Therapeutic effect of hydrogen sulfide in a parkinsons disease model

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 15    H2S was originally recognized as a proinflammatory modulator in acute pancreatitis, endotoxin-induces global inflammation, and polymicrobial sepsis-associated lung injury [195-199]. However, Hu et. al. first demonstrated that H2S attenuated neuroinflammation induced by lipopolysaccharide (LPS) in microglia cells [200]. Further investigation also indicated that H2S suppressed rotenone- and Aβ-induced inflammation in microglia cells and animal models [201, 202]. It was suggested that the anti-inflammation effects of H2S involved the inhibition of p38 mitogen-activated protein kinase [200]. Suppression of oxidative stress The anti-oxidative stress effects of H2S in neurons were first reported by Kimura’s group. They described that H2S improved the activity of γ-glutamylcysteine synthetase and elevated cystine transport to boost the glutathione levels in primary cultured neurons [203]. Furthermore, they also suggested that in supplement to upregulating glutathione levels, H2S also activated ATP-dependent K+ (KATP) and Clchannels [204]. Our group also demonstrated the anti-oxidative effects of H2S in cellular models. In neuroblastoma cell line SH-SY5Y cells, H2S protected cells against cell injuries-induced by neurotoxins including rotenone and 6-OHDA via increasing mitochondria stability and upregulating PKC/Akt pathways [205, 206]. Works on glia cells suggested that H2S rescued astrocytes from H2O2-induced injury by enhancing glutamate uptaking [207]. More direct evidence was obtained from animal models that H2S ameliorated symptoms in 6-OHDA unilateral rat model and rotenone treated rat model. The mechanism involved was concluded to be the suppression of NADPH oxidase activity and oxygen consumption in the neurons by H2S [201]. Other observations showed that H2S was also capable of reversing MPP+induced apoptosis in PC12 cells by maintaining mitochondrial membrane potential 16    and reducing intracellular ROS generation [208]. However, although the suppression of oxidative stress by H2S was confirmed by independent researches, the exact mechanism of this phenomenon was still unclear. In a recent animal-based investigation, the scientists suggested that the protective effects of H2S was not associated with KATP channels but involved uncoupling protein 2 in mice administrated with MPTP [209], which was contradicting to the previous understanding of H2S functions. Therefore, investigation to identify the exact action site of H2S in the neurons is still worthwhile. Suppression of endoplasmic reticulum (ER) stress ER is an organelle whereby the secreting proteins or membrane proteins are synthesized, folded, modified and transported. Stress conditions such as oxidative stress, nutrition deprivation, aberrant Ca2+ regulation, and viral infection, would lead to the disturbance of protein processing in ER, and induced the unfolded protein response (UPR) [210]. Overwhelming and persisting ER stress would induce apoptosis, and the existence of ER stress had been identified in PD models [211]. The role of H2S in ER stress is quite controversial recently. It was reported that H2S suppressed the cardiomyocytic ER stress induced by hyperhomocysteinemia (HHcy), thapsigargin or tunicamycin [212]. However, in INS-1E cell, which is an insulin-secreting beta cell line, upregulation of H2S induced ER stress and stimulated apoptosis [213]. In CNS, evidences support the protective effects of H2S against ER stress. Our group has reported that H2S relieved 6-OHDA-induced ER stress in SHSY5Y cells via upregulation of Hsp90 expression [214]. Similar results have also been obtained in animal models as H2S treatment significantly reduced the expression of UPR related proteins in MPTP mice [209]. Moreover, H2S has been shown to 17    sulfhydrate phosphatase PTP1B, and therefore suppressed the ER stress processes [215]. 1.2.3. H 2 S-releasing compound Although H2S has been proven to be a potential therapeutic agent in PD treatment, the challenges remain in clinical practises as how to give the H2S treatments accurately and safely. Some scientists started to seek compounds which would release H2S in the body. One group had recognized morpholin-4-ium 4 methoxyphenyl (morpholino) phosphinodithioate (GYY4137) as a H2S-slow releasing compound. Despite its slowreleasing features, GYY4137 achieved almost every physiological characteristics compared with sodium hydrosulfide (NaHS), including smooth muscle relaxation and blood pressure reduction [216]. Moreover, GYY4137 relieved LPS-induced inflammation responses in macrophage RAW264.7 and in rats [217, 218], and these effects were comparable with low-dose of NaHS treatment [218]. These researchers also believed that the slow-releasing pattern of GYY4137 made GYY4137 a better representative model for H2S investigations [217]. Another candidate of H2S-releasing compound, anetholedithiolethione (ADT), has also drawn attentions recently. ADT is a compound