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1.
Glia ; 72(2): 362-374, 2024 02.
Artigo em Inglês | MEDLINE | ID: mdl-37846809

RESUMO

Cerebral organoids (CerOrgs) derived from human induced pluripotent stem cells (iPSCs) are a valuable tool to study human astrocytes and their interaction with neurons and microglia. The timeline of astrocyte development and maturation in this model is currently unknown and this limits the value and applicability of the model. Therefore, we generated CerOrgs from three healthy individuals and assessed astrocyte maturation after 5, 11, 19, and 37 weeks in culture. At these four time points, the astrocyte lineage was isolated based on the expression of integrin subunit alpha 6 (ITGA6). Based on the transcriptome of the isolated ITGA6-positive cells, astrocyte development started between 5 and 11 weeks in culture and astrocyte maturation commenced after 11 weeks in culture. After 19 weeks in culture, the ITGA6-positive astrocytes had the highest expression of human mature astrocyte genes, and the predicted functional properties were related to brain homeostasis. After 37 weeks in culture, a subpopulation of ITGA6-negative astrocytes appeared, highlighting the heterogeneity within the astrocytes. The morphology shifted from an elongated progenitor-like morphology to the typical bushy astrocyte morphology. Based on the morphological properties, predicted functional properties, and the similarities with the human mature astrocyte transcriptome, we concluded that ITGA6-positive astrocytes have developed optimally in 19-week-old CerOrgs.


Assuntos
Células-Tronco Pluripotentes Induzidas , Transcriptoma , Humanos , Células Cultivadas , Astrócitos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Perfilação da Expressão Gênica , Organoides , Diferenciação Celular
2.
J Neuropathol Exp Neurol ; 82(9): 798-805, 2023 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-37478478

RESUMO

Neuroinflammation and microthrombosis may be underlying mechanisms of brain injury after aneurysmal subarachnoid hemorrhage (aSAH), but they have not been studied in relation to each other. In postmortem brain tissue, we investigated neuroinflammation by studying the microglial and astrocyte response in the frontal cortex of 11 aSAH and 10 control patients. In a second study, we investigated the correlation between microthrombosis and microglia by studying the microglial surface area around vessels with and without microthrombosis in the frontal cortex and hippocampus of 8 other aSAH patients. In comparison with controls, we found increased numbers of microglia (mean ± SEM 50 ± 8 vs 20 ± 5 per 0.0026 mm³, p < 0.01), an increased surface area (%) of microglia (mean ± SEM 4.2 ± 0.6 vs 2.2 ± 0.4, p < 0.05), a higher intensity of the astrocytic intermediate filament protein glial fibrillary acidic protein (GFAP) (mean ± SEM 184 ± 28 vs 92 ± 23 arbitrary units, p < 0.05), and an increased GFAP surface area (%) (mean ± SEM 21.2 ± 2.6 vs 10.7 ± 2.1, p < 0.01) in aSAH tissue. Microglia surface area was approximately 40% larger around vessels with microthrombosis than those without microthrombosis (estimated marginal means [95% CI]; 6.1 [5.4-6.9] vs 4.3 [3.6-5.0], p < 0.001). Our results show that the microglial and astrocyte surface areas increased after aSAH and that microthrombosis and microglia are interrelated.


Assuntos
Hemorragia Subaracnóidea , Humanos , Hemorragia Subaracnóidea/complicações , Hemorragia Subaracnóidea/metabolismo , Doenças Neuroinflamatórias , Autopsia , Encéfalo/metabolismo , Microglia/metabolismo
3.
Nutrients ; 14(10)2022 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-35631316

RESUMO

Evidence of the impact of nutrition on human brain development is compelling. Previous in vitro and in vivo results show that three specific amino acids, histidine, lysine, and threonine, synergistically inhibit mTOR activity and behavior. Therefore, the prenatal availability of these amino acids could be important for human neurodevelopment. However, methods to study the underlying mechanisms in a human model of neurodevelopment are limited. Here, we pioneer the use of human cerebral organoids to investigate the impact of amino acid supplementation on neurodevelopment. In this study, cerebral organoids were exposed to 10 mM and 50 mM of the amino acids threonine, histidine, and lysine. The impact was determined by measuring mTOR activity using Western blots, general cerebral organoid size, and gene expression by RNA sequencing. Exposure to threonine, histidine, and lysine led to decreased mTOR activity and markedly reduced organoid size, supporting findings in rodent studies. RNA sequencing identified comprehensive changes in gene expression, with enrichment in genes related to specific biological processes (among which are mTOR signaling and immune function) and to specific cell types, including proliferative precursor cells, microglia, and astrocytes. Altogether, cerebral organoids are responsive to nutritional exposure by increasing specific amino acid concentrations and reflect findings from previous rodent studies. Threonine, histidine, and lysine exposure impacts the early development of human cerebral organoids, illustrated by the inhibition of mTOR activity, reduced size, and altered gene expression.


