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Cataract is the leading cause of blindness among the elderly worldwide and cataract surgery is one of the most common operations performed in the United States. As the genetic etiology of cataract formation remains unclear, we conducted a multiethnic genome-wide association meta-analysis, combining results from the GERA and UK Biobank cohorts, and tested for replication in the 23andMe research cohort. We report 54 genome-wide significant loci, 37 of which were novel. Sex-stratified analyses identified CASP7 as an additional novel locus specific to women. We show that genes within or near 80% of the cataract-associated loci are significantly expressed and/or enriched-expressed in the mouse lens across various spatiotemporal stages as per iSyTE analysis. Furthermore, iSyTE shows 32 candidate genes in the associated loci have altered gene expression in 9 different gene perturbation mouse models of lens defects/cataract, suggesting their relevance to lens biology. Our work provides further insight into the complex genetic architecture of cataract susceptibility., The genetic basis of cataract is not well understood. Here, the authors perform a genome-wide association multiethnic meta-analysis of cataract, finding 37 new loci and replicating known and new loci. They additionally perform sex-specific analyses, identifying new associations specific to women.

Central corneal thickness is a quantitative trait with important associations to human health. In a phenotype-driven approach studying corneal thickness of congenic derivatives of C57BLKS/J and SJL/J mice, the critical region for a quantitative trait locus influencing corneal thickness, Cctq1a, was delimited to a 10-gene interval. Exome sequencing, RNAseq, and studying independent mutations eliminated multiple candidate genes and confirmed one. Though the causative gene, Tyr, has no obvious direct function in the transparent cornea, studies with multiple alleles on matched genetic backgrounds, both in isolation and genetic complementation crosses, confirmed allelism of Tyr-Cctq1a; albino mice lacking Tyr function had thin corneas. Albino mice also had increased axial length. Because albinism exposes eyes to increased light, the effect of dark-rearing was tested and found to rescue central corneal thickness. In sum, the results point to an epiphenomenon; developmental light exposure interacts with genotype as an important determinate of adult corneal thickness.

Cataract is the leading cause of blindness among the elderly worldwide and cataract surgery is one of the most common operations performed in the United States1-3. The etiology remains largely unclear, and to contribute to its elucidation we conducted a multiethnic genome-wide association meta-analysis of cataract, combining results from the GERA and UK Biobank cohorts, and tested for replication in the research cohort from 23andMe, Inc.. We report 54 genome-wide significant loci, 37 of which were previously unknown. Sex-stratified analyses identified two additional novel loci (CASP7 and GSTM2) specific to women and sex differences in effect sizes and significance of association at five other loci. We show that genes within or near 80% of the cataract-associated loci are significantly expressed and/or enriched-expressed in the mouse lens across various spatiotemporal stages. Further, 32 candidate genes in the associated loci have altered gene expression in 9 different gene perturbation mouse models of lens defects/cataract, suggesting their relevance to lens biology.

Variants in the LIM homeobox transcription factor 1-beta (LMX1B) gene predispose individuals to elevated intraocular pressure (IOP), a key risk factor for glaucoma. However, the effect of LMX1B mutations varies widely between individuals. To better understand the mechanisms underlying LMX1B-related phenotypes and individual differences, we backcrossed the Lmx1bV265D (also known as Lmx1bIcst) allele onto the C57BL/6J (B6), 129/Sj (129), C3A/BLiA-Pde6b+/J (C3H) and DBA/2J-Gpnmb+ (D2-G) mouse strain backgrounds. Strain background had a significant effect on the onset and severity of ocular phenotypes in Lmx1bV265D/+ mutant mice. Mice of the B6 background were the most susceptible to developing abnormal IOP distribution, severe anterior segment developmental anomalies (including malformed eccentric pupils, iridocorneal strands and corneal abnormalities) and glaucomatous nerve damage. By contrast, Lmx1bV265D mice of the 129 background were the most resistant to developing anterior segment abnormalities, had less severe IOP elevation than B6 mutants at young ages and showed no detectable nerve damage. To identify genetic modifiers of susceptibility to Lmx1bV265D-induced glaucoma-associated phenotypes, we performed a mapping cross between mice of the B6 (susceptible) and 129 (resistant) backgrounds. We identified a modifier locus on Chromosome 18, with the 129 allele(s) substantially lessening severity of ocular phenotypes, as confirmed by congenic analysis. By demonstrating a clear effect of genetic background in modulating Lmx1b-induced phenotypes, providing a panel of strains with different phenotypic severities and identifying a modifier locus, this study lays a foundation for better understanding the roles of LMX1B in glaucoma with the goal of developing new treatments., Summary: We observed that Lmx1b mutant mice of different strain backgrounds vary in onset and severity of glaucoma-related phenotypes and identified a modifier locus on Chr 18. These results could increase understanding of the mechanisms underlying glaucoma.

