RESUMEN
Recent studies have consistently demonstrated that the regulation of chromatin and gene transcription plays a pivotal role in the pathogenesis of neurodevelopmental disorders. Among many genes involved in these pathways, KMT2C, encoding one of the six known histone H3 lysine 4 (H3K4) methyltransferases in humans and rodents, was identified as a gene whose heterozygous loss-of-function variants are causally associated with autism spectrum disorder (ASD) and the Kleefstra syndrome phenotypic spectrum. However, little is known about how KMT2C haploinsufficiency causes neurodevelopmental deficits and how these conditions can be treated. To address this, we developed and analyzed genetically engineered mice with a heterozygous frameshift mutation of Kmt2c (Kmt2c+/fs mice) as a disease model with high etiological validity. In a series of behavioral analyses, the mutant mice exhibit autistic-like behaviors such as impairments in sociality, flexibility, and working memory, demonstrating their face validity as an ASD model. To investigate the molecular basis of the observed abnormalities, we performed a transcriptomic analysis of their bulk adult brains and found that ASD risk genes were specifically enriched in the upregulated differentially expressed genes (DEGs), whereas KMT2C peaks detected by ChIP-seq were significantly co-localized with the downregulated genes, suggesting an important role of putative indirect effects of Kmt2c haploinsufficiency. We further performed single-cell RNA sequencing of newborn mouse brains to obtain cell type-resolved insights at an earlier stage. By integrating findings from ASD exome sequencing, genome-wide association, and postmortem brain studies to characterize DEGs in each cell cluster, we found strong ASD-associated transcriptomic changes in radial glia and immature neurons with no obvious bias toward upregulated or downregulated DEGs. On the other hand, there was no significant gross change in the cellular composition. Lastly, we explored potential therapeutic agents and demonstrate that vafidemstat, a lysine-specific histone demethylase 1 (LSD1) inhibitor that was effective in other models of neuropsychiatric/neurodevelopmental disorders, ameliorates impairments in sociality but not working memory in adult Kmt2c+/fs mice. Intriguingly, the administration of vafidemstat was shown to alter the vast majority of DEGs in the direction to normalize the transcriptomic abnormalities in the mutant mice (94.3 and 82.5% of the significant upregulated and downregulated DEGs, respectively, P < 2.2 × 10-16, binomial test), which could be the molecular mechanism underlying the behavioral rescuing. In summary, our study expands the repertoire of ASD models with high etiological and face validity, elucidates the cell-type resolved molecular alterations due to Kmt2c haploinsufficiency, and demonstrates the efficacy of an LSD1 inhibitor that might be generalizable to multiple categories of psychiatric disorders along with a better understanding of its presumed mechanisms of action.
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Understanding environmental influences on individuals' behaviour is challenging. Here we have investigated the housing impact of 9 weeks of enriched environment (EE) and social isolation (SI) and the impact of abrupt deprivation of EE (enrichment removal: ER) on BALB/c mice. Compared with the widely used C57BL/6 strain in research, BALB/c synthesises serotonin less efficiently due to a genetic variation and thus may potentially represent human populations at higher risk of stress-related disorders. We assessed the effects of EE and SI by conducting a behavioural test battery and the effects of acute ER by monitoring homecage activities and social behaviour. We found that EE and SI impact BALB/c's physiological states and behavioural performances from lower to higher cognitive processes: increased body weight, increased rectal temperature, altered performance in motor and sensory tasks, the activity level in a novel environment and altered performance in tests of anxiety-like behaviour, stress-coping strategies and learning and memory. Furthermore, acute ER triggered stress/frustration-like behaviour in BALB/c, with increased aggression, increased social distancing and disrupted daily/nightly activities. Our results demonstrate that long-lasting housing manipulation such as EE and SI, impact behaviour via multilayered processes over a wide range of functional domains, and unforeseen change to a negative environment, ER, is a major stressor that causes behavioural and psychological consequences through environment-gene interactions, a model of direct relevance to human health.
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Conducta Exploratoria , Vivienda , Animales , Conducta Animal/fisiología , Conducta Exploratoria/fisiología , Vivienda para Animales , Aprendizaje por Laberinto/fisiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: Patients with Parkinson's disease (PD) show motor symptoms as well as various non-motor symptoms. Postmortem studies of PD have suggested that initial alpha-synuclein (α-Syn) pathology develops independently in the olfactory bulb and lower brainstem, spreading from there stereotypically. However, it remains unclear how these two pathological pathways contribute to the clinicopathological progression of PD. OBJECTIVE: The objective of this study was to examine the clinicopathological contribution of α-Syn spread from the olfactory bulb. METHODS: We conducted pathological and behavioral analyses of human α-Syn bacterial artificial chromosome transgenic mice injected with α-Syn preformed fibrils into the bilateral olfactory bulb up to 10 months postinjection. RESULTS: α-Syn preformed fibril injections induced more widespread α-Syn pathology in the transgenic mice than that in wild-type mice. Severe α-Syn pathology in the transgenic mice injected with α-Syn preformed fibrils was initially observed along the olfactory pathway and later in the brain regions that are included in the limbic system and have connections with it. The α-Syn pathology was accompanied by regional atrophy, neuron loss, reactive astrogliosis, and microglial activation, which were remarkable in the hippocampus. Behavioral analyses revealed hyposmia, followed by anxiety-like behavior and memory impairment, but not motor dysfunction, depression-like behavior, or circadian rhythm disturbance. CONCLUSION: Our data suggest that α-Syn spread from the olfactory bulb mainly affects the olfactory pathway and limbic system as well as its related regions, leading to the development of hyposmia, anxiety, and memory loss in PD. © 2021 International Parkinson and Movement Disorder Society.
