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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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Temporal resolution of single photon responses in primate rod photoreceptors and limits imposed by cellular noise
摘要： Sensory receptor noise corrupts sensory signals, contributing to imperfect perception and dictating central processing strategies. For example, noise in rod phototransduction limits our ability to detect light and minimizing the impact of this noise requires precisely tuned nonlinear processing by the retina. But detection sensitivity is only one aspect of night vision: prompt and accurate behavior also requires that rods reliably encode the timing of photon arrivals. We show here that the temporal resolution of responses of primate rods is much finer than the duration of the light response and identify the key limiting sources of transduction noise. We also find that the thermal activation rate of rhodopsin is lower than previous estimates, implying that other noise sources are more important than previously appreciated. A model of rod single-photon responses reveals that the limiting noise relevant for behavior depends critically on how rod signals are pooled by downstream neurons.
关键词： thermal activation rate,primate rod photoreceptors,single photon responses,cellular noise,rhodopsin,temporal resolution
更新于2025-09-11 14:15:04
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Müller glia phagocytose dead photoreceptor cells in a mouse model of retinal degenerative disease
摘要： Retinitis pigmentosa is a devastating, blinding disorder that affects 1 in 4000 people worldwide. During the progression of the disorder, phagocytic clearance of dead photoreceptor cell bodies has a protective role by preventing additional retinal damage from accumulation of cellular debris. However, the cells responsible for the clearance remain unidentified. Taking advantage of a mouse model of retinitis pigmentosa (RhoP23H/P23H), we clarified the roles of M ¨uller glia in the phagocytosis of rod photoreceptor cells. During the early stage of retinal degeneration, M ¨uller glial cells participated in the phagocytosis of dying or dead rod photoreceptors throughout the outer nuclear layer. Nearly 50% of M ¨uller glia engaged in phagocytosis. Among the M ¨uller phagosomes, >90% matured into phagolysosomes. Those observations indicated that M ¨uller glial cells are the primary contributor to phagocytosis. In contrast, macrophages migrate to the inner part of the outer nuclear layer during photoreceptor degeneration, participating in the phagocytosis of a limited population of dying or dead photoreceptor cells. In healthy retinas of wild-type mice, M ¨uller glial cells phagocytosed cell bodies of dead rod photoreceptors albeit at a lower frequency. Taken together, the phagocytic function of M ¨uller glia is responsible for retinal homeostasis and reorganization under normal and pathologic conditions.
关键词： retinitis pigmentosa,phagocytosis,rod photoreceptors,rhodopsin
更新于2025-09-10 09:29:36
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Targeted deletion of an NRL- and CRX-regulated alternative promoter specifically silences FERM and PDZ domain containing 1 ( <i>Frmpd1</i> ) in rod photoreceptors
摘要： Regulation of cell type-specific gene expression is critical for generating neuronal diversity. Transcriptome analyses have unraveled extensive heterogeneity of transcribed sequences in retinal photoreceptors because of alternate splicing and/or promoter usage. Here we show that Frmpd1 (FERM and PDZ domain containing 1) is transcribed from an alternative promoter specifically in the retina. Electroporation of Frmpd1 promoter region, -505 to +382 bp, activated reporter gene expression in mouse retina in vivo. A proximal promoter sequence (-8 to +33 bp) of Frmpd1 binds to NRL and CRX, two rod-specific differentiation factors, and is necessary for activating reporter gene expression in vitro and in vivo. CRISPR/Cas9-mediated deletion of the genomic region including NRL and CRX binding sites in vivo completely eliminated Frmpd1 expression in rods and dramatically reduced expression in rod bipolar cells, thereby overcoming embryonic lethality caused by germline Frmpd1 deletion. Our studies demonstrate that a cell-type-specific regulatory control region is a credible target for creating loss-of-function alleles of widely-expressed genes.
关键词： rod photoreceptors,Frmpd1,NRL,CRX,alternative promoter,CRISPR/Cas9
更新于2025-09-10 09:29:36
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Endogenous Fluorophores Enable Two-Photon Imaging of the Primate Eye
摘要： Noninvasive two-photon imaging of a living mammalian eye can reveal details of molecular processes in the retina and RPE. Retinyl esters and all-trans-retinal condensation products are two types of retinoid fluorophores present in these tissues. We measured the content of these two types of retinoids in monkey and human eyes to validate the potential of two-photon imaging for monitoring retinoid changes in human eyes.
关键词： primate retina,retinoid cycle,two-photon microscopy,rod photoreceptors,cone photoreceptors
更新于2025-09-09 09:28:46
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R9AP Overexpression Alters Phototransduction Kinetics in iCre75 Mice
摘要： PURPOSE. Determine the impact of rod photoreceptor-specific expression of Cre recombinase on the kinetics of phototransduction in the mouse eye and identify changes in gene expression that underlie any observed phenotypic differences. METHODS. Transretinal ERG and single-cell suction electrode recordings were used to measure the kinetics of phototransduction in a mouse line exhibiting rod photoreceptor–specific Cre recombinase expression, and the results were compared with those from control non–Cre-expressing littermates. Gene expression changes were evaluated using RNA sequencing transcriptome analysis. The pattern of expression of Rgs9bp was determined by mapping sequencing reads to the mouse genome and performing 30-rapid amplification of cDNA ends (30-RACE). RESULTS. Expression of the rod-specific iCre75 transgene was accompanied by accelerated phototransduction inactivation, likely due to overexpression of the Rgs9bp gene, which encodes the Rgs9 anchor protein (R9AP). R9AP upregulation stabilized the RGS9 GAP complex, altering phototransduction kinetics. 30-Race identified an abundant, unexpected Rgs9bp-Prm1 fusion mRNA in Cre-expressing mouse retinas, which was determined to be derived from a second transgene present in the iCre75 line. CONCLUSIONS. Here we report the presence of a second, R9AP-expressing transgene in the iCre75 mouse line, leading to altered kinetics of phototransduction. These results highlight an important caveat that must be considered when utilizing this mouse line for rod photoreceptor–specific gene loss of function studies.
关键词： Cre recombinase,phototransduction,mouse,rod photoreceptors,retina,R9AP
更新于2025-09-09 09:28:46