Rotenone, a naturally occurring, lipophilic compound from the roots of certain plants (Derris species) is used as the main component of many insecticides. Increasing evidence has suggested an important role for rotenone in the pathogenesis of Parkinson's disease (PD). Mechanistically, rotenoneinduced dopaminergic neurodegeneration has been associated with both its inhibition of neuronal mitochondrial complex I and the enhancement of activated microglia. [1][2][3][4][5] The activation of glia, particularly microglia is the hallmark of brain inflammation. Recently, increasing evidences from human and animal studies have suggested that neuroinflammation is an important contributor to the neuronal loss in PD. 3,[6][7][8][9] In addition, inhibition of the inflammatory reaction could attenuate degeneration of nigrostriatal dopaminecontaining neurons in several models of PD.10) Previous studies have demonstrated that microglia-enhanced rotenone neurotoxicity can be attributed to the release of NADPH oxidase-derived superoxide from activated microglia and in turn the oxidation of dopamine (DA) to the o-quinone dopaminochrome (DACHR) extracellularly. 4,11) In these cultures, superoxide was considered a dominant degenerative factor for the dopaminergic neurons. However, given that SOD and catalase afforded only a partial neuroprotection and microglia also have the ability to release pro-inflammatory and neurotoxic factors, 3,12,13) exact mechanisms underlying the role of inflammatory response involved in rotenone-induced PD remain to be identified. Though several approaches have been used to examine direct neuron-glia interactions under controlled in vitro conditions, 3,14) in these primary coculture systems it is nearly impossible to study the intracellular events in pure microglia and dopaminergic neuron and the sequence of microglia-activation and DA neurotoxicity can not be clarified specifically. As a result, the potential implications of these two cells can not be validated precisely.Recent evidence indicates that cellular redox state promi- In the present study, we adopted two consequential linking cell systems to study the mechanisms underlying the microglia-enhanced rotenone neurotoxicity. The supernatant from rotenone-stimulated human monocytic THP-1 line was then used as the culture medium for SH-SY5Y neuroblastoma which was used as target neuronal cells. Human postmortem microglia behaved similarly to THP-1 cells. 18,19) We further investigated whether rotenone can regulate the intracellular signaling of monocytic THP-1 cells. MATERIALS AND METHODS MaterialsDulbecco's modified Eagle's medium (DMEM-F12) was purchased from GibcoBRL (Gaithersburg, MD, U.S.A.). Fetal calf serum was obtained from Hyclone (Logan, UT, U.S.A). Rotenone, propidium iodide (PI), 3-(4,5-dimethylthiazal-z-yl)-2,5-diphenylterazolium (MTT), chromatin dye bisbenzimide (Hoechst 33258), RNase were purchased from Sigma (St. Louis, MO, U.S.A). Antibodies for phospho-ERK (Thr202/Tyr204), phospho-JNK (Thr183/ Tyr185), phospho-p38 (Thr180/Tyr182) we...

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