The neuroprotective effect of the heat shock proteins

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THE NEUROPROTECTIVE EFFECT OF

THE HEAT SHOCK PROTEINS

MARCUS JAMES DERMOT WAGSTAFF

A thesis submitted to the University of London

for the degree of Doctor of Philosophy.

October 1997.

Department of Molecular Pathology

Windeyer Institute of Medical Sciences

Division of Pathology

University College London Medical School

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Abstract

The work presented in this thesis investigates the protective effects o f the heat shock proteins in neurons against the cytopathic effects o f exogenous stress. The heat shock proteins (hsps) are a group of proteins that are overexpressed in cells in response to temperatures above the cells optimum growth tenq)erature.

Cerebral ischaemia in vivo increases the levels o f heat shock proteins and their mRNAs. A brief ‘sub-lethal’ ischaemic insult prior to a ‘lethal’ ischaemic stress has two effects. Firstly, hsps are overexpressed, and secondly, the extent o f neuronal damage is significantly reduced as con^ared with models that undergo the single ‘lethal’ ischaemic insult. The work in this thesis investigates whether overexpression of hsps is protective against ischaemia in neurons.

Initially, the levels o f a range o f hsp mRNA and protein overexpression are characterised over time during a focal cerebral ischaemic insult in the core region of ischaemia in rats in vivo, achieved by middle cerebral artery occlusion (MCAQ).

This thesis proceeds to describe the design, construction and characterisation of recombinant HSV-1 vectors that, after delivery, significantly overexpress hsps in a neuron-derived cell line. These vectors were created with the ultimate goal of heat shock gene delivery to the rat brain in vivo. The protective effect of hsp overexpression was subsequently assessed in neurons in response to heat shock, ischaemic stress and ap opto sis.

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Acknowledgements

I would like to thank Professor David S. Latchman for all his support, ideas and words of encouragement throughout the duration o f the project. I would also like to thank Dr. Robert S. CoflSn for his constant guidance and patience. Professor Jacqueline de BeUeroche and Dr. Yolanda Collaço-Moraes deserve a special mention for all their help and teaching with the mRNA work and Dr. M. Keith Howard for his technical advice concerning cell lines, viruses, and a whole host o f other matters. I must also thank the other members of the Medical Molecular Biology Unit, in particular Dr. Suzanne Thomas, Dr. Lynn Rose, Dr. Anastasis Stephanou and Mr. John Estridge, not only for their advice, but also for their friendship and tireless support. I am very grateful to the Dean of University College London Medical School, Prof John R Pattison, and also to Prof. Anthony Segal and Mr. Tom Wale without whose support this project would have been impossible, and to PhiHp Taylor whose friendship and bottomless pot o f coffee has just about kept me sane. Finally, I would like to thank my parents for reasons far beyond those which can be put on paper.

This work was supported by a scholarship from the Sir Jules Thom Charitable Tmst made available through University College London Medical School.

Declaration

AH the work presented in this thesis is the work o f Marcus Wagstaff. Contributions by other researchers to the work presented is acknowledged below:

1) Mr. Benjamin S. Aspey, at the Reta Lila Weston Institute o f Neurological Studies, University College London carried out the middle cerebral artery occlusion work. 2) The brain tissue mRNA extraction in Chapter Three was carried out by Dr. Yolanda

Collaço-Moraes and Professor Jacqueline de BeUeroche at the department of Biochemistry, Charing Cross Hospital Medical School, London.

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Abbreviations Ab APS ATP/ADP/AMP bp BDNF BiP BGH BHK BSA cAMP CAT cDNA CMC CMV CNS CPE cpm ddHzO DEPC DHFR DMEM DMSO DNA DRG E ECL EDTA EMCV ER ECS Antibody Ammonium persulphate

Adeno sine tripho sphate/dipho sphate/monopho sphate Base pairs

Brain derived neurotrophic factor

Immunoglobulin heavy chain binding protein Bovine growth hormone

Baby hamster kidney Bovine serum albumin

N6,2’-0-Dibutyryladenosine3 ’ : 5 ’-cychc monophosphate Chloramphenicol acetyl transferase

Conq)lementary DNA Carboxymethyl cellulose Cytomegalovirus

Central nervous system Cytopathic ejffect Counts per minute Double distilled water Diethyl pyro carbonate Dihydrofolate reductase

Dulbecco’s modified Eagle’s medium Dimethylsulfoxide

Deoxyribonucleic acid Dorsal root ganghon Early (gene)

Enhanced chemiluminescence

Diaminoethanetetra-acetic acid, disodium salt Encephalomyocarditis virus

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FGM Full growth medium

FKBP FK506 binding protein

GABA Gamma aminobutyric acid

GFAP Glial-fibrillary acidic protein

GFP Green fluorescent protein

GR Glucocorticoid receptor

HBSS Hank’s balanced salt solution

HCF Host cell factor

Hepes N- [2-hydroxyethyl] pip erazine-N ’ - [2- ethanesulfonic acid]

HMBA Hexamethylene bisacetamide

HO-1 Haem oxygenase-1

HSC Heat shock cognate

HSE Heat shock element

HSF Heat shock factor

HSP Heat shock protein

HSV Herpes Sinqplex Virus

HSV-1 Herpes Simplex Virus type 1

ICP Infected cell protein

IE Immediate-early (gene)

1RES Internal ribosome entry site

IRE Internal repeat long

1RS Internal repeat short

kb Kilobase

kDa KiloDalton

L Late (gene)

LAX Latency associated transcript

LB Luria Bertani medium

L I 5 Medium Liebowitz’s 15 medium LMP Agarose Low melting point agarose

LTR Long terminal repeat

LZ Leucine zipper

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MAP2 Microtubule associated protein 2

MWt Molecular weight

M CA(0) Middle cerebral artery (occlusion)

MOI Multiplicity of infection

MoMLV Moloney murine leukaemia virus

MR Mineralocorticoid receptor

NGF Nerve growth factor

NMDA N-methyl-D- asp artate

NO(S) Nitric oxide (synthase)

OR Oestrogen receptor

PAGE Polyacrylamide gel electrophoresis

PDF Pre-sequence binding factor

PBS Phosphate buffered saline

pfli Plaque-forming units

PPIase Peptidyl prolyl cis-trans isomerase

PR Progesterone receptor

RM Rainbow marker

RNA Ribonucleic acid

mRNA Messenger RNA

rpm Revolutions per minute

SDS Sodium dodecyl sulphate

SEM Standard error of the mean

SFM Serum jfree medium

SRP Signal recognition particle

SSC Standard saline citrate

SV40 Simian virus 40

TAE Tris- acetate-EDTA buffer

TBP TATA binding protein

TCP t-corüplex polypeptide

TdT Terminal deoxynucleotidyl transferase TEMED N,N,N’ ,N ’ - tetramethyl- ethylenediamine

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TRL Termmal repeat long

TRS Terminal repeat short

TTC 2,3,5-triphenyltetrazoUum chloride TUNEL TdT-mediated dUTP nick-end labelUng Tween 20 Polyoxy ethylene- sorbitan monolaurate UCLMS University CoUege London Medical School

UL Unique long

US Unique short

UV Ultraviolet

VSCC Voltage-sensitive calcium channels

Publications

1) Wagstaff Collaço-Moraes Y., Aspey B.S., CoflSn R.S., Harrison

T .at cil man D.S., de BeUeroche J.S. (1996) Focal cerebral ischaemia increases the levels o f several classes of heat shock proteins and their corresponding mRNAs. In Press. Mol. Brain. Res. 42 (2), 236-244.

