Protein In Rod Photoreceptors Research Articles

ATF6 is required for efficient rhodopsin clearance and retinal homeostasis in the P23H rho retinitis pigmentosa mouse model

Retinitis Pigmentosa (RP) is a blinding disease that arises from loss of rods and subsequently cones. The P23H rhodopsin knock-in (P23H-KI) mouse develops retinal degeneration that mirrors RP phenotype in patients carrying the orthologous variant. Previously, we found that the P23H rhodopsin protein was degraded in P23H-KI retinas, and the Unfolded Protein Response (UPR) promoted P23H rhodopsin degradation in heterologous cells in vitro. Here, we investigated the role of a UPR regulator gene, activating transcription factor 6 (Atf6), in rhodopsin protein homeostasis in heterozygous P23H rhodopsin (Rho+/P23H) mice. Significantly increased rhodopsin protein levels were found in Atf6−/−Rho+/P23H retinas compared to Atf6+/−Rho+/P23H retinas at early ages (~ P12), while rhodopsin mRNA levels were not different. The IRE1 pathway of the UPR was hyper-activated in young Atf6−/−Rho+/P23H retinas, and photoreceptor layer thickness was unchanged at this early age in Rho+/P23H mice lacking Atf6. By contrast, older Atf6−/−Rho+/P23H mice developed significantly increased retinal degeneration in comparison to Atf6+/−Rho+/P23H mice in all retinal layers, accompanied by reduced rhodopsin protein levels. Our findings demonstrate that Atf6 is required for efficient clearance of rhodopsin protein in rod photoreceptors expressing P23H rhodopsin, and that loss of Atf6 ultimately accelerates retinal degeneration in P23H-KI mice.

Transducin Partners Outside the Phototransduction Pathway.

Transducin mediates signal transduction in a classical G protein-coupled receptor (GPCR) phototransduction cascade. Interactions of transducin with the receptor and the effector molecules had been extensively investigated and are currently defined at the atomic level. However, partners and functions of rod transducin α (Gαt1) and βγ (Gβ1γ1) outside the visual pathway are not well-understood. In particular, light-induced redistribution of rod transducin from the outer segment to the inner segment and synaptic terminal (IS/ST) allows Gαt1 and/or Gβ1γ1 to modulate synaptic transmission from rods to rod bipolar cells (RBCs). Protein-protein interactions underlying this modulation are largely unknown. We discuss known interactors of transducin in the rod IS/ST compartment and potential pathways leading to the synaptic effects of light-dispersed Gαt1 and Gβ1γ1. Furthermore, we show that a prominent non-GPCR guanine nucleotide exchange factor (GEF) and a chaperone of Gα subunits, resistance to inhibitors of cholinesterase 8A (Ric-8A) protein, is expressed throughout the retina including photoreceptor cells. Recent structures of Ric-8A alone and in complexes with Gα subunits have illuminated the structural underpinnings of the Ric-8A activities. We generated a mouse model with conditional knockout of Ric-8A in rods in order to begin defining the functional roles of the protein in rod photoreceptors and the retina. Our analysis suggests that Ric-8A is not an obligate chaperone of Gαt1. Further research is needed to investigate probable roles of Ric-8A as a GEF, trafficking chaperone, or a mediator of the synaptic effects of Gαt1.

Gene Therapy Successfully Delays Degeneration in a Mouse Model of PDE6A-Linked Retinitis Pigmentosa (RP43).

Retinitis pigmentosa type 43 (RP43) is a blinding disease caused by mutations in the gene for rod phosphodiesterase 6 alpha (PDE6A). The disease process begins with a dysfunction of rod photoreceptors, subsequently followed by a currently untreatable progressive degeneration of the entire outer retina. Aiming at a curative approach via PDE6A gene supplementation, a novel adeno-associated viral (AAV) vector was developed for expression of the human PDE6A cDNA under control of the human rhodopsin promotor (rAAV8.PDE6A). This study assessed the therapeutic efficacy of rAAV8.PDE6A in the Pde6anmf363/nmf363-mutant mouse model of RP43. All mice included in this study were treated with sub-retinal injections of the vector at 2 weeks after birth. The therapeutic effect was monitored at 1 month and 6 months post injection. Biological function of the transgene was assessed in vivo by means of electroretinography. The degree of morphological rescue was investigated both in vivo using optical coherence tomography and ex vivo by immunohistological staining. It was found that the novel rAAV8.PDE6A vector resulted in a stable and efficient expression of PDE6A protein in rod photoreceptors of Pde6anmf363/nmf363 mice following treatment at both the short- and long-term time points. The treatment led to a substantial morphological preservation of outer nuclear layer thickness, rod outer segment structure, and prolonged survival of cone photoreceptors for at least 6 months. Additionally, the ERG analysis confirmed a restoration of retinal function in a group of treated mice. Taken together, this study provides successful proof-of-concept for the cross-species efficacy of the rAAV8.PDE6A vector developed for use in human patients. Importantly, the data show stable expression and rescue effects for a prolonged period of time, raising hope for future translational studies based on this approach.

