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1.
Nat Commun ; 12(1): 5282, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34489418

RESUMO

Homeostasis is one of the fundamental concepts in physiology. Despite remarkable progress in our molecular understanding of amino acid transport, metabolism and signaling, it remains unclear by what mechanisms cytosolic amino acid concentrations are maintained. We propose that amino acid transporters are the primary determinants of intracellular amino acid levels. We show that a cell's endowment with amino acid transporters can be deconvoluted experimentally and used this data to computationally simulate amino acid translocation across the plasma membrane. Transport simulation generates cytosolic amino acid concentrations that are close to those observed in vitro. Perturbations of the system are replicated in silico and can be applied to systems where only transcriptomic data are available. This work explains amino acid homeostasis at the systems-level, through a combination of secondary active transporters, functionally acting as loaders, harmonizers and controller transporters to generate a stable equilibrium of all amino acid concentrations.


Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos/metabolismo , Homeostase/genética , Modelos Estatísticos , Neuroglia/metabolismo , Células A549 , Sistemas de Transporte de Aminoácidos/química , Sistemas de Transporte de Aminoácidos/classificação , Sistemas de Transporte de Aminoácidos/genética , Animais , Transporte Biológico , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Simulação por Computador , Expressão Gênica , Humanos , Cinética , Metabolômica/métodos , Neuroglia/citologia , Oócitos/citologia , Oócitos/metabolismo , Xenopus laevis
2.
Nat Commun ; 12(1): 4730, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354063

RESUMO

Brain organoids derived from human pluripotent stem cells provide a highly valuable in vitro model to recapitulate human brain development and neurological diseases. However, the current systems for brain organoid culture require further improvement for the reliable production of high-quality organoids. Here, we demonstrate two engineering elements to improve human brain organoid culture, (1) a human brain extracellular matrix to provide brain-specific cues and (2) a microfluidic device with periodic flow to improve the survival and reduce the variability of organoids. A three-dimensional culture modified with brain extracellular matrix significantly enhanced neurogenesis in developing brain organoids from human induced pluripotent stem cells. Cortical layer development, volumetric augmentation, and electrophysiological function of human brain organoids were further improved in a reproducible manner by dynamic culture in microfluidic chamber devices. Our engineering concept of reconstituting brain-mimetic microenvironments facilitates the development of a reliable culture platform for brain organoids, enabling effective modeling and drug development for human brain diseases.


Assuntos
Encéfalo/crescimento & desenvolvimento , Encéfalo/fisiologia , Dispositivos Lab-On-A-Chip , Neurogênese/fisiologia , Organoides/crescimento & desenvolvimento , Organoides/fisiologia , Animais , Encéfalo/citologia , Meios de Cultura , Fenômenos Eletrofisiológicos , Matriz Extracelular/fisiologia , Estudos de Viabilidade , Perfilação da Expressão Gênica , Humanos , Hidrogéis , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Modelos Anatômicos , Modelos Neurológicos , Neurogênese/genética , Neuroglia/citologia , Neuroglia/fisiologia , Técnicas de Cultura de Órgãos/instrumentação , Técnicas de Cultura de Órgãos/métodos , Organoides/citologia , Suínos
3.
Methods Mol Biol ; 2352: 13-29, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34324177

RESUMO

Spontaneous neuronal replacement is almost absent in the postnatal mammalian nervous system. However, several studies have shown that both early postnatal and adult astroglia can be reprogrammed in vitro or in vivo by forced expression of proneural transcription factors, such as Neurogenin-2 or Achaete-scute homolog 1 (Ascl1), to acquire a neuronal fate. The reprogramming process stably induces properties such as distinctly neuronal morphology, expression of neuron-specific proteins, and the gain of mature neuronal functional features. Direct conversion of astroglia into neurons thus possesses potential as a basis for cell-based strategies against neurological diseases. In this chapter, we describe a well-established protocol used for direct reprogramming of postnatal cortical astrocytes into functional neurons in vitro and discuss available tools and approaches to dissect molecular and cell biological mechanisms underlying the reprogramming process.


Assuntos
Astrócitos/citologia , Astrócitos/metabolismo , Reprogramação Celular , Neurônios/citologia , Neurônios/metabolismo , Animais , Diferenciação Celular/genética , Separação Celular/métodos , Células Cultivadas , Reprogramação Celular/genética , Camundongos , Neocórtex/citologia , Neuroglia/citologia , Neuroglia/metabolismo , Cultura Primária de Células , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
4.
Methods Mol Biol ; 2352: 117-126, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34324183

RESUMO

Somatic cell nuclear transfer and in vitro induction of pluripotency in somatic cells by defined factors provided unambiguous evidence that the epigenetic state of terminally differentiated somatic cells is not static and can be reversed to a more primitive one. Inspired by these results, stem cell biologists have identified approaches to directly convert fibroblasts into induced neuronal (iN) cells, indicating that direct lineage conversions are possible between distantly related cell types. More recently, we took advantages of pro-neurogenic capacity of iN factors and developed methods to rapidly derive functionally mature neurons directly from human pluripotent stem cells (hPSCs) through a brief induction of defined transcription factors. In this chapter, we describe the detailed methods used to attain the direct conversion from hPSCs to glutamatergic and GABAergic iN cells.


Assuntos
Diferenciação Celular , Neurônios/citologia , Neurônios/metabolismo , Células-Tronco Pluripotentes/citologia , Linhagem Celular , Separação Celular , Fibroblastos/citologia , Fibroblastos/metabolismo , Imunofluorescência , Neurônios GABAérgicos/citologia , Neurônios GABAérgicos/metabolismo , Vetores Genéticos/administração & dosagem , Vetores Genéticos/biossíntese , Vetores Genéticos/genética , Humanos , Lentivirus/genética , Neurogênese , Neuroglia/citologia , Neuroglia/metabolismo , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição , Transdução Genética
5.
Int J Mol Sci ; 22(14)2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-34299185

RESUMO

Nervous system development involves proliferation and cell specification of progenitor cells into neurons and glial cells. Unveiling how this complex process is orchestrated under physiological conditions and deciphering the molecular and cellular changes leading to neurological diseases is mandatory. To date, great efforts have been aimed at identifying gene mutations associated with many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Mutations in the RNA/DNA binding protein Fused in Sarcoma/Translocated in Liposarcoma (FUS/TLS) have been associated with motor neuron degeneration in rodents and humans. Furthermore, increased levels of the wild-type protein can promote neuronal cell death. Despite the well-established causal link between FUS mutations and ALS, its role in neural cells remains elusive. In order to shed new light on FUS functions we studied its role in the control of neural stem progenitor cell (NSPC) properties. Here, we report that human wild-type Fused in Sarcoma (WT FUS), exogenously expressed in mouse embryonic spinal cord-derived NSPCs, was localized in the nucleus, caused cell cycle arrest in G1 phase by affecting cell cycle regulator expression, and strongly reduced neuronal differentiation. Furthermore, the expression of the human mutant form of FUS (P525L-FUS), associated with early-onset ALS, drives the cells preferentially towards a glial lineage, strongly reducing the number of developing neurons. These results provide insight into the involvement of FUS in NSPC proliferation and differentiation into neurons and glia.


Assuntos
Mutação , Células-Tronco Neurais/citologia , Neuroglia/citologia , Neurônios/patologia , Proteína FUS de Ligação a RNA/metabolismo , Medula Espinal/citologia , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Animais , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Camundongos , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Neuroglia/metabolismo , Neuroglia/patologia , Neurônios/metabolismo , Proteína FUS de Ligação a RNA/genética , Medula Espinal/embriologia , Medula Espinal/metabolismo , Medula Espinal/patologia
6.
PLoS Genet ; 17(6): e1009618, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34115759

RESUMO

Coordination of neurite extension with surrounding glia development is critical for neuronal function, but the underlying molecular mechanisms remain poorly understood. Through a genome-wide mutagenesis screen in C. elegans, we identified dyf-4 and daf-6 as two mutants sharing similar defects in dendrite extension. DAF-6 encodes a glia-specific patched-related membrane protein that plays vital roles in glial morphogenesis. We cloned dyf-4 and found that DYF-4 encodes a glia-secreted protein. Further investigations revealed that DYF-4 interacts with DAF-6 and functions in a same pathway as DAF-6 to regulate sensory compartment formation. Furthermore, we demonstrated that reported glial suppressors of daf-6 could also restore dendrite elongation and ciliogenesis in both dyf-4 and daf-6 mutants. Collectively, our data reveal that DYF-4 is a regulator for DAF-6 which promotes the proper formation of the glial channel and indirectly affects neurite extension and ciliogenesis.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Genoma Helmíntico , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas do Tecido Nervoso/genética , Neurogênese/genética , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Comunicação Celular , Cílios/genética , Cílios/metabolismo , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mutagênese , Proteínas do Tecido Nervoso/metabolismo , Neuritos/metabolismo , Neuroglia/citologia , Neuroglia/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
7.
Int J Mol Sci ; 22(11)2021 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-34073140

RESUMO

Transcription factors Satb1 and Satb2 are involved in the processes of cortex development and maturation of neurons. Alterations in the expression of their target genes can lead to neurodegenerative processes. Molecular and cellular mechanisms of regulation of neurotransmission by these transcription factors remain poorly understood. In this study, we have shown that transcription factors Satb1 and Satb2 participate in the regulation of genes encoding the NMDA-, AMPA-, and KA- receptor subunits and the inhibitory GABA(A) receptor. Deletion of gene for either Satb1 or Satb2 homologous factors induces the expression of genes encoding the NMDA receptor subunits, thereby leading to higher amplitudes of Ca2+-signals in neurons derived from the Satb1-deficient (Satb1fl/+ * NexCre/+) and Satb1-null mice (Satb1fl/fl * NexCre/+) in response to the selective agonist reducing the EC50 for the NMDA receptor. Simultaneously, there is an increase in the expression of the Gria2 gene, encoding the AMPA receptor subunit, thus decreasing the Ca2+-signals of neurons in response to the treatment with a selective agonist (5-Fluorowillardiine (FW)). The Satb1 deletion increases the sensitivity of the KA receptor to the agonist (domoic acid), in the cortical neurons of the Satb1-deficient mice but decreases it in the Satb1-null mice. At the same time, the Satb2 deletion decreases Ca2+-signals and the sensitivity of the KA receptor to the agonist in neurons from the Satb1-null and the Satb1-deficient mice. The Satb1 deletion affects the development of the inhibitory system of neurotransmission resulting in the suppression of the neuron maturation process and switching the GABAergic responses from excitatory to inhibitory, while the Satb2 deletion has a similar effect only in the Satb1-null mice. We show that the Satb1 and Satb2 transcription factors are involved in the regulation of the transmission of excitatory signals and inhibition of the neuronal network in the cortical cell culture.


Assuntos
Sinalização do Cálcio , Proteínas de Ligação à Região de Interação com a Matriz/fisiologia , Neuroglia , Receptores de Glutamato/metabolismo , Transmissão Sináptica , Fatores de Transcrição/fisiologia , Animais , Células Cultivadas , Feminino , Masculino , Camundongos , Camundongos Knockout , Neuroglia/citologia , Neuroglia/metabolismo , Receptores de GABA/metabolismo
8.
ACS Appl Mater Interfaces ; 13(20): 23423-23437, 2021 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-33978398

RESUMO

Transplanted glial-restricted progenitor (GRP) cells have potential to focally replace defunct astrocytes and produce remyelinating oligodendrocytes to avert neuronal death and dysfunction. However, most central nervous system cell therapeutic paradigms are hampered by high initial cell death and a host anti-graft immune response. We show here that composite hyaluronic acid-based hydrogels of tunable mechanical strengths can significantly improve transplanted GRP survival and differentiation. Allogeneic GRPs expressing green fluorescent protein and firefly luciferase were scaffolded in optimized hydrogel formulations and transplanted intracerebrally into immunocompetent BALB/c mice followed by serial in vivo bioluminescent imaging and chemical exchange saturation transfer magnetic resonance imaging (CEST MRI). We demonstrate that gelatin-sensitive CEST MRI can be exploited to monitor hydrogel scaffold degradation in vivo for ∼5 weeks post transplantation without necessitating exogenous labeling. Hydrogel scaffolding of GRPs resulted in a 4.5-fold increase in transplanted cell survival at day 32 post transplantation compared to naked cells. Histological analysis showed significant enhancement of cell proliferation as well as Olig2+ and GFAP+ cell differentiation for scaffolded cells compared to naked cells, with reduced host immunoreactivity. Hence, hydrogel scaffolding of transplanted GRPs in conjunction with serial in vivo imaging of cell survival and hydrogel degradation has potential for further advances in glial cell therapy.


Assuntos
Sobrevivência Celular/fisiologia , Hidrogéis/química , Neuroglia , Imagem Óptica/métodos , Células-Tronco , Animais , Diferenciação Celular/fisiologia , Rastreamento de Células , Ácido Hialurônico/química , Imageamento por Ressonância Magnética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neuroglia/citologia , Neuroglia/fisiologia , Neuroglia/transplante , Transplante de Células-Tronco , Células-Tronco/citologia , Células-Tronco/fisiologia
9.
Cell Prolif ; 54(6): e13042, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33955094

RESUMO

OBJECTIVES: The effects of general anaesthetics on fetal brain development remain elusive. Radial glial progenitors (RGPs) generate the majority of neurons in developing brains. Here, we evaluated the acute alterations in RGPs after maternal sevoflurane exposure. METHODS: Pregnant mice were exposed to 2.5% sevoflurane for 6 hours on gestational day 14.5. Interkinetic nuclear migration (INM) of RGPs in the ventricular zone (VZ) of the fetal brain was evaluated by thymidine analogues labelling. Cell fate of RGP progeny was determined by immunostaining using various neural markers. The Morris water maze (MWM) was used to assess the neurocognitive behaviours of the offspring. RNA sequencing (RNA-Seq) was performed for the potential mechanism, and the potential mechanism validated by quantitative real-time PCR (qPCR), Western blot and rescue experiments. Furthermore, INM was examined in human embryonic stem cell (hESC)-derived 3D cerebral organoids. RESULTS: Maternal sevoflurane exposure induced temporary abnormities in INM, and disturbed the cell cycle progression of RGPs in both rodents and cerebral organoids without cell fate alternation. RNA-Seq analysis, qPCR and Western blot showed that the Notch signalling pathway was a potential downstream target. Reactivation of Notch by Jag1 and NICD overexpression rescued the defects in INM. Young adult offspring showed no obvious cognitive impairments in MWM. CONCLUSIONS: Maternal sevoflurane exposure during neurogenic period temporarily induced abnormal INM of RGPs by targeting the Notch signalling pathway without inducing long-term effects on RGP progeny cell fate or offspring cognitive behaviours. More importantly, the defects of INM in hESC-derived cerebral organoids provide a novel insight into the effects of general anaesthesia on human brain development.


Assuntos
Anestésicos Inalatórios/efeitos adversos , Córtex Cerebral/efeitos dos fármacos , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Receptores Notch/metabolismo , Sevoflurano/efeitos adversos , Animais , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Córtex Cerebral/patologia , Feminino , Feto/efeitos dos fármacos , Feto/metabolismo , Feto/patologia , Humanos , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/patologia , Neurogênese/efeitos dos fármacos , Neuroglia/citologia , Neuroglia/efeitos dos fármacos , Neuroglia/patologia , Gravidez , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Efeitos Tardios da Exposição Pré-Natal/patologia , Transdução de Sinais/efeitos dos fármacos
10.
Int J Mol Sci ; 22(9)2021 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-33946667

RESUMO

Transplantation of various types of stem cells as a possible therapy for stroke has been tested for years, and the results are promising. Recent investigations have shown that the administration of the conditioned media obtained after stem cell cultivation can also be effective in the therapy of the central nervous system pathology (hypothesis of their paracrine action). The aim of this study was to evaluate the therapeutic effects of the conditioned medium of hiPSC-derived glial and neuronal progenitor cells in the rat middle cerebral artery occlusion model of the ischemic stroke. Secretory activity of the cultured neuronal and glial progenitor cells was evaluated by proteomic and immunosorbent-based approaches. Therapeutic effects were assessed by overall survival, neurologic deficit and infarct volume dynamics, as well as by the end-point values of the apoptosis- and inflammation-related gene expression levels, the extent of microglia/macrophage infiltration and the numbers of formed blood vessels in the affected area of the brain. As a result, 31% of the protein species discovered in glial progenitor cells-conditioned medium and 45% in neuronal progenitor cells-conditioned medium were cell type specific. The glial progenitor cell-conditioned media showed a higher content of neurotrophins (BDNF, GDNF, CNTF and NGF). We showed that intra-arterial administration of glial progenitor cells-conditioned medium promoted a faster decrease in neurological deficit compared to the control group, reduced microglia/macrophage infiltration, reduced expression of pro-apoptotic gene Bax and pro-inflammatory cytokine gene Tnf, increased expression of anti-inflammatory cytokine genes (Il4, Il10, Il13) and promoted the formation of blood vessels within the damaged area. None of these effects were exerted by the neuronal progenitor cell-conditioned media. The results indicate pronounced cytoprotective, anti-inflammatory and angiogenic properties of soluble factors secreted by glial progenitor cells.


Assuntos
Meios de Cultivo Condicionados/metabolismo , Meios de Cultivo Condicionados/farmacologia , AVC Isquêmico/terapia , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/terapia , Infusões Intra-Arteriais , AVC Isquêmico/metabolismo , AVC Isquêmico/patologia , Masculino , Neuroglia/citologia , Neuroglia/metabolismo , Ratos , Ratos Wistar
11.
Int J Mol Sci ; 22(7)2021 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-33805573

RESUMO

Human dental pulp stem cells (hDPSCs) are some of the most promising stem cell types for regenerative therapies given their ability to grow in the absence of serum and their realistic possibility to be used in autologous grafts. In this review, we describe the particular advantages of hDPSCs for neuroregenerative cell therapies. We thoroughly discuss the knowledge about their embryonic origin and characteristics of their postnatal niche, as well as the current status of cell culture protocols to maximize their multilineage differentiation potential, highlighting some common issues when assessing neuronal differentiation fates of hDPSCs. We also review the recent progress on neuroprotective and immunomodulatory capacity of hDPSCs and their secreted extracellular vesicles, as well as their combination with scaffold materials to improve their functional integration on the injured central nervous system (CNS) and peripheral nervous system (PNS). Finally, we offer some perspectives on the current and possible future applications of hDPSCs in neuroregenerative cell therapies.


Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Polpa Dentária/citologia , Regeneração Nervosa/fisiologia , Células-Tronco/citologia , Diferenciação Celular , Vesículas Extracelulares/fisiologia , Humanos , Neuroglia/citologia , Transplante de Células-Tronco , Células-Tronco/fisiologia , Engenharia Tecidual/métodos , Tecidos Suporte
12.
PLoS One ; 16(4): e0249954, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33852623

RESUMO

Cells have a regulatory mechanism known as heat shock (HS) response, which induces the expression of HS genes and proteins in response to heat and other cellular stresses. Exposure to moderate HS results in beneficial effects, such as thermotolerance and promotes survival, whereas excessive HS causes cell death. The effect of HS on cells depends on both exogenous factors, including the temperature and duration of heat application, and endogenous factors, such as the degree of cell differentiation. Neural stem cells (NSCs) can self-renew and differentiate into neurons and glial cells, but the changes in the HS response of symmetrically proliferating NSCs in culture are unclear. We evaluated the HS response of homogeneous proliferating NSCs derived from mouse embryonic stem cells during the proliferative phase and its effect on survival and cell death in vitro. The number of adherent cells and the expression ratios of HS protein (Hsp)40 and Hsp70 genes after exposure to HS for 20 min at temperatures above 43°C significantly increased with the extension of the culture period before exposure to HS. In contrast, caspase activity was significantly decreased by extension of the culture period before exposure to HS and suppressed the decrease in cell viability. These results suggest that the culture period before HS remarkably affects the HS response, influencing the expression of HS genes and cell survival of proliferating NSCs in culture.


Assuntos
Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Células-Tronco Neurais/metabolismo , Animais , Caspase 3/genética , Caspase 3/metabolismo , Diferenciação Celular , Proliferação de Células , Autorrenovação Celular , Sobrevivência Celular , Células Cultivadas , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP70/genética , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Neurais/citologia , Neuroglia/citologia , Neuroglia/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Temperatura
13.
Am J Physiol Regul Integr Comp Physiol ; 320(6): R994-R1003, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33826442

RESUMO

In vertebrates, the energy balance process is tightly controlled by complex neural circuits that sense metabolic signals and adjust food intake and energy expenditure in line with the physiological requirements of optimal conditions. Within neural networks controlling energy balance, tanycytes are peculiar ependymoglial cells that are nowadays recognized as multifunctional players in the metabolic hypothalamus. However, the physiological function of hypothalamic tanycytes remains unclear, creating a number of ambiguities in the field. Here, we review data accumulated over the years that demonstrate the physiological function of tanycytes in the maintenance of metabolic homeostasis, opening up new research avenues. The presumed involvement of tanycytes in the pathophysiology of metabolic disorders and age-related neurodegenerative diseases will be finally discussed.


Assuntos
Metabolismo Energético/fisiologia , Células Ependimogliais/metabolismo , Hipotálamo/metabolismo , Neuroglia/citologia , Neurônios/citologia , Animais , Homeostase/fisiologia , Humanos
14.
Int J Mol Sci ; 22(9)2021 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-33919317

RESUMO

Induced pluripotent stem cells (iPSCs) are a self-renewable pool of cells derived from an organism's somatic cells. These can then be programmed to other cell types, including neurons. Use of iPSCs in research has been two-fold as they have been used for human disease modelling as well as for the possibility to generate new therapies. Particularly in complex human diseases, such as neurodegenerative diseases, iPSCs can give advantages over traditional animal models in that they more accurately represent the human genome. Additionally, patient-derived cells can be modified using gene editing technology and further transplanted to the brain. Glial cells have recently become important avenues of research in the field of neurodegenerative diseases, for example, in Alzheimer's disease and Parkinson's disease. This review focuses on using glial cells (astrocytes, microglia, and oligodendrocytes) derived from human iPSCs in order to give a better understanding of how these cells contribute to neurodegenerative disease pathology. Using glia iPSCs in in vitro cell culture, cerebral organoids, and intracranial transplantation may give us future insight into both more accurate models and disease-modifying therapies.


Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Doenças Neurodegenerativas/terapia , Neuroglia/citologia , Neurônios/citologia , Animais , Humanos , Doenças Neurodegenerativas/patologia
15.
Proc Natl Acad Sci U S A ; 118(10)2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-33649223

RESUMO

In the mammalian neocortex, projection neuron types are sequentially generated by the same pool of neural progenitors. How neuron type specification is related to developmental timing remains unclear. To determine whether temporal gene expression in neural progenitors correlates with neuron type specification, we performed single-cell RNA sequencing (scRNA-Seq) analysis of the developing mouse neocortex. We uncovered neuroepithelial cell enriched genes such as Hmga2 and Ccnd1 when compared to radial glial cells (RGCs). RGCs display dynamic gene expression over time; for instance, early RGCs express higher levels of Hes5, and late RGCs show higher expression of Pou3f2 Interestingly, intermediate progenitor cell marker gene Eomes coexpresses temporally with known neuronal identity genes at different developmental stages, though mostly in postmitotic cells. Our results delineate neural progenitor cell diversity in the developing mouse neocortex and support that neuronal identity genes are transcriptionally evident in Eomes-positive cells.


Assuntos
Diferenciação Celular , Neocórtex/embriologia , Células-Tronco Neurais/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteína HMGA2/metabolismo , Camundongos , Neocórtex/citologia , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/citologia , Neuroglia/citologia , Neuroglia/metabolismo , Fatores do Domínio POU/metabolismo , Proteínas Repressoras/metabolismo , Proteínas com Domínio T/metabolismo
16.
Nat Rev Gastroenterol Hepatol ; 18(8): 571-587, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33731961