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Bulbo Olfatorio , alfa-Sinucleína , Animales , Anosmia , Ansiedad/etiología , Modelos Animales de Enfermedad , Humanos , Trastornos de la Memoria/etiología , Ratones , Ratones Transgénicos , Bulbo Olfatorio/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMEN
Heterochromatin protein (HP) 1γ, a component of heterochromatin in eukaryotes, is involved in H3K9 methylation. Although HP1γ is expressed strongly in neural tissues and neural stem cells, its functions are unclear. To elucidate the roles of HP1γ, we analyzed HP1γ -deficient (HP1γ KO) mouse embryonic neurospheres and determined that HP1γ KO neurospheres tended to differentiate after quaternary culture. Several genes normally expressed in neuronal cells were upregulated in HP1γ KO undifferentiated neurospheres, but not in the wild type (WT). Compared to that in the control neurospheres, the occupancy of H3K27me3 was lower around the transcription start sites (TSSs) of these genes in HP1γ KO neurospheres, while H3K9me2/3, H3K4me3, and H3K27ac amounts remained unchanged. Moreover, amounts of the H3K27me2/3 demethylases, UTX, and JMJD3, were increased around the TSSs of these genes. Treatment with GSK-J4, an inhibitor of H3K27 demethylases, decreased the expression of genes upregulated in HP1γ KO neurospheres, along with an increase of H3K27me3 amounts. Therefore, in murine neurospheres, HP1γ protected the promoter sites of differentiated cell-specific genes against H3K27 demethylases to repress the expression of these genes. A better understanding of central cellular processes such as histone methylation will help elucidate critical events such as cell-specific gene expression, epigenetics, and differentiation.
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Proteínas Cromosómicas no Histona/metabolismo , Histonas/metabolismo , Animales , Proliferación Celular/genética , Proliferación Celular/fisiología , Inmunoprecipitación de Cromatina , Proteínas Cromosómicas no Histona/genética , Técnica del Anticuerpo Fluorescente , Ontología de Genes , Imagen por Resonancia Magnética , Ratones , Ratones Noqueados , Regiones Promotoras Genéticas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sitio de Iniciación de la Transcripción/fisiologíaRESUMEN
It is uncertain which ß4-galactosyltransferase (ß4GalT; gene name, B4galt), ß4GalT-5 and/or ß4GalT-6, is responsible for the production of lactosylceramide (LacCer) synthase, which functions in the initial step of ganglioside biosynthesis. Here, we generated conditional B4galt5 knockout (B4galt5 cKO) mice, using Nestin-Cre mice, and crossed these with B4galt6 KO mice to generate B4galt5 and 6 double KO (DKO) mice in the central nervous system (CNS). LacCer synthase activity and major brain gangliosides were completely absent in brain homogenates from the DKO mice, although LacCer synthase activity was about half its normal level in B4galt5 cKO mice and B4galt6 KO mice. The DKO mice were born normally but they showed growth retardation and motor deficits at 2 weeks and died by 4 weeks of age. Histological analyses showed that myelin-associated proteins were rarely found localized in axons in the cerebral cortex, and axonal and myelin formation were remarkably impaired in the spinal cords of the DKO mice. Neuronal cells, differentiated from neurospheres that were prepared from the DKO mice, showed impairments in neurite outgrowth and branch formation, which can be explained by the fact that neurospheres from DKO mice could weakly interact with laminin due to lack of gangliosides, such as GM1a. Furthermore, the neurons were immature and perineuronal nets (PNNs) were poorly formed in DKO cerebral cortices. Our results indicate that LacCer synthase is encoded by B4galt5 and 6 genes in the CNS, and that gangliosides are indispensable for neuronal maturation, PNN formation, and axonal and myelin formation.
