Études des mécanismes d’adaptation du métabolisme énergétique dans le syndrome de Leigh de type canadien français : vers l’identification des cibles thérapeutiques

Leigh syndrome French-Canadian type (LSFC) is an infantile mitochondrial disease caused by mutations in the LRPPRC gene resulting in a tissue-specific decrease in cytochrome c oxidase (COX) and fatal lactic acidosis crises which occur during stressful situations. The pathophysiology underlying this disease and the factors that precipitate these crises remain unknown. Thus, the main objective of this study was to explore adaptive mechanisms of energy metabolism. Our group demonstrated that COX deficiency in patients’ LSFC fibroblasts was associated with a reduction in oxidative phosphorylation (OXPHOS) capacity in the absence of any changes in ATP levels. On the other hand, we have demonstrated that LSFC cells showed premature cell death when treated with 10 mM lactate and 1 mM palmitate (LP), a model of nutrient overload. We hypothesized that LSFC cells may display metabolic reprogramming under the control of the mamalian target of rapamycin complex 1 (mTORC1), allowing for extra mitochondrial ATP synthesis. We also thought that alteration of the AMP activated protein kinase (AMPK) activity, an energy sensor, may contribute to the greater susceptibility of the LSFC cells to LP.  Our results show that the Akt/mTORC1/p70S6K pathway is upregulated in LSFC cells relative to controls. This is associated with an increase in the expression of hypoxia inducible factor (HIF-1α) and its target pyruvate dehydrogenase kinase (PDHK1) resulting in an increase in the phosphorylated and inhibitory form of PDH1, the gatekeeper of mitochondrial glucose oxidation. Consistent with this, glucose contribution to the formation of pyruvate, alanine and lactate is increased in LSFC cells compared to controls. Unexpectedly, inhibition of mTORC1 by rapamycin has no effect on the expression of HIF-1α and PDHK1, nor on ATP levels in LSFC cells. However, it increases PDH1 phosphorylation and reduces the expression of LRPPRC and COX in LSFC cells. This is the first time that LRPPRC regulation by mTORC1 is reported.  We have also studied the AMPK activation in response to various stresses. AMPK is activated in response to 2,4-dinitrophenol similarly both in control and LSFC cells. Treatment with LP, a nutritional stress, does not decrease ATP levels; but does induce phosphorylation of AMPK, and overexpression of SIRT1, COXIV and LRPPRC proteins in the control cells. Pretreatment with ZMP, an AMPK-specific activator, further increases the effects of LP. All these changes in response to LP are abolished in LSFC cells. Interestingly, treatment with 2-Bromopalmitate, a non-metabolizable analog of palmitate, does not induce the phosphorylation of AMPK in the control cells. These data suggest that palmitate metabolism is required for AMPK activation and this is prevented in LSFC cells.  In conclusion, LSFC cells exhibit a Warburg-like metabolic reprogramming. mTORC1 doesn’t play a key role in maintaining ATP levels but its activation is required for LRPPRC and COX expression. Moreover, adaptive mechanisms in response to nutrient stress via AMPK, appear altered in the LSFC cells.

Keyphrases: LSFC, LRPPRC, COX, mTOR, Rapamycin, AMPK, SIRT1, Palmitate