Study On The Mechanism Of Primary Open-angle Glaucoma Induced By MYOC Mutations

MYOC is a common pathogenic gene for primary open-angle glaucoma（POAG）.Multiple MYOC variations have been found with different clinical significance.However,the pathogenesis of POAG induced by MYOC mutations has not been fully clarified.Here,we analyzed the molecular and cellular biological differences caused by multiple MYOC variants,and a possible correlation between steric clash,secretion property and pathogenicity of MYOC variants was found.Cell lines that stably expressing wild-type or mutant（G367R）MYOC were constructed to further explore the pathogenic mechanism of MYOC mutations.MYOC/p.G367R disrupted protein degradation mechanism and promoted chronic endoplasmic reticulum（ER）stress and cell apoptosis in trabecular meshwork（TM）cells.Meanwhile,MYOC/p.G367R induced autophagy dysfunction and oxidative stress in retinal cell lines.In addition,the autophagic degradation pathway performed protective effects on the maintenance of TM and retinal cell homeostasis,which is expected to be a potential therapeutic target for MYOC mutation-related POAG.Part I Correlation Between MYOC Variant Sites and PathogenicityPurpose:To explore the correlation between MYOC variant sites and pathogenicity.Methods:Denaturing and non-denaturing SDS-PAGE were used to detect the molecular characterization of intracellular and extracellular myocilin.The wild-type MYOC plasmid and 23 variant MYOC plasmids were constructed and transiently transfected into HEK 293T cells,following with secretion analysis using Western blot and immunofluorescence.Glycosylation of myocilin was inhibited by site mutation and tunicamycin,and Western blot was used to detect the effect on myocilin secretion.Swiss Pd B Viewer was applied to explore the effect of MYOC variations on the 3D structure of myocilin.Immunochemistry was used to analyzed ER localization of different MYOC variants and cellular autophagy activity;MTT assay was applied to detect the sensitivity of cells to hydrogen peroxide（H₂O₂）;Mito-Tracker staining was used to detect mitochondrial membrane potential（MMP）and DCFH-DA probe was used to detect the content of cellular reactive oxygen species（ROS）.Results:Denaturing and non-denaturing SDS-PAGE showed that myocilin was a secreted and glycosylated protein,with the tendency to self-oligomerization.The full-length myocilin and its C-terminal cleavage fragment could be secreted outside the cell.Secretion analysis of 23 MYOC variants indicated that secretion defect was closely related to the pathogenicity of MYOC variants.Glycosylation inhibition by genetic mutation and tunicamycin did not block the secretion of myocilin.Structure analysis showed that the change of steric clash was associated with the secretion characterization and pathogenicity of variant myocilin.Results from immunocytochemistry demonstrated that MYOC mutants retained in ER and induced autophagy disruption.MTT assay,Mito-Tracker staining,and DCFH-DA staining showed decreased resistance to H₂O₂,depolarized MMP and increased ROS generation in cells expressing MYOC mutants.Conclusion:Steric clash changes in MYOC variants are associated with its secretion and pathogenicity.Abnormal steric clash induces secretion defect of myocilin,which retains inside ER and promotes autophagy disruption and oxidative stress.Part II MYOC/p.G367R Induced Trabecular Meshwork Cells DysfunctionPurpose:To explore the effect of MYOC/p.G367R on the function of TM cells.Methods:i HTMCs that stably expressing wild-type myocilin（WT-MYOC）or G367R mutant myocilin（G367R-MYOC）were constructed by lentivirus infection.Immunocytochemistry assay was used to detect the expression or localization of myocilin,fibronectin,Grp94 and LC3.Cytoskeleton was labeled by phalloidin.q RT-PCR was used to detect the m RNA expression of genes that related to Wnt signaling pathway and ER stress,and Western blot was used to detect the level of proteins that associated with ER stress,autophagy and apoptosis.The interaction between myocilin and chaperones（Grp94 and CRYAB）were analyzed by immunoprecipitation.To further explore the role of autophagy in G367R mutant-induced i HTMCs dysfunction,autophagy activator（rapamycin）or inhibitor（chloroquine,CQ）was added in the culture medium for 24 hours.Then Western blot,immunocytochemistry,Hoechst 33342 staining and DHE staining were applied to detect cell viability,ER stress and oxidative stress.Results:Compared to WT-MYOC,G367R mutant did not significantly change m RNA expression levels of genes that involved in Wnt signaling pathway,fibronectin production and cytoskeletal arrangement.G367R mutant induced chronic ER stress and impaired autophagy in TM cells,according to results from q RT-PCR,Western blot and immunofluorescence.Immunoprecipitation result revealed the interaction between G367R-mutated myocilin and chaperones（Grp94and CRYAB）.Rapamycin reduced intracellular myocilin accumulation,while CQ promoted the aggregation of myocilin inside i HTMCs,and exacerbated ER stress,abnormal cell morphology,cell death and oxidative stress in i HTMCs.Conclusion:MYOC/p.G367R induces TM cell dysfunction by inhibiting autophagic degradation pathway of mutated myocilin.Part III MYOC/p.G367R Induced Retinal Cells Dysfunction and Related ResearchPurpose:To explore the role of MYOC/p.G367R on the function of retinal cells.Meanwhile,we further investigate the regulation of mitophagy on oxidative stress in retinal cells.Methods:661W cells that stably expressing wild-type myocilin（WT-MYOC）or G367R mutant myocilin（G367R-MYOC）were constructed by lentivirus infection.Western blot,DHE staining and Mito-Tracker staining were used to detect the expression of autophagic markers,oxidative stress as well as MMP.In H₂O₂-induced oxidative stress model of 661W cells,nicotinamide riboside（NR）and oligomycin/antimycin A（O/A）were used as mitophagy inducers in this study,whereas CQ and cyclosporin A（Cs A）were used as mitophagy inhibitors.The activity or content of oxidative stress markers（SOD,CAT,GSH）were examined.Cell viability was analyzed using MTT assay and Hoechst 33342 staining.The expression of proteins that related to mitophagy and apoptosis was detected using Western blot.Immunofluorescence and lentiviral vector containing LC3-GFP were applied to detect the activity of mitophagy.Furthermore,DCFH-DA,Mito-Tracker and JC-1 probe were used to detect ROS generation and mitochondrial function.Results:MYOC/p.G367R induced autophagy impairment,oxidative stress and mitochondrial injury in 661W cells.In H₂O₂-treated 661W cells,NR activated PINK1/Parkin-mediated mitophagy,and protected 661W cells against H₂O₂-induced cell death via enhancing mitochondrial function and antioxidant capability.By contrast,the inhibition of mitophagy by CQ or Cs A reversed the beneficial effect of NR in the H₂O₂-treated 661W cells.O/A activated mitophagy,however,it did not improve mitochondrial function,and accelerated cell death.Conclusion:MYOC/p.G367R induces autophagy disruption and oxidative injury of retinal cells.PINK1/Parkin-mediated mitophagy that targeting mitochondrial injury repair contributes to enhanced mitochondrial function and antioxidant capability in retinal cells.