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Microphysiological systems (MPSs) are cellular models that replicate aspects of organ and tissue functions in vitro. In contrast with conventional cell cultures, MPSs often provide physiological mechanical cues to cells, include fluid flow and can be interlinked (hence, they are often referred to as microfluidic tissue chips or organs-on-chips). Here, by means of examples of MPSs of the vascular system, intestine, brain and heart, we advocate for the development of standards that allow for comparisons of quantitative physiological features in MPSs and humans. Such standards should ensure that the in vivo relevance and predictive value of MPSs can be properly assessed as fit-for-purpose in specific applications, such as the assessment of drug toxicity, the identification of therapeutics or the understanding of human physiology or disease. Specifically, we distinguish designed features, which can be controlled via the design of the MPS, from emergent features, which describe cellular function, and propose methods for improving MPSs with readouts and sensors for the quantitative monitoring of complex physiology towards enabling wider end-user adoption and regulatory acceptance.
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Dispositivos Lab-On-A-Chip , Humanos , Animais , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Microfluídica/métodos , Modelos Biológicos , Encéfalo/fisiologia , Desenho de Equipamento , Sistemas MicrofisiológicosRESUMO
Astrocytes are in constant communication with neurons during the establishment and maturation of functional networks in the developing brain. Astrocytes release extracellular vesicles (EVs) containing microRNA (miRNA) cargo that regulates transcript stability in recipient cells. Astrocyte released factors are thought to be involved in neurodevelopmental disorders. Healthy astrocytes partially rescue Rett Syndrome (RTT) neuron function. EVs isolated from stem cell progeny also correct aspects of RTT. EVs cross the blood-brain barrier (BBB) and their cargo is found in peripheral blood which may allow non-invasive detection of EV cargo as biomarkers produced by healthy astrocytes. Here we characterize miRNA cargo and sequence motifs in healthy human astrocyte derived EVs (ADEVs). First, human induced Pluripotent Stem Cells (iPSC) were differentiated into Neural Progenitor Cells (NPCs) and subsequently into astrocytes using a rapid differentiation protocol. iPSC derived astrocytes expressed specific markers, displayed intracellular calcium transients and secreted ADEVs. miRNAs were identified by RNA-Seq on astrocytes and ADEVs and target gene pathway analysis detected brain and immune related terms. The miRNA profile was consistent with astrocyte identity, and included approximately 80 miRNAs found in astrocytes that were relatively depleted in ADEVs suggestive of passive loading. About 120 miRNAs were relatively enriched in ADEVs and motif analysis discovered binding sites for RNA binding proteins FUS, SRSF7 and CELF5. miR-483-5p was the most significantly enriched in ADEVs. This miRNA regulates MECP2 expression in neurons and has been found differentially expressed in blood samples from RTT patients. Our results identify potential miRNA biomarkers selectively sorted into ADEVs and implicate RNA binding protein sequence dependent mechanisms for miRNA cargo loading.
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Astrócitos , Vesículas Extracelulares , Células-Tronco Pluripotentes Induzidas , MicroRNAs , Neurônios , Humanos , Vesículas Extracelulares/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , MicroRNAs/metabolismo , MicroRNAs/genética , Astrócitos/metabolismo , Neurônios/metabolismo , Diferenciação Celular , Células Cultivadas , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/citologiaRESUMO
Human brain organoid technology has the potential to generate unprecedented insight into normal and aberrant brain development. It opens up a developmental time window in which the effects of gene or environmental perturbations can be experimentally tested. However, detection sensitivity and correct interpretation of phenotypes are hampered by notable batch-to-batch variability and low reproducibility of cell and regional identities. Here, we describe a detailed, simplified protocol for the robust and reproducible generation of brain organoids with cortical identity from feeder-independent induced pluripotent stem cells (iPSCs). This self-patterning approach minimizes media supplements and handling steps, resulting in cortical brain organoids that can be maintained over prolonged periods and that contain radial glial and intermediate progenitors, deep and upper layer neurons, and astrocytes.
