Investigating the molecular basis of motor neuron vulnerability in mouse models of spinal muscular atrophy
Courtney, Natalie Louise
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Spinal Muscular Atrophy (SMA) is a motor neuron disease that predominantly affects children, with the most severe form of SMA resulting in death before the age of 2 years. It is an autosomal recessive disorder that leads to progressive paralysis and muscle atrophy due to the degeneration of lower motor neurons. Furthermore, a loss of the connections between motor neurons and muscle (neuromuscular junctions; NMJs) occurs early in the disease. Importantly, not all motor neurons are equally affected. For example, in specific mouse models of SMA, there is a high level of NMJ loss in the abdominal muscles while NMJs within cranial muscles remain intact, even at late stages of disease. Prior to this study, RNAseq analysis of differentially vulnerable motor neurons in the Smn2B/- mouse model of SMA, and in wild-type mice, was carried out at a pre-symptomatic stage. In this project, there are two main aims. Firstly, transcriptional changes that occur between differentially vulnerable motor neurons were further investigated. qRT-PCR was used to detect transcriptional changes that occur between Smn2B/- and wild type mice and immunofluorescence was used to detect protein markers of transcripts that were altered in vulnerable motor neurons. qRT-PCR confirmed changes in Ubiquitin as well as transcripts associated with cell death. Immunofluorescence analysis showed no significant change in protein markers of DNA repair between differentially vulnerable motor neuron populations. Secondly, the activation of a cell death pathway in a mouse model of SMA was investigated. For this aim, the Smn2B/- mouse line was re-derived and preliminary phenotypic assessment was preformed. The re-derived model displays similar features to the original Smn2B/- mouse model of SMA. In addition, the incidence and timing of cell death pathway activation was investigated by qRT-PCR. This showed an up-regulation of transcripts involved in cell death pre-symptomatically. Up-regulation of these transcripts at this time point suggests that the activation of cell death pathways may contribute to motor neuron degeneration and therefore warrants further investigation.