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.
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