PPAR Agonists Regulate Brain Gene Expression: Relationship to Their 66 2 67 3 Effects on Ethanol Consumption 68 4 69 A, B, * A, B a a 5 Q2 Laura B
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NP5538_proof ■ 18 July 2014 ■ 1/11 Neuropharmacology xxx (2014) 1e11 55 Contents lists available at ScienceDirect 56 57 Neuropharmacology 58 59 60 journal homepage: www.elsevier.com/locate/neuropharm 61 62 63 64 65 1 PPAR agonists regulate brain gene expression: Relationship to their 66 2 67 3 effects on ethanol consumption 68 4 69 a, b, * a, b a a 5 Q2 Laura B. Ferguson , Dana Most , Yuri A. Blednov , R. Adron Harris 70 6 a 71 7 Waggoner Center for Alcohol and Addiction Research, The University of Texas at Austin, Austin, TX 78712, United States b The Institute for Neuroscience (INS), The University of Texas at Austin, Austin, TX 78712, United States 72 8 73 9 74 10 article info abstract 75 11 76 12 Article history: Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that act as ligand- 77 13 Received 20 March 2014 activated transcription factors. Although prescribed for dyslipidemia and type-II diabetes, PPAR ago- 78 14 Received in revised form nists also possess anti-addictive characteristics. PPAR agonists decrease ethanol consumption and reduce 79 6 June 2014 15 withdrawal severity and susceptibility to stress-induced relapse in rodents. However, the cellular and 80 Accepted 24 June 2014 16 molecular mechanisms facilitating these properties have yet to be investigated. We tested three PPAR Available online xxx 81 17 agonists in a continuous access two-bottle choice (2BC) drinking paradigm and found that tesaglitazar 82 fi 18 (PPARa/g; 1.5 mg/kg) and feno brate (PPARa; 150 mg/kg) decreased ethanol consumption in male 83 Keywords: fi 19 PPAR C57BL/6J mice while beza brate (PPARa/g/b; 75 mg/kg) did not. We hypothesized that changes in brain fi 84 20 Microarray gene expression following feno brate and tesaglitazar treatment lead to reduced ethanol drinking. We fi 85 21 Alcohol studied unbiased genomic pro les in areas of the brain known to be important for ethanol dependence, the prefrontal cortex (PFC) and amygdala, and also profiled gene expression in liver. Genomic profiles 86 22 Prefrontal cortex Amygdala from the non-effective bezafibrate treatment were used to filter out genes not associated with ethanol 87 23 Mice consumption. Because PPAR agonists are anti-inflammatory, they would be expected to target microglia 88 24 and astrocytes. Surprisingly, PPAR agonists produced a strong neuronal signature in mouse brain, and 89 25 fenofibrate and tesaglitazar (but not bezafibrate) targeted a subset of GABAergic interneurons in the 90 26 amygdala. Weighted gene co-expression network analysis (WGCNA) revealed co-expression of 91 27 treatment-significant genes. Functional annotation of these gene networks suggested that PPAR agonists 92 28 might act via neuropeptide and dopaminergic signaling pathways in the amygdala. Our results reveal 93 29 gene targets through which PPAR agonists can affect alcohol consumption behavior. 94 © 2014 Elsevier Ltd. All rights reserved. 30 95 31 96 32 97 33 1. Introduction PPARg that each have distinct expression patterns, tissue distribu- 98 34 tion and physiological functions (Heneka and Landreth, 2007; 99 35 PPARs belong to the nuclear hormone receptor superfamily, one Moreno et al., 2004). Endogenous ligands of PPARs include endo- 100 36 of the largest families of transcription factors (Mangelsdorf et al., cannabinoids, fatty acids and fatty acid derivatives (e.g., poly- 101 37 1995). PPARs heterodimerize with Retinoid X Receptor (RXR), unsaturated fatty acids, eicosanoids and oxidized phospholipids) 102 38 another nuclear hormone receptor, and act as ligand-regulated (Krey et al., 1997; Sun and Bennett, 2007). 103 39 104 transcription factors (Kliewer et al., 1994). There are three known There is increased interest in PPAR agonists for the treatment of 40 isotypes of PPARs: PPARa,PPARb also named PPARd (PPARb/d), and CNS diseases including Alzheimer's, Parkinson's and Huntington's 105 41 106 disease, ischemic brain injury, schizophrenia, obesity and metabolic 42 disorders. Most research analyzing PPARs as therapeutics for brain 107 43 108 disorders has focused on PPAR , the most abundant isotype in 44 g Abbreviations: PPAR, Peroxisome proliferator-activated receptor; RXR, Retinoid microglia, because of its well-documented anti-inflammatory 109 45 X receptor; CNS, Central nervous system; VTA, Ventral tegmental area; PFC, Pre- 110 properties and its potential therapeutic use in neurodegenerative 46 frontal cortex; WGCNA, Weighted gene co-expression network analysis; ORA, 111 overrepresentation analysis; GABA, Gamma-aminobutyric acid; INIA IT-GED, INIA diseases and brain injury (Heneka and Landreth, 2007). All PPAR 47 112 Texas gene expression database; 2BC, Two-bottle choice; PPI, Proteineprotein isotypes are expressed in neurons, oligodendrocytes, microglia and 48 interaction. astrocytes (Heneka and Landreth, 2007), and PPAR activity in the 113 49 * Corresponding author. The University of Texas at Austin, Waggoner Center for 114 brain is relatively high (Ciana et al., 2007; Kao et al., 2012). PPARg 50 Alcohol and Addiction Research, 2500 Speedway, Austin, TX 78712, United States. 