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1.
Mol Neurobiol ; 58(4): 1755-1768, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33387302

RESUMO

Ethanol consumption during pregnancy or lactation period can induce permanent damage to the development of the central nervous system (CNS), resulting in fetal alcohol spectrum disorders (FASD). CNS development depends on proper neural cells and blood vessel (BV) development and blood-brain barrier (BBB) establishment; however, little is known about how ethanol affects these events. Here, we investigated the impact of ethanol exposure to endothelial cells (ECs) function and to ECs interaction with astrocytes in the context of BBB establishment. Cerebral cortex of newborn mice exposed in utero to ethanol (FASD model) presented a hypervascularized phenotype, revealed by augmented vessel density, length, and branch points. Further, aberrant distribution of the tight junction ZO-1 protein along BVs and increased rates of perivascular astrocytic endfeet around BVs were observed. In vitro exposure of human brain microcapillary ECs (HBMEC) to ethanol significantly disrupted ZO-1 distribution, decreased Claudin-5 and GLUT-1 expression and impaired glucose uptake, and increased nitric oxide secretion. These events were accompanied by upregulation of angiogenesis-related secreted proteins by ECs in response to ethanol exposure. Treatment of cortical astrocytes with conditioned medium (CM) from ethanol exposed ECs, upregulated astrocyte's expression of GFAP, Cx43, and Lipocalin-2 genes, as well as the pro-inflammatory genes, IL-1beta, IL-6, and TNF-alpha, which was accompanied by NF-kappa B protein nuclear accumulation. Our findings suggest that ethanol triggers a dysfunctional phenotype in brain ECs, leading to impairment of cortical vascular network formation, and promotes ECs-induced astrocyte dysfunction, which could dramatically affect BBB establishment in the developing brain.

2.
Glia ; 69(6): 1429-1443, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33497496

RESUMO

Central nervous system (CNS) function depends on precise synaptogenesis, which is shaped by environmental cues and cellular interactions. Astrocytes are outstanding regulators of synapse development and plasticity through contact-dependent signals and through the release of pro- and antisynaptogenic factors. Conversely, myelin and its associated proteins, including Nogo-A, affect synapses in a inhibitory fashion and contribute to neural circuitry stabilization. However, the roles of Nogo-A-astrocyte interactions and their implications in synapse development and plasticity have not been characterized. Therefore, we aimed to investigate whether Nogo-A affects the capacity of astrocytes to induce synaptogenesis. Additionally, we assessed whether downregulation of Nogo-A signaling in an in vivo demyelination model impacts the synaptogenic potential of astrocytes. Our in vitro data show that cortical astrocytes respond to Nogo-A through RhoA pathway activation, exhibiting stress fiber formation and decreased ramified morphology. This phenotype was associated with reduced levels of GLAST protein and aspartate uptake, decreased mRNA levels of the synaptogenesis-associated genes Hevin, glypican-4, TGF-ß1 and BDNF, and decreased and increased protein levels of Hevin and SPARC, respectively. Corroborating these findings, conditioned medium from Nogo-A-treated astrocytes suppressed the formation of structurally and functionally mature synapses in cortical neuronal cultures. After cuprizone-induced acute demyelination, we observed reduced immunostaining for Nogo-A in the visual cortex accompanied by higher levels of Hevin expression in astrocytes and an increase in excitatory synapse density. Hence, we suggest that interactions between Nogo-A and astrocytes might represent an important pathway of plasticity regulation and could be a target for therapeutic intervention in demyelinating diseases in the future.

3.
J Neurosci Methods ; 343: 108806, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32574642

RESUMO

BACKGROUND: Astrocytes, one of the main glial cell types, play critical roles in the central nervous system (CNS) development and function, including support of neuronal survival and differentiation, blood brain barrier formation, synapse homeostasis and injury response. Cell isolation and culture techniques have been proved to be a powerful tool to study astrocyte physiology and function. Due to financial constraints and rigid biosafety and ethics rules to use animal models, freezing techniques and the creation of cell banks emerged as alternatives to optimize the use of experimental animals. One of the main challenges, however, of these techniques is to guarantee that conserved cells keep their biological properties. NEW METHOD: In this work, we characterized morphologically and functionally murine secondary astrocyte cultures that have been submitted to freezing/thawing procedures. RESULTS: Morphological characterization of SAC (secondary astrocyte culture) and SFAC (secondary frozen-astrocyte culture) did not reveal significant differences on astrocyte morphology, confluence time and cell number along culture period. Functionally, SAC and SFAC did not reveal differences in their potential to support neuronal survival, maturation, neuritogenesis and synapse formation. CONCLUSIONS: Our results suggest that murine astrocytes that are submitted to freezing/thawing procedure maintain morphological and functional characteristics when compared with non-frozen astrocytes. Thus, this methodological approach is a valuable tool for in vitro research and might allow experimental optimization and reduction of animal use.

