Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 4.051
Filtrar
1.
Ecotoxicol Environ Saf ; 183: 109498, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31377521

RESUMO

2',2',4,4'-tetrabromo diphenyl ether (BDE-47), one of the most abundant congeners of commercial pentaBDE utilized as flame retardants, has been phased out of production due to its potential neural toxicity and endocrine disrupting activities, and yet still present in the environment. Several alternatives to BDE-47, including tetrabromobisphenol A (TBBPA), tetrabromobisphenol S (TBBPS), tetrachlorobisphenol A (TCBPA) and decabromodiphenyl ether (BDE-209), are presently employed without restrictions and their potential toxic effects on human neural development are still unclear. In this study, we utilized a human neural stem cell (hNSC)-based system to evaluate the potential developmental neurotoxic effects of the above-mentioned five chemicals, at environment and human exposure relevant concentrations. We found that those compounds slightly altered the expression of hNSC identity markers (SOX2, SOX3 and NES), without impairing cell viability or proliferation, in part by either modulating glycogen synthase kinase 3 beta (GSK3ß) signaling (TBBPS, TCBPA and BDE-47), and slightly disturbing the NOTCH pathway (TBBPA, TBBPS and TCBPA). Moreover, the five chemicals seemed to alter hNSC differentiation by perturbing triiodothyronine (T3) cellular signaling. Thus, our findings suggest that the five compounds, especially TBBPS, TCBPA, and BDE-47, may affect hNSC self-renewal and differentiation abilities and potentially elicit neural developmental toxicity.


Assuntos
Retardadores de Chama/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Glicogênio Sintase Quinase 3 beta/metabolismo , Células-Tronco Neurais/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Tri-Iodotironina/metabolismo , Humanos , Hidrocarbonetos Halogenados/toxicidade , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Neurogênese/efeitos dos fármacos , Neurogênese/genética
2.
Emerg Microbes Infect ; 8(1): 1003-1016, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31282298

RESUMO

Zika virus (ZIKV) is a mosquito-borne Flavivirus that causes Zika disease with particular neurological complications, including Guillain-Barré Syndrome and congenital microcephaly. Although ZIKV has been shown to directly infect human neural progenitor cells (hNPCs), thereby decreasing their viability and growth, it is as yet unknown which of the cellular pathways involved in the disruption of neurogenesis are affected following ZIKV infection. By comparing the effect of two ZIKV strains in vitro on hNPCs, the differentiation process of the latter cells was found to lead to a decreased susceptibility to infection and cell death induced by each of the ZIKV strains, which was associated with an earlier and stronger antiviral innate immune response in infected, differentiated hNPCs, as compared to undifferentiated cells. Moreover, ZIKV modulated, both in hNPCs and in vivo in fetal brain in an experimental mouse model, the expression of the Notch pathway which is involved in cellular proliferation, apoptosis and differentiation during neurogenesis. These results show that the differentiation state of hNPCs is a significant factor contributing to the outcome of ZIKV infection and furthermore suggest that ZIKV infection might initiate early activation of the Notch pathway resulting in an abnormal differentiation process, implicated in ZIKV-induced brain injury.


Assuntos
Células-Tronco Neurais/virologia , Neurogênese , Receptor Notch1/metabolismo , Infecção por Zika virus/virologia , Zika virus/fisiologia , Animais , Apoptose , Feminino , Humanos , Camundongos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Receptor Notch1/genética , Transdução de Sinais , Zika virus/genética , Infecção por Zika virus/genética , Infecção por Zika virus/metabolismo , Infecção por Zika virus/fisiopatologia
3.
Artif Cells Nanomed Biotechnol ; 47(1): 2492-2499, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31208217

RESUMO

Neonatal hypoxia-ischemia is a troublesome disease. Angelica polysaccharide (AP) is proved to have antioxidant effects. Our study was performed to confirm the effects of AP in hypoxia-exposed neural stem cells (NSCs). NSCs were pre-treated with AP and then stimulated with hypoxia. Viability of NSCs was examined by Cell Counting Kit-8 assay. Hypoxia-introduced apoptosis was observed by flow cytometry. Essential regulators of mTOR and Notch signalling pathways were examined by Western blot. mRNA expression was accessed using qRT-PCR. Bcl2/adenovirus EIB 19kD-interacting protein 3 (BNIP3) was altered by transfection. We noticed that NSCs were sensitive to hypoxia-induced apoptosis and showed decreased viability. Moreover, Beclin and light chain 3-II was upregulated while p62 was downregulated. However, AP reversed all these results. Similarly, hypoxia decreased the phosphorylation of mTOR and p70S6K and Notch1 expression while AP increased the phosphorylation of mTOR and p70S6K as well as the expression of Notch1. BNIP3 was upregulated by hypoxia while downregulated by AP. Further experiments demonstrated that overexpression of BNIP3 broken all the effects induced by AP shown in cell viability, apoptosis, autophagy and signalling pathways. Collectively, AP alleviated hypoxia-introduced NSCs damages by maintaining cell viability, blocking apoptosis and autophagy via downregulation of BNIP3 with the activation of mTOR and Notch signalling pathways.


Assuntos
Angelica/química , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo , Células-Tronco Neurais/efeitos dos fármacos , Polissacarídeos/farmacologia , Animais , Hipóxia Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Ratos , Receptores Notch/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Fatores de Tempo
4.
J Stroke Cerebrovasc Dis ; 28(9): 2555-2562, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31248739

RESUMO

OBJECTIVE: The present study aimed to investigate the effects of Mesenchymal stem cells/glial cell line derived neurotrophic factor (MSCs/GDNF) transplantation on nerve reconstruction in rats with intracerebral hemorrhage. METHODS: GDNF transduction to MSCs was using adenovirus vector pAdEasy-1-pAdTrack-CMV prepared. Intracerebral hemorrhage (ICH) was induced by injection of collagenase and heparin into the caudate putamen. At the third day after a collagenase-induced ICH, adult male SD rats were randomly divided into saline group, MSCs group and MSCs/GDNF group. Immunofluorescence and RT-PCR were performed to detect the differentiation of MSCs or MSCs with an adenovirus vector encoding GDNF gene in vivo and in vitro. RESULT: After 6 hours of induction, both MSCs and MSCs/GDNF expressed neuro or glial specific markers and synaptic-associated proteins (SYN, GAP-43, PSD-95); additionally, they secreted bioactive compounds (BDNF, NGF-ß). MSCs/GDNF transplantation, compared to MSCs and saline solution injection, significantly improved neurological functions after ICH. The grafted MSCs or MSCs/GDNF survived in the striatum after 2 weeks of transplantation and expressed the neural cell-specific biomarkers NSE, MAP2, and GFAP. CONCLUSION: These findings demonstrate that MSCs/GDNF transplantation contributes to improved neurological function in experimental ICH rats. The mechanisms are possibly due to neuronal replacement and enhanced neurotrophic factor secretion.


Assuntos
Encéfalo/fisiopatologia , Hemorragia Cerebral/cirurgia , Terapia Genética/métodos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/biossíntese , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/metabolismo , Regeneração Nervosa , Células-Tronco Neurais/transplante , Transfecção , Animais , Biomarcadores/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Células Cultivadas , Hemorragia Cerebral/genética , Hemorragia Cerebral/metabolismo , Hemorragia Cerebral/fisiopatologia , Modelos Animais de Doenças , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Masculino , Células-Tronco Neurais/metabolismo , Neurogênese , Fenótipo , Ratos Sprague-Dawley , Recuperação de Função Fisiológica , Fatores de Tempo
5.
Nat Commun ; 10(1): 2396, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31160561

RESUMO

Modern genetic studies indicate that human brain evolution is driven primarily by changes in gene regulation, which requires understanding the biological function of largely non-coding gene regulatory elements, many of which act in tissue specific manner. We leverage chromatin interaction profiles in human fetal and adult cortex to assign three classes of human-evolved elements to putative target genes. We find that human-evolved elements involving DNA sequence changes and those involving epigenetic changes are associated with human-specific gene regulation via effects on different classes of genes representing distinct biological pathways. However, both types of human-evolved elements converge on specific cell types and laminae involved in cerebral cortical expansion. Moreover, human evolved elements interact with neurodevelopmental disease risk genes, and genes with a high level of evolutionary constraint, highlighting a relationship between brain evolution and vulnerability to disorders affecting cognition and behavior. These results provide novel insights into gene regulatory mechanisms driving the evolution of human cognition and mechanisms of vulnerability to neuropsychiatric conditions.


Assuntos
Córtex Cerebral/embriologia , Epigênese Genética/genética , Evolução Molecular , Regulação da Expressão Gênica no Desenvolvimento/genética , Células-Tronco Neurais/metabolismo , Transtornos do Neurodesenvolvimento/genética , Encéfalo/embriologia , Encéfalo/metabolismo , Córtex Cerebral/metabolismo , Predisposição Genética para Doença , Humanos , Elementos Reguladores de Transcrição/genética
6.
Nat Commun ; 10(1): 2400, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31160565

RESUMO

BET-bromodomain inhibition (BETi) has shown pre-clinical promise for MYC-amplified medulloblastoma. However, the mechanisms for its action, and ultimately for resistance, have not been fully defined. Here, using a combination of expression profiling, genome-scale CRISPR/Cas9-mediated loss of function and ORF/cDNA driven rescue screens, and cell-based models of spontaneous resistance, we identify bHLH/homeobox transcription factors and cell-cycle regulators as key genes mediating BETi's response and resistance. Cells that acquire drug tolerance exhibit a more neuronally differentiated cell-state and expression of lineage-specific bHLH/homeobox transcription factors. However, they do not terminally differentiate, maintain expression of CCND2, and continue to cycle through S-phase. Moreover, CDK4/CDK6 inhibition delays acquisition of resistance. Therefore, our data provide insights about the mechanisms underlying BETi effects and the appearance of resistance and support the therapeutic use of combined cell-cycle inhibitors with BETi in MYC-amplified medulloblastoma.


Assuntos
Azepinas/farmacologia , Ciclo Celular/efeitos dos fármacos , Neoplasias Cerebelares/tratamento farmacológico , Meduloblastoma/tratamento farmacológico , Neurogênese/efeitos dos fármacos , Proteínas/antagonistas & inibidores , Triazóis/farmacologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/efeitos dos fármacos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sistemas CRISPR-Cas , Proteínas de Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Linhagem da Célula , Neoplasias Cerebelares/genética , Ciclina D2/efeitos dos fármacos , Ciclina D2/metabolismo , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos , Perfilação da Expressão Gênica , Humanos , Meduloblastoma/genética , Camundongos , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Fase S/efeitos dos fármacos
7.
BMC Complement Altern Med ; 19(1): 113, 2019 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-31159797

RESUMO

BACKGROUND: Embryonic neural stem cells (eNSCs) are immature precursors of the central nervous system (CNS), with self-renewal and multipotential differentiation capacities. These are regulated by endogenous and exogenous factors such as alpha-linolenic acid (ALA), a plant-based essential omega-3 polyunsaturated fatty acid. METHODS: In this study, we investigated the effects of various concentrations of Alyssum homolocarpum seed oil (AHSO), containing natural ALA, stearic acid (SA), myristic acid (MA), and ß-sitosterol, on proliferation and differentiation of eNSCs, in comparison to controls and to synthetic pure ALA. RESULTS: Treatment with natural AHSO (25 to 75 µM), similar to synthetic ALA, caused a significant ~ 2-fold increase in eNCSs viability, in comparison to controls. To confirm this proliferative activity, treatment of NSCs with 50 or 75 µM AHSO resulted in a significant increase in mRNA levels of notch1, hes-1 and Ki-67and NICD protein expression, in comparison to controls. Moreover, AHSO administration significantly increased the differentiation of eNSCs toward astrocytes (GFAP+) and oligodendrocytes (MBP+) in a dose dependent manner and was more potent than ALA, at similar concentrations, in comparison to controls. Indeed, only high concentrations of 100 µM AHSO, but not ALA, caused a significant increase in the frequency of neurons (ß-III Tubulin+). CONCLUSION: Our data demonstrated that AHSO, a rich source of ALA containing also other beneficial fatty acids, increased the proliferation and stimulated the differentiation of eNSCs. We suggest that AHSO's effects are caused by ß-sitosterol, SA and MA, present within this oil. AHSO could be used in diet to prevent neurodevelopmental syndromes, cognitive decline during aging, and various psychiatric disorders.


Assuntos
Brassicaceae/química , Células-Tronco Neurais/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Óleos Vegetais/farmacologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Antígeno Ki-67/metabolismo , Camundongos , Ácido Mirístico/análise , Células-Tronco Neurais/metabolismo , Óleos Vegetais/química , Sementes/química , Sitosteroides/análise , Ácidos Esteáricos/análise , Ácido alfa-Linoleico/análise
8.
Int J Mol Sci ; 20(11)2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31159418

RESUMO

Human tonsil-derived mesenchymal stem cells (T-MSCs) are newly identified MSCs and present typical features of MSCs, including having the differentiation capacity into the three germ layers and excellent proliferation capacity. They are easily sourced and are useful for stem cell therapy in various disease states. We previously reported that T-MSCs could be differentiated into skeletal myocytes and Schwann-like cells; therefore, they are a promising candidate for cell therapies for neuromuscular disease. Motor neurons (MNs), which regulate spontaneous behavior, are affected by a wide range of MN diseases (MNDs) for which there are no effective remedies. We investigated the differentiation potential of MN-like cells derived from T-MSCs (T-MSC-MNCs) for application to therapy of MNDs. After the process of MN differentiation, the expression of MN-related markers, including Islet 1, HB9/HLXB9 (HB9), and choline acetyltransferase (ChAT), was increased when compared with undifferentiated T-MSCs. The secretion of acetylcholine to the conditioned medium was significantly increased after MN differentiation. We cocultured T-MSC-MNCs and human skeletal muscle cells, and confirmed the presence of the acetylcholine receptor clusters, which demonstrated the formation of neuromuscular junctions. The potential functional improvements afforded by these T-MSC-MNCs could be useful in the treatment of MNDs caused by genetic mutation, viral infection, or environmental problems.


Assuntos
Diferenciação Celular , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Neurônios Motores/citologia , Neurônios Motores/fisiologia , Junção Neuromuscular/fisiologia , Tonsila Palatina/citologia , Acetilcolina/metabolismo , Biomarcadores , Células Cultivadas , Expressão Gênica , Humanos , Imuno-Histoquímica , Fibras Musculares Esqueléticas/metabolismo , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo
9.
Nat Commun ; 10(1): 2835, 2019 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-31249377

RESUMO

During embryogenesis cells make fate decisions within complex tissue environments. The levels and dynamics of transcription factor expression regulate these decisions. Here, we use single cell live imaging of an endogenous HES5 reporter and absolute protein quantification to gain a dynamic view of neurogenesis in the embryonic mammalian spinal cord. We report that dividing neural progenitors show both aperiodic and periodic HES5 protein fluctuations. Mathematical modelling suggests that in progenitor cells the HES5 oscillator operates close to its bifurcation boundary where stochastic conversions between dynamics are possible. HES5 expression becomes more frequently periodic as cells transition to differentiation which, coupled with an overall decline in HES5 expression, creates a transient period of oscillations with higher fold expression change. This increases the decoding capacity of HES5 oscillations and correlates with interneuron versus motor neuron cell fate. Thus, HES5 undergoes complex changes in gene expression dynamics as cells differentiate.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células-Tronco Neurais/metabolismo , Neurogênese , Proteínas Repressoras/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos/embriologia , Camundongos/metabolismo , Camundongos Endogâmicos ICR , Camundongos Knockout , Células-Tronco Neurais/química , Células-Tronco Neurais/citologia , Proteínas Repressoras/química , Proteínas Repressoras/genética , Análise de Célula Única
10.
BMC Genomics ; 20(1): 477, 2019 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-31185909

RESUMO

BACKGROUND: Global RNA sequencing technologies have revealed widespread RNA polymerase II (Pol II) transcription outside of gene promoters. Small 5'-capped RNA sequencing (Start-seq) originally developed for the detection of promoter-proximal Pol II pausing has helped improve annotation of Transcription Start Sites (TSSs) of genes as well as identification of non-genic regulatory elements. However, apart from the most well studied genomes of human and mouse, mammalian transcription has not been profiled with sufficiently high precision. RESULTS: We prepared and sequenced Start-seq libraries from rat (Rattus norgevicus) primary neural progenitor cells. Over 48 million uniquely mappable reads from two independent biological replicates allowed us to define the TSSs of 7365 known genes in the rn6 genome, reannotating 2503 TSSs by more than 5 base pairs, characterize promoter-associated antisense transcription, and profile Pol II pausing. By combining TSS data with polyA-selected RNA sequencing, we also identified thousands of potential new genes producing stable RNA as well as non-genic transcripts representing possible regulatory elements. CONCLUSIONS: Our study has produced the first Start-seq dataset for the rat. Apart from profiling transcription initiation, our data reaffirm the prevalence of Pol II pausing across the rat genome and indicate conservation of pausing mechanisms across metazoan genomes. We suggest that pausing location, at least in mammals, is constrained by a distance from initiation of transcription, whether it occurs at or outside of a gene promoter. Abundant antisense transcription initiation around protein coding genes indicates that Pol II recruited to the vicinity of a promoter is distributed to available start sites of transcription at either DNA strand. Transcriptome profiling of neural progenitors presented here will facilitate further studies of other rat cell types as well as other organisms.


Assuntos
Genômica , Células-Tronco Neurais/metabolismo , RNA Polimerase II/metabolismo , Iniciação da Transcrição Genética , Animais , Feminino , Gravidez , RNA Antissenso/genética , Ratos , Ratos Sprague-Dawley , Análise de Sequência de RNA , Sítio de Iniciação de Transcrição
11.
Genomics Proteomics Bioinformatics ; 17(2): 154-168, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-31154015

RESUMO

N6-methyladenosine (m6A), catalyzed by the methyltransferase complex consisting of Mettl3 and Mettl14, is the most abundant RNA modification in mRNAs and participates in diverse biological processes. However, the roles and precise mechanisms of m6A modification in regulating neuronal development and adult neurogenesis remain unclear. Here, we examined the function of Mettl3, the key component of the complex, in neuronal development and adult neurogenesis of mice. We found that the depletion of Mettl3 significantly reduced m6A levels in adult neural stem cells (aNSCs) and inhibited the proliferation of aNSCs. Mettl3 depletion not only inhibited neuronal development and skewed the differentiation of aNSCs more toward glial lineage, but also affected the morphological maturation of newborn neurons in the adult brain. m6A immunoprecipitation combined with deep sequencing (MeRIP-seq) revealed that m6A was predominantly enriched in transcripts related to neurogenesis and neuronal development. Mechanistically, m6A was present on the transcripts of histone methyltransferase Ezh2, and its reduction upon Mettl3 knockdown decreased both Ezh2 protein expression and consequent H3K27me3 levels. The defects of neurogenesis and neuronal development induced by Mettl3 depletion could be rescued by Ezh2 overexpression. Collectively, our results uncover a crosstalk between RNA and histone modifications and indicate that Mettl3-mediated m6A modification plays an important role in regulating neurogenesis and neuronal development through modulating Ezh2.


Assuntos
Adenosina/análogos & derivados , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Neurogênese , Neurônios/metabolismo , Adenosina/metabolismo , Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Animais , Encéfalo/metabolismo , Diferenciação Celular/genética , Proliferação de Células , Regulação da Expressão Gênica , Metiltransferases/metabolismo , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurogênese/genética , Neurônios/citologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
12.
Nat Med ; 25(5): 784-791, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31061540

RESUMO

Owing to recent medical and technological advances in neonatal care, infants born extremely premature have increased survival rates1,2. After birth, these infants are at high risk of hypoxic episodes because of lung immaturity, hypotension and lack of cerebral-flow regulation, and can develop a severe condition called encephalopathy of prematurity3. Over 80% of infants born before post-conception week 25 have moderate-to-severe long-term neurodevelopmental impairments4. The susceptible cell types in the cerebral cortex and the molecular mechanisms underlying associated gray-matter defects in premature infants remain unknown. Here we used human three-dimensional brain-region-specific organoids to study the effect of oxygen deprivation on corticogenesis. We identified specific defects in intermediate progenitors, a cortical cell type associated with the expansion of the human cerebral cortex, and showed that these are related to the unfolded protein response and changes. Moreover, we verified these findings in human primary cortical tissue and demonstrated that a small-molecule modulator of the unfolded protein response pathway can prevent the reduction in intermediate progenitors following hypoxia. We anticipate that this human cellular platform will be valuable for studying the environmental and genetic factors underlying injury in the developing human brain.


