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Charged multivesicular-body protein 4b (CHMP4B) is a core sub-unit of the endosomal sorting complex required for transport-III (ESCRT-III) machinery that serves myriad remodeling and scission processes of biological membranes. Mutation of the human CHMP4B gene underlies rare forms of early-onset lens opacities, or cataracts, and CHMP4B is required for lens growth and differentiation in mice. Here, we determine the sub-cellular distribution of CHMP4B in the lens and uncover a novel association with gap-junction alpha-3 protein (GJA3) or connexin-46 (Cx46) and GJA8 or Cx50. Immunofluorescence confocal microscopy revealed that CHMP4B localized to cell membranes of elongated fiber cells in the outer cortex of the lens - where large gap-junction plaques begin to form - particularly on the broad faces of these flattened hexagon-like cells in cross-section. Dual immunofluorescence imaging showed that CHMP4B co-localized with gap-junction plaques containing Cx46 and/or Cx50. When combined with the in situ proximity ligation assay, immunofluorescence confocal imaging indicated that CHMP4B lay in close physical proximity to Cx46 and Cx50. In Cx46-knockout (Cx46-KO) lenses, CHMP4B-membrane distribution was similar to that of wild-type, whereas, in Cx50-KO lenses CHMP4B localization to fiber cell membranes was lost. Immunoprecipitation and immunoblotting analyses revealed that CHMP4B formed complexes with Cx46 and Cx50 in vitro. Collectively, our data suggest that CHMP4B forms plasma-membrane complexes, either directly and/or indirectly, with gap-junction proteins Cx46 and Cx50 that are often associated with ‘ball-and-socket’ double-membrane junctions during lens fiber cell differentiation.

Cataract, the clinical correlate of opacity or light scattering in the eye lens, is usually caused by the presence of high-molecular-weight (HMW) protein aggregates or disruption of the lens microarchitecture. In general, genes involved in inherited cataracts reflect important processes and pathways in the lens including lens crystallins, connexins, growth factors, membrane proteins, intermediate filament proteins, and chaperones. Usually, mutations causing severe damage to proteins cause congenital cataracts, while milder variants increasing susceptibility to environmental insults are associated with age-related cataracts. These may have different pathogenic mechanisms: Congenital cataracts induce the unfolded protein response and apoptosis. By contrast, denatured crystallins in age-related cataracts are bound by α-crystallin and form light-scattering HMW aggregates. New therapeutic approaches to age-related cataracts use chemical chaperones to solubilize HMW aggregates, while attempts are being made to regenerate lenses using endogenous stem cells to treat congenital cataracts.

Charged multivesicular body protein 4B (CHMP4B) functions as a core component of the endosome sorting complex required for transport-III (ESCRT-III) machinery that facilitates diverse membrane remodeling and scission processes in eukaryotes. Mutations in the human CHMP4B gene underlie rare, inherited forms of early-onset lens opacities or cataract. Here we have characterized the lens phenotypes of mutant (knock-in) mice harboring a human cataract-associated mutation (p.D129V) in CHMP4B (Chmp4b-mutant) and conditional knockdown mice deficient in lens CHMP4B (Chmp4b-CKD). In situ hybridization localized Chmp4b transcripts to lens epithelial cells and elongating fiber cells at the lens equator. Heterozygous Chmp4b-mutant (D/V) mice were viable and fertile with lenses grossly similar to those of wild-type. However, homozygous Chmp4b-mutant (V/V) mice died by embryonic day 15.5 (E15.5) with grossly abnormal eye and brain histology. Chmp4b-CKD mice displayed variable degrees of lens dysmorphology including lens ablation. Immuno-localization of aquaporin-0 (AQP0) revealed lens fiber cell degeneration in homozygous Chmp4b-mutant (V/V) mouse embryos and in embryonic and postnatal Chmp4b-CKD mice. DNA fragmentation (TUNEL) analysis revealed global cell death in homozygous Chmp4b-mutant (V/V) embryos, whereas, cell death was confined to the lens of Chmp4b-CKD mice. Immuno-localization of the monocyte/macrophage marker macrosialin (CD68) suggested that severe lens degeneration in Chmp4b-CKD mice resulted in an ocular immune cell response. Collectively, these mouse data suggest that (1) heterozygous, germ-line mutations in Chmp4b may not manifest as cataract, (2) homozygous, germ-line mutations in Chmp4b are embryonic lethal, and (3) conditional loss of Chmp4b results in arrest of lens growth and differentiation.

