Autophagy stimulation reduces ocular hypertension in a murine glaucoma model via autophagic degradation of mutant myocilin
Ramesh B. Kasetti, Prabhavathi Maddineni, Charles Kiehlbauch, Shruti Patil, Charles C. Searby, Beth Levine, Val C. Sheffield, Gulab S. Zode
Ramesh B. Kasetti, Prabhavathi Maddineni, Charles Kiehlbauch, Shruti Patil, Charles C. Searby, Beth Levine, Val C. Sheffield, Gulab S. Zode
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Research Article Cell biology Ophthalmology

Autophagy stimulation reduces ocular hypertension in a murine glaucoma model via autophagic degradation of mutant myocilin

Abstract

Elevation of intraocular pressure (IOP) due to trabecular meshwork (TM) damage is associated with primary open-angle glaucoma (POAG). Myocilin mutations resulting in elevated IOP are the most common genetic causes of POAG. We have previously shown that mutant myocilin accumulates in the ER and induces chronic ER stress, leading to TM damage and IOP elevation. However, it is not understood how chronic ER stress leads to TM dysfunction and loss. Here, we report that mutant myocilin activated autophagy but was functionally impaired in cultured human TM cells and in a mouse model of myocilin-associated POAG (Tg-MYOCY437H). Genetic and pharmacological inhibition of autophagy worsened mutant myocilin accumulation and exacerbated IOP elevation in Tg-MYOCY437H mice. Remarkably, impaired autophagy was associated with chronic ER stress–induced transcriptional factor CHOP. Deletion of CHOP corrected impaired autophagy, enhanced recognition and degradation of mutant myocilin by autophagy, and reduced glaucoma in Tg-MYOCY437H mice. Stimulating autophagic flux via tat-beclin 1 peptide or torin 2 promoted autophagic degradation of mutant myocilin and reduced elevated IOP in Tg-MYOCY437H mice. Our study provides an alternate treatment strategy for myocilin-associated POAG by correcting impaired autophagy in the TM.

Authors

Ramesh B. Kasetti, Prabhavathi Maddineni, Charles Kiehlbauch, Shruti Patil, Charles C. Searby, Beth Levine, Val C. Sheffield, Gulab S. Zode

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Figure 7

Torin 2 promotes autophagic degradation of mutant myocilin and reduces elevated IOP in Tg-MYOCY437H mice.

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Torin 2 promotes autophagic degradation of mutant myocilin and reduces e...
(A) TM3 cells stably expressing mutant myocilin were treated with vehicle or torin 2 with or without CQ for 12 hours. Western blot analysis demonstrated that torin 2 reduced intracellular myocilin and increased LC3BII form. These effects were blocked by CQ treatment (n = 3). (B–D) TM3 cells stably expressing mutant myocilin were treated with vehicle or torin 2 along with CQ for 12 hours and fixed cells were stained with LC3B (B) and LC3B puncta were counted (C). p62 staining is shown in (D). Torin 2 treatment stimulated autophagic degradation of mutant myocilin. Scale bar: 10 μm. Data are mean ± SEM; n = 3; unpaired t test. (E) Ocular hypertensive Tg-MYOCY437H mice (4 months old) were treated with topical ocular vehicle eye drops in 1 eye; the contralateral eyes were given torin 2 eye drops for 7 days. Torin 2 treatment significantly reduced elevated IOP in Tg-MYOCY437H mice. Data are mean ± SEM; n = 7–8; paired t test. (F) Densitometric analyses of Western blot of anterior segment tissue lysates from vehicle- or torin 2–treated mice demonstrated that torin 2 significantly reduced mutant myocilin and p62. Data are mean ± SEM; n = 7, *P < 0.05, 1-way-ANOVA.