Retina-specific gene excision by targeted expression of Cre recombinase

The use of Cre recombinase for conditional targeting permits the controlled removal or activation of genes in specific tissues and at specific times of development. The Rho–Cre mice provide an improved tool for studying gene ablation in rod photoreceptor cells. To establish a robust expression of Rho–Cre transgenic mice that would be useful for the study of various protein functions in photoreceptor cells, a total 11,987kb fragment (pNCHS4 Rho–NLS–cre) containing human rhodopsin promoter was cloned. The Rho–Cre plasmid was digested with EcoR1 and I Ceu-1, and the 9.316kb fragment containing the hRho promoter and Cre recombinase gel was purified. To generate transgenic mice, the purified DNA fragment was injected into fertilized oocytes according to standard protocols. ROSA26R reported the steady expression of Rho–Cre especially in photoreceptor cells, allowing further excising proteins in rod photoreceptors across the retina. This Rho–Cre transgenic line should thus prove useful as a general deletor line for genetic analysis of diverse aspects of retinopathy.

Targeting Photoreceptors via Intravitreal Delivery Using Novel, Capsid-Mutated AAV Vectors

Development of viral vectors capable of transducing photoreceptors by less invasive methods than subretinal injection would provide a major advancement in retinal gene therapy. We sought to develop novel AAV vectors optimized for photoreceptor transduction following intravitreal delivery and to develop methodology for quantifying this transduction in vivo. Surface exposed tyrosine (Y) and threonine (T) residues on the capsids of AAV2, AAV5 and AAV8 were changed to phenylalanine (F) and valine (V), respectively. Transduction efficiencies of self-complimentary, capsid-mutant and unmodified AAV vectors containing the smCBA promoter and mCherry cDNA were initially scored in vitro using a cone photoreceptor cell line. Capsid mutants exhibiting the highest transduction efficiencies relative to unmodified vectors were then injected intravitreally into transgenic mice constitutively expressing a Rhodopsin-GFP fusion protein in rod photoreceptors (Rho-GFP mice). Photoreceptor transduction was quantified by fluorescent activated cell sorting (FACS) by counting cells positive for both GFP and mCherry. To explore the utility of the capsid mutants, standard, (non-self-complementary) AAV vectors containing the human rhodopsin kinase promoter (hGRK1) were made. Vectors were intravitreally injected in wildtype mice to assess whether efficient expression exclusive to photoreceptors was achievable. To restrict off-target expression in cells of the inner and middle retina, subsequent vectors incorporated multiple target sequences for miR181, an miRNA endogenously expressed in the inner and middle retina. Results showed that AAV2 containing four Y to F mutations combined with a single T to V mutation (quadY−F+T−V) transduced photoreceptors most efficiently. Robust photoreceptor expression was mediated by AAV2(quadY−F+T−V) −hGRK1−GFP. Observed off-target expression was reduced by incorporating target sequence for a miRNA highly expressed in inner/middle retina, miR181c. Thus we have identified a novel AAV vector capable of transducing photoreceptors following intravitreal delivery to mouse. Furthermore, we describe a robust methodology for quantifying photoreceptor transduction from intravitreally delivered AAV vectors.

