Prolonged Positive Modulation of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors Induces Calpain-Mediated PSD-95/Dlg/ZO-1 Protein Degradation and AMPA Receptor Down-Regulation in Cultured Hippocampal Slices

Abstract

Prolonged exposure of cultured hippocampal slices to CX614 [2H,3H,6aH-pyrrolidino[2Љ,1Љ-3Ј,2Ј]1,3-oxazino[6Ј,5Ј-5,4]-benzo[e]1,4-dioxan 10-one], a positive ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (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 glutamate receptor (GluR)1 and GluR2/3 AMPAr subunits and of their anchoring proteins synapse-associated protein 97 (SAP97) and glutamate receptor interacting protein 1 (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 after 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 N-methyl-D-aspartate receptor 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.AMPA-type glutamate receptors (AMPAr) mediate most of the fast excitatory neurotransmission in the mammalian central nervous system. Several recent studies have implicated the cycling of these receptors into and out of the synaptic compartment in important neurophysiological phenomena such as long-term potentiation (LTP) and long-term depression (LTD) in support of our initial hypothesis (Baudry and

Calpains and neuronal damage in the ischemic brain: The swiss knife in synaptic injury

“…Chronic, but not excitotoxic, treatment of cultured hippocampal slices with a positive AMPAR modulator (CX614) reduced the expression of the PDZ proteins GRIP1 and SAP97 by a calpain-dependent mechanism, and induced similar alterations in spectrin protein levels, contributing to a downregulation of AMPAR subunit expression (Jourdi et al, 2005). Although there is no direct evidence of SAP97 cleavage under excitotoxic/ischemic conditions, it is reasonable to hypothesize that the scaffold protein may also be a calpain substrate under toxic conditions.…”

Calpain-1 and Calpain-2: The Yin and Yang of Synaptic Plasticity and Neurodegeneration

“…Calpain was also shown to cleave β-catenin, generating an active fragment, which regulates gene transcription, thereby providing a mechanism by which NMDA receptor stimulation, which has been repeatedly linked to calpain activation [28, 29], could modify gene expression [30]. Several scaffolding proteins were found to be calpain substrates, including PSD95 [31], GRIP [32], SAP97 [33] and ARMS (ankyrin repeat-rich membrane spanning protein) or Kidins220 (kinase D-interacting substrate of 220 kDa) [34]. By degrading the translational repressor poly(A)-binding protein (PABP)-interacting protein 2A (PAIP2A), an inhibitor of PABP, calpain could also relieve translational inhibition of proteins involved in synaptic plasticity and learning and memory [35].…”

Cleavage of the Vesicular GABA Transporter under Excitotoxic Conditions Is Followed by Accumulation of the Truncated Transporter in Nonsynaptic Sites

“…Under normal physiological conditions the activation of calpains is tightly regulated and plays a key role in the cleavage of selected protein targets (Liu et al, 2008). Activation of these proteases due to a [Ca 2ϩ ] i overload under excitotoxic conditions cleaves key synaptic proteins in glutamatergic synapses, including ionotropic (AMPA and NMDA receptor subunits) and metabotropic glutamate receptors (Xu et al, 2007;Yuen et al, 2007;Gascó n et al, 2008), and postsynaptic scaffold proteins (Jourdi et al, 2005), but little information is available regarding the effect of calpains on GABAergic synapses. In the present study we evaluated the changes in VGAT protein levels and cellular distribution in pathological conditions, including excitotoxicity, brain ischemia, and following status epilepticus.…”