RESUMO
Recent large-scale collaborations are generating major surveys of cell types and connections in the mouse brain, collecting large amounts of data across modalities, spatial scales, and brain areas. Successful integration of these data requires a standard 3D reference atlas. Here, we present the Allen Mouse Brain Common Coordinate Framework (CCFv3) as such a resource. We constructed an average template brain at 10 µm voxel resolution by interpolating high resolution in-plane serial two-photon tomography images with 100 µm z-sampling from 1,675 young adult C57BL/6J mice. Then, using multimodal reference data, we parcellated the entire brain directly in 3D, labeling every voxel with a brain structure spanning 43 isocortical areas and their layers, 329 subcortical gray matter structures, 81 fiber tracts, and 8 ventricular structures. CCFv3 can be used to analyze, visualize, and integrate multimodal and multiscale datasets in 3D and is openly accessible (https://atlas.brain-map.org/).
Assuntos
Encéfalo/anatomia & histologia , Encéfalo/metabolismo , Encéfalo/fisiologia , Animais , Atlas como Assunto , Mapeamento Encefálico/métodos , Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Modern genetic approaches are powerful in providing access to diverse cell types in the brain and facilitating the study of their function. Here, we report a large set of driver and reporter transgenic mouse lines, including 23 new driver lines targeting a variety of cortical and subcortical cell populations and 26 new reporter lines expressing an array of molecular tools. In particular, we describe the TIGRE2.0 transgenic platform and introduce Cre-dependent reporter lines that enable optical physiology, optogenetics, and sparse labeling of genetically defined cell populations. TIGRE2.0 reporters broke the barrier in transgene expression level of single-copy targeted-insertion transgenesis in a wide range of neuronal types, along with additional advantage of a simplified breeding strategy compared to our first-generation TIGRE lines. These novel transgenic lines greatly expand the repertoire of high-precision genetic tools available to effectively identify, monitor, and manipulate distinct cell types in the mouse brain.
Assuntos
Encéfalo/metabolismo , Técnicas de Inativação de Genes/métodos , Genes Reporter , Animais , Encéfalo/citologia , Cálcio/metabolismo , Linhagem Celular , Hibridização in Situ Fluorescente , Luz , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Neurônios/metabolismo , Optogenética , RNA não Traduzido/genética , Transgenes/genéticaRESUMO
Visceral adipose tissue (VAT) hosts a population of regulatory T (Treg) cells, with a unique phenotype, that controls local and systemic inflammation and metabolism. Generation of a T cell receptor transgenic mouse line, wherein VAT Tregs are highly enriched, facilitated study of their provenance, dependencies, and activities. We definitively established a role for T cell receptor specificity, uncovered an unexpected function for the primordial Treg transcription-factor, Foxp3, evidenced a cell-intrinsic role for interleukin-33 receptor, and ordered these dependencies within a coherent scenario. Genesis of the VAT-Treg phenotype entailed a priming step in the spleen, permitting them to exit the lymphoid organs and surveil nonlymphoid tissues, and a final diversification process within VAT, in response to microenvironmental cues. Understanding the principles of tissue-Treg biology is a prerequisite for precision-targeting strategies.
Assuntos
Gordura Intra-Abdominal/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T Reguladores/metabolismo , Animais , Montagem e Desmontagem da Cromatina , Fatores de Transcrição Forkhead/metabolismo , Proteína 1 Semelhante a Receptor de Interleucina-1/genética , Proteína 1 Semelhante a Receptor de Interleucina-1/metabolismo , Gordura Intra-Abdominal/imunologia , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , PPAR gama/genética , PPAR gama/metabolismo , Fenótipo , RNA/química , RNA/isolamento & purificação , RNA/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Interleucina/metabolismo , Análise de Célula Única , Baço/imunologia , Baço/metabolismo , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia , TranscriptomaRESUMO
Neutralizing antibodies (NAbs) are effective in treating COVID-19, but the mechanism of immune protection is not fully understood. Here, we applied live bioluminescence imaging (BLI) to monitor the real-time effects of NAb treatment during prophylaxis and therapy of K18-hACE2 mice intranasally infected with SARS-CoV-2-nanoluciferase. Real-time imaging revealed that the virus spread sequentially from the nasal cavity to the lungs in mice and thereafter systemically to various organs including the brain, culminating in death. Highly potent NAbs from a COVID-19 convalescent subject prevented, and also effectively resolved, established infection when administered within three days. In addition to direct neutralization, depletion studies indicated that Fc effector interactions of NAbs with monocytes, neutrophils, and natural killer cells were required to effectively dampen inflammatory responses and limit immunopathology. Our study highlights that both Fab and Fc effector functions of NAbs are essential for optimal in vivo efficacy against SARS-CoV-2.
Assuntos
Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/metabolismo , Encéfalo/patologia , COVID-19/imunologia , Pulmão/patologia , SARS-CoV-2/fisiologia , Testículo/patologia , Enzima de Conversão de Angiotensina 2/genética , Animais , Anticorpos Neutralizantes/genética , Anticorpos Antivirais/genética , Encéfalo/virologia , COVID-19/terapia , Células Cultivadas , Modelos Animais de Doenças , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Luciferases/genética , Medições Luminescentes , Pulmão/virologia , Masculino , Camundongos , Camundongos Transgênicos , Testículo/virologiaRESUMO
Most animal genomes are diploid, and mammalian development depends on specific adaptations that have evolved secondary to diploidy. Genomic imprinting and dosage compensation restrict haploid development to early embryos. Recently, haploid mammalian development has been reinvestigated since the establishment of haploid embryonic stem cells (ESCs) from mouse embryos. Haploid cells possess one copy of each gene, facilitating the generation of loss-of-function mutations in a single step. Recessive mutations can then be assessed in forward genetic screens. Applications of haploid mammalian cell systems in screens have been illustrated in several recent publications. Haploid ESCs are characterized by a wide developmental potential and can contribute to chimeric embryos and mice. Different strategies for introducing genetic modifications from haploid ESCs into the mouse germline have been further developed. Haploid ESCs therefore introduce new possibilities in mammalian genetics and could offer an unprecedented tool for genome exploration in the future.
Assuntos
Células-Tronco Embrionárias/citologia , Haploidia , Animais , Blastocisto/citologia , Quimera , Transferência Embrionária , Desenvolvimento Embrionário , Genes Recessivos , Genes Reporter , Testes Genéticos/métodos , Impressão Genômica , Mutação em Linhagem Germinativa , Humanos , Camundongos , Camundongos Transgênicos , Neoplasias/genética , Partenogênese , Especificidade da Espécie , TransgenesRESUMO
Food intake and energy balance are tightly regulated by a group of hypothalamic arcuate neurons expressing the proopiomelanocortin (POMC) gene. In mammals, arcuate-specific POMC expression is driven by two cis-acting transcriptional enhancers known as nPE1 and nPE2. Because mutant mice lacking these two enhancers still showed hypothalamic Pomc mRNA, we searched for additional elements contributing to arcuate Pomc expression. By combining molecular evolution with reporter gene expression in transgenic zebrafish and mice, here, we identified a mammalian arcuate-specific Pomc enhancer that we named nPE3, carrying several binding sites also present in nPE1 and nPE2 for transcription factors known to activate neuronal Pomc expression, such as ISL1, NKX2.1, and ERα. We found that nPE3 originated in the lineage leading to placental mammals and remained under purifying selection in all mammalian orders, although it was lost in Simiiformes (monkeys, apes, and humans) following a unique segmental deletion event. Interestingly, ablation of nPE3 from the mouse genome led to a drastic reduction (>70%) in hypothalamic Pomc mRNA during development and only moderate (<33%) in adult mice. Comparison between double (nPE1 and nPE2) and triple (nPE1, nPE2, and nPE3) enhancer mutants revealed the relative contribution of nPE3 to hypothalamic Pomc expression and its importance in the control of food intake and adiposity in male and female mice. Altogether, these results demonstrate that nPE3 integrates a tripartite cluster of partially redundant enhancers that originated upon a triple convergent evolutionary process in mammals and that is critical for hypothalamic Pomc expression and body weight homeostasis.
Assuntos
Peso Corporal , Ingestão de Alimentos , Elementos Facilitadores Genéticos , Hipotálamo , Pró-Opiomelanocortina , Peixe-Zebra , Animais , Pró-Opiomelanocortina/metabolismo , Pró-Opiomelanocortina/genética , Camundongos , Hipotálamo/metabolismo , Ingestão de Alimentos/genética , Ingestão de Alimentos/fisiologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Feminino , Masculino , Camundongos Transgênicos , Humanos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Mamíferos/metabolismo , Mamíferos/genéticaRESUMO
Spermatogenesis begins when cell fate-committed prospermatogonia migrate to the basement membrane and initiate spermatogenesis in response to retinoic acid (RA) in the neonatal testis. The underlying cellular and molecular mechanisms in this process are not fully understood. Here, we report findings on the involvement of a cancer/testis antigen, PRAMEL1, in the initiation and maintenance of spermatogenesis. By analyzing mouse models with either global or conditional Pramel1 inactivation, we found that PRAMEL1 regulates the RA responsiveness of the subtypes of prospermatogonia in the neonatal testis, and affects their homing process during the initiation of spermatogenesis. Pramel1 deficiency led to increased fecundity in juvenile males and decreased fecundity in mature males. In addition, Pramel1 deficiency resulted in a regional Sertoli cell-only phenotype during the first round of spermatogenesis, which was rescued by administration of the RA inhibitor WIN18,446, suggesting that PRAMEL1 functions as an inhibitor of RA signaling in germ cells. Overall, our findings suggest that PRAMEL1 fine-tunes RA signaling, playing a crucial role in the proper establishment of the first and subsequent rounds of spermatogenesis.
Assuntos
Espermatogênese , Tretinoína , Masculino , Camundongos , Animais , Tretinoína/farmacologia , Tretinoína/metabolismo , Espermatogênese/genética , Espermatogônias/metabolismo , Testículo/metabolismo , Transdução de Sinais , Células de Sertoli/metabolismoRESUMO
Glutathione (GSH) is a highly abundant tripeptide thiol that performs diverse protective and biosynthetic functions in cells. While changes in GSH availability are associated with inborn errors of metabolism, cancer, and neurodegenerative disorders, studying the limiting role of GSH in physiology and disease has been challenging due to its tight regulation. To address this, we generated cell and mouse models that express a bifunctional glutathione-synthesizing enzyme from Streptococcus thermophilus (GshF), which possesses both glutamate-cysteine ligase and glutathione synthase activities. GshF expression allows efficient production of GSH in the cytosol and mitochondria and prevents cell death in response to GSH depletion, but not ferroptosis induction, indicating that GSH is not a limiting factor under lipid peroxidation. CRISPR screens using engineered enzymes further revealed genes required for cell proliferation under cellular and mitochondrial GSH depletion. Among these, we identified the glutamate-cysteine ligase modifier subunit, GCLM, as a requirement for cellular sensitivity to buthionine sulfoximine, a glutathione synthesis inhibitor. Finally, GshF expression in mice is embryonically lethal but sustains postnatal viability when restricted to adulthood. Overall, our work identifies a conditional mouse model to investigate the limiting role of GSH in physiology and disease.
Assuntos
Glutamato-Cisteína Ligase , Glutationa , Animais , Camundongos , Butionina Sulfoximina/farmacologia , Modelos Animais de Doenças , Glutamato-Cisteína Ligase/genética , Glutamato-Cisteína Ligase/metabolismo , Glutationa/metabolismo , Linhagem Celular Tumoral , HumanosRESUMO
OBJECTIVE: Mutations in BMPR2 (bone morphogenetic protein receptor 2) are associated with familial and sporadic pulmonary arterial hypertension (PAH). The functional and molecular link between loss of BMPR2 in pulmonary artery smooth muscle cells (PASMC) and PAH pathogenesis warrants further investigation, as most investigations focus on BMPR2 in pulmonary artery endothelial cells. Our goal was to determine whether and how decreased BMPR2 is related to the abnormal phenotype of PASMC in PAH. METHODS: SMC-specific Bmpr2-/- mice (BKOSMC) were created and compared to controls in room air, after 3 weeks of hypoxia as a second hit, and following 4 weeks of normoxic recovery. Echocardiography, right ventricular systolic pressure, and right ventricular hypertrophy were assessed as indices of pulmonary hypertension. Proliferation, contractility, gene and protein expression of PASMC from BKOSMC mice, human PASMC with BMPR2 reduced by small interference RNA, and PASMC from PAH patients with a BMPR2 mutation were compared to controls, to investigate the phenotype and underlying mechanism. RESULTS: BKOSMC mice showed reduced hypoxia-induced vasoconstriction and persistent pulmonary hypertension following recovery from hypoxia, associated with sustained muscularization of distal pulmonary arteries. PASMC from mutant compared to control mice displayed reduced contractility at baseline and in response to angiotensin II, increased proliferation and apoptosis resistance. Human PASMC with reduced BMPR2 by small interference RNA, and PASMC from PAH patients with a BMPR2 mutation showed a similar phenotype related to upregulation of pERK1/2 (phosphorylated extracellular signal related kinase 1/2)-pP38-pSMAD2/3 mediating elevation in ARRB2 (ß-arrestin2), pAKT (phosphorylated protein kinase B) inactivation of GSK3-beta, CTNNB1 (ß-catenin) nuclear translocation and reduction in RHOA (Ras homolog family member A) and RAC1 (Ras-related C3 botulinum toxin substrate 1). Decreasing ARRB2 in PASMC with reduced BMPR2 restored normal signaling, reversed impaired contractility and attenuated heightened proliferation and in mice with inducible loss of BMPR2 in SMC, decreasing ARRB2 prevented persistent pulmonary hypertension. CONCLUSIONS: Agents that neutralize the elevated ARRB2 resulting from loss of BMPR2 in PASMC could prevent or reverse the aberrant hypocontractile and hyperproliferative phenotype of these cells in PAH.
Assuntos
Hipertensão Pulmonar , Hipertensão Arterial Pulmonar , Animais , Humanos , Camundongos , beta-Arrestina 2/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Proliferação de Células , Células Cultivadas , Células Endoteliais/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Hipertensão Pulmonar/metabolismo , Hipóxia/complicações , Hipóxia/genética , Hipóxia/metabolismo , Miócitos de Músculo Liso/metabolismo , Hipertensão Arterial Pulmonar/genética , Artéria Pulmonar/metabolismo , RNA/metabolismoRESUMO
The CTNNB1 gene, encoding ß-catenin, is frequently mutated in hepatocellular carcinoma (HCC, â¼30%) and in hepatoblastoma (HB, >80%), in which DLK1/DIO3 locus induction is correlated with CTNNB1 mutations. Here, we aim to decipher how sustained ß-catenin activation regulates DLK1/DIO3 locus expression and the role this locus plays in HB and HCC development in mouse models deleted for Apc (ApcΔhep) or Ctnnb1-exon 3 (ß-cateninΔExon3) and in human CTNNB1-mutated hepatic cancer cells. We identified an enhancer site bound by TCF-4/ß-catenin complexes in an open conformation upon sustained ß-catenin activation (DLK1-Wnt responsive element [WRE]) and increasing DLK1/DIO3 locus transcription in ß-catenin-mutated human HB and mouse models. DLK1-WRE editing by CRISPR-Cas9 approach impaired DLK1/DIO3 locus expression and slowed tumor growth in subcutaneous CTNNB1-mutated tumor cell grafts, ApcΔhep HB and ß-cateninΔExon3 HCC. Tumor growth inhibition resulted either from increased FADD expression and subsequent caspase-3 cleavage in the first case or from decreased expression of cell cycle actors regulated by FoxM1 in the others. Therefore, the DLK1/DIO3 locus is an essential determinant of FoxM1-dependent cell proliferation during ß-catenin-driven liver tumorigenesis. Targeting the DLK1-WRE enhancer to silence the DLK1/DIO3 locus might thus represent an interesting therapeutic strategy to restrict tumor growth in primary liver cancers with CTNNB1 mutations.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Animais , Humanos , Camundongos , beta Catenina/genética , beta Catenina/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Carcinogênese/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Cateninas/genética , Cateninas/metabolismo , Proliferação de Células/genética , Neoplasias Hepáticas/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Regulação para CimaRESUMO
Brain-derived neurotrophic factor (BDNF) promotes neuronal survival and growth during development. In the adult nervous system, BDNF is important for synaptic function in several biological processes such as memory formation and food intake. In addition, BDNF has been implicated in development and maintenance of the cardiovascular system. The Bdnf gene comprises several alternative untranslated 5' exons and two variants of 3' UTRs. The effects of these entire alternative UTRs on translatability have not been established. Using reporter and translating ribosome affinity purification analyses, we show that prevalent Bdnf 5' UTRs, but not 3' UTRs, exert a repressive effect on translation. However, contrary to previous reports, we do not detect a significant effect of neuronal activity on BDNF translation. In vivo analysis via knock-in conditional replacement of Bdnf 3' UTR by bovine growth hormone 3' UTR reveals that Bdnf 3' UTR is required for efficient Bdnf mRNA and BDNF protein production in the brain, but acts in an inhibitory manner in lung and heart. Finally, we show that Bdnf mRNA is enriched in rat brain synaptoneurosomes, with higher enrichment detected for exon I-containing transcripts. In conclusion, these results uncover two novel aspects in understanding the function of Bdnf UTRs. First, the long Bdnf 3' UTR does not repress BDNF expression in the brain. Second, exon I-derived 5' UTR has a distinct role in subcellular targeting of Bdnf mRNA.
Assuntos
Fator Neurotrófico Derivado do Encéfalo , RNA Mensageiro , Regiões não Traduzidas , Animais , Bovinos , Ratos , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Éxons , Neurônios/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regiões não Traduzidas/fisiologiaRESUMO
Plasma thymidine levels in rodents are higher than in other mammals including humans, possibly due to a different pattern and lower level of thymidine phosphorylase expression. Here, we generated a novel knock-in (KI) mouse line with high systemic expression of human thymidine phosphorylase to investigate this difference in nucleotide metabolism in rodents. The KI mice showed growth retardation around weaning and died by 4 weeks of age with a decrease in plasma thymidine level compared with the litter-control WT mice. These phenotypes were completely or partially rescued by administration of the thymidine phosphorylase inhibitor 5-chloro-6-(2-iminopyrrolidin-1-yl) methyl-2,4(1H,3H)-pyrimidinedione hydrochloride or thymidine, respectively. Interestingly, when thymidine phosphorylase inhibitor administration was discontinued in adult animals, KI mice showed deteriorated grip strength and locomotor activity, decreased bodyweight, and subsequent hind-limb paralysis. Upon histological analyses, we observed axonal degeneration in the spinal cord, muscular atrophy with morphologically abnormal mitochondria in quadriceps, retinal degeneration, and abnormality in the exocrine pancreas. Moreover, we detected mitochondrial DNA depletion in multiple tissues of KI mice. These results indicate that the KI mouse represents a new animal model for mitochondrial diseases and should be applicable for the study of differences in nucleotide metabolism between humans and mice.
Assuntos
Encefalomiopatias Mitocondriais , Miopatias Mitocondriais , Animais , Humanos , Camundongos , DNA Mitocondrial/metabolismo , Transtornos do Crescimento/genética , Mamíferos/metabolismo , Encefalomiopatias Mitocondriais/genética , Encefalomiopatias Mitocondriais/patologia , Nucleotídeos , Timidina , Timidina Fosforilase/genética , Timidina Fosforilase/metabolismoRESUMO
In Huntington disease (HD), the mutant huntingtin (mtHTT) protein is the principal cause of pathological changes that initiate primarily along the cortico-striatal axis. mtHTT is ubiquitously expressed and there is, accordingly, growing recognition that HD is a systemic disorder with functional interplay between the brain and the periphery. We have developed a monoclonal antibody, C6-17, targeting an exposed region of HTT near the aa586 Caspase 6 cleavage site. As recently published, mAB C6-17 can block cell-to-cell propagation of mtHTT in vitro. In order to reduce the burden of the mutant protein in vivo, we queried whether extracellular mtHTT could be therapeutically targeted in YAC128 HD mice. In a series of proof of concept experiments, we found that systemic mAB C6-17 treatment resulted in the distribution of the mAB C6-17 to peripheral and CNS tissues and led to the reduction of HTT protein levels. Compared to CTRL mAB or vehicle treated mice, the mAB C6-17 treated YAC128 animals showed improved body weight and motor behaviors, a delayed progression in motor deficits and reduced striatal EM48 immunoreactivity. These results provide the first proof of concept for the feasibility and therapeutic efficacy of an antibody-based anti-HTT passive immunization approach and suggest this modality as a potential new HD treatment strategy.
Assuntos
Doença de Huntington , Camundongos , Animais , Doença de Huntington/metabolismo , Camundongos Transgênicos , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Proteína Huntingtina/genética , Imunoterapia , Modelos Animais de Doenças , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Progressão da DoençaRESUMO
Retinitis pigmentosa (RP) is a group of progressive retinal degenerations of mostly monogenic inheritance, which cause blindness in about 1:3,500 individuals worldwide. Heterozygous variants in the rhodopsin (RHO) gene are the most common cause of autosomal dominant RP (adRP). Among these, missense variants at C-terminal proline 347, such as p.Pro347Ser, cause severe adRP recurrently in European affected individuals. Here, for the first time, we use CRISPR/Cas9 to selectively target the p.Pro347Ser variant while preserving the wild-type RHO allele in vitro and in a mouse model of adRP. Detailed in vitro, genomic, and biochemical characterization of the rhodopsin C-terminal editing demonstrates a safe downregulation of p.Pro347Ser expression leading to partial recovery of photoreceptor function in a transgenic mouse model treated with adeno-associated viral vectors. This study supports the safety and efficacy of CRISPR/Cas9-mediated allele-specific editing and paves the way for a permanent and precise correction of heterozygous variants in dominantly inherited retinal diseases.
Assuntos
Edição de Genes , Retinose Pigmentar/genética , Retinose Pigmentar/terapia , Rodopsina/genética , Alelos , Animais , Sistemas CRISPR-Cas , Linhagem Celular , Dependovirus/genética , Modelos Animais de Doenças , Eletrorretinografia , Terapia Genética , Humanos , Mutação INDEL , Camundongos , Camundongos Transgênicos , Mutação de Sentido Incorreto , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/metabolismo , Retina/fisiopatologia , Rodopsina/metabolismoRESUMO
The effect of triiodothyronine (T3) on the phosphorylation of ERK and the occurrence and development of hepatocellular carcinoma (HCC) is controversial and remains to be clarified. In the present study, both in vitro (hepatoma cell lines) and in vivo (wild-type mice [WT] and mouse models of HCC [HrasG12Vand KrasG12Dtransgenic mice (Hras-Tg and Kras-Tg)]) systems were used to investigate the effect of T3 on p-ERK and hepatocarcinogenesis. The results showed that, in vitro, T3 treatment elevated the levels of p-ERK in hepatoma cells within 30 min. However, p-ERK levels returned to normal after 1 h with no significant effects on cellular proliferation or apoptosis. Interestingly, in vivo, T3 induced early rapid and transient activation of ERK and later persistent downregulation of p-ERK in liver tissues of WT. In Hras-Tg, liver weight, liver/body weight ratio, hepatic tumor numbers and sizes were significantly reduced withT3treatment compared with the untreated group. Furthermore, the levels of albumin, HrasG12V, and p-ERK in hepatic precancerous and tumor tissues were all significantly downregulated with T3 treatment; however, the levels of endogenous Hras were not affected. In WT, T3 also induced downregulation of Albumin in liver tissues, but without influence on the expression of endogenous Hras and p-MEK. Especially, the inhibitory effect of T3 on p-ERK and hepatic tumorigenesis and development without influence on the levels of KrasG12D and p-MEK was further confirmed in Kras-Tg. In conclusion, T3 suppresses hepatic tumorigenesis and development by independently and substantially inhibiting the phosphorylation of ERK in vivo.
RESUMO
AIM: CH1641 was discovered in 1970 as a scrapie isolate that was unlike all other classical strains of scrapie isolated so far. We performed bio-assays of CH1641 in mice in order to further characterise this specific isolate. METHODS: We inoculated the original CH1641 isolate into ovine and bovine prion protein (PrP) transgenic mice as well as wild-type mice. In addition, we performed cross- and back passages between the various mouse lines to examine if one identical prion strain was isolated in all mouse lines or whether multiple prion strains exist in CH1641. RESULTS: We report the first successful transmission of CH1641 to wild-type RIII mice and via RIII mice to wild-type VM mice. Unexpectedly, analysis of the protease-resistant prion protein (PrPres ) in wild-type mice showed a classical scrapie banding pattern differing from the banding pattern of the original CH1641 isolate. Cross- and back passages of CH1641 between the various mouse lines confirmed that the same prion strain had been isolated in all mouse lines. CONCLUSIONS: The CH1641 isolate consists of a single prion strain but its molecular banding pattern of PrPres differs between wild-type mice and PrP transgenic mice. Consequently, molecular banding patterns of PrPres should be used with caution in strain typing since they do not solely depend on the properties of the prion strain but also on the host prion protein.
Assuntos
Príons , Scrapie , Camundongos , Animais , Bovinos , Ovinos , Príons/metabolismo , Scrapie/metabolismo , Proteínas Priônicas/genética , Proteínas PrPSc/metabolismo , Camundongos TransgênicosRESUMO
In female mammals, the oviduct and uterus are essential sites for female and male gamete transport, fertilization, implantation, and maintenance of a successful pregnancy. To delineate the reproductive function of Mothers against decapentaplegic homolog 4 (Smad4), we specifically inactivated Smad4 in ovarian granulosa cells and, oviduct and uterine mesenchymal cells using the Amhr2-cre mouse line. Deletion of exon 8 of Smad4 results in the production of an MH2-truncated SMAD4 protein. These mutant mice are infertile due to the development of oviductal diverticula and defects during the implantation process. The ovaries are fully functional as demonstrated in an ovary transfer experiment. The development of oviductal diverticula occurs shortly after puberty and is dependent on estradiol. The diverticula interfere with sperm migration and embryo transit to the uterus, reducing the number of implantation sites. Analysis of the uterus shows that, even if implantation occurs, decidualization and vascularization are defective resulting in embryo resorption as early as the seventh day of pregnancy. Thus, Smad4 plays an important function in female reproduction by controlling the structural and functional integrity of the oviduct and uterus.
Assuntos
Estradiol , Proteína Smad4 , Animais , Feminino , Humanos , Masculino , Camundongos , Gravidez , Implantação do Embrião , Estradiol/metabolismo , Mamíferos/metabolismo , Oviductos/metabolismo , Sêmen/metabolismo , Proteína Smad4/genética , Proteína Smad4/metabolismo , Útero/metabolismoRESUMO
Calcium modulates bone cell recruitment, differentiation, and function by binding to the calcium-sensing receptor (CaSR). However, the function of CaSR induced by high extracellular calcium (Ca2+ e ) in the regulation of osteoclast formation in rheumatoid arthritis (RA) remains unknown. Here, we used TNFα-transgenic (TNFTG ) RA mice and their wildtype (WT) littermates fed a normal or a rescue diet (high calcium, high phosphorus, and high lactose diet, termed rescue diet) to compare their joint bone phenotypes. In comparison to TNFTG mice fed the normal diet, articular bone volume and cartilage area are increased, whereas inflamed area, eroded surface, TRAP+ surface, and osteoclast-related genes expression are decreased in TNFTG mice fed the rescue diet. Besides, TNFTG mice fed the rescue diet were found to exhibit more CaSR+ area and less NFATc1+ /TRAP+ area. Furthermore, at normal Ca2+ e concentrations, osteoclast precursors (OCPs) from TNFTG mice formed more osteoclasts than OCPs from WT mice, but the number of osteoclasts gradually decreased when the Ca2+ e concentration increased. Meanwhile, the expression of CaSR increased responding to a high level of Ca2+ e , whereas the expression of NF-κB/NFATc1 signaling molecules decreased. At last, the knockdown of CaSR blocked the inhibition of osteoclast differentiation attributed to high Ca2+ e . Taken together, our findings indicate that high Ca2+ e inhibits osteoclast differentiation in RA mice partially through the CaSR/NF-κB/NFATc1 pathway.
