Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 28
Filtrar
1.
Nat Commun ; 15(1): 6534, 2024 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-39095390

RESUMO

Huntington's disease (HD) causes selective degeneration of striatal and cortical neurons, resulting in cell mosaicism of coexisting still functional and dysfunctional cells. The impact of non-cell autonomous mechanisms between these cellular states is poorly understood. Here we generated telencephalic organoids with healthy or HD cells, grown separately or as mosaics of the two genotypes. Single-cell RNA sequencing revealed neurodevelopmental abnormalities in the ventral fate acquisition of HD organoids, confirmed by cytoarchitectural and transcriptional defects leading to fewer GABAergic neurons, while dorsal populations showed milder phenotypes mainly in maturation trajectory. Healthy cells in mosaic organoids restored HD cell identity, trajectories, synaptic density, and communication pathways upon cell-cell contact, while showing no significant alterations when grown with HD cells. These findings highlight cell-type-specific alterations in HD and beneficial non-cell autonomous effects of healthy cells, emphasizing the therapeutic potential of modulating cell-cell communication in disease progression and treatment.


Assuntos
Doença de Huntington , Organoides , Fenótipo , Telencéfalo , Doença de Huntington/patologia , Doença de Huntington/genética , Doença de Huntington/metabolismo , Organoides/patologia , Organoides/metabolismo , Animais , Telencéfalo/patologia , Telencéfalo/citologia , Telencéfalo/metabolismo , Humanos , Camundongos , Neurônios GABAérgicos/metabolismo , Neurônios GABAérgicos/patologia , Análise de Célula Única , Comunicação Celular , Mosaicismo , Neurônios/metabolismo , Neurônios/patologia
2.
Life Sci Alliance ; 6(11)2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37684045

RESUMO

Huntington's disease arises from a toxic gain of function in the huntingtin (HTT) gene. As a result, many HTT-lowering therapies are being pursued in clinical studies, including those that reduce HTT RNA and protein expression in the liver. To investigate potential impacts, we characterized molecular, cellular, and metabolic impacts of chronic HTT lowering in mouse hepatocytes. Lifelong hepatocyte HTT loss is associated with multiple physiological changes, including increased circulating bile acids, cholesterol and urea, hypoglycemia, and impaired adhesion. HTT loss causes a clear shift in the normal zonal patterns of liver gene expression, such that pericentral gene expression is reduced. These alterations in liver zonation in livers lacking HTT are observed at the transcriptional, histological, and plasma metabolite levels. We have extended these phenotypes physiologically with a metabolic challenge of acetaminophen, for which the HTT loss results in toxicity resistance. Our data reveal an unexpected role for HTT in regulating hepatic zonation, and we find that loss of HTT in hepatocytes mimics the phenotypes caused by impaired hepatic ß-catenin function.


Assuntos
Hepatócitos , Fígado , Animais , Camundongos , Acetaminofen , Fenótipo
3.
bioRxiv ; 2023 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-37425835

RESUMO

Huntington's disease arises from a toxic gain of function in the huntingtin ( HTT ) gene. As a result, many HTT-lowering therapies are being pursued in clinical studies, including those that reduce HTT RNA and protein expression in the liver. To investigate potential impacts, we characterized molecular, cellular, and metabolic impacts of chronic HTT lowering in mouse hepatocytes. Lifelong hepatocyte HTT loss is associated with multiple physiological changes, including increased circulating bile acids, cholesterol and urea, hypoglycemia, and impaired adhesion. HTT loss causes a clear shift in the normal zonal patterns of liver gene expression, such that pericentral gene expression is reduced. These alterations in liver zonation in livers lacking HTT are observed at the transcriptional, histological and plasma metabolite level. We have extended these phenotypes physiologically with a metabolic challenge of acetaminophen, for which the HTT loss results in toxicity resistance. Our data reveal an unexpected role for HTT in regulating hepatic zonation, and we find that loss of HTT in hepatocytes mimics the phenotypes caused by impaired hepatic ß-catenin function.

