RESUMEN
Tunneling nanotubes (TNTs) are actin-based transient tubular connections that allow direct communication between distant cells. TNTs play an important role in several physiological (development, immunity, and tissue regeneration) and pathological (cancer, neurodegeneration, and pathogens transmission) processes. Here, we report that the Wnt/Ca2+ pathway, an intracellular cascade that is involved in actin cytoskeleton remodeling, has a role in TNT formation and TNT-mediated transfer of cargoes. Specifically, we found that Ca2+ /calmodulin-dependent protein kinase II (CaMKII), a transducer of the Wnt/Ca2+ pathway, regulates TNTs in a neuronal cell line and in primary neurons. We identified the ß isoform of CaMKII as a key molecule in modulating TNT formation and transfer, showing that this depends on the actin-binding activity of the protein. Finally, we found that the transfer of vesicles and aggregated α-synuclein between primary neurons can be regulated by the activation of the Wnt/Ca2+ pathway. Our findings suggest that Wnt/Ca2+ pathway could be a novel promising target for therapies designed to impair TNT-mediated propagation of pathogens.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/fisiología , Calcio/metabolismo , Comunicación Celular , Membrana Celular/metabolismo , Nanotubos/química , Neuronas/fisiología , Proteínas Wnt/metabolismo , Actinas/metabolismo , Animales , Señalización del Calcio , Células Cultivadas , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/citología , Transducción de SeñalRESUMEN
Congenital microcephaly is highly associated with intellectual disability. Features of autosomal recessive primary microcephaly subtype 3 (MCPH3) also include hyperactivity and seizures. The disease is caused by biallelic mutations in the Cyclin-dependent kinase 5 regulatory subunit-associated protein 2 gene CDK5RAP2. In the mouse, Cdk5rap2 mutations similar to the human condition result in reduced brain size and a strikingly thin neocortex already at early stages of neurogenesis that persists through adulthood. The microcephaly phenotype in MCPH arises from a neural stem cell proliferation defect. Here, we report a novel role for Cdk5rap2 in the regulation of dendritic development and synaptogenesis of neocortical layer 2/3 pyramidal neurons. Cdk5rap2-deficient murine neurons show poorly branched dendritic arbors and an increased density of immature thin spines and glutamatergic synapses in vivo. Moreover, the excitatory drive is enhanced in ex vivo brain slice preparations of Cdk5rap2 mutant mice. Concurrently, we show that pyramidal neurons receive fewer inhibitory inputs. Together, these findings point towards a shift in the excitation - inhibition balance towards excitation in Cdk5rap2 mutant mice. Thus, MCPH3 is associated not only with a neural progenitor proliferation defect but also with altered function of postmitotic neurons and hence with altered connectivity.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Microcefalia/fisiopatología , Neocórtex/fisiopatología , Vías Nerviosas/fisiopatología , Neurogénesis/fisiología , Animales , Proteínas de Ciclo Celular/genética , Diferenciación Celular/fisiología , Ratones , Ratones Mutantes , Microcefalia/genética , Microcefalia/metabolismo , Mutación , Neocórtex/metabolismo , Vías Nerviosas/metabolismo , Células Piramidales/metabolismo , Células Piramidales/patología , Transmisión Sináptica/fisiologíaRESUMEN
Mutations in the MECP2 gene cause the neurodevelopmental disorder Rett syndrome (RTT). Previous studies have shown that altered MeCP2 levels result in aberrant neurite outgrowth and glutamatergic synapse formation. However, causal molecular mechanisms are not well understood since MeCP2 is known to regulate transcription of a wide range of target genes. Here, we describe a key role for a constitutive BDNF feed forward signaling pathway in regulating synaptic response, general growth and differentiation of glutamatergic neurons. Chronic block of TrkB receptors mimics the MeCP2 deficiency in wildtype glutamatergic neurons, while re-expression of BDNF quantitatively rescues MeCP2 deficiency. We show that BDNF acts cell autonomous and autocrine, as wildtype neurons are not capable of rescuing growth deficits in neighboring MeCP2 deficient neurons in vitro and in vivo. These findings are relevant for understanding RTT pathophysiology, wherein wildtype and mutant neurons are intermixed throughout the nervous system.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/metabolismo , Proteína 2 de Unión a Metil-CpG/metabolismo , Neuronas/fisiología , Transducción de Señal , Animales , Diferenciación Celular , Proliferación Celular , Modelos Animales de Enfermedad , Masculino , Proteína 2 de Unión a Metil-CpG/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Síndrome de Rett/fisiopatologíaRESUMEN
