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Optical coherence tomography (OCT) is widely used to probe retinal structure and function. This study investigated the outer retina band (ORB) pattern and reflective intensity for the region between bands 2 and 3 (Dip) in three mouse models of inherited retinal degeneration (Rs1KO, TTLL5KO, RPE65KO) and in human AMD patients from the A2A database. OCT images were manually graded, and reflectivity signals were used to calculate the Dip ratio. Qualitative analyses demonstrated the progressive merging band 2 and band 3 in all three mouse models, leading to a reduction in the Dip ratio compared to wildtype (WT) controls. Gene replacement therapy in Rs1KO mice reverted the ORB pattern to one resembling WT and increased the Dip ratio. The degree of anatomical rescue in these mice was highly correlated with level of transgenic RS1 expression and with the restoration of ERG b-wave amplitudes. While the inner retinal cavity was significantly enlarged in dark-adapted Rs1KO mice, the Dip ratio was not altered. A reduction of the Dip ratio was also detected in AMD patients compared with healthy controls and was also positively correlated with AMD severity on the AMD score. We propose that the ORB and Dip ratio can be used as non-invasive early biomarkers for retina health, which can be used to probe therapeutic gene expression and to evaluate the effectiveness of therapy.
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Purpose: To describe a group of patients with retinitis pigmentosa GTPase regulator (RPGR)-related retinopathy with a tapetal-like retinal sheen and corresponding changes in the reflectivity of the ellipsoid zone on optical coherence tomography (OCT) imaging. Methods: A retrospective case series of 66 patients with a disease-causing variant in RPGR was performed. An expert examiner, masked to patient demographics, clinical evaluations, and specific RPGR variant, analyzed color fundus photographs for the presence of a tapetal-like retinal sheen and assessed OCT images for the presence of an abnormally broad hyper-reflective band in the outer retina. Longitudinal reflectivity profiles were generated and compared with healthy controls. Results: Twelve patients (18.2%) had a retinal sheen on color images that cosegregated with an abnormally broad hyper-reflective ellipsoid zone band on OCT imaging. Three-fourths of these patients were male, had a cone-rod dystrophy, and had pathogenic RPGR variants located toward the 3'-end of ORF15. This group had a different longitudinal reflectivity profile signature compared with controls. After a period of prolonged dark adaptation, the abnormal hyper-reflective band on OCT became less apparent, and the outer retinal layers adopted a more normal appearance. Conclusions: RPGR-related retinopathy should be considered for males presenting with retinal sheen, abnormal ellipsoid zone hyper-reflectivity, and cone or cone-rod dysfunction on ERG, and pursued with molecular testing. Our results have implications for understanding the role of the C-terminal domain encoded by RPGR ORF15 in the phototransduction cascade. Further, the findings may be important to incorporate into both inclusion criteria and outcome measure developments in future RPGR-related cone or cone-rod dystrophy clinical trials.
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Distrofias de Conos y Bastones , Enfermedades de la Retina , Humanos , Masculino , Femenino , Distrofias de Conos y Bastones/diagnóstico , Distrofias de Conos y Bastones/genética , Estudios Retrospectivos , Retina , Células Fotorreceptoras Retinianas Conos , Proteínas del Ojo/genéticaRESUMEN
Retinal organoids (ROs) derived from human pluripotent stem cells (hPSCs) recapitulate key features of retinogenesis and provide a promising platform to study retinal development and disease in a human context. Although multiple protocols are currently in use, hPSCs exhibit tremendous variability in differentiation efficiency, with some cell lines consistently yielding few or even no ROs, limiting their utility in research. We report here that early nicotinamide (NAM) treatment significantly improves RO yield across 8 hPSC lines from different donors, including some that would otherwise fail to generate a meaningful number of ROs. NAM treatment promotes neural commitment of hPSCs at the expense of non-neural ectodermal cell fate, which in turn increases eye field progenitor generation. Further analysis suggests that this effect is partially mediated through inhibition of BMP signaling. Our data encourage a broader use of human ROs for disease modeling applications that require the use of multiple patient-specific cell lines.
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Recombinant adeno-associated viruses (rAAV) are used extensively as gene delivery vectors in clinical studies, and several rAAV based treatments have already been approved. Significant progress has been made in rAAV manufacturing; however, better and more precise capsid characterization techniques are still needed to guarantee the purity and safety of rAAV preparations. Current analytical techniques used to characterize rAAV preparations are susceptible to background signals, have limited accuracy, or require a large amount of time and material. A recently developed single-molecule technique, mass photometry (MP), measures mass distributions of biomolecules with high-resolution and sensitivity. Here we explore applications of MP for the characterization of capsid fractions. We demonstrate that MP is able to resolve and quantify not only empty and full-genome containing capsid populations but also identify partially packaged capsid impurities. MP data accurately measures full and empty capsid ratios, and can be used to estimate the size of the encapsidated genome. MP distributions provide information on sample heterogeneity and on the presence of aggregates. Sub-picomole quantities of sample are sufficient for MP analysis, and data can be obtained and analyzed within minutes. This method provides a simple, robust, and effective tool to monitor the physical attributes of rAAV vectors.
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Dependovirus , Vectores Genéticos , Dependovirus/genética , Vectores Genéticos/genética , Terapia Genética/métodos , Técnicas de Transferencia de Gen , FotometríaRESUMEN
Mutations in the gene for Retinitis Pigmentosa GTPase Regulator (RPGR) cause the X-linked form of inherited retinal degeneration, and the majority are frameshift mutations in a highly repetitive, purine-rich region of RPGR known as the OFR15 exon. Truncation of the reading frame in this terminal exon ablates the functionally important C-terminal domain. We hypothesized that targeted excision in ORF15 by CRISPR/Cas9 and the ensuing repair by non-homologous end joining could restore RPGR reading frame in a portion of mutant photoreceptors thereby correcting gene function in vivo. We tested this hypothesis in the rd9 mouse, a naturally occurring mutant line that carries a frameshift mutation in RPGRORF15, through a combination of germline and somatic gene therapy approaches. In germline gene-edited rd9 mice, probing with RPGR domain-specific antibodies demonstrated expression of full length RPGRORF15 protein. Hallmark features of RPGR mutation-associated early disease phenotypes, such as mislocalization of cone opsins, were no longer present. Subretinal injections of the same guide RNA (sgRNA) carried in AAV sgRNA and SpCas9 expression vectors restored reading frame of RPGRORF15 in a subpopulation of cells with broad distribution throughout the retina, confirming successful correction of the mutation. These data suggest that a simplified form of genome editing mediated by CRISPR, as described here, could be further developed to repair RPGRORF15 mutations in vivo.
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Degeneración Retiniana , Retinitis Pigmentosa , Animales , Sistemas CRISPR-Cas , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Edición Génica , Ratones , Mutación , Degeneración Retiniana/genética , Degeneración Retiniana/terapia , Retinitis Pigmentosa/genética , Retinitis Pigmentosa/metabolismo , Retinitis Pigmentosa/terapiaRESUMEN
The primary cilium is a ubiquitous microtubule-based organelle that senses external environment and modulates diverse signaling pathways in different cell types and tissues. The cilium originates from the mother centriole through a complex set of cellular events requiring hundreds of distinct components. Aberrant ciliogenesis or ciliary transport leads to a broad spectrum of clinical entities with overlapping yet highly variable phenotypes, collectively called ciliopathies, which include sensory defects and syndromic disorders with multi-organ pathologies. For efficient light detection, photoreceptors in the retina elaborate a modified cilium known as the outer segment, which is packed with membranous discs enriched for components of the phototransduction machinery. Retinopathy phenotype involves dysfunction and/or degeneration of the light sensing photoreceptors and is highly penetrant in ciliopathies. This review will discuss primary cilia biogenesis and ciliopathies, with a focus on the retina, and the role of CP110-CEP290-CC2D2A network. We will also explore how recent technologies can advance our understanding of cilia biology and discuss new paradigms for developing potential therapies of retinal ciliopathies.
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Antígenos de Neoplasias/genética , Proteínas de Ciclo Celular/genética , Cilios/metabolismo , Ciliopatías/genética , Proteínas del Citoesqueleto/genética , Proteínas Asociadas a Microtúbulos/genética , Fosfoproteínas/genética , Degeneración Retiniana/genética , Retinitis Pigmentosa/genética , Animales , Antígenos de Neoplasias/metabolismo , Transporte Biológico , Proteínas de Ciclo Celular/metabolismo , Centriolos/metabolismo , Centriolos/ultraestructura , Cilios/ultraestructura , Ciliopatías/metabolismo , Ciliopatías/patología , Proteínas del Citoesqueleto/metabolismo , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Humanos , Fototransducción , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Fosfoproteínas/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patología , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Retinitis Pigmentosa/metabolismo , Retinitis Pigmentosa/patologíaRESUMEN
Rab-GTPases and associated effectors mediate cargo transport through the endomembrane system of eukaryotic cells, regulating key processes such as membrane turnover, signal transduction, protein recycling and degradation. Using developmental transcriptome data, we identified Rabgef1 (encoding the protein RabGEF1 or Rabex-5) as the only gene associated with Rab GTPases that exhibited strong concordance with retinal photoreceptor differentiation. Loss of Rabgef1 in mice (Rabgef1-/-) resulted in defects specifically of photoreceptor morphology and almost complete loss of both rod and cone function as early as eye opening; however, aberrant outer segment formation could only partly account for visual function deficits. RabGEF1 protein in retinal photoreceptors interacts with Rabaptin-5, and RabGEF1 absence leads to reduction of early endosomes consistent with studies in other mammalian cells and tissues. Electron microscopy analyses reveal abnormal accumulation of macromolecular aggregates in autophagosome-like vacuoles and enhanced immunostaining for LC3A/B and p62 in Rabgef1-/- photoreceptors, consistent with compromised autophagy. Transcriptome analysis of the developing Rabgef1-/- retina reveals altered expression of 2469 genes related to multiple pathways including phototransduction, mitochondria, oxidative stress and endocytosis, suggesting an early trajectory of photoreceptor cell death. Our results implicate an essential role of the RabGEF1-modulated endocytic and autophagic pathways in photoreceptor differentiation and homeostasis. We propose that RabGEF1 and associated components are potential candidates for syndromic traits that include a retinopathy phenotype.
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Autofagia , Endocitosis , Factores de Intercambio de Guanina Nucleótido/genética , Neurogénesis , Células Fotorreceptoras/metabolismo , Degeneración Retiniana/metabolismo , Animales , Femenino , Factores de Intercambio de Guanina Nucleótido/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Células Fotorreceptoras/citología , Degeneración Retiniana/genética , TranscriptomaRESUMEN
Purpose: Disruption of proteostasis is a key event in many neurodegenerative diseases. Heat shock proteins (HSPs) participate in multiple functions associated with intracellular transport and proteostasis. We evaluated the effect of augmented HSP70 expression in mutant photoreceptors of mouse retinal degeneration models to test the hypothesis that failure to sustain HSP70 expression contributes to photoreceptor cell death. Methods: We examined HSP70 expression in retinas of wild-type and mutant mice by RNA and protein analysis. A transgenic mouse line, TgCrx-Hspa1a-Flag, was generated to express FLAG-tagged full-length HSP70 protein under control of a 2.3 kb mouse Crx promoter. This line was crossed to three distinct retinal degeneration mouse models. Retinal structure and function were evaluated by histology, immunohistochemistry, and electroretinography. Results: In seven different mouse models of retinal degeneration, we detected transient elevation of endogenous HSP70 expression at early stages, followed by a dramatic reduction as cell death ensues, suggesting an initial adaptive response to cellular stress. Augmented expression of HSP70 in RHOT17M mice, in which mutant rhodopsin is misfolded, marginally improved photoreceptor survival, whereas elevated HSP70 led to more severe retinal degeneration in rd10 mutants that produce a partially functional PDE6B. In Rpgrip1-/- mice that display a ciliary defect, higher HSP70 had no impact on photoreceptor survival or function. Conclusions: HSP70 overexpression has divergent effects in photoreceptors determined, at least in part, by the nature of the mutant protein each model carries. Additional investigations on HSP pathways and associated chaperone networks in photoreceptors are needed before designing therapeutic strategies targeting proteostasis.
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Regulación de la Expresión Génica/fisiología , Proteínas HSP70 de Choque Térmico/genética , Degeneración Retiniana/metabolismo , Células Fotorreceptoras Retinianas Bastones/metabolismo , Animales , Supervivencia Celular , Modelos Animales de Enfermedad , Electrorretinografía , Immunoblotting , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Fluorescente , Retina/fisiopatología , Degeneración Retiniana/fisiopatología , Células Fotorreceptoras Retinianas Bastones/fisiologíaRESUMEN
Retinal pigment epithelium (RPE) differentiated from human induced pluripotent stem cells, called induced retinal pigment epithelium (iRPE), is being explored as a cell-based therapy for the treatment of retinal degenerative diseases, especially age-related macular degeneration. The success of RPE implantation is linked to the use of biomimetic scaffolds that simulate Bruch's membrane and promote RPE maturation and integration as a functional tissue. Due to difficulties associated with animal protein-derived scaffolds, including sterility and pro-inflammatory responses, current practices favor the use of synthetic polymers, such as polycaprolactone (PCL), for generating nanofibrous scaffolds. In this study, we tested the hypothesis that plant protein-derived fibrous scaffolds can provide favorable conditions permissive for the maturation of RPE tissue sheets in vitro. Our natural, soy protein-derived nanofibrous scaffolds exhibited a J-shaped stress-strain curve that more closely resembled the mechanical properties of native tissues than PCL with significantly higher hydrophilicity of the natural scaffolds, favoring in vivo implantation. We then demonstrate that iRPE sheets growing on these soy protein scaffolds are equivalent to iRPE monolayers cultured on synthetic PCL nanofibrous scaffolds. Immunohistochemistry demonstrated RPE-like morphology and functionality with appropriate localization of RPE markers RPE65, PMEL17, Ezrin, and ZO1 and with anticipated histotypic polarization of vascular endothelial growth factor and pigment epithelium-derived growth factor as indicated by enzyme-linked immunosorbent assay. Scanning electron microscopy revealed dense microvilli on the cell surface and homogeneous tight junctional contacts between the cells. Finally, comparative transcriptome analysis in conjunction with principal component analysis demonstrated that iRPE on nanofibrous scaffolds, either natural or synthetic, matured more consistently than on nonfibrous substrates. Taken together, our studies suggest that the maturation of cultured iRPE sheets for subsequent clinical applications might benefit from the use of nanofibrous scaffolds generated from natural proteins. Impact statement Induced retinal pigment epithelium (iRPE) from patient-derived induced pluripotent stem cells (iPSCs) may yield powerful treatments of retinal diseases, including age-related macular degeneration. Recent studies, including early human clinical trials, demonstrate the importance of selecting appropriate biomaterial scaffolds to support tissue-engineered iRPE sheets during implantation. Electrospun scaffolds show particular promise due to their similarity to the structure of the native Bruch's membrane. In this study, we describe the use of electroprocessed nanofibrous soy protein scaffolds to generate polarized sheets of human iPSC-derived iRPE sheets. Our evaluation, including RNA-seq transcriptomics, indicates that these scaffolds are viable alternatives to scaffolds electrospun from synthetic polymers.
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Diferenciación Celular , Células Madre Pluripotentes Inducidas/citología , Nanofibras/química , Epitelio Pigmentado de la Retina/citología , Proteínas de Soja/química , Andamios del Tejido/química , Línea Celular , Módulo de Elasticidad , Perfilación de la Expresión Génica , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Nanofibras/ultraestructura , Poliésteres/química , Epitelio Pigmentado de la Retina/ultraestructura , Proteínas de Soja/ultraestructuraRESUMEN
Human pluripotent stem cells (PSCs) can be differentiated into retinal organoids with proper neural layer organization, yet the protocols are technically challenging and time consuming. We have modified a widely used differentiation protocol by switching all-trans retinoic acid with 9-cis retinal to accelerate photoreceptor differentiation and improve morphogenesis. In this report, we provide a detailed and improved protocol to generate retinal organoids from human pluripotent stem cells. For complete details on the use and execution of this protocol, please refer to Kaya et al. (2019).
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Técnicas Citológicas , Organoides/citología , Células Madre Pluripotentes/citología , Células Fotorreceptoras Retinianas Bastones/citología , Diterpenos , Humanos , RetinaldehídoRESUMEN
Purpose: Retinal organoids (ROs) derived from human pluripotent stem cells largely recapitulate key features of in vivo retinal development, thus permitting the study of retinogenesis, disease modeling, and therapeutic development. However, the complexities of current protocols limit the use of this in vitro system in applications requiring large-scale production of organoids. Currently, widely used methods require the isolation of presumed optic vesicle-like structures from adherent cultures by dissection, a labor-intensive and time-consuming step that involves extensive practice and training. Method: We report a simple and efficient method for generating ROs by scraping the entire adherent culture and growing the resulting cell aggregates in a free-floating condition. Results: Within 1 to 7 days following the procedure, emerging morphologically well-defined optic vesicles can be identified and harvested with ease. The transition from two-dimensional (2D) to 3D culture condition favored the formation of ROs from areas devoid of typical optic vesicle-like structures, thus increasing the RO yield. Moreover, ROs generated by this approach were more often associated with the pigment epithelium. Conclusions: This improved, robust, and efficient protocol should facilitate large-scale differentiation of pluripotent stem cells into retinal organoids in support of human disease modeling and therapy development.
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Técnicas de Cultivo de Célula/métodos , Células Madre Pluripotentes Inducidas/citología , Organogénesis , Organoides/citología , Retina/citología , Epitelio Pigmentado de la Retina/citología , Línea Celular , Técnica del Anticuerpo Fluorescente , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Organoides/crecimiento & desarrollo , Organoides/metabolismo , Retina/crecimiento & desarrollo , Epitelio Pigmentado de la Retina/metabolismoRESUMEN
PURPOSE: Retinal organoids generated from human pluripotent stem cells exhibit considerable variability during differentiation. Our goals are to assess developmental maturity of the neural retina in vitro and design improved protocols based on objective criteria. METHODS: We performed transcriptome analyses of developing retinal organoids from human embryonic and induced pluripotent stem cell lines and utilized multiple bioinformatic tools for comparative analysis. Immunohistochemistry, immunoblotting and electron microscopy were employed for validation. RESULTS: We show that the developmental variability in organoids was reflected in gene expression profiles and could be evaluated by molecular staging with the human fetal and adult retinal transcriptome data. We also demonstrate that the addition of 9-cis retinal, instead of the widely used all-trans retinoic acid, accelerated rod photoreceptor differentiation in organoid cultures, with higher rhodopsin expression and more mature mitochondrial morphology evident by day 120. CONCLUSION: Our studies provide an objective transcriptome-based modality for determining the differentiation state of retinal organoids and for comparisons across different stem cell lines and platforms, which should facilitate disease modeling and evaluation of therapies in vitro.
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Diferenciación Celular , Diterpenos/farmacología , Células Madre Embrionarias Humanas/citología , Organoides/citología , Retina/citología , Células Fotorreceptoras Retinianas Bastones/citología , Retinaldehído/farmacología , Transcriptoma/genética , Diferenciación Celular/efectos de los fármacos , Línea Celular , Forma de la Célula/efectos de los fármacos , Perfilación de la Expresión Génica , Células Madre Embrionarias Humanas/efectos de los fármacos , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Organoides/efectos de los fármacos , Organoides/ultraestructura , Células Fotorreceptoras Retinianas Bastones/efectos de los fármacos , Transcriptoma/efectos de los fármacosRESUMEN
Ciliopathies display extensive genetic and clinical heterogeneity, varying in severity, age of onset, disease progression and organ systems affected. Retinal involvement, as demonstrated by photoreceptor dysfunction or death, is a highly penetrant phenotype among a vast majority of ciliopathies. Photoreceptor cells possess a specialized and modified sensory cilium with membrane discs where efficient photon capture and ensuing signaling cascade initiate the visual process. Disruptions of cilia biogenesis and protein transport lead to impairment of photoreceptor function and eventually degeneration. Despite advances in elucidation of ciliogenesis and photoreceptor cilia defects, we have limited understanding of pathogenic mechanisms underlying retinal phenotype(s) observed in human ciliopathies. Patient-derived induced pluripotent stem cell (iPSC)-based approaches offer a unique opportunity to complement studies with model organisms and examine cilia disease relevant to humans. Three-dimensional retinal organoids from iPSC lines feature laminated cytoarchitecture, apical-basal polarity and emergence of a ciliary structure, thereby permitting pathogenic modeling of human photoreceptors in vitro. Here, we review the biology of photoreceptor cilia and associated defects and discuss recent progress in evolving treatment modalities, especially using patient-derived iPSCs, for retinal ciliopathies.
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Stem cell-derived retinal organoids recapitulate many landmarks of in vivo differentiation but lack functional maturation of distinct cell types, especially photoreceptors. Using comprehensive temporal transcriptome analyses, we show that transcriptome shift from postnatal day 6 (P6) to P10, associated with morphogenesis and synapse formation during mouse retina development, was not evident in organoids, and co-expression clusters with similar patterns included different sets of genes. Furthermore, network analysis identified divergent regulatory dynamics between developing retina in vivo and in organoids, with temporal dysregulation of specific signaling pathways and delayed or reduced expression of genes involved in photoreceptor function(s) and survival. Accordingly, addition of docosahexaenoic acid and fibroblast growth factor 1 to organoid cultures specifically promoted the maturation of photoreceptors, including cones. Our study thus identifies regulatory signals deficient in developing retinal organoids and provides experimental validation by producing a more mature retina in vitro, thereby facilitating investigations in disease modeling and therapies.
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Organoides/citología , Retina/citología , Transcriptoma , Animales , Diferenciación Celular , Células Cultivadas , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Ratones , Ratones Endogámicos C57BL , Organoides/crecimiento & desarrollo , Organoides/metabolismo , Células Fotorreceptoras de Vertebrados/citología , Células Fotorreceptoras de Vertebrados/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Retina/crecimiento & desarrollo , Retina/metabolismo , Transducción de SeñalRESUMEN
Combinatorial expression of Brn3 transcription factors is required for the development of cell-specific morphologies in retinal ganglion cells (RGCs). The molecular mechanisms by which Brn3s regulate RGC type specific features are largely unexplored. We previously identified several members of the Copine (Cpne) family of molecules as potential targets of Brn3 transcription factors in the retina. We now use in situ hybridization and immunohistochemistry to characterize Copine expression in the postnatal and adult mouse retina. We find that Cpne5, 6, and 9 are expressed in the ganglion cell layer (GCL) and inner nuclear layer (INL) in both amacrine cells and RGCs. Cpne4 expression is restricted to one amacrine cell population of the INL, but is specifically expressed in RGCs in the GCL. Cpne4 expression in RGCs is regulated by Brn3b both cell autonomously (in Brn3b+ RGCs) and cell nonautonomously (in Brn3b- RGCs). Copines exhibit a variety of subcellular distributions when overexpressed in tissue culture cells (HEK293), and can induce the formation of elongated processes reminiscent of neurites in these non-neuronal cells. Our results suggest that Copines might be involved in a combinatorial fashion in Brn3b-dependent specification of RGC types. Given their expression profile and previously proven role as Ca2+ sensors, they may participate in the morphogenetic processes that shape RGC dendrite and axon formation at early postnatal ages.
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Proteínas de la Membrana/análisis , Proteínas de la Membrana/biosíntesis , Retina/química , Retina/metabolismo , Fracciones Subcelulares/química , Fracciones Subcelulares/metabolismo , Secuencia de Aminoácidos , Animales , Expresión Génica , Técnicas de Sustitución del Gen/métodos , Células HEK293 , Humanos , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Células Ganglionares de la Retina/química , Células Ganglionares de la Retina/metabolismoRESUMEN
Mutations in CEP290 cause ciliogenesis defects, leading to diverse clinical phenotypes, including Leber congenital amaurosis (LCA). Gene therapy for CEP290-associated diseases is hindered by the 7.4 kb CEP290 coding sequence, which is difficult to deliver in vivo. The multi-domain structure of the CEP290 protein suggests that a specific CEP290 domain may complement disease phenotypes. Thus, we constructed AAV vectors with overlapping CEP290 regions and evaluated their impact on photoreceptor degeneration in Cep290rd16/rd16 and Cep290rd16/rd16;Nrl-/- mice, two models of CEP290-LCA. One CEP290 fragment (the C-terminal 989 residues, including the domain deleted in mutant mice) reconstituted CEP290 function and resulted in cone preservation and delayed rod death. The CEP290 C-terminal domain also improved cilia phenotypes in mouse embryonic fibroblasts and iPSC-derived retinal organoids carrying the Cep290rd16 mutation. Our study strongly argues for in trans complementation of CEP290 mutations by a cognate fragment and suggests therapeutic avenues.
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Antígenos de Neoplasias/genética , Antígenos de Neoplasias/fisiología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiología , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/fisiología , Terapia Genética , Amaurosis Congénita de Leber/terapia , Mutación , Retina/citología , Degeneración Retiniana/terapia , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/fisiología , Cilios/metabolismo , Cilios/patología , Dependovirus/genética , Modelos Animales de Enfermedad , Proteínas del Ojo/fisiología , Femenino , Humanos , Amaurosis Congénita de Leber/genética , Amaurosis Congénita de Leber/patología , Masculino , Ratones , Ratones Noqueados , Retina/metabolismo , Células Fotorreceptoras Retinianas Conos , Degeneración Retiniana/genética , Degeneración Retiniana/patologíaRESUMEN
To obtain immunogenic conjugate antigens, adipic acid dihydrazide (ADH), as a bridge, and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride (EDAC), as a coupling agent, were used to conjugate the purified fusion protein, clumping factor A-fibronectin binding protein ClfA A-FnBPA, and type 5 capsular polysaccharide (CP5). The conjugates were mixed with an adjuvant, and mice were immunized 3 times and challenged with Staphylococcus aureus 1 week later. Antibody titers were determined by indirect enzyme-linked immunosorbent assay (ELISA). At 14 days after the first immunization, antibodies against the purified protein and conjugate were detected; after 28 days, antibody levels increased; and a week after the third immunization, antibody levels continued to increase. However, the conjugate antibody titers were higher than those of the purified protein during the study, and no IgG antibodies against purified CP5 were detected during the entire experiment. The protection rate increased to 90% in the conjugate group, indicating that the conjugate imparts a relatively higher protective efficacy than the purified protein and purified CP5.
Afin d'obtenir des antigènes conjugués immunogéniques, du dihydrazide adipique acide (DHA), comme pont, et le 1-éthyl-3-(3-diméthylaminopropyl) carbodiimidehydhrochlorure (EDAC), comme agent liguant, ont été utilisés pour conjuguer la protéine de fusion purifiée ClfAA-FnBPA (facteur de coagulation A-protéine liant la fibronectine) et le polysaccharide capsulaire de type 5 (PC5). Les conjugués ont été mélangés avec un adjuvant, et des souris immunisées trois fois et soumis à une infection défi par Staphylococcus aureus 1 semaine plus tard. Les titres d'anticorps ont été déterminés par épreuve immuno-enzymatique indirecte (ELISA). Au jour 14 suivant la première immunisation, des anticorps dirigés contre la protéine purifiée et le conjugué ont été détectés; après 28 jours, les taux d'anticorps ont augmenté, et 1 semaine après la troisième immunisation, les taux d'anticorps ont continué d'augmenter. Toutefois, durant l'étude les titres d'anticorps envers le conjugué étaient supérieurs à ceux de la protéine purifiée, et aucun anticorps de type IgG dirigé contre le PC5 purifié ne fut détecté durant l'expérience entière. Le taux de protection augmenta jusqu'à 90 % dans le groupe du conjugué, indiquant que le conjugué entraîne une protection efficace supérieure à la protéine purifiée et au PC5 purifié.(Traduit par Docteur Serge Messier).
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Adhesinas Bacterianas/inmunología , Cápsulas Bacterianas/inmunología , Vacunas Bacterianas/inmunología , Coagulasa/inmunología , Infecciones Estafilocócicas/prevención & control , Staphylococcus aureus/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Animales , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/inmunología , Clonación Molecular , Femenino , Ratones , Ratones Endogámicos BALB C , Distribución Aleatoria , Infecciones Estafilocócicas/microbiologíaRESUMEN
Mutations in CEP290, a transition zone protein in primary cilia, cause diverse ciliopathies, including Leber congenital amaurosis (LCA) and Joubert-syndrome and related disorders (JSRD). We examined cilia biogenesis and function in cells derived from CEP290-LCA and CEP290-JSRD patients. CEP290 protein was reduced in LCA fibroblasts with no detectable impact on cilia; however, optic cups derived from induced pluripotent stem cells (iPSCs) of CEP290-LCA patients displayed less developed photoreceptor cilia. Lack of CEP290 in JSRD fibroblasts resulted in abnormal cilia and decreased ciliogenesis. We observed selectively reduced localization of ADCY3 and ARL13B. Notably, Hedgehog signaling was augmented in CEP290-JSRD because of enhanced ciliary transport of Smoothened and GPR161. These results demonstrate a direct correlation between the extent of ciliogenesis defects in fibroblasts and photoreceptors with phenotypic severity in JSRD and LCA, respectively, and strengthen the role of CEP290 as a selective ciliary gatekeeper for transport of signaling molecules in and out of the cilium.
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Antígenos de Neoplasias/genética , Fibroblastos/metabolismo , Proteínas de Neoplasias/genética , Factores de Ribosilacion-ADP/genética , Factores de Ribosilacion-ADP/metabolismo , Adenilil Ciclasas/genética , Adenilil Ciclasas/metabolismo , Alelos , Animales , Antígenos de Neoplasias/metabolismo , Proteínas de Ciclo Celular , Cilios , Proteínas del Citoesqueleto , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Homocigoto , Humanos , Ratones , Ratones Noqueados , Mutación/genética , Proteínas de Neoplasias/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptor Smoothened/genética , Receptor Smoothened/metabolismoRESUMEN
Bovine viral diarrhea-mucosal disease (BVD-MD) is caused by bovine viral diarrhea virus (BVDV), and results in abortion, stillbirth, and fetal malformation in cows. Here, we constructed the phage display vector pCANTAB 5E-VHH and then transformed it into Escherichia coli TG1-competent cells, to construct an initial anti-BVDV nanobody gene library. We obtained a BVDV-E2 antigen epitope bait protein by prokaryotic expression using the nucleotide sequence of the E2 gene of the BVDV-NADL strain published in GenBank. Phage display was used to screen the anti-BVDV nanobody gene library. We successfully constructed a high quality phage display nanobody library, with an initial library capacity of 4.32×105. After the rescue of helper phage, the titer of the phage display nanobody library was 1.3×1011. The BVDV-E2 protein was then expressed in Escherichia coli (DE3), and a 49.5 kDa band was observed with SDS-PAGE analysis that was consistent with the expected nanobody size. Thus, we were able to isolate one nanobody that exhibits high affinity and specificity against BVDV using phage display techniques. This isolated nanobody was then used in Enzyme Linked Immunosorbent Assay and qRT-PCR, and ELISA analyses of BVDV infection of MDBK cells indicated that the nanobodies exhibited good antiviral effect.
Asunto(s)
Anticuerpos Antivirales/biosíntesis , Antígenos Virales/genética , Virus de la Diarrea Viral Bovina/genética , Epítopos/genética , Anticuerpos de Dominio Único/biosíntesis , Proteínas del Envoltorio Viral/genética , Secuencia de Aminoácidos , Animales , Anticuerpos Antivirales/genética , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Antígenos Virales/inmunología , Secuencia de Bases , Bovinos , Línea Celular , Virus de la Diarrea Viral Bovina/inmunología , Ensayo de Inmunoadsorción Enzimática , Células Epiteliales/inmunología , Células Epiteliales/patología , Células Epiteliales/virología , Epítopos/inmunología , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Riñón/inmunología , Riñón/patología , Riñón/virología , Biblioteca de Péptidos , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Anticuerpos de Dominio Único/genética , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
The polycistronic miR-183/96/182 cluster is preferentially and abundantly expressed in terminally differentiating sensory epithelia. To clarify its roles in the terminal differentiation of sensory receptors in vivo, we deleted the entire gene cluster in mouse germline through homologous recombination. The miR-183/96/182 null mice display impairment of the visual, auditory, vestibular, and olfactory systems, attributable to profound defects in sensory receptor terminal differentiation. Maturation of sensory receptor precursors is delayed, and they never attain a fully differentiated state. In the retina, delay in up-regulation of key photoreceptor genes underlies delayed outer segment elongation and possibly mispositioning of cone nuclei in the retina. Incomplete maturation of photoreceptors is followed shortly afterward by early-onset degeneration. Cell biologic and transcriptome analyses implicate dysregulation of ciliogenesis, nuclear translocation, and an epigenetic mechanism that may control timing of terminal differentiation in developing photoreceptors. In both the organ of Corti and the vestibular organ, impaired terminal differentiation manifests as immature stereocilia and kinocilia on the apical surface of hair cells. Our study thus establishes a dedicated role of the miR-183/96/182 cluster in driving the terminal differentiation of multiple sensory receptor cells.