Sinalização por notch e galectina-3 na indução do programa de neurogênese latente de astrócitos reativos
Data
2021-07-20
Autores
Ribeiro, Tais Novaki [UNIFESP]
Orientadores
Porcionatto, Marimelia Aparecida [UNIFESP]
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
A reativação astrocitária é uma das principais respostas celulares após lesão do sistema nervoso central. Astrócitos reativos auxiliam o fechamento e reparo do tecido danificado ao adquirirem um programa celular regenerativo, porém limitado, in vivo. Entretanto, in vitro, astrócitos reativos podem readquirir características de células tronco-neurais, como a multipotência e autorrenovação. Os fatores moleculares que acionam o potencial neurogênico latente de astrócitos reativos não são totalmente conhecidos. Neste sentido, uma melhor compreensão dos mecanismos envolvidos na reativação astrocitária e sua modulação poderia esclarecer o potencial astrocitário em fornecer células tronco-neurais no sítio da lesão, ultrapassando os desafios enfrentados pela migração de células tronco-neurais provenientes dos nichos neurogênicos. Neste contexto, nós estudamos o papel da via de sinalização do Notch e Galectina-3 (Gal-3) na reativação e desdiferenciação astrocitária. Nós reportamos que astrócitos reativados pelo modelo de scratch apresentam um aumento da proteína Gal-3 no meio intracelular e ativação da via do Notch1 in vitro. Além disso, NICD1 (domínio intracelular de Notch1) e Gal-3 mudam seu padrão de distribuição de difuso para vesicular após estímulo de reativação, indicando interação NICD1/Gal-3. Nós
reforçamos esta hipótese ao verificar que astrócitos reativos Lgals3-/- não ativam a via de sinalização Notch1. Astrócitos reativos Lgals3-/- não apresentam a proteína NICD1 nuclear, nem superexpressam Hes5, Jagged1 e Delta-like1. Ao contrário, astrócitos reativos C57Bl/6J apresentam um aumento da proteína NICD1 no núcleo e superexpressam o gene-alvo Hes5 e os ligantes de Notch, sugerindo que Gal-3 é necessária para a ativação apropriada da via do Notch1. Posteriormente, verificamos que, in vitro, os astrócitos adquirem fenótipo reativo após nocaute condicional de Rbpj, indicando que Notch atua na manutenção do estado celular astrocitário. Em seguida, nós avaliamos o papel de Notch na desdiferenciação astrocitária e reportamos que a inibição da enzima y-secretase promove o crescimento das neuroesferas, mas a deleção condicional específica de Notch2 gera maior número de neuroesferas
menores. Em conclusão, Gal-3 interage com NICD1 na resposta de reativação astrocitária e Notch2 contribui para a autorrenovação de células tronco-neurais derivadas de astrócitos reativos (RA-NSC), mas impede a ativação do programa neurogênico por astrócitos reativos.
Astrocyte reactivation is one of the main cellular responses after central nervous system lesion. Reactive astrocytes support the closure and repair of damaged tissue by acquiring a limited regenerative cell program in vivo. However, in vitro, reactive astrocytes can regain neural-stem cell hallmarks, such as multipotentiality and self-renewal ability. The molecular factors responsible for triggering the latent neurogenic program of reactive astrocytes are not well-known. Yet, a better understanding of astrocyte reactivation mechanisms and their modulation would shed light on astrocyte’s potential to provide neural-stem cells at the lesion site, bypassing challenges involved in neural-stem cell migration from neurogenic niches. In this context, we studied the role of Notch signaling pathway and Galectin-3 (Gal-3) as underlying molecular factors involved in astrocyte reactivation and dedifferentiation response. We report that scratch-induced reactive astrocytes in vitro increase intracellular Gal-3 protein and have active Notch1 signaling. Moreover, NICD1 (Notch1 intracellular domain) and Gal-3 shift from diffuse to vesicular pattern distribution with astrocyte reactivation, indicating NICD1/Gal-3 interaction. We further support this hypothesis by showing that Lgals3-/- reactive astrocytes do not activate Notch1 signaling. Nuclear NICD1 protein is not reported in Lgals3-/- reactive astrocytes, neither the upregulation of Hes5, Jagged1 or Delta-like1 mRNA expression. On the contrary, C57Bl/6J reactive astrocytes have increased NICD1 protein in the nucleus and upregulate Notch ligands and the target gene Hes5, suggesting that Gal-3 is necessary for proper Notch1 signaling activation. In addition, we show that astrocytes in vitro acquire a reactive phenotype upon Rbpj conditional knockout, indicating that Notch maintain astrocyte’s mature cell state. Next, we assessed Notch role in astrocyte dedifferentiation through the neurosphere assay and noted that y-secretase enzyme inhibition promotes neurosphere growth, but specific Notch2 conditional deletion leads to a higher number of smaller neurospheres. Overall, we conclude that Gal-3 interacts with NICD1 at astrocyte reactivation response, and that Notch2 contributes to reactive astrocytes-neural stem cells (RA-NSC) self-renewal, but prevents the activation of reactive astrocyte’s neurogenic program.
Astrocyte reactivation is one of the main cellular responses after central nervous system lesion. Reactive astrocytes support the closure and repair of damaged tissue by acquiring a limited regenerative cell program in vivo. However, in vitro, reactive astrocytes can regain neural-stem cell hallmarks, such as multipotentiality and self-renewal ability. The molecular factors responsible for triggering the latent neurogenic program of reactive astrocytes are not well-known. Yet, a better understanding of astrocyte reactivation mechanisms and their modulation would shed light on astrocyte’s potential to provide neural-stem cells at the lesion site, bypassing challenges involved in neural-stem cell migration from neurogenic niches. In this context, we studied the role of Notch signaling pathway and Galectin-3 (Gal-3) as underlying molecular factors involved in astrocyte reactivation and dedifferentiation response. We report that scratch-induced reactive astrocytes in vitro increase intracellular Gal-3 protein and have active Notch1 signaling. Moreover, NICD1 (Notch1 intracellular domain) and Gal-3 shift from diffuse to vesicular pattern distribution with astrocyte reactivation, indicating NICD1/Gal-3 interaction. We further support this hypothesis by showing that Lgals3-/- reactive astrocytes do not activate Notch1 signaling. Nuclear NICD1 protein is not reported in Lgals3-/- reactive astrocytes, neither the upregulation of Hes5, Jagged1 or Delta-like1 mRNA expression. On the contrary, C57Bl/6J reactive astrocytes have increased NICD1 protein in the nucleus and upregulate Notch ligands and the target gene Hes5, suggesting that Gal-3 is necessary for proper Notch1 signaling activation. In addition, we show that astrocytes in vitro acquire a reactive phenotype upon Rbpj conditional knockout, indicating that Notch maintain astrocyte’s mature cell state. Next, we assessed Notch role in astrocyte dedifferentiation through the neurosphere assay and noted that y-secretase enzyme inhibition promotes neurosphere growth, but specific Notch2 conditional deletion leads to a higher number of smaller neurospheres. Overall, we conclude that Gal-3 interacts with NICD1 at astrocyte reactivation response, and that Notch2 contributes to reactive astrocytes-neural stem cells (RA-NSC) self-renewal, but prevents the activation of reactive astrocyte’s neurogenic program.
Descrição
Citação
RIBEIRO, T. N. Sinalização por Notch e Galectina-3 na indução do programa de neurogênese latente de astrócitos reativos. São Paulo, 2021. 68 f. Dissertação (Mestrado em Biologia Molecular) – Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, 2021.