NOVEL MECHANISM FOR GLUTAMATE-DEPENDENT EXCITOTOXICITY

描述（由申请人提供）：先前已经提出，谷氨酸的过度释放和谷氨酸受体（主要是NMDA受体，NMDAR）的过度激活是中枢神经系统在神经元损伤（包括缺血、创伤性脑损伤和癫痫）期间发生的继发性（延迟性）神经元死亡的主要原因。最近，基于我们的实验数据，我们对谷氨酸依赖性兴奋性毒性的机制提出了新的假设。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NMDARs本身的过度激活，而是神经元间隙连接（GJ；电突触）的存在和表达的增加。我们还假设这些机制具有普遍性，并参与不同类型神经元损伤的神经元死亡。然而，目前还没有明确的证据表明神经元GJ偶联的增加和减少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偶联增加的潜在机制尚不清楚。神经元GJ在神经元死亡中的中心地位使得解决这些问题势在必行，如果阻断神经元GJ偶联在临床上被利用作为神经保护的新方法。首先，我们将验证神经元GJ偶联增加和减少预防缺血引起的神经元死亡的预测。这将通过氧-葡萄糖剥夺作为体外缺血模型，从野生型和连接蛋白36敲除小鼠的体感觉皮层制备的神经元培养物，慢病毒载体细胞培养感染，电紧张偶联，western blots和神经元死亡分析进行研究。其次，我们将验证缺血期间神经元间隙连接偶联的增加是通过连接蛋白36mrna翻译的“泄漏扫描”机制调节的假设。这将通过电紧张耦合、western blotting、神经元染色、分子生物学和从野生型小鼠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针对神经元GJs的神经保护的新方法，而不是NMDARs。

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.