Figure 3

Histology and levels of optineurin, p62, PSMB5 and LC3 in the retina of viral vector injected rat eyes. A. Representative retinal sections collected following injection of PBS, and GFP-, wild-type OPTN (OPTN)- and E50K-OPTN (E50K)-GFP vectors were stained with hematoxylin and eosin (H & E). GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; and ONL, outer nuclear layer. The retinal ganglion cell number was decreased and the INL thickness was reduced in OPTN- and E50K-injected specimens compared to PBS and GFP controls. Scale bar, 25 μm. B. Retinal sections from rat eyes were stained in parallel with polyclonal anti-optineurin, p62, PSMB5, or LC3 (all in red). An enhanced optineurin (OPTN) and LC3 staining, and a declined PSMB5 and p62 staining were observed in the retinal ganglion layer of OPTN and E50K-injected eyes compared to controls. Scale bar, 25 μm. C. Western blotting for optineurin, PSMB5, LC3, and GAPDH in retinal extracts from GFP-, OPTN-GFP (OPTN)-, and E50K-OPTN-GFP (E50K)-injected eyes. The LC3-I bands were very faint and only those of LC3-II were shown. The level of optineurin overexpression was estimated by comparing the intensities of both (optineurin-GFP + optineurin) bands from optineurin treatment groups to those of the endogenous optineurin band from GFP groups (all were normalized against GAPDH). The optineurin/GAPDH ratios (n = 3) for the GFP, OPTN, and E50K groups were, respectively, 1.0 ± 0.2, 9.5 ± 0.3, and 8.0 ± 0.5. For PSMB5/GAPDH, the corresponding values (relative to the first GFP sample) were 1.1 ± 0.1, 0.5 ± 0.1, and 0.5 ± 0.1; and for LC3/GAPDH, they were 1.0 ± 0.1, 1.6 ± 0.2, and 2.1 ± 0.2. All OPTN and E50K results were significantly (P < 0.0015) different from those of GFP controls.