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Glaucoma is chronic optic neuropathy whose pathogenesis has been associated with the altered metabolism of Trabecular Meshwork Cells, which is a cell type involved in the synthesis and remodeling of the trabecular meshwork, the main drainage pathway of the aqueous humor. Starting from previous findings supporting altered ubiquitin signaling, in this study, we investigated the ubiquitin-mediated turnover of myocilin (MYOC/TIGR gene), which is a glycoprotein with a recognized role in glaucoma pathogenesis, in a human Trabecular Meshwork strain cultivated in vitro in the presence of dexamethasone. This is a validated experimental model of steroid-induced glaucoma, and myocilin upregulation by glucocorticoids is a phenotypic marker of Trabecular Meshwork strains. Western blotting and native-gel electrophoresis first uncovered that, in the presence of dexamethasone, myocilin turnover by proteasome particles was slower than in the absence of the drug. Thereafter, co-immunoprecipitation, RT-PCR and gene-silencing studies identified STUB1/CHIP as a candidate E3-ligase of myocilin. In this regard, dexamethasone treatment was found to downregulate STUB1/CHIP levels by likely promoting its proteasome-mediated turnover. Hence, to strengthen the working hypothesis about global alterations of ubiquitin-signaling, the first profiling of TMCs ubiquitylome, in the presence and absence of dexamethasone, was here undertaken by diGLY proteomics. Application of this workflow effectively highlighted a robust dysregulation of key pathways (e.g., phospholipid signaling, β-catenin, cell cycle regulation) in dexamethasone-treated Trabecular Meshwork Cells, providing an ubiquitin-centered perspective around the effect of glucocorticoids on metabolism and glaucoma pathogenesis. [ABSTRACT FROM AUTHOR]

Trabecular meshwork (TM) tissue is highly specialized, and its structural integrity is crucial for maintaining homeostatic intraocular pressure (IOP). The administration of glucocorticoids, such as dexamethasone (DEX), can perturb the TM structure and significantly increase IOP in susceptible individuals, resulting in ocular diseases such as steroid-induced glaucoma, a form of open-angle glaucoma. Although the exact mechanism involved in steroid-induced glaucoma remains elusive, increasing evidence suggests that DEX may act through various signaling cascades in TM cells. Despite uncertainty surrounding the specific process by which steroid-induced glaucoma occurs, there is growing evidence to indicate that DEX can impact multiple signaling pathways within TM cells. In this study, we examined the impact of DEX treatment on the Wnt signaling pathway in TM cells, given that Wnt signaling has been reported to play a crucial role in regulating extracellular matrix (ECM) levels in the TM. To further elucidate the role of Wnt signaling in the glaucomatous phenotype, we examined mRNA expression patterns between Wnt signaling markers AXIN2 and sFRP1 and DEX-mediated induction of myocilin (MYOC) mRNA and protein levels over 10 days in DEX-treated primary TM cells. We observed a sequential pattern of peak expression between AXIN2, sFRP1, and MYOC. Based on the study, we propose that sFRP1 upregulation could be a result of a negative feedback mechanism generated by stressed TM cells to suppress abnormal Wnt signaling activities.

Glaucoma is chronic optic neuropathy whose pathogenesis has been associated with the altered metabolism of Trabecular Meshwork Cells, which is a cell type involved in the synthesis and remodeling of the trabecular meshwork, the main drainage pathway of the aqueous humor. Starting from previous findings supporting altered ubiquitin signaling, in this study, we investigated the ubiquitin-mediated turnover of myocilin (MYOC/TIGR gene), which is a glycoprotein with a recognized role in glaucoma pathogenesis, in a human Trabecular Meshwork strain cultivated in vitro in the presence of dexamethasone. This is a validated experimental model of steroid-induced glaucoma, and myocilin upregulation by glucocorticoids is a phenotypic marker of Trabecular Meshwork strains. Western blotting and native-gel electrophoresis first uncovered that, in the presence of dexamethasone, myocilin turnover by proteasome particles was slower than in the absence of the drug. Thereafter, co-immunoprecipitation, RT-PCR and gene-silencing studies identified STUB1/CHIP as a candidate E3-ligase of myocilin. In this regard, dexamethasone treatment was found to downregulate STUB1/CHIP levels by likely promoting its proteasome-mediated turnover. Hence, to strengthen the working hypothesis about global alterations of ubiquitin-signaling, the first profiling of TMCs ubiquitylome, in the presence and absence of dexamethasone, was here undertaken by diGLY proteomics. Application of this workflow effectively highlighted a robust dysregulation of key pathways (e.g., phospholipid signaling, β-catenin, cell cycle regulation) in dexamethasone-treated Trabecular Meshwork Cells, providing an ubiquitin-centered perspective around the effect of glucocorticoids on metabolism and glaucoma pathogenesis.

