Cet ouvrage fait partie de la bibliothèque YouScribe
Obtenez un accès à la bibliothèque pour le lire en ligne
En savoir plus

Oxidativer Stress im Alter und bei der Alzheimer Krankheit [Elektronische Ressource] : eine vergleichende Studie über oxidative Schäden und antioxidative Enzymaktivitäten in Mausmodellen und humanem Gehirngewebe / von Katrin Schüssel

De
326 pages
Oxidativer Stress im Alter und bei der Alzheimer Krankheit - Eine vergleichende Studie über oxidative Schäden und antioxidative Enzymaktivitäten in Mausmodellen und humanem Gehirngewebe Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. phil. nat.) vorgelegt beim Fachbereich Chemische und Pharmazeutische Wissenschaften der Johann Wolfgang Goethe - Universität in Frankfurt am Main von Katrin Schüssel aus Passau Frankfurt (2004) (D F 1) vom Fachbereich Chemische und Pharmazeutische Wissenschaften der Johann Wolfgang Goethe – Universität als Dissertation angenommen. Dekan: Prof. Dr. Harald Schwalbe Gutachter: Prof. Dr. Walter E. Müller, PD Dr. Anne Eckert. Datum der Disputation: 20. 12. 2004 OXIDATIVE STRESS DURING AGING AND IN ALZHEIMER’S DISEASE - A COMPARATIVE STUDY OF OXIDATIVE DAMAGE AND ANTIOXIDANT ENZYMATIC ACTIVITIES IN MOUSE MODELS AND HUMAN BRAIN TISSUE THESIS FOR THE DEGREE OF DOCTOR OF NATURAL SCIENCES (DR. PHIL. NAT.) SUBMITTED TO THE FACULTY OF CHEMICAL AND PHARMACEUTICAL SCIENCES OF THE JOHANN WOLFGANG GOETHE - UNIVERSITY FRANKFURT / MAIN BY KATRIN SCHÜSSEL FROM PASSAU FRANKFURT (2004) Der Glaube versetzt Berge, der Zweifel erklettert sie. Karl Heinrich Waggerl Meiner Familie und Matthias gewidmet. TABLE OF CONTENTS TABLE OF CONTENTS 1 INTRODUCTION ..............................................
Voir plus Voir moins

Oxidativer Stress im Alter und bei der
Alzheimer Krankheit
-
Eine vergleichende Studie über oxidative Schäden
und antioxidative Enzymaktivitäten
in Mausmodellen und humanem Gehirngewebe



Dissertation
zur Erlangung des Doktorgrades der Naturwissenschaften
(Dr. phil. nat.)


vorgelegt beim Fachbereich Chemische und
Pharmazeutische Wissenschaften
der Johann Wolfgang Goethe - Universität
in Frankfurt am Main

von
Katrin Schüssel
aus Passau

Frankfurt (2004)
(D F 1)







vom Fachbereich Chemische und Pharmazeutische Wissenschaften der Johann
Wolfgang Goethe – Universität als Dissertation angenommen.







Dekan: Prof. Dr. Harald Schwalbe
Gutachter: Prof. Dr. Walter E. Müller, PD Dr. Anne Eckert.
Datum der Disputation: 20. 12. 2004 OXIDATIVE STRESS DURING AGING AND IN
ALZHEIMER’S DISEASE
-
A COMPARATIVE STUDY OF OXIDATIVE DAMAGE AND
ANTIOXIDANT ENZYMATIC ACTIVITIES
IN MOUSE MODELS AND HUMAN BRAIN TISSUE



THESIS
FOR THE DEGREE OF
DOCTOR OF NATURAL SCIENCES
(DR. PHIL. NAT.)


