Novel delivery of Bcl-2 for neuroprotection

描述(由申请人提供)：这一探索性项目的总体目标是基于蛋白质转导的新概念开发一种新的体内神经保护策略。这一模型将被用来将全长的、抗死亡的Bcl-2蛋白输送到大脑中，以检验这样一种假设，即通过将外源蛋白输送到脑细胞的现实方法，可以实现Bcl-2的缺血后神经保护。 第二个目标是阐明抗氧化剂与抗Bax机制在不同神经细胞死亡模式中的作用，并探讨磷酸化在调节这些机制和神经保护中的具体作用。该项目的具体目标是； 1)通过体外化学缺氧和葡萄糖剥夺激活的翻译后机制，验证了Bcl2的抗死亡活性受到抑制的假说。 2)与促凋亡蛋白Bax+BH3死亡结构域蛋白的相互作用相比，确定磷酸化调节在调节Bcl2保护Ce+氧化应激引起的线粒体功能障碍机制中的作用。 3)在大鼠短暂性局灶性脑缺血模型中，验证全长Bcl2作为TAT-Bcl2融合蛋白进入脑内具有保护作用的假设。 达到这些目的的方法将利用克隆正常的Bcl-2基因和特定磷酸化位点突变的基因，每个基因都连接到Tat蛋白转导结构域。将分离TATBCL-2融合蛋白，并测试其在体外抑制缺氧性神经细胞死亡和缩小大鼠局灶性脑缺血模型脑梗塞体积的能力。外源Bcl2结构对线粒体功能障碍的影响也将通过线粒体膜色素c释放、膜电位、氧化还原电位和反应性07种产物的测量来评估。磷酸化状态对内源性和外源性Bcl2保护线粒体功能障碍和细胞死亡的能力的影响也将被研究。这些研究的意义是：1.它们可以为同时针对坏死性和凋亡性细胞死亡的新的神经保护治疗策略奠定基础；2.它们将为在病理条件下调节像Bcl-2这样的蛋白的抗死亡活性的分子机制提供全新的见解。

DESCRIPTION (provided by applicant): The overall goal of this exploratory project is to develop a novel strategy for in vivo neuroprotection based on the new concept of protein transduction. This model will be used to deliver full-length, anti-death Bcl-2 proteins into the brain to test the hypothesis that postischemic neuroprotection by Bcl-2 can be achieved by a realistic method for delivering exogenous protein into brain cells. A second objective is to elucidate the contribution of the anti-oxidant vs. anti-Bax mechanisms in different neural cell death paradigms and to investigate the specific role of phosphorylation in regulating these mechanisms and in neuroprotection. The specific aims of the project are to; 1) Test the hypothesis that the anti-death activity of Bcl-2 is inhibited by post-translational mechanisms activated in response to chemical hypoxia and glucose deprivation in vitro. 2) Determine the role of phosphoQrlation in regulating the mechanisms by which Bcl-2 protects against mitochondrial dysfunction caused by Ce plus oxidative stress compared to the interaction of proapoptotic proteins, i.e., Bax plus BH3 death domain only protein. 3) Test the hypothesis that delivery of full-length Bcl-2, as a TAT-Bcl-2 fusion protein, into the brain is protective in a rat transient focal cerebral ischemia model. The methods of approach to these aims will utilize cloning of both the normal Bcl-2 gene and the gene with mutations in specific phosphorylation sites, each ligated to the TAT protein transduction domain. TATBcl-2 fusion proteins will be isolated and tested for their ability to inhibit hypoxic neural cell death in vitro and to reduce cerebral infarct volume in a rat reversible focal ischemia model. The effects of exogenous Bcl-2 constructs on mitochondrial dysfunction will also be assessed using measures of mitochondrial cvtochrome c release, membrane potential, redox potential, and reactive 07 species production. The influence of phosphorylation state on the ability of endogenous and exogenous Bcl-2 to protect against mitochondrial dysfunction and cell death will also be investigated. The significance of these studies is 1. They could establish the foundation for a novel neuroprotective treatment strategy targeting both necrotic and apoptotic cell death and, 2. They will provide completely new insight into the molecular mechanisms by which the antideath activities of proteins like Bcl-2 are regulated under pathological conditions.