Early onset of age-related changes in the retina of cystine/glutamate antiporter knockout mice.

To determine the role of the cystine/glutamate antiporter on retinal structure and function, retinas of C57Bl/6J wild-type and xCT knockout mice, lacking the xCT subunit of the cystine/glutamate antiporter were examined from 6 weeks to 12 months of age. Fundoscopy, optical coherence tomography (OCT), and whole mount retinal autofluorescence imaging were used to visualise age-related retinal spots. Glial fibrillary acidic protein (GFAP) immunolabelling was used to assess retinal stress. Retinal function was evaluated using full-field and focal electroretinograms. Examinations revealed retinal spots in both wild-type and xCT knockout mice with the number of spots greater at 9 months in the knockout compared to wild-type. OCT confirmed these discrete spots were located at the retinal pigment epithelium (RPE)-photoreceptor junction and did not label with drusen markers. Whole mount lambda scans of the 9 month xCT knockout retinas revealed that the photoreceptor autofluorescence matched the spots, suggesting these spots were retinal debris. GFAP labelling was increased in knockout retinas compared to wild-type indicative of retinal stress, and the discrete spots were associated with migration of microglia/macrophages to the RPE-retina intersection. OCT revealed that the superior retina was thinner at 9 months in knockout compared to wild-type mice due to changes to the outer nuclear and photoreceptor layers. While global retinal function was not affected by loss of xCT, focal changes in retinal function were detected in areas where spots were present. Tother these results suggest that the xCT KO mice exhibit features of accelerated ageing and suggests that this mouse model may be useful for studying the underlying cellular pathways in retinal ageing.

Early Onset of Age-Related Cataracts in Cystine/Glutamate Antiporter Knockout Mice.

Purpose: The purpose of this study was to determine the importance of the xCT is a subunit. The cystine/glutamate antiporter is actually system xc-xCT subunit of the cystine/glutamate antiporter in maintaining redox balance by investigating the effects of the loss of xCT on lens transparency and cystine/cysteine balance in the aqueous humour. Methods: C57Bl/6 wild-type and xCT knockout mice at five age groups (6 weeks to 12 months) were used. Lens transparency was examined using a slit-lamp and morphological changes visualized by immunolabelling and confocal microscopy. Quantification of glutathione in lenses and cysteine and cystine levels in the aqueous was conducted by liquid chromatography tandem mass spectrometry (LC-MS/MS). Results: Slit-lamp examinations revealed that 3-month-old wild-type mice and xCT knockout mice lenses exhibited an anterior localized cataract. The frequency of this cataract significantly increased in the knockout mice compared to the wild-type mice. Morphological studies revealed a localized swelling of the lens fiber cells at the anterior pole. Glutathione levels in whole lenses were similar between wild-type and knockout mice. However, glutathione levels were significantly decreased at 3 months in the knockout mice in the lens epithelium compared to the wild-type mice. Aqueous cysteine levels remained similar between wild-type and knockout mice at all age groups, whereas cystine levels were significantly increased in 3-, 9-, and 12-month-old knockout mice compared to wild-type mice. Conclusions: Loss of xCT resulted in the depletion of glutathione in the epithelium and an oxidative shift in the cysteine/cystine ratio of the aqueous. Together, these oxidative changes may contribute to the accelerated development of an anterior cataract in knockout mice, which appears to be a normal feature of aging in wild-type mice.

Identification and Functional Characterization of a GSH Conjugate Efflux Pathway in the Rat Lens.

PURPOSE: To identify and functionally characterize transporters involved in the release of glutathione (GSH) conjugates from the rat lens. METHODS: Polymerase chain reaction and Western blotting were used to screen for the presence of multidrug resistance-associated protein (Mrp) and organic anion transporting polypeptide (Oatp) isoforms, and immunohistochemistry used to localize Mrp isoforms. To test for Mrp function, lenses were loaded with 5-chloromethylfluorescein diacetate and monochlorobimane to form the fluorescent GSH conjugates glutathione methylfluorescein (GS-MF) and glutathione bimane (GS-B), respectively, and cultured in artificial aqueous humour (AAH) in the presence or absence of MK571, an Mrp-specific inhibitor, or benzbromarone, a nonspecific organic anion transporter inhibitor. Glutathione-MF and GS-B fluorescence were measured in the AAH media and lenses. RESULTS: Multidrug resistance-associated proteins 1, 4, 5, and Oatp1a4 were present at the transcript level, but only Mrp1, 4, and 5 were detected at the protein level. Multidrug resistance-associated proteins 1 and 5 localized to the epithelium and peripheral fiber cells, whereas Mrp4 strongly labeled the nuclei. Glutathione-MF and GS-B efflux was significantly decreased and accumulation in the lens significantly increased in the presence of MK571, indicating that the Mrps are the predominant transporters involved in GSH conjugate release from the lens. Glutathione-B conjugate efflux was further inhibited in the presence of benzbromarone, suggesting that alternative organic anion pathways were involved in mediating GS-B efflux. CONCLUSIONS: Multidrug resistance-associated proteins are present in the lens and may be used to remove endogenous and exogenous compounds from the lens via GSH conjugation. This may represent an important pathway of detoxification required to minimize oxidative stress and maintain lens homeostasis.