Expression of Myocilin/TIGR in Normal and Glaucomatous Primate Optic Nerves (original) (raw)
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Glaucoma-associated myocilin: A better understanding but much more to learn
Experimental Eye Research, 2009
Over a decade has passed since myocilin was identified as the first gene linked to early and late-onset primary open-angle glaucoma. During this time, considerable effort has been put forth to understand the functional role myocilin has in normal and glaucomatous eyes. Myocilin is expressed in many ocular and non-ocular tissues, is found in both intracellular and extracellular spaces, and has been linked to elevations in intraocular pressure. Mutations in the myocilin gene that have been associated with glaucoma appear to confer a gain-of-functional activity rather than loss of function. Unfortunately, what the normal function of myocilin is and how alterations in the function can confer a glaucoma phenotype have yet to be elucidated. We will review the current understanding of myocilin with special emphasis on the structural makeup of the myocilin gene and protein, its possible physiological roles internal and external to ocular cells, the regulation of intraocular pressure as evidenced through the use of perfusion culture systems and animal models, and as a causative agent in some forms of glaucoma.
Investigative Ophthalmology & Visual Science, 2004
PURPOSE. To determine the cDNA sequences and analyze the expression of porcine optineurin and myocilin in trabecular meshwork cells (TMCs) and astrocytes from the optic nerve head under normal and experimental conditions. METHODS. Both porcine optineurin and myocilin were cloned to determine the cDNA sequences. Porcine TMCs and astrocytes were isolated and treated with dexamethasone (500 nM) for 2 weeks, incubated under hypoxic conditions (7% O 2) for 72 hours, or exposed to 33 mm Hg hydrostatic pressure for 72 hours. A 10% mechanical stretch for 24 hours was also performed on TMCs. The expression level of the optineurin and myocilin transcripts was analyzed by real-time quantitative PCR. RESULTS. The sequences of porcine optineurin and myocilin cDNA were determined, and the expression of both genes was confirmed in both TMCs and astrocytes. Amino acid sequences of porcine optineurin and myocilin were homologous to those of humans by 84% and 82%, respectively, and shared protein motifs and modification sites. The expression of myocilin mRNA by TMCs and astrocytes was increased by 8.0-and 5.5-fold, respectively, after exposure to dexamethasone. In contrast, the expression of optineurin was suppressed to 68% in TMCs and 48% in astrocytes after exposure to dexamethasone. A significant reduction of myocilin expression was observed after 72 hours of incubation under hypoxic conditions in both types of cells, whereas optineurin was not affected. Hydrostatic pressure for 72 hours and mechanical stretching for 24 hours had minimal affects on gene expression of both optineurin and myocilin. CONCLUSIONS. The high homology of porcine optineurin and myocilin to the comparable human genes indicates that pigs can be used to study changes in gene expression in hyperten-sive eyes. The alterations in expression of myocilin but not of optineurin under stress suggest that different mechanisms in the phenotype of glaucoma associated with the two genes are involved in development of glaucoma.
Human Molecular Genetics, 2001
Until recently, very little was known about the molecular mechanisms responsible for the development of glaucoma, a leading cause of blindness worldwide. Mutations in the glaucoma gene myocilin (MYOC, GLC1A) are associated with elevated intraocular pressure and the development of autosomal dominant juvenile glaucoma and a subset of adult-onset glaucoma. MYOC is expressed in the trabecular meshwork (TM), a tissue responsible for drainage of aqueous humor from the eye, and the tissue involved in elevated intraocular pressure associated with glaucoma. To better understand the role of MYOC in glaucoma pathogenesis, we examined the expression of normal and mutant myocilin in cultured ocular (TM) and non-ocular cells as well as in the aqueous humor of patients with and without MYOC glaucoma. Normal myocilin was secreted from cultured cells, but very little to no myocilin was secreted from cells expressing five different mutant forms of MYOC. In addition, no mutant myocilin was detected in the aqueous humor of patients harboring a nonsense MYOC mutation (Q368X). Co-transfection of cultured cells with normal and mutant myocilin led to suppression of normal myocilin secretion. These studies suggest that MYOC glaucoma is due either to insufficient levels of secreted myocilin or to compromised TM cell function caused by congestion of the TM secretory pathway.
