Novel localization of peripherin 2, the photoreceptor-specific retinal degeneration slow protein, in retinal pigment epithelium

Abstract

Retinal pigment epithelium (RPE) builds the outer blood-retinal barrier of the eye. Since one typical feature of the autoimmune disease, equine recurrent uveitis (ERU), is the breakdown of this barrier, we recently performed comparative analysis of healthy and uveitic RPE. We identified for the first time peripherin 2, which is responsible for visual perception and retina development, to be localized in RPE. The purpose of this study was therefore to validate our findings by characterizing the expression patterns of peripherin 2 in RPE and retina. We also investigated whether peripherin 2 expression changes in ERU and if it is expressed by the RPE itself. Via immunohistochemistry, significant downregulation of peripherin 2 in uveitic RPE compared to the control was detectable, but there was no difference in healthy and uveitic retina. A further interesting finding was the clear distinction between peripherin 2 and the phagocytosis marker, rhodopsin, in healthy RPE. In conclusion, changes in the expression pattern of peripherin 2 selectively affect RPE, but not retina, in ERU. Moreover, peripherin 2 is clearly detectable in healthy RPE due to both phagocytosis and the expression by the RPE cells themselves. Our novel findings are very promising for better understanding the molecular mechanisms taking place on RPE in uveitis.

Figure 1

Expression pattern of peripherin 2 (red) and rhodopsin (green) in representative retinas of healthy (left) and equine recurrent uveitis (ERU) cases (right). Differential interference contrast image of healthy (A) and diseased (E) equine retina. Peripherin 2 (red) was equally distributed in the photoreceptor outer segments of healthy (B) and diseased (F) equine retina. Cell nuclei were stained with 4',6-diamidino-2-phenylindol (DAPI) (blue); Rhodopsin (green) was also equally distributed in photoreceptor outer segments of healthy (C) and uveitic (G) retinas. The overlap of peripherin 2 and rhodopsin in healthy (D) and uveitic (H) retina show overlapping expression pattern of both proteins (yellow).

Figure 1

Expression pattern of peripherin 2 (red) and rhodopsin (green) in representative retinas of healthy (left) and equine recurrent uveitis (ERU) cases (right). Differential interference contrast image of healthy (A) and diseased (E) equine retina. Peripherin 2 (red) was equally distributed in the photoreceptor outer segments of healthy (B) and diseased (F) equine retina. Cell nuclei were stained with 4',6-diamidino-2-phenylindol (DAPI) (blue); Rhodopsin (green) was also equally distributed in photoreceptor outer segments of healthy (C) and uveitic (G) retinas. The overlap of peripherin 2 and rhodopsin in healthy (D) and uveitic (H) retina show overlapping expression pattern of both proteins (yellow).

Figure 2

Peripherin 2 (red) and rhodopsin (green) expression in retinal pigment epithelium (RPE) of representative healthy (left) and ERU cases (right). Differential interference contrast image of healthy (A) and diseased (E) equine RPE. In healthy (B) RPE, peripherin 2 was expressed over the entire RPE cell layer, and the intensity was enhanced in some spots. In uveitic RPE cells (F), peripherin 2 was significantly reduced to few small spots; Rhodopsin was limited to few isolated spots, distributed throughout the whole RPE layer, in healthy (C), as well as in diseased (G) RPE. The overlap of peripherin 2 and rhodopsin in healthy (D) RPE indicates significant higher expression of peripherin 2 compared to rhodopsin (the yellow overlap is only displayed in a few spots). In uveitic RPE (H), peripherin 2 was only expressed at rhodopsin expression sites (yellow spots).

Figure 3

(A) Quantification of the fluorescence intensity of peripherin 2 expression in photoreceptor outer segments revealed no significant difference between healthy (grey column) and ERU cases (black column); (B) There was also no significant difference between the fluorescence intensity of rhodopsin in photoreceptor outer segments of healthy (grey column) and uveitic (black column) retinas detectable; (C) A significant reduction of the fluorescence intensity of peripherin 2 in uveitic RPE to 17.9% (black column), compared to RPE of negative controls (** p ≤ 0.01); (D) Quantification of the fluorescence intensity of rhodopsin in RPE showed no significant difference between healthy (grey column) and ERU cases (black column).

Figure 4

Investigation of peripherin 2 (red) and rhodopsin (green) expression on cultivated healthy RPE cells at different confluency states. Differential interference contrast image of healthy equine RPE cells at different confluency states; from single cells (A) to 40% confluent cells (B) up to 90%–100% confluent cells (C); (D–F) Peripherin 2 (red) was expressed over whole RPE cells with an even intensity (cell nuclei = blue); (G–I) No rhodopsin (green) expression was found in primary healthy equine RPE cells (cell nuclei = blue).

Figure 5

Peripherin 2 (red) expression in healthy human RPE. (A) Differential interference contrast image of healthy human RPE; (B) peripherin 2 (red) was expressed over the whole RPE layer with an even intensity.