Cells

Review

17 pages, 3120 KiB

Open AccessFeature PaperReview

Multiscale and Multimodal Approaches to Study Autophagy in Model Plants

by Jessica Marion, Romain Le Bars, Laetitia Besse, Henri Batoko and Béatrice Satiat-Jeunemaitre

Cells 2018, 7(1), 5; https://doi.org/10.3390/cells7010005 - 9 Jan 2018

Cited by 15 | Viewed by 7959

Autophagy is a catabolic process used by eukaryotic cells to maintain or restore cellular and organismal homeostasis. A better understanding of autophagy in plant biology could lead to an improvement of the recycling processes of plant cells and thus contribute, for example, towards [...] Read more.

Autophagy is a catabolic process used by eukaryotic cells to maintain or restore cellular and organismal homeostasis. A better understanding of autophagy in plant biology could lead to an improvement of the recycling processes of plant cells and thus contribute, for example, towards reducing the negative ecological consequences of nitrogen-based fertilizers in agriculture. It may also help to optimize plant adaptation to adverse biotic and abiotic conditions through appropriate plant breeding or genetic engineering to incorporate useful traits in relation to this catabolic pathway. In this review, we describe useful protocols for studying autophagy in the plant cell, taking into account some specificities of the plant model. Full article

(This article belongs to the Special Issue Assays to Monitor Autophagy in Model Systems)

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Open AccessReview

Standard Assays for the Study of Autophagy in the Ex Vivo Retina

by Raquel Gómez-Sintes, Beatriz Villarejo-Zori, Ana Serrano-Puebla, Lorena Esteban-Martínez, Elena Sierra-Filardi, Ignacio Ramírez-Pardo, Natalia Rodríguez-Muela and Patricia Boya

Cells 2017, 6(4), 37; https://doi.org/10.3390/cells6040037 - 22 Oct 2017

Cited by 13 | Viewed by 7092

Abstract

Autophagy is a catabolic pathway that mediates the degradation and recycling of intracellular components, and is a key player in a variety of physiological processes in cells and tissues. Recent studies of autophagy in the eye suggest that this pathway is fundamental for [...] Read more.

Autophagy is a catabolic pathway that mediates the degradation and recycling of intracellular components, and is a key player in a variety of physiological processes in cells and tissues. Recent studies of autophagy in the eye suggest that this pathway is fundamental for the preservation of retinal homeostasis. Given its accessible location outside the brain, the retina is an ideal organ in which to study the central nervous system and a wide range of neuronal processes, from development to neurodegeneration. Here we review several methods used to assess autophagy in the retina in both physiological and pathological conditions. Full article

(This article belongs to the Special Issue Assays to Monitor Autophagy in Model Systems)

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Open AccessReview

Monitoring Autophagy in the Model Green Microalga Chlamydomonas reinhardtii

by María Esther Pérez-Pérez, Inmaculada Couso, Luis G. Heredia-Martínez and José L. Crespo

Cells 2017, 6(4), 36; https://doi.org/10.3390/cells6040036 - 22 Oct 2017

Cited by 34 | Viewed by 8314

Abstract

Autophagy is an intracellular catabolic system that delivers cytoplasmic constituents and organelles in the vacuole. This degradative process is mediated by a group of proteins coded by autophagy-related (ATG) genes that are widely conserved from yeasts to plants and mammals. Homologs [...] Read more.

Autophagy is an intracellular catabolic system that delivers cytoplasmic constituents and organelles in the vacuole. This degradative process is mediated by a group of proteins coded by autophagy-related (ATG) genes that are widely conserved from yeasts to plants and mammals. Homologs of ATG genes have been also identified in algal genomes including the unicellular model green alga Chlamydomonas reinhardtii. The development of specific tools to monitor autophagy in Chlamydomonas has expanded our current knowledge about the regulation and function of this process in algae. Recent findings indicated that autophagy is regulated by redox signals and the TOR network in Chlamydomonas and revealed that this process may play in important role in the control of lipid metabolism and ribosomal protein turnover in this alga. Here, we will describe the different techniques and approaches that have been reported to study autophagy and autophagic flux in Chlamydomonas. Full article

(This article belongs to the Special Issue Assays to Monitor Autophagy in Model Systems)

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Open AccessReview

Assessing Autophagy in Sciatic Nerves of a Rat Model that Develops Inflammatory Autoimmune Peripheral Neuropathies

by Susana Brun, Nicolas Schall, Hélène Jeltsch-David, Jérôme de Sèze and Sylviane Muller

Cells 2017, 6(3), 30; https://doi.org/10.3390/cells6030030 - 18 Sep 2017

Cited by 9 | Viewed by 11922

Abstract

The rat sciatic nerve has attracted widespread attention as an excellent model system for studying autophagy alterations in peripheral neuropathies. In our laboratory, we have developed an original rat model, which we used currently in routine novel drug screening and to evaluate treatment [...] Read more.

The rat sciatic nerve has attracted widespread attention as an excellent model system for studying autophagy alterations in peripheral neuropathies. In our laboratory, we have developed an original rat model, which we used currently in routine novel drug screening and to evaluate treatment strategies for chronic inflammatory demyelinating polyneuropathy (CIDP) and other closely related diseases. Lewis rats injected with the S-palmitoylated P0(180-199) peptide develop a chronic, sometimes relapsing-remitting type of disease. Our model fulfills electrophysiological criteria of demyelination with axonal degeneration, confirmed by immunohistopathology and several typical features of CIDP. We have set up a series of techniques that led us to examine the failures of autophagy pathways in the sciatic nerve of these model rats and to follow the possible improvement of these defects after treatment. Based on these newly introduced methods, a novel area of investigation is now open and will allow us to more thoroughly examine important features of certain autophagy pathways occurring in sciatic nerves. Full article

(This article belongs to the Special Issue Assays to Monitor Autophagy in Model Systems)

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Open AccessReview

Approaches for Studying Autophagy in Caenorhabditis elegans

by Yanfang Chen, Vincent Scarcelli and Renaud Legouis

Cells 2017, 6(3), 27; https://doi.org/10.3390/cells6030027 - 30 Aug 2017

Cited by 34 | Viewed by 8927

Abstract

Macroautophagy (hereafter referred to as autophagy) is an intracellular degradative process, well conserved among eukaryotes. By engulfing cytoplasmic constituents into the autophagosome for degradation, this process is involved in the maintenance of cellular homeostasis. Autophagy induction triggers the formation of a cup-shaped double [...] Read more.

Macroautophagy (hereafter referred to as autophagy) is an intracellular degradative process, well conserved among eukaryotes. By engulfing cytoplasmic constituents into the autophagosome for degradation, this process is involved in the maintenance of cellular homeostasis. Autophagy induction triggers the formation of a cup-shaped double membrane structure, the phagophore, which progressively elongates and encloses materials to be removed. This double membrane vesicle, which is called an autophagosome, fuses with lysosome and forms the autolysosome. The inner membrane of the autophagosome, along with engulfed compounds, are degraded by lysosomal enzymes, which enables the recycling of carbohydrates, amino acids, nucleotides, and lipids. In response to various factors, autophagy can be induced for non-selective degradation of bulk cytoplasm. Autophagy is also able to selectively target cargoes and organelles such as mitochondria or peroxisome, functioning as a quality control system. The modification of autophagy flux is involved in developmental processes such as resistance to stress conditions, aging, cell death, and multiple pathologies. So, the use of animal models is essential for understanding these processes in the context of different cell types throughout the entire lifespan. For almost 15 years, the nematode Caenorhabditis elegans has emerged as a powerful model to analyze autophagy in physiological or pathological contexts. This review presents a rapid overview of physiological processes involving autophagy in Caenorhabditis elegans, the different assays used to monitor autophagy, their drawbacks, and specific tools for the analyses of selective autophagy. Full article

(This article belongs to the Special Issue Assays to Monitor Autophagy in Model Systems)

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Open AccessReview

Assessment of Autophagy in Neurons and Brain Tissue

by Irene Benito-Cuesta, Héctor Diez, Lara Ordoñez and Francisco Wandosell

Cells 2017, 6(3), 25; https://doi.org/10.3390/cells6030025 - 23 Aug 2017

Cited by 43 | Viewed by 9376

Abstract

Autophagy is a complex process that controls the transport of cytoplasmic components into lysosomes for degradation. This highly conserved proteolytic system involves dynamic and complex processes, using similar molecular elements and machinery from yeast to humans. Moreover, autophagic dysfunction may contribute to a [...] Read more.

Autophagy is a complex process that controls the transport of cytoplasmic components into lysosomes for degradation. This highly conserved proteolytic system involves dynamic and complex processes, using similar molecular elements and machinery from yeast to humans. Moreover, autophagic dysfunction may contribute to a broad spectrum of mammalian diseases. Indeed, in adult tissues, where the capacity for regeneration or cell division is low or absent (e.g., in the mammalian brain), the accumulation of proteins/peptides that would otherwise be recycled or destroyed may have pathological implications. Indeed, such changes are hallmarks of pathologies, like Alzheimer’s, Prion or Parkinson’s disease, known as proteinopathies. However, it is still unclear whether such dysfunction is a cause or an effect in these conditions. One advantage when analysing autophagy in the mammalian brain is that almost all the markers described in different cell lineages and systems appear to be present in the brain, and even in neurons. By contrast, the mixture of cell types present in the brain and the differentiation stage of such neurons, when compared with neurons in culture, make translating basic research to the clinic less straightforward. Thus, the purpose of this review is to describe and discuss the methods available to monitor autophagy in neurons and in the mammalian brain, a process that is not yet fully understood, focusing primarily on mammalian macroautophagy. We will describe some general features of neuronal autophagy that point to our focus on neuropathologies in which macroautophagy may be altered. Indeed, we centre this review around the hypothesis that enhanced autophagy may be able to provide therapeutic benefits in some brain pathologies, like Alzheimer’s disease, considering this pathology as one of the most prevalent proteinopathies. Full article

(This article belongs to the Special Issue Assays to Monitor Autophagy in Model Systems)

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2757 KiB

Open AccessFeature PaperReview

Induced Pluripotent Stem Cell Neuronal Models for the Study of Autophagy Pathways in Human Neurodegenerative Disease

by Natalia Jiménez-Moreno, Petros Stathakos, Maeve A. Caldwell and Jon D. Lane

Cells 2017, 6(3), 24; https://doi.org/10.3390/cells6030024 - 11 Aug 2017

Cited by 18 | Viewed by 11800

Abstract

Human induced pluripotent stem cells (hiPSCs) are invaluable tools for research into the causes of diverse human diseases, and have enormous potential in the emerging field of regenerative medicine. Our ability to reprogramme patient cells to become hiPSCs, and to subsequently direct their [...] Read more.

Human induced pluripotent stem cells (hiPSCs) are invaluable tools for research into the causes of diverse human diseases, and have enormous potential in the emerging field of regenerative medicine. Our ability to reprogramme patient cells to become hiPSCs, and to subsequently direct their differentiation towards those classes of neurons that are vulnerable to stress, is revealing how genetic mutations cause changes at the molecular level that drive the complex pathogeneses of human neurodegenerative diseases. Autophagy dysregulation is considered to be a major contributor in neural decline during the onset and progression of many human neurodegenerative diseases, meaning that a better understanding of the control of non-selective and selective autophagy pathways (including mitophagy) in disease-affected classes of neurons is needed. To achieve this, it is essential that the methodologies commonly used to study autophagy regulation under basal and stressed conditions in standard cell-line models are accurately applied when using hiPSC-derived neuronal cultures. Here, we discuss the roles and control of autophagy in human stem cells, and how autophagy contributes to neural differentiation in vitro. We also describe how autophagy-monitoring tools can be applied to hiPSC-derived neurons for the study of human neurodegenerative disease in vitro. Full article

(This article belongs to the Special Issue Assays to Monitor Autophagy in Model Systems)

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Open AccessFeature PaperReview

Assays to Monitor Autophagy in Saccharomyces cerevisiae

by Raffaela Torggler, Daniel Papinski and Claudine Kraft

Cells 2017, 6(3), 23; https://doi.org/10.3390/cells6030023 - 13 Jul 2017

Cited by 45 | Viewed by 13795

Abstract

Autophagy is an intracellular process responsible for the degradation and recycling of cytoplasmic components. It selectively removes harmful cellular material and enables the cell to survive starvation by mobilizing nutrients via the bulk degradation of cytoplasmic components. While research over the last decades [...] Read more.

Autophagy is an intracellular process responsible for the degradation and recycling of cytoplasmic components. It selectively removes harmful cellular material and enables the cell to survive starvation by mobilizing nutrients via the bulk degradation of cytoplasmic components. While research over the last decades has led to the discovery of the key factors involved in autophagy, the pathway is not yet completely understood. The first studies of autophagy on a molecular level were conducted in the yeast Saccharomyces cerevisiae. Building up on these studies, many homologs have been found in higher eukaryotes. Yeast remains a highly relevant model organism for studying autophagy, with a wide range of established methods to elucidate the molecular details of the autophagy pathway. In this review, we provide an overview of methods to study both selective and bulk autophagy, including intermediate steps in the yeast Saccharomyces cerevisiae. We compare different assays, discuss their advantages and limitations and list potential applications. Full article

(This article belongs to the Special Issue Assays to Monitor Autophagy in Model Systems)

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2013 KiB

Open AccessFeature PaperReview

Exploring Autophagy in Drosophila

by Péter Lőrincz, Caroline Mauvezin and Gábor Juhász

Cells 2017, 6(3), 22; https://doi.org/10.3390/cells6030022 - 12 Jul 2017

Cited by 56 | Viewed by 11961

Abstract

Autophagy is a catabolic process in eukaryotic cells promoting bulk or selective degradation of cellular components within lysosomes. In recent decades, several model systems were utilized to dissect the molecular machinery of autophagy and to identify the impact of this cellular “self-eating” process [...] Read more.

Autophagy is a catabolic process in eukaryotic cells promoting bulk or selective degradation of cellular components within lysosomes. In recent decades, several model systems were utilized to dissect the molecular machinery of autophagy and to identify the impact of this cellular “self-eating” process on various physiological and pathological processes. Here we briefly discuss the advantages and limitations of using the fruit fly Drosophila melanogaster, a popular model in cell and developmental biology, to apprehend the main pathway of autophagy in a complete animal. Full article

(This article belongs to the Special Issue Assays to Monitor Autophagy in Model Systems)

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Open AccessFeature PaperReview

Studying Autophagy in Zebrafish

by Benan John Mathai, Annemarie H. Meijer and Anne Simonsen

Cells 2017, 6(3), 21; https://doi.org/10.3390/cells6030021 - 9 Jul 2017

Cited by 54 | Viewed by 12030

Abstract

Autophagy is an evolutionarily conserved catabolic process which allows lysosomal degradation of complex cytoplasmic components into basic biomolecules that are recycled for further cellular use. Autophagy is critical for cellular homeostasis and for degradation of misfolded proteins and damaged organelles as well as [...] Read more.

Autophagy is an evolutionarily conserved catabolic process which allows lysosomal degradation of complex cytoplasmic components into basic biomolecules that are recycled for further cellular use. Autophagy is critical for cellular homeostasis and for degradation of misfolded proteins and damaged organelles as well as intracellular pathogens. The role of autophagy in protection against age-related diseases and a plethora of other diseases is now coming to light; assisted by several divergent eukaryotic model systems ranging from yeast to mice. We here give an overview of different methods used to analyse autophagy in zebrafish—a relatively new model for studying autophagy—and briefly discuss what has been done so far and possible future directions. Full article

(This article belongs to the Special Issue Assays to Monitor Autophagy in Model Systems)

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Open AccessFeature PaperReview

Assays to Monitor Autophagy Progression in Cell Cultures

by Idil Orhon and Fulvio Reggiori

Cells 2017, 6(3), 20; https://doi.org/10.3390/cells6030020 - 7 Jul 2017

Cited by 45 | Viewed by 8230

Abstract

The vast number of implications of autophagy in multiple areas of life sciences and medicine has attracted the interest of numerous scientists that aim to unveil the role of this process in specific physiological and pathological contexts. Cell cultures are one of the [...] Read more.

The vast number of implications of autophagy in multiple areas of life sciences and medicine has attracted the interest of numerous scientists that aim to unveil the role of this process in specific physiological and pathological contexts. Cell cultures are one of the most frequently used experimental setup for the investigation of autophagy. As a result, it is essential to assess this highly regulated molecular pathway with efficient and reliable methods. Each method has its own advantages and disadvantages. Here, we present a review summarizing the most established assays used to monitor autophagy induction and progression in cell cultures, in order to guide researchers in the selection of the most optimal solution for their experimental setup and design. Full article

(This article belongs to the Special Issue Assays to Monitor Autophagy in Model Systems)

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Open AccessFeature PaperReview

Methods to Monitor and Quantify Autophagy in the Social Amoeba Dictyostelium discoideum

by Eunice Domínguez-Martín, Elena Cardenal-Muñoz, Jason S. King, Thierry Soldati, Roberto Coria and Ricardo Escalante

Cells 2017, 6(3), 18; https://doi.org/10.3390/cells6030018 - 3 Jul 2017

Cited by 22 | Viewed by 10757

Abstract

Autophagy is a eukaryotic catabolic pathway that degrades and recycles cellular components to maintain homeostasis. It can target protein aggregates, superfluous biomolecular complexes, dysfunctional and damaged organelles, as well as pathogenic intracellular microbes. Autophagy is a dynamic process in which the different stages [...] Read more.

Autophagy is a eukaryotic catabolic pathway that degrades and recycles cellular components to maintain homeostasis. It can target protein aggregates, superfluous biomolecular complexes, dysfunctional and damaged organelles, as well as pathogenic intracellular microbes. Autophagy is a dynamic process in which the different stages from initiation to final degradation of cargo are finely regulated. Therefore, the study of this process requires the use of a palette of techniques, which are continuously evolving and whose interpretation is not trivial. Here, we present the social amoeba Dictyostelium discoideum as a relevant model to study autophagy. Several methods have been developed based on the tracking and observation of autophagosomes by microscopy, analysis of changes in expression of autophagy genes and proteins, and examination of the autophagic flux with various techniques. In this review, we discuss the pros and cons of the currently available techniques to assess autophagy in this organism. Full article

(This article belongs to the Special Issue Assays to Monitor Autophagy in Model Systems)

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4852 KiB

Open AccessFeature PaperReview

Standard Immunohistochemical Assays to Assess Autophagy in Mammalian Tissue

by Wim Martinet, Lynn Roth and Guido R. Y. De Meyer

Cells 2017, 6(3), 17; https://doi.org/10.3390/cells6030017 - 30 Jun 2017

Cited by 28 | Viewed by 7918

Abstract

Autophagy is a highly conserved lysosomal degradation pathway with major impact on diverse human pathologies. Despite the development of different methodologies to detect autophagy both in vitro and in vivo, monitoring autophagy in tissue via immunohistochemical techniques is hampered due to the lack [...] Read more.

Autophagy is a highly conserved lysosomal degradation pathway with major impact on diverse human pathologies. Despite the development of different methodologies to detect autophagy both in vitro and in vivo, monitoring autophagy in tissue via immunohistochemical techniques is hampered due to the lack of biomarkers. Immunohistochemical detection of a punctate pattern of ATG8/MAP1LC3 proteins is currently the most frequently used approach to detect autophagy in situ, but it depends on a highly sensitive detection method and is prone to misinterpretation. Moreover, reliable MAP1LC3 immunohistochemical staining requires correct tissue processing and high-quality, isoform-specific antibodies. Immunohistochemical analysis of other autophagy-related protein targets such as SQSTM1, ubiquitin, ATG5 or lysosomal proteins is not recommended as marker for autophagic activity in tissue for multiple reasons including aspecific labeling of cellular structures and a lack of differential protein expression during autophagy initiation. To better understand the role of autophagy in human disease, novel biomarkers for visualization of the autophagic process with standard histology techniques are urgently needed. Full article

(This article belongs to the Special Issue Assays to Monitor Autophagy in Model Systems)

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Open AccessFeature PaperReview

Assessing Autophagy in Mouse Models and Patients with Systemic Autoimmune Diseases

by Fengjuan Wang, Baihui Li, Nicolas Schall, Maud Wilhelm and Sylviane Muller

Cells 2017, 6(3), 16; https://doi.org/10.3390/cells6030016 - 28 Jun 2017

Cited by 32 | Viewed by 8418

Abstract

Autophagy is a tightly regulated mechanism that allows cells to renew themselves through the lysosomal degradation of proteins, which are misfolded or produced in excess, and of damaged organelles. In the context of immunity, recent research has specially attempted to clarify its roles [...] Read more.

Autophagy is a tightly regulated mechanism that allows cells to renew themselves through the lysosomal degradation of proteins, which are misfolded or produced in excess, and of damaged organelles. In the context of immunity, recent research has specially attempted to clarify its roles in infection, inflammation and autoimmunity. Autophagy has emerged as a spotlight in several molecular pathways and trafficking events that participate to innate and adaptive immunity. Deregulation of autophagy has been associated to several autoimmune diseases, in particular to systemic lupus erythematosus. Nowadays, however, experimental data on the implication of autophagy in animal models of autoimmunity or patients remain limited. In our investigations, we use Murphy Roths Large (MRL)/lymphoproliferation (lpr) lupus-prone mice as a mouse model for lupus and secondary Sjögren’s syndrome, and, herein, we describe methods applied routinely to analyze different autophagic pathways in different lymphoid organs and tissues (spleen, lymph nodes, salivary glands). We also depict some techniques used to analyze autophagy in lupus patient’s blood samples. These methods can be adapted to the analysis of autophagy in other mouse models of autoinflammatory diseases. The understanding of autophagy implication in autoimmune diseases could prove to be very useful for developing novel immunomodulatory strategies. Our attention should be focused on the fact that autophagy processes are interconnected and that distinct pathways can be independently hyper-activated or downregulated in distinct organs and tissues of the same individual. Full article

(This article belongs to the Special Issue Assays to Monitor Autophagy in Model Systems)

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Open AccessReview

Methods for Measuring Autophagy in Mice

by Manon Moulis and Cécile Vindis

Cells 2017, 6(2), 14; https://doi.org/10.3390/cells6020014 - 8 Jun 2017

Cited by 53 | Viewed by 8802

Abstract

Autophagy is a dynamic intracellular process that mediates the degradation of damaged cytoplasmic components by the lysosome. This process plays important roles in maintaining normal cellular homeostasis and energy balance. Measuring autophagy activity is critical and although the determination of autophagic flux in [...] Read more.

Autophagy is a dynamic intracellular process that mediates the degradation of damaged cytoplasmic components by the lysosome. This process plays important roles in maintaining normal cellular homeostasis and energy balance. Measuring autophagy activity is critical and although the determination of autophagic flux in isolated cells is well documented, there is a need to have reliable and quantitative assays to evaluate autophagy in whole organisms. Because mouse models have been precious in establishing the functional significance of autophagy under physiological or pathological conditions, we present in this chapter a compendium of the current available methods to measure autophagy in mice, and discuss their advantages and limitations. Full article

(This article belongs to the Special Issue Assays to Monitor Autophagy in Model Systems)

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