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(Investigative Ophthalmology and Visual Science. 2000;41:729-740.)
© 2000 by The Association for Research in Vision and Ophthalmology, Inc.

Localization of Myocilin/Trabecular Meshwork–Inducible Glucocorticoid Response Protein in the Human Eye

Anastasia Karali1, Paul Russell2, Fritz H. Stefani3 and Ernst R. Tamm1

1 From the Department of Anatomy II, University of Erlangen-Nürnberg, Erlangen, Germany; the 2 Laboratory of Mechanisms of Ocular Diseases, National Institutes of Health, National Eye Institute, Bethesda, Maryland; and the 3 Eye Hospital of the University of Munich, Munich, Germany.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To study distribution and cellular localization of myocilin/trabecular meshwork–inducible glucocorticoid response protein (TIGR) in the human eye.

METHODS. A peptide antibody against a portion of the myosin-like domain of myocilin/TIGR was developed. Different ocular tissues from three human donors were investigated by one- and two-dimensional gel electrophoresis and Western blot analysis. Immunohistochemistry was performed on 25 human eyes enucleated because of posterior choroidal melanoma and on 7 normal human donor eyes.

RESULTS. By Western blot analysis, a band at approximately 57 kDa was visualized in cornea, trabecular meshwork, lamina cribrosa, optic nerve, retina, iris, ciliary body, and vitreous humor. By immunohistochemistry, immunoreactivity for myocilin/TIGR was observed in cells of the corneal epi- and endothelium and extracellularly in the corneal stroma and sclera. In the trabecular meshwork, cells of the uveal and corneoscleral meshwork were stained, as was the cribriform area directly adjacent to Schlemm’s canal. Positive staining was seen in cells of the ciliary epithelium, ciliary muscle, lens epithelium, and in stromal and smooth muscle cells of the iris. Throughout the entire vitreous body, fine filamentous material was positively labeled. In the retina, staining was seen along the outer surface of rods and cones, in neurons of the inner and outer nuclear layer, and in the axons of optic nerve ganglion cells. Optic nerve axons were stained in the prelaminar, laminar, and postlaminar parts of the nerve. In the region of the lamina cribrosa, astrocytes in the glial columns and cribriform plates were positively labeled.

CONCLUSIONS. Myocilin/TIGR is expressed in almost every ocular tissue. Depending on the respective tissue, it is observed extra- or intracellularly. The presence of myocilin/TIGR in optic nerve axons and lamina cribrosa astrocytes indicates that the trabecular meshwork might not be the only target of abnormal myocilin/TIGR in GLC1A-linked open-angle glaucoma.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Myocilin, a protein that is also known as trabecular meshwork–inducible glucocorticoid response protein (TIGR), has been shown to be involved in at least some forms of primary open-angle glaucoma (POAG). Stone et al.1 identified mutations in the gene for myocilin/TIGR, which lies within the interval on chromosome 1 that was originally associated with juvenile open-angle glaucoma (GLC1A).2 3 4 Subsequently, mutations in the same gene of patients with GLC1A-linked juvenile open-angle glaucoma were reported by other researchers.5 6 7 8 9 10 11 12 13 Juvenile open-angle glaucoma refers to a subset of POAG that has an earlier age of onset and a highly penetrant mode of inheritance and that is usually associated with high intraocular pressure (IOP) that requires early surgical treatment.14 15 16 In addition, mutations in the myocilin/TIGR gene are present in approximately 4.6% of patients with randomly screened adult forms of POAG.17

