New roles of aralar, the brain mitochondrial aspartate-glutamate carrier, in dopamine handling, glutamate excitotoxicity and regulation of mitochondrial respiration

Résumé

Aralar/AGC1, the neuronal aspartate-glutamate mitochondrial carrier, is the regulatory component of the malate-aspartate NADH shuttle (MAS), and it is regulated by extramitochondrial Ca2+ in the nM range. Aralar deficiency in mice and human causes a shutdown of brain shuttle activity and global cerebral hypomyelination. The lack of Aralar also induces motor and behavioral impairments in mice, similar to those described in human AGC1 deficiency, suggestive of alterations in monoaminergic systems. We have found a severe affectation in dopaminergic projection-enriched areas, particularly in striatum. Specific reduction of size, significant decrease in dopamine content and drop in vesicular monoamine transporter-2 (VMAT2) levels together with an increased metabolism of dopamine through MAO activity (increased DOPAC/DA ratio) were observed in Aralar KO striatum. Aralar-hemizygous adult mice presented also increased DOPAC/DA ratio in this region and enhanced sensitivity to amphetamine. Therefore, our results suggest that the failure to produce mitochondrial NADH due to the lack of Aralar would increase reactive oxygen species levels which in turn might cause a fall in GSH/GSSG ratio and VMAT2, particularly in striatal dopaminergic terminals which are more vulnerable to oxidative stress. Glutamate excitotoxicity is a form of neuronal death in which calcium overload in both mitochondria and cytosol and oxidative stress play prominent role. Surprisingly, we find that the lack of Aralar does not alter neuronal viability to glutamate-mediated death, both in the absence or presence of glucose. However, during glutamate exposure stimulation of mitochondrial respiration is lower, the initial Ca2+ peak is higher, delayed Ca2+ deregulation (DCD) occurs earlier the fall in GSH is larger in Aralar KO neurons. Moreover, glutamate-induced increase in 8-OHdG in mitochondrial DNA is the same regardless the genotype. This suggests that Aralar deficiency, by reducing the capacity to take up calcium in mitochondria, may decrease mitochondrial ROS production. In contrast to the effects in vitro, adult hemizygous Aralar mice are more vulnerable to kainic acid-induced seizures and neuronal damage, perhaps because in vivo factors, other than glutamate excititicicity, contribute to the final outcome. One of these factors may be lactate. We find that the protection by lactate during in vitro excitotoxicity in the presence of glucose requires Aralar. It is well-known that calcium and ATP demand control neuronal respiration but the role played by each one is unknown, as any Ca2+ signal also impacts on ATP demand. We have studied the Ca2+-regulation of mitochondrial respiration, which involves Ca2+ entry in mitochondria through the Ca2+ uniporter (MCU) but also the activation of the mitochondrial metabolite transporters by extramitochondrial Ca2+, in intact cortical neurons. We have analyzed the neuronal response in basal conditions and in response to augmented workload caused by increases in [Na+]cyt and/or [Ca2+]cyt. Respiration in non-stimulated neurons in physiological glucose concentrations depends on Aralar/MAS. All stimulation conditions induced increased OCR in the presence of Ca2+, which was prevented by BAPTA-AM loading (to preserve the workload) or in Ca2+-free medium (which also lowers cell workload). The results suggest the requirement of Aralar/MAS in priming pyruvate entry in mitochondria, a step needed to activate respiration by Ca2+ in response to moderate workloads.