The Role Of MtCB1 Receptor In Neuroprotection

Background Cardiac arrest-induced neurologic injury is a vital cause of death among the patients with a successful cardiopulmonary resuscitation. American Heart Association and related organizations have made long-term efforts to improve resuscitation guidelines. However, the hospital mortality remains proximately 70 % in patients who were resuscitated successfully. Unfortunately, about 2/3 of the survivors have moderate to severe cognitive deficits. Therefore, searching for novel effective therapeutics which can alleviate neurologic injury induced by cerebral ischemia/reperfusion(I/R) is of great importance. Mitochondria are cellular energy factories, participating in oxidative stress, calcium homeostasis, generation of reactive oxygen species(ROS) and programmed cell death, which are crucial in regulating brain functions. In cerebral I/R injury, mitochondrial dysfunction leads to neuronal death, which provides a promising strategy for targeting mitochondria to develop new therapeutics against cerebral I/R injury. However, approaches that regulate mitochondria effectively after cerebral I/R injury are still poorly understood. A recent study published in ‘Nature Neuroscience’ found that CB1 receptor is also expressed in neuronal mitochondrial membranes(mt CB1), and activation of which regulates mitochondrial respiration and energy metabolism under physiological conditions. However, whether activation of mt CB1 receptor modulates mitochondrial functions and exerts neuroprotection against I/R injury is still unknown. In the present study, we used models of primary cultured hippocampal neurons oxygen-glucose deprivation/ reoxygenation(OGD/R), mouse global cerebral I/R and Ca2+-induced injury in purified intact mitochondria from hippocampal neurons to investigate the roles of mt CB1 receptor in neuroprotection.Experiment I Selective activation of mt CB1 receptor Objective: To find the suitable method of activating mt CB1 receptor.Methods:(1) To detect the expression of mt CB1 receptor at different time points after I/R, fifty-five C57BL/6 mice were randomly divided into three groups: Sham, BCCAO and ACEA+BCCAO groups. Mitochondria protein were isolated at 2 h, 6 h, 24 h, 48 h and 72 h after reperfusion respectively and the expression of mt CB1 receptor was assessed by Western blot analysis.(2) To determine the effect of drugs on CB1 receptor expression, five groups of hippocampal neurons were defined as follows(n = 5): Control, ACEA, AM251+ACEA, Hemo+ACEA, and Vehicle groups. Mitochondrial proteins were extracted from all groups of cells for Western blot analysis at 2 h after administration of drugs. In vivo, forty C57BL/6 mice were randomly divided into five groups: Control, ACEA, AM251+ACEA, Hemo+ACEA, and Vehicle groups. The expressions of mt CB1 receptor were detected by Western blot analysis(n = 5) and immunocytochemistry for electron microscopy(n = 3) at 2 h after administration of drugs.(3) To evaluate the effect of OGD/R or cerebral I/R on mt CB1 receptor expression, there were six groups of hippocampal neurons: Control, OGD, ACEA+OGD, AM251+ACEA+OGD, Hemo+ACEA+OGD, and Vehicle+OGD groups. Mitochondrial protein was extracted from neurons for Western blot analysis at 2 h after reoxygenation. In vivo, thirty C57BL/6 mice were randomly divided into six groups(n = 5): Sham, BCCAO, ACEA+BCCAO, AM251+ACEA+BCCAO, Hemo+ACEA+BCCAO, and Vehicle+BCCAO groups. Mitochondrial protein was extracted from hippocampus for Western blot analysis at 2 h after reperfusion.Results:(1) mt CB1 receptor expression was significantly increased at 2 h after reperfusion compared with Sham group and ACEA increased the mt CB1 R expression more obviously(P < 0.05). ACEA also up-regulated the mt CB1 receptor expression at 6 h after reperfusion compared with Sham group(P < 0.05). However, there was no significant difference in mt CB1 receptor expression at the other time points.(2) mt CB1 receptor expression was significantly increased at 2 h after ACEA treatment(P < 0.05). The effects were reversed by the cell-permeant CB1 receptor antagonist, AM251(P < 0.05), but not by the cell-impermeant CB1 receptor antagonist, hemopressin. In the remaining samples without mitochondria protein, CB1 receptor expression was increased just in ACEA group(P < 0.05).