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Outbreaks of West Nile virus (WNV) occur periodically, affecting both human and equine populations. There are no vaccines for humans, and those commercialised for horses do not have sufficient coverage. Specific antiviral treatments do not exist. Many drug discovery studies have been conducted, but since rodent or primate cell lines are normally used, results cannot always be transposed to horses. There is thus a need to develop relevant equine cellular models. Here, we used induced pluripotent stem cells to develop a new in vitro model of WNV-infected equine brain cells suitable for microplate assay, and assessed the cytotoxicity and antiviral activity of forty-one chemical compounds. We found that one nucleoside analog, 2'C-methylcytidine, blocked WNV infection in equine brain cells, whereas other compounds were either toxic or ineffective, despite some displaying anti-viral activity in human cell lines. We also revealed an unexpected proviral effect of statins in WNV-infected equine brain cells. Our results thus identify a potential lead for future drug development and underscore the importance of using a tissue- and species-relevant cellular model for assessing the activity of antiviral compounds.
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Doenças dos Cavalos , Células-Tronco Pluripotentes Induzidas , Febre do Nilo Ocidental , Vírus do Nilo Ocidental , Animais , Cavalos , Humanos , Febre do Nilo Ocidental/veterinária , Febre do Nilo Ocidental/epidemiologia , Encéfalo , Antivirais/farmacologia , Antivirais/uso terapêutico , Doenças dos Cavalos/tratamento farmacológicoRESUMO
Congenital infection of the central nervous system by human cytomegalovirus (HCMV) is a leading cause of permanent sequelae, including mental retardation or neurodevelopmental abnormalities. The most severe complications include smooth brain or polymicrogyria, which are both indicative of abnormal migration of neural cells, although the underlying mechanisms remain to be determined. To gain better insight on the pathogenesis of such sequelae, we assessed the expression levels of a set of neurogenesis-related genes, using HCMV-infected human neural stem cells derived from embryonic stem cells (NSCs). Among the 84 genes tested, we found dramatically increased expression of the gene PAFAH1B1, encoding LIS1 (lissencephaly-1), in HCMV-infected versus uninfected NSCs. Consistent with these findings, western blotting and immunofluorescence analyses confirmed the increased levels of LIS1 in HCMV-infected NSCs at the protein level. We next assessed the migratory abilities of HCMV-infected NSCs and observed that infection strongly impaired the migration of NSCs, without detectable effect on their proliferation. Moreover, we observed increased immunostaining for LIS1 in brains of congenitally infected fetuses, but not in control samples, highlighting the clinical relevance of our findings. Of note, PAFAH1B1 mutations (resulting in either haploinsufficiency or gain of function) are primary causes of hereditary neurodevelopmental diseases. Notably, mutations resulting in PAFAH1B1 haploinsufficiency cause classic lissencephaly. Taken together, our findings suggest that PAFAH1B1 is a critical target of HCMV infection. They also shine a new light on the pathophysiological basis of the neurological outcomes of congenital HCMV infection, by suggesting that defective neural cell migration might contribute to the pathogenesis of the neurodevelopmental sequelae of infection. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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1-Alquil-2-acetilglicerofosfocolina Esterase/metabolismo , Infecções por Citomegalovirus/congênito , Infecções por Citomegalovirus/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/virologia , Encéfalo/metabolismo , Encéfalo/virologia , Infecções por Citomegalovirus/complicações , HumanosRESUMO
BACKGROUND: Tick-borne encephalitis virus (TBEV) is a member of the Flaviviridae family, Flavivirus genus, which includes several important human pathogens. It is responsible for neurological symptoms that may cause permanent disability or death, and, from a medical point of view, is the major arbovirus in Central/Northern Europe and North-Eastern Asia. TBEV tropism is critical for neuropathogenesis, yet little is known about the molecular mechanisms that govern the susceptibility of human brain cells to the virus. In this study, we sought to establish and characterize a new in vitro model of TBEV infection in the human brain and to decipher cell type-specific innate immunity and its relation to TBEV tropism and neuropathogenesis. METHOD: Human neuronal/glial cells were differentiated from neural progenitor cells and infected with the TBEV-Hypr strain. Kinetics of infection, cellular tropism, and cellular responses, including innate immune responses, were characterized by measuring viral genome and viral titer, performing immunofluorescence, enumerating the different cellular types, and determining their rate of infection and by performing PCR array and qRT-PCR. The specific response of neurons and astrocytes was analyzed using the same approaches after enrichment of the neuronal/glial cultures for each cellular subtype. RESULTS: We showed that infection of human neuronal/glial cells mimicked three major hallmarks of TBEV infection in the human brain, namely, preferential neuronal tropism, neuronal death, and astrogliosis. We further showed that these cells conserved their capacity to mount an antiviral response against TBEV. TBEV-infected neuronal/glial cells, therefore, represented a highly relevant pathological model. By enriching the cultures for either neurons or astrocytes, we further demonstrated qualitative and quantitative differential innate immune responses in the two cell types that correlated with their particular susceptibility to TBEV. CONCLUSION: Our results thus reveal that cell type-specific innate immunity is likely to contribute to shaping TBEV tropism for human brain cells. They describe a new in vitro model for in-depth study of TBEV-induced neuropathogenesis and improve our understanding of the mechanisms by which neurotropic viruses target and damage human brain cells.
