Stress-induced phospho-ubiquitin formation causes parkin degradation

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

A recent study found that phospho-Ub levels are elevated in the substantia nigra of patients with Lewy body disease, a category that includes PD [128] . Given our findings, this observation suggests that parkin levels may be lowered in these patients due to enhanced phospho-Ub-induced turnover. This decrease could in turn contribute to PD pathology. As such, finding ways to uncouple parkin's association with phospho-Ub from its degradation may be an attractive therapeutic avenue for PD treatment. To differentiate PC12 cells, undifferentiated cells from one 10-cm dish were first resuspended in 4mL RPMI with 1% horse serum and 1% penicillin-streptomycin. Then, cells were passed through a 5-mL repeat pipettor tip to disperse them, counted using a hemacytometer, and plated at a density of ~3x10 5 cells/mL (1mL/well in a 12-well plate) in RPMI with 1% horse serum, 1% penicillin-streptomycin, and 50 ng/mL human recombinant NGF (kind gift of Genentech, Gemini Bio-Products #300-174P). The remaining undifferentiated PC12 cells were plated on a freshly-coated 10 cm dish. Cells were used in experiments after 5-10 days of differentiation. In experiments with lentiviral transduction, cells were transduced after 3-5 days of differentiation; for shRNA constructs, cells were treated with drugs 4-5 days after transduction; for overexpression constructs, cells were treated with drugs 3-5 days after transduction.