RNA metabolism and RNA binding proteins play a critical role in neurodegenerative disorders, in particular, amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). TDP-43 is the most common RNA- binding protein associated with ALS/FTD and is a secondary pathology in other neurodegenerative diseases, including Alzheimer's disease. TDP-43 aggregation is thought to cause neurodegeneration through a combination of loss of function and gain of toxic mechanisms.
Our research aims to elucidate a major gap in understanding ALS/FTD pathogenesis: how is TDP-43 homeostasis maintained and what factors trigger aggregation. We found a dual TDP-43 phosphorylation site (pT153/Y155) that modulates TDP-43 activity, decreases misfolding, and is associated with specific RNA granule localization in human cells. pT153/Y155 localizes in nucleolar bodies, while the non-phosphorylated form accumulates in cytoplasmic stress granules. This is the first example of a TDP-43 phosphorylation epitope associated with specific RNA granule dynamics. These are ribonucleoprotein granules associated with the recruitment and aggregation of ALS/FTD-associated RNA-binding proteins, including TDP-43. Therefore, stress granules are viewed as key precursors of TDP-43 pathology and neurotoxicity in ALS and FTD. Stress granule formation is triggered by proteotoxic conditions. Recently, dipeptide repeats linked to the ALS/FTD C9orf72 hexanucleotide repeat expansion (C9-HRE) were found to increase TDP-43 accumulation in stress granules. We observe that C9-HRE peptide expression disrupts nucleolar pT153/Y155 and increases stress granule localization of non-phosphorylated TDP-43. Based on these findings, our central hypothesis is that pT153/Y155 controls TDP-43 cellular dynamics and may affect protein homeostasis. We posit that, under proteotoxic stress, non-phosphorylated T153/Y155 upregulates interactions and processes that mediate stress granule association. The goal of our project is to test the role of this pathway in pathogenesis by determining: 1) the structural and functional mechanisms by which pT153/Y155 modulates TDP-43 localization in stress granules, 2) whether phosphorylation at T153/Y155 decreases TDP-43 misfolding and aggregation under proteotoxic conditions, and 3) how pT153/Y155 impacts stress granule recruitment of TDP-43 in ALS/FTD disease models, including C9-HRE and TDP-43 mutant-derived iPS neurons. We will combine our unique expertise in the analysis of recombinant TDP-43 assembly in vitro with cellular approaches to measure TDP-43 function and cellular dynamics. We have well-established collaborations to test the significance of our findings in ALS/FTD disease models. In completing the proposed work, we will define molecular mechanisms that mediate a critical process in seeding TDP-43 pathology. We will also establish how TDP-43 posttranslational modification affects function and homeostasis under normal and pathogenic conditions. Importantly, this work will elucidate convergent disease mechanisms in TDP-43 proteinopathies.
TDP-43 is a protein central to devastating and neurological disorders including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). In addition, TDP-43-associated pathology is also present in Alzheimer's disease and other neurodegenerative disorders. Our investigation on the specific processes that regulate TDP-43 function and homeostasis will improve our understanding of the role that this protein plays in pathogenesis and contribute to the discovery of new and effective treatment for these disorders.