Expression of Mutated Mouse Myocilin Induces Open-Angle Glaucoma in Transgenic Mice
Journal of Neuroscience, 2006
We developed a genetic mouse model of open-angle glaucoma by expression of mutated mouse myocilin (Myoc) in transgenic (Tg) mice. The Tyr423His point mutation, corresponding to the severe glaucoma-causing Tyr437His mutation in the human MYOC gene, was introduced into bacterial artificial chromosome DNA encoding the full-length mouse Myoc gene and long flanking regions. Both wildtype (Wt) and Tg animals expressed Myoc in tissues of the irido-corneal angle and the sclera. Expression of mutated Myoc induced the accumulation of Myoc in cell cytoplasm and prevented its secretion into the extracellular space. The levels of ATPase-1 were reduced in the irido-corneal angle of Tg mice compared with Wt animals. Tg mice demonstrated a moderate elevation of intraocular pressure, the loss of ϳ20% of the retinal ganglion cells (RGCs) in the peripheral retina, and axonal degeneration in the optic nerve. RGC depletion was associated with the shrinkage of their nuclei and DNA fragmentation in the peripheral retina. Pathological changes observed in the eyes of Tg mice are similar to those observed in glaucoma patients.
Unaltered myocilin expression in the blood of primary open angle glaucoma patients
Molecular vision, 2012
To investigate the expression of the myocilin gene (MYOC) in the blood of primary open angle glaucoma (POAG) patients to determine if altered systemic expression is playing a role. Patients (n=47) were eligible for inclusion if they met standard clinical criteria for POAG. Control subjects (n=27) were recruited who were free from glaucoma by examination. RNA was extracted from leukocytes of patients and controls and converted to cDNA by reverse transcriptase enzyme, and quantitative PCR was used to assess expression levels of MYOC and the house keeping gene β-globulin (HBB). The ratio of MYOC expression to HBB expression for POAG patients was compared to that of controls and to clinical characteristics of POAG patients. Mean gene expression values were statistically similar in POAG patients and controls for both MYOC (p≤0.55) and HBB (p≤0.48). MYOC/HBB ratios were also statistically indistinguishable between POAG patients and controls (p≤0.90). MYOC/HBB ratios were not significantly...
Investigative ophthalmology & visual science, 2001
To isolate the rat Myoc/Tigr gene and investigate changes in its expression pattern in normal eyes and in eyes with either pressure-induced optic nerve damage or optic nerve transection. Expression pattern of the rat Myoc/Tigr gene was investigated by Northern blot hybridization. Optic nerve damage and death of ganglion cells in the retina were induced unilaterally, by injection of hypertonic saline solution, episcleral vein cauterization, or optic nerve transection. The levels of mRNA for Myoc/Tigr were compared between several tissues of the control and surgically altered eyes, by using semiquantitative RT-PCR, real-time PCR, and Northern blot analysis. The rat Myoc/Tigr gene is 10 kb long and contains three exons. Among the eye tissues analyzed, Myoc/Tigr mRNA was detected in the combined tissues of the eye angle, sclera, cornea, retina, and optic nerve head. With pressure-induced optic nerve degeneration, the level of Myoc/Tigr mRNA decreased in the retina and the combined tissu...
