Expression of potassium-dependent sodium-calcium exchanger in the murine lens.

Loss of intracellular calcium homeostasis may contribute to the opacification of lens tissue during cortical cataract formation. In healthy lenses, the concentration of intracellular calcium is maintained at levels far below electrochemical equilibrium but the identity of the calcium extrusion mechanism in lens fiber cells has remained elusive. Previous studies focused on the role of plasma membrane calcium ATPases and sodium-calcium exchangers. Here, we examined the expression of mRNA transcripts encoding potassium-dependent sodium-calcium exchangers (Nckx's, encoded by the Slc24 gene family) in the mouse lens. The most abundant of the five Slc24 family members was Slc24a4 (Nckx4). Notably, Slc24a4 was the only family member with increased expression in fiber cells. Using an antibody raised against recombinant mouse Nckx4, we showed that the protein is expressed strongly in the outer cortical fibers, consistent with results of in situ hybridization experiments and earlier mass spectrometry analysis. To test the role of Nckx4 directly, we generated mice in which Slc24a4 was deleted conditionally in lens tissue. In conditional knockout animals, the level of Nckx4 protein was reduced to background levels without a discernible effect on lens growth or transparency. Thus, despite its relative abundance in the lens, Nckx4 does not appear to have an indispensable role in the maintenance of lens clarity.

Compositional Analysis of Extracellular Aggregates in the Eyes of Patients With Exfoliation Syndrome and Exfoliation Glaucoma.

Purpose: Exfoliation syndrome (XFS) is a condition characterized by the production of insoluble fibrillar aggregates (exfoliation material; XFM) in the eye and elsewhere. Many patients with XFS progress to exfoliation glaucoma (XFG), a significant cause of global blindness. We used quantitative mass spectrometry to analyze the composition of XFM in lens capsule specimens and in aqueous humor (AH) samples from patients with XFS, patients with XFG and unaffected individuals. Methods: Pieces of lens capsule and samples of AH were obtained with consent from patients undergoing cataract surgery. Tryptic digests of capsule or AH were analyzed by high-performance liquid chromatography-mass spectrometry and relative differences between samples were quantified using the tandem mass tag technique. The distribution of XFM on the capsular surface was visualized by SEM and super-resolution light microscopy. Results: A small set of proteins was consistently upregulated in capsule samples from patients with XFS and patients with XFG, including microfibril components fibrillin-1, latent transforming growth factor-ß-binding protein-2 and latent transforming growth factor-ß-binding protein-3. Lysyl oxidase-like 1, a cross-linking enzyme associated with XFS in genetic studies, was an abundant XFM constituent. Ligands of the transforming growth factor-ß superfamily were prominent, including LEFTY2, a protein best known for its role in establishing the embryonic body axis. Elevated levels of LEFTY2 were also detected in AH from patients with XFG, a finding confirmed subsequently by ELISA. Conclusions: This analysis verified the presence of suspected XFM proteins and identified novel components. Quantitative comparisons between patient samples revealed a consistent XFM proteome characterized by strong expression of fibrillin-1, lysyl oxidase-like-1, and LEFTY2. Elevated levels of LEFTY2 in the AH of patients with XFG may serve as a biomarker for the disease.

Increased Aqueous Humor GDF15 Is Associated with Worse Visual Field Loss in Pseudoexfoliative Glaucoma Patients.

