RESUMO
To investigate how the fatty acid composition of brain phospholipids influences brain-specific processes, we leveraged the AdipoR2 (adiponectin receptor 2) knockout mouse model in which the brain is enlarged, and cellular membranes are excessively rich in saturated fatty acids. Lipidomics analysis of brains at 2, 7, and 18 months of age showed that phosphatidylcholines, which make up about two-thirds of all cerebrum membrane lipids, contain a gross excess of saturated fatty acids in AdipoR2 knockout mice, and that this is mostly attributed to an excess palmitic acid (C16:0) at the expense of oleic acid (C18:1), consistent with a defect in fatty acid desaturation and elongation in the mutant. Specifically, there was a ~12% increase in the overall saturated fatty acid content within phosphatidylcholines and a ~30% increase in phosphatidylcholines containing two palmitic acids. Phosphatidylethanolamines, sphingomyelins, ceramides, lactosylceramides, and dihydroceramides also showed an excess of saturated fatty acids in the AdipoR2 knockout mice while nervonic acid (C24:1) was enriched at the expense of shorter saturated fatty acids in glyceroceramides. Similar defects were found in the cerebellum and myelin sheaths. Histology showed that cell density is lower in the cerebrum of AdipoR2 knockout mice, but electron microscopy did not detect reproducible defects in the ultrastructure of cerebrum neurons, though proteomics analysis showed an enrichment of electron transport chain proteins in the cerebellum. Behavioral tests showed that older (33 weeks old) AdipoR2 knockout mice are hyperactive and anxious compared to control mice of a similar age. Also, in contrast to control mice, the AdipoR2 knockout mice do not gain weight in old age but do have normal lifespans. We conclude that an excess fatty acid saturation in brain phospholipids is accompanied by hyperactivity but seems otherwise well tolerated.
Assuntos
Envelhecimento , Encéfalo , Ácidos Graxos , Camundongos Knockout , Receptores de Adiponectina , Animais , Camundongos , Encéfalo/metabolismo , Ácidos Graxos/metabolismo , Envelhecimento/metabolismo , Receptores de Adiponectina/metabolismo , Receptores de Adiponectina/genética , Masculino , Camundongos Endogâmicos C57BL , Fosfatidilcolinas/metabolismo , Fosfolipídeos/metabolismoRESUMO
CRISPR-Cas genome-editing nucleases hold substantial promise for developing human therapeutic applications1-6 but identifying unwanted off-target mutations is important for clinical translation7. A well-validated method that can reliably identify off-targets in vivo has not been described to date, which means it is currently unclear whether and how frequently these mutations occur. Here we describe 'verification of in vivo off-targets' (VIVO), a highly sensitive strategy that can robustly identify the genome-wide off-target effects of CRISPR-Cas nucleases in vivo. We use VIVO and a guide RNA deliberately designed to be promiscuous to show that CRISPR-Cas nucleases can induce substantial off-target mutations in mouse livers in vivo. More importantly, we also use VIVO to show that appropriately designed guide RNAs can direct efficient in vivo editing in mouse livers with no detectable off-target mutations. VIVO provides a general strategy for defining and quantifying the off-target effects of gene-editing nucleases in whole organisms, thereby providing a blueprint to foster the development of therapeutic strategies that use in vivo gene editing.
Assuntos
Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Edição de Genes/normas , Genoma/genética , Mutação , Especificidade por Substrato/genética , Animais , Proteínas Associadas a CRISPR/genética , Feminino , Humanos , Mutação INDEL , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pró-Proteína Convertase 9/genética , Transgenes/genéticaRESUMO
Aging, obesity, and insulin resistance are associated with low levels of PGC1α and PGC1ß coactivators and defective mitochondrial function. We studied mice deficient for PGC1α and PGC1ß [double heterozygous (DH)] to investigate their combined pathogenic contribution. Contrary to our hypothesis, DH mice were leaner, had increased energy dissipation, a pro-thermogenic profile in BAT and WAT, and improved carbohydrate metabolism compared to wild types. WAT showed upregulation of mitochondriogenesis/oxphos machinery upon allelic compensation of PGC1α4 from the remaining allele. However, DH mice had decreased mitochondrial OXPHOS and biogenesis transcriptomes in mitochondria-rich organs. Despite being metabolically healthy, mitochondrial defects in DH mice impaired muscle fiber remodeling and caused qualitative changes in the hepatic lipidome. Our data evidence first the existence of organ-specific compensatory allostatic mechanisms are robust enough to drive an unexpected phenotype. Second, optimization of adipose tissue bioenergetics is sufficient to maintain a healthy metabolic phenotype despite a broad severe mitochondrial dysfunction in other relevant metabolic organs. Third, the decrease in PGC1s in adipose tissue of obese and diabetic patients is in contrast with the robustness of the compensatory upregulation in the adipose of the DH mice.
Assuntos
Tecido Adiposo/metabolismo , Mitocôndrias/genética , Proteínas Nucleares/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Fatores de Transcrição/genética , Envelhecimento/genética , Animais , Modelos Animais de Doenças , Metabolismo Energético/genética , Heterozigoto , Resistência à Insulina/genética , Masculino , Camundongos , Obesidade/genética , Termogênese/genética , Transcriptoma/genéticaRESUMO
INTRODUCTION: While the vast majority of research investigating the role of ghrelin or its receptor, GHS-R1a, in growth, feeding, and metabolism has been conducted in male rodents, very little is known about sex differences in this system. Furthermore, the role of GHS-R1a signaling in the control of pulsatile GH secretion and its link with growth or metabolic parameters has never been characterized. METHODS: We assessed the sex-specific contribution of GHS-R1a signaling in the activity of the GH/IGF-1 axis, metabolic parameters, and feeding behavior in adolescent (5-6 weeks old) or adult (10-19 weeks old) GHS-R KO (Ghsr-/-) and WT (Ghsr+/+) male and female mice. RESULTS: Adult Ghsr-/- male and female mice displayed deficits in weight and linear growth that were correlated with reduced GH pituitary contents in males only. GHS-R1a deletion was associated with reduced meal frequency and increased meal intervals, as well as reduced hypothalamic GHRH and NPY mRNA in males, not females. In adult, GH release from Ghsr-/- mice pituitary explants ex vivo was reduced independently of the sex. However, in vivo pulsatile GH secretion decreased in adult but not adolescent Ghsr-/- females, while in males, GHS-R1a deletion was associated with reduction in pulsatile GH secretion during adolescence exclusively. In males, linear growth did not correlate with pulsatile GH secretion, but rather with ApEn, a measure that reflects irregularity of the rhythmic secretion. Fat mass, plasma leptin concentrations, or ambulatory activity did not predict differences in GH secretion. DISCUSSION/CONCLUSION: These results point to a sex-dependent dimorphic effect of GHS-R1a signaling to modulate pulsatile GH secretion and meal pattern in mice with different compensatory mechanisms occurring in the hypothalamus of adult males and females after GHS-R1a deletion. Altogether, we show that GHS-R1a signaling plays a more critical role in the regulation of pulsatile GH secretion during adolescence in males and adulthood in females.
Assuntos
Grelina , Receptores de Grelina/metabolismo , Animais , Comportamento Alimentar , Feminino , Grelina/metabolismo , Hormônio do Crescimento/metabolismo , Hipotálamo/metabolismo , Masculino , Camundongos , Hipófise/metabolismo , Receptores de Grelina/genéticaRESUMO
Marginal zone (MZ) B cells are innate-like B cells that produce polyreactive antibodies with an affinity for microbial molecular patterns and carbohydrate ligands. MZ B cells have been shown to be important in mediating immunity to various bacteria including Streptococcus pneumoniae and are also implicated in inflammatory syndromes including lupus erythematosus. The intestinal microbiota is responsible for producing short-chain fatty acids, which can regulate immune cell function by several mechanisms including ligation of the G-protein-coupled receptor (GPR)43. Herein, we show that MZ B cells express Gpr43 messenger RNA and that the absence of this receptor impacts on MZ B-cell surface marker expression and antibody production. In T-cell-independent responses to the hapten 4-hydroxy-3-nitrophenylacetic acid (NP), mice deficient in GPR43 displayed higher serum titers of NP-specific antibodies. Moreover, in response to a pneumococcal polysaccharide vaccine, GPR43-deficient mice developed robust serum antibody responses and had markedly increased numbers of splenic antibody-secreting cells, compared with control mice. Finally, serum immunoglobulin M autoantibodies to double-stranded DNA and phosphatidylcholine were increased in resting 10-15-week-old mice lacking GPR43. Taken together, mice lacking GPR43 have heightened antibody responses to T-cell-independent antigens, which may be a result of impaired regulation of MZ B cells.
Assuntos
Linfócitos B , Ácidos Graxos Voláteis , Animais , Células Produtoras de Anticorpos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
AMP-activated protein kinase (AMPK) plays a key role in the cellular response to low energy stress and has emerged as an attractive therapeutic target for tackling metabolic diseases. Whilst significant progress has been made regarding the physiological role of AMPK, its function in the kidney remains only partially understood. We use a mouse model expressing a constitutively active mutant of AMPK to investigate the effect of AMPK activation on kidney function in vivo. Kidney morphology and changes in gene and protein expression were monitored and serum and urine markers were measured to assess kidney function in vivo. Global AMPK activation resulted in an early-onset polycystic kidney phenotype, featuring collecting duct cysts and compromised renal function in adult mice. Mechanistically, the cystic kidneys had increased cAMP levels and ERK activation, increased hexokinase I (Hk I) expression, glycogen accumulation and altered expression of proteins associated with autophagy. Kidney tubule-specific activation of AMPK also resulted in a polycystic phenotype, demonstrating that renal tubular AMPK activation caused the cystogenesis. Importantly, human autosomal dominant polycystic kidney disease (ADPKD) kidney sections revealed similar protein localisation patterns to that observed in the murine cystic kidneys. Our findings show that early-onset chronic AMPK activation leads to a polycystic kidney phenotype, suggesting dysregulated AMPK signalling is a contributing factor in cystogenesis.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Rim/enzimologia , Doenças Renais Policísticas/enzimologia , Proteínas Quinases Ativadas por AMP/genética , Adulto , Fatores Etários , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , AMP Cíclico/metabolismo , Metabolismo Energético , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Predisposição Genética para Doença , Hexoquinase/metabolismo , Humanos , Rim/patologia , Masculino , Camundongos Transgênicos , Fenótipo , Doenças Renais Policísticas/genética , Doenças Renais Policísticas/patologia , Rim Policístico Autossômico Dominante/enzimologia , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/patologia , Transdução de SinaisRESUMO
BACKGROUND AND AIMS: The proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a key role in cholesterol homeostasis, and its inhibition represents an effective therapy to lower low-density lipoprotein cholesterol (LDL-C) levels. In this study, we examined the impact of the PCSK9 rs11591147 loss-of-function (LOF) variant on liver damage in a multicenter collection of patients at risk of nonalcoholic steatohepatitis (NASH), in clinical samples and experimental models. METHODS: We considered 1874 consecutive individuals at risk of NASH as determined by histology. The SNP rs11591147, encoding for the p.R46L variant of PCSK9, was genotyped by TaqMan assays. We also evaluated 1) PCSK9 mRNA hepatic expression in human liver, and 2) the impact of a NASH-inducing diet in mice with hepatic overexpression of human PCSK9. RESULTS: Carriers of PCSK9 rs11591147 had lower circulating LDL-C levels and were protected against nonalcoholic fatty liver disease (NAFLD) (OR: 0.42; 95% CI: 0.22-0.81; P = .01), NASH (OR: 0.48; 95% CI: 0.26-0.87; P = .01) and more severe fibrosis (OR: 0.55; 95% CI: 0.32-0.94; P = .03) independently of clinical, metabolic and genetic confounding factors. PCSK9 hepatic expression was directly correlated with liver steatosis (P = .03). Finally, liver-specific overexpression of human PCSK9 in male mice drives NAFLD and fibrosis upon a dietary challenge. CONCLUSIONS: In individuals at risk of NASH, PCSK9 was induced with hepatic fat accumulation and PCSK9 rs11591147 LOF variant was protective against liver steatosis, NASH and fibrosis, suggesting that PCSK9 inhibition may be a new therapeutic strategy to treat NASH.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Pró-Proteína Convertase 9 , Animais , LDL-Colesterol , Humanos , Fígado , Masculino , Camundongos , Hepatopatia Gordurosa não Alcoólica/genética , Pró-Proteína Convertase 9/genéticaRESUMO
Cell senescence is an important tumour suppressor mechanism and driver of ageing. Both functions are dependent on the development of the senescent phenotype, which involves an overproduction of pro-inflammatory and pro-oxidant signals. However, the exact mechanisms regulating these phenotypes remain poorly understood. Here, we show the critical role of mitochondria in cellular senescence. In multiple models of senescence, absence of mitochondria reduced a spectrum of senescence effectors and phenotypes while preserving ATP production via enhanced glycolysis. Global transcriptomic analysis by RNA sequencing revealed that a vast number of senescent-associated changes are dependent on mitochondria, particularly the pro-inflammatory phenotype. Mechanistically, we show that the ATM, Akt and mTORC1 phosphorylation cascade integrates signals from the DNA damage response (DDR) towards PGC-1ß-dependent mitochondrial biogenesis, contributing to aROS-mediated activation of the DDR and cell cycle arrest. Finally, we demonstrate that the reduction in mitochondrial content in vivo, by either mTORC1 inhibition or PGC-1ß deletion, prevents senescence in the ageing mouse liver. Our results suggest that mitochondria are a candidate target for interventions to reduce the deleterious impact of senescence in ageing tissues.
Assuntos
Envelhecimento/fisiologia , Mitocôndrias/fisiologia , Animais , Linhagem Celular , Humanos , Camundongos , Modelos Biológicos , FenótipoRESUMO
Direct in vivo reprogramming of cardiac fibroblasts into myocytes is an attractive therapeutic intervention in resolving myogenic deterioration. Current transgene-dependent approaches can restore cardiac function, but dependence on retroviral delivery and persistent retention of transgenic sequences are significant therapeutic hurdles. Chemical reprogramming has been established as a legitimate method to generate functional cell types, including those of the cardiac lineage. Here, we have extended this approach to generate progenitor cells that can differentiate into endothelial cells and cardiomyocytes using a single inhibitor protocol. Depletion of terminally differentiated cells and enrichment for proliferative cells result in a second expandable progenitor population that can robustly give rise to myofibroblasts and smooth muscle. Deployment of a genome-wide knockout screen with clustered regularly interspaced short palindromic repeats-guide RNA library to identify novel mediators that regulate the reprogramming revealed the involvement of DNA methyltransferase 1-associated protein 1 (Dmap1). Loss of Dmap1 reduced promoter methylation, increased the expression of Nkx2-5, and enhanced the retention of self-renewal, although further differentiation is inhibited because of the sustained expression of Cdh1. Our results hence establish Dmap1 as a modulator of cardiac reprogramming and myocytic induction. Stem Cells 2019;37:958-972.
Assuntos
Benzamidas/farmacologia , Sistemas CRISPR-Cas , Reprogramação Celular/efeitos dos fármacos , Dioxóis/farmacologia , Fibroblastos/efeitos dos fármacos , Pirazóis/farmacologia , Piridinas/farmacologia , Proteínas Repressoras/genética , Células-Tronco/efeitos dos fármacos , Animais , Caderinas/genética , Caderinas/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Reprogramação Celular/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Edição de Genes/métodos , Proteína Homeobox Nkx-2.5/genética , Proteína Homeobox Nkx-2.5/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso/citologia , Músculo Liso/metabolismo , Miocárdio/citologia , Miocárdio/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Cultura Primária de Células , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Proteínas Repressoras/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
The mutation patterns at Cas9 targeted sites contain unique information regarding the nuclease activity and repair mechanisms in mammalian cells. However, analytical framework for extracting such information are lacking. Here, we present a novel computational platform called Rational InDel Meta-Analysis (RIMA) that enables an in-depth comprehensive analysis of Cas9-induced genetic alterations, especially InDels mutations. RIMA can be used to quantitate the contribution of classical microhomology-mediated end joining (c-MMEJ) pathway in the formation of mutations at Cas9 target sites. We used RIMA to compare mutational signatures at 15 independent Cas9 target sites in human A549 wildtype and A549-POLQ knockout cells to elucidate the role of DNA polymerase θ in c-MMEJ. Moreover, the single nucleotide insertions at the Cas9 target sites represent duplications of preceding nucleotides, suggesting that the flexibility of the Cas9 nuclease domains results in both blunt- and staggered-end cuts. Thymine at the fourth nucleotide before protospacer adjacent motif (PAM) results in a two-fold higher occurrence of single nucleotide InDels compared to guanine at the same position. This study provides a novel approach for the characterization of the Cas9 nucleases with improved accuracy in predicting genome editing outcomes and a potential strategy for homology-independent targeted genomic integration.
Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Reparo do DNA por Junção de Extremidades , Mutação INDEL , Software , Células A549 , Algoritmos , Sequência de Bases , Linhagem Celular , DNA Polimerase Dirigida por DNA/deficiência , DNA Polimerase Dirigida por DNA/metabolismo , Conjuntos de Dados como Assunto , Francisella/enzimologia , Humanos , Motivos de Nucleotídeos , Polimorfismo de Nucleotídeo Único , Proteínas Recombinantes/metabolismo , Streptococcus pyogenes/enzimologia , Especificidade por Substrato , DNA Polimerase tetaRESUMO
BACKGROUND: Plasma concentration of low-density lipoprotein (LDL) cholesterol is a well-established risk factor for cardiovascular disease. Inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9), which regulates cholesterol homeostasis, has recently emerged as an approach to reduce cholesterol levels. The development of humanized animal models is an important step to validate and study human drug targets, and use of genome and base editing has been proposed as a mean to target disease alleles. RESULTS: To address the lack of validated models to test the safety and efficacy of techniques to target human PCSK9, we generated a liver-specific human PCSK9 knock-in mouse model (hPCSK9-KI). We showed that plasma concentrations of total cholesterol were higher in hPCSK9-KI than in wildtype mice and increased with age. Treatment with evolocumab, a monoclonal antibody that targets human PCSK9, reduced cholesterol levels in hPCSK9-KI but not in wildtype mice, showing that the hypercholesterolemic phenotype was driven by overexpression of human PCSK9. CRISPR-Cas9-mediated genome editing of human PCSK9 reduced plasma levels of human and not mouse PCSK9, and in parallel reduced plasma concentrations of total cholesterol; genome editing of mouse Pcsk9 did not reduce cholesterol levels. Base editing using a guide RNA that targeted human and mouse PCSK9 reduced plasma levels of human and mouse PCSK9 and total cholesterol. In our mouse model, base editing was more precise than genome editing, and no off-target editing nor chromosomal translocations were identified. CONCLUSIONS: Here, we describe a humanized mouse model with liver-specific expression of human PCSK9 and a human-like hypercholesterolemia phenotype, and demonstrate that this mouse can be used to evaluate antibody and gene editing-based (genome and base editing) therapies to modulate the expression of human PCSK9 and reduce cholesterol levels. We predict that this mouse model will be used in the future to understand the efficacy and safety of novel therapeutic approaches for hypercholesterolemia.
Assuntos
Colesterol/sangue , Hipercolesterolemia/genética , Fígado/metabolismo , Pró-Proteína Convertase 9/genética , Animais , Modelos Animais de Doenças , Edição de Genes , Genoma , Humanos , Hipercolesterolemia/metabolismo , Camundongos , Camundongos TransgênicosRESUMO
We previously investigated whether inhibition of AMP-metabolizing enzymes could enhance AMP-activated protein kinase (AMPK) activation in skeletal muscle for the treatment of type 2 diabetes. Soluble 5'-nucleotidase II (NT5C2) hydrolyzes IMP and its inhibition could potentially lead to a rise in AMP to activate AMPK. In the present study, we investigated effects of NT5C2 deletion in mice fed a normal-chow diet (NCD) or a high-fat diet (HFD). On a NCD, NT5C2 deletion did not result in any striking metabolic phenotype. On a HFD however, NT5C2 knockout (NT5C2-/-) mice displayed reduced body/fat weight gain, improved glucose tolerance, reduced plasma insulin, triglyceride and uric acid levels compared with wild-type (WT) mice. There was a tendency towards smaller and fewer adipocytes in epididymal fat from NT5C2-/- mice compared to WT mice, consistent with a reduction in triglyceride content. Differences in fat mass under HFD could not be explained by changes in mRNA expression profiles of epididymal fat from WT versus NT5C2-/- mice. However, rates of lipolysis tended to increase in epididymal fat pads from NT5C2-/- versus WT mice, which might explain reduced fat mass. In incubated skeletal muscles, insulin-stimulated glucose uptake and associated signalling were enhanced in NT5C2-/- versus WT mice on HFD, which might contribute towards improved glycemic control. In summary, NT5C2 deletion in mice protects against HFD-induced weight gain, adiposity, insulin resistance and associated hyperglycemia.
Assuntos
5'-Nucleotidase/genética , Dieta Hiperlipídica/efeitos adversos , Deleção de Genes , Resistência à Insulina , Aumento de Peso , Animais , Glucose/metabolismo , Lipólise , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/metabolismo , Obesidade/genética , Obesidade/prevenção & controleRESUMO
BACKGROUND: Oxytocin (Oxt) and its receptor (Oxtr) gene system has been implicated in cardiomyogenesis and cardioprotection; however, effects of chronic activation of Oxtr are not known. We generated and investigated transgenic (TG) mice that overexpress Oxtr specifically in the heart. METHODS AND RESULTS: Cardiac-specific overexpression of Oxtr was obtained by having the α-major histocompatibility complex promoter drive the mouse Oxtr gene (α-Mhc-Oxtr). Left ventricular (LV) function and remodeling were assessed by magnetic resonance imaging and echocardiography. In α-Mhc-Oxtr TG mice, LV ejection fraction was severely compromised at 14 weeks of age compared with wild-type (WT) littermates (25 ± 6% vs 63 ± 3%; P < .001). LV end-diastolic volume was larger in the TG mice (103 ± 6 µL vs 67 ± 5 µL; P < .001). α-Mhc-Oxtr TG animals displayed cardiac fibrosis, atrial thrombus, and increased expression of pro-fibrogenic genes. Mortality of α-Mhc-Oxtr TG animals was 45% compared with 0% (P < .0001) of WT littermates by 20 weeks of age. Most cardiomyocytes of α-Mhc-Oxtr TG animals but not WT littermates (68.0 ± 12.1% vs 5.6 ± 2.4%; P = .008) were positive in staining for nuclear factor of activated T cells (NFAT). To study if thrombin inhibitor prevents thrombus formation, a cohort of 7-week-old α-Mhc-Oxtr TG mice were treated for 12 weeks with AZD0837, a potent thrombin inhibitor. Treatment with AZD0837 reduced thrombus formation (P < .05) and tended to attenuate fibrosis and increase survival. CONCLUSIONS: Cardiac-specific overexpression of Oxtr had negative consequences on LV function and survival in mice. The present findings necessitate further studies to investigate potential adverse effects of chronic Oxt administration. We provide a possible mechanism of Oxtr overexpression leading to heart failure by nuclear factor of activated T cell signaling. The recapitulation of human heart failure and the beneficial effects of the antithrombin inhibitor render the α-Mhc-Oxtr TG mice a promising tool in drug discovery for heart failure.
Assuntos
Cardiomiopatias/genética , Regulação da Expressão Gênica , Miocárdio/metabolismo , RNA/genética , Receptores de Ocitocina/genética , Animais , Cardiomiopatias/diagnóstico , Cardiomiopatias/metabolismo , Modelos Animais de Doenças , Ecocardiografia , Imagem Cinética por Ressonância Magnética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miocárdio/patologia , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Ocitocina/biossínteseRESUMO
AMP-activated protein kinase (AMPK) plays a key role in energy homeostasis and is activated in response to contraction-induced ATP depletion in skeletal muscle via a rise in intracellular AMP/ADP concentrations. AMP can be deaminated by AMP-deaminase (AMPD) to IMP, which is hydrolyzed to inosine by cytosolic 5'-nucleotidase II (NT5C2). AMP can also be hydrolyzed to adenosine by cytosolic 5'-nucleotidase 1A (NT5C1A). Previous gene silencing and overexpression studies indicated control of AMPK activation by NT5C enzymes. In the present study using gene knockout mouse models, we investigated the effects of NT5C1A and NT5C2 deletion on intracellular adenine nucleotide levels and AMPK activation in electrically stimulated skeletal muscles. Surprisingly, NT5C enzyme knockout did not lead to enhanced AMP or ADP concentrations in response to contraction, with no potentiation of increases in AMPK activity in extensor digitorum longus (EDL) and soleus mouse muscles. Moreover, dual blockade of AMP metabolism in EDL using an AMPD inhibitor combined with NT5C1A deletion did not enhance rises in AMP and ADP or increased AMPK activation by electrical stimulation. The results on muscles from the NT5C knockout mice contradict previous findings where AMP levels and AMPK activity were shown to be modulated by NT5C enzymes.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Contração Muscular/fisiologia , Músculo Esquelético/fisiologia , 5'-Nucleotidase , Animais , Ativação Enzimática , Deleção de Genes , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Nucleotídeos/metabolismo , SolubilidadeRESUMO
Despite enormous advances in translational biomedical research, there remains a growing demand for improved animal models of human disease. This is particularly true for diseases where rodent models do not reflect the human disease phenotype. Compared to rodents, pig anatomy and physiology are more similar to humans in cardiovascular, immune, respiratory, skeletal muscle, and metabolic systems. Importantly, efficient and precise techniques for genetic engineering of pigs are now available, facilitating the creation of tailored large animal models that mimic human disease mechanisms at the molecular level. In this article, the benefits of genetically engineered pigs for basic and translational research are exemplified by a novel pig model of Duchenne muscular dystrophy and by porcine models of cystic fibrosis. Particular emphasis is given to potential advantages of using these models for efficacy and safety testing of targeted therapies, such as exon skipping and gene editing, for example, using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system. In general, genetically tailored pig models have the potential to bridge the gap between proof-of-concept studies in rodents and clinical trials in patients, thus supporting translational medicine.
Assuntos
Pesquisa Biomédica , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Engenharia Genética , Suínos , Animais , Sistemas CRISPR-Cas , Fibrose Cística/genética , Distrofia Muscular de Duchenne/genéticaRESUMO
BACKGROUND: Global deletion of microsomal prostaglandin E synthase 1 (mPGES-1) in mice attenuates the response to vascular injury without a predisposition to thrombogenesis or hypertension. However, enzyme deletion results in cell-specific differential use by prostaglandin synthases of the accumulated prostaglandin H(2) substrate. Here, we generated mice deficient in mPGES-1 in vascular smooth muscle cells, endothelial cells, and myeloid cells further to elucidate the cardiovascular function of this enzyme. METHODS AND RESULTS: Vascular smooth muscle cell and endothelial cell mPGES-1 deletion did not alter blood pressure at baseline or in response to a high-salt diet. The propensity to evoked macrovascular and microvascular thrombogenesis was also unaltered. However, both vascular smooth muscle cell and endothelial cell mPGES-1-deficient mice exhibited a markedly exaggerated neointimal hyperplastic response to wire injury of the femoral artery in comparison to their littermate controls. The hyperplasia was associated with increased proliferating cell nuclear antigen and tenascin-C expression. In contrast, the response to injury was markedly suppressed by myeloid cell depletion of mPGES-1 with decreased hyperplasia, leukocyte infiltration, and expression of proliferating cell nuclear antigen and tenascin-C. Conditioned medium derived from mPGES-1-deficient macrophages less potently induced vascular smooth muscle cell proliferation and migration than that from wild-type macrophages. CONCLUSIONS: Deletion of mPGES-1 in the vasculature and myeloid cells differentially modulates the response to vascular injury, implicating macrophage mPGES-1 as a cardiovascular drug target.
Assuntos
Células Endoteliais/enzimologia , Oxirredutases Intramoleculares/metabolismo , Macrófagos/enzimologia , Microssomos/enzimologia , Músculo Liso Vascular/enzimologia , Células Mieloides/enzimologia , Animais , Aorta Torácica/citologia , Pressão Sanguínea/fisiologia , Células Endoteliais/citologia , Feminino , Oxirredutases Intramoleculares/genética , Macrófagos/citologia , Masculino , Camundongos , Camundongos Knockout , Músculo Liso Vascular/citologia , Células Mieloides/citologia , Cultura Primária de Células , Prostaglandina-E Sintases , Especificidade por Substrato/fisiologia , Trombose/metabolismo , Trombose/patologia , Doenças Vasculares/metabolismo , Doenças Vasculares/patologiaRESUMO
The cellular membrane in male meiotic germ cells contains a unique class of phospholipids and sphingolipids that is required for male reproduction. Here, we show that a conserved membrane fluidity sensor, AdipoR2, regulates the meiosis-specific lipidome in mouse testes by promoting the synthesis of sphingolipids containing very-long-chain polyunsaturated fatty acids (VLC-PUFAs). AdipoR2 upregulates the expression of a fatty acid elongase, ELOVL2, both transcriptionally and post-transcriptionally, to synthesize VLC-PUFA. The depletion of VLC-PUFAs and subsequent accumulation of palmitic acid in AdipoR2 knockout testes stiffens the cellular membrane and causes the invagination of the nuclear envelope. This condition impairs the nuclear peripheral distribution of meiotic telomeres, leading to errors in homologous synapsis and recombination. Further, the stiffened membrane impairs the formation of intercellular bridges and the germ cell syncytium, which disrupts the orderly arrangement of cell types within the seminiferous tubules. According to our findings we propose a framework in which the highly-fluid membrane microenvironment shaped by AdipoR2-ELOVL2 underpins meiosis-specific chromosome dynamics in testes.