Adult Ciliary Epithelial Cells, Previously Identified as Retinal Stem Cells with Potential for Retinal Repair, Fail to Differentiate into New Rod Photoreceptors

The ciliary margin in lower vertebrates is a site of continual retinal neurogenesis and a stem cell niche. By contrast, the human eye ceases retinal neuron production before birth and loss of photoreceptors during life is permanent and a major cause of blindness. The discovery of a proliferative cell population in the ciliary epithelium (CE) of the adult mammalian eye, designated retinal stem cells, raised the possibility that these cells could help to restore sight by replacing lost photoreceptors. We previously demonstrated the feasibility of photoreceptor transplantation using cells from the developing retina. CE cells could provide a renewable source of photoreceptors for transplantation. Several laboratories reported that these cells generate new photoreceptors, whereas a recent report questioned the existence of retinal stem cells. We used Nrl.gfp transgenic mice that express green fluorescent protein in rod photoreceptors to assess definitively the ability of CE cells to generate new photoreceptors. We report that CE cells expanded in monolayer cultures, lose pigmentation, and express a subset of eye field and retinal progenitor cell markers. Simultaneously, they continue to express some markers characteristic of differentiated CE and typically lack a neuronal morphology. Previously reported photoreceptor differentiation conditions used for CE cells, as well as conditions used to differentiate embryonic retinal progenitor cells (RPCs) and embryonic stem cell-derived RPCs, do not effectively activate the Nrl-regulated photoreceptor differentiation program. Therefore, we conclude that CE cells lack potential for photoreceptor differentiation and would require reprogramming to be useful as a source of new photoreceptors.

Pias3-Dependent SUMOylation Directs Rod Photoreceptor Development

Specification of retinal rod photoreceptors is determined by several different transcription factors that activate expression of rod-specific genes and repress expression of cone photoreceptor-specific genes. The mechanism by which this dual regulation occurs is unclear. We have found that Pias3, a transcriptional coregulator and E3 SUMO ligase that is selectively expressed in developing photoreceptors, promotes the differentiation of rod photoreceptors while preventing rods from adopting cone photoreceptor-like characteristics. Pias3 binds the photoreceptor-specific transcription factors Crx and Nr2e3 and is specifically targeted to the promoters of photoreceptor-specific genes. Pias3 SUMOylates Nr2e3, converting it into a potent repressor of cone-specific gene expression. Rod- and cone-specific promoters are bound by hyperSUMOylated proteins in rod photoreceptors, and blocking SUMOylation in photoreceptors results in cells with morphological and molecular features of cones and an absence of rod-specific markers. Our data thus identify Pias3-mediated SUMOylation of photoreceptor-specific transcription factors as a key mechanism of rod specification.

Preferential silencing of a common dominant rhodopsin mutation does not inhibit retinal degeneration in a transgenic model

Autosomal dominant retinitis pigmentosa caused by the frequent rhodopsin P23H mutation is characterized by progressive photoreceptor cell death eventually leading to blindness and for which no therapies are available. Considering the gain-of-function effect exerted by the P23H mutation, strategies aimed at silencing the expression of the mutated allele, like RNA interference, are desirable. We have designed small interfering RNAs (siRNA) to silence specifically the P23H rhodopsin allele expressed by a transgenic rat model of the disease. We have selected in vitro one siRNA and generated an adeno-associated viral (AAV) vector expressing the short hairpin RNA (shRNA) based on the selected siRNA. In vitro the shRNA significantly inhibits the expression of the P23H but not the wild-type rhodopsin allele. Subretinal administration of the AAV2/5 vector encoding the shRNA in P23H transgenic rats results in inhibition of rhodopsin P23H expression that is not able to prevent or block photoreceptor degeneration. Since rhodopsin is the most abundant rod photoreceptor protein, systems resulting in more robust shRNA expression in the retina may be required to achieve therapeutic efficacy in vivo.

The Glutamic Acid-rich Protein-2 (GARP2) Is a High Affinity Rod Photoreceptor Phosphodiesterase (PDE6)-binding Protein That Modulates Its Catalytic Properties