4.
Neuron ; 110(20): 3318-3338.e9, 2022 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-36265442

RESUMO

Brain tissue transcriptomes may be organized into gene coexpression networks, but their underlying biological drivers remain incompletely understood. Here, we undertook a large-scale transcriptomic study using 508 wild-type mouse striatal tissue samples dissected exclusively in the afternoons to define 38 highly reproducible gene coexpression modules. We found that 13 and 11 modules are enriched in cell-type and molecular complex markers, respectively. Importantly, 18 modules are highly enriched in daily rhythmically expressed genes that peak or trough with distinct temporal kinetics, revealing the underlying biology of striatal diurnal gene networks. Moreover, the diurnal coexpression networks are a dominant feature of daytime transcriptomes in the mouse cortex. We next employed the striatal coexpression modules to decipher the striatal transcriptomic signatures from Huntington's disease models and heterozygous null mice for 52 genes, uncovering novel functions for Prkcq and Kdm4b in oligodendrocyte differentiation and bipolar disorder-associated Trank1 in regulating anxiety-like behaviors and nocturnal locomotion.


Assuntos
Doença de Huntington , Transcriptoma , Animais , Camundongos , Proteína Quinase C-theta/genética , Redes Reguladoras de Genes , Doença de Huntington/genética , Encéfalo
5.
J Med Chem ; 65(18): 12445-12459, 2022 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-36098485

RESUMO

Huntington's disease (HD) is a lethal autosomal dominant neurodegenerative disorder resulting from a CAG repeat expansion in the huntingtin (HTT) gene. The product of translation of this gene is a highly aggregation-prone protein containing a polyglutamine tract >35 repeats (mHTT) that has been shown to colocalize with histone deacetylase 4 (HDAC4) in cytoplasmic inclusions in HD mouse models. Genetic reduction of HDAC4 in an HD mouse model resulted in delayed aggregation of mHTT, along with amelioration of neurological phenotypes and extended lifespan. To further investigate the role of HDAC4 in cellular models of HD, we have developed bifunctional degraders of the protein and report the first potent and selective degraders of HDAC4 that show an effect in multiple cell lines, including HD mouse model-derived cortical neurons. These degraders act via the ubiquitin-proteasomal pathway and selectively degrade HDAC4 over other class IIa HDAC isoforms (HDAC5, HDAC7, and HDAC9).


Assuntos
Histona Desacetilases , Doença de Huntington , Animais , Modelos Animais de Doenças , Desenvolvimento de Medicamentos , Histona Desacetilases/metabolismo , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Doença de Huntington/genética , Camundongos , Neurônios/metabolismo , Proteólise , Ubiquitinas
6.
J Huntingtons Dis ; 10(3): 405-412, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34397420

RESUMO

HDinHD (Huntington's Disease in High Definition; HDinHD.org) is an open online portal for the HD research community that presents a synthesized view of HD-related scientific data. Here, we present a broad overview of HDinHD and highlight the newly launched HDinHD Explorer tool that enables researchers to discover and explore a wide range of diverse yet interconnected HD-related data. We demonstrate the utility of HDinHD Explorer through data mining of a single collection of newly released in vivo therapeutic intervention study reports alongside previously published reports.


Assuntos
Doença de Huntington , Humanos , Doença de Huntington/genética
7.
Elife ; 102021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33618800

RESUMO

Loss of cellular homeostasis has been implicated in the etiology of several neurodegenerative diseases (NDs). However, the molecular mechanisms that underlie this loss remain poorly understood on a systems level in each case. Here, using a novel computational approach to integrate dimensional RNA-seq and in vivo neuron survival data, we map the temporal dynamics of homeostatic and pathogenic responses in four striatal cell types of Huntington's disease (HD) model mice. This map shows that most pathogenic responses are mitigated and most homeostatic responses are decreased over time, suggesting that neuronal death in HD is primarily driven by the loss of homeostatic responses. Moreover, different cell types may lose similar homeostatic processes, for example, endosome biogenesis and mitochondrial quality control in Drd1-expressing neurons and astrocytes. HD relevance is validated by human stem cell, genome-wide association study, and post-mortem brain data. These findings provide a new paradigm and framework for therapeutic discovery in HD and other NDs.


