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 3

Inhibition of autophagy exacerbates WT and mutant myocilin accumulation in cultured TM cells.

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Inhibition of autophagy exacerbates WT and mutant myocilin accumulation ...
(A) TM3 cells stably expressing WT or mutant myocilin (Y437H or G364V) were treated with BA1 (200 nM) for 6 hours and cellular lysates were examined for intracellular myocilin accumulation and autophagy markers. Treatment with BA1 inhibited autophagic degradation of both WT and mutant myocilin in TM3 cells. n = 3. (B and C) TM3 cells stably expressing WT or mutant myocilin were treated with BA1 for 6 hours and cells were stained with LC3B (B) and p62 (C). BA1 treatment resulted in increased myocilin (WT and mutant), LC3B puncta, and P62 levels. n = 3. Scale bar: 10 μm. (D and E) TM3 cells stably expressing WT or various mutants of myocilin were treated with CQ (50 μM) for 12 hours. Triton X-100–soluble and –insoluble fractions were examined for intracellular myocilin accumulation and autophagy markers. CQ treatment inhibited autophagic degradation of mutant myocilin and increased accumulation of Triton X-100–soluble and –insoluble mutant myocilin. n = 3. (F and G) TM3 cells stably expressing WT or Y437H mutant myocilin were transfected with CRISPR/Cas9 targeting ATG5 and stained with LC3B and P62. ATG5 knockdown prevented autophagy induction and increased WT and mutant myocilin accumulation. n = 3. Scale bar: 10 μm.