Identification of intrinsic and extrinsic regulators of TDP43 splicing function

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Project Summary Identification of intrinsic and extrinsic regulators of TDP43 splicing function. Dementias are devastating and incurable neurodegenerative diseases that represent an escalating burden on patients, their families and health systems throughout the world. Disease-modifying therapies for dementias are urgently needed, but their development has been hampered by our lack of knowledge about the vulnerable molecular networks that drive their pathogenesis. Dysfunction of the RNA-binding protein TDP43 has emerged pathological signature in many dementias, including Alzheimer’s disease (AD), limbic-predominant age-related TDP43 encephalopathy (LATE) and frontotemporal dementia (FTD). TDP43 functions as a guardian of proteome integrity by regulating the splicing of cryptic exons (CEs), exons that contain premature stop codons and frameshift mutations, present in hundreds of pre-mRNAs. However, the regulatory network that controls TDP43 function is poorly understood and the mechanism by which TDP43 dysfunction leads to neurodegeneration remains unknown. To help address this critical gap in knowledge, we have recently developed a quantitative, fluorescence-based reporter to measure TDP43-dependent splicing in single live cells. Combining this reporter with our expertise in large-scale, genome-wide screening technologies, we propose two screens designed to chart the molecular network that regulates TDP43 function in a comprehensive and unbiased manner. First, we will use recently developed deep mutational scanning methods to identify sequence features of TDP43 required for its splicing function. Mutant libraries will encompass over 50,000 single and double point mutations focused on the intrinsically disordered domain (IDR) of TDP43, since the IDR has been implicated in both splicing function and aggregation. To identify trans-acting factors and co-factors that regulate TDP43-dependent splicing, we will combine the reporter with genome-scale loss-of-function CRISPR screens. The top hits from both screens will be rigorously validated for their effects on TDP43-dependent splicing and aggregation in neurons derived from mouse embryonic stem cells. The successful completion of this project will uncover the intrinsic IDR sequence elements and extrinsic cellular components that directly or indirectly influence the splicing function of TDP43, thereby providing a foundation for the mechanistic studies required to develop new therapeutic strategies.