Central corneal thickness (CCT) is one of the most heritable human traits, with broad-sense heritability estimates ranging between 0.68 to 0.95. Despite the high heritability and numerous previous association studies, only 8.5% of CCT variance is currently explained. Here, we report the results of a multiethnic meta-analysis of available genome-wide association studies in which we find association between CCT and 98 genomic loci, of which 41 are novel. Among these loci, 20 were significantly associated with keratoconus, and one (RAPSN rs3740685) was significantly associated with glaucoma after Bonferroni correction. Two-sample Mendelian randomization analysis suggests that thinner CCT does not causally increase the risk of primary open-angle glaucoma. This large CCT study explains up to 14.2% of CCT variance and increases substantially our understanding of the etiology of CCT variation. This may open new avenues of investigation into human ocular traits and their relationship to the risk of vision disorders., Hélène Choquet et al. report the largest genome-wide analysis of central corneal thickness (CCT) to date, finding novel associations at 41 loci. The study, which includes individuals from 4 ethnic groups, including African Americans and Hispanic/Latino individuals, increases the variance explained for CCT from 8.5% to 14.2%. Study findings also suggest that thinner CCT does not causally increase the risk of primary open-angle glaucoma.

Refractive errors are the most common ocular disorders and are a leading cause of visual impairment worldwide. Although ocular axial length is well established to be a major determinant of refractive errors, the molecular and cellular processes regulating ocular axial growth are poorly understood. Mutations in genes encoding the PRSS56 and MFRP are a major cause of nanophthalmos. Accordingly, mouse models with mutations in the genes encoding the retinal factor PRSS56 or MFRP, a gene predominantly localized in the retinal pigment epithelial (RPE) exhibit ocular axial length reduction and extreme hyperopia. However, the precise mechanisms underlying PRSS56- and MFRP-mediated ocular axial growth remain elusive. Here, we show that Adamts19 expression is significantly upregulated in retina of mice lacking either Prss56 or Mfrp. Using a combination of genetic approaches and mouse models, we show that while ADAMTS19 is not required for ocular growth during normal development, its inactivation exacerbates ocular axial length reduction in both Prss56 or Mfrp mutant mice. These results suggest that the upregulation of retinal Adamts19 expression is part of an adaptive molecular response to counteract impaired ocular growth. Using a complementary genetic approach. We further demonstrate that loss of PRSS56 or MFRP function prevents excessive ocular axial growth in a mouse model of developmental myopia caused by a null mutation in Irpb, demonstrating that ocular axial elongation in Irbp-/- mice is fully dependent on PRSS56 and MFRP functions. Collectively, our findings provide insight into the molecular network involved in ocular axial growth regulation and refractive development and support the notion that relay of the signal between the retina and RPE could be critical for promoting ocular axial elongation.

We conducted a large multi-ethnic meta-analysis of genome-wide association studies for primary open-angle glaucoma (POAG) on a total of 34,179 cases vs 349,321 controls, and identified 127 independent risk loci, almost doubling the number of known loci for POAG. The majority of loci have broadly consistent effect across European, Asian and African ancestries. We identify a link, both genome-wide and at specific loci, between POAG and Alzheimer’s disease. Gene expression data and bioinformatic functional analyses provide further support for the functional relevance of the POAG risk genes. Several drug compounds target these risk genes and may be potential candidates for developing novel POAG treatments.

