Margaret J. McLaren

X-arrestin: a new retinal arrestin mapping to the X chromosome.

We have been using a differential cDNA cloning approach to isolate human retina‐specific and retina‐enriched genes [1]. A 1,314 bp cDNA was isolated by this approach, representing a highly retina‐specific message encoding a 388 amino acid protein showing 58%, 50%, and 49% homology to bovine β‐arrestin, and bovine and human retinal arrestin (S‐antigen), respectively. Chromosomal mapping localized this new arrestin genc to the proximal long arm of the X chromosome, hence it was named X‐arrestin. In situ hybridization demonstrated its expression in the inner and outer segments and the inner plexiform regions of the retina.

Cloning of the cDNA for a Novel Photoreceptor Protein

A subtractive cDNA cloning strategy was used to isolate a 1381-base pair human retina-specific cDNA, human retinal gene 4 (HRG4), which hybridized to a 1.4-kilobase message in the retina and encoded a 240-amino acid acidic protein with a calculated molecular mass of 26,964 Da. The proximal of the conceptual protein sequence was rich in glycine (18%) and proline (20%), had a predicted secondary structure of turns, and showed a loose similarity (19-24%) to various α-collagen sequences, while the distal consisted of a mixture of α-helices, β-sheets, and turns. Genomic Southern analysis with HRG4 showed cross-hybridizing sequences in six different species, and HRG4 was 92% homologous with a 1264-base pair rat cDNA (rat retinal gene 4; RRG4) at the protein level. The region of 100% identity between the two sequences corresponded to the distal of the protein sequence consisting of mixed secondary structures, suggesting a functionally important domain. In vitro transcription and translation corroborated the open reading frames corresponding to HRG4 and RRG4 in the cDNAs. Expression of HRG4 in the retina was localized to the photoreceptors by in situ hybridization. Developmentally, RRG4 began to be highly expressed around postnatal day 5 in the rat outer retina when the photoreceptors begin to differentiate and rapidly increased in expression to reach the mature adult level by postnatal day 23. No diurnal fluctuation in expression of RRG4 was seen.

Photoreceptor synaptic protein HRG4 (UNC119) interacts with ARL2 via a putative conserved domain

Human retinal gene 4 (HRG4) (UNC119) is a photoreceptor synaptic protein of unknown function, shown when mutated to cause retinal degeneration in a patient and in a confirmatory transgenic model. ADP‐ribosylation factor‐like protein 2 (ARL2) was identified as an interactor of HRG4 by the yeast two‐hybrid strategy. The presence of ARL2 in the retina and co‐localization with HRG4 was confirmed by Western blot and double immunofluorescence analysis, respectively. The interaction of ARL2 with HRG4 was further confirmed by co‐immunoprecipitation and direct binding analysis. Phosphodiesterase δ (PDEδ) is an ARL2‐binding protein homologous to HRG4. Amino acid residues of PDEδ involved in binding ARL2 and forming a hydrophobic pocket were shown to be highly conserved in HRG4, suggesting similarity in binding mechanism and function.

Immunolocalization of X-arrestin in human cone photoreceptors

X‐arrestin is a recently identified retina‐specific gene of unknown function. Affinity‐purified anti‐peptide antibody to human X‐arrestin was prepared, and used in Western blot analysis of human retinal proteins and for immunohistochemistry on human retinal sections. By Western blot analysis, the antibody specifically bound to an ≈47 kDa protein, and by indirect immunofluorescence specifically labeled cone photoreceptors with greatest intensity in their outer segments. In single and double label experiments, the localization of X‐arrestin immunoreactivity was compared with immunolabelling patterns obtained with antibodies to red/green cone opsin, rhodopsin, and S‐antigen. The results showed that X‐arrestin is expressed in red‐, green‐ and blue‐sensitive cones in the human retina.

INHERITED RETINAL DEGENERATION : BASIC FGF INDUCES PHAGOCYTIC COMPETENCE IN CULTURED RPE CELLS FROM RCS RATS

In RCS rats, the retinal pigment epithelium (RPE) is defective in phagocytosis of photoreceptor membranes. We have previously shown reduced expression of basic fibroblast growth factor (bFGF) in the RPE of 7–10‐day‐old RCS rats. This study using primary RPE cultures from rats of this age demonstrates that the phagocytic defect in the mutant RPE can be overcome by treatment with bFGF, by a mechanism involving gene transcription and that normal RPE phagocytosis, also requiring transcription, is blocked by a bFGF neutralizing antibody. The combined data point to a role for bFGF in the normal mechanism of RPE phagocytosis and the RCS defect.

ANALYSIS OF BASIC FIBROBLAST GROWTH FACTOR IN RATS WITH INHERITED RETINAL DEGENERATION

In RCS rats, photoreceptors degenerate between postnatal days 20 and 60, secondary to a genetic defect expressed in the neonatal retinal pigmented epithelium (RPE). Previous work has shown delay of the photoreceptor degeneration in this model by intraocular injection of basic fibroblast growth factor (bFGF). Evidence is presented here, from bFGF immunostaining and Northern analysis of bFGF mRNA, for reduced bFGF expression in uncultured RPE of dystrophic RCS pups. It is also shown that in the mutant eyes angiogenesis in the underlying choroid, which normally occurs between postnatal days 7 and 10, is markedly delayed, with irregular distribution of vessels, consistent with a reduction in this known angiogenesis factor. Mutational analysis of the bFGF transcript and gene by denaturing gradient gel electrophoresis and Southern analysis did not, however, reveal abnormalities in the coding sequence of this gene in RCS rats.

Isolation of Candidate Genes for Retinal Degenerations

The indentification of the molecular etiology is the first key step in understanding inherited retinal degenerations. Knowledge of the causative gene allows one to begin to investigate the mechanism by which the gene defect leads to the retinal degeneration. Understanding the pathophysiological mechanism of the disease may lead to finding the best treatment or cure for the retinal degeneration.

Defective Choroidal Angiogenesis Precedes Retinal Pigment Epithelial Phagocytic Defect in Neonatal Rcs Rats

The well known defect in phagocytosis of rod outer segment (ROS) membranes by the retinal pigment epithelium (RPE) of RCS rats, the subject of numerous investigations, has long been considered to be the primary pathology leading to retinal degeneration in this mutant (1–3). Light microscopic and ultrastructural studies of the developing photoreceptors of these animals have determined that the phagocytic defect is first evident around postnatal day 12, coincident with the onset of retinal phagocytosis in the normal animals (4). Recently, our laboratory has provided evidence that there is a second defect in dystrophic rat eyes that appears earlier in development than the phagocytic defect: this abnormality involves failed or delayed development of the choroidal vasculature, which normally proceeds rapidly between postnatal days 7–10 (5). In this chapter, I briefly review the data revealing the choroidal angiogenesis defect in mutant RCS rat pups, as well as our recent evidence indicating a reduction in the mRNA and protein for basic fibroblast growth factor (bFGF) in intact RPE of neonatal RCS rats (5). This new data is discussed in the context of previous conflicting investigations into bFGF expression in RCS rat retinas. Finally, the hypothesis is explored that the observed downregulation of bFGF in the mutant RPE during this period in postnatal ocular development may play a central role in the pathology, adversely affecting choroidal angiogenesis, RPE phagocytic function and trophic support of the photoreceptors.