Myocilin/TIGR was originally isolated from cultured human TM cells that had been treated for a long time with dexamethasone18 19 20 21 and, independently, from normal human retina.22 In addition to TM and retina, mRNA for myocilin/TIGR is expressed in various intraocular and extraocular tissues, such as cornea, sclera, ciliary body, iris, heart, skeletal muscle, thymus, small intestine, colon, stomach, thyroid, and trachea.5 23 24 25 26 The normal role of myocilin/TIGR and the mechanisms by which mutations in this gene cause glaucoma are unknown. In addition, there is controversy about the exact cellular localization of myocilin/TIGR. Some authors reported that myocilin/TIGR is secreted by trabecular meshwork cells and hypothesized that myocilin/TIGR might act extracellularly on aqueous humor outflow.18 19 20 21 Others could not find evidence for such an extracellular localization of myocilin/TIGR, but observed myocilin/TIGR in the cytoplasm of trabecular meshwork cells27 28 or in association with the connecting cilium of the photoreceptors.22 In the present study, we developed a peptide antibody against myocilin/TIGR and used it as a tool to study the distribution and cellular localization of myocilin/TIGR in the human eye.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
A polyclonal rabbit antibody was developed against the peptide sequence TRDTARAVPPGSREVST (corresponding to positions 188 to 204) of human myocilin/TIGR (AnaSpec, San Jose, CA).

Ten pairs of normal human donor eyes (age range, 51–84 years) obtained after autopsy and 25 eyes enucleated because of posterior choroidal melanoma (age range, 54–81 years) were investigated. The melanoma eyes were obtained from the Eye Hospital of the University of Munich, Germany. After enucleation, normal donor eyes were cut equatorially behind the ora serrata. In eyes from three of the normal donors, cornea, trabecular meshwork, ciliary body, iris, retina, vitreous humor, lamina cribrosa, and optic nerve were isolated, deep-frozen, and processed for gel electrophoresis and Western blot analysis. In eyes from seven of the normal donors, the anterior segment was dissected in quadrants. From each quadrant, wedge-shaped specimens of 2 mm circumferential width, containing cornea, iris, ciliary body, sclera, and trabecular meshwork were cut and immersed in 4% paraformaldehyde for 24 hours. In addition, lens, retina, lamina cribrosa, and postlaminar optic nerve were cut free and immersed in the same fixative. Specimens from five pairs of the normal human autopsy eyes were placed in fixative within 4 hours after death; specimens from two pairs of the eyes were fixed within 10 hours after death. Melanoma eyes were processed within 10 minutes after surgical removal and fixed as a whole in 10% formalin.

Methods for securing human tissue were humane, included proper consent and approval, and complied with the Declaration of Helsinki.

Gel Electrophoresis and Western Blot Analysis
The sample was homogenized in 8 M urea with 2% Nonidet P-40 (American Bioanalytical, Natick, MA), centrifuged at 14000g for 10 minutes, and the supernatant was taken. Protein concentrations were determined with the Coomassie protein assay (Pierce, Rockford, IL). For one-dimensional (1D) gels, proteins (3 µg) were subjected to SDS-PAGE on 12.5% gels using the Pharmacia PhastGel System (Pharmacia LKB, Piscataway, NJ) and the gels were silver stained. Polypeptides were transferred to nitrocellulose membranes according to the manufacturer’s protocols and blocked for 1 hour. Membranes were incubated with rabbit antibody to myocilin, and the blots were subsequently incubated with CSPD chemiluminescence system (Tropix Inc., Bedford, MA). For two-dimensional (2D) gel electrophoresis and immunoblotting, 3 µg of a trabecular meshwork homogenate was run on isoelectric focusing gels and then on 12.5% sodium dodecylsulfate—polyacrylamide gel electrophoresis (SDS-PAGE), exactly as described previously.29 The immunoblotting was performed using the Super Signal chemiluminescent method (Pierce), using an Image Station 440 (NEN, Boston, MA) accoring to the manufacturer’s instructions.

