THERAPEUTIC EFFECT OF AN HYDROGEN SULFIDERELEASING COMPOUND IN A PARKINSON’S
DISEASE MODEL

XIE LI
(B.SC) FUDAN UNIVERSITY

DEPARTMENT OF PHARMACOLOGY
YONG LOO LIN SCHOOL OF MEDICINE
NATIONAL UNIVERSITY OF SINGAPORE
2012


i 
 

Acknowledgements
I would like to express my heartfelt gratitude to my supervisor, Prof Bian
Jin-Song, for giving me the opportunity to work on this research project. I
would like to thank him for his generous instruction and support, in both
my research and my life.
I am also grateful to my seniors, Dr Hu Li Fang, Dr Lu Ming, Ms
Tiong Chi Xin and all other laboratory members for their encouragement,
technical help and critical comments. I would like to thank Shoon Mei
Leng for her technical support. With the presence of these adorable
colleagues, my experiences in research for the past three years have been
enjoyable. I would also like to thank my family and friends for their
constant support and encouragement.
 


ii 

 

Table of Contents
 
Acknowledgements .................................................................................................................... i 
Publications ............................................................................................................................... iv 
Summary .................................................................................................................................... v 
List of Tables ............................................................................................................................. vi 
List of Figures .......................................................................................................................... vii 
Abbreviations .......................................................................................................................... viii 
1. 

Introduction ....................................................................................................................... 1 
1.1. 

1.1.1. 

Genetics ............................................................................................................. 1 

1.1.2. 

The pathogenesis of Parkinson’s disease ........................................................... 4 

1.1.3. 

Treatments of Parkinson’s disease .................................................................... 7 

1.1.4. 

Experimental models for Parkinson’s disease ................................................. 10 


1.2. 

Hydrogen sulfide (H2S) ........................................................................................... 13 

1.2.1. 

Endogenous production of H2S ....................................................................... 13 

1.2.2. 

The physiological role of H2S in CNS ............................................................ 14 

1.2.3. 

H2S-releasing compound ................................................................................. 17 

1.3. 
2. 

Parkinson’s disease .................................................................................................... 1 

Research objectives ................................................................................................. 19 

Materials and Methods .................................................................................................... 20 
2.1. 

Chemicals and reagents ........................................................................................... 20 

2.2. 


Cell culture and treatment ....................................................................................... 20 

2.3. 

Cell viability assay .................................................................................................. 20 

2.4. 

Lactate dehydrogenase (LDH) release assay ........................................................... 21 

2.5. 

Reactive oxygen species (ROS) measurement ........................................................ 21 

2.6. 

Superoxide Dismutase (SOD) activity Determination ............................................ 21 

2.7. 

Reverse Transcription-PCR ..................................................................................... 22 

2.8. 

Western blot ............................................................................................................ 23 

2.9. 

Nuclear and cytoplasmic protein fractionation ........................................................ 23 


2.10.  6-OHDA induced PD rat model .............................................................................. 24 


iii 
 
2.11.  Behavioural test ....................................................................................................... 24 
2.12.  Immunohistofluorescence staining .......................................................................... 25 
2.13.  Lipid peroxidation assessment ................................................................................ 25 
2.14.  Concentration determination of dopamine and its metabolites ............................... 26 
2.15.  Statistical analysis ................................................................................................... 26 
3. 

Results ............................................................................................................................. 27 
3.1. 

Protective effect of ACS84 on 6-OHDA-induced cell injury .................................. 27 

3.2. 

ACS84 reduced the oxidative stress induced by 6-OHDA ...................................... 27 

3.3. 

ACS84 promoted anti-oxidative stress associated gene expression ........................ 29 

3.4. 

ACS84 ameliorated behaviour symptom in the unilateral 6-OHDA rat model ....... 32 


3.5. 

ACS84 attenuated the degeneration of dopaminergic neurons in both SN and

striatum ................................................................................................................................ 33 
3.6. 

ACS84 reversed the declined dopamine level in the 6-OHDA-injured striatum .... 33 

3.7. 

ACS84 suppressed the oxidative stress in the injured striatum ............................... 35 

4. 

Discussion ....................................................................................................................... 36 
4.1. 

ACS84 significantly reversed 6-OHDA-induced oxidative stress in SH-SY5Y cells.

 

…………………………………………………………………………………………………………………………….36 

4.2. 

ACS84 suppressed pathological progresses and improved symptoms in unilateral 6-

OHDA rat models ................................................................................................................ 37 
4.3. 


Limitations of the study and future directions ......................................................... 38 

4.4. 

Conclusion ............................................................................................................... 40 

References ............................................................................................................................... 42 
 


iv 
 

Publications

Xie L, Tiong CX, Bian JS. Hydrogen sulfide protects SH-SY5Y cells
against 6-hydroxydopamine-induced endoplasmic reticulum (ER) stress.
Am J Physiol Cell Physiol. 2012. (In Press)
Xie L, Hu LF, Tiong CX, Sparatore A, Del Soldato P, Dawe GS, Bian JS.
Therapeutic effect of ACS84 on 6-OHDA-induced Parkinson’s disease
rat model. (Ready for submission)


v 
 

Summary
Parkinson’s disease (PD), characterized by loss of dopaminergic neurons in the
substantia nigra, is a neurodegenerative disorder of the central nervous system. The

present study was designed to investigate the effect of ACS84, an H2S-releasing LDopa derivate, in a 6-hydroxydopamine (6-OHDA)-induced PD model. ACS84
protected SH-SY5Y cells against 6-OHDA-induced cell injury and oxidative stress.
The protective effect resulted from the stimulation of Nrf-2 nuclear translocation and
the promotion of anti-oxidant enzymes expression. In the 6-OHDA-induced PD
model, intragastric administration of ACS84 relieved the movement dysfunction of
the model rats. Immunohistochemistry and HPLC analysis showed that ACS84
reversed the loss of tyrosine-hydroxylase positive (TH+) neurons in the substantia
nigra and striatum, and the decline of dopamine concentration in the injured striatums
of the 6-OHDA-induced PD model. Moreover, ACS84 reversed the elevated
malondialdehyde level in model animals.
In conclusion, ACS84 may prevent neurodegeneration via the anti-oxidative
mechanisms and has potential therapeutic values for Parkinson’s disease.


vi 
 

List of Tables
 

Table 1.1 Loci associated with PD …………………………………………………...1
Table 3.1 Effect of ACS84 on dopamine and its metabolites in 6-OHDA-lesioned
striatum ………………………………………………………………………………35


vii 
 

List of Figures
Fig 1.1 Chemical structure of ACS84. ……...…………………………………...…..19

Fig. 3.1 Protective effect of ACS84 against cell injury induced by 6-OHDA in SHSY5Y cells…………………………………………………………………………....28
Fig. 3.2 Effect of ACS84 on oxidative stress induced by 6-OHDA in SH-SY5Y
cells …………………………………………………………………………………..30
Fig. 3.3 Effect of ACS84 on antioxidant enzyme expression in SH-SY5Y cells…... 31
Fig. 3.4 Treatment with ACS84 ameliorated the rotational behavior in the unilateral 6OHDA-lesioned rats………………………………………………………………… 32
Fig. 3.5 Effect of ACS84 on 6-OHDA-induced TH+ neuronal degeneration………………. 34
Fig. 3.6 Effect of ACS84 on oxidative stress in the striatum of unilateral 6-OHDA-lesioned
PD rat model. ………………………………………………………………………………...35


