Abstract
Aberrant NMDA receptor (NMDAR) activity contributes to several neurological disorders, but direct antagonism is poorly tolerated therapeutically. The GluN2B cytoplasmic C-terminal domain (CTD) represents an alternative therapeutic target since it potentiates excitotoxic signaling. The key GluN2B CTD-centred event in excitotoxicity is proposed to involve its phosphorylation at Ser-1303 by Dapk1, that is blocked by a neuroprotective cell-permeable peptide mimetic of the region. Contrary to this model, we find that excitotoxicity can proceed without increased Ser-1303 phosphorylation, and is unaffected by Dapk1 deficiency in vitro or following ischemia in vivo. Pharmacological analysis of the aforementioned neuroprotective peptide revealed that it acts in a sequence-independent manner as an open-channel NMDAR antagonist at or near the Mg2+ site, due to its high net positive charge. Thus, GluN2B-driven excitotoxic signaling can proceed independently of Dapk1 or altered Ser-1303 phosphorylation.
Highlights
NMDA receptor (NMDAR) -mediated excitotoxicity plays a key role in acute neurological disorders such as stroke and traumatic brain injury, neuronal loss in Huntington’s disease, and is implicated in synapto-toxicity in Alzheimer’s disease (Choi, 1988; Lipton and Rosenberg, 1994; Berliocchi et al, 2005; Lau and Tymianski, 2010; Hardingham and Lipton, 2011; Parsons and Raymond, 2014; Tu et al, 2014)
GluN2 subunits have long, evolutionarily divergent cytoplasmic C-terminal domains (CTDs) which we have shown can differentially associate with signalling molecules (Martel et al, 2012; Ryan et al, 2008, 2013; Frank et al, 2016) and differentially signal to cell death: the CTD of GluN2B (CTD2B) potentiates excitotoxicity more strongly than that of GluN2A (Martel et al, 2012)
At the late timepoint (60 min, 50 mM NMDA) we observed a decline in Ser-1303 phosphorylation (Figure 1a,b, Figure 1—figure supplement 1e,f, Figure 1—source data 3) as well as a decline in total levels of GluN2B, consistent with observations of others who have reported partial calpain-mediated cleavage and degradation of the NMDAR CTD (Dong et al, 2006; Gascon et al, 2008)
Summary
NMDA receptor (NMDAR) -mediated excitotoxicity plays a key role in acute neurological disorders such as stroke and traumatic brain injury, neuronal loss in Huntington’s disease, and is implicated in synapto-toxicity in Alzheimer’s disease (Choi, 1988; Lipton and Rosenberg, 1994; Berliocchi et al, 2005; Lau and Tymianski, 2010; Hardingham and Lipton, 2011; Parsons and Raymond, 2014; Tu et al, 2014). The mechanism is centred on Ser-1303 of CTD2B, within a region of the CTD unique to GluN2B, and with which CaMKIIa is known to interact and phosphorylate (Bayer et al, 2001; Mao et al, 2014). It was reported that in response to ischemia or excitotoxic insults, a different kinase, Dapk, causes Ser-1303 phosphorylation which increases NMDARdependent ionic flux (Tu et al, 2010). Consistent with this, Dapk1-/- neurons were reported to be resistant to excitotoxicity, and a cell-permeable peptide mimetic of the CTD2B region around Ser1303 disrupted Ser-1303 phosphorylation and was neuroprotective (Tu et al, 2010)
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