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Background: Myocilin is a gene linked directly to juvenile- and adult-onset open angle glaucoma. Mutations including Gln368stop (Q368X) and Pro370Leu (P370L) have been identified in patients. The exact role of myocilin and its functional association with glaucoma are still unclear. In the present study, we established tetracycline-inducible (Tet-on) wild type and mutant myocilin-green fluorescence protein (GFP) expressing RGC5 stable cell lines and studied the changes in cell migration and barrier function upon induction. Methodology/Principal Findings: After several rounds of selection, clones that displayed low, moderate, or high expression of wild type, Q368X or P370L myocilin-GFP upon doxycycline (Dox) induction were obtained. The levels of wild type and mutant myocilin-GFP in various clones were confirmed by Western blotting. Compared to non-induced controls, the cell migration was retarded, the actin stress fibers were fewer and shorter, and the trypsinization time needed for cells to round up was reduced when wild type or mutant myocilin was expressed. The barrier function was in addition aberrant following induced expression of wild type, Q368X or P370L myocilin. Immunoblotting further showed that tight junction protein occludin was downregulated in induced cells. Conclusions/Significance: Tet-on inducible, stable RGC5 cell lines were established. These cell lines, expressing wild type or mutant (Q368X or P370L) myocilin-GFP upon Dox induction, are valuable in facilitating studies such as proteomics, as well as functional and pathogenesis investigations of disease-associated myocilin mutants. The barrier function was found impaired and the migration of cells was hindered with induced expression of wild type and mutant myocilin in RGC5 cell lines. The reduction in barrier function might be related to the declined level of occludin. The retarded cell migration was consistent with demonstrated myocilin phenotypes including the loss of actin stress fibers, lowered RhoA activities and compromised cell-matrix adhesiveness. [ABSTRACT FROM AUTHOR]

Background: Glaucoma is the leading cause of irreversible blindness in the world. Recent evidence indicates a role for genetic susceptibility to primary open-angle glaucoma (POAG). The relation between myocilin polymorphisms and POAG susceptibility has been studied in different populations. Methods: A meta-analysis of 32 published genetic association case-control studies, which examined the relation between POAG and the R46X, R76K, Y347Y, T353I, and Q368X polymorphisms of the myocilin gene, was carried out. Results: In meta-analysis, significant associations were observed between POAG risk and two myocilin polymorphisms with summarized odds ratio of 4.68 (95%CI, 2.02-10.85) for Q368X and 2.17 (95% CI, 1.32-3.57) for T353I. Both Q368X and T353I were significantly associated with high-tension glaucoma, with summarized odds ratio of 4.26 (1.69, 10.73) and 2.26 (1.37- 3.72). In Westerners, significant association was observed for Q368X mutation (odds ratio, 5.17; 95% CI, 2.16-12.40). However, in Asians it was for T353I (odds ratio, 2.17; 95% CI, 1.32-3.57). Conclusions: There is strong evidence that myocilin polymorphisms are associated with POAG susceptibility, and the prevalence of myocilin mutations might be ethnicity-dependent in Caucasians for Q368X and in Asians for T353I. [ABSTRACT FROM AUTHOR]

Background: Myocilin is a gene linked to the most prevalent form of glaucoma, a major blinding disease. The trabecular meshwork (TM), a specialized eye tissue, is believed to be involved, at least in part, in the development of glaucoma. The Pro370 to Leu (P370L) mutation of myocilin is associated with severe glaucoma phenotypes and Gln368 stop (Q368X) is the most common myocilin mutation reported. Myocilin, upon overexpression, has been shown to induce phenotypes that include a loss of actin stress fibers, an increase in the cAMP level and protein kinase A (PKA) activity, as well as a reduction in the RhoA activity. We examined herein whether Wnt signaling pathway is involved in the myocilin phenotypes and whether P370L and Q368X mutants also display biological effects similar to those of the wild type myocilin. Methodology/Principal Findings: Wild type myocilin, when transfected into cultured human TM cells, induced a loss of actin stress fibers as judged by phalloidin staining. Such a loss was averted by treatment of secreted Frizzled-related protein 1 (sFRP1), an inhibitor of Wnt signaling. Consistent with the notion that Wnt pathway mediates the myocilin phenotype, Wnt activation was demonstrated by TOP/FOP-Flash reporter assays. Treatment of human TM cells of a Wnt activator, SB216763, as well as transfection of myocilin P370L and Q368X mutants all resulted in actin stress fiber loss, PKA activation and RhoA inactivation. The PKA elevation was obviated by the sFRP1 treatment, indicating that Wnt signaling was upstream that of PKA. Conclusions/Significance: The present study demonstrated that following forced expression of wild type myocilin, Wnt was activated, triggering in turn other myocilin-related alterations. P370L and Q368X mutations induced similar phenotypes, suggesting one possible mechanism how the mutants may lead to TM cell damage and pathology. [ABSTRACT FROM AUTHOR]

Abstract: Myocilin is a protein found in the extracellular matrix of trabecular meshwork tissue, the anatomical region of the eye involved in regulating intraocular pressure. Wild-type (WT) myocilin has been associated with steroid-induced glaucoma, and variants of myocilin have been linked to early-onset inherited glaucoma. Elevated levels and aggregation of myocilin hasten increased intraocular pressure and glaucoma-characteristic vision loss due to irreversible damage to the optic nerve. In spite of reports on the intracellular accumulation of mutant and WT myocilin in vitro, cell culture, and model organisms, these aggregates have not been structurally characterized. In this work, we provide biophysical evidence for the hallmarks of amyloid fibrils in aggregated forms of WT and mutant myocilin localized to the C-terminal olfactomedin (OLF) domain. These fibrils are grown under a variety of conditions in a nucleation-dependent and self-propagating manner. Protofibrillar oligomers and mature amyloid fibrils are observed in vitro. Full-length mutant myocilin expressed in mammalian cells forms intracellular amyloid-containing aggregates as well. Taken together, this work provides new insights into and raises new questions about the molecular properties of the highly conserved OLF domain, and suggests a novel protein-based hypothesis for glaucoma pathogenesis for further testing in a clinical setting. [Copyright &y& Elsevier]

Abstract: Elevated intraocular pressure (IOP) is a causative risk factor for the development and progression of glaucoma. Glaucomatous mutations in myocilin (MYOC) damage the trabecular meshwork and elevate IOP in humans and in mice. Animal models of glaucoma are important to discover and better understand molecular pathogenic pathways and to test new glaucoma therapeutics. Although a number of different animal models of glaucoma have been developed and characterized, there are no true models of human primary open angle glaucoma (POAG). The overall goal of this work is to develop the first inducible mouse model of POAG using a human POAG relevant transgene (i.e. mutant MYOC) expression in mouse eyes to elevate IOP and cause pressure-induced damage to the optic nerve. Four mouse strains (A/J, BALB/cJ, C57BL/6J, and C3H/HeJ) were used in this study. Ad5.MYOC.Y437H (5 × 107 pfu) was injected intravitreally into one eye, with the uninjected contralateral eye serving as the control eye. Conscious IOP measurements were taken using a TonoLab rebound tonometer. Optic nerve damage was determined by scoring PPD stained optic nerve cross sections. Retinal ganglion cell and superior colliculus damage was assessed by Nissl stain cell counts. Intravitreal administration of viral vector Ad5.MYOC.Y437H caused a prolonged, reproducible, and statistically significant IOP elevation in BALB/cJ, A/J, and C57BL/6J mice. IOPs increased to approximately 25 mm Hg for 8 weeks (p < 0.0001). In contrast, the C3H/HeJ mouse strain was resistant to Ad5.MYOC.Y437H induced IOP elevation for the 8-week time period. IOPs were stable (12–15 mm Hg) in the uninjected control eyes. We also determined whether there were any strain differences in pressure-induced optic nerve damage. Even though IOP was similarly elevated in three of the strains tested (BALB/cJ, C57BL/6J, and A/J) only the A/J strain had considerable and significant optic nerve damage at the end of 8 weeks with optic nerve damage score of 2.64 ± 0.19 (n = 18, p < 0.001) in the injected eye. There was no statistical difference in retinal ganglion cell death or superior colliculus damage at the 8-week time point in any of the strains tested. These results demonstrate strain dependent responses to Ad5.MYOC.Y437H-induced ocular hypertension and pressure-induced optic nerve damage. [Copyright &y& Elsevier]

