Alzheimer's disease amyloid-beta links lens and brain pathology in Down syndrome.

Down syndrome (DS, trisomy 21) is the most common chromosomal disorder and the leading genetic cause of intellectual disability in humans. In DS, triplication of chromosome 21 invariably includes the APP gene (21q21) encoding the Alzheimer's disease (AD) amyloid precursor protein (APP). Triplication of the APP gene accelerates APP expression leading to cerebral accumulation of APP-derived amyloid-beta peptides (Abeta), early-onset AD neuropathology, and age-dependent cognitive sequelae. The DS phenotype complex also includes distinctive early-onset cerulean cataracts of unknown etiology. Previously, we reported increased Abeta accumulation, co-localizing amyloid pathology, and disease-linked supranuclear cataracts in the ocular lenses of subjects with AD. Here, we investigate the hypothesis that related AD-linked Abeta pathology underlies the distinctive lens phenotype associated with DS. Ophthalmological examinations of DS subjects were correlated with phenotypic, histochemical, and biochemical analyses of lenses obtained from DS, AD, and normal control subjects. Evaluation of DS lenses revealed a characteristic pattern of supranuclear opacification accompanied by accelerated supranuclear Abeta accumulation, co-localizing amyloid pathology, and fiber cell cytoplasmic Abeta aggregates (approximately 5 to 50 nm) identical to the lens pathology identified in AD. Peptide sequencing, immunoblot analysis, and ELISA confirmed the identity and increased accumulation of Abeta in DS lenses. Incubation of synthetic Abeta with human lens protein promoted protein aggregation, amyloid formation, and light scattering that recapitulated the molecular pathology and clinical features observed in DS lenses. These results establish the genetic etiology of the distinctive lens phenotype in DS and identify the molecular origin and pathogenic mechanism by which lens pathology is expressed in this common chromosomal disorder. Moreover, these findings confirm increased Abeta accumulation as a key pathogenic determinant linking lens and brain pathology in both DS and AD.

Multiple and additive functions of ALDH3A1 and ALDH1A1: cataract phenotype and ocular oxidative damage in Aldh3a1(-/-)/Aldh1a1(-/-) knock-out mice.

ALDH3A1 (aldehyde dehydrogenase 3A1) is abundant in the mouse cornea but undetectable in the lens, and ALDH1A1 is present at lower (catalytic) levels in the cornea and lens. To test the hypothesis that ALDH3A1 and ALDH1A1 protect the anterior segment of the eye against environmentally induced oxidative damage, Aldh1a1(-/-)/Aldh3a1(-/-) double knock-out and Aldh1a1(-/-) and Aldh3a1(-/-) single knock-out mice were evaluated for biochemical changes and cataract formation (lens opacification). The Aldh1a1/Aldh3a1- and Aldh3a1-null mice develop cataracts in the anterior and posterior subcapsular regions as well as punctate opacities in the cortex by 1 month of age. The Aldh1a1-null mice also develop cataracts later in life (6-9 months of age). One- to three-month-old Aldh-null mice exposed to UVB exhibited accelerated anterior lens subcapsular opacification, which was more pronounced in Aldh3a1(-/-) and Aldh3a1(-/-)/Aldh1a1(-/-) mice compared with Aldh1a1(-/-) and wild type animals. Cataract formation was associated with decreased proteasomal activity, increased protein oxidation, increased GSH levels, and increased levels of 4-hydroxy-2-nonenal- and malondialdehyde-protein adducts. In conclusion, these findings support the hypothesis that corneal ALDH3A1 and lens ALDH1A1 protect the eye against cataract formation via nonenzymatic (light filtering) and enzymatic (detoxification) functions.

Age-related cataracts in alpha3Cx46-knockout mice are dependent on a calpain 3 isoform.

