Development and validation of a model for investigation of transcellular trafficking of the ALS gene product TDP-43
thesisposted on 2022-03-28, 14:57 authored by Erin Lynch
Amyotrophic lateral sclerosis (ALS) is the most common form of motor neuron disease that affects the upper and lower motor neurons. ALS is an adult onset disease, which progressively worsens after onset, leading to severe loss of motor function and eventually death. Mutations in the gene encoding TDP-43, an RNA-binding protein, have been identified in patients with heritable ALS. The mutant form of TDP-43 is associated with neuronal degeneration and intracellular aggregate formation in vitro. Aggregates have also been identified within patient tissues: it is proposed that mutant TDP-43 drives aggregate formation and that the aggregates play a role in the neuronal dysfunction and eventual death that underlie the clinical features of ALS.One paradox in the field of ALS research is that neuronal loss is largely restricted to neurons related to motor control, but that degeneration is progressive and occurs at all levels of the neuraxis. A mechanism that could potentially underlie the selective spread of dysfunction through functionally discrete pathways is self-assembly and propagation of cytotoxic proteins. We hypothesize the mutant TDP-43 may drive aggregate formation in affected cells and could potentially move between cells via a prion-like mechanism.The specific aims of this thesis are to; (1) generate a plasmid construct that encodes a GFP fusion protein of wild-type and mutant TDP-43, allowing the direct visualization of TDP-43 distribution in live cells. (2) To generate vectors that drive stable and permanent integration of those fusion proteins. (3) To test whether GFP is detectable in cells that are co-cultured with TDP-43 mutants.