The glutamic acid-rich protein-2 (GARP2) is a splice variant of the beta-subunit of the cGMP-gated ion channel of rod photoreceptors. GARP2 is believed to interact with several membrane-associated phototransduction proteins in rod photoreceptors. In this study, we demonstrated that GARP2 is a high affinity PDE6-binding protein and that PDE6 co-purifies with GARP2 during several stages of chromatographic purification. We found that hydrophobic interaction chromatography succeeds in quantitatively separating GARP2 from the PDE6 holoenzyme. Furthermore, the 17-kDa prenyl-binding protein, abundant in retinal cells, selectively released PDE6 (but not GARP2) from rod outer segment membranes, demonstrating the specificity of the interaction between GARP2 and PDE6. Purified GARP2 was able to suppress 80% of the basal activity of the nonactivated, membrane-bound PDE6 holoenzyme at concentrations equivalent to its endogenous concentration in rod outer segment membranes. However, GARP2 was unable to reverse the transducin activation of PDE6 (in contrast to a previous study) nor did it significantly alter catalysis of the fully activated PDE6 catalytic dimer. The high binding affinity of GARP2 for PDE6 and its ability to regulate PDE6 activity in its dark-adapted state suggest a novel role for GARP2 as a regulator of spontaneous activation of rod PDE6, thereby serving to lower rod photoreceptor "dark noise" and allowing these sensory cells to operate at the single photon detection limit.

A Role for Cytoskeletal Elements in the Light-Driven Translocation of Proteins in Rod Photoreceptors

Light-driven protein translocation is responsible for the dramatic redistribution of some proteins in vertebrate rod photoreceptors. In this study, the involvement of microtubules and microfilaments in the light-driven translocation of arrestin and transducin was investigated. Pharmacologic reagents were applied to native and transgenic Xenopus tadpoles, to disrupt the microtubules (thiabendazole) and microfilaments (cytochalasin D and latrunculin B) of the rod photoreceptors. Quantitative confocal imaging was used to assess the impact of these treatments on arrestin and transducin translocation. A series of transgenic tadpoles expressing arrestin truncations were also created to identify portions of arrestin that enable arrestin to translocate. Application of cytochalasin D or latrunculin B to disrupt the microfilament organization selectively slowed only transducin movement from the inner to the outer segments. Perturbation of the microtubule cytoskeleton with thiabendazole slowed the translocation of both arrestin and transducin, but only in moving from the outer to the inner segments. Transgenic Xenopus expressing fusions of green fluorescent protein (GFP) with portions of arrestin implicates the C terminus of arrestin as an important portion of the molecule for promoting translocation. This C-terminal region can be used independently to promote translocation of GFP in response to light. The results show that disruption of the cytoskeletal network in rod photoreceptors has specific effects on the translocation of arrestin and transducin. These effects suggest that the light-driven translocation of visual proteins at least partially relies on an active motor-driven mechanism for complete movement of arrestin and transducin.

Declines in Arrestin and Rhodopsin in the Macula with Progression of Age-Related Macular Degeneration

Biochemical analysis of age-related macular degeneration (AMD) at distinct stages of the disease will help further understanding of the molecular events associated with disease progression. This study was conducted to determine the ability of a new grading system for eye bank eyes, the Minnesota Grading System (MGS), to discern distinct stages of AMD so that retinal region-specific changes in rod photoreceptor protein expression from donors could be determined. Donor eyes were assigned to a specific level of AMD by using the MGS. Expression of the rod photoreceptor proteins rhodopsin and arrestin was evaluated by Western immunoblot analysis in the macular and peripheral regions of the neurosensory retina from donors at different stages of AMD. A significant linear decline in both arrestin and rhodopsin content correlated with progressive MGS levels in the macula. In contrast, the peripheral region showed no significant correlation between MGS level and the content of either protein. The statistically significant relationship between decreasing macular rod photoreceptor proteins and progressive MGS levels of AMD demonstrates the utility of the clinically based MGS to correspond with specific protein changes found at known, progressive stages of degeneration. Future biochemical analysis of clinically characterized donor eyes will further understanding of the pathobiochemistry of AMD.

The role of subunit assembly in peripherin-2 targeting to rod photoreceptor disk membranes and retinitis pigmentosa.

