Mutation in TDP-43 causes severe motor dysfunction through elevation of intracellular calcium in a C. elegans model of ALS
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Background: In Amyotrophic lateral sclerosis (ALS), motor neurons are affected and eventually these neurons are degenerated. ALS patients lose voluntary movement. Usually death occurs within 3-5 years after diagnosis. A missense mutation, A315T in the TDP-43 gene was discovered by Gitcho at el. This mutation leads to severe neuronal dysfunction in patients. This research found the evidence that the mutation in TDP-43 is causing calcium level to rise inside the cell in C elegans model of ALS. This elevated intracellular calcium level is hindering neuronal function and eventually may lead to cell death. Methods: Three different strains of transgenic C elegans were used in this research. These included a wild-type N2 strain, a strain expressing wild type copy of the human TDP-43 gene and a strain expressing a mutant TDP-43 gene respectively. The mutation is located at a single amino acid residue, Ala-315-Thr, in the TDP-43 gene. All strains expressed a fluorescent G-CaMP construct in a subset of the motor neurons, allowing in vivo optical measurement of intracellular calcium levels. Calcium levels between the worms of different strains were compared at the L4 and 2 days old adult stages of their life cycle. In order to measure their calcium levels fluorescence microscopy was used. First the calcium values were measured at the L4 stage for the worms of each strain. Then the worms were synchronized to 2 days old adults and their calcium levels were measured. A calcium crawling assay was conducted for the mutant TDP-43 worms in order to see if buffering calcium with BAPTA and EGTA would alleviate their movement disorder. Results: Calcium level was significantly elevated for the mutant TDP-43 worms compared to the wild type TDP-43 and N2 worms when measurement were taken at 2 days old adult stage. While when the same measurements were done at L4 stage, no significance difference was found. Based on this results, calcium crawling assay was conducted in order to see if buffering calcium would help the worms move better and the results shows that it was. Conclusion: This research concludes that, the mutation in TDP-43 gene is causing an increased in calcium level within the neurons in which it is expressed. The elevated calcium levels are affecting the worm motor neurons causing neuronal degeneration and partial or full paralysis of the animals. When the calcium level was buffered degeneration appeared slowed within treated animals which moved faster and better. These results within our C. elegans model, suggest a possible mode of action of this same TDP-43 mutation within human occurrences of ALS.
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