PURPOSE: Previous studies have demonstrated that in 129alpha3Cx46-/- mice, age-related nuclear cataract is formed. In the present study, a more in vivo-relevant model was generated to test the hypothesis that the calpain 3 gene is involved in age-related nuclear cataractogenesis in alpha3Cx46 knockout mice. METHODS: To test the hypothesis that the calpain 3 gene is involved in age-related nuclear cataractogenesis in alpha3Cx46 knockout mice, 129alpha3Cx46-/- and CAPN3-/- mice were mated to generate homozygous double-knockout (dKO) mice. Lenses from the mice were examined by visual observation, laser scan analysis, and histologic and biochemical methods. RESULTS: In the absence of the CAPN3 gene, the formation of a cataract was delayed, and its appearance was changed to a more diffuse, pulverulent type. Unlike in the 129alpha3Cx46-/- mouse, cleavage of gamma-crystallin was not detected in the dKO mouse. In both 129alpha3Cx46-/- and dKO mice, total Ca2+ increased. CONCLUSIONS: The present study shows for the first time that calpain 3 is necessary for the formation of age-dependent nuclear cataracts in alpha3Cx46-/- mice. Evidence that the calpain 3 gene is directly involved in, or part of the pathway that leads to, gamma-crystallin cleavage is presented. These results are consistent with the hypothesis that the loss of alpha3Cx46 leads to increased levels of Ca2+ ions, and this increase activates the CAPN3 isoform, Lp82/85, which results in the formation of a nuclear cataract.

Computer modeling of secondary fiber development and growth: I. Nonprimate lenses.

PURPOSE: The purpose of this study was to use qualitative and quantitative structural data from nonprimate lenses with branched (Y and line) sutures to generate computer models (animations) of secondary fiber development and suture formation. METHODS: A minimum of 12-18 adult lenses/species (mice, cows, frogs, and rabbits) were used in this study. Lenses were analyzed by light (LM), transmission (TEM), and scanning electron microscopy (SEM). Fiber width, thickness, and length were ascertained from micrographs and by using formulations to calculate distances between degrees of latitude and longitude on asymmetrical oblate spheroids. This information was then used to create scale computer assisted drawings (CADs) of fibers at different stages of their development. The CADs were then placed on a timeline and animated to produce dynamic representations of secondary fiber development and growth. RESULTS: Animating secondary fiber development and suture formation with the inclusion of quantifiable differences in fiber dimensions at progressive stages of their differentiation revealed the following: first, there is the presumption that fibers migrate, rotate, and elongate until they reach their sutural destinations is not likely to be correct. When developing fibers reach approximately 60-65% of their eventual total length, their migration and rotation is complete. The remaining fiber elongation (the production of end segments) occurs without either concomitant cellular migration or rotation. Second, it is presumed that suture branches originate peripherally and are then constructed sequentially until all of the branches come to confluence at the poles is also not likely to be correct. While suture branches do originate peripherally, if the rate of elongation is constant in the anterior and posterior directions (intrafiber elongation speed) and between developing fibers within a forming growth shell (interfiber elongation speed), then only a part of their construction proceeds sequentially toward the poles. A second suture branch origin will be established at the poles resulting in a short distal portion of suture branches being formed sequentially in the reverse direction. Suture formation will conclude when a long proximal and a short distal portion of branches come to confluence within unequal anterior and posterior polar cap regions. This segmented suture formation scheme will be more pronounced in line suture lenses than in Y suture lenses. Third, because lenses with branched sutures have growth shells consisting of fibers of unequal length, fiber maturation is likely to be initiated in these lenses before a growth shell as well as suture formation is completed and would proceed in distinct patterns over a period of time. This is in marked contrast to avian lens fiber maturation which does not begin until growth shell and suture (branchless umbilical) formation is completed and then occurs rapidly and essentially simultaneously across the entire growth shell. CONCLUSIONS: Animations of secondary fiber development and suture formation based on quantitative analysis of electron micrographs reveals important novel aspects of these processes that have not been apparent from the results of previous mechanistic studies. The more complex schemes of fiber differentiation and suture formation presented herein are consistent with the notion that lens function (dynamic focusing) is interdependent on lens structure and physiology. The animations confirm that while all vertebrate lenses have a similar structure, differences in the level of their structural complexity established early in development and maintained throughout life can account for the varying amount of optical quality known to exist between species.