Deep neural network-based programs can be applied to protein structure modeling by inputting amino acid sequences. Here, we aimed to evaluate the AlphaFold2-modeled myocilin wild-type and variant protein structures and compare to the experimentally determined protein structures. Molecular dynamic and ligand binding properties of the experimentally determined and AlphaFold2-modeled protein structures were also analyzed. AlphaFold2-modeled myocilin variant protein structures showed high similarities in overall structure to the experimentally determined mutant protein structures, but the orientations and geometries of amino acid side chains were slightly different. The olfactomedin-like domain of the modeled missense variant protein structures showed fewer folding changes than the nonsense variant when compared to the predicted wild-type protein structure. Differences were also observed in molecular dynamics and ligand binding sites between the AlphaFold2-modeled and experimentally determined structures as well as between the wild-type and variant structures. In summary, the folding of the AlphaFold2-modeled MYOC variant protein structures could be similar to that determined by the experiments but with differences in amino acid side chain orientations and geometries. Careful comparisons with experimentally determined structures are needed before the applications of the in silico modeled variant protein structures. [ABSTRACT FROM AUTHOR]

Purpose: To identify germline variants in myocilin (MYOC) and other known monogenic glaucoma genes in Finnish patients with juvenile open‐angle glaucoma (JOAG). Methods: Finnish patients with JOAG treated between 2010 and 2018 at the Department of Ophthalmology, Helsinki University Hospital, Finland, were enrolled. We sequenced all exonic regions and flanking splice sites of MYOC for five patients and one healthy relative using Sanger sequencing. In 48 patients, we performed exome sequencing to identify variants also in 28 other glaucoma‐related genes. Results: Fifty‐three individuals with JOAG from 50 pedigrees, and one healthy relative, participated. The mean age at diagnosis was 30.8 years [SD 7.6; range 11 to 39]. Five probands had probably pathogenic variants in MYOC: c.1102C>T p.(Gln368Ter), c.1109C>T p.(Pro370Leu), c.1130C>T p.(Thr377Met), c.1132G>A p.(Asp378Asn) and c.1456C>T p.(Leu486Phe). Four of these patients had a family history of dominantly inherited JOAG. The frequency of MYOC variants was 10% (5 of 50 families). One patient and his mother with JOAG had a novel loss‐of‐function variant in the FOXC1 gene, c.366G>A p.(Trp122Ter). A patient with sporadic JOAG had a homozygous likely pathogenic variant in the LTBP2 gene, c.3938G>A p.(Cys1313Tyr). The genetic variants explained 14% (7 out of 50 families; 95% CI, 6%–23%) of JOAG in our cohort. Conclusions: The frequency of pathogenic variants in previously known glaucoma‐associated genes is low in Finnish patients with JOAG. Because of the distinct genetic background of Finns, it might be possible to identify novel glaucoma genes through our JOAG series in the future. [ABSTRACT FROM AUTHOR]

The trabecular meshwork (TM) is the leading site of aqueous humor outflow in the eye and plays a critical role in maintaining normal intraocular pressure. When the TM fails to maintain normal intraocular pressure, glaucoma may develop. Mitochondrial damage has previously been found in glaucomatous TM cells; however, the precise metabolic activity of glaucomatous TM cells has yet to be quantitatively assessed. Using dexamethasone (Dex) treated primary human TM cells to model glaucomatous TM cells, we measure the respiratory and glycolytic activity of Dex-treated TM cells with an extracellular flux assay. We found that Dex-treated TM cells had quantifiably altered metabolic profiles, including increased spare respiratory capacity and ATP production rate from oxidative phosphorylation. Therefore, we propose that reversing or preventing these metabolic changes may represent an avenue for future research.

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.

Purpose: This study focused on the genetic screening of Myocilin (MYOC), Cytochrome P450 family 1 subfamily B member 1 (CYP1B1), Optineurin (OPTN), and SIX homeobox 6 (SIX6) genes in a family with coexistence of primary congenital glaucoma (PCG) and juvenile open-angle glaucoma (JOAG). Methods: Sanger sequencing was used to examine the coding region of all four genes. Six different online available algorithms were used for the pathogenicity prediction of missense variant. Structural analysis was done using Garnier-Osguthorpe-Robson (GOR), PyMol, ChimeraX, and Molecular Dynamic (MD) Simulations (using Graphics Processing Unit (GPU)-enabled Desmond module of Schrödinger). Results: There were a total of three sequence variants within the family. All seven algorithms determined that a single mutation, G538E, in the OPTN gene is pathogenic. The loops connecting the strands became more flexible, as predicted structurally and functionally by pathogenic mutations. Mutations create perturbations and conformational rearrangements in proteins, hence impairing their functioning. Conclusion: In this study, we describe a North Indian family in which members were having JOAG and PCG due to a rare homozygous/heterozygous mutation in OPTN. The coexistence of two types of glaucoma within a single pedigree suggests that certain OPTN mutations may be responsible for the onset of different glaucoma phenotypes. [ABSTRACT FROM AUTHOR]

