Dark-adapted Rod Research Articles

Dominant role for pigment epithelial CRALBP in supplying visual chromophore to photoreceptors

Cellular retinaldehyde-binding protein (CRALBP) supports production of 11-cis-retinaldehyde and its delivery to photoreceptors. It is found in the retinal pigment epithelium (RPE) and Müller glia (MG), but the relative functional importance of these two cellular pools is debated. Here, we report RPE- and MG-specific CRALBP knockout (KO) mice and examine their photoreceptor and visual cycle function. Bulk visual chromophore regeneration in RPE-KO mice is 15-fold slower than in controls, accounting for their delayed rod dark adaptation and protection against retinal phototoxicity, whereas MG-KO mice have normal bulk visual chromophore regeneration and retinal light damage susceptibility. Cone pigment regeneration is significantly impaired in RPE-KO mice but mildly affected in MG-KO mice, disclosing an unexpectedly strong reliance of cone photoreceptors on the RPE-based visual cycle. These data reveal a dominant role for RPE-CRALBP in supporting rod and cone function and highlight the importance of RPE cell targeting for CRALBP gene therapies.

WITHDRAWN: Investigating the ERG a-wave and Retinal Diseases with Rod Equivalent Circuit Model Based on the APD

Most empirically supported mathematical models of rod cells lack theoretical support from actual physical devices. Therefore, this paper proposes an equivalent circuit model for the rod is proposed based on the photoconductive properties of the avalanche photodetector (APD) and combined with the electrical properties of the rod. The model employs the photodetector to simulate the source of the photocurrent in outer segments of rod cells and takes into account the electrical properties of the inner and outer segments, the nucleus, and the synaptic terminals. It successfully simulates the trans-retinal voltage generated by the intracellular and extracellular flow of photocurrent in the outer segment of dark-adapted rods. Moreover, the typical waveform characteristics of two retinal diseases, the enhanced short wavelength sensitivity (SWS) cone syndrome and retinitis pigmentosa (RP), are investigated on the basis of electroretinogram (ERG) a-wave. This will further elucidate the function of the visual system and the ERG a-wave characteristics of the related diseases. Comparison with published experimental results validates the reliability of the model presented. Our study provides new ideas and strategies for the diagnosis of retinal diseases and provides some theoretical support for the application of photodetectors in the fabrication of artificial retinal devices.

Retinal functional and structural changes in patients with Parkinson’s disease

BackgroundVisual dysfunction have been well reported as one of the non-motor symptoms in Parkinson’s disease (PD). The aim of this study was to evaluate the functional and structural changes in the retina in patients with PD, and to correlate these changes with disease duration and motor dysfunction.MethodsFor this case–control study, we recruited patients fulfilling the diagnostic criteria for idiopathic PD according to British Brain Bank criteria, aged between 50 and 80 years. Age- and sex-matched healthy controls aged between 50 and 80 years were also recruited. Motor function for PD patients was assessed using Modified Hoehn and Yahr staging scale (H & Y staging) and Unified Parkinson’s Disease Rating Scale (UPDRS). Optical Coherence Tomography (OCT) and full field electroretinogram (ff-ERG) were done to all participants.ResultsData from 50 patients and 50 healthy controls were included in the analysis. Patients with idiopathic Parkinson's had significantly reduced peripapillary retinal nerve fiber layer (RNFL) thickness and macular ganglion cell complex (GCC) thickness compared to healthy controls (P-value < 0.05 in all parameters). They also had significantly delayed latency and reduced amplitude in both dark-adapted rods and the light-adapted cone for both a & b waves compared to healthy controls (P-value < 0.001 in all parameters). There were statistically significant negative correlations between disease duration, and left superior, right inferior and right & left average RNFL thickness [(r) coef. = -0.327, -0.301, -0.275, and -0.285 respectively]. UPDRS total score was negatively correlated with the amplitude of light-adapted of both RT and LT a & b wave and with dark-adapted RT b-wave latency [(r) coef. = -0.311, -0.395, -0.362, -0.419, and -0.342].ConclusionThe retinal structure and function were significantly affected in patients with PD in comparison to healthy controls. There was a significant impact of disease duration on retinal thickness, and there was a significant negative correlation between the degree of motor dysfunction in patients with PD and retinal function.

