Background: The major protein constituent that makes up the inclusions found in amyotrophic lateral sclerosis (ALS) was recently identified as TAR DNA-binding protein-43 (TDP-43), an obscure nuclear protein known to be involved in exon splicing. Subsequent to this finding, mutations in the gene encoding TDP-43 (TARDBP) were identified as a direct cause of neurodegeneration in sporadic and familial patients with ALS. Work from our laboratory has shown that increased cell death signaling induced cleavage of TDP-43 in cell culture systems. In the presence of mutations in TARDBP, TDP-43 gets cleaved, which leads to translocation from the nucleus to the cytosol, a pathologic phenotype that resembles what happens to TDP-43 in patients with FTLD-U or ALS. Thus, loss of TDP-43 function due to inappropriate cleavage, translocation, or inclusion formation could play an important role in neurodegeneration.Objective/Hypothesis: We hypothesize that the TDP-43 pathobiology is regulated by caspase activity and that the accumulation of the cleaved TDP-43 is neurotoxic in vitro and in vivo.Specific Aims: Aim 1: Determine whether TDP-43 proteolytic fragments can enhance cellular toxicity. We have recently shown that caspase activity is sufficient to induce TDP-43 proteolytic cleavage and redistribution from the nucleus to the cytoplasm. This aim will test if overexpression of TDP-43 proteolytic C-terminal fragments can form cytoplasmic TDP-43 aggregates leading to increased cellular toxicity and the sequestration of nuclear TDP-43 in the cytoplasm and loss of functional TDP-43. Aim 2: Determine the neurodegenerative disease mechanism associated with TARDBP mutations. Extensive mutation analysis of TARDBP by us and others has recently resulted in the identification of missense mutations in TARDBP in sporadic and familial patients with ALS. These mutations each affect highly conserved amino acid residues in the C-terminal part of TDP-43 known to be involved in protein-protein interactions. We will test if TARDBP mutants can increase cellular toxicity and alter the biological properties and function including inappropriate cleavage and distribution, splicing, and inclusion formation. Aim 3: Generate and characterize constitutive and conditional transgenic mice overexpressing wild-type and M337V mutant TDP-43. We intend to produce two unique transgenic models for ALS to allow us to determine the behavioral, biochemical, and neuropathological impact of mutant TDP-43 expression.Study Design: The work in Aims 1 and 2 will utilize cell culture models including primary cultures of mouse hippocampal neurons to assess neuronal loss, exon splicing assays to access TDP-43 function, and confocal microscopy to assess TDP-43 distribution and biochemical approaches to access TDP-43 function. For Aim 3, we propose to utilize the mouse prion promoter (MoPrP) to produce (1) a constitutive transgenic mouse model and (2) a conditional mouse model of mutant (M337V) TDP-43. Transgenic mice expressing wild-type human TDP-43 will be generated as controls. We will determine if mutant TDP-43 can drive (1) increased caspase-cleavage and relocalization of TDP-43, (2) motor deficits, and (3) neurodegeneration in both the mouse models. Finally, we will determine if TDP-43opathy can be reversed by suppression of mutant TDP-43 expression in the conditional mouse model after its development of salient features of ALS.Impact: Our goal is to gain better understanding of the underlying biology and developing insights causing TDP-43 proteinopathies by generating a novel model of ALS, which might ultimately lead to improved therapies.
|Effective start/end date||1/1/08 → 9/30/12|
- U.S. Department of Defense: $918,000.00
- Congressionally Directed Medical Research Programs: $918,000.00