Purpose: To determine whether increased growth differentiation factor 15 (GDF15) in aqueous humor (AH) is associated with worse visual field loss in patients with pseudoexfoliative glaucoma (PXG). Methods: We recruited 12 patients (6 males, 6 females) with primary open-angle glaucoma (POAG) or PXG who were scheduled to undergo glaucoma surgery. AH was obtained from the initial peripheral paracentesis for the planned glaucoma surgery, and GDF15 levels were quantified with enzyme-linked immunosorbent assay by an investigator masked to clinical information. Humphrey visual field testing was performed as a part of routine care; results were obtained by reviewing the medical record. Results: AH GDF15 was detectable in patients with POAG and PXG. Increased AH GDF15 was significantly associated with worse mean deviation in patients with POAG (r = -0.94; 95% confidence interval [CI], -0.99 to -0.33; P = 0.02) and PXG (r = -0.92; 95% CI, -0.99 to -0.41; P = 0.01). Conclusions: AH GDF15 is detectable in patients with PXG and POAG. Elevated AH GDF15 is strongly associated with worse mean deviation in both subgroups. These findings suggest that GDF15 may be a molecular marker of glaucoma severity that is generalizable to multiple types of glaucoma regardless of the underlying etiology. Translational Relevance: This study provides proof of concept that GDF15, a molecular marker of retinal ganglion stress that was initially identified in rodent models, may have clinical utility as a measure of glaucoma severity not only in POAG but also in PXG.

DNase IIbeta distribution and activity in the mouse lens.

PURPOSE: To map the cellular and subcellular distribution of DNase IIbeta activity in the mouse lens. METHODS: DNase IIbeta-specific activity was determined by assaying lens lysates prepared from wild-type or DNase IIbeta-null mice. Regional nuclease activity was determined by microdissection of lens samples or a tissue-imprinting assay. Subcellular distribution was determined by density-gradient ultracentrifugation. RESULTS: DNase IIbeta transcripts increased 200-fold in abundance during fiber cell formation, and DNase IIbeta activity accounted for approximately 50% of the acid nuclease activity in the cortical fiber cells. Examination of lenses from DNase IIbeta-null mice confirmed that the enzyme was required for denucleation. In wild-type lenses, nuclei were TUNEL positive before denucleation, indicating that 3'-OH DNA ends had accumulated. However, DNase IIbeta-mediated scission generates 3'-PO(4)(-) DNA ends only. This paradoxical finding was explained by the presence of phosphatases that converted the 3'-PO(4)(-) ends produced by DNase IIbeta into 3'-OH ends. DNase IIbeta activity was strongest early in differentiation, where it was associated with the lysosomal fraction. Later, an increasing proportion of DNase IIbeta activity was found in the cytosol. CONCLUSIONS: DNase IIbeta activity correlated with and was necessary for fiber denucleation and was most likely contained initially within fiber cell lysosomes before release into the cytoplasm.

Proteomic Analysis of the Bovine and Human Ciliary Zonule.

Purpose: The zonule of Zinn (ciliary zonule) is a system of fibers that centers the crystalline lens on the optical axis of the eye. Mutations in zonule components underlie syndromic conditions associated with a broad range of ocular pathologies, including microspherophakia and ectopia lentis. Here, we used HPLC-mass spectrometry to determine the molecular composition of the zonule. Methods: Tryptic digests of human and bovine zonular samples were analyzed by HPLC-mass spectrometry. The distribution of selected components was confirmed by immunofluorescence confocal microscopy. In bovine samples, the composition of the equatorial zonule was compared to that of the hyaloid zonule and vitreous humor. Results: The 52 proteins common to the zonules of both species accounted for >95% of the zonular protein. Glycoproteins constituted the main structural components, with two proteins, FBN1 and LTBP2, constituting 70%-80% of the protein. Other abundant components were MFAP2, EMILIN-1, and ADAMTSL-6. Lysyl oxidase-like 1, a crosslinking enzyme implicated in collagen and elastin biogenesis, was detected at significant levels. The equatorial and hyaloid zonular samples were compositionally similar to each other, although the hyaloid sample was relatively enriched in the proteoglycan opticin and the fibrillar collagens COL2A1, COL11A1, COL5A2, and COL5A3. Conclusions: The zonular proteome was surprisingly complex. In addition to structural components, it contained signaling proteins, protease inhibitors, and crosslinking enzymes. The equatorial and hyaloid zonules were similar in composition, but the latter may form part of a composite structure, the hyaloid membrane, that stabilizes the vitreous face.

