Dulbecco’s modified Eagle medium (DMEM), 0.05% Trypsin-EDTA, NEUROBASALTM Media, and B27 Supplement were purchased from Invitrogen (Grand Island, NY, USA). 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 [Ca2+]i elevation, CaMKII phosphorylation and neuronal apoptosis. Together, the results indicate that the crosstalk between Ca2+ signaling and mitochondrial H2O2 is required for rotenone inhibition of mTOR-mediated S6K1 and 4E-BP1 pathways. Our findings suggest that how to control over-elevation of intracellular Ca2+ and overproduction of mitochondrial H2O2 may be a new approach to deal with the neurotoxicity of rotenone. inhibiting mitochondrial respiratory chain complex I [3, 4]. Excessive ROS in turn will further inhibit complex I . The vicious cycle eventually causes apoptosis of dopaminergic neurons, leading to Parkinson’s disease (PD) [7-14]. Thus, rotenone is a possible etiological factor in PD. However, the molecular mechanism underlying the neurotoxicity of rotenone is still not fully understood. Calcium ion (Ca2+) is important for many cellular events, such as proliferation/growth, differentiation, development and cell death . When properly controlled, Ca2+ fluxes across the plasma membrane and between intracellular compartments play critical roles in fundamental functions of neurons, including the regulation of neurite outgrowth and synaptogenesis, synaptic transmission and plasticity, and cell survival . However, disturbances in cellular Ca2+ homeostasis cause synaptic dysfunction, impaired plasticity and neuronal degeneration [16-19]. Especially, abnormally high levels of intracellular free Ca2+ ([Ca2+]i) induces overproduction of free radicals such as ROS, which can activate stress cascades, resulting in apoptosis [20, 21]. In turn, excessive or sustained ROS can also exacerbate Ca2+ overload and sensitize the bioactivity of Ca2+ [20, 22, 23]. The interconnection between Ca2+ and ROS alters the structures and functions of cellular proteins, and also activates or inhibits related signaling pathways, leading to neuronal apoptosis [20, 24-27]. Mammalian/mechanistic target of rapamycin (mTOR), a serine/threonine (Ser/Thr) protein kinase, regulates differentiation, development and survival in neurons [28-30]. Thus, mTOR exerts a crucial role in synaptic plasticity, learning and memory, and food uptake in adult brain [28-30]. Increasing evidence reveals that mTOR could be activated or inhibited depending on the pathologic status of the nervous system, e.g. brain tumors, tuberous sclerosis, cortical dysplasia and neurodegenerative diseases such as PD, Pexidartinib (PLX3397) Alzheimer’s disease (AD), and Huntington’s disease (HD) [28, 30]. Our group has observed that cadmium, a heavy metal polluted in the environment, induces neuronal cell death by [Ca2+]i- and/or ROS-dependent activation of mTOR signaling [31-34], whereas hydrogen peroxide (H2O2), a major radical of ROS, elicits neuronal cell death suppression of mTOR pathway . Recently, we have also found that rotenone evokes neuronal apoptosis H2O2-dependent inhibition of mTOR-mediated phosphorylation of ribosomal p70 S6 kinase (S6K1) and eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) [14, 36]. Intracellular Ca2+ elevation is a major factor for rotenone-induced apoptosis in neuronal cells . Hence, in this study, we investigated whether rotenone induces apoptosis by Ca2+/ROS-dependent inhibition of mTOR pathway. RESULTS Pexidartinib (PLX3397) Rotenone-induced neuronal apoptosis is associated with its induction of [Ca2+]i elevation Increased [Ca2+]i levels have been documented in many experimental models of apoptosis [37-39]. To understand how Ca2+ signaling participates in rotenone-induced neuronal apoptosis, first of all, we investigated the relationship between the [Ca2+]i level and the apoptosis in our neuronal cell models treated with rotenone. After PC12 cells and mouse primary neurons were treated with 0-1 Pexidartinib (PLX3397) M rotenone for 24 h, [Ca2+]i was measured by using an intracellular Ca2+ indicator dye, Fluo-3/AM. We found that rotenone elicited strong [Ca2+]i fluorescence (in green) (Figure S1A), and the intensity of the fluorescence was rotenone concentration-dependent (Figure ?(Figure1A).1A). Concurrently, rotenone decreased cell viability (Figure ?(Figure1B),1B), and increased nuclear fragmentation and condensation (arrows), Rabbit Polyclonal to BEGIN a hallmark of apoptosis , as well as TUNEL-positive cells (in green) in PC12 cells and primary neurons (Figure S1B, Figure 1C and 1D), respectively. Besides, treatment with rotenone for 24 h induced robust cleavages of caspase-3 and poly (ADP-ribose) polymerase (PARP) in the cells (data not shown). Collectively, these data imply that rotenone-induced neuronal apoptosis is associated with the induction of [Ca2+]i elevation. Open in a separate window Figure 1 Rotenone-induced [Ca2+]i elevation is associated with cell viability reduction and apoptosis in neuronal cellsPC12 cells and primary neurons were treated with rotenone (0-1 M) for 24 h. [Ca2+]i fluorescence intensity was imaged and quantified using an intracellular Ca2+ indicator dye Fluo-3/AM (A). Cell viability was determined by the MTS assay.