Parkinson's disease (PD) is characterized by a preferential loss of nigrostriatal dopaminergic neurons, and the resultant depletion of dopamine innervation to the striatum is believed to be responsible for the hallmark symptoms of PD. The goal of this study is to test the hypothesis that regulation of the plasma membrane dopamine transporter (DAT) and the neuronal vesicular monoamine transporter (VMAT2) proteins mediate cellular susceptibility in experimental models of PD. It has been proposed that the varying susceptibility observed among different dopaminergic cell groups in idiopathic PD and in MPTP-induced parkinsonism is governed by the relative concentrations of DAT and VMAT2 protein. Specifically, the ratio of DAT to VMAT2, rather than the absolute levels of proteins, controls the cytosolic levels of the putative toxin and thus cellular susceptibility. Here, the applicants capitalize on in vitro and in vivo models recently developed in their laboratories using stable neuronal cell lines and transgenic animals expressing different levels of these transporters to further understand how DAT and VMAT2 expression participate in normal physiological function, as well as disease susceptibility. Therefore, they will test the following hypotheses: Hypothesis I: Stable neuronal cell lines expressing high ratios of DAT to VMAT2 will display increased vulnerability to the parkinsonism-inducing neurotoxin MPP+. Hypothesis II. Transgenic mice expressing high DAT to VMAT2 ratios will exhibit increased susceptibility to MPTP-induced parkinsonism. To test these hypotheses, the applicants propose the following aims:
Specific aim 1. Produce stable neuron cell lines that express different ratios of DAT/VMAT2 and determine their susceptibility to MPP+.
Specific aim 2. Examine the mechanisms by which MPP+ causes cellular damage in stable cell lines expressing different ratios of DAT/VMAT2 using microphysiology, fluorometry, and confocal microscopy.
Specific aim 3. Assess dopamine function and MPTP susceptibility in transgenic mice expressing different levels of DAT and VMAT2 protein. The collective expertise of the participating laboratories will allow successful completion of these aims and thorough testing of these hypotheses, providing crucial data regarding how these transporters regulate both the normal function of dopaminergic neurons and the vulnerability of isolated cells in vitro and in intact animals, furthering our understanding of their role in the pathogenesis of PD. Furthermore, the tools and methods developed in this study will be invaluable to researchers studying other disorders in which monoaminergic function is perturbed, such as cocaine and amphetamine abuse, schizophrenia and Tourette syndrome.
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