Intervention with exercise restores motor deficits but not nigrostriatal loss in a progressive MPTP mouse model of Parkinson’s disease

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INTERVENTION WITH EXERCISE RESTORES MOTOR DEFICITS BUT NOT NIGROSTRIATAL LOSS IN A PROGRESSIVE MPTP MOUSE MODEL OF PARKINSON’S DISEASE I

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M. D. SCONCE, a M. J. CHURCHILL, a R. E. GREENE a AND C. K. MESHUL a,b,c*

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a Research Services, VA Medical Center/Portland, Mail Code: RD-29, Research Services, 3710 SW Veterans Hospital Road, Portland, OR 97239, United States

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b Department of Behavioral Neuroscience, Oregon Health & Science University, 3181 S.W. Sam Jackson Park Road, Portland, Oregon 97239, United States

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c Department of Pathology, Oregon Health & Science University, 3181 S.W. Sam Jackson Park Road, Portland, Oregon 97239, United States

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Abstract—Many studies have investigated exercise therapy in Parkinson’s disease (PD) and have shown benefits in improving motor deficits. However, exercise does not slow down the progression of the disease or induce the revival of lost nigrostriatal neurons. To examine the dichotomy of behavioral improvement without the slowing or recovery of dopaminergic cell or terminal loss, we tested exercise therapy in an intervention paradigm where voluntary running wheels were installed half-way through our progressive PD mouse model. In our model, 1-methyl-4-phenyl-1,2,3,6-tetra hydropyridine (MPTP) is administered over 4 weeks with increased doses each week (8, 16, 24, 32-kg/mg). We found that after 4 weeks of MPTP treatment, mice that volunteered to exercise had behavioral recovery in several measures despite the loss of 73% and 53% tyrosine hydroxylase (TH) within the dorsolateral (DL) striatum and the substantia nigra (SN), respectively which was equivalent to the loss seen in the mice that did not exercise but were also administered MPTP for 4 weeks. Mice treated with 4 weeks of MPTP showed a 41% loss of vesicular monoamine

transporter II (VMAT2), a 71% increase in the ratio of glycosylated/non-glycosylated dopamine transporter (DAT), and significant increases in glutamate transporters including VGLUT1, GLT-1, and excitatory amino acid carrier 1. MPTP mice that exercised showed recovery of all these biomarkers back to the levels seen in the vehicle group and showed less inflammation compared to the mice treated with MPTP for 4 weeks. Even though we did not measure tissue dopamine (DA) concentration, our data suggest that exercise does not alleviate motor deficits by sparing nigrostriatal neurons, but perhaps by stabilizing the extraneuronal neurotransmitters, as evident by a recovery of DA and glutamate transporters. However, suppressing inflammation could be another mechanism of this locomotor recovery. Although exercise will not be a successful treatment alone, it could supplement other pharmaceutical approaches to PD therapy. Ó 2015 Published by Elsevier Ltd. on behalf of IBRO.

Key words: exercise, motor behavior, MPTP, Parkinson’s disease, glutamate transporters, tyrosine hydroxylase. 17

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This work was supported by Merit Review #1BX 001643 to CKM from the United States (U.S.) Department of Veterans Affairs Biomedical Laboratory Research and Development. The contents do not represent the views of the U.S. Department of Veterans Affairs or the United States Government. *Correspondence to: C. K. Meshul, VA Medical Center/Portland, Mail Code: RD-29, Research Services, 3710 SW Veterans Hospital Road, Portland, OR 97239, United States. Tel: +1-503-220-8262x56788. E-mail address: [email protected] (C. K. Meshul). Abbreviations: BDNF, brain-derived neurotrophic factor; DA, dopamine; DAT, dopamine transporter; DL, dorsolateral; EAAC1, excitatory amino acid carrier 1; GFAP, glial fibrillary acidic protein; GLAST, glutamate aspartate transporter; GLT-1, glutamate transporter 1; IHC, immunohistochemistry; ir, immunoreactive; NFATc3, nuclear factor of activated T-cells cytoplasmic 3; PD, Parkinson’s disease; pTrkB, phosphorylated tyrosine kinase receptor B; MPTP, 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine; SN, substantia nigra; SNpc, substantia nigra pars compacta; TBST, tris-buffered saline tween 20; TH, tyrosine hydroxylase; TrkB, tyrosine kinase receptor B; VGLUT1, vesicular glutamate transporter 1; VGLUT2, vesicular glutamate transporter 2; VMAT2, vesicular mono amine transporter 2. http://dx.doi.org/10.1016/j.neuroscience.2015.04.069 0306-4522/Ó 2015 Published by Elsevier Ltd. on behalf of IBRO. 1

INTRODUCTION

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In the clinic and in animal models, exercise has been consistently shown to alleviate some of the motor deficits associated with Parkinson’s disease (PD). PD is a common neurodegenerative disorder caused by the loss of dopaminergic neurons in the nigrostriatal pathway. This loss of dopamine (DA) also results in changes in striatal glutamate (Klockgether et al., 1991; Meshul et al., 1999; Robinson et al., 2003; Walker et al 2009) which creates an imbalance between DA and glutamate neurotransmitters. Whether this imbalance of DA and glutamate is a cause or a result of PD is unknown, and unfortunately, as the degeneration progresses over time so does the impairment of basal ganglion-stimulated motor behaviors (Meshul et al., 1999; Touchon et al., 2004; Holmer et al., 2005; Smith et al., 2011). As seen clinically and in animal models, exercise has shown to attenuate motor deficits in PD, but it does not seem to provoke recovery of the lost neurons nor prevent the progressive nature of the disease (Fisher et al., 2004; Fisher et al., 2008; Al-Jarrah et al., 2007; Petzinger et al., 2007; Pothakos et al., 2009; Petzinger et al., 2010; Vucˇkovic´ et al., 2010; Petzinger et al., 2013). This suggests that the behavioral recovery observed must be

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due to exercise inducing other compensatory mechanisms that are separate from the mechanisms of dopaminergic survival and recovery. In this study, we looked at how intervening with voluntary exercise using running wheels would affect behavioral deficits as well as dopaminergic, glutamatergic and inflammatory biomarkers in our progressive 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. Of interest is a previous study reporting exercise-induced increases in both extracellular glutamate and DA levels within the striatum (Meeusen et al., 1997). However, following exercise training for several weeks, there was an overall decrease in the basal levels of both striatal DA and glutamate compared to the control group. MPTP has been extensively used in neurotoxin animal models of PD because it selectively lesions and causes neuronal death within the dopaminergic neurons in the basal ganglia (Meredith et al., 2008; Tieu, 2011; Blandini and Armentero, 2012). In contrast to other more acute/subacute MPTP/toxin models that lesion a large percent of the dopaminergic neurons in a short period of time, our progressive model better mimics the progressive temperament of PD by long-term gradual neurodegeneration of the nigrostriatal pathway where mice are injected with MPTP for 4 weeks (5 days/week) with increasing doses for the given week (8, 16, 24, and 32-mg/kg). In order to prevent inflammation due to possible stress of forced treadmill running (Howells et al., 2005), running wheels were installed into each singly housed mouse cage after the 2nd week of MPTP treatment (i.e. intervention) so that animals had the voluntary option to exercise. Although exercise has been shown to be protective against nigrostriatal degeneration in animal models (Lau et al., 2011; Gerecke et al., 2010), most patients upon diagnosis show a substantial amount of nigrostriatal terminal and cell loss (Bernheimer et al., 1973; Riederer and Wuketich, 1976; Kordower et al., 2013) which cedes rodent protection studies to be clinically irrelevant. Testing a therapy via intervention in our progressive mouse model is a more clinically appropriate approach because like the patients, the mice will already have dopaminergic cell and terminal loss; therefore, we can gauge if exercise therapy in our model can prevent or slow the progression of the nigrostriatal loss. Furthermore, due to the progressive nature of our animal model compared to the more acute/subacute models that have been used in previous exercise studies, we hypothesized that intervention with voluntary exercise would attenuate motor deficits and be disease modifying. To our knowledge, this is the first time that intervention with voluntary exercise therapy has been tested in a progressive mouse model of PD.

EXPERIMENTAL PROCEDURES

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Animals

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31 male C57BL/6J mice (Jackson Labs, Bar Harbor, ME, USA; 8-weeks old at arrival) were housed 3–4/cage and maintained on a 12-h light/dark cycle throughout (lights on 0600). They had ad libitum access to food and water. Mice were randomized into six groups: four mice

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in the vehicle group (VEH), eight mice in the MPTP group (4WK_MPTP), six mice in the vehicle/exercise group (VEH + Ex), five mice in the MPTP/exercise (4WK_MPTP + Ex), four mice in the 2-week vehicle group (2WK_VEH), and four mice in the 2-week MPTP group (2WK_MPTP). For the exercise groups, mice were housed 1/cage and running wheels (Mini-Mitter Co., Inc, Bend, OR, USA) were installed. Mice were not forced to exercise but were able to voluntarily and at anytime. Each wheel had a digital attachment that counted the number of revolutions regardless of direction. The number of revolutions was recorded each week day and averaged over the number of hours between each recording. Handling and care of mice was consistent with federal guidelines of the Public Health Service Policy on the Humane Care and Use of Laboratory Animals and protocols were approved by the Portland VA IACUC.

