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1.
Medicine (Baltimore) ; 98(42): e17591, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31626131

RESUMO

BACKGROUND: Spinal cord ischemia-reperfusion injury (SCII) is a common complication of spinal surgery as well as thoracic and abdominal surgery. Acute cytotoxic edema is the key pathogenic alteration. Therefore, avoiding or decreasing cellular edema has become the major target for SCII treatment. METHODS: The antiedema activity of ginsenoside Rb1 on aquaporin (AQP) 4, nerve growth factor (NGF), and brain-derived neurotrophic factor expression was detected by western blot and real-time polymerase chain reaction under conditions of oxygen-glucose deprivation/reoxygenation (OGD/R) in a rat astrocyte model in vitro. In addition, the cellular membrane permeability of AQP4 overexpressing cells or AQP4 small interfering RNA-transfected cells was detected. RESULTS: Ginsenoside Rb1 significantly prevented OGD/R-induced AQP4 downregulation in rat astrocytes. In addition, ginsenoside Rb1 treatment or AQP4 overexpression in rat astrocytes significantly attenuated the OGD/R-induced increase of cellular membrane permeability. Moreover, ginsenoside Rb1 obviously prevented the OGD/R-induced decrease of NGF and BDNT expression in rat astrocytes. CONCLUSION: These findings demonstrate that ginsenoside Rb1 can relieve spinal cord edema and improve neurological function by increasing AQP4 expression.


Assuntos
Aquaporina 4/genética , Astrócitos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Ginsenosídeos/farmacologia , Glucose/metabolismo , Oxigênio/metabolismo , Traumatismo por Reperfusão/genética , Animais , Animais Recém-Nascidos , Aquaporina 4/biossíntese , Astrócitos/efeitos dos fármacos , Astrócitos/patologia , Western Blotting , Células Cultivadas , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , RNA/genética , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Medula Espinal/metabolismo , Medula Espinal/patologia
2.
Rev Assoc Med Bras (1992) ; 65(9): 1174-1180, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31618333

RESUMO

OBJECTIVE: The study aims to explore the relationship between preoperative anxiety and chronic postoperative pain. METHODS: A total of forty rats were divided into four groups, control, single-prolonged stress alone, Hysterectomy alone, and SPS+ Hysterectomy. The paw withdrawal mechanical thresholds (PWMT) were examined. qRT-PCR and western blotting assay were performed to detect the GFAP expression in astrocytes isolated from the anterior cingulate cortex (ACC) region. In addition, the long-term potentiation (LTP) in ACC was examined. RESULTS: Rats in the SPS group or the Hysterectomy alone group had no significant effect on chronic pain formation, but SPS can significantly induce chronic pain after surgery. Astrocytes were still active, and the LTP was significantly increased three days after modeling in the SPS+Hysterectomy group. CONCLUSIONS: anxiety can induce chronic pain by activating astrocytes in the ACC region.


Assuntos
Ansiedade/complicações , Astrócitos/metabolismo , Dor Crônica/etiologia , Dor Pós-Operatória/etiologia , Animais , Dor Crônica/psicologia , Modelos Animais de Doenças , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Giro do Cíngulo/metabolismo , Membro Posterior , Histerectomia , Potenciação de Longa Duração/fisiologia , Limiar da Dor/fisiologia , Dor Pós-Operatória/psicologia , Período Pré-Operatório , Distribuição Aleatória , Ratos Sprague-Dawley , Estresse Psicológico/etiologia , Fatores de Tempo
3.
Nat Neurosci ; 22(10): 1731-1742, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31501572

RESUMO

Mitochondria vary in morphology and function in different tissues; however, little is known about their molecular diversity among cell types. Here we engineered MitoTag mice, which express a Cre recombinase-dependent green fluorescent protein targeted to the outer mitochondrial membrane, and developed an isolation approach to profile tagged mitochondria from defined cell types. We determined the mitochondrial proteome of the three major cerebellar cell types (Purkinje cells, granule cells and astrocytes) and identified hundreds of mitochondrial proteins that are differentially regulated. Thus, we provide markers of cell-type-specific mitochondria for the healthy and diseased mouse and human central nervous systems, including in amyotrophic lateral sclerosis and Alzheimer's disease. Based on proteomic predictions, we demonstrate that astrocytic mitochondria metabolize long-chain fatty acids more efficiently than neuronal mitochondria. We also characterize cell-type differences in mitochondrial calcium buffering via the mitochondrial calcium uniporter (Mcu) and identify regulator of microtubule dynamics protein 3 (Rmdn3) as a determinant of endoplasmic reticulum-mitochondria proximity in Purkinje cells. Our approach enables exploring mitochondrial diversity in many in vivo contexts.


Assuntos
Encéfalo/citologia , Mitocôndrias/metabolismo , Neurônios/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Animais , Astrócitos/metabolismo , Sinalização do Cálcio/genética , Sinalização do Cálcio/fisiologia , Células Cultivadas , Cerebelo/citologia , Ácidos Graxos/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Membranas Mitocondriais/metabolismo , Proteômica , Células de Purkinje/metabolismo
4.
J Agric Food Chem ; 67(34): 9618-9629, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31381342

RESUMO

Astrocytes provide nutritional support, regulate inflammation, and perform synaptic functions in the human brain. Although butylated hydroxyanisole (BHA) is a well-known antioxidant, several studies in animals have indicated BHA-mediated liver toxicity, retardation in reproductive organ development and learning, and sleep deficit. However, the specific effects of BHA on human astrocytes and the underlying mechanisms are yet unclear. Here, we investigated the antigrowth effects of BHA through cell cycle arrest and downregulation of regulatory protein expression. The typical cell proliferative signaling pathways, phosphoinositide 3-kinase/protein kinase B and extracellular signal-regulated kinase 1/2, were downregulated in astrocytes after BHA treatment. BHA increased the levels of pro-apoptotic proteins, such as BAX, cytochrome c, cleaved caspase 3, and cleaved caspase 9, and decreased the level of anti-apoptotic protein BCL-XL. It also increased the cytosolic calcium level and the expression of endoplasmic reticulum stress proteins. Treatment with BAPTA-AM, a calcium chelator, attenuated the increased levels of ER stress proteins and cleaved members of the caspase family. We further performed an in vivo evaluation of the neurotoxic effect of BHA on zebrafish embryos and glial fibrillary acidic protein, a representative astrocyte biomarker, in a gfap:eGFP zebrafish transgenic model. Our results provide clear evidence of the potent cytotoxic effects of BHA on human astrocytes, which lead to disruption of the brain and nerve development.


Assuntos
Astrócitos/efeitos dos fármacos , Hidroxianisol Butilado/toxicidade , Cálcio/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Neurotoxinas/toxicidade , Animais , Astrócitos/metabolismo , Caspase 3/metabolismo , Caspase 9/metabolismo , Linhagem Celular , Citocromos c/metabolismo , Citosol/efeitos dos fármacos , Citosol/metabolismo , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Peixe-Zebra
5.
Chem Biol Interact ; 309: 108686, 2019 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-31152735

RESUMO

Acetylcholinesterase (EC3.1.1.7; AChE) is a key enzyme in the cholinergic system. Emerging evidence has shown that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a typical persistent organic pollutant, suppressed neuronal AChE activity via dysregulation of different biosynthesis processes in human and rat neuronal cells. In the nervous system, astrocytes protect neurons from environmental pollutants. As a known target cell of TCDD, the astrocyte might be involved in TCDD effects on neuronal AChE. Therefore, in the present study, we found astrocyte-derived conditioned medium (ACM) could induce AChE activity preferentially in mature neurons in the absence of TCDD. The enzymatic activity of AChE was generally decreased in cultured cortical neurons upon direct treatment with TCDD (0.003-0.01 nM). This trend of changes in AChE activity was not significantly altered in immature neurons exposed to ACM produced in the presence of TCDD (TACM group), but reversed in mature neurons. Compared with effects of treatment with ACM plus TCDD (ACMT), a significant differential effect on AChE activity was found in the TACM group in response to TCDD treatment specifically in immature neurons, suggesting the presence of a TCDD-specific active component derived from the astrocyte. Inconsistent alterations in expression and enzymatic activities of the AChE T subunit (AChET) and the proline-rich membrane anchor (PRiMA) were found, suggesting that a mechanism of action beyond the transcriptional level might be involved. These data indicate that the astrocyte might play a protective role in TCDD-induced alterations of neuronal AChE in certain stages of differentiation.


Assuntos
Acetilcolinesterase/metabolismo , Meios de Cultivo Condicionados/química , Expressão Gênica/efeitos dos fármacos , Dibenzodioxinas Policloradas/farmacologia , Acetilcolinesterase/genética , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Dibenzodioxinas Policloradas/química , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley
6.
Life Sci ; 232: 116501, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31163175

RESUMO

AIMS: Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS). The disease mechanisms driving progressive MS remain unresolved. Without this information, current therapeutic strategies are unsatisfactory in preventing disease progression. Our previous work revealed that DL-3-n-butylphthalide (NBP) treatment reduced demyelination in an ethidium bromide mouse model of demyelination. Here, we examine the effect of NBP in the cuprizone model of demyelination by evaluating the pathologic, functional, and behavioral consequences of treatment with NBP. MATERIALS AND METHODS: Forty mice were divided randomly into 4 groups: a normal diet group, a cuprizone diet group, and two NBP groups (10 and 20 mg/kg). CNS infiltration by microglia, axon health and myelination were assessed using immunohistochemistry and electron microscopy, and the levels of cytoplasmic complexes were assessed by Western blotting. KEY FINDINGS: The results showed the neuroprotective effects of the NBP included suppressing the microglia activation through inhibition of nuclear factor-κB (NF-κB) expression, thus decreasing activation of the NF-κB signaling pathway. In particular, myelin density was increased due to an increased mean number of mature oligodendrocytes (OLs) in the high-dose NBP (20 mg/kg) subgroup through reduced oligodendrocyte apoptosis. Meanwhile, increased expression of myelin sheath proteins, including proteolipid protein (PLP) and myelin basic protein (MBP), was observed in the same subgroup. SIGNIFICANCE: These data suggest that NBP may not only have anti-inflammatory properties but also promote the survival of OLs in a mouse cuprizone model of demyelination. NBP may have a potential role in the treatment of MS.


Assuntos
Benzofuranos/farmacologia , Doenças Desmielinizantes/tratamento farmacológico , Esclerose Múltipla/metabolismo , Animais , Astrócitos/metabolismo , Axônios/patologia , Corpo Caloso/efeitos dos fármacos , Corpo Caloso/fisiologia , Cuprizona/farmacologia , Doenças Desmielinizantes/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/fisiopatologia , Bainha de Mielina/metabolismo , NF-kappa B/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Oligodendroglia/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
7.
Toxicol Lett ; 313: 42-49, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31154016

RESUMO

Astrocytes are the major glial cell type in the central nervous system (CNS), and the distal part of the astrocyte forms the blood-brain barrier with nearby blood vessels. They maintain the overall metabolism, growth, homeostasis of neurons, and signaling in the CNS. Ochratoxin A is considered a carcinogen and immunotoxic, nephrotoxic, and neurotoxic mycotoxin. Specifically, it exhibits neurotoxicity with high affinity for the brain. Despite some previous studies about the effects of ochratoxin A in glial cells, the intracellular working mechanism in astrocytes is not fully understood. In this study, we studied the specific working mechanism of ochratoxin A in the human astrocyte cell line, NHA-SV40LT. Ochratoxin A reduced cell proliferation with sub G0/G1 cell cycle arrest by inhibiting CCND1, CCNE1, CDK4, and MYC expression. It induced apoptosis of NHA-SV40LT cells through mitochondrial membrane potential (MMP) loss and up-regulation of BAX and TP53. In addition, ochratoxin A increased cytosolic and mitochondrial calcium levels, resulting in an increase in MMP2 and PLAUR mRNA expression in NHA-SV40LT cells. Furthermore, ochratoxin A regulated the phosphorylation of AKT, ERK1/2, and JNK signal molecules of human astrocytes. Collectively, ochratoxin A exerts neurotoxicity through anti-proliferation and mitochondria-dependent apoptosis in human astrocytes.


Assuntos
Apoptose/efeitos dos fármacos , Astrócitos/efeitos dos fármacos , Cálcio/metabolismo , Mitocôndrias/efeitos dos fármacos , Síndromes Neurotóxicas/etiologia , Ocratoxinas/toxicidade , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Astrócitos/metabolismo , Astrócitos/patologia , Sinalização do Cálcio/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação da Expressão Gênica , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Síndromes Neurotóxicas/genética , Síndromes Neurotóxicas/metabolismo , Síndromes Neurotóxicas/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo
8.
Biophys Chem ; 252: 106195, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31195340

RESUMO

Astrocytes, the most common type of glial cell, are critical to the health of the central nervous system. Evidence implies that changes in the astrocyte's cytosolic calcium concentration is part of a central mechanism by which information is passed and processed in the cell, and it is linked to both external stimuli impacting the cell as well as downstream events such as metabolism and neurotransmitter release. This work proposes a novel chemical model to further the understanding of how extracellular signals could affect intracellular calcium dynamics and metabolic processes within the cell.


Assuntos
Astrócitos/metabolismo , Cálcio/metabolismo , Glucose/metabolismo , Algoritmos , Animais , Astrócitos/química , Citosol/química , Citosol/metabolismo , Humanos , Modelos Biológicos
9.
Cell Mol Biol Lett ; 24: 37, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31168302

RESUMO

Background: Accumulating evidence has shown that altered microRNA (miR) modulation is implicated in the pathologies of ischemic stroke. However, it is unclear whether and how hsa-miR-19a-3p mediates cerebral ischemic injury. Herein, we investigated the functional role of miR-19a-3p in cerebral ischemic injury and explored its underlying regulatory mechanism. Methods: In vivo ischemic/reperfusion (I/R) neuronal injury and in vitro oxygen-glucose deprivation (OGD) were established. Expression of miR-19a-3p was determined by quantitative real-time polymerase chain reaction (qRT-PCR). Glucose uptake, lactate production, and apoptosis were determined. ADIPOR2 was predicted as a target of miR-19a-3p in silico and experimentally validated by qRT-PCR, Western blot analysis and luciferase assay assays. Results: MiR-19a expression was significantly downregulated and upregulated in rat neurons and astrocytes, respectively (P < 0.01). A significantly elevated level of miR-19a-3p was found in I/R and OGD models in comparison to sham/control groups (P < 0.01). Expression of the glycolysis enzyme markers LDHA, PKM2, HK2, Glut1 and PDK1, apoptosis-related factors levels, apoptosis, glucose uptake, and lactate production were significantly repressed by both I/R and OGD (P < 0.01 in each case). Moreover, miR-19a-3p mimic aggravated, while miR-19a-3p inhibitor alleviated, the above observations. Adipor2 was predicted and confirmed to be a direct target of miR-19a. Furthermore, restoration of Adipor2 reversed miR-19a-3p-induced effects. Conclusions: Collectively, our results indicate that elevated miR-19a-3p mediates cerebral ischemic injury by targeting ADIPOR2. MiR-19a-3p attenuation thus might offer hope of a novel therapeutic target for ischemic stroke injury treatment.


Assuntos
Apoptose , Isquemia Encefálica/patologia , Glucose/metabolismo , MicroRNAs/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Neuroproteção , Acidente Vascular Cerebral/patologia , Animais , Animais Recém-Nascidos , Astrócitos/metabolismo , Sequência de Bases , Modelos Animais de Doenças , MicroRNAs/genética , Oxigênio , Ratos Sprague-Dawley , Receptores de Adiponectina/metabolismo , Regulação para Cima/genética
10.
Nat Commun ; 10(1): 2541, 2019 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-31186414

RESUMO

Reactive astrocytes evolve after brain injury, inflammatory and degenerative diseases, whereby they undergo transcriptomic re-programming. In malignant brain tumors, their function and crosstalk to other components of the environment is poorly understood. Here we report a distinct transcriptional phenotype of reactive astrocytes from glioblastoma linked to JAK/STAT pathway activation. Subsequently, we investigate the origin of astrocytic transformation by a microglia loss-of-function model in a human organotypic slice model with injected tumor cells. RNA-seq based gene expression analysis of astrocytes reveals a distinct astrocytic phenotype caused by the coexistence of microglia and astrocytes in the tumor environment, which leads to a large release of anti-inflammatory cytokines such as TGFß, IL10 and G-CSF. Inhibition of the JAK/STAT pathway shifts the balance of pro- and anti-inflammatory cytokines towards a pro-inflammatory environment. The complex interaction of astrocytes and microglia cells promotes an immunosuppressive environment, suggesting that tumor-associated astrocytes contribute to anti-inflammatory responses.


