Topical ocular sodium 4-phenylbutyrate rescues glaucoma in a myocilin mouse model of primary open-angle glaucoma - PubMed (original) (raw)

Topical ocular sodium 4-phenylbutyrate rescues glaucoma in a myocilin mouse model of primary open-angle glaucoma

Gulab S Zode et al. Invest Ophthalmol Vis Sci. 2012.

Abstract

Purpose: Mutations in the myocilin gene (MYOC) are the most common known genetic cause of primary open-angle glaucoma (POAG). The purpose of this study was to determine whether topical ocular sodium 4-phenylbutyrate (PBA) treatment rescues glaucoma phenotypes in a mouse model of myocilin-associated glaucoma (Tg-MYOC(Y437H) mice).

Methods: Tg-MYOC(Y437H) mice were treated with PBA eye drops (n = 10) or sterile PBS (n = 8) twice daily for 5 months. Long-term safety and effectiveness of topical PBA (0.2%) on glaucoma phenotypes were examined by measuring intraocular pressure (IOP) and pattern ERG (PERG), performing slit lamp evaluation of the anterior chamber, analyzing histologic sections of the anterior segment, and comparing myocilin levels in the aqueous humor and trabecular meshwork of Tg-MYOC(Y437H) mice.

Results: Tg-MYOC(Y437H) mice developed elevated IOP at 3 months of age when compared with wild-type (WT) littermates (n = 24; P < 0.0001). Topical PBA did not alter IOP in WT mice. However, it significantly reduced elevated IOP in Tg-MYOC(Y437H) mice to the level of WT mice. Topical PBA-treated Tg-MYOC(Y437H) mice also preserved PERG amplitudes compared with vehicle-treated Tg-MYOC(Y437H) mice. No structural abnormalities were observed in the anterior chamber of PBA-treated WT and Tg-MYOC(Y437H) mice. Analysis of the myocilin in the aqueous humor and TM revealed that PBA significantly improved the secretion of myocilin and reduced myocilin accumulation as well as endoplasmic reticulum (ER) stress in the TM of Tg-MYOC(Y437H) mice. Furthermore, topical PBA reduced IOP elevated by induction of ER stress via tunicamycin injections in WT mice.

Conclusions: Topical ocular PBA reduces glaucomatous phenotypes in Tg-MYOC(Y437H) mice, most likely by reducing myocilin accumulation and ER stress in the TM. Topical ocular PBA could become a novel treatment for POAG patients with myocilin mutations.

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Figures

Figure 1.

Figure 1.

Topical ocular PBA rescues ocular hypertension in Tg-MYOCY437H mice. (A) IOP measurements of 3-month-old WT and Tg-MYOCY437H mice. Tg-MYOCY437H mice developed elevated IOP compared with WT littermates. (B) Topical ocular PBA reduced IOP in the left eye compared with the contralateral control eye in 9-month-old Tg-MYOCY437H mice. Topical ocular PBA was applied to the left eye of Tg-MYOCY437H mice, and the right eye served as the control. One week after PBA treatment, IOP in the left eye was significantly reduced compared with the contralateral eye. n = 3 WT and n = 5 Tg-MYOCY437H mice. (C) IOP measurements of vehicle or PBA-treated WT and Tg-MYOCY437H mice for 5 months. WT and Tg-MYOCY437H mice (3 months old) were divided into two groups: the first received topical ocular PBA (0.2%) twice daily, and the second was given sterile PBS (vehicle) twice daily. The IOP of these mice was measured every month. Vehicle-treated Tg-MYOCY437H mice showed elevated IOP compared with that of the WT littermates; however, PBA treatment normalized the IOP of the Tg-MYOCY437H mice to WT levels. *P < 0.05, **P < 0.01, ***P < 0.0001, versus vehicle-treated Tg-MYOCY437H mice. Data are the mean ± SEM.

Figure 2.

Figure 2.

