RESUMEN

Axon branching is crucial for proper formation of neuronal networks. Although originally identified as an angiogenic factor, VEGF also signals directly to neurons to regulate their development and function. Here we show that VEGF and its receptor VEGFR2 (also known as KDR or FLK1) are expressed in mouse hippocampal neurons during development, with VEGFR2 locally expressed in the CA3 region. Activation of VEGF/VEGFR2 signaling in isolated hippocampal neurons results in increased axon branching. Remarkably, inactivation of VEGFR2 also results in increased axon branching in vitro and in vivo. The increased CA3 axon branching is not productive as these axons are less mature and form less functional synapses with CA1 neurons. Mechanistically, while VEGF promotes the growth of formed branches without affecting filopodia formation, loss of VEGFR2 increases the number of filopodia and enhances the growth rate of new branches. Thus, a controlled VEGF/VEGFR2 signaling is required for proper CA3 hippocampal axon branching during mouse hippocampus development.

Asunto(s)

Axones/fisiología , Hipocampo/crecimiento & desarrollo , Hipocampo/metabolismo , Transducción de Señal/fisiología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Efrina-B2/genética , Regulación del Desarrollo de la Expresión Génica , Hipocampo/citología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Animales , Neurogénesis/genética , Neurogénesis/fisiología , Neuronas/citología , Neuronas/metabolismo , Seudópodos/metabolismo , Transducción de Señal/genética , Sinapsis/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética

RESUMEN

Vascular endothelial growth factor (VEGF) is an angiogenic factor that play important roles in the nervous system, although it is still unclear which receptors transduce those signals in neurons. Here, we show that in the developing hippocampus VEGFR2 (also known as KDR or FLK1) is expressed specifically in the CA3 region and it is required for dendritic arborization and spine morphogenesis in hippocampal neurons. Mice lacking VEGFR2 in neurons (Nes-cre Kdrlox/-) show decreased dendritic arbors and spines as well as a reduction in long-term potentiation (LTP) at the associational-commissural - CA3 synapses. Mechanistically, VEGFR2 internalization is required for VEGF-induced spine maturation. In analogy to endothelial cells, ephrinB2 controls VEGFR2 internalization in neurons. VEGFR2-ephrinB2 compound mice (Nes-cre Kdrlox/+ Efnb2lox/+) show reduced dendritic branching, reduced spine head size and impaired LTP. Our results demonstrate the functional crosstalk of VEGFR2 and ephrinB2 in vivo to control dendritic arborization, spine morphogenesis and hippocampal circuitry development.

Asunto(s)

Dendritas/metabolismo , Efrina-B2/metabolismo , Hipocampo/metabolismo , Neurogénesis/fisiología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Región CA3 Hipocampal , Espinas Dendríticas/metabolismo , Células Endoteliales/metabolismo , Efrina-B2/genética , Regulación del Desarrollo de la Expresión Génica , Potenciación a Largo Plazo/fisiología , Ratones , Neurogénesis/genética , Plasticidad Neuronal/fisiología , Neuronas/fisiología , Sinapsis/fisiología , Transcriptoma , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética

RESUMEN

The low-density lipoprotein receptor-related protein 4 (LRP4) is essential in muscle fibers for the establishment of the neuromuscular junction. Here, we show that LRP4 is also expressed by embryonic cortical and hippocampal neurons, and that downregulation of LRP4 in these neurons causes a reduction in density of synapses and number of primary dendrites. Accordingly, overexpression of LRP4 in cultured neurons had the opposite effect inducing more but shorter primary dendrites with an increased number of spines. Transsynaptic tracing mediated by rabies virus revealed a reduced number of neurons presynaptic to the cortical neurons in which LRP4 was knocked down. Moreover, neuron-specific knockdown of LRP4 by in utero electroporation of LRP4 miRNA in vivo also resulted in neurons with fewer primary dendrites and a lower density of spines in the developing cortex and hippocampus. Collectively, our results demonstrate an essential and novel role of neuronal LRP4 in dendritic development and synaptogenesis in the CNS.

Asunto(s)

Corteza Cerebral/metabolismo , Dendritas/metabolismo , Hipocampo/metabolismo , Receptores de LDL/metabolismo , Sinapsis/metabolismo , Animales , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/embriología , Técnicas de Inactivación de Genes , Hipocampo/citología , Hipocampo/embriología , Proteínas Relacionadas con Receptor de LDL , Ratones , Ratones Endogámicos C57BL , Rabia/patología , Virus de la Rabia/crecimiento & desarrollo , Receptores de LDL/genética

RESUMEN

Vascular endothelial growth factor (VEGF) is a major driver of blood vessel formation. However, the signal transduction pathways culminating in the biological consequences of VEGF signaling are only partially understood. Here, we show that the Hippo pathway effectors YAP and TAZ work as crucial signal transducers to mediate VEGF-VEGFR2 signaling during angiogenesis. We demonstrate that YAP/TAZ are essential for vascular development as endothelium-specific deletion of YAP/TAZ leads to impaired vascularization and embryonic lethality. Mechanistically, we show that VEGF activates YAP/TAZ via its effects on actin cytoskeleton and that activated YAP/TAZ induce a transcriptional program to further control cytoskeleton dynamics and thus establish a feedforward loop that ensures a proper angiogenic response. Lack of YAP/TAZ also results in altered cellular distribution of VEGFR2 due to trafficking defects from the Golgi apparatus to the plasma membrane. Altogether, our study identifies YAP/TAZ as central mediators of VEGF signaling and therefore as important regulators of angiogenesis.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales/metabolismo , Neovascularización Fisiológica , Fosfoproteínas/metabolismo , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/metabolismo , Citoesqueleto de Actina/genética , Animales , Animales Recién Nacidos , Encéfalo/patología , Proteínas de Ciclo Celular , Línea Celular Tumoral , Membrana Celular/metabolismo , Movimiento Celular/genética , Núcleo Celular/metabolismo , Inmunoprecipitación de Cromatina , Desarrollo Embrionario/genética , Células Endoteliales/metabolismo , Eliminación de Gen , Técnicas de Inactivación de Genes , Silenciador del Gen , Aparato de Golgi/metabolismo , Ratones , Modelos Biológicos , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Neovascularización Fisiológica/genética , Transducción de Señal/genética , Transactivadores , Transcripción Genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Proteínas Señalizadoras YAP

RESUMEN

Loss of the tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a prerequisite for tumor cell-specific expression of vascular endothelial growth factor receptor (VEGFR)-2 in glioblastoma defining a subgroup prone to develop evasive resistance towards antiangiogenic treatments. Immunohistochemical analysis of human tumor tissues showed VEGFR-2 expression in glioma cells in 19% of specimens examined, mainly in the infiltration zone. Glioma cell VEGFR-2 positivity was restricted to PTEN-deficient tumor specimens. PTEN overexpression reduced VEGFR-2 expression in vitro, as well as knock-down of raptor or rictor. Genetic interference with VEGFR-2 revealed proproliferative, antiinvasive and chemoprotective functions for VEGFR-2 in glioma cells. VEGFR-2-dependent cellular effects were concomitant with activation of 'kappa-light-chain-enhancer' of activated B-cells, protein kinase B, and N-myc downstream regulated gene 1. Two-photon in vivo microscopy revealed that expression of VEGFR-2 in glioma cells hampers antiangiogenesis. Bevacizumab induces a proinvasive response in VEGFR-2-positive glioma cells. Patients with PTEN-negative glioblastomas had a shorter survival after initiation of bevacizumab therapy compared with PTEN-positive glioblastomas. Conclusively, expression of VEGFR-2 in glioma cells indicates an aggressive glioblastoma subgroup developing early resistance to temozolomide or bevacizumab. Loss of PTEN may serve as a biomarker identifying those tumors upfront by routine neuropathological methods.

Asunto(s)

Inhibidores de la Angiogénesis/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Resistencia a Antineoplásicos , Glioma/tratamiento farmacológico , Neovascularización Patológica , Fosfohidrolasa PTEN/deficiencia , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Bevacizumab/farmacología , Neoplasias Encefálicas/enzimología , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/mortalidad , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Dacarbazina/análogos & derivados , Dacarbazina/farmacología , Supervivencia sin Enfermedad , Relación Dosis-Respuesta a Droga , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Glioma/enzimología , Glioma/genética , Glioma/mortalidad , Glioma/patología , Humanos , Estimación de Kaplan-Meier , Ratones Desnudos , Invasividad Neoplásica , Fosfohidrolasa PTEN/genética , Transducción de Señal/efectos de los fármacos , Temozolomida , Factores de Tiempo , Transfección , Carga Tumoral/efectos de los fármacos , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

The influenza virus (IFV) acquires its envelope by budding from host cell plasma membranes. Using quantitative shotgun mass spectrometry, we determined the lipidomes of the host Madin-Darby canine kidney cell, its apical membrane, and the IFV budding from it. We found the apical membrane to be enriched in sphingolipids (SPs) and cholesterol, whereas glycerophospholipids were reduced, and storage lipids were depleted compared with the whole-cell membranes. The virus membrane exhibited a further enrichment of SPs and cholesterol compared with the donor membrane at the expense of phosphatidylcholines. Our data are consistent with and extend existing models of membrane raft-based biogenesis of the apical membrane and IFV envelope.

Asunto(s)

Membrana Celular/química , Lípidos de la Membrana/análisis , Orthomyxoviridae/química , Animales , Línea Celular , Colesterol/análisis , Perros , Espectrometría de Masas , Esfingolípidos/análisis