As detailed in the preliminary data, we have demonstrated that mishandling of dopamine viareduced VMAT2 expression causes a progressive loss of dopamine terminals and cell bodies in thesubstantia nigra, synuclein aggregation, and L-DOPA responsive behavioral deficits, making the VMAT2deficient mice a unique progressive model of PD. WE have shown that several environmental toxicants alterVMAT2 function. We hypothesize that the reduced ability to store dopamine causes oxidative stress in thepresynaptic dopamine neuron, damage to cellular constituents, and the death of the neuron. Thus, thesxidative damage that occurs following improper storage of dopamine may underlie the disease process.We have recently shown that the redox state (dynamic balance between reduced and oxidized components)of neurons can be spatially resolved by subcellular compartment in that compounds used in animal modelsof Parkinson's disease can preferentially oxidize cytoplasmic, mitochondria!, or nuclear redox components,such as thioredoxin (Trx) or glutathione (GSH). Thus, the perturbation of redox state by altered dopaminehomeostasis may be a key mediator of toxicity. Given the critical role of VMAT2, a-synuclein, and redoxstate the dopamine system, the goal of this project is to identify the interactions among these threecomponents in order to better understand the underlying pathogenic processes that mediate cellular damagen Parkinson's disease. Hypothesis: Altered compartmentalization of dopamine disrupts the redox balance ofthe neuron resulting in enhanced vulnerability of the dopamine neuron Aim 1. To determine the role of asynucleinin regulation of the localization and function of VMAT2. We have preliminary evidence of a directregulatory interaction of VMAT2 and a-synuclein. In this aim we will determine the nature of this molecularand functional interaction using site-directed mutagenesis, confocal microscopy, and vesicular uptakeassays.
Aim 2. t To determine if a-synuclein expression alters the vulnerability of dopamine-mediated injurydue to reduced VMAT2 expression. By harvesting midbrain dopamine neurons from animals with differentialexpression of VMAT2 and a-synuclein, we will examine the effects of increased cytoplasmic dopamine anda-synuclein on dopamine-mediated cell death.
Aim 3. To determine the effects of reduced or increasedexpression of VMAT2 on vulnerability to exogenous dopamine or MPTP.
Aim 4 : To expand our currentkinetic-dynamic model of dopamine metabolism to encompass targets of environmental toxicants. We willextend our model to account for the oxidative stress that occurs with mitochondria! dysfunction and theimpact of a-synculein on key components of the dopamine system. Completion of the above specific aimswill improve our mechanistic understanding of how environmental and genetic factors disrupt proper storageof dopamine and contribute to the pathogenesis of Parkinson's disease
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