Cell-type specific cyclophilin D gene silencing in the brain

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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.