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MPTP administration

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MPTP (Santa Cruz Biotechnology, Dallas, TX, USA)treated animals received intraperitoneal injections 5 days/week for 4 weeks in total. The dosing of MPTP (calculated as the base) increased each week with 8 mg/kg for week 1, 16 mg/kg for week 2, 24 mg/kg for week 3, and 32 mg/kg for week 4 (4WK_MPTP). Normal saline (SA) (0.1 ml/0.1 kg) was used as the vehicle (VEH) for MPTP. After the first two weeks of MPTP treatment, a group of mice treated with MPTP and another group of mice treated with vehicle were separated and housed individually with cages that had running wheels (4WK_MPTP + Ex or VEH + Ex). Two additional groups of animals were separated and euthanized after the first two weeks of MPTP or saline treatment for striatal and midbrain/nigral analysis (2WK_MPTP or 2WK_VEH).

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Grip test analysis

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Mice were tested for grip strength at the conclusion of either the 2 weeks MPTP/vehicle treatment or at the end of the MPTP/exercise treatment with the Grip Strength Meter (Columbus Instruments, Columbus, Ohio, USA). Using the Mesh Pull Bar attachment, mice were handled by the tail so that only the forepaws could touch the apparatus. When the forepaws were clearly latched to the Mesh Pull Bar, the mouse was slowly and steadily pulled away from the apparatus exactly parallel from the counter top until the mouse released its forepaws from the Mesh Pull Bar. Each animal’s grip strength was an average of five grip strength tests and animals were given a 5-min rest in between each trial. The protocol was repeated additionally, as explained above, for all the paws latching on the Mesh Pull Bar together. Grip strength was measured in Newtons (N). Mice in the 2WK_MPTP and 2WK_VEH groups were tested 10 days after the last 16 mg/kg dose of MPTP. All of the other groups of mice (VEH, 4WK_MPTP, VEH + Ex, and 4WK_MPTP + Ex) were tested 25 days after the last 32 mg/kg dose of MPTP.

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Motor behavior analysis

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Gait was assessed using a DigiGait apparatus (Mouse Specifics, Quincy, MA, USA). Ventral plane videography captured the gait of each mouse through a transparent, motor-driven treadmill belt (Kale et al., 2004; Amende et al., 2005). Digital images of the paws of each mouse were taken at 150 frames/s as mice ran at a speed of 24 cm/s. The area of each paw relative to the treadmill belt at each frame was used for spatial and temporal measurements. Mice in the 2WK_MPTP and 2WK_VEH groups were tested 9 days after the last 16 mg/kg dose of MPTP. All the other groups of mice (VEH, 4WK_MPTP, VEH + Ex, and 4WK_MPTP + Ex) were tested 24 days after the last 32 mg/kg dose of MPTP.

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Harvesting & tissue fixation

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All the animals were euthanized by cervical dislocation. The fresh brain was cut coronally in half at the level of the hypothalamus, where the rostral half containing the striatum was fixed with 2.5% glutaraldehdye/0.5% paraformaldehyde/0.1% picric acid in 0.1 M phosphate buffer (pH 7.3) using a microwave tissue processor (Pelco BioWave Ted Pella, Inc, Redding, CA, USA) as follows: the tissue was placed in a temperature controlled bath with fixative using a thermoelectric recirculating chiller (Pelco SteadyTemp Pro, Ted Pella, Inc) in the microwave for 90 min total [45 min., 150 watts(W) at 30 °C/30 min., 150 W at 25 °C/15 min., 650 W at 25 °C]. The tissue was then left in 2% paraformaldehyde/0.1 M phosphate buffer (pH 7.3) at 4 °C for 48-72 hrs, then rinsed and left in 0.1 M phosphate buffer at 4 °C until cut for future tyrosine hydroxylase (TH) immunohistochemistry (IHC) (see below). For the caudal portion of the brain, the left and right substantia nigra (SN) were micro-dissected, using a stereomicroscope and a 3-mm microdissection knife, and frozen at 80 °C for future protein/western blot analysis (see below).

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Immunohistochemistry

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A vibratome (Ted Pella Inc., Redding, CA, USA) was used to cut consecutive 60-lm thick sections of the striatum (starting at Bregma + 1.2 mm and ending at the level of the anterior commissure). Six sections were chosen that extended throughout the rostrocaudal portion of the striatum and these sections were matched anatomically in each animal to verify that the coronal sections used were similar in all groups of mice. The following incubations were carried out in the PELCO BioWaveÒ Pro microwave (Ted Pella Inc., Redding, CA, USA) with the temperature limited to 35 °C. Rinsing solutions were under normal pressure unless otherwise stated. Sections were incubated in 10 mM sodium citrate (pH 6) for 5 min at 550 W for antigen retrieval in a vacuum chamber that cycles the pressure down to 20 Hg and back to atmosphere repeatedly during this step (cycling vacuum), rinsed in 0.01 M phosphate-buffered saline (PBS) at 150 W for 1 min, rinsed with 0.3% hydrogen peroxide (150 W, 1 min), two PBS rinses for 1 min, and

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then incubated in 0.5% Triton X-100 in PBS (550 W, 5 min, cycling vacuum). Sections were incubated with tyrosine hydroxylase (TH) antibody (mouse monoclonal, 1:2000 for the striatum; Immunostar, Hudson, WI, USA) or with ionizing calcium-binding adaptor molecule 1 (Iba-1) antibody (rabbit polyclonal, 1:200 for the striatum; Proteintech, Chicago, IL, USA) at 200 W for 36 min and 20 s under continuous vacuum (20 Hg, cycling the magnetron for 2 min on/3 min off/2 min on/5 min off repeating). Sections were incubated in blocking solution [10% goat serum/0.5% Triton-X 100/0.1 M phosphate buffer; pH 7.4] for two, 1 min washes at 150 W, and then exposed to biotinylated goat anti-mouse secondary antibody (1:400; Vector, Burlingame, CA, USA) for 16 min and 20 sec under continuous vacuum (10 s at 150 W, 4 min at 200 W, 3 min at 0 W, 4 min at 200 W, 5 min at 0 W, and 10 s at 150 W), rinsed with PBS, then washed with imidazole working buffer [5% Imidazole buffer(0.2 M), pH 9.0/16% sodium acetate (0.1 M), pH7.2] (1 min at 150 W), and finally incubated with avidin-biotin complex solution (ABC) (diluted according to manufacturer instruction; Vector) for 16 min and 10 sec under continuous vacuum (4 min at 200 W, 3 min at 0 W, 4 min at 200 W, 5 min at 0 W, and 10 sec at 200 W). Tissue was then rinsed with imidazole working buffer (1 min at 150 W), incubated with diaminobenzidine (DAB) (0.1% (Sigma Chemical Co., St Louis, MO; Cat #: D5637) + 1.5% hydrogen peroxide in 0.1 M phosphate buffer) for 10 min 20 sec under continuous vacuum (10 s at 200 W, 10 min at 200 W, 10 s at 0 W), rinsed with imidazole working buffer, and finally in PBS. Tissue was mounted on gel-coated slides, dehydrated at room temperature overnight and cover-slipped using Pro-TexxÒ medium (Lerner, Pittsburgh, PA, USA). Tissue from all treatment groups was processed on the same day, and all reacted with DAB for the same length of time. Optical density of the dorsolateral (DL) region of the striatum for each section for every animal was analyzed using light microscopy (1.25x magnification, images analyzed using ImagePro Plus 6.3, Media Cybernetics, Rockville, MD, USA). Treatment groups were analyzed in a blinded manner.

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Western blots

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Dissected SN tissue was thawed on ice. Protein was extracted from the tissue by sonication in lysis buffer [5% 1 M Tris, 2% 0.5 M EDTA, 1% Triton-X 100, 0.5% Protease Inhibitor Cocktail III (EMD Millipore’s Calbiochem, Darmstadt, Germany)]. Protein concentrations of the tissue from each individual animal were measured using the BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA). 10 lg of protein from each sample was mixed with XT Sample Buffer and XT Reducing Agent (1:10; Bio-Rad, Hercules, CA, USA) and underwent electrophoresis on a 4-12% Bis-Tris XT Precast Gel (Bio-Rad, Hercules, CA, USA). Separated proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, MA, USA). The membranes were blocked in either a 5% non-fat dry milk in Tris–buffered saline with Tween-20 (TBST) for

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Please cite this article in press as: Sconce MD et al. Intervention with exercise restores motor deficits but not nigrostriatal loss in a progressive MPTP mouse model of Parkinson’s disease. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.04.069