Development of lens sutures.

Cylindrical map projections (CMPs) have been used for centuries as an effective means of plotting the features of a 3D spheroidal surfaces (e.g. the earth) on a 2D rectangular map. We have used CMPs to plot primate fiber cell organization from selected growth shells as a function of growth, development and aging. Lens structural parameters and features were derived from slit-lamp, light and transmission and scanning electron micrographs. This information was then used to create CMPs of lenses that were then correlated with azimuthal map projections (AMPs; projections that are radially symmetric around a central point [the poles]) to reveal different suture patterns during distinct time periods. In this manner, both lens fiber and suture branch locations are defined by degrees of longitude and latitude. CMPs and AMPs confirm that throughout defined periods of development, growth and ageing, increasingly complex suture patterns are formed by the precise ordering of straight and opposite end curvature fibers. However, the manner in which additional suture branches are formed anteriorly and posteriorly is not identical. Anteriorly, new branches are added between extant branches. Posteriorly, pairs of new branches are formed that progressively overlay extant branches. The advantage of using CMPs is that the shape and organization of every fiber in a growth shell can be observed in a single image. Thus, the use of CMPs to plot primate fiber cell organization has revealed more complex aspects of fiber formation that may explain, at least in part, changes in lens optical quality as a function of age and pathology. In addition, more accurate measurements of fiber length will be possible by incorporating the latitudinal and longitudinal locations of fibers.

Lens structure in MIP-deficient mice.

In this study we used correlative light, scanning, and transmission (freeze-etch) electron microscopy to characterize lens structure in normal mice and compare it with that in mice deficient in the major intrinsic protein (MIP) of fiber cells. Grossly, wild-type lenses were transparent and had typical Y sutures at all of the ages examined. These lenses had fibers of uniform shape (hexagonal in cross section) arranged in ordered concentric growth shells and radial cell columns. In addition, these fibers had normal opposite end curvature and lateral interdigitations regularly arrayed along their length. Ultrastructural evaluation of these fibers revealed anterior and posterior end segments characterized by square array membrane on low-amplitude wavy fiber membrane. Approximately 13% of the equatorial or mid segments of these same fibers were specialized as gap junctions (GJs). In contrast, heterozygote lenses, while initially transparent at birth, were translucent by 3 weeks of age, except for a peripheral transparent region that contained fibers in the early stages of elongation. This degradation in clarity was correlated with abnormal fiber structure. Specifically, although the mid segment of these fibers was essentially normal, their end segments lacked normal opposite end curvature, were larger than normal, and had a distinct non-hexagonal shape. As a result, these fibers failed to form typical Y sutures. Furthermore, the nuclear fibers of heterozygote lenses were even larger and lacked any semblance of an ordered packing arrangement. Grossly, homozygote lenses were opaque at all ages examined, except for a peripheral transparent region that contained fibers in the early stages of elongation. All fibers from homozygote lenses lacked opposite end curvature, and thus failed to form any sutures. Also, these fibers were essentially devoid of interlocking devices, and only 7% of their mid segment was specialized as GJs. The results of this study suggest that MIP has essential roles in the establishment and maintenance of uniform fiber structure, and the organization of fibers, and as such is essential for lens function.

Morphology and organization of posterior fiber ends during migration.

