An in vivo and behavioral study of ALS-associated protein Tdp-43 in zebrafish

Amyotrophic lateral sclerosis (ALS) is a progressive muscle wasting disorder, characterized by the degeneration of upper and lower motor neurons. There is currently no cure and the disease is fatal 3-5 years post symptom onset. An improved understanding of ALS at the cellular and molecular levels are essential in order to identify disease pathways, leading to motor dysfunction. Zebrafish (Danio rerio) are powerful model organisms in scientific research and have great potential for use in high-throughput drug screening and in the analysis of cellular and molecular mechanisms of disease in vivo and in real-time, as well as possible resulting musculoskeletal phenotypes. The DNA/RNA binding protein, Transactive Response DNA-binding protein 43kDa (TDP-43) is a component of ubiquitinated cytoplasmic inclusions, present in around 97% of ALS cases. A number of causative mutations in the TARDBP gene have been identified in both familial and sporadic ALS patients. This thesis will study the transient and transgenic expression of TDP-43 in zebrafish. The initial investigation of mRNA overexpression gave us rationale to pursue the development of stable transgenic zebrafish specifically expressing the zebrafish ortholog Tdp-43 in motor neurons. In this study, overexpression (OE) of a mutant Tdp-43 construct in a conserved residue associated with sporadic ALS, Q331K, Tg (-3mnx: mVenus: Tdp-43 ^Q331K), was compared to OE of wild-type (wt) Tdp-43 Tg (-3mnx: mVenus: Tdp-43 ^wt) and non-transgenic (nTg) controls. We conducted cellular and molecular analysis of these transient and transgenic zebrafish, assessing the localization of Tdp-43 in motor neurons, as well as examining motor phenotypes at larval and adult stages. Our results show Tdp-43 mislocalization in the motor neurons of larvae expressing mVenus: Tdp-43 bearing the conserved point mutation Q331K. This mislocalization is increased on induction of cellular stress, accompanied byreduced locomotion. We also identified progressive degeneration in adult transgenic fish, abnormal neuromuscular junction (NMJ) integrity, decreased swimming ability and reduced survival, compared to nTg and mVenus: Tdp-43^wt OE controls. This thesis presents a larval and adult model of ALS, recapitulating multiple facets of disease. Further elucidating the links between the observed cellular dysfunction and motor phenotypes may better our understanding of ALS and take a step toward unveiling the complex neurobiology underlying the disease.