Extrasynaptic NMDA Receptors Couple Preferentially to Excitotoxicity via Calpain-Mediated Cleavage of STEP

Abstract

NMDA receptor (NMDAR)-mediated excitotoxicity plays an important role in several CNS disorders, including epilepsy, stroke, and ischemia. Here we demonstrate the involvement of striatal-enriched protein tyrosine phosphatase (STEP) in this critical process. STEP 61 is an alternatively spliced member of the family that is present in postsynaptic terminals. In an apparent paradox, STEP 61 regulates extracellular signal-regulated kinase 1/2 (ERK1/2) and p38, two proteins with opposing functions; activated p38 promotes cell death, whereas activated ERK1/2 promotes cell survival. We found that synaptic stimulation of NMDARs promoted STEP 61 ubiquitination and degradation, concomitant with ERK1/2 activation. In contrast, extrasynaptic stimulation of NMDARs invoked calpain-mediated proteolysis of STEP 61 , producing the truncated cleavage product STEP 33 and activation of p38. The calpain cleavage site on STEP was mapped to the kinase interacting motif, a domain required for substrate binding. As a result, STEP 33 neither interacts with nor dephosphorylates STEP substrates. A synthetic peptide spanning the calpain cleavage site efficiently reduced STEP 61 degradation and attenuated p38 activation and cell death in slice models. Furthermore, this peptide was neuroprotective when neurons were subjected to excitotoxicity or cortical slices were exposed to ischemic conditions. These findings suggest a novel mechanism by which differential NMDAR stimulation regulates STEP 61 to promote either ERK1/2 or p38 activation and identifies calpain cleavage of STEP 61 as a valid target for the development of neuroprotective therapy.

Synaptic NMDA receptor stimulation activates PP1 by inhibiting its phosphorylation by Cdk5

“…In a control experiment, subsequent bath NMDA application led, as expected, to CREB dephosphorylation ( Fig. S1 f), as reported previously (Hardingham et al, 2002;Xu et al, 2009), thus confirming the activation of extrasynaptic NMDA receptors (eNMDAR Stim). Notably, extrasynaptic NMDA receptor stimulation alone did not lead to PP1 dephosphorylation at T320 (Fig.…”

“…Stimulation of synaptic and extrasynaptic NMDA receptors Synaptic and extrasynaptic NMDAR stimulations were performed according to published protocols (Hardingham et al, 2001(Hardingham et al, , 2002Ivanov et al, 2006;Chung et al, 2009;Hoey et al, 2009;Xu et al, 2009). Three protocols were used for synaptic NMDAR stimulation.…”

Role of the ubiquitin–proteasome system in brain ischemia: Friend or foe?

“…Not all NMDAR contribute to neuronal cell death in excitotoxicity, since the synaptic and extrasynaptic receptor populations are differentially coupled to the activation of intracellular signaling mechanisms: the latter receptors mediate the influx of calcium ions leading to mitochondrial injury (Stanika et al, 2009), activation of pro-apoptotic genes (Leveille et al, 2010), cleavage of fodrin and the sodium-calcium exchanger type-3 (NCX3) by calpains (Xu et al, 2009a), and cell death (Hardingham et al, 2002). On the other hand, activation of synaptic NMDAR is not coupled to hippocampal neuron damage (Hardingham et al, 2002), but rather promotes resistance to oxidative insults and prevents accumulation of reactive oxygen species in cortical neurons (Papadia et al, 2008) (Fig.…”

Augmented generation of protein fragments during wakefulness as the molecular cause of sleep: a hypothesis

“…Calpain-generated Leu-STEP 33 fragment lacks phosphatase activity. 162 #22. Leu-b-catenin is the Ct-fragment of b-catenin, a conditionally short-lived cytoskeletal protein and transcriptional regulator.…”