RESUMO
Glutathione peroxidase 4 (GPX4) is unique as it is the only enzyme that can prevent detrimental lipid peroxidation in vivo by reducing lipid peroxides to the respective alcohols thereby stabilizing oxidation products of unsaturated fatty acids. During reticulocyte maturation, lipid peroxidation mediated by 15-lipoxygenase in humans and rabbits and by 12/15-lipoxygenase (ALOX15) in mice was considered the initiating event for the elimination of mitochondria but is now known to occur through mitophagy. Yet, genetic ablation of the Alox15 gene in mice failed to provide evidence for this hypothesis. We designed a different genetic approach to tackle this open conundrum. Since either other lipoxygenases or non-enzymatic autooxidative mechanisms may compensate for the loss of Alox15, we asked whether ablation of Gpx4 in the hematopoietic system would result in the perturbation of reticulocyte maturation. Quantitative assessment of erythropoiesis indices in the blood, bone marrow (BM) and spleen of chimeric mice with Gpx4 ablated in hematopoietic cells revealed anemia with an increase in the fraction of erythroid precursor cells and reticulocytes. Additional dietary vitamin E depletion strongly aggravated the anemic phenotype. Despite strong extramedullary erythropoiesis reticulocytes failed to mature and accumulated large autophagosomes with engulfed mitochondria. Gpx4-deficiency in hematopoietic cells led to systemic hepatic iron overload and simultaneous severe iron demand in the erythroid system. Despite extremely high erythropoietin and erythroferrone levels in the plasma, hepcidin expression remained unchanged. Conclusively, perturbed reticulocyte maturation in response to Gpx4 loss in hematopoietic cells thus causes ineffective erythropoiesis, a phenotype partially masked by dietary vitamin E supplementation.
Assuntos
Eritropoese , Ferro , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/genética , Reticulócitos , Vitamina E , Animais , Homeostase , Camundongos , CoelhosRESUMO
Tetrahydrobiopterin (BH4) is an essential cofactor for the aromatic amino acid hydroxylases, alkylglycerol monooxygenase, and nitric oxide synthases (NOS). Inborn errors of BH4 metabolism lead to severe insufficiency of brain monoamine neurotransmitters while augmentation of BH4 by supplementation or stimulation of its biosynthesis is thought to ameliorate endothelial NOS (eNOS) dysfunction, to protect from (cardio-) vascular disease and/or prevent obesity and development of the metabolic syndrome. We have previously reported that homozygous knock-out mice for the 6-pyruvolytetrahydropterin synthase (PTPS; Pts-ko/ko) mice with no BH4 biosynthesis die after birth. Here we generated a Pts-knock-in (Pts-ki) allele expressing the murine PTPS-p.Arg15Cys with low residual activity (15% of wild-type in vitro) and investigated homozygous (Pts-ki/ki) and compound heterozygous (Pts-ki/ko) mutants. All mice showed normal viability and depending on the severity of the Pts alleles exhibited up to 90% reduction of PTPS activity concomitant with neopterin elevation and mild reduction of total biopterin while blood L-phenylalanine and brain monoamine neurotransmitters were unaffected. Yet, adult mutant mice with compromised PTPS activity (i.e., Pts-ki/ko, Pts-ki/ki or Pts-ko/wt) had increased body weight and elevated intra-abdominal fat. Comprehensive phenotyping of Pts-ki/ki mice revealed alterations in energy metabolism with proportionally higher fat content but lower lean mass, and increased blood glucose and cholesterol. Transcriptome analysis indicated changes in glucose and lipid metabolism. Furthermore, differentially expressed genes associated with obesity, weight loss, hepatic steatosis, and insulin sensitivity were consistent with the observed phenotypic alterations. We conclude that reduced PTPS activity concomitant with mildly compromised BH4-biosynthesis leads to abnormal body fat distribution and abdominal obesity at least in mice. This study associates a novel single gene mutation with monogenic forms of obesity.
Assuntos
Tecido Adiposo/metabolismo , Biopterinas/análogos & derivados , Distribuição da Gordura Corporal , Obesidade Abdominal/genética , Fósforo-Oxigênio Liases/genética , Alelos , Animais , Biopterinas/biossíntese , Biopterinas/genética , Peso Corporal/genética , Colesterol/genética , Feminino , Genótipo , Glucose/genética , Heterozigoto , Homozigoto , Metabolismo dos Lipídeos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação/genética , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo III/genética , Fenilalanina/genética , Transcriptoma/genéticaRESUMO
The majority of amyotrophic lateral sclerosis (ALS) cases as well as many patients suffering from frontotemporal lobar dementia (FTLD) with ubiquitinated inclusion bodies show TDP-43 pathology, the protein encoded by the TAR DNA-binding protein (Tardbp) gene. We used recombinase-mediated cassette exchange to introduce an ALS patient cDNA into the mouse Tdp-43 locus. Expression levels of human A315T TDP-43 protein were 300% elevated in heterozygotes, whereas the endogenous mouse Tdp-43 was decreased to 20% of wild type levels as a result of disturbed feedback regulation. Heterozygous TDP-43(A315TKi) mutants lost 10% of their body weight and developed insoluble TDP-43 protein starting as early as 3 months after birth, a pathology that was exacerbated with age. We analyzed the splicing patterns of known Tdp-43 target genes as well as genome-wide gene expression levels in different tissues that indicated mitochondrial dysfunction. In heterozygous mutant animals, we observed a relative decrease in expression of Parkin (Park2) and the fatty acid transporter CD36 along with an increase in fatty acids, HDL cholesterol, and glucose in the blood. As seen in transmission electron microscopy, neuronal cells in motor cortices of TDP-43(A315TKi) animals had abnormal neuronal mitochondrial cristae formation. Motor neurons were reduced to 90%, but only slight motoric impairment was detected. The observed phenotype was interpreted as a predisease model, which might be valuable for the identification of further environmental or genetic triggers of neurodegeneration.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Mitocôndrias/patologia , Alelos , Esclerose Lateral Amiotrófica/genética , Animais , Comportamento Animal , Glicemia/metabolismo , Peso Corporal , Antígenos CD36/metabolismo , HDL-Colesterol/metabolismo , DNA Complementar/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/citologia , Ácidos Graxos/metabolismo , Feminino , Técnicas de Introdução de Genes , Genoma , Genótipo , Heterozigoto , Humanos , Masculino , Aprendizagem em Labirinto , Camundongos , Camundongos Transgênicos , Neurônios Motores/metabolismo , Mutagênese Sítio-Dirigida , Mutação , Fenótipo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Aging is a major risk factor for a large number of disorders and functional impairments. Therapeutic targeting of the aging process may therefore represent an innovative strategy in the quest for novel and broadly effective treatments against age-related diseases. The recent report of lifespan extension in mice treated with the FDA-approved mTOR inhibitor rapamycin represented the first demonstration of pharmacological extension of maximal lifespan in mammals. Longevity effects of rapamycin may, however, be due to rapamycin's effects on specific life-limiting pathologies, such as cancers, and it remains unclear if this compound actually slows the rate of aging in mammals. Here, we present results from a comprehensive, large-scale assessment of a wide range of structural and functional aging phenotypes, which we performed to determine whether rapamycin slows the rate of aging in male C57BL/6J mice. While rapamycin did extend lifespan, it ameliorated few studied aging phenotypes. A subset of aging traits appeared to be rescued by rapamycin. Rapamycin, however, had similar effects on many of these traits in young animals, indicating that these effects were not due to a modulation of aging, but rather related to aging-independent drug effects. Therefore, our data largely dissociate rapamycin's longevity effects from effects on aging itself.
Assuntos
Envelhecimento/efeitos dos fármacos , Longevidade/efeitos dos fármacos , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Transformação Celular Neoplásica/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Granuloma/prevenção & controle , Imunoglobulinas/sangue , Contagem de Leucócitos , Fígado/efeitos dos fármacos , Fígado/patologia , Cirrose Hepática/prevenção & controle , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Força Muscular/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Fenótipo , Contagem de Plaquetas , Desempenho Psicomotor/efeitos dos fármacos , Análise de Sobrevida , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Glândula Tireoide/efeitos dos fármacos , Glândula Tireoide/patologiaRESUMO
Neurobeachin (Nbea) regulates neuronal membrane protein trafficking and is required for the development and functioning of central and neuromuscular synapses. In homozygous knockout (KO) mice, Nbea deficiency causes perinatal death. Here, we report that heterozygous KO mice haploinsufficient for Nbea have higher body weight due to increased adipose tissue mass. In several feeding paradigms, heterozygous KO mice consumed more food than wild-type (WT) controls, and this consumption was primarily driven by calories rather than palatability. Expression analysis of feeding-related genes in the hypothalamus and brainstem with real-time PCR showed differential expression of a subset of neuropeptide or neuropeptide receptor mRNAs between WT and Nbea+/- mice in the sated state and in response to food deprivation, but not to feeding reward. In humans, we identified two intronic NBEA single-nucleotide polymorphisms (SNPs) that are significantly associated with body-mass index (BMI) in adult and juvenile cohorts. Overall, data obtained in mice and humans suggest that variation of Nbea abundance or activity critically affects body weight, presumably by influencing the activity of feeding-related neural circuits. Our study emphasizes the importance of neural mechanisms in body weight control and points out NBEA as a potential risk gene in human obesity.
Assuntos
Índice de Massa Corporal , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Comportamento Alimentar , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Obesidade/genética , Tecido Adiposo/metabolismo , Adolescente , Animais , Tronco Encefálico/metabolismo , Criança , Privação de Alimentos , Regulação da Expressão Gênica/genética , Estudos de Associação Genética , Humanos , Hipotálamo/metabolismo , Masculino , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo ÚnicoRESUMO
High attrition rates of novel anti-cancer drugs highlight the need for improved models to predict toxicity. Although polo-like kinase 1 (Plk1) inhibitors are attractive candidates for drug development, the role of Plk1 in primary cells remains widely unexplored. Therefore, we evaluated the utility of an RNA interference-based model to assess responses to an inducible knockdown (iKD) of Plk1 in adult mice. Here we show that Plk1 silencing can be achieved in several organs, although adverse events are rare. We compared responses in Plk1-iKD mice with those in primary cells kept under controlled culture conditions. In contrast to the addiction of many cancer cell lines to the non-oncogene Plk1, the primary cells' proliferation, spindle assembly and apoptosis exhibit only a low dependency on Plk1. Responses to Plk1-depletion, both in cultured primary cells and in our iKD-mouse model, correspond well and thus provide the basis for using validated iKD mice in predicting responses to therapeutic interventions.
Assuntos
Antineoplásicos/toxicidade , Proteínas de Ciclo Celular/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Interferência de RNA/efeitos dos fármacos , Testes de Toxicidade/métodos , Animais , Apoptose/genética , Northern Blotting , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Primers do DNA/genética , Avaliação Pré-Clínica de Medicamentos , Citometria de Fluxo , Imunofluorescência , Dosagem de Genes/genética , Técnicas de Silenciamento de Genes , Engenharia Genética/métodos , Humanos , Camundongos , Camundongos Transgênicos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Quinase 1 Polo-LikeRESUMO
OBJECTIVE: It is difficult to identify a single causative factor for inflammatory arthritis because of the multifactorial nature of the disease. This study was undertaken to dissect the molecular complexity of systemic inflammatory disease, utilizing a combined approach of mutagenesis and systematic phenotype screening in a murine model. METHODS: In a large-scale N-ethyl-N-nitrosourea mutagenesis project, the Ali14 mutant mouse strain was established because of dominant inheritance of spontaneous swelling and inflammation of the hind paws. Genetic mapping and subsequent candidate gene sequencing were conducted to find the causative gene, and systematic phenotyping of Ali14/+ mice was performed in the German Mouse Clinic. RESULTS: A novel missense mutation in the phospholipase Cγ2 gene (Plcg2) was identified in Ali14/+ mice. Because of the hyperreactive external entry of calcium observed in cultured B cells and other in vitro experiments, the Ali14 mutation is thought to be a novel gain-of-function allele of Plcg2. Findings from systematic screening of Ali14/+ mice demonstrated various phenotypic changes: an abnormally high T cell:B cell ratio, up-regulation of Ig, alterations in body composition, and a reduction in cholesterol and triglyceride levels in peripheral blood. In addition, spermatozoa from Ali14/+ mice failed to fertilize eggs in vitro, despite the normal fertility of the Ali14/+ male mice in vivo. CONCLUSION: These results suggest that the Plcg2-mediated pathways play a crucial role in various metabolic and sperm functions, in addition to initiating and maintaining the immune system. These findings may indicate the importance of the Ali14/+ mouse strain as a model for systemic inflammatory diseases and inflammation-related metabolic changes in humans.