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Grant Abstract

Grant Number: 1R21EY020963-01

Abstract: DESCRIPTION (provided by applicant): Maintaining the function of the lens ?A- and ?B-crystallins with aging is the key event in preventing cataracts. Although it is well known that ?A- and ?B-crystallins undergo extensive modifications (crosslinking, glycation, oxidation, phosphorylation and truncation) over a lifetime, it is less clear when these modifications occur or in which zone of the lens (nuclear and cortical regions). This application takes advantage of two experimental approaches: (1) the ICR/f rat, a model of age-related cataracts, that spontaneously forms cataracts witihin 10- 11 weeks of life, and (2) mass spectrometry imaging of sections of the lens that allows determination of the spatial distribution (at 100 micron resolution) of individual ?A- and ?B-crystallins. In preliminary experiments we have successfully developed methods for obtaining tissue sections of the rat lens, for spotting with matrix robotically and obtaining images of the distribution of lens proteins. The goal of this application is to examine two hypotheses: (1) ?A- and ?B-crystallins undergo modifications, particularly truncation, that cause them to be specifically localized within the lens at particular stages of development, processes that are altered by intervention with genistein and disulfiram, and (2) that the modifications of the ?A- and ?B-crystallins decrease their solubility, thereby accounting for their localization. In the first aim, ICR/f rats and mass spectrometry imaging will be used to determine the effect of genistein in the diet and disulfiram by eye drops on the type and timing of ?A- and ?B-crystallins' truncation/modifications and distribution during cataract development. Lenses from untreated ICR/f rats and Wistar rats (the latter do not have cataracts) will be examined to determine the onset of the appearance of specific cataract-associated ?A- and ?B-crystallin fragments. In the second aim, selected truncated ?A- and ?B-crystallins (e.g., m/z 6409), showing marked regional distribution by mass spectrometry imaging, will be extracted from the lens (into water-soluble and water-insoluble/urea- soluble fractions). These will be purified using chromatographic and electrophoretic techniques and then fully characterized by determination of their accurate molecular weights, sequence and modifications. These studies will highlight the solubility/function information embedded in the sequence of a protein that has such an extended lifetime and also establish the ICR/f rat as a convenient test bed for exploring the mechanism of interventions to prevent/delay cataracts. PUBLIC HEALTH RELEVANCE: 50% of the general population who reach 70 years of age will have required lens replacement surgery as well as having previous diminished vision. Given the increasing proportion of the elderly in the USA and the importance of sustaining quality of life, interventions to reduce the risk of cataracts are warranted. The ICR/f rat is a model to evaluate both new therapeutic interventions as well as risk factors in dietary supplements.

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