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Glia ; 68(7): 1396-1409, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32003513


Astrogliosis comprises a variety of changes in astrocytes that occur in a context-specific manner, triggered by temporally diverse signaling events that vary with the nature and severity of brain insults. However, most mechanisms underlying astrogliosis were described using animals, which fail to reproduce some aspects of human astroglial signaling. Here, we report an in vitro model to study astrogliosis using human-induced pluripotent stem cells (iPSC)-derived astrocytes which replicate temporally intertwined aspects of reactive astrocytes in vivo. We analyzed the time course of astrogliosis by measuring nuclear translocation of NF-kB, production of cytokines, changes in morphology and function of iPSC-derived astrocytes exposed to TNF-α. We observed NF-kB p65 subunit nuclear translocation and increased gene expression of IL-1ß, IL-6, and TNF-α in the first hours following TNF-α stimulation. After 24 hr, conditioned media from iPSC-derived astrocytes exposed to TNF-α exhibited increased secretion of inflammation-related cytokines. After 5 days, TNF-α-stimulated cells presented a typical phenotype of astrogliosis such as increased immunolabeling of Vimentin and GFAP and nuclei with elongated shape and shrinkage. Moreover, ~50% decrease in aspartate uptake was observed during the time course of astrogliosis with no evident cell damage, suggesting astroglial dysfunction. Together, our results indicate that human iPSC-derived astrocytes reproduce canonical events associated with astrogliosis in a time dependent fashion. The approach described here may contribute to a better understanding of mechanisms governing human astrogliosis with potential applicability as a platform to uncover novel biomarkers and drug targets to prevent or mitigate astrogliosis associated with human brain disorders.

Sci Rep ; 7: 40780, 2017 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-28112162


Zika virus (ZIKV) has been associated with microcephaly and other brain abnormalities; however, the molecular consequences of ZIKV to human brain development are still not fully understood. Here we describe alterations in human neurospheres derived from induced pluripotent stem (iPS) cells infected with the strain of Zika virus that is circulating in Brazil. Combining proteomics and mRNA transcriptional profiling, over 500 proteins and genes associated with the Brazilian ZIKV infection were found to be differentially expressed. These genes and proteins provide an interactome map, which indicates that ZIKV controls the expression of RNA processing bodies, miRNA biogenesis and splicing factors required for self-replication. It also suggests that impairments in the molecular pathways underpinning cell cycle and neuronal differentiation are caused by ZIKV. These results point to biological mechanisms implicated in brain malformations, which are important to further the understanding of ZIKV infection and can be exploited as therapeutic potential targets to mitigate it.

Proteoma , Transcriptoma , Infecção por Zika virus/genética , Infecção por Zika virus/metabolismo , Zika virus/fisiologia , Biomarcadores , Ciclo Celular/genética , Genômica/métodos , Humanos , Imuno-Histoquímica , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Neurônios/virologia , Filogenia , Infecção por Zika virus/virologia
Sci Rep ; 7: 40920, 2017 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-28098253


Zika virus (ZIKV) is a member of the Flaviviridae family, along with other agents of clinical significance such as dengue (DENV) and hepatitis C (HCV) viruses. Since ZIKV causes neurological disorders during fetal development and in adulthood, antiviral drugs are necessary. Sofosbuvir is clinically approved for use against HCV and targets the protein that is most conserved among the members of the Flaviviridae family, the viral RNA polymerase. Indeed, we found that sofosbuvir inhibits ZIKV RNA polymerase, targeting conserved amino acid residues. Sofosbuvir inhibited ZIKV replication in different cellular systems, such as hepatoma (Huh-7) cells, neuroblastoma (SH-Sy5y) cells, neural stem cells (NSC) and brain organoids. In addition to the direct inhibition of the viral RNA polymerase, we observed that sofosbuvir also induced an increase in A-to-G mutations in the viral genome. Together, our data highlight a potential secondary use of sofosbuvir, an anti-HCV drug, against ZIKV.

Antivirais/farmacologia , Sofosbuvir/farmacologia , Replicação Viral/efeitos dos fármacos , Zika virus/fisiologia , Antivirais/uso terapêutico , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , RNA Polimerases Dirigidas por DNA/metabolismo , Genoma Viral , Humanos , Mutação , Sofosbuvir/uso terapêutico , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/metabolismo , Zika virus/genética , Zika virus/isolamento & purificação , Infecção por Zika virus/tratamento farmacológico , Infecção por Zika virus/patologia , Infecção por Zika virus/virologia
Science ; 352(6287): 816-8, 2016 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-27064148


Since the emergence of Zika virus (ZIKV), reports of microcephaly have increased considerably in Brazil; however, causality between the viral epidemic and malformations in fetal brains needs further confirmation. We examined the effects of ZIKV infection in human neural stem cells growing as neurospheres and brain organoids. Using immunocytochemistry and electron microscopy, we showed that ZIKV targets human brain cells, reducing their viability and growth as neurospheres and brain organoids. These results suggest that ZIKV abrogates neurogenesis during human brain development.

