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dissertation entitled

NEUROCHEMICAL CHARACTERIZATION OF
NIGROSTRIATAL DOPAMINE NEURONAL FUNCTION
IN THE 1-METHYL-4-PHENYL-l,2,3,6-
TETRAHYDROPYRIDINE (MPTP) MODEL OF
PARKINSON’S DISEASE

presented by

Robert E. Drolet

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NEUROCHEMICAL CHARACTERIZATION OF NIGROSTRIATAL
DOPAMINE NEURONAL FUNCTION IN THE l-METHYL-4-
PHENYL-l,2,3,6-TETRAHYDROPYRIDINE (MPTP) MODEL OF
PARKINSON’S DISEASE

By

Robert Edward Drolet

A Dissertation
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

Doctor of Philosophy

Neuroscience Program

2006

Abstract

NEUROCHEMICAL CHARACTERIZATION OF NIGROSTRIATAL
DOPAMINE NEURONAL FUNCTION IN THE l-METHYL-4-PHENYL-l,2,3,6—
TETRAHYDROPYRIDINE (MPTP) MODEL OF PARKINSON’S DISEASE
By

Robert Edward Drolet

The gradual decline in the quality of life for Parkinson’s disease (PD) patients is
the result of a progressive worsening of motor function. The manifestation and gradual
worsening of motor symptoms is due to the progressive loss of nigrostriatal dopamine
(NSDA) neurons. Preventing the progressive loss of NSDA neurons therefore, should
halt the worsening of motor symptoms. The primary goal of the research conducted in
this dissertation was to identify detrimental mechanisms that may contribute to the
progressive degeneration of NSDA neurons. The strategy employed was to use a
neurotoxin to induce NSDA cell death similar to PD. Pharmacological approaches and
neurochemical and immunohistochemical techniques were then used to identify
detrimental mechanisms that may cause degeneration of the neurons that survive the
initial neurotoxic insult.

The compensatory increase in the activity of neurons that survive neurotoxic
insult is one potential mechanism that is likely to contribute to the progressive loss of
NSDA neurons because of the consequential increase in exposure to the toxic byproducts
derived from DA metabolism. Dissection of the neurochemical events at the axon
terminal revealed the dysfunctional regulation of the DA biosynthetic pathway was

responsible for the increased metabolism of DA that occurs during compensatory

activation. The rate of DA synthesis was accelerated as a result of increased expression
of the rate limiting enzyme tyrosine hydroxylase (TH). The over-activation of DA
synthesis, coupled with decreased storage capability, results in increased metabolism of
the newly synthesized DA.

The findings from this research suggested that therapeutic strategies that enhance
vesicular packaging capability in NSDA axon terminals may not only enhance the release
of newly synthesized DA but also protect these neurons from the toxic byproducts that
result from DA metabolism. Manipulating the expression of the a-synuclein protein may
be one such therapeutic target. In the absence of this protein, NSDA axon terminals are
less susceptible to neurotoxic lesion and have enhanced vesicular packaging capability
that results in increased storage of the newly synthesized DA and decreased metabolism
following a neurotoxic lesion. Taken together, the results from the studies described in
this dissertation have: 1) identified and characterized the dysfunctional regulation of the
DA biosynthetic pathway as a potential mechanism responsible for the progressive loss of
NSDA neurons in PD, 2) clarified the role that the a-synuclein protein in NSDA neuronal
demise and 3) identified a number of potential therapeutic targets that may ultimately

slow or halt the progressive nature of PD.

This dissertation and all of the work that it is comprised of, is dedicated to my
wife Alexis, without your continual support, guidance and encouragement this would not
have been possible,

and to my daughter Laynie, I look forward to the day I make you to read this.

iv

Acknowledgements

Mom and Dad, I could not have accomplished this without you. Thank you for
the support and encouragement throughout the years.

I sincerely thank my advisors Drs. John L. Goudreau and Keith J. Lookingland for
their help and guidance. I cannot convey how much I appreciate your dedication,
constructive criticism and motivation regarding my scientific training over the past few
years. I look forward to the years of collaboration in the future.

I would also like to thank the members of my guidance committee, Drs. Kathleen
Gallo, Puliyur MohanKumar, and Hongbing Wang for their thoughtful advice and
critique in the preparation of this work.

I wish to thank the Neuroscience program faculty and students for guiding my
scientific development and providing an excellent training atmosphere here at MSU.

Last, but certainly not least, to the members of the lab, I hope you realize how
important your friendship and scientific feedback has been over the past few years. All
of you have made this a truly amazing graduate school experience. What a long, strange

trip it has been.

Table of Contents

List of Tables .................................................................................... x
List of Figures ................................................................................... xi-xii
List of Abbreviations ........................................................................... xiii
Chapter 1. Introduction ...................................................................... 1-45
A. Statement of Purpose ................................................................ 1
B. Parkinson’s disease (PD) ............................................................ 2-3
C. Pathophysiology of Motor Symptoms ........................................... 3-1 3
1. The Loss of Nigrostriatal Dopamine Neurons is the
Pathological Hallmark of Parkinson’s disease” ........3
2. NSDA Neurons are part of the Basal Ganglia Motor System ........ 3-8
3. NSDA Neurons Regulate Basal Ganglia Motor Processing. ...9
4. Basal Ganglia Dysfunction in PD ........................................ 12
D. Progressive Loss of NSDA Neurons in PD .................................... 14-16
E. NSDA Neurophysiology ........................................................... 17-23
1. NSDA Neurochemistry ................................................... 17-19
2. The Role of DA Metabolism in NSDA Neurons ..................... 20-23
F. NSDA Neurophysiology in PD .................................................... 24-26
G. PD Animal Models ................................................................. 27-43
1. 6-OHDA .................................................................... 29-33
2. Paraquat ..................................................................... 33-35
3. Rotenone ................................................................... 36-38
4. MPTP ........................................................................ 39-44
H. Thesis Objective .................................................................... 45
Chapter 2. Materials and Methods ........................................................ 46-73
A. Animals .............................................................................. 46-49

vi

1. Generation of wild-type and homozygous a-synuclein

knock-out mice ............................................................ 46
2. Determining Animal Genotype .......................................... 46-48
3. Animal Housing ........................................................... 49
B. Drugs ................................................................................ 49-51
1. Prolonged Chronic MPTP ................................................ 49
2. Sub-chronic MPTP ........................................................ 50
3. m-Hydroxybenzylhydrazine (N SD-1015) .............................. 50
4. Raclopride ................................................................... 50
5. Gamma-butyrolactone (GBL) ........................................... 50
6. L-Tyrosine .................................................................. 50
7. Chronic Raclopride and Quinelorane ................................... 51
C. Preparation and Implantation of Alzet Osmonic Mini-pumps ............... 51-52
D. Neurochemistry .................................................................... 52-59
1. Tissue Preparation ........................................................ 52-55
2. Neurochemical Analysis ................................................ 56-59
B. Western Blot Analysis ............................................................ 60-65
1. Tissue Preparation ......................................................... 60
2. Protein extraction/isolation/determination ............................ 6O
3. Electrophoresis/Transfer Conditions ................................... 60-61
4. Immunoblot Detection of Striatal Protein ............................. 61-64
5. Quantification of Protein Immunoreactivity .......................... 65
F. In vitro Monoamine Oxidase Activity Assay ................................. 65-67
G. Subcellular Fractionation ........................................................ 68-72
1. Tissue Preparation ......................................................... 68
2. Centrifugation Speeds and Conditions ................................. 68-70
2A. Isolation of Plasma Membrane Protein ...................... 68
2B. Isolation of Cytosolic, Synaptic Vesicle ................... 69
and Endosomal Protein
3. Protein detection/quantification ....................................... 71
H. Statistical Analysis ............................................................... 73

vii

Chapter 3. The Effects of Prolonged-Chronic MPT P Administration on NSDA

Neurons ............................................................................... 74-109
A. Introduction ....................................................................... 74
B. Experimental Design ............................................................ 75-83
C. Results ............................................................................. 84-98
D. Discussion ........................................................................ 99-109
Chapter 4. Identification of the Neurochemical Changes that Underlie the
Compensatory Activation of NSDA Neurons ......................................... 110-138
A. Introduction ..................................................................... 110-112
B. Experimental Design and Results ............................................. 113-130
C. Discussion ....................................................................... 131-138
Chapter 5. Dysfunctional Regulation of Tyrosine Hydroxylase in NSDA Neurons
that Survive MPT P Administration ................................................... 139-169
A. Introduction ..................................................................... 139-143
B. Experimental Design and Results ........................................... 144-162
C. Discussion ...................................................................... 163-169
Chapter 6. Dopamine Receptor (D2) Regulation of DA Synthesis in NSDA
Neurons ...................................................................................... 170-211
A. Introduction ..................................................................... 170-211
B. Experimental Design and Results ........................................... 173-199
C. Discussion ...................................................................... 200-211
Chapter 7. The Role of a-Synuclein in MPTP-Induced Toxicity and the
Compensatory Activation of NSDA Neurons ........................................ 212-256
A. Introduction ..................................................................... 212-214
B. Experimental Design and Results ........................................... 215-242
C. Discussion ...................................................................... 243-256
Chapter 8. Concluding Remarks ...................................................... 257-262
References ................................................................................... 263-284

viii

List of Tables

Table # Title Page
2-1 List of primary antibodies ................................................... 62
2-2 Efficiency of sub-cellular fiactionation using differential

centrifugation .................................................................. 72
3-1 Prolonged-chronic MPTP dose-response experimental design. . . . . . ...76
3-2 The primary experimental end-points used for indices of

NSDA neuron function ...................................................... 78
3-3 Nucleus accumbens and median eminence DA concentrations

in mice treated with prolonged-chronic MPTP ............................ 96
3-4 Nucleus accumbens and median eminence DOPAC

concentrations in mice treated with prolonged-chronic MPTP. . . . . . ....97
5-1 Experimental design used to determine TH sub-cellular

distribution in the striatum ................................................... 155
6-1 Total TH levels in the striatum of mice treated with either

saline, tyrosine, raclopride or tyrosine plus raclopride ................... 182
6-2 Total TH levels in the striatum of mice treated with either

saline, tyrosine, GBL or tyrosine plus GBL ................................ 192
6-3 Experimental design for determining whether D2 receptors

regulate the activity of NSDA neurons following MPTP

administration .................................................................. 194
7-1 Total TH levels in the striatum of a-synuclein knock-out mice

treated with either saline, tyrosine, raclopride or

tyrosine plus raclopride ....................................................... 226
7-2 Quantification of the effect of MPTP phosphorylated TH in

the striatum of a-synuclein knock-out mice ................................ 233
7-3 Quantification of the effect of MPTP on striatal TH

protein levels in a-synuclein knock-out mice .............................. 234
7-4 The efi‘ects of MPTP on TH protein distribution in the striatum

of a-synuclein knock-out mice ................................................ 241

ix

List of Figures

FigILre # Title Page
1-1 Schematic sagittal view of the rat brain depicting

catecholamine nuclei distribution ........................................... 5
1-2 Sagittal view of the rat brain depicting the projections of

A8/A9 dopamine neurons .................................................... 6
1-3 Coronal section of a human brain depicting the circuitry of

the basal ganglia direct and indirect pathways ............................ 9
1-4 Functional segregation of medium spiny GABAergic neurons

in the striatum .................................................................. 11
1-5 The rate of NSDA neuronal loss in the general population

and PD patients ................................................................ 16
1-6 Schematic diagram of the neurochemical events in a NSDA

axon terminal .................................................................. 19
1-7 DA Enzymatic and non-enzymatic metabolic pathways ................. 22
1-8 The oxidation reaction for two molecules of 6-OHDA .................. 31
1-9 Generation of 02' radicals following the oxidation of paraquat. . . . . ....35
1-10 The mechanism of rotenone induced neurotoxicity ...................... 38
1-11 Mechanisms of MPTP bioconversion and transport into

DA neurons .................................................................... 40
1-12 Effects of MPTP once inside the axon terminal .......................... 42
2-1 Ethidium bromide stained agarose gel showing PCR

reaction products from wild-type, a-synuclein knock-out

and heterozygous mice ...................................................... 48
2-2 Sagittal and coronal views of a Nissl stained mouse brain

depicting the rostral-caudal and dorsal ventral location of

nucleus accumbens dissections ............................................. 53
2-3 Sagittal and coronal views of a Nissl stained mouse brain

depicting the rostral-caudal and dorsal ventral location of

striatum dissections .......................................................... 55

2-4

3-9

3-10

HPLC-EC chromatogram depicting the retention times
of a lng six mix and representative striatal sample ..................... 58

Logarithmic scale demonstrating the linear range for
determining DA content using HPLC-EC ................................ 59

Tyrosine hydroxylase and B-III immunoreactivity and
quantification of mouse striatal samples using western
blot technique ................................................................ 64

In vitro rate of monoamine oxidase enzymatic activity ................ 67

Subcellular fractionation of striatal tissue using
differential centrifugation .................................................. 70

Experimental design for MPTP time-course experiments ............. 81

Dose response effect of prolonged-chronic MPTP
administration on DA concentrations in the striatum ................... 85

Dose response effect of prolonged-chronic MPTP
administration on DOPAC concentrations and the
DOPAC to DA ratio in the striatum ....................................... 86

Correlation between the DOPAC/DA ratio and DA
concentrations following MPTP ........................................... 87

Dose response effect of prolonged-chronic MPTP
administration on striatum VMAT-2 immunoreactivity ................ 88

Time-course of striatal DA concentrations in mice treated
with prolonged-chronic MPTP ............................................. 9O

Time-course of striatal DOPAC concentrations and the
DOPAC/DA ratio in mice treated with prolonged-chronic
MPTP .......................................................................... 91

The effects of prolonged-chronic MPTP on striatal TH
protein expression ............................................................ 93

The effects of prolonged-chronic MPTP on striatal DAT
protein expression ............................................................ 94

The effects of prolonged chronic MPTP on the DOPAC
to DA ratio in the nucleus accumbens and median eminence .......... 98

xi

4-1

4-2

4-3

4-6

4-7

4-8

4-9

5-2

5-3

5-7

Schematic diagram of a NSDA axon terminal depicting the
two primary sources of cytoplasmic DA ................................. 112

Strategy for determining the amount of recaptured DA
metabolized under basal and activated conditions ...................... 115

Effects of sub-chronic MPTP administration on striatal DA
and DOPAC concentrations and DOPAC/DA ratios ................... 117

Effects of sub-chronic MPTP on striatal TH and DAT protein
expression in the striatum ................................................... 118

Effects of GER-12909 on striatal DOPAC concentrations
under basal conditions and activated conditions ........................ 120

Effects of AMT and NSD-1015 on MAO enzymatic activity... .......123

Strategy for determining the amount of newly synthesized DA
metabolized under basal and activated conditions ...................... 125

Effects of a-methylparatyrosine on striatal DOPAC concentrations
under basal and activated conditions ....................................... 127

Concentrations of DOPA, DA and the DOPA to DA ratio in

the striatum of mice treated with MPTP ................................... 130
Schematic diagram depicting the DA biosynthetic pathway

in the axon terminals of NSDA neurons ................................... 141
Mechanisms underlying the accelerated rate of DA synthesis ......... 143

Experimental design and isolation of NSDA axon terminals for
neurochemical analysis of striatal DOPA concentrations and
phosphorylated TH ........................................................... 145
Effects of sub-chronic MPTP on TH phosphorylation state... .. . .......148

Effects of sub-chronic MPTP on the relative proportion of
phosphorylated TH in the striatum .......................................... 149

Striatal DOPA concentration, TH protein, and the ratio of
DOPA to TH in the striatum of mice treated with MPTP ............... 150

Effects of tyrosine supplementation on TH enzymatic
activity in mice treated with MPTP ......................................... 152

xii

5-8

5-9

5-10

5-11

5-12

6-1

6-5

6-6

6-7

6-8

6-9

Differential centrifugation technique used to isolate
striatal protein into sub-cellular fractions ................................. 156

TH protein, DA concentrations and the TH to DA ratio
in the striatum of mice treated with MPTP ................................ 158

Representative Western blots depicting the effects of MPTP
on TH in the cytoplasmic, endosomal, synaptic vesicle
and plasma membrane sub-cellular fractions ............................. 160

Quantification of the effects of MPTP on the sub-cellular
localization of striatal TH .................................................... 161

Effects of MPTP on the relative proportion of TH in each
sub-cellular compartment .................................................... 162

Schematic diagram depicting the neurochemical events in the
axon terminal following administration of raclopride ................... 174

Concentrations of DOPAC, and the DOPAC/DA ratio
in the striatum of mice treated with saline, tyrosine, raclopride
or raclopride plus tyrosine ................................................... 177

Phosphorylated TH in the striatum of mice treated with saline,
tyrosine, raclopride or raclopride plus tyrosine ........................... 179

Concentrations of DOPA in the striatum of mice treated with
saline, tyrosine, raclopride or raclopride plus tyrosine .................. 181

Schematic diagram depicting the neurochemical events in the
axon terminal following administration of GBL .......................... 184

Concentrations of DOPAC, DA and the DOPAC/DA ratio
in the striatum of mice treated with saline, tyrosine, GBL
or GBL plus tyrosine ......................................................... 187

Phosphorylated TH in the striatum of mice treated with
saline, tyrosine, GBL or GBL plus tyrosine .............................. 189

Concentrations of DOPA in the striatum of mice treated
with saline, tyrosine, GBL or GBL plus tyrosine ........................ 191

Effects of raclopride and quinelorane on the concentrations of

DOPAC, DA and the DOPAC to DA ratio in mice treated with
MPTP ........................................................................... 196

xiii

6-10

7-1

7-2

7-3

7-4

7-5

7-6

7-8

7-9

7-10

7-11

8-1

Concentrations of DOPAC, DA and the DOPAC/DA ratio
in the striatum of mice treated chronically with raclopride. . . . . . . . .....199

Dose response effect of prolonged-chronic MPTP administration
on DA, DOPAC concentrations and the DOPAC to DA ratio in
the striatum of a-synuclein knock-out mice .............................. 217

Dose-response effect of prolonged-chronic MPTP administration
on VMAT-2 levels in a-synuclein knock-out mice ...................... 218

Effects of prolonged-chronic MPTP on concentrations of DOPAC
and DA and the DOPAC/DA ratio in the striatum of a-synuclein
knock-out mice ............................................................... 221

Phosphorylated TH in the striatum of a-synuclein knock-out
mice treated with saline, tyrosine, raclopride or raclopride
plus tyrosine ................................................................... 223

Concentrations of DOPA in the striatum of a-synuclein
knock-out mice after administration of saline, tyrosine,
raclopride or raclopride plus tyrosine ...................................... 225

Effects of sub-chronic MPTP on striatal DA and DOPAC

concentrations and DOPAC/DA ratios in a-synuclein
knock-out mice ............................................................... 229

Effects of MPTP on Striatal DOPA and DA concentrations
and the DOPA to DA ratio in a-synuclein knock-out mice ............ 231

Effects of MPTP on the relative proportion of phosphorylated
TH in the striatum ............................................................ 235

Effects of MPTP on TH enzymatic activity and protein
expression in the striatum of a-synuclein knock-out mice ............. 237

Effects of tyrosine supplementation on striatal DOPA
concentrations in a-synuclein knock-out mice treated
with MPTP ..................................................................... 239

Effects of MPTP on the sub-cellular localization of TH in
a-synuclein knock-out mice ................................................. 242

NSDA axon terminal neurochemistry under basal conditions
and during compensatory activation ........................................ 260

xiv

6-OHDA
AADC
AD
AMT
Ca+2
CarnPKII
Complex I
DAT
DOPA
DOPAC
DOPAL
GPe

GPi

GSH
H202

HPLC-EC

LNAA
MAO
Mito
MLDA
MPDP

MPP+

List of Abbreviations
6-hydroxydopamine
L-Aromatic amino acid decarboxylase
Aldehyde dehydrogenase
Alpha-methylparatyrosine
Calcium
Ca2+—calmodulin-dependent protein kinase
Nicotinamide adenine dinucleotide (NADH)-ubiquinone reductase
Dopamine transporter
Dihydroxyphenylalanine
Dihydroxyphenylacetic acid
Dihydroxyphenylacetaldehyde
Globus pallidus external segment
Globus pallidus internal segment
Glutathione
Hydrogen peroxide

High performance liquid chromatography with electrochemical
detection

Large neutral amino-acid transporter
Monoamine oxidase

Mitochondria

Mesolimbic dopamine

1 -Methyl-4-phenyl-2,3-dihydropyridinium

1-Methyl-4-phenyl pyridinium

XV

MPTP
NADH
NSDA
02'
OH'
OONO'
PD
PKA

PKC

ROS
SEM
SNpc
STN
TH
TIDA
UPS
VL

VMAT-2

1-Methyl-4 phenyl-1,2,3,6 tetrahydropyridine
Nicotinamide adenine dinucleotide
Nigrostriatal dopamine

Superoxide

Hydroxyl radical

Peroxinitrate

Parkinsons's disease

CAMP-dependent protein kinase A
Ca2+—calmodulin-dependent protein kinase
Relative density unit

Reactive oxygen species

Standard error of the mean

Substantia nigra pars compacta
Subthalamic nucleus

Tyrosine hydroxylase

Tuberoinfundibular dopamine

Ubiquitin proteosome system

Ventral lateral thalamic nucleus

Vesicular monoamine transporter

xvi

Chapter 1. General Introduction

A. Statement of Purpose

Parkinson’s disease (PD) is a chronic, progressive, neurodegenerative disorder
that is diagnosed clinically by the development of motor abnormalities. The
manifestation of motor symptoms is attributed to the primary pathological hallmark of the
disease, the degeneration of nigrostriatal dopamine (N SDA) neurons. Interestingly,
motor symptoms do not appear until approximately 50% of NSDA neurons have
degenerated. Therefore, at the time of clinical diagnosis there is already extensive
damage to the NSDA system. Throughout the disease process, the remaining 50% of
these neurons will progressively degenerate causing a gradual worsening of motor
symptoms ultimately resulting in patient disability and death. To this end, a primary
therapeutic goal for treating PD is to halt or delay the progressive degeneration of NSDA
neurons. The task has been hindered by a lack of understanding of what contributes to
the progressive demise of these neurons.

The overall aim of the research that comprises this dissertation is to identify
mechanisms that contribute to the progressive degeneration of NSDA neurons. The
research described herein utilizes pharmacological, neurochemical and
immunohistochemical approaches to test the hypothesis that the initial death of NSDA
neurons causes compensatory increases in the activity of surviving neurons and the
sustained increase in activity contributes to the progressive, secondary death of NSDA
neurons in PD. If this hypothesis is true, then novel therapeutic targets can be identified
to prevent prolonged, compensatory increases in neuronal activity to inhibit secondary,

progressive NSDA death in PD.

B. Parkinson’s Disease

PD is a major source of disability and death for people over the age of sixty. PD
incidence rates for people under 60 are 10/100,000, but rise dramatically to 40/ 1 00,000
for people 60-69 years of age and over 100/ 100,000 for people seventy and over (Mayeux
et al., 1995; Fall et al., 1996; Bower et al., 1999; Chen et al., 2001). The prevalence rates
(or number of people affected by the disease) also dramatically increase fi'om 99/100,000
under the age of 60 to 1 193/ 100,000 for people seventy and older (Mayeux et al., 1995).
The cause of PD is unknown and currently there is no preventative or curative therapy
available for the millions of people living with the disorder. Taken together, it is not
surprising that there is a tremendous research effort to understand the pathogenesis of PD
and develop effective therapeutic strategies for the treatment of this disorder.

The clinical symptoms of PD were originally described in 1817 by James
Parkinson in his essay “The Shaking Palsy” (Parkinson, 1817). The disease is
characterized clinically by the development of cardinal motor symptoms that include;
resting tremor in the distal limbs, bradykinesia, rigidity, and postural instability.
Disturbances in facial expression, sleep, speech, chewing and gut motility are also
common(Borek et al., 2006). Traditionally, symptoms are initially mild and more of an
inconvenience for the patient rather than a significant source of disability. However, over
time, the symptoms will gradually become more severe, affect greater areas of the body
and become a major source of disability, eventually resulting in death. The average life-
span of a patient from time of diagnosis to time of death is 11 :t 2 years (Birkmayer,

1974). The progressive worsening of PD symptoms is a distinguishing feature of the

 

N3

disease that warrants additional research in order to identify the underlying pathogenic

mechanisms responsible for the disability and death of millions of people.

C. Pathophysiology of PD Motor Symptoms
The Loss of Nigrostriatal Dopamine Neurons is the Pathological Hallmark of PD

The earliest report of a distinct pathological hallmark to accompany the clinical
features of PD came in 1895 when Blocq and Marinesco described the loss of pigmented
cells in the substantia nigra pars compacta (SNpc) of human PD patients (Alvord, 1987).
Subsequently, neuroanatomical track tracing studies identified projections from the
pigmented cells in the SNpc to the corpus striatum (Rosegay, 1944). Dopamine (DA)
was determined to be a neurotransmitter proper, and moreover, the principle
neurotransmitter of nigrostriatal projections (Bertler, 1959a, 1959b; Carlsson, 1959). In
PD patients the loss of pigmented SNpc cells corresponds to a dramatic loss of striatal
DA (Ehringer, 1960; Homykiewicz, 1963). Moreover, there is a strong correlation
between NSDA neuronal loss and severity of PD symptoms (Homykiewicz, 1972).
Finally, the motor complications associated with the disease can be alleviated by
strategies that restore brain DA concentrations (Homykiewicz, 1975). It is now well
accepted that the specific loss of NSDA neurons is the primary pathology responsible for

the development of PD motor symptoms.

NSDA Neurons are a part of the Basal Ganglia Motor System
As depicted in Figure 1-1, NSDA neurons (A8/A9) reside in the midbrain (Figure

1-1) and project axons to through the medial forebrain bundle to the forebrain where they

terminate in the striatum (Figure 1-2) (Rosegay, 1944; Anden, 1964; Dahlstrom and
Fuxe, 1964; Moore et al., 1987). The release of DA into the striatum plays a critical role
in regulating the activity of the basal ganglia. The basal ganglia are comprised of
subcortical nuclei including the striatum (caudate nucleus and putamen), globus pallidus
(internal and external segments) and subthalamic nucleus (Bergman, 1998). Connections
between these subcortical nuclei serve as a critical processing loop for signals relayed
from the motor cortex to the ventral lateral thalamus (V L) and back to the motor cortex
that allows descending output from the primary motor cortex to be focused onto a
particular set of muscles required to perform a specific task (Kaji, 2001). Primary output
neurons in the motor cortex descend in the corticospinal tract and synapse directly onto
motor neurons in the spinal cord that reside in layers VIII and IX of the ventral horn.
Motor neurons that innervate proximal muscles are located in the medial portion of the
ventral horn, while motor neurons that innervate distal muscles that control limb

movement are located in the lateral portion of the ventral horn (Kandel, 1991).

 

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Cortical input to the basal ganglia can be processed through one of two primary
basal ganglia circuits before being relayed to the VL thalamic nucleus, the direct or the
indirect pathway (Figure 1-3). In the direct pathway, excitatory cortical input to the
striatum increases firing of striatal medium spiny GABA neurons. Increased firing of
striatal GABA neurons inhibits the firing of GABA neurons in the globus pallidus
internal segment (GPi). Decreased GPi firing results in a disinhibition of glutamate
neurons in the VL thalamic nucleus. Increased firing of neurons in the VL thalamic
nucleus increases excitatory input back to the motor cortex (Parent and Hazrati, 1993;
Herrero et al., 2002). Excitation of motor cortex neurons increases excitatory
corticospinal output to primary motor neurons in the spinal cord, which facilitates muscle
movement. Thus, excitation of the direct pathway reinforces subsequent cortical activity
and facilitates the execution of voluntary movement.

In contrast to the direct pathway, cortical signals projected to indirect pathway
medium spiny GABA neurons are first relayed to GABA neurons in the globus pallidus
external segment (GPe) and then to glutamate neurons in the subthalamic nucleus (STN)
before being sent to the GPi. The shunting of information from the striatum through the
GPe and STN changes the net effect of basal ganglia output such that diminished STN
firing decreases the firing of glutamate neurons in the VL thalamic nucleus. Inhibition
of VL neuronal firing diminishes excitatory input back to the motor cortex and decreases
the activity of neurons in the motor cortex, thus, reducing corticospinal output to primary
motor neurons in the spinal cord and prohibiting muscle movement (Parent and Hazrati,

1993; Herrero et al., 2002). Therefore, the direct and indirect pathways have opposing

actions and balance between these pathways is critical for coordinating muscle

movement.

Direct Pathway Indirect Pathway

 

Figure 1-3. (Left side of brain) Coronal section of a human brain depicting the circuitry
of the basal ganglia direct pathway. (Right side of brain) Coronal section of a human
brain depicting the circuitry of the basal ganglia indirect pathway. Glutatrnatergic
excitatory connections are shown as solid lines. GABAergic inhibitory connections are
shown as dashed lines. Abbreviations: VL ventral lateral thalamic nucleus, GPi globus
pallidus internal segment, GPe globus pallidus external segment, STN subthalamic
nucleus. Images in this thesis/dissertation are presented in color.

NSDA Neurons Regulate Basal Ganglia Motor Processing

Both the direct and indirect pathways are regulated by NSDA neurons. NSDA
neurons synapse onto medium-spiny projection neurons in the striatum (Lavoie et al.,
1989). As depicted in Figure 1-4, striatal projection neurons in the direct pathway are
distinguished from striatal projection neurons in the indirect pathway via expression of
two subtypes of DA receptors (Herrero et al., 2002). Striatal neurons in the direct
pathway express stimulatory D1 -like receptors (D1,D5) that are G-protein receptors
coupled to the Gs subunit which stimulates adenylate cyclase and the activity of medium
spiny striatal neurons (Dearry et al., 1990; Monsma et al., 1990; Zhou et al., 1990; Herve
et al., 1993). Thus, DA binding to D1 receptors excites the direct pathway and promotes
voluntary movement (Jackson et al., 1994).

Medium spiny striatal neurons in the indirect pathway express inhibitory D2-like
receptors (D2, D3, D4). These are G-protein coupled receptors coupled to the Gi/o
subunit which inhibits adenylate cyclase activity and the activity of medium spiny
projection neurons (Onali et al., 1985). Thus, DA binding to D2 receptors inhibits the
indirect pathway. Since the indirect pathway inhibits voluntary movement, DA binding
to D2 receptors in effect promotes voluntary movement (Jackson and Westlind-
Danielsson, 1994). Therefore, DA released by NSDA neurons terminating in the striatum

promotes voluntary movement regardless of which DA receptor it binds (Wooten, 1997).

10

Direct Pathway Indirect Pathway

I Preeynaptlc f
Dopamine Projections

 

 

 

 

   

GABAergic Neurons

Figure 1-4. Expression of DA receptor subtypes distinguishes post-synaptic medium
spiny GABAergic neurons in the direct pathway (Left Panel) from the indirect pathway
(Right Panel). Striatal neurons in the direct pathway express stimulatory (Gs-coupled)
Dl-like receptors. Medium spiny striatal neurons in the indirect pathway express
inhibitory (Gi-coupled) D2-like receptors. D2 receptors are also expressed by the pre-
synaptic NSDA neurons. Abbreviations: DAT=dopamine transporter. (Missale et al.,
1998)

11

Basal Ganglia Dysfunction in PD

In a simplified model of basal ganglia function, the loss of NSDA neurons
decreases DA binding to D1 and D2 receptors on medium spiny GABA neuron in the
striattun. This results in decreased direct pathway activation and increased activation of
the indirect pathway. The effect of the imbalance between these pathways is a reduction
in excitatory thalamic-cortical neurotransmission and diminished firing of motor cortex
output neurons. Inhibition of motor cortex projections inhibits firing of primary motor
neurons found in the spinal cord, thus reducing activation of peripheral muscles and
prohibiting voluntary movement. Decreased voluntary movement in the limbs can
explain the characteristic slowness of movement (bradykinesia) and loss of movement
fluidity (rigidity) that are hallmarks of PD. Postural instability is thought to reflect
inhibition of primary motor neurons in the medial ventral horn of the spinal cord which
innervate proximal muscles that stabilize the trunk against unwanted directional
movement. The manifestation of resting tremor is more complex.

Tremor is defined as the involuntary rhythmic oscillation of a body region that is
produced by alternating contractions of reciprocally innervated muscles (Zesiewicz and
Hauser, 2001). Interestingly, resting tremor in PD patients poorly correlates with NSDA
damage (Otsuka et al., 1996), suggesting that tremor is not the direct result of DA cell
loss. However, recent evidence suggests that abnormal activity of the STN and globus
pallidus due to NSDA neuron loss is responsible for tremor manifestation. Under normal
conditions, when there is sufficient DA input into the basal ganglia, STN and the pallidal
firing rates fire at approximately 70 Hz. However, in untreated PD patients firing rates

between these nuclei synchronize at much lower frequencies (4-8 Hz). Synchronization

12

directly corresponds to the very uniform 4-8 Hz limb tremor that occurs in PD patients
(Brown, 2001). In fact, 4-8 Hz STN discharge is time-locked to the EMG-determined 4-
8 Hz tremor in the hand (Krack et al., 1998; Rodriguez et al., 1998).

L-DOPA therapy, that restores brain DA concentrations, or deep brain stimulation
of the STN or GPi eliminates low frequency STN-globus pallidus synchronization and
alleviates tremor (Lozano et al., 1995; Burchiel et al., 1999). Additionally, lesions of the
non-human primate SNpc results in a dramatic shifi to 4-8 Hz synchronization between
GPi and STN and tremor manifestation (Bergman, 1994). Thus, a critical role for NSDA
neurons may be preventing low-frequency synchronization between basal ganglia nuclei.
In the absence of NSDA neuronal function rhythmic low frequency (4-8 Hz) firing of the
motor cortex output neurons would cause activation of primary motor neurons in the
spinal cord, rhythmic excitation of reciprocally innervated muscles in the hand, and
tremor.

NSDA neuronal loss can cause dysfunction of basal ganglia motor information
processing which accounts for the manifestation of the cardinal motor complications
associated with PD. Therefore, it is reasonable to conclude that preventing the
progressive degeneration of NSDA neurons will avert the continued decline in basal

ganglia function and the gradual worsening of PD symptoms.

13

D. Progressive Loss of NSDA Neurons in PD

The gradual worsening of PD symptoms closely corresponds to the progressive
loss of NSDA neurons (Homykiewicz, 1972; Bernheimer et al., 1973). The rate of
NSDA cell loss in a PD patient is approximately 4.5% per year (Fearnley and Lees,
1991). Accordingly, the rate of NSDA axon terminal loss has been estimated at
approximately 5-10% per year using several different indices to quantify axon terminal
number (Riederer and Wuketich, 1976; Morrish et al., 1998; Pirker et al., 2003; Au et al.,
2005; Hilker et al., 2005). As depicted in Figure 1-5, the rate of NSDA neuronal loss in a
PD patient over the 11 year average time course of the disease (Birkmayer, 1974), is
greatly accelerated when compared to the 0.45% per year rate of NSDA neuronal loss
observed in healthy age-matched controls (Riederer and Wuketich, 1976).

NSDA neuronal number is depleted to approximately 50% of controls at the time
of symptom onset (Fearnley and Lees, 1991). It is not known if the initial 50% loss of
NSDA neurons follows a similar linear progression, however if a linear slope is assumed
then a pre-clinical period of 5-10 years can be predicted. Regardless, the progressive
nature of PD can be attributed to an exponential loss of NSDA neurons that appears to
begin several years before motor abnormalities arise. Symptom onset likely occurs when
DA input to the basal ganglia decreases below a threshold point, when activation of the
inhibitory indirect pathway outweighs activation of the excitatory direct pathway. This
would decrease excitation of the VL thalamic nucleus and inhibit motor cortical activity.
Thus, cortical output to the corticospinal tract would decrease, ultimately reducing
voluntary movement in the peripheral muscles. The research described in this

dissertation was conducted to identify mechanisms that contribute to the progressive

14

degeneration of NSDA neurons. The research strategy employed was to utilize
neurochemical, immunohistochemical, and pharmacological techniques to compare
NSDA neurophysiology under basal conditions and during conditions that recapitulate
PD to identify pathogenic mechanisms that could contribute to the progressive loss of

these neurons.

15

Accelerated NSDA Cell Loss

‘O‘Control

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Figure 1-5. The rate of NSDA neuronal loss in the general population and PD patients
shown as a percentage of age-matched controls. The loss of NSDA neurons over time in
healthy controls (dashed line) is approximately 0.45% per year as determined by numbers
of SNpc dopamine neurons at autopsy. In PD patients (solid line) there is an accelerated
loss of NSDA neurons of approximately 4.5% per year (Fearnley and Lees, 1991).

16

E. NSDA Neurophysiology
NSDA Neurochemistry

The primary function of NSDA neurons is to regulate the activity of the basal
ganglia. This function is depenent upon maintenance of DA synthesis, storage, release,
reuptake and metabolism (Figure 1-6). DA synthesis begins with the uptake of dietary
tyrosine into the axon terminal via the large neutral amino-acid transporter (Oxender and
Christensen, 1963; Oldendorf and Szabo, 1976; Palacin et al., 1998). Cytoplasmic
tyrosine in the axon terminal is then converted to dihydroxyphenylalanine (DOPA) by
cytoplasmic tyrosine hydroxylase (TH), the rate limiting enzyme in catecholamine
synthesis (Levitt et al., 1965; Kumer and Vrana, 1996). DOPA is decarboxylated to DA
by L-aromatic amino acid decarboxylase (AADC) (Kumer and Vrana, 1996). Newly
synthesized DA is packaged into synaptic vesicles by the vesicular monoamine
transporter-2 (V MAT-2). Additionally, newly synthesized DA that is not packaged into
vesicles, can be degraded to dihydroxyphenylacetic acid (DOPAC) through a two-step
reaction catalyzed by mitochondrial monoamine oxidase (MAO). Cytoplasmic DA
inhibits the activity of TH via end-product inhibition (Okuno and Fujisawa, 1985;
Andersson et al., 1988; Haavik et al., 1991).

Action potential-induced Ca+2 entry into the axon terminal triggers the release of
vesicular DA into the synaptic cleft. DA released into the synapse can bind to either
post-synaptic D1 or D2 receptors, or pre-synaptic D2 autoreceptors. Activation of pre-
synaptic D2 autoreceptors inhibits further DA synthesis and release (Christiansen and
Squires, 1974; Roth, 1975). Synaptic DA is removed via the high-affinity reuptake DA

transporter (DAT) which recaptures a vast majority of synaptic DA (Kilty et al., 1991).

17

Recaptured DA can either be repackaged into synaptic vesicles for reuse, or metabolized

to DOPAC by mitochondrial MAO and aldehyde dehydrogenase (AD).

