Regulation of mammalian cone phototransduction by recoverin and rhodopsin kinase.

Cone photoreceptors function under daylight conditions and are essential for color perception and vision with high temporal and spatial resolution. A remarkable feature of cones is that, unlike rods, they remain responsive in bright light. In rods, light triggers a decline in intracellular calcium, which exerts a well studied negative feedback on phototransduction that includes calcium-dependent inhibition of rhodopsin kinase (GRK1) by recoverin. Rods and cones share the same isoforms of recoverin and GRK1, and photoactivation also triggers a calcium decline in cones. However, the molecular mechanisms by which calcium exerts negative feedback on cone phototransduction through recoverin and GRK1 are not well understood. Here, we examined this question using mice expressing various levels of GRK1 or lacking recoverin. We show that although GRK1 is required for the timely inactivation of mouse cone photoresponse, gradually increasing its expression progressively delays the cone response recovery. This surprising result is in contrast with the known effect of increasing GRK1 expression in rods. Notably, the kinetics of cone responses converge and become independent of GRK1 levels for flashes activating more than ∼1% of cone pigment. Thus, mouse cone response recovery in bright light is independent of pigment phosphorylation and likely reflects the spontaneous decay of photoactivated visual pigment. We also find that recoverin potentiates the sensitivity of cones in dim light conditions but does not contribute to their capacity to function in bright light.

Variation in rhodopsin kinase expression alters the dim flash response shut off and the light adaptation in rod photoreceptors.

PURPOSE: Rod photoreceptors are exquisitely sensitive light detectors that function in dim light. The timely inactivation of their light responses is critical for the ability of rods to reliably detect and count photons. A key step in the inactivation of the rod transduction is the phosphorylation of the rod visual pigment, rhodopsin, catalyzed by G-protein-dependent receptor kinase 1 (GRK1). Absence of GRK1 greatly prolongs the photoreceptors' light response and enhances their susceptibility to degeneration. This study examined the light responses from mouse rods expressing various levels of GRK1 to evaluate how their function is modulated by rhodopsin inactivation. METHODS: Transretinal and single-cell rod electrophysiological recordings were obtained from several strains of mice expressing GRK1 at 0.3- to 3-fold the wild-type levels. The effect of GRK1 expression level on the function of mouse rods was examined in darkness and during background adaptation. RESULTS: Altering the expression of GRK1 from 0.3- to 3-fold that in wild-type rods had little effect on the single photon response amplitude. Notably, increasing the expression level of GRK1 accelerated the dim flash response shut off but had no effect on the saturated response shut off. Additionally, GRK1 excess abolished the acceleration of saturated responses shut off during light adaptation. CONCLUSIONS: These results demonstrate that rhodopsin inactivation can modulate the kinetics of recovery from dim light stimulation. More importantly, the ratio of rhodopsin kinase to its modulator recoverin appears critical for the proper adaptation of rods and the acceleration of their response shut off in background light.

Expression characteristics of dual-promoter lentiviral vectors targeting retinal photoreceptors and Müller cells.

