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1.
Int J Mol Sci ; 21(18)2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32942636

RESUMO

The intratumor heterogeneity represents one of the most difficult challenges for the development of effective therapies to treat pediatric glioblastoma (pGBM) and diffuse intrinsic pontine glioma (DIPG). These brain tumors are composed of heterogeneous cell subpopulations that coexist and cooperate to build a functional network responsible for their aggressive phenotype. Understanding the cellular and molecular mechanisms sustaining such network will be crucial for the identification of new therapeutic strategies. To study more in-depth these mechanisms, we sought to apply the Multifluorescent Marking Technology. We generated multifluorescent pGBM and DIPG bulk cell lines randomly expressing six different fluorescent proteins and from which we derived stable optical barcoded single cell-derived clones. In this study, we focused on the application of the Multifluorescent Marking Technology in 2D and 3D in vitro/ex vivo culture systems. We discuss how we integrated different multimodal fluorescence analysis platforms, identifying their strengths and limitations, to establish the tools that will enable further studies on the intratumor heterogeneity and interclonal interactions in pGBM and DIPG.


Assuntos
Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Glioma/patologia , Neoplasias Encefálicas/metabolismo , Linhagem Celular , Glioblastoma/metabolismo , Glioma/metabolismo , Células HEK293 , Humanos , Proteínas Luminescentes/metabolismo , Pediatria , Tecnologia/métodos
2.
Brain Behav Immun ; 81: 484-494, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31279682

RESUMO

An increasing number of studies show that both inflammation and neural plasticity act as key players in the vulnerability and recovery from psychiatric disorders and neurodegenerative diseases. However, the interplay between these two players has been limitedly explored. In fact, while a few studies reported an immune activation, others conveyed an immune suppression, associated with an impairment in neural plasticity. Therefore, we hypothesized that deviations in inflammatory levels in both directions may impair neural plasticity. We tested this hypothesis experimentally, by acute treatment of C57BL/6 adult male mice with different doses of two inflammatory modulators: lipopolysaccharide (LPS), an endotoxin, and ibuprofen (IBU), a nonselective cyclooxygenase inhibitor, which are respectively a pro- and an anti-inflammatory agent. The results showed that LPS and IBU have different effects on behavior and inflammatory response. LPS treatment induced a reduction of body temperature, a decrease of body weight and a reduced food and liquid intake. In addition, it led to increased levels of inflammatory markers expression, both in the total hippocampus and in isolated microglia cells, including Interleukin (IL)-1ß, and enhanced the concentration of prostaglandin E2 (PGE2). On the other hand, IBU increased the level of anti-inflammatory markers, decreased tryptophan 2,3-dioxygenase (TDO2), the first step in the kynurenine pathway known to be activated during inflammatory conditions, and PGE2 levels. Though LPS and IBU administration differently affected mediators related with pro- or anti-inflammatory responses, they produced overlapping effects on neural plasticity. Indeed, higher doses of both LPS and IBU induced a statistically significant decrease in the amplitude of long-term potentiation (LTP), in Brain-Derived Neurotrophic Factor (BDNF) expression levels and in the phosphorylation of the AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor subunit GluR1, compared to the control group. Such effect appears to be dose-dependent since only the higher, but not the lower, dose of both compounds led to a plasticity impairment. Overall, the present findings indicate that acute treatment with pro- and anti-inflammatory agents impair neural plasticity in a dose dependent manner.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/biossíntese , Inflamação/metabolismo , Potenciação de Longa Duração/fisiologia , Plasticidade Neuronal/fisiologia , Animais , Anti-Inflamatórios/farmacologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Inibidores de Ciclo-Oxigenase/farmacologia , Citocinas/imunologia , Citocinas/metabolismo , Dinoprostona/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Ibuprofeno/farmacologia , Inflamação/imunologia , Interleucina-1beta/metabolismo , Cinurenina/metabolismo , Lipopolissacarídeos/farmacologia , Potenciação de Longa Duração/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Microglia/metabolismo , Plasticidade Neuronal/imunologia , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo
3.
Brain Behav Immun ; 55: 225-235, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-26593276

