Cell-type specific cyclophilin D gene silencing in the brain

描述（由申请人提供）：线粒体功能损伤与许多神经退行性疾病有关。然而，神经元和神经胶质中的线粒体功能障碍在多大程度上导致神经退行性变尚不清楚。线粒体功能障碍可能是由亲环蛋白D （cyp-D）调节的通透性过渡孔的长时间激活引起的。我们假设cypd调节的通透性转变在神经元和星形胶质细胞的细胞损伤和死亡中起着重要但可区分的作用。本探索性研究的主要目标是产生转基因小鼠，表达神经元和胶质特异性线粒体靶向荧光蛋白，并产生用于RNA干扰研究的microRNA，以沉默亲环蛋白D基因（cyp-D-shRNA）。荧光蛋白将能够识别cypd基因沉默的细胞。这些动物将成为研究神经元和胶质线粒体功能障碍在神经系统疾病病理生理中的特殊作用的独特工具。为了探讨cyp-D在神经元和星形细胞线粒体中的病理作用，我们提出：1。产生表达荧光标记蛋白和特异性定位于神经元或星形胶质细胞的cyp-D-shRNA的转基因小鼠。2. 确定神经元或星形细胞cypd在导致细胞死亡的线粒体机制中的作用。为了实现这些目标，我们将首先在四环素反应元件的控制下，在星形细胞或神经元中产生表达黄色荧光蛋白（eYFP）和cyp-D-shRNA的转基因动物。将确定从转基因小鼠大脑或从纯神经元或星形细胞培养物中分离的线粒体对钙诱导损伤的敏感性。cyp-D在星形胶质细胞和神经元细胞死亡中的作用将通过将细胞暴露于缺氧/葡萄糖剥夺来研究。这项工作的意义在于，神经元和胶质细胞特异性cyp-D敲低小鼠将单独使用，或者与神经疾病小鼠模型杂交，以揭示导致神经变性的亚细胞机制。我们的研究还将专门探讨cypd在体外缺血再灌注模型中神经元和星形胶质细胞死亡中的作用。叙述

DESCRIPTION (provided by applicant): Impairments in mitochondrial function have been implicated in a number of neurodegenerative diseases. However, the extent to which mitochondrial dysfunction in neurons and glia contribute to neurodegeneration is unknown. Mitochondrial dysfunction can result from prolonged activation of the permeability transition pore that is regulated by cyclophilin D (cyp-D) protein. We hypothesize that the cyp-D regulated permeability transition plays important, but distinguishable roles in cellular injury and death in neurons and astrocytes. The primary goal of this exploratory study is to generate transgenic mice that express neuronal- and glial-specific mitochondrially targeted fluorescent protein and generate microRNA for use in RNA interference studies to silence the cyclophilin D gene (cyp-D-shRNA). The fluorescent protein will enable identification of the cells where the cyp-D gene was silenced. These animals will be unique tools for studying the specific roles of neuronal and glial mitochondria dysfunction in the pathophysiology of neurologic disorders. To investigate the significance of the pathological role of cyp-D in neuronal and astrocytic mitochondria, we propose to: 1. Generate transgenic mice that express a fluorescent marker protein and cyp-D-shRNA specifically localized either to neurons or astrocytes. 2. Determine the role of neuronal or astrocytic cyp-D in mitochondrial mechanisms leading to cell death. To accomplish these aims, we will first generate transgenic animals that express yellow fluorescence protein (eYFP) and cyp-D-shRNA in astrocytes or neurons, under the control of a tetracycline responsive element. Sensitivity to calcium-induced damage of mitochondria isolated either from transgenic mice brains or from pure neuronal or astrocytic cultures will be determined. The role of cyp-D in cell death of astrocytes and neurons will be examined by exposing the cells to oxygen/glucose deprivation. The significance of this work is that the neuron- and glial-specific cyp-D knockdown mice will be useful alone, or when crossed with mouse models of neurologic disorders, to reveal the subcellular mechanisms responsible neurodegeneration. Our studies will also specifically address the roles of cyp-D in neuronal and astrocyte cell death in an in vitro model of ischemia and reperfusion.Narrative Mitochondrial dysfunction is considered an underlying factor of many neurological diseases. Since brain is comprised of two major cell populations having particular functions a cell specific drug could offer a more effective treatment. Therefore, it is important to reveal the significance of potential target proteins in individual cell types for cell death mechanisms.