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Galactosiltransferasas/fisiología , Vaina de Mielina/fisiología , Neurogénesis/genética , Animales , Axones/fisiología , Sistema Nervioso Central/fisiología , Modelos Animales de Enfermedad , Femenino , Galactosiltransferasas/genética , Laminina/fisiología , Ratones , Ratones Noqueados , Neuronas/citología , Médula Espinal/fisiologíaRESUMEN
Jmjd3 and Utx are demethylases specific for lysine 27 of histone H3. Previous reports indicate that Jmjd3 is essential for differentiation of various cell types, such as macrophages and epidermal cells in mice, whereas Utx is involved in cancer and developmental diseases in humans and mice, as well as Hox regulation in zebrafish and nematodes. Here, we report that Jmjd3, but not Utx, is involved in axial skeletal formation in mice. A Jmjd3 mutant embryo (Jmjd3Δ18/Δ18), but not a catalytically inactive Utx truncation mutant (Utx-/y), showed anterior homeotic transformation. Quantitative RT-PCR and microarray analyses showed reduced Hox expression in both Jmjd3Δ18/Δ18 embryos and tailbuds, whereas levels of Hox activators, such as Wnt signaling factors and retinoic acid synthases, did not decrease, which suggests that Jmjd3 plays a regulatory role in Hox expression during axial patterning. Chromatin immunoprecipitation analyses of embryo tailbud tissue showed trimethylated lysine 27 on histone H3 to be at higher levels at the Hox loci in Jmjd3Δ18/Δ18 mutants compared with wild-type tailbuds. In contrast, trimethylated lysine 4 on histone H3 levels were found to be equivalent in wild-type and Jmjd3Δ18/Δ18 tailbuds. Demethylase-inactive Jmjd3 mutant embryos showed the same phenotype as Jmjd3Δ18/Δ18 mice. These results suggest that the demethylase activity of Jmjd3, but not that of Utx, affects mouse axial patterning in concert with alterations in Hox gene expression.-Naruse, C., Shibata, S., Tamura, M., Kawaguchi, T., Abe, K., Sugihara, K., Kato, T., Nishiuchi, T., Wakana, S., Ikawa, M., Asano, M. New insights into the role of Jmjd3 and Utx in axial skeletal formation in mice.
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Desarrollo Óseo/fisiología , Huesos/embriología , Regulación del Desarrollo de la Expresión Génica/fisiología , Histona Demetilasas/metabolismo , Histona Demetilasas con Dominio de Jumonji/metabolismo , Animales , Desarrollo Óseo/genética , Huesos/metabolismo , Desarrollo Embrionario/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Histona Demetilasas/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Histona Demetilasas con Dominio de Jumonji/genética , Ratones , Mutación , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Germline cells reprogramme extensive epigenetic modifications to ensure the cellular totipotency of subsequent generations and to prevent the accumulation of epimutations. Notably, primordial germ cells (PGCs) erase genome-wide DNA methylation and H3K9 dimethylation marks in a stepwise manner during migration and gonadal periods. In this study, we profiled DNA and histone methylation on transposable elements during PGC development, and examined the role of DNA replication in DNA demethylation in gonadal PGCs. CpGs in short interspersed nuclear elements (SINEs) B1 and B2 were substantially demethylated in migrating PGCs, whereas CpGs in long interspersed nuclear elements (LINEs), such as LINE-1, were resistant to early demethylation. By contrast, CpGs in both LINE-1 and SINEs were rapidly demethylated in gonadal PGCs. Four major modifiers of DNA and histone methylation, Dnmt3a, Dnmt3b, Glp and Uhrf1, were actively repressed at distinct stages of PGC development. DNMT1 was localised at replication foci in nascent PGCs, whereas the efficiency of recruitment of DNMT1 into replication foci was severely impaired in gonadal PGCs. Hairpin bisulphite sequencing analysis showed that strand-specific hemi-methylated CpGs on LINE-1 were predominant in gonadal PGCs. Furthermore, DNA demethylation in SINEs and LINE-1 was impaired in Cbx3-deficient PGCs, indicating abnormalities in G1 to S phase progression. We propose that PGCs employ active and passive mechanisms for efficient and widespread erasure of genomic DNA methylation.
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Metilación de ADN , Replicación del ADN , Células Germinativas/metabolismo , Animales , Secuencia de Bases , Proteínas Potenciadoras de Unión a CCAAT , Proteínas Cromosómicas no Histona/metabolismo , Islas de CpG , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Epigénesis Genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Elementos de Nucleótido Esparcido Largo , Ratones , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Elementos de Nucleótido Esparcido Corto , Ubiquitina-Proteína LigasasRESUMEN
Ischemia and seizure cause excessive neuronal excitation that is associated with brain acidosis and neuronal cell death. However, the molecular mechanism of acidification-triggered neuronal injury is incompletely understood. Here, we show that asparagine endopeptidase (AEP) is activated under acidic condition, cuts SET, an inhibitor of DNase, and triggers DNA damage in brain, which is inhibited by PIKE-L. SET, a substrate of caspases, was cleaved by acidic cytosolic extract independent of caspase activation. Fractionation of the acidic cellular extract yielded AEP that is required for SET cleavage. We found that kainate provoked AEP activation and SET cleavage at N175, triggering DNA nicking in wild-type, but not AEP null, mice. PIKE-L strongly bound SET and prevented its degradation by AEP, leading to resistance of neuronal cell death. Moreover, AEP also mediated stroke-provoked SET cleavage and cell death in brain. Thus, AEP might be one of the proteinases activated by acidosis triggering neuronal injury during neuroexcitotoxicity or ischemia.
