Experimental Models of Parkinson’s Disease (Methods in Molecular Biology, 2322)

Preface
Contents
Contributors
Part I: Biochemical Experiments and Cellular Models of Parkinson´s Disease
	Chapter 1: α-Synuclein Seeding Assay Using RT-QuIC
		1 Introduction
		2 Materials (See Note 1)
		3 Methods
			3.1 α-Synuclein Purification from Bacteria
			3.2 Measurement of the Aggregation Using αSyn RT-QuIC Assay
			3.3 Preparation of αSyn Substrate for RT-QuIC
			3.4 Preparation of αSyn Reactions
			3.5 RT-QuIC Data Analysis: Parameters of Kinetic Change (Fig. 1, See Note 9)
			3.6 RT-QuIC Data Analysis for Sensitivity and Specificity (See Note 11)
		4 Notes
		References
	Chapter 2: Electron Microscopic Analysis of α-Synuclein Fibrils
		1 Introduction
		2 Materials
			2.1 Negative Staining
			2.2 Immunolabeling
		3 Methods
			3.1 Negative Staining of α-Syn Fibrils
			3.2 Immuno-EM of α-Syn Fibrils
		4 Notes
		References
	Chapter 3: α-Synuclein Seeding Assay Using Cultured Cells
		1 Introduction
		2 Materials
			2.1 α-Synuclein Purification from Bacteria
			2.2 α-Synuclein Seed Preparation
			2.3 Cell Culture and Transfection
			2.4 Biochemical Fractionation of α-Synuclein
			2.5 Western Blot Analysis
		3 Methods
			3.1 α-Synuclein Purification from Bacteria
			3.2 α-Synuclein Seed Preparation
			3.3 Introduction of α-Synuclein Seeds into Cultured Cells
			3.4 Sequential Extraction of α-Synuclein from Cultured Cells
			3.5 Western Blot to Monitor α-Synuclein Inclusion Formation
		4 Notes
		References
	Chapter 4: Analysis of α-Synuclein in Exosomes
		1 Introduction
		2 Materials
			2.1 Cell Culture
			2.2 Serum Preparation
			2.3 Exosome Isolation
			2.4 α-Synuclein ELISA
		3 Methods
			3.1 Preparation of Cell Culture Media
			3.2 Preparation of Serum
			3.3 Exosome Isolation
			3.4 α-Synuclein ELISA
		4 Notes
		References
	Chapter 5: Measurement of GCase Activity in Cultured Cells
		1 Introduction
		2 Materials
			2.1 Cell Culture
			2.2 Sample Preparation and GCase Assay
		3 Methods
			3.1 Cell Culture
			3.2 Sample Preparation and GCase Assay
		4 Notes
		References
	Chapter 6: Detection of Substrate Phosphorylation of LRRK2 in Tissues and Cultured Cells
		1 Introduction
		2 Materials
			2.1 Tissue Preparation and Cell Lysis
			2.2 SDS-PAGE Electrophoresis
			2.3 Immunoblotting
		3 Methods
			3.1 Sample Preparation: Cultured Cells
			3.2 Sample Preparation: Rodent Tissues
			3.3 Sample Preparation for SDS-PAGE
			3.4 Casting  Gels
			3.5 Running Gels and Electrotransfer to Membranes
			3.6 Immunoblotting
		4 Notes
		References
	Chapter 7: Two Methods to Analyze LRRK2 Functions Under Lysosomal Stress: The Measurements of Cathepsin Release and Lysosomal ...
		1 Introduction
		2 Materials
			2.1 Cell Culture and Drug Treatment
			2.2 SDS-PAGE and Immunoblotting
			2.3 Immunocytochemistry
			2.4 Antibodies
		3 Methods
			3.1 Cell Culture and CQ Treatment
			3.2 SDS-PAGE and Immunoblot Analysis of Cathepsin Secretion
			3.3 Immunocytochemical Analysis of Lysosomal Enlargement and LRRK2 Recruitment
		4 Notes
		References
	Chapter 8: Differentiation of Midbrain Dopaminergic Neurons from Human iPS Cells
		1 Introduction
		2 Materials
			2.1 Induction of Midbrain Dopaminergic Precursors
			2.2 Induction of Midbrain Dopaminergic Neurons
		3 Methods
			3.1 Differentiation of Ventral Midbrain-Specific Neurospheres from Human iPS Cells
			3.2 Differentiation into Midbrain Dopaminergic Neurons from Neurospheres
		4 Notes
		References
	Chapter 9: Monitoring PINK1-Parkin Signaling Using Dopaminergic Neurons from iPS Cells
		1 Introduction
		2 Materials
			2.1 Preparation of GFP-Parkin Lentivirus
			2.2 Lentiviral Infection of GFP-Parkin in Dopaminergic Neuron Culture
			2.3 PINK1-Parkin Activation by Mitochondrial Uncouplers
			2.4 Immunostaining and Imaging of Dopaminergic Neuron Culture
			2.5 Detection of PINK1, Parkin, and Phosphorylated Ubiquitin by Western  Blot
		3 Methods
			3.1 Preparation of GFP-Parkin Lentivirus
			3.2 Lentiviral Infection of GFP-Parkin in Dopaminergic Neuron Culture
			3.3 PINK1-Parkin Activation by Mitochondrial Uncouplers
			3.4 Immunostaining and Imaging of Dopaminergic Neuron Culture
			3.5 Detection of PINK1, Parkin, and Phosphorylated Ubiquitin by Western  Blot
		4 Notes
		References
Part II: Mammalian Models of Parkinson´s Disease
	Chapter 10: Generation of Mitochondrial Toxin Rodent Models of Parkinson´s Disease Using 6-OHDA, MPTP, and Rotenone
		1 Introduction
		2 Materials
			2.1 6-OHDA Injection
			2.2 Immunohistochemistry for Tyrosine Hydroxylase (TH)
			2.3 MPTP Administration
			2.4 Rotenone
		3 Methods
			3.1 6-OHDA Injection
			3.2 Immunohistochemistry for TH
			3.3 MPTP Administration
			3.4 Monitoring of Mice After MPTP Administration
			3.5 Histochemical Evaluation of Degenerated Dopaminergic Neurons by MPTP
			3.6 Rotenone
			3.7 Histochemical Evaluation of Degenerated Dopaminergic Neurons by Rotenone
		4 Notes
		References
	Chapter 11: Midbrain Slice Culture as an Ex Vivo Analysis Platform for Parkinson´s Disease
		1 Introduction
		2 Materials
			2.1 Apparatus for Slice Culture
			2.2 Reagents for Slice Culture
			2.3 Reagents for Immunostaining
		3 Methods
			3.1 Midbrain Slice Culture
			3.2 Immunohistochemical Staining
		4 Notes
		References
	Chapter 12: α-Synuclein Propagation Mouse Models of Parkinson´s Disease
		1 Introduction
		2 Materials
			2.1 Generation and Purification of Recombinant α-Syn Monomer
			2.2 Generation and Preparation of α-Syn Preformed Fibrils
			2.3 Stereotaxic Injections
			2.4 Inoculation of α-Syn PFFs into the Mouse Gastric  Wall
		3 Methods
			3.1 Generation and Purification of Recombinant α-Syn Monomer
			3.2 Generation and Preparation of α-Syn Preformed Fibrils
			3.3 Stereotaxic Injections of α-Syn PFFs into the Mouse Striatum and Olfactory  Bulb
			3.4 Injections of α-Syn PFFs into the Mouse Gastric  Wall
		4 Notes
		References
	Chapter 13: Common Marmoset Model of α-Synuclein Propagation
		1 Introduction
		2 Materials
			2.1 Equipment and Surgical Instruments
			2.2 Drugs
			2.3 Intracerebral Injection
			2.4 Tissue Collection
		3 Methods
			3.1 Animal Husbandry
			3.2 Preparation of the Injection
			3.3 Surgical Procedures
			3.4 Tissue Collection and Immunohistochemistry
		4 Notes
		References
	Chapter 14: Application of a Tissue Clearing Method for the Analysis of Dopaminergic Axonal Projections
		1 Introduction
		2 Materials
			2.1 Fixation
			2.2 Brain Slice Preparation
			2.3 ChemScale
			2.4 AbScale
			2.5 Brain Slice Mounting and CLSM Imaging
		3 Methods
			3.1 Fixation
			3.2 Brain Slice Preparation
			3.3 ChemScale Labeling
			3.4 AbScale Labeling
			3.5 Brain Slice Mounting and CLSM Imaging
		4 Notes
		References
	Chapter 15: Deep Brain Stimulation Using Animal Models of Parkinson´s Disease
		1 Introduction
		2 Materials
			2.1 Experimental Animal
			2.2 Surgery
			2.3 Mapping the Striatum, STN, and Globus Pallidus Interna (GPi)
			2.4 Preparation of Quadripolar Stimulation Electrodes
			2.5 Recording Experiment
			2.6 Preparation of Recording-Injection System
			2.7 High-Frequency Stimulation (HFS) of the STN or GPi
			2.8 Recording-Injection Experiments
		3 Methods
			3.1 Experimental Animals
			3.2 Surgery
			3.3 Mapping the Striatum, STN, and GPi
			3.4 Recording Preparation
			3.5 HFS of the STN or GPi
			3.6 Extracellular Neuronal Recording of the Striatum
			3.7 Recording-Injection Experiments
			3.8 Data Analysis
		4 Notes
		References
Part III: Invertebrate Models of Parkinson´s Disease
	Chapter 16: Assessment of Cytotoxicity of α-Synuclein in Budding Yeast Using a Spot Growth Assay and Fluorescent Microscopy
		1 Introduction
		2 Materials
			2.1 Budding Yeast Strains and Plasmids
			2.2 Cell Culture and Yeast Growth Media (Table 1, See Note 3)
			2.3 Transformation
			2.4 Spot Growth Assay
			2.5 Microscopy
			2.6 Digital Image Analysis
		3 Methods
			3.1 Transformation of Yeast with Plasmids for the Expression of α-Synuclein
			3.2 Spot Growth Assay to Assess the Toxicity of α-Synuclein
			3.3 Imaging of α-Synuclein-GFP in Yeast Cells
		4 Notes
		References
	Chapter 17: The Functional Assessment of LRRK2 in Caenorhabditis elegans Mechanosensory Neurons
		1 Introduction
		2 Materials
			2.1 C. elegans Strains
			2.2 Worm Culturing
			2.3 Equipment
			2.4 Reagents and Other Materials
		3 Methods
			3.1 Generation of C. elegans Strains Carrying both lrk-1 Gene Mutation and Pmec-7::gfp Transgene
			3.2 Assessment of Axon Overextension of ALM Mechanosensory Neurons
		4 Notes
		References
	Chapter 18: Analysis of Dopaminergic Functions in Drosophila
		1 Introduction
		2 Materials
			2.1 Visualization of Mitochondria in DA Neurons
			2.2 Imaging of Synaptic Release of DA Neurons
		3 Methods
			3.1 Visualization of Mitochondria in DA Neurons
			3.2 Imaging of Synaptic Release of DA Neurons
		4 Notes
		References
	Chapter 19: Evaluation of Mitochondrial Function and Morphology in Drosophila
		1 Introduction
		2 Materials
			2.1 Fly Wing Posture and Activity Assays
			2.2 ATP Level Measurement of Fly Thoracic Muscle Tissue
			2.3 Fly Mitochondria Purification
			2.4 ROS Assay for Purified Mitochondrial Sample
			2.5 MitoSox, TMRM, Rhod2, JC-1, and MitoPOP Staining in Fly Muscle
			2.6 Blue Native Gel (BNG) Analysis of Respiratory Complex Assembly
			2.7 Immunostaining of Mitochondria in Fly Neuromuscular Tissues
		3 Methods
			3.1 Wing Posture Analysis
			3.2 Jump/Flight Activity Analyses
			3.3 ATP Level Measurement of Fly Thoracic Muscle Tissue
			3.4 Fly Mitochondria Purification
			3.5 ROS Assay for Purified Mitochondrial Sample
			3.6 MitoSox, TMRM, Rhod2, JC-1, and MitoPOP Staining in Fly Muscle
			3.7 Blue Native Gel (BNG) Analysis of Respiratory Complex Assembly
			3.8 Immunostaining of Mitochondria in Fly Muscular Tissues (Fig. 1)
			3.9 Immunostaining of Mitochondria in Dopaminergic Neurons (Fig. 2)
		4 Notes
		References
	Chapter 20: Cytosolic and Mitochondrial Ca2+ Imaging in Drosophila Dopaminergic Neurons
		1 Introduction
		2 Materials
			2.1 Setting Up Imaging Tools
		3 Methods
			3.1 Ca2+ Imaging
		4 Notes
		References
Index