Soluble Guanylate Cyclase α1–Deficient Mice: A Novel Murine Model for Primary Open Angle Glaucoma

Primary open angle glaucoma (POAG) is a leading cause of blindness worldwide. The molecular signaling involved in the pathogenesis of POAG remains unknown. Here, we report that mice lacking the $α_1$ subunit of the nitric oxide receptor soluble guanylate cyclase represent a novel and translatable animal model of POAG, characterized by thinning of the retinal nerve fiber layer and loss of optic nerve axons in the context of an open iridocorneal angle. The optic neuropathy associated with soluble guanylate cyclase $α_1$–deficiency was accompanied by modestly increased intraocular pressure and retinal vascular dysfunction. Moreover, data from a candidate gene association study suggests that a variant in the locus containing the genes encoding for the $α_1$ and $β_1$ subunits of soluble guanylate cyclase is associated with POAG in patients presenting with initial paracentral vision loss, a disease subtype thought to be associated with vascular dysregulation. These findings provide new insights into the pathogenesis and genetics of POAG and suggest new therapeutic strategies for POAG

Intraocular pressure (IOP) increases with age in sGCα₁^−/− mice.

<p>IOP, measured serially at 2 time points (19±1 and 37±3 weeks) in eyes from age-matched wild-type (WT, <b>left panel</b>) and soluble guanylate cyclase α₁-deficient (sGCα₁^−/−) mice (<b>right panel</b>). While IOP remained stable in WT mice as they aged from 19 to 37 weeks (14±2 to 14±2 mmHg; <i>n</i> = 25; <i>P</i> = 0.67), IOP increased in sGCα₁^−/− mice (14±2 to 18±3 mmHg; <i>n</i> = 37; *<i>P</i> = 1.9×10⁻⁸).</p

Retinal vascular dysfunction in sGCα₁^−/− mice. A

<p>: Representative trace depicting the diameter in one segment of a retinal arteriole in a wild-type (WT) mouse before, during (arrow), and after injection of the NO-donor compound sodium nitroprusside. Dashed lines indicate the diameter before and after sodium nitroprusside injection. <b>B</b>: Quantitative analysis of the change in diameter (double arrow in fig. 6A) induced by injection of 0.8 mg/kg sodium nitroprusside in WT and sGCα₁^−/− mice. <i>n</i> = 5 mice (3–4 arterioles per mouse were assessed, see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060156#pone.0060156.s003" target="_blank">fig. S3</a>). *<i>P</i> = 4.1×10⁻³.</p

Retinal nerve fiber layer (RNFL) thinning and glaucomatous optic neuropathy in sGCα₁^−/− mice. A

<p>: Quantitative analysis, assessed by SD-OCT (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060156#pone.0060156.s001" target="_blank">fig. S1</a>), of total retinal thickness (<b>left panel</b>) and RNFL thickness, in young (6-week-old, <b>middle panel</b>) and old (70-week-old, <b>right panel</b>) wild-type (WT, <i>n</i> = 19 and 13, respectively) and soluble guanylate cyclase α₁-deficient (sGCα₁^−/−) mice (<i>n</i> = 15 and 14, respectively; *<i>P</i> = 1.2×10⁻²). <b>B</b>: Representative whole-mount retinas from age-matched young (20-week-old) and old (56-week-old) WT and sGCα₁^−/− mice, reacted with antibodies directed against SMI32, staining retinal nerve fibers yellow. Scale bars: 500 μm. <b>C</b>: Representative confocal images, taken at a similar distance from the optic nerve, of flat-mounted retinas isolated from age-matched 52-week-old WT and sGCα₁^−/− mice that were reacted with antibodies directed against βIII Tubulin, and quantitative analysis of the number of RGCs/high-powered field (<i>n</i> = 8 and 7, respectively; *<i>P</i> = 3.6×10⁻²). A retinal ganglion cell (red) is indicated by an arrow. Scale bars: 20 μm. <b>D</b>: Representative cross sections through the optic nerve of 52-week-old WT and sGCα₁^−/− mice stained with paraphenylenediamine, and quantitative analysis of the calculated number of axons/optic nerve (ON). The arrow indicates an injured area in the optic nerve, characterized by the absence of well-formed myelinated axons (<i>n</i> = 7 and 6, respectively; *<i>P</i> = 4.9×10⁻²). Scale bars: 25 μm.</p

Localization of sGC α₁ and β₁ subunits in the human and murine eye.

