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Mol Neurobiol (2016) 53:6251–6269 DOI 10.1007/s12035-015-9527-1

N-Methyl, N-propynyl-2-phenylethylamine (MPPE), a Analog, Attenuates MPTP-induced 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 -B (MAO-B) in- significantly attenuated MPTP-induced and hibitor with anti-Parkinsonian effects, but it is metabolized to MPPE-mediated antioxidant activity appeared to be more pro- . 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 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 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 , 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 , 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 and CPP induced by apytolevodopainadvancedPD[1]. In addition, it was shown (MA). that selegiline attenuates self-administration in mice [2]. Furthermore, selegiline also attenuated subjective eupho- ria produced by cocaine in [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 of selegiline to methamphetamine and 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 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 (+/−)] mice with C57BL/6J background were obtained from were adapted for 10 min before starting the recording. RIKEN BioResource Center (Tsukuba, Japan) [34]. p53 (−/−) Data were collected and analyzed between 09:00 and mice were maintained as heterozygous breeding pairs, and 17:00 h. To evaluate whether selegiline or MPPE in- neonates were genotyped by polymerase chain reaction duces behavioral side effects, mice received daily injec- (PCR) of DNA extracted from the tail, according to the infor- tion of selegiline (2.5 or 5.0 mg/kg, i.p.) or MPPE (2.5 mation provided by the RIKEN BioResource Center. Addi- or 5 mg/kg, i.p.) for seven consecutive days. MA (0.5 tional details regarding the gene characterization were given or 1.0 mg/kg, i.p.) was used as a control drug. Imme- in the Supplementary Information. diately after each injection, mice were introduced into the test box. The experimental schedule was shown in Conditioned Place Preference Supplementary Fig. 1b. To examine the effect of selegiline or MPPE on the behavioral sensitization in- CPP was examined as described previously [35–37]. The CPP duced by MA, 40 min after the first (day 4), fourth apparatus was described in the Supplementary Information. (day 10), and seventh (day 16) injection of MA (i.e., As a control, mice received an i.p. injection of saline just after the conditioning in the white compartment of CPP before entering the white or black compartment. On days 1 apparatus), locomotor activity was analyzed in a 30-min and 2, the mice were pre-exposed to the test apparatus for monitoring period. After a withdrawal period for 6 days 5 min. The guillotine doors were raised, and the mice were (day 22), mice received MA (1 mg/kg, i.p.), and loco- allowed to move freely between the two compartments. On motor activity was measured for 30 min. Mice received day 3, the that each mouse spent in each compartment selegiline (0.25 or 0.5 mg/kg, i.p.) or MPPE (0.25 or was recorded for 15 min. On days 4, 6, 8, 10, 12, and 14, the 0.5 mg/kg, i.p.) every other day (i.e., day 18 and 20) mice were injected with drugs before being confined to the during the MA withdrawal period and 30 min before white compartment, the non-preferred side, for 40 min. On MA treatment on day 22. The experimental design days 5, 7, 9, 11, and 13, the mice were injected with saline was shown in Supplementary Fig. 1c. before being confined to the black compartment, the preferred side, for 40 min. On day 15, the guillotine doors were raised. Drug Treatment The mice were initially placed in the tunnel and the time spent by mice in each compartment was recorded for 15 min. The MPTP (15 mg/kg, s.c.) was dissolved in sterile saline (1 ml/ scores were calculated from the differences in the time spent in kg) immediately before use. MPPE (0.25 mg/kg/day, i.p.), the white compartment between post-test and pre-test periods. selegiline (0.25 mg/kg/day, i.p.; Tocris Bioscience, Bristol, Data were analyzed between 09:00 and 17:00 h. UK), or pifithrin-μ (2 mg/kg/day, i.p.; Sigma–Aldrich, St. To evaluate whether selegiline or MPPE induces behavior- Louis, MO, USA) were dissolved in dimethyl sulfoxide al side effects, selegiline (2.5 or 5.0 mg/kg, i.p.) or MPPE (2.5 (DMSO) as a stock solution and then stored at 4 °C. These or 5 mg/kg, i.p.) dissolved in saline was administered imme- stock solutions were diluted in sterile saline (1 ml/kg) imme- diately before mice were placed in the white compartment. diately before use, and the final DMSO concentration was 5 % Methamphetamine (MA; 0.5 or 1.0 mg/kg, i.p.) was used as (v/v). The dose of selegiline or pifithrin- μ was determined a control drug. The experimental design was shown in Sup- based on previous studies [39, 40]. plementary Fig. 1a. To examine the effect of selegiline or Mice received MPTP once daily for seven consecutive MPPE on CPP induced by MA, selegiline (0.25 or 0.5 mg/kg, days. MPPE or selegiline was administered once a day for i.p.) or MPPE (0.25 or 0.5 mg/kg, i.p.) was administered 28 days before MPTP treatment (day 1–28). During MPTP 30 min before each MA treatment. MA (1 mg/kg, i.p.) dis- treatment (day 29–35), MPPE, selegiline, or pifithrin-μ were solved in saline was administered immediately before mice administered 2 h prior to each MPTP treatment. One day after were placed in the white compartment. The experimental de- the final MPTP treatment, behavioral evaluation was per- sign was shown in Supplementary Fig. 1c. formed, and then the mice were sacrificed. To examine mito- chondrial translocation of p53, cytosolic cytochrome c release, Locomotor Activity and Behavioral Sensitization and subsequent caspase-3 cleavage, wild-type mice were treated with MPTP as described above and sacrificed 1, 6, Locomotor activity was measured for 30 min as de- 12, and 24 h after the final MPTP treatment. The experimental scribed previously [36, 38] using an automated video- design was shown in Supplementary Fig. 1d, e. 6254 Mol Neurobiol (2016) 53:6251–6269

