RESUMEN
Inflammasomes are cytosolic multiprotein complexes that assemble in response to a variety of infectious and noxious insults. Inflammasomes play a critical role in the initiation of innate immune responses, primarily by serving as platforms for the activation of inflammatory caspase proteases. One such caspase, CASPASE-1 (CASP1), initiates innate immune responses by cleaving pro-IL-1ß and pro-IL-18, leading to their activation and release. CASP1 and another inflammatory caspase termed CASP11 can also initiate a rapid and inflammatory form of cell death termed pyroptosis. Several distinct inflammasomes have been described, each of which contains a unique sensor protein of the NLR (nucleotide-binding domain, leucine-rich repeat-containing) superfamily or the PYHIN (PYRIN and HIN-200 domain-containing) superfamily. Here we describe the surprisingly diverse mechanisms by which NLR/PYHIN proteins sense bacteria and initiate innate immune responses. We conclude that inflammasomes represent a highly adaptable scaffold ideally suited for detecting and initiating rapid innate responses to diverse and rapidly evolving bacteria.
Asunto(s)
Bacterias/patogenicidad , Inflamasomas/metabolismo , Animales , Bacillus anthracis/patogenicidad , Proteínas Adaptadoras de Señalización CARD/metabolismo , Proteínas Adaptadoras de Señalización CARD/fisiología , Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión al Calcio/fisiología , Flagelos/metabolismo , Flagelos/fisiología , Humanos , Inflamasomas/genética , Inflamasomas/fisiología , Legionella pneumophila/patogenicidad , Listeria monocytogenes/patogenicidad , Salmonella typhimurium/patogenicidadRESUMEN
Social impairment is frequently associated with mitochondrial dysfunction and altered neurotransmission. Although mitochondrial function is crucial for brain homeostasis, it remains unknown whether mitochondrial disruption contributes to social behavioral deficits. Here, we show that Drosophila mutants in the homolog of the human CYFIP1, a gene linked to autism and schizophrenia, exhibit mitochondrial hyperactivity and altered group behavior. We identify the regulation of GABA availability by mitochondrial activity as a biologically relevant mechanism and demonstrate its contribution to social behavior. Specifically, increased mitochondrial activity causes gamma aminobutyric acid (GABA) sequestration in the mitochondria, reducing GABAergic signaling and resulting in social deficits. Pharmacological and genetic manipulation of mitochondrial activity or GABA signaling corrects the observed abnormalities. We identify Aralar as the mitochondrial transporter that sequesters GABA upon increased mitochondrial activity. This study increases our understanding of how mitochondria modulate neuronal homeostasis and social behavior under physiopathological conditions.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Proteínas de Drosophila/metabolismo , Mitocondrias/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Animales Modificados Genéticamente , Ácido Aspártico/metabolismo , Calcio/metabolismo , Proteínas de Unión al Calcio/fisiología , Proteínas de Drosophila/fisiología , Drosophila melanogaster/metabolismo , Glucosa/metabolismo , Homeostasis , Humanos , Masculino , Mitocondrias/genética , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas Mitocondriales/metabolismo , Neuronas/metabolismo , Conducta Social , Transmisión Sináptica , Ácido gamma-Aminobutírico/genéticaRESUMEN
Varying pH of luminal fluid along the female reproductive tract is a physiological cue that modulates sperm motility. CatSper is a sperm-specific, pH-sensitive calcium channel essential for hyperactivated motility and male fertility. Multi-subunit CatSper channel complexes organize linear Ca2+ signaling nanodomains along the sperm tail. Here, we identify EF-hand calcium-binding domain-containing protein 9 (EFCAB9) as a bifunctional, cytoplasmic machine modulating the channel activity and the domain organization of CatSper. Knockout mice studies demonstrate that EFCAB9, in complex with the CatSper subunit, CATSPERζ, is essential for pH-dependent and Ca2+-sensitive activation of the CatSper channel. In the absence of EFCAB9, sperm motility and fertility is compromised, and the linear arrangement of the Ca2+ signaling domains is disrupted. EFCAB9 interacts directly with CATSPERζ in a Ca2+-dependent manner and dissociates at elevated pH. These observations suggest that EFCAB9 is a long-sought, intracellular, pH-dependent Ca2+ sensor that triggers changes in sperm motility.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Motilidad Espermática/fisiología , Animales , Calcio/metabolismo , Canales de Calcio/metabolismo , Señalización del Calcio/fisiología , Proteínas de Unión al Calcio/fisiología , Línea Celular , Membrana Celular/metabolismo , Fertilidad , Células HEK293 , Humanos , Concentración de Iones de Hidrógeno , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Espermatozoides/metabolismoRESUMEN
Neurogenesis in the adult brain gives rise to functional neurons, which integrate into neuronal circuits and modulate neural plasticity. Sustained neurogenesis throughout life occurs in the subgranular zone (SGZ) of the dentate gyrus in the hippocampus and is hypothesized to be involved in behavioral/cognitive processes such as memory and in diseases. Genomic imprinting is of critical importance to brain development and normal behavior, and exemplifies how epigenetic states regulate genome function and gene dosage. While most genes are expressed from both alleles, imprinted genes are usually expressed from either the maternally or the paternally inherited chromosome. Here, we show that in contrast to its canonical imprinting in nonneurogenic regions, Delta-like homolog 1 (Dlk1) is expressed biallelically in the SGZ, and both parental alleles are required for stem cell behavior and normal adult neurogenesis in the hippocampus. To evaluate the effects of maternally, paternally, and biallelically inherited mutations within the Dlk1 gene in specific behavioral domains, we subjected Dlk1-mutant mice to a battery of tests that dissociate and evaluate the effects of Dlk1 dosage on spatial learning ability and on anxiety traits. Importantly, reduction in Dlk1 levels triggers specific cognitive abnormalities that affect aspects of discriminating differences in environmental stimuli, emphasizing the importance of selective absence of imprinting in this neurogenic niche.