RESUMO

Repeated stimulation of TLR4 signaling by lipopolysaccharide (LPS) in microglia induces a state of tolerance/sensitization consisting in the reprogramming of the expression of pro-inflammatory genes in favor of anti-inflammatory ones. The molecular mechanisms underlying this adaptive response are far to be elucidated. Glycogen synthase kinase 3 (GSK3) has emerged as crucial regulator of TLR signaling, mediating the balance between pro- and anti-inflammatory functions in both periphery and central nervous system. The present study extends this notion identifying GSK3 as part of the molecular machinery regulating the LPS-adaptive response in microglial cells, by using primary microglial cultures and organotypic hippocampal slices (OHSCs). We found that lithium chloride (LiCl), a widely used GSK3 inhibitor and the mainstay treatment for bipolar disorder, reinforced the LPS adaptive response by enhancing both downregulation of pro-inflammatory genes (inducible nitric oxide synthase, interleukin 1ß, interleukin 6, tumor necrosis factor α), and upregulation of genes typically associated to anti-inflammatory functions (interleukin 10 and MRC1). The effects of GSK3 inhibition were mimicked by Wnt3a, added exogenously, and reversed by Inhibitor of Wnt-Response-1-endo, a pharmacological disruptor of the canonical Wnt/ß-catenin pathway, and GW9662, a selective peroxisome proliferator activated receptor γ antagonist, suggesting that these two pathways are involved in the regulation of LPS-tolerance/sensitization by GSK. Finally, LiCl treatment of OHSCs enhanced the protective functional consequences of the microglial adaptive response to LPS on oligodendrocyte maturation, as indicated by MBP mRNA upregulation. These results further indicate GSK3 as key component in the orchestration of neuroinflammation and target for neuroprotective strategies.


Assuntos
Endotoxinas/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Hipocampo/metabolismo , Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Cloreto de Lítio/farmacologia , Microglia/metabolismo , PPAR gama/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteína Wnt3A/metabolismo , Animais , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Ratos , Ratos Wistar
4.
J Neurochem ; 135(1): 147-56, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26173855

RESUMO

Microglial activation is a dynamic process, central to neuroinflammation, which can have beneficial or pathogenic effects to human health. Mitochondria are key players in neuroinflammatory and neurodegenerative processes, common to most brain diseases. To the best of our knowledge on the role of mitochondria in the modulation of neuroinflammation, we focused on the mitochondrial uncoupling protein-2 (UCP2), known to control mitochondrial functions and to be implicated in a variety of physiological and pathological processes. In primary microglial cultures, the M1 stimulus lipopolysaccharide induced an early and transitory decrease in UCP2 levels. The initial UCP2 down-regulation was paralleled by mitochondrial inner membrane potential (mMP) depolarization and increased mitochondrial reactive oxygen species production. The key role of UCP2 in controlling mMP and reactive oxygen species production was confirmed by both pharmacological inhibition and down-regulation by RNA interference. Additionally, UCP2-silenced microglia stimulated with lipopolysaccharide showed an enhanced inflammatory response, characterized by a greater production of nitric oxide and interleukin-6. UCP2 was differently regulated by M2 stimuli, as indicated by its persistent up-regulation by interleukin-4. In UCP2-silenced microglia, interleukin-4 failed to induce M2 genes (mannose receptor 1 and interleukin-10) and to reduce M1 genes (inducible nitric oxide synthase and tumour necrosis factor-α). Our findings indicate that UCP2 is central to the process of microglial activation, with opposite regulation of M1 and M2 responses, and point to UCP2 manipulation as a potential strategy for redirecting microglial response towards protective phenotypes in several brain diseases where neuroinflammation is recognized to contribute to neurodegeneration. We show that the mitochondrial uncoupling protein-2 (UCP2) is central to the process of microglial activation, with opposite regulation of M1 and M2 responses. In UCP2-silenced microglia, lipopolysaccharide (LPS) triggers an enhanced inflammatory response characterized by a greater expression of M1 genes, whereas interleukin-4 (IL-4) fails in inducing M2 genes and reducing M1 genes. We propose UCP2 manipulation as a potential strategy for redirecting microglial response towards protective phenotypes.


Assuntos
Canais Iônicos/metabolismo , Microglia/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Animais , Interleucina-4/metabolismo , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Microglia/metabolismo , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína Desacopladora 2 , Regulação para Cima
5.
Biochim Biophys Acta ; 1832(5): 650-9, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23402925

RESUMO

The branched-chain amino acids (BCAAs) valine, leucine and isoleucine are essential amino acids involved in several important brain functions. Although commonly used as nutritional supplements, excessive intake of BCAAs might favour the establishment of neurotoxic conditions as indicated by the severe neurological symptoms characterising inherited disorders of BCAA catabolism such as maple syrup urine disease (MSUD). Recent evidence indicates that BCAAs induce excitotoxicity through mechanisms that require the presence of astrocytes. In the present study, we evaluated the effects of BCAAs on microglia, the main immune cells of the brain. As an experimental model we used primary microglial cells harvested from mixed glial cultures that had been kept in normal or high BCAA medium (H-BCAA). We show that H-BCAA microglial cells exhibit a peculiar phenotype characterized by a partial skewing toward the M2 state, with enhanced IL-10 expression and phagocytic activity but also increased free radical generation and decreased neuroprotective functions. We suggest that such an intermediate M1/M2 phenotype might result in a less efficient microglial response, which would promote the establishment of a low grade chronic inflammation and increase the likelihood of neurodegeneration. Although based on in vitro evidence, our study adds on to an increasing literature indicating that the increasing use of dietary integrators might deserve consideration for the possible drawbacks. In addition to excitotoxicity, the altered immune profile of microglia might represent a further mechanism by which BCAAs might turn into toxicants and facilitate neurodegeneration.


