Date of Award


Degree Type


Degree Name

Master of Science (MS)




Sandra Hewett


Astrocyte;Glutamate Cysteine Ligase (GCL);Glutathione (GSH);Nrf2;oxidative stress;System xc- (xCT)

Subject Categories

Medical Sciences | Medicine and Health Sciences | Neurosciences


In the central nervous system, astrocytes are the primary source of glutathione (GSH), an important and abundant non-protein thiol antioxidant. GSH biosynthesis takes place in the cytosol and involves two sequential steps, the first catalyzed by glutamate cysteine ligase (GCL) and the subsequent by glutathione synthetase (GSS). The process relies heavily on the availability of cysteine, which is obtained through the action of the glutamate/cystine antiporter, System xc-. This transporter imports cystine into the cells, where it is rapidly reduced to cysteine intracellularly for GSH synthesis. Furthermore, the multidrug resistance protein 1 (Mrp1) facilitates the export of GSH from astrocytes into the extracellular space, contributing to the overall antioxidant defense of the CNS. Oxidative stress triggers an increase in GSH production in numerous cells and tissues, including astrocytes, with Nrf2 acting as a critical transcription factor. However, the extent to which Nrf2 controls constitutive biosynthesis of GSH and regulates the associated synthetic and release mechanisms, including System xc-, GSH synthetic enzymes, and Mrp1, in astrocytes remains incompletely investigated. To address this, we cultured astrocytes from the cerebral cortices of Nrf2 wildtype (Nrf2+/+) and null mutant (Nrf2-/-) 1-3 day old pups, derived from heterozygous breeders, and quantified intracellular and extracellular GSH concentrations in cells between 14 to 33 days in vitro. Data show a significant decrease in both intracellular (p < 0.0001) and extracellular (p = 0.0250) GSH levels in Nrf2-/- astrocytes when compared to Nrf2+/+ cells. Concomitant with this reduction, qRT-PCR analysis demonstrates that Nrf2-/- astrocytes have significantly lower basal mRNA levels of xCT, the substrate specific light chain of System xc- (p < 0.0001), as well as both GCL subunits [catalytic (p = 0.0117); modifier (p = 0.0092)], while no significant changes were observed in GSS (p = 0.7662) or Mrp1 (p = 0.5697). Western blot analysis further confirmed these findings, demonstrating reduced protein levels of both subunits of GCL [catalytic (p = 0.0017); modifier (p = 0.0269)] but not GSS (p = 0.8533). (Seleno)cystine uptake assay confirmed decreased System xc- activity (p = 0.0170) in Nrf2-/- astrocytes. Surprisingly, tert-butyl hydroperoxide (t-BOOH) shows that Nrf2-/- astrocytes are more resistant to oxidative stress-induced toxicity than Nrf2+/+ cells (p < 0.0001). Taken together, our findings provide evidence that Nrf2 regulates basal production of GSH in astrocytes, possibly through constitutive transcriptional regulation of GCL and xCT. However, decreased GSH levels do not render Nrf2-/- astrocytes vulnerable to oxidative stress. Instead, compensatory mechanisms may exist that confer protection against oxidant insults.


Open Access

Available for download on Saturday, June 21, 2025

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