RESUMEN
Protein synthesis must be finely tuned in the developing nervous system as the final essential step of gene expression. This study investigates the architecture of ribosomes from the neocortex during neurogenesis, revealing Ebp1 as a high-occupancy 60S peptide tunnel exit (TE) factor during protein synthesis at near-atomic resolution by cryoelectron microscopy (cryo-EM). Ribosome profiling demonstrated Ebp1-60S binding is highest during start codon initiation and N-terminal peptide elongation, regulating ribosome occupancy of these codons. Membrane-targeting domains emerging from the 60S tunnel, which recruit SRP/Sec61 to the shared binding site, displace Ebp1. Ebp1 is particularly abundant in the early-born neural stem cell (NSC) lineage and regulates neuronal morphology. Ebp1 especially impacts the synthesis of membrane-targeted cell adhesion molecules (CAMs), measured by pulsed stable isotope labeling by amino acids in cell culture (pSILAC)/bioorthogonal noncanonical amino acid tagging (BONCAT) mass spectrometry (MS). Therefore, Ebp1 is a central component of protein synthesis, and the ribosome TE is a focal point of gene expression control in the molecular specification of neuronal morphology during development.
Asunto(s)
Proteínas de Unión al ADN/genética , Regulación del Desarrollo de la Expresión Génica , Neocórtex/metabolismo , Neuronas/metabolismo , Biosíntesis de Proteínas , Proteostasis/genética , Proteínas de Unión al ARN/genética , Subunidades Ribosómicas Grandes de Eucariotas/genética , Animales , Animales Recién Nacidos , Sitios de Unión , Moléculas de Adhesión Celular Neuronal/química , Moléculas de Adhesión Celular Neuronal/genética , Moléculas de Adhesión Celular Neuronal/metabolismo , Línea Celular Tumoral , Microscopía por Crioelectrón , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Embrión de Mamíferos , Femenino , Masculino , Ratones , Neocórtex/citología , Neocórtex/crecimiento & desarrollo , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Neurogénesis/genética , Neuronas/citología , Cultivo Primario de Células , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Subunidades Ribosómicas Grandes de Eucariotas/ultraestructura , Partícula de Reconocimiento de Señal/química , Partícula de Reconocimiento de Señal/genética , Partícula de Reconocimiento de Señal/metabolismoRESUMEN
The seat of higher-order cognitive abilities in mammals, the neocortex, is a complex structure, organized in several layers. The different subtypes of principal neurons are distributed in precise ratios and at specific positions in these layers and are generated by the same neural progenitor cells (NPCs), steered by a spatially and temporally specified combination of molecular cues that are incompletely understood. Recently, we discovered that an alternatively spliced isoform of the TrkC receptor lacking the kinase domain, TrkC-T1, is a determinant of the corticofugal projection neuron (CFuPN) fate. Here, we show that the finely tuned balance between TrkC-T1 and the better known, kinase domain-containing isoform, TrkC-TK+, is cell type-specific in the developing cortex and established through the antagonistic actions of two RNA-binding proteins, Srsf1 and Elavl1. Moreover, our data show that Srsf1 promotes the CFuPN fate and Elavl1 promotes the callosal projection neuron (CPN) fate in vivo via regulating the distinct ratios of TrkC-T1 to TrkC-TK+. Taken together, we connect spatio-temporal expression of Srsf1 and Elavl1 in the developing neocortex with the regulation of TrkC alternative splicing and transcript stability and neuronal fate choice, thus adding to the mechanistic and functional understanding of alternative splicing in vivo.
