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  • Histopathology and functional correlations in a patient with a mutation in RPE65, the gene for retinol isomerase. 21931134

    Here the authors describe the structural features of the retina and retinal pigment epithelium (RPE) in postmortem donor eyes of a 56-year-old patient with a homozygous missense RPE65 mutation (Ala132Thr) and correlate the pathology with the patient's visual function last measured at age 51.Eyes were enucleated within 13.5 hours after death. Representative areas from the macula and periphery were processed for light and electron microscopy. Immunofluorescence was used to localize the distribution of RPE65, rhodopsin, and cone arrestin. The autofluorescence in the RPE was compared with that of two normal eyes from age-similar donors.Histologic examination revealed the loss of rods and cones across most areas of the retina, attenuated retinal vessels, and RPE thinning in both eyes. A small number of highly disorganized cones were present in the macula that showed simultaneous labeling with cone arrestin and red/green or blue opsin. RPE65 immunoreactivity and RPE autofluorescence were reduced compared with control eyes in all areas studied. Rhodopsin labeling was observed in rods in the far periphery. The optic nerve showed a reduced number of axons.The clinical findings of reduced visual acuity, constricted fields, and reduced electroretinograms (ERGs) 5 years before death correlated with the small number of cones present in the macula and the extensive loss of photoreceptors in the periphery. The absence of autofluorescence in the RPE suggests that photoreceptor cells were probably missing across the retina for extended periods of time. Possible mechanisms that could lead to photoreceptor cell death are discussed.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Repair of rhodopsin mRNA by spliceosome-mediated RNA trans-splicing: a new approach for autosomal dominant retinitis pigmentosa. 25619725

    The promising clinical results obtained for ocular gene therapy in recent years have paved the way for gene supplementation to treat recessively inherited forms of retinal degeneration. The situation is more complex for dominant mutations, as the toxic mutant gene product must be removed. We used spliceosome-mediated RNA trans-splicing as a strategy for repairing the transcript of the rhodopsin gene, the gene most frequently mutated in autosomal dominant retinitis pigmentosa. We tested 17 different molecules targeting the pre-mRNA intron 1, by transient transfection of HEK-293T cells, with subsequent trans-splicing quantification at the transcript level. We found that the targeting of some parts of the intron promoted trans-splicing more efficiently than the targeting of other areas, and that trans-splicing rate could be increased by modifying the replacement sequence. We then developed cell lines stably expressing the rhodopsin gene, for the assessment of phenotypic criteria relevant to the pathogenesis of retinitis pigmentosa. Using this model, we showed that trans-splicing restored the correct localization of the protein to the plasma membrane. Finally, we tested our best candidate by AAV gene transfer in a mouse model of retinitis pigmentosa that expresses a mutant allele of the human rhodopsin gene, and demonstrated the feasibility of trans-splicing in vivo. This work paves the way for trans-splicing gene therapy to treat retinitis pigmentosa due to rhodopsin gene mutation and, more generally, for the treatment of genetic diseases with dominant transmission.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MABN15
    Nombre del producto:
    Anti-Rhodopsin Antibody, clone 4D2

  • Morphological alterations in retinal neurons in the S334ter-line3 transgenic rat. 20127257

    The S334ter-line-3 rat is a transgenic model of retinal degeneration developed to express a rhodopsin mutation similar to that found in human retinitis pigmentosa (RP) patients. Previous studies have focused on physiological changes in retinal cells and higher centers of the visual system with this model of retinal degeneration. However, little is known about the morphological changes in retinal cells during the development of the S334ter-line-3 rat. In order to understand and aid vision-rescue strategies, our aim has been to describe the retinal degeneration pattern in this model. We focus on changes in the morphologies of horizontal, bipolar, and amacrine cells in developing S334ter-line-3 rat retinas. Degeneration of photoreceptors begins in the central retina and progresses toward the periphery. In retinas at post-natal day 15 (P15), horizontal and rod bipolar cells show normal morphology. However, at P21, horizontal and rod bipolar cells exhibit abnormal processes at the outer plexiform layer, whereas the outer nuclear layer is significantly thinner. A glial reaction occurs concomitantly. In contrast, modifications in cone-bipolar and amacrine cells are much slower and do not occur until P90 and P180, respectively. The density of horizontal and rod-bipolar cells significantly drops after P60. Overall, the S334ter-line-3 model exhibits the hallmarks of cellular remodeling caused by photoreceptor degeneration. Its moderately fast time course makes the S334ter-line-3 a good model for studying vision-rescue strategies.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AB1778

