Résumé :
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Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease targeting motoneurons and leading to muscle atrophy and death. The most common familial form and best characterized mouse model of ALS are linked to mutations in the superoxide dismutase 1 (SOD1). We previously identified a new motoneuron-restricted cell death pathway triggered by activation of the cell surface death receptor Fas and involving upregulation of nNOS (neuronal nitric oxide synthase) leading to nitric oxide (NO) production. Interestingly, purified embryonic motoneurons from mutated SOD1(mSOD1) mice showed increased susceptibility to activation of Fas and its intracellular relay NO.In order to identify potential effectors in this death pathway, a proteomic screen on motoneuron cultures was performed. A diminution of Calreticulin (CRT) was found only in motoneurons isolated from mSOD1 embryos treated with NO or Fas ligand. CRT is the main protein controlling calcium storage in the endoplasmic reticulum (ER) of neurons. Diminution of CRT in mSOD1 motoneurons could lead to a modification of calcium homeostasis and accumulation of unfolded protein, two events that could trigger ER stress activation and contribute to motoneuron death. Using immunofluorescence techniques, we confirmed a 50% diminution in CRT expression specifically in Fas/NO treated mSOD1 motoneurons in vitro. Importantly, we observed the same diminution in CRT in vivo, in mSOD1 mice spinal cord motoneurons from an asymptomatic stage to the end stage of the disease. We showed that a moderate ER stress induction in wildtype motoneurons had the same effect as the presence of mSOD1 in increasing the sensitivity of motoneurons to Fas-triggered death. Moreover, ER stress inhibitors blocked death induced by FasL/NO in mSOD1 motoneurons. Finally, a decrease of CRT by RNA interference induced death of wildtype motoneurons, while on the contrary, its overexpression significantly prevented the sFasL/NO-induced death of mSOD1 motoneurons. These results indicate that the level of CRT expression can modulate motoneuron survival and suggest that CRT could be a good therapeutic target in ALS. We are now using genetically-modified mice to modulate negatively or positively CRT expression in vivo to determine the neuroprotective potential of CRT in mSOD1 mice.
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