Mechanistic dissection of novel regulators of TDP-43 aggregation identified in a genome-wide CRISPR-Cas9 knockout screen

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Project Summary The mislocalization and aggregation of RNA-binding proteins (RBP) is a pathological hallmark of Amyotrophic Lateral Sclerosis (ALS). The RBP TAR DNA-binding protein 43 (TDP-43) is of particular interest, as pathological aggregation of TDP-43 is observed in nearly 97% of ALS patients, despite the fact that disease-causing mutations in TDP-43 explain fewer than 5% of disease incidence. This observation suggests that misregulation or mutation of other genes converge on TDP-43 pathology, however it remains poorly understood how the aggregates arise or whether they can be reversed or prevented. To this end, we have developed Pulse-Shape Analysis-based aggregation reporters for the TDP-43 that, when coupled to fluorescence activated cell sorting, quantifies aggregation at the single cell level. This reporter was leveraged against a genome-wide CRISPR- Cas9 knockout screen to identify regulators of TDP-43 aggregation. Reassuringly, this screen revealed known interactors and pathways involved in TDP-43 regulation and pathology. Additionally, this work revealed several novel proteins not previously implicated in TDP-43 biology or aggregation. Initial follow up on one top hit, SRRD, revealed that this protein reduces TDP-43 aggregation in a mammalian aggregation model, and reduces TDP- 43-associated toxicity in a yeast model, indicating that these genes indeed modify TDP-43 aggregation. The goal of this proposal is to mechanistically dissect how these novel regulators modify TDP-43 aggregation. Using an induced motor neuron model of TDP-43 aggregation, yeast toxicity models, and in vitro studies, I will assess the interactors, signaling pathways, and mode of TDP-43 modification for the following four genes: METTL5, EIF2AK2, XPO4, and SRRD. This work will not only reveal the function of uncharacterized proteins, but dissect their heretofore unknown mechanisms in regulating TDP-43 aggregation. Through better understanding of how TDP-43 aggregation is regulated, the field can begin to answer questions regarding the role of TDP-43 aggregation in disease pathogenesis and progression. By understanding the regulatory network of underlying TDP-43 aggregation, we can begin to therapeutically modulate the pathways that contribute to disease.