RESUMEN
Astrotactin 2 (ASTN2) is a transmembrane neuronal protein highly expressed in the cerebellum that functions in receptor trafficking and modulates cerebellar Purkinje cell (PC) synaptic activity. Individuals with ASTN2 mutations exhibit neurodevelopmental disorders, including autism spectrum disorder (ASD), attention-deficit/hyperactivity disorder (ADHD), learning difficulties, and language delay. To provide a genetic model for the role of the cerebellum in ASD-related behaviors and study the role of ASTN2 in cerebellar circuit function, we generated global and PC-specific conditional Astn2 knockout (KO and cKO, respectively) mouse lines. Astn2 KO mice exhibit strong ASD-related behavioral phenotypes, including a marked decrease in separation-induced pup ultrasonic vocalization calls, hyperactivity, repetitive behaviors, altered behavior in the three-chamber test, and impaired cerebellar-dependent eyeblink conditioning. Hyperactivity and repetitive behaviors are also prominent in Astn2 cKO animals, but they do not show altered behavior in the three-chamber test. By Golgi staining, Astn2 KO PCs have region-specific changes in dendritic spine density and filopodia numbers. Proteomic analysis of Astn2 KO cerebellum reveals a marked upregulation of ASTN2 family member, ASTN1, a neuron-glial adhesion protein. Immunohistochemistry and electron microscopy demonstrate a significant increase in Bergmann glia volume in the molecular layer of Astn2 KO animals. Electrophysiological experiments indicate a reduced frequency of spontaneous excitatory postsynaptic currents (EPSCs), as well as increased amplitudes of both spontaneous EPSCs and inhibitory postsynaptic currents in the Astn2 KO animals, suggesting that pre- and postsynaptic components of synaptic transmission are altered. Thus, ASTN2 regulates ASD-like behaviors and cerebellar circuit properties.
Asunto(s)
Trastorno del Espectro Autista , Cerebelo , Ratones Noqueados , Células de Purkinje , Animales , Ratones , Trastorno del Espectro Autista/metabolismo , Trastorno del Espectro Autista/genética , Trastorno del Espectro Autista/fisiopatología , Células de Purkinje/metabolismo , Cerebelo/metabolismo , Conducta Animal/fisiología , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Modelos Animales de Enfermedad , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , MasculinoRESUMEN
Astrotactin 1 (Astn1) and Astn2 are membrane proteins that function in glial-guided migration, receptor trafficking, and synaptic plasticity in the brain as well as in planar polarity pathways in the skin. Here we used glycosylation mapping and protease protection approaches to map the topologies of mouse Astn1 and Astn2 in rough microsomal membranes and found that Astn2 has a cleaved N-terminal signal peptide, an N-terminal domain located in the lumen of the rough microsomal membranes (topologically equivalent to the extracellular surface in cells), two transmembrane helices, and a large C-terminal lumenal domain. We also found that Astn1 has the same topology as Astn2, but we did not observe any evidence of signal peptide cleavage in Astn1. Both Astn1 and Astn2 mature through endoproteolytic cleavage in the second transmembrane helix; importantly, we identified the endoprotease responsible for the maturation of Astn1 and Astn2 as the endoplasmic reticulum signal peptidase. Differences in the degree of Astn1 and Astn2 maturation possibly contribute to the higher levels of the C-terminal domain of Astn1 detected on neuronal membranes of the central nervous system. These differences may also explain the distinct cellular functions of Astn1 and Astn2, such as in membrane adhesion, receptor trafficking, and planar polarity signaling.
