Rat Model of Type 2 Diabetes Mellitus Recapitulates Human Disease in the Anterior Segment of the Eye.

Changes in the anterior segment of the eye due to type 2 diabetes mellitus (T2DM) are not well-characterized, in part due to the lack of a reliable animal model. This study evaluated changes in the anterior segment, including crystalline lens health, corneal endothelial cell density, aqueous humor metabolites, and ciliary body vasculature, in a rat model of T2DM compared with human eyes. Male Sprague-Dawley rats were fed a high-fat diet (45% fat) or normal diet, and rats fed the high-fat diet were injected with streptozotocin intraperitoneally to generate a model of T2DM. Cataract formation and corneal endothelial cell density were assessed using microscopic analysis. Diabetes-related rat aqueous humor alterations were assessed using metabolomics screening. Transmission electron microscopy was used to assess qualitative ultrastructural changes ciliary process microvessels at the site of aqueous formation in the eyes of diabetic rats and humans. Eyes from the diabetic rats demonstrated cataracts, lower corneal endothelial cell densities, altered aqueous metabolites, and ciliary body ultrastructural changes, including vascular endothelial cell activation, pericyte degeneration, perivascular edema, and basement membrane reduplication. These findings recapitulated diabetic changes in human eyes. These results support the use of this model for studying ocular manifestations of T2DM and support a hypothesis postulating blood-aqueous barrier breakdown and vascular leakage at the ciliary body as a mechanism for diabetic anterior segment pathology.

Structure of native lens connexin 46/50 intercellular channels by cryo-EM.

Gap junctions establish direct pathways for cell-to-cell communication through the assembly of twelve connexin subunits that form intercellular channels connecting neighbouring cells. Co-assembly of different connexin isoforms produces channels with unique properties and enables communication across cell types. Here we used single-particle cryo-electron microscopy to investigate the structural basis of connexin co-assembly in native lens gap junction channels composed of connexin 46 and connexin 50 (Cx46/50). We provide the first comparative analysis to connexin 26 (Cx26), which-together with computational studies-elucidates key energetic features governing gap junction permselectivity. Cx46/50 adopts an open-state conformation that is distinct from the Cx26 crystal structure, yet it appears to be stabilized by a conserved set of hydrophobic anchoring residues. 'Hot spots' of genetic mutations linked to hereditary cataract formation map to the core structural-functional elements identified in Cx46/50, suggesting explanations for many of the disease-causing effects.

Capsule-epithelium-fibre unit ultrastructure in the human lens.

This study aimed to investigate the capsule-epithelium-fibre unit ultrastructure of the human lens, particularly the interfaces of the epithelium with the capsule and the epithelium with the fibre cell. A total of 12 lenses from donor humans who died of trauma without systemic and ocular diseases were investigated by transmission electron microscopy (TEM), combined with immunofluorescence staining for localising certain specific proteins. Some of the results were further studied in the anterior lens capsules of cataract patients. Our results revealed capsule protrusion into the epithelium in some areas and potential processing of capsule components. The young elongating fibre cells directly adjacent to the epithelium with a high stain density strongly expressed CD24. Numerous extracellular vesicles could be seen in the space between human lens epithelial cells (HLECs) and between HLECs and the capsule. Mitophagy and autophagy were also observed in the HLECs. Our research may be beneficial in better understanding the function of the human lens.

The mgΔlpn mouse model for Marfan syndrome recapitulates the ocular phenotypes of the disease.