The Na+/Ca2+, K+ exchanger NCKX4 is required for efficient cone-mediated vision.

Calcium (Ca2+) plays an important role in the function and health of neurons. In vertebrate cone photoreceptors, Ca2+ controls photoresponse sensitivity, kinetics, and light adaptation. Despite the critical role of Ca2+ in supporting the function and survival of cones, the mechanism for its extrusion from cone outer segments is not well understood. Here, we show that the Na+/Ca2+, K+ exchanger NCKX4 is expressed in zebrafish, mouse, and primate cones. Functional analysis of NCKX4-deficient mouse cones revealed that this exchanger is essential for the wide operating range and high temporal resolution of cone-mediated vision. We show that NCKX4 shapes the cone photoresponse together with the cone-specific NCKX2: NCKX4 acts early to limit response amplitude, while NCKX2 acts late to further accelerate response recovery. The regulation of Ca2+ by NCKX4 in cones is a novel mechanism that supports their ability to function as daytime photoreceptors and promotes their survival.

Birc7: A Late Fiber Gene of the Crystalline Lens.

PURPOSE: A distinct subset of genes, so-called "late fiber genes," is expressed in cells bordering the central, organelle-free zone (OFZ) of the lens. The purpose of this study was to identify additional members of this group. METHODS: Fiber cells were harvested from various layers of the lens by laser micro-dissection and subjected to microarray, in situ hybridization, and Western blot analysis. RESULTS: Expression of Livin, a member of the inhibitor of apoptosis protein (IAP) family encoded by Birc7, was strongly upregulated in deep cortical fiber cells. The depth-dependent distribution of Livin mRNA was confirmed by quantitative PCR and in situ hybridization. The onset of Livin expression coincided with loss of organelles from primary fiber cells. Livin expression peaked at 1 month but was sustained even in aged lenses. Antibodies raised against mouse Livin labeled multiple bands on immunoblots, reflecting progressive proteolysis of the parent molecule during differentiation. Mice harboring a floxed Birc7 allele were generated and used to conditionally delete Birc7 in lens. Lenses from knockout mice grew normally and retained their transparency, suggesting that Livin does not have an indispensable role in fiber cell differentiation. CONCLUSIONS: Birc7 is a late fiber gene of the mouse lens. In tumor cells, Livin acts as an antiapoptotic protein, but its function in the lens is enigmatic. Livin is a RING domain protein with putative E3 ubiquitin ligase activity. Its expression in cells bordering the OFZ is consistent with a role in organelle degradation, a process in which the ubiquitin proteasome pathway has been implicated previously.

DNase I and fragmented chromatin during nuclear degradation in adult bovine lens fibers.

PURPOSE: Nuclear loss is a most remarkable organelle disappearance during terminal differentiation of lens fiber cells given that it implicates the full degradation of a major molecular component, DNA. Consequently, to gain insight into the progression of DNA cleavage we analyzed the appearance of single strand breaks in relationship with chromatin condensation. To assess a possible involvement of DNase I in DNA fragmentation we explored its localization in lens fibers having different degrees of nuclear breakdown, evaluated by the state of chromatin, nuclear envelope, and DNA. METHODS: Whole mounts of adult bovine lens epithelium as well as lens cryosections were utilized to examine, using antibodies or specific molecular probes, the localization of DNase I, nuclear membrane, lamins, and DNA 3'-OH-free termini. Nuclease activity gel and western blot assays were used to characterize DNase I in different lens fiber extracts. RESULTS: Nuclear morphology was found to undergo significant changes from the onset of fiber differentiation. Initial spherical nuclei present at early fibergenesis stages evolve to elongated ones in mature fibers. Chromatin did not present signs of condensation in these nuclei. However, nuclei from fibers located deeper in lens volume exhibited some chromatin condensation and fragmentation while the nuclear lamina appeared undamaged. At more advanced stages, different patterns of nuclear envelope integrity and chromatin condensation and cleavage were observed. DNase I was found in the cytoplasm in the very initial fibers and then in the nuclear territory. DNase I appeared closely associated with fully condensed and fragmented chromatin at the final phases of nuclear breakdown. CONCLUSIONS: DNase I is a nuclease present in bovine lens fibers and can be considered as an enzyme producing final DNA cleavage since it is closely associated with highly fragmented DNA in disintegrating nuclei.

