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The Jackson Laboratory,1 The Howard Hughes Medical Institute, Bar Harbor, Maine,2 Section of Molecular Mechanisms of Glaucoma, Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland,3 Department of Ophthalmology, Tufts University School of Medicine, Boston, Massachusetts4
Received 9 April 2004/ Returned for modification 26 June 2004/ Accepted 2 July 2004
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ABSTRACT |
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INTRODUCTION |
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Multiple genetic loci are reported to contribute to POAG (32, 34, 42, 49, 51, 52). Disease-associated mutations have been identified in the myocilin gene, MYOC (14, 43). Studies from a broad range of ethnic backgrounds generally agree that MYOC mutations are responsible for approximately 3% of adult-onset POAG and a greater proportion of juvenile-onset open-angle glaucoma (2, 14). Despite this, neither the normal function(s) of MYOC nor how MYOC mutations result in IOP elevation and glaucoma has been defined.
MYOC has also been implicated in steroid-induced ocular hypertension with glaucoma. Steroid-induced ocular hypertension in response to glucocorticoid treatment occurs in as many as 40% of people treated with glucocorticoids (37, 53). MYOC is up regulated in cultured trabecular meshwork (TM; an ocular drainage structure) cells treated with glucocorticoids. Thus, MYOC was identified as a candidate to mediate glucocorticoid-induced glaucoma (26, 28). Supporting this, IOP elevation (assessed as an increase in drainage structure resistance) is glucocorticoid inducible in some but not all human anterior segment perfusion cultured eyes. In these cultures, IOP elevation correlates with MYOC induction (8) and ultrastructural changes (9) in the TM. Cultured anterior segments that developed elevated IOP had MYOC induction, while those that did not develop elevated IOP had no MYOC induction (8). Similarly, monkeys treated with glucocorticoids develop ocular hypertension (13), and in at least some eyes glucocorticoids induce MYOC and cause ultrastructural changes in the TM (8).
Further studies, which do not involve steroid use, also can support a role for elevated MYOC levels in IOP elevation and glaucoma. In some glaucoma patients, the TM has elevated MYOC levels and broadened MYOC distribution (24). Cultured human anterior segments perfused with recombinant MYOC are reported to develop elevated IOP, whereas those perfused with an equal amount of other proteins or denatured MYOC do not (12). Finally, in the albino Wistar rat strain, experimental IOP elevation did not induce ocular MYOC, suggesting that MYOC induction does not occur secondary to IOP elevation (1).
Despite these circumstantial data, there is no direct in vivo evidence that elevated MYOC levels cause IOP elevation and glaucoma. The rat study mentioned above (1) does not exclude MYOC elevation as a secondary response to increased IOP in different settings (either in different genetic contexts or in response to more chronic IOP elevation, as occurs in human patients). In fact, in some cultured human anterior segments, MYOC expression is elevated in response to IOP elevation (3). Thus, it is not clear if MYOC elevation causes IOP elevation and glaucoma or if MYOC is simply induced because of the stress of elevated IOP, secondary tissue damage, or glaucoma treatments. Whether or not increased MYOC levels cause IOP elevation and glaucoma needs to be experimentally tested in an in vivo model. This knowledge is important for understanding pathogenic mechanisms associated with MYOC.
Determining whether or not increased MYOC levels cause glaucoma also is important for assessing the risk of disease associated with noncoding mutations. Some investigators suggest that MYOC promoter variants may influence the progression of glaucoma (10, 11, 29), while others disagree with this conclusion (2). Since human patients are currently offered genetic testing based on the presence of noncoding variants, the issue is very important. In the present study, we used transgenic mice to directly test if genetically elevating MYOC is sufficient to cause elevated IOP and glaucoma. Mice with increased MYOC levels did not develop elevated IOP or glaucoma. This clearly indicates that MYOC elevation alone is not sufficient to cause glaucoma. These results question the hypotheses that MYOC is involved in steroid-induced glaucoma and that promoter sequence variants alone are involved in POAG.
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MATERIALS AND METHODS |
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Quantitative PCR. Relative RNA quantification was performed by using standard TaqMan protocols (TaqMan; PE Biosystems, Foster City, Calif.). Total RNA was extracted from eyes of transgenic and nontransgenic control mice that were18 to 23 days old (RNeasy; QIAGEN, Valencia, Calif.). RNA was fluorescently quantified (Ribogreen; Molecular Probes, Eugene, Oreg.), and 1.8 ng was used to make cDNA (Retroscript; Ambion, Austin, Tex.). Generation of cDNA was performed in triplicate for each mouse. The cDNA was used for a fluorescent quantitative PCR assay (TaqMan; PE Biosystems). Briefly, each of the three samples from each transgenic line was assayed for the number of PCR cycles required to cross the threshold of the linear range of the reaction (Ct). The three Ct values for each mouse were averaged. Samples were normalized for the total template amount by subtracting the average GAPDH Ct from the average Myoc Ct (delta Ct). The normalized Myoc expression (delta Ct) for the control mice was then subtracted from the normalized expression from the transgenic mice (delta delta Ct). The relative level of expression for transgenic mice compared to control mice equals 2(delta delta Ct). The expression value reported is the average of all of the mice of that genotype tested. The oligonucleotides (Sigma-Aldrich, St. Louis, Mo.) used for quantitative PCR were Myoc F (5'GAATTTGGACACGTTGGCCT3'), Myoc R (5'CCAGTGTCCTTCCACCCAGTA3'), GAPDH F (5'TTCACCACCATGGAGAAGGC3'), and GAPDH R (5'GGCATGGACTGTGGTCATGA3'. TaqMan probe sequences were as follows: Myoc probe, 5'TGCTGCCAGGCTCCAGGGAAGT3'; GAPDH probe, 5'TGCATCCTGCACCACCAACTGCTTAG3'.
Western analysis. Polyclonal rabbit antiserum was raised against a glutathione S-transferase fusion protein containing amino acids 100 to 187 of the first exon of mouse MYOC (21). To enrich for the drainage structures, a narrow ring of tissue centered over the iridocorneal-angle drainage structures was dissected from freshly harvested eyes (ring includes iris root and TM). The tissue was homogenized in 50 mM Tris (pH 7.5)-5 mM EDTA-20 mM dithiothreitol-1 mM phenylmethylsulfonyl fluoride-10 µg of leupeptin per ml-1 µg of pepstatin per ml-1% NP-40-1% Triton X-100-0.2% sodium dodecyl sulfate and sonicated for 1 min.
Tissue was harvested from each eye of eight animals of each genotype (control, hemizygous transgenic, and homozygous transgenic). For each genotype, two pools of protein extracts (four animals per pool) were analyzed. Twenty-five micrograms of total protein was loaded onto the gel and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Equivalent protein loading was confirmed by Coomassie staining of additional gels (data not shown). Following separation, proteins were transferred to a nitrocellulose membrane. Membranes were incubated with Western blocking reagent (Roche Applied Science, Indianapolis, Ind.) and 5% skim milk in 0.05% Tween 20-Tris-buffered saline (pH 7.4) overnight at 4°C. They were next incubated for 1 h at room temperature with the affinity-purified antibody at a 1:3,000 dilution made in 0.05% Tween 20-Tris-buffered saline (pH 7.4) containing 1% skim milk. Anti-rabbit horseradish peroxidase-linked secondary antibody (Amersham Biosciences, Piscataway, N.J.) was used at a 1:25,000 dilution. The SuperSignal chemiluminescent detection system (Pierce Biotechnology, Rockford, Ill.) was used in accordance with the manufacturer's instructions.
IOP measurement and clinical examination. IOPs were measured as previously described (18, 33). Statistical significance was tested with a two-tailed Student t test. For the C57BL/6J mouse strain, an IOP of >20 mmHg is greater than 3 standard deviations above the mean and is considered an elevated IOP in this study (33). Anterior segment clinical examinations were performed with a slit lamp biomicroscope (Haag-Streit USA, Mason, Ohio) (38). Retinal and optic nerve head examinations were performed with an indirect ophthalmoscope and a 60- or 90-diopter lens on eyes with pupils dilated with a drop of 1% cyclopentolate (15).
Histological analysis. Enucleated eyes were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2), dehydrated in graded ethanol, and embedded in fresh Historesin (Leica, Heidelberg, Germany). Twenty-four 1.5-µm sections were analyzed from each of three different ocular regions, with the lens as a landmark (region A, periphery of lens; region B, halfway between lens center and periphery; region C, center of lens and optic nerve head), and stained with hematoxylin and eosin (H&E) (40).
