Parkinson's Disease: Molecular and Therapeutic Insights From Model Systems

1

Brief Table of Contents

Copyright

Brief Table of Contents

Table of Contents

List of Figures

List of Tables

List of Contributors

Preface

Foreword

I. Clinical Overview

Chapter 1. Clinical Aspects of Parkinson Disease

Chapter 2. Genetics of Parkinson's Disease

Chapter 3. Neuropathology of Parkinson's Disease[1]

II. Non-Human Primate Models

Chapter 4. Introduction to: Neurotoxin-based Nonhuman Primate Models of Parkinson's Disease

Chapter 5. Systems Level Physiology of the Basal Ganglia, and Pathophysiology of Parkinson's Disease

Chapter 6. Neuroprotection for Parkinson's Disease

Chapter 7. The use of aged monkeys to study pd: important roles in pathogenesis and experimental therapeutics

Chapter 8. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Mammalian Models of Parkinson's Disease

Chapter 9. Non-human Primate Models of Parkinson's Disease and Experimental Therapeutics

III. Rodent Toxin Models

Chapter 10. Rodent Toxin Models of PD

Chapter 11. The MPTP Mouse Model of Parkinson's Disease: the True, the False, and the Unknown

Chapter 12. Acute and chronic administration of 1-methyl-4-phenylpyridinium

Chapter 13. Endogenous Defenses that Protect Dopamine Neurons

Chapter 14. Complex I Inhibition, Rotenone and Parkinson's Disease

Chapter 15. Paraquat-induced Neurodegeneration: a Model of Parkinson's Disease Risk Factors

IV. Rodent And Other Vertebrate Genetic Models

Chapter 16. Overview: rodent and fish models of parkinson's disease

Chapter 17. Genetic Models of Familial Parkinson's Disease

Chapter 18. The Dynamics of α-Synuclein at the Nerve Terminal

Chapter 19. The Bac Transgenic Approach to Study Parkinson's Disease in Mice

Chapter 20. Viral Vectors

Chapter 21. Environmental Explorations of Parkinson's Disease Using Rodent Genetic Models

Chapter 22. α-Synuclein, CSPα, SNAREs and Neuroprotection in vivo

Chapter 23. Insights from Zebrafish PD Models and Their Potentials for Identifying Novel Drug Targets and Therapeutic Compounds

V. Multicellular Invertebrate Models

Chapter 24. Parkinson's Disease

Chapter 25. Drosophila Models for Parkinson's Disease Research

Chapter 26. Caenorhabditis Elegans Models of Parkinson's Disease

Chapter 27. C. Elegans Genetic Strategies to Identify Novel Parkinson's Disease-associated Therapeutic Targets and Leads

VI. Cell-Based Models

Chapter 28. Overview: cell-based models of parkinson's disease

Chapter 29. PC12 Cells as a model for parkinson's disease research

Chapter 30. Dissociated mesencephalic cultures

Chapter 31. Selective Autophagy in the Pathogenesis of Parkinson's Disease

Chapter 32. The Role of LRRK2 Kinase Activity in Cellular PD Models

Chapter 33. Using yeast as a model system for the genetic dissection of α-synuclein toxicity

Chapter 34. The role of the foxa2 gene in the birth and death of dopamine neurons

Chapter 35. Embryonic stem cell-based models of parkinson's disease

Chapter 36. Neuroprotective and Neurotoxic Properties of α-Synuclein in Cell Culture Models of Dopaminergic Degeneration

Chapter 37. Postnatally Derived Ventral Midbrain Dopamine Neuron Cultures as a Model System for Studying Neurotoxicity and Parkinson's Disease

Chapter 38. Yeast Cells as a Discovery Platform for Parkinson's Disease and other Protein Misfolding Diseases

VII. Cell-Free Models

Chapter 39. Overview of cell-free systems for the study of parkinson's disease

Chapter 40. Microglial activation in a mouse model of α-synuclein overexpression

Chapter 41. Purification and Quantification of Neural α-synuclein

Chapter 42. Protein Folding and Aggregation in in vitro Models of Parkinson's Disease

Chapter 43. The use of cell-free systems to characterize parkinson's disease-related gene products

Table of Contents

Copyright

Brief Table of Contents

Table of Contents

List of Figures

List of Tables

List of Contributors

Preface

Foreword

I. Clinical Overview

Chapter 1. Clinical Aspects of Parkinson Disease

Clinical Features of Parkinson Disease

Diagnosis and Differential Diagnosis

Treatment

Uncited References

Chapter 2. Genetics of Parkinson's Disease

Genetics of Familial Parkinson's Disease

α-Synuclein (PARK1/ 4)

Parkin (PARK2)

Ubiquitin Carboxyl-Terminal Hydrolase L1 (PARK5)

PTEN-Induced Kinase 1 (PARK6)

Oncogene DJ-1 (PARK7)

Leucine-Rich Repeat Kinase 2 (PARK8)

ATPase Type 13A2 (PARK9)

OMI/HTRA Serine Peptidase 2 (PARK13)

Parkinsonism-plus genes

Microtubule-Associated Protein Tau

Progranulin

GTP Cyclohydrolase I

Na+/K+-ATPase α3 Subunit

Sporadic PD, Genetic Associations and Familial Genes

Perspectives

Acknowledgements

Uncited References

Chapter 3. Neuropathology of Parkinson's Disease[1]

Introduction

Gross and Microscopic Findings in PD

Neuropathologic Staging of PD

Molecular Biology of LBs and Neuronal Loss in PD

LBs in Other Disorders

Incidental LBs

Pure Autonomic Failure

Dementia with LBs

Dementia in PD

Relevance of Neuropathology to PD Models

II. Non-Human Primate Models

Chapter 4. Introduction to: Neurotoxin-based Nonhuman Primate Models of Parkinson's Disease

Conclusions

Acknowledgements

Uncited References

Chapter 5. Systems Level Physiology of the Basal Ganglia, and Pathophysiology of Parkinson's Disease

Introduction

Circuit Anatomy of the Basal Ganglia

General Composition of Basal Ganglia Circuits

Cell Types and Basic Neurochemistry

Direct and Indirect Pathways

Functions of Dopamine

Pathophysiology of parkinsonism

Conclusion

Chapter 6. Neuroprotection for Parkinson's Disease

A Chronic (Stepwise) MPTP Primate Model

Time Course of Nigrostriatal Degeneration after Chronic MPTP Treatment

How does that Fit with Human PD?

Additional in vivo and ex vivo Markers

A Clinically Relevant Design that takes Advantage of the Model

Disclosure

Uncited References

Chapter 7. The use of aged monkeys to study pd: important roles in pathogenesis and experimental therapeutics

Age-related Decline of Motor Activity in Primates: Relationship to PD

Age-related Downregulation of Dopaminergic Phenotype in Nigrostriatal System: Relationship to PD

Age-related Increase of α-synuclein: Relationship to PD

Aged Monkeys as a Model of PD: Relationship to Function

So Why has not Aging Processes Become a More Central Factor in PD Studies?

Conclusions

Chapter 8. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Mammalian Models of Parkinson's Disease

Introduction

MPTP Mouse Models of Parkinsonism

Similarities and Differences Between Acute and Sub-acute MPTP Mouse Models

MPTP Monkey Models

Biochemistry of Acute and Chronic Parkinsonism in Monkeys: Similarities and Differences

Striatal Dopaminergic Innervation

Striatal DA D1 and D2 Receptors

Preproenkephalin Gene Expression

Preprotachykinin Gene expression

Nicotinic Acetylcholine Receptors

Microglial Activation

Do Different Models Respond Differently to Anti-parkinsonian Therapies?

Response to Levodopa and Development of Levodopa-Induced Dyskinesias

Conclusions

Uncited References

Chapter 9. Non-human Primate Models of Parkinson's Disease and Experimental Therapeutics

Introduction

Assessment of motor behavior in non-human primate models of PD

Clinical Rating Scales

Automated Behavioral Observation Methods

Data Analysis

Non-motor features in MPTP-lesioned non-human primates

The mptp-lesioned non-human primate model

A brief retrospective of mptp in monkeys

The subtypes of the MPTP-lesion non-human primate model

The adverse effects of MPTP-lesioning

Testing pharmacological therapies for neuroprotective and symptomatic benefit

Dyskinesia and motor complications in non-human primates

Intrinsic neuroplasticity and behavioral recovery

Electrophysiological studies of basal ganglia function in the non-human primate model of PD

Other neurotoxicants in non-human primates

6 Hydroxydopamine

Methamphetamine

Proteasome Inhibition

Conclusion

Acknowledgements

Uncited References

III. Rodent Toxin Models

Chapter 10. Rodent Toxin Models of PD

Introduction

An Overview of the Neurotoxin Models

6-OHDA

MPTP

Rotenone

Paraquat

What are the Strengths of the Neurotoxin Models?