4.
Neurochem Int ; 138: 104758, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32439533

RESUMO

α-Synuclein protein (α-syn) is a central player in Parkinson's disease (PD) and in a spectrum of neurodegenerative diseases collectively known as synucleinopathies. These diseases are characterized by abnormal motor symptoms, such as tremor at rest, slowness of movement, rigidity of posture, and bradykinesia. Histopathological features of PD include preferential loss of dopaminergic neurons in the substantia nigra and formation of fibrillar intraneuronal inclusions called Lewy bodies and Lewy neurites, which are composed primarily of the α-syn protein. Currently, it is well accepted that α-syn oligomers (αSO) are the main toxic agent responsible for the etiology of PD. Glutamatergic excitotoxicity is associated with several neurological disorders, including PD. Excess glutamate in the synaptic cleft can be taken up by the astrocytic glutamate transporters GLAST and GLT-1. Although this event is the main defense against glutamatergic excitotoxicity, the molecular mechanisms that regulate this process have not yet been investigated in an early sporadic model of synucleinopathy. Here, using an early sporadic model of synucleinopathy, we demonstrated that the treatment of astrocytes with αSO increased glutamate uptake. This was associated with higher levels of GLAST and GLT-1 in astrocyte cultures and in a mouse model of synucleinopathy 24 h and 45 days after inoculation with αSO, respectively. Pharmacological inhibition of the TGF-ß1 (transforming growth factor beta 1) pathway in vivo reverted GLAST/GLT-1 enhancement induced by αSO injection. Therefore, our study describes a new neuroprotective role of astrocytes in an early sporadic model of synucleinopathy and sheds light on the mechanisms of glutamate transporter regulation for neuroprotection against glutamatergic excitotoxicity in synucleinopathy.

5.
Cerebellum ; 18(6): 1017-1035, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31218566

RESUMO

Astrocytes, initially described as merely support cells, are now known as a heterogeneous population of cells actively involved in a variety of biological functions such as: neuronal migration and differentiation; regulation of cerebral blood flow; metabolic control of extracellular potassium concentration; and modulation of synapse formation and elimination; among others. Cerebellar glial cells have been shown to play a significant role in proliferation, differentiation, migration, and synaptogenesis. However, less evidence is available about the role of neuron-astrocyte interactions during cerebellar development and their impact on diseases of the cerebellum. In this review, we will focus on the mechanisms underlying cellular interactions, specifically neuron-astrocyte interactions, during cerebellar development, function, and disease. We will discuss how cerebellar glia, astrocytes, and Bergmann glia play a fundamental role in several steps of cerebellar development, such as granule cell migration, axonal growth, neuronal differentiation, and synapse formation, and in diseases associated with the cerebellum. We will focus on how astrocytes and thyroid hormones impact cerebellar development. Furthermore, we will provide evidence of how growth factors secreted by glial cells, such as epidermal growth factor and transforming growth factors, control cerebellar organogenesis. Finally, we will argue that glia are a key mediator of cerebellar development and that identification of molecules and pathways involved in neuron-glia interactions may contribute to a better understanding of cerebellar development and associated disorders.


