Figure 1. CaMKII Binding to GluN2B Mediates Neuronal Cell Death after Resuscitation from Cardiac Arrest.

Error bars indicate SEM in all panels.

(A) Schematic representation of GluN2B, including the sequence of the overlapping binding region for DAPK1 and CaMKII.

(B) Experimental timeline for the CA/CPR model of global cerebral ischemia.

(C) Representative micrographs of the hippocampal CA1 region, with cell death visualized by H&E staining.

(D) Quantification of neuronal cell death in the CA1 region after CA/CPR. GluN2B^DCaMKII mutant mice showed significantly less cell death compared to WT (*p < 0.05, unpaired two-tailed t test).

(E–L) The GluN2B^ΔCaMKII mutation (L1298A/R1300Q) prevents binding of CaMKII, but not DAPK1.

(E) The GluN2B^ΔCaMKII mutation blocks the Ca²⁺/CaM-induced binding of CaMKII in vitro, as assessed by western analysis of GluN2B-bound CaMKII (***p < 0.001, unpaired two-tailed t test; WT, n = 5; mutant, n = 4).

(F) HEK cells co-expressing labeled CaMKII and GluN2Bc WT or mutant, imaged before and after 5 min Ca²⁺ stimuli induced with ionomycin.

(G) Quantification showed no significant difference in CaMKII/GluN2B colocalization (assessed by Pearson’s correlation) compared to mCherry/GluN2B control(not significant [NS], p > 0.05, one-way ANOVA; WT, n = 42, mutant, n = 38, mCh control, n = 36).

(H) Quantification after ionomycin treatment showed a dramatic increase (two-way ANOVA followed by Bonferroni test) of CaMKII colocalization with GluN2B WT(***p < 0.001, compared to 0 min), but not with the GluN2B^ΔCaMKII mutant (^#p < 0.05, compared to 0 min; ^###p < 0.001, compared to WT).

(I) The GluN2B^ΔCaMKII mutation allows normal DAPK1 binding (in absence of Ca²⁺/CaM) in vitro, as assessed by western analysis of GluN2B-bound DAPK1 (NS, p > 0.05, unpaired two-tailed t test; n = 4).

(J) HEK cells co-expressing labeled DAPK1 and GluN2Bc WT or mutant, imaged before and after 5 min Ca²⁺ stimuli induced with ionomycin.

(K) Quantification showed indistinguishable DAPK1 colocalization with GluN2B WT and GluN2B^DCaMKII mutant (NS, p > 0.05, Kruskal-Wallis followed by Bonferroni test; WT, n = 34, mutant, n = 41), compared to mCherry control (*p < 0.05; n = 36).

(L) Quantification after ionomycin treatment showed the same dispersal of DAPK1 from GluN2B, with no differences detected between WT and GluN2B^ΔCaMKII mutant.