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• Editorial Volume 13, Issue 10 pp 13376-13377

  When aging switches on Alzheimer’s

  Relevance score: 8.104046

  Yue Dong, Benjamin A. Harlan, Gregory J. Brewer

  Keywords: aging, Alzheimer’s disease, NAD+/NADH, oxidative shift, mitochondrial impairment, neuroinflammation

  Published in Aging on May 20, 2021

• Research Paper Volume 12, Issue 13 pp 13437-13462

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

  Relevance score: 7.8011584

  Xing-Sheng Shu, Huazhang Zhu, Xiaoyan Huang, Yangfan Yang, Dandan Wang, Yiling Zhang, Weizhen Zhang, Ying Ying

  Keywords: GSK3β/β-catenin signaling, oxidative stress-driven mitochondrial impairment, diabetic retinal neurodegeneration

  Published in Aging on June 23, 2020

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  Synaptic neurodegeneration of retinal ganglion cells (RGCs) is the earliest event in the pathogenesis of diabetic retinopathy. Our previous study proposed that impairment of mitochondrial trafficking by hyperphosphorylated tau is a potential contributor to RGCs synapse degeneration. However, other molecular mechanisms underlying mitochondrial defect in diabetic retinal neurodegeneration remain to be elucidated. Here, using a high-fat diet (HFD)-induced diabetic mouse model, we showed for the first time that downregulation of active β-catenin due to abnormal GSK3β activation caused synaptic neurodegeneration of RGCs by inhibiting ROS scavenging enzymes, thus triggering oxidative stress-driven mitochondrial impairment in HFD-induced diabetes. Rescue of β-catenin via ectopic expression of β-catenin with a recombinant adenoviral vector, or via GSK3β inhibition by a targeted si-GSK3β, through intravitreal administration, abrogated the oxidative stress-derived mitochondrial defect and synaptic neurodegeneration in diabetic RGCs. By contrast, ablation of β-catenin by si-β-catenin abolished the protective effect of GSK3β inhibition on diabetic RGCs by suppression of antioxidant scavengers and augmentation of oxidative stress-driven mitochondrial lesion. Thus, our data identify β-catenin as a part of an endogenous protective system in diabetic RGCs and a promising target to develop intervention strategies that protect RGCs from neurodegeneration at early onset of diabetic retinopathy.

  

  Mitochondrial impairment is associated with HFD-induced diabetic retinal neurodegeneration. (A) Representative images of retinal Evans Blue (EB) angiography from mice fed with regular chow (RD) or HFD for 20 weeks. Scale bar, 150 μm. (B) Retinal vascular leakage was quantified and normalized by total retinal protein concentrations, and expressed as μg of EB per mg of proteins. NS, no significant difference. (C) Representative waveforms of visual evoked potential (VEP). The differences in peak amplitude (N1-P1) were quantified. (D) Representative retinal immunofluorescence staining for synaptophysin (green). Nuclei were counterstained with DAPI (blue). Scale bar, 100 μm. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer. (E) Representative scanning electron microscopy (SEM) of retinal sections. Lower panels are high-power magnification of the areas indicated by the boxes. Scale bar, 10 μm. (F) Activities of retinal mitochondrial complex I-IV (MRCC I-IV) were measured by spectrophotometry and expressed as nmol/min/mg protein. Two retinae from 2 respective mice in the same group were pooled and preceded for each experiment. Three independent experiments were performed in duplicate for each group. (G) Representative transmission electron microscopy (TEM) of neural retinal sections. Mitochondria in IPL are indicated with arrows. Lower panels are high-power magnification of the areas indicated by the boxes. Scale bar, 0.5 μm. Data are means ± SEM. n = 4 (A, B) or n = 6 (C–G) mice per group. ^**P < 0.01 vs age-match RD controls. See also Supplementary Figure 1; Supplementary Figures 2A and 3A.

  
  
  

  

  Oxidative stress causes mitochondrial defect and synaptic neurodegeneration in diabetic retinae. (A) Contents of 4-HNE in retinae. (B) Activities of antioxidant enzymes. (C) Expression of genes encoding ROS scavengers was determined by quantitative RT-PCR in retinae. The mRNA level of each gene was normalized to the internal GAPDH control and expressed as fold changes of mRNA abundance in the retina from HFD groups relative to their age-matched RD controls. (D–H) 1 μl of NAC (500 nM) was injected intravitreally into the right eye of HFD-induced diabetic mice (HFD-R/+NAC), while PBS was injected into the contralateral left eye as a control (HFD-L/-NAC). (D) Representative waveforms of VEP and quantification of differences in peak amplitude (N1-P1). (E) Contents of retinal 4-HNE. (F) Representative images of retinal TEM with mitochondria in IPL (arrows). Areas boxed in are shown at higher magnification in the lower panels. Scale bar, 0.5 μm. (G) Representative SEM of retinal sections. Areas boxed are shown at higher magnification. Scale bar, 10 μm. (H) Representative synaptophysin (green; scale bar, 100 μm) immunostaining in retinae. Areas boxed in are shown at higher magnification in the lower panels. Data are means ± SEM. n = 4 mice (A–C) or n = 4 eyes (D–H) per group. ^**P < 0.01 vs age-match RD controls; ^*P < 0.05 and ^**P < 0.01 vs contralateral eye injected with PBS. See also Supplementary Figures 2B and 3B.

  
  
  

  

  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. (C–I) 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 (A–B), n = 4 eyes (C–E; G–I), 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.

