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

Mol Neurobiol. 2016 Jan;53(1):285-298. doi: 10.1007/s12035-014-9001-5. Epub 2014 Nov 28.

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.

Keywords: Apoptosis; DNA damage; Endoplasmic reticulum stress; Reactive oxygen species; Rotenone; Salubrinal.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Apoptosis / drug effects*
  • Blotting, Western
  • Caspases / metabolism
  • Cell Line, Tumor
  • Cell Survival / drug effects
  • Cinnamates / pharmacology
  • Comet Assay
  • DNA Damage
  • Endoplasmic Reticulum Stress / drug effects*
  • Eukaryotic Initiation Factor-2 / metabolism
  • Fluoresceins / metabolism
  • Fluorescence
  • Heat-Shock Proteins / metabolism
  • L-Lactate Dehydrogenase / metabolism
  • Membrane Potential, Mitochondrial / drug effects
  • Mice
  • Neurons / drug effects
  • Neurons / metabolism
  • Neurons / pathology*
  • Nitrites / metabolism
  • Phosphorylation / drug effects
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Reactive Oxygen Species / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Rotenone / toxicity*
  • Thiourea / analogs & derivatives
  • Thiourea / pharmacology

Substances

  • Cinnamates
  • Eukaryotic Initiation Factor-2
  • Fluoresceins
  • Heat-Shock Proteins
  • Nitrites
  • RNA, Messenger
  • Reactive Oxygen Species
  • salubrinal
  • Rotenone
  • diacetyldichlorofluorescein
  • L-Lactate Dehydrogenase
  • Caspases
  • Thiourea
  • molecular chaperone GRP78