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THE NATURE OF MYOCILIN

Myocilin is a 55–57-kDa glycoprotein that is highly expressed in several tissues of the anterior eye such as iris, ciliary body, cornea, and sclera (5–7). An extremely high expression has been observed in the trabecular meshwork (5,6,8). As a matter of fact, according to data of an un-normalized cDNA library, myocilin is one of the most highly expressed genes in the human trabecular meshwork (9). Outside the eye, considerable amounts of myocilin and its mRNA are synthesized by Schwann cells of peripheral nerves (10), whereas apparently lower levels are found in skeletal muscle, heart, optic nerve, brain, and podocytes of the kidney (11–18). Most studies have focused on the expression pattern and localization of myocilin in humans and common laboratory animals such as mouse and rat, and the available results indicate that there are no substantial differences between these species (19–25). Regarding the subcellular localization of myocilin, some earlier studies reported on an intracellular location and the association of myocilin with microtubules (26,27), mitochondria (28), actin, vimentin, or the myosin regulatory light chain (29,30). Currently, the overall consensus of multiple laboratories appears to be that myocilin is an extracellularsecreted protein that is found in the supernatant of cultured cells in vitro (31–39) and in the aqueous humor of various species including that of humans in vivo (31,34,36,37,40–43). Consistent with an extracellular role of myocilin are data that show binding of myocilin to other extracellular proteins such as fibronectin (44), optimedin (21), or hevin, a secretory protein of the BM-40/SPARC/osteonectin family (45).

Myocilin is defined by two major domains: a coiled-coil domain containing a leucine zipper motif near the N-terminal and an olfactomedin-like domain near the C-terminal (2). The olfactomedin-like domain is common to a family of extracellular matrix molecules called olfactomedins (46–49). Myocilin exists mainly in a multimeric structure, which is caused by non-covalent interactions between the leucine zipper motifs (50) and extensive disulfide bond formation using five cysteine residues (40). Consequently, myocilin is found in the human aqueous humor in complexes of 120 and 180 kDa (50) and in bovine and monkey aqueous humor in complexes of more than 200 kDa (41). The presence of disulfide bonds in myocilin multimers also strongly argues for a role of myocilin as secretory protein, as disulfide bonds, which are usually formed in the endoplasmic reticulum, are frequently observed in secretory proteins but not in proteins of the cytosol.

Some peculiarities as to the secretion of myocilin have been reported. Hardy and coworkers observed that in cultured trabecular meshwork cells, myocilin associates with the extracellular membrane of lipid particles that have some biochemical characteristics of exosomes and that the release of myocilin in the extracellular space occurs in association with exosome-like vesicles (51). The same group provided evidence that myocilin is associated with exosome-like material in the human aqueous humor (52). It was suggested that this mode of secretion is specific for the trabecular meshwork, which appears to be unlikely as myocilin in the aqueous humor very likely does not derive from the chamber angle but rather from other sources such as iris and ciliary body. The binding of myocilin to the exosome membrane was reported to involve the coiled-coil domain but not the olfactomedin domain (53). Some data from other

laboratories give support to the observation of an association between myocilin and cell membranes. Ricard and co-workers found an association of canine myocilin with lipids of putatively cell membrane origin (54) while Joe and colleagues reported on data indicating an interaction between myocilin and flotillin-1, an integral membrane protein and constituent of lipid rafts (55). Another peculiarity of the secretory mechanism of myocilin refers to the fact that recombinant myocilin in transfected immortalized laboratory cell lines (e.g., COS1 and HEK293) is often not only secreted as full-length protein but also together with a C-terminal cleavage product that appears to result from intracellular endoproteolytic cleavage of myocilin (34,38,56). The site of cleavage differs between different cell lines and experimental settings and has been reported to be between amino acids 214/215 (38,56) or amino acids 226/227 (34). C-terminal cleavage fragments should not associate with exosome-like membranes during their secretion, as they do not contain the coiled-coil domain of the full length protein. So far, the relevance (if any) of myocilin cleavage products for the living organism is unclear. Smaller fragments that stain with myocilin antibodies have been observed in aqueous humor samples (34,41), but it is not clear whether these fragments result from intracellular endoproteolytic cleavage as in immortalized cell lines or from extracellular proteolytic cleavage inside or outside the organism. No such cleavage products have been observed in fresh human trabecular meshwork (7,38), fresh human iris and ciliary body (34), in the supernatant of monolayer cultured trabecular meshwork cells (57,58), nor in tissues (59) or the aqueous humor (42) of transgenic mice with experimental overexpression of myocilin.

Very limited data are available that point to a specific function of myocilin. Interesting data were provided by Peters and colleagues who investigated the effects of recombinant myocilin on spreading and substrate adhesion of fibroblasts (60). Fibroblast attached but failed to spread on myocilin as substrate. In addition, spreading of fibroblasts on the Hep II domain of fibronectin as substrate was significantly inhibited in the presence of myocilin, as was focal adhesion formation and the incorporation of paxillin into focal adhesions. The data appear to indicate that myocilin could act as matricellular protein that modifies the number of contacts between cells and extracellular matrix in the trabecular meshwork. In general, matricellular proteins are secreted proteins that influence cell function by modulating cell–matrix interactions (61,62). Prominent matricellular proteins are thrombospondin-1 and SPARC, and it is interesting to note that, similar to myocilin, both proteins inhibit spreading of fibroblasts under culture conditions (63,64) and are constitutively expressed (albeit at much lower amounts than myocilin) in the trabecular meshwork (65,66). The expression of counteradhesive matricellular molecules in the trabecular meshwork might be important to facilitate a continuous remodelling of cell–matrix contacts in the juxtacanalicular and inner wall regions. Such a remodelling should facilitate the continuous formation of interand intracellular pores and/or giant vacuoles in the inner wall of Schlemm’s canal endothelium and constitute an important mechanism to modulate flow through the trabecular meshwork (67,68). Whatever will turn out to be the function of myocilin in vivo, it is obviously not a critical factor for the function of the outflow pathways (at least in the mouse eye) as knockout mice with a targeted deletion of myocilin do not develop an obvious phenotype (69).

THE ROLE OF NORMAL OR WILDTYPE MYOCILIN

FOR GLAUCOMA

Some experimental evidence supports the hypothesis that secreted myocilin plays a role in modulating the hydrodynamic outflow resistance in the trabecular meshwork and that elevated amounts of myocilin could obstruct the outflow system. The expression of myocilin is induced by treatment with dexamethasone in cultured trabecular meshwork cells and perfusion organ cultures in a time-dependant manner and comparable to the time course that is observed during the development of steroid-induced ocular hypertension and glaucoma (15). Moreover, an increase in immunostaining for myocilin has been reported in the trabecular meshwork of patients with POAG (70). Furthermore, recombinant myocilin is very effective at blocking polycarbonate filters with a pore size similar to that of the TM (38). In addition, myocilin in the aqueous humor is tightly bound to polycarbonate filters that become obstructed after perfusion with aqueous humor (41). Still, the strongest support for the hypothesis that elevated myocilin obstructs the outflow system comes from data provided by Fautsch and coworkers (56). The authors purified human recombinant myocilin from an eukaryotic expression system and perfused human anterior eye segment organ cultures with 2 μg/mL of recombinant myocilin. When myocilin was perfused in porcine aqueous humor, a significant increase in outflow resistance was observed. Similar effects were observed when myocilin was preincubated with porcine aqueous humor. The maximum outflow resistance was obtained 5–17 h after infusion and remained above baseline for more than 3 days. In contrast, only minimal effects were observed when myocilin was perfused with regular cell-culture medium, indicating that myocilin needs to form a complex with proteins in the aqueous humor that enables it to bind specifically within the trabecular meshwork. So far, the protein(s) that interact(s) with myocilin in porcine aqueous humor have not been identified, but experimental data suggest that albumin, a very abundant protein in aqueous humor, is not the binding partner that is necessary for myocilin-induced outflow resistance. In those eyes that showed an increase in outflow resistance, myocilin accumulated in high amounts in the juxtacanalicular trabecular meshwork supporting the concept that outflow resistance can be modified by extracellular matrix compounds in the juxtacanalicular outflow pathways. The results with eukaryotic myocilin differ markedly with that of a previous study using myocilin from a bacterial source (71) and strongly emphasize that functional data on myocilin need to be obtained with eukaryotic myocilin.