Glaucoma is a leading cause of blindness, affecting up to 70 million people worldwide. High intraocular pressure (IOP) is a major risk factor for glaucoma. It is well established that inefficient aqueous humor (AqH) outflow resulting from structural or functional alterations in ocular drainage tissues causes high IOP, but the genes and pathways involved are poorly understood. We previously demonstrated that mutations in the gene encoding the serine protease PRSS56 induces ocular angle closure and high IOP in mice and identified reduced ocular axial length as a potential contributing factor. Here, we show that Prss56−/− mice also exhibit an abnormal iridocorneal angle configuration characterized by a posterior shift of ocular drainage structures relative to the ciliary body and iris. Notably, we show that retina-derived PRSS56 is required between postnatal days 13 and 18 for proper iridocorneal configuration and that abnormal positioning of the ocular drainage tissues is not dependent on ocular size reduction in Prss56−/− mice. Furthermore, we demonstrate that the genetic context modulates the severity of IOP elevation in Prss56 mutant mice and describe a progressive degeneration of ocular drainage tissues that likely contributes to the exacerbation of the high IOP phenotype observed on the C3H/HeJ genetic background. Finally, we identify five rare PRSS56 variants associated with human primary congenital glaucoma, a condition characterized by abnormal development of the ocular drainage structures. Collectively, our findings point to a role for PRSS56 in the development and maintenance of ocular drainage tissues and IOP homeostasis, and provide new insights into glaucoma pathogenesis., Summary: This study reports a novel role of PRSS56 in the proper developmental positioning of ocular drainage tissues, establishing it as an important genetic factor involved in iridocorneal angle configuration and intraocular pressure homeostasis.

Precise regulation of ocular size is a critical determinant of normal visual acuity. Although it is generally accepted that ocular growth relies on a cascade of signaling events transmitted from the retina to the sclera, the factors and mechanism(s) involved are poorly understood. Recent studies have highlighted the importance of the retinal secreted serine protease PRSS56 and transmembrane glycoprotein MFRP, a factor predominantly expressed in the retinal pigment epithelium (RPE), in ocular size determination. Mutations in PRSS56 and MFRP constitute a major cause of nanophthalmos, a condition characterized by severe reduction in ocular axial length/extreme hyperopia. Interestingly, common variants of these genes have been implicated in myopia, a condition associated with ocular elongation. Consistent with these findings, mice with loss of function mutation in PRSS56 or MFRP exhibit a reduction in ocular axial length. However, the molecular network and cellular processes involved in PRSS56- and MFRP-mediated ocular axial growth remain elusive. Here, we show that Adamts19 expression is significantly upregulated in the retina of mice lacking either Prss56 or Mfrp. Importantly, using genetic mouse models, we demonstrate that while ADAMTS19 is not required for ocular growth during normal development, its inactivation exacerbates ocular axial length reduction in Prss56 and Mfrp mutant mice. These results suggest that the upregulation of retinal Adamts19 is part of an adaptive molecular response to counteract impaired ocular growth. Using a complementary genetic approach, we show that loss of PRSS56 or MFRP function prevents excessive ocular axial growth in a mouse model of early-onset myopia caused by a null mutation in Irbp, thus, demonstrating that PRSS56 and MFRP are also required for pathological ocular elongation. Collectively, our findings provide new insights into the molecular network involved in ocular axial growth and support a role for molecular crosstalk between the retina and RPE involved in refractive development., Author summary During ocular refractive development, the eye’s growth is modulated, such that the ocular axial length matches the optical power enabling the eyes to achieve optimal focus. Alterations in ocular growth mainly contribute to refractive errors. Mutations in human PRSS56 and MFRP are responsible for nanophthalmos that exhibit a severe reduction in ocular axial length, and high hyperopia. Importantly, mutant mouse models lacking either Müller glia expressed PRSS56, or retinal pigment epithelium (RPE) localized MFRP exhibit ocular axial length reduction. Here, we have identified Adamts19 as a factor whose levels were significantly upregulated in the retina of mice lacking either Prss56 or Mfrp. Importantly, utilizing Adamts19 knockout mice we demonstrate that upregulation of retinal Adamts19 expression constitutes a compensatory mechanism that provides partial protection against ocular axial reduction due to mutation in Prss56 and Mfrp. Next, utilizing a mouse model of early-onset myopia, we demonstrate that the mutant Irbp induced ocular axial elongation is completely dependent on Prss56 as well as Mfrp, suggesting an interplay between Müller glia and RPE in the regulation of ocular axial growth. Collectively, these findings suggest that ocular refractive development relies on complex interactions occurring between genetic factors in the retina and RPE.

