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Juvenile-onset open-angle glaucoma (JOAG) is a subset of primary open-angle glaucoma that is diagnosed before 40 years of age. The disease may be familial or non-familial, with proportions varying among different populations. Myocilin mutations are the most commonly associated. JOAG is characterized by high intraocular pressures (IOP), with many patients needing surgery. The mean age at diagnosis is in the 3(rd) decade, with a male preponderance. Myopia is a common association. The pathophysiology underlying the disease is immaturity of the conventional outflow pathways, which may or may not be observed on gonioscopy and anterior segment optical coherence tomography. The unique optic nerve head features include large discs with deep, steep cupping associated with high IOP-induced damage. Progression rates among JOAG patients are comparable to adult primary glaucomas, but as the disease affects younger patients, the projected disability from this disease is higher. Early diagnosis, prompt management, and life-long monitoring play an important role in preventing disease progression. Gene-based therapies currently under investigation offer future hope.

Background: Glaucoma is defined as progressive optic neuropathy leading to irreversible blindness if not treated on time. Primary open angle glaucoma (POAG) is most common form of glaucoma. Mutations in myocilin gene (MYOC) account for 2–4% of POAG cases. Purpose: To identify and evaluate MYOC variants alleles among patients with POAG and their healthy first degree relatives. Materials and Methods: 66 POAG patients and 26 healthy first degree relatives recruited for study. All patients underwent complete ophthalmic examination followed by genomic DNA (deoxyribonucleic acid) isolation from peripheral blood and quantification of DNA on spectrophotometer. All samples were amplified with each primer by PCR (Polymerase Chain Reaction) technique and amplified DNA and primer sequence checked again by electrophoresis for confirmation of specified MYOC gene mutation. Results: We identified a known MYOC missense mutation, Pro370leu in 16 POAG cases and found consistent genotypic but not phenotypic correlation in 4 of their first degree relatives. Out of 16 cases, pathogenic MYOC gene variant was found in 12 adult onset POAG, 3 juvenile onset POAG, and 1 case of OHT. Conclusion: This study is first of its kind in North India. Our study showed frequency of MYOC gene mutation in POAG cases was 24.24% which is much higher than found elsewhere in India and other countries (2-5%). Frequency of transmission of pathogenic MYOC gene variant in first degree relatives was 25%. The future outcome of our study is promising since early diagnosis and management of high risk family members is possible. Keywords: Primary open angle glaucoma, Myocilin, Mutation, Pro370leu.

ConspectusNumerous human disorders arise due to the inability of a particular protein to adopt its correct three-dimensional structure in the context of the cell, leading to aggregation. A new addition to the list of such protein conformational disorders is the inherited subtype of glaucoma. Different and rare coding mutations in myocilin, found in families throughout the world, are causal for early onset ocular hypertension, a key glaucoma risk factor. Myocilin is expressed at high levels in the trabecular meshwork (TM) extracellular matrix. The TM is the anatomical region of the eye that regulates intraocular pressure, and its dysfunction is associated with most forms of glaucoma. Disease variants, distributed across the 30 kDa olfactomedin domain (mOLF), cause myocilin to be sequestered intracellularly instead of being secreted to the TM extracellular matrix. The working hypothesis is that the intracellular aggregates cause a toxic gain of function: TM cell death is thought to lead to TM matrix dysfunction, hastening elevated intraocular pressure and subsequent vision loss.Our lab has provided molecular underpinnings for myocilin structure and misfolding, placing myocilin-associated glaucoma within the context of amyloid diseases like Alzheimer and diabetes. We have dissected complexities of the modular wild-type (WT) myocilin structure and associated misfolded states. Our data support the model that full-length WT myocilin adopts a Y-shaped dimer-of-dimers conferred by two different coiled-coil regions, generating new hypotheses regarding its mysterious function. The mOLF β-propellers are paired at each tip of the Y. Disease-associated variants aggregate because mOLFs are less stable, leading to facile aggregation under physiological conditions (37 °C, pH 7.2). Mutant myocilin aggregates exhibit numerous characteristics of amyloid in vitro and in cells, and aggregation proceeds from a partially folded state accessed preferentially by disease variants at physiological conditions. Interestingly, destabilization is not a universal consequence of mutation. We identified counterintuitive, stabilizing point variants that adopt a non-native structure and do not aggregate; however, these variants have not been identified in glaucoma patients. An ongoing effort is predicting the consequence of any given mutation. This effort is relevant to interpreting data from large-scale sequencing projects where clinical and family history data are not available. Finally, our work suggests avenues to develop disease-modifying precision medicines for myocilin-associated glaucoma.

This study aims to characterize disease-causing variants in the myocilin gene (MYOC), which is associated with autosomal dominant primary open-angle glaucoma (adPOAG).Case-control study.MYOC variants were collected from in-house exome sequencing data of 7092 individuals. Genotype-phenotype analysis and bioinformatics evaluation were used to distinguish potential pathogenic variants for POAG from others. MYOC mutations in published works of literature were also systemically analyzed.In total, 53 variants in MYOC were detected in the 7092 subjects, including 45 rare variants (MAF 0.01) and 8 polymorphisms (MAF ≥ 0.01), or 48 missense variants and 5 truncation variants. There was no difference in the frequency of the 8 polymorphisms between subjects with and without POAG (P 0.05). The total number of rare MYOC variants was significantly higher in POAG than that in in-house controls (P = 3.31E-14). The pathogenic/likely pathogenic variants (p.P254T, p.S341P, p.G367R, p.P370L, p.D378G, p.C433Y, and p.L486F) were exclusively present in 8 POAG but absent in in-house controls (P = 2.79E-10). Rare truncation MYOC variants were not enriched in POAG as compared with those in in-house controls (P = 0.28). Further analysis of previously reported MYOC variants suggested that pathogenic/likely pathogenic variants were enriched in the conserved olfactomedin domain. Truncation MYOC variants were scattered in the coding region, where only p.Q368∗ had relatively strong evidence to be causative for adPOAG, whereas most others are questionable.Most MYOC variants contributing to adPOAG could be characterized as rare missense variants located in OLF-domain and predicted to be damaging through multiple tools. The effect of other variants, especially for truncation variants (except for p.Q368∗) need further clarification.

Family study is an effective way to identify disease-causing mutations (DCMs) and characterize the clinical phenotype of genetic diseases. In this study we recruited a Chinese primary open-angle glaucoma (POAG) family spanning six generations and consisting 112 individuals, in which 63 were participated in. Targeted exome sequencing on the proband identified a heterozygous mutation (c.752T>C, p.Val251Ala) in MYOC gene. Sanger sequencing performed on all participants found that fourteen family members carried this mutation. Ten (71.4%) of them were diagnosed with POAG, two (14.3%) with ocular hypertension (OHT) and two (14.3%) without manifestations of glaucoma. According to the results of ophthalmic examinations of the family members and their medical history, we found that the Val251Ala mutation was associated with clinical phenotype including intermediate penetrance, high intraocular pressure (IOP), severe visual defects and requirement of surgery.

Glaucoma is a leading cause of irreversible blindness worldwide. Reducing intraocular pressure is currently the only effective treatment. Elevated intraocular pressure is associated with increased resistance of the outflow pathway, mainly the trabecular meshwork (TM). Despite great progress in the field, the development of novel and effective treatment for glaucoma is still challenging. In this study, we reported that human induced pluripotent stem cells (iPSCs) can be cultured as colonies and monolayer cells expressing OCT4, alkaline phosphatase, SSEA4 and SSEA1. After induction to neural crest cells (NCCs) positive to NGFR and HNK1, the iPSCs can differentiate into TM cells. The induced iPSC-TM cells expressed TM cell marker CHI3L1, were responsive to dexamethasone treatment with increased expression of myocilin, ANGPTL7, and formed CLANs, comparable to primary TM cells. To the best of our knowledge, this is the first study that induces iPSCs to TM cells through a middle neural crest stage, which ensures a stable NCC pool and ensures the high output of the same TM cells. This system can be used to develop personalized treatments using patient-derived iPSCs, explore high throughput screening of new drugs focusing on TM response for controlling intraocular pressure, and investigate stem cell-based therapy for TM regeneration.