Immunohistochemistry
The localization of myocilin/TIGR was studied in paraffin sections from both melanoma and normal donor eyes. The sections were placed on slides covered with 0.1% poly-L-lysine and preincubated for 45 minutes in dry milk solution.30 After preincubation, the sections were incubated overnight at room temperature with the myocilin/TIGR antibody diluted 1:50–1:100 in phosphate-buffered saline (PBS). After overnight incubation, the sections were washed in PBS, reacted for 1 hour with biotinylated secondary antibodies against rabbit immunoglobulin (Vector Laboratories, Burlingame, CA), washed again, and covered with streptavidin-fluorescein isothiocyanate (FITC; Vector). Double-labeling experiments were performed in specimens from eyes fixed in 4% paraformaldehyde within 3 hours after enucleation. Sections were incubated with myocilin/TIGR antibody in combination with mouse anti-glial fibrillary acidic protein, mouse anti-neurofilament (1:25 and 1:5; Dako, Carpinteria, CA), and mouse anti-protein gene product (PGP) 9.5 (1:100; UltraClon Ltd., Isle of Wight, UK). Binding of rabbit antibodies was visualized using biotinylated secondary antibodies and streptavidin-FITC. Mouse antisera were stained with Cy 3–conjugated anti-mouse IgG (Dianova, Hamburg, Germany).

After washing in PBS, the sections were mounted with fluorescent mounting medium (Dako) and viewed with a Leitz Aristoplan microscope (Ernst Leitz GmbH, Wetzlar, Germany). A Kodak T-max 400 film (Eastmann Kodak, Rochester, NY) was used for photography.

Control experiments were performed by incubating the myocilin/TIGR antibody with the specific peptide (1 µg/ml) or by using either PBS or preimmune serum from the same host species (rabbit, mouse) substituted for the primary antibody.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Western Blot Analysis
Using SDS-PAGE and Western blot analysis, the peptide antibody against myocilin/TIGR recognized a distinct major band at approximately 57 kDa in fresh samples from trabecular meshwork, cornea, lamina cribrosa, and postlaminar optic nerve (Fig. 1) . A similar band was observed in ciliary body, iris, vitreous humor, and retina (not shown). This size corresponds to the electrophoretic mobility of myocilin/TIGR that has been reported by others.20 21 Samples from some donors showed in some tissues additional weak bands at approximately 45 and 30 kDa (Fig. 1) . These bands tended to be more intense at longer postmortem times of the samples and in certain tissues, and were regarded as possible degradation products of myocilin/TIGR. Some samples from ciliary body and trabecular meshwork showed an additional weak band at approximately 68 kDa, which might correspond to the electrophoretic mobility of glycosylated myocilin/TIGR.20 21 In 2D Western blot analysis of trabecular meshwork samples, the antibody recognized a distinct band at approximately 57 kDa and with comparable isoelectric point, as previously reported for myocilin/TIGR (Fig. 2) .20 The band in the 2D Western blot corresponded to a protein spot in the silver-stained 2D gel. The fact that the 2D Western blot showed a band rather than a distinct spot correlates with previous observations on heterogeneity of the 57-kDa form of myocilin/TIGR, which might be due to posttranslational modifications.20 21



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  		Figure 1. SDS-PAGE (left) and Western blot analysis (right) of urea-soluble proteins from trabecular meshwork (1), cornea (2), optic nerve by lamina cribrosa (3) and posterior optic nerve (4). The Western blot was hybridized with antibody to human myocilin.

 


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  		Figure 2. Silver-stained, two-dimensional gel electrophoresis (top) of human trabecular meshwork and Western blot analysis using antibody to myocilin (bottom). The acidic side of the isoelectric focusing gel was on the left. The Western blot shows a band that corresponds to a spot in the silver-stained gel (arrow).

 
Immunohistochemistry
No differences in immunoreactivity for myocilin/TIGR could be observed between eyes from different donors or between normal donor eyes and those enucleated because of posterior choroidal melanoma. However, in the two donor eyes with a postmortem time of 10 hours, positive staining was considerably weaker and barely detectable in some of the tissues.

Cornea
Intense staining for myocilin/TIGR was observed in cells of the corneal epithelium (Fig. 3A ). The staining was most pronounced in the basal cells and was seen throughout their entire cytoplasm.