RESUMO

One of the most transformative developments in neurogastroenterology is the realization that many functions normally attributed to enteric neurons involve interactions with enteric glial cells: a large population of peripheral neuroglia associated with enteric neurons throughout the gastrointestinal tract. The notion that glial cells function solely as passive support cells has been refuted by compelling evidence that demonstrates that enteric glia are important homeostatic cells of the intestine. Active signalling mechanisms between enteric glia and neurons modulate gastrointestinal reflexes and, in certain circumstances, function to drive neuroinflammatory processes that lead to long-term dysfunction. Bidirectional communication between enteric glia and immune cells contributes to gastrointestinal immune homeostasis, and crosstalk between enteric glia and cancer stem cells regulates tumorigenesis. These neuromodulatory and immunomodulatory roles place enteric glia in a unique position to regulate diverse gastrointestinal disease processes. In this Review, we discuss current concepts regarding enteric glial development, heterogeneity and functional roles in gastrointestinal pathophysiology and pathophysiology, with a focus on interactions with neurons and immune cells. We also present a working model to differentiate glial states based on normal function and disease-induced dysfunctions.


Assuntos
Sistema Nervoso Entérico/fisiologia , Gastroenteropatias/fisiopatologia , Neuroglia/fisiologia , Animais , Sistema Nervoso Entérico/citologia , Gastroenteropatias/tratamento farmacológico , Motilidade Gastrointestinal/fisiologia , Homeostase , Humanos , Neuroglia/citologia , Transdução de Sinais
17.
Methods Mol Biol ; 2269: 233-244, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33687683

RESUMO

We describe the protocol for the efficient in vitro differentiation of human neural stem cells (NSCs) from human-induced pluripotent stem cells (iPS cells). NSCs differentiate via neural epithelial progenitors enabling the analysis of early neuronal development. They represent neural progenitor cells, which are capable of differentiating into neurons and glia.


Assuntos
Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Neurais/metabolismo , Neurogênese , Neuroglia/metabolismo , Neurônios/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Neurais/citologia , Neuroglia/citologia , Neurônios/citologia
18.
Nat Rev Neurosci ; 22(4): 237-255, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33712727

RESUMO

The brain is arguably the most complex organ. The branched and extended morphology of nerve cells, their subcellular complexity, the multiplicity of brain cell types as well as their intricate connectivity and the scattering properties of brain tissue present formidable challenges to the understanding of brain function. Neuroscientists have often been at the forefront of technological and methodological developments to overcome these hurdles to visualize, quantify and modify cell and network properties. Over the last few decades, the development of advanced imaging methods has revolutionized our approach to explore the brain. Super-resolution microscopy and tissue imaging approaches have recently exploded. These instrumentation-based innovations have occurred in parallel with the development of new molecular approaches to label protein targets, to evolve new biosensors and to target them to appropriate cell types or subcellular compartments. We review the latest developments for labelling and functionalizing proteins with small localization and functionalized reporters. We present how these molecular tools are combined with the development of a wide variety of imaging methods that break either the diffraction barrier or the tissue penetration depth limits. We put these developments in perspective to emphasize how they will enable step changes in our understanding of the brain.


Assuntos
Encéfalo/citologia , Microscopia/métodos , Neuroglia/citologia , Neurônios/citologia , Coloração e Rotulagem/métodos , Animais , Humanos
19.
J Vis Exp ; (168)2021 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-33645578

RESUMO

Neuronal axon initial segments (AIS) are sites of initiation of action potentials and have been extensively studied for their molecular structure, assembly and activity-dependent plasticity. Giant ankyrin-G, the master organizer of AIS, directly associates with membrane-spanning voltage gated sodium (VSVG) and potassium channels (KCNQ2/3), as well as 186 kDa neurofascin, a L1CAM cell adhesion molecule. Giant ankyrin-G also binds to and recruits cytoplasmic AIS molecules including beta-4-spectrin, and the microtubule-binding proteins, EB1/EB3 and Ndel1. Giant ankyrin-G is sufficient to rescue AIS formation in ankyrin-G deficient neurons. Ankyrin-G also includes a smaller 190 kDa isoform located at dendritic spines instead of the AIS, which is incapable of targeting to the AIS or rescuing the AIS in ankyrin-G-deficient neurons. Here, we described a protocol using cultured hippocampal neurons from ANK3-E22/23-flox mice, which, when transfected with Cre-BFP exhibit loss of all isoform of ankyrin-G and impair the formation of AIS. Combined a modified Banker glia/neuron co-culture system, we developed a method to transfect ankyrin-G null neurons with a 480 kDa ankyrin-G-GFP plasmid, which is sufficient to rescue the formation of AIS. We further employ a quantification method, developed by Salzer and colleagues to deal with variation in AIS distance from the neuronal cell bodies that occurs in hippocampal neuron cultures. This protocol allows quantitative studies of the de novo assembly and dynamic behavior of AIS.


Assuntos
Segmento Inicial do Axônio/metabolismo , Hipocampo/citologia , Neurônios/citologia , Animais , Anquirinas/metabolismo , Células Cultivadas , Edição de Genes , Hipocampo/metabolismo , Integrases/metabolismo , Camundongos , Neuroglia/citologia , Neurônios/metabolismo
20.
Int J Mol Sci ; 22(3)2021 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-33525421

RESUMO

The considerable post-traumatic brain recovery in fishes makes them a useful model for studying the mechanisms that provide reparative neurogenesis, which is poorly represented in mammals. After a mechanical injury to the telencephalon in adult fish, lost neurons are actively replaced due to the proliferative activity of neuroepithelial cells and radial glia in the neurogenic periventricular zone. However, it is not enough clear which signaling mechanisms are involved in the activation of adult neural stem cells (aNSC) after the injury (reactive proliferation) and in the production of new neurons (regenerative neurogenesis) from progenitor cells (NPC). In juvenile Pacific salmon, the predominant type of NSCs in the telencephalon are neuroepithelial cells corresponding to embryonic NSCs. Expression of glutamine synthetase (GS), a NSC molecular marker, was detected in the neuroepithelial cells of the pallium and subpallium of juvenile chum salmon, Oncorhynchus keta. At 3 days after a traumatic brain injury (TBI) in juvenile chum salmon, the GS expression was detected in the radial glia corresponding to aNSC in the pallium and subpallium. The maximum density of distribution of GS+ radial glia was found in the dorsal pallial region. Hydrogen sulfide (H2S) is a proneurogenic factor that reduces oxidative stress and excitotoxicity effects, along with the increased GS production in the brain cells of juvenile chum salmon. In the fish brain, H2S producing by cystathionine ß-synthase in neurogenic zones may be involved in maintaining the microenvironment that provides optimal conditions for the functioning of neurogenic niches during constitutive neurogenesis. After injury, H2S can determine cell survivability, providing a neuroprotective effect in the area of injury and reducing the process of glutamate excitotoxicity, acting as a signaling molecule involved in changing the neurogenic environment, which leads to the reactivation of neurogenic niches and cell regeneration programs. The results of studies on the control of the expression of regulatory Sonic Hedgehog genes (Shh) and the transcription factors Paired Box2 (Pax2) regulated by them are still insufficient. A comparative analysis of Pax2 expression in the telencephalon of intact chum salmon showed the presence of constitutive patterns of Pax2 expression in neurogenic areas and non-neurogenic parenchymal zones of the pallium and subpallium. After mechanical injury, the patterns of Pax2 expression changed, and the amount of Pax2+ decreased (p < 0.05) in lateral (Dl), medial (Dm) zones of the pallium, and the lateral zone (Vl) of the subpallium compared to the control. We believe that the decrease in the expression of Pax2 may be caused by the inhibitory effect of the Pax6 transcription factor, whose expression in the juvenile salmon brain increases upon injury.


Assuntos
Lesões Encefálicas/genética , Regeneração do Cérebro/genética , Cistationina beta-Sintase/genética , Proteínas de Peixes/genética , Glutamato-Amônia Ligase/genética , Fator de Transcrição PAX2/genética , Telencéfalo/metabolismo , Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Animais , Lesões Encefálicas/metabolismo , Lesões Encefálicas/patologia , Diferenciação Celular , Proliferação de Células , Cistationina beta-Sintase/metabolismo , Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica , Glutamato-Amônia Ligase/metabolismo , Ácido Glutâmico/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Sulfeto de Hidrogênio/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Células Neuroepiteliais/citologia , Células Neuroepiteliais/metabolismo , Neurogênese/genética , Neuroglia/citologia , Neuroglia/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Oncorhynchus keta , Fator de Transcrição PAX2/metabolismo , Fator de Transcrição PAX6/genética , Fator de Transcrição PAX6/metabolismo , Telencéfalo/lesões , Telencéfalo/patologia
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