Angle-closure glaucoma (ACG) is a severe form of glaucoma affecting up to 16 million people worldwide. In ACG, physical blockage of the ocular drainage tissue by the peripheral iris impedes the drainage of aqueous humor resulting in elevated intraocular pressure (IOP) and subsequent optic nerve damage. Despite the high prevalence of ACG, the precise mechanism(s) underlying pathogenesis are only partially understood. We have previously demonstrated that a mutation in the gene encoding the serine protease PRSS56 causes an ACG phenotype in mice. Notably, Prss56 mutant mice exhibit a reduced ocular axial length and a lens occupying a larger ocular volume compared to WT mice, recapitulating characteristic features of human AGC. Our findings utilizing mouse genetic models demonstrate that loss of PRSS56 function results in altered configuration of ocular angle structures characterized by a posterior shift in the positioning of the ocular drainage tissue relative to the ciliary body and iris during development, leading to a physical blockage of drainage structure (angle closure) and high IOP. Utilizing a previously employed genetic strategy of rescuing mutant Prss56 mediated reduction in ocular size by inactivation of EGR1 (Egr1;Prss56 double mutants) we determined the influence of ocular size on developmental positioning of the ocular angle tissues. Our findings suggest that abnormal positioning of the drainage structure as a result of loss of PRSS56 function is uncoupled from its effect on ocular axial length reduction. Furthermore, we demonstrate that the IOP elevation observed in Prss56 mutant mice is genetic context-dependent and identify a dominant modifier locus on Chromosome 2 of the C3H/HeJ genome conferring susceptibility to high IOP. Overall, our findings reveal a novel role for PRSS56 in the proper configuration of the iridocorneal angle and provide new insight into the developmental pathways implicated in glaucoma pathogenesis.

While all forms of glaucoma are characterized by a specific pattern of retinal ganglion cell death, they are clinically divided into several distinct subclasses, including normal tension glaucoma, primary open angle glaucoma, congenital glaucoma, and secondary glaucoma. For each type of glaucoma there are likely numerous molecular pathways that control susceptibility to the disease. Given this complexity, a single animal model will never precisely model all aspects of all the different types of human glaucoma. Therefore, multiple animal models have been utilized to study glaucoma but more are needed. Because of the powerful genetic tools available to use in the laboratory mouse, it has proven to be a highly useful mammalian system for studying the pathophysiology of human disease. The similarity between human and mouse eyes coupled with the ability to use a combination of advanced cell biological and genetic tools in mice have led to a large increase in the number of studies using mice to model specific glaucoma phenotypes. Over the last decade, numerous new mouse models and genetic tools have emerged, providing important insight into the cell biology and genetics of glaucoma. In this review, we describe available mouse genetic models that can be used to study glaucoma-relevant disease/pathobiology. Furthermore, we discuss how these models have been used to gain insights into ocular hypertension (a major risk factor for glaucoma) and glaucomatous retinal ganglion cell death. Finally, the potential for developing new mouse models and using advanced genetic tools and resources for studying glaucoma are discussed.

