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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 5, 2001 as doi:10.1096/fj.00-0663fje. |
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,
Glaucoma Research, Alcon Research, Ltd., Fort Worth, Texas 76134, USA;
* Department of Ophthalmology, Gifu University, Gifu, Japan; and
Department of Ophthalmology and Visual Sciences,
Department of Pediatrics, and
Howard Hughes Medical Institute, University of Iowa, Iowa City, Iowa, USA
2Correspondence: Glaucoma Research R2–41, Alcon Research, Ltd., 6201 South Freeway, Fort Worth, TX 76134, USA. E-mail: abe.clark{at}alconlabs.com
SPECIFIC AIMS
Glaucoma is a leading cause of blindness in the world, and the discovery of the glaucoma gene myocilin (MYOC) prompted numerous studies of its role in the molecular pathogenesis of glaucoma. In this study, we examined the expression of MYOC in the optic nerve head (ONH) of human eyes, a tissue involved in glaucomatous loss of vision. In addition, we screened for MYOC mutations in normal tension glaucoma (NTG) patients, a subset of glaucoma patients in which intraocular pressure is not appreciably elevated.
PRINCIPAL FINDINGS
1. MYOC mRNA is expressed in many human ocular tissues,
including the optic nerve head, and in two different cell types
cultured from the human optic nerve head
Analysis of MYOC mRNA expression using RT-PCR
demonstrated that many tissues of the eye express MYOC mRNA.
In addition to the expected locations—the trabecular meshwork (TM),
ciliary body, and retina—MYOC was found to be expressed in
the optic nerve and optic nerve head. In addition, MYOC mRNA
was expressed in cultured human lamina cribrosa (LC) cells and ONH
astrocytes, two different cell types found in the optic nerve head. To
further investigate MYOC expression in the optic nerve head,
we localized MYOC transcripts in the optic nerve head using
in situ hybridization. MYOC transcripts were found in high
abundance in cells lining the laminar sheets of the LC as well as in
the surrounding sclera, dura mater, arachnoid, pia mater, and the
perivascular connective tissue surrounding the central retinal artery
and vein.
2. Myocilin protein is expressed in the human ONH and in cells
cultured from the human ONH
Human optic nerve head tissues and cells were examined for
myocilin expression by immunofluorescence analysis. Intense labeling of
the optic nerve head and the pial septa of the postlaminar optic nerve
was detected with myocilin antibodies (Fig. 1A
). This labeling included, but was not restricted to, cells
aligned horizontally at the lamina cribrosa. No labeling of the choroid
or sclera was apparent, with the exception of the smooth muscle cells
that surrounded some large vessels. Two different types of cells
cultured from the optic nerve head—LC cells and optic nerve head
astrocytes—expressed myocilin protein (Fig. 1B
C
D
E
).
Myocilin was seen in a punctate staining pattern in vesicular
structures surrounding the nuclei of many, but not all, of these cells
(Fig. 1B
, D
E
). The location of some of these vesicles
appeared to overlap with the Golgi protein ß-COP (Fig. 1D
), suggesting that myocilin is located in the secretory
pathway. Extracellular staining of myocilin could also be seen in both
cultured LC cells and ONH astrocytes (Fig. 1C
Fig. 1, E
). In many
cases, LC cells and ONH astrocytes expressed both intracellular
and extracellular forms of myocilin (Fig. 1E
).
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The expression of myocilin in cultured ONH cells was confirmed by
2D-PAGE Western immunoblot analysis (Fig. 2
). Four to six different myocilin protein isoforms could be detected in
the media of cultured LC cells (Fig. 2A
) as well as in
lysates of cultured ONH astrocytes (Fig. 2B
), further
confirming intracellular and extracellular myocilin expression. The
immunoblotted myocilin proteins showed isoforms varying in apparent
mass from 55,000 to 57,000, with isoelectric points of 
5.2–5.3.
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3. Variations in the MYOC coding sequence are not
commonly associated with NTG
The fact that MYOC is expressed in the optic nerve
prompted us to investigate whether mutations in this gene are involved
in normal tension glaucoma, a form of glaucoma in which intraocular
pressure is not appreciably elevated. We screened the MYOC
gene in 213 patients and 102 normal subjects from Japan, in addition to
94 NTG patients and 91 normal subjects from Iowa. Overall, we observed
98 instances of 22 different variations in the MYOC gene.