Assuntos
Lesões Encefálicas/etiologia , Hipóxia Encefálica/etiologia , Modelos Neurológicos , Lesões Encefálicas/metabolismo , Lesões Encefálicas/patologia , Hipóxia Celular/genética , Hipóxia Celular/fisiologia , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Humanos , Hipóxia Encefálica/metabolismo , Hipóxia Encefálica/patologia , Lactente Extremamente Prematuro , Recém-Nascido , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Neurogênese/genética , Neurogênese/fisiologia , Organoides/metabolismo , Organoides/patologia , Proteínas com Domínio T/metabolismo , Resposta a Proteínas não Dobradas
13.
Nat Med ; 25(6): 988-1000, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31086348

RESUMO

An aged circulatory environment can activate microglia, reduce neural precursor cell activity and impair cognition in mice. We hypothesized that brain endothelial cells (BECs) mediate at least some of these effects. We observe that BECs in the aged mouse hippocampus express an inflammatory transcriptional profile with focal upregulation of vascular cell adhesion molecule 1 (VCAM1), a protein that facilitates vascular-immune cell interactions. Concomitantly, levels of the shed, soluble form of VCAM1 are prominently increased in the plasma of aged humans and mice, and their plasma is sufficient to increase VCAM1 expression in cultured BECs and the hippocampi of young mice. Systemic administration of anti-VCAM1 antibody or genetic ablation of Vcam1 in BECs counteracts the detrimental effects of plasma from aged individuals on young brains and reverses aging aspects, including microglial reactivity and cognitive deficits, in the brains of aged mice. Together, these findings establish brain endothelial VCAM1 at the blood-brain barrier as a possible target to treat age-related neurodegeneration.


Assuntos
Envelhecimento/sangue , Encéfalo/metabolismo , Células-Tronco Neurais/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo , Adolescente , Adulto , Idoso , Envelhecimento/imunologia , Envelhecimento/metabolismo , Animais , Barreira Hematoencefálica/imunologia , Barreira Hematoencefálica/metabolismo , Encéfalo/citologia , Células Cultivadas , Células Endoteliais/metabolismo , Feminino , Deleção de Genes , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Microglia/metabolismo , Células-Tronco Neurais/citologia , Molécula 1 de Adesão de Célula Vascular/sangue , Molécula 1 de Adesão de Célula Vascular/genética , Adulto Jovem
14.
Cell Mol Life Sci ; 76(18): 3553-3570, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31101934

RESUMO

Neural stem cells present in the subventricular zone (SVZ), the largest neurogenic niche of the mammalian brain, are able to self-renew as well as generate neural progenitor cells (NPCs). NPCs are highly migratory and traverse the rostral migratory stream (RMS) to the olfactory bulb, where they terminally differentiate into mature interneurons. NPCs from the SVZ are some of the few cells in the CNS that migrate long distances during adulthood. The migratory process of NPCs is highly regulated by intracellular pathway activation and signaling from the surrounding microenvironment. It involves modulation of cell volume, cytoskeletal rearrangement, and isolation from compact extracellular matrix. In malignant brain tumors including high-grade gliomas, there are cells called brain tumor stem cells (BTSCs) with similar stem cell characteristics to NPCs but with uncontrolled cell proliferation and contribute to tumor initiation capacity, tumor progression, invasion, and tumor maintenance. These BTSCs are resistant to chemotherapy and radiotherapy, and their presence is believed to lead to tumor recurrence at distal sites from the original tumor location, principally due to their high migratory capacity. BTSCs are able to invade the brain parenchyma by utilizing many of the migratory mechanisms used by NPCs. However, they have an increased ability to infiltrate the tight brain parenchyma and utilize brain structures such as myelin tracts and blood vessels as migratory paths. In this article, we summarize recent findings on the mechanisms of cellular migration that overlap between NPCs and BTSCs. A better understanding of the intersection between NPCs and BTSCs will to provide a better comprehension of the BTSCs' invasive capacity and the molecular mechanisms that govern their migration and eventually lead to the development of new therapies to improve the prognosis of patients with malignant gliomas.


Assuntos
Neoplasias Encefálicas/patologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neurais/metabolismo , Neoplasias Encefálicas/metabolismo , Movimento Celular , Proteínas do Citoesqueleto/metabolismo , Humanos , Ventrículos Laterais/citologia , Ventrículos Laterais/metabolismo , Invasividade Neoplásica , Células-Tronco Neoplásicas/citologia , Células-Tronco Neurais/citologia , Neurogênese , Nicho de Células-Tronco
15.
Gene ; 707: 136-142, 2019 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-31054361