Peripherin-2 is a member of the tetraspanin family of membrane proteins that plays a critical role in photoreceptor outer segment disk morphogenesis. Mutations in peripherin-2 are responsible for various retinal degenerative diseases including autosomal dominant retinitis pigmentosa (ADRP). To identify determinants required for peripherin-2 targeting to disk membranes and elucidate mechanisms underlying ADRP, we have generated transgenic Xenopus tadpoles expressing wild-type and ADRP-linked peripherin-2 mutants as green fluorescent fusion proteins in rod photoreceptors. Wild-type peripherin-2 and P216L and C150S mutants, which assemble as tetramers, targeted to disk membranes as visualized by confocal and electron microscopy. In contrast the C214S and L185P mutants, which form homodimers, but not tetramers, were retained in the rod inner segment. Only the P216L disease mutant induced photoreceptor degeneration. These results indicate that tetramerization is required for peripherin-2 targeting and incorporation into disk membranes. Tetramerization-defective mutants cause ADRP through a deficiency in wild-type peripherin-2, whereas tetramerization-competent P216L peripherin-2 causes ADRP through a dominant negative effect, possibly arising from the introduction of a new oligosaccharide chain that destabilizes disks. Our results further indicate that a checkpoint between the photoreceptor inner and outer segments allows only correctly assembled peripherin-2 tetramers to be incorporated into nascent disk membranes.

Isolation of a Zebrafish Rod Opsin Promoter to Generate a Transgenic Zebrafish Line Expressing Enhanced Green Fluorescent Protein in Rod Photoreceptors

To exploit zebrafish as a transgenic model, tissue-specific promoters must be identified. We isolated a 20-kilobase (kbp) zebrafish rod opsin genomic clone, which consists of 18 kbp of 5'-flanking region, the entire coding region, and 0.5 kbp of 3'-flanking sequence. Polymerase chain reaction, Southern blotting, and DNA sequencing revealed the rod opsin gene lacks introns. The transcription start site was localized 94 nucleotides upstream of the translation initiation site. Sequence alignment with orthologous promoters revealed conserved cis-elements including glass, NRE, OTX/Bat-1, Ret-1/PCE-1, Ret-4, and TATA box. A 1.2-kbp promoter fragment was cloned upstream of the enhanced green fluorescent protein (EGFP) cDNA and microinjected into 1- to 2-cell stage zebrafish embryos. EGFP expression was detected in the ventral-nasal eye at 3 days postfertilization and spread throughout the eye. Progeny of the positive founder fish, which were identified by polymerase chain reaction amplification of fin genomic DNA, exhibited EGFP expression in the retina, confirming the germline transmission of the transgene. Frozen eye sections demonstrated the EGFP expression was rod-specific and exhibited a similar developmental expression profile as the rod opsin protein. This stable transgenic line provides a novel tool for identification of genes regulating development and maintenance of rod photoreceptors.

Mutation of the Stargardt Disease Gene (ABCR) in Age-related Macular Degeneration

Age-related macular degeneration (AMD) is the leading cause of severe central visual impairment among the elderly and is associated both with environmental factors such as smoking and with genetic factors. Here, 167 unrelated AMD patients were screened for alterations in ABCR, a gene that encodes a retinal rod photoreceptor protein and is defective in Stargardt disease, a common hereditary form of macular dystrophy. Thirteen different AMD-associated alterations, both deletions and amino acid substitutions, were found in one allele of ABCR in 26 patients (16%). Identification of ABCR alterations will permit presymptomatic testing of high-risk individuals and may lead to earlier diagnosis of AMD and to new strategies for prevention and therapy.

Mutation of the Stargardt disease gene (ABCR) in age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of severe central visual impairment among the elderly and is associated both with environmental factors such as smoking and with genetic factors. Here, 167 unrelated AMD patients were screened for alterations in ABCR, a gene that encodes a retinal rod photoreceptor protein and is defective in Stargardt disease, a common hereditary form of macular dystrophy. Thirteen different AMD-associated alterations, both deletions and amino acid substitutions, were found in one allele of ABCR in 26 patients (16%). Identification of ABCR alterations will permit presymptomatic testing of high-risk individuals and may lead to earlier diagnosis of AMD and to new strategies for prevention and therapy.

A homozygous 1-base pair deletion in the arrestin gene is a frequent cause of Oguchi disease in Japanese.