Asunto(s)
Neocórtex , Receptor trkC , Animales , Empalme Alternativo , Mamíferos/metabolismo , Neocórtex/metabolismo , Neuronas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptor trkC/química , Receptor trkC/genética , Receptor trkC/metabolismo , Ratones , Línea Celular TumoralRESUMEN
Abnormal development of corpus callosum is relatively common and causes a broad spectrum of cognitive impairments in humans. We use acallosal Neurod2/6-deficient mice to study callosal axon guidance within the ipsilateral cerebral cortex. Initial callosal tracts form but fail to traverse the ipsilateral cingulum and are not attracted towards the midline in the absence of Neurod2/6. We show that the restoration of Ephrin-A4 (EfnA4) expression in the embryonic neocortex of Neurod2/6-deficient embryos is sufficient to partially rescue targeted callosal axon growth towards the midline. EfnA4 cannot directly mediate reverse signaling within outgrowing axons, but it forms co-receptor complexes with TrkB (Ntrk2). The ability of EfnA4 to rescue the guided growth of a subset of callosal axons in Neurod2/6-deficient mice is abolished by the co-expression of dominant negative TrkBK571N (kinase-dead) or TrkBY515F (SHC-binding deficient) variants, but not by TrkBY816F (PLCγ1-binding deficient). Additionally, EphA4 is repulsive to EfnA4-positive medially projecting axons in organotypic brain slice culture. Collectively, we suggest that EfnA4-mediated reverse signaling acts via TrkB-SHC and is required for ipsilateral callosal axon growth accuracy towards the midline downstream of Neurod family factors.
Asunto(s)
Neocórtex , Neuropéptidos , Ratones , Animales , Humanos , Cuerpo Calloso/metabolismo , Axones/fisiología , Neocórtex/metabolismo , Fibras Nerviosas , Fosfotransferasas/metabolismo , Neuropéptidos/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismoRESUMEN
Primary autosomal recessive microcephaly and Seckel syndrome spectrum disorders (MCPH-SCKS) include a heterogeneous group of autosomal recessive inherited diseases characterized by primary (congenital) microcephaly, the absence of visceral abnormalities, and a variable degree of cognitive impairment, short stature and facial dysmorphism. Recently, biallelic variants in the nuclear pore complex (NPC) component nucleoporin 85 gene (NUP85) were reported to cause steroid-resistant nephrotic syndrome (SRNS). Here, we report biallelic variants in NUP85 in two pedigrees with an MCPH-SCKS phenotype spectrum without SRNS, thereby expanding the phenotypic spectrum of NUP85-linked diseases. Structural analysis predicts the identified NUP85 variants cause conformational changes that could have an effect on NPC architecture or on its interaction with other NUPs. We show that mutant NUP85 is, however, associated with a reduced number of NPCs but unaltered nucleocytoplasmic compartmentalization, abnormal mitotic spindle morphology, and decreased cell viability and proliferation in one patient's cells. Our results also indicate the link of common cellular mechanisms involved in MCPH-SCKS spectrum disorders and NUP85-associated diseases. In addition to the previous studies, our results broaden the phenotypic spectrum of NUP85-linked human disease and propose a role for NUP85 in nervous system development.
Asunto(s)
Enanismo/diagnóstico , Enanismo/genética , Microcefalia/diagnóstico , Microcefalia/genética , Mutación , Proteínas de Complejo Poro Nuclear/genética , Fenotipo , Encéfalo/anomalías , Niño , Preescolar , Análisis Mutacional de ADN , Femenino , Fibroblastos/metabolismo , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Humanos , Lactante , Recién Nacido , Masculino , Proteínas de Complejo Poro Nuclear/química , Linaje , SíndromeRESUMEN
Epilepsy is one of the common neurological diseases that affects not only adults but also infants and children. Because epilepsy has been studied for a long time, there are several pharmacologically effective anticonvulsants, which, however, are not suitable as therapy for all patients. The genesis of epilepsy has been extensively investigated in terms of its occurrence after injury and as a concomitant disease with various brain diseases, such as tumors, ischemic events, etc. However, in the last decades, there are multiple reports that both genetic and epigenetic factors play an important role in epileptogenesis. Therefore, there is a need for further identification of genes and loci that can be associated with higher susceptibility to epileptic seizures. Use of mouse knockout models of epileptogenesis is very informative, but it has its limitations. One of them is due to the fact that complete deletion of a gene is not, in many cases, similar to human epilepsy-associated syndromes. Another approach to generating mouse models of epilepsy is N-Ethyl-N-nitrosourea (ENU)-directed mutagenesis. Recently, using this approach, we generated a novel mouse strain, soc (socrates, formerly s8-3), with epileptiform activity. Using molecular biology methods, calcium neuroimaging, and immunocytochemistry, we were able to characterize the strain. Neurons isolated from soc mutant brains retain the ability to differentiate in vitro and form a network. However, soc mutant neurons are characterized by increased spontaneous excitation activity. They also demonstrate a high degree of Ca2+ activity compared to WT neurons. Additionally, they show increased expression of NMDA receptors, decreased expression of the Ca2+-conducting GluA2 subunit of AMPA receptors, suppressed expression of phosphoinositol 3-kinase, and BK channels of the cytoplasmic membrane involved in protection against epileptogenesis. During embryonic and postnatal development, the expression of several genes encoding ion channels is downregulated in vivo, as well. Our data indicate that soc mutation causes a disruption of the excitation-inhibition balance in the brain, and it can serve as a mouse model of epilepsy.
Asunto(s)
Epilepsia Refleja , Niño , Animales , Humanos , Ratones , Epilepsia Refleja/genética , Epilepsia Refleja/metabolismo , Etilnitrosourea/toxicidad , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Anticonvulsivantes/farmacología , Encéfalo/metabolismo , Modelos Animales de EnfermedadRESUMEN
Kaufman oculocerebrofacial syndrome (KOS) is a severe autosomal recessive disorder characterized by intellectual disability, developmental delays, microcephaly, and characteristic dysmorphisms. Biallelic mutations of UBE3B, encoding for a ubiquitin ligase E3B are causative for KOS. In this report, we characterize neuronal functions of its murine ortholog Ube3b and show that Ube3b regulates dendritic branching in a cell-autonomous manner. Moreover, Ube3b knockout (KO) neurons exhibit increased density and aberrant morphology of dendritic spines, altered synaptic physiology, and changes in hippocampal circuit activity. Dorsal forebrain-specific Ube3b KO animals show impaired spatial learning, altered social interactions, and repetitive behaviors. We further demonstrate that Ube3b ubiquitinates the catalytic γ-subunit of calcineurin, Ppp3cc, the overexpression of which phenocopies Ube3b loss with regard to dendritic spine density. This work provides insights into the molecular pathologies underlying intellectual disability-like phenotypes in a genetically engineered mouse model.
Asunto(s)
Discapacidad Intelectual , Microcefalia , Animales , Calcineurina , Espinas Dendríticas , Anomalías del Ojo , Facies , Discapacidad Intelectual/genética , Deformidades Congénitas de las Extremidades , Ratones , Ratones Noqueados , Microcefalia/genética , Mutación/genética , Sinapsis , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Neocortical projection neurons are generated by neural progenitor cells (NPCs) within the ventricular and subventricular zone. While early NPCs can give rise to both deep and upper layer neurons, late progenitors are restricted to upper layer neurogenesis. The molecular mechanisms controlling the differentiation potential of early versus late NPCs are unknown. Here, we report a novel function for TrkC-T1, the non-catalytic isoform of the neurotrophin receptor TrkC, that is distinct from TrkC-TK+, the full-length isoform. We provide direct evidence that TrkC-T1 regulates the switch in NPC fate from deep to upper layer neuron production. Elevated levels of TrkC-T1 in early NPCs promote the generation of deep layer neurons. Conversely, downregulation of TrkC-T1 in these cells promotes upper layer neuron fate. Furthermore, we show that TrkC-T1 exerts this control by interaction with the signaling adaptor protein ShcA. TrkC-T1 prevents the phosphorylation of Shc and the downstream activation of the MAP kinase (Erk1/2) pathway. In vivo manipulation of the activity of ShcA or Erk1/2, directly affects cortical neuron cell fate. We thus show that the generation of upper layer neurons by late progenitors is dependent on the downregulation of TrkC-T1 in late progenitor cells and the resulting activation of the ShcA/Erk1/2 pathway.
Asunto(s)
Neocórtex , Células-Madre Neurales , Neocórtex/metabolismo , Células-Madre Neurales/metabolismo , Isoformas de Proteínas/metabolismo , Receptor trkC , Transducción de Señal/fisiologíaRESUMEN
OBJECTIVE: This study aimed to investigate the connection between the mutation of the Sip1 transcription factor and impaired Ca2+-signaling, which reflects changes in neurotransmission in the cerebral cortex in vitro. METHODS: We used mixed neuroglial cortical cell cultures derived from Sip1 mutant mice. The cells were loaded with a fluorescent ratiometric calcium-sensitive probe Fura-2 AM and epileptiform activity was modeled by excluding magnesium ions from the external media or adding a GABA(A) receptor antagonist, bicuculline. Intracellular calcium dynamics were recorded using fluorescence microscopy. To identify the level of gene expression, the Real-Time PCR method was used. RESULTS: It was found that cortical neurons isolated from homozygous (Sip1fl/fl) mice with the Sip1 mutation demonstrate suppressed Ca2+ signals in models of epileptiform activity in vitro. Wild-type cortical neurons are characterized by synchronous high-frequency and high-amplitude Ca2+ oscillations occurring in all neurons of the network in response to Mg2+-free medium and bicuculline. But cortical Sip1fl/fl neurons only single Ca2+ pulses or attenuated Ca2+ oscillations are recorded and only in single neurons, while most of the cell network does not respond to these stimuli. This signal deficiency of Sip1fl/fl neurons correlates with a suppressed expression level of the genes encoding the subunits of NMDA, AMPA, and KA receptors; protein kinases PKA, JNK, CaMKII; and also the transcription factor Hif1α. These negative effects were partially abolished when Sip1fl/fl neurons are grown in media with anti-inflammatory cytokine IL-10. IL-10 increases the expression of the above-mentioned genes but not to the level of expression in wild-type. At the same time, the amplitudes of Ca2+ signals increase in response to the selective agonists of NMDA, AMPA and KA receptors, and the proportion of neurons responding with Ca2+ oscillations to a Mg2+-free medium and bicuculline increases. CONCLUSION: IL-10 restores neurotransmission in neuronal networks with the Sip1 mutation by regulating the expression of genes encoding signaling proteins.
Asunto(s)
Calcio , Interleucina-10/metabolismo , Animales , Bicuculina/farmacología , Calcio/metabolismo , Células Cultivadas , Corteza Cerebral/fisiología , Ratones , N-Metilaspartato , Receptores de Glutamato/metabolismo , Factores de Transcripción/metabolismo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol PropiónicoRESUMEN
Chromosomal microdeletion syndromes present with a wide spectrum of clinical phenotypes that depend on the size and gene content of the affected region. In a healthy carrier, epigenetic mechanisms may compensate for the same microdeletion, which may segregate through several generations without any clinical symptoms until the epigenetic modifications no longer function. We report 2 novel cases of Xq24 microdeletions inherited from mothers with extremely skewed X-chromosome inactivation (sXCI). The first case is a boy presenting with X-linked mental retardation, Nascimento type, due to a 168-kb Xq24 microdeletion involving 5 genes (CXorf56, UBE2A, NKRF, SEPT6, and MIR766) inherited from a healthy mother and grandmother with sXCI. In the second family, the presence of a 239-kb Xq24 microdeletion involving 3 additional genes (SLC25A43, SLC25A5-AS1, and SLC25A5) was detected in a woman with sXCI and a history of recurrent pregnancy loss with a maternal family history without reproductive wastages or products of conception. These cases provide evidence that women with an Xq24 microdeletion and sXCI may be at risk for having a child with intellectual disability or for experiencing a pregnancy loss due to the ontogenetic pleiotropy of a chromosomal microdeletion and its incomplete penetrance modified by sXCI.
Asunto(s)
Aborto Habitual/genética , Deleción Cromosómica , Cromosomas Humanos X/genética , Madres , Enzimas Ubiquitina-Conjugadoras/deficiencia , Enzimas Ubiquitina-Conjugadoras/genética , Inactivación del Cromosoma X/genética , Adulto , Preescolar , Epigénesis Genética , Femenino , Humanos , Lactante , Recién Nacido , Discapacidad Intelectual/genética , Masculino , Fenotipo , Síndrome , Adulto JovenRESUMEN
Neocortical neurons have highly branched dendritic trees that are essential for their function. Indeed, defects in dendritic arborization are associated with human neurodevelopmental disorders. The molecular mechanisms regulating dendritic arbor complexity, however, are still poorly understood. Here, we uncover the molecular basis for the regulation of dendritic branching during cortical development. We show that during development, dendritic branching requires post-mitotic suppression of the RhoGTPase Cdc42. By generating genetically modified mice, we demonstrate that this is catalyzed in vivo by the novel Cdc42-GAP NOMA-GAP. Loss of NOMA-GAP leads to decreased neocortical volume, associated specifically with profound oversimplification of cortical dendritic arborization and hyperactivation of Cdc42. Remarkably, dendritic complexity and cortical thickness can be partially restored by genetic reduction of post-mitotic Cdc42 levels. Furthermore, we identify the actin regulator cofilin as a key regulator of dendritic complexity in vivo. Cofilin activation during late cortical development depends on NOMA-GAP expression and subsequent inhibition of Cdc42. Strikingly, in utero expression of active cofilin is sufficient to restore postnatal dendritic complexity in NOMA-GAP-deficient animals. Our findings define a novel cell-intrinsic mechanism to regulate dendritic branching and thus neuronal complexity in the cerebral cortex.
Asunto(s)
Factores Despolimerizantes de la Actina/metabolismo , Dendritas/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Neocórtex/crecimiento & desarrollo , Neocórtex/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Animales , Células Cultivadas , Femenino , Proteínas Activadoras de GTPasa/genética , Ratones , Ratones TransgénicosRESUMEN
Acquisition of cis-regulatory elements is a major driving force of evolution, and there are several examples of developmental enhancers derived from transposable elements (TEs). However, it remains unclear whether one enhancer element could have been produced via cooperation among multiple, yet distinct, TEs during evolution. Here we show that an evolutionarily conserved genomic region named AS3_9 comprises three TEs (AmnSINE1, X6b_DNA and MER117), inserted side-by-side, and functions as a distal enhancer for wnt5a expression during morphogenesis of the mammalian secondary palate. Functional analysis of each TE revealed step-by-step retroposition/transposition and co-option together with acquisition of a binding site for Msx1 for its full enhancer function during mammalian evolution. The present study provides a new perspective suggesting that a huge variety of TEs, in combination, could have accelerated the diversity of cis-regulatory elements involved in morphological evolution.
Asunto(s)
Elementos Transponibles de ADN/genética , Elementos de Facilitación Genéticos/genética , Factor de Transcripción MSX1/genética , Secuencias Reguladoras de Ácidos Nucleicos , Proteína Wnt-5a/biosíntesis , Animales , Sitios de Unión , Proteínas de Unión al ADN/genética , Evolución Molecular , Regulación de la Expresión Génica , Humanos , Factor de Transcripción MSX1/metabolismo , Mamíferos , Ratones , Ratones Noqueados , Hueso Paladar/crecimiento & desarrollo , Transgenes , Proteína Wnt-5a/genéticaRESUMEN
The Sip1 mutation plays the main role in pathogenesis of the Mowat-Wilson syndrome, which is characterized by the pronounced epileptic symptoms. Cortical neurons of homozygous mice with Sip1 mutation are resistant to AMPA receptor activators. Disturbances of the excitatory signaling components are also observed on such a phenomenon of neuroplasticity as hypoxic preconditioning. In this work, the mechanisms of loss of the AMPA receptor's ability to precondition by episodes of short-term hypoxia were investigated on cortical neurons derived from the Sip1 homozygous mice. The preconditioning effect was estimated by the level of suppression of the AMPA receptors activity with hypoxia episodes. Using fluorescence microscopy, we have shown that cortical neurons from the Sip1fl/fl mice are characterized by the absence of hypoxic preconditioning effect, whereas the amplitude of Ca2+-responses to the application of the AMPA receptor agonist, 5-Fluorowillardiine, in neurons from the Sip1 mice brainstem is suppressed by brief episodes of hypoxia. The mechanism responsible for this process is hypoxia-induced desensitization of the AMPA receptors, which is absent in the cortex neurons possessing the Sip1 mutation. However, the appearance of preconditioning in these neurons can be induced by phosphoinositide-3-kinase activation with a selective activator or an anti-inflammatory cytokine interleukin-10.
