High-fat diet exacerbates MPTP-induced dopaminergic degeneration in mice

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<ul><li><p>High-fat diet exacerbates MPTP-induced do</p><p>M. Bousquet a,b, I. St-Amour a,b,c, M. Vandal a,b, P. Juliena Centre de Recherche du CHUL (CHUQ), Axe Neurosciences, Qubec, QC, Canadab Facult de Pharmacie, Universit Laval, Qubec, QC, Canadac Dpartement de Recherche et Dveloppement, Hma-Qubec, Qubec, Canadad Centre de Recherche sur les Maladies Lipidiques, CHUL (CHUQ), Qubec, QC, Canadae ec,</p><p>QC,</p><p>le nsordcal)pathological features of PD. HFD-fedmice signicantly gainedweight (+41%), devel-</p><p>plasmatic cytokines/chemokines (interleukin-1, MCP-1, MIP-1). As expected, the MPTP treatment producednigral dopaminergic degeneration as evidenced by</p><p>2006). A progressive loss of dopaminergic neurons within the substantia tions such as autonomic system failure, depression and dementia, for</p><p>Neurobiology of Disease 45 (2012) 529538</p><p>Contents lists available at SciVerse ScienceDirect</p><p>Neurobiology</p><p>j ourna l homepage: www.e lnigra pars compacta (SNpc) accompaniedwith a drastic decrease in stria-tal dopamine (DA) are part of the pathological features discernible in the</p><p>which the pathophysiology is still unclear (Chaudhuri and Schapira,2009; Lees et al., 2009). For the vast majority of PD patients, the originof neurodegeneration remains unknown and less than 15% of PD casescan be attributed to specic genetic mutations as identied in -synu-clein (SNCA), LRRK2, PINK1, parkin and DJ1 genes (Meissner et al., 2011).A number of environmental risk factors have been suggested to play akey role in the development of PD but because they are readily modi-able, nutritional factors have received considerable attention over theyears. Several preclinical and epidemiological studies have indeed under-lined the impact of nutrients such as dairy products, numerous vitamins,caffeine and iron on PD pathogenesis despite the fact that some of these</p><p>Abbreviations: ARA, arachidonic acid; BDNF, brain-derived neurotrophic factor; CD,control diet; DA, dopamine; DAT, dopamine transporter; DHA, docosahexaenoic acid;DOPAC, 3,4-dihydroxyphenylacetic acid; DPA, docosapentaenoic acid; DTA, docosate-traenoic acid; GDNF, glial cell line-derived neurotrophic factor; GFAP, glial brillaryacidic protein; GM-CSF, granulocyte macrophage colony stimulating factor; HFD,high-fat diet; IFN, interferon; IL, interleukin; LA, linoleic acid; MCP, monocyte chemo-tactic protein; MIP, macrophage inammatory protein; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; n3/n6 PUFA, omega-3/omega-6 polyunsaturatedfatty acid; PD, Parkinson's disease; PDS-95, post-synaptic protein-95; SNpc, substantia</p><p>nigra pars compacta; TH, tyrosine hydroxylase; TNF, tum Correspondence to: F. Cicchetti, Centre de Rech</p><p>Neurosciences, T2-05, 2705, boulevard Laurier, QuFax: +1 418 654 2753. Correspondence to: F. Calon, CentredeRechercheduC</p><p>T2-05, 2705, boulevard Laurier, Qubec, QC, Canada G1V 4GE-mail addresses: Francesca.Cicchetti@crchul.ulaval.c</p><p>Frederic.Calon@crchul.ulaval.ca (F. Calon).1 Co-senior authors.</p><p>Available online on ScienceDirect (www.scienced</p><p>0969-9961/$ see front matter 2011 Elsevier Inc. Alldoi:10.1016/j.nbd.2011.09.009rative disorder affectingge (de Lau and Breteler,</p><p>tant motor perturbations such as resting tremors, bradykinesia, akinesiaand muscular rigidity. PD is also characterized by non-motor manifesta-Parkinson's disease (PD) is a neurodegeneover 1% of the population over 60 years of aKeywords:Parkinson's diseaseObesityInsulin resistanceDopamineFlow cytometryCytokinesLeukocytesDopaminergic neuronsNutrition</p><p>Introductionnigral tyrosine hydroxylase (TH)- and dopamine transporter-expressing neurons (23% and 25%, respectively).However, exposure to HFD exacerbated the effects of MPTP on striatal TH (23%) and dopamine levels (32%), indi-cating that diet-induced obesity is associated with a reduced capacity of nigral dopaminergic terminals to copewith MPTP-induced neurotoxicity. Since high-fat consumption is commonplace in our modern society, dietaryfat intake may represent an important modiable risk factor for PD.</p><p> 2011 Elsevier Inc. All rights reserved.</p><p>brain of PD patients (Calon et al., 2003; Damier et al., 1999; Lees et al.,2009). The dopaminergic neurodegeneration seemingly leads to impor-associations are sEtminan et al., 20we have previouhigh intake of omouse model of P</p><p>Increasing evroot of a pletho1996;Willett et a</p><p>or necrosis factor.erche du CHUL (CHUQ), Axebec, QC, Canada G1V 4G2.</p><p>HUL (CHUQ), AxeNeurosciences,2. Fax: +1 418 654 2761.a (F. Cicchetti),</p><p>irect.com).</p><p>rights reserved.the loss of striatal dopamine and the decreased number ofAvailable online 21 September 2011</p><p>oped insulin resistance and a systemic immune response characterizedby an increase in circulating leukocytes andAccepted 13 September 2011either a control (CD 12%kwhich recreates a number ofDpartement de Psychiatrie &amp; Neurosciences, Facult de Mdecine, Universit Laval, Qubf Institut des nutraceutiques et des aliments fonctionnels (INAF), Universit Laval, Qubec,</p><p>a b s t r a c ta r t i c l e i n f o</p><p>Article history:Received 26 June 2011Revised 31 August 2011</p><p>The identication of modiabgies for neurodegenerative dipaminergic degeneration in miced, F. Cicchetti a,e,,1, F. Calon a,b,f,,1</p><p>QC, CanadaCanada</p><p>utritional risk factors is highly relevant to the development of preventive strate-ers including Parkinson's disease (PD). In this study, adult C57BL/6micewere fedor a high-fat diet (HFD 60%kcal) for 8 weeks prior to MPTP exposure, a toxin</p><p>of Disease</p><p>sev ie r .com/ locate /ynbd itill being debated (Chen et al., 2007; Costa et al., 2010;05; Powers et al., 2009). In line with the present study,sly reported the neuroprotective effects of a long-termmega-3 polyunsaturated fatty acid (n3 PUFA) in aD (Bousquet et al., 2008; Calon and Cicchetti, 2008).idence suggest that diet-induced weight gain is at thera of health problems (Colditz et al., 1995; Felson,l., 1995, 1999).While obesity andmetabolic disorders,</p></li><li><p>plex assay detection limit: interleukin (IL)-1 14 pg/ml, IL-1 50 pg/ml, IL-2 16 pg/ml, IL-3 14 pg/mg, IL-4 14 pg/ml,IL-5 12 pg/ml, IL-6 12 pg/ml, IL-10 8 pg/mg, IL-12 28 pg/ml, IL-17 32 pg/ml, interferon (IFN) 32 pg/ml, tumor ne-crosis factor (TNF) 22 pg/ml, macrophage inammatory protein(MIP-1) 12 pg/ml, monocyte chemotactic protein (MCP-1) 12 pg/ml, granulocyte macrophage colony stimulating factor (GM-CSF) 14 pg/ml and RANTES 30 pg/ml) from Quansys Biosciences(Logan, UT) according to the manufacturer's instructions.</p><p>Tissue preparation for post-mortem analyses</p><p>Fourteen days following the last MPTP injection, animals were sacri-ced under deep anesthesiawith ketamine/xylazine and perfused via in-tracardiac infusion with 1X PBS containing protease inhibitors (Sigma)and phosphatase inhibitors (1 mM tetrasodium pyrophosphate and50 mM sodium uoride). Brains were collected, the frontal cortex wasdissected for fatty acid analyses and the two hemispheres were separat-ed. The left hemisectionwas snap-frozen in 2-methyl-butane and storedat 80 C for cryostat coronal brain sections of 20 m for high perfor-mance liquid chromatography (HPLC) analysis. The right hemisectionwas separated at the level of bregma 1.70 mm, the caudal sectionwas post-xed in 4% paraformaldehyde pH 7.4, while the striatum con-</p><p>530 M. Bousquet et al. / Neurobiology of Disease 45 (2012) 529538such as diabetes, are well known outcomes of unhealthy high-fat diet(HFD), more recent studies have added neurodegenerative diseases tothe list of possible deleterious consequences of high-fat consumption(Bayer-Carter et al., 2011; Gao et al., 2007; Hiltunen et al., 2011; Julienet al., 2010; Mody et al., 2011). For example, retrospective and prospec-tive epidemiological studies suggest an associationwith ahigh consump-tion of saturated fat from animalmeat and a higher risk of developing PD(Gao et al., 2007). Obesity, a predictable effect of high-fat intake, has alsobeen studied in relation to PD in prospective epidemiological studies.While a signicant weight loss is observed 2 to 4 years prior to diagnosis(Chen et al., 2003), a greater bodymass index (BMI),midlife triceps skin-fold thickness, waist circumference and waist-to-hip ratio have beenlinked to increased risk of developing PD in never smokers (Abbottet al., 2002; Chen et al., 2004; Hu et al., 2006). Other studies, however,have not reported a signicant link between obesity and PD (Logroscinoet al., 2007). With a prevalence increasing with age (Wild et al., 2004),diet-induced obesity often leads to metabolic disorders, including typeII diabetes, and chronic peripheral inammation (Das, 2001). Interest-ingly, a recently published prospective study evaluating the relation-ship between diabetes and the development of PD found an overall41% increased risk (odd ratio) among individuals who have sufferedfrom diabetes for 10 years or more (Xu et al., 2011).</p><p>High-fat intake in the general population and resultingmetabolic pe-ripheral perturbations arewidespread. Since high-fat consumption couldbe a modiable risk factor, it is critical to further investigate their effectson the development of neurodegenerative diseases. We evaluated theimpact of a 2-month high intake in calories from fat on insulin resistanceand immune systemic response as well as in the brain of a mousemodelof PD. Our main a priori hypothesis was that a HFD exacerbates the ef-fects of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP) in mice compared to mice fed a control diet (CD).</p><p>Materials and methods</p><p>Experimental design, diet and MPTP treatment</p><p>Mice were housed in groups of 3 to 4 per cage and were kept understandard conditions throughout the experiments, with free access tofood and water. All procedures were approved by the Laval UniversityAnimal Welfare Committee (#09-098). Forty-nine C57BL/6 male micewere either fed a HFD (60% of calories) or a CD (12% of calories) from2months of age (corresponding to adulthood) until the time of sacrice(see Table 1 for complete description of the diets) (Julien et al., 2010).The MPTP treatment was performed at 4 months of age and consistedof 7 i.p. injections of a MPTPHCl solution (20 mg/kg, free base; Sigma,St. Louis, MO) freshly dissolved in 0.9% saline. MPTP was administeredtwice on therst two days of the experimental protocol at 12-h intervals,and once a day on the three subsequent days, as previously described(Bousquet et al., 2008; Gibrat et al., 2009). Remaining animals received0.9% saline i.p. in replacement of the MPTP injections. To ensure equiva-lent dosage of the neurotoxin to eachmouse and to prevent any lethal in-toxication of obese mice, MPTP doses were calculated for each mousewith respect to the body surface area (BSA) instead of body weight(Cheung et al., 2009). Briey, the BSA is described as: k*m0.667, m beingtheweight (g) of themouse and k (Meeh constant) is determined empir-ically as 9.822 for the C57B/L6 mouse strain (Cheung et al., 2009). Themean weight of the mice on the control diet was 31 g for a calculatedBSA of 97.04 (BSAmean); these mice received 0.62 mg MPTP (on a20 mg/kg basis). The BSA of all mice was determined prior to calculationof MPTP doses (mg) using this formula: BSA0.62/BSAmean.</p><p>Insulin sensitivity assessment and plasmatic insulin concentration</p><p>Insulin sensitivity was evaluated after 8 weeks of diet. Mice wereinjected with human insulin (1 g/kg) after 6 h of fasting. Blood glucose</p><p>wasmonitored (OneTouch UltraMini, LifeScan, Milpitas, CA, USA) at 15,30, 45, 60 and 90 min post-insulin injection. Plasma insulin was mea-sured using an Ultrasensitive Insulin ELISA assay (Mercodia, Uppsala,Sweden) according to the manufacturer's protocol.</p><p>Flow cytometry analyses and Q-plex cytokine assay</p><p>Blood was collected via the saphenous vein using EDTA-coatedcapillary tubes. Whole blood cell populations were analyzed usingPE conjugated-anti-CD45, AF647 conjugated anti-CD3, AF647conjugated-anti-CD4 and PE conjugated anti-CD8 for T cells, PE-Cy7conjugated anti-B220 for B cells and AF488 conjugated anti-Gr1 forgranulocytes detection (all antibodies are from eBioscience, SanDiego, CA). Briey, 5 l of whole blood cells was stained for 15 minat room temperature in 800 l PBS with 1% fetal bovine serum (Ther-mo Scientic Hyclone, Logan, UT) and immediately analyzed by owcytometry using a CyFlow ML (Partec, Swedesboro, NJ). Serum cyto-kine concentrations (1:2 plasma dilution) were evaluated with anELISA-based fully quantitative Q-PlexMouse Cytokine-Screen (16-</p><p>Table 1Diet composition</p><p>Content Diets</p><p>CD HFD</p><p>Protein (% w/w) 20.3 27.4Carbonhydrate (% w/w) 66.0 25.3Fat (% w/w) 5.0 35.1Calories (kcal/g) 3.9 5.3</p><p>Ingredients (g/kg)Casein 200.0 200.0DL-methionine 3.0 3.0Corn starch 150.0 125.0Sucrose 500.0 52.5Cellulose, BW200 50.0 50.0Corn oil 30.0 0.0Safower oil 0.0 125.0Soybean oil 10.0 0.0Lard 0.0 135.0Canola oil 10.0 0.0Cholesterol, USP 0.6 3.1</p><p>Fatty acid content as determined by gas chromatography (g/kg)n3 PUFAs 2.3 1.5n6 PUFAs 25.5 111.9n6/n3 PUFA ratio 11.1 74.0tained in the rostral section was dissected for Western immunoblotting.</p></li><li><p>531M. Bousquet et al. / Neurobiology of Disease 45 (2012) 529538Lipid extraction and gas chromatography</p><p>Approximately 20 mg of frozen frontal cortex tissue from eachmousewas used for fatty acid prole analyses. The frontal cortex was selectedbecause it isminimally affected byMPTP treatment and allowedus to iso-late the effect of the diet on brain fatty acid proles (Bousquet et al., 2008,2011). Weighed brain tissues were homogenized successively with 0.9%NaCl, butylhydroxytoluene-methanol (Sigma, St. Louis, MO) and chloro-form (J.T. Baker, Phillipsburg, NJ) with 22:3n-3 methyl ester as internalstandard (Nu-Chek Prep, Elysian, MN) at a concentration of 500 g/g oftissue. After centrifugation at 2400 g for 7 min, the lower layer was col-lected (Folch et al., 1957). This procedure was repeated and the two ex-tracts were pooled and dried under a stream of N2. Lipid extracts weretransmethylated with methanol:benzene (4:1) and acetyl chloride at98 C for 90 min. After cooling down, 6% K2CO3was added. A 15-min cen-trifugation at 514 g allowed phase separation and the upper layer wascollected in a gas chromatography autosampler vial and capped underN2. Fatty acid methyl ester proles in brain tissue were obtained by cap-illary gas chromatography using a temperature gradient on a HP5890 gaschromatograph (Hewlett Packard, Toronto, Canada) equippedwith aHP-88 capillary column (100 m0.25 mm i.d.0.20 m lm thickness; Agi-lent Technologies) coupled with a ame ionization detector. Heliumwasused as a carrier gas (split ratio 1:80). Fatty acids were identied accord-ing to their retention time, using the following standard mixtures as abasis for comparison: the FAME 37 mix (Supelco Inc., Bellefonte, PA)and the GLC-411 fatty acid mix (Nu-Chek Prep Inc, Elysian, MN), aswell as the following methylated fatty acids C22:5 n6 (Larodan AB,Malm, Sweden) and C22:5 n3 (Supelco Inc., Bellefonte, PA). Amixtureof trans fatty acids containing C18:2 n6 cis/trans (Supelco IncBellefonte, PA), a mixture of cis/...</p></li></ul>