Preface
Contents
Contributors
Part I: Native Tau Protein: Conformational Studies and Protein-Protein Interactions
Chapter 1: Characterization of Posttranslationally Modified PHF-1 Tau Peptides Using Gaussian Accelerated Molecular Dynamics S...
1 Introduction
1.1 Tau: Structural Significance in Neurodegenerative Diseases
1.2 Application of Molecular Dynamics Simulations in Characterizing Protein Folding
1.3 Theory
1.3.1 Gaussian Accelerated Molecular Dynamics (GaMD)
1.3.2 Energetic Reweighting of GaMD for Free Energy Calculations
1.3.3 Characterization of Boost Potential
2 Materials
2.1 Hardware
2.2 Software
3 Methods
3.1 System Preparation
3.1.1 Creation of Linear PDB Structure of Peptide
3.1.2 Modification of Peptide Structure
3.1.3 Preparation of Simulation System in CHARMM-GUI
3.2 Minimization Using AMBER20 Software
3.3 Gaussian Accelerated MD Simulation
3.4 Analysis of GaMD Simulation
3.4.1 Preparation of Input Files for Reweighting
3.4.2 Generation of MD Trajectory Files
3.4.3 Preparation of Two-Dimensional (2D) Data for Reweighting Calculation
3.4.4 Python-Based Reweighting of GaMD Simulations and Plotting of Free-Energy Landscapes
3.4.5 Clustering Analysis of GaMD Trajectories
3.4.6 Determination of CD Profile from GaMD Simulation
3.5 Visualization of GaMD Trajectories and the Secondary Structures of Peptides
4 Notes
References
Chapter 2: Interaction of Tau with G-Protein-Coupled Purinergic P2Y12 Receptor by Molecular Docking and Molecular Dynamic Simu...
1 Introduction
2 Materials
3 Methods
3.1 Modeling of TauRD and P2Y12 Receptor
3.2 Molecular Docking of TauRD and P2Y12 Receptor
3.3 MD Simulation of P2Y12-TauRD Complex
3.4 Analysis of the Trajectory
4 Notes
References
Chapter 3: Structural Flexibility of Tau in Its Interaction with Microtubules as Viewed by Site-Directed Spin Labeling EPR Spe...
1 Introduction
1.1 Site-Directed Spin Labeling (SDSL)
1.2 CW-EPR Spectroscopy of Nitroxide Labels Grafted to Proteins
1.3 EPR Spectral Shape Analysis of Labeled Tau
1.3.1 EPR Spectra Analysis of Labeled TauProxyl
1.3.2 EPR Spectra Analysis of TauProxyl with Tubulin Dimers and Paclitaxel-Stabilized MTs
1.3.3 EPR Spectra Analysis of TauMTSL Alone and with Tubulin Dimers and Paclitaxel-Stabilized MTs
2 Materials
2.1 Spin Labeling
2.1.1 Reagents and Solutions
2.1.2 Special Equipment
2.2 Microtubule Formation
2.2.1 Reagents and Solutions
2.2.2 Special Equipment and Software
2.3 Electronic Microscopy
2.3.1 Reagents and Solutions
2.3.2 Special Equipment
2.4 EPR Spectroscopy
3 Methods
3.1 Tau and Tubulin Purifications
3.1.1 Tubulin Preparation
3.1.2 Tau Preparation
3.2 Site-Directed Spin Labeling (SDSL)
3.3 Preparation of Microtubules
3.3.1 Tau-Induced MTs
3.3.2 Paclitaxel-Stabilized MTs
3.3.3 Co-sedimentation Assay on TauMTSL and TauProxyl
3.3.4 Transmission Electronic Microscopy
3.4 EPR Spectroscopy
3.4.1 Optimizing Parameters of Acquisition
3.4.2 Labeling Yield Quantification
3.4.3 Spectral Shape Analyses
EPR Spectral Shape Analysis of TauProxyl
EPR Spectral Shape Analysis of TauMTSL
4 Notes
References
Chapter 4: Cryo-electron Microscopy and Molecular Modeling Methods to Characterize the Dynamics of Tau Bound to Microtubules
1 Introduction
2 Materials
2.1 Software
2.2 Cryo-EM Data
3 Methods
3.1 System Preparation
3.2 MD Equilibration
3.3 Prepare and Run EMMI
3.4 Analysis
4 Notes
References