Asunto(s)
Proteínas de Unión al Calcio/genética , Cognición/fisiología , Dosificación de Gen , Neurogénesis/fisiología , Alelos , Animales , Proteínas de Unión al Calcio/fisiología , Hipocampo/metabolismo , RatonesRESUMEN
The immunosuppressive and hypoxic tumor microenvironment (TME) remains a major obstacle to impede cancer immunotherapy. Here, we showed that elevated levels of Delta-like 1 (DLL1) in the breast and lung TME induced long-term tumor vascular normalization to alleviate tumor hypoxia and promoted the accumulation of interferon γ (IFN-γ)-expressing CD8+ T cells and the polarization of M1-like macrophages. Moreover, increased DLL1 levels in the TME sensitized anti-cytotoxic T lymphocyte-associated protein 4 (anti-CTLA4) treatment in its resistant tumors, resulting in tumor regression and prolonged survival. Mechanically, in vivo depletion of CD8+ T cells or host IFN-γ deficiency reversed tumor growth inhibition and abrogated DLL1-induced tumor vascular normalization without affecting DLL1-mediated macrophage polarization. Together, these results demonstrate that elevated DLL1 levels in the TME promote durable tumor vascular normalization in a CD8+ T cell- and IFN-γ-dependent manner and potentiate anti-CTLA4 therapy. Our findings unveil DLL1 as a potential target to persistently normalize the TME to facilitate cancer immunotherapy.
Asunto(s)
Vasos Sanguíneos/patología , Linfocitos T CD8-positivos/inmunología , Proteínas de Unión al Calcio/fisiología , Neoplasias/irrigación sanguínea , Neoplasias/patología , Animales , Femenino , Células HEK293 , Humanos , Inmunoterapia , Ratones , Ratones Endogámicos C57BL , Neoplasias/inmunología , Neoplasias/terapia , Microambiente TumoralRESUMEN
Organogenesis requires precise interactions between a developing tissue and its environment. In vertebrates, the developing eye is surrounded by a complex extracellular matrix as well as multiple mesenchymal cell populations. Disruptions to either the matrix or periocular mesenchyme can cause defects in early eye development, yet in many cases the underlying mechanism is unknown. Here, using multidimensional imaging and computational analyses in zebrafish, we establish that cell movements in the developing optic cup require neural crest. Ultrastructural analysis reveals that basement membrane formation around the developing eye is also dependent on neural crest, but only specifically around the retinal pigment epithelium. Neural crest cells produce the extracellular matrix protein nidogen: impairing nidogen function disrupts eye development, and, strikingly, expression of nidogen in the absence of neural crest partially restores optic cup morphogenesis. These results demonstrate that eye formation is regulated in part by extrinsic control of extracellular matrix assembly.This article has an associated 'The people behind the papers' interview.
Asunto(s)
Membrana Basal/embriología , Ojo/embriología , Cresta Neural/embriología , Alelos , Animales , Sistemas CRISPR-Cas , Proteínas de Unión al Calcio/fisiología , Movimiento Celular , Electroforesis Capilar , Matriz Extracelular/fisiología , Proteínas de la Matriz Extracelular/fisiología , Factores de Transcripción Forkhead/fisiología , Regulación del Desarrollo de la Expresión Génica , Genotipo , Mesodermo/embriología , Microscopía Electrónica de Transmisión , Morfogénesis , Mutación , Cresta Neural/citología , Organogénesis , Retina/embriología , Epitelio Pigmentado de la Retina/embriología , Transducción de Señal , Factor de Transcripción AP-2/fisiología , Pez Cebra , Proteínas de Pez Cebra/fisiologíaRESUMEN
Ca2+ uptake by mitochondria regulates bioenergetics, apoptosis, and Ca2+ signaling. The primary pathway for mitochondrial Ca2+ uptake is the mitochondrial calcium uniporter (MCU), a Ca2+-selective ion channel in the inner mitochondrial membrane. MCU-mediated Ca2+ uptake is driven by the sizable inner-membrane potential generated by the electron-transport chain. Despite the large thermodynamic driving force, mitochondrial Ca2+ uptake is tightly regulated to maintain low matrix [Ca2+] and prevent opening of the permeability transition pore and cell death, while meeting dynamic cellular energy demands. How this is accomplished is controversial. Here we define a regulatory mechanism of MCU-channel activity in which cytoplasmic Ca2+ regulation of intermembrane space-localized MICU1/2 is controlled by Ca2+-regulatory mechanisms localized across the membrane in the mitochondrial matrix. Ca2+ that permeates through the channel pore regulates Ca2+ affinities of coupled inhibitory and activating sensors in the matrix. Ca2+ binding to the inhibitory sensor within the MCU amino terminus closes the channel despite Ca2+ binding to MICU1/2. Conversely, disruption of the interaction of MICU1/2 with the MCU complex disables matrix Ca2+ regulation of channel activity. Our results demonstrate how Ca2+ influx into mitochondria is tuned by coupled Ca2+-regulatory mechanisms on both sides of the inner mitochondrial membrane.
Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Mitocondrias/metabolismo , Apoptosis , Transporte Biológico , Calcio/fisiología , Canales de Calcio/fisiología , Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión al Calcio/fisiología , Proteínas de Transporte de Catión/metabolismo , Proteínas de Transporte de Catión/fisiología , Citoplasma/metabolismo , Citosol/metabolismo , Células HEK293 , Células HeLa , Humanos , Potencial de la Membrana Mitocondrial/fisiología , Mitocondrias/fisiología , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/fisiología , Membranas Mitocondriales/metabolismo , Membranas Mitocondriales/fisiología , Oxidación-Reducción , Multimerización de Proteína , Transducción de SeñalRESUMEN
Presynaptic neurotransmitter release is strictly regulated by SNARE proteins, Ca2+ and a number of Ca2+ sensors including synaptotagmins (Syts) and Double C2 domain proteins (Doc2s). More than seventy years after the original description of spontaneous release, the mechanism that regulates this process is still poorly understood. Syt-1, Syt7 and Doc2 proteins contribute predominantly, but not exclusively, to synchronous, asynchronous and spontaneous phases of release. The proteins share a conserved tandem C2 domain architecture, but are functionally diverse in their subcellular location, Ca2+-binding properties and protein interactions. In absence of Syt-1, Doc2a and -b, neurons still exhibit spontaneous vesicle fusion which remains Ca2+-sensitive, suggesting the existence of additional sensors. Here, we selected Doc2c, rabphilin-3a and Syt-7 as three potential Ca2+ sensors for their sequence homology with Syt-1 and Doc2b. We genetically ablated each candidate gene in absence of Doc2a and -b and investigated spontaneous and evoked release in glutamatergic hippocampal neurons, cultured either in networks or on microglial islands (autapses). The removal of Doc2c had no effect on spontaneous or evoked release. Syt-7 removal also did not affect spontaneous release, although it altered short-term plasticity by accentuating short-term depression. The removal of rabphilin caused an increased spontaneous release frequency in network cultures, an effect that was not observed in autapses. Taken together, we conclude that Doc2c and Syt-7 do not affect spontaneous release of glutamate in hippocampal neurons, while our results suggest a possible regulatory role of rabphilin-3a in neuronal networks. These findings importantly narrow down the repertoire of synaptic Ca2+ sensors that may be implicated in the spontaneous release of glutamate.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Proteínas de Unión al Calcio/fisiología , Calcio/metabolismo , Hipocampo/metabolismo , Proteínas del Tejido Nervioso/fisiología , Plasticidad Neuronal/fisiología , Neuronas/fisiología , Sinaptotagmina I/fisiología , Proteínas de Transporte Vesicular/fisiología , Potenciales de Acción , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Aminoácidos , Animales , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/deficiencia , Proteínas de Unión al Calcio/genética , Células Cultivadas , Secuencia Conservada , Ácido Glutámico/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Potenciales Postsinápticos Miniatura/fisiología , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Técnicas de Placa-Clamp , Dominios Proteicos , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Sinaptotagmina I/química , Sinaptotagmina I/deficiencia , Sinaptotagmina I/genética , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/deficiencia , Proteínas de Transporte Vesicular/genética , Rabfilina-3ARESUMEN
Aging drives a progressive decline in cognition and decreases synapse numbers and synaptic function in the brain, thereby increasing the risk for neurodegenerative disease. Pioneering studies showed that introduction of blood from young mice into aged mice reversed age-associated cognitive impairments and increased synaptic connectivity in brain, suggesting that young blood contains specific factors that remediate age-associated decreases in brain function. However, whether such factors in blood from young animals act directly on neurons to enhance synaptic connectivity, or whether they act by an indirect mechanism remains unknown. Moreover, which factors in young blood mediate cognitive improvements in old mice is incompletely understood. Here, we show that serum extracted from the blood of young but not old mice, when applied to neurons transdifferentiated from human embryonic stem cells, directly increased dendritic arborization, augmented synapse numbers, doubled dendritic spine-like structures, and elevated synaptic N-methyl-d-aspartate (NMDA) receptors, thereby increasing synaptic connectivity. Mass spectrometry revealed that thrombospondin-4 (THBS4) and SPARC-like protein 1 (SPARCL1) were enriched in serum from young mice. Strikingly, recombinant THBS4 and SPARCL1 both increased dendritic arborization and doubled synapse numbers in cultured neurons. In addition, SPARCL1 but not THBS4 tripled NMDA receptor-mediated synaptic responses. Thus, at least two proteins enriched in young blood, THBS4 and SPARCL1, directly act on neurons as synaptogenic factors. These proteins may represent rejuvenation factors that enhance synaptic connectivity by increasing dendritic arborization, synapse formation, and synaptic transmission.
Asunto(s)
Envejecimiento/sangre , Proteínas de Unión al Calcio/sangre , Proteínas de la Matriz Extracelular/sangre , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapsis/fisiología , Trombospondinas/sangre , Factores de Edad , Animales , Proteínas de Unión al Calcio/fisiología , Células Cultivadas , Proteínas de la Matriz Extracelular/fisiología , Femenino , Humanos , Masculino , Ratones , Transmisión Sináptica , Trombospondinas/fisiologíaRESUMEN
Emerging evidence supports roles for secreted extracellular matrix proteins in boosting synaptogenesis, synaptic transmission, and synaptic plasticity. SPARCL1 (also known as Hevin), a secreted non-neuronal protein, was reported to increase synaptogenesis by simultaneously binding to presynaptic neurexin-1α and to postsynaptic neuroligin-1B, thereby catalyzing formation of trans-synaptic neurexin/neuroligin complexes. However, neurexins and neuroligins do not themselves mediate synaptogenesis, raising the question of how SPARCL1 enhances synapse formation by binding to these molecules. Moreover, it remained unclear whether SPARCL1 acts on all synapses containing neurexins and neuroligins or only on a subset of synapses, and whether it enhances synaptic transmission in addition to boosting synaptogenesis or induces silent synapses. To explore these questions, we examined the synaptic effects of SPARCL1 and their dependence on neurexins and neuroligins. Using mixed neuronal and glial cultures from neonatal mouse cortex of both sexes, we show that SPARCL1 selectively increases excitatory but not inhibitory synapse numbers, enhances excitatory but not inhibitory synaptic transmission, and augments NMDAR-mediated synaptic responses more than AMPAR-mediated synaptic responses. None of these effects were mediated by SPARCL1-binding to neurexins or neuroligins. Neurons from triple neurexin-1/2/3 or from quadruple neuroligin-1/2/3/4 conditional KO mice that lacked all neurexins or all neuroligins were fully responsive to SPARCL1. Together, our results reveal that SPARCL1 selectively boosts excitatory but not inhibitory synaptogenesis and synaptic transmission by a novel mechanism that is independent of neurexins and neuroligins.SIGNIFICANCE STATEMENT Emerging evidence supports roles for extracellular matrix proteins in boosting synapse formation and function. Previous studies demonstrated that SPARCL1, a secreted non-neuronal protein, promotes synapse formation in rodent and human neurons. However, it remained unclear whether SPARCL1 acts on all or on only a subset of synapses, induces functional or largely inactive synapses, and generates synapses by bridging presynaptic neurexins and postsynaptic neuroligins. Here, we report that SPARCL1 selectively induces excitatory synapses, increases their efficacy, and enhances their NMDAR content. Moreover, using rigorous genetic manipulations, we show that SPARCL1 does not require neurexins and neuroligins for its activity. Thus, SPARCL1 selectively boosts excitatory synaptogenesis and synaptic transmission by a novel mechanism that is independent of neurexins and neuroligins.
Asunto(s)
Proteínas de Unión al Calcio/fisiología , Moléculas de Adhesión Celular Neuronal/fisiología , Proteínas de la Matriz Extracelular/fisiología , Moléculas de Adhesión de Célula Nerviosa/fisiología , Sinapsis/fisiología , Animales , Corteza Cerebral/citología , Femenino , Masculino , Ratones , Ratones Noqueados , Neuroglía/metabolismo , Neuronas/metabolismo , Cultivo Primario de Células , Receptores de Superficie Celular , Receptores de N-Metil-D-Aspartato/metabolismo , Transmisión Sináptica/fisiologíaRESUMEN
Neurexins (Nrxns) and LAR-RPTPs (leukocyte common antigen-related protein tyrosine phosphatases) are presynaptic adhesion proteins responsible for organizing presynaptic machineries through interactions with nonoverlapping extracellular ligands. Here, we report that two members of the LAR-RPTP family, PTPσ and PTPδ, are required for the presynaptogenic activity of Nrxns. Intriguingly, Nrxn1 and PTPσ require distinct sets of intracellular proteins for the assembly of specific presynaptic terminals. In addition, Nrxn1α showed robust heparan sulfate (HS)-dependent, high-affinity interactions with Ig domains of PTPσ that were regulated by the splicing status of PTPσ. Furthermore, Nrxn1α WT, but not a Nrxn1α mutant lacking HS moieties (Nrxn1α ΔHS), inhibited postsynapse-inducing activity of PTPσ at excitatory, but not inhibitory, synapses. Similarly, cis expression of Nrxn1α WT, but not Nrxn1α ΔHS, suppressed the PTPσ-mediated maintenance of excitatory postsynaptic specializations in mouse cultured hippocampal neurons. Lastly, genetics analyses using male or female Drosophila Dlar and Dnrx mutant larvae identified epistatic interactions that control synapse formation and synaptic transmission at neuromuscular junctions. Our results suggest a novel synaptogenesis model whereby different presynaptic adhesion molecules combine with distinct regulatory codes to orchestrate specific synaptic adhesion pathways.SIGNIFICANCE STATEMENT We provide evidence supporting the physical interactions of neurexins with leukocyte common-antigen related receptor tyrosine phosphatases (LAR-RPTPs). The availability of heparan sulfates and alternative splicing of LAR-RPTPs regulate the binding affinity of these interactions. A set of intracellular presynaptic proteins is involved in common for Nrxn- and LAR-RPTP-mediated presynaptic assembly. PTPσ triggers glutamatergic and GABAergic postsynaptic differentiation in an alternative splicing-dependent manner, whereas Nrxn1α induces GABAergic postsynaptic differentiation in an alternative splicing-independent manner. Strikingly, Nrxn1α inhibits the glutamatergic postsynapse-inducing activity of PTPσ, suggesting that PTPσ and Nrxn1α might control recruitment of a different pool of postsynaptic machinery. Drosophila orthologs of Nrxns and LAR-RPTPs mediate epistatic interactions in controlling synapse structure and strength at neuromuscular junctions, underscoring the physiological significance in vivo.
Asunto(s)
Proteínas de Unión al Calcio/fisiología , Antígenos Comunes de Leucocito/fisiología , Moléculas de Adhesión de Célula Nerviosa/fisiología , Animales , Proteínas de Unión al Calcio/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster , Potenciales Postsinápticos Excitadores/fisiología , Espacio Extracelular/metabolismo , Femenino , Células HEK293 , Humanos , Larva , Masculino , Ratones , Conformación Molecular , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Embarazo , Terminales Presinápticos/metabolismo , Ratas , Proteínas Tirosina Fosfatasas Similares a Receptores/genética , Transmisión Sináptica/fisiologíaRESUMEN
The central nervous system of the Ciona larva contains only 177 neurons. The precise regulation of neuron subtype-specific morphogenesis and differentiation observed during the formation of this minimal connectome offers a unique opportunity to dissect gene regulatory networks underlying chordate neurodevelopment. Here we compare the transcriptomes of two very distinct neuron types in the hindbrain/spinal cord homolog of Ciona, the Motor Ganglion (MG): the Descending decussating neuron (ddN, proposed homolog of Mauthner Cells in vertebrates) and the MG Interneuron 2 (MGIN2). Both types are invariantly represented by a single bilaterally symmetric left/right pair of cells in every larva. Supernumerary ddNs and MGIN2s were generated in synchronized embryos and isolated by fluorescence-activated cell sorting for transcriptome profiling. Differential gene expression analysis revealed ddN- and MGIN2-specific enrichment of a wide range of genes, including many encoding potential "effectors" of subtype-specific morphological and functional traits. More specifically, we identified the upregulation of centrosome-associated, microtubule-stabilizing/bundling proteins and extracellular guidance cues part of a single intrinsic regulatory program that might underlie the unique polarization of the ddNs, the only descending MG neurons that cross the midline. Consistent with our predictions, CRISPR/Cas9-mediated, tissue-specific elimination of two such candidate effectors, Efcab6-related and Netrin1, impaired ddN polarized axon outgrowth across the midline.
