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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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Complex Structural <i>PPT1</i> Variant Associated with Non-syndromic Canine Retinal Degeneration
摘要： Rod and cone photoreceptors are specialized retinal neurons that have a fundamental role in visual perception, capturing light and transducing it into a neuronal signal. Aberrant functioning of rod and/or cone photoreceptors can ultimately lead to progressive degeneration and eventually blindness. In man, many rod and rod-cone degenerative diseases are classified as forms of retinitis pigmentosa (RP). Dogs also have a comparable disease grouping termed progressive retinal atrophy (PRA). These diseases are generally due to single gene defects and follow Mendelian inheritance. We collected 51 DNA samples from Miniature Schnauzers affected by PRA (average age of diagnosis ~3.9 ±1 years), as well as from 56 clinically normal controls of the same breed (average age ~6.6 ±2.8 years). Pedigree analysis suggested monogenic autosomal recessive inheritance of PRA. GWAS and homozygosity mapping defined a critical interval in the first 4,796,806 bp of CFA15. Whole genome sequencing of two affected cases, a carrier and a control identified two candidate variants within the critical interval. One was an intronic SNV in HIVEP3, and the other was a complex structural variant consisting of the duplication of exon 5 of the PPT1 gene along with a conversion and insertion (named PPT1dci). PPT1dci was confirmed homozygous in a cohort of 22 cases, and 12 more cases were homozygous for the CFA15 haplotype. Additionally, the variant was found homozygous in 6 non-affected dogs of age higher than the average age of onset. The HIVEP3 variant was found heterozygous (n=4) and homozygous wild-type (n=1) in cases either homozygous for PPT1dci or for the mapped CFA15 haplotype. We detected the wildtype and three aberrant PPT1 transcripts in isolated white blood cell mRNA extracted from a PRA case homozygous for PPT1dci, and the aberrant transcripts involved inclusion of the duplicated exon 5 and novel exons following the activation of cryptic splice sites. No neurological signs were detected among the dogs homozygous for the PPT1dci variant. Therefore, we propose PPT1dci as causative for a non-syndromic form of PRA (PRAPPT1) that shows incomplete penetrance in Miniature Schnauzers, potentially related to the presence of the wild-type transcript. To our knowledge, this is the first case of isolated retinal degeneration associated with a PPT1 variant.
关键词： progressive retinal atrophy,palmitoyl protein thioesterase,dog,complex variant,PRA,retinal degeneration,whole genome sequencing
更新于2025-09-23 15:22:29
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Manganese-Enhanced MRI for Preclinical Evaluation of Retinal Degeneration Treatments
摘要： PURPOSE. Apply manganese-enhanced magnetic resonance imaging (MEMRI) to assess ion channel activity and structure of retinas from mice subject to light-induced retinal degeneration treated with prophylactic agents. METHODS. Abca4?/?Rdh8?/? double knockout mice with and without prophylactic retinylamine (Ret-NH2) treatment were illuminated with strong light. Manganese-enhanced MRI was used to image the retina 2 hours after intravitreous injection of MnCl2 into one eye. Contrast-enhanced MRIs of the retina and vitreous humor in each experimental group were assessed and correlated with the treatment. Findings were compared with standard structural and functional assessments of the retina by optical coherence tomography (OCT), histology, and electroretinography (ERG). RESULTS. Manganese-enhanced MRI contrast in the retina was high in nonilluminated and illuminated Ret-NH2–treated mice, whereas no enhancement was evident in the retina of the light-illuminated mice without Ret-NH2 treatment (P < 0.0005). A relatively high signal enhancement was also observed in the vitreous humor of mice treated with Ret-NH2. Strong MEMRI signal enhancement in the retinas of mice treated with retinylamine was correlated with their structural integrity and function evidenced by OCT, histology, and a strong ERG light response. CONCLUSIONS. Manganese-enhanced MRI has the potential to assess the response of the retina to prophylactic treatment based on the measurement of ion channel activity. This approach could be used as a complementary tool in preclinical development of new prophylactic therapies for retinopathies.