A mismatch between optical power and ocular axial length results in refractive errors. Uncorrected refractive errors constitute the most common cause of vision loss and second leading cause of blindness worldwide. Although the retina is known to play a critical role in regulating ocular growth and refractive development, the precise factors and mechanisms involved are poorly defined. We have previously identified a role for the secreted serine protease PRSS56 in ocular size determination and PRSS56 variants have been implicated in the etiology of both hyperopia and myopia, highlighting its importance in refractive development. Here, we use a combination of genetic mouse models to demonstrate that Prss56 mutations leading to reduced ocular size and hyperopia act via a loss of function mechanism. Using a conditional gene targeting strategy, we show that PRSS56 derived from Müller glia contributes to ocular growth, implicating a new retinal cell type in ocular size determination. Importantly, we demonstrate that persistent activity of PRSS56 is required during distinct developmental stages spanning the pre- and post-eye opening periods to ensure optimal ocular growth. Thus, our mouse data provide evidence for the existence of a molecule contributing to both the prenatal and postnatal stages of human ocular growth. Finally, we demonstrate that genetic inactivation of Prss56 rescues axial elongation in a mouse model of myopia caused by a null mutation in Egr1. Overall, our findings identify PRSS56 as a potential therapeutic target for modulating ocular growth aimed at preventing or slowing down myopia, which is reaching epidemic proportions., Author summary Refractive errors mainly occur when changes in ocular size (ocular axial length) prevent light from focusing directly on the retina. Myopia (nearsightedness) is the most common form of refractive errors in which the focused image falls in front of the retina. The recent unprecedented rise in the incidence of myopia has significant implications as individuals with high myopia are at an increased risk of developing irreversible blinding conditions, including retinal detachment, macular degeneration, and glaucoma. Ocular axial growth is a key determinant of normal refractive development. Although the retina has been established as a central player involved in the regulation of ocular growth, the specific retinal cell type(s) and molecular pathways involved are poorly defined. Here, we have utilized genetic mouse models to provide significant insight into spatial and temporal requirements of the retinal factor PRSS56 in ocular size determination. Importantly, we have uncovered a previously unrecognized role for retinal Müller glia in ocular growth and demonstrated that Prss56 inactivation has translational potential to rescue axial length elongation in a mouse model of myopia. Collectively, our findings suggest that therapeutic strategies targeting PRSS56 to modulate ocular growth could have important clinical implications to prevent or slowdown the progression of myopia and associated blinding conditions in humans.

Anterior chamber depth (ACD) is a key anatomical risk factor for primary angle closure glaucoma (PACG). We conducted a genome-wide association study (GWAS) on ACD to discover novel genes for PACG on a total of 5,308 population-based individuals of Asian descent. Genome-wide significant association was observed at a sequence variant within ABCC5 (rs1401999; per-allele effect size = −0.045 mm, P = 8.17×10−9). This locus was associated with an increase in risk of PACG in a separate case-control study of 4,276 PACG cases and 18,801 controls (per-allele OR = 1.13 [95% CI: 1.06–1.22], P = 0.00046). The association was strengthened when a sub-group of controls with open angles were included in the analysis (per-allele OR = 1.30, P = 7.45×10−9; 3,458 cases vs. 3,831 controls). Our findings suggest that the increase in PACG risk could in part be mediated by genetic sequence variants influencing anterior chamber dimensions., Author Summary The anterior chamber is the space within the eye which is bound by the cornea, and the anterior surfaces of the iris and lens. Anterior chamber depth (ACD) is the distance measured along the eye's optical axis, from the cornea to the lens surface. ACD is an important risk factor for primary angle closure glaucoma (PACG), a major cause of irreversible blindness worldwide, and in particular, individuals of Asian ethnicity. In order to identify the genes that underlie PACG susceptibility, we conducted a two-staged study. We first conducted a large scale genetic study on a total of 5,308 population-based individuals of Asian descent to identify the genetic variants that influence ACD. This was followed by testing for associations between the identified genetic variant and PACG in another independent collection of 4,276 PACG cases and 18,801 controls. We found that a genetic variant within ABCC5 was associated with an increased risk of having PACG. Our findings suggest that the increase in PACG risk could in part be mediated by genetic sequence variants that influence the anterior chamber dimensions of the eye.

Angle-closure glaucoma (ACG) is a subset of glaucoma affecting 16 million people. Although 4 million people are bilaterally blind from ACG, the causative molecular mechanisms of ACG remain to be defined. High intraocular pressure induces glaucoma in ACG. High intraocular pressure traditionally was suggested to result from the iris blocking or closing the angle of the eye, thereby limiting aqueous humor drainage. Eyes from individuals with ACG often have a modestly decreased axial length, shallow anterior chamber and relatively large lens, features that predispose to angle closure. Here we show that genetic alteration of a previously unidentified serine protease (PRSS56) alters axial length and causes a mouse phenotype resembling ACG. Mutations affecting this protease also cause a severe decrease of axial length in individuals with posterior microphthalmia. Together, these data suggest that alterations of this serine protease may contribute to a spectrum of human ocular conditions including reduced ocular size and ACG.

The precise transcriptional regulation of gene expression is essential for vertebrate development, but the role of posttranscriptional regulatory mechanisms is less clear. Cytoplasmic RNA granules (RGs) function in the posttranscriptional control of gene expression, but the extent of RG involvement in organogenesis is unknown. We describe two human cases of pediatric cataract with loss-of-function mutations in TDRD7 and demonstrate that Tdrd7 nullizygosity in mouse causes cataracts, as well as glaucoma and an arrest in spermatogenesis. TDRD7 is a Tudor domain RNA binding protein that is expressed in lens fiber cells in distinct TDRD7-RGs that interact with STAU1-ribonucleoproteins (RNPs). TDRD7 coimmunoprecipitates with specific lens messenger RNAs (mRNAs) and is required for the posttranscriptional control of mRNAs that are critical to normal lens development and to RG function. These findings demonstrate a role for RGs in vertebrate organogenesis.

Arrestins regulate the activity and subcellular localization of G protein-coupled receptors and other signaling molecules. Here we demonstrate that arrestins bind microtubules (MTs) in vitro and in vivo. The MT-binding site on arrestins significantly overlaps with the receptor-binding site, but the conformations of MT-bound and receptor-bound arrestin are different. Arrestins recruit ERK1/2 and the E3 ubiquitin ligase Mdm2 to microtubules in cells, similar to the arrestin-dependent mobilization of these proteins to the receptor. Arrestin-mediated sequestration of ERK to MTs reduces the level of ERK activation. In contrast, recruitment of Mdm2 to microtubules by arrestin channels Mdm2 activity toward cytoskeleton-associated proteins, dramatically increasing their ubiquitination. The mobilization of signaling molecules to microtubules is a novel biological function of arrestin proteins.

SummaryIn rod photoreceptors, arrestin localizes to the outer segment (OS) in the light and to the inner segment (IS) in the dark. Here, we demonstrate that redistribution of arrestin between these compartments can proceed in ATP-depleted photoreceptors. Translocation of transducin from the IS to the OS also does not require energy, but depletion of ATP or GTP inhibits its reverse movement. A sustained presence of activated rhodopsin is required for sequestering arrestin in the OS, and the rate of arrestin relocalization to the OS is determined by the amount and the phosphorylation status of photolyzed rhodopsin. Interaction of arrestin with microtubules is increased in the dark. Mutations that enhance arrestin-microtubule binding attenuate arrestin translocation to the OS. These results indicate that the distribution of arrestin in rods is controlled by its dynamic interactions with rhodopsin in the OS and microtubules in the IS and that its movement occurs by simple diffusion.

Many lines of evidence show that membranes contain microdomains, "lipid rafts", that are different from the rest of the membrane in specific lipid and protein composition. In several biological systems, they were shown to be necessary for trafficking and signal transduction. Here, we investigate if lipid rafts have a role in the regulation of the G protein-mediated pathway underlying vertebrate phototransduction. Photoreceptor membranes contain detergent-resistant membrane (DRM) rafts. Rhodopsin and cGMP phosphodiesterase are found in raft and nonraft portions of the membrane; guanylate cyclase is found exclusively in the raft. Distribution of these proteins does not change in the light or dark. In contrast, the G protein transducin, the RGS9-1-Gbeta5L complex, and the p44 isoform of arrestin undergo dramatic translocation to the raft upon illumination. Phosphorylation of RGS9-1 occurs exclusively in the raft. GTPgammaS or pertussis toxin prevent the light-mediated translocation of transducin and RGS9-1, whereas AlF(minus sign)(4) causes both proteins to move to the raft in the dark. This shows that the Galphat-RGS9-1-Gbeta5L complex has the highest affinity to rafts in the transition state of the GTPase. GTPgammaS binds to transducin at a significantly slower rate in the raft, indicating that this translocation results in a reduced rhodopsin-transducin coupling. Thus, an external signal can rearrange components of a G protein pathway in specific domains of the cell membrane, changing its signaling properties. These findings could reveal a novel mechanism utilized by the cells for regulation of G protein-mediated signal transduction.