Congenital cataracts cause approximately one-third of blindness in infants worldwide. If untreated they can cause permanent blindness by interfering with the sharp focus of light onto the retina and thus fail to establish appropriate synaptic connections between the retina and the visual cortex. Between 8 and 30% (see later) of congenital cataracts are inherited, and our understanding of their genetic architecture is increasing. Delineating the relationship between the genes and mutations causing cataracts and their phenotypic presentation can help us to understand the biology of the lens and provide a framework for the clinical approach to diagnosis and treatment.

Transient-receptor-potential cation channel, subfamily M, member 3 (TRPM3) serves as a polymodal calcium sensor in diverse mammalian cell-types. Mutation of the human TRPM3 gene (TRPM3) has been linked with inherited forms of early-onset cataract with or without other eye abnormalities. Here we have characterized the ocular phenotypes of germline ‘knock-in’ mice that harbor a human cataract-associated isoleucine-to-methionine mutation (p.I65M) in TRPM3 (Trpm3-mutant) compared with germline ‘knock-out’ mice that functionally lack TRPM3 (Trpm3-null). Despite strong expression of Trpm3 in lens epithelial cells, neither heterozygous (Trpm3(+/−)) nor homozygous (Trpm3(−/−)) Trpm3-null mice developed cataract; however, the latter exhibited a mild impairment of lens growth. By contrast, homozygous Trpm3-M/M mutants developed severe, progressive, anterior pyramid-like cataract with microphthalmia, whereas, heterozygous Trpm3-I/M and hemizygous Trpm3-M/- mutants developed anterior pyramidal cataract with delayed onset and progression - consistent with a semi-dominant lens phenotype. Histochemical staining revealed abnormal accumulation of calcium phosphate-like deposits and collagen fibrils in Trpm3-mutant lenses and immunoblotting detected increased αII-spectrin cleavage products consistent with calpain hyper-activation. Immunofluorescent confocal microscopy of Trpm3-M/M mutant lenses revealed fiber cell membrane degeneration that was accompanied by accumulation of alpha-smooth muscle actin positive (α-SMA+ve) myofibroblast-like cells and macrosialin positive (CD68+ve) macrophage-like cells. Collectively, our mouse model data support an ocular disease association for TRPM3 in humans and suggest that (1) Trpm3 deficiency impaired lens growth but not lens transparency and (2) Trpm3 dysfunction resulted in progressive lens degeneration and calcification coupled with pro-fibrotic (α-SMA+ve) and immune (CD68+ve) cell responses.

Lens opacities or cataracts in children are usually congenital and/or hereditary and less often are secondary to trauma, infections, or systemic disorders such as diabetes or galactosemia. Congenital cataracts cause approximately one-third of blindness in infants worldwide. Between 8% and 25% of congenital cataracts are inherited, and knowledge of their genetic architecture is increasing. Delineating the relationship between the genes and mutations causing cataracts and their phenotypic presentation can help us to understand the biology of the lens and provide a framework for the clinical approach to diagnosis and therapy.

Ephrin type-A receptor 2 (EPHA2) and one of its ligands, ephrin-A5 (EFNA5), have been associated with loss of eye lens transparency, or cataract, - an important cause of visual impairment. Here we show that mice functionally lacking EPHA2 (Epha2-null), EFNA5 (Efna5-null), or both receptor and ligand (Epha2/Efna5-null) consistently develop mostly transparent lenses with an internal refractive disturbance and a grossly disturbed cellular architecture. In situ hybridization localized Epha2 and Efna5 transcripts to lens epithelial cells and nascent fiber cells at the lens equator. In vivo labeling of Epha2-null lenses with a thymidine analog detected a significant decrease in lens epithelial cell proliferation within the germinative zone resulting in impaired early lens growth. Ex vivo imaging of Epha2-null, Efna5-null, and Epha2/Efna5-null lenses labelled in vivo with a membrane-targeted red fluorescent protein revealed misalignment of elongating fiber cells at the lens equator and loss of Y-suture pattern formation near the anterior and posterior poles of the lens. Immuno-fluorescent labeling of lens major intrinsic protein or aquaporin-0 (MIP/AQP0) showed that the precise, radial column patterning of hexagonal fiber cells throughout the cortex region was disrupted in Epha2-null, Efna5-null and Epha2/Efna5-null lenses. Collectively, these data suggest that Epha2 and Efna5 participate in the complex, global patterning of lens fiber cells that is necessary for maximal optical quality.