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Asparaginasa/metabolismo , Proteínas Cromosómicas no Histona/antagonistas & inhibidores , Proteínas de Unión al GTP/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Factores de Transcripción/antagonistas & inhibidores , Animales , Asparaginasa/deficiencia , Asparaginasa/genética , Muerte Celular , Proteínas de Unión al ADN , Granzimas/metabolismo , Hipocampo/enzimología , Chaperonas de Histonas , Humanos , Concentración de Iones de Hidrógeno , Isquemia/enzimología , Isquemia/fisiopatología , Ácido Kaínico/farmacología , Cinética , Ratones , Ratones Noqueados , Neuronas/patología , Fármacos Neuroprotectores/metabolismo , Células PC12 , Biosíntesis de Proteínas , Ratas , Linfocitos T Citotóxicos/enzimología , Transcripción GenéticaRESUMEN
Glycosylation is a major protein modification. Although proteins are glycosylated/further modulated by several glycosyltransferases during trafficking from the endoplasmic reticulum to the Golgi apparatus, a certain glycan epitope has only been detected on a limited number of proteins. Of these glycan epitopes, Lewis X is highly expressed in the early stage of a developing brain and plays important roles in cell-cell interaction. The Lewis X epitope is comprised of a trisaccharide (Galß1-4 (Fucα1-3) GlcNAc), and a key enzyme for the expression of this epitope is α1,3-fucosyltransferase 9. However, the scaffolding glycan structure responsible for the formation of the Lewis X epitope as well as its major carrier protein has not been fully characterized in the nervous system. Here we showed that the Lewis X epitope was mainly expressed on phosphacan/receptor protein tyrosine phosphatase ß (RPTPß) in the developing mouse brain. Expression of the Lewis X epitope was markedly reduced in ß1,4-galactosyltransferase 2 (ß4GalT2) gene-deficient mice, which indicated that ß4GalT2 is a major galactosyltransferase required for the Lewis X epitope. We also showed that the Lewis X epitope almost disappeared due to the knockout of protein O-mannose ß1,2-N-acetylglucosaminyltransferase 1, an N-acetylglucosaminyltransferase essential for the synthesis of O-mannosylated glycans, which indicated that the O-mannosylated glycan is responsible for presenting the Lewis X epitope. Since O-mannosylated glycans on phosphacan/RPTPß could also present human natural killer-1, another glycan epitope specifically expressed in the nervous system, our results revealed the importance of O-mannosylated glycan chains in the presentation of functional glycan epitopes in the brain.
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Encéfalo/enzimología , Antígeno Lewis X/metabolismo , Manosa/metabolismo , Proteínas Tirosina Fosfatasas Clase 5 Similares a Receptores/metabolismo , Animales , Vías Biosintéticas , Encéfalo/embriología , Encéfalo/crecimiento & desarrollo , Células COS , Conformación de Carbohidratos , Chlorocebus aethiops , Glicosilación , Mananos/metabolismo , Ratones Noqueados , Procesamiento Proteico-PostraduccionalRESUMEN
Mutant alleles of EXT1 or EXT2, two members of the EXT gene family, are causative agents in hereditary multiple exostoses, and their gene products function together as a polymerase in the biosynthesis of heparan sulfate. EXTL2, one of three EXT-like genes in the human genome that are homologous to EXT1 and EXT2, encodes a transferase that adds not only GlcNAc but also N-acetylgalactosamine to the glycosaminoglycan (GAG)-protein linkage region via an α1,4-linkage. However, both the role of EXTL2 in the biosynthesis of GAGs and the biological significance of EXTL2 remain unclear. Here we show that EXTL2 transfers a GlcNAc residue to the tetrasaccharide linkage region that is phosphorylated by a xylose kinase 1 (FAM20B) and thereby terminates chain elongation. We isolated an oligosaccharide from the mouse liver, which was not detected in EXTL2 knock-out mice. Based on structural analysis by a combination of glycosidase digestion and 500-MHz (1)H NMR spectroscopy, the oligosaccharide was found to be GlcNAcα1-4GlcUAß1-3Galß1-3Galß1-4Xyl(2-O-phosphate), which was considered to be a biosynthetic intermediate of an immature GAG chain. Indeed, EXTL2 specifically transferred a GlcNAc residue to a phosphorylated linkage tetrasaccharide, GlcUAß1-3Galß1-3Galß1-4Xyl(2-O-phosphate). Remarkably, the phosphorylated linkage pentasaccharide generated by EXTL2 was not used as an acceptor for heparan sulfate or chondroitin sulfate polymerases. Moreover, production of GAGs was significantly higher in EXTL2 knock-out mice than in wild-type mice. These results indicate that EXTL2 functions to suppress GAG biosynthesis that is enhanced by a xylose kinase and that the EXTL2-dependent mechanism that regulates GAG biosynthesis might be a "quality control system" for proteoglycans.
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Regulación Enzimológica de la Expresión Génica , Glicosaminoglicanos/metabolismo , Proteínas de la Membrana/metabolismo , N-Acetilglucosaminiltransferasas/metabolismo , Fosfotransferasas/metabolismo , Xilosa/química , Alelos , Animales , Clonación Molecular , Fibroblastos/metabolismo , Genes Supresores de Tumor , Genómica , Glicosiltransferasas/metabolismo , Células HeLa , Humanos , Hígado/metabolismo , Ratones , Ratones Noqueados , Mutación , N-Acetilglucosaminiltransferasas/genética , Proteoglicanos/metabolismo , ARN Mensajero/metabolismoRESUMEN
BACKGROUND & AIMS: In epithelial cells, protein sorting mechanisms regulate localization of plasma membrane proteins that generate and maintain cell polarity. The clathrin-adaptor protein (AP) complex AP-1B is expressed specifically in polarized epithelial cells, where it regulates basolateral sorting of membrane proteins. However, little is known about its physiological significance. METHODS: We analyzed the intestinal epithelia of mice deficient in Ap1m2 (Ap1m2(-/-) mice), which encodes the AP-1B µ1B subunit, and compared it with 129/B6/CD1 littermates (controls). Notch signaling was inhibited by intraperitoneal injection of dibenzazepine, and ß-catenin signaling was inhibited by injection of IWR1. Intestinal tissue samples were collected and analyzed by immunofluorescence analysis. RESULTS: Ap1m2(-/-) mice developed intestinal epithelial cell hyperplasia. The polarity of intestinal epithelial cells was disrupted, as indicated by the appearance of ectopic microvilli-like structures on the lateral plasma membrane and mislocalization of basolateral membrane proteins, including the low-density lipoprotein receptor and E-cadherin. The E-cadherin-ß-catenin complex therefore was disrupted at the adherens junction, resulting in nuclear translocation of ß-catenin. This resulted in up-regulation of genes regulated by ß-catenin/transcription factor 4 (Tcf4) complex, and increased the proliferation of intestinal epithelial cells. CONCLUSIONS: AP-1B is required for protein sorting and polarization of intestinal cells in mice. Loss of AP-1B in the intestinal epithelia results in mislocalization of E-cadherin, activation of ß-catenin/Tcf4 complex, proliferation, and hyperplasia.
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Complejo 1 de Proteína Adaptadora/deficiencia , Subunidades mu de Complejo de Proteína Adaptadora/deficiencia , Polaridad Celular , Proliferación Celular , Células Epiteliales/metabolismo , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Complejo 1 de Proteína Adaptadora/fisiología , Subunidades mu de Complejo de Proteína Adaptadora/fisiología , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Biomarcadores/metabolismo , Cadherinas/metabolismo , Células Epiteliales/patología , Células Epiteliales/fisiología , Femenino , Técnica del Anticuerpo Fluorescente , Mucosa Intestinal/patología , Mucosa Intestinal/fisiopatología , Intestino Delgado/patología , Intestino Delgado/fisiopatología , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Noqueados , Factor de Transcripción 4 , beta Catenina/metabolismoRESUMEN
During meiosis, specific histone modifications at pericentric heterochromatin (PCH), especially histone H3 tri- and dimethylation at lysine 9 (H3K9me3 and H3K9me2, respectively), are required for proper chromosome interactions. However, the molecular mechanism by which H3K9 methylation mediates the synapsis is not yet understood. We have generated a Cbx3-deficient mouse line and performed comparative analysis on Suv39h1/h2-, G9a- and Cbx3-deficient spermatocytes. This study revealed that H3K9me2 at PCH depended on Suv39h1/h2-mediated H3K9me3 and its recognition by the Cbx3 gene product HP1γ. We further found that centromere clustering and synapsis were commonly affected in G9a- and Cbx3-deficient spermatocytes. These genetic observations suggest that HP1γ/G9a-dependent PCH-mediated centromere clustering is an axis for proper chromosome interactions during meiotic prophase. We propose that the role of the HP1γ/G9a axis is to retain centromeric regions of unpaired homologous chromosomes in close alignment and facilitate progression of their pairing in early meiotic prophase. This study also reveals considerable plasticity in the interplay between different histone modifications and suggests that such stepwise and dynamic epigenetic modifications may play a pivotal role in meiosis.
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Proteínas Cromosómicas no Histona/genética , Emparejamiento Cromosómico , N-Metiltransferasa de Histona-Lisina/genética , Histonas/química , Meiosis , Animales , Centrómero/ultraestructura , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/fisiología , Epigénesis Genética , Femenino , N-Metiltransferasa de Histona-Lisina/fisiología , Masculino , Metilación , Ratones , Ratones Noqueados , Microscopía Fluorescente/métodos , Modelos Genéticos , Espermatocitos/citologíaRESUMEN
BACKGROUND: Sphingosine-1-phosphate receptor 2 (S1P(2)) is expressed in vascular endothelial cells (ECs). However, the role of S1P(2) in vascular barrier integrity and anaphylaxis is not well understood. Endothelial nitric oxide synthase (eNOS) generates nitric oxide to mediate vascular leakage, compromising survival in patients with anaphylaxis. We recently observed that endothelial S1P(2) inhibits Akt, an activating kinase of eNOS. OBJECTIVE: We tested the hypothesis that endothelial S1P(2) might suppress eNOS, exerting a protective effect against endothelial barrier disruption and anaphylaxis. METHODS: Mice deficient in S1P(2) and eNOS underwent antigen challenge or platelet-activating factor (PAF) injection. Analyses were performed to examine vascular permeability and the underlying mechanisms. RESULTS: S1pr2 deletion augmented vascular leakage and lethality after either antigen challenge or PAF injection. PAF injection induced activation of Akt and eNOS in the aortas and lungs of S1pr2-null mice, which were augmented compared with values seen in wild-type mice. Consistently, PAF-induced increase in cyclic guanosine monophosphate levels in the aorta was enhanced in S1pr-null mice. Genetic Nos3 deletion or pharmacologic eNOS blockade protected S1pr2-null mice from aggravation of barrier disruption after antigen challenge and PAF injection. ECs isolated from S1pr2-null mice exhibited greater stimulation of Akt and eNOS, with enhanced nitric oxide production in response to sphingosine-1-phosphate or PAF, compared with that seen in wild-type ECs. Moreover, S1pr2-deficient ECs showed more severe disassembly of adherens junctions with augmented S-nitrosylation of ß-catenin in response to PAF, which was restored by pharmacologic eNOS blockade. CONCLUSION: S1P(2) diminishes harmful robust eNOS stimulation and thereby attenuates vascular barrier disruption, suggesting potential usefulness of S1P(2) agonists as novel therapeutic agents for anaphylaxis.
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Anafilaxia/metabolismo , Óxido Nítrico Sintasa de Tipo III/antagonistas & inhibidores , Receptores de Lisoesfingolípidos/metabolismo , Uniones Adherentes/metabolismo , Anafilaxia/genética , Anafilaxia/mortalidad , Animales , Aorta/inmunología , Aorta/metabolismo , Permeabilidad Capilar/genética , Permeabilidad Capilar/inmunología , Modelos Animales de Enfermedad , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Activación Enzimática , Eliminación de Gen , Pulmón/inmunología , Pulmón/metabolismo , Ratones , Ratones Noqueados , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa de Tipo III/genética , Óxido Nítrico Sintasa de Tipo III/metabolismo , Factor de Activación Plaquetaria/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Lisoesfingolípidos/genética , Transducción de Señal , beta Catenina/metabolismoRESUMEN
Rats are multiparous rodents that have been used extensively in research; however, the low reproductive performance of some rat strains hampers the broader use of rats as a biomedical model. In this study, the possibility of increasing the litter size after natural mating in rats through superovulation using an anti-inhibin monoclonal antibody (AIMA) was examined. In outbred Wistar rats, AIMA increased the number of ovulated oocytes by 1.3-fold. AIMA did not affect fertilization and subsequent embryonic development, resulting in a 1.4-fold increase in litter size and a high pregnancy rate (86%). In contrast, conventional superovulation by eCG/hCG administration decreased the pregnancy rate to 6-40% and did not increase the litter size. In inbred Brown Norway rats, AIMA increased the litter size by 1.2-fold, and the pregnancy rate increased more than twice (86% versus 38% in controls). AIMA also increased the litter size by 1.5-fold in inbred Tokai High Avoiders and Fischer 344 rats. AIMA increased the efficiency of offspring production by 1.5-, 2.7-, 1.4-, and 1.4-fold, respectively, in the four rat strains. Thus, AIMA may consistently improve the reproductive performance through natural mating in rats, which could promote the use of AIMA in biomedical research.
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Anticuerpos Monoclonales , Inhibinas , Tamaño de la Camada , Superovulación , Animales , Femenino , Tamaño de la Camada/efectos de los fármacos , Embarazo , Ratas , Superovulación/efectos de los fármacos , Anticuerpos Monoclonales/farmacología , Índice de Embarazo , Ratas Wistar , Reproducción/efectos de los fármacos , Masculino , Ratas Endogámicas F344RESUMEN
BACKGROUND & AIMS: Oligosaccharide modifications induce various functional changes in immune cells. The galactose-deficient fraction of fucosylated IgG oligosaccharides is increased, whereas that of ß-1,4-galactosyltransferase I (B4GalTI) is reduced, in patients with Crohn's disease. We investigated the role of oligosaccharide modification in the pathophysiology of colitis using B4galt1-deficient mice. METHODS: Colitis severity was compared between B4galt1(+/-) and B4galt1(+/+) mice. B cells isolated from B4galt1(+/-) and B4galt1(+/+) mice were adoptively transferred to recombination activating gene 2(-/-) mice, in which colitis was induced by administration of CD4(+)CD62L(+) T cells. Cell-surface glycan profiles were determined by lectin microarray analysis. Cytokine production was determined in a coculture of various types of cells isolated from either B4galt1(+/-) or B4galt1(+/+) mice. RESULTS: Colitis induction by dextran sodium sulfate or trinitrobenzene sulfonic acid was significantly reduced in B4galt1(+/-) mice, which had galactose deficiency in IgG oligosaccharides (similar to patients with Crohn's disease) compared with B4galt1(+/+) mice. Amelioration of colitis was associated with increased production of interleukin-10 by macrophages in B4galt1(+/-) mice. Colitis induction in recombination activating gene 2(-/-) mice by administration of CD4(+)CD62L(+) T cells was reduced by cotransfer of B cells isolated from B4galt1(+/-), but not from B4galt1(+/+) mice. Lectin microarray analysis revealed increased expression of polylactosamines on B4galt1(+/-) B cells and macrophages, compared with B4galt1(+/+) cells. The production of interleukin-10 from macrophages was induced via their direct interaction with B4galt1(+/-) B cells. CONCLUSIONS: Altered oligosaccharide structures on immune cells modulate mucosal inflammation. Oligosaccharides in immune cells might be a therapeutic target for inflammatory bowel diseases.
