RESUMO
The choroid plexus (CP) consists of specialized ependymal cells and underlying blood vessels and stroma producing the bulk of the cerebrospinal fluid (CSF). CP epithelial cells are considered the site of the internal blood-cerebrospinal fluid barrier, show epithelial characteristics (basal lamina, tight junctions), and express aquaporin-1 (AQP1) apically. In this study, we analyzed the expression of aquaporins in the human CP using immunofluorescence and qPCR. As previously reported, AQP1 was expressed apically in CP epithelial cells. Surprisingly, and previously unknown, many cells in the CP epithelium were also positive for aquaporin-4 (AQP4), normally restricted to ventricle-lining ependymal cells and astrocytes in the brain. Expression of AQP1 and AQP4 was found in the CP of all eight body donors investigated (3 males, 5 females; age 74-91). These results were confirmed by qPCR, and by electron microscopy detecting orthogonal arrays of particles. To find out whether AQP4 expression correlated with the expression pattern of relevant transport-related proteins we also investigated expression of NKCC1, and Na/K-ATPase. Immunostaining with NKCC1 was similar to AQP1 and revealed no particular pattern related to AQP4. Co-staining of AQP4 and Na/K-ATPase indicated a trend for an inverse correlation of their expression. We hypothesized that AQP4 expression in the CP was caused by age-related changes. To address this, we investigated mouse brains from young (2 months), adult (12 months) and old (30 months) mice. We found a significant increase of AQP4 on the mRNA level in old mice compared to young and adult animals. Taken together, we provide evidence for AQP4 expression in the CP of the aging brain which likely contributes to the water flow through the CP epithelium and CSF production. In two alternative hypotheses, we discuss this as a beneficial compensatory, or a detrimental mechanism influencing the previously observed CSF changes during aging.
Assuntos
Aquaporina 4/metabolismo , Astrócitos/metabolismo , Encéfalo/metabolismo , Plexo Corióideo/metabolismo , Epêndima/metabolismo , Células Epiteliais/metabolismo , Idoso , Animais , Aquaporina 4/genética , Cadáver , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-IdadeRESUMO
The cuprizone model is a widely used model to study the pathogenesis of multiple sclerosis (MS). Due to the selective loss of mature oligodendrocytes and myelin, it is mainly being used to study demyelination and the mechanisms of remyelination, as well as the efficiency of compounds or therapeutics aiming at remyelination. Although early investigations using high dosages of cuprizone reported the occurrence of hydrocephalus, it has long been assumed that cuprizone feeding at lower dosages does not induce changes at the blood-brain barrier (BBB). Here, by analyzing BBB ultrastructure with high-resolution electron microscopy, we report changes at astrocytic endfeet surrounding vessels in the brain parenchyma. Particularly, edema formation around blood vessels and swollen astrocytic endfeet already occurred after feeding low dosages of cuprizone. These findings indicate changes in BBB function that will have an impact on the milieu of the central nervous system (CNS) in the cuprizone model and need to be considered when studying the mechanisms of de- and remyelination.
Assuntos
Cuprizona , Doenças Desmielinizantes , Animais , Camundongos , Cuprizona/toxicidade , Astrócitos/patologia , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/patologia , Camundongos Endogâmicos C57BL , Modelos Animais de DoençasRESUMO
Claudins (cldns) represent the largest family of transmembrane tight junction (TJ) proteins, determining organ-specific epithelial barrier properties. Because methods for the analysis of multiple cldn interaction are limited, we have established the heterologous Xenopus laevis oocyte expression system for TJ protein assembly and interaction analysis. Oocytes were injected with cRNA encoding human cldn-1, -2, or -3 or with a combination of these and were incubated in pairs for interaction analysis. Immunoblotting and immunohistochemistry were performed, and membrane contact areas were analyzed morphometrically and by freeze fracture electron microscopy. Cldns were specifically detected in membranes of expressing oocytes, and coincubation of oocytes resulted in adhesive contact areas that increased with incubation time. Adjacent membrane areas revealed specific cldn signals, including "kissing-point"-like structures representing homophilic trans-interactions of cldns. Contact areas of oocytes expressing a combination markedly exceeded those expressing single cldns, indicating effects on adhesion. Ultrastructural analysis revealed a self-assembly of TJ strands and a cldn-specific strand morphology.-Vitzthum, C., Stein, L., Brunner, N., Knittel, R., Fallier-Becker, P., Amasheh, S. Xenopus oocytes as a heterologous expression system for analysis of tight junction proteins.
