A CTRP5 gene S163R mutation knock-in mouse model for late-onset retinal degeneration

Venkata R M Chavali, Naheed W Khan, Catherine A Cukras, Dirk-Uwe Bartsch, Monica M Jablonski, Radha Ayyagari, Venkata R M Chavali, Naheed W Khan, Catherine A Cukras, Dirk-Uwe Bartsch, Monica M Jablonski, Radha Ayyagari

Abstract

Late-onset retinal macular degeneration (L-ORD) is an autosomal dominant inherited disorder caused by a single missense mutation (S163R) in the CTRP5/C1QTNF5 protein. Early phenotypic features of L-ORD include: dark adaptation abnormalities, nyctalopia, and drusen deposits in the peripheral macular region. Apart from posterior segment abnormalities, these patients also develop abnormally long anterior lens zonules. In the sixth decade of life the rod and cone function declines, accompanied by electroretinogram (ERG) abnormalities. Some patients also develop choroidal neovascularization and glaucoma. In order to understand the disease pathology and mechanisms involved in retinal dystrophy, we generated a knock-in (Ctrp5(+/-)) mouse model carrying the disease-associated mutation in the mouse Ctrp5/C1QTNF5 gene. These mice develop slower rod-b wave recovery consistent with early dark adaptation abnormalities, accumulation of hyperautofluorescence spots, retinal pigment epithelium abnormalities, drusen, Bruch's membrane abnormalities, loss of photoreceptors, and retinal vascular leakage. The Ctrp5(+/-) mice, which have most of the pathological features of age-related macular degeneration, are unique and may serve as a valuable model both to understand the molecular pathology of late-onset retinal degeneration and to evaluate therapies.