The penny pusher: a cellular model of lens growth.

PURPOSE: The mechanisms that regulate the number of cells in the lens and, therefore, its size and shape are unknown. We examined the dynamic relationship between proliferative behavior in the epithelial layer and macroscopic lens growth. METHODS: The distribution of S-phase cells across the epithelium was visualized by confocal microscopy and cell populations were determined from orthographic projections of the lens surface. RESULTS: The number of S-phase cells in the mouse lens epithelium fell exponentially, to an asymptotic value of approximately 200 cells by 6 months. Mitosis became increasingly restricted to a 300-µm-wide swath of equatorial epithelium, the germinative zone (GZ), within which two peaks in labeling index were detected. Postnatally, the cell population increased to approximately 50,000 cells at 4 weeks of age. Thereafter, the number of cells declined, despite continued growth in lens dimensions. This apparently paradoxical observation was explained by a time-dependent increase in the surface area of cells at all locations. The cell biological measurements were incorporated into a physical model, the Penny Pusher. In this simple model, cells were considered to be of a single type, the proliferative behavior of which depended solely on latitude. Simulations using the Penny Pusher predicted the emergence of cell clones and were in good agreement with data obtained from earlier lineage-tracing studies. CONCLUSIONS: The Penny Pusher, a simple stochastic model, offers a useful conceptual framework for the investigation of lens growth mechanisms and provides a plausible alternative to growth models that postulate the existence of lens stem cells.

Development, composition, and structural arrangements of the ciliary zonule of the mouse.

PURPOSE: Here, we examined the development, composition, and structural organization of the ciliary zonule of the mouse. Fibrillin 1, a large glycoprotein enriched in force-bearing tissues, is a prominent constituent of the mouse zonule. In humans, mutations in the gene for fibrillin 1 (FBN1) underlie Marfan syndrome (MS), a disorder characterized by lens dislocation and other ocular symptoms. METHODS: Fibrillin expression was analyzed by in situ hybridization. The organization of the zonule was visualized using antibodies to Fbn1, Fbn2, and microfibril-associated glycoprotein-1 (Magp1) in conjunction with 5-ethynyl-2'-deoxyuridine (EdU), an S-phase marker. RESULTS: Microfibrils, enriched in Fbn2 and Magp1, were prominent components of the temporary vascular tunic of the embryonic lens. Fbn2 expression by nonpigmented ciliary epithelial cells diminished postnatally and there was a concomitant increase in Fbn1 expression, especially in cells located in valleys between the ciliary folds. Zonular fibers projected from the posterior pars plicata to the lens in anterior, equatorial, and posterior groupings. The attachment point of the posterior zonular fibers consisted of a dense meshwork of radially oriented microfibrils that we termed the fibrillar girdle. The fibrillar girdle was located directly above the transition zone, a region of the lens epithelium in which cells commit to terminal differentiation. CONCLUSIONS: The development and arrangement of the murine ciliary zonule are similar to those of humans, and consequently the mouse eye may be a useful model in which to study ocular complications of MS.

A role for epha2 in cell migration and refractive organization of the ocular lens.

