Autophagy in Disease and Clinical Applications, Part C (eBook)

Front Cover 1
Autophagy in Disease and Clinical Applications, Part C 4
Copyright Page 5
Contents 6
Contributors 14
Preface 22
Methods in Enzymology 24
Chapter 1: Initiation of Autophagy by Photodynamic Therapy 52
1. Introduction 53
2. Photosensitizing Agents 54
3. Additional Factors Unique to PDT 56
4. A Typical PDT Protocol 56
5. Identification and Characterization of Autophagy after PDT 57
6. Effects of Autophagy on PDT Responses 60
Acknowledgments 66
References 66
Chapter 2: Autophagic Cell Death 68
1. Introduction 69
2. Methods to Quantify Cell Death 70
3. Methods to Measure Autophagy 77
4. Methods to Establish Autophagy as the Cause of Cell Death 78
5. Autophagy Genes for RNAi Silencing 80
6. Transfect RNAi by Electroporation (Amaxa Nucleofection) 80
7. Conclusion 80
References 81
Chapter 3: Autophagic Neuron Death 84
1. Introduction 85
2. Experimental Models of Neurodegeneration 86
3. Measurements of Neuron Death 92
References 99
Chapter 4: Assessing Metabolic Stress and Autophagy Status in Epithelial Tumors 104
1. Introduction 106
2. Mouse Epithelial Cell Models for Studying the Role of Autophagy in Cancer 108
3. Protocols for Monitoring Autophagy in iBMK Cells and iMMECs In Vitro 112
4. Protocols for Monitoring Autophagy in Tumors In Vivo 115
5. Monitoring Chromosomal Instability Due to Autophagy Defects 118
6. Concluding Remarks and Future Perspectives 129
References 130
Chapter 5: Autophagic Clearance of Aggregate-Prone Proteins Associated with Neurodegeneration 134
1. Introduction 135
2. Aggregate-Prone Intracytoplasmic Proteins Associated with Neurodegenerative Disorders are Autophagy Substrates 137
3. Assays for The Clearance of Aggregate-Prone Proteins 138
4. Measurement of Autophagic Flux Using Bafilomycin A1 153
5. Concluding Remarks 158
Acknowledgments 158
References 158
Chapter 6: Monitoring Autophagy in Alzheimer´s Disease and Related Neurodegenerative Diseases 162
1. Introduction 163
2. General Approaches to Investigations of Human Neurodegeneration 164
3. Characterization of Autophagic Vacuoles, Evaluation of Autophagosome and Autolysosome Formation, and Autolysosomal Clearance 170
4. Metabolic Analyses of Autophagy in Neuronal and Nonneuronal Cell Models 183
5. Isolation and Characterization of Autophagic Vacuoles and Lysosomes from Cell Cultures and Brain Tissue 186
6. Western Blot Analysis of Autophagy Components and Substrates 187
References 191
Chapter 7: Live-Cell Imaging of Autophagy Induction and Autophagosome-Lysosome Fusion in Primary Cultured Neurons 196
1. Introduction 197
2. Cultured Cerebellar Purkinje Neurons as a Model to Study Neuronal Autophagy 199
3. Characterization of Autophagic Vacuole Size and Number in Purkinje Neurons 202
4. Using Colocalization of Fluorescent Tags to Measure Autophagosome-Lysosome Fusion 205
5. Concluding Remarks 207
Acknowledgments 208
References 208
Chapter 8: Using Genetic Mouse Models to Study the Biology and Pathology of Autophagy in the Central Nervous System 210
1. Introduction 211
2. Methods 215
3. Analysis of GFP-LC3 Expression and Subcellular Localization in the CNS 218
4. Analysis of p62/SQSTM1 and Ubiquitinated Protein Inclusions in the CNS 222
5. Transmission Electron Microscopy (TEM) Analysis of Autophagosomes 224
6. Conclusion 228
Acknowledgments 229
References 229
Chapter 9: Biochemical and Morphological Detection of Inclusion Bodies in Autophagy-Deficient Mice 232
1. Introduction 233
2. Detection of Ubiquitinated Proteins and p62 in Autophagy-Deficient Mice by Western Blot Analysis 234
3. Detection of Ubiquitinated Proteins and p62 in Cultured Hepatocytes Derived from Autophagy-Deficient Mice 238
4. Detection of Ubiquitin- and p62-Positive Inclusions at the Light Microscopy Level 239
5. Detection of Ubiquitin- and p62-Positive Inclusions at the Electron Microscopy Level 243
6. Conclusion 245
Acknowledgments 246
References 246
Chapter 10: Analyzing Autophagy in Clinical Tissues of Lung and Vascular Diseases 248
1. Introduction 249
2. Methods for Preparation of Lung and Vascular Cells 250
3. Analysis of Autophagy 256
4. Chromatin Immunoprecipitation 262
5. Conclusions 265
References 266
Chapter 11: Autophagy in Neurite Injury and Neurodegeneration: In Vitro and In Vivo Models 268
1. Introduction 269
2. Studying Neuronal Autophagy In Vitro 271
3. Studying Brain Autophagy In Vivo 289
4. Future Perspectives and Challenges 295
Acknowledgments 295
References 295
Chapter 12: Monitoring the Autophagy Pathway in Cancer 302
1. Introduction 303
2. LC3: A Phenotypic and Functional Marker of Autophagy 305
3. Assessing the Role of Autophagy in Emu-Myc-Driven Lymphoma 315
4. Concluding Remarks and Future Perspectives 320
Acknowledgments 320
References 320
Chapter 13: Autophagy Pathways in Glioblastoma 324
1. Introduction: Autophagy and Gliomas 325
2. Prioritization of Methods to Characterize Autophagy in Gliomas 326
3. In Vitro Cellular Markers 327
4. In Vitro Biochemical Markers 330
5. Electron Microscopy to Monitor the Autophagic Vacuoles 332
6. In Vivo Analysis of Biochemical Markers 332
7. Autophagy Indicators as Surrogate Markers of Treatment Effect in Clinical Trials 335
8. Future Directions 335
References 336
Chapter 14: Autophagy in Lung Cancer 338
1. Introduction 339
2. Methods 342
3. Conclusion 352
References 352
Chapter 15: Signal-Dependent Control of Autophagy-Related Gene Expression 356
1. Introduction 357
2. Overview: Signal Transduction and Chromatin-Associated Kinases 358
3. The p38 Pathway in Colorectal Cancer Cells 359
4. Methods to Test Kinase Activity 360
5. Profiling Gene Expression Pattern 362
6. Transcriptional Control of ATG Genes 363
7. Analysis of Transcriptional Multiprotein Complexes 369
8. Concluding Remarks 373
Acknowledgments 374
References 374
Chapter 16: Novel Methods for Measuring Cardiac Autophagy In Vivo 376
1. Introduction 377
2. In Vivo Models of Autophagy in the Myocardium 380
3. Discussion 390
Acknowledgments 392
References 392
Chapter 17: Autophagy in Load-Induced Heart Disease 394
1. Introduction 395
2. Mouse Models of Load-Induced Heart Disease 396
3. Analysis of Ventricular Remodeling 397
4. In Vitro Models of Load-Induced Hypertrophy 400
5. Techniques to Analyze Cardiomyocyte Autophagy 401
6. Immunohistochemistry for LAMP-1 or Cathepsin D to Monitor Changes in Lysosomal Abundance 403
7. Isolation of LC3 Proteins from NRVM in Culture 407
8. Isolation of LC3 Protein from Heart or Skeletal Muscle Tissue 408
9. Soluble/Insoluble Fractionation of NRVM 410
10. Perspective 411
Acknowledgments 411
References 411
Chapter 18: Evaluation of Cell Death Markers in Severe Calcified Aortic Valves 416
1. Clinical Importance of Degenerative Aortic Valve Disease 417
2. Pathological Appearances of AVD 417
3. Mechanism of Progression of AVD 418
4. Autophagy: Major Player in the Progression of Aortic Valve Disease? 419
5. Methods for Detection of Cell Death Markers in the Degenerated Aortic Valve 421
6. Methods for Quantification of Calcified Aortic Valves 425
7. Discussion 427
8. Prospects and Concluding Remarks 427
References 428
Chapter 19: Monitoring Autophagy in Muscle Diseases 430
1. Introduction 431
2. Histological Observation of Skeletal Muscles 432
3. Measuring Autophagy by Protein Quantification 438
4. Monitoring Autophagy in Cultured Skeletal Myocytes 439
5. Electron Microscopy Observation of Skeletal Muscles 443
6. Immunoelectron Microscopy 445
7. Conclusion 446
References 446
Chapter 20: Analyzing Macroautophagy in Hepatocytes and the Liver 448
1. The Pathophysiological Relevance of Macroautophagy in the Liver 449
2. Analysis of Autophagy in Isolated Hepatocytes 449
3. Analysis of Autophagy in the Liver 461
4. Summary 464
Acknowledgments 465
References 465
Chapter 21: Monitoring Autophagy in Lysosomal Storage Disorders 468
1. Introduction 469
2. General Techniques to Monitor Autophagy In LSDs 470
3. LSDs Analyzed for Autophagic Involvement 479
4. Conclusion 495
References 496
Author Index 502
Subject Index 522
Color Plates Section 534