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 London ProQuest Number: 10105625

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ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Abstract

The work presented in this thesis investigates the protective effects of the heat shock proteins in neurons against the cytopathic effects of 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 of 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 of 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 of a range of 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.

The heat shock transcription factor, HSFl, stimulates the transcription of the heat shock genes in response to stress. Using a constitutively active deletion mutant of HSFl, this thesis profiles the expression of the hsps in response to recombinant HSV-1 vector mediated delivery and overexpression of the H-BH gene in neuronal cells, and also characterises the protective effects of overexpression of the mutant in similar conditions as described above. Acknowledgements

I would like to thank Professor David S. Latchman for all his support, ideas and words of encouragement throughout the duration of 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 of 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 of 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 of Marcus Wagstaff. Contributions by other researchers to the work presented is acknowledged below: 1) Mr. Benjamin S. Aspey, at the Reta Lila Weston Institute of 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. 3) Rat dorsal root ganghon ceUs were dissected and initiaUy cultured by Ms. Elizabeth Ensor at the department of Molecular Pathology, University CoUege London Medical School. Abbreviations

Ab Antibody APS Ammonium persulphate ATP/ADP/AMP Adeno sine tripho sphate/dipho sphate/monopho sphate bp Base pairs BDNF Brain derived neurotrophic factor BiP Immunoglobulin heavy chain binding protein BGH Bovine growth hormone BHK Baby hamster kidney BSA Bovine serum albumin cAMP N6,2’-0-Dibutyryladenosine3 ’ : 5 ’-cychc monophosphate CAT Chloramphenicol acetyl transferase cDNA Conq)lementary DNA CMC Carboxymethyl cellulose CMV Cytomegalovirus CNS Central nervous system CPE Cytopathic ejffect cpm Counts per minute ddHzO Double distilled water DEPC Diethyl pyro carbonate DHFR Dihydrofolate reductase DMEM Dulbecco’s modified Eagle’s medium DMSO Dimethylsulfoxide DNA Deoxyribonucleic acid DRG Dorsal root ganghon E Early (gene) ECL Enhanced chemiluminescence EDTA Diaminoethanetetra-acetic acid, disodium salt EMCV Encephalomyocarditis virus ER Endoplasmic reticulum ECS Foetal calf serum 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 I5 Medium Liebowitz’s 15 medium LMP Agarose Low melting point agarose LTR Long terminal repeat LZ Leucine zipper MAP Mitogen-activated protein MAP2 Microtubule associated protein 2 MWt Molecular weight MCA(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 TRiC TCP-1 ring complex 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 of several classes of heat shock proteins and their corresponding mRNAs. In Press. Mol. Brain. Res. 42 (2), 236-244.

2) CoflSn R.S., Thomas S.K., Thomas N.S.B., LUley C.E., Pizzey A.R., Griffiths C.H., Gibb B.J., Wagstaff M.J.D., Inges S.J., Binks M.H., Chain B.M., Thrasher A T, Rutault K., Latchman D.S. (1997) Pure populations of transduced primary human ceUs can be produced using GFP expressing herpes virus vectors and flow cytometry. Submitted to Gene Therapy. 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 of the Heat Shock Proteins in Cell Stress 43 1.1.10 The Heat Shock Transcription Factors and HSP Gene Regulation 46

1.2 Cerebral Ischaemia 53 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 of 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 of 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 of Bacteria 104 2.2.3 E. coli Transformation 105 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 of 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 of 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 of 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 of 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 2.6.1 Mammahan Cell Lines and Primary Neuronal Cells 125

10 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 of Viral Recombinants Expressing lacZ Reporter Gene 130 2.6.7 Detection of 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 of 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 of 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 of 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 3.1.1 Aims 144

11 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 4.3.2 Preparation of HSV-1 Regions Flanking IE2 182

12 4.3.3a Insertion of 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 oflacZ 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 of 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 oflacZ 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 Chloramphenicol Acetyltransferase (CAT) Assay 227

13 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+pMl 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

REFERENCES 292

14 INDEX OF FiGURES

Chapter 1: 1.1.2 Model for the Reaction Cycle of 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 of Ischaemia During Permanent MCAO 148 3.2.2 Hsp27 and (3-tubulin Protein Levels m the Core Region of Ischaemia During Permanent MCAO 148 3.3.1 Hsp56 mRNA Levels in the Core Region of 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 Permanent MCAO 154

15 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 of 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 of IE2-Deleted Flanking Regions 183 4.3.3 The Construction of 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 of 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 B130/2 CeUs Infected with the 17+pR16R 90 and 17+pR16R 70 Recombinant HSV-1 Vectors 197

Chapter 5: 5.3 Structure of the pR19 lacZ Plasmid 206 5.3.1 Construction of the pNot3. 5cDNA3 Plasmid 208 5.3.2 Maps of the pR19 Constructs Containing Transgenes 212

16 5.4.1 Detection of 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 of the HumanHSP70B Promoter by Transient Overexpression of the HSFl Mutant H-BH 229 5.6.1 Characterisation of Expression of 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 of the pMl Plasmid 245 5.7.5 Detection of 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 of 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 H-BH Genes 265

17 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 of 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

Chapter 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 of Exp erimental Conditions for Ischaemic Tolerance Experiments 66 1.3 Methods o f/« v/vo Gene Delivery to the Central Nervous System 76

Chapter 2: 2.1 DNA Plasmids 102 2.2 Antibodies Used in this Thesis, their Sources and Conditions of Use 123

Chapter 3: 3.1 cDNA Templates Used for Random Prime Probe Generation 146

Chapter 5: 5.1 Antibodies Used for detection of HSP Transgene Expression on 217 Western Blots

18 Chapter 1

Introduction

19 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 of overexpressing the heat shock proteins (hsps) in neurons, against various insults such as heat shock and ischaemia. Overexpression of 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 of 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 of 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 of the heat shock proteins (hsps) m cerebral ischaemia, an understanding of the properties of the different famihes and their regulation is necessary.

The heat shock proteins are, by definition, proteins expressed in greater quantities in response to elevated temperatures relative to the organism’s optimum growth temperature. It is now well demonstrated that the levels of all the hsps can also be increased in response to other cellular insults such as metal ions and ischaemia. Some have distinct expression profiles during development, cellular differentiation and the cell cycle. The more recent title of ‘stress proteins’ therefore appears to be more suitable, but not fiiUy satisfactory. Members of aU the hsp families are expressed constitutively and some are essential to cell survival and are involved in signal

20 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 of them share similar functions and some are intimately linked in events along the same cellular pathway.

The hsps have been grouped into famihes, classified by their molecular weight in kiloDaltons. Conflicting data concerning the molecular weights has led to a somewhat confiising nomenclature. To avoid confusion, a summary of the hsp nomenclature used throughout this thesis along with its synonyms is illustrated in Table 1.1.

21 Table 1.1 - The Function and Nomenclature of the Heat Shock Proteins Heat Shock Protein Other Names Proposed Function(s) I Hsp27 Hsp25 (murine) Binds actin filaments. Chaperones folding of citrate synthase. Role in thermotolerance. Hsp32 Heme oxygenase 1 Catalyses degradation of (HO-1) haem. Hsp47 Collagen-specific chaperone. Hsp56 P59, FKBP59 Peptidylprolyl cis-trans isomerase. Maintains inactive form of steroid receptor. FK506 binding protein. Hsp60 Chaperonin Provides environment for GroEL (prokaryotic) mitochondrial protein folding. Hsp72 Hsp70, hsp70i Role in thermotolerance. Hsp73 Hsc70, hsc73, clathrin Binds unfolded proteins. uncoating ATPase Uncoats clathrin baskets. Targets proteins for lysosomal degradation. BiP Grp78, Binds to unfolded Immunoglobulin heavy proteins in endoplasmic chain binding protein 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 demonstrated with the cellular homologue pp60"^'". Binds actin, tubulin. Role in thermotolerance. Hsp110 Role in thermotolerance Ubiquitin Protein Degradation

22 The phenomenon of 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 of a range of proteins on heat shock (Tissieres et al., 1974). Since then, the wealth of 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 of the individual hsps and their regulation. It is worth noting at this stage, however, that many of the hsps function as ‘molecular chaperones’. R. John EUis defined ‘molecular chaperone’ as ‘...a family of unrelated classes of protein that mediate the correct assembly of 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 of polypeptide chains into functional native proteins foUowing their de novo synthesis and during recovery fi*om ceUular insult (Gething and Sambrook, 1992).

In this first part of the introduction, the hsps wiU be discussed indrviduaUy. They are not reviewed m order of molecular weight. TTiere are several reasons for this. Firstly, the hsp70 family is the most studied of the hsps and is therefore discussed first. Hsp60 or GroEL works downstream of hsp70 in the protein folding pathway and is therefore discussed second. The steroid receptor associated hsps (hsp90 and hsp56) are then discussed consecutively, and subsequently the smaU hsps (hsp27 and hsp32) are

23 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 of the hsps in cell stress. A discussion of 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 of the most characterised of 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 of 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 of 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 of hsps from the same family, the functions of any overexpressed hsp may overlap with the other members.

In mammals, cytosohc hsp70 (hsc70, hsp73, hsc73) is constitutively expressed and is involved in the translocation of non-native precursor proteins from the ribosome into the endoplasmic reticulum (ER) (Wiech et al., 1993; Zimmermann et al., 1988). Native proteins cannot cross organellar membranes, and the cytosohc hsp70s have been demonstrated to be required to maintain the polypeptide chain in an unfolded, protease

24 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 of the ER The SRP is then displaced and translation continues simultaneously with translocation of the newly formed polypeptide into the lumen of the ER

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 of the N- terminus of 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 of 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 of 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).

The constitutive cytoplasmic member of the human hsp family, heat shock cognate 73 (hsc73), has been demonstrated to bind preferentially to denatured proteins bearing the

25 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 of its polygonal basket-like structure. The basket is composed of clathrin hght and heavy chains in heptamers (three of 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 of 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).

Cytoplasmic hsp70 may also be in^ortant in enabling steroid aporeceptor assembly into a high-affinity hgand binding conformation (see Section 1.1.4).

26 A member of 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% of mitochondrial proteins are encoded in the nucleus. The mechanisms of 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’ of the precursor from folded to unfolded states on the cytoplasmic side, therefore, would lead to a step-by-step transfer of 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.

The mechanism by which denatured proteins bind to members of the hsp70 family throughout the cell and its compartments is essentially the same and has been best characterised in the prokaryote, E. coli and is illustrated in Figure 1.1.2 (Frydman and Hartl, 1994). ATP is required for the binding/release cycle, but its hydrolysis is not, as was originally thought, the mechanism for substrate release. Each DnaK molecule (hsp70 in eukaryotes) is bound to ATP at its N-terminal domain. The cycle commences when unfolded protein interacts with the DnaK and the protein/hsp conq)lex recruits a smaller chaperone, DnaJ. The bound ATP is hydrolysed to ADP which causes the formation of a tight ternary complex. GrpE then causes the dissociation of ADP from the DnaK, weakening the interaction between DnaK and the unfolded protein allowing protein release and potential transfer to GroEL (hsp60) if this is required for correct folding, or it may return to the DnaK/J conq)lex. The DnaJ molecule dissociates, ATP is bound, and the cycle starts again. DnaJ itself has been demonstrated to co­

27 precipitate with nascent chains of firefly luciferase and chloramphenicol acetyltransferase (CAT) early in their translation, and this is thought to target binding of DnaK, therefore it can be proposed that DnaJ may possess a direct chaperone fimction of its own (Langer et a l, 1992).

28 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 of a tight ternary complex. 3. GrpE causes the dissociation of ADP from DnaK, which weakens the interaction between DnaK and U allowing protein transfer to GroEL. 4. Upon ATP binding to DnaK, the ternary complex dissociates. U may fold to the native state (N), rebind to DnaJ/DnaK, or be transferred to hsp60, GroEL, dependent on the folding properties of the substrate protein.

29 DnaK

ADPfOrpE

GroEL

30 The afBnity of 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 of 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 of 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 of 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 of 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 of two heptameric rings stacked one on top of 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.

Protein folding is enabled with the binding of GroES, an hsp with a single heptameric ring structure (GoloubinofiF et al., 1989a). In the eukaryotic mitochondria, it is interesting to note that the hsp60 subunits require the hsp60 ohgomer for their own

31 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 of 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 Mdjlp, respectively) (Rowley et al., 1994). Imported dihydrofolate reductase (DHFR) was demonstrated to be less thermally stable at 3TC in Mdjlp 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 of 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 of subunits to make a functional conplex.

As a result of the various studies inE. 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.

Although no hsp60 homologue has been isolated from the eukaryotic cytosol, yeast. Drosophila, and plants have all been demonstrated to contain a cytoplasmic protein

32 entitled t-complex polypeptide (TCP-1) (Hendrick and Hartl, 1993). This protein is part of a hetero-oligomeric con^lex, TCP-1 ring con^lex (TRiC) which is comprised of eight to ten different polypeptides (Frydman et aL, 1992; Lewis et a l, 1992). Temperature-sensitive yeast mutants for TCP-1 are defective in mitotic spindle assembly (Ursic and Culbertson, 1991) and are therefore concluded to be important in microtubule-associated processes. These proteins bear a weak homology with GroEL/GroES, which is stronger in the putative nucleotide-binding sites (Frydman et al., 1992; Gupta, 1990). No associated protein homologous to GroES has yet been identified. TRiC is not inducible by heat stress, and is therefore not classed as an hsp, but its constituents may form members of a new class of proteins.

33 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 of a heptameric GroEL ring. D (hghtface), low ADP-afiBnity binding state. T, ATP-binding state. U and N, unfolded and native protein, respectively. In this model, the GroEL/GroES conçlex is favoured under physiological conditions (1). Upon binding of unfolded protein to GroEL, ADP and consequently GroES dissociate (2). ATP binding weakens the interaction between the GroEL and substrate protein and causes GroES to rebind (3). ATP hydrolysis causes release of the protein within the GroEL cavity, allowing it to fold (4). Generation of the ADP state increases binding afiBnity of GroEL for GroES. Folding to the native state is either con^leted, or the protein is rebound by GroEL in a partially folded form (5), re-entering the cycle at step 2.

34 7+7 ADP

7+7 ATP

7+7 Pi

N

y

35 1.1.4 The Heat Shock Protein 90 Family and the Untransformed Steroid Receptor Complex

The gene encoding the E. coli homologue of 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 of 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 of 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 of a particular amino acid sequence (Whitelaw et a l, 1993; Chambraud et a l, 1990).

The majority of 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 of hsp90 (Picard et a l, 1990). The interaction between hsp70 and the complex is discussed below.

When the hormone Hgand binds to the receptor, the hsps dissociate from the complex, producing an activated receptor/hormone complex that is capable of DNA binding. In

36 the dioxin receptor (DR), the hgand binds within a 200 amino acid domain that also binds hsp90, and deletions in the hsp90 binding domain of 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 of mutants of 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 of 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 of 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 3TC (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 of 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 of 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 of ATP-binding proteins. Indeed, lysate depleted of hsp70 only form hsp90- receptor complexes on the addition of 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, 1993).

37 1.1.5 Hsp56 and the Peptidyl Prolyl Isomerases

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 of the slow steps in protein folding, and so hsp56 plays a catalytic role in the process of 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 al, 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.

The other notable property of hsp56 is as an immunophilin in that it binds to the synthetic immunosuppressant FK506. Glucocorticoids are also immunosuppressants and hsp56 therefore, has been inq)hcated in the immunosuppressant effect of FK506. Indeed, the response to glucocorticoids is ten times more sensitive in the presence of FK506 than in untreated cells, but the level of maximal transcription is unchanged (Ning and Sanchez, 1993). The effect of FK506 on hormone-mediated receptor dissociation is a matter of debate. Hutchison et a l, demonstrated no effect on treatment with the immunosuppressant in vitro, on dissociation, nuclear locahsation, or maximal transcription, but these studies were carried out at saturating hormone concentrations (Hutchison et a l, 1993). FK506 has, however, been shown to attenuate the activity of the mdrl transmembrane P-glycoprotein, which has been shown to export dexamethasone, and therefore FK506 may potentiate the glucocorticoid response, by increasing concentrations of the hgand in the cell (Ueda et a l, 1992). Other FK506 binding proteins exist. One such protein is FKBP12 which stabihses the calcium releasing ryanodine receptor in muscle cehs (Brûlantes et a l, 1994). It has also been shown that FK506/FKBP12 complexes inactivate the serine/threonine phosphatase calcineurin, which blocks Ca^-dependent T- and B-lymphocyte immune responses, in a similar fashion to the immunosuppressant cyclosporin A (Kay, 1996). Whether the direct binding of FK506 to hsp56 has any immunosuppressive or other effects on cehs has not been demonstrated.

38 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 of the sequences of the small hsps of 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 of the protein, possibly highhghting the importance of these regions (Ingoha and Craig, 1982; Wistow, 1985; Southgate et al., 1983). These sections include the phosphorylation sites of the proteins. Further analysis of the small hsps of several species suggests that they are derived from a common ancestral gene (Wistow, 1985). The quartemary structure of 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.

The induction of the small hsps appears to occur later than hsp70 in response to heat stress. The constitutive level of expression is low and varied amongst differing mammahan cell lines, and can be virtually undetectable in some e.g. murine NIH3T3 (Klemenz et al., 1993). Mammahan cells show an increase in levels of hsp27 on the administration of steroids, and an oestrogen response element has been located in the promoter in close proximity to the TATA box (Fuqua et al., 1989; Gaestel et al..

39 1993). Hsp27 has also been shown to accumulate in various tissues during development, in the absence of stress, e.g. in neurons of 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 of citrate synthase, a-glucosidase and P-L-crystallin, however its range of specificity is not known, and it therefore may chaperone many more non­ native proteins (Jakob et al., 1993; Merck et al., 1993).

A second proposed role of hsp27 in unstressed cells is as a capping protein for actin (Miron et al., 1991; Miron et al., 1988). Overexpression of hsp27 in Chinese hamster cells induces an increase in filamentous actin (F-actin) and pinocytic activity in the cell cortex (Lavoie et al., 1993b). Constitutively, hsp27 exists as an unphosphorylated monomer. Phosphorylation (primarily of serine residues) of hsp27 however appears to be essential for its function (Crete and Landry, 1990; Landry et al., 1992). On amino acid sequence analysis, up to 20% of the protein structure is susceptible to phosphorylation (21 serine, 13 threonine and 5 tyrosine residues) (Hickey et al., 1986). Overexpression of a non-phosphorylatable mutant of hsp27 reduced cortical F-actin concentrations and pinocytic activity, which is dependent on actin dynamics. Overexpression of hsp27 in fibroblasts doubled the concentration of F-actin in response to the addition of the growth factors fibroblast growth factor (FGF) and thrombin. Growth factors stimulate the downstream phosphorylation of hsp27 through pp45-54 kinase {vide infra), and dissociation of hsp27 fiom the barbed end of F-actin occurs on phosphorylation. Therefore, it has been proposed that, in cells overexpressing hsp27, a greater proportion of hsp27 is bound to the barbed ends of F- actin than other cap-binding proteins. Thus on growth factor stimulation, the resultant dissociation of phosphorylated hsp27 frees more barbed ends to enable further elongation of the F-actin polymer. Phosphorylation of hsp27 however, has been demonstrated by one group not to be essential for its chaperone function or its abihty to induce thermotolerance (Knauf e/ al., 1994).

40 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 of 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 of 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 of 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

Hsp47 is a collagen-specific molecular chaperone that is raised in response to heat shock in chick embryo fibroblasts (Nagata et al., 1986). It binds to collagen type I and rv and gelatin. Hsp47 is a phosphoprotein, but it has been shown that unlike hsp27, heat shock does not affect the degree of phosphorylation whereas during mahgnant transformation hsp47 is increasingly phosphorylated but the mRNA levels decrease

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

Despite being one of 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 of a subfamily of the hsp70 group of proteins (Lee-Yoon et al., 1995). Particular amino acid sequence similarity with members of the hsp 70 family is found in the ATP-binding domain, with further similarity in conserved regions in the carboxyl- terminal two thirds of 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 of 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.

Ubiquitin is a highly conserved hsp of 7-8kDa that is expressed in ah eukaryotic cehs (Jentsch, 1992). It functions to target proteins for degradation by the 26S protease. Heat shock induces a burst of protein degradation which coincides with an increase in multi-ubiquitin-protein conjugates (Parag et al., 1987). Ubiquitin-conjugating enzymes catalyse the conjugation of denatured proteins to a terminal glycine residue on ubiquitin in an ATP dependent manner. In a mammahan ceh line, expressing a temperature-dependent mutation in one of the ubiquitin-activating enzymes in the pathway (El), the increase in temperature did not produce the characteristic increase in proteolytic activity, and therefore its role is to dispose of denatured protein, in contrast with the other hsps which prevent protein aggregation and promote correct refolding (Parag et al., 1987). Hsc70 has been demonstrated to be required for ubiquitin- targeted degradation of some, but not ah, proteins (e.g. actin, a-crystahin), possibly acting by unfolding peptides to expose a ubiquitin hgase-binding site (Bercovich et al., 1997). Ubiquitin also has been shown to conjugate with histones H2A and H2B, neutrahsing the charged lysine residue on the molecule, reducing the net positive charge (Busch and Goldknopf^ 1981). The ubiquitinated H2A molecule exists as a minority but has been demonstrated in Drosophila to be preferentiahy locahsed in

42 nucleosomes containing active genes and therefore ubiquitination may serve to alter nncleo some-nncleo some interactions preventing the formation of 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 of 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 of these two facts suggests that hsps may be protective against cell stress and therefore responsible for this thermotolerant’ phenomenon. There are several lines of evidence to substantiate this theory (reviewed by Lindquist and Craig, 1988). 1) On heat shock, the induction of 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. 5) Mammahan ceh lines, e.g. Chinese hamster fibroblasts, selected for their abihty to survive exposure to high tenq)eratures constitutively express hsp70 at higher levels than the parental line; whereas E. coli and Dictyostelium that are selected for their inabihty to acquire thermotolerance, are defective in hsp synthesis.

43 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 of 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 of 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 of 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; Kruuvet al., 1983). Mild heat pre-treatment reduces the extent of these perturbations or increases the rate of their repair.

The roles of 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.

HspTO’s afBnity for the exposed hydrophobic regions of denatured proteins and its abihty to communicate these proteins to the ER, mitochondria, hsp60 and lysosomes link it to these mechanisms. On heat shock, hspTO is distributed around ceh membranes, and in the nucleus - particularly in the nucleolus. Indeed, constitutive expression of Drosophila hsp70 accelerates the repair of nucleolar morphology after

44 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 of 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 al, 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 of the steroid aporeceptor complex.

Thermotolerant Chinese hamster cells have been shown to express higher levels of hsp27 (Chretien and Landry, 1988). In hsp27 transformants the actin network was dramatically protected from disruption during heat shock (Landry et al., 1989; Lavoie et al., 1993a; Lavoie et al., 1993b). On heat shock, hsp27 is phosphorylated and therefore may promote actin polymerisation thus stabihsing the cyto skeleton and enabling its rapid reconstruction. In vitro, the murine analogue of hsp27 (hsp25) suppresses the heat-induced aggregation of a-glucosidase and P-L-crystallin and hsp27 has been shown to prevent aggregation and promote the refolding of other denatured substrates (Mercket al., 1993; Jakob et al., 1993). These chaperone fimctions of hsp27 are unique in that they are independent of ATP. Hsp27 has also been demonstrated to inhibit serine proteases such as elastase, which may be hpportant in preventing ceh degradation foUowing heat shock (Merck et al., 1993).

45 Hsp27 has also been demonstrated to block the signals for ap opto sis conferred by the stimulation of the Fas/APOl receptor and addition of 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 Gene Regulation

Heat shock gene transcription is induced by a variety of factors. In particular a family of transcription factors, the heat shock factors (HSFs) have been identified which stimulate or repress heat shock gene transcription under different circumstances. Until recently three HSFs had been described - HSFl, 2 and 3. The first two have been

46 demonstrated in humans, S. cerevisiae, Drosophila and mice, the third has been isolated from chickens (Schuetz et al, 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).

HSFl is constitutively expressed and exists in the cytosol and nucleus as a 57kD monomer. The 529 amino acid (in humans) protem has four leucine zipper motifs, one in the carboxy terminus and three downstream of the DNA-binding domain in the amino terminus. In unstressed cells, the monomer exists in a coiled-coil form through hydrophobic interactions between the heptad repeats of the leucine zipper motifs in the amino and carboxy regions (Clos et a l, 1990; Sorger and Nelson, 1989). Deletion studies suggest that the transcriptional activation domain of human HSFl is located downstream of residue 378 (total residues = 529) in the carboxy terminus region (Zuo et a l, 1995). The first step of HSFl activation as a result of cell stress is the disruption of the leucine zipper interactions (see Figure 1.1.10a). The amino terminus leucine zipper motifs between three uncoiled HSFl monomers can then interact resulting in trimérisation (Rabindran et a l, 1993). The trimer is phosphorylated and translocates to the nucleus to initiate transcription (Rabindran et a l, 1993; Sarge et a l, 1993; Westwood et a l, 1991). HSF2 activation, however, is induced by the hemin-induced differentiation of erythroleukaemic cells, and is proposed to have a developmental role (Sistonen et a l, 1994; Sistonen et a l, 1992). HSF3 activation in avian cells, like HSFl, is induced by heat shock sodium arsenite and which stimulates nuclear translocation, trimérisation and acquisition of DNA-binding activity. The threshold temperature for HSF3 activation, however, is much higher (45°C as opposed to 41®C for HSFl) and the sodium arsenite concentration required is greater compared to HSFl. Once activated, the transcriptional activity of HSF3 is as strong as that of HSFl (Tanabe et a l, 1997).

47 LLLL LLLL LLLL LLLL

HSF MONOMER LLLL LLLL

HEAT SHOCK ENZYMATIC MODIFICATION? CHAPERONES?

LLLL* HSF TRIMER 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. Maintenance of the monomeric state depends on the C-terminal hydrophobic repeats, which may interact directly with the N-terminal repeats to suppress trimérisation. Upon heat shock, monomer structure is disrupted, and trimers form, presumably through a triple-stranded coiled coil. The mechanism of control of the monomer-trimer transition is unknown, but it could be dependent on the modifications of HSF, on interactions of HSF with chaperones, and on physical changes in the environment.

48 The action of 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 of three of these 5 base pair (bp) elements are required for high afiBnity HSFl binding (Xiao et al, 1991). Dififerent HSEs have dififerent numbers of these 5bp sequences but they usually range firom three to six, and dififerent heat shock promoters have dififerent numbers of HSEs, with varying distance between them (Amin et a i, 1988; Xiao et al., 1991). Analysis of deletion mutants of the human HSFl has demonstrated that the trimérisation and DNA binding events are regulated separately jfrom the transactivation of 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 of DNA binding. It is therefore beheved that this disruption of the monomeric structure is the first event, triggered in some way by cell stress, in the activation of HSFl and the transactivation of 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 of 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 of HSE controlled genes in particular tissues.

In Drosophila, it has been demonstrated that the heat shock region is maintained in a nucleosome-firee state, maintained by the constitutive binding of GAGA factor to its consensus sequence in the heat shock promoter (Wu, 1984). The TATA binding protein (TBP) of the transcription initiation coroplex TFBD is bound constitutively to

49 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 of 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 of 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.

The factors responsible for phosphorylating HSF have not been defined. The regulation of the heat shock response is, however, in part regulated by hsp70 (Mosser et at., 1993; Abravaya et a l, 1992). The activation of HSF may be due to an autoregulatory cycle of interactions between itself and hsp70. When misfolded proteins accumulate in the cytosol of stressed cells, hsp70 is sequestered thus freeing HSF for activation (see Figure 1.1.10b, (Morimoto et at., 1994)). Addition of exogenous hsp70 to HeLa cells blocks the in vitro activation of HSF, which is reheved by the addition of ATP, which in the hsp70 binding model {vide supra) causes complex dissociation. It is not known whether hsp70 binds directly to HSF, but evidence suggests that HSF exists as a coiled-coil monomer, not a complex, therefore any interaction is probably transient. It may be that hsp70 only binds to activated HSF, as has been demonstrated with hsp90 (Nadeau et a l, 1993). Once the hsp70 is sequestered, the HSF can trimerise and therefore acquire DNA binding activity. It then undergoes serine phosphorylation, by an unidentified factor and activates transcription. As the levels of hsp70 rise, they may bind to the DNA-bound HSF which then dissociates from the DNA and the trimer may subsequently dissociate, returning to the monomeric constitutive state.

50 I H hsp70

P P

Heat Shock Factor Cycle

5* nnGAAnnTTCnnGAAnn 3*

HSF p Trim er 4 - HSF (______) M onom er Heat Shock

\ hsp70 \T hsp70 binding to non-native poiypeptide Figure 1.1.10b - The HSF Cycle; A Model of HSF Regulation (Morimoto et aU 1994) lu the unstressed cell, HSFl is maintained in a monomeric, non-DNA binding form through its interactions with hsp70 (1). Upon heat shock or other forms of stress, HSFl assembles into a trimer (2). HSFl trimers bind to a specific sequence element, the heat shock element in heat shock gene promoters (3) and becomes phosphorylated (4). Transcriptional activation of the heat shock genes leads to increased levels of hsp70 and to formation of an HSFl-hsp70 complex (5). Finally, HSFl dissociates and is eventually converted to non-DNA-binding monomers (6).

51 If the deletion mutant of HSFl (H-BH) activates the HSP70B promoter in the absence of stress, then overexpression of this mutant in neurons achieved through gene delivery may stimulate the transcription and subsequent overexpression of the hsps and therefore protect the cells from insult. H-BH is therefore a candidate, along with the separate hsps for studying the protective effect of the hsps in neurons.

52 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 of 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 of ischaemic tolerance and the protective effects of overexpressing the hsps in vitro are reviewed.

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

As the result of a mild, transient global ischaemic insult, such as during cardiac arrest, the particularly vulnerable pyramidal neurons of the CAl and CA4 zones of the hippocampus are destroyed (Kirino et al., 1985). The CAl neurons exhibit a characteristic delayed neuronal death visible on microscopy after at least two days

53 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 of 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 of cerebral ischaemia and the consequent reperfiision is a comphcated process with multiple pathways. The deprivation of 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 of 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 of global ischaemia (Pulsinelh et al., 1982).

As the energy demands of the brain exceed its cap abihty to synthesise ATP the brain utihses its low stores of glycogen and glucose and it metabohses these anaerobicahy and lactic acid accumulates (Combs et al., 1990). The free hydrogen ions released as a result of this acidosis facihtate ferrous iron-mediated fiee-radical mechanisms and is thought to be inq)ortant in astroghal injury (Siesjo et al., 1989).

54 As a result of the loss of ATP the energy-dependent mechanisms of the brain, such as ion pun^s, faü (Siesjo, 1992). Sodium, chloride and calcium ions enter and potassium ions leave the cell. The membrane ion gradients consequently deteriorate and the membrane depolarises (anoxic depolarisation). This influx of sodium, chloride and calcium and efidux of potassium ions causes the release of neurotransmitters such as glutamate, aspartate, dopamine and GAB A in greatly increased concentrations (Benveniste et al., 1984; Hagberg et a i, 1985; Globus et a l, 1988; Hagberg et a l, 1987).

In the normal state, the ratio of intracellular to extracellular Ca^^ concentration is 1:10"^-10^. This gradient is maintained by ATP dependent calcium pumps in the cell membrane, endoplasmic reticulum (ER) and mitochondria. On energy depletion, these punq)s fail and the gradient is compromised (Siesjo and Bengtsson, 1989). The collapse of calcium homeostasis has been proposed as the major cause of cell death during severe ischaemia. The increased levels of extracellular glutamate activate NMDA hgand-regulated calcium channels further potentiating the influx (Meldrum, 1990). The depolarisation also activates voltage sensitive calcium channels (VSCC). The increasing intracellular concentration of sodium drives the sodium-calcium exchange punq)s in the mitochondrial and cell membrane further increasing the intracellular calcium concentration. Although this calcium influx has been shown not to be the sole mechanism for cell death in anoxia and energy-depletion, it is thought to accelerate many of the injurious processes (Choi, 1987).

Excitatory amino acids bind to the AMPA metabotropic receptor, hydrolysing phosphatidyl inositol diphosphate into the second messengers diacylglycerol and inositol triphosphate, which stimulates the release of calcium ions sequestered in non- mitochondrial stores (Ross et a l, 1989). Phosphohpases are also activated by calcium, and they catalyse the release of free fatty acids from membrane-bound glycerophosphohpids and these in turn facihtate free-radical peroxidation of other membrane hpids.

55 The action of phospholipase A 2 on the arachidonic acid pathway causes the production of leukotrienes, thromboxanes and prostaglandins that are catabohsed by calcium- mediated process to generate free-radical species (Pulsinelli, 1992). The increased levels of the inflammatory mediators of the phosphohpase A 2 pathway also induce vasoconstriction, platelet aggregation and the infiltration of leukocytes into the ischaemic area. Calcium also activates proteases that lyse structural proteins (Seubert et a l, 1989). Calpain I, a calcium-dependent cysteine protease which breaks down spectrin, and a calcium-dependent protease converts xanthine dehydrogenase into an oxidase which causes the production of more free radicals from hypoxanthine (McCord, 1985). Nitric oxide synthase 1 is also activated by calcium which also initiates free-radical mechanisms (Garthwaite, 1991).

Free radicals are directly toxic to the cell and therefore may be the final stage in many of the above pathways that lead to cell death. O 2 and OH and H 2 O2 in the presence of free iron are imphcated in the non-specific damage of lipids, proteins and nucleic acids in ischaemia. It is the peroxidation of membrane polyunsaturated fatty acids that is thought to be the major candidate for the final stage in cell death (Harrison, 1992). Although present in low quantities constitutively, the calcium-dependent processes leading to the cataboHsm of prostaglandins and activation of xanthine oxidase and nitric oxide synthase increase free radical generation (McCord, 1985; Garthwaite, 1991). Free radical levels are difficult to detect, and so only indirect evidence of their role in cell death is available (Schmidley, 1990). Free radical scavengers exist in the cell, for example vitamins C (ascorbate) and E (a-tocopherol), and protective enzymes have also been characterised (superoxide dismutase, catalase and glutathione reductase). Transgenic mice overexpressing superoxide dismutase levels are protected from cell death in ischaemia, supporting the theoretical role of free radicals in ischaemic cell death (Chan et a l, 1990; Imaizumi et a l, 1990).

The cell death mechanism described above, caused by the loss of ATP the resultant action of exogenous factors is termed necrosis. A second mechanism of cell death recognised in neurons, particularly during the developmental modelling of the nervous system, occurs when endogenous factors cause the cell to enter a sequence of events.

56 that result in DNA fragmentation and ‘cell suicide’. This programmed cell death or apoptosis is now considered a cause of cell death in the event of ischaemic insult (Linniket al., 1993; Li et al., 1995).

Apoptosis is characteristically associated with a native endonuclease-induced laddering of DNA into 180-200 base pair multiples and the formation of intact cellular fragments called ‘apoptotic bodies’ (Wylhe, 1980). Apoptosis usually occurs after cells arrest in the early stage of the cell cycle, and even post-mitotic neurons still contain low levels of cell cycle regulatory proteins such as cyclin D1 which may be expressed during programmed cell death in neurons (Freeman et al., 1994). p53, a cell cycle regulator that can function to arrest growth at this early stage can also arrest tumour growth and induce apoptosis (Levine et al., 1991; Clarke et al., 1993). p53 expression is increased during middle cerebral artery occlusion in the rat and it has therefore been inq)hcated in the induction of apoptosis m post-ischaemic neurons (Chopp et al., 1992; Li et al., 1994). Transgenic mice lacking in p53 demonstrate smaller infarct volumes following middle cerebral artery occlusion (Crumrine et al., 1994). Over-expression of the apoptotic inhibitor gene bcl-2 reduces neuronal cell death both in vitro by decreasing the generation of oxygen reactive free radicals, and from axotomy in vivo (Garcia et al., 1992; Dubois Dauphin et al., 1994; Kane et al., 1993). p53 activates the transcription of hax genes and a p53-dependent negative response element is located in the bcl-2 gene (Miyashita et al., 1994). Bax genes are part of the same family of proteins as bcl-2 and the balance between these and other members of this gene family {bad, bcl-x) may exert positive and negative effects on cell death (Reed, 1994; Raff et al., 1993). The interactive roles of these genes and their products have not as yet been defined in regards to cell death during ischaemia.

1.2.3 Changes in Gene Expression During Cerebral Ischaemia

Despite the inhibition of protein synthesis that has been demonstrated during delayed neuronal death, such as occurs in CAl neurons as a result of global ischaemia, three

57 categories of genes are specifically expressed and many products have been demonstrated to be raised (Tbilmann et al., 1986; Matsushima et al., 1996). Fnstly the ‘immediate early genes’ (lEGs) are expressed. Ibis group is mainly corrq)rised of proto-oncogenic products such as c-fos, fos B, c-jun and jun B which homo- and heterodimerise to form transcription factors that bind to AP I or CRE (cychc AMP response elements) sites and may regulate the expression of the second category of genes, the ‘late effector genes’ (LEGs) (Morgan and Curran, 1991). These ultimately include enzymes, neurotransmitters, structural proteins and growth factors such as nerve growth factor (NGF) (Hengerer et al., 1990; Sheng and Greenberg, 1990). The third group of genes expressed is conq)rised of the heat shock genes. The protein products of these are thought to be protective against cell death not only during high tenq)eratures but also during other stresses such as ischaemia.

The levels of lEG expression have been extensively characterised during transient global cerebral ischaemia. C-fos expression is highest in two areas on observation using in situ hybridisation in the rat hippocanq)us and these areas correlated with the distribution of delayed death of neurons in this region (Jorgensen et al., 1989). It was therefore concluded that the expression c-fos may have some role in delayed cell death. Two peaks of c-fos and c-jun expression locahsed to the CAl region of the rat hippocan^us have also been demonstrated with the second peak occurring 24-48 hours after focal ischaemia (Wessel et a l, 1991). In the gerbil hippocanq)us, it has been noted that the increased transcription of c-fos and Krox-24 was not followed by translation into protein in the vulnerable CAl neurons, but increased protein levels were detected in the less vulnerable neuronal populations in the hippocampus (Kiessling et al., 1993). It has been speculated that some of these lEGs may contribute to the regulation of suicide genes, whilst others may have a downstream neuroprotective effect (Dragunow et a l, 1993; Smeyne et a l, 1993).

In transient focal ischaemia in rats, Uemura et al. demonstrated a long-lasting c-fos expression in regions adjacent to the ischaemic core, and An et al. showed that the increase in lEG expression was associated with a four- to sixfold increase in the activity of the transcription factor AP-1, a product of the dimérisation of the protein

58 products oïc-fos and c-jun (An et a i, 1993; Uemura et a l, 1991). AP-1 may therefore have a role in regulating the expression of the LEGs, inducing the production of factors that may be neuroprotective or may even induce apoptosis (Sheng and Greenberg, 1990; Smeyne a/., 1993).

The major late effector genes (LEG) that have been characterised are the neurotrophins, a neuronal family of growth factors. The principle components of this family are NGF, brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3) and neurotrophin 4/5 (NT-4/5). NGF is transiently raised in the granule cells and the CAl neurons of the hippocampus during ischaemia (Lindvall et al., 1992; Hashimoto et al., 1992; Shozuhara et al., 1992; Takeda et al., 1993). The local action of a bolus dose of NGF bas been demonstrated in gerbüs to protect the vulnerable CAl neurons from ischaemic cell death, despite the levels of high or low aflOnity NGF receptors being too low for detection (Lindsay et al., 1994; Shigeno et al., 1991; Yamamoto et al., 1992). One group, however, has not been able to demonstrate this protective effect on intraventricular infusion of NGF in rats (Becket al., 1992). However, in this case the dose of NGF was a continuous low dose infusion before, during and after the insult. Ischaemia upregulates BDNF mRNA in the rat hippocampus, although it has not been shown whether this is translated into a protein product, and the mRNA of the BDNF receptor TrkB is also upregulated after ischaemia, supporting a possible protective role for BDNF (Lindvall et al., 1992; Merho et al., 1993). Apphcation of BDNF has been demonstrated to partially protect rat CAl neurons from ischaemic cell damage (Beck et al., 1994; Tsukahara et al., 1994). Ischaemia downregulates NT-3 mRNA in the rat hippocampus, which suggests that the neuronal response to ischaemia may be specifically regulated (Takeda et al., 1992). Exogenous NT-3 has not been apphed to the hippocampus. NT-4/5 has not been characterised during cerebral ischaemia.

59 1.2.4 Heat Shock Protein Expression in Cerebral Ischaemia

The papers described in the following text are smnmarised in Table 1.2.1.

Table 1.2.1 - Summary of Reported Heat Shock Protein Expression Following Cerebral Ischaemia Reference Animal and Nature and mRNA Time of Heat Shock Duration of Protein study Protein Ischaem ia after Ischaemia (Nowak, Jr. Gerbil, hsp70? Global, 5 mins Protein 0-24 hours 1985) (Vass et a/., Gerbil, hsp70 Global, 10 mins Protein 0-96 hours 1988) (Gonzalez at Rat, hsp70 Focal, 24 hours Protein 0 hours a/., 1989) (Abe at a/., Gerbil, hsp70 Global, 10 mins mRNA 0-7 days 1991) (Simon at a/., Rat, hsp72 Global, 2-30 Protein 24 hours 1991) mins (Kawagoe at Gerbil, hsp70 Global, 0.5, 1 mRNA 1, 3 hours a/., 1992) and hsc70 and 2 mins and 2 days (Abe at a/., Gerbil, hsp70 Global, 10 mins mRNA 0-7 days 1993b) and hsc70 (Kawagoe at Rat, hsp70 and Global, 20 mins mRNA 0-7 days a/., 1993b) hsc70 protein (Abe at a/., Gerbil, hsp72, Global, 3.5 mins mRNA 0-7 days 1993a) hsc73 and hspSO (Kawagoe at Gerbil, hsp90a Global, 10 mins mRNA 0-7 days a/., 1993a) (Higashi at a/., Rat, hsp27, Focal, 0-48 mRNA 0 hours 1994) hsp47, hsp70, hours GRP78, hsp90 (Kinouchi at a/., Rat, hsp70 Focal, 4 and 24 Protein 4 and 24 1993b) hours mRNA hours (Kinouchi at a/., Rat, hsp70 Focal, 30, 60 Protein 0-7 days 1993a) and 90 mins mRNA (Kato at a/., Rat, hsp27 and Focal, 1 hour Protein 0-14 days 1995) hsp70 (Nimura at ai, Rat, hsp32 Focal, 30 mins Protein 24 hours 1996) and 2 hours

60 (Geddes et al., Rat, hsp32 Focal, 24 hours Protein 0 hours 1996) Global, 20 mins 0-72 hours (Takeda et al., Rat, hsp32 Global, 20 mins Protein 0-7 days 1996) mRNA (Takeda et al., Rat, hsp32 Global, 20 mins mRNA 0-7 days 1994) (States et al., Rat, hsp70 Focal, 1, 3 and 7 Protein 0 hours 1996) days (Higashi et al., Rat, HSFl Focal, 0-24 hours HSF DNA 0 hours 1995) binding

The induction of hsps in vivo as a result of cerebral ischaemia is well documented. Most studies have demonstrated the increased levels of the inducible member of the hsp70 family (hsp72) and its constitutive counterpart hsc70. In 1985 Thaddeus Nowak demonstrated the synthesis of a 70kDa stress protein in the gerbil in response to 5 minutes of transient global ischaemia (Nowak, Jr. 1985). The increased translation of this protein was first detectable on protein synthesis assay of whole brain preparation at 2 hours recirculation and translation reached maximal levels at 8 hours. This ‘stress’ protein was speculated to be hsp72 on the basis of migration on two dimensional protein electrophoresis. Nowak’s group went on to localise the protein induction after 10 minutes of transient global ischaemia to the regions destined to survive the insult, namely the CA3 and dentate granule cells, and this expression preceded the characteristic loss of the CAl neurons up to 96 hours reperfusion (Vass et al., 1988). In 1989 Gonzalez et al. detected increases in hsp72 immunohistochemical activity at 24 but not 4 hours after permanent MCAO in rats, and neurons were specifically stained in regions bordering the infarction zone (Gonzalez et al., 1989). This was the first detection of hsps relating to focal cerebral ischaemia. mRNA detection of hsp70 and hsc70 up to 7 days after transient (10 minutes) global ischaemia was carried out by Abe et al. who detected constitutive hsc70 expression up to 2 days with increases in hsc70 and hsp70 between 4 hours and 1 day in the parietal cortex and CAl neurons (Abe et a l, 1991). Peak expression of hsp70 was higher in the cortex, and it was postulated that CAl neurons are not raising enough hsp70 to confer protection. This study, however, did not take into account CAl neuron

61 destruction during the timecourse in parallel although the tubulin levels do appear to fall in both the cortex and CAl samples at 7 days, possibly caused by cell death. Immunohistochemical analysis by Simon et al., however demonstrated that hsp72 levels 24 hours after global ischaemia differed according to the region tested and the length of the ischaemic insult (Simon et a l, 1991). The CAl region’s peak expression was following 4 minutes of ischaemia, whereas the CA3 and cortex levels were highest at 15 and 20 minutes respectively, so it appears that the CAl neurons are expressing significant hsp70 levels during mild ischaemia.

Three papers by Kogure et al. characterised the differences in induction of hsc70 and hsp70 mRNA levels in gerbils and rats (Kawagoe et a i, 1992; Abe et al., 1993b; Kawagoe et al., 1993b). These in situ hybridisation studies demonstrated several important points: 1) Hsc70 mRNA, although constitutively expressed could be induced in the gerbil hippocanq)us by 1 minute of global ischaemia, with detectable levels at 1 hour recirculation in the dentate gyrus and in the remaining hippocampal cells at 3 hours. No change was detected m hsp70 mRNA levels up to two days recirculation. 5 minutes of ischaemia produced a co-induction of both hsps mRNAs and after 2 days hsp70 mRNA levels were localised only to the CAl neurons of the hippocampus. 2) Although both hsp mRNAs were inducible in the gerbil as the result of a 10 minute transient global ischaemic insult, at 8 hours recirculation there was a strong induction of hsp70 mRNA in the vulnerable CAl neurons, whereas in other regions the two mRNAs were induced co-operatively. This contrasts with the study in the gerbil by Vass et al. discussed previously where only minimal hsp70 immunoreactivity was detected in the CAl neurons after 10 minutes of ischaemia, however significant levels were only detected after 16 hours (Vass et al., 1988). 3) 20 minutes of global ischaemia in the rat produced a similar co-induction of both hsp mRNAs and hsp70 protein, particularly in the CAl region at one and two days, whereas the CA3 regional levels of protein had decreased by the second day. It is difficult to compare this result to the Vass et al. results because the analysis was carried out in gerbils and the duration of the insult was 10 minutes.

62 It was therefore proposed that hsp70 levels could specifically be used as a marker for ischaemic neuronal death. The reduced levels of hsp70 protein in the CAl neurons noted by Vass et al. and Simon et al may be due to a deficit in translation, whereas the studies of Kogure et al show that mRNA is increased (Simon et a l, 1991; Vass et al, 1988). As previously mentioned, CAl neurons exhibit delayed neuronal death in response to ischaemia and this is associated with an inhibition of protein synthesis in the gerbil (Tbilmann et a l, 1986).

The same group then characterised the levels of hsp60 and hsp90a mRNA in global cerebral ischaemia in the gerbil hippocampus (Abe et a l, 1993a; Kawagoe et a l, 1993a). Using in situ hybridisation, they demonstrated a transient increase of hsp60 mRNA in the CAl region between 3 hours and 2 days and in the dentate granule region between 3 hours and 1 day of recirculation afl;er 3.5 minutes of ischaemia. 10 minutes of ischaemia increased hsp90a mRNA levels in all regions of the hippocampus, particularly in the CAl region, peaking at 8 hours recirculation. No protein analysis was made for either hsp.