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Astrócitos/imunologia , Astrócitos/virologia , Encefalite Transmitida por Carrapatos/imunologia , Encefalite Transmitida por Carrapatos/virologia , Neurônios/imunologia , Neurônios/virologia , Técnicas de Cultura de Células/métodos , Células Cultivadas , Suscetibilidade a Doenças , Vírus da Encefalite Transmitidos por Carrapatos/fisiologia , Humanos , Imunidade Inata , Tropismo ViralRESUMO
Congenital infection by human cytomegalovirus (HCMV) is a leading cause of permanent sequelae of the central nervous system, including sensorineural deafness, cerebral palsies or devastating neurodevelopmental abnormalities (0.1% of all births). To gain insight on the impact of HCMV on neuronal development, we used both neural stem cells from human embryonic stem cells (NSC) and brain sections from infected fetuses and investigated the outcomes of infection on Peroxisome Proliferator-Activated Receptor gamma (PPARγ), a transcription factor critical in the developing brain. We observed that HCMV infection dramatically impaired the rate of neuronogenesis and strongly increased PPARγ levels and activity. Consistent with these findings, levels of 9-hydroxyoctadecadienoic acid (9-HODE), a known PPARγ agonist, were significantly increased in infected NSCs. Likewise, exposure of uninfected NSCs to 9-HODE recapitulated the effect of infection on PPARγ activity. It also increased the rate of cells expressing the IE antigen in HCMV-infected NSCs. Further, we demonstrated that (1) pharmacological activation of ectopically expressed PPARγ was sufficient to induce impaired neuronogenesis of uninfected NSCs, (2) treatment of uninfected NSCs with 9-HODE impaired NSC differentiation and (3) treatment of HCMV-infected NSCs with the PPARγ inhibitor T0070907 restored a normal rate of differentiation. The role of PPARγ in the disease phenotype was strongly supported by the immunodetection of nuclear PPARγ in brain germinative zones of congenitally infected fetuses (N = 20), but not in control samples. Altogether, our findings reveal a key role for PPARγ in neurogenesis and in the pathophysiology of HCMV congenital infection. They also pave the way to the identification of PPARγ gene targets in the infected brain.