The crystalline lens plays an important role in the refractive vision of vertebrates by facilitating variable fine focusing of light onto the retina. Loss of lens transparency, or cataract, is a frequently acquired cause of visual impairment in adults and may also present during childhood. Genetic studies have identified mutations in over 30 causative genes for congenital or other early-onset forms of cataract as well as several gene variants associated with age-related cataract. However, the pathogenic mechanisms resulting from genetic determinants of cataract are only just beginning to be understood. Here, we briefly summarize current concepts pointing to differences in the molecular mechanisms underlying congenital and age-related forms of cataract.

First discovered in a light-sensitive retinal mutant ofDrosophila, the transient receptor potential (TRP) superfamily of non-selective cation channels serve as polymodal cellular sensors that participate in diverse physiological processes across the animal kingdom including the perception of light, temperature, pressure, and pain. TRPM3 belongs to the melastatin sub-family of TRP channels and has been shown to function as a spontaneous calcium channel, with permeability to other cations influenced by alternative splicing and/or non-canonical channel activity. Activators of TRPM3 channels include the neurosteroid pregnenolone sulfate, calmodulin, phosphoinositides, and heat, whereas inhibitors include certain drugs, plant-derived metabolites, and G-protein subunits. Activation of TRPM3 channels at the cell membrane elicits a signal transduction cascade of mitogen-activated kinases and stimulus response transcription factors. The mammalian TRPM3 gene hosts a non-coding microRNA gene specifying miR-204 that serves as both a tumor suppressor and a negative regulator of post-transcriptional gene expression during eye development in vertebrates. Ocular co-expression of TRPM3 and miR-204 is upregulated by the paired box 6 transcription factor (PAX6) and mutations in all three corresponding genes underlie inherited forms of eye disease in humans including early-onset cataract, retinal dystrophy, and coloboma. This review outlines the genomic and functional complexity of the TRPM3_miR-204 locus in mammalian eye development and disease.

Age-related cataract is a leading cause of vision loss and the most common reason for eye surgery in the elderly. Epidemiological studies of affected siblings and twins indicate that genetic factors may account for as much as 50% of the risk for age-related cataract. Family-based linkage studies have identified a number of genetic susceptibility loci for age-related cataract, and case–control association studies have implicated a number of diverse candidate genes involved in: antioxidant metabolism, galactose metabolism, the heat-shock stress response, and the ephrin signaling pathway. Rigorous characterization of the underlying genetic causes may lead to nonsurgical treatments for age-related cataract.

Major intrinsic protein or aquaporin-0 (MIP/AQP0) functions as a water channel and a cell-junction molecule in the vertebrate eye lens. Loss of MIP function in the lens leads to degraded optical quality and cataract formation by pathogenic mechanisms that are unclear. Here we have used microarray-hybridization analysis to detect lens transcriptome changes during cataract formation in mice that are functionally null for MIP (Mip−/−). In newborn Mip−/− lenses (P1) 11 genes were up-regulated and 18 were down-regulated (>2-fold, p=6-fold) in the Mip−/− lens at P1 included those coding for a mitochondrial translocase (Timmdc1), a matrix metallopeptidase (Mmp2), a Rho GTPase-interacting protein (Ubxn11) and a transcription factor (Twist2). Apart from Mip, the most down-regulated genes (>4-fold) in the Mip−/− lens at P1 included those coding for a proteasome sub-unit (Psmd8), a ribonuclease (Pop4), and a heat-shock protein (Hspb1). Lens fiber cell degeneration in the Mip−/− lens was associated with increased numbers of TUNEL-positive cell nuclei and dramatically elevated levels of calpain-mediated proteolysis of αII-spectrin. However red-ox status, measured by glutathione and free-radical levels, was similar to that of wild-type. These data suggest that while relatively few genes (~1.5% of the transcriptome) were differentially regulated >2-fold in the Mip−/− lens, calpain hyper-activation acts as a terminal pathogenic event during lens fiber cell death and cataract formation.

