Dibyendu Chakraborty

Differential requirements for retinal degeneration slow intermolecular disulfide-linked oligomerization in rods versus cones

It is commonly assumed that the ultrastructural organization of the rim region of outer segment (OS) discs in rods and lamellae in cones requires functional retinal degeneration slow/rod outer segment membrane protein 1 (Rds/Rom-1) complexes. Cysteine-150 (C150) in Rds has been implicated in intermolecular disulfide bonding essential for functional Rds complexes. Transgenic mice containing the Rds C150S mutation (C150S-Rds) failed to form higher-order Rds oligomers, although interactions between C150S-Rds and Rom-1 occurred in rods, but not in cones. C150S-Rds mice exhibited marked early-onset reductions in cone function and abnormal OS structure. In contrast, C150S-Rds expression in rods partly rescued the rds(+/-) phenotype. Although C150S-Rds was detected in the OSs in rods and cones, a substantial percentage of C150S-Rds and cone opsins were mislocalized to different cellular compartments in cones. The results of this study provide novel insights into the importance of C150 in Rds oligomerization and the differences in Rds requirements in rods versus cones. The apparent OS structural differences between rods and cones may cause cones to be more susceptible to the elimination of higher-order Rds/Rom-1 oligomers (e.g. as mediated by mutation of the Rds C150 residue).

Outer segment oligomerization of Rds: evidence from mouse models and subcellular fractionation.

Retinal degeneration slow (Rds) is a photoreceptor-specific tetraspanin glycoprotein essential for photoreceptor outer segment (OS) morphogenesis. Over 80 mutations in this protein are associated with several different retinal diseases. Rds forms a mixture of disulfide-linked homomeric dimers, octamers, and higher-order oligomers, with Cys150 playing a crucial role in its oligomerization. Rds also forms noncovalent homo- and hetero-tetramers with its nonglycosylated homologue, Rom-1. Here, we evaluated the subcellular site of Rds oligomerization and the pattern of Rds/Rom-1 complex assembly in several types of knockout mice, including rhodopsin (Rho-/-, lacking rod OS), Rom-1 (Rom-1-/-), neural retina leucine zipper (Nrl-/-, cone-dominant), and in comparison with wild-type (WT, rod-dominant) mice. Oligomerization and the pattern of complex assembly were also evaluated in OS-enriched vs OS-depleted preparations from WT and Rom-1-/- retinas. Velocity sedimentation under reducing- and nonreducing conditions and co-immunoprecipitation experiments showed the presence of Rds mainly as homo- and hetero-tetramers with Rom-1 in the photoreceptor inner segment (IS), while higher-order, disulfide-linked intermediate complexes and oligomers were exclusively present in the photoreceptor OS. Rom-1-independent oligomerization of Rds was observed in Rom-1-/- retinas. The pattern of Rds complexes in cones from Nrl-/- mice was comparable to that in rods from WT mice. On the basis of these findings, we propose that Rds traffics from the IS to the OS as homo- and hetero-tetramers, with subsequent disulfide-linked oligomerization occurring concomitant with OS disc morphogenesis (at either the base of OS or the tip of the connecting cilium). These results suggest that Rds mutations that interfere with tetramer formation can block Rds trafficking to the OS, leading to loss-of-function defects.

Retinal abnormalities associated with the G90D mutation in opsin

Several mutations in the opsin gene have been associated with congenital stationary night blindness, considered to be a relatively nonprogressive disorder. In the present study, we examined the structural and functional changes induced by one of these mutations, i.e., substitution of aspartic acid for glycine at position 90 (G90D). Transgenic mice were created in which the ratio of transgenic opsin transcript to endogenous was 0.5:1, 1.7:1, or 2.5:1 and were studied via light and electron microscopy, immunocytochemistry, electroretinography (ERG), and spectrophotometry. Retinas with transgenic opsin levels equivalent to one endogenous allele (G0.5) appeared normal for a period of about 3–4 months, but at later ages there were disorganized, shortened rod outer segments (ROS), and a loss of photoreceptor nuclei. Higher levels of G90D opsin expression produced earlier signs of retinal degeneration and more severe disruption of photoreceptor morphology. Despite these adverse effects, the mutation had a positive effect on the retinas of rhodopsin knockout (R−/−) mice, whose visual cells fail to form ROS and rapidly degenerate. Incorporation of the transgene in the null background (G+/−/R−/− or G+/+/R−/−) led to the development of ROS containing G90D opsin and prolonged survival of photoreceptors. Absorbance spectra measured both in vitro and in situ showed a significant reduction of more than 90% in the amount of light‐sensitive pigment in the retinas of G+/+/R−/− mice, and ERG recordings revealed a >1 log unit loss in sensitivity. However, the histological appearances of the retinas of these mice show no significant loss of photoreceptors and little change in the lengths of their outer segments. These findings suggest that much of the ERG sensitivity loss derives from the reduced quantal absorption that results from a failure of G90D opsin to bind to its chromophore and form a normal complement of light‐sensitive visual pigment. J. Comp. Neurol. 478:149–163, 2004.