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CONTENTS

Abstract 2

Acknowledgements 3

Declaration 3

Abbreviations 4

Publications 7

Index of Figures 15

Index of Tables 18

CHAPTER 1 - INTRODUCTION 19

1.0 Introduction 20

1.1 The Biology of the Heat Shock Proteins 20

1.1.1 Overview 20

1.1.2 The Heat Shock Protein 70 Family 24

1.1.3 The Heat Shock Protein 60 Family 31

1.1.4 The Heat Shock Protein 90 Family and the Untransformed Steroid Receptor

Complex 36

1.1.5 Hsp 5 6 and the Peptidyl Prolyl Isomerases 3 8

1.1.6 Heat Shock Protein 27 39

1.1.7 Heat Shock Protein 32 41

1.1.8 Other Heat Shock Proteins 41

1.1.9 The Role o f the Heat Shock Proteins in Cell Stress 43 1.1.10 The Heat Shock Transcription Factors and HSP Gene Regulation 46

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1.2.1 Introduction 53 1.2.2 The Appearance and Pathogenesis of Cell Death During Severe Cerebral

Ischaemia. 53

1.2.3 Changes in Gene Expression During Cerebral Ischaemia 57 1.2.4 Heat Shock Protein Expression in Cerebral Ischaemia 60 1.2.5 ‘Ischaemic Tolerance’ and the Heat Shock Proteins in Cerebral Ischaemia 66 1.2.6 Heat shock proteins and the stress response in neurons in vitro 70

1.2.7 Summary 74

1.3 Gene Delivery to the Brain 75

1.3.1 Introduction 7 5

1.3.2 The Choice of Vector 75

1.3.3 The Biology o f Herpes Simplex Virus Type 1 81

1.3.4 HSV-1 Based Vectors 87

1.3.5 Defective HSV-1 Vectors 90

1.3.6 Disabled HSV-1 Vectors 92

1.3.7 Expression and Regulation o f the Transgene 93

1.3.8 Insertion Site of the Transgene 95

1.3.9 Summary 97

1.4 Project Aims 98

CHAPTER 2 - MATERIALS AND METHODS 99

2.1 Laboratory Reagents 100

2.1.1 Chemicals 100

2.1.2 General Solutions 101

2.1.3 Enzymes 101

2.2 Bacterial Strains and Growth Conditions 104

2.2.1 B acterial Strains 104

2.2.2 Propagation and Storage o f Bacteria 104

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2.3 DNA Isolation and Analysis 106

2.3.1 Small Scale ‘Mini-Prep’ Plasmid DNA Extraction from£'. coli 106 2.3.2 Large Scale ‘Midi-Prep’ Plasmid DNA Extraction 6om E . coli 107

2.3.3 Restriction Site Analysis o f DNA 107

2.3.4 Purification and Precipitation of DNA 108

2.3.5 Agarose Gel Electrophoresis 108

2.3.6 Blunt Ending Reactions 109

2.3.7 Phosphatase Treatment of Plasmid DNA 109

2.3.8 Ligation of DNA 109

2.3.9 Screening E. coli colonies positive for plasmid containing insert 110

2.3.10 Preparation of cDNA for Radiolabelling 110

2.3.11 Radiolabelliug of DNA 111

2.3.12 Hybridisation 112

2.4 RNA Isolation and Analysis 114

2.4.1 Preparation o f RNase fi^ee materials 114

2.4.2 RNA Extraction from Brain Samples 114

2.4.3 Quantitation of RNA 115

2 .4 .4 Formaldehyde Gel Electrophoresis of RNA 115

2.4.5 Northern Blotting 116

2.4.6 Slot Blotting 117

2.5 Protein Isolation and Analysis 117

2.5.1 Protein Extraction firom Dissected Rat Brain 117

2.5.2 Quantitation o f Total Protein fi*om Homogenised Sandies 118

2.5.3 Protein Extraction from Cultured Cells 119

2.5.4 Polyacrylamide Gel Electrophoresis of Protein Extracts 119

2.5.5 Equahsation o f Protein Loading 120

2.5.6 Transfer of Protein to Nitrocellulose by Western Blotting 121

2.5.7 Immunodetection of Proteins on Western Blots 121

2.6 Tissue Culture 125

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2.6.2 Growth Conditions and Storage of Mammalian Cell Lines 125

2.6.3 DNA Transfections 127

2.6.4 Chloramphenicol Acetyl Transferase (CAT) Assay 129

2.6.5 Titration of Virus on Complementing Cells 130

2.6.6 Detection o f Viral Recombinants Expressing lacZ Reporter Gene 130 2.6.7 Detection o f Viral Recombinants Expressing Green Fluorescent Protein (GFP)

Reporter Gene 131

2.6.8 Purification of Viral Recombinants by Plaque Selection 131

2.6.9 Large Scale Vbal Culture 132

2.6.10 Large Scale Vbal DNA Extraction 132

2.6.11 Large Scale Vbal Purification 133

2.6.12 Infection o f Mammalian Cell Lines and Primary Neuronal Cultures with

Recombinant Virus 134

2.6.13 Mini-Vbal DNA Preparation for Southern Blot Analysis 135

2 .6 .14 Southern Blot o f Vbal DNA 136

2.7 Induction of Cell Stress 137

2.7.1 Heat Shock 137

2.7.2 Simulated Ischaemia 137

2.7.3 Induction o f Ap opto sis in ND7 Cells and DRG Neurons 138

2.8 Cell Viability Assays 139

2.8.1 Trypan Blue Exclusion Assay 139

2.8.2 /n Situ Programmed Cell Death Detection 139

2.8.3 Visuahsation of Healthy/Unhealthy Neurons by Light Microscopy 140

2.9 Middle Cerebral Artery Occlusion 141

CHAPTER 3 - CHARACTERISATION OF HSP mRNA AND PROTEIN

LEVELS DURING FOCAL CEREBRAL ISCHAEMIA 143

3.1 Introduction 144

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3.1.2 Method Details 144

3.2 Hsp27 mRNA and Protein Levels During Focal Cerebral Ischaemia 147

3.2.1 Hsp27 mRNA 147

3.2.2 Hsp27 Protein 147

3.3 Hsp56 mRNA and Protein Levels During Focal Cerebral Ischaemia 151

3.3.1 Hsp56 mRNA 151

3.3.2 Hsp56 Protein 151

3.4 Hsp60 mRNA and Protein Levels During Focal Cerebral Ischaemia 153

3.4.1 Hsp60 mRNA 153

3.4.2 Hsp60 Protein 153

3.5 Hsp70 mRNA and Protein Levels During Focal Cerebral Ischaemia 156

3.5.1 Hsp70 mRNA 156

3.5.2 Hsp70 Protein 156

3.6 Hsp90 mRNA and Protein Levels During Focal Cerebral Ischaemia 159

3.6.1 Hsp90 mRNA 159

3.6.2 Hsp90 Protein 159

3.7 Discussion 162

CHAPTER 4 - CONSTRUCTION AND CHARACTERISATION OF

RECOMBINANT HSV-1 VECTORS CO EXPRESSING INDIVIDUAL HEAT

SHOCK PROTEINS WITH g-GALACTOSIDASE 168

4.1 Introduction 169

4.2 Design of the 17+pR16R HSV-1 Recombinant Vectors 172

4.3 pR16R Plasmid and Virus Construction 174

4.3.1 Construction of Expression Plasmid 177

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4.3.3a Insertion o f SLP90 into HSV-1 Regions Flanking IE2 185

4.3.3b 5’ Extension of the LAT PI Promoter 185

4.3.4 Construction of pR16R 70 189

4.3.5 Recombination of pR16R 70 and pR16R 90 Plasmids into HSV-1 DNA 192

4.3.6 Purification of 17+pR16R Viral Recombinants 192

4.4 Characterisation of the 17+pR16R Recombinant HSV-1 Vectors 192

4.4.1 Visualisation o f lacZ Expression 192

4.4.2 Characterisation of HSP70 and HSP90 Protein Expression 193 4.4.3 Southern Blot Analysis of 17+pR16R 90 Infected B 130/2 Cells 196

4.5 Discussion 198

CHAPTER 5 - CONSTRUCTION AND CHARACTERISATION OF

RECOMBINANT HSV-1 VECTORS EXPRESSING SINGLE TRANSGENES

FROM THE LAT REGION 200

5.1 Introduction 201

5.2 17+pR19 Vector Design 202

5.3 pR19 Plasmid and 17+pR19 Virus Construction 206

5.3.1 Construction o f Expression Plasmid and HSV-1 RL Flanking Redons 207

5.3.2 Insertion of the transgenes 210

5.3.3 Recombination of the pR19 Plasmids into HSV-1 DNA 214 5.3.4 Purification of 17+pR19 HSP Viral Recombinants 214

5.4 Characterisation of the 17+pR19 Recombinant HSV-1 Vectors 215

5.4.1 Visualisation o f lacZ and Green Fluorescent Protein Expression 215 5.4.2 Characterisation of Heat Shock Protein Expression 217

5.5 The Heat Shock Transcription Factor Mutant, H-BH 226

5.5.1 Introduction 226

5.5.2 Characterisation of HSP70 promoter activity during H-BH overexpression by

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5.5.3 Construction of an H-BH Expressing 17+pR19 Recombinant HSV-1

