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

Emmanuelle Goubert,Luigi Palmieri,Julie Sutera,Yanina Mircheva,Christophe Melon,Ferdinando Palmieri,Francesco M Lasorsa,Florence Molinari,Emanuela Profilo,Hélène Becq,Laurent Aniksztejn

doi:10.3389/fncel.2017.00149

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

Highlights

The mitochondrial solute carrier (SLC) family 25 is composed of 53 members that transport a large diversity of metabolites, nucleotides and cofactors across the inner mitochondrial membrane (IMM; Palmieri, 2004, 2013; Palmieri and Monné, 2016)
Glutamate homeostasis is crucial for the proper functioning of the central nervous system and its extracellular concentration is maintained low thanks to the astroglial glutamate transporters GLT-1 and GLAST (EAAT 1 and 2; Takahashi et al, 1997; Danbolt, 2001; Robinson and Jackson, 2016)
The balance between the extent of oxidative consumption of glutamate and synthesis of glutamine by glutamine synthetase (GS) is dependent on extracellular glutamate concentration, with relatively more glutamate being oxidized at higher glutamate concentrations

Summary

Introduction

The mitochondrial solute carrier (SLC) family 25 is composed of 53 members that transport a large diversity of metabolites, nucleotides and cofactors across the inner mitochondrial membrane (IMM; Palmieri, 2004, 2013; Palmieri and Monné, 2016). These transporters are essential for mitochondria where several metabolic pathways occur including the Krebs cycle and the β-oxidation of fatty acids. The Slc25a13-null mice showed a reduced mitochondrial aspartate transport in the liver but no apparent in vivo phenotype (Sinasac et al, 2004). A complete loss of activity of the mitochondrial glutamate carrier 1 (GC1, SLC25A22) has been associated with early epileptic encephalopathy (EEE; Molinari et al, 2005, 2009; Cohen et al, 2014) and migrating partial seizures in infancy (MPSI; Poduri et al, 2013), but, to the best of our knowledge, no animal model has been developed for this human pathology

Methods

Results

Conclusion