Introduction
There is a consensus that neurorestorative strategies need to be developed for the treatment of Parkinson’s disease (PD), which could be prescribed to slow disease progression after the diagnosis (Fox et al., 2018; Bloem et al., 2021). Current treatment options for PD, such as dopaminomimetics or deep brain stimulation, improve symptoms but do not exert direct disease-modifying effects (Verschuur et al., 2015; Mao et al., 2019). There is a growing understanding that lifestyle changes, including diet and exercise, may be useful in the prevention of PD (Bousquet et al., 2009, 2011; Archer and Kostrzewa, 2016; Reynolds et al., 2016; Marras et al., 2019; Paul et al., 2019; Xu et al., 2019; Hantikainen et al., 2022), but their disease-modifying potential remains to be explored.
Omega-3 (n-3) and omega-6 (n-6) polyunsaturated fatty acids (PUFA) form two major classes of essential fatty acids, the main n-3 PUFA present in the central nervous system being docosahexaenoic acid (DHA; 22:6 n-3). Epidemiological prospective and retrospective investigations have pinpointed an association between higher consumption of n-3 PUFA and vegetables, and low meat intake with a lower risk of PD (Jackson et al., 2019; Maraki et al., 2019; Yemula et al., 2021). Moderate to vigorous exercise in midlife has been associated with a lower risk of developing PD (LaHue et al., 2016; Jang et al., 2018; Muller and Myers, 2018; Schenkman et al., 2018; Arellanes et al., 2020; Knight et al., 2022). However, these epidemiological findings remain silent on cause-effect relationships or the potential disease-modification of n-3 PUFA intake or exercise.
Animal model studies allow substantial control over the environment over a fixed period and thus provide experimental paradigms to investigate neuroprotective (before the lesion) and/or neurorestorative (after the lesion) mechanisms. Previous preclinical studies revealed that n-3 PUFA had a neuroprotective action when administered before a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) insult in mice (Bousquet et al., 2008, 2009). While MPTP administration induced a 30% neurodegeneration of dopamine (DA) nigral cells in C57BL/6 mice fed a control high n-6 PUFA diet, no signs of cell death and higher DA concentrations in the striatum were found in mice fed a high n-3 PUFA diet (Bousquet et al., 2008). Additional studies in animal models highlighted a potential neuroprotective action of exercise, assessed notably by tyrosine-hydroxylase positive (TH+) cell count and striatal DA content (Lau et al., 2011; LaHue et al., 2016; Hsueh et al., 2018; Jang et al., 2018; Crowley et al., 2019). The effect of DHA dietary treatment on synucleinopathy has been more equivocal, but DHA intake has been shown to extend the longevity of α-synuclein transgenic mice (Thyl-αSyn), with a limited disease-modifying impact (Coulombe et al., 2018). n-3 PUFA and physical exercise combine several potential mechanisms such as upregulation of neurotrophic factors, stimulation of antioxidant defenses and synaptogenesis, and reduction of neuroinflammation (Calon et al., 2004; Zigmond et al., 2009; Gerecke et al., 2010; Lalancette-Hebert et al., 2011; Lau et al., 2011; Zigmond and Smeyne, 2014; Speck et al., 2019). Therefore, both exercise and DHA carry the potential to act in patients already diagnosed with PD, when the neurodegenerative process is advanced (Kalia et al., 2015).
Treating animals already bearing a profound dopaminergic (DAergic) denervation is an interesting preclinical strategy to probe neurorestorative mechanisms, more relevant to patients already affected with severe DA loss. We recently reported that DHA, which crosses the blood–brain barrier (BBB) (Ouellet et al., 2009; Chen et al., 2015; Chouinard-Watkins et al., 2018) leads to a recovery of the DAergic system after an extensive 6-hydroxydopamine (6-OHDA)-induced lesion (Coulombe et al., 2016) hinting toward neurorestorative effects of DHA.
In this study, we aimed to determine if the combination of physical exercise and n-3 PUFA diet could synergistically exert a neurorestorative action on the murine DAergic system. DHA-enriched diet and exercise regimen were initiated following the induction of depletion of DAergic nigral cells by intrastriatal 6-OHDA administration. Behavioral assessment, as well as post-mortem DA-related endpoints and mechanistic pathways were assessed to investigate neurorestoration in vivo.
Methods
Animals
Evidence indicates that the risk of developing PD is higher in men (Cerri et al., 2019) and preclinical studies have shown sex differences in PD models, mainly due to estrogen-induced neuroprotective effects in different PD models (Isenbrandt et al., 2023; Lamontagne-Proulx et al., 2023). Furthermore, female mice run longer distances than males in running wheels (Manzanares et al., 2018). We thus limited our study, which already contained eight groups, to males to reduce variability. A total of eighty mature male C57/BL6 mice of 9 weeks of age weighing an average of 23 g were purchased from Charles River Laboratories (Québec, Canada) and initially housed four per ventilated cage (Tecniplast: 39.1 × 19.9 × 16 cm3, surface: 501 cm2). Thirty-eight mice were lesioned with 6-OHDA and 42 were not lesioned. Four weeks after the 6-OHDA lesion (see below), lesioned and unlesioned animals were given their respective diets (see section “Diets” below; Table 1) and exercise wheels were placed in the cage for selected groups of animals for 8 weeks until sacrifice. Mice were kept under a 12-hour light/dark cycle during the entire protocol and had free access to water. All experiments were performed in accordance with the Canadian Council on Animal Care guidelines. All procedures were approved by the Animal Research Committee of the Centre de Recherche du CHU de Québec-Université Laval (CPAUL number: 14-141-2, approved on November 3, 2014).
