Alvarez, D., Sullivan, P., Holmes, C., Jinsmaa, Y., Cooney, A., Goldstein, D.S.
Clinical Neurocardiology Section, CNP/DIR/NINDS/NIH
Background: Parkinson’s Disease (PD) is characterized by progressive loss of dopaminergic neurons in the nigrostriatal system. A histopathologic hallmark of PD is Lewy bodies in substantia nigra neurons. These inclusions contain an abundance of the protein alpha-synuclein. Alpha-synuclein is implicated in the pathogenesis of PD, based on rare families with inherited PD from genotypic abnormalities of the alpha-synuclein gene and from genome-wide association studies. Catecholamine neurons are rare in the nervous system, and why they are lost relatively specifically remains mysterious. One possibility is that alpha-synuclein over-expression interferes with vesicular sequestration of cytosolic catecholamines, resulting in a buildup of toxic catecholaldehydes by the action of monoamine oxidase in the outer mitochondrial membrane. We recently found evidence that patients with Lewy body diseases do have decreased vesicular uptake, but whether this is from alpha-synucleinopathy has been unknown.
Objective: In this study we examined whether alpha-synuclein over-expression impedes vesicular or extra-vesicular storage of cytosolic catecholamines, by tracking the fate of exogenous 6F-dopamine in rat pheochromocytoma PC12 cells engineered to over-express alpha-synuclein conditionally in a doxycycline-dependent manner. Since reserpine blocks vesicular uptake and drastically decreases cell 6F-dopamine content (by more than 95%) in other PC12 cells, residual 6F-dopamine in reserpine-treated cells in the present study indicated extra-vesicular storage.
Methods: PC12 cells conditionally over-expressing alpha-synuclein were grown in medium with or without added doxycycline. Absence of doxycycline enables alpha-synuclein over-expression. The cells were incubated for 3 hours with 6F-dopamine, with or without reserpine.
Results: Reserpine-treated PC12 cells used in this experiment contained much higher levels of dopamine and 6F-dopamine than did other PC12 cells previously studied. Absence of doxycycline was not associated with changes in the amount of endogenous or 6F-dopamine.
Conclusions: PC12 cells engineered to conditionally over-express alpha-synuclein have an ability to store catecholamines and both vesicular and extra-vesicular sites. Alpha-synuclein over-expression does not inhibit vesicular uptake or extra-vesicular storage of catecholamines
Last updated December 14, 2012