Assuntos
Aminoácidos , Histidina , Aminoácidos/metabolismo , Histidina/farmacologia , Humanos , Lisina/farmacologia , Organoides , Serina-Treonina Quinases TOR , Treonina
4.
Nat Commun ; 13(1): 1036, 2022 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-35210419

RESUMO

Following the decline of neurogenesis at birth, progenitors of the subventricular zone (SVZ) remain mostly in a quiescent state in the adult human brain. The mechanisms that regulate this quiescent state are still unclear. Here, we isolate CD271+ progenitors from the aged human SVZ for single-cell RNA sequencing analysis. Our transcriptome data reveal the identity of progenitors of the aged human SVZ as late oligodendrocyte progenitor cells. We identify the Wnt pathway antagonist SFRP1 as a possible signal that promotes quiescence of progenitors from the aged human SVZ. Administration of WAY-316606, a small molecule that inhibits SFRP1 function, stimulates activation of neural stem cells both in vitro and in vivo under homeostatic conditions. Our data unravel a possible mechanism through which progenitors of the adult human SVZ are maintained in a quiescent state and a potential target for stimulating progenitors to re-activate.


Assuntos
Ventrículos Laterais , Células-Tronco Neurais , Idoso , Encéfalo/metabolismo , Diferenciação Celular/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Ventrículos Laterais/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Células-Tronco Neurais/metabolismo , Neurogênese/genética , Transcriptoma
5.
Sci Rep ; 12(1): 424, 2022 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-35013418

RESUMO

Glioma is the most common form of malignant primary brain tumours in adults. Their highly invasive nature makes the disease incurable to date, emphasizing the importance of better understanding the mechanisms driving glioma invasion. Glial fibrillary acidic protein (GFAP) is an intermediate filament protein that is characteristic for astrocyte- and neural stem cell-derived gliomas. Glioma malignancy is associated with changes in GFAP alternative splicing, as the canonical isoform GFAPα is downregulated in higher-grade tumours, leading to increased dominance of the GFAPδ isoform in the network. In this study, we used intravital imaging and an ex vivo brain slice invasion model. We show that the GFAPδ and GFAPα isoforms differentially regulate the tumour dynamics of glioma cells. Depletion of either isoform increases the migratory capacity of glioma cells. Remarkably, GFAPδ-depleted cells migrate randomly through the brain tissue, whereas GFAPα-depleted cells show a directionally persistent invasion into the brain parenchyma. This study shows that distinct compositions of the GFAPnetwork lead to specific migratory dynamics and behaviours of gliomas.


Assuntos
Neoplasias Encefálicas/patologia , Encéfalo/patologia , Movimento Celular , Proteína Glial Fibrilar Ácida/metabolismo , Glioma/patologia , Animais , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Feminino , Glioma/metabolismo , Microscopia Intravital , Masculino , Camundongos Endogâmicos C57BL , Invasividade Neoplásica , Isoformas de Proteínas
6.
Brain Behav Immun ; 100: 219-230, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34896594

RESUMO

Dysregulation of microglial function contributes to Alzheimer's disease (AD) pathogenesis. Several genetic and transcriptome studies have revealed microglia specific genetic risk factors, and changes in microglia expression profiles in AD pathogenesis, viz. the human-Alzheimer's microglia/myeloid (HAM) profile in AD patients and the disease-associated microglia profile (DAM) in AD mouse models. The transcriptional changes involve genes in immune and inflammatory pathways, and in pathways associated with Aß clearance. Aß oligomers have been suggested to be the initial trigger of microglia activation in AD. To study the direct response to Aß oligomers exposure, we assessed changes in gene expression in an in vitro model for microglia, the human monocyte-derived microglial-like (MDMi) cells. We confirmed the initiation of an inflammatory profile following LPS stimulation, based on increased expression of IL1B, IL6, and TNFα. In contrast, the Aß1-42 oligomers did not induce an inflammatory profile or a classical HAM profile. Interestingly, we observed a specific increase in the expression of metallothioneins in the Aß1-42 oligomer treated MDMi cells. Metallothioneins are involved in metal ion regulation, protection against reactive oxygen species, and have anti-inflammatory properties. In conclusion, our data suggests that exposure to Aß1-42 oligomers may initially trigger a protective response in vitro.


