Inhibition of chronic endoplasmic reticulum stress rescues myocilin-associated glaucoma via stimulation of autophagic flux in the TM

Purpose: We have previously demonstrated that expression of mutant myocilin leads to chronic ER-stress induced transcriptional factors such as ATF4 and CHOP, which may lead to TM dysfunction and IOP elevation. Here, we explored whether and how genetic knockdown of ATF4 or CHOP rescues myocilin-associated glaucoma Methods: Autophagic flux was monitored in Tg-MYOCY437H mice and primary human TM cells using adenoviral expression of RFP-GFP-LC3 and analysis of key autophagy markers LC3B and P62. Key mediators of chronic ER stress, ATF4 or CHOP were knocked down using CRISPR-Cas9. Myocilin accumulation and autophagic flux was examined in TM cells and Tg-MYOCY437H mice. We further examined whether stimulation of autophagy via TatBeclin 1 peptide or Torin 2 on glaucoma phenotype and mutant myocilin accumulation using mice and TM cells Results: Expression of mutant myocilin impaired autophagic flux in primary TM cells and in the TM of Tg-MYOC Y437H mice as evident from increased LC3BI/II ratio, p62 and decreased red/yellow puncta of RFP-GFP-LC3B constructs. CRISPR-Cas9 mediated ATF4 or CHOP knockdown in TM cells stably expressing mutant myocilin improved autophagic mediated degradation of mutant myocilin, as evident by an increased LC3II/LC3I ratio, decreased P62, decreased mutant myocilin accumulation. Increased colocalization of LC3 with mutant myocilin in lysosomes was observed in ATF4 or CHOP deleted TM cells demonstrating active autophagic degradation of mutant myocilin. A dose dependent decrease in mutant myocilin accumulation was observed upon Tat-Beclin 1 treatment of TM cells expressing mutant myocilin. Tat-Beclin 1 or Torin 2 (autophagy inducers) treatment reduced intraocular pressure in Tg-MYOCY437H mice. Moreover, Western blot analysis of mice anterior segment lysates demonstrated induction of autophagy markers and reduction of mutant myocilin accumulation Conclusions: Genetic knockdown of ATF4 or CHOP increased autophagy flux, and facilitated autophagy-mediated clearance of accumulated mutant myocilin. Moreover, chemical induction of autophagy reduced mutant myocilin and rescued glaucoma in mice further suggesting the feasibility of targeting autophagy for the treatment of glaucoma