Drivers of TDP43 Dyshomeostasis in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder in which the progressive loss of motor neurons results in paralysis and respiratory failure. Though the study of ALS is complicated by its heterogeneous biochemical, genetic, and clinical features, dysregulation of the RNA-binding protein TDP43 is observed in the vast majority of ALS cases. Although TARDBP mutations account for only a small proportion of the disease burden (2-5%), cytoplasmic TDP43 mislocalization and accumulation are observed in >90% of individuals with ALS. Moreover, mutations in several other ALS-associated genes result in TDP43 pathology. TDP43 is an essential protein involved in several RNA processing events, including splicing, translation, and degradation, and small changes in its localization and expression level are sufficient to disrupt critical cell processes (Chapter 1). As such, accumulating evidence implicates TDP43 and TDP43-dependent RNA processing in neurodegenerative disease (Chapter 2), but drivers of TDP43 accumulation and mislocalization remain fundamentally unclear. Here, we seek to identify phenomena that initiate TDP43 dyshomeostasis and develop techniques to better monitor TDP43 metabolism in the context of ALS. Much like TDP43 pathology, neuronal hyperexcitability is a conserved feature observed in both familial and sporadic ALS. However, its relation to neurodegeneration and TDP43 deposition in disease remains unknown. In Chapter 3, we show that hyperexcitability recapitulates TDP43 pathology by upregulating shortened (s) TDP43 splice isoforms. These truncated isoforms accumulate in the cytosol, where they form insoluble inclusions that sequester full-length TDP43 via preserved N-terminal interactions. Consistent with these findings, sTDP43 overexpression is toxic to mammalian neurons, suggesting that neurodegeneration results from complementary gain- and loss-of-function mechanisms. In humans and mice, sTDP43 transcripts are enriched in vulnerable motor neurons, and we observed a striking accumulation of sTDP43 within neurons and glia of ALS patients. These studies uncover a hitherto unknown role of alternative TDP43 isoforms, and indicate that sTDP43 production may be a key contributor to the susceptibility of motor neurons in ALS. In Appendix A, we establish a technique to monitor TDP43 metabolism at the endogenous level. To do so, we developed induced pluripotent stem cell (iPSC)-derived neurons in which we can monitor the synthesis and degradation of native TDP43 in a non-invasive manner. Following these measurements, each neuron is tracked over time to determine its time of death via longitudinal fluorescence microscopy (Appendix B), enabling us to determine how TDP43 synthesis and decay rates impact neuronal survival. Future work can utilize this methodology to determine if TDP43 metabolism is altered in neurons derived from ALS patients with C9orf72 and TARDBP mutations to further elucidate mechanisms of TDP43 dyshomeostasis. Chapter 4 concludes the dissertation and describes future studies to better understand mechanisms of sTDP43 toxicity and determine if sTDP43 is a viable therapeutic target for ALS. Appendix C further explores the identification of novel therapies and the development of a medium-throughput screen to identify novel compounds that stop or attenuate neurodegeneration. Taken together, this dissertation uncovers a novel disease pathway that may be targeted for therapeutic development, and establishes a technique to determine how TDP43 dyshomeostasis contributes to neurodegeneration in ALS.PhDNeuroscienceUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/153387/1/kweskamp_1.pd