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Infecções por Citomegalovirus/congênito , Infecções por Citomegalovirus/complicações , Infecções por Citomegalovirus/metabolismo , Células-Tronco Neurais/virologia , Neurogênese/fisiologia , PPAR gama/metabolismo , Western Blotting , Diferenciação Celular/fisiologia , Imunoprecipitação da Cromatina , Cromatografia Líquida de Alta Pressão , Imunofluorescência , Humanos , Microscopia Eletrônica de Transmissão , Células-Tronco Neurais/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas em TandemRESUMO
It is well established that persistent viral infection may impair cellular function of specialized cells without overt damage. This concept, when applied to neurotropic viruses, may help to understand certain neurologic and neuropsychiatric diseases. Borna disease virus (BDV) is an excellent example of a persistent virus that targets the brain, impairs neural functions without cell lysis, and ultimately results in neurobehavioral disturbances. Recently, we have shown that BDV infects human neural progenitor cells (hNPCs) and impairs neurogenesis, revealing a new mechanism by which BDV may interfere with brain function. Here, we sought to identify the viral proteins and molecular pathways that are involved. Using lentiviral vectors for expression of the bdv-p and bdv-x viral genes, we demonstrate that the phosphoprotein P, but not the X protein, diminishes human neurogenesis and, more particularly, GABAergic neurogenesis. We further reveal a decrease in pro-neuronal factors known to be involved in neuronal differentiation (ApoE, Noggin, TH and Scg10/Stathmin2), demonstrating that cellular dysfunction is associated with impairment of specific components of the molecular program that controls neurogenesis. Our findings thus provide the first evidence that a viral protein impairs GABAergic human neurogenesis, a process that is dysregulated in several neuropsychiatric disorders. They improve our understanding of the mechanisms by which a persistent virus may interfere with brain development and function in the adult.
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Vírus da Doença de Borna/fisiologia , Regulação para Baixo , Neurônios GABAérgicos/metabolismo , Interações Hospedeiro-Patógeno , Neurogênese , Fosfoproteínas/metabolismo , Proteínas Estruturais Virais/metabolismo , Transporte Ativo do Núcleo Celular , Apolipoproteínas E/antagonistas & inibidores , Apolipoproteínas E/metabolismo , Biomarcadores/química , Biomarcadores/metabolismo , Doença de Borna/metabolismo , Doença de Borna/patologia , Doença de Borna/virologia , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/metabolismo , Proliferação de Células , Células Cultivadas , França , Neurônios GABAérgicos/citologia , Neurônios GABAérgicos/patologia , Neurônios GABAérgicos/virologia , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Embrionárias Humanas/patologia , Células-Tronco Embrionárias Humanas/virologia , Humanos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/metabolismo , Fosfoproteínas/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidade , Estatmina , Tirosina 3-Mono-Oxigenase/antagonistas & inibidores , Tirosina 3-Mono-Oxigenase/metabolismo , Proteínas Estruturais Virais/genéticaRESUMO
Human pluripotent stem cell-derived neural stem cells offer unprecedented opportunities for producing specific types of neurons for several biomedical applications. However, to achieve it, protocols of production and amplification of human neural stem cells need to be standardized, cost effective, and safe. This means that small molecules should progressively replace the use of media containing cocktails of protein-based growth factors. Here we have conducted a phenotypical screening to identify pathways involved in the regulation of hNSC self-renewal. We analyzed 80 small molecules acting as kinase inhibitors and identified compounds of the 5-isoquinolinesulfonamide family, described as protein kinase A (PKA) and protein kinase G inhibitors, as candidates to support hNSC self-renewal. Investigating the mode of action of these compounds, we found that modulation of PKA activity was central in controlling the choice between self-renewal or terminal neuronal differentiation of hNSC. We finally demonstrated that the pharmacological inhibition of PKA using the small molecule HA1004 was sufficient to support the full derivation, propagation, and long-term maintenance of stable hNSC in absence of any other extrinsic signals. Our results indicated that tuning of PKA activity is a core mechanism regulating hNSC self-renewal and differentiation and delineate the minimal culture media requirement to maintain undifferentiated hNSC in vitro.
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Diferenciação Celular/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Células-Tronco Neurais/enzimologia , Inibidores de Proteínas Quinases/farmacologia , Animais , Humanos , Células-Tronco Neurais/citologia , Neurônios/citologia , Neurônios/enzimologia , Inibidores de Proteínas Quinases/químicaRESUMO
Patients with myotonic dystrophy type 1 exhibit a diversity of symptoms that affect many different organs. Among these are cognitive dysfunctions, the origin of which has remained elusive, partly because of the difficulty in accessing neural cells. Here, we have taken advantage of pluripotent stem cell lines derived from embryos identified during a pre-implantation genetic diagnosis for mutant-gene carriers, to produce early neuronal cells. Functional characterization of these cells revealed reduced proliferative capacity and increased autophagy linked to mTOR signaling pathway alterations. Interestingly, loss of function of MBNL1, an RNA-binding protein whose function is defective in DM1 patients, resulted in alteration of mTOR signaling, whereas gain-of-function experiments rescued the phenotype. Collectively, these results provide a mechanism by which DM1 mutation might affect a major signaling pathway and highlight the pertinence of using pluripotent stem cells to study neuronal defects.
