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1.
Genet Med ; 26(6): 101105, 2024 06.
Artigo em Inglês | MEDLINE | ID: mdl-38430071

RESUMO

PURPOSE: To describe a recessively inherited cerebral small vessel disease, caused by loss-of-function variants in Nitrilase1 (NIT1). METHODS: We performed exome sequencing, brain magnetic resonance imaging, neuropathology, electron microscopy, western blotting, and transcriptomic and metabolic analyses in 7 NIT1-small vessel disease patients from 5 unrelated pedigrees. RESULTS: The first identified patients were 3 siblings, compound heterozygous for the NIT1 c.727C>T; (p.Arg243Trp) variant and the NIT1 c.198_199del; p.(Ala68∗) variant. The 4 additional patients were single cases from 4 unrelated pedigrees and were all homozygous for the NIT1 c.727C>T; p.(Arg243Trp) variant. Patients presented in mid-adulthood with movement disorders. All patients had striking abnormalities on brain magnetic resonance imaging, with numerous and massively dilated basal ganglia perivascular spaces. Three patients had non-lobar intracerebral hemorrhage between age 45 and 60, which was fatal in 2 cases. Western blotting on patient fibroblasts showed absence of NIT1 protein, and metabolic analysis in urine confirmed loss of NIT1 enzymatic function. Brain autopsy revealed large electron-dense deposits in the vessel walls of small and medium sized cerebral arteries. CONCLUSION: NIT1-small vessel disease is a novel, autosomal recessively inherited cerebral small vessel disease characterized by a triad of movement disorders, massively dilated basal ganglia perivascular spaces, and intracerebral hemorrhage.


Assuntos
Hemorragia Cerebral , Doenças de Pequenos Vasos Cerebrais , Transtornos dos Movimentos , Linhagem , Humanos , Feminino , Masculino , Doenças de Pequenos Vasos Cerebrais/genética , Doenças de Pequenos Vasos Cerebrais/patologia , Doenças de Pequenos Vasos Cerebrais/diagnóstico por imagem , Pessoa de Meia-Idade , Hemorragia Cerebral/genética , Hemorragia Cerebral/patologia , Hemorragia Cerebral/diagnóstico por imagem , Transtornos dos Movimentos/genética , Transtornos dos Movimentos/patologia , Transtornos dos Movimentos/diagnóstico por imagem , Imageamento por Ressonância Magnética , Alelos , Adulto , Idoso , Sistema Glinfático/patologia , Sistema Glinfático/diagnóstico por imagem , Sequenciamento do Exoma , Encéfalo/patologia , Encéfalo/diagnóstico por imagem , Aminoidrolases/genética
2.
Stem Cell Reports ; 18(9): 1793-1810, 2023 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-37541258

RESUMO

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.


Assuntos
Células-Tronco Pluripotentes Induzidas , Degeneração Retiniana , Retinose Pigmentar , Animais , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Retina/metabolismo , Degeneração Retiniana/genética , Retinose Pigmentar/genética , Mutação , Organoides/metabolismo , Proteínas do Olho/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo
3.
Neuropathol Appl Neurobiol ; 48(1): e12751, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34297860

RESUMO

AIMS: CADASIL, the most prevalent hereditary cerebral small vessel disease, is caused by cysteine-altering NOTCH3 variants (NOTCH3cys ) leading to vascular NOTCH3 protein aggregation. It has recently been shown that variants located in one of NOTCH3 protein epidermal growth-factor like repeat (EGFr) domains 1-6, are associated with a more severe phenotype than variants located in one of the EGFr domains 7-34. The underlying mechanism for this genotype-phenotype correlation is unknown. The aim of this study was to analyse whether NOTCH3cys variant position is associated with NOTCH3 protein aggregation load. METHODS: We quantified vascular NOTCH3 aggregation in skin biopsies (n = 25) and brain tissue (n = 7) of CADASIL patients with a NOTCH3cys EGFr 1-6 variant or a EGFr 7-34 variant, using NOTCH3 immunohistochemistry (NOTCH3 score) and ultrastructural analysis of granular osmiophilic material (GOM count). Disease severity was assessed by neuroimaging (lacune count and white matter hyperintensity volume) and disability (modified Rankin scale). RESULTS: Patients with NOTCH3cys EGFr 7-34 variants had lower NOTCH3 scores (P = 1.3·10-5 ) and lower GOM counts (P = 8.2·10-5 ) than patients with NOTCH3cys EGFr 1-6 variants in skin vessels. A similar trend was observed in brain vasculature. In the EGFr 7-34 group, NOTCH3 aggregation levels were associated with lacune count (P = 0.03) and white matter hyperintensity volume (P = 0.02), but not with disability. CONCLUSIONS: CADASIL patients with an EGFr 7-34 variant have significantly less vascular NOTCH3 aggregation than patients with an EGFr 1-6 variant. This may be one of the factors underlying the difference in disease severity between NOTCH3cys EGFr 7-34 and EGFr 1-6 variants.


Assuntos
CADASIL , Encéfalo/patologia , CADASIL/genética , CADASIL/metabolismo , CADASIL/patologia , Humanos , Imageamento por Ressonância Magnética , Mutação , Neuroimagem , Fenótipo , Receptor Notch3/genética , Receptor Notch3/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo
4.
Sci Adv ; 7(45): eabg8583, 2021 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-34739320

RESUMO

Osteoarthritis is the most prevalent joint disease worldwide, yet progress in development of effective disease-modifying treatments is slow because of lack of insight into the underlying disease pathways. Therefore, we aimed to identify the causal pathogenic mutation in an early-onset osteoarthritis family, followed by functional studies in human induced pluripotent stem cells (hiPSCs) in an in vitro organoid cartilage model. We demonstrated that the identified causal missense mutation in the gelatin-binding domain of the extracellular matrix protein fibronectin resulted in significant decreased binding capacity to collagen type II. Further analyses of formed hiPSC-derived neo-cartilage tissue highlighted that mutated fibronectin affected chondrogenic capacity and propensity to a procatabolic osteoarthritic state. Together, we demonstrate that binding of fibronectin to collagen type II is crucial for fibronectin downstream gene expression of chondrocytes. We advocate that effective treatment development should focus on restoring or maintaining proper binding between fibronectin and collagen type II.

5.
Int J Mol Sci ; 22(7)2021 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-33808129

RESUMO

Mutations in the Crumbs homologue 1 (CRB1) gene cause inherited retinal dystrophies, such as early-onset retinitis pigmentosa and Leber congenital amaurosis. A Brown Norway rat strain was reported with a spontaneous insertion-deletion (indel) mutation in exon 6 of Crb1. It has been reported that these Crb1 mutant rats show vascular abnormalities associated with retinal telangiectasia and possess an early-onset retinal degenerative phenotype with outer limiting membrane breaks and focal loss of retinal lamination at 2 months of age. Here, we further characterized the morphological phenotype of new-born and adult Crb1 mutant rats in comparison with age-matched Brown Norway rats without a mutation in Crb1. A significantly decreased retinal function and visual acuity was observed in Crb1 mutant rats at 1 and 3 months of age, respectively. Moreover, in control rats, the subcellular localization of canonical CRB1 was observed at the subapical region in Müller glial cells while CRB2 was observed at the subapical region in both photoreceptors and Müller glial cells by immuno-electron microscopy. CRB1 localization was lost in the Crb1 mutant rats, whereas CRB2 was still observed. In addition, we determined the tropism of subretinal or intravitreally administered AAV5-, AAV9- or AAV6-variant ShH10Y445F vectors in new-born control and Crb1 mutant rat retinas. We showed that subretinal injection of AAV5 and AAV9 at postnatal days 5 (P5) or 8 (P8) predominantly infected the retinal pigment epithelium (RPE) and photoreceptor cells; while intravitreal injection of ShH10Y445F at P5 or P8 resulted in efficient infection of mainly Müller glial cells. Using knowledge of the subcellular localization of CRB1 and the ability of ShH10Y445F to infect Müller glial cells, canonical hCRB1 and hCRB2 AAV-mediated gene therapy were explored in new-born Crb1 mutant rats. Enhanced retinal function after gene therapy delivery in the Crb1 rat was not observed. No timely rescue of the retinal phenotype was observed using retinal function and visual acuity, suggesting the need for earlier onset of expression of recombinant hCRB proteins in Müller glial cells to rescue the severe retinal phenotype in Crb1 mutant rats.


Assuntos
Proteínas de Ligação ao Cálcio/genética , Dependovirus/fisiologia , Terapia Genética/métodos , Proteínas do Tecido Nervoso/genética , Distrofias Retinianas/genética , Animais , Animais Recém-Nascidos , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte/genética , Dependovirus/genética , Células Ependimogliais/metabolismo , Proteínas do Olho/genética , Vetores Genéticos/administração & dosagem , Vetores Genéticos/farmacologia , Injeções Intravítreas , Proteínas de Membrana/genética , Mutação , Proteínas do Tecido Nervoso/metabolismo , Fenótipo , Ratos , Ratos Mutantes , Retina/fisiopatologia , Distrofias Retinianas/etiologia , Distrofias Retinianas/terapia , Epitélio Pigmentado da Retina/metabolismo , Tropismo Viral
6.
Haematologica ; 106(4): 1138-1147, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32336681

RESUMO

Von Willebrand factor (VWF) is a multimeric hemostatic protein that is synthesized in endothelial cells, where it is stored for secretion in elongated secretory organelles, so-called Weibel-Palade bodies (WPBs). Hemostatic activity of VWF is strongly tied to WPB length, but how endothelial cells control the dimensions of their WPBs is unclear. In this study we used a targeted shRNA screen to identify the longin-SNARE Sec22b as a novel determinant of WPB size and VWF trafficking. We found that Sec22b depletion resulted in loss of the typically elongated WPB morphology along with disintegration of the Golgi and dilation of rough ER (rER) cisternae. This was accompanied by reduced proteolytic processing of VWF, accumulation of VWF in the dilated rER and reduced basal and stimulated VWF secretion. Our data demonstrate that the elongation of WPBs, and thus adhesive activity of its cargo VWF, is determined by the rate of anterograde transport between ER and Golgi, which depends on Sec22b-containing SNARE complexes.


Assuntos
Células Endoteliais , Corpos de Weibel-Palade , Células Cultivadas , Exocitose , Fator de von Willebrand/genética
7.
Cell Stem Cell ; 26(6): 862-879.e11, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32459996

RESUMO

Cardiomyocytes (CMs) from human induced pluripotent stem cells (hiPSCs) are functionally immature, but this is improved by incorporation into engineered tissues or forced contraction. Here, we showed that tri-cellular combinations of hiPSC-derived CMs, cardiac fibroblasts (CFs), and cardiac endothelial cells also enhance maturation in easily constructed, scaffold-free, three-dimensional microtissues (MTs). hiPSC-CMs in MTs with CFs showed improved sarcomeric structures with T-tubules, enhanced contractility, and mitochondrial respiration and were electrophysiologically more mature than MTs without CFs. Interactions mediating maturation included coupling between hiPSC-CMs and CFs through connexin 43 (CX43) gap junctions and increased intracellular cyclic AMP (cAMP). Scaled production of thousands of hiPSC-MTs was highly reproducible across lines and differentiated cell batches. MTs containing healthy-control hiPSC-CMs but hiPSC-CFs from patients with arrhythmogenic cardiomyopathy strikingly recapitulated features of the disease. Our MT model is thus a simple and versatile platform for modeling multicellular cardiac diseases that will facilitate industry and academic engagement in high-throughput molecular screening.


Assuntos
Cardiopatias , Células-Tronco Pluripotentes Induzidas , Diferenciação Celular , Células Endoteliais , Humanos , Miócitos Cardíacos , Células Estromais
8.
Sci Rep ; 10(1): 5499, 2020 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-32218519

RESUMO

Research on acute and chronic lung diseases would greatly benefit from reproducible availability of alveolar epithelial cells (AEC). Primary alveolar epithelial cells can be derived from human lung tissue but the quality of these cells is highly donor dependent. Here, we demonstrated that culture of EpCAM+ cells derived from human induced pluripotent stem cells (hiPSC) at the physiological air-liquid interface (ALI) resulted in type 2 AEC-like cells (iAEC2) with alveolar characteristics. iAEC2 cells expressed native AEC2 markers (surfactant proteins and LPCAT-1) and contained lamellar bodies. ALI-iAEC2 were used to study alveolar repair over a period of 2 weeks following mechanical wounding of the cultures and the responses were compared with those obtained using primary AEC2 (pAEC2) isolated from resected lung tissue. Addition of the Wnt/ß-catenin activator CHIR99021 reduced wound closure in the iAEC2 cultures but not pAEC2 cultures. This was accompanied by decreased surfactant protein expression and accumulation of podoplanin-positive cells at the wound edge. These results demonstrated the feasibility of studying alveolar repair using hiPSC-AEC2 cultured at the ALI and indicated that this model can be used in the future to study modulation of alveolar repair by (pharmaceutical) compounds.


Assuntos
Células Epiteliais Alveolares/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Modelos Biológicos , Células Epiteliais Alveolares/citologia , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem Celular , Células Cultivadas , Humanos , Técnicas In Vitro , Células-Tronco Pluripotentes Induzidas/citologia , Alvéolos Pulmonares/lesões , Alvéolos Pulmonares/fisiologia , Alvéolos Pulmonares/fisiopatologia , Regeneração/fisiologia , Cicatrização/fisiologia
9.
Hum Mol Genet ; 29(11): 1853-1863, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-31960911

RESUMO

CADASIL is a vascular protein aggregation disorder caused by cysteine-altering NOTCH3 variants, leading to mid-adult-onset stroke and dementia. Here, we report individuals with a cysteine-altering NOTCH3 variant that induces exon 9 skipping, mimicking therapeutic NOTCH3 cysteine correction. The index came to our attention after a coincidental finding on a commercial screening MRI, revealing white matter hyperintensities. A heterozygous NOTCH3 c.1492G>T, p.Gly498Cys variant, was identified using a gene panel, which was also present in four first- and second-degree relatives. Although some degree of white matter hyperintensities was present on MRI in all family members with the NOTCH3 variant, the CADASIL phenotype was mild, as none had lacunes on MRI and there was no disability or cognitive impairment above the age of 60 years. RT-PCR and Sanger sequencing analysis on patient fibroblast RNA revealed that exon 9 was absent from the majority of NOTCH3 transcripts of the mutant allele, effectively excluding the mutation. NOTCH3 aggregation was assessed in skin biopsies using electron microscopy and immunohistochemistry and did not show granular osmiophilic material and only very mild NOTCH3 staining. For purposes of therapeutic translatability, we show that, in cell models, exon 9 exclusion can be obtained using antisense-mediated exon skipping and CRISPR/Cas9-mediated genome editing. In conclusion, this study provides the first in-human evidence that cysteine corrective NOTCH3 exon skipping is associated with less NOTCH3 aggregation and an attenuated phenotype, justifying further therapeutic development of NOTCH3 cysteine correction for CADASIL.


Assuntos
CADASIL/genética , Cisteína/genética , Agregação Patológica de Proteínas/genética , Receptor Notch3/genética , Substância Branca/metabolismo , Adulto , Idoso , Biópsia , CADASIL/diagnóstico por imagem , CADASIL/metabolismo , CADASIL/fisiopatologia , Sistemas CRISPR-Cas/genética , Éxons/genética , Feminino , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Agregação Patológica de Proteínas/diagnóstico por imagem , Agregação Patológica de Proteínas/metabolismo , Agregação Patológica de Proteínas/patologia , Índice de Gravidade de Doença , Pele/química , Pele/diagnóstico por imagem , Substância Branca/diagnóstico por imagem , Substância Branca/patologia
10.
Transl Stroke Res ; 11(3): 517-527, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-31667734

RESUMO

CADASIL is a NOTCH3-associated cerebral small vessel disease. A pathological ultrastructural disease hallmark is the presence of NOTCH3-protein containing deposits called granular osmiophilic material (GOM), in small arteries. How these GOM deposits develop over time and what their role is in disease progression is largely unknown. Here, we studied the progression of GOM deposits in humanized transgenic NOTCH3Arg182Cys mice, compared them to GOM deposits in patient material, and determined whether GOM deposits in mice are associated with a functional CADASIL phenotype. We found that GOM deposits are not static, but rather progress in ageing mice, both in terms of size and aspect. We devised a GOM classification system, reflecting size, morphology and electron density. Six-month-old mice showed mostly early stage GOM, whereas older mice and patient vessels showed predominantly advanced stage GOM, but also early stage GOM. Mutant mice did not develop the most severe GOM stage seen in patient material. This absence of end-stage GOM in mice was associated with an overall lack of histological vascular pathology, which may explain why the mice did not reveal functional deficits in cerebral blood flow, cognition and motor function. Taken together, our data indicate that GOM progress over time, and that new GOM deposits are continuously being formed. The GOM staging system we introduce here allows for uniform GOM deposit classification in future mouse and human studies, which may lead to more insight into a potential association between GOM stage and CADASIL disease severity, and the role of GOM in disease progression.


Assuntos
Encéfalo/irrigação sanguínea , Encéfalo/patologia , CADASIL/genética , CADASIL/patologia , Receptor Notch3/genética , Animais , Encéfalo/fisiopatologia , Encéfalo/ultraestrutura , Circulação Cerebrovascular , Humanos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos
11.
Int J Mol Sci ; 20(17)2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31438467

RESUMO

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.


Assuntos
Sensibilidades de Contraste/fisiologia , Amaurose Congênita de Leber/metabolismo , Proteínas de Membrana/metabolismo , Retina/metabolismo , Retina/patologia , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Animais , Sensibilidades de Contraste/genética , Modelos Animais de Doenças , Eletrorretinografia , Imuno-Histoquímica , Amaurose Congênita de Leber/patologia , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Retinose Pigmentar/metabolismo , Retinose Pigmentar/patologia
12.
Stem Cell Reports ; 12(5): 906-919, 2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-30956116

RESUMO

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.


Assuntos
Proteínas de Transporte/metabolismo , Células Ependimogliais/metabolismo , Proteínas do Olho/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Organoides/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/metabolismo , Adulto , Proteínas de Transporte/genética , Células Cultivadas , Dependovirus/genética , Células Ependimogliais/citologia , Células Ependimogliais/ultraestrutura , Proteínas do Olho/genética , Feminino , Feto , Humanos , Imuno-Histoquímica , Células-Tronco Pluripotentes Induzidas/citologia , Proteínas de Membrana/genética , Microscopia Imunoeletrônica , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteínas do Tecido Nervoso/genética , Organoides/citologia , Células Fotorreceptoras de Vertebrados/ultraestrutura , Gravidez , Retina/citologia , Retina/embriologia
13.
Hum Mol Genet ; 28(1): 105-123, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30239717

RESUMO

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 Müller 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.


Assuntos
Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Retinose Pigmentar/genética , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Modelos Animais de Doenças , Eletrorretinografia , Células Ependimogliais/metabolismo , Células Ependimogliais/fisiologia , Proteínas do Olho/genética , Proteínas do Olho/fisiologia , Amaurose Congênita de Leber/genética , Amaurose Congênita de Leber/fisiopatologia , Macaca fascicularis , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Proteínas do Tecido Nervoso/fisiologia , Neuroglia/fisiologia , Fenótipo , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/metabolismo , Distrofias Retinianas/metabolismo , Retinose Pigmentar/metabolismo , Retinose Pigmentar/fisiopatologia
14.
Arterioscler Thromb Vasc Biol ; 38(7): 1549-1561, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29880488

RESUMO

OBJECTIVE: Endothelial cells store VWF (von Willebrand factor) in rod-shaped secretory organelles, called Weibel-Palade bodies (WPBs). WPB exocytosis is coordinated by a complex network of Rab GTPases, Rab effectors, and SNARE (soluble NSF attachment protein receptor) proteins. We have previously identified STXBP1 as the link between the Rab27A-Slp4-a complex on WPBs and the SNARE proteins syntaxin-2 and -3. In this study, we investigate the function of syntaxin-3 in VWF secretion. APPROACH AND RESULTS: In human umbilical vein endothelial cells and in blood outgrowth endothelial cells (BOECs) from healthy controls, endogenous syntaxin-3 immunolocalized to WPBs. A detailed analysis of BOECs isolated from a patient with variant microvillus inclusion disease, carrying a homozygous mutation in STX3(STX3-/-), showed a loss of syntaxin-3 protein and absence of WPB-associated syntaxin-3 immunoreactivity. Ultrastructural analysis revealed no detectable differences in morphology or prevalence of immature or mature WPBs in control versus STX3-/- BOECs. VWF multimer analysis showed normal patterns in plasma of the microvillus inclusion disease patient, and media from STX3-/- BOECs, together indicating WPB formation and maturation are unaffected by absence of syntaxin-3. However, a defect in basal as well as Ca2+- and cAMP-mediated VWF secretion was found in the STX3-/- BOECs. We also show that syntaxin-3 interacts with the WPB-associated SNARE protein VAMP8 (vesicle-associated membrane protein-8). CONCLUSIONS: Our data reveal syntaxin-3 as a novel WPB-associated SNARE protein that controls WPB exocytosis.


Assuntos
Células Endoteliais/metabolismo , Exocitose , Síndromes de Malabsorção/metabolismo , Microvilosidades/patologia , Mucolipidoses/metabolismo , Proteínas Qa-SNARE/metabolismo , Corpos de Weibel-Palade/metabolismo , Fator de von Willebrand/metabolismo , Cálcio/metabolismo , Células Cultivadas , AMP Cíclico/metabolismo , Células Endoteliais/ultraestrutura , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Síndromes de Malabsorção/diagnóstico , Síndromes de Malabsorção/genética , Microvilosidades/genética , Microvilosidades/metabolismo , Mucolipidoses/diagnóstico , Mucolipidoses/genética , Mutação , Proteínas Qa-SNARE/genética , Proteínas R-SNARE/metabolismo , Via Secretória , Transdução de Sinais , Corpos de Weibel-Palade/ultraestrutura
15.
Neurobiol Aging ; 62: 231-242, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29195086

RESUMO

Previous MRI studies reported cortical iron accumulation in early-onset (EOAD) compared to late-onset (LOAD) Alzheimer disease patients. However, the pattern and origin of iron accumulation is poorly understood. This study investigated the histopathological correlates of MRI contrast in both EOAD and LOAD. T2*-weighted MRI was performed on postmortem frontal cortex of controls, EOAD, and LOAD. Images were ordinally scored using predefined criteria followed by histology. Nonlinear histology-MRI registration was used to calculate pixel-wise spatial correlations based on the signal intensity. EOAD and LOAD were distinguishable based on 7T MRI from controls and from each other. Histology-MRI correlation analysis of the pixel intensities showed that the MRI contrast is best explained by increased iron accumulation and changes in cortical myelin, whereas amyloid and tau showed less spatial correspondence with T2*-weighted MRI. Neuropathologically, subtypes of Alzheimer's disease showed different patterns of iron accumulation and cortical myelin changes independent of amyloid and tau that may be detected by high-field susceptibility-based MRI.


Assuntos
Doença de Alzheimer/diagnóstico por imagem , Doença de Alzheimer/patologia , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Imagem de Difusão por Ressonância Magnética , Ferro/metabolismo , Bainha de Mielina/metabolismo , Bainha de Mielina/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Peptídeos beta-Amiloides/metabolismo , Autopsia , Suscetibilidade a Doenças , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas tau/metabolismo
16.
Med Sci Educ ; 28(2): 451-455, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-31007968

RESUMO

Physician-scientists are urgently needed to make progress in the dynamic world of medical healthcare. Currently, there is a worldwide shortage in physicians pursuing a scientific career. Actively engaging students in research in early stages of medical training could help to direct students towards a scientific career and contribute to creating the next generation of physician-scientists. Leiden University Medical Center (LUMC) implemented an extracurricular Honors program with a fundamental orientation towards research. The program starts in the second year of medical training and is comprised of four different tracks in order to attract multiple types of students with different interests. All four tracks offer students scholarly experiences, but differ in content and amount of provided structure. The LUMC Honors program has a clear goal to develop future physician-scientists, and combined with its unique multiple-track model, the program accommodates about 70 students (25%) each year. The number of students in the program has grown and students' experiences are positive.

17.
Sci Rep ; 6: 24968, 2016 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-27113436

RESUMO

The transforming growth factor-ß (TGF-ß) family is known to play critical roles in cancer progression. While the dual role of TGF-ß is well described, the function of bone morphogenetic proteins (BMPs) is unclear. In this study, we established the involvement of Smad6, a BMP-specific inhibitory Smad, in breast cancer cell invasion. We show that stable overexpression of Smad6 in breast cancer MCF10A M2 cells inhibits BMP signalling, thereby mitigating BMP6-induced suppression of mesenchymal marker expression. Using a zebrafish xenograft model, we demonstrate that overexpression of Smad6 potentiates invasion of MCF10A M2 cells and enhances the aggressiveness of breast cancer MDA-MB-231 cells in vivo, whereas a reversed phenotype is observed after Smad6 knockdown. Interestingly, BMP6 pre-treatment of MDA-MB-231 cells induced cluster formation at the invasive site in the zebrafish. BMP6 also stimulated cluster formation of MDA-MB-231 cells co-cultured on Human Microvascular Endothelial Cells (HMEC)-1 in vitro. Electron microscopy illustrated an induction of cell-cell contact by BMP6. The clinical relevance of our findings is highlighted by a correlation of high Smad6 expression with poor distant metastasis free survival in ER-negative cancer patients. Collectively, our data strongly indicates the involvement of Smad6 and BMP signalling in breast cancer cell invasion in vivo.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Neoplasias da Mama/metabolismo , Proteína Smad6/genética , Proteína Smad6/metabolismo , Animais , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Movimento Celular , Técnicas de Cocultura , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Invasividade Neoplásica , Transplante de Neoplasias , Receptores de Estrogênio/metabolismo , Transdução de Sinais , Análise de Sobrevida , Regulação para Cima , Peixe-Zebra
18.
IEEE Trans Med Imaging ; 31(2): 461-73, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21997250

RESUMO

Fluorescence loss in photobleaching (FLIP) is a method to study compartment connectivity in living cells. A FLIP sequence is obtained by alternatively bleaching a spot in a cell and acquiring an image of the complete cell. Connectivity is estimated by comparing fluorescence signal attenuation in different cell parts. The measurements of the fluorescence attenuation are hampered by the low signal to noise ratio of the FLIP sequences, by sudden sample shifts and by sample drift. This paper describes a method that estimates the attenuation by modeling photobleaching as exponentially decaying signals. Sudden motion artifacts are minimized by registering the frames of a FLIP sequence to target frames based on the estimated model and by removing frames that contain deformations. Linear motion (sample drift) is reduced by minimizing the entropy of the estimated attenuation coefficients. Experiments on 16 in vivo FLIP sequences of muscle cells in Drosophila show that the proposed method results in fluorescence attenuations similar to the manually identified gold standard, but with standard deviations of approximately 50 times smaller. As a result of this higher precision, cell compartment edges and details such as cell nuclei become clearly discernible. The main value of this method is that it uses a model of the bleaching process to correct motion and that the model based fluorescence intensity and attenuation estimates can be interpreted easily. The proposed method is fully automatic, and runs in approximately one minute per sequence, making it suitable for unsupervised batch processing of large data series.


Assuntos
Algoritmos , Artefatos , Recuperação de Fluorescência Após Fotodegradação/métodos , Aumento da Imagem/métodos , Interpretação de Imagem Assistida por Computador/métodos , Fibras Musculares Esqueléticas/citologia , Reconhecimento Automatizado de Padrão/métodos , Animais , Drosophila melanogaster , Movimento (Física) , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Técnica de Subtração
19.
Int J Dev Biol ; 56(9): 701-9, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23319346

RESUMO

In some myopathies, hypoxia can be the result of pathologic effects like muscle necrosis and abnormal blood flow. At the molecular level, the consequence of hypoxic conditions is not yet fully understood. Under stress conditions, many housekeeping gene mRNAs are translationally silenced, while translation of other mRNAs increases. Alterations to the pool of mRNAs available for translation lead to the formation of so-called stress granules containing both mRNAs and proteins. Stress granule formation and dynamics have been investigated using cells in culture, but have not yet been examined in vivo. In Drosophila embryonic muscles, we found that hypoxia induces the formation of sarcoplasmic granules containing the established stress granule markers RIN and dFMR1. Upon restoration of normoxia, the observed granules were decreased in size, indicating that their formation might be reversible. Employing photobleaching approaches, we found that a cytoplasmic reporter mRNA rapidly shuttles in and out of the granules. Hence, stress granules are highly dynamic complexes and not simple temporary storage sites. Although mRNA rapidly cycles through the granules, its movement throughout the muscle is, remarkably, spatially restricted by the presence of yet undefined myofiber domains. Our results suggest that in hypoxic muscles mRNA remains highly mobile; however, its movement throughout the muscle is restricted by certain boundaries. The development of this Drosophila hypoxia model makes it possible to study the formation and dynamics of stress granules and their associated mRNAs and proteins in a living organism.


Assuntos
Grânulos Citoplasmáticos/genética , Drosophila/genética , Embrião não Mamífero/metabolismo , Hipóxia/genética , Músculo Esquelético/embriologia , RNA Mensageiro/genética , Animais , Grânulos Citoplasmáticos/metabolismo , Drosophila/metabolismo , Hipóxia/metabolismo , Músculo Esquelético/metabolismo , RNA Mensageiro/metabolismo
20.
PLoS One ; 4(8): e6663, 2009 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-19684860

RESUMO

BACKGROUND: In skeletal muscle each muscle cell, commonly called myofiber, is actually a large syncytium containing numerous nuclei. Experiments in fixed myofibers show that mRNAs remain localized around the nuclei in which they are produced. METHODOLOGY/PRINCIPAL FINDINGS: In this study we generated transgenic flies that allowed us to investigate the movement of mRNAs in body wall myofibers of living Drosophila embryos. We determined the dynamic properties of GFP-tagged mRNAs using in vivo confocal imaging and photobleaching techniques and found that the GFP-tagged mRNAs are not free to move throughout myofibers. The restricted movement indicated that body wall myofibers consist of three domains. The exchange of mRNAs between the domains is relatively slow, but the GFP-tagged mRNAs move rapidly within these domains. One domain is located at the centre of the cell and is surrounded by nuclei while the other two domains are located at either end of the fiber. To move between these domains mRNAs have to travel past centrally located nuclei. CONCLUSIONS/SIGNIFICANCE: These data suggest that the domains made visible in our experiments result from prolonged interactions with as yet undefined structures close to the nuclei that prevent GFP-tagged mRNAs from rapidly moving between the domains. This could be of significant importance for the treatment of myopathies using regenerative cell-based therapies.


Assuntos
Drosophila/embriologia , Músculos/metabolismo , Miofibrilas/metabolismo , RNA Mensageiro/metabolismo , Animais , Animais Geneticamente Modificados , Sequência de Bases , Primers do DNA , Proteínas de Fluorescência Verde/genética , Imuno-Histoquímica , Microscopia Confocal , Microscopia Eletrônica , RNA Mensageiro/genética
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