Inhibition of the Mitochondrial Glutamate Carrier SLC25A22 in Astrocytes Leads to Intracellular Glutamate Accumulation
Résumé
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
Domaines
Neurosciences [q-bio.NC]| Origine | Fichiers éditeurs autorisés sur une archive ouverte |
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