Assuntos
Astrócitos/fisiologia , Diferenciação Celular/fisiologia , Cerebelo/embriologia , Cerebelo/crescimento & desenvolvimento , Neurogênese/fisiologia , Animais , Cerebelo/citologia , Humanos
6.
Front Aging Neurosci ; 11: 59, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30941031

RESUMO

Astrocytes, one of the largest glial cell population in the central nervous system (CNS), play a key function in several events of brain development and function, such as synapse formation and function, control of neurotransmitters release and uptake, production of trophic factors and control of neuronal survival. Initially described as a homogenous population, several evidences have pointed that astrocytes are highly heterogeneous, both morphologically and functionally, within the same region, and across different brain regions. Recent findings suggest that the heterogeneity in the expression profile of proteins involved in astrocyte function may predict the selective vulnerability of brain regions to specific diseases, as well as to the age-related cognitive decline. However, the molecular mechanisms underlying these changes, either in aging as well as in brain disease are scarce. Neuroinflammation, a hallmark of several neurodegenerative diseases and aging, is reported to have a dubious impact on glial activation, as these cells release pro- and anti-inflammatory cytokines and chemokines, anti-oxidants, free radicals, and neurotrophic factors. Despite the emerging evidences supporting that reactive astrocytes have a duality in their phenotype, neurotoxic or neuroprotective properties, depending on the age and stimuli, the underlying mechanisms of their activation, cellular interplays and the impact of regional astrocyte heterogeneity are still a matter of discussion. In this review article, we will summarize recent findings on astrocyte heterogeneity and phenotypes, as well as their likely impact for the brain function during aging and neural diseases. We will focus on the molecules and mechanisms triggered by astrocyte to control synapse formation in different brain regions. Finally, we will discuss new evidences on how the modulation of astrocyte phenotype and function could impact the synaptic deficits and glial dysfunction present in aging and pathological states.

7.
J Neurochem ; 150(2): 138-157, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31009074

RESUMO

Parkinson's disease (PD) is characterized by selective death of dopaminergic neurons in the substantia nigra, degeneration of the nigrostriatal pathway, increases in glutamatergic synapses in the striatum and aggregation of α-synuclein. Evidence suggests that oligomeric species of α-synuclein (αSO) are the genuine neurotoxins of PD. Although several studies have supported the direct neurotoxic effects of αSO on neurons, their effects on astrocytes have not been directly addressed. Astrocytes are essential to several steps of synapse formation and function, including secretion of synaptogenic factors, control of synaptic elimination and stabilization, secretion of neural/glial modulators, and modulation of extracellular ions, and neurotransmitter levels in the synaptic cleft. Here, we show that αSO induced the astrocyte reactivity and enhanced the synaptogenic capacity of human and murine astrocytes by increasing the levels of the known synaptogenic molecule transforming growth factor beta 1 (TGF-ß1). Moreover, intracerebroventricular injection of αSO in mice increased the number of astrocytes, the density of excitatory synapses, and the levels of TGF-ß1 in the striatum of injected animals. Inhibition of TGF-ß1 signaling impaired the effect of the astrocyte-conditioned medium on glutamatergic synapse formation in vitro and on striatal synapse formation in vivo, whereas addition of TGF-ß1 protected mesencephalic neurons against synapse loss triggered by αSO. Together, our data suggest that αSO have important effects on astrocytic functions and describe TGF-ß1 as a new endogenous astrocyte-derived molecule involved in the increase in striatal glutamatergic synaptic density present in early stages of PD. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. Cover Image for this issue: doi: 10.1111/jnc.14514.


Assuntos
Astrócitos/metabolismo , Transtornos Parkinsonianos/metabolismo , Sinapses/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , alfa-Sinucleína/metabolismo , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Neurogênese/fisiologia , Transdução de Sinais/fisiologia
8.
Mol Neurobiol ; 56(7): 4653-4679, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30377983

RESUMO

Transforming growth factor betas (TGF-ßs) are known as multifunctional growth factors that participate in the regulation of key events of development, disease, and tissue repair. In the brain, TGF-ß1 has been widely recognized as an injury-related cytokine, particularly associated with astrocyte scar formation in response to brain injury. In the last decade, however, evidence has indicated that in addition to its role in brain injury, TGF-ß1 might be a crucial regulator of cell survival and differentiation, brain homeostasis, angiogenesis, memory formation, and neuronal plasticity. In this review, we will discuss the emerging scenario of TGF-ß1 as a key regulator of astrocyte differentiation and function and the implications of TGF-ß1 as a novel mediator of cellular interactions in the central nervous system. First, we will discuss the cellular and molecular basis underlying the effect of TGF-ß on astrocyte generation and its impact on angiogenesis and blood-brain barrier function. Then, we will focus on the role of astrocytes in the development and remodeling of synapses and the role of TGF-ß1 as a new mediator of these events. Furthermore, we present seminal data that contributed to the emerging concept that astrocyte dysfunction might be associated with neurodegenerative diseases, with a special focus on Alzheimer's disease, and discuss the pros and cons of TGF-ß signaling deficits in these processes. Finally, we argue that understanding how astrocytic signals, such as TGF-ß1, regulate brain function might offer new insights into human learning, memory, and cognition, and ultimately, this understanding may provide new targets for the treatment of neurological diseases.


Assuntos
Astrócitos/metabolismo , Encefalopatias/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Fator de Crescimento Transformador beta1/metabolismo , Envelhecimento/metabolismo , Animais , Humanos , Neovascularização Fisiológica
9.
Mol Neurobiol ; 55(1): 751-762, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28050794

RESUMO

Astrocytes, the most abundant glial cells in the central nervous system (CNS), comprise a heterogeneous population of cells. However, how this heterogeneity impacts their function within brain homeostasis and response to injury and disease is still largely unknown. Recently, astrocytes have been recognized as important regulators of synapse formation and maturation. Here, we analyzed the synaptogenic property of astrocytes from different regions of the CNS. The effect of conditioned medium derived from astrocytes (astrocyte-conditioned medium (ACM)) from cerebral cortex, hippocampus, midbrain and cerebellum, in synapse formation, was evaluated. Synapse formation was analyzed by quantification of pre- and postsynaptic proteins, synaptophysin, and postsynaptic density protein 95 (PSD-95). ACM from the four regions increased significantly the number of synaptophysin/PSD-95 puncta on neurons from the same and different brain regions. Differences on astrocytic synaptogenic potential between the regions were observed according to ACM protein concentration. Thus, cerebellar astrocytes have higher synaptogenic effect when ACM is less concentrated. Also, heterotypical co-culture assays revealed that neurons from cerebral cortex and midbrain equally respond to ACM, indicating that differences in synapse effect are unlike to be neuron-autonomous. The expression profile of the synaptogenic molecules secreted by astrocytes from distinct brain regions was analyzed by qPCR. Gene expression of glypicans 4 and 6, hevin, and secreted protein-acidic and rich in cysteine (SPARC) greatly varies between astrocytes from different brain regions. Furthermore, in vivo analysis of hevin protein confirmed that variance. These findings highlight the heterogeneity of astrocytes and suggest that their synaptogenic potential may be different in each brain region, mainly due to distinct gene expression profiles.


Assuntos
Astrócitos/metabolismo , Encéfalo/metabolismo , Sinapses/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Proteínas de Ligação ao Cálcio/metabolismo , Córtex Cerebral/metabolismo , Meios de Cultivo Condicionados/farmacologia , Proteínas da Matriz Extracelular/metabolismo , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sinapses/efeitos dos fármacos
10.
Mol Neurobiol ; 55(5): 3660-3675, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-28523566

RESUMO

Neuroangiogenesis in the developing central nervous system is controlled by interactions between endothelial cells (ECs) and radial glia (RG) neural stem cells, although RG-derived molecules implicated in these events are not fully known. Here, we investigated the role of RG-secreted TGF-ß1, in angiogenesis in the developing cerebral cortex. By isolation of murine microcapillary brain endothelial cells (MBECs), we demonstrate that conditioned medium from RG cultures (RG-CM) promoted MBEC migration and formation of vessel-like structures in vitro, in a TGF-ß1-dependent manner. These events were followed by endothelial regulation of GPR124 and BAI-1 gene expression by RG-CM. Proteome profile of RG-CM identified angiogenesis-related molecules IGFBP2/3, osteopontin, endostatin, SDF1, fractalkine, TIMP1/4, Ang-1, pentraxin3, and Cyr61, some of them modulated by TGF-ß1 induction. In vivo gain and loss of function assays targeting RG cells demonstrates a specific TGF-ß1-dependent control of blood vessels branching in the cerebral cortex. Together, our results point to TGF-ß1 signaling pathway as a potential mediator of the RG-EC interactions and shed light to the key role of RG in paving the brain vascular network.


