Chronic mild stress augments MPTP induced neurotoxicity in a murine model of Parkinson's disease

Accepted Manuscript Chronic mild stress augments MPTP induced neurotoxicity in a murine model of Parkinson's disease

Udaiyappan Janakiraman, Thamilarasan Manivasagam, Arokiasamy Justin Thenmozhi, Chinnasamy Dhanalakshmi, Musthafa Mohamed Essa, Byoung-Joon Song, Gilles J Guillemin PII: DOI: Reference:

S0031-9384(16)30864-2 doi: 10.1016/j.physbeh.2017.01.046 PHB 11668

To appear in:

Physiology & Behavior

Received date: Revised date: Accepted date:

30 September 2016 5 January 2017 23 January 2017

Please cite this article as: Udaiyappan Janakiraman, Thamilarasan Manivasagam, Arokiasamy Justin Thenmozhi, Chinnasamy Dhanalakshmi, Musthafa Mohamed Essa, Byoung-Joon Song, Gilles J Guillemin , Chronic mild stress augments MPTP induced neurotoxicity in a murine model of Parkinson's disease. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Phb(2017), doi: 10.1016/j.physbeh.2017.01.046

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ACCEPTED MANUSCRIPT Chronic mild stress augments MPTP induced neurotoxicity in a murine model of Parkinson’s disease Udaiyappan Janakiraman1, Thamilarasan Manivasagam1,*, Arokiasamy Justin Thenmozhi1, Chinnasamy Dhanalakshmi1, Musthafa Mohamed Essa2,3, ByoungJoon Song4, Gilles J Guillemin5 Department of Biochemistry and Biotechnology, Annamalai University, Annamalainagar

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608002, Tamilnadu, India

Department of Food Science and Nutrition, Sultan Qaboos University, Muscat, Oman

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Ageing and Dementia Research Group, Sultan Qaboos University, Muscat, Oman,

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NIAA, National Institute of Health, Rockville, MD, USA,

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Neuroinflammation group, Faculty of Medicine and Health Sciences, Macquarie

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University, Sydney, Australia.

Neuropharmacology Group, ASAM, Macqurie University, Sydney, NSW, Australia

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Corresponding author:

Thamilarasan Manivasagam, PhD, Department of Biochemistry and Biotechnology,

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Annamalai University, Annamalainagar 608002, Tamilnadu, India. E. mail: [email protected]; [email protected]

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Keywords

Experimental Parkinson’s disease, chronic mild stress, neurodegeneration, oxidative stress, inflammation, signalling proteins.

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ACCEPTED MANUSCRIPT Abstract Depression is frequently encountered during Parkinson's disease (PD) as a non-motor feature, which has been reported to cause and exaggerate motor deficits and neurodegenerative events in experimental PD models. We studied the effect of chronic mild stress (CMS) (pre, post and pre&post) exposure mediated depression on motor and

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non-motor symptoms, oxidative stress, inflammation and brain derived neurotrophic

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factor (BDNF) levels and its related signalling molecules against the 1-methyl-4-phenyl-

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1,2,3,6-tetrahydropyridine/probenecid (MPTP/p) induced neurotoxicity in mice. CMS and MPTP/p-coexposed C57BL/6 mice exhibited low neuromuscular strength and stride

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length with enhanced oxidative stress and inflammation as compared to CMS or MPTP/p

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alone exposed mice. Coexposure diminished the levels of BDNF and expressions of pTrkB, p-ERK/ERK, p-AKT/AKT and p-CREB in nigrostriatal regions as compared to

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those of the alone exposure. CMS alone exposed mice showed more anxiety related

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behaviour with diminished expression of serotonin transporter as compared to MPTP/p alone injected group. Post-stress exposure to MPTP/p mice exhibited lowest motor and

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reflecting higher anxiety state with greatest enhancement in inflammation and reduction in the protein expression of stress and cell signalling markers as compared to pre and

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pre&post stress exposed PD mice. However, pre- and pre&post CMS exposed PD animals are more vulnerable to oxidative stress as compared with post-stress experienced MPTP/p mice. CMS mediated depression exacerbates motor/non-motor symptoms in MPTP/p- PD animals by modulating oxidative stress and various signalling molecules. Our results suggested that stress induced NMS can accelerate neurodegenerative processes in the PD in a progressive or expedited manner.

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ACCEPTED MANUSCRIPT 1. Introduction

Parkinson’s disease (PD) is a movement disorder, caused by loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) due to depletion of striatal dopamine (DA) (Hornykiewicz, 1975). However, clinical and basic neuroscientists have

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recently started to concentrate more on non-motor symptoms (NMS) such as depression,

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anxiety, memory deficits, apathy, insomnia, sleep disturbances, etc., since those deficits

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are frequently observed during all stages of disease (Chaudhuri et al., 2006; Chaudhuri and Schapira, 2009). It was reported that the stress played a vital role in the cause

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(Gowers, 1888) and progression of PD (Smith, 2002). Moreover post (Smith, 2008) and

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pre&post-stress exposure to 6-hydroxydopamine (6-OHDA) (Hemmerle et al., 2014) exaggerated motor impairments and accelerated the loss of dopamine producing neurons

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in experimental rats. We recently reported that the exposure of chronic mild stress (CMS)

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worsened few motor and non-motor features, depleted the levels of dopamine and serotonin, diminished the expressions of tyrosine hydroxylase (rate limiting enzyme

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involved in the dopamine biosynthesis), dopamine transporter, vesicular monoamine transporter 2 (proteins involved in the transport of dopamine) and α-synuclein (plays a

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vital role in the pathogenesis and progression of PD) in the MPTP/p-exposed PD model (Janakiraman et al., 2016). However the precise mechanism(s) still remains indefinable.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is commonly used to induce experimental PD by selectively damaging the dopaminergic nigrostriatal system through inducing mitochondrial dysfunction (Selvakumar et al., 2015), inflammation (Anandhan et al., 2013) and oxidative stress (Kavitha et al., 2013). Probenecid (p) increased the

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ACCEPTED MANUSCRIPT MPTP toxicity, by blocking the excretion MPTP and their metabolites through urine (Lau e al., 2011). It also diminished the levels of brain derived neurotrophic factor (BDNF), which is essential for the survival of dopaminergic neurons (Deierborg et al., 2008). BDNF could bind with TrkB receptor (Kaplan and Stephens, 1994), resulting in the activation of the downstream signalling pathways including MAPK, PI3K and PLCγ that

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is shown to phosphorylate CREB, a transcription factor down stream to these signalling

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markers, which promotes transcription by recruiting CREB-interacting proteins to the

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CREs on CREB target genes, such as BDNF (Shaywitz and Greenberg, 1999 ).

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Evidence from various studies have demonstrated that the pathological processes in

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PD are not only confined to the dopaminergic system but also involved in other nondopaminergic neurons such as the serotonergic system (Langston, 2006). Serotonergic

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neurotransmission in the central nervous system is believed to be involved in the

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pathogenesis of depression (Spreux-Varoquaux et al., 2001). The results of our previous study (Janakiraman et al., 2016) also proved that the levels of serotonin are reduced after

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exposure of mice to CMS for four week schedule. CMS induced depression could increase the generation of reactive oxygen species (ROS) in rat brain (Duman and Voleti,

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2012). Especially, basic and clinical studies demonstrated that BDNF related signalling pathways were also involved in the pathogenesis of depression (Fontella et al., 2001).

Though the brain monoamine levels, antioxidant systems, inflammation and signalling molecules interact closely during both PD (Anandhan et al., 2012a; Tamilselvam et al., 2013; Nie et al., 2015) and stress exposure (Ahmad et al., 2010; Wang et al., 2015), the reports directly related to the two systems are rather limited. As the stress is reported to

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ACCEPTED MANUSCRIPT have causative (Smith, 2008) and progressive (Hemmerle et al., 2014) role in PD, the present study is aimed to test the exposure of stress before, after or concomitant (Fig 1) MPTP/p toxicity by analysing few motor and non-motor features, oxidative imbalance, the expressions of inflammation, serotoninergic and signalling markers in striatum (ST)

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and SN.

