protease induced plasticity: matrix metalloproteinase-1 activation of protease activated receptor 1

115 Pages·2017·9.25 MB·English

PROTEASE INDUCED PLASTICITY: MATRIX METALLOPROTEINASE-1
ACTIVATION OF PROTEASE ACTIVATED RECEPTOR 1 INDUCES NEURONAL
STRUCTURAL PLASTICITY

A Dissertation
Submitted to the Faculty of the
Graduate School of Arts and Sciences
of Georgetown University
in partial fulfillment of the requirements for the
degree of
Doctor of Philosophy
in Neuroscience

By

Megan Allen, B.A.

Washington, D.C.
January 13, 2017
Copyright 2017 by Megan Allen
All Rights Reserved
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PROTEASE INDUCED PLASTICITY: MATRIX METALLOPROTEINASE-1
ACTIVATION OF PROTEASE ACTIVATED RECEPTOR 1 INDUCES NEURONAL
STRUCTURAL PLASTICITY

Megan Allen, B.A.

Thesis Advisors: Katherine E. Conant, M.D. & Kathleen A. Maguire-Zeiss, Ph.D.

ABSTRACT

The brain continues to change long after development ceases. Structural
modifications to mature cells in the brain as well as the integration newborn neurons
through adult hippocampal neurogenesis contribute to plasticity found in the healthy brain.
Neurological disorders and injury, however, can disrupt proper plasticity processes leading
to deleterious outcomes. Elucidating the exact mechanisms of plasticity in varied contexts
remains a highly researched topic. The brain is comprised of a complex system of neurons,
glia, and other supporting cells. In neurons, modifications can occur on processes that
extend from the neuronal cell body (dendrites) as well as tiny membranous protrusions
stemming from the dendrites also known as spines. This study reveals how a specific
extracellular matrix remodeling protein, matrix metalloproteinase 1 (MMP-1), directs
neuronal structure complexity through activation of a G-protein coupled receptor called
protease activated receptor 1 (PAR1). MMP-1 mediated activation of PAR1 increases
neuronal dendritic arborization, spine number, and increased proliferation of adult neural
progenitor cells, which in turn stimulates aberrant behaviors associated with disrupted
plasticity in vivo.
Matrix metalloproteinases (MMPs) are a family of secreted endopeptidases
expressed by neurons and glia. Regulated MMP activity contributes to physiological
synaptic plasticity, while dysregulated activity can stimulate injury. Disentangling the role
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individual MMPs play in neuroplasticity is difficult due to overlapping structure and
function as well as cell-type specific expression. To overcome these challenges, this study
uses a novel system to investigate the selective overexpression of MMP-1 in GFAP
expressing cells in vivo. More specifically, application of exogenous MMP-1, in vitro,
stimulates PAR1 dependent increases in intracellular Ca2+ concentration and dendritic
arborization. Overexpression of MMP-1, in vivo, increases dendritic complexity and
induces biochemical and behavioral endpoints consistent with increased GPCR signaling.
Further, data suggest that activation of the MMP-1/PAR1 axis increases both progenitor
cell proliferation and neuronal fate specification in adult neurogenesis. These data are
exciting because they demonstrate that an astrocyte-derived protease can influence
neuronal plasticity through an extracellular matrix independent mechanism.

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ACKNOWLEDGEMENTS

I would like to thank all of the students and faculty at Georgetown University and
Georgetown University Medical Center, especially those in the Neuroscience and
Pharmacology and Physiology departments as well as Interdisciplinary Program in
Neuroscience. Discussions with you have challenged my ability to critically evaluate
scientific data and encouraged me to design innovative experiments to address fundamental
questions in the field of neuroscience.
Further, I would like to express extreme gratitude to my advisors, Dr. Katherine
Conant and Dr. Kathleen Maguire-Zeiss. I have benefited immensely from a strong female
influence and this has helped me believe that as a woman in science I too can flourish at a
senior position in academia. Drs. Conant and Maguire-Zeiss have taught me that there are
different ways of thinking about experiments and this diversity creates innovation. Both
have supported a collaborative lab environment where I felt free to create, design, and learn
without limits. I would also like to thank present and past members of the lab for
challenging me during data presentations and discussions about our work. Stefano Daniele,
Amanda Edwards, Lorenzo Bozzelli, Carissa Winland, and Dr. Yan Li were especially
helpful throughout my time in the lab. In addition, I have collaborated with great scientists
while a student at Georgetown University that has led to exciting projects and publications.
I would like to thank Drs. Maria Maddalena Valente, Mariagrazia Grilli, Jian Young Wu,
Kenneth Kellar, and Stefano Vicini.
I would also like to acknowledge the faculty within the core facilities at
Georgetown University Medical Center, who without their help, this body of work would
not be possible. Throughout the years I have worked with Mr. Peter Johnson of the the
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Microscopy & Imaging Shared Resource, Dr. Jan Blancato of the Cytogenetics Shared
Resource, Dr. Karen Creswell of the Flow Cytometry and Sorting facility, and Drs. Olga
Rodriguez and Yi Chien of the Preclinical Imaging Resource Laboratory.
Additionally, I thank my thesis committee members, Drs. Barry Wolfe, Gerard
Ahern, Rhonda Dzakpasu, and Morley Hollenberg, for their thoughtful comments and
critical evaluation of my progress over the years. Your commitment to the training of young
scientists has left a lasting impression.
Last but certainly not least, thank you to my friends and family who provided
constant love and support throughout the past five years. You helped me stay focused when
times got tough!

Many thanks,
Megan Allen

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TABLE OF CONTENTS
CHAPTER I: INTRODUCTION ........................................................................................ 1
A. Matrix metalloproteinases .................................................................................................. 4
B. G-protein coupled receptors and their role plasticity ...................................................... 6
C. Protease activated receptors and central nervous system plasticity .............................. 8
CHAPTER II: MATERIALS AND METHODS .............................................................. 16
A. Animals .............................................................................................................................. 16
i. Generation of human matrix metalloproteinase-1 transgenic animals. ................. 16
ii. PAR1 KO animals. ............................................................................................... 17
B. Genomic polymerase chain reaction (PCR) ................................................................... 17
C. Preparation of primary cultures enriched for neurons .................................................. 17
D. Preparation of primary astrocytes ................................................................................... 18
E. Dendritic Sholl analysis .................................................................................................... 19
F. Ca2+ imaging ....................................................................................................................... 20
G. Western blot ....................................................................................................................... 20
H. Enzyme linked immunosorbent assay (ELISA) ............................................................ 21
I. RNAscope fluorescent in situ hybridization combined with ......................................... 22
immunohistochemistry .......................................................................................................... 22
J. Matrix metalloproteinase-1 activity assay ....................................................................... 23
K. In vivo magnetic resonance imaging and spectroscopy ................................................ 23
L. Golgi staining and dendritic spine analysis .................................................................... 24
M. Behavioral testing ............................................................................................................. 25
i. Three-chambered sociability assay. ....................................................................... 25
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ii. Elevated plus maze. .............................................................................................. 26
iii. Morris water maze. .............................................................................................. 26
N. Isolation and culture of adult neural progenitor cells (aNPCs) from murine
hippocampus ........................................................................................................................... 27
O. Adult neural progenitor cell differentiation and proliferation ..................................... 28
P. Flow cytometry .................................................................................................................. 29
Q. Statistical analyses ............................................................................................................ 30
CHAPTER III: GENERATION AND CHARACTERIZATION OF AN IN VIVO
MODEL TO STUDY THE EFFECTS OF GLIAL-DERIVED MMP-1 ON NEURONAL
STRUCTURE ................................................................................................................... 31
A. Results ................................................................................................................................ 31
i. Generation and characterization human matrix metalloproteinase-1 transgenic
mouse (hMMP-1 Tg) ................................................................................................ 31
ii. Concurrent expression of human matrix metalloproteinase messenger ribonucleic
acid and glial fibrillary acidic protein in situ ............................................................ 33
iii. Human matrix metalloproteinase-1 transgenic animals displayed deficits in
behaviors associated with synaptic plasticity ........................................................... 34
iv. Human matrix metalloproteinase-1 transgenic animals show changes in brain
metabolites relevant to astrogliosis as determined by in vivo magnetic resonance
spectroscopy imaging ................................................................................................ 37
v. Human matrix metalloproteinase-1 transgenic animals display dendritic changes
indicative of structural remodeling at the synapse .................................................... 38
B. Summary ............................................................................................................................ 38
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CHAPTER IV: MMP-1 INDUCED ACTIVATION OF PAR1 INCREASES
COMPLEXITY OF DENDRITIC ARBOR IN HIPPOCAMPAL NEURONS ............... 45
A. Results ................................................................................................................................ 46
i. Human recombinant matrix metalloproteinase-1 application increases dendritic
tree complexity as well as intracellular Ca2+ in primary culture .............................. 46
ii. Pharmacological inhibition and genetic deletion of protease activated receptor 1
reverse human recombinant matrix metalloproteinase-1 effects on neuronal
morphology and Ca2+ flux ......................................................................................... 48
iii. Evidence supporting protease activated receptor 1 activation in human matrix . 49
metalloproteinase transgenic animals ....................................................................... 49
B. Summary ............................................................................................................................ 50
CHAPTER V: MMP-1 INDUCED ACTIVATION OF PAR1 IN ADULT
HIPPOCAMPAL NEUROGENESIS ............................................................................... 52
A. Results ................................................................................................................................ 56
i. Adult neural progenitor cells from human matrix metalloproteinase- transgenic
animals proliferate significantly more than adult neural progenitors cultured from
wild-type littermate controls ..................................................................................... 56
ii. Adult neural progenitor cells derived from human matrix metalloproteinase-1
transgenic animals show increased differentiation towards a neuronal lineage ....... 57
iii. Sex determining region Y-box 2 (SOX2) positive adult neural progenitor cells
are increased in the hippocampus of human matrix metalloproteinase-1 transgenic
animals ...................................................................................................................... 58
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iv. Inhibition of protease activated receptor 1 activity reverses increases in adult
neural progenitor cell proliferation and biases a neuronal cell fate .......................... 58
B. Summary ............................................................................................................................ 59
CHAPTER VI: DISCUSSION ......................................................................................... 64
A. Matrix metalloproteinase-1 mediated activation of protease activated receptor 1
influences neuronal structure, which may have consequences for plasticity-related
behaviors ................................................................................................................................. 65
B. Matrix metalloproteinase-1 mediated activation of protease activated receptor 1
increases adult neural progenitor cell proliferation and biases neural cell fate
specification ............................................................................................................................ 70
C. Conclusions and overall significance ............................................................................. 72
CHAPTER VII: FUTURE DIRECTIONS ....................................................................... 75
CHAPTER VIII: BIBLIOGRAPHY................................................................................. 78

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