A variety of diseases lead to the atrophy and/or death of skeletal muscle. To better understand the molecular mechanisms that mediate these processes, I have taken advantage of the intersegmental muscles (ISMs) of the tobacco hawkmoth Manduca sexta, which undergo sequential programs of atrophy and programmed cell death at the end of metamorphosis. ISM death is mediated by changes in gene expression and numerous cell-death associated transcripts have been identified. MicroRNAs (miRs) are small (~22 nucleotide) non-coding RNAs that bind to sequences in messenger RNAs (mRNAs) and either cause translational arrest or mRNA degradation. To test the hypothesis that developmentally regulated miRs may control the stability and/or translatability of target mRNAs in the ISMs, putative mRNA targets for the test miRs have been identified and their 3’ untranslated region (UTR) have been cloned into a dual luciferase reporter plasmid. The microRNA mir-92b binds to the 3’ UTR of the Small Cytoplasmic Leucine Rich repeat Protein (SCLP) mRNA. Expression of miR-92b declines during development and SCLP expression increases with the commitment to die. I found that the miR-92b inhibits luciferase mRNA translation (spectrophotometric plate assays), but does not lead to transcript degradation (quantitative polymerase chain reaction; qPCR). miR-92 plays a survival role in several mammalian tissues and is repressed in two types of cardiomyopathy. Consequently, understanding how miRs regulate mRNA translation and stability may provide a better understanding of the regulation of muscle atrophy and death as well as provide novel tools for diagnostics or therapeutics.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第21695号 / 医科博第99号 / 新制||医科||7(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 生田 宏一, 教授 Shohab YOUSSEFIAN, 教授 齊藤 博英 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Ferroptosis is a non-apoptotic, regulated form of cell death that is characterized by the iron-dependent lethal accumulation of lipid peroxides and lipid peroxide byproducts. Huntington’s disease (HD) is an autosomal neurodegenerative disease with characteristic motor, psychiatric, and cognitive signs and symptoms caused by the expansion of CAG repeats in the Huntingtin gene, resulting in the production of pathogenic protein with an extended polyglutamine tract that is prone to aggregation. Accumulating evidence has identified links between ferroptosis and HD suggesting that ferroptosis inhibition may provide therapeutic benefit. However, the ability to evaluate this potential has been limited by the unavailability of potent, brain-penetrant specific ferroptosis inhibitors. This dissertation evaluates two different types of ferroptosis inhibitors and increases the available molecular tools to investigate the role of ferroptosis in the etiology of HD. In the second and third chapters, two new classes of ferrostatin analogs, termed fourth and fifth generation ferrostatins, are developed and their in vitro and in vivo properties characterized. These efforts identify three, fifth generation analogs that are potent, brain-penetrant, and stable and can be administered chronically to symptomatic HD mice. The fourth chapter provides molecular insights into the mechanism of action of the hypocholesterolemic drug probucol in inhibiting ferroptosis and identifies cellular cholesterol levels and cholesterol import as regulators of ferroptosis. In sum, this work provides new molecular tools and insights that can be utilized to elucidate the contribution of ferroptosis to HD and other disease states.

<p>Atherosclerosis is a multi-factorial disease and is the major cause of death in the western world. Numerous risk factors, including hyperlipidemia, obesity, diabetes, smoking, hypertension, and family history increase the risk of atherosclerosis and death from cardiovascular disease (CVD). Clinical and epidemiological studies have now shown that hyperhomocysteinemia (HHcy) is an independent risk factor for CVD. Further, we and others have demonstrated that HHcy accelerates atherosclerosis in apolipoprotein Edeficient ( apoff1-) mice. Although several studies have reported that homocysteineinduced endoplasmic reticulum (ER) stress causes growth arrest and programmed cell death (PCD) in cultured vascular endothelial cells, the cellular factors responsible for this effect and their relevance to atherosclerosis have not been completely elucidated.</p><p>Previously, we have demonstrated that homocysteine induces the expression of Tcell death associated gene 51 (TDAG51), a member of the pleckstrin homology-related domain family, in cultured human vascular endothelial cells. Transient overexpression of TDAG51 elicited significant changes in cell morphology, decreased cell adhesion and promoted detachment-mediated PCD. In support of these in vitro findings, TDAG51 expression was increased and correlated with PCD in the atherosclerotic lesions from apoff1-mice fed hyperhomocysteinemic diets, compared to mice fed control diet. To investigate the in vivo significance of TDAG51 on atherosclerotic lesion development and progression, knockout mice deficient in both TDAG51 and apoE genes were generated. Our findings show that TDAG51-1-/apoff1-double knockout (DKO) mice fed control chow diet have significantly reduced atherosclerotic lesion size, compared to ageand sex-matched apoff1-control mice. Atheroprotective function of TDAG51 deficiency may be explained in part by the observation that there is a significant upregulation of peroxisome proliferator-activated receptor y (PPAR-y) in TDAG51-deficient (TDAG51 _1_) cells including mouse embryonic fibroblasts (MEFs), compared to control wildtype MEFs. Given that PPAR-y has both atheroprotective and anti-inflammatory properties, TDAG51 may represent a unique negative regulator of PPAR-y and its downstream gene targets. Taken together, my findings demonstrate that TDAG51 is a novel cellular mediator involved in the development and progression of atherosclerosis.</p><p>In addition to its anti-atherogenic properties, I have demonstrated that TDAG5 l _1_ MEFs have increased migratory properties following monolayer disruption or in response to chemotaxis on fibronectin-coated Boyden chambers, compared to wildtype control MEFs. Although TDAG51-induced cell migration could potentially affect atherosclerotic lesion development, our recent observations suggest that TDAG51 may also have a role in wound healing. Our studies have shown that dorsal skin wounds within TDAG5 l _ 1_ mice healed slowly, compared to those in control mice through a mechanism involving impaired myofibroblast differentiation. Since the underlying mechanisms of wound healing and fibrosis are similar, it is conceivable that TDAG51 may have role in fibrosis.</p><p>In summary, this thesis provides novel evidence that TDAG5 l is involved in the pathogenesis of atherosclerosis and wound healing. Furthermore, TDAG51 may represent a novel therapeutic target for attenuating atherosclerotic lesion development, thereby reducing the risk of cardiovascular disease and its complications.</p> / Thesis / Doctor of Philosophy (PhD)