Assuntos
Astrócitos/metabolismo , Citocinas/metabolismo , Glioblastoma/imunologia , Microglia/metabolismo , Astrócitos/citologia , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Humanos , Mediadores da Inflamação , Janus Quinases/metabolismo , Microglia/citologia , Fenótipo , Fatores de Transcrição STAT/metabolismo , Análise de Sequência de RNA , Transdução de Sinais , Técnicas de Cultura de Tecidos
11.
Mol Cells ; 42(4): 321-332, 2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-31085806

RESUMO

The brain is the most common metastatic site of lung adenocarcinoma; however, the mechanism of this selective metastasis remains unclear. We aimed to verify the hypothesis that exposure of tumor cells to the brain microenvironment leads to changes in their gene expression, which promotes their oriented transfer to the brain. A549 and H1299 lung adenocarcinoma cells were exposed to human astrocyte-conditioned medium to simulate the brain microenvironment. Microarray analysis was used to identify differentially expressed genes, which were confirmed by quantitative real-time PCR and western blotting. Knockdown experiments using microRNAs and the overexpression of genes by cell transfection were performed in addition to migration and invasion assays. In vitro findings were confirmed in clinical specimens using immunohistochemistry. We found and confirmed a significant increase in insulin-like growth factor binding protein-3 (IGFBP3) levels. Our results also showed that the up-regulation of IGFBP3 promoted A549 cell epithelial-mesenchymal transition, migration, and invasion, while the knockdown of IGFBP3 resulted in decreased cell motility. We also found that Transforming growth factor-ß (TGF-ß)/Mothers against decapentaplegic homolog 4 (Smad4)-induced epithelial-mesenchymal transition was likely IGFBP3-dependent in A549 cells. Finally, expression of IGFBP3 was significantly elevated in pulmonary cancer tissues and intracranial metastatic tissues. Our data indicate that up-regulation of IGFBP3 might mediate brain metastasis in lung adenocarcinoma, which makes it a potential therapeutic target.


Assuntos
Adenocarcinoma de Pulmão/metabolismo , Astrócitos/citologia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/secundário , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Neoplasias Pulmonares/metabolismo , Regulação para Cima , Células A549 , Adenocarcinoma de Pulmão/genética , Astrócitos/metabolismo , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Transição Epitelial-Mesenquimal , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos
12.
Mol Med Rep ; 19(6): 4597-4602, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31059032

RESUMO

Ubiquilin­1 (Ubqln), a ubiquitin­like protein, regulates degradation of misfolded proteins and has been reported to have a crucial role in multiple pathologic and physiologic conditions. The current study was undertaken to investigate the expression of Ubqln in the brain of a neonatal hypoxia­ischemic (HI) brain injury model induced using the Rice method with some modifications. Mouse pups at postnatal day 7 day were used in this study. Pups underwent permanent ligation of the left common carotid artery and a consecutive hypoxic challenge (8% O2 and 92% N2 for 120 min). The expression of Ubqln in the brain of pups following HI was analyzed by immunofluorescence staining and western blot analysis. Immunofluorescence staining demonstrated that Ubqln was extensively distributed in the cerebral cortex and hippocampus, and Ubqln was expressed in neurons, astrocytes and microglia in the brains of the HI brain injury model mice. Western blot analyses revealed decreased expression of Ubqln in the HI penumbra of the mouse model compared with Ubqln in the sham control group. The results of this study revealed that HI alters the expression of Ubqln, thus may provide a novel understanding of role of Ubqln in neonatal hypoxic ischemic encephalopathy.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/genética , Regulação para Baixo , Hipóxia-Isquemia Encefálica/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Animais Recém-Nascidos , Astrócitos/metabolismo , Western Blotting , Lesões Encefálicas/genética , Lesões Encefálicas/metabolismo , Córtex Cerebral/metabolismo , Modelos Animais de Doenças , Feminino , Hipocampo/metabolismo , Hipóxia-Isquemia Encefálica/metabolismo , Masculino , Camundongos , Microglia/metabolismo , Neurônios/metabolismo
13.
Int J Mol Sci ; 20(9)2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-31052177

RESUMO

Autism spectrum disorders (ASD) are a group of neurodevelopmental disorders characterized by deficits in social interaction and communication, and repetitive behaviors. In addition, co-morbidities such as gastro-intestinal problems have frequently been reported. Mutations and deletion of proteins of the SH3 and multiple ankyrin repeat domains (SHANK) gene-family were identified in patients with ASD, and Shank knock-out mouse models display autism-like phenotypes. SHANK3 proteins are not only expressed in the central nervous system (CNS). Here, we show expression in gastrointestinal (GI) epithelium and report a significantly different GI morphology in Shank3 knock-out (KO) mice. Further, we detected a significantly altered microbiota composition measured in feces of Shank3 KO mice that may contribute to inflammatory responses affecting brain development. In line with this, we found higher E. coli lipopolysaccharide levels in liver samples of Shank3 KO mice, and detected an increase in Interleukin-6 and activated astrocytes in Shank3 KO mice. We conclude that apart from its well-known role in the CNS, SHANK3 plays a specific role in the GI tract that may contribute to the ASD phenotype by extracerebral mechanisms.


Assuntos
Transtorno do Espectro Autista/microbiologia , Microbioma Gastrointestinal , Proteínas do Tecido Nervoso/genética , Animais , Astrócitos/metabolismo , Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/patologia , Interleucina-6/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Lipopolissacarídeos/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL
14.
Int J Mol Sci ; 20(9)2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31052389

RESUMO

Apolipoprotein E (apoE), a key lipid transport protein in the brain, is predominantly produced by astrocytes. Astrocytes are the most numerous cell type in the brain and are the main support network for neurons. They play a critical role in the synthesis and delivery of cholesterol in the brain. Humans have three common apoE isoforms, apoE2, apoE3 and apoE4, that show a strong genotype effect on the risk and age of onset for sporadic and late onset forms of Alzheimer's disease (AD). Carriers of an ε4 allele have an increased risk of developing AD, while those with an ε2 allele are protected. Investigations into the contribution of apoE to the development of AD has yielded conflicting results and there is still much speculation about the role of this protein in disease. Here, we review the opposing hypotheses currently described in the literature and the approaches that have been considered for targeting apoE as a novel therapeutic strategy for AD. Additionally, we provide our perspective on the rationale for targeting apoE and the challenges that arise with respect to "drug-ability" of this target.


Assuntos
Doença de Alzheimer/metabolismo , Apolipoproteínas E/metabolismo , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/genética , Animais , Apolipoproteínas E/química , Apolipoproteínas E/genética , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Humanos , Terapia de Alvo Molecular , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico
15.
Int J Mol Sci ; 20(10)2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31096580

RESUMO

Vascular cognitive impairment (VCI) is the second most common cause of cognitive deficit after Alzheimer's disease. Since VCI patients represent an important target population for prevention, an ongoing effort has been made to elucidate the pathogenesis of this disorder. In this review, we summarize the information from animal models on the molecular changes that occur in the brain during a cerebral vascular insult and ultimately lead to cognitive deficits in VCI. Animal models cannot effectively represent the complex clinical picture of VCI in humans. Nonetheless, they allow some understanding of the important molecular mechanisms leading to cognitive deficits. VCI may be caused by various mechanisms and metabolic pathways. The pathological mechanisms, in terms of cognitive deficits, may span from oxidative stress to vascular clearance of toxic waste products (such as amyloid beta) and from neuroinflammation to impaired function of microglia, astrocytes, pericytes, and endothelial cells. Impaired production of elements of the immune response, such as cytokines, and vascular factors, such as insulin-like growth factor 1 (IGF-1), may also affect cognitive functions. No single event could be seen as being the unique cause of cognitive deficits in VCI. These events are interconnected, and may produce cascade effects resulting in cognitive impairment.


Assuntos
Cognição , Disfunção Cognitiva/metabolismo , Disfunção Cognitiva/patologia , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Astrócitos/metabolismo , Encéfalo/metabolismo , Citocinas/metabolismo , Células Endoteliais/metabolismo , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Microglia/metabolismo , Modelos Animais , Óxido Nítrico , Estresse Oxidativo , Pericitos/metabolismo
16.
Artif Cells Nanomed Biotechnol ; 47(1): 1917-1923, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31079497

RESUMO

OBJECTIVE: To investigate the effect of Sulfiredoxin-1 (Srxn1) on astrocyte injury induced by hydrogen peroxide (H2O2). METHODS: Observing the changes of H2O2 on contents of lactate dehydrogenase (LDH), malondialdehyde (MDA), superoxide dismutase (SOD) and apoptosis after transfected Srxn1 siRNA into astrocytes. The protein expression of Notch 1, NICD and Hes1, the content of LDH and MDA, the activity of SOD and apoptosis rate of astrocytes after inhibiting or activation of Notch signalling pathway were detected by Western blot, ELISA and flow cytometry, respectively. RESULTS: Knockdown of Srxn1 could promote the secretion of LDH and MDA, decrease the activity of SOD and aggravate apoptosis of astrocytes induced by H2O2. The results of Western blot, ELISA assay and flow cytometry indicated that activation of the Notch signalling pathway attenuated the effect of Srxn1 on H2O2-induced oxidative damage and apoptosis of astrocytes. CONCLUSION: Srxn1 may protect astrocytes from oxidative stress injury induced by H2O2 by activation of Notch signalling pathway.


Assuntos
Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Astrócitos/metabolismo , Proliferação de Células/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , L-Lactato Desidrogenase/metabolismo , Malondialdeído/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/deficiência , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Ratos , Ratos Sprague-Dawley , Superóxido Dismutase/metabolismo
17.
BMC Res Notes ; 12(1): 225, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30987672

RESUMO

OBJECTIVE: Delivery of constructs for silencing or over-expressing genes or their modified versions is a crucial step for studying neuronal cell biology. Therefore, efficient transfection is important for the success of these experimental techniques especially in post-mitotic cells like neurons. In this study, we have assessed the transfection rate, using a previously established protocol, in both primary cortical cultures and neuroblastoma cell lines. Transfection efficiencies in these preparations have not been systematically determined before. RESULTS: Transfection efficiencies obtained herein were (10-12%) for neuroblastoma, (5-12%) for primary astrocytes and (1.3-6%) for primary neurons. We also report on cell-type specific transfection efficiency of neurons and astrocytes within primary cortical cultures when applying cell-type selective transfection protocols. Previous estimations described in primary cortical or hippocampal cultures were either based on general observations or on data derived from unspecified number of biological and/or technical replicates. Also to the best of our knowledge, transfection efficiency of pure primary neuronal cultures or astrocytes cultured in the context of pure or mixed (neurons/astrocytes) population cultures have not been previously determined. The transfection strategy used herein represents a convenient, and a straightforward tool for targeted cell transfection that can be utilized in a variety of in vitro applications.


Assuntos
Astrócitos/metabolismo , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Plasmídeos/metabolismo , Transfecção/métodos , Animais , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Biomarcadores/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Técnicas de Cocultura , Expressão Gênica , Genes Reporter , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Lipídeos/química , Lipídeos/farmacologia , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Especificidade de Órgãos , Plasmídeos/química , Cultura Primária de Células
18.
Nat Neurosci ; 22(5): 741-752, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30936556

RESUMO

Despite expanding knowledge regarding the role of astroglia in regulating neuronal function, little is known about regional or functional subgroups of brain astroglia and how they may interact with neurons. We use an astroglia-specific promoter fragment in transgenic mice to identify an anatomically defined subset of adult gray matter astroglia. Using transcriptomic and histological analyses, we generate a combinatorial profile for the in vivo identification and characterization of this astroglia subpopulation. These astroglia are enriched in mouse cortical layer V; express distinct molecular markers, including Norrin and leucine-rich repeat-containing G-protein-coupled receptor 6 (LGR6), with corresponding layer-specific neuronal ligands; are found in the human cortex; and modulate neuronal activity. Astrocytic Norrin appears to regulate dendrites and spines; its loss, as occurring in Norrie disease, contributes to cortical dendritic spine loss. These studies provide evidence that human and rodent astroglia subtypes are regionally and functionally distinct, can regulate local neuronal dendrite and synaptic spine development, and contribute to disease.


