Studies On Mechanisms Of Rotenone-induced Apoptosis Via Calcium Signaling Inhibition Of MTOR Pathway In Neuronal Cells

The present study, using cell culture, fluorescent staining for [Ca2+]i, DAPI staining for cell apoptosis, Western blotting, etc., systematically investigated change of intracellular free [Ca2+]i and its effect on mTOR signaling pathway during progress of rotenone-induced neuronal apoptosis, discussed the regulative role of rotenone triggering neuronal apoptosis and mechanism of [Ca2+]i elevation, and demonstrated signaling molecular mechanisms of CaM in rotenone-elevated [Ca2+]i contributing to neuronal apoptosis. The detailed results were summarized as follows:1Rotenone-induced [Ca2+]i elevation is involved in inhibition of mTOR pathway and apoptosis in neuronal cellsPC12cells and primary neurons were chosen and treated with different dose of rotenone (0,0.05,0.1,0.3,0.5,1μM) for24h, or exposed to rotenone (0.5and1μM) for4or24h following pretreatment with/without BAPTA/AM (30μM) for1h. Fluo-3/AM, a fluorescent probe, was used to evaluate fluorescent intensity of [Ca2+]i, Cell viability and apoptosis was assayed using One Solution and DAPI staining, respectively. Meanwhile, the effect of BAPTA/AM on mTOR signaling pathway during progress of rotenone-induced neuronal apoptosis were detected by Western blotting. The results showed that rotenone triggered [Ca2+]i elevation contributing to neuronal cell apoptosis in a concentration-dependent manner, which was associated with inhibition of mTOR and its mediation of S6K1and4E-BP1pathways. BAPTA/AM significantly attenuated rotenone-induced apoptosis in PC12cells and primary neurons via blocking inhibition of mTOR pathway. These findings suggest that rotenone-induced [Ca2+]i elevation is involved in inhibition of mTOR pathway and apoptosis in neuronal cells.2Mechanism of rotenone-induced [Ca2+]i elevation and its relation to inhibition of mTOR pathway involved in neuronal apoptosisEndoplasmic reticulum IP3receptor (IP3R) inhibitor2-APB and calcium chelator EGTA were chosen to study whether extracellular and/or intracellular calcium participated in rotenone-induced [Ca2+]i elevation. PC12and primary neurons, seeded in6-and96-well plates, were exposed to rotenone (0.5and1μM) for4or24h following pretreatment with/without2-APB (100μM) or EGTA (100μM) for1h, respectively. Cell viability was assayed using One Solution. Fluo-3/AM, a fluorescent probe, was used to evaluate effect of2-APB or EGTA on [Ca2+]i, and using DAPI staining, protective effect of2-APB or EGTA on rotenone-induced neuronal apoptosis was observed. Meanwhile, the effect of2-APB or EGTA on neuronal mTOR signaling pathway after2-APB or EGTA blockage of intracellular calcium store release and extracellular calcium source were detected by Western blotting. The results showed that2-APB or EGTA obviously decreased rotenone-induced [Ca2+]i level, and attenuated rotenone-induced viability decrease and apoptosis via reversing phosphorylated inhibition of mTOR, S6K1and4E-BP1in PC12cells and primary neurons. These data give us a hint that activation of IP3R on endoplasmic reticulum leading to open of calcium release channel EGTA as well as of extracellular [Ca2+]i inflow may be an important mechanism of rotenone-induced [Ca2+]i elevation. There exists a significant protection of2-APB or EGTA on neuronal cell apoptosis.3Rotenone-mediated CaM excitation inhibits mTOR pathway contributing to apoptosis via its induction of intracellular [Ca2+]i elevation in neuronal cellsPC12and primary neurons, seeded in6-and96-well plates, were exposed to rotenone (0.5and1μM) for4or24h following pretreatment with/without TFP (10μM), BAPTA/AM (30μM),2-APB (100μM) or EGTA (100μM) for1h, respectively. Cell viability and apoptosis were assayed using One Solution and DAPI staining, respectively. Effect of TFP on neuronal mTOR signaling pathway and effects of BAPTA/AM,2-APB, EGTA or TFP on expression of caspase-3and PARP in neuronal cells were detected by Western blotting. The results showed that TFP significantly reversed rotenone-induced phosphorylated inhibition of mTOR, S6K1and4E-BP1, and attenuated rotenone-induced viability decrease and apoptosis in PC12cells and primary neurons. Rotenone-elevated [Ca2+]i mediated caspase-dependent apoptosis in neuronal cells. BAPTA/AM, EGTA,2-APB or TFP prevented rotenone-induced activation of caspase pathway in primary neurons. Our findings suggest that rotenone-mediated CaM excitation inhibits mTOR pathway involved in apoptosis via its induction of intracellular [Ca2+]i elevation in neuronal cells.