Optic nerves were harvested as follows. Immediately after death, the top of the skull and most of the brain were removed, leaving approximately 1 mm of brain overlying the intact optic nerves. The nerves were fixed in the head overnight in 0.8% paraformaldehyde and 1.2% glutaraldehyde in 0.1 M phosphate buffer and removed on the following day. The postorbital-prechiasmal sections of nerves were then embedded in EMbed 812 resin (Electron Microscopy Sciences, Ft. Washington, Pa.), and 1-µm sections were stained with p-phenylenediamine.
Electron microscopy. Electron microscopy procedures were performed as described previously (21, 39). Briefly, eyes were fixed for 1.5 h with 0.8% paraformaldehyde and 1.2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) at 4°C. The anterior segment was cut into multiple wedge-shaped blocks and fixed further at 4°C for 12 h. Tissues were washed in phosphate-buffered saline, postfixed with 1% osmium tetroxide, and embedded. Sections were cut and stained with uranyl acetate and lead citrate.
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RESULTS |
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To determine how the increased Myoc expression affects the protein level, an investigator (unaware of the results of the quantitative PCR) performed a Western analysis with protein extracts from tissue enriched for ocular drainage structures (see Materials and Methods). The relative amounts of MYOC in control, hemizygous, and Tg/Tg mice were determined by densitometric analysis of Western blots normalized to the control sample. The results of the Western analysis are in agreement with those obtained by quantitative PCR. Tg/Tg mice had robust overexpression (15-fold, SD = 2.8) compared to that of control mice, and hemizygous mice had an intermediate level of expression (6-fold, SD = 2.2) (Fig. 1B). The level of overexpression in Tg/Tg mice was confirmed by comparison of dilutions of Tg/Tg protein to control protein.
Anterior segment and IOP. Anterior and posterior segments of Tg/Tg mice between 14.5 and 27 months of age were examined (n = 16). Structures of the ocular anterior segment (pupil, iris, cornea, and lens) in Tg/Tg mice were indistinguishable from those of controls (Fig. 2A and B). To test if MYOC overexpression altered IOP, we assessed IOP in control and Tg/Tg mice at approximately 4, 8, 12 to 18, and >19 months of age (Fig. 2C). There was no significant difference in IOP between control and Tg/Tg mice at any of the ages (the oldest mice were approximately 2 years of age) (Fig. 2C).
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Optic nerve and optic nerve head. MYOC is expressed in the optic nerve. Thus, in addition to altering IOP, MYOC mutations may have a direct detrimental effect in the optic nerve (7, 20, 44). To assess the possibility of glaucomatous damage in the absence of IOP elevation, we examined Tg/Tg mice for hallmarks of glaucoma including cupping of the optic nerve head and unhealthy or dying retinal ganglion cell axons. Fundus examinations did not reveal optic nerve head cupping or any other abnormalities. Histologically, there were no signs of nerve fiber layer loss (retinal ganglion cell axons in the eye) or optic nerve cupping in eight Tg/Tg mice between 18 and 30 months of age (Fig. 4A and B). p-Phenylenediamine darkly stains unhealthy retinal ganglion cell axons in optic nerve cross sections. Mice with glaucoma have darkly stained dying axons and/or gliosis and scarring indicating previous or ongoing damage. In advanced cases there is substantial axon loss. Aged Tg/Tg mice have optic nerves that are as healthy as those of age-matched controls (Fig. 4C and D).
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAddendum in proofReferences |
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Despite the association between increased MYOC levels and high IOP (in steroid-induced glaucoma and other glaucomas), our data argue that increased MYOC by itself is not the cause of IOP elevation. One possible explanation for the association of elevated MYOC with glaucoma is that MYOC is turned on in response to stress (24, 28). This could be the stress of elevated IOP, tissue damage, or glaucoma treatments. Data from experiments with cultured cells or organ-cultured anterior segments, where MYOC is up regulated in response to oxidative stress, mechanical stress, or IOP elevation, agree with this interpretation (3, 28, 45).
