Physiological and Optical Alterations Precede the Appearance of Cataracts in Cx46fs380 Mice.

Purpose: Cx46fs380 mice model a human autosomal-dominant cataract caused by a mutant lens connexin46, Cx46. Lenses from Cx46fs380 mice develop cataracts that are first observed at ∼2 months in homozygotes and at ≥4 months in heterozygotes. The present studies were conducted to determine whether Cx46fs380 mouse lenses exhibited abnormalities before there are detectable cataracts. Methods: Lenses from wild-type and Cx46fs380 mice were studied at 1 to 3 months of age. Connexin levels were determined by immunoblotting. Gap junctional coupling was calculated from intracellular impedance studies of intact lenses. Optical quality and refractive properties were assessed by laser scanning and by photographing a 200-mesh electron microscopy grid through wild-type and Cx46fs380 mouse lenses. Results: Connexin46 and connexin50 levels were severely reduced in mutant lenses. Gap junctional coupling was decreased in differentiating and mature fibers from Cx46fs380 lenses; in homozygotes, the mature fibers had no detectable coupling. Homozygous lenses were slightly smaller and had reduced focal lengths. Heterozygous and homozygous lenses significantly distorted the electron microscopy grid pattern as compared with wild-type lenses. Conclusions: Before cataract appearance, Cx46fs380 lenses have decreased gap junctional conductance (at least in heterozygotes) and alterations in refractive properties (heterozygotes and homozygotes). The decreased focal distance of Cx46fs380 homozygous lenses is consistent with an increase in refractive index due to changes in cellular composition. These data suggest that Cx46fs380 lenses undergo a sequence of changes before the appearance of cataracts: low levels of connexins, decreased gap junction coupling, alterations in lens cell homeostasis, and changes in refractive index.

Connexin23 deletion does not affect lens transparency.

While connexin46 (Cx46) and connexin50 (Cx50) are crucial for maintaining lens transparency and growth, the contributions of a more recently identified lens fiber connexin, Cx23, are poorly understood. Therefore, we studied the consequences of absence of Cx23 in mouse lenses. Cx23-null mice were generated by homologous Cre recombination. Cx23 mRNA was abundantly expressed in wild type lenses, but not in Cx23-null lenses. The transparency and refractive properties of Cx23-null lenses were similar to wild type lenses when examined by darkfield microscopy. Neither the focusing ability nor the light scattering was altered in the Cx23-null lenses. While both Cx46 and Cx50 localized to appositional fiber cell membranes (as in wild type lenses), their levels were consistently (but not significantly) decreased in homozygous Cx23-null lenses. These results suggest that although Cx23 expression can influence the abundance of the co-expressed lens fiber connexins, heterozygous or homozygous expression of a Cx23-null allele does not alter lens transparency.

Comparison of fixation disparity measured by saladin card and disparometer.

PURPOSE: The purpose of this study was to compare the fixation disparity (FD) measurements taken with the Saladin Near Point Balance Card (Saladin card) to those made with the Sheedy Disparometer, and to determine if the same clinical norms used with the Disparometer can be applied to the newer Saladin card. METHODS: Subjects were 44 optometry students (aged 23 to 34 years) without strabismus, amblyopia, or asthenopia during near work. They were randomized to begin at one of three examiners' stations: dissociated near phoria using Modified Thorington card; FD with Saladin card; and FD with Disparometer. Subjects proceeded to each station in turn. FD was measured for each subject through forced vergence demands of 0, 3, 6, and 9 base-in (BI), and 3, 6, 9, 16, and 20 base-out (BO), alternating BI and BO demands. Examiners were masked to subjects' results from the other stations. RESULTS: FD curve (FDC) types were the same with the two instruments in most cases. However, statistically significant differences were found for FDC slopes (p = 0.048), y intercepts (p < 0.0001), and FD values through BI prisms (p < 0.0001), with the Disparometer finding the FD to be more eso/less exo than did the Saladin card. FD values through BO prisms showed no statistically significant differences but great variability. CONCLUSIONS: The two instruments generally produce similar types of FDCs. However, the Disparometer tends to yield more eso/less exo FD measurements compared with the Saladin card. Although the newer Saladin card frequently produces FDC slopes and y intercepts within the expected range (as published for the Disparometer) for asymptomatic subjects, slopes and y intercepts obtained by the Saladin card are not sufficiently similar to those obtained by the Disparometer to warrant use of the same norms. Further study is needed to establish appropriate norms for the Saladin card.

Tropomodulin1 is required for membrane skeleton organization and hexagonal geometry of fiber cells in the mouse lens.

Hexagonal packing geometry is a hallmark of close-packed epithelial cells in metazoans. Here, we used fiber cells of the vertebrate eye lens as a model system to determine how the membrane skeleton controls hexagonal packing of post-mitotic cells. The membrane skeleton consists of spectrin tetramers linked to actin filaments (F-actin), which are capped by tropomodulin1 (Tmod1) and stabilized by tropomyosin (TM). In mouse lenses lacking Tmod1, initial fiber cell morphogenesis is normal, but fiber cell hexagonal shapes and packing geometry are not maintained as fiber cells mature. Absence of Tmod1 leads to decreased gammaTM levels, loss of F-actin from membranes, and disrupted distribution of beta2-spectrin along fiber cell membranes. Regular interlocking membrane protrusions on fiber cells are replaced by irregularly spaced and misshapen protrusions. We conclude that Tmod1 and gammaTM regulation of F-actin stability on fiber cell membranes is critical for the long-range connectivity of the spectrin-actin network, which functions to maintain regular fiber cell hexagonal morphology and packing geometry.

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.

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.

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.