Assuntos
Membrana Celular/metabolismo , Oócitos/metabolismo , Proteínas de Junções Íntimas/metabolismo , Animais , Claudina-1/genética , Claudina-1/metabolismo , Claudina-2/genética , Claudina-2/metabolismo , Claudina-3/genética , Claudina-3/metabolismo , Técnica de Fratura por Congelamento , Humanos , Immunoblotting , Imuno-Histoquímica , Microscopia Eletrônica , Ligação Proteica , Proteínas de Junções Íntimas/genética , Xenopus laevisRESUMO
Angioimmunoblastic T-cell lymphoma is a peripheral T-cell lymphoma derived from follicular T-helper cells. High-throughput genomic sequencing studies have shown that angioimmunoblastic T-cell lymphoma carries frequent mutations in RHOAG17V and IDH2R172 genes. The clinico-pathological features of angioimmunoblastic T-cell lymphoma cases with RHOAG17V mutations have been addressed; however, similar studies for IDH2 mutated cases are lacking. Therefore, the aim of the present study was to evaluate the pathological features of angioimmunoblastic T-cell lymphoma with IDH2 mutations. In order to identify cases with IDH2 mutations, 50 cases previously diagnosed as angioimmunoblastic T-cell lymphoma were subjected to next-generation sequencing analysis using a custom panel covering four genes frequently mutated in angioimmunoblastic T-cell lymphoma including DNMT3A, TET2, IDH2 and RHOA. All cases were analyzed for PD1, ICOS, CXCL13, CD10, BCL6, CD21, CD23 and EBER in situ hybridization. Mutational analysis recognized three groups. Group 1: IDH2R172 mutations were identified in 20 cases (40%). All cases carried RHOAG17V mutations. Group 2: RHOAG17V mutations without IDH2R172 mutation were identified in 16 cases (32%), and Group 3: 14 cases (28%) without RHOAG17V or IDH2R172 mutations. Morphologically, angioimmunoblastic T-cell lymphoma cases with IDH2R172 mutations were characterized by the presence of medium to large clear cells (p = 0.00001), and a follicular T-helper phenotype with the particular feature of strong CD10 (p = 0.0268) and CXCL13 expression (p = 0.0346). Interestingly, TET2 mutations were identified in 32 of 33 (97%) cases with IDH2R172 and/or RHOAG17V mutations whereas only 55% of angioimmunoblastic T-cell lymphoma cases wild-type for these two genes carried TET2 mutations (p = 0.0022). In contrast, DNMT3A mutations were found in 48% of the cases and were equally distributed in the three groups. In conclusion, our results support the results of gene expression profiling studies suggesting that IDH2R172 mutations define a unique subgroup within angioimmunoblastic T-cell lymphoma with strong follicular T-helper-like phenotype and characteristic morphological features.
Assuntos
Biomarcadores Tumorais/genética , Linfadenopatia Imunoblástica/genética , Isocitrato Desidrogenase/genética , Linfoma de Células T Periférico/genética , Mutação , Animais , Análise Mutacional de DNA , Perfilação da Expressão Gênica , Predisposição Genética para Doença , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Linfadenopatia Imunoblástica/imunologia , Linfadenopatia Imunoblástica/patologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/patologia , Linfoma de Células T Periférico/imunologia , Linfoma de Células T Periférico/patologia , Fenótipo , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/patologia , TranscriptomaRESUMO
The protease HtrA2 has a protective role inside mitochondria, but promotes apoptosis under stress. We previously identified the G399S HtrA2 mutation in Parkinson's disease (PD) patients and reported mitochondrial dysfunction in vitro. Mitochondrial dysfunction is a common feature of PD and related to neurodegeneration. Complete loss of HtrA2 has been shown to cause neurodegeneration in mice. However, the full impact of HtrA2 overexpression or the G399S mutation is still to be determined in vivo. Here, we report the first HtrA2 G399S transgenic mouse model. Our data suggest that the mutation has a dominant-negative effect. We also describe a toxic effect of wild-type (WT) HtrA2 overexpression. Only low overexpression of the G399S mutation allowed viable animals and we suggest that the mutant protein is likely unstable. This is accompanied by reduced mitochondrial respiratory capacity and sensitivity to apoptotic cell death. Mice overexpressing WT HtrA2 were viable, yet these animals have inhibited mitochondrial respiration and significant induction of apoptosis in the brain leading to motor dysfunction, highlighting the opposing roles of HtrA2. Our data further underscore the importance of HtrA2 as a key mediator of mitochondrial function and its fine regulatory role in cell fate. The location and abundance of HtrA2 is tightly controlled and, therefore, human mutations leading to gain- or loss of function could provide significant risk for PD-related neurodegeneration.
