京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第25025号 / 生博第516号 / 新制||生||69(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 鈴木 淳, 教授 中野 雄司, 教授 野田 岳志 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第22473号 / 工博第4734号 / 新制||工||1739(附属図書館) / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 梅田 眞郷, 教授 森 泰生, 教授 秋吉 一成 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18181号 / 医博第3901号 / 新制||医||1004(附属図書館) / 31039 / 京都大学大学院医学研究科医学専攻 / (主査)教授 岩田 想, 教授 松田 道行, 教授 楠見 明弘 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Doctor of Philosophy / Division of Biology / Sherry D. Fleming / Ischemia/reperfusion (IR) events result in severe tissue damage and often death. The complex network of molecular and cellular mechanisms that contributes to intestinal IR-induced pathology has hindered a comprehensive understanding of IR-induced injury and limited the success of medical intervention. Although several of the mechanisms contributing to intestinal IR-induced injury have been identified, the initiating event(s) remains unclear. Mouse models have been instrumental in the unraveling of the many components and interactions that ultimately result in tissue damage. It is clear that leukocyte infiltration, complement activation, eicosanoid and pro-inflammatory cytokine production are involved. Toll-like receptors and antibodies also play critical roles. Based on the literature, and especially data demonstrating a significant role for anti-phospholipid antibodies, we hypothesized that ischemia induces phospholipid alterations that result in the exposure of a neoantigen which is recognized by anti-phospholipid antibodies. Furthermore, we hypothesized that endothelial cells are the primary cell type involved in the initial molecular events that result in intestinal IR-induced pathology. A mouse model of intestinal IR as well as an in vitro cell culture system was used to explore these hypotheses. Mass spectrometry-based lipidomics was utilized to assess lipid responses to IR and hypoxia/re-oxygenation (HR). No inherent differences in intestinal phospholipid composition were found between wildtype and several strains of knock-out mice. It was determined that the lack of antibody production by Rag-1[superscript]-[superscript]/[superscript]- mice is responsible for protection against intestinal IR-induced injury, as antibody is needed to induce prostaglandin E[subscript]2 production, through up-regulation of cyclooxygenase 2 transcription. Unexpectedly, the presence or absence of toll-like receptor 9 was found to be inconsequential for tissue damage caused by intestinal IR. The results of several analyses point to endothelial cells as being directly involved in IR-induced pathology. Importantly, the activation of phospholipid scramblase 1 has been identified as a potential molecular mechanism by which subsequent molecular and cellular responses are elicited as a consequence of IR.

Phospholipid scramblase 4 (PLSCR4) is a member of a conserved enzyme family with high relevance for the remodeling of phospholipid distribution in the plasma membrane and the regulation of cellular signaling. While PLSCR1 and -3 are involved in the regulation of adipose-tissue expansion, the role of PLSCR4 is so far unknown. PLSCR4 is significantly downregulated in an adipose-progenitor-cell model of deficiency for phosphatase and tensin homolog (PTEN). PTEN acts as a tumor suppressor and antagonist of the growth and survival signaling phosphoinositide 3-kinase (PI3K)/AKT cascade by dephosphorylating phosphatidylinositol-3,4,5-trisphosphate (PIP3). Patients with PTEN germline deletion frequently develop lipomas. The underlying mechanism for this aberrant adipose-tissue growth is incompletely understood. PLSCR4 is most highly expressed in human adipose tissue, compared with other phospholipid scramblases, suggesting a specific role of PLSCR4 in adipose-tissue biology. In cell and mouse models of lipid accumulation, we found PLSCR4 to be downregulated. We observed increased adipogenesis in PLSCR4-knockdown adipose progenitor cells, while PLSCR4 overexpression attenuated lipid accumulation. PLSCR4 knockdown was associated with increased PIP3 levels and the activation of AKT. Our results indicated that PLSCR4 is a regulator of PI3K/AKT signaling and adipogenesis and may play a role in PTENassociated adipose-tissue overgrowth and lipoma formation.

Spinal Muscular Atrophy with Progressive Myoclonic Epilepsy (SMA-PME), Farber Lipogranulomatosis (FL), and a rare variant form of Neonatal Progeroid Syndrome (NPS) are three monogenetic rare disorders caused by pathogenic variation in genes encoding lipid modifying proteins. FL and SMA-PME are caused by loss of function mutations in ASAH1, encoding the acid ceramidase (aCDase) enzyme. It is not, however, known how aCDase deficiency can produce either the isolated neurological symptoms of SMA-PME or the predominantly systemic symptoms of FL. Further, a recently identified variant form of NPS has been attributed to variants in ANO6, encoding a dual function calcium-activated chloride channel and glycerophosphoserine (GPS) scramblase. Here, it is not known how ANO6 mutation causes the premature aging phenotype that defines NPS. To address these questions, I sought to elucidate pathogenic changes in lipid metabolism that associate clinical phenotype. I show here that the different patient mutations in ANO6 cause a non-physiological gain of channel function and either a loss or gain of scramblase function depending on the variant expressed. Both variants, however, alter GPS metabolic homeostasis suggesting a common mechanism of action. To provide in vivo insight, I characterized a novel mouse model based on our NPS patient genetics, showing extremely low penetrance of disease symptoms in terms of live births yet confirming that affected animals show impaired GPS metabolism in affected organs. Next, I characterized the clinical presentation of six new patients with SMA-PME and identified distinct sphingolipid metabolic fingerprints in FL and SMA-PME cells. I show that FL is defined by a hypometabolic sphingolipid phenotype with cellular and molecular features of a classic lysosomal storage disorder. By contrast, SMA-PME has a hypermetabolic sphingolipid phenotype with features of non-classic lysosomal trafficking disorders. To provide clinical insight, I assessed the potential of enzyme replacement therapy, demonstrating a rescue of sphingolipid metabolism in SMA-PME patient cells. Together, this thesis identified changes in the cellular and tissue lipid profiles of patients with ANO6-NPS, SMA-PME, or FL, elucidating some of the lipid-centric pathomechanisms of these diseases.