Journal of Undergraduate Research
Volume 8, Issue 3 - January / February 2007

Myocilin Localization in the Canine Eye by Confocal Microscopy

Heba Tajwar

ABSTRACT

In this study, the presence of myocilin protein has been investigated in a colony of Beagles with inherited glaucoma. The localization of Myocilin protein in the normal and glaucomatous was examined in canine eyes of beagles using immuno-histochemistry and confocal microscopy. Paraffin-embedded specimens from the anterior uveas of 5 beagles with inherited glaucoma and age-matched normal beagles were sectioned and incubated with a primary antibody. Labeled specimens were analyzed using confocal microscopy. Within the normal, moderate, and advanced glaucomatous beagle specimens, the cytoplasm of the nonpigmented epithelium of the ciliary body labeled positively but unevenly for myocilin protein. The ICA and trabecular meshwork cells were thoroughly labeled for myocilin as well. Intensity of the labeling was more intense for advanced and moderate glaucomatous specimens of beagles than the normal specimens. The same was true with regard to the ciliary body, but in the glaucomatous canines the intense labeling was uneven. The intense and uneven labeling of the nonpigmented epithelium in moderate and advanced specimens suggests that myocilin protein is produced in the nonpigmented epithelium and released through vesicles abnormally into the aqueous humor. Changes in myocilin protein may alter the dynamics of the aqueous humor outflow pathway and thus create an excessive rise in intraocular pressure and intensify the effects in individuals with spontaneous glaucoma.

INTRODUCTION

According to the Center for Disease Control (CDC), glaucoma was diagnosed as the cause of eye disease for 76.9 percent of patients seen by ophthalmologists in 1992. Glaucoma is a disease of the eye that causes a permanent loss of vision by progressive destruction of the inner retina and the optic nerve head. Primary open angle glaucoma (POAG) accounts for the majority of cases of glaucoma among humans and canines, as opposed to secondary glaucoma that is a result of extraneous ocular complications that can lead to elevated intraocular pressure and thus glaucoma.  The prevalent risk associated with POAG is elevated pressure in the eye. According to a 2002 study, immediate relatives of POAG-positive individuals have an eight-fold increased susceptibility rate of developing POAG as compared to the general public (Fingert, Stone, Sheffield, & Alward, 2002). Recently, human POAG was associated with defects in myocilin protein (Johnson, 2000). Since then numerous other mutations in 16 genes have been identified with POAG (Tamm & Polansky, 2001), among which GLC1A, associated with the manufacturing of myocilin protein, has been predominantly reported (Fingert et al., 2002).  Among 40 breeds of dogs that are genetically predisposed to developing POAG, beagles exhibit the trait as autosomal recessive (Gelatt, Brooks, & Samuelson, 1998).

In normal human and canine specimens, myocilin protein is found throughout ocular tissues. Myocilin is produced largely in the trabecular meshwork (TM) as well as scleral areas involved in aqueous humor outflow (Gelatt, MacKay, Kallberg, Samuelson, Barrie, Ollivier, Plummer, Tenorio, Kligman, Gartlidge, Miller, Andrew, & Dennis, 2005). Myocilin has been discovered in canine aqueous humor as well; however, its origin has remained unknown (Russell P, Tamm, Grehn, Picht, & Johnson, 2001). One quality of myocilin protein is that it is produced in the greatest amount during certain kinds of stress. For example, myocilin is induced during mechanical stretch, oxidative stretch, and treatment with tissue growth factor or glucocorticosteroids (Fingert, Stone, Sheffield, & Alward, 2002). Based on the findings, myocilin’s role within the body seems to be protective. It is proposed that myocilin can function as a molecular chaperone, by binding to certain proteins and preventing them from denaturing. Within the glaucomatous eye, it is proposed that myocilin is expressed in high amounts predominantly within the TM where it is thought to interfere with aqueous humor outflow by binding to extracellular molecules or the cell membrane (Tamm, 2002).

Myocilin is a glycoprotein that contains 504 amino acids and has a molecular weight of 55-57 kDa. The protein consists of two major domains: an olfactomedin domain towards the C terminal (which is highly conserved) and a myosin-like domain towards the N terminal. Within the N terminal is a leucine zipper between nucleic acids 117 and 166. Mutations that occur in exon 3 of the olfactomedin domain are generally associated with inherited juvenile glaucoma and POAG (Tamm, 2002).

The main objective of this study was to determine where myocilin protein resides within the canine eye, and to observe any new relationships  with inherited primary open angle glaucoma by using the method known as confocal microscopy. Tissue samples from a colony of beagles with an inherited hypertensive form of glaucoma were used in this study (Gelatt, Gum, Gwin, Bromberg, Merideth, & Samuelson, 1981). In a previous study conducted by our lab, Western blot analysis has been used on the colony of beagles, and we have found myocilin in the aqueous humor of members in the colony. Our findings revealed that colony members with moderate and advanced forms of POAG exhibited a large amount of myocilin within their aqueous humors (MacKay, Hart, Gelatt, Samuelson, Esson, & Sherwood, 2004).          