However, only 10 instances of 6 different sequence variations would be
expected to alter the charge, size or polarity of the MYOC
gene product. In total, these variations were equally divided
(P=1.0, Fisher’s exact test) between the NTG patients
(6/307) and normal controls (4/193), although three of these
polymorphisms, Leu215Pro (n=2), Thr256Met (n=1),
and Trp286Arg (n=1), were seen only in NTG patients. Of the
22 different sequence variants we observed, 9 were seen only in
Japanese patients and 10 were seen only in patients ascertained in
Iowa.
CONCLUSIONS
Glaucoma is a term used to refer to a heterogeneous group of optic
neuropathies that cause a progressive loss of vision. It is a prevalent
disease (1–2% of the population over the age of 40 years) and a
leading cause of blindness worldwide. Glaucomatous damage to the eye
involves pathogenic changes in the trabecular meshwork, the ganglion
cell layer of the retina, and the optic nerve head (Fig. 3
). In many glaucomatous eyes, there is a progressive loss of TM
cells and a buildup of extracellular debris in the TM that results in
increased resistance to aqueous humor outflow and elevated intraocular
pressure. Elevated IOP causes a backward bowing of the optic nerve head
associated with compression and remodeling of the lamina cribrosa,
which occurs in almost all eyes affected with glaucoma.
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Myocilin (MYOC) was the first glaucoma gene discovered. It plays a very important role in the pathogenesis of autosomal dominant juvenile glaucoma and is involved in a small but significant subset of adult onset POAG. The prevalence of probable disease-causing MYOC mutations in POAG patients with a family history of glaucoma is 4.0% in the Japanese population and 4.4% in Caucasian patients, whereas the frequency of mutations in unselected POAG patients ranges from 2.6% to 4.4%. As a result, the majority of work on the role of myocilin in glaucoma has concentrated on its expression in the trabecular meshwork. The MYOC gene encodes a 57 kDa protein expressed in the trabecular meshwork, and its aberrant expression in the TM is thought to be responsible for the elevated IOP associated with some forms of glaucoma. Increased myocilin expression has been detected in the trabecular meshwork of patients with several different types of glaucoma, including primary open-angle glaucoma, pigmentary glaucoma, and pseudoexfoliation glaucoma.
This report demonstrates that myocilin is also expressed in the optic nerve head and in cultured cells derived from this region. These observations raise the possibility that myocilin could be involved in glaucomatous damage to the optic nerve at the level of the optic nerve head. There are many parallels in the expression of myocilin in ONH cells and in TM cells. Myocilin expression in cultured lamina cribrosa cells and ONH astrocytes occurs in discrete, intracellular vesicle-like particles that surround the nucleus. A similar intracellular localization of myocilin occurs in the TM. Myocilin is secreted from TM cells and is found associated with the TM extracellular matrix. Extracellular myocilin also is found in cultured LC cells and ONH astrocytes. These optic nerve cells are responsible for making and maintaining the complex lamina cribrosa tissue structure that structurally supports the RGC axons as they exit the rigid scleral coating of the eye. In addition, these cells generate and secrete neurotrophic factors that may provide trophic support to the RGC axons. It is quite possible that myocilin expression and/or function are involved in the normal homeostasis of the optic nerve head and that myocilin defects may be involved in glaucomatous optic neuropathy.
To determine whether MYOC is directly involved in NTG, a form of glaucoma in which IOP is not appreciably elevated, we screened NTG patients for mutations in MYOC. The data in our study suggest that coding sequence variations in the MYOC are not commonly involved in the NTG phenotype. Of course, this observation does not preclude a role for myocilin in the pathogenesis of NTG. That is, it is possible that other genes or even nongenetic factors act upstream from myocilin in the pathogenic path-way for NTG and that myocilin is a critical intermediate in this process. It is noteworthy that MYOC coding sequence variations are also extremely rare in patients with steroid-induced glaucoma despite the ample evidence for the glucocorticoid inducibility of the MYOC gene.
The precise roles that myocilin plays in the pathogenesis of glaucoma remain unknown. Additional studies are under way to explore the effects of normal and mutant myocilin expression in transfected cells and to determine the role of myocilin isoforms in the regulation of TM and ONH functions.
FOOTNOTES
1 To read the full text of this article, go to
http://www.fasebj.org/cgi/doi/10.1096/fj.00-0663fje ; to cite this
article, use FASEB J. (March 5, 2001)
10.1096/fj.00-0663fje 
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