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1.
Int J Mol Sci ; 22(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34445509

RESUMO

Ischemia-like conditions reflect almost the entire spectrum of events that occur during cerebral ischemia, including the induction of oxidative stress, Ca2+ overload, glutamate excitotoxicity, and activation of necrosis and apoptosis in brain cells. Mechanisms for the protective effects of the antioxidant enzyme peroxiredoxin-6 (Prx-6) on hippocampal cells during oxygen-glucose deprivation/reoxygenation (OGD/R) were investigated. Using the methods of fluorescence microscopy, inhibitory analysis, vitality tests and PCR, it was shown that 24-h incubation of mixed hippocampal cell cultures with Prx-6 does not affect the generation of a reversible phase of a OGD-induced rise in Ca2+ ions in cytosol ([Ca2+]i), but inhibits a global increase in [Ca2+]i in astrocytes completely and in neurons by 70%. In addition, after 40 min of OGD, cell necrosis is suppressed, especially in the astrocyte population. This effect is associated with the complex action of Prx-6 on neuroglial networks. As an antioxidant, Prx-6 has a more pronounced and astrocyte-directed effect, compared to the exogenous antioxidant vitamin E (Vit E). Prx-6 inhibits ROS production in mitochondria by increasing the antioxidant capacity of cells and altering the expression of genes encoding redox status proteins. Due to the close bond between [Ca2+]i and intracellular ROS, this effect of Prx-6 is one of its protective mechanisms. Moreover, Prx-6 effectively suppresses not only necrosis, but also apoptosis during OGD and reoxygenation. Incubation with Prx-6 leads to activation of the basic expression of genes encoding protective kinases-PI3K, CaMKII, PKC, anti-apoptotic proteins-Stat3 and Bcl-2, while inhibiting the expression of signaling kinases and factors involved in apoptosis activation-Ikk, Src, NF-κb, Caspase-3, p53, Fas, etc. This effect on the basic expression of the genome leads to the cell preconditions, which is expressed in the inhibition of caspase-3 during OGD/reoxygenation. A significant effect of Prx-6 is directed on suppression of the level of pro-inflammatory cytokine IL-1ß and factor TNFα, as well as genes encoding NMDA- and kainate receptor subunits, which was established for the first time for this antioxidant enzyme. The protective effect of Prx-6 is due to its antioxidant properties, since mutant Prx-6 (mutPrx-6, Prx6-C47S) leads to polar opposite effects, contributing to oxidative stress, activation of apoptosis and cell death through receptor action on TLR4.


Assuntos
Astrócitos/citologia , Hipocampo/citologia , Peroxirredoxina VI/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Cálcio/metabolismo , Células Cultivadas , Citosol/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Interleucina-1beta/metabolismo , Microscopia de Fluorescência , Peroxirredoxina VI/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
2.
Int J Mol Sci ; 22(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34445514

RESUMO

Oxaliplatin, a well-known chemotherapeutic agent, can induce severe neuropathic pain, which can seriously decrease the quality of life of patients. JI017 is an herb mixture composed of Aconitum carmichaelii, Angelica gigas, and Zingiber officinale. Its anti-tumor effect has been reported; however, the efficacy of JI017 against oxaliplatin-induced allodynia has never been explored. Single oxaliplatin injection [6 mg/kg, intraperitoneal, (i.p.)] induced both cold and mechanical allodynia, and oral administration of JI017 (500 mg/kg) alleviated cold but not mechanical allodynia in mice. Real-time polymerase chain reaction (PCR) analysis demonstrated that the upregulation of mRNA of spinal transient receptor potential vanilloid 1 (TRPV1) and astrocytes following oxaliplatin injection was downregulated after JI017 treatment. Moreover, TRPV1 expression and the activation of astrocytes were intensely increased in the superficial area of the spinal dorsal horn after oxaliplatin treatment, whereas JI017 suppressed both. The administration of TRPV1 antagonist [capsazepine, intrathecal (i.t.), 10 µg] attenuated the activation of astrocytes in the dorsal horn, demonstrating that the functions of spinal TRPV1 and astrocytes are closely related in oxaliplatin-induced neuropathic pain. Altogether, these results suggest that JI017 may be a potent candidate for the management of oxaliplatin-induced neuropathy as it decreases pain, spinal TRPV1, and astrocyte activation.


Assuntos
Astrócitos/metabolismo , Hiperalgesia/tratamento farmacológico , Oxaliplatina/efeitos adversos , Compostos Fitoquímicos/administração & dosagem , Canais de Cátion TRPV/metabolismo , Aconitum/química , Administração Oral , Angelica/química , Animais , Astrócitos/efeitos dos fármacos , Temperatura Baixa , Modelos Animais de Doenças , Regulação para Baixo , Gengibre/química , Hiperalgesia/induzido quimicamente , Hiperalgesia/genética , Hiperalgesia/metabolismo , Camundongos , Compostos Fitoquímicos/farmacologia , Coluna Vertebral/metabolismo , Canais de Cátion TRPV/genética
3.
Int J Mol Sci ; 22(16)2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34445395

RESUMO

Glutathione (GSH) is the most abundant intrinsic antioxidant in the central nervous system, and its substrate cysteine readily becomes the oxidized dimeric cystine. Since neurons lack a cystine transport system, neuronal GSH synthesis depends on cystine uptake via the cystine/glutamate exchange transporter (xCT), GSH synthesis, and release in/from surrounding astrocytes. Transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), a detoxifying master transcription factor, is expressed mainly in astrocytes and activates the gene expression of various phase II drug-metabolizing enzymes or antioxidants including GSH-related molecules and metallothionein by binding to the antioxidant response element (ARE) of these genes. Accumulating evidence has shown the involvement of dysfunction of antioxidative molecules including GSH and its related molecules in the pathogenesis of Parkinson's disease (PD) or parkinsonian models. Furthermore, we found several agents targeting GSH synthesis in the astrocytes that protect nigrostriatal dopaminergic neuronal loss in PD models. In this article, the neuroprotective effects of supplementation and enhancement of GSH and its related molecules in PD pathology are reviewed, along with introducing new experimental findings, especially targeting of the xCT-GSH synthetic system and Nrf2-ARE pathway in astrocytes.


Assuntos
Sistema y+ de Transporte de Aminoácidos/metabolismo , Glutationa/metabolismo , Transtornos Parkinsonianos/metabolismo , Transdução de Sinais , Animais , Astrócitos/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica , Humanos , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo
4.
EBioMedicine ; 70: 103512, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34333238

RESUMO

BACKGROUND: Neurologic manifestations are well-recognized features of coronavirus disease 2019 (COVID-19). However, the longitudinal association of biomarkers reflecting CNS impact and neurological symptoms is not known. We sought to determine whether plasma biomarkers of CNS injury were associated with neurologic sequelae after COVID-19. METHODS: Patients with confirmed acute COVID-19 were studied prospectively. Neurological symptoms were recorded during the acute phase of the disease and at six months follow-up, and blood samples were collected longitudinally. Healthy age-matched individuals were included as controls. We analysed plasma concentrations of neurofilament light-chain (NfL), glial fibrillary acidic protein (GFAp), and growth differentiation factor 15 (GDF-15). FINDINGS: One hundred patients with mild (n = 24), moderate (n = 28), and severe (n = 48) COVID-19 were followed for a median (IQR) of 225 (187-262) days. In the acute phase, patients with severe COVID-19 had higher concentrations of NfL than all other groups (all p < 0·001), and higher GFAp than controls (p < 0·001). GFAp was also significantly increased in moderate disease (p < 0·05) compared with controls. NfL (r = 0·53, p < 0·001) and GFAp (r = 0·39, p < 0·001) correlated with GDF-15 during the acute phase. After six months, NfL and GFAp concentrations had normalized, with no persisting group differences. Despite this, 50 patients reported persistent neurological symptoms, most commonly fatigue (n = 40), "brain-fog" (n = 29), and changes in cognition (n = 25). We found no correlation between persistent neurological symptoms and CNS injury biomarkers in the acute phase. INTERPRETATION: The normalization of CNS injury biomarkers in all individuals, regardless of previous disease severity or persisting neurological symptoms, indicates that post COVID-19 neurological sequelae are not accompanied by ongoing CNS injury. FUNDING: The Swedish State Support for Clinical Research, SciLifeLab Sweden, and the Knut and Alice Wallenberg Foundation have provided funding for this project.


Assuntos
Astrócitos/patologia , Astrócitos/virologia , COVID-19/patologia , COVID-19/virologia , SARS-CoV-2/patogenicidade , Idoso , Astrócitos/metabolismo , Biomarcadores/sangue , Biomarcadores/metabolismo , COVID-19/sangue , COVID-19/metabolismo , Progressão da Doença , Feminino , Seguimentos , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Proteínas de Neurofilamentos/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Neurônios/virologia , Suécia
5.
Nat Biomed Eng ; 5(8): 847-863, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34385693

RESUMO

The therapeutic efficacy of stem cells transplanted into an ischaemic brain depends primarily on the responses of the neurovascular unit. Here, we report the development and applicability of a functional neurovascular unit on a microfluidic chip as a microphysiological model of ischaemic stroke that recapitulates the function of the blood-brain barrier as well as interactions between therapeutic stem cells and host cells (human brain microvascular endothelial cells, pericytes, astrocytes, microglia and neurons). We used the model to track the infiltration of a number of candidate stem cells and to characterize the expression levels of genes associated with post-stroke pathologies. We observed that each type of stem cell showed unique neurorestorative effects, primarily by supporting endogenous recovery rather than through direct cell replacement, and that the recovery of synaptic activities is correlated with the recovery of the structural and functional integrity of the neurovascular unit rather than with the regeneration of neurons.


Assuntos
AVC Isquêmico/terapia , Dispositivos Lab-On-A-Chip , Transplante de Células-Tronco , Astrócitos/citologia , Astrócitos/metabolismo , Barreira Hematoencefálica/química , Barreira Hematoencefálica/metabolismo , Técnicas de Cocultura , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Humanos , Microglia/citologia , Microglia/metabolismo , Microvasos/citologia , Modelos Biológicos , Neurônios/citologia , Neurônios/metabolismo , Pericitos/citologia , Pericitos/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo
6.
Int J Mol Sci ; 22(15)2021 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-34360699

RESUMO

Reactive astrocytes are a hallmark of neurodegenerative disease including multiple sclerosis. It is widely accepted that astrocytes may adopt alternative phenotypes depending on a combination of environmental cues and intrinsic features in a highly plastic and heterogeneous manner. However, we still lack a full understanding of signals and associated signaling pathways driving astrocyte reaction and of the mechanisms by which they drive disease. We have previously shown in the experimental autoimmune encephalomyelitis mouse model that deficiency of the molecular adaptor Rai reduces disease severity and demyelination. Moreover, using primary mouse astrocytes, we showed that Rai contributes to the generation of a pro-inflammatory central nervous system (CNS) microenvironment through the production of nitric oxide and IL-6 and by impairing CD39 activity in response to soluble factors released by encephalitogenic T cells. Here, we investigated the impact of Rai expression on astrocyte function both under basal conditions and in response to IL-17 treatment using a proteomic approach. We found that astrocytes and astrocyte-derived extracellular vesicles contain a set of proteins, to which Rai contributes, that are involved in the regulation of oligodendrocyte differentiation and myelination, nitrogen metabolism, and oxidative stress. The HIF-1α pathway and cellular energetic metabolism were the most statistically relevant molecular pathways and were related to ENOA and HSP70 dysregulation.


