RESUMEN
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.
Asunto(s)
Empalme Alternativo , Neoplasias Encefálicas/patología , Fosfatasas de Especificidad Dual/fisiología , Matriz Extracelular/patología , Proteína Ácida Fibrilar de la Glía/genética , Glioma/patología , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/fisiología , Neoplasias Encefálicas/metabolismo , Sistemas CRISPR-Cas , Línea Celular Tumoral , Fosfatasas de Especificidad Dual/genética , Matriz Extracelular/metabolismo , Técnicas de Silenciamiento del Gen , Glioma/metabolismo , Humanos , Laminina/metabolismo , MAP Quinasa Quinasa 4/metabolismo , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/genética , FosforilaciónRESUMEN
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.
Asunto(s)
Astrocitos/citología , Forma de la Célula , Adhesiones Focales/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Filamentos Intermedios/metabolismo , Actinas/metabolismo , Adulto , Anciano , Astrocitos/metabolismo , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Supervivencia Celular , Femenino , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Imagenología Tridimensional , Microtúbulos/metabolismo , Nestina/metabolismo , Isoformas de Proteínas/metabolismo , Vimentina/metabolismoRESUMEN
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 GFAPδâ¶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.
Asunto(s)
Enfermedad de Alexander/metabolismo , Astrocitos/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Histona Desacetilasas/metabolismo , Acetilación/efectos de los fármacos , Enfermedad de Alexander/genética , Empalme Alternativo/efectos de los fármacos , Astrocitos/efectos de los fármacos , Ácido Butírico/farmacología , Línea Celular Tumoral , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Epigénesis Genética , Regulación de la Expresión Génica/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/genética , Inhibidores de Histona Desacetilasas/farmacología , Histonas/metabolismo , Humanos , Ácidos Hidroxámicos/farmacología , Agregado de Proteínas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerización de Proteína/efectos de los fármacosRESUMEN
Glioblastoma multiforme (GBM) is the most common primary brain tumor and is without exception lethal. GBMs modify the immune system, which contributes to the aggressive nature of the disease. Particularly, cells of the monocytic lineage, including monocytes, macrophages and microglia, are affected. We investigated the influence of GBM-derived extracellular vesicles (EVs) on the phenotype of monocytic cells. Proteomic profiling showed GBM EVs to be enriched with proteins functioning in extracellular matrix interaction and leukocyte migration. GBM EVs appeared to skew the differentiation of peripheral blood-derived monocytes to alternatively activated/M2-type macrophages. This was observed for EVs from an established cell line, as well as for EVs from primary cultures of GBM stem-like cells (GSCs). Unlike EVs of non-GBM origin, GBM EVs induced modified expression of cell surface proteins, modified cytokine secretion (e.g., an increase in vascular endothelial growth factor and IL-6) and increased phagocytic capacity of the macrophages. Most pronounced effects were observed upon incubation with EVs from mesenchymal GSCs. GSC EVs also affected primary human microglia, resulting in increased expression of Membrane type 1-matrix metalloproteinase, a marker for GBM microglia and functioning as tumor-supportive factor. In conclusion, GBM-derived EVs can modify cells of the monocytic lineage, which acquire characteristics that resemble the tumor-supportive phenotypes observed in patients.
Asunto(s)
Neoplasias Encefálicas/patología , Glioblastoma/patología , Leucocitos Mononucleares/patología , Neoplasias Encefálicas/metabolismo , Diferenciación Celular/fisiología , Línea Celular Tumoral , Exosomas/metabolismo , Exosomas/patología , Glioblastoma/metabolismo , Humanos , Leucocitos Mononucleares/metabolismo , Macrófagos/metabolismo , Macrófagos/patología , Microglía/metabolismo , Microglía/patología , FenotipoRESUMEN
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.
