Effect Of OPTN Gene Mutations Associated With Primary Open Angle Glaucoma On Mitophagy

Objective: OPTN mutations are associated with primary open angle glaucoma(POAG),and functional studies have shown that OPTN mediates mitophagy regulation.Whether POAG-related OPTN mutations participate in POAG by damaging mitophagy is not well understood.This study mainly explored the effect of POAG-related OPTN gene mutations on PINK1-mediated mitophagy,providing experimental basis for the intervention of mitophagy in the prevention and treatment of POAG induced by OPTN mutations.Methods: To forecast the effect of POAG-related OPTN gene mutations H26 D,E50K,M98 K,E103D,T202 R,A336G,A377 T,H486R and R545 Q on function of OPTN protein,the conserved sequences of OPTN and conservativeness of POAG-related OPTN gene mutations and conformation change of POAG-related OPTN gene mutations were analyzed.Human OPTN WT plasmid and POAG-related OPTN gene mutations plasmid were constructed and overexpressed in HEK293 cells,respectively.To analyze whether POAG-related OPTN gene mutations affected PINK1-mediated mitophagy,PINK1-mediated mitophagy was induced by CCCP.Mitochondrion DNA was detected by q PCR.PINK1 and p-Ub were detected by WB.Immunofluorescence was used to detect the co-localization of each mutation with mitochondria.Results: 1.POAG-related OPTN mutations E50 K,E103D,A336 G,A377T,and H486 R were located in the conserved OPTN sequence,and the sites of E50 K,E103D,and H486 R mutations were highly conserved.Mutations of E50 K,M98K,E103 D,T202R and A377 T lead to changes in the spatial conformation of OPTN protein.Conserved and structural analysis suggested that po AG-related mutations might affect the protein function of OPTN.2.POAG-related OPTN mutations all resulted in mt DNA clearance disorders,and there were statistical differences between them and OPTN WT(P<0.05),suggesting that these mutants all affected mitochondrial autophagy levels.3.There was no significant statistical difference between POAG-related OPTN mutations and OPTN WT in the effect of PINK1,indicating that POAG-related OPTN mutations had no effect on the expression level of PINK1,especially on the expression of full-length PINK1,which verified that OPTN was downstream of PINK1 in the mitophagy pathway.4.POAG-related OPTN mutations resulted in a decrease in the content of p-Ub,which was significantly different from that of OPTN WT(P<0.01),suggesting that the mutants effect on mitophagy may be mediated by affecting Ub phosphorylation.5.There was a statistical difference in the number of point green fluorescence between E50 K,M98K,E103 D,T202R,A377 T and R545 Q co-localization with mitochondria and OPTN WT(P<0.01),indicating that these mutations were associated with mitochondrial co-localization disorders.These results suggest that the functional regions of these mutants are crucial for OPTN localization of mitophagy.6.The degradation rate of E50 K protein was accelerated,which was significantly different from that of OPTN WT(P<0.01),indicating that E50 K mutation resulted in shorter half-life of the protein,suggesting that the mutation may reduce the stability of the protein.Conclusion: 1.Mutations of E50 K,M98K,E103 D,T202R,A336 G,A377T,and H486 R may be more harmful to OPTN function than other OPTN mutations associated with POAG.2.POAG-related OPTN mutations lead to PINK1-mediated mitophagy dysfunction.