The studies proposed here follow 8 years of NEI supported work on the causes and mechanisms of age- related cataract (ARC). This work has shown the mechanisms at work in the development of ARC, in mice and rats, and still underway, in dogs and humans. The findings in ARC of detachment of the lens epithelial cells (LEC) from the lens surface and migration to the interior at abnormal sites, with bits of nuclear DNA, free DNA, and reactive oxygen species (ROS) at those sites we find to be characteristic of ARC. These characteristics are present in all 4 species examined, and are very similar in mice that develop delayed cataracts some 5-11 months after x-irradiation to the head only (thus providing a known ROS caused reference cataract). Our overall hypothesis is that ARC development begins with oxidative damage to LEC. We now seek answers for the following questions, using specific fluorescent dyes with confocal readout in living extirpated lenses, protein specific antibodies in lens tissue sections, clonal growth capacity in LEC cultures, LEC chromosome spreads with c-banding, G-banding and chromosome painting (SKY and SCAN procedures). The first studies will be in young, old and cataractous old mice (however, we will extend the studies into the larger species as initial findings warrant). We will ask the following questions: 1) Does the loss of surface LEC allow O2 penetration into the lens, resulting in the development of ROS? 2) We know that overall mitochondrial activity on the lens surface is reduced with age and ARC development, does this contribute to ROS formation? 3) Since cell excess organelle debris accumulates in ARC at both the Bow region and at abnormal interior sites- is there a debris removal failure of lysosomes and lysosomal enzyme activity? 4) Is the abnormal loss of LEC from the lens surface (a possible initiator of the other mechanistic changes) a result of ROS-produced chromosomal DNA damage (and consequent message) that accumulates over the lifetime of the animal? 5) Is the mis-migration allowed by altered cell-to-cell adhesion factors? 6) If ROS damage to LEC is the initiating factor in ARC, can transgenic mice with either added anti-oxidant enzymes, on anti-oxidant diets, or with altered metabolism that reduces ROS release from mitochondrial activity show a significant delay in the development of ARC? Age-related (senile) cataract is the leading cause of blindness in the world. Although cataract surgery is an effective treatment available in the """"""""developed"""""""" nations, it is not without risk and involves a period of visual disadvantage until surgery is chosen, as well as a large expense, either to the individual, his or her health plan, or both. This expense has been estimated as several billion dollars per year. If the advancement from minor lens opacity to frank cataract could be delayed by as little as 10 years, a great saving in inconvenience, risk, and cost could be largely avoided. My laboratory is engaged in the determination of the cause(s) of age-related cataract and the mechanism(s) of its formation. We have found that these mechanisms and the cellular events included in them are the same in several mammalian species (mouse, rat, dog, and human). These involve 1) the loss of lens epithelial cells from the surface by migration to the interior of the lens at inappropriate sites (not at the lens equator, where they normally enter to form the lens fibers), 2) a failure of these cells to dissolve and disperse their internal content of nucleus, mitochondria, and other cellular organelles to become the clear lens fibers, and 3) the development of large quantities of free oxygen radicals (reactive oxygen species, ROS) at the sites of mis-migrating lens cells. These ROS containing areas develop into cataracts with aggregated lens proteins obstructing the passage of light through the lens. The above-described events become more and more advanced after the subject passes middle age and moves to old age. My laboratory is studying the entry of local environmental oxygen into the lens at sites at which the surface cells have migrated to the lens interior and left an uncovered lens surface, and its proposed entry there, with initiation of the ROS that is found at the cataract sites. To do this we use fluorescent dyes specific for DNA and ROS, the presence of which is determined quantitatively by use of a confocal microscope. As a part of these studies, we also plan to determine the reasons why surface lens cells in old animals breakaway from their normal sites at improper places and migrate as strands of cells into the lens interior, contributing to age- related cataract there. In related work we are studying the effect of ROS on chromosomal DNA strands in lens epithelial cells of living aging animals of the above-mentioned 4 species, suspecting that essential gene messages in ROS-damaged cells is distorted and causes their abnormal behavior and function. We have available several mutant mouse strains with increased resistance to the development of ROS, by means of enzymes that convert ROS to harmless compounds, such as water. We will determine if they show a reduction of age-related cataract development and in the mechanisms that initiate it. It is known that ROS induces deletions, exchanges of chromatin exchanges between sister chromatids, and the doubling of the number of chromosomes (tetraploidy) in such cells, and that these events are accelerated in senescent cells and in old animals. We intend to match the development of oxidized areas of DNA (called adducts) on the lens cell chromosomes, and the attempted repair of this DNA damage by so-called repair proteins that are sequestered to sites of DNA damage, with the lens cell changes that accompany the development of age- related cataract. Here, our ROS protection mutants should be resistant and will help the determine cause and effect. We have also developed a model receiving head-only x-ray, known to produce ROS in the target tissue. This (young) mouse model develops cataracts that are indistinguishable from age-related cataracts, but only several months after the radiation. Thus we have two sets of models, one with an anti-oxidant delay in apparently ROS-initiated age-related cataracts, and a second with x-ray induced, ROS initiated early developing cataracts. These will aid in the search for initiating events and the mechanisms leading up to the cataract formation.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY011733-10
Application #
7531035
Study Section
Special Emphasis Panel (ZRG1-BDCN-F (02))
Program Officer
Araj, Houmam H
Project Start
1998-03-01
Project End
2010-11-30
Budget Start
2008-12-01
Budget End
2009-11-30
Support Year
10
Fiscal Year
2009
Total Cost
$390,000
Indirect Cost
Name
University of Washington
Department
Pathology
Type
Schools of Medicine
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
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Pendergrass, William; Penn, Philip; Possin, Daniel et al. (2005) Accumulation of DNA, nuclear and mitochondrial debris, and ROS at sites of age-related cortical cataract in mice. Invest Ophthalmol Vis Sci 46:4661-70
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Wolf, Norman; Galecki, Andrzej; Lipman, Ruth et al. (2004) Quantitative trait locus mapping for age-related cataract severity and synechia prevalence using four-way cross mice. Invest Ophthalmol Vis Sci 45:1922-9

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