Assuntos
Movimento Celular/fisiologia , Córtex Cerebral/metabolismo , Células Ependimogliais/metabolismo , Neovascularização Fisiológica/fisiologia , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta1/metabolismo , Animais , Linhagem Celular , Córtex Cerebral/citologia , Córtex Cerebral/embriologia , Células Endoteliais/metabolismo , Células Ependimogliais/citologia , Humanos , Camundongos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurogênese/fisiologia
11.
Infect Immun ; 85(10)2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28784928

RESUMO

Clostridium difficile, the main cause of diarrhea in hospitalized patients, produces toxins A (TcdA) and B (TcdB), which affect intestinal epithelial cell survival, proliferation, and migration and induce an intense inflammatory response. Transforming growth factor ß (TGF-ß) is a pleiotropic cytokine affecting enterocyte and immune/inflammatory responses. However, it has been shown that exposure of intestinal epithelium to a low concentration of TcdA induces the release of TGF-ß1, which has a protective effect on epithelial resistance and a TcdA/TGF-ß signaling pathway interaction. The activation of this pathway in vivo has not been elucidated. The aim of this study was to investigate the role of the TGF-ß1 pathway in TcdA-induced damage in a rat intestinal epithelial cell line (IEC-6) and in a mouse model of an ileal loop. TcdA increased the expression of TGF-ß1 and its receptor, TßRII, in vitro and in vivo TcdA induced nuclear translocation of the transcription factors SMAD2/3, a hallmark of TGF-ß1 pathway activation, both in IEC cells and in mouse ileal tissue. The addition of recombinant TGF-ß1 (rTGF-ß) prevented TcdA-induced apoptosis/necrosis and restored proliferation and repair activity in IEC-6 cells in the presence of TcdA. Together, these data show that TcdA induces TGF-ß1 signaling pathway activation and suggest that this pathway might play a protective role against the effect of C. difficile-toxin.


Assuntos
Toxinas Bacterianas/toxicidade , Enterotoxinas/toxicidade , Mucosa Intestinal/microbiologia , Transdução de Sinais , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular , Enterotoxinas/metabolismo , Íleo/imunologia , Íleo/microbiologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Intestinos/imunologia , Intestinos/microbiologia , Camundongos , Fator de Crescimento Transformador beta1/genética
12.
J Neurosci ; 37(28): 6797-6809, 2017 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-28607171

RESUMO

Alzheimer's disease (AD) is characterized by progressive cognitive decline, increasingly attributed to neuronal dysfunction induced by amyloid-ß oligomers (AßOs). Although the impact of AßOs on neurons has been extensively studied, only recently have the possible effects of AßOs on astrocytes begun to be investigated. Given the key roles of astrocytes in synapse formation, plasticity, and function, we sought to investigate the impact of AßOs on astrocytes, and to determine whether this impact is related to the deleterious actions of AßOs on synapses. We found that AßOs interact with astrocytes, cause astrocyte activation and trigger abnormal generation of reactive oxygen species, which is accompanied by impairment of astrocyte neuroprotective potential in vitro We further show that both murine and human astrocyte conditioned media (CM) increase synapse density, reduce AßOs binding, and prevent AßO-induced synapse loss in cultured hippocampal neurons. Both a neutralizing anti-transforming growth factor-ß1 (TGF-ß1) antibody and siRNA-mediated knockdown of TGF-ß1, previously identified as an important synaptogenic factor secreted by astrocytes, abrogated the protective action of astrocyte CM against AßO-induced synapse loss. Notably, TGF-ß1 prevented hippocampal dendritic spine loss and memory impairment in mice that received an intracerebroventricular infusion of AßOs. Results suggest that astrocyte-derived TGF-ß1 is part of an endogenous mechanism that protects synapses against AßOs. By demonstrating that AßOs decrease astrocyte ability to protect synapses, our results unravel a new mechanism underlying the synaptotoxic action of AßOs in AD.SIGNIFICANCE STATEMENT Alzheimer's disease is characterized by progressive cognitive decline, mainly attributed to synaptotoxicity of the amyloid-ß oligomers (AßOs). Here, we investigated the impact of AßOs in astrocytes, a less known subject. We show that astrocytes prevent synapse loss induced by AßOs, via production of transforming growth factor-ß1 (TGF-ß1). We found that AßOs trigger morphological and functional alterations in astrocytes, and impair their neuroprotective potential. Notably, TGF-ß1 reduced hippocampal dendritic spine loss and memory impairment in mice that received intracerebroventricular infusions of AßOs. Our results describe a new mechanism underlying the toxicity of AßOs and indicate novel therapeutic targets for Alzheimer's disease, mainly focused on TGF-ß1 and astrocytes.


Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Astrócitos/metabolismo , Sinapses/metabolismo , Sinapses/patologia , Fator de Crescimento Transformador beta1/metabolismo , Peptídeos beta-Amiloides , Animais , Células Cultivadas , Humanos , Masculino , Camundongos , Espécies Reativas de Oxigênio/metabolismo
13.
Front Aging Neurosci ; 9: 184, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28659786

RESUMO

Synapse formation and function are critical events for the brain function and cognition. Astrocytes are active participants in the control of synapses during development and adulthood, but the mechanisms underlying astrocyte synaptogenic potential only began to be better understood recently. Currently, new drugs and molecules, including the flavonoids, have been studied as therapeutic alternatives for modulation of cognitive processes in physiological and pathological conditions. However, the cellular targets and mechanisms of actions of flavonoids remain poorly elucidated. In the present study, we investigated the effects of hesperidin on memory and its cellular and molecular targets in vivo and in vitro, by using a short-term protocol of treatment. The novel object recognition test (NOR) was used to evaluate memory performance of mice intraperitoneally treated with hesperidin 30 min before the training and again before the test phase. The direct effects of hesperidin on synapses and astrocytes were also investigated using in vitro approaches. Here, we described hesperidin as a new drug able to improve memory in healthy adult mice by two main mechanisms: directly, by inducing synapse formation and function between hippocampal and cortical neurons; and indirectly, by enhancing the synaptogenic ability of cortical astrocytes mainly due to increased secretion of transforming growth factor beta-1 (TGF-ß1) by these cells. Our data reinforces the known neuroprotective effect of hesperidin and, by the first time, characterizes its synaptogenic action on the central nervous system (CNS), pointing astrocytes and TGF-ß1 signaling as new cellular and molecular targets of hesperidin. Our work provides not only new data regarding flavonoid's actions on the CNS but also shed light on possible new therapeutic alternative based on astrocyte biology.

14.
Sci Rep ; 7: 45091, 2017 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-28345587

RESUMO

Astrocytes play a critical role in the development and homeostasis of the central nervous system (CNS). Astrocyte dysfunction results in several neurological and degenerative diseases. However, a major challenge to our understanding of astrocyte physiology and pathology is the restriction of studies to animal models, human post-mortem brain tissues, or samples obtained from invasive surgical procedures. Here, we report a protocol to generate human functional astrocytes from cerebral organoids derived from human pluripotent stem cells. The cellular isolation of cerebral organoids yielded cells that were morphologically and functionally like astrocytes. Immunolabelling and proteomic assays revealed that human organoid-derived astrocytes express the main astrocytic molecular markers, including glutamate transporters, specific enzymes and cytoskeletal proteins. We found that organoid-derived astrocytes strongly supported neuronal survival and neurite outgrowth and responded to ATP through transient calcium wave elevations, which are hallmarks of astrocyte physiology. Additionally, these astrocytes presented similar functional pathways to those isolated from adult human cortex by surgical procedures. This is the first study to provide proteomic and functional analyses of astrocytes isolated from human cerebral organoids. The isolation of these astrocytes holds great potential for the investigation of developmental and evolutionary features of the human brain and provides a useful approach to drug screening and neurodegenerative disease modelling.


