Assessing Autophagy in Microglia: A Two-Step Model to Determine Autophagosome Formation, Degradation, and Net Turnover

Autophagy is a complex process that encompasses the enclosure of cytoplasmic debris or dysfunctional organelles in membranous vesicles, the autophagosomes, for their elimination in the lysosomes. Autophagy is increasingly recognized as a critical process in macrophages, including microglia, as it fi...

ver descrição completa

Detalhes bibliográficos
Autores: Plaza Zabala, Ainhoa, Sierra Torre, Virginia, Sierra Saavedra, Amanda
Formato: artículo
Fecha de publicación:2021
País:España
Recursos:Universidad del País Vasco
Repositorio:Addi. Archivo Digital para la Docencia y la Investigación
OAI Identifier:oai:addi.ehu.eus:10810/50582
Acesso em linha:http://hdl.handle.net/10810/50582
Access Level:acceso abierto
Palavra-chave:autophagy
autophagosome
formation
degradation
LC3
microglia
id ES_a9bf43aebb59b7a25dd27c8fac5644e6
oai_identifier_str oai:addi.ehu.eus:10810/50582
network_acronym_str ES
network_name_str España
repository_id_str
spelling Assessing Autophagy in Microglia: A Two-Step Model to Determine Autophagosome Formation, Degradation, and Net TurnoverPlaza Zabala, AinhoaSierra Torre, VirginiaSierra Saavedra, AmandaautophagyautophagosomeformationdegradationLC3microgliaAutophagy is a complex process that encompasses the enclosure of cytoplasmic debris or dysfunctional organelles in membranous vesicles, the autophagosomes, for their elimination in the lysosomes. Autophagy is increasingly recognized as a critical process in macrophages, including microglia, as it finely regulates innate immune functions such as inflammation. A gold-standard method to assess its induction is the analysis of the autophagic flux using as a surrogate the expression of the microtubule-associated light chain protein 3 conjugated to phosphatidylethanolamine (LC3-II) by Western blot, in the presence of lysosomal inhibitors. Therefore, the current definition of autophagy flux actually puts the focus on the degradation stage of autophagy. In contrast, the most important autophagy controlling genes that have been identified in the last few years in fact target early stages of autophagosome formation. From a biological standpoint is therefore conceivable that autophagosome formation and degradation are independently regulated and we argue that both stages need to be systematically analyzed. Here, we propose a simple two-step model to understand changes in autophagosome formation and degradation using data from conventional LC3-II Western blot, and test it using two models of autophagy modulation in cultured microglia: rapamycin and the ULK1/2 inhibitor, MRT68921. Our two-step model will help to unravel the effect of genetic, pharmacological, and environmental manipulations on both formation and degradation of autophagosomes, contributing to dissect out the role of autophagy in physiology and pathology in microglia as well as other cell types.This work was supported by grants from the Spanish Ministry of Science and Innovation (https://www.ciencia.gob.es/) with FEDER funds (RTI2018- 099267- B- I00) and a Tatiana Foundation project (P-048-FTPGB 2018) to AS. VS-T holds a predoctoral fellowship from the Basque Government.Frontiers Media202120212021info:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/10810/50582reponame:Addi. Archivo Digital para la Docencia y la Investigacióninstname:Universidad del País VascoInglésinfo:eu-repo/grantAgreement/MICINN/RTI2018- 099267- B- I00/https://www.frontiersin.org/articles/10.3389/fimmu.2020.620602/fullinfo:eu-repo/semantics/openAccesshttp://creativecommons.org/licenses/by/3.0/es/2021 Plaza-Zabala, Sierra-Torre and Sierra. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.Atribución 3.0 Españaoai:addi.ehu.eus:10810/505822026-06-18T09:23:17Z
dc.title.none.fl_str_mv Assessing Autophagy in Microglia: A Two-Step Model to Determine Autophagosome Formation, Degradation, and Net Turnover
