Autophagy in Health and Disease

Autophagy in Health and Disease, Second Edition provides a comprehensive overview of the process of autophagy and its impact on human physiology and pathophysiology. It expands on the scope of the first edition by covering a wider range of cell types, developmental processes, and organ systems. The second edition is an international effort by investigators from 15 different countries whose many contributions are comprised in 28 chapters organized into six sections. The first section (Chapters 1-7) covers foundational concepts, including history, trajectory of the research field, mechanisms of autophagy, and autophagy regulation. The second section (Chapters 8-11) details developmental aspects, including stem cells, embryogenesis, hematopoiesis, and paligenosis. The subsequent sections are devoted to the role of autophagy in specific organ systems involved in metabolic control and diabetes (Chapters 12-15), the cardiovascular system (Chapters 16-18), and the nervous system (Chapters 19-20). The final section (Chapters 21-28) addresses autophagy in other organ systems vital to human health and longevity. Also included are chapters on microautophagy, chaperone-mediated autophagy, and the potential for autophagy as a therapeutic target.

Autophagy in Health and Disease is invaluable to anyone new to the field as well as established investigators looking for a broader understanding of autophagy from outside their specific field of study.

• Provides a comprehensive overview of the process of autophagy and its impact on human physiology and pathology
• Offers extended coverage of the mechanisms that mediate autophagy
• Covers the role of autophagy in stem cells and induced pluripotent stem cells, as well as the regenerative process of paligenosis
• Highlights important questions that remain to be addressed

• Language: English
• Publisher: Academic Press
• Release date: Sep 22, 2021
• ISBN: 9780128220047

Book preview

Autophagy in Health and Disease

Second Edition

Editors

Beverly A. Rothermel

Division of Cardiology, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, United States

Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, United States

Abhinav Diwan

Center for Cardiovascular Research and Cardiovascular Division, Department of Medicine, Washington University School of Medicine, St. Louis, MO, United States

Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO, United States

Department of Obstetrics and Gynecology, Washington University School of Medicine, St. Louis, MO, United States

John Cochran VA Medical Center, St. Louis, MO, United States

Table of Contents

Cover image

Title page

Copyright

Contributors

About the editors

Foreword

Preface

Section I. Overview

Chapter 1. Introduction

A historical perspective

Developing a tool kit

An overview of mechanisms

Introduction to the second edition

Chapter 2. Mechanisms of autophagy: the machinery of macroautophagy and points of control

Origin of the phagophore membrane

Initiation of autophagosome biogenesis by the Atg1/ULK1 kinase complex

PI3K complex activation and PI3P production at the phagophore assembly site

Recruitment of PI3P-binding proteins to the phagophore assembly site

Cycling of Atg9/ATG9 vesicles

Phagophore expansion facilitated by ubiquitin-like conjugation systems

Fusion of autophagosomes with lysosomes/vacuoles

Concluding remarks

Chapter 3. Regulation of autophagy—transcriptional, posttranscriptional, translational, and posttranslational mechanisms

Introduction

Posttranslational regulation of autophagy

Transcriptional regulation of autophagy

Epigenetic regulation of autophagy

Translational regulation of autophagy

Conclusions

List of abbreviations

Chapter 4. Selectivity and trafficking of autophagic cargoes

Introducing autophagy—the machinery and the mechanism

Selective autophagy and cargo selection

Ubiquitin and cargo recognition

Selectivity determinants

ATG8 family (MAP1LC3A and GABARAP)

The plausible role of membrane sources in selectivity

Selective autophagy machinery and vesicular trafficking

Conclusion

Chapter 5. The role of lysosomes in autophagy

Lysosome overview

Lysosomes as metabolic sensors

Environment-sensing machinery at lysosomes

Cholesterol sensing and lysosomal machinery

Glucose sensing at the lysosome

GTPase Rheb, core for growth factor, and energy sensing at the lysosomal surface

Lysosomes regulate autophagy initiation

Lysosomes orchestrate their own biogenesis to drive autophagy

Lysosomal distribution regulates autophagy

Concluding remarks

Chapter 6. Methods for measuring autophagy

Measuring changes in levels of autophagic proteins to assess activity

Assessing cellular signaling cascades indicative of autophagic activation

Special considerations and issues

Using microscopy to measure autophagy

Measuring autophagy in vivo

Measuring organelle-specific autophagy

Concluding remarks

Chapter 7. Effects of physiologic inputs on autophagy

Autophagy during fasting

Autophagy during aging

Regulation of autophagy during exercise

Circadian regulation of autophagy

Sex differences in autophagy regulation

Section II. Development

Chapter 8. Autophagy in germ cells, stem cells, and induced pluripotent stem cells

Introduction

Autophagy during the formation of germ cells

Autophagy in embryonic stem cells

Autophagy in cell fate decisions and differentiation

Autophagy in stem cell aging and senescence

Chapter 9. Role of autophagy in embryogenesis

Introduction

Role of bulk autophagic degradation

Mouse

Role of autophagy in early embryonic development

Mechanism of autophagy induction during preimplantation embryonic development

Role of autophagy in fetal development

Role of selective autophagic degradation

Role of autophagy in gametogenesis

Concluding remarks

Chapter 10. Autophagy in hematopoiesis and leukemogenesis

Introduction

Conclusions

Chapter 11. Autophagy in cell plasticity with particular focus on paligenosis

Defining cell plasticity

Autophagic induction and mammalian target of rapamycin complex 1 in postinjury cell plasticity across diverse species

Overview of paligenosis: a conserved mechanism of cell plasticity

Component recycling and metabolic reprogramming to fuel regeneration

Cell plasticity and autophagy in tissue maintenance and disease

Conclusions

Section III. Metabolic control and diabetes

Chapter 12. Autophagy in the liver

General liver anatomy and functions

Physiological role of autophagy in the liver

Hepatic autophagy in obesity and nonalcoholic fatty liver disease

Nonhepatocyte roles of autophagy in the liver

Conclusion and future perspectives

Chapter 13. Autophagy in adipose tissue

Introduction

Autophagy and adipose tissue development and maintenance

Adipose autophagy in obesity and diabetes

Adipose lysosomal function in obesity and diabetes

Autophagy and lipodystrophy

Chapter 14. Autophagy in the pancreas

Introduction

Autophagy in the exocrine pancreas

Autophagy in the endocrine pancreas

Conclusions

List of abbreviations

Chapter 15. Skeletal muscle

Introduction

Autophagy in skeletal muscle

Exercise related autophagy

Autophagy in skeletal muscle disease

Conclusions and perspectives

Section IV. Cardiovascular system

Chapter 16. Autophagy in the cardiovascular system

Introduction

Autophagy in cardiac growth and repair

Autophagy in cardiomyocyte homeostasis and aging

Autophagy in myocardial infarction

Autophagy in arrhythmia

Autophagy in heart failure

Concluding remarks

Chapter 17. Lungs

Lungs

Autophagy in lung health

Autophagy in lung pathologies

Bronchopulmonary dysplasia

Lung cancer

Chronic obstructive pulmonary disease

Cystic fibrosis

Pulmonary hypertension

Interstitial lung diseases

Autophagy in lung infection and inflammation

Perspective

Chapter 18. Autophagy in the vasculature

Introduction

Autophagy as a mechanism for maintaining vascular function

The role of autophagy in atherosclerosis

The role of autophagy in vascular calcification

Autophagy in vascular aging: arterial stiffness and essential hypertension

Conclusion

Section V. The nervous system and neurodegeneration

Chapter 19. Altered autophagy on the path to Parkinson's disease

Introduction to Parkinson's disease

Mitophagy and mitochondrial quality control in Parkinson's disease

The function of Parkinson's disease–related proteins in macroautophagy

Role of the lysosome biogenesis program in Parkinson's disease

Role of chaperone-mediated autophagy in Parkinson's disease

Synaptic autophagy and Parkinson's disease

Future directions: targeting autophagy to treat Parkinson's disease

Chapter 20. Autophagic processes in early- and late-onset Alzheimer's disease

Introduction

Oxidized nicotinamide adenine dinucleotide: a regulator of autophagy and mitophagy

Impaired autophagy in Alzheimer's disease

Promoting autophagy as a therapeutic strategy against Alzheimer's disease

Future perspectives and concluding remarks

Section VI. Homeostasis and disease in other organ systems

Chapter 21. Autophagy as an integral immune system component

Introduction

Role of autophagy in cellular immunity in blood

Role of autophagy in antigen processing and presentation

Cross talk between autophagy and inflammatory signal transduction

Role of autophagy in autoimmune diseases

Xenophagy

Summary

List of abbreviations

Chapter 22. Autophagy in the gastrointestinal system and cross talk with microbiota

Introduction

Role of autophagy in intestinal pathology

Role of autophagy in intestinal epithelial cells and barrier function

Role of autophagy in antimicrobial defense

Role of autophagy in innate immune signaling and inflammatory control

Role of autophagy in adaptive immunity

Cross talk between autophagy and intestinal microbiota

Conclusion

Chapter 23. Role of autophagy in building and maintaining the skeletal system

Introduction

Autophagy in osteoblasts

Autophagy in osteocytes

Autophagy in osteoclasts

Autophagy in bone pathologies

Conclusion

Chapter 24. Autophagy on the road to longevity and aging

General modes of the autophagy pathway

Upstream signaling: mammalian target for rapamycin complex 1, AMP-activated protein kinase, and transcription factor EB

Signaling at the nucleation step: Unc-51-like kinase and vacuolar protein sorting 34 complexes

Formation of autophagosomes during nonselective autophagy

Formation of autophagosomes during selective autophagy

Autophagosome delivery of cargo to the lysosome, a central metabolic hub

Physiological functions of autophagy

Physiological characteristics of aging

Autophagy integration with all seven pillars of aging

Exercise, caloric restriction, and fasting mimetics: the search for antiaging interventions that exploit autophagy

Summary and future perspective

Chapter 25. Autophagy in cancer: friend or foe?

Introduction

Autophagy in tumorigenesis: dual role of autophagy in cancer

Mutations in autophagy genes in cancer

Oncogenes and tumor-suppressor genes in autophagy regulation

Genetically engineered mouse models of autophagy and cancer

Targeting autophagy for cancer treatment

Cancer and other types of autophagy

Concluding remarks

Chapter 26. Mammalian microautophagy: mechanism and roles in disease

Microautophagy in yeast

Identification and mechanism of mammalian microautophagy

Assessment of microautophagic activity in mammalian cells

Relationships with other intracellular pathways

Mammalian microautophagy in organelle homeostasis

Physiological functions of mammalian microautophagy

Role of microautophagy in disease

Concluding remarks

Chapter 27. Chaperone-mediated autophagy—mechanisms and disease role

Overview

Chaperone-mediated autophagy

Components and molecular mechanism of chaperone-mediated autophagy

The role of chaperone-mediated autophagy in pathophysiological mechanisms

Concluding remarks

Chapter 28. Targeting autophagy: lifestyle and pharmacological approaches

Introduction

The key stages of autophagy

Health implications of modulating autophagy

Concluding remarks

List of abbreviations

Index

Copyright

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Contributors

Mahmoud Abdellatif,     Department of Cardiology, Medical University of Graz, Graz, Austria

Nuzhat Ahsan,     Cell Biology and Infectious Diseases Unit, Institute of Life Sciences, Bhubaneswar, Odisha, India

Amelina Albornoz

Fundación Ciencia & Vida, Ñuñoa, Santiago, Chile

Universidad San Sebastián, Providencia, Santiago, Chile

Yahyah Aman,     Department of Clinical Molecular Biology, University of Oslo and Akershus University Hospital, Lørenskog, Norway

Allen Andres,     Arima Genomics, Inc., San Diego, CA, United States

Catherine Arden,     Biosciences Institute, Newcastle University, Newcastle upon Tyne, United Kingdom

Daniel Asa,     Department of Chemistry and Biochemistry, North Dakota State University, Fargo, ND, United States

Belén Bellido,     Antioxidant Biochemistry Lab, Biology Department, Faculty of Chemistry, National Autonomous University of Mexico, Mexico City, Mexico

Sihem Boudina,     Department of Nutrition and Integrative Physiology and Program in Molecular Medicine, University of Utah College of Health, Salt Lake City, UT, United States

Alfredo Briones-Herrera,     Antioxidant Biochemistry Lab, Biology Department, Faculty of Chemistry, National Autonomous University of Mexico, Mexico City, Mexico

Mauricio Budini,     Institute in Dentistry Sciences, Dentistry Faculty, University of Chile, Santiago, Chile

Elizabeth Bueno,     Department of Chemistry and Biochemistry, North Dakota State University, Fargo, ND, United States

Olivier Camuzard

UMR E-4320 TIRO-MATOs CEA/DRF/Institut des sciences du vivant Frédéric Joliot, Université Côte d'Azur, Faculté de Médecine Nice, Nice, France

Service de Chirurgie Réparatrice et de la Main, CHU de Nice, Nice, France

Georges F. Carle

UMR E-4320 TIRO-MATOs CEA/DRF/Institut des sciences du vivant Frédéric Joliot, Université Côte d'Azur, Faculté de Médecine Nice, Nice, France

CNRS, Paris, France

Santosh Chauhan,     Cell Biology and Infectious Diseases Unit, Institute of Life Sciences, Bhubaneswar, Odisha, India

Ramon Corbalan,     Division of Cardiovascular Diseases, Faculty of Medicine, Pontificia Universidad Catolica de Chile, Santiago, Chile

Alfredo Criollo

Instituto de Investigación en Ciencias Odontológicas, Facultad de Odontología, Universidad de Chile, Santiago, Chile

Autophagy Research Center, Universidad de Chile, Santiago, Chile

Advanced Center for Chronic Disease (ACCDiS), Facultad de Ciencias Químicas & Farmacéuticas and Facultad de Medicina, Universidad de Chile, Santiago, Chile

Srinivasulu Dasanna,     Department of Chemistry and Biochemistry, North Dakota State University, Fargo, ND, United States

Guido R.Y. De Meyer,     Laboratory of Physiopharmacology, University of Antwerp, Antwerp, Belgium

Abhinav Diwan

Center for Cardiovascular Research and Cardiovascular Division, Department of Medicine, Washington University School of Medicine, St. Louis, MO, United States

Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO, United States

Department of Obstetrics and Gynecology, Washington University School of Medicine, St. Louis, MO, United States

John Cochran VA Medical Center, St. Louis, MO, United States

Maria Jose Donate-Lagartos,     Department of Clinical Molecular Biology, University of Oslo and Akershus University Hospital, Lørenskog, Norway

Robyn Duttenhefner,     Department of Chemistry and Biochemistry, North Dakota State University, Fargo, ND, United States

Tobias Eisenberg

Institute of Molecular Biosciences, University of Graz, NAWI Graz, Graz, Austria

BioTechMed Graz, Graz, Austria

Field of Excellence BioHealth, University of Graz, Graz, Austria

Evandro F. Fang

Department of Clinical Molecular Biology, University of Oslo and Akershus University Hospital, Lørenskog, Norway

The Norwegian Centre on Healthy Ageing (NO-Age), Oslo, Norway

Johannes Frank,     Department of Clinical Molecular Biology, University of Oslo and Akershus University Hospital, Lørenskog, Norway

Valeria Garrido-Moreno,     Advanced Center for Chronic Disease (ACCDiS), Facultad de Ciencias Químicas & Farmacéuticas and Facultad de Medicina, Universidad de Chile, Santiago, Chile

Luis Garrido-Olivares,     Division of Surgery, Faculty of Medicine, Pontificia Universidad Catolica de Chile, Santiago, Chile

Merilin Georgiou,     Biosciences Institute, Newcastle University, Newcastle upon Tyne, United Kingdom

Saurav Ghimire

Université de Bordeaux, Institut des Maladies Neurodégénératives, UMR 5293, Bordeaux, France

CNRS, Institut des Maladies Neurodégénératives, UMR 5293, Bordeaux, France

Tania Gómez-Sierra,     Antioxidant Biochemistry Lab, Biology Department, Faculty of Chemistry, National Autonomous University of Mexico, Mexico City, Mexico

Ruben Gudmundsrud

Department of Clinical Molecular Biology, University of Oslo and Akershus University Hospital, Lørenskog, Norway

The Norwegian Centre on Healthy Ageing (NO-Age), Oslo, Norway

Andreas Guenther

Department of Internal Medicine, Justus-Liebig University, Giessen, Germany

Universities of Giessen and Marburg Lung Center (UGMLC), German Center for Lung Research (DZL), Giessen, Germany

European IPF/ILD Registry and Biobank, Giessen, Germany

Member of the CardioPulmonary Institute (CPI), Justus-Liebig University Giessen, Giessen, Germany

Lung Clinic, Agaplesion Evangelisches Krankenhaus Mittelhessen, Giessen, Germany

Pieter-Jan Guns,     Laboratory of Physiopharmacology, University of Antwerp, Antwerp, Belgium

Congcong He,     Department of Cell and Developmental Biology, Feinberg School of Medicine, Northwestern University, Chicago, IL, United States

G. Vignir Helgason,     Wolfson Wohl Cancer Research Centre, Institute of Cancer Sciences, University of Glasgow, Glasgow, United Kingdom

Nina Hermans,     Laboratory of Natural Products and Food-Research and Analysis (NatuRA), University of Antwerp, Antwerp, Belgium

Sergio Hernandez-Diaz

Université de Bordeaux, Institut des Maladies Neurodégénératives, UMR 5293, Bordeaux, France

CNRS, Institut des Maladies Neurodégénératives, UMR 5293, Bordeaux, France

Angela Ianniciello,     Wolfson Wohl Cancer Research Centre, Institute of Cancer Sciences, University of Glasgow, Glasgow, United Kingdom

Moydul Islam,     Cardiovascular Division, Department of Medicine, Washington University School of Medicine, St. Louis, MO, United States

Kautilya Kumar Jena,     Cell Biology and Infectious Diseases Unit, Institute of Life Sciences, Bhubaneswar, Odisha, India

Hiroshi Katsuki,     Department of Chemico-Pharmacological Sciences, Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan

Martina Korfei

Department of Internal Medicine, Justus-Liebig University, Giessen, Germany

Universities of Giessen and Marburg Lung Center (UGMLC), German Center for Lung Research (DZL), Giessen, Germany

Viktor I. Korolchuk,     Biosciences Institute, Newcastle University, Newcastle upon Tyne, United Kingdom

Nicholas T. Ktistakis,     Signalling Programme, Babraham Institute, Cambridge, United Kingdom

Sergio Lavandero

Advanced Center for Chronic Disease (ACCDiS), Facultad de Ciencias Químicas & Farmacéuticas and Facultad de Medicina, Universidad de Chile, Santiago, Chile

Cardiology Division, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, United States

Mark Li,     Department of Anatomy and Cell Biology, Fraternal Order of Eagles Diabetes Research Center Pappajohn Biomedical Institute, University of Iowa Carver College of Medicine, University of Iowa, Iowa City, IA, United States

Senka Ljubojević-Holzer

BioTechMed Graz, Graz, Austria

Department of Cardiology, Medical University of Graz, Graz, Austria

Poornima Mahavadi

Department of Internal Medicine, Justus-Liebig University, Giessen, Germany

Universities of Giessen and Marburg Lung Center (UGMLC), German Center for Lung Research (DZL), Giessen, Germany

Patrick Main

Université de Bordeaux, Institut des Maladies Neurodégénératives, UMR 5293, Bordeaux, France

CNRS, Institut des Maladies Neurodégénératives, UMR 5293, Bordeaux, France

Maria Manifava,     Signalling Programme, Babraham Institute, Cambridge, United Kingdom

Greg R. Markby,     Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, Copenhagen, Denmark

Wim Martinet,     Laboratory of Physiopharmacology, University of Antwerp, Antwerp, Belgium

Elena Martínez-Klimova,     Antioxidant Biochemistry Lab, Biology Department, Faculty of Chemistry, National Autonomous University of Mexico, Mexico City, Mexico

Subhash Mehto,     Cell Biology and Infectious Diseases Unit, Institute of Life Sciences, Bhubaneswar, Odisha, India

Jason C. Mills

Department of Developmental Biology, Washington University School of Medicine, St. Louis, MO, United States

Department of Pathology and Immunology, Washington University School of Medicine St. Louis, MO, United States

Cédric HG. Neutel,     Laboratory of Physiopharmacology, University of Antwerp, Antwerp, Belgium

Ngoc Uyen Nhi Nguyen,     Division of Cardiology, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, United States

José Pedraza-Chaverri,     Antioxidant Biochemistry Lab, Biology Department, Faculty of Chemistry, National Autonomous University of Mexico, Mexico City, Mexico

Daniel Peña-Oyarzún

Departamento de Fisiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile

Centro de Investigación Interdisciplinario en Salud Territorial del Valle de Aconcagua (CIISTe Aconcagua), Facultad de Medicina, Campus San Felipe, Universidad de Valparaíso, Valparaiso, Chile

Instituto de Investigación en Ciencias Odontológicas, Facultad de Odontología, Universidad de Chile, Santiago, Chile

Autophagy Research Center, Universidad de Chile, Santiago, Chile

Valérie Pierrefite-Carle

UMR E-4320 TIRO-MATOs CEA/DRF/Institut des sciences du vivant Frédéric Joliot, Université Côte d'Azur, Faculté de Médecine Nice, Nice, France

INSERM, Paris, France

Felipe X. Pimentel-Muiños,     Instituto de Biología Molecular y Celular del Cáncer, Centro de Investigación del Cáncer, CSIC-Universidad de Salamanca, Salamanca, Spain

Thomas Pulinilkunnil,     Department of Biochemistry and Molecular Biology, Dalhousie University, Dalhousie Medicine New Brunswick, Saint John, NB, Canada

Yoana Rabanal-Ruiz,     Department of Medical Sciences, Ciudad Real Medical School, Oxidative Stress and Neurodegeneration Group, Regional Center for Biomedical Research, University of Castilla-La Mancha, Ciudad Real, Spain

Megan D. Radyk,     Division of Gastroenterology, Department of Medicine, Washington University School of Medicine, St. Louis, MO, United States

Christian Rodríguez,     Institute in Dentistry Sciences, Dentistry Faculty, University of Chile, Santiago, Chile

Lynn Roth,     Laboratory of Physiopharmacology, University of Antwerp, Antwerp, Belgium

Beverly A. Rothermel

Division of Cardiology, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, United States

Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, United States

Kei Sakamoto,     Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, Copenhagen, Denmark

Irene Sanchez-Mirasierra

Université de Bordeaux, Institut des Maladies Neurodégénératives, UMR 5293, Bordeaux, France

CNRS, Institut des Maladies Neurodégénératives, UMR 5293, Bordeaux, France

Simon Sedej

BioTechMed Graz, Graz, Austria

Department of Cardiology, Medical University of Graz, Graz, Austria

Faculty of Medicine, University of Maribor, Maribor, Slovenia

Takahiro Seki,     Department of Chemico-Pharmacological Sciences, Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan

Alvaro Sequeida,     Institute in Dentistry Sciences, Dentistry Faculty, University of Chile, Santiago, Chile

Liu Shi,     Department of Psychiatry, University of Oxford, Oxford, United Kingdom

Sangita C. Sinha,     Department of Chemistry and Biochemistry, North Dakota State University, Fargo, ND, United States

Logan Slade,     Department of Biochemistry and Molecular Biology, Dalhousie University, Dalhousie Medicine New Brunswick, Saint John, NB, Canada

Sandra-Fausia Soukup

Université de Bordeaux, Institut des Maladies Neurodégénératives, UMR 5293, Bordeaux, France

CNRS, Institut des Maladies Neurodégénératives, UMR 5293, Bordeaux, France

Lillian B. Spatz,     Division of Gastroenterology, Department of Medicine, Washington University School of Medicine, St. Louis, MO, United States

Bieke Steenput

Laboratory of Physiopharmacology, University of Antwerp, Antwerp, Belgium

Laboratory of Natural Products and Food-Research and Analysis (NatuRA), University of Antwerp, Antwerp, Belgium

Takayuki Tatsumi,     Department of Pediatrics, Perinatal and Maternal Medicine (Ibaraki), Graduate School, Tokyo Medical and Dental University, Tokyo, Japan

Kateřina Čechová

Memory Clinic, Department of Neurology, 2nd Faculty of Medicine and Motol University Hospital, Charles University, Prague, Czech Republic

International Clinical Research Center, St. Anne's University Hospital Brno, Brno, Czech Republic

Marie-Charlotte Trojani

UMR E-4320 TIRO-MATOs CEA/DRF/Institut des sciences du vivant Frédéric Joliot, Université Côte d'Azur, Faculté de Médecine Nice, Nice, France

Service de Rhumatologie, CHU de Nice, Nice, France

Satoshi Tsukamoto,     Laboratory Animal and Genome Sciences Section, National Institute for Quantum and Radiological Science and Technology, Chiba, Japan

Silvia Vega-Rubín-de-Celis,     Institute for Cell Biology (Cancer Research), University Hospital Essen, Essen, Germany

Vishaka Vinod,     Department of Nutrition and Integrative Physiology and Program in Molecular Medicine, University of Utah College of Health, Salt Lake City, UT, United States

Martin Vyhnalek

Memory Clinic, Department of Neurology, 2nd Faculty of Medicine and Motol University Hospital, Charles University, Prague, Czech Republic

International Clinical Research Center, St. Anne's University Hospital Brno, Brno, Czech Republic

Amelia Williams,     Biosciences Institute, Newcastle University, Newcastle upon Tyne, United Kingdom

Samuel Wyatt,     Department of Chemistry and Biochemistry, North Dakota State University, Fargo, ND, United States

Chenglong Xie,     Department of Clinical Molecular Biology, University of Oslo and Akershus University Hospital, Lørenskog, Norway

Ling Yang,     Department of Anatomy and Cell Biology, Fraternal Order of Eagles Diabetes Research Center Pappajohn Biomedical Institute, University of Iowa Carver College of Medicine, University of Iowa, Iowa City, IA, United States

About the editors

Beverly A. Rothermel, PhD, is an Associate Professor at the University of Texas Southwestern Medical Center in Dallas, Texas, with appointments in the Departments of Internal Medicine (Cardiology) and Molecular Biology. Her laboratory was directly involved in some of the first studies demonstrating the dual nature of autophagy in the cardiovascular system. She has lectured on the role of autophagy in human disease as a component of the graduate school's Integrative Biology program for more than 10   years. Current studies by her lab seek to understand the circadian regulation of cardiac mitophagy and identify the causes and consequences of suppressed autophagy in Down syndrome. Her research is supported by the National Institutes of Health's INCLUDE Project, the Wellstone Muscular Dystrophy Research Network, the American Heart Association, and the Australian National Health and Medical Research Council.

Abhinav Diwan, MBBS, is a physician-scientist and a board-certified cardiologist and directs a laboratory-based research program focused on basic and translational studies that therapeutically target the autophagy–lysosome pathway of human disease. He is a Professor of Medicine, Cell Biology and Physiology, and Obstetrics and Gynecology at Washington University in St. Louis, Missouri, and Staff Physician at the John Cochran Veterans Affairs Medical Center in St. Louis, Missouri. Studies from his laboratory have uncovered evidence of acquired lysosome dysfunction as a common cellular mechanism in cardiomyopathy and heart failure, Alzheimer disease, and diabetes. Translational research from his program has established the autophagy–lysosome pathway as a viable therapeutic target, with activation of the lysosome biogenesis program as an exciting strategy under these conditions. He has also proven to be an outstanding mentor to the next generation of physician-scientists, an effort he leads as the Program Director of the Investigator Training Pathway in the Cardiovascular Division supported by an NIH T32 training grant. Research in his program is funded by the National Institutes of Health, the Department Veterans Affairs, American Heart Association and Alzheimer's Association.

Foreword

I am delighted to offer a foreword for the second edition of Autophagy in Health and Disease, edited by my colleagues and friends, Abhinav Diwan and Beverly Rothermel. I am delighted with the scope of topics and the quality of the chapters. This new edition compiles our current state of knowledge on each of the topics. The field of autophagy has advanced considerably since the first edition, and this edition brings many new topics to light. The importance of autophagy was underscored by the selection of Yoshinori Ohsumi for the 2016 Nobel Prize in Physiology or Medicine. What began as a cataloguing of genes that regulate autophagy in yeast has expanded to all mammals, leading to an appreciation for the importance of autophagy in most if not all tissues in mammals, and notably, the recognition that dysfunctional autophagy contributes to a host of diseases that afflict humans, and functional autophagy is a major determinant of life span. The field continues to expand apace as new tools become available and new genes are identified that govern specific aspects of the process or regulate it in specific tissues. Excitingly, recent work has provided a more detailed understanding of how selective autophagy is accomplished. With so much new information emerging, this new edition provides a comprehensive survey of the field, covering both what is known and pointing out gaps in knowledge where additional work is needed. Both newcomers to the subject and experts will find value in this compendium, which covers the basic mechanisms, tools used for its investigation, as well as detailed chapters on a host of specific topics. The editors, Abhinav Diwan and Beverly Rothermel, have done an outstanding job of recruiting chapter authors who are world-renowned experts in their areas, as well as editing each chapter to achieve a high level of scholarship matched with readability and compelling illustrations. Their dedication and attention to detail have resulted in an outstanding work that will benefit the field.

This edition is divided into six sections. The Overview covers autophagy machinery, its regulation, and analytical methods of analysis. Section Two provides a current summary of the many roles autophagy plays in development. Section Three covers autophagy in metabolic control at the organism level, with chapters on each major metabolic organ. The cardiopulmonary and vascular systems are addressed in Section Four, while Section Five covers the nervous system. The final part of the book, Section Six, addresses the role of autophagy in homeostasis in other organ systems and its role in cancer, aging, and the immune system, as well as two chapters on the underappreciated systems of chaperone-mediated autophagy and microautophagy (most research to date has focused on macroautophagy).

