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Anand Swaroop - One of the best experts on this subject based on the ideXlab platform.

  • retinal degeneration associated with rpgrip1 a review of natural history mutation spectrum and genotype phenotype correlation in 228 patients
    Frontiers in Cell and Developmental Biology, 2021
    Co-Authors: Avigail Beryozkin, Anand Swaroop, Frans P M Cremers, Hamzah Aweidah, Roque Daniel Carrero Valenzuela, Myriam Berman, Oscar Iguzquiza, Muhammad Imran Khan, Radgonde Amer, Samer Khateb
    Abstract:

    Purpose:RPGRIP1 encodes a ciliary protein expressed in the Photoreceptor Connecting Cilium. Mutations in this gene cause ∼5% of Leber congenital amaurosis (LCA) worldwide, but are also associated with cone-rod dystrophy (CRD) and retinitis pigmentosa (RP) phenotypes. Our purpose was to clinically characterize RPGRIP1 patients from our cohort, collect clinical data of additional RPGRIP1 patients reported previously in the literature, identify common clinical features, and seek genotype-phenotype correlations. Methods: Clinical data were collected from 16 patients of our cohort and 212 previously reported RPGRIP1 patients and included (when available) family history, best corrected visual acuity (BCVA), refraction, comprehensive ocular examination, optical coherence tomography (OCT) imaging, visual fields (VF), and full-field electroretinography (ffERG). Results: Out of 228 patients, the majority (197, 86%) were diagnosed with LCA, 18 (7%) with RP, and 13 (5%) with CRD. Age of onset was during early childhood (n = 133, average of 1.7 years). All patients but 6 had moderate hyperopia (n = 59, mean of 4.8D), and average BCVA was 0.06 Snellen (n = 124; only 10 patients had visual acuity [VA] > 0.10 Snellen). On funduscopy, narrowing of blood vessels was noted early in life. Most patients had mild bone spicule-like pigmentation starting in the midperiphery and later encroaching upon the posterior pole. OCT showed thinning of the outer nuclear layer (ONL), while cystoid changes and edema were relatively rare. VF were usually very constricted from early on. ffERG responses were non-detectable in the vast majority of cases. Most of the mutations are predicted to be null (363 alleles), and 93 alleles harbored missense mutations. Missense mutations were identified only in two regions: the RPGR-interacting domain and the C2 domains. Biallelic null mutations are mostly associated with a severe form of the disease, whereas biallelic missense mutations usually cause a milder disease (mostly CRD). Conclusion: Our results indicate that RPGRIP1 biallelic mutations usually cause severe retinal degeneration at an early age with a cone-rod pattern. However, most of the patients exhibit preservation of some (usually low) BCVA for a long period and can potentially benefit from gene therapy. Missense changes appear only in the conserved domains and are associated with a milder phenotype.

  • rpgr orf15 which is mutated in retinitis pigmentosa associates with smc1 smc3 and microtubule transport proteins
    Journal of Biological Chemistry, 2005
    Co-Authors: Hemant Khanna, Concepcion Lillo, David S. Williams, Toby W Hurd, Sunil K Parapuram, Shirley He, Masayuki Akimoto, Alan F Wright, Ben Margolis, Anand Swaroop
    Abstract:

    Abstract Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene account for almost 20% of patients with retinitis pigmentosa. Most mutations are detected in alternatively spliced RPGR-ORF15 isoform(s), which are primarily but not exclusively expressed in the retina. We show that, in addition to the axoneme, the RPGR-ORF15 protein is localized to the basal bodies of Photoreceptor Connecting Cilium and to the tip and axoneme of sperm flagella. Mass spectrometric analysis of proteins that were immunoprecipitated from the retinal axoneme-enriched fraction using an anti-ORF15 antibody identified two chromosome-associated proteins, structural maintenance of chromosomes (SMC) 1 and SMC3. Using pulldown assays, we demonstrate that the interaction of RPGR with SMC1 and SMC3 is mediated, at least in part, by the RCC1-like domain of RPGR. This interaction was not observed with phosphorylation-deficient mutants of SMC1. Both SMC1 and SMC3 localized to the cilia of retinal Photoreceptors and Madin-Darby canine kidney cells, suggesting a broader physiological relevance of this interaction. Additional immunoprecipitation studies revealed the association of RPGR-ORF15 isoform(s) with the intraflagellar transport polypeptide IFT88 as well as microtubule motor proteins, including KIF3A, p150Glued, and p50-dynamitin. Inhibition of dynein function by overexpressing p50 abrogated the localization of RPGR-ORF15 to basal bodies. Taken together, these results provide novel evidence for the possible involvement of RPGR-ORF15 in microtubule organization and regulation of transport in primary cilia.

  • rpgr orf15 which is mutated in retinitis pigmentosa associates with smc1 smc3 and microtubule
    2005
    Co-Authors: Hemant Khanna, Concepcion Lillo, David S. Williams, Toby W Hurd, Sunil K Parapuram, Masayuki Akimoto, Alan F Wright, Ben Margolis, Xinhua Shu, Anand Swaroop
    Abstract:

    Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene account for almost 20% of patients with retinitis pigmentosa. Most mutations are detected in alternatively spliced RPGR-ORF15 isoform(s), which are primarily but not exclusively expressed in the retina. We show that, in addition to the axoneme, the RPGR-ORF15 protein is localized to the basal bodies of Photoreceptor Connecting Cilium and to the tip and axoneme of sperm flagella. Mass spectrometric analysis of proteins that were immunoprecipitated from the retinal axoneme-enriched fraction using an anti-ORF15 antibody identified two chromosome-associated proteins, structural maintenance of chromosomes (SMC) 1 and SMC3. Using pulldown assays, we demonstrate that the interaction of RPGR with SMC1 and SMC3 is mediated, at least in part, by the RCC1-like domain of RPGR. This interaction was not observed with phosphorylation-deficient mutants of SMC1. Both SMC1 and SMC3 localized to the cilia of retinal Photoreceptors and Madin-Darby canine kidney cells, suggesting a broader physiological relevance of this interaction. Additional immunoprecipitation studies revealed the association of RPGR-ORF15 isoform(s) with the intraflagellar transport polypeptide IFT88 as well as microtubule motor proteins, including KIF3A, p150 Glued , and p50-dynamitin. Inhibition of dynein function by overexpressing p50 abrogated the localization of RPGR-ORF15 to basal bodies. Taken together, these results provide novel evidence for the possible involvement of RPGR-ORF15 in microtubule organization and regulation of transport in primary cilia.

Hemant Khanna - One of the best experts on this subject based on the ideXlab platform.

  • gelsolin dysfunction causes Photoreceptor loss in induced pluripotent cell and animal retinitis pigmentosa models
    Nature Communications, 2017
    Co-Authors: Roly Megaw, Hemant Khanna, Hashem Abuarafeh, Melissa Jungnickel, Carla B Mellough, Christine B Gurniak, Walter Witke, Wei Zhang, Pleasantine Mill, Baljean Dhillon
    Abstract:

    Mutations in the Retinitis Pigmentosa GTPase Regulator (RPGR) cause X-linked RP (XLRP), an untreatable, inherited retinal dystrophy that leads to premature blindness. RPGR localises to the Photoreceptor Connecting Cilium where its function remains unknown. Here we show, using murine and human induced pluripotent stem cell models, that RPGR interacts with and activates the actin-severing protein gelsolin, and that gelsolin regulates actin disassembly in the Connecting Cilium, thus facilitating rhodopsin transport to Photoreceptor outer segments. Disease-causing RPGR mutations perturb this RPGR-gelsolin interaction, compromising gelsolin activation. Both RPGR and Gelsolin knockout mice show abnormalities of actin polymerisation and mislocalisation of rhodopsin in Photoreceptors. These findings reveal a clinically-significant role for RPGR in the activation of gelsolin, without which abnormalities in actin polymerisation in the Photoreceptor Connecting cilia cause rhodopsin mislocalisation and eventual retinal degeneration in XLRP. Mutations in the Retinitis Pigmentosa GTPase Regulator (RPGR) cause retinal dystrophy, but how this arises at a molecular level is unclear. Here, the authors show in induced pluripotent stem cells and mouse knockouts that RPGR mediates actin dynamics in Photoreceptors via the actin-severing protein, gelsolin.

  • rpgr orf15 which is mutated in retinitis pigmentosa associates with smc1 smc3 and microtubule transport proteins
    Journal of Biological Chemistry, 2005
    Co-Authors: Hemant Khanna, Concepcion Lillo, David S. Williams, Toby W Hurd, Sunil K Parapuram, Shirley He, Masayuki Akimoto, Alan F Wright, Ben Margolis, Anand Swaroop
    Abstract:

    Abstract Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene account for almost 20% of patients with retinitis pigmentosa. Most mutations are detected in alternatively spliced RPGR-ORF15 isoform(s), which are primarily but not exclusively expressed in the retina. We show that, in addition to the axoneme, the RPGR-ORF15 protein is localized to the basal bodies of Photoreceptor Connecting Cilium and to the tip and axoneme of sperm flagella. Mass spectrometric analysis of proteins that were immunoprecipitated from the retinal axoneme-enriched fraction using an anti-ORF15 antibody identified two chromosome-associated proteins, structural maintenance of chromosomes (SMC) 1 and SMC3. Using pulldown assays, we demonstrate that the interaction of RPGR with SMC1 and SMC3 is mediated, at least in part, by the RCC1-like domain of RPGR. This interaction was not observed with phosphorylation-deficient mutants of SMC1. Both SMC1 and SMC3 localized to the cilia of retinal Photoreceptors and Madin-Darby canine kidney cells, suggesting a broader physiological relevance of this interaction. Additional immunoprecipitation studies revealed the association of RPGR-ORF15 isoform(s) with the intraflagellar transport polypeptide IFT88 as well as microtubule motor proteins, including KIF3A, p150Glued, and p50-dynamitin. Inhibition of dynein function by overexpressing p50 abrogated the localization of RPGR-ORF15 to basal bodies. Taken together, these results provide novel evidence for the possible involvement of RPGR-ORF15 in microtubule organization and regulation of transport in primary cilia.

  • rpgr orf15 which is mutated in retinitis pigmentosa associates with smc1 smc3 and microtubule
    2005
    Co-Authors: Hemant Khanna, Concepcion Lillo, David S. Williams, Toby W Hurd, Sunil K Parapuram, Masayuki Akimoto, Alan F Wright, Ben Margolis, Xinhua Shu, Anand Swaroop
    Abstract:

    Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene account for almost 20% of patients with retinitis pigmentosa. Most mutations are detected in alternatively spliced RPGR-ORF15 isoform(s), which are primarily but not exclusively expressed in the retina. We show that, in addition to the axoneme, the RPGR-ORF15 protein is localized to the basal bodies of Photoreceptor Connecting Cilium and to the tip and axoneme of sperm flagella. Mass spectrometric analysis of proteins that were immunoprecipitated from the retinal axoneme-enriched fraction using an anti-ORF15 antibody identified two chromosome-associated proteins, structural maintenance of chromosomes (SMC) 1 and SMC3. Using pulldown assays, we demonstrate that the interaction of RPGR with SMC1 and SMC3 is mediated, at least in part, by the RCC1-like domain of RPGR. This interaction was not observed with phosphorylation-deficient mutants of SMC1. Both SMC1 and SMC3 localized to the cilia of retinal Photoreceptors and Madin-Darby canine kidney cells, suggesting a broader physiological relevance of this interaction. Additional immunoprecipitation studies revealed the association of RPGR-ORF15 isoform(s) with the intraflagellar transport polypeptide IFT88 as well as microtubule motor proteins, including KIF3A, p150 Glued , and p50-dynamitin. Inhibition of dynein function by overexpressing p50 abrogated the localization of RPGR-ORF15 to basal bodies. Taken together, these results provide novel evidence for the possible involvement of RPGR-ORF15 in microtubule organization and regulation of transport in primary cilia.

Michael Adamian - One of the best experts on this subject based on the ideXlab platform.

  • replacement gene therapy with a human rpgrip1 sequence slows Photoreceptor degeneration in a murine model of leber congenital amaurosis
    Human Gene Therapy, 2010
    Co-Authors: Basil S Pawlyk, Eliot L. Berson, O V Bulgakov, Xinyu Liu, Michael Adamian, Xun Sun, Shahrokh C Khani, Michael A Sandberg
    Abstract:

    RPGR-interacting protein-1 (RPGRIP1) is localized in the Photoreceptor-Connecting Cilium, where it anchors the RPGR (retinitis pigmentosa GTPase regulator) protein, and its function is essential for Photoreceptor maintenance. Genetic defect in RPGRIP1 is a known cause of Leber congenital amaurosis (LCA), a severe, early-onset form of retinal degeneration. We evaluated the efficacy of replacement gene therapy in a murine model of LCA carrying a targeted disruption of RPGRIP1. The replacement construct, packaged in an adeno-associated virus serotype 8 (AAV8) vector, used a rhodopsin kinase gene promoter to drive RPGRIP1 expression. Both promoter and transgene were of human origin. After subretinal delivery of the replacement gene in the mutant mice, human RPGRIP1 was expressed specifically in Photoreceptors, localized correctly in the Connecting cilia, and restored the normal localization of RPGR. Electroretinogram and histological examinations showed better preservation of rod and cone Photoreceptor function and improved Photoreceptor survival in the treated eyes. This study demonstrates the efficacy of human gene replacement therapy and validates a gene therapy design for future clinical trials in patients afflicted with this condition. Our results also have therapeutic implications for other forms of retinal degenerations attributable to a ciliary defect.

  • retinitis pigmentosa gtpase regulator rpgr interacting protein is stably associated with the Photoreceptor ciliary axoneme and anchors rpgr to the Connecting Cilium
    Journal of Biological Chemistry, 2001
    Co-Authors: Donghyun Hong, Michael Adamian, Tiansen Li
    Abstract:

    Abstract Retinitis pigmentosa (RP) is a blinding retinal disease in which the Photoreceptor cells degenerate. Mutations in the gene for retinitis pigmentosa GTPase regulator (RPGR) are a frequent cause of RP. The function of RPGR is not well understood, but it is thought to be a putative guanine nucleotide exchange factor for an unknown G protein. Ablation of the RPGR gene in mice suggested a role in maintaining the polarized distribution of opsin across the cilia. To investigate its function, we used a protein interaction screen to identify candidate proteins that may interact physiologically with RPGR. One such protein, designated RPGR-interacting protein (RPGRIP), is expressed specifically in rod and cone Photoreceptors. It consists of an N-terminal region predicted to form coiled coil structures linked to a C-terminal tail that binds RPGR. In vivo, both proteins co-localize in the Photoreceptor Connecting cilia. RPGRIP is stably associated with the ciliary axoneme independent of RPGR and is resistant to extraction under conditions that partially solubilized other cytoskeletal components. When over-expressed in heterologous cell lines, RPGRIP appears in insoluble punctate and filamentous structures. These data suggest that RPGRIP is a structural component of the ciliary axoneme, and one of its functions is to anchor RPGR within the Cilium. RPGRIP is the only protein known to localize specifically in the Photoreceptor Connecting Cilium. As such, it is a candidate gene for human Photoreceptor disease. The tissue-specific expression of RPGRIP explains why mutations in the ubiquitously expressed RPGR confer a Photoreceptor-specific phenotype.

Tiansen Li - One of the best experts on this subject based on the ideXlab platform.

  • retinitis pigmentosa gtpase regulator rpgr interacting protein is stably associated with the Photoreceptor ciliary axoneme and anchors rpgr to the Connecting Cilium
    Journal of Biological Chemistry, 2001
    Co-Authors: Donghyun Hong, Michael Adamian, Tiansen Li
    Abstract:

    Abstract Retinitis pigmentosa (RP) is a blinding retinal disease in which the Photoreceptor cells degenerate. Mutations in the gene for retinitis pigmentosa GTPase regulator (RPGR) are a frequent cause of RP. The function of RPGR is not well understood, but it is thought to be a putative guanine nucleotide exchange factor for an unknown G protein. Ablation of the RPGR gene in mice suggested a role in maintaining the polarized distribution of opsin across the cilia. To investigate its function, we used a protein interaction screen to identify candidate proteins that may interact physiologically with RPGR. One such protein, designated RPGR-interacting protein (RPGRIP), is expressed specifically in rod and cone Photoreceptors. It consists of an N-terminal region predicted to form coiled coil structures linked to a C-terminal tail that binds RPGR. In vivo, both proteins co-localize in the Photoreceptor Connecting cilia. RPGRIP is stably associated with the ciliary axoneme independent of RPGR and is resistant to extraction under conditions that partially solubilized other cytoskeletal components. When over-expressed in heterologous cell lines, RPGRIP appears in insoluble punctate and filamentous structures. These data suggest that RPGRIP is a structural component of the ciliary axoneme, and one of its functions is to anchor RPGR within the Cilium. RPGRIP is the only protein known to localize specifically in the Photoreceptor Connecting Cilium. As such, it is a candidate gene for human Photoreceptor disease. The tissue-specific expression of RPGRIP explains why mutations in the ubiquitously expressed RPGR confer a Photoreceptor-specific phenotype.

Lisette Hetterschijt - One of the best experts on this subject based on the ideXlab platform.

  • NINL and CC2D2A co-localize with MICAL3 and are required for correct MICAL3 localization.
    2015
    Co-Authors: Ruxandra Bachmann-gagescu, Ian G. Phelps, Erik De Vrieze, Lisette Hetterschijt, Margo Dona, Edith Tonnaer, Theo Peters, Dorus A. Mans, Sylvia E. C. Van Beersum, Heleen H Arts
    Abstract:

    (a) Schematic of a Photoreceptor for orientation. (b-d’) Co-localization of endogenous MICAL3 (green signal; b) and polyglutamylated tubulin (red signal; c) in rat retina (P20) by co-immunostaining radial cryo-sections. The yellow signal in the merged image (d’) indicates co-localization at the base of the Photoreceptor Connecting Cilium. (b’-d’) are high magnification images of the boxed areas in (b-d). (e-f”) Centrosomal co-localization of NINLisoB, CC2D2A and MICAL3 in hTERT-RPE1 cells. mRFP-NINLisoB (red signal, e) localizes to the basal body of the cilia marked with polyglutamylated tubulin (cyanid signal; e) and overlaps with GFP-tagged MICAL3 (green signal, e’) at the ciliary base when co-expressed (yellow signal, e”). Co-expression of eCFP-CC2D2A (green signal, f) and mRFP-MICAL3 (red signal, f’) resulted in partial overlap at the base of the cilia (yellow signal, f”). (g) Endogenous MICAL3 (green signal) detected by immunostaining clusters at the ciliary base (white arrows; Cilium marked with anti-acetylated tubulin in red) of hTERT-RPE1 cells treated with non-targeting siRNA. (h, i) Knockdown of NINL (h) or CC2D2A (i) expression by siRNA results in dispersed distribution of MICAL3 throughout the cell body (brackets) with retention of some MICAL3 puncta at the ciliary base (arrows). qPCR analysis of NINL (j) and CC2D2A (k) siRNA treated hTERT-RPE1 cells. Cells were transfected with 10nM siRNA and all qPCR data were normalized against GUSB levels. Bar and error bars refer to mean and standard deviation, respectively (n = 3, on two biologicial replicates). *: P

