Manifestation of mutant myocilin resulted in impaired autophagy and chronic ER stress, while evident from increased intracellular myocilin, p62, LC3BI, GRP78, and CHOP in TM cells transduced with mutant myocilin. POAG by correcting impaired autophagy in the TM. mice communicate myocilin in the TM and display early-onset glaucoma phenotypes (23). We while others have shown that disease-causing myocilin mutant proteins are secretion incompetent and accumulate in the ER, inducing ER stress (16, 21C28). Chronic ER stress is known to play a key part in the elevation of IOP in myocilin-associated glaucoma (23). Specifically, induction of chronic ER stressCinduced transcription element, CCAAT/enhancer-binding protein homologous protein (CHOP), is definitely associated with a loss of TM cells (23). Cd200 However, it is not recognized how CHOP prospects to TM loss/dysfunction and IOP elevation. When the levels of misfolded proteins overwhelm normal protein degradation pathways, cells PROTAC MDM2 Degrader-2 can activate autophagy, a lysosomal-mediated degradation pathway. Several studies have exposed interaction between the ER stress response pathway and autophagy (29C31). Autophagy is responsible for the removal of protein aggregates, long-lived proteins, and defective organelles (32C34). During the autophagic process, cargos are engulfed within a double membrane vesicle called an autophagosome. These autophagosomes fuse with lysosomes, and cargos are degraded via lysosomal enzymes. Beclin 1, a protein coded from the gene, forms a complex with phosphoinositide 3-kinase (PI3K), which is required for the initiation of autophagosome formation. Microtubule-associated protein light chain 3 (LC3) is definitely a major regulator of autophagosome formation, and conversion of LC3B-I to LC3B-II is an indication of autophagosome formation (35). In addition, PROTAC MDM2 Degrader-2 the ubiquitin binding protein p62, also known as sequestosome 1 (SQSTM1), binds directly to LC3B, which is definitely then degraded by autophagy and may serve as a marker to study autophagic degradation PROTAC MDM2 Degrader-2 and autophagic flux. Autophagy takes on an important part in PROTAC MDM2 Degrader-2 preventing harmful build up PROTAC MDM2 Degrader-2 of disease-associated mutant proteins (28, 36C38). Several previous studies have shown that ER stress induces autophagic response and ER stressCinduced autophagy is definitely cytoprotective (29, 30, 39C41). Despite activation of the protecting unfolded protein response (UPR) pathway and autophagy, misfolded proteins continue to accumulate in disease conditions. It is not yet recognized whether chronic and prolonged ER stress can lead to impaired autophagy. A previous study has shown involvement of both ubiquitin-proteasome and lysosomal pathways in turnover of endogenous WT myocilin in cultured TM cells (42). However, proteosomal degradation of mutant myocilin is definitely impaired and autophagy is definitely activated. It is not recognized why autophagy fails to degrade mutant myocilin and whether autophagy takes on a critical part in mutant myocilinCinduced ocular hypertension. We have recently demonstrated that induction of chronic ER stress prospects to glaucoma by increasing protein synthesis and ER client protein weight in TM cells (43). These events can further lead to jeopardized autophagy. We propose that induction of CHOP is definitely associated with jeopardized autophagy, leading to mutant myocilin build up in TM cells, causing TM dysfunction/loss and IOP elevation inside a mouse model of myocilin POAG. Here, we investigated whether mutant myocilinCinduced chronic ER stress prospects to impaired autophagy and whether correction of autophagic flux via deletion of CHOP or pharmacological autophagy inducers promotes autophagic degradation of mutant myocilin and reduces elevated IOP inside a mouse model of myocilin-associated glaucoma. Results Mutant myocilinCinduced chronic ER stress is definitely associated with impaired autophagy in TM cells in vitro and in vivo. To study the effect of mutant myocilin on chronic ER stress and autophagy, we generated TM3 cells stably expressing DsRed-tagged WT or numerous individual myocilin mutations (Y437H, G364V, and Q368X). Y437H and G364V myocilin mutations.