PURPOSE: Growing evidence suggests that successful treatment of many inherited photoreceptor diseases will require multi-protein therapies that not only correct the genetic defects linked to these diseases but also slow or halt the related degenerative phenotypes. To be effective, it is likely that therapeutic protein expression will need to be targeted to specific cell types. The purpose of this study was to develop dual-promoter lentiviral vectors that target expression of two proteins to retinal cones and rods, rods only, or Müller cells. METHODS: Dual-promoter lentivectors were constructed using the following promoters: Xenopus opsin promoter (XOPS)1.3, murine opsin promoter (MOPS), interphotoreceptor retinoid binding protein promoter (IRBP156), rhodopsin kinase (RK), neural retina leucine zipper (NRLL), vimentin (VIM), cluster differentiation (CD44), and glial fibrillary acidic protein (GFAP). Vectors were packaged and injected into the neural tubes of chicken embryos. The activities of the promoters alone, in duplicate, or when paired with a different promoter were analyzed in transduced, fully-developed retinas, using direct fluorescent and immunofluorescent microscopy. RESULTS: IRBP156, NRLL, and RK were active in cones and rods while XOPS1.3 was active only in rods. Of the glial promoters, only GFAP activity was restricted to Müller cells; both VIM and CD44 were active in Müller and neural cells. Dual-promoter vectors carrying IRBP156 and RK or XOPS1.3 and MOPS, in the order listed, exhibited robust expression of both reporter transgenes in cones and rods or rods only, respectively. Expression of the upstream transgene was much lower than the downstream transgene in dual-promoter vectors constructed using two copies of either RK or IRBP156. Analyses of the expression of a dual-promoter vector carrying CD44 and VIM in the order listed showed that the activity of the VIM promoter was more restricted to glial cells when paired with the CD44 promoter, while the activity of the CD44 promoter was inhibited to the extent that no CD44-driven reporter protein was detected in transduced cells. CONCLUSIONS: We have identified two dual-promoter vectors, one that targets cones and rods and one that targets rods alone. Both vectors reliably express the two proteins encoded by the transgenes they carry. When two well matched promoters are not available, we found that it is possible to target expression of two proteins to single cells using dual-promoter vectors carrying two copies of the same promoter. These vectors should be useful in studies of retina when co-delivery of a reporter protein with an experimental protein is desired or when expression of two exogenous proteins in targeted cells is required.

Replacement gene therapy with a human RPGRIP1 sequence slows photoreceptor degeneration in a murine model of Leber congenital amaurosis.

RPGR-interacting protein-1 (RPGRIP1) is localized in the photoreceptor-connecting cilium, where it anchors the RPGR (retinitis pigmentosa GTPase regulator) protein, and its function is essential for photoreceptor maintenance. Genetic defect in RPGRIP1 is a known cause of Leber congenital amaurosis (LCA), a severe, early-onset form of retinal degeneration. We evaluated the efficacy of replacement gene therapy in a murine model of LCA carrying a targeted disruption of RPGRIP1. The replacement construct, packaged in an adeno-associated virus serotype 8 (AAV8) vector, used a rhodopsin kinase gene promoter to drive RPGRIP1 expression. Both promoter and transgene were of human origin. After subretinal delivery of the replacement gene in the mutant mice, human RPGRIP1 was expressed specifically in photoreceptors, localized correctly in the connecting cilia, and restored the normal localization of RPGR. Electroretinogram and histological examinations showed better preservation of rod and cone photoreceptor function and improved photoreceptor survival in the treated eyes. This study demonstrates the efficacy of human gene replacement therapy and validates a gene therapy design for future clinical trials in patients afflicted with this condition. Our results also have therapeutic implications for other forms of retinal degenerations attributable to a ciliary defect.

Effect of g protein-coupled receptor kinase 1 (Grk1) overexpression on rod photoreceptor cell viability.

PURPOSE: Photoreceptor rhodopsin kinase (Rk, G protein-dependent receptor kinase 1 [Grk1]) phosphorylates light-activated opsins and channels them into an inactive complex with visual arrestins. Grk1 deficiency leads to human retinopathy and heightened susceptibility to light-induced photoreceptor cell death in the mouse. The goal of this study was to determine whether excess Grk1 activity is protective against photoreceptor cell death. METHODS: Grk1-overexpressing transgenic mice (Grk1(+)) were generated by using a bacterial artificial chromosome (BAC) construct containing mouse Grk1, along with its flanking sequences. Quantitative reverse transcription-PCR, immunoblot analysis, immunostaining, and activity assays were combined with electrophysiology and morphometric analysis, to evaluate Grk1 overexpression and its effect on physiologic and morphologic retinal integrity. Morphometry and nucleosome release assays measured differences in resistance to photoreceptor cell loss between control and transgenic mice exposed to intense light. RESULTS: Compared with control animals, the Grk1(+) transgenic line had approximately a threefold increase in Grk1 transcript and immunoreactive protein. Phosphorylated opsin immunochemical staining and in vitro phosphorylation assays confirmed proportionately higher Grk1 enzyme activity. Grk1(+) mice retained normal rod function, normal retinal appearance, and lacked evidence of spontaneous apoptosis when reared in cyclic light. In intense light, Grk1(+) mice showed photoreceptor damage, and their susceptibility was more pronounced than that of control mice with prolonged exposure times. CONCLUSIONS: Enhancing visual pigment deactivation does not appear to protect against apoptosis; however, excess flow of opsin into the deactivation pathway may actually increase susceptibility to stress-induced cell death similar to some forms of retinal degeneration.

AAV-mediated expression targeting of rod and cone photoreceptors with a human rhodopsin kinase promoter.

PURPOSE: Gene therapy for retinal degeneration requires well-defined promoters that drive expression in rod and cone photoreceptors. This study was undertaken to develop short, active derivatives of the human rhodopsin kinase (RK) gene promoter for targeting transgene expression in rods and cones. RK, also known as G protein-coupled receptor kinase 1 (GRK1), is a component of the light adaptation pathway expressed in rods and cones. METHODS: Human RK (hRK) promoter and its concatemers or derivatives extending into the conserved 5' untranslated region (5'-UTR) were assayed for promoter activity in WERI retinoblastoma or Crx/Sp1-supplemented HEK-293 cells. The derivative displaying the highest activity was linked to a GFP reporter and packaged in a pseudotyped adenoassociated viral vector (AAV2/5). The AAV vector was tested in vivo by subretinal injections in wild-type mice, in the all-cone Nrl(-/-) mice, and in the cone-rich diurnal Nile grass rat (Arvicanthis niloticus). Control eyes received a similar AAV2/5 vector carrying a mouse rod opsin (mOps) promoter-controlled GFP reporter. RESULTS: The hRK promoter with the full 5' untranslated sequence (-112 to +180) was the most active in cell culture. Delivered by the AAV2/5 vector, RK promoter drove GFP expression specifically in photoreceptors. In rods, hRK promoter-mediated expression was as efficient as, but appeared more uniform than, mOps promoter-mediated expression. In cones, the hRK promoter drove expression, whereas the mOps promoter did not. CONCLUSIONS: The hRK promoter is active and specific for rod and cone photoreceptors. Because of its small size and proven activity in cones, it is a promoter of choice for somatic gene transfer and gene therapy targeting rods and cones.

Conserved interactions of a compact highly active enhancer/promoter upstream of the rhodopsin kinase (GRK1) gene.

Rhodopsin kinase (RK) is a conserved component of the light adaptation and recovery pathways shared among rod and cone photoreceptors of a variety of species. To gain insight into transcriptional mechanisms driving RK and potentially other genes of similar spatial profile, the components and the interactions of the highly compact enhancer/promoter region (E/P) upstream of the human RK gene were examined. Cross-species comparison outlined an active 49-bp widely shared E/P core as the major site of conservation in the entire 5' flanking sequence. The area consisted of a bicoid-type homeodomain recognition cassette and a unique T-rich module interacting with TATA-binding proteins. Homeodomain interactions involved primarily Crx and secondarily Otx2. Both strongly stimulated the E/P. In the absence of Crx, persistent E/P activity shifted from the outer retina to the inner to follow the Otx2 pattern. The spatial patterns were largely unaffected by the absence of rod transcription factors, Nrl and Nr2e3, and the RK transcriptional activity preceded the surge in rod-specific transcription. Conserved bicoid homeodomain factors thus appear to be the key factors governing localization of RK E/P activity in retina and photoreceptors.

Conserved structure and spatiotemporal function of the compact rhodopsin kinase (GRK1) enhancer/promoter.

PURPOSE: To demonstrate that the crucial elements responsible for the spatial and temporal expression patterns of rhodopsin kinase (Rk) are contained within a narrow conserved segment immediately flanking the Rk transcription start sites. METHODS: Sequences upstream of the mouse Rk gene were compared to the human sequence to identify areas of conservation. Transgenic mice carrying a segment of the conserved human DNA sequence linked upstream of the green fluorescent protein (GFP) gene were examined by fluorescence microscopy and RT-PCR to localize GFP expression in retina and pineal gland. Rk and GFP temporal expression patterns were further compared by immunostaining and real-time RT-PCR in transgenic eyes during development. RESULTS: Comparison of the mouse and human 5' flanking sequence revealed only a small island of conserved sequence upstream of the respective Rk start sites. Uniform GFP expression was supported by a 0.2 kb fragment of the conserved human sequence in the transgenic mouse rods, cones, and pinealocytes. Developmental studies revealed an exponential rise in Rk and GFP transcripts in the first ten day postnatal period followed by a plateau later extending to adulthood. Rk and GFP proteins were first detected after postnatal day 10 and rose in parallel afterwards, overlapping in time with the maturation of photoreceptor outer segments and eye opening. CONCLUSIONS: The conserved short enhancer/promoter immediately upstream of the Rk gene contains the key elements required for appropriate response to spatial and temporal cues during photoreceptor cell differentiation and fate determination. The above studies narrow the core sequences that govern gene expression in photoreceptors in vivo.

A short, highly active photoreceptor-specific enhancer/promoter region upstream of the human rhodopsin kinase gene.

PURPOSE: Rhodopsin kinase (Rk or GRK1) is a photoreceptor-specific enzyme that mediates adaptation of photoreceptors to light and protects these cells against light-induced injury. This study examined the transcriptional mechanisms that maintain physiologic levels of this essential enzyme in photoreceptors. METHODS: The 2.0-kb region flanking the 5' end of the human Rk gene was isolated, mapped, and sequenced. The sequence was fused upstream of the luciferase gene and was tested for promoter activity in retinoblastoma cells by transient transfection. Transcriptionally active segments were identified by deletion and site-directed mutagenesis. Transgenic mice were generated that carried the immediate 5' flanking segment linked upstream of the enhancerless green fluorescent protein (GFP) gene. GFP expression was analyzed by RT-PCR, fluorescence microscopy, and immunohistochemistry. RESULTS: Mapping and sequence analysis uncovered a TATA-less promoter with several recognizable elements concentrated proximally. A conserved putative homeodomain response element H1 and a GC- and a GA-rich motif were noted within a 0.11-kb region. Another putative homeodomain binding site, H2, and a stretch of C-rich repeats were present distally. Mutagenesis in conjunction with transient transfection in retinoblastoma cells identified the 0.11-kb region and H1 sequence as the key active enhancer-promoter sequences. The distal sequences were inhibitory. Transgenic mice that carried the 0.11-kb DNA segment with the GFP gene linked downstream showed GFP transcript, fluorescence, and immunoreactivity that were restricted to photoreceptors. CONCLUSIONS: The experiments defined a short, highly active photoreceptor-specific enhancer-promoter region upstream of the Rk gene. The H1 element contributed substantially but not exclusively to the transcriptional activity of the region. The findings support a transcriptional basis for photoreceptor-specific expression of Rk.

Late-onset autosomal dominant macular dystrophy with choroidal neovascularization and nonexudative maculopathy associated with mutation in the RDS gene.

PURPOSE: To examine the molecular genetic basis and phenotypic characteristics of an unusual late-onset autosomal dominant macular dystrophy with features of age-related macular degeneration (AMD) in a large family (SUNY901), by using linkage and mutation analyses. METHODS: Blood samples were collected from 17 affected members, 17 clinically unaffected members, and 5 unrelated spouses. Clinical analyses included a review of medical history and standard ophthalmic examination with fundus photography, fluorescein angiography, and electroretinography. Linkage and haplotype analyses were performed with microsatellite markers. Mutation analysis was performed by amplification of exons followed by sequencing. RESULTS: A wide spectrum of clinical phenotypes including exudative and nonexudative maculopathy was observed, with onset in the late fifth decade. Linkage analysis excluded most of the previously known maculopathy loci. Markers D6S1604 (Z(max) of 3.18 at theta = 0), and D6S282 (Z(max) of 3.18 at theta = 0) gave significant positive LOD scores and haplotype analysis localized the disease gene to a 9-centimorgan (cM) interval between markers D6S1616 and D6S459. Mutation analysis excluded the GUCA1A and GUCA1B genes and revealed a missense mutation in the RDS/peripherin gene leading to a Tyr141Cys substitution. A phenotype and haplotype comparison between this and a separate family with the Tyr141Cys mutation suggested the presence of a common ancestral haplotype. CONCLUSIONS: The RDS mutation in codon 141 is associated with an unusual AMD-like late-onset maculopathy. An apparent selective bias was noted favoring the transmission of the mutant allele. These observations broaden the spectrum of phenotypes associated with RDS gene mutations.