Ascorbic acid export from human donor lenses: Is the lens a source of ascorbic acid in the ocular humors?

In previous work, we have shown that the lens acts a reservoir of the antioxidant glutathione (GSH), capable of exporting this antioxidant into the ocular humors and potentially protecting the tissues of the eye that interface with these humors from oxidative stress. In this study, we have extended this work by examining whether the lens acts as a source of ascorbic acid (AsA) to maintain the high levels of AsA known to be present in the ocular humors either by the direct export of AsA into the humors and/or by functioning as a recycling site for AsA, via the direct uptake of oxidised ascorbate (DHA) from the humors, its regeneration to AsA in the lens and then its subsequent export back into the humors. To test this, human lenses of varying ages were cultured for 1 h under hypoxic conditions and AsA/DHA levels measured in the media and in the lens. Human lenses were also cultured in compartmentalised chambers to determine whether efflux of AsA/DHA occurs at the anterior or posterior surface. Immunohistochemistry was performed on human donor lenses and sections labelled with antibodies against GLUT1, a putative DHA uptake transporter. Vitreous humor was collected from patients undergoing vitrectomy who either had a natural clear lens, an artificial intraocular implant (IOL) or a cataractous lens, and AsA/DHA and GSH and oxidised GSH (GSSG) measured. We found that cultured human donor lenses released both AsA and DHA into the media. Culturing of lenses in a compartmentalised chamber revealed that AsA and DHA efflux occurs at both surfaces, with relatively equal amounts of AsA and DHA released from each surface. The posterior surface of the lens was shown to express the GLUT1 transporter. Analysis of vitreous samples from patients undergoing vitrectomy revealed that vitreous GSH and AsA levels were similar between the natural lens group, IOL and cataractous lens group. Taken together, while human donor lenses were shown to export AsA and DHA into the surrounding media, the amount of AsA and DHA released from donor lenses was low and not sufficient to sustain the high levels of total AsA normally present in the humors. This suggests that although the lens is not the main source for maintaining high levels of AsA in the ocular humors, the lens may help to support local AsA levels close to the lens.

Changes in glutamate and glutamine distributions in the retinas of cystine/glutamate antiporter knockout mice.

Purpose: The cystine/glutamate antiporter is involved in the export of intracellular glutamate in exchange for extracellular cystine. Glutamate is the main neurotransmitter in the retina and plays a key metabolic role as a major anaplerotic substrate in the tricarboxylic acid cycle to generate adenosine triphosphate (ATP). In addition, glutamate is also involved in the outer plexiform glutamate-glutamine cycle, which links photoreceptors and supporting Müller cells and assists in maintaining photoreceptor neurotransmitter supply. In this study, we investigated the role of xCT, the light chain subunit responsible for antiporter function, in glutamate pathways in the mouse retina using an xCT knockout mouse. As xCT is a glutamate exporter, we hypothesized that loss of xCT function may influence the presynaptic metabolism of photoreceptors and postsynaptic levels of glutamate. Methods: Retinas of C57BL/6J wild-type (WT) and xCT knockout (KO) mice of either sex were analyzed from 6 weeks to 12 months of age. Biochemical assays were used to determine the effect of loss of xCT on glycolysis and energy metabolism by measuring lactate dehydrogenase activity and ATP levels. Next, biochemical assays were used to measure whole-tissue glutamate and glutamine levels, while silver-intensified immunogold labeling was performed on 6-week and 9-month-old retinas to visualize and quantify the distribution of glutamate, glutamine, and related neurochemical substrates gamma-aminobutyric acid (GABA) and glycine in the different layers of the retina. Results: Biochemical analysis revealed that loss of xCT function did not alter the lactate dehydrogenase activity, ATP levels, or glutamate and glutamine contents in whole retinas in any age group. However, at 6 weeks of age, the xCT KO retinas revealed altered glutamate distribution compared with the age-matched WT retinas, with accumulation of glutamate in the photoreceptors and outer plexiform layer. In addition, at 6 weeks and 9 months of age, the xCT KO retinas also showed altered glutamine distribution compared with the WT retinas, with glutamine labeling significantly decreased in Müller cell bodies. No significant difference in GABA or glycine distribution were found between the WT and xCT KO retinas at 6 weeks or 9 months of age. Conclusion: Loss of xCT function results in glutamate metabolic disruption through the accumulation of glutamate in photoreceptors and a reduced uptake of glutamate by Müller cells, which in turn decreases glutamine production. These findings support the idea that xCT plays a role in the presynaptic metabolism of photoreceptors and postsynaptic levels of glutamate and derived neurotransmitters in the retina.