Assuntos
Doença de Alzheimer , Microglia , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Humanos , Camundongos , Microglia/metabolismo , Monócitos/metabolismo , Fragmentos de Peptídeos , Transcriptoma
7.
FASEB J ; 35(3): e21389, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33583081

RESUMO

The glial fibrillary acidic protein (GFAP) is a type III intermediate filament (IF) protein that is highly expressed in astrocytes, neural stem cells, and in gliomas. Gliomas are a heterogeneous group of primary brain tumors that arise from glia cells or neural stem cells and rely on accurate diagnosis for prognosis and treatment strategies. GFAP is differentially expressed between glioma subtypes and, therefore, often used as a diagnostic marker. However, GFAP is highly regulated by the process of alternative splicing; many different isoforms have been identified. Differential expression of GFAP isoforms between glioma subtypes suggests that GFAP isoform-specific analyses could benefit diagnostics. In this study we report on the differential expression of a new GFAP isoform between glioma subtypes, GFAPµ. A short GFAP transcript resulting from GFAP exon 2 skipping was detected by RNA sequencing of human glioma. We show that GFAPµ mRNA is expressed in healthy brain tissue, glioma cell lines, and primary glioma cells and that it translates into a ~21 kDa GFAP protein. 21 kDa GFAP protein was detected in the IF protein fraction isolated from human spinal cord as well. We further show that induced GFAPµ expression disrupts the GFAP IF network. The characterization of this new GFAP isoform adds on to the numerous previously identified GFAP splice isoforms. It emphasizes the importance of studying the contribution of IF splice variants to specialized functions of the IF network and to glioma research.


Assuntos
Processamento Alternativo , Neoplasias Encefálicas/metabolismo , Proteína Glial Fibrilar Ácida/biossíntese , Glioma/metabolismo , Encéfalo/metabolismo , Linhagem Celular Tumoral , Proteína Glial Fibrilar Ácida/química , Proteína Glial Fibrilar Ácida/genética , Humanos , Biossíntese de Proteínas , Isoformas de Proteínas , Vimentina/química
8.
Brain Commun ; 2(2): fcaa150, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33376983

RESUMO

Neurogenesis continues throughout adulthood in specialized regions of the brain. One of these regions is the subventricular zone. During brain development, neurogenesis is regulated by a complex interplay of intrinsic and extrinsic cues that control stem-cell survival, renewal and cell lineage specification. Cerebrospinal fluid (CSF) is an integral part of the neurogenic niche in development as it is in direct contact with radial glial cells, and it is important in regulating proliferation and migration. Yet, the effect of CSF on neural stem cells in the subventricular zone of the adult human brain is unknown. We hypothesized a persistent stimulating effect of ventricular CSF on neural stem cells in adulthood, based on the literature, describing bulging accumulations of subventricular cells where CSF is in direct contact with the subventricular zone. Here, we show by immunohistochemistry on post-mortem adult human subventricular zone sections that neural stem cells are in close contact with CSF via protrusions through both intact and incomplete ependymal layers. We are the first to systematically quantify subventricular glial nodules denuded of ependyma and consisting of proliferating neural stem and progenitor cells, and showed that they are present from foetal age until adulthood. Neurosphere, cell motility and differentiation assays as well as analyses of RNA expression were used to assess the effects of CSF of adult humans on primary neural stem cells and a human immortalized neural stem cell line. We show that human ventricular CSF increases proliferation and decreases motility of neural stem cells. Our results also indicate that adult CSF pushes neural stem cells from a relative quiescent to a more active state and promotes neuronal over astrocytic lineage differentiation. Thus, CSF continues to stimulate neural stem cells throughout aging.

9.
Brain Pathol ; 30(6): 1071-1086, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32876357

RESUMO

Pericytes are vascular mural cells that surround capillaries of the central nervous system (CNS). They are crucial for brain development and contribute to CNS homeostasis by regulating blood-brain barrier function and cerebral blood flow. It has been suggested that pericytes are lost in Alzheimer's disease (AD), implicating this cell type in disease pathology. Here, we have employed state-of-the-art stereological morphometry techniques as well as tissue clearing and two-photon imaging to assess the distribution of pericytes in two independent cohorts of AD (n = 16 and 13) and non-demented controls (n = 16 and 4). Stereological quantification revealed increased capillary density with a normal pericyte population in the frontal cortex of AD brains, a region with early amyloid ß deposition. Two-photon analysis of cleared frontal cortex tissue confirmed the preservation of pericytes in AD cases. These results suggest that pericyte demise is not a general hallmark of AD pathology.