METHODS

Paraffin-embedded specimens from the anterior uveas of 5 beagles with inherited glaucoma (two moderate glaucomatous at 2.5 and 6-yrs-old, and one advanced glaucomatous at 13-yrs-of age) and age-matched normal beagles (one preglaucomatous at 3-mos-old, and one early glaucomatous at 1-yr-old) were sectioned and incubated with a primary antibody (rabbit polyclonal anti-human MYOC IgG, made by Santa Cruz Biotechnology), diluted with 3% BSA blocker for 1 hour at RT. Specimens were successively washed in three baths of TBS and then incubated with a secondary antibody (goat anti-rabbit Texas Red made by Molecular Probes), diluted with Tris buffer for one hour at RT. Labeled specimens were analyzed using confocal microscopy.

RESULTS

The Confocal microscope contains an argon laser that functions to illuminate the fluorescent tags on the tissue. Confocal microscopy (CM) is also a useful tool when determining three dimensional structures that would otherwise not be visible through light microscopy. Also, the intensity of the signal is very distinct, as shown by our samples. Through successive staining and analysis of our results it is clear that myocilin protein has an affinity for distinct parts of the eye, mainly the ciliary body, the ICA and surrounding tissues (figs. 1 & 2). Within the ciliary body, the cells of the nonpigmented epithelium are distinctly illuminated for myocilin protein (figs 1A & 1D). Vesicles protruding from the ciliary body were strongly positive for myocilin in the advanced glaucomatous beagle. It is interesting to note the near linear fashion in which these vesicles seem to be moving from the inner parts of the tissue to the outer membrane of the ciliary body (fig. 1B). The intensity of illumination was also pronounced when animals of normal, moderate, to advanced glaucoma were analyzed (figs. 1A – 1D).

Figure 1. Analyzation of Advanced Glaucoma

Figure 1. Analyzation of Advanced Glaucoma. Fig. 1A. Localization of myocilin by CM in the ciliary process of a young beagle with POAG (X1000). Fig. 1B. Localization of myocilin by CM in the CB of an adult beagle with advanced POAG (X1800). Fig. 1C. Localization of myocilin in CB of adult beagle with moderate POAG (X1000). Fig. 1D. of myocilin protein by CM in CB of an adult beagle with moderate POAG. Notice the intensity of color in the nonpigmented epithelium (X400).

The intensity of the staining of the ICA of the normal specimen compared to the moderate and advanced specimens was relatively low (fig. 2A & 2B-2D). It is important to note that the strongly labeled structures in the center of the normal specimen (fig. 2A) are red blood cells that auto-fluoresce and must not be mistaken for a positive label for myocilin protein. Within the ICA, myocilin seems to attach to the ridges of the tissue rather than uniformly throughout the tissue.

Figure 2. Staining of the ICA of the normal specimen

Figure 2. Staining of the ICA of the normal specimen. Figure 2a. Localization of myocilin in the ICA of a young beagle with POAG (X400). Figure 2b. Localization of myocilin protein in the ICA of an adult beagle with advanced POAG (X400). Figure 3b. Localization of myocilin in the ICA of an adult beagle with moderate POAG (X400). Figure 2d. Localization of myocilin protein in the ICA of an adult beagle with moderate POAG (X400).

DISCUSSION

The findings of this study support the possibility that changes in the GLC1A gene, and thus myocilin protein, inhibits the normal activity of myocilin within the aqueous humor outflow pathway. In previous studies it has been thought that an overproduction of myocilin caused an abnormal accumulation of protein in TM cells, and thus inhibited aqueous humor outflow. However, subsequent studies have shown that the actual mutation in the GLC1A gene may be responsible for the accumulation of a faulty form of myocilin that is now accumulating within the trabecular meshwork (Tamm, 2002). Known for its ‘sticky’ property, it is thought that myocilin may be causing resistance to aqueous humor outflow by binding to extracellular components of the ECM or to the cell membrane of TM cells (Ueda, & Yue, 2003. & Fautsch, & Johnson, 2001. & Fautsch, Bahler, Jewison, & Johnson, 2000. & Fautsch, Vrabel, Peterson, & Johnson, 2004). The protein isolated from aqueous humor exhibits a unique form of myocilin protein where the leucine zipper component of the N -- terminal of one myocilin protein interacts by forming non-covalent bonds with another myocilin molecule. In this manner, the individual proteins form a kind of dimer within the eyes (Fautsch, Vrabel, et. al., 2005).

From this study we have gathered that myocilin protein exhibits a significantly stronger signal within the ciliary body, ICA and trabecular meshwork for individuals who have POAG compared to normal individuals, as seen in previous studies mentioned above. The intensity of myocilin within the ciliary body is extensive within the nonpigmented epithelium, specifically in moderate and advanced glaucomatous specimens. Within the angle and trabecular meshwork, again the illumination of myocilin protein is much greater in moderate and advanced individuals as opposed to the normal control. The presence of large amounts of myocilin protein within secretory vesicles of the nonpigmented epithelium indicates that the protein is produced within the ciliary body, especially selected cells of the nonpigmented epithelium of glaucomatous individuals.

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