Exercise Alters Glial Cell Line-Derived Neurotrophic Factor (GDNF)

205 Pages·2017·3.09 MB·English

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Dissertations Graduate College
4-2013
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Monica Janine McCullough
Western Michigan University, [email protected]
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McCullough, Monica Janine, "Exercise Alters Glial Cell Line-Derived Neurotrophic Factor (GDNF) Protein
Content in the Spinal Cord" (2013). Dissertations. 151.
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EXERCISE ALTERS GLIAL CELL LINE-DERIVED NEUROTROPHIC FACTOR
(GDNF) PROTEIN CONTENT IN THE SPINAL CORD
by
Monica J. McCullough
A dissertation submitted to the Graduate College
in partial fulfillment of the requirements
for the degree of Doctor of Philosophy
Department of Biological Sciences
Western Michigan University
April 2013
Doctoral Committee:
John M. Spitsbergen, Ph.D., Chair
Christine Byrd-Jacobs, Ph.D.
Cindy Linn, Ph.D.
Damon Miller, Ph.D.
EXERCISE ALTERS GLIAL CELL LINE-DERIVED NEUROTROPHIC FACTOR
(GDNF) PROTEIN CONTENT IN THE SPINAL CORD
Monica Janine McCullough, Ph.D.
Western Michigan University, 2013
Neurotrophic factors may play a role in exercise-induced neuroprotective effects;
however, it is not known if exercise mediates changes in glial cell line-derived
neurotrophic factor (GDNF) protein levels in the spinal cord. The aim of the studies
described herein was to determine if exercise alters GDNF protein expression in the
lumbar spinal cord of healthy and diseased animals. The lumbar spinal cord was
analyzed from adult rats aged 6-, 12-, 18- and 24-months, and from transgenic
amyotrophic lateral sclerosis (ALS) mice and wild-type mice aged 3 months. Animals
had undergone either forced wheel running, voluntary wheel running or swimming for
either 2- or 4-weeks or 6 months in duration. GDNF protein was quantified via enzyme-
linked immunosorbent assay and Western blot. Immunohistochemical analysis localized
GDNF in choline acetyltransferase (ChAT)-positive motor neurons and cell body areas
were measured. Results indicate that short-term exercise (2 weeks) increases GDNF
protein content in the rodent lumbar spinal cord. Chronic exercise (4 weeks to 6 months)
has no effect on GDNF protein content in the rodent lumbar spinal cord. All exercise
protocols increased the size of ChAT-positive motor neuron cell body areas of healthy,
non-diseased animals. Treatment with neutralizing antibodies for GDNF in the
transgenic ALS animals blocked the beneficial effects of exercise on motor neurons.
These results suggest that short-term exercise elicits an increase in neurotrophic factors
via an activity-dependent relationship that occurs with GDNF protein expression in spinal
cord. Understanding how neurotrophic factors are regulated by physical activity is
crucial for maintaining a healthy motor nervous system and for developing therapies for
individuals with compromised motor nervous systems, e.g. due to aging or disease.
ACKNOWLEDGMENTS
I would like to being by acknowledging the excellent guidance and mentoring that
I have received during these past years from Dr. John Spitsbergen. I could not have
asked for a better mentor and am grateful for all the opportunities that I've had because of
him.
I would also like to thank my committee members, Dr. Christine Byrd-Jacobs, Dr.
Cindy Linn and Dr. Damon Miller, who have taken the time out of their busy schedules
to help me succeed as a teacher and a researcher.
I would like to thank all of my family and friends for their constant support and
for always being positive. Most importantly, I would like to thank my wonderful
husband, Matt, for his continued support during this process, and for enduring all my
piles of articles in every room of our house. He has been so supportive and patient with
me, and I could not have asked for a better husband and friend.
Monica Janine McCullough
ii
TABLE OF CONTENTS
ACKNOWLEDGMENTS ................................................................................................ . ii
LIST OF TABLES................................................................................................... xi
LIST OF FIGURES.................................................................................................. xii
LIST OF ABBREVIATIONS................................................................................. xv
CHAPTER
I. INTRODUCTION................................................................................... 1
Spinal cord....................................................................................... 1
Motor neurons.................................................................................. 4
Development of motor neurons........................................................ 5
Neurotrophic factors........................................................................ 6
GDNF.............................................................................................. 7
GDNF signaling............................................................................... 8
GDNF distribution........................................................................... 10
GDNF is essential for motor neurons.............................................. 11
Exercise alters neurotrophic factors in the motor nervous system 14
II. EXPERIMENTAL DESIGN.................................................................. 18
iii
Table of Contents - Continued
CHAPTER
Specific aims.................................................................................... 18
Rationale and development.............................................................. 20
Murine animals as model organisms for studying
neurotrophic factors............................................................. 20
Optimal time for euthanasia after the last bout of exercise 22
Duration of exercise............................................................. 28
Spinal cord selection............................................................ 32
GDNF protein expression in the spinal cord following
exercise................................................................................ 35
Differences between Fischer 344 and Sprague-Dawley
rats....................................................................................... 40
Mouse studies ...................................................................... 44
III. SHORT-TERM EXERCISE INCREASES GDNF PROTEIN
LEVELS IN THE SPINAL CORD OF YOUNG AND OLD RATS 47
Introduction...................................................................................... 47
Experimental procedures.................................................................. 49
Subjects................................................................................ 49
Training protocol................................................................. 49
iv
Table of Contents - Continued
CHAPTER
Tissue processing................................................................. 50
GDNF protein quantification............................................... 51
Western blot......................................................................... 52
Immunohistochemistry........................................................ 53
Measurement of motor neuron cell body size..................... 54
Statistical analysis............................................................... 54
Results ............................................................................................. 55
Short-term exercise increases GDNF protein in the
lumbar spinal cord of 6-month-old rats............................... 55
Short-term exercise increases GDNF protein in the
spinal cord of 24-month-old rats......................................... 61
Discussion........................................................................................ 67
Motor neuron size increases at the same time as
GDNF levels following short term exercise........................ 67
Low-intensity exercise is a potent stimulus for
enhancing neurotrophic factor levels.................................. 68
v
Table of Contents - Continued
CHAPTER
Punctate immunoreactivity for GDNF is altered with
exercise and age................................................................... 69
Molecular weight of GDNF changes with age.................... 70
GDNF transport following exercise.................................... 71
Exercise stressors................................................................. 72
Conclusion........................................................................... 73
IV.CHRONIC EXERCISE DOES NOT ALTER GDNF PROTEIN
CONTENT IN ADULT RAT SPINAL CORD..................................... 74
Introduction...................................................................................... 74
Materials and methods..................................................................... 77
Experimental design............................................................ 77
Tissue processing................................................................. 78
GDNF protein quantification............................................... 78
Western blot......................................................................... 79
Immunohistochemistry......................................................... 80
Measurement of motor neuron cell body size..................... 81
vi
Table of Contents - Continued
CHAPTER
Statistical analysis................................................................ 81
Results.............................................................................................. 82
Four weeks of voluntary running........................................ 82
Six months of voluntary running......................................... 88
Discussion........................................................................................ 92
Aging increases molecular weight of GDNF in the
spinal cord............................................................................ 93
Chronic exercise does not increase neurotrophic
factor protein levels.............................................................. 93
Hypertrophy of motor neurons with chronic exercise....... 96
Conclusion........................................................................... 97
V. INVOLUNTARY EXERCISE INCREASES GDNF PROTEIN
CONTENT IN THE LUMBAR SPINAL CORD OF WILD
TYPE MICE........................................................................................... 98
Introduction...................................................................................... 98
Materials and methods..................................................................... 99
Training protocol.................................................................. 99
Tissue processing................................................................. 100
GDNF protein quantification............................................... 101
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