Our understanding of selective autophagy and cargo selection has advanced considerably, revealing a host of new adaptor proteins and mediators of trafficking (see Chapter 4: Selectivity and Trafficking of Autophagic Cargoes). Other chapters in this section address the machinery itself, its regulation, lysosomes, methods for measuring autophagy, and the impact of physiologic cues (fasting, exercise, circadian rhythm, sex, and age).

While early studies suggested that autophagy was not essential for embryonic development, subsequent studies in model organisms (Caenorhabditis elegans, Drosophila, and mice) have revealed a role for autophagy—in C. elegans in early events after fertilization and in maintenance of the dauer state, in Drosophila during the late larval stage and pupation, and in mice during early embryonic development and in the first postnatal hours until suckling is established. Additional genetic studies of autophagy gene knockouts with embryonic lethal phenotypes have implicated specific autophagic factors in developmental processes at various stages. Particularly interesting are new insights into elimination of paternal mitochondria after fertilization (see Chapter 9: Role of Autophagy in Embryogenesis). Whether this autophagy deficit explains the reported persistence of paternal mitochondrial genomes in several human families remains to be elucidated. Additional chapters in the section on development address autophagy in stem cells, hematopoiesis, and cell plasticity.

The third section addresses autophagy in metabolic control and diabetes, covering the major organs controlling metabolic homeostasis—liver, pancreas, adipose tissue, and skeletal muscle—while the fourth section addresses autophagy in the heart, lungs, and vasculature. While a great deal of information has been published on autophagy in the heart, less is known about its role in lungs and vasculature. These chapters provide updated information. Particularly noteworthy is the chapter on autophagy in vasculature, which is now known to be important for normal vascular function. The absence of autophagy in vasculature affects endothelial cell homeostasis and vascular smooth muscle cells; it is also associated with atherosclerosis. These chapters reveal the importance of autophagy and how its impairment contributes to the development of some of the most widespread diseases affecting the population, including metabolic syndrome, atherosclerosis, and ischemic heart disease.

Autophagy has long been recognized as important to the nervous system, where its failure leads to neurodegenerative diseases, including Parkinson disease (Chapter 19) and Alzheimer disease (Chapter 20). A series of chapters address autophagy in immune regulation, the GI tract, the skeletal system, cancer, and aging, as well as providing updates on microautophagy and chaperone-mediated autophagy. Finally, there is a forward-looking chapter on approaches to targeting autophagy through lifestyle and pharmacological interventions. Taken together, these chapters implicate autophagy as a central antidote to many diseases that afflict aging adults and point to approaches that can be adopted to preserve health and extend life spans.

Why Autophagy?

When there's food aplenty, organisms thrive.

But in hard times, autophagy helps them survive.

If feasts are good and famine so bad,

Why does food in excess make cells sad?

Turns out autophagy helps save the day,

Doing its work to keep disease at bay:

Clearing clumps of proteinaceous dust

And organelles that have begun to rust.

Find a way to turn self-eating on,

If you want to see your ninth decade dawn.

Roberta A. Gottlieb, MD

July 20, 2021

Preface

When Roberta Gottlieb asked us to take on the task of editing the second edition of her book, Autophagy in Health and Disease, we were both, frankly, more than a little bit apprehensive. Fortunately, the choice of putting the two of us together on the job turned out to be genius on Robbie's part. We kept each other sane, learned a lot, and most of all enjoyed getting to know each other.

Recruiting authors to write chapters for a book can be a daunting task, particularly since we wanted the book to be comprehensive in its subject matter as well as have an international point of view. This meant that we had to reach far outside our familiar circle of cardiovascular colleagues. We are extremely grateful for the positive response we received from the autophagy community at large and amazed by the quality and comprehensive nature of the work submitted. We were able to include contributions from scientists working in fifteen different countries. We would particularly like to thank Dr. Silvia Vega-Rubín-de-Celis from the Institute for Cell Biology at the University Hospital Essen, Germany, who stepped in on very short notice to write Chapter 25, which covers the role of autophagy in cancer. Beth Levine had originally and graciously agreed to contribute that chapter. Her death last summer of breast cancer was a great loss to the field. She was fearless in her scientific endeavors and generous with her invaluable insights. We would humbly like to dedicate this volume to her memory.

A good measure of gratitude is owed to our mentors, trainees, and colleagues who collectively enable our pursuits in science. We would also like to acknowledge the patience, support, and love we received from our families as we toiled through compiling this edition.

Beverly A. Rothermel, PhD

University of Texas Southwestern Medical Center, Dallas, TX, United States

and

Abhinav Diwan, MBBS

Washington University School of Medicine, St. Louis, MO, United States

Section I

Overview

Outline

Chapter 1. Introduction

Chapter 2. Mechanisms of autophagy: the machinery of macroautophagy and points of control

Chapter 3. Regulation of autophagy—transcriptional, posttranscriptional, translational, and posttranslational mechanisms

Chapter 4. Selectivity and trafficking of autophagic cargoes

Chapter 5. The role of lysosomes in autophagy

Chapter 6. Methods for measuring autophagy

Chapter 7. Effects of physiologic inputs on autophagy

Chapter 1: Introduction

Beverly A. Rothermel ¹ , ² , and Abhinav Diwan ³ , ⁴ , ⁵ , ⁶       ¹ Division of Cardiology, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, United States      ² Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, United States      ³ Center for Cardiovascular Research and Cardiovascular Division, Department of Medicine, Washington University School of Medicine, St. Louis, MO, United States      ⁴ Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO, United States      ⁵ Department of Obstetrics and Gynecology, Washington University School of Medicine, St. Louis, MO, United States      ⁶ John Cochran VA Medical Center, St. Louis, MO, United States

Abstract

Autophagy, a term coined by Christian de Duve in 1955 to describe his observations of the cell eating itself within lysosomes, is now recognized as a fundamental cellular process that enables living organisms to maintain homeostasis and respond to stress. The last 3 decades have witnessed an explosion in our understanding of the molecular mechanisms that orchestrate autophagic activity, which both functions to break down intracellular constituents and works in conjunction with endocytic pathways to internalize and degrade extracellular material or invading pathogens. As such, autophagy is required for cell survival, and its deficiency or dysfunction is now implicated in an ever-growing list of human diseases. The second edition of this book provides a contemporary snapshot of advances in the field and focuses on a range of topics from basic mechanisms to therapeutic targeting of autophagy pathways in human disease. Experts from 15 countries present state-of-the-art knowledge in their respective areas of focus, with engaging illustrations and current references that will serve to guide future investigation in autophagy. Through this book, we hope to communicate the excitement in this vibrant field and beckon the next generation of scientists to make an impact on human health and society through their work in Autophagy.

Keywords

Autophagy; Lysosome; Nutrients; Organelles; Transcriptio; n factors

Conserving and optimizing the use of resources is critical to all biological processes. Over the course of evolution, a significant advantage was gained by an ability to sense the availability of resources and respond appropriately. Organisms that could shift their dependence from external to internal sources and vice versa better survived the extreme stresses of starvation, limited oxygen supply, and toxic environments. Indeed, strategies evolved specifically to optimize the mobilization and reuse of internal resources as well as to improve intake of nutrients from the environment. Autophagy (or self-devouring, from the Greek "auto, meaning self, and phagein, meaning to eat") emerged in its various forms as a means to recycle endogenous cellular constituents, providing both an economical way to meet homeostatic cellular needs and a mechanism to support life when nutrients in the environment became limiting. Autophagy is now recognized as a fundamental cellular pathway for degrading and recycling organic matter in all eukaryotes. It is estimated that over the course of less than 2 months, almost all the proteins in our bodies are degraded and replaced without a perceptible change in function. ¹ Furthermore, in adults, most amino acids used for new protein synthesis are obtained through the breakdown of our own existing cellular proteins. This is a remarkable feat and points to the central role that autophagy, along with other degradative processes, must play in the building and maintenance of eukaryotic organisms. The goal of this book is to highlight the wealth of knowledge that recent research has uncovered regarding the impact of autophagy on human health and disease. A better understanding of the mechanisms that control and carry out autophagy will aid in the development of therapeutic approaches that harness autophagy's beneficial properties while limiting its deleterious aspects. Such an approach holds tremendous potential for treating and preventing a diversity of human diseases as well as the promise of prolonging a heathy life span.

A historical perspective

The recognition of autophagy as a fundamental cellular process is built upon experimental observations spanning many decades. The importance of autophagy on human physiology and health was acknowledged by the 2016 Nobel Prize in Physiology or Medicine awarded to Yoshinori Ohsumi, whose laboratory made key mechanistic discoveries in the field. Our current understanding of the turnover of cellular proteins grew from the recognition that all proteins have a finite life span and are degraded by enzymatic processes. Insight into how cells manage to separate the highly lytic enzymes that carry out this job from the rest of the cytoplasm to prevent widespread proteolysis and cellular breakdown came with the initial biochemical isolation and characterization of lysosomes by Christian de Duve in 1955. ² The lysosome is a membrane-bound organelle that compartmentalizes a variety of hydrolytic enzymes, including proteases, nucleases, glycosidases, lipases, phospholipases, and phosphatases, in an acidic milieu to provide optimal conditions for their degradative activity. Any enzymes escaping from lysosomes are quickly inactivated in the neutral pH environment of the cytosol, thereby providing an additional layer of protective sequestration. Alex Novikoff is credited with the first electron microscopic images of lysosomes as distinct membrane-bound organelles. ³ Subsequent observations by multiple investigators described the presence of an increasing variety of cytoplasmic constituents within lysosomes, including components of other organelles such as mitochondria, endoplasmic reticulum, and ribosomes. ⁴–⁸ This led de Duve to coin the term Autophagy to describe the process of delivering cytoplasmic components to lysosomes for degradation. ¹ Discovery of the ubiquitin–proteasome system in the late 1970s revealed another important mechanism for degrading specific cellular proteins without damaging the rest of the cell. ² In this context, it is perhaps not surprising that the discoveries of the lysosome and the ubiquitin–proteasome pathway have been the subjects of Nobel Prizes awarded in Physiology or Medicine and Chemistry, respectively.

Developing a tool kit

Early observations in the field of autophagy included the discovery of dynamic changes in autophagic activity in response to physiologic inputs such as fasting and feeding, ⁹ circadian rhythms, ¹⁰ circulating hormones, ⁵ , ⁸ and the presence or deficit of specific amino acids. ¹¹ Dr. Ohsumi's laboratory is credited with one of the first descriptions of autophagic structures inside a yeast vacuole, the yeast equivalent of a lysosome. ¹² , ¹³ Subsequently, he and his team applied powerful genetic approaches to identify autophagy-defective mutants that yielded the first collection of autophagy-related Apg genes, or what are now termed ATGs. ¹⁴ This initial set of genes has been greatly expanded by the Ohsumi laboratory and many others, with Atg43 identified most recently. ¹⁵ These genes and their mutants provided the essential tool kit for the subsequent rapid unraveling of the intricate machinery that orchestrates the process of autophagy. Another major advance was the identification of the MiT/TFE family of transcription factors as the master regulators of the autophagy–lysosome pathway. ¹⁶ , ¹⁷ This discovery opened the door to understanding transcriptional regulation of the autophagy–lysosome machinery, which plays a critical role in sustaining autophagy signaling during stress. ¹⁸–²⁰

Prior to 2000, fewer than 50 scientific papers had been published in any year on subjects relevant to autophagy. Since that time, the number has grown exponentially such that in 2020 alone, more than 9000 autophagy-related papers were published. A wealth of investigators too numerous to detail here have made seminal contributions to the field; however, two specific investigators should be mentioned beyond those already discussed, as their contributions made this astronomical expansion of the field possible. These investigators are Daniel Klionsky and Beth Levine. The Klionsky laboratory elucidated a number of the key molecular mechanisms involved in autophagy, starting with their early studies of cytoplasm-to-vacuole targeting. ²¹ However, Dan Klionsky’s contribution with the most impact may be as a community builder. He is the founding editor of the journal Autophagy, which has grown to be a premier journal in the field of biology. He has also worked tirelessly to organize researchers in order to standardize nomenclature and update guidelines for the use and interpretation of assays to monitor autophagy. ²² These efforts have been invaluable to both established investigators and those from other fields wanting to examine autophagy in new settings. In 1998 and 1999, Dr. Levine cloned Beclin 1, the human homologue of yeast ATG6, and rapidly went on to make the critical connection to human disease, specifically cancer. ²³ , ²⁴ This key connection, coupled with the genetic tools and deep mechanistic insights from yeast, initiated the exponential growth that has occurred in the study of autophagy in all aspects of human disease.

An overview of mechanisms

Here we provide a very brief overview of autophagy as a point of orientation. Subsequent chapters provide comprehensive details and many more informative insights. Autophagy is the process by which the cell sequesters and delivers intracellular material for degradation in the lysosome (Fig. 1.1). Autophagic proteins and structures also participate in the degradation of extracellular material through interactions with processes that take up material from the plasma membrane and outside the cell, such as endocytosis, micropinocytosis, and phagocytosis (Fig. 1.1). Broadly speaking, autophagy can be classified as either general autophagy, to indicate relative nonselectivity of the cargo, or selective autophagy, in which specific substrates are targeted. Selectivity is conferred by specific adaptors and autophagy receptors that direct the autophagy cargo into autophagosomes. ²⁵

In macroautophagy (often referred to simply as autophagy), an early phagophore membrane surrounds the cargo to be degraded to form a double-membrane autophagosome. The autophagosome then fuses with a lysosome, or first with an endosome and then the lysosome, to generate an active autolysosome where the cargo is degraded. In specific instances, cargo can enter an already formed autophagosome via the assistance of chaperone proteins, such as BAG3 and HSPB8, through a process termed chaperone-assisted autophagy. ²⁶ The early steps in the formation of the phagophore and the initial source of autophagosome membranes continue to be the focus of intense research. ²⁷ , ²⁸ Subsequent biogenesis and extension of autophagic membranes involves a series of conjugation steps similar to the process of ubiquitination, in which various ATG proteins act both as ligases and conjugated moieties to ultimately converge on lipidation of ATG8 (or in mammals, LC3), which then decorates the surface of autophagic membranes. ²⁹ Targeting an intracellular structure for selective autophagic degradation frequently involves ubiquitination of the protein or organelle being targeted. ³⁰ Lipidated ATG8/LC3, embedded in the phagophore membrane then provides an anchoring point for adaptor proteins, such as p62, to direct selected cargos to autophagosomes by binding both ubiquitin and LC3. Recent discoveries indicate a role for liquid–liquid phase separation and the formation of condensates in regulating autophagic membrane and protein assemblies. ³¹ , ³²

Figure 1.1 Schematic depicting subcellular processes that constitute autophagy.Depiction of autophagic sequestration and movement of intracellular cargos (both general and selective) into the lysosome for degradation as well as the intersection of the autophagic machinery with processes that degrade extracellular material. General autophagy includes macroautophagy, mediated by sequestration of subcellular material into double-membrane-bound autophagosomes; chaperone-assisted autophagy, involving the assistance of specific chaperone proteins, such as BAG3 and HSPB8, to uptake cargo into double-membrane-bound autophagosomes; chaperone-mediated autophagy, in which chaperones assist in the uptake of proteins carrying a specific KFERQ motif via a LAMP2A receptor in the lysosomal membrane; and microautophagy, where material is taken up directly by invagination of the lysosomal membrane. Selective autophagy involves specific targeting of intracellular cargo such as organelles (mitochondria by mitophagy, nucleus by nucleophagy, endoplasmic reticulum by ER-phagy, and peroxisomes by pexophagy), nutrient stores (lipid droplets by lipophagy and glycogen deposits by glycophagy), protein assemblies (ribosomes by ribophagy and inflammasomes by inflammasomophagy), protein aggregates (by aggrephagy), or invading pathogens (by xenophagy). In heterophagy, autophagic proteins participate in the uptake of extracellular material as autophagosomes fuse with membrane-bound structures generated by ligand–receptor interactions (endocytosis), sampling of extracellular material (macropinocytosis), and engagement of pathogens with scavenger receptors (phagocytosis) or dead cells (efferocytosis). Lysosomal degradation of autophagosized material releases basic building blocks (such as amino acids, simple sugars, lipids such as triglycerides and cholesterol, nucleic acids, heme, and minerals), which may be further catabolized as nutrients or recycled back into anabolic processes. Peptides are generated and released for antigen presentation. Release of signaling moieties such lipids and ions can activate downstream signaling pathways. The lysosome membrane incorporates numerous transporters and ion channels that facilitate the transfer of these end products. Lysosomal degradation of autophagic cargo also controls cellular nutrient signaling via the dynamic assembly of the LYNUS complex on the surface of lysosomes, and it triggers the autophagic lysosome reformation process to promote lysosome biogenesis and restoration of the lysosome compartment.

In chaperone-mediated autophagy, cellular proteins containing a specific KFERQ motif are translocated directly across the lysosomal membrane into the lumen assisted by the hsp90 and hsc70 chaperones via an interaction with the lysosome membrane protein LAMP2A. ³³ Direct invagination of the lysosomal membrane is another well-described mechanism for internalization of autophagic cargo via a process termed microautophagy. Mammalian cells carry out a version of microautophagy called endosomal microautophagy that involves KFERQ-mediated delivery of endosomes directly to lysosomes. Noncanonical roles for autophagy-signaling proteins also continue to be uncovered ³⁴ that govern the intersection of autophagy with heterophagic pathways ¹⁸ as well as with cellular secretion. ³⁵

The overwhelming majority of evidence indicates that autophagy is a prosurvival pathway that plays critical roles in homeostasis and cytoprotection under stress by generating nutrients and degrading damaged organelles and harmful mediators, such as invading pathogens. Furthermore, enhancing autophagy signaling has been shown to delay aging-related outcomes and to prolong life spans across an evolutionary spectrum of living organisms ranging from the unicellular yeast to mammals. ³⁶ , ³⁷ On the flip side, autophagy has also been described as directing cell death in a developmental context ³⁸ as well as in specific disease scenarios where autophagic cell death is an established pathway of cell death with specific discernible characteristics. ³⁹

Lysosomal degradation of autophagic cargo releases simple nutrients and basic building blocks that are catabolized for energy or recycled back into anabolic pathways. Lysosomes also store nutrients and ions that are released via transporters to activate signaling processes. Indeed, the lysosome surface is the site for scaffolding of the lysosome nutrient-sensing (LYNUS) complex that controls anabolic signaling through the mammalian target of rapamycin (mTOR) kinase, ⁴⁰ , ⁴¹ the key regulator of cell growth. Lysosomes also store and release ions that drive signaling process, such as activation of calcineurin with lysosomal calcium release via the mucolipin channel that dephosphorylates and activates TFEB to activate autophagy signaling. ⁴² Activation of autophagy also dynamically controls mTOR signaling in a pulsatile fashion, ⁴³ and triggers autophagic lysosome reformation, ⁴⁴ a mechanism to restore the cellular complement of lysosomes via the budding of vesicles from existing autolysosomes through the action of lipid kinases. An exciting area of inquiry is the emerging role of lysosomes in generating signaling moieties that may activate downstream signaling pathways upon the release of key signaling mediators from lysosomes and may control critical biological processes including life span. ⁴⁵ Indeed, while the lysosome was primarily believed to be an incinerator that indiscriminately degraded autophagic cargo, lysosomal degradation of foreign material such as invading pathogens is rarely complete and serves a critical role in preserving antigenic identity, which enables the adaptive immune response through antigen presentation by innate immunity cellular machinery. ⁴⁶ Therefore, our evolving understanding of autophagy–lysosome biology is nascent and being enriched by the tremendous interest generated in this area of investigation by the above-described discoveries.

Introduction to the second edition

Understanding the role of autophagy in human diseases is critical if therapeutic targeting of this pathway is to be realized. Mutations in autophagy pathway proteins have been implicated as causes of neurodegeneration, ⁴⁷ inflammatory conditions such as Crohn's disease, ⁴⁸ and cancer ⁴⁹ among a growing list of human diseases causatively linked to genetic alterations in autophagy pathway genes. ⁵⁰ This book takes the mantle from the previous edition and provides a comprehensive and state-of-the-art update on the field. The first section, titled the Overview, takes a deep dive into the mechanisms and regulation of autophagy, the molecular basis for selectivity of cargo and the role of lysosomes in this process. Also included is a discussion of the methods and various physiologic inputs that modulate autophagy in living systems. The next section focuses on the role of autophagy in Development, followed by a section on the role of autophagy in Metabolism through its control of organ function in the liver, adipose tissue, pancreas, and skeletal muscle. Subsequent sections focus on the role of autophagy in the Cardiovascular and Nervous systems and in the regulation of Gastrointestinal processes and pathology. This section is followed by a compilation of chapters focused on the role of autophagy in critical processes such as immune regulation, cancers, and longevity as well as on the therapeutic targeting of autophagy to treat disease.

It is our ardent hope that this book serves as a timely compendium to inform all readers across the spectrum, from novice to expert, of the rapid advances in the field of autophagy. We hope that the insights included herein spur further investigation toward the goal of preventing and curing human ailments and prolonging life span. Indeed, many key questions that are apparent today remain unanswered. For example, the current understanding of autophagy in regulating cellular signaling remains piecemeal. It will be critical to understand how autophagic processes integrate inputs via intracellular signals and extracellular cues to drive cellular signaling and to shape an adaptive response. The concept of lysosomes as signal processors ⁴⁵ has the potential to revolutionize our understanding of cell biology. Indeed, the adage we are what we eat may also apply to how cells are affected by what they eat via lysosomal digestion. A unique instance of such signal integration is the requirement for activation of HLH-30, the Caenorhabditis elegans ortholog of the MiT/TFE family of transcription factors, in responding to starvation ⁵¹ or infectious stimuli. ⁵² By wiring these responses together, this transcription factor (or this family) engages the autophagy–lysosome pathway to assist the organism in making key decisions on whether the ingested food is safe to eat or potentially dangerous based on its pathogenic potential. Such decisions are critical to the ability of organisms to survive and have shaped evolution for millennia. Another critical question will be to understand how the autophagy–lysosome pathway helps organisms differentiate self-from non-self and engage the adaptive immune response to fight invading pathogens. Understanding how compartmentalization is achieved within cells, by corralling cargo within membranes or by membraneless condensate formation along phase-separation principles, will likely be central to understanding how cells traffic specific cargo for degradation along various pathways. Inherent in this argument is the notion that not all lysosomes are created equal, and such diversity is likely to facilitate context- and substrate-specific degradation toward unique outcomes. Such granular understanding of the autophagy–lysosome pathway and the molecular players involved therein is likely to facilitate development of specific small molecules to harness its benefits. In a nutshell, the future of this field is bright for investigators who are just getting interested in understanding autophagy. Welcome aboard!

