Microglia and Complement in Alzheimer's Disease with Cerebral Amyloid Angiopathy

134 Pages·2016·3.7 MB·English

Loma Linda University
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Loma Linda University Electronic Theses, Dissertations & Projects
6-1-2013
Microglia and Complement in Alzheimer's Disease
with Cerebral Amyloid Angiopathy
Matthew K. Zabel
Loma Linda University
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LOMA LINDA UNIVERSITY
School of Medicine
in conjunction with the
Faculty of Graduate Studies

____________________

Microglia  and  Complement  in  Alzheimer’s  Disease
with Cerebral Amyloid Angiopathy

by

Matthew K Zabel

____________________

A Dissertation submitted in partial satisfaction of
the requirements for the degree of
Doctor of Philosophy in Anatomy

____________________

June 2013
© 2013

Matthew K Zabel
All Rights Reserved

ACKNOWLEDGEMENTS

I have many people to thank for the success of this doctoral work. I would first
like to express my deepest gratitude to my mentor, Wolff M. Kirsch, MD, who has been
tremendously supportive of me as a PhD student in his laboratory and of my future goals
as a physician-scientist. Dr. Kirsch, you have an undying passion for science and the joy I
gain from my work stems from the excitement you bring to the lab everyday. It has not
felt like a job for the last four years. Among the many aspects of science and medicine in
which you have mentored me, none surpass the perseverance you have instilled in my
daily life.
I would also like to thank those members of the Kirsch Center for Neurosurgery
Research that have stood by me during the long (and sometimes frustrating) hours in the
lab. Thank you to Jackie Knecht, who has been irreplaceable in keeping The Center
running so smoothly; and to my present and future colleagues, Andrew Crofton and
Tanya Freeman—my  comrades  in  the  metaphorical  “trenches”  of  science.
I am very thankful to my committee members for their help in making this work
possible and hope to continue working with each of them in some capacity in the future. I
would especially like to thank Dr. Vinters for his ongoing guidance and encouragement
as I progress through my career as a scientist.
I would additionally like to express my gratitude to the Department of Pathology
and Human Anatomy, including Dr. Nava, Dr. Oberg, Dr. Escobar and Dr. Wright, who
have provided me with this wonderful opportunity and trusted in my abilities. They are
truly a second family to me.
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Finally, I want to thank my immensely supportive and wonderful wife, Aubrey,
and family who have watched me go from collegiate student-athlete to research scientist
and have been with me during the whole journey.
This work has been published in the journal Brain Pathology and funded, in part,
by NIH grant AG20948 and NSF Grant No. MRI-DBI 0923559 (S.M. Wilson) and by
Loma Linda University School of Medicine.

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CONTENTS

Approval Page .................................................................................................................... iii

Acknowledgements ............................................................................................................ iv

Table of Contents ............................................................................................................... vi

List of Tables ................................................................................................................... viii

List of Figures .................................................................................................................... ix

List of Abbreviations ...........................................................................................................x

Abstract .............................................................................................................................. xi

Chapter

1. Introduction ..............................................................................................................1

Epidemiology  and  Genetics  of  Alzheimer’s  Disease .........................................1
Clinical & Pathological Diagnosis .....................................................................3

Summary of AD Pathological Hallmarks ....................................................5
Clinical Diagnosis: The Need for Early Biomarkers ...................................9

Introduction to Cerebral Amyloid Angiopathy ................................................12

Detection and Diagnosis of CAA ..............................................................12
Aβ  Deposition:  Cerebral  Topology  and  Vessel  Distribution .....................14
Production  and  Clearance  of  Aβ  in  CAA ..................................................18

Enzymatic Digestion ............................................................................21
Clearance  of  Aβ  Through  the  BBB ......................................................22
Retrograde, Perivascular Drainage ......................................................24

Microglia  and  the  Complement  Cascade  in  Aβ  Clearance ........................26

The Role of Microglia in Neurodegeneration ......................................26
Complement and AD/CAA ..................................................................29

2. A  Shift  In  Microglial  β-Amyloid  Binding  in  Alzheimer’s  Disease Is
Associated With Cerebral Amyloid Angiopathy ...................................................35

Abstract ............................................................................................................37
Introduction ......................................................................................................38
vi
Methods............................................................................................................40

Tissue Selection .........................................................................................40
Microvessel Enrichment ............................................................................41
Western Blot Analysis ...............................................................................41
Immunohistochemistry ..............................................................................43
Immunoprecipitation ..................................................................................44
Statistical Analysis .....................................................................................45

Results ..............................................................................................................45

APOE4 and CR1 Genotyping Analysis .....................................................45
α2M  and  C3b  Levels  in  Occipital  Lobe .....................................................48
Microglial  Aβ  Binding  Via  CD11b ...........................................................51
Co-Immunoprecipitation of CD11b ...........................................................52
MAC on Occipital Lobe Cerebral Vasculature ..........................................57

Discussion ........................................................................................................67
References ........................................................................................................75

3. Complement  Receptor  1  in  Alzheimer’s  disease  and  cerebral amyloid
angiopathy ..............................................................................................................80

Introduction ......................................................................................................80
Materials and Methods .....................................................................................85

Human Postmortem Tissue ........................................................................85
Immunohistochemistry ..............................................................................85

Results and Discussion ....................................................................................86
References ........................................................................................................92

4. Conclusion .............................................................................................................93

Discussion ........................................................................................................93
Future Directions .............................................................................................96

References ........................................................................................................................100

Appendix ..........................................................................................................................119
A. Supplementary Figure 1 ...................................................................................119
B. Supplementary Figure 2 ...................................................................................120
C. Supplementary Figure 3 ...................................................................................121
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TABLES

Tables Page

1. Patient Demographics ............................................................................................47

viii
FIGURES

Figures Page

1. APP Metabolism ......................................................................................................8
2. CAA histopathology ..............................................................................................16
3. Aβ  Clearance  Mechanisms ....................................................................................20
4. Western blot analysis of a2M and C3b ..................................................................50
5. Confocal  microscopic  analysis  of  the  CD11b/Aβ  complex ...................................54
6. CD11b Co-Immunoprecipitation ...........................................................................56
7. Western blot analysis of C5b-9 and CD59 ............................................................60
8. Confocal microscopic analysis of C5b-9 and thioflavin ........................................62
9. Colocalization of smooth muscle actin and C5b-9 ................................................64
10. Western blot and immunohistochemical analysis of C6 ........................................66
11. Proposed mechanism for complement-mediated amyloid clearance .....................72
12. Complement Receptor 1 ........................................................................................84
13. CR1 immunohistochemistry ..................................................................................90

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