(3) mt CB1 receptor expression was significantly increased at 2 h after reoxygenation or reperfusion and ACEA increased the mt CB1 expression further(P < 0.05). The effects were abolished by AM251(P < 0.05), but not by hemopressin. In the remaining samples without mitochondria protein, CB1 receptor expression was also increased at 2 h after reoxygenation or reperfusion and ACEA increased the expression further(P < 0.05). While treatment with AM251 or hemopressin abolished the effects of ACEA(P < 0.05).Conclusion: mtCB1 receptor can be activated selectively by using the cell-permeant CB1 receptor agonist ACEA and the cell-impermeant CB1 receptor antagonist hemopressin.Experiment II The effect of mtCB1 activation on neuroprotectionObjective: To investigate the effect of mt CB1 receptor activation on neuroprotection.Methods:(1) There were six groups of hippocampal neurons: Control, OGD, ACEA+OGD, AM251+ACEA+OGD, Hemo+ACEA+OGD, and Vehicle+OGD groups. The cells received the drugs administration at the onset of reoxygenation. Cell injury was then evaluated in terms of WST-8 cell viability(n = 8), LDH release(n = 6) and apoptotic rate(n = 5) at 24 h after reoxygenation.(2) Sixty C57BL/6 mice were randomly divided into six groups(n = 10): Sham, BCCAO, ACEA+BCCAO, AM251+ACEA+BCCAO, Hemo+ACEA+BCCAO and Vehicle+BCCAO groups. Drugs were administered intraperitoneally at the onset of reperfusion. Neurological tests were conducted at 24, 48 and 72 h after reperfusion. Neuronal apoptosis was assessed by TUNEL staining(n = 5) and apoptosis markers(n = 5) at 72 h after reperfusion.Results:(1) ACEA increased cell viability, attenuated LDH release and decreased apoptotic rate(P < 0.05). The protective effects of ACEA were abolished by AM251(P < 0.05), while not or just partially reversed by hemopressin.(2) The neurological scores in ACEA+BCCAO group were significantly higher than those of BCCAO group at 24, 48, and 72 h after reperfusion respectively(P < 0.05). AM251 reversed the ACEA-induced benefits at the three time-points(P < 0.05), while hemopressin just partially reversed the benefits. ACEA diminished the number of TUNEL-positive neurons in the hippocampal CA1 region and decreased the expression of cleaved caspase-3 induced by BCCAO(P < 0.05). The benefits of ACEA were blocked by AM251(P < 0.05), but just partially by hemopressin.Conclusion: Activation of mt CB1 receptor reduces neuronal injury induced by OGD/R and cerebral I/R.Experiment III The protective effects of mt CB1 activation on mitochondrial functionObjective: To determine the effects of mt CB1 activation in improving mitochondrial function after cerebral I/R, OGD/R and Ca2+-induced mitochondrial dysfunction.Methods:(1) To observe the effect of mt CB1 activation on mitochondrial ultrastructure after cerebral I/R, eighteen C57BL/6 mice were randomly divided into six groups(n = 3): Sham, BCCAO, ACEA+BCCAO, AM251+ACEA+BCCAO, Hemo+ACEA+BCCAO and Vehicle+BCCAO groups. Drugs were administered intraperitoneally at the onset of reperfusion. The mouse CA1 hippocampal neurons were observed by transmission electron microscopy at 72 h after reperfusion.(2) To detect the effect of mt CB1 activation on intracellular ROS level and mitochondrial functions after OGD/R, there were six groups of hippocampal neurons: Control, OGD, ACEA+OGD, AM251+ACEA+OGD, Hemo+ACEA+OGD, and Vehicle+OGD groups. The cells received the drugs administration at the onset of reoxygenation. Intracellular ROS level was detected at 24 h after reoxygenation and neuronal mitochondria were purified at 2 h and 24 h after reoxygenation respectively to assay the mitochondrial function after OGD/R.(3) To perceive the effect of mt CB1 activation on mitochondrial functions after Ca2+ induced injury, intact mitochondria were obtained from normal hippocampal neurons and were divided into five groups(n = 6). Except for the Control and the Ca2+ only groups, the other three groups of mitochondrial suspensions received 0.1 μM, 1 μM and 10 μM ACEA respectively at 30 min before Ca2+ administration. Mitochondrial swelling and mitochondrial membrane potential(MMP) were detected at 20 min after Ca2+ administration.Results:(1) The mitochondria in Sham group were elongated or round and had numerous cristae with parallel alignment. The outer and inner membranes of mitochondria were clearly discernible. Mitochondria from BCCAO, AM251+ACEA+BCCAO and Vehicle+BCCAO groups showed swelling, vacuolization, and disruption of cristae. In ACEA+BCCAO and Hemo+ACEA+BCCAO groups, the mitochondrial structure was almost normal, but the cristae were slightly disrupted.