The Experts below are selected from a list of 13932 Experts worldwide ranked by ideXlab platform
Jan Wijnholds - One of the best experts on this subject based on the ideXlab platform.
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aav crb2 protects against vision loss in an inducible CRB1 retinitis pigmentosa mouse model
Molecular therapy. Methods & clinical development, 2021Co-Authors: Thilo Matthias Buck, Jan Wijnholds, Rogier M Vos, Henrique C AlvesAbstract:Loss of Crumbs homolog 1 (CRB1) or CRB2 proteins in Muller cells or photoreceptors in the mouse retina results in a CRB dose-dependent retinal phenotype. In this study, we present a novel Muller cell-specific CRB1KOCrb2LowMGC retinitis pigmentosa mouse model (complete loss of CRB1 and reduced levels of CRB2 specifically in Muller cells). The Crb double mutant mice showed deficits in electroretinography, optokinetic head tracking, and retinal morphology. Exposure of retinas to low levels of dl-α-aminoadipate acid induced gliosis and retinal disorganization in CRB1KOCrb2LowMGC retinas but not in wild-type or CRB1-deficient retinas. CRB1KOCrb2LowMGC mice showed a substantial decrease in inner/outer photoreceptor segment length and optokinetic head-tracking response. Intravitreal application of rAAV vectors expressing human CRB2 (hCRB2) in Muller cells of CRB1KOCrb2LowMGC mice subsequently exposed to low levels of dl-α-aminoadipate acid prevented loss of vision, whereas recombinant adeno-associated viral (rAAV) vectors expressing human CRB1 (hCRB1) did not. Both rAAV vectors partially protected the morphology of the retina. The results suggest that hCRB expression in Muller cells is vital for control of retinal cell adhesion at the outer limiting membrane, and that the rAAV-cytomegalovirus (CMV)-hCRB2 vector is more potent than rAAV-minimal CMV (CMVmin)-hCRB1 in protection against loss of vision.
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AAV-CRB2 protects against vision loss in an inducible CRB1 retinitis pigmentosa mouse model
Molecular therapy. Methods & clinical development, 2020Co-Authors: Thilo Matthias Buck, Celso Henrique Alves, Rogier M Vos, Jan WijnholdsAbstract:Abstract Loss of CRB1 or CRB2 proteins in Muller cells or photoreceptors in the mouse retina results in a CRB-dose-dependent retinal phenotype. Here, we present a novel Muller cell-specific CRB1KOCrb2LowMGC retinitis pigmentosa mouse model (Complete loss of CRB1 and reduced levels of CRB2 specifically in Muller cells). The Crb double mutant mice showed deficits in electroretinography, optokinetic head tracking, and retinal morphology. Exposure of retinas to low levels of DL-α-aminoadipate acid induced gliosis and retinal disorganization in CRB1KOCrb2LowMGC but not in wild-type or CRB1-deficient retinas. CRB1KOCrb2LowMGC mice showed a substantial decrease in inner/outer photoreceptor segments length and optokinetic head-tracking response. Intravitreal application of rAAV vectors expressing human CRB2 in Muller cells of CRB1KOCrb2LowMGC mice subsequently exposed to low levels of DL-α-aminoadipate acid prevented loss of vision, whereas rAAV vectors expressing human CRB1 did not. Both rAAV-vectors partially protected the morphology of the retina. The results suggest that human CRB expression in Muller cells is vital for control of retinal cell adhesion at the outer limiting membrane, and that the rAAV-CMV-hCRB2 vector is more potent than the rAAV-CMVmin-hCRB1 in protection against loss of vision.
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Research Models and Gene Augmentation Therapy for CRB1 Retinal Dystrophies
Frontiers in Neuroscience, 2020Co-Authors: Nanda Boon, Jan Wijnholds, Lucie P. PellissierAbstract:Retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) are inherited degenerative retinal dystrophies with vision loss that ultimately lead to blindness. Several genes have been shown to be involved in early onset retinal dystrophies, including CRB1 and RPE65. Gene therapy recently became available for young RP patients with variations in the RPE65 gene. Current research programs test adeno-associated viral gene augmentation or editing therapy vectors on various disease models mimicking the disease in patients. These include several animal and emerging human-derived models, such as human-induced pluripotent stem cell (hiPSC)-derived retinal organoids or hiPSC-derived retinal pigment epithelium (RPE), and human donor retinal explants. Variations in the CRB1 gene are a major cause for early onset autosomal recessive RP with patients suffering from visual impairment before their adolescence and for LCA with newborns experiencing severe visual impairment within the first months of life. These patients cannot benefit yet from an available gene therapy treatment. In this review, we will discuss the recent advances, advantages and disadvantages of different CRB1 human and animal retinal degeneration models. In addition, we will describe novel therapeutic tools that have been developed, which could potentially be used for retinal gene augmentation therapy for RP patients with variations in the CRB1 gene.
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Microglial Cell Dysfunction in CRB1-Associated Retinopathies
Advances in experimental medicine and biology, 2019Co-Authors: C Henrique Alves, Jan WijnholdsAbstract:Inherited retinal diseases encompass a large group of clinically and genetically heterogeneous diseases estimated to affect two million people worldwide. Among these people, approximately 80,000 are or will become blind in their first decades of life due to mutations in both alleles of the Crumbs homologue-1 (CRB1) gene. Microglia are the resident immune surveyor cells in the retina, and their roles have been heavily studied in several retinal diseases, including retinitis pigmentosa (RP), age-related macular degeneration, and diabetic retinopathy. However, very little is known about the role of microglia in CRB1-associated retinopathies. Thus, we here summarize the main findings described in the literature concerning inflammation and the role of microglia in CRB1-patients and CRB1-rodent models.
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CRB2 Loss in Rod Photoreceptors Is Associated with Progressive Loss of Retinal Contrast Sensitivity.
International journal of molecular sciences, 2019Co-Authors: C Henrique Alves, Nanda Boon, Aat A. Mulder, Abraham J. Koster, Carolina R. Jost, Jan WijnholdsAbstract:Variations in the Crumbs homolog-1 (CRB1) gene are associated with a wide variety of autosomal recessive retinal dystrophies, including early onset retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA). CRB1 belongs to the Crumbs family, which in mammals includes CRB2 and CRB3. Here, we studied the specific roles of CRB2 in rod photoreceptor cells and whether ablation of CRB2 in rods exacerbates the CRB1-disease. Therefore, we assessed the morphological, retinal, and visual functional consequences of specific ablation of CRB2 from rods with or without concomitant loss of CRB1. Our data demonstrated that loss of CRB2 in mature rods resulted in RP. The retina showed gliosis and disruption of the subapical region and adherens junctions at the outer limiting membrane. Rods were lost at the peripheral and central superior retina, while gross retinal lamination was preserved. Rod function as measured by electroretinography was impaired in adult mice. Additional loss of CRB1 exacerbated the retinal phenotype leading to an early reduction of the dark-adapted rod photoreceptor a-wave and reduced contrast sensitivity from 3-months-of-age, as measured by optokinetic tracking reflex (OKT) behavior testing. The data suggest that CRB2 present in rods is required to prevent photoreceptor degeneration and vision loss.
Rogier M Vos - One of the best experts on this subject based on the ideXlab platform.
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aav crb2 protects against vision loss in an inducible CRB1 retinitis pigmentosa mouse model
Molecular therapy. Methods & clinical development, 2021Co-Authors: Thilo Matthias Buck, Jan Wijnholds, Rogier M Vos, Henrique C AlvesAbstract:Loss of Crumbs homolog 1 (CRB1) or CRB2 proteins in Muller cells or photoreceptors in the mouse retina results in a CRB dose-dependent retinal phenotype. In this study, we present a novel Muller cell-specific CRB1KOCrb2LowMGC retinitis pigmentosa mouse model (complete loss of CRB1 and reduced levels of CRB2 specifically in Muller cells). The Crb double mutant mice showed deficits in electroretinography, optokinetic head tracking, and retinal morphology. Exposure of retinas to low levels of dl-α-aminoadipate acid induced gliosis and retinal disorganization in CRB1KOCrb2LowMGC retinas but not in wild-type or CRB1-deficient retinas. CRB1KOCrb2LowMGC mice showed a substantial decrease in inner/outer photoreceptor segment length and optokinetic head-tracking response. Intravitreal application of rAAV vectors expressing human CRB2 (hCRB2) in Muller cells of CRB1KOCrb2LowMGC mice subsequently exposed to low levels of dl-α-aminoadipate acid prevented loss of vision, whereas recombinant adeno-associated viral (rAAV) vectors expressing human CRB1 (hCRB1) did not. Both rAAV vectors partially protected the morphology of the retina. The results suggest that hCRB expression in Muller cells is vital for control of retinal cell adhesion at the outer limiting membrane, and that the rAAV-cytomegalovirus (CMV)-hCRB2 vector is more potent than rAAV-minimal CMV (CMVmin)-hCRB1 in protection against loss of vision.
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AAV-CRB2 protects against vision loss in an inducible CRB1 retinitis pigmentosa mouse model
Molecular therapy. Methods & clinical development, 2020Co-Authors: Thilo Matthias Buck, Celso Henrique Alves, Rogier M Vos, Jan WijnholdsAbstract:Abstract Loss of CRB1 or CRB2 proteins in Muller cells or photoreceptors in the mouse retina results in a CRB-dose-dependent retinal phenotype. Here, we present a novel Muller cell-specific CRB1KOCrb2LowMGC retinitis pigmentosa mouse model (Complete loss of CRB1 and reduced levels of CRB2 specifically in Muller cells). The Crb double mutant mice showed deficits in electroretinography, optokinetic head tracking, and retinal morphology. Exposure of retinas to low levels of DL-α-aminoadipate acid induced gliosis and retinal disorganization in CRB1KOCrb2LowMGC but not in wild-type or CRB1-deficient retinas. CRB1KOCrb2LowMGC mice showed a substantial decrease in inner/outer photoreceptor segments length and optokinetic head-tracking response. Intravitreal application of rAAV vectors expressing human CRB2 in Muller cells of CRB1KOCrb2LowMGC mice subsequently exposed to low levels of DL-α-aminoadipate acid prevented loss of vision, whereas rAAV vectors expressing human CRB1 did not. Both rAAV-vectors partially protected the morphology of the retina. The results suggest that human CRB expression in Muller cells is vital for control of retinal cell adhesion at the outer limiting membrane, and that the rAAV-CMV-hCRB2 vector is more potent than the rAAV-CMVmin-hCRB1 in protection against loss of vision.
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Human iPSC-Derived Retinas Recapitulate the Fetal CRB1 CRB2 Complex Formation and Demonstrate that Photoreceptors and Müller Glia Are Targets of AAV5
Stem cell reports, 2019Co-Authors: Peter M. Quinn, Rogier M Vos, C Henrique Alves, Aat A. Mulder, Thilo Matthias Buck, Charlotte Ohonin, Monika Bialecka, Tessa Van Herwaarden, Elon H. C. Van Dijk, Mays TalibAbstract:Human retinal organoids from induced pluripotent stem cells (hiPSCs) can be used to confirm the localization of proteins in retinal cell types and to test transduction and expression patterns of gene therapy vectors. Here, we compared the onset of CRB protein expression in human fetal retina with human iPSC-derived retinal organoids. We show that CRB2 protein precedes the expression of CRB1 in the developing human retina. Our data suggest the presence of CRB1 and CRB2 in human photoreceptors and Muller glial cells. Thus the fetal CRB complex formation is replicated in hiPSC-derived retina. CRB1 patient iPSC retinal organoids showed disruptions at the outer limiting membrane as found in CRB1 mutant mice. Furthermore, AAV serotype 5 (AAV5) is potent in infecting human Muller glial cells and photoreceptors in hiPSC-derived retinas and retinal explants. Our data suggest that human photoreceptors can be efficiently transduced by AAVs in the presence of photoreceptor segments.
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Gene therapy into photoreceptors and Müller glial cells restores retinal structure and function in CRB1 retinitis pigmentosa mouse models
Human Molecular Genetics, 2015Co-Authors: Lucie Pellissier, John G. Flannery, Rogier M Vos, Jan Klooster, C Henrique Alves, Peter M. Quinn, J. Alexander Heimel, Jan WijnholdsAbstract:Mutations in the Crumbs-homologue-1 (CRB1) gene lead to severe recessive inherited retinal dystrophies. Gene transfer therapy is the most promising cure for retinal dystrophies and has primarily been applied for recessive null conditions via a viral gene expression vector transferring a cDNA encoding an enzyme or channel protein, and targeting expression to one cell type. Therapy for the human CRB1 disease will be more complex, as CRB1 is a structural and signaling transmembrane protein present in three cell classes: Müller glia, cone and rod photoreceptors. In this study, we applied CRB1 and CRB2 gene therapy vectors in CRB1-retinitis pigmentosa mouse models at mid-stage disease. We tested if CRB expression restricted to Müller glial cells or photoreceptors or co-expression in both is required to recover retinal function. We show that targeting both Müller glial cells and photoreceptors with CRB2 ameliorated retinal function and structure in CRB1 mouse models. Surprisingly, targeting a single cell type or all cell types with CRB1 reduced retinal function. We show here the first pre-clinical studies for CRB1-related eye disorders using CRB2 vectors and initial elucidation of the cellular mechanisms underlying CRB1 function.
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Targeted ablation of Crb2 in photoreceptor cells induces retinitis pigmentosa
Human Molecular Genetics, 2014Co-Authors: Celso Henrique Alves, Rogier M Vos, Susanne C Beck, Jan Klooster, V Sothilingam, M. Garcia Garrido, Lucie Pellissier, Christina Seide, Takahisa Furukawa, John G. FlanneryAbstract:In humans, the Crumbs homolog-1 (CRB1) gene is mutated in autosomal recessive Leber congenital amaurosis and early-onset retinitis pigmentosa. In mammals, the Crumbs family is composed of: CRB1, CRB2, CRB3A and CRB3B. Recently, we showed that removal of mouse Crb2 from retinal progenitor cells, and consequent removal from Müller glial and photoreceptor cells, results in severe and progressive retinal degeneration with concomitant loss of retinal function that mimics retinitis pigmentosa due to mutations in the CRB1 gene. Here, we studied the effects of cell-type-specific loss of CRB2 from the developing mouse retina using targeted conditional deletion of Crb2 in photoreceptors or Müller cells. We analyzed the consequences of targeted loss of CRB2 in the adult mouse retina using adeno-associated viral vectors encoding Cre recombinase and short hairpin RNA against Crb2. In vivo retinal imaging by means of optical coherence tomography on retinas lacking CRB2 in photoreceptors showed progressive thinning of the photoreceptor layer and cellular mislocalization. Electroretinogram recordings under scotopic conditions showed severe attenuation of the a-wave, confirming the degeneration of photoreceptors. Retinas lacking CRB2 in developing photoreceptors showed early onset of abnormal lamination, whereas retinas lacking CRB2 in developing Müller cells showed late onset retinal disorganization. Our data suggest that in the developing retina, CRB2 has redundant functions in Müller glial cells, while CRB2 has essential functions in photoreceptors. Our data suggest that short-term loss of CRB2 in adult mouse photoreceptors, but not in Müller glial cells, causes sporadic loss of adhesion between photoreceptors and Müller cells.
Jan Klooster - One of the best experts on this subject based on the ideXlab platform.
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Loss of CRB2 in Müller glial cells modifies a CRB1-associated retinitis pigmentosa phenotype into a Leber congenital amaurosis phenotype.
Human molecular genetics, 2018Co-Authors: Peter M. Quinn, Jan Klooster, C Henrique Alves, Aat A. Mulder, Abraham J. Koster, Carolina R. Jost, Mélissa Desrosiers, Sharon I. De Vries, Deniz Dalkara, Jan WijnholdsAbstract:Variations in the human Crumbs homolog-1 (CRB1) gene lead to an array of retinal dystrophies including early onset of retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) in children. To investigate the physiological roles of CRB1 and CRB2 in retinal Muller glial cells (MGCs), we analysed mouse retinas lacking both proteins in MGC. The peripheral retina showed a faster progression of dystrophy than the central retina. The central retina showed retinal folds, disruptions at the outer limiting membrane, protrusion of photoreceptor nuclei into the inner and outer segment layers and ingression of photoreceptor nuclei into the photoreceptor synaptic layer. The peripheral retina showed a complete loss of the photoreceptor synapse layer, intermingling of photoreceptor nuclei within the inner nuclear layer and ectopic photoreceptor cells in the ganglion cell layer. Electroretinography showed severe attenuation of the scotopic a-wave at 1 month of age with responses below detection levels at 3 months of age. The double knockout mouse retinas mimicked a phenotype equivalent to a clinical LCA phenotype due to loss of CRB1. Localization of CRB1 and CRB2 in non-human primate (NHP) retinas was analyzed at the ultrastructural level. We found that NHP CRB1 and CRB2 proteins localized to the subapical region adjacent to adherens junctions at the outer limiting membrane in MGC and photoreceptors. Our data suggest that loss of CRB2 in MGC aggravates the CRB1-associated RP-like phenotype towards an LCA-like phenotype.
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Gene therapy into photoreceptors and Müller glial cells restores retinal structure and function in CRB1 retinitis pigmentosa mouse models
Human Molecular Genetics, 2015Co-Authors: Lucie Pellissier, John G. Flannery, Rogier M Vos, Jan Klooster, C Henrique Alves, Peter M. Quinn, J. Alexander Heimel, Jan WijnholdsAbstract:Mutations in the Crumbs-homologue-1 (CRB1) gene lead to severe recessive inherited retinal dystrophies. Gene transfer therapy is the most promising cure for retinal dystrophies and has primarily been applied for recessive null conditions via a viral gene expression vector transferring a cDNA encoding an enzyme or channel protein, and targeting expression to one cell type. Therapy for the human CRB1 disease will be more complex, as CRB1 is a structural and signaling transmembrane protein present in three cell classes: Müller glia, cone and rod photoreceptors. In this study, we applied CRB1 and CRB2 gene therapy vectors in CRB1-retinitis pigmentosa mouse models at mid-stage disease. We tested if CRB expression restricted to Müller glial cells or photoreceptors or co-expression in both is required to recover retinal function. We show that targeting both Müller glial cells and photoreceptors with CRB2 ameliorated retinal function and structure in CRB1 mouse models. Surprisingly, targeting a single cell type or all cell types with CRB1 reduced retinal function. We show here the first pre-clinical studies for CRB1-related eye disorders using CRB2 vectors and initial elucidation of the cellular mechanisms underlying CRB1 function.
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Targeted ablation of Crb2 in photoreceptor cells induces retinitis pigmentosa
Human Molecular Genetics, 2014Co-Authors: Celso Henrique Alves, Rogier M Vos, Susanne C Beck, Jan Klooster, V Sothilingam, M. Garcia Garrido, Lucie Pellissier, Christina Seide, Takahisa Furukawa, John G. FlanneryAbstract:In humans, the Crumbs homolog-1 (CRB1) gene is mutated in autosomal recessive Leber congenital amaurosis and early-onset retinitis pigmentosa. In mammals, the Crumbs family is composed of: CRB1, CRB2, CRB3A and CRB3B. Recently, we showed that removal of mouse Crb2 from retinal progenitor cells, and consequent removal from Müller glial and photoreceptor cells, results in severe and progressive retinal degeneration with concomitant loss of retinal function that mimics retinitis pigmentosa due to mutations in the CRB1 gene. Here, we studied the effects of cell-type-specific loss of CRB2 from the developing mouse retina using targeted conditional deletion of Crb2 in photoreceptors or Müller cells. We analyzed the consequences of targeted loss of CRB2 in the adult mouse retina using adeno-associated viral vectors encoding Cre recombinase and short hairpin RNA against Crb2. In vivo retinal imaging by means of optical coherence tomography on retinas lacking CRB2 in photoreceptors showed progressive thinning of the photoreceptor layer and cellular mislocalization. Electroretinogram recordings under scotopic conditions showed severe attenuation of the a-wave, confirming the degeneration of photoreceptors. Retinas lacking CRB2 in developing photoreceptors showed early onset of abnormal lamination, whereas retinas lacking CRB2 in developing Müller cells showed late onset retinal disorganization. Our data suggest that in the developing retina, CRB2 has redundant functions in Müller glial cells, while CRB2 has essential functions in photoreceptors. Our data suggest that short-term loss of CRB2 in adult mouse photoreceptors, but not in Müller glial cells, causes sporadic loss of adhesion between photoreceptors and Müller cells.
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CRB1 is a determinant of retinal apical muller glia cell features
Glia, 2007Co-Authors: Serge A Van De Pavert, Inge Versteeg, Wendy M. Aartsen, Alicia Sanz Sanz, Rogier M Vos, Susanne C Beck, Jan Klooster, Mathias W Seeliger, Jan WijnholdsAbstract:Mutations in the human Crumbs homologue-1 (CRB1) gene cause retinal blinding diseases, such as Leber congenital amaurosis and retinitis pigmentosa. In the previous studies we have shown that CRB1 resides in retinal Muller glia cells and that loss of CRB1 results in retinal degeneration (particularly in the inferior temporal quadrant of the mouse eye). Degeneration is increased by exposure to white light. Here, we studied the role of light and aging to gain a better understanding of the factors involved in the progress of retinal disease. Our data reveal that light is neither sufficient nor required to induce retinal disorganization and degeneration in young CRB1(-/-) mutant mice, suggesting that it rather modulates the retinal phenotype. Gene expression profiling showed that expression of five genes is altered in light-exposed CRB1(-/-) mutant retinas. Three of the five genes are involved in chromosome stabilization (Pituitary tumor transforming gene 1 or Pttg1, Establishment of cohesion 1 homolog 1 or Esco1, and a gene similar to histone H2B). In aged retinas, degeneration of photoreceptors, inner retinal neurons, and retinal pigment epithelium was practically limited to the inferior temporal quadrant. Loss of CRB1 in Muller glia cells resulted in an irregular number and size of their apical villi. We propose that CRB1 is required to regulate number and size of these Muller glia cell villi. The subsequent loss of retinal integrity resulted in neovascularization, in which blood vessels of the choroid protruded into the neural retina.
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CRB1 is a Determinant of Retinal Apical M€ uller
2007Co-Authors: Inge Versteeg, Susanne C Beck, Jan Klooster, Mathias W Seeliger, Jan WijnholdsAbstract:KEY WORDSMueller glia cells; retinal degeneration; retinal disease;adherens junctions; photoreceptorsABSTRACTMutations in the human Crumbs homologue-1 (CRB1) genecause retinal blinding diseases, such as Leber congenitalamaurosis and retinitis pigmentosa. In the previous studieswe have shown that CRB1 resides in retinal M€uller glia cellsand that loss of CRB1 results in retinal degeneration (partic-ularly in the inferior temporal quadrant of the mouse eye).Degeneration is increased by exposure to white light. Here,we studied the role of light and aging to gain a betterunderstanding of the factors involved in the progress of ret-inal disease. Our data reveal that light is neither sufficientnor required to induce retinal disorganization and degener-ation in young CRB1
Celso Henrique Alves - One of the best experts on this subject based on the ideXlab platform.
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AAV-CRB2 protects against vision loss in an inducible CRB1 retinitis pigmentosa mouse model
Molecular therapy. Methods & clinical development, 2020Co-Authors: Thilo Matthias Buck, Celso Henrique Alves, Rogier M Vos, Jan WijnholdsAbstract:Abstract Loss of CRB1 or CRB2 proteins in Muller cells or photoreceptors in the mouse retina results in a CRB-dose-dependent retinal phenotype. Here, we present a novel Muller cell-specific CRB1KOCrb2LowMGC retinitis pigmentosa mouse model (Complete loss of CRB1 and reduced levels of CRB2 specifically in Muller cells). The Crb double mutant mice showed deficits in electroretinography, optokinetic head tracking, and retinal morphology. Exposure of retinas to low levels of DL-α-aminoadipate acid induced gliosis and retinal disorganization in CRB1KOCrb2LowMGC but not in wild-type or CRB1-deficient retinas. CRB1KOCrb2LowMGC mice showed a substantial decrease in inner/outer photoreceptor segments length and optokinetic head-tracking response. Intravitreal application of rAAV vectors expressing human CRB2 in Muller cells of CRB1KOCrb2LowMGC mice subsequently exposed to low levels of DL-α-aminoadipate acid prevented loss of vision, whereas rAAV vectors expressing human CRB1 did not. Both rAAV-vectors partially protected the morphology of the retina. The results suggest that human CRB expression in Muller cells is vital for control of retinal cell adhesion at the outer limiting membrane, and that the rAAV-CMV-hCRB2 vector is more potent than the rAAV-CMVmin-hCRB1 in protection against loss of vision.
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Targeted ablation of Crb2 in photoreceptor cells induces retinitis pigmentosa
Human Molecular Genetics, 2014Co-Authors: Celso Henrique Alves, Rogier M Vos, Susanne C Beck, Jan Klooster, V Sothilingam, M. Garcia Garrido, Lucie Pellissier, Christina Seide, Takahisa Furukawa, John G. FlanneryAbstract:In humans, the Crumbs homolog-1 (CRB1) gene is mutated in autosomal recessive Leber congenital amaurosis and early-onset retinitis pigmentosa. In mammals, the Crumbs family is composed of: CRB1, CRB2, CRB3A and CRB3B. Recently, we showed that removal of mouse Crb2 from retinal progenitor cells, and consequent removal from Müller glial and photoreceptor cells, results in severe and progressive retinal degeneration with concomitant loss of retinal function that mimics retinitis pigmentosa due to mutations in the CRB1 gene. Here, we studied the effects of cell-type-specific loss of CRB2 from the developing mouse retina using targeted conditional deletion of Crb2 in photoreceptors or Müller cells. We analyzed the consequences of targeted loss of CRB2 in the adult mouse retina using adeno-associated viral vectors encoding Cre recombinase and short hairpin RNA against Crb2. In vivo retinal imaging by means of optical coherence tomography on retinas lacking CRB2 in photoreceptors showed progressive thinning of the photoreceptor layer and cellular mislocalization. Electroretinogram recordings under scotopic conditions showed severe attenuation of the a-wave, confirming the degeneration of photoreceptors. Retinas lacking CRB2 in developing photoreceptors showed early onset of abnormal lamination, whereas retinas lacking CRB2 in developing Müller cells showed late onset retinal disorganization. Our data suggest that in the developing retina, CRB2 has redundant functions in Müller glial cells, while CRB2 has essential functions in photoreceptors. Our data suggest that short-term loss of CRB2 in adult mouse photoreceptors, but not in Müller glial cells, causes sporadic loss of adhesion between photoreceptors and Müller cells.
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Targeted ablation of CRB1 and Crb2 in retinal progenitor cells mimics Leber congenital amaurosis
PLoS Genetics, 2013Co-Authors: Lucie Pellissier, Rogier M Vos, Dm Lundvig, Celso Henrique Alves, Peter M. Quinn, Naoyuki Tanimoto, Jacobus J. Dudok, Berend Hooibrink, Fabrice Richard, Susanne C BeckAbstract:Development in the central nervous system is highly dependent on the regulation of the switch from progenitor cell proliferation to differentiation, but the molecular and cellular events controlling this process remain poorly understood. Here, we report that ablation of CRB1 and Crb2 genes results in severe impairment of retinal function, abnormal lamination and thickening of the retina mimicking human Leber congenital amaurosis due to loss of CRB1 function. We show that the levels of CRB1 and CRB2 proteins are crucial for mouse retinal development, as they restrain the proliferation of retinal progenitor cells. The lack of these apical proteins results in altered cell cycle progression and increased number of mitotic cells leading to an increased number of late-born cell types such as rod photoreceptors, bipolar and Müller glia cells in postmitotic retinas. Loss of CRB1 and CRB2 in the retina results in dysregulation of target genes for the Notch1 and YAP/Hippo signaling pathways and increased levels of P120-catenin. Loss of CRB1 and CRB2 result in altered progenitor cell cycle distribution with a decrease in number of late progenitors in G1 and an increase in S and G2/M phase. These findings suggest that CRB1 and CRB2 suppress late progenitor pool expansion by regulating multiple proliferative signaling pathways.
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Loss of CRB2 in the mouse retina mimics human retinitis pigmentosa due to mutations in the CRB1 gene.
Human Molecular Genetics, 2013Co-Authors: Celso Henrique Alves, Lucie P. Pellissier, Alicia Sanz Sanz, Susanne C Beck, Naoyuki Tanimoto, Fabrice Richard, Bokyung Park, Gesine Huber, Mariyam Murtaza, Iswariyaraja Sridevi GurubaranAbstract:In humans, the Crumbs homolog-1 (CRB1) gene is mutated in progressive types of autosomal recessive retinitis pigmentosa and Leber congenital amaurosis. However, there is no clear genotype-phenotype correlation for CRB1 mutations, which suggests that other components of the CRB complex may influence the severity of retinal disease. Therefore, to understand the physiological role of the Crumbs complex proteins, we generated and analysed conditional knockout mice lacking CRB2 in the developing retina. Progressive disorganization was detected during late retinal development. Progressive thinning of the photoreceptor layer and sites of cellular mislocalization was detected throughout the CRB2-deficient retina by confocal scanning laser ophthalmoscopy and spectral domain optical coherence tomography. Under scotopic conditions using electroretinography, the attenuation of the a-wave was relatively stronger than that of the b-wave, suggesting progressive degeneration of photoreceptors in adult animals. Histological analysis of newborn mice showed abnormal lamination of immature rod photoreceptors and disruption of adherens junctions between photoreceptors, Müller glia and progenitor cells. The number of late-born progenitor cells, rod photoreceptors and Müller glia cells was increased, concomitant with programmed cell death of rod photoreceptors. The data suggest an essential role for CRB2 in proper lamination of the photoreceptor layer and suppression of proliferation of late-born retinal progenitor cells.
C Henrique Alves - One of the best experts on this subject based on the ideXlab platform.
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Microglial Cell Dysfunction in CRB1-Associated Retinopathies
Advances in experimental medicine and biology, 2019Co-Authors: C Henrique Alves, Jan WijnholdsAbstract:Inherited retinal diseases encompass a large group of clinically and genetically heterogeneous diseases estimated to affect two million people worldwide. Among these people, approximately 80,000 are or will become blind in their first decades of life due to mutations in both alleles of the Crumbs homologue-1 (CRB1) gene. Microglia are the resident immune surveyor cells in the retina, and their roles have been heavily studied in several retinal diseases, including retinitis pigmentosa (RP), age-related macular degeneration, and diabetic retinopathy. However, very little is known about the role of microglia in CRB1-associated retinopathies. Thus, we here summarize the main findings described in the literature concerning inflammation and the role of microglia in CRB1-patients and CRB1-rodent models.
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CRB2 Loss in Rod Photoreceptors Is Associated with Progressive Loss of Retinal Contrast Sensitivity.
International journal of molecular sciences, 2019Co-Authors: C Henrique Alves, Nanda Boon, Aat A. Mulder, Abraham J. Koster, Carolina R. Jost, Jan WijnholdsAbstract:Variations in the Crumbs homolog-1 (CRB1) gene are associated with a wide variety of autosomal recessive retinal dystrophies, including early onset retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA). CRB1 belongs to the Crumbs family, which in mammals includes CRB2 and CRB3. Here, we studied the specific roles of CRB2 in rod photoreceptor cells and whether ablation of CRB2 in rods exacerbates the CRB1-disease. Therefore, we assessed the morphological, retinal, and visual functional consequences of specific ablation of CRB2 from rods with or without concomitant loss of CRB1. Our data demonstrated that loss of CRB2 in mature rods resulted in RP. The retina showed gliosis and disruption of the subapical region and adherens junctions at the outer limiting membrane. Rods were lost at the peripheral and central superior retina, while gross retinal lamination was preserved. Rod function as measured by electroretinography was impaired in adult mice. Additional loss of CRB1 exacerbated the retinal phenotype leading to an early reduction of the dark-adapted rod photoreceptor a-wave and reduced contrast sensitivity from 3-months-of-age, as measured by optokinetic tracking reflex (OKT) behavior testing. The data suggest that CRB2 present in rods is required to prevent photoreceptor degeneration and vision loss.
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Human iPSC-Derived Retinas Recapitulate the Fetal CRB1 CRB2 Complex Formation and Demonstrate that Photoreceptors and Müller Glia Are Targets of AAV5
Stem cell reports, 2019Co-Authors: Peter M. Quinn, Rogier M Vos, C Henrique Alves, Aat A. Mulder, Thilo Matthias Buck, Charlotte Ohonin, Monika Bialecka, Tessa Van Herwaarden, Elon H. C. Van Dijk, Mays TalibAbstract:Human retinal organoids from induced pluripotent stem cells (hiPSCs) can be used to confirm the localization of proteins in retinal cell types and to test transduction and expression patterns of gene therapy vectors. Here, we compared the onset of CRB protein expression in human fetal retina with human iPSC-derived retinal organoids. We show that CRB2 protein precedes the expression of CRB1 in the developing human retina. Our data suggest the presence of CRB1 and CRB2 in human photoreceptors and Muller glial cells. Thus the fetal CRB complex formation is replicated in hiPSC-derived retina. CRB1 patient iPSC retinal organoids showed disruptions at the outer limiting membrane as found in CRB1 mutant mice. Furthermore, AAV serotype 5 (AAV5) is potent in infecting human Muller glial cells and photoreceptors in hiPSC-derived retinas and retinal explants. Our data suggest that human photoreceptors can be efficiently transduced by AAVs in the presence of photoreceptor segments.
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Loss of CRB2 in Müller glial cells modifies a CRB1-associated retinitis pigmentosa phenotype into a Leber congenital amaurosis phenotype.
Human molecular genetics, 2018Co-Authors: Peter M. Quinn, Jan Klooster, C Henrique Alves, Aat A. Mulder, Abraham J. Koster, Carolina R. Jost, Mélissa Desrosiers, Sharon I. De Vries, Deniz Dalkara, Jan WijnholdsAbstract:Variations in the human Crumbs homolog-1 (CRB1) gene lead to an array of retinal dystrophies including early onset of retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) in children. To investigate the physiological roles of CRB1 and CRB2 in retinal Muller glial cells (MGCs), we analysed mouse retinas lacking both proteins in MGC. The peripheral retina showed a faster progression of dystrophy than the central retina. The central retina showed retinal folds, disruptions at the outer limiting membrane, protrusion of photoreceptor nuclei into the inner and outer segment layers and ingression of photoreceptor nuclei into the photoreceptor synaptic layer. The peripheral retina showed a complete loss of the photoreceptor synapse layer, intermingling of photoreceptor nuclei within the inner nuclear layer and ectopic photoreceptor cells in the ganglion cell layer. Electroretinography showed severe attenuation of the scotopic a-wave at 1 month of age with responses below detection levels at 3 months of age. The double knockout mouse retinas mimicked a phenotype equivalent to a clinical LCA phenotype due to loss of CRB1. Localization of CRB1 and CRB2 in non-human primate (NHP) retinas was analyzed at the ultrastructural level. We found that NHP CRB1 and CRB2 proteins localized to the subapical region adjacent to adherens junctions at the outer limiting membrane in MGC and photoreceptors. Our data suggest that loss of CRB2 in MGC aggravates the CRB1-associated RP-like phenotype towards an LCA-like phenotype.
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AAV Gene Augmentation Therapy for CRB1-Associated Retinitis Pigmentosa.
Methods in molecular biology (Clifton N.J.), 2017Co-Authors: C Henrique Alves, Jan WijnholdsAbstract:Mutations in the CRB1 gene account for around 10,000 persons with Leber congenital amaurosis (LCA) and 70,000 persons with retinitis pigmentosa (RP) worldwide. Therefore, the CRB1 gene is a key target in the fight against blindness. A proof-of-concept for an adeno-associated virus (AAV)-mediated CRB2 gene augmentation therapy for CRB1-RP was recently described. Preclinical studies using animal models such as knockout or mutant mice are crucial to obtain such proof-of-concept. In this chapter we describe a technique to deliver AAV vectors, into the murine retinas, via the subretinal route. We also present protocols to detect expression of the therapeutic protein by fluorescence immunohistochemistry and to perform histological studies using ultra-thin sections stained with toluidine blue. These techniques in combination with electroretinography and visual behavior tests are in principle sufficient to obtain proof-of-concept for new gene therapies.