RESUMO

Neural stem/progenitor cells (NSPCs) can enhance regeneration after spinal cord injury (SCI), but survival of transplanted cells remains poor. Understanding how NSPCs respond to the chemical mediators of secondary injury thus is essential for treating SCI. Thymosin ß4 (Tß4) has physiological functions that are highly relevant to SCI. We exposed NSPCs to oxidative stress and found reduced expression of Tß4 in H2O2-injured NSPCs. Using an MTT assay, we found that Tß4 dose dependently increased viability of the injured NSPCs. Tß4 also reversed the decreases of intracellular Ca2+ concentration and increases of lactate dehydrogenase in NSPCs induced by H2O2 treatment. H2O2 exposure increased NSPC apoptosis, which Tß4 decreased. In H2O2-induced NSPCs, ROS production and pro-inflammatory cytokines increased, and again, Tß4 reversed these effects. We investigated the toll-like receptor 4 (TLR4) and myeloid differentiation primary response 88 (MyD88) signaling pathway as an underlying mechanism in Tß4's protective effect on H2O2-exposed NSPCs. Our results showed that Tß4 reduced expression of TLR4 and MyD88. Moreover, H2O2-exposed NSPCs that were treated with the TLR4/MyD88 pathway inhibitor showed a reversal of all the effects caused by H2O2, similar to Tß4's effects. In conclusion, our study determined that Tß4 attenuated H2O2-induced oxidative stress injury in NSPCs via the TLR4/MyD88 pathway.


Assuntos
Células-Tronco Neurais/citologia , Transdução de Sinais , Traumatismos da Medula Espinal/metabolismo , Timosina/metabolismo , Animais , Sobrevivência Celular , Células Cultivadas , Modelos Animais de Doenças , Regulação para Baixo , Peróxido de Hidrogênio/efeitos adversos , Masculino , Fator 88 de Diferenciação Mieloide/metabolismo , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Estresse Oxidativo , Ratos , Ratos Sprague-Dawley , Receptor 4 Toll-Like/metabolismo
16.
PLoS Genet ; 15(4): e1008097, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30973875

RESUMO

Maintenance of a healthy proteome is essential for cellular homeostasis and loss of proteostasis is associated with tissue dysfunction and neurodegenerative disease. The mechanisms that support proteostasis in healthy cells and how they become defective during aging or in disease states are not fully understood. Here, we investigate the transcriptional programs that are essential for neural stem and progenitor cell (NSPC) function and uncover a program of autophagy genes under the control of the transcription factor FOXO3. Using genomic approaches, we observe that FOXO3 directly binds a network of target genes in adult NSPCs that are involved in autophagy, and find that FOXO3 functionally regulates induction of autophagy in these cells. Interestingly, in the absence of FOXO activity, aggregates accumulate in NSPCs, and this effect is reversed by TOR (target of rapamycin) inhibition. Surprisingly, enhancing FOXO3 causes nucleation of protein aggregates, but does not increase their degradation. The work presented here identifies a genomic network under the direct control of a key transcriptional regulator of aging that is critical for maintaining a healthy mammalian stem cell pool to support lifelong neurogenesis.


Assuntos
Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Proteína Forkhead Box O3/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Animais , Autofagia/genética , Autofagia/fisiologia , Células Cultivadas , Proteína Forkhead Box O3/antagonistas & inibidores , Proteína Forkhead Box O3/genética , Técnicas de Inativação de Genes , Redes Reguladoras de Genes , Camundongos , Neurogênese/genética , Neurogênese/fisiologia , Agregados Proteicos/genética , Agregados Proteicos/fisiologia , Proteoma/genética , Proteoma/metabolismo , Proteostase/genética , Proteostase/fisiologia
17.
Nat Commun ; 10(1): 1465, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30931944

RESUMO

Embryonic stem cells (ESCs) can propagate in an undifferentiated state indefinitely in culture and retain the potential to differentiate into any somatic lineage as well as germ cells. The catabolic process autophagy has been reported to be involved in ESC identity regulation, but the underlying mechanism is still largely unknown. Here we show that EPG5, a eukaryotic-specific autophagy regulator which mediates autophagosome/lysosome fusion, is highly expressed in ESCs and contributes to ESC identity maintenance. We identify that the deubiquitinating enzyme USP8 binds to the Coiled-coil domain of EPG5. Mechanistically, USP8 directly removes non-classical K63-linked ubiquitin chains from EPG5 at Lysine 252, leading to enhanced interaction between EPG5 and LC3. We propose that deubiquitination of EPG5 by USP8 guards the autophagic flux in ESCs to maintain their stemness. This work uncovers a novel crosstalk pathway between ubiquitination and autophagy through USP8-EPG5 interaction to regulate the stemness of ESCs.


