Neuronal Cell Death: Methods and Protocols

Description

This volume covers comprehensive methods on ways to assess structural and ultrastructural changes in the mitochondria, cytoskeleton, and microglia using state-of-the-art microscopy techniques including super-resolution imaging, electron microscopy, and ultra-high field MRI. The chapters in this book cover topics such as analysis of neurodegeneration in the post-mortem characterization of preclinical animal models, in vivo modeling in cell death in different model systems and brain organoids, single cell clonal analysis using Mosaic Analysis with Double Markers in genetic mouse models, and genome and proteomic methods for analysis of mRNA dynamics and quantitation of targeted peptides. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Cutting-edge and thorough, Neuronal Cell Death: Methods and Protocols is a valuable resource for any scientist and researcher interested in learning more about this developing field. Preface Contents Contributors Chapter 1: Monitoring Mitochondrial Respiration in Mouse Cerebellar Granule Neurons 1 Introduction 2 Materials 2.1 Equipment and Tools 2.2 Reagents 3 Methods 3.1 Mouse CGN Isolation and Culturing 3.2 Seeding of CGNs in XF96 Microplates 3.3 Transfection of CGNs with siRNA 3.4 Hydrate Sensor Cartridge 3.5 Assessment of OCR in Mouse CGNs Using the XF96 Extracellular Flux Analyzer 3.6 Run the Seahorse XF Cell Mito Stress Test Assay 3.7 Data Analysis 4 Notes References Chapter 2: Examining Mitochondrial Morphology in Mouse Brains 1 Introduction 2 Materials 2.1 Immunocytochemistry to Visualize Mitochondria in Hippocampal Neuronal Cultures 2.2 Immunohistochemistry to Visualize Mitochondria in Mouse Brain Sections 2.3 Confocal Imaging and Analysis of Immunostained Mitochondria 2.4 Assessing Mitochondria in Brain Slices by Electron Microscopy 3 Methods 3.1 Assessing Mitochondria by Tom20 Immunocytochemistry in Primary Hippocampal Neuronal Cultures 3.2 Perfusion and Tissue Sectioning for Immunohistochemistry to Assess Mitochondria 3.3 Assessing Mitochondria in Mouse Brain Sections Using Tom20 Immunohistochemistry 3.4 Confocal Imaging of Tom20 Immunostained Mitochondria 3.5 Perfusion for Electron Microscopy on Brain Sections 3.6 Transmission Electron Microscope (TEM) Imaging of Mitochondria in Brain Sections 4 Notes References Chapter 3: Investigating Microglial Ultrastructural Alterations and Intimate Relationships with Neuronal Stress, Dystrophy, an... 1 Introduction 2 Materials 2.1 Animal Perfusion 2.2 Brain Sectioning 2.3 Tissue Preparation for Immunohistochemistry Staining 2.4 Postfixation and Embedding 2.5 Ultramicrotomy and SEM Imaging 3 Methods 3.1 Animal Perfusion 3.2 Brain Sectioning 3.3 IBA1 Staining 3.4 Postfixation and Embedding 3.5 Ultramicrotomy 3.6 SEM Imaging 3.7 Ultrastructural Analysis of Microglia, AD Hallmarks and Synapses 3.7.1 Identification of Stained and Unstained Microglial Cells 3.7.2 Identification of Dark Microglia 3.7.3 Density Analysis of Dark and Typical Microglia Using Chip Mapping 3.7.4 Ultrastructural Analysis of Microglia 3.7.5 Ultrastructural Analysis of Microglial Interactions with Amyloid Beta Plaques, Stressed, Degenerating or Dystrophic Neur... 4 Notes References Chapter 4: Performing Single-Cell Clonal Analysis in the Mouse Brain Using Mosaic Analysis with Double Markers (MADM) 1 Introduction 2 Materials 2.1 Mouse Lines and Genotyping 2.2 Mouse Brain Perfusion and Dissection 2.3 Immunofluorescence Staining 3 Methods 3.1 Design and Collect Mouse Lines for the MADM Experiment 3.2 Genotyping of the MADM Reporters 3.3 Recombine the Mutant Allele with the MADM-TG Allele 3.4 Introduce the Cre Line into the MADM-GT Mice 3.5 Perform the MADM Experiment and Collect Brain Samples 3.6 Visualization of MADM Clones with Immunofluorescence Staining 4 Notes References Chapter 5: Combination of Endothelin-1 (ET-1) and L-NAME to Induce Murine Focal Cortical Stroke with Persistent Sensorimotor D... 1 Introduction 2 Materials 2.1 ET-1/L-NAME Vasoconstrictor Solution 2.2 Anesthesia and Animal Preparation 2.3 Stereotactic Injection Surgery 2.4 Perfusion and Tissue Sectioning 2.5 Cresyl Violet Stroke Volume Analysis 2.6 Horizontal Ladder Test 2.7 Adhesive Tape Test 3 Methods 3.1 Anesthesia and Animal Surgical Preparation (See Note 4) 3.2 Stereotaxic Surgery and Stroke Induction 3.3 Perfusion and Tissue Sectioning 3.4 Cresyl Violet Staining and Stroke Volume Analysis 3.5 Horizontal Ladder Test 3.6 Adhesive Tape Test 4 Notes References Chapter 6: Modeling Axonal Degeneration Using Motor Nerve Organoids 1 Introduction 2 Materials 2.1 Coating 2.2 hiPSC Culture 2.3 Motor Neuron Differentiation and Maintenance 2.4 Tissue Culture Device Preparation 2.5 Drug Treatment 3 Methods 3.1 Preparation of Human iPS Cells for Motor Neuron Differentiation 3.1.1 Differentiation Should Be Initiated When the Undifferentiated Human iPS Cells Reaches a Confluency of Approximately 80% 3.2 Spheroid Formation and Motor Neuron Differentiation 3.3 Preparation of Tissue Culture Devices 3.4 Motor Nerve Organoid Formation 3.5 Accelerated Axonal Degeneration with H2O2 Treatment 3.6 Imaging Analysis 4 Notes References Chapter 7: Monitoring Autophagy in Neural Stem and Progenitor Cells 1 Introduction 1.1 Autophagy: Mechanisms and Functions 1.2 Role of Autophagy in Neural Stem and Progenitor Cells 1.3 Methods for Monitoring Autophagy 2 Materials 2.1 Measuring Autophagic Flux: LC3B Lipidation and p62 Degradation Assays 2.1.1 Inducing Autophagy in Cells and Inhibiting Autophagic Flux 2.1.2 LC3B Lipidation and p62 Degradation Immunoblotting Assays 2.1.3 Assessing p62 Degradation by Simple Western 2.2 LC3B Immunofluorescence to Detect Autophagosomes 2.3 Transcriptional Analysis of Autophagy Genes 3 Methods 3.1 Measuring Autophagic Flux: LC3B Lipidation and p62 Degradation Assays 3.1.1 Inducing Autophagy in Cells and Inhibiting Autophagic Flux 3.1.2 LC3B Lipidation and p62 Degradation Immunoblotting Assays 3.1.3 Assessing p62 Degradation by Simple Western 3.2 LC3B Immunofluorescence to Detect Autophagosomes 3.3 Transcriptional Analysis of Autophagy Genes 4 Notes References Chapter 8: Direct FACS Isolation of Neural Stem/Progenitor Lineages from the Adult Brain 1 Introduction 2 Materials 2.1 Equipment and Reagents 2.2 Preparation of Solutions 3 Methods 3.1 Animal Models 3.2 Setup 3.3 Brain Dissection 3.4 Brain Slicing 3.5 Microdissection of SVZ and SGZ 3.6 Enzymatic Digestion of each Condition 3.7 FACS of NSPCs (Immunostain-Based) 3.8 FACS of NSPCs (Reporter-Based) 4 Notes References Chapter 9: Analysis of mRNA Dynamics Using RNA Sequencing Data 1 Introduction 1.1 Overview of RNA Dynamics and Its Regulatory Factors 1.2 Theoretical Background 2 Materials 2.1 Software 2.2 Data Files and Additional Scripts 3 Methods 3.1 Generating Intronic and Exonic Annotation 3.2 