To investigate the characteristics of mutation myocilin proteins and glaucoma pathological phenotype in transgenic mice with full-length human Pro370Leu mutant myocilin gene (Tg-MYOCP370L). Tg-MYOCP370L mice were established using the CRISPR/Cas9 system. Long-term intraocular pressure (IOP) was measured, myocilin protein expressions in anterior chamber angle, retina, optic nerve tissues and aqueous humor were detected by western blot. RBPMS, myocilin, Iba-1 and GFAP expression were visualized by immunofluorescence. H&E staining was applied to assess the ocular angle and retinal morphology. Aqueous humor dynamics were visualized by Gadolinium magnetic resonance imaging (Gd-MRI). TUNEL assay was used to evaluate the specific cell apoptosis in trabecular meshwork and retina. Optomotor and electroretinography tests were employed to evaluate the visual function in Tg-MYOCP370L and wild-type (WT) mice. Homozygous myocilin mutation at position 503 (C > T) was identified by PCR and sequencing in Tg-MYOCP370L mice. Myocilin protein expression was overexpressed in eye tissues of Tg-MYOCP370L mice with reduced myocilin secretion in aqueous humor. H&E staining showed normal histological morphology of anterior chamber angle whereas decreased thickness and nuclei in ganglion cell layer were found (P < 0.05). Gd signals were significantly increased in the anterior chamber of Tg-MYOCP370L compared with WT eyes (P < 0.05). IOP was elevated in Tg-MYOCP370L mice starting at 5 months of age, with significant RGC loss (P < 0.05). Upregulation of caspase-3 and caspase-9 expressions and increased TUNEL-positive cells were found in eyes of Tg-MYOCP370L mice. Excessive activation of retinal glial cells and impaired visual function were detected in Tg-MYOCP370L mice. Tg-MYOCP370L mice can induce the phenotype of open-angle glaucoma, featured as IOP elevation, activated retinal glial cells, loss of RGCs and impaired visual function. These pathologic changes may arise from the abnormal mutant myocilin protein accumulation in the trabecular meshwork and injured aqueous humor drainage. Therefore, Tg-MYOCP370L mice model can serve as an effective animal model for glaucoma research, especially for glaucoma-associated myocilin mutation studies. [ABSTRACT FROM AUTHOR]

Glaucoma, where increased intraocular pressure (IOP) leads to damage to the optic nerve and loss of sight, is amongst the foremost causes of irreversible blindness worldwide. In primary open angle glaucoma, the increased IOP is a result of the malfunctioning human trabecular meshwork (HTM) cells' inability to properly regulate the outflow of aqueous humor from the eye. A potential future treatment for glaucoma is to replace damaged HTM cells with a tissue-engineered substitute, thus restoring proper fluid outflow. Polycaprolactone (PCL) is a versatile, biodegradable, and implantable material that is widely used for cell culture and tissue engineering. In this work, PCL scaffolds were lithographically fabricated using a sacrificial process to produce submicron-thick scaffolds with openings of specific sizes and shapes (e.g., grid, hexagonal pattern). The HTM cell growth on gelatin-coated PCL scaffolds was assessed by scanning electron microscopy, tetrazolium metabolic activity assay, and cytoskeletal organization of F-actin. Expression of HTM-specific markers and ECM deposition were assessed by immunocytochemistry and qPCR analysis. Gelatin-coated, micropatterned, ultrathin, porous PCL scaffolds with a grid pattern supported proper HTM cell growth, cytoskeleton organization, HTM-marker expression, and ECM deposition, demonstrating the feasibility of using these PCL scaffolds to tissue-engineer implantable, healthy ocular outflow tissue. [ABSTRACT FROM AUTHOR]

Glaucoma is a series of linked optic diseases resulting in progressive vision loss and total blindness due to the acquired loss of retinal ganglion cells. This harm to the optic nerve results in visual impairment and, ultimately, total blindness if left untreated. Primary open-angle glaucoma (POAG) is the most frequent variety within the large family of glaucoma. It is a multifaceted and heterogeneous condition with several environmental and genetic variables aiding in its etiology. By 2040, there will be 111.8 million glaucoma patients globally, with Asia and Africa accounting for the vast majority. The goal of this review is to elaborate on the role of genes (nuclear and mitochondrial) as well as their variants in the pathogenesis of POAG. PubMed and Google Scholar databases were searched online for papers until September 2022. Prevalence and inheritance patterns vary significantly across different ethnic and geographic populations. Numerous causative genetic loci may exist; however, only a few have been recognized and characterized. Further investigation into the genetic etiology of POAG is expected to uncover novel and intriguing causal genes, allowing for a more precise pathogenesis pattern of the disease. [ABSTRACT FROM AUTHOR]

