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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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Mutation of <i>wrb</i> , a Component of the Guided Entry of Tail-Anchored Protein Pathway, Disrupts Photoreceptor Synapse Structure and Function
摘要： PURPOSE. Tail-anchored (TA) proteins contain a single hydrophobic domain at the C-terminus and are posttranslationally inserted into the ER membrane via the GET (guided entry of tail-anchored proteins) pathway. The role of the GET pathway in photoreceptors is unexplored. The goal of this study was to characterize the zebra?sh pinball wizard mutant, which disrupts Wrb, a core component of the GET pathway. METHODS. Electroretinography, optokinetic response measurements (OKR), immunohistochemistry, and electron microscopy analyses were employed to assess ribbon synapse function, protein expression, and ultrastructure in 5-day-old zebra?sh larvae. Expression of wrb was investigated with real-time qRT-PCR and in situ hybridization. RESULTS. Mutation of wrb abolished the OKR and greatly diminished the ERG b-wave, but not the a-wave. Ribeye and SV2 were partially mislocalized in both photoreceptors and hair cells of wrb mutants. Fewer contacts were seen between photoreceptors and bipolar cells in wrb(cid:2)/(cid:2) mutants. Expression of wrb was observed throughout the nervous system and Wrb localized to the ER and synaptic region of photoreceptors. Morpholino knockdown of the cytosolic ATPase trc40, which targets TA proteins to the ER, also diminished the OKR. Overexpression of wrb fully restored contrast sensitivity in mutants, while overexpression of mutant wrbR73A, which cannot bind Trc40, did not. CONCLUSIONS. Proteins Wrb and Trc40 are required for synaptic transmission between photoreceptors and bipolar cells, indicating that TA protein insertion by the TRC pathway is a critical step in ribbon synapse assembly and function.
关键词： photoreceptors,zebra?sh,optokinetic response,ribbon synapse
更新于2025-09-10 09:29:36
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Inner Segment Remodeling and Mitochondrial Translocation in Cone Photoreceptors in Age-Related Macular Degeneration With Outer Retinal Tubulation
摘要： PURPOSE. To quantify impressions of mitochondrial translocation in degenerating cones and to determine the nature of accumulated material in the subretinal space with apparent inner segment (IS)-like features by examining cone IS ultrastructure. METHODS. Human donor eyes with advanced age-related macular degeneration (AMD) were screened for outer retinal tubulation (ORT) in macula-wide, high-resolution digital sections. Degenerating cones inside ORT (ORT cones) and outside ORT (non-ORT cones) from AMD eyes and unaffected cones in age-matched control eyes were imaged using transmission electron microscopy. The distances of mitochondria to the external limiting membrane (ELM), cone IS length, and cone IS width at the ELM were measured. RESULTS. Outer retinal tubulation and non-ORT cones lose outer segments (OS), followed by shortening of IS and mitochondria. In non-ORT cones, IS broaden. Outer retinal tubulation and non-ORT cone IS myoids become undetectable due to mitochondria redistribution toward the nucleus. Some ORT cones were found lacking IS and containing mitochondria in the outer fiber (between soma and ELM). Unlike long, thin IS mitochondria in control cones, ORT and non-ORT IS mitochondria are ovoid or reniform. Shed IS, some containing mitochondria, were found in the subretinal space. CONCLUSIONS. In AMD, macula cones exhibit loss of detectable myoid due to IS shortening in addition to OS loss, as described. Mitochondria shrink and translocate toward the nucleus. As reflectivity sources, translocating mitochondria may be detectable using in vivo imaging to monitor photoreceptor degeneration in retinal disorders. These results improve the knowledge basis for interpreting high-resolution clinical retinal imaging.
关键词： ellipsoid,outer retinal tubulation,age-related macular degeneration,histology,photoreceptors,cones,myoid,transmission electron microscopy,Müller cells
更新于2025-09-10 09:29:36
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Functional optical coherence tomography of retinal photoreceptors
摘要： Age-related macular degeneration (AMD) is the leading cause of severe vision loss and legal blindness. It is known that retinal photoreceptors are the primary target of AMD. Therefore, a reliable method for objective assessment of photoreceptor function is needed for early detection and reliable treatment evaluation of AMD and other eye diseases such as retinitis pigmentosa that are known to cause photoreceptor dysfunctions. Stimulus-evoked intrinsic optical signal (IOS) changes promise a unique opportunity for objective assessment of physiological function of retinal photoreceptor and inner neurons. Instead of a comprehensive review, this mini-review is to provide a brief summary of our recent in vitro and in vivo optical coherence tomography (OCT) studies of stimulus-evoked IOS changes in animal retinas. By providing excellent axial resolution to differentiate individual retinal layers, depth-resolved OCT revealed rapid IOS response at the photoreceptor outer segment. The fast photoreceptor-IOS occurred almost right away ((cid:1) 2 ms) after the onset of retinal stimulation, differentiating itself from slow IOS changes correlated with inner neural and hemodynamic changes. Further development of the functional IOS instruments and retinal stimulation protocols may provide a feasible solution to pursue clinical application of functional IOS imaging for objective assessment of human photoreceptors.
