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Mechanistic Target of Rapamycin Complex 1 (mTORC1) is a master metabolic regulator that is active in nearly all proliferating eukaryotic cells; however, it is unclear whether mTORC1 activity changes throughout the cell cycle. We find that mTORC1 activity oscillates from lowest in mitosis/G1 to highest in S/G2. The interphase oscillation is mediated through the TSC complex but is independent of major known regulatory inputs, including Akt and Mek/Erk signaling. By contrast, suppression of mTORC1 activity in mitosis does not require the TSC complex. mTORC1 has long been known to promote progression through G1. We find that mTORC1 also promotes progression through S and G2 and is important for satisfying the Chk1/Wee1-dependent G2/M checkpoint to allow entry into mitosis. We also find that low mTORC1 activity in G1 sensitizes cells to autophagy induction in response to partial mTORC1 inhibition or reduced nutrient levels. Together, these findings demonstrate that mTORC1 is differentially regulated throughout the cell cycle, with important phase-specific consequences for proliferating cells.
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Autism spectrum disorder (ASD) is defined by common behavioral characteristics, raising the possibility of shared pathogenic mechanisms. Yet, vast clinical and etiological heterogeneity suggests personalized phenotypes. Surprisingly, our iPSC studies find that six individuals from two distinct ASD subtypes, idiopathic and 16p11.2 deletion, have common reductions in neural precursor cell (NPC) neurite outgrowth and migration even though whole genome sequencing demonstrates no genetic overlap between the datasets. To identify signaling differences that may contribute to these developmental defects, an unbiased phospho-(p)-proteome screen was performed. Surprisingly despite the genetic heterogeneity, hundreds of shared p-peptides were identified between autism subtypes including the mTOR pathway. mTOR signaling alterations were confirmed in all NPCs across both ASD subtypes, and mTOR modulation rescued ASD phenotypes and reproduced autism NPC-associated phenotypes in control NPCs. Thus, our studies demonstrate that genetically distinct ASD subtypes have common defects in neurite outgrowth and migration which are driven by the shared pathogenic mechanism of mTOR signaling dysregulation.
Although the clinical presentation of individuals with autism spectrum disorder (ASD) can vary widely, the core features are repetitive behaviors and difficulties with social interactions and communication. In most cases, the cause of autism is unknown. However, in some cases, such as a form of ASD known as 16p11.2 deletion syndrome, specific genetic changes are responsible. Despite this variability in possible causes and clinical manifestations, the similarity of the core behavioral symptoms across different forms of the disorder indicates that there could be a shared biological mechanism. Furthermore, genetic studies suggest that abnormalities in early fetal brain development could be a crucial underlying cause of ASD. In order to form the complex structure of the brain, fetal brain cells must migrate and start growing extensions that ultimately become key structures of neurons. To test for shared biological mechanisms, Prem et al. reprogrammed blood cells from people with either 16p11.2 deletion syndrome or ASD with an unknown cause to become fetal-like brain cells. Experiments showed that both migration of the cells and their growth of extensions were similarly disrupted in the cells derived from both groups of individuals with autism. These crucial developmental changes were driven by alterations to an important signaling molecule in a pathway involved in brain function, known as the mTOR pathway. However, in some cells the pathway was overactive, whereas in others it was underactive. To probe the potential of the mTOR pathway as a therapeutic target, Prem et al. tested drugs that manipulate the pathway, finding that they could successfully reverse the defects in cells derived from people with both types of ASD. The discovery that a shared biological process may underpin different forms of ASD is important for understanding the early brain changes that are involved. A common target, like the mTOR pathway, could offer hope for treatments for a wide range of ASDs. However, to translate these benefits to the clinic, further research is needed to understand whether a treatment that is effective in fetal cells would also benefit people with autism.