Myocilin is a widely expressed protein with no known function; however, mutations in myocilin appear to manifest uniquely as ocular hypertension and the blinding disease of glaucoma. Using the protein homology/analogy recognition engine (Phyre), we find that the olfactomedin domain of myocilin is similar in sequence motif and structure to a six-blade, kelch repeat motif based on the known crystal structures of such proteins. Additionally, using sequence analysis, we identify a coiled-coil segment of myocilin with homology to human Q-SNARE proteins (inset). Using COS-7 cells expressing full-length human myocilin and a version lacking the C-terminal olfactomedin domain, we identified a membrane-associated protein complex containing myocilin by hydrodynamic analysis. The myocilin construct that included the coiled-coil but lacked the olfactomedin domain formed complexes similar to the full-length protein, indicating that the coiled-coil domain of myocilin is sufficient for myocilin binding to the large detergent-resistant complex. In human retina and retinal pigment epithelium, which express myocilin, we detected the protein in a large, sodium dodecyl sulfate-resistant, membrane-associated complex. We characterized myocilin in human tissues as either a 15 S complex with an Mr of 405000-440000 yielding a slightly elongated globular shape similar to that of known SNARE complexes or a 6.4 S dimer with an Mr of 108000. By identifying the Q-SNARE homology within the second coil of myocilin and documenting its participation in a SNARE-like complex, we provide evidence of a SNARE domain-containing protein associated with a human disease. [ABSTRACT FROM AUTHOR]

Myocilin (MYOC) is a 504 aa secreted glycoprotein induced by stress factors in the trabecular meshwork tissue of the eye, where it was discovered. Mutations in MYOC are linked to glaucoma. The glaucoma phenotype of each of the different MYOC mutation varies, but all of them cause elevated intraocular pressure (IOP). In cells, forty percent of wild-type MYOC is cleaved by calpain II, a cysteine protease. This proteolytic process is inhibited by MYOC mutants. In this study, we investigated the molecular mechanisms by which MYOC mutants cause glaucoma. We constructed adenoviral vectors with variants Q368X, R342K, D380N, K423E, and overexpressed them in human trabecular meshwork cells. We analyzed expression profiles with Affymetrix U133Plus2 GeneChips using wild-type and null viruses as controls. Analysis of trabecular meshwork relevant mechanisms showed that the unfolded protein response (UPR) was the most affected. Search for individual candidate genes revealed that genes that have been historically connected to trabecular meshwork physiology and pathology were altered by the MYOC mutants. Some of those had known MYOC associations (MMP1, PDIA4, CALR, SFPR1) while others did not (EDN1, MGP, IGF1, TAC1). Some, were top-changed in only one mutant (LOXL1, CYP1B1, FBN1), others followed a mutant group pattern. Some of the genes were new (RAB39B, STC1, CXCL12, CSTA). In particular, one selected gene, the cysteine protease inhibitor cystatin A (CSTA), was commonly induced by all mutants and not by the wildtype. Subsequent functional analysis of the selected gene showed that CSTA was able to reduce wild-type MYOC cleavage in primary trabecular meshwork cells while an inactive mutated CSTA was not. These findings provide a new molecular understanding of the mechanisms of MYOC-causative glaucoma and reveal CSTA, a serum biomarker for cancer, as a potential biomarker and drug for the treatment of MYOC-induced glaucoma. [ABSTRACT FROM AUTHOR]

Genetic studies have linked myocilin to open angle glaucoma, but the functions of the protein in the eye and other tissues have remained elusive. The purpose of this investigation was to elucidate myocilin function(s). We identified α1-syntrophin, a component of the dystrophin-associated protein complex (DAPC), as a myocilin-binding candidate. Myocilin interacted with α1-syntrophin via its N-terminal domain and co-immunoprecipitated with α1-syntrophin from C2C12 myotubes and mouse skeletal muscle. Expression of 15-fold higher levels of myocilin in the muscles of transgenic mice led to the elevated association of α1-syntrophin, neuronal nitric-oxide synthase, and α-dystroglycan with DAPC, which increased the binding of laminin to α-dystroglycan and Akt signaling. Phosphorylation of Akt and Forkhead box O-class 3, key regulators of muscle size, was increased more than 3-fold, whereas the expression of muscle-specific RING finger protein-1 and atrogin-1, muscle atrophy markers, was decreased by 79 and 88%, respectively, in the muscles of transgenic mice. Consequently, the average size of muscle fibers of the transgenic mice was increased by 36% relative to controls. We suggest that intracellular myocilin plays a role as a regulator of muscle hypertrophy pathways, acting through the components of DAPC. [ABSTRACT FROM AUTHOR]

Glaucoma is a group of diseases with progressive optic disk and /or visual fields changes. The mainstays of current therapies (medical, laser, surgery) are focused of lowering intraocular pressure (IOP). The problems and failures with such therapies and the studies on physiopathology and genetics of glaucoma started explorations for new therapeutic modalities. Advances in our understanding of the genetic basis of glaucoma include the discovery of 14 linked sites (GLC 1A through GLC 1N) in primary open-angle glaucoma with three genes identified at these loci: myocilin, optineurin, and WDR 36. In the secondary glaucomas such as pseudoexfoliation syndromes, LOXL1 gene polymorphisms have been identified. Gene therapy can be used to replace a mutated gene that causes disease with a healthy copy of the gene, to inactivate or wipe out the aberrant gene that is responsible for the disease. This therapy can also be used to add or remove genes that are indirectly related to the pathophysiology of the disease. Ocular gene therapy can also provide the advantage of delivery of genes that express therapeutic proteins in a localized and sustained way. Gene therapy studies for the treatment of glaucoma are focused on lowering intraocular pressure and neuroprotection. An ideal gene delivery system is one that is able to efficiently deliver the genes to the target tissue with specificity and safety. Both viral and nonviral vector delivery systems have been tested in the animal eyes. The target tissues of gene therapy are trabecular meshwork, ciliary body, ciliary muscle, retina and optic disk. [ABSTRACT FROM AUTHOR]

Abstract: Myocilin and optineurin are two genes linked to glaucoma, a major blinding disease characterized by progressive loss of retinal ganglion cells and their axons. This review describes the characteristics of myocilin and optineurin protein products and summarizes the consequences of ectopically expressed wild-type and mutant myocilin and optineurin in trabecular meshwork and/or neuronal cells. Myocilin and optineurin exhibit differential characteristics and have divergent functional consequences. They contribute to the development of glaucoma probably via distinct mechanisms. [Copyright &y& Elsevier]

