Crosstalk between Ca2+ signaling and mitochondrial H2O2 is required for rotenone inhibition of mTOR signaling pathway leading to neuronal apoptosis.

// Chunxiao Liu 1,* , Yangjing Ye 1,* , Qian Zhou 1 , Ruijie Zhang 1 , Hai Zhang 1 , Wen Liu 1 , Chong Xu 1 , Lei Liu 2 , Shile Huang 2,3 and Long Chen 1 1 Jiangsu Key Laboratory for Molecular and Medical Biotechnology, Jiangsu Key Laboratory for Microbes and Functional Genomics, College of Life Sciences, Nanjing Normal University, Nanjing, PR China 2 Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, LA, USA 3 Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, LA, USA * These authors have contributed equally to this work Correspondence to: Long Chen, email: // Shile Huang, email: // Keywords : rotenone, apoptosis, calcium ion, CaMKII, H2O2, mTOR, Pathology Received : October 11, 2015 Accepted : January 24, 2016 Published : February 03, 2016 Abstract 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.