Cell-type specific cyclophilin D gene silencing in the brain

描述(由申请人提供)：线粒体功能受损与许多神经退行性疾病有关。然而，神经元和神经胶质细胞中的线粒体功能障碍在多大程度上促进了神经退行性变尚不清楚。线粒体功能障碍可能是由亲环素D(CYP-D)蛋白调节的通透性转换孔的长时间激活引起的。我们推测，CYP-D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。这项探索性研究的主要目标是建立表达神经元和神经胶质特异性线粒体靶向荧光蛋白的转基因小鼠，并产生用于RNA干扰研究的microRNA，以沉默亲环素D基因(CYP-D-shRNA)。这种荧光蛋白将能够识别CYP-D基因沉默的细胞。这些动物将是研究神经元和神经胶质线粒体功能障碍在神经疾病病理生理学中的具体作用的独特工具。为了研究CYP-D在神经元和星形胶质细胞线粒体中的病理作用，我们建议：1.建立表达荧光标记蛋白和CYP-D-shRNA的转基因小鼠，其定位于神经元或星形胶质细胞。2.确定神经元或星形细胞CYP-D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，我们首先将在四环素反应元件的控制下，产生在星形胶质细胞或神经元中表达黄色荧光蛋白(EYFP)和CYP-D-shRNA的转基因动物。无论是从转基因小鼠的大脑中分离出来的线粒体，还是从纯神经元或星形胶质细胞培养中分离出来的线粒体，都将被确定对钙诱导的损伤的敏感性。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在缺氧/葡萄糖剥夺中来检测。这项工作的意义在于，神经元和神经胶质特异性的CYP-D基因敲除小鼠将单独有用，或者当与神经系统疾病的小鼠模型杂交时，将有助于揭示导致神经退行性变的亚细胞机制。我们的研究还将在体外脑缺血和再灌流模型中专门探讨CYP-D在神经元和星形胶质细胞死亡中的作用。 线粒体功能障碍被认为是许多神经系统疾病的潜在因素。由于大脑由两个具有特定功能的主要细胞群组成，因此针对细胞的药物可以提供更有效的治疗方法。因此，揭示单个细胞类型中潜在的靶蛋白对于细胞死亡机制的重要性是很重要的。

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.