with a unique thiol group and is originally developed as a choleretic and sialogogue [219]. Since the last two decades, it had emerged that ADT was an effective anti-oxidant, suppressing oxidative damage in astrocytes [220], Jurkat T cells [221], and endothelial cells[222]. Evidence has also revealed that ADT significantly suppressed the MAO-B activity but not MAO A in astrocytes [223]. Recently, some researches started to consider that the 18    effects of ADT come from the H2S released from its thiol group, and they combined ADT with other widely-accepted drugs to enhance the therapeutic effects. For instances, the ADT-diclofenac hybrid (ACS 15), suggested anti-inflammatory effects, protected hearts against ischemia-reperfusion injury, suppressed vascular smooth muscle cell proliferation, as well as inhibited breast cancer-induced osteoclastogenesis and preventedosteolysis [224-227]. ACS14, the hybrid of ADT and aspirin, has also been found to modulate thiol homeostasis in cells, protected the heart from ischemia/reperfusion, suppressed microglia activation and neuroinflammation, and suppressed breast cancer cellsproliferation [228-232]. Overall, all of these observations indicated the potential application of ADT as an H2S-releasing agent for disease treatments. It was speculated that the combination of L-Dopa and H2S may have potential therapeutic value [233, 234]. ACS84, which is also a family member among ACS14 and 15, is a hybrid compound derived from L-Dopa and ADT, and is permeable to BBB and release H2S in cells [233]. Although the effect of ACS84 on PD is not known yet, ACS84 and other H2S-releasing L-Dopa derivatives have been proven to suppress neuroinflammation and inflammation-induced cell injury, and elevate glutathione (GSH) level while inhibit MAO B activity [233]. Further investigation also suggested that ACS84 protected cells against Aβ-induced cell injury via attenuation of inflammation and preservation of mitochondrial function, a similar effect like ACS14 [235]. As discussed previously, L-Dopa treatment in PD would enhance the oxidative stress and lead to the worsening of dopaminergic neuron loss. It is worthwhile to investigate whether the combination of L-Dopa and H2S ameliorates the oxidative stress and improve the therapeutic effect of L-Dopa. 19    1.3. Research objectives L-Dopa is still the most widely used treatment for PD since 1970s. Although L-Dopa efficiently compensates for the dopamine deficit in the brain, it fails to reverse the disease progresses. Long-term treatment of L-Dopa would enhance the oxidative stress and promote the neurodegeneration. H2S, which is a powerful anti-oxidant and neuroprotector, has been proven to reverse the cell injuries and disease progresses in PD cell and animal models. Evidences also suggest that H2S-releasing compound, including ADT and ACS84, may also protect the cells against neural damages. ACS84 is a hybrid compound derivate from L-Dopa and ADT moiety. Reports have indicated that it suppressed glia cells activation and relieved inflammation-induced cell injury. However, the anti-oxidative stress effects of ACS84 have not been investigated. Therefore, in this present study, we will examine the suppression of oxidative stress in 6-OHDA cell model and the amelioration of disease progress in unilateral 6-OHDA rat model. Fig 1.1 Chemical structure of ACS84. ACS84 is a hybrid of L-Dopa (left part) and ADT (right part). The dithiol thione group on ADT moiety is believed to release H2S in cells. 20    2. Materials and Methods 2.1. Chemicals and reagents All chemicals, antibodies for detecting tyrosine hydroxylase (TH) and LDH assay kit were purchased from Sigma (Sigma, St. Louis, MO). Antibodies for detecting Nrf-2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The Glutathione Assay Kit, TBARS Assay Kit and Superoxide Dismutase Assay Kit were purchased from Cayman Chemical (Ann Arbor, Michigan). ACS84 was prepared as previously described [233]. 2.2. Cell culture and treatment The human neuroblastoma cell line, SH-SY5Y, was obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were maintained in Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 10% foetal bovine serum (FBS) and 0.05 U•mL-1 penicillin and 0.05 mg•mL-1 streptomycin at 37°C in a humidified atmosphere containing 5% CO2/95% air. Cells were plated onto 96-well plates for viability tests and ROS generation assay, or 35 mm dishes and incubated overnight. Regular medium was replaced with low-serum medium (0.5% FBS/DMEM) before treatment. Note that for Nrf-2 translocation, medium was changed to non-serum medium and incubated for another 12 h. Cells were treated with ACS84, L-Dopa or NaHS for 1 – 8 h. 2.3. Cell viability assay Cell viability was measured using the MTT reduction assay as described previously [205]. At the end of each treatment, MTT was added to each well at a final concentration of 0.5 mg·mL-1 and the cells were further incubated at 37°C for 4 h. 21    Then, the insoluble formazan was dissolved in dimethyl sulphoxide (DMSO). Colorimetric