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  		Figure 3. Myocilin/TIGR immunoreactivity in the human cornea (magnification, x1300). (A) Basal cells of the corneal epithelium show intense immunoreactivity throughout their entire cytoplasm (arrow). Wing cells are more weakly stained than basal cells, and no staining is seen in cells at the corneal surface. (B) In the corneal stroma, myocilin/TIGR stains in thin lines that are seen at regular distances of 0.6 µm and are strictly in parallel to the stromal collagen bundles (arrows). (C, D) Intense staining for myocilin/TIGR is seen in the cytoplasm of corneal endothelial cells. The staining appears to be more intense close to the cell membrane (arrows). No staining is seen in Bowman’s (A) or Descemet’s membranes (C).

 
Wing cells were more weakly stained than basal cells and no staining was observed in superficial cells at the corneal surface. In the corneal stroma (Fig. 3B) , myocilin/TIGR immunoreactivity was visualized in thin extracellular lines that were seen at regular distances of 0.6 µm and that were strictly in parallel to the stromal collagen bundles. Weak staining of keratocytes was seen in tangential sections through the corneal stroma. Intense staining for myocilin/TIGR was seen in the cytoplasm of corneal endothelial cells (Figs. 3C 3D) . Staining of corneal endothelial cells appeared to be more intense close to their cell membrane. No staining was seen in Bowman’s or Descemet’s membranes (Figs. 3A 3C) .

Iris and Ciliary Body
In the iris, smooth muscle cells of the sphincter and dilator muscle were positively stained for myocilin/TIGR, as were almost all resident cells in the iris stroma (Figs. 4A 4B ). In addition, positive staining was seen in vascular endothelial cells of iris vessels. In contrast to corneal epithelial and endothelial cells, immunoreactivity for myocilin/TIGR of iris cells appeared to be more intense in the periphery of the cytoplasm. In the ciliary muscle, smooth muscle cells were labeled for myocilin/TIGR in all parts of the muscle with equal intensity (Fig. 4C) . Similar to cells of the iris, positive staining of ciliary muscle cells was not seen in all parts of the cytoplasm, but was more intense close to the cell membrane (Fig. 4D) . A similar pattern of staining was seen in the vascular smooth muscle cells that surround ciliary body arteries and arterioles (Fig. 4C) . No staining for myocilin/TIGR was observed extracellularly between individual ciliary muscle bundles or in cells of the fibroblast sheaths that surround the muscle bundles.



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  		Figure 4. Immunoreactivity for myocilin/TIGR in iris (A, B) and ciliary muscle (C, D). (A) Smooth muscle cells of the dilator muscle are positively stained for myocilin/TIGR (arrows). In addition, almost all resident cells in the iris stroma and the endothelial cells of the iris vessels (arrowheads) are immunoreactive (magnification, x600). (B) In cells of the iris stroma, staining is seen not in all parts of the cytoplasm, but predominantly in the periphery close to the cell membrane (arrows, magnification, x1300). (C) Ciliary muscle cells are labeled for myocilin/TIGR throughout the entire muscle. In addition, staining is seen in the vascular smooth muscle cells that surround ciliary body arteries and arterioles (arrow). No staining for myocilin/TIGR is observed extracellularly between individual ciliary muscle bundles or in cells of the fibroblast sheaths that surround the muscle bundles (magnification, x340). (D) Positive staining of ciliary muscle cells is not seen in all parts of the cytoplasm but is more intense close to the cell membrane (arrows, magnification, x1300).

 
Ciliary Epithelium, Lens, and Vitreous Humor
In the ciliary epithelium, intense labeling was seen in the cells of the nonpigmented layer (Fig. 5A ). Staining of ciliary epithelial cells was seen throughout the entire cytoplasm and was weaker in ciliary epithelial cells of the pars plana region than in those of the pars plicata. No labeling was observed in the stroma of the ciliary processes. The anterior epithelial cells of the lens expressed positive cytoplasmic staining for myocilin/TIGR (Fig. 5B) . In addition, nucleated lens fibers in the bow region showed positive immunoreactivity (Fig. 5B) . No staining was seen in mature lens fibers without a nucleus and in the lens capsule. No staining was seen in fibers of the zonular apparatus. At the posterior surface of the lens, fine filamentous extracellular material that appeared to be attached to the posterior capsule was intensely labeled (Fig. 5C) . Fine filamentous material that was immunoreactive for myocilin/TIGR was also seen in other parts of the vitreous body (Fig. 5D) . This material was dispersed throughout the entire vitreous humor, but was more dense in region of the vitreous limiting membrane.



