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.
In humans, the Crumbs homologue-1 (CRB1) gene is mutated in progressive types of autosomal recessive retinitis pigmentosa and Leber congenital amaurosis. The severity of the phenotype due to human ...CRB1 or mouse Crb1 mutations is dependent on the genetic background. Mice on C57BL/6J background with Crb1 mutations show late onset of retinal spotting phenotype or no phenotype. Recently, we showed that conditional deletion of mouse Crb2 in the retina results in early retinal disorganization leading to severe and progressive retinal degeneration with concomitant visual loss that mimics retinitis pigmentosa due to mutations in the CRB1 gene. Recent studies in the fruit fly and zebrafish suggest roles of the Crumbs (CRB) complex members in the regulation of cellular signalling pathways including the Notch1, mechanistic target of rapamycin complex 1 (mTORC1) and the Hippo pathway. Here, we demonstrate that mice backcrossed to C57BL/6J background with loss of CRB2 in the retina show a progressive disorganization and degeneration phenotype during late retinal development. We used microarray gene profiling to study the transcriptome of retinas lacking CRB2 during late retinal development. Unexpectedly, the retinas of newborn mice lacking CRB2 showed no changes in the transcriptome during retinal development. These findings suggest that loss of CRB2 in the developing retina results in retinal disorganization and subsequent degeneration without major changes in the transcriptome of the retina. These mice might be an interesting model to study the onset of retinal degeneration upon loss of CRB proteins.
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 Müller 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 Müller 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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•iPSC-derived retinas recapitulate the fetal human Crumbs complex•CRB2 is expressed earlier than CRB1 in fetal or iPSC-derived retina at the SAR•CRB1-RP patient iPSC-derived retinas show disruptions at the OLM•AAV5 infects photoreceptors and Müller glial cells in adult and iPSC-derived retina
Wijnholds and colleagues show that the key Crumbs complex members, CRB1 and CRB2, are recapitulated between human fetal retina and iPSC-derived retinal organoids. CRB2 is expressed earlier than CRB1 in fetal and iPSC-derived human retina. CRB1-RP patient iPSC-derived retinas show a morphological phenotype. In addition, they show that AAV5 infects photoreceptors and Müller glial cells in adult and iPSC-derived human retina.
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.
Loss of Crumbs homolog 1 (CRB1) or CRB2 proteins in Müller cells or photoreceptors in the mouse retina results in a CRB dose-dependent retinal phenotype. In this study, we present a novel Müller ...cell-specific Crb1KOCrb2LowMGC retinitis pigmentosa mouse model (complete loss of CRB1 and reduced levels of CRB2 specifically in Müller 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 Müller 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 Müller 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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The authors show that reduced endogenous mouse CRB2 levels in Müller cells lacking CRB1 (Crb1KOCrb2LowMGC) results in a retinitis pigmentosa (RP) phenotype, the CRB1-RP mouse retina is more sensitive to chemically induced stress (DL-AAA), and AAV-hCRB2 therapy to Müller cells protects against retinal degeneration and vision deficits in DL-AAA-exposed CRB1-RP mice.
Mutations in the CRB1 gene lead to retinal dystrophies ranging from Leber congenital amaurosis (LCA) to early-onset retinitis pigmentosa (RP), due to developmental defects or loss of adhesion between ...photoreceptors and Müller glia cells, respectively. Whereas over 150 mutations have been found, no clear genotype-phenotype correlation has been established. Mouse Crb1 knockout retinas show a mild phenotype limited to the inferior quadrant, whereas Crb2 knockout retinas display a severe degeneration throughout the retina mimicking the phenotype observed in RP patients associated with CRB1 mutations. Crb1Crb2 double mutant retinas have severe developmental defects similar to the phenotype observed in LCA patients associated with CRB1 mutations. Therefore, CRB2 is a candidate modifying gene of human CRB1-related retinal dystrophy. In this study, we studied the cellular localization of CRB1 and CRB2 in human retina and tested the influence of the Crb2 gene allele on Crb1-retinal dystrophies in mice. We found that in contrast to mice, in the human retina CRB1 protein was expressed at the subapical region in photoreceptors and Müller glia cells, and CRB2 only in Müller glia cells. Genetic ablation of one allele of Crb2 in heterozygote Crb1(+/-) retinas induced a mild retinal phenotype, but in homozygote Crb1 knockout mice lead to an early and severe phenotype limited to the entire inferior retina. Our data provide mechanistic insight for CRB1-related LCA and RP.
Systemic sclerosis (SSc) is characterized by multiple clinical manifestations. Vasculopathy is a main disease hallmark and ranges in severity from an exacerbated Raynaud phenomenon to pulmonary ...arterial hypertension (PAH). The potential involvement of immune system in SSc associated vascular abnormalities is not clear. Here, we set out to study SSc-related immune parameters and determine whether and which peripheral T cell subsets associate with vascular severity in SSc patients.
Peripheral blood and clinical data were collected from 30 SSc patients, 5 patients with idiopathic pulmonary arterial hypertension (IPAH) and 15 age and sex-matched healthy donors (HD). In this cross-sectional cohort SSc patients with PAH (n = 15) were matched for their age, sex and medication with SSc patients with no signs of PAH (n = 15). Lymphocyte subsets were quantified by multi-colour flow cytometry.
SSc patients exhibited elevated percentages of T peripheral helper cells (Tph), CD4+GZMB+ T cells and decreased levels of Th1 cells compared with HD. Increased presence of both CD4+ and CD8+ exhausted-like (CD28-) T cells, characterized by raised cytokine and cytotoxic signature, was also observed in SSc compared with HD blood. Furthermore, IL-4 expressing CD4+CD8+ T cells were significantly increased in SSc peripheral blood. Interestingly, the presence of PAH in SSc was accompanied by a distinct T helper profile, characterized by raised percentages of Th17 and Tph cells.
SSc patients with severe vasculopathy (presence of PAH) exhibited a distinct T cell profile, suggesting for a potential role of auto-immune inflammation in SSc vascular complications.
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.
To determine whether the correlation between the mean change in disease activity and the mean change in synovial sublining (sl) CD68 expression could be demonstrated across different academic ...centers.
Synovial biopsies obtained at arthroscopy from patients with rheumatoid arthritis before and 160 days after rituximab therapy were selected and coded. Paired sections were processed independently at Amsterdam Medical Center (AMC) and at St. Vincent's University Hospital (SVUH), Dublin. Digital image analysis (DIA) was employed at both centers to quantify sublining CD68 expression.
After analysis of CD68sl expression at centers in 2 different countries, high levels of intracenter and intercenter agreement were observed. For the pooled sections stained at AMC, the correlation between 2 investigators was R = 0.942, p = 0.000, and for sections stained at SVUH, R = 0.899, p = 0.001. Similarly, the intracenter correlations for DeltaCD68sl expression after treatment were R = 0.998, p = 0.000, for sections stained at AMC and R = 0.880, p = 0.000, for sections stained at SVUH. The intercenter correlation for the pooled scores of sections stained at AMC was R = 0.85, p = 0.000, and for the sections stained at SVUH, R = 0.62, p = 0.001. The consistent correlation between DeltaDAS (Disease Activity Score) and DeltaCD68sl expression across different studies (Pearson correlation = 0.895, p < 0.001) was confirmed. The standardized response mean values for DeltaCD68sl, calculated from analyses at both AMC and SVUH, were consistently 0.5 or greater, indicating a moderate to high potential to detect change.
The correlation between mean DeltaDAS and mean DeltaCD68sl expression was confirmed across 2 centers. Examination of serial biopsy samples can be used reliably to screen for interesting biological effects at the site of inflammation at an early stage of drug development.
CRB1 gene mutations can cause early- or late-onset retinitis pigmentosa, Leber congenital amaurosis, or maculopathy. Recapitulating human CRB1 phenotypes in animal models has proven challenging, ...necessitating the development of alternatives. We generated human induced pluripotent stem cell (iPSC)-derived retinal organoids of patients with retinitis pigmentosa caused by biallelic CRB1 mutations and evaluated them against autologous gene-corrected hiPSCs and hiPSCs from healthy individuals. Patient organoids show decreased levels of CRB1 and NOTCH1 expression at the retinal outer limiting membrane. Proximity ligation assays show that human CRB1 and NOTCH1 can interact via their extracellular domains. CRB1 patient organoids feature increased levels of WDFY1+ vesicles, fewer RAB11A+ recycling endosomes, decreased VPS35 retromer complex components, and more degradative endolysosomal compartments relative to isogenic control organoids. Taken together, our data demonstrate that patient-derived retinal organoids enable modeling of retinal degeneration and highlight the importance of CRB1 in early endosome maturation receptor recycling in the retina.
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•CRB1 is associated with retinitis pigmentosa•We made organoids from these patients and studied their biology•The extracellular domains of CRB1 and NOTCH1 interact•Our data link to altered endosomal maturation and increased autophagy
Wijnholds, Buck, and colleagues detect upregulation of the endolysosome-associated gene Wdfy-1 in a Crb1 mouse model. They also show that the extracellular domains of human CRB1 and NOTCH1 interact and levels of WDFY1 increased in human retinal organoids, whereas levels of CRB1, NOTCH1, VPS35, and RAB11A decreased in CRB1-associated retinitis pigmentosa organoids. CRB1 organoids repressed early endosome maturation and increased endosomal degradative compartments.
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