Vector 231

5.6 Characterisation of the 17+pR19 HSF Recombinant HSV-1 Vector 231

5.6.1 Characterisation of Heat Shock Factor Expression 231 5.6.2 Characterisation of Heat Shock Protein Expression 234

5.6.3 Discussion 240

5.7 The 17+pM l Bicistronic Recombinant HSV-1 Vector 242

5.7.1 Introduction - Vector Design 242

5.7.2 pM l Plasmid and 17+pMl Recombinant HSV-1 Construction 243 5.7.3 Characterisation of GFP and /acZ Expression of the 17+pMl Recombinant

HSV-1 Vector 247

5.7.4 Discussion - The Potential Use of Bicistronic Vectors in Recombinant HSV-1-

Mediated Transgene Dehvery 252

5.8 Discussion 252

CHAPTER 6 - THE NEUROPROTECTIVE EFFECT OF THE HEAT SHOCK

PROTEINS IN VITRO 256

6.1 Introduction 257

6.2 Effect Of Overexpression hsp27, hsp56, hsp70 and H-BH in ND7 and DRG

Cells on Heat Shock, Ischaemia and Apoptosis 259

6.2.1 Heat Shock 259

6.2.2 Simulated Ischaemia 264

6.2.3 Serum-Withdrawal/NGF-Withdrawal 268

6.3 Discussion 279

CHAPTER 7 - DISCUSSION 283

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INDEX OF FiGURES

Chapter 1:

1.1.2 Model for the Reaction Cycle o f DnaK, DnaJ, and GrpE in

Protein Folding 29

1.1.3 Model for the Reaction Cycle of GroEL and GroES in

Chap eronin-assisted Protein Folding 34

1.1.10a HSF Trimérisation During Heat Shock 48

1.1.10b The HSF Cycle: A Model of HSF Regulation 51

1.3.3a Herpes Simplex Virus Type 1 82

1.3.4 Schematic Representation of Growth of Disabled and Defective

Herpes Simplex Virus Vectors 88

Chapter 2:

2.9 Schematic Diagram of the Middle Cerebral Artery Occlusion

(MCAO) Model 142

Chapter 3:

3.1.1 Cerebral Blood Supply in Rats 145

3.2.1 Hsp27 and p-tubulin mRNA Levels m the Core Region o f Ischaemia

During Permanent MCAO 148

3.2.2 Hsp27 and (3-tubulin Protein Levels m the Core Region o f Ischaemia

During Permanent MCAO 148

3.3.1 Hsp56 mRNA Levels in the Core Region o f Ischaemia During

Permanent MCAO 152

3.3.2 Hsp 5 6 Protein Levels in the Core Region of Ischaemia During

Permanent MCAO 152

3 .4.1 Hsp60 mRNA Levels in the Core Region of Ischaemia During

Permanent MCAO 154

3.4.2 Hsp60 Protein Levels in the Core Region of Ischaemia During

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3.5.1 Hsp70 mRNA Levels in the Core Region of Ischaemia During

Permanent MCAO 157

3.5.2 Hsp70 Protein Levels in the Core Region of Ischaemia During

Permanent MCAO 157

3.6.1 Hsp90 mRNA Levels in the Core Region of Ischaemia During

Permanent MCAO 160

3.6.2 Hsp90 Protein Levels in the Core Region of Ischaemia During

Permanent MCAO 160

Chapter 4:

4.1 The 17+ Strain HSV-1 Genome and Maps o f its EcoB and Not3.5

Fragments 170

4.3a Structure of the pR16R 90 Plasmid 174

4.3b Summary of the Construction of pR16R 90 175

4.3.1a The Construction of SLP 178

4.3.1b The Construction of SLP 90 180

4.3.2 The Construction o f IE2-Deleted Flanking Regions 183

4.3.3 The Construction o f the pR16R 90 Plasmid 187

4.3.4 The Construction of the pR 16R 70 Plasmid 190

4.4.1 Detection of (3-Galactosidase Activity in B 130/2 CeUs Infected

with the 17+pR16R 70 Recombinant HSV-1 Vector 194 4.4.2 Characterisation o f Heat Shock Protein Expression in B 130/2 CeUs

Infected with the 17+pR16R 70 and 17+pR16R 90 Recombinant

HSV-1 Vectors 194

4.4.3 Southern Blot of DNA Extracted from B 130/2 CeUs Infected

with the 17+pR16R 90 and 17+pR16R 70 Recombinant HSV-1 Vectors 197

Chapter 5:

5.3 Structure o f the pR19 lacZ Plasmid 206

5.3.1 Construction of the pNot3. 5cDNA3 Plasmid 208

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5.4.1 Detection o f Reporter Gene Product Activity in B 130/2 Cells Infected with the 17+pR19 lacZ and 17+pR19 GFP Recombinant

HSV-1 Vectors 216

5.4.2a Characterisation of Heat Shock Protein Expression in B 130/2 Cells

Infected with the 17-HpRI9 HSP Recombinant HSV-1 Vectors 219 5.4.2b Characterisation of Heat Shock Protein Expression in BHK Cells

Infected with the 17+pR19 HSP Recombinant HSV-1 Vectors 221 5.4.2c Characterisation of Heat Shock Protein Expression in ND7 Cells

Infected with the 17+pR19 HSP Recombinant HSV-1 Vectors 223 5.5.2 Stimulation o f the Human HSP70B Promoter by Transient

Overexpression of the HSFl Mutant H-BH 229

5.6.1 Characterisation of Expression o f the H-BH Gene Product in B 130/2, BHK and ND7 Cell Lines Infected with the 17+pR19 HSF

Recombinant HSV-1 Vector 232

5.7.3 Characterisation of Heat Shock Protein Expression in ND7 CeUs

Infected with the 17+pR19 HSF Recombinant HSV-1 Vector 236

5.7.4 Construction o f the pM l Plasmid 245

5.7.5 Detection o f Reporter Gene Product Activity in B 130/2 CeUs

Infected with Recombinant HSV-1 Vectors 249

Chapter 6:

6.2.1a Percentage Survival of ND7 CeUs After Varying Durations o f

48°C Heat Stress 259

6.2. lb ND7 CeU Survival FoUowing Severe Heat Shock when Infected with Recombinant HSV-1 Vectors Expressing Heat Shock and

H-BH Genes 261

6.2. Ic Primary Rat DRG Neuron Survival FoUowing Severe Heat Shock when Infected with Recombinant HSV-1 Vectors Expressing Heat

Shock and H-BH Genes 262

6.2.2a ND7 CeU Survival FoUowing Simulated Ischaemia when Infected with Recombinant HSV-1 Vectors Expressing Heat Shock and

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6.2.2b Rat DRG Neuron Cell Survival Following Simulated Heat Shock when Infected with Recombinant HSV-1 Vectors Expressing Heat

Shock and H-BH Genes 266

6.2.3i ND7 Cell Survival Over Time Following Serum-Withdrawal in the

Presence o f Retinoic Acid 269

6.2.3Ü Neonatal Rat DRG Neuron Survival Over Time Following

NGF-Withdrawal 272

6.2.3iii Number of ND7 Cells Undergoing Programmed Cell Death Following 48 hours of Serum-Withdrawal in the Presence

of diWrtrans Retinoic Acid 277

INDEX OF TABLES

C hapter 1;

1.1 The Function and Nomenclature of the Heat Shock Proteins 22 1.2.1 Summary of Reported Heat Shock Protein Expression Following

Cerebral Ischaemia 60

1.2.2 Summary o f Exp erimental Conditions for Ischaemic Tolerance

Experiments 66

1.3 Methods o f /« v/vo Gene Delivery to the Central Nervous System 76

C hapter 2:

2.1 DNA Plasmids 102

2.2 Antibodies Used in this Thesis, their Sources and Conditions o f Use 123

Chapter 3:

3.1 cDNA Templates Used for Random Prime Probe Generation 146

Chapter 5:

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Chapter 1

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1.0 Introduction

The work presented in this thesis as outlined in the abstract and the project aims in Section 1.4, investigates the protective effects o f overexpressing the heat shock proteins (hsps) in neurons, against various insults such as heat shock and ischaemia. Overexpression o f the heat shock genes is achieved through recombinant herpes sin^lex virus type 1 (HSV-1) vector-mediated transgene delivery to the neurons prior to insult. The introduction to this thesis is therefore divided into four main parts. The first part (Section 1.1) discusses the biology o f the heat shock proteins, their roles m cell stress and their regulation. The second part (Section 1.2) discusses cerebral ischaemia, its pathogenesis, the changes in gene expression and accordingly, heat shock protein expression, the ‘ischaemic tolerance’ phenomenon and also the evidence for the involvement of the hsps in protection against cell death. The third part (Section 1.3) reviews the current strategies in transgene delivery to the central nervous system and discusses the choice of HSV-1 as a vector and continues to outline what is known about the design and use o f HSV-1 in transgene delivery.

