RESUMO
Astrocytes are intimately linked with brain blood vessels, an essential relationship for neuronal function. However, astroglial factors driving these physical and functional associations during postnatal brain development have yet to be identified. By characterizing structural and transcriptional changes in mouse cortical astrocytes during the first two postnatal weeks, we find that high-mobility group box 1 (Hmgb1), normally upregulated with injury and involved in adult cerebrovascular repair, is highly expressed in astrocytes at birth and then decreases rapidly. Astrocyte-selective ablation of Hmgb1 at birth affects astrocyte morphology and endfoot placement, alters distribution of endfoot proteins connexin43 and aquaporin-4, induces transcriptional changes in astrocytes related to cytoskeleton remodeling, and profoundly disrupts endothelial ultrastructure. While lack of astroglial Hmgb1 does not affect the blood-brain barrier or angiogenesis postnatally, it impairs neurovascular coupling and behavior in adult mice. These findings identify astroglial Hmgb1 as an important player in postnatal gliovascular maturation.
Assuntos
Astrócitos , Barreira Hematoencefálica , Proteína HMGB1 , Animais , Camundongos , Aquaporina 4 , Encéfalo , Morfogênese , Proteína HMGB1/metabolismoRESUMO
Astroglial cells are key players in the development and maintenance of neurons and neuronal networks. Astroglia express steroid hormone receptors and show rapid responses to hormonal manipulations. However, despite important sex differences in the cortex and hippocampus, few studies have examined sex differences in astroglial cells in telencephalic development. To characterize the cortical astroglial translatome in male and female mice across postnatal development, we use translating ribosome affinity purification together with RNA sequencing and immunohistochemistry to phenotype astroglia at six developmental time points. Overall, we find two distinct astroglial phenotypes between early (P1-P7) and late development (P14-adult), independent of sex. We also find sex differences in gene expression patterns across development that peak at P7 and appear to result from males reaching a mature astroglial phenotype earlier than females. These developmental sex differences could have an impact on the construction of neuronal networks and windows of vulnerability to perturbations and disease.
Assuntos
Astrócitos/metabolismo , Neurogênese/fisiologia , Neurônios/metabolismo , Caracteres Sexuais , Animais , Células Cultivadas , Feminino , Masculino , Camundongos Endogâmicos C57BL , Neocórtex/metabolismoRESUMO
Organoids (ORGs) are increasingly used as models of cerebral cortical development. Here, we compared transcriptome and cellular phenotypes between telencephalic ORGs and monolayers (MONs) generated in parallel from three biologically distinct induced pluripotent stem cell (iPSC) lines. Multiple readouts revealed increased proliferation in MONs, which was caused by increased integrin signaling. MONs also exhibited altered radial glia (RG) polarity and suppression of Notch signaling, as well as impaired generation of intermediate progenitors, outer RG, and cortical neurons, which were all partially reversed by reaggregation of dissociated cells. Network analyses revealed co-clustering of cell adhesion, Notch-related transcripts and their transcriptional regulators in a module strongly downregulated in MONs. The data suggest that ORGs, with respect to MONs, initiate more efficient Notch signaling in ventricular RG owing to preserved cell adhesion, resulting in subsequent generation of intermediate progenitors and outer RG, in a sequence that recapitulates the cortical ontogenetic process.
