NOVEL MECHANISM FOR GLUTAMATE-DEPENDENT EXCITOTOXICITY

描述（由申请人提供）：之前已经提出，谷氨酸的过度释放和谷氨酸受体（主要是NMDA受体，NMDAR）的过度活化是神经元损伤（包括缺血、创伤性脑损伤和癫痫）期间中枢神经系统中发生的继发性（迟发性）神经元死亡的主要原因。最近，我们根据实验数据，对谷氨酸依赖性兴奋毒性的机制提出了新的假说。我们认为，在神经元损伤过程中，谷氨酸依赖性神经元死亡的主要原因不是NMDAR本身的过度激活，而是神经元间隙连接（GJ;电突触）表达的存在和增加。我们还推测，这些机制具有普遍性，并参与不同类型的神经元损伤的神经元死亡。然而，目前还没有确切的证据表明神经元GJ偶联的增加和减少将分别直接决定神经元死亡的增加和减少。此外，神经元损伤期间神经元GJ耦合增加的潜在机制尚不清楚。神经元GJ对神经元死亡的中心地位使得解决这些问题势在必行，如果神经元GJ偶联的阻断在临床上被开发为神经保护的新方法。首先，我们将测试神经元GJ耦合增加增加和减少防止缺血引起的神经元死亡的预测。这将使用氧-葡萄糖剥夺作为缺血的体外模型、从野生型和连接蛋白36敲除小鼠的体感皮层制备的神经元培养物、用慢病毒载体的细胞培养物感染、电紧张偶联、蛋白质印迹和神经元死亡分析来研究。其次，我们将测试的假设，增加在缺血期间的神经元间隙连接偶联调节通过连接蛋白36 mRNA翻译的“漏扫描”的机制。这将使用电紧张耦合、蛋白质印迹、神经元染色、分子生物学和从野生型小鼠的躯体感觉皮层制备的神经元培养物进行测试。我们提出的分析符合R21机制的标准：我们关于GJ在神经元死亡中的作用的假设可能是错误的。然而，如果我们是正确的，我们将确定谷氨酸介导的兴奋性毒性的新机制和靶向神经元GJ而不是NMDAR的神经保护的新方法。 公共卫生相关性：本研究与公共卫生相关。它解决了神经元间隙连接（电突触）在缺血性神经元死亡中的作用和缺血期间神经元间隙连接耦合的调节机制。这项研究可能具有临床意义，因为缺血期间神经元间隙连接偶联增加，但间隙连接在缺血中的作用和调节尚未了解。

DESCRIPTION (provided by applicant): It has been suggested previously, that excessive release of glutamate and overactivation of glutamate receptors (mainly NMDA receptors, NMDAR) are primarily responsible for the secondary (delayed) neuronal death that occurs in the central nervous system during neuronal injuries, including ischemia, traumatic brain injury and epilepsy. Recently, based on our experimental data, we proposed novel hypothesis for the mechanisms of glutamate-dependent excitotoxicity. We suggested that, during neuronal injury, the main reason for glutamate-dependent neuronal death is not an overactivation of NMDARs per se, but rather the presence and increase in expression of neuronal gap junctions (GJ; electrical synapses). We also postulated that these mechanisms have universal character and are involved in neuronal death in different types of neuronal injuries. Currently, however, there i no definitive evidence that increase and decrease in neuronal GJ coupling would directly determine the increased and decreased neuronal death, respectively. In addition, the underlying mechanism for increase in neuronal GJ coupling during neuronal injury is not known. The centrality of neuronal GJs to neuronal death makes addressing these issues imperative, if blockade of neuronal GJ coupling is to be exploited clinically as a novel approach for neuroprotection. First, we will test the prediction that increase in neuronal GJ coupling augments, and decrease prevents, neuronal death caused by ischemia. This will be studied using oxygen-glucose deprivation as an in vitro model of ischemia, neuronal cultures prepared from the somatosensory cortex of wild-type and connexin 36 knockout mice, cell culture infections with lentiviral vectors, electrotonic coupling, western blots and analysis of neuronal death. Second, we will test the hypothesis that increase in neuronal gap junction coupling during ischemia is regulated via the mechanism of "leaky scanning" of connexin 36 mRNA translation. This will be tested using electrotonic coupling, western blotting, neuronal staining, molecular biology and neuronal cultures prepared from the somatosensory cortex of wild-type mice. The analysis we propose fits the criteria of the R21 mechanism: there is risk that our hypothesis on the role of GJs in neuronal death might be wrong. However, if we are correct, we will have identified novel mechanism for glutamate-mediated excitotoxicity and novel approach for neuroprotection that targets neuronal GJs, rather than NMDARs. PUBLIC HEALTH RELEVANCE: This study is relevant to public health. It addresses the role of neuronal gap junctions (electrical synapses) in ischemic neuronal death and the mechanisms of regulation of neuronal gap junction coupling during ischemia. This research may have clinical importance given that neuronal gap junction coupling increases during ischemia, but the role and regulation of gap junctions in ischemia are not yet understood.