18

NSDA Axon Terminal
DOPAC

DOPAC ® DA
IN) 119/
DOPAL

Tyrosine-—o DOPA— DA
TH AADC 5

 

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Tyrosine

Figure 1-6. Schematic diagram depicting the neurochemical events in a NSDA axon
terminal. Tyrosine is taken up into the axon terminal by the large neutral amino-acid
transporter (LNAA). Tyrosine is then converted to DOPA by tyrosine hydroxylase (TH).
DOPA is decarboxylated to DA by L-aromatic amino-acid decarboxylase (AADC).
Newly synthesized DA is packaged by vesicular monoamine transporter-2 (VMAT) into
synaptic vesicles for release. Alternatively, newly synthesized DA can be degraded by
mitochondrial (Mito) MAO or feedback to inhibit TH activity. DA released into the
synapse can bind to post-synaptic D1 or D2 receptors (DA-R) or D2 autoreceptors (D2-
R). Synaptic DA is removed via the DAT. Recaptured DA can be repackaged into
synaptic vesicles or degraded by MAO and aldehyde dehydrogenase (AD) to DOPAC.
DOPAC is removed from the axon terminal through diffusion.

19

The Role of DA Metabolism in NSDA Neurons

It is critical that NSDA neurons tightly regulate DA synthesis, storage, release and
reuptake because byproducts of DA metabolism are toxic to neurons and may contribute
to the progressive degeneration of NSDA neurons in PD (Graham, 1978; Graham et al.,
1978; LaVoie and Hastings, 1999b, 1999a; Perez and Hastings, 2004). As depicted in
Figure 1-7, DA oxidation by mitochondrial MAO results in the formation of
dihydroxyphenylacetaldehyde (DOPAL) which is a highly toxic metabolite (Burke et al.,
2004; Legros et al., 2004; Ebadi et al., 2005). As such, the activity of AD is normally
sufficient to quickly convert DOPAL to DOPAC, a less toxic species. However,
mitochondrial impairment or AD inhibition leads to the accumulation of DOPAL in DA
neurons. Also, AD inhibition exacerbates neurotoxicity induced by mitochondrial
inhibition in DA neurons (Lamensdorf et al., 2000), suggesting that this DA metabolite
can directly decrease neuronal viability.

The enzymatic conversion of DOPAL to DOPAC also produces reactive oxygen
species (ROS, Figure 1-6). ROS can cause oxidative damage to neurons by reacting with
lipids, protein or DNA, and ultimately causing cell death. The conversion of DOPAL to
DOPAC by AD results in the formation of a hydrogen peroxide molecule (H202). H202
can react with F e+2 to produce the neurotoxic hydroxyl radical (OH') through a reaction
known as the Fenton reaction.

Cytoplasmic DA can also undergo non-enzymatic auto-oxidation which produces
ROS. In this case, the catecholamine ring of DA is oxidized resulting in the formation of
a DA-quinone molecule, superoxide (02') and H202 radicals. In addition to the H202-

mediated OH‘ generation, the 02' radical can interact with nitric oxide to form

20

 

 

 

 

 

mo.

peroxynitrite (OONO') a free radical that causes substantial oxidative damage to
intracellular proteins and lipids. The DA-quinone molecule, formed from DA auto-
oxidation, can also cause neuronal dysfimction. DA-quinones have an affinity for
cysteine residues on proteins, and bind to these residues causing covalent protein

modification and loss of function of intracellular proteins (LaVoie and Hastings, 1999b).

21

Enzymatic DA Metabolism
DA

MAO

DOPAL

AD

DOPAC +H202

Fe+2

OH'

Oxidative Damage

DA Auto-oxidation
DA

Oxidation of
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DA-Quinone + H20 2 + 02"

l l

011' oouo‘
CysteinyI-DA \ /
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Oxrdatlve
Damage

Loss of Protein
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Figure 1-7. DA enzymatic (left) and non-enzymatic (right) metabolic pathways. The
enzymatic metabolism of DA results in the formation of DOPAC and a hydrogen
peroxide (H202) molecule. H202 can be scavenged by glutathione (GSH) or react with
iron to form hydroxyl radical (OH') through the F enton reaction (Dringen et al., 2000).
DA auto-oxidation results in the formation of DA-quinone molecules and the reactive
oxygen species H202 and superoxide (02'). H202 can produce OH' while 02' can react
with nitric oxide to form peroxynitrite (OONO'). ROS cause oxidative damage by
reacting with lipids, DNA and proteins, and may ultimately result in neuronal death.

22

 

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Neurons are protected from ROS-induced oxidative damage by endogenous
intracellular antioxidant proteins that scavenge free radicals. Glutathione is the primary
antioxidant defense protein in neurons (Cooper and Kristal, 1997). Glutathione (GSH)
donates an electron to free radicals (02', OH', OONO' and H202) reducing the ROS to a
less toxic molecule, but consequently oxidizing glutathione to glutathione disulfide.
Glutathione disulfide can be converted back to GSH by glutathione reductase (Dringen et
al., 2000). However, GSH also participates in non-enzymatic reactions, whereby the
GSH molecule is consumed in the reaction. In this case, de novo protein synthesis is
required to replenish depleted levels of GSH.

If ROS formation increases and overwhelms antioxidant proteins or if antioxidant
capacity decreases, ROS can accumulate in neurons and cause oxidative damage in the
form of lipid peroxidation, macromolecular damage and protein dysfunction. If the
oxidative damage is severe, it can cause neuronal death. This is important in the context
of PD, because GSH levels are depleted in NSDA neurons of PD patients, suggesting
their ability to manage ROS is compromised (Jenner and Olanow, 1996). Taken together,
either enzymatic or non-enzymatic DA metabolism can produce ROS, the toxic aldehyde
DOPAL, and DA-quinone molecules that can cause intracellular damage to NSDA
neurons through a number of mechanisms. Thus, the regulation of DA synthesis, storage,
release and reuptake is critical to controlling DA metabolism and formation of toxic

byproducts.

23

f} ‘

F. NSDA Physiology in PD

There is considerable evidence to suggest that the regulation of DA homeostasis is
dysfunctional in early PD. At the time of clinical diagnosis there is a loss of
approximately 50% of SNpc neurons and 70-80% of striatal DA axon terminals
(Bernheimer et al., 1973; Homykiewicz, 1975; Feamley and Lees, 1991; Homykiewicz,
1998). Interestingly, the clinical symptoms of the disease do not appear until there is
already an extensive loss of NSDA neurons. The delayed appearance of motor
complications is thought to reflect compensatory changes in the activity of surviving
neurons that maintains DA output at the near control levels, thus upholding the balance
between direct and indirect basal ganglia pathways (Zigmond, 1997). Evidence from
both human PD patients and animal models of NSDA damage support this hypothesis.

Positron emission tomography (PET) using 6-[18F]-fluoro-L-dopa (F-DOPA)
provides an in vivo index of the rate of DA synthesis and release in patients with PD. F-
DOPA imaging can be used as a reliable estimate of the rate of DA synthesis by
quantifying the conversion of F -DOPA to [18F ]-DA by AADC and its subsequent storage
into synaptic vesicles (Patlak et al., 1983; Vingerhoets et al., 1994; Vingerhoets et al.,
1996). Measuring the rate of decline in striatal ['sF]-DA is an index of the rate of DA
release and thus NSDA neuronal activity (Barrio et al., 1990; Pate et al., 1993; Yee et al.,
2001). F -DOPA imaging studies have demonstrated that early in PD, there is a
compensatory increase in the rate of DA synthesis and release in residual NSDA neurons
(Ribeiro et al., 2002; Sossi et al., 2002). Post-mortem neurochemical studies have
supported this hypothesis. DA neuronal activity is defined neurochemically by the ratio

of the DA metabolite, DOPAC to stored DA and is a reliable index of the rate of DA,

24

 

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also

release, reuptake and metabolism (Westerink and Spaan, 1982; Gopalan et al., 1993).
There is an increase in NSDA neuron activity, as defined by the DOPAC to DA ratio in
post-mortem PD patients that died during the early stages of the disease (Homykiewicz,
1975; Lloyd et al., 1975; Ribeiro et al., 2002; Sossi et al., 2002; Sossi et al., 2004).

Animal studies have supported in vivo imaging and post-mortem neurochemical
data obtained in humans. Neurotoxin-induced partial lesions of the NSDA pathway
increase the activity of surviving neurons, as reflected by an increased DOPAC to DA
ratio (Homykiewicz, 1975; Zigmond and Stricker, 1984; Zigmond et al., 1990;
Homykiewicz, 1998; Thiffault et al., 2000). Microdialysis experiments, which provide a
more direct measurement of DA release demonstrate that synaptic DA concentrations are
maintained at near normal levels despite the loss of axon terminals (Zigmond and
Stricker, 1984; Stachowiak et al., 1987; Snyder et al., 1990). While decreased DA
clearance may be partially responsible for increased synaptic DA, pre-synaptic changes
that enhanced stimulus-evoked DA release also occur (McCallum et al., 2006). Thus,
partial lesions of the NSDA pathway cause compensatory changes that increase DA
synthesis, release, reuptake and metabolism in surviving neurons that is associated with
delaying the onset of PD motor symptoms.

Compensatory increases in DA synthesis and release are likely beneficial in
delaying symptom onset in PD, however, due to the toxic nature of DA metabolism, it is
also possible that prolonged increases in DA synthesis, release, reuptake and metabolism
results in increased exposure to DOPAL, OH', 02' and DA-quinone molecules (Figure l-
7). Thus, ROS-induced oxidative stress and DA-quinone-induced covalent protein

modification, derived from sustained increases in DA metabolism, may contribute to the

25

 

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progressive degeneration of NSDA neurons in PD. Consistent with this hypothesis,
several studies suggest that ROS damage, decreased antioxidant defense, and
mitochondrial impairment play key roles in the pathogenesis of PD. There is extensive
ROS-mediated damage in the striatum of post-mortem PD brains, reflected by high levels
of lipid peroxidation and protein carbonyls, an index of oxidative damage (Dexter et al.,
1989; Dexter et al., 1994; Yoritaka et al., 1996). Additionally, there is a specific decrease
in GSH content in the SNpc of PD patients, suggesting the ability of NSDA neurons to
scavenge the free radicals generated by DA metabolism is compromised (Sofic et al.,
1992; Sian et al., 1994). Finally, mitochondrial complex I deficits have been reported in
the SNpc of PD patients (Schapira et al., 1990; Swerdlow et al., 1996). Mitochondrial
deficits could result in NSDA damage due to bioenergetic deficits, generation of ROS, or
accumulation of the highly toxic metabolite DOPAL (Lamensdorf et al., 2000).
Pathological hallmarks found in the post-mortem PD brain suggest DA metabolism may
contribute to NSDA neuron loss. Yet, the neurochemical changes that underlie the
acceleration in DA release, reuptake and metabolism remain unclear.

The goal of the research described herein is to identify and characterize the
biochemical mechanisms that are responsible for compensatory changes in DA neuron
function in response to partial lesions of the NSDA pathway. Accomplishing this task
could lead to the development of novel therapeutic targets that can be exploited to slow
or halt the progressive loss of NSDA neurons. To this end, the regulation of DA release,
reuptake and metabolism in surviving neurons, will be studied using animal models of

PD that recapitulate damage to the NSDA pathway that occurs in PD.

26

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G. PD Animal Models

The cause(s) of idiopathic PD remain unknown. As such, there is no perfect
animal model of PD that is able to recapitulate every aspect of the disorder. There exists
however, a variety of animal models that are used for reproducing different features of
the disease. Animal models are evaluated based on their ability to reproduce the cardinal
clinical and pathological hallmarks of the disease. For the purposes of the research
described in this dissertation, selection of an animal model of PD will be based on the
ability to produce reliable, reproducible lesions of the NSDA pathway that are in keeping
with the severity of NSDA loss observed in PD.

A munber of genetic mutations are associated with heritable/familial PD. While
familial PD only accounts for approximately 10% of all reported cases (Payami and
Zareparsi, 1998), the identification of genes that are associated with PD pathogenesis has
been instrumental to understanding the neurodegenerative process that occurs in the
disease. In particular, mutations, duplications, or triplications in the gene encoding the a-
synuclein protein are reported to result in heritable PD and cause degeneration of NSDA
neurons (Polymeropoulos et al., 1997; Kruger et al., 1998; Singleton et al., 2003).

a-Synuclein is also important in idiopathic PD. Abnormal aggregation of a-
synuclein into Lewy-bodies is a primary pathological hallmark of PD (Spillantini et al.,
1997). As such, several transgenic mouse animal models have been developed that
incorporate known a-synuclein mutations into the genome (F eany and Bender, 2000;
Masliah et al., 2000; van der Putten et al., 2000; Matsuoka et al., 2001; Giasson et al.,
2002; Lee et al., 2002). These studies have provided insight into the molecular events

that lead to Lewy-body formation and cell death in PD. However, the a-synuclein

27

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transgenic models, as well as transgenic mice expressing mutations in other genes linked
to PD, fail to reproduce the primary pathological hallmark of PD, the loss of NSDA
neurons (Matsuoka et al., 2001; Greene et al., 2003; Pesah et al., 2004; Goldberg et al.,
2005; Park et al., 2005). As such, these models are not ideal for studying compensatory
changes that occur in NSDA neurons in response to neurodegeneration.

Alternatively, a number of neurotoxin-based animal models of PD have been
developed that produce specific NSDA neuron loss. The most commonly used
neurotoxins for this purpose are 6-hydroxydopamine (6-OHDA), N,N’-dimethyl-4-4’-
bipiridinium (paraquat), rotenone, and l-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(MPTP). Paraquat, rotenone and MPTP were identified as risk factors for PD in humans
and subsequently developed for use in animals to recapitulate the disease in the
laboratory. Epidemiology studies have highlighted the importance of human exposure to
pesticides and herbicides in increasing risk for PD (Tanner, 1992). These studies
provided a basis for the development of animal models of PD using rotenone, a widely
used pesticide, and paraquat, a naturally occurring herbicide. The unintentional use of
MPTP, which caused a syndrome that was clinically indistinguishable from idiopathic PD
in a number of people in the 1980’s, provided the basis for development of MPTP as an
animal model of PD (Langston et al., 1983). The use of these neurotoxin-based animal
models of PD has advanced the understanding of PD etiology and pathogenesis.
However, there are a number of advantages and disadvantages to each neurotoxin-based
animal model of PD and selection of the proper animal model is critical to experimental

design and data interpretation.

28

6-0HDA

6-0HDA was the first neurotoxin used to produce a specific loss of NSDA
neurons (Ungerstedt, 1968). 6-OHDA cannot cross the blood-brain barrier, and thus
must be directly injected into the substantia nigra, medial forebrain bundle or striatum,
the sites of NSDA neuronal cell bodies, axon projections and axon terminals,
respectively. 6-0HDA is a hydroxylated analogue of DA (Blum et al., 2001) and as
such, following its injection 6-0HDA is transported into DA neurons through the DAT
where it accumulates in the cytoplasm.

ROS and reactive quinone molecules are primarily responsible for the toxicity of
cytoplasmic 6-OHDA. 6-0HDA can be directly oxidized to H202 and para-quinone in
the presence of molecular oxygen and transition metals (Przedborski and Ischiropoulos,
2005). As depicted in Figure 1-8, 6-OHDA can react with molecular oxygen to yield
semi-quinone and 02'. The 02' molecule formed from this reaction can react with
additional 6-0HDA molecules to produce semi-quinone and H202. Semi-quinone can
react with molecular oxygen to produce para-quinone and 02'. The 02' formed in this
reaction can react with additional semi-quinone producing more para-quionone and H202.
The ROS and quinones generated from these four reactions can cause oxidative damage
to lipids, proteins and DNA and are responsible for the toxicity induced by 6-0HDA
(Heikkila and Cohen, 1973). Additionally, 6-0HDA may also directly inhibit
mitochondrial complex I (Betarbet et al., 2002; Schober, 2004), suggesting neurotoxicity
may be due in part, to a bioenergetic defect. However, it is not clear if complex 1
inhibition is due to a direct binding of 6-0HDA to the enzyme, or indirect effect of

macromolecular damage to mitochondria due to ROS-generated from the intracellular

29

0X11

pro.‘

oxidation of the neurotoxin. The latter may be the case, since inhibition of MAO activity

provides almost complete protection f'rom 6-0HDA toxicity (Knoll, 1986).

30

Fig.2: }
m-riscui;
2.1:.‘26 ti;
21:02, 5
0‘4} gen c

I»: ,
disrupt;

OH HO 0'
+02 — vm/\+C)2“+H+
HO N42 NH:

6—OHDA Semiquinone
OH HO 0‘
M: NH:
6-OHDA Semiquinone
HO O 0
m +02 —m/\ +02' + H+
. . NH: NH:
Semrqurnone Para-quinone
O O
NH, NH:
Semiquinone Para-quinone

Figure 1-8. The oxidation reaction for two molecules of 6-0HDA. The first 6-0HDA
molecule reacts with molecular oxygen to form semi-quinone and 02-. The 02- formed
in the first reaction reacts with the second 6-0HDA molecule to form semi-quinone and
H202. Semi-quinones generated from the first two reactions can react with molecular
oxygen or 02- to form para-quinone and either 02- or H202 (Przedborski and
Ischiropoulos, 2005).

31

 

 

OI

6-0HDA injection produces a reliable, rapid and severe loss of NSDA neurons
(J eon et al., 1995). The extent of the lesion is dependent on the amount of 6-0HDA
injected. The advantage of the 6-OHDA model is that the neurotoxin can be injected
unilaterally to destroy SNpc neurons on one side of the brain, while sparing contralateral
neurons to be used as a control. Moreover, a unilateral NSDA lesion produces
asymmetric tuming behavior to the contralateral side of the lesion. The contralateral
turns are the result of an imbalance between the lesioned and unlesioned striatum that
causes basal ganglia dysfunction on one side of the brain and decreases motor cortex
output to the opposite side of the body. The degree of the circling behavior directly
corresponds with the extent of the lesion (Ungerstedt, 1968). As such, quantification of
the circling provides an in vivo index of the NSDA lesion severity.

The disadvantages of the 6-OHDA model is the specificity, time-course and
mechanism of cell death are not similar to what is observed in PD. 6-0HDA enters cells
via active transport mediated by the DAT and once inside the cell, the neurotoxin causes
ROS formation, oxidative damage and neuron death. However, the neurotoxicity induced
by 6-0HDA injection is not selective to NSDA neurons. 6-0HDA can be taken up into
other DA neurons or be transported into norepinepherine neurons via the norepinepherine
transporter and causes the destruction of these neurons (Luthman et al., 1989). Thus,
NSDA neurons are not more vulnerable to 6-OHDA, the selective loss of these neurons is
a function of the location of toxin administration. Additionally, the time-course of cell
death induced by 6-0HDA is very rapid (within 24 hours), which does not match the
slow progressive loss of NSDA neurons in PD. Neuronal cell death following 6-0HDA

is primarily necrotic with no evidence of apoptotic cell death as seen in PD (J eon eta1.,

32

1995). Due to a lack of selectivity, and rapid necrotic cell death induced by 6-OHDA,
this neurotoxin is not ideal for identifying mechanisms that may contribute to the

progressive cell death of NSDA neurons in PD.

Paraquat

Paraquat is an herbicide that is naturally found in the environment and is
associated with increased risk for PD (Liou et al., 1997). Paraquat enters DA neurons via
the DAT (Shimizu et al., 2001; Shimizu et al., 2003), however, non-DAT-mediated
transport may also occur (Richardson et al., 2005). Once inside the neuron paraquat
reacts with nicotinamide adenine dinucleotide (N ADPH, NADH) in a reaction catalyzed
by diaphorases to form reactive paraquat and NADP+ (Figure 1-9). Diaphorases are
enzymes that transfer electrons from electron donors (NADH, NADPH) to other
molecules (Dicker and Cederbaum, 1991; Liochev and F ridovich, 1994; Liochev et al.,
1994; Shirnada et al., 1998). The reactive paraquat formed in the first reaction can then
react with molecular oxygen to form 02' radicals (Day et al., 1999; Przedborski and
Ischiropoulos, 2005).

Paraquat-induced 02' generation leads to oxidative stress, the destruction of
NSDA neurons, and the formation of neuronal inclusions in the frontal cortex reminiscent
of Lewy-bodies (Manning-Bog et al., 2002; McCormack etal., 2002). However, the
degeneration of cortical neurons has not been reported. The advantage of the paraquat
model is that it causes the formation of protein inclusions in neurons similar to Lewy-
bodies, allowing the molecular mechanisms that result in the formation of Lewy-bodies

to be investigated. The disadvantage of this model is that NSDA cell loss has not been

33

consistently reported and it is not known if NSDA neurons are particularly vulnerable to

paraquat toxicity (Thiruchelvam et al., 2000).

34

 

“30-" HH3+ NAD< )H p H30“ wD—Cuf—CH3+ NAD(P)"

 

Paraquat Paraquat radical
H30- ”G‘CN—CHy o2 >H,c-N' HHa” 02 '
Paraquat radical Paraquat

Figure 1-9. Generation of 02- radicals following the oxidation of paraquat. Paraquat
reacts with NADPH to form paraquat radical and NADP+. The paraquat radical
generated in the first reaction can react with molecular oxygen to form paraquat and 02-.

35

Rotenone

Rotenone is a naturally occurring mitochondrial complex I inhibitor developed for
use as an insecticide. Rotenone is highly lipophillic and can easily cross the blood-brain
barrier and gain access to all cells. Following systemic injection, rotenone is found
evenly distributed throughout the brain and highest concentrations are achieved within 15
minutes (Talpade et al., 2000). As shown in Figure 1-10, once rotenone enters cells it
accumulates in mitochondria where it binds to nicotinamide adenine dinucleotide
(N ADH)-ubiquinone reductase (complex I) and inhibits the transfer of electrons, thus
inhibiting mitochondrial respiration and ATP production. Additionally, binding to
complex I inhibits the transfer of electrons and allows these electrons to leak out of the
mitochondria which can then react with molecular oxygen to form ROS (Betarbet et al.,
2002)

Cellular toxicity following rotenone is due to ROS generation and ATP depletion.
Despite the ability of rotenone to enter all cell types, chronic (28 day) intravenous
administration of the drug produces a relatively selective degeneration of NSDA neurons
and intraneuronal inclusions similar to Lewy-bodies (Betarbet et al., 2000; Thiffault et
al., 2000). These results suggest that the primary pathological hallmarks of PD, the
unique vulnerability of NSDA neurons and Lewy-body formation, can be produced as a
result of systemic inhibition of mitochondrial complex I. Also, the chronic nature of
NSDA cell loss induced by rotenone more accurately reflects the progressive nature of
PD and causes motor dysfunctions including; abnormal animal posture and decreased
spontaneous movement. Thus, rotenone recapitulates the primary aspects of idiopathic

PD, however, the use of rotenone has a munber of disadvantages as well. While rotenone

36

 

indu.‘

dame

10.1611

induces a relatively selective loss of NSDA neurons, other brain regions that are not
damaged in PD (i.e. post-synaptic striatal neurons), show pathology following chronic
rotenone treatment. Moreover, NSDA lesions only occurred in approximately 50% of the
animals that received rotenone and the lesion severity was highly variable (Hoglinger et
al., 2003). Thus, rotenone requires further investigation and characterization before

being used as a reliable animal model of PD.

37

 

Figure 1
mantra
nicorénzr
12515315 I
prrductir

ROSuBc

Cytoplasm Rotenone

 
 

Complex V

 
 
 

ATP

NADH + H* NAA H’ -
R O S ADP + PI

Mitochondrion

Figure 1-10. The highly lipophillic rotenone molecule can freely pass through cellular
membranes. Once inside the cell, it accumulates in mitochondria where it binds to
nicotinamide adenine dinucleotide (NADH)-ubiquinone reductase (complex I) and
inhibits the transfer of electrons, inhibiting mitochondrial respiration and decreasing ATP
production. Additionally, binding to complex I causes electron leak and the formation of

ROS (Betarbet et al., 2002).

38

MPT P

MPTP was discovered to be a neurotoxin in the 1980’s when a number of people
developed a syndrome clinically indistinguishable from idiopathic PD as a result of
inadvertent administration of the compound (Langston et al., 1983). Since then,
administration of MPTP to non-human primates and mice has been the most widely used
animal models of PD. However, for the purposes and aims of this dissertation, only
MPTP mouse models will be discussed.

As depicted in Figure 1-1 1, following systemic administration of MPTP, the
lipophillic MPTP molecule crosses the blood brain barrier. Once inside the central
nervous system, MPTP is able to gain access to all cells, but the majority of the toxin is
taken up by glial cells due to a much greater number of these cells compared to neurons
(Markey et al., 1984). Inside glial cells MPTP is converted to 1-methyl-4-phenyl-2,3-
dihydropyridinium (MPDP) by mitochondrial MAO-B and then oxidized to the toxic
metabolite 1-methy1-4-phenyl pyridinium (MPP'). MPP+ is released f'rom glial cells
through unknown mechanisms (Dauer and Przedborski, 2003). The positively charged

MPP' ion has an affinity for the DAT and is actively transported into DA neurons.

39

Blood brain
barrier

 

Dopamine
Neuron

Figure 1-11. MPTP transport into the brain, conversion to MPP+ and transport into DA
neurons. MPTP readily crosses the blood brain barrier and is taken up predominantly by
glial cells and converted though a two-step reaction to MPP' that is catalyzed by MAO-
B. MPP+ is then released from glial cells and is taken up primarily by DA neurons due to
its affinity for the DAT. (Panel B) The axon terminal effects of MPP'. MPP+ enters the
axon terminals of DA neurons and can either; bind to mitochondrial complex I, interfere
with cytosolic enzyme function, or be sequestered into synaptic vesicles (Dauer and
Przedborski, 2003).

40

As depicted in Figure 1-12 , once inside DA neurons, MPP+ can bind to
mitochondrial complex I and similar to rotenone, produce DA neuron death through
impairment of mitochondrial respiration and ROS generation. Alternatively, cytoplasmic
MPP+ can be sequestered into synaptic vesicles, preventing it from binding to complex I
or react with axon terminal proteins. MPP+-induced toxicity is not restricted to the DA
neurons. MPTP also causes toxicity in glial cells that bio-activate the toxin (Di Monte et
al., 1999). However, overt glial cell loss is not observed because glial cells that take up
the toxin are distributed evenly throughout the brain, and as such, there is not a
concentrated loss of these cells in any region and, unlike neurons, new glial cells may be

produced to replace lost cells.

41

 

MPP+

Figure 1-12. The effects of MPP+ on the axon terminal of NSDA neurons. MPP" enters
the axon terminals of DA neurons and can either; bind to mitochondrial complex I,
interfere with cytosolic enzyme function, or be sequestered into synaptic vesicles (Dauer
and Przedborski, 2003). Binding to mitochondrial complex I and cytosolic proteins
results in the neurotoxic effects of MPP'. Sequestration into synaptic vesicles protects
the axon terminals from the neurotoxicity of MPP'.

42

Systemic MPTP administration in mice causes a loss of NSDA neurons and the
development of motor deficits that resemble PD symptoms (Jackson-Lewis et al., 1995;
Sedelis et al., 2001). NSDA neurons are more vulnerable to the toxic effects of MPTP,
compared to norepinephrine neurons and even other DA neurons that express the DAT
(Chiueh et al., 1985). The effects of MPTP administration on the time course, extent, and
mechanism of' cell death is highly dependent on the dosing regimen. In general, acute
administration paradigms where high doses of the neurotoxin are administered over a
brief period (usually a cumulative dose of 80 mg/kg in one day) result in very rapid and
severe NSDA cell loss that is primarily necrotic (Bezard et al., 1997; Smeyne and
Jackson-Lewis, 2005). However, the rapidity and severity of cell loss in the acute
treatment model does not match the slowly progressive nature of early PD where
apoptotic cell death is known to occur (Mochizuki et al., 1996; Anglade et al., 1997).

Sub-chronic dosing regimens (25 mg/kg per day for five consecutive days) and
prolonged-chronic regimens (20 mg/kg, twice a week for five consecutive weeks) have
been developed that distribute the total dose of MPTP over several days, causing a
prolonged impairment of mitochondrial function and chronic exposure to ROS. Using
these dosing regimens cell death occurs from both apoptotic and necrotic mechanisms,
which more accurately reflects cell death in PD (Bezard et al., 1997; Tatton and Kish,
1997; Novikova et al., 2006). Also, despite a higher cumulative dose of MPTP the lesion
produced by the neurotoxin is less severe than in the acute model and more reminiscent
of the extent of NSDA neuron loss observed in early PD.

Sub-chronic or prolonged-chronic MPTP dosing regimens both produce reliable

and reproducible lesions of NSDA neurons (Petroske et al., 2001). NSDA neurons are

43

more vulnerable to degeneration in PD, and the time course and mechanisms of cell death
closely resemble what occurs in PD. Taken together, chronic administration regimens of
MPTP recapitulate the key pathological features of idiopathic PD, and as such, are ideal

for studying NSDA neuronal fimction during conditions that are similar to PD.

H. Thesis Objective

The studies described herein are designed to test the hypothesis that increased DA
metabolism secondary to compensatory increases in DA synthesis, release and reuptake
contributes to the progressive degeneration of NSDA neurons in PD. To test this
hypothesis, pharmacological, neurochemical and immunohistochemical techniques will
be used to address the following Specific Aims: 1) determine if chronic MPTP
administration regimens cause compensatory increases in the activity of surviving
unlesioned neurons, 2) characterize the neurochemical changes that allow NSDA neurons
to maintain the increased neuronal activity, 3) determine if sustained increases in DA
metabolism can cause a progressive loss of NSDA neurons. Identification of the
mechanisms that underlie compensatory changes in NSDA neuron function should lead
to the development of therapeutic strategies that can halt or delay the progressive loss of

these neurons in PD.

45

 

 

LII
. ._

Chapter 2. Materials and Methods

A. Animals
Generation of wild-type and homozygous a-synuclein knock-out mice

Homozygous u-synuclein knock-out mice were obtained in breeding pairs from
Jackson Labs (B6;129X-SncatmlRosl, stock #3692, Bar Harbor, MA). The generation,
viability, fertility and basic biochemical features of this strain have been previously
described (Abeliovich et al., 2000). a-Synuclein knock-out mice were crossed with the
inbred strain (C57/Bl6) used as the host for the blastocyst during the original generation
of the a-synuclein knock-out mice (Abeliovich et al., 2000) to yield heterozygous mice
(F1). Heterozygous mice were then crossed and the offspring of this cross (F2) were
used to maintain a stable breeding colony. Additional heterozygous (F2 x F2 and F3 x
F3) crosses were performed to expand and maintain the colony for the experiments
described herein. University-trained technicians maintained the breeding colony.
Determining Animal Genotype

Animal genotype was confirmed by PCR analysis of genomic DNA (isolated
from tail samples) using primers specific for Exon 2 of the a-synuclein gene to identify
native a-synuclein, and the neomycin resistance gene insert to identify the knock-out
sequence (Abeliovich et al., 2000). Mouse tail snips were dissolved in 600 111 of nuclei
lysis buff'er with proteinase K (25 mM EDTA, 50 mM NaCl, 0.8 mg/ml proteinase K) at
55°C for 4 hours. Following digestion, RNase (0.05 mg/ml) was added to the lysate and
incubated for 30 min. Subsequently, 200111 protein precipitation solution (4.2 M NaCl,
0.63 M KCL, 10 mM Tris base, pH 8.0) was added to the mixture, incubated on ice for 5

minutes, and centrifuged (15,000 x g 10 min) to pellet protein. The supernatant was

46

 

 

amp

hl:)m

‘h5 e:

placed into a fresh microcentrifuge tube containing 600 111 room temperature isopropanol.
The solution was gently mixed by inversion until white strands of DNA appeared. The
insoluble DNA strands were isolated by centrifugation at 15,000 x g 60 sec. The
supernatant was removed, and the DNA-containing pellet was washed in ethanol and
resuspended in 100 111 of DNA rehydration solution (10 mM tris-HCL, 1 mM EDTA, pH
7.4).

The concentration of DNA in each sample was determined by measuring the
absorbance at 260/280 nm with a nanodrop ND-1000 spectrophotometer (Nanodrop,
Wilmington, DE). The concentration of each sample was adjusted to 50 ng/ul by adding
the appropriate amount of DNA rehydration solution. For the PCR reaction, 5 111 of the
DNA sample (50 ng/ul) was added to 20 ul of PCR reaction solution (1 mM PCR buffer,
200 mM dNTP, 2 mM MgCl, 0.18 pm/ul oligo primers, 0.4 u/ul taq polymerase,
Invitrogen Carlsbad, CA). Primers were selected to bracket exon 2 of the a-synuclein
gene, or the neomycin resistance gene sequences, as described previously (Drolet et al.,
2004). Samples were vortexed and placed into the PCR machine. PCR run parameters
were 94°C x 5 min, then 30 cycles of 94°C, 65°C, 72°C x one minute each, followed by
72°C x 5 min. Subsequently, gel electrophoresis (75 mV, one hour) on a 3.0% agarose
gel was used to separate amplified DNA (10 111) from each sample. As depicted in Figure
2-1, animal genotype was confirmed for all animals through visualization of the
amplified DNA using a UV transilluminator (UV C co., San Gabriel, CA). Only
homozygous a-synuclein knock-out mice or homozygous wild-type mice were used in

the experiments described herein.

47

figure
lifli’m,‘

1 ' 1‘
gel lOl:

£35326 1.‘

 

Figure 2-1. Genotyping of Wild-type (+/+), homozygous a-synuclein knock-out (-/-),
heterozygous (+/-) mice, and control blanks (0). Ethidium bromide stained 3% agarose
gel following electrophoresis of PCR amplification products of exon 2 of the a-synuclein
gene (320 base pairs) and the neomycin resistance construct (280 base pairs).

48

Animal Housing

Animals were housed two to four per cage, maintained in a temperature- (22 :t
1°C) and light-controlled (12-h light—dark cycle, lights on at 0600 hr) room, and provided
with food and tap water ad Iibitum. The Michigan State University, All University

Committee on Animal Use and Care approved all experiments using live animals.

B. Drugs

All drugs were purchased from Sigma-Aldrich (St. Louis, MO). Concentrations
were calculated from the free base of the respective drug.
Prolonged Chronic MPT P Administration

MPTP was dissolved in 0.9% sterile saline. For dose-response experiments,
dipropylsulfamoyl-benzoic acid (probenecid) was dissolved in dimethyl sulfoxide
(DMSO). To improve solubility of the probenecid, for time-course experiments,
probenecid was dissolved in 0.1 N NaOH and the pH was adjusted to 7.4 using HCL.
Male 8—12 week old mice received so injections of either vehicle (1 kag) or MPTP (1,
5, 10, or 20 mg/kg) twice a week, alternating morning and afiemoon every 3.5 days, for a
total of 10 total injections over 5 weeks. Probenecid (250 mg/kg; i.p.) was co-
administered with each dose of MPTP to increase the plasma and central nervous system
half-life of MPTP and MPP', respectively (Petroske et al., 2001). Neither DMSO nor
probenecid produces an effect per se on NSDA neurons (Lau et al., 1990). All
experiments using MPTP were performed using previously published safety guidelines
(Przedborski et al., 2001). Experiments were terminated three weeks following the last

injection of MPTP.

49

 

Sub-chronic MPTP Administration

MPTP was dissolved in 0.9% sterile saline. Male 8—12 weeks old mice received
s.c. injections of vehicle (1 ml/kg) or MPTP (1, 5, or 25 mg/kg) once daily for five
consecutive days. Experiments were tenninated three days following the last MPTP.
Acute m-Hydroxybenzylhydrazine (NSD-1015) Administration

NSD-1015 was dissolved in 0.9% saline. For determining TH catalytic activity in
NSDA neurons, mice were injected with the L-AADC inhibitor NSD-1015 (100 mg/kg,
i.p.) thirty min prior to decapitation and striatal DOPA concentrations were measured
(Figure 2-4). The accumulation of DOPA following blockade of L-AADC with NSD-
1015 is an in vivo assay for TH catalytic activity (Carlsson et al., 1972).
Acute Raclopride Administration

Raclopride was dissolved in 0.9% saline. Mice were injected with raclopride (1.0
mg/kg, i.p.) and killed by decapitation either 30, 60, or 120 min later.
Acute Gamma-butyrolactone (GBL) Administration

GBL was diluted in 0.9% saline. Mice were injected with GBL (750 mg/kg, i.p.)
and killed by decapitation either 1, 2, or 3 h later.
Acute Tyrosine Administration

L-tyrosine dihydrochloride was dissolved in 0.1 N HCL diluted in 0.9% saline.
Mice were injected with tyrosine-HCL (100 mg/kg, i.p.) and killed by decapitation 1 h
after administration. In some experiments, mice were injected with L-tyrosine disodium
salt, since it dissolves in 0.9% saline. Mice were injected with tyrosine disodium salt and

killed by decapitation 1 h after administration.

50

Chronic Raclopride and Quinelorane Administration

Alzet osmotic minipumps (#2004, Durect Corporation, Cupertino, CA) were filled
with either raclopride hydrochloride (Sigma) dissolved in sterile saline (2.5 mg/ml) or
quinelorane hydrochloride (Sigma) dissolved in sterile saline (2.5 mg/ml). Mice received
0.015 mg/day (0.5 mg/kg/day) of either raclopride or quinelorane based on the pump

delivery rate (0.25 ul/hr, 28 days) and the average weight of a mouse (0.030 kg).

C. Preparation and Implantation of Alzet Osmotic Mini-pumps

Prior to implantation, Alzet osmotic mini-pumps were filled with saline,
raclopride or quinelorane, and primed by placing the pumps in saline at 37°C for 24 h.
0n the day of implantation, mice were anesthetized with ketaminezxylazine cocktail (10:2
mg/kg, i.p.). The incision site was shaved and cleaned with 10% povidone iodine
solution. An incision (3 mm) was made across the width of the animals back. Forceps
were used to prepare a s.c. pocket for the pump. Pumps were placed under the skin in the
prepared pocket and the wound was sealed with wound-clips. The animals were placed
on a heating pad and monitored until fully recovered from anesthesia (approximately 2-3
h).

Alzet pumps (#2004) deliver drugs at a constant rate of 6 til/day for 28 days.
Pumps were left in mice for 21 days to prevent any loss of drug delivery due to improper
pump filling or decreased diffusion rate. For a 21 day experiment, 126 111 is the ideal
volume delivered. The actual volume delivered was determined by removing residual

fluid from the inner chamber of the pumps following completion of the experiment and

51

subtracting the value from the 220 111 mean filling volume. If the actual and ideal volume

delivered differed by greater than 50 ul, the sample was not used for analysis.

D. Neurochemistry
Tissue Preparation

Following drug administration, mice were decapitated and their brains were
rapidly removed and placed on a glass dissecting surface (4°C) on its dorsal surface. The
median eminence was microdissected with the aid of a dissecting microscope and placed
into ice-cold tissue buffer (0.1M phosphate-citrate buffer pH 2.5) for neurochemical
analysis. The brain was then quickly frozen over dry ice. Consecutive 500 pm thick
coronal sections were prepared from the frozen brains using a cryostat set at -10 °C
(CTD-Model Harris, International Equipment Co., Needham, MA). Sections were
collected through the rostro-caudal extent of the forebrain beginning approximately 2.5
mM anterior to bregma (Paxinos and Watson 1986). Sections were thaw-mounted onto
glass slides, refrozen and the nucleus accumbens and striatum were dissected using a
modification of the method described by Palkovits (Palkovits, 1973, 1978).

