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A High-Throughput Drug Screening Strategy for Detecting Rhodopsin P23H Mutant Rescue and Degradation
摘要： Inherent instability of the P23H mutant opsin accounts for approximately 10% of autosomal dominant retinitis pigmentosa cases. Our purpose was to develop an overall set of reliable screening strategies to assess if either stabilization or enhanced degradation of mutant rhodopsin could rescue rod photoreceptors expressing this mutant protein. These strategies promise to reveal active compounds and clarify molecular mechanisms of biologically important processes, such as inhibition of target degradation or enhanced target folding. METHODS. Cell-based bioluminescence reporter assays were developed and validated for high-throughput screening (HTS) of compounds that promote either stabilization or degradation of P23H mutant opsin. Such assays were further complemented by immunoblotting and image-based analyses. RESULTS. Two stabilization assays of P23H mutant opsin were developed and validated, one based on b-galactosidase complementarity and a second assay involving bioluminescence resonance energy transfer (BRET) technology. Moreover, two additional assays evaluating mutant protein degradation also were employed, one based on the disappearance of luminescence and another employing the ALPHA immunoassay. Imaging of cells revealed the cellular localization of mutant rhodopsin, whereas immunoblots identi?ed changes in the aggregation and glycosylation of P23H mutant opsin. CONCLUSIONS. Our ?ndings indicate that these initial HTS and following assays can identify active therapeutic compounds, even for dif?cult targets such as mutant rhodopsin. The assays are readily scalable and their function has been proven with model compounds. High-throughput screening, supported by automated imaging and classic immunoassays, can further characterize multiple steps and pathways in the biosynthesis and degradation of this essential visual system protein.
关键词： ocular pharmacology,retinal degeneration,GPCR,phototransduction,rod photoreceptors,rhodopsin,misfolded protein
更新于2025-11-21 11:20:48
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Simvastatin Protects Photoreceptors from all-trans-retinal Induced Oxidative Stress with Up-regulation of Interphotoreceptor Retinoid Binding Protein
摘要： Background and Purpose Simvastatin is a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor with multiple targets and effects. It protects neurons in the brain but its protective effects on photoreceptors are unclear. In this study, we evaluated the neuroprotective effect of simvastatin on photoreceptors exposed to stress induced by all-trans-retinal (atRAL). Experimental approach AlamarBlue and lactate dehydrogenase assays were used to evaluate the viability and metabolic activity of Y79 cells (a retinoblastoma cell line) exposed to atRAL-induced stress with or without simvastatin pre-treatment. Changes in cellular reactive oxygen species were evaluated using flow cytometry and mitochondrial stress markers JC-1 and HSP60. Changes in levels of the photoreceptor-specific markers cone-rod homeobox protein (CRX) and Interphotoreceptor Retinoid Binding Protein (IRBP) were evaluated with Western blotting. The results were validated in ex vivo human retinal explants and a mouse model of photoreceptor degeneration. Key results Simvastatin improved mitochondrial function, alleviated oxidative stress and upregulated the photoreceptor specific markers IRBP and its upstream regulator CRX in Y79 cells and ex vivo human retinal explants under atRAL-induced stress. Simvastatin attenuated photoreceptor degeneration in association with upregulation of IRBP and CRX expression after knockdown of IRBP in a murine model. Conclusion and implications Our findings suggest that simvastatin has a novel role in protecting photoreceptors from atRAL-induced stress. Simvastatin treatment resulted in upregulation of IRBP and its upstream transcription factor CRX in Y79 cells, ex vivo human retinal explants and murine retinas in vivo. Further studies of simvastatin to treat photoreceptor degeneration are warranted.
关键词： photoreceptors,IRBP,CRX,oxidative stress,all-trans-retinal,Simvastatin
更新于2025-11-21 11:08:12
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RefMoB, a Reflectivity Feature Model-Based Automated Method for Measuring Four Outer Retinal Hyperreflective Bands in Optical Coherence Tomography
摘要： PURPOSE. To validate a model-driven method (RefMoB) of automatically describing the four outer retinal hyperreflective bands revealed by spectral-domain optical coherence tomography (SDOCT), for comparison with histology of normal macula; to report thickness and position of bands, particularly band 2 (ellipsoid zone [EZ], commonly called IS/OS). METHODS. Foveal and superior perifoveal scans of seven SDOCT volumes of five individuals aged 28 to 69 years with healthy maculas were used (seven eyes for validation, five eyes for measurement). RefMoB determines band thickness and position by a multistage procedure that models reflectivities as a summation of Gaussians. Band thickness and positions were compared with those obtained by manual evaluators for the same scans, and compared with an independent published histological dataset. RESULTS. Agreement among manual evaluators was moderate. Relative to manual evaluation, RefMoB reported reduced thickness and vertical shifts in band positions in a band-specific manner for both simulated and empirical data. In foveal and perifoveal scans, band 1 was thick relative to the anatomical external limiting membrane, band 2 aligned with the outer one-third of the anatomical IS ellipsoid, and band 3 (IZ, interdigitation of retinal pigment epithelium and photoreceptors) was cleanly delineated. CONCLUSIONS. RefMoB is suitable for automatic description of the location and thickness of the four outer retinal hyperreflective bands. Initial results suggest that band 2 aligns with the outer ellipsoid, thus supporting its recent designation as EZ. Automated and objective delineation of band 3 will help investigations of structural biomarkers of dark-adaptation changes in aging.
关键词： age-related macular degeneration,retina,ellipsoid,segmentation,optical coherence tomography,photoreceptors,interdigitation,reflectivity,retinal pigment epithelium
更新于2025-09-23 15:23:52
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Lack of cone mediated retinal function increases susceptibility to form-deprivation myopia in mice
摘要： Retinal photoreceptors are important in visual signaling for normal eye growth in animals. We used Gnat2 cplf3/cplf3 (Gnat2-/-) mice, a genetic mouse model of cone dysfunction to investigate the influence of cone signaling in ocular refractive development and myopia susceptibility in mice. Refractive development under normal visual conditions was measured for Gnat2-/- and age-matched Gnat2+/+ mice, every 2 weeks from 4 to 14 weeks of age. Weekly measurements were performed on a separate cohort of mice that underwent monocular form-deprivation (FD) in the right eye from 4 weeks of age using head-mounted diffusers. Refraction, corneal curvature, and ocular biometrics were obtained using photorefraction, keratometry and optical coherence tomography, respectively. Retinas from FD mice were harvested, and analyzed for dopamine (DA) and 3,4-dihydroxyphenylacetate (DOPAC) using high-performance liquid chromatography. Under normal visual conditions, Gnat2+/+ and Gnat2-/- mice showed similar refractive error, axial length, and corneal radii across development (p>0.05), indicating no significant effects of the Gnat2 mutation on normal ocular refractive development in mice. Three weeks of FD produced a significantly greater myopic shift in Gnat2-/- mice compared to Gnat2+/+ controls (-5.40 ± 1.33 D vs -2.28 ± 0.28 D, p=0.042). Neither the Gnat2 mutation nor FD altered retinal levels of DA or DOPAC. Our results indicate that cone pathways needed for high acuity vision in primates are not as critical for normal refractive development in mice, and that both rods and cones contribute to visual signalling pathways needed to respond to FD in mammalian eyes.
关键词： mouse,form-deprivation,dopamine,Gnat2,Refractive error,cone photoreceptors
更新于2025-09-23 15:23:52
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Cell death mechanisms in a mouse model of retinal degeneration in Spinocerebellar ataxia 7
摘要： Spino-cerebellar ataxia type 7 (SCA7) is a polyglutamine (polyQ) disorder characterized by neurodegeneration of the brain, cerebellum, and retina caused by a polyglutamine expansion in ataxin 7. The presence of an expanded polyQ tract in a mutant protein is known to induce protein aggregation, cellular stress, toxicity, and finally cell death. However, the consequences of the presence of mutant ataxin7 in the retina and the mechanisms underlying photoreceptor degeneration remain poorly understood. In this study, we show that in a retinal SCA7 mouse model, polyQ ataxin7 induces stress within the retina and activates Muller cells. Moreover, Unfolded Protein Response and autophagy are activated in SCA7 photoreceptors. We have also shown that the photoreceptor death does not involve a caspase-dependent apoptosis but instead involves apoptosis inducing factor (AIF) and Leukocyte Elastase Inhibitor (LEI/L-DNase II). When these two cell death effectors are downregulated by their siRNA, a significant reduction of photoreceptor death is observed. These results highlight the consequences of polyQ protein expression in the retina and the role of caspase-independent pathways involved in photoreceptor cell death.
关键词： retina,toxicity,Spinocerebellar ataxia type 7,caspase-independent cell death,photoreceptors,polyglutamine disorder,autophagy,unfolded protein response
更新于2025-09-23 15:22:29
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Intravitreal Injection of Proinsulin-Loaded Microspheres Delays Photoreceptor Cell Death and Vision Loss in the <i>rd10</i> Mouse Model of Retinitis Pigmentosa
摘要： PURPOSE. The induction of proinsulin expression by transgenesis or intramuscular gene therapy has been shown previously to retard retinal degeneration in mouse and rat models of retinitis pigmentosa (RP), a group of inherited conditions that result in visual impairment. We investigated whether intraocular treatment with biodegradable poly (lactic-co-glycolic) acid microspheres (PLGA-MS) loaded with proinsulin has cellular and functional neuroprotective effects in the retina. METHODS. Experiments were performed using the Pde6brd10 mouse model of RP. Methionylated human recombinant proinsulin (hPI) was formulated in PLGA-MS, which were administered by intravitreal injection on postnatal days (P) 14 to 15. Retinal neuroprotection was assessed at P25 by electroretinography, and by evaluating outer nuclear layer (ONL) cellular preservation. The attenuation of photoreceptor cell death by hPI was determined by TUNEL assay in cultured P22 retinas, as well as Akt phosphorylation by immunoblotting. RESULTS. We successfully formulated hPI PLGA-MS to deliver the active molecule for several weeks in vitro. The amplitude of b-cone and mixed b-waves in electroretinographic recording was significantly higher in eyes injected with hPI-PLGA-MS compared to control eyes. Treatment with hPI-PLGA-MS attenuated photoreceptor cell loss, as revealed by comparing ONL thickness and the number of cell rows in this layer in treated versus untreated retinas. Finally, hPI prevented photoreceptor cell death and increased AktThr308 phosphorylation in organotypic cultured retinas. CONCLUSIONS. Retinal degeneration in the rd10 mouse was slowed by a single intravitreal injection of hPI-PLGA-MS. Human recombinant proinsulin elicited a rapid and effective neuroprotective effect when administered in biodegradable microspheres, which may constitute a future potentially feasible delivery method for proinsulin-based treatment of RP.
关键词： photoreceptors,neuroprotection,retinitis pigmentosa
更新于2025-09-23 15:22:29
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Formation and Clearance of All- <i>Trans</i> -Retinol in Rods Investigated in the Living Primate Eye With Two-Photon Ophthalmoscopy
摘要： PURPOSE. Two-photon excited fluorescence (TPEF) imaging has potential as a functional tool for tracking visual pigment regeneration in the living eye. Previous studies have shown that all-trans-retinol is likely the chief source of time-varying TPEF from photoreceptors. Endogenous TPEF from retinol could provide the specificity desired for tracking the visual cycle. However, in vivo characterization of native retinol kinetics is complicated by visual stimulation from the imaging beam. We have developed an imaging scheme for overcoming these challenges and monitored the formation and clearance of retinol. METHODS. Three macaques were imaged by using an in vivo two-photon ophthalmoscope. Endogenous TPEF was excited at 730 nm and recorded through the eye’s pupil for more than 90 seconds. Two-photon excited fluorescence increased with onset of light and plateaued within 40 seconds, at which point, brief incremental stimuli were delivered at 561 nm. The responses of rods to stimulation were analyzed by using first-order kinetics. RESULTS. Two-photon excited fluorescence resulting from retinol production corresponded to the fraction of rhodopsin bleached. The photosensitivity of rhodopsin was estimated to be 6.88 ± 5.50 log scotopic troland. The rate of retinol clearance depended on intensity of incremental stimulation. Clearance was faster for stronger stimuli and time constants ranged from 50 to 300 seconds. CONCLUSIONS. This study demonstrates a method for rapidly measuring the rate of clearance of retinol in vivo. Moreover, TPEF generated due to retinol can be used as a measure of rhodopsin depletion, similar to densitometry. This enhances the utility of two-photon ophthalmoscopy as a technique for evaluating the visual cycle in the living eye.
关键词： photoreceptors,visual cycle,ophthalmic imaging,pigment regeneration
更新于2025-09-23 15:22:29
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Aberrant hiPSCs-Derived from Human Keratinocytes Differentiates into 3D Retinal Organoids that Acquire Mature Photoreceptors
摘要： Human induced pluripotent stem cell (hiPSC)-derived three-dimensional retinal organoids are a new platform for studying the organoidogenesis. However, recurrent genomic aberration, acquired during generation of hiPSCs, limit its biomedical application and/or aberrant hiPSCs has not been evaluated for generation of differentiated derivatives, such as organoids and retinal pigment epithelium (RPE). In this study, we efficiently differentiated mosaic hiPSCs into retinal organoids containing mature photoreceptors. The feeder-free hiPSCs were generated from the human epidermal keratinocytes that were rapid in process with improved efficiency over several passages and maintained pluripotency. But, hiPSCs were cytogenetically mosaic with normal and abnormal karyotypes, while copy number variation analysis revealed the loss of chromosome 8q. Despite this abnormality, the stepwise differentiation of hiPSCs to form retinal organoids was autonomous and led to neuronal lamination. Furthermore, the use of a Notch inhibitor, DAPT, at an early timepoint from days 29–42 of culture improved the specification of the retinal neuron and the use of retinoic acid at days 70–120 led to the maturation of photoreceptors. hiPSC-derived retinal organoids acquired all subtypes of photoreceptors, such as RHODOPSIN, B-OPSIN and R/G-OPSIN. Additionally, the advanced maturation of photoreceptors was observed, revealing the development of specific sensory cilia and the formation of the outer-segment disc. This report is the first to show that hiPSCs with abnormal chromosomal content are permissive to the generation of three-dimensional retinal organoids.
关键词： genomic aberrations,keratinocytes,photoreceptors,retinal organoids,hiPSCs
更新于2025-09-23 15:22:29
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Cone Photoreceptor Dysfunction in Early-Stage Diabetic Retinopathy: Association Between the Activation Phase of Cone Phototransduction and the Flicker Electroretinogram
摘要： PURPOSE. To define the nature and extent of cone photoreceptor abnormalities in diabetic individuals who have mild or no retinopathy by assessing the activation phase of cone phototransduction and the flicker ERG in these individuals. METHODS. Light-adapted single-flash and flicker ERGs were recorded from 20 diabetic individuals who have no clinically apparent retinopathy (NDR), 20 diabetic individuals who have mild nonproliferative diabetic retinopathy (NPDR), and 20 nondiabetic, age-equivalent controls. A-waves elicited by flashes of different retinal illuminance were fit with a delayed Gaussian model to derive Rmp3 (maximum amplitude of the massed photoreceptor response) and S (phototransduction sensitivity). Fundamental amplitude and phase of ERGs elicited by full-field sinusoidal flicker were obtained across a frequency range of 6 to 100 Hz. RESULTS. ANVOA indicated that both diabetic groups had significant S losses compared with the controls, whereas mean Rmp3 did not differ significantly among the groups. ANOVA also indicated significantly reduced flicker ERG amplitude for frequencies ≥56 Hz for both diabetic groups compared with the controls. Flicker ERG timing (phase) did not differ significantly among the groups. Log Rmp3 + log S was significantly correlated with the patients’ high-frequency (62.5 Hz) flicker ERG amplitude loss (r = 0.69, P < 0.001). CONCLUSIONS. The delayed Gaussian a-wave model is useful for characterizing abnormalities in the activation phase of cone phototransduction and can help explain flicker ERG abnormalities in early-stage diabetic retinopathy. Reduced cone sensitivity and attenuated high-frequency flicker ERGs provide evidence for impaired cone function in these individuals.
关键词： electroretinogram,diabetic retinopathy,flicker electroretinogram,photoreceptors,a-wave
更新于2025-09-23 15:22:29
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Outer Retinal Changes Including the Ellipsoid Zone Band in Usher Syndrome 1B due to <i>MYO7A</i> Mutations
摘要： PURPOSE. To study transition zones from normal to abnormal retina in Usher syndrome IB (USH1B) caused by myosin 7A (MYO7A) mutations. METHODS. Optical coherence tomography (OCT) scattering layers in outer retina were segmented in patients (n ? 16, ages 2–42; eight patients had serial data, average interval 4.5 years) to quantify outer nuclear layer (ONL) and outer segments (OS) as well as the locus of EZ (ellipsoid zone) edge and its extent from the fovea. Static perimetry was measured under dark-adapted (DA) and light-adapted (LA) conditions. RESULTS. Ellipsoid zone edge in USH1B-MYO7A could be located up to 238 from the fovea. Ellipsoid zone extent constricted at a rate of 0.518/year with slower rates at smaller eccentricities. A well-de?ned EZ line could be associated with normal or abnormal ONL and/or OS thickness; detectable ONL extended well beyond EZ edge. At the EZ edge, the local slope of LA sensitivity loss was 2.6 (61.7) dB/deg for central transition zones. At greater eccentricities, the local slope of cone sensitivity loss was shallower (1.1 6 0.4 dB/deg for LA) than that of rod sensitivity loss (2.8 6 1.2 dB/deg for DA). CONCLUSIONS. In USH1B-MYO7A, constriction rate of EZ extent depends on the initial eccentricity of the transition. Ellipsoid zone edges in the macula correspond to large local changes in cone vision, but extramacular EZ edges show more pronounced losses on rod-based vision tests. It is advisable to use not only the EZ line but also other structural and functional parameters for estimating natural history of disease and possible therapeutic effects in future clinical trials of USH1B-MYO7A.
关键词： myosin 7A,retinitis pigmentosa,optical coherence tomography,photoreceptors
更新于2025-09-23 15:21:21