Assuntos
Proteína Huntingtina/genética , Doença de Huntington/genética , Mutação , Proteostase , Animais , Modelos Animais de Doenças , Feminino , Proteína Huntingtina/metabolismo , Masculino , Camundongos
8.
Nat Commun ; 11(1): 4529, 2020 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-32913184

RESUMO

Although Huntington's disease (HD) is a well studied Mendelian genetic disorder, less is known about its associated epigenetic changes. Here, we characterize DNA methylation levels in six different tissues from 3 species: a mouse huntingtin (Htt) gene knock-in model, a transgenic HTT sheep model, and humans. Our epigenome-wide association study (EWAS) of human blood reveals that HD mutation status is significantly (p < 10-7) associated with 33 CpG sites, including the HTT gene (p = 6.5 × 10-26). These Htt/HTT associations were replicated in the Q175 Htt knock-in mouse model (p = 6.0 × 10-8) and in the transgenic sheep model (p = 2.4 × 10-88). We define a measure of HD motor score progression among manifest HD cases based on multiple clinical assessments. EWAS of motor progression in manifest HD cases exhibits significant (p < 10-7) associations with methylation levels at three loci: near PEX14 (p = 9.3 × 10-9), GRIK4 (p = 3.0 × 10-8), and COX4I2 (p = 6.5 × 10-8). We conclude that HD is accompanied by profound changes of DNA methylation levels in three mammalian species.


Assuntos
Metilação de DNA , Epigênese Genética , Proteína Huntingtina/genética , Doença de Huntington/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Animais Geneticamente Modificados , Comportamento Animal , Ilhas de CpG/genética , Estudos Transversais , Modelos Animais de Doenças , Progressão da Doença , Feminino , Seguimentos , Técnicas de Introdução de Genes , Loci Gênicos , Estudo de Associação Genômica Ampla , Carga Global da Doença , Humanos , Doença de Huntington/sangue , Doença de Huntington/diagnóstico , Doença de Huntington/epidemiologia , Estudos Longitudinais , Masculino , Camundongos , Pessoa de Meia-Idade , Mutação , Estudos Prospectivos , Proteínas Recombinantes/genética , Sistema de Registros/estatística & dados numéricos , Índice de Gravidade de Doença , Ovinos , Adulto Jovem
9.
Biochem Biophys Res Commun ; 521(3): 549-554, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31677786

RESUMO

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by an expansion of a CAG triplet repeat (encoding for a polyglutamine tract) within the first exon of the huntingtin gene. Expression of the mutant huntingtin (mHTT) protein can result in the production of N-terminal fragments with a robust propensity to form oligomers and aggregates, which may be causally associated with HD pathology. Several lines of evidence indicate that N17 phosphorylation or pseudophosphorylation at any of the residues T3, S13 or S16, alone or in combination, modulates mHTT aggregation, subcellular localization and toxicity. Consequently, increasing N17 phosphorylation has been proposed as a potential therapeutic approach. However, developing genetic/pharmacological tools to quantify these phosphorylation events is necessary in order to subsequently develop tool modulators, which is difficult given the transient and incompletely penetrant nature of such post-translational modifications. Here we describe the first ultrasensitive sandwich immunoassay that quantifies HTT phosphorylated at residue S13 and demonstrate its utility for specific analyte detection in preclinical models of HD.


Assuntos
Proteína Huntingtina/análise , Animais , Células Cultivadas , Técnicas de Introdução de Genes , Células HEK293 , Humanos , Proteína Huntingtina/genética , Doença de Huntington/genética , Camundongos , Mutação , Neurônios/química , Neurônios/metabolismo , Fosforilação , Agregados Proteicos , Processamento de Proteína Pós-Traducional
10.
PLoS One ; 13(1): e0190550, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29324753

RESUMO

In Huntington's disease (HD) patients and in model organisms, messenger RNA transcriptome has been extensively studied; in contrast, comparatively little is known about expression and potential role of microRNAs. Using RNA-sequencing, we have quantified microRNA expression in four brain regions and liver, at three different ages, from an allelic series of HD model mice with increasing CAG length in the endogenous Huntingtin gene. Our analyses reveal CAG length-dependent microRNA expression changes in brain, with 159 microRNAs selectively altered in striatum, 102 in cerebellum, 51 in hippocampus, and 45 in cortex. In contrast, a progressive CAG length-dependent microRNA dysregulation was not observed in liver. We further identify microRNAs whose transcriptomic response to CAG length expansion differs significantly among the brain regions and validate our findings in data from a second, independent cohort of mice. Using existing mRNA expression data from the same animals, we assess the possible relationships between microRNA and mRNA expression and highlight candidate microRNAs that are negatively correlated with, and whose predicted targets are enriched in, CAG-length dependent mRNA modules. Several of our top microRNAs (Mir212/Mir132, Mir218, Mir128 and others) have been previously associated with aspects of neuronal development and survival. This study provides an extensive resource for CAG length-dependent changes in microRNA expression in disease-vulnerable and -resistant brain regions in HD mice, and provides new insights for further investigation of microRNAs in HD pathogenesis and therapeutics.


Assuntos
Proteína Huntingtina/genética , Doença de Huntington/genética , MicroRNAs/genética , Repetições de Trinucleotídeos , Animais , Encéfalo/metabolismo , Humanos , Camundongos , Transcriptoma
11.
Eur J Prev Cardiol ; 24(5): 492-504, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27940953

RESUMO

Aims Darapladib, a potent inhibitor of lipoprotein-associated phospholipase A2 (Lp-PLA2), has not reduced risk of cardiovascular disease outcomes in recent randomized trials. We aimed to test whether Lp-PLA2 enzyme activity is causally relevant to coronary heart disease. Methods In 72,657 patients with coronary heart disease and 110,218 controls in 23 epidemiological studies, we genotyped five functional variants: four rare loss-of-function mutations (c.109+2T > C (rs142974898), Arg82His (rs144983904), Val279Phe (rs76863441), Gln287Ter (rs140020965)) and one common modest-impact variant (Val379Ala (rs1051931)) in PLA2G7, the gene encoding Lp-PLA2. We supplemented de-novo genotyping with information on a further 45,823 coronary heart disease patients and 88,680 controls in publicly available databases and other previous studies. We conducted a systematic review of randomized trials to compare effects of darapladib treatment on soluble Lp-PLA2 activity, conventional cardiovascular risk factors, and coronary heart disease risk with corresponding effects of Lp-PLA2-lowering alleles. Results Lp-PLA2 activity was decreased by 64% ( p = 2.4 × 10-25) with carriage of any of the four loss-of-function variants, by 45% ( p < 10-300) for every allele inherited at Val279Phe, and by 2.7% ( p = 1.9 × 10-12) for every allele inherited at Val379Ala. Darapladib 160 mg once-daily reduced Lp-PLA2 activity by 65% ( p < 10-300). Causal risk ratios for coronary heart disease per 65% lower Lp-PLA2 activity were: 0.95 (0.88-1.03) with Val279Phe; 0.92 (0.74-1.16) with carriage of any loss-of-function variant; 1.01 (0.68-1.51) with Val379Ala; and 0.95 (0.89-1.02) with darapladib treatment. Conclusions In a large-scale human genetic study, none of a series of Lp-PLA2-lowering alleles was related to coronary heart disease risk, suggesting that Lp-PLA2 is unlikely to be a causal risk factor.


Assuntos
Benzaldeídos/uso terapêutico , Doença das Coronárias/tratamento farmacológico , Doença das Coronárias/genética , Terapia de Alvo Molecular , Oximas/uso terapêutico , Inibidores de Fosfolipase A2/uso terapêutico , 1-Alquil-2-acetilglicerofosfocolina Esterase/efeitos dos fármacos , 1-Alquil-2-acetilglicerofosfocolina Esterase/genética , Adulto , Idoso , Alelos , Estudos de Casos e Controles , Doença das Coronárias/diagnóstico , Feminino , Regulação da Expressão Gênica , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Ensaios Clínicos Controlados Aleatórios como Assunto , Valores de Referência , Reprodutibilidade dos Testes , Medição de Risco , Resultado do Tratamento
12.
Aging (Albany NY) ; 8(7): 1485-512, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27479945

RESUMO

Age of Huntington's disease (HD) motoric onset is strongly related to the number of CAG trinucleotide repeats in the huntingtin gene, suggesting that biological tissue age plays an important role in disease etiology. Recently, a DNA methylation based biomarker of tissue age has been advanced as an epigenetic aging clock. We sought to inquire if HD is associated with an accelerated epigenetic age. DNA methylation data was generated for 475 brain samples from various brain regions of 26 HD cases and 39 controls. Overall, brain regions from HD cases exhibit a significant epigenetic age acceleration effect (p=0.0012). A multivariate model analysis suggests that HD status increases biological age by 3.2 years. Accelerated epigenetic age can be observed in specific brain regions (frontal lobe, parietal lobe, and cingulate gyrus). After excluding controls, we observe a negative correlation (r=-0.41, p=5.5×10-8) between HD gene CAG repeat length and the epigenetic age of HD brain samples. Using correlation network analysis, we identify 11 co-methylation modules with a significant association with HD status across 3 broad cortical regions. In conclusion, HD is associated with an accelerated epigenetic age of specific brain regions and more broadly with substantial changes in brain methylation levels.


Assuntos
Envelhecimento/genética , Encéfalo/metabolismo , Metilação de DNA , Doença de Huntington/genética , Adolescente , Adulto , Fatores Etários , Idade de Início , Idoso , Envelhecimento/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem
13.
J Am Soc Nephrol ; 27(10): 3204-3219, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27005919

RESUMO

Two common missense variants in APOL1 (G1 and G2) have been definitively linked to CKD in black Americans. However, not all individuals with the renal-risk genotype develop CKD, and little is known about how APOL1 variants drive disease. Given the association of APOL1 with HDL particles, which are cleared by the kidney, differences in the level or quality of mutant APOL1­HDL particles could be causal for disease and might serve as a useful risk stratification marker. We measured plasma levels of G0 (low risk), G1, and G2 APOL1 in 3450 individuals in the Dallas Heart Study using a liquid chromatography-MS method that enabled quantitation of the different variants. Additionally, we characterized native APOL1­HDL from donors with no or two APOL1 risk alleles by size-exclusion chromatography and analysis of immunopurified APOL1­HDL particles. Finally, we identified genetic loci associated with plasma APOL1 levels and tested for APOL1-dependent association with renal function. Although we replicated the previous association between APOL1 variant status and renal function in nondiabetic individuals, levels of circulating APOL1 did not associate with microalbuminuria or GFR. Furthermore, the size or known components of APOL1­HDL did not consistently differ in subjects with the renal-risk genotype. Genetic association studies implicated variants in loci harboring haptoglobin-related protein (HPR), APOL1, and ubiquitin D (UBD) in the regulation of plasma APOL1 levels, but these variants did not associate with renal function. Collectively, these data demonstrate that the risk of renal disease associated with APOL1 is probably not related to circulating levels of the mutant protein.


Assuntos
Apolipoproteínas/sangue , Lipoproteínas HDL/sangue , Insuficiência Renal Crônica/sangue , Adulto , Apolipoproteína L1 , Apolipoproteínas/genética , Estudos de Coortes , Estudos Transversais , Feminino , Variação Genética , Genótipo , Humanos , Lipoproteínas HDL/genética , Masculino , Insuficiência Renal Crônica/epidemiologia , Insuficiência Renal Crônica/genética , Fatores de Risco
14.
Nat Genet ; 46(4): 352-6, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24531328