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Transtorno do Espectro Autista , Transtorno Autístico , Células-Tronco Neurais , Humanos , Transtorno Autístico/genética , Transtorno do Espectro Autista/genética , Neuritos , Serina-Treonina Quinases TORRESUMO
Mechanistic Target of Rapamycin Complex 1 (mTORC1) is a master metabolic regulator that stimulates anabolic cell growth while suppressing catabolic processes such as autophagy. mTORC1 is active in most, if not all, proliferating eukaryotic cells. However, it remains unclear whether and how mTORC1 activity changes from one cell cycle phase to another. Here we tracked mTORC1 activity through the complete cell cycle and uncover oscillations in its activity. We find that mTORC1 activity peaks in S and G2, and is lowest in mitosis and G1. We further demonstrate that multiple mechanisms are involved in controlling this oscillation. The interphase oscillation is mediated through the TSC complex, an upstream negative regulator of mTORC1, but is independent of major known regulatory inputs to the TSC complex, including Akt, Mek/Erk, and CDK4/6 signaling. By contrast, suppression of mTORC1 activity in mitosis does not require the TSC complex, and instead involves CDK1-dependent control of the subcellular localization of mTORC1 itself. Functionally, we find that in addition to its well-established role in promoting progression through G1, mTORC1 also promotes progression through S and G2, and is important for satisfying the Wee1- and Chk1- dependent G2/M checkpoint to allow entry into mitosis. We also find that low mTORC1 activity in G1 sensitizes cells to autophagy induction in response to partial mTORC1 inhibition or reduced nutrient levels. Together these findings demonstrate that mTORC1 is differentially regulated throughout the cell cycle, with important phase-specific functional consequences in proliferating cells.
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Abnormalities in neocortical and synaptic development are linked to neurodevelopmental disorders. However, the molecular and cellular mechanisms governing initial synapse formation in the prenatal neocortex remain poorly understood. Using polysome profiling coupled with snRNAseq on human cortical samples at various fetal phases, we identify human mRNAs, including those encoding synaptic proteins, with finely controlled translation in distinct cell populations of developing frontal neocortices. Examination of murine and human neocortex reveals that the RNA binding protein and translational regulator, CELF4, is expressed in compartments enriched in initial synaptogenesis: the marginal zone and the subplate. We also find that Celf4/CELF4-target mRNAs are encoded by risk genes for adverse neurodevelopmental outcomes translating into synaptic proteins. Surprisingly, deleting Celf4 in the forebrain disrupts the balance of subplate synapses in a sex-specific fashion. This highlights the significance of RNA binding proteins and mRNA translation in evolutionarily advanced synaptic development, potentially contributing to sex differences.
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Proteínas CELF , Neocórtex , Animais , Feminino , Humanos , Masculino , Camundongos , Gravidez , Neocórtex/metabolismo , Neurônios/metabolismo , Polirribossomos/metabolismo , Biossíntese de Proteínas , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Sinapses/metabolismo , Proteínas CELF/genética , Proteínas CELF/metabolismoRESUMO
Neural precursor cell (NPC) dysfunction has been consistently implicated in autism. Induced pluripotent stem cell (iPSC)-derived NPCs from two autism groups (three idiopathic [I-ASD] and two 16p11.2 deletion [16pDel]) were used to investigate if proliferation is commonly disrupted. All five individuals display defects, with all three macrocephalic individuals (two 16pDel, one I-ASD) exhibiting hyperproliferation and the other two I-ASD subjects displaying hypoproliferation. NPCs were challenged with bFGF, and all hyperproliferative NPCs displayed blunted responses, while responses were increased in hypoproliferative cells. mRNA expression studies suggest that different pathways can result in similar proliferation phenotypes. Since 16pDel deletes MAPK3, P-ERK was measured. P-ERK is decreased in hyperproliferative but increased in hypoproliferative NPCs. While these P-ERK changes are not responsible for the phenotypes, P-ERK and bFGF response are inversely correlated with the defects. Finally, we analyzed iPSCs and discovered that 16pDel displays hyperproliferation, while idiopathic iPSCs were normal. These data suggest that NPC proliferation defects are common in ASD.
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Transtorno Autístico , Células-Tronco Pluripotentes Induzidas , Transtorno Autístico/genética , Proliferação de Células/genética , Deleção Cromossômica , Humanos , Mitógenos , FenótipoRESUMO
Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder that is remarkably heterogeneous at the clinical, neurobiological, and genetic levels. ASD can also affect language, a uniquely human capability, and is caused by abnormalities in brain development. Traditionally obtaining biologically relevant human cells to study ASD has been extremely difficult, but new technologies including iPSC-derived neurons and high-throughput omic techniques now provide new, exciting tools to uncover the cellular and signaling basis of ASD etiology.
