Neuroprotective Mechanisms of Neuroglobin in Stroke

描述（由申请人提供）：脑红蛋白（Ngb）是最近发现的脊椎动物脑中的组织珠蛋白。初步研究表明，NGB对缺氧缺血性损伤具有保护作用。然而，Ngb神经保护的机制仍有待确定。 我们的总体假设是，在缺氧/缺血后，升高的Ngb通过（a）促进氧扩散从而改善线粒体呼吸和功能，（B）清除神经元一氧化氮（NO）从而减少活性氮物质（RNS）和活性氧物质（ROS）的形成，以及（c）下调细胞死亡信号传导并减少谷氨酸释放来调节神经元存活。 在目的1中，我们将研究Ngb在体外缺氧/复氧（H/R）后调节线粒体呼吸和神经元死亡信号的作用。我们假设Ngb在小鼠大脑皮层神经元H/R后上调，并且过度表达Ngb改善线粒体呼吸并下调神经元死亡信号，如细胞色素c释放和半胱天冬酶激活。在目的2中，我们将记录体外H/R后Ngb清除NO的特定作用。Ngb将在从nNOS敲除小鼠和匹配的野生型小鼠分离的神经元中过表达。将检查和比较RNS和ROS形成、线粒体呼吸、神经元死亡信号传导和谷氨酸释放的变化。在目的3中，我们将比较Ngb过表达转基因小鼠与野生型小鼠在体内局灶性脑缺血后的情况。将测量神经学结局、ATP水平、RNS和ROS形成、细胞死亡信号传导和谷氨酸释放的变化。为了进一步剖析Ngb在调节神经元NO介导的脑损伤中的作用，将Ngb转基因小鼠与nNOS敲除小鼠杂交。 这些拟议的实验应该提供新的见解Ngb如何保护神经元缺氧/缺血，并可能最终导致新的治疗策略，用于治疗中风。

DESCRIPTION (provided by applicant): Neuroglobin (Ngb) is a recently discovered tissue globin found in the vertebrate brain. Initial study suggests that NGB is protective against hypoxia-ischemic insults. However, the mechanisms underlie Ngb neuroprotection remain to be defined. Our over all hypothesis is that after hypoxia/ischemia, elevated Ngb modulates neuron survival by (a) facilitating oxygen diffusion thus improving mitochondrial respiration and function, (b) scavenges neuronal nitric oxide (NO) thus reducing the formation of reactive nitrogen species (RNS) and reactive oxygen species (ROS), and (c) down-regulating cell death signaling and diminishing glutamate release. In Aim 1, we will examine the role of Ngb in regulating mitochondria respiration and neuronal death signaling after hypoxia/reoxygenation (H/R) in vitro. We hypothesize that Ngb is upregulated after H/R in mouse cerebral cortical neurons and over-expressing Ngb improves mitochondria respiration and down-regulates neuronal death signaling such as cytochrome c release and caspase activation. In Aim 2, we will document the specific role of NO-scavenging by Ngb after H/R in vitro. Ngb will be overexpressed in neurons isolated from nNOS knockout mice and matching wild-type mice. Changes of RNS and ROS formation, mitochondrial respiration, neuronal death signaling and glutamate release will be examined and compared. In Aim 3, we will compare Ngb overexpressing transgenic mice with wild type mice after focal cerebral ischemia in vivo. Alterations of neurological outcomes, ATP levels, RNS and ROS formations, cell death signaling, and glutamate release will be measured. To further dissect the role of Ngb in regulating neuronal NO-mediated brain damage, Ngb transgenic mice will be crossed with nNOS knockout mice. These proposed experiments should provide new insight of how Ngb protect neurons from hypoxia/ischemia and may ultimately lead to novel therapeutic strategies for the treatment of stroke.