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Evaluation of miR-124 expression in the human hippocampus with focus on Alzheimer’s Disease
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Resumo(s)
A Doença de Alzheimer (DA) é uma doença neurodegenerativa progressiva cujos mecanismos patológicos ainda não estão totalmente estabelecidos. A doença é caraterizada pela deposição de placas de Beta Amilóide (Aß) e tangles de proteína Tau hiperfosforilada, que geram ativação de microglia e neurodegeneração. Uma das áreas mais afetadas é o hipocampo, uma região relacionada com a memória e cognição, levando à demência e perda de memória, sintomas característicos da DA. Na DA registam-se níveis alterados de microRNA-124 (miR-124), tal como em outras doenças neurodegenerativas. Apesar de o miR- 124 ser considerado um microRNA (miRNA) principalmente neuronal, encontra-se também presente em microglia e astrócitos. Contudo, a sua expressão regional em células gliais nunca foi descrita em amostras humanas de DA. Além disso, ainda há muito para ser compreendido acerca do seu papel na DA, existindo relatos controversos no que respeita à alteração dos seus níveis ou do seu efeito na microglia. Neste contexto, estudámos a distribuição de miR- 124 em amostras de hipocampo humano provenientes de autópsias de DA, através de In Situ Hybridization (ISH). A ISH é uma técnica histológica que permite a deteção de ácidos nucleicos em tecidos, através do uso de uma sonda complementar. Neste trabalho, descrevemos em detalhe o protocolo de ISH e discutimos as suas principais vantagens e desvantagens, evidenciando a sua aplicação em amostras de hipocampo, provenientes de doentes com DA e controlos saudáveis. Especificamente, mostramos co-marcações para marcadores celulares e respetiva análise semi-quantitativa. Os resultados elucidam a presença de miR-124 em células microgliais HLA-DR/DP/DQ-positivas à volta de placas Aß num doente com DA num estadio avançado da doença. É visível co-localização em toda a matéria branca e no dentate gyrus, quer na DA como no controlo. Na DA, observámos a sua presença em astrócitos GFAP-positivos, onde parece ter uma localização nuclear preferencial. A ISH apresenta a característica singular de permitir o estudo e semi- quantificação da distribuição celular de um miRNA, sendo uma ferramenta valiosa que pode ser aplicada para elucidar a função de miRNAs na desregulação da comunicação célula-a- célula em contextos patológicos e na DA.
Alzheimer’s Disease (AD) is a progressive neurodegenerative disorder whose pathological mechanisms are still not fully elucidated. The disease is characterized by the deposition of Amyloid Beta (Aß) and hyperphosphorylated Tau protein tangles, leading to microglia activation and ultimately neurodegeneration. One of the most affected areas is the hippocampus, an area related to memory and cognition, leading to dementia and memory loss which are characteristic AD symptoms. Abnormal microRNA-124 (miR-124) levels are observed in AD, as well as in other neurodegenerative disorders. Although miR-124 is regarded as a neuron-enriched microRNA (miRNA), it has been found in microglia and astrocytes. However, its regional expression in glial cells has never been identified in human AD samples. Moreover, much remains to be understood about its role in AD, with controversial reports about it being up or down regulated and scarce information on its effects on microglia. In this context, we studied the distribution of miR-124 in the human hippocampus of AD autopsy samples, by In Situ Hybridization (ISH). ISH is a histological technique that allows the detection of nucleic acids in whole tissues by use of a complementary probe. In this work, we fully describe the ISH protocol, discuss its main advantages and disadvantages and give data examples from its application on human AD and control hippocampal samples. Specifically, we show co-stainings for cell-specific markers and semi-quantitative analysis of co-staining intensity. Results elucidate the miR-124 presence in HLA-DR/DP/DQ-positive microglial cells surrounding Aß plaques in an AD patient at a late disease stage. Co-localization throughout white matter and dentate gyrus was visible in both patient and control samples. In AD, we observed its presence in GFAP-positive astrocytes, where it seems to have a strong nuclear localization. This technique has the unique characteristics of allowing the study of cell-type distribution and semi-quantification, while preserving morphological aspects, which is essential in studying complex cell-to-cell communication in pathological contexts. Thus, ISH is a valuable tool that can be used to further elucidate the role of miRNAs in AD and in cell-to-cell communication deregulation.
Alzheimer’s Disease (AD) is a progressive neurodegenerative disorder whose pathological mechanisms are still not fully elucidated. The disease is characterized by the deposition of Amyloid Beta (Aß) and hyperphosphorylated Tau protein tangles, leading to microglia activation and ultimately neurodegeneration. One of the most affected areas is the hippocampus, an area related to memory and cognition, leading to dementia and memory loss which are characteristic AD symptoms. Abnormal microRNA-124 (miR-124) levels are observed in AD, as well as in other neurodegenerative disorders. Although miR-124 is regarded as a neuron-enriched microRNA (miRNA), it has been found in microglia and astrocytes. However, its regional expression in glial cells has never been identified in human AD samples. Moreover, much remains to be understood about its role in AD, with controversial reports about it being up or down regulated and scarce information on its effects on microglia. In this context, we studied the distribution of miR-124 in the human hippocampus of AD autopsy samples, by In Situ Hybridization (ISH). ISH is a histological technique that allows the detection of nucleic acids in whole tissues by use of a complementary probe. In this work, we fully describe the ISH protocol, discuss its main advantages and disadvantages and give data examples from its application on human AD and control hippocampal samples. Specifically, we show co-stainings for cell-specific markers and semi-quantitative analysis of co-staining intensity. Results elucidate the miR-124 presence in HLA-DR/DP/DQ-positive microglial cells surrounding Aß plaques in an AD patient at a late disease stage. Co-localization throughout white matter and dentate gyrus was visible in both patient and control samples. In AD, we observed its presence in GFAP-positive astrocytes, where it seems to have a strong nuclear localization. This technique has the unique characteristics of allowing the study of cell-type distribution and semi-quantification, while preserving morphological aspects, which is essential in studying complex cell-to-cell communication in pathological contexts. Thus, ISH is a valuable tool that can be used to further elucidate the role of miRNAs in AD and in cell-to-cell communication deregulation.
Descrição
Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2019
Palavras-chave
Doença de Alzheimer Células gliais Hipocampo humano In Situ Hybridization MicroRNA-124 Mestrado Integrado - 2019