Asunto(s)
Astrocitoma/metabolismo , Adhesión Celular/genética , Movimiento Celular/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Laminina/metabolismo , Isoformas de Proteínas/metabolismo , Astrocitos/metabolismo , Astrocitos/patología , Astrocitoma/genética , Astrocitoma/patología , Encéfalo/metabolismo , Encéfalo/patología , Línea Celular , Línea Celular Tumoral , Proliferación Celular , Regulación hacia Abajo/genética , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Proteína Ácida Fibrilar de la Glía/genética , Células HEK293 , Humanos , Integrinas/genética , Integrinas/metabolismo , Laminina/genética , Isoformas de Proteínas/genéticaRESUMEN
Multiple sclerosis is a serious neurological disorder, resulting in e.g., sensory, motor and cognitive deficits. A critical pathological aspect of multiple sclerosis (MS) is the influx of immunomodulatory cells into the central nervous system (CNS). Identification of key players that regulate cellular trafficking into the CNS may lead to the development of more selective treatment to halt this process. The multifunctional enzyme tissue Transglutaminase (TG2) can participate in various inflammation-related processes, and is known to be expressed in the CNS. In the present study, we question whether TG2 activity contributes to the pathogenesis of experimental MS, and could be a novel therapeutic target. In human post-mortem material, we showed the appearance of TG2 immunoreactivity in leukocytes in MS lesions, and particular in macrophages in rat chronic-relapsing experimental autoimmune encephalomyelitis (cr-EAE), an experimental MS model. Clinical deficits as observed in mouse EAE were reduced in TG2 knock-out mice compared to littermate wild-type mice, supporting a role of TG2 in EAE pathogenesis. To establish if the enzyme TG2 represents an attractive therapeutic target, cr-EAE rats were treated with TG2 activity inhibitors during ongoing disease. Reduction of TG2 activity in cr-EAE animals dramatically attenuated clinical deficits and demyelination. The mechanism underlying these beneficial effects pointed toward a reduction in macrophage migration into the CNS due to attenuated cytoskeletal flexibility and RhoA GTPase activity. Moreover, iNOS and TNFα levels were selectively reduced in the CNS of cr-EAE rats treated with a TG2 activity inhibitor, whereas other relevant inflammatory mediators were not affected in CNS or spleen by reducing TG2 activity. We conclude that modulating TG2 activity opens new avenues for therapeutic intervention in MS which does not affect peripheral levels of inflammatory mediators.
Asunto(s)
Encefalomielitis Autoinmune Experimental/enzimología , Proteínas de Unión al GTP/metabolismo , Esclerosis Múltiple/enzimología , Transglutaminasas/metabolismo , Anciano , Anciano de 80 o más Años , Animales , Movimiento Celular/efectos de los fármacos , Corteza Cerebral/enzimología , Corteza Cerebral/patología , Encefalomielitis Autoinmune Experimental/patología , Células Endoteliales/enzimología , Células Endoteliales/patología , Femenino , Proteínas de Unión al GTP/antagonistas & inhibidores , Proteínas de Unión al GTP/genética , Humanos , Mediadores de Inflamación/metabolismo , Isoxazoles/farmacología , Macrófagos/enzimología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Esclerosis Múltiple/patología , Vaina de Mielina/enzimología , Proteína Glutamina Gamma Glutamiltransferasa 2 , ARN Mensajero/metabolismo , Ratas , Médula Espinal/enzimología , Médula Espinal/patología , Bazo/metabolismo , Linfocitos T/metabolismo , Transglutaminasas/antagonistas & inhibidores , Transglutaminasas/genéticaRESUMEN
Astrocytes emerge as crucial cells for proper neuronal functioning in the developing and adult brain. Neurons and astrocytes are sequentially generated from the same pool of neural stem cells (NSCs). Tight regulation of the neuron-to-astrocyte switch is critical for (1) the generation of a balanced number of astrocytes and neurons and (2) neuronal circuit formation, since newborn astrocytes regulate synapse formation. This review focuses on signaling pathways that instruct astrogenesis, incorporating recently discovered intrinsic and extrinsic regulators. The canonical pathway of astrocytic gene expression, JAK/STAT signaling, is inhibited during neurogenesis to prevent premature astrocyte differentiation. At the onset of astrogenesis, Notch signaling induces epigenetic remodeling of astrocytic genes like glial fibrillary acidic protein to change NSC competence. In turn, astrogenesis is initiated by signals received from newborn neurons. We highlight how key molecular pathways like JAK/STAT and Notch are integrated in a complex network of environmental signals and epigenetic and transcriptional regulators to determine NSC differentiation. It is essential to understand NSC differentiation in respect to future NSC-based therapies for brain diseases, as transplanted NSCs preferentially become astrocytes. As emphasized in this review, many clues in this respect can be learned from development.