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Células-Tronco Embrionárias/citologia , Distrofia Miotônica/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Apoptose/genética , Apoptose/fisiologia , Western Blotting , Linhagem Celular , Proliferação de Células , Senescência Celular/genética , Senescência Celular/fisiologia , Eletroforese em Gel de Poliacrilamida , Humanos , Imuno-Histoquímica , Hibridização In Situ , Distrofia Miotônica/genética , Reação em Cadeia da Polimerase em Tempo Real , Serina-Treonina Quinases TOR/genéticaRESUMO
The role of microRNAs (miRNAs) as coordinators of stem cell fate has emerged over the last decade. We have used human embryonic stem cells to identify miRNAs involved in neural lineage commitment induced by the inhibition of TGFß-like molecule-mediated pathways. Among several candidate miRNAs expressed in the fetal brain, the two isoforms of miR-125 alone were detected in a time window compatible with a role in neural commitment in vitro. Functional analysis indicated that miR-125 isoforms were actively involved in the promotion of pluripotent cell conversion into SOX1-positive neural precursors. miR-125 promotes neural conversion by avoiding the persistence of non-differentiated stem cells and repressing alternative fate choices. This was associated with the regulation by miR-125 of SMAD4, a key regulator of pluripotent stem cell lineage commitment. Activation of miR-125 was directly responsive to the levels of TGFß-like molecules, placing miR-125 at the core of mechanisms that lead to the irreversible neural lineage commitment of pluripotent stem cells in response to external stimuli.
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Células-Tronco Embrionárias/citologia , MicroRNAs/metabolismo , Neurônios/metabolismo , Ativinas/metabolismo , Animais , Proteína Morfogenética Óssea 4/metabolismo , Diferenciação Celular , Linhagem Celular , Separação Celular , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , Neurônios/citologia , Células-Tronco Pluripotentes/citologia , Isoformas de Proteínas , Proteína Smad4/metabolismoRESUMO
Phelan-McDermid syndrome (PMDS) arises from mutations in the terminal region of chromosome 22q13, impacting the SHANK3 gene. The resulting deficiency of the postsynaptic density scaffolding protein SHANK3 is associated with autism spectrum disorder (ASD). We examined 12 different PMDS patient and CRISPR-engineered stem cell-derived neuronal models and controls and found that reduced expression of SHANK3 leads to neuronal hyperdifferentiation, increased synapse formation, and decreased neuronal activity. We performed automated imaging-based screening of 7,120 target-annotated small molecules and identified three compounds that rescued SHANK3-dependent neuronal hyperdifferentiation. One compound, Benproperine, rescued the decreased colocalization of Actin Related Protein 2/3 Complex Subunit 2 (ARPC2) with ß-actin and rescued increased synapse formation in SHANK3 deficient neurons when administered early during differentiation. Neuronal activity was only mildly affected, highlighting Benproperine's effects as a neurodevelopmental modulator. This study demonstrates that small molecular compounds that reverse developmental phenotypes can be identified in human neuronal PMDS models.
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Deleção Cromossômica , Transtornos Cromossômicos , Proteínas do Tecido Nervoso , Neurônios , Fenótipo , Sinapses , Humanos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Transtornos Cromossômicos/genética , Sinapses/efeitos dos fármacos , Cromossomos Humanos Par 22/genética , Masculino , Feminino , Diferenciação Celular/efeitos dos fármacos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/metabolismo , CriançaRESUMO
Periodically, the European Space Agency (ESA) updates scientific roadmaps in consultation with the scientific community. The ESA SciSpacE Science Community White Paper (SSCWP) 9, "Biology in Space and Analogue Environments", focusses in 5 main topic areas, aiming to address key community-identified knowledge gaps in Space Biology. Here we present one of the identified topic areas, which is also an unanswered question of life science research in Space: "How to Obtain an Integrated Picture of the Molecular Networks Involved in Adaptation to Microgravity in Different Biological Systems?" The manuscript reports the main gaps of knowledge which have been identified by the community in the above topic area as well as the approach the community indicates to address the gaps not yet bridged. Moreover, the relevance that these research activities might have for the space exploration programs and also for application in industrial and technological fields on Earth is briefly discussed.
