Cloning of the cDNA for a novel photoreceptor membrane protein (rom-1) identifies a disk rim protein family implicated in human retinopathies.

The molecules essential to the continual morphogenesis and shedding of the opsin-containing disks of vertebrate photoreceptors are largely unknown. We describe a 37 kd protein, rom-1, which is 35% identical and structurally similar to peripherin/retinal degeneration slow (rds). Like peripherin, rom-1 is a retina-specific integral membrane protein localized to the photoreceptor disk rim. The two proteins are similarly oriented in the membrane, and each has a highly conserved (15/16 residues) cysteine- and proline-rich domain in the disk lumen. Although both rom-1 and peripherin form disulfide-linked dimers, they do not form heterodimers with each other, but appear to associate noncovalently. These results suggest both that rom-1 and peripherin are functionally related members of a new photoreceptor-specific protein family and that rom-1, like peripherin, is likely to be important to outer segment morphogenesis. The association of mutations in RDS with retinitis pigmentosa indicates that ROM1 is a strong candidate gene for human retinopathies.     

Folding and subunit assembly of photoreceptor peripherin/rds is mediated by determinants within the extracellular/intradiskal EC2 domain: implications for heterogeneous molecular pathologies.

Peripherin/rds is an integral membrane protein required for the elaboration of rod and cone photoreceptor outer segments in the vertebrate retina; it causes a surprising variety of progressive retinal degenerations in humans and dysmorphic photoreceptors in murine models if defective or absent. (Peripherin/rds is also known as photoreceptor peripherin, peripherin/rds, rds/peripherin, rds, and peripherin-2.) Peripherin/rds appears to act as a structural element in outer segment architecture. However, neither its function at the molecular level nor its role in retinal disease processes are well understood. This report initiates a systematic investigation of protein domain structure and function by examining the molecular and cellular consequences of a series of 14 insertional mutations distributed throughout the polypeptide sequence. Protein expression, disulfide bonding, sedimentation velocity, and subcellular localization of the COS-1 cell-expressed mutant variants were examined to test the hypothesis that protein folding and tetrameric subunit assembly are mediated primarily by EC2, a conserved extracellular/intradiskal domain. Protein folding and tetrameric subunit assembly were not affected by insertion of either an uncharged dipeptide (GA) or a highly charged hendecapeptide (GDYKDDDDKAA) into any one of nine sites residing outside of EC2 as assayed by nonreducing Western blot analysis, sedimentation velocity, and subcellular localization. In contrast, insertions at five positions within the EC2 domain did cause either gross protein misfolding (two sites) or a reduction in protein sedimentation coefficient (two sites) or both (one site). These results indicate that although the vast majority of extramembranous polypeptide sequence makes no measurable contribution to protein folding and tetramerization, discrete regions within the EC2 domain do contain determinants for normal subunit assembly. These findings raise the possibility that multiple classes of structural perturbation are produced by inherited defects in peripherin/rds and contribute to the observed heterogeneity of retinal disease phenotypes.     

Localization of peripherin/rds in the disk membranes of cone and rod photoreceptors: relationship to disk membrane morphogenesis and retinal degeneration

The outer segments of vertebrate rod photoreceptor cells consist of an ordered stack of membrane disks, which, except for a few nascent disks at the base of the outer segment, is surrounded by a separate plasma membrane. Previous studies indicate that the protein, peripherin or peripherin/rds, is localized along the rim of mature disks of rod outer segments. A mutation in the gene for this protein has been reported to be responsible for retinal degeneration in the rds mouse. In the present study, we have shown by immunogold labeling of rat and ground squirrel retinas that peripherin/rds is present in the disk rims of cone outer segments as well as rod outer segments. Additionally, in the basal regions of rod and cone outer segments, where disk morphogenesis occurs, we have found that the distribution of peripherin/rds is restricted to a region that is adjacent to the cilium. Extension of its distribution from the cilium coincides with the formation of the disk rim. These results support the model of disk membrane morphogenesis that predicts rim formation to be a second stage of growth, after the first stage in which the ciliary plasma membrane evaginates to form open nascent disks. The results also indicate how the proteins of the outer segment plasma membrane and the disk membranes are sorted into their separate domains: different sets of proteins may be incorporated into membrane outgrowths during different growth stages of disk morphogenesis. Finally, the presence of peripherin/rds protein in both cone and rod outer segment disks, together with the phenotype of the rds mouse, which is characterized by the failure of both rod and cone outer segment formation, suggest that the same rds gene is expressed in both types of photoreceptor cells.     