PURPOSE: Fibrillin-1 and -2 are major components of tissue microfibrils that compose the ciliary zonule and cornea. While mutations in human fibrillin-1 lead to ectopia lentis, a major manifestation of Marfan syndrome (MFS), in mice fibrillin-2 can compensate for reduced/lack of fibrillin-1 and maintain the integrity of ocular structures. Here we examine the consequences of a heterozygous dominant-negative mutation in the Fbn1 gene in the ocular system of the mgΔlpn mouse model for MFS. METHODS: Eyes from mgΔlpn and wild-type mice at 3 and 6 months of age were analyzed by histology. The ciliary zonule was analyzed by scanning electron microscopy (SEM) and immunofluorescence. RESULTS: Mutant mice presented a significantly larger distance of the ciliary body to the lens at 3 and 6 months of age when compared to wild-type, and ectopia lentis. Immunofluorescence and SEM corroborated those findings in MFS mice, revealing a disorganized mesh of microfibrils on the floor of the ciliary body. Moreover, mutant mice also had a larger volume of the anterior chamber, possibly due to excess aqueous humor. Finally, losartan treatment had limited efficacy in improving ocular phenotypes. CONCLUSIONS: In contrast with null or hypomorphic mutations, expression of a dominant-negative form of fibrillin-1 leads to disruption of microfibrils in the zonule of mice. This in turn causes lens dislocation and enlargement of the anterior chamber. Therefore, heterozygous mgΔlpn mice recapitulate the major ocular phenotypes of MFS and can be instrumental in understanding the development of the disease.

The importance of the epithelial fibre cell interface to lens regeneration in an in vivo rat model and in a human bag-in-the-lens (BiL) sample.

Human lens regeneration and the Bag-in-the-Lens (BIL) surgical treatment for cataract both depend upon lens capsule closure for their success. Our studies suggest that the first three days after surgery are critical to their long-term outcomes. Using a rat model of lens regeneration, we evidenced lens epithelial cell (LEC) proliferation increased some 50 fold in the first day before rapidly declining to rates observed in the germinative zone of the contra-lateral, un-operated lens. Cell multi-layering at the lens equator occurred on days 1 and 2, but then reorganised into two discrete layers by day 3. E- and N-cadherin expression preceded cell polarity being re-established during the first week. Aquaporin 0 (AQP0) was first detected in the elongated cells at the lens equator at day 7. Cells at the capsulotomy site, however, behaved very differently expressing the epithelial mesenchymal transition (EMT) markers fibronectin and alpha-smooth muscle actin (SMA) from day 3 onwards. The physical interaction between the apical surfaces of the anterior and posterior LECs from day 3 after surgery preceded cell elongation. In the human BIL sample fibre cell formation was confirmed by both histological and proteome analyses, but the cellular response is less ordered and variable culminating in Soemmerring's ring (SR) formation and sometimes Elschnig's pearls. This we evidence for lenses from a single patient. No bow region or recognisable epithelial-fibre cell interface (EFI) was evident and consequently the fibre cells were disorganised. We conclude that lens cells require spatial and cellular cues to initiate, sustain and produce an optically functional tissue in addition to capsule integrity and the EFI.

Cell compaction is not required for the development of gradient refractive index profiles in the embryonic chick lens.

The development of the eye requires the co-ordinated integration of optical and neural elements to create a system with requisite optics for the given animal. The eye lens has a lamellar structure with gradually varying protein concentrations that increase towards the centre, creating a gradient refractive index or GRIN. This provides enhanced image quality compared to a homogeneous refractive index lens. The development of the GRIN during ocular embryogenesis has not been investigated previously. This study presents measurements using synchrotron X-ray Talbot interferometry and scanning electron microscopy of chick eyes from embryonic day 10: midway through embryonic development to E18: a few days before hatching. The lens GRIN profile is evident from the youngest age measured and increases in magnitude of refractive index at all points as the lens grows. The profile is parabolic along the optic axis and has two distinct regions in the equatorial plane. We postulate that these may be fundamental for the independent central and peripheral processes that contribute to the optimisation of image quality and the development of an eye that is emmetropic. The spatial distributions of the distinct GRIN profile regions match with previous measurements on different fibre cell groups in chick lenses of similar developmental stages. Results suggest that tissue compaction may not be necessary for development of the GRIN in the chick eye lens.

