RESUMO
MicroRNAs (miRNAs) are known to regulate most biological processes and have been found dysregulated in a variety of diseases, including multiple sclerosis (MS). In this study, we characterized miRNAs that associate with susceptibility to develop experimental autoimmune encephalomyelitis (EAE) in rats, a well-established animal model of MS. Using Illumina next-generation sequencing, we detected 544 miRNAs in the lymph nodes of EAE-susceptible Dark Agouti and EAE-resistant Piebald Virol Glaxo rats during immune activation. Forty-three miRNAs were found differentially expressed between the two strains, with 81% (35 out of 43) showing higher expression in the susceptible strain. Only 33% of tested miRNAs displayed differential expression in naive lymph nodes, suggesting that a majority of regulated miRNAs are EAE dependent. Further investigation of a selected six miRNAs indicates differences in cellular source and kinetics of expression. Several of the miRNAs, including miR-146a, miR-21, miR-181a, miR-223, and let-7, have previously been implicated in immune system regulation. Moreover, 77% (33 out of 43) of the miRNAs were associated with MS and other autoimmune diseases. Target genes likely regulated by the miRNAs were identified using computational predictions combined with whole-genome expression data. Differentially expressed miRNAs and their targets involve functions important for MS and EAE, such as immune cell migration through targeting genes like Cxcr3 and cellular maintenance and signaling by regulation of Prkcd and Stat1. In addition, we demonstrated that these three genes are direct targets of miR-181a. Our study highlights the impact of multiple miRNAs, displaying diverse kinetics and cellular sources, on development of pathogenic autoimmune inflammation.
Assuntos
Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/patologia , MicroRNAs/genética , Análise de Sequência de RNA , Animais , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/imunologia , Feminino , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , MicroRNAs/biossíntese , Ratos , Ratos Endogâmicos , Análise de Sequência de RNA/métodos , Análise de Sequência de RNA/tendências , Especificidade da EspécieRESUMO
Mice lacking the thermogenic mitochondrial membrane protein UCP1 (uncoupling protein 1)--and thus all heat production from brown adipose tissue--can still adapt to a cold environment (4 degrees C) if successively transferred to the cold. The mechanism behind this adaptation has not been clarified. To examine possible adaptive processes in the skeletal muscle, we isolated mitochondria from the hind limb muscles of cold-acclimated wild-type and UCP1(-/-) mice and examined their bioenergetic chracteristics. We observed a switch in metabolism, from carbohydrate towards lipid catabolism, and an increased total mitochondrial complement, with an increased total ATP production capacity. The UCP1(-/-) muscle mitochondria did not display a changed state-4 respiration rate (no uncoupling) and were less sensitive to the uncoupling effect of fatty acids than the wild-type mitochondria. The content of UCP3 was increased 3-4 fold, but despite this, endogenous superoxide could not invoke a higher proton leak, and the small inhibitory effect of GDP was unaltered, indicating that it was not mediated by UCP3. Double mutant mice (UCP1(-/-) plus superoxide dismutase 2-overexpression) were not more cold sensitive than UCP1(-/-), bringing into question an involvement of reactive oxygen species (ROS) in activation of any alternative thermogenic mechanism. We conclude that there is no evidence for an involvement of UCP3 in basal, fatty-acid- or superoxide-stimulated oxygen consumption or in GDP sensitivity. The adaptations observed did not imply any direct alternative process for nonshivering thermogenesis but the adaptations observed would be congruent with adaptation to chronically enhanced muscle activity caused by incessant shivering in these mice.
