Functional Defects Caused By Glaucoma-associated Mutations Of Optineurin

Abstract

Purpose: : Optineurin is involved in vesicle trafficking, signal transduction and autophagy. We had shown earlier that it regulates endocytic trafficking of transferrin receptor to the recycling endosomes, but its glaucoma-associated mutant E50K displays defective endocytic recycling due to its altered interactions with Rab8 and transferrin receptor. Over-expression of E50K in the retinal ganglion cell line RGC-5 induced cell death, mediated by oxidative stress. We examine here some functional defects generated by E50K and the other glaucoma-associated mutant H468R. Methods: : Plasmids expressing optineurin mutants were transfected in the retinal ganglion cell line RGC-5. Endocytic trafficking in cells was examined using fluorescently labeled transferrin. Proteins interacting with optineurin were identified by yeast two-hybrid assay. Optineurin knockdown was done by using shRNA. NF-kappaB activity was measured by using a luciferase reporter. Results: : We find that over-expression of transferrin receptor protected against E50K-induced cell death. We have also identified several novel optineurin-interacting proteins including CYLD, the ubiquitin carboxy-terminal hydrolase enzyme involved in signal transduction to the transcription factor NF-kappaB. We show that optineurin is required for CYLD-dependent inhibition of TNFa-induced NF-kappaB activation. The mutant, H486R is defective in regulating NF-kappaB activation due to its impaired interaction with CYLD. Conclusions: : The E50K mutant of optineurin causes defective endocytic recycling of transferrin receptor which contributes to the death of retinal ganglion cells. The H486R mutant is defective in regulating signaling to NF-kappaB activation.