- 标题
- 摘要
- 关键词
- 实验方案
- 产品
-
Conditional deletion of <i>Des1</i> in the mouse retina does not impair the visual cycle in cones
摘要： Cone photoreceptors are essential for vision under moderate to high illuminance and allow color discrimination. Their fast dark adaptation rate and resistance to saturation are believed to depend in part on an intra-retinal visual cycle that supplies 11-cis-retinaldehyde to cone opsins. Candidate enzymes of this pathway have been reported, but their physiologic contribution to cone photoresponses remains unknown. Here, we evaluate the role of a candidate retinol isomerase of this pathway, sphingolipid d4 desaturase 1 (Des1). Single-cell RNA sequencing analysis revealed Des1 expression not only in M ¨uller glia but also throughout the retina and in the retinal pigment epithelium. We assessed cone functional dependence on M ¨uller cell–expressed Des1 through a conditional knockout approach. Floxed Des1 mice, on a guanine nucleotide-binding protein subunit a transducin 1 knockout (Gnat1-/-) background to allow isolated recording of cone-driven photoresponses, were bred with platelet-derived growth factor receptor a (Pdgfra)-Cre mice to delete Des1 in M ¨uller cells. Conditional knockout of Des1 expression, as shown by tissue-selective Des1 gene recombination and reduced Des1 catalytic activity, caused no gross changes in the retinal structure and had no effect on cone sensitivity or dark adaptation but did slightly accelerate the rate of cone phototransduction termination. These results indicate that Des1 expression in M ¨uller cells is not required for cone visual pigment regeneration in the mouse.
关键词： Degs1,sphingolipid d(4) desaturase,retinoid cycle,photoreceptor,M ¨uller glia
更新于2025-09-23 15:22:29
-
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
-
Early Alteration of Retinal Neurons in <i>Aipl1</i> <sup>?/?</sup> Animals
摘要： PURPOSE. Mutations in the photoreceptor cell-specific gene encoding aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) lead to Leber congenital amaurosis (LCA4), retinitis pigmentosa, and cone–rod dystrophy. Gene therapy appears to be promising in the treatment for AIPL1-mediated vision loss in humans. Prior to initiating these treatments, however, it is crucial to understand how the retinal neurons remodel themselves in response to photoreceptor cell degeneration. In this study, using an animal model for AIPL1-LCA, Aipl1(cid:2)/(cid:2) mice, we investigate the changes in postreceptoral retinal neurons during the course of photoreceptor cell loss. METHODS. Morphology of the Aipl1(cid:2)/(cid:2) retina from postnatal day 8 to 150 was compared to that of age-matched, wild-type C57Bl6/J retina (WT) by immunocytochemistry using cell-specific markers. RESULTS. Expression of postsynaptic proteins in bipolar cells is reduced prior to photoreceptor cell degeneration at postnatal day 8. Bipolar and horizontal cells retract their dendrites. Cell bodies and axons of bipolar and horizontal cells are disorganized during the course of degeneration. M¨uller cell processes become hypertrophic and form a dense fibrotic layer outside the inner nuclear layer. CONCLUSIONS. An early defect in photoreceptor cells in the AIPL1-LCA mouse model affects the expression of postsynaptic markers, suggesting abnormal development of bipolar synapses. Once degeneration of photoreceptor cells is initiated, remodeling of retinal neurons in the Aipl1(cid:2)/(cid:2) animal is rapid.
关键词： childhood blindness,photoreceptor degeneration,retina,LCA,remodeling
更新于2025-09-23 15:22:29
-
Light-induced opening of the TRP channel in isolated membrane patches excised from photosensitive microvilli from Drosophila photoreceptors
摘要： Drosophila phototransduction occurs in light-sensitive microvilli arranged in a longitudinal structure of the photoreceptor, termed the rhabdomere. Rhodopsin, isomerized by light, couples to G-protein, which activates phospholipase C (PLC), which in turn cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) generating diacylglycerol (DAG), inositol trisphosphate and H+. This pathway opens the light-dependent channels, TRP and TRPL. PLC and TRP are held together in a protein assembly by the scaffold protein INAD. We report that the channels can be photoactivated in on-cell rhabdomeric patches and in excised patches by DAG. In excised patches, addition of PLC-activator, m-3M3FBS, or G-protein-activator, GTP-γ-S, opened TRP. These reagents were ineffective in PLC-mutant norpA and in the presence of PLC inhibitor U17322. However, DAG activated TRP even when PLC was pharmacologically or mutationally suppressed. These observations indicate that PLC, G-protein, and TRP were retained functional in these patches. DAG also activated TRP in the protein kinase C (PKC) mutant, inaC, excluding the possibility that PKC could mediate DAG-dependent TRP activation. Labeling diacylglycerol kinase (DGK) by fusion of fluorescent mCherry (mCherry-DGK) indicates that DGK, which returns DAG to dark levels, is highly expressed in the microvilli. In excised patches, TRP channels could be light-activated in the presence of GTP, which is required for G-protein activation. The evidence indicates that the proteins necessary for phototransduction are retained functionally after excision and that DAG is necessary and sufficient for TRP opening. This work opens up unique possibilities for studying, in sub-microscopic native membrane patches, the ubiquitous phosphoinositide signaling pathway and its regulatory mechanisms in unprecedented detail.
关键词： rhabdomere,photoreceptor,phototransduction,Drosophila,diacylglycerol,phospholipase C
更新于2025-09-23 15:21:21
-
Arl13b Interacts With Vangl2 to Regulate Cilia and Photoreceptor Outer Segment Length in Zebrafish
摘要： Mutations in the gene ARL13B cause the classical form of Joubert syndrome, an autosomal recessive ciliopathy with variable degrees of retinal degeneration. As second-site modifier alleles can contribute to retinal pathology in ciliopathies, animal models provide a unique platform to test how genetic interactions modulate specific phenotypes. In this study, we analyzed the zebrafish arl13b mutant for retinal degeneration and for epistatic relationships with the planar cell polarity protein (PCP) component vangl2. Photoreceptor and cilia structure was examined by light and electron microscopy. Immunohistochemistry was performed to examine ciliary markers. Genetic interactions were tested by pairwise crosses of heterozygous animals. Genetic mosaic animals were generated by blastula transplantation and analyzed by fluorescence microscopy. At 5 days after fertilization, photoreceptor outer segments were shorter in zebrafish arl13b?/? mutants compared to wild-type larvae, no overt signs of retinal degeneration were observed by light or electron microscopy. Starting at 14 days after fertilization (dpf) and continuing through 30 dpf, cells lacking Arl13b died following transplantation into wild-type host animals. Photoreceptors of arl13b?/?;vangl2?/? mutants were more compromised than the photoreceptors of single mutants. Finally, when grown within a wild-type retina, the vangl2?/? mutant cone photoreceptors displayed normal basal body positioning. We show that arl13b?/? mutants have shortened cilia and photoreceptor outer segments and exhibit a slow, progressive photoreceptor degeneration that occurs over weeks. The data suggest that loss of Arl13b leads to slow photoreceptor degeneration, but can be exacerbated by the loss of vangl2. Importantly, the data show that Arl13b can genetically and physically interact with Vangl2 and this association is important for normal photoreceptor structure. The loss of vangl2, however, does not affect basal body positioning.
关键词： Arl13b,planar polarity,photoreceptor,cilia,zebrafish,retina,Vangl2
更新于2025-09-23 15:21:21
-
Kif17 phosphorylation regulates photoreceptor outer segment turnover
摘要： Background: KIF17, a kinesin-2 motor that functions in intraflagellar transport, can regulate the onset of photoreceptor outer segment development. However, the function of KIF17 in a mature photoreceptor remains unclear. Additionally, the ciliary localization of KIF17 is regulated by a C-terminal consensus sequence (KRKK) that is immediately adjacent to a conserved residue (mouse S1029/zebrafish S815) previously shown to be phosphorylated by CaMKII. Yet, whether this phosphorylation can regulate the localization, and thus function, of KIF17 in ciliary photoreceptors remains unknown. Results: Using transgenic expression in zebrafish photoreceptors, we show that phospho-mimetic KIF17 has enhanced localization along the cone outer segment. Importantly, expression of phospho-mimetic KIF17 is associated with greatly enhanced turnover of the photoreceptor outer segment through disc shedding in a cell-autonomous manner, while genetic mutants of kif17 in zebrafish and mice have diminished disc shedding. Lastly, cone expression of constitutively active tCaMKII leads to a kif17-dependent increase in disc shedding. Conclusions: Taken together, our data support a model in which phosphorylation of KIF17 promotes its photoreceptor outer segment localization and disc shedding, a process essential for photoreceptor maintenance and homeostasis. While disc shedding has been predominantly studied in the context of the mechanisms underlying phagocytosis of outer segments by the retinal pigment epithelium, this work implicates photoreceptor-derived signaling in the underlying mechanisms of disc shedding.
关键词： Disc shedding,Retina,Phagocytosis,Photoreceptor,Cilia,Intraflagellar transport,Kinesin
更新于2025-09-23 15:21:01
-
Photoreceptor assessment in focal laser-treated central serous chorioretinopathy using adaptive optics and fundus autofluorescence
摘要： This study analyzed cone density, cone mosaic, and fundus autofluorescence (FAF) images in patients with focal laser-treated central serous chorioretinopathy (CSC). Observational case series. Forty-two eyes of 21 patients with unilateral treated CSC and bilateral best-corrected visual acuity of 1.0 (decimal fraction) were included. FAF and cone mosaic images were obtained in all patients with an adaptive optics fundus camera. Densities were recorded at 20 points throughout the macula, and choroidal thicknesses were measured. Mean choroidal thicknesses were 419.95 ± 110.33 mm in normal eyes, 459.09 ± 90.07 mm in eyes with active CSC, and 438.61 ± 107.57 mm in treated eyes. The highest density of cones in healthy eyes was 38146 cones/mm2, with a 5.66-mm intercellular space (IS), at 700 mm temporal to the center. In eyes with treated CSC, the highest density was 32749 cones/mm2, with a 6.13-mm IS, at 500 mm nasal to the center. In all quadrants, median values of maximum cone density were significantly higher in healthy eyes (P = .02, P = .003, P = .0001, and P = .001). Three types of lesions were identified on FAF and were correlated with those on cone mosaic images. Strong correlations were detected between the presence of hypoautofluorescent lesions on the first FAF image and a greater difference between maximum values of photoreceptor density (r2 = 0.46, P = .03), as well as between the presence of hypoautofluorescent lesions and the duration of pathology (r2 = 0.68, P < .001). The presence of hypoautofluorescent lesions and the duration of pathology were negative prognostic factors in CSC. Laser treatment could prevent photoreceptor loss.
关键词： photoreceptor density,cone mosaic,fundus autofluorescence,laser-treated central serous chorioretinopathy,adaptive optics
更新于2025-09-23 15:19:57
-
ARS2 is required for retinal progenitor cell S-phase progression and Müller glial cell fate specification.
摘要： During a developmental period that extends postnatally in the mouse, proliferating multipotent retinal progenitor cells produce one of seven major cell types (rod, cone, bipolar, horizontal, amacrine, ganglion, and Müller glial cells) as they exit the cell cycle in consecutive waves. Cell production in the retina is tightly regulated by intrinsic, extrinsic, spatial, and temporal cues and is coupled to the timing of cell cycle exit. ARS2 (also known as SRRT) is a component of the nuclear cap-binding complex involved in RNA Polymerase II transcription, and is required for cell cycle progression. We show that postnatal RPCs require ARS2 for proper progression through S phase, and ARS2 disruption leads to early exit from the cell cycle. Furthermore, we observe an increase in the proportion of cells expressing a rod photoreceptor marker, and a loss of Müller glia marker expression, indicating a role for ARS2 in regulating cell fate specification or differentiation. Knockdown of FLASH, which interacts with ARS2 and is required for cell cycle progression and 3’-end processing of replication-dependent histone transcripts, phenocopies ARS2 knockdown. These data implicate ARS2/FLASH-mediated histone mRNA processing in regulating RPC cell cycle kinetics and neuroglial cell fate specification during postnatal retinal development.
关键词： photoreceptor cells,Müller cells,retina,retinal progenitor cells,cell cycle,Ars2
更新于2025-09-19 17:15:36
-
Photoreceptor Fate Determination in the Vertebrate Retina
摘要： Photoreceptors are highly specialized primary sensory neurons that sense light and initiate vision. This critical role is well demonstrated by the fact that visual impairment accompanies photoreceptor loss or dysfunction in many human diseases. With the remarkable advances in stem cell research, one therapeutic approach is to use stem cells to generate photoreceptors and then engraft them into diseased eyes. Knowledge of the molecular mechanisms that control photoreceptor genesis during normal development can greatly aid in the production of photoreceptor cells for this approach. This article will discuss advances in our understanding of the molecular mechanisms that regulate photoreceptor fate determination during development. Recent lineage studies have shown that there are distinct retinal progenitor cells (RPCs) that produce specific combinations of daughter cell types, including photoreceptors and other types of retinal cells. Gene regulatory networks, in which transcription factors interact via cis-regulatory DNA elements, have been discovered that operate within distinct RPCs, and/or newly postmitotic cells, to direct the choice of photoreceptor fate.
关键词： retinal development,photoreceptor fate determination,transcription factors,gene regulatory network,cell lineages
更新于2025-09-19 17:15:36
-
Occurrence and mechanism of visual phosphenes in external photon beam radiation therapy and how to influence them
摘要： Background and purpose: Two plausible mechanisms to explain the appearance of visual phosphenes are: direct activation of the photochemicals in the retina and the generation of Cherenkov radiation in the vitreous humour. In this clinical trial we investigated the occurrence of visual phosphenes in external photon beam radiation therapy. Material and methods: Logistic regression analysis is used to examine whether seeing light flashes and seeing steady light depended on the ambient light intensity and the dose. Results: In total, 465 treatments of 25 patients were analysed. The odds of seeing light flashes multiply by 0,926 as the ambient light intensity increases by 10 lux. Similarly, the odds multiply by 1,604 as the dose to the retina increases by 10 cGy. The odds of seeing steady light multiply by 1,540 as the dose to the vitreous humour increases by 10 cGy. Conclusions: We postulate that one should reduce the dose rate, instruct patients to keep the eyes open and increase the illuminance in the treatment room to reduce the probability of experiencing visual phosphenes. We hypothesize that melanopsin is involved in the visual phosphenes and that fatigue of patients might be correlated with the observation of visual phosphenes.
关键词： Light flashes,Visual phosphenes,Photoreceptor proteins,Cherenkov radiation,Photon beam radiation therapy
更新于2025-09-19 17:15:36