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Colitis/prevención & control , Galactosiltransferasas/fisiología , Oligosacáridos/fisiología , Animales , Antígeno CD11b/análisis , Comunicación Celular , Colitis/inmunología , Galectinas/fisiología , Interleucina-10/biosíntesis , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Oligosacáridos/químicaRESUMEN
Septic shock is a severe systemic response to bacterial infection. Receptor for advanced glycation end products (RAGE) plays a role in immune reactions to recognize specific molecular patterns as pathogen recognition receptors. However, the interaction between LPS, the bioactive component of bacterial cell walls, and RAGE is unclear. In this study, we found direct LPS binding to RAGE by a surface plasmon resonance assay, a plate competition assay, and flow cytometry. LPS increased TNF-α secretion from peritoneal macrophages and an NF-κB promoter-driven luciferase activity through RAGE. Blood neutrophils and monocytes expressed RAGE, and TLR2 was counterregulated in RAGE(-/-) mice. After LPS injection, RAGE(+/+) mice showed a higher mortality, higher serum levels of IL-6, TNF-α, high mobility group box 1, and endothelin-1, and severe lung and liver pathologies compared with RAGE(-/-) mice without significant differences in plasma LPS level. Administration of soluble RAGE significantly reduced the LPS-induced cytokine release and tissue damage and improved the LPS-induced lethality even in RAGE(-/-) as well as RAGE(+/+) mice. The results thus suggest that RAGE can associate with LPS and that RAGE system can regulate inflammatory responses. Soluble RAGE would be a therapeutic tool for LPS-induced septic shock.
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Productos Finales de Glicación Avanzada/metabolismo , Lipopolisacáridos/metabolismo , Lipopolisacáridos/toxicidad , Receptores Inmunológicos/metabolismo , Choque Séptico/inmunología , Secuencia de Aminoácidos , Animales , Unión Competitiva/inmunología , Línea Celular Tumoral , Humanos , Mediadores de Inflamación/sangre , Mediadores de Inflamación/metabolismo , Mediadores de Inflamación/fisiología , Ligandos , Lipopolisacáridos/antagonistas & inhibidores , Masculino , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , FN-kappa B/metabolismo , Unión Proteica/genética , Unión Proteica/inmunología , Ratas , Receptor para Productos Finales de Glicación Avanzada , Receptores Inmunológicos/deficiencia , Receptores Inmunológicos/fisiología , Choque Séptico/mortalidad , Choque Séptico/patología , Choque Séptico/terapia , Transducción de Señal/genética , Transducción de Señal/inmunología , Análisis de Supervivencia , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
CD38, a transmembrane glycoprotein with ADP-ribosyl cyclase activity, catalyses the formation of Ca2+ signalling molecules, but its role in the neuroendocrine system is unknown. Here we show that adult CD38 knockout (CD38-/-) female and male mice show marked defects in maternal nurturing and social behaviour, respectively, with higher locomotor activity. Consistently, the plasma level of oxytocin (OT), but not vasopressin, was strongly decreased in CD38-/- mice. Replacement of OT by subcutaneous injection or lentiviral-vector-mediated delivery of human CD38 in the hypothalamus rescued social memory and maternal care in CD38-/- mice. Depolarization-induced OT secretion and Ca2+ elevation in oxytocinergic neurohypophysial axon terminals were disrupted in CD38-/- mice; this was mimicked by CD38 metabolite antagonists in CD38+/+ mice. These results reveal that CD38 has a key role in neuropeptide release, thereby critically regulating maternal and social behaviours, and may be an element in neurodevelopmental disorders.
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ADP-Ribosil Ciclasa 1/metabolismo , Conducta Materna/fisiología , Oxitocina/metabolismo , Conducta Social , ADP-Ribosil Ciclasa 1/deficiencia , ADP-Ribosil Ciclasa 1/genética , Amnesia/genética , Amnesia/metabolismo , Animales , Calcio/metabolismo , Señalización del Calcio , Femenino , Regulación de la Expresión Génica , Humanos , Inyecciones , Masculino , Memoria/fisiología , Ratones , Actividad Motora/fisiología , Oxitocina/administración & dosificación , Oxitocina/sangre , Oxitocina/farmacología , Vasopresinas/sangreRESUMEN
Protein tyrosine phosphatase receptor-type T (PTPRT) is the most frequently mutated tyrosine phosphatase in human cancers. However, the cell signaling pathways regulated by PTPRT largely remain to be elucidated. Here, we show that paxillin is a direct substrate of PTPRT and that PTPRT specifically regulates paxillin phosphorylation at tyrosine residue 88 (Y88) in colorectal cancer (CRC) cells. We engineered CRC cells homozygous for a paxillin Y88F knock-in mutant and found that these cells exhibit significantly reduced cell migration and impaired anchorage-independent growth, fail to form xenograft tumors in nude mice, and have decreased phosphorylation of p130CAS, SHP2, and AKT. PTPRT knockout mice that we generated exhibit increased levels of colonic paxillin phosphorylation at residue Y88 and are highly susceptible to carcinogen azoxymethane-induced colon tumor, providing critical in vivo evidence that PTPRT normally functions as a tumor suppressor. Moreover, similarly increased paxillin pY88 is also found as a common feature of human colon cancers. These studies reveal an important signaling pathway that plays a critical role in colorectal tumorigenesis.