Cystinosin-deficient rats recapitulate the phenotype of nephropathic cystinosis.

The lysosomal storage disease cystinosis is caused by mutations in CTNS, encoding the cystine transporter cystinosin, 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. Preclinical testing in cystinotic rodents is required to evaluate new therapies; however, the current models are suboptimal. To solve this problem, we generated a new cystinotic rat model using CRISPR/Cas9-mediated gene editing to disrupt exon 3 of Ctns and measured various parameters over a 12-mo time course. Ctns-/- rats display hallmarks of cystinosis by 3-6 mo of age, as demonstrated by a failure to thrive, excessive thirst and urination, cystine accumulation in tissues, corneal cystine crystals, loss of LDL receptor-related protein 2 in proximal tubules, and immune cell infiltration. High levels of glucose, calcium, albumin, and protein were excreted at 6 mo of age, consistent with the onset of Fanconi syndrome, with a progressive diminution of urine urea and creatinine from 9 mo of age, indicative of chronic kidney disease. 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. The Ctns-/- rat provides an excellent new rodent model of nephropathic cystinosis that is ideally suited for conducting preclinical drug testing and is a powerful tool to advance cystinosis research.NEW & NOTEWORTHY Animal models of disease are essential to perform preclinical testing of new therapies before they can progress to clinical trials. The cystinosis field has been hampered by a lack of suitable animal models that fully recapitulate the disease. Here, we generated a rat model of cystinosis that closely models the human condition in a timeframe that makes them an excellent model for preclinical drug testing as well as being a powerful tool to advance research.

Characterisation of Glutathione Export from Human Donor Lenses.

Purpose: 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.

Identification, Expression, and Roles of the Cystine/Glutamate Antiporter in Ocular Tissues.

The cystine/glutamate antiporter (system x c -) 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.

Corneal opacities in mice exposed to repeated contact procedures during ocular examinations.

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 age-related 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.

Mapping of the cystine-glutamate exchanger in the mouse eye: a role for xCT in controlling extracellular redox balance.

The cystine-glutamate exchanger (system xc-) is responsible for the exchange of extracellular cystine for intracellular glutamate. In this study, we mapped the expression of xCT, the light chain subunit of system xc- in the different tissues of 3-6-week-old mouse (C57BL/6J) eye and have used an xCT knockout mouse to verify labelling specificity. Moreover, using the xCT knockout mouse, we investigated whether xCT was involved in maintaining extracellular redox balance in the eye. xCT transcript and protein were present in the cornea, lens and retina of wild-type mice, but not knockout mice. xCT was localised to the corneal epithelium, and the lens epithelium and cortical fibre cells but was absent in the iris. xCT localisation could not be determined in the ciliary body or retina, since xCT labelling was also detected in the knockout indicating a lack of specificity of the xCT antibody in tissues of a neural origin. Intracellular cysteine and cystine concentrations were similar in the wild-type and xCT knockout mouse for the cornea, lens, and retina. While extracellular cysteine levels were similar between the plasma, aqueous humour, and vitreous humour of the wild-type and xCT knockout mouse, extracellular cystine levels in the plasma and aqueous were significantly elevated in the xCT knockout mouse relative to the wild type. This suggests that loss of xCT results in an increased oxidative environment, particularly within the anterior chamber of the eye in which the aqueous humour resides. How this oxidative shift impacts ocular tissues that interface with the aqueous humour over time will be the focus of future work.