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  		Figure 5. Immunoreactivity for myocilin/TIGR in ciliary epithelium, lens and vitreous. (A) In the ciliary epithelium, intense staining is seen in the cytoplasm of cells of the nonpigmented layer (magnification, x1300). (B) The anterior epithelial cells of the lens express positive cytoplasmic staining for myocilin/TIGR (arrow, magnification, x1300). In addition, nucleated lens fibers in the bow region show positive immunofluorescence (arrowheads). (C) At the posterior surface of the lens (L), fine filamentous material that appears to be attached to the posterior lens capsule is intensely labeled (magnification, x600). (D) Filamentous material that is immunoreactive for myocilin/TIGR is seen throughout the vitreous body (magnification, x300).

 
Trabecular Meshwork and Sclera
Trabecular meshwork cells covering the lamellae of the uveal and corneoscleral meshwork were homogeneously positively labeled. No immunoreactivity for myocilin/TIGR was observed in the connective tissue core of the trabecular lamellae (Figs. 6A 6B ). In the cribriform or juxtacanalicular meshwork, staining for myocilin/TIGR showed differences between the eyes of different donors. In the eyes of some donors, staining was patchy and was only seen in some parts of the cribriform meshwork. In other donors, the cribriform meshwork was continuously labeled (Fig. 6C) . Positive staining in the cribriform area appeared to be both cellular and extracellular, but a clear distinction between both was not possible at the level of light microscopy that was used in the present study. Endothelial cells of Schlemm’s canal did not stain for myocilin/TIGR. In some areas, however, a thin line that was immunoreactive for myocilin/TIGR could be observed on the luminal side of Schlemm’s canal endothelial cells. Positive staining for myocilin/TIGR was seen in the sclera adjacent to Schlemm’s canal, as well as in other parts of the sclera (Fig. 6A) . Scleral staining was seen extracellularly, between the collagen bundles. Vascular smooth muscle cells of arteries and arterioles passing through the sclera into uveal tissues were invariably labeled for myocilin/TIGR. Similar to the staining of ciliary muscle and iris smooth muscle cells, positive staining was not seen throughout the entire cytoplasm, but was more intense in the periphery close to the cell membrane (Fig. 6D) .



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  		Figure 6. Immunoreactivity for myocilin/TIGR in trabecular meshwork and sclera. (A) Intense staining for myocilin/TIGR is seen in the trabecular meshwork (arrow) and between the collagen bundles of the adjacent sclera (magnification, x240). (B) Trabecular meshwork cells covering the lamellae of the corneoscleral and uveal meshwork are positively labeled (arrows). No immunoreactivity is observed in the connective tissue core of the lamellae (magnification, x1300). (C) Positive immunoreactivity is seen in large areas of the cribriform or juxtacanalicular meshwork (arrows) close to Schlemm’s canal (magnification, x600). (D) Vascular smooth muscle cells of arteries and arterioles passing through the sclera into the uvea are labeled for myocilin/TIGR (arrow). S, sclera; SC, Schlemm’s canal.

