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The exon junction complex (EJC), nucleated by EIF4A3, is indispensable for mRNA fate and function throughout eukaryotes. We discover that EIF4A3 directly controls microtubules, independent of RNA, which is critical for neural wiring. While neuronal survival in the developing mouse cerebral cortex depends upon an intact EJC, axonal tract development requires only Eif4a3. Using human cortical organoids, we show that EIF4A3 disease mutations also impair neuronal growth, highlighting conserved functions relevant for neurodevelopmental pathology. Live imaging of growing neurons shows that EIF4A3 is essential for microtubule dynamics. Employing biochemistry and competition experiments, we demonstrate that EIF4A3 directly binds to microtubules, mutually exclusive of the EJC. Finally, in vitro reconstitution assays and rescue experiments demonstrate that EIF4A3 is sufficient to promote microtubule polymerization and that EIF4A3-microtubule association is a major contributor to axon growth. This reveals a fundamental mechanism by which neurons re-utilize core gene expression machinery to directly control the cytoskeleton.
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Mutations in components of the exon junction complex (EJC) are associated with neurodevelopment and disease. In particular, reduced levels of the RNA helicase EIF4A3 cause Richieri-Costa-Pereira syndrome (RCPS) and copy number variations are linked to intellectual disability. Consistent with this, Eif4a3 haploinsufficient mice are microcephalic. Altogether, this implicates EIF4A3 in cortical development; however, the underlying mechanisms are poorly understood. Here, we use mouse and human models to demonstrate that EIF4A3 promotes cortical development by controlling progenitor mitosis, cell fate and survival. Eif4a3 haploinsufficiency in mice causes extensive cell death and impairs neurogenesis. Using Eif4a3;p53 compound mice, we show that apoptosis has the most impact on early neurogenesis, while additional p53-independent mechanisms contribute to later stages. Live imaging of mouse and human neural progenitors reveals that Eif4a3 controls mitosis length, which influences progeny fate and viability. These phenotypes are conserved, as cortical organoids derived from RCPS iPSCs exhibit aberrant neurogenesis. Finally, using rescue experiments we show that EIF4A3 controls neuron generation via the EJC. Altogether, our study demonstrates that EIF4A3 mediates neurogenesis by controlling mitosis duration and cell survival, implicating new mechanisms that underlie EJC-mediated disorders.
Asunto(s)
Variaciones en el Número de Copia de ADN , Proteína p53 Supresora de Tumor , Animales , Humanos , Ratones , ARN Helicasas DEAD-box/metabolismo , Factor 4A Eucariótico de Iniciación/genética , Factor 4A Eucariótico de Iniciación/metabolismo , Exones/genética , Mitosis/genética , Neurogénesis/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
mRNA localization and local translation enable exquisite spatial and temporal control of gene expression, particularly in polarized, elongated cells. These features are especially prominent in radial glial cells (RGCs), which are neural and glial precursors of the developing cerebral cortex and scaffolds for migrating neurons. Yet the mechanisms by which subcellular RGC compartments accomplish their diverse functions are poorly understood. Here, we demonstrate that mRNA localization and local translation of the RhoGAP ARHGAP11A in the basal endfeet of RGCs control their morphology and mediate neuronal positioning. Arhgap11a transcript and protein exhibit conserved localization to RGC basal structures in mice and humans, conferred by the 5' UTR. Proper RGC morphology relies upon active Arhgap11a mRNA transport and localization to the basal endfeet, where ARHGAP11A is locally synthesized. This translation is essential for positioning interneurons at the basement membrane. Thus, local translation spatially and acutely activates Rho signaling in RGCs to compartmentalize neural progenitor functions.
