Autophagy stimulation reduces ocular hypertension in a murine glaucoma model via autophagic degradation of mutant myocilin
Ramesh B. Kasetti, … , Val C. Sheffield, Gulab S. Zode
Ramesh B. Kasetti, … , Val C. Sheffield, Gulab S. Zode
Published February 4, 2021
Citation Information: JCI Insight. 2021;6(5):e143359. https://doi.org/10.1172/jci.insight.143359.
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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 1

Mutant myocilin leads to impaired autophagy, which is associated with chronic ER stress in human cultured TM cells.

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Mutant myocilin leads to impaired autophagy, which is associated with ch...
(A and B) TM3 cells stably expressing DsRed-tagged WT or individual myocilin mutations (Y437H, G364V, and Q368X) were stained with ER marker, calreticulin (A), or ER stress marker GRP78 (B). All mutants of myocilin colocalized strongly with calreticulin and GRP78, indicating that mutant myocilin accumulated in the ER and induced ER stress. n = 3. Scale bar: 5 μm. (C and D) TM3 cells stably expressing WT or Y437H, G364V, and Q368X mutants of myocilin were stained with LC3B (C) and p62 (D). Compared with WT myocilin, TM cells stably expressing mutant myocilin exhibit increased LC3B and p62 staining. n = 3. Scale bar: 5 μm. (E and F) Primary human TM cells were transduced with Ad5 expressing empty (ctl), WT, or Y437H mutant myocilin. Western blot (E) and densitometric analysis (F) were performed to determine autophagy and ER stress. Expression of mutant myocilin resulted in impaired autophagy and chronic ER stress, as evident from increased intracellular myocilin, p62, LC3BI, GRP78, and CHOP in TM cells transduced with mutant myocilin. n = 3 cell strains; data are mean ± SEM; * P ≤ 0.05; ** P ≤ 0.01; ***P ≤ 0.01; 2-way ANOVA. (G and H) Primary human TM cells were transduced with Ad5 expressing WT or Y437H mutant myocilin for 48 hours and again transduced with Ad5.mRFP-GFP-LC3 for 24 hours. Autophagosomes (yellow puncta) and autolysosomes (red puncta) were counted. Expression of mutant myocilin resulted in impaired autophagic flux as evident from increased yellow puncta and significantly reduced red puncta compared with WT myocilin. n = 3 cell strains; data are mean ± SEM; 2-way ANOVA; Scale bar: 50 μm.