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Defining the Role of TDP-43 in Neurodegenerative Disease Pathways
Date of Award
Doctor of Philosophy (PhD)
Amyotrophic lateral sclerosis (ALS) is a progressive, fatal disorder characterized by muscle weakness and spasticity due to selective degeneration of upper and lower motor neurons, with no effective treatment available. Interestingly, ALS and Frontotemporal lobar degeneration (FTLD) show considerable clinical and pathologic overlap. Mutations in the TAR DNA-binding protein (TDP-43) are a major component of ubiquitinated neuronal cytoplasmic inclusions in patients with familial as well as sporadic ALS, and TDP-43 aggregates are found in both ALS and FTLD-U (FTLD with ubiquitin aggregates), suggesting a common underlying mechanism. TDP-43 is a DNA/RNA binding protein that has been shown to play a role in transcription and mRNA processing, but whose physiologic function is not completely understood. We have characterized mice expressing the disease associated A315T TDP-43 mutant that develop a progressive neurodegenerative disease and recapitulate selective ubiquitin pathology and degeneration of layer V and spinal motor neurons. Surprisingly, TDP-43 is rarely present in large cytoplasmic ubiquitinated aggregates, and is not required for TDP-43 induced neurodegeneration, and instead suggested that altered DNA/RNA binding protein function is important to TDP-43-related neurodeegenration.
To compare wild type (WT) and mutant TDP-43 and examine expression in various cell types at various levels, we have created new inducible transgenic mice expressing FLAG-tagged sequence following a transcriptional stop sequence flanked by loxP sites. By crossing to mice expressing universal Cre under the actin promoter, we created matched WT and mutant lines expressing the transgene at about one fold over endogenous levels. Neither WT nor mutant lines developed overt degeneration, behavioral abnormalities, or disease-associated pathology. However, the comparison of the dosage in the low expressing lines and original high expressing A315T line with corresponding phenotypes indicated that overexpression of TDP-43 produced neurodegeneration in a dose-dependent manner with expression above a threshold level. Furthermore, neurodegeneration correlated with the reduction of endogenous mouse TDP-43, which suggests that dysregulation of TDP-43 is taking place once this threshold of disease is crossed as well.
In order to elucidate the mechanisms that drive TDP-43 mediated neurodegeneration, information on RNA binding targets combined with global expression and splicing changes is essential to understand the resulting misregulation that eventually leads to pathologic aggregate formation, cell loss, and axon degeneration. By immunoprecipitating and sequencing cross-linked RNAs bound to TDP-43 (known as CLIP-seq) we identified a long list of TDP-43 target RNAs in our TDP-43 based model of ALS/FTLD. Our analysis identified a TDP-43 auto-regulatory mechanism disrupted in neurodegeneration caused by altered splicing the 3' UTR of TDP-43's own transcript. Additional disease relevant targets whose splicing patterns was affected when overexpression of TDP-43 is driving neurodegeneration includes disease-related Fus/Tls, as well as others. While we are just beginning to understand the network of genes whose alternative splicing contributes to neurodegeneration, ongoing and future studies of TDP-43 mediated RNA processing will be important for the development of therapeutic interventions in patients.
Chair and Committee
Robert H Baloh
Timothy M Miller, Rakesh Nagarajan, Robert E Schmidt, Conrad C Weihl
Wegorzewska, Iga Natalia, "Defining the Role of TDP-43 in Neurodegenerative Disease Pathways" (2014). Arts & Sciences Electronic Theses and Dissertations. 143.