determination of MTT reduction was measured at 570 nm with a reference wavelength of 630 nm. 2.4. Lactate dehydrogenase (LDH) release assay At the end of treatment, cell culture medium was collected and briefly centrifuged. The supernatants were transferred into wells in 96-well plates. Equal amounts of lactate dehydrogenase assay substrate, enzyme and dye solution were mixed. A half volume of the above mixture was added to one volume of medium supernatant. After incubating at room temperature for 30 min, the reaction was terminated by the addition of 1/10 volume of 1N HCl to each well. Spectrophotometrical absorbance was measured at a wavelength of 490 nm and reference wavelength of 690 nm. 2.5. Reactive oxygen species (ROS) measurement Formation of reactive oxygen species (ROS) was evaluated using non-fluorescent dye 2’, 7’- dichlorofluorescindiacetate (DCFH-DA), which freely penetrates cells and yields the highly fluorescent product dichlorofluorescein (DCF) by ROS oxidation. Following ACS84, L-Dopa or NaHS treatment, cells were rinsed with PBS solution and incubated with Hank's Buffered Salt Solution (HBSS) containing DCFH-DA dye (10 μM final concentration) 30 minutes in the dark. 6-OHDA was added then and fluorescence was read immediately for 1 h, at an excitation wavelength (Ex) of 490 nm and an emission wavelength (Em) of 520 nm. 2.6. Superoxide Dismutase (SOD) activity Determination SOD activity was measured in cells using the Cayman Chemical Superoxide Dismutase Assay Kit (Cayman Chemicals, Inc, Ann Arbor, MI). Briefly, cells were sonicated in 20 mM HEPES buffer, pH 7.2, containing 1 mM EGTA, 210 22    mMmannitol and 70 mM sucrose, on ice. After centrifugation, the supernatant was collected. Reaction was initiated by adding diluted xanthine oxidase to all wells, and then the plate was incubated on a shaker at room temperature for 20 min. The absorbance was read at 450 nm. 2.7. Reverse Transcription-PCR The mRNA levels of GclC, GclM, HO-1 and β-Actin were determined by two-step reverse transcription PCR. In brief, total RNA was extracted using TRIzol® reagent (Invitrogen, Carlsbad, CA, USA). Homogenized samples were then incubated at room temperature for 5 min. Chloroform was added and tubes were shaked vigorously by hand for 15 min followed by incubation for 3 min at room temperature once more. Samples were centrifuged at 12000g for 15 min at 4°C. Colourless upper aqueous phase was transferred to a new tube containing isopropanol and incubated for 10 min at 25°C followed by centrifugation at 12000g for 10 min at 4°C. Supernatant was thrown away and RNA pellet was washed with 70% ethanol. RNA concentration was determined with NanoDrop Spectrophotometer (ND-1000, NanoDrop Technology). Equal amounts of RNA samples obtained were reverse transcribed into cDNA using iScriptTMcDNA synthesis kit (Bio-Rad). Reverse transcription was performed at 25°C (for 5 min), 42°C (for 30 min) and 85°C (for 5 min). The resulting cDNAs were PCR-amplified using Taq DNA polymerase kit (i-DNA Biotechnology). The specific PCR primer sequences used were as follows: GclC (forward primer 5′-TGAGATTTAAGCCCCCTCCT-3′ and reverse primer 5′TTGGGATCAGTCCAGGAAAC-3′) [NM_001498.3], and GclM (forward primer 5’TTTGGTCAGGGAGTTTCCAG-3’and reverse primer 5’-ACACAGCAGGAGGCA AGATT-3’) [NM_002061.2] [236]; HO-1 (forward primer 5′-CAGGCA GAGAATGCTGAGTTC-3′ and reverse primer 5′-GCTTCACATAGCGCTGCA-3′) 23    [NM_002133.2] [237]; and β-actin forward primer (5’-AAGAGAGG CATCCTCACCCT-3’) and β-actin reverse primer (5’-TACATGGCTGGGG TGTTGAA-3’) [NM_001101.3] [238]. PCR conditions were set as 95oC (for 30 sec), 58oC (for 30 sec), and 72oC (for 30 sec) for 30 cycles. PCR products were separated on a 1% agarose gel and stained with ethidium bromide. The optical densities of the mRNA bands were analyzed with GelDoc-It Imaging Systems. 2.8. Western blot For Western blot analysis, the cells were washed with ice-cold PBS and homogenized with lysis buffer containing 150 mMNaCl, 25 mMTris (pH7.5), 5 mM EDTA, 1% Nonidet P-40, (additional 10 mMNaF and 1 mM Na3VO4 were immediately added before detection of phosphorylation) and protease inhibitor cocktail tablet (Roche Diagnostics, Penzberg, Germany). The lysates were then vigorously shaken on ice for one hour and centrifuged at 13,200 g at 4°C for 10 min. After that, the supernatant was collected and denatured by SDS-sample buffer. Epitopes were exposed by boiling the protein samples at 100°C for 5 min. The protein samples were separated by SDSPAGE gel and subsequently transferred onto the nitrocellulose membrane (Whatman). Next, the membrane were blocked with 10% milk/TBST buffer for one hour and incubated with appropriate primary antibodies at 4°C overnight. Finally, the membrane were washed and incubated with appropriate HRP-conjugated second antibody. Visualization was performed using ECL® (plus/advanced chemiluminescence) kit (GE healthcare, UK). The density of the bands on film was quantified by Image J software (National Institute of Health, USA). 2.9. Nuclear and cytoplasmic protein fractionation The preparation of cytoplasmic and nuclear extracts was performed using the Nuclear Extract kit (Active Motif) according to manufacturer’s instruction. Briefly, cells were 24    scraped using cell lifter in ice-cold PBS. Cell pellet obtained after centrifugation was re-suspended in a hypertonic buffer and incubated on ice for 10 min. After the addition of detergent, the suspension was centrifuged. The supernatant (cytoplasmic fraction) was collected. The remaining nuclear pellet was re-suspended in complete lysis buffer. After vortex and centrifugation, the supernatant (nuclear fraction) was collected. 2.10. 6-OHDA induced PD rat model Male Sprague-Dawley (SD) rats (180-220 g) were anesthetized with ketamine (75 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.). After that, the rats were placed in a stereotaxic apparatus (Stoelting Instruments, Wood Dale, IL, USA). 6-OHDA (8 µg 6-OHDA hydrobromide dissolved in 4 µl sterile saline containing 0.02% ascorbic acid) was unilaterally injected into the left striatum (coordinates from bregma: AP, +1.0 mm; ML, +3.0 mm; DV, -4.5 mm) with a Hamilton syringe (0.46 mm in diameter, blunt tip) at a rate of 0.5 µl per minute. The needle was left in place for 3 min and then slowly withdrawn in the subsequent two to three minutes. Shamoperated rats were injected with 4 µl saline containing 0.02% ascorbic acid into the left striatum and served as controls in this study. After surgery, the rats were kept in cages and exposed to a 12: 12 h light-dark cycle with unrestricted access to tap water and food. 2.11. Behavioural test Three weeks after surgery, the animals’ tendency to rotate in response to apomorphine (0.5 mg/kg, s.c.) was tested. This test was re-performed one week later, i.e. four weeks after surgery. Only those rats consistently showing at least 7 turns per min in both tests were considered as the successfully induced PD-like model. These PD-like rats were then divided into different groups receiving different treatments, vehicle- or 25    ACS84- administered group. In addition, the sham-operated rats also received vehicle treatment. These treatments continued for another 3 weeks. The rotational behaviour was monitored at one week interval till the end of treatment. 2.12. Immunohistofluorescence staining The immunohistofluorescence staining was performed according to the procedures as previously described with little modification [201]. At the end of behavioural test for the last time, the animals were anesthetized and perfused with sterile saline and subsequently with 4% paraformaldehyde (PFA). After that, the animals were decapitated. The brain were then collected and immersed into 4%PFA for postfix at 4°C overnight. These brain samples were transferred into 15% sucrose in phosphate buffered saline (PBS) overnight at 4°C and subsequently to 30% sucrose solution till the brain sunk to the tube bottom. Thereafter, the brain were sectioned on a cryostat at a thickness of 30 µm and mounted onto the poly-l-lysine coated slides. These sections were stored at -70°C for further experimentation. The sections were permeabilized with 0.3% Triton X-100/PBS for 10 min and blocked with 10% BSA in PBS for another 30 min. After that, the sections were incubated with mouse monoclonal anti-TH antibody (1:500, Sigma, St. Louis, MO, USA) for 2 h at room temperature and followed with appropriate goat anti-mouse secondary antibody incubation for one hour. 2.13. Lipid peroxidation assessment The level of MDA, a marker of oxidative stress, was measured using TRABS assay kit (Cayman Chemical). The assay was performed according to the manufacturer’s instructions. In brief, brain tissues were lyzed with chilled RIPA buffer and sonicated for 15 s at 40 V over ice. After centrifugation at 1600 g for 10 min at 4 oC, the supernatant was collected for further analysis. The MDATBA adduct formed by the 26    reaction of MDA in samples and TBA supplied in the assay kit under high temperature (100 oC) and acidic conditions. Reaction product was measured colorimetrically at 540 nm with a spectrophotometer (Tecan M200). The content of MDA in samples expressed as micromolar of MDA produced per gram of protein. 2.14. Concentration determination of dopamine and its metabolites HPLC was used to detect concentration of dopamine and its metabolites in the brain tissues. The methods had been described in the previous reports (Zhu et al. 2007). The striatum was sonicated in 0.1M perchloric acid. Homogenates were centrifuged at 14,000g for 20 min at 4 °C. The supernatants were collected and adjusted the pH value around 3. After that the supernatants were subjected to HPLC (HTEC-500; Eicom, Kyoto, Japan) equipped with the column (EICOMPAK SC-3ODS; Eicom, Kyoto, Japan) and electrochemical detector (AD Instruments Pty Ltd., Castle Hill, NSW, Australia). Data was analyzed using PowerChrom (eDAQ, Australia). 2.15. Statistical analysis Statistical significance was assessed with one-way analysis of variance (ANOVA) followed by a post hoc (Bonferroni) test for multiple group comparison using PASW 18 (IBM, NY). Differences with p value less than 0.05 were considered statistically significant. 27    3. Results 3.1. Protective effect of ACS84 on 6-OHDA-induced cell injury To evaluate the protective effect of ACS84 against 6-OHDA-induced cell injury in SH-SY5Y cells, cells were pretreated with ACS84 at different concentrations for 1 h before the treatment of 6-OHDA (50 μM) for another 6 h or 12 h. As shown in Fig 1A and 1B, ACS84 at 0.1 nM to 10 μM concentration-dependently increased cell viability (Fig 3.1 A) and decreased LDH release (Fig 3.1 B) in cells treated with 6-OHDA. As ACS84 is a compound constituted by L-Dopa and H2S-releasing moiety, we investigated whether L-Dopa or H2S alone would be able to produce similar protective effect as ACS84 did. As shown in Fig 3.1 C & 3.1 D, neither L-Dopa nor NaHS (an H2S donor) at the equal molar concentration (10 μM) was sufficient to exert the similar protective effects against 6-OHDA-induced cell injury as ACS84 did (Fig 3.1 C and 3.1 D). This is consistent with our previous findings that NaHS produced significant protective effects only when its concentration higher than 100 μM [206]. These data suggest that ACS84 may produce stronger protective effects than either LDopa or NaHS alone. 3.2. ACS84 reduced the oxidative stress induced by 6-OHDA As it is well-accepted that 6-OHDA selectively killed dopaminergic neuron via generating reactive oxygen species (ROS) and inducing oxidative stress in the cells, we proceeded to examine the effect of ACS84 on 6-OHDA-induced ROS formation in SH-SY5Y cells. As shown in Fig. 3.2 A, ACS84 at the concentration of 10 μM significantly reduced ROS production induced by 6-OHDA (50 M). It was also 28    found that both L-Dopa and NaHS failed to suppress the 6-OHDA-induced ROS formation (Fig 3.2 B). Fig. 3.1 Protective effect of ACS84 against cell injury induced by 6-OHDA in SH-SY5Y cells. Dose dependent effect of ACS84 on (A) cell viability and (B) LDH release in the 6-OHDA-treated (50 μM) SH-SY5Y cells. Cells were pretreated with ACS84 at different concentrations for 1 h and 6-OHDA was added. The results were obtained at 12 h (MTT assay) or 6 h (LDH release assay) after 6-OHDA treatment. Effect of ACS84, L-Dopa and NaHS at 10 μM pretreatment on cell viability (C) and LDH release (D) in SH-SY5Y cells treated with 6-OHDA. Data are presented as mean SEM, n = 5 - 9, ###P < 0.001 versus control; ***P < 0.001, *P < 0.05 versus 6-OHDA-treated cells; †P < 0.05, †††P < 0.001 versus ACS84-treated cells. 29    Superoxide dismutases (SODs) are a family of enzymes which catalyze the dismutation of superoxide and play important roles in cell homeostasis. As shown in Figure 3.2 C, ACS84, but not L-Dopa and NaHS, at 10 μM completely abolished the inhibitory effect of 6-OHDA on SOD activity. 3.3. ACS84 promoted anti-oxidative stress associated gene expression Cells express anti-oxidant enzymes to protect against oxidative stress and most of these enzyme-coding genes contain anti-oxidant reaction element (ARE). NF-E2related factor 2 (Nrf-2) is an important transcription factor which binds to ARE and initiates the expression of anti-oxidant enzymes, including glutamate cysteine ligase (GCL) and heme oxidase-1 (HO-1). Western blotting analysis shows that ACS84 treatment for 4 h promote the translocation of Nrf-2 from cytosol to nuclear (Fig 3.3 A). RT-PCR also indicated that the mRNA level of three important Nrf-2 target genes: Glutamate cysteine ligase catalytic subunit (GclC), Glutamate cysteine ligase modifier subunit (GclM) and HO-1, were significantly elevated after 4 h treatment of ACS84 (Fig 3.3 B). These data suggest that ACS84 induced Nrf-2 nuclear translocation and promoted the expression of anti-oxidant enzymes, which contributed to the protection against 6-OHDA-induced oxidative stress. 30    Fig. 3.2 Effect of ACS84 on oxidative stress induced by 6-OHDA in SH-SY5Y cells. (A) Dose dependent effect of ACS84 on ROS generation in the 6-OHDA-treated (50 μM) SH-SY5Y cells. Cells were pretreated with ACS84 at different concentrations for 4 h. DCFDAH2 (10 μM) was given 30 min before the 6-OHDA (50 μM) treatment. The results were obtained after 1 h of 6OHDA treatment. (B) Effect of ACS84, L-Dopa and NaHS at 10 μM pretreatment on ROS generation in SH-SY5Y cells treated with 6-OHDA. (C) Effect of ACS84, L-Dopa and NaHS at 10 μM pretreatment on SOD activity in 6-OHDA-treated SH-SY5Y cells. SOD activity was measured 4 h after 6-OHDA treatment. Data are presented as mean SEM, n = 4 - 8, #P < 0.05, ### versus control; ***P < 0.001, *P < 0.05 versus 6-OHDA-treated cells; †P < 0.05, ††† vesus ACS84-treated cells. P < 0.001 P < 0.001 31    Fig. 3.3 Effect of ACS84 on antioxidant enzyme expression in SH-SY5Y cells. (A) Immunoblotting results showed that 4 h treatment of ACS84 promote the nuclear accumulation of Nrf-2 in SH-SY5Y cells. Densitometric analysis performed by normalizing nuclear Nrf-2 to cytosol Nrf-2 signals. Data were expressed as mean ±SEM, * P < 0.05, n = 5 (B) Reverse transcription PCR results suggested that ACS84 treatment induced the mRNA expression of GclC, GclM and HO-1 after 4 h. Representative results were obtained from three independent experiments. 32    3.4. ACS84 ameliorated behaviour symptom in the unilateral 6-OHDA rat model To evaluate the therapeutic effect of ACS84 on Parkinson’s disease, we established the unilateral 6-OHDA lesion rat model. Four weeks after 6-OHDA lesion, the PD rats were injected intragastrically with vehicle or ACS84 (10mg/kg) daily and the treatment continued for 3 weeks. As shown in Fig 3.4, ACS84 significantly ameliorated the rotation behaviour after 2 weeks of treatment, which indicated that the administration of ACS84 may alleviate the behaviour disorder in Parkinson’s disease. Fig. 3.4 Treatment with ACS84 ameliorated the rotational behavior in the unilateral 6-OHDAlesioned rats. ACS84 (10mg kg-1 day-1, i.g) was given daily from the 4th to 6th week after 6-OHDA lesion. Mean ± SEM. n = 11–12. *P < 0.05 vs. the values in Vehicle group in the same time point. 33    3.5. ACS84 attenuated the degeneration of dopaminergic neurons in both SN and striatum The movement dysfunction of the PD model is mainly associated with the loss of dopaminergic neurons in the SN and striatum. From the immunostaining results (Fig 5), unilateral 6-OHDA lesion destroyed most of the tyrosine hydroxylase positive (TH+) neurons in both SN pars compacta (SNc, Fig 3.5A) and striatum (Fig 3.5B) of the injured hemisphere, while the administration of ACS84 remarkably attenuated the effect. As tyrosine hydroxylase is the rate-limiting enzyme in dopamine synthesis, this data suggests that ACS84 may preserve the function of dopaminergic neurons in 6OHDA-injured SN and striatum. 3.6. ACS84 reversed the declined dopamine level in the 6-OHDA-injured striatum We further examined the dopamine level in the injured striatum. The concentrations of dopamine and its metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were measured with HPLC. As suggested in Table 3.1, 6OHDA lesion significantly decreased the concentrations of dopamine in the injured striatum, while ACS84 treatment reversed these effects. These data were comparable with the results of behaviour test and immunohistofluorescence stain, indicating that ACS84 efficiently alleviated the loss of dopaminergic neurons and the deficient of dopamine in the striatum. 34    Fig. 3.5 Effect of ACS84 on 6-OHDA-induced TH+ neuronal degeneration. Immunohistochemistry showing ACS84 (10mg kg-1 day-1, i.g) alleviated TH+ neuron loss in both SN pars compacta (SNc) (A) and striatum (B) of 6-OHDA-lesioned PD rats. Photos were taken at x50 magnification. Samples were collected from two independent experiments. 35    Table 3.1 Effect of ACS84 on dopamine and its metabolites in 6-OHDA-lesioned striatum. The concentration of dopamine and its metabolites in 6-OHDA-lesioned striatum Treatment Sham Vehicle ACS84 DA (ng/g tissue) 8.25±1.01 1.61±0.45# 7.35±1.62* DOPAC (ng/g tissue) 2.54±0.71 1.00±0.24 3.81±0.89* HVA (ng/g tissue) 1.47±0.23 0.71±0.10 2.15±0.41* DA/DOPAC DA/HVA 4.90±0.74 2.03±0.72# 2.15±0.41 5.96±0.46 2.24±0.50# 4.90±0.67* Dopamine, DOPAC and HVA concentrations were measured using HPLC. Data indicated that 6OHDA lesion reduced the concentration of dopamine and its metabolites in the injured striatum. ACS84 tratment (10mg kg-1 day-1, i.g) alleviated the dopamine deficient but not HVA. Data are presented as mean ± SEM, n = 6 - 8. #P < 0.05 versus Sham group and *P < 0.05 versus Vehicle group.  3.7. ACS84 suppressed the oxidative stress in the injured striatum Malondialdehyde (MDA) is a marker for lipid peroxidation to indicate the oxidative stress level in the striatum. As shown in Fig 3.6, 6-OHDA induced the elevation of MDA production in the injured striatum, when compared to sham and healthy striatum. ACS84 treatment significantly suppressed this effect. This data suggested that ACS84 may protect dopaminergic neurons degeneration by suppressing oxidative stress. Fig. 3.6 Effect of ACS84 on oxidative stress in the striatum of unilateral 6-OHDA-lesioned PD rat model. ACS84 treatment (10mg kg-1 day-1, i.g) alleviated the MDA formation, Data are presented as mean SEM, n = 4 - 6. *P < 0.05 versus lesion site of Sham group and #P < 0.05 versus lesion site of Vehicle group. 36    4. Discussion 4.1. ACS84 significantly reversed 6-OHDA-induced oxidative stress in SH-SY5Y cells. The symptoms of Parkinson’s disease are associated with the loss of dopaminergic neurons and the deficiency of dopamine in the SN and striatum, and oxidative stress plays a crucial role in the pathology of neurodegeneration [6, 239]. Though traditional L-Dopa treatment for PD patients could compensate for dopamine deficiency and alleviate the behaviour disorder, long-term usage of L-Dopa has its disadvantages and has been proven to enhance oxidative stress [124-127]. H2S has been recognized as an anti-oxidant [207, 240, 241] and our group has demonstrated the protective effect of H2S in 6-OHDA and rotenone-induced PD models [205]. ACS84 is a hybrid compound which is derived from L-Dopa and one H2S-releasing moiety, ADT [233]. ACS84 and other H2S-releasing L-Dopa derivatives have been shown to have therapeutic potential as they suppress microglia activation [233]. In the present study, we used 6-OHDA-induced PD model to investigate the therapeutic effects of ACS84. We found that ACS84 showed significant protective effect against 6-OHDAinduced cell injury and oxidative stress in SH-SY5Y cells, while at equal molar concentration of both L-Dopa and NaHS failed to achieve. Although it has been reported that NaHS was able to protect the cells against apoptosis and oxidative stress in a higher concentration, our results suggested that ACS84 showed a better therapeutic potential as it produced protective effect at a lower dose, at which NaHS failed to protect neuronal cells. We postulated that the better effect of ACS84 may due to the slower H2S-releasing rate. Another possibility is that ACS84 releases H2S 37    intracellularly by mitochondria [234]. More experiments are warranted to investigate the exact underlying mechanism. Gcl and HO-1 are anti-oxidant enzymes involving in the cellular stress defence system. Both coding gene contain ARE cis-element. When activated, transcript factor Nrf-2 translocates from the cytoplasm to the nuclear and binds to the ARE. This initiates the gene expression of anti-oxidant enzymes [242-245]. Our results showed that ACS84 treatments induced nuclear translocation of Nrf-2 and promoted the gene transcription of GclC, GclM and HO-1, which further indicated that ACS84 may attenuate oxidative stress via stimulating Nrf-2/ARE pathway to increase anti-oxidant enzymes in the cells. 4.2. ACS84 suppressed pathological progresses and improved symptoms in unilateral 6-OHDA rat models We used unilateral 6-OHDA PD rat model to evaluate the protective effect of ACS84 in vivo. This model is mainly designed to study the oxidative injury in PD pathogenesis process [246]. Our group has demonstrated that the protective effects of H2S involved suppression of NADPH oxidase in this model [205]. In the present study, we also showed that post-treatments of ACS84 preserved TH+ neurons in both SN and striatum, maintained the dopamine levels and relieved the movement dysfunction. These data suggested that ACS84 is of potential therapeutic value for Parkinson’s disease. We believe that the suppression of oxidative stress is the main mechanism in the protective effect of ACS84. We have proven that ACS84 was efficient in reducing ROS formation and was able to stimulate the expression of antioxidant enzymes in vitro. Along with these results obtained from SH-SY5Y cells, we also found that ACS84 treatment reversed the effect of 6-OHDA on MDA levels. 38    Having such an effect on the oxidative stress indicator, these data proved that ACS84 plays a role as an anti-oxidant in the dopaminergic neurons. It was reported that ACS84 intravenous injection increased the dopamine level in the rat brain [233]. Our results also indicated that ACS84 elevated dopamine levels in the 6-OHDA-injured striatum. ACS84 may increase dopamine in two aspects: firstly, ACS84 may release L-Dopa which is further catalysed into dopamine by DOPA decarboxylase; secondly, ACS84 protected dopaminergic neuron degeneration, thus alleviated dopamine deficient. It had been reported that ACS84 and other similar H2S-releasing compound would inhibit the Monoamine oxidase B activity in neonatal rat striatal astroglial cell primary cultures and SH-SY5Y cells. [223, 233] We also get similar results from primary cultured astroglial cells (data no shown). However, from our HPLC results, we did not observe the significant decline of DOPAC/DA ratio in the striatum homogenates from ACS84-treated rats (Table 3.1). These data may suggest that MAO B activity inhibition of ACS84 was not as efficient under physiological conditions as in cell cultures. However, the HVA/DA ratio decreased in the ACS84 treated rats compared to vehicle rats, suggesting that ACS84 might acts as a COMT inhibitor, rather than MAO B inhibitor in vivo. The inhibition of COMT would prolong the LDopa effects, which may be another mechanism of ACS84 to improve PD-like symptoms in animal models. 4.3. Limitations of the study and future directions ACS84 is the combination of H2S-releasing moiety and L-Dopa. In this present study we investigated the protective effects against oxidative stress in vitro and in vivo. However, we only compared the effects among ACS84, L-Dopa and NaHS in cell cultures. Although we demonstrated that ACS84 displayed a better effects to reverse 39    the cell injury and suppress the ROS formation among the three compounds, it would be more interesting to examine whether ACS84 would achieve the improvement of dopamine deficits similar to L-Dopa, while ameliorate the disease progresses in animal models. Moreover, it would be better to confirm our findings in other PD animal models like MPTP mice model or some genetic models. Despite the enhancement of oxidative stress, one major adverse effect of LDopa treatment is the dyskinesia induced. The mechanisms of dyskinesia are associated with the disruption of dopaminergic system and involve maladaptive plastic changes and misregulation of neurotransmitter receptors in neurons [247, 248]. As it has been reported that H2S is capable to modulate the neuron activities [249], the role of H2S in modulation L-Dopa-induced dyskinesia would worth investigation. Therefore, it would be exciting to examine whether ACS84 would also reduce the prevalence of dyskinesia in long-term treatment. Although we reported that ACS84 promote the nuclear translocation of Nrf-2 and elevated ARE-associated gene expression in SHSY5Y cell, the exact activation mechanism is still unrevealed. More importantly, we are not sure whether the protective effect of ACS84 comes from the H2S it released or ACS84 itself. Looking for the direct reaction site of ACS84 in neurons would be helpful to recognise the role of H2S in ACS84 pharmacology. It was interesting that in our results, ACS84 seemed to be more effective in cell viability assays than LDH release assays and ROS determinations. We speculated that cell viability assay was mitochondria-dependent. As a strong reducing agent, ACS84 might efficiently preserve mitochondria functions during oxidative stress. Therefore, ACS84 performed better in cell viability assays. However, the exact 40    protective effect of ACS84 against oxidative stress might be verified with other measurements. Although our results suggested a promising protective effect of ACS84 against neurodegeneration, unfortunately there is no report indicating the toxicity, pharmacodynamics and pharmacokinetics of ACS84 in animal models. Although the administration of ACS84 in our experiments did not display obvious adverse effects, more toxicology investigation would be necessary. Recently, a process called sulfhydration, which is the directly modification of H2S on cysteine residues, has drawn researchers’ attentions. Through sulfhydration, H2S would alter the protein conformations and modulate protein functions. GAPDH, β-actin, β-tubulin, KATP channel, PIP1B, NF-κB were all proved to be sulfhydration substrates [215, 250-253]. Sulfhydration has emerged to be the key mechanism of H2S physiology and responsible for the complicate effects of H2S in cells [254]. Therefore, it could be a standard to examine the H2S-releasing compounds through detecting the amount of sulfhydration in cells. As there is no reliable measurement available recently, this method could indirectly provide us some important information about the H2S-releasing rate and bioavailiblity of these compounds, as well as the data for pharmacodynamics and pharmacokinetics. 4.4. Conclusion We have demonstrated the neuroprotective effect of ACS84, one H2S-releasing LDopa derivative, in the 6-OHDA models of Parkinson’s disease. ACS84 suppressed 6-OHDA-induced cell injury and ROS generation and induced anti-oxidant enzymes expression via Nrf-2 stimulation. Moreover, ACS84 also ameliorated the movement dysfunction and dopaminergic neuron degeneration in unilateral 6-OHDA PD rat 41    model by suppressing oxidative injury. 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suppressed microglia activation and neuroinflammation, and suppressed breast cancer cellsproliferation [228-232] Overall, all of these observations indicated the potential application of ADT as an H2S-releasing agent for disease treatments It was speculated that the combination of L-Dopa and H2S may have potential therapeutic value [233, 234] ACS84, which is also a family... 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... 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),... 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... 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 15    H2S was originally recognized as a proinflammatory modulator in acute pancreatitis, endotoxin-induces global inflammation, and polymicrobial sepsis-associated lung injury [195-199]... 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... However, Hu et al first demonstrated that H2S attenuated neuroinflammation induced by lipopolysaccharide (LPS) in microglia cells [200] Further investigation also indicated that H2S suppressed rotenone- and A -induced inflammation in microglia cells and animal models [201, 202] It was suggested that the anti-inflammation effects of H2S involved the inhibition of p38 mitogen-activated protein kinase [200]... 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. .. 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... 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 ... NSW, Australia) Data was analyzed using PowerChrom (eDAQ, Australia) 2.15 Statistical analysis Statistical significance was assessed with one-way analysis of variance (ANOVA) followed by a post... potential application of ADT as an H2S-releasing agent for disease treatments It was speculated that the combination of L-Dopa and H2S may have potential therapeutic value [233, 234] ACS84, which... [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