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60 min. Membranes were then washed three times for 5 min each in TBST, and probed with the following primary antibodies: tyrosine hydroxylase (TH at 62 kDa; Immunostar, Inc., Hudson, WI, 1:40,000, mouse monoclonal), phospho-tyrosine repeat kinase B (pTrkB at 145 kDa; Abcam (Cambridge, MA) 1:1000; rabbit polyclonal), vesicular monoamine transporter 2 (VMAT2 at 57 kDa; Synaptic Systems (Goettingen Germany); 1:200 rabbit polyclonal), dopamine transporter (DAT at 80 and 50 kDa; Proteintech (Chicago, IL); 1:1000; rabbit polyclonal), vesicular glutamate transporter 1 and 2 (VGLUT1 at 62 kDa, 1:20,000, and VGLUT2 at 52 kDa, Synaptic Systems; 1:2000 rabbit polyclonal), glutamate transporter 1 (GLT-1 or known as EAAT2 at 70 kDa, Santa Cruz Biotech Inc (Dallas, TX); 1:1000, rabbit polyclonal), excitatory amino acid carrier 1 (EAAC1 or known as EAAT3 at 57 kDa, Santa Cruz Biotech Inc; 1:1000; rabbit polyclonal), glutamate aspartate transporter (GLAST or known as EAAT1 at 64 kDa, Santa Cruz Biotech Inc; 1:1000; rabbit polyclonal) glial fibrillary acidic protein (GFAP at 55 kDa, Sigma Aldrich; 1:6000; mouse monoclonal), nuclear factor of activated T-cells cytoplasmic 3 (NFATc3 at 190 and 130 kDa, Santa Cruz Biotech Inc; 1:1000; rabbit polyclonal), and b-actin (at 47 kDa, Sigma Aldrich; 1:65,000; mouse monoclonal) . After three 5-min washes in TBST, membranes were probed with secondary antibodies for 1 h (ap-bovine anti-goat IgG H + L, Jackson ImmunoResearch Laboratories Inc (West Grove, PA); ap-goat anti-mouse IgG H + L, Bio-Rad, ap-goat anti-rabbit IgG H + L, Bio-Rad) then washed again in TBST for 3  5 min. Enhanced chemifluoresence (ECF) substrate (GE Healthcare, Piscataway, NJ, USA) was added to the membrane prior to visualization. Visualization and quantification of the antigen-antibody binding density were performed using the Typhoon HLA7000 imaging system (GE Healthcare, Piscataway, NJ, USA), and ImagePro Plus 6.3 software (Media Cybernetics, Rockville, MD, USA), respectively. Protein densities were analyzed relative to individual b-actin densities and the normalized optical density was determined for each animal. Each sample was at least duplicated on separate membranes and averaged for each animal. All the animals were averaged according to their group.

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Statistical analysis

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To analyze the amount of exercise at the end of each week, repeated measures analysis of variance (ANOVA) was used to compare the amount of exercise pursued between the VEH + Ex and 4WK_MPTP + Ex groups. A two-way ANOVA was then used for each group to compare the averages at the end of each week. Significant interactions were subject to post hoc Tukey– Kramer HSD tests for multiple comparisons. For behavioral, IHC, and western blot analysis, two-way ANOVA’s were used to compare the MPTP groups to each other as well as their respective vehicle groups. All significant interactions were subjected to post hoc Tukey–Kramer HSD tests for multiple comparisons. If there were no differences between the vehicle groups,

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then those groups were combined before calculating the percent of vehicle against the MPTP-treated groups. In the analyses for which significant interactions were not observed, the a priori hypothesis that the population (exercise versus no exercise) had a greater effect in the MPTP-compared to the vehicle-treated animals was addressed in planned comparisons between the exercise (4WK_MPTP + Ex/VEH + Ex) and no exercise pairs (4WK_MPTP/vehicle) by the Student’s paired t-tests. Pearson correlations with significant r-values were used to evaluate the correlations between motor behaviors and biochemical markers (Fig. 7). All statistical analyses were considered significant at p < 0.05.

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RESULTS

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Behavioral Analysis

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Despite 4 weeks of MPTP administration, mice that exercised showed behavioral recovery. Voluntary wheel running exercise was available to the mice starting during the 3rd week of MPTP administration (i.e. intervention). There was a main effect of MPTP and the distance the mice voluntarily exercised (Fig.1; F(4,5) = 6.3634; p = 0.0337). Compared to the vehicle group, mice administered MPTP ran 90% less during the 3rd week of the treatment (p = 0.0062) and 74% less during the 4th week of treatment (p = 0.0256). The 4WK_MPTP + Ex group then recovered to similar distances of voluntary exercise of the vehicle group after the last dosing week of MPTP. Furthermore, comparing among the MPTP group to itself after each week pre/post toxin administration, there was a

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Fig. 1. Voluntary exercise running rate during and following the final two weeks of the MPTP dosing paradigm. While mice were administered MPTP (n = 5), their rate of wheel running decreased 90% and 74% compared to the vehicle mice (n = 6) (3rd week and 4th week, respectively). After MPTP administration (off_1WK-3WK), voluntary wheel running recovered to the rates of the vehicle mice. Values are mean ± S.E.M. ⁄p = 0.0062 vs VEH + Ex (3rd week), ⁄⁄p = 0.0256 vs VEH + Ex (4th week), dp = 0.0169 vs 4WK_MPTP + Ex (3rd week), and ddp = 0.0190 vs 4WK_MPTP + Ex (4th week).

Please cite this article in press as: Sconce MD et al. Intervention with exercise restores motor deficits but not nigrostriatal loss in a progressive MPTP mouse model of Parkinson’s disease. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.04.069

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significant increase in the amount of voluntary exercise observed between the first week off treatment and the 3rd week of MPTP treatment (p = 0.0169) as well as with the 4th week of MPTP treatment (p = 0.0190). Because there was recovery in the amount of exercise for mice treated with MPTP, other physical, behavioral, and biochemical measures were analyzed including the weight gained from the beginning to end of the experiment. All mice were weighed before and after any treatment. Analysis showed that there was a main effect of MPTP on the difference in weight gain/loss from the beginning to the end of the study (Fig. 2A; F(5,20) = 3.213; p = 0.0273). Mice treated with MPTP for 2 weeks had an averaged loss of 0.15 g of weight, which was statistically significant compared to the vehicle group’s averaged weight gain of 1.11 g (p = 0.0054). The mice treated with MPTP for 4 weeks gained 0.43 g on average, which was trending from the weight difference in the vehicle group (p = 0.054), though not statistically different. Mice that were treated with MPTP and given the option to exercise on free-running wheels gained 1.5 g of weight after the study which was similar to the vehicle group as well as statically different from the 2WK_MPTP (p = 0.0015) and the 4WK_MPTP (p = 0.0119) groups. Despite 4 weeks of MPTP treatment, animals allowed to exercise had similar weight gain to the vehicle group. Further analyses of grip test behavior were then elucidated. The grip test investigates the force of grip strength. The grip strength in the forepaws and all paws together of all mice was tested before harvesting the animals. Statistically there were no differences in the grip strength of all four paws together within the groups. However, there was a main effect of MPTP on the percent of forepaw grip strength (Fig. 2B; F(5,19) = 7.1535; p = 0.0006) with respect to the strength in all the paws together. The animals treated with MPTP for 4 weeks showed an increase of 31% in the grip strength of the forepaws compared to the vehicle group (p < 0.0001). The 4WK_MPTP group increased 27% and 19% compared to the 2WK_MPTP group (p = 0.0031) and the 4WK_MPTP + Ex group (p = 0.0361), respectively. The 2WK_MPTP and 4WK_MPTP + Ex groups were not statistically different compared to the vehicle group. Animals that were administered MPTP for 4 weeks with the option to exercise showed a grip strength similar to that of the vehicle group. In additional to analyzing motor deficits by physical weight changes and grip strength, different parameters of gait dynamics were also measured. Looking at the paws while the mice were running revealed that MPTP had a main effect of the paw area at peak stance (Fig. 2C; F(5,16) = 8.4496; p = 0.0005) as well as the maximal rate of change of paw area in contact with the treadmill belt during the breaking phase (MAX dA/dT) (Fig. 2D; F(5,16) = 7.9748; p = 0.0006). Mice treated with MPTP for 4 weeks had a significant 45% increase in the overall paw area at peak stance compared to the vehicle group (p = 0.0045), which was also significantly

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increased compared to the 2WK_MPTP (p = 0.0125) and 4WK_MPTP + Ex (p = 0.0014) groups. The MAX dA/dT in the 4WK_MPTP mice also significantly increased by 72% compared to the vehicle group (p < 0.0001), which was similarly increased compared to the 2WK_MPTP (p = 0.0019) and the 4WK_MPTP + Ex (p = 0.0002) groups. An increase in the paw area at peak stance and in the rate that the animal decelerates may suggest that the behavior deficits in the animals administered MPTP include a decrease in the typical agile running dynamics. Furthermore, MPTP had a main effect on the stance width (Fig. 2E; F(4,15) = 8.6484; p = 0.0056) of the mice as well as the percent of shared stance (Fig. 2F; F(5,15) = 9.0554; p = 0.0004). In animals treated with MPTP for 4 weeks, stance width increased 27% compared to the vehicle (p = 0.003), which was also similarly increased compared to the 2WK_MPTP (p = 0.0084) and 4WK_MPTP + Ex (p = 0.0291) groups. The percent of stance that was shared or on the treadmill belt at the same time while the mice were running had significantly decreased by 50% in the 4WK_MPTP animals compared to the vehicle group (p = 0.0220). The 4WK_MPTP + Ex mice had a recovery of this deficit to vehicle levels and was significantly increased compared to the 4WK_MPTP mice (p = 0.0062). An increase in stance width and a decrease in the percent of shared stance while running suggest that the motor deficits in our progressive model include postural changes and instability which would correlate with the PD patients who experience changes in their stance and posture. Taken together, among all the measures of behavior, animals who were administered MPTP and volunteered to exercise showed a recovery in all the motor deficits to vehicle group levels.

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TH-immunoreactivity (TH-ir) Analysis

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Exercise did not recover or slow down the progressive loss of TH in the DL striatum, nor the SN. The levels of the dopaminergic biomarker TH were assessed and compared within the DL striatum by IHC and within the SN by western blot. There was a main effect of MPTP on the levels of TH-ir within the DL striatum (Fig. 3A, C; F(2,12) = 8.924; p = 0.0042) as well as within the SN (Fig. 3B, D; F(5,19) = 41.7; p < 0.0001). Within the DL striatum, mice administered MPTP for 2 weeks showed a significant decrease of 42% compared to the respective vehicle group (p = 0.0019). Progressively, the animals administered MPTP for 4 weeks had a significant decrease of 70% compared to the respective vehicle group (p < 0.0001) with the DL striatum. Similarly, the mice that were administered MPTP for 4 weeks but also exercised showed a significant 73% loss of TH compared to the respective vehicle group (p < 0.0001) within the DL striatum. However, despite the continued loss of TH expression within the DL striatum similar to that observed in the MPTP-treated animals, the animals that were administered MPTP and

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Fig. 2. Weight changes and behavioral analysis by grip test and gait dynamics. (A) The 2WK_MPTP group (n = 4) lost 0.15 g of body weight while the 4WK_MPTP group (n = 8) gained 0.43 g of body weight, which is a 61% decrease compared to the 1.11 g gained in the vehicle group (n = 14). The 4WK_MPTP + Ex group (n = 5) showed a weight gain of 1.5 g which was significantly different compared to the other MPTP groups but similar to the vehicle group. (B) Grip strength in the forepaw increased by 31% in the 4WK_MPTP group compared to the vehicle group. The 2WK_MPTP, 4WK_MPTP + Ex, and vehicle groups had similar averaged forepaw grip strength. (C) 4WK_MPTP increased their paw area at peak stance by 45%, while the 2WK_MPTP and 4WK_MPTP + Ex groups had similar paw areas compared to the vehicle group. (D) Assessing how quickly the mice decelerate, 4WK_MPTP mice showed a 72% increase in MAX dA/dT compared to the vehicle group, while the 2WK_MPTP and 4WK_MPTP + Ex mice did not show a change with respect to the vehicle group. (E) The stance width of the mice increased 27% in 4WK_MPTP animals compared to the vehicle and there were no changes in the 2WK_MPTP and 4WK_MPTP + Ex groups. (F) The percent of the stride duration that the paw was in contact with the belt decreased 50% in the 4WK_MPTP mice compared to the vehicle group. The 2WK_MPTP and 4WK_MPTP + Ex animals maintained a similar % stride in stance compared to the vehicle group. Values are mean ± S.E.M.

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exercised showed behavioral recovery. Additional sections of the DL striatum were analyzed for Iba-1, to determine the relative expression of the microglia marker by IHC. There was a main effect of MPTP on

the relative optical density of Iba-1 levels within the DL striatum (F(3,13) = 5.1509; p = 0.0145). The animals treated with MPTP had a statistically significant 65 ± 14.8% (values are means of %/respective

Please cite this article in press as: Sconce MD et al. Intervention with exercise restores motor deficits but not nigrostriatal loss in a progressive MPTP mouse model of Parkinson’s disease. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.04.069

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DL striatum

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B TH (62kDa) β-actin (43 kDa)

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D

Fig. 3. TH-ir IHC of the DL striatum and western blot analysis of the SN. Changes in the relative optical density within the (A) DL striatum by IHC and (B) SN by western blot. (C) Compared to the respective vehicle group (n = 3-4), the 2WK_MPTP group (n = 4) showed a 42% loss in TH of the DL striatum and the 4WK_MPTP group (n = 8) showed a 70% loss in TH. Similarly, the 4WK_MPTP + Ex group (n = 5) had a 73% loss. (D) By western blot analysis of the SN, only a 9% loss of TH was observed in the 2WK_MPTP group compared to the respective vehicle group. The 4WK_MPTP and 4WK_MPTP + Ex displayed similar losses of 67% and 53%, respectively, compared to their respective vehicle group. Values are mean ± S.E.M.

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vehicle ± S.E.M) increase in Iba-1 expression with respect to its vehicle group (p = 0.0287). The animals treated with MPTP and exercised had a slight but nonsignificant 21% ± 3.7 increase in Iba-1 levels compared to its respective vehicle group and was trending toward a 44% decrease compared to the MPTP animals (p = 0.0925). Within the SN, mice that were administered MPTP for 4 weeks, with or without exercise, showed a significant decrease of 52% and 63%, respectively, in TH compared to the vehicle group (p < 0.0001). The 2WK_MPTP group had similar levels of TH within the SN compared to the vehicle group. Additionally, the 2WK_MPTP group was significantly different verses the 4WK_MPTP and 4WK_MPTP + Ex groups (p < 0.0001). Because the levels of TH expression in the DL striatum and SN did not recovery via voluntary exercise, this suggests that exercise did not induce restoration or prevent loss of the nigro-striatal neurons. In contrast, the effects of exercise on levels of brain-derived neurotrophic factor (BDNF) have been extensively studied within the hippocampus (Cotman and Berchtold, 2002). BDNF binds to tyrosine repeat kinase (TrkB) causing the dimerization and autophosphorylation (pTrkB) of the receptor which leads to the signaling cascades that promotes cell survival and growth (Guillin et al., 2001; Carvalho et al., 2008; Cobb, 1999; Grewal et al., 1999; Chao, 2003; Minichiello., 2009; Skaper., 2012). To investigate if exercise had an effect

on the levels of TrkB activation, pTrkB expression was analyzed by western blot. There was a main effect of MPTP administration on the expression of pTrkB (F(5,18) = 3.4243; p = 0.0239). The 2WK_MPTP mice maintained similar levels of pTrkB compared to the respective vehicle group and the 4WK_MPTP mice had a 27% ± 4.5 (values are means of %/respective vehicle ± S.E.M) decrease of pTrkB compared to the respective vehicle group (p = 0.0296). Additionally, the 4WK_MPTP + Ex group had a similar decrease of 32% ± 11.5 compared to the respective vehicle group (p = 0.0112). Our data suggest that at least within the SN, exercise does not rescue pTrkB levels.

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Despite dopaminergic cell/terminal loss, exercise maintained other regulatory biomarkers of dopamine in the SN. VMAT2 is a vesicular transmembrane protein responsible for uptake of DA into the synaptic vesicles. Within the SNpc cell bodies, there is also dendritic release of DA, meaning that VMAT2 can be detected within the SN (Geffen et al., 1976; Cheramy et al., 1981; Greenfield, 1985). MPTP had a main effect on the expression levels of VMAT2 within the SN (Fig. 4A, C; F(5,16) = 3.8144; p = 0.0182). There was a 41% loss of VMAT2 in the 4WK_MPTP mice compared to the

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VMAT2 (57kDa)

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β-actin (43 kDa)

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DAT<

(80kDa) (50kDa) β-actin (43 kDa)

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Fig. 4. SN analysis of VMAT2 and DAT by western blot. (A) Resulting western blot of the SN for VMAT2 and (B) DAT. (C) The 2WK_MPTP mice (n = 3) maintained VMAT2 protein levels similar to the vehicle group (n = 11) while the 4WK_MPTP mice (n = 8) had a 41% loss compared to the vehicle group. Similar to the vehicle as well as the 2WK_MPTP group, the 4WK_MPTP + Ex mice (n = 4) maintained VMAT2 levels which were increased by 44% compared to animals administered MPTP for 4 weeks. (D) Taking the ratio of glycosylated DAT to non-glycolsylated DAT showed that the 2WK_MPTP mice had a significant 43% decrease compared to the respective vehicle group (n = 3–4) while the 4WK_MPTP mice had a significant 71% increase in the ratio compared to the respective vehicle group. The 4WK_MPTP + Ex group had a similar ratio of DAT compared to the respective vehicle group. Values are mean ± S.E.M.

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respective vehicle group (p = 0.0203). The 2WK_MPTP and 4WK_MPTP + Ex groups had similar levels of VMAT2 compared to the respective vehicle groups and were significantly increased compared to the 4WK_MPTP group by 50% (p = 0.0014) and 44% (p = 0.0008), respectively. Mice administered MPTP for 4 weeks and were allowed to exercise maintained VMAT2 expression levels where the mice administered MPTP for 4 weeks without exercise had decreased expression levels. Further analysis of potential mechanisms of DA homeostasis was determined by investigating levels of DAT expression. The amount of post-translational protein modification of DAT by glycosylation can play a role in the activity of the transporter’s function of clearing extraneuronal DA as well as the degradation of the protein (Li et al., 2004). The relative levels of glycosylated and non-glycosylated DAT were analyzed and expressed as a ratio. There was a main effect of MPTP on the ratio of glycosylated to non-glycosylated DAT within the SN (Fig. 4B, D; F(5,20 = 3.0354; p = 0.0337). The 2WK_MPTP mice showed a significant 43% decrease in the ratio compared to the respective vehicle group (p = 0.0076) while the 4WK_MPTP showed a trending 71% increase in the ratio compared to the respective vehicle (p = 0.0730). There was a significant 114% difference between the 2- and 4-week-treated MPTP groups (p = 0.0026). The ratio of glycosylated to

non-glycosylated DAT in the 4WK_MPTP + Ex mice was similar to the respective vehicle group, and 84% less compared to the 4WK_MPTP group (p = 0.0257). These data suggest that despite 4 weeks of MPTP administration, animals who exercised maintained comparable levels of VMAT2 and the ratio of glycosylated to un-glycosylated DAT as the vehicle group in the remaining dopaminergic neurons within the SN. This could help explain behavioral recovery without dopaminergic cell/terminal recovery.

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Exercise may help achieve glutamate homeostasis through glutamate transporters. Aside from VMAT2 and DAT, exercise could also affect the glutamatergic system. A debated hypothesis in PD is that over stimulation of the STN results in excess glutamate levels at the SNpc dendrites and soma. Excess glutamate could then induce toxicity and perpetuate additional DA cell loss. Because the previous results showed behavioral recovery despite continued dopaminergic cell/terminal loss, it is possible that exercise may be evoking regulation of glutamatergic mechanisms instead of contributing to the survival of dopaminergic cells/terminals. VGLUT1 is a vesicular transmembrane protein responsible for uptake of glutamate into the synaptic vesicles for transmission. The motor cortex projects terminals onto the SNpc and SNpr (pars reticulata) and utilizes VGLUT1 as the

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protein associated with vesicular glutamate uptake. The STN also has a glutamatergic projection to the SNpc/SNpr but uses the vesicular glutamate transporter-2 (VGLUT2) instead of the analogous VGLUT1 (Kaneko et al., 2002). Despite lack of any

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VGLUT1 (61.6 kDa)

differences between the vehicle and MPTP groups in terms of VGLUT2 expression levels within the SN (data not shown), there was a main effect of VGLUT1 expression following MPTP administration (Fig. 5A, C; F(5,18) = 4.4476; p = 0.0082). The 2WK_MPTP mice

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β-actin (43 kDa)

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GLT-1 (70 kDa) β-actin (43 kDa)

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EAAC1 (57 kDa)

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GLAST (64 kDa) β-actin (43 kDa)

β-actin (43 kDa)

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H

Fig. 5. SN analysis of glutamate biomarkers by western blot. (A) Resulting western blot of the SN for VGLUT1 and (B) GLT-1. (C) Changes in VGLUT1 levels were seen in the 4WK_MPTP (n = 5) group with a significant 120% increase compared to the vehicle group (n = 12). The 2WK_MPTP (n = 3) group was similar to the vehicle group with only a 22% non-significant increase. The 4WK_MPTP + Ex (n = 4) was statistically between the other two MPTP groups with a 54% non-significant increase compared to the vehicle group. (D) GLT-1 protein levels were both significantly increased in the 2WK_MPTP (n = 4) and 4WK_MPTP (n = 7) groups by 60% and 89%, respectively, compared to the vehicle group (n = 10). The 4WK_MPTP + Ex (n = 4) group maintained similar levels of GLT-1 compared to the respective vehicle group. (E) Resulting western blot of the SN pc for EAAC1 and (F) GLAST. (G) Expression of EAAC1 maintained similar levels compared to the respective vehicle group for the 2WK_MPTP (n = 3) and 4WK_MPTP + Ex (n = 3) groups. The 4WK_MPTP (n = 6) mice showed a 101% increase in EAAC1 levels, which were significantly different compared to the respective vehicle groups. (H) Levels of GLAST in the 2WK_MPTP (n = 3) and 4WK_MPTP (n = 7) groups were not different compared to the vehicle group (n = 11). The 4WK_MPTP + Ex mice had a 31% decrease compared to the vehicle group. Values are mean ± S.E.M. Please cite this article in press as: Sconce MD et al. Intervention with exercise restores motor deficits but not nigrostriatal loss in a progressive MPTP mouse model of Parkinson’s disease. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.04.069

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showed a 22% increase in VGLUT1 expression which was not statistically different compared to the vehicle group. The 4WK_MPTP mice had a 120% increase in VGLUT1 levels compared to the vehicle group (p = 0.0010), which was also increased compared to the 2WK_MPTP (p = 0.0133) and 4WK_MPTP + Ex (p = 0.0469) groups. The 4WK_MPTP + Ex group also had elevated VGLUT1 expression with a 49% increase compared to the vehicle group, which was not statistically significant (p = 0.0993). A decrease in VGLUT1 expression in the 4WK_MPTP + Ex mice relative to the 4WK_MPTP mice suggests that exercise helps alleviate increased glutamate input from the cortex. Other plasma membrane transporters of glutamate, such as GLT-1, EAAC1, and GLAST, could be regulated if there was increased activity of the cortico-nigral pathway. These different subtypes of glutamate transporters all function to clear extracellular glutamate. The GLT-1 transporter is located on microglia and astrocytes (Rothstein et al., 1994; Shashidharan et al., 1997; Maragakis and Rothstein, 2004). There was a main effect of MPTP on the expression of GLT-1 (Fig. 5B, D; F(5,19) = 3.3005; p = 0.0259). The 2WK_MPTP mice had a 60% increase in GLT-1 levels compared to the vehicle group (p = 0.0150), while the 4WK_MPTP mice had an 89% increase compared to the vehicle group (p = 0.0155). The 4WK_MPTP + Ex mice had similar

A

+phos~ (190 kDa) NFATc3<

-phos~ (130 kDa)

levels of GLT-1 compared to the vehicle group and had decreased levels compared to the 4WK_MPTP (p = 0.0419). Additionally, EAAC1 regulates neurotransmitter homeostasis by clearing extraneuronal glutamate and is found on the SNpc cell bodies and dendrites (Shashidharan et al., 1997; Plaitakis., 2000). After administration of MPTP, there was a main effect on levels of EAAC1 (Fig. 5E, G; F(5,18) = 4.9386; p = 0.0051). The 2WK_MPTP and 4WK_MPTP + Ex group preserved similar levels of EAAC1 compared to their respective vehicle groups. The 4WK_MPTP group had increased EAAC1 levels to 101% compared to the respective vehicle group (p = 0.0021) and the 2WK_MPTP (p = 0.0063) and 4WK_MPTP + Ex (p = 0.0049) groups. Increased expression of GLT-1 and EAAC1, along with VGLUT1, would be consistent with the suggestion that there is an increase in extracellular glutamate levels within the SN of the 4WK_MPTP mice, as we have previously reported in another MPTP model (Meredith et al., 2009). Additionally, the expression of GLAST was determined because it is also involved in regulating glutamate transport and can be found in both astroglia and neurons (Rothstein et al., 1994; Shashidharan et al., 1997; Plaitakis, 2000). There was a main effect of exercise on the expression of GLAST (Fig. 5F, H; F(5,9) = 6.1421; p = 0.0208). The 2WK_MPTP and 4WK_MPTP groups had similar and non-significant changes in the expression

B

β-actin (43 kDa)

β-actin (43 kDa)

C

GFAP (55 kDa)

D

Fig. 6. SN analysis of GFAP and NFATc3 by western blot. (A) Resulting western blot of the SN for NFATc3 and (B) GFAP. (C) Taking the ratio of phosphorylated to non-phosphorylated NFATc3 showed that the 4WK_MPTP + Ex mice had a significant increase of 25% compared to the vehicle group while the 2WK_MPTP and 4WK_MPTP were similar to the vehicle group. The 4WK_MPTP + Ex was also significantly increased by 42% compared to the 4WK_MPTP mice. (D) There were no differences between the 2WK_MPTP (n = 4) and the respective vehicle group (n = 3-4) in GFAP expression. The 4WK_MPTP mice (n = 8) showed a significant 116% increase compared to the respective vehicle group, while the 4WK_MPTP + Ex group (n = 4) had a 41% increase in GFAP protein expression compared to the respective vehicle group. Additionally, the 4WK_MPTP group was significantly increased by 108% and 75% compared to the 2WK_MPTP and 4WK_MPTP + Ex groups, respectively. Values are mean ± S.E.M. Please cite this article in press as: Sconce MD et al. Intervention with exercise restores motor deficits but not nigrostriatal loss in a progressive MPTP mouse model of Parkinson’s disease. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.04.069