Rod and Cone Dark Adaptation in Congenital Aniridia and Its Association With Retinal Structure.

To characterize the association between dark-adapted rod and cone sensitivity and retinal structure in PAX6-related aniridia. Dark-adaptation curves were measured after a 5-minute exposure to bright light with red (625 nm) and green (527 nm) 2° circular light stimuli presented at ≈20° temporal retinal eccentricity in 27 participants with aniridia (nine males; 11-66 years old) and 38 age-matched healthy controls. A two-stage exponential model was fitted to each participant's responses to determine their cone and rod thresholds over time. The thicknesses of macular inner and outer retinal layers were obtained from optical coherence tomography images in 20 patients with aniridia and the 38 healthy controls. Aniridia-associated keratopathy (AAK) grade (0-3) and lens opacities were quantified by clinical examination of the anterior segment. The rod-cone break time was similar between patients with aniridia and healthy controls. Dark-adapted cone and the rod thresholds were higher in aniridia compared with healthy controls. In aniridia, foveal outer retinal layer thickness correlated with both final cone and rod thresholds. A multiple regression model indicated that foveal outer retinal layer thickness and age were the main explanatory variables to predict both final cone and rod thresholds in aniridia when the AAK grade was 2 or less. The results show that both rod- and cone-related functions are affected in PAX6-related aniridia and suggest that retinal anatomical and physiological changes extend beyond the area commonly studied in this condition: the central macula.

Investigating the Role of Rhodopsin F45L Mutation in Mouse Rod Photoreceptor Signaling and Survival.

Rhodopsin is the critical receptor molecule which enables vertebrate rod photoreceptor cells to detect a single photon of light and initiate a cascade of molecular events leading to visual perception. Recently, it has been suggested that the F45L mutation in the transmembrane helix of rhodopsin disrupts its dimerization in vitro To determine whether this mutation of rhodopsin affects its signaling properties in vivo, we generated knock-in mice expressing the rhodopsin F45L mutant. We then examined the function of rods in the mutant mice versus wild-type controls, using in vivo electroretinography and transretinal and single cell suction recordings, combined with morphologic analysis and spectrophotometry. Although we did not evaluate the effect of the F45L mutation on the state of dimerization of the rhodopsin in vivo, our results revealed that F45L-mutant mice exhibit normal retinal morphology, normal rod responses as measured both in vivo and ex vivo, and normal rod dark adaptation. We conclude that the F45L mutation does not affect the signaling properties of rhodopsin in its natural setting.

Deletion of Protein Phosphatase 2A Accelerates Retinal Degeneration in GRK1- and Arr1-Deficient Mice.

PurposeLight detection in retinal rod photoreceptors is initiated by activation of the visual pigment rhodopsin. A critical, yet often-overlooked, step enabling efficient perception of light is rhodopsin dephosphorylation mediated by protein phosphatase 2A (PP2A). PP2A deficiency has been reported to impair rhodopsin regeneration after phosphorylation by G protein receptor kinase 1 (GRK1) and binding of arrestin (Arr1), thereby delaying rod dark adaptation. However, its effects on the viability of photoreceptors in the absence of GRK1 and Arr1 remain unclear. Here, we investigated the effects of PP2A deficiency in the absence of GRK1 or Arr1, both of which have been implicated in Oguchi disease, a form of night blindness.MethodsRod-specific mice lacking the predominant catalytic Cα-subunit of PP2A were crossed with the Grk1−/− or Arr1−/− strains to obtain double knockout lines. Rod photoreceptor viability was analyzed in histological cross-sections of the retina stained with hematoxylin and eosin, and rod function was evaluated by ex vivo electroretinography.ResultsPP2A deficiency alone did not impair photoreceptor viability up to 12 months of age. Retinal degeneration was more pronounced in rods lacking GRK1 compared to rods lacking Arr1, and degeneration was accelerated in both Grk1−/− or Arr1−/− strains where PP2A was also deleted. In Arr1−/− mice, rod maximal photoresponse amplitudes were reduced by 80% at 3 months, and this diminution was enhanced further with concomitant PP2A deficiency.ConclusionsThese results suggest that although PP2A is not required for the survival of rods, its deletion accelerates the degeneration induced by the absence of either GRK1 or Arr1.