SUBMITTED TO THE FACULTY OF CHEMICAL AND
PHARMACEUTICAL SCIENCES OF THE
JOHANN WOLFGANG GOETHE - UNIVERSITY
FRANKFURT / MAIN

BY
KATRIN SCHÜSSEL
FROM PASSAU

FRANKFURT (2004)


Der Glaube versetzt Berge, der Zweifel erklettert sie.
Karl Heinrich Waggerl


















Meiner Familie und Matthias gewidmet. TABLE OF CONTENTS
TABLE OF CONTENTS
1 INTRODUCTION .................................................................................................... 1
1.1 OXYGEN AND OXIDATIVE STRESS............................................................................. 2
1.1.1 Formation of reactive oxygen species ............................................................. 2
1.1.2 Physiological roles of reactive oxygen species................................................ 4
1.1.3 Oxidative stress................................................................................................ 5
1.1.3.1 Increased production of ROS from endogenous sources.......................... 5
1.1.3.2 Antioxidant defense .................................................................................. 7
1.1.3.3 Oxidative attack on cellular molecules................................................... 10
1.1.3.4 Intracellular signaling and transcription factors activated by ROS ........ 13
1.1.3.5 Role of ROS in cell death ....................................................................... 15
1.2 OXIDATIVE STRESS IN BRAIN AGING 18
1.2.1 Aging theories................................................................................................ 18
1.2.2 The free radical theory of aging .................................................................... 19
1.2.3 A role for oxidative stress in brain aging? .................................................... 20
1.3 OXIDATIVE STRESS AND ALZHEIMER’S DISEASE 23
1.3.1 Alzheimer’s disease – clinical symptoms, pathology and risk factors........... 23
1.3.1.1 Clinical picture of Alzheimer’s disease.................................................. 23
1.3.1.2 AD neuropathology................................................................................. 24
1.3.1.3 Genetic and non-genetic risk factors for AD .......................................... 26
1.3.2 Risk factors for sporadic AD ......................................................................... 27
1.3.3 Familial AD mutations and the amyloid beta hypothesis.............................. 29
1.3.3.1 The amyloid precursor protein APP – physiological processing and effect
of APP mutations................................................................................................ 29
1.3.3.2 Presenilins............................................................................................... 35
1.3.4 A role for oxidative stress in AD?.................................................................. 39
1.3.4.1 Oxidative stress in sporadic AD patients................................................ 39
1.3.4.2 Oxidative stress and Aβ toxicity............................................................. 40
1.3.4.3 Oxidative stress and presenilins.............................................................. 41
1.4 AIMS OF THESIS...................................................................................................... 42
2 MATERIALS AND METHODS ........................................................................... 45
2.1 MATERIALS............................................................................................................ 46
2.1.1 Chemicals....................................................................................................... 46
I TABLE OF CONTENTS
2.1.2 Kits .................................................................................................................48
2.1.3 Antibodies.......................................................................................................48
2.1.4 Buffers and media ..........................................................................................48
2.1.4.1 Buffers used for brain tissue preparation and homogenization...............49
2.1.4.2 Buffers used for lymphocyte preparation................................................49
2.1.4.3 Buffers for Western blots ........................................................................49
2.1.4.4 Buffers for DNA gel electrophoresis ......................................................50
2.1.5 Apparatus and other materials.......................................................................50
2.1.6 Computer software .........................................................................................52
2.1.7 Mice................................................................................................................52
2.1.7.1 C57BL/6J mice........................................................................................53
2.1.7.2 Transgenic mice ......................................................................................53
2.1.7.3 Summary of mice used in this thesis.......................................................55
2.1.8 Human brain tissue56
2.1.8.1 Cohort #1.................................................................................................56
2.1.8.2 Cohort #257
2.2 METHODS...............................................................................................................58
2.2.1 Preparation of tissues and cells .....................................................................58
2.2.1.1 Preparation of murine brain tissue ..........................................................58
2.2.1.2 Homogenization of human and murine brain tissue for determination of
antioxidant enzyme activities and lipid peroxidation products...........................59