RESUMO

Nonalcoholic fatty liver disease (NAFLD) is the most common form of liver disease. To elucidate the molecular basis of NAFLD, we performed an exome-wide association study of liver fat content. Three variants were associated with higher liver fat levels at the exome-wide significance level of 3.6 × 10(-7): two in PNPLA3, an established locus for NAFLD, and one (encoding p.Glu167Lys) in TM6SF2, a gene of unknown function. The TM6SF2 variant encoding p.Glu167Lys was also associated with higher circulating levels of alanine transaminase, a marker of liver injury, and with lower levels of low-density lipoprotein-cholesterol (LDL-C), triglycerides and alkaline phosphatase in 3 independent populations (n > 80,000). When recombinant protein was expressed in cultured hepatocytes, 50% less Glu167Lys TM6SF2 protein was produced relative to wild-type TM6SF2. Adeno-associated virus-mediated short hairpin RNA knockdown of Tm6sf2 in mice increased liver triglyceride content by threefold and decreased very-low-density lipoprotein (VLDL) secretion by 50%. Taken together, these data indicate that TM6SF2 activity is required for normal VLDL secretion and that impaired TM6SF2 function causally contributes to NAFLD.


Assuntos
Tecido Adiposo/metabolismo , Fígado Gorduroso/genética , Predisposição Genética para Doença/genética , Fígado/metabolismo , Proteínas de Membrana/genética , Alanina Transaminase/sangue , Sequência de Aminoácidos , Animais , Sequência de Bases , Cromatografia Líquida , Dependovirus , Exoma/genética , Técnicas de Silenciamento de Genes , Estudos de Associação Genética , Hepatócitos , Humanos , Lipoproteínas VLDL/metabolismo , Camundongos , Dados de Sequência Molecular , Mutação de Sentido Incorreto/genética , Hepatopatia Gordurosa não Alcoólica , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Triglicerídeos/metabolismo
15.
Am J Physiol Endocrinol Metab ; 302(2): E209-17, 2012 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-22045313

RESUMO

The liver is a crossroad for metabolism of lipid and carbohydrates, with acetyl-CoA serving as an important metabolic intermediate and a precursor for fatty acid and cholesterol biosynthesis pathways. A better understanding of the regulation of these pathways requires an experimental approach that provides both quantitative metabolic flux measurements and mechanistic insight. Under conditions of high carbohydrate availability, excess carbon is converted into free fatty acids and triglyceride for storage, but it is not clear how excessive carbohydrate availability affects cholesterol biosynthesis. To address this, C57BL/6J mice were fed either a low-fat, high-carbohydrate diet or a high-fat, carbohydrate-free diet. At the end of the dietary intervention, the two groups received (2)H(2)O to trace de novo fatty acid and cholesterol synthesis, and livers were collected for gene expression analysis. Expression of lipid and glucose metabolism genes was determined using a custom-designed pathway focused PCR-based gene expression array. The expression analysis showed downregulation of cholesterol biosynthesis genes and upregulation of fatty acid synthesis genes in mice receiving the high-carbohydrate diet compared with the carbohydrate-free diet. In support of these findings, (2)H(2)O tracer data showed that fatty acid synthesis was increased 10-fold and cholesterol synthesis was reduced by 1.6-fold in mice fed the respective diets. In conclusion, by applying gene expression analysis and tracer methodology, we show that fatty acid and cholesterol synthesis are differentially regulated when the carbohydrate intake in mice is altered.


Assuntos
Colesterol/biossíntese , Dieta com Restrição de Carboidratos , Dieta Hiperlipídica , Ácidos Graxos/biossíntese , Fígado/metabolismo , Animais , Colesterol/genética , Carboidratos da Dieta/metabolismo , Gorduras na Dieta/metabolismo , Ácidos Graxos/genética , Expressão Gênica , Perfilação da Expressão Gênica , Masculino , Camundongos
16.
J Cardiovasc Transl Res ; 4(6): 801-10, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21822774

