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 6

Tat-beclin 1 peptide promotes autophagic degradation of mutant myocilin and reduces elevated IOP in Tg-MYOCY437H mice.

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Tat-beclin 1 peptide promotes autophagic degradation of mutant myocilin ...
(A) Western blot analysis of TM3 cells stably expressing Y437H mutant myocilin treated with various concentrations of tat-scrambled (TS) or tat-beclin 1 (TB1) peptide. n = 3. (B and C) Immunostaining of LC3B (B) and counting of individual LC3B puncta (C) in TM3 cells expressing Y437H mutant myocilin treated with TS or TB1 peptide. Scale bar: 10 μm; n = 3; unpaired t test. (D) LC3B and mutant myocilin analysis in human primary TM cells (n = 3 cell strains) transduced with Ad5.DsRed-tagged mutant myocilin and treated with TS or TB1. Scale bar: 10 μm. (E) Human primary TM cells were transduced with Ad5 expressing mutant myocilin and mRFP-GFP-LC3 and treated with either TS or TB1 peptide for 3 hours. Autophagosome (yellow puncta) and autolysosomes (red puncta) were counted and represented graphically. n = 3 cell strains; data are mean ± SEM; 2-way ANOVA. (F) Three-month-old WT and ocular hypertensive Tg-MYOCY437H littermates were treated with topical ocular TS in 1 eye; the contralateral eyes were treated with TB1 eye drops for 7 days. Nighttime IOP measurement demonstrated that TB1 treatment significantly reduced elevated IOP in Tg-MYOCY437H mice. Data are mean ± SEM; n = 6 in each group; 2-way ANOVA with Bonferroni’s multiple-comparison test, *P < 0.05, ***P < 0.001. (G) Densitometric analyses of Western blot of anterior segment tissue lysates from TS- or TB1-treated mice demonstrated that TB1 peptide treatment significantly reduced myocilin in Tg-MYOCY437H mice. Data are mean ± SEM; n = 3, paired t test. (H) LC3-GFP analysis in C57BL/6J mice injected intravitreally with lentiviral particles expressing LC3-GFP and treated with TS (250 μM) or TB1 peptide (250 μM) eye drops for 7 days. The rectangular box indicates the TM region. n = 3. Scale bar: 50 μm.