Focal Cerebral Ischemia and Reperfusion Induce Brain Injury Through α2δ-1-Bound NMDA Receptors

Abstract

Background and Purpose- Glutamate NMDARs (N-methyl-D-aspartate receptors) play a major role in the initiation of ischemic brain damage. However, NMDAR antagonists have no protective effects in stroke patients, possibly because they impair physiological functions of NMDARs. α2δ-1 (encoded by Cacna2d1) is strongly expressed in many brain regions. We determined the contribution of α2δ-1 to NMDAR hyperactivity and brain injury induced by ischemia and reperfusion. Methods- Mice were subjected to 90 minutes of middle cerebral artery occlusion followed by 24 hours of reperfusion. Neurological deficits, brain infarct volumes, and calpain/caspase-3 activity in brain tissues were measured. NMDAR activity of hippocampal CA1 neurons was measured in an in vitro ischemic model. Results- Middle cerebral artery occlusion increased α2δ-1 protein glycosylation in the cerebral cortex, hippocampus, and striatum. Coimmunoprecipitation showed that ischemia rapidly enhanced the α2δ-1-NMDAR physical interaction in the mouse brain tissue. Inhibiting α2δ-1 with gabapentin, uncoupling the α2δ-1-NMDAR interaction with an α2δ-1 C terminus-interfering peptide, or genetically ablating Cacna2d1 had no effect on basal NMDAR currents but strikingly abolished oxygen-glucose deprivation-induced NMDAR hyperactivity in hippocampal CA1 neurons. Systemic treatment with gabapentin or α2δ-1 C-terminus-interfering peptide or Cacna2d1 genetic knock-out reduced middle cerebral artery occlusion-induced infarct volumes, neurological deficit scores, and calpain/caspase-3 activation in brain tissues. Conclusions- α2δ-1 is essential for brain ischemia-induced neuronal NMDAR hyperactivity, and α2δ-1-bound NMDARs mediate brain damage caused by cerebral ischemia. Targeting α2δ-1-bound NMDARs, without impairing physiological α2δ-1-free NMDARs, may be a promising strategy for treating ischemic stroke.

Keywords: 3; NMDA receptor; brain; caspase; gabapentin; hippocampus; ischemia; neurons; pregabalin.

Figure 1.. α2δ−1 and its interaction with NMDARs are essential for OGD-induced potentiation of NMDAR activity in hippocampal CA1 neurons.

(A and B) Representative recording traces (A) and quantification (B) of puff NMDA-elicited NMDAR currents show the effects of gabapentin (GBP, 30 µM for 60 min, n = 10 neurons; 100 µM for 60 min, n = 12 neurons), vehicle (n = 11 neurons), α2δ−1Tat peptide (1 µM for 60 min, n = 10 neurons), control peptide (cont peptide; 1 µM for 60 min, n = 10 neurons), and Cacna2d1 KO (n = 12 neurons for baseline and OGD groups) on NMDAR activity in hippocampal CA1 neurons subjected to 5 min of OGD. (C) Representative gel images and quantification of co-IP show the effect of OGD on the α2δ−1–GluN1 or α2δ−1–Cav2.2 association in mouse brain tissues (n = 6 mice per group). (D) Original gel images and quantification of co-IP show the effect of α2δ−1Tat peptide (P) and control peptide (C; both 1 µM for 30 min) on the α2δ−1–GluN1 or α2δ−1–Cav2.2 association in mouse brain slices subjected to OGD (O, n = 6 mice per group). Data are shown as means ± SEM. *p < 0.05 compared with the baseline current amplitude before OGD or with the vehicle or control peptide-treated group. ^#p < 0.05 compared with the OGD+vehicle or OGD+control peptide group.

Figure 2.. Focal cerebral ischemia increases the protein level and glycosylation of α2δ−1.

(A) Representative gel images and quantification of α2δ−1 protein levels in the cerebral cortex, hippocampus (Hippo), and striatum from sham control mice and mice subjected to 90 min of MCAO followed by 24 h of reperfusion (n = 8 mice per group). (B) Upper: Representative gel images of α2δ−1 protein bands in different regions of brain tissues from wild-type (WT) and Cacna2d1 knockout (KO) mice; Lower: α2δ−1 protein bands detected in 2 sets of cortical tissue lysates from control and MCAO mice treated with PNGase F or vehicle (Veh). (C) Original gel images and quantification of GluN1 protein levels in brain tissues from WT and Cacna2d1 KO mice (n = 8 mice per group). (D) Quantification of α2δ−1 mRNA levels in the cerebral cortex, hippocampus (Hippo), and striatum in MCAO and sham control mice (n = 6 mice per group). Data are shown as means ± SEM. *p < 0.05 compared with the sham group in the corresponding brain region.

