Reduction of ER stress via a chemical chaperone prevents disease phenotypes in a mouse model of primary open angle glaucoma

Abstract

Mutations in myocilin (MYOC) are the most common genetic cause of primary open angle glaucoma (POAG), but the mechanisms underlying MYOC-associated glaucoma are not fully understood. Here, we report the development of a transgenic mouse model of POAG caused by the Y437H MYOC mutation; the mice are referred to herein as Tg-MYOC(Y437H) mice. Analysis of adult Tg-MYOC(Y437H) mice, which we showed express human MYOC containing the Y437H mutation within relevant eye tissues, revealed that they display glaucoma phenotypes (i.e., elevated intraocular pressure [IOP], retinal ganglion cell death, and axonal degeneration) closely resembling those seen in patients with POAG caused by the Y437H MYOC mutation. Mutant myocilin was not secreted into the aqueous humor but accumulated in the ER of the trabecular meshwork (TM), thereby inducing ER stress in the TM of Tg-MYOC(Y437H) mice. Furthermore, chronic and persistent ER stress was found to be associated with TM cell death and elevation of IOP in Tg-MYOC(Y437H) mice. Reduction of ER stress with a chemical chaperone, phenylbutyric acid (PBA), prevented glaucoma phenotypes in Tg-MYOC(Y437H) mice by promoting the secretion of mutant myocilin in the aqueous humor and by decreasing intracellular accumulation of myocilin in the ER, thus preventing TM cell death. These results demonstrate that ER stress is linked to the pathogenesis of POAG and may be a target for treatment in human patients.

Figure 1. Increased transgene expression in TM of Tg-MYOC^Y437H mice.

Transgene (Y437H MYOC) and WT MYOC expression were examined in 6-month-old WT and Tg-MYOC^Y437H littermates by (A) RT-PCR, (B) Western blot analysis, and (C) immunostaining. PCR products were sequenced to confirm the presence of the transgene. Transgene and WT MYOC were expressed only in the iridocorneal angle and sclera of Tg-MYOC^Y437H mice but were absent in the retina of WT and Tg-MYOC^Y437H mice (n = 3). MYOC protein levels were increased in angle and sclera of Tg-­MYOC^Y437H mice compared with WT littermates. Note that MYOC protein was absent in retinal lysates in Tg-MYOC^Y437H and WT mice (n = 3). GAPDH was used as a loading control. Immunostaining revealed that MYOC was localized to TM and is increased in Tg-MYOC^Y437H mice (bottom panels) compared with WT littermates (top panels). The TM is shown by the arrow. Scale bars: 20 μm. Open iridocorneal angle and normal morphology of anterior chamber structures in Tg-MYOC^Y437H mice (D–G). Slit lamp examination of anterior chambers of 6-month-old (D) WT mice and (E) Tg-MYOC^Y437H mice reveal no abnormalities in the iris, cornea, and lens. TEM images of the iridocorneal angle of 12-month-old (F) Tg-­MYOC^Y437H mice were compared with (G) WT littermates. The iridocorneal angle is open in Tg-MYOC^Y437H mice (n = 6) compared with WT littermates (n = 5). Iris, ciliary body (CB), TM, and Schlemm canal (SC) are shown.

Figure 2. Glaucoma phenotypes of Tg-MYOC^Y437H mice.

(A) Elevated IOP in Tg-MYOC^Y437H mice. IOP of Tg-MYOC^Y437H (n = 69) and WT littermates (n = 62) were measured day and night at various ages. (B) Progressive RGC loss in Tg-MYOC^Y437H mice. Mean RGC numbers were counted from whole-mount γ-synuclein staining of retina in Tg-MYOC^Y437H (n = 40) and WT littermates (n = 30). (C) Functional deficit in RGCs of Tg-­MYOC^Y437H mice. PERG amplitudes (P50-N95) in Tg-MYOC^Y437H (n = 20) were compared with WT littermates (n = 14) at various ages. (D) Progressive optic nerve degeneration in Tg-MYOC^Y437H mice. Optic nerve sections were stained with PPD, and mean axon counts in Tg-MYOC^Y437H (n = 15) were compared with those of WT littermates (n = 12). Data are shown as mean ± SEM. *P < 0.05; **P < 0.005; ***P < 0.0001 versus WT.

Figure 3. Mutant MYOC accumulates in the ER, induces ER stress, and activates UPR.