viii 
 

Abbreviations
3MST

3-mercaptopyruvate sulfurtransferase

6-OHDA

6-hydroxydopamine

ADT

Anetholedithiolethione

AIMP2

Aminoacyl-tRNA-synthetase-interactingmultifunctional protein type 2


BAC

Bacterial artificialchromosome

BBB

Blood brain barrier

cAMP

Cyclic-AMP

CBS

Cystathionine β-synthase

CMA

Chaperon-medicated autophagy

CNS

Central nervous system

CO

Carbon monoxide

COMT


Catechol-O-methyltransferase

COX2

Cyclo-oxygenase 2

CSE

Cystathionine γ-lyase

DA

Dopamine

DAT

Dopamine active transporter

DBS

Deep brain stimulation

DOPAC

3,4-Dihydroxyphenylacetic acid

ER

Endoplasmic reticulum


FBP-1

Far upstreamelement-binding protein 1

Gclc

Glutamate-cysteine ligase catalytic subunit

GclM

Glutamate-cysteine ligase modulatory subunit

GPi

globuspallidusinterna

GSH

Glutathione

GWAS

Genome wide association study


ix 
 
H2S
HHcy


Hydrogen sulfide
Hyperhomocysteinemia

HO-1

Heme oxygenase -1

HPRT

Hypoxanthine-guanine phosphoribosyltransferase

HVA

Homovanillic acid

iNOS

Inducible form of nitric oxide synthase

KATP

ATP-sensitive potassium channel

LBs

Lewy Bodies

LDH

Lactate dehydrogenase


L-Dopa

Levodopa

LPS

Lipopolysaccharide

LRRK2

Leucine-rich repeat kinase 2

LTP

Long-term potentiation

MAO

Monoamine oxidase

MDA

Malondialdehyde

MPO

Andmyeloperoxidase

MTPT


1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

MTT

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

NaHS

Sodium hydrosulfide

NET

Norepinephrine transporter

NMDA

N-methyl-D-aspartate

NO

Nitric oxide

Nrf-2

Nuclear factor (erythroid-derived 2)-like 2

PD

Parkinson’s disease


PGC-1α

PPARγ coactivator-1α

PLP

pyridoxal-5’-phosphate

PPARγ

peroxisomeproliferator-activated receptor gamma

ROS

Reactive oxygen species


x 
 
SN
SOD

Superoxide Dismutase

Substantia nigra

SQR

Sulfidequinonereductase


STN

Subthalamic nucleus

UPR

Unfolded protein response

UPS

Ubiquitin-proteasome system


1 
 

1. Introduction
1.1. Parkinson’s disease
Parkinson’s disease (PD) is an age-related progressive degenerative movement
disorder, which was firstly described by James Parkinson in 1817 [1]. As the second
most common neurodegenerative disease, PD affects nearly 1% of the population
aged above 65 years [2-5]. PD patients suffer from symptoms such as bradykinesia,
resting tremor, rigidity, and postural instability, which is associated with the loss of
dopaminergic neurons and the decrease of dopamine (DA) in the substantia nigra (SN)
[6]. One hallmark of PD pathology is the presence of Lewy Bodies (LBs) in the
dopaminergic neurons, which is the inclusion of misfolding proteins [7].
1.1.1. Genetics
Although most PD cases are sporadic and largely influenced by environmental factors,
PD has already been recognised as a disorder with a significant genetic component [6,

8-11]. As listed in Table 1.1, more than ten loci have been identified associated with
different types of PD and parkinsonism.
Table 1.1
Loci associated with PD
Locus

Mode of inheritance

Chromosomal

Gene

Reference

location
PARK1 (4)

Autosomal dominant

4q21–q23

SNCA

[12, 13]

PARK2

Autosomal recessive

6q25.2–q27


parkin

[14]

PARK3

Autosomal dominant

2p13

Unknown

[15]

PARK5

Autosomal dominant

4p14

UCHL1

[16]


2 
 

PARK6


Autosomal recessive

1p35–p36

PINK1

[17]

PARK7

Autosomal recessive

1p36

DJ1

[18]

PARK8

Autosomal dominant

12p11.2–q13.1

LRRK2

[19]

PARK9


Autosomal recessive

1p36

ATP13A2

[20]

PARK10

Unknown

1p32

Unknown

[9]

PARK11

Unknown

2q36–q37

GIGYF2

[21]

Besides that, recently Genome-wide Association Studies (GWAS) and metaanalysis also provided a huge amount of information indicating the suspicious loci

associated with PD [22-29]. All these investigations contributed greatly to the
understanding of molecular mechanisms of PD pathogenesis. Here we will discuss the
roles of several genes as listed above.
α-Synuclein
α-Synuclein is encoded by gene SNCA, which is the first gene found to be linked to
PD. Three mutations (A53T [12], A30P [30], E46K [31]) and genome triplication of
SNCA[13] have been identified in familial PD patients. The physiological role of αsynuclein remains unknown, though it is highly expressed in the brain. α-Synuclein is
mainly located in the presynaptic terminal and is involved in the maintenance of
membrane structures [32, 33]. Some scientists speculated that α-synuclein might be
involved in the DA neurotransmission and synaptic vesicle recycling [34]. αSynuclein is the main component of the LBs. The mutations and over-expressions of
α-synuclein are believed to promote the formation of LBs. It has been shown that
compared to wild-type α-synuclein, A53T and A30P mutants exhibit increased
propensity to form oligomers and fibrils in vitro [35]. Moreover, the A30P mutant
expressed in transgenic mice or flies indicated inclusions formation as well as


3 
 

neurodegeneration [36, 37]. However, the mechanism of wild-type α-synuclein
accumulation in LBs inclusion is less elucidated. It was speculated to be associated
with the mitochondria complex-I malfunction [38-41], tyrosine nitration [42] and the
impairment of proteasome function [43, 44]. It is also worth noting that α-synuclein
may directly suppress proteasome function in cells. Reports had suggested that αsynuclein filaments and oligomers were resistant to proteasome degradation and
inhibit proteasome activity by directly binding to 20/26S proteasomal subunits [4547]. Overexpression of mutant α-synuclein was also proved to induce proteasome
impairment in cells [48, 49]. The impairment of proteasome function induced by αsynuclein may be a crucial pathological process in PD.
Parkin and PINK1
Parkin is a ubiquitin E3 ligase, which is responsible for tagging proteins for
proteasome degradation. The function of Parkin can be disrupted by parkin mutations
[50, 51] as well as the nitrosative and oxidative stress in sporadic PD [52]. The

dysfunction of Parkin leads to the accumulation of its substrates, including aminoacyltRNA-synthetase-interacting multifunctional protein type 2 (AIMP2) [53, 54], far
upstream element-binding protein 1 (FBP-1) [55] and most importantly, PARIS
(parkin-interacting substrate) [56]. In conditional parkin knock-out mice, PARIS
accumulated in the brain and suppressed the expression of peroxisome proliferatoractivated receptor gamma (PPARγ) coactivator-1α (PGC-1α), leading to the
degeneration of DA neurons [56].
Like Parkin, PINK1 mutations are also associated with familial PD. PINK1
is a protein kinase with a mitochondria-targeting domain [57], which was believed to
be involved in mitochondria quality control with Parkin [58]. Flies with PINK1 or


4 
 

parkin deficits suggested the vulnerability of DA neurons to oxidative stress [59, 60].
It has been recognised that PINK1 and Parkin may play crucial roles in the turnover
of damaged mitochondria. PINK1 is cleaved during mitochondria depolarization,
leading to the recruitment of Parkin and proceeding to mitophagy [61-63].
LRRK2
Leucine-rich repeat kinase 2 (LRRK2) is a serine/threonine kinase with a GTPase
modulation domain. Mutations on LRRK2 had been isolated from familial PD
patients, which would lead to the late-onset of PD symptoms [64]. The G2019S
mutation is the most common mutation in familial PD, and it is also recognised as a
significant risk factor in sporadic PD patients [65]. Several pathogenic mutations on
LRRK2 promote the formation of dimers and LRRK2 kinase activity is dependent on
the dimer formation [66]. Evidences had also suggested that the applications of
compounds which blocked LRRK2 kinase reversed LRRK2 toxicity in neurons [67].
Recent studies indicated that LRRK2 was involved in the modulation of neurite
outgrowth in neurons development [68-70], and the regulation of protein translation
via protein-microRNA interaction [71].
1.1.2. The pathogenesis of Parkinson’s disease

Although the specific molecular mechanisms for PD are still uncertain, scientists have
concluded several theories, including mitochondria dysfunction and oxidative stress,
ubiquitin-proteasome

system

malfunction

and

neuroinflammation to explain the pathogenesis of PD.
Mitochondria dysfunction and oxidative stress

autophagy

failure,

and


5 
 

Oxidative damage in sporadic PD brains has been observed in post-mortem studies,
and the source of oxidative stress might be induced by mitochondria dysfunction and
DA metabolism [72]. In order to maintain the oxidation phosphorylation, there is a
highly oxidative environment inside mitochondria. During mitochondria dysfunction,
especially the defects in complex-I, the production of ATP is reduced and the release
of reactive oxygen species (ROS) is elevated in the cells, resulting in oxidative stress
in PD brains [6]. This speculation has been supported by the observation that

complex-I activity was decreased in the SN of sporadic PD patients [73]. The
cytoplasmic hybrid cells containing mitochondria DNA (mtDNA) from PD patients,
which displayed the deficits of complex-I and increased ROS generation [74, 75], also
indicated the role of complex-I deficits in PD pathogenesis. Moreover, some
neurotoxins like MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) and rotenone,
which are the inhibitors of mitochondria complex-I, are used to induce Parkinson
mimetic symptoms in animal models [38, 76-78]. Another source of ROS generation
in dopaminergic neurons is the metabolism of DA. Under physiology condition, DA
can be degraded non-enzymatically into quinone by oxygen and enzymatically into
3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) by
monoamine

oxidase

(MAO)

and

Catechol-O-methyl

transferase

(COMT),

respectively. Both ways of degradation would generate H2O2 [79-82].
Ubiquitin-proteasome system malfunction and autophagy failure
Some scientists have also focused their research on the ubiquitin-proteasome system
(UPS) and autophagy, which are the main intracellular degradation methods [83-85].
As the existence of LBs is a major clinical hallmark for sporadic PD and some
familial PD, it is believed that UPS impairment may be a crucial process in PD

pathology [6]. Both structural and functional deficits of 20/26S proteasome have been


6 
 

observed in sporadic PD patients [43, 86]. Besides, animals treated with proteasome
inhibitors displayed PD-like symptoms, which includes DA neurons degeneration and
LB-like inclusion formation [87]. Moreover, overexpression of molecular chaperones
by transgenic or pharmacological methods reversed the pathological progresses in
Drosophila models [88, 89], which further indicated the importance of UPS activity in
PD pathogenesis.
Autophagy has emerged to be a hot-spot in neurodegenerative diseases
research. It is the pathway by which cells degrade the long-lived, stable proteins and
recycle the organelles [85]. Three types of autophagy have been introduced:
marcoautophagy, microautophagy and chaperon-medicated autophagy (CMA) [90].
Autophagy is believed to be closely related to PD pathogenesis. Numerous
investigations have demonstrated that α-synuclein could also be cleared by autophagy
in addition to the UPS [91-93]. More evidences also presented that mutations on
ATP13A2, which encodes a lysosomal ATPase, led to autophagy failure and αsynuclein aggregation [20, 94]. Moreover, both UPS and autophagy activity are
reduced during aging [95-98]. Therefore, it is understandable that age is one of the
key risk factors in PD.
Glial activation and neuroinflammation
The activation of glia cells and the neuroinflammation have been recognised as a
keynote contributor in the processes of neurodegeneration [99, 100]. Activated
microglia cells [101-103] and the increment of astrocyte density [104] have been
observed in SN of PD patients in post-mortem studies. Alongside with these findings,
it was also reported that the concentrations of cytokines such as TNFα, interleukins 1β,
6, and 2, β2-microglobulin, TGFα and β1, and interferon γ were upregulated in