Assuntos
Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Mutação , Doença de Parkinson/genética , Serina Endopeptidases/genética , Animais , Apoptose , Encéfalo/metabolismo , Encéfalo/patologia , Respiração Celular , Modelos Animais de Doenças , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Dosagem de Genes , Regulação da Expressão Gênica , Serina Peptidase 2 de Requerimento de Alta Temperatura A , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteínas Mitocondriais/metabolismo , Atividade Motora , Neurônios/metabolismo , Neurônios/patologia , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Fenótipo , Serina Endopeptidases/metabolismoRESUMO
BACKGROUND: Echovirus (E) 30 (E-30) meningitis is characterized by neuroinflammation involving immune cell pleocytosis at the protective barriers of the central nervous system (CNS). In this context, infection of the blood-cerebrospinal fluid barrier (BCSFB), which has been demonstrated to be involved in enteroviral CNS pathogenesis, may affect the tight junction (TJ) and adherens junction (AJ) function and morphology. METHODS: We used an in vitro human choroid plexus epithelial (HIBCPP) cell model to investigate the effect of three clinical outbreak strains (13-311, 13-759, and 14-397) isolated in Germany in 2013, and compared them to E-30 Bastianni. Conducting transepithelial electrical resistance (TEER), paracellular dextran flux measurement, quantitative real-time polymerase chain reaction (qPCR), western blot, and immunofluorescence analysis, we investigated TJ and AJ function and morphology as well as strain-specific E-30 infection patterns. Additionally, transmission electron and focused ion beam microscopy electron microscopy (FIB-SEM) was used to evaluate the mode of leukocyte transmigration. Genome sequencing and phylogenetic analyses were performed to discriminate potential genetic differences among the outbreak strains. RESULTS: We observed a significant strain-dependent decrease in TEER with strains E-30 Bastianni and 13-311, whereas paracellular dextran flux was only affected by E-30 Bastianni. Despite strong similarities among the outbreak strains in replication characteristics and particle distribution, strain 13-311 was the only outbreak isolate revealing comparable disruptive effects on TJ (Zonula Occludens (ZO) 1 and occludin) and AJ (E-cadherin) morphology to E-30 Bastianni. Notwithstanding significant junctional alterations upon E-30 infection, we observed both para- and transcellular leukocyte migration across HIBCPP cells. Complete genome sequencing revealed differences between the strains analyzed, but no explicit correlation with the observed strain-dependent effects on HIBCPP cells was possible. CONCLUSION: The findings revealed distinct E-30 strain-specific effects on barrier integrity and junctional morphology. Despite E-30-induced barrier alterations leukocyte trafficking did not exclusively occur via the paracellular route.
Assuntos
Barreira Hematoencefálica/virologia , Líquido Cefalorraquidiano/virologia , Plexo Corióideo/virologia , Surtos de Doenças , Enterovirus Humano B/isolamento & purificação , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/ultraestrutura , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Células Cultivadas , Líquido Cefalorraquidiano/metabolismo , Plexo Corióideo/metabolismo , Plexo Corióideo/ultraestrutura , Enterovirus Humano B/metabolismo , Humanos , Filogenia , Especificidade da EspécieRESUMO
OBJECTIVE: The objective of this report of a fatal propofol-related infusion syndrome in a young adult was to present-to our knowledge for the first time-direct ultrastructural evidence for the central role of mitochondrial damage in the pathogenesis of this syndrome. DATA SOURCES: Histological and electron microscopical analysis of liver, skeletal, and heart muscle obtained by autopsy and blood obtained from patient. STUDY SELECTION: Case report. DATA EXTRACTION: In addition to conventional macroscopical and histological investigations, electron-microscopical analysis of myocardial- and skeletal muscle and liver tissue obtained at autopsy from a young man was performed in order to search for ultrastructural changes of mitochondria. Acylcarnitine concentrations of his blood were determined by ultra-high performance liquid chromatography mass spectrometry. DATA SYNTHESIS: A 19-year-old male was admitted with acute left-side hemiparesis. The patient was intubated, then propofol infusion started, and a craniotomy was performed to remove an intracerebral hematoma. In the postoperative period, the patient presented with elevated intracranial pressure and brain edema. After repeat surgery, the patient showed impaired systolic left ventricular function, increasing fever, anuria, hyperkalemia, and metabolic acidosis, and he finally expired. Electron microscopy revealed dark, electron dense amorphous structures associated with mitochondria in heart muscle and liver tissue obtained at autopsy. Peripheral blood analysis revealed increased levels of acetyl-, propionyl-, butyryl-, malonyl-, and valeryl-carnitine as an indicator for propofol-related infusion syndrome, as well as for propofol-mediated inhibition of free fatty acid uptake into mitochondria, affecting beta-oxidation. CONCLUSIONS: Electron dense bodies found in association with mitochondria in muscle and liver cells probably correspond to accumulation of free fatty acid provide direct morphological evidence for the mitochondrial damage in propofol-related infusion syndrome.