Scanning and transmission electron microscopy study of anterior lens epithelium in presenile cataract.

PURPOSE: To study the structure of lens epithelial cells (LECs) in the anterior lens epithelium of presenile cataract and to further explore the possible reasons for presenile cataract development. METHODS: The anterior lens capsules (aLCs) of patients with presenile cataracts and patients with ordinary age-related cataracts were obtained from routine cataract surgery, and the 5-5.5 mm circles of the central aLC were cut in half and prepared for transmission electron microscopy (TEM) and scanning electron microscopy (SEM). RESULTS: The most obvious structural changes in the LECs observed in both cataract groups by TEM were uneven thickness of the anterior lens epithelium, vacuolated cytoplasm and elongated nuclei. SEM showed abnormal structural changes in the LECs, with swollen cells and spheres on the anterior lens epithelium observed in both groups and holes formed by the LECs stretching observed only in the presenile cataract patients. The degeneration of the anterior lens epithelium and the structural changes in the LECs were observed more prominently in presenile cataract patients. CONCLUSIONS: Abnormal and prominently affected structural features of LECs were observed in the presenile compared to age-related cataract patients by TEM and SEM. We suppose that ultrastructural pathological changes in the anterior lens epithelial cells are one of the important reasons for the development of presenile and age-related cataract.

Chicken GRIFIN: binding partners, developmental course of localization and activation of its lens-specific gene expression by L-Maf/Pax6.

Tissue lectins appear to be involved in a broad range of physiological processes, as reflected for the members of the family of galectins by referring to them as adhesion/growth-regulatory effectors. In order to clarify the significance of galectin presence, key challenges are to define their binding partners and the profile of localization. Having identified the chicken galectin-related interfiber protein (C-GRIFIN) as lens-specific protein present in the main body of adult lens, we here report its interaction with lens proteins in ligand blotting. The assumption for pairing with α-, ß- and δ-crystallins was ascertained by mass spectrometric detection of their presence in eluted fractions obtained by affinity chromatography. Biochemical and immunohistochemical monitoring revealed protein presence from about 3-day-old embryos onwards, mostly in the cytoplasm of elongated posterior cells, later in secondary lens fiber cells. On the level of gene expression, its promoter was activated by transcription factor L-Maf alone and together with Pax6 like a crystallin gene, substantiating C-GRIFIN's status as lens-specific galectin. Using this combined strategy for counterreceptor and expression profiling by bio- and histochemical methods including light, electron and fluorescence microscopy, respective monitoring in lens development can now be taken to the level of the complete galectin family.

Visualization of adult fish lens fiber cells.

The crystalline lens of a vertebrate eye is a gradient-index lens and grows throughout life by addition of new lens fiber cells in the periphery. In fish, the growing ball-shaped lens maintains sophisticated optical properties throughout life by maintaining the distribution of refractive index relative to the increasing radius of the lens. During this process, the central fibers must increase refractive index by increasing the cytosolic concentration of crystallin proteins. However, only the youngest, most peripheral lens fiber cells have the ability to synthesize proteins. Unfortunately, the hardness of fish lenses makes investigation of the cellular anatomy impossible with traditional histological methods. We have developed a method for visualizing lens fiber cells across the diameter of the lens in adult fish. The method relies on sectioning embedded lenses with a high-speed power saw and observing the cut surface with a scanning electron microscope (SEM). The combination of SEM and image analysis allowed for precise tracking of the positions of individual cell fiber cells. As an application of the method, we present a cell thickness profile, i.e. the distribution of cells thicknesses and their relative positions along the lens's radius. Combined with detailed optical studies, which by mathematical reasons only are possible on ball-shaped lenses, our method can lead to new insights into the mechanism governing the functional and cellular development of vertebrate lenses.

In vivo assessment of the mechanical properties of crystalline lenses in a rabbit model using ultrasound elastography: Effects of ultrasound frequency and age.