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Transtorno do Espectro Autista , Transtorno Autístico , Células-Tronco Pluripotentes Induzidas , Transtorno do Espectro Autista/genética , Transtorno Autístico/genética , Humanos , Neurônios , FenótipoRESUMO
Despite decades of study, elucidation of the underlying etiology of complex developmental disorders such as autism spectrum disorder (ASD), schizophrenia (SCZ), intellectual disability (ID), and bipolar disorder (BPD) has been hampered by the inability to study human neurons, the heterogeneity of these disorders, and the relevance of animal model systems. Moreover, a majority of these developmental disorders have multifactorial or idiopathic (unknown) causes making them difficult to model using traditional methods of genetic alteration. Examination of the brains of individuals with ASD and other developmental disorders in both post-mortem and MRI studies shows defects that are suggestive of dysregulation of embryonic and early postnatal development. For ASD, more recent genetic studies have also suggested that risk genes largely converge upon the developing human cerebral cortex between weeks 8 and 24 in utero. Yet, an overwhelming majority of studies in autism rodent models have focused on postnatal development or adult synaptic transmission defects in autism related circuits. Thus, studies looking at early developmental processes such as proliferation, cell migration, and early differentiation, which are essential to build the brain, are largely lacking. Yet, interestingly, a few studies that did assess early neurodevelopment found that alterations in brain structure and function associated with neurodevelopmental disorders (NDDs) begin as early as the initial formation and patterning of the neural tube. By the early to mid-2000s, the derivation of human embryonic stem cells (hESCs) and later induced pluripotent stem cells (iPSCs) allowed us to study living human neural cells in culture for the first time. Specifically, iPSCs gave us the unprecedented ability to study cells derived from individuals with idiopathic disorders. Studies indicate that iPSC-derived neural cells, whether precursors or "matured" neurons, largely resemble cortical cells of embryonic humans from weeks 8 to 24. Thus, these cells are an excellent model to study early human neurodevelopment, particularly in the context of genetically complex diseases. Indeed, since 2011, numerous studies have assessed developmental phenotypes in neurons derived from individuals with both genetic and idiopathic forms of ASD and other NDDs. However, while iPSC-derived neurons are fetal in nature, they are post-mitotic and thus cannot be used to study developmental processes that occur before terminal differentiation. Moreover, it is important to note that during the 8-24-week window of human neurodevelopment, neural precursor cells are actively undergoing proliferation, migration, and early differentiation to form the basic cytoarchitecture of the brain. Thus, by studying NPCs specifically, we could gain insight into how early neurodevelopmental processes contribute to the pathogenesis of NDDs. Indeed, a few studies have explored NPC phenotypes in NDDs and have uncovered dysregulations in cell proliferation. Yet, few studies have explored migration and early differentiation phenotypes of NPCs in NDDs. In this chapter, we will discuss cell migration and neurite outgrowth and the role of these processes in neurodevelopment and NDDs. We will begin by reviewing the processes that are important in early neurodevelopment and early cortical development. We will then delve into the roles of neurite outgrowth and cell migration in the formation of the brain and how errors in these processes affect brain development. We also provide review of a few key molecules that are involved in the regulation of neurite outgrowth and migration while discussing how dysregulations in these molecules can lead to abnormalities in brain structure and function thereby highlighting their contribution to pathogenesis of NDDs. Then we will discuss whether neurite outgrowth, migration, and the molecules that regulate these processes are associated with ASD. Lastly, we will review the utility of iPSCs in modeling NDDs and discuss future goals for the study of NDDs using this technology.
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Transtorno do Espectro Autista , Transtorno Autístico , Células-Tronco Neurais , Transtornos do Neurodesenvolvimento , Animais , Transtorno do Espectro Autista/genética , Transtorno Autístico/genética , Movimento Celular , Humanos , Transtornos do Neurodesenvolvimento/genética , Crescimento NeuronalRESUMO
Human brain development proceeds through a series of precisely orchestrated processes, with earlier stages distinguished by proliferation, migration, and neurite outgrowth; and later stages characterized by axon/dendrite outgrowth and synapse formation. In neurodevelopmental disorders, often one or more of these processes are disrupted, leading to abnormalities in brain formation and function. With the advent of human induced pluripotent stem cell (hiPSC) technology, researchers now have an abundant supply of human cells that can be differentiated into virtually any cell type, including neurons. These cells can be used to study both normal brain development and disease pathogenesis. A number of protocols using hiPSCs to model neuropsychiatric disease use terminally differentiated neurons or use 3D culture systems termed organoids. While these methods have proven invaluable in studying human disease pathogenesis, there are some drawbacks. Differentiation of hiPSCs into neurons and generation of organoids are lengthy and costly processes that can impact the number of experiments and variables that can be assessed. In addition, while post-mitotic neurons and organoids allow the study of disease-related processes, including dendrite outgrowth and synaptogenesis, they preclude the study of earlier processes like proliferation and migration. In neurodevelopmental disorders, such as autism, abundant genetic and post-mortem evidence indicates defects in early developmental processes. Neural precursor cells (NPCs), a highly proliferative cell population, may be a suitable model in which to ask questions about ontogenetic processes and disease initiation. We now extend methodologies learned from studying development in mouse and rat cortical cultures to human NPCs. The use of NPCs allows us to investigate disease-related phenotypes and define how different variables (e.g., growth factors, drugs) impact developmental processes including proliferation, migration, and differentiation in only a few days. Ultimately, this toolset can be used in a reproducible and high-throughput manner to identify disease-specific mechanisms and phenotypes in neurodevelopmental disorders.