Oguchi disease is a rare autosomal recessive form of congenital stationary night blindness with all other visual functions, including visual acuity, visual field, and colour vision being usually normal. A typical clinical feature of the disorder is a golden or gray-white discolouration of the fundus which disappears in the dark-adapted state and reappears shortly after the onset of light ('Mizuo phenomenon'; Fig. 1). The course of dark adaptation of rod photoreceptors is extremely retarded in Oguchi disease while that of cones appears to proceed normally. The locus for Oguchi disease was recently mapped between D2S172 and D2S345 on distal chromosome 2q by linkage analysis. Interestingly, the gene for arrestin, an intrinsic rod photoreceptor protein implicated in the recovery phase of light transduction, also maps to this region of chromosome 2q (refs 6, 7). Here we report that in five out of six unrelated Japanese patients with Oguchi disease, we have identified a homozygous deletion of nucleotide 1147 (1147delA) in codon 309 of the arrestin gene, predicting a shift in the reading frame and a premature termination of translation which may result in 'functional null alleles.'

Interaction between G-protein beta and gamma subunit types is selective.

Signal-transducing guanine nucleotide-binding proteins (G proteins) are made up of three subunits, alpha, beta, and gamma. Each of these subunits comprises a family of proteins. The rules for association between members of one family with members of another to form a multimer are not known; it is not clear whether associations are specific or nonspecific. Other than transducin (Gt), the G protein in rod photoreceptors, most purified G proteins contain more than one subtype of beta or gamma subunits. The Gt alpha subunit is associated only with beta 1 and gamma 1. It is not known whether this specificity is due to the differential expression of these subunit types in a cell type or due to intrinsically different affinities between different beta and gamma subunit types. We have used a transfected cell assay system to examine the association of the beta 1, beta 2, and beta 3 proteins with the gamma 1 and gamma 2 proteins. Results show that gamma 1 does not associate with beta 2 and that beta 3 does not associate with gamma 1 or gamma 2. Differences in affinities between types of G protein subunits will impose restrictions on the formation of certain heterotrimers and determine which G protein will be active in a cell. A chimeric molecule of beta 1 and beta 2 was used to broadly map the regions on these subunits that determine specificity of association.

Ca2+ binding capacity of cytoplasmic proteins from rod photoreceptors is mainly due to arrestin.

Arrestin (also called S-antigen or 48-kDa protein) binds to photoexcited and phosphorylated rhodopsin and, thereby, blocks competitively the activation of transducin. Using Ca2+ titration in the presence of the indicator arsenazo III and 45Ca2+ autoradiography, we show that arrestin is a Ca2(+)-binding protein. The Ca2+ binding capacity of arresting-containing protein extracts from bovine rod outer segments is about twice as high as that of arrestin-depleted extracts. The difference in the Ca2+ binding of arrestin-containing and arrestin-depleted protein extracts was attributed to arrestin. Both, these difference-measurements of protein extracts and the measurements of purified arrestin yield dissociation constants for the Ca2+ binding of arrestin between 2 and 4 microM. The titration curves are consistent with a molar ratio of one Ca2+ binding site per arrestin. No Ca2+ binding in the micromolar range was found in extracts containing mainly transducin and cGMP-phosphodiesterase. Since arrestin is one of the most abundant proteins in rod photoreceptors occurring presumably up to millimolar concentrations in rod outer segments, we suggest that aside from its function to prevent the activation of transducin, arrestin acts probably as an intracellular Ca2+ buffer.

Human interstitial retinoid-binding protein: Gene structure and primary structure

Abstract Interstitial retinoid-binding protein (IRBP) is synthesized and secreted by rod photoreceptor cells into the interphotoreceptor matrix and is known to bind retinoids and fatty acids. We have used cDNA clones encoding human IRBP to isolate a 15-kilobase genomic fragment that encompasses the complete human IRBP gene. The IRBP gene spans more than 11 kilobases and is interrupted by three introns, all of which are positioned near the 3'-end of the coding sequence. The 3741-base pair coding region of IRBP appears to have been generated by quadruplication of an approximately 900 base pair long ancestral gene. The deduced amino acid sequence predicts a mature protein of 1,230 residues (calculated molecular weight 133,000). The protein sequence can be aligned into four homologous segments, each consisting of about 300 residues. Sequence similarity between segments is as high as 60% when conservative substitutions are taken into account. Two putative N-linked glycosylation sites are located in highly conserved domains in the center of the first and second segment of IRBP. A domain consisting of 41 residues at the COOH-terminal end of the third segment has 15 matching residues (38%) with an intradiscal loop of rhodopsin, a retinal-binding protein in rod photoreceptors.