(A) Increased ER stress markers including Ire1, Grp78, Grp94, spliced Xbp-1, activated Atf-6α, and pelF-2α were detected in 6-month-old Tg-MYOC^Y437H anterior segment tissues by Western blot. These markers were also increased in 12-month-old Tg-MYOC^Y437H mice. (B) Immunostaining for KDEL (recognizes Grp78 and Grp94) in the iridocorneal sections of WT and Tg-MYOC^Y437H mice demonstrated increased ER stress marker localization in the TM of 6-month-old Tg-MYOC^Y437H mice compared with WT littermates (n = 4). (C) Decreased myocilin secretion in the aqueous humor of Tg-MYOC^Y437H mice. Representative Western blot of myocilin secretion in aqueous humor of Tg-MYOC^Y437H mice is compared with that in WT mice. (n = 6). (D) Mutant MYOC accumulates in the ER of primary TM cells. Colocalization of MYOC (red) with KDEL (green) was examined using immunostaining and confocal imaging in TM cells (n = 4). Scale bars: 20 μm. (E and F) Expression of mutant MYOC in the TM induces ER stress and elevates IOP. WT MYOC, Y437H MYOC, and G364V MYOC were expressed in the TM by adenoviral injections. Western blots of ER stress markers were examined in the iridocorneal angle tissues after 24 hours of injections (E), and IOP was examined (F). ***P < 0.005 versus WT, n = 12 WT MYOC, and 20 Y437HMYOC. Data are shown as mean ± SEM.

Figure 4. Mutant MYOC upregulates ER stress–induced apoptotic proteins, which is associated with loss of TM.

(A) Distention of rER in the TM of Tg-MYOC^Y437H mice. TEM of both genotypes demonstrated that rER (arrows) of 12-month-old Tg-MYOC^Y437H was dilated compared with that of WT littermates. Similar findings were observed in 6-month-old Tg-­MYOC^Y437H mice. Scale bars: 0.2 μm. (B) Induction of ER stress–initiated apoptotic proteins in 12-month-old Tg-MYOC^Y437H mice. Western blot analysis of anterior segment tissue lysate for ER stress–induced apoptotic proteins Chop and cleaved caspase 12 (n = 3). (C) Loss of TM cells in 12-month-old Tg-MYOC^Y437H mice examined by TEM analysis. Data are shown as an average number of TM cells/iridocorneal angle sections of WT (n = 5) and Tg-MYOC^Y437H mice (n = 6). Data are shown as mean ± SEM. *P < 0.02 versus WT. (D) Y437H MYOC leads to TM cell death in cultured TM cells. Percentage of DAPI-positive cells expressing either WT or Y437H MYOC represented over control cells expressing empty virus. Data are shown as mean ± SEM. n = 4. ***P < 0.0004.

Figure 5. Chemical induction of ER stress in the TM elevates IOP.

(A) Induction of ER stress in the TM of C57BL/6J by tunicamycin leads to elevated IOP when compared with vehicle control (DMSO). n = 12 for each group. ***P < 0.005 versus control. Data are shown as mean ± SEM. (B) Increased ER stress markers including Grp78, Grp94, Chop, and pelF-2α were detected in the iridocorneal angle tissues of tunicamycin-injected mice by Western blot (n = 4).

Figure 6. Small chemical chaperone rescues glaucoma phenotypes in Tg-­MYOC^Y437H mice.

(A) Nocturnal IOP measurements, (B) RGC counts, (C) PERG amplitudes, and (D) neuronal degeneration in PBA-treated mice compared with untreated littermates. 2-month-old WT and Tg-MYOC^Y437H mice were treated with PBA (20 mM, ~10 mg/mouse/day) in drinking water for 5 weeks and glaucoma phenotypes were compared with those of untreated littermates. n = 8 WT and 16 Tg-MYOC^Y437H mice. *P < 0.05 vs. WT; **P < 0.005 vs. WT; ^†P < 0.005 versus untreated Tg-MYOC^Y437H. Data are shown as mean ± SEM.

Figure 7. PBA promotes trafficking and secretion of mutant MYOC, thus reducing ER stress in vitro and in vivo.

MYOC secretion in the (A) medium or (B) aqueous humor was detected by Western blotting, as shown in the top panel, and analyzed by densitometric analysis, as shown in the bottom panel (n = 4 WT, 6 untreated Tg-MYOC^Y437H, and 8 PBA treated Tg-MYOC^Y437H mice). For myocilin secretion in the aqueous humor, samples were on the same gel but rearranged in the order presented. PBA effect on colocalization of MYOC with ER marker was performed using immunostaining and confocal imaging (C). Scale bars: 20 μm. PBA treatment rescued loss of TM cells due to mutant MYOC (n = 3) (D). Densitometric analysis of ER stress markers in the iridocorneal angle tissue lysates of untreated and treated Tg-MYOC^Y437H mice (n = 3) (E). *P < 0.05; **P < 0.005; ***P < 0.0005 versus untreated Tg-MYOC^Y437H. Data are shown as mean ± SEM.

Figure 8. ER stress is induced in fibroblasts from a POAG patient with the Y437HMYOC mutation.

Primary fibroblasts from a POAG patient with the Y437HMYOC mutation and age-matched control fibroblasts were examined for ER stress markers by Western blot analysis.