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Progress in mechanobiology allowed us to better understand the important role of mechanical forces in the regulation of biological processes. Space research in the field of life sciences clearly showed that gravity plays a crucial role in biological processes. The space environment offers the unique opportunity to carry out experiments without gravity, helping us not only to understand the effects of gravitational alterations on biological systems but also the mechanisms underlying mechanoperception and cell/tissue response to mechanical and gravitational stresses. Despite the progress made so far, for future space exploration programs it is necessary to increase our knowledge on the mechanotransduction processes as well as on the molecular mechanisms underlying microgravity-induced cell and tissue alterations. This white paper reports the suggestions and recommendations of the SciSpacE Science Community for the elaboration of the section of the European Space Agency roadmap "Biology in Space and Analogue Environments" focusing on "How are cells and tissues influenced by gravity and what are the gravity perception mechanisms?" The knowledge gaps that prevent the Science Community from fully answering this question and the activities proposed to fill them are discussed.
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Introduction: Alteration in the development, maturation, and projection of dopaminergic neurons has been proposed to be associated with several neurological and psychiatric disorders. Therefore, understanding the signals modulating the genesis of human dopaminergic neurons is crucial to elucidate disease etiology and develop effective countermeasures. Methods: In this study, we developed a screening model using human pluripotent stem cells to identify the modulators of dopaminergic neuron genesis. We set up a differentiation protocol to obtained floorplate midbrain progenitors competent to produce dopaminergic neurons and seeded them in a 384-well screening plate in a fully automated manner. Results and Discussion: These progenitors were treated with a collection of small molecules to identify the compounds increasing dopaminergic neuron production. As a proof-of-principle, we screened a library of compounds targeting purine- and adenosine-dependent pathways and identified an adenosine receptor 3 agonist as a candidate molecule to increase dopaminergic neuron production under physiological conditions and in cells invalidated for the HPRT1 gene. This screening model can provide important insights into the etiology of various diseases affecting the dopaminergic circuit development and plasticity and be used to identify therapeutic molecules for these diseases.
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Lesch-Nyhan disease (LND) is a X-linked genetic disease affecting boys characterized by complex neurological and neuropsychiatric symptoms. LND is caused by loss of function mutations in the HPRT1 gene leading to decrease activity of hypoxanthine-guanine phosphoribosyl transferase enzyme (HGPRT) and altered purine salvage pathway (Lesch and Nyhan, 1964). This study describes the generation of isogenic clones with deletions in HPRT1 produced from one male human embryonic stem cell line using CRISPR/Cas9 strategy. Differentiation of these cells into different neuronal subtypes will help elucidating the neurodevelopmental events leading to LND and develop therapeutic strategies for this devastating neurodevelopmental disorder.
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Células-Tronco Embrionárias Humanas , Síndrome de Lesch-Nyhan , Humanos , Masculino , Síndrome de Lesch-Nyhan/genética , Síndrome de Lesch-Nyhan/metabolismo , Hipoxantina Fosforribosiltransferase/genética , Hipoxantina Fosforribosiltransferase/metabolismo , Sistemas CRISPR-Cas/genética , Técnicas de Inativação de Genes , Células-Tronco Embrionárias Humanas/metabolismoRESUMO
Phelan-McDermid syndrome (PMS) is a rare genetic disease characterized by a global developmental delay with autism spectrum disorder. PMS is caused by loss of function mutations in the SHANK3 gene leading to SHANK3 protein haploinsufficiency. This study describes the generation of isogenic clones produced from one male human embryonic stem cell line with deletions in SHANK3, in a heterozygous or homozygous manner, using CRISPR/Cas9 indel methodology. Differentiation of these clones into different neuronal lineages will help understanding PMS etiology and find treatments for PMD patients. (85/100 words).