Primary congenital glaucoma (PCG) is one of the primary causes of blindness in children and is characterized by congenital trabecular meshwork and anterior chamber angle dysplasia. While being a rare condition, PCG severely impairs the quality of life of affected patients. However, the pathogenesis of PCG remains to be fully elucidated. It has previously been indicated that genetic factors serve a critical role in the pathogenesis of PCG, although patients with PCG exhibit significant genetic heterogeneity. Mutations in the cytochrome P450 family 1 subfamily B member 1 gene have been implicated in PCG and further genes that have been reported to be involved in PCG are myocilin, forkhead box C1, collagen type I α1 chain and latent transforming growth factor β binding protein 2. The present review aims to provide an up to date understanding of the genes associated with PCG and the use of molecular technologies in the identification of such genes and mutations. This may pave the way for the development of preventative methods, early diagnosis and improved therapeutic strategies in PCG.

PURPOSE: Juvenile onset primary open angle glaucoma (JOAG) is a rare disorder associated with high IOP and progressive optic neuropathy in patients diagnosed before the age of 40 years. While in some populations it has primarily an autosomal dominant pattern of inheritance, in others it occurs in a primarily sporadic form. The main aim of the study was to assess the relative prevalence of Myocilin (MYOC) mutations in familial versus sporadic cases of JOAG. METHODS: We screened 92 unrelated (sporadic) JOAG patients, and 22 affected families (70 affected members and 36 unaffected) for variations in the MYOC gene. We also analyzed the clinical features associated with these variations. RESULTS: Three coding sequence variants were identified as mutations causing JOAG. Four families segregated distinct mutations at Gly367Arg, and two families at Gln337Arg, while only two sporadic JOAG cases harbored MYOC mutations (Gly367Arg and Gln48His). The frequency of MYOC mutations in familial cases (27%) was significantly higher than in sporadic JOAG cases (2%); p = 0.001. A 90% penetrance for the Gly367Arg variant was seen by the age of 40 years in our patients. Characteristic allele signatures, indicative of specific founder effects, were not observed for the Gly367Arg mutation that was looked for in 12 patients among 2 geographically close families, which harbored this mutation. CONCLUSION: Our data demonstrated that genetic screening for MYOC mutations should be focused toward cases with familial rather than sporadically occurring JOAG.

Objective: To identify the interaction between the MYOC Y437H mutation and TGF-β2 in a family with primary open-angle glaucoma (POAG). Methods: The MYOC Y437H mutation was identified in a family with POAG; the family was a fourth-generation family with 27 members, of which 6 members were affected. Analysis focused on the secreted myocilin protein and TGF-β2 found in the aqueous humor. Samples were taken both from normal controls and MYOC mutant carriers and cross-talk between MYOC Y437H and TGF-β2 were evaluated in the trabecular meshwork (TM) cells. Results: Aqueous humor secreted myocilin protein levels were reduced while TGF-β2 levels were increased in patients with the MYOC (c.1309T>C) mutation. This inverse relationship indicated that elevated TGF-β2 may be an important pathogenic mechanism in the progression of myocilin-related POAG. In TM cells expressing the MYOC Y437H mutant, exogenous TGF-β2 also significantly increased myocilin expression as well as the endoplasmic reticulum (ER) stress markers GRP94 and CHOP. This increase in TGF-β2 was also associated with increased cell death in cells carrying the MYOC Y437H mutation. Conclusion: These data collectively suggest that the mutual interaction between glaucomatous MYOC mutation and TGF-β2 contributed to the cell death of TM cells. This relationship also provides a new, therapeutic targets for the treatment of glaucoma.

Glaucoma is the leading cause of irreversible blindness worldwide, with elevated intraocular pressure (IOP) as the only known modifiable risk factor. Trabecular meshwork (TM)-inducible myocilin (the MYOC gene) was the first to be identified and linked to juvenile and primary open-angle glaucoma. It has been suggested that mutations in the MYOC gene and the aggregation of mutant myocilin in the endoplasmic reticulum (ER) of TM may cause ER stress, resulting in a reduced outflow of aqueous humor and an increase in IOP. We selected 20 MYOC mutations with experimentally determined melting temperatures of mutated myocilin proteins. We included 40 published studies with at least one glaucoma patient with one of these 20 MYOC mutations and information on age at glaucoma diagnosis. Based on data from 458 patients, we found that a statistically significant but weak correlation was present between age and melting temperature based on various assumptions for age. We therefore conclude that genetic analysis of MYOC mutations alone cannot be used to accurately predict age at glaucoma diagnosis. However, it might be an important prognostic factor combined with other clinical factors for critical and early detection of glaucoma.

Juvenile open-angle glaucoma is a disease with complex pathogenesis affecting young people of working age that can lead to disability. The article describes modern concepts of diagnosis, classification and approaches to the treatment of juvenile glaucoma with special attention paid to the differential diagnostic criteria of juvenile open-angle glaucoma and congenital glaucoma.Ювенильная открытоугольная глаукома является инвалидизирующим заболеванием со сложным патогенезом, поражающим людей молодого, трудоспособного возраста. В работе представлены современные представления о диагностике, классификации и подходах к лечению ювенильной глаукомы. Особое внимание уделено дифференциально-диагностическим критериям ювенильной открытоугольной глаукомы и врожденной глаукомы.

Myocilin, a modular multidomain protein, is expressed broadly in the human body but is best known for its presence in the trabecular meshwork extracellular matrix, and myocilin misfolding is associated with glaucoma. Despite progress in comprehending the structure and misfolding of the myocilin olfactomedin domain, the structure and function of full-length myocilin, and contextual changes in glaucoma, remain unknown. Here we expressed and purified milligram-scale quantities of full-length myocilin from suspension mammalian cell culture (Expi293F), enabling molecular characterization in detail not previously accessible. We systematically characterized disulfide-dependent and -independent oligomerization as well as confirmed glycosylation and susceptibility to proteolysis. We identified oligomeric states with glycosylation sites that are inaccessible to enzymatic removal. Low-resolution single particle 2D class averaging from conventional transmission electron microscopy imaging confirms an extended arrangement of tetramers, truncated products consistent with dimers, and a higher-ordered state consistent with octamer. Taken together, our study reveals new myocilin misfolded states and layers of intrinsic heterogeneity, expands our knowledge of olfactomedin-family proteins and lays the foundation for a better molecular understanding of myocilin structure and its still enigmatic biological function.

Glaucoma is the leading cause of irreversible blindness with trabecular meshwork (TM) dysfunction resulting in elevated intraocular pressure and retinal ganglion cell (RGC) damage leading to vision loss. In this study, we discovered that secretome, derived from human TM stem cells, via minimal invasive periocular injection, can reduce intraocular pressure, restore TM homeostasis, protect RGC, and restore RGC function in both steroid-induced and genetic myocilin mutant mouse models of glaucoma. The secretome upregulated the COX2-PGE2 axis via mitochondrial TMEM177 and led to activation of endogenous stem cells and TM regeneration. Inhibition of COX2 abolished the protective effect of secretome on TM cells. Secretome treatment also enhanced RGC survival and function. Proteomic analysis revealed that the secretome is enriched with proteins involved in extracellular matrix modulation leading to the remodeling of TM to restore homeostasis. This study highlights the feasibility of stem cell-free therapy for glaucoma with minimal invasive administration and the involvement of multiple novel pathways for a cumulative regenerative effect on the TM to protect RGC.Brief summaryThis study describes a cell-free treatment using stem cell secretome in two animal models of glaucoma and explores the potential mechanisms