6-OHDA

MPTP

Rotenone

Paraquat

What are the Weaknesses of These Models?

Conclusion

Chapter 11. The MPTP Mouse Model of Parkinson's Disease: the True, the False, and the Unknown

Introduction

Assumption 1: MPTP is Neurotoxic

Assumption 2: Dopamine Transporters Explain MPTP Neurotoxic Specificity

Assumption 3: MPTP Causes a Pure Dopaminergic Neurodegeneration in Mice

Assumption 4: MPTP Mice Depart from PD on Several Important Aspects

Toxin-Related Shortcomings of the MPTP Mouse Model of PD

Mouse-Related Shortcomings of the MPTP Mouse Model of PD

Assumption 5: There is no Effect of Age on MPTP Susceptibility

Assumption 6: Different Mouse Strains Exhibit Different Sensitivities To MPTP

Conclusion

Acknowledgements

Chapter 12. Acute and chronic administration of 1-methyl-4-phenylpyridinium

Introduction

Chronic mpp+ model

Dosing and Selectivity of Damage

Progressive Degeneration

Inclusion Bodies and Abnormal Mitochondria

Variability and Mortality

Further Characterization

Limitations of the Model

Acute intracerebral infusions of mpp+ or malonate as pd models

Acute Intrastriatal or Intranigral Infusions of Metabolic Inhibitors

Intrastriatal MPP+ or Malonate Delivery via Striatal Microdialysis

Intrastriatal Infusions of Metabolic Inhibitors Coupled with Microdialysis in the SN

Advantages and Limitations of Acute Intracerebral Infusions as Models of PD

Conclusions

Chapter 13. Endogenous Defenses that Protect Dopamine Neurons

Endogenous Defenses

Antioxidant Systems

Protein-Folding Chaperones

Proteasomes and Autophagy as Endogenous Defense Systems

Trophic Factors as Endogenous Defense

Section Summary

6-ohda As a model of PD

Characteristics of 6-OHDA

Injection Protocols

In Vitro Application of 6-OHDA

Loss of Phenotype

Strengths of Model

Weaknesses of Model

Self-protection in Response to 6-ohda

Response of Neurotrophic Factors to 6-OHDA-Induced Injury

Preconditioning-Induced Protection

Possible Mechanisms Underlying Defensive Responses to 6-OHDA Injury

Summary and Conclusions

Chapter 14. Complex I Inhibition, Rotenone and Parkinson's Disease

Introduction

Mitochondrial Defects, Oxidative Stress and PD

What is Rotenone?

Rotenone as an Agricultural Chemical

Rotenone as an Inhibitor of Mitochondrial Complex I

The Rotenone Model

Previous History

Rotenone Administration

Characteristics of Rotenone-Induced Toxicity

Variability in the Rotenone Model

Use of Rotenone to Model PD in Other Species

Advantages and Disadvantages of Using Rotenone to Model PD

Converging Mechanisms

Chapter 15. Paraquat-induced Neurodegeneration: a Model of Parkinson's Disease Risk Factors

The Paraquat Model

Exposure Paradigms

Microglial Activation

Redox Cycling

Oxidative Stress

Co-exposure Paradigms

Developmental Exposure

Gene–toxicant Interactions

Conclusions

Acknowledgements

Uncited References

IV. Rodent And Other Vertebrate Genetic Models

Chapter 16. Overview: rodent and fish models of parkinson's disease

Introduction

Synuclein biology

Novel modeling strategies

Summary

Chapter 17. Genetic Models of Familial Parkinson's Disease

Introduction

Autosomal Dominant Inherited PD

α-Synuclein

Leucine Rich Repeat Kinase-2

Autosomal Recessive Inherited PD

Parkin

DJ-1

PINK1

α-Synuclein Transgenic Mouse Models

Ideal Animal Models of PD?

Thy-1 Promoter and Murine Prion Promoter α-Synuclein Transgenic Mice

Murine Prion Promoter Models of α-Synucleinopathies

Modulation of α-Synuclein Pathology in α-Synuclein Transgenic Models

Utility of Models of α-Synucleinopathies for Drug Discovery

Conclusions

Acknowledgements

Chapter 18. The Dynamics of α-Synuclein at the Nerve Terminal

Introduction

What do we know about synuclein?

Membrane binding in vitro

PD-Associated Mutations and Membrane Binding

Membrane interactions in vivo

Synaptic Localization of α-Synuclein: Role of Membranes

Transient Interactions Revealed by Fluorescence Recovery After Photobleaching

Activity-Dependent Dynamics of α-Synuclein

Relationship Between Membrane Binding and Aggregation

Concluding remarks

Chapter 19. The Bac Transgenic Approach to Study Parkinson's Disease in Mice

Introduction

Generation of Bac Transgenic Mice

Generation of BAC Transgenic Mice

Applications of BAC Transgenesis to Study Gene Expression and Gene Function in the Brain

Bac Transgenic Mouse Models of Neurodegeneration

The Advantages of BACs to Model Dominant Neurodegenerative Disorders

A BAC Transgenic Mouse Model of HD Recapitulates Phenotypic Features of Adult-Onset HD

Applications of Bac Transgenesis to Study PD

A BAC Model Reveals Dominant Toxicity of Truncated Parkin Mutant in DA Neurons

BAC Transgenic Approach to Model Dominant PD Mutant Genes

BAC Transgenic Approach to Study Pathogenic Mechanisms in PD

BAC-GFP Reporter Mice to Study Pathological Neuronal Circuits in PD

Potential Pitfalls and Solutions in Generating Bac Models of Neurodegeneration

One BAC May Contain Multiple Genes

Summary and Future Perspective

Acknowledgements

Uncited References

Chapter 20. Viral Vectors

Introduction

Genetic Modeling of Parkinson's Disease

Transgenic Animal Models

Viral Vectors for Targeted and Differential Expression in the SN

Methodology

The Choice of Viral Vector

Lentivirus

Adeno-Associated Virus

AAV Serotypes

Adenovirus

Genes Used for Viral Modeling

Targeted Species

State of the Art

Rat Models

AAV2 α-Synuclein

Mouse α-Synuclein Model

Primate Model: AAV2 α-Synuclein

Alternative Genes

Cell Specificity

Testing for Neuroprotective Approaches

Conclusion and perspectives

Abbreviations

Chapter 21. Environmental Explorations of Parkinson's Disease Using Rodent Genetic Models

Introduction

Rationale for Using Genetic Rodent Models

Rationale for Exploring Environmental Factors in PD

Rationale for Combining Exploration of Environmental and Genetic Factors

Most Frequently Used Genetic Rodent Models of PD for Environmental Explorations

Results

MPTP in Genetic Rodent Models of PD

Paraquat Exposure in Mice Overexpressing Alpha-Synuclein

Combined Paraquat and Maneb Exposure in Mice Overexpressing Alpha-Synuclein

Other Genetic Rodent Models

Conclusions

Source of Conflicting Results in the Exploration of Environmental Factors in Genetic Models of PD

Future Studies of Environmental Factors in Genetic Rodent Models of PD

Future Studies of Environmental Factors in Genetic Rodent Models of Risk Factors for PD

Therapeutic and Public Health Implications

Chapter 22. α-Synuclein, CSPα, SNAREs and Neuroprotection in vivo

Introduction

α-Synuclein and PD

Synucleins

Duality of α-Synuclein Function

α-Synuclein transgenic mice

CSPα

CSPα/α-Synuclein Transgenic Crosses

CSPα/Synuclein KO crosses

Summary of CSPα/α-Synuclein genetic interactions

Biochemical relationship between α-Synuclein and CSPα

SNAREs and neurodegeneration

Neuroprotective Functions of synucleins and PD

Open Questions

Chapter 23. Insights from Zebrafish PD Models and Their Potentials for Identifying Novel Drug Targets and Therapeutic Compounds

Introduction

The History of Zebrafish as A Vertebrate Genetic Model Organism

Molecular Genetics in Zebrafish

Chemical Genetics in Zebrafish

PD Models in Zebrafish

Why Zebrafish?

