Regulation of DNA double-strand break repair by parkin

Mutations in the E3 ubiquitin ligase parkin cause an autosomal recessive juvenile form of Parkinsonism. Parkinson's disease (PD) is characterized clinically by a classic triad of symptoms including resting tremor, bradykinesia, and muscle rigidity. These symptoms derive from a profound loss of dopaminergic (DA) neurons within the substantia nigra pars compacta. The precise molecular mechanisms underlying the degeneration of DA neurons in patients with parkin mutations remain unknown. An attractive hypothesis is that loss of parkin function leads to a deleterious accumulation of one or more substrates normally targeted for proteasome degradation. This dissertation provides another hypothesis for the mechanism of disease by demonstrating a novel function for the E3 ubiquitin ligase parkin in regulating the response to DNA double-strand breaks (DSBs). Specifically, parkin-deficient ES cells are found to have increased levels of γH2AX--a histone variant phosphorylated in response to DNA DSBs--and sensitivity to genotoxic stress. Parkin over-expression decreases the levels of γH2AX induced by DNA damaging agents in a variety of cell types, including primary neurons. This effect appears to result from parkin-mediated stimulation of DSB repair activity. A disease-causing mutation in parkin abrogates this DNA repair function. In addition, over-expression of parkin diminishes the induction of a DNA damage response and promotes survival in cells exposed to genotoxic agents. Although proteasome activity is not necessary for the mechanism by which parkin functions, the nonhomologous end-joining pathway of DSB repair is required. Finally, parkin-deficient mice display elevated levels of γH2AX in the substantia nigra pars compacta region. There is also increased γH2AX in DA neurons of the substantia nigra in a patient with Parkinsonism caused by mutations in parkin. These results depict a novel in vivo role for parkin in regulating DNA DSB repair and suggest that loss of this function may play an important role in the pathogenesis of Parkinson's disease.