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2. Materials and Methods

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2.1. Animals

Male C57BL/6J mice (25-30 g) were purchased from the National Institute of Nutrition,

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Hyderabad and acclimatized to laboratory conditions for one week before initiating

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experiments. The animals were kept under standard conditions with food and water ad libitum. The protocol was approved by the Institutional Animal Ethics Committee

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(Approval no: 893/2012) and in accordance with the guidelines of Indian National

Chemicals

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2.2.

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Science Academy.

MPTP, probenecid, 3,5-dithio-bisnitrobenzoic acid (DTNB)and thiobarbituric acid (TBA)

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were obtained from Sigma-Aldrich, Bangalore, India. Anti-ERK1/2&phospho-ERK1/2 (Thr202/Tyr204), anti-AKT & phospho-AKT (Ser473), anti-TrkB (Tyr516), anti-CREB &phospho-CREB (Ser133), anti-BDNF, anti-β-actin, anti-GFAP primary antibodies (Cell signalling) and anti-serotonin transporter (Sigma chemical, USA), and anti-iba-1 (Santa Cruz Biotechnology, USA)

produced from rabbit and anti-rabbit HRP conjugated

secondary antibody (Sigma chemical, USA) were used in the study. 2.3.

Experimental procedure

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ACCEPTED MANUSCRIPT 2.3.1. Grouping Mice were randomly divided into eight groups (n = 12/group). The Group I, control mice were treated with saline (Control); Group II,MPTP/p alone treated mice were injected with MPTP (25 mg/kg b.w. in saline, s.c.) and probenecid (250 mg/kg in 0.03 ml DMSO, i.p.) for 5 weeks (ten injections consisting of twice a week) (Anandhan et al., 2012b)

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(MPTP/p alone); Group III, stress alone exposed mice were exposed to CMS for 4 weeks

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(CMS alone); Group IV, post stress exposed mice were exposed with stressors after

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MPTP/p treatment (Post CMS exposed and MPTP/p injected); Group V, poststress unexposed MPTP/p control mice were injected with MPTP/p followed by non-exposure

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of CMS (post CMS unexposed and MPTP/p injected); Group VI, pre stress exposed mice

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were exposed to CMS and injected with MPTP/p (Pre CMS exposed and MPTP/p injected); Group VII, pre&post stress exposed mice were exposed to CMS for 2 weeks

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before and after MPTP/p injection (Pre&Post CMS exposed and MPTP/p injected); and

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Group VIII, pre&post stress unexposed MPTP/p control mice were non-exposed to CMS for 2 weeks before and after MPTP/p injection (Pre&Post CMS unexposed and MPTP/p

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injected) (Fig 1A).

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2.3.2. CMS Protocol

Experimental mice in indicated groups were subjected to different kinds of stressors to induce the CMS by exposed the following: cage tilting, damp sawdust, placement in an empty cage, placement in an empty cage with water on the bottom, inversion of the light/dark cycle, food or water deprivation and lights on for a short period of time during the dark phase for four weeks (Willner et al., 1987) (Fig 1B). 2.4.

Behavioural assessments

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ACCEPTED MANUSCRIPT Behavioural studies such as hang test, stride length measurement, elevated plus maze and hole-board test were performed 48 h after the last injection of MPTP/p, to minimize nacrotic effect of MPTP. To confirm the stress induced anxiety/depressive-like phenotype and MPTP/p intoxicated movement impairment, hang test were performed once in 28th (after stress exposure completion in IVth group) and 35th (after the completion

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of MPTP/p injection schedule in VIth group) days throughout the experimental period and

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stride length measurement test, elevated plus maze, hole board test and biochemical

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experiments were performed at the end of the experiment. 2.4.1. Hang test

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Mice were placed in a grid and allowed to hold it. Then, the grid was inverted so that the

al., 2012a).

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2.4.2. Stride length measurement

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mice were allowed to hang vertically and the hanging time was measured (Anandhan et

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The experimental mice were trained to walk along a straight line. Forepaws were dipped in black ink and the length of forepaw steps during normal walking on a clean

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sheet of paper was measured. Stride length was determined by measuring the distance between each step of the same side of the animal. Stride length was measured from the

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middle toe of the first step to the heel of the second step (Tillerson et al., 2002). 2.4.3. Elevated Plus Maze The elevated-plus maze apparatus contains two open arms and two enclosed arms (30x5cm). Each mouse was placed onto the maze facing one of the opened arms. Over 5 min, the percentage of total time spent in the open arms and of total entries in open arms were measured (Berry et al., 2012).

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ACCEPTED MANUSCRIPT 2.4.4. Hole-board test The plastic apparatus having square area of 50 x 50 cm is made up of grey floor, which contained four, equally spaced holes (3 cm in diameter) and 40-cm-high opaque walls were used. Mice were placed in the centre of the apparatus and allowed to freely explore the apparatus for 5 min. The total number of head-dip behaviours was counted in control

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and experimental mice (Saitoh et al., 2006).

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2.4.5. Body weight

Mice were weighed at the beginig and end of the experiment. The body weight gain was

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calculated.

2.4.6. Head bobbing like behaviour in open field test

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The apparatus (W100 cm×D100 cm×H40 cm) is made of wood and resin. The floor of

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this chamber was divided into 25 cm (5 × 5) squares. The mice were placed into one corner of an open field chamber and their behaviour was observed for 3 min for the

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occurrence of stereotyped behaviour like head bobbing (Molloy et al., 1986). The occurrence of stereotyped behavior in each mouse was scored as follows:

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1–2: 1 3–4: 2

5 or >5: 3 2.5.

Sample collection

After performing behavioural studies, all mice were sacrificed by terminal anaesthesia and then perfused with saline followed by 4% paraformaldehyde (pH 7.4) transcardially.

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ACCEPTED MANUSCRIPT Then ST and SN were collected based on the mouse brain atlas (Franklin and Paxinos, 2007). 2.6.

Biochemical estimation

2.6.1. Estimation of TBARS ST and SN homogenates were incubated with 0.2 ml of phenylmethosulfate at 37 ºC for 1

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h and then tricarboxylic acid (0.4 ml) and thiobarbituric acid (0.4 ml) were added. The

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reaction mixture was centrifuged at 4000 rpm for 15 min, and the supernatant was boiled

535 nm, as reported (Rajasankar et al., 2009).

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for 10 min. After cooling, the optical densitities of the individual samples were read at

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2.6.2. Measurement of Superoxide dismutase (SOD) activity

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To 20 µl of the brain supernatants, 960 µl of sodium carbonate buffer containing 0.1 mM xanthine, 0.025 mM nitro-blue tetrazolium and 0.1 mM EDTA and 20 µl of xanthine

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oxidase were added and the absorbance was measured at 560 nm (Selvakumar et al.,

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2014).

2.6.3. Determination of catalase activity

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To 20 µl of the brain tissue extracts, 50 µl of 1 M Tris–HCl buffer (pH 8.0) with 5 mM EDTA, 900 µl of 10 mM H2O2, 30µl of Milly-Q water were added. The rate of

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disintegration of hydrogen peroxide was measured at 240 nm (Anandhan et al., 2013). 2.6.4. Estimation of GSH The brain supernatants (0.5 ml) were added to 4 ml of ice cold 0.1 mM solution of DTNB in 1 M phosphate buffer (pH 8). The optical density was read at 412 nm in a spectrophotometer (Lowry et al., 1951).

