Received for publication January 19, 2005.
Revised March 17, 2005.
Accepted for publication March 18, 2005.

Prolonged positive modulation of AMPA receptors induces calpain-mediated PDZ protein degradation and AMPA receptor down-regulation in cultured hippocampal slices

Abstract

Prolonged exposure of cultured hippocampal slices to CX614, a positive AMPA receptor (AMPAr) modulator, decreases receptor response to synaptic stimulation, an effect that could reflect reduced receptor expression. The present study investigates this down-regulation and its underlying mechanisms using cultured rat hippocampal slices. Chronic treatment with CX614 gradually reduced levels of GluR1 and GluR2/3 AMPAr subunits and of their anchoring proteins, SAP97 and GRIP1 through 48 h. Decline in SAP97 and GRIP1 levels was associated with increased abundance of lower molecular weight bands, suggesting degradation of these proteins. CX614 effects were partially reversible following drug removal. GluR1 and GluR2/3 down-regulation and their slow recovery were associated with similar changes in SAP97 and GRIP1 levels. Treatment with CX614 for 48 h significantly reduced AMPAr mRNA levels in hippocampus whereas 8 h exposure did not. Blockade of ionotropic glutamate receptors prevented CX614-induced decrease in AMPAr subunits and mRNA, with regional selectivity, although an AMPAr blocker was more efficacious than an NMDAr blocker. Blockade of calpain activity reduced CX614-induced degradation of SAP97 and GRIP1 and prevented decreases in AMPAr subunit but not mRNA levels. Treatment with CX614 alone or in combination with glutamate receptor blockers or calpain inhibitor III did not modify lactate dehydrogenase release into culture medium, implying the absence of cell toxicity. We conclude that CX614-induced AMPAr protein loss is primarily mediated by AMPAr activation, and involves calpain-dependent proteolysis of SAP97 and GRIP1. CX614-induced suppression of AMPAr gene expression is, however, calpain-independent and all these effects are not associated with cell damage.

Key words: AMPA receptor, Ampakine, Calpain, GRIP1, Hippocampus, SAP97