1.1 The Biology of the Heat Shock Proteins

1.1.1 Overview

In order to interpret the possible significance o f the heat shock proteins (hsps) m cerebral ischaemia, an understanding of the properties o f the different famihes and their regulation is necessary.

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transduction. Their Amotions vary considerably throughout the cell, and some of them have been attributed more than one function; but their inq)ortance in the cell is highhghted by the remarkable conservation of their protein-coding sequences throughout evolution, even between prokaryotes and eukaryotes. It has become evident that, although these proteins have been grouped together under a seemingly vague definition, many o f them share similar functions and some are intimately linked in events along the same cellular pathway.

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Table 1.1 - The Function and Nomenclature of the Heat Shock Proteins

Heat S h o c k Protein O ther N am es P ro p o se d F unction(s)

I

Hsp27 Hsp25 (murine) Binds actin filaments.

C haperones folding of citrate synthase. Role in thermotolerance.

Hsp32 Heme oxygenase 1 (HO-1)

Catalyses degradation of haem.

Hsp47 Collagen-specific

chaperone.

Hsp56 P59, FKBP59 Peptidylprolyl cis-trans isom erase.

Maintains inactive form of steroid receptor.

FK506 binding protein.

Hsp60 Chaperonin

GroEL (prokaryotic)

Provides environment for mitochondrial protein folding.

Hsp72 Hsp70, hsp70i Role in thermotolerance. Hsp73 Hsc70, hsc73, clathrin

uncoating ATPase

Binds unfolded proteins. Uncoats clathrin baskets. Targets proteins for lysosomal degradation.

BiP Grp78,

Immunoglobulin heavy chain binding protein

Binds to unfolded

proteins in endoplasmic reticulum lumen.

Mt-hsp70 Binds to unfolded

proteins in mitochondrial lumen.

Hsp90 (a and (3) Maintains inactive form of steroid receptor.

Maintains inactive form of retrovirally encoded proteins (ppBO'' ®'''). Little or no co-precipitation dem onstrated with the cellular homologue pp60"^'".

Binds actin, tubulin. Role in thermotolerance.

Hsp110 Role in thermotolerance

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The phenomenon o f the heat shock response was first noted in Drosophila busckii in 1962 (Ritossa, 1962). In this study puffs were preferentially induced in the salivary gland polytene chromosomes in response to heat, sodium saHcylate and dinitrophenol, indicating specific transcriptional activity. Subsequent studies by several groups demonstrated that other insults could also induce puffs along with the synthesis of RNA in other Drosophila tissues and species (Berendes, 1968; Ashbumer, 1970; Leenders and Berendes, 1972). It was not until 1974 that Tissières et al, observed a dramatically increased preferential expression o f a range o f proteins on heat shock (Tissieres et al., 1974). Since then, the wealth o f hterature has increased exponentially, not only in Drosophila, but also in Saccharomyces cerevisiae, Dictyostelium, Escherichia coli and vertebrates. In short, hsps have been demonstrated in all organisms examined thus far (Lindquist and Craig, 1988).

The increase in hsp synthesis observed as a result of cellular insult is now universally accepted to be protective. These phenomena and their use as a strategy to prevent neuronal cell death during cerebral ischaemia is well characterised and will be discussed in a separate section, following the accounts o f the individual hsps and their regulation. It is worth noting at this stage, however, that many o f the hsps function as ‘molecular chaperones’. R. John EUis defined ‘molecular chaperone’ as ‘...a family of unrelated classes o f protein that mediate the correct assembly o f other polypeptides, but are not themselves components of the final fimctional structures’ (EUis and van der Vies, 1991). They are proteins that not only bind to denatured, non-native proteins preventing their aggregation and misfolding during ceU stress but also ensure the correct folding o f polypeptide chains into functional native proteins foUowing their de novo synthesis and during recovery fi*om ceUular insult (Gething and Sambrook, 1992).

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reviewed as their functions remain discrete from the rest. There then follows a short section on other hsps, prior to a discussion of the role o f the hsps in cell stress. A discussion o f heat shock gene regulation and the heat shock transcription factor (HSF) then concludes the section.

1.1.2 The Heat Shock Protein 70 Family

The hsp70 family has been one o f the most characterised o f the hsp famihes. It has been shown to be highly conserved in ah organisms studied so far, not only in amino acid identity, but also their protein coding sequences; for exanq)le the human hsp70 protein is 73% identical to the Drosophila protein, which is 48% identical to the prokaryotic DnaK, the sole E. coli hsp70 (Bardweh and Craig, 1984). Many o f the differences are homologous substitutions (Lindquist, 1986). The similarity between the protein sequences of different organisms is most striking in the N-terminal ATPase domain, suggesting a high inq)ortance of this aspect o f the protein throughout evolution.

In eukaryotes, there are several members of the hsp70 family, each bearing a different function related to its subcehular locahsation, but they ah function as molecular chaperones. Those identified thus far reside in the cytoplasm, the mitochondrial lumen and the endoplasmic reticulum, and each will be discussed below. Each hsp70 family member has a highly conserved amino-terminal ATPase domain and a less conserved carboxy terminal peptide binding region (Chappell et al., 1987; Milarski and Morimoto, 1989). As a result of the amino acid and functional homology o f hsps from the same family, the functions of any overexpressed hsp may overlap with the other members.

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sensitive conformation. In mammals, newly formed secretory precursor proteins are imported into the lumen of the ER cotranslationally (Walter and Lingappa, 1986). As the N-terminal signal peptide emerges from the mRNA/ribosome con^lex, it is bound by the ribonucleoprotein signal recognition particle (SRP). Translation then pauses until the SRP-bound ribosome complex binds to the SRP receptor protein on the surface o f the E R The SRP is then displaced and translation continues simultaneously with translocation o f the newly formed polypeptide into the lumen o f the E R

The lumenal ER member of the hsp70 family named immunoglobulin heavy chain binding protein (BiP), in yeast, binds to the receptor on the inner membrane and binds to mahblded and unassembled secretory proteins as they enter the lumen of the ER to prevent misfoldmg (Sanders and Schekman, 1992). The ATPase action o f the N- terminus o f BiP is thought to provide the energy for the translocation process. BiP is found mammalian cells also, but it is unclear as to whether it is essential in the translocation o f denatured proteins. It has been speculated that the energy for this process comes from the ribosome complex. BiP, however, has been shown in

mammalian cells to bind transiently to newly synthesised wild-type transmembrane and

secretory protein precursors until they fold or assemble in the ER (Gething and Sambrook, 1992; Gething et a i, 1986). BiP binds more permanently with misfolded, unglycosylated, or unassembled proteins (Domer et al., 1987; Hurtley et al., 1989). Peptides that bind to the peptide binding domain in the carboxy terminal region of BiP show extensive sequence variabihty (Blond Elguindi et al., 1993). Markedly hydrophobic peptides bind, suggesting that hsp70 family members bind to regions of proteins normally located in the core of the folded protein. BiP is an abundant, constitutively expressed protein, but levels mcrease substantially when levels o f mutant proteins in the ER lumen increase, or with other stresses such as glucose starvation, amino acid analogues and calcium ionophores, which are all thought to be linked with accumulation of unfolded polypeptides (Lee, 1992; Lee, 1987; Kozutsumi et al.,

1988).

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KFERQ-like amino acid motii^ e.g. the RNase S-peptide of RNase A, prior to their lysosomal import for proteolysis (Dice et al., 1986; Chiang et al., 1989; Terlecky et al., 1992). This process is ATP-dependent. As not all proteins bear the KFERQ-like moti^ this may be a mechanism, during cell stress, for the selective degradation of accumulating denatured proteins that may not be essential for protein synthesis. Hsc73 may serve a role for maintaining these proteins in the unfolded state to enable their translocation into the ly so some, in a similar manner to the translocation across ER or mitochondrial membranes, it may also actively unfold proteins for their selective degradation. The mechanisms for selective protein translocation and their regulation is not yet known. Hsc73 is itself found in lysosomes, and bears two KFERQ-like motifs, and so may itself be a target for proteolysis, or it may act as a protein transporter. Its high levels in lysosomes indicates that it may be resistant to lysosomal proteolysis (Terlecky, 1994). As unfolded, denatured proteins are known to be protease sensitive, it is possible that hsc73 maintains proteins in a denatured conformation to enhance their proteolysis.

