Astrocyte activation and neurotoxicity: A study in different rat brain regions and in rat C6 astroglial cells
Introduction
Astrocytes are the most abundant type of glial cells in the brain that play multiple roles in brain during physiological and pathological conditions. These cells are the key regulators in brain function, characterized predominantly due to their close interaction with neurons. Astrocyte metabolism, including energy generating pathways and amino acid homoeostasis are tightly coupled to neurons, and is critical for energy metabolism as well as for synthesis of neurotransmitters (Bak et al., 2006). Neurons are functionally dependent on the astrocytes for the replenishment of intermediates of glucose metabolism (Sidoryk-Wegrzynowicz and Aschner, 2013). The astrocytes can also promote neurogenesis and synaptogenesis in response to ischemia-induced brain damage (Song et al., 2002, Ullian et al., 2004). These cells release multiple neurotrophic factors, such as glia derived neurotrophic factor (GDNF) to protect them and neurons from adverse environment (Akerud et al., 1999, Burke et al., 1998, Oo et al., 2003). Takuma et al. (2004) have reported that dysfunctional astrocytes can enhance neuronal degeneration by decreasing the secretion of trophic factors. The astrocytes also play a dynamic role in CNS function by maintaining the restrictive properties of the blood-brain barrier (Liebner et al., 2011). Under ischemic condition, the astrocytes can remove excess glutamate and potassium to protect neurons from glutamate – mediated cytotoxicity and depolarization (Danbolt, 1994, Barreto et al., 2011). The study of the astrocytes is particularly important, in light of the coexistence of apoptotic death of neurons and astrocytes in the brain, affected by ischemia, neurodegenerative diseases or any neuronal injury. The activated astrocytes are consider as hallmark of neurodegenerative diseases which release different factors like array of pro and anti-inflammatory cytokines, free radicals, antioxidants and neurotrophic factors during pathological conditions which further contribute to neuronal death as well as in survival mechanisms (Singh et al., 2010). Despite their high levels of antioxidative activity, astrocytes exhibit a high degree of vulnerability and are not resistant to the effects of reactive oxygen species (ROS). They respond to substantial or sustained oxidative stress with increased intracellular Ca2+, loss of mitochondrial potential and decreased oxidative phosphorylation (Robb et al., 1999). Since the astrocytes determine the brain's vulnerability to oxidative injury and form a tight functional unit with neurons, impaired astrocytic energy metabolism, antioxidant capacity and the death of astrocytes may critically impair neuronal survival (Feeney et al., 2008, Lu et al., 2008).
Rotenone is a broad-spectrum pesticide and it causes neurotoxicity via mitochondrial complex-I inhibition. It interferes with the electron transport chain in mitochondria by inhibiting the transfer of electrons from iron-sulfur centers in complex-I to ubiquinone thus consequently affect the ATP synthesis (Betarbet et al., 2002, Liu et al., 2003, Lapointe et al., 2004, Bove et al., 2005). Rotenone causes neurotoxicity due to its ability to generate the reactive oxygen species (ROS), which leads to various adverse effects in cellular physiology (Trojanowski, 2003, Uversky, 2004, Radad et al., 2006. Chronic intraperitoneal injection of the rotenone causes dose dependent alterations in behavioral responses and symptoms like Parkinson's disease in rodents (Betarbet et al., 2000, Alam and Schmidt, 2002). Earlier we have reported the different susceptibility of the rotenone in different brain areas (Swarnkar et al., 2009) and cerebral damage by diminished mitochondrial enzyme activity and increased ROS levels (Swarnkar et al., 2010). Activation of proapoptotic factor like BAD and caspases have also been reported in rotenone treated neuroblastoma SHSY5Y cells (Kitamura et al., 2002, Watabe and Nakaki, 2004). Rotenone induced neuronal death is reported however, in context to neuronal death its effects on astrocytes are not well studied.
Reports have indicated the activation of astrocytes in neurological disorders but still it is not known whether these activated astrocytes are the consequence or the cause of the neuronal death. Rotenone causes neurotoxicity and therefore used in the present study to explore the correlation between astrocyte activation and degeneration of neurons in different rat brain regions. In addition to investigate the specific mechanistic effect of rotenone on astrocytes, the study was also performed in single population of astrocytes by using rat C6 cells.
Section snippets
Chemicals
Low melting agarose, bovine serum albumin, copper sulphate, calcium chloride, dichlorofluorescein diacetate (DCF-DA), dihydroethidium (DHE), disodium hydrogen phosphate, dimethyl sulphoxide (DMSO), ethidium bromide, Folin-Ciocalteu reagent, glucose, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 3-4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide dye (MTT), magnesium chloride, melatonin, NADH, paraformaldehyde, penicillin-streptomycin, phenylenediamine, potassium chloride,
Effect of rotenone on astrocytes activation in rat brain regions and C6 cells
Bilateral intracerebroventricular injection of rotenone caused activation of astrocytes in frontal cortex, hypothalamus, hippocampus, substantia nigra, cerebellum, cerebellar nucleus as estimated by GFAP immunostaining (Fig. 1 regions encircled in green circle). However, in other regions like corpus callosum, entopeduncular nucleus, inferior colliculus, LP, Me, PO, PCRt, olfactory tubercle, ventroposterior thalamus no GFAP staining was observed (Fig. 1 regions encircled in white circle). In
Discussion
Significant astrocyte activation was observed in the different rat brain regions after intracerebroventricular administration of rotenone and in in vitro treated rat C6 cells. However, the degeneration of neurons was not concurrent with astrocytes activation in rat brain regions suggesting that astrocyte activation might be the initiatory event in neuronal death. The astrocytes activation may also be a parallel event to neuronal death. This diverse observation and response toward rotenone
Conclusion
In conclusion, study indicates that rotenone induces significant astrocyte activation along with degeneration of neurons. The astrocyte activation was not specific to neuronal death though it was parallel to neuronal degeneration. Study in C6 cells showed that rotenone causes significantly decreased astrocyte-viability that involves the oxidative stress, nitric oxide and oxidative DNA damage.
Conflict of interest
Authors state that there is no conflict of interest.
Acknowledgements
We wish to acknowledgment Department of Biotechnology, India for funding support. We wish to thank Dr. A. K. Balapure, Head, Tissue and Cell Culture Unit, CDRI and his team for providing cells for experimentation. Poonam Goswami wishes to thank Indian Council of Medical Research, India for providing the senior research fellowship and Academy of Scientific and Innovative Research for providing opportunity to conduct research in CSIR-CDRI.
References (47)
- et al.
Rotenone destroys dopaminergic neurons and induces parkinsonian symptoms in rats
Behav. Brain Res.
(2002) - et al.
Toxin-induced models of Parkinson's disease
NeuroRx
(2005) - et al.
Bleomycin genotoxicity and amifostine (WR-2721) cell protection in normal leukocytes vs K562 tumoral cells
Biochem. Pharmacol.
(2002) - et al.
Oxidative stress and signal transduction in Saccharomyces cerevisiae: insights into ageing, apoptosis and diseases
Mol. Aspects Med.
(2001) The high affinity uptake system for excitatory amino acids in the brain
Prog. Neurobiol.
(1994)- et al.
Vulnerability of glial cells to hydrogen peroxide in cultured hippocampal slices
Brain Res.
(2008) - et al.
The effects of diazinon and cypermethrin on the differentiation of neuronal and glial cell lines
Toxicol. Appl. Pharmacol.
(2007) - et al.
Repair of oxidized DNA bases in the yeast Saccharomyces cerevisiae
Biochimie
(1997) - et al.
Thimerosal neurotoxicity is associated with glutathione depletion protection with glutathione precursors
Neurotoxicology
(2005) - et al.
Possible involvement of both mitochondria- and endoplasmic reticulum-dependent caspase pathways in rotenone-induced apoptosis in human neuroblastoma SH-SY5Y cells
Neurosci. Lett.
(2002)
Hydrogen sulfide protects astrocytes against H2O2-induced neural injury via enhancing glutamate uptake
Free Radic. Biol. Med.
Rotenone induces cell death in primary dopaminergic culture by increasing ROS production and inhibiting mitochondrial respiration
Neurochem. Int.
Microglial activation and cell death induced by the mitochondrial toxin 3-nitropropionic acid in vitro and in vivo studies
Neurobiol. Dis.
The interacting pathways for prevention and repair of oxidative DNA damage
Mutat. Res.
A study to correlate rotenone induced biochemical changes and cerebral damage in brain areas with neuromuscular coordination in rats
Toxicology
A comparative study on oxidative stress induced by LPS and rotenone in homogenates of rat brain regions
Environ. Toxicol. Pharmacol.
Astrocyte apoptosis: implications for neuroprotection
Prog. Neurobiol.
Rotenone neurotoxicity: a new window on environmental causes of Parkinson's disease and related brain amyloidoses
Exp. Neurol.
Possible involvement of Ca2+ signaling in rotenone-induced apoptosis in human neuroblastoma SH-SY5Y cells
Neurosci. Lett.
In utero treatment to nicotine and chlorpyrifos alone and in combination produces persistent sensorimotor deficits and Purkinje neuron loss in the cerebellum of adult offspring rats
Arch. Toxicol.
Differential effects of glial cell line-derived neurotrophic factor and neurturin on developing and adult substantia nigra dopaminergic neurons
J. Neurochem.
The glutamate/GABA-glutamine cycle: aspects of transport, neurotransmitter homeostasis and ammonia transfer
J. Neurochem.
Astrocytes: targets for neuroprotection in stroke
Cent. Nerv. Syst. Agents Med. Chem.
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These authors contributed equally to this work.