Mutations in parkin underlie an autosomal recessive form of Parkinson's disease, the second most common neurodegenerative disease. To test a working model of parkin as a component of a multi-subunit, SCF-like ubiquitin ligase complex that protects dopamine neurons from apoptosis, other components of the complex, including sel-10 and cullin-1, will be dowregulated by RNA interference in murine primary neuronal cultures. Downregulation of these components will be evaluated for potentiation of dopamine neuron apoptosis and compared to the effects of downregulating parkin itself. To test the hypothesis that a candidate substrate of the parkin-associated complex, cyclin E, is a key mediator of the apoptotic cascade(s) against which wildtype parkin normally protects neurons, pharmacological inhibition of cyclin E-associated activity will be evaluated for rescue of dopamine neurons in the context of parkin, sel-10, or cullin-1 downregulation. Finally, lentivirus-mediated overexpression of parkin in the same primary culture system will be assessed for protection of dopamine neurons from neurotoxins as compared to overexpression of mutant forms of parkin, including clinically defined mutations and forms deleted in the ubiquitin homology and RING domains.