Assuntos
Aminoácidos de Cadeia Ramificada/farmacologia , Citocinas/metabolismo , Microglia/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Western Blotting , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Citocinas/genética , Radicais Livres/metabolismo , Expressão Gênica/efeitos dos fármacos , Imunossupressores/farmacologia , Interleucina-10/genética , Interleucina-10/metabolismo , Lipopolissacarídeos/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Microglia/citologia , Microglia/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sirolimo/farmacologia
6.
Glia ; 61(10): 1698-711, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23918452

RESUMO

Increasing evidence indicates that "functional plasticity" is not solely a neuronal attribute but a hallmark of microglial cells, the main brain resident macrophage population. Far from being a univocal phenomenon, microglial activation can originate a plethora of functional phenotypes, encompassing the classic M1 proinflammatory and the alternative M2 anti-inflammatory phenotypes. This concept overturns the popular view of microglial activation as a synonym of neurotoxicity and neurogenesis failure in brain disorders. The characterization of the alternative programs is a matter of intense investigation, but still scarce information is available on the course of microglial activation, on the reversibility of the different commitments and on the capability of preserving molecular memory of previous priming stimuli. By using organotypic hippocampal slice cultures as a model, we developed paradigms of stimulation aimed at shedding light on some of these aspects. We show that persistent stimulation of TLR4 signaling promotes an anti-inflammatory response and microglial polarization toward M2-like phenotype. Moreover, acute and chronic preconditioning regimens permanently affect the capability to respond to a later challenge, suggesting the onset of mechanisms of molecular memory. Similar phenomena could occur in the intact brain and differently affect the vulnerability of mature and newborn neurons to noxious signals.


Assuntos
Polaridade Celular/fisiologia , Hipocampo/citologia , Microglia/fisiologia , Animais , Animais Recém-Nascidos , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Arginase/genética , Arginase/metabolismo , Movimento Celular/efeitos dos fármacos , Polaridade Celular/efeitos dos fármacos , Ciclo-Oxigenase 1/genética , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Citocinas/genética , Citocinas/metabolismo , Dinoprostona/metabolismo , Ensaio de Imunoadsorção Enzimática , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Lipopolissacarídeos/farmacologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microglia/efeitos dos fármacos , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Técnicas de Cultura de Órgãos , Fagocitose/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores Imunológicos , Fatores de Tempo
7.
Transl Psychiatry ; 13(1): 399, 2023 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-38105264

RESUMO

Maternal obesity has been recognized as a stressor affecting the developing fetal brain, leading to long-term negative outcomes comparable to those resulting from maternal psychological stress, although the mechanisms have not been completely elucidated. In this study, we tested the hypothesis that adverse prenatal conditions as diverse as maternal stress and maternal obesity might affect emotional regulation and stress response in the offspring through common pathways, with a main focus on oxidative stress and neuroplasticity. We contrasted and compared adolescent male and female offspring in two mouse models of maternal psychophysical stress (restraint during pregnancy - PNS) and maternal obesity (high-fat diet before and during gestation - mHFD) by combining behavioral assays, evaluation of the hypothalamic-pituitary-adrenal (HPA) axis reactivity, immunohistochemistry and gene expression analysis of selected markers of neuronal function and neuroinflammation in the hippocampus, a key region involved in stress appraisal. Prenatal administration of the antioxidant N-acetyl-cysteine (NAC) was used as a strategy to protect fetal neurodevelopment from the negative effects of PNS and mHFD. Our findings show that these two stressors produce overlapping effects, reducing brain anti-oxidant defenses (Nrf-2) and leading to sex-dependent impairments of hippocampal Bdnf expression and alterations of the emotional behavior and HPA axis functionality. Prenatal NAC administration, by restoring the redox balance, was able to exert long-term protective effects on brain development, suggesting that the modulation of redox pathways might be an effective strategy to target common shared mechanisms between different adverse prenatal conditions.


Assuntos
Obesidade Materna , Efeitos Tardios da Exposição Pré-Natal , Animais , Feminino , Masculino , Camundongos , Gravidez , Hipocampo/metabolismo , Sistema Hipotálamo-Hipofisário/metabolismo , Obesidade Materna/metabolismo , Sistema Hipófise-Suprarrenal/metabolismo , Estresse Psicológico/metabolismo
8.
Nutrients ; 14(15)2022 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-35956326

RESUMO

Obesity is a main risk factor for the onset and the precipitation of many non-communicable diseases. This condition, which is associated with low-grade chronic systemic inflammation, is of main concern during pregnancy leading to very serious consequences for the new generations. In addition to the prominent role played by the adipose tissue, dysbiosis of the maternal gut may also sustain the obesity-related inflammatory milieu contributing to create an overall suboptimal intrauterine environment. Such a condition here generically defined as "inflamed womb" may hold long-term detrimental effects on fetal brain development, increasing the vulnerability to mental disorders. In this review, we will examine the hypothesis that maternal obesity-related gut dysbiosis and the associated inflammation might specifically target fetal brain microglia, the resident brain immune macrophages, altering neurodevelopmental trajectories in a sex-dependent fashion. We will also review some of the most promising nutritional strategies capable to prevent or counteract the effects of maternal obesity through the modulation of inflammation and oxidative stress or by targeting the maternal microbiota.