PURPOSE: The occurrence of presbyopia and cataract is closely related to changes in the mechanical properties of the crystalline lens. There are no established methods so far for in vivo assessment. By introducing ultrasound elastography, we proposed group velocity (Vg) of an induced shear wave as a new biomarker to characterize the mechanical properties of the lens in our previous study. Here, we investigated the effect of the ultrasound frequency on measurement accuracy and validated the results with a conventional ex vivo compression testing. We also demonstrated a change trend in Vg and its correlation with age in a rabbit model. METHODS: Eight New Zealand white rabbits were fed normally from the fourth to seventh month. An ultrasound elastography system was developed to measure Vgin vivo on every eye once per month. The performances when using a high-frequency (L22-11v) and low-frequency (L11-4v) probe were compared. Rabbits were sacrificed after in vivo measurements by the end of the seventh month and this was followed by ex vivo ultrasound measurements and conventional compression tests on the extracted lenses. RESULTS: The results demonstrated that there were no significance differences in Vg between measurements with high-frequency (USE-HF) and low-frequency (USE-LF) probes in the same month-age group. The mean Vg and the standard deviation of four rabbits that were 7 months old were 2.37 ±â€¯0.24  m/s, 2.36 ±â€¯0.25 m/s, 2.43 ±â€¯0.26 m/s and 2.44 ±â€¯0.38 m/s, with USE-HF for ex vivo and in vivo measurements and USE-LF for ex vivo and in vivo measurements, respectively. There were no significant differences (p > 0.05) and they were all in agreement with the results of compression tests, which was 16.16 ±â€¯1.84 kPa in Young's modulus. The results also showed that Vg increased with age. In combination with the results of our previous study, Vg showed a relatively sharp increase from 2 to 5 months, while it had a slight increase from 5 to 7 months. CONCLUSIONS: The USE-HF and USE-LF has comparable accuracy in Vg measurements while USE-HF had an advantage regarding better spatial resolution. The change trend of Vg was in accord with the growth phase of New Zealand white rabbits, which usually results in sexual maturity at 5 months old. This implies that Vg can be used as a biomarker parameter for evaluating the mechanical properties of the lens undergoing physiological changes.

RETRACTED: Peptide-induced formation of protein aggregates and amyloid fibrils in human and guinea pig αA-crystallins under physiological conditions of temperature and pH.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the authors. The senior author contacted the journal in a forthright manner, in an effort to preserve the scientific integrity of the literature, after discovering a significant error in the results reported in the article. The authors were recently made aware of a paper by Kim et al. (Nature Commun. 2019) which shows a spirosome structure (the enzyme aldehyde-alcohol dehydrogenase) present in E. coli (Fig. 5a) that is very similar to the structure the authors thought formed when synthetic alpha A crystallin (66-80) peptide was incubated for 24 h with recombinant guinea pig alpha A insert crystallin (see Kumarasamy et al., Figs. 7C and F, and Fig. 9). Subsequent to publication of their report, the authors later found a number of images that showed what appeared to be the same structure present in samples of their presumably purified recombinant guinea pig alpha A insert crystallin which had been incubated without peptide for 24 h. Hence, the authors now conclude that the structures shown in Figs. 7C and F, and Fig. 9 of their article published in this journal are actually due to E. coli contaminant aldehyde-alcohol dehydrogenase. The authors deeply regret this error and any inconvenience it may have caused.

Transgenic lysyl oxidase homolog 1 overexpression in the mouse eye results in the formation and release of protein aggregates.

