Mol Neurobiol (2016) 53:6251–6269 DOI 10.1007/s12035-015-9527-1 N-Methyl, N-propynyl-2-phenylethylamine (MPPE), a Selegiline Analog, Attenuates MPTP-induced Dopaminergic Toxicity with Guaranteed Behavioral Safety: Involvement of Inhibitions of Mitochondrial Oxidative Burdens and p53 Gene-elicited Pro-apoptotic Change Eun-Joo Shin1 & Yunsung Nam 1 & Ji Won Lee1,2 & Phuong-Khue Thi Nguyen1 & Ji Eun Yoo1 & The-Vinh Tran1 & Ji Hoon Jeong3 & Choon-Gon Jang 4 & Young J. Oh 5 & Moussa B. H. Youdim6 & Phil Ho Lee7 & Toshitaka Nabeshima8,9 & Hyoung-Chun Kim1 Received: 1 September 2015 /Accepted: 5 November 2015 /Published online: 13 November 2015 # Springer Science+Business Media New York 2015 Abstract Selegiline is a monoamine oxidase-B (MAO-B) in- significantly attenuated MPTP-induced oxidative stress and hibitor with anti-Parkinsonian effects, but it is metabolized to MPPE-mediated antioxidant activity appeared to be more pro- amphetamines. Since another MAO-B inhibitor N-Methyl, N- nounced in mitochondrial-fraction than in cytosolic-fraction. propynyl-2-phenylethylamine (MPPE) is not metabolized to Because MPTP promoted mitochondrial p53 translocation amphetamines, we examined whether MPPE induces behav- and p53/Bcl-xL interaction, it was also examined whether ioral side effects and whether MPPE affects dopaminergic mitochondrial p53 inhibitor pifithrin-μ attenuates MPTP neu- toxicity induced by 1-methyl-4-phenyl-1,2,3,6- rotoxicity. MPPE, selegiline, or pifithrin-μ significantly atten- tetrahydropyridine (MPTP). Multiple doses of MPPE (2.5 uated mitochondrial p53/Bcl-xL interaction, impaired mito- and 5 mg/kg/day) did not show any significant locomotor chondrial transmembrane potential, cytosolic cytochrome c activity and conditioned place preference, whereas selegiline release, and cleaved caspase-3 in wild-type mice. Subsequent- (2.5 and 5 mg/kg/day) significantly increased these behavioral ly, these compounds significantly ameliorated MPTP-induced side effects. Treatment with MPPE resulted in significant at- motor impairments. Neuroprotective effects of MPPE ap- tenuations against decreases in mitochondrial complex I activ- peared to be more prominent than those of selegiline. MPPE ity, mitochondrial Mn-SOD activity, and expression induced or selegiline did not show any additional protective effects by MPTP in the striatum of mice. Consistently, MPPE against the attenuation by p53 gene knockout, suggesting that Electronic supplementary material The online version of this article (doi:10.1007/s12035-015-9527-1) contains supplementary material, which is available to authorized users. * Hyoung-Chun Kim 5 Department of Systems Biology, Yonsei University College of Life [email protected] Science and Biotechnology, Seoul 120-749, Republic of Korea 6 Eve Topf Centers of Excellence for Neurodegenerative Diseases Research, Faculty of Medicine, Technion-Israel Institute of 1 Neuropsychopharmacology and Toxicology Program, College of Technology, Haifa 31096, Israel Pharmacy, Kangwon National University, Chunchon 200-701, 7 Republic of Korea National Creative Research Initiative Center for Catalytic Organic Reactions, Department of Chemistry, Kangwon National University, 2 Hutecs Korea Pharm Co., Ltd., Osan 18111, Republic of Korea Chunchon 200-701, Republic of Korea 8 3 Department of Regional Pharmaceutical Care and Sciences, Graduate Department of Pharmacology, College of Medicine, Chung-Ang School of Pharmaceutical Sciences, Meijo University, University, Seoul 156-756, Republic of Korea Nagoya 468-8503, Japan 4 Department of Pharmacology, School of Pharmacy, Sungkyunkwan 9 NPO, Japanese Drug Organization of Appropriate Use and Research, University, Suwon 440-746, Republic of Korea Nagoya 468-8503, Japan 6252 Mol Neurobiol (2016) 53:6251–6269 p53 gene is a critical target for these compounds. Our results MPTP-induced dopaminergic neurotoxicity, it remains un- suggest that MPPE possesses anti-Parkinsonian potentials known whether p53 mitochondrial translocation is involved with guaranteed behavioral safety and that the underlying in its neurotoxic process. mechanism of MPPE requires inhibition of mitochondrial ox- In the present study, we examined the effect of MPPE on idative stress, mitochondrial translocation of p53, and pro- MPTP-induced neurotoxicity in comparison with selegiline. apoptotic process. In addition, it was also investigated whether pifithrin-μ,a mitochondrial p53 inhibitor, attenuates dopaminergic toxicity Keywords N-Methyl, N-propynyl-2-phenylethylamine . in this model. We found that MPPE significantly attenuates Mitochondria . Selegiline . Oxidative stress . Behavioral dopaminergic toxicity induced by MPTP, that protective ef- safety . p53 gene knockout mice . Pro-apoptosis . Parkinson’s