Crosstalk between Ca(2+) signaling and mitochondrial H(2)O(2) is required for rotenone inhibition of mTOR signaling pathway leading to neuronal apoptosis

Rotenone, a neurotoxic pesticide, induces loss of dopaminergic neurons related to Parkinson's disease. Previous studies have shown that rotenone induces neuronal apoptosis partly by triggering hydrogen peroxide (H(2)O(2))-dependent suppression of mTOR pathway. However, the underlying mechanism is not fully understood. Here, we show that rotenone elevates intracellular free calcium ion ([Ca(2+)](i)) level, and activates CaMKII, resulting in inhibition of mTOR signaling and induction of neuronal apoptosis. Chelating [Ca(2+)](i) with BAPTA/AM, preventing extracellular Ca(2+) influx using EGTA, inhibiting CaMKII with KN93, or silencing CaMKII significantly attenuated rotenone-induced H(2)O(2) production, mTOR inhibition, and cell death. Interestingly, using TTFA, antimycin A, catalase or Mito-TEMPO, we found that rotenone-induced mitochondrial H(2)O(2) also in turn elevated [Ca(2+)](i) level, thereby stimulating CaMKII, leading to inhibition of mTOR pathway and induction of neuronal apoptosis. Expression of wild type mTOR or constitutively active S6K1, or silencing 4E-BP1 strengthened the inhibitory effects of catalase, Mito-TEMPO, BAPTA/AM or EGTA on rotenone-induced [Ca(2+)](i) elevation, CaMKII phosphorylation and neuronal apoptosis. Together, the results indicate that the crosstalk between Ca(2+) signaling and mitochondrial H(2)O(2) is required for rotenone inhibition of mTOR-mediated S6K1 and 4E-BP1 pathways. Our findings suggest that how to control over-elevation of intracellular Ca(2+) and overproduction of mitochondrial H(2)O(2) may be a new approach to deal with the neurotoxicity of rotenone.