Dysfunction of Unc119, a Transducin-Binding Protein, Leads to Cone–Rod Dystrophy through Activating JAK-Stat and NF-κB Inflammatory Pathways in the Mouse Retina

Decreased cone light responses in Unc119^−/− mice at 1M. A–F, ERGs analysis of Unc119^−/− mice. ERGs were recorded from Unc119^+/+ and Unc119^−/− mice at 1M (n = 5 per each genotype). A, Representative scotopic ERGs elicited by four stimulus intensities (−4.0 to 1.0 log cd s/m²) from Unc119^+/+ and Unc119^−/− mice at 1M. B, C, Scotopic amplitudes of a- (B) and b-waves (C) are shown as a function of stimulus intensity. Data are presented as the mean ± SD. n = 5 per each genotype. n.s., not significant (two-way repeated-measures ANOVA, multiple comparisons). D, Representative photopic ERGs elicited by four stimulus intensities (−0.5 to 1.0 log cd s/m²) from Unc119^+/+ and Unc119^−/− mice at 1M. E, F, Photopic amplitudes of a- (E) and b-waves (F) are shown as a function of the stimulus intensity. Data are presented as the mean ± SD. n = 5 per each genotype. *p < 0.05, ***p < 0.001, n.s., not significant (two-way repeated-measures ANOVA, multiple comparison). G, H, Scotopic and photopic b-wave implicit times of ERGs were recorded from Unc119^+/+ and Unc119^−/− mice at 1M. The scotopic implicit times of the b-wave (G) reflect synaptic transmission from rod photoreceptors to rod bipolar rod cells. The photopic implicit times of the b-wave (H) reflect synaptic transmission from the cone photoreceptor to the cone ON bipolar cells. Data are presented as mean ± SD. n = 5 per each genotype. n.s., not significant (two-way repeated-measures ANOVA, multiple comparison). I, Toluidine blue staining of Unc119^+/+ and Unc119^−/− retinas at 1M. The thicknesses of the ONL, OPL, INL, IPL, and GCL were measured using ImageJ. Data are presented as the mean ± SD. n = 4 per each genotype. n.s., not significant (unpaired t test). GCL, ganglion cell layer; ONL, outer nuclear layer; INL, inner nuclear layer; IPL, inner plexiform layer. J–R, Immunofluorescence analysis of retinal sections from Unc119^+/+ and Unc119^−/− at 1M using marker antibodies as follows: Rhodopsin (rod outer segments, J), M-opsin (M-cone outer segments, K), S-opsin (S-cone outer segments, L), Chx10 (bipolar cells, M), Pax6 (amacrine and ganglion cells, N), Calbindin (horizontal cells and a subset of amacrine cells, O), Ctbp2 (photoreceptor synapses, P), Rbpms (ganglion cells, Q), and S100β (Müller glial cells, R). The nuclei were stained with DAPI (blue). Rhodopsin length was measured in four images from the retina of one mouse. In one image, Rhodopsin length was measured at three points and averaged (J). The signal intensity of M-opsin and S-opsin in OPL were measured. Arrowheads indicate mislocalization of M-opsin and S-opsin signals (K, L). Data are presented as the mean ± SD. n = 3 per each genotype. n.s., not significant (unpaired t test). GCL, ganglion cell layer; ONL, outer nuclear layer; INL, inner nuclear layer; OS, outer segment.

Decrease in cone and rod light responses of Unc119^−/− mice at 12M. A–F, ERG analysis of Unc119^−/− mice at 12M. ERGs were recorded from Unc119^+/+ and Unc119^−/− mice at 12M (n = 5 and 4 mice, Unc119^+/+ and Unc119^−/−, respectively). A, Representative scotopic ERGs elicited by four stimulus intensities (−4.0 to 1.0 log cd s/m²) from Unc119^+/+ and Unc119^−/− mice at 12M. B, C, The scotopic amplitudes of a- (B) and b-waves (C) are shown as a function of the stimulus intensity. Data are presented as the mean ± SD. n = 5 and 4 mice (Unc119^+/+ and Unc119^−/− mice, respectively). **p < 0.01, ***p < 0.001, n.s., not significant (two-way repeated-measures ANOVA, multiple comparison). D, Representative photopic ERGs elicited by four stimulus intensities (−0.5 to 1.0 log cd s/m²) from Unc119^+/+ and Unc119^−/− mice at 12M. E, F, The photopic amplitudes of a- (E) and b-waves (F) are shown as a function of the stimulus intensity. Data are presented as mean ± SD. n = 5 and 4 mice (Unc119^+/+ and Unc119^−/−, respectively). *p < 0.05, **p < 0.01, n.s., not significant (two-way repeated-measures ANOVA, multiple comparison). G, H, Scotopic (G) and photopic (H) b-wave implicit times of ERGs were recorded from Unc119^+/+ and Unc119^−/− mice at 12M. Data are presented as the mean ± SD. n = 5 and n = 4 mice, Unc119^+/+ and Unc119^−/−, respectively. n.s., not significant (two-way repeated-measures ANOVA, multiple comparisons). I, Toluidine blue staining of Unc119^+/+ and Unc119^−/− retinas at 12M. The thicknesses of the ONL, OPL, INL, IPL, and GCL were measured using ImageJ. Data are presented as the mean ± SD. n = 3 and 4 (Unc119^+/+ and Unc119^−/−, respectively). *p < 0.05, ***p < 0.01, ****p < 0.001, n.s., not significant [unpaired t test (top bar graph) and one-way ANOVA, multiple comparisons (bottom bar graph)]. GCL, ganglion cell layer; ONL, outer nuclear layer; INL, inner nuclear layer; IPL, inner plexiform layer; OS, outer segment. J–M, Immunofluorescence analysis of retinal sections from Unc119^+/+ and Unc119^−/− at 12M using marker antibodies as follows: Rhodopsin (rod outer segments, J), M-opsin (M-cone outer segments, K), S-opsin (S-cone outer segments, L), and Arr3 (cone photoreceptor cells, M). Nuclei were stained with DAPI (blue). Rhodopsin length was measured in four images from the retina of one mouse. In one image, Rhodopsin length was measured at three points and averaged (J). The number of M-opsin-, S-opsin-, and Arr3-positive cells was counted (K, L, M). Data are presented as mean ± SD. n = 3 and 4 (Unc119^+/+ and Unc119^−/−, respectively). *p < 0.05, **p < 0.01, ***p < 0.001 (unpaired t test). GCL, ganglion cell layer; ONL, outer nuclear layer; INL, inner nuclear layer; OS, outer segment.

Unc119 interacts with Gnat2 in cones. A, Generation of Nrl^−/− mice and Western blotting analysis of Nrl^+/+ and Nrl^−/− mouse retinas using the following antibodies: Rhodopsin, S-opsin, and M-opsin. α-Tubulin was used as a loading control. B, Scheme of pull-down assay and LC-MS/MS analysis. A pull-down assay was performed using GST and GST-fused Unc119 proteins. GST- and GST-fused Unc119 proteins were reacted with Nrl^−/− retinal lysates or lysis buffer. Venn diagram of the number of identified proteins in the indicated experimental conditions by pull-down assay and subsequent LC-MS/MS analysis (left). C, Fifty-eight proteins were identified using total LC-MS/MS analysis. Four proteins, Gnat2, Ytdc2, Tba1a, and Hmmr, were detected only in GST-fused mUnc119 that reacted with Nrl^−/− retinal lysates. The normalized total spectra of Gnat2 are shown. D, Immunoprecipitation of Unc119 with Gnat1 or Gnat2. Plasmids expressing FLAG-tagged Gnat1 or Gnat2, and HA-tagged Unc119 were cotransfected into HEK293T cells. Cell lysates were subjected to immunoprecipitation using an anti-FLAG antibody. The immunoprecipitated proteins were detected by Western blot analysis using anti-FLAG and anti-HA antibodies. E, F, Immunofluorescence analysis of Unc119^+/+ and Unc119^−/− retinas at 1M (D) and P14 (E), using an anti-Gnat2 antibody. Nuclei were stained with DAPI (blue). Gnat2 intensity was measured in the retinal sections of all photoreceptor layers (OS, IS, ONL, and OPL) using ImageJ software. Data are presented as the mean ± SD. n = 5 per each genotype at 1M and n = 3 per each genotype at P14. ***p < 0.001 (unpaired t test). GCL, ganglion cell layer; ONL, outer nuclear layer; INL, inner nuclear layer; OS, outer segment. G, Western blotting analysis of Gnat2 protein in retinas from Unc119^+/+ and Unc119^−/− mice at P14 using an anti-Gnat2 antibody. α-Tubulin was used as a loading control. Relative Gnat2 protein levels in Unc119^+/+ and Unc119^−/− retinas were determined by quantification of Gnat2 band intensity (normalized to α-tubulin). Data are presented as the mean ± SD. n.s., not significant (unpaired t test); n = 5 mice per genotype. H, I, Immunoprecipitation analysis of Unc119 and Gnat1, Gnat1-G2A, Gnat2, or Gnat2-G2A. Plasmids expressing FLAG-tagged Gnat1, Gnat1-G2A, Gnat2, or Gnat2-G2A, and HA-tagged Unc119 were cotransfected into HEK293T cells. Cell lysates were subjected to immunoprecipitation with an anti-FLAG antibody. Immunoprecipitated proteins were detected by Western blot analysis with anti-FLAG (H) and anti-HA (I) antibodies.

Transcriptional changes by Unc119 deficiency in the retina. A, Heatmaps of differentially expressed genes (fold change >1.4, less than −1.4; p < 0.05, unpaired t test) between Unc119^+/+ and Unc119^−/− retinas. Normalized FPKM from the RNA-seq dataset was used for heatmap visualization. B, C, Immunofluorescence analysis of Unc119^+/+ and Unc119^−/− retinas at 1M using antibodies against GFAP and C1q. Nuclei were stained with DAPI (blue). The intensities of GFAP (B) and C1q (C) signals in Unc119^+/+ and Unc119^−/− retinas at 1M were measured. Data are presented as the mean ± SD. ∗∗p < 0.01 (unpaired t test). n = 3 mice per group. GCL, ganglion cell layer; ONL, outer nuclear layer; INL, inner nuclear layer. D, qRT-PCR analysis of Nxnl2 and Nxnl1 mRNA expression levels in the Unc119^+/+ and Unc119^−/− mouse retinas. Data are presented as the mean ± SD. ∗p < 0.05, ∗∗p < 0.01 (unpaired t test). n = 3 mice per group. E, Gene set enrichment analysis of upregulated genes in the Unc119^−/− mouse retina. F, Ingenuity Pathway Analysis (IPA) to predict the upstream factors affecting gene expression changes (fold change >1.4, less than −1.4; p < 0.05, unpaired t test) in the Unc119^−/− retina. The predicted upstream factors with an activation Z score >3.0 are shown. G, H, IPA networks showing interferon regulatory factor 1 (IRF1; G) and RELA proto-Oncogene (H), NF-kB subunit (RELA) as upstream regulators. IRF1 and RELA were predicted to be activated in the Unc119^−/− mouse retina.

Curcumin suppresses cone degeneration and inflammation in the Unc119^−/− retina. A, Experimental design for curcumin administration to Unc119^−/− mice. B–G, ERG analysis of curcumin-treated Unc119^−/− mice at 1M. ERGs were recorded from control (DMSO) and curcumin-treated Unc119^−/− mice at 1M (n = 4 per each genotype). B, Representative scotopic ERGs elicited by four stimulus intensities (−4.0 to 1.0 log cd s/m²) from control (DMSO) and curcumin-treated Unc119^−/− mice at 1M. C, D, The scotopic amplitudes of a- (C) and b-waves (D) are shown as a function of the stimulus intensity. Data are presented as the mean ± SD. n = 4 per each genotype. n.s., not significant (two-way repeated-measures ANOVA, multiple comparisons). E, Representative photopic ERGs elicited by four stimulus intensities (−0.5 to 1.0 log cd s/m²) from control (DMSO) and curcumin-treated Unc119^−/− mice at 1M. F, G, Photopic amplitudes of a- (F) and b-waves (G) are shown as functions of stimulus intensity. Data are presented as the mean ± SD. n = 4 mice per each genotype. *p < 0.05, n.s., not significant (two-way repeated-measures ANOVA, multiple comparison). H–M, Immunofluorescence analysis of retinal sections from control and curcumin-injected Unc119^−/− mice was performed using antibodies against Socs3 (H), RelA (I), GFAP (J), C1q (K), M-opsin (L), and S-opsin (M). Nuclei were stained with DAPI (blue). The intensities of the Socs3, RelA, GFAP, and C1q signals in the retina were measured (H–K). The number of M-opsin- and S-opsin-positive cells was counted. The signal intensities of M-opsin and S-opsin in the OPL were measured. Arrowheads indicate mislocalization of M-opsin and S-opsin signals (L, M). Data are presented as mean ± SD. n = 4 per each genotype. *p < 0.05, **p < 0.01, ***p < 0.001, n.s., not significant (unpaired t test). GCL, ganglion cell layer; ONL, outer nuclear layer; INL, inner nuclear layer; OS, outer segment.