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Gene editing through repair of CRISPR-Cas9-induced chromosomal breaks offers a means to correct a wide range of genetic defects. Directing repair to produce desirable outcomes by modulating DNA repair pathways holds considerable promise to increase the efficiency of genome engineering. Here, we show that inhibition of non-homologous end joining (NHEJ) or polymerase theta-mediated end joining (TMEJ) can be exploited to alter the mutational outcomes of CRISPR-Cas9. We show robust inhibition of TMEJ activity at CRISPR-Cas9-induced double-strand breaks (DSBs) using ART558, a potent polymerase theta (PolÏ´) inhibitor. Using targeted sequencing, we show that ART558 suppresses the formation of microhomology-driven deletions in favor of NHEJ-specific outcomes. Conversely, NHEJ deficiency triggers the formation of large kb-sized deletions, which we show are the products of mutagenic TMEJ. Finally, we show that combined chemical inhibition of TMEJ and NHEJ increases the efficiency of homology-driven repair (HDR)-mediated precise gene editing. Our work reports a robust strategy to improve the fidelity and safety of genome engineering.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Sistemas CRISPR-Cas/genética , Quebras de DNA de Cadeia Dupla , Mutação/genética , Reparo do DNA por Junção de ExtremidadesRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with various neurological complications. Although the mechanism is not fully understood, several studies have shown that neuroinflammation occurs in the acute and post-acute phase. As these studies have predominantly been performed with isolates from 2020, it is unknown if there are differences among SARS-CoV-2 variants in their ability to cause neuroinflammation. Here, we compared the neuroinvasiveness, neurotropism and neurovirulence of the SARS-CoV-2 ancestral strain D614G, the Delta (B.1.617.2) and Omicron BA.1 (B.1.1.529) variants using in vitro and in vivo models. The Omicron BA.1 variant showed reduced neurotropism and neurovirulence compared to Delta and D614G in human induced pluripotent stem cell (hiPSC)-derived cortical neurons co-cultured with astrocytes. Similar differences were obtained in Syrian hamsters inoculated with D614G, Delta and the Omicron BA.1 variant 5 days post infection. Replication in the olfactory mucosa was observed in all hamsters, but most prominently in D614G inoculated hamsters. Furthermore, neuroinvasion into the CNS via the olfactory nerve was observed in D614G, but not Delta or Omicron BA.1 inoculated hamsters. Furthermore, neuroinvasion was associated with neuroinflammation in the olfactory bulb of hamsters inoculated with D614G. Altogether, our findings suggest differences in the neuroinvasive, neurotropic and neurovirulent potential between SARS-CoV-2 variants using in vitro hiPSC-derived neural cultures and in vivo in hamsters during the acute phase of the infection.
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COVID-19 , Células-Tronco Pluripotentes Induzidas , Animais , Cricetinae , Humanos , Mesocricetus , SARS-CoV-2RESUMO
Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) infection is associated with a diverse spectrum of neurological complications during the acute and postacute stages. The pathogenesis of these complications is complex and dependent on many factors. For accurate and consistent interpretation of experimental data in this fast-growing field of research, it is essential to use terminology consistently. In this article, we outline the distinctions between neuroinvasiveness, neurotropism, and neurovirulence. Additionally, we discuss current knowledge of these distinct features underlying the pathogenesis of SARS-CoV-2-associated neurological complications. Lastly, we briefly discuss the advantages and limitations of different experimental models, and how these approaches can further be leveraged to advance the field.
