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

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.........................................................