2.2.1.3 Isolation of murine splenic lymphocytes ................................................59
2.2.2 Assays of antioxidant enzymes .......................................................................59
2.2.2.1 Superoxide dismutase assay....................................................................59
2.2.2.2 Glutathione peroxidase assay..................................................................60
2.2.2.3 Glutathione reductase assay61
2.2.3 Lipid peroxidation measurement62
2.2.4 Determination of reactive oxygen species in isolated splenic lymphocytes...63
2.2.4.1 Flow cytometric analysis of splenic lymphocytes...................................63
2.2.4.2 Staining of lymphocytes with ROS-sensitive fluorescent dyes ..............67
2.2.4.3 FACS analysis of splenic lymphocytes...................................................70
2.2.5 Western Blot analysis of APP and Aβ............................................................71
2.2.5.1 Preparation of brain samples for determination of soluble Aβ ...............71
2.2.5.2 Preparation of brain samples for determination of insoluble Aβ ............72
II TABLE OF CONTENTS
2.2.5.3 SDS PAGE and Western Blotting of brain extracts ............................... 72
2.2.5.4 Detection of APP, C99 and Abeta .......................................................... 73
2.2.5.5 Detection of actin as loading control ...................................................... 73
2.2.6 Quantification of Aβ by ELISA ................................................................. 73 1-40
2.2.7 Genotyping of transgenic mice ...................................................................... 74
2.2.7.1 DNA isolation from rodent tails ............................................................. 74
2.2.7.2 PCR reaction........................................................................................... 75
2.2.7.3 DNA gel electrophoresis......................................................................... 75
2.2.8 RT-PCR analysis of APP and PS1 expression in splenic lymphocytes.......... 76
2.2.9 Determination of protein content................................................................... 77
2.2.10 Calculations and Statistics........................................................................... 78
3 RESULTS ................................................................................................................ 81
3.1 EFFECTS OF AGING AND GENDER ON ROS METABOLISM IN BRAIN TISSUE AND
PERIPHERAL CELLS OF MICE ......................................................................................... 82
3.1.1 ROS metabolism in C57BL/6J mice during aging......................................... 82
3.1.1.1 Oxidative damage and enzymatic antioxidant defense in murine brains
during aging ........................................................................................................ 82
3.1.1.2 ROS levels in splenic lymphocytes during aging ................................... 86
3.1.2 Gender differences in ROS metabolism in C57BL/6J mice during aging ..... 89
3.1.2.1 Gender differences in oxidative damage and enzymatic antioxidant
defense in murine brains..................................................................................... 89
3.1.2.2 Gender differences in ROS levels in splenic lymphocytes..................... 92
3.2 ROS METABOLISM IN TRANSGENIC AD MOUSE MODELS ....................................... 94
3.2.1 ROS metabolism in PDGF-APP and/or PS1 transgenic mice....................... 95
3.2.1.1 Oxidative damage and enzymatic antioxidant defense in transgenic
mouse brains ....................................................................................................... 96
3.2.1.2 Analysis of splenic lymphocytes from PS1 transgenic mice.................. 98
3.2.2 ROS metabolism in brains from Thy1-APP transgenic mice....................... 107
3.2.2.1 ROS metabolism in brains fromice during aging
.......................................................................................................................... 108
3.2.2.2 Gender differences in ROS metabolism in Thy1-APP transgenic mice111
3.2.3 Cu/Zn-SOD activity in brains from APP23 mice......................................... 116
3.2.4 Comparative analysis of human APP expression and Aβ expression levels in
PDGF-APP, PDGF-APP/PS1 and Thy1-APP mouse models..................... 117
III TABLE OF CONTENTS
3.2.4.1 Western blot analysis of human APP and Aβ expression.....................117
3.2.4.2 Quantitative analysis of soluble Aβ levels in APP transgenic mice 1-40
with ELISA .......................................................................................................122
3.3 ROS METABOLISM IN BRAINS FROM AD PATIENTS ..............................................126
3.3.1 Cohort #1 .....................................................................................................126
3.3.1.1 Increased antioxidant metabolism in AD patients.................................126
3.3.1.2 Effect of gender on antioxidant metabolism in AD patients.................131
3.3.2 Cohort #2133
3.3.2.1 Changes in oxidative stress parameters in AD patients ........................133
3.3.2.2 Levels of soluble Aβ in brains from AD patients – correlation with 1-40
Apo E4 genotype...............................................................................................137
3.3.2.3 Correlations of lipid peroxidation products and antioxidant enzyme
activities with levels of soluble Aβ ..............................................................140 1-40
3.3.2.4 Correlations of lipid peroxidatie
activities with Apo E genotype .........................................................................141
3.3.2.5 Correlations of lipid peroxidatie
activities with mini mental status (MMSE) score .............................................143
4 DISCUSSION ........................................................................................................147
4.1 EFFECTS OF AGING AND GENDER ON ROS METABOLISM IN C57BL/6J MICE........148
4.1.1 Effect of aging on oxidative stress parameters in brain tissue ....................148
4.1.2 Effect of aging on ROS levels in peripheral cells.........................................158
4.1.3 Gender differences in oxidative stress-related parameters in mice.............163
4.1.4 Summary of aging-induced effects on ROS and oxidative stress parameters in
C57BL/6J mice.............................................................................................168
4.2 OXIDATIVE STRESS-RELATED PARAMETERS IN TRANSGENIC MICE BEARING FAD
MUTATIONS ................................................................................................................172
4.2.1 Effect of PS1 mutations on oxidative stress-parameters in transgenic mice172
4.2.2 Effects of APP mutations on oxidative stress-related parameters ...............186
4.2.2.1 Analysis of PDGF-APP and PDGF-APP/PS1 double transgenic mice 186
4.2.2.2 Analysis of Thy1-APP transgenic mice ................................................188
4.2.2.3 Reduced activity of Cu/Zn-SOD in APP23 transgenic mice ................196
4.2.3 Comparison of different AD transgenic mouse models................................198
IV TABLE OF CONTENTS
4.3 CHANGES IN OXIDATIVE STRESS-RELATED PARAMETERS IN SPORADIC AD PATIENTS
204
4.3.1 Cohort #1 ..................................................................................................... 204
4.3.1.1 Upregulation of antioxidant defense prevents oxidative damage......... 205
4.3.1.2 Gender differences in antioxidant metabolism and oxidative damage in
AD patients ....................................................................................................... 209
4.3.1.3 Summary of observations in cohort #1................................................. 211
4.3.2 Cohort #2 211
4.3.2.1 Increased oxidative damage despite partial upregulation of antioxidant
defense .............................................................................................................. 211
4.3.2.2 Correlation of oxidative stress-related parameters with levels of amyloid
beta, Apo E genotype and clinical severity of dementia................................... 212
4.3.2.3 Summary of observations in cohort #2................................................. 218
4.3.3 Comparison of results from cohort #1 and #2............................................. 218
4.4 COMPARATIVE SUMMARY AND PERSPECTIVES ..................................................... 226
4.4.1 Suitability of transgenic mice to study AD-relevant pathogenic mechanisms
..................................................................................................................... 226
4.4.2 Is HNE a major toxic factor in AD? ............................................................ 229
4.4.3 Gender differences in AD and hormone replacement therapy .................... 231
4.4.4 Is it possible to prevent AD by antioxidants? .............................................. 233
5 SUMMARY / ZUSAMMENFASSUNG.............................................................. 235
5.1 SUMMARY............................................................................................................ 236
5.1.1 The influence of aging studied in mice ........................................................ 236
5.1.2 Effects of FAD mutations on oxidative stress parameters........................... 237
5.1.3 Studies on human brain tissue from sporadic AD patients.......................... 238
5.1.4 Comparative summary and conclusions...................................................... 240
5.2 ZUSAMMENFASSUNG ........................................................................................... 242
5.2.1 Alterseffekte in Mäusen................................................................................ 242
5.2.2 Einfluß von familiären Alzheimer-Mutationen ............................................ 243
5.2.3 Untersuchungen an humanem Gehirngewebe von sporadischen
Alzheimerpatienten...................................................................................... 245
5.2.4 Vergleichende Zusammenfassung und Fazit................................................ 247
6 REFERENCES...................................................................................................... 249
V TABLE OF CONTENTS
7 ABBREVIATIONS ...............................................................................................297
8 INDEX OF FIGURES AND TABLES ................................................................301
8.1 INDEX OF FIGURES................................................................................................302
8.2 INDEX OF TABLES .................................................................................................304
9 BIBLIOGRAPHY307
9.1 ORIGINAL PUBLICATIONS AND REVIEWS...............................................................308
9.2 POSTERS...............................................................................................................308
9.3 ORAL PRESENTATIONS..........................................................................................309
10 ACKNOWLEDGMENTS / DANKSAGUNG ...................................................311
11 CURRICULUM VITAE.......................................................................................315
VI

Un pour Un
Permettre à tous d'accéder à la lecture
Pour chaque accès à la bibliothèque, YouScribe donne un accès à une personne dans le besoin