115 þ þ and a are expressed in the midbrain, including tyrosine- 51 Tel.: 1 512 947 5752; fax: 1 512 232 2525. 116 E-mail address: [email protected] (L.B. Ferguson). hydroxylase positive neurons (Plaza-Zabala et al., 2010; Sarruf 52 117 53 http://dx.doi.org/10.1016/j.neuropharm.2014.06.024 118 54 0028-3908/© 2014 Elsevier Ltd. All rights reserved. 119 Please cite this article in press as: Ferguson, L.B., et al., PPAR agonists regulate brain gene expression: Relationship to their effects on ethanol consumption, Neuropharmacology (2014), http://dx.doi.org/10.1016/j.neuropharm.2014.06.024 NP5538_proof ■ 18 July 2014 ■ 2/11 2 L.B. Ferguson et al. / Neuropharmacology xxx (2014) 1e11 1 et al., 2009). Although PPARb/d is the most abundant isotype in Briefly, two drinking bottles were continuously available to 66 2 brain, we know the least about its physiological function. individually-housed mice. One contained water and the other 15% 67 3 Recent evidence suggests that drugs targeting PPARs might be ethanol (v/v). Once stable ethanol consumptions were reached, we 68 4 effective in treating drug dependence (for review see Le Foll et al., measured ethanol intake after two days of saline injections and 69 5 2013). Retinoic acid is the only known RXR ligand and, interest- grouped mice (8 mice per group) to provide similar levels of 70 6 ingly, retinoic acid signaling was implicated in acute ethanol re- ethanol intake and preference. We administered PPAR agonists or 71 7 sponses in mice (Kerns et al., 2005). Also, pioglitazone and saline depending on their group assignment. We measured con- 72 8 rosiglitazone (PPARg agonists) reduced ethanol consumption in sumption (g/kg body weight/24 h) and calculated preference as the 73 9 rats (Stopponi et al., 2011, 2013). Intracerebroventricular adminis- amount of ethanol consumed divided by the total amount of fluids 74 10 tration of a PPARg antagonist blocked the reduction in ethanol consumed per day (a value >50% indicates a preference for 75 11 consumption, suggesting that this effect is mediated by central ethanol). Data are reported as the mean ± S.E.M. We used the 76 12 PPARg receptors (Stopponi et al., 2011). Additionally, pioglitazone statistics software program GraphPad Prism (Jandel Scientific, 77 13 and rosiglitazone reduced ethanol withdrawal severity and stress- Costa Madre, CA) to perform a two-way ANOVA with repeated 78 14 induced reinstatement of ethanol consumption in dependent rats measurements and Bonferroni post hoc test. 79 15 without altering food or saccharine self-administration (Stopponi 80 16 et al., 2011). Clofibrate, a PPARa agonist, prevented acquisition of 2.3. Drugs 81 17 nicotine dependence in naïve rats and monkeys and decreased 82 18 nicotine self-administration in nicotine-dependent rats and mon- The PPARa agonist fenofibrate (feno) (SigmaeAldrich, St. Louis, 83 19 keys (Panlilio et al., 2012). Thus, in addition to their known use- MO), pan-PPAR agonist bezafibrate (beza) (SigmaeAldrich, St. 84 20 fulness in cardiovascular disease and type II diabetes mellitus Louis, MO) and dual PPARaePPARg agonist tesaglitazar (tesa) 85 21 (Rosenson et al., 2012), PPAR agonists might be a potential treat- (Tocris Bioscience, Minneapolis, MN) were freshly prepared as 86 22 ment for alcohol dependence and other addictions. suspensions in saline with 4e5 drops of Tween-80 and injected in a 87 23 Based on the evidence for PPARs in regulating ethanol intake in volume of 0.05 ml/10 g of body weight. We administered (p.o.) 88 24 rodents, we assessed the effects of different PPAR agonists on PPAR agonist (150 mg/kg, 75 mg/kg, or 1.5 mg/kg of feno, beza, and 89 25 voluntary ethanol consumption in a mouse strain that consumes tesa, respectively) or saline for eight days. Doses of drugs and routes 90 26 large amounts of alcohol and examined their effects on gene of administration were based on published biological activity 91 27 expression in brain regions important for reducing ethanol con- in vivo. Tesa was only delivered for six days in the 2BC experiment 92 28 sumption. We show that fenofibrate and tesaglitazar decrease because its decreased effect on ethanol drinking behavior pla- 93 29 ethanol consumption in mice without affecting overall fluid intake, teaued at that time-point. 94 30 while bezafibrate did not change ethanol consumption. We 95 31 reasoned that the PPAR treatment-responsive transcript changes in 2.4. Tissue collection 96 32 the brain important for reducing ethanol consumption should 97 33 reflect treatment effectiveness. To the best of our knowledge, this is Twenty-four hours after administration of the last PPAR agonist 98 34 the first study to demonstrate brain gene expression changes for the microarray study, mice were euthanized by cervical dislo- 99 35 induced by PPAR agonists at doses that decrease alcohol con- cation.