Studies on bovine brain vesicles have demonstrated that hsc73 binds clathrin (Schlossman et al., 1984; Chappell et al., 1986). Clathrin is a protein that coats vesicles which mediate membrane bound receptor transport in the cytoplasm. It is thought to introduce the curvature in the organellar membrane and formation of a uniform vesicle through formation o f its polygonal basket-like structure. The basket is composed o f clathrin hght and heavy chains in heptamers (three o f each) called a triskehon. 36 triskehons form a basket. Once the vesicle is formed, the clathrin basket is disassembled by a ‘clathrin uncoating ATPase’ which has now been identified as hsc73. The model for disassembly has been postulated as a two stage process. First, ATP hydrolysis displaces part o f the triskelion, which is thought to expose a binding site for hsc73, which stabihses the displacement. The uncoating complex is released when all the con^onents of the basket have been displaced (Schmid and Rothman,

1985b; Schmid and Rothman, 1985a).

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A member o f the hsp70 family has been isolated from the mitochondrial lumen (mt- hsp70). Despite mitochondria containing their own DNA and systems for rephcation and protein synthesis, about 95% o f mitochondrial proteins are encoded in the nucleus. The mechanisms o f interaction between hsp70 proteins and denatured polypeptides will be discussed in more detail below and in Figure 1.1.2. These chaperones may stabihse proteins in a translocation-competent state, or in a conformation that renders N- terminal pre-sequences available to bind to the receptor conqplex on the mitochondrial outer membrane. ATP has been shown to be required both in the cytoplasm, for maintaining proteins in a translocation-competent state, and also in the mitochondrial lumen, to drive the translocation process (Neupert et al., 1990). The ATP dependent mt-hsp70 binding cycle is thought to provide the energy to drive this translocation process. As the folded/unfolded equihbrium on the outside is altered by the mt-hsp70 binding cycles on the inside, the protein is transported stepwise into the mitochondrion. The spontaneous reversible ‘breathing’ o f the precursor from folded to unfolded states on the cytoplasmic side, therefore, would lead to a step-by-step transfer o f the protein during translocation. The co-operation between mt-hsp70 and mitochondrial hsp60 and the subsequent pathway to folded protein will be discussed below in the hsp60 review.

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Figure 1.1.2 - Model for the Reaction Cycle of DnaK. DnaJ, and GrpE in Protein

Folding (Frydman and Hartl, 1994)

1. Unfolded protein (U) interacts with DnaJ.

2. DnaK is recruited by DnaJ and hydrolyses bound ATP to ADP, which results in the formation o f a tight ternary complex.

3. GrpE causes the dissociation o f ADP from DnaK, which weakens the interaction between DnaK and U allowing protein transfer to GroEL.

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DnaK

A D PfO rp E

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The afBnity o f protein substrates for hsp70 is dependent on their amino acid sequence. Hsp70 does not only bind unfolded proteins. Non-polar sequences that are rich in glycine and proline residues in a mutant o f the p53 tumour antigen and the clathrin light chain have been demonstrated to bind hsp70 (Lam and Calderwood, 1992; DeLuca Flaherty et a i, 1990). These sequences also appeared to be resident in turns or extended loops in the protein, and it is beheved that the interaction with the carboxy terminal o f hsp70 is with these conformationaUy flexible and probably temporarily

exposed hydrophobic regions.

In summary, the hsp 70 family is involved in several essential functions in the prokaryotic and eukaryotic cell. Although their functions vary, the hsp70s principally fimction by preventing the folding o f denatured proteins, untü they are in an appropriate environment to do so (by enabling transport across an organellar membrane, or passing on the polypeptide chain to hsp60). In stress therefore, increasing the levels o f cytoplasmic hsp70 may prevent the aggregation and misfolding of denatured proteins and then target them to the appropriate environments for correct folding and resunq)tion of function.

1.1.3 The Heat Shock Protein 60 Family

Hsp60 is present in chloroplasts and mitochondria in eukaryotes, and in the cytosol of bacteria. The majority of work concerning the hsp60 family (chaperonins) has been on the E. coli homologue, GroEL. GroEL bears 54% homology with the amino acid sequence of eukaryotic hsp60 and exists in the bacterial cytosol as a homomeric structure o f two heptameric rings stacked one on top o f the other, in a double toroid structure with a central cavity (Reading et al., 1989; Hutchinson et al., 1989). It is into this cavity that unfolded proteins can enter, and gain their appropriate tertiary structure, sequestered fi*om the influences of the matrix outside.

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assembly (Cheng et al., 1990). HspôO requires functional mt-hsp70 for folding of newly imported proteins, and the folding reaction is ATP-dependent (Cheng et al.,

1989; Manning Krieg et al., 1991). In bacteria, the transfer o f proteins from DnaK to GroEL appears to be directed by their binding specifrcity, and involves GrpE and DnaJ in the cycle previously described (vide supra). In eukaryotes, more specifically in yeast, GrpE and DnaJ homologues have been isolated from the mitochondrial matrix (Ygelp and M djlp, respectively) (Rowley et al., 1994). Imported dihydrofolate reductase (DHFR) was demonstrated to be less thermally stable at 3T C in M djlp mutant strains of yeast, suggesting a role for this protein in stabihsing mitochondrial proteins against heat dénaturation (Rowley et al., 1994).

HspôOs bind to proteins by recognition o f hydrophobic regions in the non-native chain, and are released in vitro in the presence of Mg/ATP. It is beheved that the hsp60 stabihses proteins in an ‘assembly-coup etent’ state, enabling the spontaneous association o f subunits to make a functional conplex.

As a result of the various studies in E. coli, the mechanism for the action of GroEL in the bacterial cytoplasm has been proposed as ihustrated in Figure 1.1.3 (Martin et al.,

1993).

1) GroEL exists under physiological conditions asymmetricaUy, with GroES at one end. Each subunit on the GroEL heptamer proximal to GroES is tightly bound to ADP, the distal subunits bind ADP with lower afiBnity.

2) Unfolded protein binds to the nucleotide-free ring, and ADP and GroES dissociate. 3) ATP is bound to the nucleotide binding sites on both GroEL heptamers, which

weakens the interactions between GroEL and the substrate and GroES rebinds. 4) ATP hydrolysis releases the protein from the cavity wall, ahowing it to fold.

5) The GroEL, now in an ADP state, binds GroES with greater afiBnity and either the substrate is completely folded and released, or rebinds in a partially folded form This model for protein folding, although derived from bacterial studies, may represent the process that occurs in the eukaryotic mitochondria and cytosol.

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Figure 1.1.3 - Model for the Reaction Cycle of GroEL and GroES in

Chaperonin-assisted Protein Folding (Martin et a/., 1993)

D (bold), high-afiBnity ADP-binding state o f a heptameric GroEL ring. D (hghtface), low ADP-afiBnity binding state.

T, ATP-binding state.

U and N, unfolded and native protein, respectively.

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7+7 ADP

7+7 ATP

N

7+7 Pi

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1.1.4 The Heat Shock Protein 90 Family and the Untransformed Steroid Receptor Complex

The gene encoding the E. coli homologue o f hsp90, HtpG, can be deleted almost without biological consequence, and yet the constitutive abundance of this protein in the eukaryotic cytosol and ER suggests its importance (Bardwell and Craig, 1988). Indeed, in S. cerevisiae, hsp90 is essential for growth at aU ten^eratures (Borkovich et al., 1989). Its role however, is still uncertain. It has been shown to associate with certain tyrosine kinases e.g. the ppbO''"*'^'^ oncogene product; serine/threonine kinases e.g. c-Raf-1; transcription factors, e.g. steroid receptors; actin, tubulin, hsp56 {vide infra) and hsp70 (vide supra) (Stancato et a l, 1993; Koyasu et a l, 1986; Sanchez et a l, 1990; Catelh et a l, 1985; Perdew and Whitelaw, 1991). In its absence the ppôO''""'^'' oncogene kinase is not functional, whereas it only mildly affects the activity of the cellular homologue pp60‘^'^'^‘^ (Xu and Lindquist, 1993). Addition o f exogenous, purified hsp90 has been demonstrated to prevent the aggregation of a variety of denatured polypeptides in an ATP-independent manner, increasing the yields of the native enzymes (Wiech et a l, 1992). In this respect, therefore, hsp90 fulfils the role o f

a molecular chaperone. Its polypeptide specificity, which appears to be highly selective, has however yet to be determined given that hsp90 binding to various steroid receptors does not appear to be through recognition o f a particular amino acid sequence (Whitelaw et a l, 1993; Chambraud et a l, 1990).

The majority o f studies on hsp90 have been on its role as a steroid hormone receptor binding protein. The transcriptionally inactive steroid receptor (aporeceptor) complex exists as a receptor monomer, bound directly to an hsp90 dimer, which is itself bound to an hsp56 (FKBP59) monomer (Rexin et a l, 1991; Rehberger et a l, 1992). The glucocorticoid receptor is non-functional in the absence o f hsp90 (Picard et a l, 1990). The interaction between hsp70 and the complex is discussed below.

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the dioxin receptor (DR), the hgand binds within a 200 amino acid domain that also binds hsp90, and deletions in the hsp90 binding domain o f the glucocorticoid receptor (GR) produces defects in hgand binding (Whitelaw et a l, 1993). Taken together, these results suggest an interaction or exchange between the hsp and hgand on activation. Aporeceptor complex assembly requires ATP and both hsp90 and hsp70 possess ATPase activity. It has been proposed that hsp90 and hsp70 may have some role in ensuring the correct conformation of aporeceptors until the hgand is bound. Indeed, analysis o f mutants o f hsp90 demonstrated that functional hsp90 was essential for signal transduction at the hgand binding level, not at the transcriptional regulation level (Bohen and Yamamoto, 1993). The deleterious effect o f these mutants has been shown to be more profound on the GR and mineralocorticoid receptors (MR), than on the progesterone and oestrogen receptors (PR and OR respectively). Dissociation of hsp90 from untransformed GR, MR and DR complexes in vitro results in the loss o f high- afBnity hgand binding (Bresnick et a l, 1989; Schuhnan et a l, 1992; Pongratz et a l,

1992). OR, PR, and androgen aporeceptors (AR) bind their hgands with high afBnity at 0-4”C, but PR requires hsp90 for high afBnity hgand binding at 3T C (Smith, 1993; Eul et a l, 1989; Chambraud et a l, 1990; Nemoto et a l, 1992). Therefore, different aporeceptors have different requirements for hsp90, although the reasons for this paradox are not known.

The detection o f hsp70 associated with untransformed progesterone receptors represents a possible fmction for this hsp in steroid receptor assembly, but it may also be artefactual, if hsp70 is merely acting as a chaperone binding to hydrophobic sequences on the surface o f the receptor (Kost et a l, 1989). Purified hsp70, however, has been shown to restore conq)lex reconstitution in reticulocyte lysates that have been depleted o f ATP-binding proteins. Indeed, lysate depleted o f hsp70 only form hsp90- receptor complexes on the addition o f hsp70 (Hutchison et a l, 1994). If this is case in vivo, then it has also been shown that the association of hsp70 with the progesterone aporeceptor is transient and prior to hsp90 binding (Smith, 1993; Smith and Toft,

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1.1.5 Hsp56 and the Peptidyl Prolyl Isom erases

Hsp56 (p59, FKBP59) is a peptidyl prolyl cis-tram isomerase (PPIase) (Chambraud et al., 1993). It is expressed constitutively and is present in the cytoplasm The cis-trans isomérisation of proline residues in proteins is one o f the slow steps in protein folding, and so hsp56 plays a catalytic role in the process o f ensuring the correct folding of newly synthesised and denatured proteins. Like hsp70, it may bind to exposed hydrophobic sequences on proteins in the non-native conformation. Bound to hsp90, hsp56 is associated with the untransformed steroid receptor cortq)lex (Kost et a l, 1989; Tai et al., 1986). Its role in receptor activation is unclear, but, as previously discussed, the dissociation of these molecules from the corrq)lex occurs to enable high afiBnity ligand binding, rendering the receptor transcriptionally active.

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1.1.6 Heat Shock Protein 27

In mammals, hsp27 is a member of the small heat shock protein family, which has at least three members, hsp27 and aA- and aB-crystallin. The small hsps are less conserved between species than those with a larger molecular weight. In humans, the hsp27 gene has been located on chromosome 7 (Hickey et a i, 1986). Analysis o f the sequences o f the small hsps o f mammals and Drosophila has demonstrated that they all possess a conserved domain, referred to as the a-crystallin domain, which consists of about 80 residues in the carboxy half of the protein, and fiirther conservation between species has been locahsed to small sections o f the protein, possibly highhghting the importance o f these regions (Ingoha and Craig, 1982; Wistow, 1985; Southgate et al., 1983). These sections include the phosphorylation sites of the proteins. Further analysis o f the small hsps o f several species suggests that they are derived from a common ancestral gene (Wistow, 1985). The quartemary structure o f hsp27 has been proposed as a cylindrical conq)lex of 32 monomers 15-18nm in diameter (Behlke et al.,

1991).

Mammalian hsp27 in unstressed cells is primarily localised around the nucleus and also in the motile cytoplasm of fibroblasts (Arrigo et al., 1988; Lavoie et al., 1993b). On heat shock, or other cell stress, it appears on immunoflourescence to migrate into the nucleus, whilst remaining absent from the nucleolar structures. This nuclear hsp27 is seen to aggregate into large structures (>10^ Daltons) (Arrigo and Welch, 1987), but this does not occur in pre-treated thermotolerant cells {vide infra). The purpose of the super-aggregation, and the migration into the nucleus has not yet been resolved.

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1993). Hsp27 has also been shown to accumulate in various tissues during development, in the absence o f stress, e.g. in neurons o f the murine spinal cord and Purkinje cells (Gemold et al., 1993). Hsp27 levels have also been shown to directly correlate with tumorigenicity in breast cancer, and have therefore been proposed as a possible prognostic indicator (Thor et a l, 1991).

Hsp27 functions as a molecular chaperone, due to its ability to prevent the heat- induced aggregation o f citrate synthase, a-glucosidase and P-L-crystallin, however its range o f specificity is not known, and it therefore may chaperone many more non­ native proteins (Jakob et al., 1993; Merck et al., 1993).

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Several hsp27 kinases have been isolated. IL-1 induces an hsp27 kinase and may be homologous to the pp45-54 kinase known to phosphorylate hsp27 in response to growth factors (Guesdon et al., 1993; Huot et at., 1995). pp45-54 kinase is inactivated by protein phosphatases, and so is itself activated by phosphorylation. Possible activators o f this kinase are protein kinases A and C, and the mitogen- activated protein (MAP) kinases, which are phosphorylated by similar stimuli to hsp27 (Dubois and Bensaude, 1993; Lavoie a/., 1995; Knauf a/., 1994).

1.1.7 Heat Shock Protein 32

Haem oxygenase-1 (HO-1), is induced by heat shock, and therefore by definition is a heat shock protein (hsp32) (Shibahara et al., 1987). It contains a heat shock consensus element (HSE, see below) within its promoter. It is a microsomal enzyme that catalyses the oxidative degradation of haem (F e-protop orphyrin IX) into biliver din, carbon monoxide (CO) and iron (Stocker, 1990). Bihverdin and its catalytic product bilirubin have been shown to function as antioxidants (Stocker, 1990; Stocker et al.,

1987). Iron can be a prooxidant and regulates the expression of various genes, for example transferrin, nitric oxide synthase and hsp32 itself (Maines and Kappas, 1977; Weis et a i, 1994). CO is a putative neurotransmitter that regulates cGMP levels through activation o f guanyl cyclase and may also have a vasodilatory efifect like nitric oxide (Stevens and Wang, 1993; Maines, 1993). Raised levels of hsp32 protein have been detected in the neurofibrillary tangles seen in Alzheimer’s disease and therefore hsp32 may be associated with the pathophysiology o f neurodegeneration in this disease (Smith et al., 1994). It does not appear that hsp32 functions as a molecular chaperone.

1.1.8 Other Heat Shock Proteins

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(Nagata and Yamada, 1986; Nagata et al., 1988). During transformation the mRNA levels o f hsp47 have been demonstrated to be raised following focal cerebral ischaemia in the rat (Higashi et at., 1994).

Despite being one o f the first hsps to be characterised, httle is known about the large hsp, hsp 110. Subsequent to amino acid sequence analysis it is now beheved that hsp 110 is part o f a subfamily o f the hsp70 group o f proteins (Lee-Yoon et al., 1995). Particular amino acid sequence similarity with members o f the hsp 70 family is found in the ATP-binding domain, with further similarity in conserved regions in the carboxyl- terminal two thirds o f the protein. It is a normal constituent of mammahan cehs, it is associated with the nucleolus, is induced on heat shock, and its induction correlates strongly with the expression o f thermotolerance (Subjeck et al., 1983). It is constitutively expressed in ah mouse tissues and is highly expressed in brain (Lee-Yoon et al., 1995). Its function remains unknown.

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nucleosomes containing active genes and therefore ubiquitination may serve to alter nncleo some-nncleo some interactions preventing the formation o f higher order chromosomal structure (Levinger and Varshavsky, 1982). However ubiquitination does not prevent normal histone octamer and core particle reconstitution (Davies and Lindsey, 1994).

1.1.9 The Role of the Heat Shock Proteins in Cell Stress

The hsps, as previously mentioned, are elevated in response to cellular insults, such as heat shock. In cultured cells from a wide variety o f organisms e.g. mouse, yeast, bacteria, fruit flies etc., heat shock or other toxic stress is lethal. Pre-treatment with a mild, sub-lethal raised temperature for a period of time raises the levels of the hsps. These pre-treated cells are resistant to cell death on subsequent ‘lethal’ heat shock. The combination o f these two facts suggests that hsps may be protective against cell stress and therefore responsible for this thermotolerant’ phenomenon. There are several lines o f evidence to substantiate this theory (reviewed by Lindquist and Craig,

1988).

1) On heat shock, the induction o f hsps is very rapid, inq)hcating them in an emergency response, and hsp accumulation closely parallels the development of thermotolerance, hsp70 levels exhibit the closest correlation.

2) The induction temperature is dependent on the organisms environment, e.g. in thermophilic bacteria that grow at 50°C, hsps are induced at 60‘’C, whereas in mammals they are induced at fever terrq)eratures.

3) Sub-lethal thermal pre-treatment protects cells against death from other toxic insults and vice versa, as long as the hsps are induced.

4) During development, prior to their abihty to induce hsps, hypersensitivity to thermal killing is observed in many organisms. Once the hsps can be induced, the thermotolerant phenomenon is demonstrable.

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Abnormal and denatured proteins accumulate during cell stress, and denatured proteins aggregate. The artificial accumulation of abnormal proteins has been demonstrated to stimulate hsp synthesis (Ananthan et al., 1986). In heat shock the cytoskeleton is disrupted, in particular the intermediate filaments collapse, but the other con^onents o f the network are also affected. Actin-containing structures appear in the nucleus, or in other cell lines are destroyed (Welch and Suhan, 1985; lida et at., 1986). Microtubules are also damaged, and these being con^onents o f the mitotic spindle, explains the sensitivity of mitotic cells to heat shock (Coss et at., 1982). Protein synthesis is disrupted, and this is thought to be a regulatory rather than toxic process, in order to eliminate the competition for components of the synthetic machinery between the rapidly accumulating hsps and other proteins. mRNA sphcing is disrupted on heat shock. This does not effect hsp synthesis however, as most o f the hsp genes do not contain introns. Heat shock also has deleterious effects on rRNA synthesis in the nucleolus, RNA polymerase I transcription, DNA synthesis, chromatin assembly, and it increases the fluidity of the phosphohpid bilayers throughout the cell (Bell et at., 1988; EUgaard and Clever, 1971; Waiters and Roti Roti, 1981; Waiters and Stone, 1983; Kruuv et al., 1983). Mild heat pre-treatment reduces the extent o f these perturbations or increases the rate of their repair.

The roles o f the individual hsps on these various perturbations during stress is less well defined. There are several proposed mechanisms by which the hsps afford protection. Firstly, they act as molecular chaperones, binding to denatured proteins preventing their aggregation and misfolding and promoting the correct refolding of proteins to enable the subsequent recovery; and secondly they facihtate the degradation of abnormal and possibly non-essential proteins.

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heat shock and has been impHcated as a catalyst for the reassembly of ribonucleoproteins and damaged ribosomes after heat shock (Pelham, 1984).

Hsp60 is required in vitro to enable the folding o f chemically denatured ribulose bisphosphate carboxylase (Rubisco) and rhodanese at high tenq)eratures, but not at physiological ten^eratures. This infers a role for hsp60 during cell stress which may be shared by its cytosohc counterpart TRiC (Mendoza et al., 1991a; Mendoza et a l, 1991b; Goloubinofif e/ at., 1989b; Viitanen et a l, 1990).

Hsp90 binds both actin and tubulin {vide supra) and may serve to chaperone these - enabling cyto skeletal reformation, and it may also chaperone other cell signalling structures, despite its higher substrate specificity than hsp70, thus preventing their degradation and misfolding, ensuring proper fimction on recovery.

Hsp56 may act to fold proteins into their correct isoforms and, along with hsp90, may prevent dénaturation o f the steroid aporeceptor complex.

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Hsp27 has also been demonstrated to block the signals for ap opto sis conferred by the stimulation o f the Fas/APOl receptor and addition o f staurosporine in a murine fibrosarcoma cell line (Mehlen et al., 1996).

Hsp32 has not been shown to be a molecular chaperone, so it is beheved that protection, if any is conferred through its enzymic activity. Hsp 3 2 is induced not only by heat shock and cerebral ischaemia, but also oxidative stress (Dwyer et al., 1992; Stocker, 1990; Nimura et al., 1996; Applegate et al., 1991; Keyse and Tyrell, 1989). The proposed protective effect works on two levels. Firstly it reduces the levels of potential pro-oxidants haem and haem proteins such as cytochrome-P450 and protoporphyrinogen oxidase and secondly, the degradation products, the bile pigments possess antioxidant properties (Stocker, 1990). It has been suggested therefore, that hsp32 may be protective against the oxidative stresses on neurons incurred by both the ischaemia and subsequent reperfusion.

Overe?q)ression of hsp27, hsp70 and hsp90 have all been demonstrated to confer thermotolerance to cells. In particular, transfection-mediated overexpression of hsp70 and hsp90 confer thermotolerance to neurons, and neuron-derived cell lines in culture but they are not protective against serum- or growth factor-withdrawal induced ap opto sis and this effect is discussed in more detail in Section 1.2 (Amin et al., 1996; Mailhos et al., 1994; Uney et al., 1993). The noted protection from ap opto sis noted in fibrosarcoma cells overexpressing hsp27, along with its thermotolerance inductive properties make it a putative candidate for protecting neurons against death during stress. The protective effects of overexpressing hsp27, hsp56, hsp60 and hsp32 in neurons or neuron-derived cell lines has not previously been studied.

1.1.10 The Heat Shock Transcription Factors and HSP G ene Regulation

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demonstrated in humans, S. cerevisiae, Drosophila and mice, the third has been isolated from chickens (Schuetz et a l, 1991; Rabindran et a l, 1991; Clos et a l, 1990; Wiederrecht et a l, 1988; Sarge et a l, 1991; Nakai and Morimoto, 1993).

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LLLL LLLL

HSF MONOMER

LLLL LLLL

LLLL LLLL

HEAT SHOCK

HSF TRIMER

LLLL

ENZYMATIC MODIFICATION?

CHAPERONES?

LLLL*

LLLL LLLL

LLLL

LLLL

Figure 1.1.10a - HSF Trimérisation During Heat Shock (Lis and Wu, 1993)

Closed Oval (DNA-binding domain).

LLLL (not necessary leucines), hydrophobic heptad repeats.

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The action o f all the heat shock factors is mediated through the heat shock element (HSE) which is conserved throughout all eukaryotes and consists of inverted repeats of the sequence NGAAN (Lis and Wu, 1993). A minimum o f three of these 5 base pair (bp) elements are required for high afiBnity HSFl binding (Xiao et a l, 1991). Dififerent HSEs have dififerent numbers o f these 5bp sequences but they usually range firom three to six, and dififerent heat shock promoters have dififerent numbers o f HSEs, with varying distance between them (Amin et a i, 1988; Xiao et al., 1991). Analysis of deletion mutants o f the human HSFl has demonstrated that the trimérisation and DNA binding events are regulated separately jfrom the transactivation o f the heat shock gene (Zuo et at., 1995). One particular mutant, H-BH, was demonstrated to constitutively bind to the hsp70B promoter and drive the chloramphenicol acetyl transferase gene transcription without activation by heat shock. The H-BH contains a deletion in the second leucine zipper region which it is proposed, disrupts the coiled-coil monomeric state and encourages trimérisation, into a form capable o f DNA binding. It is therefore beheved that this disruption o f the monomeric structure is the first event, triggered in some way by cell stress, in the activation o f HSFl and the transactivation o f the heat shock genes.

Recently, a fourth HSF (HSF4) has been characterised in humans (Nakai et at., 1997). HSF4 is preferentially expressed in the human heart, brain, skeletal muscle and pancreas. The unique quahty o f HSF4 is that it lacks any transactivation properties. It has been shown to possess DNA binding activity with the HSE, but it lacks the carboxy-terminal hydrophobic repeat that all the other vertebrate HSFs posses, and its constitutive expression has been demonstrated in transfected cell lines to reduce the basal expression of the hsp90, hsp70 and hsp27 genes. It is postulated that this HSF may serve to regulate the expression o f HSE controlled genes in particular tissues.