fectsofMPPEappeartobemorepronouncedthanthoseof disease selegiline, and that MPPE, selegiline, or pifithrin-μ do not significantly affect MPTP toxicity in p53 gene knockout [p53 (−/−)] mice. Importantly, we observed that locomotor Introduction facilitation and conditioned place preference (CPP) are less pronounced in mice treated with MPPE than in mice treated Selegiline, one of the propargylamine-based monoamine oxi- with selegiline, suggesting that MPPE possesses behavioral dase (MAO)-B inhibitors, has long been used as a monother- safety. Furthermore, MPPE appeared to be more effective than apy in early Parkinson’s disease (PD) or as an adjunctive ther- selegiline against behavioral sensitization and CPP induced by apytolevodopainadvancedPD[1]. In addition, it was shown methamphetamine (MA). that selegiline attenuates cocaine self-administration in mice [2]. Furthermore, selegiline also attenuated subjective eupho- ria produced by cocaine in human [3, 4]. In spite of the ben- Materials and Methods eficial effects of selegiline, psychiatric and cardiovascular ad- verse effects have presented a problem with its use [5–7], and Synthesis of MPPE earlier studies suggested that the metabolism of selegiline to methamphetamine and amphetamine accounts for these ad- AsolutionofN-methylphenethylamine (676.0 mg, 5.0 mmol) verse psychotropic effects [8–10]. in water (10.0 mL) added 1.0 M NaOH (6.0 mL, 6.0 mmol) at N-Methyl, N-propynyl-2-phenylethylamine (MPPE) is a room temperature. The mixture was stirred for 20 min, and propargylamine-based MAO-B inhibitor [11], but it is not propargyl bromide (714.0 mg, 6.0 mmol) was added. After the metabolized to amphetamine derivatives [12]. Like other mixture was stirred at room temperature for 2.5 h, the reaction propargyl-containing MAO-B inhibitors possessing neuropro- mixture was quenched with water. The aqueous layer was tective properties, MPPE exerted neuroprotective effects in extracted with diethyl ether, and the combined layers were the animal model of thiamine deficient encephalopathy [11]. washed with water, brine, and dried over anhydrous MgSO4. However, it has not been reported whether MPPE provides The crude product was purified by column chromatography neuroprotection against any other neurotoxic conditions. In on silica gel (hexane:ethyl acetate, 1:1) to yield N-methyl-N- addition to the MAO-B inhibitory effect, several studies have propargyl-2-phenylethylamine (533.1 mg, 62 %); 1H NMR suggested that antioxidant and anti-apoptotic effects of pro- (CDCl3, 300 MHz) δ 7.28–7.14 (m, 5H), 3.37 (d, J=2.4, pargyl moiety are important for the neuroprotection provided 2H), 2.78–2.72 (m, 2H), 2.69–2.63 (m, 2H), 2.35 (s, 3H), 13 by propargylamine-based MAO-B inhibitors [13–18]. 2.21 (t, J=2.4, 1H); C NMR (CDCl3, 75 MHz) δ 140.2, p53, a tumor-suppressor gene, has been suggested to play a 128.7, 128.4, 126.1, 78.5, 73.3, 57.4, 45.6, 41.8, 34.3. key role in the apoptotic processes found in various neurode- A solution of N-methyl-N-propargyl-2-phenylethylamine generative conditions, including PD [19–22]. Elevated protein (87.0 mg, 0.5 mmol) in dichloromethane (1.25 mL) added level of p53 was reported in the postmortem brain of PD 1.0 M HCl (1.0 mL, 1.0 mmol) at 0 °C. The reaction was patients [23, 24] or 1-methyl-4-phenyl-1,2,3,6- allowedtowarmuptoroomtemperatureandstirredfor tetrahydropyridine (MPTP)-treated animal model of PD [25] 2.5 h. After evaporation of the solvents in vacuo, N-methyl- as well. In addition to the well-known transcriptional regula- N-propargyl-2-phenylethylamine∙HCl (91.0 mg, 87 %) was tion of pro-apoptotic and anti-apoptotic factors, p53 could produced. mediate apoptotic cell death via mitochondrial translocation [26–28]. Interaction of mitochondrial p53 with Bcl-2 or Bcl- Animals xL impairs mitochondrial membrane integrity and induces consequent cytosolic release of cytochrome c [26, 29–31]. All animals were treated in accordance with the National In- Although it has been reported that p53 gene knockout [32] stitutes of Health (NIH) Guide for the Humane Care and Use or pifithrin-α, a p53 transcription inhibitor [33], attenuates of Laboratory Animals (NIH Publication No. 85-23, 1985; Mol Neurobiol (2016) 53:6251–6269 6253 www.dels.nas.edu/ila). The present study was performed in tracking system (Noldus Information Technology, accordance with the Institute for Laboratory Research Wagenin, The Netherlands). Eight test boxes (40×40× (ILAR) guidelines for the care and use of laboratory animals. 30 cm high) were operated simultaneously by an IBM Mice were maintained under a 12-h light:12-h dark cycle and computer. Animals were studied individually during fed ad libitum. Breeding pairs of p53 gene heterozygous [p53 measurement of locomotion in each test box, where they (+/−)]