Altered expression of retinal molecular markers in the canine RPE65 model of Leber congenital amaurosis

Abstract

Purpose: Leber congenital amaurosis (LCA) is a group of childhood-onset retinal diseases characterized by severe visual impairment or blindness. One form is caused by mutations in the RPE65 gene, which encodes the retinal pigment epithelium (RPE) isomerase. In this study, the retinal structure and expression of molecular markers for different retinal cell types were characterized, and differences between control and RPE65 mutant dogs during the temporal evolution of the disease were analyzed.

Methods: Retinas from normal and mutant dogs of different ages were examined by immunofluorescence with a panel of 16 different antibodies.

Results: Cones and rods were preserved in the mutant retinas, and the number of cones was normal. However, there was altered expression of cone arrestin and delocalization of rod opsin. The ON bipolar cells showed sprouting of the dendritic arbors toward the outer nuclear layer (ONL) and retraction of their axons in the inner nuclear layer (INL). A decreased expression of GABA, and an increased expression of intermediate filament glial markers was also found in the mutant retinas. These changes were more evident in the adult than the young mutant retinas.

Conclusions: The structure of the retina is well preserved in the mutant retina, but several molecular changes take place in photoreceptors and in bipolar and amacrine cells. Some of these changes are structural, whereas others reflect a change in localization of the examined proteins. This study provides new information that can be applied to the interpretation of outcomes of retinal gene therapy in animal models and humans.

Figure 1.

Immunohistochemical localization of hCAR and RPE65 in normal (wt) and young and adult affected retinas. The proteins were labeled in green (RPE65) and red (hCAR) and the cell nuclei were stained with DAPI (blue). The images were taken from the central (D–F), midpoint (G–I), and peripheral (J–L) retinal regions. RPE65 was present in the wt retinas (A, D, G, J), but absent in the affected retinas, regardless of age. The hCAR antibody labeled the entire cone in the wt retinas, whereas in the affected retinas, the distribution was polarized with labeling primarily in the OS and pedicles. Note that in both the wt and mutant retinas, the peripheral cones were short and broad. Representative images from different animals have been used to illustrate the salient findings: normal (wt) (dog 7560, 18 months of age, A; dog 7559, 27 months of age, D, G, J), young affected RPE65^−/− retinas (dog Br129, 4 months of age, B, E, H, K), and adult affected RPE65^−/− retinas (dog Br118, 15 months of age, C; dog Br113, 10.5 months of age, F, I, L). Scale bar, 20 μm.

Figure 2.

Immunohistochemical localization of RPE65, M/L- and S-cone opsins, and rod opsin in normal (wt) and affected retinas. The proteins were labeled in green (RPE65) and red (different opsins), and the cell nuclei were stained with DAPI (blue). RPE65 was present in the wt retinas (A, D, G) but absent in the affected retinas regardless of age. Cone opsin antibodies labeled cone OS in the control as well as in the mutant retinas, and the number of cones labeled were comparable in both groups. Rod opsin antibodies labeled rod OS in the wt retinas, but in the mutant retinas opsin was also delocalized into the perinuclear and axonal regions of the cells. These changes were similar in the young and in adult affected retinas. Representative images from different animals were used to illustrate the salient findings: normal retinas (wt) (dog EM171, 4 months of age, A, D; dog 7561, 24 months of age, G), young affected retinas (dog Br129, 4 months of age, B, E, H), and adult affected retinas (dog Br113, 10.5 months of age, C, I; dog Br118, 15 months of age, F). Scale bar, 20 μm.

Figure 3.

The number of hCAR-labeled cones in wt and mutant retinas at different ages. There were no differences in the number of cones between the wt and mutant, but the number of cones was comparably decreased between the young and adult retinas of both genotypes (**P ≤ 0.01).

Figure 4.

Immunohistochemical localization of PKCα and Goα in bipolar cells from normal (wt) and affected retinas. The proteins were labeled in green (A, G, RPE65) and red (A– PKCα; G–L, Goα), and the cell nuclei were stained with DAPI (A, G, blue). Retraction of the axonal terminals of the rod bipolar cells was seen in the affected retinas labeled with PKCα (B, C). (D–F) Higher resolution of the bipolar terminals by confocal microscopy. In the affected retinas, there was sprouting of dendritic arbors of Goα ON-bipolar cells that extended toward the ONL in the affected retinas (compare H, I, K, L with G, J, and in confocal microscope detail in J–L). Representative images from different animals are used to illustrate the salient findings: normal retinas (wt) (dog 7560, 18 months of age, A, D, G, J), young affected retinas (dog Br129, 4 months of age, B, E, H, K), and adult affected retinas (dog Br118, 15 months of age, C, F; dog Br89, 17 months of age, I, L). Scale bar, 20 μm.

Figure 5.

Confocal fluorescent immunohistochemical localization of PKCα and synaptophysin in normal (wt), young, and adult affected retinas. The proteins were labeled in green (synaptophysin) and red (PKCα), and the cell nuclei were stained with DAPI (blue). Note the retraction of the axonal terminals of the bipolar cells double labeled with PKCα and synaptophysin, in yellow, in the affected retina. Representative images are used to illustrate the salient findings: normal retina (wt) (dog 7559; 27 months of age, A) and adult affected retina (dog Br118; 15 months of age, B). Scale bar, 20 μm.

Figure 6.

Immunohistochemical localization of RPE65, TH, calretinin, and GABA in normal (wt) and affected retinas. The proteins were labeled in green (A, D, G, RPE65) and red (TH, A–C; calretinin, D–F; GABA, G–I), and the cell nuclei were stained with DAPI (A, D, G, blue). The GABA label was lower than the control in the young affected retina (H), and immunolabeling was further decreased in the adult affected retinas (I). There were no differences between normal and affected retinas with TH and calretinin. Representative images from different animals are used to illustrate the salient findings: normal retinas (wt) (dog 7561, 24 months of age, A, D, G), young affected retinas (dog Br129, 4 months of age, B, E, H), and adult affected retinas (dog Br89, 17 months of age, C, F, I). Scale bar, 20 μm.

Figure 7.

Immunohistochemical localization of BDNF, p75, vimentin, CRALBP, RPE65, and GFAP in normal (wt) and affected retinas. (A–C) RGCs labeled with BDNF (green) and Müller cells with p75 (red). (B, C) Retinal ganglion cells were enveloped by p75-labeled processes in the affected dogs. Vimentin expressed in Müller cells labeled in red (D–F) was increased in the affected dogs (E, F). CRALBP expressed in Müller cells and RPE labeled in green (G–I) was disorganized in the affected dogs (H, I). Red label shows an increase in GFAP expression in affected dogs (J–L), and green shows RPE65 expression (J–L). Representative images from different animals were used to illustrate the salient findings: normal (wt) (dog 7560, 18 months of age, A, D, G, J), young affected retinas (dog Br129, 4 months of age, B, E, H, K), and adult affected retinas (dog Br118, 15 months of age, C, F, I, L). Scale bar, 20 μm.