Assuntos
Adesão Celular , Córtex Cerebral/metabolismo , Células Ependimogliais/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurogênese , Organoides/metabolismo , Transcriptoma , Diferenciação Celular , Córtex Cerebral/citologia , Células Ependimogliais/citologia , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Integrinas/metabolismo , Masculino , Neurônios/citologia , Neurônios/metabolismo , Técnicas de Cultura de Órgãos/métodos , Organoides/citologia , Proteoma , RNA-Seq , Receptores Notch/metabolismo , Transdução de SinaisRESUMO
Ectonucleotide phosphatase/phosphodiesterase 1 (ENPP1) deficiency results in either lethal arterial calcifications ('Generalized Arterial Calcification of Infancy' - GACI), phosphate wasting rickets ('Autosomal Recessive Hypophosphatemic Rickets type 2' - ARHR2), early onset osteoporosis, or progressive spinal rigidity ('Ossification of the Posterior Longitudinal Ligament' - OPLL). As ENPP1 generates a strong endogenous mineralization inhibitor - extracellular pyrophosphate (PPi) - ENPP1 deficiency should not result in reduced bone volume, and therefore the mechanism ENPP1 associated osteoporosis is not apparent given current understanding of the enzyme's function. To investigate genetic pathways driving the skeletal phenotype of ENPP1 deficiency we compared gene expression in Enpp1asj/asj mice and WT sibling pairs by RNAseq and qPCR in whole bones, and in the liver and kidney by qPCR, directly correlating gene expression with measures of bone microarchitectural and biomechanical phenotypes. Unbiased analysis of the differentially expressed genes compared to relevant human disease phenotypes revealed that Enpp1asj/asj mice exhibit strong signatures of osteoporosis, ARHR2 and OPLL. We found that ENPP1 deficient mice exhibited reduced gene transcription of Wnt ligands in whole bone and increased transcription of soluble Wnt inhibitors in the liver and kidney, suggestive of multiorgan inhibition of Wnt activity. Consistent with Wnt suppression in bone, Collagen gene pathways in bone were significantly decreased and Fgf23 was significantly increased, all of which directly correlated with bone microarchitectural defects and fracture risk in Enpp1asj/asj mice. Moreover, the bone findings in 10-week old mice correlated with Enpp1 transcript counts but not plasma [PPi], suggesting that the skeletal phenotype at 10 weeks is driven by catalytically independent ENPP1 function. In contrast, the bone findings in 23-week Enpp1asj/asj mice strongly correlated with plasma PPi, suggesting that chronically low PPi drives the skeletal phenotype in older mice. Finally, correlation between Enpp1 and Fgf23 transcription suggested ENPP1 regulation of Fgf23, which we confirmed by dosing Enpp1asj/asj mice with soluble ENPP1-Fc and observing suppression of intact plasma FGF23 and ALP. In summary, our findings suggest that osteoporosis associated with ENPP1 deficiency involves the suppression of Wnt via catalytically independent Enpp1 pathways, and validates Enpp1asj/asj mice as tools to better understand OPLL and Paradoxical Mineralization Disorders.
Assuntos
Osteomalacia , Osteoporose , Calcificação Vascular , Animais , Fator de Crescimento de Fibroblastos 23 , Camundongos , Osteoporose/genética , Diester Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases , Pirofosfatases/genéticaRESUMO
The neuroplasticity hypothesis of depression proposes that major depressive disorders are related to decreased hippocampal and cortical neural plasticity, which is reversed by antidepressant treatment. Astroglial cells have emerged as key mediators of neural plasticity and are involved in the cause and treatment of depression and anxiety-like behaviors. One of the ways that astroglia modulate neuroplasticity is through the formation and maintenance of perineuronal nets (PNNs). Perineuronal nets are important extracellular matrix components that respond to stress and are implicated in anxiety-like behaviors. Normally, astroglial cells continuously turnover PNNs by degrading and donating PNN proteins; however, chronic stress slows PNN protein degradation and increases cortical PNN expression overall. In this report, we used weighted gene co-expression network analysis and eigengene analysis to further delineate the pathways and key regulators involved in the astroglial-PNN relationship following chronic stress. Our analyses indicate that chronic variable stress induces the expression of PNNs through inhibition of trophic pathways and key transcription factors in astroglial cells. These data further support the integral role of astroglial cells in the neuroplasticity hypothesis of depression through their modulation of anxiety-like behaviors and PNNs.
RESUMO
Genes implicated in neuropsychiatric disorders are active in human fetal brain, yet difficult to study in a longitudinal fashion. We demonstrate that organoids from human pluripotent cells model cerebral cortical development on the molecular level before 16 weeks postconception. A multiomics analysis revealed differentially active genes and enhancers, with the greatest changes occurring at the transition from stem cells to progenitors. Networks of converging gene and enhancer modules were assembled into six and four global patterns of expression and activity across time. A pattern with progressive down-regulation was enriched with human-gained enhancers, suggesting their importance in early human brain development. A few convergent gene and enhancer modules were enriched in autism-associated genes and genomic variants in autistic children. The organoid model helps identify functional elements that may drive disease onset.
Assuntos
Córtex Cerebral/embriologia , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Modelos Neurológicos , Neurogênese/genética , Organoides/embriologia , Elementos Facilitadores Genéticos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , TranscriptomaRESUMO
Recent studies have suggested that cortical astroglia play an important role in depressive-like behaviors. Potential astroglial contributions have been proposed based on their known neuroplastic functions, such as glutamate recycling and synaptic plasticity. However, the specific mechanisms by which astroglial cells may contribute or protect against a depressive phenotype remain unknown. To delineate astroglial changes that accompany depressive-like behavior, we used astroglial-specific bacTRAP mice exposed to chronic variable stress (CVS) and profiled the astroglial translatome using translating ribosome affinity purification (TRAP) in conjunction with RNAseq. As expected, CVS significantly increased anxiety- and depressive-like behaviors and corticosterone levels and decreased GFAP expression in astroglia, although this did not reflect a change in the total number of astroglial cells. TRAPseq results showed that CVS decreased genes associated with astroglial plasticity: RhoGTPases, growth factor signaling, and transcription regulation, and increased genes associated with the formation of extracellular matrices such as perineuronal nets (PNNs). PNNs inhibit neuroplasticity and astroglia contribute to the formation, organization, and maintenance of PNNs. To validate our TRAPseq findings, we showed an increase in PNNs following CVS. Degradation of PNNs in the prefrontal cortex of mice exposed to CVS reversed the CVS-induced behavioral phenotype in the forced swim test. These data lend further support to the neuroplasticity hypothesis of depressive behaviors and, in particular, extend this hypothesis beyond neuronal plasticity to include an overall decrease in genes associated with cortical astroglial plasticity following CVS. Further studies will be needed to assess the antidepressant potential of directly targeting astroglial cell function in models of depression.
Assuntos
Astrócitos/metabolismo , Córtex Pré-Frontal/metabolismo , Biossíntese de Proteínas , Estresse Psicológico/metabolismo , Animais , Ansiedade/metabolismo , Ansiedade/patologia , Astrócitos/patologia , Doença Crônica , Corticosterona/metabolismo , Depressão/metabolismo , Depressão/patologia , Expressão Gênica , Proteína Glial Fibrilar Ácida/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Plasticidade Neuronal/fisiologia , Córtex Pré-Frontal/patologia , Estresse Psicológico/patologia , IncertezaRESUMO
We currently have a poor understanding of the pathogenesis of neurodevelopmental disorders, owing to the fact that postmortem and imaging studies can only measure the postnatal status quo and offer little insight into the processes that give rise to the observed outcomes. Human induced pluripotent stem cells (hiPSCs) should, in principle, prove powerful for elucidating the pathways that give rise to neurodevelopmental disorders. hiPSCs are embryonic-stem-cell-like cells that can be derived from somatic cells. They retain the unique genetic signature of the individual from whom they were derived, and thus enable researchers to recapitulate that individual's idiosyncratic neural development in a dish. In the case of individuals with disease, we can re-enact the disease-altered trajectory of brain development and examine how and why phenotypic and molecular abnormalities arise in these diseased brains. Here, we review hiPSC biology and possible experimental designs when using hiPSCs to model disease. We then discuss existing hiPSC models of neurodevelopmental disorders. Our hope is that, as some studies have already shown, hiPSCs will illuminate the pathophysiology of developmental disorders of the CNS and lead to therapeutic options for the millions that are affected by these conditions.
Assuntos
Encéfalo/crescimento & desenvolvimento , Células-Tronco Pluripotentes Induzidas , Modelos Biológicos , Rede Nervosa/crescimento & desenvolvimento , Transtornos do Neurodesenvolvimento , HumanosRESUMO
Few studies have been conducted to understand post-zygotic accumulation of mutations in cells of the healthy human body. We reprogrammed 32 skin fibroblast cells from families of donors into human induced pluripotent stem cell (hiPSC) lines. The clonal nature of hiPSC lines allows a high-resolution analysis of the genomes of the founder fibroblast cells without being confounded by the artifacts of single-cell whole-genome amplification. We estimate that on average a fibroblast cell in children has 1035 mostly benign mosaic SNVs. On average, 235 SNVs could be directly confirmed in the original fibroblast population by ultradeep sequencing, down to an allele frequency (AF) of 0.1%. More sensitive droplet digital PCR experiments confirmed more SNVs as mosaic with AF as low as 0.01%, suggesting that 1035 mosaic SNVs per fibroblast cell is the true average. Similar analyses in adults revealed no significant increase in the number of SNVs per cell, suggesting that a major fraction of mosaic SNVs in fibroblasts arises during development. Mosaic SNVs were distributed uniformly across the genome and were enriched in a mutational signature previously observed in cancers and in de novo variants and which, we hypothesize, is a hallmark of normal cell proliferation. Finally, AF distribution of mosaic SNVs had distinct narrow peaks, which could be a characteristic of clonal cell selection, clonal expansion, or both. These findings reveal a large degree of somatic mosaicism in healthy human tissues, link de novo and cancer mutations to somatic mosaicism, and couple somatic mosaicism with cell proliferation.
Assuntos
Evolução Clonal , Variações do Número de Cópias de DNA , Fibroblastos/citologia , Mosaicismo , Acúmulo de Mutações , Proliferação de Células , Células Cultivadas , Fibroblastos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Pele/citologiaRESUMO
BACKGROUND: Genome-wide association studies have not revealed any risk-conferring common genetic variants in Tourette syndrome (TS), requiring the adoption of alternative approaches to investigate the pathophysiology of this disorder. METHODS: We obtained the basal ganglia transcriptome by RNA sequencing in the caudate and putamen of nine TS and nine matched normal control subjects. RESULTS: We found 309 downregulated and 822 upregulated genes in the caudate and putamen (striatum) of TS individuals. Using data-driven gene network analysis, we identified 17 gene coexpression modules associated with TS. The top-scoring downregulated module in TS was enriched in striatal interneuron transcripts, which was confirmed by decreased numbers of cholinergic and gamma-aminobutyric acidergic interneurons by immunohistochemistry in the same regions. The top-scoring upregulated module was enriched in immune-related genes, consistent with activation of microglia in patients' striatum. Genes implicated by copy number variants in TS were enriched in the interneuron module, as well as in a protocadherin module. Module clustering revealed that the interneuron module was correlated with a neuronal metabolism module. CONCLUSIONS: Convergence of differential expression, network analyses, and module clustering, together with copy number variants implicated in TS, strongly implicates disrupted interneuron signaling in the pathophysiology of severe TS and suggests that metabolic alterations may be linked to their death or dysfunction.
Assuntos
Interneurônios/metabolismo , Putamen/metabolismo , Síndrome de Tourette/genética , Transcriptoma , Acetilcolina/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Variações do Número de Cópias de DNA , Feminino , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Estudo de Associação Genômica Ampla , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência de RNA , Ácido gama-Aminobutírico/metabolismoRESUMO
Autism spectrum disorder (ASD) is a disorder of brain development. Most cases lack a clear etiology or genetic basis, and the difficulty of re-enacting human brain development has precluded understanding of ASD pathophysiology. Here we use three-dimensional neural cultures (organoids) derived from induced pluripotent stem cells (iPSCs) to investigate neurodevelopmental alterations in individuals with severe idiopathic ASD. While no known underlying genomic mutation could be identified, transcriptome and gene network analyses revealed upregulation of genes involved in cell proliferation, neuronal differentiation, and synaptic assembly. ASD-derived organoids exhibit an accelerated cell cycle and overproduction of GABAergic inhibitory neurons. Using RNA interference, we show that overexpression of the transcription factor FOXG1 is responsible for the overproduction of GABAergic neurons. Altered expression of gene network modules and FOXG1 are positively correlated with symptom severity. Our data suggest that a shift toward GABAergic neuron fate caused by FOXG1 is a developmental precursor of ASD.
Assuntos
Transtornos Globais do Desenvolvimento Infantil/genética , Transtornos Globais do Desenvolvimento Infantil/patologia , Fatores de Transcrição Forkhead/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurogênese , Telencéfalo/embriologia , Feminino , Perfilação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas , Masculino , Megalencefalia/genética , Megalencefalia/patologia , Modelos Biológicos , Neurônios/citologia , Neurônios/metabolismo , Organoides/patologia , Telencéfalo/patologiaRESUMO
Autism spectrum disorder (ASD) affects as many as 1 in 68 children and is said to be the fastest-growing serious developmental disability in the United States. There is currently no medical cure or diagnostic test for ASD. Furthermore, the U.S. Food and Drug Administration has yet to approve a single drug for the treatment of autism's core symptoms. Despite numerous genome studies and the identification of hundreds of genes that may cause or predispose children to ASD, the pathways underlying the pathogenesis of idiopathic ASD still remain elusive. Post-mortem brain samples, apart from being difficult to obtain, offer little insight into a disorder that arises through the course of development. Furthermore, ASD is a disorder of highly complex, human-specific behaviors, making it difficult to model in animals. Stem cell models of ASD can be generated by performing skin biopsies of ASD patients and then dedifferentiating these fibroblasts into human-induced pluripotent stem cells (hiPSCs). iPSCs closely resemble embryonic stem cells and retain the unique genetic signature of the ASD patient from whom they were originally derived. Differentiation of these iPSCs into neurons essentially recapitulates the ASD patient's neuronal development in a dish, allowing for a patient-specific model of ASD. Here we review our current understanding of the underlying neurobiology of ASD and how the use of stem cells can advance this understanding, possibly leading to new therapeutic avenues.
Assuntos
Transtorno do Espectro Autista/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Modelos Biológicos , Animais , Transtorno do Espectro Autista/terapia , Humanos , Sistema Nervoso/patologia , Transplante de Células-TroncoRESUMO
Intimal hyperplasia (IH) is a leading cause of obstruction of vascular interventions, including arterial stents, bypass grafts and arteriovenous grafts and fistulae. Proposals to account for arterial stent-associated IH include wall damage, low wall shear stress (WSS), disturbed flow and, although not widely recognized, wall hypoxia. The common non-planarity of arterial geometry and flow, led us to develop a bare-metal, nitinol, self-expanding stent with three-dimensional helical-centreline geometry. This was deployed in one common carotid artery of healthy pigs, with a straight-centreline, but otherwise identical (conventional) stent deployed contralaterally. Both stent types deformed the arteries, but the helical-centreline device additionally deformed them helically and caused swirling of intraluminal flow. At sacrifice, one month post stent deployment, histology revealed significantly less IH in the helical-centreline than straight-centreline stented vessels. Medial cross-sectional area was not significantly different in helical-centreline than straight-centreline stented vessels. By contrast, luminal cross-sectional area was significantly larger in helical-centreline than straight-centreline stented vessels. Mechanisms considered to account for those results include enhanced intraluminal WSS and enhanced intraluminal blood-vessel wall mass transport, including of oxygen, in the helical-centreline stented vessels. Consistent with the latter proposal, adventitial microvessel density was lower in the helical-centreline stented than straight-centreline stented vessels.
Assuntos
Artérias Carótidas/patologia , Stents/efeitos adversos , Animais , Velocidade do Fluxo Sanguíneo , Artérias Carótidas/cirurgia , Hiperplasia/patologia , Modelos Cardiovasculares , Sus scrofa , Túnica Íntima/patologiaRESUMO
The recent introduction of the induced pluripotent stem cell technology has made possible the derivation of neuronal cells from somatic cells obtained from human individuals. This in turn has opened new areas of investigation that can potentially bridge the gap between neuroscience and psychopathology. For the first time we can study the cell biology and genetics of neurons derived from any individual. Furthermore, by recapitulating in vitro the developmental steps whereby stem cells give rise to neuronal cells, we can now hope to understand factors that control typical and atypical development. We can begin to explore how human genes and their variants are transcribed into messenger RNAs within developing neurons and how these gene transcripts control the biology of developing cells. Thus, human-induced pluripotent stem cells have the potential to uncover not only what aspects of development are uniquely human but also variations in the series of events necessary for normal human brain development that predispose to psychopathology.
Assuntos
Encéfalo , Desenvolvimento Humano , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurônios/metabolismo , Encéfalo/embriologia , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Diferenciação Celular/genética , Epigênese Genética , Humanos , Modelos GenéticosRESUMO
Human induced pluripotent stem cells (hiPSCs) are emerging as a tool for understanding human brain development at cellular, molecular, and genomic levels. Here we show that hiPSCs grown in suspension in the presence of rostral neuralizing factors can generate 3D structures containing polarized radial glia, intermediate progenitors, and a spectrum of layer-specific cortical neurons reminiscent of their organization in vivo. The hiPSC-derived multilayered structures express a gene expression profile typical of the embryonic telencephalon but not that of other CNS regions. Their transcriptome is highly enriched in transcription factors controlling the specification, growth, and patterning of the dorsal telencephalon and displays highest correlation with that of the early human cerebral cortical wall at 8-10 wk after conception. Thus, hiPSC are capable of enacting a transcriptional program specifying human telencephalic (pallial) development. This model will allow the study of human brain development as well as disorders of the human cerebral cortex.
Assuntos
Córtex Cerebral , Células-Tronco Pluripotentes Induzidas , Modelos Biológicos , Neurônios , Transcriptoma/fisiologia , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/embriologia , Perfilação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The development of atherosclerosis has been shown to correlate with regions of low wall shear stress and seemingly reduced mass transport. The local tortuosity of the arteries and local secondary flow oscillation also seem to be negatively correlated with the local occurrence of the disease. However there is currently no tool or physiological parameter that can be measured non-invasively to assess the local oscillation of the flow. Standard Colour Doppler imaging of secondary flow patterns during the blood pulse is studied and illustrated, and the local oscillation of the secondary flow pattern is proposed as an index, which could be an indicator of the likelihood of future disease development. Preliminary results are presented using a basic estimator developed for the proof of concept in the case of swirling flow, and based on colour-coded video signals collected in different configurations. In vitro results show that there is a correspondence between the Doppler patterns and the secondary flow patterns, the repeatability of the measures, and that the proposed index and its estimator reflect a joint influence of the local oscillation of the secondary flow pattern and of the flow rate. On another hand, while in vivo results still suffer from instabilities, noise and from scanners and processing limitations, they demonstrate that it is possible to use Colour Doppler imaging to image and characterize in vivo the secondary flow patterns and their oscillations non-invasively, and that it is possible for a trained clinician to perform manually such Doppler measurements for processing.