Assuntos
Fluidez de Membrana , Telômero , Animais , Masculino , Camundongos , Proteínas de Transporte/metabolismo , Meiose , Membrana Nuclear/metabolismo , Esfingolipídeos/metabolismo , Telômero/genética , Telômero/metabolismoRESUMO
Modern methods of molecular diagnostics and therapy have revolutionized the field of medicine in recent years by providing more precise and effective tools for detecting and treating diseases. This progress includes a growing exploration of the body's secreted vesicles, known as extracellular vesicles (EVs), for both diagnostic and therapeutic purposes. EVs are a heterogeneous population of lipid bilayer vesicles secreted by almost every cell type studied so far. They are detected in body fluids and conditioned culture media from living cells. EVs play a crucial role in communication between cells and organs, both locally and over long distances. They are recognized for their ability to transport endogenous RNA and proteins between cells, including messenger RNA (mRNA), microRNA (miRNA), misfolded neurodegenerative proteins, and several other biomolecules. This review explores the dual utilization of EVs, serving not only for diagnostic purposes but also as a platform for delivering therapeutic molecules to cells and tissues. Through an exploration of their composition, biogenesis, and selective cargo packaging, we elucidate the intricate mechanisms behind RNA transport between cells via EVs, highlighting their potential use for both diagnostic and therapeutic applications. Finally, it addresses challenges and outlines prospective directions for the clinical utilization of EVs.
RESUMO
Increased saturated fatty acid (SFA) levels in membrane phospholipids have been implicated in the development of metabolic disease. Here, we tested the hypothesis that increased SFA content in cell membranes negatively impacts adipocyte insulin signaling. Preadipocyte cell models with elevated SFA levels in phospholipids were generated by disrupting the ADIPOR2 locus, which resulted in a striking twofold increase in SFA-containing phosphatidylcholines and phosphatidylethanolamines, which persisted in differentiated adipocytes. Similar changes in phospholipid composition were observed in white adipose tissues isolated from the ADIPOR2-knockout mice. The SFA levels in phospholipids could be further increased by treating ADIPOR2-deficient cells with palmitic acid and resulted in reduced membrane fluidity and endoplasmic reticulum stress in mouse and human preadipocytes. Strikingly, increased SFA levels in differentiated adipocyte phospholipids had no effect on adipocyte gene expression or insulin signaling in vitro. Similarly, increased adipocyte phospholipid saturation did not impair white adipose tissue function in vivo, even in mice fed a high-saturated fat diet at thermoneutrality. We conclude that increasing SFA levels in adipocyte phospholipids is well tolerated and does not affect adipocyte insulin signaling in vitro and in vivo.
Assuntos
Insulina , Fosfolipídeos , Camundongos , Humanos , Animais , Insulina/metabolismo , Adipócitos/metabolismo , Ácidos Graxos/metabolismo , Membrana Celular/metabolismo , Proteínas de Transporte/metabolismoRESUMO
Streptococcus pyogenes Cas9 (SpCas9) and derived enzymes are widely used as genome editors, but their promiscuous nuclease activity often induces undesired mutations and chromosomal rearrangements. Several strategies for mapping off-target effects have emerged, but they suffer from limited sensitivity. To increase the detection sensitivity, we develop an off-target assessment workflow that uses Duplex Sequencing. The strategy increases sensitivity by one order of magnitude, identifying previously unknown SpCas9's off-target mutations in the humanized PCSK9 mouse model. To reduce off-target risks, we perform a bioinformatic search and identify a high-fidelity Cas9 variant of the II-B subfamily from Parasutterella secunda (PsCas9). PsCas9 shows improved specificity as compared to SpCas9 across multiple tested sites, both in vitro and in vivo, including the PCSK9 site. In the future, while PsCas9 will offer an alternative to SpCas9 for research and clinical use, the Duplex Sequencing workflow will enable a more sensitive assessment of Cas9 editing outcomes.