GSK3β and endoplasmic reticulum stress mediate rotenone-induced death of SK-N-MC neuroblastoma cells

Rotenone, an environmental toxin that inhibits mitochondrial complex I, has been used to induce experimental Parkinsonism in animals and cell cultures. We investigated the mechanism underlying rotenone-induced death of SK-N-MC neuroblastoma cells. Rotenone-induced cell death preceded intracellular accumulation of reactive oxygen species, and antioxidants failed to protect cells, indicating that oxidative stress was minimally involved in rotenone-induced death of SK-N-MC cells. Glycogen synthase kinase 3 beta (GSK3 beta), a multifunctional serine/threonine kinase, has been implicated in the pathogenesis of neurodegeneration. We showed that rotenone activated GSK3 beta by enhancing its phosphorylation at tyrosine 216 while inhibiting phosphorylation at serine 9. Inhibitors of GSK3 beta and dominant negative (kinase deficient) GSK3 beta partially protected SK-N-MC cells against rotenone cytotoxicity. Rotenone also induced endoplasmic reticulum (ER) stress which was evident by an increase in phosphorylation of PERK, PKR, and eIF2 alpha as well as the expression of GRP78. Rotenone had a modest effect on the expression of CHOP. An eIF2 alpha siRNA significantly reduced rotenone cytotoxicity, ER stress was experimentally induced by tunicamycin and thapsigargin, but tunicamycin/thapsigargin did not activate GSK3 beta in SK-N-MC cells. Down-regulation of eIF2 alpha also offered partial protection against rotenone cytotoxicity. Combined treatment of GSK3 beta inhibitors and eIF2 alpha siRNA provided much greater protection than either treatment alone. Taken together, the results suggest that GSK3 beta activation and ER stress contribute separately to rotenone cytotoxicity. (c) 2008 Elsevier Inc. All rights reserved.