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Chapter 2: Mechanisms of autophagy: the machinery of macroautophagy and points of control

Congcong He     Department of Cell and Developmental Biology, Feinberg School of Medicine, Northwestern University, Chicago, IL, United States

Abstract

Identification of autophagy-related (ATG) genes has been transiting autophagy research from morphological characterization to mechanistic investigation since 1993. ATG genes were first discovered by three genetic screens independently performed in the budding yeast Saccharomyces cerevisiae. Later, conserved orthologs of ATG yeast genes were found in higher organisms including Caenorhabditis elegans, Drosophila, Arabidopsis, and mammals. The core autophagy machinery essential for autophagosome formation and maturation contains more than 20 ATG genes categorized into the following major groups: (1) the Atg1/ULK1 kinase complex; (2) the class III phosphatidylinositol 3-kinase complex; (3) Atg2–Atg18/WIPIs (WD-repeat domain phosphoinositide-interacting proteins); (4) Atg9/ATG9 vesicles; (5) the Atg12–Atg5–Atg16 complex; and (6) Atg8/LC3–PE (phosphatidylethanolamine). Recent advances in structural biology, imaging, and biochemical techniques have led to characterization of the molecular and architectural details of each step in autophagy. This chapter summarizes progress over the last decade on the origin, initiation, expansion, and maturation of autophagosomal membranes.

Keywords

Autophagy-related (ATG) gene; Atg1/ULK1 kinase complex; Atg2–Atg18/WIPIs complex; ATG9 vesicle; Autophagosome biogenesis; Autophagosome–lysosome fusion; Class III phosphatidylinositol 3-kinase complex; Phagophore; Phagophore assembly site; Ubiquitin-like conjugation system

Origin of the phagophore membrane

In macroautophagy (hereafter referred to as autophagy), double-membrane autophagosomes of approximately 0.5–2   μm in diameter sequestrate and deliver cargos to lysosomes for degradation. Lysosomes fuse with the outer autophagosomal membrane and degrade the inner autophagosomal membrane along with autophagic cargos. An autophagosome emerges as a cup-shaped double-membrane structure known as the phagophore. In yeast, autophagosomes are formed at a single perivacuolar structure called the phagophore assembly site (PAS), whereas in mammalian cells, autophagosome biogenesis occurs at multiple PASs. ¹ Under autophagy-inducing conditions, many autophagy-related (ATG) proteins cluster at the PAS, which is required for phagophore membrane expansion, supporting the concept that autophagosome formation is a de novo process orchestrated by a cohort of autophagy proteins rather than a budding event from existing membranes (Fig. 2.1).

Accordingly, a fundamental question arises: What is the origin and source of autophagosomal membranes? The question has been intensively studied in recent years, and accumulating evidence suggests that autophagosomes are likely to originate from multiple sources. A variety of organelles in contact with the phagophore have been detected by scanning electron microscopy and electron tomography studies ² and have been proposed as the membrane source for phagophores, including the ER, the plasma membrane, mitochondria, recycling and late endosomes, and lipid droplets. Among them, ER subdomains in contact with other organelles have been thus far demonstrated as the primary origin of phagophore formation, including the ER–mitochondria contact site and the ER–Golgi intermediate compartment (ERGIC), a dynamic tubulovesicular membrane cluster labeled by the lectin ERGIC-53). Three-dimensional electron tomography studies reveal direct membrane contacts between the rough ER and the phagophore. Disrupting the ERGIC pharmacologically by drugs or disrupting ER–mitochondria contact sites genetically by depleting proteins required for contact formation, such as mitofusin 2 and phosphofurin acidic cluster sorting protein-2, impairs autophagosome biogenesis. ³–⁵

The PASs on ER subdomains are also known as omegasomes, named after their tubular morphology resembling the Greek letter omega (Ω). Omegasomes are dynamic punctate structures enriched in phosphatidylinositol 3-phosphate (PI3P). Two PI3P effectors (binding proteins), double FYVE domain-containing protein 1 (DFCP1) and WD-repeat domain phosphoinositide-interacting proteins (WIPIs)/Atg18 family proteins, serve as the markers of omegasomes. Notably, because DFCP1 is not an essential protein in autophagy, it can be used as an independent marker for omegasomes.

Phospholipid synthetases are enriched at ER–mitochondria contact sites, raising the possibility that de novo phospholipid synthesis at these contact sites may be an important mechanism to provide newly synthesized lipids for phagophore expansion. Indeed, the hypothesis is supported by recent evidence showing that PASs on ER subdomains are enriched in phosphatidylinositol synthetase (PIS) and that PIS is localized close to the phagophore. ⁶ More directly, in yeast, the acyl-CoA synthetase Faa1 is found accumulated on forming phagophores, and Faa1-catalyzed acyl-CoA formation from fatty acids is required for autophagosome formation. ⁷ Acyl-CoA is an essential precursor for the biosynthesis of phospholipids, a key component of the phagophore membrane.

Figure 2.1 Schematic model of autophagosome biogenesis at the phagophore assembly site.Phagophore formation is initiated at the ER subdomain omegasome, labeled DFCP1. Under autophagy-inducing conditions, the Atg1/ULK1 kinase complex phosphorylates and activates the PI3K complex I at the phagophore assembly site. PI3P produced by the PI3K recruits PI3P-binding proteins Atg18/WIPIs to the phagophore. Together with Atg18/WIPIs, Atg2/ATG2 transports additional phospholipids synthesized at the ER membrane to the phagophore. Atg9/ATG9-containing vesicles shuttles between the PAS and Atg9/ATG9 reservoirs, such as the trans-Golgi network, to facilitate phospholipid transfer by ATG2–Atg18/WIPIs and to deliver lipid kinases to the PAS for phospholipid production. Phosphatidylethanolamine-conjugated Atg8/LC3 is localized to the phagophore and further recruits autophagy cargo receptors and cargos to the forming autophagosomes.

Initiation of autophagosome biogenesis by the Atg1/ULK1 kinase complex

A hierarchical recruitment of ATG proteins to the PAS has been established in both yeast and mammalian cells. The Atg1/ULK1 (Unc-51-like 1) serine/threonine kinase complex is the first complex to be recruited to the PAS on the ER membrane. This is considered the first step and initiator of autophagosome formation, even prior to omegasome formation. So far, five components have been identified in the yeast Atg1 complex: Atg1, Atg13, Atg17, Atg29, and Atg31; and four components have been identified in the mammalian ULK1 complex: yeast Atg1 homolog (ULK1/2), yeast Atg13 homolog (ATG13), FIP200/RB1CC1 (a proposed functional mammalian counterpart of yeast Atg17 despite limited sequence homology), and ATG101. Under basal conditions, Atg1/ULK1 complex components primarily localize to the cytosol. Upon autophagy induction, regulated by various upstream signals including target of rapamycin complex 1 (TORC1) inhibition, cAMP-dependent protein kinase A (PKA) activation, and AMP-activated protein kinase (Snf1/AMPK) activation, ¹ the Atg1/ULK1 complex assembles, associates with membranes, and is recruited to PIS-enriched ER subdomains independently from other downstream ATG proteins. ⁶ , ⁸–¹⁰

The kinase activity