This research program addresses the hypothesis that extensive damage to lens crystallins by amino-carbonyl reactions between reducing sugars, ascorbic acid and proteins (so-called Maillard reaction) contributes to the aging of the human lens. Our key hypothesis is that the accumulation of these glycation products predisposes the lens toward cataractogenesis, and that much of the reaction is catalyzed by ascorbic acid oxidation products. Toward this hypothesis progress several breakthroughs have been achieved in the previous funding period. First, the structure of the fluorophore LM-1 was identified as Vesperlysine A, an ascorbate catalyzed protein crosslink that is specifically increased in diabetic human lenses. Second, we discovered carboxymethyl-lysine, a major ascorbylation product, bindsredox active Cu 2+ in the human lens. Third, we have pioneered a novel """"""""metabolomics"""""""" approach based on fluorine-labeled ascorbic acid (F-ASA) and high resolution19F-NMR spectroscopy and found that oxidatively stressed lens epithelial cells and cataracts accumulate F-ASA degradation products. Fourth, we discovered, cloned and elucidated the catalytic mechanism of deglycating enzymes for future use in transgenic animal models of excessive glycation. Finally and most recently, we discovered evidence of extensive glycation-catalyzed conversion of arginine into ornithine in the aging human lens. In the coming years we propose an entirely new and, we believe, highly promising approach to the problem of lenticular aging by ascorbylation and glycation through the creation of three transgenic/conditionalknockout models of accelerated damage to the lens. Based on the discovery that rodent lenses are deficient in ascorbic acid uptake, we propose to first create a mouse model of the human lens by overexpressing the human Vitamin C Transporter 2 (hSVCT2) in the lens. The impact of this transgene on lenticular homoestasis, protein pigmentation, fragmentation and crosslinkingwill be studied. This humanized hSVCT+/+ mouse will be then be crossbred with a mouse deficient in glutathionesynthesis obtained by conditionally knocking out y-glutamyl cysteine ligase (yGclc)from the lens. The impact of the knockouton lens crystallin changes with be similarly studied. The homozygous hSVCT^/yGcIc""""""""'"""""""" hybrid mouse lens is expected to accumulate substantial levels of reactive ascorbylation products due to impairment of ascorbic acid recycling. The approach taken will allow us to study separately the impact of multipleforms of protein damage, i.e. ascorbylation, glycation and oxidation on lens chaperone and other properties. With the help of these genetic manipulations, it is hoped that the 70-yr long aging process of the human lens can be condensed into 24 mos. This mouse model is expected to become a potent tool for the development of novel genetic and pharmacological interventions against lenticular aging.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY007099-20
Application #
7780359
Study Section
Anterior Eye Disease Study Section (AED)
Program Officer
Araj, Houmam H
Project Start
1987-08-01
Project End
2012-02-28
Budget Start
2010-03-01
Budget End
2012-02-28
Support Year
20
Fiscal Year
2010
Total Cost
$371,299
Indirect Cost
Name
Case Western Reserve University
Department
Pathology
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
Whitson, Jeremy A; Wilmarth, Phillip A; Klimek, John et al. (2017) Proteomic analysis of the glutathione-deficient LEGSKO mouse lens reveals activation of EMT signaling, loss of lens specific markers, and changes in stress response proteins. Free Radic Biol Med 113:84-96
Feng, Weiyi; Rosca, Mariana; Fan, Yuxuan et al. (2017) Gclc deficiency in mouse CNS causes mitochondrial damage and neurodegeneration. Hum Mol Genet 26:1376-1390
Whitson, Jeremy A; Sell, David R; Goodman, Michael C et al. (2016) Evidence of Dual Mechanisms of Glutathione Uptake in the Rodent Lens: A Novel Role for Vitreous Humor in Lens Glutathione Homeostasis. Invest Ophthalmol Vis Sci 57:3914-25
Sell, David R; Sun, Wanjie; Gao, Xiaoyu et al. (2016) Skin collagen fluorophore LW-1 versus skin fluorescence as markers for the long-term progression of subclinical macrovascular disease in type 1 diabetes. Cardiovasc Diabetol 15:30
Fan, Xingjun; Zhou, Sheng; Wang, Benlian et al. (2015) Evidence of Highly Conserved ?-Crystallin Disulfidome that Can be Mimicked by In Vitro Oxidation in Age-related Human Cataract and Glutathione Depleted Mouse Lens. Mol Cell Proteomics 14:3211-23
Fessel, Gion; Li, Yufei; Diederich, Vincent et al. (2014) Advanced glycation end-products reduce collagen molecular sliding to affect collagen fibril damage mechanisms but not stiffness. PLoS One 9:e110948
Monnier, Vincent M; Sun, Wanjie; Sell, David R et al. (2014) Glucosepane: a poorly understood advanced glycation end product of growing importance for diabetes and its complications. Clin Chem Lab Med 52:21-32
Linetsky, Mikhail; Raghavan, Cibin T; Johar, Kaid et al. (2014) UVA light-excited kynurenines oxidize ascorbate and modify lens proteins through the formation of advanced glycation end products: implications for human lens aging and cataract formation. J Biol Chem 289:17111-23
Monnier, Vincent M; Sell, David R; Strauch, Christopher et al. (2013) The association between skin collagen glucosepane and past progression of microvascular and neuropathic complications in type 1 diabetes. J Diabetes Complications 27:141-9
Fan, Xingjun; Liu, Xiaoqin; Hao, Shuyu et al. (2012) The LEGSKO mouse: a mouse model of age-related nuclear cataract based on genetic suppression of lens glutathione synthesis. PLoS One 7:e50832

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