2.6.5. Determination of GPx activity

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ACCEPTED MANUSCRIPT The GPx assay mixture consisted of 100 ml of 1 M Tris–HCl (pH 8.0) containing 5 mM EDTA, 20 ml of 0.1 M GSH, 100 ml of GSH reductase solution (10 U/ml), 100 ml of 2 mM NADPH, 650 ml of distilled water, 10 ml of 7 mM tertbutyl hydroperoxide and 10 ml of the brain supernatant. Oxidation of NADPH was determined spectrophotometrically

BDNF assessment

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2.7.

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oxidize 1 mM of NADPH per min (Anandhan et al., 2013).

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at 340 nm. One unit of GPx activity was defined as the amount of enzyme utilized to

Fresh brain tissues were lysed with RIPA buffer, kept on ice for 20 min and then the

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mixture was centrifuged at 4°C for 30 min. The supernatants were collected and frozen

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at-80°C for further analysis.The contents of BDNF in the brain tissues were determined by using ELISA method according to the manufacture’s instruction (PEPROTECH,

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USA). Briefly, brain homogenates from individual samples were plated in the 96-well

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plate and the corresponding antibody (anti-BDNF) was added for incubation overnight at 4°C. After removal of the primary antibody followed by washing steps, 1% BSA was

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used to block for 1 h at room temperature. Subsequently, biotin-labeled secondary antibody was added to incubate for 1 h and then 50 µL horse radish peroxidase (HRP) for

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45 min incubation at room temperature. Finally, the optical density of each sample was determined by an ELISA reader at 450 nm and the contents were expressed as pg/mg protein.

2.8.

Western blot

The choice of the animals was made randomly for performing the WB. SN and ST of brains were homogenized in an ice-cold RIPA buffer and were centrifuged at 12,000 rpm

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ACCEPTED MANUSCRIPT for 15 min at 4oC. Protein concentration was measured by the method of Lowry et al.,(1951). Samples containing 50 μg total proteins were loaded on 10% SDS– polyacrylamide gel and separated. The gel-separated proteins were then transferred onto nitrocellulose membranes (Millipore). The membranes were incubated with the blocking buffer containing 5% non-fat dry milk powder for 2 h to reduce non-specific binding sites

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and then incubated with β-actin (rabbit polyclonal; 1:500 dilution in 5% BSA in Tris-

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buffered saline and 0.05% Tween-20 (TBST)), 5-HTT (1:1000) (Sigma Aldrich); anti-

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GFAP, anti- iba-1, ERK1/2, p-ERK1/2, p-AKT, anti-pTrkB (1:2000;) and anti-CREB (1:1000; Cell Signalling) primary antibodies produced from rabbits with gentle shaking

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overnight at 4oC. After removal of the primary antibodies followed by three separate steps

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of washing steps, membranes were incubated with their corresponding secondary antibodies (anti-rabbit IgG conjugated with HRP) for 2 h at room temperature. The

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membrane was again washed thrice with TBST for 30 min, before protein bands were

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visualized by an enhanced chemiluminescence (ECL) using the kit (GenScript ECL kit,

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USA).

2.8.1. Image acquisition and densitometry analysis

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In the present study, Total Lab 1.11 software version was used for image acquisition and densitometry analysis of the immnunoblots. The software analyses the raw data in three dimensions and the density was calculated as the total volume by forming two dimensions peak. It also contains a tool to adjust the precise width of the band to account for the area under the peak of interest. Then the blot intensities were compared their control values, which was set as ‘100’.

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ACCEPTED MANUSCRIPT 2.9.

Statistical analysis

The results were expressed as mean ± SEM. Statistical analysis was carried out using two-way analysis of variance (ANOVA) using Sigma plot version 10.0 software and all pair wise comparisons were obtained using Duncan’s Multiple Range Test (DMRT). The use of DMRT is potentially problematic as it is established to be a very “liberal” test and

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can underestimate Type 1 errors.

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3. RESULTS

3.1. Changes in the body weight gain and behavioural patterns of control and

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experimental mice

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CMS alone, pre, post and pre&post CMS exposed and MPTP/p injected mice showed significantly [F(5,71) = 237.45, p<0.001] diminished weight gain in comparison

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to the control and MPTP/p alone injected groups (Fig 2). Post CMS exposed and MPTP/p

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injected mice exhibited very less gain in body weight as compared to all other groups. To evaluate the motor and non-motor disabilities, various behavioural studies

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including hang test, stride length measurement test, hole board test, head bobbing in open field test and elevated plus maze test were performed. Stress exposed animals (alone, pre,

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post and pre&post) exhibited significantly enhanced head bobbing [F(5,71) = 1.00, p<0.42] behaviour (Fig 3A) as compared to normal and MPTP/p alone treated mice, which indicated that anxiety was induced by CMS exposure. Post stress exposed MPTP/p injected animal showed more significant bobbing behaviour as compared to pre and pre&post stress exposed mice. MPTP/p alone injected group exhibited a significant decrease in hanging time in hang test [F(5,71) = 283.9, 430.93 and 935.08, p<0.001] (Fig3B) and stride length in stride measurement test [F(5,71) = 343.35 and 331.76,

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ACCEPTED MANUSCRIPT p<0.001] (Figs 3C and D) as compared to those of the CMS alone exposed mice. CMS alone exposed mice spent less time [F(5,71) = 53.76, p<0.001] (Fig 4A) and showed a decreased number of entry in open arms of elevated plus maze [F(5,71) = 11.10, p<0.001] (Fig 4B) with an increased head dip score in hole-board test [F(5,71) = 78.94, p<0.001] (Fig 4C) as compared to MPTP/p alone, CMS unexposed and MPTP/p injected mice (Fig

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4), consistent with increased anxiety-like behavior. However post CMS exposed and

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MPTP/p injected mice showed further worsened motor activities and pre CMS exposed

Variations in the levels and activities of oxidative and anti-oxidative indices in

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3.2.

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and MPTP/p injected mice exhibited more anxiety, which is the new finding in this study.

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control and experimental mice

Brain is abnormally sensitive to oxidative damage because it consumes about 20%

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of the total oxygen consumption, high content of easily peroxidizable fatty acids and

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lowered anti-oxidant defences (superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GSH) and catalase) (Floyd 1999). In Particular, SN

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appears to be most vulnerable regions to oxidative stress because it mainly operates under a pro-oxidative state due to increased content of oxidizable species such as dopamine,

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dopamine-derived ROS, neuromelanin, poly unsaturated fatty acids, iron and a low content of antioxidants (particularly glutathione) (Selvakumar et al., 2014). MPTP/p alone treatment increased the oxidative stress by significantly enhancing the levels of TBARS [F(5,71) =115.8 and 119.3, p<0.001] (a marker of lipid peroxidation processes) and activities of SOD [F(5,71) =11.82 and 26.43, p<0.001] and catalase [F(5,71) = 49.77 and 68.26, p<0.001] and by depleting the levels of GSH [F(5,71) =352.3 and 349.2, p<0.001] and the activites of GPx [F(5,71) =365.15 and 348.49, p<0.001]. Stress alone

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ACCEPTED MANUSCRIPT exposure increased the levels of TBARS and diminished the levels of GSH and activities of antioxidant enzymes. Pre&post CMS exposed and MPTP/p injected mice showed greater induction of oxidative stress as compared to mice exposed to stress MPTP/p alone. Pre and pre&post CMS exposed and MPTP/p injected mice showed enhanced oxidative stress as compared to post CMS exposed and MPTP/p injected animals (Table

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1).