As depicted in Figure 2-2, using a dissecting microscope the nucleus accumbens
was removed using a 22-gauge punch tool fashioned from a 22-gauge hypodermic
needle. Sections located approximately 1.8 mm anterior to bregma were collected for
dissection. The nucleus accumbens was dissected bilaterally and samples were placed

into ice-cold tissue buffer and stored at -20°C until neurochemical analysis.

52

Rostral Caudal

 

 

Figure 2-2. A. Sagittal view of a Nissl stained mouse brain depicting the rostro-caudal
location of the sections used to dissect the nucleus accumbens. B. Coronal brain sections
stained for Nissl that depicts the section used to dissect the nucleus accumbens. C.
Diagram of the location and size of the Palkovits micropunch (approximately 0.5 mm
id.) used to dissect the nucleus accumbens (Paxinos and Watson, 1986).

53

As depicted in Figure 2-3, using a dissecting microscope the striatum from the left
side of the brain was removed using an 18-gauge punch tool, fashioned from an 18 gauge
hypodermic needle. Striata from two consecutive sections, located approximately 1.1
mm and 0.6 mM anterior to bregma, respectively, were dissected and placed into 100 111
of ice-cold tissue buffer and stored at -20°C until neurochemical analysis. Striata from
the right side of the brain were dissected using a 12-gauge punch tool and placed into ice-
cold homogenization buffer for Western blot analysis, described in further detail below.
This method discretely isolates the terminal regions of NSDA, mesolimbic dopamine
(MLDA) and tuberinfundibular dopamine (TIDA) neurons that can be used for

neurochemical and Western blot analysis.

Rostral Caudal

 

 

Figure 2-3. A. Sagittal view of a Nissl stained mouse brain depicting the rostro-caudal
location of the sections used to dissect the striatum. B. Coronal brain sections stained for
Nissl that depicts the first section (left) and second section (right) used to dissect the
striatum. C. Diagram of the location and size of the Palkovits micropunch
(approximately 1 mm i.d.) used to dissect the striatum for neurochemistry (Left Side of
Brain) and Western blot analysis (approximately 1.5 mm i.d., Right Side of Brain)
(Paxinos and Watson, 1986).

Neurochemical Analysis

On the day of assay, samples were thawed and sonicated (Heat Systems
Ultrasonics, Plainview NY) using three one sec bursts. Samples were then centrifuged at
18,000 x g (Beckman Coulter microfuge, Palo Alto, CA) for one min. The supernatant
was removed using a 100 111 Hamilton syringe and the final volume of the supernatant
was adjusted to 100 111 and placed into a fresh microcentrifuge tube for high pressure
liquid chromatography with electrochemical detection (HPLC-EC) analysis.

The pellet from the initial centrifugation was resuspended and dissolved in 100 111
of 1N sodium hydroxide (NaOH). 0n the following day, striatal protein concentrations
were determined from these tissue pellets using the Lowry protein method (Lowry et al.,
1951). Aliquots of striatal protein samples (50 111) were transferred from the
microcentrifuge tube to a 12 x 75 glass culture tube using a 100 111 Hamilton syringe.
NaOH (50 111) was added to the protein sample in each tube yielding a final volume of
100 1.0/culture tube. Standard solutions with either 12.5, 25, or 50 11g of bovine serum
albumin (Sigma) were dissolved in 100 111 of NaOH and transferred to individual 12 x 75
culture tubes. For colorimetric determination of protein concentrations in the culture
tubes, 1.0 ml of reagent A (0.2 M sodium carbonate, 40 mM cupric sulfate, 70 mM
sodium potassium tartrate) was added to each tube and allowed to incubate for 10 min.
Subsequently, 100 111 of reagent B (folin reagent diluted 1:2 with dH2O) was added to the
culture tubes and incubated for 30 min. Following incubation, protein concentrations
were determined by normalizing the absorbance of each sample, read at 700 nm on a

Gilford spectrophotometer (Gilford Instruments, Oberlin, OH) to the absorbance of the

56

known protein standards assayed concurrently. A single 18-gauge striatal punch yields
an average of 40 ug of protein.

Striatal DA, DOPAC and DOPA was determined with HPLC-EC using a Waters
515 HPLC pump (Waters) with a flow rate of 1.0 ml/min and a ESA Coulochem 5100A
electrochemical detector with an oxidation potential of +0.4V. Neurochemical standards
containing 1.0 ng of DOPAC, DA and DOPA and striatal supernatant samples were
injected onto a C18 reverse phase analytical column (Bioanalytical systems, West
Lafayette, IN). The HPLC-EC mobile phase (0.5M sodium phosphate, 0.03M citrate, 0.1
mM EDTA, sodium octylsulfate, 15-20% methanol, pH 2.5) was adjusted by varying the
amounts of sodium octylsulfate and the methanol concentration to optimize DA,
DOPAC, and DOPA separation.

DA, DOPAC, and DOPA content was quantified by normalizing the peak heights
of each sample to the peak heights of these compounds measured in the known 1.0 ng
standards (Figure 2-4). The linear range for measuring DA content in the striatum is
approximately 0.15-4.0 ng using this HPLC-EC technique (Figure 2-5). A single 18-
gauge striatal punch yields approximately 3,000 pg of striatal DA. The broad range of
this technique allows for an approximately 200-fold increases or decreases from baseline
to be detected while remaining within the linear range of the curve. The DA, DOPAC
and DOPA content of each sample was normalized to the amount of striatal protein

dissected to yield a concentration of striatal DA, DOPAC, and DOPA per striatal protein

(ng/mg protein).

57

 

F lg
1161‘
Rs;
011
R61
011
(‘N

A. Standard B. Striatum C. Striatum
(1 ng) -NSD-1015 +NSD-1015

DA
DO PAC ‘ DA . DA
/

/

 

 

DOPAC DOPA

 

 

 

Peak Height

 

 

 

Retention Time

Figure 2-4. A. HPLC-EC chromatogram depicting the retention times (x-axis) and peak
heights (y-axis) for a standard mixture containing 1 ng of DOPAC and DA (B)
Representative HPLC-EC chromatogram depicting the peak heights and retention times
of DOPAC and DA from a striatal sample from un-treated (-NSD-1015). (C)
Representative HPLC-EC chromatogram depicting the retention times and peak heights
of DOPA and DA from striatal sample taken from a mouse injected with NSD-1015
(+NSD-1015) 30 min prior to sacrifice.

58

Dopamine Content

Peak Height (x 1000)

 

 

 

10000 P
1000 -
100
0.13 0.25 0.5 1 2 4 8
DA (ng)

Figure 2-5. Logarithmic scale plot demonstrating the linear range for determining DA
content using HPLC-EC. DA content was determined by comparing peak heights in
samples with increasing known DA concentrations (ng). Peak height was determined at
an oxidation potential of +0.4 V, and gain of 500 with a constant flow rate of 1.0 ml/min.

59

E. Western Blot Analysis
Tissue Preparation

Striata to be used for Western blotting were dissected from the contralateral side
of section 1 and section 2 (Figure 2-3) using a 12-gauge punch tool approximately 1.5
mm i.d., and were placed into ice-cold homogenization buffer (320 mM sucrose, 5.0 mM
HEPES, with Complete Mini Protease Inhibitor Cocktail Tablets, Roche Diagnostics,
Mannheim, Germany, pH 7 .4).
Protein Extraction/Isolation/Determination

Striata were homogenized by sonication and centrifuged (1000 x g 10 min).
Nuclear and plasma membrane containing pellets (P1) were saved for further analysis.
Supematants (81) were removed, placed into a fresh microcentrifuge tubes, and
centrifuged (22,000 x g 30 min). Supematants from this second high-speed
centrifugation (82) were removed, placed into a flesh microcentrifuge tubes, and assayed
for protein content using the bicinchoninic acid protein method (BCA, Sigma). 12-gauge
striatal punches yield approximately 100 11g of protein. Following determination of
protein concentrations, 10 111 of loading buff'er (250 mM tris base pH 6.8, 20% glycerol,
5% SDS, 0.01% bromophenol blue) were added to samples. Samples were vortexed,
boiled at 95°C for 10 min, and cooled to 4°C.
Electrophoresis/Transfer Conditions

Two precast polyacrylamide gels (7.5-12%; Bio-Rad, Hercules, CA, USA) were
snapped into the inner chamber of a mini-protean-3 electrophoresis cell (BioRad). The
inner chamber was filled with 125 m1 of Laemli running buffer (25 mM Tris base, 192

mM glycine, 0.1% SDS). Isolated striatal protein samples were loaded into 9 of 10 lanes

60

within the gel. The volume of each sample loaded was adjusted to ensure loading of
equal concentrations of protein. A standard containing proteins of known molecular
weights was loaded into the tenth lane. The inner chamber was then placed into the outer
chamber containing 350 m1 of Laemli running buffer. Striatal protein was separated by
applying a 200 mV current for approximately 45 min. Electrophoresis was stopped when
the lightest band of the protein standard reached the bottom of the gel. Proteins were
then transferred to 0.45-11m nitrocellulose membranes (Fisher Scientific, Pittsburgh, PA,
USA) by electrophoresis at 30 mV for 12 h.
Immunoblot Detection of Striatal Protein

Following protein transfer, nitrocellulose membranes were washed in 25 mM
TBS (4 x 5 min) and incubated in blocking buffer (Li Cor, Inc., Lincoln, NE, USA) for
one h. Membranes were reacted with primary antiserum overnight. Primary antiserum
consisted of blocking buffer containing the primary antibody to the protein of interest as
well as a loading control primary antibody, i.e. a protein not affected by experimental
conditions. The dilution and source of each primary antibody used in the studies

described in this dissertation is listed in Table 2-1.

61

 

 

 

 

 

 

 

 

 

 

 

 

 

Antigen Species Dilution Source
a-Synuclein Mouse 123000 80 Transduction Laboratories
San Jose, CA(610786)
B-lll Tubulin Mouse 1:5000 Chemioon
(MAB1637)
Dopamine transporter (DAT) Rabbit 1:2000 Chemicon
(MAB369)
Early Endosomal Antigen-1 Rabbit 123000 Affinity Bioreagents
Golden, CO (PA1 -063)

SNAP-25 Mouse 1 24000 Chemicon
(MA8331)

Synaptophysin Mouse 1 :4000 Chem icon
(MAB368)

Tyrosine hydroxylase (TH) Rabbit 1:2000 Chemicon, Temecula, CA
(#A81 52)
Senna-40 phospho-TH Rabbit 1:1500 Cell Signaling Technology,
Beverly, MA (#2791)

Senna-31 phospho-TH Rabbit 1:1500 Chernicon
(A85423)

Scone-19 phospho-TH Rabbit 1:1500 Chernicon
(ABS425)

Vesicular Monoamine Rabbit 121000 Chernicon
Transporter-2 (V MAT-2) (AB1767)

 

 

Table 2-1. Description of each primary antibody (antigen) used in the studies described in
this dissertation. Antigen characterization is based on the animal used to generate the
antibody (species), the standard dilution used for Western blotting and the company the
antibody was purchased from (source).

62

 

ba

111

fon
this

in 11

Following incubation with primary antibodies, membranes were washed in TBS
(4 x 5 min). Subsequently, membranes were incubated with both IRDye 800-conjugated
goat anti-rabbit (Rockland, Gilbertsville, PA, USA) and Alexa Fluor 680-labeled goat
anti-mouse (Molecular Probes, Eugene, OR, USA) secondary antibodies diluted 1:20,000
in blocking buffer for 1 h. Membranes were then washed in TBS (4 x 5 min) and bound
antibodies were visualized using the Odyssey infrared imager. Band density was
quantified by measuring the infrared absorbance of each band using an Odyssey infrared
imager and Odyssey software (Li-Cor). In order to determine if band density increased
proportionally to increasing concentrations of striatal protein, TH and B-III tubulin were
measured with increasing concentrations of post-nuclear striatal protein (Figure 2-6). TH
band density is linear when 10-320 11g of striatal protein is loaded per lane. However,
when 160 or 320 11g of protein is loaded, band smearing and non-specific
immunoreactivity is observed. B-III tubulin detection and visualization is linear when
10-80 11g of striatal protein is loaded, with band smearing and non-specific band
formation becoming apparent when 160 or 320 11g of protein is loaded. Therefore, using
this Western blot technique changes in band density can be interpreted as linear changes

in the amount of protein in each sample.

63

A. Tyrosine Hydroxylase B-III Tubulin

 

SOkDA ’ "7"" .
~ ~

Ladder 30 60 120 240 Ladder 30 60 120 240

"1000 -
§

4
I

Absorbanoe(x1000) tn
3
Abeorbance (x
a '8'

_.
L-
r
l.

 

 

3O 60 120 240 30 60 120 240

Striatal Protein (119) Striatal Protein (119)

Figure 2-6. (A). Immunoreactivity of TH (~60 kDA, Left Panel) and B-III tubulin (~50
kDA, Right Panel) with either 30, 60, 120 or 240 11g of striatal protein separated on 10%
polyacrylamide gels. (B) Quantification of TH (left) and B-III tubulin (right) band
density using the Odyssey Imaging software and shown in absorbance units (x 1000).

64

Quantification of Protein Immunoreactivity

Relative density units (RDU) are obtained by normalizing the band density
(absorbance) of the protein of interest to the band density of the control protein. Each
nitrocellulose membrane contains samples that represent all experimental condition such
that, only samples run at the same time, transferred onto the same nitrocellulose
membrane, exposed to the same primary and secondary conditions, and visualized at the

same time can be compared.

F. In vitro Monoamine Oxidase Activity Assay

MAO catalyzes the enzymatic conversion of DA to DOPAL. Therefore, the
enzymatic activity of MAO can be determined by incubating MAO enzyme with known
concentrations of DA and measm'ing the rate of the DOPAL product formed using
HPLC-EC (Legros et al., 2004). DA was dissolved in phosphate buffer (140 mM NaCl,
8.1 mM Na2HPO4, 1.4 mM KH2PO4, pH 7.5) to a final concentration of 5 11M. Phosphate
buffered DA (500 111) was incubated in 12x75 culture tubes at 37 C for 15 min. The
reaction was started by adding 11.25U of MAO to the culture tube and gently mixing.
The reaction was stopped at either 15, 30, 45 or 60 min by adding 1 ml of tissue buffer to
the culture tube and placing the tube on ice. Samples were transferred to a 1.5 ml
centrifuge tube and centrifuged at 12,000 x g for 10 min. Supematants were removed
and placed into a fresh tube for HPLC-EC analysis of DA and DOPAL. Concentrations
of DA were compared to lng standards of DA. Since DOPAL is not available

commercially for the preparation of standards, concentrations of DOPAL were

65

determined by normalizing the DOPAL peak height to the amount of DA lost at each

time point (Figure 2-7).

66

DOPAL Synthesis - .. m

 

 

 

A 7 ' +DOPAL
'5. 6
U)
N 5 ---I
E 4
—' 3
E 2
O 9 ........
o 1 .,
o L L I
60

 

Time (min)

Figure 2-7. The rate of DOPAL formation (solid line) and DA depletion (dashed line)
after incubating DA (511M) with MAO (11.25 U). DA and DOPAL were measured using
HPLC-EC. Concentrations of DA were determined by comparing to 1 ng DA standards.
Concentrations of DOPAL were determined by comparing DOPAL peak heights to the
amount of DA decreased at each time point.

67

G. Subcellular Fractionation of TH
Tissue Preparation

Striatal protein used for subcellular fractionation was dissected from fresh tissue.
Following decapitation, the brain was rapidly removed and placed ventral side up in TBS
(4°C). A 1 mm coronal section containing the striatum was made with a razor blade.
The striatal section was then placed flat in the ice-cold TBS and a 12-gauge punch tool
was used to bilaterally dissect the striatum. The dissected striata from two mice were
placed into a 1.5 m1 microcentrifuge tube containing ice-cold homogenization buffer with
protease inhibitors and phosphatase inhibitor (1 mM sodium orthovanadate). Tissue was
then homogenized in the 1.5 ml tube with 20 strokes with a plastic pestle. Homogenized
striatal tissue was centrifuged at 1,000 x g for 10 min (4°C Beckman Coulter microfuge).
Isolation of Plasma Membrane Protein

The pellet from this initial centrifugation (P1) was resuspended in 500 111 of
homogenization buffer. A sucrose gradient was created by adding 830 111 of high sucrose
homogenization buffer (2.5 M sucrose, 5.0 mM HEPES, Protease Inhibitor Cocktail, pH
7.4) to P1. Samples were vortexed and the sucrose gradient was centrifuged at 20,000 x
g for 5 h (4°C Beckman Coulter microfuge). Nuclear protein will pellet in the high-
density sucrose buffer. Plasma membrane protein will pellet in the low density sucrose
but not in the high-density sucrose buffer such that membrane protein locates at the
interface between the two buffers. Low molecular weight proteins such as microsomes
remain in the upper portion of the low density sucrose buffer. Plasma membrane protein

(200 111) was extracted and placed into a fresh tube and frozen (-80°C).

Isolation of C ytosolic, Synaptic Vesicle and Endosomal Protein

As depicted in Figure 2-8, the supernatant from the initial low-speed
centrifugation (Sl) was removed and placed into a fresh tube and centrifuged at 20,000 x
g for 20 min at 4°C. The supernatant from this high-speed centrifugation (S2) was placed
into ultracentrifuge tubes and centrifuged at 100,000 x g for 60 min using a Sorvall
ULTRA PRO 80 Ultracentn'fuge with a swinging-bucket rotor (#TH-660). The
supernatant from this ultracentrifugation (S3) was removed and placed into a fresh tube.
The pellet from this ultracentrifugation (P3) was resuspended in 50 111 of homogenization
buffer and frozen on dry ice. The S3 supernatant was then centrifirged at 200,000 x g for
90 min. The supernatant from this centrifugation (S4) was removed and frozen on dry
ice. The pellet (P4) was resuspended in 50 111 of homogenization buffer and frozen on
dry ice. This method has previously been used to produce fractions enriched in plasma
membrane (Pl), synaptic vesicle (P3), endosomal (P4), and cytosolic (S4) protein

(Bennett 2003).

69

    

Homogenized
f Striatal Protein

 

  

 

 

 

  
 

Sucrose I
Grad'em : ; _ _. Synaptic Vesicle
' ‘ Enriched Fraction
Plasma Membrane
Enriched Fraction
. Cytoplasmic Enriched
""""" " Fraction
.. Early Endosome
Enriched Fraction

Figure 2-8. Subcellular fractionation of striatal tissue using differential centrifugation.
Homogenized striatal tissue was centrifuged at 1,500 g. The pellet (Pl) from this low-
speed centrifugation was separated using a sucrose gradient into plasma membrane and
nuclear enriched fractions. The supernatant ($1) from the initial low-speed centrifugation
was centrifuged at 20,000 g. The supernatant (82) from this high-speed centrifiigation
was centrifuged at 100,000 g. The pellet (P3) from this ultracentrifugation was isolated
and used as the synaptic vesicle enriched fraction. The supernatant (S3) from the
ultracentrifugation was centrifuged at 200,000 g to pellet light vesicles (P4). The
supernatant (S4) f'rom the second ultracentrifugation was isolated as the cytoplasmic
enriched fraction.

70

Protein Detection/Quantification

Protein concentrations in each fraction were determined using the BCA protein
method using 10 111 from each sample. Equal concentrations of protein from each
fraction were separated using Western blot technique as described above. TH subcellular
fractionation was determined by normalizing TH protein to the following fraction
specific proteins; SNAP-25 (plasma membrane), synaptophysin (synaptic vesicle), early
endosomal antigen 1 (early endosome), and monomeric B-III tubulin (cytosolic). The
efficiency of subcellular fiactionation was determined by measuring the
immunoreactivity of fiaction specific proteins in each subcellular compartment (Table 2-
2). B-III tubulin, EEAl, and synaptophysin were enriched in the S4, P4, and P3
fractions, respectively, suggesting differentential centrifugation successfully isolated
fractions enriched in cytosolic, early endosomal and synaptic vesicle protein. SNAP-25
however, was enriched in both P1 and P3, fractions indicative of incomplete separation of
the plasma membrane fraction. Data obtained from the plasma membrane sub-cellular

compartment should therefore be interpreted cautiously.

71

 

Tubulin EEA1 Synaptophysin SNAP-25

 

Cytosol 140 :t 2 5.0 a: 0.3 n.d. n.d.
Endosome 124 :1: 1 41 t1 n.d. 5.5 :1 0.5
Synaptic Vesicle 71 :1 2 1.5 :1: 0.1 137 :t 2 66 :1: 2
Membrane 60 1 2 3.0 :t 0.1 105 t 5 56 t 3

 

Table 2-2. Efficiency of sub-cellular fractionation using differential centrifugation.
Protein immunoreactivity is shown in absorbance units (x 1000) determined using
Odyssey imagining software. Results demonstrated B-III tubulin and EEA1, and
synaptophysin proteins were enriched in the cytosolic, endosomal, and synaptic vesicle
fractions, respectively, indicative of a good sub-cellular fractionation. SNAP-25 was
enriched in both S.V. and membrane fractions, indicative of incomplete separation of the

plasma membrane fraction. Values represent means of groups of 5-7 determinants i
SEM.

72

H. Statistical Analysis

Power analyses were conducted to determine optimal sample size required for
each experiment. For experiments that involve Western blot analysis, a minimum sample
size of 7 mice per group was determined to be necessary to obtain 80% power to detect a
25 % difference in means using a standard deviation of 10% that was determined from
preliminary studies. One-way analysis of variance (ANOVA) tests were used to detect
statistical significance between two or more groups on a single independent variable.
Two-way ANOVA’s were used to detect statistical significance between two or more
groups when there were two independent variables in the study. An alpha value of less
than or equal to 0.05 was considered statistically significant. If the two-way AN OVA
revealed an interaction of statistical significance a one-way AN OVA was used for

multiple comparisons among groups.

73

Chapter 3. The Effects of Prolonged-Chronic MPTP Administration on

NSDA Neurons

A. Introduction

It is unknown why NSDA neurons progressively degenerate in PD. It is
hypothesized that the loss of NSDA neurons triggers compensatory increases in the
activity of surviving neurons in order to maintain sufficient levels of DA release in the
striatum. The compensatory increase in activity likely contributes to the progressive
demise of NSDA neurons due to increased exposure to ROS derived from enzymatic or
non-enzymatic DA metabolism. To test this hypothesis, the studies described in this
chapter utilized the prolonged-chronic MPTP administration to induce NSDA cell death
similar to what is observed in PD. The activity of surviving neurons was assessed using
pharmacological, neurochemical and immunohistochemical techniques. If this
hypothesis is true then: A) MPTP will cause a compensatory increase in the activity of
surviving NSDA neurons; B) changes in neuronal activity should be long-term, such that
surviving neurons are chronically exposed to ROS; and C) compensatory changes in DA
neuron activity should be more pronounced in NSDA neurons, as compared to other

central DA neurons systems, since these neurons progressively degenerate in PD.

74

B. Experimental Design

To determine if MPTP-induced loss of NSDA neurons causes a compensatory
increase in the activity of residual neurons a dose-response experiment was designed to
identify the MPTP dose that causes axon terminal loss similar to PD. As depicted in
Table 3-1, mice were injected with either MPTP (1, 5, 10, or 20, mg/kg, s.c.) or saline
(1.0 ml/kg, s.c.) every 3.5 days for 5 consecutive weeks. Probenecid (250 mg/kg; i.p.)
was co-administered with each MPTP injection. Probenecid inhibits the transport of
organic acids across epithelial membranes, thus preventing the excretion of MPP' from
the central nervous system (Petroske et al., 2001). Mice were killed by decapitation three
weeks following the last injection and their brains were processed for neurochemical and

Western blot analysis.

75

 

MPTP Dose (mg/kg, s.c.)
0 1 5 10 20

 

Male Mice n=8 n=8 n=8 n=8 n=8
(age 10-12 weeks)

1

Table 3-1. Prolonged—chronic MPTP dose-response experimental design. Male mice (8
per group) were injected with either MPTP (1, 5, 10, or 20 mg/kg, s.c.) or saline (1.0
ml/kg, s.c.) every 3.5 days for 5 consecutive weeks for a total of 10 injections.
Probenecid (250 mg/kg, i.p.) was co-administered with each injection of saline or MPTP.
Mice were killed by decapitation three weeks following the last injection.

76

 

 

 

 

As depicted in Table 3-2, the primary endpoint used to determine NSDA axon
terminal loss was striatal DA concentrations. Striatal DA concentrations reflect vesicular
stored DA at the axon terminal, which represents greater than 99% of striatal DA as
compared to extracellular DA measured by microdialysis and can be eliminated by the
vesicular depletor, reserpine (Carlsson, 1975; Carboni et al., 1992; Di Chiara et al.,
1996). Vesicular DA concentrations are used as an index of NSDA axon terminal
integrity that reflects the ability of the axon terminal to synthesize and store DA.
Additionally, striatal VMAT-2 protein was measured using Western blot analysis.
VMAT-2 is a vesicular protein unique to catecholamine neurons in the brain. VMAT-2
protein levels are a reliable indicator of vesicular concentrations and provide an
additional measure of axon terminal density (Wilson etal., 1996; Kilbourn et al., 2000;
Reveron et al., 2002). The activity of surviving neurons was assessed by measuring
striatal DOPAC concentrations and the ratio of DOPAC to DA. DOPAC is the primary
metabolite of DA, and concentrations of DOPAC are an index of DA metabolism. The
ratio of DOPAC to DA reflects the ratio of DA metabolism to stored DA and is an index
of DA neuron activity that reflects the rate of DA release, reuptake and metabolism in

NSDA neurons (Westerink and Spaan, 1982; DeMaria et al., 1999).

77

End-point Indices of NSDA Axon Terminal Neurotoxicity

 

 

Measurement Reflects Index of
DA Vesicular DA Axon terminal integrity
DOPAC DA metabolism Axon terminal function
DOPAC! DA ratio DA release, reuptake and NSDA activity
metabolism
VMAT-2 Vesicle content DA storage capacity
TH TH protein DA synthesis capacity
DAT DAT protein DA reu ptake capacity
TH I DA ratio TH expression Amount of protein per
axon terminals
DAT I DA ratio DATexpression Amount of protein per

axon terminals

 

Table 3-2. The primary experimental end-points used for indices of NSDA neuron
function. Striatal DA and DOPAC concentrations and the DOPAC to DA ratio were
determined by HPLC-EC. Striatal VMAT-2, TH and DAT protein levels were

determined by Western blot analysis.

78

If the compensatory increase in NSDA activity (and consequently accelerated
DA metabolism) contributes to NSDA cell death in PD, then prolonged-chronic MPTP
should cause a sustained increase in the activity of surviving neurons. If the increase in
neuronal activity is sustained, then it is likely that chronic exposure to the toxic by-
products of DA metabolism contribute to progressive neuron death in PD. To determine
if this is the case, time-course experiments were performed to measure the activity of
surviving NSDA axon terminals either 0.5, 1, 3, or 8 weeks following the last MPTP
injection. As depicted in Figure 3-1, male mice (age 10-12 weeks) were injected with
either MPTP (20 mg/kg, s.c.) or saline (1.0 ml/kg, s.c.), and probenecid (250 mg/kg, i.p.)
twice a week for 5 consecutive weeks. Mice were killed by decapitation at 0.5, 1, 3, or 8
weeks following the last MPTP injection and their brains were processed for
neurochemical and Western blot analysis. Saline-treated mice were used as 0 time
controls and were sacrificed 3 weeks after the 10th saline injection. The time of the first
MPTP injection was staggered so that all mice were killed on the same day. NSDA
activity in surviving neurons was determined by measuring the DOPAC to DA ratio.

In order to determine what neurochemical changes occur in NSDA neurons that
permits prolonged accelerations in activity, the present study measured the expression of
TH and DAT protein in surviving neurons using Western blot analysis. TH and DAT
regulate DA synthesis and reuptake at the axon terminal, respectively, the two primary
sources of cytoplasmic DA that can be metabolized to produce ROS. Thus, changes in
the expression of TH or DAT may reflect compensatory changes in DA synthesis or
reuptake, respectively that underlies the compensatory increase in activity. TH and DAT

protein content were determined in the striatum by normalizing protein expression,

79

determined through Western blot analysis, to striatal DA concentrations to account for
the loss of axon terminals following MPTP. TH and DAT protein content are an index of

the relative amount of these proteins expressed in the axon terminals of NSDA neurons.

80

— Saline Injections

— MPTP Injections
.. ..... Post-injection time Sac Animals
Time (weeks) 1
0 1 2 3 4 5 6 7 8 9 10 11 12 13
1 l 1 l l l I I l l l l 1 l

 

: 0.5 weeks
1 1 weeks

 

 

: 3 weeks

 

: 3 weeks

 

: 8 weeks

Figure 3-1. Experimental design for MPTP time-course experiments. Mice were injected
with either MPTP (red, 20 mg/kg, s.c.) or saline (blue, 1.0 ml/kg, s.c.) and killed by
decapitation at 0.5, 1, 3, or 8 weeks (dashed line, n=8 per group) following the last
injection. Probenecid (250 mg/kg, i.p.) was co-administered with each injection of saline
or MPTP. The day of the first MPTP injection was staggered so that all animals were
killed on the same day.

81

It is unknown why NSDA neurons are more vulnerable than other populations of
DA neurons in PD. One strategy used to answer this question is to compare NSDA
neurons to other populations of DA neurons that are less vulnerable in PD. Thus, the
compensatory increase in activity may be an attribute that is unique to NSDA neurons
that renders them vulnerable in PD. In this case, axon terminal loss induced by MPTP
should only cause a compensatory increase in the activity of these neurons as compared
to other DA neuron populations. To test this, the effect of prolonged-chronic MPTP
administration on DA neuron activity was compared between NSDA, mesolimbic
(MLDA) and tuberoinfundibular (TIDA) neurons.

MLDA neurons reside in the ventral tegmental nucleus in the midbrain and
project axons through the medial forebrain bundle to terminate primarily in the limbic
structures including the nucleus accumbens, arnygdala, olfactory tubercles, and septum.
Similar to NSDA neurons, MLDA neurons synthesize and release DA at the axon
terminal and are regulated by D2 autoreceptors. The synaptic action of DA is terminated
via reuptake at the DAT (Lookingland, 2005). There is evidence that MLDA neurons
also degenerate in PD, however to a lesser extent than NSDA neurons (German et al.,
1989). Thus, it is hypothesized that MPTP will cause a less severe loss of MLDA axon
terminals and a less robust increase in neuronal activity compared to NSDA neurons.

TIDA neurons are distinct from NSDA and MLDA neurons in several respects.
TIDA neurons reside in the arcuate nucleus (A12) and project ventrally a short distance
to the median eminence. Median eminence DA is released into the hypophysial portal
vasculature and is transported to the anterior lobe of the pituitary where it binds to DA

receptors on lactotrophs and tonically inhibits prolactin secretion (Ben-Jonathan, 1985).

82

TIDA neurons do not express D2 autoreceptors and as such, DA synthesis and release are
not controlled at the axon terminal by autoreceptors (Gudelsky and Moore, 1976;
Demarest and Moore, 1979b; Lookingland and Moore, 1984). Since DA released from
TIDA neurons travels through the hypophysial portal system to the anterior pituitary, the
relevance of DAT-mediated reuptake in these neurons is questionable since DA is
transporter away (Demarest and Moore, 1979a). Cell bodies in the arcuate nucleus stain
very weakly for DAT protein, while axon projections to the external layer of the median
eminence have relatively strong immunoreactivity (Revay et al., 1996). Tritiated-DA
uptake occurs in the median eminence, however it has a much lower affinity for the DAT
in these neurons. Taken together, these results suggest that these neurons express the
DAT but it is less functional than in other DA neurons (Cuello and Iversen, 1973;
Demarest and Moore, 1979b; Annunziato et al., 1980). Thus, in the axon terminals of
TIDA neurons, the metabolism of newly synthesized, un-released DA is the primary
source of DOPAC (Lindley etal., 1990). Unlike NSDA and MLDA neurons, TIDA
neurons are spared in PD (Langston and Fomo, 1978; Braak and Braak, 2000).
Therefore, it is hypothesized that MPTP will not cause TIDA axon terminal loss and

subsequently, no change in the activity of these neurons.

83

B. Results
The Eflects of Prolonged-C hronic MPTP on NSDA Neurons

Prolonged-chronic MPTP treatment produced a dose-dependent decrease in
striatal DA concentrations in mice (Figure 3-2). Administration of 10 mg/kg and 20
mg/kg of MPTP decreased striatal DA concentrations by approximately 50% and 90%,
respectively, when compared to saline-treated control mice. There was also a dose-
dependent decrease in DOPAC concentrations in the striatum of mice treated with MPTP
(Figure 3-3, A). Administration of 10 and 20 mg/kg of MPTP decreased striatal DOPAC
concentrations by 30% and 80%, respectively. In both the 10 and 20 mg/kg MPTP
groups the loss of DOPAC was less severe than the loss of DA. As such, there was an
increase in the DOPAC to DA ratio in these mice (Figure 3-3, B). The increased DOPAC
to DA ratio was significantly correlated (0.72) with the MPTP-induced decrease in
striatal DA (Figure 3-4). Prolonged-chronic MPTP also produced a dose-dependent

decrease in striatal VMAT-2 immunoreactivity (Figure 3-5)

84

Striatal DA Concentrations

 

 

0 1 5 10 20
MPTP Dose (mg/kg)

Figure 3-2. Dose response effect of prolonged-chronic MPTP administration on DA
concentrations in the striatum. Mice were treated with either MPTP (1, 5, 10 or 20
mg/kg, s.c.) or its saline vehicle (1 ml/kg, s.c.) every 3.5 days for 5 consecutive weeks
and killed by decapitation 3 weeks following the last injection. Probenecid (250 mg/kg,
i.p.) was co-administered with each injection of saline or MPTP. Columns represent
means of groups, vertical lines represent 1 SEM. of six to eight determinants. (*)
indicate a significant difference from saline treatment group. (p S 0.05).

85

P

30 _ StriataIDOPAC Concentrations

DOPAC (rig/mg)

 

 

0 1 5 10 20
MPTP Dose (mg/kg)

8' 050 _ NSDA Neuronal Activity
’6 *
E 0.40
a 0.30
2 0.20
l
8 0.10

0.00

 

 

0 1 5 10 20
MPTP Dose (mgfltg) '

Figure 3-3. Dose response effect of prolonged-chronic MPTP administration on striatal
DOPAC concentrations (A) and the DOPAC/DA ratio (B). Mice were injected with
either MPTP (1, 5, 10, or 20 mg/kg, s.c.) or its saline vehicle (1 ml/kg, s.c.) and killed by
decapitation 3 weeks following the last injection. Probenecid (250 mg/kg, i.p.) was co-
administered with each injection of saline or MPTP. Columns represent means of
groups, vertical lines represent 1 SEM. of six to eight determinants. (*) indicate a
significant difference from saline treatment group. (p S 0.05).

86

Striatal DA is Correlated with NSDA Activity

r = 0.72 °
2.5 -

DOPAC/DA (Fold Increase)

 

 

Striatal DA (% Depletion)

Figure 3-4. Correlation between the DOPAC/DA ratio (shown as fold increases over
saline-treated controls) and striatal DA concentrations (shown as a percent depletion of
control mice) in response to prolonged-chronic MPTP (1, 5, 10, or 20 mg/kg, s.c.). There
was a significant correlation between the DOPAC/DA ratio and DA concentrations. r =
0.72. p5 0.001.

87

-~ ~-~.__

O D. I I i
| | l J l l
Saline 10 20

 

 

 

Saline 10 20
MPTP Dose (mg/kg) MPTP Dose (mg/kg)

Figure 3-5. Dose response effect of prolonged-chronic MPTP administration on striatum
VMAT-2 immunoreactivity. Mice were injected with either MPTP (10 or 20 mg/kg, s.c.)
or its saline vehicle (1.0 ml/kg, s.c.) every 3.5 days for 5 consecutive weeks and killed by
decapitation 3 weeks following the last injection. Probenecid (250 mg/kg, i.p.) was co-
administered with each injection of saline or MPTP. (A) Two representative irnmunoblot
samples of striatal VMAT-2 (80 kDa) and b-llI-Tubulin (50 kDa) protein following
prolonged, chronic MPTP or saline treatment. (B) Quantification of striatal VMAT-2
protein. Relative density units (RDU) were obtained by normalizing the B-III tubulin
signal from the VMAT-2 protein signal. Columns represent means of groups, vertical
lines represent 1 SEM. of six to eight determinants. (*) indicate a significant difference
from saline treatment group. (p S 0.05).

Prolonged-Chronic MPT P T ime-Cozyrgg

Compared to saline-treated mice, DA concentrations were significantly reduced in
all mice treated with prolonged-chronic MPTP (20 mg/kg, s.c.), however, the degree of
DA loss was different depending on whether mice were killed 0.5, 1, 3 or 8 weeks after
the last MPTP injection (Figure 3-6). In the group of mice killed 0.5 weeks after the last
MPT P injection, DA concentrations were decreased to approximately 90% of saline-
treated controls. This degree of loss was similar in mice killed at 1 or 3 weeks after the
last MPTP injection (90% and 80%, respectively). However, DA concentrations were
significantly increased in mice killed 8 weeks after the last MPTP injection compared to
mice killed 3 weeks after the last MPTP treatment.

The MPTP-induced loss of striatal DOPAC concentrations was similar to the loss
of DA, but less severe. In mice killed 0.5 weeks after the last MPTP injection, striatal
DOPAC concentrations were reduced to approximately 40% of saline-treated mice.
These levels were similar to mice killed land 3 weeks after the last MPTP treatment
(Figure 3-7, A). However, there was a significant increase in DOPAC concentrations
between the 3 and 8 week MPTP treatment groups. Due to the less severe loss of
DOPAC compared to DA, the DOPAC to DA ratio was increased in all mice that were

treated with MPTP (Figure 3-7, B).

180 Striatal DA Concentrations

*#

l l l l l L l j j l l j I I

01 2 3 4 5 8 910111213
1111111111

MPTP

DA(ng/mg)
o s a a s 's'

 

Time (weeks)

Figure 3-6. Time-course of striatal DA concentrations in mice treated with prolonged-
chronic MPTP (20 mg/kg, s.c.) or its saline vehicle (1.0 ml/kg, s.c.). DA concentrations
were measured 0.5, 1, 3 or 8 weeks following the last MPTP injection. Saline-treated
mice were used as zero time controls and killed 3 weeks following the last injection.
Data points represent means of groups, vertical lines represent 1 SEM. of six to eight
determinants. Arrows represent MPTP injections. (*) indicate a significant difference
from saline treatment group. # indicate a significant difference from mice killed 0.5, 1 or
3 weeks following MPTP treatment. pS 0.05.

90

A- Striatal DOPAC Concentrations

 

 

10-
’6:
5. 8' *#
5’ 6‘
E 4- * * *
8 ,_
o nnnnnnnnnnnnnnnnnnnnnnnnnnnn
012345 678910111213
Ti Ti 11 11 11
MPTP Time (weeks)
B. ' .
0'20 _ NSDA Neuronal Actrvrty
’6‘ * *
‘5 0.15 - *
I *
Q 0.10 -
2
§ 0.05 2
0w ............................

 

 

12345678910111213

1111111111

MPTP Time (weeks)

Figure 3-7. Time-course of striatal DOPAC concentrations (A) and the DOPAC/DA
ratio (B) in mice treated with prolonged-chronic MPTP (20 mg/kg, s.c.) or its vehicle (1.0
ml/kg, s.c.). DOPAC concentrations and DOPAC/DA ratios were measured 0.5, l, 3 or 8
weeks following the last MPTP injection. Saline-treated mice were used as zero time
controls and killed 3 weeks following the last injection. Data points represent means of
groups, vertical lines represent 1 SEM. of six to eight determinants. Arrows indicate
MPTP injections. (*) indicates a significant difference from saline treatment group. #
indicate a significant difference from mice killed 3 weeks following MPTP. pS 0.05.

91

B. TH and DA T Expression Following MPT P

Striatal TH protein expression was significantly decreased in all mice that were
treated with prolonged-chronic MPTP (Figure 3-8). TH was decreased 50% in mice
killed 0.5 weeks afler the last MPTP injection (Figure 3-8, B). The degree of TH loss
was consistent in mice killed at 1, 3 or 8 weeks following MPTP treatment. When
compared to the loss of striatal DA concentrations, there was an increase in the ratio of
TH to DA in mice killed 3 weeks after the last MPTP injection (Figure 3-8, C). Striatal
DAT protein was also significantly decreased in mice that were treated with prolonged-
chronic MPTP. However, the degree of DAT loss was different depending on whether
mice were killed 0.5, 1, 3 or 8 weeks after drug treatment (Figure 3-9, B). In mice killed
0.5 weeks after MPTP, DAT was decreased by approximately 50%. DAT protein levels
were consistent between the 0.5 and 1 week groups. However, in mice killed 3 weeks
afier MPTP, DAT was significantly decreased as compared to mice killed at 0.5 and 1
week after MPTP. DAT expression was increased in mice killed 8 weeks after MPTP to
levels comparable with the 3 week groups. In contrast, to TH, the ratio of DAT protein to
striatal DA concentrations did not change in mice killed 3 weeks after the last MPTP

injection (Figure 3-9, C).

92

Striatal TH Protein

 

 

 

ladder 012345075910111213

it it it it 11
MPTP Time (weeks)

.0

Protein Content
0.25 l" *

0.20
0.15
0.10
0.5
one

TH/DA (ratio)

 

 

Saline MPTP

Figure 3-8. (A) Representative immune-blot demonstrating the effects of prolonged-
chronic MPTP (20 mg/kg, s.c.) or its saline vehicle (1.0 ml/kg, s.c.) on striatal TH (~55
kDa), and B-III tubulin (~50 kDa) protein expression. Striatal TH and B-III tubulin
protein are shown for two mice per group. (B) Quantification of TH immunoreactive
bands following prolonged-chronic MPTP administration. TH was measured in mice
sacrificed 0.5, l, 3 or 8 weeks following the last MPTP injection. Saline-treated mice
were used as zero time controls and killed 3 weeks after the last injection. Relative
density units (RDU) for TH were obtained by normalizing band density of the TH signal
to the band density of B-III tubulin. (C) TH protein content in the striatum of mice
treated with either saline or MPTP and killed 3 weeks after the last MPTP injection. TH
protein content was determined by normalizing TH RDU to striatal DA concentrations
for each mouse. Values represent means of groups (n=6-8) i: 1 S.E.M. of six to eight
determinants. (*) indicate a significant difference from saline treatment group. p_<_ 0.05.

93

Striatal DAT Protein

A. __ 3.3.

75kDa : d“ ' ' ' 3 6

50kDa — 00:4. * * *
fl , *#

25koa"\‘.___—__—’

 

 

o 1 j A j 4 A441 I

ladder 0 5 6 8 13 012345878910111213

it H H H it
Week Number MPTP Time (weeks)

Protein Content

000

O

DAT/DA (ratio)
..°. .°. ..
8388288

 

O

Saline MPTP

Figure 3-9. (A) Representative immuno-blot demonstrating the effects of prolonged-
chronic MPTP (20 mg/kg, s.c.) or its saline vehicle (1.0 ml/kg, s.c.) on striatal DAT (~80
kDa), and SNAP-25 (~25 kDa) protein expression. Striatal DAT and SNAP-25protein
are shown for two mice per group. (B) Quantification of DAT immunoreactive bands
following prolonged-chronic MPTP administration. DAT was measured in mice
sacrificed 0.5, 1, 3 or 8 weeks following the last MPTP injection. Saline-treated mice
were used as zero time controls and killed 3 weeks after the last injection. Relative
density units (RDU) for DAT were obtained by normalizing band density of the DAT
signal to the band density of SNAP-25. (C) DAT protein content in the striatum of mice
treated with either saline or MPTP and killed 3 weeks afler the last MPTP injection.
DAT protein content was determined by normalizing DAT RDU to striatal DA
concentrations for each mouse. Values represent means of groups A: l S.E.M. of six to
eight determinants. (*) indicate a significant difference from saline treatment group. #
indicate a significant difference from mice killed at 6 weeks, 1 week after the last MPTP
injection. p: 0.05.

94

C. The Eflect of Prolonged-Chronic MPT P on MLDA and T IDA Neurons

As depicted in Table 3-3, prolonged-chronic MPTP administration produced a
less severe loss of DA concentrations in the nucleus accumbens than in the striatum. In
mice killed 0.5 weeks after MPTP administration, nucleus accumbens DA concentrations
were reduced to approximately 40% of saline-treated controls. This degree of nucleus
accumbens DA loss was similar in all mice treated with MPTP. In contrast, prolonged-
chronic MPTP administration had no effect on DA concentrations in the median
eminence of mice killed at any time point (Table 3-3).

As depicted in Table 3-4, compared to saline-treated mice, nucleus accumbens
DOPAC concentrations were significantly decreased in all mice treated with MPTP. In
mice killed 0.5 weeks after MPTP, DOPAC was decreased by approximately 50%. The
degree of DOPAC loss was similar in mice killed 1 and 3 weeks following MPTP.
However, DOPAC concentrations were significantly increased in mice killed 8 weeks
after MPTP compared to the 0.5, l, or 3 week groups. There was no change in DOPAC
concentrations in the median eminence in mice treated with MPTP and sacrificed at any
time point (Table 3-4). As shown in Figure 3-10, the DOPAC to DA ratio was
significantly increased in the nucleus accumbens of mice killed 0.5, 3, and 8 weeks

following MPTP, however, there was no effect in the median eminence.

95

Time after MPTP

 

 

(weeks)
0 0.5 1 3 8
Nucleus Accumbens 99 1 5 37 1 3* 44 t 6* 45 1 4* 45 :1; 5*

MedianEminence 10016 9817 109:9 10318 113111

 

Table 3-3. Nucleus accumbens and median eminence DA concentrations in mice treated
with prolonged-chronic MPTP (20 mg/kg, s.c.) or its saline vehicle (1.0 ml/kg, s.c.). DA
concentrations were measured 0.5, 1, 3 or 8 weeks following the last injection. Saline-
treated mice were used as zero time controls and were killed 3 weeks after the last
injection. Values represent means of groups 3: l S.E.M.. (*) indicate a significant
difference from saline treatment group. p S 0.05.

96

Time after MPTP
(weeks)

 

0 0.5 1 3 8

 

* * *
Nucleus Accumbens 13.4 1 0.6 6.7 1 0.3 6.4 1 0.7 7.1 1 0.6 8.3 1 0.7*#

Median Eminence 3.2 :i: 0.4 2.2 :t 0.4 3.1 :l: 0.4 3.1 t 0.2 3.1 1 0.3

 

Table 3-4. Nucleus accumbens and median eminence DOPAC concentrations in mice
treated with prolonged-chronic MPTP (20 mg/kg, s.c.) or its saline vehicle (1.0 ml/kg,
s.c.). DOPAC concentrations were measured 0.5, l, 3 or 8 weeks following the last
MPTP injection. Saline-treated mice were used as zero time controls and were killed 3
weeks after the last injection. Values represent means of groups 1 1 SEM. (*) indicate a
significant difference from saline treatment group. # indicate a significant difference from
mice killed 3 weeks afier the last MPTP injection. p S 0.05.

97

MLDA Activity TIDA Activity

 

 

 

 

 

0.05p
A 0.25. A
O O
'3 z: 0.04.
g 0.20- * * g
v * V
a 0.15 M <91: (“WV —%
2 t °
l 0.10 E 0.02,
o o
‘3 °~°5t 0 0.01»
o.m J I l 1 1 L L J L L4 14 l o.m LLLLL I 4 l l ‘ L n ‘
012345678910111213 012345678910111213
Time (weeks) Time (weeks)

Figure 3—10. The DOPAC to DA ratio in the nucleus accumbens and median eminence in
mice treated with prolonged-chronic MPTP (20 mg/kg, s.c.) or its saline vehicle (1.0
ml/kg, s.c.). DOPAC to DA ratios were measured 0.5, 1, 3 or 8 weeks following the last
MPTP injection. Saline-treated mice were used as zero time controls and were killed 3
weeks after the last injection. Values represent means of groups 1 1 S.E.M. (*) indicate
a significant difference from saline treatment group. p S 0.05.

C. Discussion

The present studies have demonstrated that prolonged-chronic MPTP I
administration causes a loss of NSDA axon terminals that is similar to what is observed
in PD. The loss of NSDA axon terminals was associated with an increase in DA
metabolism in surviving neurons. MPTP induced long-term changes in the activity of
surviving neurons and this effect was not restricted to NSDA neurons. The results from
these studies are consistent with the finding that in PD, the loss of NSDA neurons
triggers a compensatory increase in the activity of NSDA neurons (Ribeiro et al., 2002;
Sossi et al., 2002). Due to the toxic nature of DA metabolism, the increased activity may

contribute to the progressive degeneration of NSDA neurons in PD.

The Eflects of Prolonged-Chronic MPT P on NSDA Axon Terminal Loss

Following systemic administration of MPTP, the lipophillic molecule crosses the
blood brain barrier and is absorbed primarily by glial cells, where it is converted to its
active metabolite MPP+ and released. MPP+ is primarily taken into the axon terminals of
DA neurons, due to its affinity for the DAT, where it accumulates in mitochondria and
binds to and inhibits complex I of the electron transport chain. Axon terminal death was
originally believed to result from ATP depletion and overproduction of ROS (N icklas et
al., 1985). Recently it was determined that MPP+ is a weak inhibitor of mitochondrial
complex I (Ramsay et al., 1991) and furthermore, greater than 72% reductions in
complex I activity are required before mitochondrial function and ATP levels are
compromised (Davey and Clark, 1996). These results suggest that MPTP toxicity is not

the result of compromised mitochondrial function. The primary mechanism of MPTP

toxicity is believed to be due to ROS and DA-quinones generated from DA oxidation.
Following MPP+ uptake into the axon terminal there is a robust displacement of vesicular
DA into the cytoplasm and this is associated with an increase in ROS. If DA stores are
depleted prior to MPTP administration, there is no increase in ROS and cellular toxicity
is completely blocked (Lotharius and O'Malley, 2000). Once the ROS generated from
DA oxidation exceeds the capability of intracellular antioxidant proteins, oxidative
damage to the axon terminals occurs. If the oxidative damage is severe enough it may
destroy the axon terminals.

In the present study, the effects of repeated MPTP injections was assessed by
measuring NSDA axon terminal loss. NSDA axon terminal loss in the striatum was
determined by measuring concentrations of striatal DA three weeks following the last
injection of MPTP. DA concentrations reflect DA storage at the axon terminal and are
used as an index of axon terminal integrity. MPTP caused a dose-dependent decrease in
NS DA concentrations, which is indicative of a dose-dependent destruction of NSDA
axon terminals. Alternatively, since MPTP displaces vesicular DA, the decreased DA
concentrations could reflect a deficit in DA storage as opposed to loss of terminals.
However, this is unlikely because DA concentrations were measured 3 weeks after the
last MPTP injection, long afier the neurotoxin has been excreted from the body and direct
effects on vesicular storage have subsided (Przedborski et al., 2001). Repeated MPTP
injections (20 mg/kg) caused NSDA axon terminal loss similar to what is observed in PD
(Homykiewicz, 1975), suggesting that prolonged, exposure to ROS derived fiom DA

oxidation likely plays a key role in PD pathogenesis. This hypothesis is strengthened by

100

severe oxidative damage found in the striatum of post-mortem PD brains (Dexter et al.,
1989; Schapira et al., 1990; Dexter et al., 1994).

Prolonged administration of MPTP caused severe damage to NSDA axon
terminals that persisted for several weeks after the last insult. In mice treated with 20
mg/kg, of MPTP there was a significant loss of NSDA axon terminals up to 8 weeks
following the last injection. Thus, prolonged complex I inhibition in mice causes a
robust, chronic loss of axon terminals, similar to what is observed in PD. Axon terminal
recovery, as reflected by an increase in striatal DA concentrations, was observed between
mice killed 3 and 8 weeks after the last MPTP injection, suggesting that in those 5 weeks,
new axon terminals sprouted from existing NSDA neurons and re-innervated the
striatum. It is unlikely that the recovery was the result of neurogenesis, since under basal
conditions and following toxic insult there are no cells in the substantia nigra that co-
label with TH and bromodeoxyuridine a marker for proliferating cells (F rielingsdorf et
al., 2004).

Interestingly, sprouting of new axon terminals and recovery does not occur in PD,
or in non-human primates treated with MPTP. Thus, recovery is a phenomenon that is
specific to the mouse MPTP model of PD. Identifying the mechanisms in the mouse that
facilitate recovery has become an interesting area of PD research that may lead to new
therapeutic targets for human PD. However, in mice killed at either 0.5, l or 3 weeks
following MPTP administration, NSDA axon terminals loss is severe and long lasting
and there is no evidence of axonal sprouting. Thus, prolonged-chronic MPTP
administration recapitulates the key pathological features of idiopathic PD at these time

points.

101

The Effect of Prolonged-Chronic MPTP on the Activity of Surviving NSDA Neurons

The purpose of these studies was to determine if a lesion of the NSDA pathway
similar to what occurs in PD causes a compensatory increase in the activity of un-
lesioned neurons. The activity of surviving neurons was assessed by measuring the ratio
of DOPAC to DA in the striatum of mice treated with MPTP. The ratio of DOPAC to
DA reflects the rate of DA release, reuptake and metabolism, and is used as an index of
DA neuron activity (Westerink and Spaan, 1982; DeMaria et al., 1999). Chronic
exposure to ROS derived from DA oxidation produced a robust lesion of the NSDA
pathway and a dose-dependent increase in the activity of surviving neurons. The degree
of the lesion was significantly correlated with activity of residual, un-lesioned neurons.
These results suggest that as NSDA axon terminals are destroyed by MPTP, surviving
neurons compensate by increasing DA release in order to maintain sufficient synaptic
concentrations of DA capable of activating post-synaptic D1 and D2 receptors. This is
particularly relevant in the context of PD because during the early stages of the disease
there is a compensatory increase in the rate of DA release in surviving NSDA neurons
(Ribeiro et al., 2002; Sossi et al., 2002).

PD symptom onset likely reflects a balance between NSDA loss and the ability of
remaining neurons to compensate. When neuronal compensation is not sufficient to
activate post-synaptic D1 and D2 receptors and regulate the direct and indirect pathways
the onset of motor symptoms occurs. Unfortunately, the compensatory increase in DA
release, reuptake and metabolism likely contributes to the gradual, progressive loss of
NSDA neurons as a result of the toxic nature of DA oxidation. Increased DA oxidation is

associated with an increased exposure to the ROS; OH', 02', ONOO' and DA-quinone

102

molecules. If the increase in neuronal activity is prolonged, exposure to ROS becomes
chronic and macromolecular damage and lipid peroxidation may become severe enough
to cause axon terminal death. However, if the increase in activity is only transient,
neuronal antioxidant defense mechanisms are likely capable of scavenging the ROS and
there would be very little adverse consequences on neuron survival.

To determine if axon terminal loss induced by prolonged-chronic MPTP causes a
long-term change in the activity of surviving NSDA neurons, a time-course experiment
was designed to measure NSDA activity from 3 days to 8 weeks following the last MPTP
injection. The results from this experiment demonstrated that NSDA activity was
accelerated for at least 8 weeks following MPTP administration. Therefore, as a result of
the compensatory activation, surviving NSDA neurons are chronically exposed to ROS
derived fi'om DA oxidation. The chronic exposure to R08 is likely detrimental to
neuronal survival and may ultimately be responsible for the progressive loss of NSDA
neurons in PD.

It is unknown what neurochemical changes occur in NSDA neurons that permits
them to sustain a prolonged increase in activity. Likely, increased DA synthesis is
necessary to supply the increased demand for release, and increased reuptake is likely
occurs as a result of more DA being released. To determine what neurochemical changes
underlie the compensatory increase in NSDA activity, TH and DAT protein expression
were measured in the striatum of mice treated with prolonged-chronic MPTP using
Western blot analysis. TH and DAT are responsible for regulating the rate of DA

synthesis and reuptake, respectively.

103

The MPTP-induced loss of NSDA axon terminals was associated with decreased
striatal TH protein expression. In mice treated with prolonged-chronic MPTP and killed
0.5, 1, 3 or 8 weeks following MPTP, TH was decreased by approximately 50%.
However, the loss of TH was attenuated when compared to the loss of axon terminals as
indicated by DA concentrations. As such, TH protein content, an index of the amount of
TH per axon terminal, was increased following MPTP. Consistent with this, there is an
increase in TH mRNA in NSDA neurons following MPTP administration (Jakowec et al.,
2004). Thus, one mechanism surviving neurons use to increase neuronal activity is
inducing expression of the rate limiting enzyme responsible for synthesizing the
neurotransmitter. Presumably, this allows the rate of DA synthesis to be accelerated to
supply the increased demand for release.

Interestingly, the loss of striatal DAT protein was not as uniform as TH. Initially,
in mice killed 0.5 weeks following MPTP the loss of DAT was approximately 50%,
similar to TH. However, in mice killed 3 weeks following the last injection of MPTP,
DAT protein levels were decreased by approximately 85%. This is consistent with
previous reports describing a more robust loss of DAT compared to TH protein, and no
change in DAT mRN A following MPTP (Jakowec et al., 2004). The loss of DAT at this
time point was consistent with the loss of striatal DA concentrations. As such, DAT
protein content, an index of the amount of transporter at the axon terminal, did not
change in surviving neurons. These results suggest that unlike DA synthesis, there is no
change in reuptake in NSDA neurons that survive chronic MPTP administration.
However, it is also possible that the expression of TH and DAT protein do not reflect the

actual rate of DA synthesis and reuptake, respectively. Further experimentation is

104

required to determine if changes in protein expression correspond with functional

changes in the rate of DA synthesis and reuptake.

The Eflects of MPT P on MLDA and T IDA Neurons

PD is characterized by a more pronounced loss of NSDA neurons compared to
other populations of DA neurons in the brain. The vulnerability of central DA neurons to
ROS derived from DA oxidation was assessed by comparing axon terminal loss in the
terminal fields of NSDA, MLDA and TI DA neurons following MPTP administration.
Chronic exposure to MPTP caused a differential effect on DA neuronal populations, with
NSDA neurons being the most vulnerable (~80% loss), MLDA neurons being moderately
(~60% loss) affected, and TIDA neurons being completely spared. Interestingly, the
differential susceptibility to neurotoxic insult was similar to the degree of DA neuronal
loss observed in the populations of neurons in PD (Homykiewicz, 1975; Langston and
Fomo, 1978; German et al., 1989). Therefore, these results suggest that inherent
differences in the susceptibility to ROS-mediated oxidative damage are responsible for
the vulnerability of NSDA neurons in PD.

Differential DAT expression, between NSDA, MLDA, and TIDA neurons could
also account for the differential susceptibility to MPTP as a consequence of altered
neurotoxin uptake. DAT expression is higher in NSDA neurons compared to MLDA or
TIDA neurons (Shimada et al., 1992; Lorang et al., 1994; Freed et al., 1995). However,
this is not the case, since these neuronal populations are also differentially sensitive to the

neurotoxic effects of rotenone, a complex I inhibitor that is not dependent on DAT uptake

105

(Betarbet et al., 2000). Therefore, it is more likely that NSDA neurons are more
vulnerable to oxidative stress-induced cell death than MLDA or TIDA neurons.

A number of mechanisms may account for the vulnerability of NSDA neurons to
oxidative stress induced cell death. NSDA neurons may have higher concentrations of
stored DA capable of being oxidized to ROS following displacement by MPP+. MLDA
or TIDA neurons may express higher levels of antioxidant defense proteins (such as
glutathione), or proteins that prevent apoptosis (such as BCL-2). Alternatively, MLDA
or TIDA neurons may react differently than NSDA neurons following exposure to ROS
and oxidative damage. For example, the compensatory increase in NSDA activity may
render these neurons more vulnerable

To determine if compensatory increases in activity is a phenomenon specific to
NSDA neurons, the activity of MLDA and TIDA neurons was assessed following MPTP
administration. Results demonstrated that the loss of approximately 60% of MLDA axon
terminals was associated with increased neuronal activity in surviving, un-lesioned
neurons. No change in TIDA activity was observed, supporting the hypothesis that
neuronal activity increases as a compensatory mechanism in response to axon terminal
loss, rather than a direct effect of MPTP. These results suggest that similar to NSDA
neurons, MLDA neurons also respond to axon terminal loss by increasing DA release,
reuptake and metabolism. Therefore, compensatory increases in neuronal activity may
contribute to the progressive loss of NSDA neurons in PD, but they are not unique to
NSDA neurons and thus, are unlikely to be responsible for the enhanced vulnerability of

these neurons to ROS-mediated cell death. Future studies will have to determine if

106

differences in DA storage or expression of protective proteins determines the
vulnerability of central DA neurons to MPTP-induced cell death.

Interestingly, as the time from the last MPTP injection increased, MLDA activity
also gradually increased. Whereas NSDA activity peaked in mice killed 0.5 weeks after
MPTP and continued to decline. Although compensatory changes in neuronal activity
occurred in both NSDA and MLDA neurons, the time-course was different between the
two populations of neurons. This may be due to differential regulation of proteins
responsible for synthesizing DA at the axon terminal. Studies have demonstrated several
key differences in TH gene regulation between NSDA and MLDA neurons (Pasinetti et
al., 1990; Leviel et al., 1991; Sturtz et al., 1994) that may highlight the importance of
increasing expression of TH protein to permit the compensatory increase in activity. It is
unknown what functional consequence this has on MLDA neurons but it may affect axon
terminal recovery from neurotoxic insult.

In NSDA neurons there was a recovery of axon terminals between 3 and 8 weeks
after the last MPTP injection and this likely reflected axon terminal sprouting from
existing neurons (Bezard et al., 2000). Recovery was associated with a decrease in the
activity of surviving neurons. However; it is difficult to interpret cause and effect from
these results. One possibility is that, as new axon terminals sprouted from surviving
and/or lesioned neurons, the burden on individual axon terminals was lessened and
activity gradually declined. Alternatively, the decrease in neuronal activity may have
facilitated neuronal recovery by decreasing exposure to ROS and alleviating oxidative
stress in surviving neurons. The present study cannot distinguish between these two

alternatives, yet it was interesting that in MLDA neurons no axon terminal recovery was

107

observed, and neuronal activity gradually increased. It is possible that MPTP did not
cause a sufficient lesion in MLDA neurons to induce recovery mechanisms or there may
be inherent differences in NSDA versus MLDA axon terminal sprouting. The latter
seems more likely since a 60% decrease in NSDA axon terminals induces recovery in
NSDA neurons (Petroske et al., 2001). Taken together, the results from the present
experiments highlight differences between NSDA and MLDA neurons in axonal
sprouting and neuronal activity in response to a neurotoxic lesion. While the present
studies are not sufficient to explain the precise mechanisms that underlie differences
between NSDA and MLDA neurons, they propose a link between neuronal recovery and

activity that highlight a need for additional research.

Summary

The results from the studies described in this chapter have demonstrated that
chronic exposure to MPTP (and presumably ROS derived from DA oxidation), causes
NSDA and MLDA axon terminal loss similar to what is observed in PD. Moreover, in
response to the loss of axon terminals, surviving NSDA and MLDA neurons compensate
by increasing DA release, reuptake and metabolism. As a result of ROS generated by
DA oxidation, it is likely the sustained acceleration in NSDA activity causes oxidative
damage and contributes to the progressive loss of NSDA neurons. Thus, preventing the
compensatory activation of NSDA neurons or inhibiting DA oxidation may protect these
neurons in PD. However, the neurochemical mechanisms that underlie the sustained
activation of NSDA neurons are unclear. Western blot analysis of protein expression

suggested changes in the DA biosynthetic pathway occur in surviving neurons. However,

108

further experimentation is required in order to understand the neurochemical changes that
occur in NSDA neurons that permit a prolonged acceleration in DA release, reuptake and

metabolism.

109

Chapter 4. Identification of the Neurochemical Changes that Underlie

the Compensatory Activation of NSDA Axon terminals

A. Introduction

The results from Chapter 3 demonstrated that NSDA axon terminal loss causes a
sustained increase in the activity of surviving axon terminals that maintains sufficient
concentrations of released DA in the striatum. The accelerated activity was associated
with increased DA metabolism in surviving axon terminals, reflected by an increase in
the ratio of DOPAC to DA. DOPAC is an index of the enzymatic metabolism of
cytoplasmic DA, thus, these results are indicative of an increase in cytoplasmic DA
concentrations in surviving axon terminals. Alternatively, the increased in DA
metabolism may reflect an increase in MAO activity, however this is unlikely since MAO
activity is decreased following MPTP administration (J ohannessen et al., 1991). Excess
cytoplasmic DA can increase ROS formation in axon terminals due to enzymatic or non-
enzymatic DA oxidation, suggesting that there is increased ROS exposure in surviving
axon terminals. If the ROS are not scavenged by intracellular antioxidant proteins,
oxidative damage to the axon terminal will occur. If oxidative damage is severe, it may
cause axon terminal death. Identifying the mechanisms that are responsible for the
compensatory changes that occur in NSDA axon terminals that culminate in neurotoxic
levels of cytoplasmic DA levels should provide novel therapeutic targets to protect
NSDA axon terminals in PD. To this end, the purpose of the experiments described in
this chapter is to identify the source of the cytoplasmic DA that is metabolized in

surviving NSDA axon terminals following MPTP.

110

As depicted in Figure 4-1, newly synthesized and recaptured DA are the two
primary sources of cytoplasmic DA, and thus DA metabolism in axon terminals. An
increase in the rate of either DA synthesis or reuptake could account for the increased
metabolism of DA in NSDA axon terminals that survive MPTP treatment. Results from
Chapter 3 showed there was an increase in TH protein expression in surviving axon
terminals, suggesting an increase in newly synthesized DA may be largely responsible for
the excess cytoplasmic DA and accelerated metabolism in NSDA axon terminals that
survive MPTP administration. Therefore, it was hypothesized that DA synthesis is the
primary source of cytoplasmic DA and increased metabolism. To test this hypothesis,
pharmacological, neurochemical and immunohistochemical techniques were used to
distinguish between the metabolism of newly synthesized versus recaptured DA in
NSDA axon terminals under basal conditions and following activation induced by MPTP

administration.

111

 

 

 

 

 

Tyrosine—>DOPA——>DA ——\—’—->MA DA
TH AADC

 

Figure 4-1. Schematic diagram of a NSDA axon terminal depicting the two primary
sources of cytoplasmic DA (synthesis and reuptake). Both newly synthesized and
recaptured DA can be metabolized by mitochondrial (mito) MAO to DOPAL. DOPAL is
then converted to DOPAC by aldehyde dehydrogenase (AD).

112

B. Experimental Design and Results
Determining the Contribution of DA T-Mediated Reuptake to DA Metabolism under Basal
and Activated Conditions

Blocking DAT-mediated reuptake of synaptic DA and measuring the change in
striatal DOPAC concentrations provides an index of the proportion of recaptured DA that
is metabolized (Figure 4-2 A,B). The contribution of recaptured DA to metabolism was
compared under basal and activated conditions. To determine the amount of recaptured
DA that is metabolized under basal conditions, mice were injected with either saline (1.0
ml/kg, i.p.) or the DAT blocker GER-12909 (GBR, 10 mg/kg, i.p.) and killed 30 min
later (Figure 4-2, C). This GBR administration regimen maximally inhibits DA reuptake
in NSDA axon terminals (Camarero et al., 2002). Striatal DOPAC concentrations in
GER-treated mice were compared to saline-treated mice and the difference between these
groups was used as an index of the proportion of recaptured DA that is metabolized.

The amount of recaptured DA metabolized under basal conditions was compared
to activated conditions. Activated conditions were induced by injecting mice with MPTP
in a sub-chronic regimen which consisted of MPTP injections (25 mg/kg, s.c.) once a day
for 5 consecutive days. Mice were killed 3 days after the last injection. The effects of
MPTP on NSDA neuron axon terminal loss were determined by measuring striatal DA
concentrations. The effect of sub-chronic MPTP on the activity of surviving axon
terminals was determined by measuring the ratio of DOPAC to DA, and TH and DAT
protein expression. The sub-chronic MPTP model was chosen because it produces a
similar loss of NSDA axon terminals as the prolonged-chronic administration regimen

(but in a much shorter time frame), allowing several neurochemical experiments to be

113

conducted simultaneously. Control and MPTP-treated mice were then injected with
either saline or GER-12909 (10 mg/kg, i.p.) 30 min prior to sacrifice (Figure 4-2, C).
Striatal DOPAC concentrations in GER-treated mice were compared to saline-treated
controls and the difference between these groups was used as a measure of the amount of

recaptured DA that is metabolized under activated conditions.

114

A- C o Iasmic DA

DA Reuptake D—AT—>

DOPAC

DA Synthesis m

 

 

 

 

8' GBR C o Iasmic DA
DA Reuptake *0
DOPAC
DA Synthesis m
C.
Basal Conditions Compensatory Activation
Saline n=7 Saline n=7
(1.0 mllkg, i.p.) (1.0 ml/kg. i.p.)
GBR n=7 GBR n=7
(10 mg/kg, i.p.) (10 mg/kg, i.p.)

 

 

Figure 4-2. Strategy for determining the amount of recaptured DA metabolized under
basal and activated conditions. (A,B) Blockade of DAT-mediated reuptake under basal
and activated conditions allows changes in striatal DOPAC concentrations to be
measured as an index of the amount of recaptured DA that is metabolized. (C) Mice
were injected with either saline (1.0 ml/kg, i.p.) or GBR-12909 (GBR, 10 mg/kg, i.p.) and
killed 30 min later. Activated conditions were induced by injecting mice with MPTP (25
mg/kg, s.c.) once a day for 5 consecutive days. Mice were killed by decapitation 3 days
after the last injection.

115

The Efilects of Sub-Chronic MPT P on NSDA Axon terminals

Sub-chronic MPTP administration produced a dose-dependent decrease in striatal
DA concentrations (Figure 4-3 A), resulting in 80% depletion in mice treated with the
highest dose (25 mg/kg). Concentrations of DOPAC changed in parallel with that of DA,
with a 65% decrease observed in mice treated with 25 mg/kg MPTP (Figure 4-3 B). The
DOPAC to DA ratio was two-fold higher in mice treated with the highest MPTP dose
compared to saline-treated controls (Figure 4-3 C).

Sub-chronic MPTP (25 mg/kg) also caused a 50% decrease in striatal TH protein
expression (Figures 4-4 A,B, Left Panel) and a 75% decrease in striatal DAT protein
expression (Figure 4-4 A,B, Right Panel). There was a more robust loss of ST DA
concentrations compared to striatal TH expression. As such, there was an increase in TH
protein content in mice treated with MPTP (Figure 4-4 C, Lefi Panel). Alternatively, the
loss of DA concentrations was comparable to the loss of striatal DAT expression. As
such, there was no change in DAT protein content (Figure 4-4 C) in the striattun of mice

treated with MPTP.

116

Striatal DA

.>

DA (rig/mg)
ii

50 *
0
0 1 5 25
MPTP Dose (mg/kg)
B. Striatal DOPAC
20
”a"
a 15
5 10
$2 *
(L 5
O
D 0
O 1 5 25
MPTP Dose (mg/kg)
0 NSDA Activity
§ 0.15
g 0.12
g 0.09
6 0.06
E 003
O .
O 0.00
O 1 5 25

MPTP Dose (mg/kg)

Figure 4—3. Effects of sub-chronic MPTP administration on striatal DA (A) and DOPAC
(B) concentrations and DOPAC/DA ratios (C). Mice were treated with saline (1.0 ml/kg,
s.c.) or MPTP (1,5, or 25 mg/kg, s.c.) once a day for five consecutive days and killed by
decapitation three days following the last injection. Columns represent means of groups,
vertical lines represent 1 S.E.M. of six to eight determinants. (*) indicate a significant
difference from saline treatment group. (P5 0.05).

117

Striatal TH Protein

75 kDa ....

~~ m...—
501(08 —- ————

25 kDa - .
ladder

Saline MPTP

TH Protein

RDU
8 8

Seine MPTP

TH Protein Content
*

.0
5i

TH/DA (ratio)
2

.0
at

Seine MPTP

DAT/DA (ratio)

Striatal DAT Protein

7.... ...,Hi

50kDa~

25 kDa \ .
ladder
Saline

u ‘ All. .—
MPTP

DAT Protein

*
MPTP

Saline

DAT Protein Content

 

0.40

0.20

   

0.00
MPTP

Seine

Figure 4-4. (A, LEFT PANEL) Representative immuno-blot demonstrating the effects of
sub-chronic MPTP (25 mg/kg, s.c.) or saline (1.0 ml/kg, s.c.) on striatal TH (~55 kDa)
and B-III tubulin (~50 kDa) or (RIGHT PANEL) DAT (~80 kDa) and SNAP-25 (~25
kDa) protein expression. (B) Quantification of TH (LEFT PANEL) and DAT (RIGHT
PANEL) protein expression. Relative density units (RDU) were obtained by normalizing
the band density of TH to B-III tubulin band density and DAT to SNAP-25 band density.
(C) TH (LEFT PANEL) and DAT (RIGHT PANEL) protein content in the striatum
determined by normalized TH or DAT RDU to striatal DA concentrations. Columns
represent means of groups, vertical lines represent 1 S.E.M. of six to eight determinants.
(*) indicate a significant difference from saline treatment group. (P5 0.05).

The Contribution of DA Reuptake to Striatal DA Metabolism

Under basal conditions, (vehicle pre-treated mice), GBR administration decreased
striatal DOPAC concentrations by approximately 30% compared to saline-treated mice
(Figure 4-5, Left Panel). Under activated conditions (MPTP pre-treated mice), GBR

administration had a similar effect on striatal DOPAC concentrations as it did under basal

conditions (Figure 4-5, Right Panel).

119

Basal Conditions Activated Conditions

 

 

   

‘5 ’ (Vehicle Pro-treated) (MPTP Pro-treated)
A ’5 5
E“ E
a. B» 4
5 5
o 0 3
E E 2 .
0 O
0 D
1
O
Saline GBR Saline GBR

Figure 4-5. The effect of saline or GBR-12909 on striatal DOPAC concentrations under
basal conditions (vehicle pre-treated, Left Panel) and activated conditions (MPTP pre-
treated, Right Panel. Mice were injected with saline (1.0 ml/kg, s.c.) or MPTP (25
mg/kg, s.c.) once a day fro 5 consecutive days and killed 3 days after the last injection.
Mice were injected with saline (1.0 ml/kg, i.p.) or GBR-12909 (GBR, 10 mg/kg, i.p.) 30
min prior to sacrifice. Striatal DOPAC was determined by HPLC-EC. Columns
represent means of groups, vertical lines represent 1 S.E.M. of seven determinants. (*)
indicate a significant difference from saline treatment group. (P5 0.05).

120

Determining the Contribution of DA Synthesis to Metabolism of DA

The amount of newly synthesized DA that is metabolized under basal and
activated conditions was determined by blocking DA synthesis and measuring the change
in striatal DOPAC concentrations. Unlike DA reuptake, synthesis is a two-step reaction
that occurs within the axon terminal, the location of MAO. DA synthesis can be blocked
by inhibiting TH catalytic activity with a-methylparatyrosine (AMT). AMT competes
with the normal substrate tyrosine for binding to TH and decreases the conversion of
tyrosine to DOPA. Alternatively, DA synthesis can be blocked by inhibiting the catalytic
activity of AADC with m-hydroxybenzylhydrazine (NSD-1015) (Carlsson et al., 1972).
In order to use DOPAC concentrations as an index of the rate of DA metabolism, AMT
or NSD-1015 must not directly alter the enzymatic activity of MAO.

To determine if AMT or NSD—1015 alters MAO catalytic activity, purified MAO
enzyme (11.5 U) was incubated with its substrate DA (5 uM) along with either; vehicle,
AMT (10 (1M, or 25 uM) or NSD-1015 (5 uM, 10 (1M). Doses ofAMT and NSD—lOlS
were chosen based on the expected brain concentrations of the drug following systemic
injection that maximally inhibit TH and AADC activity, respectively (Widerlov and
Lewander, 1978; Bongiovanni et al., 2003). MAO enzymatic activity was quantified by
measuring the elimination of substrate DA, and the formation of product DOPAL using
HPLC-EC. Results demonstrated that AMT had no effect on MAO activity, whereas,
NSD-lOl 5 caused a dose-dependent decrease in MAO enzymatic activity (Figure 4-6).
As such, AMT was used to inhibit TH activity and DA synthesis in subsequent

experiments.

121

Eflects of AMT or NSD-1015 on MAO Activity

As depicted in Figure 4-6 (Left Panel), basal DA concentrations were determined
by incubating DA with vehicle for 60 min. Incubation of DA with MAO enzyme
decreased DA concentrations. The addition of AMT (10 or 25 uM) to this reaction had
no effect on DA concentrations when compared to MAO and DA alone. However,
incubating MAO and DA with NSD-l 015 (5 or 10 uM) caused a dose-dependent increase
in DA concentrations when compared to MAO and DA alone.

Consistent with DA concentrations, DOPAL concentrations were not detectable
when DA was incubated in the absence of MAO. Incubation of DA with MAO enzyme
dramatically increased DOPAL concentrations. The addition of AMT (10 or 25 M) to
this reaction had no effect on DOPAL concentrations. However, incubating MAO and
DA with NSD-1015 (5 or 10 uM) caused a dose-dependent decrease in DOPAL

concentrations compared to MAO and DA alone.

122

. , Substrate Concentrations
. . T *e
*0
s "
g a
5
< r
o 2 * * *
t t

 

 

 

 

0
DA (5 W) + +

 

+ + + «1-
MAO(11.5 0) - + + + +
AMT (11M) - - 10 25 . .
NSD—1015(uM) . . . .