Assuntos
Doenças Mitocondriais/induzido quimicamente , Doenças Mitocondriais/patologia , Síndrome da Infusão de Propofol/patologia , Carnitina/análogos & derivados , Carnitina/sangue , Craniotomia , Hematoma Subdural Intracraniano/cirurgia , Humanos , Infusões Intravenosas , Masculino , Microscopia Eletrônica , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/patologia , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/patologia , Mitocôndrias Musculares/efeitos dos fármacos , Mitocôndrias Musculares/patologia , Complicações Pós-Operatórias/induzido quimicamente , Complicações Pós-Operatórias/patologia , Adulto JovemRESUMO
AIMS: To explore the ultrastructure of interstitial cells in the upper lamina propria of the human bladder, to describe the spatial relationships and to investigate cell-cell contacts. METHODS: Focused ion beam scanning electron microscopy (FIB-SEM), 3-View SEM and confocal laser scanning microscopy were used to analyze the 3D ultrastructure of the upper lamina propria in male and female human bladders. RESULTS: 3View-SEM image stacks as large as 59 × 59 × 17 µm3 (xyz) at a resolution of 16 × 16 × 50 nm3 and high resolution (5 × 5 × 10 nm3 ) FIB-SEM stacks could be analyzed. Interstitial cells with myoid differentiation (mIC) and fibroblast like interstitial cells (fIC) were the major cell types in the upper lamina propria. The flat, sheet-like ICs were oriented strictly parallel to the urothelium. No spindle shaped cells were present. We furthermore identified one branched cell (bIC) with several processes contacting urothelial cells by penetrating the basal membrane. This cell did not make any contacts to other ICs within the upper lamina propria. We found no evidence for the occurrence of telocytes in the upper lamina propria. CONCLUSIONS: Comprehensive 3D-ultrastructural analysis of the human bladder confirmed distinct subtypes of interstitial cells. We provide evidence for a foremost unknown direct connection between a branched interstitial cell and urothelial cells of which the functional role has still to be elucidated. 3D-ultrastructure analyses at high resolution are needed to further define the subpopulations of lamina propria cells and cell-cell interactions.
Assuntos
Células Epiteliais/ultraestrutura , Junções Intercelulares/ultraestrutura , Microscopia/métodos , Mucosa/ultraestrutura , Bexiga Urinária/ultraestrutura , Urotélio/ultraestrutura , Células Epiteliais/citologia , Feminino , Humanos , Imageamento Tridimensional , Imuno-Histoquímica , Masculino , Microscopia Confocal , Microscopia Eletrônica de Varredura , Mucosa/citologia , Bexiga Urinária/citologia , Urotélio/citologiaRESUMO
Brain-intrinsic degenerative cascades have been proposed to be an initial factor driving lesion formation in multiple sclerosis (MS). Here, we identify neurodegeneration as a potent trigger for peripheral immune cell recruitment into the mouse forebrain. Female C57BL/6 mice were fed cuprizone for 3 weeks, followed by a period of 2 weeks on normal chow to induce the formation of lesion foci in the forebrain. Subsequent immunization with myelin oligodendrocyte glycoprotein 35-55 peptide, which induces myelin autoreactive T cells in the periphery, resulted in massive immune cell recruitment into the affected forebrain. Additional adoptive transfer experiments together with flow cytometry analysis underline the importance of brain-derived signals for immune cell recruitment. This study clearly illustrates the significance of brain-intrinsic degenerative cascades for immune cell recruitment and MS lesion formation. Additional studies have to address the signaling cascades and mechanistic processes that form the top-down communication between the affected brain area, neurovascular unit, and peripheral immune cells. SIGNIFICANCE STATEMENT: We identify neurodegeneration as a potent trigger for peripheral immune cell recruitment into the forebrain. Thus, immune cell recruitment might be a second step during the formation of new inflammatory lesions in multiple sclerosis. A better understanding of factors regulating neurodegeneration-induced immune cell recruitment will pave the way for the development of novel therapeutic treatment strategies.