Previous studies have argued against a pathogenic role of elevated MYOC levels in steroid-induced glaucoma. Mutations in MYOC were not identified in patients with steroid-induced ocular hypertension (2, 13). These previous studies do not rule out the possibility that mutations in genes responsible for the regulation of MYOC result in abnormally high MYOC levels after steroid use and that this causes glaucoma. Our study confirms and extends these previous studies. It suggests not only that it is unlikely that noncoding MYOC mutations cause steroid-induced glaucoma but that mutations in genes that increase MYOC levels are also unlikely to cause either steroid-induced glaucoma or other glaucomas. Therefore, we suggest that other genes that are found to be differentially regulated in response to glucocorticoid treatment (8, 23, 28) be considered as candidates for causing steroid-induced ocular hypertension. The corticosteroid receptor genes that mediate glucocorticoid effects are interesting candidates. Since the steroid response may involve extracellular matrix accumulation in the iridocorneal angle (8, 25) and the ocular hypertension is reversible (13), matrix metalloproteinases (responsible for extracellular matrix turnover) and matrix metalloproteinase inhibitor genes are also good candidates.
This study shows that increasing the amount of normal MYOC protein by itself is unlikely to cause glaucoma. A previous mouse study shows that the absence of MYOC has no phenotypic consequences (21). Similarly for humans, individuals who are hemizygous for MYOC (50) or who are either homozygous (22) or heterozygous (27) for a very early truncating mutation (Arg46Stop, which is presumed to be a null) are not affected. Together, these studies suggest that altering the level of MYOC by itself is not important and that disease manifestation is dependent on the presence of abnormal protein molecules.
The mutant proteins may inhibit specific cellular processes or may have gained some undefined detrimental properties. Mutant proteins appear to form intracellular aggregates and to inhibit the secretion of normal MYOC molecules (4, 5, 16, 17). Accumulation of MYOC aggregates in the endoplasmic reticulum of TM or other drainage structure cells may alter cellular metabolism or kill cells and cause IOP elevation and subsequent glaucoma. This model suggests that genes or other factors (including cellular stress) that affect the ability of the endoplasmic reticulum to handle misfolded proteins, or that affect the efficiency of the proteosome in clearing misfolded proteins, may modify the course of glaucoma caused by mutations in MYOC. Some of these factors may provide therapeutic targets.
Owing to the induction of MYOC by different stresses, a feature of pathogenic models involving detrimental effects of mutant proteins is that the magnitude of the detrimental effect may increase at times of stress as a consequence of increased production of mutant MYOC molecules. Drugs, infection, or other factors that stress drainage structure cells therefore may influence the onset, progression, and severity of POAG. It is even possible that fluctuations in stress levels may explain clinically observed fluctuations in IOP, since stress fluctuations may result in peaks and troughs in IOP related to the abundance of abnormal MYOC molecules.
Since the level of MYOC is not important by itself, promoter sequence variants are unlikely to be sufficient to cause glaucoma. Nonetheless, similar to the suggested implications of stress, promoter variants that increase or decrease transcription of MYOC alleles that contain coding mutations may alter glaucoma presentation. One study addressed this possibility for the mt.1 promoter variant (a variant suggested to be a marker for worsening glaucoma over time and for which a genetic test is available). This group found no evidence that mt.1 had any impact on the course of the disease even in the presence of coding mutations (2). This suggests that these promoter variants are unlikely to contribute to glaucoma. Further studies including promoter variants that are known to alter MYOC transcription, however, are necessary to assess the effects on the disease of altering the transcriptional level of specific alleles that contain disease-causing coding mutations.
In summary, our data suggest that noncoding sequence variants in MYOC are by themselves unlikely to influence steroid-induced or non-steroid-induced glaucoma. Disease pathogenesis appears to be dependent upon the expression of abnormal mutant proteins. Further work remains to define the pathogenic mechanisms of the abnormal proteins and the potential role of intracellular aggregates. The production of mice with human disease-associated alleles will be a useful tool to address these issues.
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ADDENDUM IN PROOF |
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ACKNOWLEDGMENTS |
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We are supported by the National Eye Institute (grant EY11721 to S.W.M.J.), the Canadian Institute of Health Research and the Heart and Stroke Foundation of Canada (D.B.G.), and the National Eye Institute Intramural Program (S.I.T). Scientific support services at The Jackson Laboratory are subsidized by a core grant from the National Cancer Institute (CA34196). S.W.M.J. is an Associate Investigator of The Howard Hughes Medical Institute.
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FOOTNOTES |
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