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Background and Purpose –The mammalian target of rapamycin (mTOR) pathway has been implicated in cellular responses to hypoxia and inflammation. Cerebral palsy (CP) is a neurodevelopmental disorder often linked to hypoxic and inflammatory injury to the brain, however, a role for mTOR modulation in CP has not been investigated. We hypothesized that mTOR inhibition would prevent neuronal death and diminish inflammation in a mouse model of CP. Methods – Post-natal day 6 mouse pups were subjected to hypoxia-ischemia and lipopolysaccharide-induced inflammation (HIL), a model of CP causing injury to several brain areas. Mice received rapamycin (5mg/kg) following HIL, and then daily for 3 subsequent days. The phospho-activation of the mTOR effector mTOR effector proteins S6, S6K and 4EBP as well as upstream negative regulators, TSC1 and Redd1, were assessed as an in vivo measure of the mTOR signaling cascade. Expression of hypoxia inducible factor 1 (HIF-1 alpha) was assayed as an indicator of hypoxia-mediated cellular injury. Neuronal cell death was defined with Fluoro-Jade C (FJC) and cleaved-caspase 3 (CC3), a marker of apoptosis. Autophagy was measured using Beclin-1 and LC3II expression. Lastly, neuroninflammation following HIL was evaluated by examining Iba-1 labeled microglia number and morphology, as well as P-STAT3 expression. Results – Neuronal death, HIF-1alpha expression, and numerous Iba-1 labeled microglia were evident at 24 and 48 hours following HIL. Basal mTOR signaling was unchanged by HIL. Coincident with persistent mTOR signaling, a decreased in Redd1 expression but not TSC1 was observed in HIL. Increased P-STAT3 expression was observed at 24 and 48 hours post-HIL. Rapamycin treatment following HIL significantly reduced neuronal death, decreased HIF-1 alpha and P-STAT3 expression, and microglial activation, coincident with enhanced expression of Beclin-1 and LC3II, markers of autophagy induction. Increase in neuronal death was observed with concomitant administration of rapamycin and chloroquine, an autophagy inhibitor. Administration of a S6K inhibitor, PF-4708671, following HIL also decreased FJC staining further supporting an mTOR-dependent effect of HIL. Conclusions – mTOR inhibition prevented neuronal cell death and diminished neuroinflammation in this model of CP. Persistent mTOR signaling following HIL suggests a failure of autophagy induction, which may contribute to neuronal death in CP. These results suggest that mTOR signaling may be a novel therapeutic target to reduce neuronal cell death in CP. / Biomedical Neuroscience

Influenza A viruses (IAV) are negative sense RNA viruses that naturally infect a wide variety of animals. Different subtypes of the virus infect waterfowl, poultry, pigs, horses, ferrets, bats, dogs, cats and humans, causing zoonotic outbreaks and pandemics. In humans, IAV strains cause seasonal epidemics that can result in up to 50000 deaths and 700000 hospitalizations each year, and ranks in the top ten causes of death in the United States. In addition, virulent strains of IAV have caused pandemic outbreaks triggering numerous fatalities. While tropism to the upper respiratory tract is important for virus transmission, infection of the lower lung is most correlated with pathogenesis. The mammalian lung has multiple structural cell types, of which two classes are considered most important for IAV pathogenesis. These are (1) alveolar unit cells and (2) cells of the conducting airways, primarily those of the bronchi and bronchioles. Alveolar unit cells, or pneumocytes, chiefly comprise type I and type II alveolar epithelial cells, and are involved in gas exchange and surfactant production. Cells of the bronchi and bronchioles (e.g., basal, secretory, ciliated, club, goblet and neuroendocrine cells) perform numerous functions related to tissue repair/renewal, and mucous production. Upon IAV infection the regulated (or programmed) death of the infected cell represents an important pathogen clearance mechanism. Programmed cell death can be largely non-inflammatory (e.g., apoptosis) or pro-inflammatory (e.g., necroptosis). In this dissertation, I outline experiments carried out to identify the role of pro-inflammatory programmed cell death in influenza A virus clearance and pathogenesis both in vitro and in vivo. My work outlines the role of necroptosis in IAV clearance, and how this controlled form of cell death, particularly in alveolar unit cells, can be exploited as a potential new therapeutic avenue for severe influenza disease. / Cancer Biology & Genetics