Cerebral Vascular Dysfunction in Alzheimer's Disease by Robert D. Bell Submitted in Partial ...

187 Pages·2011·5.01 MB·English

Cerebral Vascular Dysfunction in Alzheimer’s Disease

by

Robert D. Bell

Submitted in Partial Fulfillment
Of the
Requirements for the Degree
Doctor of Philosophy

Supervised by
Professor Berislav V. Zlokovic
Department of Pathology
School of Medicine and Dentistry

University of Rochester
Rochester, New York
2010
ii

Dedication
I would like to dedicate this thesis to my family for all of their love and support.
iii
Curriculum Vitae
Robert Donald Bell was born in Rochester, New York on September 18, 1982.
He attended The Aquinas Institute in Rochester from 1996-2000 earning a New York
State High School Regents Diploma. Robert pursued his undergraduate education at
St. Bonaventure University, St. Bonaventure NY in 2000. During the summer of 2002
he traveled to Oxford, England as a participant in the Francis E. Kelley Oxford
Program, one of St. Bonaventure’s most prestigious summer programs, to study the
history of natural sciences at Somerville College, Oxford University. Robert
graduated St. Bonaventure University with a Bachelors of Science in Psychology,
cum laude, in the spring of 2004 and soon after accepted a position as a research
technical assistant at Socratech Research Laboratories LLC in Rochester, New York.
He worked at Socratech Research Laboratories from 2004-2006. Robert began his
graduate studies in Pathology at the University of Rochester Medical Center in the
summer of 2006. Shortly thereafter, he decided to pursue research focused on
studying the role of neurovascular dysfunction during neurodegenerative disease
pathogenesis in the Laboratory of Dr. Berislav V. Zlokovic. Robert received his
Masters of Science degree in 2009. The Department of Pathology at the University of
Rochester School of Medicine and Dentistry awarded Robert with Travel Awards for
Outstanding Research Poster Presentation in 2008 and 2010, and an Award for
Outstanding Research Publication by a Pathology Graduate Student in 2009. In 2009,
Robert was also honored to be elected the Student Ambassador of the University of
Rochester Pathways of Human Disease Pathology Graduate Program Admissions
Committee and was also awarded "Outstanding Student Contribution to the Pathology
Graduate Program" by the Pathways of Human Disease Graduate Program. Robert
has presented at the Society of Neuroscience Conference annually since 2005. In
2010, Robert attended the 2010 St. Jude National Graduate Student Symposium.
iv
Publications
1. Bell RD, Winkler EA, Sagare AP, Singh I, Larue B, Deane R, Zlokovic BV.
Pericytes Control Key Neurovascular Functions and Neuronal Phenotype in
the Adult Brain and during Brain Aging. Neuron. 2010;68(3):409-27.

2. Zlokovic BV, Deane R, Sagare AP, Bell RD, Winkler EA. Low-density
lipoprotein receptor-related protein-1: a serial clearance homeostatic
mechanism controlling Alzheimer's amyloid beta-peptide elimination from the
brain. J Neurochem. 2010;115(5):1077-89. PMCID: 2972355.

3. Winkler EA, Bell RD, Zlokovic BV. Pericyte-specific expression of PDGF
beta receptor in mouse models with normal and deficient PDGF beta receptor
signaling. Mol Neurodegener. 2010;5:32. PMCID: 2936891.

4. Zhu D, Wang Y, Singh I, Bell RD, Deane R, Zhong Z, Sagare A, Winkler EA,
Zlokovic BV. Protein S controls hypoxic/ischemic blood-brain barrier
disruption through the TAM receptor Tyro3 and sphingosine 1-phosphate
receptor. Blood. 2010;115(23):4963-72. PMCID: 2890172.

5. Bell RD, Zlokovic BV. Neurovascular mechanisms and blood-brain barrier
disorder in Alzheimer's disease. Acta Neuropathol. 2009;118(1):103-13.
PMCID: 2853006.

6. Deane R, Bell RD, Sagare A, Zlokovic BV. Clearance of amyloid-beta
peptide across the blood-brain barrier: implication for therapies in Alzheimer's
disease. CNS Neurol Disord Drug Targets. 2009;8(1):16-30. PMCID:
2872930.