MYOC encodes a secretary glycoprotein of 504 amino acids named myocilin. MYOC is the first gene to be linked to juvenile open-angle glaucoma (JOAG) and some forms of adult-onset primary open-angle glaucoma (POAG). The gene was identified as an up-regulated molecule in cultured trabecular meshwork (TM) cells after treatment with dexamethasone and was originally referred to as trabecular meshwork-inducible glucocorticoid response (TIGR). Elevated intraocular pressure (IOP), due to decreased aqueous outflow, is the strongest known risk factor for POAG. Increasing evidence showed that the modulation of the wild-type (wt) myocilin protein expression is not causative of glaucoma while some misfolded and self-assembly aggregates of mutated myocilin may be associated with POAG in related or unrelated populations. The etiology of the disease remains unclear. Consequently, a better understanding of the molecular mechanisms underlyingPOAG is required to obtain early diagnosis, avoid potential disease progression, and develop new therapeutic strategies. In the present study, we review and discuss the most relevant studies regarding structural characterizations, expressions, molecular interactions, putative functions of MYOC gene and/or its corresponding protein in POAG etiology. [ABSTRACT FROM AUTHOR]

Abstract: To better understand the role of exosomes in the trabecular meshwork (TM), the site of intraocular pressure control, the exosome proteome from primary cultures of human TM cell monolayers was analyzed. Exosomes were purified from urine and conditioned media from primary cultures of human TM cell monolayers and subjected to a two dimensional HPLC separation and MS/MS analyses using the MudPIT strategy. Spectra were searched against a human protein database using Sequest. Protein profiles were compared to each other and the Exocarta database and the presence of specific protein markers confirmed by Western blot analyses of exosomes from aqueous humor and human TM cell strains (n=5) that were untreated, or exposed to dexamethasone and/or ionomycin. TM cell exosomes contained 108 of the 143 most represented exosome proteins in ExoCarta, including previously characterized markers such as membrane organizing and tetraspanin proteins. Several cell-specific proteins in TM exosomes were identified including myocilin, emilin-1 and neuropilin-1. All TM exosome proteins had flotation densities on sucrose gradients and release responses to ionomycin typical for exosomes. Taken together, TM exosomes have a characteristic exosome protein profile plus contain unique proteins, including the glaucoma-causing protein, myocilin; suggesting a role for exosomes in the control of intraocular pressure. [Copyright &y& Elsevier]

Abstract: Myocilin, a protein associated with the development of glaucoma, is expressed in most eye tissues with highest expression observed in trabecular meshwork cells. In culture, primary human trabecular meshwork cells incubated in 10% fetal bovine serum have reduced myocilin expression compared to in vivo, but incubation in human aqueous humor, their normal in vivo nutrient source, restores myocilin expression to near in vivo levels. To investigate the mechanism by which human aqueous humor stimulates myocilin accumulation in conditioned media from normal human trabecular meshwork cells, three independent trabecular meshwork cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing various supplements: fetal bovine serum (10%), human serum (0.2%), porcine aqueous humor (50%), bovine serum albumin (0.1%), dexamethasone (10−7 M), human aqueous humor (50%) or heat-inactivated human aqueous humor (50%). Conditioned media from cultured primary trabecular meshwork cells following incubation in human aqueous humor showed significant accumulation of myocilin in a time- (15min) and dose-dependent manner (half maximal effective concentration ∼ 30%) while intracellular myocilin levels decreased. Minimal myocilin accumulation was observed in conditioned media isolated from trabecular meshwork cells cultured in DMEM containing fetal bovine or human serum, bovine serum albumin, porcine aqueous humor, dexamethasone or DMEM alone. Heat inactivation of human aqueous humor nearly eliminated human aqueous humor-stimulated myocilin secretion. Inhibitors of new protein synthesis, gene transcription, the endoplasmic reticulum/Golgi system and endocytic/exocytic secretory pathways failed to inhibit human aqueous humor-stimulated myocilin secretion. Using immunolabeling and transmission electron microscopy, myocilin was found associated with 70–90nm vesicle-like structures within the cytoplasm of human aqueous humor treated trabecular meshwork cells. These studies suggest that myocilin secretion from trabecular meshwork cells occurs in a Golgi-independent manner following human aqueous humor treatment. Heat-labile factors in human aqueous humor are responsible for the time- and dose-dependent release of myocilin from vesicle-like structures within the cytoplasm of trabecular meshwork cells. [Copyright &y& Elsevier]

Coding variants in both myocilin (MYOC) and optineurin (OPTN) are reported risk factors for primary open-angle glaucoma (POAG) in many populations. This study investigated the contribution of MYOC and OPTN coding variants in Hispanics of Mexican descent with and without POAG. We conducted a case-control study of unrelated POAG cases and nonglaucomatous controls in a population of Hispanics of Mexican descent. Ascertainment criteria for POAG included the presence of glaucomatous optic neuropathy with associated visual field loss and the absence of secondary causes of glaucoma. Controls had normal optic nerves, visual fields and intraocular pressure. All coding exons of MYOC and OPTN were sequenced. The data set consisted of 88 POAG cases and 93 controls. A novel nonsynonymous coding variant (R7H) in the first exon of MYOC was identified. Other identified variants in MYOC and OPTN have been previously described and do not seem to contribute to POAG risk. This is the first comprehensive study of MYOC and OPTN in Hispanics of Mexican descent with POAG. Neither MYOC nor OPTN sequence variants seem to have a major role in the etiology of POAG in this population. [ABSTRACT FROM AUTHOR]

Purpose: To assess the frequency of mutations in the Myocilin (MYOC) gene in Iranian patients affected with primary congenital glaucoma (PCG). Methods: The individuals evaluated herein are among a larger cohort of 100 patients who had previously been screened for CYP1B1 mutations. Eighty subjects carried mutations in CYP1B1, but the remaining 20 patients who did not, underwent screening for MYOC mutations for the purpose of the study. MYOC exons in the DNA were polymerase chain reaction (PCR) amplified and sequenced. Sequencing was performed using PCR primers, the ABI big dye chemistry and an ABI3730XL instrument. Sequences were analyzed by comparing them to reference MYOC sequences using the Sequencher software. Results: Four MYOC sequence variations were observed among the patients, but none of them were considered to be associated with disease status. Three of these variations were single nucleotide polymorphisms already reported not to be disease causing, the fourth variation created a synonymous codon and did not affect any amino acid change. Conclusion: In this cohort, MYOC mutations were not observed in any Iranian subject with PCG. It is possible that in a larger sample, a few subjects carrying disease causing MYOC mutations could have been observed. But our results show that the contribution of MYOC to PCG status in Iran is small if any. [ABSTRACT FROM AUTHOR]

Abstract: Glaucoma is a progressive, age-related optic neuropathy and a leading cause of irreversible blindness in the world. Animal models of glaucoma are essential to our continued efforts of elucidating the natural course of the disease and to developing therapeutic interventions to halt or reverse the progression of the condition. Over the past 10–15 years, rodents have become a popular model organism to study glaucoma, because of their high degree of availability, relatively low cost, short life-span, and amenability to experimental and genetic manipulation. In this review, we examine the numerous in vivo and in vitro rodent models of glaucoma, discuss the methods used to generate them, summarize some of the major findings obtained in these models, and identify individual strengths and weaknesses for the various systems. [Copyright &y& Elsevier]