Hsp mRNA levels have been characterised following permanent focal cerebral ischaemia in rats. Higashi et al demonstrated by Northern blot analysis increases in the message levels of hsps 27, 47 and 70 and GRP78 (BiP) (Higashi et a l, 1994). Hsp27 and 47 mRNA levels were induced between 16 and 24 hours, inducible hsp70 and GRP78 levels were increased at only 2 hours, but the inducible levels had disappeared by 48 hours. They claimed hsp90a mRNA levels were not significantly raised, but the data was not presented. In situ hybridisation studies showed the distribution of inducible hsp70 and hsp27 during focal cerebral ischaemia. At 2 hours the ischaemic core was overexpressing hsp70 mRNA, but after 4 hours this overexpression was limited to the ischaemic penumbra. Protein levels (not shown) were claimed to be absent from the core at 2 hours, and only in the penumbra after 24 hours. Increased levels of hsp27 mRNA were initially detected in the ischaemic core at 24 hours, and then only in the penumbra at 48 hours (Higashi et a l, 1994).

63 Kinouchi et al. also demonstrated increased hsp70 protein levels exclusively in neurons in the ischaemic penumbra at 24 hours after permanent MCAO but also noted staining in endothehal cells within the infarct, whereas mRNA induction was noted at the ischaemic penumbra both within and outside the infarct (Kinouchi et al., 1993b; Kinouchi et al., 1993a). In these studies, no hsp27 protein studies were carried out. Following transient unilateral MCAO (1 hour) hsp27 protein induction was demonstrated to be locahsed to the microgha in the ischaemic centre after 4 hours recirculation and then distributed widely in astrocytes in the ipsilateral cortex with some spread into the contralateral cortex from 1 through to 14 days reperfusion (Kato et al., 1995). No specific staining was detected in the developing infarct. This study also confirmed the induction of hsp70 in the endothehal and perivascular ceUs in the ischaemic centre at 1 day reperfiision and in neurons in the penumbra at 1 and 3 days reperfiision.

Hsp32 (haemoxygenase-1 or HO-1) has also been demonstrated to be raised in both focal and global cerebral ischaemia in the rat (Nimura et al., 1996; Geddes et al., 1996; Takeda et al., 1996). 24 hours foUowing 30 minutes of transient unilateral focal ischaemia, raised hsp32 and hsp70 protein levels were noted in the gha surrounding the infarct, and the endothehal ceUs in the ischaemic core. Hsp70, unlike hsp32 was detectable in the cortical neurons in the MCA distribution region. 24 hours recovery fohowing 2 hours of focal ischaemia caused a widespread increase m hsp32 levels in the gha of the ipsilateral cortex, even outside the MCA region. Neurons only stained positive for hsp32 and hsp70 within the MCA region. Hsp32 mRNA and protein levels were raised most prominently in the gha of the hippocampus foUowing 20 minutes of global ischaemia with maximal protein levels after 12 hours recirculation (Takeda et al., 1994; Takeda et al., 1996). Hsp70 levels, as previously discussed, are raised primarily in neurons, suggesting a different pathway for induction, however hke hsp70, the CAl subfield of the hippocampus showed limited hsp32 protein induction, despite the increase in mRNA - suggesting a common deficit in translation.

Recent evidence using DNA nick-end labelling (TUNEL) to examine DNA fragmentation in the ischaemic penumbra of rats foUowing 3 days of permanent MCA

64 occlusion demonstrates that very few hsp70 stained neurons undergo DNA fragmentation (an indicator of apoptosis), which could suggest that either hsp70 is protective against apoptosis or it protects the cells from injurious processes that may result in apoptosis (States et al., 1996), Hsp70 may, however, merely be a marker for hving ceUs or cehs capable of translation and these cells may therefore survive for alternative reasons.

Higashi et al. demonstrated the increased binding of the heat shock transcription factor, heat shock factor 1 (HSFl) to its consensus sequence, the heat shock element (HSE) after 1 hour of permanent focal cerebral ischaemia, by gel mobihty shift assay (Higashi et al., 1995). They claimed this demonstrated increased activation of HSFl during focal cerebral ischaemia, however it should be remembered that HSFl activation is regulated at several stages, and increased DNA-binding cap abihty does not necessarily represent activation (Zuo et al., 1995).

65 1.2.5 ‘Ischaemic Tolerance’ and the Heat Shock Proteins in Cerebral Ischaemia

For a summary of the experimental regimens described below, please refer to Table

1. 2.2

Table 1.2.2 - Summary of Experimental Conditions for Ischaemic Tolerance Experiments Reference Animal Nature and Time Nature and Duration of Between Duration of pre-treatment T reatm ents Second Insult (Kitagawa et al., Gerbil Global, 2x2 48 hours Global, 5 mins 1990) mins at one day intervals (Kitagawa et al., Gerbil Global, 2 mins 48 hours Global, 10 1991) 5 mins mins (Nishi et al., 1993) Rat Global, 5 mins 48 hours Global, 30 mins (Liu et al., 1993) Rat Global, 3 mins 72 hours Global, 6 mins (Kirino et al., 1991) Gerbil Global, 2 mins 24 hours, Global, 5 mins 48 hours, 96 hours (Kato et al., 1992) Gerbil Global, 2 mins 72 hours Global, 3 mins (Aoki et al., 1993a) Gerbil Global, 2 mins 48 hours Global, 3.5 mins (Aoki et al., 1993b) Gerbil Global, 2 mins 48 hours Global, 3.5 mins (Kato et al., 1994) Rat Global, 3 mins 72 hours Global, 6 mins (Glazier et al., Rat Focal, 20 mins 24 hours Global, 10 1994) mins (Simon et al., Rat Global, 2x2 48 hours Focal, 1993) mins at one Permanent day intervals (Chen et al., 1996) Rat Focal, Variable 24 hours Focal, 100 Variable 72 hours mins 3x10 mins Varied

In 1990, Kitagawa et al. discovered that two 2 minute non-lethal global ischaemic insults, at one day intervals, to the gerbil brain two days prior to a ‘lethal’ 5 minute insult drastically protected the brain from the necrotic effect of the ‘lethal’ insult (Kitagawa et al., 1990). Necrosis was detected 7 days after reperfiision by absence of

66 binding of a microtubule associated protein 2 (MAP2) antibody, and the region most protected from necrosis was the vulnerable CAl region of the hippocampus. The same group proceeded to investigate this phenomenon in other regions of the brain (CA2, CA3, and CA4 regions of the hippocampus; the frontoparietal and parietal lobes of the cerebral cortex, the caudoputamen and the thalamus) using a gerbil model of 10 minute global ischaemia (Kitagawa et al., 1991). The results demonstrated that tolerance was notable in all the regions tested, with significant differences noted in the CA3 region of the hippocampus, the frontoparietal lobe of the cerebral cortex, the caudoputamen and the thalamus.

This phenomenon has been reproduced in rat models - 5 minutes global ischaemia pre­ treatment followed by 30 minutes ischaemia after 2 days reperfiision - and the reduced neuronal CAl necrosis compared to controls was noted using cresyl violet staining (Nishi et al., 1993). This study also detected increased levels of hsp70 mRNA and protein 4 and 24 hours respectively after the 5 minute pre-treatment and immunostaining revealed that the highest levels were in the CAl region of the hippocampus. This was further confirmed by Liu et al, who additionally demonstrated that hsp70 immunoreactivity could be detected in the CAl region up to seven days after 6 minutes of global ischaemia provided there was a 3 minute pre-treatment three days before (Liu et al., 1993). These conditions were selected from previous work that optimised the length of lethal insult to neuronal density in the CAl region alone, but did not take into account other regions of neurons (Liu et al., 1992). It was therefore proposed that hsp70 may be responsible for the induction of the tolerance phenomenon.

Kirino et al. however, optimised conditions for tolerance in the gerbil according to CAl neuronal density and discovered that if the window of protection was lengthened to four days between a 2 minute global pre-treatment and a 5 minute insult, a higher CAl neuronal density was observed (Kirino et al., 1991). They also demonstrated a characteristic CAl immunostaining pattern for hsp70, however in the 1-day window ischaemic groups hsp70 immunostaining was minimal and yet moderate CAl

67 protection was detected. It is possible that hsp70 levels may be subtly raised below the threshold for antibody detection.

Interestingly MK-801, the NMDA-receptor antagonist, and anisomycin, a reversible protein synthesis inhibitor, both reduce the inducible hsp 72 protein induction after mild ischaemic pre-treatment, but only MK-801 inhibits the tolerance to subsequent ischaemia in gerbil CAl neurons (Kato et al., 1992). Although it could be concluded that de novo synthesis of hsps following preconditioning is not protective, the authors argue that anisomycin activity is transient and a rapid induction of hsps may be occurring after the secondary insult as the result of a ‘priming effect’ fi*om the first. Indeed, a preconditioning insult of 2 minutes grossly accelerates the expression of hsp70 and hsc70 mRNA and protein m gerbils following a subsequent 3.5 minute insult two days later, suggesting that preconditioning ‘primes’ the transcription machinery (Aoki et al., 1993a; Aoki et al., 1993b). Also some protein synthesis inhibitors have themselves been demonstrated to induce a stress response in non-CNS cells, and therefore other pathways may be conq)ensating for the low levels of hsp72. Reduction of protein synthesis may also prevent the production of suicide gene products that may trigger programmed cell death. The synthesis of some proteins, if not hsp72, may be resistant to anisomycin treatment and confer tolerance.

Hsp27 levels have also been characterised m rats during tolerance to global ischaemia (Kato et al., 1994). Prior to a 6 minute global insult enzyme immunoassay and immunohistochemistry for hsp27 demonstrated significant increases hi the gha of the preconditioned hippocampus (3 nhnutes three days prior to the 6 minute msult) as compared to the unpreconditioned control. In both experiments levels had dropped by 2 hours after the 6 minute insult. It is possible that after the lethal insult hsp27 is redistributed firom the cytoplasm to the nucleus as occurs in heat shock (Arrigo et al., 1988). On immunohistochemistry, CAl neurons in both preconditioned and unpreconditioned hippocampi stained intensely for hsp27 1 day after the ischaemia. At 1 and 3 days the preconditioned hippocampus returned to the pre-ischaemic levels, whereas the unpreconditioned control levels increased considerably. This increase was noted at 3 and 7 days on immunohistochemistry to be in ghal cells. The CAl neurons.

68 however, had mostly been destroyed. The preconditioned, CAl region demonstrated scattered glial irnmunoreactivity resembling the pattern of the pre-ischaemic hippocanq)us after preconditioning. The principle difference between the hsp70 and hsp27 staining patterns was that hsp70 was induced in CAl neurons after the preconditioning insult, whereas hsp27 was induced in the gha. Therefore, any protective effect of hsp27 in the ischaemic tolerance phenomenon is not likely to be direct, but an indirect action of tolerant gha in support of the vulnerable regions of neurons.

Ischaemic tolerance is not exclusive to global ischaemia. In the rat, 20 minutes of unilateral focal cerebral ischaemia by middle cerebral artery occlusion (MCAO) protected the ipsilateral parietal cortex from 10 minutes of global ischaemia 24 hours later (Glazier et a l, 1994), and hsp72 mRNA and protein detection mapped to these protected regions. Interestingly, raised hsp72 mRNA and protein levels and ischaemic tolerance were detected in the cortex not only in the pre-treated models, but also in the sham operated models, where the MCA was manipulated, but not occluded. It may be that hsp72 regulation is highly sensitive to minor perturbations in blood flow. Conversely, Simon et al. subjected rats to two 2 minute global ischaemic insults at 24 hour intervals, foUowed by a subsequent permanent MCA occlusion 48 hours later (Simon et a l, 1993). The infarct size as assessed by tetrazohum chloride (TTC) staining 48 hours after MCAO was significantly reduced in ah sections, the protected region being around the rim of the infarct, and a widespread immunostaining pattern for hsp72 was detected 24 hours after the second global insult. Subsequently, preconditioning with 2 or 3x10 minute MCA occlusions separated by 45 minutes reperfusion has been shown in rats to reduce infarct size 24 and 72 hours after a subsequent 100 minute MCA occlusion performed 72 hours after the primary insult {OiQnet al, 1996).

It is impossible to say from these in vivo data whether hsp overexpression is a marker for surviving cells that have undergone stress, or whether this overexpression is contributory to that survival. These lines of in vivo evidence give weight to the theory that hsps may play a role in the characteristic delayed neuronal death phenomenon seen

69 in the CAl region and also may be the intracellular mediators of ischaemic tolerance. Perhaps the most direct evidence of their role in protection arises from the in vitro data on hsps and their effects on neuronal survival.

1.2.6 Heat shock proteins and the stress response in neurons in vitro

Rordorf et al demonstrated in 1991 that a heat shock of 42.5°C for 20 mins protected rat cortical cultures from 10 minutes of glutamate exposure (Rordorfet a l, 1991). Viabihty was measured 20-24 hours after glutamate exposure and protection was observed when glutamate was introduced 3 and 24 hours after the shock, but not at 15 minutes or 48 hour timepoints. Addition of actinomycin D (RNA synthesis inhibitor) and cycloheximide (protein synthesis inhibitor) inhibited this protection and therefore it was concluded that this protection required RNA and protein synthesis. Raised levels of hsp70 mRNA was demonstrated by Northern blotting and f^S]methionine incorporation produced two bands at 85 and 72 kDa, indicating overexpression of proteins of these molecular weights in response to glutamate exposure. The inducible member of the hsp70 family has a molecular weight of 72kDa, and therefore this band may represent an increase in the translated product of the increased hsp70 mRNA levels.

Lowenstein et al also demonstrated increased levels of proteins in mammahan cerebellar granule cells (source not identified) on 30-180 minutes of 42.5°C heat shock including 72 and 90 kDa proteins (Lowenstein et a l, 1991). Inducible hsp72 was immunodetected from 60-180 minutes after heat shock (30 minute levels were not shown). 90 minutes of heat shock protected these neurons from the excitotoxic effects of 6 hours of glutamate exposure 14 hours after heat shock. Cell viabihty was assessed by trypan blue exclusion and lactate dehydrogenase release. Protection was inhibited by the NMDA receptor antagonist MK-801, suggesting that the protective mechanism hes downstream of the activation of the NMDA receptor by glutamate. Although mutually supportive, neither of these studies directly proved the hypothesis that hsps are protective against glutamate toxicity. Defective HSV-1 mediated gene delivery (see

70 Section 1.3) and subsequent overexpression of hsp72 to rat hippocampal neurons in vitro protected them from severe heat stress (47°C for 25 or 40 mins) (Fink et al., 1997). In the same study however, the group were unable to demonstrate significant protection from the toxic effects of additional glutamate, suggesting that hsp72 may not be involved in the protection phenomenon noted after mild heat shock.

Uney et al. demonstrated that transient overexpression after transfection of inducible hsp70 protected rat dorsal root ganghon (DRG) neurons from a severe heat stress of 46°C for 10 minutes 24 hours prior to counting (Uney et al., 1993). Surviving neurons and gha were identified by staining blue with X-gal (4-Cl,5-bromo,3-indolyl-/?- galactosidase) to detect /^-galactosidase activity from the lacZ gene product present on the transfected plasmid. Although a study on thermotolerant properties of raised inducible hsp70, this was the first direct evidence that hsp70 confers protection in neurons. Our laboratory focused on the preconditioned stress response in vitro in primary neuronal cultures and on the ND7 ceh line, a fusion of non-dividing rat DRG cells with the N18 mouse neuroblastoma cell line (Wood et al., 1990). The ND7 ceh line is a useful neuron-derived model when experiments are to be repeated several times as they can proliferate, and therefore numerous experiments using large numbers of cehs can be performed simultaneously. Like immature neuronal cehs, on transfer to a defined serum-free medium they undergo either apoptosis or differentiate into cehs resembling post-mitotic neurons dictated by the presence of eiWrtrans retinoic acid or cychc AMP respectively (Howard et al., 1993).

Mailhos et al. demonstrated that a mild pre-treatment with heat shock (42^'C for 30 minutes) protected ND7 cehs from apoptosis stimulated by transfer mto serum-free medium containing retinoic acid at 24 and 48 hours post treatment. Ceh viabihty was assessed by trypan blue exclusion (Mailhos et al., 1993). They also demonstrated that the increasing degree of protection conferred by increasing periods of thermal pre­ treatment correlated to the levels of hsp 90 induced, and that other pre-treatments that are known to mduce hsps such as ethanol and propanol also protected ND7 cehs from apoptosis.

71 These results were confirmed when similar experiments were performed on primary neuronal cultures. DRG survival against the apoptotic effects of NGF withdrawal was also enhanced by prior heat shock (42.5°C for 20 minutes) (Mailhos et al., 1994).

ND7 stable cell lines over-expressing human hsp90 and the inducible form of human hsp70 exhibited enhanced survival against a lethal thermal stress (48°C for 30-60 minutes), however they were not protected from serum-withdrawal/retinoic acid induced apoptosis (Mailhos et al., 1994). Likewise, murine trigeminal ganghon neurons micro-injected with human inducible hsp70 or human hsp90 both showed enhanced survival 12 and 24 hours afl;er a 90 minute, 46®C heat shock, but were also not protected fi*om apoptosis (Wyatt et al., 1996). To date, no single hsp has been demonstrated to protect against apoptosis in neurons. Mehlen et al. have demonstrated that murine L929 fibrosarcoma cehs over-expressing human hsp27 do not undergo ceh death on stimulation of the Fas/APO-1 receptors or on addition of staurosporine which has previously been shown to induce death by apoptosis (Mehlen et al., 1996). To date, hsp27 has not been over-expressed in neurons and it is therefore not known whether it is a putative mediator of the protection from apoptosis observed after heat shock pre-treatment.

Amin et al. demonstrated that a mild thermal pre-treatment of 42°C for 40 minutes induced greater levels of the inducible hsp70 protein in DRG neurons than a pre­ treatment with simulated in vitro ischaemia (Anhn et al., 1995). Although, on subsequent severe heat and ischaemic stress, both treated groups exhibited some degree of tolerance, the thermally pre-treated neurons exhibited greater viabihty than those pre-treated with ischaemia. It was therefore concluded that the degree of protection conferred to neuronal cehs in vitro by pre-conditioning stimuh is correlated to the amount of inducible hsp70 protein that is produced as a result, rather than the nature of the subsequent stress. The same group proceeded to show that over­ expression of hsp70 by transient transfection protected DRG neurons from a ‘lethal’ simulated ischaemia of 2 hours (Amin et al., 1996). The degree of protection from ischaemia, however, was not as substantial as that seen against ‘lethal’ heat stress.

72 Ischaemic tolerance has also been reported in the heart. Pre-treatment with thermal stress and transfection-mediated overexpression of hsp 70 into a rat heart myogenic cell line (H9c2) both protect against cell death in response to subsequent heat stress, simulated ischaemia, and hypoxia in vitro (Heads et al., 1994; Mestril et al., 1994; Heads et al., 1995). Similar lines overexpressing hsp90 were protected against heat shock, but not hypoxia, and those overexpressing hsp60 were not protected from either of these two msults. Primary cardiocytes overexpressing hsp70 and hsp90 were resistant to cell death in response to heat shock, but only hsp70 protected against ischaemia (Gumming et al., 1996). This data concurs with that noted in neuron-derived cell lines {vide supra). Overexpression of hsp60 was not protective against either stress. Adenoviral-mediated delivery and overexpression of the hsp70i gene protects the H9c2 myogenic cell line against subsequent simulated ischaemia in vitro (Mestril et al., 1996). In vivo pre-treatment of rabbit hearts with whole body hyperthermia or sub- lethal ischaemia substantially reduces infarct size after subsequent severe ischaemia, and hsp72 levels were elevated in the pre-treated models (Marber et al., 1993). Ex vivo buffer-perfused hearts from transgenic mice that overexpress hsp70 exhibit reduced infarct volume following severe ischaemia, which was also associated with increased coronary flow and contractihty, and the limited infarct size was also demonstrated in vivo in blood-perfused hearts (Marber et al., 1995; Hutter et al., 1996). No reports on brain ischaemia have been made with these transgenic mice. These studies are the only ones as yet that demonstrate a direct in vivo effect of overexpressing hsp70, or any other hsp, on cell death following ischaemia, and are therefore encouraging when considering the in vivo gene delivery of hsps to the rat brain in order to examine their roles in protection following lethal ischaemia.

To summarise, it seems likely that hsp70 is protective against ceh death in response to ischaemia, but whether in vivo in the central nervous system it is a positive regulator of the ischaemic tolerance phenomenon stiU remains unproved. In vitro, over­ expression of hsp90 appears to protect against heat shock but not ischaemia, and neither of these hsps have been demonstrated to protect against apoptosis. Over­ expression of hsp27 protects fibrosarcoma cehs from apoptosis but this has not been

73 characterised in neurons or neural-derived cell lines, in relation to its effects on heat shock, ischaemia or programmed cell death.

1.2.7 Summary

In consideration of the above data, therefore, one way of determining whether the hsps do prevent cell death as the result of ischaemia in vivo is to overexpress them specifically, without heat or ischaemic stress, and observe any reduction in pathology following the subsequent apphcation of a ‘lethal’ ischaemic insult.

In order to overexpress the hsp genes in vivo, they need to be delivered using a suitable vector to the central nervous system. This field of study is discussed in the following section of the introduction.

74 1.3 Gene Delivery to the Brain

1.3.1 Introduction

This section of the introduction applies what is known about in vivo transgene delivery to the purpose of delivering hsp transgenes to the central nervous system for overexpression. The first section discusses why HSV-1 is the most suitable vector for this purpose, and the second part reviews what is currently known about HSV-1 mediated transgene delivery.

1.3.2 The Choice of Vector

Designing the ideal vector for in vivo gene transfer is a challenge. It must be efficient in its delivery, effective, non-toxic, non-repbcating, non-immunogenic, and it shouldn’t unintentionally disrupt the host cell’s normal fimction.

Transgene delivery vector research to the nervous system is still in its infancy, and new, safer and more effective vectors are constantly being produced (reviewed by Latchman, 1996). The three major vectors currently in development are viruses, bposomes, and direct DNA injections. Of the viruses, those that are most commonly used are herpes simplex virus type 1 (HSV-1), adenovirus, adeno-associated virus (AAV) and retroviruses (see Table 1.3). Selection of an appropriate system for heat shock transgene delivery to the mature central nervous system is achieved by addressing the requnements bsted above and applying them to that particular envnonment.

75 Table 1.3 - Methods of in vivo Gene Delivery to the Central Nervous System Type of Vector Description Direct DNA Injection Expression vector DNA injected directly into the tissue. Microinjection mediated delivery extremely inefficient, hence development of delivery mediators, currently the most effective are cationic liposomes. Cationic Liposomes Positively charged unilamellar liposomes that e.g. N-[1 -(2,3-Dioleyloxy) interact with plasmid DNA through ionic propyl]-A/,A/,A/- interaction. Cytotoxicity observed with duration trimethyiammonium chloride of exposure to cationic lipids, but less cytotoxic (DOTMA) than other direct DNA delivery mediators. Retroviruses RNA viruses. Action of virally encoded reverse transcriptase produces viral DNA which is incorporated into host genome. Will not infect non-dividing neurons. Can be used in ex vivo gene delivery to immature dividing neuronal progenitor cells in culture prior to transplantation into the CNS. Lentiviruses Family of retroviruses that can integrate into the genome of non-proliferating cells, as the result of karyophilic determinants in the virion proteins MA (matrix), and Vpr. Non- immunogenic. Herpes Simplex Virus Type 1 Double stranded DNA virus. Large genome (HSV-1) (150kb pairs). Large cloning capacity (20kb). Enters state of latency in neurons. Two types of vector: defective (amplicon), and disabled (recombinant). In the former, circular concatamers of promoter/transgene sequences are packaged in HSV-1 capsids. In the latter, promoter /transgene sequence recombined into HSV-1 genome deleted for essential gene disabling replication. Adenovirus Double stranded DNA virus. Small genome (36kb pairs). Cloning capacity low (7-8kb). No latent state characterised in neurons. Different generations of vectors defined by deletions of regions to disable replication and increase cloning capacity. Immunogenic. Adeno-associated Virus Single stranded DNA parvovirus (genome = (AAV) 4.7kb). Requires co-infection with helper virus (adenovirus) for replication. Incorporated into host cell genome in absence of helper virus in q13.4 of chromosome 19, when rep gene intact. Most of genome can be deleted for transgene insertion. Has very limited cloning capacity (4.5kb).

76 The transgene must be delivered efficiently. Despite the constant development of less cytotoxic and more efficient liposomal DNA delivery systems, in vivo injections of DNA into the mouse brain has produced disappointing results. Firstly, the numbers of cells expressing the transgene are very low, and secondly these positive cells also stain positive for ghal-fibrillary acidic protein (GFAP) indicating uptake into astrocytes and not neurons (Imaokaet al., 1995; Roessler and Davidson, 1994). It appears from other studies that more efficient hposomal uptake occurs in dividing cells. For example, Takeshita et al. demonstrated a 10-fold increase of gene transfer in arterial smooth muscle cells which was achieved by the stimulation of proliferation by balloon angioplasty (Takeshita et al., 1994). For the purposes of this study therefore, hposomal delivery at its current level is unsuitable.

The efficacy of a transgene to be expressed in a tissue is dependent on many factors. Non-retroviral transgene expression decays with time, and no vectors have been demonstrated to express transgenes indefinitely. Retroviral vectors are incorporated into the host genome and give long-term expression in vitro, but this only occurs in dividing cells (Miller et al., 1990). Mature neurons are post-mitotic, and so these viruses are therefore not suitable for the purposes of this study. Non-rephcating lentiviral vectors based on the human immunodeficiency virus (HIV) genome have recently been demonstrated to efficiently infect and integrate into the genome of terminally differentiated neurons in vivo as the result of a karyophihc capabihty determined by two proteins MA (matrix) and Vpr. Stable expression of the (3- galactosidase reporter gene has been documented in neurons after six months post­ inoculation, no cellular or potent antibody immune response has been detected (Blomer et al., 1997; Naldmi et al., 1996). In the same study, a lentiviral vector infected a greater area of tissue than similar concentrations of adenoviral and adeno-associated viral vectors. Currently in their infancy, lentiviral vectors may prove to be valuable vectors for gene delivery to the CNS. For the purpose of delivery to the CNS prior to ischaemic insult, however, transgene expression need only occur for a few days prior to, and during the ischaemic insult and therefore the virus would only be required to express the transgene transiently during this period.

77 Thirdly, irrespective of the time scale required for transgene expression, the method of delivery should be as non-toxic as possible. Viruses are cytopathic, much of which is due to their utihsation of the cell DNA rephcation machinery and the viral lytic cycle, resultmg m death of the cell as the vhal progeny M s the cytoplasm to be released either by cell lysis or budding from the cell membrane. Vhal gene products can also compromise the host cell’s function, for exanq)le by shutting down host macromolecular synthesis (Oroskar and Read, 1989). Vhal vectors must therefore be amended to disable theh own rephcation, preventing theh uncontroUed spread and ceU lysis throughout the tissue and its neighbouring structures, and the cytopathic effect of vhal gene products must also be reduced to a minimum

The deletion of identified essential genes disables vhal rephcation. Homologous recombination of essential genes into the genome of the mutant strain after delivery can occur, resulting in a reversion to virulence. The frequency of these recombination events is greatly reduced when more than one essential gene is deleted, but this in turn increases the diflhculty of preparation. Essential gene products can be cytopathic, via the toxic effects caused through theh regulation of the vhal lytic transcription cascade, and interference with host ceh function and for this reason deleting more than one may also be advantageous. Mutants are therefore being developed with this in mind (Johnson et a l, 1994). For examining the effect of hsps in animal models, under controUed sterile conditions, where the aim is to gain maximal expression and effect, the deletion of one essential gene is sufi&cient.

An ideal system for transgene expression in vitro can be compromised in vivo by the hosts immune system Adenovhal studies have highhghted the inqiortance of this and the characteristic decay of expression with vhal vectors is in part attributable to the efficiency of the hosts immune response against capsid polypeptides (Simon et al., 1993; Yang et al., 1994). Viruses have theh own methods of evading the immune system, for example four of the gene products from the adenovirus E3 region have functions in counteracting the host’s immune system (Wold and Gooding, 1991). Previously, the E3 region was deleted as a non-essential region, to provide an increase

78 in the recombinant capacity of the vector, but subsequent generations of vectors now include this in their genome.

Infected cell protein 47 (ICP47) is an immediate early gene product expressed by HSV-1 in the opening stages of infection. It has been shown that cells expressing ICP47 retain their major histoconq)atabihty complex I (MHCI) molecules in the cytoplasm, preventing the presentation of viral antigens to CD8^ cytotoxic T lynq)hocytes (York et al., 1994). HSV-1 enters a latent state in neurons, interacting with nucleosomes as it enters latency {vide infra). It can remain in this dormant state for potentially long periods of time, expressing one group of transcripts, the latency- associated transcripts (LATs) whose functions remain unknown (Deshmane and Fraser, 1989; Stevens et al., 1987). Exploiting this latent state could potentially provide a means of viral gene delivery that was non-toxic and minimally immunogenic with a long term expression of the delivered gene.

Viruses express a host of genes, some cytopathic and some apparently harmless. For example, residing in the tegument of HSV-1 is a late gene product, the virion host shut-oflf protein (vhs) which disrupts host and viral protein synthesis by increasing destabihsation and degradation of mRNA viral entry into the cell and therefore conq)romises the host cell’s function (Oroskar and Read, 1989). Elimination of vhs alone does not render the virus non-cytopathic, as other HSV-1 gene products are themselves cytopathic, for exanq)le the inhibition of host cell mRNA sphcing by ICP27 {vide infra). This will be discussed in more detail later. In designing a viral vector therefore, these cytotoxic gene products should be minimised where possible. However, when HSV-1 enters latency in neurons it solely transcribes the LATs, therefore with appropriate modifications that minimise host cell disruption on infection, iahibit the lytic cascade, and encourage entry into latency, HSV-1 is potentially a very safe virus for use in the CNS. The latter two can be achieved by deletion of immediate early (IE) genes which initiate the lytic gene transcription cascade, and are discussed in more detail below.

79 As a footnote, the practicality of preparation is in^ortant. Once pure, disabled HSV-1 vectors can be grown in bulk on^ cell lines stably expressing the essential genes necessary for then rephcation (contpl^nienting ceUs), and remain unable to rephcate on non-con^lementing ceUs. They can be titrated for functional dose by counting the number of plaque forming units (pfu) per unit volumein \itro on a complementing ceh line, prior to use in vivo. Defective HSV-1 vectors (amphcons) are HSV-1 packaged cncular DNA repeats of promoter/transgene sequence with an HSV-1 origin of rephcation, and an HSV-1 packaging signal {vide infra). Both these and adeno- associated virus (AAV) vectors requne helper viruses for their growth and the helper/vector ratio varies considerably on passage and reproducible vector populations are difficult to prepare.

In conclusion, on consideration of the above factors, viral vectors seem the most appropriate delivery system to enable expression of the hsps in vivo in the CNS. Adenovirus, lentiviruses and herpes simplex virus type 1 are currently the three major candidates for transgene delivery to non-dividing cehs. In this study HSV-1 has been used, for the fohowing additional reasons: 1) HSV-1 is a virus that infects neurons. The prevalence of antibodies hi the community (80%) makes it a common infectious agent. Although it is the cause of cold sores, HSV-1 infection is rarely lethal despite its abihty to cause encephahtis. 2) Its large genome (~150kb) means that large sections of cDNA can be inserted into the virus for delivery (<20kb) (Roizman, 1979). Adenovirus can only carry at most Skb and adeno-associated virus 4.5kb. 3) The induction of latency of HSV-1 exclusively in neurons means that viral DNA can stay resident in neurons for the lifetime of the cell without causing any pathological eflfects (reviewed by Latchman, 1990). The shut down of viral DNA during latency, however, makes it potentially difficult to enable the foreign DNA to be continuously expressed. Exploitation of the elements that enable LAT transcription should enable stable expression during latency. 4) Previous use of HSV-1 in gene dehvery to the brain has produced encouraging results, in particular the introduction of the tyrosine hydroxylase gene to the

80 striatum in Parkinsonian rats using a defective HSV-1 vector (amplicon) has been shown to reheve extra-pyramidal syn^toms {vide infra) (During et al., 1994).

1.3.3 The Biology of Herpes Simplex Virus Type 1

HSV-1 is a double-stranded DNA herpesvirus. The viral particle is surrounded by an envelope derived from host nuclear membrane, and is formed as the particle buds from the nucleus. The capsid is icosahedral-shaped and the region between the capsid and the envelope, the tegument, contains the proteins VP 16 (Vmw65 or aTIF) and vhs (Roizman and Sears, 1996).

The DNA exists in a toroid structure of approximately 150kb pairs. Approximately 75 genes have been identified. The HSV-1 genome consists of a long and short unique region (UL and US, respectively), each flanked by two shorter repeat regions (RL and RS, respectively) (see Figure 1.3.3a). There are four possible genomic isoforms as each unique region can be recombined during assembly in either orientation (Roizman, 1979).

81 Figure 1.3.3a - Herpes Simplex Virus Type 1 (Fink et a/., 1996) (A) Schematic representation of structure of HSV-1 particle, showing the capsid, tegument, and glycoprotein-containing Hpid envelope, surrounding DNA in toroid structure. (B) Organisation of the HSV-1 genome, showing the unique long and short regions (UL and US, respectively) and the internal and terminal repeats flanking the long and short regions (IRL = internal repeat long, 1RS = internal repeat short, TRL terminal repeat long, TRS = terminal repeat short). (C) The HSV-1 lytic gene regulatory cascade. Expression of essential IE (immediate early, a) genes is required for subsequent expression of E (early, P) and L (late, y) class genes, but the establishment of latency does not require viral rephcation or IE class gene expression.

82 (A)

TEGUMENT

ENVELOPE DNA CAPSID

(B) HSV-1 Genome (ISOkbp) a' TRL UL IRL a’ 1RS US TRS a' b' q' c,

(C) ICP4

Viral DNA Synthesis (y) ICPO VP16 ICP27

83 HSV-1 gains entry into the ceU by an unknown mechanism, although non-specific charge interactions have been demonstrated between the envelope glycoproteins gB, gC and gD and heparan sulphate moieties on the plasma membrane (Roizman and Sears, 1996). Fusion of the envelope with the plasma membrane releases the tegument proteins vhs and VP 16 which enter the nucleus with the uncoated particle. The natural sequelae of HSV-1 infection depends on whether the virus enters latency or the lytic infection.

During the lytic cycle, the host cell transcription factor, Oct-1 (a member of the POU- domain transcription factor family) binds to VP 16 and a host cell factor (HCF). This complex then binds the TAATGArAT (r denotes a purine) consensus in the HSV-1 immediate-early (IE) gene promoter enhancers (Gafifiiey et al., 1985). Vhs assists viral rephcation by degrading cellular mRNA and interfering with host protein synthesis (Oroskar and Read, 1989). Viral protein synthesis then ensues with an ordered cascade of IE gene products, then early (E or (3) and finally late (L or y) (see Figure 1.3.3a).

There are five IE genes - infected cell polypeptide 4 (ICP4), ICP22, ICP27, ICP47 and ICPO. ICP27 and ICP4 are both essential for viral rephcation, whereas viruses deleted for the other three can stih rephcate. ICP4 is a DNA-binding protein that recognises ATCGTC consensus sites and activates the expression of E and L genes and downregulates the expression of IE genes including its own (Batchelor and O'Hare, 1990; DeLuca et al., 1985; Roberts et al., 1988). ICP4 has also been demonstrated to repress the activity of the LAT PI promoter and ICP4 and ICPO repress the activity of the LAT P2 promoter (Batchelor and O'Hare, 1990; Goins et al., 1994). Mutational analyses have attributed several fimctions to ICP27 (Rice and Knipe, 1990). It stimulates L-1 gene expression, induces L-2 gene expression, down regulates IE and E genes late in infection and stimulates viral DNA rephcation. It also contributes to the host protein shutofF seen during productive infection, through impairment of host ceh pre-mRNA sphcing (Hardwicke and Sandri Goldin, 1994; Hardy and Sandri Goldin, 1994). ICPO upregulates IE (in the absence of virion proteins such as VP 16), E and L

84 gene transcription, and viral mutants deficient in ICPO express lower levels of all three classes of genes (Cai and Schaffer, 1992; Chen and Sfiverstein, 1992). It is probable that, for this reason, ICPO viral mutants exhibit impaired growth in vitro (Sacks and Schaffer, 1987). The IE gene activation demonstrated in the absence of virion proteins may suggest a role for ICPO in the reactivation of HSV-1 from latency. 1CP22 has been demonstrated to promote efficient late gene expression in a cell-type dependent manner and is involved in the production of a novel form of cellular RNA polymerase 11 (Sears et a i, 1985; Rice et a i, 1995). 1CP47, as described previously, inhibits the presentation of viral antigens in the context of MHCI molecules on the surface of the infected cell, and therefore reduces CD8^ T lyn^hocyte mediated cytotoxicity (York et a l, 1994).

E gene products are mainly involved in viral DNA synthesis - DNA binding proteins, polymerases, thymidine kinase etc. L products are responsible for the structural proteins of the virion - the capsid, tegument, envelope and so forth. DNA synthesis occurs in a rolling circle, forming long head-to-tail concatamers of UL and US separated by the repeat regions (Roizman and Sears, 1996). As a result of these flanking repeat regions being homologous, recombination occurs. This results in the unique regions being in either orientation and subsequently the genome can be one of four isoforms. The concatamers are cleaved into genome length units which are subsequently packaged into a capsid. The cleavage and packaging signal derives from ‘a’ repeat sequences that lie between the repeat regions of the DNA concatamer (see Figure 1.3.3a). The capsids then bud through the nuclear envelope in areas that have been modified with viral glycoproteins, thus forming the viral envelope, and pass through the cytoplasm and into the extracellular space. The whole lytic cycle takes about 10 hours.

The specific mechanisms involved in the induction of latency and reactivation are unclear, but neuronal cells express an additional POU-domain transcription factor Oct- ^w hose splice variants Oct 2.4 and 2.5 also bind to the TAATGArAT consensus sequence and therefore may repress IE gene activation by direct repression or competition with the Oct-1 ATP 16 complex for binding to the consensus sequence

85 (Lillycrop et a l, 1993). A deeper understanding of this may aid in the manipulation of the virus into a purely latent variety. As previously described, the viral genome becomes methylated and associates with nucleosomes to form minichromosomes with only one region of the genome being transcribed (Deshmane and Fraser, 1989; Stevens etal., 1987).

The resulting LATs are coded for in the long repeat regions of the viral genome and the transcript exists in two forms. One is a large, apparently unstable ~8.3kb polyadenylated transcript and the other is a 2kb non-polyadenylated, nuclear transcript which may be an intron sphced from the larger transcript, or be a direct transcript from the LAT gene (Dobson et al., 1989; Devi Rao et al., 1991; Zwaagstra et al., 1990; Farrell et al., 1991). The 2kb species is fiufher sphced into transcripts of ~1.5kb transcripts. LAT transcription is driven by a combination of two promoters, LAT PI and LAT P2. The TATA-box containing promoter, LAT PI Ues 660bp upstream of the y terminus of the 2kb LAT, it confers enhanced activity in neurons as weh as giving activity in most ceh-types (Batchelor and O'Hare, 1990; Zwaagstra et al., 1990). The TATA-less promoter LAT P2 has been described in the region between the start of the large LAT and the start of the 2kb transcript (Goins et al., 1994). Also referred to as the long-term expression element (LTE), the LAT P2 region has recently been demonstrated to be required to maintain the upstream LAT PI promoter transcriptionahy active during latency (Lokensgard et al., 1997). The function of the LATs are unknown, it does not appear that LAT is essential for the induction of latency, but viruses with the LAT region deleted show different reactivation kinetics, either whd-type or delayed (Ho and Mo car ski, 1989; Trousdale et al., 1991; Leib et al., 1989). No protein products have, as yet, been rehably identified as translation products of any of the LATs. They may therefore perform an antisense role in the inhibition of translation of the ICPO IE transcript, the third exon of which is coded for antiparallel to the smaller LAT species and is therefore complementary to them (Devi Rao et al., 1991). They may be continuously expressed to be used in signal transduction of an exogenous message for reactivation. Reactivation of virus into the lytic cycle can be caused by stresses such as hyperthermia, steroids and axotomy, and

86 is the cause of recurring cold sores as the latent virus in the trigeminal gangha reactivate and are transported anterogradely to re-infect skin cells.

1.3.4 HSV-1 Based Vectors

Two systems are currently being used to deliver transgenes to the CNS using HSV-1 (reviewed by CofiSn and Latchman, 1996). These are defective (an^Hcon) vectors, and recombinant viruses with an essential gene(s) deleted to disable the capacity for rephcation. A schematic representation of the preparation disabled and defective vectors is illustrated in Figure 1.3.4.

87 Figure 1.3.4 - Schematic Representation of Growth of Disabled and Defective Herpes Simplex Virus Vectors (Fink et a/., 1996)

(A) Production of a defective fiill-length HSV- 1-based vectors is carried out on cell lines that are engineered to provide the deleted essential gene in tram. These vectors are incapable of repHcating in neurons because of the missing essential genes. (B) Amphcons are propagated in bacteria (using the bacterial origin of rephcation) and then transfected into a conq)lementing ceh line that is infected with a defective helper HSV-1, thus producing particles consisting either of anq)hcon concatamers (about 150kb in length) or defective HSV-1.

88 (B) Amplicon System

(A) Disabled HSV Vector Dj^a^ed Helper Disabled Virus HSV Vector

Transfection

Infection Infection Helper Virus Lytic Cycle Virus Lytic Cycle Amplicon DNA Replication (Cleavage at Particle Production Viral DNA Viral DNA J Replication Replication

(Cleavage at (Cleavage at a" sequence) "a"sequence) Packaging

Complementing Packaging Cell Line Complementing Cell Line Disabled HSV Vector (100%)

Amplicon Vector (2-10%)

Defective Helper Virus (90-98%)

89 1.3.5 Defective HSV-1 Vectors

Amplicon vectors are created from plasmids that contain the transgene under the control of a promoter, an HSV-1 ‘a’ packaging sequence, an HSV-1 origin of rephcation site, and an E. coli origin of rephcation (to enable rephcation of the plasmid in preparation) (Spaete and Frenkel, 1982). The plasmid is co-transfected into ceh lines with an HSV-1 helper virus. UsuaUy, this is a recombinant HSV-1 that is unable to grow at 3TC with a tenq)erature-sensitive mutation (ts k) in the IE3 gene (ICP4) (Davison et al., 1984). Subsequently, an HSV-1 mutant with a deletion in IE3 has been used (D30EBA) and the packaging occurs on a stable ceh line that complements ICP4 (M64A) (Geher et al., 1990). The plasmid can rephcate into concatamers up to ~150kb using the rephcation machinery from the helper virus acting on its origin of rephcation, and these concatamers are packaged into capsids by recognition of the ‘a’ sequence residing in their coding sequence. Subsequently, on harvesting, 90-98% of virus obtained is defective helper virus, and 2 - 1 0 % is packaged amphcons that can enter neurons by the surface moieties on the envelope and transcribe the delivered cDNA at amplified levels without amplifying any of the other viral genes. The advantage of these vectors is that they produce high levels of expression from a high copy number within each anq)hcon, and it appears that the duration of expression is not as dependent on the promoter as with disabled recombinant viruses {vide infra). One disadvantage of anphcons is the need for repeated passage of amphcons to obtain an effective dose. High passage increases the chance of recombination of the complementing gene from the ceh line with the helper virus and the plasmid, or a temperature-permissive mutation in the ts K helper virus resulting in a rephcation- competent virus. This has thought to be the cause of deaths of animal models during in vivo experiments. The other disadvantage is the unrehable ratios of defective virus:helper virus on repeated passage which limits reproducibihty.

Studies on the amphcon vector expressing (3-galactosidase pHSVlac {lacL under the control of the HSV-1 IE 4/5 promoter) have demonstrated dehvery with stable expression of the gene in primary neuronal culture and in rat DRGs and superior cervical ganghon neurons in vivo for at least two weeks with no evidence of cytopathic effect (Geller and Freese, 1990; Geller and Breakefield, 1988). It has also been

90 successfiilly introduced and characterised in non neuronal cells, in particular cancer- prone and tumour cells, as a putative candidate for investigative gene delivery (Boothman et al., 1989).

Defective HSV-1 vectors have been used with ts K helper virus to successfiiUy express neurotrophic growth factor (NGF) in cultured neurons; the NGF receptor p75*'^^^ in fibroblasts producing functional high-afiSnity binding of NGF; the glucose transporter gene in vitro and in vivo in rats to reduce rat hippocampal CA3 neuron loss resulting from kainic acid delivery; and growth associated protein 43 (GAP43) to non neuronal cells to produce neurite-like processes (Geschwind et at., 1994; Battleman et al., 1993; Ho et al., 1993; Lawrence et al., 1995; Verhaagen et al., 1994). In one study lacZ under the control of the preproenkephalin promoter was delivered to the rat brain where the pattern of expression followed the previously observed patterns of endogenous prepro enkephalin expression up to two months, and it was suggested that defective vectors may prove a useful tool m analysing promoters that will target expression to particular areas or cell-types in the brain (Kaphtt et al., 1994).

The more recent work with the D30EBA helper virus has also proved successful. The most dramatic results were presented by During et al., who demonstrated a behavioural recovery in Parkinsonian rats by delivery of the tyrosme hydroxylase gene to the striatum of 6 -hydroxydopamine unilaterally lesioned rats (During et al., 1994). Behavioural recovery was assessed by reduction in apomorphine induced rotation rate, and significant reduction was noted after two weeks after gene transfer, which remained significantly reduced for 1 year. However, <10% of rats died within two weeks of gene transfer and it was reported that the ratio of wüd type HSV-1 to D30EBA helper virus was 1x10'^ and the dose was equivalent to approximately 1x10^ infectious particles of helper virus, meaning that on average, each dose would contain a wild-type particle. Federofif et al. have reported that gene transfer using a defective HSV-1 vector expressing nerve growth factor (NGF) can maintain tyrosine hydroxylase levels in the syrcpathetic neurons of the rat superior cervical ganghon after axotomy compared to lacZ expressing controls (Federofif et al., 1992). Geller et al. also demonstrated successfid delivery of adenylate cyclase in synq)athetic neurons in

91 vitro and observed long term increases in cyclic AMP synthesis, protein kinase A activity, protein phosphorylation, and catecholamine release (Geller et a l, 1993).

The most important recent studies, in relation to the work presented in this thesis have been carried out by Robert Sapolsky’s group at Stanford University, USA. They demonstrated that defective HSV-1 delivery of the glucose transporter gene could protect striatal neurons in the rat brain from cell death following 48 hours after a 1 hour MCAO (Lawrence et al., 1996). This group used a vector expressing the lacZ gene and the glucose transporter gene {glut-\) under the control of the HSV-1 ICP22 and ICP4 promoters respectively. The number of surviving lacZ positive cells was counted after X-gal and cresyl violet staining and related to controls from a lacZ only expressing vector. No report was made of the ratio of wild-type to helper virus in either vector population, which, if different might have affected the result. No deaths were reported but in the During et at. study, deaths were only reported as having occurred within the first two weeks following gene transfer. They have also infected primary cultures of rat hippocampal neurons in vitro with a defective HSV-1 vector

CO-expressing P-galactosidase and hsp72 and demonstrated that overexpression of hsp72 by this method can protect cultured neurons from severe heat shock, but not glutamate toxicity (Fink et al., 1997).

1.3.6 Disabled HSV-1 Vectors

The second method for the creation of viral vectors is the recombination of the transgene and its regulatory sequences directly into the HSV-1 genome by co­ transfection into complementing cells (CofBn and Latchman, 1996). The recombination site is dictated by HSV-1 flanking sequences in the plasmid 5’ and 3’ of the gene and its regulatory sequences. Essential IE genes can be ‘knocked-out’, so aft progeny of the recombined virus are replication-deficient. The gene can therefore enter a non- essential locus of a virus already disabled from replication, or be recombined into an essential gene. Selection and purification of recombined against unrecombined virus is aided by the use of reporter genes such as the bacterial lacZ, or green fluorescent

92 protein (GFP) that fluoresces on exposure to ultra-violet light. The reporter gene may be ‘knocked-out’ from the unrecombined virus or be present in the transfected plasmid along with the transgene. Therefore, selection of blue, green or colourless ‘white’ plaques as appropriate enables purification of recombinants. The co-transfection, purification from unrecombined virus, and subsequent growth of vector takes place on a cell line that complements the deleted essential gene. The end product is a disabled HSV-1 that codes for the desired protein and once pure, this virus can be extracted from the ceU line, and used as a vector for experimentation.