DOPAL(09125 in)

)-

 

0
DA(5PM)+ + + + + +
+ MAO(11.5U). + ...
AMT(uM).
5 10 use-1015mm -

Product Formation
*

*1:

  
   

 

n.d.

ll
'3
.a
(In
3|

Figure 4-6. The effects of AMT and NSD-1015 on MAO enzymatic activity. DA (5 11M)
was incubated with either vehicle (no MAO enzyme) or MAO (11.5 U) for 60 nrin. The
elimination of substrate DA (LEFT PANEL) and formation of product DOPAL (RIGHT
PANEL) was measured using HPLC-EC after 60 min. DA and MAO were then
incubated with either AMT (10 or 25 uM) or NSD-lOl 5 (5 or 10 (1M) and DA and
DOPAL concentrations were measured 60 min after the start of the reaction. Columns
represent means of groups, vertical lines represent 1 S.E.M. of 4 determinants. (*)
indicate a significant difference from DA -MAO group. # indicate a significant difference

from DA+MAO group. (p S 0.05).

123

Blocking DA synthesis with AMT and measuring the decrease in striatal DOPAC
concentrations should provide an index of the proportion of newly synthesized DA that is
metabolized in NSDA axon terminals (Figure 4-7, AB). To determine the contribution of
newly synthesized DA to metabolism under basal conditions, mice were injected with
either saline (1.0 ml/kg, i.p.) or AMT (250 mg/kg, i.p.) and killed 30 min later (Figure 4-
7, C). This AMT administration regimen maximally inhibits TH catalytic activity in
NSDA axon terminals (Widerlov and Lewander, 1978). Striatal DOPAC concentrations
in AMT-treated mice were compared to saline-treated mice and the difference between
these groups was used as an index of the amount of newly synthesized DA that is
metabolized under basal conditions.

To determine the proportion of newly synthesized DA that is metabolized under
activated conditions mice were pre-treated with MPTP (25 mg/kg, s.c.) in a sub-chronic
regimen to cause axon terminal loss and the compensatory activation of surviving axon
terminals. Control and MPTP-treated mice were injected with either saline or AMT (250
mg/kg, i.p.) thirty minutes prior to sacrifice (Figure 4-7, C). Striatal DOPAC
concentrations in AMT-treated mice were compared to vehicle-treated mice and the
difference between these groups was used as an index of the amount of newly
synthesized DA that is metabolized during the compensatory activation of NSDA axon

terminals.

124

A- ,. lasmic DA

DA Reuptake L»

DA Synthesis m

B' C o lasmic DA

DA Reuptake —D—AT——v

DA Synthesis m

 

 

 

AMT
C.
Basal Conditions Compensatory Activation
Saline n=7 Saline n=7
(1.0 mllkg, i.p.) (1.0 mllkg, i.p.)
AMT n=7 AMT n=7
(250 mg/kg, i.p.) (250 mg/kg, i. p.)

 

 

Figure 4-7. Strategy for determining the amount of newly synthesized DA metabolized
under basal and activated conditions. (A,B) TH inhibition under basal and activated
conditions allows changes in striatal DOPAC concentrations to be measured as an index
of the amount of newly synthesized DA that is metabolized. (C) Mice were injected with
either saline (1.0 mllkg, i.p.) or AMT (10 mg/kg, i.p.) and killed 30 min later. The
decrease in striatal DOPAC concentrations was used to reflect the amount of newly
synthesized DA that is metabolized under basal conditions and during the compensatory
activation of NSDA axon terminals.

' 125

The Contribution of Newly Synthesized DA to Striatal DA Metabolism

Under basal conditions, (vehicle pre-treated mice), AMT administration had no
effect on striatal DOPAC concentrations (Figure 4-8, Lefi Panel). However, under
activated conditions, (MPTP pre-treated mice), AMT decreased striatal DOPAC

concentrations by approximately 40% (Figure 4-8, Right Panel).

126

Basal Conditions Activated Conditions

 

 

 

 

15 - (Vehicle Pre-treated) 6 [ (MPTP Pre-treated)
’5 a 5
E E
B) B: 4
5 S
O O 3
E E
O O 2
D D
1
0
Saline AMT Saline AMT

Figure 4-8. The effect of saline or a-methylparatyrosine (AMT) on striatal DOPAC
concentrations under basal conditions (vehicle pre-treated, LEFT PANEL) and activated
conditions (MPTP pre-treated, RIGHT PANEL). Mice were injected with saline (1.0
ml/kg, s.c.) or MPTP (25 mg/kg, s.c.) once a day fro 5 consecutive days and killed 3 days
after the last injection. Mice were injected with saline (1.0 ml/kg, i.p.) or AMT (250
mg/kg, i.p.) 30 min prior to sacrifice. Striatal DOPAC was determined by HPLC-EC.
Columns represent means of groups, vertical lines represent 1 S.E.M. of seven
determinants. (*) indicate a significant difference from saline treatment group. (P: 0.05).

127

Determining the Rate of DA Synthesis in NSDA Axon terminals

It is hypothesized that accelerated DA synthesis increases cytoplasmic DA in
surviving NSDA axon terminals following MPTP. To determine if this is the case, the
rate of striatal DA synthesis was measured in surviving NSDA axon terminals following
MPTP. Mice were injected with either saline (1.0 ml/kg, s.c.) or MPTP (25 mg/kg, s.c.)
in a sub-chronic regimen to cause axon terminal loss and the compensatory activation of
surviving axon terminals. NSD-lOl 5 (100 mg/kg, i.p.) was injected 30 min prior to
sacrifice and striatal DOPA concentrations were measured with HPLC-EC. The
accumulation of DOPA following blockade of AADC with NSD-1015 is an in vivo assay
for TH catalytic activity (Carlsson et al., 1972). DOPA concentrations were normalized
to striatal DA concentrations to control for MPTP-induced axon terminal loss. The ratio
of TH catalytic activity relative to DA concentrations is an index of the rate of DA

synthesis following neurotoxic lesion (Zigmond et al., 1984).

128

The Rate of Striatal DA Synthesis under Basal and Activated Conditions

Sub-chronic MPTP administration caused a decrease in striatal DOPA (Figure 4-
9, A) and DA concentrations (Figure 4-9, B). However, the loss of striatal DA
concentrations was much more severe than the loss of DOPA concentrations. As such,
MPTP caused a robust increase in the ratio of DOPA to DA in the striatum of mice

treated with MPTP (Figure 4-9, C).

129

A. 15 TH Catalytic Activity

     

   

12
9 *
6
3
0
Saline MPTP
B 160 DA Storage
120
80
4o *
Saline MPTP
C" 04 . Rate of DA Synthesis
*
0.3 -
0.2 -
0.1 -
0.0

 

 

Saline MPTP

Figure 4-9. Concentrations of DOPA (A), DA (B) and the DOPA to DA ratio (C) in the
striatum of mice injected with either saline (1.0 ml/kg, s.c.) or MPTP (25 mg/kg, s.c.)
once a day for 5 consecutive days. DOPA concentrations were determined 30 min afier
NSD-1015 (100 mg/kg, i.p.) injection. Columns represent means of groups, vertical lines
represent 1 S.E.M. of seven determinants. (*) indicate a significant difference from
saline treatment group. (P5 0.05).

130

C. Discussion
The Effect of Sub-Chronic MPTP Administration on NSDA Axon terminals

Sub-chronic MPTP administration produced similar effects on NSDA axon
terminals as the prolonged-chronic administration regimen despite a decreased
cumulative dose and time-course. Sub-chronic MPTP caused an 80% loss of NSDA axon
terminals and a compensatory increase in the activity of surviving axon terminals.
Moreover, TH and DAT protein expression were decreased in the striatum by 50 and
85% respectively, which was consistent with changes in the expression of these proteins
in the striatum following prolonged-chronic MPTP. A less severe decrease in TH protein
expression compared to loss of axon terminals suggests there is an increase in TH
expression in surviving axon terminals following MPTP. The equal loss of DAT protein
and axon terminals induced by MPTP is indicative of no change in DAT protein
expression in surviving axon terminals. Thus, the toxicity to NSDA axon terminals,
induced by sub-chronic MPTP is comparable in extent to the prolonged-chronic
administration regimen and surviving axon terminals appear to compensate through
similar mechanisms.

The sub-chronic administration regimen offers several advantages over the
prolonged-chronic administration regimen, including a shortened time-course which
allows several neurochemical experiments to be conducted simultaneously. Also, there
was no animal mortality associated with sub-chronic MPTP administration and as such,
fewer animals can be used in experiments. The disadvantage of the sub-chronic regimen
is that, unlike prolonged-chronic administration, axon terminals recover between 3 and

30 days after-the last MPTP injection (Petroske et al., 2001). Thus, this model is not

131

ideal for testing neuroprotective/restorative treatments since interpretations of results may
be confounded by recovery at later time points. For experiments that are designed to test
the effects of axon terminal loss on the function of surviving axon terminals, sub-chronic
MPTP administration is ideal for causing a rapid, reliable and reproducible lesions of

NSDA neurons and a compensatory increase in the activity of surviving axon terminals.

Contribution of DA Reuptake to Metabolism of DA

The purpose of the present experiments was to determine the contribution of
recaptured DA to its enzymatic metabolism under basal conditions and during the
compensatory activation of NSDA axon terminals. Under basal conditions, blocking DA
reuptake decreased striatal DOPAC concentrations by approximately 30%, suggesting
that roughly one third of enzymatic DA metabolism is due to DA recaptured from the
synaptic clefi. The amount of recaptured DA that is metabolized to DOPAC reflects a
balance between vesicular storage capabilities versus the enzymatic activity of MAO.
Vesicular storage capability is determined by number of synaptic vesicles available for
storage and the activity of VMAT-2 which transports cytoplasmic DA into the vesicle
protecting it from metabolism. Thus under basal conditions, roughly 30% of the
recaptured DA escapes repackaging and is metabolized by MAO.

It was interesting that DA metabolism only decreased by 30%, since DA release
and reuptake is thought to be the primary source of metabolism in the axon terminal
(Lindley et al., 1990). Several potential explanations may account for this. Residual
DOPAC (due to metabolism prior to blockade of DA reuptake) may mask more robust

decreases in DA metabolism. However, this is not the case since the half-life of DOPAC

132

is 11.3 min (Cumming et al., 1992), suggesting most DOPAC at the axon terminal prior
to DAT blockade would have been eliminated in the 30 min time frame between GBR
administration and animal decapitation. Also, the dose of GBR may not have been
sufficient to completely block DA reuptake in NSDA axon terminals. However, this
seems unlikely, because this administration regimen causes a dramatic increase in
extracellular DA concentrations (as measured using microdialysis), indicative of effective
blockade of DA reuptake (Camarero et al., 2002). Alternatively, there may be an
additional source for cytoplasmic DA and DOPAC levels in NSDA axon terminals such
as leakage from synaptic vesicles. Although the present experiments cannot rule out
these alternative explanations for the degree to which DOPAC is decreased following
blockade of DA reuptake, results demonstrate the relative contribution of DA reuptake to
enzymatic metabolism by blocking DA transport for 30 min. The results can, therefore,
be interpreted to reflect the relative amount of recaptured DA that is metabolized in this
time frame and whether this relative amount changes across treatment conditions.

The amount of recaptured DA that is metabolized to DOPAC under basal
conditions was compared to that metabolized under activated conditions. Activated
conditions were induced by treating mice with MPTP to cause a compensatory activation
of NSDA axon terminals. Under these conditions, blocking DA reuptake had a similar
effect as under basal conditions. This data is consistent with the finding that there was no
change in DAT protein expression levels in surviving NSDA axon terminals after MPTP
administration. This was interesting because chronic elevation in neuronal activity
(induced by blocking D2 receptors) decreases DAT mRNA and protein expression

(Meiergerd et al., 1993; Kimmel et al., 2001). In the present study, the acceleration of

133

NSDA activity induced by MPTP was not associated with decreased DAT expression or
the amount of recaptured DA that is metabolized to DOPAC. These results suggest the
compensatory activation of NSDA neurons is not mediated by D2 receptors, although a
loss of D2 receptor regulation of DAT protein expression could occur in NSDA axon
terminals following MPTP. Alternatively, decreased DAT mRNA and protein expression
following blockade of D2 receptors may not reflect actual changes in the rate of DA
reuptake. Nevertherless, the present study utilized a functional assay to determine there
is no change in the amount of recaptured DA that is metabolized in NSDA axon
terminals. Therefore, altered DA reuptake does not contribute to the increased
metabolism of cytoplasmic DA that is a neurochemical hallmark of the compensatory

activation of NSDA axon terminals.

The Contribution of DA Synthesis to Metabolism of DA

In the present experiments, the relative amount of newly synthesized DA that is
metabolized by MAO under basal and activated conditions was determined. Normally,
DA synthesis is tightly coupled to release, such that as DA molecules are released, new
molecules are synthesized and packaged into vesicles where they are protected from
metabolism. The coupling of DA synthesis to release minimizes the metabolism of
newly synthesized DA and maintains stable concentrations of vesicular DA available for
release. Consistent with this, in the present experiments blockade of DA synthesis with
AMT had no effect on striatal DOPAC concentrations under basal conditions. Therefore,
unlike DA reuptake, synthesis does not contribute to metabolism in NSDA axon

terminals under basal conditions.

134

The amount of newly synthesized DA metabolized under basal conditions was
compared to activated conditions induced by pro-treating mice with MPTP in a sub-
chronic dosing regimen to cause compensatory activation of surviving axon terminals. In
contrast to basal conditions, blockade of DA synthesis in activated NSDA axon terminals
decreased striatal DOPAC concentrations by approximately 40%, suggesting newly
synthesized DA was a primary source for metabolism. Thus, the MPTP-induced
compensatory increase in NSDA activity (reflected by an increase in the ratio of DOPAC
to DA) is due in large part to increased metabolism of newly synthesized DA. These
results could suggest there is no compensatory increase in DA release from surviving
axon terminals. Rather the increased DOPAC to DA ratio is strictly due to increased
metabolism of newly synthesized DA. However, this is not the case, since in response to
MPTP administration there is an increase in the firing rate of NSDA neurons and synaptic
DA concentrations are maintained at near baseline levels despite a dramatic loss of axon
terminals (Albert et al., 1984; Zigmond et al., 1984; Stachowiak et al., 1987; Snyder et
al., 1990; McCallum et al., 2006). More likely, the present study suggests that, while
there is increased DA release from surviving NSDA axon, synthesis is accelerated to a
much greater extent. The newly synthesized DA exceeds vesicular packaging capability

and a great majority of the newly synthesized DA is metabolized rather than utilized.

The Rate of DA Synthesis in NSDA Axon terminals Following MPTP Treatment
The results from the previous experiments suggested that there is a dramatic
increase in newly synthesized DA in NSDA axon terminals that survive MPTP treatment.

The increased synthesis exceeded vesicular packaging capability and, as such, was

135

metabolized within axon terminals without ever being released. Therefore, accelerated
synthesis is likely responsible for the increased metabolism of cytoplasmic DA that is a
neurochemical hallmark of the compensatory activation of NSDA axon terminals. To
verify these results, the rate of striatal DA synthesis was determined in mice treated with
either saline or MPTP. Results from this experiment demonstrated that MPTP
administration caused a dramatic increase in the rate of striatal DA synthesis in surviving
axon terminals.

It is unclear why the rate of DA synthesis is accelerated to an extent that exceeds
vesicular packaging capability. Normally, DA synthesis and release are tightly coupled,
suggesting there may be a deficit in the regulatory mechanisms that govern this coupling.
At the axon terminal, the rate of DA synthesis and release are controlled by D2
autoreceptors. D2 autoreceptors are G-protein coupled receptors that inhibit adenylate
cyclase activity at the axon terminal (Missale et al., 1998). Adenylate cyclase regulates
the CAMP levels and thus the activity of CAMP-dependent protein kinase A (PKA). D2
autoreceptor—PKA signaling cascade regulates both short and long-term changes in the
rate of DA synthesis by regulating the phosphorylation state and de novo synthesis of TH,
the rate limiting enzyme, respectively (Haycock, 1990; Leviel et al., 1991; Missale et al.,
1998). Therefore the present results may indicate the rate of DA synthesis is accelerated
due to a loss of D2-autoreceptor function at the axon terminal of NSDA axon terminals
that survive MPTP. Consistent with this, there was an increase in TH protein expression
in surviving axon terminals following MPTP administration. Further experiments are

required to determine if the regulation of TH is dysfunctional and whether there is a loss

136

of D2 autoreceptor regulation of the rate of DA synthesis that is ultimately responsible
for the uncoupling between DA synthesis and release.

The unregulated acceleration of DA synthesis is not restricted to mouse NSDA
axon terminals lesioned with MPTP. Other neurotoxin-induced lesions of the NSDA
pathway in the rat cause an increase in the amount of TH protein per terminal and an
acceleration in the rate of DA synthesis (Zigmond et al., 1984). Thus, this phenomenon
is not species or neurotoxin specific, but more likely typical of the compensatory
activation of mammalian NSDA axon terminals. This is particularly relevant to the
pathogenesis of PD, where there appears to be a multitude of factors that cause primary
damage to the NSDA pathway, yet it is unknown what causes secondary, progressive,
loss of NSDA neurons. The uncoupling of DA synthesis and release at the axon terminal
(and the corresponding increase in cytoplasmic DA) may be a primary pathogenic factor

that contributes to the progressive loss of NSDA neurons in PD.

Summary

The results from the experiments described in this chapter have successfully
identified DA synthesis as the source of the increased cytoplasmic DA and accelerated
metabolism that is typical of the compensatory activation of NSDA axon terminals. The
rate of DA synthesis is accelerated to an extent that the newly synthesized DA exceeds
packaging capabilities and is subsequently metabolized rather than released. The
uncoupling of DA synthesis and release is likely due to dysfunctional regulation of the

rate limiting enzyme, TH. It is unclear what changes in TH regulation are responsible for

137

the excessive rate of DA synthesis, but identification of these underlying mechanisms

may lead to novel therapeutic targets to protect NSDA neurons in PD.

138

Chapter 5. Dysfunctional Regulation of Tyrosine Hydroxylase in NSDA

Axon Terminals that Survive MPTP Administration.

A. Introduction

The results from the experiments summarized in Chapter 4 demonstrated that
regulation of the DA biosynthetic pathway is dysfunctional in NSDA axon terminals that
survive MPTP administration. Dysfunction in the DA synthesis pathway causes an
uncoupling between synthesis and vesicular packaging capability, such that there is an
increase in the metabolism of newly synthesized DA. The rate of DA synthesis is
regulated by controlling the activity of TH, the rate limiting enzyme. TH catalytic
activity and expression are tightly regulated at the axon terminal. Acceleration of TH
catalytic activity mediates short-terrn changes in the rate of DA synthesis, while long-
term changes in the rate of DA synthesis are achieved by increasing expression of the
protein (Kumer and Vrana, 1996).

As depicted in Figure 5-1, the catalytic activity of TH is tightly regulated at the
axon terminal through activation and inactivation signaling mechanisms. TH activation
occurs through phosphorylation of critical serine residues (40, 31 and 19) located in the
N-terminus of the enzyme (Campbell et al., 1986; Haycock, 1990). TH can be
phosphorylated by cyclic AMP (cAMP)-dependent protein kinase A (Joh et al., 1978;
Yamauchi and Fujisawa, 1979; Vulliet et al., 1980), Ca+2—phospholipid-dependent
protein kinase (Albert et al., 1984; Vulliet, 1985) and Ca+2—calmodulin-dependent protein
kinase (Yamauchi and F ujisawa, 1981; Vulliet et al., 1984). As such, TH enzyme

activity is accelerated in response to increased neuronal activity (Ca+2 regulated) and

139

increased adenylate cyclase activity (CAMP regulated). Phosphorylation of TH decreases
the Michaelis constant (Km) for the co-factor tetrahydrobiopterin and increases the
inhibitory constant (Ki) for DA (Lovenberg and Bruckwick, 1975; Haavik et al., 1990;
Daubner et al., 1992). These kinetic changes result in increased enzymatic efficiency,
decreased susceptibility to end-product inhibition, but also lead to decreased enzyme
stability (Lazar et al., 1981; Vrana and Roskoski, 1983; Gahn and Roskoski, 1995).

TH inactivation occurs through at least two activity-dependent mechanisms.
Diminished NSDA firing (impulse-flow) results in reduced TH phosphorylation by
activity-dependent kinases, thus allowing the enzyme to be de-phosphorylated by protein
phosphatases 2A and 2C (Haavik et al., 1989). TH inactivation in this manner is
reversible, such that subsequent phosphorylation can re-activate enzyme catalytic activity
(V rana et al., 1981; Vrana and Roskoski, 1983; Roskoski et al., 1990). Second, in the
presence of a constant stimulus phosphorylated TH can undergo long-term inactivation
through destabilization of secondary and tertiary protein structure (Lazar et al. 1981;

Vrana and Roskoski 1983; Gahn and Roskoski 1995).

140

8H4 Q‘BHz
0 H20

Tyrosine fi—P DOPA AADC: DA

 

 

 

g CAMP-dependent PKA

a3 Cat2/phospholipid-dependent PK

2 Cati’lcalmodulin-dependent PK

‘6 .

< TH P

C

.9.

iii

.2

2‘5

‘1’

d) . -

O P Recycling]
TH TH Degradation

Figure 5-1. Schematic diagram depicting the DA biosynthetic pathway in the axon
terminals of NSDA neurons. The activity of the rate limiting enzyme TH is controlled
through activation and de-activation mechanisms. TH is activated through
phosphorylation at critical serine residues within the N-terrninus of the enzyme.
Phosphorylation of TH increases the catalytic activity of the enzyme. De-activation of
TH occurs through two mechanisms. TH can be de-phosphorylated by protein
phosphatases or by destabilization which results in either recycling or degradation of the
protein.

141

In contrast to the phosphorylation-dependent short-term acceleration in DA
synthesis, long-term (stable) acceleration in synthesis is dependent on increased
expression of the rate limiting enzyme, TH. Increased impulse-driven neuronal activity
stimulates expression of the immediate early genes c-Fos and c-Jun (Morgan and Curran,
1989). These transcription factors bind to the promoter region of TH and induce gene
transcription, protein translation, enzyme expression and accelerated DA synthesis.
Thus, the increased rate of striatal DA synthesis in surviving axon terminals following
MPTP administration may be due to increased TH catalytic activity (short-term), protein
expression (long-term), or both (Figure 5-2). Results from Chapter 3 demonstrated there
is an increase in the amount of TH protein in surviving axon terminals following MPTP.
To this end, it was hypothesized that an increase in TH expression, rather than increased
catalytic activity mediates the increased rate of DA synthesis. To test this hypothesis the
present studies investigated the regulation of TH catalytic activity and protein content in
NSDA neurons under basal and activated conditions induced by MPTP administration.

The present experiments were designed to identify changes in the regulation of
TH catalytic activity and expression in NSDA axon terminals following MPTP
administration. Neurochemical, Western blot and cell biological techniques were used to
test the hypothesis that dysfunctional regulation of TH is responsible for the increased
rate of DA synthesis that is a characteristic of the compensatory activation of NSDA
neurons. Mice were treated with sub-chronic MPTP to destroy NSDA axon terminals
and cause a compensatory activation of surviving neurons. The regulation of TH;
phosphorylation state, enzymatic activity, substrate limitations, and sub-cellular

localization were all assessed in the striatum of mice following MPTP treatment.

142

BH4 q-BH2
O2 H20

Tyrosine —-—-> DOPA AADC’ DA
TH

 

1 Rate of Strialtal DA Synthesis

1 l

lAmount of TH 1TH Catalytic Activity

Figure 5-2. Following the compensatory activation of NSDA neurons the DA
biosynthetic pathway was accelerated such that there is an increase in newly synthesized
DA (TOP PANEL). Increasing the rate of this reaction requires an increase in either the
amount, or catalytic activity of the rate limiting enzyme TH (BOTTOM PANEL).

143

B. Experimental Design and Results
Characterizing the Regulation of T H Catalytic Activity in NSDA Axon Terminals

The regulation of TH activity was assessed by measuring the phosphorylation
state of the enzyme at serine-19, 31 or 40, using Western blot analysis and antibodies
specific for TH phosphorylated at each specific serine residue. Phosphorylated TH
protein was assessed from striatal protein isolated from mice treated with either saline
(1.0 ml/kg, s.c.) or sub-chronic MPTP (25 mg/kg, s.c.). TH phosphorylated at serine-19,
31, or 40 was normalized to total striatal TH protein levels in each animal and was used
as an index of the amount of enzyme phosphorylated at each residue relative to the total
amount of enzyme in the axon terminal. Phosphorylated TH and total TH protein were
measured from one half of the striatum (Figure 5-3).

In one experiment, MPTP-induced changes in TH phosphorylation were
compared to the catalytic activity of the enzyme in the striatum. The catalytic activity of
TH was determined by injecting mice with the AADC inhibitor NSD-1015 (100 mg/kg,
i.p.) and measuring striatal DOPA concentrations using HPLC-EC from the contralateral
striatum not used for Western blotting. The acute 30 min NSD-1015 administration
regimen is unlikely to cause changes in TH protein expression during this short time
frame. Striatal DOPA concentrations were normalized to the amount of TH protein,
determined through Western blot analysis. The ratio of TH catalytic activity (DOPA) to
TH protein was used to reflect the enzymatic activity of TH in mice treated with either

saline (1.0 ml/kg, s.c.) or sub-chronic MPTP (25 mg/kg, s.c.).

144

 

Saline n=7
(1.0 mllkg, s.c.)

MPTP n=7
(25 mg/kg, s.c.)

 

 

 

DOPA Concentrations Phosphorylated TH
DA Concentrations Total TH

Figure 5-3. Experimental design (TOP PANEL) and isolation of NSDA axon terminals
for neurochemical analysis of striatal DOPA concentrations or Western blot analysis of
phosphorylated and total TH (BOTTOM PANEL). Mice were injected with either saline
(1.0 ml/kg, s.c.) or MPTP (25 mg/kg, s.c.) once a day for 5 consecutive days and killed
by decapitation 3 days following the last injection. The striatum was dissected from
frozen brain sections and processed for HPLC-EC determination of striatal DOPA and
DA concentrations or Western blot determination of phosphorylated and total striatal TH.

145

In a second experiment, TH catalytic activity was further evaluated by assessing
the relationship between the enzyme and its substrate tyrosine under basal conditions and
following MPTP administration. When TH catalytic activity is accelerated, substrate
levels diminish and may become rate limiting, such that administration of tyrosine
augments DOPA formation (Sved and F emstrom, 1981). Thus, if the activity of TH is
accelerated in NSDA neurons following MPTP, administration of supplemental tyrosine
should enhance TH catalytic activity if substrate limiting conditions exist. To determine
if this is the case, mice were injected with either saline (1.0 ml/kg, s.c.) or MPTP (25
mg/kg, s.c.) once a day for 5 consecutive days and killed 3 days following the last
injection. Mice were injected with tyrosine (100 mg/kg, i.p.) 60 min prior to sacrifice
and NSD-1015 (100 mg/kg, i.p.) 30 min prior to sacrifice. TH catalytic activity was
determined by measuring the ratio of DOPA concentrations to TH protein as described

above.

146

The Efi'ects of MPTP on TH Phosphorylation and Catalytic Activity in the Striatum

Sub-chronic MPTP administration caused a differential loss of serine-19, serine-
31 and serine-40 phosphorylated TH (Figure 5-4). MPTP caused a 63% decrease in
serine-19 phosphorylated TH levels and 75% decrease in serine-31 phosphorylated TH
levels. However, there was no change in serine-40 phosphorylated TH levels in the
striatum of mice treated with MPTP as compared to saline-treated mice. Phosphorylated
serine-19, serine-31, or serine-40 TH levels were normalized to total TH protein levels in
the striatum, as an index of the relative proportion of TH phosphorylated at each residue
(Figure 5-5). Total striatal TH protein was decreased by 50% following MPTP
administration. MPTP had no effect on the amount of TH phosphorylated at serine-19 in
the axon terminal and decreased the amount of serine-31 phosphorylated TH. In contrast,
MPTP caused a robust increase in the proportion of serine-40 phosphorylated TH in
NSDA axon terminals of mice treated with MPTP.

Sub-chronic MPTP administration caused a decrease in striatal DOPA
concentrations (Figure 5-6, A) and TH protein expression (Figure 5-6, B). However, the
decrease in DOPA concentrations paralleled that of TH protein. As such, MPTP
administration did not alter the enzymatic activity of TH, as reflected by the ratio of

DOPA to TH protein (Figure 5-6, C).

147

A. Serine-19 15

3 10
o
Serine 19 TH —-——> -- -— -- ~—-
Tubulin —-0 —- '- — —' 0
ladder SL—laline Ml—IPTP Saline MPTP
B. Serine-31 10
. 8
Serine 31 TH——~> —- -- . g 2
- —— _ —
Tubulin—"4 | | | l 2 *
Saline MPTP 0
ladder Saine MPTP
9' Serine-40
2.5
*
2.0
Serine40TH—> _______ :15
Tubulin—-—-fl -— — — —. 01.0
; |_l L_J “0'5
Saline MPTP '
ladder 0.0
Saline MPTP

Figure 5-4. LEFT PANEL: Representative immunoblots depicting the band intensity of
serine-19 (A), serine-31 (B) or serine-40 (C) (~ 55 kDA) and B-III tubulin (~ 50 kDA) of
samples from two mice treated with saline and two mice treated with MPTP. RIGHT
PANEL: Quantification of the effect of MPTP (25 mg/kg, i.p.) on serine-l9 (A), serine-
31 (B), or serine-40 (C) phosphorylated TH in the striatum. Relative density units for
serine-19, 31, or 40 phosphorylated TH were obtained by normalizing the respective band
intensities to B-III tubulin band intensity. Columns represent mean i S.E.M. of seven
mice/group. * indicates a significant difference from saline-treated controls. p: 0.05.

148

2.00 - Serine-19

1.50 -
1.00 -

0.50 -

Ser19/Total TH (ratio)

 

 

0.00
Saline MPTP

0-12 ' Serine—31
0.09
0.06
0.03

Ser31/Total TH (ratio)

 

 

0.00
Saline MPTP

0.12 Serine-40 *

0.08

0.04

 

Ser40/Total TH (ratio)

Saline MPTP

Figure 5-5. Quantification of the effect of MPTP (25 mg/kg, s.c.) on the relative
proportion of serine-19 (A), serine-31 (B), or serine-40 (C) phosphorylated TH compared
to total TH in the striatum. Phosphorylated serine-19, 31, or 40 phosphorylated TH
relative density units were normalized to total TH relative density units. Columns
represent mean i S.E.M. of seven mice/group. * indicates a significant change from
saline-treated controls. p: 0.05.

149

A. 15 TH Activity
3 12 i
E 9
O)
5
if 6 *
8 3
0 e
Saline MPTP
3 60 TH Protein
E
Q; 40
E *
g 20
Saline MPTP
C. . . .
0 3 TH Enzymatic Actlwty
g
g 0.2
E
(L 0.1
o
O
0.0

Saline MPTP

Figure 5-6. Striatal DOPA concentration (A), TH protein (B), and the ratio of DOPA t0
TH in the striatum of mice treated with either saline (1 ml/kg, s.c.) or MPTP (25 mg/kg,
s.c.). Striatal DOPA accumulation was measured 30 min following injection of NSD-
1015 (100 mg/kg, i.p.). Columns represent means :t S.E.M. of seven mice/group. *
indicates a significant change from saline-treated controls. p5 0.05.

150

Effects of Tyrosine Administration on Striatal TH Catalytic Activity in Mice Pre-Treated
with Saline or MPT P

As depicted in Figure 5-7, under basal conditions (saline-treated controls) tyrosine
administration 60 min prior to decapitation had no effect striatal DOPA concentrations or
TH protein. As such, tyrosine had no effect on TH enzymatic activity as reflected by the
ratio of DOPA to TH. Similarly, under activated conditions (mice pre-treated with

MPTP) tyrosine administration did not alter TH enzymatic activity.

151

Saline-treated MPTP-treated
0.30 -

0.20 -

DOPA/T H (ratio)

 

 

0.00
Vehicle Tyrosine Vehicle Tyrosine

Figure 5-7. The effect of tyrosine supplementation (100 mg/kg, i.p., 60 min) on TH
enzymatic activity in mice pre-treated with either saline (1 ml/kg, s.c.) or MPTP (25
mg/kg, s.c.). Striatal TH enzymatic activity was determined by normalizing striatal
DOPA concentrations, determined through Western blot analysis, to striatal DA
concentrations, determined through neurochemical analysis. Striatal DOPA
concentrations were measured following NSD-1015 (100 mg/kg, i.p., 30 min)
administration. Columns represent means i S.E.M. of seven mice/group.

152

Characterizing the Regulation of TH Protein Expression in NSDA Axon Terminals

TH protein expression in NSDA axon terminals following MPTP administration
were determined by normalizing TH protein expression (determined through Western
blot analysis) to striatal DA concentrations (determined through neurochemical analysis)
to account for the loss of axon terminals due to MPTP. The regulation of TH protein
expression at the axon terminal was further addressed by determining the sub-cellular
localization of the enzyme. The enzymatic action of TH occurs primarily within the
cytoplasm, and as such, the enzyme is located primarily within this sub-cellular
compartment. However, TH may also been suggested to be associated with synaptic
vesicles and plasma membrane at the axon terminal (Kumer and Vrana, 1996). TH
compartrnentalization is thought to reflect segregation of functional TH at the axon
terminal near the site of vesicular packaging. It is possible that a deficit in TH
compartrnentalization could cause an increase in cytoplasmic DA due to the synthesis
without vesicular packaging. To test this, sub-cellular fractionation was performed to
determine if there are changes in the localization of TH during compensatory activation
of NSDA axon terminals. Differential centrifugation of striatal tissue was used to isolate
sub-cellular fractions enriched in cytoplasmic, endosomal, synaptic vesicle and plasma
membrane protein. The amount of TH associated with each fiaction was compared under
basal conditions and following MPTP administration.

Mice were injected with either saline (1.0 mllkg, s.c., 8 mice) or MPTP (25
mg/kg, s.c., 8 mice) once a day for 5 consecutive days and killed 3 days following the last
injection. In order to preserve sub-cellular compartrnentalization, following decapitation

striatal protein was freshly dissected rather than frozen. Striatal tissue from two mice

153

was pooled into a single micro-centrifuge tube resulting in 4 samples from saline-treated
mice and 4 samples from MPTP-treated mice (Table 5-1). Tissue was homogenized and
differentially centrifuged as depicted in Figure 5-8, and described in greater detail in
Chapter 2. Centrifugation of homogenized striatal protein using this technique isolates
fractions enriched in cytoplasmic protein (S4), early endosomal vesicles (P4), synaptic
vesicles (P3) and plasma membrane protein (P1) (Slowiejko et al., 1996; Leng et al.,
2001). Following differential centrifugation, equal protein concentrations from each
fraction were loaded onto 10% PAGE gels by varying the volume loaded (IO-20 ill).
Resulting Western blots were probed for TH protein immunoreactivity. TH band density
was normalized to the band densities of proteins enriched in each sub-cellular fraction; [3-
III tubulin, early endosomal antigen 1 (EEA1), synaptophysin and SNAP-25,
respectively, to yield relative density units for TH. To account for axon terminal loss
following MPTP, TH relative density units of in each fraction was normalized to total
striatal TH relative density units to yield a relative proportion of TH in each fraction to

total TH following MPTP administration.

154

 

Treatment # of Animals # of Samples

 

Saline n=8 n=4
(1.0 mllkg, s.c.)

MPTP n=8 n=4
(25 mg/kg, s.c.)

Table 5-1. Experimental design used to determine TH sub-cellular distribution in the
striatum. Mice were injected with either saline (1.0 ml/kg, s.c.) or MPTP (25 mg/kg, s.c.)
once a day for 5 consecutive days and killed by decapitation 3 days following the last
injection. The striatum was freshly dissected, placed into micro-centrifuge tubes, and
homogenized. Striata dissected from 2 mice were pooled into a single micro-centrifuge
tube for analysis, resulting a total of 4 striatal samples from mice treated with saline and
4 striatal samples from mice treated with MPTP.

155

 
   

Homogenized
f Striatal Protein

  

Spin
1,500 g

 

Sucrose
Gradient |

I
v

Plasma Membrane
Enriched Fraction

_ _. Synaptic Vesicle
' Enriched Fraction

-. Cytoplasmic Enriched
*: """"" " Fraction

 

Early Endosome
Enriched Fraction

Figure 5-8. Differential centrifugation technique used to isolate plasma membrane,
synaptic vesicle, endosomal and cytoplasmic enriched fractions from homogenized
striatal protein. Homogenized striatal tissue was centrifuged at 1,500 g. The pellet fi'om
this low-speed centrifugation was separated using a sucrose gradient into plasma
membrane and nuclear enriched fractions. The supernatant (S1) from the initial low-
speed centrifugation was centrifuged at 20,000 g. The supernatant (S2) from this high-
speed centrifugation was centrifuged at 100,000 g. The pellet (P3) from this
ultracentrifugation was isolated and used as the synaptic vesicle enriched fraction. The
supernatant (S3) from the first ultracentrifugation was centrifuged at 200,000 g to pellet
early endosomal vesicles (P4). The supernatant (S4) from the second ultracentrifugation
was isolated as the cytoplasmic enriched fraction.

156

The Eflects of MPTP on TH Protein Expression in the Striatum

MPTP caused a decrease in striatal TH protein (Figure 5-9, A) and striatal DA
concentration (Figure 5-9, B), however the decrease in TH protein expression was not as
severe as the loss of striatal DA concentrations. As such, sub-Chronic MPTP
administration caused a robust increase in TH protein content, as reflected by the ratio of

TH to DA (Figure 5-9, C).

157

.>
8

. Striatal TH Protein

 

   

 

 

S
o
5 40 .
E *
3.5. 20
O
1.
o .
Saline MPTP
8' 130 Axon Terminal Loss
3150
5,120
i so -
60 _.
o 30 . *
o
Saline MPTP
C. TH Protein Conte*nt
g 1.5 .
E 10
a .
E 0.5
0.0

 

 

Saline MPTP

Figure 5-9. TH protein (A), DA concentrations (B), and the TH to DA ratio (C) in the
striatum of mice treated with either saline (1 ml/kg, s.c.) or MPTP (25 mg/kg, s.c.).
Relative density units (RDU) for TH were obtained by normalizing the TH band intensity
to B-III tubulin band intensity. Columns represent means 1: S.E.M. of seven mice/group.
"‘ indicate a significant difference from saline treated mice. p5 0.05.

158

The Eflects of MPT P on the Sub-Cellular Localization of TH in the Striatum
Sub-Chronic MPTP administration caused a differential loss of TH protein in the
striatum depending on which sub-cellular compartment the enzyme was associated with
(Figure 5-10, 5-11). MPTP caused a 35% decrease in TH protein immunoreactivity in
the cytoplasmic fraction and a 29% decrease in TH protein in the early endosomal
fraction. The loss of TH expression associated with the synaptic vesicle and plasma
membrane enriched fractions were more severe. MPTP caused a 65 and 75% loss of TH
associated with the synaptic vesicle and plasma membrane enriched fractions,
respectively. To quantify the relative change in TH localization relative to total TH at the
axon terminal, relative density units of TH in each fraction were normalized to relative
density units for total striatal TH. Sub-Chronic MPTP increased the proportion of TH
associated with the cytoplasmic and early endosomal vesicle fractions, but decreased TH
associated with the synaptic vesicle and plasma membrane enriched fractions (Figure 5-

12).

159

A. B.

T
cytosolic H -~ ...____, ...... ...... EndosomalTHe‘ __ __ ..-...
Tubulin
l___J L___l l__l l__l
Saline MPTP Saline MPTP
C' D. ‘ f: !
Synaptic “81619 TH - — W _____ Membrane TH ..L j J Lsi
Synaptophysin _ — — — SNAP-25 ~ fl ~ he
l_l l___l I J L__l
Saline MPTP Saline MPTP

Figure 5-10. Representative Western blots of TH protein in the cytosolic (A), endosomal
(B), synaptic vesicle (C), and plasma membrane (D) sub-cellular compartments from
mice treated with either saline (1 mllkg, s.c.) or MPTP (25 mg/kg, s.c.). Differential
centrifugation was used to isolate TH associated with sub-cellular fractions enriched in
either cytosolic (A), early endosomal vesicles (B), synaptic vesicles (C), or plasma
membrane (D) protein. TH associated with each compartment was normalized to
proteins enriched in each fraction B-III tubulin, early endosome antigen 1 (EEA1),
synaptophysin, or Snap-25, respectively to control for variations in loading.

160

A' 15 Cytosolic Fraction 8. Endosomal Fraction
2‘ g 50
5 1o * E5, 40 *
*5 '- 30
i— t“ 10
o 0
Saline MPTP Saline MPTP
C. . . . D. .
S Synaptic Ves1cle Fraction 70 Plasma Membrane Fraction
0 A
E, 20 g 60
g 5 50
E 15 g, 40
g 10 * 2 30
a s 5 2°
‘L’ E 10
E 0 0
Saline MPTP Saline MPTP

Figure 5-11. Quantification of the TH protein distribution in the striatum of mice treated
with either saline (1 ml/kg, s.c.) or MPTP (25 mg/kg, s.c.). TH protein in each fraction
was normalized to either B-III tubulin (A), EEA1 (B), synaptophysin (C), or SNAP-25
(D). Columns represent means 1 S.E.M. of four samples per group. * indicate a
significant difference from saline treated mice. p S 0.05.

161

          

A. Cytosolic Fraction B. Early Endosomal Fraction
g 0.8 E *
E 0.4 E 0'8
:g 35
E 0.2 E 0'4
,_ l-
s.
O 0.0 g 0.0
Saline MPTP Saline MPTP
C. Synaptic Vesicle Fraction D. Plasma Membrane Fraction
Tg‘ 0.6 7S °~°
3?. 0.5 g ‘15
.1: 0,4 * E 0.4 *
g 0.3 g 0.3
E 0.2 E 0.2
t; 0.1 " 0"
to 0.0 E 0.0
Saline MPTP Saline MPTP

Figure 5-12. The effects of MPTP (25 mg/kg, s.c.) on the proportion of TH associated
with sub-cellular fractions enriched in either cytoplasmic (A), early endosomal vesicles
(B), synaptic vesicles (C) or plasma membrane (D) protein. The relative amount of TH
protein associated with each sub-cellular fraction was determined by normalizing relative
density units for TH in each fraction to the relative density units of total striatal TH.
Columns represent means i S.E.M. of four samples per group. * indicate a significant
difference from saline treated mice. p 5 0.05.

162

C. Discussion

The present studies have employed neurochemical, immunohistochemical and cell
biological techniques to characterize changes in the regulation of TH at the axon terminal
following MPTP administration. Results describe abnormal regulation of TH
phosphorylation, protein expression and sub-cellular compartmentalization, suggesting
that deficits in TH expression and localization underlie the uncoupling of DA synthesis

and release at the axon terminal following MPTP.

The Eflect of MPT P on the Regulation of TH Catalytic Activity in NSDA Axon Terminals

Following MPTP-induced loss of NSDA axon terminals there is a sustained
increase in the rate of DA release from surviving axon terminals. In order to meet the
increased demand for release, the rate of DA synthesis is accelerated. However, DA
synthesis is accelerated to an extent that supersedes storage resulting in metabolism of the
newly synthesized DA. TH is the rate limiting enzyme in the DA biosynthetic pathway.
As such, over-activation of DA synthesis is likely the result of changes in the regulation
of TH. The present experiments characterized the regulation of TH catalytic activity in
NSDA axon terminals.

To determine if the compensatory activation of NSDA neurons causes a change in
the enzymatic activity of TH, the phosphorylation state of was determined at three serine
residues within the N-terminus of the enzyme. Results demonstrated that MPTP caused a
differential loss of various forms of phosphorylated TH. MPTP decreased serine-19 and
serine-31 phosphorylated TH, however there was no change in serine-40 phosphorylated

TH. When the proportion of TH phosphorylated at each residue was compared to total

163

TH, the percentage of serine-40 phosphorylated TH increased at the axon terminal, while
there was no change and a decrease in serine-19 and serine-31 phosphorylated TH,
respectively.

Serine-40 can be phosphorylated by a number of kinase-second messenger
systems, however, CAMP-dependent PKA is the primary protein kinase that
phosphorylates this residue (Kumer and Vrana, 1996). Thus, increased levels of serine-
40 phosphorylated TH suggest there is an increase in adenylate cyclase activity, cAMP
levels and PKA activation. D2 autoreceptors inhibit adenylate cyclase activity and thus
PKA-mediated phosphorylation of TH at serine-40. It is possible that there is a loss of
D2 autoreceptor regulation in NSDA neurons that survive MPTP induced neurotoxicity.

Serine-40 is the primary regulatory serine residue that controls TH catalytic
activity (Campbell et al., 1986; Haycock, 1990). Phosphorylation at this residue causes a
conformational change in the enzyme that increases activity by increasing its affinity for
substrate tyrosine and decreasing susceptibility to end-product inhibition from DA.
Therefore, increased serine-40 phosphorylated TH levels predicted accelerated TH
activity in surviving NSDA axon terminals following MPTP. This was not the case
however, as there was no increase in TH enzymatic activity as reflected by the ratio of
DOPA to TH. To confirm these results, tyrosine was administered to either saline-treated
controls or MPTP-treated mice. When TH activity is accelerated, supplemental tyrosine
enhances the rate of DOPA formation. Tyrosine administration did not increase product
formation, suggesting that there is no increase in the enzymatic activity of TH in axon

terminals that surviving MPTP neurotoxicity.

164

Interestingly, there was a discrepancy between increased serine-4O
phosphorylated TH and a lack of increase in the enzymes catalytic activity. One potential
explanation for this discrepancy is that the increase in serine-40 phosphorylated TH,
which accelerates enzymatic activity, may have been compensated for by decreased
phosphorylation at serine-31, such that there was no net change in the enzymes activity.
The Western blot and neurochemical techniques used in the present study did not allow
determination of the phosphorylation state of individual TH molecules. It is possible the
activity of molecules phosphorylated at serine-40 were accelerated while the decreased
phosphorylation at serine-31 decreased the activity of other molecules resulting in no net
change in overall enzymatic activity. Alternatively, if the changes in serine-40 and
serine-31 phosphorylation occur on the same TH molecules, the acceleration in
enzymatic activity induced by serine-4O phosphorylation may be blocked by decreased
phosphorylation at the serine-31 residue. Further biochemical experiments are required
to determine the phosphorylation state of individual TH molecules. Site-directed
mutagenesis of the key serine residues on TH or mass-spectrometry analysis of the
individual phosphorylation state of the enzyme under these conditions may provide
critical information regarding the role of second messenger systems involved in
regulating TH at the axon terminal.

A second potential explanation for the discrepancy between increased serine-4O
phosphorylated TH but no change in the enzymes activity may be that there is an increase
in de-activated TH at the axon terminal. Phosphorylation of the enzyme causes an
increase in catalytic activity but consequently decreases stability. Thus, the increased

serine-40 phosphorylated TH may represent de-activated enzyme at the axon terminal

165

that is being recycled. Further co-immunoprecipitation experiments that directly measure
levels of ubiquitinated TH are required to determine whether there is an increase in
destabilized TH at the axon terminal following MPTP and whether TH associated with
the early endosomal sub-cellular fraction is indeed de-activated enzyme.

If TH is degraded by the ubiquitin-proteosome system (UPS) (as most
intracellular proteins are) the accumulation of de-activated TH may reflect deficits in
protein degradation following MPTP. This is relevant to PD pathogenesis since Lewy-
body formation is thought to result from dysfunctional protein degradation. Additionally,
mutations in genes encoding UPS proteins are associated with inheritable PD (Kitada et
al., 1998). Accordingly, there may be an important link to abnormal protein degradation
and the dysfunctional regulation of TH. Nevertheless, using two separate functional
assays of TH enzymatic activity the present experiments demonstrate there is no change
in TH catalytic activity in NSDA axon terminals that survive MPTP treatment.

Therefore, the accelerated rate of DA synthesis that occurs during the compensatory
activation of NSDA axon terminals is likely due to long-term changes in the expression

of TH.

The Eflect of MPT P on TH Protein Expression in NSDA Axon Terminals

To determine if the loss of NSDA axon terminals induced by MPTP causes an
increase in TH protein expression in surviving neurons, TH protein levels were
determined in the striatum of mice treated with saline or sub-chronic MPTP. MPTP
decreased total striatal TH protein but there was a more severe loss of NSDA axon

terminals as reflected by striatal DA concentrations. As such, there was a robust increase

166

in TH protein in surviving axon terminals following MPTP. Several lines of evidence
support this conclusion; 1) following the loss of axon terminals induced by MPTP there is
a prolonged acceleration in NSDA activity and rate of DA synthesis, 2) long-term
changes in the rate of DA synthesis are mediated by increasing protein expression rather
than a change in catalytic activity (Dunkley et al., 2004), and 3) following MPTP there is
an increase in TH mRN A and protein expression in surviving NSDA neurons (J akowec et
al., 2004). Taken together, these results suggest the accelerated rate of DA synthesis in
NSDA axon terminals following MPTP is due to an increase in the expression of TH.

Sub-chronic MPTP caused a decrease in TH associated with the synaptic vesicle
and plasma membrane sub-cellular fractions. TH is thought to associate with synaptic
vesicles to localize newly synthesized DA to the site of vesicular packaging. This
function presumably enhances storage and consequently decreases the metabolism of
newly synthesized DA. The loss of this function therefore, may be responsible for the
increased metabolism of newly synthesized DA and uncoupling between DA synthesis
and storage capability in NSDA axon terminals following MPTP.

MPTP also decreased TH associated with the plasma membrane. TH associated
with the membrane may reflect reserve populations of TH at the axon terminal that may
be utilized to increase DA synthesis during activated conditions. The loss of plasma
membrane associated TH may reflect a recruitment of reserve populations of TH to meet
the increased demand for synthesis. It is unclear if this loss of function represents protein
translocation to the cytosol or a dysfunction in the ability to compartmentalize protein

under activated conditions.

167

In contrast to the synaptic vesicle and plasma membrane fractions, MPTP caused
an increase in cytoplasmic and endosomal TH. The enzymatic activity of TH takes place
within the cytoplasm at the axon terminal, suggesting increased expression of the enzyme
in this fraction contributes to the increased rate of DA synthesis. Since the increased
cytoplasmic TH is not associated with synaptic vesicles, the increase in newly
synthesized DA is likely metabolized rather than released.

MPTP also increased the amount of TH associated with early endosomal vesicle
fraction, suggesting there was an increase in deactivated TH being recycled through the
endosomal pathway. The endosome-lysosome pathway is generally associated with
recycling/degradation of macromolecules rather than cytoplasmic protein. However, a-
synuclein, is also associated with endosome recycling (Leng et al., 2001), suggesting
small cytoplasmic proteins at the axon terminal may also be recycled through the
endosome-lysosome pathway. It is also possible that TH associated with this light vesicle
fraction represents TH bound to a complex of proteins involved in recycling degradation
such as the ubiquitin-proteosome system (UPS) which primarily recycles cytoplasmic
protein. Nevertheless, these results suggest there is an increase in deactivated TH at the
axon terminal of NSDA neurons that survive MPTP administration. Further biochemical
experiments are necessary to identify the mechanisms that are responsible for TH
recycling and turnover at the axon terminal and whether TH associated with the light

vesicle fraction is indicative of protein being recycled.

168

Summary

The experiments described in this chapter demonstrate changes in the regulation
of TH at axon terminals of NSDA neurons that survive MPTP neurotoxic insult. Results
showed that increased cytoplasmic TH (rather than enzyme catalytic activity) causes an
accelerated rate of DA synthesis that exceeds vesicular packaging and is metabolized,
rather than released. Changes in the sub-cellular localization of TH at the axon terminal
suggest deficits in compartmentalization of TH causes the increased metabolism of newly
synthesized DA. The net effect of these compensatory changes in TH protein content and
uncoupling between DA synthesis and release are not clear. Identification of the
regulatory mechanisms that govern TH phosphorylation, protein expression and sub-
cellular localization however, may provide targets to prevent ROS-mediated damage and

the progressive loss of NSDA neurons in PD.

169

Chapter 6. Dopamine Receptor (D2) Regulation of Dopamine Synthesis

in NSDA Neurons

A. Introduction

Results from the experiments conducted in the previous chapters have identified
and characterized the neurochemical mechanisms that underlie the compensatory
activation of NSDA neurons. The dysfimctional regulation of the DA biosynthetic
pathway is responsible for the accelerated metabolism of cytoplasmic DA, and is likely a
pathogenic factor that contributes to the progressive loss of NSDA neurons in PD.
Understanding how the DA synthetic pathway and the activity of NSDA neurons are
regulation will be crucial to devising strategies to protect surviving neurons from ROS-
mediated oxidative damage derived from DA metabolism.

The activity of NSDA neurons is regulated by feedback inhibition which controls
the firing rate of these neurons in concert with the rate of DA synthesis and release at the
axon terminal. Feedback inhibition is mediated by D2 receptors that are located on post-
synaptic target neurons and pre-synaptic axon terminals. The compensatory activation of
NSDA neurons is the result of decreased inhibitory feedback mediated by D2 receptors.
D2 receptors are G-protein receptors that are coupled to inhibitory G-proteins (Ga/0)
which decrease the activity of adenylate cyclase and reduce intracellular CAMP levels
(Sibley, 1999). Activation of post-synaptic D2 receptors by DA released from a NSDA
axon terminal elicits long-loop, multi-synaptic, feedback inhibition to NSDA cell bodies
in the substantia nigra which inhibits subsequent cell firing (Imperato and Di Chiara,

1988). Following MPTP administration, the loss of axon terminals decreases the amount

170

of released DA capable of activating post-synaptic D2 receptors and, as such, NSDA
firing rate increases due to the loss of feedback inhibition to NSDA cell bodies.

The rate of DA synthesis and release are also regulated by short-loop feedback
inhibition mediated by pre-synaptic D2 autoreceptors directly at the axon terminal.
Activation of D2 autoreceptors inhibits adenylate cyclase activity and cAMP
concentrations, which decreases, cAMP-dependent PKA phosphorylation of TH (Kehr et
al., 1972). Phosphorylation of TH at serine-4O by PKA increases the enzymes catalytic
activity (Haycock, 1990). Additionally, D2 autoreceptors modulate DA release directly
at the axon terminal. Action potential-stimulated DA release activates D2 autoreceptors
which inhibits DA release in response to subsequent action potentials by accelerating K+
efflux and hyperpolarizing the axon terminal (Koeltzow et al., 1998; Missale et al., 1998;
Sibley, 1999). Therefore, following MPTP administration, the loss of axon terminals
decreases the amount of released DA capable of activating pre-synaptic D2 autoreceptors
and as such, increases TH phosphorylation and enzymatic activity as well as increases
action potential-stimulated DA release.

It is clear there is a dysfunctional regulation of DA synthesis in NSDA axon
terminals following MPTP administration. This may be due to the loss of D2 receptor-
mediated inhibitory feedback. However, it is unclear what effect a loss of long and short-
loop feedback inhibition has on the regulation of TH phosphorylation, enzymatic activity
and the rate of DA synthesis at the axon terminal. To address this, the experiments
described in this chapter were designed to; l) characterize the regulation of TH following
the loss of D2 receptor-mediated feedback inhibition, 2) determine if there is a loss of D2

receptor regulation of the activity of NSDA neurons following MPTP administration and

171

3) determine if prolonged loss of D2 receptor-mediated feedback inhibition (and the
consequent chronic activation of NSDA neurons) causes the degeneration of NSDA axon

terminals.

172

B. Experimental Design and Results
Characterizing the Eflects of a Loss of Long and Short-Loop Feedback Inhibition on the
Regulation of DA Synthesis in NSDA Axon Terminals

Pharmacological blockade of post- and pre-synaptic D2 receptors was used to
recapitulate the loss of feedback inhibition following MPTP administration. Raclopride,
a selective D2 antagonist (Hall et al., 1988), was used to block both post-synaptic D2
receptors (long-loop feedback inhibition) and pre-synaptic D2 autoreceptors (short-loop
feedback inhibition). As depicted in Figure 6-1, raclopride increases NSDA cell firing,
action potential generation, and Ca+2 entry into the axon terminal. Activation of
Ca+2/Calmodulin-dependent protein kinase can accelerate DA synthesis by
phosphorylating TH at serine-19 (Yamauchi and F ujisawa, 1981; Vulliet et al., 1984).
Raclopride blockade of pre-synaptic D2 autoreceptors increases adenylate cyclase
activity, cAMP production and activation of cAMP-dependent PKA. Activation of PKA
can accelerate DA synthesis by phosphorylating TH at serine-40 (Joh et al., 1978;
Yamauchi and F ujisawa, 1979; Vulliet et al., 1980). Thus, the loss of both long and
short-loop feedback inhibition can accelerate TH enzymatic activity. Under these
conditions the enzymatic activity of TH can become limited by a lack of substrate

availability (Sved and Femstrom, 1981).

173

Raclopride Model

 

c *2
0:2 Ca+2
Action Potentids : __, Ca+2
H ca+2

 

 

 

Raclopride

Tyrosine

Figure 6-1 Schematic diagram depicting the neurochemical events in the axon terminal
following administration of raclopride. Raclopride administration blocks both post-
synaptic and pre-synaptic D2 receptors. Blockade of D2 receptors increases cell firing
and disinhibits adenylate cyclase activity at the axon terminal. This results in TH
phosphorylation by activity and cAMP-dependent kinases and acceleration in TH

enzymatic activity.

174

In the first set of experiments, mice were injected with either saline (1 ml/kg, i.p.),
tyrosine (100 mg/kg, i.p.), raclopride (1.0 mg/kg, i.p.), or tyrosine plus raclopride and
decapitated 1 h following injection. Supplemental tyrosine was administered to prevent
substrate limitations on TH activity in activated NSDA neurons. Administration of
raclopride using this dose and time course maximally activates NSDA neurons (Eaton et
al., 1992). The effects of raclopride on NSDA activity was assessed by measuring striatal
DOPAC and DA concentrations, and the DOPAC to DA ratio. To evaluate the effects of
raclopride on TH enzymatic activity, mice were injected as described above and killed 1
h after drug administration. Mice were injected with NSD-lOl 5 30 min prior to sacrifice
and striatal DOPA concentrations were measured as an index of TH catalytic activity.

TH phosphorylation state, enzymatic activity and protein expression were also

determined in the striatum as described previously.

175

The Eflect of Acute Raclopride on NSDA Activity

As depicted in Figure 6-2 (A), tyrosine administration had no effect on DA
metabolism, as reflected by striatal DOPAC concentrations. Raclopride administration
caused a 4-fold increase in striatal DOPAC concentrations Tyrosine supplementation in
raclopride-treated mice increased striatal DOPAC concentrations when compared to mice
treated with raclopride alone. As depicted in Figure 6-2 (B), tyrosine administration
slightly increased basal levels of striatal DA storage, as reflected by concentrations of
DA. Raclopride administration decreased striatal DA concentrations by approximately
25% in both vehicle and tyrosine-treated mice. As depicted in Figure 6-2 (C), tyrosine
administration had no effect on NSDA activity, as reflected by the DOPAC to DA ratio.
Raclopride administration caused a 5-fold increase in the DOPAC to DA ratio and
tyrosine supplementation caused a further increase over mice treated with raclopride

alone.

176

Striatal DA Metabolism

 

100
’6 *1:
E 75
E *
50
2 I
IL
8 25
o -
Saline Saline Raclopride Raclopride
+ + + +

Vehicle Tyrosine Vehicle Tyrosine

Striatal DA Storage

350
300 *
ET 250 *
a. 200
5
< 150
0 100
50
o
Saline Saline Raclopride Raclopride
+ + + +
Vehicle Tyrosine Vehicle Tyrosine
NSDA Neuronal Activity
0.50
3 * #
- 0.40
E *
0.30
s
< 0.20
l
8 0.10
0.00
Saline Saline Raclopride Raclopride
+ + + +

Vehicle Tyrosine Vehicle Tyrosine

Figure 6-2. Concentrations of DOPAC (A), and DA (B), and the DOPAC/DA ratio (C)
in the striatum of mice 60 min after administration of either saline (1 mllkg, i.p.) +
vehicle (saline 1 ml/kg, i.p.), saline + tyrosine (100 mg/kg, i.p.), raclopride (1.0 mg/kg,
i.p.) + vehicle, or raclopride + tyrosine. Columns represent means of groups i 1.0 SEM.
of seven mice per group. (*) Values significantly different from saline + vehicle treated
controls. # Values significantly different from raclopride + vehicle-treated mice. p50.05.

177

The Eflect of Acute Raclopride on TH Phosphorylation State

As depicted in Figure 6-3 (A), tyrosine administration had no effect on basal
levels of serine-19 phosphorylated TH. Raclopride administration produced an
approximately 1.5-fold increase in serine-19 phosphorylated TH levels. Tyrosine plus
raclopride administration increased serine-l9 phosphorylated TH levels over mice treated
with raclopride alone.

As depicted in Figure 6-3 (B), tyrosine administration did not change basal levels
of serine-31 phosphorylated TH. Raclopride administration produced an approximately
1.6-fold increase in serine-31 phosphorylated TH levels. Tyrosine supplementation in
raclopride-treated mice did not affect serine-31 phosphorylated TH levels compared with
raclopride treatment alone.

As depicted in Figure 6-3 (C), tyrosine administration had no effect on basal
levels of serine-40 phosphorylated TH. Raclopride administration produced an
approximately 6.8-fold increase in serine-4O phosphorylated TH. Tyrosine plus
raclopride administration significantly increased serine-4O phosphorylated TH levels an

additional 25% over mice treated with raclopride alone.

178

RDU
o88888

RDU

300
200
100

O

Serine-19
r * #
*

   

 

Saline Saline Raclopride Raclopride
+ + + +

Vehicle Tyrosine Vehicle Tyrosine

Serine-31

    

*

 

Saline Saline Raclopride Raclopride
+ + + 4»
Vehicle Tyrosine Vehicle Tyrosine

Serine-40
. * #

*

 

 

Saline Saline Raclopride Raclopride
+ + + 4-
Vehicle Tyrosine Vehicle Tyrosine

Figure 6-3. Serine-l9 (A), serine-31 (B) and serine-40 (C) phosphorylated TH levels in
the striatum of mice 60 min after treatment with either saline (1 ml/kg, i.p.) + vehicle (1
ml/kg, i.p.), saline + tyrosine (100 mg/kg, i.p.), raclopride (1.0 mg/kg, i.p.) + vehicle, or
raclopride + tyrosine. Mice were killed 1 hr after drug administration. Relative density
units (RDU) were obtained by normalizing the phosphorylated TH band density to B-III
tubulin band density. Columns represent means of groups :1: S.E.M. of seven mice/group.
* indicate a significant difference from saline + vehicle-treated mice. # Values
significantly different from raclopride + vehicle-treated mice. p S 0.05.

179

The Eflect of A cute Raclopride on TH Enzymatic Activity and Protein Expression

The accumulation of DOPA following blockade of AADC is an in vivo assay for
TH catalytic activity (Carlsson et al., 1972). As depicted in Figure 6-4, tyrosine
administration alone did not change basal TH catalytic activity, as reflected by striatal
DOPA concentrations. Raclopride administration produced an approximately 3-fold
increase in TH catalytic activity and tyrosine supplementation resulted in an
approximately 5-fold increase in TH catalytic activity that was significantly increased
from mice treated with raclopride alone. As depicted in Table 6-1, levels of total TH did

not vary in response to any treatment condition.

180

TH Enzymatic Activity

60
50 * #

40 *
30
20
1 0

DOPA (nglmg)

 

Saline Saline Raclo pride Raclopride

+ + + +
Vehicle Tyrosine Vehicle Tyrosine

Figure 6-4. Concentrations of DOPA in the striatum of mice 60 min afier administration
of either saline (1 mllkg, i.p.) + vehicle (saline, 1 mllkg, i.p.), saline + tyrosine (100
mg/kg, i.p.), raclopride (1.0 mg/kg, i.p.) + vehicle, or raclopride + tyrosine. All mice
received an injection of NSD-1015 (100 mg/kg, i.p.) 30 minutes prior to decapitation.
Columns represent means of groups a: 1.0 S.E.M. of seven mice per group. (*) Values
significantly different from saline + vehicle treated controls. (#) Values for tyrosine plus
raclopride-treated mice significantly different from raclopride + vehicle-treated mice.
p50.05. '

181

 

Saline Saline Raclopride Raclopride

+ + + +
Vehicle Tyrosine Vehicle Tyrosine

 

 

THProtein 32:3 27:4 24:3 33:3
(RDU)

 

Table 6-1. Total TH levels measured in relative density units (RDU) in the striattun of
mice 60 min after administration of either saline (1 mllkg, i.p.) + vehicle (saline, 1 ml/kg,
i.p.), saline + tyrosine (100 mg/kg, i.p.), raclopride (1.0 mg/kg, i.p.) + vehicle, or
raclopride + tyrosine. Relative density units were obtained by normalizing total TH band
intensity to B-III tubulin band intensity. Values represent means of groups :1: SEM.

182

Characterizing the Effects of Loss of Short-Loop Feedback Inhibition on the Regulation
of DA Synthesis in NSDA Axon Terminals

Pharmacological inhibition of pre-synaptic D2 autoreceptors was used as a model
to study the loss of short-loop feedback inhibition on DA synthesis. Administration of y-
butyrolactone (GBL) is a well documented pharmacological model that allows for
autoreceptor-specific functions to be evaluated in the absence of activity-dependent
compensation. As depicted in Figure 6-5, GBL inhibits NSDA cell firing (Howard and
Feigenbaum, 1997) and impulse driven release of DA (Bettini et al., 1987; Imperato et
al., 1987), which decreases DA binding to pre-synaptic and post-synaptic D2 receptors.
Similar to the raclopride, inhibiting DA release functionally blocks both pre-synaptic and
post-synaptic D2 receptors. The inhibition of cell firing however, prevents long-loop
feedback from stimulating DA synthesis. This allows D2 autoreceptor-stimulated DA
synthesis to be evaluated. GBL-induced disinhibition of pre-synaptic D2 autoreceptors
increases adenylate cyclase activity, cAMP production and activation of cAMP-
dependent PKA. Activation of PKA can accelerate DA synthesis by phosphorylating TH
at serine-40 (Joh et al., 1978; Yamauchi and Fujisawa, 1979; Vulliet et al., 1980). Thus,

the loss of short-loop feedback inhibition can accelerate DA synthesis.

183

GBL Model

Action Potentials Ca Channels
.‘. A

   
 
  

 

GBL

TyrosinHDOPA—>DA
TH'P DD

96
K 1....
pchMP

 

 

Tyrosine

Figure 6-5. Schematic diagram depicting the neurochemical events in the axon terminal
following administration of GBL. GBL administration blocks NSDA cell firing and
decreases DA release and occupation of pre-synaptic D2 autoreceptors. This results in
disinhibition of adenylate cyclase, TH phosphorylation by cAMP-dependent kinases and
acceleration of TH enzymatic activity. Because DA release does not occur, DA stores
increase in axon terminasl of NSDA neurons following GBL.

184

In the second set of experiment, mice were injected with either saline (1 ml/kg,
i.p.) or GBL (750 mg/kg, i.p.) and decapitated 1 h following injection. Administration of
GBL using this dose and time course activates DA synthesis (Demarest and Moore,
1979b). The effect of GBL on NSDA activity was assessed by measuring striatal
concentrations of DOPAC and DA, and the DOPAC to DA ratio. To determine the
effects of GBL on TH enzymatic activity and phosphorylation state, mice were injected
with either saline (1 ml/kg, i.p.), tyrosine (100 mg/kg, i.p.), GBL (750 mg/kg, i.p.) or
GBL + tyrosine and sacrificed 1 h following injection. Supplemental tyrosine was
administered to prevent substrate limitations on TH activity in activated NSDA neurons
(Sved and F emstrom, 1981). Mice were then injected with NSD-lOlS 30 min prior to
sacrifice. TH phosphorylation state was measured using Western blot analysis whereas,
TH enzymatic activity was determined by measuring striatal DOPA concentration as

described previously.

185

The Eflects of GBL on DA Metabolism and Storage

GBL administration increased striatal DOPAC concentrations approximately 2-
fold (Figure 6-6, A). Striatal DA concentrations were also increased by approximately 2-
fold following GBL administration. Due to equivalent increases in striatal DOPAC and
DA concentrations, there was no change in the ratio of DOPAC to DA in the striatum of

mice treated with GBL (C).

186

Striatal DA Metabolism
*

   

15
a
E
a 10
S
2
§ 5
o
Saline GBL
Striatal DA Storage
200

*

DA (09/ "19)
0i 8 a:
o o o

     

O

Saline GBL

o 10 DA Metabolism IStorage

0.08
0%
0.04
0.02
0.00

DOPAC/DA (ratio)

   

Saline GBL

Figure 6-6. Concentrations of DOPAC (A), and DA (B), and the DOPAC/DA ratio (C)
in the striatum of mice 60 min after administration of either saline (1 ml/kg, i.p.) or GBL
(750 mg/kg, i.p.). Columns represent means of groups i 1.0 S.E.M. of seven mice per
group. (*) Values significantly different from saline treated controls. pS0.05.

187

The Effect of Acute GBL on TH Phosphorylation State

As depicted in Figure 6—7 (A), neither tyrosine nor GBL altered basal levels of
serine-19 phosphorylated TH. Administration of tyrosine plus GBL increased serine-19
phosphorylated TH levels 2-fold. As depicted in Figure 6-7 (B), there was no change in
serine-31 phosphorylated TH in response to any drug treatment. As depicted in Figure 6-
7 (C), tyrosine administration had no effect on basal levels of serine-40 phosphorylated
TH. GBL administration produced an approximately 3-fold increase in serine-40
phosphorylated TH. Tyrosine plus GBL administration significantly increased serine-40

phosphorylated TH levels over raclopride treatment alone.

188

Serine-19

 

6 *
5
4
E 3
2
1
0
Saline Saline GBL GBL
+ + + +
Vehicle Tyrosine Vehicle Tyrosine
15 Serine-31
D 10
0
tr
5
Saline Saline GBL GBL
+ + + +
Vehicle Tyrosine Vehicle Tyrosine
15 Serine-40 *#
12 *
:3 9
o
‘r 6
3
0
Saline Saline GBL GBL
+ + + +
Vehicle Tyrosine Vehicle Tyrosine

Figure 6—7. Serine-19 (A), serine-31 (B) and serine-40 (C) phosphorylated TH levels in
the striatum of mice treated with either saline (1 ml/kg, i.p.) + vehicle (saline, 1 ml/kg,
i.p.), saline + tyrosine (100 mg/kg, i.p.), raclopride (1.0 mg/kg, i.p.) + vehicle, or
raclopride + tyrosine. Mice were killed 1 hr after drug administration. Relative density
units (RDU) were obtained by normalizing the phosphorylated TH band density to B-III
tubulin band density. Columns represent means of groups i S.E.M. of seven mice/group.
* indicate a significant difference from saline + vehicle-treated mice. # indicate a
significant difference from GBL + vehicle-treated mice. p S 0.05.

189

The Eflects of GBL on TH Enzymatic Activity and Expression

As depicted in Figure 6-8, tyrosine administration did not change basal TH
enzymatic activity, as reflected by striatal DOPA concentrations. GBL administration
produced an approximately 3-fold increase in TH catalytic activity and tyrosine
supplementation resulted in an approximately 5-fold increase in TH catalytic activity that
was significantly increased from mice treated with GBL alone. In contrast, levels of total

TH did not vary in response to any treatment condition (Table 6-2).

190

TH Enzymatic Activity
20 i * #

15

I

*

DOPA (nglmg)

 

 

Saline Saline GBL GBL
+ + + +

Vehicle Tyrosine Vehicle Tyrosine

Figure 6-8. Concentrations of DOPA in the striatum of mice afier administration of
either saline (1 ml/kg, i.p.) + vehicle (saline 1 mllkg, i.p.), saline + tyrosine (100 mg/kg,
i.p.), GBL (750 mg/kg, i.p.) + vehicle, or GBL + tyrosine. All mice received an injection
of NSD-lOlS (100 mg/kg, i.p.) 30 minutes prior to decapitation. Columns represent
means of groups (7 mice/group). Vertical lines represent 3: 1.0 SEM. (*) Values that are
significantly different from saline treated controls. (#) Values for tyrosine plus
raclopride-treated animals that are significantly different from raclopride-treated animals.
pS0.05.

191

 

Saline Saline GBL GBL
+ + + +

Vehicle Tyrosine Vehicle Tyrosine

 

THProtein 23:4 28:2 29:3 33:4
(RDU)

 

 

Table 6-2. Total TH levels measured in relative density units (RDU) in the striatum of
mice after administration of either saline (1 ml/kg, i.p.) + vehicle (saline 1 ml/kg, i.p.),
saline + tyrosine (100 mg/kg, i.p.), GBL (750 mg/kg, i.p.) + vehicle, or GBL + tyrosine.
Relative density units were obtained by normalizing total TH band intensity to B-III
tubulin band intensity. Values represent means of groups :1: SEM.

192

Characterizing D2 Receptor Regulation of NSDA Activity Under Basal Conditions and
Following MPT P Administration

The loss of D2 receptor function following MPTP could account for the
dysfunctional regulation of DA synthesis in surviving axon terminals. To determine if
this is the case, mice were pre-treated with either saline (basal conditions) or MPTP
(compensatory activation). Subsequently, mice were treated with either raclopride, a D2
receptor antagonist, or quinelorane, a D2 receptor agonist, to determine if D2 receptors
can accelerate or inhibit NSDA activity, respectively.

As depicted in Table 6-3, mice were injected with either saline (1 ml/kg, s.c.) or
MPTP (20 mg/kg, s.c.) twice a week for 5 consecutive weeks. Three days following the
last injection mice were anesthetized and Alzet osmotic mini-pumps filled with either
saline, raclopride or quinelorane were implanted s.c. Mice were treated with either saline
(150 til/day), raclopride (0.5 mg/kg/day), or quinelorane (0.5 mg/kg/day) for 21
consecutive days. NSDA activity was assessed by measuring striatal DA metabolism,
storage and activity by determining striatal concentrations of DOPAC, DA and the

DOPAC to DA ratio, respectively.

193

Post-Treatment

 

 

*5 Saline Raclopride Quinelorane
g (150 pllday) (0.5 mglkglday) (0.5 mglkglday)
5 Vehicle n=7 n=7 n=7
I: (1 mllkg)

I _ ._ _.

9 M pr n-7 n—7 n—7
°' (20 mg/kg)

 

Table 6-3. Experimental design for determining whether D2 receptors regulate the
activity of NSDA neurons following MPTP administration. Mice were pre-treated with
either vehicle saline (1 ml/kg, s.c.) or MPTP (20 mg/kg, s.c.) twice a week for 5
consecutive weeks. Three days after the last injection osmotic mini-pumps were
implanted s.c. filled with either saline, raclopride or quinelorane. Mice were treated with
either saline (150 til/day), raclopride (0.5 mg/kg/day) or quinelorane (0.5 mg/kg/day) for
21 consecutive days.

194

The Eflects of Chronic Loss of D2 Receptor Regulation of NSDA Activity Under Basal
Conditions and Following MPTP Administration

As depicted in Figure 6-9, in saline-treated mice (basal conditions), treatment
with raclopride had no effect on striatal DOPAC (A) or DA (B) concentrations or the
DOPAC to DA ratio (C). Under basal conditions, treatment with quinelorane decreased
striatal DOPAC concentrations by approximately 25% (A), but did not alter striatal DA
concentrations (B). Due to the decrease in striatal DOPAC, quinelorane decreased the
DOPAC to DA ratio by approximately 25% (C).

MPTP treatment increased the DOPAC to DA ratio in all mice as compared to
mice treated with saline. In MPTP-treated mice, raclopride had no effect on striatal
DOPAC concentrations (A), but decreased striatal DA concentrations approximately 50%
compared to MPTP + vehicle-treated mice. As such, raclopride treatment caused a
dramatic increase in NSDA activity, as reflected by the DOPAC to DA ratio (C). In
MPTP-treated mice, quinelorane treatment decreased striatal DOPAC (A) and DA (B)
concentrations by approximately 50%. Due to the equivalent changes in DOPAC and
DA concentrations, quinelorane treatment in MPTP-treated mice had no effect on the

DOPAC to DA ratio (C).

195

Striatal DA Metabolism

 

 

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Striatal DA Storage

Dung/ma)
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Pro-treatment Sline saine Saline WT? MPTP mp
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Post-treatment Vehicle Rec Quin Vehicle Rac Quin

NSDA Activity

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9 o .0
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PMW Saline sumo sumo MPTP MPTP wrp

+ 0 O O + #-
Post-treatment Vehicle Rec Quin Vehicle Rec Qu‘n

Figure 6-9. Concentrations of DOPAC (A), and DA (B), and the DOPAC/DA ratio (C)
in the striatum of mice injected with either saline (1 ml/kg, s.c.) or MPTP (20 mg/kg, s.c.)
twice a week for 5 consecutive weeks and post-treated with either vehicle (saline, 150
ill/day, s.c.), raclopride (Rac, 0.5 mg/kg/day, s.c.) or quinelorane (Quin 0.5 mg/kg/day,
s.c.) for 21 consecutive days. Columns represent means i SEM of seven mice/group. "'
indicate a significant difference from saline + vehicle treated mice. # indicate a
significant difference from MPTP + vehicle treated mice. p5 0.05.