title Assessing Autophagy in Microglia: A Two-Step Model to Determine Autophagosome Formation, Degradation, and Net Turnover
spellingShingle Assessing Autophagy in Microglia: A Two-Step Model to Determine Autophagosome Formation, Degradation, and Net Turnover
Plaza Zabala, Ainhoa
autophagy
autophagosome
formation
degradation
LC3
microglia
title_short Assessing Autophagy in Microglia: A Two-Step Model to Determine Autophagosome Formation, Degradation, and Net Turnover
title_full Assessing Autophagy in Microglia: A Two-Step Model to Determine Autophagosome Formation, Degradation, and Net Turnover
title_fullStr Assessing Autophagy in Microglia: A Two-Step Model to Determine Autophagosome Formation, Degradation, and Net Turnover
title_full_unstemmed Assessing Autophagy in Microglia: A Two-Step Model to Determine Autophagosome Formation, Degradation, and Net Turnover
title_sort Assessing Autophagy in Microglia: A Two-Step Model to Determine Autophagosome Formation, Degradation, and Net Turnover
dc.creator.none.fl_str_mv Plaza Zabala, Ainhoa
Sierra Torre, Virginia
Sierra Saavedra, Amanda
author Plaza Zabala, Ainhoa
author_facet Plaza Zabala, Ainhoa
Sierra Torre, Virginia
Sierra Saavedra, Amanda
author_role author
author2 Sierra Torre, Virginia
Sierra Saavedra, Amanda
author2_role author
author
dc.subject.none.fl_str_mv autophagy
autophagosome
formation
degradation
LC3
microglia
topic autophagy
autophagosome
formation
degradation
LC3
microglia
description Autophagy is a complex process that encompasses the enclosure of cytoplasmic debris or dysfunctional organelles in membranous vesicles, the autophagosomes, for their elimination in the lysosomes. Autophagy is increasingly recognized as a critical process in macrophages, including microglia, as it finely regulates innate immune functions such as inflammation. A gold-standard method to assess its induction is the analysis of the autophagic flux using as a surrogate the expression of the microtubule-associated light chain protein 3 conjugated to phosphatidylethanolamine (LC3-II) by Western blot, in the presence of lysosomal inhibitors. Therefore, the current definition of autophagy flux actually puts the focus on the degradation stage of autophagy. In contrast, the most important autophagy controlling genes that have been identified in the last few years in fact target early stages of autophagosome formation. From a biological standpoint is therefore conceivable that autophagosome formation and degradation are independently regulated and we argue that both stages need to be systematically analyzed. Here, we propose a simple two-step model to understand changes in autophagosome formation and degradation using data from conventional LC3-II Western blot, and test it using two models of autophagy modulation in cultured microglia: rapamycin and the ULK1/2 inhibitor, MRT68921. Our two-step model will help to unravel the effect of genetic, pharmacological, and environmental manipulations on both formation and degradation of autophagosomes, contributing to dissect out the role of autophagy in physiology and pathology in microglia as well as other cell types.
publishDate 2021
dc.date.none.fl_str_mv 2021
2021
2021
dc.type.none.fl_str_mv info:eu-repo/semantics/article
format article
dc.identifier.none.fl_str_mv http://hdl.handle.net/10810/50582
url http://hdl.handle.net/10810/50582
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv info:eu-repo/grantAgreement/MICINN/RTI2018- 099267- B- I00/
https://www.frontiersin.org/articles/10.3389/fimmu.2020.620602/full
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
http://creativecommons.org/licenses/by/3.0/es/
Atribución 3.0 España
eu_rights_str_mv openAccess
rights_invalid_str_mv http://creativecommons.org/licenses/by/3.0/es/
Atribución 3.0 España
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Frontiers Media
publisher.none.fl_str_mv Frontiers Media
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
_version_ 1869416066063532032
score 15.300724

Registos relacionados