Assuntos
Microbiota , Transtornos do Neurodesenvolvimento , Obesidade Materna , Disbiose/complicações , Feminino , Humanos , Inflamação/complicações , Transtornos do Neurodesenvolvimento/etiologia , Obesidade/complicações , Obesidade Materna/complicações , Gravidez , Fatores de Risco
9.
Transl Psychiatry ; 12(1): 384, 2022 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-36104346

RESUMO

Autism Spectrum Disorder (ASD) is a sex-biased neurodevelopmental disorder with a male to female prevalence of 4:1, characterized by persistent deficits in social communication and interaction and restricted-repetitive patterns of behavior, interests or activities. Microbiota alterations as well as signs of neuroinflammation have been also reported in ASD. The involvement of immune dysregulation in ASD is further supported by evidence suggesting that maternal immune activation (MIA), especially during early pregnancy, may be a risk factor for ASD. The present study was aimed at characterizing the effects of MIA on behavior, gut microbiota and neuroinflammation in the mouse offspring also considering the impact of MIA in the two sexes. MIA offspring exhibited significant ASD-like behavioral alterations (i.e., deficits in sociability and sensorimotor gating, perseverative behaviors). The analysis of microbiota revealed changes in specific microbial taxa that recapitulated those seen in ASD children. In addition, molecular analyses indicated sex-related differences in the neuroinflammatory responses triggered by MIA, with a more prominent effect in the cerebellum. Our data suggest that both sexes should be included in the experimental designs of preclinical studies in order to identify those mechanisms that confer different vulnerability to ASD to males and females.


Assuntos
Transtorno do Espectro Autista , Transtorno Autístico , Microbioma Gastrointestinal , Animais , Transtorno do Espectro Autista/complicações , Transtorno Autístico/etiologia , Comportamento Animal , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Gravidez
10.
Nutrients ; 14(9)2022 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-35565817

RESUMO

Research in both animals and humans shows that some nutrients are important in pregnancy and during the first years of life to support brain and cognitive development. Our aim was to evaluate the role of selenium (Se) in supporting brain and behavioral plasticity and maturation. Pregnant and lactating female rats and their offspring up to postnatal day 40 were fed isocaloric diets differing in Se content-i.e., optimal, sub-optimal, and deficient-and neurodevelopmental, neuroinflammatory, and anti-oxidant markers were analyzed. We observed early adverse behavioral changes in juvenile rats only in sub-optimal offspring. In addition, sub-optimal, more than deficient supply, reduced basal glial reactivity in sex dimorphic and brain-area specific fashion. In female offspring, deficient and sub-optimal diets reduced the antioxidant Glutathione peroxidase (GPx) activity in the cortex and in the liver, the latter being the key organ regulating Se metabolism and homeostasis. The finding that the Se sub-optimal was more detrimental than Se deficient diet may suggest that maternal Se deficient diet, leading to a lower Se supply at earlier stages of fetal development, stimulated homeostatic mechanisms in the offspring that were not initiated by sub-optimal Se. Our observations demonstrate that even moderate Se deficiency during early life negatively may affect, in a sex-specific manner, optimal brain development.


Assuntos
Selênio , Animais , Antioxidantes/farmacologia , Dieta , Feminino , Glutationa Peroxidase/metabolismo , Humanos , Lactação , Fígado/metabolismo , Masculino , Fenômenos Fisiológicos da Nutrição Materna , Gravidez , Ratos
11.
Neuro Oncol ; 24(7): 1150-1163, 2022 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34964902

RESUMO

BACKGROUND: Diffuse midline gliomas (DMG) H3K27M-mutant, including diffuse intrinsic pontine glioma (DIPG), are pediatric brain tumors associated with grim prognosis. Although GD2-CAR T-cells demonstrated significant anti-tumor activity against DMG H3K27M-mutant in vivo, a multimodal approach may be needed to more effectively treat patients. We investigated GD2 expression in DMG/DIPG and other pediatric high-grade gliomas (pHGG) and sought to identify chemical compounds that would enhance GD2-CAR T-cell anti-tumor efficacy. METHODS: Immunohistochemistry in tumor tissue samples and immunofluorescence in primary patient-derived cell lines were performed to study GD2 expression. We developed a high-throughput cell-based assay to screen 42 kinase inhibitors in combination with GD2-CAR T-cells. Cell viability, western blots, flow-cytometry, real time PCR experiments, DIPG 3D culture models, and orthotopic xenograft model were applied to investigate the effect of selected compounds on DIPG cell death and CAR T-cell function. RESULTS: GD2 was heterogeneously, but widely, expressed in the tissue tested, while its expression was homogeneous and restricted to DMG/DIPG H3K27M-mutant cell lines. We identified dual IGF1R/IR antagonists, BMS-754807 and linsitinib, able to inhibit tumor cell viability at concentrations that do not affect CAR T-cells. Linsitinib, but not BMS-754807, decreases activation/exhaustion of GD2-CAR T-cells and increases their central memory profile. The enhanced anti-tumor activity of linsitinib/GD2-CAR T-cell combination was confirmed in DIPG models in vitro, ex vivo, and in vivo. CONCLUSION: Our study supports the development of IGF1R/IR inhibitors to be used in combination with GD2-CAR T-cells for treating patients affected by DMG/DIPG and, potentially, by pHGG.