Although the data obtained with myocilin perfusion of anterior eye segment organ cultures are impressive, experimental evidence for a direct role of myocilin on outflow resistance in the living organism is lacking. Zillig and co-workers developed transgenic mice that strongly express myocilin in their lenses (42). The transgenic expression of myocilin from the lens resulted in an almost fivefold increase of secreted normal myocilin in the aqueous humor of the transgenic mice. Nevertheless, the intraocular pressure of the transgenic animals did not differ from that of control mice. Comparable data were obtained in a different laboratory with another set of transgenic mice that had been genetically modified to overexpress myocilin and that did not show significant changes in intraocular pressure (59). A possible explanation for the different results between transgenic animals and organ culture studies could be that myocilin

is present at much lower concentrations in the living transgenic eye (0.2 μg/mL) (42) than in perfused organ cultures (2 μg/mL) (56). Still, the relevance of these amounts for glaucoma in humans remains unclear, as it is not known whether eyes treated with dexamethasone and/or suffering from glaucoma can produce myocilin at those high amounts that were used for organ culture experiments. Another explanation for the differences between the living mouse eye and the organ-cultured human anterior segment could be species-related structural and/or biochemical differences between the trabecular meshwork of mice and men.

THE ROLE OF MUTANT MYOCILIN FOR GLAUCOMA

Data from multiple laboratories provide overwhelming evidence that recombinant myocilin-harboring mutations, which would cause a severe glaucoma phenotype in humans, are not secreted from cultured cells (31,33,35,36,42,72,73). The predominant subcellular localization of mutated myocilin in vitro has been shown to be the endoplasmic reticulum (32,33,39,73,74). Comparable data were observed in transgenic mice in vivo with ectopic expression of mutated human Y437H myocilin in the lens (see Fig. 1) (42). In patients, this mutation causes an aggressive form of juvenileonset POAG (75). In contrast to wildtype myocilin, mutated myocilin was not secreted into the aqueous humor of transgenic animals but accumulated in the endoplasmic reticulum of lens fibers (see Fig. 1) (42). The reason for the lack of secretion appears to be misfolding of mutated myocilin resulting in a highly aggregation-prone protein that forms large aggregates (73,76). Interestingly, culturing cells at 30°C, a condition known to facilitate protein folding, promotes secretion of mutant myocilin and normalizes cell morphology (73). Part of the mutated myocilin seems to aggregate with wildtype myocilin and to form heteromeric wildtype/mutant aggregates (31,32,35) that are not secreted, an effect that results in a diminished secretion of extracellular wildtype myocilin (31,36,77). Overall, intracellular sequestration and temperaturesensitive secretion are very characteristic and have been shown to be associated with the vast majority of glaucoma-causing mutations in myocilin (35,78). Similar effects as in human-mutated myocilin were observed in studies introducing glaucoma-causing mutations in mouse myocilin (39).

