RESUMO
Wnt signaling regulates neural stem cell (NSC) function and development throughout an individual's lifetime. Intriguingly, adult hippocampal progenitors (AHPs) produce several Wnts, and the intracellular machinery necessary to respond to them, creating the potential for an active autocrine-signaling loop within this stem cell niche. However, the standard luciferase-based Wnt assay failed to detect this signaling loop. This assay is inherently less temporally sensitive to activity among a population of unsynchronized proliferating cells because it relies on the rapidly degrading reporter luciferase. We circumvented this limitation using a promoter assay that employs green fluorescent protein (GFP), as a relatively long-lived reporter of canonical Wnt activity. We found that at baseline, AHPs secreted functional Wnt that self-stimulates low-level canonical Wnt signaling. Elimination baseline Wnt activity, via application of an extracellular Wnt antagonist promoted neurogenesis, based on a combination of unbiased gene expression analysis and cell-fate analysis. A detailed clonal analysis of progenitors transduced with specific intracellular antagonists of canonical signaling, either Axin or truncated cadherin (beta-catenin sequestering), revealed that loss of baseline signaling depletes the population of multipotent precursors, thereby driving an increasing fraction to assume a committed cell fate (i.e., unipotent progenitors). Similarly, baseline Wnt signaling repressed differentiation of human NSCs. Although the specific Wnts produced by neural precursors vary with age and between species, their effects remain remarkably consistent. In sum, this study establishes that autonomous Wnt signaling is a conserved feature of the neurogenic niche that preserves the delicate balance between NSC maintenance and differentiation.
Assuntos
Neurônios/citologia , Neurônios/metabolismo , Transdução de Sinais , Células-Tronco/citologia , Células-Tronco/metabolismo , Proteínas Wnt/metabolismo , Animais , Comunicação Autócrina , Diferenciação Celular , Linhagem Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Hipocampo/citologia , Humanos , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/metabolismo , Ratos , Especificidade da Espécie , beta Catenina/metabolismoRESUMO
The recent Zika virus (ZIKV) outbreak in the Western hemisphere is associated with severe pathology in newborns, including microcephaly and brain damage. The mechanisms underlying these outcomes are under intense investigation. Here, we show that a 2015 ZIKV isolate replicates in multiple cell types, including primary human fetal neural progenitors (hNPs). In immortalized cells, ZIKV is cytopathic and grossly rearranges endoplasmic reticulum membranes similar to other flaviviruses. In hNPs, ZIKV infection has a partial cytopathic phase characterized by cell rounding, pyknosis, and activation of caspase 3. Despite notable cell death, ZIKV did not activate a cytokine response in hNPs. This lack of cell intrinsic immunity to ZIKV is consistent with our observation that virus replication persists in hNPs for at least 28 days. These findings, supported by published fetal neuropathology, establish a proof-of-concept that neural progenitors in the developing human fetus can be direct targets of detrimental ZIKV-induced pathology.
Assuntos
Efeito Citopatogênico Viral/imunologia , Feto/patologia , Células-Tronco Neurais/imunologia , Células-Tronco Neurais/virologia , Infecção por Zika virus/imunologia , Infecção por Zika virus/virologia , Zika virus/imunologia , Linhagem Celular , Humanos , Fatores de Tempo , Replicação Viral , Zika virus/isolamento & purificação , Zika virus/fisiologia , Zika virus/ultraestruturaAssuntos
Fatores de Transcrição Forkhead/metabolismo , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Envelhecimento/fisiologia , Animais , Modelos Animais de Doenças , Fatores de Transcrição Forkhead/genética , Humanos , Degeneração Neural/genética , Doença de Parkinson/genéticaRESUMO
Neural stem cells have been adopted to model a wide range of neuropsychiatric conditions in vitro. However, how well such models correspond to in vivo brain has not been evaluated in an unbiased, comprehensive manner. We used transcriptomic analyses to compare in vitro systems to developing human fetal brain and observed strong conservation of in vivo gene expression and network architecture in differentiating primary human neural progenitor cells (phNPCs). Conserved modules are enriched in genes associated with ASD, supporting the utility of phNPCs for studying neuropsychiatric disease. We also developed and validated a machine learning approach called CoNTExT that identifies the developmental maturity and regional identity of in vitro models. We observed strong differences between in vitro models, including hiPSC-derived neural progenitors from multiple laboratories. This work provides a systems biology framework for evaluating in vitro systems and supports their value in studying the molecular mechanisms of human neurodevelopmental disease.
