NEUROPROTECTIVE ACTION OF CYCLOHEXIMIDE INVOLVES INDUCTION OF BCL-2 AND ANTIOXIDANT PATHWAYS

The ability of the protein synthesis inhibitor cycloheximide (CHX) to prevent neuronal death in different paradigms has been interpreted to indicate that the cell death process requires synthesis of ''killer'' proteins. On the other hand, data indicate that neurotrophic factors p rotect neurons in the same death paradigms by inducing expression of n europrotective gene products. We now provide evidence that in embryoni c rat hippocampal cell cultures, CHX protects neurons against oxidativ e insults by a mechanism involving induction of neuroprotective gene p roducts including the antiapoptotic gene bcl-2 and antioxidant enzymes . Neuronal survival after exposure to glutamate, FeSO4, and amyloid be ta-peptide was increased in cultures pretreated with CHX at concentrat ions of 50-500 nM; higher and lower concentrations were ineffective. N europrotective concentrations of CHX caused only a moderate (20-40%) r eduction in overall protein synthesis, and induced an increase in c-fo s, c-jun, and bcl-2 mRNAs and protein levels as determined by reverse transcription-PCR analysis and immunocytochemistry, respectively. At n europrotective CHX concentrations, levels of c-fos heteronuclear RNA i ncreased in parallel with c-fos mRNA, indicating that CHX acts by indu cing transcription. Neuroprotective concentrations of CHX suppressed a ccumulation of H2O2 induced by FeSO4, suggesting activation of antioxi dant pathways. Treatment of cultures with an antisense oligodeoxynucle otide directed against bcl-2 mRNA decreased Bcl-2 protein levels and s ignificantly reduced the neuroprotective action of CHX, suggesting tha t induction of Bcl-2 expression was mechanistically involved in the ne uroprotective actions of CHX. In addition, activity levels of the anti oxidant enzymes Cu/Zn-superoxide dismutase, Mn-superoxide dismutase, a nd catalase were significantly increased in cultures exposed to neurop rotective levels of CHX. Our data suggest that low concentrations of C HX can promote neuron survival by inducing increased levels of gene pr oducts that function in antioxidant pathways, a neuroprotective mechan ism similar to that used by neurotrophic factors.