  
  
  

  

  Restoring β-catenin by GSK3β inhibition protects diabetic retinae from mitochondrial and synaptic defect. (A) Western blotting analyses of phosphorylated-Akt (S473), Akt, phosphorylated-GSK3β (S9), and GSK3β in retinae from mice fed with RD or HFD, respectively. Relative intensities were quantified and normalized against the level of Akt or GSK GSK3β, respectively. (B–I) si-GSK3β was intravitreally injected in the left eye of HFD-fed mice (HFD-L/si-GSK3β), while a scramble si-sc was injected in the contralateral right eye as a control (HFD-R/si-sc). (B) Western blotting for GSK3β and active β-catenin. Relative intensities were quantified. (C) Representative waveforms of VEP and quantification of differences in peak amplitude (N1-P1). (D) Retinal immunostaining for synaptophysin (green; scale bar, 100 μm). Areas boxed in are shown at higher magnification. (E) Representative images of retinal SEM. Lower panels are high-power magnification of boxed areas. Scale bar, 10 μm. (F) Relative mRNA expression of ROS scavenging genes in retinae. (G) Activities of antioxidant enzymes in retinae. (H) Contents of retinal 4-HNE. (I) Representative images of retinal TEM with mitochondria in IPL (arrows). Areas boxed in are shown at higher magnification. Scale bar, 0.5 μm. Data are means ± SEM. n = 4 mice (A) or n = 4 eyes (B–I) per group. ^*P < 0.05 and ^**P < 0.01 vs age-match RD controls; ^*P < 0.05 and ^**P < 0.01 vs contralateral eye injected with scramble si-sc. NS, no significant difference. See also Supplementary Figures 2D and 3D.

  
  
  

  

  Knock-down of β-catenin abrogated the protective effect of GSK3β depletion on HFD-induced diabetic retinal neurodegeneration. A Cy5-labeled si-β-catenin was intravitreally co-injected with si-GSK3β into the right eye of HFD-induced diabetic mice (HFD+si- GSK3β/R-si-β-catenin), while scramble si-sc was co-administrated with si-GSK3β in the contralateral left eye as a control (HFD+si-GSK3β/L-si-sc). (A) Representative immunofluorescence for active β-catenin (green, active β-catenin; red, Cy5; blue, DAPI; scale bar, 100 μm). Areas boxed in are shown at higher magnification. (B) Western blotting for GSK3β and active β-catenin. Relative intensities were quantified. (C) Representative retinal immunofluorescence staining for synaptophysin (green; scale bar, 100 μm). Areas boxed in are shown at higher magnification. (D) Representative SEM of retinal sections. Areas boxed are shown at higher magnification. Scale bar, 10 μm. (E) Representative waveforms of VEP and quantification of differences in peak amplitude (N1-P1). Data are means ± SEM. n = 4 eyes per group. ^**P < 0.01 vs contralateral eye injected with scramble si-sc. See also Supplementary Figures 2E, 3E and 4C.

  
  
  

  

  β-catenin is required for effects of GSK3β inhibition against oxidative stress-induced mitochondrial damage. (A) Relative mRNA expression of ROS scavenging genes in retinae from eyes treated with si-GSK3β+si-β-catenin (HFD+si-GSK3β/R-si-β-catenin) or si-GSK3β+si-sc (HFD+si-GSK3β/L-si-sc), respectively. (B) Activities of antioxidant enzymes in retinae. (C) Amounts of retinal 4-HNE. (D) 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. (E) Representative images of retinal TEM with mitochondria in IPL indicated with arrows. Areas boxed in are shown at higher magnification in lower panels. Scale bar, 0.5 μm. Data are means ± SEM. n = 4 eyes (A–C; E) or n = 6 eyes (D) per group. ^*P < 0.05 and ^**P < 0.01 vs contralateral eye injected with scramble si-sc.

  
  
  

  

  Dysregulated GSK3β/β-catenin signaling caused oxidative stress-associated mitochondrial and synaptic damage of primary RGCs upon glucolipotoxicity. Primary RGCs were exposed to conditioned medium (HG+PA) for 24 h, in the absence or presence of TWS119. Alternatively, RGCs were transfected by si-β-catenin or si-sc and treated with HG+PA in the presence of TWS119. (A) Western blotting for active β-catenin in cytosolic and nuclear fraction of primary RGCs. Intensities were quantified and normalized against the level of GAPDH or Histone-3 and expressed as fold changes of protein abundance relative to controls. Relative intensities of the bands are shown below. (B) Intracellular ROS production was measured by a flow cytometer. Representative curvilineal profiles of fluorescence are shown in upper panels. Quantification of intracellular ROS is shown in lower panels. Values are expressed as the fold changes relative to controls. (C) Contents of 4-HNE in primary RGCs. (D) The mitochondrial membrane potential (MMP) was determined with JC-1 using a confocal microscope. Representative images are shown (green, JC-1 monomer; red, JC-1 polymer; scale bar, 50 μm). (E) The ratio of red to green fluorescence intensity which reflects the levels of the MMP was quantified using Image-Pro Plus software. (F) Representative synaptophysin (green; scale bar, 20 μm) immunostaining in primary RGCs. Data are means ± SEM of three independent experiments. ^*P < 0.05 and ^**P < 0.01 vs normal control; ^#P < 0.05 and ^##P < 0.01 vs HG+PA; ^&P < 0.05 vs si-sc. See also Supplementary Figures 6, 8A and 8B.

  
  
  

  

  Schematic of molecular mechanisms underlying oxidative stress-driven mitochondrial impairment in HFD-induced diabetic retinal neurodegeneration. See the text for a detailed description.