关键词： manganese-enhanced MRI,efficacy evaluation,retinal degeneration,retinylamine
更新于2025-09-23 15:22:29
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Long-term Characterization of Retinal Degeneration in Royal College of Surgeons Rats Using Spectral-Domain Optical Coherence Tomography
摘要： PURPOSE. Prospective treatments for age-related macular degeneration and inherited retinal degenerations are commonly evaluated in the Royal College of Surgeons (RCS) rat before translation into clinical application. Historically, retinal thickness obtained through postmortem anatomic assessments has been a key outcome measure; however, utility of this measurement is limited because it precludes the ability to perform longitudinal studies. To overcome this limitation, the present study was designed to provide a baseline longitudinal quantification of retinal thickness in the RCS rat by using spectral-domain optical coherence tomography (SD-OCT). METHODS. Horizontal and vertical linear SD-OCT scans centered on the optic nerve were captured from Long-Evans control rats at P30, P60, P90 and from RCS rats between P17 and P90. Total retina (TR), outer nuclear layer+ (ONL+), inner nuclear layer (INL), and retinal pigment epithelium (RPE) thicknesses were quantified. Histologic sections of RCS retina obtained from P21 to P60 were compared to SD-OCT images. RESULTS. In RCS rats, TR and ONL+ thickness decreased significantly as compared to Long-Evans controls. Changes in INL and RPE thickness were not significantly different between control and RCS retinas. From P30 to P90 a subretinal hyperreflective layer (HRL) was observed and quantified in RCS rats. After correlation with histology, the HRL was identified as disorganized outer segments and the location of accumulated debris. CONCLUSIONS. Retinal layer thickness can be quantified longitudinally throughout the course of retinal degeneration in the RCS rat by using SD-OCT. Thickness measurements obtained with SD-OCT were consistent with previous anatomic thickness assessments. This study provides baseline data for future longitudinal assessment of therapeutic agents in the RCS rat.
关键词： MERTK,spectral-domain optical coherence tomography,inherited retinal degeneration,animal models,Royal College of Surgeons Rat
更新于2025-09-23 15:22:29
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In vivo MRI assessment of bioactive magnetic iron oxide/human serum albumin nanoparticle delivery into the posterior segment of the eye in a rat model of retinal degeneration
摘要： Background: Retinal degeneration diseases affect millions of patients worldwide and lead to incurable vision loss. These diseases are caused by pathologies in the retina and underlying choroid, located in the back of the eye. One of the major challenges in the development of treatments for these blinding diseases is the safe and efficient delivery of therapeutics into the back of the eye. Previous studies demonstrated that narrow size distribution core–shell near infra-red fluorescent iron oxide (IO) nanoparticles (NPs) coated with human serum albumin (HSA, IO/HSA NPs) increase the half-life of conjugated therapeutic factors, suggesting they may be used for sustained release of therapeutics. In the present study, the in vivo tracking by MRI and the long term safety of IO/HSA NPs delivery into the suprachoroid of a rat model of retinal degeneration were assessed. Results: Twenty-five Royal College of Surgeons (RCS) pigmented rats received suprachoroidal injection of 20-nm IO/HSA NPs into the right eye. The left eye was not injected and used as control. Animals were examined by magnetic resonance imaging (MRI), electroretinogram (ERG) and histology up to 30 weeks following injection. IO/HSA NPs were detected in the back part of the rats’ eyes up to 30 weeks following injection by MRI, and up to 6 weeks by histology. No significant differences in retinal structure and function were observed between injected and non-injected eyes. There was no significant difference in the weight of IO/HSA NP-injected animals compared to non-injected rats. Conclusions: MRI could track the nanoparticles in the posterior segment of the injected eyes demonstrating their long-term persistence, and highlighting the possible use of MRI for translational studies in animals and in future clinical studies. Suprachoroidal injection of IO/HSA NPs showed no sign of adverse effects on retinal structure and function in a rat model of retinal degeneration, suggesting that suprachoroidal delivery of IO/HSA NPs is safe and that these NPs may be used in future translational and clinical studies for extended release drug delivery at the back of the eye.
