Melatonin protects against behavioral deficits, dopamine loss and oxidative stress in homocysteine model of Parkinson's disease

Accepted Manuscript Melatonin protects against behavioral deficits, dopamine loss and oxidative stress in homocysteine model of Parkinson's disease

Rajib Paul, Banashree Chetia Phukan, Arokiasamy Justin Thenmozhi, Thamilarasan Manivasagam, Pallab Bhattacharya, Anupom Borah PII: DOI: Reference:

S0024-3205(17)30593-3 doi:10.1016/j.lfs.2017.11.016 LFS 15428

To appear in:

Life Sciences

Received date: Accepted date:

27 October 2017 10 November 2017

Please cite this article as: Rajib Paul, Banashree Chetia Phukan, Arokiasamy Justin Thenmozhi, Thamilarasan Manivasagam, Pallab Bhattacharya, Anupom Borah , Melatonin protects against behavioral deficits, dopamine loss and oxidative stress in homocysteine model of Parkinson's disease. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Lfs(2017), doi:10.1016/ j.lfs.2017.11.016

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ACCEPTED MANUSCRIPT Revised Article (LFS-D-17-00595)

October, 2017

Title: Melatonin protects against behavioral deficits, dopamine loss and oxidative stress in homocysteine model of Parkinson’s disease Authors: Rajib Paul1,2, Banashree Chetia Phukan1, Arokiasamy Justin Thenmozhi3,

Cellular and Molecular Neurobiology Laboratory, Department of Life Science and

Department of Zoology, Pandit Deendayal Upadhyaya Adarsha Mahavidyalaya (PDUAM),

Eraligool-788723, Karimganj, Assam, India

Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University,

Annamalainagar, Tamil Nadu, India

Department of Pharmacology and Toxicology, National Institute of Pharmaceutical

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Bioinformatics, Assam University, Silchar, Assam, India

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Thamilarasan Manivasagam3, Pallab Bhattacharya4, Anupom Borah1#

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Address for correspondence: Anupom Borah, Ph.D.

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Education and Research (NIPER)-Ahmedabad, Gandhinagar-382355, Gujarat, India

Cellular and Molecular Neurobiology Laboratory Department of Life Science and Bioinformatics Assam University, Silchar ‒ 788011, Assam, India E‒mail: [email protected]; [email protected]

Running Title: Melatonin protects against homocysteine-induced Parkinsonism.

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ACCEPTED MANUSCRIPT Abstract Aim: Hyperhomocysteinemia and homocysteine (Hcy) mediated dopaminergic neurotoxicity is a matter of concern in the pathophysiology of Parkinson’s disease (PD). Our previous study established the involvement of oxidative stress in the substantia nigra (SN) of Hcy rat

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model of PD; however, the role of antioxidants, such as melatonin, was not tested in this model.

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Main methods: Melatonin (10, 20 and 30 mg/kg, i.p.) was administered to rats injected with

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Hcy in right SN (1.0 μmol in 2 μl saline) to investigate its potency in attenuating the behavioral abnormalities, dopamine depletion and oxidative stress prompted by Hcy.

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Key findings: Treatment of melatonin protected against nigral dopamine loss and replenished

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the striatal dopamine loss that resulted in amelioration of rotational behavioral bias in Hcy denervated animals. Melatonin administration significantly improved mitochondrial complex-

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I activity and protected the SN neurons from the toxic insults of oxidative stress induced by

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Hcy. Amelioration of oxidative stress by melatonin in Hcy-infused SN was bought by dosedependently scavenging of hydroxyl radicals, restoration of glutathione level and elevation in the activity of antioxidant enzymes.

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Significance: The observations bring into light the significant neuroprotective potentials of

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melatonin in Hcy model of PD which is attributed to the attenuation of oxidative stress in SN.

Key Words: Antioxidant; Rotational bias; Dopamine; Complex-I; Hydroxyl radical; Neuroprotection

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ACCEPTED MANUSCRIPT Introduction Amongst the various pathologies, mitochondrial dysfunction and resulting oxidative stress are known contributing factors to the destruction of dopaminergic neurons in substantia nigra (SN) pars compacta region of midbrain in Parkinson’s disease (PD) [1,2]. Moreover,

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age-dependent elevation of some endogenous molecules in brain, such as homocysteine (Hcy), further contributes to neurotoxicity by causing oxidative stress which might lead to the

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progression of disease [3-5]. Hcy, apart from other endogenous molecules, is in the limelight

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of PD research. This is because Hcy level is known to increase in blood and dopaminergic regions of brain of patients and animal models of PD under prolonged treatment with

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levodopa, the gold-standard drug of PD [5-8]. Significant reports of last decade provided

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evidence of neurotoxicity and selectivity of Hcy towards dopaminergic neurons [9-12]. This includes parkinsonian neurotoxins, such as 6-hydroxydopamine (6-OHDA), rotenone and

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MPTP [13-16]. The neurotoxic potency of Hcy is mainly attributed to oxidative stress-

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induced damages as it autoxidizes and thus acts as a pro-oxidant [11,12,17]. However, treatment with antioxidant molecules, such as ascorbic acid and folic acid, reported preventing Hcy-induced neuronal damages [18,19]. Recently, we have proclaimed the Hcy-

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induced hemiparkinsonian model and demonstrated the involvement of mitochondrial

in rat [12].

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dysfunction and oxidative stress mechanism in Hcy-induced dopaminergic neurodegeneration

Several antioxidant molecules have been screened for their neuroprotective effect in different animal models of PD [20,21]. Parting few contradictory observations [22,23], melatonin has shown potential anti-parkinsonian effect in cellular as well as animal models of PD induced by neurotoxins, such as 6-OHDA, rotenone and MPTP [13,24-27]. Melatonin, a pineal indoleamine, implicated to play a pivotal role in the regulation of cellular antioxidant homeostasis [28,29]. The antioxidant and free-radical scavenging properties of melatonin are 3

ACCEPTED MANUSCRIPT the contributing factors for protection against various disease processes where ROS-induced damages play the pivotal role such as neurodegenerative diseases [24,29-32]. Interestingly, administration of melatonin is reported to ameliorate the elevated levels of Hcy in plasma in animal models [33-35]. However, reports on the effect of melatonin on Hcy-induced parkinsonian pathologies are limited. Thus, the antioxidant and neuroprotective potentials of

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melatonin were validated in the hemiparkinsonian rat model induced by unilateral intranigral

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injection of Hcy.

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2. Materials and methods

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2.1. Animals

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Adult male rats of Sprague-Dawley strain (225 ± 25 g) used in the present study were

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provided with food and purified water ad libitum and maintained under standard laboratory conditions. All the experimental protocols of the study have been approved by the Animal

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Ethics Committee, Assam University, Silchar, India (IEC/AUS/2013-052; dt-20/3/13).

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2.2. Drugs and chemicals Hcy (H4628), dopamine (H8502), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 2,3-dihydroxybenzoic acid (2,3-DHBA; 126209), 2,5dihydroxybenzoic acid (2,5-DHBA; 78069), reduced glutathione (GSH; G4251), damphetamine, apomorphine, ethylenediaminetetraacetic acid disodium salt (EDTA), heptane sulfonic acid, triethylamine, orthophosphoric acid, poly-L-lysine, perchloric acid, hydrogen peroxide (H2O2), poly-L-lysine, Triton X-100, 3,3-diaminobenzidine (DAB) liquid substrate system (D3939) kit and water for high-performance liquid chromatography (HPLC) were 4

ACCEPTED MANUSCRIPT purchased from Sigma-Aldrich Co. USA. Melatonin (70902), acetonitrile, salicylic acid, pyrogallol, sodium borohydride and n-amyl alcohol were obtained from SISCO Research Laboratories, India. Rabbit anti-tyrosine hydroxylase (TH; ab112) primary antibody and donkey serum (ab7475) were purchased from Abcam, Cambridge, UK. Horseradish peroxidase (HRP) conjugated anti-rabbit goat secondary antibody (ap307p) was purchased

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from Millipore Co., USA.

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2.3. Injection of Hcy in SN by Stereotaxic surgery

Hcy model of PD was generated by injecting a single dose of Hcy at a dose of 1.0

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μmol in 2.0 μl of saline in the SN region contralateral to the side of saline injection as

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described by Bhattacharjee and Borah, [12]. Briefly, rats were deeply anesthetized with ketamine and xylazine at 80 mg/kg and 8 mg/kg body weight and placed in a stereotaxic frame (Stoelting, USA). The incisor bar was kept at 3.5 mm below the interaural line. Hcy

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was injected in the right SN at the stereotaxic coordinates: antero-posterior - 0.58 cm; lateral -

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0.22 cm; and dorso-ventral - 0.85 cm from the Bregma point [36] at flow rate of 0.5 μl/min. Proper postoperative cares were taken to minimize sufferings of animals.

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2.4. Experimental Design

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Hcy injected rats were administered with or without melatonin for 18 consecutive days following 2 h post-surgery. Melatonin dissolved in 5% ethanol solution and injected intraperitoneally at 10, 20 and 30 mg/kg dose based on previous reports on neuroprotection without any adverse side effects [24,25,27]. Mitochondrial complex-I activity, hydroxyl radical (•OH), GSH and activity of antioxidant enzymes (SOD, CAT) were analyzed from the contra- and ipsilateral SN on the 5th day following surgery (6 animals/assay) following Bhattacharjee and Borah, [12]. The drug-induced rotational behavioral test was performed on 14th and 16th-day post-surgery with Hcy (8 animals/tests) by an experimenter naive to the 5

ACCEPTED MANUSCRIPT animal groups. The animals were sacrificed on 19th-day for analysis of dopamine and its metabolites (DOPAC and HVA) levels [37] and perfused with paraformaldehyde for THimmunohistochemistry (5 animals/assay).

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2.5. Rotational behavior

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A cylindrical cage of 50 cm2 wide was used to measure drug-induced rotational

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behavior following Bhattacharjee and Borah [12]. Amphetamine was injected (5 mg/kg, i.p.) on the 14th day post-injection of Hcy and the complete turns toward ipsilateral side were

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recorded for 240 min. On the 16th day of post-surgery, the same sets of animals were injected

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with apomorphine (1 mg/kg, s.c.) and the complete turns in contralateral side were recorded for 120 min [37].

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2.6. Analysis of brain catecholamines

The levels of dopamine and its metabolites (DOPAC and HVA) were estimated from

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the contralateral and ipsilateral striatum on the 19th day of post-injection with Hcy. The striatum was processed and the levels of catecholamines were analyzed using HPLC-

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electrochemical detection procedure [38,39]. Briefly, the contralateral and ipsilateral striatum were individually sonicated in 0.1 M perchloric acid containing 0.01% EDTA followed by centrifugation at 10,000 × g for 5 min. 10 l of the supernatant was injected into the Rheodyne injector equipped with the HPLC-ECD system (WATERS, Austria). The flow rate of mobile phase was maintained at 0.8 ml/min and the electrochemical detection was performed at +740 mV. 100 ml of mobile phase contains heptane sulfonic acid (0.19 g), acetonitrile (13 ml), triethylamine (0.43 ml), orthophosphoric acid (0.32 ml) and EDTA (10 mg) in HPLC grade water. 6

ACCEPTED MANUSCRIPT 2.7. Tyrosine hydroxylase-immunohistochemistry Animals from all the experimental groups were perfused intracardially with cold phosphate buffered saline (0.1 M, pH 7.4) and 4% paraformaldehyde on the 19th day postsurgery. Brains were post-fixed overnight in the same fixative followed by cryoprotection in

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30% sucrose. Twenty-micron coronal sections of SN were taken in well-plates containing Tris-buffered saline (TBS; 0.1 M, pH 7.4) and processed for TH-immunohistochemistry

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following Paul et al. [38]. After washing the sections in TBS the internal endogenous

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activities were blocked by incubating the sections in 3% H2O2 for 5 min followed by blocking of non-specific binding of antibodies by incubating the sections in TBS containing

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10% donkey serum and 0.3% Triton X-100 for 1 h at room temperature. Following this, the

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sections were washed in TBS and incubated for overnight at 4°C with primary antibody (1:500) in TBS containing 2% donkey serum and 0.3% Triton X-100. The sections were then washed with TBS and were incubated at room temperature for 1 h with HRP-conjugated

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secondary antibody (1:1000) in TBS containing 2% donkey serum and 0.3% Triton X-100. After washing in TBS, the sections were subjected to DAB liquid substrate system for colour development. The sections were processed for mounting and photographed using a digital

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SLR camera attached to the microscope (Ci-L, Nikon, Japan).

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TH-positive nigral dopaminergic neurons were counted using ImageJ (Fiji version) software following Paul et al. [39]. TH-immunoreactive photographs of serial sections of SN pars compacta from five brain samples of each group were used for counting the TH-positive neurons.

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ACCEPTED MANUSCRIPT 2.8. Mitochondrial complex-I activity

Hcy infused rats treated with or without melatonin were sacrificed on the 5th day following surgery and complex-I activity was assessed from contralateral and ipsilateral SN.

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We followed the method of Saravanan et al. [40] with slight modifications [12]. The reaction mixture contains 100 mg mitochondrial fractions, 5 mM sodium azide and 50 mM coenzyme

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Q0 in 10 mM potassium phosphate buffer. After incubating the reaction mixture at 32 °C for

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3 min, the reaction was started by adding 100 μM NADH. The rate of decrease in absorbance

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was measured at 340 nm for 3 min using Spectrophotometer (MulitiskanGO, Thermo Fisher

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Scientific, Finland).

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2.9. Estimation of hydroxyl radical

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For investigating the effect of melatonin on Hcy-induced generation of •OH in SN, the animals were injected with salicylic acid (100 mg/kg; i.p) on 5th day post-surgery after 2 h

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of melatonin treatment. Animals were sacrificed by decapitation after 90 min of salicylic acid treatment and its adducts: 2,3- and 2,5-DHBA, were estimated from contralateral and

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ipsilateral SN to quantify the amount of •OH using HPLC-ECD system [38]. The micropunched SN from contralateral and ipsilateral side were processed similarly as used for detection of catecholamines. The supernatant was injected (10 μl) into the HPLC-ECD system to check the levels of 2,3- and 2,5-DHBA against the standards.

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ACCEPTED MANUSCRIPT 2.10. Estimation of reduced glutathione

Contralateral and ipsilateral SN was micro-punched from 1 mm frozen sections and processed for the analyses of GSH by employing an HPLC-ECD procedure on 5th day

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following unilateral infusion of Hcy [38,41]. 20 µl of the tissue homogenate was incubated at 40 °C for 30 min in a reaction mixture containing sodium borohydride (1.43 mM), sodium

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hydroxide (66 mM), n-amyl alcohol (10 µl) and EDTA (1.5 mM). Following precipitation of

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proteins using perchloric acid (0.4 M), the reaction mixture was centrifuged at 14,000 x g for 25 min at 4 °C. 10 μl of supernatant was injected in HPLC-ECD system. The flow rate of

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mobile phase was maintained at 0.8 ml/min and the electrochemical detection was performed

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at +850 mV. The mobile phase (100 ml, pH 2.7) composed of sodium phosphate monobasic (50 mM), 1-octanesulfonic acid (1.0 mM) and acetonitrile (2%).

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2.11. Determination of SOD activity

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Pyrogallol autoxidation method of Marklund and Marklund [42] was employed to assess the activity of SOD with minor modifications [12]. SOD activity was analyzed from

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cytosolic fraction of the contralateral and ipsilateral SN on 5th day post-surgery. The reaction mixture composed of freshly prepared pyrogallol (0.2 mM) and EDTA (1 mM) in Tris-HCl

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buffer (50 mM, pH 8.2). In presence or absence of the enzyme, pyrogallol auto-oxidation was recorded at 420 nm for 3 min using Spectrophotometer. The inhibition of pyrogallol oxidation was linear with the activity of the enzyme present.

2.12. Determination of catalase activity Catalase activity was analyzed in the cytosolic fractions of contralateral and ipsilateral SN [12]. The enzyme extract used in the determination of SOD was diluted in phosphate buffer (50 mM, pH 7.0) depending on the content of protein in the supernatant. In the assay

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ACCEPTED MANUSCRIPT mixture, H2O2 (30 mM) was added and the rate of decomposition of hydrogen peroxide was measured at 240 nm for 30 s.

2.13. Statistical analysis Statistical analysis was performed using the GraphPad Prism version 7 software. The data were analyzed employing one-way analysis of variance (ANOVA) followed by

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Newman-Keuls post-hoc test. Results are given as mean ± S.E.M. Values of P≤ 0.05 were

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considered significant.

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3. Results

3.1. Melatonin on drug-induced rotations in Hcy model of PD

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To examine the effect of melatonin on rotational behavior in Hcy model of PD, the animals infused unilaterally with Hcy were injected with amphetamine and apomorphine on

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14th and 16th day respectively following surgery. Melatonin administration in Hcy-induced

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parkinsonian rats resulted in significant decrease (P<0.05) in apomorphine- and amphetamine-induced rotational bias (Fig 2). Amphetamine administration caused ipsilateral

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rotations in Hcy-infused animals that lasted for 240 mins. The total number of rotations in the ipsilateral side exhibited by Hcy infused rat after amphetamine administration was 835 ±

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50.14. Melatonin at the dose of 30 mg/kg significantly decreased the Hcy-induced rotational bias by 35.71% after amphetamine administration (Fig 2A). The lower doses of melatonin (10 and 20 mg/kg) also decreased the total number of ipsilateral rotations by 4.43% and 18.93% respectively, but was not statistically significant. Administration of apomorphine in Hcy-infused caused contralateral rotations (318.4 ± 29.33) which lasted for 120 mins. Melatonin at the dose of 30 mg/kg and 20 mg/kg significantly decreased the apomorphine-

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ACCEPTED MANUSCRIPT induced ipsilateral rotational bias in Hcy-infused animals by 54% and 47% respectively, while the lowest dose (10 mg/kg) of melatonin did not cause a significant alteration (Fig 2B).

3.2. Melatonin on Hcy-induced striatal dopamine depletion Intranigral injection of Hcy caused a significant reduction in striatal dopamine,

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DOPAC and HVA content by 64%, 57%, and 50% respectively, while dopamine turnover was increased significantly by 20%, as compared to the vehicle-injected side (Fig 3; P<0.05).