PURPOSE: The Epha2 receptor is a surprisingly abundant component of the membrane proteome of vertebrate lenses. In humans, genetic studies have linked mutations in EPHA2 to inherited and age-related forms of cataract, but the function of Epha2 in the lens is obscure. To gain insights into the role of Epha2, a comparative analysis of lenses from wild-type and Epha2(-/-) mice was performed. METHODS: Epha2 distribution was examined using immunocytochemistry and Western blot analysis. Lens optical quality was assessed by laser refractometry. Confocal microscopy was used to analyze cellular phenotypes. RESULTS: In wild-type lenses, Epha2 was expressed by lens epithelial cells and elongating fibers but was degraded during the later stages of fiber differentiation. Epha2-null lenses retained their transparency, but two key optical parameters, lens shape and internal composition, were compromised in Epha2(-/-) animals. Epha2-null lenses were smaller and more spherical than age-matched wild-type lenses, and laser refractometry revealed a significant decrease in refractive power of the outer cell layers of mutant lenses. In the absence of Epha2, fiber cells deviated from their normal course and terminated at sutures that were no longer centered on the optical axis. Patterning defects were also noted at the level of individual cells. Wild-type fiber cells had hexagonal cross-sectional profiles with membrane protrusions extending from the cell vertices. In contrast, Epha2(-/-) cells had irregular profiles, and protrusions extended from all membrane surfaces. CONCLUSIONS: These studies indicate that Epha2 is not required for transparency but does play an indispensable role in the cytoarchitecture and refractive quality of the lens.

Cadm1 expression and function in the mouse lens.

PURPOSE: The immunoglobulin superfamily member Cadm1 is a single-pass, type 1 membrane protein that mediates calcium-independent, cell-cell adhesion. Cadm1 has been implicated in tumor formation and synaptogenesis. A recent analysis of mouse lens cell membranes identified Cadm1 as a major constituent of the fiber cell membrane proteome. Here the authors examined the expression and function of Cadm1 in the mouse lens. METHODS: Cadm1 expression was analyzed by Western blotting and immunofluorescence. The morphology of individual wild-type and Cadm1-null lens cells was visualized by confocal microscopy. RESULTS: Cadm1 was present in epithelial and superficial fiber cells as a heavily glycosylated protein with an apparent molecular mass of ≈80 kDa. Analysis of proteins extracted from various strata of the lens indicated that Cadm1 was degraded during fiber cell differentiation, at approximately the same time as the lens organelles, an observation confirmed by confocal microscopy. In epithelial cells, Cadm1 was enriched in basolateral membranes, whereas, in fiber cells, expression was restricted to the lateral membranes. Lenses from Cadm1-null mice were of normal size and transparency. The three-dimensional morphology of the cells in the epithelial layer was unaltered in the absence of Cadm1. However, in contrast to wild-type lens fiber cells, Cadm1-null fiber cells had an irregular, highly undulating morphology. CONCLUSIONS: Cadm1 is an abundant component of the lens fiber cell membrane. Although not essential for lens transparency, Cadm1 has an indispensable role in establishing and maintaining the characteristic three-dimensional architecture of the lens fiber cell mass.

Further analysis of the lens phenotype in Lim2-deficient mice.

PURPOSE: Lim2 (MP20) is the second most abundant integral protein of lens fiber cell membranes. A comparative analysis was performed of wild-type and Lim2-deficient (Lim2(Gt/Gt)) mouse lenses, to better define the anatomic and physiologic roles of Lim2. METHODS: Scanning electron microscopy (SEM) and confocal microscopy were used to assess the contribution of Lim2 to lens tissue architecture. Differentiation-dependent changes in cytoskeletal composition were identified by mass spectrometry and immunoblot analysis. The effects on cell-cell communication were quantified using impedance analysis. RESULTS: Lim2-null lenses were grossly normal. At the cellular level, however, subtle structural alterations were evident. Confocal microscopy and SEM analysis revealed that cortical Lim2(Gt/Gt) fiber cells lacked the undulating morphology that characterized wild-type fiber cells. On SDS-PAGE analysis the composition of cortical fiber cells from wild-type and Lim2-null lenses appeared similar. However, marked disparities were evident in samples prepared from the lens core of the two genotypes. Several cytoskeletal proteins that were abundant in wild-type core fiber cells were diminished in the cores of Lim2(Gt/Gt) lenses. Electrophysiological measurements indicated a small decrease in the membrane potential of Lim2(Gt/Gt) lenses and a two-fold increase in the effective intracellular resistivity. In the lens core, this may have reflected decreased expression levels of the gap junction protein connexin 46 (Cx46). In contrast, increased resistivity in the outer cell layers of Lim2(Gt/Gt) lenses could not be attributed to decreased connexin expression and may reflect the absence of cell fusions in Lim2(Gt/Gt) lenses. CONCLUSIONS: Comparative analysis of wild-type and Lim2-deficient lenses has implicated Lim2 in maintenance of cytoskeletal integrity, cell morphology, and intercellular communication.