Part II: In Vitro Self-Association of Tau Protein: Oligomer and Fibril Formation
Chapter 5: Assays for the Screening and Characterization of Tau Aggregation Inhibitors
1 Introduction
2 Materials
2.1 Cell-Free Immunoassay
2.2 Cell-Based Assay
2.3 Cytotoxicity Measurement
3 Methods
3.1 Cell-Free Tau-Tau Binding Immunoassay
3.2 Cell-Based Tau Aggregation Assay
3.3 Cellular Toxicity
3.4 Therapeutic Index
4 Notes
References
Chapter 6: Photo-Excited Dyes: Emerging Technique Against Tau Protein Aggregation
1 Introduction
2 Materials
2.1 Reagents
2.2 Instruments
2.3 Softwares
3 Methods
3.1 Preparation of Photodynamic Therapy (PDT) for Tau Aggregates
3.1.1 Preparation of Photosensitizer
3.1.2 Preparation of Tau Filaments
3.1.3 Preparation of the Dark Chamber and the Light Source
3.2 Aggregation of RB-Induced Tau by ThS
3.3 RB-Induced Tau Conformation by Circular Dichroism Spectroscopy
3.4 RB-Induced Tau Aggregate Morphology by Transmission Electron Microscopy
3.5 Disaggregation Potency of Photo-Excited RB (PE-RB)
4 Notes
References
Chapter 7: Sedimentation and Laser Light Scattering Methods for Quantifying Synthetic Tau Aggregation Propensity
1 Introduction
2 Materials
2.1 Tau Aggregation Reagents and Supplies
2.2 Sedimentation Assay
2.3 Light Scattering Assay
2.4 Modified TEM Assay
3 Methods
3.1 Tau Sedimentation Assay
3.2 Laser Light Scattering (LLS)
3.3 Electron Microscopy Grid Preparation
4 Notes
References
Chapter 8: Recombinant Production and Characterization of VHHs/Nanobodies Targeting Tau to Block Fibrillar Assembly
1 Introduction
2 Materials
2.1 VHH Production
2.2 VHH Extraction and Purification
2.3 Preparation of Recombinant 15N-Labeled and Non-labeled Tau
2.4 NMR Screening
2.5 In Vitro Aggregation Assay
3 Methods
3.1 VHH Recombinant Production
3.2 VHH Periplasmic Extraction and Purification
3.2.1 VHH Periplasmic Extraction
3.2.2 VHH Affinity Purification
3.2.3 VHH Size Exclusion Chromatography
3.3 Production of Recombinant 15N-Labeled Tau and Non-labeled Tau
3.3.1 Day 1: Bacterial Transformation
3.3.2 Day 2: Bacterial Growth
3.3.3 Day 3: Induction Phase
3.3.4 Purification of 15N-Labeled and Non-labeled Tau
3.4 NMR Screening
3.4.1 Sample Preparation
3.4.2 NMR Data Acquisition and Analysis
3.5 In Vitro Aggregation Assay
4 Notes
References
Chapter 9: Tau Oligomers as Pathogenic Seeds: Preparation, Characterization, and Propagation In Vitro and In Vivo
1 Introduction
2 Materials
2.1 Purification of Aβ42 and α-Synuclein
2.2 Purification of TDP-43
2.3 Preparation of Amyloid Oligomeric Seeds (HFIP)
2.4 Preparation of Amyloid Oligomeric Seeds (NaOH/PBS)
2.5 Preparation of Amyloid Oligomeric Seeds (TDP-43)
2.6 Preparation of Tissue Extracts
2.7 Isolation of Tau Oligomers from Brain Tissues
2.7.1 Isolation of Tau Oligomers by Immunoprecipitation Using Magnetic Beads
2.7.2 Isolation of Tau Oligomers by Co-immunoprecipitation Kit
2.7.3 Isolation of Tau Oligomers by Sucrose Gradient
2.8 Preparation of Tau Oligomer
2.8.1 In Vitro Seeding of Tau with Amyloid Oligomeric Seed
2.8.2 In Vitro Seeding of Tau with Brain-Derived Tau Oligomers
2.9 Biochemical Characterization of Tau Oligomer Seed
2.9.1 Indirect ELISA
2.9.2 Sandwich ELISA
2.9.3 Dot Blot for Tau Oligomers
2.9.4 Western Blot for Tau Oligomers
2.9.5 Filter Trap Assay (FTA)
2.9.6 Proteinase K (PK) Digestion
2.9.7 Immunohistochemistry in Frozen Sections
