Processing of Optineurin in Retinal Ganglion Cells

Abstract

Purpose: : The ubiquitin-proteasome system (UPS) and autophagy are two major routes for protein clearance. This study investigated their roles in processing of optineurin, the product of a gene associated principally with normal tension glaucoma, in retinal ganglion RGC5 cells.

Methods: : Levels of the endogenous optineurin in RGC5 cells treated with proteasomal inhibitors lactacystin and epoxomicin, autophagic inhibitor 3-methyladenine (3-MA), or lysosomal inhibitor NH₄Cl for 16 hours were assessed by Western blotting. To examine ubiquitination, RGC5 cell lysates were immunoprecipitated with anti-optineurin and the collected pool was probed with monoclonal anti-ubiquitin. To study the processing of overexpressed wild type and E50K (Glu50Lys) mutated optineurin, RGC5 cells were transfected with pEGFP-N1 (mock control), pOptineurin_WT-GFP or pOptineurin_E50K-GFP for 24 or 48 hours, treated with inhibitors or activator, and stained with antibodies against LC3 and proteasome regulatory β5 subunit. Formation of autophagosomes was examined by electron microscopy.

Results: : The endogenous optineurin level in RGC5 cells was increased by 2-3 fold by treatment of proteasome inhibitors, but only by 1.1-1.4 fold by autophagic and lysosomal inhibitors. Multiple bands immunoreactive to anti-ubiquitin were seen in the optineurin pool in RGC5 cells, indicating that optineurin was ubiquitinated. The endogenous optineurin thus appeared to be processed mainly by the UPS pathway. In optineurin transfected cells, the staining for autophagy marker LC3 was enhanced compared to mock controls and that for proteasome regulatory β5 subunit (indicative of proteasome activity) was reduced. These patterns were more dramatic with E50K than the wild type. In addition, colocalization of the optineurin foci with LC3 was noted and autophagosome formation was observed by electron microscopy. The optineurin foci accumulation in transfectants was increased after the treatment of autophagic inhibitor 3-MA, but decreased by treatment of autophagic inducer rapamycin.

Conclusions: : It appears that in normal homeostatic situation, the turnover of endogenous optineurin involves UPS. When optineurin is overexpressed or mutated, the UPS is compromised and the autophagy comes into play, similar to that observed in neurodegenerative Alzheimer and Parkinson diseases.

Keywords: ganglion cells • proteins encoded by disease genes • gene/expression