Calpain expression and activity during lens fiber cell differentiation.

In animal models, the dysregulated activity of calcium-activated proteases, calpains, contributes directly to cataract formation. However, the physiological role of calpains in the healthy lens is not well defined. In this study, we examined the expression pattern of calpains in the mouse lens. Real time PCR and Western blotting data indicated that calpain 1, 2, 3, and 7 were expressed in lens fiber cells. Using controlled lysis, depth-dependent expression profiles for each calpain were obtained. These indicated that, unlike calpain 1, 2, and 7, which were most abundant in cells near the lens surface, calpain 3 expression was strongest in the deep cortical region of the lens. We detected calpain activities in vitro and showed that calpains were active in vivo by microinjecting fluorogenic calpain substrates into cortical fiber cells. To identify endogenous calpain substrates, membrane/cytoskeleton preparations were treated with recombinant calpain, and cleaved products were identified by two-dimensional difference electrophoresis/mass spectrometry. Among the calpain substrates identified by this approach was alphaII-spectrin. An antibody that specifically recognized calpain-cleaved spectrin was used to demonstrate that spectrin is cleaved in vivo, late in fiber cell differentiation, at or about the time that lens organelles are degraded. The generation of the calpain-specific spectrin cleavage product was not observed in lens tissue from calpain 3-null mice, indicating that calpain 3 is uniquely activated during lens fiber differentiation. Our data suggest a role for calpains in the remodeling of the membrane cytoskeleton that occurs with fiber cell maturation.

The stratified syncytium of the vertebrate lens.

The fusion of cells to generate syncytial tissues is a crucial event in the development of many organisms. In the lens of the vertebrate eye, proteins and other macromolecules diffuse from cell to cell via the large molecule diffusion pathway (LMDP). We used the tamoxifen-induced expression of GFP to investigate the nature and role of the LMDP in living, intact lenses. Our data indicate that the LMPD preferentially connects cells lying within a stratum of the lens cortex and that formation of the LMPD depends on the expression of Lim2, a claudin-like molecule. The conduits for intercellular protein exchange are most likely regions of partial cellular fusion, which are commonly observed in wild-type lenses but rare or absent in Lim2-deficient lenses. The observation that lens tissue constitutes a stratified syncytium has implications for the transparency, refractive function and pathophysiology of the tissue.

Bovine DNase I: gene organization, mRNA expression, and changes in the topological distribution of the protein during apoptosis in lens epithelial cells.

Genomic DNA sequencing and alignment with the known DNase I mRNA showed that the bovine gene consists of 9 exons and that only the last 8 encode the protein, since initial ATG was found at exon II. RT-PCR was used to identify DNase I mRNA in lens epithelium in vivo and in cultured epithelial cells. We found DNase I transcripts having the same nucleotide sequence as the pancreas form and others lacking almost all exon V. The lens protein presented a slightly higher relative molecular weight than the pancreatic enzyme. Lens DNase I was located in secretory pathway organelles and excluded from the nucleus. Nevertheless, in apoptotic lens epithelial cells in vitro, DNase I translocated to the nucleus and co-localized with TUNEL positive chromatin aggregates. These results indicate that cells in the lens epithelium constitutively express DNase I, and suggest a direct involvement of this nuclease in the final phases of chromatin degradation.