Arap1 loss causes retinal pigment epithelium phagocytic dysfunction and subsequent photoreceptor death

Abstract

Retinitis pigmentosa (RP), a retinal degenerative disease, is the leading cause of heritable blindness. Previously, we described that Arap1-/- mice develop a similar pattern of photoreceptor degeneration. Arap1 is an Arf-directed GTPase-activating protein shown to modulate actin cytoskeletal dynamics. Curiously, Arap1 expression was detected in Müller glia and retinal pigment epithelium (RPE), but not the photoreceptors themselves. In this study, we generated conditional knockout mice for Müller glia/RPE, Müller glia and RPE via targeting Rlbp1, Glast and Vmd2 promoters, respectively, to drive Cre recombinase expression to knock out Arap1. Vmd2-Cre Arap1tm1c/tm1c and Rlbp1-Cre Arap1tm1c/tm1c mice, but not Glast-Cre Arap1tm1c/tm1c mice, recapitulated the phenotype originally observed in germline Arap1-/- mice. Mass spectrometry analysis of human ARAP1 co-immunoprecipitation identified candidate binding partners of ARAP1, revealing potential interactants involved in phagocytosis, cytoskeletal composition, intracellular trafficking and endocytosis. Quantification of outer segment phagocytosis in vivo demonstrated a clear phagocytic defect in Arap1-/- mice compared to Arap1+/+ controls. We conclude that Arap1 expression in RPE is necessary for photoreceptor survival due to its indispensable function in RPE phagocytosis. This article has an associated First Person interview with the first author of the paper.

Keywords: Arap1; Phagocytosis; Pigmentosa; RPE; Retinitis.

Fig. 1.

Characterization of albino Arap1^−/− retina. (A-B′) Haematoxlin and Eosin (H&E) staining of representative retinal sections of pigmented and albino Arap1^−/− (A,A′) and Arap1^+/+ (B,B′) mice. Quantifications of retinal layers in albino and pigmented wild-type (WT) and mutant animals are shown in Fig. S1. Outer retinal dysplasia and photoreceptor degeneration are prominent in both pigmented and albino Arap1^−/− retinas but not seen in Arap1^+/+ retinas. (C,C′,D,D′) Representative retinal sections of albino Arap1^−/− (C,D) and Arap1^+/+ (C′,D′) mice were used for cleaved PARP-1 (cPARP, red; C,C′) fluorescent staining immunohistochemistry and terminal deoxynucleotidyl transferase dUTP nick end labeling assay (TUNEL, green; D,D′) to assess programmed cell death. DAPI counterstaining (blue) was used to visualize the nuclei of the retinal layers. Both cPARP and TUNEL signals were prominent in the outer nuclear layer (ONL) of Arap1^−/− retinas but minimal to nonexistent in control Arap1^+/+ retinas. (C″,D″) Quantification of cPARP and TUNEL immunosignal is shown for both Arap1^−/− (KO) and WT animals per 40× high-power field (HPF). (E) Retinal sections of Arap1^+/− mice were stained with X-gal to assess LacZ histochemical reaction. Blue X-gal signal was detected in the inner nuclear layer (INL) and retinal pigment epithelium (RPE), with weaker signal in the outermost aspect of the ONL and ganglion cell layer (GCL; arrows). cPARP, TUNEL and X-gal analysis were performed on tissue harvested from mice aged P24; H&E staining was performed on tissue harvested from mice aged 6 weeks postnatal. The GCL, inner plexiform layer (IPL), INL, outer plexiform layer (OPL), ONL, inner segment (IS), outer segment (OS) and RPE are labeled in E. All images were taken at 40× magnification. Scale bars: 100 μm. n≥3 for each group, tissue was collected from three different animals of each respective genotype, average values represent the mean, error bars represent s.e.m. Significance calculated by two-tailed, unpaired Student's t-test, *P<0.05.

Fig. 2.

Quantification of Cre function in conditional knockout (cKO) mice. (A-C″) Immunohistochemistry was performed using anti-Sox9 (green) and anti-TdTomato (red) to quantify Cre function in Rlbp1-Cre, Glast-Cre and Vmd2-Cre mice at P84. (A,B,C) Sox9 immunosignal was detected in all of the Müller glia and RPE cells in all Cre lines. (A′,B′,C′) TdTomato signal was visualized in Rlbp1-Cre, Glast-Cre and Vmd2-Cre mouse lines. Channels were merged to create a composite image. TdTomato signal was present in the Müller glia in the Rlbp1-Cre and Glast-Cre strains, but not the Vmd2-Cre strain. Conversely, TdTomato signal was present in RPE in the Rlbp1-Cre and Vmd2-Cre strains, but not the Glast-Cre strain. (A″,B″,C″) Merged TdTomato/Sox9 images are shown. (D,E) Graphical quantification of the proportion of Sox9-positive cells that were also TdTomato positive in Müller glia (D) and RPE (E) for each Cre line. The IPL, INL, OPL, ONL and RPE are labeled in A. Images were taken at 40× magnification. Scale bar: 100 μm. n=3 for each group, tissue collected from three different animals of each respective genotype, error bars represent s.e.m.

Fig. 3.