<p>Panels <b>A–C</b> depict tissue sections from human eyes. Panels <b>D–F</b> depict tissue sections from mouse eyes. <b>A</b>: Ciliary muscle (CM), stained for α-smooth muscle actin (red), sGCα₁ (green, <b>upper panel</b>), or sGCβ₁ (green, <b>lower panel</b>). Both sGCα₁ and sGCβ₁ co-localized with α-smooth muscle actin in CM (yellow in merged images). Scale bars: 100 μm. <b>B</b>: An arteriole in the iris (IA) and an arteriole in the retina (RA) were stained for α-smooth muscle actin (red), sGCα₁ (green, <b>upper panels</b>), or sGCβ₁ (green, <b>lower panels</b>). Both sGCα₁ and sGCβ₁ co-localized with α-smooth muscle actin in the smooth muscle cell layer of arterioles in the iris and retina (yellow in merged images). ONL: outer nuclear layer, INL: inner nuclear layer. Scale bars: 20 μm. <b>C</b>: sGCα₁ (<b>left panel</b>) and sGCβ₁ (<b>right panel</b>) expression was detected histologically in the outer nuclear layer (ONL), inner nuclear layer (INL), and ganglion cell layer (GCL, white arrow) of the retina. sGCα₁ and sGCβ₁ are visualized by green fluorescence. Scale bars: 20 μm. <b>D</b>: Adjacent sections of a wild-type (WT) murine eye were stained for α-smooth muscle actin (green, <b>left panel</b>) or sGCα₁ (red, <b>right panel</b>). sGCα₁ co-localized with α-smooth muscle actin in ciliary muscle (CM) and in arterioles in the ciliary body (CA). The iridocorneal angle is indicated. Scale bars: 50 μm. <b>E</b>: Adjacent sections of a WT murine eye were stained for α-smooth muscle actin (green, <b>left panel</b>) or sGCα₁ (red, <b>right panel</b>). sGCα₁ co-localized with α-smooth muscle actin in retinal arterioles (RA). Scale bars: 50 μm. <b>F</b>: sGCα₁ (<b>left panel</b>) and sGCβ₁ (<b>right panel</b>) expression was detected histologically in the outer nuclear layer (ONL), inner nuclear layer (INL), and ganglion cell layer (GCL, white arrow) of the mouse retina. sGCα₁ is visualized by brown peroxidase stain and sGCβ₁ is visualized by green fluorescence. Scale bars: 20 μm.</p

Association between <i>GUCY1A3/GUCY1B3</i> single nucleotide polymorphisms (SNPs) and POAG in the Glaucoma Gene and Environment (GLAUGEN) study.

<p>Most significant single nucleotide polymorphisms (SNPs) stratified by gender and type of visual field (VF) loss. 51 SNPs were analyzed within the GUCY1A3/GUCY1B3 locus and 50 kb upstream and downstream of the region. A Bonferroni correction of 3.3×10⁻⁴, correcting for analyzing 51 SNPs and 3 subgroups, was applied to define statistical significance. The top SNP (rs11722059) reached significance in women with paracentral visual field (VF) loss (P value of 3.1×10⁻⁴, bold and italicized). MA: minor allele; OR: multivariable odds ratio associated with each minor allele dose, generated by multiple logistic regression analyses adjusting for age, gender, study site, DNA source, DNA extraction method, and three eigenvectors; CI: confidence interval; Chr: chromosome; N: number.</p