Mobility test to assess functional vision in dark-adapted patients with Leber congenital amaurosis

BackgroundInherited retinal degenerations (IRDs) affect daylight and night vision to different degrees. In the current work, we devise a method to quantify mobility under dark-adapted conditions in patients with severe childhood blindness due to Leber congenital amaurosis (LCA). Mobility thresholds from two different LCA genotypes are compared to dark-adapted vision measurements using the full-field stimulus test (FST), a conventional desktop outcome measure of rod vision.MethodsA device consisting of vertical LED strips on a plane resembling a beaded curtain was programmed to produce a rectangular pattern target defining a ‘door’ of varying luminance that could appear at one of three positions. Mobility performance was evaluated by letting the subject walk from a fixed starting position ~ 4 m away from the device with instructions to touch the door. Success was defined as the subject touching within the ‘door’ area. Ten runs were performed and the process was repeated for different levels of luminance. Tests were performed monocularly in dark-adapted and dilated eyes. Results from LCA patients with the GUCY2D and CEP290 genotypes and normal subjects were analyzed using logistic regression to estimate the mobility threshold for successful navigation. The relation of thresholds for mobility, FST and visual acuity were quantified using linear regression.ResultsNormal subjects had mobility thresholds near limits of dark-adapted rod vision. GUCY2D-LCA patients had a wide range of mobility thresholds from within 1 log of normal to greater than 8 log abnormal. CEP290-LCA patients had abnormal mobility thresholds that were between 5 and 6 log from normal. Sensitivity loss estimates using FST related linearly to the mobility thresholds which were not correlated with visual acuity.ConclusionsThe mobility task we developed can quantify functional vision in severely disabled patients with LCA. Taken together with other outcome measures of rod and cone photoreceptor-mediated vision, dark-adapted functional vision should provide a more complete understanding of the natural history and effects of treatment in patients with LCA.

Acyl-CoA:wax alcohol acyltransferase 2 modulates the cone visual cycle in mouse retina.

The daylight and color vision of diurnal vertebrates depends on cone photoreceptors. The capability of cones to operate and respond to changes in light brightness even under high illumination is attributed to their fast rate of recovery to the ground photosensitive state. This process requires the rapid replenishing of photoisomerized visual chromophore (11-cis-retinal) to regenerate cone visual pigments. Recently, several gene candidates have been proposed to contribute to the cone-specific retinoid metabolism, including acyl-CoA wax alcohol acyltransferase 2 (AWAT2, aka MFAT). Here, we evaluated the role of AWAT2 in the regeneration of visual chromophore by the phenotypic characterization of Awat2-/- mice. The global absence of AWAT2 enzymatic activity did not affect gross retinal morphology or the rate of visual chromophore regeneration by the canonical RPE65-dependent visual cycle. Analysis of Awat2 expression indicated the presence of the enzyme throughout the murine retina, including the retinal pigment epithelium (RPE) and Müller cells. Electrophysiological recordings revealed reduced maximal rod and cone dark-adapted responses in AWAT2-deficient mice compared to control mice. While rod dark adaptation was not affected by the lack of AWAT2, M-cone dark adaptation both in isolated retina and in vivo was significantly suppressed. Altogether, these results indicate that while AWAT2 is not required for the normal operation of the canonical visual cycle, it is a functional component of the cone-specific visual chromophore regenerative pathway.

