Perfil de expressão de pequenos RNAs provenientes de exossomos de pacientes em Primeiro Episódio Psicótico
Data
2021-06-28
Autores
Xavier, Gabriela [UNIFESP]
Orientadores
Belangero, Sintia Iole Nogueira [UNIFESP]
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
A Esquizofrenia é uma doença psiquiátrica grave, multifatorial e poligênica. O primeiro episódio psicótico é considerado o início desta doença e é uma fase extremamente interessante para estudo, uma vez que os pacientes apresentam altos níveis de sintomas e, muitas vezes, encontram-se livres de tratamento antipsicótico. Genes regulatórios, como de pequenos RNAs tem sido cada vez mais relacionados à doença. Estes pequenos RNAs podem ser encontrados não apenas dentro das células, mas também no meio extracelular (em fluidos biológicos) tanto na sua forma livre quanto encerrados em vesículas extracelulares. Os exossomos são pequenas vesículas extracelulares capazes de carregar estes RNAs, proteínas e lipídeos, através dos fluidos biológicos, por todo o corpo. Estas vesículas apresentam ainda capacidade de atravessar a barreira hematoencefálica. Nesta tese, buscamos investigar o conteúdo de pequenos transcritos presente em vesículas extracelulares séricas, além de investigar a expressão de pequenos RNAs nestas vesículas e de correlacionar os RNAs diferencialmente expressos às escalas diagnósticas. Dentre os pequenos RNAs, pudemos observar a predominância de miRNAs e tRNAs no conteúdo de vesículas extracelulares. Eles representam, em média, 16 e 4%, respectivamente, dos transcritos encontrados nas vesículas. Dois miRNAs mostraram-se diferencialmente expressos na comparação entre grupos (miR-335-5p e miR-30d-5p), sendo o miR-30d-5p correlacionado negativamente com a escala clínica CDSS. Além disso, ambos apresentaram AUC maior que 90% no teste de sensibilidade e especificidade. Encontramos também tRNAs diferencialmente expressos em nossas amostras. O tRNALeuAAG-7-1, apresentou maior expressão em pacientes (antes e após tratamento) quando comparados aos controles, no entanto, o uso de antipsicótico parece ter um efeito sobre sua expressão. Outros quatro tRNAs mostraram-se diferencialmente expressos entre pacientes antes e após tratamento e o uso da risperidona pareceu ter efeito sobre a expressão destes, assemelhando-as à de controles. Investigamos ainda o conteúdo proteico presente em membranas de vesículas extracelulares liberadas por diversos tipos de células neurais, geradas a partir de células pluripotentes induzidas de pacientes com esquizofrenia e doença de Alzheimer de acometimento tardio. A análise de 34 epítopos revelou, em doadores saudáveis, o aumento dos epítopos CD29, CD44, CD49e, CD146, MSCP e SSEA-4 em vesículas extracelulares coletadas em meio de cultura de astrócitos, microglia e células neurais progenitoras (NPCs) quando comparados a outros tipos celulares. Os dois estudos contidos nesta tese apresentam resultados relevantes e de alto potencial na busca por marcadores tanto para a esquizofrenia (em suas fases mais precoces ou em fase crônica) quanto para o acompanhamento do tratamento desta doença, mas necessitam ainda de replicação em outras amostras e confirmação por diferentes metodologias.
Schizophrenia is a severe, multifactorial and polygenic psychiatric illness. The first psychotic episode is considered the beginning of this disease and is an extremely interesting phase for studies, as patients present high levels of symptoms and are often free from antipsychotic treatment. Regulatory genes such as small RNAs have been increasingly linked to the disease. These small RNAs can be found not only inside cells, but also in the extracellular environment (in biological fluids) either in their free form or enclosed in extracellular vesicles. Exosomes are small extracellular vesicles capable of carrying these RNAs, proteins and lipids through biological fluids throughout the body. These vesicles also have the ability to cross the blood-brain barrier. In this thesis, we sought to investigate the content of small transcripts present in serum exosomes, in addition to investigating the expression of small RNAs in these vesicles and to correlate the differentially expressed RNAs to diagnostic scales. Among the small RNAs, we could observe the predominance of miRNAs and tRNAs in the content of exosomes. They represent, on average, 16 and 4%, respectively, of transcripts found in vesicles. Two miRNAs were differentially expressed in the comparison between groups (miR-335-5p and miR-30d-5p), with miR-30d-5p being negatively correlated with the CDSS clinical scale. Furthermore, both had AUC greater than 90% in the sensitivity and specificity test. We also found differentially expressed tRNAs in our samples. The tRNALeuAAG-7-1 showed greater expression in patients (before and after treatment) when compared to controls, however, the use of antipsychotics seems to have an effect on its expression. Another four tRNAs were differentially expressed between patients before and after treatment, and the use of risperidone seemed to have an effect on their expression, similar to that of controls. In addition, we performed the treatment of neurons originating from pluripotent stem cells from patients with schizophrenia and control in order to investigate the action of these drugs on the disease's target cells. However, the results of this study were inconclusive. We also investigated the protein content present in exosomal membranes released by different types of neural cells, generated from induced pluripotent cells from patients with schizophrenia and late-onset Alzheimer's disease. Analysis of 34 epitopes revealed, in healthy donors, an increase in CD29, CD44, CD49e, CD146, MSCP and SSEA-4 epitopes in exosomes collected from astrocytes, microglia and neural progenitor cells (NPCs) when compared to other cell types. The three studies contained in this thesis present relevant and high potential results in the search for markers both for schizophrenia (in its earlier stages or in the chronic phase) and for monitoring the treatment of this disease, but they still need replication in different samples and confirmation by different methodologies.
Schizophrenia is a severe, multifactorial and polygenic psychiatric illness. The first psychotic episode is considered the beginning of this disease and is an extremely interesting phase for studies, as patients present high levels of symptoms and are often free from antipsychotic treatment. Regulatory genes such as small RNAs have been increasingly linked to the disease. These small RNAs can be found not only inside cells, but also in the extracellular environment (in biological fluids) either in their free form or enclosed in extracellular vesicles. Exosomes are small extracellular vesicles capable of carrying these RNAs, proteins and lipids through biological fluids throughout the body. These vesicles also have the ability to cross the blood-brain barrier. In this thesis, we sought to investigate the content of small transcripts present in serum exosomes, in addition to investigating the expression of small RNAs in these vesicles and to correlate the differentially expressed RNAs to diagnostic scales. Among the small RNAs, we could observe the predominance of miRNAs and tRNAs in the content of exosomes. They represent, on average, 16 and 4%, respectively, of transcripts found in vesicles. Two miRNAs were differentially expressed in the comparison between groups (miR-335-5p and miR-30d-5p), with miR-30d-5p being negatively correlated with the CDSS clinical scale. Furthermore, both had AUC greater than 90% in the sensitivity and specificity test. We also found differentially expressed tRNAs in our samples. The tRNALeuAAG-7-1 showed greater expression in patients (before and after treatment) when compared to controls, however, the use of antipsychotics seems to have an effect on its expression. Another four tRNAs were differentially expressed between patients before and after treatment, and the use of risperidone seemed to have an effect on their expression, similar to that of controls. In addition, we performed the treatment of neurons originating from pluripotent stem cells from patients with schizophrenia and control in order to investigate the action of these drugs on the disease's target cells. However, the results of this study were inconclusive. We also investigated the protein content present in exosomal membranes released by different types of neural cells, generated from induced pluripotent cells from patients with schizophrenia and late-onset Alzheimer's disease. Analysis of 34 epitopes revealed, in healthy donors, an increase in CD29, CD44, CD49e, CD146, MSCP and SSEA-4 epitopes in exosomes collected from astrocytes, microglia and neural progenitor cells (NPCs) when compared to other cell types. The three studies contained in this thesis present relevant and high potential results in the search for markers both for schizophrenia (in its earlier stages or in the chronic phase) and for monitoring the treatment of this disease, but they still need replication in different samples and confirmation by different methodologies.
Descrição
Citação
XAVIER, G. Perfil de expressão de pequenos RNAs provenientes de exossomos de pacientes em Primeiro Episódio Psicótico. São Paulo, 2021. 303 f. Tese (Doutorado em Biologia Estrutural e Funcional) – Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, 2021.