RESUMO
Variants within the high copy number mitochondrial genome (mtDNA) can disrupt organelle function and lead to severe multisystem disease. The wide range of manifestations observed in patients with mitochondrial disease results from varying fractions of abnormal mtDNA molecules in different cells and tissues, a phenomenon termed heteroplasmy. However, the landscape of heteroplasmy across cell types within tissues and its influence on phenotype expression in affected patients remains largely unexplored. Here, we identify nonrandom distribution of a pathogenic mtDNA variant across a complex tissue using single-cell RNA-Seq, mitochondrial single-cell ATAC sequencing, and multimodal single-cell sequencing. We profiled the transcriptome, chromatin accessibility state, and heteroplasmy in cells from the eyes of a patient with mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) and from healthy control donors. Utilizing the retina as a model for complex multilineage tissues, we found that the proportion of the pathogenic m.3243A>G allele was neither evenly nor randomly distributed across diverse cell types. All neuroectoderm-derived neural cells exhibited a high percentage of the mutant variant. However, a subset of mesoderm-derived lineage, namely the vasculature of the choroid, was near homoplasmic for the WT allele. Gene expression and chromatin accessibility profiles of cell types with high and low proportions of m.3243A>G implicate mTOR signaling in the cellular response to heteroplasmy. We further found by multimodal single-cell sequencing of retinal pigment epithelial cells that a high proportion of the pathogenic mtDNA variant was associated with transcriptionally and morphologically abnormal cells. Together, these findings show the nonrandom nature of mitochondrial variant partitioning in human mitochondrial disease and underscore its implications for mitochondrial disease pathogenesis and treatment.
Assuntos
Síndrome MELAS , Doenças Mitocondriais , Doenças Retinianas , Humanos , Heteroplasmia , Síndrome MELAS/genética , Síndrome MELAS/metabolismo , Síndrome MELAS/patologia , Doenças Mitocondriais/genética , DNA Mitocondrial/genética , Retina/patologia , CromatinaRESUMO
The effects of radiation retinopathy on the retinal vasculature have been well established; however, the literature describing the pathologic changes in the choriocapillaris is relatively lacking. In this report, we describe the histologic findings of a donor eye with a choroidal melanoma with special attention to the choriocapillaris. Clinical and histological findings, including immunohistochemistry and transmission electron microscopy, are described for the retina and choroid of a donor eye affected by radiation retinopathy secondary to treatment of choroidal melanoma. Cells within the tumor exhibited an epithelioid structure and balloon melanosomes. Notable infiltration of macrophages with elongated morphology was also observed. Atrophy of photoreceptors, retinal pigmented epithelium, and choriocapillaris was observed on the inferior edge of the lesion and extending past the tumor. The choriocapillaris endothelium showed more severe dropout at the periphery of the lesion where loss of fenestration, thickened cytosol, and degenerated pericytes were observed. Morphologic analysis revealed choriocapillaris loss with pronounced degeneration of choroidal pericytes. Understanding the differences in sensitivity to radiation injury between different cell types and different patients will provide better insight into radiation retinopathy.
RESUMO
The m.3243A>G mutation in the mitochondrial genome commonly causes retinal degeneration in patients with maternally inherited diabetes and deafness and mitochondrial encephalopathy, lactic acidosis and stroke-like episodes. Like other mitochondrial mutations, m.3243A>G is inherited from the mother with a variable proportion of wild type and mutant mitochondrial genomes in different cells. The mechanism by which the m.3243A>G variant in each tissue relates to the manifestation of disease phenotype is not fully understood. Using a digital PCR assay, we found that the % m.3243G in skin derived dermal fibroblasts was positively correlated with that of blood from the same individual. The % m.3243G detected in fibroblast cultures remained constant over multiple passages and was negatively correlated with mtDNA copy number. Although the % m.3243G present in blood was not correlated with severity of vision loss, as quantified by Goldmann visual field, a significant negative correlation between % m.3243G and the age of onset of visual symptoms was detected. Altogether, these results indicate that precise measurement of % m.3243G in clinically accessible tissues such as skin and blood may yield information relevant to the management of retinal m.3243A>G-associated disease.
Assuntos
Diabetes Mellitus Tipo 2 , Síndrome MELAS , Doenças Mitocondriais , DNA Mitocondrial/genética , Surdez , Diabetes Mellitus Tipo 2/genética , Humanos , Síndrome MELAS/genética , Doenças Mitocondriais/genética , MutaçãoRESUMO
The endothelium responds to numerous chemical and mechanical factors in regulating vascular tone, blood pressure, and blood flow. The endothelial volume-regulated anion channel (VRAC) has been proposed to be mechanosensitive and thereby sense fluid flow and hydrostatic pressure to regulate vascular function. Here, we show that the leucine-rich repeat-containing protein 8a, LRRC8A (SWELL1), is required for VRAC in human umbilical vein endothelial cells (HUVECs). Endothelial LRRC8A regulates AKT-endothelial nitric oxide synthase (eNOS) signaling under basal, stretch, and shear-flow stimulation, forms a GRB2-Cav1-eNOS signaling complex, and is required for endothelial cell alignment to laminar shear flow. Endothelium-restricted Lrrc8a KO mice develop hypertension in response to chronic angiotensin-II infusion and exhibit impaired retinal blood flow with both diffuse and focal blood vessel narrowing in the setting of type 2 diabetes (T2D). These data demonstrate that LRRC8A regulates AKT-eNOS in endothelium and is required for maintaining vascular function, particularly in the setting of T2D.
Assuntos
Endotélio/fisiologia , Proteínas de Membrana/genética , Óxido Nítrico Sintase Tipo III/genética , Proteínas Proto-Oncogênicas c-akt/genética , Animais , Feminino , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Óxido Nítrico Sintase Tipo III/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: Endothelial cells (ECs) are essential regulators of the vasculature, lining arteries, veins, and capillary beds. While all ECs share a number of structural and molecular features, heterogeneity exists depending on their resident tissue. ECs lining the choriocapillaris in the human eye are lost early in the pathogenesis of age-related macular degeneration (AMD), a common and devastating form of vision loss. In order to study the mechanisms leading to choroidal endothelial cell (CEC) loss and to develop reagents for repairing the choroid, a reproducible in vitro model, which closely mimic CECs, is needed. While a number of protocols have been published to direct induced pluripotent stem cells (iPSCs) into ECs, the goal of this study was to develop methods to differentiate iPSCs into ECs resembling those found in the human choriocapillaris specifically. METHODS: We transduced human iPSCs with a CDH5p-GFP-ZEO lentiviral vector and selected for transduced iPSCs using blasticidin. We generated embryoid bodies (EBs) from expanded iPSC colonies and transitioned from mTESR™1 to EC media. One day post-EB formation, we induced mesoderm fate commitment via addition of BMP-4, activin A, and FGF-2. On day 5, EBs were adhered to Matrigel-coated plates in EC media containing vascular endothelial cell growth factor (VEGF) and connective tissue growth factor (CTGF) to promote CEC differentiation. On day 14, we selected for CECs using either zeocin resistance or anti-CD31 MACS beads. We expanded CECs post-selection and performed immunocytochemical analysis of CD31, carbonic anhydrase IV (CA4), and RGCC; tube formation assays; and transmission electron microscopy to access vascular function. RESULTS: We report a detailed protocol whereby we direct iPSC differentiation toward mesoderm and utilize CTGF to specify CECs. The CDH5p-GFP-ZEO lentiviral vector facilitated the selection of iPSC-derived ECs that label with antibodies directed against CD31, CA4, and RGCC; form vascular tubes in vitro; and migrate into empty choroidal vessels. CECs selected using either antibiotic selection or CD31 MACS beads showed similar characteristics, thereby making this protocol easily reproducible with or without lentiviral vectors. CONCLUSION: ECs generated following this protocol exhibit functional and biochemical characteristics of CECs. This protocol will be useful for developing in vitro models toward understanding the mechanisms of CEC loss early in AMD.
Assuntos
Células-Tronco Pluripotentes Induzidas , Diferenciação Celular , Células Cultivadas , Corioide , Células Endoteliais , Endotélio , HumanosRESUMO
Advances in the discovery of the causes of monogenic retinal disorders, combined with technologies for the delivery of DNA to the retina, offer enormous opportunities for the treatment of previously untreatable blinding diseases. However, for gene augmentation to be most effective, vectors that have the correct cell-type specificity are needed. While animal models are very useful, they often exhibit differences in retinal cell surface receptors compared to the human retina. This study evaluated the use of an ex vivo organotypic explant system to test the transduction efficiency and tropism of seven different adeno-associated virus type 2 (AAV2) serotypes in the human retina and retinal pigment epithelium-choroid-AAV2/1, AAV2/2, AAV2/4, AAV2/5, AAV2/6, AAV2/8, and AAV2/9-all driving expression of GFP under control of the cytomegalovirus promoter. After 7 days in culture, it was found that AAV2/4 and AAV2/5 were particularly efficient at transducing photoreceptor cells and that AAV2/5 was highly specific to the outer nuclear layer, whereas AAV2/8 displayed consistently low transduction of photoreceptors. To validate the authenticity of the organotypic culture system, the transduction of the same set of AAVs was also compared in a pig model, in which sub-retinal injections in vivo were compared to cultured and transduced organotypic cultures ex vivo. This study shows how different AAV serotypes behave in the human retina and provides insight for further investigation of each of these serotypes for gene augmentation-based treatment of inherited retinal degeneration.
Assuntos
Dependovirus/genética , Vetores Genéticos , Retina/metabolismo , Degeneração Retiniana/terapia , Animais , Técnicas de Transferência de Genes , Humanos , Camundongos , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/patologia , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patologia , Retina/fisiopatologia , Degeneração Retiniana/genética , Degeneração Retiniana/patologia , Epitélio Pigmentado da Retina , Suínos , Transdução Genética , Tropismo/genéticaRESUMO
Objective: Mutations in myocilin (MYOC) may cause either juvenile open angle glaucoma (JOAG) or adult-onset primary open angle glaucoma (POAG). MYOC encodes a glycoprotein that is normally secreted from trabecular meshwork cells that regulate intraocular pressure. Prior in vitro, transgenic rodent, and organ culture experiments have suggested that abnormal accumulation of MYOC protein within trabecular meshwork cells is a key step in glaucoma pathophysiology. We investigated the pathogenesis of MYOC glaucoma by examining a donor eye from a patient with JOAG caused by a Tyr437His MYOC mutation. Design: Case-control, immunohistochemical study of a donor eye from a patient with JOAG caused by a Tyr437His MYOC mutation and age-matched control donor eyes. Subjects: An eye from a 59-year-old male with JOAG caused by a Tyr437His MYOC mutation and eyes from five donors (ages 51-66) with no known ocular disease were examined. Methods: Frozen fixed sections of the iridocorneal angle were prepared from the donor eyes of the MYOC glaucoma patient and control eyes. We used antibodies directed against MYOC, collagen IV, and BiP/GRP78 as well as wheat germ agglutinin and concanavalin A lectins to localize MYOC protein in the trabecular meshwork. Main Outcome Measure: Qualitative comparison of MYOC protein labeling and localization in the trabecular meshwork of donor eyes from a glaucoma patient with a MYOC mutation and from control subjects. Results: Using immunohistochemistry, we detected more abundant MYOC protein within the trabecular meshwork of the MYOC glaucoma patient's eye than in control eyes. We further localized MYOC protein within the trabecular meshwork cells of the MYOC glaucoma patient's eye by co-labeling with the endoplasmic reticulum (ER) marker GRP78 (BiP). Little to no MYOC was identified within the trabecular meshwork cells of control eyes. Minimal extracellular MYOC was detected in both MYOC glaucoma eyes and control eyes. Conclusions: This is the first histopathological analysis of an eye from a glaucoma patient with a MYOC mutation. Furthermore, this analysis supports our model of MYOC-associated glaucoma, in which MYOC mutations cause abnormal intracellular retention of MYOC within the ER of trabecular meshwork cells as a key step towards development of glaucoma.
Assuntos
Humor Aquoso/metabolismo , Proteínas do Citoesqueleto/genética , DNA/genética , Proteínas do Olho/genética , Glaucoma de Ângulo Aberto/genética , Glicoproteínas/genética , Imuno-Histoquímica/métodos , Pressão Intraocular/fisiologia , Mutação , Doadores de Tecidos , Malha Trabecular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Chaperona BiP do Retículo Endoplasmático , Proteínas do Olho/metabolismo , Feminino , Glaucoma de Ângulo Aberto/metabolismo , Glaucoma de Ângulo Aberto/fisiopatologia , Glicoproteínas/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
Age-related macular degeneration (AMD) is a leading cause of irreversible blindness in the Western world. Although, the majority of stem cell research to date has focused on production of retinal pigment epithelial (RPE) and photoreceptor cells for the purpose of evaluating disease pathophysiology and cell replacement, there is strong evidence that the choroidal endothelial cells (CECs) that form the choriocapillaris vessels are the first to be lost in this disease. As such, to accurately evaluate disease pathophysiology and develop an effective treatment, production of patient-specific, stem cell-derived CECs will be required. In this study, we report for the first time a stepwise differentiation protocol suitable for generating human iPSC-derived CEC-like cells. RNA-seq analysis of the monkey CEC line, RF/6A, combined with two statistical screens allowed us to develop media comprised of various protein combinations. In both screens, connective tissue growth factor (CTGF) was identified as the key component required for driving CEC development. A second factor tumor necrosis factor (TNF)-related weak inducer of apoptosis receptor was also found to promote iPSC to CEC differentiation by inducing endogenous CTGF secretion. CTGF-driven iPSC-derived CEC-like cells formed capillary tube-like vascular networks, and expressed the EC-specific markers CD31, ICAM1, PLVAP, vWF, and the CEC-restricted marker CA4. In combination with RPE and photoreceptor cells, patient-specific iPSC derived CEC-like cells will enable scientists to accurately evaluate AMD pathophysiology and develop effective cell replacement therapies. Stem Cells Translational Medicine 2017;6:1533-1546.
Assuntos
Corioide/citologia , Células Endoteliais/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Degeneração Macular/terapia , Animais , Diferenciação Celular , Células Cultivadas , Técnicas de Reprogramação Celular/métodos , Corioide/fisiologia , Fator de Crescimento do Tecido Conjuntivo/farmacologia , Células Endoteliais/efeitos dos fármacos , Endotélio/citologia , Endotélio/fisiologia , Haplorrinos , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Regeneração , Transplante de Células-Tronco/métodosRESUMO
Endothelial cells (ECs) of the choriocapillaris are one of the first cell types lost during age-related macular degeneration (AMD), and cell replacement therapy is currently a very promising option for patients with advanced AMD. We sought to develop a reliable method for the production of human choroidal extracellular matrix (ECM) scaffolds, which will allow for the study of choroidal EC (CEC) replacement strategies in an environment that closely resembles the native tissue. Human RPE/choroid tissue was treated sequentially with Triton X-100, SDS, and DNase to remove all native cells. While all cells were successfully removed from the tissue, collagen IV, elastin, and laminin remained, with preserved architecture of the acellular vascular tubes. The ECM scaffolds were then co-cultured with exogenous ECs to determine if the tissue can support cell growth and allow EC reintegration into the decellularized choroidal vasculature. Both monkey and human ECs took up residence in the choriocapillary tubes of the decellularized tissue. Together, these data suggest that our decellularization methods are sufficient to remove all cellular material yet gentle enough to preserve tissue structure and allow for the optimization of cell replacement strategies. STATEMENT OF SIGNIFICANCE: Age-related macular degeneration (AMD) is a devastating disease affecting more than 600 million people worldwide. Endothelial cells of the choriocapillaris (CECs) are among the first cell types lost in early AMD, and cell replacement therapy is currently the most promising option for restoring vision in patients with advanced AMD. In order to study CEC replacement strategies we have generated a 3D choroid scaffold using a novel decellularization method in human RPE/choroid tissue. To our knowledge, this is the first report describing decellularization of human RPE/choroid, as well as recellularization of a choroid scaffold with CECs. This work will aid in our development and optimization of cell replacement strategies using a tissue scaffold that is similar to the in vivo environment.
Assuntos
Corioide/química , Proteínas da Matriz Extracelular/química , Matriz Extracelular/química , Alicerces Teciduais/química , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Degeneração Macular/terapia , MasculinoRESUMO
Age-related macular degeneration (AMD) is a common, blinding disease of the elderly in which macular photoreceptor cells, retinal pigment epithelium and choriocapillaris endothelial cells ultimately degenerate. Recent studies have found that degeneration of the choriocapillaris occurs early in this disease and that endothelial cell drop-out is concomitant with increased deposition of the complement membrane attack complex (MAC) at the choroidal endothelium. However, the impact of MAC injury to choroidal endothelial cells is poorly understood. To model this event in vitro, and to study the downstream consequences of MAC injury, endothelial cells were exposed to complement from human serum, compared to heat-inactivated serum, which lacks complement components. Cells exposed to complement components in human serum showed increased labelling with antibodies directed against the MAC, time- and dose-dependent cell death, as assessed by lactate dehydrogenase assay and increased permeability. RNA-Seq analysis following complement injury revealed increased expression of genes associated with angiogenesis including matrix metalloproteinase (MMP)-3 and -9, and VEGF-A. The MAC-induced increase in MMP9 RNA expression was validated using C5-depleted serum compared to C5-reconstituted serum. Increased levels of MMP9 were also established, using western blot and zymography. These data suggest that, in addition to cell lysis, complement attack on choroidal endothelial cells promotes an angiogenic phenotype in surviving cells.
Assuntos
Corioide/imunologia , Complexo de Ataque à Membrana do Sistema Complemento/imunologia , Proteínas do Sistema Complemento/farmacologia , Células Endoteliais/imunologia , Degeneração Macular/etiologia , Idoso , Idoso de 80 Anos ou mais , Anticorpos/metabolismo , Morte Celular/fisiologia , Células Cultivadas , Corioide/irrigação sanguínea , Ativação do Complemento/fisiologia , Relação Dose-Resposta Imunológica , Feminino , Humanos , Degeneração Macular/imunologia , Degeneração Macular/patologia , Masculino , Metaloproteinase 3 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
PURPOSE: Age-related macular degeneration (AMD), the most common cause of incurable blindness in the western world, is characterized by the dysfunction and eventual death of choroidal endothelial (CECs), RPE, and photoreceptor cells. Stem cell-based treatment strategies designed to replace photoreceptor and RPE cells currently are a major scientific focus. However, the success of these approaches likely also will require replacement of the underlying, supportive choroidal vasculature. The purpose of this study was to generate stem cell-derived CECs to develop efficient differentiation and transplantation protocols. METHODS: Dermal fibroblasts from the Tie2-GFP mouse were isolated and reprogrammed into two independent induced pluripotent stem cell (iPSC) lines via viral transduction of the transcription factors Oct4, Sox2, Klf4, and c-Myc. Tie2-GFP iPSCs were differentiated into CECs using a coculture method with either the RF6A CEC line or primary mouse CECs. Induced pluripotent stem cell-derived CECs were characterized via RT-PCR and immunocytochemistry for EC- and CEC-specific markers. RESULTS: Induced pluripotent stem cells generated from mice expressing green fluorescent protein (GFP) under control of the endothelial Tie2 promoter display classic pluripotency markers and stem cell morphology. Induced pluripotent stem cell-derived CECs express carbonic anhydrase IV, eNOS, FOXA2, PLVAP, CD31, CD34, ICAM-1, Tie2, TTR, VE-cadherin, and vWF. CONCLUSIONS: Induced pluripotent stem cell-derived CECs will be a valuable tool for modeling of choriocapillaris-specific insults in AMD and for use in future choroidal endothelial cell replacement approaches.
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Corioide/patologia , Células Endoteliais/patologia , Células-Tronco Pluripotentes Induzidas/citologia , Degeneração Macular/metabolismo , Animais , Animais Recém-Nascidos , Diferenciação Celular , Células Cultivadas , Células Endoteliais/metabolismo , Imuno-Histoquímica , Células-Tronco Pluripotentes Induzidas/metabolismo , Fator 4 Semelhante a Kruppel , Degeneração Macular/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
BACKGROUND: Autosomal dominant radial drusen (ADRD), also termed Malattia Leventinese and Doyne honeycomb retinal dystrophy, causes early-onset vision loss because of mutation in EFEMP1. Drusen in an exceedingly rare ADRD human donor eye was compared with eyes affected with age-related macular degeneration (AMD). This study also elucidated whether variations in high-risk AMD genotypes modify phenotypic severity of ADRD. METHODS: Morphologic and histochemical analyses of drusen in one ADRD donor and seven AMD donors. Evaluation of complement factor H (CFH) and ARMS2/HTRA1 alleles in a cohort of 25 subjects with ADRD. RESULTS: Autosomal dominant radial drusen had unique onion skin-like lamination but otherwise shared many compositional features with hard, nodular drusen and/or diffuse soft drusen with basal deposits. Autosomal dominant radial drusen also possessed collagen type IV, an extracellular matrix protein that is absent in age-related drusen. Antibodies directed against the membrane attack complex showed robust labeling of ADRD. Vitronectin and amyloid P were present in drusen of both types. High-risk alleles in the CFH and ARMS2/HTRA1 genes were not associated with increasing ADRD severity. CONCLUSION: Drusen from ADRD and AMD exhibit overlap of some major constituents, but ADRD exhibit distinct alterations in the extracellular matrix that are absent in AMD.
Assuntos
Fator H do Complemento/genética , Distrofias Hereditárias da Córnea/genética , Polimorfismo de Nucleotídeo Único , Proteínas/genética , Drusas Retinianas/genética , Serina Endopeptidases/genética , Degeneração Macular Exsudativa/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Colágeno Tipo IV/metabolismo , Distrofias Hereditárias da Córnea/metabolismo , Distrofias Hereditárias da Córnea/patologia , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Feminino , Técnicas de Genotipagem , Serina Peptidase 1 de Requerimento de Alta Temperatura A , Humanos , Masculino , Pessoa de Meia-Idade , Drusas do Disco Óptico/congênito , Drusas Retinianas/metabolismo , Drusas Retinianas/patologia , Componente Amiloide P Sérico/metabolismo , Doadores de Tecidos , Vitronectina/metabolismo , Degeneração Macular Exsudativa/metabolismo , Degeneração Macular Exsudativa/patologia , Adulto JovemRESUMO
PURPOSE: The gene that causes normal tension glaucoma (NTG) in a large pedigree was recently mapped to a region of chromosome 12q14 (GLC1P) that contains the genes TBK1, XPOT, RASSF3, and GNS. We sought to investigate the structure of the chromosome 12q14 duplication and explore the ocular expression of GLC1P locus genes. METHODS: The location of the chromosome 12q14 duplication in this pedigree was examined with fluorescent in situ hybridization (FISH) using probes for TBK1 and GNS. The expression pattern of XPOT, TBK1, RASSF3, and GNS was investigated with immunohistochemistry of human eyes. RESULTS: The karyotype of an NTG patient from pedigree GGO-414 was normal and FISH studies demonstrated that the duplicated DNA is organized as a tandem repeat on chromosome 12q14. Of the genes in or near the chromosome 12q14 duplication, TBK1 showed expression in the retina that is specific to the retinal ganglion cells and the retinal nerve fiber layer. Expression of RASSF3 and XPOT was relatively uniform throughout the retina, while GNS expression was expressed in a pattern consistent with Müller cells. CONCLUSIONS: Previous studies demonstrated that chromosome 12q14 duplications are associated with NTG inherited as an autosomal dominant trait. FISH studies now demonstrate that the duplicated segments are tandemly organized on chromosome 12q14 in close proximity. The specific expression of TBK1 in human retinal ganglion cells compared to the widespread pattern of expression of neighboring genes provides additional evidence that TBK1 is the glaucoma gene in the chromosome 12q14 duplication within the GLC1P locus.
Assuntos
Regulação da Expressão Gênica/fisiologia , Glaucoma de Baixa Tensão/genética , Proteínas Serina-Treonina Quinases/genética , Células Ganglionares da Retina/metabolismo , Trissomia/genética , Idoso , Idoso de 80 Anos ou mais , Células Cultivadas , Cromossomos Humanos Par 12/genética , Sondas de DNA , Feminino , Fibroblastos/metabolismo , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Cariotipagem , Masculino , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas de Transporte Nucleocitoplasmático/genética , Linhagem , Pele/citologia , Sulfatases/genéticaRESUMO
Next-generation and Sanger sequencing were combined to identify disease-causing USH2A mutations in an adult patient with autosomal recessive RP. Induced pluripotent stem cells (iPSCs), generated from the patient's keratinocytes, were differentiated into multi-layer eyecup-like structures with features of human retinal precursor cells. The inner layer of the eyecups contained photoreceptor precursor cells that expressed photoreceptor markers and exhibited axonemes and basal bodies characteristic of outer segments. Analysis of the USH2A transcripts of these cells revealed that one of the patient's mutations causes exonification of intron 40, a translation frameshift and a premature stop codon. Western blotting revealed upregulation of GRP78 and GRP94, suggesting that the patient's other USH2A variant (Arg4192His) causes disease through protein misfolding and ER stress. Transplantation into 4-day-old immunodeficient Crb1 (-/-) mice resulted in the formation of morphologically and immunohistochemically recognizable photoreceptor cells, suggesting that the mutations in this patient act via post-developmental photoreceptor degeneration. DOI:http://dx.doi.org/10.7554/eLife.00824.001.
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Células-Tronco Pluripotentes Induzidas/citologia , Células Fotorreceptoras de Vertebrados/patologia , Retinose Pigmentar/patologia , Animais , Western Blotting , Diferenciação Celular , Códon de Terminação , Chaperona BiP do Retículo Endoplasmático , Humanos , Camundongos , Camundongos Knockout , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Retinose Pigmentar/genéticaRESUMO
Ocular involvement in muscular dystrophy ranges from structural defects to abnormal electroretinograms. While the mechanisms underlying the abnormal retinal physiology in patients are not understood, it is thought that alpha-dystroglycan extracellular interactions are critical for normal visual function. Here we show that beta-dystroglycan anchors dystrophin and the inward rectifying K(+) channel Kir4.1 at glial endfeet and that disruption of dystrophin and potassium channel clustering in dystroglycan mutant mice is associated with an attenuation of the electroretinogram b-wave. Glial-specific inactivation of dystroglycan or deletion of the cytoplasmic domain of beta-dystroglycan was sufficient to attenuate the electroretinogram b-wave. Unexpectedly, deletion of the beta-dystroglycan cytoplasmic domain did not disrupt the laminar structure of the retina. In contrast to the role of alpha-dystroglycan extracellular interactions during early development of the CNS, beta-dystroglycan intracellular interactions are important for visual function but not the laminar development of the retina.