Assuntos
Linfócitos/fisiologia , Monócitos/fisiologia , Doenças Neurodegenerativas/patologia , Prosencéfalo/patologia , Transferência Adotiva , Animais , Complexo CD3/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Quelantes/toxicidade , Cuprizona/toxicidade , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/patologia , Feminino , Adjuvante de Freund/toxicidade , Linfonodos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/metabolismo , Glicoproteína Mielina-Oligodendrócito/imunologia , Doenças Neurodegenerativas/induzido quimicamente , Fragmentos de Peptídeos/imunologia , Toxina Pertussis/toxicidadeRESUMO
Brain-intrinsic degenerative cascades are a proposed factor driving inflammatory lesion formation in multiple sclerosis (MS) patients. We recently described a model combining noninflammatory cytodegeneration (via cuprizone) with the classic active experimental autoimmune encephalomyelitis (Cup/EAE model), which exhibits inflammatory forebrain lesions. Here, we describe the histopathological characteristics and progression of these Cup/EAE lesions. We show that inflammatory lesions develop at various topographical sites in the forebrain, including white matter tracts and cortical and subcortical grey matter areas. The lesions are characterized by focal demyelination, discontinuation of the perivascular glia limitans, focal axonal damage, and neutrophil granulocyte extravasation. Transgenic mice with enhanced green fluorescent protein-expressing microglia and red fluorescent protein-expressing monocytes reveal that both myeloid cell populations contribute to forebrain inflammatory infiltrates. EAE-triggered inflammatory cerebellar lesions were augmented in mice pre-intoxicated with cuprizone. Gene expression studies suggest roles of the chemokines Cxcl10, Ccl2, and Ccl3 in inflammatory lesion formation. Finally, follow-up experiments in Cup/EAE mice with chronic disease revealed that forebrain, but not spinal cord, lesions undergo spontaneous reorganization and repair. This study underpins the significance of brain-intrinsic degenerative cascades for immune cell recruitment and, in consequence, MS lesion formation.
Assuntos
Progressão da Doença , Encefalite/etiologia , Encefalite/patologia , Encefalomielite Autoimune Experimental/complicações , Sesquiterpenos/toxicidade , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Modelos Animais de Doenças , Encefalite/genética , Encefalomielite Autoimune Experimental/imunologia , Feminino , Adjuvante de Freund/toxicidade , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Proteína Glial Fibrilar Ácida/metabolismo , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/patologia , Microglia/ultraestrutura , Monócitos/patologia , Monócitos/ultraestrutura , Glicoproteína Mielina-Oligodendrócito/imunologia , Glicoproteína Mielina-Oligodendrócito/toxicidade , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/toxicidade , Receptores CCR2/genética , Receptores CCR2/metabolismo , Receptores de Interleucina-8A/genética , Receptores de Interleucina-8A/metabolismoRESUMO
Diabetes mellitus and neurodegeneration are common diseases for which shared genetic factors are still only partly known. Here, we show that loss of the BiP (immunoglobulin heavy-chain binding protein) co-chaperone DNAJC3 leads to diabetes mellitus and widespread neurodegeneration. We investigated three siblings with juvenile-onset diabetes and central and peripheral neurodegeneration, including ataxia, upper-motor-neuron damage, peripheral neuropathy, hearing loss, and cerebral atrophy. Exome sequencing identified a homozygous stop mutation in DNAJC3. Screening of a diabetes database with 226,194 individuals yielded eight phenotypically similar individuals and one family carrying a homozygous DNAJC3 deletion. DNAJC3 was absent in fibroblasts from all affected subjects in both families. To delineate the phenotypic and mutational spectrum and the genetic variability of DNAJC3, we analyzed 8,603 exomes, including 506 from families affected by diabetes, ataxia, upper-motor-neuron damage, peripheral neuropathy, or hearing loss. This analysis revealed only one further loss-of-function allele in DNAJC3 and no further associations in subjects with only a subset of the features of the main phenotype. Our findings demonstrate that loss-of-function DNAJC3 mutations lead to a monogenic, recessive form of diabetes mellitus in humans. Moreover, they present a common denominator for diabetes and widespread neurodegeneration. This complements findings from mice in which knockout of Dnajc3 leads to diabetes and modifies disease in a neurodegenerative model of Marinesco-Sjögren syndrome.