Strength and Weakness of Using Zebrafish to Model PD

Chemically Induced Models of PD in Zebrafish

Genetic Models of PD in Zebrafish

Studying the Development and Regeneration of DA Neurons in Zebrafish

DA Neurons in Zebrafish

Development of DA Neurons in Zebrafish

Conclusions

Uncited References

V. Multicellular Invertebrate Models

Chapter 24. Parkinson's Disease

Advantages of Invertebrate Models

Invertebrate Neurodegeneration

Model system: C. elegans

Model System: D. melanogaster

Flies Versus Worms

Generating Invertebrate Disease Models

Important c. elegans Models

Important drosophila Models

Modifier Screens

Drug Discovery

Potential Limitations with Invertebrate Models

Summary

Uncited References

Chapter 25. Drosophila Models for Parkinson's Disease Research

Introduction

Models of parkinson's disease in the fly

α-Synuclein

Parkin

Pink1

DJ-1

Toxin Models for Environmentally Triggered Parkinsonism

Perspectives

The Precision of the Models for Providing Insight into Genes and the Integration of Genes and Toxins

Genes to Therapeutic Targets and Drugs

Closing comments

Acknowledgements

Chapter 26. Caenorhabditis Elegans Models of Parkinson's Disease

Introduction

C. elegans as a genetic model

Toxin-associated C. elegans models of PD

Gene-associated C. elegans models of PD

Genetic screens to identify novel PD-associated genes and pathways

Tools to identify exogenous compounds that may contribute to DA neuron degeneration

Limitations of C. elegans models of PD-associated DA neuron cell death

Perspectives

Uncited References

Chapter 27. C. Elegans Genetic Strategies to Identify Novel Parkinson's Disease-associated Therapeutic Targets and Leads

Introduction

C. Elegans: Additional Attributes Amendable to Drug Discovery and Target Validation for PD

C. Elegans Forward and Reverse Genetic Screens to Identify Pd-associated Therapeutic Targets

C. Elegans HTS to Identify DA Neuroprotective Compounds

C. Elegans and MOA Genetic Screens

Perspectives

1.. Uncited References

VI. Cell-Based Models

Chapter 28. Overview: cell-based models of parkinson's disease

The challenge of unraveling the etiology of parkinson's disease

Cell-based models and pathogenic mechanisms in Pd

Insights from the studies of cell-based models

Chapter 29. PC12 Cells as a model for parkinson's disease research

Introduction

What are PC12 Cells?

What are the potential advantages of pc12 cells for pd research?

How have pc12 cells been used to model pd?

Toxin Models

Oxidative Stress Models

Catecholamines

Metal Ions

Overexpression of Wild-Type and Mutant α-Synucleins

Additional Genetic Models

What are the potential drawbacks of using PC12 cells to model and study PD?

Relevance

Cell–Cell Interactions

Metabolic Properties of PC12 Cells

Death Versus Dysfunction

Genetic Drift and Reproducibility

Applications of the PC12 cell model to PD

Transcriptional Changes Associated with PD

α-Synuclein

Summary and perspectives

Acknowledgements

Chapter 30. Dissociated mesencephalic cultures

Da cell death caused by mitochondrial complex i inhibitors

The MPP+ Model of DA Cell Death

DA Cell Death Induced by Complex I Inhibitors Unrelated Structurally to MPP+: Rotenone and Annonacin

Implication of ros in da cell death

Role of the ubiquitin–proteasome system in da cell death

Involvement of cell cycle-dependent mechanisms in da cell death

Prevention of da cell death by trophic peptides

Role of activity-dependent mechanisms in da cell death

Control of DA Cell Survival by Voltage-Gated Ca2+ Channels

Control of DA Cell Survival by Ligand-Gated Ion Channels

Role of inflammatory processes in da cell death

Mechanisms Underlying Microglial Cell Proliferation

Mechanisms of DA Cell Death Facilitation by Microglial Cells

Acknowledgements

Chapter 31. Selective Autophagy in the Pathogenesis of Parkinson's Disease

Introduction: Autophagy, Types and Functions

Autophagy in Neurodegeneration

Intracellular Proteolytic Systems and Neurodegeneration

Autophagy and Protein Inclusions

Autophagic Failure in Neurodegenerative Disorders?

α-Synuclein and Autophagy

Chaperone-Mediated Autophagy: What Sets It Apart from the Other Types of Autophagy?

CMA of α-Synuclein

Experimental Models for the Study of the Interplay between α-Synuclein and Autophagy

Autophagy in Cultured Cells

In Vivo Models with Disregulated Autophagy

Manipulations of Autophagy as Future Therapeutics for PD

Concluding Remarks

Chapter 32. The Role of LRRK2 Kinase Activity in Cellular PD Models

Cell-based Models for Parkinson Disease Research

LRRK2: Genetic and Sequence Analyses

LRRK2 Mutations Cause Dominant PD

Sequence and Predicted Domain Structure of LRRK2

Enzymatic Activities and the Normal Function of LRRK2

Pathogenic LRRK2 Mutations

Toxic Effects of Mutant LRRK2 and Kinase Activity

LRRK2 as a Therapeutic Target

Summary and the Pros and Cons of Cell Models

Acknowledgement

Chapter 33. Using yeast as a model system for the genetic dissection of α-synuclein toxicity

α-Synuclein: a key player in parkinson's disease and synucleinopathies

Why use yeast to model cell-autonomous aspects of neurological diseases associated with protein misfolding?

Modeling α-synucleinopathy in yeast

Genetic and pharmacologic dissection of α-synuclein toxicity

Yeast as Platform for the Discovery of Therapeutic Drugs and Drug Targets

Advantages and Disadvantages of Using Yeast Models for the Discovery of Drugs and Drug targets

Genetic Analyses in Yeast Implicate Two Major Pathways Mediating α-Synuclein Toxicity

Conclusions

Chapter 34. The role of the foxa2 gene in the birth and death of dopamine neurons

Introduction

The Floor Plate Origin of Midbrain Dopamine Neurons

The Interaction of Foxa2 and Other Transcription Factors in the Development of Dopamine Neurons

Spontaneous Degeneration of Dopamine Neurons in Foxa2 Mutant Mice

Towards a Unified View of Genetic and Spontaneous Risk Factors

Uncited References

Chapter 35. Embryonic stem cell-based models of parkinson's disease

Introduction

Advantages of ESCs

ESC Genetic modification

Making authentic MDA neurons

Neural Induction

Patterning

DA Neuron Specification

DA Neuron Survival

Examples of ESC-Based PD approaches

From development to human disease

Engrailed

FoxA1 and A2

Pitx3

Nurr1

WNTs

Lmx1a and b

Future applications for ESC-based da neurons

Uncited References

Chapter 36. Neuroprotective and Neurotoxic Properties of α-Synuclein in Cell Culture Models of Dopaminergic Degeneration

Physiological function of α-synuclein

Chaperone activity of α-synuclein

Tyrosine hydroxylase and dopamine synthesis

Vesicular transport and trafficking

Da transporter function

α-Synuclein mutations in PD

α-Synuclein phosphorylation

α-Synuclein aggregation

Neuroprotective effect of α-synuclein in dopaminergic neurons

Neurotoxic effect of α-synuclein

Immortalized mesencephalic cell line (n27) as a model system for elucidating α-synuclein function

Acknowledgements

Abbreviations

Uncited References

Chapter 37. Postnatally Derived Ventral Midbrain Dopamine Neuron Cultures as a Model System for Studying Neurotoxicity and Parkinson's Disease

Introduction

Characteristics of Postnatal VM Cultures

Neurochemical Identification

Role of Astrocytes

Longevity

Response to Neurotrophic Factors

Neurotransmitter Receptors in Postnatal DA Neurons

Calcium Channels, Calbindin, and Spontaneous Activity

Synaptic Morphology

Assay Methods

DA Pools

Toxicity Studies

Optical Probes

Electron Microscopy

Single-Cell RT-PCR

Acknowledgements

Uncited References

Chapter 38. Yeast Cells as a Discovery Platform for Parkinson's Disease and other Protein Misfolding Diseases

Introduction: Yeast as a Model System for Understanding Human Disease

Scientific Achievements Using Yeast Models

Protein Homeostasis and Human Disease

The Protein Folding Problem

Neurodegenerative Protein Folding Diseases

Current PD Genetics and Cellular Mechanisms of Toxicity

Yeast PD Models Based on α-Syn Expression

Expression of a Low Dose of α-Syn May Provide a Model of Normal α-Syn Function

Expression of a High Dose of α-Syn Provides a Model of α-Syn Toxicity

Other Models of α-Syn Toxicity

Success of Yeast PD Models in High-throughput Screening

Yeast Two-Hybrid System

Synthetic Lethal Screen

Overexpression Screens

Chemical Screens

A Proposed Mechanism for the Sensitivity of DA Neurons to α-Syn

Schizosaccharomyces Pombe: A Fission Yeast Model?