  • Modeling and mutational analysis of a putative sodium-binding pocket on the dopamine D2 receptor. 11455025

    A homology model of the dopamine D2 receptor was constructed based on the crystal structure of rhodopsin. A putative sodium-binding pocket identified in an earlier model (PDB ) was revised. It is now defined by Asn-419 backbone oxygen at the apex of a pyramid and Asp-80, Ser-121, Asn-419, and Ser-420 at each vertex of the planar base. Asn-423 stabilizes this pocket through hydrogen bonds to two of these residues. Highly conserved Asn-52 is positioned near the sodium pocket, where it hydrogen-bonds with Asp-80 and the backbone carbonyl of Ser-420. Mutation of three of these residues, Asn-52 in helix 1, Ser-121 in helix 3, and Ser-420 in helix 7, profoundly altered the properties of the receptor. Mutants in which Asn-52 was replaced with Ala or Leu or Ser-121 was replaced with Leu exhibited no detectable binding of radioligands, although receptor immunoreactivity in the membrane was similar to that in cells expressing the wild-type D2L receptor. A mutant in which Asn-52 was replaced with Gln, preserving hydrogen-bonding capability, was similar to D2L in affinity for ligands and ability to inhibit cAMP accumulation. Mutants in which either Ser-121 or Ser-420 was replaced with Ala or Asn had decreased affinity for agonists (Ser-121), but increased affinity for the antagonists haloperidol and clozapine. Interestingly, the affinity of these Ser-121 and Ser-420 mutants for substituted benzamide antagonists showed little or no dependence on sodium, consistent with our hypothesis that Ser-121 and Ser-420 contribute to the formation of a sodium-binding pocket.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AB5084P
    Nombre del producto:
    Anti-Dopamine D2 Receptor Antibody

  • Nanoparticle-mediated rhodopsin cDNA but not intron-containing DNA delivery causes transgene silencing in a rhodopsin knockout model. 26564956

    Previously, we compared the efficacy of nanoparticle (NP)-mediated intron-containing rhodopsin (sgRho) vs. intronless cDNA in ameliorating retinal disease phenotypes in a rhodopsin knockout (RKO) mouse model of retinitis pigmentosa. We showed that NP-mediated sgRho delivery achieved long-term expression and phenotypic improvement in RKO mice, but not NP housing cDNA. However, the protein level of the NP-sgRho construct was only 5-10% of wild-type at 8 mo postinjection. To have a better understanding of the reduced levels of long-term expression of the vectors, in the present study, we evaluated the epigenetic changes of subretinal delivering NP-cDNA vs. NP-sgRho in the RKO mouse eyes. Following the administration, DNA methylation and histone status of specific regions (bacteria plasmid backbone, promoter, rhodopsin gene, and scaffold/matrix attachment region) of the vectors were evaluated at various time points. We documented that epigenetic transgene silencing occurred in vector-mediated gene transfer, which were caused by the plasmid backbone and the cDNA of the transgene, but not the intron-containing transgene. No toxicity or inflammation was found in the treated eyes. Our results suggest that cDNA of the rhodopsin transgene and bacteria backbone interfered with the host defense mechanism of DNA methylation-mediated transgene silencing through heterochromatin-associated modifications.
    Tipo de documento:
    Referencia
    Referencia del producto:
    17-371
    Nombre del producto:
    EZ-ChIP™

