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

描述(申请人提供)：缺血性脑损伤会导致认知障碍、长期残疾，甚至死亡。当心脏手术、中风或心肌梗塞期间大脑血液供应停止时，这种损害就会发生。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都失败了。因此，迫切需要为脑缺血期间的药物干预寻找新的靶点。我们的初步数据表明，我们可能能够识别这样的新靶点(S)。脑缺血的毒性阶段是在神经元被高度升高的细胞外K+([K+]o)深度去极化的时候执行的。这种[K+]o升高是由K+从胞浆流出到细胞外介质引起的，并与Na+的相互内流相一致。结果，缺血神经元去极化，并含有升高的胞浆Na+。在前期工作中，我们模拟了培养神经元的这种缺血性去极化，发现细胞内[Ca~(2+)]_c的升高是由安娜依赖的Ca~(2+)内流引起的，而阻断NMDa通道不能阻止这种升高。有趣的是，钠依赖的[钙]c升高激活了一条潜在的更具神经毒性的锌离子内流途径。去极化缺血神经元Na~(2+)依赖性[Ca~(2+)]_c升高可能是由质膜K~+/Ca~(2+)交换器NCKX介导的。由于NCKX2是大脑皮层表达的一种主要的NCKX亚型，我们想探讨NCKX2介导的钙内流与激活的锌内流之间存在因果联系的观点。为了验证这一想法，我们计划使用从野生型和NCKX2基因敲除小鼠中获得的培养的皮质神经元。我们的项目有2个具体目标，将在2年内完成。在目标1中，我们将确定NCKX2基因敲除对缺血神经元胞浆锌离子([Zn2+]c)升高速率的影响。在目标2中，我们将确定NCKX2基因敲除和锌离子对缺血性神经元死亡的影响。如果我们发现NCKX2操作与神经毒性锌离子内流的激活有因果关系，我们的数据将为开发NCKX2激活锌离子内流的抑制剂提供支持。这类药物可能成为神经保护性药物，可用于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%会导致脑缺血事件，并存在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依赖的Na+/Ca~(2+)交换器的异常操作引起的，该交换器激活了神经毒性的锌离子内流。这项研究可能导致开发一种新的治疗方法，该方法可以在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.