Asunto(s)
Corteza Cerebral/fisiopatología , Hipoxia/fisiopatología , Interleucina-10/fisiología , Mutación , Proteínas del Tejido Nervioso/fisiología , Neuronas/fisiología , Receptores AMPA/fisiología , Alanina/análogos & derivados , Alanina/farmacología , Animales , Calcio/metabolismo , Señalización del Calcio , Activación Enzimática , Agonistas de Aminoácidos Excitadores/farmacología , Ratones , Microscopía Fluorescente , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Pirimidinas/farmacología , Receptores AMPA/agonistasRESUMEN
Cortical progenitors undergo progressive fate restriction, thereby sequentially producing the different layers of the neocortex. However, how these progenitors precisely change their fate remains highly debatable. We have previously shown the existence of cortical feedback mechanisms wherein postmitotic neurons signal back to the progenitors and promote a switch from neurogenesis to gliogenesis. We showed that Sip1 (Zeb2), a transcriptional repressor, controls this feedback signaling. A similar mechanism was also suggested to control neuronal cell type specification; however, the underlying mechanism was not identified. Here, we provide direct evidence that in the developing mouse neocortex, Ntf3, a Sip1 target neurotrophin, acts as a feedback signal between postmitotic neurons and progenitors, promoting both apical progenitor (AP) to basal progenitor (BP) and deep layer (DL) to upper layer (UL) cell fate switches. We show that specific overexpression of Ntf3 in neocortical neurons promotes an overproduction of BP at the expense of AP. This shift is followed by a decrease in DL and an increase in UL neuronal production. Loss of Ntf3, by contrast, causes an increase in layer VI neurons but does not rescue the Sip1 mutant phenotype, implying that other parallel pathways also control the timing of progenitor cell fate switch.
Asunto(s)
Linaje de la Célula , Retroalimentación Fisiológica , Mitosis , Proteínas del Tejido Nervioso/metabolismo , Células-Madre Neurales/citología , Neuronas/citología , Neurotrofina 3/metabolismo , Transducción de Señal , Animales , Recuento de Células , Corteza Cerebral , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Femenino , Eliminación de Gen , Masculino , Ratones , Mosaicismo , Mutación/genética , Neurogénesis , Fenotipo , Regulación hacia ArribaRESUMEN
Kv10.1 (Eag1), member of the Kv10 family of voltage-gated potassium channels, is preferentially expressed in adult brain. The aim of the present study was to unravel the functional role of Kv10.1 in the brain by generating knockout mice, where the voltage sensor and pore region of Kv10.1 were removed to render non-functional proteins through deletion of exon 7 of the KCNH1 gene using the '3 Lox P strategy'. Kv10.1-deficient mice show no obvious alterations during embryogenesis and develop normally to adulthood; cortex, hippocampus and cerebellum appear anatomically normal. Other tests, including general health screen, sensorimotor functioning and gating, anxiety, social behaviour, learning and memory did not show any functional aberrations in Kv10.1 null mice. Kv10.1 null mice display mild hyperactivity and longer-lasting haloperidol-induced catalepsy, but there was no difference between genotypes in amphetamine sensitization and withdrawal, reactivity to apomorphine and haloperidol in the prepulse inhibition tests or to antidepressants in the haloperidol-induced catalepsy. Furthermore, electrical properties of Kv10.1 in cerebellar Purkinje cells did not show any difference between genotypes. Bearing in mind that Kv10.1 is overexpressed in over 70% of all human tumours and that its inhibition leads to a reduced tumour cell proliferation, the fact that deletion of Kv10.1 does not show a marked phenotype is a prerequisite for utilizing Kv10.1 blocking and/or reduction techniques, such as siRNA, to treat cancer.
Asunto(s)
Canales de Potasio con Entrada de Voltaje/genética , Canales de Potasio con Entrada de Voltaje/metabolismo , Potenciales de Acción , Anfetamina/administración & dosificación , Anfetamina/metabolismo , Animales , Antidepresivos/administración & dosificación , Conducta Animal/efectos de los fármacos , Encéfalo/metabolismo , Catalepsia/inducido químicamente , Catalepsia/tratamiento farmacológico , Cerebelo/metabolismo , Técnicas de Inactivación de Genes , Orden Génico , Marcación de Gen , Genotipo , Haloperidol/efectos adversos , Ratones , Ratones Noqueados , FenotipoRESUMEN
Netrin-1 induces repulsive axon guidance by binding to the mammalian Unc5 receptor family (Unc5A-Unc5D). Mouse genetic analysis of selected members of the Unc5 family, however, revealed essential functions independent of Netrin-1, suggesting the presence of other ligands. Unc5B was recently shown to bind fibronectin and leucine-rich transmembrane protein-3 (FLRT3), although the relevance of this interaction for nervous system development remained unclear. Here, we show that the related Unc5D receptor binds specifically to another FLRT protein, FLRT2. During development, FLRT2/3 ectodomains (ECDs) are shed from neurons and act as repulsive guidance molecules for axons and somata of Unc5-positive neurons. In the developing mammalian neocortex, Unc5D is expressed by neurons in the subventricular zone (SVZ), which display delayed migration to the FLRT2-expressing cortical plate (CP). Deletion of either FLRT2 or Unc5D causes a subset of SVZ-derived neurons to prematurely migrate towards the CP, whereas overexpression of Unc5D has opposite effects. Hence, the shed FLRT2 and FLRT3 ECDs represent a novel family of chemorepellents for Unc5-positive neurons and FLRT2/Unc5D signalling modulates cortical neuron migration.
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Glicoproteínas de Membrana/fisiología , Neuronas/metabolismo , Receptores de Superficie Celular/fisiología , Animales , Axones/metabolismo , Movimiento Celular , Células Cultivadas , Femenino , Regulación del Desarrollo de la Expresión Génica , Hipocampo/citología , Hipocampo/metabolismo , Humanos , Immunoblotting , Integrasas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factores de Crecimiento Nervioso/metabolismo , Receptores de Netrina , Netrina-1 , Neuronas/citología , Unión Proteica , Transducción de Señal , Proteínas Supresoras de Tumor/metabolismoRESUMEN
First insights into the molecular programs orchestrating the progression from neural stem cells to cortical projection neurons are emerging. Loss of the transcriptional regulator Ski has been linked to the human 1p36 deletion syndrome, which includes central nervous system defects. Here, we report critical roles for Ski in the maintenance of the neural stem cell pool and the specification of callosal neurons. Ski-deficient callosal neurons lose their identity and ectopically express the transcription factor Ctip2. The misspecified callosal neurons largely fail to form the corpus callosum and instead redirect their axons toward subcortical targets. We identify the chromatin-remodeling factor Satb2 as a partner of Ski, and show that both proteins are required for transcriptional repression of Ctip2 in callosal neurons. We propose a model in which Satb2 recruits Ski to the Ctip2 locus, and Ski attracts histone deacetylases, thereby enabling the formation of a functional nucleosome remodeling and deacetylase repressor complex. Our findings establish a central role for Ski-Satb2 interactions in regulating transcriptional mechanisms of callosal neuron specification.
Asunto(s)
Ensamble y Desensamble de Cromatina/genética , Cuerpo Calloso/citología , Proteínas de Unión al ADN/fisiología , Proteínas de Unión a la Región de Fijación a la Matriz/fisiología , Proteínas del Tejido Nervioso/fisiología , Células-Madre Neurales/metabolismo , Neuronas/metabolismo , Proteínas Proto-Oncogénicas/fisiología , Proteínas Represoras/biosíntesis , Factores de Transcripción/fisiología , Proteínas Supresoras de Tumor/biosíntesis , Agenesia del Cuerpo Calloso/embriología , Agenesia del Cuerpo Calloso/genética , Agenesia del Cuerpo Calloso/patología , Animales , Axones/ultraestructura , Proteínas de Unión al ADN/genética , Regulación del Desarrollo de la Expresión Génica , Histona Desacetilasas/metabolismo , Proteínas de Unión a la Región de Fijación a la Matriz/deficiencia , Proteínas de Unión a la Región de Fijación a la Matriz/genética , Ratones , Ratones Noqueados , Ratones Mutantes Neurológicos , Modelos Genéticos , Complejos Multiproteicos , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Neurogénesis/genética , Nucleosomas/metabolismo , Mapeo de Interacción de Proteínas , Proteínas Proto-Oncogénicas/genética , Proteínas Represoras/genética , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
Establishment of long-range fiber tracts by neocortical projection neurons is fundamental for higher brain functions. The molecular control of axon tract formation, however, is still poorly understood. Here, we have identified basic helix-loop-helix (bHLH) transcription factors Neurod2 and Neurod6 as key regulators of fasciculation and targeted axogenesis in the mouse neocortex. In Neurod2/6 double-mutant mice, callosal axons lack expression of the cell adhesion molecule Contactin2, defasciculate in the subventricular zone, and fail to grow toward the midline without forming Probst bundles. Instead, mutant axons overexpress Robo1 and follow random trajectories into the ipsilateral cortex. In contrast to long-range axogenesis, generation and maintenance of pyramidal neurons and initial axon outgrowth are grossly normal, suggesting that these processes are under distinct transcriptional control. Our findings define a new stage in corpus callosum development and demonstrate that neocortical projection neurons require transcriptional specification by neuronal bHLH proteins to execute an intrinsic program of remote connectivity.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Corteza Cerebral/fisiología , Proteínas del Tejido Nervioso/fisiología , Vías Nerviosas/fisiología , Neuronas/fisiología , Neuropéptidos/fisiología , Animales , Animales Recién Nacidos/fisiología , Axones/fisiología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Contactina 2/genética , Contactina 2/fisiología , Cuerpo Calloso/citología , Cuerpo Calloso/crecimiento & desarrollo , Cuerpo Calloso/fisiología , Electroporación , Embrión de Mamíferos/citología , Embrión de Mamíferos/fisiología , Genotipo , Glutamatos/fisiología , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Hibridación in Situ , Captura por Microdisección con Láser , Ratones , Fibras Nerviosas/fisiología , Proteínas del Tejido Nervioso/genética , Vías Nerviosas/citología , Vías Nerviosas/crecimiento & desarrollo , Neurogénesis/fisiología , Reacción en Cadena de la Polimerasa , Receptores Inmunológicos/genética , Receptores Inmunológicos/fisiología , Sinapsis/fisiología , Proteínas RoundaboutAsunto(s)
Envejecimiento/genética , Elementos Transponibles de ADN , Epigénesis Genética , Plasticidad Neuronal/genética , Neuronas/metabolismo , Reparación del ADN por Recombinación , Animales , Encéfalo/citología , Encéfalo/metabolismo , ADN/genética , ADN/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Histonas/genética , Histonas/metabolismo , Humanos , Ratones , Red Nerviosa , Neuronas/citologíaRESUMEN
Neural stem cells (NSCs) generate new granule cells throughout life in the mammalian hippocampus. Canonical Wnt signaling regulates the differentiation of NSCs towards the neuronal lineage. Here we identified the prospero-related homeodomain transcription factor Prox1 as a target of ß-catenin-TCF/LEF signaling in vitro and in vivo. Prox1 overexpression enhanced neuronal differentiation whereas shRNA-mediated knockdown of Prox1 impaired the generation of neurons in vitro and within the hippocampal niche. In contrast, Prox1 was not required for survival of adult-generated granule cells after they had matured, suggesting a role for Prox1 in initial granule cell differentiation but not in the maintenance of mature granule cells. The data presented here characterize a molecular pathway from Wnt signaling to a transcriptional target leading to granule cell differentiation within the adult brain and identify a stage-specific function for Prox1 in the process of adult neurogenesis.
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Diferenciación Celular/fisiología , Hipocampo/crecimiento & desarrollo , Proteínas de Homeodominio/metabolismo , Células-Madre Neurales/metabolismo , Neurogénesis/fisiología , Transducción de Señal/fisiología , Proteínas Supresoras de Tumor/metabolismo , Proteínas Wnt/metabolismo , Animales , Secuencia de Bases , Western Blotting , Células Cultivadas , Inmunoprecipitación de Cromatina , Cartilla de ADN/genética , Hipocampo/citología , Proteínas de Homeodominio/genética , Inmunohistoquímica , Hibridación in Situ , Luciferasas , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Supresoras de Tumor/genéticaRESUMEN
Disruption of neocortical circuitry and architecture in humans causes numerous neurodevelopmental disorders. Neocortical cytoarchitecture is orchestrated by various transcription factors such as Satb2 that control target genes during strict time windows. In humans, mutations of SATB2 cause SATB2 Associated Syndrome (SAS), a multisymptomatic syndrome involving epilepsy, intellectual disability, speech delay, and craniofacial defects. Here we show that Satb2 controls neuronal migration and callosal axonal outgrowth during murine neocortical development by inducing the expression of the GPI-anchored protein, Semaphorin 7A (Sema7A). We find that Sema7A exerts this biological activity by heterodimerizing in cis with the transmembrane semaphorin, Sema4D. We could also observe that heterodimerization with Sema7A promotes targeting of Sema4D to the plasma membrane in vitro. Finally, we report an epilepsy-associated de novo mutation in Sema4D (Q497P) that inhibits normal glycosylation and plasma membrane localization of Sema4D-associated complexes. These results suggest that neuronal use of semaphorins during neocortical development is heteromeric, and a greater signaling complexity exists than was previously thought.