Purpose: This study focused on the genetic screening of Myocilin (MYOC), Cytochrome P450 family 1 subfamily B member 1 (CYP1B1), Optineurin (OPTN), and SIX homeobox 6 (SIX6) genes in a family with coexistence of primary congenital glaucoma (PCG) and juvenile open-angle glaucoma (JOAG). Methods: Sanger sequencing was used to examine the coding region of all four genes. Six different online available algorithms were used for the pathogenicity prediction of missense variant. Structural analysis was done using Garnier–Osguthorpe–Robson (GOR), PyMol, ChimeraX, and Molecular Dynamic (MD) Simulations (using Graphics Processing Unit (GPU)-enabled Desmond module of Schrödinger). Results: There were a total of three sequence variants within the family. All seven algorithms determined that a single mutation, G538E, in the OPTN gene is pathogenic. The loops connecting the strands became more flexible, as predicted structurally and functionally by pathogenic mutations. Mutations create perturbations and conformational rearrangements in proteins, hence impairing their functioning. Conclusion: In this study, we describe a North Indian family in which members were having JOAG and PCG due to a rare homozygous/heterozygous mutation in OPTN. The coexistence of two types of glaucoma within a single pedigree suggests that certain OPTN mutations may be responsible for the onset of different glaucoma phenotypes.

Glaucoma is a series of linked optic diseases resulting in progressive vision loss and total blindness due to the acquired loss of retinal ganglion cells. This harm to the optic nerve results in visual impairment and, ultimately, total blindness if left untreated. Primary open-angle glaucoma (POAG) is the most frequent variety within the large family of glaucoma. It is a multifaceted and heterogeneous condition with several environmental and genetic variables aiding in its etiology. By 2040, there will be 111.8 million glaucoma patients globally, with Asia and Africa accounting for the vast majority. The goal of this review is to elaborate on the role of genes (nuclear and mitochondrial) as well as their variants in the pathogenesis of POAG. PubMed and Google Scholar databases were searched online for papers until September 2022. Prevalence and inheritance patterns vary significantly across different ethnic and geographic populations. Numerous causative genetic loci may exist; however, only a few have been recognized and characterized. Further investigation into the genetic etiology of POAG is expected to uncover novel and intriguing causal genes, allowing for a more precise pathogenesis pattern of the disease.

Deep neural network-based programs can be applied to protein structure modeling by inputting amino acid sequences. Here, we aimed to evaluate the AlphaFold2-modeled myocilin wild-type and variant protein structures and compare to the experimentally determined protein structures. Molecular dynamic and ligand binding properties of the experimentally determined and AlphaFold2-modeled protein structures were also analyzed. AlphaFold2-modeled myocilin variant protein structures showed high similarities in overall structure to the experimentally determined mutant protein structures, but the orientations and geometries of amino acid side chains were slightly different. The olfactomedin-like domain of the modeled missense variant protein structures showed fewer folding changes than the nonsense variant when compared to the predicted wild-type protein structure. Differences were also observed in molecular dynamics and ligand binding sites between the AlphaFold2-modeled and experimentally determined structures as well as between the wild-type and variant structures. In summary, the folding of the AlphaFold2-modeled MYOC variant protein structures could be similar to that determined by the experiments but with differences in amino acid side chain orientations and geometries. Careful comparisons with experimentally determined structures are needed before the applications of the in silico modeled variant protein structures.

Pedigree analysis showed that a large proportion of Leber hereditary optic neuropathy (LHON) family members who carry a mitochondrial risk variant never lose vision. Mitochondrial haplotype appears to be a major factor influencing the risk of vision loss from LHON. Mitochondrial variants, including m.14484T>C and m.11778G>A, have been added to gene arrays, and thus many patients and research participants are tested for LHON mutations. Analysis of the UK Biobank and Australian cohort studies found more than 1 in 1,000 people in the general population carry either the m.14484T>C or the m.11778G>A LHON variant. None of the subset of carriers examined had visual acuity at 20/200 or worse, suggesting a very low penetrance of LHON. Haplogroup analysis of m.14484T>C carriers showed a high rate of haplogroup U subclades, previously shown to have low penetrance in pedigrees. Penetrance calculations of the general population are lower than pedigree calculations, most likely because of modifier genetic factors. This Matters Arising Response paper addresses the Watson et al. (2022) Matters Arising paper, published concurrently in The American Journal of Human Genetics. [ABSTRACT FROM AUTHOR]

