Novel delivery of Bcl-2 for neuroprotection

描述（由申请人提供）：该探索性项目的总体目标是开发一种基于蛋白转导新概念的体内神经保护新策略。该模型将用于将全长的抗死亡Bcl-2蛋白递送到大脑中，以测试Bcl-2的缺血后神经保护可以通过将外源蛋白递送到脑细胞中的现实方法来实现的假设。 第二个目的是阐明抗氧化剂与抗Bax机制在不同神经细胞死亡范例中的贡献，并研究磷酸化在调节这些机制和神经保护中的特定作用。该项目的具体目标是： 1)检验Bcl-2的抗死亡活性被体外化学缺氧和葡萄糖剥夺激活的翻译后机制抑制的假设。 2)确定与促凋亡蛋白的相互作用相比，磷酸化在调节Bcl-2保护免受由Ce加氧化应激引起的线粒体功能障碍的机制中的作用，即，Bax加BH 3死亡结构域唯一蛋白。 3)在大鼠短暂性局灶性脑缺血模型中，测试将全长Bcl-2作为TAT-Bcl-2融合蛋白递送到脑中具有保护作用的假设。 达到这些目的的方法将利用正常Bcl-2基因和在特定磷酸化位点具有突变的基因的克隆，每个基因连接到达特蛋白转导结构域。将分离TATBcl-2融合蛋白，并测试它们在体外抑制缺氧神经细胞死亡和在大鼠可逆局灶性缺血模型中减少脑梗塞体积的能力。外源Bcl-2构建体对线粒体功能障碍的影响也将使用线粒体细胞色素c释放、膜电位、氧化还原电位和反应性07物质产生的测量来评估。还将研究磷酸化状态对内源性和外源性Bcl-2防止线粒体功能障碍和细胞死亡的能力的影响。这些研究的意义在于1.他们可以建立一个新的神经保护治疗策略的基础，针对坏死和凋亡细胞死亡，2。他们将提供全新的见解的分子机制，其中蛋白质如Bcl-2的抗死亡活动在病理条件下进行调节。

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.