7 
 

striatum [105-109] and SN [110] of PD patients. Moreover, enzymes which are
involved in neuroinflammation, including inducible form of nitric oxide synthase
(iNOS), NADPH oxidase, cyclo-oxygenase 2 (COX2), andmyeloperoxidase (MPO),
were found to be upregulated in PD patients and PD models [111-114]. All these lines
of evidences suggested the crucial role of neuroinflammation in the pathogenesis of
PD. Some scientists believed that the release of protein aggregates from neurons [115,
116] or even nitrated extracellular α-synuclein [117] triggered microglia activation
during the progress of PD. Others suggested the possible influences of environmental
factors on neuroinflammation. Animals exposed to neurotoxins such as MPTP and
rotenone were observed to exhibit glia activation and neuroinflammation [118, 119].
Apart from that, although the role of infection in neuroinflammation still remains
unclear, injection of Lipopolysaccharide (LPS) intracranially would induce PD-like
symptom in rodents [120].
1.1.3. Treatments of Parkinson’s disease
There is no cure for PD so far. However, numerous medications had been developed
to supplement the DA deficit and to improve the life qualities of the patients.
Clinically, there are pharmacologic and surgical treatments being adopted to relieve
PD symptoms.
Levodopa (L-Dopa)
L-Dopa is the most widely used treatment for PD since its first development about 30
years ago. L-Dopa is able to pass through the blood-brain-barrier (BBB) and is
uptaken by dopaminergic neurons to transform into DA by dopa-decarboxylase to
compensate for the decline of DA in the brain [121]. Administration of L-Dopa
efficiently reverses the motor dysfunction in the patients. However, only 1-5% of L-



8 
 

Dopa is distributed to the centre nerves system (CNS), and the rest of the L-Dopa
would induce side-effects peripherally. In clinical practise, L-Dopa is administrated
with carpidopa, which is a BBB impermeable dopa-decarboxylase inhibitor to block
the L-Dopa metabolism in peripheral systems.
Although L-Dopa is effective in relieving the PD symptoms in patients,
chronic treatment with L-Dopa would lead to the suppression of endogenous synthesis
of DA and the disruption of DA system. Patients would experience the wear-off
effects when the effective period of the drug begins to reduce. Half of the patient may
even develop dyskinesia after years of medication [122, 123]. Moreover, L-Dopa does
not arrest the progression of PD and long-term treatment accelerates the neuron
degeneration due to oxidative stress [124-127].
Etilevodopa, which is an L-Dopa derivative, has also been developed for PD
treatment. However, the clinical trial reports suggested that little advantages were
observed in patients with motor fluctuations [128].
Dopamine agonist
DA agonist is designed to activate DA receptors, which can be a supplementary
treatment for L-Dopa medication and used to treat early PD patients. The most
commonly prescribed DA agonists are pramipexole, ropinirole and rotigotine. Clinical
trials have suggested that initial treatment of PD with pramipexole would reduce the
incidence of dopaminergic motor complications like dyskinesia compared with LDopa [129-131]. Rotigotine is also reported to relief symptoms in early PD patients in
clinical research [132, 133]. However, DA agonists also produce similar side effects
compared with L-Dopa, although they might postpone the occurrence of involuntary
movements [134, 135].


9 
 


Monoamine oxidase-B (MAO-B) inhibitor
MAO-B is the main enzyme in dopaminergic neurons which breaks down dopamine.
Therefore, the inhibition of MAO-B would increase the level of dopamine in the brain.
Two MAO-B inhibitors had been developed, namely selegiline and rasagiline.
Numerous clinical researches have revealed that monotherapy of rasagiline or
combined with L-Dopa have effectively improved the motor function decline in early
PD patients [136-139]. Experimental investigations also indicated that rasagiline
protected neurons against injuries via maintenance of mitochondria integrity and
induction of neurotropic factors [140]. Based on these observations, rasagiline has
been recognized as a promising potential therapy for PD, although more information
about the safety and further side effects are still required.
Catechol-O-methyl transferase (COMT) inhibitor
COMT is also an enzyme involved in the degradation of DA in the dopaminergic
neurons. The usage of COMT inhibitor is to prolong the effects of L-Dopa. The
adjunction of entacapone, which is a COMT inhibitor, used in combination with LDopa in PD patients with motor fluctuation, although did not significantly reverse the
symptoms, but it improved the life quality of the patients [141]. However, one adverse
effect of COMT inhibitors is that they may enhance the dyskinesia induced by LDopa.
Deep brain stimulation (DBS)
DBS is a surgical treatment using implanted electrodes to give electrical pulses to
specific brain regions. In PD patients, DBS would manage PD symptoms and improve
patients’ life quality, as well as reverse the side effects of PD medication.


