Multiplexed high throughput screening identifies broadly-active rescuers of proteotoxicity

The accumulation of misfolded proteins within intracellular aggregates is a distinctive feature observed within multiple neurodegenerative diseases (NDDs), yet the genes and pathways that regulate this process remain poorly understood. Here we describe a high-throughput discovery platform that enables 35 models of neurodegeneration and protein misfolding to be simultaneously screened to rapidly uncover genetic modifiers that alter the solubility and toxicity of a wide variety of aggregation-prone proteins associated with neurodegeneration. From these studies, we identify the human HSP40 chaperone, DNAJB6 as a potent rescuer of the cellular toxicity and aggregation of multiple RNA-binding proteins implicated in Frontotemporal dementia (FTD) and Amyotrophic Lateral Sclerosis (ALS) including FUS, TDP-43, and hnRNPA1. We further demonstrate that DNAJB6 has an intrinsic ability to phase separate under physiologic conditions and can alter the properties of FUS containing condensates by maintaining them in a unique gel like state. By conducting domain mapping studies and saturating mutagenesis on DNAJB6 we are able to further elucidate its mechanism of action, while also uncovering a series of novel DNAJB6 variants with increased activity that can serve as starting points for future gene therapy efforts.