Assuntos
Diabetes Mellitus Tipo 1/genética , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico/genética , Atrofia de Múltiplos Sistemas/genética , Adolescente , Adulto , Ataxia/genética , Diabetes Mellitus Tipo 1/diagnóstico por imagem , Chaperona BiP do Retículo Endoplasmático , Exoma/genética , Feminino , Fibroblastos , Proteínas de Choque Térmico HSP40/metabolismo , Homozigoto , Humanos , Masculino , Modelos Moleculares , Atrofia de Múltiplos Sistemas/diagnóstico por imagem , Mutação , Linhagem , Fenótipo , Radiografia , Análise de Sequência de DNA , Adulto JovemRESUMO
We previously demonstrated that the co-cultivation of endothelial cells with neural cells resulted in an improved integrity of the in vitro blood-brain barrier (BBB), and that this model could be useful to evaluate the transport properties of potential central nervous system disease drugs through the microvascular brain endothelial. In this study we have used real-time PCR, fluorescent microscopy, protein arrays and enzyme-linked immunosorbent assays to determine which neural- and endothelial cell-derived factors are produced in the co-culture and improve the integrity of the BBB. In addition, a further improvement of the BBB integrity was achieved by adjusting serum concentrations and growth factors or by the addition of brain pericytes. Under specific conditions expression of angiogenic, angiostatic and neurotrophic factors such as endostatin, pigment epithelium derived factor (PEDF/serpins-F1), tissue inhibitor of metalloproteinases (TIMP-1), and vascular endothelial cell growth factor (VEGF) closely mimicked the in vivo situation. Freeze-fracture analysis of these cultures demonstrated the quality and organization of the endothelial tight junction structures and their association to the two different lipidic leaflets of the membrane. Finally, a multi-cell culture model of the BBB with a transendothelial electrical resistance up to 371 (±15) Ω×cm2 was developed, which may be useful for preliminary screening of drug transport across the BBB and to evaluate cellular crosstalk of cells involved in the neurovascular unit.
Assuntos
Proteínas Angiogênicas/metabolismo , Barreira Hematoencefálica/metabolismo , Permeabilidade Capilar , Comunicação Celular , Células Endoteliais/metabolismo , Fatores de Crescimento Neural/metabolismo , Neurônios/metabolismo , Junções Íntimas/metabolismo , Animais , Barreira Hematoencefálica/citologia , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Técnicas de Cocultura , Impedância Elétrica , Humanos , Acoplamento Neurovascular , Fenótipo , Transdução de Sinais , Sus scrofa , Proteínas de Junções Íntimas/metabolismoRESUMO
The astrocytic endfoot membranes of the healthy blood-brain barrier-contacting the capillary-are covered with a large number of the water channel aquaporin 4 (AQP4). They form orthogonal arrays of particles (OAPs), which consist of AQP4 isoform M1 and M23. Under pathologic conditions, AQP4 is distributed over the whole cell and no or only small OAPs are found. From cell culture experiments, it is known that cells transfected only with AQP4-M1 do not form OAPs or only small ones. We hypothesized that in astrocytomas the situation may be comparable to the in vitro experiments expecting an upregulation of AQP4-M1. Quantitative Real-time PCR (qRT-PCR) of different graded astrocytomas revealed an upregulation of both isoforms AQP4 M1 and M23 in all astrocytomas investigated. In freeze fracture replicas of low-grade malignancy astrocytomas, more OAPs than in high-grade malignancy astrocytomas were found. In vitro, cultured glioma cells did not express AQP4, whereas healthy astrocytes revealed a slight upregulation of both isoforms and only a few OAPs in freeze fracture analysis. Taken together, we found a correlation between the decrease of OAPs and increasing grade of malignancy of astrocytomas but this was not consistent with an upregulation of AQP4-M1 in relation to AQP4 M23.
Assuntos
Aquaporina 4/genética , Astrócitos/citologia , Astrocitoma/patologia , Membrana Celular/metabolismo , Animais , Astrócitos/metabolismo , Astrocitoma/genética , Barreira Hematoencefálica/metabolismo , Encéfalo/citologia , Encéfalo/metabolismo , Células Cultivadas , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Imageamento Tridimensional , Camundongos , Microscopia de Fluorescência , Isoformas de Proteínas , Reação em Cadeia da Polimerase em Tempo Real , Regulação para CimaRESUMO
The main water channel of the brain, aquaporin-4 (AQP4), is one of the classical water-specific aquaporins. It is expressed in many epithelial tissues in the basolateral membrane domain. It is present in the membranes of supporting cells in most sensory organs in a specifically adapted pattern: in the supporting cells of the olfactory mucosa, AQP4 occurs along the basolateral aspects, in mammalian retinal Müller cells it is highly polarized. In the cochlear epithelium of the inner ear, it is expressed basolaterally in some cells but strictly basally in others. Within the central nervous system, aquaporin-4 (AQP4) is expressed by cells of the astroglial family, more specifically, by astrocytes and ependymal cells. In the mammalian brain, AQP4 is located in high density in the membranes of astrocytic endfeet facing the pial surface and surrounding blood vessels. At these locations, AQP4 plays a role in the maintenance of ionic homeostasis and volume regulation. This highly polarized expression has not been observed in the brain of fish where astroglial cells have long processes and occur mostly as radial glial cells. In the brain of the zebrafish, AQP4 immunoreactivity is found along the radial extent of astroglial cells. This suggests that the polarized expression of AQP4 was not present at all stages of evolution. Thus, a polarized expression of AQP4 as part of a control mechanism for a stable ionic environment and water balanced occurred at several locations in supporting and glial cells during evolution. This initially basolateral membrane localization of AQP4 is shifted to highly polarized expression in astrocytic endfeet in the mammalian brain and serves as a part of the neurovascular unit to efficiently maintain homeostasis.