PurposeTo identify the prevalence of myocilin gene mutations in a UK glaucoma cohort.MethodsPrimary open-angle (POAG) and normal tension glaucoma patients were recruited from the Southampton University Hospital Trust Eye Clinic and satellite regional glaucoma clinics. Phenotype data relating to disease history and other potential risk factors were recorded and blood samples collected for each consenting participant. Point mutation analysis of the myocilin gene was carried out using six overlapping PCR fragments covering the entire coding sequence of the gene. A total of 316 POAG samples were examined of which 7 (2.2 %) tested positive for disease-causing mutations in this gene. One of these seven non-synonymous mutations represented a previously unreported amino-acid substitution of cysteine for arginine at codon 296 (p.R296C) of the myocilin protein.ConclusionsThis study identifies a 2.2% prevalence of myocilin mutations in a cohort of ethnically homogenous glaucoma patients selected from a UK ophthalmic clinic. A novel myocilin mutation is also described. This study identifies that myocilin genetic screening is feasible in NHS glaucoma clinics for genetic counselling and cascade testing of relatives of patients affected by myocilin glaucoma. [ABSTRACT FROM AUTHOR]

Purpose: To describe the clinical phenotype in a family with primary open angle glaucoma harboring a p.Gln368X mutation in MYOC. Materials and Methods: We identified a proband with primary open angle glaucoma and the p.Gln368X MYOC mutation. She and her six siblings were examined clinically, including Heidelberg Retina Tomography II, and venous blood samples were screened for other variants in MYOC, WDR36, OPTN, and CYP1B1. Results: Four individuals showed the p.Gln368X MYOC mutation, no other genetic variations were assessed. Two of these four siblings had glaucomatous optic disc changes with corresponding visual field losses and abnormal Heidelberg Retina Tomography results by the Moorfields regression analysis, one had abnormal results by the Moorfields regression analysis but no visual field loss, and one showed no glaucomatous signs or symptoms at all. These findings did not correlate with the age of the affected individuals. Conclusion: In the primary open angle glaucoma family described here, we documented a wide range in clinical symptoms, demonstrating a highly variable penetrance of the MYOC p.Gln368X mutation. [ABSTRACT FROM AUTHOR]

Mutations in myocilin, a 55–57 kDa secreted glycoprotein, are causative for some forms of primary open-angle glaucoma (POAG). In vitro studies indicate that myocilin can modulate the hydrodynamic outflow resistance in the trabecular meshwork (TM) and that elevated amounts of myocilin can obstruct the TM outflow system in POAG. In this study, we analyzed the localization of myocilin in the trabecular meshwork (TM) of eyes with primary open-angle glaucoma (POAG), and compared it with that of normal eyes. Immunohistochemistry for myocilin was performed in the eyes of human donors (nine normal and 14 with POAG, including one with steroid-induced glaucoma). Staining for myocilin was observed in the extracellular spaces of the juxtacanalicular tissue (JCT) in all normal eyes. Some normal eyes did also show cytoplasmic staining for myocilin in TM cells. In the eyes of six donors with POAG, staining of the JCT was more widespread and intense than in normal eyes. In the other eyes with POAG, immunoreactivity for myocilin in the JCT was not markedly different to that of normal eyes. Staining intensity in the JCT of POAG eyes did not obviously correlate with intraocular pressure or clinical severity. In the eyes of one patient with steroid-induced POAG, cells of the TM, Schlemm’s canal endothelium, and the anterior stroma of the iris showed an immunoreactivity for myocilin which was considerably more intense than in normal eyes, or in the eyes with other forms of POAG. In some cases of POAG, the structural changes in the JCT include an increase in myocilin in the extracellular pathways of aqueous humor. Treatment with steroids appears to increase myocilin synthesis in TM and iris of human eyes in situ. [ABSTRACT FROM AUTHOR]

Juvenile open-angle glaucoma (JOAG) is a rare sub-type of primary open-angle glaucoma that occurs in individuals ranging from 3-40 years of age. It is characterized by high intraocular pressure and typically a strong family history. Treating a patient with JOAG may present unique challenges depending on the age of presentation, due to differences in side effect and efficacy profiles for children. Medical therapy alone is often insufficient in controlling IOP. The diagnosis, treatment, and management of one sixteen year old male presenting for his first eye exam will be presented. The underlying pathophysiology of JOAG is not fully known, however several genes and other molecular components have been linked to its development. This molecular and genetic research is paramount in predictive diagnosis and treatment for high risk, asymptomatic individuals. [ABSTRACT FROM AUTHOR]

A family of olfactomedin domain-containing proteins consists of at least 13 members in mammals. Although the first protein belonging to this family, olfactomedin, was isolated and partially characterized from frog olfactory neuroepithelim almost 20 years ago, the functions of many family members remain elusive. Most of the olfactomedin domain-containing proteins, similar to frog olfactomedin, are secreted glycoproteins that demonstrate specific expression patterns. Other family members are membrane-bound proteins that may serve as receptors. More than half of the olfactomedin domain-containing genes are expressed in neural tissues. Data obtained over the last several years demonstrate that olfactomedin domain-containing proteins play important roles in neurogenesis, neural crest formation, dorsal ventral patterning, cell–cell adhesion, cell cycle regulation, and tumorigenesis and may serve as modulators of critical signaling pathways (Wnt, bone morphogenic protein). Mutations in two genes encoding myocilin and olfactomedin 2 were implicated in glaucoma, and a growing number of evidence indicate that other genes belonging to the family of olfactomedin domain-containing proteins may contribute to different human disorders including psychiatric disorders. In this review, we summarize recent advances in understanding the possible roles of these proteins with special emphasis on the proteins that are preferentially expressed and function in neural tissues. [ABSTRACT FROM AUTHOR]

Abstract: Glaucoma is the major cause of irreversible blindness worldwide. Primary open-angle glaucoma (POAG), as the most prevalent form of glaucoma, is a complex inherited disorder and affects more than 2 million individuals in the United States. It has become increasingly clear that a host of genetic as well as environmental factors are likely to contribute to the phenotype. A number of chromosomal and genetic associations have been reported for POAG. This review examines what is currently known about the underlying genetic structure, what remains to be learned, and how this may affect our medical management of this major blinding disease. [Copyright &y& Elsevier]

Abstract: Over a decade has passed since myocilin was identified as the first gene linked to early and late-onset primary open-angle glaucoma. During this time, considerable effort has been put forth to understand the functional role myocilin has in normal and glaucomatous eyes. Myocilin is expressed in many ocular and non-ocular tissues, is found in both intracellular and extracellular spaces, and has been linked to elevations in intraocular pressure. Mutations in the myocilin gene that have been associated with glaucoma appear to confer a gain-of-functional activity rather than loss of function. Unfortunately, what the normal function of myocilin is and how alterations in the function can confer a glaucoma phenotype have yet to be elucidated. We will review the current understanding of myocilin with special emphasis on the structural makeup of the myocilin gene and protein, its possible physiological roles internal and external to ocular cells, the regulation of intraocular pressure as evidenced through the use of perfusion culture systems and animal models, and as a causative agent in some forms of glaucoma. [Copyright &y& Elsevier]