Yeast Models of HD

Specificity of the Models

Conclusions

Acknowledgements

VII. Cell-Free Models

Chapter 39. Overview of cell-free systems for the study of parkinson's disease

Introduction

Cell-Free system

Chapter content

Critical insight provided by cell-free system

Interpretation of cell-free system findings

Conclusion and perspective

Chapter 40. Microglial activation in a mouse model of α-synuclein overexpression

Introduction

Role of synuclein in PD

Microglia-directed proinflammatory events and PD

SYNWT+/+ Mice: a model to study syn and microglia as proinflammatory co-conspirators in pd pathogenesis

Conclusion and future directions: SYNwt+/+ mice

Chapter 41. Purification and Quantification of Neural α-synuclein

Introduction

Purification of Brain αs and Identification of Interacting Proteins

Affinity Enrichment of αS from Mouse Brain

Identification of αS-Interacting Proteins from Mouse Brain

Purification of CSF αs and Identification of co-eluting Proteins

Quantification of αS by Elisa using Concentrated CSF

Development of Second Generation Sandwich ELISA

Characterization of Second Generation Sandwich ELISA

Biochemical Optimization of ELISA to Quantify αS in Native CSF

Quantification of CSF αS in Living Subjects and Postmortem Brain

Collection of CSF from Clinically Well Characterized, Living Donors

Cross-sectional Measurement of CSF αS in 165 Donors

Statistical Analyses of CSF αS ELISA Data

Postmortem Analyses of Nine Study Participants

Discussion

Uncited References

Chapter 42. Protein Folding and Aggregation in in vitro Models of Parkinson's Disease

α-Synuclein in Parkinson's Disease

Potential Role for Aggregation of α-Synuclein in PD

Potential Role for Normal Function of α-Synuclein in PD

A Role for In Vitro Structural Studies of α-Synuclein in PD Models

Structure in the Free State of α-Synuclein

Wild-Type α-Synuclein Contains Residual Structure

PD-Linked α-Synuclein Mutations Influence Its Conformation

Intramolecular Contacts in α-Synuclein may Regulate Aggregation

Structure in the Lipid-bound State of α-Synuclein

α-Synuclein Binds to Lipid Membranes In Vivo and In Vitro

Detergent Micelle-Bound α-Synuclein Adopts a Non-Compact Helical Structure

α-Synuclein Remains on the Surface of Detergent Micelles

Does Lipid Vesicle-Bound α-Synuclein Resemble the Micelle-Bound Protein?

PD-Linked α-Synuclein Mutations Affect Its Lipid-Bound Structure Differently

Membrane-Bound Structure May Play a Role in α-Synuclein Aggregation

Membrane-Bound Structure is Linked to α-Synuclein Function

Role of α-Synuclein Structure in PD Models

Testing the Importance of α-Synuclein Aggregation

Testing the Importance of α-Synuclein Function

Future directions

Chapter 43. The use of cell-free systems to characterize parkinson's disease-related gene products

α-Synuclein

Effects of Familial Mutations on α-Synuclein Self-assembly

Effects of Post-translational Modifications on α-Synuclein Self-assembly

Effects of Phospholipids on α-Synuclein Self-assembly

Characterization of α-Synuclein Fibrillar Structure

Identification of Fibrillization Inhibitors via High-Throughput Screening

Advantages and Limitations of Using Cell-Free Systems to Study α-Synuclein

DJ-1

Characterization of DJ-1 Structure

Investigations of DJ-1 Function in Cell-Free Systems

Effects of Familial Substitutions and Oxidative Modifications on DJ-1 Stability and Function

Advantages and Limitations of Using Cell-Free Systems to Study DJ-1

Parkin, uch-l1, pink1, lrrk2

Parkin

UCH-L1

PINK1

LRRK2

Advantages and Limitations

Conclusions

Use of Cell-Free Systems to Explore Mechanisms of PD Pathogenesis

Use of Cell-Free Systems to Identify Therapeutic Targets and Drug Leads

Strategies to Validate Findings Obtained Using Cell-Free Systems

Acknowledgements

List of Figures

Chapter 2. Genetics of Parkinson's Disease

Figure 2.1. α-Synuclein gene and protein in PD.

Figure 2.2. Structure of PARKIN gene and protein.

Figure 2.3. Structure of PINK1 gene and protein.

Figure 2.4. Structure of DJ-1 gene and protein.

Figure 2.5. Structure of LRRK2 gene and protein.

Chapter 3. Neuropathology of Parkinson's Disease[1]

FIGURE 3.1. Lewy bodies (a) are hyaline intracytoplasmic inclusions that are found in neuromelanin containing neurons in the SN. A related inclusion is the pale body (b), which is less compact and considered by some to represent a precursor to classic hyaline-type LBs. In the end, neurons in the SN die and undergo phagocytosis, a process referred to as neuronophagia (c) (a, b, c ×400).

FIGURE 3.2. Transverse sections of the midbrain at the level of the third nerve in an elderly normal individual (a) and a patient with long standing PD (b). The major difference is loss of neuromelanin pigment that is most marked in the lateral parts of the SN (arrow).

FIGURE 3.3. Sections of vulnerable nuclei in an elderly normal individual (a, c, e, and g) and a patient with long standing PD (b, d, f, and h). Neuronal loss in PD is selective and affects the dorsal motor nucleus of the vagus (b), locus coeruleus (d), substantia nigra, (f) and basal nucleus of Meynert (h). Neuronal loss is almost total in both the LC and the SN (d and f) compared to the normal (c and e). Neuronal loss is substantial in the dorsal motor nucleus of the vagus and the basal nucleus of Meynert, but not total (all figures are ×200).

FIGURE 3.4. LBs contain α-synuclein. Typical hyaline inclusions, sometimes more than one within the same neuron (a), show α-synuclein immunoreactivity (b). Swollen neuronal processes, most notably in the dorsal motor nucleus of the vagus (c), also contain compact dense α-synuclein-immunoreactive inclusions (so-called intraneuritic Lewy bodies) (d). In the cortex less well defined cytoplasmic inclusions, so-called cortical Lewy bodies, (e) also have α-synuclein immunoreactivity (f). Note also the presence of fine neuritic processes (all figures are ×400).

FIGURE 3.5. LNs are abundant in the hippocampus CA2 sector (a), but also present in the anterior olfactory nucleus in the olfactory bulb (b) and the amygdala (c). In the latter location many of the neurites have a dot-like morphology (c). In the midbrain and basal ganglia (d) in addition to dot-like and short neurites, α-synuclein-immunoreactive glial cytoplasmic inclusions (arrows) are not uncommon (inset) (all figures are ×400).

FIGURE 3.6. Granular swollen axons, axonal spheroids, are common in the SN and globus pallidus in PD (a). Spheroids are immuno-positive for α-synuclein (b) (a and b ×400).

Chapter 5. Systems Level Physiology of the Basal Ganglia, and Pathophysiology of Parkinson's Disease

Figure 5.1. (a) Anatomical structure of the basal ganglia circuitry, and (b) changes in the activity of basal ganglia nuclei associated with the development of parkinsonism. Gray arrows indicate excitatory (glutamatergic) connections, black arrows indicate inhibitory (GABAergic) connections. Changes in thickness of the arrows indicate changes in firing rates. Abbreviations: GPe, external pallidal segment; STN, subthalamic nucleus; GPi, internal pallidal segment; SNr, substantia nigra pars reticulata; SNc, substantia nigra pars compacta; PPN, pedunculopontine nucleus; CM, centromedian nucleus of the thalamus; VA/VL, ventral anterior and ventrolateral nucleus of the thalamus.

Figure 5.2. Examples of traces of neuronal activity, recorded with standard extracellular electrophysiologic recording methods in a monkey before (a) and after (b) induction of parkinsonism with MPTP. The animal was awake and sitting quietly in a primate chair during the recordings. The traces of these different neurons are each 1-s long. Treatment with MPTP resulted in an increased firing rate, and obvious changes in firing patters in this animal.

Figure 5.3. Changes in (a) discharge rates and (b) burst discharges (proportion of spikes occurring in bursts; and changes in the proportion of cells with oscillatory discharge in the (c) 3–8 Hz range and (d) 8–15 Hz ranges. Data from Soares et al. (2004).

Chapter 6. Neuroprotection for Parkinson's Disease

Figure 6.1. (a) Evolution of mean daily clinical scores (solid line)±standard deviation (dashed lines) while daily injecting MPTP 0.2 mg/kg i.v. (small arrows) until reaching a score of 8 on our clinical scale (horizontal dashed line) (Imbert et al., 2000). The dashed vertical line symbolizes the transition between the pre-symptomatic and the symptomatic periods. Large arrows correspond to time points at which animals were killed in order to characterize kinetics of neurodegeneration (see Figure 6.2; at D0, D6, D12, D15 and D25). (b) Historical record of the number of MPTP injection days required for reaching the clinical score of 8 (Imbert et al., 2000). It shows the reproducibility of the model in developing symptoms.

Figure 6.2. Time course of nigrostriatal degeneration. The dashed red line indicates the transition between the pre-symptomatic and symptomatic period in these animals. (a) Examples of cell counting maps showing the typical patterns of degeneration in the SNc (n=5 at D0, D6, D12, D15 and D25) (Bezard et al., 2001d). The number of TH-IR and Nissl-stained neurons in the SNc were counted in one representative section corresponding to a median plane of the SNc by one examiner blinded to the experimental condition. TH-IR neurons are shown in red whereas the blue symbols represent the Nissl-stained cells that were not TH positive. The horizontal line above each map indicates the mean percentage of surviving cells (i.e., Nissl stained). Note the selective disappearance of the dorsal tier of the SNc with time. (b) Representative examples of DAT binding autoradiographs showing the progression of striatal denervation at the caudal level of the same animals (Bezard et al., 2001d). Note the homogenous degeneration and the severe lesion in the D25 group. The horizontal line under each example indicates the mean percentage of DAT binding in striatal sections. Non-specific binding is shown on the bottom left corner of the figure (c) Representative examples of 123I-PE2I SPECT as a marker of DAT binding in living animals during disease progression between D0 and D25 (n=2) (Prunier et al., 2003). In agreement with the DAT binding in striatal sections, there is a homogenous degeneration and severe lesion in the D25 group. The inferior border of each image corresponds to the mean percentage of striatal 123I-PE2I binding potential as determined by Logan's graphical method. Changes affecting D2 DAR binding (d) (Bezard et al., 2001d), PPE-A mRNA expression (e) of the same animals (Bezard et al., 2001 c) and GPi multi-unit electrophysiological activity (f) in two additional animals (Bezard et al., 1999). D2 DAR binding (a) and PPE-A mRNA (c) autoradiographs have been taken at the rostral and caudal level of the striatum, respectively.

Figure 6.3. Example of a clinically driven experimental design in the MPTP macaque model. Data are the mean of daily clinical scores in the course of the intoxication protocol. All animals are daily treated with MPTP, from Day 1 onward, until they individually reach a score of 8 on the clinical rating scale. In addition to MPTP, animals receive either vehicle (solid line), tested drug (zz) from day 1 onward (dashed line) or tested drug (zz) from day 8 onward (punctiform line – delayed application). The only pertinent parameter is the day of reaching a score of 8 on the clinical scores. Delay denotes a significant difference in reaching this predefined threshold between zz-treated groups and vehicle-treated group. In this theoretical case, both concomitant and delayed application of the zz drug proved to be efficacious. Since animals are treated with MPTP until they reach that score of 8, they ALL become parkinsonian. Thus the putative difference in clinical scores once syndrome is stabilized does not reflect necessarily a protection effect. This emphasizes the fact that only the day for symptom reaching a score of 8 must be taken as the relevant parameter in the behavioral part of the experiment. DAT SPECT (bottom left examples) could be performed at specific time points of the intoxication protocol to maximize the data collection and to differentiate between symptomatic-like from neuroprotective-like activity of a given drug. Finally, these experiments should then be associated to post-mortem measures at different time points (arrows). A difference in the extent of nigrostriatal denervation at stages 2 and 3 would be indicative of neuroprotection.

Chapter 7. The use of aged monkeys to study pd: important roles in pathogenesis and experimental therapeutics

Figure 7.1. A pickup test to measure the speed of movement showing the increased latency (i.e., decreased motor function) in aged monkeys relative to young (***p<0.0001).

Figure 7.2. (a) The number of α-synuclein-ir nigral neurons significantly increases in monkeys as a function of age (*p<0.05, **p<0.01 compared with young group). (b) The optical density (OD) of TH-ir nigral neurons was significantly decreased in nigral neurons with detectable α-synuclein-ir when compared with the OD of TH-ir nigral neurons without detectable α-synuclein-ir profiles (*p<0.05 compared with young group). Modified from Chu and Kordower (2007) with permission.

Figure 7.3. (a) In human nigra, the number of α-synuclein-ir nigral neurons significantly increases as a function of age (**p<0.01). (b) The optical density (OD) of TH-ir nigral neurons was significantly decreased in the neurons with α-synuclein-ir as compared with the neurons without detectable α-synuclein-ir (**p<0.01 compared with young group, – p<0.05 compared with middle-aged group). Modified from Chu et al. (2007) with permission.

Figure 7.4. Hypothetical process by which α-synuclein influences nigrostriatal function during normal aging and PD. (top) During normal aging, soluble α-synuclein accumulates within nigral perikarya and this causes a phenotypic downregulation of dopamine. The level of dopamine insufficiency is not severe enough to induce the cardinal symptoms of PD. (bottom) In PD, the same process occurs, but at some point, lysosomal function is overwhelmed, the progressive loss of dopaminergic function becomes more severe, and the magnitude of dopamine dysfunction is sufficient for the cardinal symptoms of PD to occur. Used with permission from Chu et al. (2007).

Figure 7.5. With aging process, the capacity for nigrostriatal neurons to compensate in response to MPTP insult is significantly lost in the oldest monkeys. After MPTP exposure, a large increase in the HVA/DA ratio is seen in young monkeys indicating their ability to compensate for lost nigrostriatal function. Although middle-aged monkeys do not show a statistically significant difference from young monkeys, the standard errors are much greater indicating that some middle-aged animals can compensate while others cannot. In contrast, no aged monkeys can compensate for the loss of nigrostriatal function (Caudate: *p<0.005 for comparison of intact to lesioned hemispheres, +p<0.009 for comparison of middle-age lesion to old lesion, ++p<0.04 for comparison of young lesion to both middle-age and old lesion. Putamen: *p<0.005 for comparison of intact to lesioned hemispheres, +p<0.003 for comparison of young and middle-age lesion to old lesion). Modified from Collier et al. (2007) with permission.

Chapter 8. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Mammalian Models of Parkinson's Disease

Figure 8.1. A comparison of the changes in striatal DA levels over time, in acute and sub-acute MPTP-lesioned mice. Acute MPTP administration produced a greater reduction in DA concentration when compared with sub-acute MPTP administration at all post-lesion time points, with significant differences between the two models apparent at 2 (*p<0.01), 7 (**p<0.001), and 14 days (***p<0.001).

Figure 8.2. Effects of acute or sub-acute MPTP administration on substantia nigra pars compacta (SNc) neurons. Cell numbers were estimated by unbiased stereological analysis of the region spanning the entire rostro-caudal extent of SNc. Loss of both TH- and NeuN-immunoreactive cells in the two MPTP administration paradigms was significantly different between the models only at 2 days after acute MPTP administration (highlighted by box, p<0.01), possibly indicating a loss of TH phenotype or expression preceding actual cell loss.

Figure 8.3. Microglial activation in the SN of acute and sub-acute MPTP-lesioned mice. Note the reactive microglia (MAC1+) at 48 h after toxin exposure in the acute MPTP condition, with no visible activation in animals with sub-acute exposure. At 1-week post-MPTP, activated microglia are seen in the SNc in both acute and sub-acute MPTP-treated animals. Acute MPTP-treated animals have reduced numbers of activated microglia at 1 week post-MPTP compared with the number of activated microglia observed at 48 h.

Figure 8.4. Microglial activation in the SN of acute MPTP-treated mice co-administered minocycline. Reactive microglia cells (MAC1+ immunoreactive cells) are visible throughout the SN of acute MPTP-treated animals at both 48 h and 1 week. Co-administration of minocycline with MPTP inhibited microglial activation for up to 48 h, but not at 1 week following the last MPTP injection.

Chapter 9. Non-human Primate Models of Parkinson's Disease and Experimental Therapeutics

Figure 9.1. Altered levels of serum MPP+ levels with successive injections of MPTP. Squirrel monkeys were administered a series of six injections of MPTP (i.p., 2.0 mg/kg free-base, 2 weeks between each injection). Blood was collected at days 1, 4, and 10 after each injection of MPTP and the level of MPP+ determined by HPLC analysis. These data demonstrate that with successive injections of MPTP there is an increase in the serum level of MPP+ indicating systemic conversion of MPTP to MPP+ most likely due to induction of metabolic enzymes in peripheral organs especially the liver. Since MPP+ cannot cross the blood–brain barrier the degree of lesioning in the brain is reduced with later injections of MPTP.

Figure 9.2. Time course in motor recovery in the MPTP-lesioned non-human primate model. Squirrel monkeys (N=6 per group) were administered saline, three or six injections of MPTP (2.0 mg/kg free-base, 2 weeks between injections). A CRS was administered to monitor parkinsonian features (Petzinger et al., 2006). A score greater than 4 is considered the threshold for parkinsonian motor features. Animals receiving three injections of MPTP displayed mild transient parkinsonian features for only a few weeks while those receiving six injections showed moderate to severe parkinsonian features and showed full motor behavioral recovery 12 weeks after the last injection of MPTP. The open and gray bars represent the time frame of 6 and 3 injections of MPTP, respectively.

Figure 9.3. Electrophysiological evidence for MPTP-induced changes in synaptic transmission. (a) Input (stimulus intensity applied to corpus callosum) – output (excitatory post-synaptic current or EPSC amplitude) relationships were determined for cortico-putamen synapses using whole cell voltage clamp with GABAA receptor blocked by picrotoxin. Note a greater tendency for larger amplitude EPSCs at 6 weeks post-MPTP (left panel), with a return to normal amplitude EPSCs by 9 months post-MPTP (right panel). (b) Example of synaptic currents recorded from a putamen neuron in response to corpus callosum stimulation before and after addition of picrotoxin. Synaptic currents were evoked in cells clamped at membrane potentials of −60 mV to maximize AMPA current activation and 0 mV to maximize GABAA current activation. (c) Ratios of synaptic currents illustrate a shift in synaptic function. At 6-week post-MPTP there was a reduction in the GABAA/AMPA ratio, a reduction in the NMDA/AMPA ratio, an increase in the NR2B/total NMDA ratio, and a decrease in the GYKI 52466 sensitive/CNQX sensitive AMPA ratio. Interestingly, these trends returned to saline control levels by 9 months post-MPTP.

Figure 9.4. Changes in dopamine release and synaptic plasticity in the MPTP-lesioned non-human primate. (a) Fast-scan cyclic voltammetry revealed regional differences in evoked dopamine release in the putamen that is markedly reduced even after 9 months post-MPTP when animals are motorically recovered. Letters in the graph correspond to putamen brain slice sites. (b) Comparison of short-term (3 min post-tetanus) and long-term (30 min post-tetanus) synaptic plasticity at corticostriatal synapses from saline and 9 post-MPTP-lesioning. Intracellular recording of EPSPs Excitatory Post-Synaptic potential evoked at cortico-putamen synapses was used to monitor changes in strength induced by tetanic activation of the corpus callosum. Medial cortico-putamen synapses produced short- and long-term potentiation (LTP) in both groups. Lateral cortico-putamen synapses expressed short- and long-term depression (LTD) in saline and MPTP exposed monkeys, but the MPTP group tended toward greater LTD. (c) Example of LTD induced at lateral cortico-putamen synapses and tetanic activation of lateral cortico-putamen synapses in the presence of the D2 dopamine receptor antagonist l-sulpiride that enabled the expression of profound LTP. Insert shows the response of the putamen neuron recorded during the l-sulpiride experiment to current injection, which is typical of a medium spiny projection neuron.

Chapter 12. Acute and chronic administration of 1-methyl-4-phenylpyridinium

FIGURE 12.1. Dose-response effect of chronic icv MPP+ infusion on striatal content of DA, serotonin (5HT), GABA and glutamate. After 28 days of MPP+ infusion into the left cerebral ventricle, striatal contents of DA, 5HT, GABA, and glutamate were measured. Results (mean ± SEM from 4–5 rats/group) are presented in ratios of neurotransmitter content in left to right striata. No significant changes in neurotransmitter levels were observed in the right striata at any of the MPP+ doses tested (data not shown). *Statistically different (p < 0.05) from vehicle controls. Reprinted from Exp Neurol, 200 (1); Yazdani et al. (2006). Rat model of Parkinson's disease: Chronic central delivery of MPP+, with permission from Elsevier.

FIGURE 12.2. Striatal neuropathology after 28 days of icv infusion of MPP+ (0.142 mg/kg/day). TH immunostaining in striatum of rat infused with (a) vehicle or (b) MPP+. Tr = cannula track; V = ventricle. Note loss of TH immunostaining in dorsomedial region of striatum in MPP+-treated rat. (c) Staining for striatal choline acetyltransferase. Note that staining for choline acetyltransferase is normal in nerve endings and cell bodies (arrow) except for a small region adjacent to the cerebral ventricle that shows depletion. (d) Presence of ubiquitin stained inclusion bodies. Inclusion bodies (arrow) were found immediately adjacent to the lateral ventricle close to where the MPP+ was infused. Scale bar in panels a and b = 450 μm, panel c = 150 μm and in panel d = 14 μm. Reprinted from Exp Neurol, 200 (1); Yazdani et al. (2006). Rat model of Parkinson's disease: Chronic central delivery of 1-methyl-4-phenylpyridinium (MPP+ ), with permission from Elsevier.

FIGURE 12.3. Striatal inclusion bodies are found 28 days after icv infusion of MPP+ (0.142 mg/kg/day). Striatal sections were stained for ubiquitin (black) and counterstained with cresyl violet. Inclusion bodies in (a–c) are depicted by arrows. A ubiquitin stained Lewy body in a nigral DA neuron from a patient with PD is shown in (d). Note the presence of neuromelanin pigment in the DA neuron. Scale bar = 10 μm. Reprinted from Exp Neurol, 200 (1), Yazdani et al. (2006). Rat model of Parkinson's disease: Chronic central delivery of 1-methyl-4-phenylpyridinium (MPP+), with permission from Elsevier.

FIGURE 12.4. Loss of DA neurons and presence of microgliosis in SN of a rat chronically infused with MPP+ (0.142 mg/kg/day). SN sections from rostral (a) to caudal (d) illustrate the loss of TH staining in fibers and cell bodies on the side ipsilateral to the icv infusion of MPP+ as compared to the contralateral side. Note prominent loss of medial SN pars compacta TH-positive neurons (arrows). VTA = ventral tegmental area. Scale bar = 300 μm. (e) Activation of microglia in SN of MPP+-treated rat is illustrated by immunostaining with the microglial marker OX-24. Insert is a higher power view. Scale = 130 μm; insert scale = 30 μm. Reprinted from Exp Neurol, 200 (1), Yazdani et al. (2006). Rat model of Parkinson's disease: Chronic central delivery of 1-methyl-4-phenylpyridinium (MPP+), with permission from Elsevier.

FIGURE 12.5. Abnormal mitochondria in nigral DA neurons of a rat chronically infused with MPP+. Electron micrographs are from a representative rat treated with MPP+ (0.142 mg/kg/day) for 28 days followed by a 2-week recovery period. (a) Two DA dendrites (d1 and d2) are identified using an antibody to the DAT. Arrow points to one of the DAT-labeled gold particles. (b) Arrow points to a very large mitochondrion located next to a normal sized mitochondrion (arrowhead). (c) An enlarged mitochondrion (arrow) which contains a clear space within its center. Note normal mitochondria in lower half of the field. (d) Mitochondrion with condensation particles. (e) Electron dense (1) and translucent (2, 3) mitochondria. (f) Mitochondria with electron dense accumulation (arrows). (g) Normal appearing mitochondria in the red nucleus. (h) Rare mitochondrion (arrow) in the red nucleus exhibit swelling. Scale bar: a, b, d–f = 0.125 μm; C = 0.25 μm; G = 0.5 μm; and h = 0.175 μm. Reprinted from Exp Neurol, Vol 200 (1); Yazdani et al. (2006). Rat model of Parkinson's disease: Chronic central delivery of 1-methyl-4-phenylpyridinium (MPP+), with permission from Elsevier.

FIGURE 12.6. Glutathione monoethyl ester (GEE) partially protects against striatal DA loss produced by chronic icv infusion of MPP+. Rats received icv infusions of vehicle, GEE (10 mg/kg/day), MPP+ (0.142 mg/kg/day), or GEE plus MPP+ for 28 days delivered from osmotic pumps. Results are the mean striatal DA ± SEM (5–9 rats/group). aSignificantly different from vehicle control; bsignificantly different from MPP+ group. Reprinted from Exp Neurol, 203 (2); Zeevalk et al. (2007). Characterization of intracellular elevation of glutathione (GSH) with GEE and GSH in brain and neuronal cultures: Relevance to Parkinson's disease, with permission from Elsevier.

Chapter 13. Endogenous Defenses that Protect Dopamine Neurons

Figure 13.1. Mechanism of action and sites of application of 6-OHDA. 6-OHDA is concentrated within the intracellular milieu by the high affinity dopamine transporter (DAT) and oxidizes to form hydrogen peroxide, 6-OHDA quinone, or dihydroxyphenylacetic acid (DOPAC) by action of monoamine oxidase (MAO). Inset: 6-OHDA is typically applied to one of three sites – the medial forebrain bundle (mfb), striatum (STR), or substantia nigra (SN). The Paxinos rat brain is shown from a sagittal view (Paxinos and Watson, 2006).

Figure 13.2. Quantification of striatal loss of TH immunoreactivity. (a) A coronal section of a rat infused into the striatum unilaterally with 3 ?g 6-OHDA, perfused after 7 days, immunostained with an infrared secondary against mouse anti-TH, and scanned with an Odyssey Infrared Imager (Li-Cor Biosciences, Lincoln, NA). The grayscale images have a bit depth of 16, allowing 65,536 levels of gray, increasing the linear range and sensitivity of the system. The relative lack of background in the infrared range also increases sensitivity. The image in (b) was thresholded in MetaMorph (Molecular Devices, Sunnyvale, CA) relative to the fluorescent intensity in the contralateral control hemisphere. Thus, the area of loss of TH (striatal area lacking fluorescent label in thresholded image) shows greater than 75% loss of staining relative to the contralateral, intact striatum. This technique nullifies variations in immunohistochemical staining between animals. A blind observer then traces the lesion area in sections centered around the needle track. Scale bar = 3 mm.

Figure 13.3. 6-OHDA degradation as a function of time and different vehicles. Samples were prepared with 6-OHDA (100 μM) in media alone (■), media plus 0.015% ascorbic acid (*), or vehicle containing ascorbic acid, DETAPAC, and flushed with nitrogen (▴). Samples were incubated for indicated times at 37 C and analyzed by HPLC. Data represent the mean and SEM of two or three replicates for each condition. Reprinted with permission from Ding et al. (2004).

Figure 13.4. The effects of 15-min exposure to 40, 100, and 500 mM 6-OHDA on the survival of DA neurons. After 3–4 days in vitro, cultures were exposed to vehicle or 6-OHDA. After 48 h, cells were fixed and immunostained for TH and GABA. Note the specific loss of DA neurons, but not of GABA neurons at lower 6-OHDA concentrations. Data represent the mean and SEM for 8–10 experiments, performed in duplicate or triplicate. *p < 0.01 versus control (0 μM 6-OHDA), Bonferroni post hoc following ANOVA. Representative photomicrographs in color of TH versus GABA cells are shown in Ding et al. (2004), and this figure is reprinted with permission.

Figure 13.5. Preconditioning MN9D cells with sublethal doses of 6-OHDA protects them against subsequent, otherwise lethal doses of 6-OHDA. (a) MN9D cells were pretreated with indicated concentrations of 6-OHDA or vehicle 6 h prior to post-treatment with 50 μM 6-OHDA and Hoechst stained 24 h thereafter. All pretreatments were 20 min and post-treatments were 30 min in duration. Cell counts of viable nuclei are shown as a percentage of controls treated twice with vehicle in the same manner. (b) MN9D cells were preconditioned with indicated sublethal 6-OHDA for 20 min 6 h before a 24 h challenge with 1 μM MG-132. Cells were stained with Hoechst reagent 24 h after initiation of MG-132 treatment. Data are presented as means and SEM of three independent experiments. *p < 0.05 versus vehicle, Bonferroni post hoc following ANOVA. Reprinted with permission from Leak et al. (2006).

Figure 13.6. Decreasing DA levels in MN9D cells exacerbates 6-OHDA toxicity. MN9D cells were treated with PBS or 1 mM α-methyl-p-tyrosine (AMPT) for 24 h prior to a 30 min treatment with 250 μM 6-OHDA and stained for viable nuclei with the Hoechst reagent 24 h thereafter. Viable cells are expressed as live/total. The effect upon basal viability was not statistically significant. This treatment dropped DA levels down by 93.5%, as assessed by HPLC. *p < 0.05 versus PBS. **p < 0.05 versus 0 μM 6-OHDA, Bonferroni post hoc following ANOVA.

Figure 13.7. Sublethal 6-OHDA activates the kinases ERK1/2, Akt, and JNK and inhibitors of all three kinases blocks preconditioning-induced protection in MN9D cells. (a) Western blotting data shows ERK1/2, Akt, and JNK phosphokinase and total kinase levels in untreated cells (UT) or 15 min after the onset of treatment with vehicle (Veh) or 5 μM 6-OHDA (Pre), with or without phospho-ERK (p-ERK) inhibitor U0126 (5 μM), p-Akt inhibitor LY294002 (10 μM) and p-JNK inhibitor SP600125 (5 μM). Blots are representative of three independent experiments. Other time points examined showed no changes. (b) Effect of p-ERK, p-Akt, and p-JNK inhibition on preconditioning. MN9D cells were preconditioned with sublethal 6-OHDA (5 μM; Pre) for 20 min in the absence or presence of U0126, LY294002, or SP600125, as done for Westerns. Six hours later, cells were post-treated with 50 μM 6-OHDA for 30 min.Cells were stained with Hoechst reagent 24 h after post-treatment. Data are presented as means ± SEM of five independent experiments. *p < 0.05 compared to vehicle, represented by previous black bar. **p <0.05 compared to non-preconditioned cells, represented by previous white bar, Bonferroni post hoc following ANOVA. Reprinted with permission from Leak et al. (2006).

Figure 13.8. Decreasing phospho-ERK1/2 levels with U0126 exacerbates 6-OHDA toxicity. MN9D cells were treated with U0126 1 h prior to and during 6-OHDA treatment, resulting in decreased cell viability at all 6-OHDA concentrations. Viable cells are expressed as a fraction of total (live plus dead). This concentration of U0126 decreased phospho-ERK1/2 to undetectable levels by Western blotting. *p < 0.05, significant difference between 0 μM and 5 μM U0126 at each 6-OHDA concentration; Bonferroni post hoc following ANOVA. Reprinted from Lin et al. (2008) with permission.

Chapter 15. Paraquat-induced Neurodegeneration: a Model of Parkinson's Disease Risk Factors

Figure 15.1. Role of microglia in paraquat-induced neurodegeneration. Microglial activation, which can be induced by an initial paraquat exposure or via administration of pro-inflammatory agents (lipopolysaccharide, LPS), enhances the vulnerability of dopaminergic cells to injury (priming effect). Once reactive microglia are present, paraquat exposure triggers neurodegeneration.

Figure 15.2. Mechanism of formation of ROS after paraquat exposure. Upon microglial activation, membrane-bound NADPH oxidase can catalyze the redox cycling of paraquat, generating significant amounts of deleterious superoxide anion. This sequence of toxic events is supported by in vitro evidence and may also characterize paraquat-induced neurotoxicity in the mouse model.

Chapter 19. The Bac Transgenic Approach to Study Parkinson's Disease in Mice

Figure 19.1. Advantages of BAC transgenic approach. Zinc-finger transcription factor Zipro1 is used to illustrate the advantages of BAC transgenic approach. Zipro1 is mainly expressed in cerebellum, hippocampus, and olfactory granule cell precursors as shown by in situ hybridization. Conventional transgenic approach with 10 kb genomic construct containing the Zipro1 promoter led to ectopic expression of lacZ transgene due to the positional effects (Yang and Heintz, unpublished data). BAC transgenic approach with 131 kb Zipro1 BAC construct results in the accurate lacZ transgene expression (Yang et al., 1997).

Figure 19.2. Timeline for generation of BAC transgenic mice.

Figure 19.3. Basic BAC transgenic construct designs. Construct 1 (CS-1) is designed to overexpress a transgenic open reading frame (Tg-ORF), such as the GFP, lacZ, Cre, and others, but the target gene (a cognate gene on the BAC) itself is not overexpressed. CS-2 uses an internal ribosomal entry sequence (IRES) which allows the expression of both the target gene and a Tg-ORF (i.e., GFP, lacZ, or Cre) from the same promoter.

Figure 19.4. Engineering BACs by homologous recombination in bacteria. (a) The RecA method. The shuttle vector used for modification contains a R6Kγ origin of replication, a RecA gene to support homologous recombination, and a recombination cassette containing two small homology sequences A and B (∼500 bp each) flanking the modification to be introduced (e.g., marker insertion, deletion, or point mutation). The shuttle vector plasmid is electroporated into the BAC-host bacteria. Some of the shuttle vectors can undergo homologous recombination with the BAC through either the A or B box, resulting in a complete integration of the shuttle vector into the BAC to form a co-integrate BAC. Bacteria containing co-integrate BACs can be selected by growth on chloramphenicol (chlor) and ampicillin (Amp). If co-integration and resolution occur through different homology boxes, the result is a correctly modified BAC with precise placement of the EGFP marker gene on a chosen locus on the BAC. (b) Recombineering is another BAC modification method based on the bacteriophage-encoded recombination enzymes. The BAC is transformed into an E. coli strain that can inducibly express γ-Red recombination enzymes and is deficient in galK. A recombination cassette is generated to flank the galK gene with two short homology arms. The cassette is then transformed into the BAC in the specialized recombination-competent bacteria, and subsequent induction of homologous recombination leads to precise integration of galK into the BAC. A second recombination step, using a cassette containing the intended modifications (i.e., marker gene insertion, deletion, or point mutation) flanked by the same short homology arms, can introduce the modification into a precise location on the BAC.