Transgenic Analysis of Rds/Peripherin N-Glycosylation

Abstract : Rds/peripherin is an integral membrane glycoprotein that is present in the rims of photoreceptor outer segment disks. In mammals, it is thought to stabilize the disk rim through heterophilic interactions with the related nonglycosylated protein rom1. Glycosylation of rds/peripherin at asparagine 229 is widely conserved in vertebrates. In this study, we investigated the role of rds/peripherin N -glycosylation. We generated transgenic mice that expressed only S231A-substituted rds/peripherin in their retinas. This protein was not glycosylated but formed covalent dimers with itself and with glycosylated rds/peripherin. Nonglycosylated rds/peripherin also interacted noncovalently with rom1 homodimers to form a heterooligomeric complex. The glycosylated rds/peripherin ·· rom1 complex bound to concanavalin A-Sepharose, suggesting that the glycan is not directly involved in the interaction between these proteins. In double transgenic mice expressing normal and S231A-substituted rds/peripherin, the mRNA-to-protein ratios were similar for both transgenes, indicating no effect of N -glycosylation on rds/peripherin stability. Finally, expression of nonglycosylated rds/peripherin in transgenic mice rescued the phenotype of outer segment nondevelopment in retinal degeneration slow (rds-/-) null mutants. These observations indicate that N -glycosylation of rds/peripherin is not required for its normal processing, stability, or in vivo function.     

Analysis of the rds/peripherin.rom1 complex in transgenic photoreceptors that express a chimeric protein.

Mice homozygous for the retinal degeneration slow (rds) mutation completely lack photoreceptor outer segments. The rds gene encodes rds/peripherin (rds), a membrane glycoprotein in the rims of rod and cone outer segment discs. rds is present as a complex with the related protein, rom1. Here, we generated transgenic mice that express a chimeric protein (rom/D2) containing the intradiscal D2 loop of rds in the context of rom1. rom/D2 was N-glycosylated, formed covalent homodimers, and interacted non-covalently with itself, rds, and rom1. The rds.rom/D2 interaction was significantly more stable than the non-covalent interaction between rds and rom1 by detergent/urea titration. Analysis of mice expressing rom/D2 revealed that rds is 2.5-fold more abundant than rom1, interacts non-covalently with itself and rom1 via the D2 loop, and forms a high order complex that may extend the entire circumference of the disc. Expression of rom/D2 fully rescued the ultrastructural phenotype in rds+/- mutant mice, but it had no effect on the phenotype in rds-/- mutants. Together, these observations explain the striking differences in null phenotypes and frequencies of disease-causing mutations between the RDS and ROM1 genes.     

The C terminus of peripherin/rds participates in rod outer segment targeting and alignment of disk incisures

Protein targeting is essential for domain specialization in polarized cells. In photoreceptors, three distinct membrane domains exist in the outer segment: plasma membrane, disk lamella, and disk rim. Peripherin/retinal degeneration slow (rds) and rom-1 are photoreceptor-specific members of the transmembrane 4 superfamily of transmembrane proteins, which participate in disk morphogenesis and localize to rod outer segment (ROS) disk rims. We examined the role of their C termini in targeting by generating transgenic Xenopus laevis expressing green fluorescent protein (GFP) fusion proteins. A GFP fusion containing residues 317-336 of peripherin/rds localized uniformly to disk membranes. A longer fusion (residues 307-346) also localized to the ROS but exhibited higher affinity for disk rims than disk lamella. In contrast, the rom-1 C terminus did not promote ROS localization. The GFP-peripherin/rds fusion proteins did not immunoprecipitate with peripherin/rds or rom-1, suggesting this region does not form intermolecular interactions and is not involved in subunit assembly. Presence of GFP-peripherin/rds fusions correlated with disrupted incisures, disordered ROS tips, and membrane whorls. These abnormalities may reflect competition of the fusion proteins for other proteins that interact with peripherin/rds. This work describes novel roles for the C terminus of peripherin/rds in targeting and maintaining ROS structure and its potential involvement in inherited retinal degenerations.     

