Effect of Rds abundance on cone outer segment morphogenesis, photoreceptor gene expression, and outer limiting membrane integrity

Abstract

We examined the molecular, structural, and functional consequences on cone photoreceptors of the neural retinal leucine zipper knockout (Nrl −/− ) mice when only one allele of retinal degeneration slow (Rds) is present (Rds +/− /Nrl −/− ). Quantitative RT-PCR and immunoblot analysis were used to assess the expression levels of several phototransduction genes; electroretinography was used to assess quantitatively the retinal responsiveness to light; and immunohistochemistry and ultrastructural analysis were used to examine retinal protein distribution and morphology, respectively. In Rds/Nrl double-null mice, S-cones form dysmorphic outer segments that lack lamellae and fail to associate properly with the cone matrix sheath and the outer limiting membrane. In Rds +/− /Nrl −/− mice, cones form oversized and disorganized outer segment lamellae; although outer limiting membrane associations are maintained, normal interactions with cone matrix sheaths are not, and photoreceptor rosette formation is observed. These retinas produce significantly higher photopic a-wave and b-wave amplitudes than do those of Rds −/− /Nrl −/− mice, and the levels of several cone phototransduction genes are significantly increased coincidently with the presence of Rds and partial lamellae formation. Thus, as in rod photoreceptors, expression of only one Rds allele is unable to support normal outer segment morphogenesis in cones. However, cone lamellae assembly, albeit disorganized, concomitantly permits outer limiting membrane association, and this appears to be linked to photoreceptor rosette formation in the rodless (cone-only) Nrl −/− retina. In addition, photoreceptor gene expression alterations occur in parallel with changes in Rds levels. Indexing termsRds; Nrl; retina; cone photoreceptor; rosette; gene expression Visual transduction begins when a photon of light is absorbed by a visual pigment chromophore in the outer segment of the photoreceptor cell. In rod photoreceptors, the outer segment consists of stacks of membranous "discs" that are physically separated from the plasma membrane by cytosol; however, in cone photoreceptors, these discs are contiguous with the plasma membrane and are called "lamellae" (Arikawa et al., 1992;Steinberg et al., 1980). Outer segment morphogenesis is an active process. In mammals, nearly 10% of these discs/lamellae are shed by the photoreceptor cells and subsequently phagocytosed by the retinal pigment NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript epithelium (RPE) each day, and a comparable amount of new discs/lamellae must be produced and added to maintain the constant length of the outer segment. The proper formation of rod and cone outer segment discs/lamellae is proposed to be governed by a series of evagination and invagination events that are reliant on the function of the retinal degeneration slow (Rds) protein (Boesze-Battaglia and Goldberg, 2002;Damek-Poprawa et al., 2005;Eckmiller, 1987Eckmiller, , 1990Goldberg, 2006;Ritter et al., 2004;Steinberg et al....

Differences in RDS trafficking, assembly and function in cones versus rods: insights from studies of C150S-RDS

“…Retinal degeneration slow (rds 2/2 ) mutant mice (in the roddominant wild-type background) fail to develop OSs and have no detectable retinal function (4 -6). In contrast, we have reported that cones lacking RDS (in the cone-dominant neural retina leucine zipper knockout, nrl 2/2 , background) (7 -9) retain some OS function and structure, albeit abnormal (3,10). This divergence in the role of RDS in rods versus cones is underscored by the observation that mutations in the human Rds gene are associated with a variety of rod-or conedominant blinding retinal diseases, including autosomal dominant retinitis pigmentosa and multiple classes of macular degeneration (11,12).…”

“…The methods employed for tissue collection and processing for plastic-embedment light and electron microscopy and immunogold labeling were as described previously (3,10,15). For light microscopy, 0.75 mm sections were viewed and photographed with an Olympus BH-2 photomicroscope with a Nikon digital camera system.…”

“…All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org segment (IS), RDS forms non-covalently bound homo-and hetero-tetramers with its homolog ROM1 (3,10,(15)(16)(17), another tetraspanin protein with similar localization and structure to RDS (18,19). After trafficking to the OS, homo-and hetero-tetrameric RDS complexes are linked to octomers while RDS homo-tetramers are linked to higher order oligomers through C150-mediated disulfide bonds.…”

Gene Therapy for PRPH2-Associated Ocular Disease: Challenges and Prospects

“…Studies in rds 2/2 /Nrl 2/2 mice have highlighted the differential role of peripherin-2 in rods and cones. In contrast to rods lacking peripherin-2 (in the WT background), cones lacking peripherin-2 (in the rds 2/2 /Nrl 2/2 mice) form OSs, albeit dysmorphic ones (Farjo et al 2006b(Farjo et al , 2007. These OSs lack the elaborate folded lamellae structure of normal cones and have no rims at all, but nonetheless retain the ability to mediate phototransduction.…”

Outer Segment Oligomerization of Rds: Evidence from Mouse Models and Subcellular Fractionation

“…Eyes were fixed in Davidson's fixative [30 mL of 95% (v/v) ethanol, 20 mL of 10% neutral buffered formalin, 10 mL of glacial acetic acid and 30 mL of distilled water] overnight at 4 °C (28). The eyes were then washed three times in phosphate-buffered saline (PBS) and stored in 70% ethanol for subsequent serial dehydration and embedment in paraffin.…”