A novel neuroprotective strategy

描述(申请人提供)：这项申请旨在调查病毒用来阻止肝细胞凋亡的机制是否可以被翻译和优化以阻止损伤后的神经细胞死亡。尽管中风和相关疾病后导致神经退行性变化的几个信号级联反应已经得到了很好的描述，但治疗人类疾病的有效神经保护疗法仍然是我们无法找到的。因此，旨在发现新的神经保护策略的研究，如本文所述，可能具有非常重要的意义。我们的实验室正在测试一个普遍的假设，即不同的细胞死亡路径，由不同的损伤刺激触发或由独特的生化信号级联组成，需要一组共同的条件才能以最佳方式运行。在过去的十年里，我们定义了一种神经元促凋亡信号级联反应，其特征是电压依赖性K电流的强劲增强。这种现象通过为细胞质K的丢失提供场所，为蛋白酶和核酸酶的激活建立一个允许的环境，从而确保细胞死亡程序的完成。干扰导致细胞凋亡性钾电流激增的过程可以有效地阻止神经细胞死亡。在哺乳动物的大脑皮层和中脑神经元中，电流峰是由依赖于新的SNARE的Kv2.1编码的K通道胞吐插入细胞膜所介导的。值得注意的是，丙型肝炎病毒基因组RNA的翻译和加工产物非结构蛋白5A(NS5A)最近被证明能有效地干扰Kv2.1介导的肝细胞凋亡K电流，抑制肝细胞死亡。在初步研究中，我们观察到NS5A也可以用于修复损伤后的神经元，并且该蛋白干扰Kv2.1介导的神经元凋亡性K电流。在本申请中，我们打算(I)研究NS5A干扰Kv2.1功能表达的机制，以及(Ii)确定NS5A限制通道功能所必需的分子结构域。这些后面的实验将建立最小的NS5A衍生序列，可以用于设计新的神经保护探针。我们研究计划的首要目标是设计新的治疗策略，以保护损伤后的神经元。我们正在探索一种新颖的、可能具有开创性的方法来实现这一目标，方法是建设性地利用一种生物策略，该策略逐渐阻止肝脏中的细胞死亡，并将其转化为治疗中风和其他形式的神经退化的新方法的产生。 公共卫生相关性：本申请中提出的研究将帮助我们了解钾通道功能在神经细胞死亡中的作用。最重要的是，在该项目期间进行的研究可能会揭示开发一类新的神经保护药物的新途径，以防止中风和相关疾病中的脑损伤。

DESCRIPTION (provided by applicant): This application aims to investigate whether a mechanism used by a virus to block hepatocyte apoptosis can be translated and optimized to block neuronal cell death following injury. Although several signaling cascades responsible for neurodegenerative changes following stroke and related disorders have been well characterized, effective neuroprotective therapies to treat human conditions continue to elude us. Thus, investigations designed to uncover novel neuroprotective strategies, such as those describe here, are potentially of very high significance. Our laboratory is testing the general hypothesis that distinct cell death pathways, triggered by diverse injurious stimuli or composed of unique biochemical signaling cascades, require a set of common conditions to operate optimally. Over the last ten years, we have defined a neuronal pro-apoptotic signaling cascade characterized by a robust enhancement of voltage dependent K+ currents. This phenomenon ensures the completion of cell death programs by providing a venue for the loss of cytoplasmic K+, establishing a permissive environment for protease and nuclease activation. Interfering with the processes responsible for the apoptotic K+ current surge can effectively block neuronal cell death. In mammalian cortical and midbrain neurons, the current surge is mediated by a de novo SNARE-dependent exocytotic insertion of Kv2.1-encoded K+ channels into the cell membrane. Remarkably, a product of the translation and processing of the hepatitis C virus genomic RNA, the non-structural protein 5A (NS5A), was recently shown to effectively interfere with Kv2.1-mediated apoptotic K+ currents in liver cells and inhibit hepatocyte cell death. In preliminary studies we observed that NS5A could also be employed to rescue neurons following injury and that this protein interferes with the neuronal Kv2.1-mediated apoptotic K+ current surge. In this application we intend to (i) investigate the mechanism responsible for NS5A interference with Kv2.1 functional expression, and (ii) define the molecular domains of NS5A necessary for restricting channel function. These latter experiments will establish the minimal NS5A-derived sequences that can be used for the design of novel neuroprotective probes. The overarching goal of our research program is to devise new therapeutic strategies to protect neurons following injury. We are exploring a novel, possibly groundbreaking approach to achieve this goal by constructively harnessing a biological strategy that evolved to block cell death in the liver and translating it towards the generation of novel methods to treat stroke and other forms of neurodegeneration. PUBLIC HEALTH RELEVANCE: The research proposed in this application will help us understand the role of potassium channel function in neuronal cell death. Most importantly, research conducted during this project may reveal novel avenues for developing a new class of neuroprotective drugs to prevent brain damage during stroke and related conditions.