Characterization of Rlbp1-Cre Arap1^tm1c/tm1c, Glast-Cre Arap1^tm1c/tm1c and Vmd2-Cre Arap1^tm1c/tm1c cKO mouse lines. Cre cKO mice were analyzed with fundus photography, histology and optical coherence tomography (OCT) at 3 months of age (Glast-Cre, Rlbp1-Cre) and 1 month of age (Vmd2-Cre). (A,I) Fundus photography demonstrated pigmentary changes, optic nerve pallor and vascular attenuation in the Rlbp1-Cre Arap1^tm1c/tm1c and Vmd2-Cre Arap1^tm1c/tm1c strains. (E) Conversely, the Glast-Cre Arap1^tm1c/tm1c strain demonstrated no significant differences from WT littermates. (B,F,J) Representative retinal sections from the Cre strains were stained with H&E. (C,G,K) Quantification of retinal layers on histology is shown for each cKO line compared to conditional heterozygote controls. (B,C) Rlbp1-Cre Arap1^tm1c/tm1c retinas demonstrated significant degeneration of the ONL with relative preservation of all other retinal layers. (F,G) Glast-Cre Arap1^tm1c/tm1c retinas were indistinguishable from Glast-Cre Arap1^tm1c/+ littermate retinas. (J,K) Vmd2-Cre Arap1^tm1c/tm1c retinas demonstrated more severe degeneration of the ONL (J), and quantification of retinal layers revealed significant degeneration of the IPL, INL and OS layers compared to those of heterozygous littermates (K). (D,H,L) These changes were consistent with in vivo OCT imaging of retinal layers. The GCL, IPL, INL, OPL, ONL, IS, OS and RPE are labeled in F. Scale bars: 100 μm. Images in B, F and J were taken at 40× magnification. n=3, tissue collected from three different animals of each respective genotype, average values represent the mean, error bars represent s.e.m. Significance calculated by two-tailed, unpaired Student's t-test, *P<0.05, **P<0.01, ***P<0.001.

Fig. 4.

External limiting membrane degeneration in germline and conditional Arap1 knockout retinas. Transmission electron microscopy of representative retinal sections in WT, Arap1^−/−, Glast-Cre Arap1^tm1c/tm1c and Rlbp1-Cre Arap1^tm1c/tm1c mice. WT and Arap1^−/− retinas were assessed at P12. Glast-Cre Arap1^tm1c/tm1c and Rlbp1-Cre Arap1^tm1c/+ retinas were assessed at 8 months of age. (A) Arap1^−/− retinas demonstrated fewer adherens junction (AJ) complexes with increased space between each junction (indicated by the white arrowheads) as well as loss of their linear morphology and arrangement. (B) WT retinas demonstrated normal architecture of AJ complexes (white arrowheads) with typical linear arrangement. (C) Glast-Cre Arap1^tm1c/tm1c retinas also demonstrated frequent gaps between AJ complexes (indicateds by the white arrowheads) alternating with regions of normal AJ complexes (black arrowheads) and loss of linear arrangement. (D) Littermate Glast-Cre Arap1^tm1c/+ retinas did not demonstrate abnormalities in the external limiting membrane (ELM). (E) Rlbp1-Cre Arap1^tm1c/tm1c retinas also demonstrated abnormal ELM junction structure with significant gaps between AJs, although linear arrangement was relatively preserved. (F) These abnormalities were not observed in Rlbp1-Cre Arap1^tm1c/+ littermate retinas. Images were taken at 4300× magnification.

Fig. 5.

ARAP1 co-immunoprecipitation. (A) Western blot analysis validated that the anti-Arap1 antibody detected a band at ∼136 kD in Arap1^+/+, but not Arap1^−/− mouse liver lysate. β-actin was included as an endogenous control. (B-D) Fetal RPEs (FRPEs) were harvested from donor tissue and grown in culture. Cells were lysed with NP-40 lysis buffer when mature, as defined by pigmentation and hexagonal ‘cobblestone’ morphology (passage 1, culture day 98). (E,F) Immunoprecipitation of FRPE lysate with anti-ARAP1 was performed with a parallel goat IgG control immunoprecipitation. Immunoprecipitates were analyzed with western blot analysis along with FRPE lysate. A band was detected at ∼136 kDa in both FRPE lysate (E) and anti-ARAP1 immunoprecipitate, but not the control goat IgG immunoprecipitate (F). (G) Coomassie Blue analysis of the anti-ARAP1 immunoprecipitation, with a faint blue band at ∼136 kDa (arrow). Uncropped blots are shown in Fig. S6. Scale bars: 300 μm (B), 100 μm (C). Images were taken at 4× (B) and 10× magnification (C,D).

Fig. 6.

Reduction of RPE rod phagocytosis in Arap1^−/− retinas. (A-D) Immunohistochemistry using anti-rhodopsin (green) and anti-M- and anti-L-opsin (red, not pictured, shown in Fig. S6) was performed to quantify rod and cone RPE phagosomes, respectively, with DAPI counterstaining (blue) to visualize the RPE nuclei. Only merged images of anti-rhodopsin immunosignal and DAPI staining are shown. (A,B) Rod phagosomes (white arrows) were counted in eyes sectioned at P24 in both Arap1^−/− mice and WT littermates. Only phagosomes present in the RPE layer were counted (first monolayer of nuclei). Examples of counted boundaries are shown (white dashed lines) and in Fig. S8. Scale bar: 20 μm. (E) Arap1^−/− retinas demonstrated reduced numbers of rod phagosomes (18.4±2.7 per 200 μm retina) compared to WT littermates (79.1±13.3 per 200 μm retina). (C,D) Phagosomes were also quantified in P16 Arap1^−/− and WT littermates. (F) Arap1^−/− retinas at P16 also demonstrated reduced numbers of rod phagosomes (18.2±2.8 per 200 μm retina) compared to their WT littermates (73.1±5.8 per 200 μm retina). Uncropped low-power images of the same sections with RPE boundaries defined are shown in Fig. S8. Quantifications of cone phagosomes are shown in Fig. S7. n=3 for each group, tissue was collected from three different animals of each respective genotype, average values represent the mean, error bars represent s.e.m. Significance calculated by two-tailed, unpaired Student's t-test, *P<0.05.