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its consensus sequence, the TATA box, and RNA polymerase H is also constitutively sited up to 65bp downstream from the transcription start site, engaged in transcription, but paused after elongation of about a 25 nucleotide mRNA chain (Thomas and Elgin, 1988; Gilmour and Lis, 1986). There are two preferred positions for this paused RNA polymerase II which are separated by approximately one turn o f the DNA helix. RNA polymerase II binds DNA when the carboxy terminal domain is unphosphorylated and transcription is initiated on hyperphosphorylation. Trimerised HSF can bind to its consensus sequence, but remains inactive unless serine phosphorylated. HSF has several phosphorylation sites. It has been shown that the carboxy terminal o f paused RNA polymerase II is unpho sphorylated, and it is beheved that activated, phosphorylated HSF has some upstream effect in causing the phosphorylation of the polymerase to initiate transcription (Layboum and Dahmus, 1989; O'Brien et al., 1994; Layboum and Dahmus, 1990). The significance of the nucleosome-free state and the paused polymerase phenomenon suggests that the mRNA synthesis machinery is primed for rapid transcription when HSF is bound.

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H

I

hsp70

P P

Heat Shock Factor Cycle

H SF T rim er

5* nnG AAnnTTCnnG AAnn 3*

Heat Shock

H SF

(________

)

M o n o m e r

p

4

-\

hsp70

\T hsp70 binding to non-native poiypeptide

Figure 1.1.10b - The HSF Cycle; A Model of HSF Regulation (Morimoto et aU 1994)

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1.2 Cerebral Ischaemia

1.2.1 Introduction

Ischaemia may be defined as ‘the state existing when an organ or tissue has its arterial perfiision lowered relative to its metabohc needs’ (Wool^ 1986). In the brain, ischaemia can be either focal, for exanq)le due to emboH or thrombotic events occluding individual cerebral arteries; or it can be global, due to an overall lack of perfiision such as occurs during cardiac arrest (Kawai et al., 1992; Nagasawa and Kogure, 1989).

If all the symptoms and signs of a focal ischaemic insult resolve within 24 hours post­ occlusion, then it is clinically defined as a transient ischaemic attack (TIA). If any symptoms or signs persist, then condition is defined as a stroke. The abihty of the central nervous system (CNS) to recover from TIAs and strokes, and the discovery of the ‘ischaemic tolerance’ phenomenon in the brain strongly suggest that the CNS has mechanisms that afford protection against neuron loss during stress and for a limited duration afterwards (Kitagawa et al., 1990). The heat shock proteins (hsps) have been proposed as contributory to this protection (Nishi et al., 1993).

In this section, the mechanisms o f cell death during cerebral ischaemia will be discussed followed by the subsequent changes in gene expression and heat shock protein expression and finally the phenomenon o f ischaemic tolerance and the protective effects of overexpressing the hsps in vitro are reviewed.

1.2.2 The A ppearance and Pathogenesis of Cell Death During Severe Cerebral Ischaemia.

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following the insult. From five minutes to one hour of focal ischaemia, a similar selective neuron destruction occurs to the region proximal to the occlusion (Pulsinelli, 1992). During a focal ischaemic period greater than one hour, such as might occur during a stroke, the zone supphed by the occluded vascular bed is infarcted and neurons, gha and supportive cells are non-selectively destroyed. The abundance of collateral circulation in the CNS ensures that the ischaemic zone can still be supphed with some oxygen and glucose. Therefore, during a focal ischaemic insult, the areas proximal to the coUaterals and distal to the area o f lowest blood flow form a rim of moderate ischaemia. This marginal rim or penumbra contains hving , but electrically silent neurons (Astrup et al., 1981). As time progresses past one hour when a focal infarct has formed, its size increases into the penumbra to reach a maximal volume over 3-4 hours in rodents (Kaplan et al., 1991). In the ischaemic penumbra, dynamic pathophysiological processes are taking place - the final event being cell death. Synaptic transmission is blocked or suppressed and this electrophysiological suppression is interspersed with random depolarisation/repolarisation events which is related to glucose phosphorylation in neurons (Nedergaard and Astrup, 1986).

The mechanism for cell death during and as a result o f cerebral ischaemia and the consequent reperfiision is a comphcated process with multiple pathways. The deprivation o f oxygen and glucose results in a drop in oxidative metabohsm and subsequently, a fall in adenosine triphosphate (ATP) levels occurs. The majority of neurons and gha can recover from one hour of complete ischaemic insult and brain cells can tolerate a transient loss o f ATP (Hossmann and Kleihues, 1973). Once these pathways are initiated, however, the initial ischaemic stimulus may not be required to follow the cells through to death. CAl regional death continues at least 72 hours after 30 minutes o f global ischaemia (Pulsinelh et al., 1982).

Figure

Figure 1.1.10a - HSF Trimérisation During Heat Shock (Lis and Wu, 1993)
Figure 1 1 10a HSF Trim risation During Heat Shock Lis and Wu 1993 . View in document p.49
Figure 1.1.10b - The HSF Cycle; A Model of HSF Regulation (Morimoto et aU
Figure 1 1 10b The HSF Cycle A Model of HSF Regulation Morimoto et aU. View in document p.52
Table 2.1 - DNA Plasmids
Table 2 1 DNA Plasmids. View in document p.103
Figure 3.4.2
Figure 3 4 2. View in document p.156
Figure 4.3.3 - The Construction of the pR16R 90 Plasmid
Figure 4 3 3 The Construction of the pR16R 90 Plasmid. View in document p.188
Figure 4.3.3
Figure 4 3 3. View in document p.189
Figure 4.3.4 - The Construction of the pR16R 70 Plasmid
Figure 4 3 4 The Construction of the pR16R 70 Plasmid. View in document p.191
Figure 4.3.4
Figure 4 3 4. View in document p.192
Figure 4.4.1
Figure 4 4 1. View in document p.196
Figure 4.4.3 - Southern Blot of DNA Extracted from B130/2 Cells Infected with
Figure 4 4 3 Southern Blot of DNA Extracted from B130 2 Cells Infected with. View in document p.198
Figure 5.3 - Structure of the pR19 lacZ Plasmid
Figure 5 3 Structure of the pR19 lacZ Plasmid. View in document p.207
Figure 5.3.1 - Construction of the pNot3.5cDNA3 Plasmid
Figure 5 3 1 Construction of the pNot3 5cDNA3 Plasmid. View in document p.209
Figure 5.3.1
Figure 5 3 1. View in document p.210
Figure 5.3.2 - Maps of the pR19 Constructs Containing Transgenes
Figure 5 3 2 Maps of the pR19 Constructs Containing Transgenes. View in document p.213
Figure 5.3.2
Figure 5 3 2. View in document p.214
Figure 5.4.1 - Detection of Reporter Gene Product Activity in B130/2 CeUs
Figure 5 4 1 Detection of Reporter Gene Product Activity in B130 2 CeUs . View in document p.217
Figure 5.4.2a
Figure 5 4 2a. View in document p.221
Figure 5.4.2b
Figure 5 4 2b. View in document p.223
Figure 5.4.2c
Figure 5 4 2c. View in document p.225
Figure 5.5.2
Figure 5 5 2. View in document p.231
Figure 5.6.1
Figure 5 6 1. View in document p.234
Figure 5.6.2b) Probed with anti-hsp32 antibody
Figure 5 6 2b Probed with anti hsp32 antibody . View in document p.238
Figure 5.6.2c) Probed with anti-hspSO antibody
Figure 5 6 2c Probed with anti hspSO antibody. View in document p.239
Figure 5.6.2d) Probed with anti-hsp70 antibody
Figure 5 6 2d Probed with anti hsp70 antibody . View in document p.239
Figure 5.6.2e) Probed with anti-hsp90 antibody
Figure 5 6 2e Probed with anti hsp90 antibody. View in document p.240
Figure 5.7.2 - Construction of the pMl Plasmid
Figure 5 7 2 Construction of the pMl Plasmid. View in document p.246
Figure 5,7.2
Figure 5 7 2. View in document p.247
Figure 6.2.1b
Figure 6 2 1b. View in document p.263
Figure 6.2.1c
Figure 6 2 1c. View in document p.264
Figure 6.2.2a
Figure 6 2 2a. View in document p.267

References

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