Análise da metilação global e metilação dos genes DPYSL2, CNP, HSPA8 e HSPA9, e sua influência na expressão gênica em pacientes com Doença de Alzheimer
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Data
2012
Autores
Silva, Patricia Natalia Oliveira [UNIFESP]
Orientadores
Smith, Marília de Arruda Cardoso [UNIFESP]
Tipo
Tese de doutorado
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Objetivos: Este estudo teve como objetivo avaliar a expressao e a regulacao epigenetica dos genes DPYSL2, CNP, HSPA8 e HSPA9 na Doenca de Alzheimer (DA) e quantificar a metilacao de sequencias LINE1 no tecido cerebral de pacientes e controles. Metodos: A quantificacao de RNAm foi realizada por meio de qRT-PCR e os padroes de metilacao foram determinados por espectrometria de massa e pirosequenciamento. Foram avaliadas tres regioes cerebrais post mortem (cortex entorrinal, cortex auditivo e hipocampo) de cerca de 10 pacientes com Doenca de Alzheimer e 10 idosos saudaveis, assim como sangue periferico de cerca de 20 jovens, 20 idosos saudaveis e 30 pacientes com DA. Resultados: No sangue periferico os genes DPYSL2, CNP foram mais expressos em individuos jovens e os genes HSPA8 e HSPA9 foram mais expressos em pacientes com DA, em relacao aos demais grupos. A expressao dos genes DPYSL2, CNP, HSPA8 e HSPA9 foi menor no cortex auditivo, no cortex entorrinal e no hipocampo de pacientes com DA do que em idosos controles, nao diferindo entre si no grupo de pacientes com DA e no grupo de idosos. Entre os tecidos sangue e cerebro de pacientes e de idosos controles, os genes DPYSL2 e CNP foram mais expressos em sangue, enquanto que as chaperonas HSPA8 e HSPA9 foram mais expressas no cerebro. Conclusoes: A maior expressao dos genes DPYSL2 e CNP em sangue periferico de jovens pode estar relacionada a uma maior proliferacao do sistema imunologico ou a atuacao desses genes em vias ainda nao conhecidas. Ja a expressao aumentada das chaperonas no sangue pode estar associada a uma resposta ao estresse oxidativo observado na doenca. As diferencas nos niveis de RNAm observadas entre o sangue e o cerebro de pacientes e de controles podem ser decorrentes de funcoes tecido-especificas. A diminuicao da expressao dos quatro genes no tecido cerebral de pacientes com DA em relacao aos idosos, pode indicar uma participacao destes genes na fisiopatologia da doenca, atuando na morte neuronal, na modulacao de sinais extracelulares e no acumulo de proteinas no cerebro. Os niveis aumentados de metilacao das sequencias LINE1 devem estar relacionados ao desenvolvimento da DA, porem, a metilacao dos promotores de genes candidatos nao parece ser o mecanismo responsavel pelo controle da expressao genica em pacientes e controles
Alzheimer’s Disease (AD) is the most common type of dementia in elderly population. AD is a complex neurodegenerative disorder that shows many gene expressions and protein alterations. Thus, gene expression regulation process, such epigenetic regulation, has been investigated in AD. AIM: This study aimed to evaluate gene expression and epigenetic regulation of DPYSL2, CNP, HSPA8 e HSPA9 genes in AD and controls. Furthermore, we aimed to evaluate the methylation levels of LINE1 in brain and peripheral blood of AD patients and controls. METHODS: mRNA quantification was performed using qRT-PCR, and methylation patterns were determinated by mass spectrometry and pyrosequencing. We analyzed three post mortem brain regions (entorhinal and auditory cortices and hippocampus) of 10 AD patients and 10 healthy elderly as well as peripheral blood lymphocytes of 16 young, 23 elderly and 34 AD patients. RESULTS: In peripheral blood, DPYSL2 and CNP genes were upregulated in young controls, and HSPA8 and HSPA9 were upregulated in AD patients. DPYSL2, CNP, HSPA8 and HSPA9 genes were downregulated in auditory cortex, entorhinal cortex and hippocampus of AD patients compared to elderly control group. Between the blood and brain tissues of AD patients and elderly controls, DPYSL2 and CNP genes presented higher expression in blood, whilst the HSPA8 and HSPA9 chaperones were more expressed in brain tissue. CONCLUSIONS: DPYSL2 and CNP upregulation in peripheral blood of young controls could be related to a higher proliferation of immune system and to the activation of those genes in pathways still unknown. The upregulation of chaperones in blood may be associated with a cellular response to oxidative stress. Differences in mRNA levels observed between blood and brain of AD patients and controls could be due to tissue-specific functions. The downregulation of all four genes in brain tissue of AD patients compared to controls could indicate a role for those genes in AD pathology, acting in neuronal death, extracellular signal modulation and protein accumulation. The higher methylation levels of LINE1 sequences may be related to AD development; however, methylation of candidate genes promoters does not appear to be the mechanism regulating gene expression in AD patients and controls.
Alzheimer’s Disease (AD) is the most common type of dementia in elderly population. AD is a complex neurodegenerative disorder that shows many gene expressions and protein alterations. Thus, gene expression regulation process, such epigenetic regulation, has been investigated in AD. AIM: This study aimed to evaluate gene expression and epigenetic regulation of DPYSL2, CNP, HSPA8 e HSPA9 genes in AD and controls. Furthermore, we aimed to evaluate the methylation levels of LINE1 in brain and peripheral blood of AD patients and controls. METHODS: mRNA quantification was performed using qRT-PCR, and methylation patterns were determinated by mass spectrometry and pyrosequencing. We analyzed three post mortem brain regions (entorhinal and auditory cortices and hippocampus) of 10 AD patients and 10 healthy elderly as well as peripheral blood lymphocytes of 16 young, 23 elderly and 34 AD patients. RESULTS: In peripheral blood, DPYSL2 and CNP genes were upregulated in young controls, and HSPA8 and HSPA9 were upregulated in AD patients. DPYSL2, CNP, HSPA8 and HSPA9 genes were downregulated in auditory cortex, entorhinal cortex and hippocampus of AD patients compared to elderly control group. Between the blood and brain tissues of AD patients and elderly controls, DPYSL2 and CNP genes presented higher expression in blood, whilst the HSPA8 and HSPA9 chaperones were more expressed in brain tissue. CONCLUSIONS: DPYSL2 and CNP upregulation in peripheral blood of young controls could be related to a higher proliferation of immune system and to the activation of those genes in pathways still unknown. The upregulation of chaperones in blood may be associated with a cellular response to oxidative stress. Differences in mRNA levels observed between blood and brain of AD patients and controls could be due to tissue-specific functions. The downregulation of all four genes in brain tissue of AD patients compared to controls could indicate a role for those genes in AD pathology, acting in neuronal death, extracellular signal modulation and protein accumulation. The higher methylation levels of LINE1 sequences may be related to AD development; however, methylation of candidate genes promoters does not appear to be the mechanism regulating gene expression in AD patients and controls.
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SILVA, Patricia Natalia Oliveira. Análise da metilação global e metilação dos genes DPYSL2, CNP, HSPA8 e HSPA9, e sua influência na expressão gênica em pacientes com Doença de Alzheimer. Tese (Doutorado em Ciências) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, 2012.