IMPORTANCE The c.1102C>T, p.(Gln368Ter) variant in themyocilin (MYOC) gene is a known risk allele for glaucoma. It is the most common MYOC risk variant for glaucoma among individuals of European ancestry, and its prevalence is highest in Finland. Furthermore, exfoliation syndrome has high prevalence in Scandinavia, making the Finnish population ideal to study the association of the variant with different types of glaucoma. OBJECTIVES To examine the association and penetrance of MYOC p.(Gln368Ter) (rs74315329) variant with different types of glaucoma in a Finnish population. DESIGN, SETTING, AND PARTICIPANTS This genetic association study included individuals of Finnish ancestry in the FinnGen project. The participants were collected from Finnish biobanks, and the disease end points were defined using nationwide registries. The MYOC c.1102C>T variant was either directly genotyped or imputed with microarrays. Recruitment of samples to FinnGen was initiated in 2017, and data analysis was performed between December 2019 and May 2020. MAIN OUTCOMES AND MEASURES The main outcomes were odds ratios (ORs) and penetrance with different types of glaucoma and in different age groups. RESULTS A total of 218 792 individuals were included in this study (mean [SD] age 52.4 [17.5] years; 123 579 women [56.5%]), including 8591 (3.9%) with glaucoma, 3412 (1.6%) with primary open-angle glaucoma, 1515 (0.7%) with exfoliation glaucoma, 892 (0.4%) with normal-tension glaucoma, and 4766 (2.2%) with suspected glaucoma. The minor allele frequency of MYOC p.(Gln368Ter) was 0.28%. Individuals with the heterozygous variant had higher odds of primary open-angle glaucoma (OR, 3.36; 95% CI, 2.55-4.37), overall glaucoma (OR, 2.58; 95% CI, 2.12-3.13), suspected glaucoma (OR, 2.53; 95% CI, 1.93-3.26), exfoliation glaucoma (OR, 2.61; 95% CI, 1.60-4.02), and undergoing glaucoma-related operations (OR, 5.45; 95% CI, 2.95-9.28). The penetrance of heterozygous MYOC p.(Gln368Ter) was 5.2% in individuals with primary open-angle glaucoma, 9.6% in individuals with glaucoma, 5.4% in individuals with suspected glaucoma, and 1.9% in individuals with exfoliation glaucoma. There was no significant association with normal-tension glaucoma (OR, 1.69; 95% CI, 0.72-3.35). CONCLUSIONS AND RELEVANCE This genetic association study found that the MYOC p.(Gln368Ter) variant was associated with exfoliation glaucoma. The association with normal-tension glaucoma could not be replicated. These findings suggest that MYOC p.(Gln368Ter) was associated with open-angle glaucoma and exfoliation glaucoma in a Finnish population.

Glaucoma is a leading cause of irreversible blindness worldwide, and increased intraocular pressure (IOP) is a major risk factor. We aimed to determine if early functional and molecular differences in the glaucomatous retina manifest before significant retinal ganglion cell (RGC) loss is apparent. Adenoviral vectors expressing a pathogenic form of myocilin (Ad5.MYOC) were used to induce IOP elevation in C57BL/6 mice. IOP and pattern electroretinograms (pERG) were recorded, and retinas were prepared for RNA sequencing, immunohistochemistry, or to determine RGC loss. Ocular injection of Ad5.MYOC leads to reliable IOP elevation, resulting in significant loss of RGC after nine weeks. A significant decrease in the pERG amplitude was evident in eyes three weeks after IOP elevation. Retinal gene expression analysis revealed increased expression for 291 genes related to complement cascade, inflammation, and antigen presentation in hypertensive eyes. Decreased expression was found for 378 genes associated with the γ-aminobutyric acid (GABA)ergic and glutamatergic systems and axon guidance. These data suggest that early functional changes in RGC might be due to reduced GABAA receptor signaling and neuroinflammation that precedes RGC loss in this glaucoma model. These initial changes may offer new targets for early detection of glaucoma and the development of new interventions.

Primary open-angle glaucoma (POAG) is the most common form of glaucoma, prevalent in approximately 1–2% of Caucasians in the UK over the age of 40. It is characterised by an open anterior chamber angle, raised intraocular pressure (IOP) and optic nerve damage leading to loss of sight. The myocilin gene (MYOC) is the most common glaucoma-causing gene, accounting for ~2% of British POAG cases. 358 patients were selected for next generation sequencing (NGS) with the following selection criteria: Caucasian ethnicity, intraocular pressure (IOP) 21–40 mm Hg, cup:disc ratio ≥0.6 and visual field mean deviation ≤−3. The entire MYOC gene (17,321 bp) was captured including the promoter, introns, UTRs and coding exons. We identify 12 exonic variants (one stop-gain, five missense and six synonymous variants), two promoter variants, 133 intronic variants, two 3′ UTR variants and 23 intergenic variants. Four known or predicted pathogenic exonic variants (p.R126W, p.K216K, p.Q368* and p.T419A) were identified across 11 patients, which accounts for 3.07% of this POAG cohort. This is the first time that the entire region of MYOC has been sequenced and variants reported for a cohort of POAG patients.

Glaucoma is the second leading cause of blindness across the globe and is estimated with a rise in prevalence in 79.6 million people by the year 2020. Glaucoma, as a most complex disease it is categorized into primary and secondary. The pathogenesis is attributed with multiple molecular mechanisms underlying the association of multiple genes with varying magnitudes of effects. The hallmark genes of glaucoma being myocilin (MYOC), optineurin (OPTN) and neurotrophin-4 (NTF4) in primary open-angle glaucoma (POAG) are already well associated with glaucoma. However, the candidate genes that are associated with glaucoma at various stages of pathogenesis and their genetic heterogeneity could significantly be emphasized as a novel therapeutic target of glaucoma. In line with this, we provide GluDB ( http://glaucomahq.info ) as repository of the genes and candidate genes associated with glaucoma and also their association with various diseases. The GluDB holding nearly 700 genes associated with glaucoma would serves as ecstasy resource for the eye researcher and can be a source for the development of novel therapeutics against glaucoma.

Myocilin (MYOC) was discovered more than 20 years ago and is the gene whose mutations are most commonly observed in individuals with glaucoma. Despite extensive research efforts, the function of WT MYOC has remained elusive, and how mutant MYOC is linked to glaucoma is unclear. Mutant MYOC is believed to be misfolded within the endoplasmic reticulum, and under normal physiological conditions misfolded MYOC should be retro-translocated to the cytoplasm for degradation. To better understand mutant MYOC pathology, we CRISPR-engineered a rat to have a MYOC Y435H substitution that is the equivalent of the pathological human MYOC Y437H mutation. Using this engineered animal model, we discovered that the chaperone αB-crystallin (CRYAB) is a MYOC-binding partner and that co-expression of these two proteins increases protein aggregates. Our results suggest that the misfolded mutant MYOC aggregates with cytoplasmic CRYAB and thereby compromises protein clearance mechanisms in trabecular meshwork cells, and this process represents the primary mode of mutant MYOC pathology. We propose a model by which mutant MYOC causes glaucoma, and we propose that therapeutic treatment of patients having a MYOC mutation may focus on disrupting the MYOC–CRYAB complexes.

Purpose Juvenile-onset open-angle glaucoma (JOAG) is an uncommon type of primary open-angle glaucoma that affects individuals during childhood and early adulthood. Pathogenic variants in the myocilin gene account for varying frequencies of primary open-angle glaucoma and JOAG cases in different populations. This study has screened and identified novel and previously identified myocilin variants in a north Indian cohort of JOAG patients. Methods Eighty unrelated JOAG cases and one hundred controls have been screened for MYOC variants by PCR and DNA sequencing of exons. Results DNA sequencing revealed seventeen different variants. Out of these variants, five (p.G122A, p.R136I, p.S173T, p.K216I, and p.R200KTer*15) were novel and registered in NCBI. Pathogenic MYOC variants identified in 7.5% of JOAG cases. Conclusion Pathogenic myocilin variants account for 7.5% of cases of JOAG in our patient’s cohort. This study augments the mutation spectrum of the MYOC gene, provides population-specific information, and aids in better understanding the underlying lesions of the disease.