Recombinant HSV-1 vectors, once pure, are sicople to quantitate by titration and plaque assay, and therefore doses can be assessed. Individual amphcons contain differing numbers of concatamers and total numbers vary on differing passages with helper virus. They are therefore less consistent, particularly if experiments are to be reproduced. Master stocks of recombinant HSV-1 vectors can be stored for long periods of time at -70°C enabling the later growth m bulk on coDoplementing ceU lines for subsequent experiments. For these reasons, in the experimental delivery of hsp transgenes to the CNS, with the need for control doses and reproducibihty, the use of recombinant HSV-1 vectors seems the most practical.

1.3.7 Expression and Regulation of the Transgene

In the design of recombinant HSV-1 vectors, the next consideration is which promoters to use to drive transgene expression and where in the HSV-1 genome to insert the transgene.

TheoreticaUy, exploiting the long term transcriptional activity of the HSV-1 LAT promoters to enable expression during latency is favourable. Therefore, insertion of genes downstream of the LAT promoters in an IE gene-deleted recombinant virus may produce the desired expression. As there are two copies of LAT in each genome, in the RL regions of the genome the recombination event can therefore occur in both sites, possibly increasing the expression of the transgene. Insertion of genes downstream of the LAT PI promoter produced 3 weeks of rat beta globin RNA expression in murine

93 DRGs in vivo whereas the expression of (3-galactosidase from a lacZ gene inserted directly after the TATA box of the LAT PI promoter was considerably lower than during the lytic cycle (Dobson et al., 1989; MargoHs et al., 1993). A similar experiment inserting a lacZ gene just downstream of the LAT P2 promoter produced long term expression in murine trigeminal gangha cehs ( 8 weeks) but it was punctate and only in a smaU number of cehs (Ho and Mocarski, 1989). Expression of the p- glucuronidase gene under the control of LAT PI and the 5’ end of LAT P2 gave up to four months expression post-inoculation in the murine trigeminal gangha of mucopolysaccharidosis VII (MPS VH) mice (Wolfe et al., 1992). The rephcation- competent virus was administered by comeal inoculation, and was transported to the gangha where it estabhshed a latent infection. Lachmann et al. recombined a lacZ gene downstream of the LAT P2 promoter with a 5’ internal ribosome entry site (1RES, from encephalomyocarditis vims which ahows cap-independent ribosomal access) into wild-type HSV-1, without deleting the LAT gene. After subcutaneous injection into the mouse pinna, P-galactosidase activity was detectable for at least 140 days post­ inoculation in murine cervical DRGs and at least 307 days in specific regions in the central nervous system (facial, hypoglossal, and upper cervical spinal cord) (Lachmann and Efstathiou, 1997). It was postulated that maintaining the LAT gene in the HSV-1 genome confers some regulatory effect on transcription during latency.

However, the LAT promoters may not afford sufficient levels of expression of the transgene product to be effective, but the neuron-specific regions upstream of the LAT PI TATA box identified by Zwaagstra et al. and the apparent long term expression during latency conferred by the LAT P2 region suggests that these elements may be combined with a more powerfiil promoter to exploit ah these characteristics (Zwaagstra et al., 1990; Lokensgard et al., 1997).

Using more powerful promoters downstream of the LAT promoters to drive the transcription of the transgene, with appropriate intervening sequences (IVS) and polyadenylation sites downstream of the transgene has been explored. Exanq)les of such promoters are the cytomegalovims (CMV) immediate-early 94 promoter (CMV- IE94) or Moloney murine leukaemia virus long terminal repeat promoter (MoMLV-

94 LTR). Although the LAT promoters are not being used to drive transcription, the regulatory elements e.g. enhancers and repressors and neuron-specific elements, in these sequences and possibly at other sites in the HSV-1 genome may be utihsed to mimic the characteristic latency associated transcription of the LATs. MoMLV- LTR//flcZ insertion into the ICP4 region conferred short term activity in the CNS, up to 5 weeks (Dobsonet a i, 1990). One viral/promoter construct, of a series tested, that conferred long term activity in mouse DRGs con^rised of a TATA-less LAT PI promoter upstream of the MoMLV-LTR//acZ construct inserted into the gC locus of the HSV-1 genome which gave at least 42 days expression post-infection, suggesting that LAT promoters may indeed confer long term expression during latency in the CNS (Lokensgard et al., 1994). The LTR promoter may contain an element that enables latent transcription when the LAT PI promoter is being used m a different locus of the herpes genome, whereas a similar element may be fimctioning with LAT in its wild-type locus. The differing functions of LAT PI and LAT P2 and their relationship to each other is unknown, and therefore continued work needs to be carried out to enable a possible effective long term expression profile during latency.

1.3.8 Insertion Site of the Transgene

One problem of inserting transgene constructs into non-essential regions of the HSV-1 genome however, is that homologous recombination of an IE gene fi*om a co-infected opportunistic wild-type virus mto the deleted virus or recombination of the transgene/promoter region into the non-essential wild-type genome may produce a virus capable of rephcation that transcribes the transgene. Aside firom the experimental effect this may incur, affecting the natural history of infection and expression, it could feasibly result in the release of a transgenic rephcation-conq)etent virus into the population. In order to avoid this, the transgene and exogenous promoter/regulatory elements can be recombined into an essential gene locus, so any homologous recombination with wild-type HSV-1 would result iu the deletion of the transgene construct, and wild-type progeny would then be produced.

95 The essential genes coding for ICP4 and ICP27 (IE3 and IE2 respectively) have previously been used as insertion sites. Johnson et al. demonstrated, however, that a mutant IE3 virus was cytopathic in many cell types including neurons (Johnson et al., 1992). Cells infected with the mutant virus exhibited chromosomal aberrations, DNA fragmentation and cytoplasmic blebbing. Single viral mutants in the genes lE l (ICPO), IE2 and IE3 and double mutants of IE3/IE4 (ICP22) and IE3/IE5 (ICP47) all exhibited cytopathic effect (CPE). The only tested methods that reduced CPE were UV- irradiation of the virus and pre-treatment of cells with interferon-y, which have both been shown to reduce viral gene transcription. A triple mutant defective in ICP27, ICP4 and ICPO was visually less toxic than double mutants similar to those described above but the inhibition of host DNA synthesis seemed to impair cell survival to some degree (Wu et al., 1996). Further deletion of non-essential genes that may be responsible for cytopathic effect has been studied. An HSV-1 mutant deleted for the gene encoding the neurovirulence factor (ICP34.5) has been shown to increase the

LD50 in vivo 1 0 ^-fold compared to wild-type virus on intracranial inoculation in mice (Chou a/., 1990).

A deletion mutant of the vhs gene in an IE3 deleted virus did not significantly reduce cytopathic effect in fibroblast-derived ceU lines (Johnson et al., 1994). In the same study the gene encoding VP 16 was mutated in order to prevent the /ra« 5 -induction of IE genes. Significant inq)rovement in cell survival was noted in DE3 deleted viruses that also did not express VP 16. Insertional mutation in the gene encoding VP 16 or deletion of the gene encoding the VP 16 modifying protein coded from the UL47 region have both been shown to prevent the /raw-induction of the IE genes and therefore the virus estabhshes a latent infection (Ace et al., 1989; Steiner et al., 1990; Zhang et al., 1991). VP 16 mutant viruses can be grown in vitro on the addition of hexamethylene bisacetamide (HMBA) which stimulates IE gene expression in the absence of VP 16 (McFarlane et al., 1992). It has been demonstrated that an IE2 deleted HSV-1 proved less cytopathic at a range of multiphcity of infections (MOI) to rat primary cardiac myocytes in vitro than ICP34.5 and ICP34.5/VP16 deleted viruses according to numbers of surviving cells still expressing (3-galactosidase and beat frequencies (CofBn etal., 1996).

96 It is therefore possible, on consideration of the above, that the removal of aU of the IE genes, or the combination of removing one essential IE gene with a mutation in VP 16 or UL47 and the gene encoding vhs, which is present in the tegument, may produce a less cytopathic, latent, non-rephcating viral mutant for gene delivery to the brain.

1.3.9 Summary

The work described above is encouraging when considering the use of recombinant HSV-1 vectors for transgene delivery to the central nervous system. Our laboratory has aheady noted that the delivery of the lacZ gene 1CP27 (1E2) deleted mutants (previously described in cardiac cells - vide supra) can be extended to neuronal cehs in culture and the central nervous system in vivo (Howard et al., submitted). On the basis of this data, the recombinant HSV-1 vectors e?q)ressing heat shock genes described in this thesis were designed and constructed.

97 1.4 Project Aims

The aim of the work reported in this thesis was to study the neuroprotective effect of overexpressing the hsps in neuronal in vitro, by designing and characterising vectors that can ultimately be used for heat shock gene delivery and assessment of the neuroprotective effect of the hsps in vivo. The principle questions addressed in this thesis are: 1) What are the levels of a range of hsp mRNA and protem over time during permanent focal cerebral ischaemia induced by middle cerebral artery occlusion (MCAO)? 2) Can the heat shock genes and H-BH, the constitutively active mutant of HSFl, be successfiilly delivered to neuron-derived ND7 cell lines using recombinant HSV-1 vector technology in vitro, with a view to ultimately delivering the heat shock genes to the rat brain in vivol 3) Which heat shock proteins are overexpressed in neuron-derived cell lines following gene delivery of the H-BH gene? 4) What is the effect, if any, that pre-mfecfion with these viral vectors has on neuron- derived ND7 cells and primary neurons in vitro on heat shock and simulated ischaemia-induced cell death and serum/NGF-withdrawal induced ap opto sis?

98 Chapter 2

Materials and Methods

99 2.1 Laboratory Reagents

2.1.1 Chemicals

General laboratory chemicals were of analytical grade and purchased from either Merck Ltd, Poole, Dorset, U.K.; Boehringer Mannheim, Lewes, East Sussex, U K ; Difco Laboratories, Detroit Laboratories, Detroit, U.S.A.; Sigma Chemical Company Ltd, Poole, Dorset, U K ; Life Technologies, Paisley, Renfrewshire, U K ; or Biometra

Ltd, U.K Hexanucleotides [pd(N) 6] for random prime labelling and dNTPs were obtained from Pharmacia Biotechnology Ltd, St. Albans, Herts, U.K Radiochemicals, Hybond™-N+ nylon membranes and Hybond™-C/Hybond™-C Extra nitrocellulose membranes were obtained from Amersham International Pic., Little Chalfont, Bucks, U.K.

Kodak X-OMAT imaging photographic film was purchased from Sigma Chemical Co. Ltd., Poole, Dorset, U.K, and Hyperfihn™-MP was obtained from Amersham Litemational Pic., Little Chalfont, Bucks, U.K Film developing and fixing chemicals were obtained from Photosol, Genetic Research Instrumentation. 30% (w/v) acrylamide/ 0 .8 % bisacrylamide stock solution for polyacrylamide gels was obtained from Scotlab.

General disposable plasticware was supphed by Greiner, except 2 0 ml Universal Tubes and 10ml disposable pipettes which were supphed by Sterilin. 3MM chromatography paper was supphed by Whatman and disposable filters were obtained from Gelman Life Sciences.

Ah sohd chemicals were dissolved in ddH 2 0 , adjusted to the required pH with HCl or NaOH and autoclaved or filter sterihsed unless otherwise stated. NaOH and SDS and SSC were not autoclaved; phenol and ethidium bromide solutions were stored in the dark. Chloroform always contained 4% isoamyl alcohol. Phenol (Fisher Scientific Company) was buffered by shaking three times with an equal volume of 0.5M Tris (pHS.O), and then with 0. IM Tris (pHS.O) removing the aqueous layer each time.

100 2.1.2 General Solutions

(AU concentrations expressed at xl) Stock Concentration PBS xlO 104mM sodium, l.SmM potassium chloride, 5.4mM disodium orthophosphate dihydrate, 1.25mM potassium dihydrogen orthophosphate, pH7. Autoclaved and stored at room temperature.

SSC x20 150mM sodium chloride, 15mM sodium citrate, pH 8 . Stored at room temperature.

TE xl lOmM Tris-HCl pH7.4, ImM EDTA, pH8.0.

2.1.3 Enzymes

AU restriction Enzymes and DNA modifying enzymes were obtained from Promega, Southan^ton, U.K unless otherwise stated and stored at -20°C. DNase-free RNase-A (Boehringer Mannheim, Lewes, East Sussex, U.K) was prepared by dissolving lOmg ml'^ in 15mM NaCl, lOmM Tris-HCl, and boiling for 10 minutes to remove DNase activity. AUquots were stored at -20”C.

101 Table 2.1 - DNA Plasmids

Plasmid Description Reference/Source

pBS27 Chinese hamster hsp27 Dr. Jacques Landry cDNA in pBluescript SK+ Université Laval, Quebec, (Stratagene Ltd., Cambridge, Canada.(Lavoie etal., 1990) U.K.)

p59 pGem7Zf+ (Promega, Dr. Marie-Claire Lebeau, Southampton, U.K.) Institut national de la sante containing rabbit hsp56 cDNA et de la recherche médicale, Inserm U 33. (Lebeau etal., 1992)

pRep65 Human hsp60 cDNA in Prof. Radhay S. Gupta. pRepS RSV-LTR driven (Jindal etal., 1989) expression vector (Invitrogen Corporation)

PH2.3 Human hsp70 cDNA in American Type Culture pAT 153 vector Collection (Wu etal., 1985)

ppa90 Human hsp90 cDNA cloned Dept, of Molecular into PHbA Pr-1-neo Pathology, University expression vector College London (Twomey et al., 1993)

GM-BH pGem3Z vector (Promega, Prof. Richard Voellmy, Southampton, U.K.) University of Miami, containing Hinc\\/BamH\ U.S.A. (Zuo etal., 1995) deletion mutant of human HSF-1

pCH110 Eukaryotic assay vector Pharmacia Biotechnology containing E. coli lacZ gene Ltd, St. Albans, Herts, U.K

pEGFPNI Eukaryotic expression vector Clontech Laboratories Inc., expressing green fluorescent Palo Alto, protein (GFP) variant California, optimised for expression in U.S.A. mammalian cells under the control of a CMV IE promoter, with an SV40 polyadenylation sequence

102 (Hall et ai, 1983) pUC-tub 800 bp fragment of human p- tubulin cDNA cloned into Pst\ site of pUCS plasmid vector pJ4Q Expression vector containing the (Morgenstern and Land, Moloney murine leukaemia virus 1990) long terminal repeat (MoMLV- LTR) followed by a multiple cloning site and a 3' SV401 intervening sequence (IVS) and polyadenylation sequence pJ7Q Expression vector containing the (Morgenstern and Land, constitutive immediate early 1990) cytomegalovirus IE94 promoter (CMV IE94) with a 3' polylinker and a transcriptional termination signal

LSN HSP70 promoter driven CAT and Dr. Richard 1. Morimoto neomycin resistance gene Northwestern University, plasmid U.S.A. (Williams and Morimoto, 1990) pSP72 Ampicillin resistant E. coli cloning Promega, Southampton, vector U.K. pcDNAS Mammalian expression vector Invitrogen Corporation containing major CMV IE promoter/enhancer and 3’ bovine growth hormone (BGH) polyadenylation sequence pNot3.5 3.5kb Noti fragment of RL region Dr. Robert S. Coffin, of herpes simplex virus type 1 University College (HSV-1) DNA cloned into London pGem5Zf+ vector (Promega)

pDde rev 589bp Ddel fragment of HSV-1, Dr. Robert S. Coffin, including the LAT PI promoter, University College cloned into pGem3Z vector London (Promega)

103 pAEIsplA Adenoviral shuttle vectors Dr. Lionel Wightman, United Medical and pAEIsplB Dental Schools, U.K. pCITE-1 Encephalomyocarditis virus Dr. Jeffrey Almond, internal ribosome entry site University of Reading, sequence in plasmid vector U.K.

2.2 Bacterial Strains and Growth Conditions

2.2.1 Bacterial Strains

DH5a supE44 Alacl69 (0lacZAM15) hsdRlV recAl (Bethesda Research Labs, 1986) endAlgyrA96 thi-1 relAl

XLl-BIue recAl endAl gyrA96 thi-1 hsdR17 supE44 relAl (Stratagene Ltd., Cambridge U.K.) lac[F’proAB lacPZAMlS Tn70 (Tet)7

2.2.2 Propagation and Storage of Bacteria

Luria Bertani (LB) Media: 1% (w/v) Bacto®-tryptone 1% (w/v) NaCl 0.5% (w/v) Bacto®-yeast extract

LB Agar: LB containing 2% Bacto®-agar

Media were autoclaved at 120°C for 20 minutes at 10 lb square inch'\ Ampicillin (lOOmg ml'^ stock) [Sigma Chemical Corqpany, Poole, Dorset, U.K] was stored at - 20°C. Tetracycline (5mg ml'^ in 50% (v/v) ethanol) [Sigma Chemical Coropany, Poole, Dorset, U.K.] was stored at -20°C.

104 Starter culturesoïE. coli were grown in 5ml of LB containing no antibiotic, lOOfxg ml ^ of anq)icillin or 15 tig ml^ of tetracycline where required. Cultures were grown in a Gallenkamgp orbital shaker overnight at 37°C at 200rpm. lOOfil of the overnight starter culture was used to inoculate 100ml LB for growth in preparation of transformation- corqpetent cells. Larger volumes of LB to culture overnight for plasmid preparation were inoculated with 1 : 1 0 0 0 0 (v/v) of starter culture.

2.2.3 E. CO//Transformation

The method used for preparation and transformation of competent cells is based on the calcium chloride method outlined in Sambrook et al., 1989.

The 100ml culture of E. coli described in Section 2.2.2 was grown to an OD 580 of 0.4- 0.55 units and the bacteria were pelleted in 50 ml sterile tubes by centrifugation in a standard benchtop centrifuge at 3,000rpm at 4°C for 1 0 minutes. The supernatant was discarded and the tubes and lids wiped dry. The pellets were resuspended by shaking in 10 ml ice-cold lOOmM CaCL. The bacteria were again pelleted and drained as above, resuspended in 2ml/pellet of ice-cold lOOmM CaCL, and incubated on ice for at least 30 minutes prior to use. Conq)etent cells were stored on ice and used within 72 hours or discarded.

For transformation, 200p,l of conq)etent cell suspension was ahquotted into 15ml sterile tubes. The DNA for transformation was added to the cell suspension and mixed by flicking and incubated on ice for 30 minutes. The cells were heat shocked at 42°C for 90 seconds and cooled on ice for 2 minutes. SOOpl of LB medium was added and the cells were grown in an orbital shaker at 37°C/200rpm for 30 minutes to one hour. The cells were pelleted in a standard benchtop centrifiige at 22°C/3,000rpm, 900pl of supernatant was discarded, and the cells were resuspended in the remainder. The whole suspension was spread on selective LB plates supplemented with anq)icillin or tetracycline at 100|ig ml^ and 15pg ml'^ respectively. Plates required to demonstrate expression of (3-galactosidase were pre-treated with 50pl of 4-Cl,5-bromo,3-mdolyl-y0-

105 galactosidase (X-gal, Insight Biotechnology Ltd) [20mg ml'^ stock in dimethyl formamide, stored in the dark at -20^*0]. Plates were incubated overnight at 37°C.

2.3 DNA Isolation and Analysis

2.3.1 Small Scale ‘Mini-Prep’ Plasmid DNA Extraction from E. coli

This method for extraction and precipitation of recombinant DNA from E. coli transformants is based on the alkaline lysis method described by Bimboim and Doly, 1979.

Solution 1: 50mM Tris-HCl pH 7.5 lOmMEDTApHS lOOfxg ml^ RNase-A Solution 2: 200mM NaOH 1% (v/v) Triton X-100 Solution 3: 3M NaOAc pH5.5

5ml of LB medium was inoculated with a single colony or 5 pi of Uquid culture of DH5a E. coli and grown overnight in an orbital shaker (200rpm) at 37°C. 1.5ml was transferred to a 1.5ml microcentrifiige tube and centrifuged for Imin at 13,000rpm in a microcentrifuge. The supernatant was removed by aspiration before the sequential addition and thorough mixing by vortexing of lOOpl of Solution 1, 200pl of Solution 2, 150pl of Solution 3, to the remaining pellet. The tubes were microcentrifuged for 3mins, and the pellet removed by lancing with a hypodermic needle bent at the tip. To the supernatant was added 500pl of isopropanol and after vortexing it was microcentrifiiged for 5mins. The supernatant was drained off and the pellet was washed with 500pl of 70% ethanol in d H 2 O, air dried, and resuspended in 45pi of ddH20 containing 20pg/ml RNase A. Restriction digests were carried out on 5 pi of this DNA solution (see Section 2.3.4).

106 2.3.2 Large Scale ‘Midi-Prep’ Plasmid DNA Extraction from E. coli

400ml of LB was inoculated at 1:10,000 (v/v) with E. coli starter culture as described in Section 2.2.2 and grown overnight in an orbital shaker at 37°C/200rpm.

Large scale purification of plasmid DNA was achieved using the ‘Qiagen-tip 100’ midi-prep kit. The protocol is based on a modified alkaline lysis procedure, followed by plasmid separation on the Qiagen resin, elution and precipitation by isopropanol, and is defined with the kit. The subsequent dried, purified DNA was resuspended to

approximately Ipg pl'^ in ddH 2 0 and average yields were approximately lOOpg.

2.3.3 Restriction Site Analysis of DNA

Small scale digests were carried out to characterise the structure of a newly isolated plasmid. Digests were usually incubated in a total volume of lOpl for 1-2 hours at the temperature specified by the enzyme manufacturer. The amount of enzyme did not exceed 0.1 volumes as specified by the manufacturer. Appropriate enzyme buffers were used at the concentration specified by the manufacturer. The recipe for a typical small scale digest would be as follows: DNA Ipg or 5pi o f ‘mini-prep’ solution Enzyme(s) 10 Units total ( 1 pi) Buffer Ix(lpl) ddHzO to lOpl

Large scale digests were performed to isolate fragments of DNA for subsequent hgation or oHgonucleotide radiolabelling. The final reaction volume would normally be lOOpl, and the proportions would be the same as above with lOx the amounts of DNA, enzyme and buffer. The digest would be incubated at the manufacturer’s recommended temperature for 1-16 hours and digested DNA would be extracted by phenol/chloroform extraction and ethanol precipitation as described in Section 2.3.4.

107 2.3.4 Purification and Precipitation of DNA

To purify DNA after a restriction enzyme or blunt ending reaction, the mix was made up to 400pl with ddH20, and the same volume of phenol solution added. The mixture was vortexed and microfuged for 2 minutes at 13,000rpm and the top aqueous layer transferred to a fresh 1.5ml microfrige tube. To this, one volume of chloroform solution was added, vortexed and pulse spun in a microfuge up to 13,000rpm The aqueous layer was transferred to a fresh microfuge tube and 10% (v/v) of 3M sodium acetate (pH5.5) and 2 volumes of 100% ethanol (-20®C) and added. The mixture was vortexed and spun at 13,000rpm for 15 minutes. The pellet was washed with 70%

ethanol in H 2 O, vortexed and spun for 5 minutes at 13,000rpm The supernatant was taken off and the purified DNA peUet freeze-dried and resuspended in 5 pi ddH20.

2.3.5 Agarose Gel Electrophoresis

IxTAE: 0.4M Tris 0.2M sodium acetate 20mMEDTA pH Adjusted to 8.3 with HCl lOxLoading Buffer: IxTAE 50% (v/v) glycerol 0.025% bromophenol blue Ethidium Bromide: lOmg ml'^ dissolved in ddH20 and stored in the dark at room ten^erature. DNA M arkers: 1 kilobase (kb) DNA ladder (MBI Fermentas, Immunogen International, Tyne and Wear, U .K ) was diluted 1:5 with Ixloading buffer and stored at -20°C. Agarose (Biometra) was melted and dissolved at 1% (w/v) in IxTAE in a microwave oven. Ethidium bromide was added to a final concentration of 0.5pg ml \ After cooling to ‘hand-hot’ the melted agarose was poured into a Biometra ‘mini’ or ‘midi’ gel caster and a suitable comb(s) added. Once set, the comb(s) were removed and the DNA marker and sanq)les containing 1 x loading buffer were added and run at 100 V at room temperature. After the loading dye had travelled up to % the length of the gel

108 it was photographed on a Biometra UV transilluminator onto black and white Polaroid film. Low melting point gels for DNA fragment isolation and excision were performed as above using low melting point agarose (Biometra). Fragments were excised, removing all excess agarose, on a UV transilluminator (Biometra) and stored at -20°C until required.

2.3.6 Blunt Ending Reactions

If a restriction fragment can not be hgated into a vector by sticky end Hgation, then the 3’ overhangs of the DNA were blunted by treatment with T4 DNA polymerase (Promega, Soutbanqjton, U.K.). After the digest reaction, Ijil of a 25mM solution of each of the four dNTPs (dATP, dTTP, dCTP, dGTP), and 15 units of T4 DNA polymerase was added. The reaction was incubated for 1 hour at 3TC and then the DNA purified and precipitated by pbenol/cbloroform precipitation or treated with calf intestinal alkaline phosphatase (see Section 2.3.7).

2.3.7 Phosphatase Treatment of Plasmid DNA

To prevent rebgation of like ends of vector during insert/vector Hgation, vector DNA was treated with Calf Intestinal Alkaline Phosphatase (CIP) (Promega, Southampton, U.K). Restriction enzyme reactions/blunt ending reactions were inactivated by mcubation for 20 minutes at 65°C and the reaction volume was made up to 400pl with a final concentration of 1 x alkaline phosphatase buffer (stock lOx), ddH 2 0 and 10 units of CIP. The reaction was mcubated at 37°C for no longer than 30 minutes and the CIP was inactivated by incubation for 20 minutes at 65°C. Subsequently, the DNA was purified by two phenol extractions and one chloroform extraction, and precipitated as hi Section 2.3.4.

2.3.8 Ligation of DNA

Ligations were carried out in 6pi reactions in microcentrifiige tubes containing a final volume of 1 X Hgase buffer (Stock 5x), 1-3 units of T4 DNA Hgase (Promega,

109 Southampton, U.K.). If both insert and vector were gel purified, the gel fragments were melted at 80°C for 5 minutes and Ipl of insert suspension was mixed with 0.7p.l of vector suspension and added to the reaction mix, prior to the addition of enzyme. If only the insert was gel purified Ip-l of insert suspension was mixed with 2pi of resuspended DNA from phenol/chloroform extraction and ethanol precipitated. Final reaction volume was made up to 7pi with ddH20. The reaction was left for 2 hours at room temperature, before the reaction mix was transformed into competent cells as in Section 2.2.3.

2.3.9 Screening E. coli colonies positive for plasmid containing insert

In the absence of blue/white selection on X-gal plates, screening of colonies was carried out by blotting the colonies onto Hybond-N+ membrane (Amersham International Pic, Little Chalfont, Bucks, U.K), lysing the bacteria in alkaline solution, denaturing and fixing the DNA to the membrane using a UV Stratahnker 2400, according to the protocols provided with the membrane. cDNA radiolabelled random primers of the desired insert were prepared as described in Section 2.3.11, Method 2. Pre-hybridisation and hybridisation of the membranes was carried out as described in Section 2.3.12. Positive colonies were selected against an autoradiograph of the probed discs. Selected colonies were grown up overnight in LB medium containing the appropriate antibiotic at 37°C in a shaking incubator, and small scale DNA isolation and restriction enzyme screening was carried out as described in Sections 2.3.1 and 2.3.3.

2.3.10 Preparation of cDNAfor Radiolabelling

cDNA fragments were isolation from plasmid DNA by large scale digestion with appropriate restriction enzymes as in Section 2.3.3. The digested DNA was purified by phenol/chloroform extraction and ethanol precipitation, and run in a low melting point agarose gel. The required shce was excised, melted at 80°C for 2 minutes and mixed with 3 volumes (w/w) of ddH20 as described by Sambrook et al., 1989.

110 2.3.11 Radiolabelling of DNA

Two methods were used for radiolabelling of DNA, both of which were based on the random prime labelling reaction adapted fromFeinberg and Vogelstein, 1983.

Method 1: Preparation of Probes for Labelling RNA Blots

Oligolabelling Buffer (OLB) OLB was made by mixmg A B C in a ratio of 100:250; 150 Solution O: 1.25M Tris-HCl (pHS.O) Stored at room temp. 0.125MMgCb Solution A: 1ml Solution O Stored at -20°C ISpl (3-mercaptoetbanol 5pl0.1MdATP 5|xl0.1MdGTP 5pl0.1MdTTP Solution B: 2M HEPES pH6.6 Stored at 4°C Solution C: 90 Units ml'^ random bexamers [pd(N)6] Stored at -20°C dissolved in TE pHS

2-3 pi of DNA fragment from Section 2.3.10 was denatured in 30pl ddH20 at 97°C for 10 minutes and snap cooled on ice. To this suspension was added lOpl OLB, 2pi bovine serum albumin (BSA), 5 units of DNA polymerase large fragment 1 (Klenow), and 50pCi of a-p^P]-dCTP. The reaction was incubated either overnight at 16°C (optimum annealing temperature) or for 1-2 hours at 3TC (optimum enzyme temperature). The probe was filtered through a G50 Sepbadex column and subsequently flushed with 100pi abquots of 3xSSC. Each lOOpl filtered abquot was screened for ^-emissions on an LKB beta-counter and the most active sarqple was selected for probing.

I l l Method 2: Preparation of Probes for Colony Screening HEPES Buffer: 50mM MgCl: 40mM HEPES pH to 6.6 with NaOH 3pi of agarose/DNA suspension was added to: 5 pi of HEPES buffer 20pl ddHiO Ipl of 25mM ATP, GTP, T IP solution 3 pi of 50 Units ml^ random hexamers [pd(N6)] DNA in mixture was denatured at 97°C for 5 minutes before addition of 30pCi of a- [^^P]-dCTP and 5 Units of DNA polymerase I large fragment (Klenow). Reaction was incubated at 37®C for 1 hour.

2.3.12 Hybridisation

100 X Denhardt’s reagent: 2% (w/v) bovine serum albumin (BSA) 2% (w/v) frcoll® (type 400) 2% (w/v) polyvinylpyrrohdone Dissolved in DEPC-treated, ddH20 (see Section 2.4.1). Prepared in ahquots and stored at -20°C (Sambrook et al., 1989).

For RNA blots, the filters were pre-hybridised for 2 hours at 42°C in 5 x SSPE, 50% (w/v) deionised formamide, 5 x Denhardt’s reagent, 0.5% (w/v) SDS and lOOpg mf^ of denatured salmon sperm DNA (97°C for 5-10 minutes, snap cooled on ice) and made up to volume with DEPC-treated ddH20. The hybridisation solution was replaced containing the denatured (97°C for 5-10 minutes, snap cooled on ice) radiolabelled probe and the blots were incubated overnight at 42°C.

For DNA blots, the filters were pre-hybridised for 1 hour at 65°C in 5 x SSC, 5 x Denhardt’s reagent, 0.5% (w/v) SDS and 200pg ml'^ of denatured (as above) herring

112 sperm DNA. The probe was denatured for 5 minutes at 100°C and added to the pre­ hybridisation solution and the filters were incubated overnight at 65°C.

Post-hybridisation washes

RNA Filters: The hybridisation solution was discarded. All wash solutions were pre-heated to 65°C. Filters were washed for 30 minutes at 65®C in 3 x SSC / 0.1% (w/v) SDS. Wash solution was replaced with 1 x SSC / 0.1% (w/v) SDS and incubated for 30 minutes at 65°C. The Northern blot filters were wrapped in cling film and exposed to an X-ray film overnight in an autoradiography cassette containing an image intensifying screen at - 70°C and the film was subsequently developed. Slot blot filters were wrapped in cling film and incubated for 1 hour-overnight against a imaging screen and the detected bands quantified using a BioRad GS-250 Molecular Imager™.

DNA Filters: The hybridisation solution was discarded. Filters were washed twice for 10 minutes at room temperature in 2 x SSC / 0.1% (w/v) SDS. Wash solution was replaced with pre-heated 1 x SSC / 0.1% (w/v) SDS and incubated for 15 minutes at 65°C. The solution was replaced with 0.1 x SSC / 0.1% (w/v) SDS and incubated for 10 minutes at 65°C. The filters were wrapped in cling film and exposed to X-ray film for 4-5 hours at -70°C in an autoradiography cassette containing an image intensifying screen and the film was subsequently developed.

Stripping Filters RNA jGlters were stripped for re-probing by incubating for 1 hour at 65°C in 0.2% (w/v) SDS. To confirm that all the radioactivity was removed, filters were exposed to a phosphoimager screen and scanned usmg a BioRad GS-250 Molecular Imager™.

113 2.4 RNA Isolation and Analysis

2.4.1 Preparation of RNase free materials

Gloves were worn at aU times when handling RNA or RNase-free materials. All glassware was baked overnight at 180°C. ddH20 was incubated with 0.1% (v/v) diethyl pyrocarbonate (DEPC) for 4 hours at room temperature, then the DEPC was inactivated by autoclaving the treated solution. DEPC is an RNase inhibitor. Solutions containing amines (Tris-HCl) were not DEPC treated, but were prepared using DEPC treated ddHzO which had previously been autoclaved, and incubated overnight at 65°C to evaporate off the degradation products.

2.4.2 RNA Extraction from Brain Samples

Dénaturant: 4M guanidium thiocyanate 25mM sodium citrate pH7.0 0.5% L-lauryl sarcosine 0. IM p-mercaptoethanol (added on day of use) Made up with DEPC treated ddHzO

Brain samples were transferred from hquid nitrogen to 15ml polypropylene tubes and 1ml of Dénaturant was added. Samples were homogenised for 10 seconds and stored on ice. 150pl of 2M sodium acetate pH4.0 was added to each suspension which were subsequently vortexed. 1ml of phenol solution was added, followed by vortexing.

300|l i 1 of chloroform: iso amyl alcohol (49:1) was added and mixed by vortexing. The suspensions were incubated on ice for 15 minutes. The suspensions were centrifixged at 10,000rpm for 20 minutes at 4°C, and the aqueous phase was transferred into a fresh tube. An equal volume of ice-cold isopropanol was added to the aqueous phase and they were incubated overnight at -20°C. The samples were subsequently centrifixged at 10,000rpm for 20 minutes at 4^’C and the supernatant discarded. The pellet was resuspended in 300|il of dénaturant and transferred to 1.5ml microfixge tubes. 1 volxxme of ice-cold isopropanol was added and mixed by vortexing, and the sanples

114 were mcubated at -20°C for at least 2 hours. The samples were centrifuged at 13,000rpm for 10 minutes at 4°C in a cold room. The supernatant was discarded and the pellet was resuspended in lOOpl of DEPC treated ddH20.

2.4.3 Quantitation of RNA lOpl of sangle was diluted 5Ox in DEPC treated ddHiO, and absorbance was measured at A260 and A 280 on a spectrophotometer. Ratio of A 26 0 :A280 should be 2:1 if sanq)le is pure. Total RNA concentration in pg ml'^ was calculated using the following calculation:

[RNA] = X X 40 X 50 X 90/1000

Where x = A 260 40: 1.0 A26o= 40pg ml^ 50: Dilution factor 90/1000 = 0.009ml = Volume of remaining sample in ml Therefore, [RNA] = x x 180

70% (v/v) ethanol was added to samples and were stored at -70°C until required.

2.4.4 Formaldehyde Gel Electrophoresis of RNA

10 X MOPS Buffer: 120mM MOPS (morpholinopropane sulphonic acid) 30mM sodium acetate 60mM EDTA Made up in ddH20 Keep in the dark

Running Gel: 1. Ig Agarose 10ml 10 X MOPS buffer

75ml ddH 2 0 Microwaved, cooled to ~50°C 20% (v/v) formaldehyde added

115 Running Buffer: 1 x MOPS Buffer Made up with ddHzO

Denaturing Buffer: 250(il formaldehyde 50|il 10 X MOPS Buffer 80|xl deionised formamide

Sandies were spun in a microcentrifiige at 13,000rpm for 10 minutes at 4°C in a cold room and resuspended in running buffer to give 20|ig per 6|il per well. 6p.l sample was added to 19|xl of denaturing buffer. Duphcate samples and RNA kb ladder were run in parallel to locate 28S and 18S positions on the blot. Samples were denatured at 65°C for 5 minutes and snap cooled. 1-3p,l of loading buffer was added and 28p,l of sanple was loaded per well. Gels were run at lOOV until dye front was % down the gel. Marker lanes were cut off gel and formaldehyde washed out four times for 10 minutes in 400ml ddH20. Gel was stained with 10% (v/v) ethidium bromide in ddH20, and washed three times in ddH 2 0 for 10 minutes. Gel was soaked in ddH20 for 10 minutes and transferred to 3 x SSC for 15 minutes

2.4.5 Northern Blotting

RNA was transferred to Hybond-N nylon membrane using capillary action. The gel was placed upside down on a plastic support covered in a 3MM paper wick that was soaking in a bed of 20 x SSC. The nylon membrane was cut to the size of the gel and the edges of the gel were covered in cling film to prevent the 20 x SSC spreading to the membrane outside the gel. The membrane (pre-soaked in ddH20 and then 3 x SSC) was placed on the gel followed by 2 layers of 3MM paper (pre-soaked in 20 x SSC). These were overlayed with a stack of dry paper towels and topped with a suitable weight. The gel was blotted overnight. The membrane was then removed and crosslinked in a UV Stratalinker 2400. The membrane was wrapped in cling film and stored at 4°C prior to hybridisation as in Sections 2.3.10-2.3.12.

116 2.4.6 Slot Blotting

Slot Buffer: 7.5 x SSC 25% (v/v) formaldehyde Made up with DEPC treated ddH20

RNA samples from Section 2.4.3 were pelleted in a microcentrifuge at 13,000rpm for 10 minutes at 4°C. The supernatant was discarded an the pellets resuspended in DEPC treated ddH20 to a final concentration of 3.2p,g p,r\ 10|rl (32p,g) was added to 190^1 DEPC treated ddH20 in a 1.5ml microfuge tube and mixed by pipetting. lOOp.1 of sample was transferred into a fresh tube containing 100|il DEPC treated ddH20 (serial dilution of 1 in 2), mixed, and a second serial 1 in 2 dilution was also prepared from this. 400|il of slot buffer was added to each sample and vortexed. The sangles were denatured for 10 minutes at 65°C, snap cooling on ice. Hybond-N membrane was cut to slot apparatus size. Membrane was pre-soaked in ddH20 and then 3 x SSC. Apparatus was assembled and 240|xl was loaded into each slot and a gentle vacuum was apphed until all solution had passed through (~10 minutes). RNA was crosslinked using a UV Stratalinker 2400. Membrane was wrapped in cling film and stored at 4°C until required for hybridisation as described in Sections 2.3.10-2.3.12.

2.5 Protein Isolation and Analysis

2.5.1 Protein Extraction from Dissected Rat Brain

All procedures were carried out on ice unless otherwise stated. Frozen rat brain tissue was weighed and homogenised in approximately 5 x (v/w) of ice-cold 0.1% (w/v) SDS. 2pi of suspension was removed and added to 198pl of 0.1% (w/v) SDS for total protein assay (see Section 2.5.2). lOOpl of suspension was removed and added to 300pi of 0.1% (w/v) SDS. To this was added the same volume of 2 X Laemmli Sample Buffer (20% (v/v) glycerol, 6% (w/v) SDS, 0.12M Tris-HCl, pH6.8, stored at room tercperature) (Laemmli, 1970). Further sample buffer was added to make sanples up to 50pg 15pl'^ total protein concentration according to the

117 total protein assay (see Section 2.5.2). 5% (v/v) p-mercaptoethanol and 10% (w/v) bromophenol blue were added to the sangles before denaturing the proteins by boiling for 5-10 minutes. Sanq)les were subsequently loaded at 50pg (15pl) per well on an SDS-polyacrylamide gel (see Section 2.5.4), or stored at -20^C.

2.5.2 Quantitation of Total Protein from Homogenised Samples

The protein concentration of extracts were quantified by Pierce BCA Protein Assay (Rockford, Illinois, U.S.A.). This method is based on the reaction between protein and Cu^^ ions in the presence of BCA protein assay reagent (green in colour), OIT ions

(alkah medium) and H 2O to form a BCA-Cu^^ complex which has a purple colour and exhibits a strong absorbance at 562nm This colour change can be quantitated spectometrically and is a measure of the amount of protein present in a sample.

BCA Protein Assay Reagent: Immediately prior to use Reagent A and Reagent B were mixed at a ratio of 50A: IB Reagent A: As supphed by manufacturers (Pierce). Reagent B: 4% copper sulphate

Briefly, lOpl of sample was added to 200pl BCA Protein Assay Reagent and was incubated in a microtitre plate at 37°C for 30 minutes. Each sample was incubated in duphcate. A range of duphcate bovine serum albumin standard reactions (Omg ml^ - 0.2mg ml'^) was also set up each time the reaction was performed to give a standard curve which aUowed the calculation of protein concentration for each sample. The absorbance at 562nm was measured on a Lab systems Multiskan RC microtitre plate reader and the concentration calculated according to the standard using Lab systems Genesis Communication software.

118 2.5.3 Protein Extraction from Cultured Cells

Between procedures, cells were stored on ice. 1 X 10^ cells were washed in 1ml of 1 x PBS and harvested into 1.5ml microfuge tubes. Cells were pelleted in a microcentrifiige at 2,000rpm for 10 minutes, AU the supernatant was drained using a hypodermic needle and immediately resuspended by vortexing in 100|xl ice-cold 2 x Laemmh Sample Buffer containing 5% (v/v) )3- mercaptoethanol and 10% (w/v) bromophenol blue and left on ice for 10 minutes. The samples were incubated at 100°C for 5-10 minutes and either run in an SDS- polyacrylamide gel immediately, or stored at -20°C. Comparison of protein loading of these sanqiles was attained by running a duphcate gel and staining it with Coomassie stain solution (see Section 2.5.5).

2.5.4 Polyacrylamide Gel Electrophoresis of Protein Extracts

Total ceU protein samples were separated into polypeptide units by SDS- polyacrylamide gel electrophoresis (Sambrook et a l, 1989). The denatured proteins form a negatively charged complex with the detergent SDS present in the gel and migrate through a high porosity stacking gel to then separate according to protein size in a resolving gel (Laemmli, 1970).

Stacking Gel: 5% acrylamide mix (30% (w/v) acrylamide, 0.8% (w/v)bisacrylamide) 125mM Tris-HCl (pH6.8) 0.1% (w/v) SDS 0.1% (w/v) ammonium persulphate 0.1% (v/v) NNNN-tetraethylethalinediamine (TEMED)

Resolving Gel: 10% acrylamide mix (10%) 375mM Tris-HCl (pH8.8) 0.1% (w/v) SDS 0.05% ammonium persulphate 0.05% TEMED

119 Tris-Glycine Electrophoresis Running Buffer: 25mM Tris 250mM glycine (pH8.3) 0.1% SDS

The resolving gel was poured then immediately layered with water saturated isobutanol to prevent oxygen diffusing into the gel and thereby preventing polymerisation. After the gel had set, this layer was removed and the gel was rinsed with ddH20. Once the gel was dry through draining, the stacking gel was added to form the top layer. An appropriate comb was added prior to the polymerisation of the stacking gel.

Protein sangles were incubated at 100°C for 5-10 minutes immediately before loading on to the gel. High range molecular weight protein standards (Rainbow Markers, Amersham International Pic, Little Chalfont, Bucks, U.K.) were run on each gel to indicate the protein sizes. Air bubbles were removed fi*om the base of the gel, which was run in Tris-Glycine electrophoresis running buffer, in an acrylamide gel running tank (Life Technologies, Paisley, Renfi-ewshire, U.K.) at a constant 180Volts, 40mA (per gel), for 4-6 hours or until the dye front had reached the base.

2.5.5 Equalisation of Protein Loading

Coomassie Stain Solution: 2% (w/v) Coomassie Brilhant Blue R250 50% (v/v) methanol 50% (v/v) glacial acetic acid

Destain Solution: 10% (v/v) glacial acetic acid 30% (v/v) methanol

To equahse protein concentration in each lane, duphcate protein samples were separated by polyacrylamide gel electrophoresis (Section 2.5.5) and the gel placed in Coomassie stain solution. This was covered and incubated at room temperature overnight on a gently shaking platform. The foUowing day, any unbound stain was

120 washed off in repeated replacements of destain solution, and the gel sealed in a plastic bag. Non-specific protein levels were quantitated by densitometry (Bio-Rad GS-670 Imaging Densitometer).

2.5.6 Transfer of Protein to Nitrocellulose by Western Blotting

Blotting Buffer: 192mM glycine 20% (v/v) methanol 25mM Tris-HCl pH8.0 To transfer protein from SDS-polyacrylamide gel to nitrocellulose membrane (Hybond™-C/Hybond™-C Extra, Amersham International Pic, Little Chalfont, Bucks, U.K.), gels were blotted overnight at 175mA/55V at 4°C in a Bio-Rad Trans-blot™ Cell protein transfer apparatus. The membrane was subsequently removed dried on 3MM paper and the Rainbow Molecular Weight Marker highlighted with biro pen.

2.5.7 Immunodetection of Proteins on Western Blots

Block Buffer: 1 x PBS 0.05% polyoxyethylene sorbitan monolaurate (Tween 20) 4% (w/v) Marvel skimmed milk powder Wash Buffer: 1 x PBS 0.05% Tween 20

Following drying, filters were blocked in blocking buffer shaking gently on a platform for 1 hour at room temperature. The buffer was subsequently replaced with fiesh blocking buffer containing primary antibody at the required concentration (see Table 2.2). The filters were incubated, shaking gently, for 2 hours at the temperature specified by the antibody suppher (see Table 2.2). Unbound antibody was rinsed off by washing the membrane once in block buffer for 5 minutes, and twice for 5 minutes each in wash buffer on a shaking platform The filters were subsequently incubated for one hour at room temperature on a shaking platform in block buffer containing horseradish peroxidase-conjugated secondary antibody (see Table 2.2) diluted to the required concentration. Unbound secondary antibody was rinsed off m three 10 minute

121 washes of wash buffer at room temperature on a shaking platform The bound horseradish peroxidase was then detected using enhanced chemiluminescence (ECL™, Amersham International Pic, Little Chalfont, Bucks, U.K ) and the resultant hght emissions exposed to X-ray film for 5 seconds - 1 hour, depending on the strength of the signal. The exposed photographs were conoparatively quantified by scanning on a GS-670 imaging densitometer (Bio-Rad) and equahsed by conq>arison to the actin band on a Coomassie stained gel.

122 Table 2.2 - Antibodies Used in this Thesis, their Sources and Conditions for Use Antibody Conditions for Antibody Secondary Antibody Size of Source Hybridisation Dilution Conditions Protein Detected Anti-murine Hsp25 2 Hours Incubation at 1/1000 1/1000 dilution of HRP- 27kDa Stressgen (Polyclonal) Room Temperature swine anti-rabbit Ig. 1 hour Biotechnologies Corp., incubation at room Victoria, Canada. temperature Anti-Chinese 2 Hours Incubation at 1/1000 1/1000 dilution of HRP-rat 27kDa A Kind gift from Dr. Hamster Ovary Room Temperature anti-mouse Ig. 1 hour Jacques Landry, Hsp27 (Monoclonal) incubation at room Université Laval, Clone L2R3 temperature Quebec, Canada. Anti-Rat 2 Hours Incubation at 1/1000 1/1000 dilution of HRP- 32kDa Stressgen

N) Haemoxygenase-1 Room Temperature swine anti-rabbit Ig. 1 hour Biotechnologies Corp., U) (Polyclonal) incubation at room Victoria, Canada. temperature Anti-Rabbit Hsp56 2 Hours Incubation at 1/1000 1/1000 dilution of HRP-rat 56kDa Stressgen (Monoclonal) Room Temperature anti-mouse Ig. 1 hour Biotechnologies Corp., Clone KN382/EC1 incubation at room Victoria, Canada. temperature Anti-Chicken 2 Hours Incubation at 1/1000 1/1000 dilution of HRP-rat 56kDa Stressgen FKBP54 Room Temperature anti-mouse Ig. 1 hour Biotechnologies Corp., (Monoclonal) incubation at room Victoria, Canada. Clone FF1 temperature Anti-Human Hsp60 2 Hours Incubation at 1/1000 1/1000 dilution of HRP-rat 60kDa Affinity Bioreagents, Monoclonal Room Temperature anti-mouse Ig. 1 hour Inc., (MA3-012) incubation at room Colorado, U.S.A. temperature Antibody Conditions for Antibody Secondary Antibody Size of Source Hybridisation Dilution Conditions Protein Detected Anti-Human Hsp60 2 Hours Incubation at 1/1000 1/1000 dilution of HRP-rat BOkDa Stressgen (Monoclonal) Room Temperature anti-mouse Ig. 1 hour Biotechnologies Corp., Clone LK-1 incubation at room Victoria, Canada. temperature Anti-Human Hsp70 2 Hours Incubation at 1/1000 1/1000 dilution of HRP-rat 70kDa Stressgen (Monoclonal) Room Temperature anti-mouse Ig. 1 hour Biotechnologies Corp., Clone C92F3A-5 incubation at room Victoria, Canada. temperature AnW-Achlyia 2 Hours Incubation at 1/1000 1/1000 dilution of HRP-rat 90kDa A kind gift from Dr. D.A. ambisexualis Hsp90 Room Temperature anti-mouse Ig. 1 hour Toft, The Mayo Clinic, K) (Monoclonal) incubation at room Rochester, U.S.A. Clone AC88 temperature Anti-human HSF1 2 Hours Incubation at 1/500 1/15000 dilution of HRP- 83kDa Stressgen (Polyclonal) 37"C rabbit anti-chicken Ig Biotechnologies Corp., (Sigma). 1 hour at room Victoria, Canada. temperature Anti-rat p-tubulin 2 Hours Incubation at 1/1000 1/1000 dilution of HRP-rat 55kDa Sigma (Monoclonal) Room Temperature anti-mouse Ig. 1 hour Immunochemicals, Clone TUB 2.1 incubation at room Sigma Chemical temperature Company, Poole, Dorset. 2.6 Tissue Culture

All tissue culture preparation was carried out under sterile conditions in a Laminar Flow safety cabinet. All viral preparation was carried out under Health and Safety Executive category 2 conditions. All plasticware was obtained from Nunc, Roskilde, Denmark, unless otherwise stated. All media and supplements were obtained from Life Technologies, Paisley, Renfrewshire, U.K. unless otherwise stated. AU gas cylinders were supphed by the British Oxygen Company (BOG Ltd, GuUdford, UK).

