Autophagy in disease and clinical applications. Part C / edited by Daniel J. Klionsky.

Format:

Book

Contributor:

Klionsky, Daniel J.

Series:

Methods in enzymology ; v. 453.

Methods in enzymology ; v. 453

Language:

English

Subjects (All):

Cell death.

Autophagic vacuoles.

Eukaryotic cells.

Physical Description:

xlix, 481 pages, 4 unnumbered pages, 16 unnumbered pages of plates : illustrations (some color) ; 24 cm.

Place of Publication:

San Diego, Calif. : Elsevier/Academic Press, [2009]

Contents:

1 Initiation of Autophagy by Photodynamic Therapy / David Kessel, Nancy L. Oleinick 1

1 Introduction 2

2 Photosensitizing Agents 3

3 Additional Factors Unique to PDT 5

4 A Typical PDT Protocol 5

5 Identification and Characterization of Autophagy after PDT 6

6 Effects of Autophagy on PDT Responses 9

Acknowledgments 15

2 Autophagic Cell Death / Michael J. Lenardo, Christina K. McPhee, Li Yu 17

1 Introduction 18

2 Methods to Quantify Cell Death 19

3 Methods to Measure Autophagy 26

4 Methods to Establish Autophagy as the Cause of Cell Death 27

5 Autophagy Genes for RNAi Silencing 29

6 Transfact RNAi by Electroporation (Amaxa Nucleofection) 29

7 Conclusion 29

3 Autophagic Neuron Death / Yasuo Uchiyama, Masato Koike, Masahiro Shibata, Mitsuho Sasaki 33

1 Introduction 34

2 Experimental Models of Neurodegeneration 35

3 Measurements of Neuron Death 41

4 Assessing Metabolic Stress and Autophagy Status in Epithelial Tumors / Robin Mathew, Vassiliki Karantza-Wadsworth, Eileen White 53

1 Introduction 55

2 Mouse Epithelial Cell Models for Studying the Role of Autophagy in Cancer 57

3 Protocols for Monitoring Autophagy in iBMK Cells and iMMECs In Vitro 61

4 Protocols for Monitoring Autophagy in Tumors In Vivo 64

5 Monitoring Chromosomal Instability Due to Autophagy Defects 67

6 Concluding Remarks and Future Perspectives 78

5 Autophagic Clearance of Aggregate-Prone Proteins Associated with Neurodegeneration / Sovan Sarkar, Brinda Ravikumar, David C. Rubinsztein 83

1 Introduction 84

2 Aggregate-Prone Intracytoplasmic Proteins Associated with Neurodegenerative Disorders are Autophagy Substrates 86

3 Assays for the Clearance of Aggregate-Prone Proteins 87

4 Measurement of Autophagic Flux Using Bafilomycin A1 102

5 Concluding Remarks 107

Acknowledgements 107

6 Monitoring Autophagy in Alzheimer's Disease and Related Neurodegenerative Diseases / Dun-Sheng Yang, Ju-Hyun Lee, Ralph A. Nixon 111

1 Introduction 112

2 General Approaches to Investigations of Human Neurodegeneration 113

3 Characterization of Autophagic Vacuoles, Evaluation of Autophagosome and Autolysosome Formation, and Autolysosomal Clearance 119

4 Metabolic Analyses of Autophagy in Neuronal and Nonneuronal Cell Models 132

5 Isolation and Characterization of Autophagic Vacuoles and Lysosomes from Cell Cultures and Brain Tissue 135

6 Western Blot Analysis of Autophagy Components and Substrates 136

7 Live-Cell Imaging of Autophagy Induction and Autophagosome-Lysosome Fusion in Primary Cultured Neurons / Mona Bains, Kim A. Heidenreich 145

1 Introduction 146

2 Cultured Cerebellar Purkinje Neurons as a Model to Study Neuronal Autophagy 148

3 Characterization of Autophagic Vacuole Size and Number in Purkinje Neurons 151

4 Using Colocalization of Fluorescent Tags to Measure Autophagosome-Lysosome Fusion 154

5 Concluding Remarks 156

Acknowledgments 157

8 Using Genetic Mouse Models to Study the Biology and Pathology of Autophagy in the Central Nervous System / Zhenyu Yue, Gay R. Holstein, Brian T. Chait, Qing Jun Wang 159

1 Introduction 160

2 Methods 164

3 Analysis of GFP-LC3 Expression and Subcellular Localization in the CNS 167

4 Analysis of p62/SQSTM1 and Ubiquitinated Protein Inclusions in the CNS 171

5 Transmission Electron Microscopy (TEM) Analysis of Autophagosomes 173

6 Conclusion 177

Acknowledgments 178

9 Biochemical and Morphological Detection of Inclusion Bodies in Autophagy-Deficient Mice / Satoshi Waguri, Masaaki Komatsu 181

1 Introduction 182

2 Detection of Ubiquitinated Proteins and p62 in Autophagy-Deficient Mice by Western Blot Analysis 183

3 Detection of Ubiquitinated Proteins and p62 in Cultured Hepatocytes Derived from Autophagy-Deficient Mice 187

4 Detection of Ubiquitin-and p62-Positive Inclusions at the Light Microscopy Level 188

5 Detection of Ubiquitin-and p62-Positive Inclusions at the Electron Microscopy Level 192

6 Conclusion 194

Acknowledgments 195

10 Analyzing Autophagy in Clinical Tissues of Lung and Vascular Diseases / Hong Pyo Kim, Zhi-Hua Chen, Augustine M. K. Choi, Stefan W. Ryter 197

1 Introduction 198

2 Methods for Preparation of Lung and Vascular Cells 199

3 Analysis of Autophagy 205

4 Chromatin Immunoprecipitation 211

5 Conclusions 214

11 Autophagy in Neurite Injury and Neurodegeneration: In Vitro and In Vivo Models / Charleen T. Chu, Edward D. Plowey, Ruben K. Dagda, Robert W. Hickey, Salvatore J. Cherra, Robert S. B. Clark 217

1 Introduction 218

2 Studying Neuronal Autophagy In Vitro 220

3 Studying Brain Autophagy In Vivo 238

4 Future Perspectives and Challenges 244

Acknowledgements 244

12 Monitoring the Autophagy Pathway in Cancer / Frank C. Dorsey, Meredith A. Steeves, Stephanie M. Prater, Thomas Schröter, John L. Cleveland 251

1 Introduction 252

2 LC3: A Phenotypic and Functional Marker of Autophagy 254

3 Assessing the Role of Autophagy in Eμ-Myc-Driven Lymphoma 264

4 Concluding Remarks and Future Perspectives 269

Acknowledgments 269

13 Autophagy Pathways in Glioblastoma / Hong Jiang, Erin J. White, Charles Conrad, Candelaria Gomez-Manzano, Juan Fueyo 273

1 Introduction: Autophagy and Gliomas 274

2 Prioritization of Methods to Characterize Autophagy in Gliomas 275

3 In Vitro Cellular Markers 276

4 In Vitro Biochemical Markers 279

5 Electron Microscopy to Monitor the Autophagic Vacuoles 281

6 In Vivo Analysis of Biochemical Markers 281

7 Autophagy Indicators as Surrogate Markets of Treatment Effect in Clinical Trials 284

8 Future Directions 284

14 Autophagy in Lung Cancer / Jerry J. Jaboin, Misun Hwang, Bo Lu 287

1 Introduction 288

2 Methods 291

3 Conclusion 301

15 Signal-Dependant Control of Autophagy-Related Gene Expression / Fulvio Chiacchiera, Cristiano Simone 305

1 Introduction 306

2 Overview: Signal Transduction and Chromatin-Associated Kinases 307

3 The p38 Pathway in Colorectal Cancer Cells 308

4 Methods to Test Kinase Activity 309

5 Profiling Gene Expression Pattern 311

6 Transcriptional Control of ATG Genes 312

7 Analysis of Transcriptional Multiprotein Complexes 318

8 Concluding Remarks 322

Acknowledgments 323

16 Novel Methods for Measuring Cardiac Autophagy In Vivo / Cynthia N. Perry, Shiori Kyoi, Nirmala Hariharan, Hiromitsu Takagi, Junichi Sadoshima, Roberta A. Gottlieb 325

1 Introduction 326

2 In Vivo Models of Autophagy in the Myocardium 329

3 Discussion 339

Acknowledgments 341

17 Autophagy in Load-Induced Heart Disease / Hongxin Zhu, Beverly A. Rothermel, Joseph A. Hill 343

1 Introduction 344

2 Mouse Models of Load-Induced Heart Disease 345

3 Analysis of Ventricular Remodeling 346

4 In Vitro Models of Load-Induced Hypertrophy 349

5 Techniques to Analyze Cardiomyocyte Autophagy 350

6 Immunohistochemistry for LAMP-1 or Cathepsin D to Monitor Changes in Lysosomal Abundance 352

7 Isolation of LC3 Proteins from NRVM in Culture 356

8 Isolation of LC3 Protein from Heart of Skeletal Muscle Tissue 357

9 Soluble/Insoluble Fractionation of NRVM 359

10 Perspective 360

Acknowledgments 360

18 Evaluation of Cell Death Markers in Severe Calcified Aortic Valves / Wilhelm Mistiaen, Michiel Knaapen 365

1 Clinical Importance of Degenerative Aortic Valve Disease 366

2 Pathological Appearances of AVD 366

3 Mechanism of Progression of AVD 367

4 Autophagy: Major Player in the Progression of Aortic Valve Disease? 368

5 Methods for Detection of Cell Death Markers in the Degenerated Aortic Valve 370

6 Methods for Quantification of Calcified Aortic Valves 374

7 Discussion 376

8 Prospects and Concluding Remarks 376

19 Monitoring Autophagy in Muscle Diseases / May Christine V.

Malicdan, Satoru Noguchi, Ichizo Nishino 379

1 Introduction 380

2 Histological Observation of Skeletal Muscles 381

3 Measuring Autophagy by Protein Quantification 387

4 Monitoring Autophagy in Cultured Skeletal Myocytes 388

5 Electron Microscopy Observation of Skeletal Muscles 392

6 Immunoelectron Microscopy 394

7 Conclusion 395

20 Analyzing Macroautophagy in Hepatocytes and the Liver / Wen-Xing Ding, Xiao-Ming Yin 397

1 The Pathophysiological Relevance of Macroautophagy in the Liver 398

2 Analysis of Autophagy in Isolated Hepatocytes 398

3 Analysis of Autophagy in the Liver 410

4 Summary 413

Acknowledgments 414

21 Monitoring Autophagy in Lysosomal Storage Disorders / Nina Raben, Lauren Shea, Victoria Hill, Paul Plotz 417

1 Introduction 418

2 General Techniques to Monitor Autophagy In LSDs 419

3 LSDs Analyzed for Autophagic Involvement 428

4 Conclusion 444.

Notes:

Includes bibliographical references and indexes.

ISBN:

9780123749369

0123749360

OCLC:

309215866