Asunto(s)
Ciona intestinalis/genética , Ganglios de Invertebrados/citología , Regulación del Desarrollo de la Expresión Génica , Neuronas/clasificación , Animales , Orientación del Axón/fisiología , Sistemas CRISPR-Cas , Proteínas de Unión al Calcio/biosíntesis , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/fisiología , Sistema Nervioso Central/citología , Centrosoma/fisiología , Ciona intestinalis/citología , Ciona intestinalis/embriología , Ciona intestinalis/crecimiento & desarrollo , Conectoma , Embrión no Mamífero , Ganglios de Invertebrados/crecimiento & desarrollo , Edición Génica , Interneuronas/fisiología , Interneuronas/ultraestructura , Larva , Microtúbulos/fisiología , Neuronas Motoras/fisiología , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/fisiología , Netrina-1/biosíntesis , Netrina-1/genética , Netrina-1/fisiología , Neurogénesis , Neuronas/fisiología , Neuronas/ultraestructura , Proteínas Represoras/biosíntesis , Proteínas Represoras/genética , Proteínas Represoras/fisiología , TranscriptomaRESUMEN
Calsyntenin-3 (Clstn3) is a postsynaptic adhesion molecule that induces presynaptic differentiation via presynaptic neurexins (Nrxns), but whether Nrxns directly bind to Clstn3 has been a matter of debate. Here, using LC-MS/MS-based protein analysis, confocal microscopy, RNAscope assays, and electrophysiological recordings, we show that ß-Nrxns directly interact via their LNS domain with Clstn3 and Clstn3 cadherin domains. Expression of splice site 4 (SS4) insert-positive ß-Nrxn variants, but not insert-negative variants, reversed the impaired Clstn3 synaptogenic activity observed in Nrxn-deficient neurons. Consistently, Clstn3 selectively formed complexes with SS4-positive Nrxns in vivo Neuron-specific Clstn3 deletion caused significant reductions in number of excitatory synaptic inputs. Moreover, expression of Clstn3 cadherin domains in CA1 neurons of Clstn3 conditional knockout mice rescued structural deficits in excitatory synapses, especially within the stratum radiatum layer. Collectively, our results suggest that Clstn3 links to SS4-positive Nrxns to induce presynaptic differentiation and orchestrate excitatory synapse development in specific hippocampal neural circuits, including Schaffer collateral afferents.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Animales , Cadherinas/metabolismo , Proteínas de Unión al Calcio/fisiología , Cromatografía Liquida/métodos , Hipocampo/metabolismo , Proteínas de la Membrana/fisiología , Ratones , Proteínas del Tejido Nervioso/fisiología , Moléculas de Adhesión de Célula Nerviosa/fisiología , Neuronas/metabolismo , Sinapsis/metabolismo , Espectrometría de Masas en Tándem/métodosRESUMEN
Acute respiratory distress syndrome (ARDS) is characterized by acute lung injury (ALI) secondary to an excessive alveolar inflammatory response. Reticulocalbin 3 (Rcn3) is an endoplasmic reticulum (ER) lumen protein in the secretory pathway. We previously reported the indispensable role of Rcn3 in type II alveolar epithelial cells (AECIIs) during lung development and the lung injury repair process. In the present study, we further observed a marked induction of Rcn3 in the alveolar epithelium during LPS-induced ALI. In vitro alveolar epithelial (MLE-12) cells consistently exhibited a significant induction of Rcn3 accompanied with NF-κB activation in response to LPS exposure. We examined the role of Rcn3 in the alveolar inflammatory response by using mice with a selective deletion of Rcn3 in alveolar epithelial cells upon doxycycline administration. The Rcn3 deficiency significantly blunted the ALI and alveolar inflammation induced by intratracheal LPS instillation but not that induced by an intraperitoneal LPS injection (secondary insult); the alleviated ALI was accompanied by decreases in NF-κB activation and NLRP3 levels but not in GRP78 and cleaved caspase-3 levels. The studies conducted in MLE-12 cells consistently showed that Rcn3 knockdown blunted the activations of NF-κB signaling and NLRP3-dependent inflammasome upon LPS exposure. Collectively, these findings suggest a novel role for Rcn3 in regulating the alveolar inflammatory response to pulmonary infection via the NF-κB/NLRP3/inflammasome axis and shed additional light on the mechanism of ARDS/ALI.
Asunto(s)
Lesión Pulmonar Aguda/prevención & control , Células Epiteliales Alveolares/metabolismo , Proteínas de Unión al Calcio/fisiología , Inflamación/prevención & control , Lipopolisacáridos/toxicidad , FN-kappa B/metabolismo , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/metabolismo , Lesión Pulmonar Aguda/patología , Animales , Chaperón BiP del Retículo Endoplásmico , Femenino , Inflamasomas , Inflamación/inducido químicamente , Inflamación/metabolismo , Inflamación/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/genética , Transducción de SeñalRESUMEN
Calcium-dependent activator protein for secretion 1 (CAPS1) is a SNARE accessory protein that facilitates formation of the SNARE complex to enable neurotransmitter release. Messenger RNAs encoding CAPS1 are subject to a site-specific adenosine-to-inosine (A-to-I) editing event resulting in a glutamate-to-glycine (E-to-G) substitution in the C-terminal domain of the encoded protein product. The C-terminal domain of CAPS1 is necessary for its synaptic enrichment and Cadps RNA editing has been shown previously to enhance the release of neuromodulatory transmitters. Using mutant mouse lines engineered to solely express CAPS1 protein isoforms encoded by either the non-edited or edited Cadps transcript, primary neuronal cultures from mouse hippocampus were used to explore the effect of Cadps editing on neurotransmission and CAPS1 synaptic localization at both glutamatergic and GABAergic synapses. While the editing of Cadps does not alter baseline evoked neurotransmission, it enhances short-term synaptic plasticity, specifically short-term depression, at inhibitory synapses. Cadps editing also alters spontaneous inhibitory neurotransmission. Neurons that solely express edited Cadps have a greater proportion of synapses that contain CAPS1 than neurons that solely express non-edited Cadps for both glutamatergic and GABAergic synapses. Editing of Cadps transcripts is regulated by neuronal activity, as global network stimulation increases the extent of transcripts edited in wild-type hippocampal neurons, whereas chronic network silencing decreases the level of Cadps editing. Taken together, these results provide key insights into the importance of Cadps editing in modulating its own synaptic localization, as well as the modulation of neurotransmission at inhibitory synapses in hippocampal neurons.