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Células-Tronco Neurais/metabolismo , Transtornos do Neurodesenvolvimento/diagnóstico , Neurônios/metabolismo , Animais , Diferenciação Celular , Movimento Celular , Humanos , Camundongos , Células-Tronco Neurais/citologia , Transtornos do Neurodesenvolvimento/patologia , Fenótipo , RatosRESUMO
Late infantile neuronal ceroid lipofuscinosis (LINCL) is a fatal inherited neurodegenerative disease caused by loss of lysosomal protease tripeptidyl peptidase 1 (TPP1). We have investigated the effects of chronic intrathecal (IT) administration using enzyme replacement therapy (ERT) to the brain of an LINCL mouse model, in which locomotor function declines dramatically prior to early death. Median lifespan was significantly extended from 126 days to >259 days when chronic IT treatment was initiated before the onset of disease. While treated animals lived longer and showed little sign of locomotor dysfunction as measured by stride length, some or all (depending on regimen) still died prematurely. One explanation is that cerebrospinal fluid (CSF)-mediated delivery may not deliver TPP1 to all brain regions. Morphological studies support this, showing delivery of TPP1 to ventral, but not deeper and dorsal regions. When IT treatment is initiated in severely affected LINCL mice, lifespan was extended modestly in most but dramatically extended in approximately one-third of the cohort. Treatment improved locomotor function in these severely compromised animals after it had declined to the point at which animals normally die. This indicates that some pathology in LINCL is reversible and does not simply reflect neuronal death.
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The morphology and severity of human congenital cataract varies even among individuals with the same mutation, suggesting that genetic background modifies phenotypic penetrance. The spontaneous mouse mutant, vacuolated lens (vl), arose on the C3H/HeSnJ background. The mutation disrupts secondary lens fiber development by E16.5, leading to full penetrance of congenital cataract. The vl locus was mapped to a frameshift deletion in the orphan G protein-coupled receptor, Gpr161, which is expressed in differentiating lens fiber cells. When Gpr161vl/vl C3H mice are crossed to MOLF/EiJ mice an unexpected rescue of cataract is observed, suggesting that MOLF modifiers affect cataract penetrance. Subsequent QTL analysis mapped three modifiers (Modvl3-5: Modifier of vl) and in this study we characterized Modvl4 (Chr15; LOD = 4.4). A Modvl4MOLF congenic was generated and is sufficient to rescue congenital cataract and the lens fiber defect at E16.5. Additional phenotypic analysis on three subcongenic lines narrowed down the interval from 55 to 15Mb. In total only 18 protein-coding genes and 2 micro-RNAs are in this region. Fifteen of the 20 genes show detectable expression in the E16.5 eye. Subsequent expression studies in Gpr161vl/vl and subcongenic E16.5 eyes, bioinformatics analysis of C3H/MOLF polymorphisms, and the biological relevancy of the genes in the interval identified three genes (Cdh6, Ank and Trio) that likely contribute to the rescue of the lens phenotype. These studies demonstrate that modification of the Gpr161vl/vl cataract phenotype is likely due to genetic variants in at least one of three closely linked candidate genes on proximal Chr15.
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Catarata/congênito , Catarata/genética , Cromossomos de Mamíferos/metabolismo , Cristalino/metabolismo , Receptores Acoplados a Proteínas G/genética , Animais , Pareamento de Bases/genética , Diferenciação Celular , Cruzamentos Genéticos , Feminino , Regulação da Expressão Gênica , Genes Dominantes , Estudos de Associação Genética , Cristalino/patologia , Masculino , Camundongos , Anotação de Sequência Molecular , Mapeamento Físico do Cromossomo , Polimorfismo de Nucleotídeo Único/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Retinoic acid (RA) is an important developmental morphogen that coordinates anteroposterior and dorsoventral axis patterning, somitic differentiation, neurogenesis, patterning of the hindbrain and spinal cord, and the development of multiple organ systems. Due to its chemical nature as a small amphipathic lipid, direct detection and visualization of RA histologically remains technically impossible. Currently, methods used to infer the presence and localization of RA make use of reporter systems that detect the biological activity of RA. Most established reporter systems, both transgenic mice and cell lines, make use of the highly potent RA response element (RARE) upstream of the RAR-beta gene to drive RA-inducible expression of reporter genes, such as beta-galactosidase or luciferase. The transgenic RARE-LacZ mouse is useful in visualizing spatiotemporal changes in RA signaling especially during embryonic development. However, it does not directly measure overall RA levels. As a reporter system, the F9 RARE-LacZ cell line can be used in a variety of ways, from simple detection of RA to quantitative measurements of RA levels in tissue explants. Here we describe the quantitative determination of relative RA levels generated in embryos and neurosphere cultures using the F9 RARE-LacZ reporter cell line.
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Genes Reporter , Óperon Lac , Tretinoína , Animais , Linhagem Celular , Embrião de Mamíferos , Feminino , Camundongos Transgênicos , Gravidez , beta-GalactosidaseRESUMO
Many genes involved in brain development have been associated with human neurodevelopmental disorders, but underlying pathophysiological mechanisms remain undefined. Human genetic and mouse behavioral analyses suggest that ENGRAILED-2 (EN2) contributes to neurodevelopmental disorders, especially autism spectrum disorder. In mouse, En2 exhibits dynamic spatiotemporal expression in embryonic mid-hindbrain regions where monoamine neurons emerge. Considering their importance in neuropsychiatric disorders, we characterized monoamine systems in relation to forebrain neurogenesis in En2-knockout (En2-KO) mice. Transmitter levels of serotonin, dopamine and norepinephrine (NE) were dysregulated from Postnatal day 7 (P7) to P21 in En2-KO, though NE exhibited the greatest abnormalities. While NE levels were reduced â¼35% in forebrain, they were increased 40 -: 75% in hindbrain and cerebellum, and these patterns paralleled changes in locus coeruleus (LC) fiber innervation, respectively. Although En2 promoter was active in Embryonic day 14.5 -: 15.5 LC neurons, expression diminished thereafter and gene deletion did not alter brainstem NE neuron numbers. Significantly, in parallel with reduced NE levels, En2-KO forebrain regions exhibited reduced growth, particularly hippocampus, where P21 dentate gyrus granule neurons were decreased 16%, suggesting abnormal neurogenesis. Indeed, hippocampal neurogenic regions showed increased cell death (+77%) and unexpectedly, increased proliferation. Excess proliferation was restricted to early Sox2/Tbr2 progenitors whereas increased apoptosis occurred in differentiating (Dcx) neuroblasts, accompanied by reduced newborn neuron survival. Abnormal neurogenesis may reflect NE deficits because intra-hippocampal injections of ß-adrenergic agonists reversed cell death. These studies suggest that disruption of hindbrain patterning genes can alter monoamine system development and thereby produce forebrain defects that are relevant to human neurodevelopmental disorders.