Figure 19.5. BAC-GFP mice to study basal ganglia neuronal circuits and FACS array. (a) Schematic drawing of a sagittal mouse brain section illustrating projection of striatopallidal MSNs to the GPe and of striatonigral MSNs to the substantia nigra (SN). GENSAT-GFP mice (i.e., D1-GFP, D2-GFP, and TH-GFP) have been generated to label the neuronal types that are relevant to the pathological PD circuit (Gong et al., 2003). These mice can be obtained from Mutant Mouse Regional Resource Centers (www.mmrrc.org). (b) Schematic drawing of the FACS-array procedure to dissect live striata from BAC-GFP transgenic mice, enzymatically dissociate the striatal neurons and use FACS to purify live (GFP positive and propidium iodide negative) striatonigral or striatopallidal neurons. RNA isolated from the sorted neurons are amplified before being used in microarray studies (Lobo et al., 2006).

Figure 19.6. Conditional BAC mouse model to study pathological cell–cell interactions in PD. (a) Schematic drawing of a conditional BAC PD mice in which expression of the toxic PD gene can be switched off by Cre recombinase in the DA neurons in SNc by Cre (i.e., Dat-Cre). (b) A schematic drawing of a BAC construct in which a critical exon of a mutant PD gene (i.e., the one containing the translation initiation codon) can be flanked by two LoxP sites (floxed). When crossed to a cell-type-specific Cre mouse lines, the expression of the mutant PD gene from the conditional BAC PD model can be selectively switched off in the specific cell types. Using such a model, one may address the pathogenic role of mutant PD gene expression in specific neuronal and non-neuronal cell types in the brain.

Chapter 20. Viral Vectors

Figure 20.1. Schematic description of the procedure. Viral vectors (in most cases lentiviral or AAV vectors) are unilaterally injected in the SN to overexpress pathogenic proteins in neurons. After virus injection, an 8- to 16-week period is usually needed for the lesion to fully develop. The loss of the nigrostriatal neuronal function is analyzed by histological examination to quantify both the number of dopaminergic neurons at the level of the SNpc and the remaining innervation in the striatum, with respect of the non-injected side. Typically, dopaminergic markers including TH, vesicular monoamine transporter 2 (VMAT2), and dopamine transporter (DAT) are used to label the neurons of interest.

Chapter 22. α-Synuclein, CSPα, SNAREs and Neuroprotection in vivo

Figure 22.1. Sequence alignment of human α-synuclein, β-synuclein and γ-synuclein protein sequences. Residues are colored gray to indicate identity with human α-synuclein. The horizontal lines outline the seven 11 amino acid repeats, the arrowheads exon boundaries. The stars indicate the position of the three known PD mutations, A30P, A53T and E46K. Arrow indicates location of break in α-helical conformation. (Chandra et al., 2003; Ulmer et al., 2005)

Figure 22.2. Folded conformation of α-synuclein on SDS micelle. Ribbon diagram of SDS micelle-bound α-synuclein structure (PDB entry 1XQ8, Chandra et al., 2003, Ulmer et al., 2005). The N-terminal portion of the molecule folds on the micelle surface, with the C-terminal remaining unbound and unfolded. The break in the helix occurs at amino acid 43–45.

Figure 22.3. Model of CSPα as a synaptic vesicle chaperone. CSPα binds to Hsc70 in the presence of ATP and accelerates its intrinsic ATPase activity. The CSPα-Hsc70-ADP complex then binds denatured or misfolded substrates along with SGT, a small guanine-rich tetratricopeptide protein. This chaperone complex uses ATP hydrolysis to catalyze folding of substrates. The full repertoire of presynaptic substrates for this chaperone complex is unknown. Our evidence suggests that the membrane SNARE SNAP-25 is one substrate of the Hsc70/CSPα/SGT complex (Tobaben et al., 2001).

Figure 22.4. Effect of transgenic α-synuclein on the growth and survival CSPα KO mice. (a) Breeding strategy. Heterozygous CSPα KO mice (CSP+/−) were mated with heterozygous CSPα KO mice containing an α-synuclein transgene (CSP+/−Syntg). (b) Immunoblot analysis of brain proteins from wild type and CSPα KO mice that express or lack transgenic human wild type α-synuclein. Blots were probed with antibodies to CSPα, α-synuclein and VCP (as a loading control). (c) Photograph of CSPα KO mice lacking (CSP−/−) or containing the wild type human α-synuclein transgene (CSP−/−Synhtg), and of a wild type littermate control mouse (CSP+/+) at 10 weeks. (d) Body weights of littermate female (♀; top panels) and male mice (♂; bottom panels) as a function of age. (e) Survival of female and male mice as a function of age. In (d) and (e), the left panels describe animals that express wild type human α-synuclein, central panels wild type mouse α-synuclein and right panels A30P mutant human α-synuclein (Chandra et al., 2005); reproduced with permission.

Figure 22.5. Model for the genetic interactions between α-synuclein and CSPα in preventing neurodegeneration. Based on the genetic crosses, we hypothesize that α-synuclein acts on one or more CSPα substrates to rescue the neurodegeneration of CSPα KO mice. These presynaptic substrates are critical for maintaining synapses and neurons.

Chapter 23. Insights from Zebrafish PD Models and Their Potentials for Identifying Novel Drug Targets and Therapeutic Compounds

Figure 23.1. The life cycle of zebrafish. Hundreds of eggs can be produced from a single mating. A typical vertebrate body plan is laid out by 2 days post-fertilization. By 5 days post-fertilization, the larval zebrafish are free-living, hunt for food, and escape from predators. By 3 months post-fertilization, zebrafish are ready for reproduction. Adapted from Guo (2004).

Figure 23.2. Schemes for forward genetic screens. Adult male zebrafish are mutagenized with the chemical mutagen ENU (ethyl nitrosourea). ENU induces mutations in the spermatogonia. Mutagenized males are mated with wild-type females to produce the F1 generation. The F1 generation is heterozygous for any induced mutations, thus, only dominant mutations can be recovered if genetic screens are conducted on the F1 generation. To identify recessive mutations, F1 fish need to be crossed to obtain F3 generations, in which homozygous mutations are present (two-generation screen). Alternatively, F1 female fish can be induced to undergo gynogenesis by applying early pressure (EP); therefore, homozygous mutations can be recovered in the F2 generation. Adapted from Guo (2004).

Figure 23.3. The scheme of the reverse genetic method, TILLING. Similar to a forward genetic screen, the F1 generation that is heterozygous for any induced mutations are raised to adulthood. A frozen sperm library is from F1 fish. In addition, genomic DNA are prepared from individual F1 fish to establish a corresponding genomic DNA library. To identify a mutation in a particular gene of interest, gene-specific primers are used to amplify PCR products from the genomic DNA library. PCR products are digested with CEL1 enzyme, which cuts at mismatches. Gel electrophoresis is carried out to identify which F1 carries a mutation in the gene of interest. Once the F1 carrying a mutation in the gene of interest is identified, the line can be recovered from the frozen sperm library. Adapted from Guo (2004).

Figure 23.4. Small molecule screen in zebrafish. View of 96-well plates containing 2-day-old zebrafish embryos treated with small molecule compounds directly dissolved in the medium. Note the chemical in the top left well disrupts embryonic development.

Figure 23.5. MPTP effects on DA neurons in larval zebrafish. (a) Control 3-day-old zebrafish embryo showing DA neurons labeled in red (with TH antibody). (b) MPP+-treated 3-day-old zebrafish embryo showing a significant reduction in the appearance of DA neurons.

Figure 23.6. Developmental loss of DA neurons in the too few mutant zebrafish. Ventral view of 2-day-old zebrafish embryos showing the loss of DA neurons in the too few mutant (labeled with TH antibody).

Chapter 25. Drosophila Models for Parkinson's Disease Research

Figure 25.1. The parallel approach of the fly in defining new mechanistic insight and the foundation for new therapeutics, for human Parkinson's disease. The figure highlights how the fly was used to develop a Parkinson's disease model (Feany and Bender, 2000), and then the model used to define a role of molecular chaperones in modulating of disease phenotype in fly, then the findings extended to human synucleinopathies including Parkinson's disease (Auluck et al., 2002). From Helfand (2002). Reprinted with permission from AAAS.

Chapter 26. Caenorhabditis Elegans Models of Parkinson's Disease

Figure 26.1. Visualization of all eight DA neurons in living, adult C. elegans hermaphrodites using DAT::GFP transcriptional fusions. (a) 3D reconstruction of confocal epifluorescence from head DA neurons in a Pdat:1::GFP transgenic line. Arrows identify CEP and ADE processes. NR refers to the nerve ring. (b) DIC image of animal in panel (a). (c) Schematic drawing showing location of DA neurons in the head relative to the pharynx. In this top view two pairs of CEP neurons project dendritic endings to the tip of the nose and one pair of ADE neurons extend ciliated processes to amphids adjacent to the terminal bulb of the pharynx. (d) 3D reconstruction of confocal epifluorescence of the PDE neurons. Both PDE cell bodies are apparent. (e) DIC image of animal in panel (d). (f) Schematic drawing showing left-hand member of pair of PDE neurons in lateral location