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COVID-19 , Doenças do Sistema Nervoso , Humanos , SARS-CoV-2RESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with a wide variety of neurological complications. Even though SARS-CoV-2 is rarely detected in the central nervous system (CNS) or cerebrospinal fluid, evidence is accumulating that SARS-CoV-2 might enter the CNS via the olfactory nerve. However, what happens after SARS-CoV-2 enters the CNS is poorly understood. Therefore, we investigated the replication kinetics, cell tropism, and associated immune responses of SARS-CoV-2 infection in different types of neural cultures derived from human induced pluripotent stem cells (hiPSCs). SARS-CoV-2 was compared to the neurotropic and highly pathogenic H5N1 influenza A virus. SARS-CoV-2 infected a minority of individual mature neurons, without subsequent virus replication and spread, despite angiotensin-converting enzyme 2 (ACE2), transmembrane protease serine 2 (TMPRSS2), and neuropilin-1 (NPR1) expression in all cultures. However, this sparse infection did result in the production of type III interferons and interleukin-8 (IL-8). In contrast, H5N1 virus replicated and spread very efficiently in all cell types in all cultures. Taken together, our findings support the hypothesis that neurological complications might result from local immune responses triggered by virus invasion, rather than abundant SARS-CoV-2 replication in the CNS. IMPORTANCE Infections with the recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are often associated with neurological complications. Evidence suggests that SARS-CoV-2 enters the brain via the olfactory nerve; however, SARS-CoV-2 is only rarely detected in the central nervous system of COVID-19 patients. Here, we show that SARS-CoV-2 is able to infect neurons of human iPSC neural cultures but that this infection is abortive and does not result in virus spread to other cells. However, infection of neural cultures did result in the production of type III interferon and IL-8. This study suggests that SARS-CoV-2 might enter the CNS and infect individual neurons, triggering local immune responses that could contribute to the pathogenesis of SARS-CoV-2-associated CNS disease.
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Células-Tronco Pluripotentes Induzidas/citologia , Virus da Influenza A Subtipo H5N1/fisiologia , Neurônios/virologia , SARS-CoV-2/fisiologia , Tropismo Viral , Replicação Viral , Animais , Encefalopatias/etiologia , COVID-19/complicações , Chlorocebus aethiops , Cães , Humanos , Virus da Influenza A Subtipo H5N1/imunologia , Cinética , Células Madin Darby de Rim Canino , SARS-CoV-2/imunologia , Células VeroRESUMO
The human UBE3A gene, which is essential for normal neurodevelopment, encodes three Ubiquitin E3 ligase A (UBE3A) protein isoforms. However, the subcellular localization and relative abundance of these human UBE3A isoforms are unknown. We found, as previously reported in mice, that UBE3A is predominantly nuclear in human neurons. However, this conserved subcellular distribution is achieved by strikingly distinct cis-acting mechanisms. A single amino-acid deletion in the N-terminus of human hUBE3A-Iso3, which is homologous to cytosolic mouse mUBE3A-Iso2, results in its translocation to the nucleus. This singe amino-acid deletion is shared with apes and Old World monkeys and was preceded by the appearance of the cytosolic hUBE3A-Iso2 isoform. This hUBE3A-Iso2 isoform arose after the lineage of New World monkeys and Old World monkeys separated from the Tarsiers (Tarsiidae). Due to the loss of a single nucleotide in a non-coding exon, this exon became in frame with the remainder of the UBE3A protein. RNA-seq analysis of human brain samples showed that the human UBE3A isoforms arise by alternative splicing. Consistent with the predominant nuclear enrichment of UBE3A in human neurons, the two nuclear-localized isoforms, hUBE3A-Iso1 and -Iso3, are the most abundantly expressed isoforms of UBE3A, while hUBE3A-Iso2 maintains a small pool of cytosolic UBE3A. Our findings provide new insight into UBE3A localization and evolution and may have important implications for gene therapy approaches in Angelman syndrome.
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Síndrome de Angelman/genética , Neurônios/metabolismo , Ubiquitina-Proteína Ligases/genética , Processamento Alternativo/genética , Síndrome de Angelman/patologia , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Impressão Genômica/genética , Humanos , Camundongos , Neurônios/patologia , Isoformas de Proteínas/genéticaRESUMO
Developmental disorders (DD), characterized by malformations/dysmorphism and/or intellectual disability, affecting around 3% of worldwide population, are mostly linked to genetic anomalies. Despite clinical exome sequencing (cES) centered on genes involved in human genetic disorders, the majority of patients affected by DD remain undiagnosed after solo-cES. Trio-based strategy is expected to facilitate variant selection thanks to rapid parental segregation. We performed a second step trio-ES (not only focusing on genes involved in human disorders) analysis in 70 patients with negative results after solo-cES. All candidate variants were shared with a MatchMaking exchange system to identify additional patients carrying variants in the same genes and with similar phenotype. In 18/70 patients (26%), we confirmed causal implication of nine OMIM-morbid genes and identified nine new strong candidate genes (eight de novo and one compound heterozygous variants). These nine new candidate genes were validated through the identification of patients with similar phenotype and genotype thanks to data sharing. Moreover, 11 genes harbored variants of unknown significance in 10/70 patients (14%). In DD, a second step trio-based ES analysis appears an efficient strategy in diagnostic and translational research to identify highly candidate genes and improve diagnostic yield.
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Deficiências do Desenvolvimento/genética , Exoma/genética , Predisposição Genética para Doença/genética , Deficiência Intelectual/genética , Feminino , Genômica/métodos , Humanos , Masculino , Fenótipo , Sequenciamento do Exoma/métodosRESUMO
Noncoding RNAs have been widely recognized as essential mediators of gene regulation. However, in contrast to protein-coding genes, much less is known about the influence of noncoding RNAs on human diseases. Here we examined the association of genetic variants located in primary microRNA sequences and long noncoding RNAs (lncRNAs) with Alzheimer disease (AD) by leveraging data from the largest genome-wide association meta-analysis of late-onset AD. Variants annotated to 5 miRNAs and 10 lncRNAs (in seven distinct loci) exceeded the Bonferroni-corrected significance threshold (p < 1.02 × 10-6 ). Among these, a leading variant (rs2526377:A>G) at the 17q22 locus annotated to two noncoding RNAs (MIR142 and BZRAP1-AS) was significantly associated with a reduced risk of AD and fulfilled predefined criteria for being a functional variant. Our functional genomic analyses revealed that rs2526377 affects the promoter activity and decreases the expression of miR-142. Moreover, differential expression analysis by RNA-Seq in human iPSC-derived neural progenitor cells and the hippocampus of miR-142 knockout mice demonstrated multiple target genes of miR-142 in the brain that are likely to be involved in the inflammatory and neurodegenerative manifestations of AD. These include TGFBR1 and PICALM, of which their derepression in the brain due to reduced expression levels of miR-142-3p may reduce the risk of AD.
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Doença de Alzheimer/genética , Predisposição Genética para Doença , Variação Genética , MicroRNAs/genética , Regiões Promotoras Genéticas , Alelos , Doença de Alzheimer/metabolismo , Animais , Linhagem Celular , Mapeamento Cromossômico , Biologia Computacional/métodos , Modelos Animais de Doenças , Regulação da Expressão Gênica , Estudos de Associação Genética , Estudo de Associação Genômica Ampla , Hipocampo/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Camundongos Knockout , Modelos Biológicos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Polimorfismo de Nucleotídeo Único , Interferência de RNA , RNA não TraduzidoRESUMO
Mutations affecting the gene encoding the ubiquitin ligase UBE3A cause Angelman syndrome. Although most studies focus on the synaptic function of UBE3A, we show that UBE3A is highly enriched in the nucleus of mouse and human neurons. We found that the two major isoforms of UBE3A exhibit highly distinct nuclear versus cytoplasmic subcellular localization. Both isoforms undergo nuclear import through direct binding to PSMD4 (also known as S5A or RPN10), but the amino terminus of the cytoplasmic isoform prevents nuclear retention. Mice lacking the nuclear UBE3A isoform recapitulate the behavioral and electrophysiological phenotypes of Ube3am-/p+ mice, whereas mice harboring a targeted deletion of the cytosolic isoform are unaffected. Finally, we identified Angelman syndrome-associated UBE3A missense mutations that interfere with either nuclear targeting or nuclear retention of UBE3A. Taken together, our findings elucidate the mechanisms underlying the subcellular localization of UBE3A, and indicate that the nuclear UBE3A isoform is the most critical for the pathophysiology of Angelman syndrome.