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Neurônios Dopaminérgicos/metabolismo , Proteínas de Homeodomínio/genética , Proteínas do Tecido Nervoso/genética , Neurogênese , Prosencéfalo/metabolismo , Neurônios Serotoninérgicos/metabolismo , Animais , Neurônios Dopaminérgicos/fisiologia , Proteína Duplacortina , Feminino , Deleção de Genes , Humanos , Masculino , Camundongos , Camundongos Knockout , Norepinefrina/metabolismo , Prosencéfalo/crescimento & desenvolvimento , Prosencéfalo/patologia , Prosencéfalo/fisiopatologia , Neurônios Serotoninérgicos/fisiologia , NataçãoRESUMO
The vacuolated lens (vl) mouse mutation arose on the C3H/HeSnJ background and results in lethality, neural tube defects (NTDs) and cataracts. The vl phenotypes are due to a deletion/frameshift mutation in the orphan GPCR, Gpr161. A recent study using a null allele demonstrated that Gpr161 functions in primary cilia and represses the Shh pathway. We show the hypomorphic Gpr161(vl) allele does not severely affect the Shh pathway. To identify additional pathways regulated by Gpr161 during neurulation, we took advantage of naturally occurring genetic variation in the mouse. Previously Gpr161(vl-C3H) was crossed to different inbred backgrounds including MOLF/EiJ and the Gpr161(vl) mutant phenotypes were rescued. Five modifiers were mapped (Modvl: Modifier of vl) including Modvl5(MOLF). In this study we demonstrate the Modvl5(MOLF) congenic rescues the Gpr161(vl)-associated lethality and NTDs but not cataracts. Bioinformatics determined the transcription factor, Cdx1, is the only annotated gene within the Modvl5 95% CI co-expressed with Gpr161 during neurulation and not expressed in the eye. Using Cdx1 as an entry point, we identified the retinoid acid (RA) and canonical Wnt pathways as downstream targets of Gpr161. QRT-PCR, ISH and IHC determined that expression of RA and Wnt genes are down-regulated in Gpr161(vl/vl) but rescued by the Modvl5(MOLF) congenic during neurulation. Intraperitoneal RA injection restores expression of canonical Wnt markers and rescues Gpr161(vl/vl) NTDs. These results establish the RA and canonical Wnt as pathways downstream of Gpr161 during neurulation, and suggest that Modvl5(MOLF) bypasses the Gpr161(vl) mutation by restoring the activity of these pathways.
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Regulação da Expressão Gênica no Desenvolvimento , Neurulação , Receptores Acoplados a Proteínas G/metabolismo , Tretinoína/metabolismo , Proteínas Wnt/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Genes Reporter , Variação Genética , Proteínas Hedgehog/metabolismo , Proteínas de Homeodomínio/metabolismo , Hibridização In Situ , Camundongos , Camundongos Endogâmicos C3H , Camundongos Transgênicos , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Defeitos do Tubo Neural/genética , Fenótipo , Locos de Características Quantitativas , RNA Mensageiro/metabolismo , Transdução de Sinais , Fatores de Tempo , Via de Sinalização Wnt , Proteína Wnt3A/metabolismoRESUMO
BACKGROUND: Previous genetic studies demonstrated association between the transcription factor engrailed2 (EN2) and Autism Spectrum Disorder (ASD). Subsequent molecular analysis determined that the EN2 ASD-associated haplotype (rs1861972-rs1861973 A-C) functions as a transcriptional activator to increase gene expression. EN2 is flanked by 5 genes, serotonin receptor5a (HTR5A), insulin induced gene1 (INSIG1), canopy1 homolog (CNPY1), RNA binding motif protein33 (RBM33), and sonic hedgehog (SHH). These flanking genes are co-expressed with EN2 during development and coordinate similar developmental processes. To investigate if mRNA levels for these genes are altered in individuals with autism, post-mortem analysis was performed. METHODS: qRT-PCR quantified mRNA levels for EN2 and the 5 flanking genes in 78 post-mortem cerebellar samples. mRNA levels were correlated with both affection status and rs1861972-rs1861973 genotype. Molecular analysis investigated whether EN2 regulates flanking gene expression. RESULTS: EN2 levels are increased in affected A-C/G-T individuals (p =â.0077). Affected individuals also display a significant increase in SHH and a decrease in INSIG1 levels. Rs1861972-rs1861973 genotype is correlated with significant increases for SHH (A-C/G-T) and CNPY1 (G-T/G-T) levels. Human cell line over-expression and knock-down as well as mouse knock-out analysis are consistent with EN2 and SHH being co-regulated, which provides a possible mechanism for increased SHH post-mortem levels. CONCLUSIONS: EN2 levels are increased in affected individuals with an A-C/G-T genotype, supporting EN2 as an ASD susceptibility gene. SHH, CNPY1, and INSIG1 levels are also significantly altered depending upon affection status or rs1861972-rs1861973 genotype. Increased EN2 levels likely contribute to elevated SHH expression observed in the post-mortem samples.
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Transtorno Autístico/genética , Cerebelo/metabolismo , DNA Intergênico/genética , Predisposição Genética para Doença , Proteínas de Homeodomínio/genética , Proteínas do Tecido Nervoso/genética , Mudanças Depois da Morte , Animais , Cerebelo/patologia , Regulação da Expressão Gênica , Genoma Humano/genética , Células HEK293 , Haplótipos/genética , Proteínas Hedgehog/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
BACKGROUND: The homeobox transcription factor Engrailed2 (En2) has been studied extensively in neurodevelopment, particularly in the midbrain/hindbrain region and cerebellum, where it exhibits dynamic patterns of expression and regulates cell patterning and morphogenesis. Because of its roles in regulating cerebellar development and evidence of cerebellar pathology in autism spectrum disorder (ASD), we previously examined an ENGRAILED2 association and found evidence to support EN2 as a susceptibility gene, a finding replicated by several other investigators. However, its functions at the cell biological level remain undefined. In the mouse, En2 gene is expressed in granule neuron precursors (GNPs) just as they exit the cell cycle and begin to differentiate, raising the possibility that En2 may modulate these developmental processes. METHODS: To define En2 functions, we examined proliferation, differentiation and signaling pathway activation in En2 knockout (KO) and wild-type (WT) GNPs in response to a variety of extracellular growth factors and following En2 cDNA overexpression in cell culture. In vivo analyses of cerebellar GNP proliferation as well as responses to insulin-like growth factor-1 (IGF1) treatment were also conducted. RESULTS: Proliferation markers were increased in KO GNPs in vivo and in 24-h cultures, suggesting En2 normally serves to promote cell cycle exit. Significantly, IGF1 stimulated greater DNA synthesis in KO than WT cells in culture, a finding associated with markedly increased phospho-S6 kinase activation. Similarly, there was three-fold greater DNA synthesis in the KO cerebellum in response to IGF1 in vivo. On the other hand, KO GNPs exhibited reduced neurite outgrowth and differentiation. Conversely, En2 overexpression increased cell cycle exit and promoted neuronal differentiation. CONCLUSIONS: In aggregate, our observations suggest that the ASD-associated gene En2 promotes GNP cell cycle exit and differentiation, and modulates IGF1 activity during postnatal cerebellar development. Thus, genetic/epigenetic alterations of EN2 expression may impact proliferation, differentiation and IGF1 signaling as possible mechanisms that may contribute to ASD pathogenesis.
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In the follow-up of New Jersey newborn screens suggestive of medium chain acyl-CoA dehydrogenase deficiency (MCADD) during a 30-month period, we identified five patients of Hispanic American ethnicity. With information provided by the New Jersey Department of Health and Human Services Newborn Screening program we calculated an overall cumulative incidence of approximately 7.20/100,000 for MCADD; 7.58/100,000 among Hispanic Americans and 7.08/100,000 among non-Hispanic Americans. Among the five Hispanic American infants who screened positive, a common variant (c.443G>A [p.R148K]) was identified which accounted for 30% of the alleles; c.799G>A (p.G267R) and c.985A>G (p.K329E) each accounted for an additional 20%; and a novel variant c.302G>A (p.G101E) was identified in one patient. Although treated prospectively during interim illnesses to prevent unwanted sequelae; till date, none of the patients carrying the c.443G>A variant have been symptomatic.
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Hispânico ou Latino , Erros Inatos do Metabolismo Lipídico/diagnóstico , Triagem Neonatal , Acil-CoA Desidrogenase/deficiência , Acil-CoA Desidrogenase/genética , Humanos , Recém-Nascido , Erros Inatos do Metabolismo Lipídico/genética , Mutação , New Jersey , Estudos ProspectivosRESUMO
ENGRAILED 2 (En2), a homeobox transcription factor, functions as a patterning gene in the early development and connectivity of rodent hindbrain and cerebellum, and regulates neurogenesis and development of monoaminergic pathways. To further understand the neurobiological functions of En2, we conducted neuroanatomical expression profiling of En2 wildtype mice. RTQPCR assays demonstrated that En2 is expressed in adult brain structures including the somatosensory cortex, hippocampus, striatum, thalamus, hypothalamus and brainstem. Human genetic studies indicate that EN2 is associated with autism. To determine the consequences of En2 mutations on mouse behaviors, including outcomes potentially relevant to autism, we conducted comprehensive phenotyping of social, communication, repetitive, and cognitive behaviors. En2 null mutants exhibited robust deficits in reciprocal social interactions as juveniles and adults, and absence of sociability in adults, replicated in two independent cohorts. Fear conditioning and water maze learning were impaired in En2 null mutants. High immobility in the forced swim test, reduced prepulse inhibition, mild motor coordination impairments and reduced grip strength were detected in En2 null mutants. No genotype differences were found on measures of ultrasonic vocalizations in social contexts, and no stereotyped or repetitive behaviors were observed. Developmental milestones, general health, olfactory abilities, exploratory locomotor activity, anxiety-like behaviors and pain responses did not differ across genotypes, indicating that the behavioral abnormalities detected in En2 null mutants were not attributable to physical or procedural confounds. Our findings provide new insight into the role of En2 in complex behaviors and suggest that disturbances in En2 signaling may contribute to neuropsychiatric disorders marked by social and cognitive deficits, including autism spectrum disorders.