Figures

Figure 1.
Figure 1.
Schematic depiction of the Ctrp5+/− targeting strategy. (A) The WT Ctrp5 allele with restriction enzyme sites (Bg, BglII; R, EcoRI; RV, EcoRV; H, HindIII; K, KpnI) and the location of exons 1–3 (boxes). The restriction sites were used to analyze the recombinants (data not shown). (B) The targeting vector was generated with a C->G mutation in Ctrp5 gene at 693 bp in exon 3 and by introducing a Neo gene cassette flanked by loxP sites. The location of the loxP and FRT sites are indicated.
Figure 2.
Figure 2.
Evaluation of expression of Ctrp5 transcript and protein in Ctrp5+/− mice. (A) Expression of Ctrp5 transcripts in WT C57BL/6 mice (WT) and Ctrp5+/− mice. Restriction digestion analysis of an AluI restriction enzyme-digested RT-PCR product on a 1.5% agarose gel. Arrows indicate respective DNA fragment sizes after restriction digestion. (B) Western blot analysis of Ctrp5 protein and β-actin in the posterior eye cup protein extract of Ctrp5+/− mice in comparison with WT retina.
Figure 3.
Figure 3.
Fluorescein angiograms showing retinal vasculature in Ctrp5+/− mice. Mice injected intra-peritoneally with fluorescein-Na showed leakage. The fundus image for the WT mice looks normal, whereas foci of fluorescein dye leakage were observed in the Ctrp5+/− mice 3 min after fluorescein injection. Sites of dye leakage are represented by arrows.
Figure 4.
Figure 4.
Electroretinography analysis in Ctrp5+/− mice. (A) Representative waveforms for (i) scotopic and (ii) photopic ERGs for 21-month-old C57BL/6 mice and 10-, 16- and 21-month-old Ctrp5+/− mice. (B) Intensity response functions showing change in amplitude with age for Ctrp5+/− and WT mice at 10, 13, 16, 18 and 21 months for (i) rod Vmax amplitude, (ii) photopic b-wave amplitude, (iii) scotopic a-wave amplitude, and (iv) scotopic b-wave amplitude. Each point represents the mean ± SE.
Figure 5.
Figure 5.
Rod-b wave recovery in Ctrp5+/− mice. Recovery of rod b-wave from intense bleach in 13-month-old Ctrp5+/− mice (n= 9) and WT (n= 5) mice. Recovery was slow in the Ctrp5+/− mice, and after 60 min, b-wave amplitude recovered to 75% of the pre-bleach amplitude.
Figure 6.
Figure 6.
Accelerated accumulation of autofluorescent spots in Ctrp5+/− mice with age. AF-SLO projection images of 30 frames taken with a 55°angle lens. (A) Representative fundus images obtained by AF-SLO imaging from both Ctrp5+/− mice (n= 12) and age-matched WT mice (n= 10) genotypes at 7–8 months, 12–13 months, 15–16 months and 20–21 months. In both C57Bl/6 and Ctrp5+/− mice, the number of autofluorescent spots was increased with age; however, the accumulation of these spots was accelerated and more pronounced in Crp5+/− mice. (B) Quantification of autofluorescent spots in WT (black circles, dashed line) and Ctrp5+/− mice (black squares, continuous line) per AF-SLO images. The number of autofluorescent spots per AF-SLO image is shown as the mean number of spots ± SD of both eyes for each animal. For both genotypes, the mean number of spots was significantly correlated with the age indicating an age-related increase in autofluorescent spots as an age-related normal process (linear regression plot). However, a significantly higher number of autofluorescent spots were observed in 16–21-month-old Ctrp5+/− mice compared with aged-matched WT mice and compared with the young (7–8 months) groups of both genotypes (P< 0.0005).
Figure 7.
Figure 7.
Histological analysis of retinal structure in Ctrp5+/− mice by light microscopy. (A, C, E, G, I) Histology of the outer retinas from WT control and Ctrp5+/− heterozygote mice from 5 to 21 months. Retinas from control mice show no abnormalities at any age. (B) Retinas from heterozygous 5-month-old Ctrp5+/− mice have no apparent pathology at this magnification. (D) At 8 months of age in the heterozygous mice, rod OSs are elongated and properly organized. Cone inner segments appear swollen (black arrows) but no other pathology is visible. (F) By 12 months of age, inner segments of both rods and cones are mildly swollen and the cytoplasm is heterogeneous, suggesting a degenerative process is beginning in the Ctrp5+/− mice. In addition, chromatin is condensed in the nuclei of cones (white arrows). (H) In the outer retina of 15-month-old heterozygous mice, all inner segments are markedly swollen (asterisks). In addition, RPE apical processes fill the sub-retinal space and no longer surround photoreceptor OSs (black arrows). (J) By 21 months of age, there are marked structural abnormalities in both the RPE and photoreceptors: the basal cytoplasm of the RPE is heterogeneous and filled with vesicle-like structures (black arrows); nuclei of both RPE and photoreceptors have migrated into the area of inner and OSs (white arrows); some inner segments remain swollen (asterisk). RPE, retinal pigment epithelium; OS, outer segment; IS, inner segment; ONL, outer nuclear layer. Magnification bar= 2 µm.
Figure 8.
Figure 8.
Comparison of retinal thickness from the eyes of 21-month-old mice. The retina of WT mice (A) is thicker than that of age-matched Crp5+/− mice (B). RPE, retinal pigment epithelium; OS, outer segments; IS, inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Bar= 10 µm. Direct measurements of the outer nuclear layer (ONL) thickness (μm) in the peripheral, mid-peripheral and central regions of the retina indicate that this layer is significantly thicker in WT mice (P< 0.001) (C).
Figure 9.
Figure 9.
Ultrastructural analysis of RPE and sub-RPE areas from WT control and Ctrp5+/− mice. (A, D, G, J, M) RPE from control mice show no abnormalities at any age. In contrast, the RPE of heterozygous mice have structural aberrations as young as 5 months. (B and C) Subtle disruptions of Bruch's membrane (black arrows) and basal linear deposits consisting of long collagen and fine granular material are apparent at a low frequency (asterisks). (E and F) After 8 months of age, basal linear deposits are more prominent and more frequent (asterisks). (H, I, K, O) By 12 months, sub-RPE drusen-like deposits remain prominent (asterisks) and the RPE cytoplasm contains vesicular profiles (H, I, N). Also visible by 12 months are vesicular profiles containing an amorphous possibly lipid-like substance (white arrows). Areas enclosed in boxes in (B), (E), (H), (K) and (N) are magnified in the panels (C), (F), (I), (L) and (O), respectively. Magnification bars= 2 µm in first and second columns and 500 nm in third column.
Figure 10.
Figure 10.
Expression of marker genes in the retina of Ctrp5+/− mice. Expression profile of photoreceptor-specific markers in Ctrp5+/− mouse posterior eye cup in comparison to controls, taking the expression values of these genes in age-matched controls to be 100% in the retina and RPE. (A) Quantitative expression of Ctrp5, the rod-specific marker rhodopsin and the cone-specific markers (Cone opsin_sw, cone opsin_mw). (B) The stress response and stress-mediated markers (HO-1 and CP). *P-values< 0.002.
Figure 11.
Figure 11.
Localization of cone opsins in the mice retina. (A) Dorsoventral distribution of S-cones (red) and M-cones (green) by immunohistochemical localization in the retinas from WT mice at 21 months, and Ctrp5+/− mice at 12 and 21 months. Blue: nuclei.
Figure 12.
Figure 12.
Spectral ERG responses recorded from 18-month-old Ctrp5+/− mice. Means ± SEM of light-adapted S- (360 nm) and M- (510 nm) ERG amplitudes. Dashed horizontal lines represent ± SD for the WT S-cone responses. S-cone amplitude in Ctrp5+/− mice is significantly lower than M-cone responses.

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