Sequence variants in LOXL1 coding for the secreted enzyme lysyl oxidase homolog 1 (LOXL1) associate with pseudoexfoliation (PEX) syndrome, a condition that is characterized by the deposition of extracellular fibrillar PEX material in the anterior eye and other parts of the body. Since the specific role of LOXL1 in the pathogenesis of PEX is unclear, and an increase in its expression was reported for early stages of PEX syndrome, we generated and studied transgenic mice with ocular overexpression of its mouse ortholog Loxl1. The chicken ßB1-crystallin promoter was used to overexpress Loxl1 in the lenses of ßB1-crystallin-Loxl1 transgenic mice. Transgenic lenses contained high levels of the protein LOXL1 and its mRNA, which were both not detectable in lenses of wildtype littermates. In wildtype mice, immunoreactivity for LOXL1 was mainly seen extracellularly in region of the ciliary zonules. ßB1-crystallin-Loxl1 littermates showed an additional diffuse immunostaining in lens fibers and capsule, and in the inner limiting membrane and retina indicating secretion of soluble LOXL1 from transgenic lenses. In addition, lens fibers of transgenic animals contained multiple distinct spots of very intense LOXL1 immunoreactivity. By transmission electron microscopy, those spots correlated with electron-dense round or oval bodies of 20-50 nm in diameter which were localized in the rough endoplasmic reticulum and not seen in wildtype lenses. Immunogold electron microscopy confirmed that the electron-dense bodies contained LOXL1 indicating aggregation of insoluble LOXL1. Similar structures were seen in the extracellular lens capsule suggesting their secretion from lens fibers. Otherwise, no changes were seen between the eyes of ßB1-crystallin-Loxl1 mice and their wildtype littermates, neither by light microscopy and funduscopy of whole eyes, nor by scanning and quantitative transmission electron microscopy of ciliary epithelium and zonules. At one month of age, intraocular pressure was significantly higher in transgenic mice than in wildtype littermates. No differences in IOP were seen though at 2-5 months of age. We conclude that LOXL1 has a strong tendency to aggregate in the rER when expressed in vivo at high amounts. A similar scenario, involving intracellular aggregation of LOXL1 and secretion of LOXL1 aggregates into the extracellular space, may be involved in the early pathogenetic events in eyes of PEX patients.

[Ultrastructure of the lens anterior capsule in secondary cataract associated with uveitis under an electron microscope].

Objective: To observe the ultrastructure of lens epithelium cells in cataract associated with uveitis. Methods: A cross-sectional study. Seven patients(7 eyes) received phacoemulsification in Peking University First Hospital from August 2016 to March 2017 due to complicated cataract associated with uveitis[2 males and 5 females,with an average age of (49±20) years] and 3 patients of age-related cataract (3 eyes, females, aged 54, 71, 74 years) were enrolled. Anterior capsule samples were collected during surgery. Transmission electron microscopy was performed. The cell morphology, the proportion of cell nuclear and organelle changes and the proportion of apoptosis in cataract associated with uveitis were described statistically by x±s or median (range). Results: Spindle-shaped epithelial cells were observed in cataract associated with uveitis (100%), while 28.00%,16.67%,16.67% of spindle-shaped epithelial cells were observed in age-related cataract. The organelle changes included mitochondria swelling and damage to the tight cell junction in cataract associated with uveitis. The tight junction between two cells was damaged in different degrees, only 8.33% (0-16.67%)of the cell nuclei appeared normal, and increased chromatin density(47.07%±22.28%), nuclear pyknosis(38.02%±19.61%) and nuclear fragmentation (9.96%±8.10%) were observed in cataract secondary to uveitis. The apoptosis rate was (48.16%±26.66%) in cataract associated with uveitis and correlated to the duration of intraocular inflammation. While the apoptosis rate were 0, 8.33%, 0 in age-related cataract patients. No autophagosome was observed in cataract associated with uveitis. Conclusions: Ultrastructure changes existed in lens epithelium cells in cataract associated with uveitis. The increased rate of apoptosis and inhibition of autophagy could be the possible mechanism of cataract formation in uveitic eyes. (Chin J Ophthalmol, 2018, 54: 357-362).

Structure and function of α-crystallins: Traversing from in vitro to in vivo.

