Ca2+ is an important second messenger which modulates a variety of signalling pathways in both excitable and non-excitable cells. In the CNS, Ca2+ plays an important role in neurons both physiologically and pathologically. Ca2+ influx following synaptic activity, is important in development, plasticity, redox balance, as well as in neuroprotection, largely through activation of pro-survival pathways downstream of synaptic NMDAR activation, including upregulation of antioxidant defences. However, excessive Ca2+ influx in neurons leads to neuronal damage and excitotoxicity, in which mitochondrial Ca2+ uptake through the mitochondrial Ca2+ uniporter (Mcu) resulting in mitochondrial dysfunction is a key player. Excitotoxicity occurs due to glutamate efflux from astrocytes following stroke, traumatic brain injury and in chronic neurodegenerative diseases, leading to excessive neuronal NMDAR activation and triggering of its downstream pro-death pathways. This thesis focuses on understanding the pro-survival and pro-death effects of signalling pathways activated by Ca2+ in neurons, as well as the potential effect of neuronal synaptic activity on influencing neuroprotective gene transcription in astrocytes. I investigated the role of AMPK, a master regulator of metabolism, in NMDA excitotoxicity in cortical neurons as a potential downstream effector of Mcu-dependent excitotoxic death; and found the deletion of AMPKα1/2 to be neuroprotective against NMDA-mediated excitotoxicity, however I found AMPK activation to be independent of Mcu. I also investigated the expression pattern of Mcu and other mitochondrial calcium regulatory genes (MCRGs), and found MCRGs to be differentially expressed in different neural cells (primary neurons vs astrocytes), and neuronal subtypes (CA1 vs CA3 region of the hippocampus), suggesting differing dependence on the various MCRGs in mitochondrial Ca2+ handling in these cell types. A better functional understanding of these genes will allow for investigation of their importance in mitochondrial Ca2+ handling, including their potential role in excitotoxicity. I next investigated the neuroprotective effects of synaptic activity induced Ca2+ influx, focusing on antioxidant target genes of Nrf2, a transcription factor and major regulator of antioxidant genes. I found that unlike astrocytes, neurons express very low levels of Nrf2. However, synaptic activity increased the expression of several Nrf2 target genes in neurons, independently of astrocytes and Nrf2. Additionally, I found no effect of synaptic activity on increasing Nrf2 protein levels, despite previous reports in literature of Nrf2 pathway activation following synaptic activity. Finally, using RNA-seq I identified a list of genes strongly upregulated by a known Nrf2 activator in astrocytes, and found no evidence that neuronal activity triggers expression of these genes independently of neurons, providing further evidence that neuronal activity does not activate the Nrf2 pathway in astrocytes. This suggests that synaptic activity via pathways activated by Ca2+ signalling provides neurons with cell-autonomous antioxidant defences, independently of Nrf2; thus providing a distinct pathway for antioxidant defences in neurons from the Nrf2 pathway, which is activated in astrocytes providing neurons with non-cell autonomous antioxidant support. These results give us further insight into the mechanisms that underlie synaptic and non-synaptic Ca2+ signalling pathways mediating neuronal survival and death, which could help in identifying therapeutic targets to combat excitotoxicity and oxidative stress in various neurological diseases.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13354号 / 論医博第2200号 / 新制||医||1044(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 長船 健二, 教授 木村 剛, 教授 羽賀 博典 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive form of cancer with poor prognosis and limited treatment options. Since many patients present with metastatic disease and are thus ineligible for surgical resection, PDAC is almost ubiquitously fatal; new treatment options are therefore needed to combat this disease. A key hallmark of many cancers, including PDAC, is metabolic reprogramming and a shift towards a high glycolytic rate, known as the Warburg effect. This allows cancer cells to generate ATP in the face of hypoxia and to meet the increased metabolic requirements associated with rapid proliferation. We hypothesised that this shift towards glycolytic metabolism has important implications for the regulation of cytosolic Ca2+ ([Ca2+]i) in PDAC, since the plasma membrane Ca2+ ATPase (PMCA), which is critical for maintaining low [Ca2+]i and thus cell survival, is dependent on ATP to extrude cytosolic Ca2+. The relative contributions of mitochondrial vs glycolytic ATP in fuelling the PMCA in human PDAC cell lines (PANC-1 and MIA PaCa-2) were therefore assessed. Moreover, the effects of numerous mechanistically distinct metabolic inhibitors on key readouts of cell death, [Ca2+]i and ATP were investigated. Treatment with glycolytic inhibitors induced significant ATP depletion, PMCA inhibition, [Ca2+]i overload and cell death in both PANC-1 and MIA PaCa-2 cells, while mitochondrial inhibitors had no effect. Subsequently, these experiments were repeated on PDAC cells cultured in media formulated to "switch" their highly glycolytic phenotype back to one more reliant on mitochondrial metabolism. Culture in nominal glucose-free media supplemented with either galactose (10 mM) or alpha-ketoisocaproate (KIC, 2 mM) resulted in a switch in metabolism in MIA PaCa-2 cells, where proliferation rate and glycolysis were significantly decreased, and in the case of cells cultured in KIC, oxidative phosphorylation rate was preserved (assessed using Seahorse XF technology). Following culture of MIA PaCa-2 cells in either galactose or KIC, glycolytic inhibition failed to recapitulate the profound ATP depletion, PMCA inhibition and [Ca2+]i overload observed in glucose-cultured MIA PaCa-2 cells. These data demonstrate that in PDAC cells exhibiting a high rate of glycolysis, glycolytically-derived ATP is important for fuelling [Ca2+]i homeostasis and thus is critical for survival. Finally, using a cell surface biotinylation assay, the keyglycolytic enzymes LDHA, PFKP, GAPDH, PFKFB3 and PKM2 were all found to associate with the plasma membrane in MIA PaCa-2 cells, possibly in a tyrosine phosphorylation-dependent manner. To investigate whether the dynamic membrane-association of glycolytic enzymes provides a privileged supply of ATP to the PMCA in PDAC, the effects of tyrosine kinase inhibitors was assessed on PMCA activity. However, while these inhibited PMCA activity, this occurred without accompanying global ATP depletion. These data indicate that glycolytic ATP is critical for the regulation of [Ca2+]i by the PMCA in PDAC, and that the glycolytic regulation of the PMCA may be an important therapeutic locus. However, further research is required to determine whether membrane-bound glycolytic enzymes regulate its activity.

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La production de radicaux libres oxygénés (ROS), la surcharge calcique cytosolique et l’ouverture des pores de transition membranaires mitochondriales (mPTP) consécutives à l’ischémie myocardique (IM) sont aggravées lors de la reperfusion. Dans cette thèse, nous nous sommes intéressés : 1) à l’évaluation des effets de la trimétazidine sur le seuil électrique de fibrillation ventriculaire (VFT) et sur les lésions structurales et fonctionnelles des mitochondries lors de l’IM (étude 1, N=26 porcs); 2) à la détermination de la cinétique d’évolution des lésions d’ischémie/reperfusion (I/R) (étude 2a, N=30 porcs) ;3) à l’étude de l’impact de la ranolazine, du propranolol et de leur association dans la préventions des lésions d’I/R (étude 2b, N=30 porcs). Ces études qui ont été réalisées sur le cœur de porcs anesthésiés, ont permis de suivre l’évolution des paramètres électrophysiologiques et hémodynamiques cardiaques et, à l’échelle cellulaire, l’évolution de la structure et de la fonction des mitochondries. Les résultats montrent : 1) dans l’étude 1 : que la TMZ prévient la chute du VFT et l’ensemble des altérations structurales et fonctionnelles mitochondriales observées lors de l’IM ; 2) dans l’étude 2a : que les lésions observées durant l’IM sont significativement aggravées dans les premières dizaines de secondes de la reperfusion alors qu’une certaine amélioration est observée après 10 et surtout 45 min de reperfusion; 3) dans l’étude 2b : qu’un prétraitement par de la ranolazine, du propranolol et par leur association réduit la sévérité de ces lésions d’I/R. Les mécanismes moléculaires et cellulaires d’action des produits utilisés dans cette étude seraient en rapport avec l’amélioration des lésions de l’I/R / The generation of reactive oxygen species (ROS), the cytosolic calcium overload and the opening of mitochondrial permeability transition pores (mPTP) resulting from myocardial ischemia (MI) are aggravated during reperfusion. In the present work, the following points have been addressed: 1) the evaluation of trimetazidine effects on the electrical threshold of ventricular fibrillation (VFT) and both structural and functional alterations of mitochondria during MI (study 1, N=26 pigs); 2) the determination of the kinetics of ischemia/reperfusion (I/R) lesions (study 2a, N=30 pigs); 3) the protective effects of ranolazine and propranolol, alone or combined on I/R lesions (study 2b, N=30 pigs). All studies were performed in anesthetized pigs. They allowed to follow changes in cardiac electrophysiological and hemodynamic parameters and, at the cellular level, changes in the structure and function of mitochondria. The obtained results show: 1) in study 1, that TMZ can prevent the drop in VFT and all structural and functional alterations of mitochondria noticed during MI; 2) in study 2a, that the lesions seen during MI are significantly aggravated within the first seconds of reperfusion whereas some improvement is observed after 10 minutes and more markedly after 45 minutes of reperfusion; 3) in study 2b, that pretreatment with ranolazine or propranolol, alone or combined can reduce the severity of I/R lesions. The molecular and cellular mechanisms of action of both agents are thought to be involved in this improvement of I/R lesions

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