Assuntos
Artrite Reumatoide , Receptores de Detecção de Cálcio , Camundongos , Animais , Receptores de Detecção de Cálcio/genética , Cálcio , Osteogênese , NF-kappa B , DietaRESUMO
Nr1i2, a nuclear receptor known for its key function in xenobiotic detoxification, has emerged as a potential regulator of intestinal homeostasis and inflammation. However, the role of Nr1i2 in different intestinal segments remains poorly known. Moreover, in vivo investigations on intestinal Nr1i2 have essentially been performed in whole-body Nr1i2 knockout (Nr1i2-/- ) mice where the deletion of Nr1i2 in all tissues may affect the intestinal phenotype. To better understand the role of Nr1i2 in the intestine, we generated intestinal epithelial-specific Nr1i2 knockout (iNr1i2-/- ) mice and studied the duodenum, jejunum, ileum, and colon of these animals during steady-state conditions and lipopolysaccharide (LPS)-induced inflammation. As compared to control (iNr1i2+/+ ) mice, iNr1i2-/- mice showed normal intestinal permeability as assessed by in vivo FITC-dextran test. The expression of genes involved in inflammation, tight- and adherens-junction, proliferation, glucose, and lipid metabolism was comparable in the duodenum, jejunum, ileum, and colon of iNr1i2-/- and iNr1i2+/+ mice. In line with these findings, histological analyses of the jejunum revealed no difference between iNr1i2-/- and iNr1i2+/+ mice. When treated with LPS, the intestine of iNr1i2-/- mice had no increased inflammatory response as compared to iNr1i2+/+ mice. Moreover, the health monitoring of LPS-treated iNr1i2-/- and iNr1i2+/+ mice was similar. Taken together, our results demonstrate that the specific deletion of Nr1i2 in the intestinal epithelium does not cause major intestinal damages in mice during both steady-state and inflammatory conditions.
Assuntos
Hipersensibilidade , Animais , Camundongos , Lipopolissacarídeos , Intestinos , HomeostaseRESUMO
Spinocerebellar ataxia type 2 (SCA2) is a hereditary disorder, caused by an expansion of polyglutamine in the ataxin-2 protein. Although the mutant protein is expressed throughout all the cell and organ types, the cerebellum is primarily affected. The disease progression is mainly accompanied by a decline in motor functions. However, the disturbances in cognitive abilities and low mental state have also been reported in patients. Recent evidence suggests that the cerebellar functionality expands beyond the motor control. Thus, the cerebellum turned out to be involved into the language, verbal working, and spatial memory; executive functions such as working memory, planning, organizing, and strategy formation; and emotional processing. Here, we used the transgenic SCA2-58Q mice to evaluate their anxiety, cognitive functions, and mood alterations. The expression of the mutant ataxin-2 specifically in the cerebellar Purkinje cells (PCs) in SCA2-58Q mice allowed us to study the direct involvement of the cerebellum into the cognitive and affective control. We determined that SCA2-58Q mice exhibit anxiolytic behavior, decline in spatial memory, and a depressive-like state. Our results support the idea of cerebellar involvement in cognitive control and the handling of emotions.