellular dysfunction and disease. This study introduces a novel phenotypic assay using Caenorhabditis elegans to screen for small molecule enhancers of proteostasis, aiming at mitigating the proteotoxic stress associated with NDs. This new methodology- PRO-FitS- uses C. elegans motor activity as a proxy for the PROteome Fitness State upon a noxious protein-denaturating stimulus, while allowing a fast and experimenter-free readout. We demonstrate the efficacy of the assay by validating the role of pharmacological mTOR inhibition and serotonergic signaling activation in reducing heat shock-induced proteotoxic damage at the whole-organism level. PRO-FitS will allow the identification of novel compounds that alleviate protein aggregation disorders, potentially revealing new pathways and cellular targets not previously implicated in proteotoxicity. Significance Statement Neurodegenerative diseases remain without effective cures, in part due to the lack of scalable methods to identify compounds that improve proteostasis. We developed PRO-FitS, a whole-organism, automated phenotypic assay in C. elegans that uses motor activity recovery as a proxy for proteome fitness after proteotoxic stress. This platform enables rapid, unbiased screening of small molecules and genetic modifiers, bridging the gap between cellular assays and complex animal models. By demonstrating the assay’s robustness in both wild-type and disease-relevant contexts, we establish PRO-FitS as a versatile tool for discovering therapeutic candidates and uncovering novel pathways relevant to protein aggregation disorders.