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Síndrome de Angelman/genética , Síndrome de Angelman/psicologia , Comportamento Animal , Ubiquitina-Proteína Ligases/genética , Animais , Proteínas de Transporte/metabolismo , Núcleo Celular/enzimologia , Núcleo Celular/genética , Citosol/enzimologia , Fenômenos Eletrofisiológicos/genética , Feminino , Humanos , Isoenzimas/genética , Masculino , Camundongos , Camundongos Knockout , Mutação de Sentido Incorreto/genética , Comportamento de Nidação , Neurônios/enzimologia , Desempenho Psicomotor , Proteínas de Ligação a RNA , Natação/psicologia , Dedos de ZincoRESUMO
Schizophrenia is highly heritable, yet its underlying pathophysiology remains largely unknown. Among the most well-replicated findings in neurobiological studies of schizophrenia are deficits in myelination and white matter integrity; however, direct etiological genetic and cellular evidence has thus far been lacking. Here, we implement a family-based approach for genetic discovery in schizophrenia combined with functional analysis using induced pluripotent stem cells (iPSCs). We observed familial segregation of two rare missense mutations in Chondroitin Sulfate Proteoglycan 4 (CSPG4) (c.391G > A [p.A131T], MAF 7.79 × 10-5 and c.2702T > G [p.V901G], MAF 2.51 × 10-3). The CSPG4A131T mutation was absent from the Swedish Schizophrenia Exome Sequencing Study (2536 cases, 2543 controls), while the CSPG4V901G mutation was nominally enriched in cases (11 cases vs. 3 controls, P = 0.026, OR 3.77, 95% CI 1.05-13.52). CSPG4/NG2 is a hallmark protein of oligodendrocyte progenitor cells (OPCs). iPSC-derived OPCs from CSPG4A131T mutation carriers exhibited abnormal post-translational processing (P = 0.029), subcellular localization of mutant NG2 (P = 0.007), as well as aberrant cellular morphology (P = 3.0 × 10-8), viability (P = 8.9 × 10-7), and myelination potential (P = 0.038). Moreover, transfection of healthy non-carrier sibling OPCs confirmed a pathogenic effect on cell survival of both the CSPG4A131T (P = 0.006) and CSPG4V901G (P = 3.4 × 10-4) mutations. Finally, in vivo diffusion tensor imaging of CSPG4A131T mutation carriers demonstrated a reduction of brain white matter integrity compared to unaffected sibling and matched general population controls (P = 2.2 × 10-5). Together, our findings provide a convergence of genetic and functional evidence to implicate OPC dysfunction as a candidate pathophysiological mechanism of familial schizophrenia.
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Proteoglicanas de Sulfatos de Condroitina/genética , Proteínas de Membrana/genética , Células Precursoras de Oligodendrócitos/metabolismo , Esquizofrenia/genética , Adulto , Antígenos/genética , Diferenciação Celular/fisiologia , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Imagem de Tensor de Difusão , Família , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Mutação/genética , Células Precursoras de Oligodendrócitos/fisiologia , Oligodendroglia/metabolismo , Linhagem , Proteoglicanas/genética , Esquizofrenia/metabolismo , Substância Branca/metabolismoRESUMO
Zinc homeostasis is a highly regulated process in mammalian cells that is critical for normal growth and development. Movement of zinc across cell compartments is controlled by two classes of transporters: Slc39a family members transport zinc into the cytosol from either the extracellular space or intracellular stores such as the endoplasmic reticulum (ER), whereas the SLC30A family mediates zinc efflux from the cytosol. In this study, we report that genetic ablation of SLC39A7 (ZIP7) results in decreased cytosolic zinc levels, increased ER zinc levels, impaired cell proliferation, and induction of ER stress. Confirmatory of impaired zinc transport as the causal mechanism, both the increased ER stress and impaired cell proliferation were rescued by increasing cytosolic zinc. Furthermore, using these robust cellular phenotypes, we implemented a small-molecule library screen with 2800 compounds and identified one small molecule capable of rescuing ER stress and cell proliferation in ZIP7-deficient cells in the low micromolar range.