Assuntos
Aquaporina 4/metabolismo , Astrócitos/metabolismo , Encéfalo/metabolismo , Animais , Astrócitos/citologia , Encéfalo/citologia , Células Ependimogliais/citologia , Células Ependimogliais/metabolismo , Humanos , Mucosa Olfatória/citologia , Mucosa Olfatória/metabolismo , Água/metabolismoRESUMO
The astrocytic aquaporin-4 (AQP4) water channel is the target of pathogenic antibodies in a spectrum of relapsing autoimmune inflammatory central nervous system disorders of varying severity that is unified by detection of the serum biomarker neuromyelitis optica (NMO)-IgG. Neuromyelitis optica is the most severe of these disorders. The two major AQP4 isoforms, M1 and M23, have identical extracellular residues. This report identifies two novel properties of NMO-IgG as determinants of pathogenicity. First, the binding of NMO-IgG to the ectodomain of astrocytic AQP4 has isoform-specific outcomes. M1 is completely internalized, but M23 resists internalization and is aggregated into larger-order orthogonal arrays of particles that activate complement more effectively than M1 when bound by NMO-IgG. Second, NMO-IgG binding to either isoform impairs water flux directly, independently of antigen down-regulation. We identified, in nondestructive central nervous system lesions of two NMO patients, two previously unappreciated histopathological correlates supporting the clinical relevance of our in vitro findings: (i) reactive astrocytes with persistent foci of surface AQP4 and (ii) vacuolation in adjacent myelin consistent with edema. The multiple molecular outcomes identified as a consequence of NMO-IgG interaction with AQP4 plausibly account for the diverse pathological features of NMO: edema, inflammation, demyelination, and necrosis. Differences in the nature and anatomical distribution of NMO lesions, and in the clinical and imaging manifestations of disease documented in pediatric and adult patients, may be influenced by regional and maturational differences in the ratio of M1 to M23 proteins in astrocytic membranes.
Assuntos
Aquaporina 4/metabolismo , Astrócitos/metabolismo , Biomarcadores/metabolismo , Imunoglobulina G/metabolismo , Neuromielite Óptica/sangue , Neuromielite Óptica/patologia , Animais , Astrócitos/ultraestrutura , Biomarcadores/sangue , Western Blotting , Membrana Celular/metabolismo , Imunofluorescência , Técnica de Fratura por Congelamento , Imunoglobulina G/sangue , Camundongos , Microscopia Eletrônica , Bainha de Mielina/patologia , Oócitos/metabolismo , Ratos , Vacúolos/patologiaRESUMO
The principal central nervous system (CNS) water channel, aquaporin-4 (AQP4), is confined to astrocytic and ependymal membranes and is the target of a pathogenic autoantibody, neuromyelitis optica (NMO)-IgG. This disease-specific autoantibody unifies a spectrum of relapsing CNS autoimmune inflammatory disorders of which NMO exemplifies the classic phenotype. Multiple sclerosis and other immune-mediated demyelinating disorders of the CNS lack a distinctive biomarker. Two AQP4 isoforms, M1 and M23, exist as homotetrameric and heterotetrameric intramembranous particles (IMPs). Orthogonal arrays of predominantly M23 particles (OAPs) are an ultrastructural characteristic of astrocytic membranes. We used high-titered serum from 32 AQP4-IgG-seropositive patients and 85 controls to investigate the nature and molecular location of AQP4 epitopes that bind NMO-IgG, and the influence of supramolecular structure. NMO-IgG bound to denatured AQP4 monomers (68% of cases), to native tetramers and high order arrays (90% of cases), and to AQP4 in live cell membranes (100% of cases). Disease-specific epitopes reside in extracellular loop C more than in loops A or E. IgG binding to intracellular epitopes lacks disease specificity. These observations predict greater disease sensitivity and specificity for tissue-based and cell-based serological assays employing "native" AQP4 than assays employing denatured AQP4 and fragments. NMO-IgG binds most avidly to plasma membrane surface AQP4 epitopes formed by loop interactions within tetramers and by intermolecular interactions within high order structures. The relative abundance and localization of AQP4 high order arrays in distinct CNS regions may explain the variability in clinical phenotype of NMO spectrum disorders.