Assuntos
Inteligência Artificial , Córtex Cerebral/embriologia , Células-Tronco Embrionárias/fisiologia , Redes Reguladoras de Genes/genética , Modelos Neurológicos , Células-Tronco Neurais/fisiologia , Inteligência Artificial/tendências , Células Cultivadas , Córtex Cerebral/citologia , Feminino , Humanos , MasculinoRESUMO
Two papers address the contribution of DISC1 to neural development and schizophrenia risk in this issue of Neuron. These complementary studies elegantly bridge the gap between genetic and cellular studies of schizophrenia, providing a level of functional validation that is often lacking in the field.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Química Encefálica/genética , Encéfalo/crescimento & desenvolvimento , Quinase 3 da Glicogênio Sintase/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurogênese , Polimorfismo Genético/genética , Esquizofrenia/metabolismo , Transdução de Sinais/genética , Proteína Wnt3A/genética , Animais , Feminino , Glicogênio Sintase Quinase 3 beta , Humanos , Masculino , GravidezRESUMO
Wnt proteins are critical to mammalian brain development and function. The canonical Wnt signaling pathway involves the stabilization and nuclear translocation of ß-catenin; however, Wnt also signals through alternative, noncanonical pathways. To gain a systems-level, genome-wide view of Wnt signaling, we analyzed Wnt1-stimulated changes in gene expression by transcriptional microarray analysis in cultured human neural progenitor (hNP) cells at multiple time points over a 72-hour time course. We observed a widespread oscillatory-like pattern of changes in gene expression, involving components of both the canonical and the noncanonical Wnt signaling pathways. A higher-order, systems-level analysis that combined independent component analysis, waveform analysis, and mutual information-based network construction revealed effects on pathways related to cell death and neurodegenerative disease. Wnt effectors were tightly clustered with presenilin1 (PSEN1) and granulin (GRN), which cause dominantly inherited forms of Alzheimer's disease and frontotemporal dementia (FTD), respectively. We further explored a potential link between Wnt1 and GRN and found that Wnt1 decreased GRN expression by hNPs. Conversely, GRN knockdown increased WNT1 expression, demonstrating that Wnt and GRN reciprocally regulate each other. Finally, we provided in vivo validation of the in vitro findings by analyzing gene expression data from individuals with FTD. These unbiased and genome-wide analyses provide evidence for a connection between Wnt signaling and the transcriptional regulation of neurodegenerative disease genes.
Assuntos
Doença de Alzheimer/metabolismo , Demência Frontotemporal/metabolismo , Regulação da Expressão Gênica , Proteínas do Tecido Nervoso/biossíntese , Transcrição Gênica , Via de Sinalização Wnt , Proteína Wnt1/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Células Cultivadas , Demência Frontotemporal/genética , Demência Frontotemporal/patologia , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas do Tecido Nervoso/genética , Análise de Sequência com Séries de Oligonucleotídeos , Presenilina-1/biossíntese , Presenilina-1/genética , Progranulinas , Proteína Wnt1/genéticaRESUMO
Progranulin (GRN) mutations cause frontotemporal dementia (FTD), but GRN's function in the CNS remains largely unknown. To identify the pathways downstream of GRN, we used weighted gene coexpression network analysis (WGCNA) to develop a systems-level view of transcriptional alterations in a human neural progenitor model of GRN-deficiency. This highlighted key pathways such as apoptosis and ubiquitination in GRN deficient human neurons, while revealing an unexpected major role for the Wnt signaling pathway, which was confirmed by analysis of gene expression data from postmortem FTD brain. Furthermore, we observed that the Wnt receptor Fzd2 was one of only a few genes upregulated at 6 weeks in a GRN knockout mouse, and that FZD2 reduction caused increased apoptosis, while its upregulation promoted neuronal survival in vitro. Together, these in vitro and in vivo data point to an adaptive role for altered Wnt signaling in GRN deficiency-mediated FTD, representing a potential therapeutic target.
Assuntos
Genoma , Genômica/métodos , Peptídeos e Proteínas de Sinalização Intercelular/deficiência , Transdução de Sinais/fisiologia , Proteínas Wnt/metabolismo , Animais , Morte Celular , Diferenciação Celular , Células Cultivadas , Receptores Frizzled/genética , Receptores Frizzled/metabolismo , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Camundongos , Análise em Microsséries , Células-Tronco Neurais/fisiologia , Neurônios/fisiologia , Progranulinas , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Wnt/genéticaRESUMO
Friedreich's ataxia (FRDA) is caused by reduction of frataxin levels to 5-35%. To better understand the biochemical sequelae of frataxin reduction, in absence of the confounding effects of neurodegeneration, we studied the gene expression profile of a mouse model expressing 25-36% of the normal frataxin levels, and not showing a detectable phenotype or neurodegenerative features. Despite having no overt phenotype, a clear microarray gene expression phenotype was observed. This phenotype followed the known regional susceptibility in this disease, most changes occurring in the spinal cord. Additionally, gene ontology analysis identified a clear mitochondrial component, consistent with previous findings. We were able to confirm a subset of changes in fibroblast cell lines from patients. The identification of a core set of genes changing early in the FRDA pathogenesis can be a useful tool in both clarifying the disease process and in evaluating new therapeutic strategies.