Liberation of Intracellular Zinc and Neuronal Cell Death

描述(申请人提供)：本申请中描述的实验的主要目标是开发和评估一种高度创新的、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离子介导的使能凋亡的信号通路，最终导致p38/Src双磷酸化Kv2.1编码的K+通道依赖于SNARE插入神经元质膜。这一过程导致延迟整流性K+电流的显著增强，介导了细胞内K+的丢失，这是在受损神经元中建立允许的、最佳的caspase和核酸酶激活环境所必需的。虽然干扰导致K+电流激增的过程可以有效地阻止神经细胞死亡，但导致K+电流增强的上游信号事件都不是这一途径所特有的。在这里提出的初步研究中，我们表明，缺乏SNARE结合域的通道不支持凋亡电流激增。此外，我们发现在体外，过度表达SNARE结合的细胞内通道结构域本身就具有神经保护作用。我们推测，干扰触发Kv2.1介导的细胞凋亡K+电流激增的细胞过程可能为卒中及相关损伤的神经保护提供高度特异和有效的治疗策略。为了充分评估这一假说，我们将解决以下实验的具体目标：第一，我们将详细描述导致Kv2.1介导的K+电流凋亡的磷酸化和SNARE依赖的机制；第二，我们将调查在啮齿动物中风模型中，使用细胞穿透肽干扰SNARE/Kv2.1相互作用是否是一种可行的神经保护策略。我们研究计划的长期目标是设计新的神经保护方法来治疗中风和相关的神经退行性疾病。Kv2.1介导的K+电流的激增导致细胞内K+的丢失可能是大脑皮层和海马神经元细胞死亡的普遍条件。由于治疗人类神经系统疾病的有效神经保护策略仍然难以捉摸，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，而且迫切需要。

DESCRIPTION (provided by applicant): 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.