Rare germ-line mutations in the coding regions of the human EPHA2 gene (EPHA2) have been associated with inherited forms of pediatric cataract, whereas, frequent, non-coding, single nucleotide variants (SNVs) have been associated with age-related cataract. Here we sought to determine if germ-line EPHA2 coding SNVs were associated with age-related cataract in a case-control DNA panel (> 50 years) and if somatic EPHA2 coding SNVs were associated with lens aging and/or cataract in a post-mortem lens DNA panel (> 48 years). Micro-fluidic PCR amplification followed by targeted amplicon (exon) next-generation (deep) sequencing of EPHA2 (17-exons) afforded high read-depth coverage (1000x) for > 82% of reads in the cataract case-control panel (161 cases, 64 controls) and > 70% of reads in the post-mortem lens panel (35 clear lens pairs, 22 cataract lens pairs). Novel and reference (known) missense SNVs in EPHA2 that were predicted in silico to be functionally damaging were found in both cases and controls from the age-related cataract panel at variant allele frequencies (VAFs) consistent with germ-line transmission (VAF > 20%). Similarly, both novel and reference missense SNVs in EPHA2 were found in the post-mortem lens panel at VAFs consistent with a somatic origin (VAF > 3%). The majority of SNVs found in the cataract case-control panel and post-mortem lens panel were transitions and many occurred at di-pyrimidine sites that are susceptible to ultraviolet (UV) radiation induced mutation. These data suggest that novel germ-line (blood) and somatic (lens) coding SNVs in EPHA2 that are predicted to be functionally deleterious occur in adults over 50 years of age. However, both types of EPHA2 coding variants were present at comparable levels in individuals with or without age-related cataract making simple genotype-phenotype correlations inconclusive.

Maintenance of proper biomechanics of the eye lens is important for its structural integrity and for the process of accommodation to focus near and far objects. Several studies have shown that specialized cytoskeletal systems such as the beaded filament (BF) and spectrin-actin networks contribute to mammalian lens biomechanics; mutations or deletion in these proteins alters lens biomechanics. Aquaporin 0 (AQP0), which constitutes ∼45% of the total membrane proteins of lens fiber cells, has been shown to function as a water channel and a structural cell-to-cell adhesion (CTCA) protein. Our recent exvivo study on AQP0 knockout (AQP0 KO) mouse lenses showed the CTCA function of AQP0 could be crucial for establishing the refractive index gradient. However, biomechanical studies on the role of AQP0 are lacking. The present investigation used wild type (WT), AQP5 KO (AQP5(-/-)), AQP0 KO (heterozygous KO: AQP0(+/-); homozygous KO: AQP0(-/-); all in C57BL/6J) and WT-FVB/N mouse lenses to learn more about the role of fiber cell AQPs in lens biomechanics. Electron microscopic images exhibited decreases in lens fiber cell compaction and increases in extracellular space due to deletion of even one allele of AQP0. Biomechanical assay revealed that loss of one or both alleles of AQP0 caused a significant reduction in the compressive load-bearing capacity of the lenses compared to WT lenses. Conversely, loss of AQP5 did not alter the lens load-bearing ability. Compressive load-bearing at the suture area of AQP0(+/-) lenses showed easy separation while WT lens suture remained intact. These data from KO mouse lenses in conjunction with previous studies on lens-specific BF proteins (CP49 and filensin) suggest that AQP0 and BF proteins could act co-operatively in establishing normal lens biomechanics. We hypothesize that AQP0, with its prolific expression at the fiber cell membrane, could provide anchorage for cytoskeletal structures like BFs and together they help to confer fiber cell shape, architecture and integrity. To our knowledge, this is the first report identifying the involvement of an aquaporin in lens biomechanics. Since accommodation is required in human lenses for proper focusing, alteration in the adhesion and/or water channel functions of AQP0 could contribute to presbyopia.