Assuntos
Transtorno Autístico/fisiopatologia , Transtornos Cognitivos/fisiopatologia , Animais , Transtorno Autístico/genética , Encéfalo/fisiologia , Tronco Encefálico/fisiologia , Transtornos Cognitivos/genética , Hipocampo/fisiologia , Proteínas de Homeodomínio , Hipotálamo/fisiologia , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso , Transdução de Sinais , Comportamento Social , Córtex Somatossensorial/fisiologiaRESUMO
Both common and rare variants contribute to autism spectrum disorder (ASD) risk, but few variants have been established as functional. Previously we demonstrated that an intronic haplotype (rs1861972-rs1861973 A-C) in the homeobox transcription factor ENGRAILED2 (EN2) is significantly associated with ASD. Positive association has also been reported in six additional data sets, suggesting EN2 is an ASD susceptibility gene. Additional support for this possibility requires identification of functional variants that affect EN2 regulation or activity. In this study, we demonstrate that the A-C haplotype is a transcriptional activator. Luciferase (luc) assays in mouse neuronal cultures determined that the A-C haplotype increases expression levels (50%, P < 0.01, 24 h; 250%, P < 0.0001, 72 h). Mutational analysis indicates that the A-C haplotype activator function requires both associated A and C alleles. A minimal 202-bp element is sufficient for function and also specifically binds a protein complex. Mass spectrometry identified these proteins as the transcription factors, Cut-like homeobox 1 (Cux1) and nuclear factor I/B (Nfib). Subsequent antibody supershifts and chromatin immunoprecipitations demonstrated that human CUX1 and NFIB bind the A-C haplotype. Co-transfection and knock-down experiments determined that both CUX1 and NFIB are required for the A-C haplotype activator function. These data demonstrate that the ASD-associated A-C haplotype is a transcriptional activator, and both CUX1 and NFIB mediate this activity. These results provide biochemical evidence that the ASD-associated A-C haplotype is functional, further supporting EN2 as an ASD susceptibility gene.
Assuntos
Transtornos Globais do Desenvolvimento Infantil/genética , Haplótipos , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição NFI/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Alelos , Animais , Linhagem Celular , Criança , Regulação da Expressão Gênica , Proteínas de Homeodomínio/biossíntese , Humanos , Íntrons , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/biossíntese , Primatas , Fatores de TranscriçãoRESUMO
The severe disorders associated with a loss or dysfunction of midbrain dopamine neurons (DNs) have intensified research aimed at deciphering developmental programs controlling midbrain development. The homeodomain proteins Lmx1a and Lmx1b are important for the specification of DNs during embryogenesis, but it is unclear to what degree they may mediate redundant or specific functions. Here, we provide evidence showing that DN progenitors in the ventral midbrain can be subdivided into molecularly distinct medial and lateral domains, and these subgroups show different sensitivity to the loss of Lmx1a and Lmx1b. Lmx1a is specifically required for converting non-neuronal floor-plate cells into neuronal DN progenitors, a process that involves the establishment of Notch signaling in ventral midline cells. On the other hand, lateral DN progenitors that do not appear to originate from the floor plate are selectively ablated in Lmx1b mutants. In addition, we also reveal an unanticipated role for Lmx1b in regulating Phox2a expression and the sequential specification of ocular motor neurons (OMNs) and red nucleus neurons (RNNs) from progenitors located lateral to DNs in the midbrain. Our data therefore establish that Lmx1b influences the differentiation of multiple neuronal subtypes in the ventral midbrain, whereas Lmx1a appears to be exclusively devoted to the differentiation of the DN lineage.