Assuntos
Neoplasias do Tronco Encefálico , Glioma , Imunoterapia Adotiva , Receptor IGF Tipo 1 , Receptor de Insulina , Neoplasias do Tronco Encefálico/patologia , Criança , Glioma/tratamento farmacológico , Glioma/genética , Glioma/patologia , Humanos , Receptor IGF Tipo 1/antagonistas & inibidores , Receptor de Insulina/antagonistas & inibidores , Linfócitos T/metabolismo
12.
Cancer Discov ; 12(3): 712-729, 2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-34737188

RESUMO

The survival of children with diffuse intrinsic pontine glioma (DIPG) remains dismal, with new treatments desperately needed. In a prospective biopsy-stratified clinical trial, we combined detailed molecular profiling and drug screening in newly established patient-derived models in vitro and in vivo. We identified in vitro sensitivity to MEK inhibitors in DIPGs harboring MAPK pathway alterations, but treatment of patient-derived xenograft models and a patient at relapse failed to elicit a significant response. We generated trametinib-resistant clones in a BRAFG469V model through continuous drug exposure and identified acquired mutations in MEK1/2 with sustained pathway upregulation. These cells showed hallmarks of mesenchymal transition and expression signatures overlapping with inherently trametinib-insensitive patient-derived cells, predicting sensitivity to dasatinib. Combined trametinib and dasatinib showed highly synergistic effects in vitro and on ex vivo brain slices. We highlight the MAPK pathway as a therapeutic target in DIPG and show the importance of parallel resistance modeling and combinatorial treatments for meaningful clinical translation. SIGNIFICANCE: We report alterations in the MAPK pathway in DIPGs to confer initial sensitivity to targeted MEK inhibition. We further identify for the first time the mechanism of resistance to single-agent targeted therapy in these tumors and suggest a novel combinatorial treatment strategy to overcome it in the clinic. This article is highlighted in the In This Issue feature, p. 587.


Assuntos
Neoplasias do Tronco Encefálico , Recidiva Local de Neoplasia , Criança , Humanos , Neoplasias do Tronco Encefálico/tratamento farmacológico , Neoplasias do Tronco Encefálico/genética , Neoplasias do Tronco Encefálico/patologia , Linhagem Celular Tumoral , Dasatinibe/farmacologia , Dasatinibe/uso terapêutico , Quinases de Proteína Quinase Ativadas por Mitógeno , Recidiva Local de Neoplasia/tratamento farmacológico , Estudos Prospectivos , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico
13.
J Neurochem ; 113(4): 1060-72, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20236219

RESUMO

Inflammation is regarded as a main obstacle to brain regeneration. Major detrimental effects are attributed to microglial/macrophagic products, such as TNF-alpha and interleukin (IL)-6. The role of cytokines of the IL-1 family, particularly of IL-1alpha, in the modulation of neural precursor cell (NPC) properties is less characterized. IL-1alpha is one of the most abundant cytokines released upon acute stimulation of microglia with lipopolysaccharide and is down-regulated upon chronic stimulation. As we recently demonstrated, acutely activated microglia reduces NPC survival, prevent neuronal differentiation and promote glial differentiation. Chronically activated microglia are instead permissive to NPC survival and neuronal differentiation, and less effective in promoting astrocytic differentiation. We thus investigated whether IL-1alpha could contribute to the effects of acutely activated microglia on NPC. We found that NPC express functional IL-1 receptors and that exposure to recombinant IL-1alpha strongly enhances NPC differentiation into astrocytes, without affecting cell viability and neuronal differentiation. In the same conditions, recombinant IL-1beta has pro-gliogenic effects at concentrations 10-fold higher than those found in activated microglial conditioned media. Interestingly, immunodepletion of IL-1alpha in activated microglial conditioned media fails to revert microglial pro-gliogenic action and slightly enhances neuronal differentiation, revealing that other microglial-derived factors contribute to the modulation of NPC properties.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Células-Tronco Embrionárias/efeitos dos fármacos , Interleucina-1alfa/farmacologia , Neuroglia/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Diferenciação Celular/fisiologia , Linhagem da Célula/fisiologia , Células Cultivadas , Técnicas de Cocultura , Meios de Cultivo Condicionados/farmacologia , Citocinas/genética , Citocinas/metabolismo , Citocinas/farmacologia , Relação Dose-Resposta a Droga , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Encefalite/metabolismo , Encefalite/fisiopatologia , Gliose/metabolismo , Gliose/fisiopatologia , Interleucina-1alfa/genética , Interleucina-1alfa/metabolismo , Camundongos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Neuroglia/citologia , Neuroglia/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ratos , Receptores de Interleucina-1/efeitos dos fármacos , Receptores de Interleucina-1/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Células-Tronco/citologia , Células-Tronco/metabolismo
14.
J Neurochem ; 115(2): 450-9, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20681951

RESUMO

Nucleotides act as early signals for microglial recruitment to sites of CNS injury. As microglial motility and activation can be influenced by several local factors at the site of the lesion, we investigated the effects of interferon-gamma, lipopolysaccharide (LPS) or transforming growth factor-ß (TGF-ß) addition to mixed glial cell cultures, on microglial migration in response to ADP, P2Y12 and P2Y1 mRNA expression as well as on the expression of an array of genes associated with the process of microglial activation. First, we demonstrated, by pharmacological inhibition and by using small interfering RNAs, that in addition to P2Y12, P2Y1 is involved in ADP-stimulated microglial migration. The ability of specific agonists to induce Ca(2+) mobilization further confirmed the expression of functional P2Y receptors in microglia. Then, we found that migratory capability and expression of both P2Y receptors were abrogated in microglial cells from LPS-stimulated mixed glial cultures, while TGF-ß increased ADP-induced migration and the expression of P2Y12 and P2Y1 receptors. Interferon-gamma did not influence receptor expression or microglial migration. Finally, the patterns of gene expression induced in microglia by LPS or TGF-ß treatment of mixed glial cultures were clearly distinct. LPS induced a set of classical pro-inflammatory genes, whereas TGF-ß increased the expression of genes associated with atypical microglial phenotype, namely arginase-1 and TGF-ß genes. These results imply that both P2Y1 and P2Y12 may guide microglia toward the lesion. They also suggest that the modulation of microglial purinergic receptors expression by local factors, through direct and/or astrocyte-mediated actions, may represent a novel mechanism affecting neuroinflammatory response.


Assuntos
Movimento Celular/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Microglia/efeitos dos fármacos , Receptores Purinérgicos P2Y1/metabolismo , Receptores Purinérgicos P2/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/farmacologia , Animais , Animais Recém-Nascidos , Cálcio/metabolismo , Células Cultivadas , Córtex Cerebral/citologia , Regulação da Expressão Gênica/efeitos dos fármacos , Interferon gama/farmacologia , Microglia/metabolismo , RNA Interferente Pequeno/farmacologia , Ratos , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2Y1/genética , Receptores Purinérgicos P2Y12 , Tionucleotídeos/farmacologia , Fator de Crescimento Transformador beta/agonistas , Fator de Crescimento Transformador beta/antagonistas & inibidores
15.
J Clin Med ; 8(10)2019 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-31547098

RESUMO

Adverse psychosocial experiences have been shown to modulate individual responses to immune challenges and affect mitochondrial functions. The aim of this study was to investigate inflammation and immune responses as well as mitochondrial bioenergetics in an experimental model of Paediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus (PANDAS). Starting in adolescence (postnatal day 28), male SJL/J mice were exposed to five injections (interspaced by two weeks) with Group-A beta-haemolytic streptococcus (GAS) homogenate. Mice were exposed to chronic psychosocial stress, in the form of protracted visual exposure to an aggressive conspecific, for four weeks. Our results indicate that psychosocial stress exacerbated individual response to GAS administrations whereby mice exposed to both treatments exhibited altered cytokine and immune-related enzyme expression in the hippocampus and hypothalamus. Additionally, they showed impaired mitochondrial respiratory chain complexes IV and V, and reduced adenosine triphosphate (ATP) production by mitochondria and ATP content. These brain abnormalities, observed in GAS-Stress mice, were associated with blunted titers of plasma corticosterone. Present data support the hypothesis that challenging environmental conditions, in terms of chronic psychosocial stress, may exacerbate the long-term consequences of exposure to GAS processes through the promotion of central immunomodulatory and oxidative stress.

16.
Sci Rep ; 9(1): 4572, 2019 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-30872738

RESUMO

Mutations of Fused in sarcoma (FUS), a ribonucleoprotein involved in RNA metabolism, have been found associated with both familial and sporadic cases of amyotrophic lateral sclerosis (ALS). Notably, besides mutations in the coding sequence, also mutations into the 3' untranslated region, leading to increased levels of the wild-type protein, have been associated with neuronal death and ALS pathology, in ALS models and patients. The mechanistic link between altered FUS levels and ALS-related neurodegeneration is far to be elucidated, as well as the consequences of elevated FUS levels in the modulation of the inflammatory response sustained by glial cells, a well-recognized player in ALS progression. Here, we studied the effect of wild-type FUS overexpression on the responsiveness of mouse and human neural progenitor-derived astrocytes to a pro-inflammatory stimulus (IL1ß) used to mimic an inflammatory environment. We found that astrocytes with increased FUS levels were more sensitive to IL1ß, as shown by their enhanced expression of inflammatory genes, compared with control astrocytes. Moreover, astrocytes overexpressing FUS promoted neuronal cell death and pro-inflammatory microglia activation. We conclude that overexpression of wild-type FUS intrinsically affects astrocyte reactivity and drives their properties toward pro-inflammatory and neurotoxic functions, suggesting that a non-cell autonomous mechanism can support neurodegeneration in FUS-mutated animals and patients.


Assuntos
Astrócitos/metabolismo , Regulação da Expressão Gênica , Microglia/metabolismo , Neurônios/metabolismo , Proteína FUS de Ligação a RNA/genética , Animais , Biomarcadores , Morte Celular , Suscetibilidade a Doenças , Perfilação da Expressão Gênica , Humanos , Mediadores da Inflamação , Camundongos , Neurônios Motores/metabolismo , Mutação , Transporte Proteico , Proteína FUS de Ligação a RNA/metabolismo
17.
J Exp Clin Cancer Res ; 35: 55, 2016 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-27015814

RESUMO

BACKGROUND: The CXCL12/CXCR4 pathway regulates tumor cell proliferation, metastasis, angiogenesis and the tumor-microenvironment cross-talk in several solid tumors, including glioblastoma (GBM), the most common and fatal brain cancer. In the present study, we evaluated the effects of peptide R, a new specific CXCR4 antagonist that we recently developed by a ligand-based approach, in an in vitro and in vivo model of GBM. The well-characterized CXCR4 antagonist Plerixafor was also included in the study. METHODS: The effects of peptide R on CXCR4 expression, cell survival and migration were assessed on the human glioblastoma cell line U87MG exposed to CXCL12, by immunofluorescence and western blotting, MTT assay, flow cytometry and transwell chamber migration assay. Peptide R was then tested in vivo, by using U87MG intracranial xenografts in CD1 nude mice. Peptide R was administered for 23 days since cell implantation and tumor volume was assessed by magnetic resonance imaging (MRI) at 4.7 T. Glioma associated microglia/macrophage (GAMs) polarization (anti-tumor M1 versus pro-tumor M2 phenotypes) and expressions of vascular endothelial growth factor (VEGF) and CD31 were assessed by immunohistochemistry and immunofluorescence. RESULTS: We found that peptide R impairs the metabolic activity and cell proliferation of human U87MG cells and stably reduces CXCR4 expression and cell migration in response to CXCL12 in vitro. In the orthotopic U87MG model, peptide R reduced tumor cellularity, promoted M1 features of GAMs and astrogliosis, and hindered intra-tumor vasculature. CONCLUSIONS: Our findings suggest that targeting CXCR4 by peptide R might represent a novel therapeutic approach against GBM, and contribute to the rationale to further explore in more complex pre-clinical settings the therapeutic potential of peptide R, alone or in combination with standard therapies of GBM.


Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Quimiocina CXCL12/antagonistas & inibidores , Glioblastoma/tratamento farmacológico , Microglia/efeitos dos fármacos , Peptídeos/administração & dosagem , Receptores CXCR4/antagonistas & inibidores , Animais , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/metabolismo , Humanos , Camundongos , Camundongos Nus , Microglia/patologia , Peptídeos/farmacologia , Carga Tumoral/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
18.
J Neuroinflammation ; 2(1): 4, 2005 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-15670336

RESUMO

BACKGROUND: Nicotinic acetylcholine (Ach) receptors are ligand-gated pentameric ion channels whose main function is to transmit signals for the neurotransmitter Ach in peripheral and central nervous system. However, the alpha7 nicotinic receptor has been recently found in several non-neuronal cells and described as an important regulator of cellular function. Nicotine and ACh have been recently reported to inhibit tumor necrosis factor-alpha (TNF-alpha) production in human macrophages as well as in mouse microglial cultures. In the present study, we investigated whether the stimulation of alpha7 nicotinic receptor by the specific agonist nicotine could affect the functional state of activated microglia by promoting and/or inhibiting the release of other important pro-inflammatory and lipid mediator such as prostaglandin E2. METHODS: Expression of alpha7 nicotinic receptor in rat microglial cell was examined by RT-PCR, immunofluorescence staining and Western blot. The functional effects of alpha7 receptor activation were analyzed in resting or lipopolysaccharide (LPS) stimulated microglial cells pre-treated with nicotine. Culture media were assayed for the levels of tumor necrosis factor, interleukin-1beta, nitric oxide, interleukin-10 and prostaglandin E2. Total RNA was assayed by RT-PCR for the expression of COX-2 mRNA. RESULTS: Rat microglial cells express alpha7 nicotinic receptor, and its activation by nicotine dose-dependently reduces the LPS-induced release of TNF-alpha, but has little or no effect on nitric oxide, interleukin-10 and interleukin-1beta. By contrast, nicotine enhances the expression of cyclooxygenase-2 and the synthesis of one of its major products, prostaglandin E2. CONCLUSIONS: Since prostaglandin E2 modulates several macrophage and lymphocyte functions, which are instrumental for inflammatory resolution, our study further supports the existence of a brain cholinergic anti-inflammatory pathway mediated by alpha7 nicotinic receptor that could be potentially exploited for novel treatments of several neuropathologies in which local inflammation, sustained by activated microglia, plays a crucial role.

19.
J Neuropathol Exp Neurol ; 61(3): 237-44, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11895038

RESUMO

Exposure of phosphatidylserine (PS), an aminophospholipid normally sequestered in the inner leaflet of plasma membrane, is one of the crucial steps in the recognition and ingestion of apoptotic cells by macrophages. The recognition of PS on apoptotic cells by peripheral macrophages is mediated by a phosphatidylserine-specific receptor (PtdSerR), which has recently been cloned. In spite of the important role of apoptosis in the CNS, the process of apoptotic neuron recognition by microglia is poorly understood. Because recent studies suggest that engagement of PS with a not yet characterized microglial receptor is necessary for apoptotic neuron uptake, we investigated the expression of PtdSer-R and its functional role in neonatal rat brain microglial cultures. Semi-quantitative RT-PCR analysis revealed that PtdSerR mRNA was detectable in unstimulated cultures and enhanced in LPS activated microglia. The presence of PS-liposomes strongly reduced the release of pro-inflammatory molecules such as nitric oxide, interleukin-1beta, and tumor necrosis factor-alpha by LPS-activated microglia. At variance, the immunoregulatory cytokines interleukin-10 and transforming growth factor-beta1 were moderately decreased or unaffected. The activity of PS-liposomes was mimicked by the PS head group phospho-L-serine, but not by phosphatidylcholine-containing liposomes. Our data suggest that, as for peripheral macrophages, PS through its receptor can modulate microglial activation toward an anti-inflammatory phenotype.


Assuntos
Mediadores da Inflamação/metabolismo , Microglia/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Regulação para Baixo , Interferon gama/farmacologia , Ligantes , Lipossomos , Camundongos , Microglia/efeitos dos fármacos , Microglia/fisiologia , Fosfatidilserinas/administração & dosagem , Fosfatidilserinas/metabolismo , RNA Mensageiro/metabolismo , Ratos , Receptores de Superfície Celular/genética
20.
J Neuropathol Exp Neurol ; 62(2): 208-16, 2003 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-12578230

RESUMO

The interaction of phosphatidylserine (PS), exposed on the surface of apoptotic cells and with its specific receptor (PtdSerR) expressed by microglia, is a crucial event in the recognition and clearance of apoptotic neurons. Here, we extend our previous studies in which PS-liposomes mimicking apoptotic cells were used to investigate the functional role of PS-PtdSerR interactions on microglial functional state. Purified rat microglial cells were either incubated with PC12 cells maintained in complete medium (healthy), exposed to staurosporine or serum deprivation (apoptotic), or treated with hydrogen peroxide (necrotic). After 24 hours, supernatants from co-cultures and single cell type cultures were analyzed for nitric oxide (NO), tumor necrosis factor-alpha (TNF-alpha), interleukin-10 (IL-10), prostaglandin E2 (PGE2), transforming growth factor-beta1 (TGF-beta1), and nerve growth factor (NGF). When lipopolysaccharide (LPS)-activated microglia was cultured with apoptotic PC12 cells, NO and TNF-alpha levels significantly decreased, IL-10 was not affected, and PGE2 levels were substantially increased. In addition, TGF-beta and NGF syntheses increased when resting microglia was cultured with apoptotic but not healthy or necrotic PC12 cells. We proposed that upon interaction with PS-expressing apoptotic neurons, microglia no longer act as a promoter of the inflammatory cascade and that the specific microglial functional state induced by PS-PtdSerR may be relevant for the final outcome of neurodegenerative diseases.


Assuntos
Apoptose/imunologia , Encéfalo/metabolismo , Encefalite/metabolismo , Microglia/metabolismo , Neurônios/metabolismo , Fagocitose/imunologia , Fosfatidilserinas/metabolismo , Animais , Anti-Inflamatórios/metabolismo , Antígenos de Superfície/imunologia , Encéfalo/imunologia , Encéfalo/fisiopatologia , Comunicação Celular/imunologia , Células Cultivadas , Técnicas de Cocultura , Citocinas/imunologia , Citocinas/metabolismo , Dinoprostona/imunologia , Dinoprostona/metabolismo , Encefalite/imunologia , Encefalite/fisiopatologia , Vigilância Imunológica/imunologia , Microglia/imunologia , Necrose , Fator de Crescimento Neural/metabolismo , Neurônios/imunologia , Fármacos Neuroprotetores/metabolismo , Células PC12 , Ratos
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