2.9.8 Immunofluorescence in Frozen Sections
2.9.9 Immunohistochemistry in Paraffin-Embedded Sections
2.9.10 Immunofluorescence in Paraffin-Embedded Sections
2.9.11 Bis-ANS Fluorescence Reactivity
2.9.12 Thioflavin T Fluorescence Reactivity
2.10 Biophysical Characterization of Tau Oligomer Seed
2.10.1 Atomic Force Microscopy (AFM)
2.10.2 Transmission Electron Microscopy (TEM)
2.10.3 Circular Dichroism Spectroscopy
2.10.4 Fourier Transform Infrared Spectroscopy (FTIR)
2.10.5 Fluorescent Amyloid Multi-Emission Spectra (FLAMES) Microscopy
2.11 Bioactivity of Tau Oligomer Seeds
2.11.1 Seeding in Primary Cortical Neurons
2.11.2 Seeding in Cell Lines
2.11.3 Cytotoxicity Measurement by Lactate Dehydrogenase (LDH) Release Assay
2.11.4 Cell Viability Measurement by MTT Assay
2.11.5 Dendritic Spine Pathology Using Immunocytochemistry
2.11.6 Long-Term Potentiation (LTP) Measurement
2.12 In Vivo Seeding and Propagation of Tau Oligomer
2.13 Tau Oligomer-Specific Antibodies
3 Methods
3.1 Preparation of Tau Monomeric Samples
3.2 Preparation of Amyloid Seeds
3.2.1 Preparation of Aβ42 Protein
3.2.2 Preparation of Aβ42 Oligomers (HFIP)
3.2.3 Preparation of Aβ42 Oligomers (NaOH)
3.2.4 Preparation of α-Synuclein Protein
3.2.5 Preparation of α-Synuclein Oligomers (HFIP)
3.2.6 Preparation of α-Synuclein Oligomers (PBS)
3.2.7 Preparation of TDP-43 Oligomers
3.3 Preparation of Tissue Extracts
3.4 Isolation of Tau Oligomers from Brain Tissues
3.4.1 Isolation of Tau Oligomers by Immunoprecipitation Using Magnetic Beads
3.4.2 Isolation of Tau Oligomers by Co-immunoprecipitation Kit
3.4.3 Isolation of Tau Oligomers by Sucrose Gradient
3.5 Preparation of Tau Oligomers
3.5.1 In Vitro Seeding of Tau with Amyloid Oligomeric Seed
3.5.2 In Vitro Seeding of Tau with Brain-Derived Tau Oligomer
3.6 Biochemical Characterization of Tau Oligomer Seed
3.6.1 Indirect ELISA
3.6.2 Sandwich ELISA
3.6.3 Dot Blot for Tau Oligomers
3.6.4 Western Blot for Tau Oligomers
3.6.5 Filter Trap Assay (FTA)
3.6.6 Proteinase K (PK) Digestion
3.6.7 Immunohistochemistry in Frozen Tissue
3.6.8 Immunofluorescence in Frozen Tissue
3.6.9 Immunohistochemistry in Paraffin-Embedded Tissue
3.6.10 Immunofluorescence in Paraffin-Embedded Tissue
3.6.11 Bis-ANS Fluorescence Reactivity
3.6.12 Thioflavin T (ThT) Fluorescence Reactivity
3.7 Biophysical Characterization of Tau Oligomer Seed
3.7.1 Atomic Force Microscopy (AFM)
3.7.2 Transmission Electron Microscopy (TEM)
3.7.3 Circular Dichroism Spectroscopy
3.7.4 Fourier Transform Infrared Spectroscopy (FTIR)
3.7.5 Fluorescent Amyloid Multi-Emission Spectra (FLAMES) Microscopy
3.8 Bioactivity of Tau Oligomer Seeds
3.8.1 Seeding in Primary Cortical Neurons
3.8.2 Seeding in Cell Lines
3.8.3 Cytotoxicity by Lactate Dehydrogenase (LDH) Release Assay
3.8.4 Cell Viability by MTT Release Assay
3.8.5 Dendritic Spine Pathology
3.8.6 Immunocytochemistry
3.8.7 Long-Term Potentiation (LTP) Measurement
3.8.8 In Vivo Seeding and Propagation of Tau Oligomer
Stereotaxic Injection of Tau Oligomer Seed
Biochemical Analyses of Tau Pathology in Injected Mice Brain Tissues
Histological Measure of Tau Pathology in Injected Mice Brain Tissues
4 Notes
References
Chapter 10: Preparation of Tau Condensates by Liquid-Liquid Phase Separation to Study Tau Amyloid Aggregation
1 Introduction
2 Materials
3 Methods
3.1 Preparation of Recombinant Tau Protein Stocks
