Imaging and Quantifying Neuronal Autophagy (Neuromethods, 171)

✍ Scribed by Ben Loos (editor), Esther Wong (editor)

• Publisher: Humana
• Year: 2021
• Tongue: English
• Leaves: 155
• Category: Library

✦ Synopsis

The volume aims to explore the dynamic nature of the autophagy pathway, and the latest techniques that allow researchers to capture and quantify this process in neurons. The chapters in this volume cover topics such as fundamental, historical, and functional approaches that began in baker’s yeast; Saccharomyces cerevisiae; the role of both electron microscopy and live-cell imaging using fluorescently tagged autophagy proteins; and the rate of puncta appearance and its correlation with the rate of autophagosome formation. In the Neuromethods series style, chapters include the kind of detail and key advice from the specialists needed to get successful results in your laboratory.

Cutting-edge and practical, Imaging and Quantifying Neuronal is a valuable resource that provides insights into the power of microscopy tools, live-cell imaging, and photoactivation and correlative techniques.

✦ Table of Contents

Preface to the Series
Preface
Contents
Contributors
Chapter 1: Quantification of Autophagosome Size and Formation Rate by Electron and Fluorescence Microscopy in Baker´s Yeast
1 Introduction: Detection of Autophagy by Microscopy in Baker´s Yeast
2 Materials
2.1 Embedding and TEM
2.2 Immobilization and Imaging of GFP-Atg8 Cells Using Concavity Slides
3 Methods
3.1 Acquisition of TEM Images
3.2 Quantification of EM Micrographs
3.3 Acquisition of Time-Lapse GFP-Atg8 Movies
3.4 Quantification of Time-Lapse GFP-Atg8 Movies
4 Notes
References
Chapter 2: Ultrastructure of the Macroautophagy Pathway in Mammalian Cells
1 Introduction
2 Materials
3 Methods
3.1 Phagophores and Autophagosomes
3.2 Amphisomes and Autolysosomes
4 Notes
References
Chapter 3: Live Imaging of Autophagosome Biogenesis and Maturation in Primary Neurons
1 Introduction
1.1 Dynamics of Autophagosome Biogenesis and Maturation in Neurons
1.1.1 Cell Biology of the Neuron
1.1.2 Imaging Autophagy in the Neuron
1.1.3 Conserved Pathway for Constitutive Autophagosome Biogenesis and Maturation in the Axon
2 Materials
2.1 Selection of Model System
2.2 Isolation and Culture of Primary Rodent Neurons
2.2.1 Selection of Neuronal Type
2.2.2 Hippocampal and Cortical neurons
2.2.3 DRG Neurons
2.3 Transfecting Neurons
2.3.1 Hippocampal and Cortical Neurons
2.3.2 DRG Neurons
2.4 Preparation of Neurons Prior to Imaging
2.4.1 Labeling with Fluorescent Ligands
2.5 Microscope Selection
2.6 Selection of Fluorescent Probes to Image
2.7 Data Acquisition and Analysis
3 Methods
3.1 Imaging Parameters
3.2 Data Analysis
4 Notes
References
Chapter 4: Monitoring Autophagic Activity In Vitro and In Vivo Using the GFP-LC3-RFP-LC3DeltaG Probe
1 Introduction: Macroautophagy
2 Materials
2.1 Cell Culture
2.2 Establishment of Cells Stably Expressing the Probe Using the Retrovirus Vector
2.3 Genomic PCR
2.4 Flow Cytometry
2.5 Zebrafish Preparation
2.6 In Vitro Transcription
2.7 Injection of mRNA into Zebrafish Eggs
2.8 Confocal Microscopy
3 Methods
3.1 Principle Underlying the Novel Probe GFP-LC3-RFP-LC3DeltaG
3.2 Analysis of Autophagic Activity in Culture Cells
3.2.1 Preparation of Probe-Expressing Cells
3.2.2 Analysis by Flow Cytometry
3.3 Analysis of Autophagic Activity in Zebrafish
3.3.1 Preparation of Zebrafish Expressing the GFP-LC3-RFP-LC3DeltaG Probe
3.3.2 Analysis of Zebrafish
4 Notes
References
Chapter 5: Measuring Autophagic Flux in Neurons by Optical Pulse Labeling
1 Introduction
2 Materials
2.1 Primary Neuron Prep and Culture
2.2 Photoconvertible Fluorescent Proteins and Expression System
2.3 Positive Controls
2.4 Automated Fluorescence Microscopy
2.5 UV Photoconversion Source
2.6 Image Processing and Analysis Software
3 Methods
3.1 Expression of OPL Reporter in Primary Neurons
3.2 UV Optimization
3.3 Imaging Optimization
3.4 UV Photoconversion and Time-Lapse Imaging
3.5 Image Processing and Measurement
3.6 Single-Cell Half-Life Analysis
4 Notes
References
Chapter 6: Measuring Autophagosome Flux
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Chemicals
2.3 Image Acquisition
3 Methods
3.1 Measuring Autophagy Variables
3.2 Flux Is the Rate of Flow Through the Autophagy Pathway at Steady State
3.3 The Complete Inhibition of Fusion Between Autophagosomes and Lysosomes
4 Notes
References
Chapter 7: Measurement of Neuronal Tau Clearance In Vivo
1 Introduction
1.1 Overview of the Method
2 Materials
2.1 Zebrafish Strains
2.2 Solutions and Reagents
2.3 Equipment
3 Methods
3.1 Zebrafish Crosses and Fish Selection
3.2 Preparation of Larvae for Imaging
3.3 Confocal Imaging
3.3.1 Preparation for Live Imaging
3.3.2 Photoconversion and Live Imaging of Initial Time Point (T = 0)
3.3.3 Live Imaging at 12, 24, 36, and 48 h Time Points
3.4 Analysis of Images
3.4.1 Z-Stack Projection
3.4.2 Quantification of the Red Signal
3.4.3 Analysis of the Red Signal Values
3.5 Additional Protocols
3.5.1 Assay for Lysosomal Protein Clearance
3.5.2 Assay for Proteasomal Protein Clearance
3.5.3 Investigation of Compounds for Their Effects on Protein Clearance
3.6 Results
3.7 Conclusions and Perspectives
4 Notes
References
Chapter 8: Methods for Studying Axonal Autophagosome Dynamics in Adult Dorsal Root Ganglion Neurons
1 Introduction
2 Materials
2.1 Primary Mouse DRG Neuron Culture
2.1.1 Medias, Solutions, and Reagents
2.1.2 Dissection Tools and Other Materials
2.2 Primary Mouse DRG Neuron Transfection
2.3 Live-Cell Imaging of Autophagosomes in Primary DRG Neurons
2.4 Analysis of Autophagosome Dynamics
3 Methods
3.1 Primary Adult Mouse DRG Neuron Culture, Nucleofection, and Maintenance
3.2 Time-Lapse Live-Cell Imaging of Autophagosomes in Primary DRG Neuron Axons
3.3 Analysis of Axonal Autophagosome Dynamics
4 Notes
References
Chapter 9: Imaging and Quantifying Neuronal Autophagy to Determine the Autophagy Contribution to Neuronal and Dendritic Morpho...
1 Introduction
2 Materials
2.1 Primary Hippocampal and Cortical Neuron Culture
2.1.1 Clean and Treat Glass Coverslips with Poly-l-Lysine (for Hippocampal Neuron Culture)
2.1.2 Treat Glass 6-Well Cell Culture Plated with Poly-l-Lysine (for Cortical Neuron Culture)
2.2 Constructs for Visualization of Neuronal Morphology and Autophagy
2.3 Autophagy Induction and Inhibition with the Use of Small Molecules
2.4 Genetic Manipulation
3 Methods
3.1 Hippocampal and Cortical Neuron Culture from Wild-Type and Genetically Modified Mice
3.1.1 Dissection of Hippocampi from P0 Mouse Pups
3.1.2 Cell Dissociation and Plating of Neuronal Cells
3.2 Visualization and Quantification of Neuronal Morphology and Spine Density
3.2.1 Hippocampal Neuron Transfection Using Lipofectamine 3000
3.2.2 Sholl Analysis and Spine Counting
3.3 Manipulation and Quantification of Neuronal Autophagy
3.3.1 Autophagy Induction and Inhibition with the Use of Small Molecules
Autophagy Induction in Neurons by Nutrient Deprivation
SBI-0206965 (ULK-1 Inhibitor)
Spautin-1
3.3.2 Genetic Inactivation of Autophagy Machinery
Small Interfering RNA Knockdown in Neurons Using DharmaFECT
Day 0: Cortical Neuronal Culture
Day 2: siRNA Preparation and Transfection (According to Manufacturer´s Protocol)
Day 4: Check siRNA Knockdown Efficiency
CRISPR-Cas9-Mediated Gene Deletion in Postmitotic Neurons
Lentivirus Production Using Lipofectamine 2000
Lentivirus Infection in Primary Neuronal Culture
3.3.3 Immunofluorescence and Image Acquisition
3.3.4 Neuronal Immunofluorescence Staining
3.3.5 Lysate Preparation for Immunoblots
3.3.6 LC3-II Puncta Quantification
4 Notes
5 Conclusions
References
Chapter 10: Correlative Light and Electron Microscopy (CLEM): Bringing Together the Best of Both Worlds to Study Neuronal Auto...
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Microscopy
2.3 Sectioning
2.4 Image Processing Software
3 Methods
3.1 Fluorescence Microscopy: Sample Preparation and Image Acquisition
3.2 Electron Microscopy: Sample Preparation
3.3 Electron Microscopy: 2D Image Acquisition/Targeted Array Tomography
3.4 Electron Microscopy: Focused Ion Beam SEM Acquisition
3.5 Overlay of Fluorescence and Electron Micrographs
4 Notes
References
Index