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Paxillin/metabolismo , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/metabolismo , Tirosina/metabolismo , Animales , Azoximetano , Western Blotting , Línea Celular , Línea Celular Tumoral , Proliferación Celular , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Femenino , Células HCT116 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Desnudos , Mutación , Neoplasias Experimentales/inducido químicamente , Neoplasias Experimentales/genética , Neoplasias Experimentales/metabolismo , Paxillin/genética , Paxillin/fisiología , Fosforilación , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/genética , Especificidad por Sustrato , Transfección , Trasplante Heterólogo , Tirosina/genéticaRESUMEN
The development of ZFN, TALEN, and CRISPR/Cas9 systems has simplified the process of generating knockout (KO) and knock-in (KI) rats in addition to mice. However, in rats, an efficient genome editing technique that uses in vitro fertilized oocytes has not been established. Recently, we reported the stable generation of offspring from five standard strains of rats by superovulation and in vitro fertilization (IVF). Furthermore, genome-edited rats can be easily generated by electroporation. First, juvenile female rats are administered LHRH (luteinizing hormone-releasing hormone) to synchronize the estrous cycle and then AIS (Automatic Identification System) with PMSG (pregnant mare serum gonadotropin) before hCG (human chorionic gonadotropin) for superovulation. Sperm collected from a sexually mature male rat the following morning is then pre-cultured. Cumulus cell-oocyte complexes (COCs) are collected from female rats under anesthesia, and COCs are induced into a medium containing concentration-adjusted sperm. Thereafter, oocytes with two pronucleus are selected as fertilized oocytes. Next, fertilized oocytes are transferred into a glass chamber containing CRISPR ribonucleoprotein (RNP) complexes formed from gRNA and Cas9 protein. After electroporation, fertilized oocytes are then immediately transferred to culture medium. The next day, embryos are transferred into the oviduct of pseudopregnant female rats. Using the above method, offspring can be obtained 22 days after the day of embryo transfer. In this paper, we outline a method allowing simple and efficient generation of genetically modified rats without the need for technically difficult micromanipulation techniques.
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Oocitos , Semen , Animales , Femenino , Humanos , Masculino , Embarazo , Ratas , Transferencia de Embrión , Fertilización In Vitro/métodos , Edición Génica/métodos , CaballosRESUMEN
Background: Beta-1,4-galactosyltransferase-3 (B4GALT3) belongs to the family of beta-1,4-galactosyltransferases (B4GALTs) and is responsible for the transfer of UDP-galactose to terminal N-acetylglucosamine. B4GALT3 is differentially expressed in tumors and adjacent normal tissues, and is correlated with clinical prognosis in several cancers, including neuroblastoma, cervical cancer, and bladder cancer. However, the exact role of B4GALT3 in the tumor immune microenvironment (TIME) remains unclear. Here, we aimed to elucidate the function of B4GALT3 in the TIME. Methods: To study the functions of B4GALT3 in cancer immunity, either weakly or strongly immunogenic tumor cells were subcutaneously transplanted into wild-type (WT) and B4galt3 knockout (KO) mice. Bone marrow transplantation and CD8+ T cell depletion experiments were conducted to elucidate the role of immune cells in suppressing tumor growth in B4galt3 KO mice. The cell types and gene expression in the tumor region and infiltrating CD8+ T cells were analyzed using flow cytometry and RNA sequencing. N-glycosylated proteins from WT and B4galt3 KO mice were compared using the liquid chromatography tandem mass spectrometry (LC-MS/MS)-based glycoproteomic approach. Results: B4galt3 KO mice exhibited suppressed growth of strongly immunogenic tumors with a notable increase in CD8+ T cell infiltration within tumors. Notably, B4galt3 deficiency led to changes in N-glycan modification of several proteins, including integrin alpha L (ITGAL), involved in T cell activity and proliferation. In vitro experiments suggested that B4galt3 KO CD8+ T cells were more susceptible to activation and displayed increased downstream phosphorylation of FAK linked to ITGAL. Conclusion: Our study demonstrates that B4galt3 deficiency can potentially boost anti-tumor immune responses, largely through enhancing the influx of CD8+ T cells. B4GALT3 might be suppressing cancer immunity by synthesizing the glycan structure of molecules on the CD8+ T cell surface, as evidenced by the changes in the glycan structure of ITGAL in immune cells. Importantly, B4galt3 KO mice showed no adverse effects on growth, development, or reproduction, underscoring the potential of B4GALT3 as a promising and safe therapeutic target for cancer treatment.