关键词： Retinal degeneration,Suprachoroidal injection,RCS rats,Iron oxide nanoparticles
更新于2025-09-23 15:22:29
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Chlorogenic Acid Supplementation Improves Multifocal Electroretinography in Patients with Retinitis Pigmentosa
摘要： To evaluate the effect of chlorogenic acid supplementation in patients with retinitis pigmentosa, we evaluated objective change in visual function with multifocal electroretinography, along with visual acuity, visual field, standard electroretinography, and contrast sensitivity. Eighteen patients diagnosed with retinitis pigmentosa were enrolled in this prospective, non-comparative, single-arm study. Multifocal electroretinography, best-corrected visual acuity in Early Treatment Diabetic Retinopathy Study letters, total point score on visual field examination with Humphrey Field Analyzer II, electroretinography, and contrast sensitivity were measured and repeated after 3 months supplementation with chlorogenic acid. The amplitude of ring 5 was significantly higher on multifocal electroretinography after 3 months of chlorogenic acid supplementation (7.2 ± 9.5 vs 8.3 ± 10.8 nV/deg2, mean ± standard deviation, P = 0.022). There were no significant changes in the best-corrected visual acuity, total point score on Humphrey Field Analyzer, 30 Hz flicker amplitude on standard electroretinography, or contrast sensitivity. Chlorogenic acid may have a beneficial effect on the peripheral area at the margins of retinal degeneration, and should be considered as an anti-oxidant for the management of retinitis pigmentosa.
关键词： Retinitis Pigmentosa,Oxidative Stress,Chlorogenic Acid,Retinal Degeneration,Antioxidants
更新于2025-09-23 15:22:29
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Intravitreal Administration of Human Bone Marrow CD34+ Stem Cells in a Murine Model of Retinal Degeneration
摘要： PURPOSE. Intravitreal murine lineage-negative bone marrow (BM) hematopoietic cells slow down retinal degeneration. Because human BM CD34t hematopoietic cells are not precisely comparable to murine cells, this study examined the effect of intravitreal human BM CD34t cells on the degenerating retina using a murine model. METHODS. C3H/HeJrd1/rd1 mice, immunosuppressed systemically with tacrolimus and rapamycin, were injected intravitreally with PBS (n ? 16) or CD34t cells (n ? 16) isolated from human BM using a magnetic cell sorter and labeled with enhanced green ?uorescent protein (EGFP). After 1 and 4 weeks, the injected eyes were imaged with scanning laser ophthalmoscopy (SLO)/optical coherence tomography (OCT) and tested with electroretinography (ERG). Eyes were harvested after euthanasia for immunohistochemical and microarray analysis of the retina. RESULTS. In vivo SLO fundus imaging visualized EGFP-labeled cells within the eyes following intravitreal injection. Simultaneous OCT analysis localized the EGFP-labeled cells on the retinal surface resulting in a saw-toothed appearance. Immunohistochemical analysis of the retina identi?ed EGFP-labeled cells on the retinal surface and adjacent to ganglion cells. Electroretinography testing showed a ?at signal both at 1 and 4 weeks following injection in all eyes. Microarray analysis of the retina following cell injection showed altered expression of more than 300 mouse genes, predominantly those regulating photoreceptor function and maintenance and apoptosis. CONCLUSIONS. Intravitreal human BM CD34t cells rapidly home to the degenerating retinal surface. Although a functional bene?t of this cell therapy was not seen on ERG in this rapidly progressive retinal degeneration model, molecular changes in the retina associated with CD34t cell therapy suggest potential trophic regenerative effects that warrant further exploration.
关键词： intravitreal,stem cells,CD34t,retinal degeneration
更新于2025-09-23 15:22:29
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Retinal degeneration 3 (RD3) protein, a retinal guanylyl cyclase regulator, forms a monomeric and elongated four helix bundle
摘要： Retinal degeneration 3 (RD3) protein promotes accumulation of retinal membrane guanylyl cyclase (RetGC) in photoreceptor outer segment and suppresses RetGC activation by guanylyl cyclase activating proteins (GCAPs). Mutations truncating RD3 cause severe congenital blindness by preventing the inhibitory binding of RD3 to the cyclase. The high propensity of RD3 to aggregate in solution has prevented structural analysis. Here, we produced a highly soluble variant of human RD3 (residues 18–160) that is monomeric and can still bind and negatively regulate RetGC. The NMR solution structure of RD3 revealed an elongated backbone structure (70? long and 30? wide), consisting of a four helix bundle with a long unstructured loop between helices 1 and 2. The structure reveals that RD3 residues previously implicated in RetGC binding map to a localized and contiguous area on the structure, involving a loop between helices 2 and 3 and adjacent parts of helices 3 and 4. The NMR structure of RD3 was validated by mutagenesis. Introducing Trp85 or Phe29 to replace Cys or Leu, respectively, disrupts packing in the hydrophobic core and lowers RD3’s apparent affinity for RetGC1. Introducing a positive charge at the interface (Glu32 to Lys), also lowered the affinity. Conversely, introducing Val in place of Cys93 stabilized the hydrophobic core and increased the RD3 affinity for the cyclase. The NMR structure of RD3 presented here provides a structural basis for elucidating RD3/RetGC interactions relevant for normal vision or blindness.
关键词： phototransduction,retina,NMR spectroscopy,guanylate cyclase (guanylyl cyclase),retinal degeneration 3 (RD3),photoreceptor,blindness
更新于2025-09-23 15:22:29
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Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research
摘要： HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the subretinal space, and to evaluate toxicity and efficacy in vivo. HR-SD-OCT uses near infrared light (800-880 nm) and has optics specifically designed for the unique optics of the mouse eye with sub-2-micron axial resolution. Transgenic mouse models of outer retinal (photoreceptor) degeneration and controls were imaged to assess the disease progression. Pulled glass microneedles were used to deliver sub retinal injections of adeno-associated virus (AAV) or nanoparticles (NP) via a trans-scleral and trans-choroidal approach. Careful positioning of the needle into the subretinal space was required prior to a calibrated pressure injection, which delivers fluid into the sub retinal space. Real time subretinal surgery was conducted on our retinal imaging system (RIS). HR-SD-OCT demonstrated progressive uniform retinal degeneration due to expression of a toxic mutant human mutant rhodopsin (P347S) (RHOP347S) transgene in mice. HR-SD-OCT allows rigorous quantification of all the retinal layers. Outer nuclear layer (ONL) thickness and photoreceptor outer segment length (OSL) measurements correlate with photoreceptor vitality, degeneration, or rescue. The RIS delivery system allows real-time visualization of subretinal injections in neonatal (~P10-14) or adult mice, and HR-SD-OCT immediately determines success of delivery and maps areal extent. HR-SD-OCT is a powerful tool that can evaluate the success of subretinal surgery in mice, in addition to measuring vitality of photoreceptors in vivo. HR-SD-OCT can also be used to identify uniform animal cohorts to evaluate the extent of retinal degeneration, toxicity, and therapeutic rescue in preclinical gene therapy research studies.
关键词： Optical Coherence Tomography,Retinal Degeneration,Imaging,Real-time,Retina,Microscope,Intraocular Injection,Preclinical,In Vivo,Photoreceptors,Greenough Stereo Microscopy,Sub-retinal
更新于2025-09-23 15:21:01
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Protective effect of RIPK1-inhibitory compound in in vivo models for retinal degenerative disease
摘要： Receptor interacting protein kinase 1 (RIPK1) plays a key role in necroptosis, which is a type of programmed necrosis that is involved in ocular diseases, including glaucoma and dry age-related macular degeneration (AMD). We previously introduced RIPK1-inhibitory compound (RIC), which has biochemical characteristics and a mode of action that are distinct from those of the prototype RIPK1 inhibitor necrostatin-1. The intraperitoneal administration of RIC exerts a protective effect on retinal ganglion cells against a glaucomatous insult. In this study, we examined the protective effect of RIC on retinal pigment epithelium (RPE) against sodium iodate (SI) insult, which is associated with dry AMD pathogenesis. The eye drop administration of RIC that reached on the retina prevented RPE loss in SI-induced retinal degeneration. RIC consistently demonstrated retinal protection in the funduscopy and electroretinogram analyses in SI-injected rabbits and iodoacetic acid-treated mini-pigs. Moreover, the in vivo protective effects of RIC were superior to those of ACU-4429 and doxycycline, which are other medications investigated in clinical trials for the treatment of dry AMD, and RIC did not induce retinal toxicity following topical administration in rats. Collectively, RIC displayed excellent retinal penetration and prevented retinal degeneration in the pathogenesis of dry AMD with a high in vivo efficacy.
关键词： RPE protection,Receptor interacting protein kinase1 (RIPK1),Dry AMD,Topical application,Retinal degeneration,RIPK1-inhibitory compound (RIC)
更新于2025-09-23 15:21:01