It is not entirely clear how the intracellular sequestration of mutated myocilin should cause glaucoma in patients. One possibility could be the formation of heteromeric wildtype/mutant aggregates resulting in lower levels of wildtype myocilin. The data from knockout mice with a targeted deletion of myocilin, which do not develop an obvious phenotype and glaucoma (69), strongly argue against this possibility. Another possibility is that the intracellular sequestration of mutated myocilin initiates a cellular unfolded protein response, which results in cell stress and finally apoptotic cell death (79,80). Misfolded proteins are usually recognized by control systems in the rough endoplasmic reticulum, transported to the cytosol, and degraded by ubiquination and proteosomal degradation (81). Failure of this mechanism results in the accumulation of misfolded protein in the endoplasmic reticulum, in the congestion of the secretory pathway, and dysfunction of the endoplasmic reticulum and initiates the unfolded protein response that leads to cell death. Such a scenario is thought to cause cell death in several inherited neurodegenerative disorders that are—similar to

glaucoma associated with myocilin mutations—dominant, delayed disorders (82–85). Indeed, the accumulation of mutated myocilin in the endoplasmic reticulum in cultured cells induces cytotoxic changes, cell death, and apoptosis (32,33,73,74) but not necessarily a general block of the secretory pathway (39). Comparable findings have been reported in a transgenic animal with ectopic expression of mutant myocilin in the lens (42). The accumulation of mutant proteins in the endoplasmic reticulum of lens fibers resulted in the formation of nuclear cataracts, loss of transparency, cell death, and finally the rupture of the lens.

Clearly, a similar scenario in patients with myocilin glaucoma could lead to cell death of trabecular meshwork cells resulting in structural changes of the outflow pathways. Such changes might cause a substantial increase in outflow resistance in the trabecular meshwork and finally lead to the very high levels of intraocular pressure, which are commonly observed in patients suffering from glaucoma because of mutations in the myocilin gene (75). Still, data from mouse models do not entirely support this concept. Gould and colleagues developed mice carrying a mutant allele of the myocilin gene (86). The mutation Y423H was used, which is analogous to the particular severe human mutation (Y437H) that causes an aggressive form of juvenile-onset POAG (75). As in cell-culture studies, mutant myocilin was not secreted but accumulated in cells of the chamber angle. Nevertheless, this accumulation did not lead to the initiation of an unfolded protein response, to an increase in intraocular pressure, nor to glaucomatous changes. Taken together, the data of Gould and co-workers indicate that apparent misfolding and non-secretion of mutant myocilin is not sufficient to cause glaucoma in the mouse eye. Surprisingly, data from another mouse model are at variance with those of Gould et al. Senatorov and co-workers developed transgenic mice with overexpression of mutated myocilin by introducing a bacterial artificial chromosome encoding the human Y437H mutation into the mouse genome (87). Similar to findings in the other mouse models with this mutation (42,86), mutated myocilin was not secreted (87). Despite the absence of any significant pathological changes in the trabecular meshwork, the mice developed a moderate elevation of intraocular pressure, which was about 2 mmHg higher than in eyes of wildtype littermates. Correlated with the increase was a continuous loss of retinal ganglion cells similar to the situation in glaucoma. So far, it is completely unclear why the mouse models developed by Gould and coworkers and by Senatorov and co-workers differ so substantially in their phenotypes. Still, none of these models provides support for the unfolded protein response in the rough endoplasmic reticulum as causative for glaucoma associated with mutant myocilin.

A reasonable approach to shed some light on the role of mutant myocilin for the outflow pathways and the development of glaucoma could be a thorough

Fig. 1. (Continued) their origin from rER cisterns, which are of normal size in wildtype littermates (arrows in E). (G and H) Immunocytochemistry with antibodies specific for myocilin shows strong positive immunoreactivity in lens fiber vesicles (arrows) of transgenic animals and confirms that the vesicles are caused by an accumulation of Y437H-mutated myocilin in the rER. Scale bars: A and B, 16 μm; C and D, 690 nm; E and F, 166 nm; G, 6 μm; and H, 4 μm (adapted with the permission of the Association for Research in Vision and Ophthalmology (42)).