DOPAMINE HOMEOSTASIS AND ENVIRONMENTAL RISK FACTORS IN A
PARKINSON’S DISEASE MODEL
by
O’NEIL WRIGHT
JANIS O’DONNELL, CHAIR
MARGARET JOHNSON
HARRIETT SMITH-SOMERVILLE
JOHN VINCENT
PERRY CHURCHILL
A DISSERTATION
Submitted in partial fulfillment of the requirements
for the degree of Doctor of Philosophy in the
Department of Biological Sciences
in the Graduate School of
The University of Alabama
TUSCALOOSA, ALABAMA
2011
Copyright O’Neil Wright 2011
ALL RIGHT RESERVED
ABSTRACT
The neurotransmitter dopamine (DA) is an important factor in the regulation of many
biological processes, from pleasure and addiction to balance and locomotion. Therefore,
understanding and defining the mechanisms and factors that are required for proper DA
homeostasis is an integral component in managing and elucidating the causes of DA related
diseases. Among these diseases, Parkinson’s disease (PD) is the most notable and remains one
of the most researched yet puzzling motor system associated neurological disorders.
PD is characterized by a preferential loss of DA neurons in the substantia nigra. Though the
cause and exact mechanism of this disease remains undefined, numerous environmental factors
such as metals and pesticides have been associated with the etiology of the disease process.
In the following studies, the genetic components of DA homeostasis and environmental
risk factors in a Drosophila model of PD are investigated. The implication of metals as a
component in the pathology of PD is examined in relationship to zinc toxicity. Catecholamines’s
up (Catsup), which plays a crucial role in regulating DA homeostasis and is proposed to be a
member of the mammalian KE4 ZIP transporter family, demonstrates zinc sensitivity, with the
proposed underlying factors being a dysregulation of DA synthesis and DA transport. The
findings of this report demonstrate that loss of dopamine transporter (DAT) function, results in a
more robust sensitivity to zinc than that seen in Catsup mutants. In addition exogenous DA
increases sensitivity of wild type flies to zinc, similar to that which is seen Catsup mutants.
ii
Interestingly, LiCl ameliorates the toxic effects of zinc. The results also demonstrate a functional
relationship between paraquat toxicity and DAT, which affects DA transport.
To determine the consequences of early exposure to paraquat on lifespan and mobility,
the effect of a one time exposure to young adult flies was observed. The results of this
experiment show that a brief exposure to paraquat illicts long term detrimental affects on
survival as well as parkinsonian type phenotypes.
iii
LIST OF ABBREVIATIONS AND SYMBOLS
3-IT
3- iodo-tyrosine
6-OHDA
6-hydroxydopamine
Å
Angstrom
AADC/DDC
Aromatic-L-amino-acid decarboxylase/dopa decarboxylase
Acb
Accubens
ADHD
Attention deficit hyperactivity disorder
AD
Alzheimer’s disease
ADLH2
Aldehyde dehydrogenase
AML1
Acute Myeloid Leukemia-1
AMPA
Alpha-amino-3-hydroxy-5-methyl-4-isoaxazolepropionic acid
ANOVA
Analysis of variance
Apaf-1
Apoptosis activating factor-1
APP
Amyloid protein precursor
ATP
Adenosine triphosphate
BDNF
Brain -derived-neurotrophic factor
BH4
Tetrahydrobiopterin
o
C
Celsius
C
Carboxyl
cAMP
Cyclic adensoine monophosphate
iv
CATSUP
Catecholamines up
CD 14
Cluster of Differentiation 14
CDF
Cation diffusion facilitator
cDNA
Complementary deoxyribonucleic acid
COX 1
Cyclooxygenase -1
CNS
Central nervous system
CP
Choroid plexus
Cx
Cerebral cortex
D2 S
Dopamine receptor 2 short
D2 L
Dopamine receptor 2 long
DA
Dopamine
DAT
Dopamine transporter
dDAT
Drosophila dopamine transporter
DD2R
Drosophila D2-like receptor
Df
Deficiency
DHPR
Dihydropteridine reductase
DOPAL
3,4-dihydroxyphenylacetaldheyde
DOPAC
Dihydrophenylacetic acid
DNA
Deoxyribonucleic acid
DRD
Dopa responsive dystonia
DTH
Drosophila Tyrosine hydroxylase
DVMAT
Drosophila vesicular monoamine transporter
EOP
Early-onset Parkinsonism
v
fmn
Fumin
g
Unit of gravity
GABA
Gamma-aminobutyric acid
GABAAR
Gamma-aminobutyric acid type A receptors
GFP
Green flouresent protein
GFRP
GTPCH feedback regulatory protein
GSK-3
Glycogen synthase kinase 3
GTP
Guanosine triphosphate
GTPCH
Guanosine triphosphate cyclohydrolase
H2NTP
Dihydroneopterin triphosphate
H2 O2
Hydrogen peroxide
HPLC
High Performance Liquid Chromatography
HSP
Heat shock protein
HVA
Homovanillic acid
kDa
Kilodalton
Ki
Inhibitor constant
Km
Michaelis-Menten constant
L
Liter
L-Dopa
L-3,4-dihydroxyphenylalaine
LZT
LIV-1 subfamily of ZIP Zn2+ Transporter
M
Molar
MAO
Monoamine oxidase
MAPK
mitogen activated protein kinase
vi
MPP+
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
mм
Millimolar
MT
Methallothionein
MW
Molecular weight
N
Amino
NMDA
N-methyl-D-asparate
NS
Not significant
NO
Nitric oxide
NOS
Nitrate oxide synthase
OT
Occipitotemporal
PAH
Phenylalanine hydroxylase
PC12
Pheochromocytoma colonal cell
PCD
Pterin-4a-carbinolamine dehydratase
PD
Parkinson’s disease
PKA
cAMP-dependent protein kinase
PKC
Protein kinase C
PKU
Phenylketonuria
PQ
Paraquat
PTP
6-pyruvol-tetrahydropterin
PTPS
6-pyruvol-tetrahydropterin synthase
RNA
Ribonucleic acid
RNAi
RNA interference
ROS
Reactive oxygen species
vii
SD
Standard deviation
SEM
Standard error of the mean
Ser
Serine
SN
Substantia nigra
SNc
Substantia nigra par compacta
SR
Sepiapterin reductase
TH
Tyrosine hydroxylase
TDP-43
TAR DNA-binding protein of 43 kDa
TPH
Tryptophan hydroxylase
VMAT
Vesicular monoamine transporter
Vmax
Maximum enzyme velocity
VTA
Ventral tegmented area
µl
Microliter
µM
Micromolar
ZEN
Zinc enriched neurons
ZIP
Zrt, Irt-like protein
ZnT
Zinc transporter
Zn2+
Zinc
ZnCl2
Zinc chloride
viii
ACKNOWLEDGMENTS
I would like to acknowledge my advisor and chair of my graduate committee, Dr. Janis
M. O’Donnell, for her excellent guidance, belief in my abilities, constructive criticism, and
unrelenting support, friendship and mentorship throughout my research and academic years. It is
because of her that I considered this journey of my life. My ability to present this body of work
would not have been possible without her, and for that I am forever grateful and in her debt.
I would also like to thank Dr. Martha Powell for allowing me this opportunity and for
encouraging me to pursue the terminal degree. Your words of encouragement, support and belief
in my abilities will always be appreciated. I would also like to thank Dr. Margaret Johnson,
whose presence, concern and support have enable me to keep fighting even when I felt I could
not go on. In addition, I would be remised if I did not thank Dr. Harriett Smith-Somerville,
whose kind demeanor and support will mean much more than she will ever know. I would also
like to thank Dr. John Vincent and Dr. Perry Churchill for not only serving on my committee but
for their advice in all matters personal and professional. I am also grateful to the current chair of
the Biology Department, Dr. Patricia Sobecky. Even though you were a brand new department
chair, with so many responsibilities, you took time to get to know me and offered your full
support in seeing me through this process. You too are responsible for me being able to
complete this journey, and I will never forget your kindness.
To all the current and past members of the O’Donnell lab, I am grateful for the
intellectual support, mentorship, and friendship offered by you, my colleagues and friends. Your
ix
support both personal and professional has been an invaluable tool in this experience. A big
thank you goes out to all the members of the janitorial staff. The many late nights I spent in the
lab ensured that our paths would cross and each of you have always been kind by offering your
words of encouragement, showing interest in my research, and sometimes even offering a meal.
Thank you for being a part of this process. Much love and gratitude is extended to Mary
Musumeci and all the ladies in the Biology Department office. From day one, your interest in
my development and support throughout this process has ensured that this journey would be
possible and for that I am grateful. I would especially like to acknowledge my best friend, and
sister from another mother, Dr. Rosianna Gray. Your friendship, support, and unselfish love
have made this moment possible. There are not enough words to explain how thankful I am to
you for just being who you are to me.
Finally, I wish to thank my biological and church family for their support, advice, and
prayers throughout this process. I especially would like to acknowledge my grandparents,
Robert and Clara Anderson, for their love and encouragement throughout this process. You have
always seen the best in me, even in the times that I could not see it in myself.
x
CONTENTS
ABSTRACT .......................................................................................................................... ii
LIST OF ABBREVIATIONS AND SYMBOLS.................................................................. iv
ACKNOWLEDGMENTS..................................................................................................... ix
1. INTRODUCTION............................................................................................................ 1
Dopamine synthesis.......................................................................................................... 2
Depamine packing, transport, and metabolism ................................................................ 2
Tyrosine hydroxylase ....................................................................................................... 8
Regulation of tyrosine hydroxylase.................................................................................. 9
Drosophila tyrosine hydroxylase...................................................................................... 12
Tetrahydrobiopterin function and dysfunction................................................................. 13
Tetrahydrobiopterin biosynthesis ..................................................................................... 15
Regulation of GTP cyclohydrolase .................................................................................. 17
Drosophila GTPCH .......................................................................................................... 20
Zinc function .................................................................................................................... 21
Zinc metabolism ............................................................................................................... 23
Zinc and the brain............................................................................................................. 27
Zinc and dopamine ........................................................................................................... 30
Zinc and neurodegenerative diseases ............................................................................... 31
xi
2. ZINC AND DOPAMINE: A SYNERGISTIC RELATIONSHIP? ................................ 38
Introduction ...................................................................................................................... 38
Materials and Methods ..................................................................................................... 39
Drosophila strains and culture maintenance................................................................ 39
Zinc feeding experiments ............................................................................................. 40
Locomotion assay........................................................................................................ 40
DA analysis ................................................................................................................. 40
Statistical analysis ....................................................................................................... 46
Results ................................................................................................................................... 41
Catsup mutants are Zn2+ sensitive .................................................................................... 41
Dopamine increases Zn2+ sensitivity in Catsup mutants and wild type flies.................... 44
Zinc increases adult mobility but reduces DA pools ....................................................... 47
Loss of DAT results in increased sensitivity to Zn2+ ....................................................... 51
Simultaneous reduction in Catsup and DAT function enhances Zn2+sensitivity.............. 51
Fears of intimacy (foi) mutants are Zn2+sensitive and hypermobile ................................ 55
LiCl rescues the sensitivity of Catsup mutants to Zn2+ .................................................. 58
Discussion ............................................................................................................................. 60
3. ALTERATION IN DA HOMEOSTASIS BY A REDUCTION IN DAT
FUNCTION ATTENUATES THE TOXIC EFFECTS OF THE
HERBICIDE PARAQUAT .............................................................................................. 70
Introduction ...................................................................................................................... 70
Materials and Methods ..................................................................................................... 72
Drosophila strains and culture maintenance............................................................... 72
Behavior ..................................................................................................................... 72
xii
Paraquat and reserpine exposure ................................................................................ 73
Statistical analysis ....................................................................................................... 73
Results ................................................................................................................................... 73
Expression of a wild type Catsup transgene in dopaminergic neurons does not
ameliorate resistance to parquat ....................................................................................... 73
Catsup over-expression flies are hypermobile............................................................. 82
Over-expression of Catsup modulates VMAT function.............................................. 76
Loss of DAT function reduces sensitivity to paraquat ................................................ 78
Discussion ............................................................................................................................. 81
4. A BRIEF EXPOSURE TO PARAQUAT RESULTS IN REDUCED LIFE
SPAN ANDPARKINSONIAN RELATED PHENOTYPES IN YOUNG ADULT
DROSOPHILA.................................................................................................................. 87
Introduction ...................................................................................................................... 87
Materials and Methods ..................................................................................................... 89
Drosophila strains and culture maintenance................................................................ 89
Feeding experiments.................................................................................................... 89
Locomotion assays ...................................................................................................... 89
Statistical analysis ....................................................................................................... 90
Results ................................................................................................................................... 90
Early PQ exposure to young adult flies modulates mobility both short and long term ... 90
Early paraquat exposure reduces lifespan in adult flies ................................................... 91
Discussion ............................................................................................................................. 94
REFERENCES ...................................................................................................................... 97
xiii
LIST OF FIGURES
1.1 Components of DA synthesis, transport and metabolism ............................................... 7
1.2 Tetrahyrdrobiopterin (BH4) biosynthesis pathway.......................................................... 16
2.1 The effect of ZnCl2 on lifespan in wild type and Catsup mutant adult flies ................... 43
2.2 The effect of DA prefeeding on Zn2+ -induced mortality of wild type and Catsup
mutant flies .................................................................................................................... 46
2.3 Zn2+ modulates the metabolism of DA............................................................................ 48
2.4 Zn2+ feeding enhances mobility in wild type flies........................................................... 50
2.5 Loss of function mutations of DAT and RNAi knockdown of DAT expression
in dopaminergic neurons increase Zn2+ sensitivity ......................................................... 53
2.6 Catsup-DAT interactions in zinc toxicity........................................................................ 56
2.7 The effect of Zn2+ on lifespan in foi mutants .................................................................. 61
2.8 foi and foi/Catsup transheterozygote mutants are hypermobile ...................................... 57
2.9 LiCl suppresses the toxicity of ZnCl2 in Catsup mutant and wild type flies.................. 59
2.10 A model of the interaction of Catsup with the genetic regulators of DA
homeostasis .................................................................................................................. 64
3.1 Over-expression of Catsup in dopaminerigic cells alters parquat survival and
mobility .......................................................................................................................... 75
3.2 Expression of Catsup in dopaminergic cells confers sensitivity to reserpine ................. 77
3.3 Loss of DAT function reduces sensitivity to paraquat toxictity...................................... 80
4.1 A one time exposure to paraquat attenuates locomotor activity ..................................... 92
4.2 A one time exposure to paraquat (PQ) reduces lifespan ................................................. 93
xiv
CHAPTER ONE
INTRODUCTION
Dopamine (DA), a neurohormone/neurotransmitter that is a member of the catecholamine
family of molecules, is necessary for both neurological and non-neurological functions.
Catecholamine is characterized by the presence of a catechol nucleus and a terminal amine group
and is derived from the amino acid L-tyrosine. DA not only serves as a necessary precursor for
the important vertebrate catecholamine, epinephrine and norepinephrine, but it also is the most
abundant catecholamine in the nervous system, accounting for 50% of the total catecholamine
levels in mammals (Carlsson, 1959). Although DA is found predominately within the central
nervous system (CNS), it is also synthesized within the gastrointestinal tract and the adrenal
glands (Falck et al., 1967; Axelrod, 1971; Bloom et al., 1988).
DA has been implicated in a diverse set of functions including the regulation of mood,
learning and memory, attention, addiction, reward, stress management, melanin production,
cuticle hardening in arthropods, development, fertility and courtship, insect tracheal
morphogenesis, motor neuron activity and neuromuscular function (Kobayashi et al., 1995;
Neckameyer, 1996; Neckameyer et al., 2001; Smidt et al., 2003, Wise, 2004; Schultz, 2007;
Björklund & Dunnett, 2007, Hsouna et al., 2007). Given the role of DA in regulating so many
important processes, it is no surprise that disruption in DA synthesis and signaling has been
implicated in a variety of diseases such as Alzheimer’s disease, Parkinson’s disease, and DOPA
responsive dystonia (Bernheimer et al., 1973; Segawa et al., 1976; Reinikainen et al., 1988;
1
Ichinose et al., 1999). In addition, DA imbalance is a key component in many psychiatric
illnesses such as dementia, depression, schizophrenia and addiction (Pendelton et al., 1998;
Berman et al., 1999; Iversen & Iversen, 2007). Therefore, understanding the genetic components
of DA regulation and synthesis is an essential element in deciphering the cause and treatment of
DA related disorders.
Dopamine synthesis
The process of DA synthesis is similar in both vertebrates and invertebrates. Tyrosine
hydroxylase (TH) is the first and rate-limiting enzyme in the biosynthesis of DA (Nagatsu et al.,
1964). DA synthesis begins with the hydroxylation of L-tyrosine to 3, 4-dihydroxyphenylalanine
(L-DOPA) by TH, mediated by the binding of oxygen (O2) and the pteridine cofactor,
tetrahydrobiopterin (BH4) at this enzyme’s active site (Nagatsu et al., 1964; Axelrod, 1971). In
addition, ferrous iron is also required for catalysis to occur (Fitzpatrick, 1989; Haavik et al.,
1992, 1993). Following the production of L-DOPA, it is converted to DA by the enzyme
aromatic –L-amino acid decarboxylase or dopa decarboxylase (AADC/DDC) (Holtz et al.,
1938). In Drosophila, the synthesis of DA is the terminal point of the catecholamine pathway,
but in mammals, DA is converted to norepinephrine and epinephrine (Kumer & Vrana, 1996).
Although TH is the primary enzyme responsible for the production of L-DOPA, tyrosinase,
present in melanocytes, can also convert tyrosine to L-DOPA. However, it should be noted that
melanocytes lack AADC/DDC and are unable to convert L-DOPA into DA (Rios et al., 1999).
Dopamine packing, transport, and metabolism
Once DA is synthesized, it is stored, transported and recycled in an efficient and highly
regulated manner. Newly synthesized DA is packaged into vesicles by vesicular monoamine
transporters (VMAT). The packaging of DA is an important aspect of neuronal health because it
2
stabilizes the neurotransmitter and prevents cellular damage due to its degradation. VMAT is a
general transporter of all biogenic amines and is regulated by changes in the electrochemical
gradient by ATPase. This process is necessary because it allows for the packaging and
subsequent release of neurotransmitters (Flatmark et al., 2002; Brunk et al., 2006). In mammals,
two isoforms of VMAT, VMAT1 and VMAT2, have been identified and analyzed. VMAT2 is
expressed predominately in neurons, whereas VMAT1 is exclusive to neuroendocrine cells
(Erickson et al., 1996). One VMAT gene has been identified in Drosophila, which produces two
alternatively, spliced transcripts, VMAT A and VMAT B. These two variants are identical in
structure except for changes in the C- terminal domain. Drosophila VMAT (DVMAT) has a high
degree of conservation with its mammalian counterpart. The predicted motif of VMAT A, but
not VMAT B, shows similarity between the carboxy terminus of VMAT A with rat VMAT2 and
other mammalian VMATs. In addition, in vitro assays of VMAT A demonstrate that it is
capable of transporting, serotonin, and octopamine, and is localized to dopaminergic and
serotonergic neurons in the CNS (Greer et al., 2005). Following the packaging and transport of
DA by VMAT, it is released into the synapse.
The DA transporter (DAT) is the main enzyme responsible for clearing DA from the
synapse after completion of synaptic signaling. Located in the pre-synaptic plasma membrane,
DAT maintains proper neurotransmission of DA by recycling unused DA back into the cytosol
(Hitri et al., 1994). Elucidation of the primary structure of DAT has revealed that it is a member
of the family of NA+/Cl-- dependent transporters containing 12 putative transmembrane domains
(Amara & Kuhar, 1993; Giros & Caron, 1993). Analysis of homozygous DAT-deficient mice
demonstrated that DA persists at least 100 times longer in the extracellular space and results in
spontaneous hyper-locomotion despite major adaptive changes, such as decrease in
3
neurotransmitter and receptor levels (Giros et al., 1996). In addition, the DAT is a major target
for psychoactive drugs such as antidepressant and abused drugs including cocaine and
amphetamines (Horn, 1990; Ritz & Kuhar, 1993). In Drosophila, the cloning and
characterization of dDAT indicates striking similarities to vertebrate orthologs as it relates to
cocaine sensitivity and locomotion, and dDAT mRNA is found predominately within
dopaminergic cells in the fly nervous system (Porzgen et al., 2001).
To maintain DA homeostasis and prevent cytological insult, DA that has been transported
back into the presynaptic termini has one of two fates. It can be broken down by monoamine
oxidase (MAO) into a stable nontoxic metabolite, 3, 4-dihydroxyphenylacetic acid (DOPAC)
and reactive oxygen species (ROS), or it is repackaged into vesicles by VMAT (Blaschko, 1957;
Kopin, 1968; Henry et al., 1998; Youdim & Bakhle, 2006). Two isoforms of MAO, MAO-A
and MAO-B exists in mammals, and both have been shown to be active in the degradation of
DA. In the rodent brain, mainly MAO A accomplishes DA degradation, while MAO B is
responsible for this process in humans and other primates (reviewed in Bortolato et al., 2008).
Moreover, examination of the expression pattern of these isoforms indicated that they were
differentially expressed in mammalian tissues, suggestive of variation in functions. In humans,
MAO-A is primarily localized within catecholaminergic neurons, while MAO-B is mainly
expressed within serotonergic and histaminergic neurons, as well as in astrocytes (Westlund et
al., 1988; Saura et al., 1994). In Drosophila, a low level of MAO activity is detected within the
brain (Dewhurst et al., 1972). However, this concentration of MAO seems to be sufficient for
DA metabolism because DOPAC is detectable by HPLC analysis (Chaudhuri et al., 2007).
Receptors for DA on the surface of neurons are necessary not only for receiving
messages but are an integral part of maintaining homeostasis. DA receptors belong to the family
4
of seven transmembrane domain (7TM) G protein-coupled membrane receptors (Jackson and
Westlind-Danielsson, 1994). In mammals, five distinct DA receptors have been identified and
subdivided into two families, D1-like and D2-like, based on pharmacological properties
(Kebabian & Calne, 1979). Members of D1-like receptors are D1 and D5 receptors while the D2like includes D2, D3 and D4 receptors. Among the DA receptor genes, D1 and D2 have the
highest and most widespread level of expression (Jackson & Westlind-Danielsson, 1994).
Analysis of the D2 receptor gene within mammals indicates that is composed of eight
exons, seven of which are coding (Gingrich & Caron, 1993). Alternative splicing of the sixth
exon generates two isoforms, DA receptor 2 short and DA receptor 2 long (D2S and D2L). Both
D1 and D2 receptors are expressed in the brain. The D1 receptor is believed to be post-synaptic
and is expressed in the choroid plexus (CP), nucleus accubens (Acb), occipitotemporal (OT),
cerebral cortex (Cx) and amygdale (Jackson & Westlind-Daniellson, 1994). In contrast, the D2
receptor is primarily pre-synaptic and is found within areas of the brain such as the CP, OT, and
Acb. In addition, the D2 receptor is also expressed in the substania nigra par compacta (SNc)
and in the ventral tegmented area (VTA). Outside the brain, the D2 receptor is also found in the
kidney, retina, pituitary gland and vascular system (Ng et al., 1994; Jackson & WestlindDanielsson, 1994; Picetti et al., 1997). DA receptors regulate many chemical pathways;
however, the activation or inhibition of the cAMP pathway and modulation of calcium signaling
seem to be the most prevalent. Additionally, activation of D1-like receptors stimulates adenyl
cyclase and phosphatidylinositol-4,5 biphosphate metabolism, whereas activation of D2-like
receptors inhibits adenylyl cyclase and activates potassium channels. The inhibition of adenylyl
cyclase reduces the phosphorylation of TH, which in turn, decreases TH activity and DA
production (Jackson & Westlind-Danielsson, 1994). One of the adenosine G protein coupled
5
receptors, A2A, is restricted to DA innervated regions (Ferré et al., 1997). Analysis of adenosine
receptor function, in particular A2A and D2 receptor, demonstrates that A2A receptor stimulation
inhibits D2-mediated activation in mice (Ferré et al., 1991). Moreover, it has been shown that
stimulation of A2A receptors in neuroblastoma cells decreased the affinity of the D2 receptor for
DA. Therefore, modulation of DA receptor activity is an important part of maintaining DA
homeostasis and function. Interestingly, analysis of ADHD patients, whose symptoms are
commonly believed to be a consequence of DA dysregulation, has indicated that polymorphisms
within the D4-R and DAT-1 genes are possible factors in this disorder (Cook et al., 1995; Gill et
al., 1997). The importance of the DA receptor, in regulating DA homeostasis, is further
underscored by the use of DA receptor agonist as potential treatment options for PD.
In Drosophila, DA receptors belonging to both the D1-like and D2-like subfamilies of DA
have been cloned and characterized (Gotzes et al., 1994; Feng et al., 1996; Hearn et al., 2002).
Two D1-like receptor genes, DmDop1 and DopR99B have been isolated. Analysis of amino acid
sequence determined that DmDop1 is approximately 70% identical to human D1/D5 receptors
(Gotzes et al., 1994). When expressed in mammalian cell cultures, both DmDop1 and DopR99B
stimulate cAMP production in response to DA application (Gotzes et al., 1994; Feng et al.,
1996). Analysis of the expression pattern of DmDop1 indicates that it is expressed in the optic
lobe, whereas DopR99B is primarily expressed in the central and peripheral nervous system.
Isolation and analysis of the Drosophila D2- like receptor (DD2R) demonstrates that eight splice
variants exist for this receptor (Hearn et al., 2002). Pharmacological assessment of three of these
isoforms, DD2R-606, DD2R-506, DD2R-461, supports the classification of these proteins as D2like receptors. DD2R is expressed predominantly in adult fly head as well as the pupa and larva.
6
Figure 1.1. Components of DA synthesis, transport and metabolism (modified from
Wang et al., manuscript in progress). Once DA has been produced; it is packaged by
VMAT and released from the presynaptic terminal. DA that is located inside the synaptic
region then interacts with DA receptors on the postsynaptic or presynaptic terminal, where it
elicits a chemical response. In addition, DA within the synaptic space is reshuffled back into
the presynaptic terminal by DAT, where VMAT repackages it into synaptic vesicles or MAO
breaks it down into the metabolite DOPAC. All of these components work together to
maintain proper homeostasis of DA.
7
Tyrosine hydroxylase
Mutations within the Th gene have been associated with human disorders such as LDOPA-responsive dystonia, Parkinsonism in infancy, and progressive infantile encephalopathy
with L-DOPA – nonresponsive dystonia (Lüdecke et al., 1996; Hoffmann et al., 2003; Schiller et
al., 2004). Studies conducted with TH-deficient mice, generated either by complete or partial
knockout of the Th gene or mutation within the gene, show an array of anomalies including
reduced body size and hypokinesia, impaired locomotor activity, and defects in learning and
memory and conditioned learning (Kobayashi et al., 2000). Additionally, conditional knockout
of Th in dopaminergic neurons results in a wide range of phenotypes such as akinesia, cataleptic
behavior, loss of drug response, blockade of dopamine receptor agonist-induced motor
activation, postnatal development and death (Zhou & Palmiter, 1995; Nishii et al., 1998).
A single gene encodes TH in all organisms (Grima et al., 1987; Kobayashi & Sano,
1988). Molecular analysis of this gene indicates that it is a member of the family of
tetrahydrobiopterin-dependent aromatic amino acid hydroxylases that also includes
phenylalanine hydroxylase and tryptophan hydroxylase (Grenett et al., 1987). These proteins are
orthologous not only in protein sequence but also in catalytic mechanism (Goodwill et al., 1997).
TH is expressed in all catecholaminergic cells and has been shown to be localized to neurons of
the sympathetic nervous system and the adrenal medulla as well as neurons within the ventral
tegumentum, substania nigra, hypothalamus and the olfactory bulb in vertebrates (Wong & Tank,
2007). Analysis of purified TH has demonstrated that the enzyme exists as a tetramer in its
native form and as a homotetramer in most species. Each subunit consists of an N-terminal
regulatory domain and a C-terminal catalytic domain (reviewed in Kumer & Vrana, 1996).
Isolation and characterization of cDNA clones of rat TH has shown that the predicted molecular
8
weight of TH is about 55kDA, varying slightly among species (Grima et al., 1985). The catalytic
core of TH is highly conserved across species but the N-terminal domain is diverse (Fitzpatrick,
1999). The divergence of the N-terminal domains is believed to be an important aspect in the
regulation of TH, including phosphorylation, which occurs in this domain (Campbell et al., 1986;
Haycock, 1990). In humans, four isoforms of TH (human TH variants 1-4) are produced by
alternative splicing of mRNA (Grima et al., 1987; Kaneda et al., 1987). TH variants 1-4 are
identical in their C terminal domain but exhibit differences within the N terminal region
(Kobayashi et al., 1988). Examination of expression pattern indicates that all isoforms of human
TH are expressed in every tissue including the brain (reviewed in Kumer & Vrana, 1996).
Regulation of tyrosine hydroxylase
The diversity in the regulation of TH synthesis and activity allows for a control
mechanism of catecholamine production that is spatially and temporally suited to the
requirement of the cell. Utilizing both long-term and short-term regulation, the cell is able to
direct the production and activity of TH. Long-term regulation of TH includes modulation of
gene expression by transcription regulation, alternative splicing of mRNA, RNA stability, and
translational regulation. Conversely, TH is regulated short term by feedback inhibition,
phosphorylation, and allosteric modulation of enzyme activity (Kumer & Vrana, 1996).
One of the initial observations regarding the regulation of Th gene expression was that
cold stress could increase TH activity in chromaffin cells of the adrenal medulla (Thoenen et al.,
1969, Mueller et al., 1970; Guidotti et al., 1975). Some of these regulatory elements in the
promoter region of the Th gene include glucocorticoid, AP-1, cyclic adenosine monophosphate
(cAMP) responsive element, and hypoxia inducible regulatory elements (Lewis et al., 1983;
Icard-Liepkalns et al., 1992; Best et al., 1995). Other stressors in addition to cold, such as
9
hypoglycemia, immobilization, isolation, and excessive exercise, have been shown to lead to an
increase in TH activity (Wong & Tank, 2007). The pharmalogical manipulation of TH levels
can be accomplished through the use of drugs such as reserpine, cocaine, haloperidol, diazepam
and nicotine (Kumer & Vrana, 1996). Reserpine, which has been shown to deplete
catecholamine stores by inhibiting VMAT function, selectively increases TH levels in the
adrenal gland, peripheral sympathetic cells, and the central noradrenergic cells of the locus
coeruleus (Plescher, 1991; Alterio, et al., 2001). Initially, it was thought that reserpine had no
affect on TH activity in the dopaminergic cells of the substantia nigra (Joh et al., 1973; Zigmond
et al., 1974; Pasinetti et al., 1990). However, it has since been demonstrated that injection of
reserpine in rat subtantia nigra, inhibits TH activity (Sun et al., 1993). Chronic administration of
cocaine, which blocks biogenic amine reuptake into nerve terminals, has been shown to increases
TH mRNA levels, TH activity and TH immunoreactivity in the VTA with no change in the
relative phosphoryalation state of the enzyme (Beitner-Johnson & Nestler, 1991). Additionally,
nerve growth factor and epidermal growth factor can affect TH expression by inducing TH
activity and steady-state mRNA levels (Acheson & Thoenen, 1987; Lewis & Chikaraishi, 1987).
Phosphorylation, which increases TH activity, is a mechanism for short-term regulation
of TH and is also an example of posttranslational modification of the enzyme. Therefore, it is no
coincidence that the analysis of the mechanisms and components of TH phosphorylation is
probably the most prevalent in the study of TH regulation. These studies have identified seven
kinase systems that directly phosphorylate TH within the N- terminal regulatory domain at Ser 8,
Ser 19, Ser 31, and Ser 40 (Joh et al., 1978; Campbell et al., 1986; Zigmond et al., 1989; Haycock,
1990; Sutherland 1993; Toska et al., 2002; Kansy et al., 2004; Nakashima et al., 2011).
Moreover, these phosphorylated residues are conserved among rats and human with the
10
exception of Ser 8, which has been replaced in human isoforms with threonine (Grima et al.,
1987). In vitro studies of TH phosphorylation targets have demonstrated that phosphorylation of
Ser 19 has no direct effect on TH activity whereas the phosphorylation of Ser 31 results in a 2-fold
increase in TH activity. Of the four serines phosphorylation sites for TH, Ser 40 has been shown
to be the most prominent in the regulation of enzyme activity. The phosphorylation of Ser 40
results in a 20-fold increase in TH activity. TH is phosphorylated at Ser 40 by number of kinases,
such as CaM kinase II, PKC, cyclic GMP dependent protein kinase, MAPK-activated protein
kinase, p38-regulated/activated kinase and mitogen- and stress- activated protein kinase
(reviewed in Dunkley et al., 2004). Of these kinases, cyclic AMP-dependent protein kinase
(PKA) has been shown to the most important in phosphorylating Ser 40 and thus increasing TH
enzyme activity (Fitzpatrick, 1999). When Ser 40 is phosphorylated by PKA, it lowers the Km of
TH for BH4 and increases the inhibitory constant (Ki) for feedback inhibitors such as DA
(Zigmond et al., 1989; Goldstein et al., 1995).
TH activity can also be influenced by its association with heparin, polyanions and
phospholipids at places other than the active site of the protein (Lloyd & Kaufman, 1975; Raese
et al., 1976; Katz et al., 1976; Lloyd, 1979). Allosteric effectors increase enzyme activity by
decreasing the Km of the enzyme for its pterin cosubstrate, BH4 (Kumer & Vrana, 1996).
The stability of TH mRNA is an important factor in the regulation and function of TH.
Several studies have concluded that factors important to transcription, such as cAMP,
glucocorticords, nicotine, and hypoxia are also key components in modulating stability of TH
mRNA (Fossom et al., 1992; Craviso et al., 1992; Czyzyk-Krzeska et al., 1992, 1994). CzyzkKrzeska et al. (1992) demonstrated that hypoxia stimulated the synthesis and release of DA from
O2 sensitive cells in the mammalian carotid bodies but not in other catecholamine tissues such as
11
the adrenal gland or the sympathetic ganglia. In a follow-up study, utilizing pheochromocytoma
(PC12) clonal cell line as an experimental model, Czyzk-Krzeska and colleagues (1994) further
demonstrated that hypoxia enhances the rate of transcription of the Th gene and results in a 3fold increase in stability of TH mRNA.
To maintain TH homeostasis and prevent cellular damage, two forms of catecholamine
feedback inhibition can negatively regulate TH activity. The first method is based on the
concentrations of catecholamine product and is readily reversible. During this process,
catecholamine such as DA competitively inhibits TH activity by binding to TH at the same site
as BH4 (Goldstein, 1995). The second mechanism of inhibition involves the association of DA
with ferric iron (Fe3+) at the active site of TH. This method, which is nearly irreversible, not
only decreases enzyme activity but also stabilizes TH (Okuno & Fujisawa, 1991). The formation
of this catecholamine-metal complex results in oxidation of Fe3+ and consequently the
inactivation of the enzyme (Okuno & Fujisawa, 1985; Okuno & Fujisawa, 1991).
Drosophila tyrosine hydroxylase
As in mammals, TH is the rate-limiting enzyme in the production of DA in Drosophila,
and is encoded by the pale (ple) locus (Neckameyer & White, 1993). ple, which maps to the
same chromosomal region as Drosophila TH (DTH) was first recovered as a recessive
embryonic lethal (Nüsslein-Volhard et al., 1985). Later, the isolation and characterization of
DTH by Neckameyer and Quinn (1989) indicated that DTH is the homologue of rat TH and
shares 50% sequence identity with its mammalian counterpart. Additionally, many of the
regulatory components of mammalian TH are conserved within the fly (Vie et al., 1999). The
discovery of the Drosophila gene encoding TH has led to many studies that seek to evaluate the
molecular kinetics of TH regulation and function in insects. The pale locus encodes two
12
isoforms, DTH 1 and DTH 2, which are generated by alternative splicing (Neckameyer & White,
1992; Birman, 1994). Although both isoforms are expressed in all stages of development, the
minor isoform, DTH 1, is expressed only in the CNS where as the major isoform, DTH 2, is
expressed predominately in the hypoderm, where it participates in the synthesis of cross-linked
cuticles (Birman, 1994). There are also structural differences between both isoforms that may
affect the regulatory properties of DTH. DTH 2 adds an acidic domain of 71 amino acids in the
regulatory part of the enzyme, causing it to have a higher specific activity than unphosphorylated
DTH 1, which must be phosphorylated to achieve maximum activity (Birman, 1994). Analysis
of purified DTH recombinant proteins revealed that the mammalian equivalent of Ser40, which is
important for phosphorylation in mammals, is conserved in Drosophila at Ser32 and is
phosphorylated by PKA (Vie et al., 1999). Interestingly, site-directed mutagenesis of Ser32
abolished the phosphorylation of both isoforms. Additionally, the results of this study suggest
that there are several important regulatory differences between DTH 1 and DTH 2 that is most
likely related to differences in structure. DTH 2 in comparison to DTH 1 has a lower affinity for
BH4, a broader pH profile, increased specific activity, and the inability to be stimulated by PKA
phosphorylation (Vie et al., 1999).
Tetrahydrobiopterin function and dysfunction
The pteridine, BH4, is a required cofactor for the catalytic activities of phenylalanine
hydroxylase, tyrosine hydroxylase, tryptophan hydroxylase, all isoforms of nitric oxide synthase,
and glycerol ether monoxygenase (Kaufman, 1963; Nagatsu et al., 1964; Lovenberg et al., 1967;
Kwon et al., 1989; Kaufman et al., 1990). These enzymes are essential for the maintenance of
diverse body functions such as blood pressure regulation, immune function, neurotransmission
and the conversion of phenylalanine to tyrosine (Thony et al., 2000. Tight regulation of BH4
13
synthesis, therefore, is essential; defective synthesis is implicated in a broad spectrum of medical
conditions.
Physiological studies have demonstrated that BH4 is involved in neuronal cell
proliferation and survival (Koshimura et al., 1999). As it relates to neuronal health, several
mechanisms have been proposed for the neuroprotective capabilities of BH4. BH4 is subject to
oxidation by a number of reactive species such as the hydroxyl radical and reactive nitrogen
species (Heales et al., 1988; Heales et al., 2004). Therefore, its ability to react with oxidants
suggests antioxidant properties, a function that is supported by the evidence of Gramsbergen et
al., (2002) who noted that under conditions of glutathione depletion and incubation of
nigrostriatal cultures with the BH4 precursor, sepiapterin, enhanced production of BH4
substituted for a loss of the autoxidant. In another study, Nakumara and colleagues (2000)
demonstrated that BH4 functions as an antioxidant and prevented cell death in glutathionedepleted neurons. Moreover, it has also been suggested that BH4 functions as a scavenger of
reactive oxygen species and regulates cell survival during paraquat exposure (Kojima et al.,
1995). However, the neuroprotective function of BH4 is challenged by the reports of some
studies, that demonstrate that exogenous BH4 can elevate oxidative stress and adversely affect
neuron survival in some disease models (Choi et al., 2004; Kim et al., 2004; Choi et al., 2006).
Several of the BH4-requiring enzymes noted above catalyze the rate-limiting reactions in
the synthesis of neurotransmitters, and mis-regulation or defective synthesis of this cofactor has
been implicated in many neurological disorders and diseases. Loss of BH4, due to mutations in
the gch-1 gene, is one cause of hyperphenylalaninemia, an atypical form of phenylketunoria
(PKU), and dopa responsive dystonia (Ichinose et al., 1995; Nagatsu et al., 1997; Hirano et al.,
1998). Limited availability of this cofactor has also been implicated in features of several
14
disorders such as Alzheimer’s disease, Parkinson’s disease, depression, and autism (Hamon &
Blair, 1986; Thöny et al., 2000; Richardson et al., 2007; Nagatsu et al., 2007). Moreover, loss of
BH4 results in a reduction in TH levels, which consequently leads to neurological anomalies such
as physical and mental retardation, seizures, and hypotonia. Based on these findings, it can be
hypothesized that maintaining BH4 homeostasis is a crucial component in the regulation of cell
function and health (Longo, 2009).
Tetrahydrobiopterin biosynthesis
BH4 is synthesized via two pathways: the de novo synthesis and the ‘salvage’ pathways.
The de novo synthesis pathway requires Mg2+-, Zn2+-, and NADPH-dependent reactions, and
begins with the conversion of guanosine triphosphate (GTP) to the first intermediate,
dihydroneopterin triphosphate (H2NTP) by GTP cyclohydrolase 1 (GTPCH1) (Thöny et al.,
2000). This process is the first and rate-limiting step in the de novo biosynthesis of BH4 (Nichol
et al., 1985; Weisberg & O’Donnell 1986). Following this conversion, 6-pyruvoyltetrahydropterin synthase (PTPS) modifies H2NTP to form the second intermediate in the BH4
biosynthesis pathway, 6-pyruvol tetrahydropterin (PTP). The final step in this pathway is the
reduction of PTP, by sepiapterin reductase, to BH4 (Matsubara & Akino, 1964; Matsubara et al.,
1966).
The ‘salvage’ pathway begins with the conversion of sepiapterin into BH2 by the enzyme
sepiapterin reductase. BH2 is then is converted into BH4 by the NADPH dependent enzyme,
dihydrofolate reductase (Nichol et al., 1985). However, it appears that the salvage pathway
function cannot compensate entirely for BH4 deficiencies resulting from defects in the de novo
biosynthesis pathway (Thöny et al., 2000).
15
Figure 1.2. Tetrahydrobiopterin (BH4) biosynthesis pathway. The de novo synthesis of BH4
begins with the rate-limiting step within this pathway, the conversion of guanosine triphosphate
to dihydroneopterin triphosphate. This is followed by the modification of the two intermediates
by the enzymes 6-pyruvol-tetrahydropterin synthase (PTPS) and sepiaterin reductase (SR),
respectively, into the end product, BH4. The salvage pathway begins with the conversion of
pterin 4 a-carbinolamine by the enzymes pterin-4a-carbinolamine dehydratase (PCD) and
dihydropteridine reductase (DHPR) into BH4.
16
Regulation of GTP cyclohydrolase
Mammalian GTPCH is encoded by a single copy gene, gch1, which is comprised of six
exons spanning 30kb (Ichinose et al., 1995). As previously discussed, GTPCH is the ratelimiting enzyme in the production of BH4 which is a necessary cofactor for the catalytic activity
of TH, the rate limiting enzyme in dopamine synthesis. Structural analysis of E. coli GTP
cyclohydrolase 1 reveals that the holoenzyme is a homodecamer consisting of two tightly
associated pentamers. The protein ranges in mass from 300-500kDA, depending on the species,
but all forms of GTPCH I seem to be comprised of homodecamers (Nar et al., 1995). Each
subunit consists of a catalytic domain and an N-terminal helical domain that extends outward
from the enzyme’s core. Sequence analysis reveals that the C-terminal domain of the protein is
evolutionarily conserved with 60% residue identity between human and E. coli forms of the
enzyme (Witter et al., 1996). However, the N-terminal regulatory domain is variable among
vertebrates and invertebrates. In mammals, six alternatively spliced transcripts produce GTPCH
polypeptides, although only the longest polypeptide has been demonstrated to assemble into a
catalytically active enzyme (Gütlich et al., 1994). Because GTPCH is the rate-limiting enzyme
in BH4 synthesis, mutations in the gch1 are a major cause of disorders associated with BH4
deficiencies. More than 100 mutations have been identified in patients with GTPCH1
deficiencies, most of which are heterozygous and result in the dominant genetic disorder DOPAresponsive dystonia (Thöny & Blau, 2006). Other GTPCH mutations are recessive, and
homozygous individuals are afflicted with severe BH4 deficiencies that are characterististic of the
disorder. Analysis of these patients reveals that, in the absence of BH4 supplementation, they
suffer from convulsions, severe developmental retardation, muscular hypotonia, and frequent
hyperthermia, as a consequence of defects in the biosynthesis of catecholamine and serotonin
17
(Niederwieser et al., 1984). GTPCH1 deficiency has also been implicated in vascular diseases
such as diabetes, hypertension, and atherosclerosis (Stroes et al., 1997; Meininger et al., 2000;
Cosentino et al., 2001). Moreover, these disorders are also associated with the abnormal
function of the enzyme nitric oxide synthase, which, like the aromatic amino acid hydroxylases,
also requires the BH4 cofactor for catalytic function.
GTPCH protein levels and enzymatic activity are regulated by transcriptional and posttranslational mechanisms that range from the induction of transcription by cytokines to the
regulation of enzyme activity by phosphorylation. At the transcriptional level, it has been
demonstrated that pro-inflammatory cytokines, such as interferon-γ, interlukin-1β and tumor
necrosis factor-α can increase intracellular levels of GTPCH in a variety of mammalian cell lines
and tissue cultures (Frank et al., 1998; Katusic et al., 1998; Franscini et al., 2003). Other factors
such as lipopolysaccharides, neurotrophic factor, cAMP, the transcription factor Nurr-1 and
glucocorticoids have been shown to have positive effects on GTPCH levels (Serova et al., 1997).
Interestingly, glucocorticoid interaction with the glucocorticoid receptor has also been
demonstrated to have a negative effect on GTPCH expression (Mitchell et al., 2004). The basis
for this discrepancy is unclear. Additionally, it has been reported that alternative splicing in
human peripheral blood mononuclear cells can have both positive and negative effects on
GTPCH expression with the negative effect acting as a dominant-negative mutation (Hwu et al.,
2003). Two alternatively spliced mRNAs, type I and type II, have been reported for GTPCH
(Togari et al., 1992). Both mRNAs are identical for the 5’ region but differ in their 3’ make up
between exons 5 and 6. Type 1 represents the full length mRNA with a long noncoding 3’
region encoded by exon 6, whereas type II is a truncated mRNA due to a splice between intron 5
and exon 6. Examination of type I and type II effect on GTPCH protein levels in peripheral
18
blood mononuclear cells indicated that type II has a dominant –negative effect by disrupting the
catalytic activity of wild-type GTPCH.
The labeling of mammalian cell cultures with 32P first revealed that GTPCH activity
could be increased by phosphorylation (Hesslinger et al., 1998). A similar study conducted by
Lapize and colleagues (1998), implicated protein kinase C in this modification. These
investigators were able to decrease GTPCH activity by treating cells with the PKC-specific
inhibitor, RO-31-8220. Recently, it was proposed that Ser81 is an important phosphorylation site
in human GTPCH, and that this site is phosphorylated by casein kinase II (Widder et al., 2007).
Tumor necrosis factor-α and Janus kinase-2 were also reported to have positive effects on
GTPCH activity (Vann et al., 2002; Shimizu et al., 2008). Few of these studies, however,
included stringent structure function analysis to clearly identify activating residues in GTPCH.
While phosphorylation is an important component in the up-regulation of GTPCH
activity, end product feedback inhibition of BH4 is probably the most efficient down-regulation
mechanism. In mammals, GTP cyclohydrolase 1 feedback regulatory protein (GFRP) functions
in both positive and negative regulation of the enzyme, maintaining intracellular levels of BH4 at
or below that needed by BH4 requiring enzymes (Harada et al., 1993). Harada and colleagues
(1993) noted that the inhibition of GTPCH1 occurred by the formation of a complex between
BH4, a small protein termed “p35” in that study, and GTPCH. In the presence of BH4, p35,
which was subsequently named GFRP, inhibited GTPCH activity in a dose-dependent manner
with no inhibition occurring in the absence of BH4. Subsequent gel filtration purification and
crystallographic studies led to a model for this association, in which two pentamers of GFRP
associate with the decameric GTPCH1, lowering the Vmax for the GTP substrate in a
noncompetitive manner when BH4 is also bound to GFRP (Yoneyama & Hatakeyama, 1998;
19
Maita et al., 2001; Harada et al., 1993). As a positive regulator, the association of GFRP with
GTPCH1 and phenylalanine increases GTPCH activity (Harada et al., 1993). The inhibition of
GTPCH activity by the association of GTPCH, BH4, and GFRP can be reversed by
phenylalanine, and it is postulated that phenylalanine displaces BH4 during this process
(Yoneyama & Hatakeyama, 1998; Xie et al., 1998; Maita et al., 2004). The association of
phenylalanine with GFRP reduces the positive cooperativity of GTPCH1 and as a result,
increases the enzyme’s activity in saturating amounts of GTP. However, it should be noted that
in the absence of GFRP, phenylalanine has no effect on GTPCH1 activity (Harada et al., 1993;
Maita et al., 2001).
Drosophila GTPCH
Drosophila GTPCH is evolutionarily conserved with all other forms of the enzyme and
shares 80% sequence identity in its catalytic domain with its mammalian counterparts (McLean
et al., 1993). As in mammals, Drosophila GTPCH is the rate-limiting enzyme in the production
of BH4 (Weisburg & O’Donnell, 1986; Hatakeyama et al., 1989). There are four isoforms of
GTPCH in Drosophila, all encoded by the Punch (Pu) locus (Mackay & O’Donnell, 1983;
McLean et al., 1993). These forms, unlike alternative forms of the mammalian protein, have
identical C-termini, and distinct N-terminal domains (McLean et al., 1993), and at least three of
the four forms are catalytically active (Funderburk et al., 2006). The fourth form has not yet
been assayed in vitro to confirm its activity.
The Drosophila transcripts, as well as the isoforms encoded by them, are spatially and
temporally regulated in complex patterns (Mackay et al., 1993; McLean et al., 1993; Chen et al.,
1994). Analysis of the expression patterns of these transcripts revealed that Transcript A is
expressed primarily in the newly eclosed heads of adult flies, Transcript B is abundantly
20
expressed in the larvae and adult head, and Trancript C has been found in all stages of
development (McLean et al., 1993). Previous research in our lab demonstrated that the Nterminal domains are significantly longer than in any other organism, and they are functionally
similar to GFRP in mammals in that these domains serve to down-regulate catalytic activity
when in an unphosphorylated state, while serving as targets for activating phosphorylation events
(Funderburk et al., 2006). Additional roles of GTPCH in Drosophila include the regulation of
pigments in the developing eye, regulating brain and tracheal development, and mediating
embryonic nuclear division (O’Donnell, 1989; Chen et al., 1994; Hsouna et al., 2007).
Zinc function
The small size of zinc (Zn2+), 65Å, as well as the physical and chemical properties of this
element, makes it highly adaptable for meeting the needs of enzymes and other proteins that
carry out diverse biological functions (Caujungco & Gordan, 1997). Advances in molecular
techniques in detecting and determining Zn2+ levels in vivo as well as more efficient methods in
the isolation, purification, and characterization of macromolecules, have led to a wealth of
knowledge establishing this trace element as an integral part of many biological processes and an
essential nutrient for all organisms on earth (Caujungco & Gordan, 1997; Gaither & Eide, 2001).
The importance of Zn2+ function is further underscored by the requirement of this element for
over 300 enzymes in microorganisms, plants, and animals (Vallee & Falchuk, 1993). Thus, the
requirement of this element for many biological proecesses, means that Zn2+ is usually found
associated with proteins within the cell (Williams, 1989).
Zn2+ is an integral component in the metabolism of proteins, lipids, carbohydrates, and
nucleic acids (Valle & Falchuk, 1993). Additionally, Zn2+ has been demonstrated to have an
inhibitory and catalytic effect on several enzymes (Caujungco & Gordan, 1997). In this
21
capacity, Zn2+ is required for numerous ligases, oxioreductases, transferases, hydroxylases, and
isomerases (Valle & Falchuk, 1993). Zn2+ is also an essential structural component of many
proteins including the ubiquitous Zn2+ finger DNA binding proteins (Rhodes & Klug, 1993). In
addition, calcium-binding proteins, with helix-loop-helix motif, contain high affinity Zn2+
binding sites and have been implicated in an array of biological processes from cell membrane
rearrangement to brain-related diseases (Baudier & Kuznicki, 1983; Filipek et al., 1990; Sheng et
al., 1998; Mbele et al., 2002). Studies of brain disorders have also shown that Zn2+ is an integral
factor in the mis-regulation of protein folding (Mantyh et al., 1993; Bush et al., 1994; Clements
et al., 1996; Chattopadhyay & Valentine, 2009). As it relates to gene expression, Zn2+ modulates
DNA and RNA activity by regulating activator proteins, which in turn, control transcription and
replication of DNA.
Although there is an abundance of Zn2+ within the cell, the amount of free Zn2+ is
minimal due to the association of Zn2+ with proteins and the efficient transport and storage of
Zn2+ within vesicles (Caujungco & Gordan, 1997). To maintain Zn2+ homeostasis and prevent
oxidative damage, free Zn2+ may be sequestered by metallothioneins, taken up by organelles, or
transported in or out of the cell by Zn2+ transporter proteins (Cole et al., 1999). This tight
regulation of Zn2+ homeostasis is a contributing factor to the idea that Zn2+ is a relatively nontoxic molecule (Plum et al., 2010). Moreover, no disorder has been linked to an abundance of
Zn2+ (Vallee & Falchuk 1993; Plum et al., 2010). In contrast, studies in a variety of organisms
have concluded that Zn2+ deficient cells fail to divide and differentiate as a consequence of cell
cycle arrest during development (Vallee & Falchuk, 1993).
22
Zinc metabolism
The Zn2+ requirement of various enzymes and the need to tightly regulate the homeostatic
condition of the cell is accomplished through the manipulation of ion influx and efflux by Zn2+
transporters, and the chelation of Zn2+ by apothionein or other Zn2+ sequestering proteins and
amino acids (Caujungco & Gordan, 1997). Methallothioneins, a group of low molecular weight
proteins, aid in maintaining Zn2+ homeostasis by binding to and therefore detoxifying reactive
metals and free radicals (Caujungco & Gordan, 1997; Palmiter, 2004). However,
metallothionein apparently is not used for long-term storage of Zn2+ as it has been shown to have
a short biological half-life in mammalian cell cultures (Krezoski et al., 1988). Zn2+ transport is
tissue and condition dependent and is regulated according to the needs of particular cell types.
Under conditions of Zn2+ toxicity, when extracellular Zn2+ levels are high, Zn2+ may enter into
neurons via N-methyl-D-aspartate (NMDA) receptors, α-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid (AMPA)/kainite receptors, voltage-dependent calcium channels or
transporter-mediated exchange with intracellular sodium (Sensi et al., 1997).
Whether transporting Zn2+ into intracellular organelles, to serve as cofactors for various
enzymes, or operating as a detoxifying agent by facilitating the sequestration of Zn2+ within
organelles or the efflux of this ion across the plasma membrane, Zn2+ transporters (ZnTs) play an
essential role in modulating Zn2+ metabolism (Gaither & Eide, 2001). Much of what was known
initially regarding ZnT function is due to the results of studies conducted in yeast. These studies
have revealed the presence of at least two transport systems within this organism. One system
has a high affinity for Zn2+ and is active in Zn2+-limited cells (Zhao & Eide, 1996a), while, the
other system has a low affinity for Zn2+ and is active in Zn2+ depleted cells (1996b). Therefore,
it is suggested that both systems are specific for Zn2+ and probably do not contribute to the
23
accumulation of other metals (Zhao & Eide, 1996a). ZrT1 and ZrT2 are genes that encode the
transporter proteins of the high and low affinity systems, respectively (Zhao & Eide, 1996a, b).
Both genes are transcriptionally regulated by the transcriptional activator ZAP1 and encode
members of the Zrt, Irt-like protein (ZIP) family of metal ion transporters (Zhao & Eide, 1997).
In addition, yeast Zn2+ levels are regulated by the endocytosis of ZrT1, a plasma membrane
protein, which prevents the import of excess Zn2+ within the cell (Gitan et al., 1998).
ZnT function is conserved from prokaryotes to eukaryotes. In bacteria, two ZnT families that
function in Zn2+ uptake and export have been identified. The ABC family of ZnT proteins
functions in Zn2+ uptake. One such transporter, the ZnuABC is a major source of Zn2+
accumulation in the cells of E. coli (Patzer & Hantke, 1998). Unlike the ABC family of proteins,
the family of P-type ATPases has been shown to demonstrate Zn2+ export function in bacteria.
For example, E.coli ZntA plays an important role in Zn2+ detoxification by pumping the metal
ion out of the cell when intracellular levels are too high (Rensing et al., 1997). In eukaryotes,
ZIP family plays a prominent role in uptake and the ‘cation diffusion facilitator’ (CDF) family
promotes Zn2+ efflux. The ZIP family transports Zn2+ from outside the cell into the cytoplasm
and has been found to mobilize stored Zn2+ by transporting the metal from intracellular
compartment into the cytoplasm (Gaither & Eide, 2001). Alternatively, the CDF family pumps
Zn2+ from the cytoplasm out of the cell or into the lumen of an organelle (Gaither & Eide, 2001).
The ZIP family of proteins was first discovered in Saccharomyces cerevisae and
Arabidopsis thaliana and has been shown to transport Zn2+ and iron, respectively (Zhao & Eide
1996a; Eide et al., 1996). More than 100 members of this family have been identified in bacteria,
fungi, protozoa, insects, plants, and mammals (Gaither & Eide, 2001; Taylor & Nicholson, 2003;
Lichten & Cousins, 2009). ZIP family members are characterized by a predicted topology in
24
which the amino and carboxy termini are found on the extracellular surface of the plasma
membrane. In addition, most ZIP proteins have approximately eight transmembrane domains
and the presence of a cytoplasmic histidine rich loop (Gaither & Eide, 2001). Based on the
number of members of the ZIP family of proteins and the higher degree of sequence
conservation among groups, the ZIP family of proteins is divided into four subfamilies:
subfamilies I and II, gufA and LIV-1 subfamily of ZIP Zn2+ transporters (LZT) (Gaither & Eide,
2001; Lichten & Cousins, 2009). Subfamily I consists largely of fungal and plant members,
whereas subfamily II is a smaller group of nematode and mammalian proteins (Guerinot, 2000).
The GufA group consists of prokaryotes and eukaryotic members and are proteins of unknown
function (McGowan et al., 1993). The LZT subfamily is restricted to eukaryotes; its founding
member is a protein of unknown function that has been linked to metastatic breast cancer gene,
LIV-1 (Manning et al., 1995). Like other members of the ZIP family of proteins, the LZT
subfamily also has histidine-rich repeats present in the cytosolyic loop between transmembranes
III and IV. In addition, this subfamily also has histidine residues on the extracellular loop
between transmembranes II and III and in the extracellular N terminus (Rogers et al., 2000).
Though the function of these additional histidine residue is unknown, the presence of these
repeats on both sides of the membrane is unprecedented and are believed to be involved in the
transport of metal ions (Rogers et al., 2000). In addition, conserved amino acid regions include a
novel metalloprotease motif that is not found in other three subfamilies of ZlP transporters.
Taylor et al. (2004) has further divided this subfamily into a KE4 subgroup. The KE4 subgroup,
which was first identified in the mouse, has been demonstrated to localize to the endoplasmic
reticulum and not the plasma membrane (Suzuki & Endo, 2002). Analysis of the cellular
location of HKE4 protein, the human homologue of the mouse KE4 gene, demonstrates that it
25
also localizes to intracellular membranes, including the endoplasmic recticulum (Taylor et al.,
2004). In addition, HKE4 has been shown to increase the intracellular Zn2+. Among this group
is CATSUP from Drosophila, which is a negative regulator of tyrosine hydroxylase. Although
the exact zinc related function of CATSUP has not been determined, analysis of the KE4
subfamily has implicated this protein family in ion transport and the regulation of tyrosine
hydroxylase (Stathakis et al., 1999; Lasswell et al., 2000). The main physiological function of
ZIP proteins is yet to be determined. However, mutations within the hZIP4 gene in humans have
been linked to the Zn2+ deficiency disease acrodermatitis enteropathica, which is caused by a
defective uptake of Zn2+ in the small intestine (Kury et al.; 2002; Wang et al., 2004).
As previously stated, the CDF family of proteins is involved mostly in the transport of
Zn2+ from the cytoplasm out of the cell or into the compartments of organelles. Like the ZIP
family of ZnT, groups of protein within this family have been divided into subfamilies, I, II, and
III, based on sequence similarities. Subfamily I consists mostly of prokaryotic members, while
subfamilies II and III contain an approximately equal representation of prokaryotic and
eukaryotic members. The CDF family of proteins is characterized by six transmembrane
domains with a great degree of conservation among proteins within transmembrane domains I,
II, and V. In addition, most members of the CDF family share a similar predicted topology
where the amino and carboxy termini are cytoplasmic. Topological analysis of the CzcD protein
in bacteria has confirmed some elements of this topology (Anton et al., 1999). Several members
of the CDF family of ZnT have been identified in humans and mouse. To date, 10 ZnTs, ZnT 110, of the CDF family of proteins have been identified in humans. ZnT1 is a Zn2+ efflux protein
found within the plasma membrane of neuron and glial cells and in this capacity is believed to
play a role in cellular detoxification of Zn2+ by exporting excess metal ion out of the cell (Gaither
26
& Eide, 2001; Sekler et al., 2007). Analysis of mouse brains, demonstrates that ZnT1 is
localized to regions rich in synaptic Zn2+ and its expression is transcriptionally regulated in
correlation with the appearance of synaptic Zn2+ (Sekler et al., 2002; Nitzan et al., 2002).
Moreover, over-expression of ZnT-1 has been shown to protect against Zn2+ toxicity in neurons
and glial cells (Palmiter, 2004; Nolte et al., 2004). Znt-2 is believed to be involved in
intracellular Zn2+ sequestration and is located in the membranes of an acidic compartment that
accumulates Zn2+ when cells are grown in concentration of 100 or 200 µM Zn2+ (Gaither & Eide,
2001). Like ZnT-2, ZnT-3 also sequesters Zn2+ and is required for transport of Zn2+ into synaptic
vesicles where it is presumed to play a neuromodulatory role (Palmiter et al., 1996; Cole et al.,
1999). Similarly, ZnT4-ZnT10 are also involved in Zn2+ export (Luizzi & Cousins, 2004).
Zinc and the brain
The importance of Zn2+ for proper brain function is demonstrated by the abundance of this
element in brain tissue with respect to other organs. The average total brain Zn2+ concentration is
approximately 150 µmol/L (Takeda, 2000). However, much of this Zn2+ is associated with other
proteins and therefore estimation of free Zn2+ is sub-nanomolar and is estimated to be
approximately 500 nM in brain extracellular fluids (Weiss et al., 2000).
The main supply of Zn2+ to the brain is through the blood- brain barrier system (Pullen et
al., 1990; Franklin et al., 1992). Found within the plasma are large components of exchangeable
Zn2+ associated with albumin and amino acids such as histidine and cysteine (Hallman et al.,
1971; Harris & Keen, 1989). L-histidine, both in the plasma and in cerebrospinal fluid (CSF),
plays a significant role in transferring Zn2+ to target sites (Takedo et al., 2002). Inside the brain,
Zn2+ is transferred freely throughout the cerebrospinal and the brain extracellular fluid
compartments.
27
Once Zn2+ has been taken up by a neuron, it is transported anterogradely and retrogradely
via the axonal transporting system. These Zn2+-containing neurons sequester Zn2+ in presynaptic
vesicles and release it in a calcium-dependent manner (Takeda, 2001). Additionally, Zn2+
sequestered in synaptic vesicles is released with glutamate and may modulate neurotransmission
(Takeda, 2001). Once inside the synaptic cleft, Zn2+ may be taken up by postsynaptic and
presynaptic neurons (Takeda et al., 1997). The uptake of Zn2+ into postsynaptic neurons as a
result of excessive excitation of Zn2+-containing glutamate neurons, can lead to degeneration of
these cells (Sloviter, 1985; Choi et al., 1988; Tonder et al., 1990; Choi & Koh, 1998).
Examination of Zn2+ levels within various sections and tissues of the brain have
demonstrated that excitatory glutamatergic neurons, located in the forebrain regions, are Zn2+enriched neurons (ZEN) or cells that contain free Zn2+ ions in the vesicles of their presynaptic
boutons (Slomianka, 1992; Frederickson and Moncrieff, 1994). These regions include the
amygdala, the hippocampus, and the neocortex. ZEN have also been found in the spinal cord
and the cerebellum. However, most of the ZEN in the spinal cord are inhibitory γ-aminobutyric
acid (GABA)ergic (Danscher et al., 1994).
The storage and release of Zn2+ within glutaminerigic and GABA neurons has led to the
idea that Zn2+ may function as a neuromodulator. In this capacity, Zn2+ that is released from
synaptic vesicle is believed to interact with certain neurotransmitter receptors. Several studies
have indicated that the release of Zn2+ from synaptic vesicles results in the inhibition of NMDA
receptors (Paoletti et al., 1997; Vogt et al., 2000; Kay, 2003). Glutamate is the major excitatory
transmitter employed in the nervous system. The release of Zn2+ with glutamate reduces the
ability of glutamate to activate NMDA receptors, by direct interaction with NMDA receptors at
the post-synaptic level (Smart et al., 1994). Moreover, the Zn2+ effect on glutamate neurons is
28
significant because glutamate is the most abundant neurotransmitter in the brain and Zn2+ is
found predominately within the synaptic vesicles of glutamate neurons. In addition, Zn2+ has
been shown to inhibit GABA receptors (Ruiz et al., 2004). The results of these studies indicate
that Zn2+ has an inhibitory effect on the major excitatory and inhibitory neurotransmitters of the
CNS.
Based on the abundance of Zn2+ in brain tissues and increasing evidence of its role in
diverse cellular functions in neurons, it is now generally accepted that Zn2+ transport is an
essential component in modulating brain function. As previously mentioned, ZnTs are
responsible for transporting Zn2+ into synaptic vesicles. In mammals, ZnT-3 has been implicated
as the main transporter that functions in this capacity. Cole et al. (1999) has demonstrated that
knockout of ZnT3 in mice results in a complete loss of Zn2+ within synaptic vesicles and a 20 %
reduction of Zn2+ levels in the hippocampus and cortex (Cole et al., 1999). Loss of ZnT3 in
these animals did not affect memory or sensorimotor function, but these animals were more
prone to seizures and seizure-related neurodegeneration than controls (Cole et al., 2000, 2001).
However, the lack of phenotype in these animals remained puzzling to researchers. Recently, it
was determined that ZnT3 knockout mice exhibited age-dependent learning deficits that were
manifested at 6 months but were not present at 3 months. Additionally, manipulation of
vesicular Zn2+ by Zn2+ deprivation diets and the use of the Zn2+ chelator, cliniquol, resulted in
reduced hippocampal neurogenesis in mice and rats (Suh et al., 2009).
Neuronal mitochondria can also uptake Zn2+ as a way of maintaining Zn2+ homeostasis in
the cell (Sensi & Jeng, 2004). This finding is significant because mitochondrial dysfunction is
thought to be a key component in many neurological disorders such as Parkinson’s and
Alzheimer’s disease (Cappaso et al., 2005; Jones, 2010; Santos et al., 2010). Moreover, excess
29
mitochondrial Zn2+ can lead to dysfunction within this organelle due to prolonged ROS
generation (Sensi & Jeng, 2004; Frazzini et al., 2006; Sensi et al., 2008).
Zinc and dopamine
The DAT located at the presynaptic termini is responsible for modulating DA
neurotransmission by rapidly clearing DA from the synapse and hence, terminating DA signaling
and controlling the duration of synaptic inputs into the brain (Jones et al., 1998; Pifl et al., 1995;
Norregard et al., 1998). Structure-function analysis of endogenous Zn2+ binding sites within the
DAT has revealed that Zn2+ inhibits DAT function and attenuates DA content (Norregard et al.,
1998; Loland et al., 1999; Meinild et al., 2004). Interestingly, Zn2+ modulates DAT function not
by preventing DA binding but by inhibiting the translocation of this neurotransmitter. Moreover,
it has been demonstrated that Zn2+ inhibition of DAT function is not due to a conformational
change of the protein but rather a change in the membrane potential by the depolarization of
DAT (Meinild et al., 2004). Others studies have indicated that DAT also mediates DA efflux
(Levi & Raiteri, 1993; Pifl et al., 1995). Therefore, Zn2+ may function as a neuromodulator of
DA homeostasis by not only regulating the clearance of DA from the synapse, but also the
exocytotic release of DA.
Zn2+ binding sites have also been identified within DA receptors (Liu et al., 2006) Studies
of the molecular interaction of the DAT and Zn2+ have determined that Zn2+ allosterically
modulates D2 receptor function by preventing DA from binding to the receptor (Schetz & Sibley,
1997; Schetz et al., 1999; Liu et al., 2006). In addition, Zn2+ also affects post-synaptic dopamine
interactions by inhibiting D1 receptor function (Schetz & Sibley, 1997). Moreover, inhibition of
DA receptor function is specific to Zn2+, given the fact that other metals such as manganese had
no effect on receptor function (Schetz & Sibley, 1997).
30
Sequence analysis of proteins involved in the metabolism of DA has also identified Zn2+
binding sites on GTPCH1. Analysis of the crystal structure of GTPCH in mammals and bacteria
revealed the presence of Zn2+ ions at the active site of both the inhibitory and stimulatory
complexes of this enzyme (Maita et al., 2002; Maita et al., 2004). Moreover, Zn2+ is required for
the catalytic activity of purified GTPCH in bacteria and mammals (Auerbach et al., 2000). This
is significant because the synthesis of BH4, a necessary cofactor in the synthesis of DA, is
regulated by GTPCH1. In addition, concentrations greater than 1mM Zn2+ are capable of
inhibiting purified GTPCHs from bacteria and mammals (Kohashi et al., 1980; Blau et al., 1985;
Kwon et al., 1989). Thus, these studies suggest that Zn2+ can regulate DA neurotransmission and
function by directly interacting with components of DA homeostasis. However, further studies
need to be conducted to determine if Zn2+ is a neuromodulator of DA.
Zinc and neurodegenerative diseases
Increasingly, metals, including Zn2+, are being implicated in the etiology of many
diseases. Although alteration of Zn2+ homeostasis may be a component of brain dysfunction and
neurological diseases, the mechanism of alteration is poorly understood. Moreover, the
requirement of Zn2+ for so many biological processes and the abundance of this metal within the
brain make it difficult to decipher whether alteration in Zn2+ homeostasis is the end result of
neurological disease or a contributing factor in this process. Therefore, researchers have been on
a quest to examine the biological significance of Zn2+ metabolism in many neurological
disorders.
As previously discussed, Zn2+ is tightly regulated within the brain by the blood brain
barrier, ZnTs, metallothionein, and other Zn2+–binding proteins. The inability of a diseased cell
to maintain proper Zn2+ homeostasis subsequently leads to a toxic accumulation of Zn2+ and is
31
believed to be a causative component in the neurodegeneration of brain cells. Koh and
colleagues (1996) have demonstrated that excessive increase of intracellular Zn2+ in the brain due
to transient forebrain ischemia in rats’ results in neurodegeneration. In addition, some studies
have indicated that Zn2+ enters into post-synaptic neurons in toxic excess during seizures and
traumatic brain injury (Frederickson et al., 1989; Suh et al., 2000). Moreover, the influx of toxic
amounts of Zn2+ from pre-synaptic vesicles into post-synaptic neurons seems to be a factor in the
neurodegenerative process (Koh et al., 1996; Suh et al., 2000). All together, this body of
evidence not only demonstrates that excess Zn2+ is toxic to cells, but supports the concept that
Zn2+ may be a constituent in the onset and development of neurological disorders such as
Amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), and Parkinson’s disease (PD).
ALS is a progressive neurodegenerative disease that results in the deterioration of
anterior horn cells in the spinal cord and brain motor cortex cells, which in turn, leads to
progressive muscle degeneration, paralysis, and death (Boillée et al., 2006; Pasinelli & Brown,
2006; Banks et al., 2008). Though this disease is mostly sporadic, approximately 10% of all
cases are inherited or familial (Pasinelli & Brown, 2006). Among the few genes that have been
identified, mutation of the gene encoding the ubiquitously expressed enzyme superoxide
dismutase (SOD-1) accounts for 20% of familial ALS and 1% of sporadic ALS (Rosen et al.,
1993; Pasinelli & Brown, 2006). The causes of ALS remain undefined; however, the recent
discovery of TAR DNA binding protein 43 (TDP-43) in the inclusion bodies of ALS
degenerating neurons have led to new insights into the pathophysiology of this disease (Boillée
et al., 2006; Arai et al., 2006; Caragounis et al., 2010). As a result, ubiquitinated neuronal
aggregates with TDP-43 inclusions are now understood to be a pathological hallmark of ALS.
32
The ubiquitously expressed TDP-43 has RNA recognition and glycine rich motifs and
may normally function to regulate transcription (Ayala et al., 2005). Although, TDP-43 is a
nuclear protein, aggregates often occur in the cytoplasm and in the cerebrospinal fluid (Geser et
al., 2008). The biochemical mechanisms underlying the modification of TDP-43 in affected
neurons, is the result of C-terminal fragmentation, phosphorylation, and ubiquitination (Nonaka
et al., 2009). Analysis of molecular determinants of TDP-43 inclusion in yeast has demonstrated
that alteration in TDP-43 localization is a consequence of increased TDP-43 expression (Johnson
et al., 2008). The mis-localization of TDP-43 led to a dramatic inhibition in the growth of yeast
cells and provided initial direct evidence that TDP43 cytoplasmic aggregation may be a factor in
the pathogenesis of TDP-43 related neuropathies. Additionally, TDP-43 cytoplasmic expression
and aggregation resulted in oxidative damage and induced the generation of superoxide
dismutase, indicating that TDP-43 aggregation may be a component in the induction of cellular
toxicity. It was recently determined that Zn2+ modulates TDP-43 metabolism by decreasing
nuclear expression of endogenous TDP-43 and resulted in the formation of TDP-43 positive
inclusion in SY5Y neuronal like cells (Caragounis et al., 2010). Moreover, analysis of the role of
altered metal homeostasis in this disease process revealed that aggregation of TDP-43 was Zn2+
specific, since neither copper nor iron affected TDP-43 metabolism (Caragounis et al., 2010).
Therefore, Zn2+ is now thought to be an integral component in the neurodegenerative process in
ALS; however, further studies need to be conducted to determine if Zn2+ is indeed a contributing
factor in this disease.
The neurological disorder AD is one of the main causes of dementia in older adults and is
characterized by a loss of cholinergic neurons as well as the progressive deterioration of
cognitive function and memory (Drachman & Levitt, 1974; Davis & Maloney, 1976). The main
33
pathological hallmark of this disease is the accumulation of amyloid-β peptide (AB) within
senile plaques in the brain as well as the deposition of neurofibrillary tangles (NFT) and
neurophil threads (Cras et al., 1991; Perry et al., 1991). AB peptide is generated from the
amyloid precursor protein (APP) by the proteolytic activity of β- and γ-secretase (Checler, 1995).
Though most cases of AD are sporadic, about 5-10% of AD patients suffer from familial AD that
is marked by an autosomal dominant inheritance of a mutation within the APP gene (ChartierHarlin et al., 1991; Murrell et al., 1991). Specific and saturable binding sites for zinc and copper
have been identified within the cysteine-rich region of the APP-695 conserved ectodomain
indicating that these metals may be an intergral component in the proper regulation of this
protein (Bush et al., 1993; Hesse et al., 1994). In addition, the AB peptide, which is central to
progression of AD, has been shown to directly produce hydrogen peroxide (H202) due to metal
ion reduction (Huang et al., 1999). Analysis of the role of metal ion homeostasis in the
pathogenesis of AD indicated that the induction of H2O2 displaced Zn2+ from metalloproteins,
and metallothioneins and may be a contributing factor in the up-regulation of Zn2+ in AD brains
(Fliss & Menard, 1992; Danscher et al., 1997; Cornett et al., 1998). These findings are supported
by evidence indicating that metal chelators are able to combat the progressive decline of AD.
Cherny et al. (2001) have shown that the ingestion of the metal chelator clioquinol (CQ) in
transgenic mice expressing human Aβ peptide reduced Aβ plaque deposits and improved the
overall health and weight of the animals. Moreover, the use of metal chelators as a theraputic
agent in reducing the progression of AD has shown promising results in a pilot phase II clinical
trail of CQ among moderate to severely affected AD patients. These patients exhibited a
reduction in cognitive decline most likely due to a decrease in Aβ42 plasma levels (Ritchie et al.,
2003).
34
While the exact cause of PD is unknown, many studies have indicated that Zn2+-induced
neurotoxicity may be a significant player in the pathogenesis of this disease (Gorrel et al., 1999;
Kim et al., 2000). Post-mortem analysis of PD patients demonstrates an accumulation of free
zinc within the nigrostriatal dopaminergic system (Dexter et al., 1989). However, given the
requirement of Zn2+ for so many processes within the brain, it is currently unclear whether Zn2+
is a contributing factor in PD. In fact, some studies have suggested that Zn2+ may actually be
neuroprotective. Ahn et al. (1998) noted that the depletion of endogenous Zn2+ in mouse cortical
neuronal cultures resulted in the induction of cell death by modulating NMDA receptor function.
This finding is supported by the results of Peters et al. (1987) and others (Choi et al., 1998; Ma &
Zhao, 2001) who noted that blockage of NMDA function, attenuates NMDA receptor-mediated
neurotoxicity. Although the exact mechanism is still undefined, the blockage of NMDA
receptors by Zn2+ is believed to prevent the binding of NMDA receptor antagonists.
Consequently, activated NMDA receptors are unable to mediate glutamate-induced
excitotoxicity (Choi, 1992). As it relates to PD, dysfunction in trafficking and subunit
composition of NMDA receptors is believed to be involved in the pathogenesis of dyskinesia or
abnormal involuntary muscle movement (Bagetta et al., 2010). Moreover, NMDA receptor
antagonists have been demonstrated to inhibit the induction of nitric oxide (NO) synthesis by
acting on alpha synuclien (Radenovic & Selakovic, 2005; Adamczyk et al., 2009). Alphasynuclein (α-synuclein), a small presynaptic protein, has been found in the Lewy bodies of PD
patients and is believed to be an important component in the pathogenesis of PD. Therefore, by
blocking NMDA receptor function through the use of antagonist, alpha synuclein levels are
reduced and NO synthesis is inhibited. Studies have indicated that increased synthesis of NO
results in the activation of a neuroinflammatory response in which macromolecular oxidation as
35
well as mitochondrial and DNA damage occurs (Chalimoniuk et al., 2006; Aquilano et al.,
2008).
In contrast to the reported neuroprotective abilities of Zn2+, other studies have indicated
that Zn2+ may be an integral component of the neurodegenerative process of PD. These studies
have demonstrated that infusion of Zn2+ (10-1000 mM) induced neurotoxicity in cultured cortical
neurons and in the hippocampus and nigrostriatal dopaminergic system of mice (Yokoyama &
Koh, 1986; Choi et al., 1988; Canzoniero et al., 1997; Lin, 2001). More recently, Lin et al.
(2003) have established that zinc-induced oxidative stress may result in apoptosis and reduced
DA function in the nigrostriatal dopaminergic system of rats. Interestingly, the administration of
vitamin D3 abolished this Zn2+ -stimulated oxidative stress and prevented apoptosis.
There are two proposed mechanism for vitamin D3 neuroprotection. First, vitamin D3
reportedly up-regulates factors such as glial cell line-derived neurotrophic factor (GDNF).
Analysis of GDNF function indicates that it enhances DA uptake and promotes the survival of
DA neurons in disassociated rat embryo cultures (Lin et al., 1993). In addition, the
administration of vitamin D3 attenuated 6-OHDA induced neurodegeneration by reducing DA
and its metabolites in rats (Wang et al., 2001). Secondly, vitamin D3 prevented zinc-induced
lipid peroxidation. This is significant because the products of lipid peroxidation have been
demonstrated to inhibit DA catabolism by reducing mitochondrial aldehyde dehydrogenase
[ADLH2] (Jinsmaa et al., 2009). ADLH2 is necessary for the oxidation of 3,4dihydroxyphenylacetaldheyde (DOPAL) to DOPAC. Thus, the inhibition of ALDH2 function by
lipid peroxidation prevents the proper metabolism of DA and results in an increase in DOPAL
levels. Interestingly, DOPAL has been shown to be more toxic than DA and its metabolites and
is therefore relevant to PD pathogenesis (Burke et al., 2003).
36
Alpha synuclein is the major filamentous component of Lewy bodies and is therefore
considered an integral factor in the pathology of PD (Heintz & Zoghbi, 1997; Clayton & George,
1998). Transgenic mice over-expressing α-synuclein developed inclusions and a loss of
dopaminergic terminals. Interestingly, Zn2+ has been shown to induce the aggregation of αsynuclein in vitro by causing a conformational change in the protein (Kim et al., 2000; Lowe et
al., 2004). To examine the role of Zn2+ in the substantia nigra (SN) and determine the effect of
Zn2+ on the intrinsic electrical properties of this region of the brain, Chung et al. (2000)
employed a whole cell recording method. This study concluded that Zn2+ hyperpolarized SN
neurons and modulated their voltage-gated ion content, which contributed to an increase of
neuronal excitability to incoming stimuli. This excitatory effect may compensate for the loss of
dopaminergic neurons as PD develops, as well as the ability to relay chemical messages.
Collectively, these findings suggest that Zn2+ may be an essential factor in the pathophysiology
of PD.
37
CHAPTER 2
ZINC AND DOPAMINE: A SYNERGISTIC RELATIONSHIP?
Introduction
Dopamine (DA) regulates various processes from development to locomotion.
Therefore, the dysregulation of DA homeostasis is implicated in the pathogenesis of many
neurological diseases. Probably the most prominent of these DA related disorders is Parkinson’s
disease (PD). PD is characterized by a preferential loss of DA neurons within the substantia
nigra leading to a reduction of DA content, locomotor deficits and ultimately, death (Farrer,
2006). The rich biological evidence that exists in this area of research has implicated many
genetic and environmental factors in the etiology of this disease. Epidemiological studies have
associated environmental factors such as pesticide use, rural living, and heavy metals as potential
factors in the neurodegenerative process of PD (Tanner & Goldman, 1996; Firestone et al., 2005;
Jankovic, 2005; Anderson, 2004; Priyadarshi et al., 2001). Moreover, these studies suggest that
exposure to environmental risk factors may subsequently lead to neurodegeneration due to
biochemical abnormalities such as oxidative stress and mitochondrial dysfunction (Schapira,
2006).
Analyses of PD brains have revealed a buildup of free Zn2+ within the substantia nigra,
for which there is no clear biological requirement (Dexter et al., 1989, 1991). Based on the
evidence that high concentrations of free Zn2+ can be toxic (Revuelta et al., 2005), the role of
Zn2+ toxicity in combination with the genetic components of DA homeostasis was investigated
38
as possible mechanism in pathogenesis of PD. In this study, it was hypothesized that feeding
flies excess Zn2+ in combination with mutations in genes that are involved in DA synthesis and
transport would increase susceptibility to this transition metal. Furthermore, these mutants
provide a strong tool for modeling the in vivo conditions of extreme DA dysregulation and may
provide new insights into the disease process of PD. One of the mutants used in this study,
Catsup, is predicted to contain seven transmembrane domains and is a member of the ZIP family
of Zn2+ transporters that include the mammalian KE4 protein (Begum et al., 2002; Carbone et al.,
2006). Additionally, Catsup is a negative regulator of TH, and is therefore an important
modulator of DA synthesis (Stathakis et al., 1999). Thus, loss of Catsup protein results in an
elevation in DA pools, but these flies are surprisingly, paraquat resistant (Chaudhuri et al., 2007).
The results of this study demonstrate that Catsup mutant strains are Zn2+ sensitive.
Interestingly, the Drosophila DA transporter mutant fumin (fmn) is also Zn2+sensitive.
Moreover, Zn2+exposure at low concentrations for 12 hrs, increases mobility in wild type strains
but reduces DA content. Finally, we show that exogenous DA in combination with Zn2+
exposure increases Drosophila’s susceptibility to Zn2+ toxicity. These findings suggest that Zn2+
is a possible trigger in the etiology and pathophysiology of PD.
Materials and Methods
Drosophila strains and culture maintenance:. All strains were maintained on standard
cornmeal-agar medium at 25o C. Strains used as controls in this experiment included Canton S
and a white-eyed mutant, Df(1)w, y. Both are wild type for the DA-regulating genes under
examination. The Catsup mutant alleles used in this study, Catsup26/CyO and Catsup12/CyO
flies, have been previously described (Stathakis et al. 1999). These mutant heterozygotes were
crossed into either Df(1)w, y or Canton S backgrounds. The fumin mutant flies (fmn) were a
39
generous gift from J. Hirsh, University of Virginia. These flies have a genetic lesion abolishing
dopamine transporter function (Kume et al., 2005) and were crossed into Df(1) w, y backgrounds
unless otherwise noted. Foi mutants were obtained from Bloomington stock center and have
been previously described (Matthews et al., 2006). The UAS-DAT RNAi strains were obtained
from the Vienna Drosophila RNAi Center (Vienna, Austria). The TH-GAL-4 strain (FriggiGrelin et al., 2003) was a generous gift from J. Hirsh (University of Virginia).
Zinc feeding experiments. Adult male flies (n=10 per vial) were collected and aged 24 to 72 hrs
post-eclosion before being placed in vials of 1% agar containing 5% sucrose, or 5% sucrose and
1 mм, 5 mм, or 10 mм ZnCl2 (Sigma, St. Louis, MO). Their survival was monitored at 12 hrs
intervals until zinc-fed flies had died. Approximately, five vials and 50 flies were used per
treatment.
Locomotion assay. Adult male flies, 48 hrs post-eclosion, were collected, and each fly was
transferred, individually, to a food vial for 1 hr. The vial then was gently tapped to bring the fly
down to the bottom, and mobility was measured by counting the number of seconds the fly was
in motion during a 45 sec time period following the tapping. All assays were conducted during
the afternoon. Fifteen to twenty flies were assayed once for each data set.
DA analysis. Heads from adult male flies, 48 hrs post-eclosion, were collected by freezing the
flies in liquid nitrogen, vortexing and sieving to separate the heads from bodies. Approximately
75-100 heads were homogenized in 75-100 µl of cold 0.1 M perchloric acid by using a motorized
tissue grinder. The extracts were chilled on ice for 15 min to allow proteins to precipitate.
Samples were pelleted by centrifugation at 9300 x g for 10 min at 4oC. The supernatants were
removed and immediately stored at –80oC before HPLC analysis. Catecholamine levels were
determined using a CoulArray HPLC system (model 5600A; ESA, Chelmsford, MA) and a
40
Synergi 4 µm Hydro column (4.6 X 150 mm; Phenomenex, Torrance, CA) as described by
McClung and Hirsh (1999) with modifications. Extracts were filtered through 0.2 µm filters.
Ten µl of each filtrate were injected. The mobile phase contained 75 mм sodium phosphate, pH
3.0, 1.4 mм octanesulfonic acid, 25 µм ethylene diamine tetraacetic acid, 100 µl/L triethylamine,
and 7% acetonitrile. Separations were performed with isocratic flow at 1ml/min. Amines were
detected with an ESA electrochemical analytical cell (model 5011; channel 1 at-50mV, channel 2
at 300mV). Pool sizes were determined relative to freshly prepared standards (Sigma). Analysis
was performed using ESA CoulArray software.
Statistical analysis. Analyses of all data were conducted using GraphPad Prism (San Diego,
CA), using a two-tailed Student’s t test assuming equal variances, or using a one-way ANOVA
followed by the Dunnett’s post test. Details of analyses are described in the figure legends.
Results
Catsup mutants are Zn2+ sensitive
Since Catsup is a putative Zn2+ transporter and functions as a regulator of DA synthesis
and transport, the hypothesis that this protein is an integral component in Zn2+and DA
interactions that are suggested to play a role in the etiology of PD was tested. Specifically, the
combination of elevated DA synthesis and transport with the proposed Zn2+ transport
dysfunction expected in Catsup mutants could interact to alter neuronal function and to enhance
the susceptibility of Catsup mutants to Zn2+. This prediction was analyzed by comparing the
effects of Zn2+ ingestion by Catsup12 and Catsup26 mutants with the response of wild type adult
flies. Wild type flies that were fed Zn2+ exhibited neurological symptoms such as tremor and
paralysis, 24-48 hrs after the exhibition of these symptoms in Catsup mutants. Moreover,
survival rates of Catsup mutants compared to those of wild type flies fed 1 or 10 mм ZnCl2 were
41
significantly reduced, indicating that some component of Catsup function is an essential factor in
modulating the toxic effects of Zn2+ (see Figure 2.1).
42
43
Dopamine increases Zn2+ sensitivity in Catsup mutant and wild type flies
Catsup mutants have elevated DA pools due to the increased activity of GTPCH and TH.
Since synergistic interactions between DA and Zn2+ have been shown to lead to increased death
in PC12 cell lines (Lo et al., 2004), the hypothesis that the elevation of DA pools in Catsup
mutants contribute to their hypersensitivity to Zn2+ was tested. If the sensitivity of Catsup
mutants is associated with DA- Zn2+ interactions, then elevating DA pools in wild type flies
through ingestion of DA should increase the toxic effects of Zn2+, while ingestion of DA by
Catsup mutants might be expected to have little or no additional consequence.
Wild type and Catsup mutants were pre-fed DA for 24 hrs and then fed Zn2+ for 3 days, a
period of time resulting in approximately 50% mortality in flies fed Zn2+only. In this
experiment, flies were fed a higher concentration of Zn2+ than employed in the previous
experiment (10 mм vs 1 mм) to obtain a more rapid response in the induction of toxicity under
conditions of DA pre-feeding. Prior trials at lower concentrations of Zn2+ did not show a
synergistic effect between Zn2+ and DA, probably because DA pools had most likely returned to
basal levels by the time Zn2+ toxicity ensued. Though the concentrations of Zn2+ and assay time
points were different for this experiment, similar results in survival on Zn2+ alone was manifested
(Figs 2.1 and 2.2A). Feeding of 1 mм DA alone did not affect the survival of either wild type
control or Catsup mutant flies during the time course of these experiments (data not shown).
However, the implementation of increased DA in wild type flies dramatically increased Zn2+
toxicity in comparison to flies that had been fed only Zn2+ (see Fig 2.2 A). In contrast, the Zn2+
and DA combination resulted in a non-significant increase in sensitivity in Catsup mutants (Fig
2.2B), demonstrating that Zn2+ had a more robust effect on wild type flies than Catsup mutants
under conditions of DA supplementation. This difference in Zn2+ sensitivity between wild type
44
and Catsup mutants presumably is due to the fact that DA pools in Catsup mutants are already
elevated several folds, creating conditions in which toxic DA-Zn2+ interactions are so severe that
additional DA cannot further accelerate the damage to neurons. It is also plausible, that Catsup
mutant flies have compensated for increase DA synthesis by modulating packaging and
transport. Previous research in our lab has shown that Catsup mutants are resistant to reserpine,
which attenuates VMAT function (Wang et al, submission in progress). Therefore, as it relates
to the sensitivity that is seen in Catsup mutants, it is believed that some factor other than elevated
DA is integral in this process. Taken together, these findings demonstrate that DA in
combination with Zn2+ increased the toxicity of Zn2+ mostly in wild type flies and further support
models that predict an integral role for this metal in the neurodegenerative process of DAassociated diseases such as PD. However, the evidence from this experiment also suggests that
the sensitivity of Catsup mutant to zinc toxicity is due to some other factor other than elevated
DA synthesis.
45
46
Zinc increases adult mobility but reduces DA pools
It is believed that the metabolism of DA is an essential component in the
neurodegenerative process of PD. Moreover, it has been demonstrated that free radicals can be
generated by the autoxidation of DA (Dunnett & Björklund, 1999). Since we noted that
exogenous DA enhances zinc related mortality, here we explored possible Zn- DA interactions
by assaying for Zn- mediated effects on DA- dependent mobility and on the oxidative
metabolism of DA. For this experiment, flies were fed 1 mм ZnCl2 for 6 hrs, after which, they
were placed on regular fly medium for the duration of the experiment. Mobility assays were
performed post-Zn2+ exposure, and DA pools in the heads of these flies were then measured to
determine whether Zn2+ was affecting DA metabolism.
As seen in Figure 2.3, ingestion of excess Zn2+ for a period of only 6 hrs is associated
with reduced DA content in head extracts and increased DOPAC levels. We have previously
shown that oxidative stress increases DOPAC levels and results in DA neuron degeneration
(Chaudhuri et al., 2007). Therefore, the increased DOPAC levels as a consequence of exposure
to excess Zn2+ is suggestive of elevated oxidative stress and is in agreement with the findings of
previous studies of zinc-exposed cells (Hsiao et al., 2004). In addition, the reduced DA pools are
consistent with the conversion of cytoplasmic DA to DOPAC and to other oxidative metabolites
in the neuron.
47
48
Previous studies in our laboratory and others (Pendleton et al., 2002; Chaudhuri et al., 2007)
indicate that diminished DA pools, as detected above, are associated with decreased mobility. It
was, therefore, surprising that flies exposed to either 0.1 or 1 mм Zn2+ for 6 hrs demonstrate
increased mobility 24 hrs after Zn2+ exposure (see Figure. 2.4.A). Even more surprising, is that
mobility remained elevated in this group for three weeks post initial Zn2+ exposure (see Figure
2.4.B). Therefore, these findings suggest that Zn2+may be directly or indirectly affecting the
availability of synaptically active DA, and that the alterations in DA metabolism or transport
induced by Zn2+ are permanent synaptic changes.
49
50
Loss of DAT results in increased sensitivity to Zn2+
Even though Zn2+ decreases DA pool in wild type flies, it also increases mobility and is
most likely due to alteration in DAT function. Interestingly, high affinity Zn2+binding sites have
been identified in the human dopamine transporter (hDAT), and Zn2+ was found to act as a
potent, non-competitive blocker of DA uptake in hDAT-expressing COS cells (Norregaard et al.,
1998). Furthermore, it has been demonstrated that the inability to properly clear DA from the
synaptic region of the brain results in hypermobility in mice and flies lacking the DAT (Giros et
al., 1996). These studies, along with the fact that Catsup mutant are hypermobile and Zn2+
sensitive, suggest that DAT function may be compromised in Catsup mutants and is further
enhanced by Zn2+ exposure. Therefore, we hypothesized that loss of DAT function will result in
increased Zn2+ sensitivity. To test this hypothesis, wild type and DAT mutant (fmn) flies, as well
as flies in which DAT RNAi was expressed in dopaminergic cells, were fed 1 mм ZnCl2 and
average survival duration was measured. As seen in Figures 2.5 and 2.6, the reduction of DAT
function, in DAT mutants and DAT knock-down flies, increased Zn2+ sensitivity relative to the
effect of Zn2+on control flies. Thus, we conclude that a reduced level of DAT is an important
factor in not only modulating the physiological effects of DA but also in regulating survival
under conditions of exposure to toxic levels of Zn2+.
Simultaneous reduction in Catsup and DAT function enhances Zn2+ sensitivity
To further explore the functional relationship between Catsup and DAT, transheterozygous double mutants flies were created. These flies were fed 1 or 10 mм ZnCl2 for 4
days, the point at which approximately 50 % of the Zn2+ fed flies had died. As shown in Figure
2.6, double mutant flies that were fed 1mM ZnCl2 were significantly more sensitive to Zn2+ than
flies’ mutant for Catsup alone. In addition, the results of Figure 2.6 A demonstrate that the
51
transheterozygous fmn/Catsup26 mutants display an intermediate zinc-sensitivity relative to either
single heterozygous mutant. Thus, the findings of this study indicate that both of these proteins
are significant players in mediating the toxic effects of Zn2+and that loss of Catsup partially
rescues DAT mutant sensitivity. The fact that we saw no significant differences in survival
between DAT and transheterozygous mutants that were fed 10 mм Zn2+ is most likely a
consequence of the increased toxicity of Zn2+at high concentrations and demonstrates why it was
necessary to repeat this experiment at 1 mм ZnCl2. However, it is intriguing to note that the
sensitivity of the heterozygous mutants was similarly affected at both concentrations. The
increased sensitivity of the fmn/Catsup26 mutants in comparison to Catsup heterozygotes suggest
that there is a functional relationship between these proteins in regulating DA metabolism and
that this relationship is a significant factor in modulating the response to Zn2+ toxicity.
52
53
Figure 2.6. Catsup-DAT interactions in zinc toxicity. DAT and Catsup double mutation increases
Zn2+ sensitivity, relative to Catsup26/+ alone. DAT (fmnub), fmnub/Df(1)w, y; Casup26, Df(1)w, y;
Catsup26/+ and Df(1)w, y flies 1-3 days post-eclosion were fed continuously with 1 mм (A) or 10 mм
ZnCL2 dissolved in 1% agar and 5% sucrose. The number of dead flies was counted twice each day
for 5 days and the data were expressed as average percent mortality. Each value is the average of 5-10
replications, and each replication was made up of 10 flies. Statistical analysis was performed using
Student’s t-test and one way ANOVA (**, p<0.01). Error bars indicate ± SEM.
54
Fears of intimacy (foi) mutants are Zn2+sensitive and hypermobile
Given the proposed role of Catsup as a Zn2+ transporter, we sought to determine whether
the loss of another Zn2+ transporter function similarly affected Zn2+ sensitivity. To address this
issue, we tested another mutant member of the LIV-1 subfamily of Zn2+ transporters, fear of
intimacy (foi), for zinc sensitivity. The results of this experiment (see Figure. 2.7) demonstrate
that heterozygous foi mutants are significantly more sensitive to Zn2+ exposure than wild type
flies. In addition, loss of foi function is associated with hypermobility in comparison to wild
type flies (see Figure 2.8). These findings are similar to the phenotypes of Catsup mutant.
Although both proteins are proposed members of the ZIP family of transporters, we did not
anticipate that foi mutants would demonstrate a phenotype similar to Catsup mutants given
Catsup’s distinct function as a regulator of DA synthesis. Interestingly, reduction of both Catsup
and foi expression further enhances mobility in comparison with flies that are mutant for Catsup
only (see Figure 2.8). However, we did not note a significant difference between foi mutants and
the transheterozygous mutants. Thus, the component that is modulating mobility is not enhanced
by the loss of Catsup in a foi mutant background. Taken together, these findings suggest that
disruption of Zn2+ transporter function makes cells more susceptible to Zn2+ toxicity probably as
a result of an inability to properly regulate Zn2+ homeostasis. Thus we conclude that mutations
in modulators of DA biosynthesis, metabolism and transport, as well mutations in Zn2+ transport,
enhances susceptibility to Zn2+ toxicity and may be a useful tool in assessing the role of Zn2+
exposure in DA associated neurodegenerative diseases.
55
Figure 2.7. The effect of Zn2+ on lifespan in foi mutants. Mutation of foi increases Zn2+ sensitivity. A
strain containing the mutant foi allele, foi20.71, and Df(1)w, y flies 1-3 days post-eclosion were fed
continuously with 1 mм ZnCl2 dissolved in 1% agar and 5% sucrose. The number of dead flies was
counted twice each day for 4 days, the time period at which approximately 50% of the Zn2+ fed flies had
died and the data analyzed as average percent mortality. Each value is the average of 5-10 replications,
and each replication was made up of 10 flies. Statistical analysis was performed using Student’s t-test
(***p<0.001). Error bars indicate ± SEM.
56
Figure 2.8. foi and foi/Catsup transheterozygous mutants are hypermobile. Mobility was
determined in 2-3 day old Catsup/+, foi/+ and Catsup/foi transheterozygous mutants relative to
wild type flies. Mobility assays were conducted by measuring time in motion of each fly for a
period of 45 seconds. Each value is the average of 15-20 replications. Statistical analysis of the
difference between Catsup26/+, foi/+, and Catsup/foi was performed using one way Anova
(**, p<0.01, ***, p<0.0001). Error bars indicate ± SEM. NS: not significant
57
LiCl rescues the sensitivity of Catsup mutants to Zn2+
LiCl is used predominately as a mood stabilizer to treat mood disorders such as bipolar
disease. However, lithium (Li+) also been shown to be effective in preventing neurodegeneration
(Bhalla et al., 2010). Here, I sought to determine if LiCl is an effective agent in preventing or
reducing Zn2+ toxicity in this Drosophila model. To address the possible neuroprotective role of
LiCl, several different combinations of treatments were performed with wild type and Catsup
flies. The final concentrations used were based on preliminary dosage trials to identify non-toxic
concentrations. In the experiments presented here, 20 mм LiCl, a concentration that had almost
negligible effects on the viability of either wild type or Catsup mutant flies, was employed. As
expected, ZnCl2 alone at a concentration of 10 mм was very toxic to both wild type and Catsup
mutant flies, with the latter being more sensitive to Zn2+ exposure, displaying an average
mortality of nearly 70% by day 3, relative to approximately 20% mortality for the wild type
strain. Combining Li+ with Zn2+ resulted in a striking reduction in Zn2+toxicity, producing a 5-6fold reduction in mortality for both the wild type and Catsup mutant strains (see Figure 2.8).
This finding is particularly intriguing, suggesting that further exploration of the effects of lithium
on Zn2+-DA interactions will be a fruitful avenue for investigating the mechanisms of zinc
toxicity as well as the mechanisms of Li+ rescue of neurodegeneration observed in other models.
58
Figure 2.9. LiCl suppresses the toxicity of ZnCl2 in Catsup mutant and wild type flies.
Catsup mutant and Df(1)w, y (WT) flies, 1-3 days post eclosion, were fed continuously with 10
mм ZnCl2, 20 mм LiCl or 10 mм ZnCl2 and 20 mм LiCl together, dissolved in regular fly
media. The number of dead flies was counted twice each day for 3 days, the time point at which
approximately 50% of the flies had died, and the data were expressed as average percent
mortality. Each value is the average of 5-10 replications, and each replication was made up of
10 flies. (A) Li+ in combination with Zn2+ rescues Zn toxicity in WT. (B) Ingestion of Li+ with
Zn2+ rescues Catsup sensitivity to Zn2+. Statistical analysis were performed using one way
ANOVA ***p<0.001). Error bars indicate ± SEM.
59
Discussion
This report demonstrates several novel findings related to genes that are components of
DA synthesis and transport and their susceptibility to Zn2+ toxicity. For the first time, we show
that Catsup, DAT and foi mutants are zinc sensitive. In addition, we also demonstrate that
exogenous DA increases susceptibility to Zn2+ toxicity and that the mood stabilizer, LiCl,
enhances survival in Zn2+ fed flies. We also noted that Zn2+ reduced DA pools and caused longterm modulation of mobility resulting in hypermobile flies.
Previously, we demonstrated that Catsup mutants are resistant to oxidative stressors such
as paraquat (Chaudhuri et al., 2007). Here, we demonstrate that this resistance does not apply to
Zn2+ toxicity. In addition, exposing Catsup flies to other metals resulted in no significant
differences in sensitivity and thus confirmed the idea that Catsup mutant sensitivity is zincspecific (K. Lackey and J. O’Donnell, unpublished results; Taylor et al., 2004). This finding is
rather intriguing given the fact that the predicted topology of Catsup indicates that it is a
transmembrane protein with several histidine repeats and shares structural homology with known
members of a zinc transporter family of ZIP proteins. Interestingly, we noted that excess Zn2+
not only reduces life span, but also results in parkinsonian-like phenotypes such as tremors and
paralysis. Based on these findings, we sought to gain a better understanding of the functional
relationship between zinc toxicity and the regulation of DA homeostasis.
Catsup functions as a negative regulator of TH and GTPCH and mutations within this
gene result in elevated DA pools (Stathakis et al., 1999; Wang et al., submitted). To assess the
potential role of DA in enhancing the response of Catsup mutants to Zn2+ exposure, we pre-fed
wild type flies DA and then exposed them to Zn2+. In the present study, we noted that DA and
Zn2+ enhanced susceptibility to Zn2+ toxicity in vivo with no significant effects on survival with
60
flies fed DA alone. Previously, Zn2+ and DA have been shown to synergistically enhance the
apoptosis of PC12 cells and the depletion of striatal DA (Lo et al., 2004). The evidence from our
study suggests that the concerted action of Zn2+ and DA may be responsible for PD pathogenesis.
However, the mechanism of this reaction is still unclear. Thus, we hypothesized that the
combination of increased Zn2+ and DA enhances oxidative stress by modulating DA transport
and synthesis. This concept is supported by evidence that Zn2+ can modulate DA homeostasis by
binding to and inhibiting DA re-uptake by DAT and ligand binding to D2 DA receptors
(Richfield, 1993; Schetz & Sibley, 1997). Moreover, various substrates of DAT, including DA
and MPTP (1-methyl-4-phenyl-1, 2, 5, 6-tetrahydropyridine), have been shown to be neurotoxic
(Miller et al., 1999). Here, we demonstrate that loss of DAT enhances sensitivity to Zn2+. The
findings of our study also revealed that a one time exposure to exogenous Zn2+ results in an
increase in mobility and reduction of DA pools, and intriguingly, that these effects are long term.
Additionally, these findings suggest that this single exposure to Zn2+ elicits permanent changes in
DA regulation. However, we were surprised that these flies were hypermobile since loss of DA
pools should result in decreased mobility. Though we cannot definitively conclude from these
findings the mechanism by which Zn2+ modulation of DA content results in hypermobility, we
hypothesized that this is due to the inhibition of the DAT by Zn2+. Moreover, this hypothesis is
supported by the findings of several studies that have concluded that a loss of DAT function
creates excess extracellular synaptic DA and hypermobility in mice (Jones et al., 1998; Zhuang
et al., 2000). Therefore, the observed increase in DOPAC levels indicates an enhancement in DA
turnover and possibly signals the induction of oxidative stress.
The idea that oxidative stress is contributing to neurodegeneration is supported by the
reduction in DA pools following six hours of Zn2+exposure. Additionally, this hypothesis is
61
supported by evidence in DAT knockout mice which revealed that, under normal conditions, the
tissue content of DA metabolites was unaltered; however, inhibition of DAT by cocaine, which
also blocks DA reuptake, increased DOPAC levels and oxidative stress (Jones et al., 1998).
Though the ability of Zn2+ to modify mobility while reducing DA content was fascinating, we
did not expect this behavioral phenotype to continue three weeks following the initial exposure
to Zn2+. Therefore, if Zn2+ is truly acting on DAT to create this behavioral phenotype, this result
suggest that Zn2+ exposure has long term effects on DA transport and homeostasis, perhaps by
mechanisms similar to those involved in long-term alterations following exposure to cocaine
(Meyer et al., 1993; Jones et al., 1998; Bhide, 2009; Kubrusly & Bhide, 2010).
So, is there a connection between DAT and Catsup that contributes to the similarity in
phenotypes? We have previously shown that Catsup mutant flies are extremely hypermobile,
which appears to be the result of a combination of increased synthesis, packaging and release of
synaptically active DA (Wang et al., submitted). Based on the findings that both Catsup and
DAT mutants are Zn2+ sensitive, the effect of Zn2+ on the survival of Catsup and DAT double
mutants were examined. We hypothesized that the loss of both these genes would augment
susceptibility to Zn2+ toxicity. Transheterozygous loss-of-function mutants for DAT and Catsup
genes demonstrate enhanced sensitivity to Zn2+ at both high and low concentrations in
comparison to heterozygous Catsup mutant alone. It also could be argued that the presence of
the Catsup mutation rescued DAT sensitivity to Zn2+ at lower concentrations. However, based on
our findings that this effect is abolished at higher concentration (see Figure 2.6 B), along with the
fact that Catsup mutants are hypermobile, suggesting a deficit in DAT function, we conclude that
the double mutation most likely enhances susceptibility to Zn2+ toxicity due to a further
attenuation of DAT function. Consequently, Catsup and DAT regulation of DA homeostasis is
62
an integral factor in the toxicology of Zn2+ exposure. Although many of findings of this study
are novel and interesting, there are limits to what can be extrapolated from this information. For
example, it is unknown if the double mutation changes extracellular DA content, which would
indicate differences in the inhibition of DAT. Interestingly, mutants that are heterozygous for
DAT were more sensitive to 1mM Zn2+ exposure than double mutants and Catsup mutants. Thus,
the amount of DAT that is functionally available may be an important factor in Zn2+ induced
toxicity. Taken together, the results of this study lead to a model whereby Catsup regulates DAT
function by maintaining Zn2+ homeostasis such that a reduction in the function of either gene
causes DA dysregulation and increases susceptibility to neurotoxicity under conditions of excess
Zn2+ exposure.
63
Figure 2.10. A model of the interaction of Catsup with the genetic regulators of DA homeostasis.
Loss of Catsup results in an elevation in TH activity and thus an increase in DA synthesis. It has been
demonstrated that Catsup physically interacts with TH and thus acts a negative regulator of this enzyme
(Wang et al., submitted). As it relates to VMAT and DAT, there is no evidence that Catsup physically
associates with these proteins, however, we proposed that loss of Catsup enhances VMAT function and
down regulates DAT to compensate for the enhanced metabolism of DA. Specifically, as it relates to
DAT, our data suggest that any loss in Catsup function also compromises DAT function resulting in an
inability to properly recycle DA and a dysregulation of DA homeostasis.
64
In addition to inhibiting DAT, Zn2+ also has been shown to modulate DA receptor
antagonist interactions through allosteric mechanisms (Schetz et al., 1999). These regulatory
properties of Zn2+ are also important to endogenous Zn2+ exposure, because synaptic
concentrations of Zn2+ within the synapse can reach up to 300uM (Assaf & Chung, 1984).
Therefore maintaining Zn2+ homeostasis is an important factor in neuronal health and may be an
important component in the pathogenesis of PD.
Although Catsup mutants are Zn2+ sensitive, we cannot definitively conclude that this
phenotype is due to Catsup’s proposed role as a Zn2+ transporter, as the protein has not been
biochemically analyzed for transport function, nor has Zn2+ distribution in Catsup mutants been
examined. However, it has been shown that Catsup localizes with TH in the synaptic regions of
dopaminergic neurons and that Catsup mutants demonstrate increased synaptic activity in
comparison to wild type. Additionally, mammalian DAT is a pre-synaptic plasma membrane
protein that is susceptible to inhibition by Zn2+. Therefore, Catsup may function by modulating
the access of the DAT to Zn2+ and if so, then Catsup’s proposed role as a zinc transporter could
be a factor in Zn2+ sensitivity. This concept is supported by evidence that under toxic zinc
conditions, when extracellular Zn2+ is high, Zn2+ has many routes to enter neurons including
transporter-mediated exchange with intracellular sodium (Sensi et al., 1997). To examine the role
of Zn2+ transport in modulating the toxic response to Zn2+ exposure, flies that are mutant for the
fear of intimacy (foi) gene, which has been characterized as a Zn2+ transporter protein in
Drosophila were analyzed for Zn2 sensitivity. Interestingly, foi, like Catsup, is a member of the
ZIP family of Zn2+ transporters and has been identified as an essential component of tissue
architecture and in the regulation of development in Drosophila, particularly in the modulation
of the migration of germ cells, glia and tracheal cells (Matthews et al., 2005; Van Doren et al.,
65
2003). In addition, foi belongs to the LIV-1A group of the LIV-1 subfamily of ZIP transporters,
unlike Catsup, which belongs to the LIV-1B group (Matthews et al., 2005). The ZIP family of
zinc transporters has been identified as the main regulator of Zn2+ influx into the cytoplasm. To
determine the importance of Zn2+ transport in Zn2+ toxicity, we fed foi mutants Zn2+ and
measured survival. Like Catsup mutants, foi mutants are also Zn2+ sensitive. Thus, the ability to
regulate Zn2+ influx is an important factor in maintaining Zn2+ homeostasis and may play a
significant role in modulating oxidative stress. Interestingly, the disruption of the ZnT-3 gene,
which is responsible for zinc uptake into synaptic vesicles in mammals, revealed no
morphological differences in brain development, or any differences in Zn2+ content in other
organs such as the pancreatic B-islet cells, germ cells in the testis or the cuboidal cells of the
choroid plexus (Cole et al., 1999). However, examination of degenerating neurons after seizure
induction in ZnT3 null mice indicated zinc accumulation in both mutant and wild type strains
(Lee et al., 2000). Moreover, it in a later study, Lee et al. (2003) suggested that the release of
Zn2+ from methallothionein maybe a contributing factor in neurodegenerative process following
acute brain injury.
All together, the findings of this study have demonstrated that excess DA in combination
with Zn2+, loss of Zn2+ transporter function, loss of DAT, and loss of DAT and Catsup enhances
susceptibility to Zn2+ toxicity. Are all these processes working together under conditions of
oxidative stress or are they separate factors that are involved independently in the toxic process
of excess Zn2+ exposure? As it relates to Catsup mutants, we suggest that these are factors that
work together in increasing susceptibility to Zn2+ with DAT being the main regulator of all of
these processes. The proposed Zn2+ transporter dysfunction of Catsup mutants may result in
increased extracellular Zn2+ and ultimately affect DA neurotransmission. This idea is
66
corroborated by the increased mobility seen in Catsup mutant flies and DAT mutant mice.
However, it should be noted that Catsup mutants are heterozygous and have one functional copy
of the Catsup gene. The fact that excess Zn2+ can affect other components of DA regulation
suggests that exposure to this metal exacerbates the oxidative process. It is unlikely, however,
that Zn2+ directly causes oxidative damage like metals such as copper, because Zn2+ does not
undergo redox changes which can catalyze the generation of increased reactive oxygen species
(Fenton, 1894).
Finally, we examined the effect of lithium, which has been shown to have
neuroprotective property in both mammals and Drosophila (Berger et al., 2005; French and
Heberlein 2009, Toledo and Inestrosa, 2010), on Zn2+ -induced toxicity. Feeding flies LiCl in
combination with Zn2+ significantly reduced Zn2+ induced toxicity. For this experiment several
different concentrations of LiCl were used before determining that 20mM was the most effective
at reducing the toxic effects of Zn2+. Lithium appears to exert its neuroprotective properties by
preventing the activation of glycogen synthase –3 (GSK-3) and by inducing several pathways
including the Wnt/Wg signaling pathway in Drosophila and mammals (Phiel and Klein, 2001).
Interestingly, lithium has been shown to prevent cocaine from binding to DAT, unlike Zn2+,
which stimulates the binding of this DAT inhibitor (Wu et al., 1997). These data suggest that
lithium also regulates DAT function. The ability of both of these cations to modulate DAT
function corroborates our findings, which suggest that the modulation of DAT is an integral
component of the cytotoxic process induced by Zn2+. Our findings along with the wealth of
information on the neuroprotective quality of lithium suggest that this cation may be an
important therapeutic agent in alleviating PD induced neurodegeneration.
67
The mechanism by which lithium attenuates Zn2+ toxicity remains undefined. However,
based on an examination of the literature, we hypothesize that there are two possible mechanisms
by which lithium may exert its effect. It is possible that lithium competes with Zn2+ for binding
to DAT and thus prevent Zn2+ induced DA dysregulation. The other mechanism by which lithium
could exert it effect is by inhibiting GSK3ß under conditions of Zn2+induced toxicity due to DA
dysregulation. Interestingly, GSK3ß activation, has been shown to be a key component in the
modulation of apoptosis (Pap & Cooper, 1998; Bijur et al, 2000; Cross et al., 2001)
While the results from this study provide fascinating insights into the role of Zn2+ toxicity
as a possible contributing factor in PD, there are many questions that are yet to be answered as it
relates to the mechanism of this action. 1) Does Zn2+exposure result in the neurodegeneration of
DA neurons? Here, we noted an increase in the metabolism of DA in Zn2+ fed flies and a
reduction in DA content; however, the effect of Zn2+ on neuronal health is yet to be determined.
In addition, if there are differences in DA neuron count between the respective groups, is this
difference in DA neuron insult specific to dopaminergic neurons? Using DA specific markers
and confocal microscopy we will determine neurodegeneration of DA neuron. To determine if
Zn2+ affects other neuronal subsets, non-DA specific markers will be employed. In addition, the
proposed role of Catsup as a Zn2+ transporter may mean that it is an important component in
regulating Zn2+homoestasis in the brain. The dysregulation of Zn2+ homeostasis may be apparent
if there are differences in Zn2+ levels or localization in Catsup mutants versus wild type flies.
This could be accomplished by staining the brains of both strains with the free zinc marker,
Newport green.
The findings of this study also demonstrated that a one time exposure to Zn2+ for six
hours resulted in reduced DA content and increased mobility. 2) Does Zn2+ -induced reduction of
68
DA pools continue at week three? Having observed that mobility remains elevated three weeks
following initial Zn2+ exposure, it would be interested to note if DA pools remained reduced at
the three week time period. Also, if there is a reduction in DA content, is it progressive
reduction or is DA levels unchanged overtime? Finally, we demonstrate that lithium can be
protective against Zn2+ toxicity. The works of Berger et al. supports this finding, (2005) by
demonstrating that lithium protected against aggregate prone proteins by inhibiting GSK3ß
through the Wnt/Wg signaling pathway in Drosophila. The Drosophila homologue of GSK-3 is
Zeste-white3Shaggy and has been shown to be an essential component of the wingless signaling
pathway. 3) Is Zn2+ toxicity due to induction of apoptosis and does this occur by the modulation
of GSK-3 activity? Given the proposed mechanism by which lithium is thought to be
neuroprotective and based on our evidence that lithium protects against Zn2+ induced toxicity, it
would be rather informative to determine the mechanism by which Zn2+ may trigger
neurodegeneration. Both a mutant strain and an over-expression line of Shaggy is available and
would be a wonderful tool in dissecting the mechanism by which lithium protects against Zn2+,
as well as, provide information regarding the signaling pathway that is involved in this process.
69
CHAPTER THREE
ALTERATION IN DA HOMEOSTASIS BY A REDUCTION IN DAT FUNCTION
ATTENUATES THE TOXIC EFFECTS OF THE HERBICIDE PARAQUAT
Introduction
Dopamine (DA) plays an essential role in proper brain function and is required for
learning and memory, reward, cognitive processing, regulating movement, and the sleep wake
cycle (Graybiel et al., 1994; Schultz et al., 2000; Nieoullon, 2002; Wise, 2004; Dzirasak, et al.,
2006). The dopamine transporter (DAT) is an important component in maintaining DA
homeostasis by modulating DA synthesis and transport (Amara & Kuhar, 1993). This significant
role is underscored by the implication of DAT function in regulating psychological and
neurological processes. Loss of DAT function has been associated with cognitive disorders such
as attention deficit hyperactivity disorder (ADHD) and schizophrenia, as well as
neurodegenerative diseases such as Alzheimer and Parkinson’s (Cohen & Servan-Schreiber,
1992; Cerruti et al., 1993; Swanson et al., 1997; Uhl, 2003; Pritchard et al., 2008). As it relates
to Parkinson’s disease, DAT is believed to be responsible for regulating the access of
neurotoxins such as MPTP (1-methyl-1, 2, 3, 6-tetrahydropyridine), and paraquat to
dopaminergic neurons (Shimizu et al., 2001, 2003; Yang & Tiffany-Castiglioni, 2005; Ritz et al.,
2009; Kumar et al., 2010). Therefore, deciphering the mechanisms by which environmental
toxins gain access to dopaminergic neurons, and particularly, their interactions with the DAT, are
critical to understanding the roles of environmental interactions with gene products implicated in
idiopathic PD, approximately 90% of all PD cases.
70
The herbicide paraquat has been shown to be a DA neuron-specific neurotoxin and is
thought to be an environmental trigger in the neurodegeneration of DA neurons in some PD
patients (McCormack et al., 2002; Li et al., 2005). Structural similarities between DAT and the
toxic MPTP derivative, MPP+, has led some investigator to suggest that paraquat gains access to
DA neurons through the DAT (Shimuzu et al., 2001, 2003). However, whether this hypothesis is
correct and if so, the mechanism by which this is accomplished, remains undefined and
contested. The notion that paraquat exerts its affect on DA neuron through transport by DAT is
refuted by the findings of Richardson et al. (2005), which suggest that paraquat is neither a
substrate nor an inhibitor of DAT.
Analysis of DAT function within Drosophila indicates that this gene is essential to the
regulation of the circadian process and may be critical to DA homeostasis (Kume et al., 2005).
As in mammals, the Drosophila DAT mutants, fumin (fmn), exhibit hyperactivity and decreased
periods of rest and arousal threshold. The conservation of fly DAT function is supported by
recent data that demonstrated that the neurochemical function of fly DAT in regulating DA
synthesis and transport is similar to that of its mammalian counterparts. In this study,
electrochemical analysis of exogenously applied DA indicated a significant elevation in synaptic
DA in fmn mutants, due to a failure to properly recycle DA, compared to wild type controls
(Makos et al., 2009). In addition, clearance of exogenously applied DA was significantly
reduced in wild type flies following cocaine treatment, but no differences were noted in fmn flies
with identical treatments (Makos et al., 2010). Both of these studies provide neurochemical
evidence that dopamine uptake is decreased in fmn flies, and makes this mutant a useful tool to
investigate the role of the DAT in DA associated diseases. Previously, we have shown that
Drosophila is a good model organism to study the susceptibility of DA neurons to paraquat
71
based on genetic variation in DA associated genes. In addition, the results of this study
demonstrated that loss of Catsup, which results in elevation of DA synthesis coupled to enhanced
VMAT activity, which increases the efficiency of DA packaging in vesicles, reduces sensitivity
to paraquat. Due to phenotypic similarities between DAT and Catsup mutants in locomotor
response and in regulating sensitivity to paraquat toxicity, we hypothesized that loss of Catsup
results in a reduction of DAT function. The results of this experiment show that over-expression
of Catsup in TH neurons does not alleviate resistance. Furthermore, the findings of this report
demonstrate that a reduction in DAT function attenuates sensitivity to paraquat and provides
evidence of DAT-Catsup functional interactions.
Materials and Methods
Drosophila strains and culture maintenance. Drosophila strains were cultured on standard
corn-yeast-sugar based media at room temperature (23-25ºC). The Catsup mutants used have
been previously described (Stathakis et al., 1999). The w; TH-Gal4 and fmn (DAT) [Kume et al.,
2005] stocks were a generous gift from J. Hirsh, University of Virginia. The w, UAS-DAT-RNAi
was obtained from the Vienna Drosophila RNAi Center (Vienna, Austria). Dr. Kevin Bowling, a
former graduate student in our lab, generated the UAS-Catsup strain. The strains used as controls
were: Df (1) w, y, w; UAS-DAT RNAi, and w; TH-Gal4.
Behavior. Locomotion was assayed by time-in-motion as described previously (Carbone et al.,
2006) with modifications. Adult males (15-20 individuals of each genotype), 3-7 day old posteclosion, were collected and each fly was transferred to a food vial for 1 hour. Each test vial was
given a gentle mechanical disturbance before beginning the locomotor assay. The mobility of
the fly was measured by counting the number of seconds the fly was in motion during the 45 sec
period immediately following the disturbance. All assays were conducted during the afternoon.
72
Negative geotaxis assays were conducted for some experiments by placing a single fly in an
empty plastic vial. After a ten-minute rest period, the fly was tapped to the bottom of the vial,
and the time to cross 5 cm (within a 60 second time frame) was recorded. The assays were
repeated for 15-20 flies and the scores for each replication were averaged.
Paraquat and reserpine exposure. Two slightly different methods were used for paraquat and
reserpine exposure. For paraquat, separated adult male flies, 24 to 48 hrs old after eclosion, were
placed in vials containing 10 mΜ paraquat mixed with 5% sucrose and 1% agar. For reserpine
exposure, adult male flies, 24-48 hrs after eclosion and aged for 72 hrs, were placed on filter
paper saturated with 5% sucrose or 5% sucrose, 60 mM reserpine. Feeding was continued for a
period of 8-10 hrs.
Statistical analysis. Analyses of all data were conducted using GraphPad Prism (San Diego,
CA) using two-tailed Student’s T-test assuming equal variances or one way ANOVA followed
by Bonferroni’s post-test as required. Details of analyses are described in figure legends.
Results
Expression of a wild type Catsup transgene in dopaminergic neurons does not
ameliorate resistance to paraquat
Having observed previously that loss of Catsup decreased sensitivity to paraquat
(Chaudhuri et al., 2007), the effects of expressing a Catsup transgene in the dopaminergic
neurons of Catsup+ adults in combination with feeding the toxic herbicide, paraquat was
evaluated. If loss-of-function Catsup mutants resulted in elevated DA pools, increased synaptic
release of DA as determined by hypermobility, and resistance to paraquat, then over-expression
of wild type Catsup should result in opposing phenotypes. Thus, Catsup O.E. flies should
demonstrate decreased DA synthesis, less efficient synaptic release and increased sensitivity to
paraquat. As expected, the positive control (Catsup26) demonstrated decreased sensitivity to
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paraquat relative to wild type flies (see Figure 3.1A). However, expression of a Catsup+
transgene in dopaminergic neurons did not result in increased sensitivity to paraquat, but
demonstrated resistance to the herbicide similar to that of Catsup mutants (Fig 3.1 A). Based on
this unexpected result and the knowledge that Catsup is a negative regulator of DA synthesis, the
mobility of the Catsup over-expression (O.E.) strain was assayed to determine if the
hypermobility phenotype of Catsup mutants would also be observed when Catsup is expressed in
dopaminergic neurons.
Catsup over-expression flies are hypermobile
Since Catsup mutants are hypermobile, the hypothesis was formulated that overexpression of Catsup in dopaminergic neurons would result in decreased mobility. This
hypothesis seemed plausible based on the fact that a loss of Catsup results in elevation of DA
synthesis due to increase TH and GTPCH activity. To ascertain differences in mobility, flies
were subjected to a time in motion assay in which the amount of time each individual fly was in
motion in a 45 sec interval for both Catsup transgenic lines and wild type controls was
determined (Fig 3.2 B). Unexpectedly, O.E. of the Catsup protein significantly potentiated
mobility in comparison to controls. In addition, the hypothesis of lower rates of mobility was not
supported by the findings of this report. Therefore, the conclusion that O.E. of Catsup modulates
some component of DA synthesis resulting in phenotypes similar to that of Catsup mutants
seems plausible.
74
A
B
Figure 3.1. Over-expression of Catsup in dopaminergic cells alters paraquat survival and mobility.
(A) Over-expression of Catsup in dopaminergic neurons increases resistance to paraquat (PQ). Catsup
over-expression (O.E.) flies, Catsup 26/+ and wild type (WT) controls [Df(1)w,y transgenic strains
UAS-Catsup (without the Gal4 driver) and TH-Gal4 (without the UAS promoter element)] were fed 10
mM PQ to determine differences in survival. Catsup O.E and Catsup mutants demonstrate decreased
sensitivity to PQ in comparison to WT controls. Ten male flies were scored per replication, and the
mean values represent the average of 5 replications. (B) Over-expression of Catsup in dopaminergic
neurons increases mobility. Time-in-motion assays were conducted for a period of 45 sec for 20 adult
male flies from the Catsup O.E. strain and WT control to determine differences in mobililty. The
assays were performed for each individual fly and the mean values represent the average of 20
independent replication per strain. The * indicates the significance of differences between Catsup O.E.
flies and control strains. Statistical analysis was performed using one way Anova (**, p < 0.01, ***, p
< 0.001). Error bars indicate ± SEM.
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Over-expression of Catsup modulates VMAT function
Previously, we have demonstrated that loss of Catsup results in increased VMAT activity
as determined by the hypermobility of Catsup mutant flies after reserpine treatment (Wang et al.,
manuscript in progress). Reserpine is an inhibitor of VMAT function, which prevents the
packaging of DA within synaptic vesicles. The attenuation of VMAT activity by reserpine
feeding results in decreased mobility in Drosophila (Chang et al., 2006). Based on the known
behavioral phenotype of Catsup mutants, we hypothesized that increased Catsup expression
would down-regulate VMAT function, resulting in flies that were sensitive to reserpine. The
Catsup O.E. flies and wild type controls were fed 60 mM resperpine for 8 hrs and a negative
geotaxis-climbing assay was conducted. As expected, reserpine caused reduced mobility in both
wild type flies and Catsup O.E. flies, relative to the sucrose-fed controls (see Figure.3.2). Since
the Catsup O.E. strain and Catsup mutant adults both had increased resistance to paraquat, we
anticipated that resistance to the effects of reserpine would be comparable for these strains.
Surprisingly, this expectation was not met. Whereas the climbing rate of control strain was
decreased approximately 2.75 fold with reserpine ingestion, the Catsup over-expression flies
were slowed by nearly 5-fold, and are approximately 20% slower in the time required for them to
cross 5cm than are the control flies. This observation suggests that Catsup over-expression
reduces VMAT activity and increases susceptibility to the pharmacological manipulation of
VMAT function.
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Figure 3.2. Expression of Catsup in dopaminergic cells confers sensitivity to reserpine. To
determine effects on VMAT function, 15 to 20 male Catsup O.E. flies and WT strains were fed 60
mм reserpine and 5 % sucrose solutions for 8 hrs. Flies were removed and mobility calculated by
measuring the time to cross 5cm. Both wild type and Catsup O.E. flies were significantly more
sensitive to reserpine than their sucrose fed counterparts. In addition, reserpine-fed Catsup
overexpression flies were significantly slower than wild type flies on reserpine. No significant
difference was noted between Catsup O.E. and wild type sucrose fed flies. Each value is the
average of 15-20 replications and replication was made up of one fly per strain. Statistical analysis
was performed using one way Anova (*, p<0.05, ***, p<0.001). The * indicates the significance of
differences between Catsup over-expression and WT controls and the differences between
resperpine fed and sucrose fed flies. Error bars indicate ± SEM.
77
Loss of DAT function reduces sensitivity to paraquat
Though the insecticide paraquat has been implicated in the pathophysiology of
Parkinson’s disease (PD), the mechanism by which this compound triggers neurodegeneration is
unclear. Some studies have suggested that paraquat may gain access to DA neurons through the
DAT. This hypothesis is based on the structural similarities between paraquat and a neurotoxic
metabolite of MPTP, MPP+, which gains access to DA neurons through the DAT. Utilizing the
availability of a Drosophila DAT mutant strain fumin (fmn), we sought to explore the possible
relevance of DAT to PQ toxicity and to examine the proposed functional interactions between
Catsup and DAT. Here, we hypothesized that reduction of DAT function would decrease
susceptibility to paraquat toxicity based on the concept that dimishing DAT would decrease
access of PQ to DA neurons.
To determine susceptibility to paraquat, survival in heterozygous DAT (fumin) mutants
and DAT RNAi flies fed 10 mM paraquat was measured. Reduction of DAT function reduced
susceptibility to paraquat relative to wild type controls (Fig 3.3A). In addition, DAT mutants
survived almost twice as long as wild type flies. A similar resistance to paraquat was observed
when DAT product was knocked down by expression of DAT RNAi in dopaminergic neurons
(see Figure 3.3B). Although we cannot conclude that access of paraquat to the dopaminergic
neurons is reduced in DAT mutants, in the absence of transport studies, it appears that the amount
of DAT that is functionally available is an important factor in the toxic response to paraquat
exposure. These results suggest either that DAT function is an important component in regulating
paraquat access to the neuron or that neuronal homeostatic responses to reduction in DAT levels
trigger protective mechanisms to detoxify paraquat or otherwise modulate oxidative stress
responses. Moreover, these findings also reveal another instance in which genetic variation
78
within DA homeostasis interacts with environmental factors and recapitulates PD related
phenotypes.
79
B
A
Figure 3.3. Loss of DAT function reduces sensitivity to paraquat toxicity. (A, B) Loss of DAT
function enhances resistance to paraquat. The results show that attenuation of DAT function
enhances survival against paraquat toxicity in comparison to wild type and transgenic controls. Ten
flies were scored per replication and the mean values represent the average of 5 independent
replication. The * indicates the significance of differences DAT deficient and control flies.
Statistical analysis was performed using Student’s t-test and one way Anova (*, p < 0.05, ***, p <
0.001). Error bars indicate ± SEM.
80
Discussion
Given the proposed role of Catsup as a negative regulator of TH and GTPCH, a UASCatsup transgenic line was generated by a former graduate student, Dr. Kevin Bowling, with the
intent of better defining the role of Catsup in regulating DA homeostasis by driving expression
of the transgene specifically in dopaminergic cells. The expression of the transgene clearly
affects DA-dependent characteristics; however, it should be noted here, that we are currently
uncertain of the degree to which these flies are expressing Catsup. Western blot analysis of this
Catsup transgenic line indicated that the Catsup band appeared to have a more intense signal
than the 50kD band of Catsup expressed by the wild type control, suggesting an increase in the
concentration of this protein. Unfortunately, this analysis was conducted without proper controls
and will need to be repeated with proper loading controls so that the relative signals from
transgenic and wild type control flies can be quantified. Thus, we can only speculate about the
relationship of Catsup over-expression and the phenotypes observed within this study. However,
our knowledge of Catsup function and the evidence presented with this study are very suggestive
that this protein is being over-expressed and modulating components of DA homeostasis.
The phenotypes observed in this study, related to dopamine synthesis and function
resulting from expression of the Catsup transgene, such as increased resistance to paraquat and
increased mobility relative to wild type controls, were unexpected. Both phenotypes were
observed previously in heterozygous loss of function Catsup mutants; we expected the opposite
result from over-expressing the wild type Catsup protein, decreased sensitivity to paraquat and
decreased mobility. There are two possible explanations for the observed phenotypes of the
Catsup over-expression flies. 1) If this is a true over-expression line, then a compensatory
mechanism to deal with the increased expression of Catsup protein may alter the expected
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outcome. Such a mechanism is very plausible given the importance of Catsup expression for
development, survival, and the regulation of DA homeostasis (Stathakis et al., 1999). 2) If
Catsup expression is not up regulated, there may be an auto-regulatory response to Catsup
transgene over-expression that down-regulates the expression of the endogenous Catsup gene.
This mechanism may be a necessary adaptive response in an effort to maintain proper DA
homeostasis. If this is so, then Catsup expression levels within the over-expression lines may be
comparable to wild type or even mutant, in the case of an excessive response. Based on the
observed phenotypes of increased mobility in time-in-motion assays and resistance to paraquat
toxicity within these flies, the down-regulation would most likely mimic mutant expression
levels.
Previously, we have shown that loss of Catsup results in increased VMAT activity, which
in turn decreases the effect of reserpine on locomotor activity (Wang et al., manuscript in
progress). In this study, it was hypothesized that Catsup functioned as a negative regulator of
VMAT (either via direct or indirect mechanisms) and that the increase in VMAT activity
diminished the susceptibility of Catsup mutants to paraquat and reserpine exposure by efficiently
packaging DA and preventing the production of toxic metabolites. Based on this proposed
mechanistic interaction between Catsup and VMAT in the regulation of DA homeostasis, we
hypothesized that over-expression of Catsup should result in decreased VMAT activity as
measured by increased sensitivity to reserpine. The findings of this experiment are consistent
with this model; we noted that exposing the presumed over-expression line to reserpine treatment
increased sensitivity to this drug. While TH>Catsup transgenic flies exhibited elevated mobility
in time-in motion assays and increased paraquat sensitivity relative to control flies, they
nevertheless were far more strongly affected by reserpine exposure. However, time in motion
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and negative geotaxis are two different assays used to determine locomotor activity. Time in
motion is geared towards determining differences in any form of mobility while negative
geotaxis measures climbing ability. For this experiment, negative geotaxis was a more
conclusive assay to assess the effect of reserpine on locomotor activity. What is clear from
either assays, is that any modulation in Catsup activity results in a change in locomotor
phenotype due to dysregulation in DA homeostasis. Consequently, the time required for the
transgenic flies to cross 5 cm increased approximately 4.33 fold, from 1.5 to 6.5 sec while the
control flies increased from 2.0 sec to 5.5 sec or a 2.75% increase. These data suggest that the
initial elevated activity of the transgenic Catsup line results from a compensatory response in DA
synthesis with a concentration dependent synaptic release, while the role of Catsup in modulating
VMAT function is not subject to the same degree of compensation. We observed previously that
feeding adult Drosophila L-DOPA or DA similarly results in somewhat elevated activity, but
also strongly elevated DOPAC pools, indicating that much of the supplied DA remains in the
cytoplasm where it is subject to oxidation.
Loss of VMAT activity, which also elevates cytoplasmic DA and oxidative load, has
been shown to enhance DA neurodegeneration (Hanson et al., 2004). Given the enhanced
sensitivity of VMAT to reserpine, indicating a decrease in VMAT activity, when Catsup is overexpressed, we would expect that these flies would exhibit heightened sensitivity to paraquat.
Surprisingly, these flies demonstrate resistance to paraquat toxicity. Indeed, it is plausible that
VMAT activity in conjunction with DAT function may play a role in modulating the phenotypic
response seen here as consequence of paraquat exposure. It has been shown that interruption of
DAT function reduces sensitivity to paraquat, presumably by diminishing the accessibility of
paraquat to DA neurons (Shimizu et al., 2003). Behavioral analysis of loss of function alleles of
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DVMAT-A indicated a reduction in sensitivity to cocaine, which binds to DAT, reduced DA
content and increased locomotion in adult flies (Simon et al., 2009). This study not only
demonstrates the ability of flies to adapt to loss of VMAT function, but provides evidence that
one component of such adaptation is a reduction of DAT function. Recently it has been
determined that VMAT (2), which is responsible for loading of dopamine into synaptic vesicles,
associates with components of DA synthesis, TH and AADC, at synaptic vesicles (Cartier et al.,
2010). Moreover, the authors of this study provided evidence that the formation of this complex
has a functional role in regulating DA synthesis and transport. This finding is in line with
previous results in our lab that demonstrates that the coupling of TH and GTPCH has a direct
effect on the synthesis of DA in Drosophila (Bowling et al., 2008). In addition, Catsup directly
associates with both GTPCH and TH and this association is an integral component in the
regulation of DA synthesis. Therefore, we propose that over-expression of Catsup downregulates VMAT and DAT function, resulting in enhanced sensitivity to reserpine,
hyperlocomotion, and resistance to paraquat. It is also likely that DA synthesis is attenuated by
the over-expression of this protein. Interestingly, lost of DAT function, which prevents
clearance of DA within the synapse, in mammals has been linked to increased mobility
notwithstanding the modulation of DA production and release (Jones et al., 1998). Interruption of
Drosophila DAT function has been credited with modulation of sleep pattern (Kume et al.,
2005). Moreover, the idea that the DAT function is compromised within Catsup mutants is
further supported by evidence demonstrating that polymorphism within Catsup contributes to
variation in sleep pattern (Harbison et al., 2009).
To determine if Catsup is really being over-expressed, Western blot analysis will be
performed for these flies with the proper controls. In addition, FLAG and Venus epitope tagged
84
lines for the over-expression of Catsup will be used to evaluate mobility and paraquat sensitivity.
Another benefit of theses fusion protein tag lines will be the ability to quantify differences in the
relative levels of endogenous Catsup protein in the transgenic lines compared to wild type,
allowing an assessment of possible autoregulatory responses to transgene over-expression. To
ascertain whether phenotypes demonstrated by Catsup O.E. are DA specific the pan-neuronal
driver, Elav-Gal4 will be used to over-express Catsup. Finally, based on the evidence that
VMAT activity is down regulated in Catsup O.E. flies, Western blot analysis will be performed
to determine levels of VMAT expression within these lines.
Is there a real link between DAT function and paraquat toxicity? Arguments for, as well
as against, the requirement of the DAT in regulating paraquat exposure to DA neurons (Shimizu
et al., 2003; Richardson et al., 2005) have been proposed. Based on the suggestions that DAT
function is compromised in both Catsup mutants and Catsup O.E. lines, we hypothesized that
loss of DAT function reduces sensitivity to paraquat toxicity. This was confirmed by the results
of this experiment, which indicated that DAT mutants as well DAT-RNAi flies are less
susceptible to morbidity as a consequence of paraquat exposure. Moreover, post-mortem
neuroanatomical analysis of control and PD patient brain revealed that areas with high DAT
expression are most sensitive to damage in PD (Miller et al., 1997). To determine the effect of
of paraquat exposure on DA neuron survival, immunohistochemistry analysis will be performed.
This is information is vital because loss of DA neurons within the substantia nigra is the main
pathological hallmark of PD. In addition, we have previously shown that paraquat selectively
destroys subsets of DA neurons by inducing oxidative stress and apoptosis (Chaudhuri et al.,
2007). Thus, it is possible that Catsup O.E. may reduce the susceptibility of DA neurons to
paraquat insult. Given the proposed role of oxidative stress in contributing to the neurotoxic
85
process of paraquat exposure, differences in DOPAC levels as well as catalase activity will be
determined. Both of these assays are important indicator of oxidative stress. In addition, Catsup
has been proposed to modulate both DAT and VMAT function. Therefore, exposing DAT
mutants to reserpine and analyzing locomotor activity will evaluate the effect of loss of DAT
activity on VMAT function. Finally, western blot analysis with VMAT antibody will be
conducted to determine if VMAT levels are modulated in DAT mutants.
86
CHAPTER FOUR
A BRIEF EXPOSURE TO PARAQUAT RESULTS IN REDUCED LIFE SPAN AND
PARKINSONIAN-REKLATED PHENOTYPES IN YOUNG ADULT DROSOPHILA
This work is a continuation of a project begun by Arati Inamdar who contributed Fig. 4.1 D and
Fig.4.2 B. I conducted all remaining experiments with the help of my undergraduate assistant
Eric Cook.
Introduction
Parkinson’s disease (PD) is the second most common progressive neurological disorder,
which results in a preferential loss of dopamine (DA) neurons in the substantia nigra (Olanow &
Tatton, 1999). Although cell culture and animal models have provided important information
regarding some of the genetic and environmental factors that may result in Parkinsonism, the
exact mechanisms and cause of this disease remain elusive. Moreover, the misregulation of DA
homeostasis is thought to play an important role in the etiology of PD (Hastings et al., 1996;
Guillot & Miller, 2009). Loss of DA neurons results in DA deficiency and has been attributed to
early symptoms of the disease such as tremor and rigidity (Fahn, 2003). Additionally, DA
dysregulation, which results in the release of DA into the cytoplasm, contributes to increase in
oxidative cascades and may ultimately lead to the demise of DA neurons (Stokes et al., 1999).
Although several genes have been linked to rare cases of familial Parkinsonism, such as
mutations in genes encoding alpha synuclein, DJ-1, and ubiquitin C-terminal hydroxylase L1,
more than 90% of the cases of PD are the result of sporadic PD (Miller et al., 2005). As it relates
to sporadic PD, a combination of environmental factors and genes involved in DA regulation are
believed to components in the neurodegenerative process. The factors that may ultimately lead
87
to a diagnosis of PD vary among individuals; however, there are some factors that are universal
in the pathophysiology of PD. First, PD selectively targets DA neurons in the midbrain. Second,
mitochondrial dysfunction leads to the activation of apoptosis and is often implicated in the
neurodegenerative process (Parker et al., 1989; Bywood & Johnson, 2003; Tretter et al., 2004).
Third, the loss of DA neurons results in motor impairment (Fahn, 2003).
Parkinson’s disease primarily affects individuals between the ages of 50 and 60.
However, there have been documented cases of young adults and even children being affected by
this dreadful disease (Lang & Lozano, 1998). Cases of PD in individuals under the age of 50 are
classified as early-onset Parkinsonism (EOP). Patients who exhibit symptoms under the age of
21 are classified as belonging to the EOP subset Juvenile Parkinsonism, while patients over the
age of 21 are classified as belonging to the EOP subset Young-Onset PD.
Neurotoxins, such as MPTP, and pesticides such as rotenone and paraquat (PQ), have
been implicated as environmental contributors of PD (Liou et al., 1997; Betarbet et al., 2000;
Uversky, 2004). Previously, we have shown that the herbicide PQ induced neurodegeneration of
DA neurons and resulted in PD related phenotypes. Based on these results and epidemiological
studies that suggest that PQ is an environmental trigger of PD, the goal of this study was to
determine if a one-time exposure to PQ at early stages of development is sufficient to elicit
behavioral phenotypes that are associated with PD or to increase risk later in life. Currently, it is
undetermined if a one time exposure to PQ is sufficient to mimic or accelerate the PD
neurodegenerative process. This is an important issue because there is a lag time between onset
of neuronal loss and diagnosis, and considerable time may have elapsed between initial exposure
to an environmental agent and neuron loss. Moreover, the probable time between the initial
triggering event and onset of observable symptoms can be a period of several decades, making it
88
difficult to effectively treat or delay the progressive neurodegenerative process. Therefore, by
the time the disease process has been diagnosed, PD patients would have lost approximately 80%
of their DA neuron. In addition, this study may provide useful insights into the pathogenesis of
PD as it pertains to the correlation between PQ exposure and Juvenile Parkinsonism.
The results of this study demonstrate that a brief exposure to low doses of PQ among
newly eclosed young adult flies reduces lifespan and mobility. Both of these phenotypes have
been previously identified as key components in the pathogenesis of PD and in a Drosophila
model of PD exposure to PQ (Jellinger, 2000; Chaudhauri et al. 2007).
Material and Methods
Drosophila strains and culture maintenance. All Drosophila stocks were maintained at 25º C
on standard cornmeal-yeast-sugar media, and all the experiments were performed at the same
temperature. A transgenic reporter strain, TH-GAL4; UAS-eGFP was employed in all
experiments. The transgenic strain UAS- 2X eGFP (Chromosome II) was obtained from the
Bloomington, IN Drosophila stock center and a TH-GAL-4 strain (Friggi-Grelin et al., 2003) was
a generous gift from Jay Hirsh (University of Virginia).
Feeding experiments. Newly eclosed flies (0-6 hours) were fed 5% sucrose and 0.1 mм, 1 mм,
and 10 mм PQ. The feeding solution were mixed with 1% agar. For all experiments, flies were
fed for 12 hours.
Locomotion assays. A time in motion assay was conducted as described previously (Carbone et
al., 2006) with a few modifications. The flies were immediately removed from their respective
feeding vials and allowed to acclimate in vials with regular fly media for 24 hours. The assay
was then performed by gently tapping the vial and measuring the length of time the fly remained
in motion for a period of 45 seconds. Mobility for each treatment set was calculated as the
89
average of 20 independent replications. These assays were performed subsequently for the same
treatment sets at two and three weeks with the original set of flies.
Statistical analysis. All data were analyzed by one-way ANOVA with Dunnett’s post-test or by
one-tailed Student’s t-test using GraphPad Prism (San Diego, CA). Details of the analyses are
described in the figure legends.
Results
Early PQ exposure to young adult flies modulates mobility both short-term and long-term
Motor impairment is a pathological hallmark of PD due to loss of DA neurons (Fahn,
2003). To ascertain whether a one time exposure to low and high does of PQ could affect
mobility, a time in motion assay was performed immediately following PQ and sucrose
exposures. As seen in Fig 4.1 A-C, transient exposure of young adult flies to 10 mм PQ causes a
significant reduction in mobility 24 hrs after the 12 hr ingestion period. Ingestion of 0.1 mм or 1
mм PQ did not affect mobility 24 hrs post PQ exposure. Similarly, Inamdar (manuscript in
progress) noted a concentration dependent reduction in locomotion in a negative geotaxis assay,
due to one time exposure to PQ for 12 hrs (Fig 4.1 D). Though our assays were different, these
results concur in demonstrating that a high concentration of PQ for a 12 hr period, affects the
locomotor activity of PQ treated flies.
To determine if locomotor activity is progressively reduced over time as a consequence
of early exposure to PQ, time in motion assays were conducted two weeks following the initial
PQ exposure. Here, we noted no significant difference in mobility between control flies and
those exposed to 0.1 mм PQ, but saw a significant and progressive reduction in mobility among
flies that were fed 1 mм and 10 mм PQ. Interestingly, we also noted that 1 mм PQ fed flies
appeared to be more sensitive than flies that had been exposed to 10 mм PQ. Based on this
90
result, we also did a three-week follow up. Here we noted that flies that were initially fed 10 mм
PQ continued to show a significant reduction in mobility compared to non-fed controls but we
saw no significant differences between sucrose fed flies and flies that were fed 0.1 mм and 1 mм
PQ. It should be noted, however, that by 5 weeks post-eclosion, control flies were beginning to
slow dramatically as they experienced normal age-dependent loss of DA neurons. Thus, we do
not observe evidence of accelerated onset of symptoms after exposure to the lower doses of PQ
at these much later times. Overall, these results demonstrate that transient exposure to PQ
reduces locomotor activity with flies fed 10 mм PQ and results in a robust and progressive
decline in mobility.
Early paraquat exposure reduces lifespan in adult flies
We have previously demonstrated that the environmental toxin PQ is capable of initiating
Parkinsonian type phenotypes in flies such as loss of DA neurons and reduced lifespan
(Chaudhuri et al., 2007). To determine the effect of a one time exposure on long-term survival,
we fed flies 0.1 mм, 1 mм or 10 mм PQ for 12 hrs, then placed them on regular fly media and
measured lifespan. The same procedures were followed for controls, which were fed 5% sucrose
for 12 hrs. All PQ-fed flies showed a reduction in lifespan by approximately twenty days in
comparison to control. However, flies that were fed 10 mм PQ appeared to have the most
detrimental effect on longevity with these flies surviving approximately 30 days less than wildtype controls. We should note, however, that the survival of 10 mM PQ fed flies were not
significantly different than 0.1 mM and 1 mM PQ fed flies. As seen in figure 4.2 B, the results
of this experiment are supported by the findings of Inamdar (manuscript in progress) who also
demonstrated that one time exposure to PQ has a significant effect on lifespan.
91
A
B
D
C
Figure 4.1. A one time exposure to paraquat attenuates locomotor activity. Twenty newly eclosed
(between 0hrs and 6hrs post eclosion) TH-Gal4-UAS GFP flies were fed 5% sucrose, 0.1 mм, 1 mм and 10
mм PQ for 12 hrs. After feeding for 12 hrs, flies were then removed and allowed to acclimate on regular fly
media for 24 hrs. Following the acclimation period, time in motion assays were performed
for each feeding set. The assays were performed for each individual fly and the mean value represents the
average of 20 independent replications per strain. Flies were placed back on regular media and transferred
twice per week for the duration of the experiment. Time in motion assays were also determined at two
weeks and three weeks post PQ exposure. (A) At 24 hrs post-PQ exposure, 10 mм
PQ flies exhibited reduced mobility compared to sucrose fed controls. (B) 1 mм and 10 mм PQ fed flies
demonstrated reduced mobility two weeks following initial exposure. (C) 10 mм PQ reduces mobility
compared to sucrose control three weeks following PQ exposure. (D) Young adult male flies
demonstrate reduced mobility after 12 hrs of exposure to PQ (1 mм and 10 mм). Mobility was determined
by the negative geotaxis assay. The * indicates the significance of differences between control and PQ-fed
flies. Statistical analysis was performed using one way Anova (*, p< 0.05, **, p <. 0.01, ***, P < 0.001).
Error bars represent standard error of the mean.
92
A
B
Figure 4.2. A one time exposure to paraquat (PQ) reduces lifespan. (A) Newly eclosed (0-6 hrs)
TH-Gal4-UAS-GFP flies were fed 5% sucrose, 0.1 mм PQ, 1 mм PQ, and 10 mм PQ for 12hrs,
removed and then placed on regular fly media. Lifespan was determined by counting the number of
dead flies for each data set over the duration of the experiment at 2-3 day intervals. Each data point
represents five replications of 10-15 flies each. (B) Young adult flies, aged to 6 hrs post-eclosion,
were exposed to 10 mм and 1 mм PQ, or 5% sucrose for 12 hrs and then transferred to the normal
food. The flies exposed to PQ in early adulthood showed decreased survival duration. Each data point
represents at least 5 replications of 10-15 flies each. The * indicates the significance of differences
between control and PQ-fed flies. Statistical analysis was performed using one way Anova,
(*p<0.05, **, p < 0.01, ***p < 0.001). Error bars represent standard error of the mean.
93
Discussion
Epidemiological studies associate PQ exposure to increased incidents of PD cases in
agricultural communities (Lai et al., 2002; Landrigan et al., 2005; Brown et al., 2006). However,
there are discrepancies as to the actual effect of PQ on PD. In addition, many of these studies are
based on the premise of repeated exposure to the herbicide and the implicit effect of the impact
of short-term exposure to PQ has not been fully explored. For example, what length of exposure
is sufficient to result in parkinsonian phentoypes? Moreover, is age of exposure a determining
factor in the impact of PQ on PD related phenotypes? In this report we demonstrate that a one
time exposure to PQ, early in life, is sufficient to elicit the parkinsonian-related phenotype of
loss of mobility, as well as a subsequent shortening of life span. While it has been previously
demonstrated that PQ exposure resulted in PD related symptoms in Drosophila (Chaudhuri et al.,
2007), the previous study was conducted using higher concentrations of PQ and acute dosing. In
addition, it was undetermined whether a one time exposure could mimic the disease process.
Taken together, the findings of this study indicated that PQ exposure at the young adult stage of
development has detrimental long-term effects.
Analysis of locomotor activity following the initial PQ feeding (see Figure 4.1 A) showed
a decreased in mobility for 10 mм PQ but no effect for 0.1 mм and 1 mм PQ. Therefore, we
assumed there was a concentration dependent effect occurring for this time period of exposure.
This finding is slightly contradicted by the findings of Inamdar (manuscript in progress), who
noted a difference in mobility at 1 mм and 10 mм PQ. In addition, this difference in motor
impairment is probably due in part to the different assays used to detect mobility in flies exposed
to PQ for these two experiments. Inamdar measured mobility by determining the time it took a
fly to cross 5cm, and we measured mobility by determining the time in motion of the fly over a
94
45 sec period. These two assays, while corresponding in general, have differences in the type of
neural responses being assessed, and therefore, outcomes can diverge somewhat with respect to
time of onset in locomotion deficits and in sensitivity to some treatments. In addition, Inamdar
performed the assay immediately following PQ exposure, unlike our experiment in which we
waited 24 hrs before measuring mobility. The decision to allow for an acclimation period of 24
hrs prior to performing the mobility assays was based on the findings of previous experiments in
which we noted great fluctuation in numbers among individual groups following PQ treatment.
Upon allowing for a 24 hr acclimation period prior to measuring mobility, the results were more
consistent for individual groups. In addition, the recovery time should not negatively impact the
results of this experiment because PQ toxicity is believed to be a progressive process. However,
it is possible that the difference in assay and the time period of measurement employed by
Inamdar, could have been more sensitive in detecting mobility deficits at 1 mм PQ.
The findings of this report also demonstrated that a one time exposure to the herbicide
PQ, at the highest concentrations, resulted in a progressive decline in locomotor activity, as seen
in the increased decline in activity for flies fed 10 mм PQ at two and three weeks following the
initial PQ exposure. Interestingly, we also saw a decline in mobility for 1 mм PQ fed flies at two
weeks post exposure, but noted no difference in mobility at three weeks post exposure.
Additionally, we saw no difference in mobility for 0.1 mм PQ fed flies compared to 5% sucrose
control at any time point. Therefore, it is possible that these flies have developed resistance to
oxidative stress as the flies responded to the short term, low dose treatment, or they are simply
unaffected by this low dose. Oxidative stress measurements such as catalase or lipid
peroxidation assays can be used to distinguish these alternatives.
95
Here we sought to determine if a one time exposure to PQ for a relatively short period
(12 hrs) could also generate a reduction in lifespan. Interestingly, a one time exposure to PQ at
0.1 mм, 1 mм, and 10 mм significantly reduced in lifespan compared to sucrose fed control. As
in mobility deficit, 10 mм PQ had the most pronounced effect. These finding are also supported
by the results of Inamdar (manuscript in progress). All together, these results suggest that a one
time exposure to PQ could be a factor in Juvenile Parkinsonism.
Although the findings of this report are intriguing, there are several questions that are yet
to determined. For example, is there a direct correlation with mobility deficit and DA? To
address this question, we will repeat the parameters of the experiment and follow-up with HPLC
analysis in which DA and DOPAC levels will be determined. Determining DOPAC levels will
be a good gauge of oxidative damage because DA is a highly reactive amine that generates
neurotoxic quinones and reactive oxgen species. To determine if DA neurons are undergoing
apoptosis, as a result of PQ exposure, we will do immunohistochemistry and confocal analysis.
If there are indications that DA, DOPAC and DA neurons are affected by PQ exposure, a will
follow-up catalase assays to ascertain the role of oxidative response will be performed.
We also report here that PQ exposure at all concentrations significantly reduced lifespan
in adults. It would be interesting to determine if a two-week follow-up hit of PQ at 0.1 mм and 1
mм will further reduce lifespan. We anticipate that re-exposure of these flies after a two weeks
post initial exposure will accelerate the decline in lifespan compared to flies that were subjected
to only one hit of PQ exposure.
96
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