Prolonged glutamate excitotoxicity increases GluR1 immunoreactivity but decreases mRNA of GluR1 and associated regulatory proteins indissociated rat retinae invitro.

Abstract

Glutamate excitotoxicity contributes to damage following injury to the central nervous system via mechanisms including changes in the expression of receptors, calcium overload, oxidative stress and cell death. Excitotoxicity is triggered by glutamate binding to receptors, including calcium permeable AMPA receptors, which can be upregulated under injury conditions. However, the transcriptional response of AMPA receptor regulatory proteins to excitotoxic conditions is unknown. Here, we use real-time quantitative PCR (RT-qPCR), to determine the effect of prolonged glutamate excitotoxicity on the expression of mRNA encoding for GluR1 and AMPA receptor regulatory proteins in dissociated rat retinal cultures that include neuronal (retinal ganglion cell (RGCs)) and glial (Müller) cell populations. mRNA levels of GluR1 and regulators of the GluR1 subunit of AMPA receptors, including Sap97, Cnih2 and Cnih3, decreased following 6, 24 and 48h incubation with 5mM glutamate: related regulators not associated with GluR1 were unaffected. In contrast, GluR1 protein, assessed immunohistochemically, was increased in both RGCs and Müller cells after 24h glutamate exposure: western blotting analysis was inconclusive. Reductions in mRNA of GluR1 and associated regulators occurred prior to cell death, which was first detected at 24h, and substantial by 48h. Exposure to glutamate acutely increased the intracellular calcium concentration in the cultures and by 24h, reactive oxygen species were increased. Our data suggest a negative feedback mechanism in retinal cells, that down-regulates transcription of genes encoding GluR1 regulatory proteins in response to glutamate exposure. Despite this mechanism, GluR1 protein levels remain increased, and are associated with increased reactive species and cell death. Therapeutic strategies targeting calcium permeable AMPA receptors should take into account that increases in GluR1 protein are not necessarily associated with increases in associated mRNA levels over time.