TAR DNA-binding protein-43 (TDP-43) is an essential DNA/RNA-binding protein that plays a major role in RNA processing and stability. Although predominantly nuclear in localization, over 95% of amyotrophic lateral sclerosis (ALS) cases and up to half of frontotemporal dementia (FTD) cases show mislocalization of TDP-43 to the cytoplasm. Substantial evidence links TDP-43 mislocalization to the pathogenesis of neurodegeneration in ALS/FTD, and TDP-43 mutations have been identified in subset of familial ALS cases, further supporting a primary role for TDP-43 disruption in disease. However, the cause of TDP-43 nuclear clearance in ALS/FTD remains unknown. A major barrier has been lack of understanding of the mechanism of TDP-43 nuclear export. Until recently, TDP-43 was thought to be a cargo of the nuclear export receptor, exportin-1 (XPO1). However, multiple recent studies demonstrate that XPO1 does not bind TDP-43 or mediate its export. Alternative export mechanisms may include passive diffusion from the nucleus, or active co-export with RNA, such as via the NXF1/TREX mRNA export complex. Our preliminary data show that TDP-43 nuclear export is ATP- and RNA- dependent, but the energy may be required upstream of the pore, for TDP-43 intranuclear mobility, rather than translocation across the nuclear pore complex (NPC). Indeed, a permeabilized cell assay suggests that, once displaced from nuclear RNA, TDP-43 likely diffuses out of the nucleus. Moreover, studies in an NXF1 auxin- inducible degron (AID) cell line suggest that the bulk mRNA pathway is not required for TDP-43 export. Based on these data, we hypothesize that ATP-dependent nuclear tethering of TDP-43, most likely to nuclear RNA, critically dictates the rate at which it diffuses into the cytoplasm. In the proposed studies, we will perform an arrayed CRISPRi and CRISPRa screen of a targeted library to determine the mechanism of energy-dependent nuclear tethering of TDP-43. Hits will be validated in nuclear transport and photobleaching assays, and also evaluated in ALS/FTD patient tissue. We will then combine high content microscopy with nucleocytoplasmic transport assays, including a newly developed permeabilized cell export assay, to confirm the mechanism of TDP-43 translocation across the nuclear pore during export. Additional AID cell lines and knockdown studies will be used to definitively exclude a role for the mRNA export pathway. Finally, we will test the therapeutic efficacy of a novel approach to promote TDP-43 nuclear retention, identified during our preliminary studies. Testing will include TDP-43 mutant Drosophila (in collaboration with the Daniela Zarnescu lab), TDP-43 mutant iNeurons, and mouse sciatic nerve axotomy, to determine if we can promote TDP-43 nuclear retention in vivo, and whether this strategy will attenuate molecular and neuropathologic features of TDP-43 proteinopathy. These studies will enable a precise mechanistic understanding of the mechanism and regulation of ...