Elevated intraocular pressure (IOP) is a major risk factor in developing primary open angle glaucoma (POAG), which is the most common form of glaucoma. Transforming growth factor-beta 2 (TGFβ2) is a pro-fibrotic cytokine that plays an important role in POAG pathogenesis. TGFβ2 induced extracellular matrix (ECM) production, deposition and endoplasmic reticulum (ER) stress in the trabecular meshwork (TM) contribute to increased aqueous humor (AH) outflow resistance and IOP elevation. Drugs which alter the glaucomatous fibrotic changes and ER stress in the TM may be effective in reducing ocular hypertension. Astragaloside IV (AS.IV), a novel saponin isolated from the roots of Astragalus membranaceus, has demonstrated antifibrotic and ER stress lowering effects in various tissues during disease conditions. However, the effect of AS.IV on glaucomatous TM fibrosis, ER stress and ocular hypertension has not been studied. Primary human TM cells treated with AS.IV decreased TGFβ2 induced ECM (FN, Col-I) deposition and ER stress (KDEL, ATF4 and CHOP). Moreover, AS.IV treatment reduced TGFβ2 induced NF-κB activation and αSMA expression in TM cells. We found that AS.IV treatment significantly increased levels of matrix metalloproteases (MMP9 and MMP2) and MMP2 enzymatic activity, indicating that the antifibrotic effects of AS.IV are mediated via inhibition of NF-κB and activation of MMPs. AS.IV treatment also reduced ER stress in TM3 cells stably expressing mutant myocilin. Interestingly, the topical ocular AS.IV eye drops (1 mM) significantly decreased TGFβ2 induced ocular hypertension in mice, and this was associated with a decrease in FN, Col-1 (ECM), KDEL (ER stress) and αSMA in mouse TM tissues. Taken together, the results suggest that AS.IV prevents TGFβ2 induced ocular hypertension by modulating ECM deposition and ER stress in the TM. [ABSTRACT FROM AUTHOR]

Abstract: Although the glaucoma-associated protein myocilin has been the focus of intensive research, its biological function is still unknown. One of the limiting factors has been the lack of well-characterized antibodies, particularly monoclonal antibodies. We describe the development of six monoclonal antibodies specific to myocilin and characterize their suitability in Western blot and immunohistochemical applications. Three of the six monoclonal antibodies recognize the N-terminus of myocilin (amino acids 33–214), two antibodies recognize the middle third of the protein (amino acids 215–368), and one antibody recognizes the C-terminus (amino acids 369–504). Isotyping revealed that all antibodies are of the IgG1κ class except one, which is IgG2bκ. Purified myocilin monoclonal antibodies were able to recognize myocilin in human aqueous humor separated on denatured/reduced and native gels, and human trabecular meshwork lysate by Western blot. Myocilin was also detected by immunohistochemistry in trabecular meshwork, ciliary body, iris, cornea, sclera, choroid, and retinal pigment epithelial cells. [Copyright &y& Elsevier]

Objective To compare aqueous humor myocilin protein levels in dogs with the primary glaucomas to those with the secondary glaucomas, primary cataracts, and diabetic cataracts. Materials and methods Four groups were selected, based on diagnosis by the attending veterinary ophthalmologists and included: primary glaucoma (primary open-angle glaucoma (POAG) and primary closed angle glaucoma (PCAG); n = 155); secondary glaucoma ( n = 94); primary (presumed inherited) cataract ( n = 142), and diabetic cataract ( n = 83). A total of 474 samples (187 males, 263 females, 24 unreported) with average ages of 117 months for the males and 101 months for the females were analyzed. Myocilin protein was measured using the Coomassie staining and Western blot methods relative to a myocilin control. Results Differences were seen between nonglaucomatous (cataractous) and glaucomatous dogs with myocilin levels in glaucomatous eyes being many times higher than those in the cataractous dogs. Primary glaucomatous dogs were found to have an aqueous humor myocilin protein level of 17.30 ± 1.03 units. Secondary glaucomas had the highest level of myocilin in the aqueous humor with 19.27 ± 1.41 units. Diabetic cataractous dogs had the lowest levels of myocilin reported with 6.60 ± 0.88 (mean ± SEM) units. Normal (cataractous) dogs had a myocilin level in the aqueous humor of 8.05 ± 0.86 units. Conclusion Aqueous humor protein levels were elevated, relative to the myocilin control, in both the primary and secondary glaucoma groups compared to the cataract and diabetic cataract groups. Like in the Beagle POAG, aqueous humor myocilin protein levels are increased. Further studies are indicated to investigate the exact role of the aqueous humor myocilin protein in the genesis in increased IOP in these primary glaucomatous breeds. [ABSTRACT FROM AUTHOR]

WE Gillies was a major contributor to research in glaucoma, notably pseuodexfoliation (XFS), as well as strabismus, particularly in relation to axial length (AL). The latter work involved breaking down the geometry of the eye to its basic components and using the measured AL to tailor the amount of strabismus surgery required. Similarly, the search for glaucoma genes requires us to break down glaucoma into its component measures and associated risk factors. Over the last 14 years, our data from the Glaucoma Inheritance Study in Tasmania have shown the following: that a family history is present in 60% of glaucoma cases; that 27% of members of large glaucoma families were unaware of their family history of glaucoma; and that familial glaucoma is more severe than sporadic glaucoma. Myocilin mutations account for 3% of cases of primary open angle glaucoma. Some genotype–phenotype correlations have been identified. Notably, with respect to earlier age of onset, higher maximum recorded intraocular pressure and need for surgery, the Gln368Stop mutation confers mild risk, Thr377Met and Gly252Arg mutations intermediate risk, and the Pro370Leu mutation severe risk. To identify the other genes associated with glaucoma, we have examined normal twins in the Twins Eye Study to determine the heritability of parameters that are abnormal in glaucoma – intraocular pressure and cup-to-disc ratio and confounding factors for glaucoma such as central corneal thickness, disc area, refraction and AL. We have identified high heritabilities for all of these as well as a gene locus associated with AL on chromosome 5. Recently, the LOXL1 gene was associated with XFS. Identification of further genes will improve our understanding of glaucoma and allow cascade genetic screening. [ABSTRACT FROM AUTHOR]

Objective The presence of myocilin was investigated in a colony of Beagles, a canine model for inherited primary open-angle glaucoma (POAG). The myocilin protein was localized in the normal and glaucomatous canine eyes by immunohistochemistry and immunocytochemistry. Methods Paraffin- and plastic-embedded specimens from the anterior uveas of 10 Beagles with inherited glaucoma (3 months to 13 years old) and 6 age-matched normal dogs were sectioned, and were then incubated with primary antibody, rabbit polyclonal antihuman MYOC IgG, overnight at 4 °C. Specimens were incubated with secondary antibody with one of the following: biotinylated link followed by peroxidase-labeled streptavidin and then by substrate-chromogen for light microscopy; fluorescent marker Texas red; or 18 nm colloidal gold-labeled goat antirabbit IgG for transmission electron microscopy. Results With normal, pre- and early glaucomatous canine specimens, cell membranes of smooth muscle cells of the iris and ciliary body stained positively, as well as most resident stromal and vascular endothelial cells. The cytoplasm of cells within the nonpigmented ciliary epithelium of the ciliary body processes stained intensely, being weaker along the pars plana. Trabecular meshwork (TM) cells and surrounding extracellular matrix labeled, as well as the sclera adjacent to the angular aqueous plexus. In specimens with moderate and advanced glaucoma, greater intensity of staining was observed within TM cells and adjacent sclera, and portions of the nonpigmented epithelium of the ciliary processes. Fibrinous material labeled intensely within the posterior chamber. Conclusions Myocilin in the normal and glaucomatous canine eye was successfully immunolocalized. These findings with regard to the normal eye are nearly identical to those previously reported in humans, and support the original hypothesis that there is an increase in both accumulation and localization of myocilin in glaucomatous canine eyes. It also supports the possibility that changes in the activity of myocilin within the aqueous humor outflow pathway of individuals with spontaneous glaucoma are associated with the rise of intraocular pressure and subsequent development of this disease, but may not be the primary event in the initial raise in intraocular pressure in POAG in the Beagle. [ABSTRACT FROM AUTHOR]

Objective To identify canine myocilin cDNA and compare its sequence in glaucomatous and nonglaucomatous Shiba Inu dogs with closed and open iridocorneal angles (ICAs). Procedures Total RNA was extracted from the ciliary body of the eyes of a healthy Beagle, and the canine myocilin gene was cloned and sequenced. Of the Shiba Inu dogs tested, five were glaucomatous with closed ICA, three were nonglaucomatous with open ICA, and two were nonglaucomatous with closed ICA. The genomic DNA of these dogs was extracted from peripheral blood leukocytes. The exons of the canine myocilin gene were amplified using the polymerase chain reaction (PCR), and sequenced. The frequency of mutation in canine myocilin DNA was verified in these dogs by using the myocilin cDNA of a Beagle. Results The canine myocilin cDNA was 1452 bp long and contained the entire open reading frame encoding 483 amino acids. A leucine zipper-like motif and olfactomedin-like domain were conserved in the amino acid residues. The presence of sequence variants in the genomic DNA of Shiba Inu dogs was independent of the occurrence of glaucoma and ICA grading. Conclusions Myocilin RNA was detected in the ciliary body and trabecular meshwork (TM) of a Beagle. The myocilin sequence of Shiba Inu dogs suggests that myocilin mutations are unlikely to play a significant role in the pathogenesis of primary closed-angle glaucoma in this breed. However, several mutations in the myocilin gene in exon 1 of Shiba Inu dogs may predispose them to an obstruction in the anterior aqueous outflow. [ABSTRACT FROM AUTHOR]

Abstract: Myocilin (MYOC, TIGR) variations are associated with juvenile and primary open angle glaucoma (POAG). To investigate consequences of MYOC wildtype overexpression and selected mutations, we established a heterologous insect cell system (High Five™). Wildtype, Pro370Leu, Gln368X and Lys423Glu were cloned into a modified pIB/V5-His (pEXIV) vector with and without downstream GFP in frame fusion. Mutations were introduced by in vitro mutagenesis. Heterologous expression was shown and analysed by RT-PCR, Western blotting, immunocytochemistry and fluorescence microscopy. Extended cultivation (>14 days) resulted in accumulation of MYOC protein for all variants in growing dilated cisterns of the rough endoplasmic reticulum. Finally cell death for overexpressed wildtype and mutants occurs. A direct attachment of ribosomes to these growing vesicles preceding the cell death was observed by electron microscopy. Our observations indicate that this system is suitable to trace the intracellular effects of MYOC mutants. [Copyright &y& Elsevier]

Myocilin is a 55-57-kDa protein that is a member of the olfactomedin protein family. It is expressed in the cornea, sclera and trabecular network of the eye, myelinated peripheral nerves, heart, skeletal muscle, trachea and other tissues. Myocilin binds to a domain of fibronectin, type IV collagen and laminen in the trabecular meshwork of the eye, and its expression is influenced by transforming growth factor beta. Because these extracellular matrix components also are common in the intervertebral disc, the objective of our study was to determine whether the matricellular protein myocilin could be detected in the human or sand rat intervertebral disc using immunohistochemistry and to assess its localization. We investigated 16 specimens of human disc tissue and discs from six sand rats. Three human disc cell cultures grown in three-dimensional culture also were evaluated. Immunocytochemical annulus analysis showed the presence of myocilin within the disc cell cytoplasm in some, but not all, cells. Extracellular matrix in both the human and sand rat disc was negative for myocilin localization. Myocilin is believed to play a role in cell-cell adhesion and/or signaling. Myocilin may have such functions within the disc cell population in a manner similar to tenascin, SPARC and thrombospondin, which are other matricellular proteins recently shown to be present in the disc. [ABSTRACT FROM AUTHOR]

Glaucoma, which is a complex heterogeneous disease, presents an ideal case for genetic investigation. Primary open-angle glaucoma (POAG) is the commonest subtype and will be the focus of this review. When detected early, POAG is amenable to therapeutic intervention. Unfortunately, current population-based clinical screening lacks efficacy. If individuals with a genetic predisposition for developing POAG can be identified, then efficient and cost-effective population-based screening programs could be designed. Although considerable inroads have been made in understanding the natural history of POAG caused by mutations in the myocilin and optineurin genes, other POAG genes accounting for most cases remain to be identified. This review explores the genetic mechanisms that have been unequivocally linked to the glaucomatous process and then discusses potential avenues for future breakthroughs. [ABSTRACT FROM AUTHOR]

Abstract: Myocilin forms high molecular weight complexes in vivo presumably due to interaction with itself and other myocilin binding proteins. To identify myocilin interacting proteins, yeast 2-hybrid analysis was performed on >1×106 human trabecular meshwork cDNA clones. Coimmunoprecipitation and Far Western analysis were also performed on cell lysates obtained from fresh human trabecular meshworks or cultured human monolayer trabecular cell lines. Among the different methods, 46 candidate myocilin-associated proteins were identified, including molecules associated with the extracellular matrix, cytoskeleton, signaling, and metabolism. The most consistent interaction was myocilin–myocilin binding. Yeast-2 hybrid and Far Western analysis also found an association between myocilin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). None of the other candidate myocilin interacting proteins were identified in more than one method. Characterization of these potential interacting proteins may help to better understand the function of myocilin in the trabecular meshwork and aqueous outflow pathway. [Copyright &y& Elsevier]

Abstract: Gene silencing by siRNAs offers the potential for reducing the expression of mutated genes that cause disease. It has been shown that the folding or secondary structure of a specific mRNA was significantly correlated to the silencing observed. Since this base pairing is dependent on free energy, the temperature of the cells may influence the effectiveness of a particular siRNA. The aqueous humor of the human eye has been measured to be around 34°C with the lens acting as a thermal barrier in the eye. The trabecular meshwork, bathed by the aqueous humor, probably has a temperature lower than body temperature under normal conditions. Mutated myocilin, that is associated with primary open angle glaucoma, would appear to be a candidate for silencing by siRNAs. Silencing of the mutated myocilin might prevent additional accumulation of this protein in the rough endoplasmic reticulum. These experiments were undertaken to determine the influence of lowered temperatures on the silencing of myocilin by five siRNAs. Three different patterns of silencing emerged when the silencings were compared with cells grown at 33, 35, and 37°C. For one of the siRNAs, the silencing was increased at lower temperatures. For two siRNAs, no significant changes in silencing were observed with different temperatures. Two of the siRNAs were significantly influenced by temperature with little if any silencing occurring at the lowest temperature. These data indicate that siRNAs directed to tissues in the anterior chamber of the eye should be checked at temperatures lower than 37°C to determine their effectiveness. [Copyright &y& Elsevier]