Assuntos
Astrócitos/citologia , Córtex Cerebral/citologia , Crescimento Neuronal , Organoides/citologia , Animais , Astrócitos/metabolismo , Sinalização do Cálcio , Células Cultivadas , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Ácido Glutâmico/metabolismo , Humanos , Camundongos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Proteoma/genética , Proteoma/metabolismo
15.
Neurochem Int ; 95: 85-91, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26845377

RESUMO

In the last decade, there have been major advances in the understanding of the role of glial cells as key elements in the formation, maintenance and refinement of synapses. Recently, the discovery of natural compounds capable of modulating nervous system function has revealed new perspectives on the restoration of the injured brain. Among these compounds, flavonoids stand out as molecules easily obtainable in the diet that have remarkable effects on cognitive performance and behavior. Nevertheless, little is known about the cellular and molecular mechanisms underlying the actions of flavonoids in the nervous system. The present review presents recent advances in the effects of natural compounds, particularly flavonoids, in the nervous system. We shed light on astrocytes as targets of flavonoids and discuss how this interaction might contribute to the effects of flavonoids on neuronal survival, differentiation and function. Finally, we discuss how the effects of flavonoids on astrocytes might contribute to the development of alternative therapeutic approaches to the treatment of neural diseases.


Assuntos
Astrócitos/efeitos dos fármacos , Produtos Biológicos/administração & dosagem , Sistema Nervoso Central/efeitos dos fármacos , Flavonoides/administração & dosagem , Animais , Astrócitos/metabolismo , Produtos Biológicos/metabolismo , Sistema Nervoso Central/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Flavonoides/metabolismo , Humanos
17.
Mol Cancer ; 14: 105, 2015 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-25976744

RESUMO

BACKGROUND: Na/K-ATPase (NKA) is inhibited by perillyl alcohol (POH), a monoterpene used in the treatment of tumors, including brain tumors. The NKA α1 subunit is known to be superexpressed in glioblastoma cells (GBM). This isoform is embedded in caveolar structures and is probably responsible for the signaling properties of NKA during apoptosis. In this work, we showed that POH acts in signaling cascades associated with NKA that control cell proliferation and/or cellular death. METHODS: NKA activity was measured by the amount of non-radioactive Rb(+) incorporation into cultured GBM cell lines (U87 and U251) and non-tumor cells (mouse astrocytes and VERO cells). Cell viability was measured by lactate dehydrogenase levels in the supernatants of POH-treated cells. Activated c-Jun N-terminal Kinase (JNK) and p38 were assessed by western blotting. Apoptosis was detected by flow cytometry and immunocytochemistry, and the release of interleukins was measured by ELISA. RESULTS: All four cell types tested showed a similar sensitivity for POH. Perillic acid (PA), the main metabolite of POH, did not show any effect on these cells. Though the cell viability decreased in a dose-dependent manner when cells were treated with POH, the maximum cytotoxic effect of PA obtained was 30% at 4 mM. 1.5 mM POH activated p38 in U87 cells and JNK in both U87 and U251 cells as well as mouse astrocytes. Dasatinib (an inhibitor of the Src kinase family) and methyl ß-cyclodextrin (which promotes cholesterol depletion in cell membranes) reduced the POH-induced activation of JNK1/2 in U87 cells, indicating that the NKA-Src complex participates in this mechanism. Inhibition of JNK1/2 by the JNK inhibitor V reduced the apoptosis of GBM cells that resulted from POH administration, indicating the involvement of JNK1/2 in programmed cell death. 1.5 mM POH increased the production of interleukin IL-8 in the U251 cell supernatant, which may indicate a possible strategy by which cells avoid the cytotoxic effects of POH. CONCLUSIONS: A signaling mechanism mediated by NKA may have an important role in the anti-tumor action of POH in GBM cells.


Assuntos
Antineoplásicos/farmacologia , Terapia de Alvo Molecular , Monoterpenos/farmacologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cicloexenos/farmacologia , Citocinas/metabolismo , Dasatinibe/farmacologia , Ativação Enzimática/efeitos dos fármacos , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Modelos Biológicos , beta-Ciclodextrinas/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
18.
Mol Neurobiol ; 52(1): 653-63, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25257696