BACKGROUND: The two α-crystallins (αA- and αB-crystallin) are major components of our eye lenses. Their key function there is to preserve lens transparency which is a challenging task as the protein turnover in the lens is low necessitating the stability and longevity of the constituent proteins. α-Crystallins are members of the small heat shock protein family. αB-crystallin is also expressed in other cell types. SCOPE OF THE REVIEW: The review summarizes the current concepts on the polydisperse structure of the α-crystallin oligomer and its chaperone function with a focus on the inherent complexity and highlighting gaps between in vitro and in vivo studies. MAJOR CONCLUSIONS: Both α-crystallins protect proteins from irreversible aggregation in a promiscuous manner. In maintaining eye lens transparency, they reduce the formation of light scattering particles and balance the interactions between lens crystallins. Important for these functions is their structural dynamics and heterogeneity as well as the regulation of these processes which we are beginning to understand. However, currently, it still remains elusive to which extent the in vitro observed properties of α-crystallins reflect the highly crowded situation in the lens. GENERAL SIGNIFICANCE: Since α-crystallins play an important role in preventing cataract in the eye lens and in the development of diverse diseases, understanding their mechanism and substrate spectra is of importance. To bridge the gap between the concepts established in vitro and the in vivo function of α-crystallins, the joining of forces between different scientific disciplines and the combination of diverse techniques in hybrid approaches are necessary. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.

Human alpha A-crystallin missing N-terminal domain poorly complexes with filensin and phakinin.

The aim of this study was to determine relative importance of N-terminal domain and C-terminal extension of αA-crystallin during their in vitro complex formation with phakinin and filensin (the two lens-specific intermediate filament [IF] proteins). Cloned phakinin, filensin and vimentin were purified under a denaturing conditions by consecutive DEAE-cellulose-, hydroxyapatite- and Sephadex G-75-column chromatographic methods. WTαA-crystallin, αA-NT (N-terminal domain [residue number 1-63])-deleted and αA-CT (C-terminal terminal extension [residue number 140-173]-deleted), were cloned in pET 100 TOPO vector, expressed in BL-21 (DE3) cells using 1% IPTG, and purified using a Ni2+-affinity column. The following two in vitro methods were used to determine complex formation of WT-αA, αA-NT, or αA-CT with phakinin, filensin or both phakinin plus filensin together: an ultracentrifugation sedimentation (centrifugation at 80,000 × g for 30 min at 20 °C) followed by SDS-PAGE analysis, and an electron microscopic analysis. In the first method, the individual control proteins (WT-αA, αA-NT and αA-CT crystallin species) remained in the supernatant fractions whereas phakinin, filensin, and vimentin were recovered in the pellet fractions. On complex formation by individual WT-αA-, αA-NT or αA-CT-species with filensin, phakinin or both phakinin and filensin, WT-αA and αA-CT were recovered in the pellet fraction with phakinin, filensin or both filensin and phakinin, whereas αA-NT remained mostly in the supernatant, suggesting its poor complex formation property. EM-studies showed filamentous structure formation between WT-αA and αA-CT with phakinin or filensin, or with both filensin and phakinin together but relatively poor filamentous structures with αA-NT. Together, the results suggest that the N-terminal domain of αA-crystallin is required during in vitro complex formation with filensin and phakinin.

Factors Determining the Oxygen Permeability of Biological Membranes: Oxygen Transport Across Eye Lens Fiber-Cell Plasma Membranes.

Electron paramagnetic resonance (EPR) spin-label oximetry allows the oxygen permeability coefficient to be evaluated across homogeneous lipid bilayer membranes and, in some cases, across coexisting membrane domains without their physical separation. The most pronounced effect on oxygen permeability is observed for cholesterol, which additionally induces the formation of membrane domains. In intact biological membranes, integral proteins induce the formation of boundary and trapped lipid domains with a low oxygen permeability. The effective oxygen permeability coefficient across the intact biological membrane is affected not only by the oxygen permeability coefficients evaluated for each lipid domain but also by the surface area occupied by these domains in the membrane. All these factors observed in fiber cell plasma membranes of clear human eye lenses are reviewed here.