Assuntos
Astrócitos/metabolismo , Encefalomielite Autoimune Experimental/metabolismo , Vesículas Extracelulares/metabolismo , Interleucina-17/farmacologia , Neuroproteção , Oligodendroglia/fisiologia , Proteína 3 de Transformação que Contém Domínio 2 de Homologia de Src/genética , Animais , Diferenciação Celular , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/fisiopatologia , Regulação da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Bainha de Mielina , Proteômica , Proteína 3 de Transformação que Contém Domínio 2 de Homologia de Src/metabolismo
7.
Int J Mol Sci ; 22(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34360942

RESUMO

The exact mechanism underlying selective dopaminergic neurodegeneration is not completely understood. The complex interplay among toxic alpha-synuclein aggregates, oxidative stress, altered intracellular Ca2+-homeostasis, mitochondrial dysfunction and disruption of mitochondrial integrity is considered among the pathogenic mechanisms leading to dopaminergic neuronal loss. We herein investigated the molecular mechanisms leading to mitochondrial dysfunction and its relationship with activation of the neuroinflammatory process occurring in Parkinson's disease. To address these issues, experiments were performed in vitro and in vivo in mice carrying the human mutation of α-synuclein A53T under the prion murine promoter. In these models, the expression and activity of NCX isoforms, a family of important transporters regulating ionic homeostasis in mammalian cells working in a bidirectional way, were evaluated in neurons and glial cells. Mitochondrial function was monitored with confocal microscopy and fluorescent dyes to measure mitochondrial calcium content and mitochondrial membrane potential. Parallel experiments were performed in 4 and 16-month-old A53T-α-synuclein Tg mice to correlate the functional data obtained in vitro with mitochondrial dysfunction and neuroinflammation through biochemical analysis. The results obtained demonstrated: 1. in A53T mice mitochondrial dysfunction occurs early in midbrain and later in striatum; 2. mitochondrial dysfunction occurring in the midbrain is mediated by the impairment of NCX3 protein expression in neurons and astrocytes; 3. mitochondrial dysfunction occurring early in midbrain triggers neuroinflammation later into the striatum, thus contributing to PD progression during mice aging.


Assuntos
Mesencéfalo/metabolismo , Mitocôndrias/metabolismo , Doença de Parkinson/metabolismo , Trocador de Sódio e Cálcio/metabolismo , alfa-Sinucleína/genética , Animais , Astrócitos/metabolismo , Cálcio/metabolismo , Células Cultivadas , Neurônios Dopaminérgicos/metabolismo , Mesencéfalo/citologia , Camundongos , Camundongos Endogâmicos C57BL , Mutação de Sentido Incorreto , Doença de Parkinson/genética , Trocador de Sódio e Cálcio/genética , alfa-Sinucleína/metabolismo
8.
Int J Mol Sci ; 22(15)2021 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-34360719

RESUMO

Agonists of the Gi protein-coupled A3 adenosine receptor (A3AR) have shown important pain-relieving properties in preclinical settings of several pain models. Active as a monotherapy against chronic pain, A3AR agonists can also be used in combination with classic opioid analgesics. Their safe pharmacological profile, as shown by clinical trials for other pathologies, i.e., rheumatoid arthritis, psoriasis and fatty liver diseases, confers a realistic translational potential, thus encouraging research studies on the molecular mechanisms underpinning their antinociceptive actions. A number of pathways, involving central and peripheral mechanisms, have been proposed. Recent evidence showed that the prototypical A3AR agonist Cl-IB-MECA and the new, highly selective, A3AR agonist MRS5980 inhibit neuronal (N-type) voltage-dependent Ca2+ currents in dorsal root ganglia, a known pain-related mechanism. Other proposed pathways involve reduced cytokine production, immune cell-mediated responses, as well as reduced microglia and astrocyte activation in the spinal cord. The aim of this review is to summarize up-to-date information on A3AR in the context of pain, including cellular and molecular mechanisms underlying this effect. Based on their safety profile shown in clinical trials for other pathologies, A3AR agonists are proposed as novel, promising non-narcotic agents for pain control.


Assuntos
Agonistas do Receptor A3 de Adenosina/uso terapêutico , Sinalização do Cálcio/efeitos dos fármacos , Gânglios Espinais , Dor , Receptor A3 de Adenosina/metabolismo , Animais , Astrócitos/metabolismo , Gânglios Espinais/metabolismo , Gânglios Espinais/fisiopatologia , Humanos , Microglia/metabolismo , Dor/tratamento farmacológico , Dor/metabolismo , Dor/fisiopatologia
9.
Int J Mol Sci ; 22(15)2021 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-34360652

RESUMO

Recent studies implicate astrocytes in Alzheimer's disease (AD); however, their role in pathogenesis is poorly understood. Astrocytes have well-established functions in supportive functions such as extracellular ionic homeostasis, structural support, and neurovascular coupling. However, emerging research on astrocytic function in the healthy brain also indicates their role in regulating synaptic plasticity and neuronal excitability via the release of neuroactive substances named gliotransmitters. Here, we review how this "active" role of astrocytes at synapses could contribute to synaptic and neuronal network dysfunction and cognitive impairment in AD.


Assuntos
Doença de Alzheimer/patologia , Astrócitos/patologia , Sinalização do Cálcio , Comunicação Celular , Neurônios/patologia , Doença de Alzheimer/metabolismo , Animais , Astrócitos/metabolismo , Humanos , Plasticidade Neuronal , Neurônios/metabolismo
10.
Methods Mol Biol ; 2352: 31-43, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34324178

RESUMO

Astrocytes play an important role in maintaining brain homeostasis and their dysfunction is involved in a number of neurological disorders. An accessible source of astrocytes is essential to model neurological diseases and potential cell therapy approaches. Cell reprogramming techniques offer possibilities to reprogram terminally differentiated cells into other cell types. By overexpressing the three astrocytic transcription factors NFIA, NFIB, and SOX9, we showed that it is possible to directly transdifferentiate fibroblasts into functional astrocytes. These induced astrocytes (iAstrocytes) express glial fibrillary acidic protein (GFAP) and S100 calcium binding protein B (S100B), as well as other astrocytic markers. Moreover, electrophysiological properties indicate that iAstrocytes are functionally comparable to native brain astrocytes. Here we describe an optimized protocol to generate iAstrocytes starting from skin fibroblasts and this approach can be adapted for a wide range of somatic cell types.


Assuntos
Astrócitos/citologia , Astrócitos/metabolismo , Transdiferenciação Celular/genética , Reprogramação Celular/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Fatores de Transcrição/genética , Animais , Cálcio , Linhagem Celular , Células Cultivadas , Expressão Gênica , Vetores Genéticos/biossíntese , Vetores Genéticos/genética , Humanos , Lentivirus/genética , Camundongos , Imagem Molecular , Fatores de Transcrição NFI/genética , Fatores de Transcrição SOX9/genética , Fatores de Transcrição/metabolismo
11.
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
12.
Methods Mol Biol ; 2352: 45-55, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34324179

RESUMO

Astrocytes play important roles in neurodevelopment and diseases. Previous studies described ways to derive astrocytes from somatic cells by going through iPSC or iNSC/iNPC intermediates. Here we describe a method to directly convert mouse fibroblasts into functional astrocytes using small molecules without transgenes or viral transduction. The direct chemical reprogramming method described in this study provides a more rapid way to derive astrocytes from fibroblasts.


Assuntos
Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Técnicas de Reprogramação Celular , Reprogramação Celular/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Animais , Astrócitos/metabolismo , Biomarcadores , Diferenciação Celular/genética , Separação Celular , Células Cultivadas , Reprogramação Celular/genética , Fibroblastos/metabolismo , Expressão Gênica , Humanos , Imuno-Histoquímica , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Fatores de Transcrição/genética , Transgenes
13.
Methods Mol Biol ; 2352: 57-71, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34324180

