Separation of brain glial and neuronal mitochondria

描述（由申请人提供）：脑线粒体的研究因中枢神经系统的区域、细胞和亚细胞异质性而变得复杂。本探索性研究的目标是产生能够在线粒体中表达细胞特异性荧光蛋白的转基因小鼠，并利用这些动物开发一种独特的技术，从脑匀浆中分离神经元和胶质线粒体。这种技术将使研究不同线粒体种群在神经系统疾病机制中的作用成为可能。根据我们的初步数据，我们建议：产生转基因小鼠，表达神经元或星形细胞线粒体特异性荧光标记蛋白。2. 建立从脑匀浆中分离神经元和胶质线粒体的分离技术。利用Percoll梯度离心可以从脑匀浆中分离出神经元和胶质来源的线粒体。在实现这些目标的过程中，我们将首先产生在星形细胞中表达黄色荧光蛋白（EYFP）和在神经元线粒体中表达红色荧光蛋白（HcRed1）的转基因动物。这些蛋白的表达将受到四环素反应元件的控制。这些动物的大脑将用于改进我们的星形细胞和神经元线粒体的分离技术。为了检测胶质线粒体富集的分离组分，我们将确定EYFP的存在，并通过HcRed1鉴定神经元线粒体。EYFP和HcRed1的表达将与内源性细胞特异性线粒体标记物的存在相关。丙酮酸羧化酶和普遍存在的线粒体肌酸激酶将分别用于鉴定星形细胞和神经元线粒体。这些酶对分离组分的富集将通过免疫印迹和酶活性测定来确定。作为对照，从转基因动物获得的纯原代神经元和星形细胞培养物中分离的线粒体将被使用。利用氮空化新技术分离细胞线粒体。

DESCRIPTION (provided by applicant): Studies of brain mitochondria are complicated by the regional, cellular and subcellular heterogeneity of the central nervous system. The goal of this exploratory study is to generate transgenic mice that will express cell specific fluorescent proteins in mitochondria and use these animals to develop a unique technique for separating neuronal and glial mitochondria from brain homogenate. Such a technique would make studying the role of different mitochondrial population in the mechanisms of neurological disease possible. Based on our preliminary data we propose to: 1. Generate transgenic mice that will express fluorescent marker proteins specific either to neuronal or to astrocytic mitochondria. 2. Establish isolation techniques that will separate neuronal and glial mitochondria from brain homogenate. Mitochondria of neuronal and glial origin can be separated from brain homogenate by using Percoll gradient centrifugation. In the approach of these aims we will first generate transgenic animals that will express yellow fluorescent protein (EYFP) in astrocytic and red fluorescent protein (HcRed1) in neuronal mitochondria. The expression of these proteins will be under the control of tetracycline responsive element. Brains of these animals will be used to improve our separation technique of astrocytic and neuronal mitochondria. For examining the enrichment of separated fractions by glial mitochondria we will determine the presence of EYFP and neuronal mitochondria will be identified by HcRed1. The expression of EYFP and HcRed1 will be correlated with the presence of endogenous cell specific mitochondrial markers. Pyruvate carboxylase and ubiquitous mitochondrial creatine kinase will be used to identify astrocytic and neuronal mitochondria, respectively. Enrichment of separated fractions by these enzymes will be determined by both immunoblotting and enzyme activity assays. As a control, mitochondria isolated from pure primary neuronal and astrocytic cell culture obtained from transgenic animals will be employed. Cell mitochondria will be isolated by using a new technique of nitrogen cavitation.