Electrical responses from human retinal cone pathways associate with a common genetic polymorphism implicated in myopia

Myopia is the commonest visual impairment. Several genetic loci confer risk, but mechanisms by which they do this are unknown. Retinal signals drive eye growth, and myopia usually results from an excessively long eye. The common variant most strongly associated with myopia is near the GJD2 gene, encoding connexin-36, which forms retinal gap junctions. Light-evoked responses of retinal neurons can be recorded noninvasively as the electroretinogram (ERG). We analyzed these responses from 186 adult twin volunteers who had been genotyped at this locus. Participants underwent detailed ERG recordings incorporating international standard stimuli as well as experimental protocols aiming to separate dark-adapted rod- and cone-driven responses. A mixed linear model was used to explore association between allelic dosage at the locus and international standard ERG parameters after adjustment for age, sex, and family structure. Significant associations were found for parameters of light-adapted, but not dark-adapted, responses. Further investigation of isolated rod- and cone-driven ERGs confirmed associations with cone-driven, but not rod-driven, a-wave amplitudes. Comparison with responses to similar experimental stimuli from a patient with a prior central retinal artery occlusion, and from two patients with selective loss of ON-bipolar cell signals, was consistent with the associated parameters being derived from signals from cone-driven OFF-bipolar cells. Analysis of single-cell transcriptome data revealed strongest GJD2 expression in cone photoreceptors; bipolar cell expression appeared strongest in OFF-bipolar cells and weakest in rod-driven ON-bipolar cells. Our findings support a potential role for altered signaling in cone-driven OFF pathways in myopia development.

Extraction of phase-based optoretinograms (ORG) from serial B-scans acquired over tens of seconds by mouse retinal raster scanning OCT system.

Several specialized retinal optical coherence tomography (OCT) acquisition and processing methods have been recently developed to allow in vivo probing of light-evoked photoreceptors function, focusing on measurements in individual photoreceptors (rods and cones). Recent OCT investigations in humans and experimental animals have shown that the outer segments in dark-adapted rods and cones elongate in response to the visible optical stimuli that bleach fractions of their visual photopigment. We have previously successfully contributed to these developments by implementing OCT intensity-based "optoretinograms" (ORG), the paradigm of using near-infrared OCT (NIR OCT) to measure bleaching-induced back-scattering and/or elongation changes of photoreceptors in the eye in vivo. In parallel, several groups have successfully implemented phase-based ORGs, mainly in human studies, exploiting changes in the phases of back-scattered light. This allowed more sensitive observations of tiny alterations of photoreceptors structures. Applications of the phase-based ORG have been implemented primarily in high speed and cellular resolution AO-OCT systems that can visualize photoreceptor mosaic, allowing phase measurements of path length changes in outer segments of individual photoreceptors. The phase-based ORG in standard resolution OCT systems is much more demanding to implement and has not been explored extensively. This manuscript describes our efforts to implement a phase analysis framework to retinal images acquired with a standard resolution and raster scanning OCT system, which offers much lower phase stability than line-field or full-field OCT detection schemes due to the relatively slower acquisition speed. Our initial results showcase the successful extraction of phase-based ORG signal from the B-scans acquired at ∼100 Hz rate and its favorable comparison with intensity-based ORG signal extracted from the same data sets. We implemented the calculation of phase-based ORG signals using Knox-Thompson paths and modified signal recovery by adding decorrelation weights. The phase-sensitive ORG signal analysis developed here for mouse retinal raster scanning OCT systems could be in principle extended to clinical retinal raster scanning OCT systems, potentially opening doors for clinically friendly ORG probing.

A large animal model of RDH5-associated retinopathy recapitulates important features of the human phenotype.