Asunto(s)
Glicoproteínas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Serina Endopeptidasas/metabolismo , Animales , Biocatálisis , Retículo Endoplásmico/metabolismo , Glicoproteínas/química , Glicosilación , Membranas Intracelulares/metabolismo , Ratones , Microsomas/metabolismo , Proteínas del Tejido Nervioso/química , ProteolisisRESUMEN
Prior studies demonstrate that astrotactin (ASTN1) provides a neuronal receptor for glial-guided CNS migration. Here we report that ASTN1 binds N-cadherin (CDH2) and that the ASTN1:CDH2 interaction supports cell-cell adhesion. To test the function of ASTN1:CDH2 binding in glial-guided neuronal migration, we generated a conditional loss of Cdh2 in cerebellar granule cells and in glia. Granule cell migration was slowed in cerebellar slice cultures after a conditional loss of neuronal Cdh2, and more severe migration defects occurred after a conditional loss of glial Cdh2 Expression in granule cells of a mutant form of ASTN1 that does not bind CDH2 also slowed migration. Moreover, in vitro chimeras of granule cells and glia showed impaired neuron-glia attachment in the absence of glial, but not neuronal, Cdh2 Thus, cis and trans bindings of ASTN1 to neuronal and glial CDH2 form an asymmetric neuron-glial bridge complex that promotes glial-guided neuronal migration.
Asunto(s)
Cadherinas/fisiología , Adhesión Celular , Movimiento Celular , Cerebelo/fisiología , Glicoproteínas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuroglía/fisiología , Neuronas/fisiología , Animales , Células Cultivadas , Cerebelo/citología , Glicoproteínas/genética , Ligandos , Proteínas del Tejido Nervioso/genética , Neurogénesis , Neuroglía/citología , Neuronas/citologíaRESUMEN
Surface protein dynamics dictate synaptic connectivity and function in neuronal circuits. ASTN2, a gene disrupted by copy number variations (CNVs) in neurodevelopmental disorders, including autism spectrum, was previously shown to regulate the surface expression of ASTN1 in glial-guided neuronal migration. Here, we demonstrate that ASTN2 binds to and regulates the surface expression of multiple synaptic proteins in postmigratory neurons by endocytosis, resulting in modulation of synaptic activity. In cerebellar Purkinje cells (PCs), by immunogold electron microscopy, ASTN2 localizes primarily to endocytic and autophagocytic vesicles in the cell soma and in subsets of dendritic spines. Overexpression of ASTN2 in PCs, but not of ASTN2 lacking the FNIII domain, recurrently disrupted by CNVs in patients, including in a family presented here, increases inhibitory and excitatory postsynaptic activity and reduces levels of ASTN2 binding partners. Our data suggest a fundamental role for ASTN2 in dynamic regulation of surface proteins by endocytic trafficking and protein degradation.
Asunto(s)
Variaciones en el Número de Copia de ADN , Glicoproteínas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Trastornos del Neurodesarrollo/genética , Sinapsis/fisiología , Animales , Movimiento Celular , Células Cultivadas , Endocitosis , Glicoproteínas/genética , Humanos , Ratones , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/genética , Trastornos del Neurodesarrollo/patología , Transporte de Proteínas , Proteolisis , Células de Purkinje/metabolismoRESUMEN
Inflammation-induced release of prostaglandin E2 (PGE2) changes breathing patterns and the response to CO2 levels. This may have fatal consequences in newborn babies and result in sudden infant death. To elucidate the underlying mechanisms, we present a novel breathing brainstem organotypic culture that generates rhythmic neural network and motor activity for 3 weeks. We show that increased CO2 elicits a gap junction-dependent release of PGE2. This alters neural network activity in the preBötzinger rhythm-generating complex and in the chemosensitive brainstem respiratory regions, thereby increasing sigh frequency and the depth of inspiration. We used mice lacking eicosanoid prostanoid 3 receptors (EP3R), breathing brainstem organotypic slices and optogenetic inhibition of EP3R(+/+) cells to demonstrate that the EP3R is important for the ventilatory response to hypercapnia. Our study identifies a novel pathway linking the inflammatory and respiratory systems, with implications for inspiration and sighs throughout life, and the ability to autoresuscitate when breathing fails.