Abstract: The domestic cat (Felix domesticus) is a useful model for understanding the implications of long-term expression and function of normal and mutant myocilin. To better understand the role myocilin has in the cat eye, we isolated and characterized cat myocilin. Oligonucleotides designed against conserved nucleotide regions of myocilin mammalian orthologs were used to PCR amplify a partial cat myocilin cDNA clone. Rapid amplification of cDNA ends (5′ and 3′ RACE) was used to obtain full-length cat myocilin. The 2125 nucleotide cat myocilin cDNA contains a 490 amino acid open reading frame. Comparison of cat myocilin to human myocilin shows a 87% similarity, including conservation of the N-terminal leucine zipper, N-linked glycosylation site, C-terminal olfactomedin domain, and all five cysteine residues thought to be involved in disulfide bond formation. Expression in a transformed human trabecular cell line or in Crandall feline kidney cells showed cat myocilin was secreted from these cells, similar to human myocilin, suggesting cat myocilin contains a functional signal peptide sequence. In contrast, expression of cat myocilin containing a known human glaucoma-associated mutation (Y423H in cat; Y437H in human) was not secreted. Characterization of cat myocilin will enable long-term studies be performed in Felix domesticus to analyze changes to intraocular pressure and the aqueous outflow pathway following expression of myocilin and glaucoma causing mutations. [Copyright &y& Elsevier]

Abstract: TIGR/MYOC mutations account for 2–4% of the primary open-angle glaucoma (POAG) patients. More than 90% of the known mutations are located within its carboxy-terminus olfactomedin-homology (Olf) domain (amino acids (aa) 245–504). In vitro and in vivo studies showed that several Olf domain mutations prevented myocilin secretion. To investigate if intracellular sequestration was a characteristic feature shared by a majority of the mutations, we analyzed the secretion status of 36 myocilin variants. These encompassed 26 glaucoma-causing mutations and 10 non-disease associated or undefined polymorphisms. As several variants were found to be secreted, we tested for their adhesion to the extracellular matrix (ECM) and/or cell surface. Myocilin variants were generated by site-directed mutagenesis of a vector encoding the human MYOC cDNA. COS-7 or immortalized human trabecular meshwork cells were transfected with wild-type or mutated MYOC constructs. Myocilin levels were estimated by immunoprecipitation and/or immunoblotting. All variants showed identical behaviors in both cell lines; the truncated R46X polypeptide being the only variant which could not be detected in our assays. Of the 35 variants monitored, 20 remained sequestered intracellularly. All of them encoded disease-causing polypeptides carrying Olf domain mutations. Of the 15 variants secreted into the culture medium, six (6) were POAG mutants (of which three (3) located within the Olf domain) while the remaining nine (9) were non-disease causing or undefined polymorphisms. Three (3) of the six (6) secreted mutations caused familial POAG; these were the R126W, T377M and A427T mutants. Both, the T377M and A427T mutants located within the Olf domain. When cells were cultured at 30°C, a process known to facilitate protein folding, 11 of the 20 sequestered mutants were released in the extracellular medium. Out of the 15 secreted variants tested for their adhesion to the ECM and/or cell surface, only the R82C and L95P polypeptides displayed loss of their adhesive properties. Deletion experiments revealed that the coiled-coil (aa 78–105) and leucine zipper (aa 114–183) motifs were essential for adhesion. These experiments demonstrate that intracellular sequestration might be the primary mechanism contributing to myocilin-related POAG as it was associated with more than 80% of the disease-causing mutants tested in our study. A second mechanism may involve abnormal interaction(s) between myocilin and ECM and/or cell surface proteins. Our data further revealed the importance of the olfactomedin-homology domain for myocilin secretion and the significant role of the N-terminal region for its extracellular interactions. [Copyright &y& Elsevier]

Abstract: Wild-type TIGR/MYOC is a secreted protein implicated in the development of steroid glaucoma. Mutations in TIGR/MYOC have been linked to some patients who develop elevated intraocular pressure (IOP) and glaucoma. Because there is evidence of some other factors contributing to the TIGR/MYOC causative role in glaucoma, and because substantial increased levels of a particular cellular mRNA and protein might alter expression of other host genes, we began to investigate the effect of TIGR/MYOC overexpression on the transcriptome of human trabecular meshwork cells. We used a recombinant adenovirus carrying wild-type TIGR/MYOC cDNA, primary HTM cells, 300 viral particles per cell and U133 Affymetrix GeneChips. Our results indicate that 2361 out of the 22 284 genes (10.6%) were altered more than two-fold (p≤0.005) by the overexpression of TIGR/MYOC. A higher proportion of the altered genes were downregulated (1412 vs. 949). Potentially relevant upregulated genes include angiopoietin 2, matrix metalloproteinase 1 (MMP1) and thrombomodulin; among those downregulated we observed growth arrest specific 1, proteins involved in the ubiquitination pathway and vascular cell adhesion molecule 1. In addition, collagen type 1, one of the MMP1 substrates, was also downregulated. Genes affected by wild-type TIGR/MYOC might prove to be candidate mediators for future studies of the mechanisms of glaucoma. [Copyright &y& Elsevier]

The intraocular pressure rise that can complicate the use of topical or systemic corticosteroid has been recognised for 50 years. More recently, following isolation of the myocilin gene (previously known as the trabecular meshwork inducible glucocorticoid response or TIGR gene), there has been renewed interest in this steroid-responsive phenomenon. Furthermore, the currently fashionable use of injectable intraocular steroids in the management of clinically significant subretinal fluid and macular oedema has resulted in an increased incidence. Animal studies, cell biology, molecular biology, and an improved knowledge of genetics have provided a better understanding of the mechanisms behind the response. The purpose of this review is to describe the risk factors for developing corticosteroid-induced glaucoma, to discuss the underlying mechanisms and genetics of the condition and to present management options.Eye (2006) 20, 407–416. doi:10.1038/sj.eye.6701895; published online 6 May 2005 [ABSTRACT FROM AUTHOR]

Abstract: Cultured retinal pigment epithelial (RPE) cells are commonly used as a model of the tissue to study their involvement in visual diseases. Unfortunately, cultured RPE often lose their differentiated phenotype reducing their usefulness as a model of the RPE in vivo. In this study, we used a Ca++-switch protocol to initiate the patterned expression of several phenotypic and functional markers of RPE differentiation. Cultured RPE cells from adult donors were maintained through at least six serial passages prior to assay to minimize their differentiated properties. The cells were then subjected to the Ca++-switch protocol and maintained at confluence for up to 4 months. Paired control and Ca++-switch cells were examined for phenotype, pigmentation, and the expression of tyrosinase, CRABP, myocilin, and bestrophin by western blot analysis. The Ca++-switch protocol led to a rapid restriction of N-cadherin to lateral cell borders, and to expression of tyrosinase by day 4. After 8 weeks, the experimental RPE monolayers began to accumulate visible pigment, and after 12 weeks CRABP expression was observed. Myocilin was observed at 4 months after the Ca++-switch but bestrophin was not detected at any time point. Our results suggest this protocol may drive epithelial morphogenesis in RPE cells. We note two specific differences in cells plated in low Ca++, reduced spreading on the substrate and coordinated development of cadherin adhesion when the Ca++-concentration is returned to normal. Thus, we suggest that this method produces phenotypic changes through multiple cell signalling pathways. [Copyright &y& Elsevier]

