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

Abstract

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. Our results show that mitochondrial glutamate transport via GC1 is important in sustaining glutamate homeostasis in astrocytes.Main Points: 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

Bioinformatics Analysis Discovers Microtubular Tubulin Beta 6 Class V (TUBB6) as a Potential Therapeutic Target in Glioblastoma

“…Mitochondrial metabolic inhibitors have been reported to activate a mitochondrion-to-nucleus stress signaling network that leads to alterations in gene expression, which affects a wide variety of cellular processes. Mitochondria are key organelles for cellular bioenergetics and constantly undergo dynamic remodeling processes, and increased production of reactive oxygen products is associated with a variety of human disorders (Goubert et al, 2017). Moreover, there is a lack of research on mitochondrial metabolism in GBM.…”

Diseases Caused by Mutations in Mitochondrial Carrier Genes SLC25: A Review

“…The ammonium enters the urea cycle for clearance of excess nitrogen, whereas 2-oxoglutarate is used in gluconeogenesis (in liver and kidney) or oxidized in TCA cycle for ATP production (in non-hepatic tissues). SLC25A22 is highly expressed in most tissues including the brain, especially in areas associated with motor coordination and in astrocytes, that control the uptake of extracellular neurotransmitter glutamate [147,148]. It is likely that extracellular glutamate levels in brain are dysregulated in SLC25A22 deficiency patients [143] because it was shown that (i) reduced expression of SLC25A22 in glial cells leads to intracellular glutamate accumulation [148], and (ii) aberrant glutamate catabolism in astrocytes is associated with altered clearance of extracellular (synaptic) glutamate and early epileptic encephalopathy [149].…”

“…SLC25A22 is highly expressed in most tissues including the brain, especially in areas associated with motor coordination and in astrocytes, that control the uptake of extracellular neurotransmitter glutamate [147,148]. It is likely that extracellular glutamate levels in brain are dysregulated in SLC25A22 deficiency patients [143] because it was shown that (i) reduced expression of SLC25A22 in glial cells leads to intracellular glutamate accumulation [148], and (ii) aberrant glutamate catabolism in astrocytes is associated with altered clearance of extracellular (synaptic) glutamate and early epileptic encephalopathy [149]. Moreover, some patients with SLC25A22 deficiency manifest hyperprolinemia [150], which might be explained by assuming that dysfunctional GC1 activity diminishes or abolishes the export of proline-derived glutamate from mitochondria leading to accumulation of proline in the body.…”

Amino Acid Transport Defects in Human Inherited Metabolic Disorders

“…It catalyzes the glutamate/H+ symporter in order to transport glutamate into the mitochondria [184]. The dysregulation of extracellular glutamate and the activation of the extrasynaptic glutamate receptors would result from an absence of a functional SLC25A22 protein [185]. To date, 16 patients with EIEE3 have been reported [182][183][184]186,187].…”