PURPOSE: To characterize structural parameters of the basal membrane complex (BMC) and to determine the arrangement and organization of posterior fiber ends during elongation/migration in lenses with branched sutures. METHODS: Lenses from normal, juvenile (4-6 week old) Sprague-Dawley rats (n=16) were utilized. Posterior fiber ends were assessed on both whole mounts of lens capsules and on decapsulated lenses. The size, shape and organization of migrating fiber ends was assessed by scanning electron microscopy (SEM) and laser scanning confocal microscopy (LSCM) along the entire posterior surface. The area of the BMC was measured using image analysis software and subjected to statistical analysis. RESULTS: Posterior fiber ends had a characteristic regional arrangement during elongation and migration along the capsule. These regions were termed the equatorial, the lateral-posterior (posterior from the equator to within 150 microm of the sutures), the peri-sutural (150 microm surrounding the sutures), and the sutural. The area of fiber ends (seen by SEM) was compared to the area of fluorescent F-actin profiles (seen by LSCM). There was no significant difference (p=0.324) between the average basal end area (40.21 microm2) and the average area of F-actin profiles (40.65 microm2). The average fiber end area in the lateral-posterior, peri-sutural, and sutural regions was 63.19 microm2, 71.95 microm2, and 25.75 microm2, respectively. In the equatorial region, footprints were aligned in rows oriented toward the posterior pole, consistent with the arrangement of straight, meridional rows. Initially, fiber ends within the lateral-posterior region were arranged in short irregular rows having variable orientation with respect to the posterior pole. The remainder of these ends were randomly arranged. In the peri-sutural region, fiber ends approaching suture branches were aligned in short rows oriented at angles to the posterior pole. At the sutures, fiber ends appeared to become rounded, presumably during detachment from the capsule. CONCLUSIONS: The results confirm that F-actin profiles delineate the BMC of posterior fiber ends. Furthermore, the average area, shape and arrangement of fiber ends varies in a predictable pattern during migration. The data suggests that elongating fiber ends follow defined migration patterns along the posterior capsule to their sutural destinations. This controlled process is crucial to the formation of ordered suture patterns, thereby minimizing their adverse effects on lens optical quality.

Knockout of the intermediate filament protein CP49 destabilises the lens fibre cell cytoskeleton and decreases lens optical quality, but does not induce cataract.

In this report, the phenotype associated with the first targeted knockout of the lens specific intermediate filament gene CP49 is described. Several surprising observations have been made. The first was that no cataract was observed despite the fact that the beaded filaments of the lens fibre cells had been disrupted. Light scatter and the lens optical properties had, however, deteriorated in the CP49 knockout lenses compared to litter mate controls. These changes were accompanied by dramatic changes in plasma membrane organisation of the fibre cells as revealed by detailed morphological examinations and providing the second surprising result. The CP49 knockout mouse is therefore an important model to study the functional link between lens transparency, the cytoskeleton and plasma membrane organisation.

Analysis of nuclear fiber cell compaction in transparent and cataractous diabetic human lenses by scanning electron microscopy.

BACKGROUND: Compaction of human ocular lens fiber cells as a function of both aging and cataractogenesis has been demonstrated previously using scanning electron microscopy. The purpose of this investigation is to quantify morphological differences in the inner nuclear regions of cataractous and non-cataractous human lenses from individuals with diabetes. The hypothesis is that, even in the presence of the osmotic stress caused by diabetes, compaction rather than swelling occurs in the nucleus of diabetic lenses. METHODS: Transparent and nuclear cataractous lenses from diabetic patients were examined by scanning electron microscopy (SEM). Measurements of the fetal nuclear (FN) elliptical angles (anterior and posterior), embryonic nuclear (EN) anterior-posterior (A-P) axial thickness, and the number of EN fiber cell membrane folds over 20 microns were compared. RESULTS: Diabetic lenses with nuclear cataract exhibited smaller FN elliptical angles, smaller EN axial thicknesses, and larger numbers of EN compaction folds than their non-cataractous diabetic counterparts. CONCLUSION: As in non-diabetic lenses, the inner nuclei of cataractous lenses from diabetics were significantly more compacted than those of non-cataractous diabetics. Little difference between diabetic and non-diabetic compaction levels was found, suggesting that diabetes does not affect the degree of compaction. However, consistent with previous proposals, diabetes does appear to accelerate the formation of cataracts that are similar to age-related nuclear cataracts in non-diabetics. We conclude that as scattering increases in the diabetic lens with cataract formation, fiber cell compaction is significant.