RESUMO

Lecithin:cholesterol acyltransferase (LCAT) is the key circulating enzyme responsible for high-density lipoprotein (HDL) cholesterol esterification, HDL maturation, and potentially reverse cholesterol transport. To further explore LCAT's mechanism of action on lipoprotein metabolism, we employed adeno-associated viral vector (AAV) serotype 8 to achieve long-term (32-week) high level expression of human LCAT in hCETP;Ldlr(+/-) mice, and characterized the lipid profiles in detail. The mice had a marked increase in HDL cholesterol, HDL particle size, and significant reduction in low-density lipoprotein (LDL) cholesterol, plasma triglycerides, and plasma apoB. Plasma LCAT activity significantly increased with humanized substrate specificity. HDL cholesteryl esters increased in a fashion that fits human LCAT specificity. HDL phosphatidylcholines trended toward decrease, with no change observed for HDL lysophosphatidylcholines. Triglycerides reduction appeared to reside in all lipoprotein particles (very low-density lipoprotein (VLDL), LDL, and HDL), with HDL triglycerides composition highly reflective of VLDL, suggesting that changes in HDL triglycerides were primarily driven by the altered triglycerides metabolism in VLDL. In summary, in this human-like model for lipoprotein metabolism, AAV8-mediated overexpression of human LCAT resulted in profound changes in plasma lipid profiles. Detailed lipid analyses in the lipoprotein particles suggest that LCAT's beneficial effect on lipid metabolism includes not only enhanced HDL cholesterol esterification but also improved metabolism of apoB-containing particles and triglycerides. Our findings thus shed new light on LCAT's mechanism of action and lend support to its therapeutic potential in treating dyslipidemia.


Assuntos
Proteínas de Transferência de Ésteres de Colesterol/metabolismo , Dependovirus/genética , Dislipidemias/terapia , Técnicas de Transferência de Genes , Terapia Genética , Vetores Genéticos , Lipídeos/sangue , Fosfatidilcolina-Esterol O-Aciltransferase/genética , Receptores de LDL/deficiência , Animais , Proteínas de Transferência de Ésteres de Colesterol/genética , Modelos Animais de Doenças , Dislipidemias/enzimologia , Dislipidemias/genética , Humanos , Fígado/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Tamanho da Partícula , Fosfatidilcolina-Esterol O-Aciltransferase/metabolismo , Receptores de LDL/genética , Fatores de Tempo
17.
J Cell Sci ; 124(Pt 16): 2837-50, 2011 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-21807948

RESUMO

RAD18 is an ubiquitin ligase that is involved in replication damage bypass and DNA double-strand break (DSB) repair processes in mitotic cells. Here, we investigated the testicular phenotype of Rad18-knockdown mice to determine the function of RAD18 in meiosis, and in particular, in the repair of meiotic DSBs induced by the meiosis-specific topoisomerase-like enzyme SPO11. We found that RAD18 is recruited to a specific subfraction of persistent meiotic DSBs. In addition, RAD18 is recruited to the chromatin of the XY chromosome pair, which forms the transcriptionally silent XY body. At the XY body, RAD18 mediates the chromatin association of its interaction partners, the ubiquitin-conjugating enzymes HR6A and HR6B. Moreover, RAD18 was found to regulate the level of dimethylation of histone H3 at Lys4 and maintain meiotic sex chromosome inactivation, in a manner similar to that previously observed for HR6B. Finally, we show that RAD18 and HR6B have a role in the efficient repair of a small subset of meiotic DSBs.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Meiose , Testículo/metabolismo , Animais , Montagem e Desmontagem da Cromatina/genética , Metilação de DNA , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Técnicas de Silenciamento de Genes , Histonas/genética , Histonas/metabolismo , Masculino , Meiose/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , RNA Interferente Pequeno/genética , Testículo/patologia , Enzimas de Conjugação de Ubiquitina/metabolismo , Inativação do Cromossomo X/genética
18.
Cancer Res ; 71(8): 3052-65, 2011 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-21493594

RESUMO

PDK1 activates AKT suggesting that PDK1 inhibition might suppress tumor development. However, while PDK1 has been investigated intensively as an oncology target, selective inhibitors suitable for in vivo studies have remained elusive. In this study we present the results of in vivo PDK1 inhibition through a universally applicable RNAi approach for functional drug target validation in oncogenic pathway contexts. This approach, which relies on doxycycline-inducible shRNA expression from the Rosa26 locus, is ideal for functional studies of genes like PDK1 where constitutive mouse models lead to strong developmental phenotypes or embryonic lethality. We achieved more than 90% PDK1 knockdown in vivo, a level sufficient to impact physiological functions resulting in hyperinsulinemia and hyperglycemia. This phenotype was reversible on PDK1 reexpression. Unexpectedly, long-term PDK1 knockdown revealed a lack of potent antitumor efficacy in 3 different mouse models of PTEN-deficient cancer. Thus, despite efficient PDK1 knockdown, inhibition of the PI3K pathway was marginal suggesting that PDK1 was not a rate limiting factor. Ex vivo analysis of pharmacological inhibitors revealed that AKT and mTOR inhibitors undergoing clinical development are more effective than PDK1 inhibitors at blocking activated PI3K pathway signaling. Taken together our findings weaken the widely held expectation that PDK1 represents an appealing oncology target.


Assuntos
Neoplasias Experimentais/enzimologia , PTEN Fosfo-Hidrolase/deficiência , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Animais , Técnicas de Silenciamento de Genes , Inativação Gênica , Leucemia Experimental/enzimologia , Leucemia Experimental/genética , Masculino , Camundongos , Camundongos Transgênicos , Neoplasias Experimentais/genética , Proteína Oncogênica v-akt/antagonistas & inibidores , Proteína Oncogênica v-akt/metabolismo , PTEN Fosfo-Hidrolase/genética , Fosforilação , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/genética , Proteínas Serina-Treonina Quinases/genética , Piruvato Desidrogenase Quinase de Transferência de Acetil , Interferência de RNA
19.
Methods Enzymol ; 477: 415-27, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20699153

RESUMO

Expression of small hairpin RNA (shRNA) in mammalian cells can trigger potent RNAi-mediated gene silencing. The dominant-acting RNAi can often result in phenotypes similar to that of a null allele. Moreover, the generation of shRNA knockdown mice and subsequent phenotypic analysis can be achieved in a condensed timeline compared to that of conventional gene-targeting knockout strategies. Here, we discuss methods for the in vivo analysis of gene function in adult mouse tissues following tetracycline-induced RNA knockdown in a single-copy inducible polymerase III promoter-driven shRNA system.


Assuntos
Inativação Gênica/fisiologia , Interferência de RNA/fisiologia , Tetraciclina/farmacologia , Animais , Doxiciclina/farmacologia , Expressão Gênica/efeitos dos fármacos , Camundongos , Interferência de RNA/efeitos dos fármacos , RNA Interferente Pequeno/genética
20.
Dev Dyn ; 238(7): 1803-12, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19479951

RESUMO

Tight regulation of Notch pathway signaling is important in many aspects of embryonic development. Notch signaling can be modulated by expression of fringe genes, encoding glycosyltransferases that modify EGF repeats in the Notch receptor. Although Lunatic fringe (Lfng) has been shown to play important roles in vertebrate segmentation, comparatively little is known regarding the developmental functions of the other vertebrate fringe genes, Radical fringe (Rfng) and Manic fringe (Mfng). Here we report that Mfng expression is not required for embryonic development. Further, we find that despite significant overlap in expression patterns, we detect no obvious synergistic defects in mice in the absence of two, or all three, fringe genes during development of the axial skeleton, limbs, hindbrain, and cranial nerves.


Assuntos
Padronização Corporal/genética , Osso e Ossos/embriologia , Desenvolvimento Embrionário/genética , Extremidades/embriologia , Proteínas/fisiologia , Rombencéfalo/embriologia , Animais , Embrião de Mamíferos , Fertilidade/genética , Fertilidade/fisiologia , Viabilidade Fetal/genética , Viabilidade Fetal/fisiologia , Deleção de Genes , Glucosiltransferases , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Família Multigênica/genética , Família Multigênica/fisiologia , Proteínas/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA