Etude du dysfonctionnement mitochondrial dans le maintien de la biogenèse mitochondriale et la réponse à l'apoptose induite

Abstract

Mitochondria are involved in numerous cell processes, such as ATP production, calcium homeostasis, fatty acid metabolism, heme synthesis, urea cycle, redox cell status, autophagy and apoptosis. Impairment of its bioenergetic activity is thus obviously associated with numerous pathologies. However, while various origins and symptoms have been described for mitochondrial diseases over the past 10 years, only very few retrograde signalling pathways (that could be defined as communication between impaired mitochondria and nucleus) have been identified. In addition, little is still known about the molecular mechanisms leading to differential gene expression in response to chronic or acute mitochondrial dysfunction. In that research field, the generation of cells totally (r0) or partially (r-) depleted in mtDNA has been very useful to study the response of cells to a chronic energetic stress. The major aim of this work was to get a better understanding of the molecular mechanisms involved in the retrograde communication between impaired mitochondria and the nucleus that participate to the maintenance of 1) the mitochondrial membrane potential (Dym), 2) the mitochondrial biogenesis and 3) the apoptotic response to staurosporine, an alkaloïd that inhibits numerous kinases. In the first part of this work, we highlighted the role of the protein mtCLIC/CLIC4 in the maintenance of the Dym in mtDNA-depleted cells. Using a "mRNA RT-PCR differential display" approach, we first identified that the gene was over-expressed in mtDNA-depleted cells. We also show that modifications of its abundance (over expresion and silencing by siRNA) were able to modify the Dym. Finally, we evidenced that mtCLIC allows the importation of chlorine into mitochondria of r-L929 (murine fibrosarcoma cells). In the second part of this work, we characterized and compared mitochondrial populations between 143B (osteosarcoma cell line) and 143B r0 cells. We monitored the activity status of several key transcription factors known to be involved in the control of mitochondrial biogenesis and we determined the expression level of several mitochondrial proteins used as common markers of mitochondrial biogenesis. We also clearly demonstrated the role for calcium-CaMKIV-CREB pathway in the maintenance of mitochondrial biogenesis in mtDNA-depleted cells. Indeed, we show that the over-expression of cytochrome c and the higher mitochondrial NAO (Nonyl Acridine Orange) staining (two indicators for a higher abundance of mitochondrial mass) observed in mtDNA-depleted cells could be reduced in r0 cells that over-express either a dominant negative forms of CREB or CaMKIV. Moreover, we show that the importation of matrix-targeted proteins is reduced in mtDNAdepleted cells, a feature that can be explained by the lower Dym and reduced ATP content in these cells. As several evidence were reported to link mitochondrial dysfunction and apoptosis in vivo, the last part of this work has been dedicated to the characterization of the apoptotic response of mtDNAdepleted cells to staurosporine. Indeed, the higher or lower sensitivity of mtDNA-depleted cells to apoptotic stimuli is still a debated question in the literature. We first show that r0 143B cells are hypersensitive to staurosporine-induced apoptosis, a phenomenon that could most likely be explained by the constitutive down-regulation of anti-apoptotic proteins such as Bcl-2 an Bcl-XL in r0 cells. Moreover, we show that the mechanisms of r0 cells response to staurosporine seems to be different from those triggered in parental cells. Indeed, we show that cathepsin B might play a role in staurosporine-induced mtDNA-depleted cell apoptosis, despite the activation of many caspases. Finally, we show that autopahgy is also triggered by staurosporine in r0 143B cells, an upstream event of caspase activation as 3-methyladenine (3-MA) strongly reduces caspase activation. In conclusion, our results bring new information in the understanding of mechanisms and cell signalling activated in mammalian cells facing a chronic energetic stress, and thus bring new insights into the cellular consequences of mitochondrial impairment, a feature found in numerous mitochondrial diseases and pathologies associated with aging.

Date of Award	17 Mar 2008
Original language	French
Awarding Institution	University of Namur
Supervisor	Thierry ARNOULD (Supervisor), Patricia Renard (Co-Supervisor), MICHEL JADOT (Jury), Xavier De Bolle (Jury), Bernard KNOOPS (Jury) & Rudolf WIESNER (Jury)