关键词： intrinsic optical signal,photoreceptors,Optical coherence tomography,age-related macular degeneration
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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The Negative Cone Mosaic: A New Manifestation of the Optical Stiles-Crawford Effect in Normal Eyes
摘要： The purpose of this study was to describe a previously unreported manifestation of the optical Stiles-Crawford effect (oSCE) in normal eyes. In a cohort of 50 normal subjects, the directional reflectance of cones in the retinal periphery was explored by flood-illuminated adaptive optics (FIAO) and optical coherence tomography (OCT). In 32 eyes (64%), off-axis FIAO images of the retinal periphery (~15–208 from the fovea) showed variably sized patches of hyporeflective dots (called here negative mosaic) coexisting with hyperreflective (positive) cones. In nine cases, shifting the entry pupil toward the optical axis restored the positive cone mosaic, with a point-by-point correspondence between positive and negative mosaics. Rods remained hyperreflective around negative and positive cones. These changes were paralleled by changes of the OCT reflectance of the cone outer segment tips and, to a lesser extent, of the inner/outer segment limit. By en face FIAO imaging of the retina, the contrast of cones over rods may be strongly dependent on the entry pupil to such an extent that their reflectance is lower than that of rods. We hypothesized that the negative cone mosaic aspect results from the differential Stiles-Crawford effect of cones and rods. Cone reflectance by en face FIAO parallels the reflectance from the cone outer segment tip line and to a lesser extent of the inner/outer segment limit by OCT. Taking this into account, the oSCE is of importance for the interpretation of high-resolution images of photoreceptors.
关键词： photoreceptors,adaptive optics,optical coherence tomography,optical Stiles-Crawford effect
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
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Human Retinal Pigment Epithelium: In Vivo Cell Morphometry, Multispectral Autofluorescence, and Relationship to Cone Mosaic
摘要： PURPOSE. To characterize in vivo morphometry and multispectral autofluorescence of the retinal pigment epithelial (RPE) cell mosaic and its relationship to cone cell topography across the macula. METHODS. RPE cell morphometrics were computed in regularly spaced regions of interest (ROIs) from contiguous short-wavelength autofluorescence (SWAF) and photoreceptor reflectance images collected across the macula in one eye of 10 normal participants (23–65 years) by using adaptive optics scanning light ophthalmoscopy (AOSLO). Infrared autofluorescence (IRAF) images of the RPE were collected with AOSLO in seven normal participants (22–65 years), with participant overlap, and compared to SWAF quantitatively and qualitatively. RESULTS. RPE cell statistics could be analyzed in 84% of SWAF ROIs. RPE cell density consistently decreased with eccentricity from the fovea (participant mean 6 SD: 6026 6 1590 cells/mm2 at fovea; 4552 6 1370 cells/mm2 and 3757 6 1290 cells/mm2 at 3.5 mm temporally and nasally, respectively). Mean cone-to-RPE cell ratio decreased rapidly from 16.6 at the foveal center to <5 by 1 mm. IRAF revealed cells in six of seven participants, in agreement with SWAF RPE cell size and location. Differences in cell fluorescent structure, contrast, and visibility beneath vasculature were observed between modalities. CONCLUSIONS. Improvements in AOSLO autofluorescence imaging permit efficient visualization of RPE cells with safe light exposures, allowing individual characterization of RPE cell morphometry that is variable between participants. The normative dataset and analysis of RPE cell IRAF and SWAF herein are essential for understanding microscopic characteristics of cell fluorescence and may assist in interpreting disease progression in RPE cells.
关键词： autofluorescence,retinal imaging,photoreceptors,retinal pigment epithelium,adaptive optics
更新于2025-09-09 09:28:46
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The Effect of Transient Local Anti-inflammatory Treatment on the Survival of Pig Retinal Progenitor Cell Allotransplants
摘要： The development of photoreceptor degenerative disorders requires the identification of the optimal cell source and immunosuppressive regimen in a large animal model. Allotransplants are not acutely rejected in swine subretinal space, although it is not known if survival can be improved with immunosuppression. Here we investigated the survival and integration of expanded pig retinal progenitor cells (pRPCs) in normal recipients with and without transient anti-inflammatory suppression. pRPCs were derived from the neural retina of E60 GFP transgenic pigs, expanded for six passages, characterized, and transplanted into the subretinal space of 12 pigs. Six recipients received a single intravitreal injection of rapamycin and dexamethasone. pRPCs expressed the photoreceptor development genes Sox2, Pax6, Lhx2, Crx, Nrl, and Recoverin in vitro. Transplanted cells were identified in 9 out of 12 recipients 4 weeks after the injection. pRPCs integrated primarily into the photoreceptor inner segment layer and outer nuclear layer with single cells present in the inner nuclear layer. Donor cells remained recoverin-positive and acquired rhodopsin. We did not observe any signs of graft proliferation. The immunosuppression did not affect the survival or distribution of grafts. No macrophage infiltration or loss of retinal structure was observed in either group. Local immunosuppression with rapamycin and dexamethasone does not improve the outcome of pRPC allotransplantation into the subretinal space. Survival and integration of pRPC together with the lack of graft proliferation suggests that allogeneic RPC transplantation without transient immunosuppression is a favorable approach for photoreceptor cell replacement.
关键词： rapamycin,retina,retinal progenitor cells,cell therapy,photoreceptors
更新于2025-09-04 15:30:14