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levels of GLAST comparable to the vehicle group. The 4WK_MPTP + Ex group had decreased levels of GLAST by 31% compared to the vehicle group (p = 0.0265), which was also significantly less than the 2WK_MPTP (p = 0.0156) and 4WK_MPTP (p = 0.0030) groups. A decrease in GLAST expression in the 4WK_MPTP + Ex mice could imply that glutamate concentrations within the SN decreased compared to the 4WK_MPTP mice due to exercise. Animals administered MPTP and allowed to exercise had lower levels of inflammatory markers within the SN. Mechanistically, excess glutamate levels would increase inflammation due to the influx of Ca2+ upon binding to the glutamate receptors, which would lead to an increase in reactive oxygen species (Choi, 1988; Coyle and Puttfarcken, 1993; Blandini et al., 1996; Plaitakis and Shashidharan, 2000; Shashidharan et al., 2000). NFATc3 (component 3 of the nuclear factor of activated T-cells) is regulated in part by phosphorylation where it can only transverse the nucleus to turn on genes that signal apoptosis when it is non-phosphorylated (Crabtree and Olson, 2002; Macian, 2005). There was a main effect of exercise on the ratio of phosphorylated to non-phosphorylated NFATc3 (Fig. 6B, D; F(5,17) = 3.1389; p = 0.0346). Both the 2WK_MPTP and 4WK_MPTP mice maintained similar ratios of phosphorylated to non-phosphorylated NFATc3 compared to each other and their respective vehicle group. The 4WK_MPTP + Ex group had a significantly elevated ratio of 25% compared to the respective vehicle group (p = 0.0101) and a significant increase of 42% compared to the 4WK_MPTP group (p = 0.0130). An increased ratio of phosphorylated NFATc3 would mean that there is an increase in the percent of the NFATc3 protein population that cannot enter into the nucleus to regulate genes that would signal apoptosis. This piece of evidence further demonstrates that the behavioral recovery without dopaminergic cell/terminal recovery could be due in part by exercise normalizing DA release (as evidenced by DAT levels), regulating appropriate glutamate levels (as evidenced by VGLUT1, GLT-1, GLAST and EAAC1 levels) and thereby suppressing inflammation. The expression of GFAP levels was additionally assessed to indirectly gauge the relative amounts of inflammation. There was a main effect of MPTP on the levels of GFAP (Fig. 6A, C; F(5,19) = 15.4820; p < 0.0001). The 2WK_MPTP mice showed little astrocytic reaction and had similar GFAP levels compared to the vehicle group. The 4WK_MPTP mice had increased GFAP levels of 116%, which were significantly different compared to the vehicle group (p = 0.0043) as well as the 2WK_MPTP group (p = 0.0082). The 4WK_MPTP + Ex mice had GFAP expression levels that ranged between the other two MPTP groups, with an increase of 41% compared to the vehicle group. Levels of GFAP in the 4WK_MPTP + Ex mice were decreased by 75% compared to the 4WK_MPTP mice (p = 0.0373). These results with both NFATc3 and GFAP suggest that exercise may have helped alleviate the degree of inflammation within the SN; therefore, perhaps with less inflammation,

11

the remaining TH-ir cells within the SNpc can compensate by normalizing DA transmission to restore behavioral deficits without the need for either neurogenesis or axonal sprouting.

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Correlation analysis

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Dopaminergic, glutamatergic, and inflammatory biomarkers correlated with behavioral measures. By Pearson correlations, additional analysis showed a moderate negative Pearson correlation between VMAT2 and the paw area at peak stance (Fig. 7A; r = 0.5476; p = 0.0056) as well as the MAX dA/dT (Fig. 7B; r = 0.5787; p = 0.0031). There was a moderate positive correlation between VLGUT1 and the forepaw grip strength (Fig. 7C; r = 0.6433; p = 0.0007) as well as the MAX dA/dT (Fig. 7D; r = 0.5313; p = 0.0075) and a moderate positive correlation was observed between GLAST and the paw area at peak stance (Fig. 7E; r = 0.5245; p = 0.0085) as well as the MAX dA/dT (Fig. 7F; r = 0.5369; p = 0.0068). Furthermore, there was a moderate negative Pearson correlation between the ratio of phosphorylated to nonphosphorylated NFATc3 and the paw area at peak stance (Fig. 7G; r = 0.5351; p = 0.0071). Additional analysis showed a moderate negative Pearson correlation between levels of GFAP and the percent of shared stance (Fig. 7H; r = 0.5125; p = 0.0105).

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DISCUSSION

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Generally, exercise has many known benefits for overall health and has been shown to be protective against PD locomotor deficits in animal models (Lau et al., 2011) like many other pharmacological treatments (Ross and Petrovitch, 2001; Checkoway et al., 2002; Ravina et al., 2003). However, it is generally accepted that when patients are diagnosed with PD, dopaminergic loss of the nigrostriatal terminals is greater than 70%, while about 60% of the cell bodies of the SNpc are lost (Bernheimer et al., 1973; Riederer and Wuketich, 1976; Kordower et al., 2013), thus, rendering the protection study design in animal models much less translational to clinical PD. To more closely mimic the progressive dopaminergic cell loss and behavioral deficits seen clinically, we administered MPTP chronically and with increasing doses (Goldberg et al., 2011a,b; Sconce et al., 2015). Furthermore, PD patients are encouraged to exercise to help lighten motor symptoms and with the optimism that it might slow the progression of the disease. We are reporting in the current study that in our progressive mouse model of PD, intervening half-way through the progression with exercise alleviated motor deficits but did not prevent continued loss of the dopaminergic biomarker TH within either the striatum or SN. After the final administration of MPTP, the amount of exercise pursued by the mice increased back to the vehicle levels. Additionally, despite 4 weeks of MPTP administration, mice that pursued exercise gained similar weight and had similar grip strength, paw area at peak stance, MAX dA/dT, stance width, and percent shared stance, compared to the vehicle-

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Fig. 7. Pearson correlations of motor behaviors and biochemical markers. The expression of VMAT2 negatively correlated with (A) the paw area at peak stance and (B) the MAX dA/dT. The expression of VGLUT1 shows a significant positive correlation with (C) the % forepaw grip strength and (D) the MAX dA/dT. The relative expression levels of GLAST shows a significant positive correlation with (E) the paw area at peak stance and (F) the MAX dA/dT. (G) A moderate negative correlation was seen between the ratio of phosphorylated to non-phosphorylated NFATc3 and the paw area at peak stance, while (H) GFAP showed a negative correlation with the percent of shared stance (n = 24). Please cite this article in press as: Sconce MD et al. Intervention with exercise restores motor deficits but not nigrostriatal loss in a progressive MPTP mouse model of Parkinson’s disease. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.04.069

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treated mice. In comparison, mice that were administered MPTP for 4 weeks without exercise had a deficit in weight gain, which was associated with increased grip strength, stance width, paw area at peak stance, percent shared stance, and a decreased MAX dA/dT. By IHC and western blot, there were no differences in TH-ir within the DL striatum or the SN between the 4WK_MPTP and 4WK_MPTP + Ex groups despite the behavioral recovery observed in the mice that exercised. This suggests that exercise had an effect on other factors besides TH recovery that may be contributing to behavioral restoration. While 4 weeks of MPTP administration caused a decrease in VMAT2 and an increase in the ratio of glycosylated/non-glycosylated DAT within the SN, mice that were also administered 4 weeks of MPTP and pursued exercise showed the opposite trends which were similar to the vehicle group. Analysis of glutamate transporters within the SN revealed an analogous theme where the 4WK_MPTP + Ex mice maintained similar levels of VGLUT1, GLT-1, and EAAC1 compared to the vehicle and the 4WK_MPTP mice had significantly increased levels of these glutamate biomarkers, suggesting that exercised mice were capable of maintaining normal glutamate function. Additionally, animals that were administered 4 weeks of MPTP and pursued exercise showed a decrease in inflammatory markers that were increased in the 4WK_MPTP mice. Taken together, the return to vehicle levels of the glutamate markers and the association with the decrease in astrocytic/inflammatory markers suggests that following MPTP, there is an increase in extracellular glutamate within the SN via activation of the cortico-nigral/VGLUT1 pathway, which is reversed by exercise. These findings demonstrate that exercise did not modify the progressive loss of DA (as measured by TH) but perhaps alleviated motor deficits by compensating for DA release (as revealed by the changes in VMAT and DAT), maintaining glutamate homeostasis, and decreasing inflammation. Future studies will focus on measuring the extracellular levels of glutamate and DA within the SN and DL striatum using in vivo microdialysis (Meshul et al., 1999; Meredith et al., 2009). Exercise restored motor behaviors despite continued loss of TH in the DL striatum and SNpc Exercise has been known to alleviate some motor symptoms in animal models of PD (Tillerson et al., 2003; Al-Jarrah et al., 2007; Petzinger et al., 2007; Pothakos et al., 2009; Gorton et al., 2010; Vucˇkovic´ et al., 2010; Lau et al., 2011; Smith et al., 2011) as well as in the clinic (Bilowit, 1956; Szekely et al., 1982 ; Baatile et al., 2000; Toole et al., 2000; Petzinger et al., 2010). It is debated which kind of exercise is most effective and many mouse models have looked at forced exercise by treadmill running (Tillerson et al., 2003; Petzinger et al., 2007; Yoon et al., 2007; Pothakos et al., 2009; Vucˇkovic´ et al., 2010; Lau et al., 2011; Smith et al., 2011). To bypass the potential stress due to forced exercise that could interfere with exercise therapy (Howells et al., 2005), we installed running wheels into the mouse cages so each animal could volunteer to run at anytime. We found that following 2 weeks of MPTP treatment,

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when running wheels were then installed, mice volunteered to exercise 74–90% less than the vehicle-treated animals during the 3rd and 4th week of toxin administration. During the following weeks off any MPTP treatment, the amount of volunteered exercise increased back to the vehicle-treated mice. Gorton et al. (2010) compared both forced exercise via treadmill running and voluntary exercise by cage wheels in the same study. In contrast to our findings, they found that an MPTP lesion did not reduce voluntary exercise. This could be attributed to the differences in their acute MPTP model of four high doses of toxin injected in the same day, versus our progressive toxin administration over 4 weeks with increasing doses. Another difference that could explain why the MPTP mice in our study had a decrease in voluntary exercise is because we administered MPTP during the same time they were allowed to exercise versus after the MPTP lesion. It is possible that the decrease in exercise is simply due to the ill-effects of MPTP toxicity, suppressing any typical mouse movement and hence, the increased exercise after MPTP treatment. However, if the 4WK_MPTP + Ex mice were not exercising as much during the last two weeks of MPTP administration because of toxicity, the intervention experimental design still holds clinical value where patients with progressed PD have trouble getting out of bed, let alone doing cardiovascular exercise. Furthermore, other behavioral measures followed the same trend of recovery in the 4WK_MPTP + Ex mice and these tests were administered 25 days after the last injection of MPTP. As mice age, they typically gain weight. In our progressive MPTP studies, we consistently see that mice administered MPTP either gain little weight or lose it (Goldberg et al., 2012). Animals were weighed before the experiment and again before they were euthanized. The animals administered MPTP for 2 and 4 weeks did not gain the typical weight as seen with the vehicle-treated mice. Interestingly, the 4WK_MPTP + Ex mice gained the most weight compared to all the other groups. In addition to weight changes, the grip strength test was also implemented before harvesting. Nowak and Hermsdo¨rfer (2006) reported that patients increased their grip force earlier in anticipation of the opposite hand dropping a weight in a container compared to healthy controls. In addition the PD patients produced more variable force levels at the impact of the weight which, according to the authors, could indicate deficits of sensorimotor integration associated with motor dysfunction in basal ganglia disorders. For a similar correlate in our study, mice administered MPTP for 4 weeks had increased forepaw grip strength. Fellows and Noth (2004) reported that grip force was abnormally high in the hold and lifting phases for PD patients regardless of DA medication. Potentially comparable with basal ganglia dysfunction in mice, perhaps the 4WK_MPTP mice have increased forepaw grip strength because they have trouble letting go of the apparatus. Nonetheless, the mice that exercised had restored grip strength comparable to the vehicle group despite 4 weeks of MPTP treatment. Our findings also indicate that other motor behaviors associated with gait dynamics were restored in animals

Please cite this article in press as: Sconce MD et al. Intervention with exercise restores motor deficits but not nigrostriatal loss in a progressive MPTP mouse model of Parkinson’s disease. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.04.069

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that exercise and were administered MPTP. By analyzing the area of the paws, we found that there was a significant increase in the paw area at peak stance as well as the MAX dA/dT or rate that the animal decelerates via paw area in contact with the ground, in the animals treated with MPTP for 4 weeks. This could indicate that the 4WK_MPTP mice were not picking up their paws as quickly or had a decrease in agility. Additionally, we found a significant increase in the stance width and a decrease in the percent that the stance was shared during the running form with mice administered MPTP for 4 weeks. Patients with PD are reported to have motor symptoms of postural changes and instability/falling (Politis et al., 2010; Jankovic, 2008). An increase in stance width could suggest that the animals are compensating for postural instability by forming a wider stance which coincides with increased paw to ground surface area at peak stance. The deficit in the percent of shared stance further supports a decrease in the typical running agility and postural changes in the MPTP-treated mice. Taken together, animals that exercised had behavioral and motor recovery in all measures despite 4 weeks of MPTP treatment. Even though many studies have shown that exercise is of benefit in the recovery of motor deficits, it has not been shown to be disease modifying, such that it neither attenuates the progression of the disease nor induces recovery in the population of neurons within the SNpc that are degenerating (Al-Jarrah et al., 2007; Petzinger et al., 2007; Pothakos et al., 2009; Petzinger et al., 2010; Vucˇkovic´ et al., 2010). Although we used a different model of PD, our data are in agreement with the observation from other studies that exercise does not spare or result in any recovery of dopaminergic neurons when initiated following toxin administration (Al-Jarrah et al., 2007; Moroz et al., 2004; O’Dell et al., 2007). The 4WK_MPTP + Ex group had complete recovery of motor deficits but no such improvement or prevention of dopaminergic loss as gauged by TH-ir within the DL striatum and SN. Nonetheless, the 4WK_MPTP and 4WK_MPTP + Ex groups had equivalent and significant loss of TH within the DL striatum and SN compared to the vehicle group. This indicates that in our progressive model, behavioral recovery does not imply nigrostriatal sparing or recovery. To elucidate the lack of neuronal recovery further, the levels of pTrkB were analyzed within the SN. It has been repeatedly reported that BDNF levels increase in hippocampal neurons after exercise (Oliff et al., 1998; Russo-Neustadt et al., 1999; Cotman and Berchtold, 2002; Adlard et al., 2005). BDNF is needed for dopaminergic neurotransmission (Guillin et al., 2001; Carvalho et al., 2008) and binds to the TrkB receptor, causing the receptor to dimerize and autophosphorylate, which then results in the promotion of a series of signaling cascades that regulate cell growth/differentiation, neuronal survival, and synaptic plasticity (Guillin et al., 2001; Carvalho et al., 2008; Cobb, 1999; Grewal et al., 1999; Chao, 2003; Minichiello, 2009; Skaper, 2012). Instead of analyzing the expression of BDNF directly within the SN, measuring the levels of the phosphorylated form of TrkB (pTrkB) was

felt to be a more straight forward and sensitive assessment of neuronal restoration. We are reporting that even though exercise evoked behavioral recovery, it did not result in any stimulation of the TrkB receptor in animals treated with MPTP within the SN. This suggests that other compensatory mechanisms that participate in DA regulation may have played a role in the behavioral recovery that was observed.

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VMAT2 and DAT-glycosylation within the SN recovered to normal levels in animals that exercised

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Even though mice that were administered MPTP and voluntarily exercised showed just as much TH-ir loss as the mice that were administered MPTP, we looked at the relative levels of VMAT2 and DAT-glycosylation within the SN to determine if there was differential regulation between DA biomarkers. Our results show that VMAT2 levels significantly decreased in the 4WK_MPTP mice but had fully recovered in the 4WK_MPTP + Ex mice. This suggests that exercise could have evoked the remaining SN cells to increase their overall dopaminergic function as previously reported (O’Dell et al., 2007). It has previously been reported in the 6-hydroxydopamine (6-OHDA) lesion models, that after initial nigrostriatal loss, the remaining cells normalize striatal DA signaling in part by increasing DA release (Zigmond and Stricker, 1984; Zigmond et al., 1984, 1989, 1992; Abercrombie et al., 1990; Robinson et al., 1990; Snyder et al., 1990). However, with progressive MPTP lesions over 4 weeks, we previously reported that striatal DA is significantly decreased and DOPAC/DA turnover is increased (Goldberg et al., 2011a, 2012). In addition we have reported a direct correlation between levels of TH in the SN by western blot analysis and the average number of TH neurons within the SNpc (Goldberg et al., 2011a), suggesting that the loss of TH within the SN in the current study is most likely due to the loss of SNpc DA neurons. Therefore, the results of the current study suggest that within the remaining nigrostriatal neurons, exercise either induced an increase of dopaminergic function or prolonged initial normalization of striatal DA signaling rather than sparing the dopaminergic neurons. Another way to gauge dopaminergic function is by determining DAT levels. Aside from the DL striatum, DAT can also be localized to the dendrites of the SN cell bodies and is thought to be crucial for terminating DA action by clearing extraneuronal DA (Iversen, 1971). DAT is a heavily glycosylated protein (Li et al., 2004). Generally, including carbohydrates in post-translational modifications of proteins can contribute to protein folding and its stability at that conformation, regulating protein trafficking, and protecting against proteolysis (Lis and Sharon, 1998). Li et al. (2004) carefully characterized DAT stability and activity when all or individual glycosylation sites were mutated and found that prevention of N-glycosylation decreased DA transport efficiency but did not terminate function. We analyzed both the fully glycosylated DAT at 80 kDa as well as the non-glycosylated DAT at 50 kDa. Calculating a ratio between the two forms

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indicated that the 4WK_MPTP mice had elevated levels of the 80-kDa glycosylated DAT with respect to nonglycosylated DAT, whereas the 4WK_MPTP + Ex mice recovered to a DAT ratio similar to the vehicle group. These findings are consistent with the hypothesis that exercise could be provoking normal DA signaling through DAT regulation, but further studies are still needed to support this idea. Li et al. (2004) also observed that prevention of N-glycosylation reduced surface DAT, suggesting that non-glycosylated DAT could be more susceptible to degradation and that changes in DAT glycosylation are likely to impact DA clearance. Taken together with Li et al. (2004) reporting that non-glycosylated DAT still maintained transporter function, glycosylation of DAT could be a regulated mechanism contributing to normalizing DA extra/intra neuronal levels. Essentially an increase in glycosylated DAT as seen in the 4WK_MPTP mice implies that within the DAT protein population, increased levels of the transporter were more stable, efficient, and likely to reach the membrane surface. This suggests that the 4WK_MPTP mice may be compensating for abnormal DA levels by increasing the levels of a more stable DAT. Of note is a recent study in which a full unilateral nigrostriatal lesion with 6-OHDA resulted in a greater than 70% loss of striatal DAT, but with a surprising decrease in DA uptake of only 25% (Chotibut et al., 2012). As the loss of DAT increased, the degree of DA uptake continued to increase. Our findings of a more stable DAT following nigrostriatal DA loss would also be consistent with the observations of Chotibut et al. (2012). Even though DA levels were not determined in the current study, the data regarding VMAT2 and DAT recovery suggest the possibility that exercise results in increased DA levels (Hattori et al., 1994; Wilson and Marsden, 1995; Meeusen et al., 1995), resulting in behavioral improvement despite continued TH loss. Glutamate transporters were up-regulated in mice treated with MPTP for 4 weeks A common hypothesis within the PD field is that when the dopaminergic neurons degenerate, there is over activation of the glutamatergic STN neurons that project to the remaining SNpc neurons, resulting in further neuronal degeneration (Albin et al., 1989; Maesawa et al., 2004; however see Paul et al., 2004). Whether initial over activity of the STN is a cause or an effect of the DA neuronal loss is unknown, but the loss of dopaminergic function that contributes to the regulation of glutamatergic corticostriatal neurotransmission perpetuates a state of hyperexcitability (Day et al., 2006). Our results indicate that animals administered progressive doses of MPTP had significant increases in glutamate transporter expression of GLT-1, and EAAC1 within the SN. We suggest that this is due to an increase in extracellular glutamate levels. This hypothesis is consistent with our previous report that using another more chronic toxin animal model of PD, we find that following 5 weeks of MPTP/probenecid treatment, there was an increase in the basal extracellular level of glutamate within the SN (Meredith et al., 2009). However, the origin of that increased SN extracellular glutamate is not known.

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VGLUT2 is a vesicular glutamate transporter utilized in the subthalamo-nigral projection and VGLUT1 is specific for the cortico-nigral terminal projections. VGLUT2 expression levels (data not shown) revealed a nonsignificant 20% increase in the 4WK_MPTP mice. Lack of a significant increase in VGLUT2 levels within the SN following MPTP does not necessarily imply that the STN glutamate neurons are not hyperexcitable. It could simply indicate that the number of glutamatergic vesicles remained the same with an increase in glutamate concentration or there were more vesicles within the terminal but with less VGLUT2 protein per vesicle. Interestingly, VGLUT1 expression significantly increased in the 4WK_MPTP mice, suggesting the possibility of increased glutamate originating from the motor cortex. This observation is contrary to the notion that depleting DA signaling in the SN would affect the direct and indirect pathways with the same outcome of quenching the thalamus and thereby suppressing glutamate signaling from the motor cortex to at least the SN. However, it is possible that the origin of the motor cortex input to the SN is different compared to that of other parts of the basal ganglia, including the striatum and STN. Nonetheless, mice that were administered MPTP initially, followed by intervention with the wheel running, showed recovery of VGLUT1, GLT-1, and EAAC1 protein levels within the SN that were associated with behavioral recovery. Also, GLAST levels in the mice that exercised showed significantly decreased levels. Exercise may be compensating for dopaminergic loss by increasing the activity of the thalamo-corticostriatal circuit (Meeusen et al., 1997; Bezard et al., 2003; O’Dell et al., 2007; Massie et al., 2010; Petzinger et al., 2010).

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Mice treated with MPTP for 4 weeks and exercised had less inflammation in the SN

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Perhaps by better regulation of glutamate excitotoxicity, mice that exercised also had decreased inflammatory markers. It is not known whether inflammation is a cause or outcome of PD, but it does seem to play a role in the progression of the disease. Postmortem PD brain tissue and other animal models indicate that production of pro-inflammatory factors by microglia mediates continued neurodegeneration (Liu, 2006; Chen et al., 2008). Other studies have shown that suppressing inflammatory factors associated with either astrocytes or microglial are protective against the loss of dopaminergic neurons (Chung et al., 2010). NFAT proteins are hyperphosphorylated and located within the cytoplasm during the resting state of the microglia, where activation contributes to the loss of neurons (Luo et al., 2014). Calcineurin (CN) targets the NFAT family of transcription factors (Crabtree and Olson, 2002) and after activation, CN rapidly dephosphorylates the NFAT proteins, which are then able to translocate into the nucleus to regulate gene transcription (Macian, 2005). Luo et al. (2014) showed that alpha-synuclein contributed to NFATc3 nuclear import, or increased non-phosphorylated NFATc3, by instigating CN phosphotase activity which leads to dopaminergic cell loss in their alpha-synuclein A53T conditional transgenic mouse model of PD. Our

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results show that animals that were administered increased doses of MPTP over 4 weeks and volunteered to exercise showed a significant increase in the ratio of resting-state phosphorylated NFATc3 compared to the 4WK_MPTP and vehicle groups. This suggests that exercise decreased inflammation by regulating the ratio of phosphorylated to non-phosphorylated NFATc3. Furthermore, most neurological disorders show microglia activation (Chen and Trapp, 2015) and the invasion of blood-borne macrophages which leads to astrocytosis (Mirza et al., 1999), that is, the increase in number or phenotypic change in astrocytes. In MPTP mouse models or 6-OHDA lesion models, the astrocytosis response begins after initial dopaminergic cell loss and is elevated after most dopaminergic neurons have died as evidenced by an increase in GFAP immunoreactivity (Teismann and Schulz, 2004; Maragakis and Rothstein, 2006). As an intermediate filament protein, GFAP is localized to astrocytes and has been pathologically observed to be up regulated for a long time post injury (Maragakis and Rothstein, 2006). Our results show that within the SN, animals treated with increased doses of MPTP over 4 weeks had greatly increased GFAP levels, which suggests astrocytosis processes were activated. Interestingly, the 4WK_MPTP + Ex group also had elevated GFAP levels compared to the vehicle group, however, the levels had significantly decreased compared to the 4WK_MPTP group. This suggests that exercise suppressed the inflammatory response sufficiently, resulting in a decrease in astrocytosis.

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Mechanistic links between exercise and inflammation

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Generally, there are many known benefits of exercise on overall well-being, including brain health. While exercise has shown many benefits in the hippocampus including enhancements in learning and memory, and improving executive function (Cotman et al., 2007), it has also been shown to increase angiogenesis or the proliferation of new blood vessels and improvement of blood flow in some brain regions (Black et al., 1990). Ehninger and Kempermann (2003) have previously reported that voluntary running wheel exercise results in an increase of microglia in several brain regions and this increase may assist in perpetuating better brain health and function. Furthermore, microglia can either suppress neuroinflammation and restore homeostasis by producing antiinflammatory cytokines (Colton, 2009) or microglia can promote pro-inflammatory markers when activated which has been shown to mediate continued neurodegeneration (Liu, 2006; Chen et al., 2008). Since the distinction between pro- and anti-inflammatory microglia cells was not determined in the current study, we can only speculate that the exercise-induced decrease in both the ratio of phosphorylated NFATc3 and in GFAP expression could be due to increased levels of anti-inflammatory microglia cells. Aside from the astrocytes and microglia, one of the interesting findings of this study was the observation that animals which exercised while being administered MPTP behaviorally recovered despite lack of TH

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resurgence within the DL striatum and SN. It has been previously shown using in vivo microdialysis that exercise increases the DA concentration in the rat striatum (Hattori et al., 1994; Wilson and Marsden, 1995; Meeusen et al., 1995), which could be due in part to an increase in TH enzyme activity (Hattori et al., 1994). It is possible that within the remaining nigrostriatal cells of the mice administered MPTP, exercise increased DA concentration through increased TH enzyme activity, which would help explain the behavioral recovery. Additionally, mice administered MPTP alone showed significant increases in glutamate transporters indicating increased levels of extracellular glutamate, which can result in neuroinflammation in and or itself through excitotoxicity. Also by in vivo microdialysis, it has been shown that MPTP administration increases extracellular glutamate levels within the SN (Meredith et al., 2009), a finding consistent with the increase in SN glutamate transporters in the current study. Perhaps by normalizing DA output by exercise, the mice administered MPTP and exercised could suppress glutamate excitotoxicity by regulating the activity of the DA–D2 receptors located on the SN cell bodies (i.e., DA release resulting in DA–D2 inhibition of the DA neurons) or by activating pre-synaptic DA–D2 receptors on glutamate terminals within the SN, resulting in a decrease in glutamate release (Xu et al., 1999; Hatzipetros and Yamamoto, 2006). This would explain why the mice that exercised showed glutamate transporter and GFAP levels within the SN that were similar to the vehicle group. Taken together, our data indicate that intervention with exercise can alleviate behavioral deficits despite 4 weeks of MPTP and do so without restoring or even preventing continued loss of nigrostriatal TH levels. We are reporting that this dichotomy could be explained by exercise increasing DA release within the remaining cells (Hattori et al., 1994; Wilson and Marsden, 1995; Meeusen et al., 1997) as evident by the restoration of VMAT2 levels (i.e., increased uptake of DA into either more synaptic vesicles or increase in VMAT protein expression/vesicle) and glycosylated DAT [i.e., as intracellular DA levels increase due to exercise, the more stable form of DAT (i.e., glycosylated) decreases to vehicle levels]. Furthermore, our results suggest that exercise alleviated excess glutamate within the SN as indicated by restored levels of VGLUT1, EAAC1, and GLT-1. These data are consistent with the decrease in basal levels of glutamate following exercise (Meeusen et al., 1997). We also observed a suppression of the inflammatory response in the MPTP group that exercised. Future studies should not solely rely on different kinds of exercise as a means of treatment, but rather in addition to pharmaceutical approaches that could provoke recovery or sprouting of the remaining dopaminergic neurons/terminals (Sconce et al., 2015).

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(Accepted 28 April 2015) (Available online xxxx)

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