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Células Ependimogliales , Neuroglía , Humanos , Ratones , Animales , Células Ependimogliales/metabolismo , ARN Mensajero/metabolismo , Neuroglía/metabolismo , Neurogénesis , Corteza Cerebral , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismoRESUMEN
Mutations in components of the exon junction complex (EJC) are associated with neurodevelopment and disease. In particular, reduced levels of the RNA helicase EIF4A3 cause Richieri-Costa-Pereira Syndrome (RCPS) and CNVs are linked to intellectual disability. Consistent with this, Eif4a3 haploinsufficient mice are microcephalic. Altogether, this implicates EIF4A3 in cortical development; however, the underlying mechanisms are poorly understood. Here, we use mouse and human models to demonstrate that EIF4A3 promotes cortical development by controlling progenitor mitosis, cell fate, and survival. Eif4a3 haploinsufficiency in mice causes extensive cell death and impairs neurogenesis. Using Eif4a3 ; p53 compound mice, we show that apoptosis is most impactful for early neurogenesis, while additional p53-independent mechanisms contribute to later stages. Live imaging of mouse and human neural progenitors reveals Eif4a3 controls mitosis length, which influences progeny fate and viability. These phenotypes are conserved as cortical organoids derived from RCPS iPSCs exhibit aberrant neurogenesis. Finally, using rescue experiments we show that EIF4A3 controls neuron generation via the EJC. Altogether, our study demonstrates that EIF4A3 mediates neurogenesis by controlling mitosis duration and cell survival, implicating new mechanisms underlying EJC-mediated disorders. Summary statement: This study shows that EIF4A3 mediates neurogenesis by controlling mitosis duration in both mouse and human neural progenitors, implicating new mechanisms underlying neurodevelopmental disorders.
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Searches for the genetic underpinnings of uniquely human traits have focused on human-specific divergence in conserved genomic regions, which reflects adaptive modifications of existing functional elements. However, the study of conserved regions excludes functional elements that descended from previously neutral regions. Here, we demonstrate that the fastest-evolved regions of the human genome, which we term "human ancestor quickly evolved regions" (HAQERs), rapidly diverged in an episodic burst of directional positive selection prior to the human-Neanderthal split, before transitioning to constraint within hominins. HAQERs are enriched for bivalent chromatin states, particularly in gastrointestinal and neurodevelopmental tissues, and genetic variants linked to neurodevelopmental disease. We developed a multiplex, single-cell in vivo enhancer assay to discover that rapid sequence divergence in HAQERs generated hominin-unique enhancers in the developing cerebral cortex. We propose that a lack of pleiotropic constraints and elevated mutation rates poised HAQERs for rapid adaptation and subsequent susceptibility to disease.
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Hominidae , Hombre de Neandertal , Animales , Humanos , Hominidae/genética , Secuencias Reguladoras de Ácidos Nucleicos , Hombre de Neandertal/genética , Genoma Humano , GenómicaRESUMEN
Negative feedback loops represent a regulatory mechanism that guarantees that signaling thresholds are compatible with a physiological response. Previously, we established that Lrig1 acts through this mechanism to inhibit Ret activity. However, it is unclear whether other Lrig family members play similar roles. Here, we show that Lrig1 and Lrig3 are co-expressed in Ret-positive mouse dorsal root ganglion (DRG) neurons. Lrig3, like Lrig1, interacts with Ret and inhibits GDNF/Ret signaling. Treatment of DRG neurons with GDNF ligands induces a significant increase in the expression of Lrig1 and Lrig3. Our findings show that, whereas a single deletion of either Lrig1 or Lrig3 fails to promote Ret-mediated axonal growth, haploinsufficiency of Lrig1 in Lrig3 mutants significantly potentiates Ret signaling and axonal growth of DRG neurons in response to GDNF ligands. We observe that Lrig1 and Lrig3 act redundantly to ensure proper cutaneous innervation of nonpeptidergic axons and behavioral sensitivity to cold, which correlates with a significant increase in the expression of the cold-responsive channel TrpA1. Together, our findings provide insights into the in vivo functions through which Lrig genes control morphology, connectivity and function in sensory neurons.
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Axones/metabolismo , Epidermis/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Proto-Oncogénicas c-ret/metabolismo , Células Receptoras Sensoriales/metabolismo , Transducción de Señal/genética , Animales , Animales Recién Nacidos , Línea Celular Transformada , Ganglios Espinales/metabolismo , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Factor Neurotrófico Derivado de la Línea Celular Glial/farmacología , Células HEK293 , Humanos , Ligandos , Masculino , Glicoproteínas de Membrana/genética , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Neuronas Motoras/metabolismo , Proteínas del Tejido Nervioso/genética , Proyección Neuronal/genética , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos , TransfecciónRESUMEN
Embryonic development is a complex process that is unamenable to direct observation. In this study, we implanted a window to the mouse uterus to visualize the developing embryo from embryonic day 9.5 to birth. This removable intravital window allowed manipulation and high-resolution imaging. In live mouse embryos, we observed transient neurotransmission and early vascularization of neural crest cell (NCC)-derived perivascular cells in the brain, autophagy in the retina, viral gene delivery, and chemical diffusion through the placenta. We combined the imaging window with in utero electroporation to label and track cell division and movement within embryos and observed that clusters of mouse NCC-derived cells expanded in interspecies chimeras, whereas adjacent human donor NCC-derived cells shrank. This technique can be combined with various tissue manipulation and microscopy methods to study the processes of development at unprecedented spatiotemporal resolution.
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Embrión de Mamíferos/citología , Embrión de Mamíferos/fisiología , Desarrollo Embrionario , Microscopía Intravital/métodos , Cresta Neural , Animales , Encéfalo/embriología , Encéfalo/fisiología , División Celular , Movimiento Celular , Quimera/embriología , Quimera/fisiología , Electroporación , Femenino , Técnicas de Transferencia de Gen , Ratones , Ratones Transgénicos , Neovascularización Fisiológica , Cresta Neural/irrigación sanguínea , Cresta Neural/citología , Cresta Neural/embriología , Placenta/fisiología , Embarazo , Retina/embriología , Retina/fisiología , Transmisión Sináptica , ÚteroRESUMEN
De novo germline mutations in the RNA helicase DDX3X account for 1%-3% of unexplained intellectual disability (ID) cases in females and are associated with autism, brain malformations, and epilepsy. Yet, the developmental and molecular mechanisms by which DDX3X mutations impair brain function are unknown. Here, we use human and mouse genetics and cell biological and biochemical approaches to elucidate mechanisms by which pathogenic DDX3X variants disrupt brain development. We report the largest clinical cohort to date with DDX3X mutations (n = 107), demonstrating a striking correlation between recurrent dominant missense mutations, polymicrogyria, and the most severe clinical outcomes. We show that Ddx3x controls cortical development by regulating neuron generation. Severe DDX3X missense mutations profoundly disrupt RNA helicase activity, induce ectopic RNA-protein granules in neural progenitors and neurons, and impair translation. Together, these results uncover key mechanisms underlying DDX3X syndrome and highlight aberrant RNA metabolism in the pathogenesis of neurodevelopmental disease.
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Corteza Cerebral/metabolismo , ARN Helicasas DEAD-box/genética , Mutación Missense , Trastornos del Neurodesarrollo/genética , Neurogénesis , Animales , Línea Celular Tumoral , Células Cultivadas , Corteza Cerebral/anomalías , Corteza Cerebral/embriología , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Trastornos del Neurodesarrollo/patología , ARN/metabolismoRESUMEN
Genomic surveillance is crucial for shaping brain development. However, are these mechanisms always beneficial, and can they be manipulated to ameliorate neurodevelopmental disease? A recent paper by Shi et al. (Nat. Commun., 2019) sheds light on these questions and examines the consequences of both inducing genomic instability and suppressing safeguard mechanisms for the development of the cerebral cortex.
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Aneuploidia , Inestabilidad Genómica , Encéfalo , HumanosRESUMEN
Papillary thyroid carcinoma (PTC) and medullary thyroid carcinoma (MTC) are characterized by genomic rearrangements and point mutations in the proto-oncogene RET. Leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) is a suppressor of various receptor tyrosine kinases, including RET. LRIG1 expression levels are associated with patient survival in many cancer types. In the present study, we investigated whether the oncogenic RET mutants RET2A (C634R) and RET2B (M918T) were regulated by LRIG1, and the possible effects of LRIG1 expression in thyroid cancer were investigated in three different clinical cohorts and in a RET2B-driven mouse model of MTC. LRIG1 was shown to physically interact with both RET2A and RET2B and to restrict their ligand-independent activation. LRIG1 mRNA levels were downregulated in PTC and MTC compared to normal thyroid gland tissue. There was no apparent association between LRIG1 RNA or protein expression levels and patient survival in the studied cohorts. The transgenic RET2B mice developed pre-cancerous medullary thyroid lesions at a high frequency (36%); however, no overt cancers were observed. There was no significant difference in the incidence of pre-cancerous lesions between Lrig1 wild-type and Lrig1-deficient RET2B mice. In conclusion, the findings that LRIG1 is a negative regulator of RET2A and RET2B and is also downregulated in PTC and MTC may suggest that LRIG1 functions as a thyroid tumor suppressor.
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Carcinoma Neuroendocrino/genética , Carcinoma Papilar/genética , Regulación Neoplásica de la Expresión Génica/genética , Glicoproteínas de Membrana/genética , Proteínas Proto-Oncogénicas c-ret/genética , Neoplasias de la Tiroides/genética , Animales , Carcinoma Neuroendocrino/metabolismo , Carcinoma Papilar/metabolismo , Regulación hacia Abajo , Humanos , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-ret/metabolismo , Cáncer Papilar Tiroideo , Neoplasias de la Tiroides/metabolismoRESUMEN
Nerve growth factor (NGF) is a target-derived neurotrophic growth factor that controls many aspects of sensory and sympathetic neuronal development. The identification of transcription factors and downstream target genes that mediate NGF-dependent neuronal differentiation and target field innervation is currently a major challenge. Here, we show that the Pea3 transcription factor family members Etv4 and Etv5 are expressed by developing TrkA-positive dorsal root ganglion (DRG) neurons during the period of target innervation. Real-time PCR assays indicated that Etv4 and Etv5 mRNAs are significantly induced by NGF in different neuronal cells, suggesting that they could be involved in the biological responses induced by this neurotrophin. Interestingly, distal axon application of NGF in compartmentalized cultures of rat DRG sensory neurons was sufficient to induce a significant increase in Etv4 and Etv5 mRNA expression. Pharmacological assays also revealed that activation of MEK/ERK (MAPK) pathway is required for Etv4 and Etv5 gene induction in response to NGF. Downregulation of Etv4 and Etv5 using small interference RNA knockdown experiments inhibited NGF-induced neurite outgrowth of rat sensory neurons, while overexpression of full-length Etv4 or Etv5 potentiated neuronal differentiation in response to this neurotrophin. Together, these data establish Etv4 and Etv5 as essential molecules of the transcriptional program linking neurotrophin signaling to sensory neuronal differentiation, and suggest that they can be involved in NGF-mediated target innervation.
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Axones/metabolismo , Proteínas de Unión al ADN/metabolismo , Ganglios Espinales/metabolismo , Factor de Crecimiento Nervioso/farmacología , Células Receptoras Sensoriales/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Animales , Axones/efectos de los fármacos , Embrión de Pollo , Proteínas de Unión al ADN/genética , Ganglios Espinales/efectos de los fármacos , Células PC12 , Ratas , Ratas Wistar , Células Receptoras Sensoriales/efectos de los fármacos , Transactivadores/genética , Factores de Transcripción/genéticaRESUMEN
Neurotrophic growth factors control neuronal development by activating specific receptor tyrosine kinase positive signaling pathways, such as Ras-MAPK and PI3K-Akt cascades. Once activated, neurotrophic factor receptors also trigger a cascade of molecular events, named negative receptor signaling, that restricts the intensity of the positive signals and modulates cellular behavior. Thus, to avoid signaling errors that ultimately could lead to aberrant neuronal physiology and disease, negative signaling mechanisms have evolved to ensure that suitable thresholds of neuronal stimulation are achieved and maintained during right periods of time. Recent findings have revealed that neurotrophic factor receptor signaling is tightly modulated through the coordinated action of many different protein regulators that limit or potentiate signal propagation in spatially and temporally controlled manners, acting at specific points after receptor engagement. In this review, we discuss progress in this field, highlighting the importance of these modulators in axonal growth, guidance, neural connectivity, and nervous system regeneration.
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Regeneración Nerviosa/fisiología , Neurogénesis/fisiología , Receptores de Factor de Crecimiento Nervioso/metabolismo , Transducción de Señal/fisiología , Animales , HumanosRESUMEN
The Sprouty (Spry) family of proteins represents endogenous regulators of downstream signaling pathways induced by receptor tyrosine kinases (RTKs). Using real time PCR, we detect a significant increase in the expression of Spry4 mRNA in response to NGF, indicating that Spry4 could modulate intracellular signaling pathways and biological processes induced by NGF and its receptor TrkA. In this work, we demonstrate that overexpression of wild-type Spry4 causes a significant reduction in MAPK and Rac1 activation and neurite outgrowth induced by NGF. At molecular level, our findings indicate that ectopic expression of a mutated form of Spry4 (Y53A), in which a conserved tyrosine residue was replaced, fail to block both TrkA-mediated Erk/MAPK activation and neurite outgrowth induced by NGF, suggesting that an intact tyrosine 53 site is required for the inhibitory effect of Spry4 on NGF signaling. Downregulation of Spry4 using small interference RNA knockdown experiments potentiates PC12 cell differentiation and MAPK activation in response to NGF. Together, these findings establish a new physiological mechanism through which Spry4 regulates neurite outgrowth reducing not only the MAPK pathway but also restricting Rac1 activation in response to NGF.