Rhodopsin mislocalization drives ciliary dysregulation in a novel autosomal dominant retinitis pigmentosa knock-in mouse model

Abstract

Rhodopsin mislocalization encompasses various blind conditions. Rhodopsin mislocalization is the primary factor leading to rod photoreceptor dysfunction and degeneration in autosomal dominant retinitis pigmentosa (adRP) caused by class I mutations. In this study, we report a new knock-in mouse model that harbors a class I Q344X mutation in the endogenous rhodopsin gene, which causes rod photoreceptor degeneration in an autosomal dominant pattern. In Rho^Q344X/+ mice, mRNA transcripts from the wild-type (Rho) and Rho^Q344X mutant rhodopsin alleles are expressed at equal levels. However, the amount of RHO^Q344X mutant protein is 2.7 times lower than that of wild-type rhodopsin, a finding consistent with the rapid degradation of the mutant protein. Immunofluorescence microscopy indicates that RHO^Q344X is mislocalized to the inner segment and outer nuclear layers of rod photoreceptors in both Rho^Q344X/+ and Rho^Q344X/Q344X mice, confirming the essential role of the C-terminal VxPx motif in promoting OS delivery of rhodopsin. The mislocalization of RHO^Q344X is associated with the concurrent mislocalization of wild-type rhodopsin in Rho^Q344X/+ mice. To understand the global changes in proteostasis, we conducted quantitative proteomics analysis and found attenuated expression of rod-specific OS membrane proteins accompanying reduced expression of ciliopathy causative gene products, including constituents of BBSome and axonemal dynein subunit. Those studies unveil a novel negative feedback regulation involving ciliopathy-associated proteins. In this process, a defect in the trafficking signal leads to a reduced quantity of the trafficking apparatus, culminating in a widespread reduction in the transport of ciliary proteins.

Figure 1.. CRISPR/Cas9-mediated knock-in of Q344X mutation in mouse rhodopsin (Rho).

(A) Schematic representation of mouse Rho gene in chromosome 6. The PAM sequences tailored to exon 5 and microhomology template insertion site are indicated along with wild-type and Q344X alleles. The C to T conversion at the 344-position, resulting in the Q344X mutation present in exon 5 of the Rho gene, is highlighted in red. Another silent point mutation C to A in the codon for threonine (T) at the 342 position (highlighted in red) is introduced to prevent the recognition by guide RNA after microhomology template insertion. (B) Validation of CRISPR/Cas9-mediated knock-in of Q344X mutation to the mouse rhodopsin gene (Rho) by sequencing. Upper panel shows wild-type allele with the QVAPA motif. Lower panel shows the Q344X allele with C to G mutation, leading to an early insertion of a stop codon at the 344th position. (C) Partial sequences of exon 5 from the wild-type and Q344X rhodopsin alleles surrounding the BsaI restriction site (GAGACC) highlighted by the shaded rectangle. In the Rho^Q344X allele, the introduced silent point mutation in the codon for threonine (T) at position 342 resulted in the loss of the BsaI restriction site. Lane 1, undigested PCR fragment from a Rho^Q344X/+ knock-in mouse; lane 2, same PCR fragment as lane 1 digested with BsaI; lane3, undigested PCR fragment from a wild-type mouse; lane 4, same PCR fragment as lane 3 digested with BsaI. Upon a digestion with BsaI, a 598 bp PCR product of wild-type allele (lane 2, upper and lower fragments, respectively) was digested into two fragments (lane 4, 318 bp and 280 bp). (D) Ratio of wild-type and Q344X mutant rhodopsin mRNA expression in the Rho^Q344X/+ knock-in retina. PCR product from Rho^Q344X/+ retinal cDNA was either undigested or digested with BsaI (left panel, - and +, respectively). Using real-time quantitative PCR, relative amounts of wild-type and Q344X mutant mRNA were compared (n = 3 animals). There is no significant difference in the mRNA expression levels between wild-type and Q344X mutant alleles (48.6 ± 3.6 % for wild-type and 51.5 ± 8.6 % for Rho^Q344X mRNA). ns: not significant (p = 0.6164)

Figure 2.. OCT analyses of class I Rho^Q344X knock-in mutant mouse indicate degeneration of photoreceptor neurons.

(A) OCT images for the ventral and dorsal regions including the optic nerve head (ONH) were acquired for wild-type, Rho^Q344X/+, and Rho^Q344X/Q344X mice at P21, P60, and P120. ONLs are indicated by red vertical bars. Scale bars, 100 μm. (B) OCT images from P60 wild-type and Rho^Q344X/+ mice at the locations 500 μm away from the ONH are shown. ELM, external limiting membrane; RPE, retinal pigment epithelium. Scale bar, 50 μm. (C). The thicknesses of the ONL were measured at four distinct locations situated 500 μm away from the ONH, as illustrated in the fundus images of wild-type and Rho^Q344X/+ mice. (D) ONL thicknesses were compared among wild-type (blue), Rho^Q344X/+ (magenta), and Rho^Q344X/Q344X (green) mice in four distinct regions (nasal, temporal, ventral, and dorsal) as indicated in (C). The thicknesses of the ONL were plotted as a function of postnatal days (P21 – 360), and the data were presented as mean ± SD (n = 4 animals for each genotype). The data were subjected to statistical analysis using two-way ANOVA, followed by Tukey’s post-hoc test for pairwise comparisons. Significant differences (****, p < 0.0001) were observed when comparing wild-type mice to Rho^Q344X/+ or Rho^Q344X/Q344X mice.

Figure 3.. Histological analysis of photoreceptor cell loss in P35 and 90 Rho^Q344X/+ mice.

(A) Retinal sections from wild-type and Rho^Q344X/+ mice, aged P35 and P90, were labeled with Hoechst 33342 (blue) to visualize their nuclear layers. RPE: retinal pigmented epithelium, OS: outer segments, IS: inner segments, ONL: outer nuclear layer, INL: inner nuclear layer, GCL: ganglion cell layer. Scale bar, 20 μm. (B) Thicknesses in the ONL were measured every 150 μm on the dorsal and ventral sides of the ONHs. The data show a progressive reduction of ONL thickness in Rho ^Q344X/+ mice due to the loss of photoreceptor cells from P35 to P90. Data were shown as mean ± SD for P35 and P90 (n = 4). The data were analyzed using two-way ANOVA. For pair-wise comparison of wild-type and Rho^Q344/+ mice, the Šídák method was utilized to calculate p-values comparing wild-type to Rho^Q344X/+ mice. Statistical significance (****, p < 0.0001; ***, p < 0.001; **, p < 0.01) is indicated above each data point. ns: not significant (p > 0.01). (C) Retinal sections from P21 Rho^Q344X/Q344X mice were labeled with Hoechst 33342 (blue) to visualize their nuclear layers (left panel). Retinal sections from P21 Rho^Q344X/Q344X mice were subjected to immunofluorescence labeling using antibodies against rhodopsin (B6–30, green) and a photoreceptor OS marker, Pde6γ (red), to label OS (right panels). DIC, differential interference contrast. (D) Thicknesses of the OS layers from P35 wild-type mice, P35 Rho^Q344X/+ mice, and P21 Rho^Q344X/Q344X mice were measured every 150 μm on the dorsal and ventral sides of the ONH. Data were presented as mean ± SD (n = 4 mice) for all the genotypes. The data were analyzed using two-way ANOVA. For pairwise comparisons, Dunnett’s multiple comparison test was utilized to calculate p-values (wild-type vs. Rho^Q344X/+ or wild-type vs. Rho^Q344X/Q344X, ****, p < 0.0001), as indicated above each data point.

Figure 4.. Rhodopsin mislocalization in Rho^Q344X knock-in mice.

(A) Retinal sections from wild-type, and Rho^Q344X/+, and Rho^Q344X/Q344X were subjected to immunofluorescent labeling using the 1D4 antibody (red). As the 1D4 recognizes the Ct epitope only present in wild-type rhodopsin protein, signals were only observed in wild-type and Rho^Q344X/+ mice. Mice were studied at P24 and P62 or 63. Rhodopsin is observed mainly in the OS of wild-type mice, whereas it is also mildly mislocalized to IS, ONL and OPL in Rho^Q344X/+ mice both at P24 and P62/63. OPL: outer plexiform layer (B) Retinal sections from wild-type, Rho^Q344X/+, and Rho^Q344X/Q344X were subjected to immunofluorescent labeling using B6–30 antibody (red). As B6–30 recognizes the Nt epitope preserved both in wild-type and RHO^Q344X mutant rhodopsin, signals were observed in wild-type, Rho^Q344X/+, and Rho^Q344X/Q344X mice. Both at P24 and P62/63, signals are observed mainly in the OS of wild-type mice, whereas they are also observed in IS, ONL, and OPL of Rho^Q344X/+ mice. RHO^Q344X mutant rhodopsin is significantly mislocalized to IS, ONL, OPL of Rho^Q344X/Q344X mice. Nuclei were labeled with Hoechst 33342 (blue). Scale bars, 20 μm.

