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.
