Expression of SOD1 G93A or wild-type SOD1 in primary cultures of astrocytes down-regulates the glutamate transporter GLT-1: lack of involvement of oxidative stress.
Glutamate excitotoxicity is implicated in the aetiology of amyotrophic lateral sclerosis (ALS) with impairment of glutamate transport into astrocytes a possible cause of glutamate-induced injury to motor neurons. It is possible that mutations of Cu/Zn superoxide dismutase (SOD1), responsible for about 20% of familial ALS, down-regulates glutamate transporters via oxidative stress. We transfected primary mouse astrocytes to investigate the effect of the FALS-linked mutant hSOD1(G93A) and wild-type SOD1 (hSOD1wt) on the glutamate uptake system. Using western blotting, immunocytochemistry and RT-PCR it was shown that expression of either hSOD1(G93A) or hSOD1wt in astrocytes produced down-regulation of the levels of a glutamate transporter GLT-1, without alterations in its mRNA level. hSOD1(G93A) or hSOD1wt expression caused a decrease of the monomeric form of GLT-1 without increasing oxidative multimers of GLT-1. The effects were selective to GLT-1, since another glutamate transporter GLAST protein and mRNA levels were not altered. Reflecting the decrease in GLT-1 protein, [3H]d-aspartate uptake was reduced in cultures expressing hSOD1(G93A) or hSOD1wt. The hSOD1-induced decline in GLT-1 protein and [3H]d-aspartate uptake was not blocked by the antioxidant Trolox nor potentiated by antioxidant depletion using catalase and glutathione peroxidase inhibitors. Measurement of 2',7'-dichlorofluorescein (DCF)-induced fluorescence revealed that expression of hSOD1(G93A) or hSOD1wt in astrocytes does not lead to detectable increase of intracellular reactive oxygen species. This study suggests that levels of GLT-1 protein in astrocytes are reduced rapidly by overexpression of hSOD1, and is due to a property shared between the wild-type and G93A mutant form, but does not involve the production of intracellular oxidative stress.
Tortarolo
Massimo
M
Biochemical Neuropharmacology Group, GKT Centre for Neuroscience Research, King's College London, Guy's Hospital Campus, London, UK.
Crossthwaite
Andrew J
AJ
Conforti
Laura
L
Spencer
Jeremy P
JP
Williams
Robert J
RJ
Bendotti
Caterina
C
Rattray
Marcus
M
D000690:Amyotrophic Lateral Sclerosis / Q000201:enzymology* / Q000235:genetics*; D000818:Animals; D001253:Astrocytes / Q000201:enzymology*; D002478:Cells, Cultured; D015536:Down-Regulation / Q000502:physiology; D027342:Excitatory Amino Acid Transporter 2 / Q000096:biosynthesis* / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D006801:Humans; D051379:Mice; D009154:Mutation; D018384:Oxidative Stress / Q000502:physiology*; D013482:Superoxide Dismutase / Q000096:biosynthesis* / Q000235:genetics; D000072105:Superoxide Dismutase-1
D027342:Excitatory Amino Acid Transporter 2; C000606290:SOD1 protein, human; C109415:SOD1 G93A protein; C000606291:Sod1 protein, mouse; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1