196

Determining if a Prolonged Loss of D2-Receptor—Mediated Feedback Inhibition and
Chronic Activation is Detrimental to NSDA Neurons

To determine if a prolonged loss of D2 receptor-mediated feedback inhibition and
the consequential chronic activation of NSDA neurons is detrimental, osmotic mini-
pumps filled with either saline or raclopride were implanted s.c. in mice. Saline (150
ill/day) or raclopride (6 mg/kg/day) was administered to mice for 21 consecutive days.
Administration of raclopride (1.0 mg/kg, i.p.) activates NSDA neurons for at least 4 h
(Eaton et al., 1992). As such, the dose of raclopride was chosen to induce continual
maximal activation of NSDA neurons. Mice were sacrificed on the 21St day of drug
treatment. NSDA axon terminal loss was assessed by measuring striatal DA
concentrations. To discriminate between the effects of raclopride on DA vesicular stores
versus axon terminal loss, one group of mice was treated with raclopride for 21 days and
pumps were removed 3 days prior to sacrifice to allow a drug elimination period. The
half-life of raclopride following systemic administration is approximately 22 min
(V olkow et al., 1996). Behavioral deficits and NSDA neuronal activity return to baseline
by 4 h after systemic administration (Eaton et al., 1992; Ahlenius et al., 1997). Thus, 3
days after administration it is unlikely that raclopride is available to activate NSDA

neurons.

197

The Eflects of Chronic Raclopride on NSDA Axon Terminals

As depicted in Figure 6-10, chronic raclopride administration had no effect on
striatal DOPAC concentrations (A). Chronic raclopride decreased striatal DA
concentrations by approximately 50%, however this loss was transient as it was
eliminated in mice following a 3 drug elimination period (B). Due to the decrease in
striatal DA storage, chronic raclopride caused a 2-fold increase in the DOPAC to DA
ratio (C). In mice treated with raclopride for 21 days followed with a 3 day drug
elimination period, the DOPAC to DA ratio was significantly reduced compared to

raclopride-treated mice.

198

.>

DOPAC (nglmg)

in

DA (nglmg)

.0

DOPAC/0N ratio)

Striatal DA Metabolism

15

12

o

e

3

o

Saline Raclopride Raclopride
+
Elimination

35° Stnatal DA Storage
son I
250
zoo
150 *
100

so

Saline Raclopride Raclopride
+
Elimination
NSDA Activity

0.12 *

one

0.04 7

0.00

Saline Raclopride Raclopride
+

Elimination

Figure 6-10. Concentrations of DOPAC (A), and DA (B), and the DOPAC/DA ratio (C)
in the striatum of treated with either saline (150 ill/day) or raclopride (6 mg/kg/day, s.c.)
chronically for 21 days. In the 3rd group of mice, pumps were implanted 3 days prior to
the other 2 groups. Mice were treated for 21 days, and then 3 days prior to sacrifice,
pumps were removed to allow a drug washout period. Columns represent means of
groups 3: S.E.M. of seven mice/group. * indicate a significant difference from saline +
vehicle treated mice. # indicate a significant difference from raclopride-treated mice p5

199

C. Discussion

Using well-characterized pharmacological models and standard neurochemical
and immunohistochemical techniques, the present studies investigated the regulation of
TH phosphorylation, enzymatic activity and protein expression following the loss of D2
receptor-mediated feedback inhibition. Furthermore, the present studies have
characterized D2 receptor regulation of NSDA activity under basal conditions and
following MPTP-induced compensatory activation of surviving NSDA neurons. The
findings from these studies highlight differences in the regulation of DA metabolism
following D2 receptor-mediated chronic activation of NSDA neurons and the neurotoxin-
induced compensatory activation that suggest the compensatory activation of NSDA

neurons is unique and may be detrimental to the survival of these neurons.

NSDA Activity Following a Loss of D2 Receptor-Mediated Long and Short-loop
Feedback Inhibition

Under basal conditions, NSDA cell firing and DA release is governed at the axon
terminal by feedback inhibition fiom post-synaptic and pre-synaptic D2 receptors.
Blockade of post-synaptic D2 receptors diminishes feedback inhibition to NSDA cell
bodies and increases cell firing, bursts of action potentials, and DA release at the axon
terminal (Andersson et al., 1994; Andersson et al., 1995; Millan et al., 1998). Consistent
with this, the present study suggests that acute raclopride-mediated blockade of D2
receptors increased NSDA neuronal activity, as reflected by the DOPAC to DA ratio.
The findings from this study demonstrate that under basal conditions D2 receptors play a

major role in inhibiting the activity of NSDA neurons, and that the loss of this feedback

200

inhibition results in a dramatic increase in DA release, reuptake and metabolism at the
axon terminal.

The increased DOPAC to DA ratio was due to an increase in DA metabolism and
a decrease in DA storage in the striatum. Normally, newly synthesized DA is
preferentially released (Arbuthnott et al., 1990; Fairbrother et al., 1990a, 1990b; Biggs et
al., 1996a; Biggs et al., 1996b), while storage pools of DA are only depleted when
synthesis cannot keep up with the demand for release. The results from this experiment
suggest that following blockade of long and short-loop feedback inhibition, DA synthesis
cannot generate sufficient concentrations of DA to maintain the accelerated rate of

release, and as a result stored vesicular DA is released to compensate.

TH Regulation Following a Loss of Long and Short-loop Feedback Inhibition

D2 receptors inhibit the activity of adenylate cyclase and thereby decrease cAMP
concentrations. TH activation occurs predominantly through cAMP-dependent PKA
phosphorylation of the serine-40 residue (Kumer and Vrana, 1996). Consistent with this,
blockade of D2 receptors increased serine-40 phosphorylated TH levels and produced a
dramatic increase in the catalytic activity of the enzyme. Additionally, as expected, there
was no change in TH protein expression following acute raclopride since the l h time
frame is too short to allow for de novo protein synthesis. The results from this study
demonstrate that short-term (l h) regulation of the rate of DA synthesis is achieved
through D2 receptor-mediated inhibition of TH phosphorylation at serine-40.

The acceleration of NSDA neuronal activity caused by blockade of D2 receptors

also resulted in increased TH phosphorylation at serine-31 and serine-19 but to a much

201

lesser extent than the serine-40 residue. The role of these serine residues in regulating
TH activity is less well understood. Serine-31 is phosphorylated by MAP kinases, and
the results from this study are consistent with previous reports that phosphorylation of
this residue correlates with a 2-fold increase in TH activity (Haycock et al., 1992;
Sutherland et al., 1993; Halloran and Vulliet, 1994). Serine-19 is phosphorylated
predominantly by Ca";2 Calmodulin-dependent protein kinase II (Yamauchi and F ujisawa,
1981; Vulliet et al., 1984; Campbell et al., 1986) and this corresponded with an
approximately 1.5-fold increase in enzymatic activity. Results suggest that serine-31 and
serine-19 are also phosphorylated following accelerated neuronal activity and likely
contribute to the increase in enzymatic activity. However, the Western blot and
neurochemical techniques used in the present study do not resolve whether individual TH
molecules are phosphorylated specifically at serine-40, serine-31 or serine-19, or whether
multiple residues may be phosphorylated at the same time. If multiple serine residues
may be simultaneously phosphorylated, the phosphorylation sequence of each residue in
response to increased neuronal activity could provide important information as to the role
of second messenger systems involved in TH activation and resolve the physiological
consequence of phosphorylation of serine-31 and serine-19.

The present study also investigated the specific role of D2 autoreceptors in
regulating TH phosphorylation and enzymatic activity. GBL administration inhibits
NSDA cell firing thus decreasing action potential-mediated DA release. Decreased DA
release prevents DA from binding to and activating pre-synaptic autoreceptors. The
disinhibition of autoreceptors accelerates DA synthesis at the axon terminals, similar to

the raclopride, but in the absence of increased neuronal activity. Acute GBL

202

administration caused a 2-fold increased DA metabolism and storage in the striatum.
Since GBL inhibits DA release, the increased metabolism and storage of DA reflects the
metabolism of newly synthesized DA, rather than recaptured DA.

Inhibition of D2 autoreceptor occupancy following GBL caused a 3-fold increase
in serine-40 phosphorylated TH and a 2-fold increase in enzymatic activity. In contrast
to the raclopride model, there was no change in serine-19 or serine-31 phosphorylated TH
following GBL. Thus, D2 autoreceptors inhibit TH phosphorylation specifically at
serine-40 and a loss of this feedback inhibition accelerates enzymatic activity. By
comparing the effects of raclopride versus GBL on TH phosphorylation state and
enzymatic activity we can infer that kinases activated in response to increased neuronal
activity can phosphorylated TH at serine-19, serine-31 and also serine-40, whereas
kinases activated in response to blockade of D2 autoreceptors can only phosphorylate TH
at serine-40. Consistent with this, the induction of enzymatic activity by GBL was not as
robust as following raclopride.

The specific increase in serine-40 phosphorylated TH following GBL was similar
to the increase in serine-40 phosphorylated TH following MPTP administration. Thus,
the dysfunctional regulation of DA synthesis following MPTP may be due to a loss of D2
autoreceptor function. However, in the present study, the increased phosphorylation was
associated with a stimulation of catalytic activity. Following MPTP, there is no change
in TH catalytic activity. The discrepancies between these two studies may be due to the
acute GBL model versus the chronic MPTP model. D2 autoreceptor phosphorylation
may increase TH enzymatic activity on a short-term basis, but lead to changes in protein

expression for long-term acceleration in enzymatic activity. Thus, the increased

203

phosphorylation of TH following MPTP administration may reflect a loss of D2
autoreceptor function, but compensatory increases in TH protein expression which allow
the rate of DA synthesis to be elevated, without increasing the activity of individual TH

molecules.

The Eflects of Substrate Tyrosine on TH in Activated NSDA Neurons

In the present study, administration of tyrosine had no effect on basal levels of
serine-40, serine-31 or serine 19 phosphorylated TH, total TH or TH enzymatic activity.
However, tyrosine supplementation to raclopride or GBL-treated mice resulted in
enhanced TH catalytic activity over that seen following raclopride and GBL treatment
alone. This is consistent with previous reports demonstrating that when the rate of
catecholamine synthesis is increased, tyrosine administration enhances TH activity (Sved
et al., 1979; Melamed et al., 1980; Sved and Femstrom, 1981). Moreover, the effect of
tyrosine on TH catalytic activity was associated with enhanced serine-40 and serine-19
phosphorylated TH. This is the first demonstration of substrate-enhanced TH activity
corresponding enhanced phosphorylated TH levels at two distinct serine residues in
activated NSDA neurons.

The balance of kinase activity, phosphatase activity and the amount of total TH
available determine the final degree of phosphorylated TH. As such, tyrosine may have
1) facilitated TH phosphorylation, 2) hindered TH de-phosphorylation, or 3) increased
the amount of available TH for phosphorylation. Tyrosine alone did not alter
phosphorylated TH levels or TH catalytic activity. Therefore, it seems unlikely that the

effect of substrate administration on TH occurs by enhanced kinase activity, increased

204

kinase access to phosphorylation sites, or interference with phosphatase activity under
basal conditions. It is possible, that tyrosine is able to manipulate TH
phosphorylation/de-phosphorylation only under stimulated conditions.

Another possibility is that tyrosine enhanced phosphorylated TH levels in
response to raclopride and GBL by increasing the amount of TH available for
phosphorylation. While striatal levels of total TH remained consistent in all treatment
conditions, total TH protein may not reflect the actual amount of enzyme that is available
for phosphorylation. Therefore, tyrosine may have either permitted additional TH to be
phosphorylated or prevented irreversible TH inactivation. In the case of additional TH
phosphorylation, sub-cellular fractionation studies have not been performed that would
allow for distinguishing between TH protein available for phosphorylation, versus total
TH levels in activated NSDA neurons in the presence or absence of tyrosine. The second
mechanism by which tyrosine may increase the amount of TH available for
phosphorylation is by preventing the irreversible inactivation of TH, such that it cannot
be re-phosphorylated. In this case, tyrosine may enhance phosphorylated TH levels and
TH activity in response to raclopride by preventing the long-term inactivation of TH
through destabilization. The latter mechanism is supported by the observation that in rat
striatal homogenates incubated with the protein kinase catalytic subunit (constant
stimulus), increasing tyrosine concentrations prevents the irreversible inactivation of TH

(Roskoski et al., 1990).

205

DZ Receptor Regulation of NSDA Activity Following MPTP Administration

D2 receptors regulate the activity of NSDA neurons and the rate of DA synthesis.
As such, the compensatory activation of these neurons and dysfunctional regulation of
DA synthesis following MPTP administration suggests there is a loss of D2 receptor
function. To determine if this is the case, the ability of a D2 antagonist and agonist to
increase or decrease NSDA activity, respectively, was assessed under basal conditions
and following MPTP administration. This study was also used to determine if activation
of D2 receptors could be used to decrease the activity of surviving NSDA neurons and
protect them from oxidative damage due to increased DA metabolism. To this end, mice
were pre-treated with either saline (basal conditions) or MPTP to recapitulate the
compensatory activation of NSDA neurons. Mice were then treated with either vehicle,
raclopride, or the D2 agonist quinelorane for 21 consecutive days.

The results demonstrated that under basal conditions, raclopride administration
had no effect on striatal DA metabolism, storage or NSDA activity. It is possible the
dose of raclopride was not sufficient to block D2 receptors and stimulate NSDA activity.
However, treatment with quinelorane produced a 25% decrease in striatal DA
metabolism, while having no effect on DA storage. Due to the decreased DA metabolism
and no change in DA storage, quinelorane decreased the activity of NSDA neurons by
approximately 25%. These findings suggest that chronic treatment with a DA agonist can
cause a sustained decrease in DA metabolism in NSDA neurons. Thus, DA agonist
therapy may protect NSDA neurons from oxidative damage under normal conditions.
Consistent with this, chronic DA agonist administration decreases the age-related loss of

NSDA neurons in rats (F elten et al., 1992).

206

Similar to basal conditions, in MPTP treated mice, raclopride treatment had no
effect on striatal DA metabolism. However, raclopride had a very different effect on DA
storage and NSDA activity compared to basal conditions. At the same dose of raclopride
that had no effect under basal conditions, raclopride treatment in MPTP-treated mice
caused a 50% decrease in DA storage and a robust increase in the activity of surviving
NSDA axon terminals. These findings demonstrate that D2 receptors are functional in
the striatum following MPTP administration. In fact, since the blockade of D2 receptors
results in a more robust increase in NSDA activity in MPTP-treated mice, D2 receptors
play a primary role in regulating neuronal activity under activated conditions.

Interestingly, acute raclopride administration causes an increase in DA
metabolism and a decrease in DA storage. However, chronic raclopride decreased DA
storage, but had no effect on DA metabolism. These results may suggest the dose of
raclopride was not sufficient to increase DA metabolism, however this is unlikely
because the same dose caused a 50% decrease in striatal DA storage. More likely, these
results suggest there are compensatory mechanisms that occur in NSDA neurons that
allow them to sustain an accelerated rate of DA release, in the absence of increased DA
metabolism. This is particularly relevant, since this does not occur in NSDA neurons that
have been activated in response to neurotoxic lesion. The compensatory activation of
NSDA neurons is associated with a prolonged, sustained increase in DA metabolism due
to over-activation of DA synthesis coupled with decreased storage capability. These
results therefore suggest that DA synthesis and storage remain coupled during chronic

activation of NSDA neurons induced by the loss of D2 receptor-mediated long- and

207

short-loop feedback inhibition. This may be due to either; increased VMAT-2 expression
and activity, increased TH-synaptic vesicle association, or both.

Taken together, the results from the present experiments highlight differences
between the compensatory activation of NSDA neurons following MPTP and activation
of these neurons in response to a prolonged loss of D2 receptor-mediated long and short-
loop feedback inhibition. Determining the mechanisms that occur in NSDA neurons that
permit a prolonged acceleration in neuronal activity without increasing DA metabolism
may; 1) identify targets that can be used to protect NSDA neurons from the toxic
byproducts of DA metabolism, without compromising their ability to accelerate DA
release and 2) delineate dysfunctional mechanisms in NSDA neurons that may contribute
to the pathogenesis of PD.

In MPTP-treated mice, quinelorane treatment produced a similar decrease in DA
metabolism as it did under basal conditions. However, quinelorane treatment had a
different effect on DA storage and NSDA activity than it did basal conditions. The same
dose of quinelorane that failed to alter DA storage under basal conditions decreased DA
concentrations by approximately 50% in MPTP-treated mice. Since quinelorane
decreased DA metabolism and storage there was no change in the activity of NSDA
neurons compared to mice treated with MPTP and treated with vehicle. The decrease in
DA storage likely reflects an inhibition of DA synthesis by D2 autoreceptors. The
inhibition of DA synthesis decreased the amount of newly synthesized DA available for
release and stored DA was released to compensate.

Interestingly, while the activation of D2 receptors by quinelorane did not decrease

neuronal activity, it did inhibit DA metabolism. Therefore, activation of D2 receptors

208

may protect NSDA neurons from the toxic byproducts of DA metabolism and prevent the
progressive demise of these neurons in PD. However, in the MPTP model of PD this was
not the case, since quinelorane treatment did not protect NSDA axon terminals, as
reflected by striatal DA concentrations. One potential explanation for this finding is that
quinelorane was administered after MPTP had already caused significant damage to
NSDA axon terminals. It is possible that if quinelorane was co-administered with MPTP,
the inhibition of DA metabolism may slow the progressive loss of NSDA axon terminals
caused by MPTP. A number of previous studies have supported this conclusion (Lange
et al., 1994; Youdim et al., 2000; Schapira, 2002), however, since activation of D2
receptors can also decrease DAT expression and uptake of the MPP+ neurotoxin, these
results are open to interpretation. Further experiments utilizing neurotoxins that are not
dependent on the DAT (such as rotenone) or transgenic PD models may determine if DA
agonists (due to their ability to decrease DA metabolism) are neuroprotective in animal

models of PD.

Dijfirences Between Acute and Chronic Activation of NSDA Neurons

Acute blockade of D2 receptors with raclopride increases DA metabolism,
decreases DA storage and increases the DOPAC to DA ratio. However, chronic blockade
of D2 receptors with raclopride in MPTP-treated mice had no effect on DA metabolism,
but caused a decrease in DA storage and an increase in NSDA activity. These results
highlight differences between short and long-term regulation of NSDA activity and
suggest that long-term compensatory changes in NSDA regulation allow for an increase

in neuronal activity without an increase in DA metabolism. However, these findings may

209

have been due to the low dose of raclopride used in the chronic dosing study compared to
the relatively high dose used in the acute dosing study or the effects of MPTP pre-
treatment. To determine if this is the case, mice were treated with raclopride chronically
for 21 days using a dose sufficient to activate NSDA neurons for 24 consecutive h.
Results from this study demonstrated that chronic activation of NSDA neurons
with raclopride has no effect on DA metabolism, but decreased DA storage and increased
NSDA activity. Due to the decreased concentrations of striatal DA, these results could
suggest that chronic activation of NSDA neurons caused a loss of NSDA axon terminals.
However, this is not the case since the decreased DA concentrations were only transient
and were eliminated in mice that had a 3 day drug elimination period. The findings from
this study demonstrate critical differences between acute and chronic activation of NSDA
neurons using a D2 antagonist as well as differences between chronic activation with a
D2 antagonist and the compensatory activation of NSDA neurons following neurotoxin
insult. Under normal conditions, the chronic activation of NSDA neurons is unlikely to
cause axon terminal loss, since compensatory mechanisms prevent excessive DA
metabolism. However, the MPTP-induced compensatory increase in activity results in a
chronic activation of NSDA neurons that may contribute to axon terminal loss due to un-
regulated accelerations in DA metabolism and induction of cytoplasmic DA-mediated

oxidative stress.

Summary

The results from the present studies have characterized the regulation of TH

following a loss of D2 receptor-mediated feedback inhibition. The loss of D2

210

autoreceptor-mediated short-loop feedback inhibition increases TH phosphorylation at
the axon terminal similar to the MPTP-induced compensatory activation of NSDA
neurons. These findings suggest that a loss of D2 autoreceptor regulation could underlie
the dysfimctional regulation of DA synthesis following MPTP. However, further studies
using the GBL and raclopride models of D2 autoreceptor regulation and evaluation of D2
autoreceptor expression will be necessary to delineate the specific role for short-loop
feedback inhibition in the dysfunctional regulation of DA synthesis following MPTP.
The present studies also describe critical differences in acute versus chronic
activation of NSDA neurons, induced by blockade of D2 receptor-mediated feedback
inhibition. Acute loss of feedback inhibition results in activation of NSDA neurons and
increased DA metabolism that is similar to the compensatory activation of these neurons
following neurotoxin insult. However, prolonged activation of NSDA neurons results in
compensatory mechanisms that allow DA release to remain accelerated in the absence of
increased DA metabolism. Taken together, the compensatory activation of NSDA
neurons in response to MPTP administration is unique from the chronic activation of
these neurons induced by blockade of D2 receptor-mediated feedback inhibition. Further
experiments comparing the regulation of VMAT-2 protein expression and activity as well
as the coupling between DA synthesis and storage will be crucial to understanding the
differential activation of NSDA neurons in response to axon terminal loss versus the loss
of D2 receptor mediated feedback inhibition. These studies will be important for
identifying compensatory mechanisms that allow NSDA neuronal activity to be

accelerated without a corresponding increase in DA metabolism.

211

Chapter 7. The Role of a-Synuclein in MPTP-Induced Neurotoxicity

and the Compensatory Activation of NSDA Neurons

A. Introduction

a-Synuclein is a protein that is distributed ubiquitously throughout the brain and
found primarily in axon terminals (Nakajo et al., 1993; lakes et al., 1994). a-Synuclein is
a relatively small protein with 140 amino acids and a molecular weight of 19 kDa. The
biochemical structure predicts that this protein may be a molecular chaperone capable of
binding to other intracellular proteins. Within the first 87 residues of the 140 amino acid
sequence there are repetitive amino acid motifs (KTKEGV) with a strict periodicity of 11
amino acids. These repeats are reminiscent of amphipathic helices in apolipoproteins that
determine protein hydrophobicity and facilitate membrane binding (George et al., 1995).
a-Synuclein exists primarily in the cytosolic fraction as a natively unfolded protein
(Ostrerova et al., 1999; Takenouchi et al., 2001). However, this protein can readily
associate with membranes (McLean et al., 2000) and take on a a-helical conformation
upon binding to synthetic lipid membranes (Davidson et al., 1998; Jo et al., 2000).

Several lines of evidence suggest that a-synuclein is critical to NSDA neuronal
function. Degeneration of NSDA neurons is associated with the clinical motor deficits of
PD and inappropriate aggregation of a-synuclein likely plays a causative role in NSDA
cell death. Mutations, duplications, or triplications in the a-synuclein gene occur in
familial forms of PD and result in the degeneration of NSDA neurons (Polymeropoulos et
al., 1997; Kruger et al., 1998; Singleton et al., 2003). Also, degenerating NSDA neurons

in brain tissue from post-mortem PD patients contain cytoplasmic aggregations of protein

212

termed Lewy-bodies. a-Synuclein is the primary structural component of Lewy-bodies.
The roles that a—synuclein aggregation and Lewy-body formation play in NSDA neuronal
degeneration are not known, but it is clear these protein aggregates overlap PD
pathogenesis. Taken together, it is apparent that understanding the function of normal a-
synuclein in NSDA neurons will greatly enhance the relatively unknown pathology of
PD.

Comparing the function of NSDA neurons that express a-synuclein to those
lacking this protein provides a usefiil tool for determining the function of u-synuclein.
To this end, our laboratory generated a stable breeding colony of homozygous a-
synuclein knock-out mice. Mice originally obtained in breeding pairs from Jackson
Laboratories (B6;129 X-SNCA‘m'R‘m') were crossed with wild type C57/Bl6 mice to
obtain heterozygote mice (Fl). Heterozygote mice were then crossed and the offspring
were used to generate a stable breeding colony of age-matched wild-type and a-synuclein
knock-out mice. The generation, viability, and anatomical characteristics of these mice
have been described previously (Abeliovich et al., 2000). However, NSDA neuronal
physiology in these mice has not been thoroughly studied.

a-Synuclein knock-out mice are virtually indistinguishable from wild-type mice
using several indices of NSDA neuron function. a-Synuclein knock-out mice possess
normal complements of NSDA cell bodies and synapses, and there is no difference in
electrical stimulus-evoked DA release compared to wild-type mice (Abeliovich et al.,
2000). Thus, it is unlikely that a-synuclein plays an essential role in maintaining NSDA
activity under basal conditions. a-Synuclein, however, may be important in the

regulation of activated NSDA neurons. Evidence suggests a-synuclein serves as an

213

activity dependent regulator of axon terminal function. While there is no difference
electrophysiological measurements of synaptic transmission in the striatum under simple
electrically stimulated conditions, following paired pulse trains of electrical stimuli, a-
synuclein knock-out mice display enhanced neurotransmitter release (Abeliovich et al.,
2000). This suggests that a-synuclein is important to neuronal physiology under
activated conditions.

Administration of MPTP is a useful pharmacological model for evaluation of
NSDA neuron fimction under activated conditions which results from the compensatory
increase in the activity of surviving neurons. To determine if a-synuclein plays a role in
regulating NSDA activity under activated conditions, u-synuclein knock-out mice were
treated with prolonged-chronic MPTP and DA metabolism, storage and the activity of
surviving NSDA neurons were measured in the striatum. Results from this initial
experiment suggested that mice lacking a-synuclein had a deficit in NSDA neuronal
response to MPTP administration. Since the compensatory activation of NSDA neurons
is known to be due to dysfunctional regulation of DA synthesis, these experiments
suggested a-synuclein may play a role in regulating TH. To determine if this is the case,
regulation of TH phosphorylation state, enzymatic activity and expression were assessed
in the striatum following either; raclopride-induced loss of D2 receptor-mediated

feedback inhibition, or MPTP-induced compensatory activation of NSDA neurons.

214

B. Experimental Design and Results
Prolonged-Chronic MPTP Administration in a-Synuclein Knock-out Mice

To determine if a-synuclein plays a role in regulating NSDA activity under
activated conditions, mice lacking u-synuclein were injected with either saline (1 mllkg,
s.c.) or MPTP (1, 5, 10 or 20 mg/kg, s.c.) twice a week for 5 consecutive weeks.
Probenecid (250 mg/kg, i.p.) was co-administered with saline and MPTP injections.
Mice were sacrificed 3 weeks following the last MPTP injection. Striatal DOPAC and

DA concentrations were measured using HPLC-EC.

215

The Eflects of Prolonged-Chronic MPT P Administration on NSDA Axon Terminals in a-
Synuclein Knock-out Mice

In a-synuclein knock-out mice prolonged-chronic MPTP administration at the
highest dose (20 mg/kg) decreased striatal DA concentrations approximately 60%
compared to saline-treated mice (Figure 7-1, A). MPTP administration at this dose also
caused a decrease in DOPAC concentrations in the striatum (Figure 7-1, B). The
decrease in striatal DOPAC concentrations was equivalent to the loss of striatal DA
following MPTP, such that there was no change in the DOPAC to DA ratio in a-
synuclein knock-out mice treated with MPTP (Figure 7-1, C). Despite a loss of
approximately 60% of striatal DA concentrations, there was no change in striatal VMAT-

2 protein levels following MPTP (Figure 7-2).

216

 

 

A_ Axon Terminal Loss

DA (nglmg)

Saline 1 5 10 20

Striatal DA Metabolism

DOPAC (nglmg) EU
8 .

 

Saline 1 5 10 20

NSDA Activity

DOPAC/ DA (ratio) 0

F3
o
o

Saline 1 5 1 O 20

Figure 7-1. Dose response effect of prolonged-chronic MPTP administration on DA (A)
and DOPAC (B) concentrations and the DOPAC to DA ratio (C) in the striatum of a-
synuclein knock-out mice. Mice were treated with either MPTP (1 , 5, 10 or 20 mg/kg,
s.c.) or its saline vehicle (1 ml/kg, s.c.) every 3.5 days for 5 consecutive weeks and killed
by decapitation 3 weeks following the last injection. Probenecid (250 mg/kg, i.p.) was
co-administered with each injection of saline or MPTP. Columns represent means of
groups i S.E.M. of six to eight determinants. (*) indicate a significant difference from
saline treatment group. (p S 0.05).

217

 

 

   

 

 

Striatal VMAT-2

60'
D40-
0
n:

20"

0

 

 

 

 

Saline 10 20
Dose of MPTP (mg/kg)

Figure 7-2. Dose-response effect of prolonged-chronic MPTP administration on striatal
VMAT-2 immunoreactivity. Mice were injected with either MPTP (10 or 20 mg/kg, s.c.)
or its saline vehicle (1.0 ml/kg, s.c.) every 3.5 days for 5 consecutive weeks and killed by
decapitation 3 weeks following the last injection. Probenecid (250 mg/kg, i.p.) was co-
administered with each injection of saline or MPTP. Striatal VMAT-2 was measured
using Western blot analysis and are shown as relative density units (RDU). Columns
represent means of groups :I: S.E.M. of six to eight determinants.

218

Regulation of T H Activity in a-Synuclein Knock-out Mice Following Raclopride-Induced
Loss of D2 Receptor Mediated Feedback Inhibition

To determine if a-synuclein plays a role in regulating DA synthesis under basal
and/or activated conditions, a-synuclein knock-out mice were injected with either saline
or raclopride, in the presence or absence of supplemental tyrosine. Mice were injected
with either saline (1 ml/kg, i.p.) or saline plus tyrosine (100 mg/kg, i.p.) to simulate basal
conditions. Other groups of mice were injected with either raclopride (1.0 mg/kg, i.p.)
plus vehicle, or raclopride plus tyrosine to simulate activated conditions. Mice were
sacrificed 60 min after drug administration. NSDA activity was determined by
measuring striatal DOPAC and DA concentrations using HPLC-EC. To determine if a-
synuclein regulates TH catalytic activity, a separate experiment was performed in which
mice were administered either saline, tyrosine, raclopride or tyrosine plus raclopride as
described above and killed 60 min following injection. All mice were injected with
NSD-1015 (100 mg/kg, i.p.) 30 min prior to sacrifice. TH phosphorylation state was
measured using Western blot analysis. Striatal DOPA concentrations were measured

using HPLC-EC as an index of TH catalytic activity.

219

The Eflect of Acute Raclopride on NSDA Activity in a-Synuclein Knock-out Mice

As depicted in Figure 7-3 (A), tyrosine administration alone had no effect on DA
metabolism, as reflected by striatal DOPAC concentrations. Raclopride administration
alone caused a 4-fold increase in striatal DA metabolism. Tyrosine supplementation in
raclopride-treated mice further increased striatal DA metabolism compared to mice
treated with raclopride alone. As depicted in Figure 7-3 (B), tyrosine administration
alone slightly increased basal levels of striatal DA storage, as reflected by concentrations
of DA. Raclopride administration decreased striatal DA storage by approximately 25%
in normal and tyrosine supplemented mice. As depicted in Figure 7-3 (C), tyrosine
administration had no effect on NSDA activity, as reflected by the DOPAC to DA ratio.
Raclopride administration alone caused a S-fold increase in NSDA activity and tyrosine
supplementation caused a further 30% increase in the DOPAC to DA ratio over mice

treated with raclopride alone.

220

 

>

Striatal DA Metabolism

 

 

 

   

100 -
g 7.
5
3 50
a.
8 25
o i
Saline Saline Raclopride Raclopride
+ + + 4»
Vehicle Tyrosine Vehicle Tyrosine
B. Striatal DA Storage
350 - *
300 l
A 250 l-
E zoo i *
f 150 -
0 1“) l-
50 b
o
Saline Saline Raclopride Raclopride
+ + + 4-

Vehicle Tyrosine Vehicle Tyrosine

.0

0.50 _ NSDA Activity

9
S

*

_o
8

DOPAC (nglmg)
B

.o .0
an.
O

 

 

p
8

Saline Saline Raclopride Raclopride
+ + +

...

Vehicle Tyrosine Vehicle Tyrosine

Figure 7-3. Concentrations of DOPAC (A), and DA (B), and the DOPAC/DA ratio (C)
in the striatum of a-synuclein knock-out mice 60 min after administration of either saline
(1 ml/kg, i.p.) + vehicle (saline 1 ml/kg, i.p.), saline + tyrosine (100 mg/kg, i.p.),
raclopride (1.0 mg/kg, i.p.) + vehicle, or raclopride + tyrosine. Columns represent means
of groups :i: S.E.M. of seven mice per group. (*) Values significantly different from
saline treated controls. # Values significantly different from raclopride + vehicle-treated
mice. p50.05.

221

The Eflect of Acute Raclopride Administration on TH Phosphorylation State in a-
Synuclein Knock-out Mice

As depicted in Figure 7-4 (A), tyrosine administration alone had no effect on
basal levels of serine-19 phosphorylated TH. Raclopride administration alone produced
an approximately 1.5-fold increase in serine-l 9 phosphorylated TH levels. Tyrosine plus
raclopride administration did not further increase serine-19 phosphorylated TH levels
over mice treated with raclopride alone. As depicted in Figure 7-4 (B), tyrosine
administration alone did not change basal levels of serine-31 phosphorylated TH.
Raclopride administration alone produced an approximately 1.5-fold increase in serine-31
phosphorylated TH levels. Tyrosine supplementation in raclopride-treated mice did not
affect serine-31 phosphorylated TH levels compared with raclopride treatment alone. As
depicted in Figure 7-4 (C), tyrosine administration alone had no effect on basal levels of
serine-40 phosphorylated TH. Raclopride administration alone produced an
approximately 6-fold increase in serine-40 phosphorylated TH. Tyrosine plus raclopride
administration did not further increase serine-40 phosphorylated TH levels over

raclopride treatment alone.

222

 

 

 

   

 

     

10° _ Senna-19
80 l-
3 60 *
0
n: 40
20
0
Saline Saline Raclopride Raclopride
+ + + 4»
Vehicle Tyrosine Vehicle Tyrosine
B. .
25 i Serine-31
20 ~ * *
D .
D 15
n:
10 i'
5 P
Saline Saline Raclopride Raclopride
+ + + +
Vehicle Tyrosine Vehicle Tyrosine
C. Serine-40
200 r * *
D 150 i-
0
0: 100 i-
50 r

 

 

Saline Saline Raclopride Raclopride
+ + + 4-
Vehicle Tyrosine Vehicle Tyrosine

Figure 7-4. Serine-19 (A), serine-31 (B) and serine-40 (C) phosphorylated TH levels in
the striatum of a-synuclein knock-out mice treated with either saline (1 ml/kg, i.p.) +
vehicle (1 ml/kg, i.p.), saline + tyrosine (100 mg/kg, i.p.), raclopride (1.0 mg/kg, i.p.) +
vehicle, or raclopride + tyrosine. Mice were killed 1 hr afier drug administration.
Columns represent means of groups :I: S.E.M. of seven mice/group. * indicate a
significant difference from saline + vehicle-treated mice. p S 0.05.

223

The Efihct of Acute Raclopride Administration on TH Enzymatic Activity and Protein
Expression I

As depicted in Figure 7-5, tyrosine administration alone did not change basal TH
catalytic activity, as reflected by striatal DOPA concentrations. Raclopride
administration alone produced an approximately 3-fold increase in TH catalytic activity
and tyrosine supplementation did not further increase TH catalytic activity compared to
mice treated with raclopride alone. As depicted in Table 7-1, levels of total TH did not

vary in response to any treatment condition.

224

TH Enzymatic Activity

 

 

A 40 * *
a:
E 30
B:
5 20
E
o 10
D
o .
Saline Saline Raclopride Raclopride
+ + + +

Vehicle Tyrosine Vehicle Tyrosine

Figure 7-5. Concentrations of DOPA in the striatum of a-synuclein knock-out mice after
administration of either saline (1 ml/kg, i.p.) + vehicle (saline, 1 ml/kg, i.p.), saline +
tyrosine (100 mg/kg, i.p.), raclopride (1.0 mg/kg, i.p.) + vehicle, or raclopride + tyrosine.
All mice received an injection ofNSD-1015 (100 mg/kg, i.p.) 30 minutes prior to
decapitation. Columns represent means of groups :I: S.E.M of seven mice per group. (*)
Values significantly different from saline treated controls. pS0.05.

225

 

Saline Saline Raclopride Raclopride
+ + + +

Vehicle Tyrosine Vehicle Tyrosine

THProtein 31:4 39:4 29:4 31:3
(RDU)

 

Table 7-1. Total TH levels measured in relative density units (RDU) in the striatum of a-
synuclein knock-out mice after administration of either saline (1 ml/kg, i.p.) + vehicle
(saline, 1 ml/kg, i.p.), saline + tyrosine (100 mg/kg, i.p.), raclopride (1.0 mg/kg, i.p.) +
vehicle, or raclopride + tyrosine. Values represent means of groups i S.E.M of seven
mice per group.

226

Regulation of TH Activity in a-Synuclein Knock-out Mice Following Sub-Chronic MPTP
Administration

To determine if a-synuclein plays a role in regulating DA synthesis in NSDA
axon terminals activated in response to MPTP administration, o-synuclein knock-out
mice were treated with saline (1 ml/kg, s.c.) or MPTP (25 mg/kg, s.c.) once a day for 5
consecutive days. Mice were sacrificed 3 days afier the last injection. NSDA activity
was measured by determining striatal DOPAC and DA concentrations, and the DOPAC
to DA ratio. In a separate experiment, mice were injected with either saline or MPTP as
described above and 30 min prior to sacrifice mice were injected with NSD-1015 (100
mg/kg, i.p.). The rate of DA synthesis as well as TH phosphorylation state, enzymatic
activity and protein expression were measured in the striatum as described above. To
determine if a-synuclein plays a role in regulating the sub-cellular distribution of TH at
the axon terminal, a subsequent experiment was conducted in which a-synuclein knock-
out mice were injected with either saline (1 ml/kg, s.c.) or MPTP (25 mg/kg, s.c.) once a
day for 5 consecutive days. Mice were killed 3 days afier the last injection. The striatum
was freshly dissected and prepared for sub-cellular fractionation as described previously
in Chapter 5. Striatal protein was separated into cytoplasmic, early endosomal, synaptic
vesicle and plasma membrane fractions using differential centrifugation. TH protein

located in each fraction was determined using Western blot analysis.

227

Eflects of Sub-Chronic MPTP Administration on NSDA Axon terminals in a-Synuclein

Knock-out Mice

In a-synuclein knock-out mice sub-chronic MPTP administration caused a 75%
loss of NSDA axon terminals, as reflected by striatal DA concentrations (Figure 7-6, A).
MPTP also caused a 65% decrease in striatal DA metabolism, as reflected by striatal
DOPAC concentrations (Figure 7-6, B). There was a slight increase in the activity of
NSDA neurons in the striatum following MPTP administration, as reflected by the

DOPAC to DA ratio (Figure 7-6, C).

228

.>

Axon Terminal Loss

J *
w -__

Saline MPTP

150

DA (no/Lug)
‘o” 8

 

 

O

Striatal DA Metabolism

J *
-_

.3
M

DOPAC (nglmg) ED

 

 

 

 

4
O
Saline MPTP
C. NSDAActivity
is} 0.12 -
13; *
< 008 -
Q
o
8 000
Saline MPTP

Figure 7-6. Effects of sub-chronic MPTP administration on striatal DA (A) and DOPAC
(B) concentrations and DOPAC/DA ratios (C) in a-synuclein knock-out mice. Mice were
treated with saline (1.0 ml/kg, s.c.) or MPTP (25 mg/kg, s.c.) once a day for five
consecutive days and killed by decapitation three days following the last injection.
Columns represent means of groups :l: S.E.M. of six to eight determinants. (*) indicate a
significant difference from saline treatment group. (PS 0.05).