Assuntos
Aclimatação/fisiologia , Canais Iônicos/deficiência , Canais Iônicos/metabolismo , Mitocôndrias Musculares/metabolismo , Proteínas Mitocondriais/deficiência , Proteínas Mitocondriais/metabolismo , Aclimatação/genética , Trifosfato de Adenosina/biossíntese , Animais , Temperatura Baixa , Metabolismo Energético , Ácidos Graxos/metabolismo , Ácidos Graxos/farmacologia , Feminino , Guanosina Difosfato/farmacologia , Técnicas In Vitro , Canais Iônicos/genética , Peroxidação de Lipídeos , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias Musculares/efeitos dos fármacos , Proteínas Mitocondriais/genética , Músculo Esquelético/metabolismo , Estresse Oxidativo , Consumo de Oxigênio , Espécies Reativas de Oxigênio/metabolismo , Superóxidos/metabolismo , Termogênese/genética , Termogênese/fisiologia , Proteína Desacopladora 1 , Proteína Desacopladora 3 , Regulação para CimaRESUMO
ATP-synthase assembly requires coordinated control of ATP mRNA translation; this may e.g. occur through the formation of mRNA-protein complexes. In this study we aim to identify sequences in the 3'UTR of the beta-subunit F(1)-ATPase mRNA necessary for RNA-protein complex formation. We examined the interaction between a brain cytoplasmic protein extract and in vitro-synthesized beta-subunit 3'UTR probes containing successive accumulative 5'- and 3'-deletions, as well as single subregion deletions, with or without poly(A) tail. Using electrophoretic mobility shift assays we found that two major RNA-protein complexes (here called RPC1 and RPC2) were formed with the full-length 3'UTR. The RPC2 complex formation was fully dependent on the presence of both the poly(A) tail and one subregion directly adjacent to it. For RPC1 complex formation, a 3'UTR sequence stretch (experimentally divided into three subregions) adjacent to but not including the poly(A) tail was necessary. This sequence stretch includes a conserved 40-nucleotide region that, according to the structure prediction program mfold, is able to fold into a characteristic stem-loop structure. Since the formation of the RPC1 complex was not dependent on a conventional sequence motif in the 3'UTR of the beta-subunit mRNA but rather on the presence of the predicted stem-loop-forming region as such, we hypothetize that this RNA region, by forming a stem-loop in the 3'UTR beta-subunit mRNA, is necessary for formation of the RNA-protein complex.
Assuntos
Regiões 3' não Traduzidas/genética , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/metabolismo , RNA Mensageiro/genética , RNA/genética , Animais , Sequência de Bases , Primers do DNA , DNA Complementar/genética , Masculino , Camundongos , Camundongos Endogâmicos , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Plasmídeos , Reação em Cadeia da Polimerase , Biossíntese de Proteínas , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Mensageiro/química , Deleção de Sequência , Transcrição GênicaRESUMO
Despite the significance of mitochondrial ATP synthase for mammalian metabolism, the regulation of the amount of ATP synthase in mammalian systems is not understood. As brown adipose tissue mitochondria contain very low amounts of ATP synthase, relative to respiratory chain components, they constitute a physiological system that allows for examination of the control of ATP synthase assembly. To examine the role of the expression of the P1-isoform of the c-Fo subunit in the biogenesis of ATP synthase, we made transgenic mice that express the P1-c subunit isoform under the promoter of the brown adipose tissue-specific protein UCP1. In the resulting UCP1p1 transgenic mice, total P1-c subunit mRNA levels were increased; mRNA levels of other F1Fo-ATPase subunits were unchanged. In isolated brown-fat mitochondria, protein levels of the total c-Fo subunit were increased. Remarkably, protein levels of ATP synthase subunits that are part of the F1-ATPase complex were also increased, as was the entire Complex V. Increased ATPase and ATP synthase activities demonstrated an increased functional activity of the F1Fo-ATPase. Thus, the levels of the c-Fo subunit P1-isoform are crucial for defining the final content of the ATP synthase in brown adipose tissue. The level of c-Fo subunit may be a determining factor for F1Fo-ATPase assembly in all higher eukaryotes.
Assuntos
Tecido Adiposo Marrom/enzimologia , Isoenzimas/metabolismo , Mitocôndrias/enzimologia , ATPases Translocadoras de Prótons/metabolismo , Animais , Sequência de Bases , Western Blotting , Primers do DNA , Efeito Fundador , Canais Iônicos/genética , Canais Iônicos/fisiologia , Camundongos , Camundongos Transgênicos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/fisiologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Desacopladora 1RESUMO
Fragmentation of DNA is the very important first step in preparing nucleic acids for next-generation sequencing. Here we report a novel Fragmentation Through Polymerization (FTP) technique, which is a simple, robust, and low-cost enzymatic method of fragmentation. This method generates double-stranded DNA fragments that are suitable for direct use in NGS library construction and allows the elimination of the additional step of reparation of DNA ends.
Assuntos
Fragmentação do DNA , DNA/química , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Polimerização , Análise de Sequência de DNA/métodos , Biologia Computacional , DNA/genética , Desoxirribonuclease I/química , Biblioteca GênicaRESUMO
BACKGROUND: Due to limited access to brain tissue, the precise mechanisms underlying neuro-axonal dysfunction in neurological disorders such as multiple sclerosis (MS) are largely unknown. In that context, profiling DNA methylation, which is a stable and cell type-specific regulatory epigenetic mark of genome activity, offers a unique opportunity to characterize the molecular mechanisms underpinning brain pathology in situ. We examined DNA methylation patterns of neuronal nuclei isolated from post-mortem brain tissue to infer processes that occur in neurons of MS patients. RESULTS: We isolated subcortical neuronal nuclei from post-mortem white matter tissue of MS patients and non-neurological controls using flow cytometry. We examined bulk DNA methylation changes (total n = 29) and further disentangled true DNA methylation (5mC) from neuron-specific DNA hydroxymethylation (5hmC) (n = 17), using Illumina Infinium 450K arrays. We performed neuronal sub-type deconvolution using glutamate and GABA methylation profiles to further reduce neuronal sample heterogeneity. In total, we identified 2811 and 1534 significant (genome-wide adjusted P value < 0.05) differentially methylated and hydroxymethylated positions between MS patients and controls. We found striking hypo-5mC and hyper-5hmC changes occurring mainly within gene bodies, which correlated with reduced transcriptional activity, assessed using published RNAseq data from bulk brain tissue of MS patients and controls. Pathway analyses of the two cohorts implicated dysregulation of genes involved in axonal guidance and synaptic plasticity, with meta-analysis confirming CREB signalling as the most highly enriched pathway underlying these processes. We functionally investigated DNA methylation changes of CREB signalling-related genes by immunohistofluoresence of phosphorylated CREB in neurons from brain sections of a subcohort of MS patients and controls (n = 15). Notably, DNA methylation changes associated with a reduction of CREB activity in white matter neurons of MS patients compared to controls. CONCLUSIONS: Our data demonstrate that investigating 5mC and 5hmC modifications separately allows the discovery of a substantial fraction of changes occurring in neurons, which can escape traditional bisulfite-based DNA methylation analysis. Collectively, our findings indicate that neurons of MS patients acquire sustained hypo-5mC and hyper-5hmC, which may impair CREB-mediated neuro-axonal integrity, in turn relating to clinical symptoms.
Assuntos
5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Metilação de DNA , Esclerose Múltipla/genética , Neurônios/química , Adulto , Idoso , Idoso de 80 Anos ou mais , Cadáver , Estudos de Casos e Controles , Regulação para Baixo , Epigênese Genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/metabolismo , Neurônios/citologia , Fosforilação , Análise de Sequência de DNA , Transdução de Sinais , Substância Branca/química , Substância Branca/citologiaRESUMO
A physiological function of the original uncoupling protein, UCP1, is well established: UCP1 is the molecular background for nonshivering thermogenesis. The functions of the "novel" UCPs, UCP2 and UCP3, are still not established. Recent discussions imply that all UCPs may play a role in protection against reactive oxygen species (ROS). Here we examine critically the evidence that UCP1, UCP2 and UCP3 are stimulated by ROS (superoxide) or ROS products (4-hydroxy-2-nonenal), and that the UCPs actually diminish oxidative damage. We conclude that, concerning UCP1, it is unlikely that it has such a role; concerning UCP2/UCP3, most evidence for physiologically significant roles in this respect is still circumstantial.
Assuntos
Proteínas de Transporte/fisiologia , Proteínas de Membrana/fisiologia , Proteínas de Membrana Transportadoras/fisiologia , Proteínas Mitocondriais/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Aldeídos/metabolismo , Aldeídos/farmacologia , Animais , Proteínas de Transporte/antagonistas & inibidores , Humanos , Canais Iônicos , Proteínas de Membrana/antagonistas & inibidores , Proteínas Mitocondriais/antagonistas & inibidores , Estresse Oxidativo , Nucleotídeos de Purina/metabolismo , Nucleotídeos de Purina/farmacologia , Superóxidos/metabolismo , Superóxidos/farmacologia , Proteína Desacopladora 1 , Proteína Desacopladora 2 , Proteína Desacopladora 3RESUMO
In brown-fat mitochondria, fatty acids induce thermogenic uncoupling through activation of UCP1 (uncoupling protein 1). However, even in brown-fat mitochondria from UCP1-/- mice, fatty-acid-induced uncoupling exists. In the present investigation, we used the inhibitor CAtr (carboxyatractyloside) to examine the involvement of the ANT (adenine nucleotide translocator) in the mediation of this UCP1-independent fatty-acid-induced uncoupling in brown-fat mitochondria. We found that the contribution of ANT to fatty-acid-induced uncoupling in UCP1-/- brown-fat mitochondria was minimal (whereas it was responsible for nearly half the fatty-acid-induced uncoupling in liver mitochondria). As compared with liver mitochondria, brown-fat mitochondria exhibit a relatively high (UCP1-independent) basal respiration ('proton leak'). Unexpectedly, a large fraction of this high basal respiration was sensitive to CAtr, whereas in liver mitochondria, basal respiration was CAtr-insensitive. Total ANT protein levels were similar in brown-fat mitochondria from wild-type mice and in liver mitochondria, but the level was increased in brown-fat mitochondria from UCP1-/- mice. However, in liver, only Ant2 mRNA was found, whereas in brown adipose tissue, Ant1 and Ant2 mRNA levels were equal. The data are therefore compatible with a tentative model in which the ANT2 isoform mediates fatty-acid-induced uncoupling, whereas the ANT1 isoform may mediate a significant part of the high basal proton leak in brown-fat mitochondria.
Assuntos
Translocador 1 do Nucleotídeo Adenina/fisiologia , Translocador 2 do Nucleotídeo Adenina/fisiologia , Tecido Adiposo Marrom/efeitos dos fármacos , Atractilosídeo/análogos & derivados , Ácidos Graxos/farmacologia , Mitocôndrias/efeitos dos fármacos , Desacopladores/farmacologia , Translocador 1 do Nucleotídeo Adenina/antagonistas & inibidores , Translocador 1 do Nucleotídeo Adenina/biossíntese , Translocador 1 do Nucleotídeo Adenina/genética , Translocador 2 do Nucleotídeo Adenina/antagonistas & inibidores , Translocador 2 do Nucleotídeo Adenina/biossíntese , Translocador 2 do Nucleotídeo Adenina/genética , Tecido Adiposo Marrom/metabolismo , Animais , Atractilosídeo/farmacologia , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Respiração Celular/efeitos dos fármacos , Cruzamentos Genéticos , Ácidos Graxos/metabolismo , Guanosina Difosfato/farmacologia , Canais Iônicos/deficiência , Canais Iônicos/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Proteínas Mitocondriais/deficiência , Proteínas Mitocondriais/genética , Modelos Biológicos , Ácido Oleico/farmacologia , Especificidade de Órgãos , Consumo de Oxigênio/efeitos dos fármacos , Palmitatos/farmacologia , Prótons , Ácido Pirúvico/farmacologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Termogênese/efeitos dos fármacos , Termogênese/fisiologia , Proteína Desacopladora 1RESUMO
Whole-genome amplification (WGA) techniques are used for non-specific amplification of low-copy number DNA, and especially for single-cell genome and transcriptome amplification. There are a number of WGA methods that have been developed over the years. One example is degenerate oligonucleotide-primed PCR (DOP-PCR), which is a very simple, fast and inexpensive WGA technique. Although DOP-PCR has been regarded as one of the pioneering methods for WGA, it only provides low genome coverage and a high allele dropout rate when compared to more modern techniques. Here we describe an improved DOP-PCR (iDOP-PCR). We have modified the classic DOP-PCR by using a new thermostable DNA polymerase (SD polymerase) with a strong strand-displacement activity and by adjustments in primers design. We compared iDOP-PCR, classic DOP-PCR and the well-established PicoPlex technique for whole genome amplification of both high- and low-copy number human genomic DNA. The amplified DNA libraries were evaluated by analysis of short tandem repeat genotypes and NGS data. In summary, iDOP-PCR provided a better quality of the amplified DNA libraries compared to the other WGA methods tested, especially when low amounts of genomic DNA were used as an input material.
Assuntos
Primers do DNA , Dosagem de Genes , Genoma Humano , Genômica , Reação em Cadeia da Polimerase/métodos , Variações do Número de Cópias de DNA , Biblioteca Gênica , Genômica/métodos , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , HumanosRESUMO
The environmental pollutants perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) cause a dramatic reduction in the size of the major adipose tissue depots and a general body weight decrease when they are added to the food of mice. We demonstrate here that this is mainly due to a reduction in food intake; this reduction was not due to food aversion. Remarkably and unexpectedly, a large part of the effect of PFOA/PFOS on food intake was dependent on the presence of the uncoupling protein 1 (UCP1) in the mice. Correspondingly, PFOA/PFOS treatment induced recruitment of brown adipose tissue mitochondria: increased oxidative capacity and increased UCP1-mediated oxygen consumption (thermogenesis). In mice pair-fed to the food intake during PFOA/PFOS treatment in wildtype mice, brown-fat mitochondrial recruitment was also induced. We conclude that we have uncovered the existence of a regulatory component of food intake that is dependent upon brown adipose tissue thermogenic activity. The possible environmental consequences of this novel PFOA/PFOS effect (a possible decreased fitness) are noted, as well as the perspectives of this finding on the general understanding of control of food intake control and its possible extension to combatting obesity.
Assuntos
Tecido Adiposo Marrom/efeitos dos fármacos , Ácidos Alcanossulfônicos/toxicidade , Caprilatos/toxicidade , Ingestão de Energia , Fluorocarbonos/toxicidade , Canais Iônicos/metabolismo , Mitocôndrias/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , Regulação para Cima/efeitos dos fármacos , Tecido Adiposo Marrom/metabolismo , Animais , Poluentes Ambientais/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Proteína Desacopladora 1RESUMO
The sensitivity and robustness of various DNA detection and amplification techniques are to a large extent determined by the properties of the DNA polymerase used. We have compared the performance of conventional Taq and Bst DNA polymerases to a novel Taq DNA polymerase mutant (SD DNA polymerase), which has a strong strand displacement activity, in PCR (including amplification of GC-rich and complex secondary structure templates), long-range PCR (LR PCR), loop-mediated amplification (LAMP), and polymerase chain displacement reaction (PCDR). Our results demonstrate that the strand displacement activity of SD DNA polymerase, in combination with the robust polymerase activity, provides a notable improvement in the sensitivity and efficiency of all these methods.
Assuntos
DNA/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase/métodos , Taq Polimerase/genética , Animais , DNA/genética , Primers do DNA/genética , Camundongos , Mycobacterium tuberculosis/genética , Sensibilidade e Especificidade , Taq Polimerase/químicaRESUMO
XAP2 (also known as aryl hydrocarbon receptor interacting protein, AIP) is originally identified as a negative regulator of the hepatitis B virus X-associated protein. Recent studies have expanded the range of XAP2 client proteins to include the nuclear receptor family of transcription factors. In this study, we show that XAP2 is recruited to the promoter of ERα regulated genes like the breast cancer marker gene pS2 or GREB1 and negatively regulate the expression of these genes in MCF-7 cells. Interestingly, we show that XAP2 downregulates the E2-dependent transcriptional activation in an estrogen receptor (ER) isoform-specific manner: XAP2 inhibits ERα but not ERß-mediated transcription. Thus, knockdown of intracellular XAP2 levels leads to increased ERα activity. XAP2 proteins, carrying mutations in their primary structures, loose the ability of interacting with ERα and can no longer regulate ER target gene transcription. Taken together, this study shows that XAP2 exerts a negative effect on ERα transcriptional activity and may thus prevent ERα-dependent events.
Assuntos
Receptor alfa de Estrogênio/metabolismo , Estrogênios/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Transdução de Sinais , Animais , Linhagem Celular Tumoral , Estradiol/farmacologia , Receptor beta de Estrogênio/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Camundongos , Modelos Biológicos , Ligação Proteica , Isoformas de Proteínas/metabolismo , Proteínas Repressoras/metabolismo , Elementos de Resposta/genética , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
Uncoupling proteins have been ascribed a role in defense against oxidative stress, particularly by being activated by products of oxidative stress such as 4-hydroxy-2-nonenal (HNE). We have investigated here the ability of HNE to activate UCP1. Using brown fat mitochondria from UCP1+/+ and UCP1-/- mice to allow for identification of UCP1-dependent effects, we found that HNE could neither (re)activate purine nucleotide-inhibited UCP1, nor induce additional activation of innately active UCP1. The aldehyde nonenal had a (re)activating effect only if converted to the corresponding fatty acid by aldehyde dehydrogenase; the presence of a carboxyl group was thus an absolute requirement for (re)activation. The UCP1-dependent proton leak was not increased by HNE but HNE changed basal proton leak characteristics in a UCP1-independent manner. In agreement with the in vitro results, we found, as compared with UCP1+/+ mice, no increase in HNE/protein adducts in brown fat mitochondria isolated from UCP1-/- mice, irrespective of whether they were adapted to thermoneutral temperature (30 degrees C) or to the cold (4 degrees C). The absence of oxidative damage in UCP1-/- mitochondria was not due to enhanced activity of antioxidant enzymes. Thus, HNE did not affect UCP1 activity, and UCP1 would appear not to be physiologically involved in defense against oxidative stress. Additionally, it was concluded that at least in brown adipose tissue, conditions of high mitochondrial membrane potential, high oxygen tension, and high substrate supply do not necessarily lead to increased oxidative damage.