Abstract

Amyotrophic Lateral Sclerosis (ALS) is a terminal neurodegenerative disorder with a mean survival of 2-5 years after diagnosis. In ALS, the neurons innervating voluntary muscles retract and die, leading to muscle atrophy and loss of limb function. This neuronal death eventually reaches the lungs, causing death by suffocation. One protein implicated in ALS-related neuron death is TAR DNA Binding Protein 43 (TDP-43). TDP-43 is a highly conserved, RNA-binding protein that has been shown to form aggregates in the cytoplasm of cells in 90% of ALS cases. The aggregation of the protein is attributed to sequence mutations that lead to its erroneous location in the cytoplasm and enhance its ability to form the detrimental aggregates. Only two of the over fifty identified TDP-43 mutations are outside the domain responsible for protein-protein interactions. One of them is an Aspartate-169 to Glycine (D169G) mutation, which is located in the first RNA-recognition domain, suggesting that a protein with this mutation could utilize a unique pathogenic mechanism. The Alanine-315 to Threonine (A315T) mutant is located in a toxic membrane binding region within the prion-like domain of TDP-43, and if part of the subdomain is removed, there is evidence it prevents TDP- 43 toxicity. The A315T mutation is also associated with higher lethality. The aim of this research is to create two TDP-43 mutants with D169G and A315T substitutions via site-directed mutagenesis and then transfect the constructs into a motor neuron cell line NSC-34 for overexpression. Transfected cells will be imaged to document rate of morphological changes, and rate of cell death will be quantified by spectrographic analysis. These data will be entered into a databank to collect a broad range of data pairing specific mutations with the physiological effects they produce.

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