Pathogenic variants in retinol dehydrogenase 5 (RDH5) attenuate supply of 11-cis-retinal to photoreceptors leading to a range of clinical phenotypes including night blindness because of markedly slowed rod dark adaptation and in some patients, macular atrophy. Current animal models (such as Rdh5-/- mice) fail to recapitulate the functional or degenerative phenotype. Addressing this need for a relevant animal model we present a new domestic cat model with a loss-of-function missense mutation in RDH5 (c.542G > T; p.Gly181Val). As with patients, affected cats have a marked delay in recovery of dark adaptation. In addition, the cats develop a degeneration of the area centralis (equivalent to the human macula). This recapitulates the development of macular atrophy that is reported in a subset of patients with RDH5 mutations and is shown in this paper in seven patients with biallelic RDH5 mutations. There is notable variability in the age at onset of the area centralis changes in the cat, with most developing changes as juveniles but some not showing changes over the first few years of age. There is similar variability in development of macular atrophy in patients and while age is a risk factor, it is hypothesized that genetic modifying loci influence disease severity, and we suspect the same is true in the cat model. This novel cat model provides opportunities to improve molecular understanding of macular atrophy and test therapeutic interventions for RDH5-associated retinopathies.

IMPAIRMENTS IN CONE PIGMENT REGENERATION AND ABSOLUTE THRESHOLD IN MACULAR TELANGIECTASIA TYPE 2.

To test the hypothesis that Müller cell dysfunction in macular telangiectasia type 2 (MacTel) results in delayed cone adaptation kinetics and to assess absolute cone and rod thresholds in this condition. Adaptation after an approximate 63.5% full-field cone photopigment bleach was assessed for Goldmann size V (1.7° diameter) 640 nm (red) and 480 nm (blue) targets presented at a retinal locus corresponding to 2° temporal to fixation. The cone time constant of adaptation and absolute cone and rod thresholds were calculated from exponential functions fitted to the resultant dark adaptation curves. Eighteen eyes with MacTel (from 11 patients) were compared with 19 control eyes (from 16 normal subjects). Cone adaptation kinetics were significantly impaired in MacTel, as was the absolute cone threshold. Final thresholds for blue targets were also significantly elevated in MacTel, consistent with impaired rod absolute threshold. Losses in sensitivity observed in MacTel were consistent with a so-called d1/2 mechanism (i.e., receptoral) site of sensitivity loss. In addition to previously documented impairments in rod dark adaptation, MacTel results in a significant elevation in cone thresholds because of pathology at the level of the photoreceptors. The delays in cone adaptation that we found in eyes with MacTel may reflect impairment of the Müller cell-mediated cone-specific visual cycle.

Correlations Between Dark-Adapted Rod Threshold Elevations and ERG Response Deficits in Duchenne Muscular Dystrophy.

PurposeThe purpose of this study was to characterize changes in the full-field flash electroretinogram (ERG) in association with psychophysical dark-adapted visual thresholds in patients with genetically characterized Duchenne muscular dystrophy (DMD) either lacking Dp427 (Up 30) or at least Dp260 in addition to Dp427 (Down 30).MethodsTwenty-one patients with DMD and 27 age-similar controls participated in this study. Dark-adapted (0.01, 3.0, and 10 cd.s/m² flashes) and light-adapted (3.0 cd.s/m² flash) ERGs were recorded following International Society for Clinical Electrophysiology of Vision (ISCEV) standard protocols. Visual detection thresholds to 625-nm (cone function) and 527-nm (rod function) light-emitting diode (LED) flashes (2 degree diameter) were measured during a dark adaptation period after a 1-minute exposure to a bleaching light (3000 cd/m²). Initially, 8 minutes of interleaved 625-nm and 527-nm thresholds were measured. After an additional 5 minutes of dark-adaptation, a second set of threshold measurements to 527-nm stimuli was performed during the subsequent 6 minutes.ResultsDark-adapted b-wave amplitude was significantly reduced to all strengths of flash and a-wave in response to the strong flash stimulus was delayed (15.6 vs. 14.7 ms, P < 0.05) in patients with Down 30 compared with controls. Dark-adapted cone thresholds did not differ among the groups (−2.0, −1.8, and −1.7 log cd/m² for Down 30, Up 30, and controls, respectively, P = 0.21). In contrast, dark-adapted rod thresholds were elevated (F(2,36) = 8.537, P = 0.001) in patients with Down 30 (mean = −3.2 ± 1.1 log cd/m²) relative to controls (mean = −4.2 ± 0.3 log cd/m²). Dark-adapted b-wave amplitudes were correlated with dark-adapted rod sensitivity in patients with DMD (Spearman Rho = 0.943, P = 0.005). The changes were much smaller or absent in patients with intact Dp260.ConclusionsDp260 is particularly required for normal rod-system function in dark adaptation.

Function of mammalian M-cones depends on the level of CRALBP in Müller cells.

Cone photoreceptors mediate daytime vision in vertebrates. The rapid and efficient regeneration of their visual pigments following photoactivation is critical for the cones to remain photoresponsive in bright and rapidly changing light conditions. Cone pigment regeneration depends on the recycling of visual chromophore, which takes place via the canonical visual cycle in the retinal pigment epithelium (RPE) and the Müller cell–driven intraretinal visual cycle. The molecular mechanisms that enable the neural retina to regenerate visual chromophore for cones have not been fully elucidated. However, one known component of the two visual cycles is the cellular retinaldehyde-binding protein (CRALBP), which is expressed both in the RPE and in Müller cells. To understand the significance of CRALBP in cone pigment regeneration, we examined the function of cones in mice heterozygous for Rlbp1, the gene encoding CRALBP. We found that CRALBP expression was reduced by ∼50% in both the RPE and retina of Rlbp1+/− mice. Electroretinography (ERG) showed that the dark adaptation of rods and cones is unaltered in Rlbp1+/− mice, indicating a normal RPE visual cycle. However, pharmacologic blockade of the RPE visual cycle revealed suppressed cone dark adaptation in Rlbp1+/− mice in comparison with controls. We conclude that the expression level of CRALPB specifically in the Müller cells modulates the efficiency of the retina visual cycle. Finally, blocking the RPE visual cycle also suppressed further cone dark adaptation in Rlbp1−/− mice, revealing a shunt in the classical RPE visual cycle that bypasses CRALBP and allows partial but unexpectedly rapid cone dark adaptation.

Rhodopsin-mediated light-off-induced protein kinase A activation in mouse rod photoreceptor cells

Light-induced extrasynaptic dopamine release in the retina reduces adenosine 3',5'-cyclic monophosphate (cAMP) in rod photoreceptor cells, which is thought to mediate light-dependent desensitization. However, the fine time course of the cAMP dynamics in rods remains elusive due to technical difficulty. Here, we visualized the spatiotemporal regulation of cAMP-dependent protein kinase (PKA) in mouse rods by two-photon live imaging of retinal explants of PKAchu mice, which express a fluorescent biosensor for PKA. Unexpectedly, in addition to the light-on-induced suppression, we observed prominent light-off-induced PKA activation. This activation required photopic light intensity and was confined to the illuminated rods. The estimated maximum spectral sensitivity of 489 nm and loss of the light-off-induced PKA activation in rod-transducin-knockout retinas strongly suggest the involvement of rhodopsin. In support of this notion, rhodopsin-deficient retinal explants showed only the light-on-induced PKA suppression. Taken together, these results suggest that, upon photopic light stimulation, rhodopsin and dopamine signals are integrated to shape the light-off-induced cAMP production and following PKA activation. This may support the dark adaptation of rods.

Disease Progression in Patients with Autosomal Dominant Retinitis Pigmentosa due to a Mutation in Inosine Monophosphate Dehydrogenase 1 (IMPDH1).

