RESUMEN
The sophisticated function of the central nervous system (CNS) is largely supported by proper interactions between neural cells and blood vessels. Accumulating evidence has demonstrated that neurons and glial cells support the formation of blood vessels, which in turn, act as migratory scaffolds for these cell types. Neural progenitors are also involved in the regulation of blood vessel formation. This mutual interaction between neural cells and blood vessels is elegantly controlled by several chemokines, growth factors, extracellular matrix, and adhesion molecules such as integrins. Recent research has revealed that newly migrating cell types along blood vessels repel other preexisting migrating cell types, causing them to detach from the blood vessels. In this review, we discuss vascular formation and cell migration, particularly during development. Moreover, we discuss how the crosstalk between blood vessels and neurons and glial cells could be related to neurodevelopmental disorders.
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Sistema Nervioso Central , Neuronas , Neuronas/metabolismo , Sistema Nervioso Central/fisiología , Movimiento Celular/fisiología , Integrinas/metabolismo , Vasos Sanguíneos/fisiologíaRESUMEN
Rho family small GTPases, such as Rho, Rac, and Cdc42, play essential roles during brain development, by regulating cellular signaling and actin cytoskeletal reorganization. Rich2/Arhgap44, a Rac- and Cdc42-specific GTPase-activating protein, has been reported to be a key regulator for dendritic spine morphology and synaptic function. Given the essential roles of Rac and Cdc42 in brain development, Rich2 is supposed to take part in brain development. However, not only the molecular mechanism involved but also the expression profile of Rich2 during neurodevelopment has not yet been elucidated. In this study, we carried out expression analyses of Rich2 by focusing on mouse brain development. In immunoblotting, Rich2 exhibited a tissue-dependent expression profile in the young adult mouse, and the expression was increased during brain development. In immunohistochemical analyses, Rich2 was observed in the cytoplasm of cortical neurons at postnatal day (P) 0 and then came to be enriched in the nucleus with moderate distribution in neuropils at P7. Later at P30, a complex immunostaining pattern of Rich2 was observed; Rich2 was distributed in the nucleus, cytoplasm, and neuropils in many cortical neurons, whereas other neurons frequently displayed little expression. In the hippocampus at P7, Rich2 was distributed mainly in the cytoplasm of excitatory neurons in the cornu ammonis regions, while it was moderately detected in the nucleus in the dentate granule cells. Notably, Rich2 was distributed in excitatory synapses of the cornu ammonis 1 region at P30. Biochemical fractionation analyses also detected Rich2 in the postsynaptic density. Taken together, Rich2 is found to be expressed in the central nervous system in a developmental stage-dependent manner and may be involved in synapse formation/maintenance in cortical neurons.
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Proteínas Activadoras de GTPasa , Neuronas , Ratones , Animales , Proteínas Activadoras de GTPasa/metabolismo , Neuronas/metabolismo , Hipocampo/metabolismo , Sinapsis/metabolismo , NeurogénesisRESUMEN
INTRODUCTION: CtBP1 (C-terminal-binding protein 1) is a multi-functional protein with well-established roles as a transcriptional co-repressor in the nucleus and a regulator of membrane fission in the cytoplasm. Although CtBP1 gene abnormalities have been reported to cause neurodevelopmental disorders, the physiological role and expression profile of CtBP1 remains to be elucidated. METHODS: In this study, we used biochemical, immunohistochemical and immunofluorescence methods to analyze the expression of CtBP1 during mouse brain development. RESULTS: Western blotting analyses revealed that CtBP1 appeared to be expressed mainly in the central nervous system throughout the developmental process. In immunohistochemical analyses, region-specific nuclear as well as weak cytoplasmic distribution of CtBP1 was observed in telencephalon at embryonic day (E)15 and E17. It is of note that CtBP1 was barely detected in axons, but observed in the nucleus of oligodendrocytes in the white matter at E17. As to cerebellum at postnatal day 30, CtBP1 appeared to be expressed in the nucleus and cytoplasm of Purkinje cells, the nucleus of granule cells and cells in the molecular layer (ML), and the ML per se where granule cell axons and Purkinje cell dendrites are enriched. In addition, CtBP1 was detected in the cerebellar nuclei. CONCLUSION: The obtained results suggest involvement of CtBP1 in brain function.