Assuntos
Autofagia/genética , Endopeptidases/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas/metabolismo , Ubiquitina Tiolesterase/genética , Animais , Autofagossomos/metabolismo , Células-Tronco Embrionárias/metabolismo , Fibroblastos/metabolismo , Células HEK293 , Humanos , Lisina/metabolismo , Lisossomos/metabolismo , Fusão de Membrana/genética , Camundongos , Células-Tronco Neurais/metabolismo , Ubiquitinação
18.
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
19.
Int J Mol Sci ; 20(8)2019 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-31018557

RESUMO

Sonic hedgehog (Shh) signaling is a key pathway within the central nervous system (CNS), during both development and adulthood, and its activation via the 7-transmembrane protein Smoothened (Smo) may promote neuroprotection and restoration during neurodegenerative disorders. Shh signaling may also be activated by selected glucocorticoids such as clobetasol, fluocinonide and fluticasone, which therefore act as Smo agonists and hold potential utility for regenerative medicine. However, despite its potential role in neurodegenerative diseases, the impact of Smo-modulation induced by these glucocorticoids on adult neural stem cells (NSCs) and the underlying signaling mechanisms are not yet fully elucidated. The aim of the present study was to evaluate the effects of Smo agonists (i.e., purmorphamine) and antagonists (i.e., cyclopamine) as well as of glucocorticoids (i.e., clobetasol, fluocinonide and fluticasone) on NSCs in terms of proliferation and clonal expansion. Purmorphamine treatment significantly increased NSC proliferation and clonal expansion via GLI-Kruppel family member 1 (Gli1) nuclear translocation and such effects were prevented by cyclopamine co-treatment. Clobetasol treatment exhibited an equivalent pharmacological effect. Moreover, cellular thermal shift assay suggested that clobetasol induces the canonical Smo-dependent activation of Shh signaling, as confirmed by Gli1 nuclear translocation and also by cyclopamine co-treatment, which abolished these effects. Finally, fluocinonide and fluticasone as well as control glucocorticoids (i.e., prednisone, corticosterone and dexamethasone) showed no significant effects on NSCs proliferation and clonal expansion. In conclusion, our data suggest that Shh may represent a druggable target system to drive neuroprotection and promote restorative therapies.


Assuntos
Proliferação de Células/efeitos dos fármacos , Clobetasol/farmacologia , Glucocorticoides/farmacologia , Proteínas Hedgehog/metabolismo , Células-Tronco Neurais/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Células-Tronco Adultas/citologia , Células-Tronco Adultas/efeitos dos fármacos , Células-Tronco Adultas/metabolismo , Animais , Células Cultivadas , Masculino , Camundongos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo
20.
Nat Biotechnol ; 37(4): 436-444, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30936566

RESUMO

Human brain organoids generated with current technologies recapitulate histological features of the human brain, but they lack a reproducible topographic organization. During development, spatial topography is determined by gradients of signaling molecules released from discrete signaling centers. We hypothesized that introduction of a signaling center into forebrain organoids would specify the positional identity of neural tissue in a distance-dependent manner. Here, we present a system to trigger a Sonic Hedgehog (SHH) protein gradient in developing forebrain organoids that enables ordered self-organization along dorso-ventral and antero-posterior positional axes. SHH-patterned forebrain organoids establish major forebrain subdivisions that are positioned with in vivo-like topography. Consistent with its behavior in vivo, SHH exhibits long-range signaling activity in organoids. Finally, we use SHH-patterned cerebral organoids as a tool to study the role of cholesterol metabolism in SHH signaling. Together, this work identifies inductive signaling as an effective organizing strategy to recapitulate in vivo-like topography in human brain organoids.


Assuntos
Proteínas Hedgehog/metabolismo , Organoides/crescimento & desenvolvimento , Organoides/metabolismo , Prosencéfalo/crescimento & desenvolvimento , Prosencéfalo/metabolismo , Animais , Biotecnologia , Padronização Corporal , Diferenciação Celular , Colesterol/metabolismo , Humanos , Camundongos , Modelos Neurológicos , Células-Tronco Neurais/metabolismo , Neurogênese , Organoides/citologia , Células-Tronco Pluripotentes/metabolismo , Prosencéfalo/citologia , Transdução de Sinais
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA