Copyright Arati Ajinkya Inamdar 2009 - acumen - The University of ...

DISCOVERY OF A NITRIC OXIDE SYNTHASE-DEPENDENT RESPONSE AND A
FUNCTIONAL ANALYSIS OF GENES REGULATING THE RESPONSE IN A
DROSOPHILA MODEL OF PARKINSON’S DISEASE
by
ARATI AJINKYA INAMDAR
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
2009

Copyright Arati Ajinkya Inamdar 2009
ALL RIGHTS RESERVED

ABSTRACT
The processes of neuroinflammation and oxidative stress are thought to be among the
primary mechanisms playing roles in the etiology and pathophysiology of Parkinson’s disease
(PD). Recently, it has been proposed that microglia, the innate immune cells of the mammalian
brain, become hyperactivated and deregulated in response to neuronal dysfunction in PD and
thereby, accelerate dopaminergic neuronal loss. The mechanisms for neuroinflammatory
responses that exaggerate neurotoxicity are poorly understood. Moreover, the studies to date that
investigate neuroinflammatory mechanisms have utilized primarily in vitro approaches. We have
established a Drosophila PD model based on ingestion of the herbicide paraquat, which
recapitulates most behavioral and patho-physiological features of PD, including loss of
dopaminergic neurons. Using this model, we have discovered that paraquat ingestion induces
nitric oxide synthase (NOS) and a corresponding elevation of NO production in the adult
Drosophila brain. Mammalian microglia have been shown to be a source of NOS, recognized as
a major component/marker in neuron death, in mammalian PD models. Therefore, the
observation of NOS induction during Drosophila neurodegeneration parallels the mammalian
process. The paraquat-induced stimulation of NOS was further confirmed using pharmacological
approaches, which demonstrated that inhibition of NOS partially rescued adult Drosophila from
the deleterious effects of paraquat. Minocycline, a tetracycline derivative, has been reported to
act primarily on microglia, ameliorating excessive activation of NOS, in mammalian
neurodegenerative disease models including PD. Similarly, ingestion of minocycline by adult
ii

Drosophila ameliorates the effects of paraquat, including reduction of NOS activity and
protection of dopaminergic neurons. The paraquat-induced PD model was also employed to
identify the mechanisms of action that confer the neuroprotective properties of minocycline.
Components of signaling pathways that potentially could mediate DA toxicity in this PD model
were tested for their ability to modify the action of minocycline.
The discovery of a NOS-dependent paraquat response in flies provides the foundation for
future work to explore cellular and molecular mechanisms directing NOS induction and action in
Drosophila, which exhibits features resembling neuroinflammation. This research also takes
advantage of the ease of genetic and pharmacological methods in the Drosophila model to
identify important genetic components of this process.
iii

3-IT 3- iodo-tyrosine
6-OHDA 6-hydroxydopamine
o C Celsius
Apaf-1 Apoptosis activating factor-1
ATP Adenosine triphosphate
LIST OF ABBEREVIATIONS AND SYMBOLS
BDNF Brain -derived-neurotrophic factor
BH4 6(R)L-erythro-5,6,7,8-tetrahydrobiopterin or Tetrahydrobiopterin
Catsup Catecholamines up
CD 14 Cluster of Differentiation 14
cDNA Complementary deoxyribonucleic acid
COX 1 Cyclooxygenase -1
COX 2 Cyclooxygenase -2
CSF Cerebrospinal fluid
DA Dopamine
DAT Dopamine transporter
dDAT Drosophila dopamine transporter
Ddc Dopa decarboxylase
Diablo/Smac Direct IAP Binding protein with low pI/second mitochondria derived
activators of caspases
iv

DOPAC 3,4-Dihydroxy-phenylacetic acid
dTH1 Drosophila tyrosine hydroxylase 1
dTH2 Drosophila tyrosine hydroxylase 2
dsRNAi double stranded RNA interference
ERK Extracellular signal–regulated kinases
FAD Flavin adenine dinucleotide
FADD Fas associated death domain
FMN Flavin mononucleotide
GDNF Glial cell-line-derived-neurotrophic factor
GFP Green fluorescent protein
gmc Glial cells missing
gmc2 Glial cells missing 2
GSH Glutathione
GTP Guanosine triphosphate
GTPCH GTP cyclohydrolase
HPLC High pressure liquid chromatography
IAP Inhibitor of apoptosis
ICE IL-1β converting enzyme
IFN γ Interferon gamma
IKK α NF-inhibitor
iNOS Inducible nitric oxide synthase
IL-6 Interleukin 6
IL-1α Interleukin 1 alpha
v

IL-1β Interleukin 1 beta
JNK c-Jun NH2-terminal kinases
kDa kilo Dalton
L-DOPA 3, 4-dihydroxy-L-phenylalanine
L-NAME N G -nitro-L-arginine methyl ester
L-NMMA N G -monomethyl-L-arginine
MAO Monoamine oxidase
MAPKs Mitogen activated protein kinases
MKKs Mitogen-activated protein kinase kinases
MKKKs Mitogen-activated protein kinase kinase kinases
mg Milligram
MHC Major histo-compatibility complex
ml Milliliter
mM Millimolar
MPP+ 1-methyl-4-phenylpyridinium
MPTP 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
NADP Nicotinamide Adenine Dinucleotide Phosphate
NF-κβ Nuclear factor kappa beta
NH2PPP Dihydroneoperin triphosphate
nm Nanometer
NO Nitric oxide
NOS Nitric oxide synthase
NSAIDs Non steroidal anti inflammatory drugs
vi

O2 − Superoxide radicals
PBS Phosphate buffered saline
PBT Phosphate buffered saline with Tween 20
PCD Programmed cell death
PD Parkinson’s disease
PI3K Phosphoinositide 3-kinase
PKB Protein kinase B
PKC Protein kinase C
ple Pale
ppt Pointed
PQ N,N'-dimethyl-4,4'-bipyridinium dichloride or Paraquat
PQ .+ Paraquat radical
PTP 6-pyruvoyl tetra-hydropterin
Pu Punch
rpr reaper
repo Reverse polarity
RNS Reactive nitrogen species
ROI Reactive oxygen intermediate
ROS Reactive oxygen species
SN Substantia nigra
SNpc Substantia nigra pars compacta
TDC Tyrosine decarboxylase
TH Tyrosine hydroxylase
vii

TNF α Tumor necrotic factor alpha
ttk Tramtrack
VMAT Vesicular monoamine transporter
VTA Ventral tegmental area
μg Microgram
μl Microliter
μM Micromolar
μmol Micromole
μm Micrometer
viii

ACKNOWLEDGMENTS
It is my great pleasure to convey my acknowledgements to people whose valuable
presence and support has made this journey successful. First of all, I would like to thank God to
bless me with highly knowledgeable, intelligent and caring husband, Dr. Ajinkya C. Inamdar,
without whom I would have never achieved this honor. I am so fortunate to be a mother of our
son, Atharva A. Inamdar, who has made these years memorable with his smiles, babbles and big
first footsteps.
I am also thankful to God to fulfill my life with love and encouragements from my
parents, Mangala and Arun Thoke throughout my life. I will always owe you for your teachings
and blessings. My infinite thanks to my in-laws, Shilpa and Chintamani Inamdar, for their help,
care and advice since I have become member of their family. Ajinkya and I thank you all for
being so wonderful and loving grand-parents of Atharva. I would also like to thank my relatives
in India, especially my brother Samir, my sister-in-law Priya and nephew Ishaan for their
affection and support. .
I would like to thank my advisor, Dr. Janis O’ Donnell, for her timely support, guidance
and advice. Her talent, knowledge, positive criticism, persistence and understanding are
invaluable to develop me as a scientist in addition to a physician. You have given a new direction
to my life and I believe that these achievements will succeed in imparting unique contribution to
the world.
ix

I am thankful to my committee members, Drs. Guy and Kim Caldwell, Dr. Perry
Churchill and Dr. Ed Stephenson, for their guidance in my research projects throughout these
years. Many thanks to Guy for his timely help to obtain post-doctoral position at Rutgers
University, New Jersey with Dr. Joan W. Bennett. I appreciate Joan’s support for my post-
doctoral project and hope to make valuable contribution for the project.
I appreciate past and present graduate and undergraduate students of Dr. O’Donnell lab
for their friendship, advice and help. I also thank members of Dr. Bennett lab for their help
during our initial period of adjustment in New Jersey. Finally, many thanks to faculty and staff
members especially Mary Musumecci at the Department of Biological Sciences, UA and Liz
Scarpa at the Department of Plant Biology and Pathology at Rutgers University for their
friendliness, open-heartedness and time throughout these years.
x

CONTENTS
ABSTRACT……………………………………………………………..…………………….....ii
LIST OF ABBEREVIATIONS AND SYMBOLS………………………………..…………......iv
ACKNOWLEDGMENTS………………………………………………...………………..…....ix
LIST OF TABLES……………………………………………………………………...............xiv
LIST OF FIGURES……………………………………………………………………………..xv
1. INTRODUCTION……………………………………………………………………………..1
i. Dopamine and dopamine biosynthesis pathway in mammals………………………………….1
ii. Drosophila dopamine biosynthesis pathway…………………………………………………..2
iii. DA packaging and transportation……………………………………………………………..4
iv. Drosophila DA biosynthesis pathway regulators……………………………………………..4
v. Functions of DA in Drosophila………………………………………………………………..8
vi. Parkinson’s disease……………………………………….………………………………….11
vii. Parkinson’s disease and oxidative stress……………….…………………………………...12
viii. Parkinson’s disease and Neuroinflammation……………………………………………....17
ix. Glia in mammals and Drosophila…………………………………………………………...18
x. Microglia…………………………………………………….……………………………….22
xi. Neuroinflammation in experimental models of PD………………….……………………...25
xii. Inhibition of neuroinflammation in PD models…………………………………………….25
xiii. Nitric oxide………………………………………………………………………………...27
xi

xiv. NO signaling in invertebrates……………………………………………………………....30
xv. Drosophila NOS and NO signaling………………………………………………………....30
xvi. Role of NO in Drosophila immune response……………………………………………....31
xvii. Role of Signaling pathways in the pathogenesis of PD…………………………………...32
xviii. Apoptosis signaling pathway……………………………………………………………..32
xix. PCD in Drosophila…………………………………………………………………………35
xx. Mitogen activation protein kinase signaling pathway ……………………………………...36
xxi. JNK signaling pathway……………………………………………………….....................37
xxii. Drosophila JNK…………………………………………………………………………...39
xxiii. JNK signal transduction pathway and Parkinson’s disease………………………………39
xxiv. ERK pathway in mammals………………………………………………………………..40
xxv. ERK in Drosophila………………………………………………………… …………......41
xxvi. ERK signaling pathway and Parkinson’s disease ………………………………...............41
xxvii. Akt signaling pathway………………………………………………………....................42
xxviii. Drosophila Akt signaling pathway……………………………………………………....43
xxix. Akt signaling pathway and Parkinson’s disease……………………………………..........43
2. DROSOPHILA MOUNTS A NITRIC OXIDE-DEPENDENT RESPONSE TO
PARAQUAT-INDUCED DEGENERATION OF DOPAMINERGIC NEURONS…………...48
i. Introduction…………………………………………………………………………………...49
ii. Materials and Methods…………………………………………………………………..…...53
iii. Results…………………………………………………………………………………..…...57
iv. Discussion…………………………………………………..……………………………….73
3. IDENTIFICATION OF A NEUROPROTECTIVE ROLE FOR MINOCYCLINE IN A
DROSOPHILA MODEL OF PARKINSON’S DISEASE AND FUNCTIONAL
ANALYSIS OF SIGNALING PATHWAYS MEDIATING PARAQUAT TOXICITY IN
xii

A DROSOPHILA PD MODEL……………………………………………………………........78
i. Introduction…………………………………………………………………………………...79
ii. Materials and Methods……………………………………………………………………….83
iii. Results……………………………………………………………………………………….88
iv. Discussion…………………………………………………………………………………..106
4. BRIEF EXPOSURE TO PARAQUAT DURING JUVENILE AND EARLY ADULT
STAGES CAUSES PARKINSONIAN SYMPTOMS, INCREASED SENSITIVITY TO
OXIDATIVE INSULT LATER IN LIFE AND A SHORTENED LIFE
SPAN…………………………………………………………………………………………..112
i. Introduction………………………………………………………………………………….113
ii. Materials and Methods……………………………………………………………………...116
iii. Results…...…………………………………………………………………………………118
iv. Discussion...………………………………………………………………………………..135
5. IDENTIFICATION OF A NITRIC OXIDE-DEPENDENT RESPONSE IN
S. VENEZUELAE-INDUCED PARKINSON’S DISEASE IN DROSOPHILA
MELANOGASTER..…………………………………………………………………………...140
i. Introduction………………………………………………………………………………….141
ii. Materials and Methods………………………………………...……………………..……..144
iii. Results…………………………………………………………...………………………....146
iv. Discussion………………………………………………………...………………………...159
6. SUMMARY, FUTURE DIRECTIONS AND APPLICATIONS……..………....................164
i. Drosophila and neuroinflammatory-like response…………………………………………..164
ii. Drosophila and signal transduction pathway in PD………………………………………...171
iii. Drosophila as a model to study Early Onset Parkinsonism………………………………..172
7. REFERENCES………………………..…………………………………………………….174
xiii

LIST OF TABLES
1.1. Similarity in the function and distribution of vertebrate and Drosophila glia in
CNS……………………………………………………………………………………………..21
xiv

LIST OF FIGURES
1.1. Dopamine Biosynthesis Pathway in Drosophila………………………………………….....6
1.2. The BH4 biosynthesis pathway………………………………………………. ……………..7
1.3. The schematic diagram showing the location and number of DA neurons in
adult Drosophila brain viewed from anterior and posterior aspects…………………………….10
1.4. The sagittal section of human brain showing major regions of the
human brain…….……………………………………………………………………………….13
1.5. The chemical structure of paraquat (PQ)……………………….…………………………..16
1.6. The two step mechanism of PQ toxicity……………………………………….…………...16
1.7. The Fenton reaction………………………………………………………………………...16
1.8. An outcome of the sustained activation of microglia in chronic
neurodegenerative disease ……………………………………………………………………..24
1.9. A schematic diagram showing the components involved in the formation
of nitric oxide (NO)………………………………………………………………………..........29
1.10. Comparison of mammalian and Drosophila apoptotic signaling pathways……………....34
1.11. The schematic diagram showing the components of MAPK signaling pathway in
mammals and Drosophila……………………………………………………………………….38
1.12. A schematic diagram showing the signaling pathways associated with
activated Akt…………………………………………………………………………………….45
2.1. Dopamine, tetrahydrobiopterin, and nitric oxide biosynthesis pathways………………......56
2.2. L-NAME, an inhibitor of NOS, improves survival duration of adults
exposed to paraquat………………………………………………………… ………………….58
2.3. Effect of ingestion of minocycline on wild type flies with and without PQ on
survival duration of wild type flies ……………………………………………………………..60
xv

2.4. PQ induces, and minocycline suppresses, NOS activity in adult flies……………………..62
2.5. Minocycline shows differential effects on paraquat-induced damage in dopamine
regulatory mutants …………………...………………………………………………………....65
2.6. Exposure to PQ causes induction of NOS in adult brain …………………………………..68
2.7. Ingestion of PQ (1.5 mM) induces NOS-expression near and around
dopaminergic neurons in TH-GAL4; UAS-eGFP adult brain……………………………….…..70
2.8. Paraquat-induced NOS is expressed in non-glial cells in adult fly brain………..................72
3.1. Effect of minocycline and doxycycline with 10 mM paraquat on survival of adult male
flies. ………………..…………………………………………………...………........................90
3.2. Minocycline protects against PQ-induced mobility defects…………………….………….92
3.3. Minocycline confers protection to dopaminergic neurons…………………………….…...94
3.4. Minocycline delays paraquat induced selective loss of
dopaminergic neurons...………………………..……………………………………………….95
3.5. Minocycline blocks changes in DA pathway components indicative of PQ-induced
oxidative stress……………………………………………………………….............................98
3.6. Minocycline reduces PQ-generated reactive oxygen species……………………………..101
3.7. Paraquat induced inflammatory response is regulated by JNK and Akt
signaling pathways.…………………………………………………………………………….103
3.8. Over-Expression of wild type JNK and Akt provide protection against PQ……………...105
4.1. Exposure of juvenile (2nd instar larvae) and young adult (

5.2. Exposure to crude conditioned medium of S. venezualae induces
Parkinsonian-like mobility defects……………………………………………..……………...149
5.3. Exposure to crude conditioned medium of S. venezualae induces
loss of DA neuron………………………………………………………………………..…….151
5.4. Exposure of crude conditioned medium of S. venezualae induced NOS near DA
neuron in TH-GAL4; UAS-eGFP transgenic strains………………………………….…….….155
5.5. Exposure of S. venezuelae conditioned medium causes expression of NOS around
glial cells in adult brain………………………………………………………...........................158
xvii

CHAPTER 1
INTRODUCTION
Dopamine and dopamine biosynthesis pathway in mammals
Dopamine (DA) is the major neurotransmitter and neurohormone known to be present in
all invertebrates and vertebrates. A member of the catecholamine family, DA is chemically
known as 4-(2-aminoethyl) benzene-1, 2-diol. It is a precursor to norepinephrine and epinephrine
in the catecholamine biosynthesis pathway. DA neurons originate in the substantia nigra pars
compacta (SNpc), ventral tegmental area (VTA), and the hypothalamus from where axons are
projected into different regions of the brain. The four major pathways are the mesocortical,
mesolimbic, nigrostriatal and tuberoinfundibular pathways (Iversen and Iversen, 2007; Björklund
and Dunnett, 2007).
The mesocortical pathway connects the ventral tegmentum to the frontal lobes of the
cerebral cortex and is known to be involved in emotional and motivational responses.
Deregulation of this pathway is associated with psychotic behavior, including schizophrenia as
well as impairment of the memory (Berman et al., 1988; Weinberger et al., 1988).
The mesolimbic pathway connects the ventral tegmentum to the nucleus accumbens in
the striatum. This pathway is mainly involved in reward and pleasure related activities (Berridge
and Robinson, 1998). The tendency of individuals towards addiction to certain drugs such as
cocaine, morphine, amphetamines, and alcohol has been correlated with the dysfunction of the
1

mesolimbic pathway (Beitner-Johnson and Nestler, 1991; Leyton et al., 2002; Boileau et al.,
2003).
The nigrostriatal pathway is crucial pathway for locomotor function and connects the
substantia nigra to the striatum. This pathway forms a part of the basal ganglia motor loop and
thus indirectly interconnects with the cerebral cortex, thalamus and brainstem and functions in
the motor control, cognition, emotions, and learning. Loss of dopaminergic neurons in this
circuit results in Parkinson’s disease, the most common movement disorder associated with
motor deficits as well as cognitive and emotional disorders (Dunnett et al., 1981).
The tuberoinfundibular pathway originates from the arcuate nucleus of the mediobasal
hypothalamus and projects the axons to the infundibular region. The function of the
tuberoinfundibular pathway is mainly implicated in lactation and sexual arousal (Gunnet et al.,
1986; Moore et al., 1987).
Drosophila dopamine biosynthesis pathway
In Drosophila, as in mammals, DA is an essential neurotransmitter and neuromodulator
functionally important for various biological activities such as learning, behavior, locomotion,
memory, and development of the reproductive system (Neckameyer, 1996). The DA biosynthesis
pathway of Drosophila is well-conserved with that of mammals. The genes encoding the
enzymes regulating dopamine biosynthesis pathway in mammals also function in the similar
manner in Drosophila (Livingstone and Tempel, 1983). Briefly, the first step in the
catecholamine biosynthesis is the hydroxylation of tyrosine into 3,4-dihydroxy-L-phenylalanine
(L-DOPA) via the enzyme tyrosine hydroxylase (TH). This is the rate limiting step in the
pathway, and TH undergoes multiple levels of regulation as described below. L-DOPA is then
2

converted into DA by dopa decarboxylase (Ddc) (Nagatsu et al., 1964). The catecholamine
biosynthesis pathway terminates after formation of DA from tyrosine. In addition, tyrosine acts
as a precursor for tyramine, another biogenic amine in Drosophila via tyrosine decarboxylase
(TDC). Tyramine is converted into octopamine, another biogenic amine known to be a functional
homolog of norepinephrine in Drosophila using tyramine β hydroxylase (Cole et al., 2005). The
genes regulating formation of DA and tyramine from tyrosine are shown in Fig. 1.1.
A cDNA for the Drosophila gene encoding TH was isolated from adult head using a
cDNA for rat TH as a probe, indicating a strong conservation between Drosophila and mammals.
Further, Drosophila TH demonstrates 50 % amino-acid sequence identity and 80.5 % similarity
to rat TH (Neckameyer and Quinn, 1989). TH is found to be expressed from embryonic stage 15
onwards in Drosophila (Lundell and Hirsh, 1994). The mammalian TH exists as a homotetramer
with C-terminal catalytic domain and N-terminal regulatory domain (Grima et al., 1985).
Conservation of amino acid sequence with mammalian TH is restricted to the predicted catalytic
domain of Drosophila TH; the presumptive N-terminal regulatory region is largely non-
conserved. Nevertheless, fly neuronal TH can be detected by mammalian TH antibody and
corresponds to a 58 kDa band in western blots (Vié et al., 1999)
In contrast to human TH, which possesses four isoforms, Drosophila expresses two
isoforms, both encoded by pale. Drosophila TH1 (dTH1) is expressed mainly in the CNS while
Drosophila TH2 (dTH2) is expressed in non-neural tissues, predominantly in cuticular hypoderm
(Birman et al., 1994). The dTH1 mRNA is 3.7 kb long, translating into an 80 kDa protein, while
dTH2 mRNA is a 3.2 kb in length translating to a 56 kDa protein (Birman et al., 1994).
3

DA packaging and transportation
Once DA is synthesized in the neuron, Drosophila vesicular monoamine transporter
(VMAT) mediates its packaging into synaptic vesicles. Drosophila VMAT shares high sequence
similarity with mammalian VMAT (Greer et al., 2005). DA is recycled back into the cytoplasm
of the presynaptic neuron after being released into the synaptic cleft for post-synaptic
transmission by Drosophila Dopamine transporter (dDAT). dDAT is a twelve transmembrane
domain protein functionally conserved with human DAT. The dDAT gene is located on the right
arm of the second chromosome and encodes a single DAT isoform (Pörzgen et al., 2001).
In mammals, cytoplasmic DA is converted into 3, 4-Dihydroxy-Phenylacetic Acid
(DOPAC) in the presynaptic region by Monoamine oxidase (MAO). However, in Drosophila,
although MAO has been predicted by sequence analysis, its functional role is still unknown.
Nevertheless, DA in the fly-brain is converted into DOPAC via N-acetylation of DA, and level
of DOPAC can be detected by HPLC (Downer and Martin, 1987). The DA released at the
presynaptic membrane interacts with DA receptors mediating the transmission of stimulus across
post-synaptic region and hence play a key role in DA signaling. In Drosophila, there are two
classes of DA receptors: D1-like and D2-like, based on their biochemical and pharmacological
properties. These two classes of receptors show functional homology with mammalian DA
receptors (Hearn et al., 2002).
Drosophila DA biosynthesis pathway regulators
In Drosophila, DA biosynthesis is well-conserved with that of mammals. It involves two
enzymatic pathways regulating the DA synthesis at different levels as mentioned above. DA
4

synthesis is regulated by the rate-limiting enzyme, TH, encoded by pale (ple) (Neckameyer and
White, 1993).
TH activity is further regulated via tetrahydrobiopterin (BH4), a member of the pteridine
family, which acts as a crucial co-factor for DA synthesis pathway (Nagatsu et al., 1964). BH4 is
synthesized via two pathways, a de novo one where guanosine triphosphate (GTP) functions as a
precursor and other one a salvage pathway utilizing enzymatically reducible dihydropterins (Fig.
1.2). Briefly, GTP is converted into dihydroneoperin triphosphate (NH2PPP) via GTP
cyclohydrolase (GTPCH), the first and rate-limiting enzyme of the pathway. NH2PPP is then
converted into 6-pyruvoyl tetra-hydropterin (PTP) which in turn is converted into 6(R)L-erythro-
5,6,7,8-tetrahydrobiopterin (BH4) in two successive steps utilizing PTP synthase and sepiapterin
reductase, respectively (Thöny et al., 2000). GTPCH is encoded by the Punch (Pu) locus located
on the right arm of the second chromosome (Mackey and O’Donnell, 1983). Both ple and Pu
loss of function mutants are homozygous lethal, but the heterozygous mutants are viable and
fertile. These heterozygous mutants show dominant effects on DA and BH4 where Pu mutants
have low DA and BH4 levels while ple mutants have only low DA levels (Chaudhuri et al.,
2007).
Catsup is another important regulator of the DA and BH4 biosynthesis pathways, acting
as a negative regulator of both GTPCH and TH. The Catsup protein contains seven predicted
transmembrane domains and interacts with both GTPCH and TH (The O’Donnell Lab,
unpublished observations). The loss of function mutants of Catsup are homozygous lethal;
however, heterozygous mutants show elevated DA (Stathakis et al., 1999).
5

Catsup
Tyrosine
Tyrosine hydroxylase Tyramine decarboxylase
L-DOPA BH4
Tyramine
Dopa decarboxylase Tyramine β Hydroxylase
Dopamine Octapamine
Figure 1.1: Dopamine Biosynthesis Pathway in Drosophila. Tyrosine acts as a precursor to two
distinct pathways in Drosophila. The hydroxylation of tyrosine yields DA as the end product
where L-DOPA acts as an intermediate product. Catecholamines up (Catsup) acts as a negative
regulator of TH whereas tetrahydrobiopterin (BH4) functions as a cofactor for TH. Octopamine
is generated from tyramine, which in turn, is obtained from decarboxylation of tyrosine.
6

GTP
GTP
Cyclohydrolase
(GTPCH)
Dihydroneopterin
triphosphate
6-pyruvoyl
tetrahydropterin
synthase
Figure 1.2: The BH4 biosynthesis pathway. GTPCH, the rate limiting enzyme converts GTP to
dihydroneopterin triphosphate which is then dephosphorylated and reduced to 6- pyruvoyl
tetrahydropterin (PTP) via PTP synthase. PTP is reduced to tetrahydrobiopterin (BH4) by
sepiaterin reductase.
7
6-Pyruvoyl
hydropterin
Sepiaterin
reductase
Tetra-
hydro-
biopterin

Functions of DA in Drosophila
In the adult Drosophila brain, there are 200 TH positive neurons, and expression of DA
has been detected in the nerve terminals present in mushroom body, the center for memory and
learning in flies (Neckameyer, 1998b). Cell bodies are present in specific clusters and their
nomenclature is based on the region of brain where they localize (Fig. 1.3 A, B). These DA
neurons can be visualized by using anti-TH antibody or by driving the expression of Green
Fluorescent Protein (GFP) in TH neurons using the GAL4-UAS system (Brand and Perrimon,
1993, Friggi-Grelin et al., 2003).
The functions of DA in Drosophila will be discussed here considering that DA mediates
similar functions in invertebrates and vertebrates. In Drosophila, the level of DA controls
locomotion both in adult and larvae (Neckameyer, 1996; Cooper and Neckameyer, 1999). In
addition, depletion of DA levels results in defective learning in young males as assayed by the
learned ability to distinguish between mature and immature females for courtship (Neckameyer,
1998a). However, certain behavior features such as larval phototaxis, salt aversion, and heptanol
preference were not altered by depletion of DA in larvae (Neckameyer, 1996).
The loss of function mutations in the gene encoding TH, ple, are homozygous lethal.
Homozygous ple mutants lack melanin, derived from catechols and die at late embryogenesis
(Neckameyer and White, 1993). Pharmacological depletion of DA via DA inhibitor, 3-iodo-
tyrosine (3-IT) or via loss-of-function mutations leads to defective ovarian development. DA
depleted larvae also show developmental delay and decreased fertility suggesting an important
role of DA during oogensis, embryogenesis and larval development (Neckameyer, 1996).
Further, DA and L-DOPA function in cross-linking of cuticle proteins during cuticle formation
8

(Wright, 1987). DA is also implicated in regulation of sleep cycle via DAT (Kume et al., 2005)
and in responses to drugs such as amphetamine and cocaine (Pörzgen et al., 2001).
9

A B
Figure 1.3: The schematic diagram showing the location and number of DA neurons in adult
Drosophila brain viewed from anterior and posterior aspects. (A) The anterior aspect of the brain
consists of two subgroups, PAL (protocerebral anterolateral) and PAM (protocerebral
anteromedial). (B) The posterior aspect consists of five subgroups, PPM1 (unpaired), PPM2
(paired), PPM3 (paired) (protocerebral posterior medial); PPL1 and PPL2 (paired) (protocerebral
posterolateral).
10

Parkinson’s disease
Parkinson’s disease (PD) is the second most common chronic neurodegenerative disease
and the most common movement disorder (Dauer and Przedborski, 2003; Mayeux, 2003). PD is
characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars
compacta (SNpc) along with a decrease in DA levels in their terminals within the dorsal striatum
(Hornykiewicz and Kish, 1987). In addition, PD is associated with neuronal damage in regions
of the brain stem such as the locus coeruleus, raphe nuclei, and the nucleus basalis of Meynert
(Fig. 1.4). This progressive neurodegeneration of the nigrostriatal pathway causes the clinical
manifestations of PD such as rigidity, resting tremor (a rhythmic involuntary 5–7 Hz tremor in
patients at rest), slowness of voluntary movement, postural instability, and in some cases,
dementia (Jellinger, 2001; Savitt et al., 2006).
PD is an age-related neurodegenerative disease, mainly affecting the population over the
age of 55, with an age-dependent increase in incidence. Recent studies indicate that PD afflicts
about 3% of people over 65 years and 4–5% of people over 85 years of age; however, 5–10% of
PD patients are less than 40 years of age (Whitton, 2007). PD appears in two forms: familial and
sporadic. The familial form of PD contributes to only 5% of PD cases and has been linked to
mutations in several genes such as α-synuclein (Spillantini et al., 1997), parkin (Kitada et al.,
1998), DJ1 (Bonifati et al., 2003), PINK1 (Velente et al., 2004), UCHL1 (Das et al., 1998),
Omi/Htra2 (Strauss et al., 2005), and LRRK2 (Paisán-Ruíz et al., 2004). The remaining 95% of
PD cases are attributed to idiopathic causes with possible contributions from mitochondrial
dysfunction, oxidative stress and protein aggregation (Zhou et al., 2008). In addition, recently the
neuroinflammatory process has been recognized as an important factor in the onset as well as
progression of PD (Mosley et al., 2006).
11

Parkinson’s disease and oxidative stress
Oxidative stress has been considered as a major contributor for the initiation of DA
neuron loss in SN. It could be due to decreased endogenous anti-oxidant capacity and/or
excessive production of reactive oxygen species (ROS), reactive oxygen intermediates (ROI) and
reactive nitrogen species (RNS) in cells. The brain consumes almost 20% of the total oxygen in
the body and is relatively sparse in its anti-oxidant defenses such as catalase, superoxide
dismutase, glutathione, and glutathione peroxidase (Floyd, 1999). Especially, the higher
metabolic rate but relatively lower content of anti-oxidant capacity of SN than other regions of
the brain renders it highly sensitive to the effects of ROS/ROI/RNS even in healthy individuals
(Marshall et al., 1999).
The “oxidative stress hypothesis” has been further supported by many experimental PD
models generated using various neurotoxins such as rotenone, 1-methyl-4-phenyl-1,2,3,6-
tetrahydropyridine (MPTP), 6-hydroxydopamine (6-OHDA) and N,N'-dimethyl-4,4'-
bipyridinium dichloride (paraquat) (PQ), which recapitulate many of the pathological features of
PD (Mendez and Finn, 1975; Burns et al., 1983; Betarbet et al., 2000; McCormack et al., 2002).
MPTP and rotenone are well-known mitochondrial toxins. MPTP is highly lipophilic and after
crossing the blood brain barrier is converted into 1-methyl-4-phenylpyridinium (MPP+) in the
presence of monoamine oxidase B in the glia and serotonergic neurons in the central nervous
system (Heikkila et al., 1984). Once formed, MPP+ is released extracellularly and then
internalized in the dopaminergic neurons via DAT (Javitch et al., 1985). Both MPP+ and
rotenone can accumulate within the mitochondria, bind to complex I of the electron transport
12

Figure 1.4: The sagittal section of human brain showing major regions of the human brain. The
cell bodies of dopaminergic neurons, targeted in PD, are located in Substantia nigra (SN), which
is situated in the midbrain region of the brainstem. (Image source:
http://content.answers.com/main/content/img/McGrawHill/Encyclopedia/images/CE093200FG0
010.gif )
13

chain (Nicklas et al., 1985) and subsequently lead to deficient formation of mitochondrial ATP
and accumulation of ROS, such as superoxide, in mitochondria (Cleeter et al., 1992). Superoxide
radical (O2 - ) is converted into hydrogen peroxide, H2O2, initiating the process of cellular
destruction inside as well as outside the mitochondria. On the other hand, after entering into the
brain, oxidation of 6-OHDA in the presence of iron or copper in the intraneuronal and
extraneuronal regions, generates para-quinone, H2O2, superoxide and hydroxyl radicals (Saner
and Thoenen, 1972). H2O2, superoxide and hydroxyl radicals bring about the oxidation of
cellular organelle leading to cell death. Para-quinone causes depletion of vital antioxidants such
as glutathione, inactivation of critical enzymes such as catechol-O-methyltransferase and
tyrosine hydroxylase, which are required for dopamine synthesis (Borchardt et al., 1976; Kuhn et
al., 1999).
PQ or N,N'-dimethyl-4-4'-bipyridinium ion is comprised of two pyridine
rings (aromatic rings in which one carbon atom is replaced by a nitrogen atom) joined covalently
to each other and possessing methyl groups attached to both nitrogens (Fig. 1.5). PQ undergoes
a single electron, reduction-oxidation cycling with subsequent formation of superoxide radicals
(O2 − ) (Bus et al., 1974). This process occurs in two steps: the first step is single-electron
reduction of PQ, which occurs in the presence of diaphorases. PQ diaphorases are
oxidoreductase enzymes which use NADPH as a source of electron(s) to donate to PQ,
generating a PQ radical (PQ .+ ) (Dicker and Cederbaum, 1991). Recently, nitric oxide synthase
(NOS) has been shown to be one of the diaphorases capable of reacting with PQ (Day et al.,
1999). In the second step, oxidation of the PQ .+ obtained from the first step by molecular oxygen
takes place, yielding oxidized PQ or PQ and O2 − (Fig. 1.6). The PQ radical (PQ .+ ) is a powerful
reducing radical, capable not only of reacting with molecular oxygen to generate superoxide
14

adicals, but also of reacting with transitional metals such as iron and thus generates hydroxyl
radicals via the Fenton reaction as shown in Fig.1.7. Intrastriatal, intraperitoneal and intravenous
injections of PQ in the various animal models of PD have demonstrated loss of dopaminergic
neurons, possibly due to increased oxidation of DA to a greater extent than other subpopulation
of neurons (Brooks et al., 1999; McCormack et al., 2002). Aside from oxidative stress, PQ
indirectly brings about depletion of the intracellular stores of NADPH and NADH, and impairs
vital metabolic pathways such as fatty acid synthesis, inhibition of fatty acid synthesis and
increased oxidation of glucose by the pentose phosphate shunt all culminating in death of the
cells (Fisher and Reicherter, 1984).
15

Figure 1.5: The chemical structure of paraquat (PQ). PQ is comprised of two pyridine rings
(aromatic rings in which one carbon atom is replaced by a nitrogen atom) joined covalently to
each other and contain attached methyl groups to both nitrogen. (Image source: Przedborski and
Ischiropolous, 2005).
Figure 1.6: The two step mechanism of PQ toxicity. In the first step, PQ uses NADPH as a
source of electron(s) to donate to PQ, generating PQ radical (PQ .+ ). In the second step, oxidation
of the PQ .+ obtained from the first step by molecular oxygen takes place, yielding oxidized PQ or
PQ and superoxide radical (O2 − ). (Image source: Przedborski and Ischiropolous, 2005).
Figure 1.7: The Fenton reaction. PQ radical generated after reacting with NADPH also reacts
with transitional metals such as iron and thus generates hydroxyl radicals. (Image source:
Przedborski and Ischiropolous, 2005).
16

Parkinson’s disease and Neuroinflammation
Neuroinflammation is proposed to play a crucial role in neurodegenerative. Neuro-
inflammation can be broadly defined as the activation of immune cells such as microglia within
the central nervous system in response to non-self agents or abnormal environment. In general,
this response includes production of inflammatory mediators from neuronal and non-neuronal
cells such a microglia, astrocytes. The initial study by McGeer et al. (1988) demonstrated the up-
regulation of Major Histocompatibility Complex (MHC) molecules in PD patients, along with an
increase in activated microglial cells, the resident immune cells of the brain, in the substantia
nigra (SN). Elevated levels of proinflammatory cytokines such as tumor necrosis factor-alpha
(TNF-α), interleukin (IL)-1beta and IL-6 in the cerebrospinal fluid and striatum in PD brains
(Mogi et al., 1994; Muller et al., 1998). Further, up-regulation of inducible nitric oxide synthase
(iNOS) and cyclooxygenase 2 (COX-2) was found in the SN of PD patients but not in control
individuals (Knott et al., 2000). Enhanced expression of IL-1, IL-6 and TNF-α has also been
shown in the cerebrospinal fluid as well as in the basal ganglia of PD patients (Nagatsu and
Sawada, 2006). In order to understand the mechanisms of neuroinflammation, several animal
models have been generated using MPTP, rotenone and 6-OHDA (Czlonkowska et al., 1996;
Ciccetti et al., 2002; Gao et al., 2002) in which activated microglia have been observed.
Increased pro-inflammatory cytokines/chemokines such as TNF-α, IL-1, IL-6 and enzymes such
as COX-2 and NO are proposed to be toxic to dopaminergic neurons (Liberatore et al., 1999;
Block et al., 2007). Further, the injured neurons recruit more microglia and astrocytes and
influence the release of pro-inflammatory cytokines causing further activation of microglia (Le et
al., 2001). Therefore, neuroinflammatory response has been considered to amplify and/or
accelerate the loss of DA neurons in PD. However, the mechanisms for neuroinflammatory
17

esponses that exaggerate neurotoxicity are poorly understood. Moreover, the studies to date that
investigate neuroinflammatory mechanisms have mostly utilized in vitro approaches. Further, it
is difficult to identify the genes capable of modifying the neuroinflammatory response in
mammalian studies. We have established a Drosophila PD model based on ingestion of the
herbicide paraquat, which recapitulates most behavioral and patho-physiological features of PD,
including loss of dopaminergic neurons (Chaudhuri et al., 2007). Data presented in Chapter 2
demonstrates that paraquat ingestion induces a dramatic and rapid activation of nitric oxide
synthase (NOS) and a corresponding elevation of NO production in adult Drosophila brain.
Since the deleterious effects of paraquat are partially rescued by a NOS inhibitor, the data
suggest that NO plays an important role in PQ toxicity in Drosophila. These data set the
preliminary results for further exploring the neuroinflammatory response in adult Drosophila
brain.
Glia in mammals and Drosophila
In mammals, glial cells are non-neuronal cells constituting about 50 % of the volume of
the CNS where the glia to neuron ratio in CNS is about 10:1. Glia are also found in the
peripheral nervous system. Mammalian glia play a key role in support and nutrition to CNS
neurons, formation of myelin sheath and in signal transmission in the CNS. In addition, they also
perform crucial developmental roles by guiding migration of neurons and generating molecules
supporting the growth of axons and dendrites (Freeman and Doherty, 2006).
Drosophila is emerging as an excellent model to study the molecular and functional and
developmental aspects of glia. In Drosophila, there are mainly four types of glia, namely,
cortex, neuropil, surface, and peripheral glia. Cortex glia, functionally homologous to vertebrate
18

astrocytes, remain in close contact with neurons during the development of the brain (Ito et al.,
1995; Pereanu et al., 2005). Neuropil glia, similar to vertebrate oligodendrocytes, provide
insulation to the axons associated with larval and adult neurons and trophic support to the
developing neurons (Booth et al., 2000). Peripheral glia ensheath and support the peripheral
nerves mediating motor and sensory circuitry peripherally, like Schwann cells in vertebrates
(Leiserson et al., 2000). However, there has been no indication of cells acting as a functional
homolog of vertebrate microglial cells (Freeman and Doherty, 2006). The functions performed
by vertebrate and Drosophila glia are summarized in Table 1.1.
In Drosophila, almost all glial cells are derived from neuroectoderm, the peripheral
ectoderm present lateral to the ventral midline (Ito et al., 1995). The longitudinal glia
ensheathing the longitudinal axons reside along the ventral nerve cord. These lateral glia express
the glial cells missing (gmc) transcription factor, which regulates glial cell differentiation; gmc
positive cells become glia while gmc negative cells become neurons. When gmc is ectopically
expressed, neurons transform into glia (Hosoya et al., 1995). In addition to differentiation of glial
cells, gmc together with its homolog, glial cells missing 2 (gmc2) is essential for the proper
differentiation of hemocytes (Alfonso and Jones, 2002). It is to be noted that gmc expression is
required for the early differentiation of glial cells, and terminal differentiation and maintenance
of glial cell fate is carried out by other genes. Among these fate-determing genes, reverse
polarity (repo), pointed (ppt) and tramtrack (ttk) are well-characterized (Klaes et al., 1994;
Halter et al., 1995; Giesen et al., 1997). repo is a homeodomain transcription factor expressed in
nearly all glial cells and mutation in the repo gene results in glial cell defects at later stages
during embryonic development. The expression of repo and ppt follows the expression of gmc in
different cell contexts.
19

In PD, glial cell dysfunction has been reported. Microglia are mainly associated with the
neuroinflammatory process, but other glial cells have also been known to function in the
pathogenesis of PD (McGeer and McGeer, 2008). In mammalian PD models, astrocytes show
reactive morphology and function to isolate the pathological site being attacked by microglia,
thereby preventing microglial attack to the normal surrounding area. They are also shown to
secrete neurotrophic factors for DA neurons such as glial cell-line-derived-neurotrophic factor
(GDNF) and brain-derived neurotrophic factors (BDNF) (Lin et al., 1993; Knott et al., 2002;
Chen et al., 2006). Some studies have, however, reported that activation of astrocytes is
associated with amplification of the inflammatory reaction through expression of chemokines
and adhesion molecules (Miklossy et al., 2006; Ubogu et al., 2006). On the other hand, only a
few studies have elucidated the functional role of the oligodendrocyte. Overall, these studies
suggest the activation of oligodendrocytes in the SN of PD cases (Yamada et al., 1991; Yamada
et al., 1992).
20

Glial cell type in
vertebrates/Drosophila
Function Distribution in CNS
Astrocytes/Cortex glia Trophic support of CNS cell cortex,
neurons
Synapse transmission
synapse, CNS surface
Oligodendrocytes/Neurophil glia Trophic support of Ensheathment of axons
neurons,
myelination
of CNS
Microglia/?? Immune response
Macrophage function
All around the CNS
Schwann cells/peripheral glia Support of peripheral Ensheathment of
nerves and their
myelination
peripheral nerves
Table 1.1: Similarity in the function and distribution of vertebrate and Drosophila glia in CNS.
Drosophila is thought to have functional homologs for all types of vertebrate glia, except for
microglia. (The table is presented as modified version of table published in Freeman and
Doherty, 2006).
21

Microglia
Microglia are bone marrow derived macrophage-lineage cells which enter into the brain
during embryogenesis and comprise approximately 12 % of the cells in human brain (Kaur et al.,
2001). Microglia function as resident immune surveillance cells in the CNS. Cajal (1931)
defined the cells in the brain: neurons as the first element, astrocytes as the second element and
microglia as the third element. Rio-Hortega (1932) subsequently performed a systemic
investigation of microglial cells. In the normal brain, microglia, are in a resting stage, with cell
bodies having only a few fine ramified processes and low expression of surface antigens
(Lawson et al., 1990). In pathological conditions or during invasion of pathogens, microglia
become activated with morphological changes evident by an increase in cell body area and
ramifying processes exhibiting amoeboid and spider-like appearance (Fig. 1.8). In addition, once
activated, microglia increase in number and up-regulation of surface markers such as CD14,
Major Histocompatibility Complex (MHC) molecules, chemokine receptors, pro-inflammatory
cytokines, interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin 1-β (IL-1β), and
enzymes such as inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX) 1 and 2
occurs (McGeer et al., 1988; Mirza et al., 2001; Rock et al., 2004; Nimmerjahn et al., 2005). It is
well known that activation of macrophages/microglia against invading microbes and other
harmful pathogens serves as the first line of defense to the host organisms and serves as an
essential function for the survival of organisms. It is proposed that mild to moderate activation of
microglia performs a homeostatic, neurotrophic and thus neuroprotective role in neuropathology
(Batchelor et al., 1999; Batchelor et al., 2002), but sustained activation of microglia actively
participates in the self-perpetuating inflammatory processes linked to neurodegeneration in
various neurodegenerative and inflammatory diseases. Therefore, the role of microglial cells in
22

the pathogenesis of chronic neurodegenerative diseases including PD, discussed above, suggests
that maintenance of the delicate balance for the microglial beneficial and deleterious functions is
crucial (Wyss-Coray and Mucke, 2002; McGeer and McGeer, 2004) (Fig. 1.8). However, the
stimulus causing the alteration in such balance is still not well understood. In Chapter 2,
identification of NOS expressing structures in adult Drosophila brain in response to PQ ingestion
have been identified which may appear analogous to NOS expressing mammalian microglia.
23

Physiological condition
(supporting regulatory immune
and homeostatic functions)
Microglia
Figure 1.8: An outcome of the sustained activation of microglia in chronic neurodegenerative
disease. Microglia activation is associated with a delicate balance of physiological and
pathological conditions. In chronic neurodegenerative diseases, sustained activation of microglia
leads to destruction of diseased as well and as healthy neurons and thus amplification and
acceleration of neurodegenerative process. The morphology of becomes reactive one from
resting stage as shown in figure. (Image source for images showing microglial morphology:
Mosley et al., 2006).
24
Pathological condition
(reactive to destructive role)
-Excessive stimulation of
microglia
-Up-regulation of microglial
markers
-Destruction of diseased and
Resting microglia Reactive microglia

Neuroinflammation in experimental models of PD
MPTP is a potent neurotoxin known to induce depletion of dopaminergic neurons in
humans (Langston et al., 1983). Examination of post-mortem brains of patients 3-16 years after
exposure to MPTP for relatively short period and with symptoms of PD showed pathological
features of neurodegeneration and activated microglia (Langston et al., 1999). Similarly, 6-
OHDA exposure in mice induces the proinflammatory cytokine TNF-α in the striatum and CSF
of rats (Mogi et al., 1994). Signs of inflammation remained one month after 6-OHDA exposure
as evaluated by increased expression levels of mRNA for IL-1α and IL-1β in lesioned tissues
(Depino et al., 2003). Rotenone induced increased expression of Mac-1-positive reactive
microglia in the striatum and SN, even in the absence of DA neuron loss (Sherer et al., 2003). PQ
toxicity has been reported to cause an increase in cytokines, IL-1β, IL-6, IL-10 and TNF-α in
serum of rats; however, these changes are associated with a high dose (120 mg/kg) (Jian et al.,
2007). Similarly, acute poisoning with lethal and non-lethal doses of PQ involeved up-regulation
of iNOS and IL-1β, respectively in rats (Tomita et al., 1999). In in vitro studies, PQ has been
shown to generate excess superoxide radicals via NOS and NADPH oxidase enzymes in
microglia and thereby proposed to mediate PQ induced oxidative damage to nearby neurons
including DA neurons (Bonneh-Barkay et al., 2005; Wu et al., 2005). Exposure to relatively low
dose (10 mg/kg) of PQ exposure also causes an increase in Mac-1 immunoreactive cells in in
vivo mammalian model (Purisai et al., 2007).
Inhibition of neuroinflammation in PD models
Genetic and pharmacological inhibition of microglia has been shown to decrease the
induction of microglia by MPTP, 6-OHDA and lipopolysaccharide (LPS). Although these
25

studies have targeted isoforms of NOS and/or cytokines known to be up-regulated in various
toxin-induced PD models, the results are somewhat controversial since these approaches may not
produce complete inhibition of activated microglia or prevention of DA neuron loss. Especially,
although activation of iNOS is mainly reported with reactive microgliosis in PD models,
suppression of microglial activation has not been achieved with elimination iNOS genetically
(Dehmer et al., 2000; Iravani et al., 2002). More recently, inhibition of nNOS has been reported
to impart protection against MPTP induced mouse PD model (Watanabe et al., 2008).
The daily intake of Non-Steroidal Anti-inflammatory Drugs (NSAIDs) has been shown to
reduce the risk of PD in epidemiological studies (Chen et al., 2003). Inhibition of COX-2, the
rate-limiting enzyme in prostaglandin E2 synthesis markedly diminishes dopaminergic
neurodegeneration along the nigrostriatal axis when exposed to MPTP and 6-OHDA (Teismann
et al., 2001; Sanchez-Pernaute et al., 2004).
Minocycline, a second-generation semi-synthetic tetracycline derivative has been shown
to act on multiple components involved with neuroinflammatory process in chronic
neurodegenerative and neuronal injury. It has been shown to down-regulate iNOS, inhibit NOS-
mediated phosphorylation of p38 mitogen-activated protein kinase (MAPKs), and reduce IL-1β
converting enzyme (ICE) and IL-1β production, to decrease MPTP induced increase in IL-1 α,
IL-6 and other microglial markers (Yrjänheikki et al., 1999; Lin et al., 2001; Wu et al., 2002;
Sriram et al., 2006). For these properties, minocycline is currently being tested in Phase III
clinical trials for PD. Minocycline has been used in many studies as an agent of microglial
inhibition in mammalian models for PD (Peng et al., 2006; Purisai et al., 2007) and has been
shown to increase the survival of PQ-fed flies (Bonilla et al., 2006). However, neuroprotective
and anti-inflammatory roles for minocycline in flies have never been studied in detail.
26

Identification of neuroprotective and anti-inflammatory functions of minocycline in a well
known in vivo model that can be genetically manipulated, such as Drosophila melanogaster,
could define a model that could be used to identify genes modifying this response and to identify
signaling pathways that mediate this inflammatory response. We have demonstrated that
minocycline is capable of suppressing NOS activity in adult Drosophila brain. In chapter three,
we have further identified the neuroprotective properties of minocycline and presented the
preliminary results for the signaling pathways mediating the PQ-induced DA toxicity in this in
vivo model.
Nitric oxide
Nitric oxide is a gas that functions as a second messenger molecule in various metabolic
processes (Bredt and Snyder, 1994; Murad, 1998). NO is generated when L-arginine is converted
into citrulline via nitric oxide synthase (NOS) in the presence of molecular oxygen and NADPH.
NOS, which is active as a dimer, consists of an oxygenase (catalytic) domain and a reductase
domain. The catalytic domain of NOS (NOSox) contains a heme group and BH4, and binds the
substrate, L-arginine (Mayer, 1994). The cofactors, flavin mononucleotide (FMN), flavin
adenine dinucleotide (FAD) and NADPH bind to the reductase domain of the enzyme (NOSred)
(Marletta, 1989; Bredt et al., 1991). Ca 2+ /calmodulin acts to promote electron transport between
the NOSred and NOSox (Crane et al., 1999). NO generation involves a two-stage reaction via
formation of hydroxyarginine resulting in the oxidation of the guanadino nitrogen group of
arginine, to form citrulline (Fig. 1.9). Three isoforms of NOS are encoded by separate genes,
NOS1, NOS2 and NOS3, in vertebrate genomes. These genes are further classified according to
27

the tissue of origin: nNOS (neuronal NOS; encoded by NOS1); iNOS (inducible, macrophage
NOS; encoded by NOS2), and eNOS (endothelial NOS; encoded by NOS3).
In vertebrates, the function of NOS was recognized first in the maintenance of the
vasodilation, where the absence of NO signaling was implicated in the erectile dysfunction in
males (Burnett, 1995). Subsequently, various studies performed using biochemical, physiological
and transgenic approaches in vertebrates, identified diverse physiological roles of NO. It is
known that NO signaling is important in learning and memory (Hölscher, 1997), reproduction
(Rosselli et al., 1998), and in host defense mechanisms (Liew et al., 1999). Activation of NO
signaling is mediated by diverse stimuli and by rapid diffusion of the NO molecule across
cellular membrane (Davies SA, 2000).
28

Nitric oxide
synthase
Figure 1.9: A schematic diagram showing the components involved in the formation of nitric
oxide (NO). NO is generated along with Citrulline from L-Arginine and molecular oxygen. The
catalytic domain of NOS (NOSox) contains a heme group and BH4, and binds the substrate, Larginine.
The cofactors, FMN, FAD and NADPH binds to the reductase domain NOS.
Ca 2+ /calmodulin promotes electron transport between the NOSred and NOSox. (Image source:
modification of figure published in Davies SA, 2000).
29
NADPH

NO signaling in invertebrates
Many comparative physiological and biochemical studies have presented strong evidence
that NO signaling is evolutionarily and functionally conserved. Several insect NOS genes have
been cloned, including those from Drosophila melanogaster (Regulski and Tully, 1995) and
Anopheles stephensi (Luckhart and Rosenberg, 1999). Insect NOS, including Drosophila NOS,
shows highest sequence similarity to NOS1 (49%), as compared with NOS2 (44% identity) and
NOS3 (47% identity) (Luckhart and Rosenberg, 1999; Stasiv et al., 2001). The insect NOS
protein sequence shows 100% sequence identity in regions corresponding to the cofactor binding
sites for calmodulin, FAD, FMN and NADPH in vertebrate NOS. Similar to vertebrates, NO is
known to function in learning and memory, axonal guidance, locomotion and olfaction in insects.
Drosophila NOS and NO signaling
Regulski and Tully (1995) first reported the cloning and molecular characterization of the
Drosophila NOS gene. The gene consists of 19 exons extending over 34 kb of DNA, located on
the second chromosome at cytological position 32B. Using in situ hybridization and quantitative
RT-PCR, Stasiv et al. (2001) determined the expression of dNOS transcript during Drosophila
development. dNOS transcripts were detected in lower levels in the embryonic stage but were
abundantly present in the late larval and pupal stages. NO signaling in Drosophila has been
shown to be involved in the regulation of cell proliferation during imaginal disc development
(Kuzin et al., 1996; Kuzin et al., 2000), development of the nervous system (Truman et al., 1996;
Wildemann and Bicker, 1999), retinal patterning in the optic lobe (Gibbs and Truman, 1998),
epithelial fluid secretion (Dow et al., 1994), response to hypoxia (Wingrove and O’Farrel,1999;
30

DiGregorio et al., 2001), behavior (Wingrove and O’Farrel, 1999), and immunity (Nappi et al.,
2000).
In Drosophila, different methods have been used to determine the expression pattern of
NOS. A universal NOS antibody has been utilized to detect all isoforms of NOS. This antibody,
which recognizes conserved amino-acid sequences corresponding to amino acid residues 1113-
1122 of murine iNOS and nNOS, has been used to detect Drosophila NOS in studies of visual
system development and of malphigian tubules where it functions in osmoregulation and
excretion (Davies, 2000; Gibbs, 2001). Similarly, the 150 kDA NOS polypeptide has been
detected in malphigian tubules with western blot analysis using the universal antibody
(Broderick et al., 2003). Although the expression pattern for NOS in adult brain has not been
determined with this antibody, the expression of NOS in embryos and larval brain have been
studied in detail using NADPH diaphorase staining, confirming its presence in the Drosophila
nervous system (Wildemann and Bicker, 1999).
Role of NO in Drosophila immune response
Nappi and Vaas (1998) showed that wasp parasitoid L. boulardi induced production of
reactive oxygen intermediates (ROI) in larval hemocytes. Both hydrogen peroxide and
superoxide function as important messengers capable of activating transcription factor NF-κβ
(Schmidt et al., 1995). Induction of this innate immune response has been demonstrated against
parasites and bacteria, but there has been no similar study with respect to a possible neurotoxin
induction of the insect innate immune response that is comparable to microglial activation in
adults. Nappi et al. (2000) demonstrated increased production of superoxide radical and H2O2 in
immune-challenged Drosophila against the wasp parasitoid L. boulardi. They showed that upon
31

infection with wasp, D. melanogaster and D. teissieri generate reactive intermediate species of
oxygen and nitrogen, ROS and RNI, which constitutes an important step in induction of
antimicrobial peptides like Diptericin. Foley and O’Farrell (2003) used universal NOS antibody
and an antibody generated against a C-terminal peptide of dNOS to detect increased expression
of NOS protein in larval hemocytes, the innate immune cells of Drosophila in response to gram-
negative infection. Similar results were obtained with both antibodies. The presence of
hemocytes in the adult Drosophila brains has been reported (Lanot et al., 2001; Holz et al.,
2003). These results indicate the role of Drosophila hemocytes in induction of NOS against
infection suggesting future directions for determining whether these cells have a crucial role in
the response to paraquat in adult brain.
Role of signaling pathways in the pathogenesis of PD
Apoptosis signaling pathway
Apoptosis or programmed cell death (PCD) is an important and essential process in all
multicellular organisms to prevent developmental disorders and tumorigenesis (Vaux and
Korsmeyer, 1999; Yuan and Yankner, 2000). It is proposed that death signals induced by
developmental and cellular damage cause activation of the cysteine proteases, caspases, which
are known to play an important role in PCD including membrane blebbing and fragmentation of
DNA. Drosophila melanogaster has evolutionarily conserved mechanisms for PCD (Vernooy et
al., 2000). Mammalian cells possess two types of PCD pathways, extrinsic and intrinsic death
inducing pathways. The intrinsic pathway involves release of mitochondrial cytochrome c due to
cellular stress and its binding to adaptor protein, Apaf-1, which in turn recruits pro-caspases 9
and brings about autocatalytic activation (Li et al., 1997; Zou et al., 1999). The extrinsic pathway
32

involves tumor necrosis factor family with activation of caspases via Fadd (Fas-associated death
domain) (Ashkenazi and Dixit, 1998; Ashkenazi A, 2002). The Drosophila PCD pathway shares
many of the signaling components found in mammals and provides an excellent opportunity of
dissecting the functions of signaling components of PCD in vivo. The extrinsic and intrinsic
signaling pathways involved with mammalian apoptosis are compared with Drosophila apoptosis
signaling pathway in Fig. 1.10.
33

Extrinsic death pathway
Tumor Necrotic Factor
Receptor family
FADD
dFADD
Diablo/Smac
Rpr, hid, grim
p53
Dmp53
Intrinsic death
pathway
Bcl-2
Dabcl
Apaf-1
Dark
Caspase-8
Dredd
Cytochrome c
Initiator caspases
Dronc, Dredd
Figure 1.10: Comparison of mammalian and Drosophila apoptotic signaling pathways.
Drosophila possesses homologues (shown in red) for the mammalian apoptosis signaling
components (shown in black) (for details, see text). (Image source: Modification of the image
published in Richardson and Kumar, 2002)
34
IAP
Diap
Effector caspase
Drice, Caspase-3 Dcp-1
PROGRAMMED
CELL DEATH

PCD in Drosophila
Caspases are cysteine proteases found in an inactive form, pro-caspases in cells, which
upon receiving an apoptotic signal undergo proteolytic processing to generate active enzyme,
caspase. Seven caspases have been identified: Dcp-1, Dredd/Dcp-2, Drice, Dronc, Decay,
Strica/Dream and Damm/Daydream (Kumar and Doumanis, 2000). Out of the several PCD
related genes, reaper (rpr) acts as a pro-death genes in Drosophila; the protein encoded by this
gene is functionally homologous to mammalian Diablo/Smac (Direct IAP Binding protein with
low pI/second mitochondria derived activator of caspases). Like the mammalian Diablo/Smac,
Rpr, along with other Drosophila pro-death proteins, Hid and Grim, interacts with the IAP
(Inhibitor of Apoptosis) and antagonizes the action of IAP, thereby causing PCD (Vucic et al.,
1997; Vucic et al., 1998). In Drosophila, Dronc acts as an apoptosis/caspase initiator leading to
activation of Drice, the executor of PCD.
In PD, the death of DA neurons in SN has been proposed to occur through activation of
the apoptotic signaling pathway (Levy et al., 2009). Several experimental models for PD have
reported the involvement of up-regulation of Bax, Bak Bim and other apoptosis related genes
(Biswas et al., 2005; Perier et al., 2007; Fei et al., 2008). Moreover, activation of PCD
components, caspase-3 and caspase-8 in the DA neurons of SN in PD has been reported
(Hartmann et al., 2001; Tatton et al., 2003). Furthermore, many of these studies have reported
the reversal of apoptotic death using caspase-inhibitors suggesting the involvement of apoptosis
in the pathogenesis of PD. The interaction of apoptotic signaling with other signal transduction
pathways is described below.
35

Mitogen activation protein kinase signaling pathway
Mitogen activation protein kinases (MAPKs) are evolutionarily conserved signaling
pathway components that mediate numerous fundamental cellular processes, including cell
proliferation, survival, differentiation, apoptosis, motility and metabolism (Chang and Karin,
2002). Various MAPK family members are activated by different stimuli and are involved in
signaling by phosphorylating specific serines and threonines of target protein substrates such as
protein kinases, phospholipases, transcription factors, and cytoskeletal proteins. MAPKs are a
part of the phospho-relay system involving sequentially activated kinases regulated by
phosphorylation (Fig 1.11). Cells receive different stimuli from their environment and depending
on the stimuli, activation of downstream Mitogen-activated protein kinase kinase kinases
(MKKKs) occurs. MKKKs phosphorylate and activate specific MKKs. MKKKs have distinct
motifs in their sequences that selectively mediate their activation in response to different stimuli.
MKKs, in turn, catalyze the phosphorylation of MAPKs. Activation of MAPK catalyzes the
phosphorylation of its own substrates which are varied for the different MAPKs signaling
pathways. MAPKs are regulated by MAPKs phosphatases which reverse phosphorylation and
return the MAPKs to an inactive state (Johnson and Lapadat, 2002).
There are three well-characterized MAPKs signaling pathway subfamilies: extracellular
signal–regulated kinases (ERK), c-Jun NH2-terminal kinases (JNK) and p38 enzymes.
Activation of these signaling pathways depends on the stimuli and the cell type in which they are
activated. In general, the ERK subfamily, which includes ERK 1 and ERK 2, is involved in the
regulation of meiosis, mitosis, and postmitotic functions in differentiated cells. The JNK
pathway participates in stress-related signaling while p38 is activated in immune cells by
inflammatory cytokines (Kyriakis and Avruch, 2001).
36

JNK signaling pathway
JNK signaling pathway has been well studied (Davis RJ, 2000; Johnson and Lapadat,
2002). The JNK serves as phosphorylation substrates for MAP kinase kinases (MKKs) such as
MKEK4/7, which are activated in turn by phosphorylation via MAP kinase kinase kinases
(MKKKs) such as MKK4/7 (Fig. 1.11). It has been proposed that activation of JNK involves
release of cytochrome c or Smac/DIALBO into the cytoplasm and subsequent formation of a
complex with apoptosis activating factor-1 (Apaf-1) and caspase-9 causing activation of
executioner caspase-3 and cleavage of cellular substrates eventually (Tournier et al., 2000;
Chauhan et al., 2003). In addition, other studies have suggested that the pro-apoptotic cascade
induces apoptosis via a mitochondria-dependent pathway involving JNK-activated pro-apoptotic
members of the Bcl-2 family, Bim and Bmf, resulting in activation of Bax and Bak (Lei et al.,
2002; Lei and Davis, 2003). Furthermore, JNK activation results in phosphorylation of anti-
apoptotic genes, Bcl-2 and Bcl-xL and thus induction of apoptosis (Pandey et al., 1999;
Yamamoto et al., 1999).
37

Figure 1.11: A schematic diagram showing the components of MAPK signaling pathway in
mammals and Drosophila. ERK and JNK signaling pathways are sub-families of MAPK and
follow the phospho-relay system involving sequentially activated kinases regulated by
phosphorylation and results in specific biological response (for details, see text). Drosophila
possesses homologues (shown in red) for the mammalian apoptosis signaling components
(shown in black) for each component of ERK and JNK signaling pathways. (Image source:
modification of figure from www.cellsignaling.com).
38

Drosophila JNK
Drosophila JNK is a homolog of mammalian JNK and is encoded by the gene basket on
the left arm of the second chromosome (Nüsslein-Volhard et al., 1984). The JNK pathway
includes a MAPKKKs (mekk1, slipper, wallenda/MEKK, MLK3, ASK1) (Stronach and
Perrimon, 2002) and a MAPKKs (hemipterous/MKK7) (Glise et al., 1995), as well as the JNK
homolog basket. Basket, upon phosphorylation by Hemipterous, activates the AP-1 transcription
complex consisting of Jun and Fos, modifying target gene expression. Drosophila JNK has been
reported to play crucial roles in morphogenesis and mobility, immune response, wound healing
and stress response in flies.
JNK signal transduction pathway and Parkinson’s disease
Recently, the role of JNK in the pathogenesis of PD has been proposed. In mouse PD
models in which neurodegeneration is induced by MPTP or PQ, activation of JNK via
phosphorylation of c-Jun is associated with apoptosis of dopaminergic neurons (Xia et al., 2001;
Saporito et al., 2003; Peng et al., 2004). Peng et al. (2004) showed that intraperitoneal injection
of PQ induces an increase in superoxide and related ROS, subsequently causing activation of
JNK thereby triggering up-regulation of caspases-3, contributing to the apoptotic response. A
similar mechanism has been shown to be involved in the 6-OHDA induced loss of dopaminergic
neurons in PC cells where activation of JNK culminates in the induction of caspase-3
(Rodriguez-Blanco et al., 2008). More recently, using PC 12 cell culture and primary cultured
dopaminergic neurons, Klintworth et al. (2007) found that treatment of these cultures with PQ
causes the stimulation of p38 and JNK signal transduction pathways and, subsequently, loss of
dopaminergic neurons. Although these in vitro studies support the pro-apoptotic role of JNK
39

signaling pathway in PD models, the genetic redundancy resulting from the three JNK genes in
mammalian genomes, JNK1, 2 and 3 and the 10 or more isoforms of JNK expressed by these
genes, creates complexities in interpretations of the roles of JNK in the pathogenesis of PD.
In Drosophila, functional roles of JNK/Bsk have been studied in the context of neuronal
connectivity and response to oxidative and pathogenic stress. Srahna et al. (2006) demonstrated
that Bsk is required for the axonal connectivity of dorsal cluster neurons between the lobula and
medulla during the development of the optic lobe. Wang et al. (2003b) showed that heterozygous
loss of function mutant, bsk 2 , is highly sensitive to PQ-induced oxidative stress which could be
overcome by pan-neuronal over-expression of bsk employing an elav-GAL4 driver. In addition,
these investigators found that JNK is essential for longevity and combating oxidative stress. Cha
et al. (2005) detected the up-regulation of JNK in loss-of-function of parkin mutants. Moreover,
using genetic interaction between transgenic strains for signaling components of the JNK signal
transduction pathway and parkin revealed a negative relationship between JNK and parkin.
These results suggest that cell type and the context of the stimulus play an important role for
anti-apoptotic and pro-apoptotic property of JNK. Mammalian JNK appears to respond to
oxidative stress by triggering apoptosis in DA neurons in in vitro mammalian models, but
functional roles of JNK in in vivo PD models need further elucidation.
ERK pathway in mammals
ERK function is mainly concerned with cellular proliferation and differentiation of
various cells in response to growth factors and other stimuli. In addition, recently, ERK1/2 are
reported to regulate neuronal responses to both functional (learning and memory) and pathologic
40

(regulated cell death) stimuli. The cascade for the ERK mediated activation of transcription
factors includes phosphorylation of MAPKKK, MAPKK and MAPK as shown in Fig 1.11.
ERK in Drosophila
Drosophila ERK is a homolog of mammalian ERK and is encoded by the gene rolled
(Lim et al., 1999). The ERK pathway includes a MAPKKK (draf-1/RAF), and a MAPKK
(dsor1/MEK1/2), as well as the ERK1/2 homolog rolled. Rolled, upon phosphorylation,
activates the pointed transcription factor activating target gene expression (Brunner et al., 1994).
In Drosophila, rolled has been shown to mediate key roles in synaptic transmission and
regulation of developmental processes such as sensory organ development.
ERK signaling pathway and Parkinson’s disease
There are numerous controversial studies presenting data for both neuroprotective and
neurodegenerative roles of ERK in the pathogenesis of PD. The neuroprotective role of ERK1/2
in neuronal cell lines and primary neuron cultures in mammalian toxin and genetic PD models
has been reported (Hashimoto et al., 2003; Cavanaugh, 2004; Hetman and Gozdz, 2004).
However, at the same time, aberrant up-regulation of phospho-ERK1/2 in substantia nigra
neurons of patients with PD and other Lewy body diseases, as well as in the midbrains of these
patients has been documented (Zhu et al., 2002a). Furthermore, such up-regulation of ERK1/2 is
associated with excessive generation of ROS and RNS in PD (Kulich and Chu, 2001). More
recently, Miller et al. (2007) demonstrated that NADPH oxidase mediates the PQ-induced
elevation of ROS in microglial cells, which involves activation of the ERK dependent signaling
pathway.
41

In a PQ+ Maneb model for PD, association of phosphorylation at serine 112 of pro-
apoptotic protein Bad with the phosphorylation of p 42 /p 44 serine sites of ERK in the midbrain
extract has been reported. Such association suggests the up-regulation of ERK is inclined
towards apoptotic death in oxidative stress (Thiruchelvam et al., 2005). Therefore, in light of
these reports, the exact functional role of ERK is controversial in PD and could be elucidated
using a in vivo Drosophila PD model.
Akt signaling pathway
Akt is a serine/threonine protein kinase acting as a cellular homolog of the viral oncogene
v-Akt. Although the C-terminal catalytic domain of Akt is closely related to that of most of the
members of the protein kinase C (PKC) family, the N-terminal domain is distant from that of
PKC family members, and therefore, Akt is categorized as a member of the Protein kinase B
(PKB) family (Kandel and Hay, 1999). In mammals, there are three known isoforms of the Akt
kinase, Akt1, Akt2, and Akt3, each activated by various growth and survival factors. Once a
surface receptor is activated, secondary messengers are activated and in turn activate
phosphoinositide 3-kinase (PI3K) located upstream of Akt. Once phosphorylated, upon
activation of PDK1, Akt promotes cell survival through various distinct pathways. Among them,
two are commonly implicated. In the first one, activation inhibits apoptosis by phosphorylating
the Bad component of the Bad/Bcl-xL complex. The phosphorylated Bad binds to 14-3-3 causing
dissociation of the Bad/Bcl-xL complex and mediating cell survival. In the second one, Akt
directly antagonizes the activation of NF-κβ inhibitor, IKK-α, ultimately leading to NF- κβ
activation and cell survival (Burke, 2007) (Fig. 1.13). Akt also regulates cell growth,
42

proliferation, migration, glucose metabolism, transcription, protein synthesis and angiogenesis
(Brazil & Hemmings, 2001).
Drosophila Akt signaling pathway
The Drosophila genome contains a single Akt1 gene encoding a protein that is 76.5%
similar to mammalian Akt protein (Franke et al., 1994). All known components of the
mammalian Akt signaling pathway are implicated in the Drosophila Akt signaling pathway
(Scanga et al., 2000). Genetic analysis of Akt demonstrated that this kinase mediates an anti-
apoptotic mechanism that is regulated by caspases instead of the pro-apoptotic components,
reaper, hid and grim (Staveley et al., 1998).
Akt signaling pathway and Parkinson’s disease
There is compelling evidence supporting dysregulation of the mammalian Akt signaling
pathway in genetic and toxin models for PD (Seo et al., 2002 ; Rodriguez-Blanco et al., 2008;
Xiromerisiou et al., 2008 ). Yang et al. (2005) reported that knock-down of DJ1, one of the PD-
associated genes, down-regulates the PI3K/Akt signaling pathway, thereby causing loss of DA
neurons in Drosophila. Therefore, these studies suggest that inactivation of Akt results in
apoptotic death of DA neurons. It is proposed that Akt negatively regulates the phosphorylation
and activation of c-jun, a component of the JNK signaling pathway known to induce apoptotic
death in numerous PD mammalian models. Ries et al. (2006) proposed that vector based delivery
of Akt is associated with neurotrophic effects in mammals. However, it is to be noted that most
of these studies have utilized in vitro PD models and thus the functional role of Akt at the whole
43

organism level has not been well elucidated. Moreover, its role in PQ-mediated DA toxicity is
lacking.
44

Figure 1.12: A schematic diagram showing the signaling pathways associated with activated Akt.
Stimulus from growth and survival factors results in activation of Akt via PI3K. Activated Akt
promotes cell survival by inhibiting pro-apoptotic gene, Bad, and by stimulating activation of
NF-κβ (also, see text).
45

Gaps in understanding the mechanism of induction of an inflammatory response in Parkinson’s
disease and its importance in the pathogenesis of Parkinson’s disease
There are several studies correlating the induction of the inflammatory process and its
role in the pathogenesis of Parkinson’s disease in mammalian models. However, there are
several unanswered key questions. These questions are presented below briefly with possible
directions to answer them.
1. While there are compelling studies linking the process of inflammation to the pathogenesis
and progression of Parkinson’s disease, most of these studies have been performed using in vitro
approaches. It is still debated whether activation of microglia in PD is deleterious or beneficial to
the individual. Moreover, studies exploring the importance of genetic interaction in the
inflammatory process are lacking. A simple and genetically modifiable model that would allow
in vivo approaches could provide novel insights into the process of inflammation in the
Parkinson’s disease. Chapter 2 describes the discovery of the first in vivo Drosophila model
capable of inducing increased expression of NOS against PQ exposure. We have used this model
to study the role of genes modulating the inflammatory response in Parkinson’s disease using a
well-known anti-inflammatory drug, minocycline. This chapter presents the data which could be
further explored to determine the source of NOS in response to paraquat and may support the
induction of NOS expressing mammalian microglial cells in PQ-induced in vivo Drosophila PD
model.
2. A variety of signal transductions pathways have been implicated in processes leading to
pathological conditions in neurons and to the neuroinflammatory response. Most components of
these pathways are evolutionary conserved, exhibiting functional similarities in invertebrates and
vertebrates. The activation of signaling pathways in PD have been extensively studied in
46

mammalian genetic and toxic PD models. However, the functional redundancy associated with
the existence of multiple genes and numerous isoforms in mammals creates difficulties in
understanding the exact role of signaling pathway in the pathogenesis of PD and could be
eliminated in the simple in vivo Drosophila PD model, since most signaling proteins are encoded
by single genes. In Chapter 3, identification of the neuroprotective role of minocycline in a
Drosophila PD model are described. Further, this chapter also reports preliminary studies for the
signaling pathways involved with DA toxicity in this Drosophila PD model.
3. The occurrence of Parkinson’s disease in the population of less than 40 years of age is referred
to as Early Onset Parkinsonism. Chapter 3 investigates the correlation of brief exposure to
paraquat during the juvenile and young ages with the pathogenesis of PD in Drosophila PD
model. This study opens new avenues to understand the progressive alteration in dopamine
homeostasis during the entire period of life in a simple and efficient manner.
4. Although the exact etiological agents for PD pathogenesis is still not known, experimental
studies have resulted in the hypothesis that multiple factors including exposure to insecticides,
pesticides, and other environmental toxins could act as substantial etiological causes for sporadic
PD. It is noteworthy that increased incidence of PD is associated reported in some agricultural
populations. Recently, the role of bacterial pathogens in neuronal degeneration has been tested
(The Caldwell Lab, UA). They have identified Streptomyces venezuale as a pathogen responsible
for the loss of dopaminergic neurons in C. elegans., In Chapter 5, one of the possible
mechanisms of action of this toxin via induction of NOS positive structures in adult Drosophila
brain in the pathogenesis of PD has been elucidated.
47

CHAPTER 2
DROSOPHILA MOUNTS A NITRIC OXIDE-DEPENDENT RESPONSE TO PARAQUAT-
INDUCED DEGENERATION OF DOPAMINERGIC NEURONS
This work is under revision for submission to Nature Neuroscience with the following authors:
Inamdar A., Lawal H., Ferdousy F., Chaudhuri A., and O’Donnell J. Drs. Hakeem Lawal and
Faiza Ferdousy collected data presented in Fig. 2.6 and Fig. 2.8. Dr. Anathbandhu Chaudhuri
helped Arati Inamdar to collect data presented in Fig. 2.3. The remaining data were collected and
analyzed by Arati Inamdar. Dr. Janis O’Donnell and Arati Inamdar wrote the manuscript.
48

INTRODUCTION
Excessive activation of the innate inflammatory response is considered a crucial and key
factor in the pathogenesis of neurodegenerative diseases as well as in neurological conditions
associated with injury and compromised blood supply to brain. Recently, evidence has been
presented in support of an escalated neuroinflammatory response in Parkinson’s disease (PD)
both in patients and in experimental models. These studies include documentation of increased
levels of inflammatory cytokines/chemokines in PD patients and PD models (Hunot and Hirsch,
2003; Liu, 2006). In addition, generation of nitric oxide, reactive nitrogen species (RNS) and
peroxynitrites has been observed in experimental animals modeling Parkinsonian features and
symptoms (Hunot et al., 1996; Hunot et al., 1999; Mosley et al., 2006; Block et al., 2007).
Further, an increase in cyclooxygenase 2 (COX-2), the enzyme responsible for the formation of
prostanoids and thus mediating inflammatory responses in the substantia nigra and around DA
neurons in PD patients and in MPTP and 6-OHDA-induced PD animal models, has been
reported (Di Matteo et al., 2006; Tyurina et al., 2006; Vijitruth et al., 2006). Moreover,
epidemiological studies have found a correlation between long-term consumption of Non-
Steroidal-Anti-Inflammatory Drugs (NSAIDS) and a decreased incidence of PD (Esposito et al.,
2007). Most importantly, a profound increase has been observed in the number and the activity
of the microglial cells, innate immune surveillance cells resident in the CNS, in the nigral
regions of PD brains. These microglial cells normally are present in small numbers in the brain,
where they are maintained in a resting state in and around the SN (Sugama et al., 2003; Zhang et
al., 2005).
Under pathological conditions such as injury and infection, these cells mediate an inflammatory
process as a normal protective response. In addition, they perform numerous cellular
49

maintenance functions essential for neuronal survival, releasing trophic and anti-inflammatory
factors, and clearing cellular debris by initiating programmed cell death in neurons (Liao et al.,
2004; Morgan et al., 2004; Cho et al., 2006; Simard et al., 2006).
However, microglia also are considered to be the main culprits responsible for converting
their normal surveillance functions into functions detrimental to normal healthy neurons in the
brain, thereby amplifying the effects of neurodegenerative disease or neuronal injury. Under
pathological conditions such as neuronal injury or immunological stimuli, microglia are activated
and release excessive chemokines/cytoxins such as TNF-α, IL-1, IL-6 and enzymes such as
iNOS, COX-2 to combat the altered cells in such disease states (Block et al., 2007). The failure
of the normal surveillance function of microglia in neurological diseases including PD and the
derangement of the balance between neuroprotective and neurodegenerative roles is still not well
understood. Further, it is unknown whether signals from dying neuron or activated microglia
initiate a self-perpetuating condition resulting into amplified microgliosis in PD. Most studies
suggesting an adverse role for microglia in PD pathogenesis are performed in in vitro conditions,
thereby lacking strong evidence for such detrimental effects of microglia at the whole organism
level. Moreover, in the absence of a simple in vivo model in which powerful genetic
manipulation methods can applied to understand the functional role of microglia in Parkinson’s
disease, the modulatory effect of genes participating in the neuroinflammatory response cannot
be studied.
Among the several microglial-derived pro-inflammatory cytokines and enzymes
contributing to the dysfunction of DA neurons, the enzyme inducible NOS (iNOS) has been
recently recognized as a major component in DA neuron degeneration. Various mammalian PD
models have established that upon activation by neurotoxic and excitotoxic agents, glial
50

(astrocytes and microglia)-derived inducible NOS (iNOS) and NO production facilitated the
inhibition of the mitochondrial respiratory chain via cytochrome c inhibition, generation of
peroxynitrites capable of causing protein nitration, depletion of anti-oxidant pools, and
modification of cellular reactions by acting as a diaphorase (Moncada and Bolanos, 2006).
We have shown previously that upon ingestion of paraquat (PQ), Drosophila exhibits
mobility defects, loss of DA neurons, and truncation of its lifespan, the key features of PD. In
addition, mutations in genes regulating DA homeostasis, Punch (Pu), Catecholamines up
(Catsup) and pale (ple) caused differential responses to PQ (Chaudhuri et al., 2007). Pu encodes
GTP cyclohydrolase (GTPCH), the rate-limiting enzyme for the synthesis of tetrahydrobiopterin
(BH4), which acts as a cofactor for both DA and NO biosynthesis (Fig.2.1). The Drosophila
genome possesses only one NOS gene (compared to three in mammals), and the enzyme is
known to have a variety of physiological and developmental functions in insects, including
Drosophila.
In this report, we demonstrate with biochemical and immunolocalization data that NOS
synthesis is up-regulated in adult fly brain in response to PQ. To confirm the role of NOS in PQ
toxicity, we co-fed PQ with a well-known anti-inflammatory drug, minocycline, which is
capable of suppressing microglial and NOS activation (Wu et al., 2002; Ryu and McLarnon,
2006) and found that the drug improved survival duration and suppressed the induction of NOS
activity in adult. Moreover, we demonstrate that pharmacological inhibition of NOS activity
similarly increases the life span during paraquat exposure.
In an effort to characterize these NOS positive cells, we also present preliminary
evidence that NOS is not induced in glial cells, but further work is necessary to further
characterize NOS-positive structures. Therefore, we present results demonstrating the capacity of
51

Drosophila to mount a NOS-dependent response that has some similarities to mammalian
neuroinflammation mediated by microglial under neurodegenerative conditions.
52

MATERIALS AND METHODS
Drosophila strains and culture maintenance. Strains utilized in all experiments were
Canton S, a wild type strain, the Df(1)w; y, a white-eyed strain that carries wild type alleles of all
DA-regulating genes, a Pu (GTPCH) mutant strain Df(1)w, y; dp cn Pu Z22 a px sp/SM1, Cy cn 2
sp 2 , a ple (tyrosine hydroxylase) mutant strain, ple 2 /TM3, Sb e, and a Catsup mutant strain,
Catsup 26 /CyO. For all experiments, the mutant strains were mated to the appropriate wild type
strain, and all assays were performed on mutants heterozygous for the wild type genes. All
stocks were maintained at 25º C.
A second chromosome UAS-eGFP transgenic strain was obtained from the Bloomington
Drosophila Stock Center. The following Gal4 transgenes were employed to drive cell-specific
GFP expression: tyrosine hydroxylase (TH)-GAL4 (dopaminergic neuron expression) was a gift
from Jay Hirsh (University of Virginia). Reversed polarity (Repo)-GAL4 (glial expression line)
was obtained from the Bloomington Stock Center.
Feeding experiments. Male flies at 24 hr post-eclosion were fed on filter paper saturated
with 5% sucrose only or 5% sucrose containing the following chemicals separately or in
combinations as described in the Results: paraquat (1 mM, 1.5 mM, 3 mM or 10mM),
minocycline HCl (200 μM or 1mM), NG-nitro-L-arginine methyl ester (L-NAME) (200μM or 2
mM). For minocycline and L-NAME, preliminary dose response tests confirmed that the
concentrations employed with PQ co-feeding were not toxic alone within the time constraints of
each experiment. Concentrations of PQ between wild type and the Punch mutant strain were
varied because the Punch mutant strain is hypersensitive to PQ and the progression of
degeneration is too rapid at 10 mM paraquat to achieve measurable rescue with either L-NAME
53

or minocycline at non-toxic concentrations. The rate of degeneration of the wild type strain on 1
mM is slowed to the point that secondary effects from lack of normal medium become apparent.
Concentrations of minocycline and L-NAME were varied to test responses to different
concentration ratios of PQ to therapeutic drug. All reagents were obtained from Sigma. For
assays of survival, feeding was continued until all flies had died, with daily supplementation with
fresh solutions. For confocal microscopic analysis of adult brains, exposure to the appropriate
chemicals continued for 6 hrs prior to dissection of brains.
Nitric oxide synthase assay. Male and female adult flies at 24-48 hrs post-eclosion, were
fed PQ, minocycline and L-NAME according to the doses indicated in the Results for up to 24
hr. Head extracts were prepared by homogenizing in 0.1M phosphate buffer, pH 7.2, followed
by centrifugation for 10 min at 10,000 g at 4°C. The supernatants were mixed with freshly
prepared Modified Griess reagent (Sigma) in proportion of 1: 1 and incubated for 15 min. Nitrite
levels were measured spectrophotometrically at 595 nm, with concentrations of nitrite calculated
against a silver nitrite-derived standard curve and data was presented as a concentration of nitrite
in micromolars for 50 fly-heads.
Immunocytochemistry and confocal microscopy. Brains from TH-GAL4; UAS-eGFP
adults (untreated or exposed to 1.5 mM or 3 mM paraquat for 6 or 12 hrs) were fixed in 4%
paraformaldehyde for 3.5 h and washed extensively in 1x phosphate buffered saline (PBS) and
then in 0.1% Triton X-100, 0.2% bovine serum albumin, in PBS (PBT). Brains were then
blocked in 5% normal goat serum in PBT overnight at 4°C, followed by overnight incubation
with a 1:500 dilution of rabbit anti-universal NOS antiserum (Catalogue No. N127, Sigma) and
54

mouse anti-GFP antibody (Abcam). After additional washing, the brains were incubated at room
temperature for 2 h in a 1: 5000 dilutions of Cy-3-conjugated goat anti-rabbit IgG and FITC
conjugated rabbit anti-mouse IgG. Confocal studies were performed using a Leica TCS SP2
AOBS confocal microscope (Wetzlar, Germany). Each brain was scanned to include 10-15
sections for optimum visualization of NOS-positive structures. For capturing magnified images
(63X and 126X) about 5-8 Z-sections were used to obtain average image of all sections. These
imaging criteria were utilized for all the images mentioned in the Results section.
Statistical Analysis. All data were analyzed by one way ANOVA with Dunnett’s post
test or two way ANOVA or by two-tailed Student’s t-test, assuming equal variances wherever
applicable, using GraphPad Prism (San Diego, CA). Details of the analyses are described in the
figure legends.
55

Tyrosine hydroxylase
Tyrosine
L-Dopa Dopamine
GTP cyclohydrolase
GTP H2NPPP PTP BH4
Arginine
Nitric oxide synthase
Citrulline +
Figure 2.1: Dopamine, tetrahydrobiopterin, and nitric oxide biosynthesis pathways. BH4
functions as an essential cofactor for the generation of DA from tyrosine, and NO from citrulline
in separate pathways. Tyrosine hydroxylase converts tyrosine into L-Dopa which is converted
into dopamine. GTP cyclohydrolase acts as a rate limiting enzyme for synthesis of BH4 which is
formed from guanosine triphosphate (GTP) through the formation of two intermediates,
dihydroneopterin triphosphate (H2NPPP) and 6- pyruvoyl tetrahydropterin (PTP). Nitric oxide
synthase converts citrulline to arginine with generation of nitric oxide.
56
Nitric oxide

RESULTS
A NOS inhibitor, L-NAME, improves survival duration of adults exposed to paraquat
Recently, nitric oxide was proposed to be involved in PQ-induced neurotoxicity in
mammals (Djukic et al., 2007). We therefore asked if the PD-like effects from PQ exposure in
our Drosophila model are also mediated via nitric oxide. To test this, we co-fed wild type flies 2
mM L-NAME, a non-selective NOS inhibitor and 10 mM PQ continuously until all flies were
dead. We found that addition of L-NAME results in improvement of survival duration by
approximately 1.5 days when compared to the survival duration of PQ-fed wild type flies,
suggesting that truncation of life span with PQ exposure is at least partially due to NO-induced
damage and prevention of PQ-mediated NOS induction could be neuroprotective (Fig. 2.2).
57

Survival (days)
6
5
4
3
2
1
0
PQ
Figure 2.2: L-NAME, an inhibitor of NOS, improves survival duration of adults exposed to
paraquat. 48 hr old post eclosion flies were fed 10 mM PQ alone or 10 mM PQ with 2 mM L-
NAME continuously until death and the average survival duration was calculated. Co-feeding of
PQ and L-NAME extends the average survival duration by about 1.5 days, compared to flies fed
PQ alone. ** =P

Minocycline prevents PQ-induced truncation of life span in wild type flies
It is also reported recently that minocycline enhances survival of paraquat (PQ)-fed
Drosophila (Bonilla et al., 2006). Because minocycline toxicity has been reported in some
mammalian disease models, we first tested increasing concentrations of minocycline on non-PQ
treated flies. We found that ingestion of concentrations of 5 mM and above affected viability,
while lower concentrations caused no observable deleterious effects (Fig. 2.3 A). Therefore, all
subsequent experiments utilized minocycline concentrations of 1 mM or less. We asked whether
minocycline is able to rescue PQ- induced toxicity in flies. To test this, we determined the
average survival duration of wild type adult flies continuously fed PQ alone (10 mM), or 10 mM
PQ with 1 mM minocycline. We found that flies survived about 4 days when exposed to PQ
alone, but addition of 1 mM minocycline with the PQ extended the survival duration by about 2
days (Fig. 2.3 B). A similar response was detected by pre-feeding flies 1 mM minocycline for 5
days and then feeding only 10 mM paraquat until death (Fig. 2.3 B). Therefore, as in the
mammalian PD model studies, minocycline ameliorates PQ-induced truncation of life span in
our Drosophila model.
59

A B
Mortality (%)
100
90
80
70
60
50
40
30
20
10
0
0 24 48 72 96 120 144 168 192 216 240
Duration (hr)
100uM
250uM
500uM
1mM
5mM
10mM
25mM
50mM
Figure 2.3: Effect of ingestion of minocycline, with and without PQ, on survival duration of wild
type flies. (A) Effect of increasing concentrations of minocycline (100 μM-50 mM) on survival
of adult male flies. Data were collected every 24 hrs for each group until 100 % mortality was
noted with 50mM minocycline. (B) Wild type flies at 48 hrs post-eclosion were fed 10 mM PQ
alone, after 5 days of continuous ingestion of minocycline (1 mM) and along with minocycline
(1 mM). The co-feeding group was compared to the PQ alone group and the pre-feeding group
was compared with the same aged group pre-fed 5% sucrose and then PQ (10 mM). Both cofeeding
and post-feeding regimens improved the survival duration compared to PQ alone. **=
P

Minocycline reduces the PQ-induced increase in NOS activity in wild type adult flies
It is proposed that minocycline in some PD models, to be neuroprotective, prevents up-
regulation of inducible nitric oxide synthase (iNOS), an important enzyme mediating
inflammatory response in an MPTP-induced mammalian model for Parkinson’s disease (Du et
al., 2001; Wu et al., 2002). We hypothesized that minocycline could reduce the PQ-induced
toxicity by preventing the induction of NOS. We sought to test this by quantifying the level of
nitrites, the product of NO breakdown, after exposure to PQ alone or with minocycline, using
modified Griess reagent (Green et al., 1982). We found that NOS activity was induced to about
4.5 to 5 mM within the first 24 hrs of exposure to PQ in both males and females against 3 mM in
control flies. However, minocycline, as in mammalian models, inhibits production of NO in flies
that were co-fed PQ and minocycline (Fig. 2.4 A, B). These data confirm similarities in the
mechanism of action between L-NAME and minocycline in Drosophila and mammals, and they
provide evidence of the anti-inflammatory property of minocycline via suppression of NOS in
flies.
61

A
B
Nitrite (M)
Nitrite (M)
5% sucrose
6
5
4
3
2
1
0
7
6
5
4
3
2
1
0
5% sucrose
*
*
PQ
##
PQ+Min
5% sucrose
PQ
Male Female
PQ
##
PQ+Min
5% sucrose
*
PQ
Male Female
*
NS
PQ+Min
##
PQ+Min
Figure 2.4: PQ induces, and minocycline suppresses, NOS activity in adult flies. (A) After 24 hrs
of PQ exposure, the specific activity of NOS is five-fold higher in both males and females.
Minocycline, co-fed with PQ, suppresses NOS induction in males but not in females. (B)
Induction of NOS and suppression by minocycline was noted in both males and females after 48
hrs of ingestion of PQ and minocycline. All NOS activity values are averages of three
independent assays per condition. * = P < 0.05 and ** = P< 0.005 and represent the significant
difference between control and PQ fed groups; ##= P< 0.005 and represents the significant
difference between PQ, and minocycline and PQ co-fed group. Error bars represent standard
error of means and n= 250-300 fly heads for all groups.
62

Minocycline increases the life span of ple and Catsup mutants but cannot rescue Pu mutants
We previously reported the differential PQ sensitivity of Drosophila strains heterozygous
for mutations in DA-regulating genes. These included mutations in the rate-limiting genes for
BH4 and DA synthesis, Punch (Pu; GTPCH) (Mackay and O'Donnell, 1983) and pale (ple; TH)
(Neckameyer and White, 1993), respectively. These mutations result in decreased DA pools in
adult heads, even in the heterozygous state in which a copy of a wild type allele of each gene is
also present. Further, these strains are highly sensitive to PQ (Chaudhuri et al., 2007).
Conversely, heterozygous strains carrying one mutant and one wild type allele of
Catecholamines up (Catsup), a negative regulator of TH and GTPCH (Stathakis et al.,1999),
have elevated BH4 and DA pools and a strong resistance to PQ (Chaudhuri et al., 2007). We
asked whether these mutants showed altered responses to PQ in the presence of minocycline.
Wild type adult males survive approximately 4.5 days on 10 mM PQ, while Catsup
heterozygotes survive 24 hrs longer, on average (Fig.2.5 A). In contrast, the low DA Pu and ple
heterozygotes survive only 2 days (Fig. 2.5 A). Survival of Catsup and ple mutants, as well as
wild type, was extended by minocycline, by approximately the same increments. Therefore, we
detected no DA-specific interactions with minocycline in these mutants. Strikingly, however,
minocycline was unable to improve the survival of Pu mutants under these conditions. One
possibility for this result could be that oxidative damage in Pu mutants progresses too rapidly for
minocycline to impart its protective effect. We therefore, reduced the dose of PQ fed to the Pu
mutant animals to 1 mM, which, though eventually causing similar neurological symptoms,
extends life span and the time period before onset of movement disorders, consistent with a
reduction in oxidative stress compared to 10 mM paraquat. We then tested 1 mM and 200 μM
minocycline to determine whether a slower accumulation of oxidative damage with the lower PQ
63

dose would allow some amelioration of symptoms by minocycline. We found, however, that co-
feeding at these concentrations also failed to rescue the mutant flies (Fig. 2.5 B). We further
detected elevated NO production with PQ treatment in wild type and Pu mutants which was
alleviated by minocycline in wild type flies but not in Pu mutants (Fig. 2.5 C, D). Since Pu
mutants have limited BH4 production (Krishnakumar et al., 2000) and limiting BH4 is known to
cause catalytic uncoupling of NOS, producing elevated peroxides and peroxynitrites in in vitro
conditions, we hypothesized that the failure of minocycline protection in Pu mutants was linked
to BH4 deficits leading to more catastrophic oxidative damage (Presta et al., 1998; Werner et al.,
2003; Foxton et al., 2007). If this hypothesis is correct, then inhibition of NOS activity should
limit the production of ROS and RNS, and thereby, improve survival of the Pu mutant flies. We
therefore, co-fed PQ and L-NAME to Pu mutants and found extension of survival of the Pu
mutants by 3 days (Fig. 2.5 E). In addition, we observed that co-feeding of PQ and L-NAME for
24 hr resulted in a decrease in NO production, in contrast to the absence of NOS suppression
with minocycline (Fig. 2.5 F). These results suggest that the failure of minocycline to rescue Pu
mutants was due to its inability to control excessive NO production when BH4 production is
compromised (Fig 2.5 E, F). This interpretation is supported by the observation that untreated
heterozygous Pu mutants possessed low nitrite levels relative to wild type adults (Figure 2.5 F)
and stimulation of NOS activity by PQ, which would exacerbate the uncoupling effects of
limiting BH4 (Fig. 2.5 D). Therefore, these results show that our in vivo model could be used to
identify the modulatory effect of genes on the protective properties of minocycline or
minocycline-like therapeutic agents in simple and efficient manner..
64

Figure 2.5: Minocycline shows differential effects on paraquat-induced damage in dopamine
regulatory mutants. (A) Effect of 1 mM minocycline co-fed with 10 mM PQ on DA regulatory
mutants, Catsup 26 /+, Pu Z22 /+ and ple 2 /+. Mutant adult males were continuously exposed to
either 10 mM PQ only or 10 mM PQ with 1 mM minocycline. Catsup and ple mutant flies
showed significant extension of life span, incrementally similar to the wild type control strain,
while Pu mutants did not. (B) Neither 200 μM nor 1 mM minocycline improved the survival of
Pu mutants exposed to 1 mM PQ. (C) After 24 hr of PQ, or PQ with minocycline exposure,
suppression of NOS was detected in wild type heads. (D) The NO levels of non-PQ treated Pu
mutants, assayed at 48 hr post-eclosion, are significantly lower than NO levels in non-PQ treated
wild type heads. Minocycline, which prevents PQ-induced elevation of NO in wild type heads
(see C), could not modify NO levels induced by PQ in Pu. (E) The survival of Pu mutants was
improved by co-feeding of L-NAME with PQ when compared with survival of Pu mutants on
PQ alone. (F) Addition of L-NAME to PQ reduced the NO production in Pu mutants. In contrast,
200 μM L-NAME prevented PQ-induced decrease in survival duration. NS = not significant. **
= P < 0.005 and represent significant differences between PQ and PQ and minocycline in Fig
2.7A. *, ** and *** =P < 0.05, P=< 0.005 and P=

A
Survival (days)
8
7
6
5
4
3
2
1
0
**
PQ (1mM)
NS
**
**
Wild type-PQ
Wild-type-PQ+Min
Pu/+ -PQ
Pu/+ -PQ+Min
Catsup/+ -PQ
Catsup/+ -PQ+Min
Pale/+ -PQ
Pale/+ -PQ+Min
C D
E
Survival (days)
10
8
6
4
2
0
***
PQ+L-NAME (1mM+200 uM)
66
F
B
Survival (days)
Nitrite (M)
10
8
6
4
2
0
5
4
3
2
1
0
PQ (1mM)
5% sucrose (wild type)
NS
PQ+Min (1 mM+200 uM)
*
5% sucrose (Pu/+)
*
PQ+Min (1 mM+1 mM)
*
PQ (Pu/+)
PQ+Min (Pu/+)

PQ exposure causes activation of NOS as evidenced by expression of NOS positive structures in
adult fly brain
Since exposure to PQ with L-NAME and minocycline delayed PQ-induced truncation of
life span in flies, we hypothesized that the activation of NOS was contributing to PQ-induced
damage to the CNS. To test this, we sought to detect the expression of NOS in the adult brain
using universal anti-NOS antibody. This antibody was raised against a conserved amino-acid
sequences corresponding to amino acid residues 1113-1122 of murine iNOS and nNOS. A
polyclonal antibody raised against this epitope from a different commercial source has been
previously used to detect the expression of NOS during Drosophila visual system development
and in malphigian tubules (Davies, 2000; Gibbs, 2001). Similarly, a protein of 150 kDa, the
predicted molecular mass of the Drosophila NOS polypeptide was detected by immunoblotting
using another polyclonal antibody independently raised against this epitope (Broderick et al.,
2003). We detected little NOS-positive signal in normal, untreated brain (Fig. 2.6 A) with the
exception of expression restricted to the junction of the central brain complex and optic lobes
(data not shown). In contrast, upon exposure to PQ, a significant increase in signal was noted in
the central complex (Fig. 2.6 B). Further, these NOS-positive structures exhibited an array-like
pattern suggesting that they might be aligning along processes of neuronal or non-neuronal
structures. The size for these NOS-positive structures ranged from 0.5 to 2 μm.
67

5% sucrose PQ
A B
C D
Figure 2.6: Exposure to PQ causes induction of NOS in adult brain. (A) Little NOS expression
was detected in normal, 5% sucrose brains. (B) Upon exposure to 3 mM PQ for 12 hrs, a
dramatic increase in the expression of NOS was detected by anti-NOS antibody. Scale bars in A,
B=10 μm.
68

PQ increases the expression of NOS-positive structures near dopaminergic neurons
Having found that PQ increases expression of NOS in adult Drosophila brain, we sought
out to determine any association between NOS-positive cells and DA neurons in response to PQ
exposure. Using the transgenic reporter strain, TH-GAL4; UAS-eGFP, we found that ingestion of
PQ induces NOS signal in structures near the DA neuron and or processes (Fig. 2.7 A).
Moreover, we detected clusters of NOS signal around the dopaminergic neurons in the adult
brains exposed to PQ (Fig. 2.7 B). Since, we found that NOS suppression by L-NAME also
improves the survival of PQ-exposed flies; we propose that NOS mediates the loss of DA
neurons in adult Drosophila brain.
69

A B
Figure 2.7: Ingestion of PQ (1.5 mM) induces NOS-expression near and around dopaminergic
neurons in TH-GAL4; UAS-eGFP adult brain. (A) Arrows indicate NOS positive structures (red)
near the DA neurons and or processes (green) (B) The arrowhead indicates the cell body of a
dopaminergic neuron (green) surrounded by NOS positive structures (red) (arrows). Scale bar for
A= 15.87 μm and for B= 5 μm.
70

Paraquat induces expression of NOS in non-glial cells in adult brain
Induction of NOS activity in response to bacterial invasion in Drosophila has been
reported previously (Nappi et al., 2000; Foley and O’Farrell, 2003). However, there have been
no published reports of an association of NO with neurodegeneration in Drosophila models of
neurodegenerative disease. Our data suggest that generation of NO by PQ is deleterious to
Drosophila as observed in mammalian PD models. In mammals, iNOS, induced in microglia and
glia is associated with their activation and proliferation in response to inflammation (Block et al.,
2007). In Drosophila, glial cells perform various functions including phagocytosis of harmful
non-self invaders, but the presence of microglial-like cells in the CNS of Drosophila has not yet
been reported (Freeman and Doherty, 2006). Therefore, we hypothesized that glial cells could be
the source of NOS induced in response to PQ in flies. We used Repo-GAL4; UAS-eGFP flies,
where all differentiated glial cells express reversed polarity (repo), and are therefore, identified
by repo-driven expression of GFP. We found that NOS, detected by a universal anti-NOS
antibody, was not expressed in glia (Fig. 2.8 A). These results indicate that NOS-expressing cells
are a distinct population from glial cells in adult Drosophila brain.
71

A B
Figure 2.8: Paraquat-induced NOS is expressed in non-glial cells in adult fly brain. (A) NOS
expression was induced in Drosophila brain by treatment of young Repo-Gal4; UAS-GFP flies
with 1.5 mM PQ. Glial cells (green, thick arrows) are distinct from NOS-expressing cells (red,
thin arrows). Note that NOS-positive structures were seen of varied sizes, the one indicated with
notched arrow measures about 5 μm in diameter. Scale bar in A= 31.75 μm.
72
C
D

DISCUSSION
Exposure to paraquat causes induction of NOS in the adult Drosophila brain
In this report, we demonstrate for the first time, induction of NOS in adult Drosophila
brain in response to PQ. Although NOS activity has been previously reported, the response was
induced via bacterial and wasp infection in larvae (Nappi et al., 2001; Foley and O'Farrell, 2003).
PQ is one of the most potent redox-cyclers capable of generating toxicity in lung and
brain tissue in mammals (Ilett et al., 1974; Prasad et al., 2007). PQ, in fact, induces the
degeneration of DA neurons in invertebrate and vertebrate models, and such models are being
used to identify neurodegenerative mechanisms for PD pathogenesis as well as to identify
genetic susceptibility factors for PD (McCormack et al., 2002; Chaudhuri et al., 2007; Kuter et
al., 2007). In the presence of molecules with diaphorase activity, PQ transfers single electrons
and generates superoxide radicals upon reacting with oxygen (Bus et al., 1974). NOS, which
catalyzes the formation of NO from L-arginine (Palmer et al., 1987), acts as a diaphorase
molecule that also reacts with generated superoxide radicals to form highly toxic and cell
membrane permeable peroxynitrite (Nemery and van Klaveren, 1995). N G -nitro-L-arginine
methyl ester (L-NAME), but not N G -monomethyl-L-arginine (L-NMMA), has been shown to
block the PQ-induced increase in NADPH oxidation by inhibiting the formation of superoxide
radicals (Pou et al., 1992). We found that addition of L-NAME (2 mM) to PQ (10 mM)
improved the survival duration relative to age-matched PQ (10 mM) alone fed flies (Fig. 2.2).
We also found that PQ (10 mM) ingestion for 24 hrs stimulated NOS activity in adult fly
heads, as reflected in the increase of nitrite levels determined with modified Griess reagent (Fig.
2.4 A, B; 2.5 C). Moreover, our results demonstrate that co-feeding 2 mM L-NAME decreased
NOS activity levels in wild type flies (Fig.2.5 F). Our data parallel reports that L-NAME is
73

protective against PQ toxicity in rats that had been pre-fed L-NAME for 30 min prior to PQ
injections (Djukic et al., 2008).
Drosophila possesses only one NOS gene compared to three NOS genes present in
mammals and shows highest similarity with the NOS1 isoform of mammalian NOS (Regulski
and Tully, 1995). However, the single isoform in Drosophila appears to have functional
properties that are distributed amongst the three mammalian isoforms. We employed an antibody
that was raised against a conserved epitope in the C-terminus of the NOS protein to detect the
expression of NOS in brains of non-PQ treated and PQ treated adult flies (Fig. 2.6 A, B). NOS
positive signal was found to be localized primarily at the junction of the central brain complex
and the optic lobes in the absence of PQ exposure (data not shown). However, upon exposure to
PQ, expression of NOS significantly increases and the location of the signal changes to regions
near or surrounding DA neurons in the central brain as demonstrated in a reporter strain
expressing GFP in dopaminergic neurons (Fig. 2.7 A, B). The size of the NOS-positive structures
ranged from 0.5 to 2 μm. In an effort to determine the source of NOS induced in response to PQ,
we exposure Repo-GAL4; UAS-eGFP adult flies to 1.5 mM of PQ. We found that glial cells
identified by GFP expression driven by the glia-specific driver reverse polarity (repo)-GAL 4
transgenic line, were devoid of NOS expression, demonstrating that glial cells are not of the
source of NOS in PQ treated brain (Fig. 2.8 A). These findings suggest that these small NOS-
positive structures could be cellular inclusions or extracellular particles, bacteria.
It is to be noted that the increased expression of NOS-positive structures and their
variability in expression needs further validation by employing other staining techniques such as
the NADPH diaphorase colorimetric reaction. Furthermore, the increased expression of the
NOS-positive structures requires quantification to confirm the expression pattern. Nevertheless,
74

our data have pioneered the demonstration of a NOS-dependent response to PQ in adult
Drosophila brain, that appears to be similar to responses observed in mammalian PD models and
further studies will be performed to determine the source of NOS after confirming the results
obtained so far.
Minocycline imparts anti-inflammatory functions against PQ
In order to confirm the neuroinflammatory response against PQ, we took a
pharmacological approach by using a potent anti-inflammatory agent reported to delay the
inflammation-mediated pathological process in neurodegenerative diseases including PD (Kim
and Suh, 2008). We found that co-feeding and 5 days of pre-feeding 1 mM minocycline with 10
mM PQ are effective in increasing the survival duration of flies by about 2-3 days compared to
an average survival of 4 days on PQ alone after finding determining that 1 mM minocycline and
below are non-toxic in flies (Fig. 2.3 A, B). Since minocycline has been reported to inhibit the
activation of NOS in mammalian neurodegenerative models (Chen et al., 2000; Du et al., 2001),
we tested the levels of NOS induction by PQ with and without minocycline treatment. We found
that co-feeding minocycline with PQ decreases NOS activity in both males and females although
a significant effect was evident in females only after 48 hrs, while response in males was
observed after 24 hrs of the feeding regimen, implying possible gender-specificity in the action
of minocycline (Fig. 2.4 A, B). Hence, our biochemical data suggest that, as in vertebrate PD
models, minocycline imparts NOS inhibitory functions in this invertebrate model for PD, one of
the important inflammatory marker in PD.
75

Minocycline causes differential responses to DA regulatory gene mutations in PQ exposure
After establishing the protective role of minocycline during PQ exposure in wild type
Drosophila, we next tested its effects in strains mutant for DA regulatory genes. We tested three
key genes that we previously found to modulate DA synthesis and affect PQ sensitivity, pale,
Punch and Catsup, the TH, GTPCH and Catsup encoding genes, respectively (Neckameyer and
White, 1993; Mackay and O’Donnell, 1983; Stathakis et al., 1999; Chaudhuri et al., 2007). Since
these mutations are homozygous lethal, we tested heterozygous mutants of ple, Pu and Catsup to
determine whether their effects on DA homeostasis affected the efficacy of minocycline. We
found that co-feeding 1 mM minocycline with PQ extended the survival duration of ple and
Catsup mutants (low and high DA levels, respectively) to the same extent as it did wild type
Drosophila (Fig. 2.5 A). We conclude from this result that perturbations of DA pools per se do
not affect the ameliorative properties of minocycline. Interestingly, Punch mutants were not
responsive to minocycline. We further decreased the concentration of PQ to equalize the
proportion of PQ and minocycline, hypothesizing that the severity of Pu mutant phenotypes
required additional minocycline for observable rescue. However, we were still unable to detect
any protective effect of minocycline on Pu mutants. This result contrasts with the ability of L-
NAME to improve the survival duration of Pu mutants (Fig 2.5 E). Pu mutants are deficient in
BH4 synthesis and this cofactor is required by NOS as well as by TH. Because it has been
reported that limiting BH4 can result in the uncoupling of electron transfer in NOS under in vitro
conditions (Presta et al., 1998; Werner et al., 2003), we asked whether limiting cofactor
produced an elevated oxidative/nitrosative background that enhanced PQ sensitivity including
high nitrite concentrations. However, in the non-PQ treated Pu mutants, Greiss reagent assays
detected lower than normal nitrite levels consistent with lower than normal NOS activity
76

expected in Pu mutants (Fig. 2.5 D). Therefore, the 50% decrease in BH4 pools observed in
heterozygous Pu mutants (Krishnakumar et al., 2000) is not sufficient to produce elevated
peroxynitrites in the absence of PQ. We found, however, that, as in wild type flies, NOS activity
was further enhanced in the presence of PQ in Pu mutants (Fig. 2.5 D). This result suggests that
the BH4 deficit, which we previously found to be exacerbated by PQ treatment (Chaudhuri et al.,
2007) might lead to a greater imbalance between the cofactor and NOS and, in consequence,
more severe uncoupling of Drosophila NOS, leading to excessive production of highly reactive
superoxide radicals. This scenario is supported by our observation that L-NAME, but not
minocycline, was able to reduce NOS levels and extend the survival duration of Pu mutants (Fig.
2.5 E, F). A similar response is reported in BH4 limited samples of vascular smooth muscles
resulting in the elevation of blood pressure (Wang et al., 2008).
Therefore, this report presents our preliminary results of NOS-dependent response in
adult Drosophila PD model that was further supported by pharmacological study.
77

CHAPTER 3
IDENTIFICATION OF A NEUROPROTECTIVE ROLE FOR MINOCYCLINE IN A
DROSOPHILA MODEL OF PARKINSON’S DISEASE AND FUNCTIONAL ANALYSIS OF
SIGNALING PATHWAYS MEDIATING PARAQUAT TOXICITY IN A DROSOPHILA PD
MODEL
This work is a manuscript in preparation for submission to Journal of Neuroscience. Co-authors
for this work were Arati Inamdar, Anathbandhu Chaudhuri and Janis O’ Donnell.
Dr. Anathbandhu Chaudhuri contributed in collecting data presented in figures 3.1, 3.2, 3.3, 3.4
and 3.5. Arati Inamdar collected the remaining data.
78

INTRODUCTION
Parkinson disease (PD) is a debilitating neurodegenerative disease insulting initially
dopaminergic neurons in the substantia nigra (SN). Although the exact causes of PD are
unknown, pathogenic mechanisms include protein aggregation, defective mitochondrial function
and damage to other cellular membranes, associated with increased oxidative stress within the
neurons themselves. Pesticides such as rotenone and paraquat (PQ) have been implicated in the
oxidative pathogenesis of idiopathic PD (Betarbet et al., 2000; McCormack et al., 2002;
Uversky, 2004), while mutations in various genes are associated with familial PD (Mizuno et al.,
2008).
Minocycline, a second generation tetracycline drug known to be clinically safe
(MacDonald et al., 1973), has shown promising ameliorative effects in animal models for
chronic neurodegenerative diseases, including neurotoxin-induced PD models (Wu et al., 2002;
Zhu et al., 2002b; Lin et al., 2003; Wang et al., 2003a). Minocycline appears to have anti-
inflammatory property that mediates neuroprotection in PD animal models (Wu et al., 2002).
Further, antioxidant property of minocycline has been reported (Kraus et al., 2005).
It was reported recently that minocycline enhances survival of paraquat (PQ)-fed
Drosophila (Bonilla et al., 2006), but the precise mechanism of minocycline action is not well-
understood in flies. We have developed a Drosophila model based upon ingestion of PQ, which
recapitulates characteristic symptoms of PD with modulation of dopamine (DA) pools,
degeneration of dopaminergic neurons, and accompanying neurological symptoms including
resting tremors and postural instability (Chaudhuri et al., 2007). Moreover, we demonstrated that
mutations that directly alter the regulation of DA production dramatically alter susceptibility to
79

PQ, and more recently, we discovered that nitric oxide synthase (NOS) expression is induced in
response to PQ (Inamdar et al., 2009, under revision).
The mitogen activated protein kinases (MAPKs) consist of three subfamilies, namely
extracellular signal regulated kinase (ERK), c-Jun N-terminal kinases (SAPK/JNK), and p38
kinases which are known to be activated by a wide range of stimuli including hormones and
growth factors, inflammatory cytokines, and diverse environmental stresses. These signal
transduction pathways mediate a variety of downstream effects such as the regulation of gene
transcription, the cell cycle, cellular differentiation and cell death (Gotoh and Nishida, 1995;
Chang and Karin, 2001). These MAPK-mediated downstream effects depend on the stimulus and
the cell type in which they are activated. Moreover, experimental conditions employed to
investigate the roles of these signaling pathways, in particular developmental processes, or in
pathogenic responses are generally manipulated in mammalian cell culture systems.
Nevertheless, the dysregulation of MAPK has been reported in many neurodegenerative
diseases including PD (Burke et al., 2007). In these mammalian models for neuronal injury and
neurodegenerative diseases, pharmacological approaches have provided evidence that p38 and
JNK are mainly implicated in the neuronal death processes, while ERK may promote cellular
recovery/ survival from neuronal death implicated in these conditions (Xia et al., 1995; Harper
and LoGrasso, 2001). Immortalized rat brain neuroblasts (E18 cells) exposed to low doses of PQ,
up regulate phosphorylated forms of ERK, PKB and JNK, but exhibit no measurable increase in
phospho-p38. Further, when these kinases were blocked pharmacologically, only inhibition of
JNK prevented PQ-induced cell death (Niso-Santano et al., 2006). Peng et al. (2004) reported
that activated JNK induced caspase-3 dependent apoptosis in Primary Mesencephalic
dopaminergic neuron cultures, in response to PQ but detected no role for ERK in this system.
80

Although these studies present strong evidence for the key roles of activated kinases in
neuronal survival or death after exposure to PQ, there are discrepancies due to variations in the
doses, cell types and cultural conditions employed. Further, in vitro cultural conditions lack
appropriate cell-cell interactions from neighboring cells which could modulate the response to
external stimuli. Moreover, many of the commercially available pharmacological inhibitors of
kinases employed in these experiments are not well-defined with respect to their exact modes of
action and may give rise to non-specific effects due to blockage of unknown target molecules
(Sekiguchi et al., 1999; Learish et al., 2000; Waetzig and Herdegen, 2005).
All these limitations of in vitro mammalian PD models necessitate the development of a
simple in vivo model to further define the exact roles of these kinases in PD pathogenesis.
Drosophila signaling pathways are highly conserved with those of mammals (Blenis, 1993; Tan
and Kim, 1999). Among the many signal transduction pathways investigated, receptor tyrosine
kinases are the best characterized. Unlike vertebrates, the Drosophila genome contains only one
gene encoding each of the MAPK subfamilies and PKB/Akt1 (FlyBase), eliminating the
possibility of functional redundancy due to the presence of multiple genes and isoforms for these
kinases in vertebrates. Further, modulation of the inflammatory response to PQ exposure in
mammalian models by these signaling pathways has not been reported.
PQ toxicity is associated with nitric oxide (NO) production and is alleviated when NO
synthesis is reduced by a competitive inhibitor of NOS, L-NAME (Djukic et al., 2007; Inamdar
et al., 2009, under revision). Further, we have shown that minocycline suppresses the PQ-
induced activation of NOS (Inamdar et al., 2009, under revision).
In this report, we demonstrate that minocycline prolongs survival of PQ-exposed adult
Drosophila, diminishes PQ-induced mobility defects, blocks associated changes in the DA
81

pathway, diminishes levels of reactive oxygen species (ROS), and prolongs DA neuron survival.
We further sought to identify signaling pathways that mediate the PQ-induced toxicity in this in
vivo Drosophila model, as well as the signaling pathways modifying the protective response of
anti-oxidant and anti-inflammatory drug, minocycline against PQ. Using loss of function mutant
and over-expression transgenic lines, we found that JNK and Akt1 per se play an important role
in protecting DA neurons against PQ toxicity while caspase (dronc, Drosophila homolog of
caspase) mediates PQ toxicity in Drosophila. We further report the identification of signaling
pathways capable of modifying the neuroinflammatory response against PQ using
pharmacological and genetic approaches.
82

MATERIALS AND METHODS
Drosophila strains and culture maintenance. Strains utilized in all experiments were
Canton S, a wild type strain, Df(1)w, y, a white-eyed strain that carries otherwise wild type
genes, and mutant alleles of the following signal transduction genes: rolled (rl 1 ) (Morgan et al.,
1925), a weak loss-of-function allele of the gene encoding ERK, basket, bsk 1 /Cyo, a loss-of-
function allele of the gene encoding JNK (Nüsslein-Volhard et al., 1984), PKB/Akt1, ry 506
P{PZ}Akt 104226 /TM3, ry RK Sb 1 Ser 1 , carrying a loss-of-function mutation of Akt1 (Perrimon et al.,
1996), a loss-of-function allele for the gene encoding Diablo/Smac, reaper, y 1 w 67c23 ; P{SUPor-
P}KG07184 ry 506 (White et al., 1994), and a null mutant allele of Nc or Dronc, encoding a
caspase, y 1 w*; Nc 51 /TM3, Sb 1 (Chew et al., 2004). Transgenic strains employed for wild type
expression of kinases were: JNK (bsk), y 1 w 1118 ; P{UAS-bsk.A-Y}1 (Adachi-Yamada, personal
communication to Bloomington Stock Center) and Akt1, y 1 w 1118 ; P{UAS-Akt1}/Cyo (from Dr.
Tien Hsu, Medical University of South Carolina, Charleston, SC). All mutants and transgenic
strains were mated to the appropriate wild type strain, and all assays were performed on mutants
heterozygous for the wild type genes. All stocks were maintained at 25º C.
The transgenic strain UAS-2X eGFP (Chromosome II) and the above mutant strains for
signal transduction pathways were obtained from the Bloomington, IN Drosophila stock center
and a TH-Gal4 strain (Friggi-Grelin et al., 2003) was obtained from Jay Hirsh (University of
Virginia).
Feeding experiments. Separated male and female flies, 48-96 hr post-eclosion, were fed
on filter paper saturated with one of the following solutions: 5% sucrose only or 5% sucrose
with 1 or 10mM paraquat only, minocycline HCl only at varying concentrations, 10mM paraquat
83

with 1mM minocycline HCl, 10mM doxycycline only or with 10mM paraquat. Feedings were
continued for up to 48 hr. All chemicals were obtained from Sigma (St. Louis, MO).
Locomotion assay. The mobility of adult male and female flies from each treatment
group was assessed using a negative geotaxis climbing assay. A single fly was placed in an
empty plastic vial, tapped to the bottom and the time required to climb 5 cm was recorded three
times sequentially with 10 min rest periods between each measurement. Each replication value
recorded is an average of the three trials; each assay was conducted on 10 flies per test group.
HPLC analysis. Monoamine and pteridine levels were determined using an ESA
CoulArray Model 5600A high performance liquid chromatography system. Fifty adult heads
were extracted in 60 μl 0.1M perchloric acid, followed by centrifugation. Ten μl of each extract
was injected per extract. Analyses were conducted on 3 extracted replicas of each test set.
Amines and pteridines were separated on a Phenomenex Synergi 4μm Hydro-RP column (4.5 X
150 mm) according to the method of McClung and Hirsh (1999). Separations were performed
with isocratic flow at 1ml/min.
Amines were detected with the ESA CoulArray electrochemical analytical call, Model
5011 (channel 1 at -50 mV, channel 2 at 300 mV). Pteridines were detected with a Linear Model
LC305 fluorescence detector (excitation wavelength 360 nm and emission wavelength 456 nm).
Analysis was performed using ESA CoulArray software.
GTPCH assay. GTPCH activity was assayed by modification of a method described by
Viveros et al. (1981). Thirty heads of 3-5 days old adult males were collected and homogenized
84

in 100μl of lysis buffer (50mM Tris, 2.5 mM EDTA, pH 8.0). Extracts were centrifuged at
10,000 rpm for 10 min and the protein concentrations of supernatants were determined using the
BioRad Protein Assay Reagent. Seven µl of 2mM GTP and extract corresponding to 45 μg of
protein were brought to a volume of 70 μl. The mixture was incubated for 1 hr at 37° C to
convert GTP to dihydroneopterin triphosphate (dNP3), followed by the addition of 30μl of 1%
iodine and 2% potassium iodide in 1M HCl. The mixture was then incubated in the dark at room
temperature for 1 hr, which terminates the reaction and oxidizes dNP3, converting it to a
florescent form. The reaction was decolorized in 3% absorbic acid and centrifuged at 14,000 rpm
for 5 min. The supernatant were dephosphorylated with 2 units of alkaline phosphatase (Roche)
in 10X dephosphorylation buffer and incubated at 37°C for 1 hr. Neopterin peaks, which were
detected by fluorescence at excitation wavelength of 353 nm and emission wavelength of 438
nm, were identified and quantified relative to a neopterin (Sigma) standard.
Confocal microscopy. Whole mounts of dissected brains from TH-Gal4; UAS-eGFP
adults fed with sucrose alone, or with paraquat, as described in the Results were examined for
dopaminergic neuron morphology and number, detected by visualizing GFP-expressing neurons.
Each brain was scanned to include 15-18 sections for optimum visualization of DA neurons. The
Z-sections were then utilized to get the average of all sections using a Leica TCS SP2 AOBS
confocal microscope (Wetzlar, Germany).
Catalase assay. Separated males and female flies at 2-4 days post-eclosion were fed 5%
sucrose alone, 10mM paraquat, 1mM minocycline or a combination of 10mM paraquat and 1mM
minocycline in 5% sucrose solution for 24 hr prior to sacrifice. Crude enzyme extracts were
85

prepared by homogenizing 10 heads from each treatment group in 150 μl 0.1M sodium–
potassium phosphate buffer containing 0.1 M Triton X-100 (pH 7.0) following the method of
Bewley et al (1983). After homogenization, all the samples were incubated at 4ºC for 10 min
prior to centrifugation at 12,000 x g for 10 min. Assays were performed at 30 ºC following the
methods of Lubinsky and Bewley (1979). The reaction was initiated by adding 20 μl head tissue
extract to the reaction mixture containing 100 μl (48.6mM) H2O2 in 0.1M sodium-potassium
phosphate buffer (pH 6.8) to a final volume of 2.55 ml. The reaction of head extract with H2O2
was determined at absorbance wavelength 230 nm and calculated using a molar extinction
coefficient for H2O2 of 62.4. One unit of catalase activity was defined as 1μmole of H2O2
decomposed per min. All values represent the average of 6-8 replications from independently
prepared extracts.
Lipid peroxidation assay. Head extracts were prepared from female flies at 2-4 days post-
eclosion fed 5% sucrose alone, 10mM paraquat, 1mM minocycline or a combination of 10mM
paraquat and 1mM minocycline in 5% sucrose solution after 24 hr of feeding were homogenized
with 0.1M phosphate buffer and centrifuged for 10 min at 10,000g at 4°C. Two ml of reagent
TCA-TBA (thiobarbituric acid)-HCl was added to 1ml of head extract and heated for 15 min in a
boiling water bath to allow malondialdehyde, the product of the lipid peroxidase reaction, to
develop a red chromophore, detected spectrophotometrically at 535 nm, as described by Beuge
and Aust (1978).
86

Statistical Analysis. All data were analyzed by one way ANOVA with Dunnett’s post test
or by two-tailed Student’s t-test, assuming equal variances wherever applicable, using GraphPad
Prism (San Diego, CA). Details of the analyses are described in the figure legends.
87

RESULTS
Effect of minocycline on paraquat-induced truncation of life span
Minocycline has been reported to improve survival duration in animal models for
amyotrophic lateral sclerosis, Huntington’s disease, and Parkinson’s disease (Hersch et al., 2003;
Van Den Bosch et al., 2002; Quintero et al., 2005). However, this antibiotic has been reported to
have detrimental effects in some systems (Diguet et al., 2004). Therefore, we first assessed
whether ingestion of minocycline alone, at concentrations ranging from 100μM to 50 mM,
affected viability of adult Drosophila. The results for male flies are shown in Fig. 2.3A, Chapter
2; females gave comparable results (data not shown).
Employing non-toxic levels of minocycline, we then asked whether this antibiotic could
rescue the toxic effects of PQ. We first co-fed minocycline doses from 100 μM to 1 mM with 10
mM PQ and determined the average survival duration (Fig. 3.1 A). We found that co-feeding of
1 mM minocycline with 10 mM PQ extended average survival time an additional 48 hr, from
three to five days (Fig. 3.1.A). Minocycline at concentrations of 500 μM and below were unable
to modify the effects of 10 mM PQ. We then tested the efficacy of minocycline under three
different regimens, co-feeding 10 mM PQ and 1 mM minocycline, pre-feeding minocycline for 2
days prior to PQ exposure, and pre-feeding 10 mM PQ for 2 days prior to exposure to
minocycline alone. We found that neither pre-feeding nor post-treatment of minocycline were
able to modify survival duration of flies ingesting 10 mM PQ (data not shown). However,
extending the minocycline pre-feeding period to 5 days resulted in extension of life span to
almost the same degree as the co-feeding regimen (Fig. 2.3 B, Chapter 2). We also co-fed
another semi-synthetic tetracycline derivative belonging to minocycline group, doxycycline, at 1
mM concentration, to determine if the protective effect is a general property of tetracycline drugs
88

or specific to minocycline. Although minocycline and doxycycline belong to semi-synthetic
tetracycline group, minocycline possesses an extra diethylamino group at C7 and lacks
functional group at C6 (Fig. 3.1 C, D) (Kim et al., 2009). We tested both 10 mM and 5 mM
doses for PQ. However, co-feeding doxycycline with 10 mM and 5 mM was unable to affect the
survival duration of 10 mM (data not shown) and 5 mM PQ fed adults (Fig.3.1B).
Thus, altogether the above data indicates that minocycline at lower doses is not toxic to
flies and able to extend PQ-induced truncation of life span. It also suggests that this effect is not
a general effect of the whole family of tetracycline molecules, since doxycycline could not
provide such protection.
89

A B
Survival (days)
6
5
4
3
2
1
0
PQ 10mM
PQ 10mM+Min 100 uM
NS
**
PQ 10mM+Min 250 uM
PQ 10mM+Min 500 uM
PQ 10mM+Min 1mM
C D
Figure 3.1: Effect of minocycline and doxycycline with 10 mM paraquat on survival of adult
male flies. (A) Effect of different doses of minocycline co-fed with 10mM PQ on the life span of
wild type adult males. Male flies were co-fed 100 µM, 250 µM, 500 µM and 1mM minocycline
with 10mM PQ and their survival duration was monitored. Co-feeding of 1mM minocycline with
10 mM PQ significantly extends the survival duration compared with PQ alone. Addition of 100
µM, 250 μM and 500 µM minocycline with 10mM PQ had no effect on life span. ** represents
the difference between PQ-fed flies and those fed PQ with 1mM minocycline at P< 0.005. (B)
Effect of doxycycline on PQ-treated flies. Addition of doxycycline had no effect on survival
duration suggesting that the action of minocycline is specific. NS= non-significant. Error bars
represent the standard error of the mean. Each data point represents at least 10 replications of 15
flies each. (C,D) Chemical structures of minocycline and doxycycline, respectively.
90
Survival (days)
6 NS
5
4
3
2
1
0
PQ PQ+Doxycycline

Minocycline protects against PQ-induced mobility defects
We employed a climbing assay to assess whether minocycline could provide rescue of
the mobility deficits induced by PQ, similar to the improvement in motor performance after
minocycline treatment reported in ALS and Huntington’s disease models (Kriz et al., 2002; Zhu
et al., 2002b; Hersch et al., 2003) and in a 6-OHDA PD model (Quintero et al., 2003). Within 24
hr of the initiation of 10 mM PQ feeding in the absence of minocycline, tremors and
bradykinesia were apparent. At 48 hr, these flies were unable to climb and appeared frozen at
the bottom of the vial. Within 72-84 hr, 40-50% mortality was observed. In contrast, when PQ
was co-fed with 1 mM minocycline, no movement defects were apparent until after 72 hr,
establishing that minocycline is capable of rescuing the behavioral syndrome associated with PQ
ingestion (Fig.3.2).
91

Time to cross 5 cm distance (sec)
13
12
11
10
9
8
7
6
5
4
3
2
1
0
**
#
24 hrs 48 hrs
Figure 3.2: Minocycline protects against PQ-induced mobility defects. The ability of
minocycline to prevent the PQ-induced mobility defect was determined by negative geotaxis
assay. The time required by 10 flies to cross 5 cm distance at 24 and 48 hr after the initiation of
ingestion of 10mM PQ and 10mM PQ with 1mM minocycline in female flies. The ingestion of
1mM minocycline has no effect on mobility, while PQ ingestion alone adversely affects
mobility. Co-feeding minocycline with PQ results in mobility performance near control levels.
Ten flies were scored per replication and the mean values represent the average of 15
independent replications. The * and # indicate the significance of differences between control
and PQ-fed flies, and between PQ only and co-fed flies, respectively. ***/###=P< 0.001 ** = P
< 0.005 and # = P < 0.05. Error bars represent standard error of the mean. Each data point
represents at least 10 replications of 15 flies each.
92
***
###
Control
PQ
PQ+ Min
Min

Minocycline delays PQ-induced loss of dopaminergic neurons
We recently established that the onset of movement dysfunction upon PQ ingestion
coincides with the loss of specific subsets dopaminergic neurons in the adult brain (Chaudhuri et
al., 2007). In light of the ability of minocycline to ameliorate PQ-induced tremors and mobility
deficits, we next asked whether this effect is mediated through protection of at-risk dopaminergic
neurons. Dopaminergic neurons were detected by the TH promoter-directed expression of GFP
in the transgenic strain, TH-Gal4; UAS-eGFP (Friggi-Grelin et al., 2003). We compared
dopaminergic neuron morphology and numbers in brains dissected every 24 hr after the initiation
of feeding 5% sucrose only, 10 mM PQ alone, 10 mM minocycline alone or PQ in combination
with 1 mM minocycline (Fig.3.3). As we had observed previously (using both the GFP reporter
and immunolocalization of TH to identify dopaminergic neurons), these neurons display
characteristic patterns of neuronal sensitivity. Upon exposure of 10 mM PQ for 24 hr, we
observed that the PAL subgroup in the anterior region and the PPL1, PPM2 and PPM3
subgroups in the posterior brain exhibited statistically significant neuron loss relative to controls.
In the animals that were co-fed PQ and minocycline, there was no neuron loss in the PPM1 or
PPL2 subgroups at 24 hr, and neuron numbers and morphology in other clusters were almost
identical to those in control brains (Fig. 3.4). After 48 hr of PQ feeding, previously affected
clusters continued to deteriorate, while neurodegeneration was noted in the previously unaffected
PPM1 and PPL2 clusters (data not shown). At this time point, flies that ingested minocycline
with PQ exhibited loss of neurons, especially PAL (data not shown). Therefore, we conclude that
the extension of life span observed when flies were fed minocycline along with PQ correlates
with both delay of the onset of movement deficits and protection against PQ-induced
neurodegeneration.
93

A PPM2
B
Control
C PPM2
D
PQ
Figure 3.3: Minocycline confers protection to dopaminergic neurons. (A-D) The effect of 24 hr
exposure of sucrose, 1mM minocycline alone, 10mM paraquat alone, and 10mM paraquat co-fed
with 1mM minocycline on the dopaminergic neurons of TH-GAL4; UAS-eGFP adult brain. The
inset in each image demonstrates the change in the morphology and number of the PPM2
subgroup of neurons. The exposure to minocycline alone (B) does not induce any change in
number and morphology of the dopaminergic neuron when compared to the control image in
Panel A. The addition of minocycline to PQ (D) delays the loss and alteration in the shape and
neuronal process of dopaminergic neuron as opposed to paraquat only (C). Scale bar for A-
D=100 μm.
94
PPM2
PPM2
Min
PQ+Min

No of neurons
16
14
12
10
8
6
4
2
0
* #
*
#
PAL PPM1 PPM2 PPM3 PPL1 PPL2
Subgroups of DA neurons
*
Figure 3.4: Minocycline delays paraquat induced selective loss of dopaminergic neurons. The
average number of neurons per subset was determined 24 hr after the initiation of feeding. All
scoring was done on TH-Gal4:UAS-GFP adults. Each subset of dopaminergic neurons was
scored separately (n=15-25). PAL, PPM2, PPM3 and PPL1 subsets show significant reduction in
the number of neurons in the PQ-treated groups, while the number of corresponding neurons in
the co-fed animals show significantly elevated neuron numbers when compared to the brains of
animals fed PQ only, except in the most sensitive cluster, PPL1. No significant difference in the
neuron counts was noted between control, minocycline only, and co-fed individuals. The
significance of difference in each neuron cluster between the PQ-treated and control groups, and
between the PQ-treated and co-fed groups are indicated as * and #, respectively, where * = P<
0.05 and #= P < 0.05. Error bars represent standard error of the mean. Each data point represents
at least 10 replications of 15 flies each.
95
#
*
Control
PQ
PQ+Min
Min

Minocycline blocks changes in DA pathway components indicative of PQ-induced oxidative
stress
The production of DA is rate-limited by two enzymes; tyrosine hydroxylase (TH), which
converts tyrosine to L-DOPA, and GTP cyclohydrolase (GTPCH), which is rate-limiting for the
production of tetrahydrobiopterin (BH4), a cofactor for and regulator of TH catalysis. We
previously observed that sensitivity to PQ is at least partially defined by the activity of the BH4
and DA biosynthesis pathways (Chaudhuri et al., 2007). Although the precise mechanism
remains unclear, we found that mutants synthesizing abnormally low amounts of BH4 and DA
and wild type flies in which DA was depleted pharmacologically exhibited increased sensitivity
to PQ. Conversely, mutants with elevated pools of DA and wild type flies that have ingested L-
DOPA or DA exhibit increased resistance to PQ. Moreover, we found that following PQ
ingestion, but prior to loss of dopaminergic neurons (and the accompanying decrease in BH4 and
DA levels and corresponding increase in their oxidative products), there is a transient stimulation
of DA pathway activity. The observed protection by minocycline against the effects of PQ might
be mediated through interactions with the DA homeostasis machinery in at least two possible
ways. Minocycline itself might stimulate the activity of the DA biosynthetic pathway and
homeostasis components, creating a cellular environment that emulates those generated
genetically or pharmacologically. Alternatively, minocycline, which reportedly can act as a
scavenger of ROS (Kraus et al., 2006), might prevent the PQ-induced oxidative depletion of DA,
which is expected to elevate oxidative load. As shown in Fig. 5, ingestion of minocycline alone
for 24 hr has no significant effect on the production of pathway metabolites or on GTPCH
activity, ruling out the possibility that minocycline activates DA synthesis. As observed in
Chaudhuri et al. (2007), ingestion of PQ alone resulted in dynamic changes of enzyme activity,
pathway products and oxidative products. After 24 hr of PQ exposure, L-DOPA pools are
96

significantly elevated, relative to controls, indicating an increase in TH activity; however, the
oxidative environment results in depletion of DA and a corresponding increase in the oxidative
product, DOPAC (Fig. 3.5 A, B). Similarly, GTPCH activity increases (Fig. 3.5 C), and the
oxidized product biopterin increases in concentration as BH4 pools are diminished (Fig.3.5 A).
In contrast, when minocycline was co-fed with PQ for 24 hr, the effect of PQ on each of these
components was significantly less severe. Stimulation of GTPCH and TH activity with these
components was minimal and levels of the oxidized products, biopterin and DOPAC, were
indistinguishable from those in sucrose only or minocycline only controls (Fig.3.5 A, B, C).
97

Figure 3.5: Minocycline blocks changes in DA pathway components indicative of PQ-induced
oxidative stress. Minocycline blocks the changes induced by PQ the DA and BH4 biosynthesis
pathways. (A) Changes in the DA and BH4 metabolites after 24 hr of exposure of adult males to
PQ and PQ with minocycline. The addition of minocycline prevents the reduction in the PQinduced
DA and BH4 levels. The increase in L-DOPA levels, indicative of PQ-stimulated TH
activity, is reduced by minocycline. (B) The oxidized DA metabolite,DOPAC, is elevated by PQ
exposure and is significantly decreased by the co-feeding of minocycline to male adults for 24
hr. (C) The compensatory increase in GTPCH activity in PQ-fed adult males is reduced in PQminocycline
co-fed males after the 24 hr of ingestion. The significance of differences in each
subset between the PQ-treated and control groups, and PQ-treated and co-fed groups are
indicated as * and # respectively where * and #= P < 0.05, ** and ## = P < 0.005. Error bars
represent the standard error of the mean. Each data point represents at least 10 replications of 15
flies each.
98

A
B
C
DOPAC
ng/head
ng/head
2.25
2.00
1.75
1.50
1.25
1.00
0.75
0.50
0.25
0.00
0.0175
0.0150
0.0125
0.0100
0.0075
0.0050
0.0025
0.0000
GTPCH activity
(nmole/min/mg)
*
#
**
#
L-Dopa Dopamine BH4 Biopterin
Control
*
PQ
9 **
8
7
6
5
4
3
2
1
0
99
*
#
PQ+Min
#
*
Min
Control PQ PQ+Min Min
#
Control
PQ
PQ+Min
Min

Minocycline reduces PQ-generated reactive oxygen species
The ability of minocycline to block the PQ-induced changes in the DA and BH4
biosynthesis pathways indicative of oxidative stress led us to hypothesize that minocycline was
serving principally as a scavenger of PQ- generated ROS in Drosophila. In order to test this
hypothesis, we assayed several markers of PQ-induced oxidative stress that are known to be
elevated in mammalian systems. Elevation in malondialdehyde, formed from the breakdown of
polyunsaturated fatty acids and the accompanying increase lipid peroxidase level, is an important
and specific cellular response of PQ-mediated toxicity due to the ability of PQ to produce
hydroxyl radicals which react with cellular membranes (Bus et al., 1974). Therefore, we
employed a lipid peroxidation assay to test the ability of minocycline to specifically counteract
the PQ toxicity in Drosophila. As seen in Fig. 3.6A, PQ ingestion results in a two-fold elevation
of lipid peroxidation within the first 24 hr. Co-feeding of minocycline with PQ almost
completely blocks these indicators of lipid damage; lipid peroxidation levels in these flies were
indistinguishable from levels in sucrose only and minocycline only controls. Stimulation of
catalase activity in neural cell cultures (Röhrdanz et al., 2001; Yang and Tiffany-Castiglioni.,
2005) and in Drosophila (Chaudhuri et al., 2007) is another indicator of elevated ROS. We,
therefore, compared catalase activities in the heads of PQ only and PQ and minocycline co-fed
flies. Elevated catalase activities were detected in the heads of both groups (Fig. 3.6 B);
however, the catalase activity in flies that had ingested minocycline with PQ was significantly
lower than those exposed to PQ only. These results strongly suggest that minocycline has a
strong ROS suppression capacity.
100

A
B
Lipid peroxidation
Moles/mg head tissue(x10 -8 )
Catalase Specific activity
Units (10 6 )/mg protein
11
10
9
8
7
6
5
4
3
2
1
0
5
4
3
2
1
0
**
Control PQ PQ+ Min Min
**
Figure 3.6: Minocycline reduces PQ-generated reactive oxygen species. (A) The level of
malondialdehyde, formed from the breakdown of polyunsaturated fatty acids by PQ, was
determined by adding thiobarbituric acid to head extracts of males that had ingested PQ or PQ
with minocycline for 24 hr. The reaction results in a red colored product absorbing at 535nm.
Minocycline significantly decreases the amount of lipid peroxidation induced by PQ. (B)
Minocycline reduces the specific activity of catalase after 24 hr of ingestion in co-fed male flies.
Specific activity is expressed in 10 6 units/mg protein, where 1 unit is defined as 1 μmole of H2O2
decomposed per minute. The significance of differences between the PQ-treated and control
groups, and between the PQ-treated and co-fed groups was indicated as * and #, respectively,
where * and # = P < 0.05. Error bars represent standard error of the mean. The experiments were
done as 5-8 replicas of 10 head extracts.
101
##
Control PQ PQ+Min Min
#

Loss of function mutants of the genes encoding JNK and Akt are sensitive to PQ but involvement
of reaper, caspase and rolled in PQ-induced toxicity was not detected
We next used this PD model to investigate the signaling pathways associated with PQ
toxicity. Specifically, we tested JNK, encoded by the gene basket and Akt, associated with PKB
signaling pathway and encoded by Akt1. In addition, rolled, dronc and reaper which encode
ERK, caspase-9 and the pro-apoptotic protein, reaper, respectively, were tested. Mutations in
rolled gene are homozygous viable while others are homozygous lethal. These kinases function
in myriad biological processes, but are particularly known to respond to various cellular stresses.
We hypothesized that these kinases also play key roles in the DA associated toxic response
triggered by PQ. To test this hypothesis, we exposed heterozygous loss-of-function mutants,
bsk 1 , Akt 1 , rolled, dronc and reaper, to 1 mM PQ alone and to a combined dose of 1mM PQ and
1mM minocycline. When compared to wild type flies, heterozygous bsk 1 and Akt 1 mutants
showed increased sensitivity to PQ and died on average 2 days before wild type flies (Fig. 3.7).
However, heterozygous reaper and rolled mutants were indistinguishable from the wild type
controls for the effect of PQ on their survival. In contrast, the heterozygous caspase mutants,
dronc, survived 2 days more than the wild type controls on PQ (Fig.3.7). Increasing the dose of
PQ to 10 mM did not alter the outcome for these mutant strains (data not shown). Further, we
found that, unlike wild type flies, the lifespan of bsk 1 and Akt 1 mutants exposed to PQ could not
be extended by minocycline treatment, suggesting that JNK and Akt signaling pathways are
important in the protective response of minocycline in flies. In addition, the result shows lack of
regulatory effects by ERK and reaper on PQ toxicity since these heterozygous mutants were
indistinguishable from wild type flies with respect to sensitivity to paraquat and rescue by
minocycline (Fig. 3.7).
102

Figure 3.7: Paraquat induced inflammatory response is regulated by JNK and Akt signaling
pathways. Effect of 1 mM PQ and 1 mM minocycline co-fed with 1 mM PQ on loss-of-function mutants,
JNK/+ (bsk 1 /+), Akt1/+, reaper/+ , caspase/+ (dronc/+), ERK/+ (rolled/+) . rolled (ERK), reaper and
caspase mutants showed an extension of life span with minocycline treatment, while the survival of bsk 1
(JNK) and Akt 1 mutants was unmodified in the presence of minocycline. **= P < 0.005 represents
significant difference between PQ, and PQ with minocycline fed groups. ##=P< 0.005 and shows
significant difference between PQ-fed wild type, and PQ-fed bsk and Akt loss-of-function mutants NS=
not significant. Error bars represent standard error of the mean. n= 180 and each data point represents at
least three independent replications of 50-60 flies each.
103

JNK and Akt confer protection against paraquat-induced toxicity in dopaminergic neurons
The observations that JNK and Akt loss-of-function heterozygous mutants increase the
sensitivity to PQ and that these genes are important for minocycline mediated protective
responses against PQ led us to test whether this effect is reversed when JNK and Akt are over-
expressed. Since PQ toxicity is associated with DA (Peng et al., 2004), we first drove the
expression of JNK and Akt in DA neurons using the GAL4-UAS system (Brand and Perrimon,
1993). Expression of JNK and Akt in dopaminergic neurons resulted in a two-fold increase in the
survival duration in the presence of PQ (Fig. 3.8). Furthermore, over-expression of JNK and Akt
in DA neurons resulted in protection by minocycline against PQ. The exposure of 1 mM
minocycline with 10 mM PQ improved the lifespan by 30% compared to 10 mM PQ alone in
control and over-expressed strains. These results suggest that both Akt and JNK have pro-
survival function in PQ-induced DA toxicity.
104

Figure 3.8: Over-expression of wild type JNK and Akt provide protection against PQ. Adult
males of genotypes TH-GAL4/+, UAS-bsk 1 /+, UAS-Akt 1 /+, TH-GAL4;UAS-bsk 1 /+ and TH-GAL4;
UAS-Akt 1 /+ flies were fed, beginning at 48 hr post-eclosion, 10 mM PQ or 10 mM PQ and 1 mM
minocycline. The average survival duration for each group was determined. Expression of JNK
and Akt in TH neurons increased the survival duration by two fold with respect to control flies.
** =P < 0.005 and represent significant differences between the PQ and PQ with minocycline groups.
##=P< 0.005, indicating a significant difference between control and JNK or Akt expressing flies fed only
PQ. Error bars represent standard error of the mean. Each data point represents at least three independent
replications of 50-60 flies each.
105

DISCUSSION
Minocycline imparts anti-oxidant effects in a paraquat induced Drosophila model for
Parkinson’s disease
PQ is considered an oxidative stressor, generating super oxide and hydroxyl radicals (Bus
et al., 1974). Moreover, epidemiological and experimental studies point to PQ as an etiological
agent for PD (Dinis-Oliveira et al., 2006). We have used PQ ingestion to establish an in vivo
Drosophila PD model (Chaudhuri et al., 2007). This model was employed to study the gene-
environmental interactions for DA regulatory genes regulating the dopamine biosynthesis and
homeostasis that modified susceptibility to PQ.
Minocycline, a second generation semi-synthetic antibiotic derived from tetracycline, is
gaining attention due to its anti-inflammatory and anti-oxidant properties in numerous
neurodegenerative and injury induced mammalian models such as such as cerebral ischemia,
traumatic brain injury, amyotrophic lateral sclerosis, Parkinson's diseases, kainic acid treatment,
Huntington' disease, multiple sclerosis and Alzheimer’s disease (Jordan et al., 2007; Kim and
Suh, 2009). Tetracyclines are available in three groups, tetracycline as original, semi-synthstic
compounds, and modified tetracyclines. Minocycline and doxycycline belong to the semi-
synthetic group. Minocycline differs from doxycycline due to the presence of diethylamino
group at C7 carbon ring and lack of a functional group at C6 (Fig. 3.1 C, D). The hydroxyl group
at C10 and diethylamino group at C7 on the fourth carbon ring have been proposed to play a
crucial role in scavenging free radicals (Kraus et al., 2005; Kim and Suh, 2009). Moreover,
minocycline has been found to be a more potent radical scavenger than doxycycline in Fe 3+
induced oxidative stress in in vitro neuron culture (Kraus et al., 2005). Although the exact
biological targets for minocycline are still not well known, certain results have demonstrated that
minocycline causes the inhibition of the mitochondrial permeability-transition mediated
106

cytochrome c release from mitochondria, the inhibition of caspase-1 and -3 expression, and the
suppression of microglial activation (Chen et al., 2000; Wu et al., 2002; Zhu et al., 2002b).
More recently, controversial studies on the protective effects of minocycline in various
disease models have been reported. Diguet et al. (2004) reported that the protective or deleterious
effects of minocycline depend on the mode of administration and dose of minocycline. Along the
same line, we first tested different doses of minocycline ranging from 100 µm to 50 mM to wild
type flies and conducted experiments subsequently using 1 mM minocycline as this was a highly
efficient and non-toxic dose in wild type Drosophila (Fig. 2.3 A, Chapter 2). We found that this
concentration of minocycline rescued the effects of 10 mM PQ (Fig.3.1 A), and that pre-feeding
and co-feeding regimens for minocycline were equally protective (Fig. 2.3 B, Chapter 2). In
contrast, doxycycline could not extend the PQ induced truncation of life span, suggesting the
specificity of the protective role of minocycline in this PD model mainly due to favorable
chemical groups at fourth carbon ring (Fig. 3.1 B, C, D). Bonelli et al. (2006) also reported the
prevention of PQ- induced reduction of survival duration in Drosophila but the mechanism
through which minocycline imparts this effect in Drosophila was not investigated. We first
assessed the behavioral, neurochemical and neuroprotective abilities of minocycline.
Minocycline delayed PQ induced mobility defects and the loss of dopaminergic neurons (Fig. 3.2
and Fig. 3.3). Further, this neuroprotective effect was observed in all the dopaminergic
subgroups found in Drosophila (Fig. 3.4). Similar protective effects of minocycline have been
reported with respect to different PD mimetics in mammalian in vivo and in vitro models (Wu et
al., 2002; Zhu et al., 2002b; Lin et al., 2003; Wang et al., 2003a). We also assessed the
antioxidant effects of minocycline and detected a reduction in the generation of ROS as assayed
by changes in catalase activity and lipid peroxidation (Fig. 3.6 A, B). We have shown previously
107

that PQ modulates the DA biosynthesis pathway by inducing rapid oxidative turn-over of the
products (Fig. 3.5 and Chaudhuri et al. 2007). Our current results indicate that minocycline
prevents the rapid turn-over of key DA pathway metabolites induced by PQ and thus acts as a
strong anti-oxidant (Fig. 3.5).
We recently found that minocycline causes suppression of PQ-mediated increase in NOS
activity in the adult fly head (Inamdar et al., 2009, under revision). After this initial validation of
the anti-oxidant in addition to NOS-associated anti-inflammatory properties of minocycline
against PQ in Drosophila, we then turned to an investigation of the potential involvement of
signal transduction pathways associated with PQ-induced in vivo PD model.
Identification of signal transduction pathways mediating PQ-induced neurotoxic and
neuroinflammatory responses in Drosophila
Our investigation of signaling pathways mediating PQ-induced neurotoxic and NOS
associated neuroinflammatory response in Drosophila is unique in that it could be get insight
into signal transduction pathway involved in PQ mediated toxicity at the whole organism level.
Most of the previous mammalian studies have utilized in vitro (i.e., cell culture) approaches to
address this question. Moreover, these mammalian studies have used pharmacological inhibitors
to block the proposed functions of the signaling pathway, which may lack complete specificity of
function. Finally, the variations in the type of inhibitor used, inhibitor concentrations, time of
addition, as well as cell lines may affect the outcome of these experiments (Sekiguchi et al.,
1999; Learish et al., 2000; Waetzig and Herdegen, 2005).
We have tested the role of kinases known to be functionally conserved with mammals in
PQ-induced Drosophila PD. We found that heterozygous loss-of-function mutants for JNK,
ERK, Akt, caspase and reaper (pro-apoptosis gene) show differential responses to PQ. The
108

heterozygous loss-of-function mutants for JNK/bsk and Akt1exhibit extreme sensitivity to PQ
and fail to respond to minocycline, indicating crucial roles of these signaling pathways in either
the anti-inflammatory or neuronal stress responses to PQ. On the contrary, heterozygous loss-of-
function mutants for ERK and reaper mutants were equivalent to wild type flies in their
sensitivity to PQ (Fig. 3.7).
In Drosophila, Akt1, which encodes phosphoinositide-3-OH-kinase-dependent
serine/threonine protein kinase, affects cell and organ size in a cell autonomous manner (Verdu
et al., 1999). Akt1 has been shown to be regulated via the Drosophila PI(3)K, in a mechanism
similar to that of its mammalian homolog, indicating the functional conservation of Akt1’s mode
of action. In mammals, Akt1 plays a crucial role in cell survival. It is regulated by the PI3K-
mediated signaling pathway and negatively regulates JNK and caspase-mediated apoptosis
(Burke, 2007). Deregulation of Akt1 mediated signaling pathway has been well documented in
familial and sporadic forms of PD models (Dudek et al., 1997; Yang et al., 2005; Rodriguez-
Blanco et al., 2008; Xiromerisiou et al., 2008). Stimulation of the Akt1 signaling pathway in in
vitro or in vivo models resulted into neurotrophic, anti-apoptotic effects (Ries et al., 2006; Burke,
2007). Yang et al. (2005) found the suppression of ROS and survival of DA neurons in
transgenic strains over-expressing Drosophila Akt1 in DJ1 RNAi strains. Therefore, our
preliminary results indicate that, like mammalian Akt1, Drosophila Akt1 appears to have
functions in promoting survival of DA neurons in PQ-induced Drosophila PD model (Fig. 3.7,
Fig. 3.8).
In mammalian PD models, JNK has been documented to initiate PCD (programmed cell
death) by inactivating anti-apoptotic protein Bcl-xl. Moreover, phospho-JNK is detected in DA
neurons in MPTP and 6-OHDA- induced PD model (Gearan et al., 2001; Ganguly et al., 2004).
109

Furthermore, Peng et al. (2004) detected up-regulation of phospho-JNK in TH neurons in a PQ-
induced mammalian PD model. Similarly, activated JNK has been detected in parkin mutants in
Drosophila (Yang et al., 2005). However, in Drosophila, JNK, encoded by bsk, has also been
reported to play an important role in morphogenesis and innate immune response (Noselli and
Agnès, 1999; Boutros et al., 2002). Moreover, Wang et al. (2003b) also demonstrated important
roles for JNK in longevity and resistance to PQ induced-oxidative stress. Our preliminary data
suggest a specific role of JNK in the survival response of DA neurons in our PD model and could
raise the possibility that JNK may function in both neurons and innate immune cells in PQ
response (Fig. 3.7, Fig. 3.8).
ERK is proposed to play an important neuroprotective role in PD (Cavanaugh et al.,
2006). However, there are controversies since evidence for a neurodegenerative role of ERK in
PD also has been reported (Chu et al., 2004). The elucidation of a direct functional role for ERK
in in vivo PD models has not been reported. We, therefore, used the heterozygous mutant form
for ERK/rolled in PQ- induced PD model to investigate potential functions, and we found that
heterozygous loss-of-function mutant form for ERK lack any detectable functional involvement
in our model, although the result could also infer the lack of sufficient knock-down due to
heterozygous strain (Fig. 3.7). It would be interesting to test for the dominant negative form for
ERK in DA neurons to correlate direct role of ERK in our PD model.
We further analyzed the role of PCD in PQ neurotoxicity by testing the heterozygous
mutants for pro-apoptotic gene, reaper and PCD initiator, caspase-9 ortholog, dronc. We found
that the in heterozygous loss-of-function mutants for reaper and dronc, reaper mutants lack
comparable survival to wild type flies on PQ, while dronc mutants showed increase in lifespan.
These results suggest that the activation of caspase mediates apoptosis/PCD in PQ toxicity which
110

lack direct interaction with reaper gene known to inhibit the Inhibitor of Apoptosis (IAPs) in
Drosophila and mammals (Fig. 3.7) (Steller, 2008).
Thus, our preliminary results here provide in vivo evidence for essential role for kinases
in a PQ induced PD model. Furthermore, we performed primary screening using minocycline, an
agent known to possess anti-inflammatory and anti-oxidant in mammalian and Drosophila model
for PD in wild type flies (Inamdar et al., 2009, under revision). Further, we have detected the
modification of protective properties of minocycline by DA regulatory genes (Inamdar et al.,
2009, under revision). In this report, we present data showing the failure of heterozygous Akt1
and JNK loss-of-function mutants to respond to minocycline, suggesting that minocycline might
act via these signaling pathways. However, over-expression of wild type Akt1 and JNK in DA
neurons does not provide additional levels of protection. Thus, these pathways may function
parallel to minocycline, or alternatively, their role in the minocycline response is mediated in a
cell type other than dopaminergic neurons (Fig. 3.8). We must perform genetic interactions
between Atk1 and JNK signaling pathways by testing double mutants for Akt1 and JNK for PQ
and PQ with minocycline sensitivity to further assess the mechanisms by which these signaling
pathways are involved in this neurodegeneration model. In addition, it will be necessary to test
the association of these signaling pathway with the PQ-induced NOS response in CNS by
expressing the loss-of-function and/or dominant negative, and over-expression transgenic lines in
the cells inducing the NOS against PQ.
111

CHAPTER 4
BRIEF EXPOSURE TO PARAQUAT DURING JUVENILE AND EARLY STAGES CAUSES
PARKINSONIAN SYMPTOMS, INCREASED SENSITIVITY TO OXIDATIVE INSULT
LATER IN LIFE AND A SHORTENED LIFE SPAN
This work is a manuscript in preparation for submission to Journal of Neuroscience. Co-authors
for this work were Arati Inamdar, Shane Welch, Russ Alexander and Janis O’Donnell. Shane
Welch and Russ Alexander contributed in collecting data presented in figure 4.2 A, C. Arati
Inamdar collected the remaining data.
112

INTRODUCTION
Parkinson’s disease (PD), characterized by loss of dopaminergic neurons and presence of
Lewy bodies in the substantia nigra pars compacta (SNpc), is the second most common chronic
neurodegenerative disease (Schiller, 2000). The loss of dopaminergic neurons along with
degeneration of nerve terminals in the striatum eventually develops into Parkinsonian symptoms,
resting tremor, rigidity, bradykinesia, and gait disturbance (Jellinger, 2001). Unfortunately, the
exact cause of sporadic Parkinson’s disease is unknown. However, epidemiological and
experimental studies have suggested multifactorial etiology with potential roles for
environmental toxins, genetic susceptibility and aging in the pathogenesis of PD. Upon exposure
to environmental toxins such as MPTP, 6-OHDA, rotenone, and paraquat in animal models,
typical Parkinsonian features have been noted, implying that gene-environment interactions can
be modeled to understand the mechanisms of etio-pathogenesis for PD (Mendez and Finn, 1975;
Burns et al., 1983; Betarbet et al., 2000; McCormack et al., 2002; Uversky, 2004).
PD previously has been considered to be associated with geriatric populations, but it has
also been reported in populations below age 40. The appearance of Parkinsonian symptoms in
the patients below the age of 40 (in some studies the cut off age is considered as 50 yrs) are
classified as Early-Onset Parkinsonism (EOP). The incidence of EOP ranges from 0.8 per
100000 per year to 3.0 per 100000 per year in population between 0-49 yrs (Schrag and Schott,
2006). Early-onset Parkinsonism has two subsets. Juvenile Parkinsonism patients represent that
subset of EOP who present with the Parkinsonian features under age 21 while those with onset of
Parkinsonian features at or above 21 yrs but under 40/50 yrs are classified as Young-Onset PD
(YOPD). The causes for Juvenile and YOPDs are largely proposed to be genetic but the definite
cause is still unknown. Epidemiological studies have not yet reported exposure to pesticides as
113

the risk factor for the pathogenesis of YOPD although rural living associated with well water
drinking and head injury early in life have been proposed to be important factors accelerating the
occurrences of Parksinonism symptoms in such individuals (Tsai et al., 2002). However,
correlation between early exposure to toxins and early onset of PD has been proposed
(Logroscino, 2005).
Paraquat is one of the well-known Parkinson mimetic agents proposed to induce
generation of ROS, thereby causing loss of dopaminergic neurons in both mammalian and
Drosophila PD models (McCormack et al., 2002; McCormack et al., 2005; Chaudhuri et al.,
2007). Various studies have recently demonstrated that like other neurotoxins, paraquat toxicity
is partially mediated by nitric oxide where it acts as a diaphorase and increases the production of
oxygen radicals and lipid peroxidation, thereby causing dysfunction of membrane potential
(Djukic et al., 2007, Day et al., 1999). In vitro studies demonstrated that PQ toxicity is induced
in microglial cultures, but not in neuron-glia cultures implying that microglia mainly contributed
to PQ-induced ROS in PQ-treated microglia enriched cultures (Wu et al., 2005). Further, single
paraquat exposure is capable of activating microglia without DA neuron loss in mammalian PQ
induced PD model (Purisai et al., 2007).
There has been a growing incidence of YOPD and higher mortality and morbidity in
YOPD patients than the normal population (Schrag et al., 1998; Schrag A and Schott, 2006).
Therefore, it is still unknown whether a single exposure to Parkinson mimetics early in life plays
any role in etio-pathogenesis of early onset PD. Here, we have used a PQ induced Drosophila
PD model, which, as in mammalian PD models generated by continuous exposure of PQ,
shortens life-span and induces mobility defects correlating with the loss of DA neurons
(Chaudhuri et al., 2007). Further, we showed that PQ toxicity in our Drosophila model is
114

associated with NO production. Finally, we showed that NO is induced in microglial-like cells,
hemocytes, that mediate the engulfment and demise of dopaminergic neurons (Inamdar et al.,
2009, under revision).
In the present study, we present a comprehensive study employing our in vivo Drosophila
PQ-induced PD model showing that brief exposure to paraquat in young adult and juvenile/larval
stages is accompanied by loss of dopaminergic neurons, mobility defects and modified survival
duration. Moreover, differential PQ sensitivity seems to be dependent upon the age at exposure,
as well as dose of exposure. The results suggest that sporadic or brief exposure is capable of
initiating the toxic process influencing the sensitivity towards paraquat upon subsequent
exposure.
115

MATERIALS AND METHODS
Drosophila strains and culture maintenance. 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 obtained from Jay Hirsh (University of
Virginia). All stocks were maintained at 25º C, and all the experiments were performed at the
same temperature.
Feeding experiments. Separated males and females, less than six hours old post-
eclosion and second instar larvae were fed paraquat at1 mM or 10 mM concentrations soaked
on filter paper with 5% sucrose, and in 1 % agar with 5% sucrose and yeast for 12 hr for
young adult flies and larvae, respectively. The Drosophila food medium was changed every
15 days after exposure to PQ.
Locomotion assay. The mobility of larvae was performed in petri dishes containing
1% agar. The time required for the larvae to crawl a 5 cm distance towards a filter paper
piece soaked in acetone was recorded. The average of three recordings per larva was noted
for about 15-20 larvae. The mobility of adult male and female flies from each treatment
group was assessed using a negative geotaxis climbing assay. A single fly was placed in an
empty plastic vial, tapped to the bottom and the time required to climb 5 cm was recorded
three times sequentially with 10 min rest periods between each measurement. Each
replication value recorded is an average of the three trials; each assay was conducted on 10
adult flies per test group.
116

Confocal microscopy. Whole mounts of dissected brains from age matched TH-Gal4;
UAS-eGFP adults and larvae fed with sucrose alone, or with paraquat, as described in the
Results were examined for dopaminergic neuron morphology and number, detected by
visualizing GFP-expressing neurons. Each brain was scanned to include 10-15 sections for
optimum visualization of DA neurons. The Z-sections were then utilized to get the average of
all sections using a Leica TCS SP2 AOBS confocal microscope (Wetzlar, Germany).
Statistical Analysis. All data were analyzed by one way ANOVA with Dunnett’s post
test or by one-tailed Student’s t-test, assuming equal variances wherever applicable, using
GraphPad Prism (San Diego, CA). Details of the analyses are described in the figure legends.
117

RESULTS
High and low doses of PQ, fed to juvenile (2nd instar larvae) and young (

in contrast to chronic PQ exposure of sexually mature adults, where females were less
sensitive to PQ than males (Chaudhuri et al., 2007). Therefore, as is the case for chronic PQ
exposure, a single episode of PQ-exposure earlier in life has long-term consequences for
survival.
119

Figure 4.1: Exposure of juvenile (2 nd instar larvae) and young adult (

A
B
121

Exposure of juvenile and young adult (

Figure 4.2: Exposure of larva and young adult flies to high and low doses of PQ causes
mobility defects immediately after exposure to PQ and in later stages of life. (A) Second
instar stage larvae were exposed to 10 mM and 1 mM PQ, or to 5% sucrose for 12 hr and
then transferred to the normal food. The mobility of the larvae was assayed by the rate at
which they crawled towards an acetone soaked filter paper kept in a petri-dish 5 cm away
from the start-line. The larvae exposed to PQ in the larval stage demonstrate mobility defects.
(B) The PQ (10 mM and 1 mM) induced mobility defect in adult males exposed to PQ during
the larval stage and aged to one and a half months post-eclosion, was determined by the
negative geotaxis assay. (C) The PQ-mobility defect in the young adult males performed
immediately after 12 hr exposure to PQ (1 mM and 10 mM) was determined by the negative
geotaxis assay. (D) The PQ-induced mobility defects of males exposed to 1 mM PQ for 12 hr
as newly eclosed adults and subsequently aged for one and a half months on normal food.
The * indicates the significance of differences between control and PQ-fed flies. NS = not
significant ***=P< 0.005, ** = P < 0.005 and * = P < 0.05. Error bars represent standard
error of the mean. Each data point represents at least 5 replications of 15 flies each.
123

A
B
124

C
D
125

Exposure of juvenile and young adult (

for each subgroup of neurons following exposure to both PQ concentrations is shown in Fig
4.3P.
These results show that there is dose-dependent loss of DA neurons which correlates
well with the mobility defects associated with PQ exposure reported in the previous section.
127

Figure 4.3: Exposure to high and low doses of PQ induces dose-dependent loss of DA
neurons in larval and young adult brains. (A), (B). Schematic diagrams showing the positions
of DA subgroups of neurons in larval and adult brains, respectively. (C, D, E) The effect of 5
% sucrose, 1 mM PQ and 10 mM PQ on the dopaminergic neurons of w; TH-GAL4; UASeGFP
larval brain. PQ causes severe loss of almost all regions of DA neurons in the larval
brain exposed to 10 mM PQ (E) but not with 1 mM PQ (D) compared to control larval
brain(C). (F, G, H) Insets enlarged for larval DM, DL1 subgroups of DA neurons for control,
PQ (1 mM) and PQ 10 mM. (I) The average number of DA neurons after 12 hr of continuous
feeding of 5 % sucrose, 1 mM PQ and 10 mM PQ performed on of w; TH-GAL4; UAS-eGFP
larval brain. There was significant reduction in the DA neurons in DM, DL1, DL2 subgroups
in the larval brain fed with 10 mM PQ but no difference between the neuron counts between
the larval brains fed with 5 % sucrose and 1 mM PQ. (J,K,L) The effect of 5 % sucrose, 1
mM PQ and 10 mM PQ on the dopaminergic neurons of w; TH-GAL4; UAS-eGFP adult
brain. 10 mM PQ (L), but not 1 mM (K), causes alteration in the number and morphology of
DA neurons compared to control DA neurons (J), suggesting a dose-dependent loss of DA
neurons induced by PQ in both larval and adult brain. (M,N,O) Insets enlarged for PPM2
subgroup of DA neurons in adult brain for control, PQ (1 mM) and PQ (10 mM). (P)The
average number of DA neurons after 12 hr of continuous feeding of 5 % sucrose, 1 mM PQ
and 10 mM PQ, performed on of w; TH-GAL4; UAS-eGFP adult brain, shows that PAL,
PPM3 and PPL2 subgroups of DA neurons are affected by 10 mM PQ but not with 1 mM
PQ. The significance of differences between PQ-treated and control groups in each subgroup
were indicated as *, where * = P< 0.05. Error bars represent standard error of the mean. Each
data point represents at least 5-7 brains. Scale bars for C,D,E=50 μm; J,K,L =100 μm. (Image
source for A, B: Images published in Friggi-Grenin et al., 2003)
128

Wild type
F
129

I
130

J
Wild-type
K
PQ 1 mM
L
PQ 10 mM
131
M
N
O

P
132

Exposure of juvenile and young adult (

Figure 4.4: Exposure to 1 mM PQ at early stages of life sensitizes adults to subsequent PQ
exposure. A reduction in average survival duration of flies pre-exposed to 1 mM PQ at larval
and young adult age for 12 hr and continuously exposed to 1 mM PQ after 2 months on
normal food was detected. This result demonstrates that early exposure to PQ sensitizes the
flies for a second exposure of PQ. The significance of the difference between 5 % sucrose
pre-exposed and 1 mM PQ pre-exposed at larval and young adult stages are represented with
* where, P

DISCUSSION
Brief exposure to PQ early in life causes truncation of life span and mobility defects in later
stages of life
We report here the long-term effect of brief exposure early in life to PQ, one of the
potential Parkinson mimetics. The etiology of sporadic forms of PD is considered to be
multifactorial. The processes of oxidative stress and inflammation are considered the most
important factors amplifying the degenerative changes in PD. The exact stimuli capable of
initiating or enhancing this degenerative process are still unknown. However, current studies
have suggested that exposure to insecticides, pesticides, brain injury, and brain infections
early in life may serve key roles in the pathogenesis of PD (Liu et al., 2003; Liu and Hong,
2003). Idiopathic PD affects 3% of the population between ages 65-85, 4-5% of the
population over 85 years and, surprisingly, 5-10 % of all PD patients are being reported to be
less than 40 years of age. These epidemiological data suggest that the pathogenesis of
idiopathic form of PD may be associated with certain risk factors capable of altering the
homeostasis of DA in the brain via multiple mechanisms. In PD patients, the SN shows a
reduction in the levels of Glutathione (GSH), increase in lipid peroxidases, oxidative
products of damaged RNA and other products, indicating the detrimental role of oxidative
damage for DA neuron degeneration (Halliwell, 2001). Several toxin-induced PD models
have further supported the association of oxidative stress to PD.
Among many neurotoxins capable of inducing PD-like symptoms, PQ is considered
to be the most potent redox cycler and is capable of inducing oxidative damage to almost all
cells including DA neurons (McCormack et al., 2002). Noteworthy, epidemiological studies
support an increase in the incidence of PD in agricultural populations known to be exposed to
PQ (Liou et al., 1997). We have generated a Drosophila PD model based on the continuous
135

ingestion of PQ, which is able to reproduce Parkinsonian features including mobility deficits
(Chaudhuri et al., 2007). Further, we found that PQ toxicity is dependent on the dosage of
PQ, and these results are supported by the observations reported here. In this report, we have
shown that brief exposure of high (10 mM) or low (1 mM) doses of PQ is capable of
initiating processes detrimental to the organism and ultimately results in early death,
although the immediate impact of low dose of PQ was not evident immediately after
exposure as assessed by the status of the DA neurons. In contrast, feeding the higher dose of
PQ (10 mM) not only caused DA neuron loss even after only a 12 hr exposure to larvae and
young adults but also resulted in a significant reduction in average survival duration in both
groups. Similarly, although the 1 mM PQ dose did not cause degeneration of DA neurons in
larvae or young adult brains, the treatment strongly decreased their survival duration (~65-70
days) relative to control flies (~90 days) (Fig. 4.1 A, B and Fig 4.3 C, D, E, F, G, H,I, J, K, L,
M, N, O, P). Surprisingly, flies exposed to 10 mM PQ for 12 hrs soon after eclosion could
only survive about 30 days (Fig. 4.1 B). Flies eclosed after larval exposure to 10 mM PQ
fared somewhat better, surviving about 50 days (Fig. 4.1 A). It has been observed that during
metamorphosis further development and differentiation of DA neurons occurs and this could
be the reason for the differences in the total survival durations for the larvae and young flies
exposure to same concentration of PQ (Budnik and White, 1988). Moreover, loss of DA
neurons has been reported in aged flies (Neckameyer et al., 2000). Interestingly, we did not
find a significant difference in the survival duration between males and females in these
experiments as opposed to the differences we found when 20 mM PQ was exposed
continuously to 24-48 hr old flies (Chaudhuri et al., 2007) (Fig. 4.1 A, B). However, it has
previously been reported that response to the PQ-induced oxidative stress alters the TH
136

activity differentially depending upon the gender, age, reproductive status and sexual
maturity (Neckameyer and Weinstein, 2005). Our results are consistent with their
observations.
Both larvae and young adults exposed to 10 mM PQ demonstrated mobility defects as
assessed by crawling and negative geotaxis assays, respectively. Although 1 mM PQ induced
bradykinesis in larvae after 12 hr of continuous feeding, young adult flies ingesting the same
dose for 12 hr lacked significant mobility defects (Fig 4.2 A, C). Interestingly, upon transfer
to normal food these flies had obvious mobility defects until about 35 days post-eclosion,
when we noted the onset of tremors in the flies exposed to 1 mM PQ at either the larval and
young adult stages. Gradually these features became worse as assessed by negative geotaxis
assay performed after one and a half months being on normal food medium on the flies
exposed to 1 mM PQ at larval and young adult age (Fig.4.2 B, D). Surprisingly, the flies pre-
exposed to PQ during the larval stage survived longer than those exposed as young adults,
suggesting that metamorphosis may be a favorable event for removing dead DA neurons,
development and differentiation of DA neurons and DA homeostasis (Budnik and White,
1988).
Brief exposure to PQ sensitizes the DA neurons for the subsequent exposure of paraquat
Although several studies have presented evidence supporting the exposure of PQ as
an etiological agent for PD, most of these studies employ continuous exposure of PQ.
Considering the growing incidence of PD in a comparatively young population, it may be
speculated that exposure to proposed PD inducers briefly at early points in life increases the
vulnerability of individual towards exposure to PD inducers at later stages.
137

Purisai et al. (2007) found that a low dose of PQ is capable of microglial activation in SN,
even though no DA neurons are lost. However, this even sensitizes the DA neurons for
degeneration after a subsequent PQ exposure. Our experiments demonstrate the generality of
these effects. The brief exposure of PQ without any detectable of DA neuron loss by 1 mM
PQ amplifies the death process after the flies are exposed to another 1 mM dose around 2
months later possibly via generation of ROS and ROS mediated DA neuron death (Fig. 4.4).
It is to be noted that the difference in the survival of control groups (from larvae and young
adult) upon exposed to 1 mM PQ after 2 months could be due to two different time points at
which these experiments were performed. It will be important to pursue the question of
whether this early event also perturbs DA homeostasis in the adult brain as well as other
metabolic processes that may lead to increased sensitivity towards such PD inducers as well
as early death. Whether, brief exposure of PQ-like neurotoxins are capable for inducing
NOS, one of the important inflammatory mediators known to be activated in inflammatory
response in mammalian models are not known. Since, we have detected the induction of
NOS in the adult brain after exposure to PQ for 6 hr (Inamdar et al., 2009, under revision); it
would be interesting to known if exposure to high and low doses of PQ for brief (12 hr)
period of time is also associated with induction of NOS in larvae and young adults at any
time-point. Moreover, Langston et al. (1999) reported a study performed on three patients
that were known to be exposed to MPTP in which activated microglia were found around the
DA neurons after 3-16 years suggesting a correlation between inflammatory processes and
DA neuron loss at later stages of life. However, it is unknown if a similar response will be
evident with PQ exposure at early stages of life. Therefore, whether the sustained induction
of the NOS dependent response leads to long-term induction of pro-inflammatory molecules
138

as seen in mammalian models is currently unknown and will be an important topic for further
experiments.
139

CHAPTER 5
IDENTIFICATION OF A NITRIC OXIDE-DEPENDENT RESPONSE IN S. VENEZUELAE-
INDUCED PARKINSON’S DISEASE IN DROSOPHILA MELANOGASTER
This work has been in progress in O’Donnell Lab. All the data were collected by Arati Inamdar.
140

INTRODUCTION
The processes of neuroinflammation and oxidative stress are thought to be among the
primary mechanisms playing roles in the etiology and pathophysiology of Parkinson’s disease
(PD). In addition to genetic causes, environmental factors are proposed to contribute to the
sporadic, and possibly hereditary, forms of PD. Among the various candidate environmental
factors, recent concern has been raised regarding exposure to the soil bacterium, Actinobacteria,
in agricultural populations, who are known to exhibit increased incidence of PD (Semchuk et al.,
1992; Kamel et al., 2007). Actinobacteria, which consist of pathogenic species like Streptomyces
and Nocardia, are gram-positive bacteria. Actinobacteria are known to produce secondary
metabolites, which are composed of various products balanced in necessary biological elements
such as carbon, nitrogen, and phosphorus that can be reutilized by the bacteria to survive in
nutrient depleted environment. Further, these secondary metabolites could contain products toxic
to other microorganisms or possess therapeutically important properties (Shapiro, 1989).
The etiological agents responsible for Parkinsonian symptoms in patients and
experimental models provide growing evidence for an infectious cause for some cases of PD,
particularly various viruses, especially the Epstein Barr virus, influenza virus, coronavirus and
Japanese encephalitis virus (Duvoisin and Yahr, 1965; Fazzini et al., 1992; Pradhan et al., 1999).
Since the 1980’s, several additional microorganisms such as Borrelia burgdorferi, Haemophilus
influenzae, Corynebacterium diphtheriae, Cryptococcus neoformans, Mycoplasma pneumoniae
and Helicobacter pylori have been reported to be associated with PD features (Wszolek et al.,
1988; Kim et al., 1995; Altschuler, 1996).
141

Recently, the role of Nocardia in the pathogenesis of Parkinson’s disease has been
reported (Broxmeyer, 2002). Exposure of neuronal cell culture and a mouse model to media
containing the secreted metabolites from growing Nocardia caused apoptosis in dopaminergic
neurons and induced Parkinsonian symptoms such as bradykinesia and stooped posture. In
another study, it was observed that exposure to Nocardia caused inhibition of the ubiquitin
proteosome pathway, leading to apoptotic death of neurons in substantia nigra without induction
of an inflammatory response in that region (Tam et al., 2002). Díaz-Corrales et al. (2004)
showed that intravenous injection of the Nocardia species isolated from a patient suffering from
Actinomycetoma in mice reproduced Parkinsonian symptoms with alterations in dopamine (DA)
levels in the substantia nigra (SN).
Given the possibility of an infectious etiology of Parkinson’s disease, our collaborators in
The Caldwell Lab (University of Alabama), recently tested for effects of Streptomyces
venezuelae on the dopaminergic neurons of C. elegans and found that exposure to media
containing bacterial secondary metabolites from this strain of common soil bacteria cause loss of
dopaminergic neurons. They also found that ingestion of the proteosome inhibitor, MG132, also
induces loss of dopaminergic neurons in C. elegans. The results were also confirmed in human
dopaminergic neuron cell culture, SHSY5Y, employing culture media obtained from growing S.
colicolor as the control against S. venezuelae culture media (J. Armagost, K. Caldwell, and G.
Caldwell, unpublished results).
Despite these results, the mechanism through which S. venezuelae induces DA neuron
loss is still unknown. As discussed in Chapter 2, paraquat (PQ), a well-known herbicide that is
also considered an environmental risk factor, was previously found in our laboratory to produce
Parkinsonian symptoms in Drosophila, including resting tremors and region-specific
142

degeneration of dopaminergic neurons (Chaudhuri et al., 2007). Further, we have recently
discovered that upon exposure to PQ, the adult brain of Drosophila also shows induction of nitric
oxide synthase (NOS) as assayed biochemically and by immunolocalization. Further NOS-
positive structures found to be surrounding the dopaminergic neurons were not in glial cells
(Inamdar et al., 2009, under revision).
Here, I demonstrate that the ingestion of a crude conditioned medium of S. venezuelae
also causes Parkinsonian symptoms in Drosophila, similar to the behavioral and cellular changes
induced by PQ. In addition, we found that exposure to such medium induces NOS in the adult
brain. In particular, unlike PQ exposure, the induced NOS cells do not seem to surround the
dopaminergic cell bodies; rather they associate closely with glial cells suggesting that the
mechanism of action of this toxin on CNS may be distinct from PQ-associated mechanisms. I
report results that begin the process of defining the mechanism of action of this toxin, which has
the propensity to cause Parkinsonian symptoms, and may be one of the environmental causes of
PD.
143

MATERIALS AND METHODS
Drosophila strains and culture maintenance. The strain, Df (1)w; y, which is normal for
all genes functioning in DA homeostasis, was used as the wild type strain. The transgenic strains
UAS-2X eGFP (Chromosome II), Repo (reversed polarity), GAL4 (a glial cell driver) were
obtained from the Bloomington, IN Drosophila stock center and a TH-GAL4 (a DA neuron
driver) (Friggi-Grelin et al., 2003) was obtained from Jay Hirsh (University of Virginia).
Cultures were maintained at 25º C.
Feeding experiments. Separated male and female flies, 48 hr post-eclosion, were fed on
filter paper saturated with one of the following solutions: 5% sucrose only or 5% sucrose with
crude conditioned culture media collected from S. venezuelae and S. colicolor bacterial strains.
Feedings were continued until all flies were dead. The crude conditioned culture media were
obtained from Caldwell Lab, UA.
Locomotion assay. The mobility of adult male and female flies from each treatment
group was assessed using a negative geotaxis climbing assay. A single fly was placed in an
empty plastic vial, tapped to the bottom and the time required to climb 5 cm was recorded three
times sequentially with 10 min rest periods between each measurement. Each replication value
recorded is an average of the three trials; each assay was conducted on 10 flies per test group.
Immunohistochemistry. Brains from TH-GAL4; UAS-2XeGFP and Repo-GAL4; UAS-
eGFP adults (untreated or exposed to crude conditioned medium from S. venezuelae and S.
colicolor bacterial strains for 24 or 48 hr) were fixed in 4% paraformaldehyde for 3.5 hr and
144

washed extensively in 1x phosphate buffered saline (PBS) and then in 0.1% Triton X-100, 0.2%
bovine serum albumin, in PBS (PBT). Brains were then blocked in 5% normal goat serum in PBT
overnight at 4°C, followed by overnight incubation with a 1: 500 dilution of rabbit anti-NOS
antiserum (N217, Sigma Aldrich) and mouse anti-GFP antibody (Abcam). After additional
washing, the brains were incubated at room temperature for 2 hr in a 1: 5000 and 1: 5000 dilution
of Cy-3-conjugated goat anti-rabbit IgG and FITC conjugated rabbit anti-mouse IgG,
respectively. Confocal studies were performed using a Leica TCS SP2 AOBS confocal
microscope (Wetzlar, Germany). The whole mounts of dissected brains from TH-Gal4; UAS-
eGFP adults for the dopaminergic neuron morphology and number, detected by visualizing GFP-
expressing neurons was performed on the above mentioned confocal microscope. Each brain was
scanned to include 10-15 sections for optimum visualization of cells being studied. These Z-
sections were then used to get the average image of all sections as presented in Results section.
Statistical Analysis. All data were analyzed by one way ANOVA with Dunnett’s post test
or by one-tailed Student’s t-test, assuming equal variances wherever applicable, using GraphPad
Prism (San Diego, CA). Details of the analyses are described in the figure legends.
145

RESULTS
Exposure to S. venezuelae conditioned medium causes truncation of life span in wild type flies
The crude conditioned medium is derived from the media containing secondary
metabolites from the growing S. venezuelae. In order to determine whether crude conditioned
medium is functionally active and imparts detrimental effects in our Drosophila model, we
hypothesized that exposure of the conditioned medium causes truncation of life span of wild type
flies. Wild type flies were exposed to crude conditioned medium from S. venezuelae and S.
colicolor and their survival was noted every 24 hr intervals until all flies were dead. The
conditioned medium of S. colicolor, which was non-toxic in C. elegans and a human
dopaminergic cell line, SHSY5Y was used as a control (J. Armagost, K. Caldwell, and G.
Caldwell, unpublished results). We also found that exposure to S. colicolor had limited toxicity
in flies, while flies exposed to S. venezuelae showed a gradual decline in survival with a sudden
peak in the death rate after 96 hr of exposure with death of all flies by 168 hr. The death trend
observed with S. venezuelae toxin exposed was different from that of PQ exposure, in which the
rate the wild type flies died was almost the same at each time point (Fig. 5.1). This suggests that
upon exposure to conditioned medium of S. venezuelae, an accumulation of toxic substances
occur, under the influence of which flies succumb in a delayed response.
146

Figure 5.1: Exposure to crude conditioned medium of S. venezuelae causes truncation of life
span in wild type flies. The crude conditioned media from S. venezuelae and S. colicolor cultures
were fed to wild type flies aged to 48 hr post-eclosion continuously until all flies on S.
venezuelae conditioned medium were dead. There was an initial increase in death on both
conditioned mediums until 96 hr. However, no further death was seen on S. colicolor
conditioned medium while the flies on S. venezuelae exhibited a continuous death trend after 96
hr, culminating in death of all flies in the next three days. The SEM are not clearly visible in the
graph due to small error numbers. Each data point represents at least 10 replications of 10 flies
each.
147

Exposure to S. venezuelae conditioned medium causes mobility defects in wild type flies
Since our collaborators had noted the loss of DA neurons in both C. elegans and cell
culture after exposure to S. venezuelae conditioned medium, we next asked whether this bacterial
strain had a similar effect on Drosophila DA neurons. We previously found that wild type flies
exposed to 20 mM PQ began exhibiting strong tremors and bradykinesia within 24 hr, preceding
death by approximately one to two days (See supplementary videos of tremors and mobility after
PQ ingestion in Chaudhuri et al., 2007). Flies exposed to the S. venezuelae conditioned medium
for four days (a point at which approximately 50% of the flies had died) exhibited bradykinesia
(Fig. 5.2) as in PQ exposure. However, there were also significant differences between the
effects of PQ and the bacterial medium. Under PQ exposure tremors preceded the initiation of
increased mortality, while flies exposed to the bacterial medium only exhibited tremors well after
the initial increase in mortality, generally between 3-4 days after exposure to the medium (Fig.
5.2). Moreover, these tremors were not as severe as seen with PQ exposure. No obvious mobility
defects were observed in flies exposed to S. colicolor conditioned medium. These data show that
exposure to crude conditioned medium from S. venezuelae induces mobility defects, a
characteristic feature of PD.
148

Figure 5.2: Exposure to crude conditioned medium of S. venezuelae induces Parkinsonian-like
mobility defects. The movement defects induced by feeding S. venezuelae medium to wild type
flies were determined using a negative geotaxis assay, performed on the fourth day after the
initiation of continuous exposure started at 48 hr post-eclosion. The average time required by a
single fly to climb a 5 cm distance is presented. The flies exposed to S. venezuelae conditioned
medium showed predominant bradykinesia along with tremors at this time while these features
were not evident in flies fed conditioned medium of S. colicolor or 5% sucrose. There was no
significant difference in the climbing ability of wild type flies on the conditioned medium of the
non-toxic S. colicolor conditioned medium and 5% sucrose. Each data point represents at least
10 replications of 10 flies each. NS= non significant, *= P

Exposure to crude conditioned medium of S. venezuelae causes loss of dopaminergic neurons in
wild type flies
I next asked whether exposure to S. venezuelae conditioned medium was associated with
loss of DA neurons, employing the transgenic reporter strain, TH-GAL4; UAS-eGFP to monitor
the DA neurons (Friggi-Grelin et al., 2003). I observed morphological changes in each subgroup
of DA neurons at 24 hr intervals and found no significant loss of DA neurons (data not shown).
At 48 hr of continuous exposure, differential responses of DA neuron subgroups were found
(Fig. 5.3 A, B, C and Fig. 5.3 G). For example, loss of the PPM2 subgroup of DA neurons
exposed to S. venezuelae conditioned medium was found relative to the same subgroup of
neurons exposed to 5% sucrose and crude conditioned medium of S. colicolor (Fig. 5.3 D, E, F).
DA neurons exposed to S. colicolor conditioned medium showed no decrease in the number or
morphology of neurons relative to control (5 % sucrose fed) brains up to 48 hr after initiation of
exposure (Fig. 5.3 G). Therefore, exposure to crude conditioned medium of S. venezuelae is
associated with loss of DA neurons, and thus provides evidence for its potency to induce
Parkinsonism in flies.
150

Figure 5.3: Exposure to crude conditioned medium of S. venezuelae induces loss of DA neurons.
The effect of 48 hr of continuous exposure of 5% sucrose, crude conditioned media of S.
colicolor and S. venezuelae on the dopaminergic neurons of TH-GAL4; UAS-eGFP adult brain.
(A, B) The number and normal asymmetric neuron morphology was seen in brains exposed to
5% sucrose and crude conditioned medium of S. colicolor (B). (C) The brains treated with S.
venezuelae conditioned medium induced loss of DA neurons in different subgroups of neurons.
(D, E, F) The insets show the changes in the number of the PPM2 subgroup of neurons in A,B,C,
respectively. Scale in A, B, C= 100 μm. (G) The average number of neurons per subgroup was
determined 48 hr after the initiation of feeding. All scoring was done on TH-Gal4:UAS-GFP
adults. Each subset of dopaminergic neurons was scored separately (n=10-15). No difference in
the neuron counts between 5% sucrose and S. colicolor conditioned medium were found but
there was significant loss of DA neurons in PPM2, PPM3, and PPL1 was detected in S.
venezuelae conditioned medium. **=P < 0.005 and represents significant difference between S.
venezuelae conditioned medium and 5% sucrose or S. colicolor conditioned medium.
151

A
152
D
E
F

G
153

Exposure to conditioned medium of S. venezuelae causes activation of nitric oxide synthase
(NOS) near DA neurons in adult fly brain
Upon exposure to PQ, we found NOS expressing-structures surrounding the individual DA
cell bodies. These are postulated to be cells engulfing debris from dying neurons (Inamdar et al.,
2009, under revision; Fig. 2.6A, chapter 2). I hypothesized that exposure of conditioned medium
of S. venezuelae causes activation of NOS like in mammalian PD model, leading to death of DA
neurons, a response we have proposed to be crucial for the death of DA neurons upon PQ
exposure (Inamdar et al., 2009, under revision). I therefore exposed TH-GAL4; UAS-eGFP flies
to the conditioned medium of S. venezuelae and S. colicolor for 48 hr and immunohistochemistry
using anti-NOS and anti-GFP antibodies to detect NOS and DA neurons expression,
respectively. I found that normal DA neuron number and morphology were retained in brains
exposed to S. colicolor conditioned medium with no induction of NOS near DA neurons (Fig.
5.3 G, Fig. 5.4 A). In contrast, S. venezuelae exposed brains exhibited a gradual reduction in the
number of DA neurons in each subgroup from 48 hr until 72-96 hr exposure when complete loss
of DA neurons occurred (data not shown). Notably, unlike the close association of NOS signal,
which surrounded DA neurons in PQ-treated brain (Fig. 2.7 B, Chapter 2), the activated NOS
was never found to completely surround DA neurons upon exposure to S. venezuelae (Fig. 5.4B).
A similar response was detected in the brains exposed to crude conditioned medium of S.
venezuelae for 24 hr (data not shown). The size of the NOS-positive structures roughly ranges
between 0.6-2 μm in diameter. The difference observed in distribution of the NOS signal in PQ
vs. toxin exposed brains suggests the possibility that the toxin is inducing neuron death by a
different mechanism.
154

Figure 5.4: Exposure of crude conditioned medium of S. venezuelae induced NOS near DA
neurons in TH-GAL4; UAS-eGFP transgenic strains. TH-GAL4; UAS-eGFP flies were exposed
to conditioned media of S. colicolor and S. venezuelae for 48 hr. Exposure to S. colicolor
conditioned medium (A), failed to induce NOS, while exposure to S. venezuelae conditioned
medium caused induction of NOS detected by anti-NOS antibody (arrows, red) near GFP
expressing DA neurons detected using anti-GFP antibody. However, these NOS-positive
structures, while occurring in the approximate vicinity of DA neurons, were neither found to be
in close proximity to the neuronal cell bodies nor surrounding the DA neurons. The PPM1
subgroup of DA neuron is shown in the images A and B. Scale bar=15.87 μm.
155

A
B
S. colicolor
S. venezuelae
156

Exposure to conditioned medium of S. venezuelae induced NOS near glial cells
In Drosophila, glial cells constitute a significant volume of adult brain, where among
other functions they provide trophic support, metabolic and homeostatic functions necessary for
neuron survival. Death of glial cells has been proposed to result in death of neurons in
Drosophila (Parker and Auld, 2006). The distinctive pattern of NOS expression in toxin-exposed
brains suggested the possibility that NOS was localized on or in glia under these conditions.
Therefore, I tested the hypothesis that the toxin was causing glial death and thus indirectly
affecting neuron survival. I employed a GAL4 driver controlled by the promoter of the gene
reversed polarity (repo) (Halter et al., 1995) to direct the expression of GFP in all glial cells to
test this hypothesis. I exposed Repo-GAL4; UAS-eGFP flies to conditioned mediums of S.
colicolor and S. venezuelae for 24 hr. I found that in the presence of the control conditioned
medium of S. colicolor, glial cells, identified by GFP expression, showed mild induction of NOS
(Fig. 5.5 A, thin arrow). In contrast, the brains exposed to crude conditioned medium of S.
venezuelae showed the presence of NOS signal in the close vicinity of glial cell bodies or even
surrounding the glial cell bodies, but I did not observe a widespread co-localization of signal
(Fig. 5.5 B and Fig.5.5 D, dotted arrow). Occasionally, however, I did observe apparent co-
localized GFP and NOS signal, which suggests either a regionally specific induction of NOS in
glia, or alternatively, the possible engulfment of glial cells by NOS-expressing structures (Fig.
5.5 D, arrows). In either case, the positioning of the NOS signal and the occasional co-
localization of signal support the hypothesis that toxin-induced neuronal death is a by-product of
damaged glia.
157

S. colicolor
S. venezuelae
Figure 5.5: Exposure of S. venezuelae conditioned medium causes expression of NOS around
glial cells in adult brain. Repo-GAL4;UAS-eGFP flies were exposed to conditioned mediums of
S. colicolor (A) and S. venezuelae (B) for 24 hr. (A) There was minimal expression of NOS in
the presence of crude conditioned medium of S. colicolor. However, there was a increased
expression of NOS signal in brains exposed to conditioned medium of S. venezuelae (B). Arrows
(thin) in A,B indicate NOS expression. (C, D) Enlarged images of insets for A,B. Arrows
indicate co-localization (yellow) for NOS-signal (red) and glial cells (green) suggesting that
NOS may be associated with induced by glial cells or affecting glia. The dotted arrow shows the
expression of NOS around glial cell(s). Scale bar= 31.75 μm.
B
158

DISCUSSION
In this report, I have used our robust Drosophila model to identify the mechanism of
action of the crude preparation of secondary metabolites from Streptomyces venezuelae on
dopaminergic neurons in Drosophila. S. venezuelae belongs to a class of gram-positive bacteria,
Actinomycetes, well known to play a crucial role in the degradation of organic matter,
maintaining the ecosystem by forming an important soil microbial community (Kennedy, 1999;
Buckley and Schmidt, 2003). S. venezuelae is also an important species in the phylum
Actinobacteria, known to produce many bioactive molecules, many of which are currently used
as antibiotics (Bradley and Ritzi, 1968). These are chloramphenicol (He, 2001), methymycin and
neomethymycin (Donin et al., 1954; Perlman and O'Brien, 1954). Although many of the soil-
residing bacteria are commercially important for their ability to produce antibiotics, recently
some subspecies of Actinobacteria, such as Nocardia and even Streptomyces, were found to
produce neurotoxic secondary metabolites (Barry and Beaman, 2007; Tam et al., 2002;
unpublished findings of Caldwell Lab, UA). The Caldwell Lab recently discovered an effect of
S. venezuelae on the dopaminergic neurons of C. elegans and found that exposure to media
containing bacterial secondary metabolites from this strain of common soil bacteria caused loss
of dopaminergic neurons. Subsequently human DA culture cells, SHSY5Y were shown to be
similarly affected (The Standaert Lab, unpublished observations). However, the mechanism
through which the crude conditioned medium acts on DA neurons is still unknown.
Exposure to crude conditioned medium of S. venezuelae causes truncation of life and
Parkisonian features in wild type flies
We recently established a Drosophila model for PD that recapitulates essential features of
PD, such as tremors, bradykinesia, postural instability and loss of DA neurons (Chaudhuri et al.,
159

2007). I, therefore, used our in vivo fly model to test whether exposure to crude conditioned
medium induced Parkinsonian feature equivalent to those caused by PQ exposure by assessing
lifespan, mobility defects and loss of DA neurons. We used the S. colicolor species and 5%
sucrose-fed flies as the controls, since they are devoid of DA neurotoxicity in C. elegans and
human DA cell lines, SHSY5Y (unpublished data, Caldwell Lab and Standaert Lab). I also found
a similar lack of toxic effects of crude conditioned medium of S. colicolor in flies. I found that
exposure to the crude conditioned medium of S. venezuelae drastically reduces survival duration
relative to the crude conditioned medium of S. colicolor. The average survival of adults exposed
to S. venezuelae was only 4 days (Fig. 5.1). Moreover, the flies exposed to S. venezuelae
demonstrated tremors and bradykinesia, as assayed by a negative geotaxis assay (Chaudhuri et
al., 2007). The tremors we found in flies exposed to high (20 mM) dose of PQ were severe and
were seen within 6 hr of exposure to 20 mM PQ (Chaudhuri et al., 2007). Interestingly, the flies
exposed to the crude conditioned medium of S. venezuelae showed mild tremors and
bradykinesia only after 72-96 hr of exposure to the toxic conditioned medium when lethal effects
were also evident (Fig. 5.1, Fig. 5.2). This implies that the mechanism of bacterial-induced
neurotoxicity could be different from PQ-generated toxicity. In addition to the Parkinsonian
symptoms, we also found that upon continuous exposure of the crude conditioned medium from
S. venezuelae and S. colicolor up to 48 hr, a significant loss of DA neuron loss in PPM2, PPM3
and PPL1 was apparent. However, neuron loss in the PPM1 and PPL2 subgroups was evident
only after 48 hr of exposure to S. venezuelae conditioned medium relative to S. colicolor
conditioned medium treated and 5% sucrose-fed brains (data not shown). We used crude
conditioned medium in these experiments; it is possible that exposure to more purified extract
possessing a higher concentration of the biologically active toxic molecule may induce loss of
160

DA neuron loss much earlier. Our result and the studies performed on mice inoculated with
Nocardia asteroides strain GUH-2 show typical Parkinsonian features upon chronic exposure.
The results demonstrate parallel development of Parkinsonian-like symptoms by certain species
in the Actinobacteria phylum and suggest a possibility that these species play an etiological role
in PD (Kohbata and Beaman, 1991). Moreover, many other bacterial species are reported to
produce Parkinsonian symptoms (Wszolek et al., 1988; Kim et al., 1995).
Exposure to the crude conditioned medium of S. venezuelae induces NOS dependent response in
adult brain
The processes of neuroinflammation are recently being considered as a primary
mechanism in the etiology and pathophysiology of PD. We recently found that exposure to PQ is
associated with the induction of NOS activity and expression of NOS-positive cells in adult
Drosophila brains. The NOS-positive cells were also found to surround the DA neurons,
apparently mediating the loss of DA neurons in vivo (Inamdar et al., 2009, under revision).
Therefore, I hypothesized that like PQ, exposure to crude conditioned medium of S. venezuelae
also would mediate DA neuron loss by inducing NOS. To test this, I used universal NOS
antibody (Sigma) known to recognize conserved domains in NOS isoforms among different
species including Drosophila, human and mouse. As discussed in Chapter 2, this antibody has
been previously used to detect the role of NOS in the development of visual system during
metamorphosis, Drosophila malphigian tubules and hemocytes (Davies, 2000; Gibbs, 2001;
Foley and O’Farrell, 2003).
I found a significant level of induction of NOS in response to exposure to crude
conditioned medium of S. venezuelae in adult Drosophila brain, but almost no induction of NOS
with S. colicolor crude conditioned medium near DA neurons but never found to be surrounding
161

the DA neurons (Fig. 5.4 A, B). Although further quantification data for NOS expression is
needed to confirm the observation, based on these results it is to be speculated that various
species in the Actinomyces genus mediate DA toxicity with different mechanism(s).
Exposure to crude conditioned medium of S. venezuelae induces NOS near glial cells in adult
brain
Upon exposure to PQ, NOS-signal was found surrounding the DA neurons and seem to
be correlated with neuronal death (Inamdar et al., 2009, under revision). However, we never
found similar patterns around DA neurons during exposure to crude conditioned medium of S.
venezuelae (Fig. 5.4 B). Like mammalian brain, Drosophila brain contains a large population of
glial cells that play important roles in axonal guidance, ensheathment and neuronal trophic
support (Booth et al., 2000; Hidalgo and Booth, 2000). It has been proposed that in adult
Drosophila brain, glial cells generate different bio- active molecules to support neuronal growth
and development (Freeman and Doherty, 2006). We found that the NOS signal induced by the
crude conditioned medium of S. venezuelae for 24 hr surrounded glial cells and some instances
of co-localized signal suggests the possibility that NOS-expressing cells are engulfing glia or that
a regionally-specific subset of glia are producing NOS (Fig. 5.5 B, D). Even though NOS
expression was found occasionally in brains exposed to S. colicolor crude conditioned medium, I
never observed co-localization of signal (Fig. 5.5 A, C). In mammalian in vitro studies, survival
of transplanted DA neurons has been shown to be significantly improved by glial subtype,
oligodendrocyte-type 2 astrocyte-derived trophic factors (Sortwell et al., 2000). Although
reactive gliosis has been reported in PD induced via different neurotoxic agents including PQ,
the mechanism through which S. venezuelae induces PD features seems to be different and
possibly may occur indirectly via degeneration of glia. However, further studies will be required
162

to directly address the glial link to neurodegeneration in this case. Finally, it is possible that
altered response may be seen against the purified extract/component from crude conditioned
medium of S. venezuelae. Nevertheless, so far these results present preliminary data implying the
crucial role of NOS in the pathogenesis of S. venezuelae crude conditioned medium- induced DA
neuron loss.
163

CHAPTER 6
SUMMARY, FUTURE DIRECTIONS AND APPLICATIONS
Drosophila and neuroinflammatory-like response
Neuroinflammation is considered a crucial component of chronic neurodegenerative
diseases including PD. Neuroinflammation is characterized by activation of immune cells in
mammals where microglia act as a primary line of defense against non-self and pathological
invaders. Microglia are bone marrow-derived macrophage-lineage cells and constitute about
12% of the total number of cells in the human brain. These cells appear in small numbers in the
resting state, in certain regions of the brain (Sugam et al., 2003; Zhang et al., 2005). Under
potentially pathological conditions such as injury and infection, these cells alter their
morphology and number, and express myriad inflammatory cytokines/chemokines and other
enzymes to combat such pathological conditions. Moreover, neuronal and non-neuronal cell
populations are proposed to induce inflammatory mediators and further interact with each other
during the process of neuroinflammation (Mosley et al., 2006; Block et al., 2007).
Parkinson’s disease, the second most common chronic neurodegenerative disease, has
been demonstrated to be associated with altered microglial morphology and number along with
induction of cytokines/chemokines such as IL-1, IL-6, TNF-α and enzymes COX 1/2, NOS in
mammalian model and PD patients (Mogi et al., 1994; Blum-Dgen et al., 1995; Knott et al.,
2000). It is still debated whether the neuroinflammation/activation of microglia is a beneficial or
detrimental process in PD (Wyss-Coray and Mucke, 2002; McGeer and McGeer, 2004). The
current notion is that in acute pathological conditions, the mild to moderate activation of
164

microglia play a crucial role in maintaining homeostasis in CNS by scavenging toxins, removing
dying cells and debris, and thereby, promoting wound healing. However, in chronic pathological
conditions, especially in chronic neurodegenerative diseases including PD, the activated
microglia display elevated levels of response ultimately causing damage to viable host tissue in
addition to dead one. In several mammalian PD models, pharmacological and genetic
suppression of the inflammatory mediators known to be expressed by activated microglia are
reported to delay the DA neuron loss in different mammalian models suggesting that induction of
the neuroinflammatory process is detrimental in pathogenesis of PD (Wu et al., 2002; Esposito et
al., 2007; Vafeiadou et al., 2007). However, it appears that sustained activation of microglia in
PD is a downstream event. The factors or stimuli that trigger the events leading to alteration of
the beneficial roles of microglia to detrimental responses are not well understood. Moreover, it is
unknown whether the genes known to play a crucial role in the pathogenesis of PD also show
modulatory roles in the PD-associated neuroinflammatory process. Furthermore, the key signal
transduction pathways controlling the different aspects of microglia activation such as alteration
in morphology, proliferation, up-regulation of inflammatory mediators, phagocytosis etc. are not
yet identified. Finally, the characterization of the overall impact of any triggering event for
microglial activation in the development and survival of an organism is difficult in mammalian
models. Therefore, a simple genetic model for this response would be of great benefit for
analysis of the role of neuroinflammation in neurodegenerative disease.
My research project has laid the foundation for future projects for the exploration of a
previously unknown process in the Drosophila Parkinson’s disease model that seems to parallel
in some respects, mammalian neuroinflammation. My research has pioneered crucial preliminary
data supporting the presence of a PQ-induced NOS-dependent response in the adult Drosophila
165

ain. As discussed above, we recently established a PQ-induced PD model based on ingestion of
PQ in Drosophila (Chaudhuri et al., 2007). PQ has been proposed to act as a potent redox cycler
in the presence of diaphorases, electron donors, to generate superoxide radicals, which further
give rise to other reactive oxygen and nitrogen species (Dinis-Oliveira et al., 2006). NOS also is
known to function as a diaphorase to mediate PQ toxicity in vitro (Day et al., 1999); however,
the function of NOS at the whole organism level in PQ-mediated neurotoxicity has never been
identified in Drosophila. Drosophila possesses only one NOS gene (Regulski and Tully, 1995).
Since mammalian studies have identified NOS as a well-known marker for microglial activation
and as a mediator of potentially deleterious activity in PD pathogenesis, I hypothesized that NOS
also plays an important toxic effect in the PQ-induced Drosophila PD model. I found that PQ-
induced toxicity was at least partly mediated via generation of nitric oxide as L-NAME, an
inhibitor of NOS, was able to improve survival during PQ exposure. I further found that PQ
ingestion activates NOS in adult Drosophila brain. Interestingly, in control (non-treated) adult
brain, NOS signal is mainly confined to the junction of the central complex and optic lobe.
However, in PQ-treated brain, this NOS signal was found to be closely associated with the DA
neurons in the central complex in many instances. Further, we found that NOS signal was
surrounding the DA neurons and the presence of such NOS signal appeared to correlate with
neuronal death as such neurons were almost uniformly abnormal in morphology. We further
determined that NOS was not expressed in glial cells, taking advantage of reports expressed only
in glial cells. I also used minocycline, a well known drug reported to suppress the induction of
inducible NOS in mammalian models to further characterize the toxic role of NOS in PQ-
mediated toxicity in Drosophila. Moreover, minocycline was found to mediate both anti-oxidant
and anti-inflammatory properties against PQ in Drosophila. Although these results are
166

promising, further experiments are required to confirm the NOS expression in Drosophila CNS.
NO has been reported to play a important role in several biological processes including
development of the nervous system (Truman et al., 1996; Wildemann and Bicker, 1999),
synaptogenesis (Enikolopov et al., 1999) and immunity (Nappi et al., 2000). However, detection
of NOS response in the Drosophila PD model, which is seemingly analogous to the mammalian
microglial response in PD pathogenesis, has opened new avenues to explore the previously
unknown NOS mediated, presumably neuroinflammatory response, in an in vivo Drosophila
model of neurodegeneration. However, confirmation of these results by employing other NOS
detection methods such as NADPH diaphorase histochemical assay is needed. Furthermore,
increased NOS signal close to DA neurons needs quantification data to determine whether PQ
also causes increase in the number of NOS positive structures, since I was unable to determine
whether these structures were comprised of individual cells. The most likely candidate for this
activity is the Drosophila innate immune cells, the hemocytes (see below), which are known to
express NOS in response to bacterial infection and which frequently form cellular aggregates in
such responses (Foley and O’Farrell, 2003). Further studies are in progress and will set the stage
for future studies to further explore the role of NOS in our Drosophila PD model. The
preliminary result showing that glial cells lack the expression of NOS by performing
immunolocalization studies to detect the co-localization signal for glial and NOS signal has also
provided the directions for identifying the source of activated NOS in response to PQ.
Like mammals, Drosophila possesses a robust host defense system against
microorganisms. The anti-microbial peptides are present in the epidermis of the digestive system,
genital tract, tracheal and malphigian tubules. These peptides inhibit microbial growth
(Ferrandon et al., 1998; Tzou et al., 2000; Onfelt Tingvall et al., 2001). Microbes that escape the
167

ody wall-mediated first line of defense are subject to further innate immune responses after
entering into the general body cavity or hemocoele. In general, Drosophila exhibits two types of
immune responses: a humoral response involving production of antimicrobial peptides in the fat
body (equivalent to the mammalian liver), and cellular immunity dependent on phagocytosis by a
macrophage-like class of hemocytes (Drosophila blood cells), the plasmatocytes, and
encapsulation by crystal cells and/or lamellocytes, the remaining two classes of hemocytes
(Rizki, 1962; Hoffmann and Reichhart, 2002; Kim and Kim, 2005; Lemaitre and Hoffmann,
2007). Drosophila cellular immune response has been studied in detail at embryonic and larval
stages. Three classes of hemocytes corresponding to mammalian hematopoietic lineage cells
including microglia also arise from mesoderm and mediate cellular immune response in
Drosophila. The three cell types, the plasmatocytes, lamellocytes and crystal cells, are distinct in
morphology and function (Evans et al., 2003; Jung et al., 2005). Plasmatocytes comprise 90-95%
of all mature hemocytes and appear as small rounded cells with alteration to size in response to
phagocytosis. The developmental and functional similarity of plasmatocytes, known to represent
the only class of hemocytes in adult Drosophila, could be hypothesized to act like mammalian
microglia in response to chronic neurodegenerative diseases including PD. This hypothesis could
be tested by employing transgenic lines generated using the promoter regions for the genes
associated with hemocytes, hemese, collagen and hemolectin (Yasothornsrikul et al., 1997; Goto
et al., 2002; Kurucz et al., 2002; Asha et al., 2003; Zettervall et al., 2004); these experiments
have been initiated. Furthermore, detection of plasmatocytes could be confirmed by using P1
antibody (Asha et al., 2003). We hope to identify the source of NOS induction in response to PQ
so that mentioned unanswered questions discussed above from the mammalian studies could be
further determined in our in vivo Drosophila model.
168

In addition, we can further explore with this model the role of other inflammatory
mediators such as TNF and cytokines and COX-1/2 enzyme. Eiger, upd-3 and dhf, and pxt are
the homologs for TNF, cytokine and COX-1/2, respectively, known to present in Drosophila
(Igaki et al., 2002; Malagoli et al., 2008; Tootle and Spalding, 2008). It would be interesting to
know the response of these genes to PQ by using loss-of-function mutants and transgenic lines
available at Bloomington Stock center.
Having found that PQ ingestion induces activation of NOS, one of the important
inflammatory mediators for inflammatory response, I further performed studies showing the
implementation of this model to understand the NOS-associated disease mechanisms in our in
vivo disease model. In C. elegans and SHSY5Y cell lines, loss of DA neurons by medium
containing secondary metabolites (or crude conditioned medium) of S. venezuelae has been
detected (The Caldwell lab, UA, and The Standaert Lab, UAB). I further confirmed the observed
neurotoxic effects of crude conditioned medium of S. venezuelae in Drosophila. The ingestion of
crude conditioned medium of S. venezuelae to flies caused truncation of life and mobility defects
seen with PQ ingestion. However, the observed death trend with S. venezuelae crude conditioned
medium on wild type flies was different from those on PQ. The S. venezuelae crude conditioned
medium exposed flies showed a gradual decline in survival with a sudden peak in the death rate
at later half of total survival duration whereas in PQ exposed flies, the rate the wild type flies
died was almost the same at each time point (Fig. 5.1). This suggests that upon exposure to
conditioned medium of S. venezuelae, an accumulation of toxic substances occur, under the
influence of which flies succumb in a delayed response.
Interestingly, the flies exposed to the crude conditioned medium of S. venezuelae showed
mild tremors and bradykinesia only in the latter half of the total survival duration when lethal
169

effects were also evident. This result further indicates that the mechanism of bacterial-induced
neurotoxicity could be different from PQ-generated toxicity. In addition to the Parkinsonian
symptoms, we also found that continuous exposure of the crude conditioned medium from S.
venezuelae caused loss of DA neurons, but this loss was not as rapid as that observed during
exposure to 10 mM PQ. However, it is to be noted that I used crude conditioned medium in these
experiments; it is possible that exposure to more purified extract possessing a higher
concentration of the biologically active toxic molecule may induce loss of DA neuron much
earlier. Studies to determine the active toxic molecule responsible for the neurotoxic effects by S.
venezuelae crude conditioned medium are in progress.
I further explored the ability of such media to induce a NOS-dependent response in adult
fly brain. My preliminary results suggest that, as in PQ treatment, S. venezuelae secreted
secondary metabolites mediate DA neuron loss partly by causing induction of NOS. The NOS
signal was, however, not found closely associated with DA neuron cell bodies. Interestingly, the
positioning of the NOS signal and the occasional co-localization of signal with the glial cells, the
cells required for the maintaining trophic support and homeostasis for neurons including DA
neurons supports the hypothesis that toxin-induced neuronal death is a by-product of damaged
glia. Although further studies are required to directly link the DA neurodegeneration and glia in
response to crude conditioned medium of S. venezuelae by performing quantification data for
glial and the NOS positive structures, my results clearly indicate that further exploration of
disease mechanisms of S. venezuelae crude conditioned medium associated DA toxicity in in
vivo Drosophila model will be a profitable avenue for further study. These results are of
particular interest because, unlike the degenerative process in C. elegans which does not have
comparable glial populations or in DA neurons cultured in the absence of glia, they suggest a
170

potential involvement of non-neuronal cells in the CNS and thus add a new dimension to this
research.
Drosophila and signal transduction pathway in PD
Signal transduction pathways mediate myriad downstream effects such as the regulation
of gene transcription, the cell cycle, cellular differentiation and cell death (Nishida and Gotoh,
1993; Chang and Karin, 2001). More recently, the roles of MAPK and Akt mediated signal
transduction pathways have been elucidated in mammalian PD model. The results are somewhat
controversial regarding whether these roles of MAPK are neuroprotective or neurotoxic in the
pathogenesis of PD (Xia et al., 1995; Harper and LoGrasso, 2001; Peng et al., 2004; Niso-
Santano et al., 2006; Burke, 2007). Moreover, these models have mainly employed in vitro (cell-
culture) approach where the experimental conditions employed to investigate the roles of these
signaling pathways, in particular pathogenic responses, are generally manipulated. Further, in
vitro culture conditions lack appropriate cell-cell interactions from neighboring cells which could
modulate the response to external stimuli. Moreover, many of the commercially available
pharmacological inhibitors of kinases employed in these experiments are not well-defined with
respect to their exact modes of action and may give rise to non-specific effects due to blockage
of unknown target molecules (Sekiguchi et al., 1999; Learish et al., 2000; Waetzig and
Herdegen, 2005).
I used heterozygous loss-of-function mutants for JNK, Akt, ERK and the pro-apoptotic
genes, reaper and caspase-9 to determine the role of these important genes in PQ toxicity in our
in vivo Drosophila PD model. As opposed to mammalian studies, we found that ERK and reaper
lack any observable regulatory roles while Akt and JNK provide protective role in our PD model.
171

These studies could be further confirmed by expressing dominant negative form of these in
dopaminergic neurons. The protective effect of Akt parallels mammalian findings, however,
interestingly indicate a protective role for JNK in this PD model, in contrast to mammalian PD
models using PQ, 6-OHDA (Gearan et al., 2001; Ganguly et al., 2004; Peng et al., 2004). I also
present data showing the failure of herterozygous Akt1 and JNK loss-of-function mutants to
respond to minocycline action, while Akt1 and JNK wild type over-expression in DA neurons
fails to provide additional benefit, suggesting that JNK and Akt 1 may operate in separate
pathway(s) and act independently from those of minocycline. Generating the double mutants for
Akt, JNK together and with caspase-9 may provide further insight into the regulatory effects of
these pathways in PQ toxicity. It is to be noted that in Drosophila, JNK has also been reported to
play an important role in innate immune response and longevity (Boutros et al., 2002; Wang et
al., 2003b). My results present the preliminary studies to further explore the role of JNK, Akt and
other MAPK-related signaling pathway components in the NOS-dependent response against PQ
ingestion.
Drosophila as a model to study Early Onset Parkinsonism
Recently, idiopathic form of PD has been diagnosed in the under 40 age group. Such
cases are classified as Early Onset Parkinsonian cases. I postulated that brief exposure to PQ
early in life might contribute to the pathogenesis of PD at later stage of life. We used larvae and
adult flies of less that 6 hr age to represent the juvenile and young aged population. Exposure to
10 mM and 1 mM PQ for even a brief period (12 hr) at larval and young age resulted in
truncation of life span, mobility defects and DA neuron loss in a dose dependent manner at later
stage of life. It will be important to pursue the question of whether this early event also perturbs
172

DA homeostasis in the adult brain as well as other metabolic processes that may lead to
increased sensitivity towards such PD inducers as well as early death. Further, taking into
account our discovery of the NOS-dependent response in PQ treated adult brains, it would be
interesting to know if a similar response is seen in larvae, and if so whether a sustained response
is seen at the later time point. As discussed above, this NOS dependent response needs other
confirmatory experiments, and these are to be completed before further exploration of the effects
of juvenile exposure to this toxin. However, my data strongly support the current findings for the
role of exposure of neurotoxins as one of the insults inflicted early in life in triggering or
accelerating the pathogenic events for PD (Liu et al., 2003; Liu and Hong, 2003; Logroscino,
2005).
173

REFERENCES
Alfonso TB, Jones BW (2002) gcm2 promotes glial cell differentiation and is required with glial
cells missing for macrophage development in Drosophila. Dev Biol 248:369-383.
Altschuler E (1996) Gastric Helicobacter pylori infection as a cause of idiopathic Parkinson
disease and non-arteric anterior optic ischemic neuropathy. Med Hypotheses 47:413-414.
Asha H, Nagy I, Kovacs G, Stetson D, Ando I, Dearolf CR (2003) Analysis of ras-induced
overproliferation in Drosophila hemocytes. Genetics 163:203-215.
Ashkenazi A (2002) Targeting death and decoy receptors of the tumour-necrosis factor
superfamily. Nat Rev Cancer 2:420-430.
Ashkenazi A, Dixit VM (1998) Death receptors: signaling and modulation. Science 281:1305-
1308.
Barry DP, Beaman BL (2007) Nocardia asteroides strain GUH-2 induces proteasome inhibition
and apoptotic death of cultured cells. Res Microbiol 158:86-96.
Batchelor PE, Liberatore GT, Wong JY, Porritt MJ, Frerichs F, Donnan GA, Howells DW
(1999) Activated macrophages and microglia induce dopaminergic sprouting in the injured
striatum and express brain derived neurotrophic factor and glial cell line-derived
neurotrophic factor. J Neurosci 19:1708-1716.
Batchelor PE, Porritt MJ, Martinello P, Parish CL, Liberatore GT, Donnan GA, Howells DW
(2002) Macrophages and microglia produce local trophic gradients that stimulate axonal
sprouting toward but not beyond the wound edge. Mol Cell Neurosci 21:436-453.
174

Beaman BL, Canfield D, Anderson J, Pate B, Calne D (2000) Site-specific invasion of the basal
ganglia by Nocardia asteroides GUH-2. Med. Microbiol Immunol 188:161-168.
Beitner-Johnson D, Nestler EJ (1991) Morphine and cocaine exert common chronic actions on
tyrosine hydroxylase in dopaminergic brain reward regions. J Neurochem 57:344-347.
Berman KF, Illowsky BP, Weinberger DR (1988) Physiological dysfunction of dorsolateral
prefrontal cortex in schizophrenia. IV. Further evidence for regional and behavioral
specificity Arch Gen Psychiatry 45:616-622.
Berridge KC, Robinson TE (1998) What is the role of dopamine in reward: hedonic impact,
reward learning, or incentive salience? Brain Res Brain Res Rev 28:309-369.
Betarbet R, Sherer TB, MacKenzie G, Garcia-Osuna M, Panov AV, Greenamyre JT (2000)
Chronic systemic pesticide exposure reproduces features of Parkinson's disease. Nat
Neurosci 3:1301-1306.
Beuge J, Aust SD (1978) Microsomal lipid peroxidation. Methods Enzymol 52:302-310.
Bewley GC, Nahmias JA, Cook JL (1983) Developmental and tissue-specific control of catalase
expression in Drosophila melanogaster: correlations with rates of enzyme synthesis and
degradation. Dev Genet 4:49-60.
Birman S, Morgan B, Anzivino M, Hirsh J (1994) A novel and major isoform of tyrosine
hydroxylase in Drosophila is generated by alternative RNA processing. J Biol Chem
269:26559-26567.
Biswas SC, Ryu E, Park C, Malagelada C, Greene LA (2005) Puma and p53 play required roles
in death evoked in a cellular model of Parkinson disease. Neurochem Res 30:839-845.
Björklund A, Dunnett SB (2007) Dopamine neuron systems in the brain: an update. Trends
Neurosci 30:194-202.
175

Blenis J (1993) Signal transduction via the MAP kinases: proceed at your own RSK. Proc Natl
Acad Sci USA 90:5889-5892.
Block ML, Zecca L, Hong JS (2007) Microglia-mediated neurotoxicity: uncovering the
molecular mechanisms. Nat Rev Neurosci 8:57-69.
Boileau I, Assaad JM, Pihl RO, Benkelfat C, Leyton M, Diksic M, Tremblay RE, Dagher A
(2003) Alcohol promotes dopamine release in the human nucleus accumbens Synapse
49:226-231.
Bonifati V, Rizzu P, van Baren MJ, Schaap O, Breedveld GJ, Krieger E, Dekker MC, Squitieri F,
Ibanez P, Joosse M, van Dongen JW, Vanacore N, van Swieten JC, Brice A, Meco G, van
Duijn CM, Oostra BA, Heutink P (2003) Mutations in the DJ-1 gene associated with
autosomal recessive early-onset parkinsonism. Science 299:256-259.
Bonilla E, Medina-Leendertz S, Villalobos V, Molero L, Bohórquez A (2006) Paraquat-induced
oxidative stress in Drosophila melanogaster: effects of melatonin, glutathione, serotonin,
minocycline, lipoic acid and ascorbic acid. Neurochem Res 31:1425-32.
Bonneh-Barkay D, Reaney SH, Langston WJ, Di Monte DA (2005) Redox cycling of the
herbicide paraquat in microglial cultures. Brain Res Mol Brain Res 134:52-56.
Booth GE, Kinrade EF, Hidalgo A (2000) Glia maintain follower neuron survival during
Drosophila CNS development. Development 127:237-244.
Borchardt RT, Reid JR, Thakker DR (1976) Catechol Omethyltransferase. Mechanism of
inactivation by 6- hydroxydopamine. J Med Chem 19:1201-1209.
Boutros M, Agaisse H, Perrimon N (2002) Sequential activation of signaling pathways during
innate immune responses in Drosophila. Dev Cell 3:711-722.
176

Bradley SG, Ritzi D (1968) Composition and ultrastructure of Streptomyces venezuelae. J
Bacteriol 95:2358-2364.
Brand AH, Perrimon N (1993) Targeted gene expression as a means of altering cell fates and
generating dominant phenotypes. Development 118:401–415.
Brazil DP, Hemmings BA (2001) Ten years of protein kinase B signalling: a hard Akt to follow.
Trends Biochem Sci 26:657-664.
Bredt DS, Hwang PM, Glatt CE, Lowenstein C, Reed RR, Snyder SH (1991) Cloned and
expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase. Nature
351:714-718.
Bredt DS, Snyder SH (1994) Nitric oxide: a physiologic messenger molecule. Annu Rev
Biochem 63:175-195.
Broderick KE, MacPherson MR, Regulski M, Tully T, Dow JA, Davies SA (2003) Interactions
between epithelial nitric oxide signaling and phosphodiesterase activity in Drosophila. Am
J Physiol Cell Physiol 285:C1207-C1218.
Brooks AI, Chadwick CA, Gelbard HA, Cory-Slechta DA, Federoff HJ (1999) Paraquat elicited
neurobehavioral syndrome caused by dopaminergic neuron loss. Brain Res 823:1-10.
Broxmeyer L (2002) Parkinson's: another look. Med Hypotheses 59:373-377.
Brunner D, Dücker K, Oellers N, Hafen E, Scholz H, Klämbt C (1994) The ETS domain protein
pointed-P2 is a target of MAP kinase in the sevenless signal transduction Nature 370:386-
389.
Buckley DH, Schmidt TM (2003) Diversity and dynamics of microbial communities in soils
from agro-ecosystems. Environ Microbiol 5:441-452.
177

Budnik V, White K (1988) Catecholamine-containing neurons in Drosophila melanogaster:
distribution and development. J Comp Neurol 268:400-413.
Burke RE (2007) Inhibition of mitogen-activated protein kinase and stimulation of Akt kinase
signaling pathways: Two approaches with therapeutic potential in the treatment of
neurodegenerative disease. Pharmacol Ther 114:261-277.
Burnett AL (1995) Role of nitric oxide in the physiology of erection. Biol Reprod 52:485-489.
Burns RS, Chiueh CC, Markey SP, Ebert MH, Jacobowitz DM, Kopin IJ (1983) A primate
model of parkinsonism: selective destruction of dopaminergic neurons in the pars compacta
of the substantia nigra by N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Proc Natl Acad
Sci USA 80:4546-4550.
Bus JS, Aust SD, Gibson JE (1974) Superoxide- and singlet oxygen-catalyzed lipid peroxidation
as a possible mechanism for paraquat (methyl viologen) toxicity. Biochem Biophys Res
Commun 58:749-755.
Cavanaugh JE (2004) Role of extracellular signal regulated kinase 5 in neuronal survival. Eur J
Biochem 271:2056-2059.
Cavanaugh JE, Jaumotte JD, Lakoski JM, Zigmond MJ (2006) Neuroprotective role of ERK1/2
and ERK5 in a dopaminergic cell line under basal conditions and in response to oxidative
stress. J Neurosci Res 84:1367-1375.
Cha GH, Kim S, Park J, Lee E, Kim M, Lee SB, Kim JM, Chung J, Cho KS (2005) Parkin
negatively regulates JNK pathway in the dopaminergic neurons of Drosophila. Proc Natl
Acad Sci USA 102:10345-10350.
Chang L, Karin M (2001) Mammalian MAP kinase signaling cascades. Nature 410:37-40.
178

Chaudhuri A, Bowling K, Funderburk C, Lawal H, Inamdar A, Wang Z, O'Donnell JM (2007)
Interaction of genetic and environmental factors in a Drosophila parkinsonism model. J
Neurosci 27:2457-2467.
Chauhan D, Li G, Hideshima T, Podar K, Mitsiades C, Mitsiades N, Munshi N, Kharbanda S,
Anderson KC (2002) JNK-dependent release of mitochondrial protein, Smac, during
apoptosis in multiple myeloma (MM) cells. J Biol Chem 278:17593-17596.
Chen H, Zhang SM, Hernán MA, Schwarzschild MA, Willett WC, Colditz GA, Speizer FE,
Ascherio A (2003) Nonsteroidal anti-inflammatory drugs and the risk of Parkinson disease.
Arch Neurol 60:1059-1064.
Chen M, Ona VO, Li M, Ferrante RJ, Fink KB, Zhu S, Bian J, Guo L, Farrell LA, Hersch SM,
Hobbs W, Vonsattel JP, Cha JH, Friedlander RM (2000) Minocycline inhibits caspase-1
and caspase-3 expression and delays mortality in a transgenic mouse model of Huntington
disease. Nat Med 6:797-801.
Chen PS, Peng GS, Li G, Yang S, Wu X, Wang CC, Wilson B, Lu RB, Gean PW, Chuang DM,
Hong JS (2006) Valproate protects dopaminergic neurons in midbrain neuron/glia cultures
by stimulating the release of neurotrophic factors from astrocytes. Mol Psychiatry 11:1116
-1125.
Chew SK, Akdemir F, Chen P, Lu WJ, Mills K, Daish T, Kumar S, Rodriguez A, Abrams JM
(2004) The apical caspase Dronc governs programmed and unprogrammed cell death in
Drosophila. Dev Cell 7:897-907.
Cho BP, Song DY, Sugama S, Shin DH, Shimizu Y, Kim SS, Kim YS, Joh TH (2006)
Pathological dynamics of activated microglia following medial forebrain bundle
transaction. Glia 53:92-102.
179

Chu CT, Levinthal DJ, Kulich SM, Chalovich EM, DeFranco DB (2004) Oxidative neuronal
injury. The dark side of ERK1/2. Eur J Biochem 271:2060-2066.
Ciccetti F, Brownell AL, Williams K, Chen YI, Livni E, Isacson O (2002) Neuroinflammation of
the nigrostriatal pathway during progressive 6-OHDA dopamine degeneration in rats
monitored by immunohistochemistry and PET imaging. Eur J Neurosci 15:991-998.
Cleeter MW, Cooper JM, and Schapira AH (1992) Irreversible inhibition of mitochondrial
complex I by 1-methyl-4- phenylpyridinium: evidence for free radical involvement. J
Neurochem 58:786-789.
Cole SH, Carney GE, McClung CA, Willard SS, Taylor BJ, Hirsh J (2005) Two functional but
noncomplementing Drosophila tyrosine decarboxylase genes: distinct roles for neural
tyramine and octopamine in female fertility. J Biol Chem 280:14948-14955.
Cooper RL, Neckameyer WS (1999) Dopaminergic modulation of motor neuron activity and
neuromuscular function in Drosophila melanogaster. Comp Biochem Physiol B Biochem
Mol Biol 122:199-210.
Crane BR, Rosenfeld, RJ, Arvai AS, Ghosh DK, Ghosh S, Tainer JA, Stuehr DJ, Getzoff ED
(1999) N-terminal domain swapping and metal ion binding in nitric oxide synthase
dimerization. EMBO J 18:6271-6281.
Czlonkowska A, Kohuknika M, Kurkowska-Jatrzebska I, Czlonkowski A (1996) Microglial
reaction in MPTP (1-methyl-4phenyl-1,2,3, 6-tetrahydropyridine) induced Parkinson’s
disease mice model. Neurodegeneration 5:137-143.
Das C, Hoang QQ, Kreinbring CA, Luchansky SJ, Meray RK, Ray SS, Lansbury PT, Ringe D,
Petsko GA (1998) Structural basis for conformational plasticity of the Parkinson's disease-
associated ubiquitin hydrolase UCH-L1. Proc Natl Acad Sci USA 103:4675-4680.
180

Dauer W, Przedborski S (2003) Parkinson's disease: mechanisms and models. Neuron 39:889-
909.
Davies S (2000) Nitric oxide signaling in insects. Insect Biochem Mol Biol 30:1123-1138.
Davis RJ (2000) Signal transduction by the JNK group of MAP kinases. Cell 103:239-252.
Day BJ, Patel M, Calavetta L, Chang LY, and Stamler JS (1999) A mechanism of paraquat
toxicity involving nitric oxide synthase. Proc Natl Acad Sci USA 96:12760-12765.
Dehmer T, Lindenau J, Haid S, Dichgans J, Schulz JB (2000) Deficiency of inducible nitric
oxide synthase protects against MPTP toxicity in vivo. J Neurochem 74:2213-2216.
Depino AM, Earl C, Kaczmarczyk E, Ferrari C, Besedovsky H, del Rey A, et al. (2003)
Microglial activation with atypical proinflammatory cytokine expression in a rat model of
Parkinson's disease. Eur J Neurosci 18:2731-2742.
Di Matteo V, Pierucci M, Di Giovanni G, Di Santo A, Poggi A, Benigno A, Esposito E (2006)
Aspirin protects striatal dopaminergic neurons from neurotoxin-induced degeneration: an in
vivo microdialysis study. Brain Res 1095:167-177.
Díaz-Corrales FJ, Colasante C, Contreras Q, Puig M, Serrano JA, Hernández L, Beaman BL.
(2004) Nocardia otitidiscaviarum (GAM-5) induces parkinsonian-like alterations in mouse.
Braz J Med Biol Res 37:539-548.
Dicker E, Cederbaum AI (1991) NADH-dependent generation of reactive oxygen species by
microsomes in the presence of iron and redox cycling agents. Biochem Pharmacol 42:529-
535.
DiGregorio PJ, Ubersax JA, O'Farrell PH (2001) Hypoxia and nitric oxide induce a rapid,
reversible cell cycle arrest of the Drosophila syncytial divisions. J Biol Chem 276:1930-
1937.
181

Diguet E, Fernagut PO, Wei X, Du Y, Rouland R, Gross C, Bezard E, Tison F (2004)
Deleterious effects of minocycline in animal models of Parkinson's disease and
Huntington's disease. Eur J Neurosci 19:3266-3276.
Dinis-Oliveira RJ, Remião F, Carmo H, Duarte JA, Navarro AS, Bastos ML, Carvalho F (2006)
Paraquat exposure as an etiological factor of Parkinson's disease. Neurotoxicology
27:1110-1122.
Djukic M, Jovanovic MC, Ninkovic M, Vasiljevic I, Jovanovic M (2008) The role of nitric oxide
in paraquat-induced oxidative stress in rat striatum. Ann Agric Environ Med 14:247-252.
Donin MN, Pagano J, Dutcher JD, McKee CM (1954) Methymycin, a new crystalline antibiotic.
Antobiot Annu 1:179-185.
Dow JA, Maddrell SH, Davies SA, Skaer NJ, Kaiser K (1994) A novel role for the nitric oxide-
cGMP signaling pathway: the control of epithelial function in Drosophila. Am J Physiol.
266:R1716-R1719.
Downer RG, Martin RJ (1987) Analysis of monoamines and their metabolites by high
performance liquid chromatography with coulometric electrochemical detection. Life Sci
41:833-836.
Du Y, Ma Z, Lin S, Dodel RC, Gao F, Bales KR, Triarhou LC, Chernet E, Perry KW, Nelson
DL, Luecke S, Phebus LA, Bymaster FP, Paul SM (2001) Minocycline prevents
nigrostriatal dopaminergic neurodegeneration in the MPTP model of Parkinson's disease.
Proc Natl Acad Sci USA 98:14669-14674.
Dudek H, Datta SR, Franke TF, Birnbaum MJ, Yao R, Cooper GM, Segal RA, Kaplan DR,
Greenberg ME (1997) Regulation of neuronal survival by the serine-threonine protein
kinase Akt. Science 275:661-665.
182

Dunnett SB, Björklund A, Stenevi U, Iversen SD (1981) Behavioural recovery following
transplantation of substantia nigra in rats subjected to 6-OHDA lesions of the nigrostriatal
pathway. I. Unilateral lesions. Brain Res 215:147-61.
Duvoisin RC, Yahr MD (1965). Encephalitis and parkinsonism. Archives of Neurology 12:227-
239.
Enikolopov G, Banerji J, Kuzin B (1999) Nitric oxide and Drosophila development. Cell Death
Differ 6:956-963.
Esposito E, Di Matteo V, Benigno A, Pierucci M, Crescimanno G, Di Giovanni G (2007) Non-
steroidal anti-inflammatory drugs in Parkinson's disease. Exp Neurol 205:295-312.
Evans CJ, Hartenstein V, Banerjee U (2003) Thicker than blood: conserved mechanisms in
Drosophila and vertebrate hematopoiesis. Dev Cell 5:673-690.
Fazzini E, Fleming J & Fahn S (1992) Cerebrospinal fluid antibodies to coronavirus in patients
with Parkinson’s disease. Movement Disorders 7:153-158.
Fei Q, McCormack AL, Di Monte DA, Ethell DW (2008) Paraquat neurotoxicity is mediated by
a Bak-dependent mechanism. J Biol Chem 283:3357-3364.
Ferrandon D, Jung AC, Criqui M, Lemaitre B, Uttenweiler-Joseph S, Michaut L, Reichhart J,
Hoffmann JA (1998) A drosomycin-GFP reporter transgene reveals a local immune
response in Drosophila that is not dependent on the Toll pathway. EMBO J 17:1217-1227.
Fisher AB, Reicherter J (1984) Pentose pathway of glucose metabolism in isolated granular
pneumocytes. Metabolic regulation and stimulation by paraquat. Biochem Pharmacol
33:1349-1353.
Floyd RA (1999) Antioxidants, oxidative stress, and degenerative neurological disorders. Proc
Soc Exp Biol Med 222:236-245.
183

Foley E, O'Farrell PH (2003) Nitric oxide contributes to induction of innate immune responses to
gram-negative bacteria in Drosophila. Genes Dev 17:115-125.
Foxton RH, Land JM, Heales SJ (2007) Tetrahydrobiopterin availability in Parkinson's and
Alzheimer's disease; potential pathogenic mechanisms. Neurochem Res 32:751-756.
Franke TF, Tartof KD, Tsichlis PN (1994) The SH2-like Akt homology (AH) domain of c-akt is
present in multiple copies in the genome of vertebrate and invertebrate eukaryotes. Cloning
and characterization of the Drosophila melanogaster c-akt homolog Dakt1. Oncogene
9:141-148.
Freeman MR, Doherty J (2006) Glial cell biology in Drosophila and vertebrates. Trends
Neurosci 29:82-90.
Friggi-Grelin F, Coulom H, Meller M, Gomez D, Hirsh J, Birman S (2003) Targeted gene
expression in Drosophila dopaminergic cells using regulatory sequences from tyrosine
hydroxylase. J Neurobiol 54:618-627.
Ganguly A, Oo TF, Rzhetskaya M, Pratt R, Yarygina O, Momoi T, Kholodilov N, Burke RE
(2004) CEP11004, a novel inhibitor of the mixed lineage kinases, suppresses apoptotic
death in dopamine neurons of the substantia nigra induced by 6-hydroxydopamine. J
Neurochem 88:469-480.
Gao HM, Hong JS, ZhangW, Liu B (2002) Distinct role for microglia in rotenone-induced
degeneration of dopaminergic neurons. J Neurosci 22:782-790.
Gearan T, Castillo OA, Schwarzschild MA (2001) The parkinsonian neurotoxin, MPP+ induces
phosphorylated c-Jun in dopaminergric neurons of mesencephalic cultures. Parkinsonism
Relat Disord 8:19-22.
184

Gibbs SM (2001) Regulation of Drosophila Visual System Development by Nitric Oxide and
cyclic GMP. Amer Zool 41:268-281.
Gibbs SM, Truman JW (1999) Nitric oxide and cyclic GMP regulate retinal patterning in the
optic lobe of Drosophila. Neuron 20:83-93.
Giesen K, Hummel T, Stollewerk A, Harrison S, Travers A, Klämbt C (1997) Glial development
in the Drosophila CNS requires concomitant activation of glial and repression of neuronal
differentiation genes. Development 124:2307-2316.
Glise B, Bourbon H, Noselli S (1995) hemipterous encodes a novel Drosophila MAP kinase
kinase, required for epithelial cell sheet movement. Cell 83:451-461.
Goto A, Kumagai T, Kumagai C, Hirose J, Narita H, Mori H, Kadowaki T, Beck K, Kitagawa Y
(2002) A Drosophila hemocyte-specific protein, hemolectin, similar to human von
Willebrand factor. Biochem J 359:99-108.
Gotoh Y, Nishida E (1995) Activation mechanism and function of the MAP kinase cascade. Mol
Reprod Dev 42:486-492.
Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR (1982)
Analysis of nitrate, nitrite, and [15N] nitrate in biological fluids. Anal Biochem 126:131-
138.
Greer CL, Grygoruk A, Patton DE, Ley B, Romero-Calderon R, Chang HY, Houshyar R,
Bainton RJ, Diantonio A, Krantz DE (2005) A splice variant of the Drosophila vesicular
monoamine transporter contains a conserved trafficking domain and functions in the
storage of dopamine, serotonin, and octopamine. J Neurobiol 64:239-258.
Grima B, Lamouroux A, Blanot F, Biguet NF, Mallet J (1985) Complete coding sequence of rat
tyrosine hydroxylase mRNA. Proc Natl Acad Sci USA 82:617-621.
185

Gunnet JW, Lookingland KJ, Moore KE (1986) Effects of gonadal steroids on
tuberoinfundibular and tuberohypophysial dopaminergic neuronal activity in male and
female rats. Proc Soc Exp Biol Med 183:48-53.
Halliwell B (2001) Role of free radicals in the neurodegenerative diseases: therapeutic
implications for antioxidant treatment. Drugs Aging 18:685-716.
Halter DA, Urban J, Rickert C, Ner SS, Ito K, Travers AA, Technau GM (1995) The homeobox
gene repo is required for the differentiation and maintenance of glia function in the
embryonic nervous system of Drosophila melanogaster. Development 121:317-332.
Harper SJ, LoGrasso P (2001) Signalling for survival and death in neurones: the role of stress-
activated kinases, JNK and p38. Cell Signal 13:299-310.
Hartmann A, Troadec JD, Hunot S, Kikly K, Faucheux BA, Mouatt-Prigent A, Ruberg M, Agid
Y, Hirsch EC (2001) Caspase-8 Is an effector in apoptotic death of dopaminergic neurons
in Parkinson’s disease, but pathway inhibition results in neuronal necrosis. J Neurosci
21:2247-2255.
Hashimoto M, Takenouchi T, Rockenstein E, Masliah E (2003) Alpha-synuclein up-regulates
expression of caveolin-1 and down-regulates extracellular signal-regulated kinase activity
in B103 neuroblastoma cells: role in the pathogenesis of Parkinson’s disease. J Neurochem
85:1468-1479.
He J, Magarvey N, Piraee M, Vining LC (2001) The gene cluster for chloramphenicol
biosynthesis in Streptomyces venezuelae ISP5230 includes novel shikimate pathway
homologues and a monomodular non-ribosomal peptide synthetase gene. Microbiology
147:2817-2829.
186

Hearn MG, Ren Y, McBride EW, Reveillaud I, Beinborn M, Kopin AS (2002) A Drosophila
dopamine 2-like receptor: Molecular characterization and identification of multiple
alternatively spliced variants. Proc Natl Acad Sci USA 99:14554-14559.
Heikkila RE, Hess A, and Duvoisin RC (1984) Dopaminergic neurotoxicity of 1-methyl-4-
phenyl-1,2,3,6-tetrahydropyridine in mice. Science 224:1451-1453.
Hersch S, Fink K, Vonsattel JP, Friedlander RM (2003) Minocycline is protective in a mouse
model of Huntington's disease. Ann Neurol 54:841.
Hetman M, Gozdz A (2004) Role of extracellular signal regulated kinases 1 and 2 in neuronal
survival. Eur J Biochem 271:2050-2055.
Hidalgo, A. and Booth, G.E. (2000) Glia dictate pioneer axon trajectories in the Drosophila
embryonic CNS. Development 127:393-402.
Hirsh EC, Hunot S, Damier P, Faucheux B (1998). Glial cells and inflammation in Parkinson’s
disease: a role in neurodegeneration? Ann Neurol 44:S115–S120
Hoffmann JA, Reichhart JM (2002) Drosophila innate immunity: an evolutionary perspective.
Nat Immunol 3:121-126.
Hölscher C (1997) Nitric oxide, the enigmatic neuronal messenger: its role in synaptic plasticity.
Trends Neurosci. 20:298-303.
Holz A, Bossinger B, Strasser T, Janning W, Klapper R (2003) The two origins of hemocytes in
Drosophila. Development 130:4955-4962.
Hornykiewicz O, Kish SJ (1987) Biochemical pathophysiology of Parkinson's disease. Adv
Neurol 45:19-34.
Hosoya T, Takizawa K, Nitta K, Hotta Y (1995) glial cells missing: a binary switch between
neuronal and glial determination in Drosophila. Cell 82:1025-1036.
187

Hunot S, Boissiere F, Faucheux B, Brugg B, Mouatt-Prigent A, Agid Y (1996) Nitric oxide
synthase and neuronal vulnerability in Parkinson’s disease. Neuroscience 72:355-363.
Hunot S, Dugas N, Faucheux B, Hartmann A, Tardieu M, Debré P, Agid Y, Dugas B, Hirsch EC
(1999) FcepsilonRII/CD23 is expressed in Parkinson's disease and induces, in vitro,
production of nitric oxide and tumor necrosis factor-alpha in glial cells. J Neurosci
19:3440-3447.
Hunot S, Hirsch EC (2003) Neuroinflammatory processes in Parkinson's disease. Ann Neurol. 53
:S49-S58, discussion S58-S60.
Igaki T, Kanda H, Yamamoto-Goto Y, Kanuka H, Kuranaga E, Aigaki T, Miura M (2002) Eiger,
a TNF superfamily ligand that triggers the Drosophila JNK pathway. EMBO J 21:3009-
3018.
Ilett KF, Stripp B, Menard RH, Reid WD, Gillette JR (1974) Studies on the mechanism of the
lung toxicity of paraquat: comparison of tissue distribution and some biochemical
parameters in rats and rabbits. Toxicol Appl Pharmacol 28:216-226.
Inamdar A, Lawal H, Ferdousy F, Chaudhuri A, O’ Donnell (2009) Drosophila mounts a
microglial-like neuroinflammatory response to paraquat-induced degeneration of
dopaminergic neurons (under revision for Nature Neuroscience).
Iravani MM, Kashefi K, Mander P, Rose S, Jenner P (2002) Involvement of inducible nitric
oxide synthase in inflammation-induced dopaminergic neurodegeneration. Neuroscience
110:49–58.
Ito K, Urban J, Technau, GM (1995) Distribution, classification and development of Drosophila
glial cells in the late embryonic and early larval ventral nerve cord. Roux’s Arch Develop
Biol 209:289–307.
188

Iversen SD, Iversen LL (2007) Dopamine: 50 years in perspective. Trends Neurosci 30:188-193.
Javitch JA, D’Amato RJ, Strittmatter SM, and Snyder SH (1985) Parkinsonism-inducing
neurotoxin, N-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine: uptake of the metabolite N-
methyl- 4-phenylpyridinium by dopamine neurons explain selective toxicity. Proc Natl
Acad Sci USA 82:2173-2177.
Jellinger KA (2001) The pathology of Parkinson’s disease. Adv Neurol 86:55-72.
Jian XD, Sui H, Chu ZH, Zhang ZW, Kan BT, Zhang L, Zhang HT (2007) Changes of serum
cytokine caused by acute paraquat poisoning. Zhonghua Lao Dong Wei Sheng Zhi Ye Bing
Za Zhi 25:230-232.
Johnson GL, Lapadat R (2002) Mitogen-Activated Protein Kinase Pathways Mediated by ERK,
JNK, and p38 Protein Kinases. Science 298:1911-1912.
Jordan J, Fernandez-Gomez FJ, Ramos M, Ikuta I, Aguirre N, Galindo MF (2007) Minocycline
and cytoprotection: shedding new light on a shadowy controversy. Curr Drug Deliv 4:225-
231.
Jung SH, Evans CJ, Uemura C, Banerjee U (2005) The Drosophila lymph gland as a
developmental model of hematopoiesis. Development 132:2521-2533.
Kamel F, Tanner C, Umbach D, Hoppin J, Alavanja M, Blair A, Comyns K, Goldman S, Korell
M, Langston J, Ross G, Sandler D. (2007) Pesticide Exposure and Self-reported
Parkinson's Disease in the Agricultural Health Study. Am J Epidemiol 165:364-374.
Kandel ES, Hay N (1999) The regulation and activities of the multifunctional serine/threonine
kinase Akt/PKB. Exp Cell Res 253:210-229.
Kaur C, Hao AJ, Wu CH, Ling EA (2001) Origin of microglia. Microsc Res Tech 54:2-9.
Kennedy AC (1999) Bacterial diversity in agroecosystems. Agric Ecosyst Environ 74:65-76.
189

Kim HS, Suh YH (2009) Minocycline and neurodegenerative diseases. Behav Brain Res
196:168-179.
Kim JS, Choi IS, Lee MC (1995). Reversible parkinsonism and dystonia following probable
Mycoplasma pneumoniae infection. Mov Disord 10:510-512.
Kim T, Kim YJ (2005) Overview of innate immunity in Drosophila. J Biochem Mol Biol 38:
121-127.
Kitada T, Asakawa S, Hattori N, Matsumine H, Yamamura Y, Minoshima S, Yokochi M,
Mizuno Y, Shimizu N (1998) Mutations in the parkin gene cause autosomal recessive
juvenile parkinsonism. Nature 392:605-608.
Klaes A, Menne T, Stollewerk A, Scholz H, Klämbt C (1994) The Ets transcription factors
encoded by the Drosophila gene pointed direct glial cell differentiation in the embryonic
CNS. Cell 78:149-160.
Klintworth H, Newhouse K, Li T, Choi WS, Faigle R, Xia Z (2007) Activation of c-Jun N-
terminal protein kinase is a common mechanism underlying paraquat- and rotenone-
induced dopaminergic cell apoptosis. Toxicol Sci 97:149-162.
Knott C, Stern G, Kingsbury A, Welcher AA, Wilkin GP (2002) Elevated glial brain-derived
neurotrophic factor in Parkinson’s diseased nigra. Parkinsonism Rel Disord 8:329-341.
Knott C, Stern G, Wilkin GP (2000) Inflammatory regulators in Parkinson’s disease: iNOS,
Lipocortin-1, and cyclooxygenase-1 and-2. Mol cell Neurosci 16:724-739.
Kohbata S, Beaman BL (1991) L-DOPA-responsive movement disorder caused by Nocardia
asteroides localized in the brains of mice. Infect Immun 59:181-191.
190

Kraus RL, Pasieczny R, Lariosa-Willingham K, Turner MS, Jiang A, Trauger JW (2005)
Antioxidant properties of minocycline: neuroprotection in an oxidative stress assay and
direct radical-scavenging activity. J Neurochem 94:819-827.
Krishnakumar S, Burton D, Rasco J, Chen X, O'Donnell J (2000) Functional interactions
between GTP cyclohydrolase I and tyrosine hydroxylase in Drosophila. J Neurogenet 14:1-
23.
Kriz J, Nguyen MD, Julien JP (2002) Minocycline slows disease progression in a mouse model
of amyotrophic lateral sclerosis. Neurobiol Dis 10:268-278.
Kuhn DM, Arthur RE Jr, Thomas DM, Elferink LA (1999) Tyrosine hydroxylase is inactivated
by catechol-quinones and converted to a redox-cycling quinoprotein: possible relevance to
Parkinson’s disease. J Neurochem 73:1309-1317.
Kulich SM, Chu CT (2001) Sustained extracellular signal-regulated kinase activation by 6-
hydroxydopamine: implications for Parkinson's disease. J Neurochem 77:1058-1066.
Kumar S, Doumanis J (2000) The fly caspases. Cell Death Differ 7:1039-1044.
Kume K, Kume S, Park SK, Hirsh J, Jackson FR (2005) Dopamine is a regulator of arousal in
the fruit fly. J Neurosci 25:7377-7384.
Kurucz E, Zettervall CJ, Sinka R, Vilmos P, Pivarcsi A, Ekengren S, Hegedüs Z, Ando I,
Hultmark D (2002) Hemese, a hemocyte-specific transmembrane protein affects the
cellular immune response in Drosophila. Proc Natl Acad Sci USA 100:2622-2627.
Kuter K, Smiałowska M, Wierońska J, Zieba B, Wardas J, Pietraszek M, Nowak P, Biedka I,
Roczniak W, Konieczny J, Wolfarth S, Ossowska K (2007) Toxic influence of subchronic
paraquat administration on dopaminergic neurons in rats. Brain Res 1155:196-207.
191

Kuzin B, Regulski M, Stasiv Y, Scheinker V, Tully T, Enikolopov G (2000) Nitric oxide
interacts with the retinoblastoma pathway to control eye development in Drosophila. Curr
Biol 10:459-462.
Kuzin B, Roberts I, Peunova N, Enikolopov G (1996) Nitric oxide regulates cell proliferation
during Drosophila development. Cell 87:639-649.
Kyriakis JM, Avruch J (2001) Mammalian mitogen-activated protein kinase signal transduction
pathways activated by stress and inflammation. Physiol. Rev 81:807-869.
Langston JW, Ballard P, Tetrud JW, Irwin I (1983) Chronic Parkinsonism in humans due to a
product of meperidine-analog synthesis. Science 219:979-980.
Langston JW, Forno LS, Tetrud J, Reeves AG, Kaplan JA, Karluk D (1999) Evidence of active
nerve cell degeneration in the substantia nigra of humans years after 1-methyl-4-phenyl-
1,2,3,6- tetrahydropyridine exposure. Ann Neurol 46:598-605.
Lanot R, Zachary D, Holder F, Meister M (2001) Postembryonic hematopoiesis in Drosophila.
Dev Biol 230:243-257.
Lawson LJ, Perry VH, Dri P, Gordon S (1990) Heterogeneity in the distribution and morphology
of microglia in the normal adult mouse brain. Neuroscience 39:151-170.
Le W, Rowe D, Xie W, Ortiz I, He Y, Appel SH (2001) Microglial activation and dopaminergic
cell injury: an in vitro model relevant to Parkinson’s disease. J Neurosci 21:8447–8455.
Learish RD, Bruss MD, Haak-Frendscho M (2000) Inhibition of mitogen-activated protein
kinase kinase blocks proliferation of neural progenitor cells. Brain Res Dev Brain Res 122:
97-109.
Lei K and Davis RJ (2003) JNK phosphorylation of Bim-related members of the Bcl2 family
induces Bax-dependent apoptosis. Proc Natl Acad Sci USA 100:2432-2437.
192

Lei K, Nimnual A, Zong WX, Kennedy NJ, Flavell RA, Thompson CB, Bar-Sagi D, Davis RJ
(2002) The Bax subfamily of Bcl2-related proteins is essential for apoptotic signal
transduction by c-Jun NH(2)-terminal kinase. Mol Cell Biol 22:4929-4942.
Leiserson WM, Harkins EW, Keshishian H (2000) Fray, a Drosophila serine/threonine kinase
homologous to mammalian PASK, is required for axonal ensheathment. Neuron 28:793-
806.
Lemaitre B, Hoffmann J (2007) The host defense of Drosophila melanogaster. Annu Rev
Immunol 25:697-743.
Levy OA, Malagelada C, Greene LA (2009) Cell death pathways in Parkinson's disease:
proximal triggers, distal effectors, and final steps. Apoptosis 14:478-500.
Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, Wang X (1997)
Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an
apoptotic protease cascade. Cell 91:479-489.
Liao H, Bu WY, Wang TH, Ahmed S, Xiao ZC (2005) Tenascin-R plays a role in
neuroprotection via its distinct domains that coordinate to modulate the microglia function.
J Biol Chem 280:8316-8323.
Liberatore GT, Jackson-Lewis V, Vukosavic S, Mandir AS, Vila M, McAuliffe WG, Dawson
VL, Dawson TM, Przedborski S (1999) Inducible nitric oxide synthase stimulates
dopaminergic neurodegeneration in the MPTP model of Parkinson disease. Nat Med
5:1403-1409.
Liew FY, Xu D, Chan WL (1999) Immune effector mechanism in parasitic infections. Immunol
Lett. 65:101-104.
193

Lim YM, Nishizawa K, Nishi Y, Tsuda L, Inoue YH, Nishida Y (1999) Genetic analysis of
rolled, which encodes a Drosophila mitogen-activated protein kinase. Genetics 153:763-
771.
Lin LF, Doherty DH, Lile JD, Bektesh S, Collins F (1993) GDNF: a glial cell line-derived
neurotrophic factor for midbrain dopaminergic neurons. Science 260:1130-1132.
Lin S, Zhang Y, Dodel R, Farlow MR, Paul SM, Du Y (2001) Minocycline blocks nitric oxide-
induced neurotoxicity by inhibition p38 MAP kinase in rat cerebellar granule neurons.
Neurosci Lett 315:61-64.
Liou HH, Tsai MC, Chen CJ, Jeng JS, Chang YC, Chen SY, Chen RC (1997) Environmental
risk factors and Parkinson’s disease: a case-control study in Taiwan. Neurology 48:1583-
1588.
Liu B (2006) Modulation of microglial pro-inflammatory and neurotoxic activity for the
treatment of Parkinson's disease. AAPS J 8:E606-E621.
Liu B, Gao HM, Hong JS (2003) Parkinson’s Disease and Exposure to Infectious Agents and
Pesticides and the Occurrence of Brain Injuries: Role of Neuroinflammation. Environ
Health Perspect 111:1065-1073.
Liu B, Hong JS (2003) Role of microglia in inflammation-mediated neurodegenerative diseases:
mechanisms and strategies for therapeutic intervention. J Pharmacol Exp Ther 304:1-7.
Livingstone MS, Tempel BL (1983) Genetic dissection of monoamine neurotransmitter synthesis
in Drosophila. Nature 303:67-70.
Logroscino G (2005) The role of early life environmental risk factors in Parkinson disease: what
is the evidence? Environ Health Perspect 113:1234-1238.
194

Lubinsky S, Bewley GC (1979) Genetics of catalase in Drosophila melanogaster: rates of
synthesis and degradation of the enzyme in flies aneuploid and euploid for the structural
gene. Genetics 91:723-749.
Luckhart S, Rosenberg R (1999) Gene structure and polymorphism of an invertebrate nitric
oxide synthase gene. Gene 232:25-34.
Lundell MJ, Hirsh J (1994) Temporal and spatial development of serotonin and dopamine
neurons in the Drosophila CNS. Dev Biol 165:385-396.
Macdonald H, Kelly RG, Allen ES, Noble JF, Kanegis LA (1973) Pharmacokinetic studies on
minocycline in man. Clin Pharmacol Ther 14:852-861.
Mackey WJ, O’Donnell JM (1983) A genetic analysis of the pteridine biosynthetic enzyme,
guanosine triphosphate cyclohydrolase, in Drosophila melanogaster. Genetics 105:35–53.
Malagoli D, Sacchi S, Ottaviani E (2008) unpaired (upd)-3 expression and other immune-related
functions are stimulated by interleukin-8 in Drosophila melanogaster SL2 cell line.
Cytokine 44:269-274.
Marletta MA (1989) Nitric oxide: biosynthesis and biological significance. Trends Biochem Sci
14:488-492.
Marshall KA, Reist M, Jenner P, Halliwell B (1999) The neuronal toxicity of sulfite plus
peroxynitrite is enhanced by glutathione depletion: implications for Parkinson's disease.
Free Radic Biol Med 27:515-520.
Mayer B (1994) Regulation of nitric oxide synthase and soluble guanylyl cyclase. Cell Biochem
Funct 12:167-177.
Mayeux R (2003) Epidemiology of neurodegeneration. Annu Rev Neurosci 26:81-104.
195

McClung C, Hirsh J (1999) The trace amine tyramine is essential for sensitization to cocaine in
Drosophila. Curr Biol 9:853-860.
McCormack AL, Atienza JG, Johnston LC, Andersen JK, Vu S, Di Monte DA (2005) Role of
oxidative stress in paraquat-induced dopaminergic cell degeneration. J Neurochem
93:1030-1037.
McCormack AL, Thiruchelvam M, Manning-Bog AB, Thiffault C, Langston JW, Cory-Slechta
DA, Di Monte DA (2002) Environmental risk factors and Parkinson’s disease: selective
degeneration of nigral dopaminergic neurons caused by the herbicide paraquat. Neurobiol
Dis 10:119-127.
McGeer PL, Itagaki S, Botes BE, McGeer EG (1988) Reactive microglia are positive for HLA-
DR in the substantia nigra of Parkinson’s and Alzheimer’s disease brains. Neurology 38:
1285-1291.
McGeer PL, McGeer EG (2004) Inflammation and the degenerative diseases of aging. Ann N Y
Acad Sci 1035:104-116.
McGeer PL, McGeer EG (2008) Glial reactions in Parkinson's disease. Mov Disord 23: 474-483.
Mendez JS, Finn BW (1975) Use of 6-hydroxydopamine to create lesions in catecholamine
neurons in rats. J Neurosurg 42:166-173.
Miklossy J, Doudet DD, Schwab C, Yu S, McGeer EG, McGeer PL (2006) ICAM-1 in
persisting inflammation in Parkinson disease and MPTP monkeys. Exp Neurol 197:275-
283.
Miller RL, Sun GY, Sun AY (2007) Cytotoxicity of paraquat in microglial cells: Involvement of
PKCdelta- and ERK1/2-dependent NADPH oxidase. Brain Res 1167: 129-139.
196

Mirza B, Hadberg H, Thomsen P, Moos T (2001) The absence of reactive astrocytosis is
indicative of a unique inflammatory process in Parkinson's disease. Neuroscience 95:425-
432.
Mizuno Y, Hattori N, Mochizuki H (2008) Genetic aspects of Parkinson's disease. Handb Clin
Neurol 83:217-44.
Mogi M, Harada M, Riederer P, Narabyashi H, Fujita J, Nagatsu T (1994). Interleukin-1 beta
growth factor and transforming growth factor-alpha are elevated in the brain from
Parkinsonian patients. Neurosci Lett 180:147–150.
Moncada S, Bolanos JP (2005) Nitric oxide, cell bioenergetics and neurodegeneration J
Neurochem 97:1676–1689.
Moore KE, Demarest KT, Lookingland KJ (1987) Stress, prolactin and hypothalamic
dopaminergic neurons. Neuropharmacology 26:801-808.
Morgan SC, Taylor DL, Pocock JM (2004) Microglia release activators of neuronal proliferation
mediated by activation of mitogen-activated protein kinase, phosphatidylinositol-3-
kinase/Akt and delta-Notch signalling cascades. J Neurochem 90:89-101.
Mosley RL, Benner EJ, Kadiu I, Thomas M, Boska MD, Hasan K, Laurie C, Gendelman HE
(2006) Neuroinflammation, Oxidative Stress and the Pathogenesis of Parkinson's Disease
Clin Neurosci Res 6: 261-281.
Muller T, Blum-Degen D, Przuntek H, Kuhn W (1998) Interleukin-6 levels in cerebrospinal fluid
inversely correlate to severity of Parkinson’s disease. Acta Neurol Scand 98:142-144.
Murad F (1998) Nitric oxide signaling: would you believe that a simple free radical could be a
second messenger, autacoid, paracrine substance, neurotransmitter, and hormone? Recent
Prog Horm Res 53:43-59; discussion 59-60.
197

Nagatsu T, Levitt M, Udenfriend S (1964) Tyrosine hydroxylase. The initial step in
norepinephrine biosynthesis, J Biol Chem 239:2910–2917.
Nagatsu T, Sawada M (2006) Cellular and molecular mechanisms of Parkinson's disease:
neurotoxins, causative genes, and inflammatory cytokines. Cell Mol Neurobiol 26:781-802.
Nappi AJ, Vass E (1998) Hydrogen peroxide production in immune-reactive Drosophila
melanogaster. J Parasitol 84:1150-1157.
Nappi AJ, Vass E, Frey F, Carton Y (2000) Nitric oxide involvement in Drosophila immunity.
Nitric Oxide 4:423-430.
Neckameyer WS (1996) Multiple roles for dopamine in Drosophila development. Dev Biol
176:209-219.
Neckameyer WS (1998a) Dopamine modulates female sexual receptivity in Drosophila
melanogaster. J Neurogenet 12:101-114.
Neckameyer WS (1998b) Dopamine and mushroom bodies in Drosophila: experience-dependent
and -independent aspects of sexual behavior. Learn Mem 5:157-165.
Neckameyer WS, Quinn WG (1989) Isolation and characterization of the gene for Drosophila
tyrosine hydroxylase. Neuron 2:1167-1175.
Neckameyer WS, Weinstein JS (2005) Stress affects dopaminergic signaling pathways in
Drosophila melanogaster. Stress 8:117-131.
Neckameyer WS, White K (1993) Drosophila tyrosine hydroxylase is encoded by the pale locus.
J Neurogenet 8:189-199.
Neckameyer WS, Woodrome S, Holt B, Mayer A (2000) Dopamine and senescence in
Drosophila melanogaster. Neurobiol Aging 21:145-152.
198

Nemery B, van Klaveren RJ (1995). NO wonder paraquat is toxic. Hum Exp Toxicol 14:308-
309.
Nicklas WJ, Vyas I, and Heikkila RE (1985) Inhibition of NADH-linked oxidation in brain
mitochondria by 1-methyl-4-phenyl-pyridine, a metabolite of the neurotoxin, 1-methyl-4-
phenyl-1,2,5,6-tetrahydropyridine. Life Sci 36:2503-2508.
Nimmerjahn A, Kirchhoff F, Helmchen F (2005) Resting microglial cells are highly dynamic
surveillants of brain parenchyma in vivo. Science. 308:1314-1318.
Niso-Santano M, Morán JM, García-Rubio L, Gómez-Martín A, González-Polo RA, Soler G,
Fuentes JM (2006) Low concentrations of paraquat induces early activation of extracellular
signal-regulated kinase 1/2, protein kinase B, and c-Jun N-terminal kinase 1/2 pathways:
role of c-Jun N-terminal kinase in paraquat-induced cell death. Toxicol Sci 92:507-515.
Noselli S, Agnès F (1999) Roles of the JNK signaling pathway in Drosophila morphogenesis.
Curr Opin Genet Dev 9:466-472.
Nüsslein-Volhard C, Wieschaus E, Kluding H (1984) Mutations affecting the pattern of the
larval cuticle in Drosophila melanogaster. I. Zygotic loci on the second chromosome.
Onfelt Tingvall T, Roos E, Engström Y (2001) The imd gene is required for local Cecropin
expression in Drosophila barrier epithelia. EMBO Rep 2:239-243.
Paisán-Ruíz et al. (2004) Cloning of the gene containing mutations that cause PARK8-linked
Parkinson's disease. Neuron 44:595-600.
Palmer RM, Ferrige AG, Moncada S (1987) Nitric oxide release accounts for the biological
activity of endothelium-derived relaxing factor. Nature 32:524-526.
Pandey P, Avraham S, Place A, Kumar V, Majumder PK, Cheng K, Nakazawa A, Saxena S and
Kharbanda S (1999) Bcl-xL blocks activation of related adhesion focal tyrosine
199

kinase/proline-rich tyrosine kinase 2 and stress activated protein kinase/c-Jun N-terminal
protein kinase in the cellular response to methylmethane sulfonate. J. Biol Chem 274:
8618-8623.
Parker and Auld (2006) Roles of glia in the Drosophila nervous system. Semin Cell Dev Biol
17:66-77.
Peng J, Xiao OM, Stevenson FF, Hsu M, Anderson JK (2004). The Herbicide Paraquat Induces
Dopaminergic Nigral Apoptosis through Sustained Activation of the JNK Pathway. J Bio
Chem 279:32626-32632.
Peng J, Xie L, Stevenson FF, Melov S, Di Monte DA, Andersen JK (2006) Nigrostriatal
dopaminergic neurodegeneration in the weaver mouse is mediated via neuroinflammation
and alleviated by minocycline administration. J Neurosci 26:11644-11651.
Pereanu W, Shy D, Hartenstein V (2005) Morphogenesis and proliferation of the larval brain glia
in Drosophila. Dev Biol 283:191-203.
Perier C, Bové J, Wu DC, Dehay B, Choi DK, Jackson-Lewis V, Rathke-Hartlieb S, Bouillet P,
Strasser A, Schulz JB, Przedborski S, Vila M (2007) Two molecular pathwaysinitiate
mitochondria-dependent dopaminergic neurodegeneration in experimental Parkinson’s
disease. Proc Natl Acad Sci USA 104:8161-8166.
Perlman D, O'Brien E (1954) Microbiological production of methymycin and related antibiotics.
Antibiot Chemother 4:894-898.
Perrimon N, Lanjuin A, Arnold C, Noll E (1996) Zygotic lethal mutations with maternal effect
phenotypes in Drosophila melanogaster. II. Loci on the second and third chromosomes
identified by P-element-induced mutations. Genetics 144:1681-1692.
200

Pörzgen P, Park SK, Hirsh J, Sonders MS, Amara SG (2001) The antidepressant-sensitive
dopamine transporter in Drosophila melanogaster: a primordial carrier for catecholamines.
Mol Pharmacol 59:83-95.
Pou S, Pou WS, Bredt DS, Snyder SH, Rosen GM (1992) Generation of superoxide by purified
brain nitric oxide synthase. J Biol Chem 267:24173-24176.
Pradhan S, Pandey N, Shashank S, Gupta RK & Mathur A (1999) Parkinsonism due to
predominant involvement of substantia nigra in Japanese encephalitis. Neurology 53:1781-
1786.
Prasad K, Winnik B, Thiruchelvam MJ, Buckley B, Mirochnitchenko O, Richfield EK (2007)
Prolonged toxicokinetics and toxicodynamics of paraquat in mouse brain. Environ Health
Perspect 115:1448-1453.
Presta A, Siddhanta U, Wu C, Sennequier N, Huang L, Abu-Soud HM, Erzurum S, Stuehr DJ
(1998) Comparative functioning of dihydro- and tetrahydropterins in supporting electron
transfer, catalysis, and subunit dimerization in inducible nitric oxide synthase.
Biochemistry 37:298-310.
Purisai MG, McCormack AL, Cumine S, Li J, Isla MZ, Di Monte DA (2007) Microglial
activation as a priming event leading to paraquat-induced dopaminergic cell degeneration.
Neurobiol Dis 25: 392-400.
Quintero EM, Willis L, Singleton R, Harris N, Huang P, Bhat N, Granholm AC (2006)
Behavioral and morphological effects of minocycline in the 6- hydroxydopamine rat model
of Parkinson’s disease. Brain Res 1093:198-207.
Regulski M, Tully T (1995) Molecular and biochemical characterization of dNOS: a Drosophila
Ca2+/calmodulin-dependent nitric oxide synthase. Proc Natl Acad Sci USA 92:9072-9076.
201

Ries V, Henchcliffe C, Kareva T, Rzhetskaya M, Bland R, During MJ, Kholodilov N, Burke RE
(2006) Oncoprotein Akt/PKB induces trophic effects in murine models of Parkinson's
disease. Proc Natl Acad Sci USA 103:18757-18762.
Rio-Hortega Pd (1932) Microglia. In: Cytology and cellular pathology of the nervous system (W
Penfield, ed), pp 482-534, New York: Hoeber.
Rizki TM (1962) Experimental analysis of hemocyte morphology in insects. Am Zool 2:247-
256.
Rock RB, Gekker G, Hu S, Sheng WS, Cheeran M, Lokensgard JR, Peterson PK (2004) Role of
microglia in central nervous system infections. Clin Microbiol Rev 17:942-964.
Rodriguez-Blanco J, Martín V, Herrera F, García-Santos G, Antolín I, Rodriguez C (2008)
Intracellular signaling pathways involved in post-mitotic dopaminergic PC12 cell death
induced by 6-hydroxydopamine. J Neurochem 107:127-140.
Röhrdanz E, Schmuck G, Ohler S, Kahl R (2001) The influence of oxidative stress on catalase
and MnSOD gene transcription in astrocytes. Brain Res 900:128-136.
Rosselli M, Keller PJ, Dubey RK (1998) Role of nitric oxide in the biology, physiology and
pathophysiology of reproduction. Hum Reprod Update 4:3-24.
Ryu JK, McLarnon JG (2006) Minocycline or iNOS inhibition block 3-nitrotyrosine increases
and blood-brain barrier leakiness in amyloid beta-peptide-injected rat hippocampus. Exp
Neurol 198:552-557.
Sanchez-Pernaute R, Ferree A, Cooper O, Yu M, Brownell AL, Isacson O (2004) Selective
COX-2 inhibition prevents progressive dopamine neuron degeneration in a rat model of
Parkinson's disease. J Neuroinflammation 1:6.
202

Saner A, Thoenen H (1972) Model experiments on the molecular mechanism of action of 6-
hydroxydopamine. Mol Pharmacol 7:147-154.
Saporito MS, Thomas BA, Scott RW (2000) MPTP activates c-Jun NH(2)-terminal kinase (JNK)
and its upstream regulatory kinase MKK4 in nigrostriatal neurons in vivo. J Neurochem
75:1200-1208.
Savitt JM, Dawson VL, Dawson TM (2006) Diagnosis and treatment of Parkinson disease:
molecules to medicine. J Clin Invest 116:1744-1754.
Scanga SE, Ruel L, Binari RC, Snow B, Stambolic V, Bouchard D, Peters M, Calvieri B, Mak
TW, Woodgett JR, Manoukian AS (2000) The conserved PI3'K/PTEN/Akt signaling
pathway regulates both cell size and survival in Drosophila. Oncogene 19:3971-3977.
Schiller F (2000) Fritz Lewy and his bodies. J Hist Neurosci 9:148-151.
Schrag A, Ben-Shlomo Y, Brown R, Marsden CD, Quinn N (1998) Young-onset Parkinson's
disease revisited--clinical features, natural history, and mortality. Mov Disord 13:885-894.
Schrag A, Schott JM (2006) Epidemiological, clinical, and genetic characteristics of early-onset
parkinsonism. Lancet Neurol. 5:355-363.
Sekiguchi K, Yokoyama T, Kurabayashi M, Okajima F, Nagai R (1999)
Sphingosylphosphorylcholine induces a hypertrophic growth response through the
mitogen-activated protein kinase signaling cascade in rat neonatal cardiac myocytes. Circ
Res 85:1000-1008.
Semchuk KM, Love EJ, Lee RG (1992) Parkinson's disease and exposure to agricultural work
and pesticide chemicals. Neurology 42:1328-1335.
203

Seo JH, Rah JC, Choi SH, Shin JK, Min K, Kim HS, Park CH, Kim S, Kim EM, Lee SH, Lee S,
Suh SW, Suh YH (2002) Alpha-synuclein regulates neuronal survival via Bcl-2 family
expression and PI3/Akt kinase pathway. FASEB J 16:1826-1828.
Shapiro S (1989) Regulation of Secondary Metabolism in Actinomycetes. CRC Press
Sherer TB, Betarbet R, Kim JH, Greenamyre JT (2003) Selective microglial activation in the rat
rotenone model of Parkinson's disease. Neurosci Lett 341:87-90.
Simard AR, Soulet D, Gowing G, Julien JP, Rivest S (2006) Bone marrow-derived microglia
play a critical role in restricting senile plaque formation in Alzheimer’s disease. Neuron
49:489-502.
Sortwell CE, Daley BF, Pitzer MR, McGuire SO, Sladek JR Jr, Collier TJ (2000)
Oligodendrocyte-type 2 astrocyte-derived trophic factors increase survival of developing
dopamine neurons through the inhibition of apoptotic cell death. J Comp Neurol 426:143-
153.
Spillantini MG, Schmidt ML, Lee VM, Trojnowski JQ, Jakes R, Goedert M (1997) Alpha-
synuclein in Lewy bodies. Nature 388:839-840.
Srahna M, Leyssen M, Choi CM, Fradkin LG, Noordermeer JN, Hassan BA (2006) A signaling
network for patterning of neuronal connectivity in the Drosophila brain. PLoS Biol 4:e348.
Sriram K, Miller DB, O'Callaghan JP (2006) Minocycline attenuates microglial activation but
fails to mitigate striatal dopaminergic neurotoxicity: role of tumor necrosis factor-alpha. J
Neurochem 96:706-718.
Stasiv Y, Regulski M, Kuzin B, Tully T, Enikolopov G (2001) The Drosophila nitric-oxide
synthase gene (dNOS) encodes a family of proteins that can modulate NOS activity by
acting as dominant negative regulators. J Biol Chem 276:42241-42251.
204

Stathakis DG, Burton DY, McIvor WE, Krishnakumar S, Wright TR, O'Donnell JM (1999) The
catecholamines up (Catsup) protein of Drosophila melanogaster functions as a negative
regulator of tyrosine hydroxylase activity. Genetics 153:361-382.
Staveley BE, Ruel L, Jin J, Stambolic V, Mastronardi FG, Heitzler P, Woodgett JR, Manoukian
AS (1998) Genetic analysis of protein kinase B (AKT) in Drosophila. Curr Biol 8:599-602.
Steller H (2008) Regulation of apoptosis in Drosophila. Genes Dev 22:2256-2266.
Strauss KM, Martins LM, Plun-Favreau H, Marx FP, Kautzmann S, Berg D, Gasser T, Wszolek
Z, Müller T, Bornemann A, Wolburg H, Downward J, Riess O, Schulz JB, Krüger R
(2005) Loss of function mutations in the gene encoding Omi/HtrA2 in Parkinson's disease.
Hum Mol Genet 14:2099-2111.
Stronach B, Perrimon N (2002) Activation of the JNK pathway during dorsal closure in
Drosophila requires the mixed lineage kinase, slipper. Genes Dev 16:377-387.
Sugama S, Yang L, Cho BP, De Giorgio LA, Lorenzl S, Albers DS, Beal MF, Volpe BT, Joh TH
(2003) Age-related microglial activation in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(MPTP)-induced dopaminergic neurodegeneration in C57BL/6 mice. Brain Res 964:288-
294.
Tam S, Barry DP, Beaman L, Beaman BL (2002) Neuroinvasive Nocardia asteroides GUH-2
induces apoptosis in the substantia nigra in vivo and dopaminergic cells in vitro. Exp
Neurol 177:453-460.
Tan PB, Kim SK (1999) Signaling specificity: the RTK/RAS/MAP kinase pathway in
metazoans. Trends Genet 15:145-149.
Tatton WG, Chalmers-Redman R, Brown D, Tatton N (2003) Apoptosis in Parkinson’s disease:
signals for neuronal degradation. Ann Neurol 53:S61–S70. discussion S70-S72.
205

Teismann P, Ferger B (2001) Inhibition of the cyclooxygenase isoenzymes COX-1 and COX-2
provide neuroprotection in the MPTP-mouse model of Parkinson's disease. Synapse
39:167-174.
Thiruchelvam M, Prokopenko O, Cory-Slechta DA, Buckley B, Mirochnitchenko O (2005)
Overexpression of superoxide dismutase or glutathione peroxidase protects against the
paraquat + maneb-induced Parkinson disease phenotype. J Biol Chem 280:22530-22539.
Thöny B, Auerbach G, Blau N (2000) Tetrahydrobiopterin biosynthesis, regeneration and
functions. Biochem J 347:1-16.
Tomita M, Okuyama T, Hidaka K (1999) Changes in mRNAs of inducible nitric oxide synthase
and interleukin-1 beta in the liver, kidney and lung tissues of rats acutely exposed to
paraquat. Leg Med (Tokyo) 1:127-34.
Tootle TL, Spradling AC (2008) Drosophila Pxt: a cyclooxygenase-like facilitator of follicle
maturation. Development 135:839-847.
Tournier C, Hess P, Yang DD, Xu J, Turner TK, Nimnual A, Bar-Sagi D, Jones SN, Flavell RA,
Davis RJ (2000) Requirement of JNK for stress-induced activation of the cytochrome c-
mediated death pathway. Science 288:870-874.
Truman JW, De Vente J, Ball EE (1996) Nitric oxide-sensitive guanylate cyclase activity is
associated with the maturational phase of neuronal development in insects. Development
122:3949-3958.
Tsai CH, Lo SK, See LC, Chen HZ, Chen RS, Weng YH, Chang FC, Lu CS (2002)
Environmental risk factors of young onset Parkinson's disease: a case-control study. Clin
Neurol Neurosurg 104:328-33
206

Tyurina YY, Kapralov AA, Jiang J, Borisenko GG, Potapovich AI, Sorokin A, Kochanek PM,
Graham SH, Schor NF, Kagan VE (2006) Oxidation and cytotoxicity of 6-OHDA are
mediated by reactive intermediates of COX-2 overexpressed in PC12 cells. Brain Res
1093:71-82.
Tzou P, Ohresser S, Ferrandon D, Capovilla M, Reichhart JM, Lemaitre B, Hoffmann JA, Imler
JL (2000) Tissue-specific inducible expression of antimicrobial peptide genes in
Drosophila surface epithelia. Immunity 13:737-748.
Ubogu EE, Cossoy MB, Ransohoff RM (2006) The expression and function of chemokines
involved in CNS inflammation. Trends Pharmacol Sci 27:48-55.
Uversky VN (2004) Neurotoxicant-induced animal models of Parkinson's disease: understanding
the role of rotenone, maneb and paraquat in neurodegeneration. Cell Tissue Res 318:225-
241.
Vafeiadou K, Vauzour D, Spencer JP (2007) Neuroinflammation and its modulation by
flavonoids. Endocr Metab Immune Disord Drug Targets 7:211-224.
Valente et al. (2004) Hereditary early-onset Parkinson's disease caused by mutations in PINK1.
Science 304:1158-1160.
Van Den Bosch L, Tilkin P, Lemmens G, Robberecht Wm (2002) Minocycline delays disease
onset and mortality in a transgenic model of ALS. Neuroreport 13:1067-1070.
Vaux DL, Korsmeyer SJ (1999) Cell death in development. Cell 96:245-254.
Verdu J, Buratovich MA, Wilder EL, Birnbaum MJ (1999) Cell-autonomous regulation of cell
and organ growth in Drosophila by Akt/PKB. Nat Cell Biol 1:500-506.
Vernooy SY, Copeland J, Ghaboosi N, Griffin EE, Yoo SJ, Hay BA (2000) Cell death regulation
in Drosophila: conservation of mechanism and unique insights. J Cell Biol 150: F69-76.
207

Vié A, Cigna M, Toci R, Birman S (1999) Differential regulation of Drosophila tyrosine
hydroxylase isoforms by dopamine binding and cAMP-dependent phosphorylation. J Biol
Chem 274:16788-16795.
Vijitruth R, Liu M, Choi DY, Nguyen XV, Hunter RL, Bing G (2006) Cyclooxygenase-2
mediates microglial activation and secondary dopaminergic cell death in the mouse MPTP
model of Parkinson's disease. J Neuroinflammation 3:6.
Viveros OH, Lee CL, Abou-Donia MM, Nixon JC, Nichol CA (1981) Biopterin cofactor
biosynthesis: independent regulation of GTP cyclohydrolase in adrenal medulla and cortex.
Science 213:349-350.
Vucic D, Kaiser WJ, Harvey AJ, Miller LK (1997) Inhibition of reaper-induced apoptosis by
interaction with inhibitor of apoptosis proteins (IAPs). Proc Natl Acad Sci USA 94:10183-
10188.
Vucic D, Kaiser WJ, Miller LK (1998) Inhibitor of apoptosis proteins physically interact with
and block apoptosis induced by Drosophila proteins hid and grim. Mol Cell Biol 18:3300-
3309.
Waetzig V, Herdegen T (2005) MEKK1 controls neurite regrowth after experimental injury by
balancing ERK1/2 and JNK2 signaling. Mol Cell Neurosci 30:67-78.
Wang MC, Bohmann D, Jasper H (2003b) JNK signaling confers tolerance to oxidative stress
and extends lifespan in Drosophila. Dev Cell 5:811-816.
Wang S, Xu J, Song P, Wu Y, Zhang J, Chul Choi H, Zou MH (2008) Acute inhibition of
guanosine triphosphate cyclohydrolase 1 uncouples endothelial nitric oxide synthase and
elevates blood pressure. Hypertension 52:484-490.
208

Wang X, Zhu S, Drozda M, Zhang W, Stavrovskaya IG, Cattaneo E, Ferrante RJ, Kristal BS,
Friedlander RM (2003a) Minocycline inhibits caspase-independent and -dependent
mitochondrial cell death pathways in models of Huntington's disease. Proc Natl Acad Sci
USA100:10483-10487.
Watanabe Y, Kato H, Araki T (2008) Protective action of neuronal nitric oxide synthase inhibitor
in the MPTP mouse model of Parkinson's disease. Metab Brain Dis 23:51-69.
Weinberger DR, Berman KF, Illowsky BP (1988) Physiological dysfunction of dorsolateral
prefrontal cortex in schizophrenia. III. A new cohort and evidence for a monoaminergic
mechanism. Arch Gen Psychiatry 45:609-615
Werner ER, Gorren AC, Heller R, Werner-Felmayer G, Mayer B (2003) Tetrahydrobiopterin and
nitric oxide: mechanistic and pharmacological aspects. Exp Biol Med (Maywood)
228:1291-302.
White K, Tahaoglu E, Steller H (1996) Cell killing by the Drosophila gene reaper. Science
271:805-807.
Whitton PS (2007) Inflammation as a causative factor in the aetiology of Parkinson’s disease. Br
J Pharmacol 150:963-976.
Wildemann B, Bicker G (1999) Nitric oxide and cyclic GMP induce vesicle release at
Drosophila neuromuscular junction. J Neurobiol 39:337-346.
Wilhelm Roux's Arch. Dev Biol 193:267-282.
Wingrove JA, O'Farrell PH (1999) Nitric oxide contributes to behavioral, cellular, and
developmental responses to low oxygen in Drosophila. Cell 98:105-114.
Wright TR (1987) The genetics of biogenic amine metabolism, sclerotization, and melanization
in Drosophila melanogaster. Adv Genet 24:127-222.
209

Wszolek Z, Monsour H, Smith P, Pfeiffer R (1988) Cryptococcal meningoencephalitis with
parkinsonian features. Mov Disord 3:271-273.
Wu DC, Jackson-Lewis V, Vila M, Tieu K, Teismann P, Vadseth C, Choi DK, Ischiropoulos H,
Przedborski S (2002) Blockade of microglial activation is neuroprotective in the 1-methyl-
4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson disease. J Neurosci
22:1763-1771.
Wu XF, Block ML, Zhang W, Qin L, Wilson B, Zhang WQ, Veronesi B, Hong JS (2005) The
role of microglia in paraquat induced dopaminergic neurotoxicity. Antioxid Redox Signal
7: 654-661.
Wyss-Coray T, Mucke L (2002) Inflammation in neurodegenerative disease--a double-edged
sword. Neuron 35:419-432.
Xia XG, Harding T, Weller M, Bieneman A, Uney JB, Schulz JB (2001) Gene transfer of the
JNK interacting protein-1 protects dopaminergic neurons in the MPTP model of
Parkinson's disease. Proc Natl Acad Sci USA 98:10433-10438.
Xiromerisiou G, Hadjigeorgiou GM, Papadimitriou A, Katsarogiannis E, Gourbali V, Singleton
AB (2008) Association between AKT1 gene and Parkinson's disease: a protective
haplotype. Neurosci Lett 436:232-234.
Yamada T, McGeer PL, McGeer EG (1991) Relationship of complement- activated
oligodendrocytes to reactive microglia and neuronal pathology in neurodegenerative
disease. Dementia 2:71-77.
Yamada T, McGeer PL, McGeer EG (1992) Lewy bodies in Parkinson’s disease are recognized
by antibodies to complement proteins. Acta Neuropath 84:100-104.
210

Yamamoto K, Ichijo H and Korsmeyer SJ (1999) Bcl-2 is phosphorylated and inactivated by an
ASK1/Jun N-terminal protein kinase pathway normally activated at G(2)/M. Mol Cell Biol
19:8469-8478.
Yang W, Tiffany-Castiglioni E (2005) The bipyridyl herbicide paraquat produces oxidative
stress-mediated toxicity in human neuroblastoma SH-SY5Y cells: relevance to the
dopaminergic pathogenesis. J Toxicol Environ Health A 68:1939-1961.
Yang Y, Gehrke S, Haque ME, Imai Y, Kosek J, Yang L, Beal MF, Nishimura I, Wakamatsu K,
Ito S, Takahashi R, Lu B (2005) Inactivation of Drosophila DJ-1 leads to impairments of
oxidative stress response and phosphatidylinositol 3-kinase Akt signaling. Proc Natl Acad
Sci USA 102:13670-13675.
Yasothornsrikul S, Davis WJ, Cramer G, Kimbrell DA, Dearolf CR (1997) viking: identification
and characterization of a second type IV collagen in Drosophila. Gene 198:17-25.
Yrjänheikki J, Tikka T, Keinänen R, Goldsteins G, Chan PH, Koistinaho J (1999) A tetracycline
derivative, minocycline, reduces inflammation and protects against focal cerebral ischemia
with a wide therapeutic window. Proc Natl Acad Sci USA 96:13496-13500.
Yrjänheikki J, Tikka T, Keinänen R, Goldsteins G, Chan PH, Koistinaho J (1999) A tetracycline
derivative, minocycline, reduces inflammation and protects against focal cerebral ischemia
with a wide therapeutic window. Proc Natl Acad Sci USA 96: 13496-13500.
Yuan J, Yankner BA (2000) Apoptosis in the nervous system. Nature 407:802-809.
Zettervall CJ, Anderl I, Williams MJ, Palmer R, Kurucz E, Ando I, Hultmark D (2004) A
directed screen for genes involved in Drosophila blood cell activation. Proc Natl Acad Sci
USA 101:14192-14197.
211

Zhang W, Wang T, Pei Z, Miller DS, Wu X, Block ML, Wilson B, Zhang W, Zhou Y, Hong JS,
Zhang J (2005) Aggregated alpha synuclein activates microglia: a process leading to
disease progression in Parkinson's disease. FASEB J 19:533-542.
Zhou C, Huang Y, Przedborski S (2008) Oxidative stress in Parkinson's disease: a mechanism of
pathogenic and therapeutic significance. Ann N Y Acad Sci 1147:93-104.
Zhu JH, Kulich SM, Oury TD, Chu CT (2002a) Cytoplasmic aggregates of phosphorylated
extracellular signal-regulated protein kinases in Lewy body diseases. Am J Pathol 161:
2087-2098.
Zhu S, Stavrovskaya IG, Drozda M, Kim BY, Ona V, Li M, Sarang S, Liu AS, Hartley DM, Wu
DC, Gullans S, Ferrante RJ, Przedborski S, Kristal BS, Friedlander RM. (2002b)
Minocycline inhibits cytochrome c release and delays progression of amyotrophic lateral
sclerosis in mice. Nature 417:74-78.
Zou H, Henzel WJ, Liu X, Lutschg A, Wang X (1999) Apaf-1, a human protein homologous to
C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3. Cell 90:
405-413.
212