RESUMEN
Variants in RAC3, encoding a small GTPase RAC3 which is critical for the regulation of actin cytoskeleton and intracellular signal transduction, are associated with a rare neurodevelopmental disorder with structural brain anomalies and facial dysmorphism. We investigated a cohort of 10 unrelated participants presenting with global psychomotor delay, hypotonia, behavioural disturbances, stereotyped movements, dysmorphic features, seizures and musculoskeletal abnormalities. MRI of brain revealed a complex pattern of variable brain malformations, including callosal abnormalities, white matter thinning, grey matter heterotopia, polymicrogyria/dysgyria, brainstem anomalies and cerebellar dysplasia. These patients harboured eight distinct de novo RAC3 variants, including six novel variants (NM_005052.3): c.34G > C p.G12R, c.179G > A p.G60D, c.186_188delGGA p.E62del, c.187G > A p.D63N, c.191A > G p.Y64C and c.348G > C p.K116N. We then examined the pathophysiological significance of these novel and previously reported pathogenic variants p.P29L, p.P34R, p.A59G, p.Q61L and p.E62K. In vitro analyses revealed that all tested RAC3 variants were biochemically and biologically active to variable extent, and exhibited a spectrum of different affinities to downstream effectors including p21-activated kinase 1. We then focused on the four variants p.Q61L, p.E62del, p.D63N and p.Y64C in the Switch II region, which is essential for the biochemical activity of small GTPases and also a variation hot spot common to other Rho family genes, RAC1 and CDC42. Acute expression of the four variants in embryonic mouse brain using in utero electroporation caused defects in cortical neuron morphology and migration ending up with cluster formation during corticogenesis. Notably, defective migration by p.E62del, p.D63N and p.Y64C were rescued by a dominant negative version of p21-activated kinase 1. Our results indicate that RAC3 variants result in morphological and functional defects in cortical neurons during brain development through variant-specific mechanisms, eventually leading to heterogeneous neurodevelopmental phenotypes.
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Trastornos del Neurodesarrollo , Proteínas de Unión al GTP rac , Animales , Humanos , Ratones , Trastornos del Neurodesarrollo/genética , Trastornos del Neurodesarrollo/metabolismo , Neuronas/metabolismo , Fenotipo , Quinasas p21 Activadas/genética , Proteínas de Unión al GTP rac/genética , Proteínas de Unión al GTP rac/metabolismoRESUMEN
Rac3 is a member of Rho family small GTPases which regulate cellular signaling and cytoskeletal dynamics. The RAC3 gene abnormalities have been shown to cause neurodevelopmental disorders with structural brain anomalies, including polymicrogyria/dysgyria, callosal abnormalities, brainstem anomalies, and cerebellar dysplasia. Although this evidence indicates that Rac3 is essential in brain development, not only its molecular mechanism but also the expression profile is yet to be elucidated. In this study, we carried out expression analyses of Rac3 with mouse brain tissues. In immunoblotting, Rac3 exhibited a tissue-dependent expression profile in the young adult mouse and was expressed in a developmental stage-dependent manner in brain. In primary cultured hippocampal neurons, while Rac3 was distributed mainly in the cytoplasm, it was visualized in axon and dendrites with partial localization at synapses, in consistent with the observation in biochemical fractionation analyses. In immunofluorescence analyses with brain slices, Rac3 was distributed strongly and moderately in the axon and cytoplasm, respectively, of cerebral cortex at postnatal day (P) 2 and P18. Similar distribution profile was also observed in hippocampus. Taken together, the results obtained strongly suggest that Rac3 plays an important physiological role in neuronal tissues during corticogenesis, and defects in the Rac3 function induce structural brain anomalies leading to pathogenesis of neurodevelopmental disorders.
