Endoplasmic Reticulum Stress Plays a Key Role in Rotenone-Induced Apoptotic Death of Neurons

Abstract

Rotenone, a pesticide, causes neurotoxicity via the mitochondrial complex-I inhibition. The present study was conducted to evaluate the role of endoplasmic reticulum (ER) stress in rotenone-induced neuronal death. Cell viability, cytotoxicity, reactive oxygen species (ROS) generation, nitrite level, mitochondrial membrane potential (MMP), and DNA damage were assessed in rotenone-treated neuro-2A cells. Protein levels of ER stress markers glucose regulated protein 78 (GRP78), growth arrest- and DNA damage-inducible gene 153 (GADD153), and phosphorylation of eukaryotic translation initiation factor 2 subunit α (eIF2-α) were estimated to assess the ER stress. To confirm the apoptotic death of neurons, mRNA levels of caspase-9, caspase-12 and caspase-3 were estimated. Further, to confirm the involvement of ER stress, neuro-2A cells were pretreated with ER stress inhibitor salubrinal. Co-treatment of antioxidant melatonin was also given to assess the role of oxidative stress in rotenone-induced apoptosis. Rotenone (0.1, 0.5, and 1 μM) treatment to neurons caused significantly decreased cell viability, increased cytotoxicity, increased ROS generation, increased expression of GRP78 and GADD, DNA damage and activation of caspase-12 and caspase-3 which were significantly attenuated by pretreatment of salubrinal (25 μM). Rotenone-induced dephosphorylation of eIF2α was also inhibited with salubrinal treatment. However, pretreatment of salubrinal did not affect the rotenone-induced increased nitrite levels, decreased MMP and caspase-9 activation. Co-treatment of antioxidant melatonin (1 mM) did not offer attenuation against rotenone-induced increased expression of caspase-9, caspase-12 and caspase-3. In conclusion, results indicated that ER stress plays a key role in rotenone-induced neuronal death, rather than oxidative stress. Graphical Abstract Pictorial presentation showed the involvement of endoplasmic reticulum (ER) stress, increased reactive oxygen species (ROS), nitrite level, decreased mitochondrial membrane potential (MMP), caspase activation and DNA damage in neuronal cells after rotenone treatment. ER stress inhibitor-salubrinal showed significant attenuation against most of the rotenone-induced adverse effects reflecting its key involvement in rotenone-induced neuronal death.

Downregulation of miR-384-5p attenuates rotenone-induced neurotoxicity in dopaminergic SH-SY5Y cells through inhibiting endoplasmic reticulum stress

“…Wu et al (52) reported that candesartan cilexetil inhibited rotenone-evoked ER stress by downregulating the expression of ATF4 and CHOP and protected rat dopaminergic neurons. A recent study revealed that ER stress played an important role in rotenone-induced apoptosis of mouse neuroblastoma cells (10). In line with these findings, our data also indicate that rotenone induced ER stress in human dopaminergic SH-SY5Y cells, which was evident by an increase in phosphorylation of ATF4 and IRE1␣.…”

“…The results showed that the expression levels of phosphorylated ATF4 (pATF4) and phosphorylated IRE1␣ (pIRE1␣) were both significantly increased by rotenone treatment (Fig. 5, A and B), implying that rotenone treatment evoked ER stress signaling, which is consistent with the findings of Goswami et al (10). As expected, the expression levels of pATF4 and pIRE1␣ were significantly decreased by miR-384-5p inhibitor treatment or further increased by treatment with miR-384-5p mimics (Fig.…”

“…More recently, Salganik et al (40) found that GRP78 protein overexpression protected aging nigral dopamine neurons in the ␣-synuclein-induced rat model of PD. Rotenone has been reported to induce ER stress both in cell models and animal models of PD (10,11,49). Here, we showed that downregulating miR-384-5p increased the expression of GRP78 and reduced ER stress signaling induced by rotenone.…”

“…Cells were exposed to rotenone (Sigma, St. Louis, MO) at various concentrations (1,5,10,20, and 50 M) (28) for the indicated times before being harvested for analysis. For miRNA transfection, cells were transfected with miR-384-5p mimics or inhibitors using Lipofectamine 2000 (Invitrogen) at a final concentration of 50 nM for 24 h prior to rotenone (20 M) exposure.…”

“…Furthermore, ER stress has been found to be involved in cellular models of PD (39,44). It has been reported that the prolonged ER stress induced by toxic stimuli such as rotenone results in the apoptotic death of neurons (10). In a rotenone rat model of PD, suppression of ER stress provides neuroprotection (49).…”