Liberation of Intracellular Zinc and Neuronal Cell Death

项目总结 本应用程序中描述的实验的主要目标是开发和评估一个高度 创新的、机械合理的脑缺血神经保护策略。在过去的十年里 在这笔赠款的支持下，我们已经表征了一种由锌介导的使能凋亡的信号通路 最终依赖圈套插入p38/Src双磷酸化Kv2.1编码的K通道 在神经细胞质膜上。这一过程导致延迟整流器K的显著增强 电流，调节细胞内钾的损失所需的建立一个允许的，最佳的 损伤神经元半胱氨酸天冬氨酸酶和核酸酶激活的环境。尽管干扰了这些进程 负责细胞凋亡的K电流浪涌可有效阻断神经细胞死亡，无一上行 导致K电流增强的信号事件是该途径所特有的。在初步研究中 在这里，我们展示了缺乏SNARE结合域的通道不支持凋亡电流 涌动。此外，我们发现仅SNARE结合的细胞内通道结构域的过度表达是 体外神经保护作用。我们假设，干扰触发Kv2.1介导的细胞过程 细胞凋亡性钾电流波峰可能为神经保护提供高度特异和有效的治疗策略 中风和相关伤害。为了充分评估这一假设，我们将解决以下问题 实验的具体目标：首先，我们将详细描述磷酸化和圈套依赖 导致Kv2.1介导的细胞凋亡K电流激增的机制，第二，我们将调查 用细胞穿透性多肽干扰SNARE/Kv2.1相互作用是一种可行的神经保护措施 在啮齿动物中风模型中的策略。我们研究计划的长期目标是设计出新颖的 治疗中风和相关神经退行性疾病的神经保护方法。损失的损失 通过Kv2.1介导的K电流激增的细胞内K电流可能构成了细胞凋亡的普遍要求 大脑皮层和海马神经元细胞死亡。作为治疗人类的有效神经保护策略 神经疾病仍然是高度难以捉摸的，概念上的创新研究，如靶向 神经元凋亡性K电流，不仅具有潜在的高意义，而且也是迫切需要的。

PROJECT SUMMARY The main objective of the experiments described in this application is to develop and evaluate a highly innovative, mechanistically rational neuroprotective strategy in cerebral ischemia. During the last ten years with support from this grant, we have characterized a Zn2+-mediated apoptotic-enabling signaling pathway that culminates with the SNARE-dependent insertion of p38/Src dual phosphorylated Kv2.1-encoded K+ channels in the neuronal plasma membrane. This process results in a dramatic enhancement of delayed-rectifier K+ currents, mediating the loss of intracellular K+ required for the establishment of a permissive, optimal environment for caspase and nuclease activation in injured neurons. Although interfering with the processes responsible for the apoptotic K+ current surge can effectively block neuronal cell death, none of the upstream signaling events leading to the K+ current enhancement are specific for this pathway. In preliminary studies presented here, we show that channels lacking a SNARE binding domain do not support an apoptotic current surge. Moreover, we show that overexpression of the SNARE-binding intracellular channel domain alone is neuroprotective in vitro. We hypothesize that interfering with a cellular process that trigger the Kv2.1-mediated apoptotic K+ current surge may provides a highly specific and effective therapeutic strategy for neuroprotection in stroke and related injury. In order to adequately evaluate this hypothesis we will address the following experimental Specific Aims: First, we will characterize in detail the phosphorylation and SNARE-dependent mechanisms leading to Kv2.1-mediated apoptotic K+ current surges~ and second, we will investigate whether interfering with the SNARE/Kv2.1 interaction using cell-penetrating peptides is a viable neuroprotective strategy in a rodent stroke model. The long-term goal of our research program is to devise novel neuroprotective approaches for the treatment of stroke and related neurodegenerative conditions. The loss of intracellular K+ via a surge of Kv2.1-mediated K+ currents may constitute a ubiquitous requirement for apoptotic cell death of cortical and hippocampal neurons. As effective neuroprotective strategies to treat human neurological conditions continue to be highly elusive, conceptually innovative studies, such as targeting neuronal apoptotic K+ currents, are not only of potentially high significance, but also urgently needed.