 
Retina, Lamina Cribrosa, and Optic Nerve
In all parts of the retina, immunoreactivity for myocilin/TIGR was observed in the nerve fiber layer, the outer and inner nuclear layer, and most intensly in region of the outer segments of the photoreceptors (Fig. 7A ). In the outer and inner nuclear layer, the cellular surface of most if not all cells was positively labeled (Fig. 7B) . Staining of photoreceptor outer segments was equally intense in rods and cones and was always confined to the outer surface of both (Figs. 7C 7D) . No distinct positive staining was seen in region of the connecting cilium between outer and inner segments. Weak or no staining was seen in the perikarya of optic nerve ganglion cells. In contrast, the axons of optic nerve ganglion cells stained intensely for myocilin/TIGR (Figs. 8A 8B ). This axonal staining was seen in peripheral and central parts of the optic nerve fiber layer, and in the prelaminar, laminar, and postlaminar part of the optic nerve. Double-staining experiments using antibodies to both neurofilament and myocilin/TIGR showed colocalization of both in numerous optic nerve axons (Figs. 8C 8D) . Similar results were obtained when antibodies against PGP 9.5 were used in combination with myocilin/TIGR (not shown). In addition to optic nerve axons, astrocytes in the prelaminar glial columns and the laminar cribriform plates were immunoreactive for myocilin/TIGR (Figs. 9A 9B ). In contrast, astrocytes in the postlaminar part of the optic nerve did not stain. This lack of staining was confirmed by double-labeling experiments with antibodies against both GFAP and myocilin/TIGR, which showed no colocalization of both antigens (Figs. 9D 9C) .



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  		Figure 7. Immunoreactivity for myocilin/TIGR in the human retina. (A) Positive immunoreactivity for myocilin/TIGR is seen in the nerve fiber layer, the outer and inner nuclear layer and, most intense, in region of the outer segments of the photoreceptors (arrow, magnification, x240). (B) Tangential section through the inner nuclear layer. Most, if not all of the cells are positively labeled (magnification, x1300). (C, D) Sagittal (C) and tangential (D) section through the outer segments of the photoreceptors (magnification, x1300). Positive immunoreactivity is equally intense in rods and cones and always is confined to the outer surface of both.

 


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  		Figure 8. (A, B) Immunoreactivity for myocilin/TIGR in optic nerve axons. Sagittal (A, magnification, x1300) and tangential (B, magnification, x240) section through nerve fiber bundles in the nerve fiber layer. Axons bundles of optic nerve ganglion cells show positive immunoreactivity for myocilin/TIGR (arrows). Double immunofluorescence using antibodies against myocilin/TIGR (C) and neurofilament (D) in the postlaminar optic nerve. Myocilin/TIGR and neurofilament colocalize in numerous optic nerve axons (arrows, magnification, x1300).

 


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  		Figure 9. Immunoreactivity for myocilin/TIGR in lamina cribrosa and optic nerve. Magnification, (A) x600; (B) x1300. In the prelaminar region (PRL) and the lamina cribrosa (LC) labeling is observed in optic nerve axons and in astrocytes of the glial columns and cribriform plates (arrows). Magnification, (C, D) x1300. Double immunofluorescence using antibodies against glial fibrillary acidic protein (GFAP) (C) and myocilin/TIGR (D) in postlaminar optic nerve. Postlaminar astrocytes show immunioreactivity for GFAP (arrows), whereas only optic nerve axons are labeled for myocilin/TIGR (arrows).

 
Retinal pigmented epithelial cells appeared to be negative for myocilin, but were difficult to evaluate because of their pronounced autofluorescence.

Ciliary Nerves and Choroid
Similar to optic nerve axons, some larger myelinated axons in the ciliary nerves were immunoreactive for myocilin/TIGR (Fig. 10) . Axonal staining for myocilin/TIGR showed colocalization with staining for PGP 9.5 (Figs. 10C 10D) and neurofilament (not shown). Staining was also seen in cells of the perineurium and, weaker, in those of the endoneurium. Smooth muscle cells of choroidal arteries and arterioles were positively stained.



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  		Figure 10. Immunoreactivity for myocilin/TIGR in ciliary nerves. Magnification, (A) x600; (B) x1300. Several myelinated axons in a ciliary nerve show strong positive staining (arrows). Staining is also seen in the perineurium and more weakly in the endoneurium of the nerve (arrowheads). Magnification, (C, D) x1300. Double immunohistochemistry of a ciliary nerve with antibodies against myocilin/TIGR (C) and the neuronal marker PGP 9.5 (D). Axonal staining for myocilin/TIGR and PGP-9.5 is clearly colocalized (arrows).

