Retinal pigment epithelium defects accelerate photoreceptor degeneration in cell type-specific knockout mouse models of choroideremia

Abstract

Purpose: Choroideremia (CHM) is a progressive X-linked degeneration of three ocular layers (photoreceptors, retinal pigment epithelium, and choroid), with a complex and still largely unclear pathogenesis. To investigate the pathophysiology of CHM, the authors engineered mice with a cell type-specific Chm/Rep1 knockout (KO).

Methods: A mouse line carrying a conditional allele Chm(Flox) was crossed with the transgenic line IRBP-Cre to achieve Chm KO, specifically in the photoreceptor layer, and Tyr-Cre to produce Chm KO, specifically in the retinal pigment epithelial and other pigmented cells. Chm(Flox), Tyr-Cre+ and Chm(Flox), IRBP-Cre+ mice were mated to produce mice with Chm KO in both layers. All mouse lines were studied by histology, electron microscopy, electroretinography (ERG), scanning laser ophthalmoscopy (SLO), and biochemical

Results: In Chm(Flox), IRBP-Cre+ mice the authors observed the progressive degeneration of photoreceptors in the presence of normal retinal pigment epithelium (RPE). Chm(Flox), Tyr-Cre+ mice exhibited coat color dilution and pigment abnormalities of the RPE in the presence of an intact outer nuclear layer. In 6- to 8-month-old Chm(Flox), Tyr-Cre+, IRBP-Cre+ mice, the degeneration of photoreceptors was accelerated compared with Chm(Flox), IRBP-Cre+ mice but became leveled with age, such that it was comparable at 12 to 14 months. Detailed ERG and SLO analysis supported the histopathologic findings.

Conclusions: Defects in photoreceptors and RPE can arise because of intrinsic defects caused cell autonomously by the Chm KO. However, when both photoreceptors and RPE are diseased, the dynamics of the degenerative process are altered. Photoreceptor functional deficit and cell death manifest much earlier, suggesting that the diseased RPE accelerates photoreceptor degeneration.

Figure 1.

Characterization of Chm^Flox, IRBP-Cre+ model. (A) Illustration showing main features of the Chm^Flox allele and its conversion to the Chm^Null allele after Cre-recombination. (B) Neuroretina (N) and RPE (R) were isolated from the eyes of Chm^Null/WT, Chm^Flox, Chm^Flox, IRBP-Cre+ animals and analyzed by PCR using primers that could identify Chm^Null, Chm^Flox, Chm^WT alleles (top) and control primers (bottom). (C) Eyes were collected from Chm^WT, IRBP-Cre+ animals at P21, stained with Cre-specific primary antibody and Alexa-568 secondary antibody, and visualized by confocal microscopy. (D) Phase image corresponding to (C). (E) In vitro prenylation reaction was performed on the cytosolic fractions of the lysates isolated from neuroretina (NR) and the RPE of the Chm^WT, Chm^Flox, and Chm^Flox, IRBP-Cre+ animals. (F–I) Paraffin wax sections were prepared from the eyes of Chm^Flox animals at 6 months (F) and 12 months (H) and Chm^Flox, IRBP-Cre+ animals at 6 months (G) and 12 months (I), cut at 4-μm thickness, and stained with hematoxylin and eosin.

Figure 2.

Characterization of Chm^Flox, Tyr-Cre+ model (A) Photograph of 4-week-old Chm^Flox, Tyr-Cre+ and Chm^Flox littermates. (B) Immunoblot analysis of the neuroretina (NR) and RPE cells isolated from the eyes of the Chm^Flox and Chm^Flox, Tyr-Cre+ animals using Rep-specific antibody J905 and α-tubulin antibody (as a loading control). (C) In vitro prenylation reaction was performed on the cytosolic fractions of the lysates isolated from the RPE of the Chm^WT, Chm^Flox, Chm^Null/WT, TM-induced Chm^Flox, MerCreMer+ and Chm^Flox, Tyr-Cre+ animals. (D–G) Frozen section were prepared from Chm^WT, Tyr-Cre+ animals at E12.5, stained with Cre-specific primary antibody and Alexa-568 secondary antibody, and visualized by confocal microscopy. (E) Phase image corresponding to (D). (F) Enlarged area of the inset in (D). (G) Phase image corresponding to (F). (H–K) Paraffin wax sections were prepared from the eyes of Chm^Flox (H) and Chm^Flox, Tyr-Cre+ animal (J) at 1 year, cut at 4-μm thickness, and stained with hematoxylin and eosin. (I) Enlargement of the boxed area in (H). (K) Enlargement of the boxed area in (J).

