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京都大学 / 新制・課程博士 / 博士(農学) / 甲第23247号 / 農博第2454号 / 新制||農||1084(附属図書館) / 学位論文||R3||N5337(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 三芳 秀人, 教授 宮川 恒, 教授 森 直樹 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

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Mitochondria shape cytosolic Ca2+ (cCa2+) transients. Ca2+ entry into the mitochondria is driven by the highly negative mitochondrial membrane potential and through a highly selective channel, the Mitochondrial Calcium Uniporter (MCU). Mitochondrial Ca2+ (mCa2+) is utilized by the matrix dehydrogenases for maintaining cellular bioenergetics. The TCA cycle-derived NADH and FADH2 are mCa2+ dependent thus, feed into the electron transport chain (ETC) to generate ATP. Either loss of mCa2+ or metabolite uptake by the mitochondria results in a bioenergetic crisis and mitochondrial dysfunction. Reciprocally, sudden elevation of cCa2+ under conditions of stroke or ischemia/reperfusion injury (I/R) drives excessive mCa2+ overload that in turn leads to the opening of a large channel, the mitochondrial permeability transition pore (PTP) that triggers necrotic cell death. Thus, Ca2+ and metabolite equilibrium is essential to maintain a healthy mitochondrial pool. Our laboratory has previously showed that loss of mCa2+ uptake leads to decreased ATP generation and cell survival through autophagy. Although metabolite scarcity also results in similar reduction in ATP generation, the molecular mechanisms by which metabolites control mitochondrial ion homeostasis remain elusive. Deprivation of glucose or supplementation of mitochondrial pyruvate carrier (MPC) transport blocker UK5099 and or carnitine-dependent fatty acid blocker etomoxir triggered an increase in the expression of MICU1, a regulator of the mitochondrial calcium uniporter (MCU) but not the MCU core subunit. Consistently, either RNAi-mediated deletion of MPC isoforms or dominant negative human mutant MPC1 R97W showed significant induction of MICU1 protein abundance and inhibition of MCU-mediated mCa2+ uptake. Moreover, TCA cycle substrate-dependent MICU1 expression is under the control of EGR1 transcriptional regulation. Reciprocally, the MICU1 dependent inhibition of mCa2+ uptake exhibited lower NADH production and oxygen consumption and ATP production. The reduction of mitochondrial pyruvate by MPC knockdown is linked to higher production of mitochondrial ROS and elevated autophagy markers. These studies reveal an unexpected regulation of MCU-mediated mCa2+ flux machinery involving major TCA cycle substrate availability and possibly MICU1 to control cellular switch between glycolysis and oxidative phosphorylation. While mCa2+ is required for energy generation, sustained elevation of mCa2+ results in mitochondrial swelling and necrotic death. Hence, it was thought that preventing mCa2+ overload can be protective under conditions of elevated cCa2+. Contrary to this, mice knocked-out for MCU, that demonstrated no mCa2+ uptake and hence no mitochondrial swelling, however failed protect cells from I/R- mediated cell death. MCU-/- animals showed a similar infarct size comparable to that of control animals, suggesting that prevention of MCU-mediated mCa2+ overload alone is not sufficient to protect cells from Ca2+ -induced necrosis. The absence of mCa2+ entry revealed an elevation in the upstream cCa2+ transients in hepatocytes from MCUDHEP. Ultra-structural analysis of liver sections from MCU-/- (MCUDHEP) and MCUfl/fl animals revealed stark contrast in the shape of mitochondria: MCUfl/fl liver sections showed long and filamentous mitochondria (spaghetti-like) while MCUDHEP mitochondria were short and circular (donut-like). Furthermore, challenging MCUfl/fl and MCUDHEP hepatocytes with ionomycin caused a marked increase in cCa2+ and a simultaneous change in mitochondrial shape (from spaghetti to donut), a phenomenon we termed mitochondrial shape transition (MiST) that was independent of mitochondrial swelling. The cCa2+-mediated MiST is induced by an evolutionarily conserved mitochondrial surface EF-hand domain containing Miro1. Glutamate and Ca2+ -stress driven cCa2+ mobilization cause MiST in neurons that is suppressed by expression of Miro1 EF1 mutants. Miro1-dependent MiST is essential for autophagosome formation that is attenuated in cells harboring Miro1 EF1 mutants. Remarkably, loss of cCa2+ sensitization by Miro1 prevented MiST and mitigated autophagy. These results demonstrate that an interplay of ions and metabolites function in concert to regulate mitochondrial shape that in turn dictates the diverse mitochondrial processes from ATP generation to determining mechanisms of cell death. / Biomedical Sciences

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