Myocilin is an enigmatic glaucoma-associated glycoprotein whose biological role remains incompletely understood. To gain novel insight into its normal function, we used transposon-mediated transgenesis to generate the first zebrafish line stably overexpressing myocilin [Tg(actb1:myoc-2A-mCherry)]. qPCR showed an approximately four-fold increased myocilin expression in transgenic zebrafish embryos (144 hpf). Adult (13 months old) transgenic animals displayed variable and age-dependent ocular anterior segment alterations. Almost 60% of two-year-old male, but not female, transgenic zebrafish developed enlarged eyes with severe asymmetrical and variable abnormalities in the anterior segment, characterized by corneal limbus hypertrophy, and thickening of the cornea, iris, annular ligament and lens capsule. The most severe phenotype presented small or absent ocular anterior chamber and pupils, due to iris overgrowth along with dysplastic retinal growth and optic nerve hypertrophy. Immunohistochemistry revealed increased presence of myocilin in most altered ocular tissues of adult transgenic animals, as well as signs of retinal gliosis and expanded ganglion cells and nerve fibers. The preliminary results indicate that these cells contributed to retinal dysplasia. Visual impairment was demonstrated in all old male transgenic zebrafish. Transcriptomic analysis of the abnormal transgenic eyes identified disrupted expression of genes involved in lens, muscular and extracellular matrix activities, among other processes. In summary, the developed transgenic zebrafish provides a new tool to investigate this puzzling protein and provides evidence for the role of zebrafish myocilin in ocular anterior segment and retinal biology, through the influence of extracellular matrix organization and cellular proliferation. [ABSTRACT FROM AUTHOR]

Trabecular meshwork (TM) tissue is highly specialized, and its structural integrity is crucial for maintaining homeostatic intraocular pressure (IOP). The administration of glucocorticoids, such as dexamethasone (DEX), can perturb the TM structure and significantly increase IOP in susceptible individuals, resulting in ocular diseases such as steroid-induced glaucoma, a form of open-angle glaucoma. Although the exact mechanism involved in steroid-induced glaucoma remains elusive, increasing evidence suggests that DEX may act through various signaling cascades in TM cells. Despite uncertainty surrounding the specific process by which steroid-induced glaucoma occurs, there is growing evidence to indicate that DEX can impact multiple signaling pathways within TM cells. In this study, we examined the impact of DEX treatment on the Wnt signaling pathway in TM cells, given that Wnt signaling has been reported to play a crucial role in regulating extracellular matrix (ECM) levels in the TM. To further elucidate the role of Wnt signaling in the glaucomatous phenotype, we examined mRNA expression patterns between Wnt signaling markers AXIN2 and sFRP1 and DEX-mediated induction of myocilin (MYOC) mRNA and protein levels over 10 days in DEX-treated primary TM cells. We observed a sequential pattern of peak expression between AXIN2, sFRP1, and MYOC. Based on the study, we propose that sFRP1 upregulation could be a result of a negative feedback mechanism generated by stressed TM cells to suppress abnormal Wnt signaling activities.

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 3rd 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. [ABSTRACT FROM AUTHOR]

A lack of sufficient functional information exists for appropriately categorizing a large number of myocilin (MYOC) variants and their involvement in primary open angle glaucoma, hindering their clinical significance classification. Most glaucoma-causing MYOC mutations result in protein non-secretion and intracellular insoluble aggregate formation in cultured cells. Herein, we generated a Gaussia luciferase-based MYOC fusion protein to quickly and sensitively track the secretion of MYOC variants and compared these results to the better-established western blotting assay for MYOC. Fourteen clinically-derived MYOC variants with varying degrees of predicted pathogenicity were transfected into HEK-293A cells and analyzed by either a luciferase assay or western blotting. Eight of the variants (G12R, V53A, T204T, P254L, T325T, D380H, D395_E396insDP, and P481S) had not been biochemically assessed previously. Of these, P254L and D395_E396insDP demonstrated significant secretion defects reminiscent of glaucoma-causing mutations. The luciferase assay results agreed with western blotting for thirteen of the fourteen variants (93%), suggesting a strong concordance. These results suggest that the Gaussia luciferase assay may be used as a complementary or standalone assay for quickly assessing MYOC variant behavior and we anticipate that these results will be useful in MYOC variant curation and reclassification. [ABSTRACT FROM AUTHOR]

