Visual cycle protein RPE65 persists in new retinal cells during retinal regeneration of adult newt

Abstract

Adult newts can regenerate their entire retina through transdifferentiation of the retinal pigment epithelium (RPE). The objective of this study was to redescribe the retina regeneration process by means of modern biological techniques. We report two different antibodies (RPENo.112 and MAB5428) that recognize the newt homolog of RPE65, which is involved in the visual cycle and exclusively label the RPE cell-layer in the adult newt eye. We analyzed the process of retinal regeneration by immunohistochemistry and immunoblotting and propose that this process should be divided into nine stages. We found that the RPE65 protein is present in the RPE-derived new retinal rudiment at 14 days postoperative (po) and in the regenerating retinas at the 3-4 cell stage (19 days po). These observations suggest that certain characteristics of RPE cells overlap with those of retinal stem/progenitor cells during the period of transdifferentiation. However, RPE65 protein was not detected in either retinal stem/progenitor cells in the ciliary marginal zone (CMZ) of adult eyes or in neuroepithelium present during retina development, where it was first detected in differentiated RPE. Moreover, the gene expression of RPE65 was drastically downregulated in the early phase of transdifferentiation (by 10 days po), while those of Connexin43 and Pax-6, both expressed in regenerating retinas, were differently upregulated. These observations suggest that the RPE65 protein in the RPE-derived retinal rudiment may represent the remainder after protein degradation or discharge rather than newly synthesized protein.

Shared Triggering Mechanisms of Retinal Regeneration in Lower Vertebrates and Retinal Rescue in Higher Ones

“…When we compared RPE in the intact eye and RPE soon after NR removal in Urodela we found by PCR a considerable decrease of RPE65 transcript abundance (Avdonin et al, 2008). Other results strongly suggest that RPE65 in the RPE-derived cells of early retinal regenerate in Urodela is the only reminder after protein degradation or discharge (Chiba et al, 2006). When RPE and NR are disintegrated, there is not only a decrease of visual cycle protein synthesis but also protein translocation into the inner compartments of photoreceptors.…”

Cell Models to Study Regulation of Cell Transformation in Pathologies of Retinal Pigment Epithelium

“…In adult newts, for example, central RPE cells and low-differentiated cells of the peripheral growth zone account for regeneration of the retina [1–4]. After the surgical removal of the retina, RPE cells dedifferentiate, lose pigment, and proliferate; thereafter, part of cells recover RPE differentiation, while another part transdifferentiate into neural retinal cells [5]. Unlike in lower vertebrates, in which the ability to regenerate the retina via transdifferentiation is lifelong, its regeneration in some mammals is possible only during the embryonic period [6], whereas RPE plasticity retained in adults is responsible for a variety of ocular pathologies.…”

The newt reprograms mature RPE cells into a unique multipotent state for retinal regeneration

“…In the adult newt, when the NR is completely removed from the eye via surgery (i.e., retinectomy), the retina is regenerated from two cell sources56; the primary cell source is the RPE, which regenerates the NR and renews the RPE, and the secondary source is retinal stem/progenitor cells, which are present in the ciliary marginal zone (CMZ) starting at the embryonic stage. These cells extend toward the central retina along the RPE and participate in regeneration of the peripheral portion of the NR.…”

“…Therefore, by eliminating the peripheral retina, it is possible to focus on retinal regeneration that originates solely from the RPE. We previously described this process using a Japanese fire bellied newt, Cynops pyrrhogaster 56 (see Supplementary Movie 1). Upon retinectomy, RPE cells are detached from each other as well as from the basement membrane (Bruch's membrane), re-enter the cell cycle, and form cell aggregates [Stage E-1; this event occurs typically between days 5 and 10 post-operation (po).…”