The molecular mechanisms causing pigment dispersion syndrome (PDS) and the pathway(s) by which it progresses to pigmentary glaucoma are not known. Mutations in two melanosomal protein genes (Tyrp1 b and Gpnmb R150X ) are responsible for pigment dispersing iris disease, which progresses to intraocular pressure (IOP) elevation and subsequent glaucoma in DBA/2J mice. Melanosomal defects along with ocular immune abnormalities play a role in the propagation of pigment dispersion and progression to IOP elevation. Here, we tested the role of specific immune components in the progression of the iris disease and high IOP. We tested the role of NK cells in disease etiology by genetically modifying the B6.D2-Gpnmb R150X Tyrp1 b strain, which develops the same iris disease as DBA/2J mice. Our findings demonstrate that neither diminishing NK mediated cytotoxic activity (Prf1 mutation) nor NK cell depletion (Il2rg mutation) has any influence on the severity or timing of Gpnmb R150X Tyrp1 b mediated iris disease. Since DBA/2J mice are deficient in CD94, an important immune modulator that often acts as an immune suppressor, we generated DBA/2J mice sufficient in CD94. Sufficiency of CD94 failed to alter either the iris disease or the subsequent IOP elevation. Additionally CD94 status had no detected effect on glaucomatous optic nerve damage. Our previous data implicate immune components in the manifestation of pigment dispersion and/or IOP elevation in DBA/2J mice. The current study eliminates important immune components, specifically NK cells and CD94 deficiency, as critical in the progression of iris disease and glaucoma. This narrows the field of possible immune components responsible for disease progression.

Vertebrate phototransduction is mediated by cGMP, which is generated by retGC (retinal guanylate cyclase) and degraded by cGMP phosphodiesterase. Light stimulates cGMP hydrolysis via the G-protein transducin, which directly binds to and activates phosphodiesterase. Bright light also causes relocalization of transducin from the OS (outer segments) of the rod cells to the inner compartments. In the present study, we show experimental evidence for a previously unknown interaction between G(alphat) (the transducin alpha subunit) and retGC. G(alphat) co-immunoprecipitates with retGC from the retina or from co-transfected COS-7 cells. The retGC-G(alphat) complex is also present in cones. The interaction also occurs in mice lacking RGS9 (regulator of G-protein signalling 9), a protein previously shown to associate with both G(alphat) and retGC. The G(alphat)-retGC interaction is mediated primarily by the kinase homology domain of retGC, which binds GDP-bound G(alphat) stronger than the GTP[S] (GTPgammaS; guanosine 5'-[gamma-thio]triphosphate) form. Neither G(alphat) nor G(betagamma) affect retGC-mediated cGMP synthesis, regardless of the presence of GCAP (guanylate cyclase activating protein) and Ca2+. The rate of light-dependent transducin redistribution from the OS to the inner segments is markedly accelerated in the retGC-1-knockout mice, while the migration of transducin to the OS after the onset of darkness is delayed. Supplementation of permeabilized photoreceptors with cGMP does not affect transducin translocation. Taken together, these results suggest that the protein-protein interaction between G(alphat) and retGC represents a novel mechanism regulating light-dependent translocation of transducin in rod photoreceptors.

BackgroundTheGpnmbgene encodes a transmembrane protein whose function(s) remain largely unknown. Here, we assess if a mutant allele ofGpnmbconfers susceptibility to glaucoma by altering immune functions. DBA/2J mice have a mutantGpnmbgene and they develop a form of glaucoma preceded by a pigment dispersing iris disease and abnormalities of the immunosuppressive ocular microenvironment.ResultsWe find that theGpnmbgenotype of bone-marrow derived cell lineages significantly influences the iris disease and the elevation of intraocular pressure. GPNMB localizes to multiple cell types, including pigment producing cells, bone marrow derived F4/80 positive antigen-presenting cells (APCs) of the iris and dendritic cells. We show that APCs of DBA/2J mice fail to induce antigen induced immune deviation (a form of tolerance) when treated with TGFβ2. This demonstrates that some of the immune abnormalities previously identified in DBA/2J mice result from intrinsic defects in APCs. However, the tested APC defects are not dependent on a mutantGpnmbgene. Finally, we show that theGpnmbmediated iris disease does not require elevated IL18 or mature B or T lymphocytes.ConclusionThese results establish a role forGpnmbin bone marrow derived lineages. They suggest that affects ofGpnmbon innate immunity influence susceptibility to glaucoma in DBA/2J mice.