Trabecular meshwork dysfunction is the main cause of primary open angle glaucoma (POAG) associated with elevated intraocular pressure (IOP). Mutant myocilin causes glaucoma mainly via elevating IOP. Previously we have found that accumulated Asn 450 Tyr (N450Y) mutant myocilin impairs human trabecular meshwork (TM) cells by inducing chronic endoplasmic reticulum (ER) stress response in vitro. However, it is unclear how ER stress leads to TM damage and whether N450Y myocilin mutation is associated with POAG in vivo. Here we found that N450Y mutant myocilin induces autophagy, which worsens cell viability, whereas inhibition of autophagy increases viability and decreases cell death in human TM cells. Furthermore, we construct a transgenic mouse model of N450Y myocilin mutation (Tg-MYOCN450Y) and Tg-MYOCN450Y mice exhibiting glaucoma phenotypes: IOP elevation, retinal ganglion cell loss and visual impairment. Consistent with our published in vitro studies, mutant myocilin fails to secrete into aqueous humor, causes ER stress and actives autophagy in Tg-MYOCN450Y mice, and aqueous humor dynamics are altered in Tg-MYOCN450Y mice. In summary, our studies demonstrate that activation of autophagy is correlated with pathogenesis of POAG.

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. [ABSTRACT FROM AUTHOR]

Rare variants of the olfactomedin domain of myocilin are considered causative for inherited, early‐onset open‐angle glaucoma, with a misfolding toxic gain‐of‐function pathogenic mechanism detailed by 20 years of laboratory research. Myocilin variants are documented in the scientific literature and identified through large‐scale genetic sequencing projects such as those curated in the Genome Aggregation Database (gnomAD). In the absence of key clinical and laboratory information, however, the pathogenicity of any given variant is not clear, because glaucoma is a heterogeneous and prevalent age‐onset disease, and common variants are likely benign. In this review, we reevaluate the likelihood of pathogenicity for the ~100 nonsynonymous missense, insertion‐deletion, and premature termination of myocilin olfactomedin variants documented in the literature. We integrate available clinical, laboratory cellular, biochemical and biophysical data, the olfactomedin domain structure, and population genetics data from gnomAD. Of the variants inspected, ~50% can be binned based on a preponderance of data, leaving many of uncertain pathogenicity that motivate additional studies. Ultimately, the approach of combining metrics from different disciplines will likely resolve outstanding complexities regarding the role of this misfolding‐prone protein within the context of a multifactorial and prevalent ocular disease, and pave the way for new precision medicine therapeutics. [ABSTRACT FROM AUTHOR]

Adipose-derived mesenchymal stem cells (ASCs) hold promise for use in cell-based therapies. Their intrinsic anti-inflammatory properties are potentially useful for treatments of inflammatory conditions such as uveitis, while their ability to differentiate along multiple cell lineages suggests use in regenerating damaged or degenerated tissue. However, how ASCs will respond to the intraocular environment is poorly studied. We have recently reported that aqueous humor (AH), the fluid that nourishes the anterior segment of the eye, potently increases alkaline phosphatase (ALP) activity of ASCs, indicating osteogenic differentiation. Here, we expand on our previous findings to better define the nature of this response. To this end, we cultured ASCs in the presence of 0, 5, 10, and 20% AH and assayed them for ALP activity. We found ALP activity correlates with increasing AH concentrations from 5 to 20%, and that longer treatments result in increased ALP activity. By using serum free media and pretreating AH with dextran-coated charcoal, we found that serum and charcoal-adsorbable AH components augment but are not required for this response. Further, by heat-treating the AH, we established that thermally labile components are required for the osteogenic response. Finally, we showed myocilin, a protein present in AH, could induce ALP activity in ASCs. However, this was to a lesser extent than untreated 5% AH, and myocilin could only partially rescue the effect after heat treatment, documenting there were additional thermally labile constituents of AH involved in the osteogenic response. Our work adds to the understanding of the induction of ALP in ASCs following exposure to AH, providing important insight in how ASCs will be influenced by the ocular environment. In conclusion, increased osteogenic potential upon exposure to AH represents a potential challenge to developing ASC cell-based therapies directed at the eye.

Myocilin is an enigmatic glaucoma-associated glycoprotein whose biological role remains incompletely understood. To gain novel insight into its normal function, we used transposon-mediated transgenesis to generate the first zebrafish line stably overexpressing myocilin [Tg(actb1:myoc-2A-mCherry)]. qPCR showed an approximately four-fold increased myocilin expression in transgenic zebrafish embryos (144 hpf). Adult (13 months old) transgenic animals displayed variable and age-dependent ocular anterior segment alterations. Almost 60% of two-year-old male, but not female, transgenic zebrafish developed enlarged eyes with severe asymmetrical and variable abnormalities in the anterior segment, characterized by corneal limbus hypertrophy, and thickening of the cornea, iris, annular ligament and lens capsule. The most severe phenotype presented small or absent ocular anterior chamber and pupils, due to iris overgrowth along with dysplastic retinal growth and optic nerve hypertrophy. Immunohistochemistry revealed increased presence of myocilin in most altered ocular tissues of adult transgenic animals, as well as signs of retinal gliosis and expanded ganglion cells and nerve fibers. The preliminary results indicate that these cells contributed to retinal dysplasia. Visual impairment was demonstrated in all old male transgenic zebrafish. Transcriptomic analysis of the abnormal transgenic eyes identified disrupted expression of genes involved in lens, muscular and extracellular matrix activities, among other processes. In summary, the developed transgenic zebrafish provides a new tool to investigate this puzzling protein and provides evidence for the role of zebrafish myocilin in ocular anterior segment and retinal biology, through the influence of extracellular matrix organization and cellular proliferation.

Transgenic C57BL/6 mice expressing human myocilinY437 (Tg-MYOCY437H) are a well-established model for primary open-angle glaucoma (POAG). While the reduced trabecular meshwork (TM) cellularity due to severe endoplasmic reticulum (ER) stress has been characterized as the etiology of this model, there is a limited understanding of how glaucomatous phenotypes evolve over the lifespan of Tg-MyocY437H mice. In this study, we compiled the model's intraocular pressure (IOP) data recorded in our laboratory from 2017 to 2023 and selected representative eyes to measure the outflow facility (C r), a critical parameter indicating the condition of the conventional TM pathway. We found that Tg-MYOCY437H mice aged 4−12 months exhibited significantly higher IOPs than age-matched C57BL/6 mice. Notably, a decline in IOP was observed in Tg-MYOCY437H mice at 17−24 months of age, a phenomenon not attributable to the gene dosage of mutant myocilin. Measurements of the C r of Tg-MYOCY437H mice indicated that the age-related IOP reduction was not a result of ongoing TM damage. Instead, Hematoxylin and Eosin staining, immunohistochemistry analysis, and transmission electron microscopic examination revealed that this reduction might be induced by degenerations of the non-pigmented epithelium in the ciliary body of aged Tg-MYOCY437H mice. Overall, our findings provide a comprehensive profile of mutant myocilin-induced ocular changes over the Tg-MYOCY437H mouse lifespan and suggest a specific temporal window of elevated IOP that may be ideal for experimental purposes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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.

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.

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.

Deep neural network-based programs can be applied to protein structure modeling by inputting amino acid sequences. Here, we aimed to evaluate the AlphaFold2-modeled myocilin wild-type and variant protein structures and compare to the experimentally determined protein structures. Molecular dynamic and ligand binding properties of the experimentally determined and AlphaFold2-modeled protein structures were also analyzed. AlphaFold2-modeled myocilin variant protein structures showed high similarities in overall structure to the experimentally determined mutant protein structures, but the orientations and geometries of amino acid side chains were slightly different. The olfactomedin-like domain of the modeled missense variant protein structures showed fewer folding changes than the nonsense variant when compared to the predicted wild-type protein structure. Differences were also observed in molecular dynamics and ligand binding sites between the AlphaFold2-modeled and experimentally determined structures as well as between the wild-type and variant structures. In summary, the folding of the AlphaFold2-modeled MYOC variant protein structures could be similar to that determined by the experiments but with differences in amino acid side chain orientations and geometries. Careful comparisons with experimentally determined structures are needed before the applications of the in silico modeled variant protein structures.

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 CRISPRengineered a rat to have aMYOCY435H substitution that is the equivalent of the pathological human MYOC Y437H mutation. Using this engineered animal model, we discovered that the chaperone βB-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 mutantMYOCpathology. We propose a model by which mutant MYOCcauses glaucoma, and we propose that therapeutic treatment of patients having a MYOC mutation may focus on disrupting the MYOC-CRYAB complexes. [ABSTRACT FROM AUTHOR]

Myocilin is an extracellular glycoprotein with a poorly understood biological function and typically known because of its association with glaucoma. In this study, we analyzed the expression and biological activity of human myocilin in some non-ocular tissues. Western immunoblot showed the presence of myocilin in blood plasma as well as in liver and lymphoid tissues (thymus and lymph node). Quantitative PCR confirmed the expression of MYOC in these lymphoid organs and revealed that its mRNA is also present in T-lymphocytes and leukocytes. In addition, detection of 30 kDa C-terminal myocilin fragments in thymus and liver suggested that myocilin undergoes an in vivo proteolytic processing that might regulate its biological activity. The presence of myocilin in blood was further corroborated by peptide mass fingerprinting of the HPLC-isolated protein, and gross estimation of its concentration by Western immunoblot indicated that it is a medium-abundance serum protein with an approximate concentration of 0.85 mg/ml (15.5 μM). Finally, in vitro analyses indicated that myocilin acts as an anti-adhesive protein for human circulating leukocytes incubated with endothelial cell monolayers. Altogether, these data provide insightful information on new biological properties of myocilin and suggest its putative role as a blood matricellular protein. [ABSTRACT FROM AUTHOR]

Myocilin (MYOC) is the gene with mutations most common in glaucoma. In the eye, MYOC is in trabecular meshwork, ciliary body, and retina. Other tissues with high MYOC transcript levels are skeletal muscle and heart. To date, the function of wild-type MYOC remains unknown and how mutant MYOC causes high intraocular pressure and glaucoma is ambiguous. By investigating mutant MYOC in a non-ocular tissue we hoped to obtain novel insight into mutant MYOC pathology. For this study, we utilized a transgenic mouse expressing human mutant MYOC Y437H protein and we examined its skeletal (gastrocnemius) muscle phenotype. Electron micrographs showed that sarcomeres in the skeletal muscle of mutant CMV-MYOC-Y437H mice had multiple M-bands. Western blots of soluble muscle lysates from transgenics indicated a decrease in two M-band proteins, myomesin 1 (MYOM1) and muscle creatine kinase (CKM). Immunoprecipitation identified CKM as a MYOC binding partner. Our results suggest that binding of mutant MYOC to CKM is changing sarcomere ultrastructure and this may adversely impact muscle function. We speculate that a person carrying the mutant MYOC mutation will likely have a glaucoma phenotype and may also have undiagnosed muscle ailments or vice versa, both of which will have to be monitored and treated. [ABSTRACT FROM AUTHOR]

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.

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 < T (p.L486F) in MYOC and c.322G < A (p.V108I) in B4GALT3 are likely responsible for the pathogenesis of POAG in this family.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 amyloids. 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 13C solid-state NMR spectra validate that aggregated P1 exhibits amyloid signatures and is more homogeneous than aggregated P3. DMD/PRIME20 simulations provide a viable method to predict peptide aggregation propensities and aggregate structure/order which cannot be accessed by bioinformatics or readily attained experimentally. [ABSTRACT FROM AUTHOR]

Mutations in the gene MYOC account for approximately 5% of cases of primary open angle glaucoma (POAG). MYOC encodes for the protein myocilin, a multimeric secreted glycoprotein composed of N-terminal coiled-coil (CC) and leucine zipper (LZ) domains that are connected via a disordered linker to a 30 kDa olfactomedin (OLF) domain. More than 90% of glaucoma-causing mutations are localized to the OLF domain. While myocilin is expressed in numerous tissues, mutant myocilin is only associated with disease in the anterior segment of the eye, in the trabecular meshwork. The prevailing pathogenic mechanism involves a gain of toxic function whereby mutant myocilin aggregates intracellularly instead of being secreted, which causes cell stress and an early timeline for TM cell death, elevated intraocular pressure, and subsequent glaucoma-associated retinal degeneration. In this review, we focus on the work our lab has conducted over the past ∼15 years to enhance our molecular understanding of myocilin-associated glaucoma, which includes details of the molecular structure and the nature of the aggregates formed by mutant myocilin. We conclude by discussing open questions, such as predicting phenotype from genotype alone, the elusive native function of myocilin, and translational directions enabled by our work. [ABSTRACT FROM AUTHOR]

AIM:To describe the anticipation and anti-glaucoma drugs response of a Chinese family with juvenile-onset open angle glaucoma (JOAG) caused by the Pro370Leu myocilin (MYOC) mutation.METHODS: Fifteen members of a three-generation Chinese family with JOAG were recruited to this study. They all underwent ophthalmic common examinations. Patients suspected to have JOAG got an assessment of visual field and optical coherence tomography. Intraocular pressures (IOPs) of four patients were measured at 8, 10, 12, 14, 17 o’clock respectively after using anti-glaucoma drugs. Mutation screening of all MYOC gene coding exons of the participants was performed by using direct sequencing of PCR products.RESULTS: Clinical examinations and pedigree analysis revealed eight family members were suffered from JOAG. Apparent genetics anticipation phenomenon was observed in this family. Their clinical features included elevated IOP of 35-55mmHg, loss of visual field, thinning of retinal nerve fiber layer, and glaucomatous optic disc damage. Noticeably, their intraocular pressure levels could be controlled within normal range at 8 and 10 o’clock by anti-glaucoma drugs,but their IOPs would elevate >21mmHg after 12 o’clock. Seven patients received trabeculectomy produced thin-walled, pale, and saccate filtering blebs maintaining lower intraocular pressure efficiently. Mutation screening indentified a heterozygous C→T missense mutation in the MYOC gene at position 1 109 in exon 3, corresponding to a substitution of a highly conserved proline to leucine at codon 370 in the olfactomedin domain of MYOC.CONCLUSION: The clinical characteristics of JOAG in this family were 1) genetics anticipation; 2) high IOP; 3) temporay response to anti-glaucoma drugs; 4) filtering surgery produced thin-walled and saccate filtering blebs, helping maintain lower IOP.