Expression of the type VI intermediate filament proteins CP49 and filensin in the mouse lens epithelium.

PURPOSE: The differentiated lens fiber cell assembles a filamentous cytoskeletal structure referred to as the beaded filament (BF). The BF requires CP49 (bfsp2) and filensin (bfsp1) for assembly, both of which are highly divergent members of the large intermediate filament (IF) family of proteins. Thus far, these two proteins have been reported only in the differentiated lens fiber cell. For this reason, both proteins have been considered robust markers of fiber cell differentiation. We report here that both proteins are also expressed in the mouse lens epithelium, but only after 5 weeks of age. METHODS: Localization of CP49 was achieved with immunocytochemical probing of wild-type, CP49 knockout, filensin knockout, and vimentin knockout mice, in sections and in the explanted lens epithelium, at the light microscope and electron microscope levels. The relationship between CP49 and other cytoskeletal elements was probed using fluorescent phalloidin, as well as with antibodies to vimentin, GFAP, and α-tubulin. The relationship between CP49 and the aggresome was probed with antibodies to γ-tubulin, ubiquitin, and HDAC6. RESULTS: CP49 and filensin were expressed in the mouse lens epithelium, but only after 5 weeks of age. At the light microscope level, these two proteins colocalize to a large tubular structure, approximately 7 × 1 µm, which was typically present at one to two copies per cell. This structure is found in the anterior and anterolateral lens epithelium, including the zone where mitosis occurs. The structure becomes smaller and largely undetectable closer to the equator where the cell exits the cell cycle and commits to fiber cell differentiation. This structure bears some resemblance to the aggresome and is reactive with antibodies to HDAC6, a marker for the aggresome. However, the structure does not colocalize with antibodies to γ-tubulin or ubiquitin, also markers for the aggresome. The structure also colocalizes with actin but appears to largely exclude vimentin and α-tubulin. In the CP49 and filensin knockouts, this structure is absent, confirming the identity of CP49 and filensin in this structure, and suggesting a requirement for the physiologic coassembly of CP49 and filensin. CONCLUSIONS: CP49 and filensin have been considered robust markers for mouse lens fiber cell differentiation. The data reported here, however, document both proteins in the mouse lens epithelium, but only after 5 weeks of age, when lens epithelial growth and mitotic activity have slowed. Because of this, CP49 and filensin must be considered markers of differentiation for both fiber cells and the lens epithelium in the mouse. In addition, to our knowledge, no other protein has been shown to emerge so late in the development of the mouse lens epithelium, suggesting that lens epithelial differentiation may continue well into post-natal life. If this structure is related to the aggresome, it is a rare, or perhaps unique example of a large, stable aggresome in wild-type tissue.

Breakdown of interlocking domains may contribute to formation of membranous globules and lens opacity in ephrin-A5(-/-) mice.

Ephrin-A5, a ligand of the Eph family of receptor tyrosine kinases, plays a key role in lens fiber cell packing and cell-cell adhesion, with approximately 87% of ephrin-A5(-/-) mice develop nuclear cataracts. Here, we investigated the extensive formation of light-scattering globules associated with breakdown of interlocking protrusions during lens opacification in ephrin-A5(-/-) mice. Lenses from wild-type (WT) and ephrin-A5(-/-) mice between 2 and 21 weeks old were studied with light and electron microscopy, immunofluorescence labeling, freeze-fracture TEM and filipin cytochemistry for membrane cholesterol detection. Lens opacities with various densities were first observed in ephrin-A5(-/-) mice at around 60 days old. Dense cataracts in the mutant lenses were seen primarily in the nuclear region surrounded by transparent cortices from all eyes examined. We confirmed that a majority of nuclear cataracts were dislocated posteriorly and ruptured the thinner posterior lens capsule. SEM analysis indicated that numerous interlocking protrusions and wavy ridge-and-valley membrane surfaces in deep cortical and nuclear fibers did not cause lens opacity in both transparent ephrin-A5(-/-) and WT mice. In contrast, abundant isolated membranous globules of approximately 1000 nm in size were distributed randomly along the intact fiber cells during early stage of all ephrin-A5(-/-) cataracts examined. A further examination using both SEM and TEM revealed that isolated globules were generated from the disintegrated interlocking protrusions originally located along the corners of hexagonal fiber cells. Freeze-fracture TEM further revealed the association of square-array aquaporin junctions with both isolated globules and interlocking membrane domains. This study reports for the first time that disrupted interlocking protrusions are the source of numerous large membranous globules that contribute to light scattering and nuclear cataracts in the ephrin-A5(-/-) mice. Our results further suggest that dissociations of N-cadherin and adherens junctions in the associated interlocking domains may result in the formation of isolated globules and nuclear opacities in the ephrin-A5(-/-) mice.

