Goubert Emmanuelle, Mircheva Yanina, Lasorsa Francesco M, Melon Christophe, Profilo Emanuela, Sutera Julie, Becq Hélène, Palmieri Ferdinando, Palmieri Luigi, Aniksztejn Laurent, Molinari Florence
INMED, INSERM, Aix-Marseille UniversitéMarseille, France.
Centre De Recherche De L'Institut Universitaire En Santé Mentale de QuébecQuebec City, QC, Canada.
Front Cell Neurosci. 2017 May 31;11:149. doi: 10.3389/fncel.2017.00149. eCollection 2017.
The solute carrier family 25 (SLC25) drives the import of a large diversity of metabolites into mitochondria, a key cellular structure involved in many metabolic functions. Mutations of the mitochondrial glutamate carrier (also named ) have been identified in early epileptic encephalopathy (EEE) and migrating partial seizures in infancy (MPSI) but the pathophysiological mechanism of GC1 deficiency is still unknown, hampered by the absence of an model. This carrier is mainly expressed in astrocytes and is the principal gate for glutamate entry into mitochondria. A sufficient supply of energy is essential for the proper function of the brain and mitochondria have a pivotal role in maintaining energy homeostasis. In this work, we wanted to study the consequences of GC1 absence in an model in order to understand if glutamate catabolism and/or mitochondrial function could be affected. First, short hairpin RNA (shRNA) designed to specifically silence were validated in rat C6 glioma cells. Silencing in C6 resulted in a reduction of the mRNA combined with a decrease of the mitochondrial glutamate carrier activity. Then, primary astrocyte cultures were prepared and transfected with shRNA-GC1 or mismatch-RNA (mmRNA) constructs using the Neon® Transfection System in order to target a high number of primary astrocytes, more than 64%. Silencing in primary astrocytes resulted in a reduced nicotinamide adenine dinucleotide (Phosphate) (NAD(P)H) formation upon glutamate stimulation. We also observed that the mitochondrial respiratory chain (MRC) was functional after glucose stimulation but not activated by glutamate, resulting in a lower level of cellular adenosine triphosphate (ATP) in silenced astrocytes compared to control cells. Moreover, GC1 inactivation resulted in an intracellular glutamate accumulation. Our results show that mitochondrial glutamate transport via GC1 is important in sustaining glutamate homeostasis in astrocytes. The mitochondrial respiratory chain is functional in absence of GC1Lack of glutamate oxidation results in a lower global ATP levelLack of mitochondrial glutamate transport results in intracellular glutamate accumulation.
溶质载体家族25（SLC25）驱动多种代谢物进入线粒体，线粒体是参与许多代谢功能的关键细胞结构。线粒体谷氨酸载体（也称为）的突变已在早期癫痫性脑病（EEE）和婴儿期迁移性部分性癫痫发作（MPSI）中被鉴定出来，但由于缺乏该模型，GC1缺乏的病理生理机制仍不清楚。这种载体主要在星形胶质细胞中表达，是谷氨酸进入线粒体的主要通道。充足的能量供应对大脑的正常功能至关重要，而线粒体在维持能量稳态中起关键作用。在这项工作中，我们想在该模型中研究GC1缺失的后果，以了解谷氨酸分解代谢和/或线粒体功能是否会受到影响。首先，在大鼠C6胶质瘤细胞中验证了设计用于特异性沉默的短发夹RNA（shRNA）。在C6中沉默导致mRNA减少，同时线粒体谷氨酸载体活性降低。然后，制备原代星形胶质细胞培养物，并使用Neon®转染系统用shRNA-GC1或错配RNA（mmRNA）构建体进行转染，以靶向大量原代星形胶质细胞，超过64%。在原代星形胶质细胞中沉默导致谷氨酸刺激后烟酰胺腺嘌呤二核苷酸（磷酸）（NAD(P)H）形成减少。我们还观察到，葡萄糖刺激后线粒体呼吸链（MRC）功能正常，但不受谷氨酸激活，导致沉默的星形胶质细胞中细胞三磷酸腺苷（ATP）水平低于对照细胞。此外，GC1失活导致细胞内谷氨酸积累。我们的结果表明，通过GC1的线粒体谷氨酸转运在维持星形胶质细胞中的谷氨酸稳态中很重要。线粒体呼吸链在缺乏GC1时功能正常缺乏谷氨酸氧化导致整体ATP水平降低缺乏线粒体谷氨酸转运导致细胞内谷氨酸积累。
