Activated astrocytes and microglia, hallmark of neurodegenerative diseases release different factors like array of pro and anti-inflammatory cytokines, free radicals, anti-oxidants, and neurotrophic factors during neurodegeneration which further contribute to neuronal death as well as in survival mechanisms. Astrocytes act as double-edged sword exerting both detrimental and neuroprotective effects while microglial cells are attributed more in neurodegenerative mechanisms. The dual and insufficient knowledge about the precise role of glia in neurodegeneration showed the need for further investigations and thorough review of the function of glia in neurodegeneration. In this review, we consolidate and categorize the glia-released factors which contribute in degenerative and protective mechanisms during neuropathological conditions.

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.

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Astrocytes are the most abundant glial cells, which provide metabolic support for neurons. Rotenone is a botanical pesticide of natural origin, known to exhibit neurotoxic potential via inhibition of mitochondrial complex-I. This study was carried out to explore the effect of rotenone on C6 cells. The cell line C6 derived from rat glioma cells represents astrocyte-like cell. C6 cells were treated with rotenone (0.1, 1 and 10 μM) for 4 h. The effect of rotenone was studied on cell survival (MTT reduction and PI uptake); free radicals (ROS and RNS) and DNA damage (comet assay and Hoechst staining). The glial cell activation and apoptotic cell death was evaluated by expression of Glial fibrillary acidic protein (GFAP) and caspase-3 respectively. The treatment with rotenone resulted in decreased cell survival and increased free radical generation. Altered nuclear morphology and DNA damage were evident following rotenone treatment in Hoechst staining and Comet assay. Rotenone elevated expression of GFAP and caspase-3 that indicates glial cell activation and apoptosis, respectively. We further studied the effect of melatonin, an antioxidant, on the observed toxic effects. Co-incubation of antioxidant, melatonin (300 μM), significantly suppressed rotenone induced above-mentioned effects in C6 cells. Inhibitory effects of melatonin suggest that free radicals play a major role in rotenone induced astrocyte activation and cellular toxicity leading to apoptosis of astroglial cells.

Intracerebroventricular (icv) injection of streptozotocin (STZ) in rat brain causes prolonged impairment of brain energy metabolism and oxidative damage and leads to cognitive dysfunction; however, its mechanistic specific effects on neurons are not known. The present study was conducted to investigate the STZ-induced cellular and molecular alterations in mouse neuronal N2A cells. The N2A cells were treated with STZ (10, 50, 100, 1000 μM) for 48 h, and different assays were performed. STZ treatment caused significant decrease in cell viability, choline levels, increased acetylcholinesterase (AChE) activity, tau phosphorylation and amyloid aggregation. STZ treatment also led to low levels of glucose uptake, elevated mitochondrial stress, translocation of cytochrome c in cytosol, phosphatidylserine externalization, increased expression of caspase-3 and DNA damage. Co-treatment of clinically used drug donepezil (1 μM) offered significant protection against STZ induced neurotoxicity. Donepezil treatment significantly inhibited the STZ induced neurotoxicity, altered choline level, AChE activity, lowered glucose uptake and mitochondrial stress. However, the caspase-3 expression remains unaltered with co-treatment of donepezil. In conclusion, findings showed that STZ treated N2A cells exhibited the Alzheimer's disease (AD) related pathological markers which are attenuated with co-treatment of donepezil. Findings of the study suggested the potent use of STZ treated N2A cells as in vitro experimental test model to study the disease mechanism at cellular level.

The present study was conducted to evaluate the involvement of endoplasmic reticulum stress in rotenone-induced oxidative neuronal death in rat brain. Rotenone (6 μg/3 μl) was administered intranigrally, unilaterally (right side) in SD rat brain. Neuronal morphology, expression level of tyrosine hydroxylase (TH) and endoplasmic reticulum (ER) stress markers like glucose-regulated protein 78 (GRP78), growth arrest and DNA damage-inducible gene 153 (GADD153), eukaryotic translation initiation factor 2α (p-eIF2α/eIF2α) and cleaved caspase-12 were estimated in the rat brain. Levels of reactive oxygen species (ROS), reduced glutathione (GSH) and enzymatic activities of glutathione peroxidase (GPx) and glutathione reductase (GRd) were estimated to assess the rotenone induced oxidative stress. Apoptotic death of neurons was assessed by estimating the mRNA level of caspase-3. Rotenone administration caused altered neuronal morphology, decreased expression of TH, augmented ROS level, decreased level of GSH and decreased activities of GPx and GRd enzymes which were significantly attenuated with the pretreatment of ER stress inhibitor, salubrinal (1 mg/kg, intraperitoneal). Significantly increased levels of GRP78, GADD, dephosphorylated eIF2α and cleaved caspase-12 was also observed after rotenone administration, which was inhibited with the pretreatment of salubrinal. Rotenone-induced increased mRNA level of caspase-3 was also attenuated by pretreatment of salubrinal. Findings suggested that salubrinal treatment significantly inhibited the rotenone-induced neurotoxicity implicating that ER stress initiates the rotenone-induced oxidative stress and neuronal death.

Rotenone causes cytotoxicity in astrocytic cell culture by glial activation, which is linked to free radical generation. The present study is an investigation to explore whether rotenone could also cause cellular toxicity in mouse neuroblastoma cells (Neuro-2a) under treatment similar to astroglial cells. The effect of rotenone (0.1, 1, and 10 μM) on mitochondrial dehydrogenase enzyme activity by MTT reduction assay, PI uptake, total reactive oxygen species (ROS)/superoxide levels, nitrite levels, extent of DNA damage (by comet assay), and nuclear morphological alteration by Hoechst staining was studied. Caspase-3 and Ca⁺²/calmodulin-dependent protein kinase II (CaMKIIα) gene expression was determined to evaluate the apoptotic cell death and calcium kinase, respectively. Calcium level was estimated fluorometrically using fura-2A stain. Rotenone decreased mitochondrial dehydrogenase enzyme activity and generated ROS, superoxide, and nitrite. Rotenone treatment impaired cell intactness and nuclear morphology as depicted by PI uptake and chromosomal condensation of Neuro-2a cells, respectively. In addition, rotenone resulted in increased intracellular Ca⁺² level, caspase-3, and CaMKIIα expression. Furthermore, co-exposure of melatonin (300 μM), an antioxidant to cell culture, significantly suppressed the rotenone-induced decreased mitochondrial dehydrogenase enzyme activity, elevated ROS and RNS. However, melatonin was found ineffective to counteract rotenone-induced increased PI uptake, altered morphological changes, DNA damage, elevated Ca⁺², and increased expression of caspase-3 and CaMKIIα. The study indicates that intracellular calcium rather than oxidative stress is a major factor for rotenone-induced apoptosis in neuronal cells.

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