Pyrithiamine-induced
thiamine deficiency (TD) is a well-established model of
Wernicke's encephalopathy in which a
glutamate-mediated excitotoxic mechanism may play an important role in determining selective vulnerability. In order to examine this possibility, cultured astrocytes were exposed to TD and effects on
glutamate transport and metabolic function were studied. TD led to decreases in cellular levels of
thiamine and
thiamine diphosphate (TDP) after 24 h of treatment and decreased activities of the TDP-dependent
enzymes alpha-ketoglutarate dehydrogenase and
transketolase after 4 and 7 days, respectively. TD treatment for 10 days led to a reversible decrease in the uptake of [(3)H]-
D-aspartate, a nonmetabolizable analogue of
glutamate. Kinetic analysis revealed that the uptake inhibition was caused by a 47% decrease in the V(max) for uptake of [(3)H]-
D-aspartate, with no change in the K(m) value. Immunoblotting showed that this decrease in uptake was due to an 81% downregulation of the astrocyte-specific GLAST
glutamate transporter. Loss of uptake activity and GLAST
protein were blocked by treatment with the
protein kinase C inhibitor H7, while exposure to
DCG IV, a group II
metabotropic glutamate receptor (mGluR) agonist, resulted in improvement of [(3)H]-
D-aspartate uptake and a partial reversal of transporter downregulation. These results are consistent with our recent in vivo findings of a loss of astrocytic
glutamate transporters in TD and provide evidence that TD conditions may increase phosphorylation of GLAST, contributing to its downregulation. In addition, manipulation of group II mGluR activity may provide an important strategy in the treatment of this disorder.