Elevation of intraocular pressure (IOP) due to trabecular meshwork (TM) damage is associated with primary open-angle glaucoma (POAG). Myocilin mutations resulting in elevated IOP are the most common genetic causes of POAG. We have previously shown that mutant myocilin accumulates in the ER and induces chronic ER stress, leading to TM damage and IOP elevation. However, it is not understood how chronic ER stress leads to TM dysfunction and loss. Here, we report that mutant myocilin activated autophagy but was functionally impaired in cultured human TM cells and in a mouse model of myocilin-associated POAG (Tg-MYOCY437H). Genetic and pharmacological inhibition of autophagy worsened mutant myocilin accumulation and exacerbated IOP elevation in Tg-MYOCY437H mice. Remarkably, impaired autophagy was associated with chronic ER stress-induced transcriptional factor CHOP. Deletion of CHOP corrected impaired autophagy, enhanced recognition and degradation of mutant myocilin by autophagy, and reduced glaucoma in Tg-MYOCY437H mice. Stimulating autophagic flux via tat-beclin 1 peptide or torin 2 promoted autophagic degradation of mutant myocilin and reduced elevated IOP in Tg-MYOCY437H mice. Our study provides an alternate treatment strategy for myocilin-associated POAG by correcting impaired autophagy in the TM.

Ocular hypertension occurs due to increased resistance to aqueous humor removal through the conventional outflow pathway. Unlike the proximal region of the conventional outflow pathway, the distal region has not been well studied, mostly due to lack of model systems. Here we describe isolation and characterization of human primary vascular distal outflow pathway (VDOP) cells from the distal region of the conventional outflow pathway. Tissue from the distal region was isolated from human corneo-scleral rims, digested with collagenase type I (100 U/ml) and placed on gelatin coated plates to allow cellular growth in Dulbecco’s Modified Eagle’s Medium (low glucose) containing fetal bovine serum and antibiotic/antimycotic. VDOP cells showed consistent proliferation for up to 7 passages, retained endothelial-like nature of the parent tissues and showed a unique marker phenotype of Lectin+VEGFR2-CD34-NG2- that was distinct from neighboring trabecular meshwork (Lectin+VEGFR2-CD34-NG2+) and Schlemm’s canal (Lectin+VEGFR2+CD34+NG2+) cells. Dexamethasone treated VDOP cells did not express myocilin and did not form cross-linked actin networks, in contrast to trabecular meshwork cells. These data show that VDOP cells are unique to the distal outflow region and can be used as a viable in vitro model system to understand the biology of the distal outflow pathway and intraocular pressure regulation.

Simple Summary Myocilin is a protein with an incompletely understood function, mainly known because of its role in glaucoma. In this study we have analysed the normal role of this protein in vivo. To that end, we generated the first myocilin knockout zebrafish line reported to date. This zebrafish line did not show any apparent gross morphological anomaly, but unexpectedly, we observed that all knockout animals were males. Detailed analyses revealed the existence of apoptosis in the immature juvenile gonad, which is associated with male differentiation. Moreover, we demonstrate that adult knockout differentially expressed key genes involved both in male sex determination and the Wnt signalling pathway, which also plays a role in zebrafish gonad differentiation. Altogether, these results indicate that myocilin is a novel key protein involved in sex determination in zebrafish. Abstract Myocilin is a secreted glycoprotein with a poorly understood biological function and it is mainly known as the first glaucoma gene. To explore the normal role of this protein in vivo we developed a myoc knockout (KO) zebrafish line using CRISPR/Cas9 genome editing. This line carries a homozygous variant (c.236_239delinsAAAGGGGAAGGGGA) that is predicted to result in a loss-of-function of the protein because of a premature termination codon p.(V75EfsX60) that resulted in a significant reduction of myoc mRNA levels. Immunohistochemistry showed the presence of myocilin in wild-type embryonic (96 h post-fertilization) anterior segment eye structures and caudal muscles. The protein was also detected in different adult ocular and non-ocular tissues. No gross macroscopic or microscopic alterations were identified in the KO zebrafish, but, remarkably, we observed absence of females among the adult KO animals and apoptosis in the immature juvenile gonad (28 dpf) of these animals, which is characteristic of male development. Transcriptomic analysis showed that adult KO males overexpressed key genes involved in male sex determination and presented differentially expressed Wnt signalling genes. These results show that myocilin is required for ovary differentiation in zebrafish and provides in vivo support for the role of myocilin as a Wnt signalling pathway modulator. In summary, this myoc KO zebrafish line can be useful to investigate the elusive function of this protein, and it provides evidence for the unexpected function of myocilin as a key factor in zebrafish sex determination.

The purpose of the present study was to assess the differential intraocular pressure response (IOP) to dexamethasone (DEX) treatment at two dose levels (100 or 500 nM) in perfusion cultured Indian cadaveric eyes to investigate glucocorticoid (GC) responsiveness. In a human organ-cultured anterior segment (HOCAS) set-up, the eye pressure was monitored for every 24 h post DEX infusion (100 or 500 nM) or 0.1% ethanol treatment for 7 days after baseline stabilization. The expression of DEX-inducible proteins such as myocilin and fibronectin in HOCAS-TM tissues was assessed by immunostaining. Elevated IOP was observed in 6/16 eyes [Mean ± SEM (mΔIOP): 15.50 ± 1.96 mmHg; 37.5% responders] and 3/15 eyes (Mean ± SEM mΔIOP: 10 ± 0.84 mmHg; 20% responders) in 100 nM and 500 nM dose groups respectively. Elevated IOP in GC responder eyes was substantiated with a significant increase in myocilin (11.8-fold; p = 0.0002) and fibronectin (eightfold; p = 0.04) expression as compared to vehicle-treated eyes by immunofluorescence analysis. This is the first study reporting the GC responsiveness in Indian cadaveric eyes. The observed GC response rate was comparable with the previous studies and hence, this model will enable us to investigate the relationship between differential gene expression and individual GC responsiveness in our population.

Purpose To assess the clinical relevance of myocilin (MYOC) gene variants as risk factors for glaucoma in literature and to estimate their prevalence in different populations. Methods We reviewed the literature for published MYOC variants in glaucoma patients and estimated their prevalence in general population using gnomAD and BRAVO databases. We used several bioinformatics tools and the criteria of the American College of Medical Genetics and Genomics (ACMG) to assess the pathogenicity of the variants. We evaluated the carrier frequency of the variants in gnomAD, including its subpopulations. Results We found 13 missense and 5 loss-of-function (LOF) reported variants in MYOC that were both probable pathogenic or risk variants and listed in gnomAD. Six likely pathogenic missense variants were p.(Cys25Arg), p.(Gln48His), p.(Gly326Ser), p.(Thr353Ile), p.(Thr377Met) and p.(Gly399Val). They were most prevalent in East and South Asia (frequency, 0.92% and 0.81%, respectively). The most common missense variants were p.(Thr353Ile) (0.91% in East Asia) and p.(Gln48His) (0.79% in South Asia). Five LOF variants were p.(Arg46Ter), p.(Arg91Ter), p.(Arg272Ter), p.(Gln368Ter) and p.(Tyr453MetfsTer11). We considered these glaucoma risk variants. They were most prevalent in the East Asian and the Finnish population (0.93% and 0.33%, respectively). Conclusion Pathogenic MYOC variants appear to be population-associated. Our results highlight allelic heterogeneity of MYOC variants in open-angle glaucoma. Many of the probable pathogenic variants are over-represented in some of the populations causing doubt of their status as monogenic disease-causing variants.