Asunto(s)
Astrocitos/citología , Encéfalo/crecimiento & desarrollo , Diferenciación Celular/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Células-Madre Neurales/fisiología , Neurogénesis/fisiología , Transducción de Señal/fisiología , Encéfalo/embriología , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Quinasas Janus/metabolismo , Modelos Biológicos , Células-Madre Neurales/citología , Receptores Notch/metabolismoRESUMEN
Remyelination following central nervous system demyelination is essential to prevent axon degeneration. However, remyelination ultimately fails in demyelinating diseases such as multiple sclerosis. This failure of remyelination is likely mediated by many factors, including changes in the extracellular signalling environment. Here, we examined the expression of the extracellular matrix molecule fibronectin on demyelinating injury and how this affects remyelination by oligodendrocytes progenitors. In toxin-induced lesions undergoing efficient remyelination, fibronectin expression was transiently increased within demyelinated areas and declined as remyelination proceeded. Fibronectin levels increased both by leakage from the blood circulation and by production from central nervous system resident cells. In chronically demyelinated multiple sclerosis lesions, fibronectin expression persisted in the form of aggregates, which may render fibronectin resistant to degradation. Aggregation of fibronectin was similarly observed at the relapse phase of chronic experimental autoimmune encephalitis, but not on toxin-induced demyelination, suggesting that fibronectin aggregation is mediated by inflammation-induced demyelination. Indeed, the inflammatory mediator lipopolysaccharide induced fibronectin aggregation by astrocytes. Most intriguingly, injection of astrocyte-derived fibronectin aggregates in toxin-induced demyelinated lesions inhibited oligodendrocyte differentiation and remyelination, and fibronectin aggregates are barely expressed in remyelinated multiple sclerosis lesions. Therefore, these findings suggest that fibronectin aggregates within multiple sclerosis lesions contribute to remyelination failure. Hence, the inhibitory signals induced by fibronectin aggregates or factors that affect fibronectin aggregation could be potential therapeutic targets for promoting remyelination.
Asunto(s)
Enfermedades Desmielinizantes/metabolismo , Encefalomielitis Autoinmune Experimental/metabolismo , Fibronectinas/metabolismo , Esclerosis Múltiple/metabolismo , Vaina de Mielina/metabolismo , Animales , Astrocitos/metabolismo , Astrocitos/patología , Axones/metabolismo , Axones/patología , Encéfalo/metabolismo , Encéfalo/patología , Encéfalo/fisiopatología , Células Cultivadas , Enfermedades Desmielinizantes/patología , Enfermedades Desmielinizantes/fisiopatología , Encefalomielitis Autoinmune Experimental/patología , Encefalomielitis Autoinmune Experimental/fisiopatología , Femenino , Humanos , Esclerosis Múltiple/patología , Esclerosis Múltiple/fisiopatología , Vaina de Mielina/patología , Regeneración Nerviosa/fisiología , Oligodendroglía/metabolismo , Oligodendroglía/patología , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Médula Espinal/patología , Médula Espinal/fisiopatologíaRESUMEN
There are many indications that neurogenesis is impaired in Parkinson's disease, which might be due to a lack of dopamine in the subventricular zone. An impairment in neurogenesis may have negative consequences for the development of new therapeutic approaches in Parkinson's disease, as neural stem cells are a potential source for endogenous repair. In this study, we examined the subventricular zone of 10 patients with Parkinson's disease and 10 age- and sex-matched controls for proliferation and neural stem cell numbers. We also included five cases with incidental Lewy body disease, which showed Parkinson's disease pathology but no clinical symptoms and thus did not receive dopaminergic treatment. We quantified the neural stem cell number and proliferative capacity in the subventricular zone of these three donor groups. We found subventricular neural stem cells in each donor, with a high variation in number. We did not observe significant differences in neural stem cell number or in proliferation between the groups. Additionally, we were able to culture neural stem cells from post-mortem brain of several patients with Parkinson's disease, confirming the presence of viable neural stem cells in these brains. We have also examined the subventricular zone of a chronic, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinson's disease mouse model, and again found no effect of dopaminergic denervation on precursor proliferation. Lastly, we investigated the proliferation capacity of two different human neural stem cell lines in response to dopamine. Both cell lines did not respond with a change in proliferation to treatment with dopamine agonists and an antagonist. In summary, the adult neural stem cell pool in the subventricular zone was not clearly affected in the human parkinsonian brain or a Parkinson's disease mouse model. Furthermore, we did not find evidence that dopamine has a direct effect on human neural stem cell proliferation in vitro. Thus, we conclude that the number of adult neural stem cells is probably not diminished in the parkinsonian brain and that dopamine depletion most likely has no effect on human neural stem cells.
