effects of intranasally administered dnsp-11 on the central dopamine system of normal and

209 Pages·2017·7.65 MB·English

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Theses and Dissertations--Neuroscience Neuroscience
2013
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James H. Sonne
University of Kentucky, [email protected]
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Sonne, James H., "EFFECTS OF INTRANASALLY ADMINISTERED DNSP-11 ON THE CENTRAL DOPAMINE
SYSTEM OF NORMAL AND PARKINSONIAN FISCHER 344 RATS" (2013). Theses and Dissertations--
Neuroscience. 5.
https://uknowledge.uky.edu/neurobio_etds/5
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The document mentioned above has been reviewed and accepted by the student’s advisor, on
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James H. Sonne, Student
Dr. Don Marshall Gash, Major Professor
Dr. Wayne A. Cass, Director of Graduate Studies
EFFECTS OF INTRANASALLY ADMINISTERED DNSP-11
ON THE CENTRAL DOPAMINE SYSTEM
OF NORMAL AND PARKINSONIAN FISCHER 344 RATS

_______________________________________

DISSERTATION
_______________________________________

A dissertation submitted in partial fulfillment of the
requirements for the degree of Doctor of Philosophy in the
College of Medicine
at the University of Kentucky

By
James William Hendry Sonne

Lexington, Kentucky

Director: Don Marshall Gash, Ph.D., Professor of Anatomy and Neurobiology

Lexington, Kentucky

2013

Copyright © James William Hendry Sonne
ABSTRACT OF DISSERTATION

EFFECTS OF INTRANASALLY ADMINISTERED DNSP-11
ON THE CENTRAL DOPAMINE SYSTEM
OF NORMAL AND PARKINSONIAN FISCHER 344 RATS

Due to the blood-brain barrier, delivery of many drugs to the brain has
required intracranial surgery which is prone to complication. Here we show that
Dopamine Neuron Stimulating Peptide 11 (DNSP-11), following non-invasive
intranasal administration, protects dopaminergic neurons from a lesion model of
Parkinson’s disease in the rat. A significant and dose-dependent increase in an
index of dopamine turnover (the ratio of DOPAC to dopamine) was observed in
the striatum of normal young adult Fischer 344 rats by whole-tissue
neurochemistry compared to vehicle administered controls.
Among animals challenged with a moderate, unilateral 6-hydroxy-
dopamine (6-OHDA) lesion of the substantia nigra, those treated repeatedly with
intranasally administered DNSP-11 exhibited greater numbers of tyrosine
hydroxylase (TH) positive dopaminergic neuronal cell bodies in the substantia
nigra and greater TH+ fiber density in the striatum when compared to animals
treated intranasally with vehicle only or a scrambled version of the DNSP-11
sequence. Lesioned animals that received intranasal DNSP-11 treatment did not
exhibit abnormal, apomorphine-induced rotation behavior, contrasted with
animals that received only vehicle or scrambled peptide that did exhibit
significantly greater rotation behavior.
In addition, the endogenous expression of DNSP-11 from the pro-region of
GDNF was investigated by immunohistochemistry with a custom, polyclonal
antibody. Signal from the DNSP-11 antibody was found to be differentially
localized from the mature GDNF protein both spatially and temporally. While
DNSP-11-like immunoreactivity extensively colocalizes with GDNF
immunoreactivity at post-natal day 10, the day of maximal GDNF expression,
DNSP-11-like signal was found to be present in the 3 month old rat brain with
signal in the substantia nigra, ventral thalamic nucleus, dentate gyrus of the
hippocampus, with the strongest signal observed in the locus ceruleus where
GDNF is not expressed. Results from immunoprecipitation of brain homogenate
were not consistent with the synthetic, amidated 11 amino-acid rat DNSP-11
sequence. However, binding patterns in the literature of NPY, the only
homologous sequence present in the CNS, do not recapitulate the
immunoreactive patterns observed for the DNSP-11 signal.
This study provides evidence for a potential easy-to-administer intranasal
therapeutic using the DNSP-11 peptide for protection from a 6-OHDA lesion rat
model of Parkinson’s disease.

KEYWORDS: Parkinson’s disease, GDNF, 6-OHDA, aging, non-invasive

James William Hendry Sonne

May 3rd, 2013
EFFECTS OF INTRANASALLY ADMINISTERED DNSP-11
ON THE CENTRAL DOPAMINE SYSTEM
OF NORMAL AND PARKINSONIAN FISCHER 344 RATS

By

James William Hendry Sonne

Don Marshall Gash, Ph.D.
Director of Dissertation

Wayne A. Cass, Ph.D.
Director of Graduate Studies

May 3rd, 2013
For my grandparents, Jody Tharp and Lloyd Gould Hendry
ACKNOWLEDGEMENTS

I am truly grateful for the help and invaluable assistance I have received,
without which this work would not have been feasible. My success is truly not my
own. First and foremost, I am indebted to my mentor, Don Marshall Gash, Ph.D.,
for his support, faith and confidence in me, as well as his friendship. Before
beginning my endeavors, when I envisioned a translational research laboratory, it
was the goals and motivations that he shares which were the core of my vision.
It is difficult to imagine how I can take a step upward from his laboratory.
My committee members have provided excellent guidance, and I
appreciate them providing me with resources and taking the time out of their
productive schedules to support my progress. They are Michael Bardo, Ph.D.,
Luke H. Bradley, Ph.D., Wayne A. Cass, Ph.D., James Geddes, Ph.D., Greg A.
Gerhardt, Ph.D., Zhiming Zhang, M.D., my outside examiner John T. Slevin,
M.D., and Susan Barron, Ph.D. for acting as a substitute committee member for
my defense. I would like to especially thank Yi Ai, M.D., Wayne Cass, Ph.D.,
Richard Grondin, Ph.D., and Zhiming Zhang, M.D. They are fantastic
investigators and sincere and caring people who have provided technical
assistance and a deep interest in my development as a scientist. I would also
like to thank the teaching faculty, especially April D. Richardson-Hatcher, Ph.D.
and Bruce E. Maley, Ph.D., for helping me to become a better instructor and
exposing me to the joy of helping students succeed.
The members of Dr. Gash’s laboratory and his collaborating laboratories
have been more than simply invaluable resources, but true friends that I know I
can trust. My fellow denizens of the laboratory have been tremendous in their
technical assistance and comic relief, especially Ramsey Edwards, M.B.A., Eric
Forman, Hamed Haghnazar, M.P.H., April Evans, Ryan Weeks, Daisy Ramos,
Ofelia Meagan Littrell, Ph.D. and Jennifer Moorehead. Most of all, Mallory J.
Stenslik, M.S. has provided countless hours of laughs and teamwork. These
people have kept me going during tough times.
iii
I am indebted to my grandparents, Jody T. and Lloyd G. Hendry, J.D., for
teaching me so much about life. They instilled in me the meaning of hard work,
devotion, and the importance of taking joy in a job well done, and taught me that
without these things success is not possible. I believe that every act they
performed was directed at making life better for their grandchildren. Their way of
looking forward is what made them successful, and it has influenced my life
beyond measure. They are my role models in every aspect of life, while my
parents, Mary C. H. Sonne, J.D., and Kenneth G. Sonne, Jr., have provided the
support to allow me to pursue my dreams wherever they lead.
Above all of these, I owe my wife, Stephanie Lynn Sonne, C.P.A.,
everything. During my long hours and tribulations, she has stood by me,
supporting me when I needed it the most. Without her love, I would be nothing.
These people have been my fuel, the oil in my engine, and the tread on
my tires. My successes and accomplishments are because of them, while my
missteps are purely mine own.
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TABLE OF CONTENTS
ACKNOWLEDGEMENTS ..................................................................................... iii
LIST OF TABLES .................................................................................................ix
LIST OF FIGURES ............................................................................................... x
LIST OF ABBREVIATIONS ................................................................................ xiii
Chapter One: Introduction .................................................................................... 1
Parkinson’s Disease ......................................................................................... 1
A History of Parkinson’s Disease .................................................................. 1
Signs and Symptoms .................................................................................... 2
Pathogenesis of Parkinson’s Disease ........................................................... 2
The Discovery of Dopamine .......................................................................... 5
Dopamine Biosynthesis and Signaling .......................................................... 6
Dopaminergic Pathways ................................................................................ 7
Basal Ganglia ................................................................................................ 8
Direct and Indirect Pathways ....................................................................... 10
Rat Models of Parkinson’s Disease ............................................................. 11
Treatment Options ....................................................................................... 13
Neurotrophic Factors ...................................................................................... 15
History ......................................................................................................... 15
The Effects of GDNF ................................................................................... 16
Clinical Trials of GDNF ................................................................................ 17
The Search for Mimetics, Signal and Pro-Proteins ...................................... 18
Development of DNSP-11 ........................................................................... 18
Delivery of Drugs to the Central Nervous System ........................................... 20
Blood-Brain Barrier ...................................................................................... 20
Penetration .................................................................................................. 21
Circumvention ............................................................................................. 22
Intranasal Administration Bypasses the Blood-Brain Barrier ........................... 23
Goal ................................................................................................................ 27
Study Outline .................................................................................................. 27
Chapter Two: Materials and Methods ................................................................. 36
Reagents ........................................................................................................ 36
Animals ........................................................................................................... 36
Ethics Statement ......................................................................................... 36
Animal Care and Housing ............................................................................ 36
Surgical Procedures .................................................................................... 36
Unilateral 6-OHDA lesion of the substantia nigra ........................................ 37
Blinded intranasal administration ................................................................. 37
Peptide Synthesis and Dilution ....................................................................... 38
MiniMitter Automated Monitoring System ....................................................... 39
Statistical analysis ........................................................................................... 39
Chapter Three: A study of the effects of intranasally administered DNSP-11 on
tyrosine hydroxylase positive dopaminergic neurons and their projections in a
6-hydroxydopamine lesion rat model of Parkinson’s disease ......................... 45
Hypothesis ...................................................................................................... 45
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