RESUMO
Glycosylphosphatidylinositol (GPI) is a posttranslational glycolipid modification of proteins that anchors proteins in lipid rafts on the cell surface. Although some GPI-anchored proteins (GPI-APs), including the prion protein PrPC, have a glycan side chain composed of N-acetylgalactosamine (GalNAc)-galactose-sialic acid on the core structure of GPI glycolipid, in vivo functions of this GPI-GalNAc side chain are largely unresolved. Here, we investigated the physiological and pathological roles of the GPI-GalNAc side chain in vivo by knocking out its initiation enzyme, PGAP4, in mice. We show that Pgap4 mRNA is highly expressed in the brain, particularly in neurons, and mass spectrometry analysis confirmed the loss of the GalNAc side chain in PrPC GPI in PGAP4-KO mouse brains. Furthermore, PGAP4-KO mice exhibited various phenotypes, including an elevated blood alkaline phosphatase level, impaired bone formation, decreased locomotor activity, and impaired memory, despite normal expression levels and lipid raft association of various GPI-APs. Thus, we conclude that the GPI-GalNAc side chain is required for in vivo functions of GPI-APs in mammals, especially in bone and the brain. Moreover, PGAP4-KO mice were more vulnerable to prion diseases and died earlier after intracerebral inoculation of the pathogenic prion strains than wildtype mice, highlighting the protective roles of the GalNAc side chain against prion diseases.
Assuntos
Acetilgalactosamina , Glicosilfosfatidilinositóis , Doenças Priônicas , Príons , Acetilgalactosamina/química , Acetilgalactosamina/metabolismo , Animais , Encéfalo/metabolismo , Glicosilfosfatidilinositóis/química , Glicosilfosfatidilinositóis/metabolismo , Camundongos , Osteogênese , Doenças Priônicas/metabolismo , Príons/metabolismo , Relação Estrutura-AtividadeRESUMO
CaMKII is a pivotal kinase that plays essential roles in synaptic plasticity. Apart from its signaling function, the structural function of CaMKII is becoming clear. CaMKII - F-actin interaction stabilizes actin cytoskeleton in a dendritic spine. A transient autophosphorylation at the F-actin binding region during LTP releases CaMKII from F-actin and opens a brief time-window of actin reorganization. However, the physiological relevance of this finding in learning and memory was not presented. Using a knock-in (KI) mouse carrying phosphoblock mutations in the actin-binding domain of CaMKIIß, we demonstrate that proper regulation of CaMKII - F-actin interaction is important for fear conditioning memory tasks. The KI mice show poor performance in contextual and cued versions of fear conditioning test. These results suggest the importance of CaMKII - F-actin interactions in learning and memory.
Assuntos
Actinas/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Condicionamento Clássico/fisiologia , Medo/fisiologia , Actinas/genética , Animais , Feminino , Técnicas de Introdução de Genes , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , FosforilaçãoRESUMO
Gene targeting of mouse Sushi-ichi-related retrotransposon homologue 11/Zinc finger CCHC domain-containing 16 (Sirh11/Zcchc16) causes abnormal behaviors related to cognition, including attention, impulsivity and working memory. Sirh11/Zcchc16 encodes a CCHC type of zinc-finger protein that exhibits high homology to an LTR retrotransposon Gag protein. Upon microdialysis analysis of the prefrontal cortex region, the recovery rate of noradrenaline (NA) was reduced compared with dopamine (DA) after perfusion of high potassium-containing artificial cerebrospinal fluid in knockout (KO) mice. These data indicate that Sirh11/Zcchc16 is involved in cognitive function in the brain, possibly via the noradrenergic system, in the contemporary mouse developmental systems. Interestingly, it is highly conserved in three out of the four major groups of the eutherians, euarchontoglires, laurasiatheria and afrotheria, but is heavily mutated in xenarthran species such as the sloth and armadillo, suggesting that it has contributed to brain evolution in the three major eutherian lineages, including humans and mice. Sirh11/Zcchc16 is the first SIRH gene to be involved in brain function, instead of just the placenta, as seen in the case of Peg10, Peg11/Rtl1 and Sirh7/Ldoc1.
Assuntos
Cognição , Mamíferos/genética , Retroelementos , Sequências Repetidas Terminais , Animais , Comportamento Animal , Feminino , Crescimento/genética , Humanos , Masculino , Camundongos , Camundongos Knockout , Norepinefrina/metabolismo , Córtex Pré-Frontal/metabolismoRESUMO
Sirh7/Ldoc1 [sushi-ichi retrotransposon homolog 7/leucine zipper, downregulated in cancer 1, also called mammalian retrotransposon-derived 7 (Mart7)] is one of the newly acquired genes from LTR retrotransposons in eutherian mammals. Interestingly, Sirh7/Ldoc1 knockout (KO) mice exhibited abnormal placental cell differentiation/maturation, leading to an overproduction of placental progesterone (P4) and placental lactogen 1 (PL1) from trophoblast giant cells (TGCs). The placenta is an organ that is essential for mammalian viviparity and plays a major endocrinological role during pregnancy in addition to providing nutrients and oxygen to the fetus. P4 is an essential hormone in the preparation and maintenance of pregnancy and the determination of the timing of parturition in mammals; however, the biological significance of placental P4 in rodents is not properly recognized. Here, we demonstrate that mouse placentas do produce P4 in mid-gestation, coincident with a temporal reduction in ovarian P4, suggesting that it plays a role in the protection of the conceptuses specifically in this period. Pregnant Sirh7/Ldoc1 knockout females also displayed delayed parturition associated with a low pup weaning rate. All these results suggest that Sirh7/Ldoc1 has undergone positive selection during eutherian evolution as a eutherian-specific acquired gene because it impacts reproductive fitness via the regulation of placental endocrine function.
Assuntos
Parto/metabolismo , Placenta/metabolismo , Lactogênio Placentário/metabolismo , Progesterona/metabolismo , Animais , Primers do DNA/genética , Feminino , Genótipo , Hibridização In Situ , Camundongos , Camundongos Knockout , Mifepristona , Reação em Cadeia da Polimerase , Gravidez , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
We have developed an open-source database system named "Pheno-Pub" to support a series of data-handling and publication tasks, including statistical analyses, data review, and web site construction, for mouse phenotyping experiments. This system is composed of three applications. "Mou-Stat" provides semiautomatic statistical analyses for a batch of phenotypic data, including a variety of conditions for group comparisons (e.g., different scales of measurement parameters). "Genotype Viewer" and "Strain Viewer" provide representation of genotype-driven and measurement parameter-driven views of phenotypic data; they highlight significant differences in genotypes and between strains, respectively. Direct links from the Strain Viewer web site to the Genotype Viewer web site provide flexible navigation in the exploration of phenotypic data. With these publication tools, phenotypic data can be made available on the Internet by simple operations. This system is expandable for a wide range of uses in phenotypic comparative analyses, including comparisons among different genotypes and strains and comparisons among groups exposed to different environmental conditions. Finally, Pheno-Pub provides advanced usability for both producers of experimental data and consumers of phenotypic information. Therefore, Pheno-Pub contributes significantly to the publication of data in various fields of phenotyping research and to broad data sharing, thereby promoting the understanding of the functions of the entire mouse genome.
Assuntos
Bases de Dados Factuais , Camundongos/genética , Software , Animais , Genótipo , Internet , Camundongos/classificação , FenótipoRESUMO
DExH-box helicases are involved in unwinding of RNA and DNA. Among the 16 DExH-box genes, monoallelic variants of DHX16, DHX30, DHX34, and DHX37 are known to be associated with neurodevelopmental disorders. In particular, DHX30 is well established as a causative gene for neurodevelopmental disorders. Germline variants of DHX9, the closest homolog of DHX30, have not been reported until now as being associated with congenital disorders in humans, except that one de novo heterozygous variant, p.(Arg1052Gln) of the gene was identified during comprehensive screening in a patient with autism; unfortunately, the phenotypic details of this individual are unknown. Herein, we report a patients with a heterozygous de novo missense variant, p.(Gly414Arg) of DHX9 who presented with a short stature, intellectual disability, and ventricular non-compaction cardiomyopathy. The variant was located in the glycine codon of the ATP-binding site, G-C-G-K-T. To assess the pathogenicity of these variants, we generated transgenic Drosophila lines expressing human wild-type and mutant DHX9 proteins: 1) the mutant proteins showed aberrant localization both in the nucleus and the cytoplasm; 2) ectopic expression of wild-type protein in the visual system led to the rough eye phenotype, whereas expression of the mutant proteins had minimal effect; 3) overexpression of the wild-type protein in the retina led to a reduction in axonal numbers, whereas expression of the mutant proteins had a less pronounced effect. Furthermore, in a gene-editing experiment of Dhx9 G416 to R416, corresponding to p.(Gly414Arg) in humans, heterozygous mice showed a reduced body size, reduced emotionality, and cardiac conduction abnormality. In conclusion, we established that heterozygosity for a loss-of-function variant of DHX9 can lead to a new neurodevelopmental disorder.
Assuntos
Deficiência Intelectual , Transtornos do Neurodesenvolvimento , Animais , Humanos , Camundongos , RNA Helicases DEAD-box/genética , Genética Humana , Deficiência Intelectual/genética , Proteínas de Neoplasias/genética , Transtornos do Neurodesenvolvimento/genética , RNA/genética , RNA HelicasesRESUMO
Zinc plays a critical role in many physiological processes, and disruption of zinc homeostasis induces various disorders, such as growth retardation, osteopenia, immune deficiency, and inflammation. However, how the imbalance in zinc homeostasis leads to heart disease is not yet fully understood. Cardiovascular diseases are a major cause of death worldwide, and the development of novel therapeutic targets to treat it is urgently needed. We report that a zinc transporter, ZIP13, regulates cardiovascular homeostasis. We found that the expression level of Zip13 mRNA was diminished in both primary neonatal cardiomyocytes and mouse heart tissues treated with the cardiotoxic agent doxycycline. Primary neonatal cardiomyocytes from Zip13 gene-knockout (KO) mice exhibited abnormal irregular arrhythmic beating. RNA-seq analysis identified 606 differentially expressed genes in Zip13-KO mouse-derived primary neonatal cardiomyocytes and Gene ontology (GO) analysis revealed that both inflammation- and cell adhesion-related genes were significantly enriched. In addition, telemetry echocardiography analysis suggested that arrhythmias were likely to occur in Zip13-KO mice, in which elevated levels of the cardiac fibrosis marker Col1a1, vascular inflammation-related gene eNOS, and Golgi-related molecule GM130 were observed. These results indicate the physiological importance of ZIP13-it maintains cardiovascular homeostasis by resolving inflammation and stress response. Our findings suggest that optimizing ZIP13 expression and/or function may improve cardiovascular disease management.
Assuntos
Proteínas de Transporte de Cátions , Síndrome de Ehlers-Danlos , Camundongos , Animais , Proteínas de Transporte de Cátions/genética , Síndrome de Ehlers-Danlos/genética , Cardiotoxinas , Doxiciclina , Camundongos Knockout , Zinco/metabolismo , Homeostase , Inflamação , RNA MensageiroRESUMO
Protocadherin 9 (Pcdh9) is a member of the cadherin superfamily and is uniquely expressed in the vestibular and limbic systems; however, its physiological role remains unclear. Here, we studied the expression of Pcdh9 in the limbic system and phenotypes of Pcdh9-knock-out mice (Pcdh9 KO mice). Pcdh9 mRNA was expressed in the fear extinction neurons that express protein phosphatase 1 regulatory subunit 1 B (Ppp1r1b) in the posterior part of the basolateral amygdala (pBLA), as well as in the Cornu Ammonis (CA) and Dentate Gyrus (DG) neurons of the hippocampus. We show that the Pcdh9 protein was often localised at synapses. Phenotypic analysis of Pcdh9 KO mice revealed no apparent morphological abnormalities in the pBLA but a decrease in the spine number of CA neurons. Further, the Pcdh9 KO mice were related to features such as the abnormal optokinetic response, less approach to novel objects, and reduced fear extinction during recovery from the fear. These results suggest that Pcdh9 is involved in eliciting positive emotional behaviours, possibly via fear extinction neurons in the pBLA and/or synaptic activity in the hippocampal neurons, and normal optokinetic eye movement in brainstem optokinetic system-related neurons.
Assuntos
Extinção Psicológica , Medo , Animais , Camundongos , Extinção Psicológica/fisiologia , Medo/fisiologia , Hipocampo , Neurônios , ProtocaderinasRESUMO
UNLABELLED: This article reports the development of SDOP-DB, which can provide definite, detailed and easy comparison of experimental protocols used in mouse phenotypic analyses among institutes or laboratories. Because SDOP-DB is fully compliant with international standards, it can act as a practical foundation for international sharing and integration of mouse phenotypic information. AVAILABILITY: SDOP-DB (http://www.brc.riken.jp/lab/bpmp/SDOP/).
Assuntos
Bases de Dados Factuais , Genômica/métodos , Camundongos , Fenótipo , Software , Animais , Internet , Interface Usuário-ComputadorRESUMO
In the RIKEN large-scale N-ethyl-N-nitrosourea (ENU) mutagenesis project we screened mice with a dominant mutation that exhibited abnormal behavior in the open-field test, passive avoidance test and home-cage activity test. We tested 2045 progeny of C57BL/6J males treated with ENU and untreated DBA/2J females in the open-field test and isolated behavioral mutant M100174, which exhibited a significant increase in spontaneous locomotor activity. We identified a missense mutation in the Grin1 gene, which encodes NMDA receptor subunit 1, and designated the mutant gene Grin1(Rgsc174). This mutation results in an arginine to cysteine substitution in the C0 domain of the protein. Detailed analyses revealed that Grin1(Rgsc174) heterozygote exhibited increased novelty-seeking behavior and slight social isolation in comparison with the wild type. In contrast to other Grin1 mutant mice, this mutant exhibited no evidence of heightened anxiety. These results indicate that this is a unique behavioral Grin1 gene mutant mouse that differs from the known Grin1 mutant mice. The results of immunohistochemical and biochemical analyses suggested that impaired interaction between the glutamatergic pathway and dopaminergic pathway may underlie the behavioral phenotypes of the Grin1(Rgsc174) mutant.
Assuntos
Alquilantes/farmacologia , Proteínas de Transporte/genética , Etilnitrosoureia/farmacologia , Mutagênese/efeitos dos fármacos , Proteínas do Tecido Nervoso/genética , Fenótipo , Sequência de Aminoácidos , Análise de Variância , Animais , Arginina/genética , Cálcio/metabolismo , Células Cultivadas , Estimulantes do Sistema Nervoso Central/farmacologia , Córtex Cerebral/citologia , Mapeamento Cromossômico/métodos , Cisteína/genética , Embrião de Mamíferos , Comportamento Exploratório/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Metilfenidato/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Transgênicos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Atividade Motora/efeitos dos fármacos , Mutação de Sentido Incorreto , N-Metilaspartato/farmacologia , Neurônios , Fenazinas/farmacologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
Pogo transposable element derived with ZNF domain (POGZ) has been identified as one of the most recurrently de novo mutated genes in patients with neurodevelopmental disorders (NDDs), including autism spectrum disorder (ASD), intellectual disability and White-Sutton syndrome; however, the neurobiological basis behind these disorders remains unknown. Here, we show that POGZ regulates neuronal development and that ASD-related de novo mutations impair neuronal development in the developing mouse brain and induced pluripotent cell lines from an ASD patient. We also develop the first mouse model heterozygous for a de novo POGZ mutation identified in a patient with ASD, and we identify ASD-like abnormalities in the mice. Importantly, social deficits can be treated by compensatory inhibition of elevated cell excitability in the mice. Our results provide insight into how de novo mutations on high-confidence ASD genes lead to impaired mature cortical network function, which underlies the cellular pathogenesis of NDDs, including ASD.
Assuntos
Transtorno Autístico/genética , Predisposição Genética para Doença/genética , Malformações do Desenvolvimento Cortical/genética , Mutação , Fenótipo , Transposases/genética , Adolescente , Animais , Comportamento Animal , Encéfalo/patologia , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Feminino , Edição de Genes , Técnicas de Silenciamento de Genes , Heterozigoto , Humanos , Deficiência Intelectual , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Transtornos do Neurodesenvolvimento/genética , Neurogênese , Neurônios/metabolismoRESUMO
Dysfunction of glucose transporter 1 (GLUT1) proteins causes infantile epilepsy, which is designated as a GLUT1 deficiency syndrome (GLUT1DS; OMIM #606777). Patients with GLUT1DS display varied clinical phenotypes, such as infantile seizures, ataxia, severe mental retardation with learning disabilities, delayed development, hypoglycorrhachia, and other varied symptoms. Glut1Rgsc200 mutant mice mutagenized with N-ethyl-N-nitrosourea (ENU) carry a missense mutation in the Glut1 gene that results in amino acid substitution at the 324th residue of the GLUT1 protein. In this study, these mutants exhibited various phenotypes, including embryonic lethality of homozygotes, a decreased cerebrospinal-fluid glucose value, deficits in contextual learning, a reduction in body size, seizure-like behavior and abnormal electroencephalogram (EEG) patterns. During EEG recording, the abnormality occurred spontaneously, whereas the seizure-like phenotypes were not observed at the same time. In sleep-wake analysis using EEG recording, heterozygotes exhibited a longer duration of wake times and shorter duration of non-rapid eye movement (NREM) sleep time. The shortened period of NREM sleep and prolonged duration of the wake period may resemble the sleep disturbances commonly observed in patients with GLUT1DS and other epilepsy disorders. Interestingly, an in vivo kinetic analysis of glucose utilization by positron emission tomography with 2-deoxy-2-[fluorine-18]fluoro-D-glucose imaging revealed that glucose transportation was reduced, whereas hexokinase activity and glucose metabolism were enhanced. These results indicate that a Glut1Rgsc200 mutant is a useful tool for elucidating the molecular mechanisms of GLUT1DS.This article has an associated First Person interview with the joint first authors of the paper.
Assuntos
Encéfalo/metabolismo , Erros Inatos do Metabolismo dos Carboidratos/metabolismo , Erros Inatos do Metabolismo dos Carboidratos/fisiopatologia , Glucose/metabolismo , Proteínas de Transporte de Monossacarídeos/deficiência , Sono/fisiologia , Vigília/fisiologia , Animais , Aprendizagem da Esquiva , Comportamento Animal , Peso Corporal , Encéfalo/patologia , Erros Inatos do Metabolismo dos Carboidratos/genética , Modelos Animais de Doenças , Eletroencefalografia , Perda do Embrião/genética , Perda do Embrião/patologia , Glucose/líquido cefalorraquidiano , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Heterozigoto , Homozigoto , Cinética , Aprendizagem , Camundongos Mutantes , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Atividade Motora , Mutação de Sentido Incorreto/genética , Convulsões/genética , Convulsões/patologia , Convulsões/fisiopatologia , Transcrição GênicaRESUMO
METTL20 is a seven-ß-strand methyltransferase that is localised to the mitochondria and tri-methylates the electron transfer flavoprotein (ETF) ß subunit (ETFB) at lysines 200 and 203. It has been shown that METTL20 decreases the ability of ETF to extract electrons from medium-chain acyl-coenzyme A (CoA) dehydrogenase (MCAD) and glutaryl-CoA dehydrogenase in vitro. METTL20-mediated methylation of ETFB influences the oxygen consumption rate in permeabilised mitochondria, suggesting that METTL20-mediated ETFB methylation may also play a regulatory role in mitochondrial metabolism. In this study, we generated Mettl20 knockout (KO) mice to uncover the in vivo functions of METTL20. The KO mice were viable, and a loss of ETFB methylation was confirmed. In vitro enzymatic assays revealed that mitochondrial ETF activity was higher in the KO mice than in wild-type mice, suggesting that the KO mice had higher ß-oxidation capacity. Calorimetric analysis showed that the KO mice fed a ketogenic diet had higher oxygen consumption and heat production. A subsequent cold tolerance test conducted after 24 h of fasting indicated that the KO mice had a better ability to maintain their body temperature in cold environments. Thus, METTL20 regulates ETF activity and heat production through lysine methylation when ß-oxidation is highly activated.
Assuntos
Jejum/metabolismo , Corpos Cetônicos/metabolismo , Metiltransferases/metabolismo , Oxirredução , Termogênese , Animais , Sistemas CRISPR-Cas , Catálise , Flavoproteínas Transferidoras de Elétrons/metabolismo , Ácidos Graxos/metabolismo , Edição de Genes , Humanos , Mutação com Perda de Função , Lisina/metabolismo , Metabolômica/métodos , Metilação , Metiltransferases/genética , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Consumo de Oxigênio , Especificidade por SubstratoRESUMO
We have assessed the frequency of alpha-synuclein (SNCA) mutations in Japanese patients with familial or sporadic Parkinson's disease (PD) and surveyed their associated clinical manifestations. We screened SNCA exon 3 in 988 patients without SNCA multiplications (430 with autosomal dominant PD and 558 with sporadic PD). We detected 1 patient harboring a homozygous SNCA p.A53V substitution albeit with an autosomal dominant pattern of disease inheritance (frequency 2/860 = 0.2%). The proband manifested slow and progressive parkinsonism at 55 years. Later she complicated with cognitive decline and hallucinations. Several of her immediate family members also presented with parkinsonism, cognitive decline, and psychosis. Positron emission tomography imaging of 18F-6-fluoro-L-dopa (18F-DOPA) uptake, 11C(+)dihydrotetrabenzine (type 2 vesicular monoamine transporter), and 11C-d-threo-methylphenidate (a plasmalemmal dopamine transporter marker) binding in the striatum were significantly reduced. Hence, alpha-synuclein p.A53V homozygous mutation leads to a distinct phenotype of progressive parkinsonism and cognitive decline, commonly observed in patients with SNCA missense mutation or multiplications.
Assuntos
Homozigoto , Mutação de Sentido Incorreto/genética , Doença de Parkinson/genética , alfa-Sinucleína/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Disfunção Cognitiva/etiologia , Corpo Estriado/diagnóstico por imagem , Progressão da Doença , Éxons/genética , Feminino , Genes Dominantes/genética , Alucinações/etiologia , Humanos , Masculino , Pessoa de Meia-Idade , Doença de Parkinson/complicações , Doença de Parkinson/diagnóstico por imagem , Fenótipo , Tomografia por Emissão de Pósitrons , Adulto JovemRESUMO
BACKGROUND: Epidemiological studies suggest that hyponutrition during the fetal period increases the risk of mental disorders such as attention deficit hyperactivity disorder and autism-spectrum disorder, which has been experimentally supported using animal models. However, previous experimental hyponutrition or protein-restricted (PR) diets affected stages other than the fetal stage, such as formation of the egg before insemination, milk composition during lactation, and maternal nursing behavior. RESULTS: We conducted in vitro fertilization and embryo transfer in mice and allowed PR diet and folic acid-supplemented PR diet to affect only fetal environments. Comprehensive phenotyping of PR and control-diet progenies showed moderate differences in fear/anxiety-like, novelty-seeking, and prosocial behaviors, irrespective of folic-acid supplementation. Changes were also detected in gene expression and genomic methylation in the brain. CONCLUSIONS: These results suggest that epigenetic factors in the embryo/fetus influence behavioral and epigenetic phenotypes of progenies. Significant epigenetic alterations in the brains of the progenies induced by the maternal-protein restriction were observed in the present study. To our knowledge, this is first study to evaluate the effect of maternal hyponutrition on behavioral phenotypes using reproductive technology.
RESUMO
Wild-derived mice have contributed to experimental mouse genetics by virtue of their genetic diversity, which may help increase the chance of identifying novel modifier genes responsible for specific phenotypes and diseases. However, gene targeting using wild-derived mice has been unsuccessful because of the unavailability of stable embryonic stem cells. Here, we report that CRISPR/Cas9-mediated gene targeting can be applied to the Japanese wild-derived MSM/Ms strain (Mus musculus molossinus). We targeted the nonagouti (a) gene encoding the agouti protein that is localized in hair and the brain. We obtained three homozygous knockout mice as founders, all showing black coat colour. While homozygous knockout offspring were physiologically indistinguishable from wild-type litter-mates, they showed specific domesticated behaviours: hypoactivity in the dark phase and a decline in the avoidance of a human hand. These phenotypes were consistent over subsequent generations. Our findings support the empirical hypothesis that nonagouti is a domestication-linked gene, the loss of which might repress aggressive behaviour.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Proteína Agouti Sinalizadora/genética , Alelos , Animais , Animais Selvagens , Análise Mutacional de DNA , Dopamina/metabolismo , Expressão Gênica , Humanos , Mesencéfalo/metabolismo , Camundongos , Camundongos Knockout , Mutação , FenótipoRESUMO
A large body of evidence has shown the involvement of serotonin (5-HT) in anxiety. The administration of serotonergic neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) into adult rats has been shown to produce a prolonged reduction in the content of brain 5-HT along with anxiolytic effects. In this experiment, 5,7-DHT was administrated intraventricularly to neonatal and adult rats. All rats were tested in an elevated plus maze at 30, 50, 70, and 90 days old to evaluate the anxiety level. Adult treatment increased the time spent in open-arm, and decreased the brain 5-HT content in all the regions measured. In contrast, neonatal treatment decreased the time spent in open-arm, and 5-HT contents in these animals did not decrease in the hypothalamus and medulla oblongata. A 5-HT syndrome test was conducted once when the rats were 91 to 97 days old to evaluate the sensitivity of 5-HT recepotors. It was found that 5-HTP (25 mg/kg) produces a severe serotonin syndrome in the adult 5,7-DHT-treated rats, but only a moderate syndrome in the neonatal-treated animals. Significant negative correlation coefficients were obtained between the score of serotonin syndrome and 5-HT content in the hypothalamus, midbrain, medulla oblongata, and cerebellum of the neonatal 5,7-DHT-treated rats. The results suggest that neonatal 5,7-DHT treatment produces an anxiogenic effect in contrast with the anxiolytic effect with adult treatment.
Assuntos
5,7-Di-Hidroxitriptamina , Ansiolíticos , Ansiedade/induzido quimicamente , Serotoninérgicos , 5,7-Di-Hidroxitriptamina/farmacologia , 5-Hidroxitriptofano/farmacologia , Animais , Animais Recém-Nascidos , Encéfalo/metabolismo , Feminino , Masculino , Ratos , Ratos Endogâmicos F344 , Receptores de Serotonina/metabolismo , Serotonina/metabolismo , Serotoninérgicos/farmacologiaRESUMO
As part of the RIKEN large-scale N-ethyl-N-nitrosourea (ENU) mutagenesis project, we screened mice with a dominant mutation that exhibited abnormal behavior using an open-field test and a home-cage activity test. We tested 495 male progeny of C57BL/6J males treated with ENU and untreated C3H/HeJ females using the open-field test and isolated behavioral mutant M101736, which exhibited a significant increase in spontaneous locomotor activity. We identified a missense mutation in the Tuba1 gene, which encodes the TUBA1 protein, and designated the mutant gene Tuba1(Rgsc1736). This mutation results in an aspartic acid to glycine substitution in the TUBA1 protein. Detailed analyses revealed that Tuba1(Rgsc1736) heterozygotes exhibited inattention to novel objects and aberrant patterns of home-cage activity. The results of a behavioral pharmacological analysis using methylphenidate and morphological analyses of embryonic and adult brains suggested that Tuba1(Rgsc1736) is a novel animal model for neurodevelopmental disorders.
Assuntos
Comportamento Animal/fisiologia , Camundongos Mutantes/genética , Neocórtex/patologia , Neurônios/patologia , Tubulina (Proteína)/genética , Análise de Variância , Animais , Animais Recém-Nascidos , Ácido Aspártico/genética , Atenção/efeitos dos fármacos , Atenção/fisiologia , Bromodesoxiuridina , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Estimulantes do Sistema Nervoso Central/farmacologia , Mapeamento Cromossômico , Análise Mutacional de DNA , Adaptação à Escuridão/efeitos dos fármacos , Adaptação à Escuridão/genética , Deficiências do Desenvolvimento/tratamento farmacológico , Deficiências do Desenvolvimento/genética , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Embrião de Mamíferos , Etilnitrosoureia/farmacologia , Comportamento Exploratório/fisiologia , Feminino , Regulação da Expressão Gênica/genética , Glicina/genética , Comportamento de Retorno ao Território Vital/fisiologia , Masculino , Metilfenidato/farmacologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Mutantes/fisiologia , Microscopia Eletrônica de Transmissão , Mutagênese/efeitos dos fármacos , Mutagênicos/farmacologia , Neocórtex/embriologia , Neocórtex/crescimento & desenvolvimento , Neurônios/ultraestrutura , Fenótipo , Fatores de TempoRESUMO
To establish the cutoff values for screening ENU-induced behavioral mutations, normal variations in mouse behavioral data were examined in home-cage activity (HA), open-field (OF), and passive-avoidance (PA) tests. We defined the normal range as one that included more than 95% of the normal control values. The cutoffs were defined to identify outliers yielding values that deviated from the normal by less than 5% for C57BL/6J, DBA/2J, DBF(1), and N(2) (DXDB) progenies. Cutoff values for G1-phenodeviant (DBF(1)) identification were defined based on values over +/- 3.0 SD from the mean of DBF(1) for all parameters assessed in the HA and OF tests. For the PA test, the cutoff values were defined based on whether the mice met the learning criterion during the 2nd (at a shock intensity of 0.3 mA) or the 3rd (at a shock intensity of 0.15 mA) retention test. For several parameters, the lower outliers were undetectable as the calculated cutoffs were negative values. Based on the cutoff criteria, we identified 275 behavioral phenodeviants among 2,646 G1 progeny. Of these, 64 were crossed with wild-type DBA/2J individuals, and the phenotype transmission was examined in the G2 progeny using the cutoffs defined for N(2) mice. In the G2 mice, we identified 15 novel dominant mutants exhibiting behavioral abnormalities, including hyperactivity in the HA or OF tests, hypoactivity in the OF test, and PA deficits. Genetic and detailed behavioral analysis of these ENU-induced mutants will provide novel insights into the molecular mechanisms underlying behavior.