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A Method to Examine Intracellular Vesicular Uptake of Cytosolic Catecholamines

2011 Exceptional Summer Student Award Winner Rachel Sullivan Rachel Sullivan

Parkinson disease (PD) is a relatively common progressive neurodegenerative disorder of the elderly. A pathological hallmark of PD is Lewy bodies, cytosolic inclusions that contain abundant aggregates of the protein alpha-synuclein, and a neurochemical hallmark of PD is depletion of the catecholamine, dopamine (DA), in the striatum; however, the relationship between synucleinopathy and death of dopaminergic neurons remains poorly understood. PD is also characterized by decreased concentrations of the closely related catecholamine, norepinephrine (NE) in the brain and heart. Cardiac noradrenergic denervation has been demonstrated in studies using 18F-DA positron emission tomographic scanning. We recently obtained clinical laboratory evidence for decreased vesicular uptake of cytosolic catecholamines in Lewy body diseases including PD. In this project we developed an analogous method using tracer amounts of F-DA, to examine the fate of intracellular catecholamines and in particular to measure vesicular uptake in vitro. The method relies on the fact that DA-beta-hydroxylase, the enzyme converting DA to NE, is localized to vesicles. Formation of F-NE from F-DA therefore requires and provides a measure of vesicular uptake. To validate the method we examined the metabolic fate of F-DA in rat pheochromocytoma PC-12 cells exposed to reserpine, which rapidly, irreversibly blocks the vesicular monoamine transporter. Reserpine depleted endogenous DA content by 90% in 3 hours and completely prevented formation of F-NE but did not prevent formation of F-dihydroxyphenylacetic acid, a product of cytosolic F-DA metabolism that does not require vesicular uptake. The results indicate that using the exogenous tracer, F-DA, is an effective means to assess vesicular uptake in catecholaminergic cells. The method can now be applied to evaluate effects of over-expression of wild-type or mutant alpha-synuclein. Because of in vivo information demonstrating decreased vesicular uptake in patients with Lewy body diseases, having an in vitro means of assessing vesicular uptake will be important

Last updated December 23, 2013