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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...

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Autores principales: Plaza-Zabala, Ainhoa, Sierra-Torre, Virginia, Sierra, Amanda
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7878397/
https://www.ncbi.nlm.nih.gov/pubmed/33584716
http://dx.doi.org/10.3389/fimmu.2020.620602
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author Plaza-Zabala, Ainhoa
Sierra-Torre, Virginia
Sierra, Amanda
author_facet Plaza-Zabala, Ainhoa
Sierra-Torre, Virginia
Sierra, Amanda
author_sort Plaza-Zabala, Ainhoa
collection PubMed
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.
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spelling pubmed-78783972021-02-13 Assessing Autophagy in Microglia: A Two-Step Model to Determine Autophagosome Formation, Degradation, and Net Turnover Plaza-Zabala, Ainhoa Sierra-Torre, Virginia Sierra, Amanda Front Immunol Immunology 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. Frontiers Media S.A. 2021-01-29 /pmc/articles/PMC7878397/ /pubmed/33584716 http://dx.doi.org/10.3389/fimmu.2020.620602 Text en Copyright © 2021 Plaza-Zabala, Sierra-Torre and Sierra https://creativecommons.org/licenses/by/4.0/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.
spellingShingle Immunology
Plaza-Zabala, Ainhoa
Sierra-Torre, Virginia
Sierra, Amanda
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
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_short 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
topic Immunology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7878397/
https://www.ncbi.nlm.nih.gov/pubmed/33584716
http://dx.doi.org/10.3389/fimmu.2020.620602
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