Abstracts

Rationale: Reduced glutathione (GSH) and ascorbic acid (AA) are two major antioxidants in maintaining redox homeostasis and protecting against oxidative damage to central nervous system (CNS). In addition, both antioxidants have been suggested to act as neuromodulators in neurotransmitter systems. We previously performed a battery of behavioral tests in AA-deficient (Gulo?'/?' knockout) and GSH-deficient (Gclm?'/?' knockout) mice, respectively. The two mouse models exhibit subtle, but different, behavioral phenotypes. Interestingly, both show an exaggerated hyperactivity in response to a dopamine agonist. These studies imply a significant antioxidant reserve capacity in CNS, yet certain regions may be particularly vulnerable to oxidative injury. GSH and AA can interact as a redox couple, such that depletion of either one of them can be compensated for by the continued presence or compensatory rise of the other. In this current study, we tested the hypothesis that deficiency in both GSH and AA will render these mice highly susceptible to endogenous oxidative damage in CNS. Methods: We crossed the Gulo?'/?' and Gclm?'/?' mouse lines to create Gclm?'/?'/Gulo?'/?' double-knockout (DKO) mice. Given that presence of one allele of either Gclm or Gulo gene is sufficient to attain normal GSH or AA levels, respectively, we used heterozygotes as the control for mutant homozygotes of each gene. We performed following analyses: levels of GSH, AA and lipid peroxidation in selected brain regions (n=5-7/per genotype), histological and immunohistochemical examination of brain (n=3-5/per genotype), and electroencephalography recording (n=3/per genotype). In a subset group of mice (n=5-7/per genotype), we supplemented AA in the drinking water to test whether this treatment afforded neuroprotection. Results: DKO mice exhibit growth retardation and lethal spontaneous epilepsy between the 2nd and the 3rd postnatal week. Around postnatal day 18, DKO mice had significant low levels of AA and high levels of lipid peroxidation in the cerebral cortex and hippocampus. Histologically, these mice showed neuronal loss and glial proliferation in these regions. The mortality in DKO mice was prevented by AA supplementation in the drinking water. Remarkably, when AA was stopped for 2 weeks, rescued DKO mice developed epilepsy and similar pathological changes in brain; these mice died within 3 weeks of AA withdrawal. Conclusions: Thus, this lethal epileptic phenotype, associated with combined GSH and AA deficiency, implies that a significant and underestimated role of GSH-AA interaction occurs in the CNS under physiological conditions. Funding: This study was supported, in part, by NIH grants R01 ES012463 and P30 ES006096.