Unilateral 6-OHDA lesion
Administering 6-OHDA into the right striatum results in a lesion of the nigrostriatal pathway. Dopaminergic neurons internalize 6-OHDA, which damages striatal terminals before triggering degeneration in the substantia nigra pars compacta (SNpc) through oxidative stress and mitochondrial dysfunction (reviewed in (Duty and Jenner, 2011; Hamadjida et al., 2019; Salari and Bagheri, 2019; Gonzalez-Rodriguez et al., 2020). Animals were anesthetized with isoflurane (Sigma-Aldrich, St. Louis, MO, USA) 3%–4% with oxygen debit at 1 L/min. Mice were then placed in the stereotaxic frame (David Kopf Instruments, Los Angeles, CA, USA) and the isoflurane concentration was set at 2% and oxygen debit was set at 0.5 L/min during the surgical procedure. Four µg of 6-OHDA (Sigma-Aldrich) was dissolved at a concentration of 2 µg/µL in 0.9% saline and 0.02% ascorbic acid. A volume of 2 µL was injected with a Hamilton syringe in the right striatum at a rate of 0.5 µL/min and the injection was performed for 4 minutes at the following coordinates: AP (Bregma): +0.5 mm; ML: –1.8 mm; DV: –3.1 mm (Franklin and Paxinos, 2013). The needle was left in place for 3 minutes after the injection before complete retraction. Sham mice were subjected to the same surgical procedures but were injected with 2 µL of the vehicle solution (Coulombe et al., 2016).
Diets
Mature 3-month-old animals were fed a control diet (CD) or a DHA-enriched diet for 8 weeks, between weeks 4 and 12 from 12 to 21 weeks old (Figure 1A). Thus, the dietary treatment started ~4 weeks following the 6-OHDA lesion and lasted until sacrifice. DHA was obtained from DSM Nutritional Products in a microencapsulated formulation to avoid oxidation. Based on diet consumption measured every 2 weeks, the estimated dose of DHA averaged 0.5 to 1.0 g/kg daily per animal. Although DHA administration through the diet does not allow us to report the exact daily dose, we selected this approach because it more closely replicates the real-life intake of fatty acids throughout the day. Purified diet formulations were designed to be isocaloric and to contain similar concentrations of macronutrients, vitamins, and minerals. They were produced by Research Diets Inc. (NJ, USA) and were standardized to ensure consistency and eliminate batch-to-batch variation. Diet contents are provided in Table 1.
Exercise wheels
Before the 6-OHDA lesion until 3 weeks later, all the animals were housed collectively (4 per cage). Four weeks after the 6-OHDA lesion, exercised animals were housed singly for 8 weeks, between week 4 and week 12 following the 6-OHDA lesion (Figure 1), in a cage containing a running wheel (5” ID) (Lafayette Instruments, Lafayette, IN, USA). Meanwhile, due to logistical reasons, control animals were housed collectively (4 per cage) in cages of the same size but without access to a wheel. The average distance run per day, corresponding to moderate spontaneous activity, was quantified three times during the protocol with an automated counting device (Scurry Mouse Activity system, Lafayette) during the active cycle of the mouse (from 19:00 to 07:00).
Behavioral tests
Behavioral tests were performed 3 weeks after the lesion for baseline measurements before diets and exercise, then at 11 weeks post-lesion. All tests were run in the morning and on consecutive days, as previously described (Coulombe et al., 2016). Animals were first exposed to the Open field test, which allowed them to quantify locomotion in an open area for 1 hour (Coulombe et al., 2016). Speed and distance data were recorded and analyzed. The stepping test consisted in counting the number of adjusting steps made with the contralateral or ipsilateral paw (relative to the lesioned hemisphere) when the mouse was held by the base of the tail with its hindlimbs suspended above a table and moved backwards at a steady rate so that it traverses a 1-meter distance over 3–4 seconds. Spontaneous limb asymmetry is expected, translating into motor impairment of the paw contralateral to the injected hemisphere. The apomorphine-induced rotation test is a reliable physiological measure of DAergic depletion and asymmetry in DA receptor stimulation (Iancu et al., 2005). By administering apomorphine, DAergic receptors are directly stimulated, leading to rotational behavior of the animal contralateral to the DA-depleted hemisphere. Mice were injected apomorphine (Sigma-Aldrich) intraperitoneally at a dose of 0.5 mg/kg and were then placed in the apparatus for 1 hour, and the number and orientation of rotations (contraversive or ipsiversive) were recorded. The number of unclockwise rotations correlates with the extent of the lesion (Zigmond et al., 1992; Iancu et al., 2005). The apomorphine-induced rotation test was the last test to be performed in this battery of behavioral measures to avoid any bias caused by apomorphine.
Tissue processing
Animals were sacrificed 12 weeks following the 6-OHDA lesion via an intracardiac perfusion of phosphate buffer saline containing a co*cktail of protease inhibitors (SIGMAFASTTM Tablets, Sigma-Aldrich) and phosphatase inhibitors (1 mM sodium pyrophosphate and 50 mM sodium fluoride). First, animals were deeply anesthetized with a ketamine (100 mg/kg; Narketan, Vetoquinol N.-A Inc., Lavaltrie, QC, CA) and xylazine (10 mg/kg, respectively; Rompun, Elanco Canada Limited, Guelph, ON, Canada) mixture administered intraperitoneally. Brains were collected and the anterior part, corresponding to structures anterior to Bregma −2 mm (Franklin and Paxinos, 2013), was immediately frozen on dry ice and stored at –80°C for cryostat sectioning to generate striatum extracts for western blots and HPLC analyses, as well as coronal sections for immunohistochemistry (IHC, 20 μm) and autoradiography (12 μm). The part of the brain posterior to Bregma –2 mm (Franklin and Paxinos, 2013) was postfixed with 4% paraformaldehyde (PFA) pH 7.4 for 48 hours and transferred to 20% sucrose in 0.1 M PBS for cryoprotection and cut onto a freezing microtome at 25 μm (including the SNpc).
Protein fractionation from striatum and western blot analyses
The procedure for protein extraction was described previously (Coulombe et al., 2016), Briefly, samples were hom*ogenized using eight volumes of lysis buffer (150 nM NaCl, 10 nM NaH2PO4, 1% v/v Triton X-100, 0.5% SDS and 0.5 sodium deoxycholate) with a mix of protease (Roche, ON, Canada) and phosphatase inhibitors (1 mM tetrasodium pyrophosphate and 50-mM sodium fluoride). They were then sonicated (3 × 10-second pulse) and centrifuged for 20 minutes at 100,000 × g, 4°C. The supernatant was collected and frozen at –80°C. The quantification of protein concentration was performed using the bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Thirty-five μg of proteins were heated with Laemmli loading buffer for 5 minutes and separated by electrophoresis on a 12% SDS-polyacrylamide gel. Proteins were then transferred onto 0.45 μm Immobilon polyvinylidene difluoride membranes (Millipore, ON, Canada) and blocked with 5% skimmed milk and 1% BSA in PBS-0.05% Tween for 1 hour. The membranes were immunoblotted overnight at 4°C with the following primary antibodies: rabbit anti-glial cell derived neurotrophic factor (GDNF; 1:1000, Biovision, Milpitas, CA, USA), rabbit anti-neural nuclear protein (NeuN; 1:5000, ab177487, Abcam, Cambridge, UK), mouse anti-Neuroligin1 (1:2000, sc-365110, Santa Cruz Biotechnology, Dallas, TX, USA), anti-Neurexin (1:1000, sc-136001, Santa Cruz Biotechnology), rabbit anti-brain neutrophic factor pro- and mature (BDNF; 1:500, sc-20981, Santa Cruz Biotechnology), anti-vascular glutamate transporter 1 (VGLUT1; 1:4000, clone N28/9, Neuromab, Davis, CA, USA). Membranes were washed in PBS-Tween then incubated with the appropriate HRP-coupled anti-mouse (1:60,000) or anti-rabbit (1:50,000) secondary antibodies (Jackson Immunoresearch, West Grove, PA, USA) followed by detection with chemiluniscence reagent (Luminata Forte Western horseradish peroxidase substrate, Millipore) and measured with the Amersham Imager 680 (Cytiva, Mississauga, ON, Canada). The density of immunoblot bands was quantified with the ImageLab Software (Bio-Rad Laboratories, ON, Canada).
Lipid extraction and chromatography
Approximately 40 mg of frozen tissue was dissected for fatty acid analyses from the frontal cortex (six mice per group). The frontal cortex was selected because it is known to reflect fatty acid profiles in other brain regions (Brenna and Diau, 2007) and because all striatum was used in other experiments. Weighed brain tissues were hom*ogenized successively with 0.9% NaCl, butylhydroxytoluene-methanol (Sigma-Aldrich) and chloroform (J.T. Baker, NJ, USA) using C17:0 methyl ester as an internal standard (Nu-Chek Prep, MN, USA) at a concentration of 500 μg/g of C16:0. Tubes were centrifuged horizontally at 2400 × g for 7 minutes (Precision Durafuge 300R) at room temperature. Then, the lower layer was collected. This procedure was repeated twice, and the two extracts were pooled and dried under a stream of N2. Lipid extracts were transmethylated with methanol:benzene (4:1) and acetyl chloride at 98°C for 90 minutes. After cooling down, 6% K2CO3 was added. A 15-minute centrifugation at 514 × g allowed phase separation and the upper layer was collected in a gas chromatography autosampler vial and capped under N2. Fatty acid methyl esters were quantified using a model 6890 series gas chromatograph (Agilent Technologies, CA, USA) using a FAST-GC method, as previously described (Coulombe et al., 2016). Five microliters of each sample were injected at a 25:1 split ratio. Tissue fatty acid methyl ester peak identification was performed by comparison to the peak retention times of a 28-component methyl ester reference standard (GLC-462; Nu-Check Prep, Elysian, MN, USA) (Masood et al., 2005).
DA, 3,4-dihydroxyphenylacetic acid (DOPAC), serotonin (5-HT), and 5-hydroxyindolacetic acid (5-HIAA) were quantified by high-performance liquid chromatography (HPLC) using electrochemical detection, as described previously (Coulombe et al., 2016). Areas of the anterior striatum were dissected and hom*ogenized with 200 μL of perchloric acid (0.1 N). A volume of 10–30 μL of supernatant was directly injected into the chromatograph composed of a Waters 717 plus autosampler automatic injector, a Waters 1525 binary pump equipped with an Atlantis dC18 column, a Waters 2465 electrochemical detector, and a glassy carbon electrode (Waters Limited, Brossard, QC, CA). The electrochemical potential was 10 nA. The mobile phase was composed of 8% MeOH, 0.47 M EDTA, 2 M NaCl, 0.69 M octanic sulfonic acid, and 0.055 M NaH2PO4, and pH was adjusted to 2.9. The mobile phase was delivered at a flow rate of 1.2 mL/min. After identification of the peaks, HPLC quantification was normalized with respect to the protein concentration of each sample, which was determined with a BiCinchoninic Acid assay (Thermo Fisher Scientific).
Immunohistochemistry
Dissected brains posterior to Bregma −2 mm were post-fixed in 4% PFA pH 7.4 for 48 hours, followed by a 20% sucrose solution or post-fixed for 1 hour in 4% PFA. Brains were sliced in coronal sections of 25-μm thickness using a microtome (see section “Tissue processing”). Brain sections were washed in 0.1 M PBS and subsequently placed in 3% hydrogen peroxide for 30 minutes at room temperature. Sections were washed in PBS and placed in a blocking solution (PBS, 0.1% Triton X-100 (Sigma-Aldrich) containing 5% normal goat serum (NGS; Wisent, QC, Canada) for 30 minutes. The sections were then incubated overnight at 4°C with the primary antibody rabbit anti-TH (PEL-Freez, 1:5000) in the blocking solution. Slices were washed three times in PBS, incubated with a secondary biotinylated goat anti-rabbit IgG (Jackson Immunoresearch) followed by an incubation with an avidin-biotin peroxidase complex (ABC; Vector Laboratories, Burlingame, CA, USA) for 1 hour at room temperature. The antibody binding was detected by placing the sections in a solution of aminoethyl carbazole (AEC) at 2 mg/mL. The reaction was stopped by extensive washes in PBS. Following the AEC staining, sections were counterstained with hematoxylin solution, Gill No. 1 (Sigma-Aldrich), and coverslipped with Mowiol mounting medium.
Quantification of TH-immunoreactive neurons in the SNpc
The number of nigral TH+ neurons in the SNpc was determined by stereological counts under brightfield illumination (Bousquet et al., 2008). Seven sections at 125 μm intervals through the SNpc (AP: levels of –2.70 mm to –3.80 mm (Franklin and Paxinos, 2013) were analyzed using the Stereo Investigator software (MBF Bioscience, Inc., Williston, VT, USA) integrated into an E800 Nikon microscope (Nikon Canada Inc., ON, Canada). Counting was restricted to SNpc and did not include the ventral tegmental area.
DAT autoradiography and quantification of striatal TH immunostaining
Striatal dopamine transporter (DAT)-specific binding was evaluated using 125I-RTI-55 [(–)-2 β-Carbomethoxy-3 β-(4-iodophenyl)tropane] (Perkin Elmer, ON, Canada; 2200 Ci/mmol) according to previously published procedures (Calon et al., 2001; Coulombe et al., 2016). Macroscopic quantification of all autoradiograms (DAT) and slides (TH) was performed on a KODAK IS 4000MM Digital Imaging System (Carestream Molecular Imaging Software version 5.3.4.17821). Optical density was measured in both striatums and expressed as a percentage of the left striatum.
Statistical analysis
No statistical methods were used to predetermine sample sizes; however, our sample sizes are similar to those reported in previous publications (Bousquet et al., 2011; Coulombe et al., 2016, 2018). All analyses were performed blinded to treatment and lesion. Results for each group are presented as the mean ± SEM. Equality of variances was established by Bartlett’s test while normality was evaluated using Shapiro-Wilk tests. For comparisons between more than two groups one- (one independent variable), two- (two independent variables), or three-way (three independent variables) analysis of variance were performed. In case of significant interaction between variables disproving the independence of variables, one-way analysis of variance followed by Tukey’s post hoc test were used. When variances were unequal, Kruskal-Wallis tests were used (more than two groups compared, followed by a Dunn’s test. One-sample t-test was performed to compare means to a theoretical value. Linear regression was used to calculate the coefficient of correlation R2 and the P value was obtained using a generalized linear model. Outliers were identified using the ROUT method, based on the analyses of non-linear regression and evaluation of the residuals of the robust fit. All statistical analyses were performed using the JMP (version 13; SAS Institute Inc., Cary, IL, USA) and GraphPad Prism 8 (GraphPad Software Inc., La Jolla, CA, USA) softwares. A P value of 0.05 was considered statistically significant.
Results
DHA intake translates into high cortical n-3 PUFA content
The 6-OHDA injection induced a transient weight loss, although from the beginning of the behavioral tests until the sacrifice at 12 weeks, average weights were similar between groups (Figure 1B). The mean number of rotations per day on the wheel ranged between 12,000 and 18,000 (corresponding to 5–7 km per day) and were not affected by the 6-OHDA lesion (Figure 1C).
Following the 6-OHDA injection, intake of a high-DHA diet over 8 weeks led to a significant increase in DHA, total n-3 PUFA, n-3 eicosapentaenoic acid, and n-3 DPA levels in the frontal cortex (Figure 2A, C, E, and Additional Figure 1A). In contrast, levels of ARA and total n-6 fatty acids were lower in DHA-fed groups compared with those fed the control diet (P < 0.0001) (Figure 2B and D). Dietary intake of DHA also led to a significant reduction of n-6 DTA (Additional Figure 1B). Accordingly, the n-3:n-6 fatty acid ratio was significantly higher in DHA-fed animals than in those fed the control diet (Figure 2F). Lesion and exercise had no effect on cortical fatty acid profiles, with the exception that cortical n-6 DTA was higher in exercised mice under the control diet compared to non-exercised mice under the same diet (Additional Figure 1B).
Combination of DHA and exercise improves motor impairments in 6-OHDA lesioned mice
In the open field test, 6-OHDA lesioned animals traveled less distance at 3 weeks (Figure 3A), but this small difference receded at 11 weeks. Post-lesion DHA, exercise, or their combination had no significant impact on locomotor activity (Figure 3B). In the stepping test, all 6-OHDA-treated mice displayed impairment in the use of the contralateral versus ipsilateral paw at 3 weeks after lesion. At 11 weeks post-lesion, after 8 weeks of treatment, 6-OHDA-lesioned animals fed with DHA and also under exercise recovered from asymmetric forelimb akinesia to the level of unlesioned mice (P < 0.001; Figure 3C and D). The difference was statistically significant between the DHA + exercise group and all other lesioned animals, consistent with a synergistic effect. In the apomorphine-induced rotation test, all mice treated with 6-OHDA displayed significantly higher contraversive rotations at 3 weeks (P < 0.0001; Figure 4A and B). Neither DHA intake nor exercise alone had any major impact on rotational behavior as assessed at 11 weeks post-lesion, although animals under the combined treatment displayed a non-significant tendency toward reduced unclockwise rotations (P = 0.1589).
Post-lesion interventions have no effect on the number of TH+neurons in the SNpc, but DHA increases TH immunoreactivity in the striatum
Stereological counts confirmed that 6-OHDA lesions induced a significant decrease (P < 0.0001) in the number of TH+ cells in the SNpc (–49% on average; Figure 5A). However, no significant difference was observed for animals post-treated with DHA, exercise, or a combination of both (Figure 5A). Accordingly, TH+ staining in the striatum was decreased (P < 0.0001) in all lesioned animals compared to unlesioned animals (Figure 5B). Moreover, TH immunoreactivity in the lesioned animals was significantly higher in the striatum of mice that received DHA treatment versus those fed the control diet (P < 0.0001; Figure 5B), possibly reflecting an increased number of TH+ terminals following the DHA treatment.
The combination of DHA and exercise increases DAT levels in the striatum of lesioned animals
To further document changes in DAergic terminals in the striatum of mice at the end of treatments, DAT concentrations were quantified using receptor autoradiography (Figure 6). As expected, specific binding of [125I]-RTI-55 to DAT was significantly reduced in the striatum of lesioned compared to unlesioned animals (P < 0.0001; Figure 6). However, animals exposed to the combination of DHA and exercise after 6-OHDA lesions displayed significantly higher levels of DAT in the striatum compared to other lesioned animals (P < 0.001; Figure 6). Since such an effect was not detected following exercise or DHA alone, the data is consistent with a synergistic effect of combining exercise and DHA on DAergic innervation in the striatum.
DHA intake increases DA levels, while the combination of DHA and exercise lowers DOPAC/DA and 5HIAA/5HT ratios in lesioned animals
As expected, HPLC analysis of striatal catecholamines revealed a significant decrease of striatal DA 12 weeks following the 6-OHDA lesion. However, compared with the control diet, the DHA-enriched diet induced an increase in DA levels in the lesioned striatum to about half the quantity of the unlesioned striatum (Figure 7A), corroborating previous observations (Coulombe et al., 2016). Furthermore, 6-OHDA lesions increased the striatal DOPAC/DA ratio only in animals fed the control diet, whereas DOPAC/DA ratios of DHA-treated mice were similar to those of unlesioned animals (Figure 7D). Conversely, the combined DHA and exercise regimen resulted in a decreased DOPAC/DA ratio (Figure 7D). There were no significant differences in 5HT levels between lesioned and unlesioned mice, regardless of treatment and exercise (Figure 7B). In lesioned animals fed DHA and under the exercise regimen, average 5HIAA/5HT levels were significantly lower than DHA-fed lesioned animals that were not exercised (P < 0.01; Figure 7E). Correlation analyses confirmed the robust link between the DAergic status, striatal DA in particular (R2 = 0.2777, P < 0.0001), and performance in the stepping test (Figure 7C). Correlations were also performed only in the 6-OHDA groups to isolate an effect of post-lesion treatments, and the association with DAT levels remained significant (R2 = 0.1519, P < 0.05) and the inverse relationship with the DOPAC/DA ratio was strengthened (R2 = 0.3739, P < 0.001), suggesting reduced DA turnover is involved in the improvement of asymmetrical akinesia (Figure 7F).
DHA intake and exercise do not impact levels of striatal neurotrophic factors or vesicular glutamate transporter 1
To investigate mechanistic correlates behind motor improvement and dopaminergic recovery, postmortem protein levels for the neurotrophic factors BDNF/proBDNF and GDNF were measured by western blot, but no significant changes were observed in any treatment groups (Figure 8A–D). Similarly, there were no significant differences in the levels of synaptic proteins VGLUT1, neurexin, and neuroligin1 (Figure 8E–G). Neuronal nuclear protein (NeuN) levels did not change (Figure 8H).
Discussion
The future of PD therapies relies on the development of treatments that alter the progression of the disease, beyond transiently palliative effects. Large multidomain clinical trials involving dietary changes and exercise are currently ongoing in Alzheimer’s disease (Barreto et al., 2018; Rolland et al., 2019; Rosenberg et al., 2020). In addition, it has been shown that high-intensity exercise is achievable in patients with PD (Ahlskog, 2018; Schenkman et al., 2018; Landers et al., 2019). However, given the methodological challenges to demonstrate disease modification in a clinical setting (Espay et al., 2020; McFarthing et al., 2020), we opted first for a study in animal models where the environment can be better controlled over a definite period of time. The goal of our study was to determine if the combination of DHA intake and voluntary exercise could lead to signs of neurorestoration in the context of severe nigrostriatal denervation as observed in PD. Interestingly, the combination of DHA intake and voluntary exercise increased DAT levels in 6-OHDA lesioned striatum, while decreasing DA and 5-HT turnover. A synergistic effect of the DHA + exercise combination was also measured in fine forelimb dexterity detected in the stepping test. These results suggest a notable effect of DHA on DAergic recovery at the level of terminals in the striatum, with key synergistic effects on molecular and behavioral endpoints when combined with exercise.
Effects of dietary DHA treatment and exercise on brain fatty acid levels
The results obtained confirmed the ability of dietary DHA to increase cerebral levels of n-3 PUFA, including DHA itself (Coulombe et al., 2018). This dietary-induced brain accretion of DHA has been repeatedly shown by our group (Calon et al., 2004; Bousquet et al., 2008; Arsenault et al., 2011; Lalancette-Hebert et al., 2011; Coulombe et al., 2016) and others (Joffre et al., 2016; Lacombe et al., 2017). Also consistent with previous studies is the finding that levels of arachidonic acid and total n-6 PUFA remained significantly lower in animals fed a DHA-enriched diet (Bousquet et al., 2008, 2011; Coulombe et al., 2016; Joffre et al., 2016). Moreover, the 6-OHDA lesion or exercise regimen had no significant effect on cortical fatty acid profiles. Our group previously documented the absence of effects of a DAergic lesion on the levels of most fatty acids, at least in the cerebral cortex (Julien et al., 2006; Bousquet et al., 2008, 2011; Coulombe et al., 2016). However, to our knowledge, the absence of major significant impact of exercise on brain fatty acid profiles has not been reported previously.
Effects of dietary DHA treatment and exercise on motor behavior
Despite the unilateral lesion induced by 6-OHDA, mice in the open field test only showed a small decrease in the total distance traveled at 3 weeks and this effect was lost at 11 weeks, regardless of treatments. This is classically explained by compensatory mechanisms by the animal relying on the unlesioned side and an intact cerebellum (Bezard et al., 2003; Fox et al., 2016). Replicating the symptoms of PD in an animal model is a complex endeavor (Duty and Jenner, 2011). Since its establishment in the seventies (Ungerstedt and Arbuthnott, 1970), and as confirmed afterwards (Hefti et al., 1980; Zigmond et al., 1992), the 6-OHDA model has often been investigated to provide a behavioral index of nigral denervation (Coulombe et al., 2016; Su et al., 2018). The behavior most frequently generated and measured in this model is the apomorphine-induced rotations (Salari and Bagheri, 2019; Rezaee et al., 2020). Here, 6-OHDA intrastriatal administration induced a large increase of contraversive rotations, but with no difference between groups. This suggests that the effect of DHA or exercise on the DAergic system did not translate into a consistent change in the response to apomorphine.
The third paradigm used here, the stepping test (Olsson et al., 1995) is designed to assess more subtle asymmetric motor deficits and akinesia in rodents (Glajch et al., 2012), which are reversed by levodopa in MPTP (Blume et al., 2009) and 6-OHDA murine models (Olsson et al., 1995; Kaindlstorfer et al., 2019). Here, it revealed an evolution of the response over time in DA-depleted animals. Before treatment, 3 weeks after lesion, we observed a slight but significant asymmetry in all animals treated with 6-OHDA. Only the combination of voluntary exercise with DHA led to a total recovery of behavioral imbalance in lesioned mice still present at 11 weeks post-lesion. Such data provide evidence of motor recovery following exercise that is potentiated by DHA in the long run, highlighting a synergy between DHA and voluntary exercise to improve fine dexterity of forelimbs in the mouse.
Effects of dietary DHA treatment and exercise on the dopaminergic system
As expected, the striatal administration of 6-OHDA induced a loss of TH+ immunosignal in both the SN and the striatum. The number of TH+ neurons in the SNpc remained similar for all treatment groups, as expected from previous work, which also reported an increase in cell body size after DHA consumption (Coulombe et al., 2016). However, a notable increase of TH+ DAergic terminals in the striatum was observed in animals fed the DHA diet after the 6-OHDA lesion. The changes in DA, DAT, and TH+ terminals in the striatum were not associated with an increase in the number of TH+ neurons in the SNpc, as revealed by TH immunohistochemistry. This should not come as a surprise as it is reasonable not to expect the generation of new neurons after a 6-OHDA lesion. We previously observed higher DAergic neuronal counts in DHA-treated mice, but only in a neuroprotection paradigm where DHA was implemented to prevent MPTP-induced neuronal loss (Bousquet et al., 2008). Using the 6-OHDA model allowed us to begin DHA and exercise regimens after the induction of the lesion and to use the contralateral hemi-brain as a control for each animal. In addition, it should be noted that no study has demonstrated any effect of exercise on the number of DAergic neurons in the SNpc (for review Crowley et al., 2019).
While DHA intake led to a small rise in TH+ immunosignal in the striatum, a synergy between exercise and DHA was necessary to induce a significant, although partial, recovery of DAT transporter levels in the striatum. Extracellular levels of DA are mainly regulated by uptake through the DAT (Bhat et al., 2021). The observation that DHA alone did not have a significant effect on DAT levels is in agreement with our previous reports in 6-OHDA and MPTP mouse models (Bousquet et al., 2011; Coulombe et al., 2016). A previous positron emission tomography study in humans also reported higher DAT levels in the putamen and caudate nucleus in exercised patients (Shih et al., 2019), in agreement with observations from animal models (Sconce et al., 2015; Churchill et al., 2017; da Costa et al., 2017).
The present results confirmed the enhancing effects of DHA intake on striatal DA content in 6-OHDA lesioned mice, as we have previously reported (Coulombe et al., 2016), which may be due to a higher release of DA in the synaptic cleft and/or an increase of DA synthesis and subsequent storage in presynaptic vesicles. This could be explained by compensatory mechanisms that may intervene after the lesion, which are accentuated by DHA. Indeed, previous reports demonstrated the importance of such mechanisms in PD pathophysiology (Fox et al., 2016, 2018). Results from [11C]-raclopride positron emission tomography imaging studies suggest that exercise enhances dopamine release in the brain of PD patients (Sacheli et al., 2018, 2019). However, we did not observe any obvious effect of exercise on post-mortem DA levels measured directly in the striatal tissue. A previous report in MPTP mice rather suggested that the improvement in motor function observed in exercised mice may be due to an increase in levels of D2-receptor-containing medium spiny neurons (Toy et al., 2014). Therefore, these novel data indicate that DA levels were upregulated by DHA intake, not by exercise. In 6-OHDA–lesioned animals, the parallel increase in DA and DAT can be interpreted as a partial recovery of dopaminergic terminals.
In parallel to its effect on DA, DHA treatment also corrected the increase of striatal DOPAC/DA ratio after the lesion, most particularly when voluntary exercise was combined with DHA, consistent with a synergistic mechanism acting on DA metabolism. Correlation analyses indicate this reduced metabolism of DA may be a key factor behind behavioral improvement. A reduction in the 5HIAA/5HT ratio was also significant in 6-OHDA-lesioned animals exposed to both DHA and exercise. This pinpoints toward the enzymatic machinery shared by both 5HT and DA, monoamine oxidase (MAO), which catalyzes the production of DOPAC and 5-HIAA from DA and 5HT, respectively. Hence, we can reasonably hypothesize that the combination of exercise and DHA led to reduced DAergic and 5HTergic turnovers by downregulating MAO activity. Such an explanation is consistent with previous reports of a reduction of MAO-B activity in the frontal cortex of rats fed a fish-oil-enriched diet (Chalon et al., 1998). Other effects of n-3 fatty acids on the serotoninergic system have been reported such as a downregulation and upregulation of 5-HT1A receptors in the dorsal raphe and hippocampus, respectively, following fish oil intake (Farkas et al., 2002; Farioli Vecchioli et al., 2018). It is thus conceivable that DHA treatment and exercise may affect 5-HT signalization, in part by regulating MAO activity. As MAO inhibition has therapeutic effects against depression, this effect may underlie the possible benefits of n-3 PUFA and exercise on mood disorders (Reynolds et al., 2016; Deacon et al., 2017).
Absence of detectable effects of treatments on the levels of proteins linked to neurorestoration
Exercise and/or DHA supplementation have been mechanically linked to synaptic markers neurexin (Innocenzi et al., 2021), neuroligin, and VGlut1 (Sconce et al., 2015), as well as neurotrophic factors (Ceccarini et al., 2022) BDNF and GDNF (Bousquet et al., 2009; Zigmond and Smeyne, 2014; Kim et al., 2020). Using immunoblotting, we did not detect significant modulations in striatal levels of these proteins and of NeuN, a global neuronal marker. A possibility is that tissue samples were collected spatially too far from the lesion or temporally too late after these regulators may have been altered quantitatively.
Finally, in the absence of clear neurogenesis, which would have indicated neural regeneration, the treatments could have exerted their neurorestorative effects in the form of enhanced sprouting of DAergic fibres and neuroplasticity, as well as through compensatory mechanisms that sustain neurological functions, like increased receptor sensitivity or mitochondrial biogenesis (Park et al., 2021), endpoints that could be investigated in future research.
Limitations
This study has some limitations that should be noted. First, the aim of this study was to observe neurorestoration in a parkinsonian mouse model following injection of 6-OHDA. This strategy allowed us to determine the impact of an 8-week treatment on motor recovery and postmortem endpoints. Thus, it is possible that the molecular events driving DAergic recovery may have peaked during that 8-week span and then rescinded, leaving no significant increase/decrease to be detected post-mortem. More animals would have been necessary to perform measurements at different time points to identify mechanisms involved in neurorecovery. Secondly, spontaneous DAergic recovery in the striatum has been reported 4–6 months after a partial lesion (Duty and Jenner, 2011), making it difficult to distinguish between a novel effect of treatments or simply an acceleration of the normal healing process and compensatory mechanisms. Third, due to the housing capacity of our animal facility, control mice were group housed, whereas exercised mice were housed singly, which could have added a confounding factor, particularly in behavioral analyses. However, no significant difference was noted throughout the study between mice on the control diet that exercised and those that did not, lesioned or not. Fourth, the study was limited to males to reduce variability in a complex protocol already featuring 8 different groups.
Conclusion
This study supports the potential role of n-3 PUFA, and more specifically DHA, as a therapeutic approach to PD. Interestingly, synergistic effects of voluntary exercise when combined with DHA have also been evidenced, particularly on fine motor skills, DA transporter, and monoamine turnover. As treatment began after the 6-OHDA lesion, the results suggest that such interventions could be implemented after diagnosis. Administration of DHA and exercise protocol could be set up in a clinical study on patients diagnosed or at the prodromal stages of PD. Current treatments for PD are focused on symptomatic treatments (Verschuur et al., 2015; Fox et al., 2018), and consequently, so are most of the clinical studies. However, while the present study used voluntary exercise, it should be kept in mind that several types of exercise protocols could be used (LaHue et al., 2016; Muller and Myers, 2018). Large multidomain lifestyle intervention programs currently active to prevent AD and related dementias (Rosenberg et al., 2020) could serve as a blueprint to follow in the field of PD.
Acknowledgments:We would like to thank Michael J. Zigmond (University of Pittsburgh) for providing insightful discussions regarding the experimental design and editorial comments on the manuscript.
Author contributions:OK performed surgeries, animal experiments, apomorphine injections, immunohistochemistry, HPLC, autoradiography, completed the statistical analysis, interpretation of the data, and wrote the manuscript. CR contributed to tissue processing and HPLC. KC performed 6-OHDA injection surgeries jointly with OK. MSP provided expertise for 6-OHDA surgeries and behavioral experiments. VÉ contributed to HPLC and autoradiography experiments. LB and PJ performed gas chromatography analyses of fatty acids. CT provided her expertise on wheels and tissue preparation. MFOMMM contributed to the writing of the manuscript. FCa and FCi designed the study and secured the funding. FCa wrote the manuscript. All authors reviewed and approved the final manuscript.
Conflicts of interest:All the authors declare that there is no conflict of interest.
Data availability statement:The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
Additional file:
Additional figure 1:Cortical levels of fatty acid.
C-Editor: Zhao M; S-Editor: Li CH; L-Editors: Li CH, Song LP; T-Editor: Jia Y
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Keywords:
6-hydroxydopamine; dopamine; dopamine transporter; exercise; neurorestoration; Parkinson’s disease; polyunsaturated fatty acids omega-3