3.2 Preparation of Tau LLPS with Low Salt Concentration
3.3 Preparation of Tau LLPS with RNA, Single-Strand DNA, Heparin, Desulfated Heparin, or Hyaluronic Acid
3.4 Preparation of Tau LLPS with Very High Salt Concentration
3.5 Thioflavin T Assay of Tau Aggregation in the Presence of Coacervates
3.6 TEM of Tau Fibrils in the Presence of Coacervates
4 Notes
References
Chapter 11: Biochemical and Biophysical Characterization of Tau and α-Linolenic Acid Vesicles In Vitro
1 Introduction
2 Materials
3 Methods
3.1 Preparation of ALA Vesicles
3.2 Aggregation of Tau in the Presence of ALA
3.3 ALA and Tau Characterized by SDS-PAGE and TEM
3.4 Conformation of Tau and ALA by CD Spectroscopy
4 Notes
References
Chapter 12: Detection of Small-Molecule Interactions with Fibrillar Tau Protein Aggregates Using Microscale Thermophoresis
1 Introduction
2 Materials
3 Methods
3.1 Tau Fibrils Preparation
3.2 AFM Imaging of Tau Fibrils
3.3 Thioflavin S (ThS) Fluorescent Spectroscopy
3.4 Removal of Free DTT and Buffer Exchange
3.5 Tau Fibrils Labelling
3.6 Homogenization of Tau Fibrils
3.7 Preparation of Small Molecular Binding Partners
3.7.1 Thiazine Red
3.7.2 Lansoprazole
3.7.3 Compound 582407
3.7.4 PBB3
3.7.5 T807
3.8 MST Measurement of Small Molecule-Tau Fibril Molecular Systems
3.9 MST Data Analysis
4 Notes
References
Part III: Tau Post-translational Modifications
Chapter 13: Quantification of Methylation and Phosphorylation Stoichiometry
1 Introduction
2 Materials
2.1 Phospho-Tau Expression and Purification
2.2 Phosphorylation Stoichiometry
2.3 Tau Methylation Stoichiometry
3 Methods
3.1 Phospho-Tau Expression, Purification, and Estimation of Stoichiometry
3.2 Phosphorylation Stoichiometry
3.3 Tau Methylation Stoichiometry
4 Notes
References
Chapter 14: The O-GlcNAc Modification of Recombinant Tau Protein and Characterization of the O-GlcNAc Pattern for Functional S...
1 Introduction
2 Materials
2.1 Production of Recombinant 15N- and 15N,13C-Labeled Tau
2.2 Purification of Recombinant 15N- and 15N,13C-Labeled Tau
2.3 Production of Recombinant OGT
2.4 Purification of Recombinant OGT
2.5 O-GlcNAcylation Assay with Recombinant ncOGT
2.6 O-GlcNAcylation of Tau Protein with Recombinant ncOGT
2.7 Characterization of the O-GlcNAc Pattern of Tau Protein
3 Methods
3.1 Production of Recombinant 15N- and 15N, 13C-Labeled Tau
3.1.1 Day 1: Bacterial Transformation
3.1.2 Day 2: Bacterial Growth
3.1.3 Day 3: Induction Phase
3.2 Purification of 15N- and 15N, 13C-Labeled Tau
3.3 Production of Recombinant ncOGT
3.3.1 Day 1: Bacterial Transformation
3.3.2 Day 2: Bacterial Growth
3.3.3 Day 3: Induction Phase
3.4 Purification of Recombinant ncOGT
3.5 Standard Analysis of OGT Activity on a Peptide Substrate
3.6 O-GlcNAcylation of Tau by Recombinant OGT
3.7 Characterization of the O-GlcNAc Pattern of Tau Protein
3.7.1 Determination of the Overall O-GlcNAc Level of Tau by MALDI-TOF MS
3.7.2 Detecting Tau O-GlcNAcylation by Chemoenzymatic Labeling and Click Reaction with TAMRA-Alkyne
3.7.3 Determination of the O-GlcNAc Distribution by Chemoenzymatic Labeling
3.7.4 Characterization of the Site-Specific, Quantitative O-GlcNAcylation Pattern by NMR: Assignment of Resonances of the O-Gl...
4 Notes
References
Chapter 15: Phosphorylation of Tau Protein by CDK2/cyclin A and GSK3β Recombinant Kinases: Analysis of Phosphorylation Pattern...
1 Introduction
2 Materials
2.1 Production of Recombinant 15N- and 15N,13C-Labeled Tau
2.2 Purification of Recombinant 15N- and 15N, 13C-Labeled Tau
2.3 Production of Recombinant GST-CDK2/cyclin A
2.4 Purification of Recombinant GST-CDK2/cyclin A
2.5 Standard Analysis of CDK2/cyclin A Activity on a Peptide Substrate
2.6 Production of Recombinant GSK3β-His6
2.7 Purification of Recombinant GSK3β-His6
2.8 Standard Analysis of GSK3β Activity on a Primed Peptide Substrate
2.9 Phosphorylation of Tau by CDK2/cyclin A (Tau-P)
2.10 Phosphorylation of Tau-P or Tau by GSK3β
2.11 NMR Spectroscopy of Phosphorylated Tau
3 Methods
3.1 Production of Recombinant 15N- and 15N, 13C-Labeled Tau
3.1.1 Day 1: Bacterial Transformation
3.1.2 Day 2: Bacterial Growth
3.1.3 Day 3: Induction Phase
3.2 Purification of 15N- and 15N, 13C-Labeled Tau
3.3 Production of Recombinant GST-CDK2/cyclin A
3.3.1 Day 1: Bacterial Transformation
3.3.2 Day 2: Bacterial Growth
3.3.3 Day 3: Induction Phase
3.4 Purification of the Recombinant GST-CDK2/cyclin A Complex
3.5 Standard Analysis of CDK2/cyclin A Activity on a Peptide Substrate
3.6 Production of Recombinant GSK3β-His6
3.6.1 Day 1: Bacterial Transformation
3.6.2 Day 2: Bacterial Growth
3.6.3 Day 3: Induction Phase
3.7 Purification of Recombinant GSK3β-His6
3.8 Standard Analysis of GSK3β Activity on a Primed Peptide Substrate
3.9 Phosphorylation of Tau by Recombinant CDK2/cyclin A (Tau-P)
3.10 Phosphorylation of Tau-P by Recombinant GSK3β (Tau-PP)
3.11 NMR Spectroscopy: Assignment of Resonances of the Phosphorylated Residues and Identification of the Phosphorylation Patte...
4 Notes
References
Part IV: Analytical Methods to Detect Tau Proteins, Mutations, and Regulatory Molecules Associated to Tau Pathology
Chapter 16: Western Blot of Tau Protein from Mouse Brains Extracts: How to Avoid Signal Artifacts
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 17: Methods for Biochemical Isolation of Insoluble Tau in Rodent Models of Tauopathies
1 Introduction
1.1 Sarkosyl-Based Extraction Methods
1.2 Formic-Acid Based Extraction Methods
1.3 Choosing Between Sarkosyl and Formic Acid Extraction Method
2 Materials
2.1 Insoluble Tau Isolation with 1% Sarkosyl by Greenberg and Davies
2.2 Insoluble Tau Isolation with 1% Sarkosyl by Sahara et al.
2.3 Insoluble Tau Isolation with 1% Sarkosyl by Eckermann et al.
2.4 Insoluble Tau Isolation with 1% Sarkosyl by Planel et al.
2.5 Insoluble Tau Isolation with Formic Acid by Ishihara et al.
3 Methods
3.1 Insoluble Tau Isolation with 1% Sarkosyl by Greenberg and Davies (Fig. 1)
3.2 Insoluble Tau Isolation with 1% Sarkosyl by Sahara et al. (Fig. 2)
3.3 Insoluble Tau Isolation with 1% Sarkosyl by Eckermann et al. (Fig. 3)
3.4 Insoluble Tau Isolation with 1% Sarkosyl by Planel et al. (Fig. 4)
3.5 Insoluble Tau Isolation with Formic Acid by Ishihara et al. (Fig. 5)
4 Notes
References
Chapter 18: A Method to Collect Cerebrospinal Fluid from Mouse Cisterna Magna to Determine Extracellular Tau Levels
1 Introduction
2 Materials
3 Methods
3.1 Preparation of Tubing and a Roller Pump for CSF Collection
3.2 Muscle Dissection
3.3 CSF Collection
4 Notes
References
Chapter 19: Detection of Glymphatic Outflow of Tau from Brain to Cerebrospinal Fluid in Mice
1 Introduction
2 Materials
2.1 Proteins, Chemicals, and Solutions
2.2 The Equipment and Accessories for Stereotaxic Surgery
3 Methods
3.1 Stereotaxic Surgery for Injecting Labeled Recombinant Tau
3.2 Analysis of Remaining Tau in the Brain and Measurements of the Levels of Tau Effluxed into CSF
4 Notes
References
Chapter 20: Differential Regulation of Neurotrophic Factors During Pathogenic Tau-Aggregation in a Tau Transgenic Mouse Model ...
1 Introduction
1.1 Expression Pattern of BDNF and NT-3 and Their Receptors TrkB and TrkC
1.2 Cortical and Hippocampal Loss of BDNF in Tau Transgenic Mice
1.3 Increase in the Number of NGF-Positive Neurons in Hippocampus and Cortex of THY-Tau22 Mice
2 Materials
2.1 Transgenic Mice
2.2 Solutions, Reagents, and Material for Probe Generation (Synthesis of cDNA, PCR, Ligation and In Vitro Transcription)
2.3 Solutions, Buffers, and Material for ISH
2.4 Solutions, Buffer, and Materials for Tissue Preparation, IHC, Immunofluorescence (IF) and Western-Blot (WB)
3 Methods
3.1 Tissue Preparation for Histology
3.2 Generation of cDNA Templates for Riboprobes
3.3 Synthesis of DIG-Labeled Riboprobes for In Situ Hybridization
3.4 ISH
3.5 Double-Labeling Protocol for ISH and IHC
3.6 Immunofluorescence (IF)
3.7 Semiquantitative Estimation of Cell Density and Pixel Density
3.8 Western Blot Analysis
3.9 Data Analysis
4 Notes
References
Chapter 21: Intranasal Nose-to-Brain Drug Delivery via the Olfactory Region in Mice: Two In-Depth Protocols for Region-Specifi...
1 Introduction
1.1 Region-Specific Intranasal Nose-to-Brain Delivery in Mice
1.2 Brain Distribution of Antibodies After Intranasal Delivery via the Olfactory Region
1.3 Adjustment of a Colorimetric ISH Protocol to Analyze Expression in the Nasal Mucosa
1.4 Pretreatment Enhances the ISH Signals in Mucosa
1.5 Expression of Fc Receptors in the Nasal Mucosa and Their Role in IgG Transport
2 Materials
2.1 Animals
2.2 Solutions, Reagents, and Materials for Region-Specific Intranasal Administration and for Immunofluorescence
2.3 Solutions, Reagents, and Materials for Tissue Preparation and Immunofluorescence
2.4 Solutions, Reagents, and Materials for Probe Generation (Synthesis of cDNA, PCR, Ligation, and In Vitro Transcription)
2.5 Solutions, Buffers, and Materials for ISH
3 Methods
3.1 Intranasal Administration at the Olfactory Region in Mice
3.2 Tissue Preparation for Immunofluorescence and ISH
3.3 Immunofluorescence to Determine the Distribution of the Intranasally Delivered Antibody
3.4 Generation of cDNA Templates for ISH Riboprobes
3.5 Synthesis of DIG-Labeled Riboprobes for In Situ Hybridization
3.6 Optional Pretreatment for Amplified ISH Signals
3.7 ISH
4 Notes
References
Chapter 22: Detecting and Validating MAPT Mutations in Neurodegeneration Patients and Analysis of Exon Splicing Consequences
1 Introduction
1.1 Chapter Overview
1.2 Considerations for Next-Generation Sequencing Analysis of MAPT
2 Materials
2.1 PCR
2.2 PCR Purification
2.3 Sanger Sequencing
2.4 Exon-Trapping Construct
2.5 Exon-Trapping Assay
3 Methods
3.1 Polymerase Chain Reaction 1(PCR) Amplification of MAPT Exons
3.2 Purification of PCR Amplicons
3.3 Preparation of MAPT Amplicons for Sanger Sequencing
3.4 Strategy for Sequence Analysis of Electropherograms
3.5 Exon Trapping: Construction of pSPL3 Constructs
3.6 Exon-Trapping Assay
4 Notes
References
Part V: Cellular and In Vivo Models of Tau Physiopathology
Chapter 23: Measuring Antibody-Mediated Tau Fibril Uptake in Microglia by Flow Cytometry
1 Introduction
2 Materials
2.1 Tau Protein Fibrillization and Fluorescent Labeling
2.2 BV2 Cell Culture
2.3 Flow Cytometry Assay
3 Methods
3.1 Tau Fibrillization and Fluorescent Labeling
3.2 Dialyzing and Quenching Unconjugated AlexaFluor Dye
3.3 BV2 Cell Culture and Recombinant Tau Uptake Assay
3.4 Analysis by Flow Cytometry
4 Notes
References
Chapter 24: Super-Resolution Imaging of Tau Proteins in Isolated and Immobilized Brain Synaptosomes
1 Introduction
2 Materials
2.1 Synaptosome Preparation
2.2 Synaptosome Immobilization
2.3 Immunofluorescence Imaging
2.4 dSTORM Imaging
3 Methods
3.1 Synaptosome Isolation
3.2 Synaptosome Immobilization
3.3 Immunofluorescence Imaging
3.4 dSTORM Imaging
4 Notes
References
Chapter 25: Purinergic Receptor P2Y12-Mediated Tau Internalization in Microglia
1 Introduction
2 Materials
2.1 Solutions and Reagents
2.1.1 Tau Species Preparation and Labeling
2.1.2 SDS Gel Electrophoresis
2.1.3 Thioflavin-S and ANS Assay and Negative Staining
2.1.4 Cell Culture
2.1.5 Immunofluorescence Assay
2.2 Instruments
2.3 Software
3 Methods
3.1 Tau Species Preparation
3.2 Characterization of Tau Species by Various Biochemical Approaches
3.3 Culturing of N9 Microglial Cells
3.4 Immunofluorescence Assay
3.5 Imaging of Cells by Fluorescence Microscopy
4 Notes
References
Chapter 26: α-Linolenic Acid Induces Microglial Activation and Extracellular Tau Internalization
1 Introduction
2 Materials
3 Methods
3.1 Internalization of Extracellular Tau in Microglia Cells
3.2 Immunofluorescence and Western Blot Analysis of Microglia Activation in the Presence of ALA and Tau Species Through Iba-1 ...
4 Notes
References
Chapter 27: Identification of Tau Toxicity Modifiers in the Drosophila Eye
1 Introduction
2 Materials
3 Methods
3.1 UAS/Gal4-Mediated Expression of Human Tau in the Drosophila Eye
3.2 Assessment of Human Tau Expression in the Drosophila Eye by Western Blot
3.3 Assessment of Human Tau Toxicity by Eye Size Measurement
3.4 Genetic Screening of Tau Toxicity Modifier
4 Notes
References
Chapter 28: Tracking Tau in Neurons: How to Transfect and Track Exogenous Tau in Primary Neurons
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Plasmids
2.3 Transfection Reagents
3 Methods
3.1 Preparation of Primary Neurons for Transfection
3.2 Preparation of Transfection Mix
3.3 Transfection and Washing Procedure
3.4 Optional: Increasing Expression Efficiency
4 Notes
References
Chapter 29: Tracking Tau in Neurons: How to Grow, Fix, and Stain Primary Neurons for the Investigation of Tau in All Developme...
1 Introduction
2 Materials
2.1 Animals
2.2 Cell Culture Media and Reagents for Cultivation of Forebrain Neurons from Embryonic Mice (E13.5)
2.3 Cell Culture Media for Cultivation of Hippocampal and Cortical Neurons from Postnatal Mice (P0-P3)
2.4 Fixation Solution for Endogenous Tau Trafficking in Primary Neurons
2.5 Blocking and Staining Solutions for Endogenous Tau Trafficking in Primary Neurons
3 Methods
3.1 Cultivation of Forebrain Neurons from Embryonic Mice (E13.5)
3.1.1 Coating of Cell Culture Plates for Neurons
3.1.2 Dissection of Embryonic Forebrain
3.1.3 Cell Isolation and Plating
3.1.4 Neuronal Maintenance
3.2 Cultivation of Hippocampal and Cortical Neurons from Postnatal Mice (P0-P3)
3.2.1 Coating of Coverslips for Neurons
3.2.2 Dissection of Hippocampi/Cortices
3.2.3 Cell Isolation and Plating
3.2.4 Neuronal Maintenance
3.3 Fixation and Staining of Primary Neurons for Endogenous Tau Trafficking
4 Notes
References
Chapter 30: Differentiating SH-SY5Y Cells into Polarized Human Neurons for Studying Endogenous and Exogenous Tau Trafficking: ...
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Cell Culture Medium
2.3 Reagents
3 Methods
3.1 Cultivating, Passaging, and Maintaining SH-SY5Y Cultures
3.2 Seeding of Undifferentiated SH-SY5Y Cells for Differentiation Protocols
3.3 Differentiation with RA/BDNF
3.4 Differentiation with RA/TPA
3.5 Differentiation with RA
3.6 Differentiation with TPA
4 Notes
References
Chapter 31: Cultivation, Differentiation, and Lentiviral Transduction of Human-Induced Pluripotent Stem Cell (hiPSC)-Derived G...
1 Introduction
2 Materials
2.1 Cell Lines
2.2 hiPSC Maintenance Reagents
2.3 hiPSC Differentiation Reagents
2.4 Primary Glial Cell Culture Reagents
2.5 HEK293T Maintenance Reagents
2.6 Transduction Reagents
2.7 Medium Recipes
3 Methods
3.1 Cultivation of hiPSCs
3.1.1 Coating of Culture Plates
3.1.2 Thawing of hiPSCs
3.1.3 Routine Cultivation of hiPSCs
3.1.4 Freezing of hiPSCs
3.2 Doxycycline-Induced Differentiation of hiPSCs into Cortical Glutamatergic Neurons
3.2.1 Pre-differentiation
3.2.2 Coating of Culture Plates for hiPSC Differentiation
3.2.3 Differentiation of hiPSCs into Cortical Glutamatergic Neurons
3.2.4 Co-cultivation with Primary Mouse Glial Cells
Isolation of Glial Cells from Embryonic Mouse Brain
Maintenance and Freezing of Glial Cells
Seeding of Primary Mouse Glial Cells to hiPSC-Derived Neurons
3.3 Lentiviral Transduction of hiPSC-Derived Neurons
3.3.1 HEK293T Maintenance
3.3.2 Virus Particle Production
3.3.3 Transduction of hiPSC-Derived Neurons
4 Notes
References
Chapter 32: Optimized Calcium-Phosphate-Based Co-transfection of Tau and tdTomato into Human iPSC-Derived Neurons for the Stud...
1 Introduction
2 Materials
2.1 Equipment
2.2 Buffers and Reagents
3 Methods
3.1 Day 0: Prepare Cultured Cells for Transfection
3.2 Day 10 After Differentiation: Transfection
4 Notes
References
Chapter 33: Studying Microtubule Dynamics in Human Neurons: Two-Dimensional Microtubule Tracing and Kymographs in iPSC- and SH...
1 Introduction
2 Materials
2.1 Cell Lines
2.2 SH-SY5Y Cell Differentiation Reagents
2.3 HiPSC Differentiation Reagents
2.4 Transfection Reagents
2.5 Coating
2.6 Medium Recipes
2.7 Microscope and Imaging Software
3 Methods
3.1 Cultivation
3.1.1 Cultivation of SH-SY5Y-Derived Neurons
Coating
Seeding of SH-SY5Y Cells for Differentiation
3.1.2 Cultivation of iPSC-Derived Neurons
Coating
Seeding of iPSCs for Differentiation (See Note 4)
3.2 Transfection
3.2.1 Transfection of SH-SY5Y-Derived Neurons
3.2.2 Transfection of iPSC-Derived Neurons
3.3 Live-Cell Imaging
3.4 Image Analysis
3.4.1 MT Dynamics in Somata
3.4.2 MT Dynamics in Neurites
4 Notes
References
Chapter 34: A Brain Ischemia-Reperfusion Model for the Study of Tau Phosphorylation and O-GlcNAcylation
1 Introduction
2 Materials
2.1 Animals
2.2 Behavioral Assessment
2.3 Measurement of Physiological Parameters
2.4 Surgical Procedures
2.4.1 Vessel Occluders and Anesthesia
2.4.2 Drugs
2.4.3 Materials for MACo Occlusion and Equipment
2.5 Perfusion of the Rat and Brain Dissection
2.6 Slice Preparation
2.7 Immunostaining
2.8 Tissue Processing for Western Blot
2.9 SDS Polyacrylamide Gel and Immunoblotting
2.9.1 Materials and Equipment
2.9.2 Solutions
3 Methods
3.1 Behavioral Assessment (See Note 2)
3.1.1 Garcia´s Neurological Test Modified by Shimamura (See Note 3)
3.1.2 Cylinder Test
3.1.3 Adhesive Removal Test (See Note 4)
3.1.4 Open Field Test
3.2 Preparation of Animals for Surgical Procedures
3.3 Measurement of Physiological Parameters (See Note 6)
3.4 Middle Cerebral Artery Occlusion (MCAo) Surgical Procedure
3.5 Harvesting the Rat Brain
3.5.1 For Immunofluorescence
3.5.2 For Western Blot
3.6 Processing of Brain Sections for Immunostaining
3.7 Preparation of Brain Lysate
3.8 Performing Gel Electrophoresis
3.9 Western Blotting
4 Notes
References
Index