Fig. 1 Changes in locomotor activity and conditioned place preference after repeated treatment with MPPE or selegiline and the effect of MPPE or selegiline on the locomotor facilitation and conditioned place preference induced by MA. a Chemical of selegiline and MPPE. b–d Changes in locomotor activity (b)and representative locomotor patterns (c) and conditioned place preference (d)afterrepeated treatment with MPPE (2.5 or 5.0 mg/kg, i.p.) or selegiline (2.5 or 5.0 mg/kg, i.p.). Methamphetamine (MA; 0.5 or 1.0 mg/kg, i.p.) was used as a control drug. Representative locomotor patterns show the track of mouse movement during the fifth 5 min (i.e., 30–35 min after the last drug injection) of locomotor recording after repeated treatment for seven consecutive days with each drug (c). Note the typical locomotor tracing patterns (increase in marginal activity [70]) after repeated treatment with MA or selegiline (c). e–f Effect of MPPE (0.25 or 0.5 mg/kg, i.p.) or selegiline (0.25 or 0.5 mg/kg, i.p.) on the hyperlocomotor activity (e), behavioral sensitization (f), and conditioned place preference (g) induced by MA (1.0 mg/kg, i.p.). Sal Saline. Each value is the mean ± S.E.M. of 6–10 animals. *P<0.01 vs. Sal or Sal + Sal; &P<0.01 vs. MA (0.5 mg/kg); #P<0.05, ##P<0.01 vs. MA (1.0 mg/kg) or Sal + MA (1.0 mg/kg); §P<0.01 vs. Sel (2.5 mg/kg); †P<0.01 vs. Sel (5.0 mg/kg) (b, one-way ANOVA for repeated measures; d,one- way ANOVA; e,two-way ANOVAfor repeated measures; f, two-way ANOVA. Post hoc Fisher’s LSD pairwise comparisons test followed)RL Mol Neurobiol (2016) 53:6251–6269 6255

Behavioral Assessments After MPTP Treatment Monoamine Oxidase-B Activity

Locomotor activity was measured for 30 min as described MAO-B activity was examined as described previously [39, above. Rota-rod test was performed as described previously 58]. Striatal tissues were homogenized in 0.1 M potassium [41]. The apparatus (model 7650; Ugo Basile, Comerio, Va- phosphate buffer (pH 7.4), and 250 μLofhomogenatewas rese, Italy) consisted of a platform and a rotating rod with added to 1.25 mL of 0.5 mM kynuramine dissolved in potas- a non-slip surface. The rod was placed at a height of 15 cm sium phosphate buffer. Then, 250 μLof1μMclorgylinewas above the base. The rod, 30 cm in length, was divided into added to inhibit MAO-A activity. The reaction mixture was equal sections by six opaque disks so that the animals would incubated at 37 °C for 30 min, and the reaction was terminated not be distracted by one another. To assess motor perfor- by adding ice-cold 0.4 N perchloric . After centrifugation mance, the mice were first trained on the apparatus for 2 min at 7500×g for 5 min, an equal volume of 0.1 N NaOH was at a constant rate of 4 rpm. The test was performed 30 min added to the supernatant. Fluorescence intensity was recorded after training and an accelerating paradigm was applied, which with excitation and emission wavelengths of 315 and 350 nm, is starting from a rate of 4 rpm to maximal speed of 40 rpm. respectively. MAO-B activity was calculated using a standard The rotation speed was then kept constant at 40 rpm. The curve of 4-hydroxyquinoline, the resultant product of the re- latency to fall was measured with a maximal cutoff time of action. The result was expressed as nanomoles 4- 300 s. hydroxyquinoline formed per hour per milligram protein.

Brain Dissection Complex I Activity

Mice were sacrificed by decapitation, and were rap- Complex I activity was examined as described previously idly removed and placed on an ice-cold brain matrix (ASI [59]. Isolated mitochondrial sample (μL) was added to the Instruments, Warren, MI, USA). Coronal slices containing reaction mixture containing 25 mM potassium phosphate striatum or were made at 1.1 to −0.1 mm buffer (pH 7.8), 3.5 mg/mL bovine serum albumin, or at −3.0 to −3.5 mm from bregma using chilled razor 60 μM 2,6-dichloroindophenol, 70 μM decylubiquinone, blades, according to the atlas of Franklin and Paxinos and 1 μM antimycin A, and reaction mixture was incu- [42]. Dorsal striatum and substantia nigra were punched bated at 37 °C for 3 min. After adding NADH to the final bilaterally with a sample corer (2 mm inner diameter for concentration of 0.2 mM, the absorbance was recorded at striatum, 1 mm inner diameter for substantia nigra; Fine 60-s intervals for 4 min at 600 nm. Then, rotenone was Science Tools Inc., Vancouver, Canada) and a plunger [43, added to the final concentration of 1 μM, and the absor- 44]. Dissected tissues were frozen in liquid nitrogen and bance was recorded again at 60-s intervals for 4 min at stored at −80 °C until use. 600 nm. One unit of complex I activity was defined as Since it may be not available for preparing an enough 1 μmol 2,6-dichloroindophenol reduced per minute, and it amount of mitochondrial fraction from substantia nigra, we was calculated based on the coefficient for 2,6- have mainly employed striatal tissue to investigate neuro- dichloroindophenol of 19.1 mM/cm. The result was chemical changes in the mitochondrial fraction in this study expressed as a percentage of the control group. [45]. