Research Paper Advance Articles

Loss of β-catenin via activated GSK3β causes diabetic retinal neurodegeneration by instigating a vicious cycle of oxidative stress-driven mitochondrial impairment

Figure 3. β-catenin downregulation triggers oxidative stress-induced mitochondrial damage and synapse degeneration in diabetic retinae. (A) Representative retinal immunofluorescence for active β-catenin (green) from mice fed with RD or HFD. Nuclei were counterstained with DAPI (blue). Areas boxed in are shown at higher magnification in the lower panels. Scale bar, 100 μm. (B) Western blotting of active β-catenin in total retina lysate. Intensities were quantified and normalized against the level of GAPDH and expressed as fold changes of protein abundance in the retina from HFD groups relative to RD controls. (CI) An adenovirus coding for β-catenin with Flag tag was injected intravitreally into the right eye of HFD-fed mice (HFD-R/Ad-β-catenin), while an empty control vector was injected into the contralateral left eye as a control (HFD-L/Ad-Ctrl). (C) Retinal double-immunostaining for Flag (red) and active β-catenin (green). Areas boxed in are shown at higher magnification. Scale bar, 100 μm. (D) Relative mRNA expression of genes encoding ROS scavengers. (E) Contents of 4-HNE in retinae. (F) Activities of retinal mitochondrial complex MRCC I-IV. Two retinae from 2 respective eyes in one group were pooled. Three independent experiments were performed in duplicate for each group. (G) Representative retinal immunostaining for synaptophysin (green; scale bar, 100 μm). Areas boxed in are shown at higher magnification in the lower panels. (H) Representative SEM of retinal sections. Areas boxed are shown at higher magnification. Scale bar, 10 μm. (I) Representative VEP waveforms and quantification of peak amplitude difference (N1-P1). Data are means ± SEM. n = 4 mice (AB), n = 4 eyes (CE; GI), or n = 6 eyes (F) per group. **P < 0.01 vs age-match RD controls; *P < 0.05 and **P < 0.01 vs contralateral eye injected with Ad-Ctrl. See also Supplementary Figures 2C, 3C, 4A, and 4B.