京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19608号 / 医博第4115号 / 新制||医||1015(附属図書館) / 32644 / 京都大学大学院医学研究科医学専攻 / (主査)教授 大森 孝一, 教授 鈴木 茂彦, 教授 影山 龍一郎 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Spinal Muscular Atrophy (SMA) is a debilitating neurodegenerative disease resulting in death of the lower motor neurons, muscle atrophy, and in severe cases death. Due to mutations or deletions in the Survival Motor Neuron 1 (SMN1) gene, levels of functional SMN protein product are decreased. While SMA was previously described as a motor neuron exclusive disorder, recent evidence suggests that many tissue and cell types throughout the body are affected. The objective of our study was to outline the effects of varying levels of SMN depletion on two genes of interest, namely Insulin-like growth factor 1 (Igf-1) and Tumor suppressor protein 53 (Trp53) in multiple tissues throughout disease course. The severe Smn2B/- and mild Smn2B/-; SMN2+/- mouse models of SMA were utilized in our studies to determine the levels of mRNA expression and subsequent protein output for these two genes. We employed RT-qPCR, western blot, and ELISA experimental methods. In Smn2B/- mice, Igf-1 mRNA was substantially decreased in symptomatic liver tissue. This was accompanied by widespread decrease in IGF-1 protein in peripheral tissues. Interestingly, this depletion effect on Igf-1 was not observed in the mild mouse model. Our analysis also showed that Trp53 mRNA was dramatically increased within tibialis anterior skeletal muscle of symptomatic Smn2B/- mice, alongside an upregulation of factors involved in p53 mediated apoptosis. Once again, this effect was not observed in the mild Smn2B/-; SMN2+/- mouse model. Overall, we have demonstrated that the extent of SMN depletion, determines whether the expression of Igf-1 and Trp53 is perturbed, suggesting that disease severity is an important factor in what pathways are affected. Finally, we show that alterations in gene expression patterns or subsequent protein levels act in a tissue-specific fashion. More investigation is encouraged to highlight IGF-1’s role as a potential SMN-independent therapeutic for SMA.

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Skeletal muscle is the most abundant muscle in the body and performs important bodily functions such as movement, body temperature regulation, joint stabilization, etc. It requires calcium ions (Ca2+) for proper function, growth, development, and repair. Regulated Ca2+ signaling is crucial for muscle health and perturbation of Ca2+ signaling can, therefore, lead to a multitude of muscle pathophysiologies, one of which is atrophy. Skeletal muscle atrophy results from loss of proteins leading to loss of muscle mass and, unfortunately, there are no effective pharmacological therapies presently available for it. The only truly effective treatment includes exercise and proper diet. Physical activity is not a feasible option for ailing and aging populations who have the most occurrence of skeletal muscle atrophy. Thus, it is imperative to explore various molecular mechanisms which can initiate and contribute to skeletal muscle atrophy in order to elucidate possible pharmacological interventions. That is where this dissertation comes into place. The potassium (K+) channel, ERG1A, induces atrophy in the skeletal muscle of mice when ectopically expressed. Indeed, the human homolog of this atrophy-inducing K+ channel increases basal cytosolic Ca2+ concentration in cultured mouse skeletal muscle cells, C2C12. Here, we sought to discover the sources of the increased cytosolic Ca2+. To that end, we explored the effect of HERG1A overexpression on multiple important calcium signaling pathways. We discovered that HERG1A affects various Ca2+ signaling mechanisms in skeletal muscle cells. We show that HERG1A enhances the very abundant extracellular Ca2+ entry mechanism of SOCE (store operated calcium entry) to increase cytosolic Ca2+ levels. We also show that HERG1A also increases RyR1 (ryanodine receptor 1) signaling along with ECCE (excitation coupled calcium entry) in skeletal muscle cells. Most interestingly, we connected all these HERG1A-modulated pathways by showing that HERG1A modulates the global Ca2+ homeostasis regulator, Calsequestrin1 (CSQ1) both at the level of transcription and translation. This suggests that HERG1A is a global Ca2+ homeostasis regulator itself. We speculate that HERG1A is affecting the various Ca2+ signaling mechanisms in cells via its regulation of the levels of CSQ1 and thereby modulating the availability of cytosolic Ca2+ in the cells. This dissertation thus summarizes the vastness of HERG1A regulation of Ca2+ homeostasis in skeletal muscle cells. Furthermore, this dissertation also shows how HERG1A affects various mechanisms in skeletal muscle cells which could also have roles in apoptosis and cancer. Most importantly, HERG1A in skeletal muscle cells has been shown to have a real and significant impact on the functioning of multiple cellular signaling pathways and not the levels of the members of such pathways which reveals for the first time a mechanistic control of the intracellular pathways in skeletal muscle cells by this voltage gated K+ channel.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 112-138).

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 5 uppsatser.

Diss. (sammanfattning) Linköping : Univ., 2004. / Härtill 4 uppsatser.

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