UNLABELLED: Neurotransmitter release requires the formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes by SNARE proteins syntaxin-1 (Stx1), synaptosomal-associated protein 25 (SNAP-25), and synaptobrevin-2 (Syb2). In mammalian systems, loss of SNAP-25 or Syb2 severely impairs neurotransmitter release; however, complete loss of function studies for Stx1 have been elusive due to the functional redundancy between Stx1 isoforms Stx1A and Stx1B and the embryonic lethality of Stx1A/1B double knock-out (DKO) mice. Here, we studied the roles of Stx1 in neuronal maintenance and neurotransmitter release in mice with constitutive or conditional deletion of Stx1B on an Stx1A-null background. Both constitutive and postnatal loss of Stx1 severely compromised neuronal viability in vivo and in vitro, indicating an obligatory role of Stx1 for maintenance of developing and mature neurons. Loss of Munc18-1, a high-affinity binding partner of Stx1, also showed severely impaired neuronal viability, but with a slower time course compared with Stx1A/1B DKO neurons, and exogenous Stx1A or Stx1B expression significantly delayed Munc18-1-dependent lethality. In addition, loss of Stx1 completely abolished fusion-competent vesicles and severely impaired vesicle docking, demonstrating its essential roles in neurotransmission. Putative partial SNARE complex assembly with the SNARE motif mutant Stx1A(AV) (A240V, V244A) was not sufficient to rescue neurotransmission despite full recovery of vesicle docking and neuronal survival. Together, these data suggest that Stx1 has independent functions in neuronal maintenance and neurotransmitter release and complete SNARE complex formation is required for vesicle fusion and priming, whereas partial SNARE complex formation is sufficient for vesicle docking and neuronal maintenance. SIGNIFICANCE STATEMENT: Syntaxin-1 (Stx1) is a component of the synaptic vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex and is essential for neurotransmission. We present the first detailed loss-of-function characterization of the two Stx1 isoforms in central mammalian neurons. We show that Stx1 is fundamental for maintenance of developing and mature neurons and also for vesicle docking and neurotransmission. We also demonstrate that neuronal maintenance and neurotransmitter release are regulated by Stx1 through independent functions. Furthermore, we show that SNARE complex formation is required for vesicle fusion, whereas partial SNARE complex formation is sufficient for vesicle docking and neuronal maintenance. Therefore, our work provides insights into differential functions of Stx1 in neuronal maintenance and neurotransmission, with the latter explored further into its functions in vesicle docking and fusion.
Asunto(s)
Fusión de Membrana/fisiología , Neuronas/fisiología , Terminales Presinápticos/fisiología , Transmisión Sináptica/fisiología , Vesículas Sinápticas/fisiología , Sintaxina 1/metabolismo , Animales , Proliferación Celular/fisiología , Supervivencia Celular/fisiología , Células Cultivadas , Femenino , Hipocampo/citología , Hipocampo/fisiología , Masculino , Ratones , Neurogénesis/fisiología , Neuronas/citología , Terminales Presinápticos/ultraestructura , Vesículas Sinápticas/ultraestructuraRESUMEN
STX1 is a major neuronal syntaxin protein located at the plasma membrane of the neuronal tissues. Rodent STX1 has two highly similar paralogs, STX1A and STX1B, that are thought to be functionally redundant. Interestingly, some studies have shown that the distribution patterns of STX1A and STX1B at the central and peripheral nervous systems only partially overlapped, implying that there might be differential functions between these paralogs. In the current study, we generated an STX1B knockout (KO) mouse line and studied the impact of STX1B removal in neurons of several brain regions and the neuromuscular junction (NMJ). We found that either complete removal of STX1B or selective removal of it from forebrain excitatory neurons in mice caused premature death. Autaptic hippocampal and striatal cultures derived from STX1B KO mice still maintained efficient neurotransmission compared with neurons from STX1B wild-type and heterozygous mice. Interestingly, examining high-density cerebellar cultures revealed a decrease in the spontaneous GABAergic transmission frequency, which was most likely due to a lower number of neurons in the STX1B KO cultures, suggesting that STX1B is essential for neuronal survival in vitro. Moreover, our study also demonstrated that although STX1B is dispensable for the formation of the mouse NMJ, it is required to maintain the efficiency of neurotransmission at the nerve-muscle synapse.