RESUMO

Direct neuronal reprogramming is a promising strategy to generate various types of neurons that are, otherwise, inaccessible for researchers. However, the efficiency of neuronal conversion is highly dependent on the transcription factor used, the identity of the initial cells to convert, their species' background, and the neuronal subtype to which cells will convert. Regardless of these conditioning factors, the apoptotic regulator Bcl-2 acts as a pan-neuronal reprogramming enhancer. Bcl-2 mediates its effect in reprogramming by preventing an overshot of oxidative stress during the acquisition of a neuronal oxidative metabolism, thus reducing cell death by ferroptosis and facilitating the phenotypic conversion. In this chapter, we outline two methods to obtain either mouse or human neurons derived from postnatal astrocytes and skin fibroblasts, respectively. The overall reprogramming strategy is based on the co-expression of Bcl-2 and the transcription factor Neurog2 that produces mostly excitatory neurons. However, the method can be easily adapted to achieve alternative neuronal subtypes by using additional transcription factors, such as Isl1 for motor neurons. Therefore, our approaches provide solid but flexible platforms to obtain human and mouse induced neurons in vitro that can be applied to basic or translational research.


Assuntos
Astrócitos/citologia , Astrócitos/metabolismo , Técnicas de Reprogramação Celular , Reprogramação Celular/genética , Fibroblastos/citologia , Neurônios/citologia , Neurônios/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Animais , Astrócitos/efeitos dos fármacos , Técnicas de Cultura de Células , Linhagem Celular , Células Cultivadas , Reprogramação Celular/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Expressão Gênica , Vetores Genéticos/genética , Humanos , Camundongos , Neurônios/efeitos dos fármacos , Retroviridae/genética , Transdução Genética , Transfecção
14.
Methods Mol Biol ; 2352: 133-148, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34324185

RESUMO

Astrocytes are essential cells for normal brain functionality and have recently emerged as key players in many neurological diseases. However, the limited availability of human primary astrocytes for cell culture studies hinders our understanding of their physiology and precise role in disease development and progression. Here, we describe a detailed step-by-step protocol to rapidly and efficiently generate functionally mature induced astrocytes (iAs) from human embryonic and induced pluripotent stem cells (hES/iPSCs). Astrocyte induction is accomplished by ectopic lentiviral expression of two gliogenic transcription factors, Sox9 and Nfib. iAs exhibit morphology features as well as gene and protein expression similar to human mature astrocytes and display important astrocytic functions, such as glutamate uptake, propagation of calcium waves, expression of various cytokines after stimulation, and support of synapse formation and function, making them suitable models for studying the role of astrocytes in health and disease. Moreover, we describe a procedure for cryopreservation of iAs for long-term storage or shipping. Finally, we provide the required information needed to set up cocultures with human induced neurons (iNs, also described in this book), generated from hES/iPSCs, to generate cocultures, allowing studies on astrocyte-neuron interactions and providing new insights in astrocyte-associated disease mechanisms.


Assuntos
Astrócitos/citologia , Astrócitos/metabolismo , Reprogramação Celular/genética , Neurônios/citologia , Neurônios/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição/genética , Técnicas de Cultura de Células , Células Cultivadas , Técnicas de Reprogramação Celular , Técnicas de Cocultura , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Humanos , Imuno-Histoquímica
15.
Neuron ; 109(16): 2545-2555.e7, 2021 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-34245686

RESUMO

Astrocytes play an essential role in the development of neural circuits by positioning transporters and receptors near synapses and secreting factors that promote synaptic maturation. However, the mechanisms that coordinate astrocyte and neural maturation remain poorly understood. Using in vivo imaging in unanesthetized neonatal mice, we show that bursts of neuronal activity passing through nascent sound processing networks reliably induce calcium transients in astrocytes. Astrocyte transients were dependent on intense neuronal activity and constrained to regions near active synapses, ensuring close spatial and temporal coordination of neuron and astrocyte activity. Astrocyte responses were restricted to the pre-hearing period and induced by synergistic activation of two metabotropic glutamate receptors, mGluR5 and mGluR3, which promoted IP3R2-dependent calcium release from intracellular stores. The widespread expression of these receptors by astrocytes during development and the prominence of neuronal burst firing in emerging neural networks may help coordinate the maturation of excitatory synapses.