Abstract: Myocilin, a matricellular protein, is mutated in glaucoma. Here we report the identification and characterization, by the yeast two-hybrid system, of a putative interacting protein with myocilin. One of the positive clones exhibited 100% identity with the carboxyl-terminal (C-t) region of hevin, a member of the BM-40/SPARC/osteonectin family of extracellular matrix proteins. Protein interaction was assayed, in doubly transfected 293-T cells, by Western blot and fluorescent microscopy. Western blot analysis of the culture medium and lysates from cotransfected cells indicated that myocilin causes intracellular accumulation of hevin-C-t and impairs its secretion. This effect on hevin-C-t was augmented when coexpressed with the myocilin P370L mutant, known to cause a severe form of glaucoma. By fluorescent microscopy, myocilin localizes with hevin-C-t in the Golgi in cotransfected 293-T cells and with hevin-wt in the ocular ciliary epithelium. Overall, these results suggested that the C-t of hevin contains important determinants for interaction with myocilin. [Copyright &y& Elsevier]

Abstract: Mutations in MYOC gene encoding myocilin are responsible for primary open-angle glaucoma (POAG). In order to search for protein(s) that can interact with myocilin, we screened a human skeletal muscle cDNA library using yeast two-hybrid system and identified flotillin-1, a structural protein of lipid raft that is detergent-resistant and a liquid ordered microdomain, as a protein interacting with myocilin. The interaction was confirmed by in vitro glutathione S-transferase pulldown and in vivo co-immunoprecipitation studies. In yeast two-hybrid assay, the C-terminus of myocilin, an olfactomedin-like domain in which most mutations related to POAG are scattered, was found to be necessary and sufficient for the interaction. However, myocilins with mutations such as G364V, K423E, and Y437H on the domain failed to interact with flotillin-1. Although the physiological significance of the interaction has yet to be elucidated, our results showed that the alteration of the interaction by mutations in MYOC might be a key factor of the pathogenesis of POAG. [Copyright &y& Elsevier]

Abstract: The region-specific characteristics of the extracellular matrix are crucial for diverse functions in the brain. Pancortins/neuron-specific olfactomedin-related glycoproteins are components of the extracellular matrix. They comprise four alternatively spliced variants, Pancortin-1 to -4, which share a common portion, the B part, in the middle of their structure, have two pairs of alternatively spliced 5′ regions, A1 and A2, and 3′ regions, C1 and C2. Here we demonstrate that in mice, Pancortin-3 (A2-B-C1) and Pancortin-4 (A2-B-C2), which we have grouped together the A2-Pancortins, were transcribed early during the development of the brain in a region specific manner and were expressed very stably in vivo. They are N-glycosylated and secreted. Furthermore, we examined their ontogenetical expression profiles in the developing thalamus using antiserum against the common B region, since transient expressions of their mRNAs were notable there. In the developing thalami, they lasted long in oligomerized form even after the transcription of their mRNAs decreased to an undetectable level. Further analyses revealed that cysteine residues that are located in the common B part are important for homo- and hetero-oligomer formation of A2-Pancortins. When we substituted cysteine residues 45 and 47 with serine residues in that common B part, oligomerization of the A2-Pancortins was highly disturbed. [Copyright &y& Elsevier]

Glaucoma describes a group of diseases that kill retinal ganglion cells. There are different types of glaucoma, and each appears to be genetically heterogeneous. Different glaucoma genes have been identified, but these genes account for only a small proportion of glaucoma. Most glaucoma cases appear to be multifactorial, and are likely affected by multiple interacting loci. A number of genetic susceptibility factors have been suggested to contribute to glaucoma. These factors fit into two broad groups, those affecting intraocular pressure and those important in modulating retinal ganglion cell viability. Defining the complex genetics of glaucoma will require significant further study of the human disease and animal models. Genetic approaches are essential and will be enhanced by recently developed genomic and proteomic technologies. These technologies will provide valuable clues about pathogenesis for subsequent testing. In this review, we focus on endogenous genetic susceptibility factors and on how experimental studies will be valuable for dissecting the multifactorial complexity of their interactions. [ABSTRACT FROM AUTHOR]

Abstract: Phenylketonuria (PKU) is an inborn error of amino acid metabolism. Phenylalanine hydroxylase (PAH) mutations resulting reduced enzyme levels lead to accumulation of phenylalanine (Phe) in brain, if Phe diet is not restricted. Patients with PKU show neurophysiological abnormalities including demyelination and cognitive defect. How PAH defect causes events seen in PKU is not obvious. Therefore, expression analysis was performed in the brain of a mouse model for PKU. Microarray expression profile of the brain showed lower expression of myocilin (Myoc) in the PKU mouse. Reduced expression of Myoc was further confirmed by one-step real-time RT-PCR. Western blotting analysis of the brain using equal quantities of protein showed a thin band in PKU compared to a prominent band in the wild type brain. In addition, expression of genes associated with transcription was found to be altered in the PKU mouse brain as observed by microarray analysis. These data suggest that PAH defect alters other genes expression likely to contribute neurophysiological abnormalities seen in the mouse, if documented also in patients with PKU. [Copyright &y& Elsevier]

Kaur K, Reddy ABM, Mukhopadhyay A, Mandal AK, Hasnain SE, Ray K, Thomas R, Balasubramanian D, Chakrabarti S. Myocilin gene implicated in primary congenital glaucoma.Primary congenital glaucoma (PCG) has been associated withCYP1B1gene (2p21), with a predominantly autosomal recessive mode of inheritance. Our earlier studies attributedCYP1B1mutations to only 40% of Indian PCG cases. In this study, we included 72 such PCG cases whereCYP1B1mutations were detected in only 12 patients in heterozygous condition, implying involvement of other gene(s). On screening these patients for mutations in myocilin (MYOC), another glaucoma-associated gene, using denaturing high-performance liquid chromatography followed by sequencing, we identified a patient who was double heterozygous atCYP1B1(c.1103G>A; Arg368His) andMYOC(c.144G>T; Gln48His) loci, suggesting a digenic mode of inheritance of PCG. In addition, we identified the sameMYOCmutation, implicated for primary open angle glaucoma, in three additional PCG patients who did not harbor any mutation inCYP1B1. These observations suggest a possible role ofMYOCin PCG, which might be mediated via digenic interaction withCYP1B1and/or an yet unidentified locus associated with the disease. [ABSTRACT FROM AUTHOR]

Mutations in the MYOC/TIGR gene are responsible for autosomal dominant primary open angle glaucoma (POAG). Almost all mutations responsible for POAG have been detected in the coding region (in particular at exon 3). By using the techniques of PCR, SSCP, automated sequencing and restriction analysis, we have studied 79 patients suffering from glaucoma. We have found five patients with sequence variants in the consensus region of the promoter. These sequence variants might be involved in the altered association between the consensus region and the corresponding transcription factor. This possibility might be favouring the association of other transcription factors, which would operate as activators or inhibitors of the transcription, altering the MYOC/TIGR expression.European Journal of Human Genetics (2005) 13, 384-387. doi:10.1038/sj.ejhg.5201299 Published online 13 October 2004 [ABSTRACT FROM AUTHOR]