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Neuronas , Proteínas de Unión al GTP rho , Animales , Encéfalo/metabolismo , Hipocampo/metabolismo , Ratones , Neuronas/metabolismo , Sinapsis/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Abnormalities of PLEKHG2 gene, encoding a Rho family-specific guanine nucleotide exchange factor, are involved in microcephaly with intellectual disability. However, not only the role of PLEKHG2 in the developmental process but also its expression profile is unknown. In this study, we prepared a specific antibody against PLEKHG2 and carried out expression analyses with mouse tissues. In western blotting, PLEKHG2 exhibited a tissue-dependent expression profile in adult mouse and was expressed in a developmental stage-dependent manner in brain. Then, in immunohistochemical analyses, while PLEKHG2 was observed in the cortical plate and ventricular zone surface of the cerebral cortex at embryonic day 14, it came to be distributed throughout the cerebral cortex in layer II/III and V during corticogenesis. PLEKHG2 was also detected mainly in the nucleus of neurons in the hippocampal CA regions and dentate gyrus at P7. Notably, the nuclear accumulation disappeared at P30 and PLEKHG2 came to be located at the axons and/or dendrites at this time point. Moreover, in vitro immunofluorescence revealed that PLEKHG2 was at least partially localized at both excitatory and inhibitory synapses in primary cultured hippocampal neurons. These results suggest roles of PLEKHG2 in the development of the central nervous tissue and synaptic function.
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Encéfalo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Factores de Intercambio de Guanina Nucleótido/genética , Neuronas/metabolismo , Animales , Encéfalo/crecimiento & desarrollo , Células COS , Línea Celular , Línea Celular Tumoral , Chlorocebus aethiops , Hipocampo/crecimiento & desarrollo , Hipocampo/metabolismo , Inmunohistoquímica , Ratones , Especificidad de ÓrganosRESUMEN
Neuronal differentiation and cell-cycle exit are tightly coordinated, even in pathological situations. When pathological neurons re-enter the cell cycle and progress through the S phase, they undergo cell death instead of division. However, the mechanisms underlying mitotic resistance are mostly unknown. Here, we have found that acute inactivation of retinoblastoma (Rb) family proteins (Rb, p107 and p130) in mouse postmitotic neurons leads to cell death after S-phase progression. Checkpoint kinase 1 (Chk1) pathway activation during the S phase prevented the cell death, and allowed the division of cortical neurons that had undergone acute Rb family inactivation, oxygen-glucose deprivation (OGD) or in vivo hypoxia-ischemia. During neurogenesis, cortical neurons became protected from S-phase Chk1 pathway activation by the DNA methyltransferase Dnmt1, and underwent cell death after S-phase progression. Our results indicate that Chk1 pathway activation overrides mitotic safeguards and uncouples neuronal differentiation from mitotic resistance.
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División Celular , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Neuronas/citología , Neuronas/enzimología , Animales , Muerte Celular , Hipoxia de la Célula , Supervivencia Celular , ADN (Citosina-5-)-Metiltransferasa 1 , Modelos Animales de Enfermedad , Técnicas de Silenciamiento del Gen , Glucosa/deficiencia , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/metabolismo , Neurogénesis , Oxígeno , Proteína de Retinoblastoma/metabolismo , Fase S , Transducción de Señal , Accidente Cerebrovascular/patologíaRESUMEN
The precise control of neuronal migration and morphological changes during differentiation is essential for neocortical development. We hypothesized that the transition of progenitors through progressive stages of differentiation involves dynamic changes in levels of mitochondrial reactive oxygen species (mtROS), depending on cell requirements. We found that progenitors had higher levels of mtROS, but that these levels were significantly decreased with differentiation. The Prdm16 gene was identified as a candidate modulator of mtROS using microarray analysis, and was specifically expressed by progenitors in the ventricular zone. However, Prdm16 expression declined during the transition into NeuroD1-positive multipolar cells. Subsequently, repression of Prdm16 expression by NeuroD1 on the periphery of ventricular zone was crucial for appropriate progression of the multipolar phase and was required for normal cellular development. Furthermore, time-lapse imaging experiments revealed abnormal migration and morphological changes in Prdm16-overexpressing and -knockdown cells. Reporter assays and mtROS determinations demonstrated that PGC1α is a major downstream effector of Prdm16 and NeuroD1, and is required for regulation of the multipolar phase and characteristic modes of migration. Taken together, these data suggest that Prdm16 plays an important role in dynamic cellular redox changes in developing neocortex during neural differentiation.
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Proteínas de Unión al ADN/fisiología , Neocórtex/embriología , Células-Madre Neurales/citología , Células-Madre Neurales/fisiología , Factores de Transcripción/fisiología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Movimiento Celular/genética , Movimiento Celular/fisiología , Células Cultivadas , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Ratones , Ratones Endogámicos ICR , Ratones Transgénicos , Mitocondrias/metabolismo , Neocórtex/citología , Neocórtex/fisiología , Neurogénesis/genética , Neurogénesis/fisiología , Oxidación-Reducción , Embarazo , Especies Reactivas de Oxígeno/metabolismo , Imagen de Lapso de Tiempo , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genéticaRESUMEN
SAD kinases regulate presynaptic vesicle clustering and neuronal polarization. A previous report demonstrated that Sada-/- and Sadb-/- double-mutant mice showed perinatal lethality with a severe defect in axon/dendrite differentiation, but their single mutants did not. These results indicated that they were functionally redundant. Surprisingly, we show that on a C57BL/6N background, SAD-A is essential for cortical development whereas SAD-B is dispensable. Sada-/- mice died within a few days after birth. Their cortical lamination pattern was disorganized and radial migration of cortical neurons was perturbed. Birth date analyses with BrdU and in utero electroporation using pCAG-EGFP vector showed a delayed migration of cortical neurons to the pial surface in Sada-/- mice. Time-lapse imaging of these mice confirmed slow migration velocity in the cortical plate. While the neurites of hippocampal neurons in Sada-/- mice could ultimately differentiate in culture to form axons and dendrites, the average length of their axons was shorter than that of the wild type. Thus, analysis on a different genetic background than that used initially revealed a nonredundant role for SAD-A in neuronal migration and differentiation.
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Movimiento Celular/fisiología , Corteza Cerebral/embriología , Corteza Cerebral/enzimología , Neuronas/enzimología , Proteínas Serina-Treonina Quinasas/fisiología , Animales , Axones/enzimología , Células Cultivadas , Femenino , Isoenzimas , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
The hippocampus is generally considered as a brain center for learning and memory. We have recently established an electroporation-mediated gene transfer method to investigate the development of neonatal dentate granule cells in vivo. Using this new technique, we introduced knockdown vectors against Rac1 small GTPase into precursors for dentate granule cells at postnatal day 0. After 21 days, Rac1-deficient cells were frequently mispositioned between the granule cell layer (GCL) and hilus. About 60% of these mislocalized cells expressed a dentate granule cell marker, Prox1. Both the dendritic spine density and the ratio of mature spine were reduced when Rac1 was silenced. Notably, the deficient cells have immature thin processes during migrating in the early neonatal period. Knockdown of another Rac isoform, Rac3, also resulted in mislocalization of neonatally born dentate granule cells. In addition, knockdown of Cdc42, another Rho family protein, also caused mislocalization of the cell, although the effects were moderate compared to Rac1 and 3. Despite the ectopic localization, Rac3- or Cdc42-disrupted mispositioned cells expressed Prox1. These results indicate that Rho signaling pathways differentially regulate the proper localization and differentiation of dentate granule cells.
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Giro Dentado/enzimología , Giro Dentado/crecimiento & desarrollo , Neuropéptidos/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Animales , Animales Recién Nacidos , Diferenciación Celular , Movimiento Celular , Giro Dentado/citología , Técnicas de Silenciamiento del Gen , Técnicas de Transferencia de Gen , Proteínas de Homeodominio/metabolismo , Ratones , Ratones Endogámicos ICR , Neurogénesis , Neuropéptidos/deficiencia , Neuropéptidos/genética , Interferencia de ARN , Transducción de Señal , Proteínas Supresoras de Tumor/metabolismo , Proteína de Unión al GTP cdc42/deficiencia , Proteína de Unión al GTP cdc42/genética , Proteínas de Unión al GTP rac/deficiencia , Proteínas de Unión al GTP rac/genética , Proteína de Unión al GTP rac1/deficiencia , Proteína de Unión al GTP rac1/genéticaRESUMEN
POGZ is a heterochromatin protein 1 α-binding protein and regulates gene expression. On the other hand, accumulating pieces of evidence indicate that the POGZ gene abnormalities are involved in various neurodevelopmental disorders. In this study, we prepared a specific antibody against POGZ, anti-POGZ, and carried out biochemical and morphological characterization with mouse brain tissues. Western blotting analyses revealed that POGZ is expressed strongly at embryonic day 13 and then gradually decreased throughout the brain development process. In immunohistochemical analyses, POGZ was found to be enriched in cerebrocortical and hippocampal neurons in the early developmental stage. The nuclear expression was also detected in Purkinje cells in cerebellum at postnatal day (P)7 and P15 but disappeared at P30. In primary cultured hippocampal neurons, while POGZ was distributed mainly in the nucleus, it was also visualized in axon and dendrites with partial localization at synapses in consistency with the results obtained in biochemical fractionation analyses. The obtained results suggest that POGZ takes part in the regulation of synaptic function as well as gene expression during brain development.
