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 3beta (GSK3beta),
a multifunctional serine/threonine kinase, has been implicated in the pathogenesis
of neurodegeneration. We showed that rotenone activated GSK3beta by enhancing its
phosphorylation at tyrosine 216 while inhibiting phosphorylation at serine 9. Inhibitors
of GSK3beta and dominant negative (kinase deficient) GSK3beta 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
eIF2alpha as well as the expression of GRP78. Rotenone had a modest effect on the
expression of CHOP. An eIF2alpha siRNA significantly reduced rotenone cytotoxicity.
ER stress was experimentally induced by tunicamycin and thapsigargin, but tunicamycin/thapsigargin
did not activate GSK3beta in SK-N-MC cells. Down-regulation of eIF2alpha also offered
partial protection against rotenone cytotoxicity. Combined treatment of GSK3beta inhibitors
and eIF2alpha siRNA provided much greater protection than either treatment alone.
Taken together, the results suggest that GSK3beta activation and ER stress contribute
separately to rotenone cytotoxicity.