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1.
PLoS Biol ; 18(3): e3000470, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32150534

RESUMO

In the spinal cord, the central canal forms through a poorly understood process termed dorsal collapse that involves attrition and remodelling of pseudostratified ventricular layer (VL) cells. Here, we use mouse and chick models to show that dorsal ventricular layer (dVL) cells adjacent to dorsal midline Nestin(+) radial glia (dmNes+RG) down-regulate apical polarity proteins, including Crumbs2 (CRB2) and delaminate in a stepwise manner; live imaging shows that as one cell delaminates, the next cell ratchets up, the dmNes+RG endfoot ratchets down, and the process repeats. We show that dmNes+RG secrete a factor that promotes loss of cell polarity and delamination. This activity is mimicked by a secreted variant of Crumbs2 (CRB2S) which is specifically expressed by dmNes+RG. In cultured MDCK cells, CRB2S associates with apical membranes and decreases cell cohesion. Analysis of Crb2F/F/Nestin-Cre+/- mice, and targeted reduction of Crb2/CRB2S in slice cultures reveal essential roles for transmembrane CRB2 (CRB2TM) and CRB2S on VL cells and dmNes+RG, respectively. We propose a model in which a CRB2S-CRB2TM interaction promotes the progressive attrition of the dVL without loss of overall VL integrity. This novel mechanism may operate more widely to promote orderly progenitor delamination.


Assuntos
Proteínas de Membrana/metabolismo , Medula Espinal/citologia , Medula Espinal/embriologia , Animais , Adesão Celular , Embrião de Galinha , Cães , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , Células Madin Darby de Rim Canino , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Junções Íntimas/metabolismo , Imagem com Lapso de Tempo
2.
Eur J Immunol ; 50(2): 245-255, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31778214

RESUMO

IL-23 plays an important role in the development of arthritis and the IL-23 receptor (IL-23R) is expressed on different types of T cells. However, it is not fully clear which IL-23R+ T cells are critical in driving T cell-mediated synovitis. We demonstrate, using knock-in IL-23R-GFP reporter (IL-23RGFP/+ ) mice, that CD4+ CCR6+ T cells and γδ T cells, but not CD8+ T cells, express the IL-23R(GFP). During early arthritis, IL-23R(GFP)+ CD4+ CCR6+ T cells, but not IL-23R(GFP)+ γδ T cells, were present in the inflamed joints. IL-23RGFP/+ mice were bred as homozygotes to obtain IL-23RGFP/GFP (IL-23R deficient/IL-23R-/- ) mice, which express GFP under the IL-23R promotor. Arthritis progression and joint damage were significantly milder in IL-23R-/- mice, which revealed less IL-17A+ cells in their lymphoid tissues. Surprisingly, IL-23R-/- mice had increased numbers of IL-23R(GFP)+ CD4+ CCR6+ and CCR7+ CD4+ CCR6+ T cells in their spleen compared to WT, and IL-23 suppressed CCR7 expression in vitro. However, IL-23R(GFP)+ CD4+ CCR6+ T cells were present in the synovium of IL-23R-/- mice at day 4. Finally, adoptive transfer experiments revealed that CD4+ CCR6+ T cells and not γδ T cells drive arthritis progression. These data suggest that IL-23R-dependent T cell-mediated synovitis is dependent on CD4+ CCR6+ T cells and not on γδ T cells.


Assuntos
Artrite/imunologia , Linfócitos T CD4-Positivos/imunologia , Inflamação/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Receptores CCR6/imunologia , Receptores de Interleucina/imunologia , Transferência Adotiva/métodos , Animais , Modelos Animais de Doenças , Feminino , Interleucina-17/imunologia , Interleucina-23/imunologia , Tecido Linfoide/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células Th17/imunologia
3.
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
4.
Hum Mol Genet ; 27(18): 3137-3153, 2018 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-29893966

RESUMO

The mammalian apical-basal determinant Crumbs homolog-1 (CRB1) plays a crucial role in retinal structure and function by the maintenance of adherens junctions between photoreceptors and Müller glial cells. Patients with mutations in the CRB1 gene develop retinal dystrophies, including early-onset retinitis pigmentosa and Leber congenital amaurosis. Previously, we showed that Crb1 knockout mice developed a slow-progressing retinal phenotype at foci in the inferior retina, although specific ablation of Crb2 in immature photoreceptors leads to an early-onset phenotype throughout the retina. Here, we conditionally disrupted one or both alleles of Crb2 in immature photoreceptors, on a genetic background lacking Crb1, and studied the retinal dystrophies thereof. Our data showed that disruption of one allele of Crb2 in immature photoreceptors caused a substantial aggravation of the Crb1 phenotype in the entire inferior retina. The photoreceptor layer showed early-onset progressive thinning limited to the inferior retina, although the superior retina maintained intact. Surprisingly, disruption of both alleles of Crb2 in immature photoreceptors further aggravated the phenotype. Throughout the retina, photoreceptor synapses were disrupted and photoreceptor nuclei intermingled with nuclei of the inner nuclear layer. In the superior retina, the ganglion cell layer appeared thicker because of ectopic nuclei of photoreceptors. In conclusion, the data suggest that CRB2 is required to maintain retinal progenitor and photoreceptor cell adhesion and prevent photoreceptor ingression into the immature inner retina. We hypothesize, from these animal models, that decreased levels of CRB2 in immature photoreceptors adjust retinitis pigmentosa because of the loss of CRB1 into Leber congenital amaurosis phenotype.


Assuntos
Amaurose Congênita de Leber/genética , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Retina/fisiopatologia , Junções Aderentes/genética , Alelos , Animais , Adesão Celular/genética , Modelos Animais de Doenças , Células Ependimogliais/metabolismo , Células Ependimogliais/patologia , Humanos , Amaurose Congênita de Leber/fisiopatologia , Camundongos , Camundongos Knockout , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/patologia , Retina/crescimento & desenvolvimento , Sinapses/genética , Sinapses/patologia
5.
Adv Exp Med Biol ; 1185: 159-163, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31884605

RESUMO

Inherited retinal diseases encompass a large group of clinically and genetically heterogeneous diseases estimated to affect two million people worldwide. Among these people, approximately 80,000 are or will become blind in their first decades of life due to mutations in both alleles of the Crumbs homologue-1 (CRB1) gene. Microglia are the resident immune surveyor cells in the retina, and their roles have been heavily studied in several retinal diseases, including retinitis pigmentosa (RP), age-related macular degeneration, and diabetic retinopathy. However, very little is known about the role of microglia in CRB1-associated retinopathies. Thus, we here summarize the main findings described in the literature concerning inflammation and the role of microglia in CRB1-patients and CRB1-rodent models.


Assuntos
Proteínas do Olho/genética , Proteínas de Membrana/genética , Microglia/patologia , Proteínas do Tecido Nervoso/genética , Doenças Retinianas/patologia , Animais , Humanos , Mutação , Retinose Pigmentar/patologia
6.
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
7.
Hum Mol Genet ; 24(11): 3104-18, 2015 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-25701872

RESUMO

Mutations in the Crumbs-homologue-1 (CRB1) gene lead to severe recessive inherited retinal dystrophies. Gene transfer therapy is the most promising cure for retinal dystrophies and has primarily been applied for recessive null conditions via a viral gene expression vector transferring a cDNA encoding an enzyme or channel protein, and targeting expression to one cell type. Therapy for the human CRB1 disease will be more complex, as CRB1 is a structural and signaling transmembrane protein present in three cell classes: Müller glia, cone and rod photoreceptors. In this study, we applied CRB1 and CRB2 gene therapy vectors in Crb1-retinitis pigmentosa mouse models at mid-stage disease. We tested if CRB expression restricted to Müller glial cells or photoreceptors or co-expression in both is required to recover retinal function. We show that targeting both Müller glial cells and photoreceptors with CRB2 ameliorated retinal function and structure in Crb1 mouse models. Surprisingly, targeting a single cell type or all cell types with CRB1 reduced retinal function. We show here the first pre-clinical studies for CRB1-related eye disorders using CRB2 vectors and initial elucidation of the cellular mechanisms underlying CRB1 function.


Assuntos
Células Ependimogliais/fisiologia , Proteínas do Tecido Nervoso/genética , Retinose Pigmentar/genética , Animais , Proteínas de Transporte/genética , Modelos Animais de Doenças , Terapia Genética , Células HEK293 , Humanos , Injeções Intravítreas , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Retina/patologia , Retina/fisiopatologia , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Retinose Pigmentar/terapia
8.
Sci Rep ; 14(1): 15189, 2024 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-38956231

RESUMO

Variants in the retinitis pigmentosa GTPase regulator (RPGR) gene are responsible for the majority of X-linked retinitis pigmentosa cases, which not only affects male patients but also some heterozygous females. Vision-related disability and anxiety of patients with RPGR-associated retinal degeneration have never been explored before. This study aimed to evaluate self-reported visual function and vision-related anxiety in a Portuguese cohort of male and female patients with RPGR-associated retinal degeneration using two validated patient-reported outcome measures. Cross-sectional data of thirty-two genetically-tested patients was examined, including scores of the Michigan retinal degeneration questionnaire (MRDQ) and Michigan vision-related anxiety questionnaire. Patients were classified according to retinal phenotypes in males (M), females with male phenotype (FM), and females with radial or focal pattern. Both M and FM revealed higher rod-function and cone-function anxiety scores (p < 0.017). Most MRDQ disability scores were higher in M and FM (p < 0.004). Overall, positive correlations (p < 0.004) were found between every MRDQ domain and both anxiety scores. In RPGR-associated retinal degeneration, males and females with male phenotype show similar levels of increased vision-related anxiety and disability. Every MRDQ visual function domain showed a strong correlation with anxiety scores.


Assuntos
Ansiedade , Proteínas do Olho , Degeneração Retiniana , Autorrelato , Humanos , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Degeneração Retiniana/fisiopatologia , Proteínas do Olho/genética , Estudos Transversais , Retinose Pigmentar/fisiopatologia , Retinose Pigmentar/psicologia , Retinose Pigmentar/genética , Idoso , Fenótipo , Adulto Jovem , Inquéritos e Questionários
9.
Ophthalmic Genet ; 44(4): 334-340, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-36946380

RESUMO

PURPOSE: To evaluate self-reported visual function and the psychosocial impact of visual loss EYS-associated retinal degeneration (EYS-RD) using two patient-reported outcome (PRO) measures: Michigan Retinal Degeneration Questionnaire (MRDQ) and Michigan Vision-related Anxiety Questionnaire (MVAQ). METHODS: Cross-sectional, single-center study conducted at a tertiary care hospital in Portugal. Patients with biallelic EYS variants were invited to participate. Clinical data including demographics, ETDRS best-corrected visual acuity (BCVA) in the better-seeing eye and genetic testing results were collected. Interviews were carried out during clinic visits or by phone between November 2021 and February 2022. A blind grader used horizontal and vertical spectral domain optical coherence tomography (SD-OCT) scans to manually measure ellipsoid zone (EZ) width in the nasal, temporal, superior and inferior macular quadrants to calculate the EZ area. RESULTS: Forty-nine patients (53.1% males; mean age 53 ± 14 years) were included. A positive correlation (p < .05) was found between age and most MRDQ domain scores (central vision, color vision, contrast sensitivity, scotopic function, photopic peripheral vision and mesopic peripheral vision). A negative correlation was found between both BCVA and EZ area across all MRDQ domains. In MVAQ, SD-OCT EZ area negatively correlated with both rod function and cone function-related anxiety. Neither age, BCVA or gender correlated with MVAQ domains. CONCLUSIONS: This study provides strong evidence supporting a correlation between PRO measures and both functional and structural clinician-reported outcomes. The use of MRDQ and MVAQ adds a new dimension to our understanding of EYS-RD and establishes both PRO measures as important disease outcome measures.


Assuntos
Degeneração Retiniana , Masculino , Humanos , Adulto , Pessoa de Meia-Idade , Idoso , Feminino , Degeneração Retiniana/genética , Portugal , Autorrelato , Estudos Transversais , Acuidade Visual , Transtornos da Visão , Tomografia de Coerência Óptica/métodos , Proteínas do Olho/genética
10.
Ophthalmol Retina ; 7(7): 628-638, 2023 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-36764454

RESUMO

PURPOSE: To describe the natural history, genetic landscape, and phenotypic spectrum of Eyes shut homolog (EYS)-associated retinal degeneration (EYS-RD). DESIGN: Retrospective, single-center cohort study complemented by a cross-sectional examination. SUBJECTS: Patients with biallelic EYS variants were recruited at an inherited RD referral center in Portugal. METHODS: Every patient underwent a cross-sectional examination comprising a comprehensive ophthalmic examination including best-corrected visual acuity (BCVA), dilated slit-lamp anterior segment, and fundus biomicroscopy; ultrawide-field color fundus photography and fundus autofluorescence imaging; and spectral domain-OCT. In the setting of a retinitis pigmentosa (RP) diagnosis, every patient was classified as typical or atypical RP according to imaging criteria. Baseline demographics, age at onset of symptoms, family history, history of consanguinity, symptoms, age at diagnosis, BCVA at baseline and throughout follow-up, and EYS variants were collected from each individual patient file. MAIN OUTCOME MEASURES: Clinical/demographic, genetic, multimodal imaging data, and BCVA variation were compared between typical and atypical RP. Additionally, BCVA variation during follow-up was used as an endpoint to describe EYS-RD natural history. RESULTS: Fifty-eight patients (59% men; mean age 52 ± 14 years) from 48 White families of Portuguese ancestry were included. Twenty distinct EYS variants were identified, 8 of which are novel. In 32.8% of patients, onset of symptoms was in early adulthood (21-30 years). A clinical diagnosis of RP was established in 57 patients and cone-rod dystrophy in 1 patient. Regarding RP, 75.0% of the patients were graded as typical and 25.0% as atypical. Atypical EYS-RP commonly presents with inferior crescent-shaped macular atrophy with superior midperipheral sparing. In EYS-RD, a negative correlation was found between age and BCVA (r = -0.50; P < 0.001), with an average loss of 1.45 letters per year. When stratifying for RP phenotype, lower average loss of letters per year (P < 0.001), higher BCVA (P < 0.001), and larger ellipsoid zone widths (P < 0.001) were found in atypical RP. CONCLUSIONS: This study expands the genetic spectrum of EYS-RD by reporting 8 novel variants. A high frequency of atypical phenotypes was identified. These patients have better BCVA and larger ellipsoidal zone widths, thus presenting an overall better prognosis. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references.


Assuntos
Degeneração Retiniana , Retinose Pigmentar , Humanos , Degeneração Retiniana/diagnóstico , Degeneração Retiniana/genética , Estudos de Coortes , Estudos Retrospectivos , Estudos Transversais , Mutação , Proteínas do Olho/genética , Tomografia de Coerência Óptica , Retinose Pigmentar/diagnóstico , Retinose Pigmentar/genética , Fenótipo
11.
Mol Ther Methods Clin Dev ; 20: 423-441, 2021 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-33575434

RESUMO

Loss of Crumbs homolog 1 (CRB1) or CRB2 proteins in Müller cells or photoreceptors in the mouse retina results in a CRB dose-dependent retinal phenotype. In this study, we present a novel Müller cell-specific Crb1 KO Crb2 LowMGC retinitis pigmentosa mouse model (complete loss of CRB1 and reduced levels of CRB2 specifically in Müller cells). The Crb double mutant mice showed deficits in electroretinography, optokinetic head tracking, and retinal morphology. Exposure of retinas to low levels of dl-α-aminoadipate acid induced gliosis and retinal disorganization in Crb1 KO Crb2 LowMGC retinas but not in wild-type or Crb1-deficient retinas. Crb1 KO Crb2 LowMGC mice showed a substantial decrease in inner/outer photoreceptor segment length and optokinetic head-tracking response. Intravitreal application of rAAV vectors expressing human CRB2 (hCRB2) in Müller cells of Crb1 KO Crb2 LowMGC mice subsequently exposed to low levels of dl-α-aminoadipate acid prevented loss of vision, whereas recombinant adeno-associated viral (rAAV) vectors expressing human CRB1 (hCRB1) did not. Both rAAV vectors partially protected the morphology of the retina. The results suggest that hCRB expression in Müller cells is vital for control of retinal cell adhesion at the outer limiting membrane, and that the rAAV-cytomegalovirus (CMV)-hCRB2 vector is more potent than rAAV-minimal CMV (CMVmin)-hCRB1 in protection against loss of vision.

12.
Acta Neuropathol Commun ; 8(1): 189, 2020 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-33168089

RESUMO

Mutations in the PTEN-induced kinase 1 (PINK1) and Parkin RBR E3 ubiquitin-protein ligase (PARKIN) genes are associated with familial forms of Parkinson's disease (PD). PINK1, a protein kinase, and PARKIN, an E3 ubiquitin ligase, control the specific elimination of dysfunctional or superfluous mitochondria, thus fine-tuning mitochondrial network and preserving energy metabolism. PINK1 regulates PARKIN translocation in impaired mitochondria and drives their removal via selective autophagy, a process known as mitophagy. As knowledge obtained using different PINK1 and PARKIN transgenic animal models is being gathered, growing evidence supports the contribution of mitophagy impairment to several human pathologies, including PD and Alzheimer's diseases (AD). Therefore, therapeutic interventions aiming to modulate PINK1/PARKIN signalling might have the potential to treat these diseases. In this review, we will start by discussing how the interplay of PINK1 and PARKIN signalling helps mediate mitochondrial physiology. We will continue by debating the role of mitochondrial dysfunction in disorders such as amyotrophic lateral sclerosis, Alzheimer's, Huntington's and Parkinson's diseases, as well as eye diseases such as age-related macular degeneration and glaucoma, and the causative factors leading to PINK1/PARKIN-mediated neurodegeneration and neuroinflammation. Finally, we will discuss PINK1/PARKIN gene augmentation possibilities with a particular focus on AD, PD and glaucoma.


Assuntos
Inflamação/genética , Mitocôndrias/metabolismo , Doenças Neurodegenerativas/genética , Proteínas Quinases/genética , Ubiquitina-Proteína Ligases/genética , Doença de Alzheimer/genética , Esclerose Lateral Amiotrófica/genética , Animais , Técnicas de Introdução de Genes , Glaucoma/genética , Humanos , Doença de Huntington/genética , Inflamação/metabolismo , Degeneração Macular/genética , Camundongos , Mitofagia/genética , Doenças Neurodegenerativas/metabolismo , Doença de Parkinson/genética , Proteínas Quinases/metabolismo , Ratos , Transdução de Sinais , Ubiquitina-Proteína Ligases/metabolismo
13.
Cells ; 9(5)2020 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-32423062

RESUMO

The retina is a highly metabolically active tissue with high-level consumption of nutrients and oxygen. This high metabolic demand requires a properly developed and maintained vascular system. The retina is nourished by two systems: the central retinal artery that supplies the inner retina and the choriocapillaris that supplies the outer retina and retinal pigment epithelium (RPE). Pathological neovascularization, characterized by endothelial cell proliferation and new vessel formation, is a common hallmark in several retinal degenerative diseases, including age-related macular degeneration (AMD). A limited number of studies have suggested that microglia, the resident immune cells of the retina, have an important role not only in the pathology but also in the formation and physiology of the retinal vascular system. Here, we review the current knowledge on microglial interaction with the retinal vascular system under physiological and pathological conditions. To do so, we first highlight the role of microglial cells in the formation and maintenance of the retinal vasculature system. Thereafter, we discuss the molecular signaling mechanisms through which microglial cells contribute to the alterations in retinal and choroidal vasculatures and to the neovascularization in AMD.


Assuntos
Corioide/irrigação sanguínea , Degeneração Macular/patologia , Microglia/patologia , Retina/patologia , Animais , Corioide/patologia , Neovascularização de Coroide/patologia , Humanos , Modelos Biológicos
14.
J Mol Neurosci ; 38(1): 41-9, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19015999

RESUMO

The choroid plexuses (CPs) of the brain form a unique interface between the peripheral blood and the cerebrospinal fluid (CSF). CPs produce several neuroprotective peptides, which are secreted into the CSF. Despite their importance in neuroprotection, the mechanisms underlying the regulation of most of these peptides in CPs remain unknown. Androgens regulate the expression of neuroprotective peptides in several tissues where the androgen receptor (AR) is coexpressed, including the brain. The presence of AR in CPs has never been investigated, but recent studies in our laboratory show that the CP is an androgen-responsive tissue. In order to fulfill this gap, we investigated and characterized AR distribution and expression in male and female rat CPs and in primary cultures from rat CP epithelial cells. In addition, the response of AR to 5alpha-dihydrotestosterone (DHT) in castrated male and female mice subjected to DHT replacement was analyzed. We show that rat CP epithelial cells contain AR mRNA and protein. Moreover, we demonstrate that AR is downregulated by DHT in mice CPs.


Assuntos
Plexo Corióideo/efeitos dos fármacos , Plexo Corióideo/metabolismo , Di-Hidrotestosterona/farmacologia , Receptores Androgênicos/biossíntese , Androgênios/farmacologia , Animais , Plexo Corióideo/citologia , Regulação para Baixo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Expressão Gênica/efeitos dos fármacos , Masculino , Camundongos , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Ratos Wistar , Receptores Androgênicos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
15.
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
16.
Methods Mol Biol ; 1715: 135-151, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29188511

RESUMO

Mutations in the CRB1 gene account for around 10,000 persons with Leber congenital amaurosis (LCA) and 70,000 persons with retinitis pigmentosa (RP) worldwide. Therefore, the CRB1 gene is a key target in the fight against blindness. A proof-of-concept for an adeno-associated virus (AAV)-mediated CRB2 gene augmentation therapy for CRB1-RP was recently described. Preclinical studies using animal models such as knockout or mutant mice are crucial to obtain such proof-of-concept. In this chapter we describe a technique to deliver AAV vectors, into the murine retinas, via the subretinal route. We also present protocols to detect expression of the therapeutic protein by fluorescence immunohistochemistry and to perform histological studies using ultra-thin sections stained with toluidine blue. These techniques in combination with electroretinography and visual behavior tests are in principle sufficient to obtain proof-of-concept for new gene therapies.


Assuntos
Dependovirus/genética , Terapia Genética/métodos , Mutação , Proteínas do Tecido Nervoso/genética , Retina/metabolismo , Retinose Pigmentar/terapia , Animais , Proteínas do Olho/genética , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Retinose Pigmentar/genética
17.
Clin Rev Allergy Immunol ; 51(1): 27-47, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26634933

RESUMO

Throughout life, bone is continuously remodelled. Bone is formed by osteoblasts, from mesenchymal origin, while osteoclasts induce bone resorption. This process is tightly regulated. During inflammation, several growth factors and cytokines are increased inducing osteoclast differentiation and activation, and chronic inflammation is a condition that initiates systemic bone loss. Rheumatoid arthritis (RA) is a chronic inflammatory auto-immune disease that is characterised by active synovitis and is associated with early peri-articular bone loss. Peri-articular bone loss precedes focal bone erosions, which may progress to bone destruction and disability. The incidence of generalised osteoporosis is associated with the severity of arthritis in RA and increased osteoporotic vertebral and hip fracture risk. In this review, we will give an overview of different animal models of inflammatory arthritis related to RA with focus on bone erosion and involvement of pro-inflammatory cytokines. In addition, a humanised endochondral ossification model will be discussed, which can be used in a translational approach to answer osteoimmunological questions.


Assuntos
Artrite/metabolismo , Artrite/patologia , Reabsorção Óssea/metabolismo , Reabsorção Óssea/patologia , Citocinas/metabolismo , Mediadores da Inflamação/metabolismo , Animais , Animais Geneticamente Modificados , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Antígenos/imunologia , Artrite/imunologia , Artrite/terapia , Artrite Experimental , Artrite Reumatoide/imunologia , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Biomarcadores , Regeneração Óssea , Remodelação Óssea , Reabsorção Óssea/terapia , Diferenciação Celular , Reações Cruzadas/imunologia , Modelos Animais de Doenças , Humanos , Terapia de Alvo Molecular , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteoclastos/metabolismo
18.
PLoS One ; 10(11): e0142972, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26587585

RESUMO

Dendritic cells (DCs) are professional antigen presenting cells that have the dual ability to stimulate immunity and maintain tolerance. However, the signalling pathways mediating tolerogenic DC function in vivo remain largely unknown. The ß-catenin pathway has been suggested to promote a regulatory DC phenotype. The aim of this study was to unravel the role of ß-catenin signalling to control DC function in the autoimmune collagen-induced arthritis model (CIA). Deletion of ß-catenin specifically in DCs was achieved by crossing conditional knockout mice with a CD11c-Cre transgenic mouse line. Bone marrow-derived DCs (BMDCs) were generated and used to study the maturation profile of these cells in response to a TLR2 or TLR4 ligand stimulation. CIA was induced by intra-dermal immunization with 100 µg chicken type II collagen in complete Freund's adjuvant on days 0 and 21. CIA incidence and severity was monitored macroscopically and by histology. The T cell profile as well as their cytokine production were analysed by flow cytometry. Lack of ß-catenin specifically in DCs did not affect the spontaneous, TLR2- or TLR4-induced maturation and activation of BMDCs or their cytokine production. Moreover, no effect on the incidence and severity of CIA was observed in mice lacking ß-catenin in CD11c+ cells. A decreased frequency of splenic CD3+CD8+ T cells and of regulatory T cells (Tregs) (CD4+CD25highFoxP3+), but no changes in the frequency of splenic Th17 (CCR6+CXCR3-CCR4+), Th2 (CCR6-CXCR3-CCR4+) and Th1 (CCR6-CXCR3+CCR4-) cells were observed in these mice under CIA condition. Furthermore, the expression of IL-17A, IL-17F, IL-22, IL-4 or IFNγ was also not affected. Our data indicate that ablation of ß-catenin expression in DCs did not alter the course and severity of CIA. We conclude that although deletion of ß-catenin resulted in a lower frequency of Tregs, this decrease was not sufficient to aggravate the onset and severity of CIA.


Assuntos
Artrite Experimental/genética , Células Dendríticas/metabolismo , Linfócitos T Reguladores/metabolismo , beta Catenina/genética , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Artrite Experimental/imunologia , Artrite Experimental/patologia , Colágeno Tipo II/administração & dosagem , Colágeno Tipo II/imunologia , Células Dendríticas/imunologia , Humanos , Tolerância Imunológica , Camundongos , Camundongos Knockout , Transdução de Sinais/imunologia , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Células Th17/metabolismo , Receptor 2 Toll-Like/imunologia , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/imunologia , Receptor 4 Toll-Like/metabolismo , beta Catenina/biossíntese
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