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Encéfalo/metabolismo , Neurogénesis/fisiología , Transposasas/metabolismo , Animales , Encéfalo/embriología , Encéfalo/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica/fisiología , Ratones , Trastornos del Neurodesarrollo/genética , Trastornos del Neurodesarrollo/metabolismoRESUMEN
Lipid raft domains, where sphingolipids and cholesterol are enriched, concentrate signaling molecules. To examine how signaling protein complexes are clustered in rafts, we focused on the functions of glycoprotein M6a (GPM6a), which is expressed at a high concentration in developing mouse neurons. Using imaging of lipid rafts, we found that GPM6a congregated in rafts in a GPM6a palmitoylation-dependent manner, thereby contributing to lipid raft clustering. In addition, we found that signaling proteins downstream of GPM6a, such as Rufy3, Rap2, and Tiam2/STEF, accumulated in lipid rafts in a GPM6a-dependent manner and were essential for laminin-dependent polarity during neurite formation in neuronal development. In utero RNAi targeting of GPM6a resulted in abnormally polarized neurons with multiple neurites. These results demonstrate that GPM6a induces the clustering of lipid rafts, which supports the raft aggregation of its associated downstream molecules for acceleration of neuronal polarity determination. Therefore, GPM6a acts as a signal transducer that responds to extracellular signals.SIGNIFICANCE STATEMENT Lipid raft domains, where sphingolipids and cholesterol are enriched, concentrate signaling molecules. We focused on glycoprotein M6a (GPM6a), which is expressed at a high concentration in developing neurons. Using imaging of lipid rafts, we found that GPM6a congregated in rafts in a palmitoylation-dependent manner, thereby contributing to lipid raft clustering. In addition, we found that signaling proteins downstream of GPM6a accumulated in lipid rafts in a GPM6a-dependent manner and were essential for laminin-dependent polarity during neurite formation. In utero RNAi targeting of GPM6a resulted in abnormally polarized neurons with multiple neurites. These results demonstrate that GPM6a induces the clustering of lipid rafts, which supports the raft aggregation of its associated downstream molecules for acceleration of polarity determination. Therefore, GPM6a acts as a signal transducer that responds to extracellular signals.
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Líquido Extracelular/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Microdominios de Membrana/genética , Microdominios de Membrana/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Transducción de Señal/fisiología , Secuencia de Aminoácidos , Animales , Células COS , Células Cultivadas , Chlorocebus aethiops , Análisis por Conglomerados , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Noqueados , EmbarazoRESUMEN
Precise control of neuronal migration is essential for the development of the neocortex. However, the molecular mechanisms underlying neuronal migration remain largely unknown. Here we identified helix-loop-helix transcription factor Ebf3 as a Prdm8 target gene, and found that Ebf3 is a key regulator of neuronal migration via multipolar-to-bipolar transition. Ebf3 knockdown cells exhibited severe defects in the formation of leading processes and an inhibited shift to the locomotion mode. Moreover, we found that Ebf3 knockdown represses NeuroD1 transcription, and NeuroD1 overexpression partially rescued migration defects in Ebf3 knockdown cells. Our findings highlight the critical role of Ebf3 in multipolar-to-bipolar transition via positive feedback regulation of NeuroD1 in the developing neocortex.