229

 

 

 

 

 

The Rate of Striatal DA Synthesis under Basal and Activated Conditions

Sub-chronic MPTP administration caused a decrease in striatal DOPA (Figure 7-
7, A) and DA concentrations (Figure 7-7, B). However, the loss of striatal DA
concentrations was much more severe than the loss of DOPA concentrations. As such,
MPTP caused a robust increase in the ratio of DOPA to DA in the striatum of mice

treated with MPTP (Figure 7-7, C).

230

.>

TH Catalytic Activity

   

     

 

20
E 15 *
g 10
E
8 5
o
Saline MPTP
3 NSDA Axon Terminal Loss
150
3120
E
E” 90
I; 60
O 30
o
Saline MPTP
C. Rate of Striatal DA Synthesis
A 0.20 *
.9
g 0.15
E 0.10
E
o 0.05
D
0.00

   

Saline MPTP

Figure 7-7. Striatal DOPA (A) and DA (B) concentrations, and the DOPA to DA ratio in
a-synuclein knock-out mice injected with either saline (1.0 ml/kg, s.c.) or MPTP (25
mg/kg, s.c.) once a day for 5 consecutive days and killed by decapitation 3 days
following the last injection. DOPA concentrations were determined 30 min after
injection with NSD—lOl 5 (100 mg/kg, i.p.). Columns represent means of groups :t
S.E.M. of seven determinants. (*) indicate a significant difference from saline treatment
group. (P5 0.05).

Effects of MPT P Administration on TH Phosphorylation State in a-Synuclein
Knock-out Mice

Sub-chronic MPTP administration caused a differential loss of serine-19, serine-
31, and serine-40 phosphorylated TH (Table 7-2). MPTP caused a 50% decrease in
serine-l9 and serine-31 phosphorylated TH levels. However, there was no change in
serine-40 phosphorylated TH levels in the striatum of mice treated with MPTP as
compared to saline-treated mice. Phosphorylated serine-19, serine-31, or serine-40 TH
levels were normalized to total TH protein levels in the striatum (Table 7-3), as an index
of the relative proportion of TH phosphorylated at each residue (Figure 7-8). MPTP
decreased the proportion of TH phosphorylated at serine-l9 and serine-31 by
approximately 40%. MPTP had no effect on the proportion of serine-40 phosphorylated

TH in NSDA axon terminals of a-synuclein knock-out mice treated with MPTP.

232

 

 

 

 

Serine-1 9 Serine-31 Serine-40

(RDU) (RDU) (RDU)

same 118103 e1:o7 23:04
*

MPTP 6.4 : 0.3 3.1: 05* 1.3 : 0.3

 

Table 7-2. Quantification of the effect of either saline (1 ml/kg, s.c.) or MPTP (25
mg/kg, i.p.) on serine-19, serine-31, or serine-40 phosphorylated TH in the striatum of a-
synuclein knock-out mice. Columns represent mean i: S.E.M. of seven mice/group. *
indicates a significant decrease from saline-treated controls. pS 0.05.

233

 

Striatal TH Protein
(RDU)

Saline 49 i 4

MPTP 33 : 2 *

 

Table 7-3. Quantification of the effect of either saline (1 ml/kg, s.c.) or MPTP (25
mg/kg, i.p.) on striatal TH protein levels in a-synuclein knock-out mice. Columns
represent mean :l: S.E.M. of seven mice/group. * indicates a significant decrease from
saline-treated controls. p5 0.05.

234

Serine-19

*

Ser19/Total TH (ratio)

 

Saline MPTP

8. Serine-31

.3
o

Ser31/Total TH (ratio)
0 M b O O

Saline MPTP

Serine-40

Ser40frotal TH (ratio)
A

Saline MPTP

Figure 7-8. Quantification of the effect of MPTP (25 mg/kg, s.c.) on the relative
proportion of serine-19 (A), serine-31 (B), or serine-40 (C) phosphorylated TH compared
to total TH in the striatum. Phosphorylated serine-19, 31, or 40 phosphorylated TH
relative density units were normalized to total TH relative density units. Columns
represent mean :t S.E.M. of seven mice/group. * indicates a significant decrease from
saline-treated controls. p5 0.05.

235

Effects of MPT P Administration on TH Catalytic Activity and Protein Expression in a-
Synuclein Knock-out Mice

Sub-chronic MPTP administration caused a decrease in striatal DOPA
concentrations (Figure 7-7, A) and TH protein expression (Table 7-3). The decrease in
DOPA concentrations paralleled that of TH protein. As such, MPTP administration did
not alter the catalytic activity of TH, as reflected by the ratio of DOPA to TH protein
(Figure 7-9, LEFT PANEL).

MPTP caused a decrease in striatal TH protein (Table 7-3) however the decrease
in TH protein expression was not as robust as the loss of striatal DA concentrations
(Figure 7-7, B). As such, sub-chronic MPTP administration caused a marked increase in
TH protein expression in surviving axon terminals, as reflected by the ratio of TH to DA

(Figure 7-9, RIGHT PANEL).

236

TH Enzymatic Activity 15° TH Protein Expression*

DOPA/TH (ratio)

9 p .0

8 8 6
mouse)

,o
..
o

   

p
8

Saline MPTP Saline MPTP

Figure 7-9. LEFT PANEL: TH enzymatic activity in the striatum of a-synuclein knock-
out mice treated with either saline (1 ml/kg, s.c.) or MPTP (25 mg/kg, s.c.). TH
enzymatic activity was determined by normalizing striatal DOPA concentrations,
determined through neurochemical analysis to striatal TH protein content, determined
through Western blot analysis. Striatal DOPA accumulation was measured 30 minutes
following injection ofNSD-1015 (100 mg/kg, i.p.). RIGHT PANEL: TH protein content
in the striatum of mice treated with either saline or MPTP. TH protein content was
determined by normalizing striatal TH protein levels, determined through Western blot
analysis, to striatal DA concentrations, determined through neurochemical analysis.
Columns represent means i S.E.M. of seven mice/group. * indicate a significant
difference from saline treated mice. p: 0.05.

237

Effects of Tyrosine Administration on TH Catalytic Activity in a-Synuclein Knock-out
Mice Pre-Treated Under Basal and Activated Conditions

As depicted in Figure 7-10, tyrosine administration 60 min prior to decapitation
had no effect on the enzymatic activity of TH in mice pre-treated with either saline or

MPTP, as reflected by the ratio of striatal DOPA concentrations to total TH.

238

.>

Striatal DOPA Concentration

20
a 15
5 *
(5 1o *
D.
8 5
Saline Saline MPTP MPTP
+ + + +
Vehicle Tyrosine Vehicle Tyrosine
B. 75 Striatal TH Protein
5 50
o
g; * *
E 25
Saline Saline MPTP MPTP
+ + + +
Vehicle Tyrosine Vehicle Tyrosine

TH Enzymatic Activity

   

Saline Saline MPTP MPTP
+ + + +
Vehicle Tyrosine Vehicle Tyrosine

Figure 7-10. The effects of vehicle (1 .0 ml/kg, i.p.) or tyrosine (100 mg/kg, i.p.) on
striatal DOPA concentrations (A), TH protein (B) and the DOPA to TH ratio in a-
synuclein knock-out mice treated with either saline (1 ml/kg, s.c.) or MPTP (25 mg/kg,
s.c.) once a day for 5 consecutive days and killed by decapitation 3 days afier the last
injection. Striatal DOPA concentrations were measured 30 min following NSD-1015
administration (100 mg/kg, i.p.). Columns represent means 3: S.E.M. of seven
mice/group. * indicate a significant difference from saline vehicle treated control mice.

p: 0.05.

239

The Eflects of MPT P Administration on the Sub-Cellular Localization of T H at the Axon
Terminal of a-Synuclein Knock-out Mice

Sub-chronic MPTP administration caused a differential loss of TH protein in the
striatum depending on which sub-cellular compartment the enzyme was associated with
(Table 7-4). MPTP had no effect on TH protein expression in the cytoplasmic fi'action.
However, it caused a 23% decrease in TH protein associated with the early endosomal
fraction, and a 25% decrease in TH associated with the synaptic vesicle fraction. The
loss of TH associated with plasma membrane fraction was more severe. MPTP caused a
60% decrease in TH associated with the plasma membrane enriched fiaction. To
quantify the relative change in TH localization relative to total TH at the axon terminal,
TH in each fi'action was normalized to total striatal TH. Sub-chronic MPTP increased the
proportion of TH associated with the cytoplasmic fraction, but had no effect on TH
associated with the early endosomal or synaptic vesicle fractions. MPTP caused a

decreased in TH associated with the plasma membrane enriched fraction (Figure 7-11).

240

 

Cytoplasmic Endosomal Synaptic Vesicle Plasma Membrane

 

(RDU) (RDU) (RDU) (RDU)

Saline 15:2 62:8 16:2 20:2
* * *

MPTP 13:1 48:2 12:1 8:1

 

Table 7-4. Quantification of the TH protein distribution in the striatum of a-synuclein
knock-out mice treated with either saline (1 ml/kg, s.c.) or MPTP (25 mg/kg, s.c.).
Differential centrifugation was used to isolate TH associated with sub-cellular fractions
enriched in either cytosolic, early endosomal vesicles, synaptic vesicles, or plasma
membrane protein. TH associated with each compartment was normalized to proteins
enriched in each fraction B-III tubulin, early endosome antigen 1 (EEA1), synaptophysin,
or Snap-25, respectively to control for variations in loading. Values represent means :
S.E.M. of four samples per group. * indicate a significant difference from saline treated
mice. p S 0.05.

241

   

   

A- Cytosolic TH 8- Endosomal TH

E 0.75 * g ”0

g 5 1.2)

E 0.50 E '~°°

E g 08)

O t 0.”

E 025 E m

*5 -§ 0.20

O 0.00 i" 0.00

Saline MPTP Saline MPTP

C. Synaptic Vesicle TH D. plasma Membrane TH

E 0.60 .. 05°

2 g m

E 0'40 E can *

5 r

° 020

E 0.20 E

>: E 0.10

"’ 0.00 0.00

Saline MPTP Saline MPTP

Figure 7-11. The effects of MPTP (25 mg/kg, s.c.) on the proportion of TH associated
with sub-cellular fractions enriched in either cytoplasmic (A), early endosomal vesicles
(B), synaptic vesicles (C) or plasma membrane (D) protein in the striatum of a-synuclein
knock-out mice. The relative amount of TH protein associated with each sub-cellular
fraction was determined by normalizing TH in each fraction to total striatal TH.
Columns represent means : S.E.M. of four samples per group. * indicate a significant
difference from saline treated mice. p S 0.05.

242

D. Discussion
Prolonged-Chronic MPTP Toxicity in a-Synuclein Knock-out Mice

Specific deletion of the o-synuclein gene by homologous recombination
attenuates the destruction of nigrostriatal DA neurons following acute and sub-chronic
MPTP administration (Dauer et al., 2002; Schluter et al., 2003). However, Schliiter et a1.
(2003) also reported that mice with a spontaneous deletion of a 2 cM region surrounding
the native a-synuclein gene have decreases in striatal DA concentrations following a
single dose of MPTP that is similar to wild-type controls. This latter finding raises the
possibility of confounding differences in background strain as an alternative explanation
for the attenuated response to MPTP seen in the a-synuclein null mice generated by
homologous recombination.

The results of this study provide evidence that mice lacking a-synuclein have an
attenuated response to prolonged, chronic MPTP administration. Axon terminal
integrity, as reflected by striatal DA concentrations, demonstrate that a-synuclein knock-
out mice have attenuated responses to prolonged, chronic systemic MPTP exposure when
compared to wild-type control mice. Using a MPTP administration protocol that more
closely mimics the chronic time course of neurodegeneration neurodegeneration seen in
PD, our findings are in agreement with previous studies suggesting that selective deletion
of a-synuclein gene expression from either insertion of a stop codon prior to a start
codon, or the deletion of the a-synuclein gene with targeting vectors confers resistance to
either acute, sub-chronic and now prolonged-chronic MPTP administration (Dauer et al.,

2002; Schluter et al., 2003).

243

Despite supporting the hypothesis that o—synuclein is involved in MPTP-induced
toxicity, the present study does not provide an explanation why mice with a spontaneous
2 cM genomic deletion in a region containing the o-synuclein gene have a similar
response to MPTP as wild-type mice. The present study alone cannot exclude the
possibility of an unknown variation in background strain between the wild-type and a-
synuclein knock-out mice as an alternative explanation for the differential response to
MPTP described herein.

An attenuated response to MPTP however, has now been replicated independently
in three unique strains of mice with absent a-synuclein expression. Taken together, it is
more plausible that the resistance to MPTP is directly related to the loss of a-synuclein
and less likely due to an unaccounted variation in background strain coincidentally
occurring in three independent strains of a-synuclein knock-out mice. Nevertheless, the
fact that the spontaneous deletion of a segment of the genome containing the a-synuclein
gene does not result in altered sensitivity to acute MPTP exposure is an important finding
and might suggest the presence of an adjacent modifying gene that could also influence
the response to MPTP or interact with a-synuclein.

The activity of DA neurons in a-synuclein knock-out mice was assessed using the
ratio of DOPAC to DA in the striatum. Following treatment with doses of MPTP that
sufficiently deplete striatal DA levels, wild-type mice have an increase in the
DOPAC/DA ratio (Chapter 3). The increase in the ratio of DOPAC to DA observed in
wild-type mice following MPTP exposure reflects increased metabolism of newly
synthesized DA due to the dysfunctional regulation of DA synthesis. Interestingly, a-

synuclein knock-out mice do not have an increase in the DOPAC/DA ratio following a

244

loss of greater than 50% of striatal DA innervation. One potential explanation is that
following MPTP there is a similar deficit in regulating DA synthesis in a-synuclein
knock-out mice however, the newly synthesized DA does not have access to the
mitochondria, where it is metabolized by MAO to DOPAC. This could also account for
the attenuated response to MPTP.

Similar to DA, there may be an inability of MPP+ to reach mitochondrial complex
1, its primary site of neurotoxic action (Dauer et al., 2002). However, this is not the case
since following MPTP administration to wild-type and a-synuclein knock-out mice,
mitochondrial complex I activity is inhibited to the same extent (Fomai et al., 2005). A
more likely explanation for the lack of a compensatory increase in NSDA activity is that
following MPTP the rate of DA synthesis remains coupled with vesicular storage
capability. Thus, in a-synuclein knock-out mice, there may not be an over-activation of
DA synthesis, or there is an enhanced ability to package the increase in newly
synthesized DA.

Despite a significant loss of DA concentrations, mice lacking a-synuclein do not
have a loss of VMAT-2 protein in the striatum. Using VMAT-2 as an indicator of DA
vesicular storage capacity, there is a discrepancy between DA and VMAT-Z
concentrations in a-synuclein knockout mice following MPTP. Therefore, it is possible
that the lack of a compensatory increase in NSDA activity in a-synuclein knock-out mice
is due to an increased storage capability for newly synthesized DA in these mice.
However, it is unclear whether VMAT-Z protein levels reflect actual activity of the

transporter and storage capability in vivo. The lack of a compensatory increase in NSDA

245

activity following MPTP could also be explained by an inability to increase the rate of

DA synthesis.

The Regulation of NSDA Activity in a-Synuclein Knock-out Mice

a-Synuclein interacts with numerous proteins that play a role in medating the
compensatory activation of NSDA neurons. For example, a-synuclein inhibits the
activity of phospholipase D2, which may regulate monoaminergic vesicle content and
axon terminal DA storage (Jenco et al., 1998; Lotharius and Brundin, 2002). a-Synuclein
expression also alters DAT-mediated uptake of synaptic DA, suggesting a role in
terminating DA neurotransmission (Sidhu et al., 2004). Finally, a-synuclein regulates the
catalytic activity of TH, the rate-limiting enzyme in DA synthesis (Perez et al., 2002).
Taken together, there is ample evidence that a-synuclein may play a critical role in
mediating the uncoupling between DA synthesis and storage capacity that occurs
following MPTP administration.

To determine if a-synuclein plays a role in regulating DA synthesis, TH
phosphorylation state, catalytic activity and protein expression were assessed in the
striatum of a-synuclein knockout mice under basal conditions and during accelerated
neuronal activity. Mice were injected with raclopride, which increases DA neuronal
firing and stimulates DA synthesis, by decreasing D2 receptor-mediated long- and short-
loop feedback inhibition (Chapter 6). Supplemental tyrosine was administered to prevent
substrate limitations on TH activity in activated NSDA neurons (Sved and Femstrom,

1981).

246

Under basal conditions, DA storage, release, reuptake and metabolism were
similar in NSDA neurons of a-synuclein knock-out mice as in wild-type mice (Chapter
6). Therefore, it appears that a-synuclein does not affect DA release, reuptake and
metabolism or DA storage in NSDA neurons under basal conditions.

Raclopride increases NSDA neuronal activity through a loss of long and short-
loop feedback inhibition. The loss of feedback inhibition increases NSDA cell firing,
bursts of action potentials and DA release at the axon terminal (Andersson et al., 1994;
Andersson et al., 1995; Millan et al., 1998). In the present study, raclopride increased
NSDA neuronal activity in o-synuclein knockout mice to a similar extent as wild-type
mice (Chapter 6), as demonstrated by an increase in the ratio of DOPAC to DA. These
results suggest both long and short-loop feedback mechanisms are not affected by a-
synuclein. This finding is consistent with previous work demonstrating that there is no
difference in DA neurotransmission between wild-type and a-synuclein knockout mice in
response to either single-pulse or train electrical stimulation of NSDA axon terminals
(Abeliovich et al., 2000). There is an increased recovery time from a paired-pulse
depression paradigm in o-synuclein knockout mice, suggesting that a—synuclein may play
a role in the refilling of the readily releasable pool of synaptic vesicles. Although this has
implications for the present study, the differences in the time frame of sample collection
and methodological considerations in determination of DA release prevent direct

comparison of the paired-pulse depression electrical stimulation paradigm.

247

The Regulation of DA Synthesis in a-Synuclein Knock-out Mice

Under basal conditions, levels of serine-1 9, serine-31 and serine-40 as well as TH
enzymatic activity were similar in a-synuclein knock-out mice as in wild-type mice
(Chapter 6). This is in contrast to a previous report that a-synuclein inhibits the
phosphorylation and consequently the activity of TH in immortalized MN9D cells under
basal conditions (Perez et al., 2002). The discrepancy in findings may stem from the
ability of a-synuclein to bind to TH. The initial report demonstrated a clear deficit in TH
phosphorylation and activity when either wild-type or mutant (A53T) a-synuclein was
over-expressed in MN9D cells. Over-expression of the protein corresponded with a
dramatic increase in TH and u-synuclein co-immunoprecipitation. Therefore, over-
expression of wild-type or mutant a-synuclein may have resulted in non-physiological
protein—protein interactions that could have prevented kinase access to proper
phosphorylation sites, resulting in decreased enzyme phosphorylation and consequently
catalytic activity. In addition, in vitro studies may lack key microenvironmental factors
or long-loop neuronal feedback mechanisms that are physiologically relevant.
Regardless, in the present study there was no difference between wild-type and a-
synuclein knockout mice in NSDA neuronal phosphorylated TH levels
or enzyme catalytic activity in vivo under normal physiological conditions. This suggests
that a-synuclein does not play a role in regulating the phosphorylation state or catalytic
activity of TH under basal levels of neuronal activity.

TH activation occurs predominantly through phosphorylation of the serine-40
residue (Kumer and Vrana, 1996), which serves to couple neuronal activity with DA

synthesis. Consistent with this, in the present study, acceleration of NSDA neuronal

248

activity increased serine-19, serine-31, and serine-40 phosphorylated TH levels, and TH
catalytic activity in a-synuclein knock-out mice. There was no difference between wild-
type and a-synuclein knock-out mice in phosphorylated TH levels or TH catalytic
activity in response to raclopride, suggesting that feedback mechanisms that couple
neuronal activity and D2 autoreceptors to TH phosphorylation state are not affected by a-
synuclein. Accordingly, a-synuclein does not appear play a role in activity-dependent or

D2 autoreceptor-dependent TH activation.

The Ability of Tyrosine to Enhance Phosphorylated TH is Dependent on a-Synuclein

The present study reveals that a-synuclein plays a role in the ability of tyrosine to
enhance phosphorylated TH levels and TH catalytic activity in activated NSDA neurons.
In wild-type mice, tyrosine supplementation enhances phosphorylated TH levels and
enzymatic activity in raclopride-treated mice (Chapter 6). In contrast, tyrosine
supplementation does not further increase phosphorylated TH levels or enzyme catalytic
activity in activated NSDA neurons in a-synuclein knock-out mice. Tyrosine likely
stabilizes phosphorylated TH preventing it from de-activation through destabilization,
and this phenomenon is dependent on the presence of a-synuclein. Taken together, these
findings suggest that a-synuclein plays a critical role in regulating TH phosphorylation
state in an activity and substrate-dependent manner. The net result of this proposed
function for a-synuclein would be to allow DA synthesis to remain elevated in NSDA
neurons during prolonged periods of accelerated neuronal activity.

The results from the present study are congruent with the molecular chaperone

hypothesis for a-synuclein function. The activity-dependent molecular chaperone

249

hypothesis for o-synuclein is strengthened by two observations. First, a-synuclein shares
structural homology with the 14-3-3 family of molecular chaperone proteins that can bind
to phospho-serine residues on proteins and stabilize their active conformation (Tzivion et
al., 1998; Ostrerova et al., 1999). Secondly, a-synuclein expression is regulated
throughout development; i.e., during periods of accelerated neuronal activity and
plasticity, a—synuclein expression is up-regulated (George et al., 1995; Petersen et al.,
1999).

Taken together, the results from the experiments described have demonstrated
that a-synuclein knock-out mice have are less susceptible to the toxic effects of
prolonged, chronic MPTP administration and there is no increase in DA metabolism in
surviving NSDA neurons. While further experiments are necessary to determine a cause
and effect relationship, the present studies suggest that preventing excessive DA
metabolism may protect NSDA neurons from the toxic effects of MPTP. However, it is
clear that a-synuclein knock-out mice do not have a deficit in regulating TH
phosphorylation state or catalytic activity under basal or activated conditions induced by
a loss of long and short-loop feedback inhibition. As such, the lack of an increase in DA
metabolism in surviving NSDA neurons in a-synuclein knock-out mice may not reflect a
deficit in accelerating DA synthesis. However, as described in Chapter 6, there are
critical differences between the compensatory activation of NSDA neurons in response to
MPTP administration and the chronic activation of NSDA neurons induced by the loss of
D2 receptor-mediated feedback inhibiton. The possibility that a-synuclein knock-out
mice have a deficit in accelerating DA synthesis following MPTP administration cannot

be excluded.

250

The Regulation of DA Synthesis in a-Synuclein Knock-out Mice Following MPT P
Administration

To determine if a-synuclein knock-out mice have a deficit in regulating DA
synthesis following MPTP administration, mice were treated with sub—chronic MPTP and
the rate of striatal DA synthesis and TH phosphorylation state, enzymatic activity, protein
expression and sub-cellular distribution were assessed in the striatum. Sub-chronic
MPTP administration caused a 75% loss of NSDA axon terminals and a 65% decrease in
striatal DA metabolism. Interestingly, sub-chronic MPTP administration caused a slight
increase in the activity of surviving axon terminals (unlike following prolonged-chronic
administration). However, the loss of axon terminals in this study was more severe than
following prolonged, chronic MPTP administration, suggesting that NSDA neurons in o-
synuclein knock-out mice may be activated in response to more a pronounced loss of
axon terminals. Thus, o-synuclein knock-out mice may have a higher threshold of axon
terminal loss required for the compensatory activation of NSDA neuron than wild-type
mice.

The increase in DA metabolism following MPTP administration is due to an
uncoupling between DA synthesis and storage, such that synthesis is accelerated beyond
storage capability. It is clear that mice lacking a-synuclein are more capable of
maintaining the balance between synthesis and storage capability than wild-type mice.
This may be due to a less robust activation of DA synthesis or enhanced storage
capability following MPTP administration. To determine if this is the case, the rate of

striatal DA synthesis was determined in the striatum following MPTP administration.

251

Results demonstrated that there is a robust acceleration in the rate of DA synthesis
following MPTP administration that was similar in magnitude to wild-type mice. Also
similar to wild-type mice, the accelerated rate of DA synthesis was the result of an
increase in TH protein expression in surviving axon terminals, rather than an increase in
the catalytic activity of individual TH molecules. These findings demonstrate that the
primary response of NSDA neurons to axon terminal loss (i.e., up-regulation of TH
expression in surviving neurons and acceleration of DA synthesis) is not affected by the
loss of a-synuclein protein.

Sub-chronic MPTP administration also caused a differential loss of TH protein
depending on whether it was phosphorylated at serine-l9, serine-31, or serine-40.
Following MPTP, there was a 50% decrease in TH phosphorylated at serine-19 and
serine-31, but no loss of serine-40 phosphorylated TH. The amount of TH
phosphorylated at each residue was compared to the amount of total TH at the axon
terminal to reflect the relative change in TH phosphorylation state. Similar to wild-type
mice, MPTP caused a decrease in proportion of TH phosphorylated at serine-19 and
seirne-31. However, in contrast to wild-type mice, there was no increase in TH
phosphorylated at serine-40 (relative to total TH levels). The lack of effect on serine-40
was likely due to the attenuated MPTP-induced lesion since the preservation of serine-40
phosphorylated TH was consistent with what was observed in wild-type mice. Thus, it is
unlikely that the a-synuclein protein plays a role in regulating TH phosphorylation state
in the axon terminals of NSDA neurons following MPTP administration.

Regulation of TH sub-cellular compartrnentalization is also conserved in o-

synuclein knock-out mice. Under basal conditions, there were no differences between in

252

wild-type and a-synuclein knock-out mice in TH associated with the cytoplasmic,
endosomal, synaptic vesicle, or plasma membrane subcellular fractions. Thus, it does
not appear that a-synuclein performs a molecular chaperone-like function in trafficking
proteins within sub-cellular compartments under basal conditions. Following MPTP
administration, there was a differential loss of TH depending on which sub-cellular
compartment the enzyme was associated with. MPTP administration had no effect on TH
associated with the cytoplasmic fraction. When normalized to total TH at the axon
terminal prior to sub-cellular fractionation, there was an increase in TH associated with
the cytosolic fraction. This was consistent with wild-type mice and suggests increased
TH expression in surviving axon terminals following MPTP results in an increase in
cytoplasmic TH, where the enzyme is primarily active and can contribute to the
accelerated rate of DA synthesis.

In contrast to the cytoplasmic fraction, there was a robust loss of TH associated
with the plasma membrane fraction. Similar to wild-type mice, the loss of plasma
membrane associated TH was more severe than the loss of total striatal TH and as such,
there was a decrease in the proportion of total enzyme located in this sub-cellular
compartment. This may reflect either protein translocation to the cytosolic fraction or a
loss of compartmentalization capability in axon terminals that survive MPTP
administration.

In contrast to wild-type mice, MPTP administration caused a 25% decrease in TH
associated with the early endosomal vesicle and synaptic vesicle enriched fractions. The
loss of TH associated with these sub-cellular compartments was similar to the loss of

total TH, and as such, there was no change in the proportion of the enzyme located within

253

these sub-cellular compartments. In wild-type mice, there is an increase in the amount of
TH associated with the early endosomal vesicle fraction and a robust decrease in TH
associated with the synaptic vesicle fraction. These results could suggest that a-synuclein
plays a role in regulating the sub-cellular distribution of TH between early endosomal
and synaptic vesicles. Further experiments comparing the regulation of TH sub-cellular
compartmentalization in a-synuclein knock-out mice to wild-type mice (following
equivalent neurotoxic lesions) are required to determine if these mice have a deficit in
regulating TH compartmentalization. Another possibility is that the differential effect of
MPTP on endosomal and synaptic vesicle TH in wild-type versus a-synuclein knock—out
mice is due to the attenuated lesion in a-synuclein knock-out mice treated with MPTP. In
this case, the sub-cellular distribution of TH in MPTP-treated a-synuclein knock-out
mice may reflect the distribution of the enzyme in wild-type mice at a time point when
the lesion was less severe.

Thus, these findings suggest the initial changes in TH distribution following
MPTP are increased cytosolic TH and decreased plasma membrane TH. It is still unclear
whether these events reflect protein translocation from reserve pools of TH to the active
cytosolic fraction or an increase in de novo TH synthesis coupled with decreased enzyme
compartmentalization at the axon terminal. As the lesion becomes more severe, the
amount of TH associated with the endosome is increased and TH associated with
synaptic vesicles is decreased. In wild-type mice the increase in endosomal TH is
associated with increased serine-40 phosphorylated TH without an increase in enzyme
catalytic activity, indicative of an increased amount of destabilized enzyme at the axon

terminal. The fact that there is no increase in endosomal TH in a-synuclein knock~out

254

mice is consistent with a lack of an increase in serine-40 phosphorylated TH. Thus, the
increase in destabilized TH may occur later in the neurodegenerative process.

The loss of synaptic vesicle-associated TH may contribute to the uncoupling
between DA synthesis and storage capability. The localization of the rate limiting
enzyme in DA synthesis near the site of vesicular packaging is likely a mechanism used
to couple DA synthesis to storage capability which prevents the metabolism of newly
synthesized DA. In this case, the fact that synthesis-storage coupling does not occur in a-
synuclein knock-out mice is consistent with the lack of an increase in DA metabolism
following prolonged-chronic MPTP and an attenutated increase following sub-chronic
MPTP administration.

Taken together, it is clear that there is no difference between wild-type and a-
synuclein knock-out mice in the activation of DA synthesis following MPTP
administration. The fact that there is a lack of an increase in the metabolism of newly
synthesized DA in axon terminals that survive MPTP administration likely reflects
enhanced vesicular packaging capability in these mice, which may reflect enhanced TH
and synaptic vesicle association or increased VMAT-2 activity and/or expression, or
both. Several lines of evidence support this conclusion. There is no loss of VMAT-2
protein expression in the striatum, a marker of vesicular packaging capacity, despite the
loss of over 50% of striatal DA concentrations. While further experiments are required
that directly measure VMAT-2 activity and storage capability following MPTP, enhanced
VMAT-2 protein in o-synuclein knock-out mice would be consistent with the proposed
role for the protein in inhibiting the activity of phospholipase D2 which stimulates

monoaminergic vesicle formation (Lotharius and Brundin, 2002). Alternatively, the lack

255

of an increase in the metabolism of newly synthesized DA may be due to sparing of
synaptic vesicle associated TH. Further biochemical experiments that isolate and
quantify the activity of synaptic vesicle associated TH as well as further characterize the
mechanisms that govern sub-cellular compartmentalization are required to determine if
the association of TH with synaptic vesicles couples synthesis to vesicular packaging.
These experiments may define an important role for this coupling in the pathogenesis of

PD and finally determine what role a-synuclein plays in this process.

Summary

The findings from the studies described in this Chapter highlight the importance
of a-synuclein protein expression in mediating the toxic effects of MPTP and the
compensatory activation of surviving NSDA neurons. Results point towards a molecular
chaperone like function for a-synuclein that mediates the ability of tyrosine to stablize
phosphorylated TH. However, using two distinct models of NSDA activation it is clear
that a-synuclein does not play a role in the regulation of NSDA activity or DA synthesis
under basal or activated conditions. Yet, decreasing the expression of this protein may
protect NSDA neurons from the oxidative damage induced by toxic byproducts of DA
metabolism due to an enhanced ability to generate synaptic vesicles to package newly

synthesized DA.

256

Chapter 8. Concluding Remarks

PD places a tremendous burden on the American society. Greater than one-
million Americans currently suffer from PD and when primary caregivers are accounted
for, the number of people adversely affected by the disease drastically increases. The
estimated cost of health care treatment for PD patients is over 6 billion dollars per year
(Whetten-Goldstein et al., 1997), placing a tremendous economic burden on the
American society as well. These statistics draw enormous emphasis towards the
discovery of therapeutic strategies for curing the disease. The primary drawback to this
approach is that PD is not identified until significant pathological damage has already
occurred. A logical approach to treating PD therefore, is to slow or halt disease
progression once it is identified. This would drastically improve the quality of life for
both patients and primary caregivers and reduce the associated economic burden of
treating late-stage PD. A major roadblock that prevents accomplishing this task is the
mechanisms that underlie PD progression are still unknown.

The gradual decline in the quality of life for PD patients is due to the progressive
worsening of the cardinal motor symptoms. The manifestation and gradual worsening of
symptoms is due to the progressive loss of NSDA neurons. Preventing the progressive
loss of NSDA neurons should halt the worsening of motor symptoms. The primary goal
of the research conducted in this dissertation was to identify detrimental mechanisms that
may contribute to the progressive degeneration of NSDA neurons. The strategy
employed was to use a neurotoxin to induce NSDA cell death similar to PD.

Pharmacological manipulation combined with neurochemical and immunohistochemical

257

techniques were then used to identify detrimental mechanisms that may cause
degeneration of the neurons that survive the initial neurotoxic insult.

One of the primary findings of this dissertation research is that following a
neurotoxic lesion there is an increase in the activity of surviving NSDA neurons. This
compensatory increase is similar to PD, where there is an increase in the activity of the
50% of NSDA neurons that have not yet degenerated. The activation of NSDA neurons
following neurotoxic insult is a compensatory mechanism that increases the amount of
DA release from surviving neurons to continue to activate post-synaptic DA receptors
involved in basal ganglia function. The sustained increase in surviving neuron activity
likely increases ROS exposure derived fi'om accelerated DA metabolism and may cause
NSDA cell death. Oxidative damage in the striatum of post-mortem PD brains supports
this hypothesis. The compensatory activation of NSDA neurons may, therefore,
contribute to the progressive loss of NSDA neurons which underlies the progressive
nature of PD.

NSDA firing rate and DA release are regulated by feedback inhibition from D2
receptors, suggesting that the compensatory activation that occurs in response to
neurotoxicity is due to a loss of this feedback inhibition. Consistent with this, studies
demonstrated that acute D2 receptor blockade increases NSDA neuronal activity and DA
metabolism in axon terminals. Chronic D2 receptor blockade however, does not activate
NSDA neurons in a similar manner. Prolonged activation of NSDA neurons does not
result in a prolonged increase in DA metabolism and thus is not detrimental to neuronal
survival. These findings suggest compensatory activation of NSDA neurons, following

D2 receptor blockade, permits prolonged accelerated neuronal activity without increasing

258

DA metabolism. This is in stark contrast to the compensatory activation of NSDA
neurons where the chronic increase in NSDA activity does cause a chronic increase in
DA metabolism.

The finding that NSDA neurons are differentially activated in response to axon
terminal loss as compared with blockade of D2 receptors highlights the importance and
potentially detrimental nature of the compensatory activation of NSDA neurons in PD.
An important task of future research will be to 1) determine the mechanisms in NSDA
neurons that permit prolonged accelerated neuronal activity without increased DA
metabolism and 2) determine if these mechanisms are dysfunctional following a
neurotoxic lesion. Accomplishing these tasks will provide critical insight into PD
pathogenesis and potential pathways that can be manipulated to protect NSDA neurons.

A key finding from this dissertation research is that the primary source of
excessive DA metabolism during NSDA compensatory activation is the DA biosynthetic
pathway. As described in Figure 8-1 (A, B) the increased metabolism of newly
synthesized DA is due to an accelerated rate of DA synthesis. Accelerated DA synthesis
is due to increased expression of the rate limiting enzyme TH. Increasing the rate of DA
synthesis could benefit surviving neurons by boosting the amount of neurotransmitter
available for release (Figure 8-lC). This is not the case however, since the newly
synthesized DA is primarily metabolized. These findings highlight the therapeutic
potential of enhancing DA vesicular storage in preventing exposure to ROS-derived from

DA metabolism and augmenting the function of surviving NSDA neurons.

259

 

 

 

 

 

 

 

B.
mfAcoofAc DOPAC
ROS ROS R08 0

DOPAL

__ _ ___j
7W" THDOPA DA

T ne —°DOPA-—eDA—-—-i
M W AADC

 

war
Tyrosine —_. — _.
TH DOPAMDC DA

 

 
  
  

 

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260

 

 

   

 

 

Figure 8-1. (A) NSDA axon
terminal neurochemistry under
basal conditions. Normally,
newly synthesized DA is
coupled to vesicular storage,
such that metabolism of newly
synthesized DA is minimal.
(B) NSDA axon terminal
neurochemistry following
compensatory activation.
There is an increase in the
expression of TH protein in
surviving axon terminals which
accelerates the rate of DA
synthesis. The newly
synthesized DA is not
packaged into synaptic vesicles
and is metabolized as a
consequence, increasing ROS
production. (C) Enhancing DA
storage can decrease the
metabolism of newly
synthesized DA following
compensatory activation and
increase the amount of
vesicular DA available for
release

Studies also identified mechanisms that may be responsible for the inability to
package the newly synthesized DA. The uncoupling between DA synthesis and vesicular
packaging may be due to decreased expression or activity of the vesicular transporter
VMAT-Z, or both. Additionally, decreased association of TH with synaptic vesicles may
cause DA to be synthesized at a location distant from the site of vesicular storage and
thus increase the metabolism of newly synthesized DA. To this end, a major goal of
future research will be to determine the mechanisms that regulate VMAT-2 expression
and activity and TH-synaptic vesicle compartmentalization. These mechanisms may be
primary therapeutic target that can be manipulated to inhibit DA metabolism and enhance
DA release in NSDA axon terminals following compensatory activation.

The hypothesis that enhancing the association between TH and synaptic vesicles
could be neuroprotective is strengthened by another primary finding of this dissertation
research. Blocking the expression of a—synuclein, a protein that plays a primary role in
PD pathogenesis, prevents increased DA metabolism following compensatory activation
and attenuates the neurotoxin-induced lesion. Accelerated DA synthesis however, still
occurs in the absence of a-synuclein, suggesting the loss of this protein enhances
vesicular storage. These findings provide evidence that promoting DA vesicular storage
can prevent excessive DA metabolism following a neurotoxic lesion and may protect
NSDA neurons from oxidative damage. It is still unclear if enhanced vesicular storage is
due to increased VMAT-2 expression and activity, preservation of TH-synaptic vesicle
compartmentalization, or both. A goal of future research will be to determine how

expression of the a-synuclein protein affects these mechanisms. Accomplishing these

261

goals will increase the current knowledge of o-synuclein function and provide pathways
that can be exploited to treat PD.

To summarize, the research described in this dissertation was conducted under the
premise that identifying mechanisms responsible for progressive NSDA cell death will
lead to therapeutic approaches to slow or halt the gradual worsening of motor symptoms
in PD. Studies have identified and characterized dysfunctional regulation of a key
pathway in NSDA neurons that may predispose these neurons to oxidative damage and
cell death. Additionally, studies have clarified the role that a genetic factor, extensively
linked to PD pathogenesis, plays in mediating NSDA neuronal death. Moreover, a
number of potential therapeutic targets have been identified that may prevent the
progressive demise of NSDA neurons in PD, ultimately slowing or halting the

progressive nature of the disease.

262

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