TNF-alpha-induced microglia activation requires miR-342: impact on NF-kB signaling and neurotoxicity
Autor(a) principal: | |
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Data de Publicação: | 2020 |
Outros Autores: | , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | http://hdl.handle.net/10400.22/19421 |
Resumo: | Growing evidences suggest that sustained neuroinflammation, caused by microglia overactivation, is implicated in the development and aggravation of several neurological and psychiatric disorders. In some pathological conditions, microglia produce increased levels of cytotoxic and inflammatory mediators, such as tumor necrosis factor alpha (TNF-α), which can reactivate microglia in a positive feedback mechanism. However, specific molecular mediators that can be effectively targeted to control TNF-α-mediated microglia overactivation, are yet to be uncovered. In this context, we aim to identify novel TNF-α-mediated micro(mi)RNAs and to dissect their roles in microglia activation, as well as to explore their impact on the cellular communication with neurons. A miRNA microarray, followed by RT-qPCR validation, was performed on TNF-α-stimulated primary rat microglia. Gain- and loss-of-function in vitro assays and proteomic analysis were used to dissect the role of miR-342 in microglia activation. Co-cultures of microglia with hippocampal neurons, using a microfluidic system, were performed to understand the impact on neurotoxicity. Stimulation of primary rat microglia with TNF-α led to an upregulation of Nos2, Tnf, and Il1b mRNAs. In addition, ph-NF-kB p65 levels were also increased. miRNA microarray analysis followed by RT-qPCR validation revealed that TNF-α stimulation induced the upregulation of miR-342. Interestingly, miR-342 overexpression in N9 microglia was sufficient to activate the NF-kB pathway by inhibiting BAG-1, leading to increased secretion of TNF-α and IL-1β. Conversely, miR-342 inhibition led to a strong decrease in the levels of these cytokines after TNF-α activation. In fact, both TNF-α-stimulated and miR-342-overexpressing microglia drastically affected neuron viability. Remarkably, increased levels of nitrites were detected in the supernatants of these co-cultures. Globally, our findings show that miR-342 is a crucial mediator of TNF-α-mediated microglia activation and a potential target to tackle microglia-driven neuroinflammation. |
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TNF-alpha-induced microglia activation requires miR-342: impact on NF-kB signaling and neurotoxicityEnzyme-linked immunosorbent assay (ELISA)Mixed glial cellsPrimary microgliaN9 microglial cellsTNF-alpha-induced microglia activationmiR-342Growing evidences suggest that sustained neuroinflammation, caused by microglia overactivation, is implicated in the development and aggravation of several neurological and psychiatric disorders. In some pathological conditions, microglia produce increased levels of cytotoxic and inflammatory mediators, such as tumor necrosis factor alpha (TNF-α), which can reactivate microglia in a positive feedback mechanism. However, specific molecular mediators that can be effectively targeted to control TNF-α-mediated microglia overactivation, are yet to be uncovered. In this context, we aim to identify novel TNF-α-mediated micro(mi)RNAs and to dissect their roles in microglia activation, as well as to explore their impact on the cellular communication with neurons. A miRNA microarray, followed by RT-qPCR validation, was performed on TNF-α-stimulated primary rat microglia. Gain- and loss-of-function in vitro assays and proteomic analysis were used to dissect the role of miR-342 in microglia activation. Co-cultures of microglia with hippocampal neurons, using a microfluidic system, were performed to understand the impact on neurotoxicity. Stimulation of primary rat microglia with TNF-α led to an upregulation of Nos2, Tnf, and Il1b mRNAs. In addition, ph-NF-kB p65 levels were also increased. miRNA microarray analysis followed by RT-qPCR validation revealed that TNF-α stimulation induced the upregulation of miR-342. Interestingly, miR-342 overexpression in N9 microglia was sufficient to activate the NF-kB pathway by inhibiting BAG-1, leading to increased secretion of TNF-α and IL-1β. Conversely, miR-342 inhibition led to a strong decrease in the levels of these cytokines after TNF-α activation. In fact, both TNF-α-stimulated and miR-342-overexpressing microglia drastically affected neuron viability. Remarkably, increased levels of nitrites were detected in the supernatants of these co-cultures. Globally, our findings show that miR-342 is a crucial mediator of TNF-α-mediated microglia activation and a potential target to tackle microglia-driven neuroinflammation.We would like to thank Dr. João Relvas laboratory for the help with N9 microglia cell culture; Dr. Sofia Lamas for the guidance on the animal welfare and support with animal experiments (Animal facility, i3S); and to LC Sciences for the miRNA microarray data and analysis. The mass spectrometry technique was performed by Hugo Osório at the i3S Proteomics Scientific Platform with support from the Portuguese Mass Spectrometry Network, integrated in the National Roadmap of Research Infrastructures of Strategic Relevance (ROTEIRO/0028/2013; LISBOA-01–0145-FEDER-022125). This work was funded by project NORTE-01–0145-FEDER-000012, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). J.P.B. and J.B. are supported by FCT–Fundação para a Ciência e Tecnologia, through BiotechHealth PhD program fellowship (PD/BD/135490/2018) and Areas of Basic and Applied Biology PhD program fellowship (PD/BD/135450/2017), respectively.Springer NatureRepositório Científico do Instituto Politécnico do PortoBrás, João PauloBravo, JoanaFreitas, JaimeBarbosa, Mário AdolfoSantos, Susana GomesSummavielle, Teresa2022-01-12T11:35:45Z20202020-01-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/10400.22/19421engBrás, J. P., Bravo, J., Freitas, J., Barbosa, M. A., Santos, S. G., Summavielle, T., & Almeida, M. I. (2020). TNF-alpha-induced microglia activation requires miR-342: Impact on NF-kB signaling and neurotoxicity. Cell Death & Disease, 11(6), Artigo 6. https://doi.org/10.1038/s41419-020-2626-610.1038/s41419-020-2626-6info:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2023-12-20T01:54:49Zoai:recipp.ipp.pt:10400.22/19421Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-19T17:39:18.435397Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse |
dc.title.none.fl_str_mv |
TNF-alpha-induced microglia activation requires miR-342: impact on NF-kB signaling and neurotoxicity |
title |
TNF-alpha-induced microglia activation requires miR-342: impact on NF-kB signaling and neurotoxicity |
spellingShingle |
TNF-alpha-induced microglia activation requires miR-342: impact on NF-kB signaling and neurotoxicity Brás, João Paulo Enzyme-linked immunosorbent assay (ELISA) Mixed glial cells Primary microglia N9 microglial cells TNF-alpha-induced microglia activation miR-342 |
title_short |
TNF-alpha-induced microglia activation requires miR-342: impact on NF-kB signaling and neurotoxicity |
title_full |
TNF-alpha-induced microglia activation requires miR-342: impact on NF-kB signaling and neurotoxicity |
title_fullStr |
TNF-alpha-induced microglia activation requires miR-342: impact on NF-kB signaling and neurotoxicity |
title_full_unstemmed |
TNF-alpha-induced microglia activation requires miR-342: impact on NF-kB signaling and neurotoxicity |
title_sort |
TNF-alpha-induced microglia activation requires miR-342: impact on NF-kB signaling and neurotoxicity |
author |
Brás, João Paulo |
author_facet |
Brás, João Paulo Bravo, Joana Freitas, Jaime Barbosa, Mário Adolfo Santos, Susana Gomes Summavielle, Teresa |
author_role |
author |
author2 |
Bravo, Joana Freitas, Jaime Barbosa, Mário Adolfo Santos, Susana Gomes Summavielle, Teresa |
author2_role |
author author author author author |
dc.contributor.none.fl_str_mv |
Repositório Científico do Instituto Politécnico do Porto |
dc.contributor.author.fl_str_mv |
Brás, João Paulo Bravo, Joana Freitas, Jaime Barbosa, Mário Adolfo Santos, Susana Gomes Summavielle, Teresa |
dc.subject.por.fl_str_mv |
Enzyme-linked immunosorbent assay (ELISA) Mixed glial cells Primary microglia N9 microglial cells TNF-alpha-induced microglia activation miR-342 |
topic |
Enzyme-linked immunosorbent assay (ELISA) Mixed glial cells Primary microglia N9 microglial cells TNF-alpha-induced microglia activation miR-342 |
description |
Growing evidences suggest that sustained neuroinflammation, caused by microglia overactivation, is implicated in the development and aggravation of several neurological and psychiatric disorders. In some pathological conditions, microglia produce increased levels of cytotoxic and inflammatory mediators, such as tumor necrosis factor alpha (TNF-α), which can reactivate microglia in a positive feedback mechanism. However, specific molecular mediators that can be effectively targeted to control TNF-α-mediated microglia overactivation, are yet to be uncovered. In this context, we aim to identify novel TNF-α-mediated micro(mi)RNAs and to dissect their roles in microglia activation, as well as to explore their impact on the cellular communication with neurons. A miRNA microarray, followed by RT-qPCR validation, was performed on TNF-α-stimulated primary rat microglia. Gain- and loss-of-function in vitro assays and proteomic analysis were used to dissect the role of miR-342 in microglia activation. Co-cultures of microglia with hippocampal neurons, using a microfluidic system, were performed to understand the impact on neurotoxicity. Stimulation of primary rat microglia with TNF-α led to an upregulation of Nos2, Tnf, and Il1b mRNAs. In addition, ph-NF-kB p65 levels were also increased. miRNA microarray analysis followed by RT-qPCR validation revealed that TNF-α stimulation induced the upregulation of miR-342. Interestingly, miR-342 overexpression in N9 microglia was sufficient to activate the NF-kB pathway by inhibiting BAG-1, leading to increased secretion of TNF-α and IL-1β. Conversely, miR-342 inhibition led to a strong decrease in the levels of these cytokines after TNF-α activation. In fact, both TNF-α-stimulated and miR-342-overexpressing microglia drastically affected neuron viability. Remarkably, increased levels of nitrites were detected in the supernatants of these co-cultures. Globally, our findings show that miR-342 is a crucial mediator of TNF-α-mediated microglia activation and a potential target to tackle microglia-driven neuroinflammation. |
publishDate |
2020 |
dc.date.none.fl_str_mv |
2020 2020-01-01T00:00:00Z 2022-01-12T11:35:45Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://hdl.handle.net/10400.22/19421 |
url |
http://hdl.handle.net/10400.22/19421 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
Brás, J. P., Bravo, J., Freitas, J., Barbosa, M. A., Santos, S. G., Summavielle, T., & Almeida, M. I. (2020). TNF-alpha-induced microglia activation requires miR-342: Impact on NF-kB signaling and neurotoxicity. Cell Death & Disease, 11(6), Artigo 6. https://doi.org/10.1038/s41419-020-2626-6 10.1038/s41419-020-2626-6 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.publisher.none.fl_str_mv |
Springer Nature |
publisher.none.fl_str_mv |
Springer Nature |
dc.source.none.fl_str_mv |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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