Functional overlap between retinitis pigmentosa 2 protein and the tubulin-specific chaperone cofactor C

Mutations in the X-linked retinitis pigmentosa 2 gene cause progressive degeneration of photoreceptor cells. The retinitis pigmentosa 2 protein (RP2) is similar in sequence to the tubulin-specific chaperone cofactor C. Together with cofactors D and E, cofactor C stimulates the GTPase activity of native tubulin, a reaction regulated by ADP-ribosylation factor-like 2 protein. Here we show that in the presence of cofactor D, RP2 protein also stimulates the GTPase activity of tubulin. We find that this function is abolished by mutation in an arginine residue that is conserved in both cofactor C and RP2. Notably, mutations that alter this arginine codon cause familial retinitis pigmentosa. Our data imply that this residue acts as an "arginine finger" to trigger the tubulin GTPase activity and suggest that loss of this function in RP2 contributes to retinal degeneration. We also show that in Saccharomyces cerevisiae, both cofactor C and RP2 partially complement the microtubule phenotype resulting from deletion of the cofactor C homolog, demonstrating their functional overlap in vivo. Finally, we find that RP2 interacts with GTP-bound ADP ribosylation factor-like 3 protein, providing a link between RP2 and several retinal-specific proteins, mutations in which also cause retinitis pigmentosa.     

The connecting cilium inner scaffold provides a structural foundation that protects against retinal degeneration

Inherited retinal degeneration due to loss of photoreceptor cells is a leading cause of human blindness. These cells possess a photosensitive outer segment linked to the cell body through the connecting cilium (CC). While structural defects of the CC have been associated with retinal degeneration, its nanoscale molecular composition, assembly, and function are barely known. Here, using expansion microscopy and electron microscopy, we reveal the molecular architecture of the CC and demonstrate that microtubules are linked together by a CC inner scaffold containing POC5, CENTRIN, and FAM161A. Dissecting CC inner scaffold assembly during photoreceptor development in mouse revealed that it acts as a structural zipper, progressively bridging microtubule doublets and straightening the CC. Furthermore, we show that Fam161a disruption in mouse leads to specific CC inner scaffold loss and triggers microtubule doublet spreading, prior to outer segment collapse and photoreceptor degeneration, suggesting a molecular mechanism for a subtype of retinitis pigmentosa.

Inherited retinal degeneration due to loss of photoreceptor cells is a leading cause of human blindness. Ultrastructure expansion microscopy on mouse retina reveals the presence of a novel structure inside the photoreceptor connecting cilium, the inner scaffold, that protects the outer segment against degeneration.     

Functional analysis of retinitis pigmentosa 2 (RP2) protein reveals variable pathogenic potential of disease-associated missense variants

Genetic mutations are frequently associated with diverse phenotypic consequences, which limits the interpretation of the consequence of a variation in patients. Mutations in the retinitis pigmentosa 2 (RP2) gene are associated with X-linked RP, which is a phenotypically heterogenic form of retinal degeneration. The purpose of this study was to assess the functional consequence of disease-associated mutations in the RP2 gene using an in vivo assay. Morpholino-mediated depletion of rp2 in zebrafish resulted in perturbations in photoreceptor development and microphthalmia (small eye). Ultrastructural and immunofluorescence analyses revealed defective photoreceptor outer segment development and lack of expression of photoreceptor-specific proteins. The retinopathy phenotype could be rescued by expressing the wild-type human RP2 protein. Notably, the tested RP2 mutants exhibited variable degrees of rescue of rod versus cone photoreceptor development as well as microphthalmia. Our results suggest that RP2 plays a key role in photoreceptor development and maintenance in zebrafish and that the clinical heterogeneity associated with RP2 mutations may, in part, result from its potentially distinct functional relevance in rod versus cone photoreceptors.     

Disulfide-mediated oligomerization of Peripherin/Rds and Rom-1 in photoreceptor disk membranes. Implications for photoreceptor outer segment morphogenesis and degeneration

Peripherin/Rds is a tetraspanning membrane protein that has been implicated in photoreceptor outer segment morphogenesis and inherited retinal degenerative diseases. Together with the structurally related protein, Rom-1, it forms a complex along the rims of rod and cone disc membranes. We have compared the oligomeric structure of these proteins from nonreduced and dithiothreitol reduced membranes by velocity sedimentation, SDS-gel electrophoresis, immunoaffinity chromatography, and chemical cross-linking. Under reducing conditions peripherin/Rds and Rom-1 existed as homomeric and heteromeric core complexes devoid of intermolecular disulfide bonds. Under nonreducing conditions core complexes associated through intermolecular disulfide bonds to form oligomers. One intermediate-size oligomer contained monomers and disulfide-linked dimers of peripherin/Rds and Rom-1, while larger oligomers consisted only of disulfide-linked peripherin/Rds dimers when analyzed on nonreducing SDS gels. Consistent with this result, disc membranes contained twice as much peripherin/Rds as Rom-1. Peripherin/Rds individually expressed in COS-1 cells also formed disulfide-linked oligomers bridged through Cys-150 residues, whereas Rom-1 showed little tendency to form oligomers. These results indicate that peripherin/Rds and Rom-1 associate noncovalently to form multisubunit core complexes. Peripherin/Rds containing core complexes interact through specific intermolecular disulfide bonds to form oligomers which may play a crucial role in photoreceptor disc morphogenesis and retinal degenerative diseases.     

Molecular genetics of retinitis pigmentosa.

Retinitis pigmentosa is a model for the study of genetic diseases. Its genetic heterogeneity is reflected in the different forms of inheritance (autosomal dominant, autosomal recessive, or X-linked) and, in a few families, in the presence of mutations in the visual pigment rhodopsin. Clinical and molecular genetic studies of these disorders are discussed. Animal models of retinal degeneration have been investigated for many years with the hope of gaining insight into the cause of photoreceptor cell death. Recently, the genes responsible for two of these animal disorders, the rds and rd mouse genes, have been isolated and characterized. The retinal degeneration of the rd mouse is presented in detail. The possible involvement of human analogues of these mouse genes in human retinal diseases is being investigated.     

Expression and structural characterization of peripherin/RDS, a membrane protein implicated in photoreceptor outer segment morphology

Peripherin/RDS is a member of the tetraspanin family of integral membrane proteins and plays a major role in the morphology of photoreceptor outer segments. Peripherin/RDS has a long extracellular loop (hereafter referred to as the LEL domain), which is vital for its function. Point mutations in the LEL domain often lead to impaired photoreceptor formation and function, making peripherin/RDS an important drug target. Being a eukaryotic membrane protein, acquiring sufficient peripherin/RDS for biophysical characterisation represents a significant challenge. Here, we describe the expression and characterisation of peripherin/RDS in Drosophila melangolaster Schneider (S2) insect cells and in the methylotrophic yeast Pichia pastoris. The wild-type peripherin/RDS and the retinitis pigmentosa causing P216L mutant from S2 cells are characterised using circular dichroism (CD) spectroscopy. The structure of peripherin/RDS and of a pathogenic mutant is assessed spectroscopically for the first time. These findings are evaluated in relation to a three-dimensional model of the functionally important LEL domain obtained by protein threading.     

The retinal degeneration slow (rds) gene product is a photoreceptor disc membrane-associated glycoprotein

Mice homozygous for the retinal degeneration slow (rds) mutation exhibit abnormal development of photoreceptor cells, followed by their slow degeneration. We have recently cloned the rds gene and determined the structure of the wild-type rds mRNA. Here we show that the gene is expressed exclusively in photoreceptor cells. We demonstrate that it encodes a 39 kd membrane-associated glycoprotein that is restricted to photoreceptor outer segments. By electron microscopy, we show that the rds protein is distributed uniformly within outer segment discs. The developmental appearance of the rds protein coincides with outer segment disc formation. We propose that the rds protein functions as an adhesion molecule for stabilization of the outer segment discs.     

Zinc-desferrioxamine attenuates retinal degeneration in the rd10 mouse model of retinitis pigmentosa

Iron-associated oxidative injury plays a role in retinal degeneration such as age-related macular degeneration and retinitis pigmentosa. The metallo-complex zinc-desferrioxamine (Zn/DFO) may ameliorate such injury by chelation of labile iron in combination with release of zinc. We explored whether Zn/DFO can affect the course of retinal degeneration in the rd10 mouse model of retinitis pigmentosa. Zn/DFO-treated animals showed significantly higher electroretinographic responses at 3 and 4.5 weeks of age compared with saline-injected controls. Corresponding retinal (photoreceptor) structural rescue was observed by quantitative histological and immunohistochemical techniques. When administered alone, the components of the complex, Zn and DFO, showed a lesser, partial effect. TBARS, a marker of lipid peroxidation, and levels of oxidative DNA damage as quantified by 8-OHdG immunostaining were significantly lower in Zn/DFO-treated retinas compared with saline-injected controls. Reduced levels of retinal ferritin as well as reduced iron content within ferritin molecules were measured in Zn/DFO-treated retinas. The data, taken together, suggest that the protective effects of the Zn/DFO complex are mediated through modulation of iron bioavailability, leading to attenuation of oxidative injury. Reducing iron-associated oxidative stress using complexes such as Zn/DFO may serve as a “common pathway” therapeutic approach to attenuate injury in retinal degeneration.     

Ca2+/recoverin dependent regulation of phosphorylation of the rhodopsin mutant R135L associated with retinitis pigmentosa

No single molecular mechanism accounts for the effect of mutations in rhodopsin associated with retinitis pigmentosa. Here we report on the specific effect of a Ca2+/recoverin upon phosphorylation of the autosomal dominant retinitis pigmentosa R135L rhodopsin mutant. This mutant shows specific features like impaired G-protein signaling but enhanced phosphorylation in the shut-off process. We now report that R135L hyperphosphorylation by rhodopsin kinase is less efficiently inhibited by Ca2+/recoverin than wild-type rhodopsin. This suggests an involvement of Ca2+/recoverin into the molecular pathogenic effect of the mutation in retinitis pigmentosa which is the cause of rod photoreceptor cell degeneration. This new proposed role of Ca2+/recoverin may be one of the specific features of the proposed new Type III class or rhodopsin mutations associated with retinitis pigmentosa.     

Genetic Expression of Cyclic GMP Phosphodiesterase Activity Defines Abnormal Photoreceptor Differentiation in Neurological Mutants of Inherited Retinal Degeneration

We have examined cyclic GMP concentrations, guanylate cyclase activities, and cyclic GMP phosphodiesterase (PDE) activities in developing retinas of congenic mice with different allelic combinations at the retinal degeneration (rd) and retinal degeneration slow (rds) loci. Although guanylate cyclase activities were found to be uniformly low in the mutant retinas, striking differences in PDE activity and cyclic GMP levels were observed in retinas of the various genotypes. Homozygous rds mice, which lack receptor outer segments, showed reduced retinal PDE activity and cyclic GMP concentration in comparison to normal animals. In heterozygous rds/+ mice with abnormal outer segments, the levels were intermediate. In retinas of homozygous rd mice, PDE activity was lower than in rds retinas and cyclic GMP levels were much higher. In mice homozygous for both rd and rds genes, retinal PDE activities were even lower than in single homozygous rd mice; the cyclic GMP level reached the same high value as in the rd animals, persisted for a longer time at this high level, and did not correlate with the rate of photoreceptor cell loss. Thus, a marked variation in PDE activity appears to be the major manifestation of abnormal outer segment differentiation and eventual degeneration of photoreceptor cells in these neurological mutants. An increased cyclic GMP level seems to be an essential corollary in the expression of the rd gene even in the absence of outer segments, but it appears unlikely that an abnormally high nucleotide level in itself causes photoreceptor cell death.     

Effect of Purified Murine NGF on Isolated Photoreceptors of a Rodent Developing Retinitis Pigmentosa

A number of different studies have shown that neurotrophins, including nerve growth factor (NGF) support the survival of retinal ganglion neurons during a variety if insults. Recently, we have reported that that eye NGF administration can protect also photoreceptor degeneration in a mice and rat with inherited retinitis pigmentosa. However, the evidence that NGF acts directly on photoreceptors and that other retinal cells mediate the NGF effect could not be excluded. In the present study we have isolated retinal cells from rats with inherited retinitis pigmentosa (RP) during the post-natal stage of photoreceptor degenerative. In presence of NGF, these cells are characterized by enhanced expression of NGF-receptors and rhodopsin, the specific marker of photoreceptor and better cell survival, as well as neuritis outgrowth. Together these observations support the hypothesis that NGF that NGF acts directly on photoreceptors survival and prevents photoreceptor degeneration as previously suggested by in vivo studies.     

Mapping and retinal phenotype of the hugger mutation in the mouse

Hugger, hug, is a recessively expressed mutation in mice that features mildly abnormal locomotion, not yet explained, and a unique combination of developmental and degenerative retinal abnormalities. Analysis with the efficient MEV linkage testing stock established that hug is on mouse Chr 19 about 14 cM from th centromere, between the microsatellite markers D19Mit28 and D19Mit14. An abnormal retinal phenotype was recognized on the day of birth, when some retinal ganglion cells already lie in abnormal positions in the inner plexiform layer. By postnatal day 18 the number of neurons is reduced in all three cellular layers of the retina. Rod photoreceptor cells develop only rudimentory outer segments, and by 9 months of age, about 75% of the photoreceptor cells have completely disappeared. Similar photoreceptor cell abnormalities are seen in prph2 (formerly rds) homozygotes, which lack the peripherin/rds protein of the rod outer segments, but a mating of the respective homozygotes yielded normal progeny. Rom1, which codes for an outer segment protein similar to peripherin/rds, maps to a more proximal position on Chr 19. Received: 4 October 1996 / Accepted: 31 January 1997     

Uncoupling of photoreceptor peripherin/rds fusogenic activity from biosynthesis, subunit assembly, and targeting: a potential mechanism for pathogenic effects

Inherited defects in the RDS gene cause a multiplicity of progressive retinal diseases in humans. The gene product, peripherin/rds (P/rds), is a member of the tetraspanin protein family required for normal vertebrate photoreceptor outer segment (OS) architecture. Although its molecular function remains uncertain, P/rds has been suggested to catalyze membrane fusion events required for the OS renewal process. This study investigates the importance of two charged residues within a predicted C-terminal helical region for protein biosynthesis, localization, and interaction with model membranes. Targeted mutagenesis was utilized to neutralize charges at Glu(321) and Lys(324) individually and in combination to generate three mutant variants. Studies were conducted on variants expressed as 1) full-length P/rds in COS-1 cells, 2) glutathione S-transferase fusion proteins in Escherichia coli, and 3) membrane-associated green fluorescent protein fusion proteins in transgenic Xenopus laevis. None of the mutations affected biosynthesis of full-length P/rds in COS-1 cells as assessed by Western blotting, sedimentation velocity, and immunofluorescence microscopy. Although all mutations reside within a recently identified localization signal, none altered the ability of this region to direct OS targeting in transgenic X. laevis retinas. In contrast, individual or simultaneous neutralization of the charged amino acids Glu(321) and Lys(324) abolished the ability of the C-terminal domain to promote model membrane fusion as assayed by lipid mixing. These results demonstrate that, although overlapping, C-terminal determinants responsible for OS targeting and fusogenicity are separable and that fusogenic activity has been uncoupled from other protein properties. The observation that subunit assembly and OS targeting can both proceed normally in the absence of fusogenic activity suggests that properly assembled and targeted yet functionally altered proteins could potentially generate pathogenic effects within the vertebrate photoreceptor.     

RPGR-containing protein complexes in syndromic and non-syndromic retinal degeneration due to ciliary dysfunction

Dysfunction of primary cilia due to mutations in cilia-centrosomal proteins is associated with pleiotropic disorders. The primary (or sensory) cilium of photoreceptors mediates polarized trafficking of proteins for efficient phototransduction. Retinitis pigmentosa GTPase regulator (RPGR) is a cilia-centrosomal protein mutated in >70% of X-linked RP cases and 10%–20% of simplex RP males. Accumulating evidence indicates that RPGR may facilitate the orchestration of multiple ciliary protein complexes. Disruption of these complexes due to mutations in component proteins is an underlying cause of associated photoreceptor degeneration. Here, we highlight the recent developments in understanding the mechanism of cilia-dependent photoreceptor degeneration due to mutations in RPGR and RPGR-interacting proteins in severe genetic diseases, including retinitis pigmentosa, Leber congenital amaurosis (LCA), Joubert syndrome, and Senior-Loken syndrome, and explore the physiological relevance of photoreceptor ciliary protein complexes.