Figure 3.. α2δ−1 critically contributes to brain injury and neurological deficit caused by MCAO.

(A) Neurological deficit scores shown in a box-whisker plot in sham control mice (n = 8 mice) and MCAO mice treated with vehicle (n = 9 mice), gabapentin before MCAO (pre-GBP, n = 10 mice), or gabapentin after MCAO (post-GBP, n = 8 mice). (B) Representative TTC staining and quantification of brain infarct volume in sham control mice (n = 8 mice) and MCAO mice treated with vehicle (n = 9 mice), gabapentin before MCAO (n = 10 mice), or gabapentin after MCAO (n = 8 mice). (C) Neurological deficit scores shown in a box-whisker plot in Cacna2d1 knockout (KO) mice and wild-type (WT) mice subjected to MCAO and reperfusion (n = 8 mice per group). (D) Representative TTC staining and quantification of brain infarct volume in Cacna2d1 KO and WT mice (n = 8 mice per group). Data are shown as means ± SEM (neurological deficit score data shown as median ± min-max). *p < 0.05, **p < 0.01, ***p < 0.001 each compared with the vehicle MCAO or WT control group.

Figure 4.. α2δ−1–bound NMDARs contribute to brain injury and neurological deficit caused by MCAO in the brain.

(A) Original co-IP gel images show the interaction between α2δ−1 and GluN1 in two samples from mouse brain tissues. (B) Effect of α2δ−1Tat peptide (1 µM for 60 min) on the α2δ−1–GluN1 protein complex in mouse brain tissues (n = 6 mice per group). (C) Representative gel images and quantification of co-IP analysis show the effect of 15 min of MCAO followed by 2 h of reperfusion on the α2δ−1–GluN1 protein complex level in mouse brain tissues (Cont, sham control; n = 6 samples from 12 mice per group). (D) Neurological severity scores shown in a box-whisker plot (left) and representative TTC staining and quantification (right) of brain infarct volume in MCAO mice treated with α2δ−1Tat peptide or control peptide (both 200 µg/kg, i.p.) at 15 min before MCAO and immediately before reperfusion (n = 8 mice per group). Data are shown as means ± SEM (neurological deficit score data shown as median ± min-max). *p < 0.05, **p < 0.01 compared with the control peptide-treated or sham control group.

Figure 5.. Gabapentin, α2δ−1Tat peptide, or α2δ−1 genetic deletion reduces MCAO-induced calpain activity in brain tissues.

(A and B) Original gel images (A) and quantification (B) of spectrin BD levels in the cerebral cortex, hippocampus (Hippo), and striatum from sham control mice, MCAO mice treated with vehicle (Veh) or gabapentin (GBP), and Cacna2d1 knockout (KO) MCAO mice (n = 8 mice per group). (C and D) Original blotting images (C, two pairs of samples) and quantification (D) of spectrin BD levels in the cerebral cortex, hippocampus, and striatum from MCAO mice treated with α2δ−1Tat peptide or control peptide (n = 8 mice per group). Data are shown as means ± SEM. *p < 0.05 compared with the sham group. ^#p < 0.05 compared with MCAO+vehicle group or control peptide group in the same brain region.

Figure 6.. Gabapentin, α2δ−1Tat peptide, or α2δ−1 ablation suppresses MCAO-induced apoptotic signaling in brain tissues.

(A and B) Representative blotting images (A) and quantification (B) of cleaved caspase-3 protein levels in the cerebral cortex, hippocampus (Hippo), and striatum from WT sham control mice, WT MCAO mice treated with vehicle (Veh) or gabapentin (GBP), and Cacna2d1 knockout (KO) MCAO mice (n = 8 mice per group). (C and D) Original gel images (C, two pairs of samples) and quantification (D) of cleaved caspase-3 protein levels in the cerebral cortex, hippocampus, and striatum from MCAO mice treated with α2δ−1Tat peptide or control peptide (n = 8 mice per group). Data are shown as means ± SEM. *p < 0.05 compared with the sham group. ^#p < 0.05 compared with MCAO+vehicle group or control peptide group in the same brain region.