Myocilin in the trabecular meshwork of eyes with primary open-angle glaucoma
Graefe's Archive for Clinical and Experimental Ophthalmology, 2009
Background Mutations in myocilin, a 55-57 kDa secreted glycoprotein, are causative for some forms of primary open-angle glaucoma (POAG). In vitro studies indicate that myocilin can modulate the hydrodynamic outflow resistance in the trabecular meshwork (TM) and that elevated amounts of myocilin can obstruct the TM outflow system in POAG. In this study, we analyzed the localization of myocilin in the trabecular meshwork (TM) of eyes with primary open-angle glaucoma (POAG), and compared it with that of normal eyes. Methods Immunohistochemistry for myocilin was performed in the eyes of human donors (nine normal and 14 with POAG, including one with steroid-induced glaucoma). Results Staining for myocilin was observed in the extracellular spaces of the juxtacanalicular tissue (JCT) in all normal eyes. Some normal eyes did also show cytoplasmic staining for myocilin in TM cells. In the eyes of six donors with POAG, staining of the JCT was more widespread and intense than in normal eyes. In the other eyes with POAG, immunoreactivity for myocilin in the JCT was not markedly different to that of normal eyes. Staining intensity in the JCT of POAG eyes did not obviously correlate with intraocular pressure or clinical severity. In the eyes of one patient with steroid-induced POAG, cells of the TM, Schlemm's canal endothelium, and the anterior stroma of the iris showed an immunoreactivity for myocilin which was considerably more intense than in normal eyes, or in the eyes with other forms of POAG. Conclusions In some cases of POAG, the structural changes in the JCT include an increase in myocilin in the extracellular pathways of aqueous humor. Treatment with steroids appears to increase myocilin synthesis in TM and iris of human eyes in situ.
Molecular …, 2008
PurposeHeterozygous mutations in the myocilin gene (MYOC) cause glaucoma by an unknown mechanism. MYOC encodes an extracellular protein of unidentified function that undergoes intracellular endoproteolytic processing in the secretory pathway. It has been described that co-expression of wild-type/mutant myocilin reduces the secretion of the wild-type protein and that single expression of glaucoma myocilin mutants reduces its proteolytic processing. However, the effect of wild-type myocilin on mutant myocilin secretion and how mutant myocilin affects the proteolytic processing of wild-type myocilin have not been investigated. We herein analyze these two issues.MethodsWe modeled the heterozygous state for 4 missense (E323K, R346T, P370L, D380A) and 1 nonsense (Q368X) myocilin mutants by transiently co-expressing each mutant with the wild-type protein in HEK-293T cells. Recombinant mutant and wild-type myocilin in both culture media and cellular fractions were quantified by western immunoblot and densitometry.ResultsA 24 h transient co-expression of each myocilin mutant with the wild-type protein elicited an augmented secretion of the mutant forms from 1.5 fold (D380A) to 5.4 fold (E323K). Under such conditions, extracellular mutant myocilin represented up to 20% of the total mutant protein. Other than this effect, secreted wild-type myocilin significantly decreased from 2.6 fold (E323K) to 36 fold (Q368X). When myocilin proteolytic processing was enhanced (96 hour co-expression) the extracellular amount of wild-type processed myocilin diminished from approximately 2.1 fold (E323K) to 6.3 fold (P370L). Nonreducing SDS-PAGE indicated that extracellular myocilin resulting from 24 h co-expression of wild-type myocilin and each of the 4 missense mutants forms hetero-oligomers and that glaucoma mutations do not increase the size of myocilin aggregates.ConclusionsIncreased extracellular levels of mutant myocilin expressed in heterozygosis may play a relevant role in glaucoma pathogenesis. This effect is likely the result of intracellular mutant/wild-type myocilin hetero-oligomerization.
Characterization of monoclonal antibodies against the glaucoma-associated protein myocilin
Experimental Eye Research, 2008
Although the glaucoma-associated protein myocilin has been the focus of intensive research, its biological function is still unknown. One of the limiting factors has been the lack of well characterized antibodies, particularly monoclonal antibodies. We describe the development of six monoclonal antibodies specific to myocilin and characterize their suitability in Western blot and immunohistochemical applications. Three of the six monoclonal antibodies recognize the N-terminus of myocilin (amino acids 33-214), two antibodies recognize the middle third of the protein (amino acids 215-368), and one antibody recognizes the C-terminus (amino acids 369-504). Isotyping revealed all antibodies are of the IgG1κ class except one, which is IgG2bκ. Purified myocilin monoclonal antibodies were able to recognize myocilin in human aqueous humor separated on denatured/reduced and native gels, and human trabecular meshwork lysate by Western blot. Myocilin was also detected by immunohistochemistry in trabecular meshwork, ciliary body, iris, cornea, sclera, choroid, and retinal pigment epithelial cells.