Assuntos
Doença de Alzheimer/patologia , Capilares/patologia , Lobo Frontal/patologia , Pericitos/patologia , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/patologia , Capilares/metabolismo , Circulação Cerebrovascular/fisiologia , Feminino , Lobo Frontal/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Fragmentos de Peptídeos/metabolismo , Pericitos/metabolismo
10.
FASEB J ; 33(11): 12941-12959, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31480854

RESUMO

Gliomas are the most common primary brain tumors. Their highly invasive character and the heterogeneity of active oncogenic pathways within single tumors complicate the development of curative therapies and cause poor patient prognosis. Glioma cells express the intermediate filament protein glial fibrillary acidic protein (GFAP), and the level of its alternative splice variant GFAP-δ, relative to its canonical splice variant GFAP-α, is higher in grade IV compared with lower-grade and lower malignant glioma. In this study we show that a high GFAP-δ/α ratio induces the expression of the dual-specificity phosphatase 4 (DUSP4) in focal adhesions. By focusing on pathways up- and downstream of DUSP4 that are involved in the cell-extracellular matrix interaction, we show that a high GFAP-δ/α ratio equips glioma cells to better invade the brain. This study supports the hypothesis that glioma cells with a high GFAP-δ/α ratio are highly invasive and more malignant cells, thus making GFAP alternative splicing a potential therapeutic target.-Van Bodegraven, E. J., van Asperen, J. V., Sluijs, J. A., van Deursen, C. B. J., van Strien, M. E., Stassen, O. M. J. A., Robe, P. A. J., Hol, E. M. GFAP alternative splicing regulates glioma cell-ECM interaction in a DUSP4-dependent manner.


Assuntos
Processamento Alternativo , Neoplasias Encefálicas/patologia , Fosfatases de Especificidade Dupla/fisiologia , Matriz Extracelular/patologia , Proteína Glial Fibrilar Ácida/genética , Glioma/patologia , Fosfatases da Proteína Quinase Ativada por Mitógeno/fisiologia , Neoplasias Encefálicas/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Fosfatases de Especificidade Dupla/genética , Matriz Extracelular/metabolismo , Técnicas de Silenciamento de Genes , Glioma/metabolismo , Humanos , Laminina/metabolismo , MAP Quinase Quinase 4/metabolismo , Fosfatases da Proteína Quinase Ativada por Mitógeno/genética , Fosforilação
11.
J Neurol Neurosurg Psychiatry ; 89(2): 138-146, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28951498

RESUMO

OBJECTIVE: To report the clinical and immunological characteristics of 22 new patients with glial fibrillar acidic protein (GFAP) autoantibodies. METHODS: From January 2012 to March 2017, we recruited 451 patients with suspected neurological autoimmune disease at the Catholic University of Rome. Patients' serum and cerebrospinal fluid (CSF) samples were tested for neural autoantibodies by immunohistochemistry on mouse and rat brain sections, by cell-based assays (CBA) and immunoblot. GFAP autoantibodies were detected by immunohistochemistry and their specificity confirmed by CBA using cells expressing human GFAPα and GFAPδ proteins, by immunoblot and immunohistochemistry on GFAP-/- mouse brain sections. RESULTS: Serum and/or CSF IgG of 22/451 (5%) patients bound to human GFAP, of which 22/22 bound to GFAPα, 14/22 to both GFAPα and GFAPδ and none to the GFAPδ isoform only. The neurological presentation was: meningoencephalomyelitis or encephalitis in 10, movement disorder (choreoathetosis or myoclonus) in 3, anti-epileptic drugs (AED)-resistant epilepsy in 3, cerebellar ataxia in 3, myelitis in 2, optic neuritis in 1 patient. Coexisting neural autoantibodies were detected in five patients. Six patients had other autoimmune diseases. Tumours were found in 3/22 patients (breast carcinoma, 1; ovarian carcinoma, 1; thymoma, 1). Nineteen patients were treated with immunotherapy and 16 patients (84%) improved. Histopathology analysis of the leptomeningeal biopsy specimen from one patient revealed a mononuclear infiltrate with macrophages and CD8+ T cells. CONCLUSIONS: GFAP autoimmunity is not rare. The clinical spectrum encompasses meningoencephalitis, myelitis, movement disorders, epilepsy and cerebellar ataxia. Coexisting neurological and systemic autoimmunity are relatively common. Immunotherapy is beneficial in most cases.


