Monocyte-Mediated Rotenone Neurotoxicity towards Human Neuroblastoma SH-SY5Y: Role of Mitogen-Activated Protein Kinases

Abstract

Increasing evidence has suggested an important role for rotenone in the pathogenesis of Parkinson's disease (PD). In this report, sequential linking of two culture systems, monocytic THP-1 cell line and SH-SY5Y neuroblastoma, was utilized. The supernatant from rotenone-stimulated THP-1 cells was used as the incubating medium for the second culture which adopted cells of the SH-SY5Y neuroblastoma. At 6.25—50 nM, concentrations that were nontoxic to SH-SY5Y directly, rotenone induced dose-dependent cell death on SH-SY5Y through stimulating monocyte THP-1 within a period of 48 h. Cytotoxicity was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Hoechst 33258 staining revealed that the treatment of SH-SY5Y with rotenone-stimulated THP-1 supernatant resulted in condensed nuclei and a decrease in cell size. Apoptotic rate measured by flow cytometric analysis indicated that at 25 and 50 nM, the percentage of apoptotic SH-SY5Y cells accumulated to 31.5% and 37.0% respectively. We further investigated whether rotenone (50 nM) activated mitogen-activated protein kinase (MAPK) cascades, and found it had effect on p38 MAPK and ERK in THP-1 cells, but not JNK. Pretreatment of THP-1 cells with the MAPK kinase inhibitor, PD98059, inhibited THP-1 cell-mediated rotenone neurotoxicity towards SH-SY5Y, whereas the p38 MEK inhibitor, SB203580, had no effect. These results suggested that activation of microglia intracellular signaling pathway may also involve in microglia-enhanced rotenone neurotoxicity.