2.6.1 Mammalian Cell Lines and Primary Neuronal Cells

BHK CeUs - Transformed baby hamster kidney ceUs, BHK-21, Clone 13 were provided by the lnq)erial Cancer Research Fund (ICRF), London, U K (Macpherson and Stoker, 1962). ND7 CeUs - Created by the fusion of immortaUsed HGPRT-mouse neuroblastoma ceUs (N18Tg2) with rat post-mitotic neonatal dorsal root ganghon neurons (Wood et al., 1990). This ceU line was a kind gift from Dr. J. Wood, Sandoz Institute, London, U K B 130/2 - BHK ceU line stably expressing neomycin resistance gene and HSV-1 strain 17+ ICP27 under the control of the ICP27 promoter. This ceU line was created by Dr. M.K Howard in our laboratory. DRGs - Sprague-Dawley rat dorsal root ganghon ceUs dissected and transferred to glass coversHps in defined media at postnatal day 2 by Elizabeth Ensor, in our laboratory.

2.6.2 Growth Conditions and Storage of Mammalian Cell Lines

ND7 ceUs were grown in Liebovitz L I5 media containing 10% foetal calf serum (FCS), 100 units ml^ penicillin and streptomycin, was supplemented with 0.35% (w/v) glucose, 2mM L-glutamine lOOx and 0.375% (w/v) sodium bicarbonate. The ceUs were not used for more than 30 passages. CeUs were passaged by dislodging the ceUs

125 growing on the surface of the culture flask and ahquotting the cell suspension into jfresh culture flasks to a final dilution of 1:5 in Jfresh growth media.

BHK cell lines were grown in Dulbecco’s modified Eagle’s medium containing 10% FCS and 100 units ml^ penicillin and streptomycin. B 130/2 selection was achieved by supplementing growth media with 500pg ml^ of Geneticin-G418 sulphate. Both cell lines were passaged by washing in Hanks Balanced Salt Solution (HBSS) at room temperature, and incubating for two minutes with 2ml per 80cm^ culture flask of 10%

(v/v) trypsin in versene at 37®C/5% CO 2 Enzyme activity was blocked with the same volume of growth media, the cells were vortexed to prevent clumping, and ahquotted 1:8 in fi-esh growth media.

Cells were subcultured whilst in log phase, as determined by growth curves. Frozen stocks were made by mixing 0.5ml of cell suspension with 0.5ml of ice-cold freezing mixture [85% FCS, 15% dimethyl sulfoxide (DMSO; Merck Ltd, Poole, Dorset, U.K.)] in 1.5ml cryo-tubes and transferring the vials to -70°C in an insulated box to ensure gentle freezmg.

Frozen cell stocks were quickly thawed at 37°C and transferred immediately to 10ml of growth media, and pelleted in a standard benchtop centrifuge at 20°C, 2,000rpm for 10 minutes, and resuspended in 0.5ml and titrated by 1:2 serial dilutions m 8 wells of a 24 well plate in selection media. Resistant colonies were selected and grown up for subsequent use.

FoUowmg dissection, primary DRG cells were incubated at 37°C/5% CO 2 overnight on glass coversHps in defined media (Davies, 1995) in petri dishes prior to transferral of the coversHps to individual wells of a 24-well plate for infection with recombinant HSV-1 vectors.

Defined Media: 1 x F-14 containing 2g.f^ NaHCOs, pH 7, stored at 4°C. 2mM glutamine 0.35% (w/v) bovine serum albumin

126 ôOng.ml'^ progesterone lôpg.ml'^ putrescine 400ng.ml'^ L-thyroxine 38ng.ml'^ sodium selenite 340ng.ml'^ triiodothyronine

2.6.3 DNA Transfections

HEBES Transfection Buffer: 140mM NaCI Filter sterilised (0.2pm) 5mM KCl

Stored at 4°C 0.7mM Na 2HP0 4 5.5mM D-glucose 20mM Hepes pH 7.05 with NaOH The calcium phosphate-mediated transfection method was adapted by to introduce HSV-1 DNA for homologous recombination into mammahan cell lines (Stow and Wilkie, 1976). This method also gave efficient delivery of expression vectors, and was adapted for this purpose. Briefly, cells were grown in 35mm-diameter wells at

37^C/5% CO2 to 60% confluency. The next day the following mixes were set up in 1.5ml sterile microflige tubes. For Expression Vector Transfection Tube A

31 pi 2M CaCl 2 (filter sterihsed) lOpg total DNA 20pg herring sperm DNA (pre-treated by phenoFchloroform extraction and ethanol precipitation)

For Viral Homologous Recombination Tube A

31 pi 2M CaCl 2 (filter sterihsed) lOpg linearised DNA for recombination 10-30pg HSV-1 DNA

127 l|ig pre-treated herring sperm DNA

Tube B

400|li1 HEBES transfection buffer (warmed to room ten^erature)

The contents of Tube A was added dropwise to Tube B, with constant gentle mixing by flicking. The DNA was allowed to precipitate by incubating at room temperature for 20-40 minutes, before adding the mix onto the cells with the growth media removed. The cells were incubated at 37°C/5% CO 2 for a further 20-40 minutes, rockmg occasionally before adding 1ml of growth media (DMEM growth media for ND7 cells also) to each well. After 4-7 hours, the media was replaced on ND7 cells with 2ml of L I5 growth media. BHK transfection efficiency was further increased by shocking the cells with DMSO after 4-7 hours. The media was removed and the cells were washed with 1ml growth medium. The cells were shocked for no more than 2.5 minutes in 25% (v/v) DMSO in HEBES transfection buffer (exothermic reaction on mixing, so mixture cooled to room temperature prior to use). The cells were immediately washed twice in 2ml of growth media per well. 2ml of growth media was

added to each well and the cells incubated at 37°C/5% CO 2 . Viral recombination transfection media was supplemented with 3mM hexamethylene bisacetamide (HMBA) to induce HSV-1 immediate-early gene transcription (McFarlane et al., 1992).

Viral recombination transfections were incubated for 3-5 days, until full cytopathic effect (CPE), characterised by rounding of the cells, was observed. The monolayer was subsequently harvested by resuspension in the well using a P I000 Gilson micropipette, and transferred to a 15ml tube for storage at -80°C. The cells were lysed by flreeze- thawing twice in hquid nitrogen and a 37°C water bath, and titrated on conq)lementing cells as described in Section 2.6.5.

Transfections for subsequent X-gal staining or chloramphenicol acetyl transferase (CAT) assay were incubated overnight, prior to assay. Transiently transfected cells for CAT assay were harvested according to the protocol supphed by the Invitrogen Corporation by removing the media, washing with 1 x PBS and scraping the ceUs in

128 1ml TE into a 1.5ml microfuge tube. The cells were pelleted by microcentrifugation at 13,000rpm for 1 minute and resuspended in lOOpl of ice-cold 0.25mM Tris-HCl (pH7.5). The cell membranes were lysed by freezing in hquid nitrogen and then thawing by incubation at 37°C for 5 minutes for 3 cycles. The ceU suspension was microfuged at 13,000rpm for 5 minutes to remove ceU debris and the supernatant was transferred to a fresh microfuge tube. The sanq)les were stored at -20®C until required.

2.6.4 Chloramphenicol Acetyl Transferase (CAT) Assay

Promoter activity by its abihty to express the enzyme chloranq)henicol acetyl transferase (CAT). This assay measured the abihty of CAT to acetylate [^"^C]- chloramphenicol and the amount of product was determined by thin-layer chromatography (TEC).

The fohowing reaction was set up according to the protocol supphed by the Invitrogen Corporation:

IM Tris (pH7.5) 33pl [^^C] chloroamphenicol (54 mCi mmol'^) Ipl 4mM acetyl Co-enzyme-A 25pi Ceh extract (see Section 2.6.3) 50pl ddHzO 50pl

The reaction was incubated at 37°C for 1-2 hours (provided that enough enzyme was added to keep the assay linear) and then the chloranq)henicol was extracted by addition of 1ml ethyl acetate. The mix was vortexed for 1 minute and the top organic layer was transferred to a fresh 1.5ml microfuge tube and dried down under vacuum. Samples were resuspended in 30pl ethyl acetate and 5pi was spotted on to a sUica gel TEC plate (Whatman International Etd., Maidstone, Kent, U.K.). This plate was subjected to ascending chromatography with a 19:1 (v/v) nhx of chloroform:methanol until the front had nearly reached the top of the TEC plate (approximately 1 hour). The plate was air dried and then either exposed to X-ray film overnight - 1 week depending on

129 the strength of the promoter under investigation and quantified by densitometry using a Bio-Rad model GS-670 Imaging Densitometer.

2.6.5 Titration of Virus on Complementing Cells

B 130/2 cells complementing HSV-1 ICP27 were seeded in a 6 well plate and grown at

37”C/5% CO2 to 80% confluency. Virus was added to wells in a 1:10 serial dilution of virus from 1 xlO"^ - IxlO'^ml of viral suspension in 0.5ml of serum free DMEM. The plate was incubated for 1 hour at 37°C/5% CO 2 After which the media was taken off by pipette and replaced with 2ml 1:2 of 1.6% (w/v) CMC:growth medium. The plate was returned to the incubator for 48 hours.

2.6.6 Detection of Viral Recombinants Expressing lacZ Reporter Gene

X-Gal Buffer: 1 xPBS lOmM sodium phosphate ImM MgCE 150mM NaCl

3.3mM K4Fe(CN)6

3.3mM K3Fe(CN)6 X-Gal Solution: 150pg ml'^ 4-Cl,5-bromo,3-indolyl-y^galactosidase (X-gal) in DMSO. Fixing Solution: 1 x PBS 0.05% glutaraldehyde

The cells were washed twice in 1ml of 1 x PBS and incubated for 10 minutes at room temperature in 1ml of fixing solution. The cells were washed twice in 1ml of 1 x PBS. 0.2% (v/v) X-gal solution was dissolved in prewarmed (37°C) X-gal buffer, and 3ml was added to each well. The plate was returned to the 37°C/5%C02 for 2 hours- ovemight, depending on the intensity of staining. The wells were then examined under an inverted microscope for blue viral plaques indicating P-galactosidase activity and therefore lacZ expression. The buffer was discarded and replaced with 2ml 70% glycerol for storage.

130 If viral plaques staining positive or negative for p-galactosidase activity are to be picked for purification (see Section 2.6.8) then the above staining procedure was performed without fixing. Due to the toxicity of the buffer and stain, the plaques were picked within 10 hours of staining.

2.6.7 Detection of Viral Recombinants Expressing Green Fluorescent Protein (GPP) Reporter Gene

Cells infected with a GFP expressing virus were detected under UV hght. Cells were prepared by replacing the media with 1 x PBS. GFP positive cells fluoresce green when exposed to UV hght and visuahsed using fluorescein optics.

2.6.8 Purification of Viral Recombinants by Plaque Selection

After staining or UV hght visuahsation as in Sections 2.6.6/2.6.7, blue/green/white plaques were picked from the monolayer using a P20 Gilson micropipette set at 5 pi underneath an inverted microscope/fluorescent microscope. The blue cehs were isolated using the pipette tip and then drawn into the pipette and transferred to a freezing vial containing 100pi of serum free DMEM. The resulting suspensions were then freeze-thawed three times by transferring between hquid nitrogen and a 37°C water bath. lOpl and 90pi of the nhx were then added to 0.5ml of serum free DMEM in separate 35mm-diameter weUs of 80% confluent B 130/2 cehs. The cehs were incubated for 1 hour at 37°C/5% CO2 before addition of CMC/FGM and incubation for 48 hours as in Section 2.6.5. In order to purify the viruses by cloning this 2 day cycle of picking, infecting, staining, picking etc., was carried out until ah infected plaques appeared blue or white on staining. The whole weh was harvested by scraping and after freeze-thawing three times, a sarnple (lOOpl) of the mixture would be added to non-complementing BHKs as above and examined by microscopy after two days for any viral plaques indicating rephcation. If there was no detectable activity, the ‘master’ stock would be titrated and infected onto a 6-weh plate as in Section 2.6.5 and plaques were counted and the pfli (number of plaque forming units per ml of viral suspension) was calculated.

131 2.6.9 Large Scale Viral Culture

An 80cm^ tissue culture flask of 100% confluent B 130/2 cells was split as in Section 2.6.2 into 5 x 175cm^ flasks and grown at 37°C/5% CO^ until confluent. These cells were subsequently split into 10 x 850cm^ disposable roller bottles (Coming Glass Works, Coming, New York, U.S.A.) in 100ml each of growth media. The bottles were filled with 5% C02/95% an through a filtered gas line, and the hds replaced tightly. The bottles were incubated overnight in a roller bottle incubator at 37°C/0.5 rpm

Cells (at 70% confluency) were then infected with 1 x 10^-1 x 10^ pfii of vims per roller bottle in 50ml of growth media. The roller bottles were retumed to the incubator and further incubated at 3 l°C/0.5rpm for 3-5 days, until fiiU CPE was observed.

2.6.10 Large Scale Viral DNA Extraction

RSB; lOmM Tris-HCl pH7.4 lOmM KCl 15mM MgCL NTE: O.lMNaCl lOmM Tris pH7.4 ImM EDTA 10 X 850cm^ roller bottles were seeded with B 130/2 cells and infected with vims as in Section 2.6.9. When full CPE was observed, the cells were resuspended by vigorous shaking and the suspension transferred to 50ml centrifuge mbes. The cells were pelleted in a standard benchtop centrifuge at 2,000rpm for 15 minutes at 4°C and the supematants transferred to 200ml centrifuge tubes and stored on ice. The nuclei were extracted by resuspension through gentle pipetting in 1ml of RSB per roller bottle and 0.05% Nonidet-P40 (5pi of 10% Nonidet P40 stock per roller bottle) and the suspension was incubated on ice for 10 minutes. The extracted nuclei were pelleted in a standard benchtop centrifuge for 5 minutes at 2,000rpm, 4°C, and the supematant transferred to the 200ml centrifuge tubes. The nuclei were extracted a second time as above and the supematant transferred to the 200ml centrifuge tubes. The vims was

132 pelleted from the supernatant by centrifugation in a Beckman JAIO rotor for 2 hours at 12,000rpm, 4°C. The supematant was discarded and the viral pellet resuspended by gentle pipetting in 8ml of NTE. The vims was lysed by addition to a final concentration of 2% SDS and 20mM EDTA and incubation at 37°C for 5 minutes. Viral DNA was extracted three times with 1:1 (v/v) of phenol and once with 1:1 (v/v) chloroform/isoamyl alcohol (24:1 v/v), mixing by inversion at room temperature for 10 minutes and centrifugation in a standard benchtop centrifuge for 10 minutes at 2,000rpm, 20°C and transferring the aqueous top layer to a fresh centrifuge tube each time. The DNA was precipitated with 2 volumes of ethanol at room temperature by running the ethanol down the side of the tube, layering on the aqueous solution, and mixing by gentle inversion. The DNA precipitate was observed and pelleted in a standard benchtop centrifuge at 3,000rpm for 10 minutes at room temperature. The pelleted was air-dried and gently resuspended in lOOfil - 1ml ddH20 containing 50|ig ml^ RNase A depending on the size of the pellet, and a lp,l sanq)le of uncut DNA was run on an 1% agarose gel.

2.6.11 Large Scale Viral Purification

Adapted from Rixon et al., 1992. Vims was grown in 10 roller bottles as in Section 2.6.9. On full CPE the cells were resuspended by shaking and transferring to 50ml centrifuge tubes. The remaining cells were rinsed in 15ml per roller bottle in serum free medium by returning to the roller bottle incubator for 10 minutes, and the medium transferred to 50ml centrifuge tubes. Cell debris was pelleted by spinning in a standard benchtop centrifuge at 2,000rpm for 15 mins at 4°C. The combined supematants were transferred to 200ml centrifuge tubes and the vims was pelleted in a Beckman JAIO rotor at 12,000rpm for 2 hours at 4°C. The supematants were discarded and the pellets gently resuspended (with a cut-ofiF 1ml pipette) and combined in a total 3ml of Eagle’s medium without phenol red or calf serum The pellets were further resuspended by shaking overnight on ice on a platform in a cold room. The viral suspensions were subsequently layered gently on top of a 5-15% ficoll gradient. No more than 3ml was layered on each gradient.

133 Preparation of Ficoll Gradient 5% and 15% (w/v) ficoll solutions were made up in Eagle’s medium without phenol red or calf serum by shaking on a platform at room temperature and cooled to 4°C and a ficoll gradient was set up in clear 38ml tubes using a Bio-Rad gradient former and peristaltic pump. Gradients were stored at 4°C until used.

Gradients were centrifuged at 12,000rpm for 2 hours at 4°C in a Kontron Tst 41.14 swing out rotor. The viral band was visuahsed in the dark with a strong beam of hght shining into gradient from above. The sharp, opaque virion band was removed with a wide bore needle and 10ml syringe and transferred to a fresh 38ml centrifiige tube. 20ml of Eagle’s medium without phenol red and calf serum and the virions were peheted overnight at 12,000rpm, 4°C in a Kontron Tst 41.14 swing out rotor. The supematant was discarded and the side of the tube drained. The peUet was resuspended in 100pi phenol-free Eagle’s medium plus foetal calf serum by gentle overnight shaking on ice in a cold room. The vims was titrated onto B 130/2 as in Section 2.6.5, yields were commonly 1x10^ pfu.

2.6.12 Infection of Mammalian Cell Lines and Primary Neuronal Cultures with Recombinant Virus

ND7, B 130/2, and BHK ceh lines, and primary rat DRG cehs were infected with recombinant vimses for subsequentin vitro assays.

Vims was added to ceh lines in 0.5ml weh'^ (6-weh plate) or 0.25ml weh'^ (24-weh

plate) of serum free DMEM and plates were incubated at 37°C/5% CO 2 for 1 hour. The viral media was replaced with 2ml weh^ (6-weh plate) or 1ml weh'^ (24-weh

plate) of appropriate growth media. Cehs were incubated overnight at 37°C/5% CO 2 , prior to assay.

Infection titre of vims on ceh lines was defined by multiphcity of infection (MOI - number of pfu per cell, MOI optimised by titration).

134 The complementing cell line B 130/2 was infected at an MOI of Ipfu cell'^ as the virus will rephcate prior to assay. BHK and ND7 cell lines were infected at an MOI of 10 pfii cell'\

Primary cultures were infected with the same amount of virus as the cell lines (approximately lO^phi well \ Preliminary titrations of P-galactosidase expressing HSV-1 onto primary cultures in a 24-well plate demonstrated that if an MOI of lOpfu.cell’^ were used only about 70% of cells would become infected. This was due to much of the enveloped virus attaching to the plasticware (only around 2 0 0 cells per culture). However, in using the same amount of virus per well as for cell lines (80% confluent) to account for this, viral delivery efiBciency was 1 0 0 % and cells remained visibly healthy.

Primary DRG neurons were infected over one hour in defined media lacking bovine serum albumin at3 7 °C/5 %C0 2 , following which the media was replaced with complete defined media and cells were incubated overnight at 3 7 °C/5 %C0 2 prior to assay.

2.6.13 Mini-Viral DNA Preparation for Southern Blot Analysis

Adapted fi*om method described by Feldman, 1996. TES: 50mMTrispH7.8 ImM EDTA 30% sucrose

Proteinase K: 20mg ml^ proteinase K in lOmM CaCl 2 B 130/2 cells were infected with virus as in Section 2.6.12, and incubated until fidl cytopathic effect. Cells were pelleted in a microfuge at l,000rpm for 1 0 mins and the supematant discarded. The pellet was resuspended in 200pl of TES. 200|xl of SDS was added to the suspension 2 0 0 pl of lOOmM P-mercaptoethanol, and the solution incubated on ice for 30 mins. lOpl of proteinase K was added and the sample incubated overnight at 55°C. The DNA was extracted twice with phenol/chloroform (1:1), then once with chloroform:isoamyl alcohol (24:1). DNA was precipitated by adding 75pi of 7.5M NH 4 OAC and 2.5 volumes of ice-cold 95% ethanol, and the

135 sample subsequently rocked at room temperature and dried at room tenq)erature. The pellet was resuspended in 30|rl of ddHzO. 25til was digested with an appropriate restriction enzyme in a total reaction volume of 40 til. 15 pi of sample was run with Ipl of loading buffer at 75 volts in a 0.8% agarose gel (stained with ethidium bromide), alongside well containing 5 pi of kilobase ladder and appropriate DNA controls.

2.6.14 Southern Blot of Viral DNA

Denaturing Solution: 1.5M NaCl 0.5M NaOH Neutralising Solution: IM Tris pH5.5 2.5MNaCl

Once dye front of samples described in Section 2.6.13 had reached the bottom of the gel, the gel was trimmed to remove unused wells and placed on a transilluminator for 2 minutes to shear the DNA. A photograph of the gel was made at this point alongside a fluorescent ruler to ahgn kb ladder bands with those on the subsequent autoradiograph. The DNA in the gel was denatured by soaking the gel on a rocking platform at room temperature for 45 mins in denaturing solution. The solution was replaced with neutrahsing solution and incubated for a further 30 mins, and then replaced with fresh neutrahsing solution and incubated for a further 15 minutes. The gel was transferred to 2 X SSC. Hybond-N membrane (Amersham Life Sciences) was cut to the same size of the gel and pre-soaked by floating on ddHzO until wet and then onto 20 x SSC for at least 5 mins. The DNA was transferred to the gel overnight at room temperature by capillary action (as described in Section 2.4.5), using 10 x SSC. After transfer, the well positions were marked with a pen and the filtersoaked in 6 x SSC for 5 mins. The gel was placed on a paper towel and allowed to dry at room temperature for at least 30 mins. The DNA was crosslinked to a membrane using a UV Stratalinker on autosetting.

136 The filter was then pre-hybridised, incubated overnight with an appropriate random primer generated a[^^P] dCTP-radiolabelled probe, washed and exposed to X-ray film as described in Sections 2.3.10-2.3.12.

2.7 Induction of Cell Stress

2.7.1 Heat Shock

Appropriate growth media was pre-warmed to 48°C and added 1ml per well to ND7 cells/primary rat DRG cells in vitro. The plates were wrapped in Parafilm and incubated in a water bath at 48®C (lethal heat shock) for 20 minutes (ND7 cells) or 30 minutes (rat primary DRG cultures) (Finket al., 1997). The plates were subsequently transferred to a 37°C/5% CO 2 incubator for a recovery period of 1 hour (ND7 cells) or 24 hours (primary neurons). The ND7 cells were gently harvested with a 1ml pipette and transferred to 1.5ml microfuge tubes and pelleted in a microcentrifuge at 2,000rpm for 10 minutes. The pellet was gently resuspended by flicking in lOOfxl of 1 x PBS and stored on ice before Trypan blue exclusion assay for cell viabihty (see Section 2.8.1). Healthy primary DRG neurons were assessed in situ at 0, 12 and 24 hour timepoints (see Section 2.8.3) (Wyatt et at., 1996).

2.7.2 Simulated Ischaemia

This method is based on one devised for simulating ischaemia upon cardiomyocytes (Esumi et at., 1991) and has been further adapted by Dr. Jing Zhao (The Rayne Institute for Cardiovascular Studies, United Medical and Dental Schools, London, U.K.).

Control Buffer: 118mM Nad 24mM NaHCOs 4mM KCl

ImM NaH2P0 4 2.5mM CaCh

137 1.2mM MgCli 0.5mM EDTA 2mM sodium pyruvate lOmM D-glucose pH 7.4

Ischaemic Buffer: Control Buffer containing: 20mM sodium lactate 12mMKCl pH 6.2

ND7 and primary neonatal rat DRG cells were rinsed gently with 1ml well"^ of 1 x PBS. They were subsequently incubated in 1ml well'^ in control buffer or ischaemic buffer for 4 hours at 37°C/5% CO2 ND7 Cells were harvested as in Section 2.7.1 and viabihty assessed by Trypan blue exclusion (see Section 2.8.1). Primary cultures were retumed to delBned media and incubated for a further 24 hours. Healthy primary DRG neurons were assessed in situ at 0, 12 and 24 hour timep oints (see Section 2.8.3) (Wyatt et al., 1996).

2.7.3 Induction of Apoptosis in ND7 Cells and DRG Neurons

ND7 cells undergo apoptosis under conditions of serum-withdrawal and addition of ?X\-trans retinoic acid (Howard et al., 1993).

Apoptosis Media: 50% DMEM 50% Ham’s F-12 medium 5pg ml'^ human transferrin 250ng ml^ bovine insulin 30nM sodium selenite lOpM diWrtrans retinoic acid

138 ND7 cells were rinsed gently with 1ml well^ of 1 x PBS. They were subsequently incubated in 1ml well'^ apoptosis buffer for 24 or 48 hours at 37°C/5% CO 2 Cells were harvested as in Section 2.7.1 and viabihty assessed by Trypan blue exclusion (see Section 2.8.1).

Neonatal rat DRG neurons undergo apoptosis under conditions of NGF withdrawal (Hamburger et al., 1981; Wyatt et a l, 1996). Therefore rat DRG cultures (postnatal day 2) were incubated in defined media without NGF for 48 hours at 37°C/5%C02, and healthy neurons were assessed at 0, 24 and 48 hour timep oints (see Section 2.8.3).

2.8 Celt Viability Assays

2.8.1 Trypan Blue Exclusion Assay

Non-viable cells are unable to exclude trypan blue dye, and therefore stain blue.

25 pi of harvested ND7 cells in 1 x PBS (see above), were mixed gently with the same volume of 0.04% (w/v) Trypan Blue (Sigma Chemical Conq)any Ltd, Poole, Dorset, U.K.) in 1 X PBS. The suspension was incubated at room temperature for about 5 minutes and viable/non-viable cells were counted on a haemocytometer counting chamber (Weber Scientific International Ltd., U K ) at 40x magnification.

2.8.2 In Situ Programmed Cell Death Detection

Detection of programmed cell death, or apoptosis, was achieved using the fluorescein in situ cell death detection kit, supphed by Boehringer Mannheim, Lewes, East Sussex, U.K. The kit enables detection DNA fi'agmentation by labelling DNA strand breaks using terminal deoxynucleotidyl transferase (TdT) to incorporate nucleotide polymers containing fluorescein conjugated dUTP. This TdT-mediated dUTP nick end labelling (TUNEL) assay can therefore be used to detect individual apoptotic cehs in cultures of cehs in chamber shdes. The method used was adapted firom that supphed with the kit.

139 Paraformaldehyde 4% Paraformaldehyde dissolved in PBS pre-heated to 60”C Solution: Cooled to room temperature pH 7.4 Filtered through Whatman IMM paper

Briefly, ND7 cells were seeded into 8-well chamber shdes, and infected at an MOI of lOpfu cell^ with recombinant HSV-1. Subsequent to serum-withdrawaFaddition of retinoic acid as in Section 2.7.3, the cultures were incubated for 48 hours at 37°C/5%

CO2 . The media was discarded, the waUs of the chamber shdes removed and replaced with 50pl weh’^ of paraformaldehyde solution for 30 mins The shdes were washed twice in PBS at 37°C and the area around the samples dried. 50pl of TUNEL reaction mix was added (containing calf thymus TdT, reaction buffer and labehed nucleotide polymers), and the shdes placed in a sealed box with a danq) paper towel (to prevent evaporative loss), and incubated in the dark at 37°C for 60 mins The shdes were subsequently washed three times in PBS at 37°C, the rubber seals around the samples removed, mounted in glycerol and covered with a glass shp. The samples were visuahsed at 400x magnification using fluorescein optics and the numbers of positive staining, apoptotic cehs, were counted in three confluent fields of view for each sample.

2.8.3 Visualisation of Healthy/Unhealthy Neurons by Light Microscopy

Healthy primary rat DRG neurons can retain trypan blue and so this cannot be used as a ceh viabihty assay. Therefore viable neurons were characterised as surviving or dying, subsequent to the apphcation of exogenous insult by visuahsation at 400x magnification under phase-contrast microscopy. This identification has been previously used by our laboratory (Wyatt et a l, 1996). The parameters used to define a viable neuron were, phase bright bodies, intact neurites, agranular appearance, non-ruffled membranes. Non-viable neurons were defined by phase dark bodies, lack of neurites, granular/vacuolated appearance, ruffled membranes.

140 2.9 Middle Cerebral Artery Occlusion

The following procedure was performed by Mr. Benjamin S. Aspey at the Rita Lila Weston Institute of Neurological Sciences, Windeyer Institute of Medical Sciences, UCL Medical School, London. Brain dissection was performed by Prof. Jacqueline S. de Belleroche, Department of Biochemistry, Charing Cross Hospital Medical School, London.

Male Sprague-Dawley rats (wt. 300 ± 25 g at time of surgery) were anaesthetised with 4% halothane in a 1:1 mixture of nitrous oxide and oxygen after an overnight fast. With the halothane then maintained at 1.5% in an open anaesthetic system without tracheotomy, the origin of the left middle cerebral artery was occluded with an intravascular suture following a method modified from that of Nagasawa and Kogure 1989 (Nagasawa and Kogure, 1989). Briefly, this involved introducing a 4/0 nylon suture (Ethylon, Ethicon Ltd., UK) with 7mm of its shaft from the tip thickened with sihcone rubber to an outside diameter of 0.28mm, (Silastic sealant 732 RTV, Merck Ltd., UK) into the left external carotid artery in a retrograde fashion towards the carotid bifurcation and then directed distally up the left internal carotid artery to a distance of 17 to 18mm from the carotid bifurcation to the tip of the suture (Figure 2.1). The suture was secured at two points: first, to the stump of the Hgated left external carotid artery and second, within the hgated internal carotid artery, proximal to the pterygopalatine branch. The whole procedure took 20 minutes, with rectal temperature maintained throughout at 37.0 ± 0.5 °C with a heat lamp. The cutaneous wound was sutured and cleaned, and the animals were left to recover with free access to food and water. This intravascular suture model of MCAO was adopted because of its less traumatic surgical procedure compared to other focal ischaemia models and is a comparatively simple procedure and animals recovered from anaesthesia within 20 minutes of surgery. After the determined time of occlusion rats were sacrificed the brains rapidly removed, anatomically dissected and frozen in hquid nitrogen. The ‘core’ ischaemic area of the cortex lay in the central region of the middle cerebral artery territory. The tissue samples were stored at -70°C until required.

141 Anterior Cerebral Artery

Middle Cerebral Artery 7mm

Internal Carotid Artery

lOmm

Posterior Cerebral Artery 4/0 Nylon Thread I L_ Connected to Silicon Basilar Artery Rubber

Common Carotid Artery External Carotid Artery

Figure 2.9 - Schematic Diagram of the Middle Cerebral Artery Occlusion rMCAO) Model Unilateral focal cerebral ischaemia was induced by occluding the entry to the middle cerebral artery with a sihcon-tipped intra-luminal suture. The suture enters the arterial system in the external carotid artery, passes through the internal carotid artery and around the circle of Wilhs to the entry of the middle cerebral artery.

142 Chapter 3

Characterisation of HSP mRNA and Protein Levels During Focal Cerebral Ischaemia

143 3.1 Introduction

3.1.1 Aims

Prior to overexpressing exogenous heat shock proteins in the brain in order to detect the effects this may have on the pathology of focal cerebral ischaemia, an assessment of the expression of the hsps and their corresponding mRNAs during the same model of ischaemia was necessary. The core region of ischaemia following MCAO, defined as the area that excludes 2,3,5-triphenyltetrazohum chloride (TTC) staining, includes areas of rat parietal cortex, striatum, caudate, putamen and basal gangha excluding the occipital cortex, olfactory bulbs and cerebellum (Figure 3.1.1). This study describes the levels of hsp27, 56, 60, 70 and 90 and their corresponding mRNAs, over time, in the core region of ischaemia following permanent MCAO. Northern blot analysis was carried out at 0, 2, 8 and 24 hours, and this was quantitated using slot blot analysis at the same timepoints including an additional 4 hour timepoint. Protein levels at all timep oints were detected using western blot analysis.

3.1.2 Method Details

Middle Cerebral Artery Occlusion MCAO was carried out as described in section 2.8. Afl;er 2, 4, 8 and 24 hours of permanent occlusion, the rats were sacrificed by cervical dislocation, and the brains rapidly removed, anatomically dissected and frozen in hquid nitrogen. The effect of MCAO was studied in a region of the cerebral cortex which lay in the centre of middle cerebral artery territory, and in corresponding regions of the contralateral (right) hemisphere. The tissue sangles were stored at -70°C until processed. Unoperated animals serve as to controls.

144 Fed by Anterior y Cerebral Artery

Fed by Middle Cerebral Artery

Fed by Posterior Olfactory Bulb Cerebral Artery

Cerebral Cortex \

Cerebellum

Figure 3.1.1 - Cerebral Blood Supply in Rats (Yamori et a/., 1976)

145 cDNA Templates for Random Prime Probe Generation All probes were made by random priming generated from fidl length cDNA templates which were excised from plasmids as in Table 3.1. For fidl plasmid details, please refer to Table 2.1.

Table 3.1 - cDNA Templates used for Random Prime Probe Generation cDNA Template Source Restriction Size of Plasmid Enzyme(s) Used Fragment

Chinese hamster hsp 27 BS27 EcoRI 700bp Rabbit hsp 5 6 p59 EcoRI 2kb Human hsp60 pRep65 XhoVHinàSl 2.2kb Human Inducible hsp 70 pH2.3 2.3kb Human hsp 90 pPa90 Æ'coRI 2.5kb Human P-tubulin pUC-tub Pstl SOObp

The linearity of the mRNA signal on slot blots was confirmed through analysis of serial dilutions of p-tubulin mRNA, and quantitation was carried out over a linear response range. Statistical analyses were carried out using Student’s t-test.

Antibodies Used for Detection of Protein on Western Blots Please refer to Table 2.2 for antibody details. Hsp27 was detected using the L2R3 antibody (a generous gift from Jacques Landry) (Lavoie et al., 1995). Hsp56 was detected using the FKBP54 antibody (Stressgen Biotechnologies Corp., Victoria, Canada). Hsp60 was detected using the hsp60 antibody (Affinity Bioreagents). Inducible hsp70 was detected using the hsp70 antibody (Stressgen). The other antibodies were used as indicated in Table 2.2.

146 3.2 Hsp27 mRNA and Protein Levels During Focal Cerebral Ischaemia

3.2.1 Hsp27 mRNA

On Northern analysis, the hsp27 probe hybridised to a single species of approximately 0.7kb, consistent with the expected size of hsp27 mRNA as predicted from the frill length cDNA size (Lavoie et a i, 1990). No increase in hsp27 mRNA expression was detected in the contralateral cortex throughout the time period examined. Increases against contralateral and îq controls are visible at 8 hours with a further increase at 24 hours in the ischaemic sangle.

Confirmation of these observations, and quantitation in relation to P-tubulin mRNA levels was achieved by slot blot analysis. Constitutive expression of hsp27 mRNA was detectable and no significant change was detected in the contralateral hemisphere throughout the time period examined. A significant increase in hsp27 mRNA was detected in the ipsilateral cortex as early as 2 hours after MCAO, being 2.7-fold above the level seen in ^ control animals. The levels of hsp27 mRNA were higher at 2, 4, 8 and 24 hours compared to the contralateral cortex and to controls. The maximum increase against controls was seen at 24 hours where levels were 33 times greater than those in the contralateral cortex and 9.9 times greater than those in the to control cortex.

3.2.2 Hsp27 Protein

The hsp27 antibody detected a single protein species at 27kDa on western blot analysis, consistent with the molecular weight of hsp27 protein. A single weak band was detected in the ipsilateral cortex at 24 hours, but not at other times or in the contralateral cortex. The weakness of the signal may be due to low protein levels or to low-aflBnity antibody binding. This suggests, in conjunction with the Northern and slot blots in Figure 3.2.1 that an increase in hsp27 mRNA between 1 and 2 hours leads to an increase in protein levels between 8 and 24 hours. This may be a reflection of differing sensitivities between the assays, or may be attributable to a decrease in translation conferred to the cortex by ischaemic stress. In summary, the concentrations of hsp27 mRNA and protein both increase in vivo in the first 24 hours following focal cerebral ischaemia in the rat.

147 Figure 3.2.1 - Hsp27 and B-tubulin mRNA Levels in the Core Region of Ischaemia During Permanent MCAO

a) i) Hsp27 and ii) p-tubulin mRNA levels in the core region of ischaemia during middle cerebral artery occlusion as detected on Northern blot. Brain sarcples were dissected at 0, 2, 8 and 24 hours, and the ipsilateral (left) and contralateral (right) samples. mRNA was extracted and separated by electrophoresis before hybridisation with i) hsp27 cDNA and ii) P-tubulin [^^P] radiolabelled probes. The IBS (1.9kb) and 28S (4.7kb) rRNA markers are indicated on left. Loading of control samples (C) was greater to allow detection of low basal levels of hsp mRNA. L and R denote cortex ipsilateral and contralateral to the occlusion respectively Jfrom the same animal for each time point. b) Time course of hsp27 mRNA levels following at 2, 4, 8 and 24 hours after permanent MCAO. Quantitation of mRNA was carried out by slot blot analysis with reference to levels of P-tubulin mRNA. Values are means with the SEMs shown by error bars for 3-6 animals at each time point. §, §§ and §§§ indicates that MCAO significantly increased levels of hsp27 mRNA compared to ipsilateral 0 hour (to) controls (P < 0.05, P < 0.025, P < 0.005, respectively). *, ** and *** indicates that the value in cortex ipsilateral to the occlusion is significantly greater than contralateral cortex of the same animals (P < 0.05, P < 0.025, P < 0.005, respectively).

Figure 3.2.2 - Hsp27 and B-tubulin Protein Levels in the Core Region of Ischaemia During Permanent MCAO

Western blot showing i) hsp27 and ii) P-tubulin protein levels during the time course (0, 2, 8 and 24 hours) following permanent MCAO. Blots were incubated with i) L2R3 anti-Chinese hamster ovary hsp27 antibody and ii) anti-P-tubulin antibody. Molecular weights in kDa are indicated on left. L and R denote cortex ipsilateral and contralateral to the occlusion respectively fi'om the same animal for each time point.

148 Figure 3.2.1 a) i) C 2 8 24

LRLRLRLR

J ss­ • »

ii)

18 S - # # •

b) * p<0.05 * p<0.025 * * * p<0.005 § p<0.05 §§§ §§ p<0.025 §§§ p<0.005

I ipsilateral

I I contralateral

time (h)

149 Figure 3.2.2 i) C 2 8 24

LRLRLRLR

30-

Ü)

66 -

150 3.3 Hsp56 mRNA and Protein Levels During Focal Cerebral Ischaemla

3.3.1 Hsp56 mRNA

On Northern analysis, the hsp56 probe hybridised to a single species of approximately 2kb, consistent with the expected size of bsp56 mRNA as predicted from the frdl- lengtb cDNA size (Lebeau et a l, 1992). The expression of bsp56 mRNA was very low on Northern blot analysis, and no increase was detected throughout the timecourse either against the contralateral cortex or the to control animals. Levels were subsequently too low on slot blot analysis for effective quantitation by densitometric analysis.

3.3.2 Hsp56 Protein

The antibody detected a single protein species at 56kDa, consistent with the hsp56 molecular weight. Constitutive e?q)ression of hsp56 protein is detectable throughout the time period examined, but no notable increase was detected in the ipsilateral cortex up to the 8 hour timepoint. A mild increase was detected in hsp56 protein levels at 24 hours in the ipsilateral cortex. As no increase in mRNA levels was detected at any time point, however, this may suggest an increase in translation of existing mRNA species in response to the ischaemic insult, or it may be artefactual. The band of higher molecular weight on the hsp56 blot was due to the binding of the hsp70 antibody at 8 and 24 hours as part of a separate experiment (see Section 3.5.2).

In summary, the concentrations of hsp56 mRNA and protein did not significantly increase in vivo in the first 24 hours following focal cerebral ischaemia in the rat.

151 c 2 8 24

LRLRLRLR

1 8 S -

Figure 3.3.1 - Hsp56 mRNA Levels in the Core Region of Ischaemia During Permanent MCAO Hsp56 mRNA levels in the core region of ischaemia during middle cerebral aitei^ occlusion as detected on Northern blot. Brain samples were dissected at 0, 2, 8 and 24 hours, and the ipsilateral (left) and contralateral (right) samples. mRNA was extracted and separated by electrophoresis before hybridisation with hsp56 cDNA [^^P] radiolabelled probe. Tlie 18S (1.9kb) and 28S (4.7kb) iRNA markers are indicated on left. Loading of control samples (C) was greater to allow detection of low basal levels of hsp mRNA. L and R denote coitex ipsilateral and contralateral to the occlusion respectively ft om the same animal for each time point. Hsp56 levels on slot blot hybridisation were too low for effective densitometric analysis and quantitation.

C 2 8 24

LRLRLRLR

66 -

Figuie 3.3.2 - Hsp56 Protein Levels in the Core Region of Ischaemia During Permanent MCAO Western blot showing hsp56 protein levels during the time course (0, 2, 8 and 24 hours) following pennanent MCAO. Blots were incubated with anti-hsp54 antibody. Molecular weights in kDa are indicated on left. L and R denote cortex ipsilateral and contralateral to the occlusion respectively ftom the same animal for each time point. Bands in ipsilateral coitex at 8 and 24 hours at the higher molecular weight are fi om binding of an hsp70 antibody as a separate exjieriment.

52 3.4 HspôO mRNA and Protein Levels During Focal Cerebral Ischaemia

3.4.1 Hspeo mRNA

On Northern analysis, the hsp60 probe hybridised to a single species of approximately 2.2kb, consistent with the expected size of hsp60 mRNA as predicted from the full- length cDNA size (Jindal et al., 1989). The hsp60 mRNA levels in the ipsilateral cortex was markedly elevated above the constitutive levels seen in both the contralateral cortex at 8 and 24 hours after MCAO and îq controls.

The quantitation of hsp60 mRNA expression relative to P-tubulin levels over time by slot blot analysis confirmed the levels detected on the Northern blot. The levels of hsp60 mRNA in the ipsilateral cortex at 8 and 24 hours were 4.9 and 5.2-fold greater than those in the contralateral cortex respectively, and 5.5 and 6.6 times greater than those in the ^ control cortex respectively.

3.4.2 Hsp60 Protein

The hsp60 antibody detected a single protein species at approximately 60kDa, consistent with the hsp60 molecular weight. The antibody detected a constitutive expression of hsp60 with an increase in the ipsilateral cortex at 24 hours. On densitometry after P-tubulin correction, this increase was 3.3-fold compared to the tç, controls. The increase of mRNA levels occurred between 4 and 8 hours, and the increase of protein levels occurred between 8 and 24 hours, so it appears that this newly synthesised mRNA above the constitutive level is being translated into protein.

In summary, the concentrations of hsp60 mRNA and protein both increase in vivo in the first 24 hours following focal cerebral ischaemia in the rat.

153 Figure 3.4.1 - Hsp60 mRNA Levels in the Core Region of Ischaemia During Permanent MCAO a) Hsp 60 mRNA levels in the core region of ischaemia during middle cerebral artery occlusion as detected on Northern blot. Brain samples were dissected at 0, 2, 8 and 24 hours, and the ipsilateral (left) and contralateral (right) samples. mRNA was extracted and separated by electrophoresis before hybridisation with hsp60 cDNA radiolabelled probe. The IBS (1.9kb) and 28S (4.7kb) rRNA markers are indicated on left. Loading of control samples (C) was greater to allow detection of low basal levels of hsp mRNA. L and R denote cortex ipsilateral and contralateral to the occlusion respectively fi’om the same animal for each time point. b) Time course of hsp60 mRNA levels following at 2, 4, 8 and 24 hours after permanent MCAO. Quantitation of mRNA was carried out by slot blot analysis with reference to levels of p-tubulin mRNA. Values are means with the SEMs shown by error bars for 3-6 animals at each time point. §, §§ and §§§ indicates that MCAO significantly increased levels of hsp60 mRNA compared to ipsilateral 0 hour (to) controls (P < 0.05, P < 0.025, P < 0.005, respectively). *, ** and *** indicates that the value in cortex ipsilateral to the occlusion is significantly greater than contralateral cortex of the same animals (P < 0.05, P < 0.025, P < 0.005, respectively).

Figure 3.4.2 - Hsp60 Protein Levels in the Core Region of Ischaemia During Permanent MCAO

Western blot showing hsp60 protein levels during the time course (0, 2, 8 and 24 hours) following permanent MCAO. Blots were incubated with anti-hsp54 antibody. Molecular weights in kDa are indicated on left. L and R denote cortex ipsilateral and contralateral to the occlusion respectively from the same animal for each time point.

154 Figure 3.4.1 a) C 2 8 24

LRLRLRLR

28S-

I8S -

b) 0 . 3 -

z.< ce E H ipsilateral I 0.2 n I I contralateral CQ_ < I E 0. T

k m 0 2 4 8 24

time (11)

Figure 3.4.2

C 2 8 24

LRLRLRLR

9 7 -

66 - / r

155 3.5 Hsp70 mRNA and Protein Levels During Focal Cerebral Ischaemia

3.5.1 Hsp70 mRNA

On Northern analysis, the hsp70 probe hybridised to a single species of approximately 2.3kb, consistent with the expected size of hsp70 mRNA as predicted form the hill length cDNA size (Wu et al., 1985). No constitutive expression of hsp70 mRNA was detected, which was expected considering the clone was the inducible (hsp72) member of the hsp70 family. No induction of hsp70 mRNA was detected in the contralateral cortex, but an initial increase in hsp70 mRNA was detectable in the ipsilateral cortex as early as 2 hours post-occlusion. Further increases were detected at 8 and 24 hours.

The levels of hsp70 mRNA were quantitated by slot blot analysis in relation to 13- tubulin mRNA levels. The hsp70 mRNA levels in the ipsilateral cortex after MCAO showed a rapid, sustained, signihcant increase at 2, 4, 8 and 24 hours compared to the contralateral cortex of animals with MCAO (3-, 4.7-, 5.3-, and 18-fold, respectively). These levels were also signihcantly greater than the to control levels (3-, 2.8-, 3.7- and 8.3-fold, respectrvely).

3.5.2 Hsp70 Protein

The hsp70 antibody detected a single protein species at approximately 72kDa, consistent with the hsp70 molecular weight. At 8 and 24 hours post-occlusion, there are increasing levels of hsp70 protein in the ipsilateral cortex. No constitutive levels were detected in the ipsilateral cortex at 0 and 2 hours or in the contralateral cortex throughout the time period examined.

In summary, the concentrations of inducible hsp70 mRNA and protein both increase in vivo in the first 24 hours following focal cerebral ischaemia iu the rat.

