Novel delivery of Bcl-2 for neuroprotection

用于神经保护的 Bcl-2 新型递送

• 批准号: 6683609
• 负责人: GARY M FISKUM
• 金额: $ 17.63万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-12-01 至 2005-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6683609
• 关键词: BCL2 gene /protein; antioxidants; apoptosis; brain cell; calcineurin; calcium ion; cerebral ischemia /hypoxia; chimeric proteins; cytochrome c; disease /disorder model; enzyme inhibitors; free radical oxygen; glucose; laboratory rat; mitochondria; neuroprotectants; oxidative stress; phosphorylation; posttranslational modifications; protein transport; technology /technique development

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.

描述（由申请人提供）：该探索性项目的总体目标是根据蛋白质转导的新概念制定一种新型的体内神经保护策略。该模型将用于将全长的抗死Bcl-2蛋白传递到大脑中，以检验以下假设：通过将外源性蛋白输送到脑细胞中的现实方法可以实现Bcl-2的缺血后神经保护。 第二个目标是阐明在不同神经细胞死亡范式中抗氧化剂与抗BAX机制的贡献，并研究磷酸化在调节这些机制和神经保护中的特定作用。该项目的具体目的是： 1）检验以下假设：在体外对化学缺氧和葡萄糖剥夺而激活的翻译后机制抑制了Bcl-2的抗死活性。 2）确定磷酸化的作用在调节Bcl-2防止CE和氧化应激引起的线粒体功能障碍的机制中，与促凋亡蛋白的相互作用相比，即Bax Plus BH3死亡域仅蛋白质。 3）检验以下假设：在大鼠短暂性局灶性局灶性脑缺血模型中，全长Bcl-2作为TAT-BCL-2融合蛋白的递送为大脑具有保护性。 这些目标的方法方法将利用特定磷酸化位点中的正常Bcl-2基因和具有突变的基因克隆，每个基因都与TAT蛋白转导域相连。 TATBCL-2融合蛋白将被分离并测试其在体外抑制缺氧神经细胞死亡并减少大鼠可逆局灶性缺血模型中脑梗塞体积的能力。外源Bcl-2构建体对线粒体功能障碍的影响还将使用线粒体CVTOCHROME C释放，膜电位，氧化还原电位和反应性07种物种产生进行评估。还将研究磷酸化状态对内源性和外源性Bcl-2预防线粒体功能障碍和细胞死亡的能力的影响。这些研究的意义是1。它们可以为针对坏死和凋亡细胞死亡的新型神经保护治疗策略建立基础，2。它们将在病理条件下对蛋白质（如Bcl-2）进行抗蛋白质的抗毒素活性的分子机制提供全新的见解。