Asunto(s)
Tronco Encefálico/efectos de los fármacos , Tronco Encefálico/fisiología , Dióxido de Carbono/metabolismo , Dinoprostona/metabolismo , Respiración/efectos de los fármacos , Potenciales de Acción , Animales , Ratones , Red Nerviosa/efectos de los fármacos , Optogenética , Técnicas de Cultivo de ÓrganosRESUMEN
Wnt/beta-catenin signaling plays an important role in neural development, instructing both progenitor cell division and differentiation. During early corticogenesis, Wnt7b is expressed in a restricted expression pattern in the ventricular zone progenitor cells. However, its influence on progenitor cell behavior has not been fully studied. We report that transgenic overexpression of Wnt7b in neural progenitor cells impairs neuronal differentiation and the development of forebrain structures at embryonic day 10.5 (E10.5). This was accompanied by a decreased expression of T-domain transcription factors Tbr1 and Tbr2, in both progenitor cells and post-mitotic neurons. However, proliferation, apoptosis and the overall proportion of pax6(+) neural progenitor cells were similar to wild-type litter mates. These results suggest that Wnt signaling may affect early neural progenitor differentiation by regulating the expression of pro-neural transcription factors.
Asunto(s)
Proteínas de Unión al ADN/biosíntesis , Neurogénesis/fisiología , Prosencéfalo/metabolismo , Proteínas Proto-Oncogénicas/fisiología , Proteínas de Dominio T Box/biosíntesis , Proteínas Wnt/fisiología , Animales , Movimiento Celular/fisiología , Sistema Nervioso Central/embriología , Sistema Nervioso Central/metabolismo , Citoesqueleto/ultraestructura , Proteínas de Unión al ADN/genética , Regulación hacia Abajo , Elementos de Facilitación Genéticos/genética , Genes Sintéticos , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microinyecciones , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/fisiología , Nestina/genética , Células-Madre Neurales/metabolismo , Tubo Neural/ultraestructura , Neuronas/metabolismo , Prosencéfalo/embriología , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/genética , Proteínas de Dominio T Box/genética , Tubulina (Proteína)/análisis , Proteínas Wnt/biosíntesis , Proteínas Wnt/genética , CigotoRESUMEN
During neuronal maturation, the neuron-specific K-Cl co-transporter KCC2 lowers the intracellular chloride and thereby renders GABAergic transmission hyperpolarizing. Independently of its role as a co-transporter, KCC2 plays a crucial role in the maturation of dendritic spines, most probably via an interaction with the cytoskeleton-associated protein 4.1N. In this study, we show that neural-specific overexpression of KCC2 impairs the development of the neural tube- and neural crest-related structures in mouse embryos. At early stages (E9.5-11.5), the transgenic embryos had a thinner neural tube and abnormal body curvature. They displayed a reduced neuronal differentiation and altered neural crest cell pattern. At later stages (E11.5-15.5), the transgenic embryos had smaller brain structures and a distinctive cleft palate. Similar results were obtained using overexpression of a transport-inactive N-terminal-deleted variant of KCC2, implying that the effects were not dependent on KCC2's role as a K-Cl co-transporter. Interestingly, the neural tube of transgenic embryos had an aberrant cytoplasmic distribution of 4.1N and actin. This was corroborated in a neural stem cell line with ectopic expression of KCC2. Embryo phenotype and cell morphology were unaffected by a mutated variant of KCC2 which is unable to bind 4.1N. These results point to a role of KCC2 in neuronal differentiation and migration during early development mediated by its direct structural interactions with the neuronal cytoskeleton.