156 Figure 3.S.1 - Hsp70 mRNA Levels in the Core Region of Ischaemia During Permanent MCAO a) Hsp70 mRNA levels in the core region of ischaemia during middle cerebral artery occlusion as detected on Northern blot. Brain samples were dissected at 0, 2, 8 and 24 hours, and the ipsilateral (left) and contralateral (right) samples. mRNA was extracted and separated by electrophoresis before hybridisation with hsp60 cDNA [^^P] radiolabelled probe. The IBS (1.9kb) and 28S (4.7kb) rRNA markers are indicated on left. Loading of control samples (C) was greater to aUow detection of low basal levels of hsp mRNA. L and R denote cortex ipsilateral and contralateral to the occlusion respectively from the same animal for each time point. b) Time course of hsp70 mRNA levels following at 2, 4, 8 and 24 hours after permanent MCAO. Quantitation of mRNA was carried out by slot blot analysis with reference to levels of p-tubulin mRNA. Values are means with the SEMs shown by error bars for 3-6 animals at each time point. §, §§ and §§§ indicates that MCAO significantly increased levels of hsp70 mRNA compared to ipsilateral 0 hour (/o) controls (P < 0.05, P < 0.025, P < 0.005, respectively). *, ** and *** indicates that the value in cortex ipsilateral to the occlusion is significantly greater than contralateral cortex of the same animals (P < 0.05, P < 0.025, P < 0.005, respectively).

Figure 3.5.2 - H sd70 Protein Levels in the Core Region of Ischaemia During Permanent MCAO

Western blot showing inducible hsp70 protein levels during the time course (0, 2, 8 and 24 hours) following permanent MCAO. Blots were incubated with the anti-hsp70 antibody. Molecular weights in kDa are indicated on left. L and R denote cortex ipsilateral and contralateral to the occlusion respectively from the same animal for each time point.

157 Figure 3.5.1 a) C 2 8 24

LRLRLRLR

2 8 S - I I 1 8 S -

b)

B ipsilateral * * * *** *** §§§ I I contralateral §§§ §§§

0 2 4 8 24

time (11)

Figure 3.5.2

C 2 8 2 4

LRLRLRLR

9 7 -

66-

158 3.6 Hsp90 mRNA and Protein Levels During Focal Cerebral Ischaemia

3.6.1 Hsp90 mRNA

On Northern analysis, the hsp90 probe hybridised to a single species of approximately 2.5kb, consistent with the expected size of hsp90 mRNA as predicted by the full length cDNA size (Twomey et al., 1993). Constitutive hsp90 mRNA was detected in all samples throughout the time period examined. The Northern blot may suggest some increase in hsp90 mRNA levels at 8 and 24 hours m the ipsilateral cortex in corcparison to the p-tubulin mRNA levels.

The quantitation of hsp90 mRNA levels in relation to p-tubulin mRNA levels was achieved by slot blot analysis. Significant increases of hsp90 mRNA in the ipsilateral cortex against to controls were observed at 8 and 24 hours post MCAO. The only significant difference detected between ipsilateral and contralateral cortex, however, was at 24 hours. The varied ranges of expression cast some ambiguity on these results, but if this data is considered in parallel with the Northern blot results, it appears that the mRNA levels are raised in the ipsilateral cortex at 24 hours, although in comparison with constitutive expression, this increase may be shght.

3.6.2 Hsp90 Protein

The hsp90 antibody detected a single band at approximately 90 kDa, consistent with the expected molecular weight of the protein. Constitutive hsp90 protein was detected in all samples. No notable increase in hsp90 protein expression was detected throughout the time course examined in comparison to P-tubulin controls. Any increase in hsp90 translation as a result of an increase in hsp90 mRNA at 24 hours may only be detected at a later timepoint, beyond the scope of these experiments.

In summary, the concentrations of hsp90 mRNA and protein did not consistently increase in vivo in the first 24 hours following focal cerebral ischaemia in the rat.

159 Figure 3.6.1 - Hsp90 mRNA Levels in the Core Region of Ischaemia During Permanent MCAO

a) Hsp90 mRNA levels in the core region of ischaemia during middle cerebral artery occlusion as detected on Northern blot. Brain samples were dissected at 0, 2, 8 and 24 hours, and the ipsilateral (left) and contralateral (right) samples. mRNA was extracted and separated by electrophoresis before hybridisation with hsp90 cDNA [^^P] radiolabelled probe. The IBS (1.9kb) and 28S (4.7kb) rRNA markers are indicated on left. Loading of control satrples (C) was greater to allow detection of low basal levels of hsp mRNA. L and R denote cortex ipsilateral and contralateral to the occlusion respectively fi*om the same animal for each time point.

b) Time course of hsp90 mRNA levels following at 2, 4, 8 and 24 hours after permanent MCAO. Quantitation of mRNA was carried out by slot blot analysis with reference to levels of P-tubulin mRNA. Values are means with the SEMs shown by error bars for 3-6 animals at each time point. §, §§ and §§§ indicates that MCAO significantly increased levels of hsp90 mRNA compared to ipsilateral 0 hour (to) controls (P < 0.05, P < 0.025, P < 0.005, respectively). *, ** and *** indicates that the value in cortex ipsilateral to the occlusion is significantly greater than contralateral cortex of the same animals (P < 0.05, P < 0.025, P < 0.005, respectively).

Figure 3.6.2 - Hsp90 Protein Levels in the Core Region of Ischaemia During Permanent MCAO

Western blot showing hsp90 protein levels during the time course (0, 2, 8 and 24 hours) following permanent MCAO. Blots were incubated with anti-hsp90 antibody. Molecular weights in kDa are indicated on left. L and R denote cortex ipsilateral and contralateral to the occlusion respectively fi^om the same animal for each time point.

160 Figure 3.6.1 C 2 8 24 a)

LRLRLRLR

2 8 S -

1 8 S -

b)

1.00 -1

I ipsilateral

I I contralateral

§§§ §§§ L 0.50

time (h)

Figure 3.6.2 C 2 8 24

LRLRLRLR

220 -

9 7 -

161 3.7 Discussion

This study demonstrates that hsp27, hsp60 and hsp72 mRNA levels increase in the first 24 hours of permanent MCAO in the core region of ischaemia. The constitutive mRNA levels of hsp27 and hsp70 were almost undetectable. Hsp27 and hsp72 mRNAs were significantly increased against to and contralateral controls as early as 2 hours post-occlusion. Hsp60 levels on both the Northern and slot blots demonstrated a significant increase at 8 hours post-occlusion. All of these mRNA levels further increased at 24 hours. Increases in the protein levels of these hsps followed at subsequent time points, hsp27 and hsp60 were first detectable at 24 hours, and the hsp72 increase being first detectable at 8 hours. No constitutive levels of hsp27 and 72 proteins were detected. Hsp72 is the inducible member of the hsp70 family, but hsp27 is present at low levels in neurons. The low level of detection of hsp27 protein in the ipsilateral cortex at 24 hours, despite the high increase in mRNA induction may be representative of the binding of the monoclonal antibody which was raised against the Chinese hamster ovary hsp27 species, and therefore may not bind with high affinity to the rat species.

Hsp 5 6 mRNA levels did not increase but the protein levels did exhibit a mild increase at 24 hours. The hsp90 mRNA levels were not as clear-cut. Although the levels were variable on the graphic representation of the slot blot data, hsp90 mRNA levels appeared to be increased in conq)arison to the P-tubulin mRNA levels at 8 and 24 hours on the Northern blot against to and contralateral controls. The slot blot data demonstrated significantly greater levels in the ipsilateral core against the to controls at 8 and 24 hours, but was only significantly increased against the contralateral controls at 24 hours. No increases in the concentration of hsp90 mRNA can definitively be concluded m this study. The hsp90 protein levels were not notably increased against the to or contralateral controls but any increase in protein as a result of the possible increase of mRNA at 24 hours would not, on the evidence of the other hsp profiles, be detected until afl;er this time point.

162 This is the first study that examines the levels of hsp5 6 mRNA and hsp5 6, hsp60 and hsp90 protein levels in any model of cerebral ischaemia. This is also the first study of overall mRNA and protein expression of hsp27, hsp56, hsp60, hsp70, and hsp90 mRNA and protein levels exclusively in the core region of focal cerebral ischaemia. Higashi et aL, examined the mRNA levels of hsp27, hsp47, hsp70 and hsp90 during permanent MCAO, but the extraction for Northern blot analysis was taken fi'om whole brain shces, rather than exclusively the core region (Higashi et aL, 1994). In agreement with our data, Higashi et al. demonstrated an increase in hsp27 mRNA levels up to 24 hours, whereas they also demonstrated that hsp 70 mRNA levels declined afl;er 16 hours occlusion. They also stated that hsp90 mRNA levels were not raised throughout the timecourse, although the data was not presented. Transient global ischaemia (10 minutes) increases the mRNA levels of the a-isoform of hsp90 in the gerbil hippocampus with the highest levels at 8 hours post-occlusion, continuing before the levels diminished at 2 days (Kawagoe et aL, 1993a). The cDNA probe and antibody used in this study detect both isoforms of hsp90. The data iu this study suggests that hsp 90 mRNA and protein levels are not significantly increased in the core region in the first 24 hours of focal cerebral ischaemia. Hsp60 mRNA levels have only previously been investigated in the gerbil hippocampus afl;er transient global ischaemia (Abe et aL, 1993a). A 3.5 minute occlusion stimulated an increase in hsp60 mRNA after 3 hours post-occlusion and was sustained until 24 hours in the CAl neurons, and a mild increase was noted in the CA3 neurons at 3 hours. Hsp60 mRNA levels, therefore, are increased as a result of ischaemia, and this study has demonstrated that levels also increase during a severe ischaemic insult to cortical tissue and that this increase is followed by a subsequent increase in protein levels.

The areas of gerbil hippocan^us that undergo delayed neuronal death, such as the CAl subfield, show an inhibition of overall protein synthesis, throughout the recovery period from 5 minutes of global ischaemia (Thilmann et aL, 1986). The other areas of the forebrain demonstrate a transient inhibition of protein synthesis, with ^H-tyrosine incorporation reaching normal levels between 30 minutes and 12 hours post-ischaemia, and this includes the region suppHed by the middle cerebral artery. Protein synthesis during stress is believed to be impaired in part due to defects in spHcing, and the

163 increases in hsp70 mRNA demonstrated in the CAl region during ischaemia may be attributable to the lack of introns in the gene. Permanent focal cerebral ischaemia is a more severe insult than global, evident by the widespread irreversible brain injury. The data generated by this study has demonstrated that in the most severely affected region of cortex the levels of certain hsp mRNA and protein levels are still increasing 24 hours after MCAO. Unfortunately, no material from ischaemic tissue was available later than 24 hours occlusion, as it would have been interesting to study the levels at further time points.

This study is limited, in that no locahsation of expression has been defined. This could be achieved by hnmunostaining for hsp protein expression and in situ hybridisation for mRNA expression on ischaemic brain shces. Previous work has gone some way in illustrating the sub-locahsation of expression of the hsps during global and focal cerebral ischaemia. The expression of hsp27 protein has been largely locahsed to the astrocytes and microgha of the ipsilateral hemisphere from 4 hours reperfusion after 1 hour of transient MCAO (Kato et aL, 1995). There was also a corresponding difRise immunostaining for hsp27 in the gha in non-MCA territory in the ipsilateral hemisphere, including the midbrain, thalamus and hippocampus, and some in the contralateral hemisphere. One study has demonstrated that hsp is transferred from gha to neuronal axons (Tytell et aL, 1986). This might suggest that during ischaemia, the gha rnay offer some protection to neurons in repair and restoration after ischaemia through neuron-gha interactions. Increases in hsp70 protein hnmunostaining after permanent MCAO has been locahsed to the endothehal cehs in the infarct and to neurons in the ischaemic penumbra outside the infarct, whereas increased hsp70 mRNA has been locahsed to the penumbra both inside and outside the infarct (Kinouchi et aL, 1993b; Kato et aL, 1995).

The purposes, if any, of these increased expression profiles have not been defined, and are therefore speculative. Conditions that increase hsp levels have been demonstrated in cerebeUar granule and cortical cehs to protect against the cytopathic effects of increased extracehular glutamate, such as occurs during ischaemia (Abe et aL, 1993a; Rordorf g/ aL, 1991). The increased presence of hsp70 hnmunostaining as a result of

164 global and focal ischaemia may only be a by-product of ischaemia and a marker of neurons or gha that have survived by an alternative mechanism Hsp 70 may, however, be directly responsible for the survival of these cells. Dorsal root ganghon cehs overexpressing hsp 70 are resistant to ceU death in vitro as a result of simulated ischaemia (increased lactic acid in the presence of sodium dithionite at pH6.5), although the protection is not as profound as that which it confers against ‘lethal’ heat shock (Amin et aL, 1996).

Although heat shock protects neurons in vitro against ap opto sis, neuron-derived ceh lines and trigeminal neuronal cultures overexpressing hsp70 or hsp90 have been demonstrated not to be protected against the apoptotic stimuh of serum- withdrawal/retinoic acid and nerve growth factor withdrawal, respectively (Mailhos et aL, 1993; Mailhos et aL, 1994; Wyatt et aL, 1996). Therefore, any protection conferred in vivo may only be against the necrotic mechanisms induced by ischaemia.

Although many fimctions have been attributed to the various members of the hsp70 family (targeting non-native proteins for proteosomal digestion, uncoating clathrin baskets, maintaining non-native proteins in an unfolded state for bilayer translocation) the function(s) of the inducible hsp72 (hsp70i) are unclear. All the members of the hsp 70 family bind to denatured proteins, therefore the inducible member may bind to proteins in the cytosol and nucleus that have misfolded or denatured as a result of stress and prevent their aggregation, enable correct refolding during recovery, and target denatured proteins for digestion or bilayer translocation to enable correct folding in cellular organelles.

Overexpression of hsp27 protects against ‘lethal’ heat shock, but no in vitro or in vivo evidence has been presented to suggest that it may protect against ischaemia. Its role as an actin binding protein may serve to stabihse the cytoskeleton during stress, enabling recovery. Overexpression also blocks the apoptotic mechanisms stimulated by staurosporine and the Fas/APO-1 receptor in murine fibrosarcoma cells (Mehlen et aL, 1996). Ap opto sis is one of the mechanisms of cell death as a result of ischaemia, and therefore overexpression in the gha may confer a similar protection to these cehs. The

165 increased levels of hsp27 in the glia in the ischaemic core and penumbra, and surrounding cortex, may promote survival of the gha during the stress and recovery. Astrocytes maintain the ionic and neurotransmitter concentrations of the extracellular fluid and therefore survival of these gha may be important for restoration of these concentrations to prevent cytopathic effect, for example as a result of glutamate toxicity. If hsp27 is transported flrom the gha to neuronal axons, then it may serve to stabihse the structural integrity of these neurons and enable recovery. Hsp27 also has a role as a molecular chaperone. It has been demonstrated thus far to enable the folding of citrate synthase and a-glucosidase, and may therefore chaperone other proteins, possibly preventing protein aggregation and misfolding and enabling correct protein folding during recovery ifrom ischaemia.

Hsp60, the mitochondrial chaperonin and eukaryotic homologue of the prokaryotic GroEL molecule has not been demonstrated to confer tolerance to heat shock in cehs, despite its levels being raised during ceh stress. Its function has thus far been defined as providing an environment within its core to enable the correct folding of mitochondrial proteins. The increase noted in this study of mRNA and protein levels as a result of permanent MCAO and in the subregions of the hippocampus after transient global ischaemia may, like hsp72 and hsp27, serve a purpose in preventing ceh death as a result of ischaemia. Recovery of correct mitochondrial protein folding may be facihtated by raised levels of hsp 60.

Dorsal root ganghon neurons overexpressing hsp90 in vitro are resistant to ceh death under TethaT heat shock conditions, but any resistance to simulated ischaemia was not as profound as that when hsp70 is overexpressed (Amin et a/., 1996). Like hsp70, hsp90 overexpression does not confer any protection against ap opto sis (Mailhos et a/., 1994; Wyatt et aL, 1996). Any increase in expression of hsp90 may therefore confer some protection against necrotic ceh death during ischaemia, possibly by chaperoning misfolded proteins such as actin, tubulin or signal transduction molecules, preventing their aggregation, and enabling correct folding on recovery. Hsp90 and hsp5 6 may act by binding to the untransformed steroid receptor conq)lex, maintaining its correct conformation to enable response to the neuroendocrine stress pathway.

166 In summary therefore, these data show definite increases in the levels of hsp27, hsp60 and hsp70 mRNA and protein levels within the first 24 hours of focal cerebral ischaemia in the rat. The other hsps tested (hsp90 and hsp56 mRNA and protein) did not appear to be significantly raised.

In considering these results as a whole, it is clear that for greater understanding, further investigations into the role of the hsps in cell death as a result of ischaemia are required before the hsps, individually or in concert, can be named as the mediators of protection that is observed in the 'ischaemic tolerance’ phenomenon. It may therefore be usefiil to deliver the genes encoding these heat shock proteins to the brain, and assess whether overexpressing hsps in vivo affords any protection to neurons and gha during MCAO.

167 Chapter 4

Construction and Characterisation of Recombinant HSV-1 Vectors Co-expressing Individual Heat Shock Proteins and (3-Galactosidase

168 4.1 Introduction

The purpose of the techniques and data presented in this chapter and in Chapter 5 of this thesis is firstly to illustrate the methods by which disabled recombinant herpes sinq)lex virus type 1 (HSV-1) viral vectors were constructed, and secondly to characterise their expression with a view to using these vectors for subsequent transgene delivery into neurons and neuron-derived cell lines. This chapter in particular describes the design, construction and characterisation of the 17+pR16R recombinant HSV-1 vectors which were intended to co-express the reporter gene lacZ with the heat shock protein genes.

The arguments for and against the use of disabled recombinant HSV-1 as a vector overexpressing transgenes in neuronal cells are discussed in the introduction to this thesis (Section 1.3). This discussion also covers previous work concerning the fimctions and the deletion of the essential immediate-early (EE) genes, infected cell protein (ICP) 4 and/or ICP27, to disable rephcation. Previous work in our laboratory has shown that an ICP27 (IE2) deleted virus is efi&cient at gene delivery to the heart and to newborn primary cardiocytes and vascular smooth muscle cehs in vitro (Coffin et al., 1996) and also to neuronal cells both in vitro and in vivo (Howard et al., submitted). This mutant not only expressed P-galactosidase from the lacZ transgene in the in vivo and in vitro infected cells, but the primary cardiocytes infected demonstrated the highest synchronous beat frequencies after 5 days compared to VP 16 and VP16/ICP34.5 deleted HSV-1 mutants, suggesting that deleting ICP27 may minimise cytopathic effects. The viruses constructed for the purpose of this study were therefore deleted for ICP27.

As an essential gene will be deleted, the lytic cycle pathway can not ensue, and therefore, in neurons, the virus will enter a state of latency, and therefore the transgene should be expressed during the HS V-1 latent state.

All HSV-1 DNA and virus was derived fi'om the 17+ strain. The HSV-1 genome and the derivative components utilised in this chapter and also in Chapter 5 of this thesis (EcoB and Not3.5) are depicted in Figure 4.1

169 Figure 4.1 - The 17+ Strain HSV-1 Genome and Maps of its EcoB and Not3.5 Fragments

Schematic diagram depicting the sections of the 17+ strain HSV-1 genome that have been used in construction of all of the expression plasmids described in this chapter. The top line illustrates the structure of the full length HS V-1 genome.

The middle line depicts the EcoB fragment, the second largest EcoRI DNA fragment that contains part of the UL (unique long) and 1RS (internal repeat short) regions and all of the IRL (internal repeat long) region. EcoB includes the ICP27 gene (IE2, UL54), the EAT region and the ICPO and ICP4 genes.

The lower line depicts the Not3.5 fragment of EcoB containing both EAT PI and P2 promoters, part of the major (8.7kb) EAT coding sequence and aU of the 2kb EAT species coding sequence.

170 HSV-1 Genome (150kbp)

a TRL UL IRL a 1RS US IR S a

EcoB Fragment (110095-131534)

Dde\ M------UL56 ICPO ICP4 UL53^ UL54 7 1 T h Mlu\ Mlu\EcoR\ Not\ Sa/I Not\ EcoR\ Not\ IRL

LAT P1 TATA Box Not 3.5 Fragment (118439-122025)

LAT P1 ( - LAT P2 (~ 600bp)

Not\ Dde\^^^^Sty\ Sty\ BstX\ BstXl Not\

8.7kb LAT (118801-127167)

2.0kb LAT (119461-121416) 4.2 Design of the 17+pR16R HSV-1 Recombinant Vectors

The aim of the pR16R HSV-1 recombmant vector was to produce stable overexpression of the E. coli lacZ reporter gene product, P-galactosidase and a heat shock protein (hsp) in neurons in vivo and in cells in vitro during latent infection. Cells infected with the virus would therefore stain positive for p-galactosidase, on addition of the chromogenic substrate X-gal ( 5 -bromo-4- chloro- 3 -indolyl- p-D- galactopyranoside), and would also be overexpressing the required hsp transgene.

In 1994, Lokensgard et aL, tested the abihty of several promoter combinations to drive the E. coli lacZ reporter gene in the glycoprotein C (gC) locus of recombinant HSV-1, during viral latency (Lokensgard et aL, 1994). Mice were inoculated with the viruses by footpad injection. LacZ mRNA and protein expression in dorsal root gangha (DRG) was assessed by in situ hybridisation and staining with X-gal, respectively. Although all the viruses were transcriptionally active during acute infection (4 days post-infection), and all the viruses entered latency, only the LAT PI promoter (minus the TATA-box) fused with the Moloney murine leukaemia virus long terminal repeat (MoMLV-LTR) promoter remained transcriptionally active during latency (6 weeks post-infection). Neither the MoMLV-LTR or LAT PI promoters were individually active during latency. It was proposed that the LAT region provided neuronal transcription factor binding sites, and the LTR provided the TATA box for transcriptional initiation.

In a previous study the same group had recombined an LTR- (3-galacto sidase construct situated in the ICP4 locus of a mutant virus with the majority of the LAT region deleted, so that the promoter was approximately 2kb downstream of the LAT PI promoter. This recombinant virus stably expressed p-galactosidase during latency in mouse neurons and it was suggested that the continued expression may be due to transcriptional read-through from the LAT promoter (Dobson et aL, 1990). The LAT- LTR mutant virus and the above ICP4-inserted mutant virus were not corcpared directly, and so it is unclear whether the length of spacer between the LAT PI promoter and the LTR promoter are important; the presence of the LAT PI TATA- box, or the remaining fragment of the LAT P2 promoter may have had any influence

172 on the ICP4-inserted mutant virus; or whether using the LAT PI promoter in the gC locus, rather than in its wild-type site, had any specific effects.

On the basis of this data combined with the encouraging results obtained from the IE2 deleted recombinant HSV-1 in the heart, newborn primary cardiocytes and vascular smooth muscle (Coffin et aL, 1996), the pR16R construct was designed. The LAT PI promoter (including the TATA-box) was used to drive the transcription of the lacZ gene (containing a polyadenylation sequence). The MoMLV-LTR was positioned 3’ to the lacZ gene and was used to drive transcription of the hsp cDNA. Although the hsp cDNAs contain a polyadenylation sequence 3’ to the stop codon and a short untranslated sequence, there was a further simian virus 40 (SV40) intervening sequence (IVS) and polyadenylation sequence added to ensure efficient sphcing and polyadenylation of the nascent mRNA transcript in the absence of contextual elements normally present downstream in the gene. It was hoped that the 3.7kb spacer of lacZ gene between the LAT PI promoter and the MoMLV-LTR would not be too great as to lose the prolonged activity during latency as noted in Dobson et al, 1990. This whole transgene expression cassette was franked with 3-4kb of HSV-1 genome regions that frank the IE2 (UL54) gene in the UL regions of the wild-type virus, to enable homologous recombination of the cassette into genome and ‘knocking out’ of the essential gene.

As the LAT PI promoter is also present in the RL regions of the HSV-1 genome in the recombinant mutant virus, there is a possibihty of homologous recombination within the virus between the LAT PI promoters, whilst still remaining rephcation corq)etent. Therefore, the orientation of the expression cassette in the franking regions, and therefore the resultant virus genome had to be considered. It was decided to insert the cassette in the same orientation as the LAT region of the genome. Any recombination between the LAT promoter regions would therefore ‘knock out’ the whole expression region of the cassette, including the lacZ gene, and therefore undesirable recombinants would not stain blue on addition of X-gal. These recombinants would not be selected on purification of recombinant against wild-type virus.

173 4.3 pR16R Plasmid and Virus Construction

For overall plasmid structure, please refer to Figure 4.3a. For construction summary, please refer to Figure 4.3b. For individual plasmid details, please refer to Table 2.1.

pSP72 Backbone

l!t.-yglg:T.|IMiHldnî1!inT?ll=gM lîrM

(HSV-1 bp 110095) (HSV-1 bp 120900) Mlu\ Fragment Deleted (bp 113400-116800)

• Non LAT P I S’ E xtension

[M M LV LTR Xhol

ÏS V 4 0 IVS+pA

H in m H in m

Figure 4.3a - Structure of the pR16R 90 Plasmid Schematic representation of the pR16R90 plasmid illustrating the orders of promoter and gene sequences. Please refer to the following text for construction details. Tlie top plasmid shows the HSV-1 sequences flanking the deleted IE2 gene in the pSP72 backbone. Tlie second fragment shows SLP containing the LAT PI promoter (including the 5’ extension, vide infra) driving the transcription of the E. coli lacZ gene and the downstream SV40 t intervening sequence and T polyadenylation sequence from pJ4Q. Tlie third fragment shows the pJ4 90 expression vector fragment containing the MMLV-LTR drivmg transcription of the human hsp90 cDNA {Hin&\\\ and Xho\ sites mark sites for replacement with hsp70 cDNA. Tlie diagram in parentheses illustrates the overall order of promoter and gene sequences in the constmct.

174 Figure 4.3b - Summary of the Construction of pR16R 90

Due to the cono^lexity of the construction of the pR16R 90 plasmid, the various steps described and illustrated in full detail below are summarised in this figure. Plasmid and gene representations are schematic and not to scale. Numbers associated with each stage represent the sections of this chapter where the full details of the step and the plasmids involved can be located. For more details of original plasmids and their sources, please refer to Table 2.1.

175 Figure 4.3b

pBa90 + pJ4Q 4.3.1b- Exclsion & L HSP90 cDNA J Insertion MoMLVLTR MCS SV40 IVS Poly A

pJ4 90 S L P (4 .3.1a) 4.3.1b - Excision & LTR HSP90cDNA IVS PolyA insertion LATP1 lacZ SV40 IVS PolyA

SLP 90 + pSP72 iCP27 Not(-)R

LAT P1 /acZ LTR HSP90 IVS Poly A HSV-1 Region Flanking IE2 Gene

4.3.3 - Insertion of Expression Construct into Flanking Regions 5' Extension of i_AT PI Promoter

pR16R 90 4.3.2 - Insertion of Flanking Regions into Amp Resistant pSP72 HSV-1DNA LATP1+/acZ LTR HSP90 IVS Poly A HSV-1 DNA

4.3.2 - Deletion of IE2 (ICP27) Gene Deletion of Internal NoO Fragment Extension Downstream of NoA Site Blunting of NoA Site

iCP27 Not(-)R EcoB ------►

EcoB Fragment of HSV-1 (Strain 17+) HSV-1 Region Flanking IE2 Gene -*

176 4.3.1 Construction of Expression Plasmid

A plasmid containing lacZ downstream of the LAT PI promoter and followed by an SV40 rVS and polyadenylation sequence was prepared from two parental plasmids, and was prepared previously by Dr. Robert Coffin (SLP, see Figure 4.3. la). 1) The Notl fragment of the RL region of HSV-1 (base pans 118439-122025) which includes the LAT PI region and part of the major LAT region (8.7kb) was bgated into the Notl site of pGem5Zf+ (with the Apal site of the pGem5Zf+ multiple cloning site lying upstream of the 5’ end of the fragment). Named pNot5.5. 2) The lacZ gene (3.7kb) was excised from pCHllO using HindJR (5’) and BamYH (3’) and blunt-end bgated into the Kpnl site of pJ4. Named pJ4 lacZ. The Hin^inUPstl fragment of pJ4 lacZ was blunt-end bgated into the Styl sites downstream of the TATA-box of the LAT PI promoter in pNot3.5 to make SLP.

Human bsp90 cDNA (2.5kb) was excised from pBa90 using Sail and bgated into the unique Sail site of the MoMLV LTR expression vector pJ4D. Insertion and correct orientation of the gene was confirmed with a BamlAi restriction digest and named pJ4 90 (see Figure 4.3.1b).

The pJ4 90 expression cassette including the LTR promoter, the bsp90 cDNA and the SV40 IVS and polyadenylation sequence was excised using Nhel (5’) and BglQ. (3’) and blunt-end bgated into the unique BglU site of SLP. The pJ4 90 SLP construct was checked with BamYQ. and Sphl in separate digests to confirm insertion and orientation, and was named SLP 90 (see Figure 4.3. lb).

177 Figure 4.3.1a - The Construction of SLP

Diagram depicting the pathway by which SLP was constructed from the parental plasmids. For full details of restriction digests and hgations, please refer to the text in Section 4.3.1.

Briefly, the E. coli lacZ gene was excised from pCHllO (top left) and inserted downstream of the MoMLV-LTR promoter in pJ4 (top right) to make pJ4 lacZ (centre). The /

178 Figure 4.3.1a

SV 40 fifL'narV prom olar

p C H I I O p J4 7120 b p 3 70 0 bp

SV 40 T p o ly A s t t e

MoMLV4.TR ^

SV40TpulyA,

Afup

10325 bp

SV 4 0 T p o K ^ A $ r te SV401

179 Figure 4.3.1b - The Construction of SLP 90

Diagram depicting the pathway by which SLP 90 was coiistmcted fiom the parental plasmids. For full details of restriction digests and ligations, please refer to the text in Section 4.3.1.

Briefly, the hsp90 was excised fi om pBa90 (top leA) and inserted downstream of the MoMLV-LTR promoter in pJ4 (top right) to make pJ4 90 (centre). Tlie MoMLV- LTR/lisp90 cDNA cassette was excised fiom pJ4 90 and inserted downstream of the lacZ gene in SLP (bottom left), to make SLP 90 (bottom right).

The etliidium bromide stained gel below shows the confiiinatory restriction digests of pJ4 90 and SLP 90. See plasmid maps opposite for restriction site locations.

Lane 1 : Ikb ladder Lane 2: pJ4 90 digested with BamWi (band sizes - 0.5kb, 5.7kb) Lane 3: SLP 90 co-digested with BaniHl and Sph\ (band sizes - 0.5kb, 0.7kb, O.Skb, 2.5kb, 3. 7kb, 5kb, uncut) Lane 4: Ikb ladder

Ikb Ladder Fragment Sizes 1 2 3 4

lOkb — 10000

6000 5000 4000 3500 3000 2500 2000 1500 Ikb _ 1000

500

80 Figure 4.3.1b

h s p 9 0 A m p

p B a B O p J 4 1 2 5 0 0 h p 3 7 0 0 b p

SV40TpolpAs«e

b-actin pronw ter

iM L V -L T R

1 3 3 4 2 b p

SV40TpolyAsltB

SV40 I poly A s « e

81 4.3.2 Preparation of HSV-1 Regions Flanking IE2

The 21.4kb EcoB fragment of 17+ strain HSV-1 genome (the second largest EcoRI fragment - base pairs 110095-131534, see Figure 4.1) contains part of the UL region including the IE2 gene (UL54), and the whole RL region including the LAT region, and the ICP4 and ICPO genes and was cloned into the EcoKL site of the tetracycline resistant plasmid pACYC184.

This lengthy sequence had to be reduced to produce the franking regions, and therefore the 11.4kb Notl fragment (base pairs 118439-129800) was excised, and the franking regions religated. The Mlul fragments (base pairs 113200-116900 - containing the fuU length IE2 gene) were excised and the franking regions rehgated. This plasmid was named MN18 (see Figure 4.3.2).

The remainder of EcoB downstream of the rehgated Notl site in MN18 was deleted by excision of the NotVXmnl region {Xmnl site in pACYC184 backbone, 3’ to genome sequence) and replaced by blunt-end hgation with the NotUSall fragment of pNot3.5 to lengthen the 3’ franking region to approximately 4kb of continuous HSV-1 genome sequence. This plasmid was named ICP27 (see Figure 4.3.2).

The Notl site was subsequently deleted by digestion, treatment with T4 DNA polymerase and rehgation. This site was deleted to enable further 5’ extension of the LAT PI promoter {vide infra). This plasmid was named ICP27 Not(-)R (see Figure 4.3.2).

The whole franking sequence was excised from the pACYC184 backbone of ICP27 Not(-)R using Srfl (in downstream franking sequence) and EcoRI at 5’ end of upstream franking sequence, and inserted between the Ndel (3’) and EcoRI (5’) sites of the ampicillin resistant plasmid backbone pSP72 (Promega), for ease of subsequent cloning. Correct insertion and orientation was confirmed by co-digestion with Mlul and E'coRV and the plasmid was named pSP72 ICP27 Not(-)R (see Figure 4.3.2).

182 Figure 4.3.2 - The Construction of IE2-Peleted Flanking Regions

Diagram depicting the pathway by which the IE2-deleted flanking regions were prepared fiom the EcoB flagment of the 17+ strain HSV-1 genome. For full details of restriction digests and ligations, please refer to the text in Section 4.3.2.

Briefly, the EcoB flagment (top left) was shortened by deletion of the Notl fragment and the M lul fragment containing the CE2 gene to make MN18 (top riglit). Tire remaining downstream Notl-EcoK[ fragment was replaced with the Notl-Sall fragment of pNot3.5 to make ICP27 (middle left) and the remaining Notl site deleted to make ICP27 Not(-)R (middle right). Tlie flanking regions were transfened to the ampicillin resistant plasmid pSP72 (bottom left) to make pSP72 ICP27 Not(-)R.

Tlie etliidium bromide gel below shows the conflnnatory digests of pSP72 ICP27 Not(-)R. See plasmid maps opposite for restriction site locations.

Lane 1: Ikb ladder Lane 2: pSP72 ICP27 Not(-)R co-digested with Mlul and EcoKV (band sizes - 0.6kb, 1.7kb, 2.7kb, 4.7kb) Lane 2: pSP72 ICP27 Not(-)R digested with EcoVN/ (band sizes - 0.6kb, 4.4kb, 4.7kb) Lane 3: pSP72 ICP27 Not(-)R digested with Mlu\ (linearised) Lane 4: Ikb ladder

Ikb Ladder Fragment Sizes

lOkb 10000 8000

1HD —

183 Fiiîure 4.3.2

EcoB Fragm ent of HSV-1

N o ll

Motl/Sdl Fraoniwil of pHülli Nnrt/Sal Fragment nfpNofl^

NoUfSa# F raynem of pMol'J.6

p S P / ? pSP/2 ICP2/Mot<-)R 2 4 6 ? b p 9 / 2 8 b p

A m p

M lul 3 9 6 1

184 4.3.3a Insertion of SLP90 into HSV-1 Regions Flanking IE2

The expression region of SLP 90 was excised from the pGrem5Zf+ backbone using Sph\. One Sphl site hes upstream of the expression construct in the pGem5Zf+ multiple cloning site and the second hes in the HSV-1 LAT genome, just downstream of the polyadenylation sequence. Excision of the expression region with Sphl therefore deletes the unnecessary remainder of the LAT region from the SLP part of SLP 90. This excerpt was blunt-end hgated into the Mlul site of pSP72 ICP27 Not(-)K The presence of insert and orientation was confirmed by digestion with EcoRI and was named pR16R 90 Short (see Figure 4.3.3).

Plasmid DNA containing the insert oriented with the LAT PI promoter proximal to the 5’ flanking region were selected for recombination so any recombination between this inserted promoter and the HSV-1 genomic LAT PI promoter would ‘knock out’ the downstream region including the lacZ gene and hsp cDNA. CeUs infected with this virus would not turn blue on staining with X-gal, and would not be selected during plaque purification.

4.3.3b 5’ Extension of the LAT PI Promoter

The LAT PI promoter region used by Lokensgard et at., extended to a Ddel site, approximately 250bp upstream of the Notl site at the 5’ terminus of the promoter used to drive lacZ expression in the above construct (Lokensgard et a l, 1994). To recreate this region, and any possible effects it may confer, the promoter was further extended at this stage to include this upstream sequence. It was for this reason that the Notl site was deleted in the flanking regions prior to the insertion of pR16R 90 Short.

The sequence was excised from pDde rev, a plasmid containing the 589bp Ddel fragment (HSV-1 base pairs 118181-118770) including the 5’ region of the LAT PI promoter cloned into the Smal (5’) and BamHl (3’) sites of pGem3Z (Promega). The HinSQ. (in the pGem3 multiple cloning site upstream of the Ddel fragment) and Notl (the same site as that at the 5’ end of the LAT PI promoter in pR16R 90 Short) sites were cleaved to excise this fragment. It was hgated into pR16R 90 Short using Spel (in

185 the pGem5Zf+ multiple cloning site upstream of the LAT PI promoter) and Notl (at the 5’ terminus of the LAT PI promoter in pR16R 90 Short). The presence and orientation of the insert was confirmed by digestion with Xbal, and this final construct was named pR16R 90 (see Figure 4.3.3).

186 Figure 4.3.3 - The Construction of the pR16R 90 Plasmid

Diagram depicting the pathway by which the SLP 90 expression cassette was inserted into the IE2 deleted flanking regions, and how the 5’ end of the LAT PI promoter, driving the transcription of the lacZ gene, was extended. For full details of restriction digests and ligations, please refer to the text in Section 4.3.3.

Briefly, the SLP 90 expression cassette was excised (top left) and ligated into the flanking regions of pSP72 ICP27 Not(-)R (top right) to make pR16R 90 Short (middle left). Tlie region 5’ to the LAT PI promoter was excised from pDde rev (middle right) and inserted into pR16R 90 short to make pR16R 90 (bottom).

The etliidium bromide stained gel below shows the confiniiatory restriction digests of pR16R 90 Short and pR16R 90. See plasmid maps opposite for restriction site locations.

Lane 1: Ikb ladder Lane 2: pR16R Short digested with EcoKl (band sizes - 1.5kb, 1.9kb, 3.2kb, 4. Ikb, 6.9kb, uncut) Lane 3; pR16R 90 digested with Xbai (band sizes - O.Skb, 2.7kb, 4.2kb, 10.2kb) Lane 4; Ikb ladder

Ikb Ladder Fragment Sizes

lOkb-^ 10000 8000 6000 5000 4000 3500 3000 2500 2000 1500 Ikb - , 1000

500

187 Figure 4.3.3

NUIfSdl Pragment u# pNut3^

pSPT? ICP27Hfrt|)R S p tU 1 3 3 4 ? b p 9 7 2 9 h p

SV40 I pnlyAsile

BamHI M lul

Dde fraornent <119101 110770)

y HSV 1 RanKIng Sequence

HSV-1 Flanking Sequence

PHIHM rniShmi

ECORI 11444 \ V,

Amp

3' HSV-1 Flanking Sequence

5' HSV 1 Flanking Sequence Not3.5 Fragment PR16R90 i SV40TpolyAsite 17900 bp

[bal 4844 LAT Promoter Extension Xbal 12502 Hsp90 cDNA LAT P I Promoter

MoMLVLTR Xbal11744 lacZ

Xbal 7544

188 4.3.4 Construction of pR16R 70

Human hsp70 cDNA was inserted into a pR16R backbone derived from pR16R90.

Hsp70 was first excised from PH2.3 using BamHI (5’) and 7/mdin (3’) and inserted into the BamHL and Smal sites of pJ40, and named pJ4 70. Hsp70 cDNA was then excised from pJ4 70 using HindJR (5’) and EcoRI (3’) and inserted by blunt-end hgation into the EcoRV site of an adenovirus shuttle vector pAElsplA (used for its multiple cloning site sequence), and named pE170 (see Figure 4.3.4).

The hsp90 cDNA was excised from pR16R 90 using HinôIQ. (upstream of hsp90 cDNA in the pJ4 multiple cloning site) and Xhol (in 5’ terminus of hsp90 cDNA). The HindHUXhol fragment of pE170 was excised and cohesive-end hgated into the remaining pR16R backbone. The insertion of the hsp70 gene was confirmed by digestion with EcoRI, and named pR16R 70 (see Figure 4.3.4).

189 Figure 4.3.4 - The Construction of the pR16R 70 Plasmid

Diagram depicting the pathway hy wliich hsp90 was replaced in pR16R 90 with hsp70 to make pR16R 70. For full details of restriction digests and hgations, please refer to the text in Section 4.3.4.

Briefly, the hsp70 cDNA was excised fiom pJ4 70 (top left) and inserted into the adenoviral shuttle vector pElsplA (top right) to make pE170 (middle left), fiom which it was excised and inserted in-between the HinàlW and Xhol sites of pR16R 90 (middle right), thus replacing the hsp90 cDNA, to make pR16R 70 (bottom).

Tlie etliidium bromide stained gel below shows the confirmatoiy/ restriction digest of pR16R 70. See plasmid maps opposite for restriction site locations.

Lane 1 ; Ikb ladder Lane 2: pR16R 70 digested with EcoRI (band sizes - 0.9kb, 2.5kb, 3.2kb, 4.4kb, 6.9kb) Ikb Ladder Fragment Sizes 1 2

10kb _J 10000

500

190 Figure 4.3.4

B!«I

Ad El SnqiiBni:8S.

SVW IpoH/Asile

,No13.5FfaomHi1 p l 1 ? (l i SU4III pDlyAKdn 8 7 0 9 b p

\ Ad E1 Sequences

EcoR115777 Amp

3' HSV-1 Flanking Sequence

5' HSV-1 Flanking Sequence Not3.5 Fragment PR16R70 ISV40T poly A site 17870 bp S V 4 0 tlV S ! EcoRI 4844

LAT PI Extension Hsp90 5' Fragment

LAT PI Promoter Hsp7G cDNA

lacZ

MoMLVLTR

EcoRI 7314

EcoRI 8214

191 4.3.5 Recombination of pR16R 70 and pR16R 90 Plasmids into HSV-1 DNA pR16R 70 and pR16R 90 plasmid DNA was linearised at unique cloning sites in the pSP72 backbone by digestion with Sca\ and incorporated into wild-type HSV-1 strain 17+ DNA by homologous recombination according to the method outlined in Section 2.6.3.

4.3.6 Purification of 17+pR16R Viral Recombinants

Viral recombinants were selected by blue plaque selection on staining virally infected IE2 complementing cell lines with X-gal, according to the methods outlined in Sections 2.Ô.5-2.6.8. The purification of the recombinant 17+pR16R virus from unrecombined 17+ wild-type virus was (fifficult, taking 25-30 cycles of re-infection with selected plaques. This difficulty is thought to be for two reasons.

Firstly, the rephcation of recombinant virus was dependent on the overexpression of the complementing IE2 gene in the B 130/2 cell line, whereas unrecombined wild type virus was not under such limitation, and repUcated at a greater rate.

Secondly, the expression of the lacZ gene under the control of the LAT PI promoter was weak, taking approximately 6 hours after X-gal staining for the visuahsation of blue plaques without fixing. The X-gal and buffer are toxic to the cells and therefore the virus, and the longer staining time required greatly reduced the efficiency of re­ infection with selected plaques.

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

4.4.1 Visualisation oiiacZ Expression

Figure 4.4.1 illustrates the activity of P-galactosidase, the protein product of lacZ m the B 130/2 IE2-expressing cell line infected with the 17+pR16R 70 recombinant HSV- 1 vector. The plaque assay was prepared by titration of the viruses in serial dilutions of 1:10 on 6 well plates seeded with the B 130/2 cell line as described in Section 2.6.5.

192 After 48 hours of incubation, 17+pR16R 70 infected wells were fixed and stained overnight with the chromogenic substrate X-gal to detect p-galactosidase activity as described in Section 2.6.6. P-galactosidase activity was similar in B 130/2 cells infected with the 17+pR16R 90 recombinant HSV-1 vector (data not shown). The recombinant virus was pure (no rephcation was noted on non-complementing cells), yet some viraUy infected cells within the plaque remain unstained. These are most likely cells infected with virus whose LAT PI promoters have undergone homologous recombination, and ‘knocked out’ thelacZ gene.

4.4.2 Characterisation of HSP70 and HSP90 Protein Expression

The 17+pR16 recombinant viruses (17+pR16R 70 and 17+pR16R 90) were each screened for hsp70 and hsp90 protein production. This was achieved by western blot analysis of extracted protein fi’om infected plates of B 130/2 cell lines, see Sections 2.5.3-2.5.7. The monoclonal anti-hsp90 antibody, clone AC88 (a kind gift fi*om Dr. David Toft) and the monoclonal anti-hsp70, clone C92F3A-5 (Stressgen) were used for immunodetection of hsp90 and hsp70 protein, respectively. The X-ray films in Figure 4.4.2 illustrate that 17+pR16R 70 infected B 130/2 ceUs were overexpressing hsp70 against viral and mock infected controls, whereas there was no significant overexpression of hsp90.

193 Figure 4.4.1 - Detection of B-Galactosidase Activity in B130/2 Cells Infected with the 17+pR16R 70 Recombinant HSV-1 Vector

Photomicrograph of B130/2 cells infected with the 17+pR16R 70 recombinant HSV-1 vector, stained for P-galactosidase activity at lOOx magnification. For details please refer to the text in Section 4.4.1.

Figure 4.4.2 - Characterisation of Heat Shock Protein Expression in B130/2 Cells Infected with the 17+pR16R 70 and 17+pR16R 90 Recombinant HSV-1 Vectors

Western blots of protein extracted from B 130/2 cells infected with 17+pR16R 70 and 17+pR16R 90 recombinant HSV-1 vectors probed with a) anti-human hsp70 monoclonal (clone C92F3A-5, Stressgen Biotechnologies Ltd, Canada) and b) anti- hsp90 monoclonal (clone AC88, a kind gift from Dr. David Toft). For details, please refer to Section 4.4.2. Numbers on the side indicate molecular weight in kiloDaltons. ECL exposure time was 1 minute for blot a) and 5 minutes for blot b). For both blots: Lane 1 : Rainbow Marker Lane 2: 17-^R16R 90 infected B 130/2 cells Lane 3: Mock-infected control B 130/2 cells Lane 4: ICP27-deleted control virus infected B 130/2 cells Lane 5: 17+pR16R 70 infected B 130/2 cells

194 Figure 4.4.1

* * . A

- %

Figure 4.4.2 a) 12345 b) 12345

220- -220

9 7 - -9 7

66-

-66

195 4.4.3 Southern Blot Analysis of 17+pR16R 90 Infected 8130/2 Cells

The western blots demonstrated no signMcant change in hsp90 levels in JE2 coruqplementing B 130/2 cells after infection with 17+pR16R 90 virus. They did not demonstrate whether the hsp90 cDNA had been successfiilly delivered to the cells or whether or not the virus itself contained the hsp90 cDNA. The presence of the hsp90 cDNA in the 17+pR16R 90 virus was confirmed by hybridisation of a [^^P] radiolabelled hsp90 cDNA random-primed probe to a Southern blot of DNA extracted fiom 17+pR16R 90 infected B 130/2 cells. For methods, see Section 2.6.13-14. See Figure 4.4.3.

196 Figure 4.4.3 - Southern Blot of DNA Extracted from B130/2 Cells Infected with the 17+PR16R 90 and 17+pR16R 70 Recombinant HSV-1 Vectors

Tlie Southern blot was probed with a [^^P] radiolabelled random-piimed cDNA probe generated fiom the full length hsp90 cDNA and exposed for 5 hours and overnight after washhig. All DNA was digested with EcoKi. Lane 1; Ikb ladder Lane 2; 17+ wild-type DNA Lane 3; pR16R90 DNA Lane 4: pR16R 70 DNA Lane 5; 17+pR16R 90 infected B 130/2 cells Lane 6: 17+pR16R 70 infected B 130/2 cells Lane 7: lCP27-deleted control virus infected B 130/2 cells

Radioactive bands in lanes 3 and 5 correspond with the molecular weight of the hsp90 EcoKl cDNA fiagments in the pR16R 90 plasmid (1.5kb and L8kb).