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Movimiento Celular/fisiología , Desarrollo Embrionario/fisiología , N-Metiltransferasa de Histona-Lisina/metabolismo , Neocórtex/embriología , Neocórtex/fisiología , Neuronas/fisiología , Factores de Transcripción/metabolismo , Animales , Proteínas de Unión al ADN , Regulación del Desarrollo de la Expresión Génica/fisiología , Histona Metiltransferasas , Ratones , Ratones Endogámicos ICR , Neocórtex/citología , Neurogénesis/fisiología , Neuronas/citologíaRESUMEN
MACRO Domain Containing 2 (MacroD2) is a neurodevelopmental disorder-related mono-ADP-ribosylhydrolase. Molecular features of this protein in neural tissues are largely unknown. In this study, we generated a specific antibody against MacroD2, and carried out expression and morphological analyses of the molecule during mouse brain development. In Western blotting, 2 MacroD2 isoforms with molecular masses of â¼70 and â¼75 kDa started to be expressed at embryonic day 16.5, reached the maximal level at postnatal day 8, and then gradually decreased through P30. In contrast, other isoforms with molecular masses of â¼110 and â¼140 kDa gradually increased during embryonic to postnatal development. In immunohistochemical analyses, MacroD2 was strongly detected in cortical neurons in layer II-V at P0 and P7, while the protein expression decreased significantly in the neurons at P30. Immunofluorescence analyses revealed that MacroD2 was mainly distributed in the soma and to a lesser extent in the axon and dendrite of immature primary cultured mouse hippocampal neurons. On the other hand, in the matured hippocampal neurons, while MacroD2 was detected in the soma, it displayed in dendrites a punctate distribution pattern with a partial colocalization with synaptic markers, synaptophysin, and PSD95. The obtained results indicate that MacroD2 is expressed and may have a physiological role in the central nervous system during brain development.
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Enzimas Reparadoras del ADN/metabolismo , Hipocampo/patología , Hidrolasas/metabolismo , N-Glicosil Hidrolasas/metabolismo , Neuronas/metabolismo , Animales , Axones/metabolismo , Células Cultivadas , Dendritas/metabolismo , Homólogo 4 de la Proteína Discs Large/metabolismo , Hipocampo/metabolismo , Ratones , Neurogénesis/fisiología , Sinaptofisina/metabolismoRESUMEN
Migfilin, encoded by FBLIM1 at the 1p36 locus, is a multi-domain adaptor protein essential for various cellular processes such as cell morphology and migration. Small deletions and duplications at the 1p36 locus, monosomy of which results in neurodevelopmental disorders and multiple congenital anomalies, have also been identified in patients with autism spectrum disorder (ASD). However, the impact of FBLIM1, the gene within 1p36, on the pathogenesis of ASD is unknown. In this study, we performed morphological analyses of migfilin to elucidate its role in brain development. Migfilin was detected specifically in the embryonic and perinatal stages of the mouse brain. Either silencing or overexpression of migfilin in embryos following in utero electroporation disrupted Neocortical neuronal migration. Additionally, neurite elongation was impaired when migfilin was silenced in cultured mouse hippocampal neurons. We then screened FBLIM1 for rare exonic deletions/duplications in 549 Japanese ASD patients and 824 controls, detecting one case of ASD and intellectual delay that harbored a 26-kb deletion at 1p36.21 that solely included the C-terminal exon of FBLIM1. The FBLIM1 mRNA expression level in this case was reduced compared to levels in individuals without FBLIM1 deletion. Our findings indicate that tightly regulated expression of migfilin is essential for neuronal development and that FBLIM1 disruption may be related to the phenotypes associated with ASD and related neurodevelopmental disorders.
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Trastorno del Espectro Autista/genética , Moléculas de Adhesión Celular/genética , Proteínas del Citoesqueleto/genética , Adolescente , Adulto , Animales , Trastorno del Espectro Autista/metabolismo , Trastorno del Espectro Autista/patología , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Encéfalo/patología , Moléculas de Adhesión Celular/metabolismo , Movimiento Celular/fisiología , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Proteínas del Citoesqueleto/metabolismo , Femenino , Hipocampo/metabolismo , Hipocampo/patología , Humanos , Masculino , Ratones , Persona de Mediana Edad , Embarazo , ARN Mensajero/metabolismo , Eliminación de Secuencia , Adulto JovenRESUMEN
The preoptic area (POa) of the rostral diencephalon supplies the neocortex and the amygdala with GABAergic neurons in the developing mouse brain. However, the molecular mechanisms that determine the pathway and destinations of POa-derived neurons have not yet been identified. Here we show that Chicken ovalbumin upstream promoter transcription factor II (COUP-TFII)-induced expression of Neuropilin-2 (Nrp2) and its down-regulation control the destination of POa-derived GABAergic neurons. Initially, a majority of the POa-derived migrating neurons express COUP-TFII and form a caudal migratory stream toward the caudal subpallium. When a subpopulation of cells steers toward the neocortex, they exhibit decreased expression of COUP-TFII and Nrp2. The present findings show that suppression of COUP-TFII/Nrp2 changed the destination of the cells into the neocortex, whereas overexpression of COUP-TFII/Nrp2 caused cells to end up in the medial part of the amygdala. Taken together, these results reveal that COUP-TFII/Nrp2 is a molecular switch determining the pathway and destination of migrating GABAergic neurons born in the POa.
