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Character Analysis Of VMAT2 LO Mice

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C. APPROACH.
C.1. Mouse PD model to be used in this project: In VMAT2 LO, a transgenic mouse PD model, shut-down of 95% endogenous vesicular monoamine transporter 2 (VMAT2) leads to a massive oxidative deamination of monoamine neurotransmitters [43], accompanied by increased oxidative stress markers and a significant decrease of antioxidant ability [44]. Therefore, oxidative damage could be the culprit for the replication of the pathogenic features of PD [45], including substantial reductions of DA and NE levels in the brain, progressive neurodegeneration in the SNpc and LC with formation of α–synuclein containing inclusions. More importantly, neuronal loss in the LC of VMAT2 LO mice starts earlier (at 12 months) and to a greater extent
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To address this possibility, SK-N-BE(2)M17 and MN9D cells will be exposed to 50, 100, and 200 µM H2O2 for 4 days (concentration course) or 200 µM H2O2 for 1, 2 and 4 days (time course). After several sets of exposures, cells will be harvested for different measurements: intracellular ROS by DCFH2-DA assay, reduced form of glutathione (GSH) by the fluorescent probe monochlorobimane assay, levels of Cu/Zn superoxide dismutase 1 (SOD1) by western blotting and cell viability (MTT assay), respectively. Similar experiments and measurements will be carried out in primary cultures from the mouse ventral mesencephalon and LC to verify the findings in cell lines.
Anticipated results and interpretation: Based on our primary experiments, increased ROS levels and a reduced viability will be expected in noradrenergic neurons, indicating a higher vulnerability of noradrenergic neurons compared to DAergic neurons. Although most ROS are membrane permeant, O2•− does not generally cross cell membranes readily [52]. As the DCFH2-DA assay mainly measures O2•− levels [53], this methodology will provide accurately reflect the right ROS levels in the cells. We will also measure levels of the antioxidants SOD1, which catalyzes O2•− into O2 [54] and GSH. We expect levels of SOD1 and GSH will be lower in noradrenergic than DAergic neurons, as reduced antioxidant levels are always concomitantly
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