10 
 

Subthalamic nucleus (STN) and globuspallidusinterna (GPi) are two major
stimulation site for PD, but other sites like caudal zonaincerta and pallidofugal fibers
are also reported to be effective [142]. However, it should be noted that DBS would

induce psychiatric dysfunction in the patients, although this adverse effect was
reported to be reversible [143].
1.1.4. Experimental models for Parkinson’s disease
Animal models would always be the powerful tools to understand the disease
mechanisms and to seek the effective potential medications in biomedical research.
For PD, both non-genetic and genetic models have been established. However, none
of those models would be capable to represent the pathogenesis of human PD. Here,
we will discuss the advantages and imperfections of those widely used models.
6-hydroxydopamine (6-OHDA)
6-OHDA-induced PD model is the most widely used animal models for PD research.
When injected intracerebrally, 6-OHDA is selectively taken up by dopamine
transporter (DAT) and norepinephrine transporter (NET) into the dopaminergic
neurons. Consequently, 6-OHDA undergoes catalytic processes and releases reactive
oxygen species (ROS) which induces cell injury in neurons [144]. 6-OHDA-induced
PD model displays similar clinical features of human PD, including dopamine
depletion, dopaminergic neuron loss, and neurobehavioral deficits [145]. However,
the pathological protein aggregations and the deposition of LBs are neglected in this
model. Moreover, the acute lesion of dopaminergic nerve system in this model might
not represent the slow progress of clinical PD pathogenesis. In the present study,
unilateral 6-OHDA rat model was used to test the anti-oxidative effects of compound


11 
 

ACS84. The severity of the lesion can be monitored by amphetamine or
apomorphine-induced turning behaviour.
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)
MPTP animal model is a widely accepted PD model which mimics a majority of PD
features including oxidative stress, mitochondria dysfunction and neuroinflammation.

MPTP is BBB permeable and it is transformed into active form MPP+ in astrocytes by
MAO-B. Following that, MPP+ enters neurons through DAT. Once inside the neurons,
MPP+ blocks mitochondria complex-I activity, leading to the release of ROS and ATP
deficiency. This animal model would display akinesia and rigidity after MPTP
administration, although protein aggregation is rare in this model [146].
Rotenone
Rotenone is a pesticide which inhibits the mitochondria respiration chain in cells.
Although the effect is not selective, rotenone application exhibits almost all the
characteristic of human PD symptoms, especially the aggregation of α-synuclein and
the formation of LB-like inclusions [41, 147]. Moreover, rotenone also induces
microglia activation in animal models [148-150], indicating that rotenone models are
capable of mimicking the neuroinflammatory features of PD. Interestingly,
chronically administration of rotenone suggested a highly selectivity to nigrostriatal
neurons [38], while few theories could explain this selective vulnerability. Some
scientists purposed that rotenone might also inhibit the microtubule stability. The
microtubule malfunction further disrupted the transport of dopamine vesicles in the
dopaminergic neurons, leading to elevation of dopamine oxidation in the cells [151].
Genetic models


12 
 

As α-synuclein is intimately associated with PD pathogenesis, some researchers
attempted to establish transgenic mice which express mutant α-synuclein in the brain.
However, no model has been found to perfectly replicate the clinical and pathological
features of PD. Only one model, mPrP-A53T mice displayed α-synuclein pathology
including

α-synuclein


aggregation

and

age-dependent

progressive

DA

neurodegeneration [152, 153], despite that this degeneration was not L-Dopa
responsive [154].
LRRK2 mutation is also a major risk factor in late-onset PD. However,
Bacterial artificial chromosome (BAC) transgenic mice expressing R1441G or
G2019S mutants of LRRK2, and conditional knock-in of the R1141C mutation did
not exhibit significant dopaminergic neurodegeneration, although all of these models
displayed some abnormalities in the nigrostriatal system [155-157].
As discussed above, Parkin and PINK1 are involved in the mitochondria
maintenance, and mutations on Parkin and PINK1 would lead to familial PD. The
knockouts of parkin or pink1 in Drosophila lead to significant motor deficit and
mitochondria dysfunction [60, 158-160]. In contrast, parkin or pink1 knockout mice
did not show any substantial dopaminergic or behavioural abnormalities [161-166].
However, overexpression of mutant human parkin in mice induced progressive
degeneration of DA neurons [167].
Interestingly, disruption of some other genes which are not suggested to
associate with PD also leads to PD-like symptoms in mice models. For example, the
deficiency of transcription factor Pitx3 and conditional knockout of mitochondrial
transcription factor Tfam in dopaminergic neurons in mice produced progressive loss



13 
 

of dopaminergic neurons and displayed PD-like phenotypes [168, 169]. These
investigations provided novel insights into the molecular mechanisms of PD.

1.2. Hydrogen sulfide (H2S)
H2S, which is a flammable, water soluble and colourless gas with an unfavourable
odour, was traditionally thought to be a toxic gas but recently is recognized as one of
the gas-transmitter followed by NO and CO. In the last decade, numerous
investigations have focused on the physiological and pathological functions of H2S in
the body systems, especially in the centre nervous system and cardiovascular system.
1.2.1. Endogenous production of H 2 S
The identification of endogenous H2S was inspired by the detection of sulfide levels
in the brains from rats, humans and bovine [170-172] as well as in blood samples [173]
and hearts [174]. Although the exact concentration of H2S is quite controversial due to
the high variety of measurement methods, there is no doubt that H2S is endogenously
produced in many tissues.
There are two kinds of enzymes which are responsible for H2S production:
pyridoxal-5’-phosphate (PLP)-dependent enzymes including cystathionine-synthase
(CBS) and cystathionine-lyase (CSE) [175-178] and a PLP-independent enzyme,
called 3-mercaptopyruvate sulfurtransferase (3MST). The main substrates of CBS and
CSE are L-cysteine and/or homocysteine [179, 180], while 3MST facilitates the
transfer of thiol group from L-cysteine to -ketoglutarate, in combination with
cysteine aminotransferase (CAT) [181].
However, CBS is the predominant enzyme for H2S production in CNS,
suggested by the results from western and northern blots detecting the protein and



14 
 

mRNA expression levels in the rat brains [182]. Further investigations localized CBS
to astrocytes [183, 184], while 3MST was found to be expressed in neurons [185].
The endogenous levels of H2S in CNS are still controversial nowadays. Originally, it
was reported that H2S levels in brain is around 47-166 µM [170-172, 182, 186, 187].
However, with novel methods, this value had been reconsidered to be as low as few
nano molars [188, 189]. Recently, some scientists suggested that the intracellular halflife of H2S was as short as few seconds [190, 191]. They indicated the enzyme,
sulfidequinone reductase (SQR), oxidised H2S and transferred the electron to the
mitochondria respiration chain [191]. However, SQR is absent in neurons, which may
suggest a unique role of H2S in neurons.
1.2.2. The physiological roles of H 2 S in CNS
Neurophysiology modulation
In 1996, it was first reported that physiological concentration (≤130 µM) of H2S
selectively upregulated the N-methyl-D-aspartate (NMDA) receptor-mediated
responses and improved the induction of the hippocampal long-term potentiation
(LTP), which indicated the potential role of H2S in neuromodulation [182]. Further
investigation revealed that the enhancing of the NMDA receptor activity was
dependent on the H2S-induced increment of cyclic-AMP (cAMP) [192].
Other investigations also indicated that H2S elevated intracellular Ca2+ and
induced Ca2+ waves in astrocytes, via mechanisms which modulated neuron functions
[193]. This observation had been confirmed by an independent investigation which
suggested that H2S induced both Ca2+ influx and the release of Ca2+ from intracellular
stores, and this effect was cAMP/PKA dependent [194].
Suppression of neuroinflammation