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3.3 Changes in the expression of inflammatory markers in control and experimental

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mice

Invading pathogens, trauma, toxins and stroke can trigger the activation of brain resident

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macrophages known as microglias and astrocytes, that results in the production of

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reactive oxygen and nitrogen species, cytokines, chemokines and other immune factors (McGeer and McGeer, 2008) that can contribute to dopaminergic brain cell destruction

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(Selvakumar et al., 2014). MPTP/p alone treatment increased the inflammation by

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significantly enhancing the expressions of GFAP [F(5,35) =143.58 and 140.3, p<0.001] and iba-1[F(5,35) =132.8 and 128.75, p<0.001] (Fig 5) (markers of microglial and

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astrocyte activation, whereas stress alone exposure showed no significant changes as compared to control animals. MPTP/p and CMS coexposed mice showed greater

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induction of inflammation as compared to mice exposed to stress or MPTP/p alone. Post exposure of CMS to

MPTP/p injected mice showed greastest enhancement in

inflammation as compared to pre and pre&post CMS exposed and MPTP/p injected animals. 3.4.

Variations in the level of BDNF in control and experimental animals

Neurotrophins is the umbrella term for nerve growth factor, BDNF, neurotrophin-3, and neurotrophin-4, which are araised from a common ancestral gene and have an vital role in

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ACCEPTED MANUSCRIPT regulating development and maintenance of the peripheral and central nervous systems. BDNF promoted the survival of hippocampal and cortical neurons (Ghosh et al., 1994; Lindholm et al., 1996), nigral dopaminergic (Hyman et al., 1991), cholinergic (Alderson et al., 1990), and serotonergic neurons (Eaton and Whittemore, 1996). In PD, dopaminergic degeneration is linked to deficient of neurotrophins in neurons or brain

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areas

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associated with disease (Siegel and Chauhan, 2000). Moreover a close link between

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levels of serotonin and the neurotrophin has been suggested (Martinowich and Lu, 2008) in the progression of some psychiatric disorders. Till now, no studies showed the effect of

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stressors and MPTP/p on the expression of BDNF. MPTP/p alone injected mice revealed

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slightly but significantly decreased BDNF level compared to control mice, whereas stress alone exposure further decreased their level [F(5,71) = 283.09, p<0.001] (Fig 6).

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Exposure of CMS alone or MPTP/p alone injected mice did not significantly decrease

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the expression of BDNF, as compared to pre, post and pre&post CMS exposed and MPTP/p injected mice. Post exposure of CMS to MPTP/p injected animals further

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decreased the BDNF expression, as compared to pre and pre&post CMS exposed and

3.5.

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MPTP/p injected group.

Alterations in the expression of 5-HTT and signalling protein molecules in

control and experimental mice The expression of BDNF is also altered due to the abnormalities in the expressions of serotonin transporter, because serotonin regulates its expression and secretion (Bramham and Messaoudi, 2005). BDNF regulates the apoptosis by inducing autophosphorylation of TrkB receptor and activating downstream mitogen-activated protein

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ACCEPTED MANUSCRIPT kinases (MAPKs) /extracellular signal-regulated kinase (ERK) and Akt (Chao, 2003; Kaplan and Stephens 1994). MPTP/p alone injected mice showed significantly decreased levels of 5-HTT (Fig 7), p-TrkB, p-ERK, p-AKT and p-CREB (Fig 8) in ST and SN as compared to those in mice exposed to stress alone. Exposure to stress alone or treatment with MPTP/p alone did not decrease the expressions of 5-HTT, p-TrkB, p-

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ERK,p-AKT and p-CREB as compared to pre, post and pre&post CMS exposed and

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MPTP/p injected mice. Post CMS exposed and MPTP/p injected animals showed greater

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decrease in the 5-HTT [F(5,35) =125.6 and 113.41, p<0.001], p-TrkB [F(5,35) =303.7 and 282.7, p<0.001], p-ERK [F(5,35) =273.19 and 269.21, p<0.001], p-AKT [F(5,35)

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=189.15 and 231.55, p<0.001] and p-CREB [F(5,35) =208.42 and 188.76, p<0.001]

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expression, as compared to pre and pre&post CMS exposed and MPTP/p injected mice,

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which is also a new finding.

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4. Discussion

In the present study, animals of the groups exposed with stress showed low body

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weight gain than the MPTP/p alone injected group. Further, MPTP/p mice showed significant reduction in the hanging time in hang test and exhibited low stride length in

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stride length measurement test as compared with the mice exposed to stress alone, whereas post-stress exposure to PD mice worsened both the motor and non-motor symptoms. Lowered hanging time indicates lowered neuromuscular strength, whereas reduced stride length reflects Parkinsonian gait. MPTP/p treatment reduced the ability to hold the grid and reduced stride length, which may be resulted from the permanent decrease in nigrostriatal dopamine content (Fernagut et al., 2002; Tillerson and Miller, 2003). Exposure of stress to MPTP/p mice further diminished the motor behaviour. Few

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ACCEPTED MANUSCRIPT unaffected dopaminergic neurons continue to synthesize and release dopamine after 6OHDA lesion although this response becomes inadequate under chronic stress (Abercrombie, 1989; Keefe et al., 1990), which further supports our results. Recently we reported that the levels of dopamine and the depleted TH immunoreactivity of positive neurons in post-stress exposed MPTP/p mice (Janakiraman et al., 2016), which

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corroborated with the motor behaviours.

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In depressed patients, high comorbidity rates of depression and anxiety are observed and the margins between depression and anxiety become blurred, which suggests that

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these disorders may not be entirely distinct conditions in humans or animals (Kaufman

were to

be

induced

by

the

stress

procedure

(Strekalova

et

al.,

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found

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and Charney, 2000). Increases in anxiety-related behavior and locomotor disturbances

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2004; Schweizer et al., 2009). Head bobbing, hole board and elevated-plus maze test are used to measure anxiety-like behaviour (Lister, 1987). The results of our current study

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indicated that the exposure of CMS induced significant head bobbing behaviour as compared to normal and MPTP/p treated mice. Enhanced head bobbing behaviour was

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remarkably higher in post stress exposed animals. Several experimental evidence indicated that the decreased serotonergic activity plays an important role in the aetiology and severity of anxiety and depression (Aan het Rot et al., 2009; Booij et al., 2010). We also reported that the levels of serotonin were significantly diminished by stress alone exposure than MPTP/p alone injection and was further diminished by their co-exposure (Janakiraman et al., 2016). Like serotonin depletion, the depressive and anxiety symptoms were not significantly altered by MPTP/p injection, as

17

ACCEPTED MANUSCRIPT compared to the stress alone exposed rodents, but those symptoms were further amplified in pre, post and pre&post stress exposed and MPTP/p injected groups. More serotonin depletion was found in the post CMS exposed and MPTP/p injected group, which corroborates with our current behaviour alterations. CMS has been widely used in animals to mimic depression-like disorders and is regarded as model for the exposure of

PT

unexpected stressors of everyday life in humans (Henn and Vollmayr, 2005). Even

RI

though the reduction in 5HT markers is not regarded as appropriate markers for

SC

depression, the behavior test such as sucrose test for anhedonia or social interaction are needed to carry out to prove that animals exposed to stressors are experiencing depressive

NU

like symptoms. We have reported the sucrose preference task in our recent paper

MA

(Janakiraman et al., 2016). Stress exposed mice showed more anhedonic symptom as compared to MPTP/p alone treated animals, whereas post MPTP treated animals

D

illustrated most anhedonic symptoms as compared with pre and pre&post MPTP treated

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animals, which is correlated with the levels of 5HT markers (Janakiraman et al., 2016). Previous studies have indicated that oxidative stress might play a vital role in the cause,

CE

progression and pathogenesis of neurological including PD (Selvakumar et al., 2014) and psychiatric diseases including major depression (Rezin et al., 2009) and bipolar disorder.

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MPTP causes oxidative stress in neurons through its metabolism by monoamine oxidase B (MAO-B) in astrocytes to form 1-methyl-4-phenyl pyridinium (MPP+) and by the inhibition of mitochondrial respiration particularly the electron transport chain (ETC) I in mitochondria (Mytilineou et al., 2002). Inhibition of ETC-I by MPP+, results in the shunting of electrons passage through the ETC-II, which may generate the production of ROS approximately five to seven fold higher. Enhanced oxidative stress were found in rats exposed to restraint and chronic mild stress, which was consistent our study, due to

18

ACCEPTED MANUSCRIPT the inhibition of Complexes I–III and II–III of mitochondrial respiratory chain (Madrigal et al., 2001; Rezin et al., 2009). In this study, chronic MPTP/p treatment showed significantly elevated TBARS levels, catalase and SOD activities with diminished GSH levels and GPx activities, whereas a significant increase in TBARS levels, reduction in GSH levels and enzymatic antioxidant activities in stressed mice, which is in agreement

PT

with previous studies (Anandhan et al., 2012a; Anandhan et al., 2012b; Madrigal et al.,

RI

2001; Zhang et al., 2009). In contrast to elevated neurodegenerative changes and

SC

motor/non-motor impairments in post-stress exposed PD mice, enhanced oxidative stress was found in pre/pre&post-stress exposed PD animals. MPTP/p alone treatment

NU

persuaded oxidative stress more significantly than CMS alone exposed animals in

MA

nigrostriatal regions. Pre and pre&post CMS exposed and MPTP/p injected mice were more prone to oxidative stress because the measurement of oxidative stress was

D

performed on 4 and 19 days after last MPTP/p administration, respectively, as compared

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to post-stress PD animals (32 days after the last injection of MPTP/p), which reflects the diminished MPTP neurotoxicity.

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Neuroinflammation is a coordinated cellular response of defence mechanism and the key component in the progression of various neurodegenerative disorders especially in PD

AC

(Monahan et al., 2008; Vroon et al., 2007). Activated astrocytes and microgilia, the cellular manifestation of neuroinflammation, is a common response to MPTP in brain contributed significantly to neuronal degeneration. GFAP and iba-1 are well known markers of reactive microglial cells and astrocytes, and their upregulation by MPTP/p treatment indicated the astroglial activation (Selvakumar et al., 2014). Kopp et al., (2013) shown exposure

that enhanced

the

microglial

chronic activation

19

in

stress the

prefrontal

cortex

ACCEPTED MANUSCRIPT (PFC) but not in the ST. Our results also indicated that the CMS alone exposure did not induced the inflammation in the striatum or SN, but have a synergetic effect through which it enhanced the MPTP/p mediated inflammation. Neuronal damages induced during normal aging, neurodegenerative disease and physical injury results in the decreased expression of BDNF, which is involved in

PT

synaptic plasticity, neurogenesis and neuronal survival (Mora, 2013). Previous studies

RI

indicated that the reduced striatal levels of serotonin and its metabolite, transporter and

SC

synthesizing enzyme protein provide strong evidence for a serotonergic disturbance in PD (Kish et al., 2008), which may be due to loss of cell bodies in the dorsal raphe area in PD

NU

(Jellinger, 1986; Paulus and Jellinger, 1991). Recently, we also reported the diminished

MA

levels of serotonin in striatum, cortex and hippocampus of pre, post and pre&post stress exposed and MPTP/p injected animals, which confirms their influence on non-

D

dopaminergic pathways (Janakiraman et al., 2016). So we cannot expect an increase in

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BDNF levels. TrkB is the primary signal transduction receptor for BDNF. As lowered neurotrophic support or its deficiency might involved in the pathogenesis of PD (Sathiya

CE

et al., 2013), additional supplement of neurotrophic factors rescued the dopaminergic neurons and offered a protective role in PD degenerative processes (Ding et al., 2011).

AC

Many of the chronic stressors such as CMS and social defeat stress have also been found to vigorously down-regulate BDNF mRNA and protein expression in rodents (Haenisch et al., 2009; Zhang et al., 2010). TrkB receptor auto-phosphorylation elicits its downstream mitogen-activated protein kinases (MAPKs) and Akt activation, which plays a key role in preventing apoptotic cell death (Chao 2003; Kaplan and Stephens 1994). The PI3K pathway activates protein kinase B (AKT), promoting cell survival; while the MAPK/extracellular signal-regulated

20

ACCEPTED MANUSCRIPT kinase (ERK) pathway leads to cell growth and differentiation. Several lines of evidence indicated that the AKT signalling pathway responds to oxidative stress (Crossthwaite et al., 2002) through oxidative modification of active site Cys residues of various phosphatases (Song et al., 2014) and exerts a neuroprotective function (Lee et al., 2009; Sun, 2009). Our results indicated that the expressions of p-ERK and p-AKT, two

PT

important protein kinases that mediate MEK/ERK and PI3K/AKT signalling pathways,

RI

were reduced in both MPTP/p alone injected and CMS alone exposed animals and, in

SC

agreement with the previous studies (Nie et al., 2015; Wang et al., 2015). Negative effects of stress and MPTP/p alone on p-CREB expression, which is a downstream messenger of

NU

ERK and AKT, were also observed in SN and ST. Evidence showed that p-CREB

MA

expression was decreases in stressed animals and humans (Laifenfeld et al., 2009; Xu et al., 2006) and major classes of anti-depressants increase CREB levels in several brain

D

regions of stressed rodents (Tardito et al., 2009; Tiraboschi et al., 2004). Further

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reduction in the levels of BDNF and expressions of TrkB, p-ERK, p-AKT and p-CREB proteins in post CMS exposed and MPTP/p injected group indicated that the accelerated

CE

or greater neurodegeneration can be induced by stress in PD animals. Our current results showed an additive or synergistic interaction between CMS

AC

and PD. As stress induced NMS can accelerate neurodegenerative processes in the PD in a progressive or expedited manner, we can predict that reduction of day – today life stress exposure along with currently used anti-PD drugs may become more successful way for curbing PD.

21

ACCEPTED MANUSCRIPT Acknowledgments Financial

assistance

in

the

form

of

Neuroscience

Task

Force

project

(BT/PR4958/MED/30/748/2012) from the Department of Biotechnology, New Delhi, is gratefully acknowledged. Prof Guillemin is supported by the National Health and Medical Research Council (NHMRC), the Australian Research Council (ARC) and Macquarie

RI

PT

University.

SC

Conflict of interest The authors declare no conflict of interest.

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Author Contributions

MA

Conceived and designed the experiments: UJ TM. Performed the experiments: UJ TM CD. Analyzed the data: MME UJ TM CD. Contributed reagents/materials/analysis tools:

AC

CE

PT E

D

UJ TM AJT. Wrote the paper: MME TM BJS GJG AJT.

22

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ACCEPTED MANUSCRIPT Figure legends Fig 1.Experimental study design(A) and one week schedule for stress exposure (B), which is repeated for other three weeks.

Fig 2. MPTP/p alone treatment, stress alone exposure and pre, post and pre& post

PT

stress exposure to MPTP/p injected mice changed the body weight gain. Reduced

RI

body weight gain was found in experimental groups as compared to control mice. More

SC

significant reduction in body weight gain was found in post CMS exposed and MPTP/p injected animals as compared to all other groups. Values are given as mean ± SEM for six

NU

mice in each group and analysed by using two-way analysis of variance (ANOVA)

MA

followed by Duncan’s Multiple Range Test (DMRT). Results are given as statistically significant at p<0.05; compared with (a): group I, (b): group II, (c): group III, (d): group

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D

IV, (e): group V, (f): group VI, (g): group VII and (h): group VIII.

Fig 3. MPTP/p alone treatment, stress alone exposure, pre, post and pre& post stress

CE

exposure to MPTP/p mice diminished the motor behaviour.Increased head bobbing in open field test (A), diminished hanging time in hang test (B), stride lengths of fore limb

AC

(C) and hind limb (D) were observed in experimental groups as compared to control mice. More significant motor impairments were found in post-stress exposed and MPTP/p injected animals as compared to all other groups. Values are given as mean ± SEM for six mice in each group and analysed by using two-way analysis of variance (ANOVA) followed by Duncan’s Multiple Range Test (DMRT). Results are given as statistically significant at p<0.05; compared with (a): group I, (b): group II, (c): group III, (d): group IV, (e): group V, (f): group VI, (g): group VII and (h): group VIII.

33

ACCEPTED MANUSCRIPT

Fig 4. MPTP/p alone treatment, stress alone exposure and pre, post and pre& post stress exposure to MPTP/p injected mice changed the non-motor behaviour. Diminished entry (A) and time spent (B) in the open arms of the elevated plus maze test, indicating anxiety, with enhanced head tip in hole board (C), increased depression in

PT

forced swim test (D), were found in experimental groups as compared to control mice.

RI

More significant non-motor impairments were found in post-stress exposed and MPTP/p

SC

injected animals as compared to all other groups. Values are given as mean ± SEM for six mice in each group and analysed by using two-way analysis of variance (ANOVA)

NU

followed by Duncan’s Multiple Range Test (DMRT). Results are given as statistically

MA

significant at p<0.05; compared with (a): group I, (b): group II, (c): group III, (d): group

D

IV, (e): group V, (f): group VI, (g): group VII and (h): group VIII.

PT E

Fig 5. MPTP/p alone treatment, stress alone exposure and pre, post and pre& post stress exposure to MPTP/p injected mice enhanged the expression of inflammatory in striatum and Substantianigra. Increased expression of inflammatory

CE

markers

markers were found in experimental groups as compared to control mice. More

AC

significant increase in the expression of inflammatory markers were found in post-stress exposed MPTP/p injected animals as compared to all other groups. Values are given as mean ± SEM for six mice in each group and analysed by using two-way analysis of variance (ANOVA) followed by Duncan’s Multiple Range Test (DMRT). Results are given as statistically significant at p<0.05; compared with (a): group I, (b): group II, (c): group III, (d): group IV, (e): group V, (f): group VI, (g): group VII and (h): group VIII.

34

ACCEPTED MANUSCRIPT Fig 6. MPTP/p alone treatment, stress alone exposure and pre, post and pre& post stress exposure to MPTP/p injected mice reduced the levels of BDNF in striatum. Decreased levels of BDNF were found in experimental groups as compared to control mice. More significant reduction in the levels of BDNF was found in post-stress exposed MPTP/p injected animals as compared to all other groups. Values are given as mean ±

PT

SEM for six mice in each group and analysed by using two-way analysis of variance

RI

(ANOVA) followed by Duncan’s Multiple Range Test (DMRT). Results are given as

SC

statistically significant at p<0.05; compared with (a): group I, (b): group II, (c): group III,

NU

(d): group IV, (e): group V, (f): group VI, (g): group VII and (h): group VIII.

MA

Fig 7. MPTP/p alone treatment, stress alone exposure and pre, post and pre& post stress exposure to MPTP/p injected mice reduced the expression of 5-HTT in

D

striatum and Substantianigra. Decreased expression of 5-HTT was found in

PT E

experimental groups as compared to control animals. More significant reduction in the expression of 5-HTT were found in post stress exposed MPTP/p injected animals as

CE

compared to all other groups. Values are given as mean ± SEM for six mice in each group and analysed by using two-way analysis of variance (ANOVA) followed by Duncan’s

AC

Multiple Range Test (DMRT). Results are given as statistically significant at p<0.05; compared with (a): group I, (b): group II, (c): group III, (d): group IV, (e): group V, (f): group VI, (g): group VII and (h): group VIII.

Fig 8. MPTP/p alone treatment, stress alone exposure and pre, post and pre& post stress exposure to MPTP/p injected mice modulated in the levels of the signalling pathway molecules p-TrkB (A), p-ERK 1/2 (B), p-AKT (C) and p-CREB (D) in

35

ACCEPTED MANUSCRIPT Striatum and Substantianigra. Decreased levels of p-TrkB, p-ERK 1/2, p-AKT and pCREB expression were found in experimental groups as compared to control mice. More significant reduction in the expression of p-TrkB, p-ERK 1/2, p-AKT and p-CREB were found in post stress exposed MPTP/p injected animals as compared to all other groups. Values are given as mean ± SEM for six mice in each group and analysed by using twoMultiple

PT

way analysis of variance (ANOVA) followed by Duncan’s

Range

Test

RI

(DMRT). Results are given as statistically significant at p<0.05; compared with (a): group

SC

I, (b): group II, (c): group III, (d): group IV, (e): group V, (f): group VI, (g): group VII

AC

CE

PT E

D

MA

NU

and (h): group VIII.

36

ACCEPTED MANUSCRIPT Table legend Table 1.Levels of TBARS, modulated antioxidants and related enzymes.High levels of TBARS with changed levels and activities of antioxidant enzymes such as SOD, catalase, and GSH and GPx were found in experimental animals as compared to control animals. More significant elevation of the levels of TBARS, SOD and Gpx were found in

PT

pre- stress exposed MPTP/p injected animals as compared to all other groups. Values are

RI

given as mean ± SEM for six mice in each group and analysed by using two-way analysis

SC

of variance (ANOVA) followed by Duncan’s Multiple Range Test (DMRT). Results are given as statistically significant at p<0.05; compared with (a): group I, (b): group II, (c):

A

MA

NU

group III, (d): group IV, (e): group V, (f): group VI, (g): group VII and (h): group VIII.

Amount of enzyme required to inhibit 50% of NBT reduction.

nmol H2O2 consumed/minute/mg protein.

C

nmol NADPH oxidized/minute/mg protein

AC

CE

PT E

D

B

37

ACCEPTED MANUSCRIPT Table 1: Striatum

Control

TBARS

SOD

Catalase

GSH

GPx

(nmoles/g tissue)

(UA/mg protein)

(UB/mg protein)

(mg/g of tissue)

1.23 ± 0.034bcdefgh

1.83 ± 0.057bcdefgh

1.72 ± 0.045bcdefgh

T P

(UC/mg protein)

10.17 ± 0.307bcdefgh

6.40 ± 0.167bcdefgh

acdefgh

4.97 ± 0.135

acdefgh

3.15 ± 0.094

5.88 ± 0.221adfg

4.29 ± 0.131acdfg

6.19 ± 0.162adfg

5.85 ± 0.189abdefgh

4.96 ± 0.158abcefh

3.91 ± 0.112abceh

2.86 ± 0.086abcdfh

6.02 ± 0.186adfg

4.07 ± 0.143acdfg

2.51 ± 0.090abceg

4.02 ± 0.117abcdeh

3.55 ± 0.112abceh

4.11 ± 0.127

2.63 ± 0.076abcdfh

4.42 ± 0.135abceh

3.61 ± 0.103abceh

3.91 ± 0.128abcdef

2.98 ± 0.102abceg

6.21 ± 0.228adfg

4.18 ± 0.120acdfg

acdfg

MPTP/p

3.20 ± 0.118

CMS

2.67 ± 0.085abdefgh

1.00 ± 0.032abdefgh

1.14 ± 0.036abdefgh

MPTP/p+CMS

4.10 ± 0.121abcefh

4.24 ± 0.155abcgh

2.78 ± 0.081abceg

acdfg

MPTP/p+NS

3.39 ± 0.09

CMS+MPTP/p

4.77 ± 0.127abcdegh

CMS+MPTP/p+CMS 4.38 ± 0.125

abcefh

NS+MPTP/p+NS

acdefg

3.28 ± 0.102

abcgh

4.28 ± 0.124

T P E

C C

Substantianigra

A

D E

3.60 ± 0.121abcgh

abcdef

I R

C S U

N A

M

Control

1.56 ± 0.040bcdefgh

1.09 ± 0.031bcdefgh

1.94 ± 0.049bcdefgh

9.27 ± 0.280bcdefgh

8.14 ± 0.234bcdefgh

MPTP/p

3.59 ± 0.108acdfg

3.95± 0.141acdfh

3.01 ± 0.090acdeh

5.01 ± 0.140ac

4.76 ± 0.144acdfg

CMS

2.79 ± 0.085abdefgh

0.68 ± 0.022abdefgh

1.27 ± 0.036abdefgh

7.19 ± 0.239abdefgh

10.27 ± 0.316abdeh

38

ACCEPTED MANUSCRIPT MPTP/p+CMS

4.01 ± 0.117abcefgh

3.14 ± 0.090abcefgh

2.76 ± 0.090abcfg

5.65 ± 0.203ac

6.88 ± 0.225abcefgh

MPTP/p+NS

3.48 ± 0.112acdfg

3.74 ± 0.115acdf

2.88 ± 0.105abc

5.28 ± 0.165ac

5.23 ± 0.161acdfg

CMS+MPTP/p

4.89 ± 0.149abcdegh

2.76 ± 0.072abcdegh

2.41 ± 0.085acd

5.01 ± 0.153ac

6.15 ± 0.212abdeh

2.76 ± 0.085acdf

2.45 ± 0.072acd

5.29 ± 0.176ac

6.24 ± 0.228abdeh

3.86 ± 0.143abcdf

2.97 ± 0.087abc

5.64 ± 0.208ac

4.91 ± 0.161acdfg

CMS+MPTP/p+CMS 4.54 ± 0.131abcdefh NS+MPTP/p+NS

3.51 ± 0.108acdfg

C S U

N A

D E

I R

M

T P E

C C

A

39

T P

ACCEPTED MANUSCRIPT Fig 1:

T P

I R

C S U

N A

D E

M

T P E

C C

A

40

ACCEPTED MANUSCRIPT Fig 2:

bcdefgh

7 6 5 4 3 2 1 0

acdfg

C C

A

41

S+ M

PT P/

p+ NS C M +M S PT P/ p+ N S

p PT P/

S

S+ M CM

T P E

CM

P/ p+ N

M S

M PT

D E

N A

M PT

P/ p+ C

CM

S

P/ p M PT

Co nt ro

abcefgh

M

acdfg

C S U abcdeh

abdefgh

T P

I R

acdfg

abcdeh

l

Body weight gain (gm)

Body weight gain

ACCEPTED MANUSCRIPT Fig 3:

A

T P

I R

C S U

abcefgh abdeh

A

42

p

cdfg

PT P/ S+ M

C M

C C

abcdeh abcdeh

PT P/

S

S+ M

C M

P/ p+ N

M S

P/ p+ C

M PT

T P E

C M

D E

M M PT

P/ p

l

S

cdfg

cdfg

C on tr o

N A cdfh

p+ N C S+ M M S PT P/ p+ N S

2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0

M PT

No. of head bobbing (3 min)

Head bobbing

ACCEPTED MANUSCRIPT B Hang test

Hang time (sec)

350

28th day 35th day 67th day

T P

bcdefgh

300

I R

abdefgh

250 200

SC

150 acdfg

100

acdfg

abceh

50

abceg

U N

C M S M PT P/ p+ C M S M PT P/ p+ N S C M S+ M PT C M P/ S+ p M PT P/ p+ C M S N S+ M PT P/ p+ N S

C

on tr o

l

M PT P/ p

0

abceh

D E

A M

T P E

C C

A

43

acdfg

44 P/ p+ C N M S S+ C M M PT S+ M P/ p PT P/ p N +C S+ M M S PT P/ p+ N S

M PT

S

M S

C M

D E

P/ p+ C

l

P/ p

C on tr o

D

M PT

A C C

M PT

T P E

Stride length measurement of hind limb (cm)

M C M

N A

S+ M

C M

PT P/

p

S

M S

S

P/ p+ N S+ M

l P/ p C M P/ p+ C M PT

M PT

M PT

C on tr o

Stride length measurement of fore limb (cm) 8 bcdefgh abdefgh 7 acdfg acdfg 6 abcefgh 5 4 3 2 1 0

8 bcdefgh 7 abdefgh acdfg acdfg 6 5 abcefgh 4 3 2 1 0

N S+ M PT

P/

M S

p+ N

p+ C

C S U PT P/

abcdeh abcdeh

I R S

ACCEPTED MANUSCRIPT

C Stride length measurement

acdfg

T P

Stride length measurement

abcdeh abcdeh acdfg

ACCEPTED MANUSCRIPT Fig 4: A

T P

I R

SC

A

45

p+ C

S

NS +M PT

S+ M CM

C C

PT P/

PT P/

S

S+ M

P/ p+ N

M S

T P E

CM

M PT

D E

M PT

S

P/ p+ C

CM

A M

p

U N

aceh

a

p+ N

a

P/

ad ad

M S

ad

P/ p

Co nt ro

l

6 bcdefgh 5 4 3 2 1 0 M PT

Number of open arm entries

Elevated plus maze test

ad

ACCEPTED MANUSCRIPT B

adfg

adfg

abcdeh abcdeh

C C

A

46

p

PT P/

PT P/ S+ M

S+ M

S

T P E

CM

P/ p+ N

M

CM

M S

M PT

P/ p+ C

CM

M PT

D E

N A

p+ NS C M +M S PT P/ p+ N S

C S U

abcefgh

T P

I R

adfg

S

P/ p M PT

l

18 bcdefgh 16 14 adfg 12 10 8 6 4 2 0 Co nt ro

Time spent in open arm (s)

Elevated plus maze test

ACCEPTED MANUSCRIPT C

70 abcefgh abcdeh abcdeh 60 acdfg acdfg acdfg 50 abdefgh 40 bcdefgh 30 20 10 0

T P

A

47

p+ NS C M +M S PT P/ p+ N S

p

PT P/

PT P/ S+ M

S+ M

S

C C

CM

P/ p+ N

M

T P E

C S U

N A CM

M S

D E

M PT

M PT

P/ p+ C

CM

S

P/ p M PT

l

I R

Co nt ro

Number of head tips/5 mins

Hole board test

ACCEPTED MANUSCRIPT Fig 5: ST

SN

iba-1

21 k Da

GFAP

50 k Da

β-actin

C S U 42 k Da

N A

48

acdfg

p PT P/ p+ NS C M +M S PT P/ p+ N S

S

PT P/ S+ M

CM

CM

M S

abceh abceh acdfg

P/ p+ N

M PT

S

P/ p+ C

CM

M PT

l

P/ p M PT

p PT P/ p+ NS C M +M S PT P/ p+ N S

Substantia nigra

350 abceh 300 acdfg 250 200 bdefgh 150 bcdefgh 100 50 0 Co nt ro

C C

iba-1 relative intensity (% of control)

acdg

A

PT P/

S

aceh

aceh

S+ M CM

P/ p+ N

S+ M

CM

M S

M PT

S CM

P/ p+ C

M PT

l

P/ p M PT

Co nt ro

iba-1 relative intensity (% of control)

300 abceh 250 acdh acdgh 200 bdefgh 150 bdefgh 100 50 0

D E

T P E

Striatum

M

S+ M

A) iba-1

I R

T P

S+ M

S

PT P/

D E N A

T P E

A C C

49

S+ M

M

P/

p+ N

S

M S

p

S PT P/ p+ C

S+ M PT P/

CM

450 abceh 400 350 acdfg 300 250 abdefgh 200 150 bcdefgh 100 50 0

NS +M PT

CM

M S

S

P/ p+ N

P/ p+ C

CM

Striatum

M PT

M PT

P/ p

l

acdfg GFAP relative intensity (% of control)

abceh

M PT

acdfg aceh

Co nt ro

p PT P/ p+ NS C M +M S PT P/ p+ N S

S+ M

CM

P/ p+ N

M S

S

400 abceh 350 acdgh 300 250 200 abdefgh 150 bcdefgh 100 50 0

CM

l

P/ p

CM P/ p+ C

M PT

M PT

M PT

Co nt ro

GFAP relative intensity (% of control)

ACCEPTED MANUSCRIPT

B) GFAP Substantia nigra

acdh abceh abceh acdfg

C S U I R

T P

ACCEPTED MANUSCRIPT Fig 6:

A

50

p+ NS C M +M S PT P/ p+ N S

p

PT P/ S+ M

S+ M

CM

T P

I R

C S U PT P/

S

C C

CM

M

T P E

acdfg

abcdeh abcdeh

N A

P/ p+ N

M S

D E

M PT

M PT

P/ p+ C

CM

S

P/ p

Co nt ro

l

30 bcdefgh acdfg acdfg 25 abdefgh 20 abcefgh 15 10 5 0

M PT

BDNF (pg/ml)

BDNF

ACCEPTED MANUSCRIPT Fig 7: Lane 1: Control Lane 2: MPTP/p

T P

Lane 3: CMS Lane 4: MPTP/p + CMS

5-HTT

I R

Lane 5: MPTP/p+ NS

ST

83 k Da

Lane 6: CMS + MPTP/p

C S U

Lane 7: CMS + MPTP/p + CMS

β-actin

42 k Da

SN

83 k Da

β-actin

42 k Da

D E

Lane 8: NS + MPTP/p + NS

N A

M

T P E

C C

A

51

ACCEPTED MANUSCRIPT

D E

M

T P E

C C

A

52

p+ NS C +M M S PT P/ p+ N S

p

PT P/

S+ M

CM

CM

N A

T P

I R

C S U

PT P/

S

S+ M

P/ p+ N

M S

M PT

S

P/ p+ C

M PT

CM

P/ p

Co nt ro

l

120 bcdefgh 100 acdfg acdfg acdfg abdefgh 80 abcdeh abcdeh abcefgh 60 40 20 0

M PT

5-HTT relative intensity (% of control)

Striatum

ACCEPTED MANUSCRIPT

D E

M

T P E

C C

A

53

p+ NS C +M M S PT P/ p+ N S

p

PT P/

S+ M

CM

CM

N A

T P

I R

C S U

PT P/

S

S+ M

P/ p+ N

M S

M PT

S

P/ p+ C

M PT

CM

P/ p

Co nt ro

l

120 bcdefgh acdfg acdfg acdfg abdefgh 100 abcdeh 80 abcdeh abcefgh 60 40 20 0

M PT

5-HTT relative intensity (% of control)

Substantia nigra

ACCEPTED MANUSCRIPT Fig 8: ST

SN

p-TrkB

140 k Da

ERK 1/2

C S U

Da p-ERK 1/2

44 k Da/42 k

N A Da

AKT

D E

p-AKT

β-actin

60 k Da 60 k Da

T P E

CREB p-CREB

M

43 k Da

C C

43 k Da

A

42 k Da

A) p-TrkB 54

T P

I R

44 k Da/42 k

S+ M PT P/

A

120 bcdefgh 100 abdefgh 80 acdfg acdfg acdfg 60 abcdeh abcdeh abcefgh 40 20 0

55

NS +M PT

P/

p+ N

S

M S

p

S PT P/ p+ C

S+ M PT P/

CM

M S

S

120 bcdefgh 100 abdefgh 80 acdfg acdfg acdfg 60 abcdeh abcdeh abcefgh 40 20 0

D E P/ p+ N

P/ p+ C

CM

P/ p

l

Striatum

M PT

M PT

M PT

Co nt ro

T P E p-ERK/ERK (%)

B) p-ERK 1/2

S+ M

p-ERK/ERK (%)

S+ M

CM

S+ M

S PT P/

P/ p+ N

M S

S

S+ M

CM

S+ M

S PT P/

P/ p+ N

M S

S

p PT P/ p+ NS C +M M S PT P/ p+ N S

CM

l P/ p CM P/ p+ C M PT

M PT

M PT

Co nt ro

PT p P/ p+ NS C +M M S PT P/ p+ N S

CM

l

P/ p

CM P/ p+ C

M PT

M PT

M PT

Co nt ro

TrkB relative intensity (% of control)

TrkB relative intensity (% of control)

120 bcdefgh 100 abdefgh 80 60 acdfg acdfg abcdeh abcdeh acdfg abcefgh 40 20 0

CM

p+ N

C C P/ S

M S

p

S

PT P/

p+ C

S+ M

NS +M PT

CM

CM

M S

S

P/ p+ N

P/ p+ C

M PT

M PT

l

P/ p

CM

M PT

Co nt ro

ACCEPTED MANUSCRIPT

Striatum

Substantia nigra

120 bcdefgh abdefgh 100 80 acdfg acdfg acdfg 60 abcdeh abcdeh abcefgh 40 20 0

N A

C S U

Substantia nigra

M

I R T P

ACCEPTED MANUSCRIPT

C) p-AKT

p

PT P/

S+ M

CM

Substantia nigra

S P/ p+ C M M S PT P/ p CM +N S S+ CM M S+ PT M P/ PT p P/ p+ NS C +M M S PT P/ p+ N S

CM

M PT

p-CREB/CREB (%)

p+ NS C M +M S PT P/ p+ N S

PT P/

S

S+ M

P/ p+ N

CM

M S

M PT

S CM

P/ p+ C

N A

M

56

I R

C S U

120 bcdefgh 100 80 abdefgh acdfg acdfg acdfg abcdeh abcdeh 60 abcefgh 40 20 0 Co nt ro l M PT P/ p

p+ NS C M +M S PT P/ p+ N S

p PT P/ S+ M

PT P/

S CM

S+ M CM

M S

P/ p+ N

M PT

P/ p+ C

S CM

M PT

P/ p

A

l

P/ p

l

M PT

S+ M

C C

120 bcdefgh 100 80 abdefgh acdfg acdfg acdfg 60 abcdeh abcdeh abcefgh 40 20 0 M PT

M PT

p-AKT/AKT (%)

Co nt ro

p

p+ NS C M +M S PT P/ p+ N S

PT P/

S

S+ M CM

CM

M S

P/ p+ N

M PT

S

P/ p+ C

M PT

CM

P/ p

l Co nt ro

PT P/

T P E

Striatum p-CREB/CREB (%)

120 bcdefgh abdefgh 100 80 acdfg acdfg abcdeh abcdeh acdfg 60 abcefgh 40 20 0

D E

D) p-CREB

Co nt ro

T P

Substantia nigra

120 bcdefgh 100 abdefgh 80 acdfg acdfg acdfg abcdeh abcdeh 60 abcefgh 40 20 0 M PT

p-AKT/AKT (%)

Striatum

ACCEPTED MANUSCRIPT Highlights  MPTP/p treatment induced the motor dysfunction, oxidant-antioxidant imbalance and altered the expressions of BDNF, serotonin transporter and signalling molecules.

T P

 CMS exposure provoked the non-motor symptoms, antioxidant imbalance and altered the expressions of molecular indices.

I R

 Pre, post and pre&post stress exposure aggravated the MPTP/p mice induced motor and non-motor symptoms, antioxidant

C S U

imbalance and molecular expressions.

N A

 Post exposure of CMS to PD animals further worsened above said indices than pre or pre&post stress exposed PD animals.

D E

M

T P E

C C

A

57