Myocilin is a secreted glycoprotein with a poorly understood biological function and it is mainly known for its association with glaucoma. To explore the normal role of this proteinin vivowe developed amyocknockout (KO) zebrafish line using CRISPR/Cas9 genome editing. This line carries a homozygous variant (c.236_239delinsAAAGGGGAAGGGGA) that is predicted to result in a loss-of-function of the protein because of a premature termination codon p.(V75EfsX60) that resulted in a significant reduction ofmyocmRNA levels. Immunohistochemistry showed the presence of myocilin in wild-type embryonic (96 hours post-fertilization) anterior segment eye structures and caudal muscles. The protein was also detected in different adult ocular and non-ocular tissues. No gross macroscopic or microscopic alterations were identified in the KO zebrafish, but remarkably, we observed absence of females among the adult KO animals and apoptosis in the immature juvenile gonad (28 dpf) of these animals, which is characteristic of male development. Transcriptomic analysis showed that adult KO males overexpressed key genes involved in male sex determination and presented differentially expressed Wnt signalling genes. These results show that myocilin is required for ovary differentiation in zebrafish and providein vivosupport for the role of myocilin as a Wnt signalling pathway modulator. In summary, thismyocKO zebrafish line can be useful to investigate the elusive function of this protein, and it provides evidence for the unexpected function of myocilin as a key factor in zebrafish sex determination.

Glucocorticoid-induced glaucoma (GIG) is a chronic optic neuropathy caused by systemic or topical glucocorticoid (GC) treatment, which could eventually lead to permanent vision loss. To investigate the protective effects of rapamycin (RAP) on the trabecular cells during the development of GIG in mice, the effects of RAP on intraocular pressure (IOP), trabecular ultrastructure, and retinal ganglion cells (RGCs) were examined in C57BL/6J female mice treated with dexamethasone acetate (Dex-Ace). The expression of α-actin in trabecular tissue was detected by immunofluorescence, and the autophagic activity of trabecular cells and the expression of GIG-related myocilin and α-actin were detected by immunoblotting. Our results indicated that Dex-Ace significantly increased IOP at the end of the third week (p < 0.05), while RAP treatment neutralized this elevation of IOP by Dex-Ace. Dex-Ace treatment significantly decreased the RGC numbers (p < 0.05), while synchronous RAP treatment kept the number comparable to control. The outer sheath of elastic fibers became thicker and denser, and the mitochondria of lesions increased in Dex-Ace-treated groups at 4 weeks, while no significant change was observed in the RAP-treated trabecular tissues. Dex-Ace induced myocilin, α-actin, Beclin-1, and LC3-II/LC-I ratio, and lowered p62, while synchronous RAP treatment further activated autophagy and neutralized the induction of myocilin and α-actin. Our studies suggested that RAP protected trabecular meshwork cells by further inducing autophagy way from damages of GC treatment.

Previously, we have shown that Tfap2b, the gene encoding transcription factor AP-2β, is needed for normal mouse eye development. Specifically, targeted loss of Tfap2b in neural crest cells (NCCs)(1) and their derivatives, particularly the periocular mesenchyme (POM), resulted in anterior segment defects affecting the cornea and angle tissue. These defects were further associated with an increase in intraocular pressure (IOP). The present study investigates the underlying changes in embryonic and postnatal POM cell development and differentiation caused by loss of AP-2β in the NCCs, particularly in the structures that control aqueous outflow using Wnt1Cre(+/−); Tfap2b(-/lox); tdTomato(lox/+) mice (AP-2β neural crest cell knockout or AP-2β NCC KO). Toluidine blue-stained sections and ultrathin sections stained with uranyl acetate and lead citrate were used to assess morphology and ultrastructure, respectively. Immunohistochemistry of KO and control eyes was performed at embryonic day (E) 15.5, E18.5, postnatal day (P) 1, P7 and P14 using phospho-histone H3 (PH3), α-smooth muscle actin (αSMA), myocilin and endomucin antibodies, as well as a TUNEL assay. Conditional deletion of AP-2β in the NCC-derived POM resulted in defects that appeared during both embryogenesis and postnatal stages. Fate mapping of the knockout cells in the mutants revealed that the POM migrated appropriately into the eye during embryogenesis. However, during postnatal stages a significant reduction in POM proliferation in the angle region was observed in the mutants compared to controls. This was accompanied by a lack of expression of appropriate trabecular meshwork and Schlemm’s canal markers. This is the first study to show that AP-2β is required for development and differentiation of the trabecular meshwork and Schlemm’s canal. Together, these defects likely contributed to the elevated intraocular pressure (IOP) previously reported in the AP-2β NCC KO mice.

Glaucoma is a leading cause of irreversible blindness. In this study, we investigated if exogenous stem cells are able to rescue a glaucoma mouse model with transgenic myocilin Y437H mutation and explored the possible mechanisms. Human trabecular meshwork stem cells (TMSCs) were intracamerally transplanted which reduced mouse intraocular pressure, increased outflow facility, protected the retinal ganglion cells and preserved their function. TMSC transplantation also significantly increased the TM cellularity, promoted myocilin secretion from TM cells into the aqueous humor to reduce endoplasmic reticulum stress, repaired the TM tissue with extracellular matrix modulation and ultrastructural restoration. Co-culturing TMSCs with myocilin mutant TM cells in vitro promoted TMSCs differentiating into phagocytic functional TM cells. RNA sequencing revealed that TMSCs had upregulated genes related to TM regeneration and neuroprotection. Our results uncovered therapeutic potential of TMSCs for curing glaucoma and elucidated possible mechanisms by which TMSCs achieve the treatment effect.

Glaucoma is a complex disease caused by the combined effects of many genetic and environmental risk factors. Approximately 5% of glaucoma has a strong genetic basis, whereby a mutation in a single gene is responsible for disease. Many of these rare, single-gene forms of glaucoma frequently have an early age of onset and severe disease progression. Genetic research over the last few decades has led to the identification of three genes, myocilin, optineurin, and TBK1, that can cause glaucoma with little influence from other genes or the environment. Study of these genes has provided important insights into glaucoma pathogenesis and led to the discovery of specific biological pathways that are altered by disease. Furthermore, the study of disease-causing mutations lays the foundation for developing clinical genetic testing and mutation-directed therapies to improve clinical outcomes.

Contains fulltext : 218893.pdf (Publisher’s version ) (Closed access) Glaucoma, a disease characterized by progressive optic nerve degeneration, can be prevented through timely diagnosis and treatment. We characterize optic nerve photographs of 67,040 UK Biobank participants and use a multitrait genetic model to identify risk loci for glaucoma. A glaucoma polygenic risk score (PRS) enables effective risk stratification in unselected glaucoma cases and modifies penetrance of the MYOC variant encoding p.Gln368Ter, the most common glaucoma-associated myocilin variant. In the unselected glaucoma population, individuals in the top PRS decile reach an absolute risk for glaucoma 10 years earlier than the bottom decile and are at 15-fold increased risk of developing advanced glaucoma (top 10% versus remaining 90%, odds ratio = 4.20). The PRS predicts glaucoma progression in prospectively monitored, early manifest glaucoma cases (P = 0.004) and surgical intervention in advanced disease (P = 3.6 x 10(-)(6)). This glaucoma PRS will facilitate the development of a personalized approach for earlier treatment of high-risk individuals, with less intensive monitoring and treatment being possible for lower-risk groups.

OBJECTIVE: Mutations in myocilin (MYOC) may cause either juvenile open angle glaucoma (JOAG) or adult-onset primary open angle glaucoma (POAG). MYOC encodes a glycoprotein that is normally secreted from trabecular meshwork cells that regulate intraocular pressure. Prior in vitro, transgenic rodent, and organ culture experiments have suggested that abnormal accumulation of MYOC protein within trabecular meshwork cells is a key step in glaucoma pathophysiology. We investigated the pathogenesis of MYOC glaucoma by examining a donor eye from a patient with JOAG caused by a Tyr437His MYOC mutation. DESIGN: Case-control, immunohistochemical study of a donor eye from a patient with JOAG caused by a Tyr437His MYOC mutation and age-matched control donor eyes. SUBJECTS: An eye from a 59-year-old male with JOAG caused by a Tyr437His MYOC mutation and eyes from five donors (ages 51–66) with no known ocular disease were examined. METHODS: Frozen fixed sections of the iridocorneal angle were prepared from the donor eyes of the MYOC glaucoma patient and control eyes. We used antibodies directed against MYOC, collagen IV, and BiP/GRP78 as well as wheat germ agglutinin and concanavalin A lectins to localize MYOC protein in the trabecular meshwork. MAIN OUTCOME MEASURE: Qualitative comparison of MYOC protein labeling and localization in the trabecular meshwork of donor eyes from a glaucoma patient with a MYOC mutation and from control subjects. RESULTS: Using immunohistochemistry, we detected more abundant MYOC protein within the trabecular meshwork of the MYOC glaucoma patient’s eye than in control eyes. We further localized MYOC protein within the trabecular meshwork cells of the MYOC glaucoma patient’s eye by co-labeling with the endoplasmic reticulum (ER) marker GRP78 (BiP). Little to no MYOC was identified within the trabecular meshwork cells of control eyes. Minimal extracellular MYOC was detected in both MYOC glaucoma eyes and control eyes. CONCLUSIONS: This is the first histopathological analysis of an eye from a glaucoma patient with a MYOC mutation. Furthermore, this analysis supports our model of MYOC-associated glaucoma, in which MYOC mutations cause abnormal intracellular retention of MYOC within the ER of trabecular meshwork cells as a key step towards development of glaucoma.

The presence of myocilin is often used in the process of validating trabecular meshwork (TM) cells and eye tissues, but the antibody reagents used for detection are poorly characterized. Indeed, for over a century, researchers have been using antibodies to track proteins of interest in a variety of biological contexts, but many antibodies remain ill-defined at the molecular level and in their target epitope. Such issues have prompted efforts from major funding agencies to validate reagents and combat reproducibility issues across biomedical sciences. Here we characterize the epitopes recognized by four commercial myocilin antibodies, aided by structurally and biochemically characterized myocilin fragments. All four antibodies recognize enriched myocilin secreted from human TM cell media. The detection of myocilin fragments by ELISA and Western blot reveal a variety of epitopes across the myocilin polypeptide chain. A more precise understanding of myocilin antibody targets, including conformational specificity, should aid the community in standardizing protocols across laboratories and in turn, lead to a better understanding of eye physiology and disease.

Myocilin (MYOC) is a secreted protein found in human aqueous humor (AH) and mutations in the MYOC gene are the most common mutation observed in glaucoma patients. Human AH analyzed under non-reducing conditions suggests that MYOC is not normally found in a monomeric form, but rather is predominantly dimeric. Although MYOC was first reported almost 20 years ago, a technical challenge still faced by researchers is an inability to isolate full-length MYOC protein for experimental purposes. Herein we describe two methods by which to isolate sufficient quantities of human full-length MYOC protein from mammalian cells. One method involved identification of a cell line (HeLa S3) that would secrete full-length protein (15 mg/L) while the second method involved a purification approach from 293 cells requiring identification and modification of an internal MYOC cleavage site (Glu214/Leu215). MYOC protein yield from 293 cells was improved by mutation of two MYOC N-terminal cysteines (C47 and C61) to serines. Analytical size exclusion chromatography of our full-length MYOC protein purified from 293 cells indicated that it is predominantly dimeric and we propose a structure for the MYOC dimer. We hope that by providing methods to obtain MYOC protein, researchers will be able to utilize the protein to obtain new insights into MYOC biology. The ultimate goal of MYOC research is to better understand this target so we can help the patient that carries a MYOC mutation retain vision and maintain quality of life.

Mutant myocilin aggregation is associated with inherited open angle glaucoma, a prevalent optic neuropathy leading to blindness. Comprehension of mutant myocilin aggregation is of fundamental importance to glaucoma pathogenesis and ties glaucoma to amyloid diseases such as Alzheimer’s. Here we probe the aggregation properties of peptides derived from the myocilin olfactomedin domain. Peptides P1 (residues 326–337) and P3 (residues 426–442) were identified previously to form amyloid. Coarse-grained discontinuous molecular dynamics simulations using the PRIME20 force field (DMD/PRIME20) predict that P1 and P3 are aggregation-prone; P1 consistently forms fibrillar aggregates with parallel in-register β-sheets whereas P3 forms β-sheet-containing aggregates without distinct order. Natural abundance (13)C solid-state NMR spectra validate that aggregated P1 exhibits amyloid signatures and is less heterogeneous than aggregated P3. DMD/PRIME20 simulations provide a viable method to predict peptide aggregation propensities and aggregate’s structure/order which cannot be accessed by bioinformatics or readily attained experimentally.

Aim To identify the mutations of MYOC, OPTN, CYP1B1 and WDR36 in a large Chinese family affected by juvenile open angle glaucoma (JOAG). Methods Of 114 members of one family were recruited in this study. Blood samples from twelve members of this pedigree were collected for further research. As a control, 100 unrelated subjects were recruited from the same hospital. The exon and flanking intron sequences of candidate genes were amplified using the polymerase chain reaction and direct DNA sequencing. Results The proband (III:10) was a seventy-three years old woman with binocular JOAG at the age of 31. A recurrent heterozygous mutation (c.1099G>A) of MYOC was identified in the three JOAG patients and another suspect. This transition was located in the first base pair of codon 367 (GGA>AGA) in exon 3 of MYOC and was predicted to be a missense substitution of glycine to arginine (p.G367R) in myocilin. Mutations in OPTN, CYP1B1 or WDR36 were not detected in this study. The G367R mutation was not present in unaffected family members or in 100 ethnically matched controls. Other variants of the coding regions of candidate genes were not detected in all participants. To date, this family was the largest to have been identified as carrying a certain MYOC mutation in China, further evidence of a founder effect for the G367R MYOC mutant was provided by our data. Conclusion A MYOC c.1099G>A mutation in an autosomal dominant JOAG family is identified and the characteristic phenotypes among the patients are summarized. Genetic testing could be utilized in high-risk populations and be helpful not only for genetic counseling, but also for early diagnosis and treatment of affected patients or carriers of inherited JOAG.

AIM: To investigate the effect of myocilin on the expression of fibronectin(FN)and the adhesion function in trabecular meshwork cells of primary open angle glaucoma(POAG)in vitro. METHODS: The concentration of recombinant myocilin protein was 0(control group), 0.5, 1, 1.5, 2 and 2.5μg/mL, respectively. The expression of FN in the cells and the cell culture medium were detected by Western blot and ELISA. The effect of myocilin protein on the adhesion of cultured POAG trabecular meshwork cells was detected by CCK-8 method. RESULTS: Western blot showed that the ratio of FN/β-actin was 34.8±0.6, 33.4±1.0, 28.9±0.8, 21.6±0.9, 15.9±1.1 and 11.9±0.8; that of 0.5μg/mL group was not significantly different compared with that of control group(P=0.092); that of 1.0, 1.5, 2 and 2.5μg/mL group were significantly different compared with that of the control group(F=346.131, PP=0.120); the difference between of 1, 1.5, 2, 2.5μg/mL group and the control group were statistically significant(F=176.054, PP=0.300); the difference between of 1, 1.5, 2, 2.5μg/mL group and the control group were statistically significant(F=177.818, PCONCLUSION: Myocilin protein can inhibit the fibronectin expression in POAG trabecular meshwork cells, showing a dose dependent manner. Myocilin protein can reduce the adhesion of POAG trabecular meshwork cells.

Purpose The trabecular meshwork (TM) has an important role in the regulation of aqueous humor outflow and IOP. Regulation of the extracellular matrix (ECM) by TGFβ2 has been studied extensively. Bone morphogenetic protein (BMP) and activin membrane-bound inhibitor (BAMBI) has been shown to inhibit or modulate TGFβ2 signaling. We investigate the role of TGFβ2 and BAMBI in the regulation of TM ECM and ocular hypertension. Methods Mouse TM (MTM) cells were isolated from B6;129S1-Bambitm1Jian/J flox mice, characterized for TGFβ2 and dexamethasone (DEX)-induced expression of fibronectin, collagen-1, collagen-4, laminin, α-smooth muscle actin, cross-linked actin networks (CLANs) formation, and DEX-induced myocilin (MYOC) expression. MTM cells were transduced with Ad5.GFP to identify transduction efficiency. MTM cells and mouse eyes were transduced with Ad5.Null, Ad5.Cre, Ad5.TGFβ2, or Ad5.TGFβ2 + Ad5.Cre to evaluate the effect on ECM production, IOP, and outflow facility. Results MTM cells express TM markers and respond to DEX and TGFβ2. Ad5.GFP at 100 MOI had the highest transduction efficiency. Bambi knockdown by Ad5.Cre and Ad5.TGFβ2 increased fibronectin, collagen-1, and collagen-4 in TM cells in culture and tissue. Ad5.Cre, Ad5.TGFβ2, and Ad5.TGFβ2 + Ad5.Cre each significantly induced ocular hypertension and lowered aqueous humor outflow facility in transduced eyes. Conclusions We show for the first time to our knowledge that knockdown of Bambi alters ECM expression in cultured cells and mouse TM, reduces outflow facility, and causes ocular hypertension. These data provide a novel insight into the development of glaucomatous TM damage and identify BAMBI as an important regulator of TM ECM and ocular hypertension.

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.

Purpose : To explore newer computational approaches in the design of novel myocilin inhibitors for the treatment of glaucoma. Methods : An in-silico virtual screening technique based on simulation of molecular docking was utilised to design a novel myocilin inhibitors for the treatment of glaucoma. The designed novel molecules were theoretically evaluated to predict their pharmacokinetic properties and toxic effects. Lead molecules were screened out in virtual screening technique on the basis of low binding energies obtained in AutoDock based molecular docking simulation. Results : Out of ten top lead compounds screened, ZINC01729523 and ZINC04692015 were promising, having shown potent inhibition of myocilin, good pharmacokinetic properties and absence of any toxic effects. Conclusion : In-silico virtual screening of molecular libraries containing a large number of ligands is very useful for short-listing of potential lead molecules for further structure-based discovery of antiglaucoma-drugs. Keywords : Glaucoma, Myocilin, Docking, Virtual-screening, Autodock, Ligand, Drug design

Several studies have reported long-term dexamethasone application to cause glaucoma, with understanding of the underlying mechanism being incomplete. As Trabecular Meshwork (TM) cells and Retinal Ganglion Cells (RGCs) are primary targets for glaucomatous induction, studying the effect of dexamethasone on these cells becomes significant. The present work used an in vitro approach where Human Trabecular Meshwork (HTM) and RGC-5 cell lines were subjected to dexamethasone treatment and analysed. This study reports differential response of both types of cells towards same dexamethasone challenge, where HTM cells exhibited altered morphology, decreased cell survival, elevated reactive oxygen species (ROS) status along with increased expression of pro-apoptotic markers (p53, Bax) while RGC-5 cells did not show signs of stress prima facie. Since dexamethasone has been reported to increase intra-ocular pressure (IOP) in vivo thereby depleting nutrient supply to retinal neuronal cells, treatment of RGC-5 cells with serum free medium was performed. In this condition, RGC-5 cells exhibited glaucoma-associated stress features. Elevated expression of myocilin in both dexamethasone treated Human Trabecular Meshwork (HTM) cells and RGC-5 cells was observed. A significant upregulation of myocilin and downregulation of γ-synuclein were also observed in serum free medium treated RGC-5 cells. In conclusion, RGCs do not get affected directly by dexamethasone due to rich nutrient supply unlike HTM cells that are affected, as they exist in regions of less nutrient availability in vivo. Dexamethasone affects RGCs indirectly by depleting their nutrients. Thus, maintenance of rich nutrient supply to RGCs that are under dexamethasone administration may help prevent its damaging effect.

Recombinant antibodies with well-characterized epitopes and known conformational specificities are critical reagents to support robust interpretation and reproducibility of immunoassays across biomedical research. For myocilin, a protein prone to misfolding that is associated with glaucoma and an emerging player in other human diseases, currently available antibodies are unable to differentiate among the numerous disease-associated protein states. This fundamentally constrains efforts to understand the connection between myocilin structure, function, and disease. To address this concern, we used protein engineering methods to develop new recombinant antibodies that detect the N-terminal leucine zipper structural domain of myocilin and that are cross-reactive for human and mouse myocilin. After harvesting spleens from immunized mice and in vitro library panning, we identified two antibodies, 2A4 and 1G12. 2A4 specifically recognizes a folded epitope while 1G12 recognizes a range of conformations. We matured antibody 2A4 for improved biophysical properties, resulting in variant 2H2. In a human IgG1 format, 2A4, 1G12, and 2H2 immunoprecipitate full-length folded myocilin present in the spent media of human trabecular meshwork (TM) cells, and 2H2 can visualize myocilin in fixed human TM cells using fluorescence microscopy. These new antibodies should find broad application in glaucoma and other research across multiple species platforms.

Simple Summary MYOC is a secreted glycoprotein and it expresses at high levels in skeletal muscle cells. However, the function of MYOC in muscle is still unclear. Accordingly, in this study, we examined that MYOC expression increased gradually during C2C12 differentiation and it could promote the differentiation of C2C12. Furthermore, we demonstrated that MYOC could bind to CAV1. We further confirmed that CAV1 could positively regulate C2C12 differentiation through the TGF-β pathway. At last, we determined the relationship among MYOC, CAV1 and TGF-β. We found that MYOC promoted the differentiation of C2C12 cells by regulation of the TGF-β signaling pathways via CAV1. The present study is the first to demonstrate the mechanism of action of MYOC in C2C12 cells. It provides a novel method of exploring the mechanism of muscle differentiation and represents a potential novel method for the treatment of muscle diseases. Abstract Myocilin (MYOC) is a glycoprotein encoded by a gene associated with glaucoma pathology. In addition to the eyes, it also expresses at high transcription levels in the heart and skeletal muscle. MYOC affects the formation of the murine gastrocnemius muscle and is associated with the differentiation of mouse osteoblasts, but its role in the differentiation of C2C12 cells has not yet been reported. Here, MYOC expression was found to increase gradually during the differentiation of C2C12 cells. Overexpression of MYOC resulted in enhanced differentiation of C2C12 cells while its inhibition caused reduced differentiation. Furthermore, immunoprecipitation indicated that MYOC binds to Caveolin-1 (CAV1), a protein that influences the TGF-β pathway. Laser confocal microscopy also revealed the common sites of action of the two during the differentiation of C2C12 cells. Additionally, CAV1 was upregulated significantly as C2C12 cells differentiated, with CAV1 able to influence the differentiation of the cells. Furthermore, the Western blotting analysis demonstrated that the expression of MYOC affected the TGF-β pathway. Finally, MYOC was overexpressed while CAV1 was inhibited. The results indicate that reduced CAV1 expression blocked the promotion of C2C12 cell differentiation by MYOC. In conclusion, the results demonstrated that MYOC regulates TGF-β by influencing CAV1 to promote the differentiation of C2C12 cells.

Purpose Primary congenital glaucoma (PCG) accounts for 26–29% of childhood blindness in Egypt. The identification of disease causing mutations has not been extensively investigated. We aimed to examine the frequency of CYP1B1 and MYOC mutations in PCG Egyptian patients, and study a possible genotype/phenotype correlation. Methods Ninety-eight patients with PCG diagnosed at the Ophthalmology department of Alexandria Main University Hospital were enrolled. Demographic and phenotypic characteristics were recorded. Patients and 100 healthy subjects (control group) were screened for two mutations in CYP1B1 gene (G61E, R368H) and one mutation in MYOC gene (Gln48His) using polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP). Phenotypic characteristics pertaining to disease severity were compared. Results Nineteen patients (19%) with PCG were found positive for one or more of the mutations screened for. Seven patients (7%) were homozygous for the G61E mutation. Ten patients (10%) were heterozygous; 6 for the G61E mutation, 2 for the R368H mutation and 2 for the Gln48His mutation. Two patients (2%) were double heterozygotes harboring a R368H as well as a Gln48His mutation. The most common mutation observed was the G61E in 13 patients; 7 homozygotes and 6 heterozygotes for the mutation. The control group were negative for all mutations screened for. No significant correlations between the mutations and phenotype severity were detected. A statistically significant positive correlation however was found between the different mutations and each of the IOP and the cup/disk ratio. Conclusion The current study further endorses the role of CYP1B1 mutations in the etiology of PCG among Egyptian patients and is the first study to report MYOC gene mutation in Egyptian patients with PCG.

Glucocorticoid (GC)-induced ocular hypertension (OHT) is a serious adverse effect of prolonged GC therapy that can lead to iatrogenic glaucoma and permanent vision loss. An appropriate mouse model can help us understand precise molecular mechanisms and etiology of GC-induced OHT. We therefore developed a novel, simple, and reproducible mouse model of GC-induced OHT. GC-induced myocilin expression in the trabecular meshwork (TM) has been suggested to play an important role in GC-induced OHT. We further determined whether myocilin contributes to GC-OHT. C57BL/6J mice received weekly periocular conjunctival fornix injections of a dexamethasone-21-acetate (DEX-Ac) formulation. Intraocular pressure (IOP) elevation was relatively rapid and significant, and correlated with reduced conventional outflow facility. Nighttime IOPs were higher in ocular hypertensive eyes compared to daytime IOPs. DEX-Ac treatment led to increased expression of fibronectin, collagen I, and α-smooth muscle actin in the TM in mouse eyes. No changes in body weight indicated no systemic toxicity associated with DEX-Ac treatment. Wild-type mice showed increased myocilin expression in the TM on DEX-Ac treatment. Both wild-type and Myoc −/− mice had equivalent and significantly elevated IOP with DEX-Ac treatment every week. In conclusion, our mouse model mimics many aspects of GC-induced OHT in humans, and we further demonstrate that myocilin does not play a major role in DEX-induced OHT in mice.

Glaucoma is a very common disorder of the eye wherein the disturbance of the structural or functional integrity of the optic nerve causes characteristic atrophic changes in the optic nerve, which may lead to specific visual field defects over time. Primary open angle glaucoma (POAG) is most frequent among the three principle glaucoma subtypes. With well-established role of genes like Myocilin (MYOC), Optineurin (OPTN) and WD repeat Domain 36, (WDR36), at least 29 genetic loci have been found till date to be linked to POAG. Moreover, association studies have found 66 loci with 76 genes associated to POAG till date with conflicting results. This particular study is to summarize the current knowledge regarding the change in glaucoma prevalence worldwide and in India from 1993 onwards and compiles all the studied genes that are involved in POAG pathogenesis in Indian population.

Background: Glaucoma is a major cause of irreversible blindness worldwide. There is evidence showing that a subset of the disease is genetically determined. In this study, we screened for mutations in chromosome 1q-linked open-angle glaucoma (GLC1A) in a Chinese family with primary open-angle glaucoma (POAG). Methods: A total of 23 members from five generations of a family were enrolled and underwent thorough ophthalmologic examinations. In addition, 200 unrelated healthy Chinese controls were also recruited as normal control. GLC1A gene was amplified by polymerase chain reaction, and DNA sequencing was performed to screen for mutations. Results: Six members were diagnosed as POAG, with severe clinical manifestations, and history of high intraocular pressures. The mean age of disease onset was 26.3 years. However, the others were asymptomatic. In six affected and three asymptomatic members, gene sequencing revealed a mutation c.C1456T in exon 3 of myocilin gene (MYOC). Furthermore, we also identified a novel mutation c.G322A in beta-1,4-galactosyltransferase 3 (B4GALT3) gene in all six affected and three asymptomatic members, which was not reported previously in POAG patients. The two newly identified variants were absent in other family members as well as controls. Conclusion: The mutations c.1456C

Skeletal muscle wasting is a devastating consequence of cancer that contributes to increased complications and poor survival, but is not well understood at the molecular level. Herein, we investigated the role of Myocilin (Myoc), a skeletal muscle hypertrophy-promoting protein that we showed is downregulated in multiple mouse models of cancer cachexia. Loss of Myoc alone was sufficient to induce phenotypes identified in mouse models of cancer cachexia, including muscle fiber atrophy, sarcolemmal fragility, and impaired muscle regeneration. By 18 months of age, mice deficient in Myoc showed significant skeletal muscle remodeling, characterized by increased fat and collagen deposition compared with wild-type mice, thus also supporting Myoc as a regulator of muscle quality. In cancer cachexia models, maintaining skeletal muscle expression of Myoc significantly attenuated muscle loss, while mice lacking Myoc showed enhanced muscle wasting. Furthermore, we identified the myocyte enhancer factor 2 C (MEF2C) transcription factor as a key upstream activator of Myoc whose gain of function significantly deterred cancer-induced muscle wasting and dysfunction in a preclinical model of pancreatic ductal adenocarcinoma (PDAC). Finally, compared with noncancer control patients, MYOC was significantly reduced in skeletal muscle of patients with PDAC defined as cachectic and correlated with MEF2c. These data therefore identify disruptions in MEF2c-dependent transcription of Myoc as a novel mechanism of cancer-associated muscle wasting that is similarly disrupted in muscle of patients with cachectic cancer. Significance: This work identifies a novel transcriptional mechanism that mediates skeletal muscle wasting in murine models of cancer cachexia that is disrupted in skeletal muscle of patients with cancer exhibiting cachexia.

Primary open-angle glaucoma (POAG) is the leading cause of irreversible blindness worldwide. Most cases are multifactorial in etiology, but some are associated with variants in the myocilin gene, MYOC. Here, we report the identification of a novel MYOC variant, c.1153G>A, in a 24-yr-old female patient with a personal and family history of juvenile/early-onset POAG. Further genetic testing within her family demonstrated that this variant segregates with the POAG phenotype in an autosomal dominant pattern. Identification of this MYOC variant in multiple affected relatives provides evidence for its pathogenicity, supporting previous findings linking MYOC mutations, in particular in the third exon's olfactomedin domain, to juvenile-onset POAG. This case also emphasizes the potential value of genetic testing in families with histories of eye disorders.

Regeneration In glaucoma, intraocular pressure damages innervation from the eye, resulting in irreversible blindness. The most common form of glaucoma is primary open-angle glaucoma (POAG), characterized by deterioration of the trabecular meshwork (TM) cells that encircles the base of the cornea. When Xiong et al. transplanted exogenous human trabecular meshwork stem cells (TMSCs) into a POAG mouse model, aqueous humor drained normally, intraocular pressed was reduced and the function of the retinal ganglion cells was preserved. TMSC transplantation also increased TM cellularity and promoted myocilin secretion, which reduced endoplasmic reticulum stress in the cells. These signs of TM tissue repair show promise for use of stem cells in the development of clinical treatments for glaucoma. eLife 10 , e63677 (2021).

Background Genome-wide association studies (GWAS) have identified association of common alleles with primary open-angle glaucoma (POAG) and its quantitative endophenotypes near numerous genes. This study aims to determine whether rare pathogenic variants in these disease-associated genes contribute to POAG. Methods Participants fulfilled strict inclusion criteria of advanced POAG at a young age of diagnosis. Myocilin mutation carriers were excluded using direct sequencing. Whole exome sequencing was performed on 187 glaucoma cases and 103 local screened nonglaucoma controls then joint-called with exomes of 993 previously sequenced Australian controls. GWAS-associated genes were assessed for enrichment of rare predicted pathogenic variants in POAG. Significantly enriched genes were compared against Exome Aggregation Consortium (ExAC) public control. Results Eighty-six GWAS disease or trait-associated glaucoma genes were captured and sequenced. CARD10 showed enrichment after Bonferroni correction for rare variants in glaucoma cases (OR = 13.2, P = 6.94 9 10�5) with mutations identified in 4.28% of our POAG cohort compared to 0.27% in controls. CARD10 was significantly associated with optic disc parameters in previous GWAS. The whole GWAS gene set showed no enrichment in POAG overall (OR = 1.12,P = 0.51). Conclusion We report here an enrichment of rare predicted pathogenic coding variants within a GWAS-associated locus in POAG (CARD10). These findings indicate that both common and rare pathogenic coding variants in CARD10 may contribute to POAG pathogenesis.