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Transtorno do Espectro Autista , Células-Tronco Embrionárias Humanas , Humanos , Masculino , Células-Tronco Embrionárias Humanas/metabolismo , Transtorno do Espectro Autista/genética , Sistemas CRISPR-Cas/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células Clonais/metabolismoRESUMO
The present white paper concerns the indications and recommendations of the SciSpacE Science Community to make progress in filling the gaps of knowledge that prevent us from answering the question: "How Do Gravity Alterations Affect Animal and Human Systems at a Cellular/Tissue Level?" This is one of the five major scientific issues of the ESA roadmap "Biology in Space and Analogue Environments". Despite the many studies conducted so far on spaceflight adaptation mechanisms and related pathophysiological alterations observed in astronauts, we are not yet able to elaborate a synthetic integrated model of the many changes occurring at different system and functional levels. Consequently, it is difficult to develop credible models for predicting long-term consequences of human adaptation to the space environment, as well as to implement medical support plans for long-term missions and a strategy for preventing the possible health risks due to prolonged exposure to spaceflight beyond the low Earth orbit (LEO). The research activities suggested by the scientific community have the aim to overcome these problems by striving to connect biological and physiological aspects in a more holistic view of space adaptation effects.
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Although placental small extracellular vesicles (sEVs) are extensively studied in the context of pregnancy, little is known about their role during viral congenital infection, especially at the beginning of pregnancy. In this study, we examined the consequences of human cytomegalovirus (hCMV) infection on sEVs production, composition, and function using an immortalized human cytotrophoblast cell line derived from first trimester placenta. By combining complementary approaches of biochemistry, electron microscopy, and quantitative proteomic analysis, we showed that hCMV infection increases the yield of sEVs produced by cytotrophoblasts and modifies their protein content towards a potential proviral phenotype. We further demonstrate that sEVs secreted by hCMV-infected cytotrophoblasts potentiate infection in naive recipient cells of fetal origin, including human neural stem cells. Importantly, these functional consequences are also observed with sEVs prepared from an ex vivo model of infected histocultures from early placenta. Based on these findings, we propose that placental sEVs could be important actors favoring viral dissemination to the fetal brain during hCMV congenital infection.
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Infecções por Citomegalovirus , Vesículas Extracelulares , Citomegalovirus/genética , Vesículas Extracelulares/metabolismo , Feminino , Humanos , Placenta , Gravidez , ProteômicaRESUMO
One of the major obstacles to the identification of therapeutic interventions for central nervous system disorders has been the difficulty in studying the step-by-step progression of diseases in neuronal networks that are amenable to drug screening. Recent advances in the field of human pluripotent stem cell (PSC) biology offers the capability to create patient-specific human neurons with defined clinical profiles using reprogramming technology, which provides unprecedented opportunities for both the investigation of pathogenic mechanisms of brain disorders and the discovery of novel therapeutic strategies via drug screening. Many examples not only of the creation of human pluripotent stem cells as models of monogenic neurological disorders, but also of more challenging cases of complex multifactorial disorders now exist. Here, we review the state-of-the art brain cell types obtainable from PSCs and amenable to compound-screening formats. We then provide examples illustrating how these models contribute to the definition of new molecular or functional targets for drug discovery and to the design of novel pharmacological approaches for rare genetic disorders, as well as frequent neurodegenerative diseases and psychiatric disorders.
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Descoberta de Drogas , Modelos Biológicos , Doenças do Sistema Nervoso/metabolismo , Células-Tronco Pluripotentes/metabolismo , Humanos , Neurônios/patologia , Doenças Raras/genéticaRESUMO
In neurodegenerative disorders associated with primary or secondary mitochondrial defects such as Huntington's disease (HD), cells of the striatum are particularly vulnerable to cell death, although the mechanisms by which this cell death is induced are unclear. Dopamine, found in high concentrations in the striatum, may play a role in striatal cell death. We show that in primary striatal cultures, dopamine increases the toxicity of an N-terminal fragment of mutated huntingtin (Htt-171-82Q). Mitochondrial complex II protein (mCII) levels are reduced in HD striatum, indicating that this protein may be important for dopamine-mediated striatal cell death. We found that dopamine enhances the toxicity of the selective mCII inhibitor, 3-nitropropionic acid. We also demonstrated that dopamine doses that are insufficient to produce cell loss regulate mCII expression at the mRNA, protein and catalytic activity level. We also show that dopamine-induced down-regulation of mCII levels can be blocked by several dopamine D2 receptor antagonists. Sustained overexpression of mCII subunits using lentiviral vectors abrogated the effects of dopamine, both by high dopamine concentrations alone and neuronal death induced by low dopamine concentrations together with Htt-171-82Q. This novel pathway links dopamine signaling and regulation of mCII activity and could play a key role in oxidative energy metabolism and explain the vulnerability of the striatum in neurodegenerative diseases.
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Corpo Estriado/efeitos dos fármacos , Dopamina/farmacologia , Complexo II de Transporte de Elétrons/metabolismo , Proteínas do Tecido Nervoso/genética , Neurônios/efeitos dos fármacos , Proteínas Nucleares/genética , Animais , Técnicas de Cultura de Células , Dopaminérgicos/farmacologia , Antagonistas dos Receptores de Dopamina D2 , Regulação para Baixo/efeitos dos fármacos , Complexo II de Transporte de Elétrons/antagonistas & inibidores , Expressão Gênica/efeitos dos fármacos , Proteína Huntingtina , Proteínas Mutantes/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Subunidades Proteicas/metabolismo , Ratos , Receptores de Dopamina D2/metabolismoRESUMO
Lesch-Nyhan disease (LND) is a rare monogenic disease caused by deficiency of the salvage pathway enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT). LND is characterized by severe neuropsychiatric symptoms that currently cannot be treated. Predictive in vivo models are lacking for screening and evaluating candidate drugs because LND-associated neurological symptoms are not recapitulated in HGPRT-deficient animals. Here, we used human neural stem cells and neurons derived from induced pluripotent stem cells (iPSCs) of children affected with LND to identify neural phenotypes of interest associated with HGPRT deficiency to develop a target-agnostic-based drug screening system. We screened more than 3000 molecules and identified 6 pharmacological compounds, all possessing an adenosine moiety, that corrected HGPRT deficiency-associated neuronal phenotypes by promoting metabolism compensations in an HGPRT-independent manner. This included S-adenosylmethionine, a compound that had already been used as a compassionate approach to ease the neuropsychiatric symptoms in LND. Interestingly, these compounds compensate abnormal metabolism in a manner complementary to the gold standard allopurinol and can be provided to patients with LND via simple food supplementation. This experimental paradigm can be easily adapted to other metabolic disorders affecting normal brain development and functioning in the absence of a relevant animal model.
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Células-Tronco Pluripotentes Induzidas/citologia , Síndrome de Lesch-Nyhan/tratamento farmacológico , Síndrome de Lesch-Nyhan/terapia , Células-Tronco Neurais/citologia , Alopurinol/uso terapêutico , Animais , Estudos de Casos e Controles , Diferenciação Celular , Modelos Animais de Doenças , Humanos , Hipoxantina Fosforribosiltransferase/genética , Células-Tronco Neurais/enzimologia , FenótipoRESUMO
A central challenge in embryonic stem (ES) cell biology is to understand how to impose direction on primary lineage commitment. In basal culture conditions, the majority of ES cells convert asynchronously into neural cells. However, many cells resist differentiation and others adopt nonneural fates. Mosaic activation of the neural reporter Sox-green fluorescent protein suggests regulation by cell-cell interactions. We detected expression of Notch receptors and ligands in mouse ES cells and investigated the role of this pathway. Genetic manipulation to activate Notch constitutively does not alter the stem cell phenotype. However, upon withdrawal of self-renewal stimuli, differentiation is directed rapidly and exclusively into the neural lineage. Conversely, pharmacological or genetic interference with Notch signalling suppresses the neural fate choice. Notch promotion of neural commitment requires parallel signalling through the fibroblast growth factor receptor. Stromal cells expressing Notch ligand stimulate neural specification of human ES cells, indicating that this is a conserved pathway in pluripotent stem cells. These findings define an unexpected and decisive role for Notch in ES cell fate determination. Limiting activation of endogenous Notch results in heterogeneous lineage commitment. Manipulation of Notch signalling is therefore likely to be a key factor in taking command of ES cell lineage choice.