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In Hcy-injected animals, melatonin treatment at 30 mg/kg dose significantly elevated striatal

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dopamine and DOPAC content, without affecting HVA, in the ipsilateral side compared to the vehicle-injected side contralateral to Hcy infused side (Fig 3). The highest dose of

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melatonin significantly elevated the striatal dopamine and DOPAC content by 1.98-fold and

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1.59-fold respectively, compared to the ipsilateral side injected with Hcy. Melatonin at 10 and 20 mg/kg dose did not prevent Hcy-induced loss of striatal dopamine and its metabolites levels. The increased dopamine turnover in the ipsilateral striatum of Hcy injected side of SN

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was reduced significantly by 27% after melatonin treatment at 30 mg/kg dose (Fig 3).

3.3. Melatonin on Hcy-induced loss of dopaminergic neurons

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Dopaminergic neurons in SN were stained specifically with TH-immunoreactivity. The SN ipsilateral to Hcy infusion showed less number of TH-positive neurons (Fig 4), as

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compared to the contralateral side. Melatonin treatment at 30 mg/kg dose for 18 days caused a visible increase in the number of TH-positive neurons in the ipsilateral side to Hcy-injection (Fig 4E), compared to the Hcy-injected animals without melatonin treatment (Fig 4B). The TH-immunoreactive photographs were subjected to ImageJ software for counting the number of TH-positive neurons in SN and are represented as the percentage of surviving neurons to vehicle-injected contralateral side (Fig 4F). In neuronal counts, Hcy causes 60% reduction in TH-positive neurons in the side ipsilateral to Hcy infusion in SN, while melatonin at dose 30 mg/kg significantly recovered Hcy-induced loss of TH-positive neurons by 37% (P<0.05) 11

ACCEPTED MANUSCRIPT compared to the ipsilateral SN to Hcy injection without melatonin treatment. Melatonin treatment at doses 10 and 20 mg/kg did not recover the loss of TH-positive neurons in SN induced by Hcy injection (Fig 4).

3.4.

Melatonin

on

Hcy-induced

mitochondrial

complex-I

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inhibition Hcy-induced inhibition of mitochondrial complex-I activity in right SN was

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significantly recovered in animals after treatment with higher doses of melatonin (20 and 30

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mg/kg). Mitochondrial complex-I activity was found to be significantly inhibited by 31.31%

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(P<0.05) in the Hcy-infused side compared to vehicle-injected side of SN. Administration of melatonin at doses 20 and 30 mg/kg caused a significant increase in complex-I activity by

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1.36- and 1.32-fold respectively in the ipsilateral side to Hcy injection compared to Hcyinfused animals (Fig 5). However, the lower dose of melatonin (10 mg/kg) did not cause any

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significant alterations in the reduced activity of mitochondrial complex-I activity induced by

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Hcy.

3.5. Melatonin on Hcy-induced hydroxyl radical (•OH) generation

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Melatonin administration in Hcy-induced hemiparkinsonian rats attenuated the Hcyinduced •OH generation in SN (Fig 6). 2,3- and 2,5-DHBA, the indicators of •OH, levels

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were found to be increased significantly by 1.82- and 2.51-fold (P<0.05) respectively in the ipsilateral SN infused with Hcy compared to the contralateral side (Fig 6). Melatonin at the dose of 10, 20 and 30 mg/kg significantly decreased the Hcy-induced elevated levels of 2,3DHBA by 33.53%, 45%, and 46% respectively, while 2,5-DHBA was decreased by 42%, 49%, and 51% respectively. All the doses of melatonin were found to significantly decrease the level of total DHBA generated by intranigral injection of Hcy (Fig 6).

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ACCEPTED MANUSCRIPT 3.6. Melatonin on Hcy-induced GSH depletion Melatonin treatment at doses 20 and 30 mg/kg twice daily following 2 h of intranigral unilateral injection of Hcy significantly improved the Hcy-induced depleted level of GSH (P<0.05). In naïve animals, injection of Hcy caused 37.2% significant reduction of GSH

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levels in ipsilateral SN compared to vehicle-injected side. The higher doses of melatonin (20 and 30 mg/kg) significantly elevated the Hcy-induced depleted levels of GSH in ipsilateral

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SN by 1.70- and 1.78-fold respectively (Fig 7).

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3.7. Melatonin on Hcy-induced increase in activity of antioxidant

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enzymes

The activity of antioxidant enzymes, SOD and catalase, was evaluated from the

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cytosolic fraction of SN in Hcy infused animals administered with or without melatonin. There occurred a significant increase in SOD and catalase activity respectively by 1.31- and

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1.38-fold (P<0.05) in SN ipsilateral to the side of Hcy injection compared to the contralateral

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side. Melatonin at higher doses (20 and 30 mg/kg) further significantly increased the activity of SOD in the ipsilateral SN compared to the respective contralateral side (Fig 8A).

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Moreover, higher doses of melatonin caused a significant increase in activity of SOD by 1.22- and 1.25-fold (P<0.05) in the ipsilateral SN compared to the SN of the animals infused

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with Hcy only (Fig 8A).

Melatonin treatment at all doses (10, 20, 30 mg/kg) increased the activity of catalase respectively by 1.4-, 1.55- and 1.58-fold (P<0.05) in Hcy injected ipsilateral SN compared to respective contralateral side (Fig 8B). Compared to the Hcy-infused SN, there occurred significant elevation of catalase activity by 1.17-, 1.33- and 1.28-fold respectively by 10, 20 and 30 mg/kg doses of melatonin in the ipsilateral SN (Fig 8B).

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ACCEPTED MANUSCRIPT 4. Discussion The finding describes the potent neuroprotective effect of melatonin in Hcy model of PD. The potential findings of the study are that melatonin dose-dependently reverses Hcyinduced (i) motor abnormalities, (ii) dopamine depletion in striatum, (iii) loss of TH-positive

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neurons in SN, (iv) inhibition of mitochondrial complex-I activity in SN, and (v) oxidative stress in SN.

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Previous studies have convincingly demonstrated the specific dopaminergic

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neurotoxic potentials of Hcy in cellular as well as animal models [5,10,12]. Our previous report [12] and those of Chandra et al. [10] have shown that unilateral intranigral injection of

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Hcy in rat caused the stereotypic rotational behavior of PD as a result of depletion of striatal

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dopamine by more than 60%. The underlying mechanism of Hcy-induced dopaminergic neurotoxicity was found to be due to mitochondrial complex-I inhibition with subsequent

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generation of •OH and enhanced activity of antioxidant enzymes in SN [12], which is

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consistent with the present findings. Based on these findings, Hcy model of PD was proclaimed to be a viable model [12].

Melatonin has shown promising protective effect in the experimental model of PD

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[24-27]. The free radical scavenging effect and oxidative stress-ameliorating potency of

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melatonin have been reported to be the underlying mechanism of neuroprotection [28,29]. Moreover, Hcy-induced neurotoxicity was reported to be ameliorated upon treatment with antioxidants [18,19]. Since melatonin is a strong antioxidant molecule [31,32] and Hcy is known to induce free radical stress [18,19], therefore melatonin was injected 2 hours postsurgery, allowing Hcy to develop its toxicity. Mounting evidence documented the antioxidant property of melatonin in several animal models of neurodegenerative diseases, including PD [24,29-31]. However, the present study is the first demonstration on the neuroprotective potentials of melatonin in Hcy-induced rat model of PD. 14

ACCEPTED MANUSCRIPT The doses of melatonin that have shown neuroprotective efficacy in different animal models of PD is ranging from 5 mg to 30 mg/kg b.w and in most of the studies, 30 mg is the maximum dose with significant neuroprotection without any side-effects [24,25,27]. Therefore, we choose 10, 20 and 30 mg/kg doses of melatonin. Melatonin treatment at dose 30 mg/kg replenished striatal dopamine and DOPAC level and thereby reduces dopamine

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turnover in the ipsilateral striatum of Hcy-infused animal (Fig 3). Previous studies

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demonstrated that melatonin significantly restored the loss of striatal dopamine level induced

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by 6-OHDA lesion [43,44]. Interestingly, in the present study, the same dose of melatonin (30 mg/kg) was found to prevent the loss of dopaminergic neurons in Hcy model of PD as

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evident from the improved counting of TH-positive neurons in Hcy infused side of SN (Fig 4). Studies have also shown the dopaminergic neuroprotective potentials of melatonin in

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animal models of PD [27,45]. Also, several studies reported that melatonin prevents neuronal cell death from toxic insults by influencing the anti-apoptotic proteins and factors [4,46].

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Thus, the observed restoration of striatal dopamine level by melatonin at 30 mg/kg dose (Fig

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3), but not at other doses, is attributed to its ability to prevent dopaminergic neuronal loss in SN at the same dose (Fig 4) in Hcy model of PD. Melatonin by elevating the levels of striatal

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dopamine contributes to the betterment of amphetamine- and apomorphine-induced rotational bias in Hcy-induced parkinsonian rats (Fig 2), suggesting that melatonin acts at the

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postsynaptic level. In this respect, melatonin administration in 6-OHDA lesioned model of PD has shown to ameliorate stereotypic rotational behavior produced by amphetamine and apomorphine [30,47]. Functional impairment of mitochondria at complex-I is linked with PD pathology [1,2] and interestingly administration of melatonin significantly recovered reduction of complex-I activity induced by Hcy (Fig 5). Efficacy of melatonin to attenuate complex-I inhibition by specific and non-specific complex-I toxins in the experimental model of PD was

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ACCEPTED MANUSCRIPT reported earlier [25,30,48]. Our previous report [12] and from the present result it is evident that on the 19th day following intranigral injection of Hcy more than 60% loss of dopaminergic neurons in SN pars compacta occurred. Thus, there is no point of performing various assays for detection of ROS and other oxidative stress parameters at the time point when most of the neurons are dead. Since ROS generation precedes much before the neuronal

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death in PD, which is the early event in the disease pathogenesis, therefore we sacrificed the

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animals on 5th following surgery to perform the assays of oxidative stress parameters as done

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by others [12,25]. It has been found that melatonin at higher doses significantly protected the dopaminergic neurons in SN from Hcy-induced toxic insults of oxidative stress by i)

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scavenging •OH, ii) elevating the levels of the cellular antioxidant molecule, GSH, and iii) up-regulating the activity of antioxidant enzymes, SOD and catalase. Our result thus provided

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further supportive evidence of the appearance of equivalent PD-pathologies induced by intranigral injection of Hcy.

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In PD, mitochondrial complex-I inhibition is reported to elevate the levels of ROS and

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complex-I is vulnerable to ROS-induced functional impairment [1,2,49]. Furthermore, the best known parkinsonian neurotoxins elevate •OH by depleting the levels of GSH in the SN

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and thus further lead to complex-I inhibition [25,30]. Thus, melatonin treatment-induced recovery of the complex-I activity in SN (Fig 5) could be due to dose-dependent attenuation

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of •OH (Fig 6), and subsequent elevation of GSH level (Fig 7) in SN, as observed in the present study. The present study confirms the contribution of antioxidant potency of melatonin as one of the major factor of neuroprotection in Hcy model of PD. The cellular redox potential regulated by melatonin is attributed to its ability to stimulate the production of antioxidant enzymes and molecule [50,51]. Moreover, the derivatives of melatonin produced as a result of scavenging of free radicals are also known to confer antioxidant and radical scavenging activity [52,53]. In the present study, Hcy-induced

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ACCEPTED MANUSCRIPT elevated activity of the antioxidant enzymes was found to be further increased upon melatonin treatment at higher doses (Fig 8). Following melatonin treatment, increased activity of SOD and catalase in brain was found in naïve animals [54] and also in MPTPtreated animals [24]. In addition to melatonin’s influence on the SOD gene expression in naïve animals, it further stimulates the activity of SOD and catalase in degenerating SN [24].

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In short, melatonin in Hcy model of PD prevents dopamine neuronal loss and thus

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replenishes striatal dopamine level and ameliorates oxidative stress by improving complex-I function, elevating the activity of antioxidant enzymes and molecule as well as by scavenging

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so produced •OH. The significance of using melatonin in Hcy model of PD, apart from the

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present observation, is that melatonin treatment reported reducing elevated levels of homocysteine in plasma [33-35]. Thus, the present study on the neuroprotective effect of

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melatonin in Hcy model of PD is at least in part attributed to melatonin’s antioxidant and free

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Acknowledgement

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radical scavenging properties.

We acknowledge the funding and support provided by the Department of

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Biotechnology, Government of India (Sanction Order No. BT/PR6806/GBD/27/480/2012,

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dated August 05, 2013).

17

ACCEPTED MANUSCRIPT References [1] Panov A, Dikalov S, Shalbuyeva N, Taylor G, Sherer T, Greenamyre JT. Rotenone model of Parkinson disease: Multiple brain mitochondria dysfunctions after short term systemic rotenone intoxication. J Biol Chem. 2005; 280: 42026-42035.

PT

[2] Dexter DT, Jenner P. Parkinson disease: from pathology to molecular disease mechanisms. Free Radic Biol Med. 2013; 62: 132-144.

RI

[3] Doherty GH. Homocysteine and Parkinson’s Disease: A Complex Relationship. J Neurol

SC

Disord. 2013, 1: 107.

[4] Paul R, Borah A. The potential physiological crosstalk and interrelationship between two

NU

sovereign endogenous amines, melatonin and homocysteine. Life Sci. 2015; 139: 97-107.

MA

[5] Paul R, Borah A. L-DOPA-induced hyperhomocysteinemia in Parkinson's disease: Elephant in the room. Biochim Biophys Acta. 2016; 1860(9): 1989-1997.

D

[6] Zoccolella S, dell'Aquila C, Specchio LM, Logroscino G, Lamberti P. Elevated

PT E

homocysteine levels in Parkinson's Disease: is there anything besides L-dopa treatment? Curr Med Chem. 2010; 17(3): 213-221. [7] Shin JY, Ahn YH, Paik MJ, Park HJ, Sohn YH, Lee PH. Elevated homocysteine by

AC

e50496.

CE

levodopa is detrimental to neurogenesis in parkinsonian model. PLoS One. 2012; 7(11):

[8] Bhattacharjee N, Mazumder MK, Paul R, Choudhury A, Choudhury S, Borah A. LDOPA treatment in MPTP-mouse model of Parkinson's disease potentiates homocysteine accumulation in substantia nigra. Neurosci Lett. 2016; 628: 225-229. [9] Lee ES, Chen H, Soliman KF, Charlton CG. Effects of homocysteine on the dopaminergic system and behavior in rodents. Neurotoxicology. 2005; 26(3): 361-371.

18

ACCEPTED MANUSCRIPT [10]

Chandra G, Gangopadhyay PK, Senthil Kumar KS, Mohanakumar KP. Acute

intranigral homocysteine administration produces stereotypic behavioral changes and striatal dopamine depletion in Sprague-Dawley rats. Brain Res. 2006; 1075(1): 81-92. [11]

Bhattacharjee N, Paul R, Giri A, Borah A. Chronic exposure of homocysteine in mice

contributes to dopamine loss by enhancing oxidative stress in nigrostriatum and produces

Bhattacharjee N, Borah A. Oxidative stress and mitochondrial dysfunction are the

RI

[12]

PT

behavioral phenotypes of Parkinson’s disease, Biochem. Biophys. Rep. 2016; 6: 47–53.

SC

underlying events of dopaminergic neurodegeneration in homocysteine rat model of Parkinson's disease. Neurochem Int. 2016; 101: 48-55.

Borah A, Mohanakumar KP. Salicylic acid protects against chronic L-DOPA-induced

NU

[13]

6-OHDA generation in experimental model of parkinsonism. Brain Res. 2010; 1344: 192-

[14]

MA

199. s

Borah A, Mohanakumar KP. L-DOPA induced-endogenous 6-hydroxydopamine is

D

the cause of aggravated dopaminergic neurodegeneration in Parkinson's disease patients.

[15]

PT E

Med Hypotheses. 2012; 79(2): 271-273. Cicchetti F, Drouin-Ouellet J, Gross RE. Environmental toxins and Parkinson's

CE

disease: what have we learned from pesticide-induced animal models? Trends Pharmacol Sci. 2009; 30: 475-483.

Goldman SM. Environmental toxins and Parkinson's disease. Annu Rev Pharmacol

AC

[16]

Toxicol. 2014; 54: 141-64. [17]

Baydas G, Koz ST, Tuzcu M, Etem E, Nedzvetsky VS. Melatonin inhibits oxidative

stress and apoptosis in fetal brains of hyperhomocysteinemic rat dams. J Pineal Res 2007; 43: 225-231. [18]

Ataie A, Sabetkasaei M, Haghparast A, Moghaddam AH, Kazeminejad B.

Neuroprotective effects of the polyphenolic antioxidant agent, Curcumin, against

19

ACCEPTED MANUSCRIPT homocysteine-induced cognitive impairment and oxidative stress in the rat. Pharmacol Biochem Behav. 2010; 96: 378-85. [19]

Machado FR, Ferreira AG, da Cunha AA, Tagliari B, Mussulini BH, Wofchuk S,

Wyse AT. Homocysteine alters glutamate uptake and Na+,K+-ATPase activity and oxidative status in rats hippocampus: protection by vitamin C. Metab Brain Dis. 2011; 26:

Mythri RB, Bharath MM. Curcumin: a potential neuroprotective agent in Parkinson's

RI

[20]

PT

61-67.

[21]

SC

disease. Curr Pharm Des. 2012; 18 (1): 91-99.

Jin H, Kanthasamy A, Ghosh A, Anantharam V, Kalyanaraman B, Kanthasamy AG.

NU

Mitochondria-targeted antioxidants for treatment of Parkinson's disease: preclinical and clinical outcomes. Biochim Biophys Acta. 2014; 1842(8): 1282-1294. van der Schyf CJ, Castagnoli K, Palmer S, Hazelwood L, Castagnoli N Jr. Melatonin

MA

[22]

fails to protect against long-term MPTP-induced dopamine depletion in mouse striatum.

Tapias

V,

Cannon

JR,

Greenamyre

PT E

[23]

D

Neurotox Res. 2000; 1: 261–269.

JT.

Melatonin

treatment

potentiates

neurodegeneration in a rat rotenone Parkinson's disease model. J Neurosci Res. 2010; 88:

[24]

CE

420-427.

Thomas B, Mohanakumar KP. Melatonin protects against oxidative stress caused by

AC

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in the mouse nigrostriatum. J Pineal Res. 2004; 36(1): 25-32. [25]

Saravanan KS, Sindhu KM, Mohanakumar KP. Melatonin protects against rotenone-

induced oxidative stress in a hemiparkinsonian rat model. J Pineal Res. 2007; 42(3): 247253.

20

ACCEPTED MANUSCRIPT [26]

Borah A, Mohanakumar KP. Melatonin inhibits 6-hydroxydopamine production in the

brain to protect against experimental parkinsonism in rodents. J Pineal Res. 2009; 47(4): 293-300. [27]

Naskar A, Prabhakar V, Singh R, Dutta D, Mohanakumar KP. Melatonin enhances L-

DOPA therapeutic effects, helps to reduce its dose, and protects dopaminergic neurons in

PT

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinsonism in mice. J Pineal

Pohanka M, Bandouchova H, Pikula J. Melatonin influences antioxidant homeostasis

SC

[28]

RI

Res. 2015; 58(3): 262-274.

and basal metabolism in the BALB/c mouse model. Neuro Endocrinol Lett. 2012; 33

[29]

NU

Suppl 3: 183-189.

Manchester LC, Coto-Montes A, Boga JA, Andersen LP, Zhou Z, Galano A, Vriend

MA

J, Tan DX, Reiter RJ. Melatonin: an ancient molecule that makes oxygen metabolically tolerable. J Pineal Res. 2015; 59(4): 403-419.

Dabbeni-Sala F, Di Santo S, Franceschini D, Skaper SD, Giusti P. Melatonin protects

D

[30]

PT E

against 6-OHDA-induced neurotoxicity in rats: a role for mitochondrial complex I activity. FASEB J. 2001; 15(1): 164-170. Pandi-Perumal SR, BaHammam AS, Brown GM, Spence DW, Bharti VK, Kaur C,

CE

[31]

Hardeland R, Cardinali DP. Melatonin antioxidative defense: therapeutical implications

[32]

AC

for aging and neurodegenerative processes. Neurotox Res. 2013; 23(3): 267-300. Reiter RJ, Tan DX, Poeggeler B, Menendez-Pelaez A, Chen LD, Saarela S. Melatonin

as a free radical scavenger: implications for aging and age-related diseases. Ann N Y Acad Sci. 1994; 719: 1-12. [33]

Baydas G, Gursu MF, Cikim G, Canatan H. Homocysteine levels are increased due to

lack of melatonin in pinealectomized rats: is there a link between melatonin and homocysteine? J Pineal Res. 2002; 32(1): 63-64.

21

ACCEPTED MANUSCRIPT [34]

Murawska-Cialowicz E, Januszewska L, Zuwala-Jagiello J, Milczarska J, Zawadzki

M, Paprocka-Borowicz M, Wierzbicka-Damska I. Melatonin decreases homocysteine level in blood of rats. J Physiol Pharmacol. 2008; 59(4): 717-729. [35]

Kantar Ş, Türközkan N, Bircan FS, Paşaoğlu ÖT. Beneficial effects of melatonin on

serum nitric oxide, homocysteine, and ADMA levels in fructose-fed rats. Pharm Biol.

Paxinos G, Watson C. The Rat Brain in Stereotaxic Coordinates. 7th Ed 2013;

RI

[36]

PT

2015; 53(7): 1035-1041.

[37]

SC

Academic Press, San Diego.

Sindhu KM, Banerjee R, Senthilkumar KS, Saravanan KS, Raju BC, Rao JM,

NU

Mohanakumar KP. Rats with unilateral median forebrain bundle, but not striatal or nigral,

MA

lesions by the neurotoxins MPP+ or rotenone display differential sensitivity to amphetamine and apomorphine. Pharmacol Biochem Behav. 2006; 84(2): 321-329. [38]

Paul R, Choudhury A, Kumar S, Giri A, Sandhir R, Borah A. Cholesterol contributes

PT E

D

to dopamine-neuronal loss in MPTP mouse model of Parkinson's disease: Involvement of mitochondrial dysfunctions and oxidative stress. PLoS One. 2017; 12(2): e0171285. [39]

Paul R, Choudhury A, Chandra Boruah D, Devi R, Bhattacharya P, Choudhury MD,

CE

Borah A. Hypercholesterolemia causes psychomotor abnormalities in mice and alterations

AC

in cortico-striatal biogenic amine neurotransmitters: Relevance to Parkinson's disease. Neurochem Int. 2017 Feb 3. pii: S0197-0186(16)30231-5. [40]

Saravanan KS, Sindhu KM, Senthilkumar KS, Mohanakumar KP. L-deprenyl protects

against rotenone-induced, oxidative stress-mediated dopaminergic neurodegeneration in rats. Neurochem Int. 2006; 49(1): 28-40. [41]

Melnyk S, Pogribna M, Pogribny I, Hine RJ, James SJ. A new HPLC method for the

simultaneous determination of oxidized and reduced plasma aminothiols using coulometric electrochemical detection. J Nutr Biochem. 1999; 10: 490-497. 22

ACCEPTED MANUSCRIPT [42]

Marklund S, Marklund G. Involvement of the superoxide anion radical in the

autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur J Biochem. 1974; 47: 469-474. [43]

Joo WS, Jin BK, Park CW, Maeng SH, Kim YS. Melatonin increases striatal

dopaminergic function in 6-OHDA-lesioned rats. Neuroreport. 1998; 9(18): 4123-4126. Lin L, Meng T, Liu T, Zheng Z. Increased melatonin may play dual roles in the striata

PT

[44]

Antolín I, Mayo JC, Sainz RM, del Brío Mde L, Herrera F, Martín V, Rodríguez C.

SC

[45]

RI

of a 6-hydroxydopamine model of Parkinson's disease. Life Sci. 2013; 92(4-5): 311-316.

Protective effect of melatonin in a chronic experimental model of Parkinson's disease.

[46]

NU

Brain Res. 2002; 943(2): 163-173.

Jou MJ, Peng TI, Hsu LF, Jou SB, Reiter RJ, Yang CM, Chiao CC, Lin YF, Chen CC.

MA

Visualization of melatonin's multiple mitochondrial levels of protection against mitochondrial Ca(2+)-mediated permeability transition and beyond in rat brain astrocytes.

Sharma R, McMillan CR, Tenn CC, Niles LP. Physiological neuroprotection by

PT E

[47]

D

J Pineal Res. 2010; 48(1): 20-38.

melatonin in a 6-hydroxydopamine model of Parkinson's disease. Brain Res. 2006; 1068:

[48]

CE

230-236.

Tan DX, Manchester LC, Qin L, Reiter RJ. Melatonin: A Mitochondrial Targeting

AC

Molecule Involving Mitochondrial Protection and Dynamics. Int J Mol Sci. 2016; 17(12). pii: E2124. [49]

Subramaniam SR, Chesselet MF. Mitochondrial dysfunction and oxidative stress in

Parkinson's disease. Prog Neurobiol. 2013 Jul-Aug;106-107:17-32. [50]

Rodriguez C, Mayo JC, Sainz RM, Antolín I, Herrera F, Martín V, Reiter RJ.

Regulation of antioxidant enzymes: a significant role for melatonin. J Pineal Res. 2004; 36: 1-9.

23

ACCEPTED MANUSCRIPT [51]

Jimenez-Ortega V, Cano P, Cardinali DP, Esquifino AI. 24-Hour variation in gene

expression of redox pathway enzymes in rat hypothalamus: effect of melatonin treatment. Redox Rep. 2009; 14: 132-138. [52]

Onuki J, Almeida EA, Medeiros MH, Di Mascio P. Inhibition of 5-aminolevulinic

quercetin or resveratrol. J Pineal Res. 2005; 38: 107-115.

Tengattini S, Reiter RJ, Tan DX, Terron MP, Rodella LF, Rezzani R.

RI

[53]

PT

acid-induced DNA damage by melatonin, N1-acetyl-N2-formyl-5-methoxykynuramine,

[54]

SC

Cardiovascular diseases: protective effects of melatonin. J Pineal Res. 2008; 44: 16-25. Liu F, Ng TB. Effect of pineal indoles on activities of the antioxidant defense

NU

enzymes superoxide dismutase, catalase, and glutathione reductase, and levels of reduced

AC

CE

PT E

D

MA

and oxidized glutathione in rat tissues. Biochem Cell Biol 2000; 78: 447–453.

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ACCEPTED MANUSCRIPT Legends to Figures Fig 1: Schematic representation of the experimental paradigm. Abbreviations: Hcy, homocysteine; SN, substantia nigra; •OH, hydroxyl radical; GSH, glutathione; DA,

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dopamine; TH-IR, tyrosine hydroxylase-immunoreactivity.

Fig 2. Effect of melatonin on (A) amphetamine- and (B) apomorphine-induced

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rotational behavior in homocysteine (Hcy)-infused rat. Rats were treated with melatonin

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(10, 20 and 30 mg/kg; i.p.) daily following 2 h of post-surgery with Hcy. Amphetamineinduced ipsilateral and apomorphine-induced contralateral rotations were counted on 14th and

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16th day following surgery. Data represented as mean ± S.E.M., *P ≤ 0.05 as compared with

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Hcy-infused animals (n = 8).

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Fig 3. Effect of melatonin on homocysteine (Hcy)-induced changes striatal dopamine

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(DA), its metabolites and DA-turnover. Hcy infused rats were treated with or without melatonin (10, 20 and 30 mg/kg; i.p.) daily till 18th days from 2 h of post-surgery and sacrificed on 19th day. DA and its metabolites, DOPAC and HVA, were analyzed

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individually from the contra- and ipsilateral striatum by a sensitive HPLC-ECD system. DA-

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turnover was calculated as the ratio of the metabolites level to the neurotransmitter [DA turnover = (HVA + DOPAC): DA]. The data are expressed as mean ± S.E.M. *P ≤ 0.05 as compared with respective contralateral striatum and #P ≤ 0.05 as compared with ipsilateral striatum from the animals infused with Hcy (n = 5).

Fig 4. Effect of melatonin on homocysteine (Hcy)-induced loss of tyrosine hydroxylase (TH)-positive neurons in substantia nigra (SN). Animals treated with melatonin were sacrificed on 19th day post-injection with Hcy for TH-immunoreactivity. Right SN, which 25

ACCEPTED MANUSCRIPT received Hcy showed marked decrease in TH-positive neurons (B) as compared to the contralateral side infused with vehicle (A). TH-positive neurons from SN were counted using ImageJ software and are represented as the percentage of surviving neurons to the contralateral side. The data are expressed as mean ± S.E.M. *P ≤ 0.05 as compared with respective contralateral SN and #P ≤ 0.05 as compared with ipsilateral SN from the animals

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infused with Hcy (n = 5).

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Fig 5. Effect of melatonin on homocysteine (Hcy)-induced mitochondrial complex-I inhibition. Complex-I activity was analyzed in contra- and ipsilateral substantia nigra (SN)

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by using NADH as substrate. Specific activity is defined as nmol of NADH oxidized/min/mg protein and represented as mean ± S.E.M. *P ≤ 0.05 as compared with respective

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contralateral SN and #P ≤ 0.05 as compared with ipsilateral SN from the animals infused with

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Fig 6. Effect of melatonin on homocysteine (Hcy)-induced hydroxyl radical (•OH) generation in substantia nigra (SN) region. From micropunched SN, 2,3- and 2,5-

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dihydroxybenzoic acid (DHBA; •OH adducts of salicylate) formed were measured by employing HPLC-ECD system. Data are represented as mean ± S.E.M. *P ≤ 0.05 as

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compared with respective contralateral SN and #P ≤ 0.05 as compared with ipsilateral SN from the animals infused with Hcy (n = 6).

Fig 7. Effect of melatonin on depleted levels of glutathione (GSH) in homocysteine (Hcy)-induced hemiparkinsonian rats. GSH levels were analyzed from contra- and ipsilateral substantia nigra (SN) region. Data are represented as mean ± S.E.M. *P ≤ 0.05 as

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Fig 8. Effect of melatonin on homocysteine (Hcy)-induced changes in activity of (A) superoxide dismutase (SOD) and (B) catalase (CAT). SOD activity was analyzed

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employing pyrogallol oxidation method in cytosolic fractions contra- and ipsilateral SN

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which represents Cu-Zn SOD. One unit of the SOD activity is defined as 50%

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inhibition/min/mg protein. CAT activity was analyzed by monitoring the disappearance of hydrogen peroxide in presence of the enzyme. Specific activity of the CAT is described as the

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change in absorbance at 240 nm/min/mg protein. Data are represented as mean ± S.E.M. *P ≤ 0.05 as compared with respective contralateral SN and #P ≤ 0.05 as compared with ipsilateral

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SN from the animal infused with Hcy (n = 6).

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