7. Bell RD, Deane R, Chow N, Long X, Sagare A, Singh I, Streb JW, Guo H,
Rubio A, Van Nostrand W, Miano JM, Zlokovic BV. SRF and myocardin
regulate LRP-mediated amyloid-beta clearance in brain vascular cells. Nat
Cell Biol. 2009;11(2):143-53. PMCID: 2654279.

8. Zhong Z, Ilieva H, Hallagan L, Bell R, Singh I, Paquette N, Thiyagarajan M,
Deane R, Fernandez JA, Lane S, Zlokovic AB, Liu T, Griffin JH, Chow N,
Castellino FJ, Stojanovic K, Cleveland DW, Zlokovic BV. Activated protein
C therapy slows ALS-like disease in mice by transcriptionally inhibiting
SOD1 in motor neurons and microglia cells. J Clin Invest.
2009;119(11):3437-49. PMCID: 2769191.

9. Long X, Bell RD, Gerthoffer WT, Zlokovic BV, Miano JM. Myocardin is
sufficient for a smooth muscle-like contractile phenotype. Arterioscler
Thromb Vasc Biol. 2008;28(8):1505-10. PMCID: 2574857.
v
10. Sagare A, Deane R, Bell RD, Johnson B, Hamm K, Pendu R, Marky A,
Lenting PJ, Wu Z, Zarcone T, Goate A, Mayo K, Perlmutter D, Coma M,
Zhong Z, Zlokovic BV. Clearance of amyloid-beta by circulating lipoprotein
receptors. Nat Med. 2007;13(9):1029-31. PMCID: 2936449.

11. Chow N, Bell RD, Deane R, Streb JW, Chen J, Brooks A, Van Nostrand W,
Miano JM, Zlokovic BV. Serum response factor and myocardin mediate
arterial hypercontractility and cerebral blood flow dysregulation in
Alzheimer's phenotype. Proc Natl Acad Sci U S A. 2007;104(3):823-8.
PMCID: 1783398.

12. Bell RD, Sagare AP, Friedman AE, Bedi GS, Holtzman DM, Deane R,
Zlokovic BV. Transport pathways for clearance of human Alzheimer's
amyloid beta-peptide and apolipoproteins E and J in the mouse central
nervous system. J Cereb Blood Flow Metab. 2007;27(5):909-18. PMCID:
2853021.

13. Wu Z, Guo H, Chow N, Sallstrom J, Bell RD, Deane R, Brooks AI, Kanagala
S, Rubio A, Sagare A, Liu D, Li F, Armstrong D, Gasiewicz T, Zidovetzki R,
Song X, Hofman F, Zlokovic BV. Role of the MEOX2 homeobox gene in
neurovascular dysfunction in Alzheimer disease. Nat Med. 2005;11(9):959-
65.
vi
Acknowledgements

I would like to thank my Ph.D. advisor, Dr. Berislav V. Zlokovic, for teaching me
how to become a better scientist, providing me with an outstanding learning
environment and helping me understand the rewards of hard work. I would like to
thank Dr. Rashid Deane for his many helpful discussions regarding experimental
designs and statistical analysis. Next, I would like to thank Dr. Abhay P. Sagare, Dr.
Nienwen Chow, Dr. Jan Sallstrom and Dr. Xiaochun Long for their friendship,
support and help during my Ph.D. studies. Furthermore, I would like to extend my
appreciation to all former and current members of the Zlokovic Laboratory that have
made my Ph.D. training so enjoyable.
I would also like to thank the members of my Ph.D. Thesis Committee, Drs. Joseph
Miano, Edward Brown, Therese Wiedmer and Kim Tieu for their helpful scientific
and motivational comments throughout my Ph.D. training.

Finally, I would like to express my unconditional gratitude to my wife, Kristina Lynn
Bell for her love and support during my Ph.D. Training.
vii
Abstract
The neurovascular unit is comprised of brain endothelium, pericytes, vascular
smooth muscle cells (VSMC), astrocytes, microglia and neurons. Complex and
dynamic communication between the cells of the neurovascular unit is essential for
maintaining normal brain function. Recently, the role of cerebral vascular dysfunction
has been highlighted in several neurodegenerative disease processes, such as
Alzheimer’s disease (AD). Here, I utilized multiple experimental models to test the
central hypothesis that cerebral vascular dysfunction can contribute to AD
pathogenesis. We found that (1) cerebral vascular dysfunction, mediated by pericyte
deficiency in adult mice, can lead to neurodegeneration, (2) molecular changes
specifically within brain VSMC can contribute to the development of AD-like
pathologies in the cerebral cortex and (3) a known inherited genetic risk factor for AD
may contribute to the cerebral vascular damage that is present during disease
pathogenesis.

First, pericytes play a key role in the development of the cerebral
microcirculation, although the exact role of pericytes in the adult brain remains
elusive. Using adult viable mice with varying degrees of pericyte deficiency, we show
that pericyte loss leads to reduction in brain microcirculation causing chronic
perfusion stress and hypoxia, and blood-brain barrier breakdown associated with
brain accumulation of vasculotoxic and/or neurotoxic serum proteins. Age-dependent
vascular damage in pericyte-deficient mice precedes neuronal degenerative changes,
and learning and memory impairment. Thus, pericytes control key neurovascular
functions that are necessary for proper neuronal structure and function, and pericyte
loss results in progressive age-dependent vascular-mediated neurodegeneration. This
study provides proof-of-principle evidence supporting the hypothesis that vascular
damage may initiate neurodegenerative disease processes.

Next, we investigate the molecular basis of a vascular-specific insult mediated
by amyloid β-peptide (Aβ) deposition in cerebral vessels, called cerebral amyloid
angiopathy, which contributes to AD pathogenesis. Here, we report increased levels
viii
of two proteins, serum response factor (SRF) and myocardin (MYOCD), in cerebral
VSMC in AD and in two mouse models of AD generates an Aβ non-clearing VSMC
phenotype via transactivation of sterol regulatory element binding protein-2, which
downregulates low density lipoprotein receptor-related protein-1, a key Aβ clearance
receptor. Hypoxia stimulated SRF/MYOCD expression in human cerebral VSMC and
in an AD mouse model. We suggest SRF and MYOCD constitute a new
transcriptional switch controlling Αβ cerebrovascular clearance and progression of
AD. This study supports the hypothesis that molecular changes specifically in
vascular cells of the neurovascular unit may contribute to AD pathogenesis.

Finally, the apolipoprotein E4 (APOE4) allele is an inherited genetic risk
factor for the development of neurological disorders that are associated with
neurovascular dysfunction, including AD. The mechanism by which APOE affects
the neurovascular unit is largely unknown. Here, we report that APOE2 and APOE3,
but not APOE4, effectively maintain blood-brain barrier (BBB) integrity, brain
vascular density and cerebral blood flow (CBF) in Apoe-/- mice. We next found an
increase in peptidylprolyl isomerase A (PPIA), a proinflammatory cytokine that
mediates extracellular matrix degradation and endothelial and neuronal apoptosis also
known as cyclophilin A, expression in the brains of Apoe-/- and APOE4-expressing
mice. Interestingly, lack of PPIA or PPIA inhibition with cyclosporine inhibited BBB
disruption and CBF reductions and improved neuronal spine density in Apoe-/- and
APOE4-expressing mice. Our findings suggest APOE plays a role in maintaining
neurovascular integrity, and PPIA may be a therapeutic target for neurovascular
dysfunction present in AD.

In summary, cerebral vascular dysfunction, whether initiated by a loss of key
brain vascular cells, neurovascular cell-specific molecular changes and/or genetic
predisposition, may be an important target for the development of therapeutics for
AD and other neurodegenerative disease processes with associated cerebral vascular
dysfunction.
ix
Table of Contents
List of Tables xi
List of Figures xii
List of Commonly Used Abbreviations xv
Foreword 1
Chapter 1 Introduction- Neurovascular mechanisms and blood–brain 5
barrier disorder in Alzheimer’s disease
Chapter 2- Cerebral pericyte deficiency disrupts brain 23
microcirculation leading to neurodegeneration
Abstract 24
Introduction 25
Materials and Methods 27
Results 41
Discussion 62
Chapter 3- SRF and myocardin regulate LRP1-mediated amyloid-ββββ 67
clearance in brain vascular cells
Abstract 68
Introduction 69
Materials and Methods 71
Results 81
Discussion 100
x

Chapter 4- The role of apolipoprotein E genotype and peptidylprolyl 104
isomerase A in Alzheimer’s disease neurovascular uncoupling
Abstract 105
Introduction 106
Materials and Methods 108
Results 115
Discussion 125
Chapter 5- Conclusion 127
References 129
Appendices 155