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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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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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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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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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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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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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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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Western blot analysis
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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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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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VMAT2 (57kDa)
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β-actin (43 kDa)
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DAT