  • The G-protein-coupled receptor kinase GRK4 mediates homologous desensitization of metabotropic glutamate receptor 1. 11099476

    G-protein-coupled receptor kinases (GRKs) are involved in the regulation of many G-protein-coupled receptors. As opposed to the other GRKs, such as rhodopsin kinase (GRK1) or beta-adrenergic receptor kinase (beta ARK, GRK2), no receptor substrate for GRK4 has been so far identified. Here we show that GRK4 is expressed in cerebellar Purkinje cells, where it regulates mGlu(1) metabotropic glutamate receptors, as indicated by the following: 1) When coexpressed in heterologous cells (HEK293), mGlu(1) receptor signaling was desensitized by GRK4 in an agonist-dependent manner (homologous desensitization). 2) In transfected HEK293 and in cultured Purkinje cells, the exposure to glutamate agonists induced internalization of the receptor and redistribution of GRK4. There was a substantial colocalization of the receptor and kinase both under basal condition and after internalization. 3) Kinase activity was necessary for desensitizing mGlu(1a) receptor and agonist-dependent phosphorylation of this receptor was also documented. 4) Antisense treatment of cultured Purkinje cells, which significantly reduced the levels of GRK4 expression, induced a marked modification of the mGlu(1)-mediated functional response, consistent with an impaired receptor desensitization. The critical role for GRK4 in regulating mGlu(1) receptors implicates a major involvement of this kinase in the physiology of Purkinje cell and in motor learning.
    Tipo de documento:
    Referencia
    Referencia del producto:
    07-617
    Nombre del producto:
    Anti-mGluR1 Antibody

  • Functional and anatomical remodeling in human retinal detachment. 22406310

    Rhegmatogenous retinal detachment is by far the most common indication for retinal surgery and a major cause of severe vision loss. Increased levels of glutamate found in the vitreous of human patients and persistent remodeling, even after reattachment, suggest substantial neurochemical, functional and anatomical changes have occurred in the detached retina. Therefore, this study was designed to characterize the morphological changes and glutamate receptor functionality in human rhegmatogenous retinal detachment. A cation channel permeating probe, agmatine (1-amino-4-guanidobutane; AGB), was employed to track endogenous and kainate (KA) driven channel functionality combined with immunocytochemical characterization of cellular remodeling. In the detached retina increased AGB permeability was identified in the outer retina while there was a decrease in the inner retina in basal conditions. KA receptors exhibited increased AGB permeability in ON bipolar cells and decreased permeability in calbindin labeled inner retinal cells. All retinal detachment samples demonstrated ectopic synaptic protein expression, photoreceptor processes extending toward the inner retina, and other remodeling features of retinal degeneration. These anatomical changes have been demonstrated in animal studies and are novel features unreported in primary cases of human retinal detachment. We conclude that deafferentation in retinal detachment leads to alteration of the glutamatergic pathway.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB5356
    Nombre del producto:
    Anti-Rhodopsin Antibody, CT, last 9 amino acids, clone Rho 1D4

  • X-Ray diffraction analysis of three-dimensional crystals of bovine rhodopsin obtained from mixed micelles. 10806093

    Rhodopsin, a prototypic G protein-coupled receptor responsible for absorption of photons in retinal rod photoreceptor cells, was selectively extracted from bovine rod outer segment membranes, employing mixed micelles of nonyl beta-d-glucoside and heptanetriol. Highly purified rhodopsin was crystallized from solutions containing varying amounts of detergent and amphiphile. The crystals contained ground state rhodopsin molecules as judged by their red color and the linear dichroism originating from the 11-cis-retinal chromophore. However, when exposed to visible light, even at 4 degrees C, rhodopsin was bleached and the crystals decomposed. Reflections in the diffraction pattern were observed out to 3.5-A resolution at 100 K for the most ordered crystals. Diffraction data have been processed to 3.85-A resolution. The symmetry of the diffraction pattern and the systematic absences indicate that the crystals have tetragonal symmetry, space group P4(1)22 or P4(3)22, a = b = 96.51 A, c = 148.55 A. A value of 4.12 A(3)/Da for V(M) was obtained for one monomer in the asymmetric unit (eight molecules per unit cell). Our study is the first characterization of a three-dimensional crystal of a G protein-coupled receptor and may be valuable for future structural studies on related receptors of this important superfamily.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB2236

  • Immunochemical or fluorescent labeling of vesicular subcellular fractions for microscopy imaging. 20232463

    We describe a procedure for the labeling of membranous vesicular purified subcellular fractions, to image them, typically by confocal laser scanning microscopy. Being intracellular organelles, these fractions, once purified cannot be attached to glass slides as for cells. Fractions are labeled in batch without prior embedding or freezing. Each labeling step performed by passages of resuspension/centrifugation is followed by washings. Then samples are dispersed on the glass slides. Mammalian retinal rod outer segment disks, intact brain stem myelin vesicles, and brain synaptosomes were chosen, as these subcellular fractions can be purified by well established procedures. These fractions were immunolabeled with specific antibodies. Moreover, by the earlier procedure, we show that the mitochondrial vital membrane potential probe MitoTracker Deep Red 633 stains myelin vesicles and rod disks before fixation, consistently with our previous reports of a respiring capacity of these membranes. Therefore, the technique seems adequate to become an instrument to study the structure and the function of these and other subcellular fractions.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB5356
    Nombre del producto:
    Anti-Rhodopsin Antibody, CT, last 9 amino acids, clone Rho 1D4

  • An activated unfolded protein response promotes retinal degeneration and triggers an inflammatory response in the mouse retina. 25522272

    Recent studies on the endoplasmic reticulum stress have shown that the unfolded protein response (UPR) is involved in the pathogenesis of inherited retinal degeneration caused by mutant rhodopsin. However, the main question of whether UPR activation actually triggers retinal degeneration remains to be addressed. Thus, in this study, we created a mouse model for retinal degeneration caused by a persistently activated UPR to assess the physiological and morphological parameters associated with this disease state and to highlight a potential mechanism by which the UPR can promote retinal degeneration. We performed an intraocular injection in C57BL6 mice with a known unfolded protein response (UPR) inducer, tunicamycin (Tn) and examined animals by electroretinography (ERG), spectral domain optical coherence tomography (SD-OCT) and histological analyses. We detected a significant loss of photoreceptor function (over 60%) and retinal structure (35%) 30 days post treatment. Analysis of retinal protein extracts demonstrated a significant upregulation of inflammatory markers including interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1) and IBA1. Similarly, we detected a strong inflammatory response in mice expressing either Ter349Glu or T17M rhodopsin (RHO). These mutant rhodopsin species induce severe retinal degeneration and T17M rhodopsin elicits UPR activation when expressed in mice. RNA and protein analysis revealed a significant upregulation of pro- and anti-inflammatory markers such as IL-1β, IL-6, p65 nuclear factor kappa B (NF-kB) and MCP-1, as well as activation of F4/80 and IBA1 microglial markers in both the retinas expressing mutant rhodopsins. We then assessed if the Tn-induced inflammatory marker IL-1β was capable of inducing retinal degeneration by injecting C57BL6 mice with a recombinant IL-1β. We observed ~19% reduction in ERG a-wave amplitudes and a 29% loss of photoreceptor cells compared with control retinas, suggesting a potential link between pro-inflammatory cytokines and retinal pathophysiological effects. Our work demonstrates that in the context of an established animal model for ocular disease, the persistent activation of the UPR could be responsible for promoting retinal degeneration via the UPR-induced pro-inflammatory cytokine IL-1β.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo