Inhibition of the Mitochondrial Glutamate Carrier SLC25A22 in Astrocytes Leads to Intracellular Glutamate Accumulation

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 SLC25A22 (also named GC1) 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 in vivo 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 in vitro model in order to understand if glutamate catabolism and/or mitochondrial function could be affected. First, short hairpin RNA (shRNA) designed to specifically silence GC1 were validated in rat C6 glioma cells. Silencing GC1 in C6 resulted in a reduction of the GC1 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 GC1 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.

Mots clés

Abbreviations: AAT aspartate amino transferase AGC aspartate/glutamate carriers ALAT alanine amino transferase ATP aenosine triphosphate ∆ψm mitochondrial membrane potential DL-TBOA DL-threo-ß-Benzyloxyaspartic acid EAAT excitatory amino acid transporter EEE early epileptic encephalopathy FCCP Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone GABA gamma-aminobutyric acid GC1 glutamate carrier 1 GDH glutamate dehydrogenase GFAP glial fibrillary acidic protein Glc glucose Gln glutamine Glu glutamate GS glutamine synthetase GSH glutathione HPLC high-performance liquid chromatography IMM inner mitochondrial membrane L-MSO L-Methionine sulfoximine mmRNA mismatch-RNA MPSI migrating partial seizures in infancy MRC mitochondrial respiratory chain NAD(P)+/NAD(P)H nicotinamide adenine dinucleotide (Phosphate) R123 Rhodamine123 shRNA short hairpin RNA SLC25 solute carrier family 25 STC synaptic transporter current

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Neurosciences [q-bio.NC]

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Front. Cell. Neurosci. 2017 Goubert.pdf (3.25 Mo)

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https://hal.science/hal-01961870

Soumis le : jeudi 20 décembre 2018-11:31:05

Dernière modification le : mercredi 30 octobre 2024-21:45:16

Archivage à long terme le : vendredi 22 mars 2019-09:22:40

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hal-01961870 , version 1 (20-12-2018)

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HAL Id : hal-01961870 , version 1
DOI : 10.3389/fncel.2017.00149

Citer

Emmanuelle Goubert, Yanina Mircheva, Francesco M Lasorsa, Christophe Melon, Emanuela Profilo, et al.. Inhibition of the Mitochondrial Glutamate Carrier SLC25A22 in Astrocytes Leads to Intracellular Glutamate Accumulation. Frontiers in Cellular Neuroscience, 2017, 11, pp.149. ⟨10.3389/fncel.2017.00149⟩. ⟨hal-01961870⟩

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