Peripherin/rds influences membrane vesicle morphology. Implications for retinopathies

Peripherin/rds is an integral membrane glycoprotein found in the rim regions of vertebrate photoreceptor cell discs. Natural mutations of the encoding gene result in degenerative retinal disorders, such as retinitis pigmentosa. The retinal degeneration slow (rds) phenotype, observed in mice, is considered to be an appropriate model for peripherin/rds-mediated retinitis pigmentosa. Associated abnormalities in the outer segment of photoreceptor cells have implicated peripherin/rds in some aspect of disc morphology, yet it remains unclear whether such morphological effects are the cause or the result of this condition. Here we present the first direct evidence to support a role for peripherin/rds in maintaining the flattened vesicle morphology characteristic of photoreceptor outer segments. In vitro expression yields a 36-kDa immunoreactive species, which is inserted into membranes and undergoes N-glycosylation, inter- and intramolecular disulfide bonding, and dimerization. Electron microscopy reveals that peripherin/rds flattens microsomal vesicles. This effect appears to be dependent on disulfide bond formation but not N-glycosylation. The inability of two pathogenic peripherin/rds mutants (P216L and C165Y) to flatten membrane vesicles implicates such mutations as the primary cause of the retinal degeneration observed in retinitis pigmentosa.     

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.     

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.     

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.     

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.     

Membranes of retinal pigment epithelial cells in vitro are damaged in the phagocytotic process of the photoreceptor outer segment discs peroxidized by ferrous ions

The ferrous ions released from haemoglobin and storage-transferrin ions cause oxidative stress in the eyes. We observed the phagocytotic process of the photoreceptor outer segment discs peroxidized by ferrous ions in the retinal pigment epithelial (RPE) cells in vitro, and investigated how the ferrous ions influenced RPE in vitro and the photoreceptor outer segment discs. We obtained isolated photoreceptor outer segment discs using sucrose gradient of specific gravity after homogenizing porcine retinas. After bovine RPE cells were cultured with isolated photoreceptor outer segment discs containing FeCl2 for 5 and 24 h, we incubated the specimens with rhodamine phalloidin, antimouse alpha-tubulin antibody and antimouse Ig G (FITC and rhodamine labelled). We observed the specimens by a laser scanning microscopy, and made the ultrathin sections with or without 2% uranyl acetate and 2% lead acetate for examination by transmission electron microscopy. Actin filaments and microtubules of specialized cells such as RPE cells were actively involved in phagocytosis of the photoreceptor outer segment discs. Microtubules were damaged during the phagocytotic process of the photoreceptor outer segment discs peroxidized by ferrous ions. The peroxidation increased the granular and aggregated autofluorescence of the photoreceptor outer segment discs. The membranes of the disc and the phagosomes, and lysosomes in RPE cells were damaged by ferrous ions and had fine particles with high electron density staining without uranium acetate and lead citrate. The cytoskeletons such as actin filaments and microtubules, and the membranes of the phagosomes and the lysosomes in RPE cells in vitro were damaged during the phagocytotic process of the photoreceptor outer segment discs peroxidized by ferrous ions.     

Complete rescue of photoreceptor dysplasia and degeneration in transgenic retinal degeneration slow (rds) mice.

retinal degeneration slow (rds) is a semidominant mutation of mice with the phenotype of abnormal development of rod and cone photoreceptors, followed by their slow degeneration. The rds gene has been putatively cloned and its novel protein product initially characterized biochemically. In the present study we undertook to correct in vivo the retinal phenotype of mice with the rds mutation. We assembled a transgene containing a regulatory segment of the opsin gene positioned upstream of the wild-type rds coding region. Mice from three transgenic lines, homozygous for the rds mutation, were analyzed for expression of the transgene and for their retinal phenotypes. In two high expressing lines, we observed complete reversion to wild-type retinal morphology. In a third, low expressing line, we observed a retinal phenotype intermediate between wild type and rds/rds, suggesting partial rescue of the mutation. These results constitute formal proof that we have cloned the rds gene.     

Protective gene expression changes elicited by an inherited defect in photoreceptor structure

Inherited defects in retinal photoreceptor structure impair visual transduction, disrupt relationship with the retinal pigment epithelium (RPE), and compromise cell viability. A variety of progressive retinal degenerative diseases can result, and knowledge of disease etiology remains incomplete. To investigate pathogenic mechanisms in such instances, we have characterized rod photoreceptor and retinal gene expression changes in response to a defined insult to photoreceptor structure, using the retinal degeneration slow (rds) mouse model. Global gene expression profiling was performed on flow-sorted rds and wild-type rod photoreceptors immediately prior and subsequent to times at which OSs are normally elaborated. Dysregulated genes were identified via microarray hybridization, and selected candidates were validated using quantitative PCR analyses. Both the array and qPCR data revealed that gene expression changes were generally modest and dispersed amongst a variety of known functional networks. Although genes showing major (>5-fold) differential expression were identified in a few instances, nearly all displayed transient temporal profiles, returning to WT levels by postnatal day (P) 21. These observations suggest that major defects in photoreceptor cell structure may induce early homeostatic responses, which function in a protective manner to promote cell viability. We identified a single key gene, Egr1, that was dysregulated in a sustained fashion in rds rod photoreceptors and retina. Egr1 upregulation was associated with microglial activation and migration into the outer retina at times subsequent to the major peak of photoreceptor cell death. Interestingly, this response was accompanied by neurotrophic factor upregulation. We hypothesize that activation of Egr1 and neurotrophic factors may represent a protective immune mechanism which contributes to the characteristically slow retinal degeneration of the rds mouse model.     

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.     

Insights into the function of Rim protein in photoreceptors and etiology of Stargardt's disease from the phenotype in abcr knockout mice.

Rim protein (RmP) is an ABC transporter of unknown function in rod outer segment discs. The human gene for RmP (ABCR) is affected in several recessive retinal degenerations. Here, we characterize the ocular phenotype in abcr knockout mice. Mice lacking RmP show delayed dark adaptation, increased all-trans-retinaldehyde (all-trans-RAL) following light exposure, elevated phosphatidylethanolamine (PE) in outer segments, accumulation of the protonated Schiff base complex of all-trans-RAL and PE (N-retinylidene-PE), and striking deposition of a major lipofuscin fluorophore (A2-E) in retinal pigment epithelium (RPE). These data suggest that RmP functions as an outwardly directed flippase for N-retinylidene-PE. Delayed dark adaptation is likely due to accumulation in discs of the noncovalent complex between opsin and all-trans-RAL. Finally, ABCR-mediated retinal degeneration may result from "poisoning" of the RPE due to A2-E accumulation, with secondary photoreceptor degeneration due to loss of the RPE support role.     

Immunoassay of rod visual pigment (opsin) in the eyes of rds mutant mice lacking receptor outer segments

In 020/A mice, homozygous for the retinal degeneration slow (rds) gene, the photoreceptor cells fail to develop outer segments, and in the absorption spectra of retinal extracts the rhodopsin peak is lacking. Application of an enzyme-linked immunoassay using antisera against bovine opsin shows, however, that opsin is present in the homozygous mutant retina (0.010 nmol/eye) at 3% of the level of the normal retina (0.38 nmol/eye) of Balb/c mice. In the retina of heterozygous mice the opsin level (0.19 nmol/eye) is about half of the normal. Detection of opsin in the rds mutant retina demonstrates the functional basis for the reported electroretinographic response and light-mediated reduction in cyclic nucleotide levels in this mutant.     

Calreticulin reveals a critical Ca(2+) checkpoint in cardiac myofibrillogenesis

Approximately 10% of the photoreceptor outer segment (OS) is turned over each day, requiring large amounts of lipid and protein to be moved from the inner segment to the OS. Defects in intraphotoreceptor transport can lead to retinal degeneration and blindness. The transport mechanisms are unknown, but because the OS is a modified cilium, intraflagellar transport (IFT) is a candidate mechanism. IFT involves movement of large protein complexes along ciliary microtubules and is required for assembly and maintenance of cilia. We show that IFT particle proteins are localized to photoreceptor connecting cilia. We further find that mice with a mutation in the IFT particle protein gene, Tg737/IFT88, have abnormal OS development and retinal degeneration. Thus, IFT is important for assembly and maintenance of the vertebrate OS.     

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.     

Proteolytic shedding of the extracellular domain of photoreceptor cadherin. Implications for outer segment assembly

Photoreceptor cadherin (prCAD) is a distinctive cadherin family member that is concentrated at the base of rod and cone outer segments and is required for their structural integrity. During retinal development, prCAD localizes to the site of the future outer segment before rhodopsin or other phototransduction proteins. In vivo, prCAD undergoes a single proteolytic cleavage that releases the ectodomain as a soluble fragment. The C-terminal fragment containing the transmembrane and cytosolic domains remains associated with the outer segment. In rds(-/-) retinas, in which outer segment assembly is severely disrupted because of the absence of retinal degeneration slow (RDS)/peripherin, an essential outer segment structural protein, the level of prCAD is increased, whereas the levels of other outer segment proteins are decreased relative to wild type retinas. Additionally, the ratio of intact:cleaved prCAD polypeptides is increased in rds(-/-) retinas. These data imply that prCAD ectodomain cleavage is an integral part of the outer segment assembly process, and they further suggest that outer segment assembly might be driven, at least in part, by the near irreversibility of proteolysis.     