Anteriorly located zonular fibres as a tool for fine regulation in accommodation.

PURPOSE: To describe an anteriorly located system of zonular fibres that could be involved in fine-tuning of accommodation. METHODS: Forty-six human and 28 rhesus monkey eyes were dissected and special preparations were processed for scanning electron microscopy and reflected-light microscopy. Additional series of frontal and sagittal histological and ultrathin sections were analysed in respect to the origin and insertion of anteriorly located zonules. The presence of sensory terminals at the site of the originating zonules within the connective tissue of the ciliary body was studied by immunohistochemistry. For in-vivo visualization ultrasound biomicroscopy (UBM) was performed on 12 human subjects. RESULTS: Fine zonular fibres originated from the valleys and lateral walls of the most anterior pars plicata that covers the anterior and inner circular ciliary muscle portion. These most anterior zonules (MAZ) showed attachments either to the anterior or posterior tines or they inserted directly onto the surface of the lens. At the site of origin, the course of the MAZ merged into the connective tissue fibres connecting the adjacent pigmented epithelium to the ciliary muscle. Numerous afferent terminals directly at the site of this MAZ-origin were connected to the intrinsic nervous network of the ciliary muscle. CONCLUSIONS: A newly described set of zonular fibres features the capabilities to register the tensions of the zonular fork and lens capsule. The close location and neural connection towards the circular ciliary muscle portion could provide the basis for stabilization and readjustment of focusing that serves fast and fine-tuned accommodation and disaccommodation.

Deficiency of the RNA binding protein caprin2 causes lens defects and features of Peters anomaly.

BACKGROUND: It was recently demonstrated that deficiency of a conserved RNA binding protein (RBP) and RNA granule (RG) component Tdrd7 causes ocular defects including cataracts in human, mouse and chicken, indicating the importance of posttranscriptional regulation in eye development. Here we investigated the function of a second conserved RBP/RG component Caprin2 that is identified by the eye gene discovery tool iSyTE. RESULTS: In situ hybridization, Western blotting and immunostaining confirmed highly enriched expression of Caprin2 mRNA and protein in mouse embryonic and postnatal lens. To gain insight into its function, lens-specific Caprin2 conditional knockout (cKO) mouse mutants were generated using a lens-Cre deleter line Pax6GFPCre. Phenotypic analysis of Caprin2(cKO/cKO) mutants revealed distinct eye defects at variable penetrance. Wheat germ agglutinin staining and scanning electron microscopy demonstrated that Caprin2(cKO/cKO) mutants have an abnormally compact lens nucleus, which is the core of the lens comprised of centrally located terminally differentiated fiber cells. Additionally, Caprin2(cKO/cKO) mutants also exhibited at 8% penetrance a developmental defect that resembles a human condition called Peters anomaly, wherein the lens and the cornea remain attached by a persistent stalk. CONCLUSIONS: These data suggest that a conserved RBP Caprin2 functions in distinct morphological events in mammalian eye development.