Role of NCKX2 in the activation of toxic Zn2+ influx in ischemic neurons

描述（由申请人提供）：缺血性脑损伤可导致认知缺陷，长期残疾，甚至死亡。在心脏手术、中风或心肌梗塞期间，当大脑的血液供应中断时，就会发生这种损伤。NMDA通道阻滞剂和其他旨在保护大脑免受缺血性损伤的药物在临床试验中失败了。因此，迫切需要确定脑缺血期间药物干预的新靶点。我们的初步数据表明，我们可能能够确定这样的新目标。脑缺血的毒性期发生在神经元因细胞外K+ ([K+]o)高度升高而深度去极化的时候。这种[K+]o的升高是由K+从细胞质向细胞外介质的外排引起的，并与相互的Na+内流相吻合。结果，缺血神经元去极化，胞浆内Na+含量升高。在初步工作中，我们模拟了培养神经元的这种缺血性去极化，发现胞质[Ca2+] （[Ca2+]c）升高是由na依赖性Ca2+内流引起的，而阻断NMDA通道无法阻止这种内流。有趣的是，na依赖性[Ca2+]c升高激活了一条潜在的更具神经毒性的Zn2+内流途径。去极化缺血神经元中Na依赖性[Ca2+]c的升高可能是由浆体k依赖性Na+/Ca2+交换剂NCKXs介导的。由于NCKX2是在大脑皮层表达的主要NCKX亚型，我们想探索NCKX2介导的Ca2+内流和Zn2+内流激活之间存在因果关系的想法。为了验证这一想法，我们计划使用从野生型和NCKX2敲除小鼠中获得的培养皮层神经元。我们的项目有2个具体目标，将在2年内完成。在Aim 1中，我们将确定NCKX2敲除对缺血神经元胞浆Zn2+ ([Zn2+]c)升高率的影响。在Aim 2中，我们将确定NCKX2敲除和Zn2+对缺血性神经元死亡的影响。如果我们发现NCKX2操作与缺血性神经元中神经毒性Zn2+内流的激活有因果关系，我们的数据将为开发NCKX2激活Zn2+内流的抑制剂提供支持。这些药物可能成为神经保护药物，可以用来提高心脏手术的安全性。公共卫生相关性：在涉及体外循环（CPB）的非常常见的心脏手术中，约有32%会引起脑缺血事件，并具有术后认知缺陷的显著风险。该项目探讨了与CPB相关的脑损伤是由K依赖性Na+/Ca2+交换器的异常操作引起的，该交换器激活了神经毒性Zn2+内流。这项研究可能会导致一种新的治疗方法的发展，可以在CPB手术之前应用，以提高其安全性。

DESCRIPTION (provided by applicant): Ischemic brain damage leads to cognitive deficits, long-term disability, and even death. This damage occurs when the blood supply to the brain is arrested, which takes place during cardiac surgeries, stroke, or myocardial infarction. NMDA channel blockers and other drugs designed to protect the brain from ischemic damage have failed in clinical trials. Therefore, the need to identify novel targets for pharmacological intervention during brain ischemia is very urgent. Our preliminary data indicate that we may be able to identify such novel target(s). The toxic phase of brain ischemia is executed at the time when neurons are profoundly depolarized by highly elevated extracellular K+ ([K+]o). This [K+]o elevation is caused by K+ efflux from the cytosol to the extracellular medium and coincides with a reciprocal Na+ influx. As a result, ischemic neurons are depolarized and contain elevated cytosolic Na+. In preliminary work, we simulated such ischemic depolarization in cultured neurons and found that cytosolic [Ca2+] ([Ca2+]c) elevations resulted from an Na-dependent Ca2+ influx that could not be prevented by blocking NMDA channels. Interestingly, the Na-dependent [Ca2+]c elevations activated a pathway of potentially even more neurotoxic Zn2+ influx. The Na-dependent [Ca2+]c elevations in depolarized ischemic neurons are likely mediated by plasmalemmal K-dependent Na+/Ca2+ exchangers, NCKXs. Since NCKX2 is a major NCKX isoform expressed in the brain cortex, we would like to explore the idea that there is a causal link between NCKX2-mediated Ca2+ influx and an activation of Zn2+ influx. To test this idea, we plan to use cultured cortical neurons obtained from wild type and NCKX2 knockout mice. Our project has 2 specific aims and will be completed in 2 years. In Aim 1, we will determine the impact of the NCKX2 knockout on the rate of cytosolic Zn2+ ([Zn2+]c) elevation in ischemic neurons. In Aim 2, we will determine the impact of the NCKX2 knockout and Zn2+ on ischemic neuronal death. If we find that NCKX2 operation is causally linked to the activation of neurotoxic Zn2+ influx in ischemic neurons, our data will provide support for developing inhibitors of Zn2+influx activation by NCKX2. Such agents may become neuroprotective drugs that could be used to improve the safety of cardiac surgeries. PUBLIC HEALTH RELEVANCE: About 32% of the very commonly performed cardiac surgeries involving cardiopulmonary bypass (CPB) cause ischemic events in the brain and carry a significant risk of post-operative cognitive deficits. This project explores the idea that the brain damage relevant to CPB is caused by the abnormal operation of a K- dependent Na+/Ca2+ exchanger that activates a neurotoxic Zn2+ influx. This research may lead to the development of a novel therapy that could be applied prior to CPB surgeries to improve their safety.