Thioltransferase Knockout Model And The Function Of Thioltransferase In Cataractogenesis

Part1Age-dependent Morphological and Biochemical Changes inthe Eyes of Thioltransferase Knockout MicePurpose:Thioltransferase (TTase) is an important regulator for redox homeostasis in thelens. The purpose of this study was to examine the morphological andbiochemical alterations in the eyes of TTase knockout (TTase KO) and wild type(WT) mice as a function of age.Methods:TTase KO model has been established in this laboratory. TTase KO and WT miceof matching age between1-20months were examined using a slit lamp and thelens opacity was classified and graded by LOSII system. Each lens washomogenized in lysis buffer and processed for measurement of glutathione (GSH)level by DTNB colorimetric method. Examination of Protein-GSH mixed disulfides (PSSG) formation in the lens and the retina by Western blot analysisused an anti-GSH specific antibody. Immunoprecipitation was used to identify theproteins formed PSSG. Dethiolation of lens proteins was carried out usingpurified recombinant human lens TTase (RHLT).Results:The slit lamp examination showed an age-dependent nuclear cataractdevelopment in both eyes of the WT and KO mice. The onset of cataract was4months in the KO mice and8months in the WT mice. The opacity covered thewhole lens by20months in both groups. The GSH loss was also similar in bothgroups during aging. PSSG in the lenses of both groups showed progressiveelevation, whereas the lenses of the KO group had a higher level of PSSG after12months. In contrast, both groups showed a similar PSSC level, which was low at2-12months and doubled at20months. Western blot analysis showed that PSSGin the lens gradually increased with age and corroborated with the severity ofopacity of the lens. PSSG was more abundant in the retina of KO group at9months compared to that of the WT group. These GSH-conjugated proteins wereconfirmed as actin and glyceraldehyes3-phosphate dehydrogenase (GAPDH) byimmunoprecipitation and they could be eliminated when the homogenates weretreated with RHLT.Conclusions:The results showed that deletion of TTase gene in the mouse could lead to anearly age-dependent cataract formation and the PSSG formation in these lensesappeared to link directly to lens opacity. This data strengthens our hypothesis thatTTase plays an essential role in maintaining lens clarity. Part2Ultraviolet radiation-induced morphological andbiochemical changes in the mouse lenses in vivoPurpose:The purpose of the present study was to investigate the morphological andbiochemical alterations induced by in vivo exposure of ultraviolet B radiation(UVB) in the C57BL/6mouse lens at young and old age.Methods:Two age groups of C57BL/6mice (1month and16months) were exposed in vivoto UVB (302nm, at14.4kJ/m2) for15min. The lenses were examined48hoursafter exposure with a slit lamp. Lens epithelium (with the capsule) was peeled off,pooled and homogenized in lysis buffer, and processed for various biochemicalanalyses. Glutathione (GSH) level was determined by DTNB colorimetric method.Glyceraldehye3phosphate dehydrogenase (GAPDH) and thioltrnasferase (TTase)were assayed following a standard method. The level of protein-thiol mixeddisulfide, and levels of TTase and thioredoxin (Trx) were evaluated by westernblot analysis.Results: Both age groups of the mice developed cataract at the anterior subcapsular regionafter UVB radiation. GSH level and GAPDH activity were decreased in the lensepithelium. The average decrease of GSH levels was25%in the16months oldand10%in the1month old in comparison with the age-matched and untreatedcontrols. Similarly, GAPDH activity was decreased29%in the16months old and26%in the1month. TTase activity was increased16%in the1month old afterUVB radiation. Protein-GSH mixed disulfides (PSSG) was elevated after UVradiation while the whole lens lysates showed higher expression of TTase and Trxafter the radiation.Conclusions:The lens of the old mouse was more susceptible to UVB radiation than the youngone. The up-regulation of these antioxidant enzymes is likely used to protect thelens from UVB radiation-induced oxidative stress in the mouse.