Quantifying Pre-mRNA and Mature mRNA Abundances 3.3 Acquiring Δ Stability Estimates 3.4 Using Δ Stability Estimates in Downstream Analyses 4 Notes References Chapter 10: Assessment of Dopaminergic Neurodegeneration in Mice 1 Introduction 2 Materials 2.1 Reagents Required (in Order of Appearance) 2.2 Equipment and Consumables (in Order of Appearance) 3 Methods 3.1 Brain Dissection and Processing 3.2 Immunohistochemistry of Free-Floating Brain Sections 3.3 Microscopy and Stereological Quantification Using the Optical Fractionator 3.4 Tissue Processing and Paraffin Sections 3.5 Immunohistochemistry of Paraffin Brain Sections 3.6 Microscopy and Stereological Quantification Using the Physical Fractionator 3.7 Optical Densitometry in the Striatum 4 Notes References Chapter 11: Superresolution Imaging of Cytoskeletal Networks in Fixed Brain Tissue 1 Introduction 2 Materials 2.1 Equipment 2.2 Perfusion, Brain Collection, and Slicing 2.3 Slice Staining 2.4 Superresolution Imaging 3 Methods 3.1 Preparation of the Fixative 3.2 Perfusion 3.3 Brain Collection and Slicing 3.4 Slice Staining 3.5 Superresolution Imaging 4 Notes References Chapter 12: Spinal Cord Injury in the Mouse Using the Infinite Horizon Spinal Cord Impactor 1 Introduction 2 Materials 2.1 Equipment 2.2 Reagents and Consumables 3 Methods 3.1 Anesthetizing the Mice and Preoperative Care 3.2 Surgery 3.3 Postoperative Care 4 Notes References Chapter 13: Immuno-MALDI-MS for Accurate Quantitation of Targeted Peptides from Volume-Restricted Samples 1 Introduction 2 Materials 2.1 Labware and Equipment 2.2 Standards and Antibodies 2.3 Buffers 2.4 Digestion and Dephosphorylation 2.5 Spotting and Matrix Application 3 Methods 3.1 Target and Antibody Selection 3.2 Antibody-Bead Coupling 3.3 Standard Curve Preparation 3.4 Internal Standard Preparation 3.5 Sample Preparation 3.6 Tryptic Digestion, IS Addition, and Dephosphorylation 3.7 Immunoenrichment, Washing, Spotting, and Matrix Application 3.8 MALDI-MS Acquisition and Data Analysis 4 Notes References Chapter 14: Three-Dimensional Atlas of the Human Amygdala Subnuclei Constructed Using Immunohistochemical and Ultrahigh-Field ... 1 Introduction 1.1 Anatomy of the Amygdala 1.2 Amygdala Atlases 2 Materials 2.1 Human Brain Prosections 2.2 Immunohistochemistry 2.3 Antibodies 2.4 Imaging Materials 3 Methods 3.1 Medial Temporal Lobe Dissection 3.2 MR Imaging 3.3 Immunohistochemical Procedure 3.4 Manual Segmentation of the Amygdala Subnuclei 4 Notes References Chapter 15: Using Single-Molecule Fluorescence Microscopy to Uncover Neuronal Vulnerability to Protein Damage 1 Introduction 2 Materials 2.1 Neuronal Culture 2.2 Heat Shock 2.3 smFISH and Immunofluorescence (smFISH-IF) 2.3.1 smFISH 2.3.2 smFISH Reagents 2.3.3 Immunofluorescence 2.4 Wide-Field Fluorescence Microscope 2.5 Data Analysis 3 Methods 3.1 Mouse Postnatal Hippocampal Neuron Culture 3.1.1 Reagents Preparation 3.1.2 Removal of the Brain 3.1.3 Hippocampus Extraction 3.1.4 Dissociation for Neuronal Isolation 3.2 Heat Shock 3.3 Immunofluorescence (IF): smFISH 3.4 Imaging 3.5 Imaging Analysis 3.5.1 Imaging Visualization and FISH-Quant Analysis 3.5.2 Create Folders and Subfolders 3.5.3 Define the Outlines 3.5.4 Quantifying mRNA 3.5.5 Predetection 3.5.6 Fitting and Thresholding 3.5.7 Batch Processing 3.5.8 Quantifications 3.6 Programming Through Python 4 Notes References Chapter 16: Autopsy Human Brain Dissection Protocol for Common Age-Related Neurodegenerative Disorders 1 Introduction 2 Materials 2.1 Histochemical Stains 2.2 Immunohistochemistry 2.3 Dissection Materials 3 Methods 3.1 Gross Dissection 3.1.1 Removing the Brain and Dura from the Skull 3.1.2 Dissection of the Fresh/Frozen Brain (see Note 7) 3.1.3 Dissection of the Fixed Whole or Half Brain 3.1.4 Cord (see Note 12) 3.2 Staining Methods 3.3 Disease Specific Neuropathological Examination 3.3.1 Alzheimer Disease (AD) Gross Examination Microscopic Examination 3.3.2 Lewy Body Disease (LBD) Gross Examination 3.3.3 Microscopic Examination 3.3.4 Cerebrovascular Disease (CVD) Gross Examination Microscopic Examination 3.3.5 Hippocampal Sclerosis (HS) Gross Examination Microscopic Examination 3.3.6 Frontotemporal Lobar Degeneration (FTLD) Gross Examination Microscopic Examination 3.3.7 Other Tauopathies and Synucleinopathies 4 Notes References Chapter 17: In Vitro Brain Organoids and Computational Models to Study Cell Death in Brain Diseases 1 Introduction 2 Materials 2.1 Solutions 2.2 Software and Equipment 3 Methods 3.1 Mold 3D Printing and Fabrication of the Hydrogel-Based SFMWs 3.2 Cell Culture and Organoid Formation 3.2.1 Cell Culture Medium Preparation 3.2.2 Neural Progenitor Cell Growth Medium Preparation 3.2.3 Neural Progenitor Cell Differentiation Medium 3.2.4 Establishing Organoids in SFMWs Platform Using Human Cell Lines 3.2.5 Establishing Organoids in SFMWs Platform Using Neural Cells 3.3 Live/Dead Staining of the Organoids in the SFMWs 3.4 PB Analysis of the Organoids in the Microwell 3.5 Immunostaining of Organoids in the SFMWs 3.6 Measurement of Glucose Uptake and Proliferation Rates in Human Cell Lines 3.7 Modeling the Growth of Organoids 3.8 Therapeutic Analysis 4 Notes References Chapter 18: Isolation of Mouse Embryonic Neural Stem Cells and Characterization of Neural Stem Markers by Flow Cytometry 1 Introduction 2 Materials 2.1 Equipment and Tools 2.2 Reagents 3 Methods 3.1 Set-Up Prior to Tissue Dissection 3.2 Harvesting E16.5 Mouse Brain 3.3 Passaging of Embryonic NSCs 3.4 Immunostaining of Embryonic NSCs 3.5 Flow Cytometry Analysis 4 Notes References Chapter 19: Assessment of Neuronal Cell Death in Caenorhabditis elegans 1 Introduction 2 Materials 2.1 Nematode Strains 2.2 Equipment and Reagents for General Worm Manipulation 2.3 Nematode Food 2.4 Imaging 3 Methods 3.1 Sample Preperation (for Either Microscopy Method) 3.2 Locating Neuronal Cell Corpses Via Nomarski Differential Interference Contrast (DiC) Microscopy 3.3 Identifying Loss of Fluorescently Tagged Neuronal Cells Via Fluorescent Microscopy 4 Notes References Chapter 20: Emerging Methods in Modeling Brain Development and Disease with Human Pluripotent Stem Cells 1 Advantages of hPSCs in Modeling Brain Development and Function 2 Methods to Derive Neuronal Cells and Tissue from hPSCs 3 hPSC-Based Neurological Disease Models 3.1 Neurodegenerative Diseases 3.2 Cell Death-Driven Developmental Microcephaly 4 Recent Technological Advances in hPSC Brain Modeling 5 Concluding Remarks References Chapter 21: Isolation of Adult Mouse Neural Stem Cells and Assessment of Self-Renewal by ELDA 1 Introduction 2 Materials 2.1 Tools and Equipment 2.2 Reagents 3 Methods 3.1 Initial Set Up 3.2 Harvesting Adult Mouse Brain 3.3 Isolation of Adult Mouse SVZ 3.4 Adult NSC Culture 3.5 Passaging and Splitting aNSCs 3.6 Extreme Limiting Dilution Assay (ELDA) 4 Notes References Index