Asunto(s)
Encéfalo/patología , Proliferación Celular , Ventrículos Cerebrales/patología , Intoxicación por MPTP/patología , Neurogénesis/fisiología , Enfermedad de Parkinson/patología , Anciano , Anciano de 80 o más Años , Animales , Encéfalo/metabolismo , Encéfalo/fisiopatología , Células Cultivadas , Ventrículos Cerebrales/metabolismo , Ventrículos Cerebrales/fisiopatología , Femenino , Humanos , Intoxicación por MPTP/metabolismo , Intoxicación por MPTP/fisiopatología , Masculino , Ratones , Células-Madre Neurales , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/fisiopatologíaRESUMEN
During normal brain development, axons are myelinated by mature oligodendrocytes (OLGs). Under pathological, demyelinating conditions within the central nervous system (CNS), axonal remyelination is only partially successful because oligodendrocyte precursor cells (OPCs) largely remain in an undifferentiated state resulting in a failure to generate myelinating OLGs. Tissue Transglutaminase (TG2) is a multifunctional enzyme, which amongst other functions, is involved in cell differentiation. Therefore, we hypothesized that TG2 contributes to differentiation of OPCs into OLGs and thereby stimulates remyelination. In vivo studies, using the cuprizone model for de- and remyelination in TG2(-/-) and wild-type mice, showed that during remyelination expression of proteolipid protein mRNA, as a marker for remyelination, in the corpus callosum lags behind in TG2(-/-) mice resulting in less myelin formation and, moreover, impaired recovery of motor behavior. Subsequent in vitro studies showed that rat OPCs express TG2 protein and activity which reduces when the cells have matured into OLGs. Furthermore, when TG2 activity is pharmacologically inhibited, the differentiation of OPCs into myelin-forming OLGs is dramatically reduced. We conclude that TG2 plays a prominent role in remyelination of the CNS, probably through stimulating OPC differentiation into myelin-forming OLGs. Therefore, manipulating TG2 activity may represent an interesting new target for remyelination in demyelinating diseases.
Asunto(s)
Vaina de Mielina/metabolismo , Células-Madre Neurales/fisiología , Oligodendroglía/fisiología , Transglutaminasas/metabolismo , Animales , Western Blotting , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cuprizona/farmacología , Enfermedades Desmielinizantes/metabolismo , Complejo II de Transporte de Electrones/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/fisiología , Inmunohistoquímica , Hibridación in Situ , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Fluorescente , Mitocondrias Cardíacas/enzimología , Inhibidores de la Monoaminooxidasa/farmacología , Equilibrio Postural/fisiología , Proteína Glutamina Gamma Glutamiltransferasa 2 , Proteolípidos/biosíntesis , Proteolípidos/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Transglutaminasas/genética , Transglutaminasas/fisiología , Proteínas de Unión al GTP rho/metabolismo , Proteína de Unión al GTP rhoARESUMEN
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.
