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 5

Deletion of CHOP prevents IOP elevation in Tg-MYOCY437H mice by promoting autophagic degradation of mutant myocilin.

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Deletion of CHOP prevents IOP elevation in Tg-MYOCY437H mice by promotin...
(A) TM3 cells stably expressing Y437H mutant myocilin were transfected with CRISPR/Cas9 targeting CHOP. CRISPR/Cas9 targeting CHOP prominently reduced CHOP protein levels and decreased intracellular mutant myocilin. n = 3. (B and C) TM3 cells expressing mutant myocilin were transfected with CRISPR/Cas9 targeting CHOP and lentiviral particles expressing LC3-GFP for 24 hours. Cells were further treated with vehicle or CQ for 12 hours. DsRed and GFP were imaged by confocal microscopy (B) and LC3 puncta/cell are shown graphically (C). Scale bar: 10 μm. n = 3; 1-way ANOVA. (D) Immunostaining for p62 demonstrated that reduction of CHOP led to reduced p62. n = 3. Scale bar: 10 μm. (E) TM3 cells stably expressing mutant myocilin were transfected with CRISPR/Cas9 targeting CHOP and cellular lysates were examined for TM cell death. n = 3. (F) Tg-MYOCY437H mice were crossed with Chop–/– mice and IOPs were monitored (6 months old). Data are mean ± SEM; n = 4 in WT, n = 20 in Tg-MYOC, n = 12 in CHOP–/– and n = 14 in Chop–/–Tg-MYOC, * P ≤ 0.05; ** P ≤ 0.01, 2-way ANOVA. (G and H) Western blot and densitometric analyses of anterior segment tissues from WT or Chop–/– mice injected with Ad.5 expressing mutant myocilin demonstrated significant reduction in mutant myocilin. n = 4. Paired t test. (I and J) TM3 cells stably expressing mutant myocilin were transduced with lentiviral particles expressing LC3-GFP and treated either with vehicle or ISRIB (200 nM) for 12 hours. GFP and DsRed color were examined by confocal microscope (I) and LC3 puncta counted and represented graphically (J). Data are mean ± SEM; n = 3; unpaired t test.