Activation of rod photoreceptors by light induces a massive redistribution of the heterotrimeric G-protein transducin. In darkness, transducin is sequestered within the membrane-enriched outer segments of the rod cell. In light, it disperses throughout the entire neuron. We show here that redistribution of rod transducin by light requires activation, but it does not require ATP. This observation rules out participation of molecular motors in the redistribution process. In contrast to the light-stimulated redistribution of rod transducin in rods, cone transducin in cones does not redistribute during activation. Remarkably, when cone transducin is expressed in rods, it does undergo light-stimulated redistribution. We show here that the difference in subcellular localization of activated rod and cone G-proteins correlates with their affinity for membranes. Activated rod transducin releases from membranes, whereas activated cone transducin remains bound to membranes. A synthetic peptide that dissociates G-protein complexes independently of activation facilitates dispersion of both rod and cone transducins within the cells. This peptide also facilitates detachment of both G-proteins from the membranes. Together, these results show that it is the dissociation state of transducin that determines its localization in photoreceptors. When rod transducin is stimulated, its subunits dissociate, leave outer segment membranes, and equilibrate throughout the cell. Cone transducin subunits do not dissociate during activation and remain sequestered within the outer segment. These findings indicate that the subunits of some heterotrimeric G-proteins remain associated during activation in their native environments.

PURPOSE Heterotrimeric G proteins are regulated by receptors that act as guanine nucleotide exchange factors (GEFs) and by RGS proteins, which act as guanosine triphosphatase (GTPase) activating proteins (GAPs). Guanosine diphosphate (GDP) dissociation inhibitors (GDIs), such as activators of G protein signaling (AGS)-1 and -3 and Leu-Gly-Asn repeat-enriched (LGN) proteins regulate the Gi family of G proteins. AGS3 and LGN contain four characteristic G protein regulator (GPR) domains that are responsible for its GDI function. This study investigates the presence of a GDI for transducin in photoreceptor cells. METHODS Western blot analysis of bovine and mouse retina was performed using specific antibodies to AGS and LGN proteins. The subcellular localization of LGN in retina was studied by immunofluorescence microscopy of mouse retinal sections and fractionation of retinal lysates, using sucrose density gradients. The interaction of LGN with transducin was studied using pull-down assays with GST-fused LGN constructs, co-immunoprecipitation and assays for GTPgammaS binding. RESULTS LGN, but not AGS3 and AGS1, was present in the retina, where it was localized mostly in the inner segments and outer plexiform layer of photoreceptor cells in both light and dark conditions. LGN was present in the cytosol, membrane, and the detergent-resistant cytoskeletal fraction. The amount of LGN relative to transducin was at least 1:15. The alpha subunit of transducin in its GDP-bound state interacted with endogenous and recombinant LGN, and the recombinant GPR domain of LGN reduced the rate of GTP exchange. CONCLUSIONS Photoreceptor inner segments contain LGN, which can bind to the alpha subunit of transducin and potentially regulate its function.

Proper function of visual arrestin is indispensable for rapid signal shut-off in rod photoreceptors. Dramatic light-dependent changes in its subcellular localization are believed to play an important role in light adaptation of photoreceptor cells. Here we show that visual arrestin binds microtubules. The truncated splice variant of visual arrestin, p44, demonstrates dramatically higher affinity for microtubules than the full-length protein (p48). Enhanced microtubule binding of p44 underlies its earlier reported preferential localization to detergent-resistant membranes, where it is anchored via membrane-associated microtubules in a rhodopsin-independent fashion. Experiments with purified proteins demonstrate that arrestin interaction with microtubules is direct and does not require any additional protein partners. Most importantly, arrestin interactions with microtubules and light-activated phosphorylated rhodopsin are mutually exclusive, suggesting that microtubule interaction may play a role in keeping p44 arrestin away from rhodopsin in dark-adapted photoreceptors.