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1.
Nat Immunol ; 23(4): 505-517, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-35354960

RESUMEN

Intrinsic and extrinsic cues determine developmental trajectories of hematopoietic stem cells (HSCs) towards erythroid, myeloid and lymphoid lineages. Using two newly generated transgenic mice that report and trace the expression of terminal deoxynucleotidyl transferase (TdT), transient induction of TdT was detected on a newly identified multipotent progenitor (MPP) subset that lacked self-renewal capacity but maintained multilineage differentiation potential. TdT induction on MPPs reflected a transcriptionally dynamic but uncommitted stage, characterized by low expression of lineage-associated genes. Single-cell CITE-seq indicated that multipotency in the TdT+ MPPs is associated with expression of the endothelial cell adhesion molecule ESAM. Stable and progressive upregulation of TdT defined the lymphoid developmental trajectory. Collectively, we here identify a new multipotent progenitor within the MPP4 compartment. Specification and commitment are defined by downregulation of ESAM which marks the progressive loss of alternative fates along all lineages.


Asunto(s)
ADN Nucleotidilexotransferasa , Células Madre Hematopoyéticas , Células Madre Multipotentes , Animales , Diferenciación Celular , Linaje de la Célula/genética , ADN Nucleotidilexotransferasa/genética , ADN Nucleotidilexotransferasa/metabolismo , Células Madre Hematopoyéticas/fisiología , Ratones , Ratones Transgénicos
2.
Immunity ; 50(5): 1289-1304.e6, 2019 05 21.
Artículo en Inglés | MEDLINE | ID: mdl-31079916

RESUMEN

Pathogenic lymphocytes initiate the development of chronic inflammatory diseases. The cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) (encoded by Csf2) is a key communicator between pathogenic lymphocytes and tissue-invading inflammatory phagocytes. However, the molecular properties of GM-CSF-producing cells and the mode of Csf2 regulation in vivo remain unclear. To systematically study and manipulate GM-CSF+ cells and their progeny in vivo, we generated a fate-map and reporter of GM-CSF expression mouse strain (FROG). We mapped the phenotypic and functional profile of auto-aggressive T helper (Th) cells during neuroinflammation and identified the signature and pathogenic memory of a discrete encephalitogenic Th subset. These cells required interleukin-23 receptor (IL-23R) and IL-1R but not IL-6R signaling for their maintenance and pathogenicity. Specific ablation of this subset interrupted the inflammatory cascade, despite the unperturbed tissue accumulation of other Th subsets (e.g., Th1 and Th17), highlighting that GM-CSF expression not only marks pathogenic Th cells, but that this subset mediates immunopathology and tissue destruction.


Asunto(s)
Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Interleucina-1beta/inmunología , Subunidad p19 de la Interleucina-23/inmunología , Células TH1/inmunología , Células Th17/inmunología , Animales , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Inflamación/genética , Inflamación/patología , Interferón gamma/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores CXCR6/metabolismo , Receptores de Interleucina/genética , Receptores de Interleucina/inmunología , Receptores Tipo I de Interleucina-1/genética , Receptores Tipo I de Interleucina-1/inmunología , Factor de Necrosis Tumoral alfa/metabolismo
3.
Proc Natl Acad Sci U S A ; 121(11): e2318657121, 2024 Mar 12.
Artículo en Inglés | MEDLINE | ID: mdl-38446855

RESUMEN

Viral mimicry of host cell structures has been postulated to curtail the B cell receptor (BCR) repertoire against persisting viruses through tolerance mechanisms. This concept awaits, however, experimental testing in a setting of natural virus-host relationship. We engineered mouse models expressing a monoclonal BCR specific for the envelope glycoprotein of lymphocytic choriomeningitis virus (LCMV), a naturally persisting mouse pathogen. When the heavy chain of the LCMV-neutralizing antibody KL25 was paired with its unmutated ancestor light chain, most B cells underwent receptor editing, a behavior reminiscent of autoreactive clones. In contrast, monoclonal B cells expressing the same heavy chain in conjunction with the hypermutated KL25 light chain did not undergo receptor editing but exhibited low levels of surface IgM, suggesting that light chain hypermutation had lessened KL25 autoreactivity. Upon viral challenge, these IgMlow cells were not anergic but up-regulated IgM, participated in germinal center reactions, produced antiviral antibodies, and underwent immunoglobulin class switch as well as further affinity maturation. These studies on a persisting virus in its natural host species suggest that central tolerance mechanisms prune the protective antiviral B cell repertoire.


Asunto(s)
Linfocitos B , Tolerancia Central , Animales , Ratones , Anticuerpos Antivirales , Virus de la Coriomeningitis Linfocítica , Antivirales , Inmunoglobulina M
4.
Immunity ; 46(2): 245-260, 2017 02 21.
Artículo en Inglés | MEDLINE | ID: mdl-28228281

RESUMEN

Chronic inflammatory diseases are influenced by dysregulation of cytokines. Among them, granulocyte macrophage colony stimulating factor (GM-CSF) is crucial for the pathogenic function of T cells in preclinical models of autoimmunity. To study the impact of dysregulated GM-CSF expression in vivo, we generated a transgenic mouse line allowing the induction of GM-CSF expression in mature, peripheral helper T (Th) cells. Antigen-independent GM-CSF release led to the invasion of inflammatory myeloid cells into the central nervous system (CNS), which was accompanied by the spontaneous development of severe neurological deficits. CNS-invading phagocytes produced reactive oxygen species and exhibited a distinct genetic signature compared to myeloid cells invading other organs. We propose that the CNS is particularly vulnerable to the attack of monocyte-derived phagocytes and that the effector functions of GM-CSF-expanded myeloid cells are in turn guided by the tissue microenvironment.


Asunto(s)
Sistema Nervioso Central/inmunología , Sistema Nervioso Central/patología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Fagocitos/inmunología , Animales , Citometría de Flujo , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Animales , Reacción en Cadena de la Polimerasa
5.
PLoS Biol ; 21(5): e3002111, 2023 05.
Artículo en Inglés | MEDLINE | ID: mdl-37159457

RESUMEN

Atypical chemokine receptors (ACKRs) scavenge chemokines and can contribute to gradient formation by binding, internalizing, and delivering chemokines for lysosomal degradation. ACKRs do not couple to G-proteins and fail to induce typical signaling induced by chemokine receptors. ACKR3, which binds and scavenges CXCL12 and CXCL11, is known to be expressed in vascular endothelium, where it has immediate access to circulating chemokines. ACKR4, which binds and scavenges CCL19, CCL20, CCL21, CCL22, and CCL25, has also been detected in lymphatic and blood vessels of secondary lymphoid organs, where it clears chemokines to facilitate cell migration. Recently, GPR182, a novel ACKR-like scavenger receptor, has been identified and partially deorphanized. Multiple studies point towards the potential coexpression of these 3 ACKRs, which all interact with homeostatic chemokines, in defined cellular microenvironments of several organs. However, an extensive map of ACKR3, ACKR4, and GPR182 expression in mice has been missing. In order to reliably detect ACKR expression and coexpression, in the absence of specific anti-ACKR antibodies, we generated fluorescent reporter mice, ACKR3GFP/+, ACKR4GFP/+, GPR182mCherry/+, and engineered fluorescently labeled ACKR-selective chimeric chemokines for in vivo uptake. Our study on young healthy mice revealed unique and common expression patterns of ACKRs in primary and secondary lymphoid organs, small intestine, colon, liver, and kidney. Furthermore, using chimeric chemokines, we were able to detect distinct zonal expression and activity of ACKR4 and GPR182 in the liver, which suggests their cooperative relationship. This study provides a broad comparative view and a solid stepping stone for future functional explorations of ACKRs based on the microanatomical localization and distinct and cooperative roles of these powerful chemokine scavengers.


Asunto(s)
Transducción de Señal , Animales , Ratones , Quimiocina CCL19/metabolismo , Movimiento Celular
6.
Proc Natl Acad Sci U S A ; 120(48): e2315503120, 2023 Nov 28.
Artículo en Inglés | MEDLINE | ID: mdl-37988464

RESUMEN

Gasdermins (GSDMs) share a common functional domain structure and are best known for their capacity to form membrane pores. These pores are hallmarks of a specific form of cell death called pyroptosis and mediate the secretion of pro-inflammatory cytokines such as interleukin 1ß (IL1ß) and interleukin 18 (IL18). Thereby, Gasdermins have been implicated in various immune responses against cancer and infectious diseases such as acute Salmonella Typhimurium (S.Tm) gut infection. However, to date, we lack a comprehensive functional assessment of the different Gasdermins (GSDMA-E) during S.Tm infection in vivo. Here, we used epithelium-specific ablation, bone marrow chimeras, and mouse lines lacking individual Gasdermins, combinations of Gasdermins or even all Gasdermins (GSDMA1-3C1-4DE) at once and performed littermate-controlled oral S.Tm infections in streptomycin-pretreated mice to investigate the impact of all murine Gasdermins. While GSDMA, C, and E appear dispensable, we show that GSDMD i) restricts S.Tm loads in the gut tissue and systemic organs, ii) controls gut inflammation kinetics, and iii) prevents epithelium disruption by 72 h of the infection. Full protection requires GSDMD expression by both bone-marrow-derived lamina propria cells and intestinal epithelial cells (IECs). In vivo experiments as well as 3D-, 2D-, and chimeric enteroid infections further show that infected IEC extrusion proceeds also without GSDMD, but that GSDMD controls the permeabilization and morphology of the extruding IECs, affects extrusion kinetics, and promotes overall mucosal barrier capacity. As such, this work identifies a unique multipronged role of GSDMD among the Gasdermins for mucosal tissue defense against a common enteric pathogen.


Asunto(s)
Gasderminas , Infecciones por Salmonella , Animales , Ratones , Infecciones por Salmonella/prevención & control , Salmonella typhimurium , Inflamación , Células Epiteliales , Inflamasomas
7.
Immunity ; 43(3): 502-14, 2015 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-26341401

RESUMEN

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has emerged as a crucial cytokine produced by auto-reactive T helper (Th) cells that initiate tissue inflammation. Multiple cell types can sense GM-CSF, but the identity of the pathogenic GM-CSF-responsive cells is unclear. By using conditional gene targeting, we systematically deleted the GM-CSF receptor (Csf2rb) in specific subpopulations throughout the myeloid lineages. Experimental autoimmune encephalomyelitis (EAE) progressed normally when either classical dendritic cells (cDCs) or neutrophils lacked GM-CSF responsiveness. The development of tissue-invading monocyte-derived dendritic cells (moDCs) was also unperturbed upon Csf2rb deletion. Instead, deletion of Csf2rb in CCR2(+)Ly6C(hi) monocytes phenocopied the EAE resistance seen in complete Csf2rb-deficient mice. High-dimensional analysis of tissue-infiltrating moDCs revealed that GM-CSF initiates a combination of inflammatory mechanisms. These results indicate that GM-CSF signaling controls a pathogenic expression signature in CCR2(+)Ly6C(hi) monocytes and their progeny, which was essential for tissue damage.


Asunto(s)
Autoinmunidad/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Inflamación/inmunología , Monocitos/inmunología , Receptores CCR2/inmunología , Transducción de Señal/inmunología , Animales , Antígenos Ly/genética , Antígenos Ly/inmunología , Antígenos Ly/metabolismo , Autoinmunidad/genética , Subunidad beta Común de los Receptores de Citocinas/genética , Subunidad beta Común de los Receptores de Citocinas/inmunología , Subunidad beta Común de los Receptores de Citocinas/metabolismo , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Encefalomielitis Autoinmune Experimental , Citometría de Flujo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Humanos , Inflamación/genética , Inflamación/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Interleucina-1beta/metabolismo , Ratones Noqueados , Ratones Transgénicos , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Células Mieloides/efectos de los fármacos , Células Mieloides/inmunología , Células Mieloides/metabolismo , Fosforilación/efectos de los fármacos , Fosforilación/inmunología , Receptores CCR2/genética , Receptores CCR2/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT5/inmunología , Factor de Transcripción STAT5/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transcriptoma/efectos de los fármacos , Transcriptoma/genética , Transcriptoma/inmunología
8.
Circulation ; 146(21): 1610-1626, 2022 11 22.
Artículo en Inglés | MEDLINE | ID: mdl-36268721

RESUMEN

BACKGROUND: Arrhythmogenic cardiomyopathy (ACM) is characterized by progressive loss of cardiomyocytes with fibrofatty tissue replacement, systolic dysfunction, and life-threatening arrhythmias. A substantial proportion of ACM is caused by mutations in genes of the desmosomal cell-cell adhesion complex, but the underlying mechanisms are not well understood. In the current study, we investigated the relevance of defective desmosomal adhesion for ACM development and progression. METHODS: We mutated the binding site of DSG2 (desmoglein-2), a crucial desmosomal adhesion molecule in cardiomyocytes. This DSG2-W2A mutation abrogates the tryptophan swap, a central interaction mechanism of DSG2 on the basis of structural data. Impaired adhesive function of DSG2-W2A was confirmed by cell-cell dissociation assays and force spectroscopy measurements by atomic force microscopy. The DSG2-W2A knock-in mouse model was analyzed by echocardiography, ECG, and histologic and biomolecular techniques including RNA sequencing and transmission electron and superresolution microscopy. The results were compared with ACM patient samples, and their relevance was confirmed in vivo and in cardiac slice cultures by inhibitor studies applying the small molecule EMD527040 or an inhibitory integrin-αVß6 antibody. RESULTS: The DSG2-W2A mutation impaired binding on molecular level and compromised intercellular adhesive function. Mice bearing this mutation develop a severe cardiac phenotype recalling the characteristics of ACM, including cardiac fibrosis, impaired systolic function, and arrhythmia. A comparison of the transcriptome of mutant mice with ACM patient data suggested deregulated integrin-αVß6 and subsequent transforming growth factor-ß signaling as driver of cardiac fibrosis. Blocking integrin-αVß6 led to reduced expression of profibrotic markers and reduced fibrosis formation in mutant animals in vivo. CONCLUSIONS: We show that disruption of desmosomal adhesion is sufficient to induce a phenotype that fulfils the clinical criteria to establish the diagnosis of ACM, confirming the dysfunctional adhesion hypothesis. Deregulation of integrin-αVß6 and transforming growth factor-ß signaling was identified as a central step toward fibrosis. A pilot in vivo drug test revealed this pathway as a promising target to ameliorate fibrosis. This highlights the value of this model to discern mechanisms of cardiac fibrosis and to identify and test novel treatment options for ACM.