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Proteínas de Transporte de Cátions/metabolismo , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Zinco/metabolismo , Proteínas de Transporte de Cátions/genética , Linhagem Celular , Proliferação de Células/fisiologia , Retículo Endoplasmático/genética , Estresse do Retículo Endoplasmático/fisiologia , HumanosRESUMO
OBJECTIVE: To identify the clinical characteristics and genetic etiology of a family affected with hereditary spastic paraplegia (HSP). METHODS: Clinical, genetic, and functional analyses involving genome-wide linkage coupled to whole-exome sequencing in a consanguineous family with complicated HSP. RESULTS: A homozygous missense mutation was identified in the ACO2 gene (c.1240T>G p.Phe414Val) that segregated with HSP complicated by intellectual disability and microcephaly. Lymphoblastoid cell lines of homozygous carrier patients revealed significantly decreased activity of the mitochondrial aconitase enzyme and defective mitochondrial respiration. ACO2 encodes mitochondrial aconitase, an essential enzyme in the Krebs cycle. Recessive mutations in this gene have been previously associated with cerebellar ataxia. CONCLUSIONS: Our findings nominate ACO2 as a disease-causing gene for autosomal recessive complicated HSP and provide further support for the central role of mitochondrial defects in the pathogenesis of HSP.
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Recent Zika virus (ZIKV) infections have been associated with a range of neurological complications, in particular congenital microcephaly. Human neural progenitor cells (hNPCs) are thought to play an important role in the pathogenesis of microcephaly, and experimental ZIKV infection of hNPCs has been shown to induce cell death. However, the infection efficiency and rate of cell death have varied between studies, which might be related to intrinsic differences between African and Asian lineage ZIKV strains. Therefore, we determined the replication kinetics, including infection efficiency, burst size, and ability to induce cell death, of two Asian and two African ZIKV strains. African ZIKV strains replicated to higher titers in Vero cells, human glioblastoma (U87MG) cells, human neuroblastoma (SK-N-SH) cells, and hNPCs than Asian ZIKV strains. Furthermore, infection with Asian ZIKV strains did not result in significant cell death early after infection, whereas infection with African ZIKV strains resulted in high percentages of cell death in hNPCs. The differences between African and Asian lineage ZIKV strains highlight the importance of including relevant ZIKV strains to study the pathogenesis of congenital microcephaly and caution against extrapolation of experimental data obtained using historical African ZIKV strains to the current outbreak. Finally, the fact that Asian ZIKV strains infect only a minority of cells with a relatively low burst size together with the lack of early cell death induction might contribute to its ability to cause chronic infections within the central nervous system (CNS). IMPORTANCE The mechanism by which ZIKV causes a range of neurological complications, especially congenital microcephaly, is not well understood. The fact that congenital microcephaly is associated with Asian lineage ZIKV strains raises the question of why this was not discovered earlier. One possible explanation is that Asian and African ZIKV strains differ in their abilities to infect cells of the CNS and to cause neurodevelopmental problems. Here, we show that Asian ZIKV strains infect and induce cell death in human neural progenitor cells-which are important target cells in the development of congenital microcephaly-less efficiently than African ZIKV strains. These features of Asian ZIKV strains likely contribute to their ability to cause chronic infections, often observed in congenital microcephaly cases. It is therefore likely that phenotypic differences between ZIKV strains could be, at least in part, responsible for the ability of Asian ZIKV strains to cause congenital microcephaly.