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Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/fisiología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/fisiología , Edición de ARN/genética , Transmisión Sináptica/genética , Transmisión Sináptica/fisiología , Adenosina Desaminasa/metabolismo , Animales , Sistemas CRISPR-Cas , Fenómenos Electrofisiológicos , Ácido Glutámico/fisiología , Secuenciación de Nucleótidos de Alto Rendimiento , Hipocampo/citología , Hipocampo/metabolismo , Ratones , Mutación , Plasticidad Neuronal/genética , Plasticidad Neuronal/fisiología , Cultivo Primario de Células , Edición de ARN/fisiología , Ácido gamma-Aminobutírico/fisiologíaRESUMEN
Syntrophins are a family of modular adaptor proteins that are part of the dystrophin protein complex, where they recruit and anchor a variety of signaling proteins. Previously we generated mice lacking α- and/or ß2-syntrophin but showed that in the absence of one isoform, other syntrophin isoforms can partially compensate. Therefore, in the current study, we generated mice that lacked α, ß1 and ß2-syntrophins [triple syntrophin knockout (tKO) mice] and assessed skeletal and cardiac muscle function. The tKO mice showed a profound reduction in voluntary wheel running activity at both 6 and 12 months of age. Function of the tibialis anterior was assessed in situ and we found that the specific force of tKO muscle was decreased by 20-25% compared with wild-type mice. This decrease was accompanied by a shift in fiber-type composition from fast 2B to more oxidative fast 2A fibers. Using echocardiography to measure cardiac function, it was revealed that tKO hearts had left ventricular cardiac dysfunction and were hypertrophic, with a thicker left ventricular posterior wall. Interestingly, we also found that membrane-localized dystrophin expression was lower in both skeletal and cardiac muscles of tKO mice. Since dystrophin mRNA levels were not different in tKO, this finding suggests that syntrophins may regulate dystrophin trafficking to, or stabilization at, the sarcolemma. These results show that the loss of all three major muscle syntrophins has a profound effect on exercise performance, and skeletal and cardiac muscle dysfunction contributes to this deficiency.
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Proteínas de Unión al Calcio/fisiología , Proteínas Asociadas a la Distrofina/fisiología , Proteínas de la Membrana/fisiología , Proteínas Musculares/fisiología , Animales , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Distrofina/genética , Distrofina/fisiología , Proteínas Asociadas a la Distrofina/genética , Corazón/fisiología , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Actividad Motora , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiología , Miocardio/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologíaRESUMEN
The collective function of calcineurin B-like (CBL) calcium ion (Ca2+ ) sensors and CBL-interacting protein kinases (CIPKs) in decoding plasma-membrane-initiated Ca2+ signals to convey developmental and adaptive responses to fluctuating nitrate availability remained to be determined. Here, we generated a cbl-quintuple mutant in Arabidopsis thaliana devoid of these Ca2+ sensors at the plasma membrane and performed comparative phenotyping, nitrate flux determination, phosphoproteome analyses, and studies of membrane domain protein distribution in response to low and high nitrate availability. We observed that CBL proteins exert multifaceted regulation of primary and lateral root growth and nitrate fluxes. Accordingly, we found that loss of plasma membrane Ca2+ sensor function simultaneously affected protein phosphorylation of numerous membrane proteins, including several nitrate transporters, proton pumps, and aquaporins, as well as their distribution within plasma membrane microdomains, and identified a specific phosphorylation and domain distribution pattern during distinct phases of low and high nitrate responses. Collectively, these analyses reveal a central and coordinative function of CBL-CIPK-mediated signaling in conveying plant adaptation to fluctuating nitrate availability and identify a crucial role of Ca2+ signaling in regulating the composition and dynamics of plasma membrane microdomains.
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Proteínas de Arabidopsis , Arabidopsis/fisiología , Proteínas de Unión al Calcio , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiología , Calcineurina/metabolismo , Calcio/metabolismo , Proteínas de Unión al Calcio/fisiología , Membrana Celular/fisiología , Nitratos/metabolismo , Fosforilación , Raíces de Plantas/crecimiento & desarrolloRESUMEN
Early signalling events in response to elicitation include reversible protein phosphorylation and re-localization of plasma membrane (PM) proteins. Oligogalacturonides (OGs) are a class of damage-associated molecular patterns (DAMPs) that act as endogenous signals to activate the plant immune response. Previous data on early phosphoproteome changes in Arabidopsis thaliana upon OG perception uncovered the immune-related phospho-regulation of several membrane proteins, among which PCaP1, a PM-anchored protein with actin filament-severing activity, was chosen for its potential involvement in OG- and flagellin-triggered responses. Here, we demonstrate that PCaP1 is required for late, but not early, responses induced by OGs and flagellin. Moreover, pcap1 mutants, unlike the wild type, are impaired in the recovery of full responsiveness to a second treatment with OGs performed 24 h after the first one. Localization studies on PCaP1 upon OG treatment in plants expressing a functional PCaP1-GFP fusion under the control of PCaP1 promoter revealed fluorescence on the PM, organized in densely packed punctate structures, previously reported as microdomains. Fluorescence was found to be associated also with endocytic vesicles, the number of which rapidly increased after OG treatment, suggesting both an endocytic turnover of PCaP1 for maintaining its homeostasis at the PM and an OG-induced endocytosis.
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Alarminas/metabolismo , Proteínas de Arabidopsis/fisiología , Arabidopsis/inmunología , Proteínas de Unión al Calcio/fisiología , Membrana Celular/metabolismo , Flagelina/metabolismo , Polinucleótidos/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Botrytis , Proteínas de Unión al Calcio/metabolismo , Regulación de la Expresión Génica de las Plantas , Glucanos/metabolismo , Microscopía Confocal , Fosfoproteínas/metabolismo , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Especies Reactivas de Oxígeno/metabolismo , TranscriptomaRESUMEN
PURPOSE: To explore the role of nucleotide-binding oligomerization domain-like receptors (NLRs) family caspase-activation and the recruitment domain containing 4 (NLRC4) inflammasome in retinal ganglion cell (RGC) injury induced by an acute glaucoma mouse model. METHOD: A mouse model of acute ocular hypertension, which can lead to retinal ischemia-reperfusion (I/R) injury, was established. The expression level of NLRC4 was detected by polymerase chain reaction and western blotting. Localized expression of NLRC4 was detected by examining immunofluorescence in eyeball sections. Intravitreal adeno-associated virus 2(AAV2) administration was used to knockdown retinal Nlrc4. Fluoro-Gold labeled RGCs and TdT-mediated dUTP nick end labeling were used to evaluate the survival and apoptosis of RGCs. Tlr4-/- mice were utilized to explore whether NLRC4 inflammasome is influenced by Toll-like receptor4 (TLR4). RESULTS: NLRC4, expressed in RGCs and microglial cells, was actively involved in mouse retinal I/R injury. Knockdown of Nlrc4 using an AAV2 vector caused an obvious reduction in the generation of IL-1ß led by the rapidly elevated intraocular pressure, and thereby improved the RGC survival. In addition, activation of the NLRC4 inflammasome could influence the phosphorylation of p38 and Jun N-terminal kinase, which was largely dependent on TLR4 signaling. CONCLUSION: Our study demonstrated the role of NLRC4 inflammasome in promoting RGC damage in mouse retinal I/R injury. Inhibition of NLRC4 might be leveraged as a potential therapeutic target in glaucomatous retinopathy.
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Proteínas Reguladoras de la Apoptosis/fisiología , Proteínas de Unión al Calcio/fisiología , Muerte Celular/fisiología , Glaucoma/patología , Inflamasomas/metabolismo , Células Ganglionares de la Retina/patología , Enfermedad Aguda , Animales , Western Blotting , Dependovirus , Modelos Animales de Enfermedad , Glaucoma/metabolismo , Etiquetado Corte-Fin in Situ , Presión Intraocular , MAP Quinasa Quinasa 4/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Fluorescente , Hipertensión Ocular/metabolismo , Hipertensión Ocular/patología , Parvovirinae/genética , Fosforilación , Reacción en Cadena en Tiempo Real de la Polimerasa , Daño por Reperfusión/metabolismo , Retina/metabolismo , Células Ganglionares de la Retina/metabolismo , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Renal fibrosis is the common pathological process of various chronic kidney diseases (CKD). Recent studies indicate that mitochondrial fragmentation is closely associated with renal fibrosis in CKD. However, the molecular mechanisms leading to mitochondrial fragmentation remain to be elucidated. The present study investigated the role of regulators of calcineurin 1 (RCAN1) in mitochondrial fission and renal interstitial fibrosis using conditional knockout mice in which RCAN1 was genetically deleted in tubular epithelial cells (TECs). TEC-specific deletion of RCAN1 attenuated tubulointerstitial fibrosis and epithelial to mesenchymal transition (EMT)-like phenotype change after unilateral ureteral obstruction (UUO) and ischemia reperfusion injury (IRI) through suppressing TGF-ß1/Smad3 signaling pathway. TEC-specific deletion of RCAN1 also reduced the tubular apoptosis after UUO by inhibiting cytochrome c/caspase-9 pathway. Ultrastructure analysis revealed a marked decrease in mitochondrial fragmentation in TECs of RCAN1-deficient mice in experimental CKD models. The expression of mitochondrial profission proteins dynamin-related protein 1 (Drp1) and mitochondrial fission factor (Mff) was also downregulated in obstructed kidney of TEC-specific RCAN1-deficient mice. Furthermore, TEC-specific deletion of RCAN1 attenuated the dysfunctional tubular autophagy by regulating PINK1/Parkin-induced mitophagy in CKD. RCAN1 knockdown and knockout similarly improved the mitochondrial quality control in HK-2 cells and primary cultured mouse tubular cells stimulated by TGF-ß1. Put together, our data indicated that RCAN1 plays an important role in the progression of tubulointerstitial fibrosis through regulating the mitochondrial quality. Therefore, targeting RCAN1 may provide a potential therapeutic approach in CKD.