 
Controls
No positive staining was observed after preincubating the myocilin/TIGR antibody with the specific peptide or when the sections were incubated in PBS. The same was true when sections were incubated with preimmune rabbit antiserum (instead of the primary antibody) at a protein concentration comparable to that of the primary antibody. Using higher concentrations, some weak unspecific staining was observed in corneal epithelium and endothelium.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We developed and characterized an antibody against a peptide sequence of myocilin/TIGR that in 1D and 2D SDS-PAGE and Western blot analysis binds with high specificity to a protein with same electrophoretic mobility and isoelectric point as previously reported for myocilin/TIGR.20 21 We are therefore confident that our immunohistochemical data correctly visualize the in situ localization of myocilin/TIGR in the human eye. Our results show that myocilin/TIGR is found in almost every ocular tissue and with relatively strong immunoreactivity in trabecular meshwork, cornea, sclera, ciliary body, iris, retina, and optic nerve. This distribution of myocilin/TIGR largely corresponds with published Northern blot analysis hybridization data that reported a similar distribution of myocilin/TIGR mRNA in the tissues of the human eye.5 22 23 An exception appears to be the optic nerve, where we found a consistent positive staining for myocilin/TIGR in optic nerve axons, whereas Adam et al.5 could not detect any myocilin/TIGR mRNA. A likely explanation is that, like other neuronal proteins, myocilin/TIGR is translated in the perikarya of optic nerve ganglion cells in the retina and transported to the axons of the optic nerve by axoplasmatic flow. In support of this are data from in situ hybridization analyses that describe mRNA for myocilin/TIGR in mouse optic nerve ganglion cells26 and in distinct neurons in the mouse brain.31

The nature and function of myocilin/TIGR are largely unclear. Nguyen et al.21 reported that myocilin/TIGR is secreted by cultured trabecular meshwork cells into the surrounding culture medium, whereas others found only evidence for an intracellular localization of myocilin/TIGR in trabecular meshwork cells.27 28 Although our results provide for the first time clear evidence for an extracellular in situ localization of myocilin/TIGR in corneal stroma, sclera, and vitreous body, staining of the uveal and corneoscleral trabecular meshwork showed a distinct cellular staining of myocilin/TIGR. As for the myocilin/TIGR immunoreactivity in the cribriform or juxtacanalicular meshwork, it was not possible to clearly distinguish cellular from extracellular labeling, because of the technical limits of light microscopy. Clearly, studies using electron microscopy in conjunction with antibody labeling are necessary to define the exact localization of myocilin/TIGR in those parts of the trabecular meshwork that are most critical for aqueous humor outflow. Our results on myocilin/TIGR immunoreactivity in normal human trabecular meshwork in situ differ in some aspects from those previously reported by Lütjen–Drecoll et al.,27 who found only some cells in the inner parts of the meshwork positively stained for myocilin/TIGR and no immunoreactivity in the cribriform trabecular meshwork. This difference might be explained by the fact that a different antibody, generated against recombinant myocilin/TIGR, was used and that some epitopes critical for immunodetection by this antibody might have been lost during tissue processing.

Cellular staining for myocilin/TIGR was not confined to corneoscleral and uveal trabecular meshwork cells, but also was seen in other tissues of the anterior eye such as the corneal, ciliary, and lens epithelium, as well as the corneal endothelium, which all showed predominant labeling of their cytoplasm. A cytoplasmic localization of myocilin/TIGR might indicate a function of myocilin/TIGR different from those in tissues that express convincing extracellular labeling, such as the corneal stroma or the vitreous. Another likely possibility might be that these cells synthesize relatively large amounts of myocilin/TIGR and secrete it into the aqueous humor or in case of the corneal epithelium, into the inner mucous layer of the tear film. In support of the latter hypothesis appears to be the fact that myocilin/TIGR contains at its C terminus a relatively large olfactomedin domain.20 21 22 Olfactomedin is a component of the mucous layer of the frog olfactory epithelium.32 In other cell types, such as in cells of the iris stroma, and in vascular and ciliary smooth muscle cells, staining for myocilin/TIGR was predominantely associated with the peripheral cytoplasm close to the cell membrane. Direct membrane binding of myocilin/TIGR appears to be unlikely, because its protein sequence does not indicate the presence of domains that are regarded as characteristic for membrane-binding proteins. Still, myocilin/TIGR might associate with such proteins at the inner or outer surface of the cell membrane. Clearly, electron micoscopy is needed to clarify this issue.

