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.

Public Health Relevance

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.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
High Priority, Short Term Project Award (R56)
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Cellular and Molecular Biology of Neurodegeneration Study Section (CMND)
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Gubitz, Amelie
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Saint Louis University
Schools of Medicine
Saint Louis
United States
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