Mechanistic impact of the Swedish app-mutation and caspase-3 cleaved c-terminal presenilin fragment in the neurotoxic effects of beta-amyloid [Elektronische Ressource] / by Celio Azinheiro Marques

English
191 Pages
Read an excerpt
Gain access to the library to view online
Learn more

Description

MECHANISTIC IMPACT OF THE SWEDISH APP-MUTATION AND CASPASE-3 CLEAVED C–TERMINAL PRESENILIN FRAGMENT IN THE NEUROTOXIC EFFECTS OF BETA-AMYLOID Inaugural-Dissertation for the Achievement of the Doctor’s Degree of Natural Sciences submitted to the Faculty of Pharmaceutical and Chemical Sciences of the Johann Wolfgang Goethe-University Frankfurt am Main by Celio Azinheiro Marques from Frankfurt am Main Frankfurt (2004) para os meus paisMESCHANISTISCHE AUSWIRKUNGEN DER SCHWEDISCHEN APP-MUTATION UND DES CASPASE-3 GESPALTENEN C-TERMINALEN PRESENILIN FRAGMENTS AUF DIE NEUROTOXISCHEN EFFEKTE VON BETA AMYLOID Dissertation zur Erlangung des akademischen Grades vorgelegt beim Fachbereich 14 (Pharmazeutische und chemische Wissenschaften) der Johann Wolfgang-Goethe Universität Frankfurt am Main von Celio Azinheiro Marques aus Frankfurt am Main Frankfurt 2004 Vom Fachbereich Chemische und Pharmazeutische Wissenschaften der Johann Wolfgang Goethe-Universität als Dissertation angenommen. Dekan: Prof. H. Schwalbe Gutachter: Prof. W.E. Müller Pharmakologisches Institut für Naturwissenschaftler der Johann Wolfgang Goethe-Universität in Frankfurt Marie-Curie-Str. 9 60349 Frankfurt PD. Dr.

Subjects

Informations

Published by
Published 01 January 2005
Reads 31
Language English
Document size 5 MB
Report a problem

MECHANISTIC IMPACT OF THE SWEDISH APP-
MUTATION AND CASPASE-3 CLEAVED C–TERMINAL
PRESENILIN FRAGMENT IN THE NEUROTOXIC
EFFECTS OF BETA-AMYLOID



Inaugural-Dissertation
for the Achievement of the Doctor’s Degree
of Natural Sciences
submitted to the Faculty of Pharmaceutical and Chemical Sciences
of the
Johann Wolfgang Goethe-University
Frankfurt am Main


by
Celio Azinheiro Marques
from Frankfurt am Main
Frankfurt (2004)






para os meus paisMESCHANISTISCHE AUSWIRKUNGEN DER
SCHWEDISCHEN APP-MUTATION UND DES CASPASE-3
GESPALTENEN C-TERMINALEN PRESENILIN FRAGMENTS
AUF DIE NEUROTOXISCHEN EFFEKTE VON BETA
AMYLOID

Dissertation zur Erlangung des akademischen
Grades
vorgelegt beim Fachbereich 14
(Pharmazeutische und chemische
Wissenschaften)

der Johann Wolfgang-Goethe Universität
Frankfurt am Main
von Celio Azinheiro Marques
aus Frankfurt am Main



Frankfurt 2004









Vom Fachbereich Chemische und Pharmazeutische Wissenschaften der
Johann Wolfgang Goethe-Universität als Dissertation angenommen.








Dekan: Prof. H. Schwalbe

Gutachter: Prof. W.E. Müller
Pharmakologisches Institut für Naturwissenschaftler der
Johann Wolfgang Goethe-Universität in Frankfurt
Marie-Curie-Str. 9
60349 Frankfurt


PD. Dr. Anne Eckert
Neurobiologisches Forschungslabor
Psychiatrische Universitätsklinik
Wilhelm Klein-Strasse 27
4025 Basel
Schweiz



Datum der Disputation:




1Table of contents


Table of Contents



1. Introduction 6
1.1 THE IMPACT OF ALZHEIMER’S DISEASE (AD),
A CHALLENGE TO SCIENCE AND SOCIETY 6
1.2 PATHOLOGICAL CHANGES IN AD BRAIN 7
1.2.1 Changes in brain 7
1.2.2 Amyloid plaques 8
1.2.3 Neurofibrillary tangles 9
1.3 GENETICS OF ALZHEIMER’S DISEASE 10
1.3.1 Causative genes of inherited forms of AD 10
1.3.2 Apolipoprotein E 10
1.3.3 Other genetic factors 12
1.4 MOLECULAR MECHANISMS OF ALZHEIMER’S DISEASE 14
1.4.1 The Amyloid-Precursor-Protein 14
1.4.2 The beta amyloid peptide 16
1.4.3 Presenilin 19
1.5 NEURONAL DEGENERATION IN ALZHEIMER’S DISEASE 21
1.5.1 Cell death mechanisms in central nervous system 21
1.5.1.1 Necrosis 22
1.5.1.2 Apoptosis 22
1.5.2 Caspases 23
1.5.2.1 Caspase structure 23
1.5.3 Apoptotic cell signaling 26
1.5.3.1 Extrinsic apoptotic pathway 26
1.5.3.2 Intrinsic apoptotic pathway 27
1.5.4 Caspase-Substrates 29
1.5.5 Apoptosis and Alzheimer’s disease 30
1.6 OXIDATIVE STRESS IN ALZHEIMER’ S DISEASE 31
1.6.1 Generation of Reactive Oxygen Species 31
1.6.1 Stress-activated Protein Kinase and AD 32
1.7 AIM OF THESIS 35
1.8 ZIELE DER ARBEIT 37 2Table of contents

2. Material and Methods 39
2.1 APPARATUS 39
2.2 CELLBIOLOGICAL METHODS 40
2.2.1 Culture off cell lines 40
2.2.2 Used cell lines 40
2.2.3 Cell counting and viability test 43
2.2.4 Cryoconservation of cells 43
2.2.5 Transfection of cells 43
2.3 GENETIC ENGINEERING 44
2.3.1 Plasmids 45
2.3.2 Gelelektrophoresis 46
2.3.3 Quantification of Nucleic Acid Concentration 47
2.3.4. RNA- Isolation 47
2.3.5 cDNA- synthesis 48
2.3.6 Reverse Transcriptase PCR (RT-PCR) 49
2.3.7 Cloning of PS-plasmides 50
2.3.8 Transformation in competent Bacteria 51
2.3. 9 Plasmid-Minipreparation 52
2.3.10 Analytical Restrictionanalysis 53
2.4 ANALYTICAL CELLBIOLOGICAL METHODS: 54
2.4.1 Detection of cell viability 54
2.4.1.1. Induction of cell death 54
2.4.1.2. Quantification of apoptosis by flow cytometry 54
2.4.1.3 MTT-assay 56
2.4.1.4 Caspase-Assay 58
2.4.1.5 Determination of mitochondrial membrane potential 61
2.4.1.6 ATP-levels using a bioluminescence assay 62
2.4.2 Proteinchemical methods 63
2.4.2.1 Determination of protein amount by Lowry protein test: 63
2.4.2.2 Western blotting 63
2.4.2.3 Immunoblotting of cytoplasmic and mitochondrial proteins 65
2.4.2.4 Western Blot Stripping 65
2.4.3. Genomic DNA elektrophoresis 66
2.5. STATISTICAL EVALUATION OF THE DATA 66
3Table of contents

3. Results 67
3.1 CELL DEATH CASCADES IN A CELL MODEL EXPRESSING
MUTANT APP REFELECTING LOW Aß LOAD 67
3.1.1 Characterization of PC12 cell lines overexpressing APPwt and APPsw 67
3.1.2 APPsw mutation leads to an enhanced vulnerability to oxidative
stress induced apoptosis 68
3.2 CASPASE-ACTIVATION ON OXIDATIVE STRESS INDUCED CELL DEATH 70
3.2.1 Oxidative stress induces activation of caspase 2 in PC12 cells 70
3.2.2 Activation of caspase 8 in response to oxidative stress 72
3.2.3 Involvement of caspase 9 73
3.2.4 Increased caspase 3 activity of APPsw PC12 cells in response to oxidative stress 74
3.3 EFFECTS OF CASPASE INHIBITORS ON OXIDATIVE STRESS
INDUCED CELL DEATH 76
3.3.1 Prevention of apoptosis by caspase inhibitors 76
3.3.2 Reduction of caspase activity 78
3.3.2 Necrotic cell death 79
3.3.3 Protection of metabolic activity by caspase inhibitors 81
3.3.4 Protection of mitochondrial membrane potential 83
3.4 ACTIVATION OF JNK DURING OXIDATIVE STRESS
IN PC 12 CELLS 85
3.4.1 Aß doses effect of APP on JNK activation 85
3.5 EFFECTS OF JNK INHIBITOR SP600125 ON OXIDATIVE STRESS
INDUCED CELL DEATH 87
3.5.1 Effects on caspase activity 87
3.5.2 Protection of mitochondria 88
3.5.3 JNK activation in mitochondrial fraction 89
3.5.3 JNK inhibitor studies on apoptosis 89
3.6 MESSENGER RNA EXPRESSION OF APOPTOTIC FACTORS
DURING OXIDATIVE STRESS IN PC12 CELLS OVEREXPRESSING APP 90
3.6.1 Members of the Bcl-2 family 90
3.6.2 Apoptosis inducing factor 91
3.6.3 Glycogen synthesis kinase 3ß 92
3.6.4 GAPDH 93
3.7 REDUCTION OF TROPHIC SUPPORT ENHANCE APOPTOSIS
IN APPSW EXPRESSING PC12 CELLS 94
3.7.1 DNA ladder 90 4Table of contents


3.8 A CELL MODEL REFLECTING HIGH Aß LOAD 96
3.8.1 Characterization of HEK cell lines overexpressing APPwt and APPsw 96
3.8.2 Basal apoptosis is already increased in APPsw HEK cell 97
3.8.3 Oxidative stress induced cell death in HEK cells 98
3.8.4 Effects of caspase- inhibition in oxidative stress
induced cell death in HEK cells 98
3.8.5 JNK activation in HEK cells 100
3.8.6 Mitochondrial membrane potential 100
3.8.7 Effects of caspase 2 - and JNK inhibition on mitochondrial
membrane potential in HEK cells 102
3.8.8 ATP levels of APP transfected HEK cells 103
3.8.9 Messenger-RNA expression of apoptotic factors 104

3.9 APOPTOSIS IN CELL MODELS OVEREXPRESSING
PRESENILIN 1 108
3.9.1 Neuronal cell model 108
3.9.1.1 Characterization of PC12 cells expressing Presenilin 108
3.9.1.2 Antiapoptotic effects of Presenilin during oxidative stress 110
3.9.1.3 Presenilin and JNK activation 111
3.9.1.4 Presenilin 1 overexpression does not effect mitochondrial
membrane potential 112
3.9.1.5 Presenilin and Bcl-xl expression 113
3.9.1.6 Vulnerability against Staurosporine induced apoptosis 114
3.9.2 Peripheral cell model 115
3.9.2.1Fas induced apoptosis in Jurkat cells 116
3.9.2.2 Staurosporine induced apoptosis in Jurkat cells 117

4. Discussion118
4.1 APPSW MUTATION LEADS TO AN ENHANCED VULNERABILITY
TO OXIDATIVE STRESS-INDUCED APOPTOSIS IN PC12 CELLS 118
4.2 CASPASE-ACTIVATION IN OXIDATIVE STRESS
INDUCED CELL DEATH 119
4.2.1. The Swedish APP mutation induces activation of caspase 2 in PC12 cells 119
4.2.2 Enhanced caspase 8 activation by the Swedish APP mutation
during oxidative stress 123 5Table of contents

4.2.3 Oxidative stress induce mitochondrial dysfunction leading to caspase 9 activation 126
4.3 THE IMPLICATION OF THE JNK ACTIVATION IN NEUROTOXIC
MECHANISM INDUCED BY THE SWEDISH APP MUTATION 129
4.3.1 JNK inhibitor protects mitochondrial membrane potential and
reduce caspase 9 and 3 activity 133
4.4 EFFECTS OF CASPASE INHIBITORS IN OXIDATIVE STRESS
INDUCED CELL DEATH IN PC 12 CELLS 134
4.5 BASAL APOPTOSIS IS INCREASED IN APPSW HEK CELLS 136
4.5.1 The Swedish APP mutations impairs metabolism in HEK cells 137
4.5.2 Caspase inhibition in HEK cells 138
4.5.3 JNK activation in HEK cells 139
4.6 SWEDISH APP MUTATION INDUCES TRANKRIPTION OF AIF 139
4.7 NEUROTOXIC MECHANISMS CAUSED BY THE SWEDISH APP
MUTATION: MITOCHONDRIAL DYSFUNCTION, OXIDATIVE
STRESS, CASPASES AND JNK PATHWAY 141
4.8 NEUROPROTECTIVE STRATEGIES INHIBITING CASPASES AND JNK 143
4.9 IMPLICATION OF PRESENILIN 1 IN APOPTOSIS 145

5. Summary 149
6. Zusammenfassung 152
7. References 158
8. Abbreviations 181
9. Bibliography 183
10. Danksagung 186
11. Curriculum Vitae 187
Introduction 6

1. Introduction

1.1 THE IMPACT OF ALZHEIMER’S DISEASE, A CHALLENGE TO SCIENCE AND
SOCIETY

In 1907, Alois Alzheimer, a German Psychiatrist, described at a meeting in Tübingen the
symptoms as well as the neuropathological features of a strange degenerative disease of the
brain cortex (Alzheimer 1907). His original patient, a woman referred to as Auguste D. in his
report, suffered from several symptoms like progressive memory impairment; disordered
cognitive function; altered behaviour including paranoia, delusions, loss of social
appropriateness and a progressive decline in language function.
Neither he nor his audience could expect the impact of this disease for the 21th century. Due
to an improved medical care, life expectancy increased dramatically within the last century.
This resulted in a burgeoning number of individuals achieving the age at which
neurodegenerative disorders become evident. Today, Alzheimer’s disease (AD) is recognized
as a major public health problem in developed nations and the most common cause of
dementia among people aged 65 and older. The impact of AD for the major public health
acquires a growing importance for our society:
• According to the American National Institut of Aging, AD affects probably 20–30
million people worldwide.
• An estimated 10 percent of Americans over the age of 65 and half of those over age 85
have AD.
• Currently, more than four million Americans currently suffer from this disease, and
the number is projected to balloon to 10-15 million over the next several decades.
• AD is after heart disease and cancer the third most expensive disease to treat in the
USA., costing society close to $100 billion annually.
• In Germany actually about 1.000.000 people have AD. This number is expected to
duplicate within the next decade as a result of an adjustment of the age pyramid.

Besides the financial aspect for the health care, of course there is the dramatic impact of AD
for the individual families. The patients are helpless and require full-time health care.
Families, friends and caregivers struggle with great emotional and physical stress to cope with
the mental and physical changes of the concerned family member.
6