Assuntos
Astrócitos/metabolismo , Córtex Cerebral/metabolismo , Proteínas do Olho/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Animais , Células Cultivadas , Espinhas Dendríticas/fisiologia , Substância Cinzenta/metabolismo , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Córtex Motor/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Transcriptoma
19.
Int J Mol Sci ; 20(6)2019 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-30934555

RESUMO

Apolipoprotein E (apoE) is linked to the risk for Alzheimer's disease (AD) and thus has been suggested to be an important therapeutic target. In our drug screening effort, we identified Ondansetron (OS), an FDA-approved 5-HT3 antagonist, as an apoE-modulating drug. OS at low micromolar concentrations significantly increased apoE secretion from immortalized astrocytes and primary astrocytes derived from apoE3 and apoE4-targeted replacement mice without generating cellular toxicity. Other 5-HT3 antagonists also had similar effects as OS, though their effects were milder and required higher concentrations. Antagonists for other 5-HT receptors did not increase apoE secretion. OS also increased mRNA and protein levels of the ATB-binding cassette protein A1 (ABCA1), which is involved in lipidation and secretion of apoE. Accordingly, OS increased high molecular weight apoE. Moreover, the liver X receptor (LXR) and ABCA1 antagonists blocked the OS-induced increase of apoE secretion, indicating that the LXR-ABCA1 pathway is involved in the OS-mediated facilitation of apoE secretion from astrocytes. The effects of OS on apoE and ABCA1 were also observed in human astrocytes derived from induced pluripotent stem cells (iPSC) carrying the APOE ε3/ε3 and APOE ε4/ε4 genotypes. Oral administration of OS at clinically-relevant doses affected apoE levels in the liver, though the effects in the brain were not observed. Collectively, though further studies are needed to probe its effects in vivo, OS could be a potential therapeutic drug for AD by modulating poE metabolism through the LXR-ABCA1 pathway.


Assuntos
Transportador 1 de Cassete de Ligação de ATP/metabolismo , Apolipoproteínas E/metabolismo , Receptores X do Fígado/metabolismo , Ondansetron/farmacologia , Antagonistas do Receptor 5-HT3 de Serotonina/farmacologia , Animais , Apolipoproteína E3/genética , Apolipoproteína E4/genética , Astrócitos/metabolismo , Células Cultivadas , Humanos , Masculino , Camundongos Transgênicos
20.
Int J Mol Sci ; 20(6)2019 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-30934587

RESUMO

Alzheimer's disease (AD) is the most common neurodegenerative disorder affecting 5.4 million people in the United States. Currently approved pharmacologic interventions for AD are limited to symptomatic improvement, not affecting the underlying pathology. Therefore, the search for novel therapeutic strategies is ongoing. A hallmark of AD is the compromised blood-brain barrier (BBB); thus, developing drugs that target the BBB to enhance its integrity and function could be a novel approach to prevent and/or treat AD. Previous evidence has shown the beneficial effects of growth factors in the treatment of AD pathology. Based on reported positive results obtained with the product Endoret®, the objective of this study was to investigate the effect of plasma rich in growth factors (PRGF) on the BBB integrity and function, initially in a cell-based BBB model and in 5x Familial Alzheimer's Disease (5xFAD) mice. Our results showed that while PRGF demonstrated a positive effect in the cell-based BBB model with the enhanced integrity and function of the model, the in-vivo findings showed that PRGF exacerbated amyloid pathology in 5xFAD brains. At 10 and 100% doses, PRGF increased amyloid deposition associated with increased apoptosis and neuroinflammation. In conclusion, our results suggest PRGF may not provide beneficial effects against AD and the consideration to utilize growth factors should further be investigated.


Assuntos
Amiloide/metabolismo , Barreira Hematoencefálica/patologia , Peptídeos e Proteínas de Sinalização Intercelular/efeitos adversos , Plasma/química , Peptídeos beta-Amiloides/metabolismo , Animais , Astrócitos/metabolismo , Biomarcadores/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Proteína 4 Homóloga a Disks-Large/metabolismo , Mediadores da Inflamação/metabolismo , Radioisótopos do Iodo , Camundongos Transgênicos , Transporte Proteico , Fator A de Crescimento do Endotélio Vascular/metabolismo
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