RESUMO

Recent clinical studies have shown that sepsis survivors may develop long-term cognitive impairments. The cellular and molecular mechanisms involved in these events are not well understood. This study investigated synaptic deficits in sepsis and the involvement of glial cells in this process. Septic animals showed memory impairment and reduced numbers of hippocampal and cortical excitatory synapses, identified by synaptophysin/PSD-95 co-localization, 9 days after disease onset. The behavioral deficits and synaptophysin/PSD-95 co-localization were rescued to normal levels within 30 days post-sepsis. Septic mice presented activation of microglia and reactive astrogliosis, which are hallmarks of brain injury and could be involved in the associated synaptic deficits. We treated neuronal cultures with conditioned medium derived from cultured astrocytes (ACM) and microglia (MCM) that were either non-stimulated or stimulated with lipopolysaccharide (LPS) to investigate the molecular mechanisms underlying synaptic deficits in sepsis. ACM and MCM increased the number of synapses between cortical neurons in vitro, and these effects were antagonized by LPS stimulation. LPS-MCM reduced the number of synapses by 50%, but LPS-ACM increased the number of synapses by 500%. Analysis of the composition of these conditioned media revealed increased levels of IL-1ß in LPS-MCM. Furthermore, inhibition of IL-1ß signaling through the addition of a soluble IL-1ß receptor antagonist (IL-1 Ra) fully prevented the synaptic deficit induced by LPS-MCM. These results suggest that sepsis induces a transient synaptic deficit associated with memory impairments mediated by IL-1ß secreted by activated microglia.


Assuntos
Transtornos Cognitivos/etiologia , Interleucina-1beta/metabolismo , Microglia/patologia , Sepse/complicações , Sinapses/patologia , Animais , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/patologia , Transtornos Cognitivos/patologia , Gliose/etiologia , Gliose/patologia , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Lipopolissacarídeos/farmacologia , Transtornos da Memória/etiologia , Transtornos da Memória/patologia , Camundongos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/patologia , Sepse/patologia , Sinapses/efeitos dos fármacos , Sinapses/metabolismo
19.
Front Cell Neurosci ; 8: 296, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25309328

RESUMO

Lysophosphatidic acid (LPA) is one of the main membrane-derived lysophospholipids, inducing diverse cellular responses like cell proliferation, cell death inhibition, and cytoskeletal rearrangement, and thus is important in many biological processes. In the central nervous system (CNS), post-mitotic neurons release LPA extracellularly whereas astrocytes do not. Astrocytes play a key role in brain development and pathology, producing various cytokines, chemokines, growth factors, and extracellular matrix (ECM) components that act as molecular coordinators of neuron-glia communication. However, many molecular mechanisms underlying these events remain unclear-in particular, how the multifaceted interplay between the signaling pathways regulated by lysophospholipids is integrated in the complex nature of the CNS. Previously we showed that LPA-primed astrocytes induce neuronal commitment by activating LPA1-LPA2 receptors. Further, we revealed that these events were mediated by modulation and organization of laminin levels by astrocytes, through the induction of the epidermal growth factor receptor (EGFR) signaling pathway and the activation of the mitogen-activated protein (MAP) kinase (MAPK) cascade in response to LPA (Spohr et al., 2008, 2011). In the present work, we aimed to answer whether LPA affects astrocytic production and rearrangement of fibronectin, and to investigate the mechanisms involved in neuronal differentiation and maturation of cortical neurons induced by LPA-primed astrocytes. We show that PKA activation is required for LPA-primed astrocytes to induce neurite outgrowth and neuronal maturation and to rearrange and enhance the production of fibronectin and laminin. We propose a potential mechanism by which neurons and astrocytes communicate, as well as how such interactions drive cellular events such as neurite outgrowth, cell fate commitment, and maturation.

20.
Neurochem Int ; 78: 18-27, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25125369

RESUMO

Brain function depends critically on the coordinated activity of presynaptic and postsynaptic signals derived from both neurons and non-neuronal elements such as glial cells. A key role for astrocytes in neuronal differentiation and circuitry formation has emerged within the last decade. Although the function of glial cells in synapse formation, elimination and efficacy has greatly increased, we are still very far from deeply understanding the molecular and cellular mechanism underlying these events. The present review discusses the mechanisms driving astrocytic control of excitatory and inhibitory synapse formation in the central nervous system, especially the mechanisms mediated by soluble molecules, particularly those from the TGF-ß family. Further, we discuss whether and how human astrocytes might contribute to the acquisition of human cognition. We argue that understanding how astrocytic signals regulate synaptic development might offer new insights into human perception, learning, memory, and cognition and, ultimately, provide new targets for the treatment of neurological diseases.


Assuntos
Astrócitos/fisiologia , Encéfalo/fisiologia , Rede Nervosa/fisiologia , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Animais , Humanos , Sinapses/fisiologia
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