Assuntos
Aquaporina 4/imunologia , Astrócitos/imunologia , Autoanticorpos/imunologia , Neuromielite Óptica/imunologia , Aquaporina 4/metabolismo , Aquaporina 4/ultraestrutura , Membrana Celular/imunologia , Células Cultivadas , Cristalografia por Raios X , Epitopos/imunologia , Feminino , Humanos , Masculino , Proteínas Recombinantes de FusãoRESUMO
The blood-brain barrier (BBB) is a selectively permeable boundary that separates the circulating blood from the extracellular fluid of the brain and is an essential component for brain homeostasis. In glioblastoma (GBM), the BBB of peritumoral vessels is often disrupted. Pericytes, being important to maintaining BBB integrity, can be functionally modified by GBM cells which induce proliferation and cell motility via the TGF-ß-mediated induction of central epithelial to mesenchymal transition (EMT) factors. We demonstrate that pericytes strengthen the integrity of the BBB in primary endothelial cell/pericyte co-cultures as an in vitro BBB model, using TEER measurement of the barrier integrity. In contrast, this effect was abrogated by TGF-ß or conditioned medium from TGF-ß secreting GBM cells, leading to the disruption of a so far intact and tight BBB. TGF-ß notably changed the metabolic behavior of pericytes, by shutting down the TCA cycle, driving energy generation from oxidative phosphorylation towards glycolysis, and by modulating pathways that are necessary for the biosynthesis of molecules used for proliferation and cell division. Combined metabolomic and transcriptomic analyses further underscored that the observed functional and metabolic changes of TGF-ß-treated pericytes are closely connected with their role as important supporting cells during angiogenic processes.
RESUMO
In human glioblastoma, the blood-brain barrier (BBB) is disturbed. According to our concept, the glio-vascular relationships and thus the control of the BBB are essentially dependent on the polarity of astroglial cells. This polarity is characterized by the uneven distribution of the water channel protein aquaporin-4 (AQP4), dystroglycan and other molecules. Recently, we were able to show that the extracellular matrix component agrin is important for the construction and localization of the so-called orthogonal arrays of particles (OAPs), which consist in AQP4. Here, combining freeze-fracture electron microscopy, immunohistochemistry and Western blotting, we describe alterations of expression and distribution of AQP4, dystroglycan, agrin and the matrix metalloproteinases (MMP) 2, 3 and 9 in human primary glioblastomas (eight primary tumours, six recurrent tumours). Increase of MMP3- and MMP2/9 immunoreactivities went along with loss of agrin and dystroglycan respectively. On the protein level, AQP4 expression was increased in glioblastoma compared to control tissue. This was not accompanied by an increase of OAPs, suggesting that AQP4 can also occur without forming OAPs. The results underline our concept of the loss of glioma cell polarity as one of the factors responsible for the disturbance of the neurovascular unit and as an explanation for the formation of edemas in the glioblastoma.
Assuntos
Agrina/metabolismo , Aquaporina 4/metabolismo , Neoplasias Encefálicas/metabolismo , Encéfalo/metabolismo , Distroglicanas/metabolismo , Glioblastoma/metabolismo , Metaloproteinases da Matriz/metabolismo , Barreira Hematoencefálica/metabolismo , Polaridade Celular , Matriz Extracelular/metabolismo , Técnica de Fratura por Congelamento , Humanos , Imuno-Histoquímica , Microscopia EletrônicaRESUMO
The Rho GTPase Miro1, located at the mitochondrial outer membrane is known to properly distribute mitochondria to synapses, aid calcium buffering and initiate PINK1-Parkin mediated mitophagy. Several heterozygous RHOT1/Miro1 variants were identified in sporadic Parkinson's disease patients. Miro1 R272Q is located within a calcium binding domain, but the functional outcome of this point mutation and its contribution to the development of disease are unclear. To address this, we introduced a heterozygous RHOT1/Miro1 R272Q point mutation in healthy induced pluripotent stem cells. In dopaminergic neurons, Miro1 R272Q does not affect Miro1 protein levels, CCCP-induced mitophagy, nor mitochondrial movement yet causes the fragmentation of mitochondria with reduction of cristae and ATP5A. Inhibition of the mitochondrial calcium uniporter phenocopied Miro1 R272Q cytosolic calcium response to Thapsigargin in active neurons, a similar effect was observed during the calcium buffering phase in Miro1 knockdown neuroblastoma cells. Altered mitochondrial calcium regulation is associated with reduced mitochondrial respiration and reduced catecholamine neurotransmitter uptake. Synaptic changes are not coupled to dopamine distribution or dopamine transporters but are linked to Miro1 R272Q-related calcium handling via the mitochondria concomitant with defective dopamine regulation at the mitochondrial surface by monoamine oxidase. We conclude that the Miro1 R272Q heterozygous point mutation dampens mitochondrial-calcium regulation and mitochondrial capacity via events at the outer membrane that are sufficient to disrupt dopaminergic function.