Assuntos
Autoanticorpos/imunologia , Doenças Autoimunes do Sistema Nervoso/fisiopatologia , Proteína Glial Fibrilar Ácida/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Doenças Autoimunes do Sistema Nervoso/complicações , Doenças Autoimunes do Sistema Nervoso/imunologia , Doenças Autoimunes do Sistema Nervoso/terapia , Encéfalo/diagnóstico por imagem , Neoplasias da Mama/complicações , Carcinoma/complicações , Ataxia Cerebelar/complicações , Ataxia Cerebelar/imunologia , Ataxia Cerebelar/fisiopatologia , Ataxia Cerebelar/terapia , Criança , Epilepsia Resistente a Medicamentos/complicações , Epilepsia Resistente a Medicamentos/imunologia , Epilepsia Resistente a Medicamentos/fisiopatologia , Epilepsia Resistente a Medicamentos/terapia , Encefalomielite/complicações , Encefalomielite/imunologia , Encefalomielite/fisiopatologia , Encefalomielite/terapia , Feminino , Proteína Glial Fibrilar Ácida/genética , Glucocorticoides/uso terapêutico , Humanos , Imunoglobulinas Intravenosas/uso terapêutico , Fatores Imunológicos/uso terapêutico , Imunoterapia , Imageamento por Ressonância Magnética , Masculino , Meningoencefalite/complicações , Meningoencefalite/imunologia , Meningoencefalite/fisiopatologia , Meningoencefalite/terapia , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Transtornos dos Movimentos/complicações , Transtornos dos Movimentos/imunologia , Transtornos dos Movimentos/fisiopatologia , Transtornos dos Movimentos/terapia , Mielite/complicações , Mielite/imunologia , Mielite/fisiopatologia , Mielite/terapia , Mioclonia/complicações , Mioclonia/imunologia
12.
Oncotarget ; 8(50): 88104-88121, 2017 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-29152145

RESUMO

Astrocytomas are the most common malignant brain tumours and are to date incurable. It is unclear how astrocytomas progress into higher malignant grades. The intermediate filament cytoskeleton is emerging as an important regulator of malignancy in several tumours. The majority of the astrocytomas express the intermediate filament protein Glial Fibrillary Acidic Protein (GFAP). Several GFAP splice variants have been identified and the main variants expressed in human astrocytoma are the GFAPα and GFAPδ isoforms. Here we show a significant downregulation of GFAPα in grade IV astrocytoma compared to grade II and III, resulting in an increased GFAPδ/α ratio. Mimicking this increase in GFAPδ/α ratio in astrocytoma cell lines and comparing the subsequent transcriptomic changes with the changes in the patient tumours, we have identified a set of GFAPδ/α ratio-regulated high-malignant and low-malignant genes. These genes are involved in cell proliferation and protein phosphorylation, and their expression correlated with patient survival. We additionally show that changing the ratio of GFAPδ/α, by targeting GFAP expression, affected expression of high-malignant genes. Our data imply that regulating GFAP expression and splicing are novel therapeutic targets that need to be considered as a treatment for astrocytoma.

13.
Cell Mol Life Sci ; 73(21): 4101-20, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27141937

RESUMO

Glial fibrillary acidic protein (GFAP) is the characteristic intermediate filament (IF) protein in astrocytes. Expression of its main isoforms, GFAPα and GFAPδ, varies in astrocytes and astrocytoma implying a potential regulatory role in astrocyte physiology and pathology. An IF-network is a dynamic structure and has been functionally linked to cell motility, proliferation, and morphology. There is a constant exchange of IF-proteins with the network. To study differences in the dynamic properties of GFAPα and GFAPδ, we performed fluorescence recovery after photobleaching experiments on astrocytoma cells with fluorescently tagged GFAPs. Here, we show for the first time that the exchange of GFP-GFAPδ was significantly slower than the exchange of GFP-GFAPα with the IF-network. Furthermore, a collapsed IF-network, induced by GFAPδ expression, led to a further decrease in fluorescence recovery of both GFP-GFAPα and GFP-GFAPδ. This altered IF-network also changed cell morphology and the focal adhesion size, but did not alter cell migration or proliferation. Our study provides further insight into the modulation of the dynamic properties and functional consequences of the IF-network composition.


Assuntos
Astrócitos/citologia , Forma Celular , Adesões Focais/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Filamentos Intermediários/metabolismo , Actinas/metabolismo , Adulto , Idoso , Astrócitos/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Feminino , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imageamento Tridimensional , Microtúbulos/metabolismo , Nestina/metabolismo , Isoformas de Proteínas/metabolismo , Vimentina/metabolismo
14.
J Immunol ; 194(8): 3623-33, 2015 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-25769924

RESUMO

Aicardi-Goutières syndrome (AGS) is a monogenic inflammatory encephalopathy caused by mutations in TREX1, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, ADAR1, or MDA5. Mutations in those genes affect normal RNA/DNA intracellular metabolism and detection, triggering an autoimmune response with an increase in cerebral IFN-α production by astrocytes. Microangiopathy and vascular disease also contribute to the neuropathology in AGS. In this study, we report that AGS gene silencing of TREX1, SAMHD1, RNASEH2A, and ADAR1 by short hairpin RNAs in human neural stem cell-derived astrocytes, human primary astrocytes, and brain-derived endothelial cells leads to an antiviral status of these cells compared with nontarget short hairpin RNA-treated cells. We observed a distinct activation of the IFN-stimulated gene signature with a substantial increase in the release of proinflammatory cytokines (IL-6) and chemokines (CXCL10 and CCL5). A differential impact of AGS gene silencing was noted; silencing TREX1 gave rise to the most dramatic in both cell types. Our findings fit well with the observation that patients carrying mutations in TREX1 experience an earlier onset and fatal outcome. We provide in the present study, to our knowledge for the first time, insight into how astrocytic and endothelial activation of antiviral status may differentially lead to cerebral pathology, suggesting a rational link between proinflammatory mediators and disease severity in AGS.


Assuntos
Astrócitos/imunologia , Doenças Autoimunes do Sistema Nervoso/imunologia , Citocinas/imunologia , Células Endoteliais/imunologia , Interferon-alfa/imunologia , Malformações do Sistema Nervoso/imunologia , Células-Tronco Neurais/imunologia , Adenosina Desaminase/genética , Adenosina Desaminase/imunologia , Astrócitos/patologia , Doenças Autoimunes do Sistema Nervoso/genética , Doenças Autoimunes do Sistema Nervoso/mortalidade , Doenças Autoimunes do Sistema Nervoso/patologia , Citocinas/genética , Células Endoteliais/patologia , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/imunologia , Inativação Gênica , Células HEK293 , Humanos , Interferon-alfa/genética , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/imunologia , Mutação , Malformações do Sistema Nervoso/genética , Malformações do Sistema Nervoso/mortalidade , Malformações do Sistema Nervoso/patologia , Células-Tronco Neurais/patologia , Fosfoproteínas/genética , Fosfoproteínas/imunologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/imunologia , Ribonuclease H/genética , Ribonuclease H/imunologia , Proteína 1 com Domínio SAM e Domínio HD
15.
J Cell Sci ; 127(Pt 20): 4368-80, 2014 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-25128567

RESUMO

Glial fibrillary acidic protein (GFAP) is the main intermediate filament in astrocytes and is regulated by epigenetic mechanisms during development. We demonstrate that histone acetylation also controls GFAP expression in mature astrocytes. Inhibition of histone deacetylases (HDACs) with trichostatin A or sodium butyrate reduced GFAP expression in primary human astrocytes and astrocytoma cells. Because splicing occurs co-transcriptionally, we investigated whether histone acetylation changes the ratio between the canonical isoform GFAPα and the alternative GFAPδ splice variant. We observed that decreased transcription of GFAP enhanced alternative isoform expression, as HDAC inhibition increased the GFAPδ∶GFAPα ratio. Expression of GFAPδ was dependent on the presence and binding of splicing factors of the SR protein family. Inhibition of HDAC activity also resulted in aggregation of the GFAP network, reminiscent of our previous findings of a GFAPδ-induced network collapse. Taken together, our data demonstrate that HDAC inhibition results in changes in transcription, splicing and organization of GFAP. These data imply that a tight regulation of histone acetylation in astrocytes is essential, because dysregulation of gene expression causes the aggregation of GFAP, a hallmark of human diseases like Alexander's disease.


Assuntos
Doença de Alexander/metabolismo , Astrócitos/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Histona Desacetilases/metabolismo , Acetilação/efeitos dos fármacos , Doença de Alexander/genética , Processamento Alternativo/efeitos dos fármacos , Astrócitos/efeitos dos fármacos , Ácido Butírico/farmacologia , Linhagem Celular Tumoral , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Epigênese Genética , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína Glial Fibrilar Ácida/genética , Inibidores de Histona Desacetilases/farmacologia , Histonas/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Agregados Proteicos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerização Proteica/efeitos dos fármacos
16.
FASEB J ; 28(7): 2942-54, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24696300

RESUMO

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein expressed in astrocytes and neural stem cells. The GFAP gene is alternatively spliced, and expression of GFAP is highly regulated during development, on brain damage, and in neurodegenerative diseases. GFAPα is the canonical splice variant and is expressed in all GFAP-positive cells. In the human brain, the alternatively spliced transcript GFAPδ marks specialized astrocyte populations, such as subpial astrocytes and the neurogenic astrocytes in the human subventricular zone. We here show that shifting the GFAP isoform ratio in favor of GFAPδ in astrocytoma cells, by selectively silencing the canonical isoform GFAPα with short hairpin RNAs, induced a change in integrins, a decrease in plectin, and an increase in expression of the extracellular matrix component laminin. Together, this did not affect cell proliferation but resulted in a significantly decreased motility of astrocytoma cells. In contrast, a down-regulation of all GFAP isoforms led to less cell spreading, increased integrin expression, and a >100-fold difference in the adhesion of astrocytoma cells to laminin. In summary, isoform-specific silencing of GFAP revealed distinct roles of a specialized GFAP network in regulating the interaction of astrocytoma cells with the extracellular matrix through laminin.-Moeton, M., Kanski, R., Stassen, O. M. J. A., Sluijs, J. A., Geerts, D., van Tijn, P., Wiche, G., van Strien, M. E., Hol, E. M. Silencing GFAP isoforms in astrocytoma cells disturbs laminin dependent motility and cell adhesion.


Assuntos
Astrocitoma/metabolismo , Adesão Celular/genética , Movimento Celular/genética , Proteína Glial Fibrilar Ácida/metabolismo , Laminina/metabolismo , Isoformas de Proteínas/metabolismo , Astrócitos/metabolismo , Astrócitos/patologia , Astrocitoma/genética , Astrocitoma/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo/genética , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Proteína Glial Fibrilar Ácida/genética , Células HEK293 , Humanos , Integrinas/genética , Integrinas/metabolismo , Laminina/genética , Isoformas de Proteínas/genética
17.
Stem Cells Transl Med ; 3(4): 470-80, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24604282

RESUMO

Neural progenitor cells (NPCs) in the subventricular zone (SVZ) hold promise for future therapy for neurodegenerative disorders, because the stimulation of adult neurogenesis could potentially restore the function of degenerating neurons and glia. To obtain more knowledge on these NPCs, we developed a method to specifically isolate NPCs from postmortem adult human brains based on the expression of the specific human adult neural stem/progenitor cell marker glial fibrillary acidic protein δ (GFAPδ). An extensive immunophenotyping analysis for cell surface markers resulted in the observation that CD271 was limited to the SVZ-derived GFAPδ-positive cells. CD271(+) cells developed into neurospheres and could be differentiated into astrocytes, neurons, and oligodendrocytes. We are the first to show that a pure population of NPCs can be isolated from the adult human SVZ, which is highly instrumental for developing future therapies based on stimulating endogenous SVZ neurogenesis.


Assuntos
Antígenos de Diferenciação/biossíntese , Encéfalo , Separação Celular , Expressão Gênica , Proteínas do Tecido Nervoso/biossíntese , Células-Tronco Neurais , Receptores de Fator de Crescimento Neural/biossíntese , Adulto , Astrócitos/citologia , Astrócitos/metabolismo , Encéfalo/citologia , Encéfalo/metabolismo , Diferenciação Celular , Feminino , Proteína Glial Fibrilar Ácida/biossíntese , Humanos , Masculino , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurogênese/fisiologia , Neurônios/citologia , Neurônios/metabolismo , Oligodendroglia/citologia , Oligodendroglia/metabolismo
18.
Neurobiol Aging ; 35(3): 492-510, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24269023

RESUMO

In Alzheimer's disease (AD), amyloid plaques are surrounded by reactive astrocytes with an increased expression of intermediate filaments including glial fibrillary acidic protein (GFAP). Different GFAP isoforms have been identified that are differentially expressed by specific subpopulations of astrocytes and that impose different properties to the intermediate filament network. We studied transcript levels and protein expression patterns of all known GFAP isoforms in human hippocampal AD tissue at different stages of the disease. Ten different transcripts for GFAP isoforms were detected at different abundancies. Transcript levels of most isoforms increased with AD progression. GFAPδ-immunopositive astrocytes were observed in subgranular zone, hilus, and stratum-lacunosum-moleculare. GFAPδ-positive cells also stained for GFAPα. In AD donors, astrocytes near plaques displayed increased staining of both GFAPα and GFAPδ. The reading-frame-shifted isoform, GFAP(+1), staining was confined to a subset of astrocytes with long processes, and their number increased in the course of AD. In conclusion, the various GFAP isoforms show differential transcript levels and are upregulated in a concerted manner in AD. The GFAP(+1) isoform defines a unique subset of astrocytes, with numbers increasing with AD progression. These data indicate the need for future exploration of underlying mechanisms concerning the functions of GFAPδ and GFAP(+1) isoforms in astrocytes and their possible role in AD pathology.


Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Astrócitos/metabolismo , Astrócitos/patologia , Proteína Glial Fibrilar Ácida/metabolismo , Placa Amiloide/metabolismo , Células Cultivadas , Progressão da Doença , Expressão Gênica , Hipocampo/metabolismo , Humanos , Isoformas de Proteínas , Índice de Gravidade de Doença , Transcrição Gênica , Regulação para Cima
19.
PLoS One ; 7(8): e42823, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22912745

RESUMO

Glial fibrillary acidic protein (GFAP) is the main astrocytic intermediate filament (IF). GFAP splice isoforms show differential expression patterns in the human brain. GFAPδ is preferentially expressed by neurogenic astrocytes in the subventricular zone (SVZ), whereas GFAP(+1) is found in a subset of astrocytes throughout the brain. In addition, the expression of these isoforms in human brain material of epilepsy, Alzheimer and glioma patients has been reported. Here, for the first time, we present a comprehensive study of GFAP isoform expression in both wild-type and Alzheimer Disease (AD) mouse models. In cortex, cerebellum, and striatum of wild-type mice, transcripts for Gfap-α, Gfap-ß, Gfap-γ, Gfap-δ, Gfap-κ, and a newly identified isoform Gfap-ζ, were detected. Their relative expression levels were similar in all regions studied. GFAPα showed a widespread expression whilst GFAPδ distribution was prominent in the SVZ, rostral migratory stream (RMS), neurogenic astrocytes of the subgranular zone (SGZ), and subpial astrocytes. In contrast to the human SVZ, we could not establish an unambiguous GFAPδ localization in proliferating cells of the mouse SVZ. In APPswePS1dE9 and 3xTgAD mice, plaque-associated reactive astrocytes had increased transcript levels of all detectable GFAP isoforms and low levels of a new GFAP isoform, Gfap-ΔEx7. Reactive astrocytes in AD mice showed enhanced GFAPα and GFAPδ immunolabeling, less frequently increased vimentin and nestin, but no GFAPκ or GFAP(+1) staining. In conclusion, GFAPδ protein is present in SVZ, RMS, and neurogenic astrocytes of the SGZ, but also outside neurogenic niches. Furthermore, differential GFAP isoform expression is not linked with aging or reactive gliosis. This evidence points to the conclusion that differential regulation of GFAP isoforms is not involved in the reorganization of the IF network in reactive gliosis or in neurogenesis in the mouse brain.


Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Astrócitos/metabolismo , Astrócitos/patologia , Encéfalo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurogênese , Envelhecimento/genética , Envelhecimento/metabolismo , Envelhecimento/patologia , Doença de Alzheimer/genética , Animais , Especificidade de Anticorpos , Encéfalo/patologia , Modelos Animais de Doenças , Proteína Glial Fibrilar Ácida , Proteínas de Filamentos Intermediários/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/imunologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
20.
Parkinsons Dis ; 2012: 420957, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22577599

RESUMO

Dementia is a common feature in Parkinson's disease (PD) and is considered to be the result of limbic and cortical Lewy bodies and/or Alzheimer changes. Astrogliosis may also affect the development of dementia, since it correlates well with declining cognition in Alzheimer patients. Thus, we determined whether cortical astrogliosis occurs in PD, whether it is related to dementia, and whether this is reflected by the presence of glial fibrillary acidic protein (GFAP) and vimentin in cerebrospinal fluid (CSF). We have examined these proteins by immunohistochemistry in the frontal cortex and by Western blot in CSF of cases with PD, PD with dementia (PDD), dementia with Lewy bodies (DLB) and nondemented controls. We were neither able to detect an increase in cortical astrogliosis in PD, PDD, or DLB nor could we observe a correlation between the extent of astrogliosis and the degree of dementia. The levels of GFAP and vimentin in CSF did not correlate to the extent of astrogliosis or dementia. We did confirm the previously identified positive correlation between the presence of cortical Lewy bodies and dementia in PD. In conclusion, we have shown that cortical astrogliosis is not associated with the cognitive decline in Lewy body-related dementia.

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