Asunto(s)
Embrión de Mamíferos/fisiología , Transporte Iónico/fisiología , Neuronas/fisiología , Simportadores/metabolismo , Animales , Diferenciación Celular/fisiología , Movimiento Celular/fisiología , Células Cultivadas , Citoesqueleto/metabolismo , Citoesqueleto/ultraestructura , Embrión de Mamíferos/anatomía & histología , Femenino , Regulación del Desarrollo de la Expresión Génica , Humanos , Ratones , Ratones Transgénicos , Neuronas/citología , Embarazo , Simportadores/genética , Ácido gamma-Aminobutírico/metabolismo , Cotransportadores de K ClRESUMEN
The Wnt planar cell polarity (Wnt/PCP) pathway signals through small Rho-like GTPases to regulate the cytoskeleton. The core PCP proteins have been mapped to the Wnt/PCP pathway genetically, but the molecular mechanism of their action remains unknown. Here, we investigate the function of the mammalian PCP protein Vang-like protein 2 (Vangl2). RNAi knockdown of Vangl2 impaired cell-cell adhesion and cytoskeletal integrity in the epithelial cell lines HEK293T and MDCK. Similar effects were observed when Vangl2 was overexpressed in HEK293T, MDCK or C17.2 cells. The effects of Vangl2 overexpression could be blocked by knockdown of the small GTPase Rac1 or by dominant-negative Rac1. In itself, knockdown of Rac1 impaired cytoskeletal integrity and reduced cell-cell adhesion. We found that Vangl2 bound and re-distributed Rac1 within the cells but did not alter Rac1 activity. Moreover, both transgenic mouse embryos overexpressing Vangl2 in neural stem cells and loop-tail Vangl2 loss-of-function embryos displayed impaired adherens junctions, a cytoskeletal unit essential for neural tube rigidity and neural tube closure. In vivo, Rac1 was re-distributed within the cells in a similar way to that observed by us in vitro. We propose that Vangl2 affects cell adhesion and the cytoskeleton by recruiting Rac1 and targeting its activity in the cell to adherens junctions.
Asunto(s)
Uniones Adherentes/metabolismo , Proteínas de la Membrana/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Uniones Adherentes/genética , Animales , Línea Celular , Perros , Humanos , Inmunohistoquímica , Inmunoprecipitación , Hibridación in Situ , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/genética , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Unión Proteica/genética , Unión Proteica/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína de Unión al GTP rac1/genéticaRESUMEN
Wnt7a and HA-tagged Wnt7a have previously been shown to promote or delay neuronal differentiation respectively. In this study, we show that embryonic days 9.5 and 10.5 transgenic mouse embryos overexpressing Wnt7a specifically in nestin-positive neural stem/progenitor cells displayed a delay in neuronal differentiation, assayed by beta-tubulin III expression. Our results corroborate previous studies using HA-Wnt7a, and suggest a critical role for Wnt7a in control of neuronal progenitor maturation.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/fisiología , Neuronas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Células Madre/metabolismo , Tubulina (Proteína)/metabolismo , Proteínas Wnt/metabolismo , Animales , Encéfalo/citología , Encéfalo/embriología , Encéfalo/metabolismo , Diferenciación Celular , Proteínas de Filamentos Intermediarios/metabolismo , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/metabolismo , Nestina , Neuronas/citología , Proteínas Proto-Oncogénicas/genética , Células Madre/citología , Proteínas Wnt/genéticaRESUMEN
Wnt7a has been reported to signal via the canonical pathway, but also in non-canonical pathways acting on the cytoskeleton. Since Wnt7a is expressed after neurulation, we set to investigate the effects of Wnt7a on brain regionalization. We engineered transgenic mouse embryos that, under control of the nestin second intron, overexpressed Wnt7a in neural stem/progenitor cells. Surprisingly, transgenic embryos failed to complete cranial neurulation due to reduced levels and an impaired distribution of actin microfilaments, beta-catenin, and N-cadherin at the neural tube adherens junctions. These transgenic embryos expressed high levels of Vangl2, an essential component of non-canonical Wnt signaling. In agreement with a disregulation of this pathway, aberrant spinal neurulation was detected in the transgenic embryos, revealing a novel function regulated by Wnts. Thus, our findings suggest that Wnt7a overexpression disrupts normal Wnt signaling in the neural tube, resulting in defective adherens junctions and neurulation.