Astrocyte mediated protection of fetal cerebral cortical neurons from rotenone and paraquat
Highlights
► The developing brain can be damaged by rotenone (R) and paraquat (P). ► Fetal neurons are more sensitive to rotenone and paraquat than astrocytes. ► Mature astrocytes can augment neuron GSH homeostasis via the γ-glutamyl cycle. ► Co-culturing fetal neurons with astrocytes protects from R and P-related damage and death. ► This protection is provided by the γ-glutamyl cycle.
Introduction
Pesticides, substances used to repel or destroy insects and herbicides employed for weed control, are chemicals applied to our environment in enormous quantities in rural and urban settings. In the United States, pesticides are applied in quantities of at least 4.5 billion pounds per year, while herbicide application is in excess of 500 million pounds per year (Weiss et al., 2004). While the use of such compounds is arguably necessary to maintain food supplies, a striking reality is the toxicity of these compounds to humans. The scientific literature is dense with studies utilizing common pesticides to generate adult animal models for neurodegenerative diseases (Corasaniti et al., 1998, Sherer et al., 2003a). However, there is an incomplete understanding of the relative toxicities of these compounds to the developing brain and the neuroprotective systems present at various stages of brain development that could mitigate such events.
Rotenone is a pro-oxidant inhibitor of complex I (Sherer et al., 2003a) of the respiratory chain which can generate widespread toxic effects in the central nervous system (Lapointe et al., 2004). This inhibition of complex I by rotenone can recapitulate dopamine (DA) neuronal degeneration and associated neurological disabilities in experimental animals (Betarbet et al., 2000, Dawson and Dawson, 2003, Schneider et al., 1987, Sherer et al., 2003b). Paraquat toxicity is primarily generated by reactive oxygen species (ROS) elicited by redox-cycling with molecular oxygen and NADH-dependent formation of superoxide anions (Kim et al., 2008, Suntres, 2002). This herbicide induces oxidative stress in both rat astrocytes and neurons (Schmuck et al., 2002, Mollace et al., 2003). These widely used environmental toxins, with chronic treatment of rodents, induce many key features of Parkinson Disease (PD), motor deficits, loss of dopaminergic neurons, and accumulation of synuclein containing inclusion bodies (Betarbet et al., 2000, Brooks et al., 1999, Manning-Bog et al., 2002, Manning-Bog et al., 2003, McCormack and Di Monte, 2003, Sherer et al., 2003a, Sherer et al., 2003b, Thiruchelvam et al., 2000, Ferrante et al., 1997).
Astrocytes play a crucial role in the maintenance of brain GSH, which is vital for neuronal survival in baseline or enhanced pro-oxidant settings. A key mechanism underlying this function is the γ-glutamyl cycle. This cycle essentially consists of GSH being extruded from astrocytes where it is hydrolyzed to the Cysgly dipeptide by γ-glutamyl transpeptidase (γ-GT) at the external surface of the astrocyte plasma membrane. This dipeptide is subsequently hydrolyzed by aminopeptidase N (ApN) at the external neuronal plasma membrane, yielding Cys which is transported into the neuronal cytoplasm, driving the synthesis on GSH (Dringen et al., 1999a, Dringen et al., 1999b, Rathinam et al., 2006). We have previously found that this astrocyte-mediated antioxidant system provides protection of neurons from ethanol-mediated oxidative damage, likely a much milder pro-oxidant setting than those generated by either rotenone or paraquat. This neuroprotection occurs due to cerebral cortical astrocytes having a robust GSH synthesizing capacity which is reflected in high GSH content (Cooper, 1997, Schulz et al., 2000) and having numerous close contacts with neurons (Rohlmann and Wolff, 1996). This is an elegant, highly regulated system that is subject to manipulation.
The following studies illustrate the varying toxicity of rotenone and paraquat toward neurons and astrocytes, the critical role of the γ-glutamyl cycle in the maintenance of neuronal GSH homeostasis, and a central role for astrocytes in protection of neurons from rotenone and paraquat.
Section snippets
Materials
Minimum Essential Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), Fetal Bovine Serum (FBS) were from Invitrogen (Carlsbad, CA). Monochlorobimane (MCB), 2,7′-Dichlorofluorescein diacetate (DCF-DA), Horse serum (HS), Bovine serum albumin (BSA), N-acetylcysteine (NAC), rotenone, paraquat, 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), acivicin and bestatin were from Sigma (St. Louis, MO). Annexin Cy 3 was from Abcam (Cambridge, MA) and 7-Amino-Actinomycin D (7AAD)
Rotenone or paraquat alter the viability of fetal cortical neurons and astrocytes differently
Following treatment of neurons and astrocytes with varying concentrations of rotenone and paraquat, viability of the cells was determined by trypan blue exclusion. Viable cells, excluding the dye were manually counted. Rotenone treatment (0.08–0.5 μM) for 4 h (Fig. 1a) decreased TB exclusion by 25–76% (P < 0.05) and by 32–95% (P < 0.05) when treated for 24 h (0.025–1 μM), Similarly, paraquat treatment for 4 h (Fig. 1c, 30–100 μM) decreased TB exclusion by 32% and 53% at 24 h (Fig. 1d). Rotenone was
Discussion
Rotenone and paraquat are toxic to the developing brain (Choi et al., 2010). In our laboratory, we have shown that oxidative stress which can ultimately enhance apoptotic death of cortical neurons can be mitigated by the presence of astrocytes and that the underlying mechanism is maintenance of neuronal GSH homeostasis via the γ-glutamyl cycle (Rathinam et al., 2006, Watts et al., 2005). In the present study, we report that in a neuron-astrocyte co-culture setting, the toxic effects of rotenone
Conflict of interest statement
All the authors declare that there are no conflicts of interest.
Acknowledgements
This work was supported by R01 ES015700. We would like to thank Dr. Patrick Reynolds, Director of the TTUHSC Cancer Center Flow Cytometry Core Facility for use of the core, and Dr Malkanthi Mudannayake and Matthew V. Wilkin for helping us generate the FACS data.
References (43)
- et al.
Paraquat elicited neurobehavioral syndrome caused by dopaminergic neuron loss
Brain Res.
(1999) Isolation culture and use of viable central nervous system perikarya
- et al.
The use of monochlorobimane to determine hepatic GSH levels and synthesis
Anal. Biochem.
(1990) - et al.
Systemic administration of rotenone produces selective damage in the striatum and globus pallidus, but not in the substantia nigra
Brain Res.
(1997) - et al.
Oxidative DNA damage and cytotoxicity induced by copper-stimulated redox cycling of salsolinol, a neurotoxic tetrahydroisoquinoline alkaloid
Free Radic. Biol. Med.
(2001) - et al.
Different preferences in the utilization of amino acids for glutathione synthesis in cultured neurons and astroglial cells derived from rat brain
Neurosci. Lett.
(1996) - et al.
The herbicide paraquat causes up-regulation and aggregation of alpha-synuclein in mice: paraquat and alpha-synuclein
J. Biol. Chem.
(2002) - et al.
The role of oxidative stress in paraquat-induced neurotoxicity in rats: protection by non peptidyl superoxide dismutase mimetic
Neurosci. Lett.
(2003) - et al.
Selective loss of subpopulations of ventral mesencephalic dopaminergic neurons in the monkey following exposure to MPTP
Brain Res.
(1987) - et al.
Subcutaneous rotenone exposure causes highly selective dopaminergic degeneration and alpha-synuclein aggregation
Exp. Neurol.
(2003)
Role of antioxidants in paraquat toxicity
Toxicology
Changing numbers of neuronal and non-neuronal cells underlie postnatal brain growth in the rat
Proc. Natl. Acad. Sci. U. S. A.
Chronic systemic pesticide exposure reproduces features of Parkinson's disease
Nat. Neurosci.
JNK3 mediates paraquat- and rotenone-induced dopaminergic neuron death
J. Neuropathol. Exp. Neurol.
Glutathione in the brain disorders of glutathione metabolism
Paraquat: a useful tool for the in vivo study of mechanisms of neuronal cell death
Pharmacol. Toxicol.
Molecular pathways of neurodegeneration in Parkinson's disease
Science
Glutathione metabolism in brain metabolic interaction between astrocytes and neurons in the defense against reactive oxygen species
Eur. J. Biochem.
The glutathione system of peroxide detoxification is less efficient in neurons than in astroglial cells
J. Neurochem.
Synthesis of the antioxidant glutathione in neurons: supply by astrocytes of CysGly as precursor for neuronal glutathione
J. Neurosci.
Activation of the antioxidant response element in primary cortical neuronal cultures derived from transgenic reporter mice
J. Neurochem.
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