Primary open-angle glaucoma (POAG) is a clinically important and genetically heterogeneous cause of progressive vision loss as a result of retinal ganglion cell death. Here we have utilized trio-based, whole-exome sequencing to identify the genetic defect underlying an autosomal dominant form of adult-onset POAG segregating in an African-American family. Exome sequencing identified a novel missense variant (c.418C>T, p.Arg140Trp) in exon-5 of the gene coding for epidermal growth factor (EGF) containing fibulin-like extracellular matrix protein 1 (EFEMP1) that co-segregated with disease in the family. Linkage and haplotype analyses with microsatellite markers indicated that the disease interval overlapped a known POAG locus (GLC1H) on chromosome 2p. The p.Arg140Trp substitution was predicted in silico to have damaging effects on protein function and transient expression studies in cultured cells revealed that the Trp140-mutant protein exhibited increased intracellular accumulation compared with wild-type EFEMP1. In situ hybridization of the mouse eye with oligonucleotide probes detected the highest levels of EFEMP1 transcripts in the ciliary body, cornea, inner nuclear layer of the retina, and the optic nerve head. The recent finding that a common variant near EFEMP1 was associated with optic nerve-head morphology supports the possibility that the EFEMP1 variant identified in this POAG family may be pathogenic.

In order to accomplish its function of transmitting and focusing light, the crystalline lens of the vertebrate eye has evolved a unique cellular structure and protein complement. These distinct adaptations have provided a rich source of scientific discovery ranging from biochemistry and genetics to optics and physics. In addition, because of these adaptations, lens cells persist for the lifetime of an organism, providing an excellent model of the aging process. The chapters dealing with the lens will demonstrate how the different aspects of lens biology and biochemistry combine in this singular refractive organ to accomplish its critical role in the visual system.

The primary function of the lens resides in its transparency and ability to focus light on the retina. These require both that the lens cells contain high concentrations of densely packed lens crystallins to maintain a refractive index constant over distances approximating the wavelength of the light to be transmitted, and a specific arrangement of anterior epithelial cells and arcuate fiber cells lacking organelles in the nucleus to avoid blocking transmission of light. Because cells in the lens nucleus have shed their organelles, lens crystallins have to last for the lifetime of the organism, and are specifically adapted to this function. The lens crystallins comprise two major families: the βγ-crystallins are among the most stable proteins known and the α-crystallins, which have a chaperone-like function. Other proteins and metabolic activities of the lens are primarily organized to protect the crystallins from damage over time and to maintain homeostasis of the lens cells. Membrane protein channels maintain osmotic and ionic balance across the lens, while the lens cytoskeleton provides for the specific shape of the lens cells, especially the fiber cells of the nucleus. Perhaps most importantly, a large part of the metabolic activity in the lens is directed toward maintaining a reduced state, which shelters the lens crystallins and other cellular components from damage from UV light and oxidative stress. Finally, the energy requirements of the lens are met largely by glycolysis and the pentose phosphate pathway, perhaps in response to the avascular nature of the lens. Together, all these systems cooperate to maintain lens transparency over time.

Background Inherited cataract is a clinically important and genetically heterogeneous cause of visual impairment. Typically, it presents at an early age with or without other ocular/systemic signs and lacks clear phenotype-genotype correlation rendering both clinical classification and molecular diagnosis challenging. Here we have utilized trio-based whole exome sequencing to discover mutations in candidate genes underlying autosomal dominant cataract segregating in three nuclear families. Results In family A, we identified a recurrent heterozygous mutation in exon-2 of the gene encoding γD-crystallin (CRYGD; c.70C > A, p.Pro24Thr) that co-segregated with ‘coralliform’ lens opacities. Families B and C were found to harbor different novel variants in exon-2 of the gene coding for gap-junction protein α8 (GJA8; c.20T > C, p.Leu7Pro and c.293A > C, p.His98Pro). Each novel variant co-segregated with disease and was predicted in silico to have damaging effects on protein function. Conclusions Exome sequencing facilitates concurrent mutation-profiling of the burgeoning list of candidate genes for inherited cataract, and the results can provide enhanced clinical diagnosis and genetic counseling for affected families. Electronic supplementary material The online version of this article (doi:10.1186/s40246-014-0019-6) contains supplementary material, which is available to authorized users.