Topical ocular PBA treatment prevents retinal ganglion cells functional deficits in Tg-MYOCY437H mice. PERG amplitudes (P50–N95, μV) were measured to evaluate the functional deficit in the RGCs of Tg-MYOCY437H mice treated with topical PBA. PBA treatment of Tg-MYOCY437H mice for 5 months prevented a reduction in PERG amplitudes compared with those in vehicle-treated Tg-MYOCY437H mice (∼50% loss in PERG amplitudes). n = 6 vehicle-treated WT, n = 5 PBA treated WT, n = 12 vehicle-treated Tg-MYOCY437H, and n = 6 PBA-treated Tg-MYOCY437H mice. Data are the mean ± SEM.

Figure 3.

Figure 3.

Topical ocular PBA does not cause abnormalities to anterior segment structures in WT or Tg-MYOCY437H mice. Slit lamp examination of PBA-treated WT (A) and Tg-MYOCY437H (B) mice revealed no abnormalities in the anterior segment structures (iris, pupil, lens, and cornea). H&E staining of PBA-treated WT (C) and Tg-MYOCY437H (D) mice. Optical coherence tomography (OCT) shows no abnormalities in cornea of PBA-treated Tg-MYOCY437H mice (F) compared with WT littermates (E). Cornea thickness measurements are shown in (G). n = 4 WT and Tg-MYOCY437H treated with PBA or vehicle. Data are the mean ± SEM.

Figure 4.

Figure 4.

Topical ocular PBA reverses inhibition of myocilin secretion in the aqueous humor of Tg-MYOCY437H mice. (A) Western blot analysis of myocilin in the aqueous humor samples from 9-month-old PBA-treated WT and Tg-MYOCY437H mice is compared with vehicle-treated WT and Tg-MYOCY437H mice. Coomassie stain was performed to ensure equal loading of aqueous humor. (B) Densitometric analysis of myocilin secretion normalized to a loading control demonstrated a significant reduction in myocilin secretion in the Tg-MYOCY437H mice compared with that in WT littermates; however, PBA treatment of Tg-MYOCY437H mice significantly enhanced myocilin secretion in the aqueous humor. n = 3 WT, n =3 PBA-treated WT, n =5 Tg-MYOCY437H, and n =4 PBA-treated Tg-MYOCY437H mice.

Figure 5.

Figure 5.

Topical ocular PBA reduces intracellular accumulation of myocilin and ER stress in the TM of Tg-MYOCY437H mice. Myocilin levels and ER stress markers in the iridocorneal angle of PBA-treated WT (B) and Tg-MYOCY437H (D) mice were compared with those in vehicle-treated WT (A) and Tg-MYOCY437H mice (C) by immunostaining and confocal imaging. Arrows: TM, CB, and iris. Vehicle-treated Tg-MYOCY437H mice demonstrated increased myocilin staining and ER stress markers (the KDEL antibody recognizes GRP78 and GRP94) in the TM and CB compared with that in the WT mice. Of note, PBA-treated Tg-MYOCY437H mice showed reduction of myocilin staining in the TM and CB compared with vehicle-treated Tg-MYOCY437H mice. n = 3 vehicle-treated WT, n = 5 vehicle-treated Tg-MYOCY437H, n = 3 PBA-treated WT, and n = 4 PBA-treated Tg-MYOCY437H mice. Scale bar, 20 μm.

Figure 6.

Figure 6.

Topical PBA reduces IOP that has been elevated by tunicamycin in WT mice. WT mice (n = 14) injected with 0.2 μg/eye of tunicamycin had elevated IOP 1 week after injection, compared with IOP in WT mice with control vehicle injection (n = 6). Tunicamycin-injected mice that showed elevated IOP were treated with topical PBA eye drops (1%) twice daily afterward, and IOP was measured every week. A 3-week treatment with PBA significantly reduced the elevated IOP in the WT mice. Data are the mean ± SEM.

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