Asunto(s)
Encéfalo/fisiopatología , Unión Neuromuscular/fisiología , Neuronas/fisiología , Sintaxina 1/metabolismo , Animales , Western Blotting , Encéfalo/patología , Supervivencia Celular/fisiología , Células Cultivadas , Muerte , Potenciales Postsinápticos Excitadores/fisiología , Inmunohistoquímica , Potenciales Postsinápticos Inhibidores/fisiología , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Potenciales Postsinápticos Miniatura/fisiología , Proteínas Munc18/metabolismo , Neuronas/patología , Técnicas de Placa-Clamp , Sintaxina 1/genética , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Synaptic vesicles undergo sequential steps in preparation for neurotransmitter release. Individual SNARE proteins and the SNARE complex itself have been implicated in these processes. However, discrete effects of SNARE proteins on synaptic function have been difficult to assess using complete loss-of-function approaches. We therefore used a genetic titration technique in cultured mouse hippocampal neurons to evaluate the contribution of the neuronal SNARE protein Syntaxin1 (Stx1) in vesicle docking, priming, and release probability. We generated graded reductions of total Stx1 levels by combining two approaches, namely, endogenous hypomorphic expression of the isoform Stx1B and RNAi-mediated knockdown. Proximity of synaptic vesicles to the active zone was not strongly affected. However, overall release efficiency of affected neurons was severely impaired, as demonstrated by a smaller readily releasable pool size, slower refilling rate of primed vesicles, and lower release probability. Interestingly, dose-response fitting of Stx1 levels against readily releasable pool size and vesicular release probability showed similar Kd (dissociation constant) values at 18% and 19% of wild-type Stx1, with cooperativity estimates of 3.4 and 2.5, respectively. This strongly suggests that priming and vesicle fusion share the same molecular stoichiometry, and are governed by highly related mechanisms.
Asunto(s)
Exocitosis/fisiología , Sinapsis/metabolismo , Transmisión Sináptica/fisiología , Vesículas Sinápticas/metabolismo , Sintaxina 1/metabolismo , Animales , Línea Celular , Hipocampo/citología , Hipocampo/metabolismo , Fusión de Membrana/fisiología , Ratones , Neuronas/citología , Neuronas/metabolismo , Vesículas Sinápticas/genética , Sintaxina 1/genéticaRESUMEN
Borna disease virus (BDV) is a nonsegmented, negative-stranded RNA virus characterized by noncytolytic persistent infection and replication in the nuclei of infected cells. To gain further insight on the intracellular trafficking of BDV components during infection, we sought to generate recombinant BDV (rBDV) encoding fluorescent fusion viral proteins. We successfully rescued a virus bearing a tetracysteine tag fused to BDV-P protein, which allowed assessment of the intracellular distribution and dynamics of BDV using real-time live imaging. In persistently infected cells, viral nuclear inclusions, representing viral factories tethered to chromatin, appeared to be extremely static and stable, contrasting with a very rapid and active trafficking of BDV components in the cytoplasm. Photobleaching (fluorescence recovery after photobleaching [FRAP] and fluorescence loss in photobleaching [FLIP]) imaging approaches revealed that BDV components were permanently and actively exchanged between cellular compartments, including within viral inclusions, albeit with a fraction of BDV-P protein not mobile in these structures, presumably due to its association with viral and/or cellular proteins. We also obtained evidence for transfer of viral material between persistently infected cells, with routing of the transferred components toward the cell nucleus. Finally, coculture experiments with noninfected cells allowed visualization of cell-to-cell BDV transmission and movement of the incoming viral material toward the nucleus. Our data demonstrate the potential of tetracysteine-tagged recombinant BDV for virus tracking during infection, which may provide novel information on the BDV life cycle and on the modalities of its interaction with the nuclear environment during viral persistence.
Asunto(s)
Enfermedad de Borna/virología , Virus de la Enfermedad de Borna/patogenicidad , Núcleo Celular/metabolismo , Cisteína/química , Citoplasma/metabolismo , Fosfoproteínas/metabolismo , Proteínas Virales de Fusión/metabolismo , Proteínas Estructurales Virales/metabolismo , Animales , Northern Blotting , Western Blotting , Enfermedad de Borna/metabolismo , Chlorocebus aethiops , Técnica del Anticuerpo Fluorescente , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Inmunoprecipitación , Fosfoproteínas/genética , Transporte de Proteínas , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Vero , Proteínas Virales de Fusión/genética , Proteínas Estructurales Virales/genéticaRESUMEN
Borna disease virus (BDV) persistently infects neurons of the central nervous system of various hosts, including rats. Since type I IFN-mediated antiviral response efficiently blocks BDV replication in primary rat embryo fibroblasts, it has been speculated that BDV is not effectively sensed by the host innate immune system in the nervous system. To test this assumption, organotypical rat hippocampal slice cultures were infected with BDV for up to 4 weeks. This resulted in the secretion of IFN and the up-regulation of IFN-stimulated genes. Using the rat Mx protein as a specific marker for IFN-induced gene expression, astrocytes and microglial cells were found to be Mx positive, whereas neurons, the major cell type in which BDV is replicating, lacked detectable levels of Mx protein. In uninfected cultures, neurons also remained Mx negative even after treatment with high concentrations of IFN-α. This non-responsiveness correlated with a lack of detectable nuclear translocation of both pSTAT1 and pSTAT2 in these cells. Consistently, neuronal dissemination of BDV was not prevented by treatment with IFN-α. These data suggest that the poor innate immune response in rat neurons renders this cell type highly susceptible to BDV infection even in the presence of exogenous IFN-α. Intriguingly, in contrast to rat neurons, IFN-α treatment of mouse neurons resulted in the up-regulation of Mx proteins and block of BDV replication, indicating species-specific differences in the type I IFN response of neurons between mice and rats.
Asunto(s)
Enfermedad de Borna/inmunología , Virus de la Enfermedad de Borna/fisiología , Inmunidad Innata/fisiología , Neuronas/metabolismo , Animales , Astrocitos/citología , Astrocitos/metabolismo , Enfermedad de Borna/metabolismo , Enfermedad de Borna/virología , Hipocampo/citología , Hipocampo/metabolismo , Interferón-alfa/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Microglía/citología , Microglía/metabolismo , Proteínas de Resistencia a Mixovirus/metabolismo , Neuronas/citología , Neuronas/virología , Fosforilación , Ratas , Ratas Endogámicas Lew , Ratas Sprague-Dawley , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT2/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Replicación ViralRESUMEN
Infection of newborn rats with Borne disease virus (BDV) results in selective degeneration of granule cell neurons of the dentate gyrus (DG). To study cellular countermechanisms that might prevent this pathology, we screened for rat strains resistant to this BDV-induced neuronal degeneration. To this end, we infected hippocampal slice cultures of different rat strains with BDV and analyzed for the preservation of the DG. Whereas infected cultures of five rat strains, including Lewis (LEW) rats, exhibited a disrupted DG cytoarchitecture, slices of three other rat strains, including Sprague-Dawley (SD), were unaffected. However, efficiency of viral replication was comparable in susceptible and resistant cultures. Moreover, these rat strain-dependent differences in vulnerability were replicated in vivo in neonatally infected LEW and SD rats. Intriguingly, conditioned media from uninfected cultures of both LEW and SD rats could prevent BDV-induced DG damage in infected LEW hippocampal cultures, whereas infection with BDV suppressed the availability of these factors from LEW but not in SD hippocampal cultures. To gain further insights into the genetic basis for this rat strain-dependent susceptibility, we analyzed DG granule cell survival in BDV-infected cultures of hippocampal neurons derived from the F1 and F2 offspring of the crossing of SD and LEW rats. Genome-wide association analysis revealed one resistance locus on chromosome (chr) 6q16 in SD rats and, surprisingly, a locus on chr3q21-23 that was associated with susceptibility. Thus, BDV-induced neuronal degeneration is dependent on the host genetic background and is prevented by soluble protective factors in the disease-resistant SD rat strain.
Asunto(s)
Virus de la Enfermedad de Borna/fisiología , Giro Dentado/virología , Degeneración Nerviosa/virología , Neuronas/virología , Animales , Animales Recién Nacidos , Factores Biológicos/química , Factores Biológicos/farmacología , Factor Neurotrófico Derivado del Encéfalo/farmacología , Supervivencia Celular/efectos de los fármacos , Mapeo Cromosómico , Cromosomas de los Mamíferos/genética , Medios de Cultivo Condicionados/química , Medios de Cultivo Condicionados/farmacología , Giro Dentado/metabolismo , Giro Dentado/patología , Resistencia a la Enfermedad/genética , Femenino , Hipocampo/metabolismo , Hipocampo/patología , Hipocampo/virología , Interacciones Huésped-Patógeno , Masculino , Degeneración Nerviosa/genética , Degeneración Nerviosa/prevención & control , Neuronas/metabolismo , Neuronas/patología , Polimorfismo de Nucleótido Simple , Ratas , Ratas Endogámicas Lew , Ratas Sprague-Dawley , Solubilidad , Especificidad de la Especie , Técnicas de Cultivo de TejidosRESUMEN
The cytopathogenicity of vesicular stomatitis virus (VSV) has been attributed mainly to the host shut-off activity of the viral matrix (M) protein, which inhibits both nuclear transcription and nucleocytoplasmic RNA transport, thereby effectively suppressing the synthesis of type I interferon (IFN). The M protein from persistently VSV-infected cells was shown to harbour characteristic amino acid substitutions (M51R, V221F and S226R) implicated in IFN induction. This study demonstrates that infection of human fibroblasts with recombinant VSV containing the M51R substitution resulted in IFN induction, whereas neither the V221F nor the S226R substitution effected an IFN-inducing phenotype. Only when V221F was combined with S226R were the host shut-off activity of the M protein abolished and IFN induced, independently of M51R. The M33A substitution, previously implicated in VSV cytotoxicity, did not affect host shut-off activity. M-mutant VSV containing all four amino acid substitutions retained cytotoxic properties in both Vero cells and IFN-competent primary fibroblasts. Infected-cell death was associated with the formation of giant polynucleated cells, suggesting that the fusion activity of the VSV G protein was involved. Accordingly, M-mutant VSV expressing a fusion-defective G protein or with a deletion of the G gene showed significantly reduced cytotoxic properties and caused long-lasting infections in Vero cells and mouse hippocampal slice cultures. In contrast, a G-deleted VSV expressing wild-type M protein remained cytotoxic. These findings indicate that the host shut-off activity of the M protein dominates VSV cytotoxicty, whilst the fusion-active G protein is mainly responsible for the cytotoxicity remaining with M-mutant VSV.
Asunto(s)
Efecto Citopatogénico Viral , Glicoproteínas de Membrana/metabolismo , Proteínas Mutantes/toxicidad , Vesiculovirus/inmunología , Vesiculovirus/patogenicidad , Proteínas del Envoltorio Viral/metabolismo , Proteínas de la Matriz Viral/toxicidad , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Animales , Células Cultivadas , Cricetinae , Fibroblastos/virología , Células Gigantes/virología , Hipocampo/virología , Humanos , Interferones/biosíntesis , Glicoproteínas de Membrana/genética , Ratones , Datos de Secuencia Molecular , Proteínas Mutantes/genética , Proteínas Mutantes/inmunología , Técnicas de Cultivo de Órganos , Proteínas del Envoltorio Viral/genética , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/inmunologíaRESUMEN
Granule cells are major targets of entorhinal afferents terminating in a laminar fashion in the outer molecular layer of the dentate gyrus. Since Borna disease virus (BDV) infection of newborn rats causes a progressive loss of granule cells in the dentate gyrus, entorhinal fibres become disjoined from their main targets. We have investigated the extent to which entorhinal axons react to this loss of granule cells. Unexpectedly, anterograde DiI tracing has shown a prominent layered termination of the entorhinal projection, despite an almost complete loss of granule cells at 9 weeks after infection. Combined light- and electron-microscopic analysis of dendrites at the outer molecular layer of the dentate gyrus at 6 and 9 weeks post-infection has revealed a transient increase in the synaptic density of calbindin-positive granule cells and parvalbuminergic neurons after 6 weeks. In contrast, synaptic density reaches values similar to those of uninfected controls 9 weeks post-infection. These findings indicate that, after BDV infection, synaptic reorganization processes occur at peripheral dendrites of the remaining granule cells and parvalbuminergic neurons, including the unexpected persistence of entorhinal axons in the absence of their main targets.