RESUMEN
It is currently accepted that the human brain has a limited neurogenic capacity and an impaired regenerative potential. We have previously shown the existence of CD271-expressing neural stem cells (NSCs) in the subventricular zone (SVZ) of Parkinson's disease (PD) patients, which proliferate and differentiate towards neurons and glial cells in vitro. To study the molecular profile of these NSCs in detail, we performed RNA sequencing and mass spectrometry on CD271+ NSCs isolated from human post-mortem SVZ and on homogenates of the SVZ. CD271+ cells were isolated through magnetic cell separation (MACS). We first compared the molecular profile of CD271+ NSCs to the SVZ homogenate from control donors and then compared CD271+ cells to CD11b+ microglia. These results confirmed their neural stem cell identity. Finally we compared controls and PD patients to establish a specific molecular profile of NSCs and the SVZ in PD. While our transcriptome analysis did not identify any differentially expressed genes in the SVZ between control and PD patients, our proteome analysis revealed several proteins that were differentially expressed in PD. Some of these proteins are involved in cytoskeletal organization and mitochondrial function. Transcriptome and proteome analyses of NSCs from PD revealed changes in the expression of genes and proteins involved in metabolism, transcriptional activity and cytoskeletal organization. Our data suggest that NSCs may transit into a primed-quiescent state, that is in an "alert" non-proliferative phase in PD. Our results not only confirm pathological hallmarks of PD (e.g. impaired mitochondrial function), but also show that the NSCs from SVZ undergo significant changes at both transcriptome and proteome level following PD.
Asunto(s)
Ventrículos Laterales/metabolismo , Células-Madre Neurales/metabolismo , Enfermedad de Parkinson/metabolismo , Proteoma , Transcriptoma , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Proteínas del Tejido Nervioso/metabolismo , Receptores de Factor de Crecimiento Nervioso/metabolismoRESUMEN
Microglia are key players in the central nervous system in health and disease. Much pioneering research on microglia function has been carried out in vivo with the use of genetic animal models. However, to fully understand the role of microglia in neurological and psychiatric disorders, it is crucial to study primary human microglia from brain donors. We have developed a rapid procedure for the isolation of pure human microglia from autopsy tissue using density gradient centrifugation followed by CD11b-specific cell selection. The protocol can be completed in 4 h, with an average yield of 450,000 and 145,000 viable cells per gram of white and grey matter tissue respectively. This method allows for the immediate phenotyping of microglia in relation to brain donor clinical variables, and shows the microglia population to be distinguishable from autologous choroid plexus macrophages. This protocol has been applied to samples from over 100 brain donors from the Netherlands Brain Bank, providing a robust dataset to analyze the effects of age, post-mortem delay, brain acidity, and neurological diagnosis on microglia yield and phenotype. Our data show that cerebrospinal fluid pH is positively correlated to microglial cell yield, but donor age and post-mortem delay do not negatively affect viable microglia yield. Analysis of CD45 and CD11b expression showed that changes in microglia phenotype can be attributed to a neurological diagnosis, and are not influenced by variation in ante- and post-mortem parameters. Cryogenic storage of primary microglia was shown to be possible, albeit with variable levels of recovery and effects on phenotype and RNA quality. Microglial gene expression substantially changed due to culture, including the loss of the microglia-specific markers, showing the importance of immediate microglia phenotyping. We conclude that primary microglia can be isolated effectively and rapidly from human post-mortem brain tissue, allowing for the study of the microglial population in light of the neuropathological status of the donor.
Asunto(s)
Encéfalo , Separación Celular , Microglía , Factores de Edad , Anciano , Anciano de 80 o más Años , Encéfalo/metabolismo , Encéfalo/patología , Antígeno CD11b/metabolismo , Separación Celular/métodos , Células Cultivadas , Líquido Cefalorraquídeo/química , Criopreservación , Femenino , Citometría de Flujo , Expresión Génica , Perfilación de la Expresión Génica , Humanos , Concentración de Iones de Hidrógeno , Antígenos Comunes de Leucocito/metabolismo , Masculino , Microglía/metabolismo , Microglía/patología , Factores de Tiempo , Bancos de Tejidos , Donantes de TejidosRESUMEN
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.
RESUMEN
Multiple sclerosis (MS) is a chronic inflammatory disorder of the central nervous system, leading to severe neurological deficits. Current MS treatment regimens, consist of immunomodulatory agents aiming to reduce the rate of relapses. However, these agents are usually insufficient to treat chronic neurological disability. A promising perspective for future therapy of MS is the regeneration of lesions with replacement of the damaged oligodendrocytes or neurons. Therapies targeting to the enhancement of endogenous remyelination, aim to promote the activation of either the parenchymal oligodendrocyte progenitor cells or the subventricular zone-derived neural stem cells (NSCs). Less studied but highly potent, is the strategy of neuronal regeneration with endogenous NSCs that although being linked to numerous limitations, is anticipated to ameliorate cognitive disability in MS. Focusing on the forebrain, this review highlights the role of NSCs in the regeneration of MS lesions.