Assuntos
Astrócitos/metabolismo , Cálcio/metabolismo , Neurônios/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Animais , Sinalização do Cálcio/fisiologia , Camundongos , Sinapses/fisiologia
16.
Int J Mol Sci ; 22(13)2021 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-34206710

RESUMO

Astrocytes are a specific type of neuroglial cells that confer metabolic and structural support to neurons. Astrocytes populate all regions of the nervous system and adopt a variety of phenotypes depending on their location and their respective functions, which are also pleiotropic in nature. For example, astrocytes adapt to pathological conditions with a specific cellular response known as reactive astrogliosis, which includes extensive phenotypic and transcriptional changes. Reactive astrocytes may lose some of their homeostatic functions and gain protective or detrimental properties with great impact on damage propagation. Different astrocyte subpopulations seemingly coexist in reactive astrogliosis, however, the source of such heterogeneity is not completely understood. Altered cellular signaling in pathological compared to healthy conditions might be one source fueling astrocyte heterogeneity. Moreover, diversity might also be encoded cell-autonomously, for example as a result of astrocyte subtype specification during development. We hypothesize and propose here that elucidating the epigenetic signature underlying the phenotype of each astrocyte subtype is of high relevance to understand another regulative layer of astrocyte heterogeneity, in general as well as after injury or as a result of other pathological conditions. High resolution methods should allow enlightening diverse cell states and subtypes of astrocyte, their adaptation to pathological conditions and ultimately allow controlling and manipulating astrocyte functions in disease states. Here, we review novel literature reporting on astrocyte diversity from a developmental perspective and we focus on epigenetic signatures that might account for cell type specification.


Assuntos
Astrócitos/metabolismo , Epigênese Genética , Gliose/genética , Animais , Astrócitos/citologia , Astrócitos/patologia , Diferenciação Celular , Gliose/metabolismo , Humanos
17.
Int J Mol Sci ; 22(13)2021 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-34209958

RESUMO

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease whose pathophysiology is largely unknown. Despite the fact that motor neuron (MN) death is recognized as the key event in ALS, astrocytes dysfunctionalities and neuroinflammation were demonstrated to accompany and probably even drive MN loss. Nevertheless, the mechanisms priming astrocyte failure and hyperactivation are still obscure. In this work, altered pathways and molecules in ALS astrocytes were unveiled by investigating the proteomic profile and the secreted metabolome of primary spinal cord astrocytes derived from transgenic ALS mouse model overexpressing the human (h)SOD1(G93A) protein in comparison with the transgenic counterpart expressing hSOD1(WT) protein. Here we show that ALS primary astrocytes are depleted of proteins-and of secreted metabolites-involved in glutathione metabolism and signaling. The observed increased activation of Nf-kB, Ebf1, and Plag1 transcription factors may account for the augmented expression of proteins involved in the proteolytic routes mediated by proteasome or endosome-lysosome systems. Moreover, hSOD1(G93A) primary astrocytes also display altered lipid metabolism. Our results provide novel insights into the altered molecular pathways that may underlie astrocyte dysfunctionalities and altered astrocyte-MN crosstalk in ALS, representing potential therapeutic targets to abrogate or slow down MN demise in disease pathogenesis.


Assuntos
Esclerose Amiotrófica Lateral/metabolismo , Astrócitos/citologia , Metabolômica/métodos , Proteômica/métodos , Superóxido Dismutase/genética , Esclerose Amiotrófica Lateral/genética , Animais , Astrócitos/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Feminino , Glutationa/metabolismo , Humanos , Metabolismo dos Lipídeos , Masculino , Camundongos , Cultura Primária de Células , Mapas de Interação de Proteínas , Transdução de Sinais , Medula Espinal/citologia , Medula Espinal/metabolismo
18.
Int J Mol Sci ; 22(12)2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34204581

RESUMO

Parkinson's disease (PD) is considered the most common disorder of synucleinopathy, which is characterised by intracellular inclusions of aggregated and misfolded α-synuclein (α-syn) protein in various brain regions, and the loss of dopaminergic neurons. During the early prodromal phase of PD, synaptic alterations happen before cell death, which is linked to the synaptic accumulation of toxic α-syn specifically in the presynaptic terminals, affecting neurotransmitter release. The oligomers and protofibrils of α-syn are the most toxic species, and their overexpression impairs the distribution and activation of synaptic proteins, such as the SNARE complex, preventing neurotransmitter exocytosis and neuronal synaptic communication. In the last few years, the role of the immune system in PD has been increasingly considered. Microglial and astrocyte activation, the gene expression of proinflammatory factors, and the infiltration of immune cells from the periphery to the central nervous system (CNS) represent the main features of the inflammatory response. One of the actors of these processes is α-syn accumulation. In light of this, here, we provide a systematic review of PD-related α-syn and inflammation inter-players.