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The directed differentiation of patient-derived induced pluripotent stem cells into cell-type specific neurons has inspired the development of therapeutic discovery for neurodegenerative diseases. Many forms of ataxia result from degeneration of cerebellar Purkinje cells, but thus far it has not been possible to efficiently generate Purkinje neuron (PN) progenitors from human or mouse pluripotent stem cells, let alone to develop a methodology for in vivo transplantation in the adult cerebellum. Here, we present a protocol to obtain an expandable population of cerebellar neuron progenitors from mouse embryonic stem cells. Our protocol is characterized by applying factors that promote proliferation of cerebellar progenitors. Cerebellar progenitors isolated in culture from cell aggregates contained a stable subpopulation of PN progenitors that could be expanded for up to 6 passages. When transplanted into the adult cerebellum of either wild-type mice or a strain lacking Purkinje cells (L7cre-ERCC1 knockout), GFP-labeled progenitors differentiated in vivo to establish a population of calbindin-positive cells in the molecular layer with dendritic trees typical of mature PNs. We conclude that this protocol may be useful for the generation and maturation of PNs, highlighting the potential for development of a regenerative medicine approach to the treatment of cerebellar neurodegenerative diseases.
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Diferenciação Celular , Cerebelo/citologia , Células-Tronco Embrionárias/citologia , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Células de Purkinje/citologia , Células de Purkinje/metabolismo , Potenciais de Ação , Fatores Etários , Animais , Biomarcadores , Técnicas de Cultura de Células , Células Cultivadas , Meios de Cultura , Feminino , Imunofluorescência , Expressão Gênica , Genes Reporter , Imunofenotipagem , Masculino , Camundongos , Transplante de Células-TroncoRESUMO
A recent phase 1 trial of the fatty acid amide hydrolase (FAAH) inhibitor BIA 10-2474 led to the death of one volunteer and produced mild-to-severe neurological symptoms in four others. Although the cause of the clinical neurotoxicity is unknown, it has been postulated, given the clinical safety profile of other tested FAAH inhibitors, that off-target activities of BIA 10-2474 may have played a role. Here we use activity-based proteomic methods to determine the protein interaction landscape of BIA 10-2474 in human cells and tissues. This analysis revealed that the drug inhibits several lipases that are not targeted by PF04457845, a highly selective and clinically tested FAAH inhibitor. BIA 10-2474, but not PF04457845, produced substantial alterations in lipid networks in human cortical neurons, suggesting that promiscuous lipase inhibitors have the potential to cause metabolic dysregulation in the nervous system.
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Amidoidrolases/antagonistas & inibidores , Analgésicos/farmacologia , Ansiolíticos/farmacologia , Óxidos N-Cíclicos/farmacologia , Neurônios/efeitos dos fármacos , Piridinas/farmacologia , Analgésicos/efeitos adversos , Analgésicos/química , Analgésicos/metabolismo , Ansiolíticos/efeitos adversos , Ansiolíticos/química , Ansiolíticos/metabolismo , Linhagem Celular Tumoral , Ensaios Clínicos Fase I como Assunto , Reações Cruzadas , Óxidos N-Cíclicos/efeitos adversos , Óxidos N-Cíclicos/química , Óxidos N-Cíclicos/metabolismo , Humanos , Neurônios/metabolismo , Mapas de Interação de Proteínas , Piridazinas/farmacologia , Piridazinas/uso terapêutico , Piridinas/efeitos adversos , Piridinas/química , Piridinas/metabolismo , Ureia/análogos & derivados , Ureia/farmacologia , Ureia/uso terapêuticoRESUMO
Hepatitis E virus (HEV), as a hepatotropic virus, is supposed to exclusively infect the liver and only cause hepatitis. However, a broad range of extrahepatic manifestations (in particular, idiopathic neurological disorders) have been recently reported in association with its infection. In this study, we have demonstrated that various human neural cell lines (embryonic stem cell-derived neural lineage cells) induced pluripotent stem cell-derived human neurons and primary mouse neurons are highly susceptible to HEV infection. Treatment with interferon-α or ribavirin, the off-label antiviral drugs for chronic hepatitis E, exerted potent antiviral activities against HEV infection in neural cells. More importantly, in mice and monkey peripherally inoculated with HEV particles, viral RNA and protein were detected in brain tissues. Finally, patients with HEV-associated neurological disorders shed the virus into cerebrospinal fluid, indicating a direct infection of their nervous system. Thus, HEV is neurotropic in vitro, and in mice, monkeys, and possibly humans. These results challenge the dogma of HEV as a pure hepatotropic virus and suggest that HEV infection should be considered in the differential diagnosis of idiopathic neurological disorders.