Figure 3.

Electron microscopy of the Chm^WT (A), Chm^Flox (B), Chm^Flox, IRBP-Cre+ (C), Chm ^Null/WT (D), TM -induced Chm^3lox, MerCreMer+ (E), Chm^Flox, Tyr-Cre+ (F) at 5 months. In (A), (B), (C), and (E), the RPE is highly pigmented, and melanosomes are found in apical processes. In contrast, in (F) and some areas of (D), pigmentation is altered and melanosomes remain in the cell body. Scale bar, 10 μm.

Figure 4.

Histologic analysis of the retina of the 6-month old Chm^Flox (A), Chm^Flox, Tyr-Cre+ (B), Chm^Flox, IRBP-Cre+ (C), Chm^Flox, IRBP-Cre+, Tyr-Cre+ (D) and 12-month old Chm^Flox (E), IRBP-Cre+, Tyr-Cre+ (F). (G) Morphometric analysis of the number of photoreceptor rows in the central retina in the Chm^Flox, IRBP-Cre+ (purple bars) and Chm^Flox, IRBP-Cre+, Tyr-Cre+ (blue bars). Numbers of animals analyzed were: Chm^Flox, IRBP-Cre+: 6 to 8 months (n = 15), 12 to 14 months (n = 19); Chm^Flox, IRBP-Cre+, Tyr-Cre+: 6 to 8 months (n = 7), 12 to 14 months (n = 11).

Figure 5.

Quantification of the amplitude of the major components (a- and b-wave) of the dark-adapted ERG across a range of stimulus intensity. Two groups of animals (8-month- and 14-month-old) were studied. Numbers of 8-month-old animals were: Chm^Flox, Tyr-Cre+ (n = 5); Chm^Flox, IRBP-Cre+ (n = 3); Chm^Flox, Tyr-Cre+, IRBP-Cre+ (n = 5), and Chm^Flox (n = 4). Numbers of 14-month-old animals were: Chm^Flox, Tyr-Cre+ (n = 6); Chm^Flox, IRBP-Cre+ (n = 5); Chm^Flox, Tyr-Cre+, IRBP-Cre+ (n = 6), and Chm^Flox (n = 6). All genotypes are shown on each graph: Chm^Flox, Tyr-Cre+ (red line), Chm^Flox, IRBP-Cre+ (blue line), Chm^Flox, IRBP-Cre+, Tyr-Cre+ (green line). Mean amplitudes ± SEM are plotted. Values for control animals (Chm^Flox, mean ± SEM) are indicated by gray shading.

Figure 6.

Representative AF-SLO projection fundus images obtained from Chm^Flox (A, E, I), Chm^Flox, Tyr-Cre+ (B, F, J), Chm^Flox, IRBP-Cre+ (C, G, K), Chm^Flox, IRBP-Cre+, Tyr-Cre+ (D, H, L) at 8 months (A–D) and 14 months (E–L). (I–L) Enlargement of the boxed area in (E–H).

Figure 7.

(A) Retinal flat mounts from an 11-month-old Chm^Flox IRBP-Cre+, Tyr-Cre+ animal were stained with anti–Iba1 primary and anti–rabbit Alexa-488 secondary antibody. (B) Enlargement of the boxed area in (A). (C) Quantitative data of the number of Iba1-positive cells per eye cup from 11- to 12-month-old Chm^Flox (gray bar), Chm^Flox, IRBP-Cre+ (closed bar), Chm^Flox, Tyr-Cre+ (open bar), and Chm^Flox, IRBP-Cre+, Tyr-Cre+ (hatched bar) animals.