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

Cysteine 150 of retinal degeneration slow protein (RDS) mediates the intermolecular disulfide bonding necessary for large RDS complex assembly and morphogenesis of the rim region of photoreceptor outer segments. Previously, we showed that cones have a different requirement for RDS than rods, but the nature of that difference was unclear. Here, we express oligomerization-incompetent RDS (C150S-RDS) in the cone-dominant nrl(-/-) mouse. Expression of C150S-RDS leads to dominant functional abnormalities, ultrastructural changes, biochemical anomalies and protein mislocalization in cones. These data suggest that RDS complexes in cones are more susceptible to disruption than those in rods, possibly due to structural or microenvironmental differences in the two cell types. Furthermore, our results suggest that RDS intermolecular disulfide bonding may be part of RDS inner-segment assembly in cones but not in rods. These data highlight significant differences in assembly, trafficking and function of RDS in rods versus cones.

Insights into the mechanisms of macular degeneration associated with the R172W mutation in RDS.

Mutations in the photoreceptor tetraspanin gene peripherin-2/retinal degeneration slow (PRPH2/RDS) cause both rod- and cone-dominant diseases. While rod-dominant diseases, such as autosomal dominant retinitis pigmentosa, are thought to arise due to haploinsufficiency caused by loss-of-function mutations, the mechanisms underlying PRPH2-associated cone-dominant diseases are unclear. Here we took advantage of a transgenic mouse line expressing an RDS mutant (R172W) known to cause macular degeneration (MD) in humans. To facilitate the study of cones in the heavily rod-dominant mouse retina, R172W mice were bred onto an Nrl(-/-) background (in which developing rods adopt a cone-like fate). In this model the R172W protein and the key RDS-binding partner, rod outer segment (OS) membrane protein 1 (ROM-1), were properly expressed and trafficked to cone OSs. However, the expression of R172W led to dominant defects in cone structure and function with equal effects on S- and M-cones. Furthermore, the expression of R172W in cones induced subtle alterations in RDS/ROM-1 complex assembly, specifically resulting in the formation of abnormal, large molecular weight ROM-1 complexes. Fundus imaging demonstrated that R172W mice developed severe clinical signs of disease nearly identical to those seen in human MD patients, including retinal degeneration, retinal pigment epithlium (RPE) defects and loss of the choriocapillaris. Collectively, these data identify a primary disease-causing molecular defect in cone cells and suggest that RDS-associated disease in patients may be a result of this defect coupled with secondary sequellae involving RPE and choriocapillaris cell loss.

Initiation of Rod Outer Segment Disc Formation Requires RDS

Rod outer segment (OS) morphogenesis involves assembly of flattened discs circumscribed by a hairpin-like rim, however, the role of the rim and rim proteins such as retinal degeneration slow (RDS) and its homologue rod OS membrane protein-1 (ROM-1) in this process remains unclear. Here we show that without RDS, no disc/OS formation occurs, while without rhodopsin, small OS structures form containing aligned nascent discs. In the absence of both rhodopsin and RDS, RDS-associated degeneration is slowed, and ROM-1 is stabilized and trafficked to the OS. These animals (rho − /− /rds − /−) exhibit OSs slightly better than those lacking only RDS, but still without signs of disc formation. These results clearly demonstrate that OS morphogenesis is initiated by RDS-mediated rim formation, a process ROM-1 cannot recapitulate, with subsequent disc growth mediated by rhodopsin. The critical role of RDS in this process helps explain why photoreceptors are so sensitive to varied RDS levels, and why mutations in RDS cause debilitating retinal disease.

Structural characterization of the second intra-discal loop of the photoreceptor tetraspanin RDS.

Vertebrate photoreceptors contain a unique tetraspanin protein known as ‘retinal degeneration slow’ (RDS). Mutations in the RDS gene have been identified in a variety of human retinal degenerative diseases, and more than 70% of these mutations are located in the second intra‐discal (D2) loop, highlighting the importance of this region. Here we examined the conformational and thermal stability properties of the D2 loop of RDS, as well as interactions with ROM–1, a non‐glycosylated homolog of RDS. The RDS D2 loop was expressed in Escherichia coli as a fusion protein with maltose binding protein (MBP). The fusion protein, referred to as MBP–D2, was purified to homogeneity. Circular dichroism spectroscopy showed that the wild‐type (WT) D2 loop consists of approximately 21% α–helix, approximately 20% β–sheet and approximately 59% random coil. D2 loop fusion proteins carrying disease‐causing mutations in RDS (e.g. R172W, C214S, N244H/K) were also examined, and conformational changes were observed (compared to wild‐type D2). In particular, the C150S, C214S and N244H proteins showed significant reductions in α–helicity. However, the thermal stability of the mutants was unchanged compared to wild‐type, and all the mutants were capable of interacting with ROM–1, indicating that this functional aspect of the isolated D2 loop remained intact in the mutants despite the observed conformational changes. An I–TASSER model of the RDS D2 loop predicted a structure consistent with the circular dichroism experiments and the structure of the conserved region of the D2 loop of other tetraspanin family members. These results provide significant insight into the mechanism of RDS complex formation and the disease process underlying RDS‐associated retinal degeneration.

A Dominant Mutation in Rpe65, D477G, Delays Dark Adaptation and Disturbs the Visual Cycle in the Mutant Knock-In Mice

RPE65 is an indispensable component of the retinoid visual cycle in vertebrates, through which the visual chromophore 11-cis-retinal (11-cis-RAL) is generated to maintain normal vision. Various blinding conditions in humans, such as Leber congenital amaurosis and retinitis pigmentosa (RP), are attributed to either homozygous or compound heterozygous mutations in RPE65. Herein, we investigated D477G missense mutation, an unprecedented dominant-acting mutation of RPE65 identified in patients with autosomal dominant RP. We generated a D477G knock-in (KI) mouse and characterized its phenotypes. Although RPE65 protein levels were decreased in heterozygous KI mice, their scotopic, maximal, and photopic electroretinography responses were comparable to those of wild-type (WT) mice in stationary condition. As shown by high-performance liquid chromatography analysis, levels of 11-cis-RAL in fully dark-adapted heterozygous KI mice were similar to that in WT mice. However, kinetics of 11-cis-RAL regeneration after light exposure were significantly slower in heterozygous KI mice compared with WT and RPE65 heterozygous knockout mice. Furthermore, heterozygous KI mice exhibited lower A-wave recovery compared with WT mice after photobleaching, suggesting a delayed dark adaptation. Taken together, these observations suggest that D477G acts as a dominant-negative mutant of RPE65 that delays chromophore regeneration. The KI mice provide a useful model for further understanding of the pathogenesis of RP associated with this RPE65 mutant and for the development of therapeutic strategies.

The K153Del PRPH2 mutation differentially impacts photoreceptor structure and function

Peripherin 2 (Prph2) is a photoreceptor tetraspanin, and deletion of codon 153 (K153Δ) leads to retinitis pigmentosa, pattern dystrophy, and fundus flavimaculatus in the same family. To study this variability, we generated a K153Δ-Prph2 knockin mouse. K153Δ-Prph2 cannot form the complexes required for outer segment formation, and in cones cannot interact with its binding partner rod outer segment membrane protein 1. K153Δ causes dominant defects in rod and cone function; however, rod but not cone ultrastructure is improved by the presence of K153Δ-Prph2. Likewise, supplementation of K153Δ heterozygotes with WT-Prph2 results in structural but not functional improvements. These results support the idea that mutations may differentially affect Prph2s role as a structural component, and its role as a functional protein key for organizing membrane domains for cellular signalling. These roles may be different in rods and cones, thus contributing to the phenotypic heterogeneity that characterizes diseases associated with Prph2 mutations.

Overexpression of ROM-1 in the Cone-Dominant Retina

ROM-1 (rod outer segment membrane protein-1) is a tetraspanin protein present in the outer segment (OS) rims of rod and cone photoreceptors. It shares many common features with other tetraspanins, including a large intradiscal loop which contains several cysteines. This loop enables ROM-1 to associate with its homologue retinal degeneration slow (RDS) to form hetero-tetramers and hetero-octamers. Although large RDS homo-oligomers are required for building and maintaining the rim region of rod discs and cone lamellae, the function of ROM-1 is less clear. ROM-1 is not necessary for disc morphogenesis but is required for fine tuning OS disc size and structure. Furthermore, the role of ROM-1 has not been well studied in cone photoreceptors. To help alleviate this knowledge gap, we overexpressed ROM-1 via transgenesis in the cone-dominant nrl −/− mouse retina. We show that ∼20% overexpression of ROM-1 results in vacuoles in the cone OSs and significant reductions in photopic electroretinogram responses. These results indicate that overexpression of ROM-1 is harmful to cone OSs. We hypothesize that overexpression of ROM-1 alters the normal balance between RDS homo-oligomers and RDS/ROM-1 heteromeric complexes, resulting in insufficient or abnormal higher-order RDS homo-oligomers for the maintenance of proper OS structure and function.