No abstract

The cystine/glutamate antiporter (system xc-) is composed of a heavy chain subunit 4F2hc linked by a disulphide bond to a light chain xCT, which exchanges extracellular cystine, the disulphide form of the amino acid cysteine, for intracellular glutamate. In vitro research in the brain, kidney, and liver have shown this antiporter to play a role in minimising oxidative stress by providing a source of intracellular cysteine for the synthesis of the antioxidant glutathione. In vivo studies using the xCT knockout mouse revealed that the plasma cystine/cysteine redox couple was tilted to a more oxidative state demonstrating system xc- to also play a role in maintaining extracellular redox balance by driving a cystine/cysteine redox cycle. In addition, through import of cystine, system xc- also serves to export glutamate into the extracellular space which may influence neurotransmission and glutamate signalling in neural tissues. While changes to system xc- function has been linked to cancer and neurodegenerative disease, there is limited research on the roles of system xc- in the different tissues of the eye, and links between the antiporter, aging, and ocular disease. Hence, this review seeks to consolidate research on system xc- in the cornea, lens, retina, and ocular humours conducted across several species to shed light on the in vitro and in vivo roles of xCT in the eye and highlight the utility of the xCT knockout mouse as a tool to investigate the contribution of xCT to age-related ocular diseases.

Background: The lysosomal storage disease cystinosis is caused by mutations in CTNS, encoding a cystine transporter, and in its severest form leads to proximal tubule dysfunction followed by kidney failure. Patients receive the drug-based therapy cysteamine from diagnosis. However, despite long-term treatment, cysteamine only slows the progression of end-stage renal disease and a kidney transplant is inevitable. Pre-clinical testing in cystinotic rodents is required to evaluate new therapies; however, the current models are sub-optimal. To solve this problem we generated a new cystinotic rat model. Methods: We utilized CRISPR/Cas9-mediated gene editing to disrupt exon 3 of Ctns and measured various parameters over a 12-month time-course including blood and tissue cystine levels, urine and serum electrolytes, and analysed the histopathology and immunohistochemistry of the kidney. Results: Ctns-/- rats display hallmarks of cystinosis by 3-6 months of age as seen by a failure to thrive, excessive thirst and urination, cystine accumulation in tissues, corneal cystine crystals, a loss of Lrp2 in proximal tubules and immune cell infiltration. High levels of glucose, calcium, albumin and protein are excreted at 6-months of age, consistent with the onset of Fanconi syndrome, with a progressive diminution of urine urea and creatinine from 9-months of age, indicative of chronic kidney disease. The kidney histology and immunohistochemistry showed proximal tubule atrophy and glomerular damage as well as classic swan neck lesions. Overall, Ctns-/- rats show a disease progression that more faithfully recapitulates nephropathic cystinosis than existing rodent models. Conclusions: The Ctns-/- rat provides an excellent new rodent model of nephropathic cystinosis that is ideally suited for conducting pre-clinical drug testing and a powerful tool to advance cystinosis research.

Background: Cystine/glutamate exchanger (xCT) knockout mice are reported to exhibit an oxidative shift in the plasma cystine/cysteine ratio reminiscent of that seen in human plasma of ageing individuals. This suggests that the xCT knockout mouse is a model of accelerated ageing. The aim of this study was to examine the progression of age-related pathologies in the ocular tissues of wild-type mice and compare this to the xCT knockout mice. Methods: Wild-type and xCT knockout mice were examined longitudinally or as separate groups of animals at six weeks, three months, six months, nine months, and 12 months of age. All groups of mice were anaesthetised, intraocular pressure measured using the iCare TONOLAB rebound tonometer and eyes examined using the Micron IV system. Results: While the aim of the study was to determine if xCT knockout mice developed agerelated pathologies earlier than wild-type mice, it was inadvertently discovered in the longitudinal cohort of animals, that the eyes developed corneal lesions in both groups of animals by six months of age, which obscured examination of the lens and retina. These lesions were not characteristic of age-related pathologies, but rather due to an external stressor. Lesions in the xCT knockout mice developed at an earlier age compared to wild-type mice, suggesting that loss of xCT exacerbates damage to the cornea, most likely caused by the rebound tonometer. When the same ocular procedures were performed on separate cohorts of mice of specific ages, no corneal lesions were detected for both groups of mice. Conclusions: While it may seem advantageous to examine the same cohort of mice to monitor the development of age-related pathologies, the type of ophthalmic tests conducted needs to be carefully considered to avoid introducing pathologies that are inadvertently a result of the examination process itself.

To investigate whether human donor lenses are capable of exporting reduced glutathione. Methods: Human lenses of varying ages were cultured in artificial aqueous humor for 1 hour under hypoxic conditions to mimic the physiologic environment and reduced glutathione (GSH) and oxidized glutathione (GSSG) levels measured in the media and in the lens. Results: Human donor lenses released both GSH and GSSG into the media. Donor lenses cultured in the presence of acivicin, a γ-glutamyltranspeptidase inhibitor, exhibited a significant increase in GSSG levels (P < 0.05), indicating that GSSG undergoes degradation into its constituent amino acids. Screening of GSH/GSSG efflux transporters revealed Mrp1, Mrp4, and Mrp5 to be present at the transcript level, but only Mrp5 was expressed at the protein level. Blocking Mrp5 function with the Mrp inhibitor MK571 led to a significant decrease in GSSG efflux (P < 0.05), indicating that Mrp5 is likely to be involved in mediating GSSG efflux. Measurements of efflux from the anterior and posterior surface of the lens revealed that GSH and GSSG efflux occurs at both surfaces but predominantly at the anterior surface. Conclusions: Human lenses export GSH and GSSG into the surrounding ocular humors, which can be recycled by the lens to maintain intracellular GSH homeostasis or used by neighboring tissues to maintain GSH levels. Translational Relevance: Early removal of a clear lens, as occurs to treat myopia and presbyopia, would eliminate this GSH reservoir and reduce the supply of GSH to other tissues, which, over time, may have clinical implications for the progression of other ocular diseases associated with oxidative stress.