Asunto(s)
Displasia Ventricular Derecha Arritmogénica , Cardiomiopatías , Ratones , Animales , Cardiomiopatías/genética , Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Integrinas/metabolismo , Miocitos Cardíacos/metabolismo , Fibrosis , Factor de Crecimiento Transformador beta/metabolismo , Factores de Crecimiento Transformadores/metabolismo , Displasia Ventricular Derecha Arritmogénica/patología
9.
EMBO J ; 38(10)2019 05 15.
Artículo en Inglés | MEDLINE | ID: mdl-30902848

RESUMEN

Pyroptosis is a form of lytic inflammatory cell death driven by inflammatory caspase-1, caspase-4, caspase-5 and caspase-11. These caspases cleave and activate the pore-forming protein gasdermin D (GSDMD) to induce membrane damage. By contrast, apoptosis is driven by apoptotic caspase-8 or caspase-9 and has traditionally been classified as an immunologically silent form of cell death. Emerging evidence suggests that therapeutics designed for cancer chemotherapy or inflammatory disorders such as SMAC mimetics, TAK1 inhibitors and BH3 mimetics promote caspase-8 or caspase-9-dependent inflammatory cell death and NLRP3 inflammasome activation. However, the mechanism by which caspase-8 or caspase-9 triggers cell lysis and NLRP3 activation is still undefined. Here, we demonstrate that during extrinsic apoptosis, caspase-1 and caspase-8 cleave GSDMD to promote lytic cell death. By engineering a novel Gsdmd D88A knock-in mouse, we further demonstrate that this proinflammatory function of caspase-8 is counteracted by caspase-3-dependent cleavage and inactivation of GSDMD at aspartate 88, and is essential to suppress GSDMD-dependent cell lysis during caspase-8-dependent apoptosis. Lastly, we provide evidence that channel-forming glycoprotein pannexin-1, but not GSDMD or GSDME promotes NLRP3 inflammasome activation during caspase-8 or caspase-9-dependent apoptosis.


Asunto(s)
Apoptosis/fisiología , Conexinas/fisiología , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteínas del Tejido Nervioso/fisiología , Células 3T3 , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Caspasas/metabolismo , Células Cultivadas , Embrión de Mamíferos , Células HEK293 , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Complejos Multiproteicos/metabolismo , Proteínas de Unión a Fosfato/metabolismo , Unión Proteica , Multimerización de Proteína , Receptores de Estrógenos/metabolismo , Transducción de Señal/fisiología
10.
Mol Cell ; 60(4): 611-25, 2015 Nov 19.
Artículo en Inglés | MEDLINE | ID: mdl-26549683

RESUMEN

The integrity of chromatin, which provides a dynamic template for all DNA-related processes in eukaryotes, is maintained through replication-dependent and -independent assembly pathways. To address the role of histone deposition in the absence of DNA replication, we deleted the H3.3 chaperone Hira in developing mouse oocytes. We show that chromatin of non-replicative developing oocytes is dynamic and that lack of continuous H3.3/H4 deposition alters chromatin structure, resulting in increased DNase I sensitivity, the accumulation of DNA damage, and a severe fertility phenotype. On the molecular level, abnormal chromatin structure leads to a dramatic decrease in the dynamic range of gene expression, the appearance of spurious transcripts, and inefficient de novo DNA methylation. Our study thus unequivocally shows the importance of continuous histone replacement and chromatin homeostasis for transcriptional regulation and normal developmental progression in a non-replicative system in vivo.


Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Oogénesis , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Animales , Metilación de ADN , Femenino , Fertilización , Regulación de la Expresión Génica , Ratones , Oocitos/metabolismo , Transcripción Genética
11.
EMBO J ; 37(6)2018 03 15.
Artículo en Inglés | MEDLINE | ID: mdl-29459437

RESUMEN

Pathogenic and commensal Gram-negative bacteria produce and release outer membrane vesicles (OMVs), which present several surface antigens and play an important role for bacterial pathogenesis. OMVs also modulate the host immune system, which makes them attractive as vaccine candidates. At the cellular level, OMVs are internalized by macrophages and deliver lipopolysaccharide (LPS) into the host cytosol, thus activating the caspase-11 non-canonical inflammasome. Here, we show that OMV-induced inflammasome activation requires TLR4-TRIF signaling, the production of type I interferons, and the action of guanylate-binding proteins (GBPs), both in macrophages and in vivo Mechanistically, we find that isoprenylated GBPs associate with the surface of OMVs or with transfected LPS, indicating that the key factor that determines GBP recruitment to the Gram-negative bacterial outer membranes is LPS itself. Our findings provide new insights into the mechanism by which GBPs target foreign surfaces and reveal a novel function for GBPs in controlling the intracellular detection of LPS derived from extracellular bacteria in the form of OMVs, thus extending their function as a hub between cell-autonomous immunity and innate immunity.


Asunto(s)
Bacterias/inmunología , Membrana Celular/inmunología , Proteínas de Unión al GTP/inmunología , Inflamasomas/inmunología , Lipopolisacáridos/inmunología , Animales , Proteínas de Unión al GTP/genética , Ratones Endogámicos C57BL , Ratones Noqueados
12.
Proc Natl Acad Sci U S A ; 116(21): 10547-10556, 2019 05 21.
Artículo en Inglés | MEDLINE | ID: mdl-31061112

RESUMEN

There is a growing body of evidence linking maternal overnutrition to obesity and psychopathology that can be conserved across multiple generations. Recently, we demonstrated in a maternal high-fat diet (HFD; MHFD) mouse model that MHFD induced enhanced hedonic behaviors and obesogenic phenotypes that were conserved across three generations via the paternal lineage, which was independent of sperm methylome changes. Here, we show that sperm tRNA-derived small RNAs (tsRNAs) partly contribute to the transmission of such phenotypes. We observe increased expression of sperm tsRNAs in the F1 male offspring born to HFD-exposed dams. Microinjection of sperm tsRNAs from the F1-HFD male into normal zygotes reproduces obesogenic phenotypes and addictive-like behaviors, such as increased preference of palatable foods and enhanced sensitivity to drugs of abuse in the resultant offspring. The expression of several of the differentially expressed sperm tsRNAs predicted targets such as CHRNA2 and GRIN3A, which have been implicated in addiction pathology, are altered in the mesolimbic reward brain regions of the F1-HFD father and the resultant HFD-tsRNA offspring. Together, our findings demonstrate that sperm tsRNA is a potential vector that contributes to the transmission of MHFD-induced addictive-like behaviors and obesogenic phenotypes across generations, thereby emphasizing its role in diverse pathological outcomes.


Asunto(s)
Fenómenos Fisiologicos Nutricionales Maternos , Obesidad/genética , Efectos Tardíos de la Exposición Prenatal , ARN/metabolismo , Espermatozoides/metabolismo , Animales , Conducta Adictiva , Dieta Alta en Grasa/efectos adversos , Femenino , Masculino , Ratones , Fenotipo , Embarazo
13.
Proc Natl Acad Sci U S A ; 116(51): 25688-25696, 2019 12 17.
Artículo en Inglés | MEDLINE | ID: mdl-31772009

RESUMEN

Neural stem cells (NSCs) generate neurons and glial cells throughout embryonic and postnatal brain development. The role of S-palmitoylation (also referred to as S-acylation), a reversible posttranslational lipid modification of proteins, in regulating the fate and activity of NSCs remains largely unknown. We used an unbiased screening approach to identify proteins that are S-acylated in mouse NSCs and showed that bone morphogenic protein receptor 1a (BMPR1a), a core mediator of BMP signaling, is palmitoylated. Genetic manipulation of S-acylated sites affects the localization and trafficking of BMPR1a and leads to altered BMP signaling. Strikingly, defective palmitoylation of BMPR1a modulates NSC function within the mouse brain, resulting in enhanced oligodendrogenesis. Thus, we identified a mechanism regulating the behavior of NSCs and provided the framework to characterize dynamic posttranslational lipid modifications of proteins in the context of NSC biology.


Asunto(s)
Receptores de Proteínas Morfogenéticas Óseas de Tipo 1 , Lipoilación/fisiología , Células-Madre Neurales , Neurogénesis/fisiología , Animales , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/química , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/metabolismo , Células Cultivadas , Ratones , Células-Madre Neurales/química , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo
14.
J Biol Chem ; 295(30): 10331-10339, 2020 07 24.
Artículo en Inglés | MEDLINE | ID: mdl-32499372

RESUMEN

Mutations in the ryanodine receptor 1 (RYR1) gene are associated with several human congenital myopathies, including the dominantly inherited central core disease and exercise-induced rhabdomyolysis, and the more severe recessive phenotypes, including multiminicore disease, centronuclear myopathy, and congenital fiber type disproportion. Within the latter group, those carrying a hypomorphic mutation in one allele and a missense mutation in the other are the most severely affected. Because of nonsense-mediated decay, most hypomorphic alleles are not expressed, resulting in homozygous expression of the missense mutation allele. This should result in 50% reduced expression of the ryanodine receptor in skeletal muscle, but its observed content is even lower. To study in more detail the biochemistry and pathophysiology of recessive RYR1 myopathies, here we investigated a mouse model we recently generated by analyzing the effect of bi-allelic versus mono-allelic expression of the RyR1 p.A4329D mutation. Our results revealed that the expression of two alleles carrying the same mutation or of one allele with the mutation in combination with a hypomorphic allele does not result in functionally equal outcomes and impacts skeletal muscles differently. In particular, the bi-allelic RyR1 p.A4329D mutation caused a milder phenotype than its mono-allelic expression, leading to changes in the biochemical properties and physiological function only of slow-twitch muscles and largely sparing fast-twitch muscles. In summary, bi-allelic expression of the RyR1 p.A4329D mutation phenotypically differs from mono-allelic expression of this mutation in a compound heterozygous carrier.


Asunto(s)
Regulación de la Expresión Génica , Fibras Musculares de Contracción Lenta/metabolismo , Fuerza Muscular , Mutación Missense , Canal Liberador de Calcio Receptor de Rianodina/biosíntesis , Sustitución de Aminoácidos , Animales , Masculino , Ratones , Ratones Mutantes , Canal Liberador de Calcio Receptor de Rianodina/genética
15.
Hum Mol Genet ; 28(11): 1872-1884, 2019 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-30689883

RESUMEN

Here we characterized a mouse model knocked-in for a frameshift mutation in RYR1 exon 36 (p.Gln1970fsX16) that is isogenic to that identified in one parent of a severely affected patient with recessively inherited multiminicore disease. This individual carrying the RYR1 frameshifting mutation complained of mild muscle weakness and fatigability. Analysis of the RyR1 protein content in a muscle biopsy from this individual showed a content of only 20% of that present in a control individual. The biochemical and physiological characteristics of skeletal muscles from RyR1Q1970fsX16 heterozygous mice recapitulates that of the heterozygous parent. RyR1 protein content in the muscles of mutant mice reached 38% and 58% of that present in total muscle homogenates of fast and slow muscles from wild-type (WT) littermates. The decrease of RyR1 protein content in total homogenates is not accompanied by a decrease of Cav1.1 content, whereby the Cav1.1/RyR1 stoichiometry ratio in skeletal muscles from RyR1Q1970fsX16 heterozygous mice is lower compared to that from WT mice. Electron microscopy (EM) revealed a 36% reduction in the number/area of calcium release units accompanied by a 2.5-fold increase of dyads (triads that have lost one junctional sarcoplasmic reticulum element); both results suggest a reduction of the RyR1 arrays. Compared to WT, muscle strength and depolarization-induced calcium transients in RyR1Q1970fsX16 heterozygous mice muscles were decreased by 20% and 15%, respectively. The RyR1Q1970fsX16 mouse model provides mechanistic insight concerning the phenotype of the parent carrying the RYR1 ex36 mutation and suggests that in skeletal muscle fibres there is a functional reserve of RyR1.


Asunto(s)
Canales de Calcio Tipo L/genética , Debilidad Muscular/genética , Miopatías Estructurales Congénitas/genética , Oftalmoplejía/genética , Canal Liberador de Calcio Receptor de Rianodina/deficiencia , Adulto , Alelos , Animales , Modelos Animales de Enfermedad , Mutación del Sistema de Lectura/genética , Heterocigoto , Humanos , Ratones , Microscopía Electrónica , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patología , Fibras Musculares Esqueléticas/ultraestructura , Debilidad Muscular/patología , Miopatías Estructurales Congénitas/fisiopatología , Oftalmoplejía/fisiopatología , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/ultraestructura
16.
Hum Mol Genet ; 28(18): 2987-2999, 2019 09 15.
Artículo en Inglés | MEDLINE | ID: mdl-31044239

RESUMEN

Recessive ryanodine receptor 1 (RYR1) mutations cause congenital myopathies including multiminicore disease (MmD), congenital fiber-type disproportion and centronuclear myopathy. We created a mouse model knocked-in for the Q1970fsX16+A4329D RYR1 mutations, which are isogenic with those identified in a severely affected child with MmD. During the first 20 weeks after birth the body weight and the spontaneous running distance of the mutant mice were 20% and 50% lower compared to wild-type littermates. Skeletal muscles from mutant mice contained 'cores' characterized by severe myofibrillar disorganization associated with misplacement of mitochondria. Furthermore, their muscles developed less force and had smaller electrically evoked calcium transients. Mutant RyR1 channels incorporated into lipid bilayers were less sensitive to calcium and caffeine, but no change in single-channel conductance was observed. Our results demonstrate that the phenotype of the RyR1Q1970fsX16+A4329D compound heterozygous mice recapitulates the clinical picture of multiminicore patients and provide evidence of the molecular mechanisms responsible for skeletal muscle defects.


Asunto(s)
Calcio/metabolismo , Fuerza Muscular/genética , Músculo Esquelético/metabolismo , Mutación , Miopatía del Núcleo Central/etiología , Miopatía del Núcleo Central/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Alelos , Animales , Señalización del Calcio , Modelos Animales de Enfermedad , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Heterocigoto , Masculino , Ratones , Ratones Noqueados , Actividad Motora , Músculo Esquelético/fisiopatología , Músculo Esquelético/ultraestructura , Miopatía del Núcleo Central/fisiopatología , Fenotipo
17.
FASEB J ; 34(8): 11143-11167, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32627872

RESUMEN

Exercise modulates metabolism and the gut microbiome. Brief exposure to low mT-range pulsing electromagnetic fields (PEMFs) was previously shown to accentuate in vitro myogenesis and mitochondriogenesis by activating a calcium-mitochondrial axis upstream of PGC-1α transcriptional upregulation, recapitulating a genetic response implicated in exercise-induced metabolic adaptations. We compared the effects of analogous PEMF exposure (1.5 mT, 10 min/week), with and without exercise, on systemic metabolism and gut microbiome in four groups of mice: (a) no intervention; (b) PEMF treatment; (c) exercise; (d) exercise and PEMF treatment. The combination of PEMFs and exercise for 6 weeks enhanced running performance and upregulated muscular and adipose Pgc-1α transcript levels, whereas exercise alone was incapable of elevating Pgc-1α levels. The gut microbiome Firmicutes/Bacteroidetes ratio decreased with exercise and PEMF exposure, alone or in combination, which has been associated in published studies with an increase in lean body mass. After 2 months, brief PEMF treatment alone increased Pgc-1α and mitohormetic gene expression and after >4 months PEMF treatment alone enhanced oxidative muscle expression, fatty acid oxidation, and reduced insulin levels. Hence, short-term PEMF treatment was sufficient to instigate PGC-1α-associated transcriptional cascades governing systemic mitohormetic adaptations, whereas longer-term PEMF treatment was capable of inducing related metabolic adaptations independently of exercise.


Asunto(s)
Microbioma Gastrointestinal/fisiología , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Adaptación Fisiológica/fisiología , Animales , Bacteroidetes/crecimiento & desarrollo , Composición Corporal/fisiología , Ácidos Grasos/metabolismo , Femenino , Firmicutes/crecimiento & desarrollo , Estudios de Seguimiento , Expresión Génica/fisiología , Insulina/metabolismo , Campos Magnéticos , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Desarrollo de Músculos/fisiología , Músculo Esquelético/metabolismo , Condicionamiento Físico Animal/fisiología , Transcripción Genética/fisiología , Activación Transcripcional/fisiología
18.
Immunity ; 37(6): 1050-1060, 2012 Dec 14.
Artículo en Inglés | MEDLINE | ID: mdl-23177320

RESUMEN

Colony stimulating factor-1 (Csf-1) receptor and its ligand Csf-1 control macrophage development, maintenance, and function. The development of both Langerhans cells (LCs) and microglia is highly dependent on Csf-1 receptor signaling but independent of Csf-1. Here we show that in both mice and humans, interleukin-34 (IL-34), an alternative ligand for Csf-1 receptor, is produced by keratinocytes in the epidermis and by neurons in the brain. Mice lacking IL-34 displayed a marked reduction of LCs and a decrease of microglia, whereas monocytes, dermal, and lymphoid tissue macrophages and DCs were unaffected. We identified IL-34 as a nonredundant cytokine for the development of LCs during embryogenesis as well as for their homeostasis in the adult skin. Whereas inflammation-induced repopulation of LCs appears to be dependent on Csf-1, once inflammation is resolved, LC survival is again IL-34-dependent. In contrast, microglia and their yolk sac precursors develop independently of IL-34 but rely on it for their maintenance in the adult brain.


Asunto(s)
Interleucinas/fisiología , Células de Langerhans/inmunología , Microglía/inmunología , Células del Estroma/metabolismo , Animales , Encéfalo/inmunología , Encéfalo/metabolismo , Diferenciación Celular/genética , Epidermis/inmunología , Epidermis/metabolismo , Homeostasis , Humanos , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Interleucinas/genética , Interleucinas/inmunología , Interleucinas/metabolismo , Queratinocitos/inmunología , Queratinocitos/metabolismo , Células de Langerhans/citología , Células de Langerhans/metabolismo , Ratones , Microglía/citología , Microglía/metabolismo , Psoriasis/inducido químicamente , Psoriasis/inmunología , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Transducción de Señal , Piel/inmunología , Piel/metabolismo
19.
J Mammary Gland Biol Neoplasia ; 24(1): 39-45, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-30209717

RESUMEN

Genetically engineered mouse models have become an indispensable tool for breast cancer research. Combination of multiple site-specific recombination systems such as Cre/loxP and Flippase (Flp)/Frt allows for engineering of sophisticated, multi-layered conditional mouse models. Here, we report the generation and characterization of a novel transgenic mouse line expressing a mouse codon-optimized Flp under the control of the mouse mammary tumor virus (MMTV) promoter. These mice show robust Flp-mediated recombination in luminal mammary gland and breast cancer cells but no Flp activity in non-mammary tissues, with the exception of limited activity in salivary glands. These mice provide a unique tool for studying mammary gland biology and carcinogenesis in mice.


Asunto(s)
Carcinogénesis/genética , ADN Nucleotidiltransferasas/genética , Glándulas Mamarias Animales/patología , Neoplasias Mamarias Experimentales/genética , Virus del Tumor Mamario del Ratón/genética , Animales , Carcinogénesis/patología , Progresión de la Enfermedad , Células Epiteliales/patología , Femenino , Genes Reporteros/genética , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Proteínas Luminiscentes/genética , Glándulas Mamarias Animales/citología , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Transgénicos , Microinyecciones , Regiones Promotoras Genéticas/genética , Recombinación Genética , Glándulas Salivales/patología , Microambiente Tumoral/genética , Proteína Fluorescente Roja
20.
Am J Physiol Endocrinol Metab ; 315(5): E825-E832, 2018 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-29989850

RESUMEN

Brown adipose tissue (BAT) has the unique ability to convert energy stored in the form of triglycerides into heat. This property makes BAT a target tissue to increase energy expenditure and improve systemic metabolic control. TRPC1 is a founding member of the TRP protein family that also includes several temperature sensitive channels. We show that TRPC1 is highly expressed in all adipocyte depots including BAT and that Trpc1-deficient mice are prone to weight gain and manifest reduced metabolic control. We also demonstrate that knockdown of TRPC1 in cultured brown adipocytes leads to a downregulation of several metabolic genes, including UCP1 and PPARγ, as well as upregulation of a BAT-specific thermosensitive channel TRPV2, ultimately resulting in impaired respiratory function. We also show that TRPC1 is a possible target of PPARγ, suggesting that TRPC1 is a downstream component of a mechanism that translates metabolic or environmental stimuli into output in the form of BAT activity. Better understanding of the possible role of TRPC1 and other TRP channels in body temperature regulation and BAT function may help us to develop obesity therapies based on BAT activation.


Asunto(s)
Tejido Adiposo Pardo/metabolismo , Metabolismo Energético/genética , PPAR gamma/metabolismo , Canales Catiónicos TRPC/metabolismo , Aumento de Peso/genética , Animales , Canales de Calcio/genética , Canales de Calcio/metabolismo , Regulación hacia Abajo , Ratones , Ratones Noqueados , PPAR gamma/genética , Canales Catiónicos TRPC/genética , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/metabolismo , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismo , Regulación hacia Arriba
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