In the retina, we found strong staining for myocilin/TIGR along the outer surface of rods and cones, but in contrast to others,22 no evidence for an association with the connecting cilium of the photoreceptors. Again, the exact ultrastructural localization of myocilin/TIGR remains to be clarified, but it is tempting to speculate that myocilin/TIGR might be part of the interphotoreceptor matrix. Other distinct retinal structures that showed positive labeling were the axons of the optic nerve ganglion cells. This labeling of optic nerve axons was not only seen in the optic nerve fiber layer, but also in its prelaminar, laminar and postlaminar parts. Although the function of myocilin/TIGR in optic nerve axons remains unclear, it appears to be a feature that is not unique to this kind of axons, because it also was observed in larger myelinated axons in the ciliary nerves.

In addition to optic nerve axons, astrocytes in the optic nerve head showed expression of myocilin/TIGR, which is in agreement with findings of others.33 34 This expression was confined to astrocytes in the glial columns and cribriform plates of the optic nerve, but was not seen in its postlaminar parts. For the human optic nerve, two subpopulations of astrocytes (1A and 1B) have been identified in the prelaminar and laminar regions.35 Type 1A astrocytes are present at the edges of the cribriform plates, whereas type 1B astrocytes are lining the cribriform plates and form the glial columns. Type 2 astrocytes are the predominant cell type in the myelinated postlaminar axon bundles. Based on this classification, myocilin/TIGR expression appears to be largely confined to 1B astrocytes. Different astrocyte populations are mainly distinguished by their different expression of various glial cell markers, whereas true differences in function remain largely unclear. In a recent study, we showed that myocilin/TIGR expression is induced upon mechanical stretch.36 Clearly, the lamina cribrosa is the part of the optic nerve that is most prone to mechanical influences that are caused by changes in IOP or eye movements.37 The function of myocilin/TIGR expressed by lamina cribrosa astrocytes might be associated with such factors.

Recently, mutations in the gene for myocilin/TIGR, which lie within the interval on chromosome 1 that was originally associated with juvenile open angle-glaucoma (GLC1A), have been identified in patients with autosomal-dominant juvenile open-angle glaucoma, as well as in some patients with adult-onset POAG.1 5 6 9 13 Because these patients express, depending on the site of the respective mutation, a relatively high IOP that requires early surgical treatment14 15 17 and because myocilin/TIGR has originally been isolated from cultured human trabecular meshwork cells,20 21 it has been generally assumed that the function of the trabecular meshwork for aqueous humor outflow is primarily affected by mutations in the coding sequences of myocilin/TIGR. Our findings of myocilin/TIGR expression in optic nerve axons and astrocytes indicate that abnormal myocilin/TIGR might also primarily interfere with function and survival of optic nerve axons in the lamina cribrosa. The trabecular meshwork might not be the only target of abnormal myocilin/TIGR in GLC1A-linked glaucoma.


   Acknowledgements
 
The authors thank Antonia Kellenberger for expert technical assistance and Marco Gößwein for the excellent preparation of the micrographs.


   Footnotes
 
Supported by grants from the Deutsche Forschungsgemeinschaft (Ta 115/8–1 and Ta 115/11–1) and the American Health Assistance Foundation (all to ERT).

Submitted for publication May 7, 1999; revised October 13, 1999; accepted October 26, 1999.

Commercial relationships policy: N.

Presented in part at the Annual Meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May, 1999.

Corresponding author: Ernst R. Tamm, Department of Anatomy II, University of Erlangen-Nürnberg, Universitätsstr. 19, D-91054 Erlangen, Germany. ertamm{at}anatomie.uni-erlangen.de


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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