PurposeMutations in the inosine monophosphate dehydrogenase 1 (IMPDH1) gene are a common cause of inherited retinal degeneration (IRD). Due to species- and tissue-dependent expression of IMPDH1, there are no appropriate models of human IMPDH1 disease. Therefore, a limited understanding remains of disease expression and rates of progression for IMPDH1-related IRD.MethodsWe evaluated semiautomated kinetic and chromatic static perimetry, spectral-domain optical coherence tomography (SD-OCT), and ultra-wide field fundus images with autofluorescence in a cohort of 12 patients (ages 11–58 at first visit). Ten patients had longitudinal data for which rates of progression were estimated.ResultsVisual acuities were relatively stable over time and the photoreceptors within the central retina remained intact. Perifoveal photoreceptor loss measured over a period of years coincided with visual fields, which were constricted and progressed over time in all patients. Rod sensitivity showed a similar pattern of defect to that of the kinetic perimetry and the autofluorescence ultra-wide field imaging. Full-field electroretinograms were severely reduced and the dark-adapted rod and mixed responses were extinguished at earlier visits than the light-adapted cone responses.ConclusionsThere was variability in disease severity at the first visit, but results show that the peripheral retina is more susceptible to the deleterious consequences of an IMPDH1 mutation. Given the pattern of degeneration and the alternatively spliced isoforms of IMPDH1, potential interventions may consider targeting the periphery early in disease, modulating transcript expression, and/or preserving central vision at late stages of the disease.Translational RelevanceThese results inform clinical prognosis and offer evidence strategies toward therapeutic intervention.

Phosphorylation at Serine 21 in G protein-coupled receptor kinase 1 (GRK1) is required for normal kinetics of dark adaption in rod but not cone photoreceptors.

Timely recovery of the light response in photoreceptors requires efficient inactivation of photoactivated rhodopsin. This process is initiated by phosphorylation of its carboxyl terminus by G protein-coupled receptor kinase 1 (GRK1). Previously, we showed that GRK1 is phosphorylated in the dark at Ser21 in a cAMP-dependent manner and dephosphorylated in the light. Results in vitro indicate that dephosphorylation of Ser21 increases GRK1 activity, leading to increased phosphorylation of rhodopsin. This creates the possibility of light-dependent regulation of GRK1 activity and its efficiency in inactivating the visual pigment. To address the functional role of GRK1 phosphorylation in rods and cones in vivo, we generated mutant mice in which Ser21 is substituted with alanine (GRK1-S21A), preventing dark-dependent phosphorylation of GRK1. GRK1-S21A mice had normal retinal morphology, without evidence of degeneration. The function of dark-adapted GRK1-S21A rods and cones was also unaffected, as demonstrated by the normal amplitude and kinetics of their responses obtained by ex vivo and in vivo ERG recordings. In contrast, rod dark adaptation following exposure to bright bleaching light was significantly delayed in GRK1-S21A mice, suggesting that the higher activity of this kinase results in enhanced rhodopsin phosphorylation and therefore delays its regeneration. In contrast, dark adaptation of cones was unaffected by the S21A mutation. Taken together, these data suggest that rhodopsin phosphorylation/dephosphorylation modulates the recovery of rhodopsin to the ground state and rod dark adaptation. They also reveal a novel role for cAMP-dependent phosphorylation of GRK1 in regulating the dark adaptation of rod but not cone photoreceptors.

CRB2 Loss in Rod Photoreceptors Is Associated with Progressive Loss of Retinal Contrast Sensitivity.

Variations in the Crumbs homolog-1 (CRB1) gene are associated with a wide variety of autosomal recessive retinal dystrophies, including early onset retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA). CRB1 belongs to the Crumbs family, which in mammals includes CRB2 and CRB3. Here, we studied the specific roles of CRB2 in rod photoreceptor cells and whether ablation of CRB2 in rods exacerbates the Crb1-disease. Therefore, we assessed the morphological, retinal, and visual functional consequences of specific ablation of CRB2 from rods with or without concomitant loss of CRB1. Our data demonstrated that loss of CRB2 in mature rods resulted in RP. The retina showed gliosis and disruption of the subapical region and adherens junctions at the outer limiting membrane. Rods were lost at the peripheral and central superior retina, while gross retinal lamination was preserved. Rod function as measured by electroretinography was impaired in adult mice. Additional loss of CRB1 exacerbated the retinal phenotype leading to an early reduction of the dark-adapted rod photoreceptor a-wave and reduced contrast sensitivity from 3-months-of-age, as measured by optokinetic tracking reflex (OKT) behavior testing. The data suggest that CRB2 present in rods is required to prevent photoreceptor degeneration and vision loss.

Fundus-Controlled Dark Adaptometry in Young Children Without and With Spontaneously Regressed Retinopathy of Prematurity.

PurposeWe correlate dark adaptation course with foveal morphologic alterations in preterm and term-born children using a modified fundus-controlled perimeter and spectral domain–optical coherence tomography (SD-OCT) imaging.MethodsWe performed fundus-controlled chromatic dark adaptometry in premature children aged 6 to 13 years without retinopathy of prematurity (no-ROP; n = 61) and with spontaneously regressed ROP (sr-ROP, n = 29), and in 11 age-matched term-born children. The degree of macular developmental arrest (MDA), defined as a disproportion of the outer nuclear layer to inner retinal layers in the fovea (ONL+/IRL-ratio), was analyzed with the DiOCTA tool in SD-OCT scans.ResultsChildren with MDA showed a flatter dark adaptation course progression with a significant rod-mediated sensitivity recovery delay (0.0113 vs. 0.0253 dB/s; P < 0.001). Preterm-born children with regular foveal morphology reached the final rod-mediated dark-adapted threshold at 12 minutes after bleach at 18.8 dB, compared to after 18.7 minutes at 17.6 dB in children with MDA (no significant difference in final threshold; P = 0.773). The cone-mediated dark adaptation progression showed a significant lower final threshold in children with MDA (6.0 vs. 8.1 dB; P = 0.004).ConclusionsChanges in dark adaptation were seen in the presence of MDA observed in premature children in the no-ROP and sr-ROP groups. MDA in former premature children is associated with functional deficits of cone and rod photoreceptor visual pathways.Translational RelevanceMorphologic alterations in the central retina of premature children, evident in SD-OCT, are associated with long-term functional deficits in the rod and cone pathways, particularly evident in the rod dark adaptation course measured at 12° eccentricity. This indicates a more widespread retinal functional pathology not limited to the fovea, but occurring together with foveal alterations best defined as MDA.

Progression in X-linked Retinitis Pigmentosa Due to ORF15-RPGR Mutations: Assessment of Localized Vision Changes Over 2 Years.

To determine the progression rate and the variability of rod and cone sensitivities in patients with X-linked retinitis pigmentosa (XLRP) caused by mutations in ORF15-RPGR. ORF15-RPGR-XLRP patients (n = 15) were studied prospectively over 2 years with static perimetry sampling the visual field under dark-adapted and light-adapted conditions on a 12° square grid covering 168° width and 84° height. Natural history of rod and cone sensitivity loss and test-retest variability were estimated. Data were analyzed pointwise as well as averaged across small regions of neighboring loci of approximately 80 mm2 (900 deg2) in size representing the likely extent of localized gene therapy injections. Retinal loci with mild to moderate loss of sensitivity tended to be in the mid- to far-peripheral retina in most patients. When averaged across small regions, dark-adapted rod vision progressed at an average of 2 dB per year with a coefficient of repeatability (CR) of 6.3 dB, and light-adapted cone vision with white stimulus progressed at an average of 0.9 dB per year with a CR of 3.8 dB. For an average patient enrolled in an early-phase clinical trial, significant (α = 0.05) progression would be predicted to occur with 80% power in 4.5 years for rod vision and 6.1 years for cone vision. Localization of regions in the temporal hemifield and grouping of results from multiple patients would permit trial designs of shorter duration. Measurement of rod sensitivity under dark-adapted conditions averaged across a small region showed the greatest potential for detectability of progression in the shortest period.