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Encéfalo/metabolismo , Factor de Transcripción COUP II/metabolismo , Diencéfalo/metabolismo , Neuronas GABAérgicas/metabolismo , Neuropilina-2/metabolismo , Amígdala del Cerebelo/embriología , Amígdala del Cerebelo/metabolismo , Animales , Western Blotting , Encéfalo/embriología , Factor de Transcripción COUP II/genética , Movimiento Celular/genética , Diencéfalo/embriología , Neuronas GABAérgicas/citología , Regulación del Desarrollo de la Expresión Génica , Hibridación in Situ , Ratones Endogámicos ICR , Ratones Noqueados , Microscopía Confocal , Neocórtex/embriología , Neocórtex/metabolismo , Neuropilina-2/genética , Área Preóptica/embriología , Área Preóptica/metabolismo , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Técnicas de Cultivo de TejidosRESUMEN
We analyzed the role of a heterotrimeric G-protein, Gi2, in the development of the cerebral cortex. Acute knockdown of the α-subunit (Gαi2) with in utero electroporation caused delayed radial migration of excitatory neurons during corticogenesis, perhaps because of impaired morphology. The migration phenotype was rescued by an RNAi-resistant version of Gαi2. On the other hand, silencing of Gαi2 did not affect axon elongation, dendritic arbor formation or neurogenesis at ventricular zone in vivo. When behavior analyses were conducted with acute Gαi2-knockdown mice, they showed defects in social interaction, novelty recognition and active avoidance learning as well as increased anxiety. Subsequently, using whole-exome sequencing analysis, we identified a de novo heterozygous missense mutation (c.680C>T; p.Ala227Val) in the GNAI2 gene encoding Gαi2 in an individual with periventricular nodular heterotopia and intellectual disability. Collectively, the phenotypes in the knockdown experiments suggest a role of Gαi2 in the brain development, and impairment of its function might cause defects in neuronal functions which lead to neurodevelopmental disorders.
Asunto(s)
Corteza Cerebral/crecimiento & desarrollo , Corteza Cerebral/metabolismo , Subunidad alfa de la Proteína de Unión al GTP Gi2/fisiología , Discapacidad Intelectual/metabolismo , Heterotopia Nodular Periventricular/metabolismo , Animales , Reacción de Prevención/fisiología , Células COS , Corteza Cerebral/diagnóstico por imagen , Chlorocebus aethiops , Femenino , Subunidad alfa de la Proteína de Unión al GTP Gi2/deficiencia , Humanos , Discapacidad Intelectual/diagnóstico por imagen , Discapacidad Intelectual/genética , Ratones , Ratones Endogámicos ICR , Ratones Noqueados , Heterotopia Nodular Periventricular/diagnóstico por imagen , Heterotopia Nodular Periventricular/genética , EmbarazoRESUMEN
Class III phosphoinositide 3-kinase (PIK3C3 or mammalian vacuolar protein sorting 34 homolog, Vps34) regulates vesicular trafficking, autophagy, and nutrient sensing. Recently, we reported that PIK3C3 is expressed in mouse cerebral cortex throughout the developmental process, especially at early embryonic stage. We thus examined the role of PIK3C3 in the development of the mouse cerebral cortex. Acute silencing of PIK3C3 with in utero electroporation method caused positional defects of excitatory neurons during corticogenesis. Time-lapse imaging revealed that the abnormal positioning was at least partially because of the reduced migration velocity. When PIK3C3 was silenced in cortical neurons in one hemisphere, axon extension to the contralateral hemisphere was also delayed. These aberrant phenotypes were rescued by RNAi-resistant PIK3C3. Notably, knockdown of PIK3C3 did not affect the cell cycle of neuronal progenitors and stem cells at the ventricular zone. Taken together, PIK3C3 was thought to play a crucial role in corticogenesis through the regulation of excitatory neuron migration and axon extension. Meanwhile, when we performed comparative genomic hybridization on a patient with specific learning disorders, a 107 Kb-deletion was identified on 18q12.3 (nt. 39554147-39661206) that encompasses exons 5-23 of PIK3C3. Notably, the above aberrant migration and axon growth phenotypes were not rescued by the disease-related truncation mutant (172 amino acids) lacking the C-terminal kinase domain. Thus, functional defects of PIK3C3 might impair corticogenesis and relate to the pathophysiology of specific learning disorders and other neurodevelopmental disorders. Acute knockdown of Class III phosphoinositide 3-kinase (PIK3C3) evokes migration defects of excitatory neurons during corticogenesis. PIK3C3-knockdown also disrupts axon outgrowth, but not progenitor proliferation in vivo. Involvement of PIK3C3 in neurodevelopmental disorders might be an interesting future subject since a deletion mutation in PIK3C3 was detected in a patient with specific learning disorders (SLD).
