RESUMO
Neuronal remodeling is a fundamental process by which the brain responds to environmental influences, e.g., during stress. In the hippocampus, chronic stress causes retraction of dendrites in CA3 pyramidal neurons. We have recently identified the glycoprotein M6a as a stress-responsive gene in the hippocampal formation. This gene is down-regulated in the hippocampus of both socially and physically stressed animals, and this effect can be reversed by antidepressant treatment. In the present work, we analyzed the biological function of the M6a protein. Immunohistochemistry showed that the M6a protein is abundant in all hippocampal subregions, and subcellular analysis in primary hippocampal neurons revealed its presence in membrane protrusions (filopodia/spines). Transfection experiments revealed that M6a overexpression induces neurite formation and increases filopodia density in hippocampal neurons. M6a knockdown with small interference RNA methodology showed that M6a low-expressing neurons display decreased filopodia number and a lower density of synaptophysin clusters. Taken together, our findings indicate that M6a plays an important role in neurite/filopodium outgrowth and synapse formation. Therefore, reduced M6a expression might be responsible for the morphological alterations found in the hippocampus of chronically stressed animals. Potential mechanisms that might explain the biological effects of M6a are discussed.
Assuntos
Espinhas Dendríticas/fisiologia , Hipocampo/citologia , Glicoproteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Neuritos/fisiologia , Pseudópodes/fisiologia , Estresse Fisiológico/metabolismo , Animais , Células COS , Linhagem Celular Tumoral , Membrana Celular/genética , Membrana Celular/patologia , Membrana Celular/fisiologia , Células Cultivadas , Chlorocebus aethiops , Hipocampo/fisiologia , Hipocampo/fisiopatologia , Masculino , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Camundongos , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Plasticidade Neuronal/genética , Neurônios/citologia , Neurônios/metabolismo , Neurônios/fisiologia , Células PC12 , RNA Interferente Pequeno/farmacologia , Ratos , Estresse Fisiológico/patologia , Estresse Fisiológico/fisiopatologia , TransfecçãoRESUMO
Depressive disorders are among the most frequent forms of mental illness. Both genetic and environmental factors, such as stress, are involved in the etiology of depression. Therefore, chronic stress paradigms in laboratory animals constitute an important tool for research in this field. The molecular bases of chronic stress/depression are largely unknown, although a large amount of information has been accumulated during recent years. Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis as well as structural and physiological alterations in the hippocampus and neocortex are known to occur. Modifications in the expression level of some genes, such as brain-derived neurotrophic factor, cAMP-response-element binding protein, serotonin receptors and HPA axis components were consistently associated in a number of experimental models. However, recent results suggest that several synaptic proteins, transcription factors and proteins involved in neuronal growth/differentiation, are also modified in their expression in experimental models of chronic stress. In general, these alterations can be reversed by treatment with antidepressants. Thus, a complex pattern of gene expression leading to stress/depression is starting to emerge. We summarize here recent findings on the alterations of gene expression in the hippocampus of chronically stressed and antidepressant treated animals.
Assuntos
Antidepressivos/farmacologia , Transtorno Depressivo/tratamento farmacológico , Transtorno Depressivo/fisiopatologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Estresse Fisiológico/fisiopatologia , Animais , Doença Crônica , Transtorno Depressivo/etiologia , Modelos Animais de Doenças , Regulação da Expressão Gênica/fisiologia , Hipocampo/metabolismo , Humanos , Sistema Hipotálamo-Hipofisário/metabolismo , Sistema Hipotálamo-Hipofisário/fisiopatologia , Estresse Fisiológico/complicações , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/fisiologiaRESUMO
Adrenal corticosteroids influence the function of the hippocampus, the brain structure in which the highest expression of glucocorticoid receptors is found. Chronic high levels of cortisol elicited by stress or through exogenous administration can cause irreversible damage and cognitive deficits. In this study, we searched for genes expressed in the hippocampal formation after chronic cortisol treatment in male tree shrews. Animals were treated orally with cortisol for 28 days. At the end of the experiments, we generated two subtractive hippocampal hybridization libraries from which we sequenced 2,246 expressed sequenced tags (ESTs) potentially regulated by cortisol. To validate this approach further, we selected some of the candidate clones to measure mRNA expression levels in hippocampus using real-time PCR. We found that 66% of the sequences tested (10 of 15) were differentially represented between cortisol-treated and control animals. The complete set of clones was subjected to a bioinformatic analysis, which allowed classification of the ESTs into four different main categories: 1) known proteins or genes (approximately 28%), 2) ESTs previously published in the database (approximately 16%), 3) novel ESTs matching only the reference human or mouse genome (approximately 5%), and 4) sequences that do not match any public database (50%). Interestingly, the last category was the most abundant. Hybridization assays revealed that several of these clones are indeed expressed in hippocampal tissue from tree shrew, human, and/or rat. Therefore, we discovered an extensive inventory of new molecular targets in the hippocampus that serves as a reference for hippocampal transcriptional responses under various conditions. Finally, a detailed analysis of the genomic localization in human and mouse genomes revealed a survey of putative novel splicing variants for several genes of the nervous system.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Hidrocortisona/administração & dosagem , Animais , Clonagem Molecular/métodos , Esquema de Medicação , Etiquetas de Sequências Expressas , Perfilação da Expressão Gênica/métodos , Biblioteca Gênica , Hipocampo/fisiologia , Humanos , Hibridização In Situ/métodos , Masculino , RNA Mensageiro/biossíntese , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , TupaiidaeRESUMO
Analysis of differentially expressed genes in the brain is a promising tool for elucidating pathological mechanisms that lead to central nervous disorders. Stress is known to be involved in the development of psychopathologies such as depression. In the present study, we searched for differentially expressed genes in the hippocampal formation after chronic psychosocial stress and after treatment with the antidepressant clomipramine. Experiments were conducted in male tree shrews, a valid psychosocial stress model in which antidepressant drugs prevent diverse effects of stress. Because many effects of stress have been attributed to the stress-induced elevation in glucocorticoids, we screened two subtractive hippocampal cDNA libraries generated from RNA of chronic cortisol-treated animals. Using real-time PCR to measure mRNA amounts, we identified five sequences whose expression levels differed between stressed animals and controls. Transcript levels of four of them, nerve growth factor (NGF), membrane glycoprotein 6a (M6a), CDC-like kinase 1 (CLK-1) and G-protein alpha q (GNAQ) were reduced by chronic psychosocial stress. Reduced amounts of these genes, which are all related to processes of cell differentiation, is in agreement with previous findings showing a retraction of dendrites and an impairment of neurogenesis in the hippocampal formation after chronic stress. An additional expressed sequence that was also regulated by stress could not be assigned to any known gene. Treatment with the antidepressant clomipramine prevented stress effects on expression of M6a, CLK-1, GNAQ and the novel sequence, but showed no effect on NGF stress-induced down-regulation. These findings support the concept that depressive disorders are accompanied by processes of neuronal dedifferentiation, at least in the hippocampal formation, and that antidepressants prevent these processes.