RESUMEN
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.
Asunto(s)
Antígenos de Diferenciación/biosíntesis , Encéfalo , Separación Celular , Expresión Génica , Proteínas del Tejido Nervioso/biosíntesis , Células-Madre Neurales , Receptores de Factor de Crecimiento Nervioso/biosíntesis , Adulto , Astrocitos/citología , Astrocitos/metabolismo , Encéfalo/citología , Encéfalo/metabolismo , Diferenciación Celular , Femenino , Proteína Ácida Fibrilar de la Glía/biosíntesis , Humanos , Masculino , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Neurogénesis/fisiología , Neuronas/citología , Neuronas/metabolismo , Oligodendroglía/citología , Oligodendroglía/metabolismoRESUMEN
The basis of gender differences in the prevalence and clinical progression of multiple sclerosis (MS) is not understood. Here, we identify gender-specific responses in steroid synthesis and signaling in the brains of MS patients as possible contributors to these differences. We investigated gene expression changes in these pathways and of inflammatory cytokines in MS lesions and normal-appearing white matter (NAWM) of male and female patients (n=21) and control NAWM (n=14) using quantitative polymerase chain reaction (25 MS lesions, 21 MS NAWM, and 14 control NAWM) and immunohistochemistry (3-4 sections per group). In MS lesions in males, there was local upregulation of aromatase (an enzyme involved in estrogen biosynthesis), estrogen receptor-ß (ERß), and tumor necrosis factor (TNF) mRNA; whereas in females, there was local upregulation of 3ß-hydroxysteroid-dehydrogenase, a progesterone synthetic enzyme, and of progesterone receptor. Astrocytes in the rim and center of MS lesions were found to be the primary source of steroidogenic enzyme and receptor expression. Aromatase and ERα mRNA levels were positively correlated with that of TNF in primary cultures of human microglia and astrocytes; TNF caused increased ERα, suggesting that inflammatory signals stimulate estrogen signaling in this cell type. Together, these findings suggest that there are gender differences in the CNS of MS patients that may affect lesion pathogenesis, that is, in males, estrogen synthesis and signaling are induced; whereas in females, progestogen synthesis and signaling are induced. These differences may represent contributing factors to gender differences in the prevalence and course of MS.
Asunto(s)
Encéfalo/metabolismo , Estrógenos/metabolismo , Esclerosis Múltiple/patología , Progesterona/metabolismo , Caracteres Sexuales , Transducción de Señal/fisiología , Adulto , Anciano , Anciano de 80 o más Años , Aromatasa/metabolismo , Encéfalo/efectos de los fármacos , Citocinas/genética , Citocinas/metabolismo , Estrógenos/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Fibras Nerviosas Mielínicas/metabolismo , Fibras Nerviosas Mielínicas/patología , Cambios Post Mortem , Progesterona/genética , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Kinesin family member 21b (kif21b) is one of the few multiple sclerosis (MS) risk genes with a presumed central nervous system function. Kif21b belongs to the kinesin family, proteins involved in intracellular transport of proteins and organelles. We hypothesised that kif21b is involved in the neurodegenerative component of MS and Alzheimer's (AD) disease. Post-mortem kinesin expression was assessed in 50 MS, 58 age and gender matched non-demented controls (NDC) and 50 AD. Kif21b expression was five-fold increased in AD compared to MS and NDC aged below 62 years (p = 8*10(-5)), three-fold between 62-72 years (p = 0.005) and not different above 72 years. No significant differences were observed between MS and NDC. In AD, kif21b expression was two-fold increased in Braak stage 6 (scoring for density of neurofibrillary tangles) compared with stage 5 (p = 0.003). In MS patients, kif21b correlated with the extent of grey matter demyelination (Spearman's rho = 0.31, p = 0.03). Abundant kif21b, defined as expression above the median, was associated with a two-fold accelerated development of the Kurtzke Expanded Disability Status Scale (EDSS) 6.0 (median time in low kif21b group 16 years vs. high kif21b 7.5 years, log-rank test p = 0.04) in MS. Given the genetic association of kif21b with MS, the results were stratified according to rs12122721[A] single nucleotide polymorphism (SNP). No association was found between kif21b expression or the time to EDSS 6 in kif21b risk SNP carriers compared to non-risk carriers. Kif21b was expressed in astrocytes in addition to neurons. Upon astrocyte activation, kif21b increased nine-fold. Abundant kif21b expression is associated with severe MS and AD pathology and with accelerated neurodegeneration independent of the kif21b risk SNP.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Encéfalo/metabolismo , Progresión de la Enfermedad , Cinesinas/metabolismo , Esclerosis Múltiple/metabolismo , Enfermedad de Alzheimer/patología , Astrocitos/metabolismo , Encéfalo/patología , Femenino , Humanos , Cinesinas/genética , Masculino , Esclerosis Múltiple/patología , Neuronas/metabolismo , Polimorfismo de Nucleótido Simple , Factores de Riesgo , Índice de Severidad de la Enfermedad , Médula Espinal/metabolismoRESUMEN
One important pathological process in the brain of Parkinson disease (PD) patients is the degeneration of the dopaminergic neurons in the substantia nigra, which leads to a decline in striatal dopamine levels and motor dysfunction. A major clinical problem is that this degenerative process currently cannot be stopped or reversed. Expectations from the restorative capacity of neural stem cells (NSCs) are high, as these cells can potentially replace the degenerating neurons. The discovery of the presence of NSCs in the adult human brain has instigated research into the potential of these cells as a resource to promote brain repair in neurodegenerative diseases. Neural stem and progenitor cells reside in the subventricular zone (SVZ), which is closely situated to the striatum, which is affected in PD. Therefore, restoring the dopamine levels in the striatum of PD patients through stimulating endogenous NSCs in the nearby SVZ to migrate into the striatum and differentiate into dopaminergic neurons might thus be an attractive future therapeutic approach. We will review the reported changes in NSCs in the SVZ of PD animal models and PD patients, which are due to a lack of striatal dopamine. Furthermore, we will summarise the reports that describe efforts to stimulate NSCs to replace dopaminergic cells in the SN and restore striatal dopamine levels. In our opinion, mobilizing the endogenous SVZ NSCs to replenish striatal dopamine is an attractive approach to alleviate the motor symptoms in PD patients, without the ethical and immunological challenges of transplantation of NSCs and foetal brain tissue.
Asunto(s)
Células Madre Adultas/patología , Encéfalo/patología , Células-Madre Neurales/patología , Enfermedad de Parkinson/patología , Animales , Encéfalo/metabolismo , Dopamina/metabolismo , Humanos , NeurogénesisRESUMEN
Vitamin D deficiency has been implicated as a risk factor for multiple sclerosis (MS), but how vitamin D metabolism affects MS pathophysiology is not understood. We studied the expression of vitamin D receptor (VDR) and related enzymes, including 1,25(OH)(2)D-24-hydroxylase (24-OHase; CYP24A1) and 25(OH)D-1α-hydroxylase (CYP27B1), in CNS tissues of 39 MS patients and 20 controls and in primary human glial cells in vitro. In control and MS normal-appearing white matter (NAWM), nuclear VDR immunostaining was observed in oligodendrocyte-like cells, human leukocyte antigen (HLA)-positive microglia, and glial fibrillary acidic protein-positive astrocytes. There was a 2-fold increase in VDR transcripts in MS NAWM versus control white matter (p = 0.03). In chronic active MS lesions, HLA-positive microglia/macrophages showed nuclear VDR staining; astrocytes showed nuclear and cytoplasmic VDR staining. Staining for 24-OHase was restricted to astrocytes.VDR and CYP27B1 mRNA expressions were increased in active MS lesions versus NAWM (p < 0.01, p = 0.04, respectively). In primary human astrocytes in vitro, the active form of vitamin D, 1,25(OH)(2)D(3), induced upregulation of VDR and CYP24A1. Tumor necrosis factor and interferon-γ upregulated CYP27B1 mRNA in primary human microglia and astrocytes. Increased VDR expression in MS NAWM and inflammatory cytokine-induced amplified expression of VDR and CYP27B1 in chronic active MS lesions suggest increased sensitivity to vitamin D in NAWM and a possible endogenous role for vitamin D metabolism in the suppression of active MS lesions.