Assuntos
Suscetibilidade a Doenças , Doença de Parkinson/metabolismo , Sinapses/metabolismo , alfa-Sinucleína/metabolismo , Imunidade Adaptativa , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Biomarcadores , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Humanos , Imunidade Inata , Microglia/imunologia , Microglia/metabolismo , Microglia/patologia , Doença de Parkinson/etiologia , Doença de Parkinson/patologia , Sinapses/imunologia , alfa-Sinucleína/genética
19.
Croat Med J ; 62(3): 250-263, 2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-34212562

RESUMO

AIM: To investigate the cyclic guanosine monophosphate (cGMP)/guanylate cyclase C (GC-C)-independent signaling pathway in astrocytes, which are a suitable model due to their lack of GC-C expression. METHODS: Patch clamp was performed and intracellular Ca2+ concentrations and pH were measured in primary astrocyte cultures and brain slices of wild type (WT) and GC-C knockout (KO) mice. The function of GC-C-independent signaling pathway in the cerebellum was determined by behavior tests in uroguanylin (UGN) KO and GC-C KO mice. RESULTS: We showed for the first time that UGN changed intracellular Ca2+ levels in different brain regions of the mouse. In addition to the midbrain and hypothalamus, GC-C was expressed in the cerebral and cerebellar cortex. The presence of two signaling pathways in the cerebellum (UGN hyperpolarized Purkinje cells via GC-C and increased intracellular Ca2+ concentration in astrocytes) led to a different motoric function in GC-C KO and UGN KO mice, probably via different regulation of intracellular pH in astrocytes. CONCLUSION: The UGN effects on astrocytes via a Ca2+-dependent signaling pathway could be involved in the modulation of neuronal activity.


Assuntos
Astrócitos , Cálcio , Animais , Astrócitos/metabolismo , Guanilato Ciclase/metabolismo , Camundongos , Peptídeos Natriuréticos , Receptores de Peptídeos , Transdução de Sinais
20.
Int J Mol Sci ; 22(12)2021 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-34207355

RESUMO

Astrocytes (also, astroglia) consume huge amounts of glucose and produce lactate regardless of sufficient oxygen availability, indicating a high capacity for aerobic glycolysis. Glycolysis in astrocytes is activated in accordance with neuronal excitation and leads to increases in the release of lactate from astrocytes. Although the fate of this lactate remains somewhat controversial, it is believed to fuel neurons as an energy substrate. Besides providing lactate, astrocytic glycolysis plays an important role in neuroprotection. Among the minor pathways of glucose metabolism, glucose flux to the pentose-phosphate pathway (PPP), a major shunt pathway of glycolysis, is attracting research interest. In fact, PPP activity in astrocytes is five to seven times higher than that in neurons. The astrocytic PPP plays a key role in protecting neurons against oxidative stress by providing neurons with a reduced form of glutathione, which is necessary to eliminate reactive oxygen species. Therefore, enhancing astrocytic glycolysis might promote neuronal protection during acute ischemic stroke. Contrariwise, the dysfunction of astrocytic glycolysis and the PPP have been implicated in the pathogenesis of various neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis, since mitochondrial dysfunction and oxidative stress trigger and accelerate disease progression.


Assuntos
Astrócitos/metabolismo , Glicólise , Doenças Neurodegenerativas/metabolismo , Acidente Vascular Cerebral/metabolismo , Animais , Humanos , Fator 2 Relacionado a NF-E2/metabolismo , Doenças Neurodegenerativas/genética , Via de Pentose Fosfato , Acidente Vascular Cerebral/genética
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