  • disruption of the basal body protein poc1b results in autosomal recessive cone rod dystrophy
    American Journal of Human Genetics, 2014
    Co-Authors: Susanne Roosing, Carel B. Hoyng, Ideke J C Lamers, Erik De Vrieze, Ingeborgh L Van Den Born, Stanley Lambertus, Heleen H Arts, Theo A Peters, H Kremer, Lisette Hetterschijt
    Abstract:

    Exome sequencing revealed a homozygous missense mutation (c.317C>G [p.Arg106Pro]) in POC1B, encoding POC1 centriolar protein B, in three siblings with autosomal-recessive cone dystrophy or cone-rod dystrophy and compound-heterozygous POC1B mutations (c.199_201del [p.Gln67del] and c.810+1G>T) in an unrelated person with cone-rod dystrophy. Upon overexpression of POC1B in human TERT-immortalized retinal pigment epithelium 1 cells, the encoded wild-type protein localized to the basal body of the primary Cilium, whereas this localization was lost for p.Arg106Pro and p.Gln67del variant forms of POC1B. Morpholino-oligonucleotide-induced knockdown of poc1b translation in zebrafish resulted in a dose-dependent small-eye phenotype, impaired optokinetic responses, and decreased length of Photoreceptor outer segments. These ocular phenotypes could partially be rescued by wild-type human POC1B mRNA, but not by c.199_201del and c.317C>G mutant human POC1B mRNAs. Yeast two-hybrid screening of a human retinal cDNA library revealed FAM161A as a binary interaction partner of POC1B. This was confirmed in coimmunoprecipitation and colocalization assays, which both showed loss of FAM161A interaction with p.Arg106Pro and p.Gln67del variant forms of POC1B. FAM161A was previously implicated in autosomal-recessive retinitis pigmentosa and shown to be located at the base of the Photoreceptor Connecting Cilium, where it interacts with several other ciliopathy-associated proteins. Altogether, this study demonstrates that POC1B mutations result in a defect of the Photoreceptor sensory Cilium and thus affect cone and rod Photoreceptors.

  • fam161a associated with retinitis pigmentosa is a component of the cilia basal body complex and interacts with proteins involved in ciliopathies
    Human Molecular Genetics, 2012
    Co-Authors: Silvio Alessandro Di Gioia, Lisette Hetterschijt, Stef J F Letteboer, Corinne Kostic, Dikla Bandahrozenfeld, Dror Sharon, Yvan Arsenijevic, Ronald Roepman, Carlo Rivolta
    Abstract:

    Retinitis pigmentosa (RP) is a retinal degenerative disease characterized by the progressive loss of Photoreceptors. We have previously demonstrated that RP can be caused by recessive mutations in the human FAM161A gene, encoding a protein with unknown function that contains a conserved region shared only with a distant paralog, FAM161B. In this study, we show that FAM161A localizes at the base of the Photoreceptor Connecting Cilium in human, mouse and rat. Furthermore, it is also present at the ciliary basal body in ciliated mammalian cells, both in native conditions and upon the expression of recombinant tagged proteins. Yeast two-hybrid analysis of binary interactions between FAM161A and an array of ciliary and ciliopathy-associated proteins reveals direct interaction with lebercilin, CEP290, OFD1 and SDCCAG8, all involved in hereditary retinal degeneration. These interactions are mediated by the C-terminal moiety of FAM161A, as demonstrated by pull-down experiments in cultured cell lines and in bovine retinal extracts. As other ciliary proteins, FAM161A can also interact with the microtubules and organize itself into microtubule-dependent intracellular networks. Moreover, small interfering RNA-mediated depletion of FAM161A transcripts in cultured cells causes the reduction in assembled primary cilia. Taken together, these data indicate that FAM161A-associated RP can be considered as a novel retinal ciliopathy and that its molecular pathogenesis may be related to other ciliopathies.