Mertk triggers uptake of photoreceptor outer segments during phagocytosis by cultured retinal pigment epithelial cells

The RCS rat is a widely studied model of recessively inherited retinal degeneration. The genetic defect, known as rdy (retinal dystrophy), results in failure of the retinal pigment epithelium (RPE) to phagocytize shed photoreceptor outer segment membranes. We previously used positional cloning and in vivo genetic complementation to demonstrate that Mertk is the gene for rdy. We have now used a rat primary RPE cell culture system to demonstrate that the RPE is the site of action of Mertk and to obtain functional evidence for a key role of Mertk in RPE phagocytosis. We found that Mertk protein is absent from RCS, but not wild-type, tissues and cultured RPE cells. Delivery of rat Mertk to cultured RCS RPE cells by means of a recombinant adenovirus restored the cells to complete phagocytic competency. Infected RCS RPE cells ingested exogenous outer segments to the same extent as wild-type RPE cells, but outer segment binding was unaffected. Mertk protein progressively co-localized with outer segment material during phagocytosis by primary RPE cells, and activated Mertk accumulated during the early stages of phagocytosis by RPE-J cells. We conclude that Mertk likely functions directly in the RPE phagocytic process as a signaling molecule triggering outer segment ingestion.     

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.     

The tetraspanin protein peripherin-2 forms a complex with melanoregulin, a putative membrane fusion regulator

Peripherin-2, the product of the rds gene, is a tetraspanin protein. In this study, we show that peripherin-2 forms a complex with melanoregulin (MREG), the product of the Mreg locus. Genetic studies suggest that MREG is involved in organelle biogenesis. In this study, we explore the role of this protein in processes associated with the formation of disk membranes, specialized organelles of photoreceptor rod cells. MREG antibodies were generated and found to be immunoreactive with a 28 kDa protein in retinal extracts, bovine OS, ARPE-19 cells, and rat RPE. MREG colocalized with peripherin-2 in WT (CB6F1/J) and in rds+/- retinas. Western blots of serial tangential sections confirmed the close association of these two proteins within the IS and basal outer segment of rods. Immunoprecipitation (IP) of OS extracts showed formation of a complex between MREG and peripherin-2-ROM-1 hetero-oligomers. This interaction was confirmed with pulldown analyses in which the GST-PerCter protein selectively pulled down His-MREG and His-MREG selectively pulled down PerCter. Biacore analysis using peptide inhibitors and per-2 truncation mutant studies allowed us to map the MREG binding site on per-2 to the last five residues of the C-terminus (Gln341-Gly346), and kinetic data predicted a KD of 80 nM for PerCter-MREG binding. Finally, the effect of MREG on photoreceptor specific membrane fusion was assayed using a disk-plasma membrane cell free assay. Preincubation of target membranes with MREG resulted in a dose-dependent inhibition of fusion with an IC50 in the submicromolar range. Collectively, these results suggest that this newly identified protein regulates peripherin-2 function.     

CERKL interacts with mitochondrial TRX2 and protects retinal cells from oxidative stress-induced apoptosis

Mutations in the ceramide kinase-like gene (CERKL) are associated with severe retinal degeneration. However, the exact function of the encoded protein (CERKL) remains unknown. Here we show that CERKL interacts with mitochondrial thioredoxin 2 (TRX2) and maintains TRX2 in the reduced redox state. Overexpression of CERKL protects cells from apoptosis under oxidative stress, whereas suppressing CERKL renders cells more sensitive to oxidative stress. In zebrafish, CERKL protein prominently locates in the outer segment and inner segment of the photoreceptor of the retina. Knockdown of CERKL in the zebrafish leads to an increase of retinal cell death, including cone and rod photoreceptor degeneration. Signs of oxidative damage to macromolecules were also detected in CERKL deficient zebrafish retina. Our results show that CERKL interacts with TRX2 and plays a novel key role in the regulation of the TRX2 antioxidant pathway and, for the first time, provides an explanation of how mutations in CERKL may lead to retinal cell death.     

Predicted and measured disorder in peripherin/rds, a retinal tetraspanin

Vertebrate photoreceptor outer segment (OS) morphogenesis requires peripherin/rds (P/rds). We have characterized this protein's C-terminus and present evidence that suggests it is intrinsically disordered. We propose that structural flexibility may underlie the multifunctionality proposed for this domain previously. The extremely short C-termini present in other tetraspanin family members suggest that intrinsic disorder may also play a role for those proteins.     

Localization of Usher 1 proteins to the photoreceptor calyceal processes,which are absent from mice

The mechanisms underlying retinal dystrophy in Usher syndrome type I (USH1) remain unknown because mutant mice lacking any of the USH1 proteins—myosin VIIa, harmonin, cadherin-23, protocadherin-15, sans—do not display retinal degeneration. We found here that, in macaque photoreceptor cells, all USH1 proteins colocalized at membrane interfaces (i) between the inner and outer segments in rods and (ii) between the microvillus-like calyceal processes and the outer segment basolateral region in rods and cones. This pattern, conserved in humans and frogs, was mediated by the formation of an USH1 protein network, which was associated with the calyceal processes from the early embryonic stages of outer segment growth onwards. By contrast, mouse photoreceptors lacked calyceal processes and had no USH1 proteins at the inner–outer segment interface. We suggest that USH1 proteins form an adhesion belt around the basolateral region of the photoreceptor outer segment in humans, and that defects in this structure cause the retinal degeneration in USH1 patients.