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1.
Glia ; 66(8): 1724-1735, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29575211

RESUMO

The prevalent view in neuroenergetics is that glucose is the main brain fuel, with neurons being mostly oxidative and astrocytes glycolytic. Evidence supporting that astrocyte mitochondria are functional has been overlooked. Here we sought to determine what is unique about astrocyte mitochondria by performing unbiased statistical comparisons of the mitochondriome in astrocytes and neurons. Using MitoCarta, a compendium of mitochondrial proteins, together with transcriptomes of mouse neurons and astrocytes, we generated cell-specific databases of nuclear genes encoding for mitochondrion proteins, ranked according to relative expression. Standard and in-house Gene Set Enrichment Analyses (GSEA) of five mouse transcriptomes revealed that genes encoding for enzymes involved in fatty acid oxidation (FAO) and amino acid catabolism are consistently more expressed in astrocytes than in neurons. FAO and oxidative-metabolism-related genes are also up-regulated in human cortical astrocytes versus the whole cortex, and in adult astrocytes versus fetal astrocytes. We thus present the first evidence of FAO in human astrocytes. Further, as shown in vitro, FAO coexists with glycolysis in astrocytes and is inhibited by glutamate. Altogether, these analyses provide arguments against the glucose-centered view of energy metabolism in astrocytes and reveal mitochondria as specialized organelles in these cells.


Assuntos
Astrócitos/metabolismo , Metabolismo Energético/fisiologia , Ácidos Graxos/metabolismo , Glicólise/fisiologia , Mitocôndrias/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Ácido Glutâmico/metabolismo , Humanos , Metabolismo dos Lipídeos , Camundongos , Proteínas Mitocondriais/metabolismo , Neurônios/metabolismo , Oxirredução
2.
Immunol Cell Biol ; 95(6): 538-548, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28108746

RESUMO

Myeloid-derived suppressor cells (MDSCs) have an important role in controlling inflammation. As such, they are both a therapeutic target and, based on the administration of ex vivo-generated MDSCs, a therapeutic tool. However, there are relatively few reports describing methods to generate human MDSCs, and most of them rely on cells obtained from peripheral blood monocytes. We investigated alternative approaches to the generation of MDSCs from hematopoietic progenitors and monocytes. Purified CD34+ hematopoietic progenitors from apheresis products and CD14+ cells isolated from buffy coats were cultured in the presence of different combinations of cytokines. The resulting myeloid cell populations were then characterized phenotypically and functionally. Progenitor cells cultured in the presence of SCF+TPO+FLT3-L+GM-CSF+IL-6 gave rise to both monocytic (M)- and granulocytic (G)-MDSCs but production of the latter was partially inhibited by IL-3. M-MDSCs but not G-MDSCs were obtained by culturing peripheral blood monocytes with GM-CSF+IL-6 or GM-CSF+TGF-ß1 for 6 days. CD14 expression was downregulated in the cultured cells. PD-L1 expression at baseline was lower in hematopoietic progenitor cell-derived than in monocyte-derived MDSCs, but was markedly increased in response to stimulation with LPS+IFN-γ. The functionality of the two MDSC subtypes was confirmed in studies of the suppression of allogeneic and mitogen-induced proliferation and by cytokine profiling. Here we describe both the culture conditions that allow the generation of MDSCs and the phenotypical and functional characterization of these cell populations.


Assuntos
Diferenciação Celular , Células-Tronco Hematopoéticas/citologia , Monócitos/citologia , Células Supressoras Mieloides/citologia , Biomarcadores , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Citocinas/metabolismo , Citocinas/farmacologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Imunomodulação/efeitos dos fármacos , Imunofenotipagem , Mediadores da Inflamação/metabolismo , Mediadores da Inflamação/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Monócitos/imunologia , Monócitos/metabolismo , Células Supressoras Mieloides/imunologia , Células Supressoras Mieloides/metabolismo , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo
3.
Glia ; 64(5): 853-74, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26880229

RESUMO

The clinical challenge in acute injury as in traumatic brain injury (TBI) is to halt the delayed neuronal loss that occurs hours and days after the insult. Here we report that the activation of CREB-dependent transcription in reactive astrocytes prevents secondary injury in cerebral cortex after experimental TBI. The study was performed in a novel bitransgenic mouse in which a constitutively active CREB, VP16-CREB, was targeted to astrocytes with the Tet-Off system. Using histochemistry, qPCR, and gene profiling we found less neuronal death and damage, reduced macrophage infiltration, preserved mitochondria, and rescued expression of genes related to mitochondrial metabolism in bitransgenic mice as compared to wild type littermates. Finally, with meta-analyses using publicly available databases we identified a core set of VP16-CREB candidate target genes that may account for the neuroprotective effect. Enhancing CREB activity in astrocytes thus emerges as a novel avenue in acute brain post-injury therapeutics.


Assuntos
Astrócitos/metabolismo , Lesões Encefálicas/patologia , Lesões Encefálicas/terapia , Proteína de Ligação a CREB/metabolismo , Animais , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Astrócitos/efeitos dos fármacos , Proteína de Ligação a CREB/genética , Células Cultivadas , Modelos Animais de Doenças , Etoposídeo/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/genética , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Inflamação/etiologia , Inflamação/prevenção & controle , Masculino , Metanálise como Assunto , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteínas de Neurofilamentos/metabolismo
4.
Br J Pharmacol ; 178(3): 654-671, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33140839

RESUMO

BACKGROUND AND PURPOSE: Activation of type 2 imidazoline receptors has been shown to exhibit neuroprotective properties including anti-apoptotic and anti-inflammatory effects, suggesting a potential therapeutic value in Alzheimer's disease (AD). Here, we explored the effects of the imidazoline-2 ligand BU224 in a model of amyloidosis. EXPERIMENTAL APPROACH: Six-month-old female transgenic 5XFAD and wild-type (WT) mice were treated intraperitoneally with 5-mg·kg-1 BU224 or vehicle twice a day for 10 days. Behavioural tests were performed for cognitive functions and neuropathological changes were investigated by immunohistochemistry, Western blot, elisa and qPCR. Effects of BU224 on amyloid precursor protein (APP) processing, spine density and calcium imaging were analysed in brain organotypic cultures and N2a cells. KEY RESULTS: BU224 treatment attenuated spatial and perirhinal cortex-dependent recognition memory deficits in 5XFAD mice. Fear-conditioning testing revealed that BU224 also improved both associative learning and hippocampal- and amygdala-dependent memory in transgenic but not in WT mice. In the brain, BU224 reduced levels of the microglial marker Iba1 and pro-inflammatory cytokines IL-1ß and TNF-α and increased the expression of astrocytic marker GFAP in 5XFAD mice. These beneficial effects were not associated with changes in amyloid pathology, neuronal apoptosis, mitochondrial density, oxidative stress or autophagy markers. Interestingly, ex vivo and in vitro studies suggested that BU224 treatment increased the size of dendritic spines and induced a threefold reduction in amyloid-ß (Aß)-induced functional changes in NMDA receptors. CONCLUSION AND IMPLICATIONS: Sub-chronic treatment with BU224 restores memory and reduces inflammation in transgenic AD mice, at stages when animals display severe pathology.


Assuntos
Doença de Alzheimer , Imidazolinas , Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides , Precursor de Proteína beta-Amiloide/genética , Animais , Cognição , Modelos Animais de Doenças , Feminino , Imidazóis , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos
5.
EMBO Mol Med ; 13(2): e12105, 2021 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-33369245

RESUMO

Lamins are crucial proteins for nuclear functionality. Here, we provide new evidence showing that increased lamin B1 levels contribute to the pathophysiology of Huntington's disease (HD), a CAG repeat-associated neurodegenerative disorder. Through fluorescence-activated nuclear suspension imaging, we show that nucleus from striatal medium-sized spiny and CA1 hippocampal neurons display increased lamin B1 levels, in correlation with altered nuclear morphology and nucleocytoplasmic transport disruption. Moreover, ChIP-sequencing analysis shows an alteration of lamin-associated chromatin domains in hippocampal nuclei, accompanied by changes in chromatin accessibility and transcriptional dysregulation. Supporting lamin B1 alterations as a causal role in mutant huntingtin-mediated neurodegeneration, pharmacological normalization of lamin B1 levels in the hippocampus of the R6/1 mouse model of HD by betulinic acid administration restored nuclear homeostasis and prevented motor and cognitive dysfunction. Collectively, our work points increased lamin B1 levels as a new pathogenic mechanism in HD and provides a novel target for its intervention.


Assuntos
Doença de Huntington , Animais , Corpo Estriado , Doença de Huntington/genética , Lamina Tipo B/genética , Camundongos , Neurônios
7.
Sci Rep ; 7(1): 6390, 2017 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-28743894

RESUMO

The cyclic AMP response element binding protein (CREB) is a primary hub of activity-driven genetic programs in neurons controlling plasticity, neurogenesis and survival. By contrast, the gene networks coordinated by CREB in astrocytes are unknown despite the fact that the astrocytic CREB is also activity-driven and neuroprotective. Herein we identified the transcriptional programs regulated by CREB in astrocytes as compared to neurons using, as study materials, transcriptome databases of astrocyte exposed to well-known activators of CREB-dependent transcription as well as publicly available transcriptomes of neuronal cultures. Functional CREB signatures were extracted from the transcriptomes using Gene Ontology, adult-brain gene lists generated by Translating Ribosome Affinity Purification (TRAP) and CREB-target gene repositories. We found minimal overlap between CREB signatures in astrocytes and neurons. In astrocytes, the top triad of functions regulated by CREB consists of 'Gene expression', 'Mitochondria', and 'Signalling', while in neurons it is 'Neurotransmission', 'Signalling' and 'Gene expression', the latter two being represented by different genes from those in astrocytes. The newly generated databases will provide a tool to explore novel means whereby CREB impinges on brain functions requiring adaptive, long-lasting changes by coordinating transcriptional cascades in astrocytes.


Assuntos
Astrócitos/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes , Neurônios/metabolismo , Animais , Astrócitos/citologia , Células Cultivadas , Bases de Dados Genéticas , Regulação da Expressão Gênica , Neurônios/citologia , Análise de Sequência com Séries de Oligonucleotídeos , Especificidade de Órgãos , Ratos , Ratos Sprague-Dawley , Transcrição Gênica
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