Stress-induced phospho-ubiquitin formation causes parkin degradation

Lyudmila Kovalchuke; Eugene V. Mosharov; Oren A. Levy; Lloyd A. Greene

L-DOPA can generate reactive oxygen species (ROS) via non-enzymatic autoxidation and by conversion to dopamine, with subsequent autoxidation and oxidative metabolism of the latter [53] , [62] , [63] . To test whether conversion of L-DOPA to dopamine is required for its effect on parkin, we used carbidopa to block the activity of aromatic L-amino acid decarboxylase (AADC), the enzyme responsible for this conversion. As anticipated, 200 µM L-DOPA treatment alone for 16-24 hours significantly increased intracellular dopamine levels (to 3.0 ± 0.4 fold over control levels, p = 0.02, N = 5) (Fig. 2B ). Co-treatment with carbidopa robustly diminished this effect (to 1.1 ± 0.3 fold over control levels, p = 0.02 vs. L-DOPA alone, N = 5) but did not impact the loss of parkin from L-DOPA treatment (30.9 ± 5.9% parkin loss with L-DOPA alone vs. 31.4 ± 4.7% loss with L-DOPA + carbidopa, p = 0.9, N = 4) (Fig. 2C) . These results suggest that autoxidation of L-DOPA itself, rather than its conversion to dopamine, is the source of the oxidative stress that leads to parkin loss following L-DOPA treatment in our system. Consistent with this possibility, we observed "browning" of the cell culture medium after 8 24 hours of exposure to L-DOPA, even in the absence of cells (Fig. S6) , which has been shown to be a result of quinone formation from L-DOPA autoxidation [61] , [64] [65] [66] .