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,
,1,2,1,2
* Centre de Recherche en Endocrinologie Moléculaire et Oncologique, and
Centre de Recherche en Neurosciences, Centre Hospitalier de l’Université Laval (CHUL), Centre Hospitalier Universitaire de Québec (CHUQ), Québec, Canada;
Faculté de pharmacie, and
Département de Médecine, Université Laval, Québec, Canada; and
|| Section of Nutritional Neuroscience, Laboratory of Membrane Biochemistry and Biophysics, Division of Intramural Clinical and Biological Research, National Institute on Alcohol Abuse and Alcoholism, Rockville, Maryland, USA
2Correspondence: F. Calon, Centre de Recherche du CHUL (CHUQ), Endocrinologie moléculaire et Oncologique, T2–05, 2705, Blvd. Laurier, Québec, QC, G1V 4G2, Canada. E-mail: frederic.calon{at}crchul.ulaval.ca; F. Cicchetti, Centre de Recherche du CHUL (CHUQ), Neurosciences, RC-9800, 2705, Blvd. Laurier, Québec, QC, G1V 4G2, Canada. E-mail: francesca.cicchetti{at}crchul.ulaval.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: MPTP • neuroprotection • DHA • dopamine
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Epidemiological studies demonstrate that 95% of patients diagnosed with PD suffer from a sporadic form of the disease (2)
, whereas the remaining 5% of PD cases are seen in familial clusters characterized by an early onset (3)
. Several theories have been developed to explain the etiology of PD and underscore a role of environmental toxins (4)
, mitochondrial dysfunctions (5)
, neuroinflammation (6)
, and oxidative stress (7)
. PD is recognized as the second most common neurodegenerative disorder after Alzheimer’s disease (AD) and affects 1% of the population worldwide after 65 yr of age (8)
.
Polyunsaturated fatty acids (PUFAs) are known to be an important component of cell membranes in the brain (9
10
11
12)
. PUFAs are crucial to maintain cellular activity by modulating membrane order, gene transcription, cell signaling, and caspase activation (13
14
15
16
17)
. Omega-3 (n-3) and omega-6 polyunsaturated fatty acids (n-6 PUFAs) are essential fatty acids, as mammals cannot synthesize them de novo. Thus, n-3 and n-6 PUFAs must be obtained through the diet. For example, fish represents a significant source of long chain n-3 PUFAs such as eicosapentaenoic (EPA) and docosahexaenoic acid (DHA). Epidemiological studies have associated low n-3 PUFA consumption with a higher risk of developing AD (18
19
20)
. Moreover, a neuroprotective action of n-3 PUFAs has been observed in animal models of AD (15
, 16
, 21
22
23)
. However, no such studies are available for PD. Recent epidemiological studies report that high dietary intake of saturated fat and low intake of unsaturated fatty acids are associated with a higher risk of developing PD, but none have made a direct link between n-3 PUFA intake with the risk of developing PD (24
, 25)
. A recent postmortem gas chromatographic analysis of brain fatty acid profiles has revealed no significant differences in n-3 PUFA concentrations in brain cortex between PD patients and age-matched controls (9)
.
To test our hypothesis, we exposed mice to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a meperidine analog used accidentally by heroin addicts. MPTP has potent neurotoxic properties with selective effects on nigral DAergic neurons, producing a Parkinsonian syndrome similar to PD (26
, 27)
. MPTP toxicity is due to the inhibition of the complex I of the mitochondrial electron-transport chain, leading to energy failure and cell death. When administered to mice, MPTP crosses the blood-brain barrier and is converted by the monoamine oxydase B (MAO-B), mainly by glial cells, into its active form MPP+. Over the last two decades, MPTP has been widely used in rodents and nonhuman primates to generate animal models of PD (28
29
30
31)
.
