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.
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‐1β, 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.
Main Points
TNF‐α triggers astrogliosis in human induced pluripotent stem cells‐derived astrocytes.
Short‐term TNF‐α exposure causes NF‐kB translocation and increases cytokines secretion.
Long‐term TNF‐α exposure increases GFAP, Vimentin, and astrocyte polarization and decreases aspartate uptake.
Chikungunya virus (CHIKV) causes a febrile disease associated with chronic arthralgia, which may progress to neurological impairment. Chikungunya fever (CF) is an ongoing public health problem in ...tropical and subtropical regions of the world, where control of the CHIKV vector,
mosquitos, has failed. As there is no vaccine or specific treatment for CHIKV, patients receive only palliative care to alleviate pain and arthralgia. Thus, drug repurposing is necessary to identify antivirals against CHIKV. CHIKV RNA polymerase is similar to the orthologue enzyme of other positive-sense RNA viruses, such as members of the
family. Among the
, not only is hepatitis C virus RNA polymerase susceptible to sofosbuvir, a clinically approved nucleotide analogue, but so is dengue, Zika, and yellow fever virus replication. Here, we found that sofosbuvir was three times more selective in inhibiting CHIKV production in human hepatoma cells than ribavirin, a pan-antiviral drug. Although CHIKV replication in human induced pluripotent stem cell-derived astrocytes was less susceptible to sofosbuvir than were hepatoma cells, sofosbuvir nevertheless impaired virus production and cell death in a multiplicity of infection-dependent manner. Sofosbuvir also exhibited antiviral activity
by preventing CHIKV-induced paw edema in adult mice at a dose of 20 mg/kg of body weight/day and prevented mortality in a neonate mouse model at 40- and 80-mg/kg/day doses. Our data demonstrate that a prototypic alphavirus, CHIKV, is also susceptible to sofosbuvir. As sofosbuvir is a clinically approved drug, our findings could pave the way to it becoming a therapeutic option against CF.
▶ Quinacrine labels ATP-containing vesicles in Müller glia cells in culture. ▶ Glutamate induces an increase in extracellular ATP levels in these cultures. ▶ NMDA and non-NMDA receptors mediate the ...accumulation of extracellular ATP. ▶ Glutamate induces ATP release by a calcium-dependent exocytotic mechanism.
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 Ca2+-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 MgCl2 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.
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.
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.
Extracellular vesicles (EVs) represent a new axis of intercellular communication that can be harnessed for therapeutic purposes, as cell-free therapies. The clinical application of mesenchymal ...stromal cell (MSC)-derived EVs, however, is still in its infancy and faces many challenges. The heterogeneity inherent to MSCs, differences among donors, tissue sources, and variations in manufacturing conditions may influence the release of EVs and their cargo, thus potentially affecting the quality and consistency of the final product. We investigated the influence of cell culture and conditioned medium harvesting conditions on the physicochemical and proteomic profile of human umbilical cord MSC-derived EVs (hUCMSC-EVs) produced under current good manufacturing practice (cGMP) standards. We also evaluated the efficiency of the protocol in terms of yield, purity, productivity, and expression of surface markers, and assessed the biodistribution, toxicity and potential efficacy of hUCMSC-EVs in pre-clinical studies using the LPS-induced acute lung injury model.
hUCMSCs were isolated from a cord tissue, cultured, cryopreserved, and characterized at a cGMP facility. The conditioned medium was harvested at 24, 48, and 72 h after the addition of EV collection medium. Three conventional methods (nanoparticle tracking analysis, transmission electron microscopy, and nanoflow cytometry) and mass spectrometry were used to characterize hUCMSC-EVs. Safety (toxicity of single and repeated doses) and biodistribution were evaluated in naive mice after intravenous administration of the product. Efficacy was evaluated in an LPS-induced acute lung injury model.
hUCMSC-EVs were successfully isolated using a cGMP-compliant protocol. Comparison of hUCMSC-EVs purified from multiple harvests revealed progressive EV productivity and slight changes in the proteomic profile, presenting higher homogeneity at later timepoints of conditioned medium harvesting. Pooled hUCMSC-EVs showed a non-toxic profile after single and repeated intravenous administration to naive mice. Biodistribution studies demonstrated a major concentration in liver, spleen and lungs. HUCMSC-EVs reduced lung damage and inflammation in a model of LPS-induced acute lung injury.
hUCMSC-EVs were successfully obtained following a cGMP-compliant protocol, with consistent characteristics and pre-clinical safety profile, supporting their future clinical development as cell-free therapies.
Axon guidance is required for the establishment of brain circuits. Whether much of the molecular basis of axon guidance is known from animal models, the molecular machinery coordinating axon growth ...and pathfinding in humans remains to be elucidated. The use of induced pluripotent stem cells (iPSC) from human donors has revolutionized in vitro studies of the human brain. iPSC can be differentiated into neuronal stem cells which can be used to generate neural tissue-like cultures, known as neurospheres, that reproduce, in many aspects, the cell types and molecules present in the brain. Here, we analyzed quantitative changes in the proteome of neurospheres during differentiation. Relative quantification was performed at early time points during differentiation using iTRAQ-based labeling and LC-MS/MS analysis. We identified 6438 proteins, from which 433 were downregulated and 479 were upregulated during differentiation. We show that human neurospheres have a molecular profile that correlates to the fetal brain. During differentiation, upregulated pathways are related to neuronal development and differentiation, cell adhesion, and axonal guidance whereas cell proliferation pathways were downregulated. We developed a functional assay to check for neurite outgrowth in neurospheres and confirmed that neurite outgrowth potential is increased after 10 days of differentiation and is enhanced by increasing cyclic AMP levels. The proteins identified here represent a resource to monitor neurosphere differentiation and coupled to the neurite outgrowth assay can be used to functionally explore neurological disorders using human neurospheres as a model.
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•Quantitative proteomic analysis of human neurospheres during differentiation using isobaric labeling (iTRAQ).•Axon guidance proteins are increased during differentiation.•Increased neurite outgrowth during differentiation is shown in a neuron outgrowth assay.•Neuron outgrowth in neurospheres is regulated by PKA and cAMP.•Axon guidance proteins related to neurological disorders are highlighted.
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 ...Casuper2+-dependent way. In the present work, we investigated the release of ATP from Mueller glia cells of the chick embryo retina by examining quinacrine staining and by measuring the extracellular levels of ATP in purified Mueller glia cultures. Our data revealed that glial cells could be labeled with quinacrine, a reaction that was prevented by incubation of the cells with 1 microM bafilomycin A1 or 2 microM Evans blue, potent inhibitors of vacuolar ATPases and of the vesicular nucleotide transporter, respectively. Either 50 mM KCl or 1 mM 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 5 min incubation of the cells. Glutamate-induced rise in extracellular ATP could be mimicked by 100 microM kainate (81.5%) but not by 100 microM NMDA in medium without MgCl sub(2 but with 2 mM glycine. However, both glutamate- and kainate-induced increase in extracellular ATP levels were blocked by 50 microM 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 30 microM BAPTA-AM or 1 microM bafilomycin A1. These results suggest that glutamate, through activation of both NMDA and non-NMDA receptors, induces the release of ATP from retinal Mueller cells through a calcium-dependent exocytotic mechanism.)
Scientific Reports 7: Article number: 40920; published online 18 January 2017; updated on 24 April 2017 The Competing Financial Interests section in this Article is incorrect and should read: “Dr. ...Karin Brüning is a member of the BMK consortium, able to produce sofosbuvir”.
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