The long-term goal of this research is to understand at the molecular level the factors involved in the tissue-specific expression of neuronal genes. In this application the investigators propose to use this information in conjunction with advances in molecular genetics to create animal models of Parkinson's Disease (PD) in which gene expression is spatially and temporally controlled. A bottleneck in taking full advantage of these powerful tools however, lies in the lack of appropriate promoters to target genetic switches to specific populations of neurons. Thus, a prerequisite for using genetic manipulation to unravel brain function is the development of new tools to target brain regions of interest. Because preliminary experiments indicate that the reporter gene, eGFP, can be targeted to the tyrosine hydroxylase (TH) locus where it is co-expressed with endogenous TH, the investigators have hypothesized that the TH locus can be combined with inducible elements to generate a targeted switch in vivo. If subsequent experiments show that catecholaminergic properties have not been changed in the TH/eGFP heterozygotes, the tetracyline (TET) regulatory system transactivator derivative, rtTA2s-M2, will be swapped for eGFP using homologous recombination. TH/TET transactivator lines will be evaluated by crossing with TET-responsive reporter gene mouse lines. If reporter genes are appropriately regulated, these animals will serve as an invaluable resource for turning gene expression on and off in catecholaminergic cells. A second major conceptual goal is to use this resource to develop an animal model of Parkinson's disease. Defects in the ubiquitin-proteosome pathway have been genetically linked to Parkinson's disease including a loss of function of the E3 ligase, Parkin. The co-chaperone CHIP is a positive regulator of Parkin mediating its ability to ubiquitinate its substrates. Dominant-negative (DN) CHIP derivatives block endogenous CHIP activity contributing to impaired proteosome function, increased levels of aberrant proteins, and unfolded protein response. In order to test the hypothesis that overexpression of DN-CHIP in catecholaminergic cell types can lead to an animal model of Parkinson's Disease, transgenic lines carrying a TET-responsive DN-CHIP minigene will be generated and crossed with the TH-driven TET transactivator. Offspring will be evaluated for induction of unfolded protein response (BiP, CHOP10, caspase 12 induction) and the loss of catecholaminergic cells. Conceivably, these animals will serve as a useful model of Parkinson's disease and as an aid in determining the underlying mechanisms of this response. Knowledge gained from these studies may offer new routes for therapeutic intervention.
