RESUMO
Maintenance of the drug-addicted state is thought to involve changes in gene expression in different neuronal cell types and neural circuits. Midbrain dopamine (DA) neurons in particular mediate numerous responses to drugs of abuse. Long noncoding RNAs (lncRNAs) regulate CNS gene expression through a variety of mechanisms, but next to nothing is known about their role in drug abuse. The proportion of lncRNAs that are primate-specific provides a strong rationale for their study in human drug abusers. In this study, we determined a profile of dysregulated putative lncRNAs through the analysis of postmortem human midbrain specimens from chronic cocaine abusers and well-matched control subjects (n = 11 in each group) using a custom lncRNA microarray. A dataset comprising 32 well-annotated lncRNAs with independent evidence of brain expression and robust differential expression in cocaine abusers is presented. For a subset of these lncRNAs, differential expression was validated by quantitative real-time PCR and cellular localization determined by in situ hybridization histochemistry. Examples of lncRNAs exhibiting DA cell-specific expression, different subcellular distributions, and covariance of expression with known cocaine-regulated protein-coding genes were identified. These findings implicate lncRNAs in the cellular responses of human DA neurons to chronic cocaine abuse. Long noncoding RNAs (lncRNAs) regulate the expression of protein-coding genes, but little is known about their potential role in drug abuse. In this study, we identified lncRNAs differentially expressed in human cocaine abusers' midbrains. One up-regulated antisense lncRNA, tumor necrosis factor receptor-associated factor 3-interacting protein 2-antisense 1 (TRAF3IP2-AS1), was found predominantly in the nucleus of human dopamine (DA) neurons, whereas the related TRAF3IP2 protein-coding transcript was distributed throughout these cells. The abundances of these transcripts were significantly correlated (left) suggesting that TRAF3IP2-AS1 may regulate TRAF3IP2 gene expression, perhaps through local chromatin changes at this locus (right).
Assuntos
Transtornos Relacionados ao Uso de Cocaína/genética , Mesencéfalo/metabolismo , Neurônios/metabolismo , RNA Longo não Codificante/metabolismo , RNA/metabolismo , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Cocaína/farmacologia , Transtornos Relacionados ao Uso de Cocaína/metabolismo , Dopamina/genética , Dopamina/metabolismo , Humanos , Neurônios/efeitos dos fármacos , Transcrição GênicaRESUMO
Epilepsy is a disorder of recurrent seizures that affects 1% of the population. To understand why some areas of cerebral cortex produce seizures and others do not, we identified differentially expressed genes in human epileptic neocortex compared with nearby regions that did not produce seizures. The transcriptome that emerged strongly implicates MAPK signaling and CREB-dependent transcription, with 74% of differentially expressed genes containing a cAMP response element (CRE) in their proximal promoter, more than half of which are conserved. Despite the absence of recent seizures in these patients, epileptic brain regions prone to seizures showed persistent activation of ERK and CREB. Persistent CREB activation was directly linked to CREB-dependent gene transcription by chromatin immunoprecipitation that showed phosphorylated CREB constitutively associated with the proximal promoters of many of the induced target genes involved in neuronal signaling, excitability, and synaptic plasticity. A distinct spatial pattern of ERK activation was seen in superficial axodendritic processes of epileptic neocortex that colocalized with both CREB phosphorylation and CREB target gene induction in well demarcated populations of layer 2/3 neurons. These same neuronal lamina showed a marked increase in synaptic density. The findings generated in this study generate a robust and spatially restricted pattern of epileptic biomarkers and associated synaptic changes that could lead to new mechanistic insights and potential therapeutic targets for human epilepsy.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/biossíntese , Epilepsia/genética , Epilepsia/metabolismo , Regulação da Expressão Gênica , Marcação de Genes , Neocórtex/fisiologia , Adolescente , Adulto , Criança , Pré-Escolar , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Feminino , Marcação de Genes/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Neocórtex/citologia , Tempo de Reação/genética , Transcrição Gênica/fisiologiaRESUMO
Within the brain, the reduced pteridine cofactor 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) is absolutely required for the synthesis of the monoamine (MA) neurotransmitters dopamine (DA), norepinephrine, epinephrine (E), and serotonin (5-HT), the novel gaseous neurotransmitter nitric oxide and the production of yet to be identified 1-O-alkylglycerol-derived lipids. GTP cyclohydrolase I (GTPCH) catalyzes the first and limiting step in the BH4 biosynthetic pathway, which is now thought to involve up to eight different proteins supporting six alternate de novo and two alternate salvage pathways. Gene expression analysis across different regions of the human brain shows the abundance of transcripts coding for all eight of these proteins to be highly correlated with each other and to be enriched within human MA neurons. The potential for multiple routes for BH4 synthesis therefore exists within the human brain. GTPCH expression is particularly heterogeneous across different populations of human and rodent MA-containing neurons, with low expression levels and therefore BH4 being a characteristic of nigrostriatal DA (NSDA) neurons. Basic knowledge of how GCH1 gene transcription is controlled within NSDA neurons may explain the distinctive susceptibility of these neurons to human genetic mutations that result in BH4 deficiency. A model for cyclic adenosine monophosphate-dependent GCH1 transcription is described that involves a unique combination of DNA regulatory sequences and transcription factors. This model proposes that low levels of GCH1 transcription within NSDA neurons are driven by their distinctive physiology, suggesting that pharmacological manipulation of GCH1 gene transcription can be used to modify BH4 levels and therefore DA synthesis in the basal ganglia.
Assuntos
Biopterinas/análogos & derivados , Dopamina/metabolismo , Neurônios Dopaminérgicos , GTP Cicloidrolase/metabolismo , Biopterinas/biossíntese , Biopterinas/genética , Biopterinas/metabolismo , Encéfalo/metabolismo , Dopamina/química , Neurônios Dopaminérgicos/química , Neurônios Dopaminérgicos/metabolismo , Epinefrina/química , Epinefrina/metabolismo , GTP Cicloidrolase/química , Humanos , Neurobiologia , Neurotransmissores/química , Neurotransmissores/metabolismo , Serotonina/metabolismo , Transcrição GênicaRESUMO
Although recent data suggest that some long non-coding RNAs (lncRNAs) exert widespread effects on gene expression and organelle formation, lncRNAs as a group constitute a sizable but poorly characterized fraction of the human transcriptome. We investigated whether some human lncRNA sequences were fortuitously represented on commonly used microarrays, then used this annotation to assess lncRNA expression in human brain. A computational and annotation pipeline was developed to identify lncRNA transcripts represented on Affymetrix U133 arrays. A previously published dataset derived from human nucleus accumbens was then examined for potential lncRNA expression. Twenty-three lncRNAs were determined to be represented on U133 arrays. Of these, dataset analysis revealed that five lncRNAs were consistently detected in samples of human nucleus accumbens. Strikingly, the abundance of these lncRNAs was up-regulated in human heroin abusers compared to matched drug-free control subjects, a finding confirmed by quantitative PCR. This study presents a paradigm for examining existing Affymetrix datasets for the detection and potential regulation of lncRNA expression, including changes associated with human disease. The finding that all detected lncRNAs were up-regulated in heroin abusers is consonant with the proposed role of lncRNAs as mediators of widespread changes in gene expression as occur in drug abuse.
Assuntos
Química Encefálica/genética , Mineração de Dados/métodos , Dependência de Heroína/genética , Núcleo Accumbens/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA não Traduzido/biossíntese , RNA não Traduzido/genética , Analgésicos Opioides/efeitos adversos , Química Encefálica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Marcadores Genéticos/efeitos dos fármacos , Marcadores Genéticos/fisiologia , Heroína/efeitos adversos , Dependência de Heroína/metabolismo , Humanos , Núcleo Accumbens/efeitos dos fármacos , Reação em Cadeia da Polimerase/métodos , RNA não Traduzido/efeitos dos fármacos , Recompensa , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
CCAAT/Enhancer Binding Proteins (C/EBPs) play pivotal roles in the development and plasticity of the nervous system. Identification of the physiological targets of C/EBPs (C/EBP target genes) should therefore provide insight into the underlying biology of these processes. We used unbiased genome-wide mapping to identify 115 C/EBPbeta target genes in PC12 cells that include transcription factors, neurotransmitter receptors, ion channels, protein kinases and synaptic vesicle proteins. C/EBPbeta binding sites were located primarily within introns, suggesting novel regulatory functions, and were associated with binding sites for other developmentally important transcription factors. Experiments using dominant negatives showed C/EBPbeta to repress transcription of a subset of target genes. Target genes in rat brain were subsequently found to preferentially bind C/EBPalpha, beta and delta. Analysis of the hippocampal transcriptome of C/EBPbeta knockout mice revealed dysregulation of a high percentage of transcripts identified as C/EBP target genes. These results support the hypothesis that C/EBPs play non-redundant roles in the brain.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Neurônios/fisiologia , Isoformas de Proteínas/metabolismo , Animais , Sítios de Ligação , Encéfalo/citologia , Encéfalo/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Imunoprecipitação da Cromatina , AMP Cíclico/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Células PC12 , Isoformas de Proteínas/genética , Ratos , Reprodutibilidade dos TestesRESUMO
Mutations in GTP-cyclohydrolase 1 (GCH1) cause autosomal dominant dopa-responsive dystonia (DRD), characterized by childhood-onset foot dystonia that later generalizes. DRD patients frequently present with associated Parkinsonism. Conversely, early-onset Parkinson's disease (EOPD) patients commonly display dystonia. Herein, we investigated the frequency of GCH1 mutations in a series of 53 familial EOPD patients (21 with dystonia) and screened them for mutations in PRKN, PINK1, and DJ-1. In addition, we examined a matched EOPD patient-control series for association of common variability at the GCH1 locus and EOPD susceptibility. No GCH1 coding change or copy-number abnormality was identified in familial EOPD patients. A novel 18-bp deletion was found in the proximal promoter (two patients, one control), which is expected to knock out two regulatory elements previously shown to regulate GCH1 transcription. No association was found between GCH1 variability and risk of EOPD. Fourteen (26.4%) familial EOPD patients had homozygous or compound heterozygous PRKN mutations. PRKN-positive patients were 10 years younger than PRKN-negative patients and had a twofold higher prevalence of dystonia. This study does not support a significant role for genetic variation at the GCH1 locus in EOPD. However, our results further highlight the relevance of PRKN screening in familial EOPD.
Assuntos
GTP Cicloidrolase/genética , Doença de Parkinson/genética , Adulto , Idade de Início , Idoso , Idoso de 80 Anos ou mais , DNA/genética , Análise Mutacional de DNA , Feminino , Dosagem de Genes , Frequência do Gene , Variação Genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Pessoa de Meia-Idade , América do Norte/epidemiologia , Proteínas Oncogênicas/genética , Doença de Parkinson/epidemiologia , Polimorfismo de Nucleotídeo Único , Proteína Desglicase DJ-1 , Proteínas Quinases/genética , Ubiquitina-Proteína Ligases/genética , População BrancaRESUMO
The role of the proximal promoter GC-box in regulating basal and cAMP-dependent GTP Cyclohydrolase I gene transcription was investigated using a variety of cell lines and techniques. These studies show that the GC-box is composed of a triad of cis-elements that in vitro bind specificity proteins Sp1 and Sp3. Sp1 and Sp3 were found associated with the native proximal promoter in PC12 cells but were not recruited to the promoter during cAMP-dependent transcription. Studies using Drosophila SL2 cells showed that Sp3 occupies two sites within the GC-box and enhances transcription when acting alone and synergistically when combined with nuclear factor-Y (NF-Y) and CCAAT/Enhancer-Binding Protein (C/EBP)beta, cognate binding proteins for the adjacent cAMP response element (CRE) and CCAAT-box cAMP response elements. In contrast, Sp1 bound only one site within the GC-box and did not enhance transcription unless combined with NF-Y and C/EBPbeta. Studies in SL2 cells also showed that Sp1 and Sp3 do not co-occupy the GC-box, and accordingly Sp1 competes for Sp3 binding to repress Sp3-dependent transcription. In PC12 cells, complete mutation of the GC-box reduced basal but not cAMP-dependent transcription, resulting in an overall increase in the cAMP response and demonstrating that formation of this enhanceosome does not require Sp1 or Sp3. Experiments in which the GC-box was replaced with a Gal4 element and the promoter challenged with Gal4 fusion proteins support this conclusion and a role for Sp3 in maintaining high levels of basal transcription in PC12 cells. Equivalent amounts of Sp1 and Sp3 were found associated with the native proximal promoter in PC12 and Rat2 cells, which differ 10-fold in basal transcription. Similar levels of methylation of CpG dinucleotides located within the GC-box were also observed in these two cells lines. These results suggest that Sp1 and Sp3 bound to the GC-box might help to preserve an open chromatin configuration at the proximal promoter in cells which constitutively express low levels of GTP Cyclohydrolase I.
Assuntos
GTP Cicloidrolase/genética , GTP Cicloidrolase/metabolismo , Regiões Promotoras Genéticas , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp3/metabolismo , Transcrição Gênica/fisiologia , Animais , Linhagem Celular , Mutagênese Sítio-Dirigida , Células PC12 , Ligação Proteica/genética , Transporte Proteico/genética , Ratos , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/fisiologia , Fator de Transcrição Sp3/genética , Fator de Transcrição Sp3/fisiologiaRESUMO
Cyclic AMP-responsive element binding protein, hepatocyte specific (CREBH), is a liver-enriched, endoplasmic reticulum-tethered transcription factor known to regulate the hepatic acute-phase response and lipid homeostasis. In this study, we demonstrate that CREBH functions as a circadian transcriptional regulator that plays major roles in maintaining glucose homeostasis. The proteolytic cleavage and posttranslational acetylation modification of CREBH are regulated by the circadian clock. Functionally, CREBH is required in order to maintain circadian homeostasis of hepatic glycogen storage and blood glucose levels. CREBH regulates the rhythmic expression of the genes encoding the rate-limiting enzymes for glycogenolysis and gluconeogenesis, including liver glycogen phosphorylase (PYGL), phosphoenolpyruvate carboxykinase 1 (PCK1), and the glucose-6-phosphatase catalytic subunit (G6PC). CREBH interacts with peroxisome proliferator-activated receptor α (PPARα) to synergize its transcriptional activities in hepatic gluconeogenesis. The acetylation of CREBH at lysine residue 294 controls CREBH-PPARα interaction and synergy in regulating hepatic glucose metabolism in mice. CREBH deficiency leads to reduced blood glucose levels but increases hepatic glycogen levels during the daytime or upon fasting. In summary, our studies revealed that CREBH functions as a key metabolic regulator that controls glucose homeostasis across the circadian cycle or under metabolic stress.
Assuntos
Ritmo Circadiano/fisiologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Gluconeogênese/fisiologia , Glucose/metabolismo , Glicogenólise/genética , Homeostase/fisiologia , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica/fisiologia , Hepatócitos/metabolismo , Fígado/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Drug abuse is thought to induce long-term cellular and behavioral adaptations as a result of alterations in gene expression. Understanding the molecular consequences of addiction may contribute to the development of better treatment strategies. This study utilized high-throughput Affymetrix microarrays to identify gene expression changes in the post-mortem nucleus accumbens of chronic heroin abusers. These data were analyzed independently and in relation to our previously reported data involving human cocaine abusers, in order to determine which expression changes were drug specific and which may be common to the phenomenon of addiction. A significant decrease in the expression of numerous genes encoding proteins involved in presynaptic release of neurotransmitter was seen in heroin abusers, a finding not seen in the cocaine-abusing cohort. Conversely, the striking decrease in myelin-related genes observed in cocaine abusers was not evident in our cohort of heroin subjects. Overall, little overlap in gene expression profiles was seen between the two drug-abusing cohorts: out of the approximately 39,000 transcripts investigated, the abundance of only 25 was significantly changed in both cocaine and heroin abusers, with nearly one-half of these being altered in opposite directions. These data suggest that the profiles of nucleus accumbens gene expression associated with chronic heroin or cocaine abuse are largely unique, despite what are thought to be common effects of these drugs on dopamine neurotransmission in this brain region. A re-examination of our current assumptions about the commonality of molecular mechanisms associated with substance abuse seems warranted.
Assuntos
Transtornos Relacionados ao Uso de Cocaína/genética , Transtornos Relacionados ao Uso de Cocaína/patologia , Expressão Gênica/fisiologia , Dependência de Heroína/genética , Dependência de Heroína/patologia , Núcleo Accumbens/fisiopatologia , Adulto , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Mudanças Depois da Morte , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodosRESUMO
The circadian clock orchestrates diverse physiological processes critical for health and disease. CREB, hepatocyte specific (CREBH) is a liver-enriched, endoplasmic reticulum (ER)-tethered transcription factor known to regulate the hepatic acute phase response and energy homeostasis under stress conditions. We demonstrate that CREBH is regulated by the circadian clock and functions as a circadian regulator of hepatic lipid metabolism. Proteolytic activation of CREBH in the liver exhibits typical circadian rhythmicity controlled by the core clock oscillator BMAL1 and AKT/glycogen synthase kinase 3ß (GSK3ß) signaling pathway. GSK3ß-mediated phosphorylation of CREBH modulates the association between CREBH and the coat protein complex II transport vesicle and thus controls the ER-to-Golgi transport and subsequent proteolytic cleavage of CREBH in a circadian manner. Functionally, CREBH regulates circadian expression of the key genes involved in triglyceride (TG) and fatty acid (FA) metabolism and is required to maintain circadian amplitudes of blood TG and FA in mice. During the circadian cycle, CREBH rhythmically regulates and interacts with the hepatic nuclear receptors peroxisome proliferator-activated receptor α and liver X receptor α as well as with the circadian oscillation activator DBP and the repressor E4BP4 to modulate CREBH transcriptional activities. In conclusion, these studies reveal that CREBH functions as a circadian-regulated liver transcriptional regulator that integrates energy metabolism with circadian rhythm.
Assuntos
Relógios Circadianos/fisiologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Fígado/metabolismo , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Imunoprecipitação da Cromatina , Relógios Circadianos/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Ácidos Graxos/metabolismo , Quinases da Glicogênio Sintase/genética , Quinases da Glicogênio Sintase/metabolismo , Hepatócitos/metabolismo , Metabolismo dos Lipídeos/genética , Metabolismo dos Lipídeos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Sítio-Dirigida , Ligação Proteica , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Triglicerídeos/metabolismoRESUMO
BACKGROUND: We recently reported that arterial superoxide (O2-) is augmented by increased endothelin-1 (ET-1) in deoxycorticosterone acetate (DOCA)-salt hypertension, a model of low renin hypertension. Tetrahydrobiopterin (BH4), a potent reducing molecule with antioxidant properties and an essential cofactor for endothelial nitric oxide synthase, protects against O2--induced vascular dysfunction. However, the interaction between O2- and BH4 on endothelial function and the underlying mechanisms are unknown. METHODS AND RESULTS: The present study tested the hypothesis that BH4 deficiency due to ET-1-induced O2- leads to impaired endothelium-dependent relaxation and that gene transfer of human guanosine 5'-triphosphate (GTP) cyclohydrolase I (GTPCH I), the first and rate-limiting enzyme for BH4 biosynthesis, reverses such deficiency and endothelial dysfunction in carotid arteries of DOCA-salt rats. There were significantly increased arterial O2- levels and decreased GTPCH I activity and BH4 levels in DOCA-salt compared with sham rats. Treatment of arteries of DOCA-salt rats with the selective ETA receptor antagonist ABT-627, NADPH oxidase inhibitor apocynin, or superoxide dismutase (SOD) mimetic tempol abolished O2- and restored BH4 levels. Basal arterial NO release and endothelium-dependent relaxations were impaired in DOCA-salt rats, conditions that were improved by apocynin or tempol treatment. Gene transfer of GTPCH I restored arterial GTPCH I activity and BH4 levels, resulting in reduced O2- and improved endothelium-dependent relaxation and basal NO release in DOCA-salt rats. CONCLUSIONS: These results indicate that a BH4 deficiency resulting from ET-1-induced O2- via an ETA/NADPH oxidase pathway leads to endothelial dysfunction, and gene transfer of GTPCH I reverses the BH4 deficiency and endothelial dysfunction by reducing O2- in low renin mineralocorticoid hypertension.
Assuntos
Biopterinas/análogos & derivados , Biopterinas/metabolismo , Endotélio Vascular/fisiopatologia , GTP Cicloidrolase/genética , Terapia Genética/métodos , Hipertensão/terapia , Acetofenonas/uso terapêutico , Animais , Antioxidantes/uso terapêutico , Atrasentana , Biopterinas/deficiência , Artérias Carótidas/efeitos dos fármacos , Artérias Carótidas/fisiopatologia , Óxidos N-Cíclicos/uso terapêutico , Desoxicorticosterona , Modelos Animais de Doenças , Antagonistas dos Receptores de Endotelina , Endotelina-1/farmacologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Inibidores Enzimáticos/farmacologia , GTP Cicloidrolase/metabolismo , GTP Cicloidrolase/farmacologia , Técnicas de Transferência de Genes , Humanos , Hipertensão/induzido quimicamente , Hipertensão/fisiopatologia , Técnicas In Vitro , Masculino , Óxido Nítrico/metabolismo , Pirrolidinas/farmacologia , Ratos , Ratos Sprague-Dawley , Receptor de Endotelina A , Cloreto de Sódio , Marcadores de Spin , Superóxidos/metabolismo , Vasodilatação/efeitos dos fármacosRESUMO
Chronic drug abuse, craving, and relapse are thought to be linked to long-lasting changes in neural gene expression arising through transcriptional and chromatin-related mechanisms. The key contributions of midbrain dopamine (DA)-synthesizing neurons throughout the addiction process provide a compelling rationale for determining the drug-induced molecular changes that occur in these cells. Yet our understanding of these processes remains rudimentary. The postmortem human brain constitutes a unique resource that can be exploited to gain insights into the pathophysiology of complex disorders such as drug addiction. In this study, we analyzed the profiles of midbrain gene expression in chronic cocaine abusers and well-matched drug-free control subjects using microarray and quantitative PCR. A small number of genes exhibited robust differential expression; many of these are involved in the regulation of transcription, chromatin, or DA cell phenotype. Transcript abundances for approximately half of these differentially expressed genes were diagnostic for assigning subjects to the cocaine-abusing vs control cohort. Identification of a molecular signature associated with pathophysiological changes occurring in cocaine abusers' midbrains should contribute to the development of biomarkers and novel therapeutic targets for drug addiction.
Assuntos
Encéfalo/metabolismo , Transtornos Relacionados ao Uso de Cocaína/genética , Transtornos Relacionados ao Uso de Cocaína/metabolismo , Neurônios Dopaminérgicos/metabolismo , Mesencéfalo/metabolismo , Cromatina/metabolismo , Doença Crônica , Dopamina/metabolismo , Regulação da Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Análise em Microsséries , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Transcrição GênicaRESUMO
Dopa-responsive dystonia (DRD) is a familial childhood-onset disease characterized by fluctuating dystonia, associated with tremor and parkinsonism in some patients. In most families the disease displays autosomal dominant inheritance due to mutations in the GTP cyclohydrolase 1 gene (GCH1). Penetrance and symptom severity display strong female predominance for which gender-specific GCH1 expression has been hypothesized. In this study, GCH1 mRNA expression was measured in cerebellar tissue from 66 healthy human subjects (30 women), and in cerebellar and nigral tissue from eight individuals. No significant difference was found between men and women with small effect sizes observed. Although the correlation between cerebellar and nigral GCH1 expression remains to be further examined, this exploratory study does not support gender-specific GCH1 expression being the basis for the skewed gender distribution observed in DRD patients.
Assuntos
Cerebelo/enzimologia , GTP Cicloidrolase/metabolismo , Caracteres Sexuais , Idoso , Idoso de 80 Anos ou mais , Envelhecimento , Distúrbios Distônicos , Feminino , Humanos , Modelos Lineares , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , Substância Negra/metabolismoRESUMO
Cyclic-AMP stimulation of GTP cyclohydrolase I (GCH1) gene transcription was investigated in PC12 cells, the protein kinase A-deficient PC12 cell line 126-1B2 and C6 cells using transient transfection assays of proximal promoter reporter constructs and wild type or dominant negative proteins, chromatin immunoprecipitation and real-time quantitative PCR. These studies show that protein kinase A is necessary and sufficient for cAMP-dependent transcription conferred by both the cAMP regulatory element and the adjacent CCAAT-box. In intact cells these cis-elements were shown to bind cAMP response element binding protein, CCAAT-enhancer binding protein beta and nuclear factor-Y, with each protein controlling a different aspect of the cAMP response. Cyclic-AMP acting through protein kinase A stimulated promoter recruitment of CCAAT-enhancer binding protein beta, nuclear factor-Y and RNA polymerase II while depleting the promoter of cyclic-AMP response element binding protein. Stimulation of transcription by cAMP was not associated with increased acetylation of histones H3 and H4 at proximal promoter nucleosomes, indicating that histone acetyltransferases are not involved in this response. Nonetheless, pharmacological inhibition of histone deacetylase activity did increase histone H4 acetylation and the recruitment of RNA polymerase II, indicating that histone acetyltransferases are normally associated with the proximal promoter. Only in C6 cells, however, did inhibition of histone deacetylases stimulate transcription and synergize with cAMP. These experiments provide the first glimpse of the GCH1 gene promoter functioning within intact cells and supply evidence for the involvement of histone acetyltransferase-containing complexes in GCH1 gene transcription.
Assuntos
Fator de Ligação a CCAAT/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , GTP Cicloidrolase/genética , Histonas/metabolismo , Regiões Promotoras Genéticas/fisiologia , RNA Polimerase II/metabolismo , Fatores de Transcrição/metabolismo , Acetilação/efeitos dos fármacos , Animais , Imunoprecipitação da Cromatina/métodos , AMP Cíclico/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/deficiência , Interações Medicamentosas , Inibidores Enzimáticos/farmacologia , Histona Desacetilases/metabolismo , Células PC12 , Regiões Promotoras Genéticas/efeitos dos fármacos , RNA Mensageiro/biossíntese , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Transfecção/métodosRESUMO
The yeast 2-hybrid system was used to identify protein domains involved in the oligomerization of human guanosine 5'-triphosphate (GTP) Cyclohydrolase I (GCH1) and the interaction of GCH1 with its regulatory partner, GCH1 feedback regulatory protein (GFRP). When interpreted within the structural framework derived from crystallography, our results indicate that the GCH1 N-terminal alpha-helices are not the only domains involved in the formation of dimers from monomers and also suggest an important role for the C-terminal alpha-helix in the assembly of dimers to form decamers. Moreover, a previously unknown role of the extended N-terminal alpha-helix in the interaction of GCH1 and GFRP was revealed. To discover novel GCH1 protein binding partners, we used the yeast 2-hybrid system to screen a human brain library with GCH1 N-terminal amino acids 1-96 as prey. This protruding extension of GCH1 contains two canonical Type-I Src homology-3 (SH3) ligand domains located within amino acids 1-42. Our screen yielded seven unique clones that were subsequently shown to require amino acids 1-42 for binding to GCH1. The interaction of one of these clones, Activator of Heat Shock 90 kDa Protein (Aha1), with GCH1 was validated by glutathione-s-transferase (GST) pull-down assay. Although the physiological relevance of the Aha1-GCH1 interaction requires further study, Aha1 may recruit GCH1 into the endothelial nitric oxide synthase/heat shock protein (eNOS/Hsp90) complex to support changes in endothelial nitric oxide production through the local synthesis of BH4.
Assuntos
GTP Cicloidrolase/química , GTP Cicloidrolase/metabolismo , Guanosina Trifosfato/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Sequência de Aminoácidos/fisiologia , Sítios de Ligação/fisiologia , Biopterinas/análogos & derivados , Biopterinas/biossíntese , Chaperoninas , Cristalografia por Raios X , Endotélio Vascular/enzimologia , Ativação Enzimática/fisiologia , GTP Cicloidrolase/genética , Biblioteca Gênica , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Dados de Sequência Molecular , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo III/metabolismo , Polímeros/metabolismo , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína/fisiologia , Estrutura Terciária de Proteína/fisiologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Chronic cocaine abuse induces long-term neurochemical, structural and behavioural changes thought to result from altered gene expression within the nucleus accumbens and other brain regions playing a critical role in addiction. Recent methodological advances now allow the profiling of gene expression in human postmortem brain. In this article, we review studies in which we have used Affymetrix oligonucleotide microarrays to identify transcripts that are differentially expressed in the nucleus accumbens of cocaine abusers in comparison to well-matched control subjects. Of the approximately 39,000 gene transcripts interrogated, the expression of only a fraction of 1% is significantly modified in cocaine abusers. Found within this list are equivalent incidences of increased and decreased transcript abundance, including known gene transcripts clustered into several functional categories. A striking exception is a group of myelin-related genes, consisting of multiple transcripts representing myelin basic protein (MBP), proteolipid protein (PLP) and myelin-associated oligodendrocyte basic protein (MOBP), which as a group are substantially decreased in cocaine abusers compared to controls. These data, suggesting a possible dysregulation of myelin in cocaine abusers, are discussed in the context of myelin-related changes in other human brain disorders. Finally, the effects of cocaine abuse on the profile of gene expression in some other brain regions critical for addiction (the prefrontal cortex and ventral midbrain) are briefly reviewed.
Assuntos
Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Transtornos Relacionados ao Uso de Cocaína/metabolismo , Perfilação da Expressão Gênica/métodos , Humanos , Proteínas da Mielina , Proteína Proteolipídica de Mielina/efeitos dos fármacos , Proteína Proteolipídica de Mielina/genética , Glicoproteína Associada a Mielina/efeitos dos fármacos , Glicoproteína Associada a Mielina/genética , Glicoproteína Mielina-Oligodendrócito , RNA Mensageiro/biossíntese , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodosRESUMO
It is assumed that brain biopterin and dopamine loss should not be as severe in asymptomatic dopa-responsive dystonia caused by GCH1 mutations as it is in symptomatic dopa-responsive dystonia. However, the actual status of dopaminergic systems in asymptomatic cases is unknown. In the autopsied putamen of an asymptomatic GCH1 mutation carrier, we found that brain biopterin loss (-82%) paralleled that reported in dopa-responsive dystonia patients (-84%). However, tyrosine hydroxylase protein and dopamine levels (-52 and -44%, respectively) were not as severely affected as in symptomatic patients (exceeding -97 and -88%, respectively). Our data suggest that the extent of striatal tyrosine hydroxylase protein loss may be critical in determining dopa-responsive dystonia symptomatology.
Assuntos
Biopterinas/genética , Encéfalo/metabolismo , Distonia/tratamento farmacológico , Distonia/enzimologia , Levodopa/uso terapêutico , Tirosina 3-Mono-Oxigenase/genética , Antiparkinsonianos/uso terapêutico , Encéfalo/enzimologia , Encéfalo/patologia , Distonia/genética , Distonia/metabolismo , Evolução Fatal , Feminino , Humanos , Pessoa de Meia-Idade , SíndromeRESUMO
Chronic cocaine abuse induces long-term neural adaptations as a consequence of alterations in gene expression. This study was undertaken to identify those transcripts differentially regulated in the nucleus accumbens of human cocaine abusers. Affymetrix microarrays were used to measure transcript abundance in 10 cocaine abusers and 10 control subjects matched for age, race, sex, and brain pH. As expected, gene expression of cocaine- and amphetamine-regulated transcript (CART) was increased in the nucleus accumbens of cocaine abusers. The most robust and consistent finding, however, was a decrease in the expression of a number of myelin-related genes, including myelin basic protein (MBP), proteolipid protein (PLP), and myelin-associated oligodendrocyte basic protein (MOBP). The differential expression seen by microarray for CART as well as MBP, MOBP, and PLP was verified by RT-PCR. In addition, immunohistochemical experiments revealed a decrease in the number of MBP-immunoreactive oligodendrocytes present in the nucleus accumbens and surrounding white matter of cocaine abusers. These findings suggest a dysregulation of myelin in human cocaine abusers.
Assuntos
Transtornos Relacionados ao Uso de Cocaína/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Bainha de Mielina/genética , Núcleo Accumbens/metabolismo , Adulto , Contagem de Células , Transtornos Relacionados ao Uso de Cocaína/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteína Básica da Mielina/biossíntese , Proteína Básica da Mielina/genética , Proteínas da Mielina , Proteína Proteolipídica de Mielina/genética , Glicoproteína Associada a Mielina/genética , Glicoproteína Mielina-Oligodendrócito , Proteínas do Tecido Nervoso/genética , Núcleo Accumbens/patologia , Oligodendroglia/metabolismo , Oligodendroglia/patologia , RNA Mensageiro/metabolismo , Valores de Referência , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Gene expression profiles from the anterior cingulate cortex (ACC) of human, chimpanzee, gorilla, and macaque samples provide clues about genetic regulatory changes in human and other catarrhine primate brains. The ACC, a cerebral neocortical region, has human-specific histological features. Physiologically, an individual's ACC displays increased activity during that individual's performance of cognitive tasks. Of approximately 45,000 probe sets on microarray chips representing transcripts of all or most human genes, approximately 16,000 were commonly detected in human ACC samples and comparable numbers, 14,000-15,000, in gorilla and chimpanzee ACC samples. Phylogenetic results obtained from gene expression profiles contradict the traditional expectation that the non-human African apes (i.e., chimpanzee and gorilla) should be more like each other than either should be like humans. Instead, the chimpanzee ACC profiles are more like the human than like the gorilla; these profiles demonstrate that chimpanzees are the sister group of humans. Moreover, for those unambiguous expression changes mapping to important biological processes and molecular functions that statistically are significantly represented in the data, the chimpanzee clade shows at least as much apparent regulatory evolution as does the human clade. Among important changes in the ancestry of both humans and chimpanzees, but to a greater extent in humans, are the up-regulated expression profiles of aerobic energy metabolism genes and neuronal function-related genes, suggesting that increased neuronal activity required increased supplies of energy.
Assuntos
Encéfalo/fisiologia , Perfilação da Expressão Gênica , Hominidae/genética , Pan troglodytes/genética , Animais , Sequência de Bases , Mapeamento Cromossômico , Evolução Molecular , Genoma , Genoma Humano , Hominidae/classificação , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Pan troglodytes/classificação , Filogenia , Primatas/classificação , Primatas/genéticaRESUMO
Mutations in the parkin gene are common in early-onset and familial Parkinson's disease (PD), and the parkin protein interacts in the ubiquitin-proteasome system as an E3 ligase. However, the regulatory pathways that govern parkin expression are unknown. In this study, we showed that a phylogenetically conserved N-myc binding site in the bi-directional parkin promoter interacted with myc-family transcription factors in reporter assays, and N-myc bound to the parkin promoter in chromatin immunoprecipitation assays and repressed transcription activity. Parkin expression was inversely correlated with N-myc levels in the developing mouse and human brain, in human neuroblastoma cell lines with various levels of n-myc amplification, and in an inducible N-myc cell line. Although parkin and N-myc expression were dramatically altered upon retinoic acid-induced differentiation of a human neuroblastoma cell line, modulation of parkin expression did not significantly affect either rates of cellular proliferation or levels of cyclin E. Analysis of additional genes associated with familial PD revealed a shared basis of transcription regulation mediated by N-myc and the cell cycle. Our results, in combination with functional knowledge of the proteins encoded by these genes, suggest a common pathway linking together PD, the ubiquitin-proteasome system, and cell cycle control.