Since PD and AD may share common degenerative processes, we hypothesized that n-3 PUFA could have a neuroprotective action in an MPTP-induced animal model of PD as reported for AD.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Purified diet formulations were precisely determined in collaboration with Research Diets Inc. (New Brunswick, NJ, USA) to avoid batch to batch variations and to rule out the presence of contaminants such as phytoestrogens or pesticides commonly found in laboratory chow. DHA was obtained in a microencapsulated formulation (MEG-3) from Ocean Nutrition Inc. (Halifax, NS, Canada) to protect DHA molecules from oxidation and was incorporated in an isocaloric diet to obtain a daily dose of 424 mg/kg (5.3 g/kg of the diet; Table 1
). Levels of fatty acids in the diets were quantified using gas chromatography (see below). The microencapsulation process is intended to allow incorporation of DHA into common food such as milk or bread and has been designed to preserve DHA for months (32
, 33)
. Macronutrient, vitamin, and mineral concentrations were equal in both diets.
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Mice were housed five per cage under standard conditions throughout the experiments with free access to food and water and were handled under the same conditions by one investigator. All procedures were performed in accordance with the U.S. National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Policy of CHUL. Mice were fed the diets described in Table 1
from 2 to 12 months of age before the exposure to MPTP. Twenty mice received seven intraperitoneal injections of MPTP-HCl (20 mg/kg free base; Sigma, St. Louis, MO, USA) dissolved in saline 0.9%, prepared fresh. MPTP was administered twice on the first 2 days of the experimental protocol at an interval of 12 h and once a day on the 3 subsequent days. Remaining animals received saline 0.9% intraperitoneal after an identical MPTP regimen. The present MPTP regimen was selected to produce a moderate DAergic denervation (34
, 35)
.
Tissue preparation and postmortem analyses
Two weeks after the last MPTP injection, animals were sacrificed under deep anesthesia with ketamine/xylazine and perfused via transcardiac infusion with 0.1 M PBS. After intracardiac perfusion, brains were collected and the two hemispheres were separated. The left hemisphere was postfixed in 4% paraformaldehyde (PFA) for 48 h and transferred to 20% sucrose in 0.1 M PBS for cryoprotection. Coronal brain sections of 25 µm thickness were cut onto a freezing microtome (Leica Microsystems, Montreal, QC, Canada). The right hemisphere was snap-frozen in 2-methyl-butane and then stored at –80°C. Samples of the frontal cortex and anterior striatum were extracted from this hemisection for lipid and HPLC analyses. Coronal brain sections (12 µm) were cut onto a cryostat and stored at –80°C for 125I-RTI-121 autoradiography assay (36)
.
Lipid extraction and gas chromatography
Approximately 20 mg of frozen frontal cortex tissue from each mouse was used for the present study. The frontal cortex was selected because it is minimally affected by MPTP treatment and allowed us to isolate the effect of the diet on brain fatty acid profiles. Weighed brain tissues were homogenized with butylhydroxytoluene (BHT)-methanol (Sigma) and with 22:3 n-3 methyl ester internal standard (NuChek Prep company, Elysian, MN, USA) at a concentration of 500 µg/g of tissue. Two volumes of chloroform (J. T. Baker, Phillipsburg, NJ, USA) and NaH2PO4 buffer solution were added to the resulting homogenate. After centrifugation at 3500 RPM for 7 min, the lower layer was collected (37)
. This procedure was repeated twice, and the two extracts were pooled and brought to dryness with a stream of nitrogen. Lipid extracts were transmethylated with BF3-MeOH (Alltech, State College, PA, USA) at 100°C for 60 min. After the extracts were cooled, water and hexane (J. T. Baker) were added. A 3 min centrifugation allowed separation of phases, and then the upper layer was collected. These last steps were performed twice to pool the hexane extracts. Hexane was dried down to 
100 µl, transferred to a gas chromatography autosampler vial, and capped under nitrogen. Fatty acid methyl esters were quantified on a model 6890 series gas chromatograph (Agilent Technologies, Palo Alto, CA, USA) using a FAST-GC method. One microliter of each sample was 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 standard (462, Nu-Chek Prep) (38)
.
Immunohistochemical evaluation of DA cell loss
Coronal PFA-fixed brain sections were collected in cold PBS 0.1 M and subsequently placed in 3% hydrogen peroxide for 30 min at room temperature. Slices were transferred in 0.1 M PBS for several washes and then preincubated in a 0.1 M PBS solution containing 0.1% Triton X-100 (Sigma) and 5% normal goat serum (NGS; Wisent, Quebec, Canada) for another 30 min. Then, sections were incubated overnight at 4°C with mouse anti-tyrosine hydroxylase (TH; Chemicon, Temecula, CA, USA; 1:2500), 0.1% Triton X-100, and 5% NGS.
After overnight incubation at 4°C with this primary antibody, sections were washed in 0.1 M PBS and incubated for 1 h at room temperature in a 0.1 M PBS solution containing 0.1% Triton X-100, 5% NGS, and biotinylated goat anti-mouse IgG (Vector Laboratories, Burlington, ON, Canada; 1:1500). After three washes in PBS, sections were placed in a solution containing avidin-biotin peroxidase complex (ABC; Vector Laboratories) for 1 h at room temperature. Bound antibodies were visualized by placing the sections in a 0.1 M PBS solution containing 0.05% 3,3'-diaminobenzidine tetrahydrochloride (DAB; Sigma) and 0.1% of 30% hydrogen peroxide at room temperature. The reaction was stopped by washing in 0.1 M PBS. After the TH DAB reaction, sections were counterstained with cresyl violet (Sigma), dehydrated, and coverslipped (35)
.
In situ hybridization
The Nurr1 probe was produced, synthesized, and labeled using the Riboprobe System (Promega, Madison, WI, USA) with 35S-UTP (Perkin Elmer, Waltham, MA, USA) and the T3 or T7 RNA polymerase (35)
. The cRNA probe for Nurr1 was generated from a 403 bp fragment from the mouse sequence subcloned into pBluescript SK+ plasmid and linearized with HindIII/T3. The dopamine transporter (DAT) probe, a 2238-bp fragment, was cloned into pBluescript II SK+ plasmid (35)
. Linearization was made with Notl enzyme. The antisense probe was synthesized with 35S-UTP and T7 RNA polymerase.
Brain sections were mounted onto Snowcoat X-tra slides (Surgipath, Winnipeg, MB, Canada) and air-dried overnight at room temperature (or use 20°C). Brain sections were fixed in 4% PFA at 4°C for 20 min and rinsed twice with 1x PBS for 5 min. They were then submerged in a protease K solution at 37°C. After that, they were washed for a few seconds in DEPC water and incubated with a triethanolamine (100 mM TEA, pH 8.0) and anhydride acetic acid solution for 10 min in an acetylation bath. Slides were rinsed twice in a standard salt sodium citrate (SSC) solution and subsequently dehydrated into increasing concentrations of ethanol. They were further air-dried at room temperature. The 35S-UTP-radiolabeled complementary RNA (cRNA) probe was added to a hybridization mix (Denhart’s solution, dextran sulfate, and 50% deionized formamide) and heated at 80°C for 5 min. Each slide was covered with 100 µl of the hybridization solution and coverslipped. The hybridization took place on a slide warmer at 58°C overnight. After hybridization, slides were placed in 4x SSC for 30 min to remove coverslips and washed twice in SSC baths. Slides were incubated at 37°C for 1 h in a RNase A solution. Afterward, slides were washed in four SSC baths (2x, room temperature, 15 min; 0.5x, 60°C, 30 min; 0.1x, 60°C, 30 min; and 0.1x, room temperature, 5 min). Finally, tissues were dehydrated in ethanol. Tissue sections were then exposed to BiomaxMR (Kodak, New Haven, CT, USA) radioactive-sensitive films for 5 days for Nurr1 and 5 h for DAT.
Defatting was performed for DAT experimentation with four sequential baths of ethanol, two baths of xylene, and three baths of ethanol. After these steps, slides were dipped in NTB emulsion (Kodak) and prepared at 42°C, air-dried for 2 h, and stored in the dark for 7 days at 4°C. The emulsion was then developed in D-19 developer (3.5 min; Kodak), rinsed in deionized water, and fixed (5 min) in Rapid Fixer solution (Kodak). Slides were rinsed in deionized water for 1 h and then colored. Coloration was performed using thionine (1 min), followed by water, ethanol dips, and an additional three ethanol (1 min) and three xylene baths (3 min). Slides were coverslipped with DPX mounting media (35)
.
125I-RTI-121 autoradiography
DAT was evaluated with 125I-RTI-121 [3β-(4-125I-iodophenyl) tropane-2β-carboxylic acid isopropylester] (NEN-DuPont, Boston, MA, USA; 2200 Ci/mmol) according to a previously published procedure (36)
. Sections were preincubated at room temperature for 30 min in a phosphate buffer (10.1 mM NaHPO4, 1.8 mM KH2PO4, 137 mM NaCl, and 2 mM KCl pH 7.4). They were then incubated 90 min at room temperature with 20 pM 125I-RTI-121. Nonspecific binding was determined in the presence of 10 µM mazindol (Novartis, Basel, Switzerland). After two 20 min washes at room temperature in buffer, sections were then rinsed briefly (10 s) in ice-cold distilled water. Finally, the slide-mounted tissue sections were dried overnight at room temperature and exposed to 3H-sensitive film (Kodak) for 40 h (36)
.
Densitometric measurements of striatal TH fibers
Digitized brain images of the striatum were obtained with a CCD camera model XC-77 (Sony Electronics Inc., New York, NY, USA) equipped with a 60 mm f/2.8D magnification lens (Nikon Canada Inc., Mississauga, ON, Canada). The density of striatal DAergic fibers was analyzed on a MacIntosh computer using the Image NIH software (Image 1.63, W. Rasband, NIH, Bethesda, USA). The average labeling for each area was calculated from four adjacent brain sections of the same animal at the level of the anterior commissure (AP levels: –2.80 to –3.16) in the atlas of Paxinos and Franklin (39)
. The rostro-caudal extent of the analysis was comparable across animals, permitting us to average the data. All images were acquired at the same magnification (x10) to allow the visualization of the entire striatum in a single field. Striatal images converted to gray scale were then delineated, and the intensity of staining was thus assessed for the entire region of the sections sampled, subsequently averaged for each animal. Background intensities taken from the corpus callosum devoid of TH staining were subtracted from every measurement.
Quantification of TH-immunoreactive neurons and DAT-expressing cells
The loss of DAergic neurons was determined by stereological counts of TH- and DAT-immunoreactive cells (identifiable somas) under bright-field illumination. Every tenth section through the SNpc was analyzed using Stereo investigator software (MicroBrightfield, Colchester, VT, USA) attached to an E800 Nikon microscope (Nikon). After delineation of the SNpc at low magnification (x4 objective), a point grid was overlaid onto each section. Immunostained cells were counted by the optical fractionator method at higher magnification (x20 objective). The counting variables were as follows: distance between counting frames (150x150 µm), counting frame size (75 µm), and guard zone thickness (1 µm). Cells were counted only if they did not intersect forbidden lines. The optical fractionator (40)
method was used to count TH-positive (TH and cresyl violet positive) and TH-negative (cresyl violet positive only) or DAT-expressing cells. Stereological counts were performed blindly by two independent investigators. Note that the analyses of both the TH-immunoreactive profiles and the DAT-expressing cells were restricted to the SNpc and thus excluded the ventral tegmental area (VTA).
Densitometric measurements of Nurr1 and DAT levels in the SNpc and striatum
Levels of autoradiographic labeling for Nurr1 and DAT in the SNpc and striatum were quantified by computerized densitometry. Digitized brain images and their analyses were made with Kodak 4000MM Digital Imaging System and with Kodak Molecular Imaging Software (Carestream Health, Toronto, ON, Canada). Optical densities of the autoradiograms (Nurr1 and nigral DAT) were translated in microcuries per grams of tissue using 14C radioactivity standards (ARC 146-14C standards, American Radiolabeled Chemical Inc., St. Louis, MO, USA). The average labeling for each area was calculated from three adjacent brain sections (same levels) of the same mouse (AP levels: –2.80 to –3.16) (36)
. Background intensities taken from the white layer of the superior colliculus devoid of Nurr1 and DAT levels were subtracted from every measurement.
Catecholamine quantification
DA and 3,4-dihydroxyphenylacetic acid (DOPAC) were measured by HPLC with electrochemical detection (36
, 41)
. Extracts of rostral striatum were collected from mice brain, and 200 µl of perchloric acid (0.1 N; J. T. Baker) were added to generate a supernatant. Fifty microliters of supernatant from striatal tissues were directly injected into the chromatograph consisting in a Waters 717 plus autosampler automatic injector, a Waters 1525 binary pump equipped with an Atlantis dC18 (3 µl) column, a Waters 2465 electrochemical detector, and a glassy carbon electrode (Waters Limited, Lachine, QC, Canada). Electrochemical potential was set at 10 nA. The mobile phase consisted of 0.025 M citric acid (Sigma) with 1.7 mM sodium heptyl sulfate (Sigma), and 10% MeOH (J. T. Baker) at pH 4.0 and was delivered at 1.0 ml/min. Peaks were identified using Breeze software (Waters). HPLC quantifications were normalized to protein concentrations. Protein measurements were determined with a bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL, USA) as described by the manufacturer’s protocol.
Statistical analyses and image preparation of postmortem material
Statistical analyses were performed using JMP software 6.0.2 (SAS Institute Inc., Cary, IL, USA) and PRISM 4 (Graphpad Software, San Diego, CA, USA). Student’s t test was used to analyze the fatty acid profiles. One-way ANOVA was performed for all the other experimentations, and log adjustments were used to normalize variances when needed. Newman-Keuls Multiple comparison tests were performed as post hoc analyses. Photomicrographs were taken with a Microfire 1.0 camera (Optronics, Goleta, CA, USA) linked to an E800 Nikon microscope (Nikon) using the imaging software Picture Frame (MicroBrightfield) and were prepared for illustration in Adobe Photoshop 7.0.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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D) but decreased levels of the n-6 PUFA, docosapentaenoic acid (DPA; –77%; Student’s t test; P<0.0001; Fig. 1E
). The n-6 PUFA:n-3 PUFA ratio was also decreased by 48% after this treatment (Student’s t test; P<0.0001; Fig. 1F
), whereas fatty acids, linoleic (LA; Fig. 1A
), eicosapentaenoic (EPA; Fig. 1B
), and arachidonic (ARA; Fig. 1C
) levels remained unchanged. Cortical concentration of alpha-linolenic (LNA) remained under 0.12% of total fatty acid in all groups (data not shown). As reported in primates (9)
, no effect of the neurotoxin MPTP on the cortical fatty acid profile was observed (data not shown).
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Beneficial effects of the high n-3 PUFA diet on the nigral DAergic system
As expected, a 5 day systemic MPTP treatment induced a 31% decrease in the number of DAergic cells of the SNpc as assessed with TH immunostaining (Fig. 2
E) (35)
. A significant difference was observed between vehicles (Fig. 2A, B
) and MPTP-treated animals fed the control diet [Fig. 2C
; ANOVA; F(groups)3,33=6.513; P<0.01]. Stereological counts also revealed a significant difference between animals treated with MPTP fed the control diet compared to animals fed the high n-3 PUFA diet (Fig. 2D
; P<0.01), while no differences were observed between vehicle animals and MPTP-treated animals fed the high n-3 PUFA diet.
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Nurr1, a nuclear receptor important for the regulation of the survival of DAergic neurons (42)
, was quantified by in situ hybridization in the SNpc (Fig. 3
). Neuroprotection was provided by the high n-3 PUFA diet [ANOVA; F(groups)3,33=6.32;P<0.01]. Nurr1 mRNA levels of both vehicle groups (Fig. 3A, C
) and MPTP-treated animals fed the high n-3 PUFA diet (Fig. 3D
) were similar, while Nurr1 mRNA levels in MPTP-treated animals fed the control diet were decreased by 28% (P<0.01 vs. other groups). The same pattern was observed for nigral DAT mRNA expression (Fig. 4
). Quantification of this aspect of DAergic system was assessed by densitometric measurements as well as by stereological counts of DAT-expressing cells (Fig. 4A-D
). Both methods showed a significant neuroprotection of DAT mRNA levels by the high n-3 PUFA in the SNpc (P<0.05). MPTP treatment decreased DAT mRNA levels by 50% [ANOVA; F(groups)3,33=9.88; P<0.0001; Fig. 4E
], and a 40% [ANOVA; F(groups)3,32=6.64; P<0.001] decrease was observed for DAT-expressing cells (Fig. 4F
).
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Effects of high n-3 PUFA diet on the striatal DAergic terminals
Two striatal components of the DAergic system were not affected by the high n-3 PUFA diet, TH-positive fibers and DAT. MPTP treatment decreased TH fibers by 31% [ANOVA; F(groups)3,34=19.58; P<0.0001; Fig. 5
C–E] and DAT by 61% [ANOVA; F(groups)3,34=169.93; P<0.0001; Fig. 5H-J
], but no significant difference was observed between groups treated with MPTP on either diets. Despite the absence of a significant effect on striatal TH fibers and DAT levels, HPLC measures of striatal DA and its metabolite DOPAC strongly corroborate the protective effect of n-3 PUFA on nigral DAergic neurons. MPTP treatments induced an 80% decrease in striatal DA [ANOVA; F(groups)3,34=13.75; P<0.0001; Fig. 6
A] and a 79% decrease in striatal DOPAC levels [ANOVA; F(groups)3,32=4.49; P<0.0001; Fig. 6B
]. A significant difference between MPTP-treated animals fed control and high n-3 PUFA diets was found for both DA and DOPAC (P<0.05).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 21
, 43
, 44)
. To this day, the available epidemiological and postmortem data are not sufficient to draw any conclusion on the role n-3 PUFA in PD (24
, 25)
. Nevertheless, a direct effect of DHA on DA-related motor function has been reported in a nonhuman primate model of Parkinsonism (45)
. Although several treatments are available to alleviate PD symptoms, no drug, to this day, is effective to prevent the loss of DAergic neurons or to regenerate neurons already lost. Clearly, the search for a pharmaceutical tool that could offer neuroprotective or neurorestorative effects remains the holy grail of PD research. Here, we provide the first preclinical evidence that high n-3 PUFA consumption exerts a neuroprotective action against MPTP-induced toxicity as assessed by the evaluation of different aspects of the DAergic system (Fig. 7
).
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Using gas chromatography and flame ionization detection, we first demonstrated that a 10 month treatment with a diet high in n-3 PUFAs modified the fatty acid content in the mouse frontal cortex. This highly significant effect confirms that the brain fatty acid profile in mammals depends on the dietary intake, as demonstrated previously (10
, 46
, 47)
. As expected, MPTP treatment had no significant effect on the frontal cortex fatty acid profile. Significant alterations produced by the high n-3 PUFA diet included an increase in DHA as well as a decrease in the n-6/n-3 PUFA ratio and DPA. The absence of diet-dependent changes in the concentrations of ARA, LNA, and EPA, suggest that DHA is the prime n-3 PUFA involved in the neuroprotective effect observed in our paradigm. Conversely, a detrimental effect of the high n-6 PUFA content of the control diet is an equally logical interpretation of our data.
To probe for a protective effect of n-3 PUFAs, we focused our investigations on six important markers of DAergic activity, three assessing the nigral cell population (TH immunostaining, Nurr1, and DAT mRNA) and three measuring striatal axonal innervation (catecholamine concentrations, DAT autoradiography, and TH immunostaining). The MPTP treatment induced a decrease in all markers investigated, but striatal levels of DA were the most sensitive parameters. Indeed, reduction in DA and DOPAC reached 80 and 79% in the striatum, respectively, levels comparable to that observed in previous studies (36
, 48)
. This increased sensitivity of DA to MPTP compared to DAT mRNA (40%) is also in accordance with previous studies (36)
.
Preservation of DAT and Nurr1 mRNA was observed in MPTP mice fed a high n-3 PUFA diet, suggesting that nigral DAergic neurons were protected from MPTP neurotoxicity. Protection is unlikely to be attributable to an increase in gene expression of both transcripts, as the high n-3 PUFA diet had no effect on DAT and Nurr1 mRNA levels in vehicle-treated animals. Whereas DAT is used here as a marker of DAergic neuron viability (49
, 50)
, Nurr1 is involved in the development (51)
and maintenance (42)
of the DAergic phenotype in neurons of the midbrain (SN and VTA). The recently characterized regulatory role of Nurr1 on TH expression reinforces the hypothesis that Nurr1 is a susceptibility factor for DAergic neurodegeneration in PD (52)
. In addition, it has recently been shown that reduced Nurr1 expression increases the vulnerability of mesencephalic DAergic neurons to MPTP-induced injury (53)
. Thus, the fact that high dietary intake of n-3 PUFA protected against MPTP-induced decrease of Nurr1 provides a strong argument for a neuroprotective action.
The deleterious effect of MPTP on TH-positive nigral neurons was completely blunted in animals fed the high n-3 PUFA diet, suggesting an important protective effect of n-3 PUFA. In addition, we reported a concomitant loss of Nissl-stained neurons in the SNpc, which argues against a down-regulation of TH expression. In contrast to the neuroprotective effect observed in three different assessments of nigral cell number, the two distinct methodologies used to assess striatal DAergic terminals, namely TH immunostaining and 125I-RTI-121-specific binding to DAT, did not provide evidence for an effect of n-3 PUFA intake. This difference in sensitivity to MPTP and of response to n-3 PUFA treatment can result from a differential sensitivity of the two compartments of DAergic cells to the toxin or the inherent sequential action of MPTP toxicity. The MPTP sensitivity discrepancy between cell bodies and terminals has already been documented in rodents and monkeys using TH immunostaining (54
, 55)
, dopamine levels (54)
, DAT autoradiography (55
, 56)
, and vesicular monoamine transporter autoradiography (56)
. A differential level of DAergic denervation between the striatum and the SN is consistent with postmortem studies in human PD patients (57
, 58)
. DHA, presumably consequent to its incorporation in nigral cell membranes, protected cell bodies from the MPTP insult, an effect not perceptible in striatal fiber terminals at the time point chosen here. Perhaps, with a longer delay after the MPTP lesion, the levels of DAergic terminals would have matched the cell body counts. An alternative explanation comes from the fact that the striatum contains TH- and DAT-positive cells that do not originate from the SNpc and that their number increases after nigrostriatal denervation (59
60
61
62)
. A change of TH or DAT expression in these cells that is different from SNpc DAergic neurons may account, in part, for the present findings.
A diminution of axonal projections may be of limited relevance if the cell bodies are intact and if the release of striatal DA remains unimpaired. Thus, we assessed MPTP-induced toxicity by measuring the concentrations of DA and its metabolite DOPAC in the mouse striatum. The high intake of n-3 PUFA before the MPTP insult led to intermediate levels of DA between vehicle and MPTP-treated mice fed the control diet. Even though our HPLC technique does not distinguish between intracellular and extracellular DA, this suggests that n-3 PUFA had a partially protective effect against MPTP-induced reduction in DA production, storage, and/or release. Accordingly, n-3 PUFA did not affect the DOPAC/DA ratio (data not shown), suggesting no impact on the DA turnover. Again, no increase of DA concentrations was seen in vehicle-treated mice exposed to high n-3 PUFA treatment, indicating no effect on DA synthesis. Since DA depletion is closely related to the expression of the clinical symptoms of PD, the capacity of n-3 PUFA to maintain DA levels is consistent with a clinically relevant neuroprotective action.
It is not clear from the present data if n-3 PUFA protected against the MPTP neurotoxicity per se or if it stimulated nigral cell regeneration after MPTP insult. Multiple actions of DHA have been demonstrated in vitro and in vivo (11
, 63
64
65)
. Since n-3 PUFA exerts antioxidant action in vivo, prevention of oxidative damage is a likely mechanism of action underlying its neuroprotective effect (16
, 66
67
68
69)
. Indeed, DHA was shown to increase glutathione reductase activity (66)
and decrease the accumulation of oxidized proteins (16
, 67)
and levels of lipid peroxide and reactive oxygen species (ROS) (66
, 70)
in the brain. An obvious advantage for the use of MPTP models of Parkinsonism is that this neurotoxin recreates quite faithfully several pathological characteristics seen in PD such as a specific loss of DAergic cells in the SNpc associated with oxidative damages (71
, 72)
. MPTP toxicity involves an increase in ROS and damaged mitochondria DNA (73)
. Those similarities raise the possibility that ROS may play a significant role in the early pathogenesis of PD DAergic neurodegeneration as well as in MPTP neurotoxicity. Therefore, if n-3 PUFA-related antioxidant action is determinant in our MPTP model, it could be the case in idiopathic PD as well. Mechanisms related to the ability of DHA to inactivate caspase-related programmed cell death pathways through the phosphatidylinositol-3 kinase (PI3K)-Akt cascade are also plausible (13
14
15
16
, 74)
. Finally, the beneficial effects of DHA could be mediated by the activation of nuclear receptors that operate as transcription factors, such as retinoid X receptors (75)
. Through its incorporation into the brain cell membrane, DHA can affect membrane order (76)
and ion channels (77
, 78)
or may act as a second messenger in the modulation of synaptic signal transduction pathways (79)
. DHA also incorporates into brain microsomal, synaptosomal, and mitochondrial membranes (80)
. For example, n-3 PUFA can modulate voltage-dependant calcium channels (64
, 81)
, which were recently shown to play an important role in survival of DAergic nigral cells (82)
. Obviously, the present observation provides arguments for further analyses of the neuroprotective mechanisms of DHA.
It is also possible that altered metabolism of MPTP could explain the effect of n-3 PUFA on DAergic markers. For example, chronic treatment with n-3 PUFA might have decreased the conversion of MPTP into MPP+. Indeed, evidence that dietary n-3 PUFA slightly reduces MAO-B activity in the brain of rodents has been reported (83
, 84)
. However, this effect was not found on the basal ganglia and its low magnitude could hardly explain the present observation. Nevertheless, further studies are needed to completely rule out the possibility that n-3 PUFA blunted the neurotoxicity of MPTP though regulation of its metabolism.
In our modern society, an underconsumption of fish and, consequently, an underconsumption of n-3 PUFA is commonplace. The average DHA intake is between 60–80 mg per day in the United States in contrast to expert panel recommendations suggesting a daily consumption of 200–300 mg (19)
. It is likely that the majority of PD patients have been exposed to low dietary intake of DHA as well. As our present results suggest, this prevalent low consumption of DHA might be an important modifiable risk factor for PD. Fortunately, it is easy to treat n-3 PUFA deficiency by changes in dietary habits or by administration of inexpensive supplements. Indeed, n-3 PUFA are nonpatentable, widely available at low cost, and have an excellent safety profile (85
, 86)
. Randomized clinical trials have recently been initiated to test the effects of DHA in various CNS disorders (87
88
89)
. In view of the fact that there is a real possibility for therapeutic intervention, the present study indicates a need for further investigations of the neuroprotective role of n-3 PUFA in PD.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication August 20, 2007. Accepted for publication October 18, 2007.
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REFERENCES |
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