Negative regulation of autophagy in retinal ganglion cells by Opn5 through inhibiting Egr-1 nuclear translocation

Objective  To investigate whether neuropsin (Opn5) affects the autophagy in mouse retinal ganglion cells (RGCs) by regulating the nuclear translocation of early growth response 1 (Egr-1).

Methods  RGC-5 cells were cultured in vitro. Cells transfected with Opn5-short hairpin RNA (shRNA) lentivirus served as the shOpn5 group and cells transfected with scramble shRNA served as the shRNA control group. Cell proliferation was detected by Cell Counting Kit-8 (CCK-8) assay before transfection, and at 24, 48, 72, 96 and 120 hours after transfection, and apoptosis was analyzed by flow cytometry. Reverse transcription-quantitative qPCR and Western blot were performed to detect the expression of Opn5, Egr-1, and autophagy markers (LC3-Ⅱ/Ⅰ ratio, p62, Beclin-1). The colocalization of Opn5 with Egr-1, LC3, or p62 was examined by double immunofluorescence staining. Autophagic flux was evaluated using the mRFP-GFP-LC3 tandem fluorescent protein system. The interaction between Opn5 and Egr-1 was verified by co-immunoprecipitation (Co-IP), and the subcellular localization of Egr-1 was analyzed by nuclear-cytoplasmic fractionation.

Results  Compared with the shRNA control group, the cell proliferation rates were significantly reduced at 96 and 120 hours after infection in the shOpn5 group (both P<0.001). The apoptosis rates at 72 hours after infection were (9.88±0.17)% and (5.44±0.15)% in the shOpn5 group and the shRNA control group, respectively, with a significant difference between them ( P<0.001). Compared with the shRNA control group, both mRNA and protein expression levels of Opn5 and Egr-1 were significantly decreased, and the expression levels of the LC3-Ⅱ/Ⅰ ratio and Beclin-1 proteins were upregulated, while the expression level of p62 was significantly decreased in the shOpn5 group (all P<0.001). Dual fluorescence system revealed enhanced autophagic flux. Co-IP confirmed a direct binding between Opn5 and Egr-1 in the shRNA control group, and the binding was reduced in the shOpn5 group. The results of the nuclear-cytoplasmic fractionation assay showed that the ratios of nuclear to cytoplasmic distribution of Egr-1 protein in the shRNA control group and the shOpn5 group were (80.53±7.96)% and (13.47±1.48)%, respectively, with a significant difference between them ( P<0.001).

Conclusions  Opn5 negatively regulates autophagy in RGCs by mediating the nuclear translocation of Egr-1, thereby influencing Egr-1 transcriptional activity. This finding provides a novel mechanistic insight into the role of photoreceptor protein Opn5 in the development of myopia.