(2) ACEA inhibited the generation of intracellular ROS in the cells exposed to OGD/R(P < 0.05). The effect of ACEA was abolished by AM251(P < 0.05), while just partially reversed by hemopressin. At 2 and 24 h after reoxygenation, the activities of complexes I and IV and the MMP showed significant reduction in OGD group and ACEA improved the mitochondrial functions significantly(P < 0.05). The benefits of ACEA were completely reversed by AM251(P < 0.05), while not or just partially by hemopressin. The OGD-induced decrease in complex II activity was observed at 24 h after reoxygenation(P < 0.05), but not at 2 h; however, ACEA did not alleviate the decrease of complex II activity.(3) Ca2+ induced a noticeable mitochondrial swelling, which was followed by a decline of MMP(P < 0.05). ACEA markedly prevented Ca2+-induced mitochondrial swelling and increased the MMP(P <0.05).Conclusion: mt CB1 activation ameliorates mitochondrial ultrastructure after cerebral I/R, improves mitochondrial dysfunction after OGD/R, alleviate Ca2+-induced mitochondrial swelling and increased the MMP.Experiment IV The roles of mitochondrial function in neuroprotection of mt CB1 receptorObjective: To study whether activation of mt CB1 induces neuroprotection by improving mitochondrial functions.Methods:(1) To investigate the roles of mitochondrial respiratory chain in neuroprotection of mt CB1 receptor, there were five groups of hippocampal neurons: Control, OGD, ACEA+OGD, rotenone+ACEA+OGD, TTFA+ACEA+OGD groups. The cells received the administration at the onset of reoxygenation. Cell injury was then evaluated in terms of WST-8 cell viability(n = 8) and LDH release(n = 6) at 24 h after reoxygenation.(2) To evaluate the effect of mt CB1 activation on mitochondrial permeablity transition pore(m PTP), there were four groups of hippocampal neurons(n = 5): Control, OGD, ACEA+OGD, Atr+ACEA+OGD groups. The cells received the administration at the onset of reoxygenation. The levels of cytochrome c(Cyto C) and apoptosis-inducing factor(AIF) were detected at 2 h after reoxygenation.(3) To determine the role of m PTP in neuroprotection of mt CB1 receptor, there were four groups of hippocampal neurons: Control, OGD, ACEA+OGD and Atr+ACEA+OGD groups. The cells received the administration at the onset of reoxygenation. Cell injury was then evaluated in terms of WST-8 cell viability(n = 8), LDH release(n = 6) and apoptotic rate(n = 5) at 24 h after reoxygenation. Forty C57BL/6 mice were randomly divided into four groups(n = 10): Sham, BCCAO, ACEA+BCCAO, Atr+ACEA+BCCAO groups. Atractyloside(Atr), an m PTP opener, was administered intracerebroventricularly at 5 min before reperfusion and ACEA was administered intraperitoneally at the onset of reperfusion. Neurological tests were conducted at 24, 48 and 72 h after reperfusion. Neuronal apoptosis was assessed by TUNEL staining(n = 5) and apoptosis markers(n = 5) at 72 h after reperfusion.Results:(1) ACEA increased cell viability and attenuated LDH release at 24 h after reoxygenation(P < 0.05). The protective effects of ACEA were partially abolished by mitochondrial complex I inhibitor rotenone(P < 0.05), while not reversed by mitochondrial complex II inhibitor TTFA.(2) OGD/R-induced cytochrome c release and AIF translocation were significantly reduced by ACEA treatment(P < 0.05), while Atr reverse the effects of ACEA(P < 0.05).(3) ACEA increased cell viability, attenuated LDH release and decreased apoptotic rate at 24 h after reoxygenation, which were partially abolished by Atr(P < 0.05). The neurological scores in ACEA+BCCAO group were significantly higher than those of BCCAO group at 24, 48, and 72 h after reperfusion respectively(P < 0.05). ACEA diminished the number of TUNEL-positive neurons in the hippocampal CA1 region and decreased the expression of cleaved caspase-3 at 72 h after reperfusion(P < 0.05). The benefits of ACEA were partially blocked by Atr.Conclusion: The neuroprotective effects of mt CB1 receptor are partially reversed by inhibiting mitochondrial complex activity or increasing m PTP opening, suggesting activation of mt CB1 induces neuroprotection by ameliorating mitochondrial functions.Summary: In the present study, we proved that mt CB1 receptor can be activated selectively by using the cell-permeant CB1 receptor agonist ACEA and the cell-impermeant CB1 receptor antagonist hemopressin, which are able to induce neuroprotection. The mechanism involved is that mt CB1 activation improves mitochondrial complex activity, increases MMP and inhibits m PTP opening. The study suggests that mt CB1 receptor may be a potential novel target for the treatment of cerebral I/R injury.