Encéfalo/anormalidades , Encéfalo/virologia , Microcefalia/virologia , Células-Tronco Neurais/virologia , Neurogênese , Infecção por Zika virus/complicações , Zika virus/patogenicidade , Brasil , Morte Celular , Células Cultivadas , Humanos , Microcefalia/patologia , Células-Tronco Neurais/patologia , Organoides/anormalidades , Organoides/virologia , Infecção por Zika virus/patologia
Purinergic Signal ; 11(2): 183-201, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25663277


When retinal cell cultures were mechanically scratched, cell growth over the empty area was observed. Only dividing and migrating, 2 M6-positive glial cells were detected. Incubation of cultures with apyrase (APY), suramin, or Reactive Blue 2 (RB-2), but not MRS 2179, significantly attenuated the growth of glial cells, suggesting that nucleotide receptors other than P2Y1 are involved in the growth of glial cells. UTPγS but not ADPßS antagonized apyrase-induced growth inhibition in scratched cultures, suggesting the participation of UTP-sensitive receptors. No decrease in proliferating cell nuclear antigen (PCNA(+)) cells was observed at the border of the scratch in apyrase-treated cultures, suggesting that glial proliferation was not affected. In apyrase-treated cultures, glial cytoplasm protrusions were smaller and unstable. Actin filaments were less organized and alfa-tubulin-labeled microtubules were mainly parallel to scratch. In contrast to control cultures, very few vinculin-labeled adhesion sites could be noticed in these cultures. Increased Akt and ERK phosphorylation was observed in UTP-treated cultures, effect that was inhibited by SRC inhibitor 1 and PI3K blocker LY294002. These inhibitors and the FAK inhibitor PF573228 also decreased glial growth over the scratch, suggesting participation of SRC, PI3K, and FAK in UTP-induced growth of glial cells in scratched cultures. RB-2 decreased dissociated glial cell attachment to fibronectin-coated dishes and migration through transwell membranes, suggesting that nucleotides regulated adhesion and migration of glial cells. In conclusion, mechanical scratch of retinal cell cultures induces growth of glial cells over the empty area through a mechanism that is dependent on activation of UTP-sensitive receptors, SRC, PI3K, and FAK.

Movimento Celular/efeitos dos fármacos , Neuroglia/citologia , Nucleotídeos/metabolismo , Retina/efeitos dos fármacos , Animais , Apirase/farmacologia , Movimento Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Galinhas , Cromonas/farmacologia , Morfolinas/farmacologia , Neurogênese/efeitos dos fármacos , Neuroglia/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Quinolonas/farmacologia , Retina/lesões , Transdução de Sinais/efeitos dos fármacos , Sulfonas/farmacologia , Suramina/farmacologia
Neurochem Int ; 58(3): 414-22, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21193002


ATP can be released from neurons and act as a neuromodulator in the nervous system. Besides neurons, cortical astrocytes also are capable of releasing ATP from acidic vesicles in a Ca(2+)-dependent way. In the present work, we investigated the release of ATP from Müller glia cells of the chick embryo retina by examining quinacrine staining and by measuring the extracellular levels of ATP in purified Müller glia cultures. Our data revealed that glial cells could be labeled with quinacrine, a reaction that was prevented by incubation of the cells with 1µM bafilomycin A1 or 2µM Evans blue, potent inhibitors of vacuolar ATPases and of the vesicular nucleotide transporter, respectively. Either 50mM KCl or 1mM glutamate was able to decrease quinacrine staining of the cells, as well as to increase the levels of ATP in the extracellular medium by 77% and 89.5%, respectively, after a 5min incubation of the cells. Glutamate-induced rise in extracellular ATP could be mimicked by 100µM kainate (81.5%) but not by 100µM NMDA in medium without MgCl(2) but with 2mM glycine. However, both glutamate- and kainate-induced increase in extracellular ATP levels were blocked by 50µM of the glutamatergic antagonists DNQX and MK-801, suggesting the involvement of both NMDA and non-NMDA receptors. Extracellular ATP accumulation induced by glutamate was also blocked by incubation of the cells with 30µM BAPTA-AM or 1µM bafilomycin A1. These results suggest that glutamate, through activation of both NMDA and non-NMDA receptors, induces the release of ATP from retinal Müller cells through a calcium-dependent exocytotic mechanism.

Trifosfato de Adenosina/metabolismo , Sinalização do Cálcio/fisiologia , Ácido Glutâmico/fisiologia , Neuroglia/metabolismo , Animais , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Embrião de Galinha , Ácido Glutâmico/farmacologia , Neuroglia/efeitos dos fármacos , Quinacrina/química , Retina/citologia , Retina/metabolismo