title: Astrocytes Prevent Ethanol Induced Apoptosis of Nrf2 Depleted Neurons by Maintaining GSH Homeostasis.
abstract: Glutathione (GSH), a major cellular antioxidant protects cells against oxidative stress injury. Nuclear factor erythroid 2-related factor 2 (NFE2L2/Nrf2) is a redox sensitive master regulator of battery of antioxidant enzymes including those involved in GSH antioxidant machinery. Earlier we reported that ethanol (ETOH) elicits apoptotic death of primary cortical neurons (PCNs) which in partly due to depletion of intracellular GSH levels. Further a recent report from our laboratory illustrated that ETOH exacerbated the dysregulation of GSH and caspase mediated cell death of cortical neurons that are compromised in Nrf2 machinery (Narasimhanet al., 2011). In various experimental models of neurodegeneration, neuronal antioxidant defenses mainly GSH has been shown to be supported by astrocytes. We therefore sought to determine whether astrocytes can render protection to neurons against ETOH toxicity, particularly when the function of Nrf2 is compromised in neurons. The experimental model consisted of co-culturing primary cortical astrocytes (PCA) with Nrf2 downregulated PCNs that were exposed with 4 mg/mL ETOH for 24 h. Monochlorobimane (MCB) staining followed by FACS analysis showed that astrocytes blocked ETOH induced GSH decrement in Nrf2-silenced neurons as opposed to exaggerated GSH depletion in Nrf2 downregulated PCNs alone. Similarly, the heightened activation of caspase 3/7 observed in Nrf2-compromised neurons was attenuated when co-cultured with astrocytes as measured by luminescence based caspase Glo assay. Furthermore, annexin-V-FITC staining followed by FACS analysis revealed that Nrf2 depleted neurons showed resistance to ETOH induced neuronal apoptosis when co-cultured with astrocytes. Thus, the current study identifies ETOH induced dysregulation of GSH and associated apoptotic events observed in Nrf2-depleted neurons can be blocked by astrocytes. Further our results suggest that this neuroprotective effect of astrocyte despite dysfunctional Nrf2 system in neurons could be compensated by astrocytic GSH supply.
authors.affiliation: Department of Pharmacology and Neuroscience, Texas Tech University Health Sciences Center, Lubbock, USA ; South Plains Alcohol and Addiction Research Center, Texas Tech University Health Sciences Center, Lubbock, USA.
authors.forename: Madhusudhanan
authors.initials: M
authors.lastname: Narasimhan
authors.affiliation: Department of Pharmacology and Neuroscience, Texas Tech University Health Sciences Center, Lubbock, USA.
authors.forename: Marylatha
authors.initials: M
authors.lastname: Rathinam
authors.affiliation: Department of Pharmacology and Neuroscience, Texas Tech University Health Sciences Center, Lubbock, USA.
authors.forename: Dhyanesh
authors.initials: D
authors.lastname: Patel
authors.affiliation: Department of Pharmacology and Neuroscience, Texas Tech University Health Sciences Center, Lubbock, USA ; South Plains Alcohol and Addiction Research Center, Texas Tech University Health Sciences Center, Lubbock, USA.
authors.forename: George
authors.initials: G
authors.lastname: Henderson
authors.affiliation: Department of Pharmacology and Neuroscience, Texas Tech University Health Sciences Center, Lubbock, USA ; South Plains Alcohol and Addiction Research Center, Texas Tech University Health Sciences Center, Lubbock, USA.
authors.forename: Lenin
authors.initials: L
authors.lastname: Mahimainathan
keywords: Astrocyte-Neuron Co-Culture; Ethanol; GSH; Nrf2; Oxidative Stress