Essential Role of XBP1 in Maintaining Photoreceptor Synaptic Integrity in Early Diabetic Retinopathy

Abstract

Purpose: Diabetic retinopathy (DR) is a leading cause of blindness in working-age adults characterized by retinal dysfunction and neurovascular degeneration. We previously reported that deletion of X-box binding protein 1 (XBP1) leads to accelerated retinal neurodegeneration in diabetes; however, the mechanisms remain elusive. The goal of this study is to determine the role of XBP1 in the regulation of photoreceptor synaptic integrity in early DR.

Methods: Diabetes was induced by streptozotocin in retina-specific XBP1 conditional knockout (cKO) or wild-type (WT) mice to generate diabetic cKO (cKO/DM) or WT/DM mice for comparison with nondiabetic cKO (cKO/NDM) and WT/NDM mice. Retinal morphology, structure, and function were assessed by immunohistochemistry, optical coherence tomography, and electroretinogram (ERG) after 3 months of diabetes. The synapses between photoreceptors and bipolar cells were examined by confocal microscopy, and synaptic integrity was quantified using the QUANTOS algorithm.

Results: We found a thinning of the outer nuclear layer and a decline in the b-wave amplitude in dark- and light-adapted ERG in cKO/DM mice compared to all other groups. In line with these changes, cKO mice showed increased loss of synaptic integrity compared to WT mice, regardless of diabetes status. In searching for candidate molecules responsible for the loss of photoreceptor synaptic integrity in diabetic and XBP1-deficient retinas, we found decreased mRNA and protein levels of DLG4/PSD-95 in cKO/DM retina compared to WT/DM.

Conclusions: These findings suggest that XBP1 is a crucial regulator in maintaining synaptic integrity and retinal function, possibly through regulation of synaptic scaffold proteins.

Figure 1.

Reduced retinal function in cKO/DM mice. Dark- and light-adapted ERGs were performed in WT and cKO mice with or without diabetes. (A) Graph depicts the a- and b-wave amplitudes of dark-adapted ERG in WT/NDM (n = 5), cKO/NDM (n = 3), WT/DM (n = 7), and cKO/DM (n = 7) mice. A significant decrease in b-wave amplitude was observed in cKO/DM mice compared to WT/NDM, cKO/NDM, and WT/DM mice. *P < 0.05, one-way ANOVA. (B) Graph depicts the a- and b-wave amplitudes of light-adapted ERGs in WT/NDM (n = 6), cKO/NDM (n = 3), WT/DM (n = 8), and cKO/DM (n = 6) mice with a significant decrease in b-wave amplitude for cKO/DM mice compared to all other groups. *P < 0.05, one-way ANOVA with Tukey post hoc test.

Figure 2.

Thinning of retinal ONL in cKO/DM mice. OCT was performed in WT and cKO mice with or without diabetes. (A–D) Representative images of OCT in each group of mice. (E–G) Quantifications of thicknesses of the whole retina (E), inner retina (F), and ONL (G). A small and nonsignificant decrease in ONL thickness in WT/DM (n = 6) mice compared to WT/NDM (n = 5) mice and a significant decrease in ONL thickness in cKO/DM mice (n = 5) compared to WT/NDM, cKO/NDM (n = 6), and WT/DM mice were observed. *P < 0.05, one-way ANOVA with Tukey post hoc test.

Figure 3.

XBP1 deletion does not affect cone bipolar cell morphology. (A–D) Representative confocal images of retinal cryosections with immunofluorescence staining for SCGN (green) to label cone bipolar cell bodies in the INL and their dendrites and presynaptic terminals in the OPL and IPL, respectively. Nuclei were stained with DAPI (blue). Scale bar: 50 µm. (E–H) Higher-magnification images of cone bipolar cell bodies and dendrites in the INL and OPL. Scale bar: 20 µm. (I) Quantification of SCGN-positive cell bodies in the INL reveals no difference in cone bipolar cell numbers across the groups. n = 4 mice in each group. One-way ANOVA with Tukey post hoc test.

Figure 4.

Decreased number of GABAergic amacrine cells in cKO/DM mice. (A–D) Representative confocal images of retinal cryosections with immunofluorescence staining for gamma-aminobutyric acid (GABA, red) to label GABAergic amacrine cells and their processes in the INL and GCL. Nuclei were stained with DAPI (blue). Scale bar: 50 µm. (E) Quantification of GABA-positive cell bodies in the INL and GCL reveals a nonsignificant trend for fewer GABAergic amacrine cells in cKO/NDM retinas compared to WT/NDM retinas (E–G). Under diabetic conditions, there was a significant reduction in GABA-positive cells in cKO/DM mice compared to WT/DM mice in total number of cells (E) and within the INL (F). *P < 0.05. n = 4 mice in each group. One-way ANOVA with Tukey post hoc test.

Figure 5.

Confocal microscopy of ribbon synapses labeled with Ribeye and mGluR6 in mouse retinas. Cryosections (12 µm thick) of mouse retinas were stained with antibodies against metabotropic glutamate receptor 6 (mGluR6, red) and Ribeye (green) to label the presynaptic (Ribeye) and postsynaptic (mGluR6) components of ribbon synapses between photoreceptors and bipolar cells in the OPL. Ribeye also extensively labeled synapses within the IPL. Nuclei were stained with DAPI (blue). (A–D) Representative confocal images from five mice per group. Each image is a projection of confocal scans taken from five consecutive focal planes. Scale bar: 50 µm.

Figure 6.

Reduced integrity of ribbon synapses in WT/DM, cKO/NDM, and cKO/DM mice quantified by the QUANTOS algorithm. Graphs depict QUANTOS Bayes scores of individual synapses. (A) Combined QUANTOS Bayes scores for synapses from WT/NDM and cKO/NDM mice. The distribution of scores for XBP1 cKO/NDM mice is significantly shifted up and to the left when compared to WT/NDM mice. (B) The distribution of scores for WT/DM mice is significantly shifted up and to the left when compared to WT/NDM mice. (C) The distribution of scores for cKO/DM mice is significantly shifted up and to the left when compared to cKO/NDM mice. (D) Graph shows a significant shift up and to the left for cKO/DM mice compared to WT/DM mice. Lines represent linear trendlines. n = 5 mice for each group. **P < 0.01; ***P < 0.0001, Fasano–Franceschini test.

Figure 7.

Reduced levels of PSD-95 mRNA and protein in cKO/DM mice. (A) mRNA levels of Dlg4/PSD-95 in the retinas were measured by quantitative RT-PCR with β-actin as an internal control. n = 3 mice for each group. *P < 0.05. (B, C) Western blot analysis of PSD-95 and quantitation with densitometry. n = 3 mice for each group. *P < 0.05. (D–G) Confocal images of retinal cryosections with double immunofluorescence labeling for PSD-95 (green) and Ribeye (red) showing a reduction in PSD-95 in cKO/DM retina. Nuclei were stained with DAPI (blue). Scale bar: 5 µm. One-way ANOVA with Tukey post hoc test.