Genetically increasing Myoc expression supports a necessary pathologic role of abnormal proteins in glaucoma

Abstract

Despite the importance of MYOC for glaucoma, the protein's normal function(s) and the pathogenic mechanism(s) of MYOC mutations are not clear. Elevated intraocular pressure (IOP) and glaucoma are sometimes induced by corticosteroids, and corticosteroid use can result in substantially increased MYOC expression. It has been suggested, therefore, that steroid-induced MYOC protein levels cause steroid-induced glaucoma and that protein level-increasing mutations in MYOC contribute to glaucoma not associated with steroid use. A causative role of elevated MYOC levels in steroid-induced glaucoma is controversial, however, and it is not clear if elevated MYOC levels can result in IOP elevation. To directly test if increased levels of MYOC can cause IOP elevation and glaucoma, we generated bacterial artificial chromosome transgenic mice that overexpress Myoc at a level similar to that induced by corticosteroid use. These mice do not develop elevated IOP or glaucoma. Our present findings, along with the absence of glaucoma in mice completely lacking MYOC, show that changing the level of MYOC is not pathogenic (from absent to approximately 15 times normal). These findings suggest that noncoding sequence variants are unlikely to influence glaucoma and that disease pathogenesis in primary open-angle glaucoma patients is dependent upon the expression of abnormal mutant proteins. This work does not support a causative role for increased MYOC levels or the MYOC gene in steroid-induced glaucoma.

FIG. 1.

BAC transgene and protein levels. (A) Schematic of the BAC containing the entire Myoc gene and flanking genomic DNA. The BAC contains an approximately 20-kb region upstream of exon 1 and an 85-kb region downstream of exon 3. The upstream sequence includes a strain-specific sequence length polymorphism (arrow) that is approximately 4 kb from exon 1. The length of each exon is indicated in nucleotides. (B) Substantial increase in MYOC protein in transgenic mice compared to that in control mice. Total protein was isolated from ocular tissue enriched in drainage structures and analyzed by Western blotting (see Materials and Methods). Twenty-five micrograms of total protein was loaded in each lane. Lanes are homozygous transgenic (Tg/Tg), hemizygous (Tg), and control (wt [wild type]). Molecular masses are indicated on the left.

FIG. 2.

Slit lamp examination and IOP measurement. Slit lamp photographs of a normal control (wild type [WT]) eye (A) and a Tg/Tg eye (B). Visible structures, i.e., the cornea, iris, and lens, all appear normal in the Tg/Tg mouse. Slit lamp examinations were conducted on 16 Tg/Tg mice between 14.5 and 27 months of age. (C) IOPs of Tg/Tg and control mice were determined at a variety of ages. For each group, the mean and standard error of the mean are shown. Sex had no effect on the IOP of either genotype, and so the data were analyzed irrespective of sex. There were no significant differences in IOP between mice of each genotype at any age (P = 0.9, 0.1, 0.3, and 0.8 comparing each genotype at 4, 8, 12 to 18, and >19 months, respectively). Starting with 4 months and ending with >19 months, the numbers of control (C) and transgenic (Tg/Tg) mice were as follows: C, 20 and Tg/Tg, 20; C, 17 and Tg/Tg, 11; C, 9 and Tg/Tg, 11; and C, 14 and Tg/Tg, 11.

FIG. 3.

Morphology and ultrastructure of the iridocorneal angle. Representative H&E-stained sections of eyes from a 25-month-old control (wild type [WT]) mouse (A) and a 27-month-old Tg/Tg mouse (B). The iridocorneal angle, TM (arrowhead), and Schlemm's canal (between arrows) are not affected by genotype and appear normal. The eyes of mice of both genotypes contain pigment-filled cells (asterisks). This is typical in C57BL/6J mice at this very old age and is not a phenotypic consequence of the transgene. There were no detectable histological differences in the iridocorneal angles of each genotype between 18 and 30 months of age. Scale bar, 40 μm. Electron micrographs of eyes from 20-month-old control (C) and Tg/Tg (D) mice. Similar results were observed in mice more than 2 years old. The TM of mice of each genotype has a normal morphology including an endothelium-lined Schlemm's canal (SC), trabecular beams with organized collagen fibrils (filled arrowhead) and elastic tissue (open arrowhead), and open intertrabecular spaces (arrow). The anterior chamber is identified as AC. A normal myelinated nerve (N) is visible in the transgenic eye. Scale bars, 1 μm.

FIG. 4.

The optic nerve head and retinal ganglion cell axons. Representative H&E-stained sections of eyes from a 25-month-old control (wild type [WT]) (A) and a 27-month-old Tg/Tg (B) mouse. Both have a healthy optic nerve head with a thick nerve fiber layer (arrow). The transgenic mouse shows no signs of optic nerve head cupping. A blood vessel is typically present over the central optic nerve in mice (arrowhead) and is variably associated with pigment in normal mice of this genetic background. Optic nerve cross sections of a 19-month-old control mouse (C) and a17-month-old Tg/Tg mouse (D) are indistinguishable, without significant axon damage or loss. The same result was observed in mice >2 years old. No optic nerves from aged Tg/Tg mice (n = 6) showed signs of glaucomatous damage. Scale bars, 100 μm.