Abstract

Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disease primarily affecting motor neurons. There is no cure, with death typically occurring within three years of symptom onset. Dozens of genes have been implicated in ALS, including Fused in Sarcoma (FUS), a little-studied RNA/DNA binding protein. FUS mutations have been documented in both late-onset and juvenile-onset ALS, with mutations found in the C-terminus being associated with aggressive juvenile-onset. Specifically, the 1554_1557del mutation in the C-terminus of FUS has been observed in cases of unusually early onset of ALS, which is characterized by an extremely aggressive progression. The 1554_1557del mutation was introduced into a bacterial plasmid via site-directed mutagenesis and transformed into E. coli. Bacterial colonies were cultured and lysed whereupon the mutated plasmids were extracted, purified, and submitted for sequencing. Confirmed mutations were subcloned into the Promega Regulated Mammalian Expression System. However, sanger sequencing was inconclusive. Additionally, HEK-293 cells were cultured, and method development for transfection with the Promega System was begun. Future research should focus on analyzing changes in cellular structure, function, and viability due to the introduction of the 1554_57 mutation to the cell line. The collection of cytoplasmic and nuclear RNA and protein fractions from the cells will allow for analysis of changes in RNA and protein expression on a compartmental level. Once analyzed, the data will allow for greater understanding of how the cellular functions of transcription and translation are affected by the 1554_1557del mutation, possibly providing insight into the mechanism of neuronal death and loss of function. Furthermore, this understanding could potentially provide greater insight into how other C-terminus mutations in FUS can drive neurodegeneration in ALS.

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