Studying Autophagy in Microglia: Overcoming the Obstacles.

In this chapter, we provide an overview of the main techniques and experimental approaches that can be used to analyze autophagy flux in microglia, the brain-resident macrophages. For this purpose, we first briefly introduce the main peculiarities of microglial biology, describe the basic mechanisms...

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Detalles Bibliográficos
Autores: Plaza Zabala, Ainhoa, Sierra Saavedra, Amanda
Tipo de recurso: capítulo de libro
Fecha de publicación:2023
País:España
Institución:Universidad del País Vasco
Repositorio:Addi. Archivo Digital para la Docencia y la Investigación
OAI Identifier:oai:addi.ehu.eus:10810/74087
Acceso en línea:http://hdl.handle.net/10810/74087
Access Level:acceso abierto
Palabra clave:autophagy flux
microglia
autophagosomes
lysosomes
western blot
confocal microscopy
electron microscopy
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spelling Studying Autophagy in Microglia: Overcoming the Obstacles.Plaza Zabala, AinhoaSierra Saavedra, Amandaautophagy fluxmicrogliaautophagosomeslysosomeswestern blotconfocal microscopyelectron microscopyIn this chapter, we provide an overview of the main techniques and experimental approaches that can be used to analyze autophagy flux in microglia, the brain-resident macrophages. For this purpose, we first briefly introduce the main peculiarities of microglial biology, describe the basic mechanisms and functions of autophagy, and summarize the evidence accumulated so far on the role of autophagy in the regulation of microglial survival and functions, mainly phagocytosis and inflammation. Then, we highlight conceptual and technical aspects of autophagic recycling and microglial physiology that need to be taken into account for the accurate evaluation of autophagy flux in microglia. Finally, we describe the main assays that can be used to analyze the complete sequence of autophagosome formation and degradation or autophagy flux mainly in cultured microglia and in vivo. The main approaches include indirect tracking of autophagosomes by autophagic enzymes such as LC3 by western blot and fluorescence-based confocal microscopy, as well as direct analysis of autophagic vesicles by electron microscopy. We also discuss the advantages and disadvantages of using these methods in specific experimental contexts and highlight the need to complement LC3 and/or electron microscopy data with analysis of other autophagic effectors and lysosomal proteins that participate in the initiation and completion of autophagy flux, respectively. In summary, we provide an experimental guide for the analysis of autophagosome turnover in microglia, emphasizing the need of combining as many markers and complementary approaches as possible to fully characterize the status of autophagy flux in microglia.Humana202520252023info:eu-repo/semantics/bookPartapplication/pdfhttp://hdl.handle.net/10810/74087reponame:Addi. Archivo Digital para la Docencia y la Investigacióninstname:Universidad del País VascoIngléshttps://doi.org/10.1007/978-1-0716-3437-0_3info:eu-repo/semantics/openAccess© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Natureoai:addi.ehu.eus:10810/740872026-06-18T09:23:17Z
dc.title.none.fl_str_mv Studying Autophagy in Microglia: Overcoming the Obstacles.
title Studying Autophagy in Microglia: Overcoming the Obstacles.
spellingShingle Studying Autophagy in Microglia: Overcoming the Obstacles.
Plaza Zabala, Ainhoa
autophagy flux
microglia
autophagosomes
lysosomes
western blot
confocal microscopy
electron microscopy
title_short Studying Autophagy in Microglia: Overcoming the Obstacles.
title_full Studying Autophagy in Microglia: Overcoming the Obstacles.
title_fullStr Studying Autophagy in Microglia: Overcoming the Obstacles.
title_full_unstemmed Studying Autophagy in Microglia: Overcoming the Obstacles.
title_sort Studying Autophagy in Microglia: Overcoming the Obstacles.
dc.creator.none.fl_str_mv Plaza Zabala, Ainhoa
Sierra Saavedra, Amanda
author Plaza Zabala, Ainhoa
author_facet Plaza Zabala, Ainhoa
Sierra Saavedra, Amanda
author_role author
author2 Sierra Saavedra, Amanda
author2_role author
dc.subject.none.fl_str_mv autophagy flux
microglia
autophagosomes
lysosomes
western blot
confocal microscopy
electron microscopy
topic autophagy flux
microglia
autophagosomes
lysosomes
western blot
confocal microscopy
electron microscopy
description In this chapter, we provide an overview of the main techniques and experimental approaches that can be used to analyze autophagy flux in microglia, the brain-resident macrophages. For this purpose, we first briefly introduce the main peculiarities of microglial biology, describe the basic mechanisms and functions of autophagy, and summarize the evidence accumulated so far on the role of autophagy in the regulation of microglial survival and functions, mainly phagocytosis and inflammation. Then, we highlight conceptual and technical aspects of autophagic recycling and microglial physiology that need to be taken into account for the accurate evaluation of autophagy flux in microglia. Finally, we describe the main assays that can be used to analyze the complete sequence of autophagosome formation and degradation or autophagy flux mainly in cultured microglia and in vivo. The main approaches include indirect tracking of autophagosomes by autophagic enzymes such as LC3 by western blot and fluorescence-based confocal microscopy, as well as direct analysis of autophagic vesicles by electron microscopy. We also discuss the advantages and disadvantages of using these methods in specific experimental contexts and highlight the need to complement LC3 and/or electron microscopy data with analysis of other autophagic effectors and lysosomal proteins that participate in the initiation and completion of autophagy flux, respectively. In summary, we provide an experimental guide for the analysis of autophagosome turnover in microglia, emphasizing the need of combining as many markers and complementary approaches as possible to fully characterize the status of autophagy flux in microglia.
publishDate 2023
dc.date.none.fl_str_mv 2023
2025
2025
dc.type.none.fl_str_mv info:eu-repo/semantics/bookPart
format bookPart
dc.identifier.none.fl_str_mv http://hdl.handle.net/10810/74087
url http://hdl.handle.net/10810/74087
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv https://doi.org/10.1007/978-1-0716-3437-0_3
dc.rights.none.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 Humana
publisher.none.fl_str_mv Humana
dc.source.none.fl_str_mv reponame:Addi. Archivo Digital para la Docencia y la Investigación
instname:Universidad del País Vasco
instname_str Universidad del País Vasco
reponame_str Addi. Archivo Digital para la Docencia y la Investigación
collection Addi. Archivo Digital para la Docencia y la Investigación
repository.name.fl_str_mv
repository.mail.fl_str_mv
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