A novel neuroprotective strategy

描述（由申请人提供）：本申请旨在研究病毒用于阻止肝细胞凋亡的机制是否可以转化和优化以阻止损伤后神经元细胞死亡。尽管导致中风和相关疾病后神经退行性变化的几种信号级联已经得到了很好的表征，但治疗人类疾病的有效神经保护疗法仍然困扰着我们。因此，旨在发现新颖的神经保护策略（例如本文描述的策略）的研究可能具有非常重要的意义。我们的实验室正在测试一般假设，即由不同的有害刺激触发或由独特的生化信号级联组成的不同细胞死亡途径需要一组常见条件才能最佳运行。在过去的十年中，我们定义了一种神经元促凋亡信号级联，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 的损失提供场所，为蛋白酶和核酸酶的激活建立一个宽松的环境，确保细胞死亡程序的完成。干扰导致凋亡 K 电流激增的过程可以有效阻止神经元细胞死亡。在哺乳动物皮质和中脑神经元中，电流激增是由 Kv2.1 编码的 K 通道从头开始的 SNARE 依赖性胞吐插入细胞膜介导的。值得注意的是，丙型肝炎病毒基因组 RNA 翻译和加工的产物非结构蛋白 5A (NS5A) 最近被证明可以有效干扰肝细胞中 Kv2.1 介导的凋亡 K电流并抑制肝细胞死亡。在初步研究中，我们观察到 NS5A 也可用于拯救损伤后的神经元，并且该蛋白干扰神经元 Kv2.1 介导的凋亡 K 电流激增。在此应用中，我们打算（i）研究 NS5A 干扰 Kv2.1 功能表达的机制，以及（ii）定义限制通道功能所需的 NS5A 分子结构域。后面的这些实验将建立最小的 NS5A 衍生序列，可用于设计新型神经保护探针。我们研究计划的总体目标是设计新的治疗策略来保护损伤后的神经元。我们正在探索一种新颖的、可能具有突破性的方法来实现这一目标，通过建设性地利用一种生物策略来阻止肝脏细胞死亡，并将其转化为治疗中风和其他形式神经退行性疾病的新方法。 公共健康相关性：本申请中提出的研究将帮助我们了解钾通道功能在神经元细胞死亡中的作用。 最重要的是，该项目期间进行的研究可能会揭示开发新型神经保护药物的新途径，以预防中风和相关疾病期间的脑损伤。

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.