5 hours 1 2 3 4 5 6 7 Overnight 12 3 4 5 6 7

1.8kb- A I.8kb- f l.Skb- l.Skb-

197 4.5 Discussion

These expression studies show that 17-HpR16R 70 is overexpressing hsp70 protein in B 130/2 cells but 17+pR16R 90 cDNA is not significantly overexpressing hsp90 protein detectable by western blot. The X-ray film exposure time required to detect the overexpression of hsp 70 was as much as 5 minutes, and therefore the MoMLV-LTR promoter may drive downstream cDNA expression in the context of virus only weakly. Hsp90 is expressed constitutively at a high level (approximately 1% of total cell protein), and therefore the overexpression of hsp90 in cells infected with the 17+pR16R 90 virus may not be sufficient in relation to the endogenous gene to be detectable on western blot analysis.

Lokensgard et al., fiised the TATA-less LAT PI promoter with the MoMLV-LTR, and they proposed that elements in the LTR promoter activated latent expression in concert with the LAT PI promoter (Lokensgard et at., 1994). It was hoped in these recombinant vectors that the MoMLV-LTR would be in close enough proximity to the LAT PI promoter (~3.7kb) to confer this effect. This may not have been the case. The authors infected murine dorsal root gangha, whereas in this study BHK-derived cell lines were infected, and therefore expression may be different between neuronal or non-neuronal cell types. Dobson et a l, demonstrated expression of p-galactosidase during latency when the LTR-transgene construct was inserted into the ICP4 region approximately 2kb downstream of the LAT PI promoter (the majority of the LAT coding region had been deleted using ^jffill) (Dobson et a l, 1990). The remaining 5’ fragment of the LAT P2 promoter in that virus may exert some additional regulatory effect, rather than solely elements located in the LAT PI promoter. Therefore, the proximity of the LAT PI promoter to the MoMLV-LTR may be crucial to enhance transcription during latency.

The four major problems with the 17+pR16R HSV-1 vectors were considered thus:

1) The lacZ expression under the control of the LAT PI promoter was weak, being difficult to detect on B 130/2 IE2 com|)lementing cell lines, making purification

198 difficult. This may render detection of p-galactosidase activity above background in vivo, in non-con^lementing neurons problematic. 2) The recombination frequency between the LAT PI promoter regions in the expression cassette and the RL region of the HSV-1 genome will mean that some non-expressing mutant virus will be present after large scale viral culture, and therefore the resulting preparations will be inq)ure. 3) Purification of subsequent recombinant viruses containing other transgene cDNAs would be equally as time-consuming as recombination was made into the wild-type HSV-1 genome, which exhibits a much greater advantage in rephcation. 4) The relatively weak overexpression of the hsp cDNAs as driven by the MoMLV- LTR promoter in the context of HSV-1 may not be sufficient to produce any physiological and/or protective effect on exposure of neuron-derived cell lines and primary neuronal cultures to various cell stresses.

It was therefore decided to design new recombinant viruses that wiU overexpress the hsp cDNAs at a higher level. The viruses should not undergo homologous recombination that will ‘knock out’ the expression region, and the expression cassette should be recombined into a mutant virus that wiU not exhibit such a growth advantage, making preparation less labour-intensive. These viruses were named the 17+pR19 viruses and their design, construction and characterisation is covered in the following chapter.

199 Chapter 5

Construction and Characterisation of Recombinant HSV-1 Vectors Expressing Single Transgenes from the LAT Region

2 0 0 5.1 Introduction

From the experience gained in the work described in the previous chapter, the aims of the viruses whose design, construction and characterisation are covered in the first part of this chapter, was to overcome the problems encountered with the 17+pR16R recombinant HSV-1 vectors discussed in Section 4.5. Principally, these new recombinant vectors (17+pR19) were designed to overe?q)ress the transgene to a higher level during latency, without the disadvantage of homologous recombination within the mutant genome that would ‘knock out’ the transgene. These problems and potential solutions are discussed in more detail in the following section.

Consequently, this chapter describes the construction and characterisation of 17+pR19-based recombinant HSV-1 vectors that individually express the hsps 27, 56 and 70. The reporter genes lacZ and green fluorescent protein (GFP) were also individually expressed firom 17+pR19-based vectors and the use of these as markers of infection is discussed and the expression of lacZ is conq)ared to that demonstrated in similar cells infected with the 17+pR16R 70 recombinant HSV-1 vector.

This chapter also describes the construction and characterisation of a 17+pR19-based recombinant HSV-1 vector that overexpresses a constitutrvely active mutant of the human heat shock protein transcription factor, heat shock factor 1 (HSFl). Overexpression of this mutant (H-BH, kindly donated by Richard Voelhny, University of Miami, USA) has been demonstrated to increase the expression of the reporter gene encoding chloranq)henicol acetyltransferase (CAT) expressed under the control of the hsp70 promoter (Zuo et al., 1995). Although this was solely a study on the effect of expressing the H-BH mutant on the activity of the hsp70 promoter, it indicates that it may increase the expression of endogenous heat shock proteins when overexpressed in the context of a recombinant HSV-1 vector. The studies reported in this chapter demonstrate that overexpression of H-BH in this fashion does increase the expression of some endogenous heat shock proteins, namely hsp27, hsp70 and hsp32; but not others i.e. hsp90, hsp60 and hsp56 and therefore this recombinant HSV-1 vector may confer a protective effect fi’om cell death as a result of cellular insults.

201 The final section in this chapter describes the design, construction and characterisation of a 17-HpR19-based recombinant HSV-1 vector (17+pMl) that co-expresses two reporter genes, namely lacZ and GFP. The transcription of both of these genes is driven by the same promoter. This is enabled by inserting an encephalomyocarditis virus internal ribosome entry site (EMCV 1RES) between the genes, thus creating a bicistronic construct. The reasons for using this technique are discussed in more detail in Section 5.7. The purpose of creating this vector was to investigate the potential of bicistronic vectors in recombinant HSV-1-mediated transgene delivery.

5.2 17+pR19 Vector Design

Transgenes driven by the human cytomegalovirus immediate-early (CMV-IE) promoter are expressed at a greater level than those under the control of the MoMLV- LTR Therefore, in the context of a recombinant HSV-1 transgene vector, the CMV- IE promoter may increase the level of transcription of transgene cDNA as compared to the previous vectors.

The use of the LAT PI promoter in concert with the CMV-IE promoter may aid transgene expression during viral latency, as was noted with the MoMLV-LTR in Lokensgard et a i, 1994. A second promoter in the LAT region, LAT P2, situated immediately downstream of the LAT PI promoter in the 5’ coding region of the major 8.3kb LAT, has also been demonstrated to confer long-term expression of the p- glucuronidase gene (8 weeks) in murine trigeminal gangha neurons (Zwaagstra et a l, 1990; Wolfe et al., 1992). LAT P2, therefore, may also aid expression in neurons during latency. One way of preventing the homologous recombination observed in the 17+pR16R vectors between inserted and endogenous LAT promoters in the HSV-1 genome is to use the endogenous LAT promoters in the two RL regions of the HSV-1 genome. One additional advantage of this is that each recombinant virus will contain two copies of the expression region, which may also increase the level of transgene expression. The insertion of the CMV-IE promoter immediately downstream of the

2 0 2 LAT P2 promoter may therefore combine the observed effects of the LAT PI, LAT P2 and exogenous promoter to confer long term, high level, transcriptional activity during latency. This was the basis of the 17-i-pR19 recombinant HSV-1 vector design.

Using just one promoter meant that only one transgene could be expressed in each recombinant virus. Therefore, one 17+pR19 recombinant virus was made expressing the lacZ gene, and separate 17+pR19 recombinant viruses were made expressing hsp cDNAs. This had the advantage that the 17+pR19 lacZ virus could be used as vahd viral controls during characterisation of expression of the different recombinant HSV-1 vectors, and during subsequent in vitro and in vivo experimentation to assess the protective effects of overexpressing the heat shock proteins against cell stress.

As the expression construct was not to be recombined directly into the BE2 region, thus ‘knocking out’ the essential gene encoding ICP27 (as was the case with the pR16R recombination), an IE2-deleted mutant HSV-1 DNA was required for insertion of the expression constructs into the non-essential LAT region of the HSV-1 genome. This was obtained by plaque purification of the ‘white’ (i.e. recombinant virus not expressing the lacZ gene) LAT PI recombination mutants that occurred during growth of the 17-HpR16R recombinant virus. Therefore, in conq)arison to an IE2 deleted virus by ‘knocking out’ the region between the Mlu\ sites at base pairs 113200 and 116800, an additional 1.4kb deletion between the Mlu\ site at 116800 and the Ddel site at the 5’ end of the LAT PI promoter at base pairs 118181 has taken place.

This deletion mutant was considered suitable for recombination with the pR19 expression constructs for three reasons: 1) From what is currently known about the HSV-1 genome, no fiirther genes were deleted as a result of this additional recombination. 2) The other RL region at the end of the UL region distal to the IE2 gene would not be affected by this recombinatorial event and the whole of this RL sequence, prior to recombination, would be as in the wild-type HSV-1 genome. Any unidentified upstream elements that may also enhance transcription of this LAT during latency would therefore be preserved after recombination.

203 3) In both RL regions, any downstream regulatory elements would also be preserved after recombination.

The pR19 lacZ construct was recombined into the 17+pR16R-derived ‘white’ IE2 deleted HSV-1 mutant DNA, to create a ‘blue’ 17+pR19 lacZ recombinant HSV-1 vector. No unique sites were available in the backbone for the linearisation of the plasmid DNA, which lowered the efficiency of recombiuation. Once pure, the recombinant virus was cultured in roller bottles and the DNA extracted as described in Sections 2.6.9 and 2.6.10. The pR19 expression constructs containing the heat shock transgene were then recombined into this 174q)R19 lacZ recombinant HSV-1 DNA, thus replacing the lacZ expression region with the transgene expression DNA construct to create a ‘white’ virus that expressed the desired transgene.

The reporter gene encoding green fluorescent protein (EGFP, Clontech Laboratories Inc, California, U.S.A) was also inserted into the pR19 construct to make pR19 GFP (Chalfle et al., 1994). The variant of GFP used was selected and optimised for brighter fluorescence and higher expression in mammahan cells. Its excitation maxima is at 488nm and its emission maxima is at 507nm. Cells expressing the gene could therefore be visuahsed on microscopy under ultraviolet (UV) hght using fluorescein optics. The potential advantages of GFP over (3-galactosidase selection during purification are: 1) Cells infected with a recombinant HSV-1 vector expressing GFP could be selected without waiting for a stain to develop and without the toxic effects of the X-gal solution, theoretically increasing the viral yield on re-infection. 2) The GFP gene (~700bp) is considerably shorter than the lacZ gene (-3600 bp) which facflitates recombination of transgene-containing plasmid DNA into HSV-1 DNA, particularly when other transgenes are included in the plasmid. pR19 GFP DNA was also recombined into the 17+pR16R-derived ‘white’ viral DNA without linearisation, and 17-HpR19 GFP recombinant virus was purified by green plaque selection on visuahsation under UV hght usmg fluorescein optics.

The hsp cDNAs inserted into the pR19 construct were those encoding Chinese hamster hsp27 (a kind gift from Jacques Landry (Lavoie et a l, 1990)), rabbit hsp56 (a kind gift

204 from Marie-Claire Lebeau (Lebeauet al., 1992)), human hsp70 (Wu et al., 1985) and human hsp90 (Twomey et al., 1993). The constitutively active mutant of human HSFl (H-BH) was also inserted into the pR19 construct, and this is discussed m more detail in Section 5.5.

205 5.3 pR19 Plasmid and 17+pR19 Virus Construction

For overall plasmid structure, please refer to Figure 5.3. For individual plasmid details, please refer to Table 2.1.

I Ampicillin Resistance pGem5Zf+ Backbone

HSV-1 Not3.5 F ragm ent Flanking R egions 1.8Kb 1.6Kb

LAT PI Promoter -O.Skti^LAT P2 Promoter ~0.6kb

HSV-1 bp 118439 BsfXI Fragm ent Deleted (HSV-1 bp 122025) (bp 120200-120400)

3.7Kb

|CMV IE Promoter!|| IBGH poly A

LAT PI LAT P2

Figure 5.3 - Structure of the pR19 lacZ Plasmid

Schematic representation of the pR19 lacZ plasmid illustrating the orders of promoter and gene sequences. Please refer to the following text for construction details. The top plasmid shows the HSV-1 flanking sequences derived from the Not3.5 fragment containing the LAT PI and LAT P2 promoter sequences. The lower fragment shows the CMV-IE promoter (from pcDNA3) driving transcription of the E. coli lacZ gene and the bovine growth hormone (BGH) polyadenylation sequence (also from pcDNA3).

Tlie diagram in parentheses illustrates the overall order of promoter and gene sequences in the construct.

206 5.3.1 Construction of Expression Plasmid and HSV-1 RL Flanking Regions

For the positions of HSV-1 sequences in the HSV-1 genome, please refer to the schematic diagram in Figure 4.1.

The flanking regions containing the LAT PI and LAT P2 promoter were derived fi'om the Notl fi’agment of the RT region of the HSV-1 genome (118439-122025 base pairs). This is the genomic fragment previously cloned into the Not\ unique cloning site of the pGem5Zf+ cloning vector to make pNot3.5 (see Section 4.3.1). The BstXl site in the pGem5Zf+ backbone was first deleted by digestion of the flanking Sac\ and Nsil sites, and blunt-ended rehgation. The deletion was confirmed by digestion with .65^X1 and the plasmid named pNot3.5BstXI(-) (see Figure 5.3.1).

The expression region containing the CMV-IE promoter and bovine growth hormone (BGH) polyadenylation sequence was excised fi’om the eukaryotic expression vector pcDNA3 by digestion of the unique cloning sites Nru\ (5’) and Bbsl (3’) and blunt-end hgated into the two BsfX\ sites immediately downstream of the LAT P2 promoter in pNot3.5BstXI(-). The CMV-IE94 promoter was oriented proximal to the LAT P2 promoter. Insertion and orientation was confirmed using Ndel and the plasmid named pNot3.5cDNA3 (see Figure 5.3.1).

207 Figure 5.3.1 - Construction of the pNot3.5cDNA3 Plasmid

Diagram depicting the pathway by which pNot3.5cDNA3 was constructed fiom the parental plasmids. For full details of restriction digests and ligations, please refer to the text in Section 5.3.1.

Briefly, the BstX] site in the pNot3.5 backbone (top left) was deleted to make pNot3.5BstXI(-) (top right). Tire CMV-IE promoter and BGH poly A site were excised fiom pcDNA3 (bottom left) and inserted into the pNot3.5 flanking regions to make pNot3.5cDNA3 (bottom right).

Tlie ethidium bromide stained gel below shows the confinnatoiy restriction digest of pNot3.5cDNA3. See plasmid maps opposite for restriction site locations.

Lane 1 : Ikb ladder Lane 2: pNot3.5cDNA3 digested with Ndel (band sizes - 2.7kb, 4.7kb)

Ikb Ladder Fragment Sizes

I0kb_j 10000 8000 6000 5000 4000 3500 3000 2500 ir 2000 1500 Ikb _ 1000

500

208 Figure 5.3.1

pW ot3.5Bs1)a(.) 6 5 8 9 b p Sty»

B s«l deleted SI/

A m p

PCDHA3 üüHpolyAs«e\ r 5 4 8 0 b p 1

209 5.3.2 Insertion of the transgenes

The structure o f all the pR 19-derived plasmids are illustrated in Figure 5.3.2.

The lacZ gene was excised from pCHllO using ///«dm (5’) and BarnHi (3’) and cohesive-end hgated into the ///«dm(5’) and BamYH (3’) sites of the multiple cloning region in pNot3.5cDNA3 (derived from the pcDNA3 insert between the CMV-IE promoter and the BGH polyadenylation sequence). Correct insertion was confirmed by digestion with £'coRI, and the plasmid was named pR19 lacZ.

Green fluorescent protein (GFP) cDNA was excised from the eukaryotic expression vector pEGFPNl by digestion with ///«dm (5’) and Not\ (3’) and hgated into the similar sites within the multiple cloning region of pNot3.5cDNA3. Correct insertion was confirmed by co-digestion with EcoRI and ///«dm and the plasmid was named pR19 GFP.

Chinese hamster hsp27 was also excised from BS27 (pBluescript SK+ containing the hsp27 cDNA in the EcoRI unique cloning site) using ///«dm(5’) and Bam¥PL (3’) and inserted into pNot3.5cDNA3 as above. Correct insertion was confirmed by co­ digestion with BamYQ. and ///«dm, and the plasmid was named pR19 27.

Rabbit hsp56 cDNA was excised from the parent plasmid, p59, by digestion with EcoRI and cohesive-end hgated into the Eco91 site in the multiple cloning region of pNot3.5cDNA3. Correct insertion and orientation was confirmed by digestion with BamYQ. and the plasmid was named pR19 56.

Human hsp70 cDNA was excised from pE170 (see Section 4.3.4) by digestion with ///«dm (5’) and Xhoi (3’) and cohesive-end hgated into the similar sites within the multiple cloning region of pNot3.5cDNA3. Correct insertion was confirmed by co­ digestion with Xho\ and ///«dmand the plasmid was named pR19 70.

Human hsp90 cDNA was excised from pJ4 90 (see Section 4.3.1) by digestion with and Sati and blunt-end hgated into the ///«dm site within the multiple cloning region

210 of pNot3.5cDNA3. Correct insertion and orientation was confirmed by digestion with EcdRl and the plasmid was named pR19 90.

211 Figure 5.3.2 - Maps of the pR19 Constructs Containing Transgenes

For full details of the insertion of specific transgenes into pNot3 .5cDNA3, please refer to the text in Section 5.3.2 (Section 5.5.3 for pR19 HSP).

Labelled restriction sites represent those used for confinnation of insertion of the transgene, and the etliidiura bromide stained gel below shows each of the plasmids after confuTnatoiy restriction digests. See plasmid maps opposite for restriction site locations.

Lane 1 : Ikb ladder Lane 2; pR19 lacZ digested with EcoK\ (band sizes - 0.5kb, 10.6kb) Lane 3: pR19 GFP co-digested with HinàlW and EcoKi (band sizes - 0.8kb, 7.3kb) Lane 4: pR19 27 co-digested with BamYQ. and HinàlW (band sizes - 0.8kb, 7.3kb) Lane 5; pR19 56 digested with BamY^ (band sizes - 1.2kb, 8. Ikb) Lane 6; pR19 70 co-digested with Xhol and HinAYYi (band sizes - 2 3kb, 7.3kb) Lane 7: pR19 90 digested with EcoRI (band sizes - 1.5kb, 8.4kb) Lane 8: pR19 HSF co-digested with HinàlW and Xhol (band sizes - 2. Ikb, 7.4kb) Lane 9: Ikb ladder

Ikb Ladder Fragment Sizes lOkb — 10000 8000 6000 5000 4000 3500 3000 2500 2000 1500

Ikb _ 1000

500

212 Figure 5.3.2

p R I B G F P 8 1 5 4 b p

BGH polyA site

Kbai 3845

A m p

PR19 27 p H 1 9 ba 8154 bfi 9 3 5 4 b p

BGH pofy A site HGH im l^A ailfi

\

rco R I5 5 4 5

p R I O H S f 9 4 5 4 b p

FIGHpoly A sile

KlxX 5145

213 5.3.3 Recombination of the pR19 Plasmids into HSV-1 DNA

The pR19 27 and pR19 90 plasmid DNA constructs were linearised at unique Xmn\ cloning site in the pGem5Zf+ backbone, whereas like the pR19 lacZ and pR19 GFP plasmids, the pR19 56 and pR19 70 plasmids did not contain unique linearisation sites and were therefore co-transfected for recombination in a circular form.

Linearised/unlinearised DNA was incorporated into pR19 lacZ HSV-1 DNA by homologous recombination according to the method outlined hi Section 2.6.3.

5.3.4 Purification of 17+pR19 HSP Viral Recombinants

Viral recombinants were selected by ‘white’ plaque selection against the unrecombined ‘blue’ viral plaques on staining virally infected IE2 complementing cell lines (B130/2) with X-gal, according to the methods outlined in Sections 2.6.5-2.6.8. The purification of the 17+pR19 recombinant viruses from unrecombined 17+pR19 lacZ virus was considerably e a ^ r than the previous purification of the pR16R viruses, taking between 5 and 10 cycles of re-infection with selected plaques. There were two main reasons for this.

Firstly, excluding any effect that expression of the hsp transgenes may have on viral rephcation, both the unrecombined and recombinant 17+pR19 virus’ growth was dependent on the expression of the IE2 gene in the B130/2 cell line. Therefore the unrecombined 17+pR19 lacZ virus exhibited no replication advantage, unlike the previous unrecombined wild-type virus.

Secondly, the level of activity of P-galactosidase in the 17+pR19 lacZ virus under the control of the CMV-IE promoter was much stronger than the expression seen in the 17+pR16R viruses. This meant that ‘white’ plaques infected with the recombined 17+pR19 HSP virus were easy to differentiate from unrecombined ‘blue’ plaques after only 2 hours of staining with X-gal, aiding selection and picking of the plaques.

214 5.4 Characterisation of the 17+pR19 Recombinant HSV-1 Vectors

5.4.1 Visualisation of lacZ and Green Fluorescent Protein Expression

Figures 5.4.1a and 5.4.1b illustrate the level of activity of P-galactosidase and the expression of GFP by 17-kpR19 lacZ and 17-HpR19 GFP, respectively. Plaque assays were prepared by titration of the viruses in serial dilutions of 1:10 on 6 well plates seeded with the B 130/2 IE2-expressing cell line as described in Section 2.6.5. After 48 hours of incubation, 174q)R19 lacZ infected wells were fixed and stained overnight with X-gal to detect P-galactosidase activity as described in Section 2.6.6 and 17+pR19 GFP cells were visuahsed using fluorescein optics as described in Section 2.6.7.

The P-galactosidase activity in ceUs infected with the 17-HpR19 lacZ virus was considerably greater than that of similar ceUs infected with the 17+pR16R lacZ virus, indicating that the expression of the hsps in cells infected with the 17+pR19 HSP viruses may also be to a similarly high degree. The expression of GFP in ceUs infected with the 17+pR19 GFP virus was also easily visuahsed, and therefore taking into account the advantages of using GFP discussed in Section 5.2, the EGFP gene is a potentiahy usefiil candidate as a reporter gene for use in HSV-1 mediated transgene delivery and detection.

215 a)

b)

Figure 5.4.1 - Detection of Reporter Gene Product Activity in B130/2 CeUs Infected with the 17+pR19 iacZ and 17+pR19 GFP Recombinant HSV-1 Vectors a) Photomicrograph of B130/2 ceils infected with the I7+pR19 lacZ recombinant

HSV-1 vector, stained for (3-galactosidase activity, at lOx magnification. For details please refer to the text in Section 5.4.1.

b) Photomicrograph of B 130/2 cells infected with 17+pR19 GFP recombinant HSV-1 vector, visuahsed under UV hght using fluorescein optics, at lOx magnification. For details please refer to the text in Section 5.4.1.

216 5.4.2 Characterisation of Heat Shock Protein Expression a) Method Details The levels of the different hsps expressed by the 17+pR19 HSP viruses were quahtatively assessed by western blot analysis. The antibodies used for detection were :

Table 5.1 - Antibodies Used for Detection of HSP Transgene Expression on Western Blots (For fiiU details see Table 2.2)

Transgene Antibody Supplier

Chinese hamster hsp27 Polyclonal anti-murine hsp25 Stressgen, Canada Rabbit hsp 5 6 KN382/EC1 monoclonal anti-rabbit Stressgen, Canada hsp56 Human hsp70 C92F3A-5 monoclonal anti-human Stressgen, Canada hsp70 Human hsp90 AC88 monoclonal anti-hsp90 Dr. D A. Toft

In the first study B 130/2 ceUs (expressing the IE2 essential gene product, ICP27) were infected with the 17-HpR19 recombinant HSV-1 vectors to fuU cytopathic effect, at which point the virus has rephcated throughout the cell culture, the cells lysed and the protein extracted and the level of heat shock transgene expression assessed (Figure 5.4.2a).

B130/2S were derived from the baby hamster kidney (BHK) cell line and therefore the expression of the hsp transgene was studied in BHKs infected with the 17+pR19 recombinant HSV-1 vectors to confirm that expression was not due in some way to the complementary expression of the IE2 gene, and that viral rephcation was not necessary for transgene expression (Figure 5.4.2b).

The in vitro purpose of these viruses was to study the effect of overexpressing the hsps in the neuron-derived ND7 cell line (fusion of N18 neuroblastoma ceUs and rat dorsal

217 root ganglion neurons (Wood et al., 1990)) and therefore the levels of transgene expression were measured m the ND7 cell line infected with the 17-HpR19 recombinant HSV-1 vectors (Figure 5.4.2c).

Cell lines were infected with virus according to the method described in Section 2.6.12 and harvested and protein was extracted, denatured and separated by SDS-PAGE as described in Sections 2.5.3 and 2.5.4. Protein loading was equahsed by analysis of a Coomassie stained gel as described in Section 2.5.5 and the protein was transferred to nitrocellulose and immunodetected as in Sections 2.5.6 and 2.5.7. In every gel one lane contained protein extracted from mock-infected cells, i.e. cells that underwent all the experimental conditions excluding the addition of virus, and another lane contained protein extracted from cells that were infected with the 17+pR19 lacZ recombinant HSV-1 vector as a non-hsp expressing viral control.

218 Figure 5.4.2a - Characterisation of Heat Shock Protein Expression in B130/2 Cells Infected with the 17+pR19 HSP Recombinant HSV-1 Vectors

Western blots of protein extracted from B 130/2 cells infected with the 17+pR19 HSP recombinant HSV-1 vectors probed with the antibodies (Ab) detailed in Table 5.1. For method details please refer to Section 5.4.2. Numbers on the left side indicate molecular weights in kDa. ECL exposure times were all less than 30 seconds.

Legend

Blot 1: Incubated with anti-hsp25 antibody Blot 2: Incubated with anti-hsp56 antibody Blot 3: Incubated with anti-hsp70 antibody Blot 4: Incubated with anti-hsp90/anti-hsp70 antibodies

RM - Rainbow marker C - Protein extracted from mock-infected B 130/2 cells lac - Protein extracted from B 130/2 cells infected with 17-^pR19 lacZ virus 27 - Protein extracted from B 130/2 cells infected with 174^R19 27 virus 56 - Protein extracted from B 130/2 cells infected with 17+pR19 56 virus 70 - Protein extracted from B 130/2 cells infected with 17+pR19 70 virus 90 - Protein extracted from B 130/2 cells infected with 17+pR19 90 virus HSF - Protein extracted from B 130/2 cells infected with 17-i-pR19 HSF virus (see Section 5.5)

219 Figure 5.4.2a Blot 1 RM C lac 27 70 Blot2 RM C lac 56 HSF

220 -

220 -

9 7 -

9 7 -

66 -

66 - 4 7 -

4 7 -

3 0 -

3 0 -

Blot 3 RM C lac 70 90 HSF Blot 4 RM C lac 70 90 HSF

^ 220 - '

220- . 97- ^ irn • t "ni

9 7 -

66-

66 -

4 7 -

4 7 - .

30 - _

220 Figure 5.4.2b - Characterisation of Heat Shock Protein Expression in BHK Cells Infected with the 17+pR19 HSP Recombinant HSV-1 Vectors

Western blots of protein extracted from BHK cells infected with the 17+pR19 HSP recombinant HSV-1 vectors probed with the antibodies detailed in Table 5.1. For method details please refer to Section 5.4.2. Numbers on left hand side indicate molecular weights in kDa. ECL e?q)osure times were all less than 30 seconds.

Legend

Blot 1: Incubated with anti-hsp25 antibody Blot 2: Incubated with anti-hsp56 antibody Blot 3: Incubated with anti-hsp70 antibody

RM - Rainbow marker C - Protein extracted from mock-infected BHK cells lac - Protein extracted from BHK cells infected with 17-fpR19 lacZ virus 27 - Protein extracted from BHK cells infected with 17+pR19 27 virus 56 - Protein extracted from BHK cells infected with 17+pR19 56 virus 70 - Protein extracted from BHK cells infected with 174pR19 70 virus HSF - Protein extracted from BHK cells infected with 17-fpR19 HSF virus (see Section 5.5)

221 Figure 5.4.2b Blot 1 RM C 27 lac HSF Blot 2 RM C 56 lac HSF —

220- »

220 - 9 7 -

9 7 -

66 -

66 -

4 7 -

4 7 - 3 0 -

Blot 3 RM C 70 lac HSF

220-

9 7 -

- S 66

4 7 - ,

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

Western blots of protein extracted from ND7 ceUs infected with the 17-^R19 HSP recombinant HSV-1 vectors probed with the antibodies detailed in Table 5.1. For method details please refer to Section 5.4.2. Numbers on left hand side indicate molecular weights in kDa. ECL exposure times were all less than 30 seconds.

Legend

Blot 1: Incubated with anti-hsp25 antibody Blot 2: Incubated with anti-hsp 5 6 antibody Blot 3: Incubated with anti-hsp70 antibody

RM - Rainbow marker C - Protein extracted from mock-infected ND7 cells lac - Protein extracted from ND7 cells infected with 17+pR19 IacZ virus 27 - Protein extracted from ND7 cells infected with 17+pR19 27 virus 56 - Protein extracted from ND7 cells infected with 17+pR19 56 virus 70 - Protein extracted from ND7 cells infected with 17-HpR19 70 virus

223 Figure 5.4.2c Blotl RIM C iac 27 Blot 2 RIM C lac 56

220 -

220-

9 7 -

9 7 -

66 -

66 -

4 7 -

4 7 -

3 0 -

Blot3 RIM C lac 70

220-

9 7 -

66 -

4 7 -

224 b) Results It is clear from all three figures that the 17+pR19 27 recombinant HSV-1 vector is expressing high levels of immunospecific protein at the 27kDa level as detected by the anti-murine hsp25 antibody. The protocols were carried out on the three cell lines at different times with different batches of reagents so it is not possible to con^are the levels of protein expression between the cell lines. The multiple bands visible on the X- ray films may represent multiply phosphorylated forms of hsp27 or alternatively degradation products. No constitutive expression of hsp27 was detected in mock infected or 17+pR19 lacZ infected control lanes and therefore no quantitative conq)arison can be made. Any further exposure in order to detect constitutive expression saturated the film, and the data therefore would have been underepresentative.

The 17+pR19 56 and the 17+pR19 70 recombinant HSV-1 vectors expressed similarly high levels of their immunospecific transgenes at 59kDa and 72kDa, respectively, in all three cell lines. Again, at these exposure times (less than 30 seconds), no constitutive expression of hsp56 or hsp70 was detectable and therefore no quantitative coroparison can be made with the control lanes.

B 130/2 cells infected with the 17+pR19 90 recombinant HSV-1 vector did not significantly overexpress hsp90, that was detectable with the AC88 monoclonal antibody. As a result it was not tested on any other cell line. Taking into account the similar result from the 17+pR16R 90 recombinant HSV-1 vector (see Section 4.4.2), this could be for several reasons: 1) The gene is not producing a functional transcript for translation. 2) The translation of hsp90 mRNA is controlled by endogenous cellular mechanisms, preventing overexpression. 3) Overexpression of hsp90 under the control of the CMV-IE promoter may be shght relative to the high constitutive expression of hsp90 (approximately 1% of total cellular protein).

225 c) Conclusion These results show that in all the cell lines tested, the 17-HpR19 27, 17+pR19 56 and 17-kpR19 70 recombinant HSV-1 vectors all significantly overexpress their respective hsps against the constitutive levels detectable at the exposure times used. They are therefore potential gene transfer agents to study the protective effects of overexpressing hsp27, hsp56 and hsp70 against cell stresses on neuron-derived ND7 cells and rat primary dorsal root ganghon neuronal cultures in vitro. Subsequently these vectors could be administered in vivo to the rat brain to examine the protective effects, if any, that overexpression of these hsps may confer to neurons during a subsequent focal cerebral ischaemic insult. The lack of significant overexpression of hsp90 in B 130/2 cells by 17+pR19 90 (and 17+pR16R 90) make it an unsuitable candidate for these experiments as it can not be shown e?q)erimentally that the virus is overexpressing the protein.

5.5 The Heat Shock Transcription Factor Mutant, H-BH

5.5.1 Introduction

In 1994, Zuo et al., demonstrated that a deletion in the LZ2 (LZ=leucine zipper) region of the human heat shock transcription factor, heat shock factor 1 (HSFl) between the HincH and BamlU sites of the gene (coding for amino acids 186-202) created a mutant protein that exhibited constitutive trimérisation and DNA binding properties (Zuo et at., 1995). As discussed in Section 1.2j, in non-stress conditions wild-type HSFl exists as a coiled-coil monomer, through internal interactions between the hydrophobic heptad repeats in the leucine zipper regions. On stress the monomer uncoils and homotrimerises through interactions between these hydrophobic heptad repeats, and gains DNA binding activity at the heat shock element consensus sequence NGAAN (Lis and Wu, 1993). The deletion in the LZ2 region appears to disrupt the native coiled monomer configuration, without affecting the homotrimerisation event. Overexpression of the mutant was demonstrated to constitutrvely increase chloramphenicol acetyl transferase (CAT) activity under the control of the highly inducible hsp70B promoter, but it was not shown whether it could transactivate stable expression of any of the endogenous heat shock genes.

226 5.5.2 Characterisation of HSP70 promoter activity during H-BH overexpression by Chloramphenicol Acetyltransferase (CAT) Assay

This mutant (H-BH) was kindly donated by Richard Voelhny as the plasmid GM BH for insertion into the pR19 construct, and subsequent expression in recombinant HSV- 1 for gene delivery. H-BH was first excised fi*om GM BH using EcoRI (3’) and Hinàni (5’) and cohesive-end ligated into similar sites of the adenovirus shuttle vector pAElsplB (used for its arrangement of unique cloning sites), to make pAElHSF. To test whether H-BH transactrvated the heat shock promoter, the H-BH containing Hin^J5UXho\ fi*agment of pAElHSF {vide infra) was cohesive-end ligated into the Hinàni (5’)/iSa/I (S’) sites of the mammalian expression vectors pJTH and pJ5H, to be expressed under the control of the CMV IE94 promoter and the dexamethasone inducible MMTV promoter respectively to make pJ5 H-BH and pJ7 H-BH, see Figure 5.5.2a.

These constructs were co-transfected into ND7 cells to give transient expression along with ESN, a vector expressing the CAT gene under the control of the human HSP70B promoter which was kindly donated by Richard Morimoto, see Figure 5.5.2a (Williams and Morimoto, 1990). IpM dexamethasone was added as appropriate after 24 hours and CAT activity assayed after a further 24 hours according to the methods outlined in Section 2.6.4, see Figure 5.5.2b. The production of CAT was highest when the H-BH gene was expressed under the control of the CMV promoter, the MMTV promoter produced some CAT activity, which was not significantly increased in the presence of the inducible agent i.e. dexamethasone. All of the H-BH transfection reactions exhibited greater CAT activity than the controls where the ESN construct was solely transfected.

This data confirmed Zuo et al., 1994 and yet did not show whether the mutant was capable of increasing the stable expression of the heat shock genes. This may not occur for example if the stability of the hsp mRNAs are under regulation by other mechanisms and hence although transcription may be stimulated, translation or post- translational modification to produce functional hsps may be under separate endogenous control. Insertion of the H-BH mutant into the pR19 construct, and

227 subsequent purification of an H-BH expressing recombinant HSV-1 vector would provide a means of characterising any changes in the expression profiles of the various hsps under the influence of activated HSFl. Infection of cells with a recombinant HSV-1 vector overexpressing H-BH may also confer protection against cellular insult.

228 Figure 5.5.2 - Stimulation of the Human HSP70B Promoter by Transient Over expression of the HSFl Mutant H-BH a) Plasmid maps of pJ5 H-BH, pJ7 H-BH and the LSN HSP70B promoter/CAT plasmid. For details please refer to text in Section 5.5.2. b) Autoradiograph of chloramphenicol acetyltransferase (CAT) activity as a result of CAT expression from the LSN plasmid. For details please refer to the text in Section 5.5.2. Lower bands represent [^"^C]-chloramphenicol, middle and upper bands represent mono- and di-acetylated [^"^C]-chloran^henicol, respectively. Transfections and conditions were as follows:

Lane 1: LSN^J7 H-BH+Dexamethasone Lane 2: LSN+pJ7 H-BH Lane 3: LSN+pJ5 H-BH+Dexamethasone Lane 4: LSN+pJ5 H-BH Lane 5: LSN+Dexamethasone Lane 6: LSN

229 Figure 5.5.2 a)

6200 IIP

5V4U T pol^A SftG

Htvttan HSr 7ü Piwnalei

b) 1

230 5.5.3 Construction of an H-BH Expressing 17+pR19 Recombinant HSV-1 Vector

The H-BH transgene was inserted in the pR19 expression plasmid as follows. The i/m dm (5’) and Xho\ (3’) fragment of pAElHSF {vide supra) containing the H-BH mutant cDNA was then inserted into pNot3.5cDNA3 as above. Correct insertion was confirmed by co-digestion with HinôSl and Xho\ and the plasmid named pR19 HSF. pR19 HSF was subsequently recombined into 17-HpR19 lacZ viral DNA without linearisation and purified in a similar manner to the pR19 HSP vectors {vide supra). The resultant ICP27-deleted H-BH expressing HSV-1 recombinant vector was named 17+pR19 HSF (see Figure 5.3.2).

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

5.6.1 Characterisation of Heat Shock Factor Expression

B 130/2, BHK and ND7 cell lines were infected with the 17+pR19 HSF recombinant HSV-1 vector, the proteins extracted, denatured, separated by SDS-PAGE and blotted onto nitrocellulose as in Section 4.5.2. H-BH protein was detected using the polyclonal anti-human HSFl antibody (Stressgen, Canada) see Figure 5.6.1.

The X-ray films demonstrate that aU the cell lines infected with the 17-iq)R19 HSF recombinant HSV-1 vector overexpressed an immunoreactive protein at approximately 83kDa, the expected molecular weight of denatured HSFl. Low-level constitutive expression of HSFl in the mock infected and the 17-HpR19 lacZ infected control cell lanes was detectable in the ND7 and BHK but not the B 130/2 cell extracts at the exposure times of these X-ray films.

231 Figure 5.6.1 - Characterisation of Expression of the H-BH Gene Product in B130/2, BHK and ND7 Cell Lines Infected with the 17+pR19 HSF Recombinant HSV-1 Vector

Western blots demonstrating overexpression of the human HSFl antibody immunospecific mutant H-BH protein at 83kDa in a) B 130/2 cells b) BHK cells and c) ND7 cells infected with the 17+pR19 HSF recombinant HSV-1 vector. Numbers down the sides indicate molecular weights in kDa. For details please refer to the text in Figure 5.6.1. ECL exposure time was less than 1 minute in all cases.

For blot a): For blots b) and c) Lane 1: Rainbow Marker Lane 1: Rainbow Marker Lane 2: Mock-infected B 130/2 cells Lane 2: Mock infected cells Lane 3: 17+pR19 lacZ infected B130/2 cells Lane 3: 17+pR19 lacZ infected cells Lane 4: 17+pR19 70 infected B 130/2 cells Lane 4: 17+pR19 HSF infected cells Lane 5: 17+pR19 90 infected B 130/2 cells Lane 6; 17+pR19 HSF infected B 130/2 cells

232 Figure 5.6.1 a) 1 2 3 4 5 6 b) 1 2 3 4

-220 I

-9 7 e 9 7 - , -66 t i 66-1 *

-4 7 4 7 - K

e) 1 2 3 4

220 - ^

9 7 - »

66 -

4 7 -

3 0 -

233 These results show that H-BH is over-expressed following viral infection but do not demonstrate whether the overexpressed H-BH gene is producing a functional protein that will transactivate heat shock gene expression in cells in the absence of stress. Therefore the expression of the individual hsps as a result of H-BH overe?q)ression was characterised.

5.6.2 Characterisation of Heat Shock Protein Expression

The intended purpose of the 17-HpR19 HSF recombinant HSV-1 vector, and the other vectors that were made, was for delivery to the ND7 neuron-derived cell line, rat primary DRG neuronal cultures, and to the rat brain in vivo. As the above results show that this vector overexpresses H-BH in ND7 cells, the heat shock protein expression profile of ND7 cells infected with the vector was assayed. The same methods were employed as above in cellular protein and nitrocellulose blot preparation. The blots were incubated with the hsp70, hsp90, hsp56 and hsp25 antibodies detailed above and additionally with a polyclonal anti-rat haemoxygenase-1 (hsp32) antibody (Stressgen, Canada) and a monoclonal anti-human hsp60 antibody (clone LK-1, Stressgen, Canada).

Figure 5.6.2 is a graphical representation of the data compiled jfrom densitometry scans (see Section 2.5.7) showing the levels of five of these immunospecific heat shock proteins in ND7 cells infected with the 17+pR19 HSF recombinant HSV-1 vector, in conq)arison to mock infected ND7 cells and also ND7 cells infected with the 17+pR19 lacZ recombinant HSV-1 vector. The hsp56 graph is absent because no constitutive or inducible bands were detectable.

The densitometry data is represented as a proportion of the actin band density, visible on the Coomassie blue stained gel. The protein loading, according to the Coomassie stained gel, was lower in the lane containing protein firom the cells infected with the lacZ expressing virus, and lower still in the lane containing protein firom the cells infected with the H-BH expressing virus. Using con^arative densitometry with the actin band on the Coomassie stained gel, this decline in loading is taken into account.

234 The blot incubated with the anti-hsp70 antibody clearly demonstrates an increase in hsp 70 levels in ND7 cells infected with the H-BH expressing recombinant virus. The apparent levels of hsp70 protein in the mock infected and 17+pR19 lacZ infected ND7 lanes are representative of background on the blot, detected by the densitometer, however no constitutive levels of hsp70 were visible.

Incubation with the anti-hsp25 antibody reveals a faint immunospecific band at 27kDa that was only present in the cells infected with the 17+pR19 HSF virus, no band was visible in the other lanes at this level and these columns are representative of background on the blot.

The anti-hsp32 detects a higher level of immunospecific protein at the 32kDa level in the ND7 cells infected with the H-BH expressing virus than both the 17+pR19 lacZ infected ND7 cells, and the mock-infected ND7 cells (3.5- and 1.7-fold, respectively).

The anti-hsp90 and anti-hsp60 antibodies did not reveal any substantial change in the their respective heat shock proteins against the constitutive levels in the control lanes. It appears that the levels of these protems may even decrease shghtly on H-BH overexpression.

Even after a long exposure (5 minutes) by which time the non-specific background levels had developed no constitutive or H-BH inducible levels of hsp 5 6 were visible on the film (data not shown).

235 Figure 5.6.2 - Characterisation of Heat Shock Protein Expression in ND7 Cells Infected with the 17+pR19 HSF Recombinant HSV-1 Vector

Densitometry scans of western blot of protein extracted from ND7 cells infected with the 17-HpR19 HSF recombinant HSV-1 vector. Data is expressed as a proportion of the actin band density on a Coomassie stained gel of similar loading. For details of method and antibodies used please refer to the text in Section 5.6.2. Cells were infected as follows: Mock infected ND7 cells 17+pR19 lacZ infected ND7 cells 17+pR19 HSF infected ND7 cells

Primary antibodies incubated on blot: a) anti-hsp25 b) anti-hsp32 c) anti-hsp60 d) anti-hsp70 e) anti-hsp90

236 Figure 5.6.2a) Probed with anti-hsp27 antibody

M 1.4

u g 0.8

ND7 Control 17+pR19 lacZ 17+PR19 HSF

Figure 5.6.2b) Probed with anti-hsp32 antibody 6

ND7 Control 17+pR19 lacZ 17+PR19 HSF

237 Figure 5.6.2c) Probed with anti-hspSO antibody

25

to 10

ND7 Control 17+pR19 lacZ 17+pRIO HSF

Figure 5.6.2d) Probed with anti-hsp70 antibody

25

ND7 Control 17+pR19 lacZ 17+PR19 HSF

238 Figure 5.6.2e) Probed with anti-hsp90 antibody

» 30

ND7 Control 17+pR19 lacZ 17+pR19 HSF

239 5.6.3 Discussion

This is the first study that demonstrates the stable expression of heat shock proteins on overexpression of the HSFl mutant, H-BH. Endogenous factors do not appear to prevent the overexpression of at least some of hsps triggered by the transactivation pathway of HSFl.

These results show differing degrees of increase in the levels of hsp70, hsp27 and hsp32 in ND7 cells when infected with the H-BH expressing 17-^pR19 HSF recombinant HSV-1 vector. Hsp27 and hsp32 were increased to a much lesser degree than hsp70. This difference in overexpression may he cell-type dependent. Blot 3 in Figure 5.4.2a shows a marked overexpression of hsp70 m B 130/2 cells infected with the 17+pR19 HSF virus. However, in BHK cells in Blot 3, Figure 5.4.2b, the level of overexpression of hsp70 and hsp27 is almost as great as cells infected with the 17+pR19 70 and 17+pR19 27 viruses respectively. Hsp32 overexpression was mild in all three cell types (data not shown). No significant increases were noted in hsp60, hsp90 and hsp56 in any of the cell types tested. In ND7 cells, constitutive immunospecific bands were detected on the hsp60 and hsp90 blots, whereas, even during long exposure times on chemiluminescence, no constitutive immunospecific hsp56 bands were detected (data not shown). The hsp56 antibody used was specific for rabbit hsp 5 6 and therefore bound with high afiOnity to the rabbit hsp56 expressed by the 17+pR19 56 recombinant HSV-1 vector. This antibody is reported to react with human, primate, calf and mink cell lines but (being a monoclonal antibody) may not react with rat, or hamster (as in BHK cell line) hsp 56 despite the high sequence similarities characteristic of the heat shock proteins throughout evolution.

These blots do not prove that this mutant HSFl species is acting at the heat shock element to stimulate transcription, however hsp70 and 27 have been shown in Chapter Three of this thesis and in previous work, to be highly inducible in response to stress. Along with the hsp32 gene these hsp genes may therefore be more sensitive to activated HSFl stimulation, more so than other stress genes, which may be more dependent on interactions between other endogenous factors.

240 It has previously been reported that increased HSFl binding occurs in cells of ischaemic cortex during focal cerebral ischaemia (Higashi et al., 1995). This study did not demonstrate however, that the HSFl is activated, or that it is the transcription factor driving the overexpression of the heat shock proteins reported in similar ischaemic experiments (Higashi et at., 1994), and Chapter Three of this thesis. If HSFl remains functional in the ischaemic brain, then the prospects of in vivo overexpression of hsps by viral gene delivery of the H-BH mutant both prior to, and during ischaemic stress are encouraging.

The overexpression of hsp70 has been shown to protect both primary neuronal cultures as well as neuron-derived cell lines against cell death in response to ‘lethal’ heat shock, glutamate toxicity and simulated ischaemia, but not against ap opto sis (Amin et al., 1996; Uney et al., 1993; Rordorf et al., 1991; Wood et al., 1990). The protective effect of overexpression of hsp27 and hsp32 in such cultures has not been studied. Hsp27 overe?q)ression, however, has been demonstrated to confer thermoresistance to non-neuronal mammalian cells and also to protect against Fas/APO-1 stimulated ap opto sis in a murine fibrosarcoma cell line (Mehlen et al., 1996; Landry et al., 1989). Hsp27 has also been demonstrated in vivo to be overexpressed in response to a sub- lethal ischaemic pre-treatment in the rat brain in models of ischaemic tolerance (see Section 1.2) particularly in the gha in the sub-regions of the hippocampus, however the pattern of overexpression was in different locations to those associated with survival in response to ischaemic preconditioning (Kato et al., 1994). Hsp32 has been demonstrated to be overexpressed in the rat brain in response to both global and focal ischaemic insults, which may serve to augment the cytopathic oxidative stress caused by ischaemia (Nimura et al., 1996; Geddes et al., 1996; Takeda et al., 1996).

The 17-HpR19 HSF recombinant HSV-1 vector may, in stimulating the overexpression of hsp27, hsp32 and hsp70 to differing degrees, protect cells from insult both in vitro and in vivo. In controlling the stress response at the transcriptional level, overexpression of H-BH may simulate an endogenous stress response, providing the appropriate balance of stress proteins. One can speculate, therefore that this vector

241 may be more suited for cell survival than the substantial overexpression of individual hsps by their respective viruses. This study is limited, however, it examines the levels of only a few heat shock proteins whereas activated HSFl may affect the transcriptional control of many more genes that may result in a beneficial or an adverse effect on the cell.

5.7 The 17+pM1 Bicistronic Recombinant HSV-1 Vector

5.7.1 Introduction - Vector Design

The 17+pR19 recombinant HSV-1 vectors successfully express their transgenes in cells in vitro. However, the incorporation of an additional reporter gene such as lacZ or GFP into the pR19 expression would enable the identification of virally infected cells by staining with X-gal or visuahsation under fluorescein optics, respectively.

Expressing two transgenes under two different promoters in one vector as in the 17+pR16R recombinant HSV-1 vectors has its disadvantages: 1) The expression levels of the transgenes would be different if under the control of different promoters. It would be useful if the level of expression of the reporter gene could mimic that of the desired transgene for delivery. 2) Even if the reporter gene was transcribed in infected cells, this did not necessarily indicate transcription of the delivered transgene. The transgene promoter activity may be repressed by cellular factors. 3) In the context of the pR19 expression construct, transcription of one gene may be enhanced during viral latency through its proximity with the LAT PI and LAT P2 promoter regions whereas the second gene, being distal to this region, may not benefit fiom this enhancement. 4) Addition of an extra promoter increases the size of the expression construct which results in a lower frequency in recombination into HSV-1 genomic DNA.

These problems were addressed and solved by creating a bicistronic expression construct based on pR19, where the two transgenes were separated with an

242 encephalomyocarditis virus (EMCV) internal ribosome entry site (1RES), and situated downstream of the CMV-IE promoter in the pR19 backbone. A single transcript containing the mRNA sequences of both transgenes would thus be driven by the CMV- IE promoter downstream of the LAT PI and P2 promoters, through to the pcDNA3 BGH polyadenylation sequence. The 1RES provides sites for internal binding of ribosomes which directs cap-independent translation of the downstream gene transcript. (Hiattas et aL, have shown through biochemical and immunohistochemical assays that the expression of lacZ and CAT genes separated by an 1RES is more effective than a CMV-IE/LTR double promoter in expressing the two genes from a single retroviral vector (Ghattas et aL, 1991).

This bicistronic pR19 expression construct was made expressing GFP and lacZ to investigate the future possibihty of using 1RES systems in recombinant HSV-1 vector gene delivery. The construct was named pMl and when incorporated into the viral genome and purified, was named 17+pMl.

5.7.2 pM1 Plasmid and 17+pM1 Recombinant HSV-1 Construction a) Construction of Plasmid The EGFP gene was excised from pEGFPNl by digestion with 7/mdin (5’) and Notl (3’) and Hgated between the //mdlQ (5’) and BstXl (3’) sites of pNot3.5cDNA3 (see Section 5.3.1). Successful hgation was confirmed using Xhol and HindiHi and the plasmid named pR19R GFP.

The EMCV 1RES sequence was excised from pCITE-1 (kindly donated by Dr. Jeffrey Almond, University of Reading) by digestion with EcoRI (5’) and Ncol (3’) and blunt- end hgated into the Sma\ site ofpJ4 lacZ (5’ to the lacZ gene see Section 4.2.2a). Use of these sites ensured that the 1RES sequence was in frame with the downstream lacZ start codon. Correct insertion and orientation was confirmed by co-digestion with Ball and Sstl and the plasmid named pJ4 1RES lacZ (see Figure 5.7.2).

243 The 1RES lacZ cassette was excised from pJ4 1RES lacZ by digestion with BamYÜ and blunt-end hgated into the Xbal site of pR19R GFP. Correct insertion and orientation was confirmed using separate digestions of//m dlQ and EcoRI and the plasmid named pM l (see Figure 5.7.2). b) Recombination of pMl into HSV-1 DNA and Purification of the 17+pMl Recombinant HSV-1 Vector. pMl was co-transfected unlinearised and recombined into the 17-t^R16R-derived IE2 deleted ‘white’ viral DNA, and purified by staining infected B 130/2 IE2 complementing cells with X-gal and picking blue plaques. As the lacZ gene is situated downstream of the 1RES sequence, it was already evident that the 1RES was fimctioning properly.

244 Figure 5.7.2 - Construction of the pMl Plasmid

Diagiam depicting the pathway by which pMl was constmcted fiom the pJ4 1RES lacZ and pR19R GFP plasmids. For full details of restriction digests and ligations, please refer to the text in Section 5.7.2.

Briefly, EMCV IRES//acZ cassette was excised fiom pJ4 ERES lacZ (top left) and inserted downstream of the GFP gene in pR19R GFP (top right) to make pMl (bottom middle).

Tlie ethidium bromide stained gel below shows confinnatoi'y restriction digests of these plasmids. See plasmid maps opposite for restriction site locations.

Lane 1: 1 kb ladder Lane 2: pJ4 1RES lacZ co-digested with Ball and Xba\ (band sizes - 0.15kb, 0.6kb, 7.1 kb) Lane 3: pR19R GFP co-digested with Xhol and HinàlW (band sizes - O.Skb, 7.3kb) Lane 4; pMl digested with H M lll (band sizes - Ikb, 11.5kb) Lane 5: pMl digested with EcoKl (band sizes - 1.4kb, 3.2kb, 7.8kb) Lane 6: Ikb ladder

Ikb Ladder Fragment Sizes

10kb_ 10000

6000 5000 4000 3500 3000 2500 2000 1500 Ikb —, 1000

500

245 Figure 5,7.2

X hal250 Xbal 400

EMCVIRTS

ilacZ p R I O R G fP pJ4IRESIacZ 0 1 5 4 b p A m p 7895 bp

CMV-IE

BGH poly A

X h o l 3 8 4 5

f1 ori Amp lacZ

Not3.5 lacZ

pM1 CMV IE EcoRl 3045 12454 bp Not3.5 Hindlll 3045 GFP

BGH poly A site ECMVIRES

Hindlll 4045

lacZ EcoRI 4445 EcoRl 7645

246 5.7.3 Characterisation of GFP and lacZ Expression of the 17+pM1 Recombinant HSV-1 Vector

Once pure, the virus was titrated by 1:10 serial dilution onto a 6 well plate of B130/2 cells, and cultured for 48 hours (see Section 2.6.5). All plaques fluoresced green under UV hght using fluorescein optics and selected plaques were photographed (see Figure 5.7.3a). The cells were subsequently fixed with glutaraldehyde and stained overnight with X-gal (see Section 2.6.6). All the plaques stained blue, indicating the presence of functional p-galactosidase and the previously selected plaques were photographed (see Figure 5.7.3b).

The intensity of GFP fluorescence in 17+pMl infected B 130/2 cells was similar to that of similar cells infected with 17+pR19 GFP for a similar time period. This is to be expected as in both cases the mRNA transcripts are translated fi'om the 5’ cap AUG sequence.

The intensity of the blue stain in the B 130/2 cells infected with 17+pMl, representing P-galactosidase activity, was quahtatively lower than that seen in similar cells infected with 17+pR19 lacZ for a similar time period. It can therefore be concluded that the 3’ transgene (lacZ) is not expressed as efficiently as the 5’ transgene (GFP). Cap- independent translation is not as efficient as cap dependent translation, and this may account for the difference (Ho et a/., 1995a). The cells were more intensely stained than those infected in a similar manner with 17+pR16R 70. Therefore, the 1RES system under the control of the CMV-IE promoter downstream of the LAT promoters in the two HSV-1 RL regions is more efficient at transgene expression than a single LAT Pl-/flcZ system recombined into the ICP27 region of the HSV-1 genome. This may be attributable to the site of insertion, the strength of the promoters, the number of transgenes per viral genome (two in 17+pMl, one in 17+pR16R 70) or combinations of aU of these factors. As a LAT PI lacZ construct has not been recombined into the same sites as the pMl construct, these questions cannot be answered here, and would be a suitable control experiment. For the purposes of this

247 thesis, however, when the 17+pR19 recombinant HSV-1 vectors produced suitable transgene expression for delivery to ND7 cells, this avenue was not pursued.

248 Figure 5.7.3 - Detection of Reporter Gene Product Activity in B130/2 Cells Infected with Recombinant HSV-1 Vectors

Photomicrographs at lOOx magnification of B130/2 cells infected with: a), b) the 17+pMl recombinant HSV-1 vector c) the 17-i^R19 lacZ recombinant HSV-1 vector d) the 17+pR16R 70 recombinant HSV-1 vector e) the 17-HpR19 GFP recombinant HSV-1 vector

Cells in a), c) and d) were fixed and stained overnight for P-galactosidase activity. Cells in b) and e) were viewed under UV hght using fluorescein optics for detection of GFP expression. The same viral plaque is visuahsed in photomicrographs a) and b), indicating co­ expression of both the lacZ and GFP reporter genes. For details please refer to the text in Section 5.7.3.

249 After examination of the manuscript, on recommendation by the examiners, the author would like to include the following statement regarding the 17+pMl virus.

No quantitation of (3-galactosidase or green fluorescent protein (GFP) expression was carried out on B 130/2 cells infected with 17+pMl, 17+pR19 GFP, or 17+pR19 lacZ. Although qualitatively, under the descibed visualisation techniques (either under fluorescein optics [GFP], or by staining with X-gal [p-galactosidase]) the expression of both cistrons lying upstream and downstream of the 1RES in 17+pMl was at a lower level than the individual transgenes in the 17+pR19 lacZ and 17+pR19 GFP, this result cannot be confirmed without additional verification by comparative quantitation for example by galacto-light assay (p-galactosidase) or western blot (GFP)

b)

250 C)

d)

e)

251 5.7.4 Discussion - The Potential Use of Bicistronic Vectors in Recombinant HSV-1-Mediated Transgene Delivery

The purpose of constructing and characterising the 17+pMl recombinant HSV-1 vector was to investigate the potential of bicistronic constructs in recombinant HSV-1 vector mediated gene delivery. This is the first such study of bicistronic constructs in recombinant HSV-1 vectors.

Bicistronic vectors have previously been used in amphcpn vectors to express both the glucose transporter gene and the lacZ gene in cultured neurons to protect against necrotic cell death (Ho et al., 1995b; Ho et a i, 1995a). The authors also noted that the second cistron is expressed at a lower level than the first, however this study demonstrates that the first cistron is expressed at a lower level than when the 1RES and second cistron are absent (conq)arison between GFP expression in 17+pMl and pR19 GFP). This difference in expression may be due to unstable sequences in the considerably longer mRNA (5100bp as opposed to SOObp) or to the proximity of the BGH polyadenylation sequence to the GFP gene in 17+pR19 GFP. In either case, translation of the second cistron implies transcription of the first, and therefore if the second cistron is a reporter gene and the first is a gene of interest, then reporter gene product activity subsequent to infection will indicate the presence of the transcribed form of the first cistron. This elimination of the potential false positives and negatives that may occur when the two genes are under the control of separate promoters suggests the use of this system in future strategies for recombinant HSV-1 vector transgene delivery.

5.8 Discussion

The results presented in this chapter demonstrate the successfid construction of recombinant HSV-1 gene delivery vectors expressing lacZ, GFP, hsp27, hsp56, hsp70 and the constitutively active mutant of HSFl, H-BH, at the protein level. All of these vectors overexpress their transgenes in B 130/2, BHK and ND7 cell lines above the levels of constitutive expression. The expression of hsp70 in 17+pR19 70 infected B 130/2 ceUs was considerably greater than that in similar cells infected with similar

252 titres of 17+pR16R 70. Therefore, the 17+pR19-based recombinant HSV-1 vectors were the chosen vectors for the characterisation of the potential protective effects of overe?q)ressmg the hsps in cells prior to a range of insults (see Chapter 6). This increase in overexpression may be due to all or a combination of five reasons: 1) The CMV-IE promoter drives greater transcription than the MoMLV-LTR 2) The recombination of the promoter/transgene downstream of the LAT PI and LAT P2 promoters may confer increased transcriptional activity. 3) The recombination of the promoter/transgene directly into the LAT region may confer increased transcriptional activity through the action of upstream and downstream regulatory elements. 4) Each 17-HpR19-based vector contains two copies of the promoter/transgene construct in each RL region, whereas the 17+pR16R-based plasmids recombine into the sole UL region of the HSV-1 genome. 5) The homologous recombination of the LAT regions and subsequent ‘knocking out’ of the transgene during 17+pR16R 70 repUcation will decrease the yield of recombinant virus expressing the transgene, as conq)ared to the 17+pRl9-based vectors, where such recombination was not detected.

Although the hsp90 cDNA was successfiiUy delivered to B 130/2 cells using the 17+pR16R 90 recombinant HSV-1 vector, overexpression could not be detected. This may have been due to a non-fimctional cDNA sequence, masking of MoMLV-LTR and CMV-IE driven overexpression by the already high (~1% of total cellular protein) constitutive levels of hsp90, or endogenous mechanisms e.g. translation repression inhibiting further expression of the protein. An hsp90 cDNA f^P] radiolabelled northern blot and a nuclear run-on assay of virally infected cells against mock-infected and control virus infected cells would show whether hsp90 mRNA levels and transcription were increased. However, without evidence of significant overexpression of hsp90 protein, any fimctional effects that the 17+pR16R 90 or 17+pR19 90 vectors may confer on cell lines cannot be justly attributed to the overexpression of hsp90, therefore further investigations were not performed.

253 The expression of lacZ was considerably greater in cells infected with the 17+pR19 lacZ recombinant HSV-1 vector than either of the previously constructed 174^R16R 70 or 17+pR16R 90 viruses. These viruses are not directly comparable as the transgenes are under the control of different promoters and inserted in different sites of the HSV-1 genome. The same reasons as those stated above may be responsible for the observed differences in expression excepting that the LAT PI promoter is used to drive lacZ transcription in the 17+pR16R viruses instead of the MoMLV-LTR.

This study demonstrates that GFP is effectively expressed in recombinant HSV-1 vectors, and can be used for purification and subsequent detection of virally infected cells. Use of the reporter gene encoding GFP is advantageous to lacZ because of its small size (~800bp, compared to ~3700bp) which not only aids homologous recombination but also increases the remaining available space in the HSV-1 genome for the insertion of large transgenes or more than one transgene. X-gal buffer is toxic to the cell, and the longer the time taken for the blue product of X-gal to appear decreases the viral yield. GFP does not require staining, the protein product being directly visible under UV hght using fluorescein optics, and experience gained through the work described in this chapter has shown that re-infection is more successful on selection of ‘green’ GFP expressing plaques than ‘blue’ (3-galactosidase expressing plaques (data not shown). Any toxic effect of exposure of the cells and virus to this degree of UV hght is therefore outweighed by the negative effects of X-gal buffer. The gene encoding GFP is therefore a putatively better reporter gene for detection of viral infection than lacZ.

This study also demonstrates that HSV-1 vector-mediated overexpression of the constitutively active H-BH deletion mutant of the human HSFl transcription factor detectably increases the levels of the heat shock proteins 27, 32 and 70 in the absence of exogenous cell stress, in all cell lines infected (B130/2 and BFfK data not specifically shown). The levels of hsp56, hsp60 and hsp90 however, were not detectably altered. As discussed above, this 17+pR19 HSF recombinant HSV-1 vector may therefore be effective in protecting neurons in vitro and in vivo against stress, through simulation of a stress response during an unstressed state.

254 The final study in this chapter is the first to demonstrate the effective expression of two genes separated by an EMCV-IRES in a recombinant HSV-1 viral vector. Despite the lower expression of both reporter genes compared to their 17+pR19 counterparts, this system may have value in indication of transcription of the first cistron (gene of interest) in infected cells through cap-independent translation of the second cistron (reporter gene) under the influence of the ECMV-IRES, and therefore may be usefifl in designing fixture recombinant HSV-1 vectors.

255 Chapter 6

The Neuroprotective Effect of the Heat Shock Proteins in vitro

256 After examination of the manuscript, on recommendation by the examiners, the author would like to include the following statement regarding the experiments described in this chapter.

The work described in this chapter was carried out towards the end of the term of laboratory study, and it is recognised that the value for n (2) for the DRG experiments are not sufficient to draw any firm conclusions. These therefore represent preliminary studies which require repetition with a greater value for n to support the hypotheses drawn in the discussion of this chapter. Additionally, the correct model for statistical comparison between groups would be a one or two way analysis of variance followed by a post-hoc comparison with a test such as the Fisher protected least significant difference or Scheffes test. 6.1 Introduction

This Chapter reports on the protective effect of overexpression by recombinant HSV-1 delivery of three heat shock proteins (hsp27, hsp56 and hsp70), and a constitutively active mutant p f HSFl (H-BH), in a neuron-derived cell line and primary neuronal cells in vitro against a variety of insults. The heat shock and H-BH transgenes were delivered to the cells using the 17+pR19 recombinant HSV-1 vectors described in Chapter Five of this thesis.

The ND7 cell line is one of a series of cell lines prepared by fusing non-dividing rat DRGs and the N18 mouse neuroblastoma cell line, and selecting for the drug resistance of the ganglion cells (Wood et a/., 1990). The cell line proliferates indefinitely in culture and retains many characteristics of the ganglion cells not noted in the neuroblastoma line. Their ability to proliferate enables multiple experiments to be performed on large populations of cells and hence reliable assessment of significance in assessing protection fi'om cell death. When ND7 cells are cultured in the absence of foetal bovine serum they undergo either differentiation into a mature neuronal phenotype, extending dendrite-like processes, or programmed cell death (apoptosis) (Howard et al., 1993). The proportion of cells that undergo apoptosis can be increased in the presence of IpM dM-trans retinoic acid. Therefore, these cells can be used to assess the protective effects of overexpression of the heat shock and H-BH transgenes against apoptosis in neuron-derived cells.

However, it is necessary to investigate the protective effect observed in ND7 cells in real neurons. Rat DRG neurons in primary culture make this possible in vitro. Neonatal rat DRG neurons can be stimulated to undergo programmed cell death by withdrawal of nerve growth factor (NGF) from the culture medium (Mailhos et at., 1994; Hamburger et at., 1981), and therefore the protective effect of the hsps against apoptosis can be assessed in primary cultures also. Tentative comparisons can be made with apoptosis in ND7 cells on making the assun^tion that in both populations with separate stimulatory protocols the pathways to apoptosis, and the protective mechanisms (if any) of the hsps, are similar. DRG neurons are non-dividing and can

257 not be cultured in high numbers, and populations are not homogeneous in the way that ND7 cells are, therefore quantitative results can be more erratic. Therefore combining the advantages of neuron-derived cell lines and primary neuronal cultures by performing similar e^qperiments on both enables rehable assessment of effect.

Previous work has investigated the protective effects of transfection-mediated overexpression of hsp70 and hsp90 in ND7 cells and primary neuronal cultures. Hsp70 protects against a 46®C-48°C heat shock in both cultures, whereas hsp90 only protects ND7 cells (Mailhos et al., 1994; Uney et al., 1993; Wyatt et al., 1996; Amin et al., 1996). Hsp70 but not hsp90 protects ND7 cells and primary neurons (and cardiomyocytes (Mestril et al., 1994)), against simulated ischaemia (Amin et al., 1996). Although prior heat shock protects ND7 cells and dorsal root ganghon neurons from serum/NGF withdrawal-induced apoptosis, transfection-mediated overexpression of hsp70 or hsp90 does not confer protection to ND7 cells or trigeminal ganghon neurons (Mailhos et al., 1993; Mailhos et al., 1994; Wyatt et al., 1996). The protective effects of overexpression of hsp27, hsp56 and H-BH had not previously been studied in neuron-derived or primary neuronal cells. Indeed, prior to the work described in Chapter Five, no HSFl mutant had been demonstrated to overexpress hsps in the absence of stress. This study showed the combined overexpressions of hsp27, 32 and 70. The effect of overexpression of hsp56 on cells undergoing thermal, ischaemic or ap opt otic stress has not been tested, whereas hsp27 has been demonstrated to confer tolerance to both thermal and apoptotic stress in mammahan non-neuronal cell lines (Landry et al., 1989; Mehlen et al., 1996).

This study therefore has two purposes. Firstly, it serves to further investigate the effect of overexpressing the heat shock proteins and HSFl in ND7 and primary neonatal rat dorsal root ganghon cells during stress. Secondly, it examines the ef&cacy of the 17+pR19 (IE2-deleted) recombinant HSV-1 vector-mediated gene delivery to neuronal cells in vitro, in order to achieve a protective effect. These data are therefore the preliminary results required prior to introducing the recombinant 17-HpR19-based vectors to the rat brain in vivo, to examine the neuroprotective effects of the hsps during focal cerebral ischaemia.

258 6.2 Effect Of Overexpression hsp27, hsp56, hsp70 and H-BH in ND7 and DRG Ceiis on Heat Shock, ischaemia and Apoptosis

6.2.1 Heat Shock

ND7 heat shock assay conditions were determined by exposing ND7 cell cultures to 48°C for increasing durations prior to a subsequent 1 hour recovery period at 37°C/5%

CO2 Subsequently cell viabihty was quantitated by trypan blue exclusion assay, see Sections 2.7.1 and 2.8.1 (Mailhos et a i, 1994). Figure 6.2.1a shows that under similar conditions, a duration of heat stress between 20 and 40 mins should be appropriate to note changes in cell viabUity effected by overexpression of the heat shock and H-BH genes. It was thus decided to expose ND7 cells to 20 minutes heat shock at 48°C with 1 hour recovery prior to the trypan blue exclusion assay.

Figure 6.2.1a

100

75 5 ’E 3 50 (0

25

0 0 30 60 90 Time of Heat Stress (mins)

Figure 6.2.1a - Percentage Survival of ND7 cells After Varying Durations of 48"C Heat Stress The proportion of viable cells for three fields of view of a counting chamber was counted for each sample (n), and the mean calculated. The mean and standard deviation was calculated from the means of each triphcate (n). For 30 mins n=5, for aU other samples n=6. Error bars indicate standard deviations of the mean.

259 ND7 cells were subsequently infected at a multiplicity of infection (MOI) of lOpfii cell ^ with the 17-HpR19 recombinant HSV-1 vectors in 24-well plates as described in

Section 2.6.12. After overnight incubation at 37°C/5% CO 2 the cells were exposed to a 48®C heat shock for 20 mins followed by a recovery period of 1 hour. Control cells were incubated throughout at 37®C/5% CO 2 . Percentage cell survival was calculated using the trypan blue exclusion assay (see Section 2.8.1). Figure 6.2.1b shows the effect of overexpressing the separate transgenes on ND7 viabihty subsequent to lethal heat shock.

Dorsal root ganghon ceUs from neonatal (P2) Sprague Dawley rats were extracted and cultured overnight prior to iofection/mock-infection at similar amounts to the ND7 ceUs (see Section 2.6.12) with/without the 17+pR19-based viruses. After overnight incubation the ceUs were heat shocked at 48°C for 30 mins (Fink et al., 1997) and the proportion of healthy against unhealthy or dead neurons was identified under phase- contrast hght microscopy at 400x magnification at 12 and 24 hours recovery at

37°C/5% CO2 (see Section 2.8.3) (Wyatt et al., 1996). Phase bright, non-granular neurons were classed as healthy neurons. Granular neurons with ruffled membranes and lack of neurites were classed as unhealthy or dead (Wyatt et al., 1996). Non-heat shock control experiments were performed and no marked difference in survival was noted amongst individual virahy infected and control populations, the neurons remaining healthy throughout the timecourse examined (the lowest live/dead proportion at 24 hours after heat shock being 87%, data not shown). The results are ihustrated in Figure 6.2.1c.

260 Figure 6.2.1b No Heat Shock Heat Shock 90

80

70

60 w > 50 I + * W 40

30

20

10

0 (£> N 0 6 y 1 0 CM N (0 « G)G)G) X o> XXX o> Q. Q. CL q 5 + + + E Q. h - Q. + h-+

Figure 6.2.1b - ND7 Cell Survival Following Severe Heat Shock when Infected with Recombinant HSV-1 Vectors Expressing Heat Shock and H-BH Genes Black bars - Proportion of surviving mock-infected (ND7) and 17+pR19 virus infected

ND7 cells incubated at 37”C/5% CO2 throughout. Striped bars - Proportion of surviving mock-infected (ND7) and 17+pR19 virus infected ND7 cells after 20 mins heat shock at 48”C with 1 hour recovery at 37°C/5% CO2 EiTor bars represent standard deviations o f the means calculated from the means o f three counts for each sample (n). n for all experiments = 6. * - Significant difference in survival compared with mock-infected ND7 cells (p<0.01). + - Significant difference in survival compared with 17+pR19 lacZ-infected ND7 cells (p<0.01 ). Calculated using Student’s t-test.

261 Figure 6.2.1c

100 ^ ■ Mock-infected DRGs r f f k □ 17+PR19 lacZ 90 ^17+pR19 27 □ 17+PR19 56 E317+pR19 70 80 H17+PR19 HSF

60

50

40 Oh 12h 24h Time After Lethal Heat Shock (hours)

Figure 6.2.1c - Primary Rat DRG Neuron Survival Following Severe Heat Shock when Infected with Recombinant HSV-1 Vectors Expressing Heat Shock and H- BH Genes DRG neuronal cultures were infected/mock-infected with the 17+pR19-based vimses, incubated ovemiglit at 37°C/5% CO 2 prior to 30 minutes ‘lethal’ heat shock at 47‘’C. Tlie proportion of healthy against unhealthy neurons was assessed by the parameters described in the text prior to the insult (0 hours), and at 12 and 24 hours recovery at

37°C/5% CO2 after heat shock. Bars represent the means of the means of triplicate counts for each sample (n). n=2. Error bars represent the standard deviations of the sample means. Significance was not calculated due to the low value of n.

262 The ND7 graph shows significant differences (approximately two-fold) in the proportions of cells that survived the ‘lethal’ heat shock between the 17-HpR19 27-, 17+pR19 70- and 174^R19 HSF-infected ND7 cells versus the mock- and 17+pR19 lacZ-infected ND7 cells. It also reports that no significant difference was noted between the 17+pR19 56-infected ND7 cells versus the mock- and 17+pR19 lacZ- infected ND7 cells.

The primary cell data confirms these data, but also suggests that infection with the 17+pR19 56 HSV-1 recombinant vector may confer some protection against heat shock to cultured neurons. Considerably more of the mock- and 17+pR19 lacZ- infected neurons were visually unhealthy or dead at both timep oints as the result of the ‘lethal’ heat shock, the greatest difference being visible at the 24 hours timepoint.

It can be concluded therefore that infection with the recombinant HSV-1 vectors overexpressing hsp27, hsp70 and H-BH protects ND7 cells and cultured neuions from severe heat stress. It can also be concluded from the non-heat shocked controls that none of the recombinant HSV-1 vectors were notably cytopathic to ND7 cells or cultured neurons during the time period examined.

263 6.2.2 Simulated Ischaemia

Ischaemia can be simulated in vitro by incubating the cells in a physiological buffer containing raised levels of lactic acid, high potassium and decreased pH (Esumi et al., 1991). For method details, please refer to Section 2.7.2. ND7 and rat DRG cells (as in Section 6.2.1) were cultured in a 24-well plate and mock-infected or infected with the 17+pR19 viruses as in Section 2.6.12, 24 hours prior to ischaemia. The cells were incubated for 4 hours at 37°C/5% CO2 in either control buffer (pH 7.4) or ischaemic buffer (as control buffer except pH 6.2, 20mM sodium lactate, 12mM KCl), and then survival was either assessed immediately using the trypan blue exclusion assay (ND7 cells, see Section 2.8.1) or by visuahsation after 12 and 24 hours of healthy neurons by hght microscopy (DRG neurons, see Section 2.8.3). The results can be seen in Figure

6.2.2.

Primary DRG neurons were also cultured in control buffer (pH7.4) and no marked difference in survival was noted amongst individual virally- and mock-infected populations (data not shown). Although the control buffer was toxic compared to the non-heat shock controls (described in Section 2.6.1) the decrease in surviving populations was approximately one quarter that of the populations that were cultured in the ischaemic buffer, demonstrating the cytopathic effect was as the result of the additional components in the ischaemic buffer.

264 Control Buffer Figure 6.2.2a Ischaemic Buffer

95

90 + *

85

80

■(5 > 75 I W 70

65

60

55

50 ND7 17+PR19 17+pR19 17+pR19 17+pR19 17+pR19 LacZ 56 27 70 HSF

Figure 6.2.2a - ND7 CeU Survival Following Simulated Ischaemia when Infected with Recombinant HSV-1 Vectors Expressing Heat Shock and H-BH Genes Black bars - Proportion of surviving mock-infected (ND7) and 17+pR19 virus infected

ND7 cells incubated 37”C/5% CO2 in control buffer for 4 hours. Striped bars - Proportion of surviving mock-infected (ND7) and 17+pR19 virus infected ND7 cells after 4 hours incubation in ischaemic buffer at 37°C/5% CO2 Error bars represent standard deviations of the means calculated from the means of three counts for each sample (n). n for all experiments = 6.

* - Significant difference in survival compared with mock-infected ND7 cells (p<0.02). + - Significant difference in survival compared with 17+pR19 lacZ-infected ND7 cells (p<0.01). Significance calculated using Student’s t-test.

265 Figure 6.2.2b

100 ■ Mock-infected DRGs 90 □ 17+pRie lacZ ^17+pR19 27 80 □ 17+pR19 56 70 4 m i7+pR19 70 B17+PR19 HSF

m 60 ; > I 50 co ^ 40

30

20 j

10

0 12 24 Time Following Simulated Ischaemia (hours)

Figure 6.2.2b - Primary Rat DRG Neuron Survival Following Simulated Ischaemia when infected with Recombinant HSV-1 Vectors Expressing Heat Shock and H-BH Genes DRG neuronal cultures were infected/mock-infected with the 17+pR19-based viruses, incubated overnight at 37°C/5% CO 2 prior to 4 hours of simulated ischaemia. Tlie proportion of healthy against unhealthy neurons was assessed by the parameters described in Section 6.2.1 prior to the insult (0 hours), and at 12 and 24 hours recovery at 37°C/5% CO 2 in complete media after the ischaemic insult. Bars represent the means of the means of triphcate counts for each sample (n). n=2. EiTor bars represent the standard deviations of the sample means. Significance was not calculated due to the low value of n.

266 Figure 6.2.2a demonstrates that ND7 cells are significantly protected from simulated ischaemia when pre-treated by infection with 17+pR19 viruses overexpressing hsp27, hsp70 and H-BH, but not when infected with 17+pR19 lacZ or 17+pR19 56. As noted in the heat shock experiments, none of the viruses appear cytopathic to ND7 cells over the time period of infection prior to assay (28 hours). Approximately 20% more of the cells survived in all the protected samples, compared to the non-protected, and remarkably the degree of protection of cells infected with the 17-HpR19 27 virus is sufficient to totally block the toxic effects of the ischaemic buffer when con^ared with the control buffer results. The control buffer itself does not appear to be toxic to ND7 cells over four hours, with approximately 10-15% of cells dying, a similar proportion that was noted in the non-heat shock samples in Section 6.2.1. It is of interest that the same viruses that protect the cells from severe heat stress also protect against ischaemic stress.

Figure 6.2.2b confirms that this protective effect can also be shown in primary cultured rat neurons. At both 12 and 24 hour timepoints the 17+pR19 27, 70 and HSF infected cultures are markedly protected against cell death following simulated ischaemia. The most dramatic difference between these cultures and the mock- and 17+pR19 lacZ- infected controls was noted at 12 hours post-insult. Neurons infected with the 17+pR19 56 virus may be protected against ischaemia to some degree at 12 hours, but this is to a lesser extent to those infected with viruses expressing the other heat shock transgenes. The protective effect of overexpressing hsp27 also appears to weaken at 24 hours post-ischaemia, whereas the most marked protection at this timepoint is noted in cultures infected with the viruses expressing the hsp70 and H-BH transgenes.

267 6.2.3 Serum-Withdrawal/NGF-Withdrawal

To investigate whether virus-mediated overexpression of the heat shock and H-BH genes were protective to ND7 cells against apoptosis, infected cells were incubated for

0, 24 and 48 hours at 37°C/5% CO 2 in the absence of foetal calf serum and in the presence of IfiM dWrtrans retinoic acid (see Section 2.7.3). Cells were infected according to the method described in Section 2.6.12 and the 0 hour timepoint was taken after overnight incubation, prior to serum-withdrawal. At each timepoint, ND7 cells were harvested and cell survival was assessed by trypan blue exclusion assay (see Section 2,8.1).

Neonatal primary rat DRG neurons (postnatal day 2) (as described in Section 6.2.1) were infected according to the method described in Section 2.6.12 and incubated overnight. At the 0 hour timepoint, the neurons were stimulated to undergo programmed cell death by incubating them at 37°C/5% CO 2 for 48 hours in the absence of nerve growth factor (NGF) (see Section 2.7.3). The proportion healthy/unhealthy or dead primary DRG cells was assessed at 0, 24 and 48 hours according to the parameters described in Section 2.8.3.

Figure 6.2.3i and ii compares the percentage survival of mock- and 17+pR19 lacZ- infected ND7 and neonatal DRG cells respectively, with those infected with a) 17+pR19 27, b) 17+pR19 56, c) 17+pR19 70 and d) 17+pR19 HSF.

In both ND7 and neonatal DRG cultures were also incubated for a similar time period in the presence of serum and NGF, respectively. No marked differences were noted between the survival of individually virally- or mock-infected populations. In both ND7 and primary cell controls, prolonged culture did produce some degree of cell death, but this was to a lesser extent than in the serum-/NGF-withdrawal populations, and no individual population showed a greater degree of survival than the others (data not shown).

268 Figure 6.2.3: - NP7 Cell Survival Over Time Following Serum-Withdrawal in the Presence of viM-trans Retinoic Acid

Graphs a-d plot the proportion of surviving ND7 cells over time following serum- withdrawal in the presence of IpM all-/ra«is retinoic acid. Cell survival was assessed by trypan blue exclusion assay. ND7 cells either mock-infected or infected with the 17-HpR19 lacZ recombinant HSV-1 vector are represented in all graphs. Graphs a, b, c and d show the results for cells infected with 17+pR19 27, 17-HpR19 56, 17+pR19 70 and 17-HpR19 HSF, respectively. Error bars represent standard deviations of the means calculated from the means of three counts for each sample (n). n = 4-6. Significant differences in the results were calculated using Students’ t-test. + indicates significant differences in survival between hsp/H-BH overexpressing ND7 cells and 17+pR19 lacZ infected ND7 cells. (+ = p<0.05, ++ = p<0.01).

269 Figure 6.2.3:

a)17+pR19 27

90 85 Mock-infected ND7 Cells • - 17+pR19 lacZ ++ 75 ^ — 17+pR19 27 > 70 % 65 ++ 5 60

45 40 0 24 48 Time after Serum-withdrawaI (hours)

b)17+pR19 56

90 85 — Mock-infected ND7 Cells 80 - * - 17+pR19 lacZ 75 17+PR19 56 > 70 E 3 65 (0 60 55 50 45 40 0 24 48 Time after Serum-withdrawaI (hours)

270 c) 17+pR19 70

90 85 — Mock-infected ND7 Cells 75 - * - 17+pR19 lacZ l 7 0 4*— 17+pR19 70 I 65 % 60

45 40 0 24 48 Time after Serum-wIthdrawaI (hours)

d)17+pR19HSF 90 -e — Mock-infected 85 ND7 Cells 80 « - 17+pR19 lacZ 75 17+PR19 HSF « 70 I 65 ^ 60

45 40 0 24 48 Time after Serum-wIthdrawaI (hours)

271 Figure 6.2.3Ü - Neonatal Rat DRG Neuron Survival Over Time Following NGF Withdrawal Graphs a-d plot the proportion of surviving neonatal rat DRG cells over time following NGF-withdrawal. Cell survival was assessed according to the parameters described in Section 6.2.1. Neuronal cultures either mock-infected or infected with the 17+pR19 lacZ recombinant HSV-1 vector are represented in all graphs. Graphs a, b, c and d show the results for cells infected with 17^R19 27, 17-HpR19 56, 17+pR19 70 and 17-HpR19 HSF, respectively. Plots represent the means of the means of triphcate counts for each sample (n). n=2. Error bars represent the standard deviations of the sample means. Significance was not calculated due to the low value ofn.

272 Figure 6.2.311 a) 17+pR19 27 100 - - ♦ - - Mock-infected DRGs - • - 17+pR19 lacZ ■A— 17+PR19 27 80

5 70 'E 3 w CA

40

30 0 24 48 Time (hours) after NGF withdrawal

b)17+pR19 56 100 -e — Mock-infected DRGs « - 17+pR19 iacZ -A— 17+PR19 56

5 70 E 3 (0

40

0 24 48 Time (hours) after NGF withdrawal

273 c) 17+pR19 70

100 Mock-infected DRGs « - 17+pR19 lacZ 17+pR19 70 80

5 70 I ^ 60

40

30 0 24 48 Time (hours) after NGF withdrawal

d) 17+pR19 HSF

100 -e— Mock-infected DRGs 90 * - 17+pR19 lacZ

5 70 I % 60

40

0 24 48 Time (hours) after NGF withdrawal

274 Although the proportion of surviving ND7 cells infected with 17+pR19 27, 56, 70 and HSF were significantly different from the mock-infected ND7 cells at 0 hours, it is important to note that the proportion of surviving cells infected with the 17+pR19 lacZ virus was also significantly different from the mock-infected controls (p<0.05). Therefore, assuming all the virally infected ND7 cell populations are protected from other insults prior to the withdrawal of serum and the addition of retinoic acid, then the 17+pR19 lacZ infected ND7 cells would make a more suitable control, hi this case the results show significant protection from cell death conferred to ND7 cells by infection with the 174^R19 27 recombinant HSV-1 vector at both 24 and 48 hours after serum- withdrawal. Infection with 17-HpR19 70 also significantly protects ND7 cells from cell death following serum-withdrawal, however the degree of protection is less than that seen with hsp27 overexpression, in that significant differences in the proportion of surviving cells against those infected with the 17+pR19 lacZ virus were not detectable until 48 hours.

The ND7 data is confirmed in the neonatal primary neuronal experiments. The only populations that demonstrated any marked protection (against the mock- and 17-HpR19 lacZ controls) from NGF-withdrawal induced cell death were those infected with the 17+pR19 27 recombinant HSV-1 vector. No marked protection was noted in populations infected with the 17+pR19 70 virus. Therefore hsp27 overexpression is protective against growth factor withdrawal induced cell death not only in neuron- derived cells but also in primary neonatal neurons in culture.

This study does not demonstrate whether the cells are protected specifically from programmed cell death, although that is the reported mechanism of death in ND7 cells following serum-withdrawal in the presence of ?X\rtrans retinoic acid (Howard et al., 1993) and in developing neurons subsequent to NGF-withdrawal (Hamburger et al., 1981; Madhos et al., 1994).

In order to clarify whether overexpression of the hsps protect ND7 cells against apoptosis, ND7 cells were plated into 8-well chamber shdes, were infected with the

275 recombinant HSV-1 vectors as above, and cultured for 48 hours in serum-free media in the presence of retinoic acid prior to TUNEL (TdT-mediated dUTP nick end labelling) assay (see Section 2.8.2). This assay labels the ends of DNA at strand breaks with fluorescein-conjugated dUTP by terminal transferase. Therefore in cells undergoing programmed cell death, in which DNA fragmentation is characteristic, the fluorescein intensity is greater. Cell permeabiHsation was not performed as described in the protocol as this increased the levels of background. As a result, the mtensity of fluorescent, ap opt otic cells was low, making it in^ractical to take photographs. The numbers of fluorescent, ap opt otic cells were counted in three confluent fields of view of each sangle (see Figure 6.2.3iii).

276 Figure 6.2.3111

^ 120

o 100

a 2 K N

Figure 6.2.3iii - Number of NP7 Cells Undergoing Programmed Cell Death

Following 48 hours of Serum-Withdrawal in the Presence of 2M-trans Retinoic Acid ND7 cells were pre-treated by mock-infection, or by infection with 17+pR19 lacZ, 27, 56, 70 and HSF viruses. One day later the cells were incubated in serum-free media

containing lp,M 2Si-trans retinoic acid for 48 hours. The cells were fixed and fragmented DNA was nick-end labelled in situ with fluorescein-conjugated dUTP, indicating programmed cell death, or apoptosis. Fluorescent cells were counted in confluent fields at a magnification of lOOx. Three fields were counted for each sample (n). n=4 for each group. Error bars represent one standard deviation above the mean of the means of each n. + - Significant difference in survival compared with 17+pR19 lacZ-infected ND7 cells (p<0.05). Significance calculated using Student’s t-test.

277 Figure 6.2.31 shows that pre-infection with the 17-fpR19 27 virus and therefore overexpression of hsp27 confers protection to ND7 cells from death following prolonged serum-withdrawal. Figure 6.2.3iii confirms that under similar conditions overexpression of hsp27 protects ND7 cells from programmed cell death. The error bars are greater and therefore the significance lower in the second figure because the quantitation of cell viability was based on lower numbers of cells.

Figure 6.2.3iii did not however confirm that overexpression of hsp70 by pre-infection with the 174pR19 70 virus protected ND7 cells from programmed cell death following 48 hours of serum-withdrawal, as was noted in Figure 6.2.31. The effect in the trypan blue exclusion experiment was more subtle than that conferred by pre-infection with the 17+pR19 27 virus, and taking into account the error bars in Figure 6.2.3iii the TUNEL assay may not be sensitive enough at this degree of counting to detect this subtle effect. Alternatively, hsp70 may not be protecting against programmed cell death specifically, and the effect noted in Figure 6.2.31 may be due to protection from a necrotic cell death response to serum-withdrawal.

The above data in Figure 6.2.3iii also confirm that pre-infection of ND7 cells with the 17+pR19 56 and 17+pR19 HSF viruses, and therefore overexpression of hsp56 and the H-BH mutant of HSF 1 respectively, do not protect against programmed cell death following serum-withdrawal in the presence of ?X[-trans retinoic acid.

278 6.3 Discussion

The above data demonstrates that recombinant HSV-1 vector-mediated delivery and overexpression of hsp70, hsp27 and the constitutively active mutant of HSFl, H-BH, confers protection to the ND7 neuron-derived cell line and primary neuronal cells against a range of exogenous stresses. None of the viruses appear to be causing significant cytopathic effect. Overexpression of hsp27, hsp70 and H-BH all significantly protect against ‘lethal’ heat shock and simulated ischaemia. ND7 cells and neurons overexpressing hsp27 are also protected against cell death following serum- or NGF-withdrawal respectively at both 24 and 48 hour timepoints, whereas significant differences in the survival of cells overexpressing hsp70 con^ared to mock-infected and cells overexpressing P-galactosidase are only reported in ND7 cells at 48 hours, and were less profound than those overexpressing hsp27. On TUNEL assay following

48 hours of similar conditions, hsp27 was shown to significantly protect ND7 cells against programmed cell death, whereas hsp70 did not confer any detectable protection. In the TUNEL assay results however, the total counts and sample numbers were lower and the error bars greater, and therefore it may not have been sensitive enough to detect the subtle protective effect noted in the trypan blue exclusion cell viability assay, or hsp70 overexpression may not protect against apoptosis, but necrosis.

Overexpression of H-BH did not protect against cell death m either ND7 cells or primary neurons as the result of serum- or NGF-withdrawal respectively. Overexpression of hsp56 did not protect ND7 cells from any of the forms of exogenous stress that were apphed, however primary neurons infected with the 17+pR19 56 virus were tolerant to both heat shock and ischaemia, the former to a greater extent than the latter.

In summary, the results presented in this chapter clearly demonstrate that overexpression of hsps in neuron-derived cell lines and primary neurons in vitro can confer protection against stress. The way in which this differs from previous experiments involving prior heat shock or transfection-mediated overexpression is the

279 manner in which the transgene was delivered. This is the first demonstration of the use of recombinant HSV-1 vectors to overexpress transgenes in order to achieve a neuroprotective effect in vitro. Defective HSV-1 vectors or amphcons delivering the hsp70 cDNA have been delivered to primary neuronal cultures in vitro and have conferred protection firom severe heat stress, and not glutamate toxicity but no investigations were carried out on other stresses or on other hsps (Fink et al., 1997). Here, not only is the protection firom thermal stress reported, but also protection firom simulated ischaemia and serum-withdrawal, and conq>ared with the protective effects of overexpressing hsp27, hsp56 and H-BH.

The only hsps previously investigated in neuronal overexpression experiments are hsp70 and hsp90 (Mailhos et al., 1994; Wyatt et al., 1996; Amin et al., 1996; Amin et a i, 1995). Prior to this study, hsp27 had not been studied in neuron-derived cells or neurons in vitro, despite its previously reported abihty to confer thermotolerance and block apoptosis in other mammalian cell lines, and its documented overexpression in hippocampal ghal cells during cerebral ischaemia (Lavoie et al., 1993a; Mehlen et al., 1996; Kato et al., 1994). This study demonstrates that the neuroprotective effect of overexpressing hsp27 during ‘lethal’ heat shock and simulated ischaemia is at least as great as hsp70, and its effect on blocking apoptosis is more profound than hsp70. The mechanisms for the protective effects of hsp27 overexpression are purely speculative. It has been proposed that hsp27 acts as an actin-capping protein and overexpression will lead to a greater proportion binding to the barbed ends of the F-actin polymer (Miron et al., 1988; Miron et al., 1991). On heat shock hsp27 is multiply phosphorylated which may cause a conformational change either stabihsing the polymer and maintaining cytoskeletal integrity, or cause the hsp27 to dissociate to chaperone other denatured molecules, preventing their aggregation, and promoting correct refolding, whilst fi'eeing the barbed ends of F-actin to allow polymerisation and elongation. The mechanism by which overexpression of hsp27 might protect against apoptosis is unknown.

It has remained a mystery as to which stress protein was responsible for the protection against serum-withdrawal induced cell death noted on pre-treatment with a mild

280 thermal stress. This study demonstrates that hsp27 may at least be partially involved, and hsp70 may to a lesser degree exert some efifect. Whether hsp70 does exert a protective effect is therefore not clarified here, but has been shown not to have an effect in previous work from our laboratory (Mailhos et al., 1994). Overexpression of hsp70 is thought to protect cells from stress-induced necrotic cell death and promote recovery by chaperoning denatured proteins, preventing their aggregation and enabling correct refolding (Lindquist, 1986).

Although these data, along with previous transfection experiments fail to show that hspTO definitely protects neuronal cells against serum-/NGF-withdrawal induced apoptosis, hsp70 and hsp27 overexpression has recently been demonstrated to protect non-neuronal cell lines against actinomycin-D, canoptothecin and etopo side-induced apoptosis (Samah and Cotter, 1996). To further comphcate matters heat shock (44^C for 60 mins) has recently been demonstrated to cause apoptosis in non-neuronal cell lines and similar cells overexpressing hsp70 show a decreased ap opt otic fraction cotq)ared to controls (Li et at., 1996). The tenq)eratures used in the heat shock study were lower than the ‘lethal’ heat shock apphed to the neuronal cells in this chapter and therefore it can not be concluded from this study that a similar fraction of cells will be undergoing programmed cell death. It has also been recently shown that hsp70 expression inhibits the CED-3 related protease caspase-3, and somewhere upstream and downstream of the stress-activated protein kinase SAPK/JNK (c-Jun N-terminal kinase) signaling pathway (Mosser et al., 1997). However, these mechanisms of protection may be cell-type dependent, not occurring in neuronal cells.

Previous to this study the effects of overexpressing hsp56 had not been studied in neuron-derived cell lines or primary neurons. Unlike hsp27 and hsp70, overexpression of hsp56 had not previously been tested in the context of overexpression and thermotolerance in any organisms/cell lines, conversely thermotolerant cells have never been demonstrated to overexpress hsp56. It is therefore of httle surprise that in these experiments, overexpression of hsp56 in ND7 cells did not confer tolerance to ‘lethal’ heat shock, simulated ischaemia or serum-withdrawal. The negative results nevertheless provided a usefid negative control to highlight the protective effects of

281 overexpression of the other heat shock genes. It is of interest that similar overexpression in primary neurons confers protection against ‘lethal’ heat shock and, to a lesser degree, protects against simulated ischaemia. A similar protective effect may be demonstrated in ND7 cells if the recovery after the two insults was prolonged to 12 and 24 hours, as was the case with the primary neuronal experiments, or this result may indicate a cell-type specificity for tolerance to necrotic insults.

In the previous chapter the first demonstration of the heat shock gene transactivation effects of overexpressing the HSFl mutant H-BH was reported. The studies reported in this chapter are therefore the first to demonstrate neuroprotection by expressing a constitutively active mutant of a transcription factor. Chapter 5 described how hsp70 (and to a lesser degree hsp27 and hsp32) was overexpressed in ND7 cells following infection with the 17+pR19 HSF virus, whereas hsp90, 56 and 60 levels remained unchanged. These studies demonstrate that hsp70 and hsp27 both protect from ‘lethal’ heat shock and simulated ischaemia, therefore the protective effects conferred to ND7 cells and cultured neurons by overexpression of H-BH may be mediated through these proteins. HSFl may however stimulate the transcription of other genes through interaction with the heat shock elements in their promoters that may be protective or detrimental to the cell which may fiirther effect the outcome. The levels of overexpression in ND7 cells of hsp27 and 70 following infection with the 17+pR19 27 and 17+pR19 70 viruses respectively were considerably greater than those stimulated by H-BH after infection under similar conditions with the 174^R19 HSF virus. Therefore the resultant levels of these proteins may not have been enough to confer any protective effect against serum-withdrawal induced apoptotic cell death. Nevertheless, this study opens the possibihties for manipulating the stress response at the heat shock gene transcription level.

282 Chapter 7

Discussion

283 The work presented in this thesis has combined what is currently known about herpes simplex virus type 1 (HSV-1) recombinant vector technology with heat shock protein (hsp) biology to further investigate the possible roles of protection that overexpression of the hsps may confer to neuronal cells during stress. The thesis reports on the mRNA and protein levels of hsp27, 56, 60, 70, and 90 over time in the core region of ischaemia following permanent middle cerebral artery occlusion (MCAO) in the rat brain. The two subsequent chapters describe the design and construction of disabled, recombinant HSV-1 vectors that overexpress the hsps in a variety of cell lines in vitro. The effect of delivery and expression of the constitutively active mutant of HSFl (H- BH) to neuron-derived ND7 cells on hsp protein levels was also studied. The use of green fluorescent protein (GFP) and the encephalomyocarditis virus internal ribosome entry site (EMCV-IRES) were investigated as possible strategies in future recombinant HSV-1 vector design. The final chapter reports on the neuroprotective effect in vitro that prior infection with recombinant HSV-1 vectors overexpressing hsp27, 56, 70, H- BH and p-galactosidase has on the survival of the neuron-derived ND7 cell line and neonatal rat dorsal root ganglion (DRG) neurons after TethaF heat shock, simulated ischaemia and serum-withdrawal induced apoptosis.

In summary of the data in the third chapter of this thesis, the mRNA and protein levels of hsp27, 60 and 70 markedly increase over time following permanent MCAO, whereas no marked increases were noted in the mRNA or protein levels of hsp 5 6 or hsp 90. This data contributes a more comprehensive study to the field of hsp expression than has previously been reported, not only in that it reports on five heat shock proteins over 24 hours under the same conditions, but also because it demonstrates that translation of the hsps continues throughout the permanent ischaemic insult.

Further immunohistochemistry and in situ hybridisation experiments on shces of tissue throughout the time period examined would reveal where, in the ischaemic core region, this upregulation is concentrated, and whether there is any dichotomy between the locations of increases in mRNA and protein concentration. Another useful future direction would be to repeat these experiments taking the end timep oint through to 48 and 72 hours to examine at which point the overexpression of the hsps is maximal and

284 how rapidly they decrease with time. Other hsps, for exanq)le hsp32, hsp 110 and ubiquitin might also be examined to gain an overall picture of hsp expression in this particular model of stroke.

The characterisation of the 17+pR16R- and 17+pR19-based recombinant HSV-1 vectors in Chapters 4 and 5 raised some interesting points. The LAT PI promoter alone was used to drive lacZ expression in the 17-HpR16R-based vectors. The subsequent expression of (3-galactosidase was very poor in IE2 conq)lementing B 130/2 cells. In similar cells infected with the 17+pR19 lacZ where the lacZ gene was inserted downstream of the CMV-IE promoter, itself being situated downstream of the LAT P2 promoter, the expression was much greater. No conclusions can be drawn regarding the activity of the LAT promoters, the CMV-IE promoter or the sites of recombination form con^aring these results as the two vectors are too different. Despite two copies of the lacZ gene being recombined into, and expressed from, each recombinant HSV-1 genome in the 17+pR19 lacZ virus, the difference in expression appears much greater than two-fold. This could be quantitated by luminometry of a p-galactosidase assay using a fluorescent substrate. It would, however, be useful to know whether this increase in expression is due to the presence of the LAT P2 promoter, the CMV-IE promoter, the site of recombination or combinations of all of these. Recent work has shown that the LAT P2 promoter (long term expression element - LTE) is necessary for long term transgene activity during latency in neurons, hut they do not comment on the level of short-term expression (Lokensgard et al., 1997). The experiments described in these sections of this thesis are fairly crude and a detailed analysis of the activity of exogenous promoters in combination with one and/or the other of the HSV- 1 LAT promoters in different recombination sites is required for greater understanding in order to enable the optimisation of transgene expression. In the future, it may well be the case that the expression of a transgene at the highest expression level obtainable for the longest time period possible may not be desirable, and data from a study such as this would considerably contribute to the knowledge required to achieve the appropriate levels and characterisation of expression.

Long term expression studies were not carried out in vivo with the 17+pR19-based viruses, as transgene overexpression was only required for up to three days, however it

285 would be usefid to see how long the transgenes are expressed in the central nervous system for conq)arison with other pubHshed recombinant HSV-1 vectors, to see if the situation of the LAT P2 promoter upstream of the CMV-IE promoter may confer long term expression. Recombinant viruses would therefore need to be constructed without the presence of the LAT P2 promoter for comparison.

For the purposes of delivering heat shock transgenes to neuron-derived cells in vitro it is clear fi*om the western blots in Chapter 5 and the data in Chapter 6 that hsp27, hsp70 and H-BH are several-fold overexpressed in cells infected with their respective 17+pR19-based vectors. Moreover, overe?q)ression was significant enough to detect a fimctional protective effect against TethaF heat shock and simulated ischaemia in ND7 cells and neonatal rat DRG neurons. It may be that altering the levels of e?qpression may alter the level of tolerance, indeed overexpressing hsp 5 6 to a lower or higher degree may ehcit a protective response that was not noted in this study.

The data presented in Chapter 6 confirms and also extends the data reported in previous pubHshed work. Hsp70 overexpression has already been demonstrated to protect ND7 and primary neuronal cells against thermal and ischaemic insults, but not against apoptosis (Uney et al., 1993; Mailhos et a l, 1994; Wyatt et a i, 1996), however the work described in Chapter 6 was the first study that examined the consequences of overexpressing hsp70 during heat shock, ischaemia, and serum-/NGF- withdrawal together in neuron-derived and primary neuronal cells. This study also demonstrates that recombinant HSV-1 vector-mediated hsp70 transgene delivery can produce a similar effect, although direct conqjarisons were not made with expression vector transfection controls. In order to compare the fimctional effects of the two regimens the levels of transgene expression and the ef&ciency of delivery would need to be identical.

In addition, this is the first time overexpression of hsp27 has been studied in neuron- derived cells, confirming tolerance to thermal and apoptotic stimuH noted in other mammahan non-neuronal cell lines (Mehlen et al., 1996; Lavoie et al., 1993a; Samah and Cotter, 1996). The effects of hsp27 overexpression had not previously been investigated during simulated ischaemia. This study demonstrated that the recombinant

286 HSV-1 vector-mediated transgene delivery and subsequent overexpression of hsp27 confers both thermal and ischaemic tolerance and inhibits programmed cell death in response to serum- or NGF-withdrawal in ND7 cells and primary DRG neurons respectively.

H-BH and hsp 5 6 overexpression had also not previously been studied in any in vitro or in vivo tolerance experiments. This study demonstrates that overexpression of hsp 5 6 in ND7 cells by pre-infection with the recombinant 17+pR19 56 HSV-1 vector does not protect against thermal, ischaemic or serum-withdrawal induced insults. However, pre­ infection of primary neuronal cultures with the 17+pR19 56 virus confers protection against thermal insult, and to some degree against simulated ischaemia. No groups have pubHshed data to suggest that hsp 5 6 might be protective in neuronal or any other cell types. Although no tolerance was conferred to ND7 cells by pre-infection with the 17+pR19 56 vector, a marked tolerance to heat shock was conferred to primary DRG neurons, and a less marked tolerance to simulated ischaemia after similar pre­ treatment. This not only suggests that any tolerance induced by hsp 56 overexpression to necrotic insults may be ceU-type specific, but also highlights the inq)ortance of performing such experiments in primary neurons as weft as neuron-derived ceU lines.

The H-BH mutant, had previously only been demonstrated to activate the hsp 70 promoter (Zuo et a l, 1995). The study reported in Chapter 5 was the first to show that when H-BH is expressed in ceUs foUowing infection with the 17+pR19 HSF virus, hsp 70 is markedly overexpressed, with some detectable over expression in hsp 3 2 and hsp27 whereas hsp56, hsp60 and hsp90 were not detectably overexpressed. This presented the possibihty of using the 17+pR19 HSF virus to simulate a stress response in the absence of stress, which may confer protection against cell death, in particular for the interests of this thesis, in neurons.

H-BH is a deletion mutant of the human HSFl gene, and the work presented in Chapter 5 of this thesis demonstrated that overexpression of this protein increases the levels of hsps in two différent rodent cell lines, one derived from hamster kidney fibroblasts (B130/2) and the mouse/rat fusion ND7 neuron-derived cell line. The levels of expression of the hsps differed between the two ceU lines. In particular hsp27 was

287 markedly overexpressed in B 130/2 cells infected with the 17+pR19 HSF virus, whereas the level of overexpression was very mild in similarly infected ND7 cells. The stimulation of heat shock gene transcription in the presence of H-BH may therefore be ceU-type dependent and it might be useful to know to what extent across evolution this cell-type dependency may occur, thus increasing the knowledge of HSFl and its role in different eukaryotes.

In Chapter 6 it was shown that subsequent to viral gene delivery of H-BH, ND7 cells and neonatal rat DRG neurons were tolerant to TethaF heat shock and simulated ischaemia but not to serum-/NGF-withdrawal induced apoptosis. The degree of overexpression of hsp27 in response to H-BH overexpression may not have been sufficient (in corrq)arison to cells infected with the 17+pR19 27 virus) to protect against programmed cell death, and the overe?q)ression of hsp 70 may only protect against necrotic cell death, and this might explain why no protection was noted. Only hsp27, hsp32, hsp56, hsp60, hsp70 and hsp90 levels were studied in ND7 cells after infection with the 17-kpR19 HSF virus. It is probably the case that the transcription of other endogenous genes is also stimulated. To gain a more complete picture, therefore, a future study might examine what these genes are, either by analysis of a differential display or by western blot and subsequent hybridisation of specific antibodies to protein extracts of H-BH overexpressing cells.

As an additional note, similar experiments to those described in Chapter 6 are being performed in our laboratory on primary cardiac cell cultures, and preliminary data show that all the recombinant viruses tested (17+pR19 lacZ, 17-kpR19 27 and 17+pR19 70) are protecting against simulated ischaemia, indicating that \iral infection in itself may raise a protective stress response. One cardiomyocyte-derived cell line is tolerant solely after pre-infection with the 17+pR19 70 virus, whereas another is protected against similar stress by pre-infection either with the 17+pR19 27 or 17+pR19 70 recombinant HSV-1 vectors (Dr. B. Brar, pers. commun). The protective effect of overexpressing hsps may therefore be cell-type dependent, where in some cells it may be concealed by the protective effect of the stress response induced by infection with the vector alone. However, in the experiments described in Chapter 6, no such protection was noted in primary neurons infected with the 17+pR19 lacZ

288 virus, and therefore the induction of a stress response by HSV-1 vector infection may be cell-type dependent.

Further in vitro tolerance experiments could be carried out after pre-infection with the viral vectors. Primary cortical and cerebellar granule cells could be infected prior to exposure to glutamate to examine the effects of overexpressing the hsps on glutamate toxicity. Previous work has found that protection can be conferred to similar cells with a mild thermal pre-treatment, but no hsp has yet been shown to be directly responsible, despite the elevation of hsp70 in cells with the pre-treatment (Rordorf et al., 1991; Lowenstein et al., 1991). Delivery of hsp72 using an ampHcon HSV-1 vector, however, does not protect primary neurons against glutamate toxicity (Fink et al., 1997).

The use of GFP was discussed in Chapter 5 as a potential reporter gene for use in future strategies of gene delivery. It has two major advantages over lacZ. Firstly, the GFP gene is much shorter than lacZ (SOObp as opposed to 3700bp). Secondly, it can be visuahsed under fluorescein optics with UV hght throughout a timecourse experiment without the need for fixative, toxic buffer or stain such as those required for (3-galactosidase visuahsation. This was the first demonstration that GFP could be visuahsed in cells infected with a recombinant HSV-1 GFP-expressing virus.

The design, construction and characterisation of the first bicistronic recombinant HSV- 1 vector is reported in Chapter 5. An EMCV-IRES sequence was inserted between the GFP and lacZ genes so that they are both under the control of a single CMV-IE promoter, prior to recombination into the same locus as the expression cassette in the 17+pR19-based viruses, to make 17+pMl. The 1RES enables cap-independent translation of the lacZ message. Quahtative visuahsation of the intensity of fluorescence of GFP and the blue staining for P-galactosidase in B 130/2 cells infected with the 17+pMl virus demonstrated that the levels of expression of both transgenes was below those in cells infected with the 17+pR19 GFP and 17+pR19 lacZ viruses, respectively. The deficit in expression of GFP may be explained by the considerably increased length of mRNA, due to the combined message of GFP, the 1RES and lacZ (5100bp) which may contain less stable sequences than GFP alone (SOObp). The

289 differences in P-galactosidase expression in cells infected with the 17+pMl virus compared with similar cells infected with the 17+pR19 lacZ virus to may be due to the relative inefficiency of cap-independent translation of the lacZ message, as other groups have reported (Ho et a l, 1995a). Nevertheless, the expression of both transgenes was high, and considering the advantage of knowing that wherever the downstream transgene is detected then the upstream transgene must he transcribed also, and under the same intracellular controls, the EMCV-IRES should prove usefid in fixture strategies of transgene delivery.

In the light of the results presented in this thesis, if the hsp expressing viruses were to be re-designed, they would be similar to 17+pMl with the hsp/H-BH replacing the GFP gene upstream of the 1RES and a lacZ or GFP gene downstream of the 1RES. This would ensure heat shock transgene expression wherever the reporter gene product was detected. Cell survival coxdd be more easily and rehably assessed by counting p-galactosidase expressing cells, a technique currently used in similar work in the hterature (Fhik et a l, 1997; Ho et a l, 1995b). The detection of survivhig primary neurons by visuahsation under phase-contrast microscopy described in Chapter 6 is subjective and therefore limited by the potential for misinterpretation of ceU state, and does not show that the surviving neurons were infected with the vector or overexpressing the transgene. The other advantage would be that one could quantitate the amoxmt of surviving neurons that were overexpressing the transgene as a proportion to tolerant ceUs that were not.

It may be beneficial to ‘knock out’ more essential and immediate-early genes fi*om the HSV-1 genome, for exanqxle those encoding ICP4, ICPO and ICP22 as suggested by Wu et a l, 1996. ICP47 is involved in evasion of cytotoxic T-ceU mediated immunity, and therefore probably shoxxld remain in the recombmant genome. The 17+pR19-based viruses are not causing ceU death to neuron-derived ceUs or primary neurons in vitro nor to primary cardiac cells or cardiac ceU-derived ceU lines (Dr. B. Brar, our laboratory, pers. commun), however the consequences of long-term infection have not yet been assessed in vitro or in vivo. This would be an important avenue to follow with regards to learning more concerning the possible cytotoxicity of these viruses.

290 Another avenue for Anther study would be to examine the neuroprotective effects of overexpressing other hsps. For example, hsp32 expression is increased in ischaemic cortex (Nimura et al., 1996), and it was noted in Chapter 3 of this thesis that hsp60 mRNA and protein are also overexpressed. These might therefore be valuable proteins to overexpress with regards to neuroprotection from ischaemia. Other proteins that are overexpressed following cerebral ischaemia such as brain-derived neurotrophic factor, basic fibroblast growth factor and transforming growth factor beta may also confer protection on delivery (Knuckey et al., 1996; Kokaia et al., 1996; Spehotes et al., 1997).

As a result of the encouraging data generated in the work described in this thesis, the next phase of the project is to inject the 17+pR19-based recombinant viruses into the rat cerebral cortex prior to MCAO and subsequent histological examination for change in pathology and immunohistochemical analysis of expression of hsp/H-BH expression. The results of the in vitro data are encouraging for continuation into the in vivo experiments, where the question of whether hsp overexpression can protect neurons from stroke-induced ischaemic cell death will be addressed. This work is continuing in the Reta Lila Weston Institute of Neurological Sciences, UCL Medical School, however, at the time of submission of this thesis the conditions for such experiments were being set up.

The data produced from these studies, therefore, and the viral vectors presented here will be valuable assets in the fields of heat shock protein, gene delivery and cerebral ischaemic research.

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341 Reprinted from MOLECULAR BRAIN RESEARCH

Molecular Brain Research 42 (1996) 236-244

Research report Focal cerebral ischaemia increases the levels of several classes of heat shock proteins and their corresponding mRNAs

Marcus J.D. Wagstaff Yolanda Collaço-Moraes Benjamin S, Aspey , Robert S. Coffin Michael J.G. Harrison David S. Latchman Jacqueline S. de Belleroche ^

* Department of Molecular Pathology, University College London Medical School, The Windeyer Building, 46 Cleveland Street, London, WIP 6DB, UK ** Reta Lila Weston Institute o f Neurological Studies, University College London Medical School, The Windeyer Building, 46 Cleveland Street, London, WIP 6DB, UK Department of Biochemistry, Charing Cross and Westminster Medical School, Fulham Palace Road, London, W6 8RF, UK

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Research report Focal cerebral ischaemia increases the levels of several classes of heat shock proteins and their corresponding mRNAs

Marcus J.D. Wagstaff Yolanda Collaço-Moraes Benjamin S. Aspey Robert S. Coffin Michael J.G. Harrison David S. Latchman Jacqueline S. de Belleroche ^

* Department of Molecular Pathology, University College London Medical School, The Windeyer Building, 46 Cleveland Street, London, WIP 6DB, UK Reta Lila Weston Institute of Neurological Studies, University College London Medical School, The Windeyer Building, 46 Cleveland Street, London, WIP 6DB, UK Department of Biochemistry, Charing Cross and Westminster Medical School, Fulham Palace Road, London, W6 8RF, UK

Revised 7 May 1996

Abstract

The induction of focal cerebral ischaemia in rats by middle cerebral artery occlusion has previously been shown to increase, over time, the mRNA levels of the heat shock proteins (HSPs) 27 and 70. However, the levels of HSP90 mRNA remain constant. In contrast, during global ischaemia, HSP70 and HSP90 mRNA levels are both raised, particularly in the CAl neurons in the hippocampus, an area that is resistant to the insult in comparison to the surrounding regions. HSP27 mRNA is raised in the neuroglia in the subregions of the hippocampus. However, the protein levels of HSP27, 70 and 90 have not been characterised in focal ischaemia. With this data in mind, we have carried out a comparative study of HSP27, 56, 60, 70 and 90 mRNA and protein levels during focal cerebral ischaemia in rats, up to 24 h post-occlusion. We have shown that HSP70 and HSP27 mRNA levels are increased and also that HSP60 mRNA levels (which had also not previously been characterised in this model of focal ischaemia) are significantly raised. HSP90 and HSP56 mRNAs were not significantly elevated. On Western blot analysis, the inducible HSP72 protein was first detected at 8 h post-occlusion, HSP27 protein was detected only at 24 h post-occlusion and HSP60 protein, although constimtive, appeared to increase at 24 h post-occlusion. HSP56 protein levels appeared to rise on the occluded side, but HSP90 protein levels remained constant.

Keywords: Focal cerebral ischaemia; Heat shock protein; mRNA; Protein; Time course

1. Introduction raised during global ischaemia. HSP mRNA transcription and protein synthesis in neurons has also occurred in Cerebral ischaemia has been shown to alter gene ex­ response to a number of other inducing agents, for exam­ pression in the mammalian brain in the infarcted tissue and ple surgical incision or chemical-induced excitotoxicity in the surrounding cortical regions [23,24]. Sub-lethal stim­ leads to raised HSP70 levels [7,34]. uli such as heat shock and ischaemia prior to a lethal insult It has been speculated that the role of these proteins have also been demonstrated to confer protection against during ischaemia is to protect against neuronal damage and subsequent neuronal damage [25,26,40]. Many of the im­ cell death. Transfection of HSP70 cDNA into rat dorsal mediate early genes that are induced following an is­ root ganglion neurons (DRGs) has been shown to protect chaemic insult have been identified as members of the heat these cells from severe heat stress [49], whereas overex­ shock family of proteins (HSPs), in particular the HSPs 27, pressing HSP90 is not protective [3]. The most extensively 47, 70 and 90, whose mRNA levels have been charac­ characterized response to cerebral ischaemia is the induc­ terised temporally [16,22,24]. Both HSP27 and 70 mRNA tion of HSP72 mRNA, the inducible member of the HSP70 levels were shown to rise over time both during global and family, which is induced by kainic acid injury as well as focal ischaemia, whereas HSP90 mRNA levels were only ischaemia [14,38]. The regional localisation and temporal synthesis of HSP70 mRNA in the CNS during ischaemia Corresponding author. Fax: +44 (171) 387-3310. differs according to the severity of the insult [45,50].

0169-328X/96/$15.00 Copyright © 1996 Elsevier Science B.V. AH rights reserved. PI! S0169-328X(96)00127-1 M.J.D. Wagstaff et al. / Molecular Brain Research 42 (1996) 236-244 237

Furthermore, the CAl pyramidal hippocampal neurons, the previously undocumented levels of HSP56 and HSP60, which express particularly high levels of HSP70 mRNA but in measuring the full range of the heat shock proteins after as little as two min of temporary global cerebral together under similar laboratory conditions, comparisons ischaemia, maintain a higher cell density on a subsequent of the mRNA and protein levels during the timecourse can ‘lethal’ ischaemic insult than the surrounding regions which be made. It is important to measure protein levels in sustain greater damage [25,26]. Indeed, heat shock treat­ parallel with mRNA as mRNA levels may not parallel ment which increases expression of HSP70 mRNA in vitro protein levels after an ischaemic insult. Levels of mRNA in cultured neurons, has been demonstrated to induce a were examined in the core ischaemic area by Northern blot protective phenomenon which renders those cells which analysis and further quantitated by slot blot analysis, with respond with high HSP70 mRNA expression more resis­ protein levels being examined by immunodetection on tant to glutamate toxicity [40]. Greater levels of HSP70 Western blots. protein are seen in neurons in vitro by pre-treatment with heat than with ischaemia, and the former gives greater protection than the latter irrespective of the type of subse­ 2. Materials and methods quent lethal insult. Therefore it has been concluded that the degree of protection conferred to neuronal cells in vitro by 2.1. Middle cerebral artery occlusion (MCAO) pre-conditioning stimuli is correlated to the amount of Male Sprague-Dawley rats (wt. 305 ±15 g) were HSP70 protein that is produced as a result, rather than the anaesthetised with 4% halothane in a 1:1 mixture of nature of the subsequent stress [2]. The raised levels of nitrous oxide and oxygen after an overnight fast. With the HSP70 and other immediate-early gene products through halothane then maintained at 1.5% in an open anaesthetic heat shock or ischaemic pre-treatment may therefore be system without tracheotomy, the origin of the left middle responsible for the subsequent tolerance to ischaemia. cerebral artery was occluded with an intravascular suture Although most studies in this area have concentrated on following a method modified from Nagasawa and Kogure HSP70, other HSPs have been investigated to a lesser [37]. Briefly, this involved introducing a 4/0 nylon suture extent. HSP27, an aB-crystallin homologue, also induces (Ethylon, Ethicon, UK) with 7 mm of its shaft from the tip thermotolerance [28] and has subsequently been shown to thickened with silicone rubber (Silastic sealant 732 RTV, be raised in focal cerebral ischaemia at the mRNA level at Merck, UK) to an outside diameter of 0.27 mm, into the least [16]. The p-isoform of HSP90 is also raised in global left external carotid artery in a retrograde fashion towards cerebral ischaemia [22], although its elevated expression the carotid bifurcation and then directed distaUy up the left has not yet been shown to produce the ‘ischaemic toler­ internal carotid artery to a distance of 17.5 ± 0.5 mm from ance’ associated with HSP70. The protein levels of both of the carotid bifurcation to the tip of the suture. The suture these have not been characterised in focal cerebral is­ was secured at two points: first, to the stump of the ligated chaemia. left external carotid artery and second, within the ligated HSP56 is an FK506 binding protein and FK506, a internal carotid artery, proximal to the pterygopalatine synthetic immunosuppressant, has been shown to give branch. The whole procedure took 20 min, with rectal neuroprotection during focal cerebral ischaemia [44]. temperature maintained throughout at 37.0 ± 0.5°C with a HSP56 is also hnked functionally with HSP90 and 27 as a heat lamp. The cutaneous wound was sutured and cleaned, part of the untransformed steroid receptor complex, which and the animals were left to recover with free access to is formed under the influence of HSP70. However, HSP56 food and water. This intravascular suture model of MCAO levels have not as yet been characterised during cerebral was adopted because it is a comparatively simple proce­ ischaemia, HSP60 is an auto-immunogen and chaperonin dure and is less surgically traumatic than other focal and has not been measured during focal ischaemia al­ ischaemia models involving craniotomy, so that post-oper­ though its mRNA levels have been shown to be raised in ative recovery is facilitated. At 2, 4, 8 and 24 h after the hippocampus after 3 h of reperfusion subsequent to a MCAO, rats were sacrificed by cervical dislocation, and global ischaemic insult of 3.5 min [1]. the brains rapidly removed, anatomically dissected and The encouraging results from studies on HSP70 might frozen in liquid nitrogen. The effect of MCAO was studied suggest that other HSPs may also be protective. We be­ in a region of the ipsilateral cerebral cortex which lay in lieve it is necessary to understand the dynamics of the the centre of the middle cerebral artery territory, and in brain’s physiological response to ischaemia in detail before corresponding regions of the contralateral (right) hemi­ the therapeutic possibilities of manipulating this response sphere. The tissue samples were stored at — 70°C until can be effectively assessed. With this in mind, the present processed. Unoperated animals served as tg controls. study was carried out in order to examine the temporal expression of the mRNA and protein levels of HSP27, 56, 2.2. Preparation of cDNA probes 60, 70 and 90 in a rat model of focal cerebral ischaemia, produced by occlusion of the left middle cerebral artery Tissue levels of a number of HSP mRNAs were screened with an intra-luminal suture. Not only have we examined by reference to ^-tubulin mRNA using genomic cDNA 238 M.J.D. Wagstaff et al. / Molecular Brain Research 42 (1996) 236-244 probes for detection. The HSP27 probe was derived from a C 2 , 8 24 700-bp insert in pBluescript M13 + coding for Chinese L R L R L R L R hamster HSP27 (kindly donated by Jacques Landry) [30]. A 2-kb fcoRI fragment of rabbit HSP56 cDNA cloned 18 s- into pGem-7zf was used to detect HSP56 mRNA (p59, a gift from Marie Claire Lebeau) and an HSP60 probe was hsp 27 derived from a 2.2-kb cDNA insert coding for human HSP60 (a kind gift from Prof. R.S. Gupta). The HSP70 probe was obtained from a 2.3-kb human cDNA insert in pAT153, excised with BamHl plus Hindlll (supplied by the ATCC) [52]; and the HSP90 cDNA probe was derived from a 2.5-kb EcoRl insert in pBa90 coding for human 28 s- HSP90 [48]. An 800-bp fragment of ^-tubulin derived from a human foetal brain library [15] was used as a reference probe. , hsp 60 18 s- 2.3. RNA extraction and hybridization Total RNA was isolated from tissue samples by acid guanidinium thiocyanate-phenol-chloroform extraction [9]. The resulting RNA was analysed by Northern blotting 28 s- and slot blotting as previously described [11]. Northern blot filters were pre-incubated in hybridization buffer [50% deionized formamide, 5 X SSPE (0.9 M NaCl, 18 s- hsp70 0.05 M sodium pyrophosphate, pH 7.4, 5 mM EDTA), 5 X Denhardt’s solution (0.1% bovine serum albumin, 0.1% ficoll, 0.1% polyvinylpyrrolidone), 0.5% (w/v) sodium dodecyl sulphate, 100 p-g/ml salmon sperm DNA] for 2 h at 42°C. Filters were then hybridized overnight at 42°C in "T hybridization buffer containing [^^P]-labeIled cDNA (3-5 X 10^ d.p.m./ml) to a specific activity of 2.5-3.5 X 10^ 18 s- h sp56 d.p.m./p,g with [a-^^P]dCTP using the oligolabelling a method of Feinberg and Vogelstein [12,13]. Blots were 28 s- washed with 3 X SSC, 0.1% SDS for 30 min at 65°C and 1 X SSC, 0.1% SDS for a further 30 min at 65°C and exposed to Hyperfilm-MP (Amersham International PLC) hsp90 with intensifying screens at -70°C. 18 s- Quantitation of HSP mRNA levels relative to (3-tubulin mRNA levels was carried out by slot blot analysis, which enables the study of a large number of samples whilst 28 s- maintaining similar experimental conditions as follows; a ■ Identical triplicate slot blot filters were hybridized with B-tubulin either HSP or (3-tubulin [^^P]cDNA probes and then 18 s- washed and the signal measured using a Biorad phospho- imager. Prior to rehybridization the blot was stripped of !!■» labelled probe by washing in 0.1% SDS for 30 min at 65°C and reprobed with a ^-tubulin cDNA probe using the same conditions described above. Fig. 1. Northern blots showing the effect of unilateral middle cerebral Slot blot phosphoimaging screens were scanned using a artery occlusion on levels of HSP mRNAs. Cerebral cortex from the left, Biorad model GS-250 molecular imager and peak area ipsilateral (L) and right, contralateral (R) hemisphere was dissected at various times (2-24 h) after left MCAO and frozen in liquid nitrogen. integration carried out using Biorad Molecular Analyst The contralateral and ipsilateral cortical samples at each time point were software. The results are expressed as a ratio of the HSP derived from a single animal. Messenger RNA was extracted from tissue mRNA signal: P-tubulin mRNA signal. The linearity of the homogenates, separated by electrophoresis, blotted and hybridized with mRNA signal was confirmed through analysis of serial cDNA probes for HSP27, HSP56, HSP60, HSP70, HSP90 and p-tubulin. dilutions of P-tubulin mRNA. Quantitation was carried out The IBS and 28S rRNA markers are indicated by arrows. Loading in control samples (C) was greater to allow detection of low basal levels of over a linear response range. Statistical analysis was car­ HSP mRNAs. Quantitation was carried out at each time point (n = 3-6) ried out using Student’s t-test. by slot blot analysis (see Fig. 2). M.J.D. Wagstajf et al. / Molecular Brain Research 42 (1996) 236-244 239

2.4. Total protein preparation, SDS-PAGE and Western blotting the protein was transferred electrophoreticaUy over blotting 16 h to Hybond-C membranes using a Biorad blotting transblot cell at 180 mA/55 V at room temperature (buffer: The brain samples were weighed and then homogenised 25 mM Tris-HCl pH 8.0, 192 mM glycine, 20% methanol). in 5 X (v/w) of 0.1% (w/v) SDS. Protein content of each Following blotting the membranes were blocked using 4% sample was assayed using BCA protein assay kit (Pierce) (w/v) dried milk powder (Marvel) and 0.05% (v/v) Tween and the samples were resuspended in 2 X Laemmli sample 20 in phosphate buffered saline (PBS) for 1 h at room buffer (20% (w/v) glycerol; 6.0% (w/v) SDS; 120 mM temperature. Filters were then incubated for 1.5 h at room Tris-HCl, pH 6.8). Five percent P-mercaptoethanol and temperature with mAbs at a dilution of 1:1000 for anti- 10% bromophenol blue was added to each sample and the HSP72 (Stressgen), anti-HSP90 (Stressgen), anti-HSP60 samples were boiled for 7 min. (Affinity Bioreagents), anti-HSP54 (Stressgen), anti-13- Samples were loaded at 50 jxg/well and elec- tubulin (Sigma), and 1:400 for anti-HSP27 [32] in 4% trophoresed on 10% polyacrylamide SDS-PAGE gels ac­ Marvel and 0.05% (v/v) Tween 20 in PBS. Filters were cording to Laemmli [27]. Gels were stained in 0.3% washed three times for 5 min in 0.05% Tween 20 in PBS Coomassie brilliant blue R250 (BDH) in 40% (v/v) and then incubated for 1 h at room temperature with methanol; 7% (v/v) glacial acetic acid and destained in horseradish peroxidase-conjugated rabbit-anti-mouse IgG the same buffer without Coomassie blue. For Western at a dilution of 1:1000 in PBS containing 4% (w/v)

* p<0.05 1.5 * * p<0.025 * * * p<0.005 < § p<0.05 §§ p<0.025 §§§ p<0.005 H ipsilateral *** I I contralateral < H ipsilateral *** *** 0 .5 - 0.5 §§§ §§§ I I contralateral §§§

0 0 2 4 8 24 0 2 4 8 24

tim e (h) tim e (h)

0.3 n 1.00-1

< H ipsilateral

I I contralateral s H ipsilateral *** ! EH contralateral

0.25

0 2 4 8 24

tim e (h) tim e (h) Fig. 2. Time course of HSP27, HSP60, HSP70 and HSP90 mRNA levels following MCAO. At various times (2 h, 4 h, 8 h and 24 h) after MCAO, cerebral cortex from ipsilateral and contralateral hemispheres was dissected and used for preparation of mRNA. Quantitation of HSP mRNAs was carried out by slot blot analysis with reference to levels of 3-tubulin mRNA. Values are means with the SEMs shown by error bars for 3-6 animals at each time point. \ §§ §§§ indicates that MCAO significantly increased levels of HSP mRNAs compared to ipsilateral 0 h controls (P < 0.05, P < 0.025 and P < 0.005 respectively). *, * * and * * * indicates that the value in ipsilateral cortex is significantly greater than contralateral cortex of the same animals (P< 0.05, P < 0.025, and P < 0.005 respectively). 240 M.J.D. Wagstajf et al. / Molecular Brain Research 42 (1996) 236-244

Marvel and 0.05% (v/v) Tween 20. Filters were washed KDa C 2 . 8 24 as above and developed using an enhanced chemilumines- L R L R L R L R cence detection (ECL, Amersham) and exposed to Hyper­ hsp 27 film (Amersham) for between 3 s and 30 min after allow­ 30- ing 1 min for the solutions to react with the membrane and reach maximum light emission.

6 6 - 3. Results hsp 60

3.1. Effect of experimental cerebral ischaemia on the expression of HSP mRNAs

The timecourse of mRNA levels encoding several heat shock proteins was determined in cerebral cortex up to 24 66 hsp72 h after MCAO, as shown by Northern analysis (Fig. 1). A single species of mRNA was detected for HSP27, HSP56, HSP60, HSP70 and HSP90 of 0.7 kb, 2 kb, 2.2 kb, 2.3 kb, hsp72/73 and 2.5 kb respectively which is consistent with the ex­ 66 pected sizes. All HSP mRNAs were constitutively ex­ pressed except for HSP70. Levels of HSP70 mRNA in control animals were not clearly detectable despite greater RNA loading for control samples (Fig. 1). An increase in 66 hsp54 HSP27 and HSP70 mRNA was detected in the left cerebral cortex as early as 2 h after MCAO with a maximal response being visible at 24 h for both mRNAs. No induction of HSP27 and HSP70 mRNA was detected by hsp90 Northern analysis in contralateral cerebral cortex. The 97- expression of HSP56 mRNA in the cerebral cortex of tg control animals was low and no increase in expression was detected in response to MCAO. HSP60 mRNA expression in the ipsilateral cerebral cortex was markedly elevated above constitutive levels seen in both the contralateral 66 8-tubulin cortex at 8 and 24 h after MCAO and fg controls. How­ ever, HSP90 mRNA expression was constitutively ex­ pressed, unlike HSP60, and no response was observed on Fig. 3. Western blots showing the time course of levels of HSP27, Northern blots. HSP54, HSP60, HSP72/73 and HSP90 following MCAO. Protein was Quantitation of four HSP mRNAs was determined by extracted from tissue homogenates, separated by electrophoresis, blotted slot blot analysis for up to 24 h after MCAO (Fig. 2). A and incubated with antibodies for HSP27, HSP54, HSP60, HSP72, significant increase in HSP27 mRNA was detected in the HSP72/73, HSP90 and p-tubulin. Molecular weight markers in kDa are indicated by arrows. ipsilateral cortex as early as 2 h after MCAO, being 2.7-fold above the level seen in control animals. The levels of HSP27 were higher at 2, 4, 8 and 24 h compared to those in the contralateral cortex and 5.5 and 6.6 times those in the contralateral cortex and tg controls. The greater than those in the fg control cortex. A variable maximum increase was seen at 24 h where levels were 33 induction of HSP90 mRNA was seen in the ipsilateral times greater than those in the contralateral cortex and 9.9 cortex and no significant increase was detected when times greater than those in the ?g control cortex. The levels comparing ipsilateral to contralateral cortical levels. Signif­ of HSP70 mRNA in the ipsilateral cerebral cortex after icant elevation was seen at 8 and 24 h post-occlusion MCAO also showed a rapid and sustained induction at 2, against fg controls. HSP56 levels were too low for slot blot 4, 8 and 24 h compared to contralateral cortex of animals quantitation. with MCAO (3-, 4.7-, 5.3-, and 18-fold respectively) and to fg controls (3-, 2.8-, 3.7- and 8.3-fold, respectively). 3.2. Effect of experimental cerebral ischaemia on the The overall expression of HSP60 mRNA was of a much expression of heat shock proteins lower magnitude than HSP27 or HSP70 mRNA, but was significantly induced in the ipsilateral cerebral cortex at 8 The timecourse of the levels in the cerebral cortex of and 24 h where levels were 4.9 and 5.2 times greater than six heat shock proteins was determined up to 24 h after M.J.D. Wagstaff et al / Molecular Brain Research 42 (1996) 236-244 241 unilateral MCAO (Fig. 3). All the antibodies used for the increased further at 24 h, whereas the HSP27 protein was Western blots bound to protein bands of the expected not detected before 24 h. HSP60 mRNA levels increased at molecular weight. The HSP27 antibody detected a single 8 h with a visible protein increase at 24 h. The HSP56 and band in the ipsilateral cerebral cortex at 24 h but not at HSP90 mRNA levels appeared unchanged but there ap­ other times or on the contralateral side. This demonstrates peared to be more HSP54/56 protein on the occluded side in conjunction with the slot blots in Fig. 3 that an induc­ at 24 h than on the contralateral side. HSP90 protein, tion at 2 h of HSP27 mRNA leads to an increase in protein however, remained apparently unchanged. It is important synthesis which is not detectable until 24 h, probably to note the delay between an increase in mRNA levels in reflecting the differing sensitivities of the assays. As shown the HSP27, 60 and 70 slots blots and the corresponding from mRNA analysis, HSP60 was expressed constitutively increase in protein levels. and levels were increased in the ipsilateral cerebral cortex The gene for HSP70 showed, at 2 h, a rapid increase in at 24 h. On densitometry after tubulin ratio correction this mRNA and an increase in protein at 8 h unique to the was a 3.3-fold increase compared to Iq controls. The occluded side. At 24 h the protein level was further HSP72 antibody binds to the inducible form of the HSP70 markedly increased. HSP70 has already been shown to be family and was therefore not constitutively expressed but protective against stress; the chaperonin properties of was detected at 8 and 24 h in the ipsilateral cerebral HSP70 have been proposed as the mechanism responsible cortex, a similar 3.3-fold increase between the timepoints for this protection through binding to and inhibiting pro­ on densitometry. The binding of the HSP72/73 antibody tein degradation at times of stress [35]. HSP70 may bind to to the blot produced one band, in which there appears to be and inhibit the protein synthesis machinery, which has increased levels at 24 h on the occluded side. This increase previously been shown to delay neuronal death in the is probably attributable to the increase HSP72 protein hippocampus during global ischaemia [47]. MK-801, an levels bearing in mind the result with the HSP72 specific A^-methyl-D-aspartate (NMDA) receptor antagonist lowers antibody, but we cannot be sure. Thus, it appears there are the amount of inducible HSP72 produced during sublethal no gross changes in HSP73. ischaemia in the gerbil, and inhibits the resulting tolerance A small 1.2-fold ipsilateral increase was detected in supporting the above finding that protection is mediated HSP54/56 protein levels only at 24 h but with no apparent via the glutamate toxicity pathway [20]. increase in mRNA, possibly reflecting an increase in trans­ HSP27, an aB-crystallin homologue, is a member of lation. The band of higher molecular weight on the HSP54 the small heat shock protein family. We have shown blot was due to the binding of HSP72 antibody to HSP72 significant mRNA induction in great excess of the consti­ protein, as a separate experiment. The HSP90 antibody, tutive expression on the occluded side as early as 2 h however, detected little change in protein levels at the post-occlusion, along with HSP70 and an increase in pro­ timepoints investigated, in agreement with the mRNA tein levels at 24 h, the only detectable HSP27 band. The data. function of HSP27 is unknown. It has been demonstrated that HSP27 acts as a chaperonin under heat shock condi­ tions, binding to and preventing the aggregation of citrate synthase and a-glucosidase [19]. It has also been shown 4. Discussion that HSP27 prevents actin depolymerization during hyper­ thermia [31]. During heat shock, HSP27 dissociates from a In this study, we have demonstrated for the first time an 700-kDa complex with aB-crystallin due to a conforma­ increase in levels of HSP60 mRNA and protein in focal tional change [53], and is subsequently activated by phos­ ischaemia. We have not only confirmed the previously phorylation on which the degree of actin stabilisation and published HSP72 and 27 mRNA levels, but demonstrated thermotolerance may depend [29]. There is also a rapid, that increases in protein levels of both of these are also several-fold increase in the synthesis of HSP27 mRNA. apparent. We also studied these in parallel with the other This evidence suggests that activated HSP27 is intimately HSPs (HSP56 and 90) and their mRNA levels in order to involved in the development of at least some of the compare any changes that occurred and plot their time protective mechanisms during thermotolerance. Our re­ courses. The HSP90 mRNA and protein levels did not sults, along with previous data generated during experi­ appear to be raised compared to the Iq control levels, or mental ischaemia [16] suggest there may be potential for a the corresponding contralateral levels, and the protein lev­ protective role during ischaemia. In one study, however, els appeared relatively unaffected. However, the HSP56 the HSP27 raised during global cerebral ischaemia was protein levels appeared to increase over time on the oc­ shown to be localised to the glial cells in the subregions of cluded side, although with no detectable mRNA increase. the hippocampus and not to the tolerant areas of the CAl Comparing the mRNA increases of the various HSPs, it regions of the hippocampus [21]; implying that HSP27 appears that the levels of HSP70 and 27 mRNAs are the may not be involved with the mechanisms of tolerance. If first to be elevated post-occlusion, with increases visible at the glial cells are stabilised by HSP27 during ischaemia, 2 h. HSP72 protein was first detected at 8 h, and then however, this may affect local glutamate or toxin concen­ 242 M.J.D. Wagstaff et al. / Molecular Brain Research 42 {1996) 236-244 trations of the extracellular space and consequently reduce strated that HSP60 mRNA and protein is constitutively the effects of ischaemia. expressed, which suggests that its function is necessary for The function of HSP56 remains to be elucidated. It has the general housekeeping of the cell. Although mRNA been shown to possess peptidyl prolyl cis-trans isomerase levels were previously characterised after transient global activity, which serves a chaperone function [36]. It forms ischaemia [1], we have demonstrated that HSP60 mRNA is an integral part of the untransformed steroid receptor significantly inducible during the permanent occlusion complex, dissociating on the binding of steroid to the model of focal cerebral ischaemia, and shown an increase receptor [4,43]. HSP56 has not been shown to be inducible in the levels of the protein. HSP60, like HSP70 may act as during global or focal cerebral ischaemia. The poor hy­ a molecular chaperone and protect the intracellular pro­ bridization of our cDNA probe to mRNA on the slot blots teins from aggregation and degradation during stress. has left our results inconclusive, but the Western blot If an inhibition of protein synthesis is the mechanism of results suggest that some increase in HSP56 synthesis may HSP protection during ischaemia, the initial, subsequent be occurring. The synthetic immunosuppressant FK506 has neurological dysfunction may be due to an inability to been shown to induce tolerance during focal cerebral synthesise neurotransmitters or signal transduction ischaemia but the mechanism has not been uncovered. It molecules. Metabolic recovery following a transient is­ has two major binding proteins; one of which, FKBP12 chaemic insult may partly depend on the tissue’s capacity stabilises the calcium releasing ryanodine receptor and for counteracting and clearing localised toxic agents during therefore make it a putative candidate for the signalling reperfusion. This response may be aided by the increase of mechanism of this tolerance [6]. HSP56, however, is also the chaperonin activity of the HSPs, and/or it may be an FK506 binding protein (FKBP56/FKBP59) and so we involved with the interaction of these proteins with each felt it necessary, in consideration of the effects of other other and the steroid receptor complex. We have docu­ HSPs, to study the expression of this HSP. It has been mented increases in HSP60, 27 and 72 mRNA and protein disputed whether binding of FK506 to HSP56 does affect levels, the manipulation of which may therefore provide steroid receptor function, one study claims there is no the key toward a prophylactic or therapeutic approach to effect [18]; whereas another has shown that it potentiates cerebral ischaemia in the future. the transcriptional activity of the progesterone receptor [46]. The mechanism of immunosuppression has not yet been proved to be related to the steroid receptor, but it is Acknowledgements known that FK506 does act in T-cells by interacting with the cell signalling molecule calcineurin [51]. We thank Jacques Landry for kindly donating the Chi­ HSP90 is also a constituent of the untransformed steroid nese hamster HSP27 cDNA [30] and the HSP27 peptide receptor complex, its dissociation allows binding of the antibody L2R3 [32]. We also thank Marie Claire Lebeau steroid receptor to DNA, but its action is thought to be for kindly donating the rabbit p59 (HSP56) cDNA, and enabling rather than inhibitory for the activation of the Professor R.S. Gupta for the HSP60 cDNA. complex [39]. HSP90a mRNA has been shown previously to be induced in the CAl cells of the hippocampus by transient global ischaemia [22] but focal cerebral ischaemia References has not induced any change [16]. We have shown that no significant increase in HSP90 mRNA between 2 and 24 h [1] Abe, K., Kawagoe, J., Aoki, M. and Kogure, K., Changes of is visible in the slot blots compared to the contralateral mitochondrial DNA and heat shock protein gene expressions in levels or the controls, and confirmed this at the protein gerbil hippocampus after transient forebrain ischaemia, J. Cereb. level. HSP90’s properties as a protein synthesis inhibitor Blood. Flow. Metab., 13 (1993) 773-780. [41] and its affinity for tyrosine kinases (e.g. pp60'' ®'^‘^) and [2] Amin, V., Gumming, D.V.E., Coffin, R.S. and Latchman, D.S., The degree of protection provided to neuronal cells by a pre-conditioning therefore the Ras signalling pathway all indicate its role as stress correlates with the amount of heat shock protein 70 it induces a chaperone [42]. HSP70 has been demonstrated to be and not with the similarity of the subsequent stress, Neurosci. Lett., required for the incorporation of HSP90 into the steroid 200 (1995) 85-88. receptor complex [17]. HSP90 binds HSP56 and also pos­ [3] Amin, V., Gumming, D.V.E. and Latchman, D.S., Over expression sesses ATPase activity [36]. The interaction between the of heat shock protein 70 protects neuronal cells against both thermal and ischaemic stress but with different efficiencies, Neurosci. Lett., three HSPs (56, 70 and 90) involved with the steroid 206 (1996) 45-48. receptor complex may have an effect on the tolerant state [4] Bagchi, M.K., Tsai, S.Y., Tsai, M.J. and O’Malley, B.W., Proges­ of the cell. terone enhances target gene transcription by receptor free of heat HSP60 is also a molecular chaperone that binds to shock proteins hsp90, hsp56, and hsp70. Mol. Cell Biol., 11 (1991) unfolded precursors before the export of secretory proteins 4998-5004. [5] Bochkareva, E.S., Lissin, N.M. and Girshovich, A.S., Transient [5,33] and possesses some ATPase activity, preventing the association of newly synthesized unfolded proteins with the heat- aggregation of misfolded proteins, such as that which shock GroEL protein. Nature, 336 (1988) 254-257. occurs during a cellular insult [8,10]. We have demon­ [6] Brillantes, A.B., Ondrias, K., Scott, A., Kobrinsky, E., Ondriasova, M.J.D. Wagstaff et al. / Molecular Brain Research 42 (1996) 236-244 243

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