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Encéfalo/virologia , Vírus da Hepatite E/patogenicidade , Hepatite E/patologia , Neurônios/virologia , Adulto , Idoso , Animais , Antivirais/farmacologia , Encéfalo/patologia , Linhagem Celular Tumoral , Líquido Cefalorraquidiano/virologia , Feminino , Síndrome de Guillain-Barré/virologia , Hepatite E/tratamento farmacológico , Humanos , Interferon-alfa/farmacologia , Fígado/patologia , Fígado/virologia , Macaca mulatta , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Neurônios/patologia , RNA Viral/análise , Ribavirina/farmacologia , Replicação Viral/efeitos dos fármacos , Eliminação de Partículas ViraisRESUMO
Loss of function of the FMR1 gene leads to fragile X syndrome (FXS), the most common form of intellectual disability. The loss of FMR1 function is usually caused by epigenetic silencing of the FMR1 promoter leading to expansion and subsequent methylation of a CGG repeat in the 5' untranslated region. Very few coding sequence variations have been experimentally characterized and shown to be causal to the disease. Here, we describe a novel FMR1 mutation and reveal an unexpected nuclear export function for the C-terminus of FMRP. We screened a cohort of patients with typical FXS symptoms who tested negative for CGG repeat expansion in the FMR1 locus. In one patient, we identified a guanine insertion in FMR1 exon 15. This mutation alters the open reading frame creating a short novel C-terminal sequence, followed by a stop codon. We find that this novel peptide encodes a functional nuclear localization signal (NLS) targeting the patient FMRP to the nucleolus in human cells. We also reveal an evolutionarily conserved nuclear export function associated with the endogenous C-terminus of FMRP. In vivo analyses in Drosophila demonstrate that a patient-mimetic mutation alters the localization and function of Dfmrp in neurons, leading to neomorphic neuronal phenotypes.
Assuntos
Núcleo Celular , Proteína do X Frágil da Deficiência Intelectual , Síndrome do Cromossomo X Frágil , Mutação , Sinais de Localização Nuclear , Expansão das Repetições de Trinucleotídeos , Animais , Linhagem Celular Transformada , Linhagem Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Drosophila melanogaster , Proteína do X Frágil da Deficiência Intelectual/genética , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Síndrome do Cromossomo X Frágil/genética , Síndrome do Cromossomo X Frágil/metabolismo , Síndrome do Cromossomo X Frágil/patologia , Humanos , Masculino , Camundongos , Sinais de Localização Nuclear/genética , Sinais de Localização Nuclear/metabolismo , Estrutura Terciária de Proteína , Transporte Proteico/genéticaRESUMO
Silencing of the FMR1 gene leads to fragile X syndrome, the most common cause of inherited intellectual disability. To study the epigenetic modifications of the FMR1 gene during silencing in time, we used fibroblasts and induced pluripotent stem cells (iPSCs) of an unmethylated full mutation (uFM) individual with normal intelligence. The uFM fibroblast line carried an unmethylated FMR1 promoter region and expressed normal to slightly increased FMR1 mRNA levels. The FMR1 expression in the uFM line corresponds with the increased H3 acetylation and H3K4 methylation in combination with a reduced H3K9 methylation. After reprogramming, the FMR1 promoter region was methylated in all uFM iPSC clones. Two clones were analyzed further and showed a lack of FMR1 expression, whereas the presence of specific histone modifications also indicated a repressed FMR1 promoter. In conclusion, these findings demonstrate that the standard reprogramming procedure leads to epigenetic silencing of the fully mutated FMR1 gene.