Figure 5.. Rhodopsin expression is remarkably downregulated in the Rho^Q344X/+ retina at P35.

Quantitative immunoblotting analysis was conducted using anti-rhodopsin monoclonal antibodies, B6–30 (Nt-specific) and 1D4 (Ct-specific) (n = 4 mice per genotype). Anti-β-tubulin was employed for loading controls. For quantification of signals originating from rhodopsin, we selected the regions corresponding to monomeric and dimeric forms of rhodopsin. (A) B6–30 recognizes wild-type, RHO^Q344X, and RHO^P23H mutant rhodopsin on retinal homogenates from wild-type, Rho^Q344X/+, and Rho^P23H/+ mice. Based on the analysis, total rhodopsin in the retina is significantly decreased in Rho^Q344X/+, and Rho^P23H/+ retinas. (B) The 1D4 antibody recognizes wild-type and RHO^P23H but is incapable of binding to RHO^Q344X, which lacks the Ct 5 amino acids. Based on the analysis, wild-type and RHO^P23H are significantly decreased in Rho^Q344X/+, and Rho^P23H/+ retinas. (C) The quantities of RHO^Q344X in Rho^Q344X/+ retinas were calculated and compared to those of wild-type rhodopsin based on the difference in immunoblot signal intensities generated by B6–30 and 1D4 antibodies.

Figure 6.. NKA expression is downregulated and GFAP expression is upregulated in the P35 Rho^Q344X/+ retinas.

(A and B) Retinal sections from wild-type (A) and Rho^Q344X/+ (B) mice were subjected to immunofluorescence labeling using antibodies against NKA (red) and ROM1 (green), a photoreceptor OS marker. Nuclei were labeled with Hoechst 33342 (blue). NKA is localized to the ISs and other regions of photoreceptors but is not observed in the OSs. (C) Immunofluorescence originating from NKA was quantitated in IS, INL and GCL spanning 227 μm of retina length. Average intensities measured for Rho^Q344X/+ were normalized to those for wild-type and the data expressed as mean ± SD (n = 4 animals for each genotype). The data were subjected to statistical analysis using unpaired t-test for comparisons. Significant differences (*, p < 0.05) were observed when comparing fluorescence intensity from inner segment of wild-type mice to Rho^Q344X/+ mice. ns: not significant (p > 0.05). (D and E) Retinal sections from wild-type (C) and Rho^Q344X/+ (D) mice were subjected to immunofluorescence labeling using antibodies against GFAP (green), a marker for astrocytes and Müller cell-mediated reactive gliosis. Nuclei were labeled with Hoechst 33342 (blue). Müller cell-mediated gliosis was activated in the P35 Rho^Q344X/+ retina. Scale bars, 20 μm.

Figure 7.. Differentially expressed proteins show rod outer segment proteins are downregulated in the Rho^Q344x/+ retina at P35.

(A) A volcano plot for the identified proteins. X-axis shows log2-transformed fold change (differential protein expression between wild-type and Rho^Q344X/+ retinas), and y-axis shows −log10-transformed q-value. For instance, the log2-transformed fold change for Pdap1 is 1.2. This equates to a linear scale fold change of 2.4. A total of 89 proteins (shown in red) were differentially expressed with 2-fold change (log2-transformed change of < −1 or > 1) and q-value < 0.05 thresholds indicated by the vertical and horizontal dashed lines. (B) Pathway analysis for GO terms using the protein abundance with 60% threshold (a total of 93 proteins). Top 20 significantly changed pathways are shown in dot plots from top to bottom for biological process (left, adjusted p < 0.0001), cellular component (middle, adjusted p < 0.005), and molecular function (right, adjusted p < 0.05). The size of each dot within the plots (Count) represents the level of protein enrichment, while the color coding indicates statistical significance (p.adjust). These markers are explained within each plot. (C) Networks illustrating the outcomes of the hypergeometric test and pathway analysis based on gene enrichment are presented. The size of each dot (Count) represents the level of gene enrichment (number of genes), while the color coding indicates statistical significance (p.adjust), as indicated in the bottom right corner of the panel.