Asunto(s)
Proteínas Quinasas Dependientes de 3-Fosfoinosítido/genética , Proteínas Quinasas Dependientes de 3-Fosfoinosítido/metabolismo , Encéfalo/enzimología , Encéfalo/crecimiento & desarrollo , Discapacidades para el Aprendizaje/genética , Animales , Axones , Encéfalo/embriología , Movimiento Celular/genética , Corteza Cerebral/embriología , Corteza Cerebral/enzimología , Corteza Cerebral/crecimiento & desarrollo , Ventrículos Cerebrales/citología , Ventrículos Cerebrales/enzimología , Ventrículos Cerebrales/crecimiento & desarrollo , Niño , Exones/genética , Femenino , Eliminación de Gen , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Humanos , Pruebas de Inteligencia , Discapacidades para el Aprendizaje/psicología , Ratones , Células-Madre Neurales , Hibridación de Ácido Nucleico , Embarazo , Interferencia de ARNRESUMEN
During the development of the cerebral cortex, excitatory neurons are produced in the ventricular zone lining the lateral ventricle or in the adjacent subventricular zone and migrate toward the brain surface in a process known as radial migration. During radial migration, neurons undergo multiple steps including a multipolar cell phase, a multipolar-bipolar transition, and a locomotion phase. Many genes tightly regulate the cell behavior in each phase. We have established an in utero electroporation method as a rapid in vivo gene transfer system, and this system has greatly contributed to recent advances in our knowledge of the molecular mechanisms underlying each migration phase. Here, we review the cell behaviors of neurons during each phase of radial migration and the molecular mechanisms involved in these phases. Knockdown or functional blocking of these genes using in utero electroporation results in various migration defects and abnormal cell morphologies. Here, we describe these phenotypes as much as possible so that this review can be used as a chart to evaluate the phenotypes of novel gene knockdown experiments. We also discuss the recent application of in utero electroporation in studies examining the functions of neurodevelopmental disorder-related genes.
Asunto(s)
Movimiento Celular , Electroporación/métodos , Neuronas/citología , Adhesión Celular , Polaridad Celular , Humanos , Microtúbulos/metabolismo , Neuronas/metabolismoRESUMEN
The mammalian Class III phosphoinositide 3-kinase (PIK3C3, also known as mammalian vacuolar protein sorting 34 homologue, Vps34) is a regulator of vesicular trafficking, autophagy, and nutrient sensing. In this study, we generated a specific antibody against PIK3C3, and carried out expression and morphological analyses of PIK3C3 during mouse brain development. In Western blotting, PIK3C3 was detected throughout the developmental process with higher expression in the early embryonic stage. In immunohistochemical analyses with embryonic day 16 mouse brain, PIK3C3 was detected strongly in the axon of cortical neurons. While PIK3C3 was distributed at the soma, nucleus, axon, and dendrites in primary cultured mouse hippocampal neurons at 3 days in vitro (div), it was also found in a punctate distribution with partial colocalization with synaptic marker, synaptophysin, at 21 div. The obtained results indicate that PIK3C3 is expressed and may have a physiological role in central nervous system during corticogenesis.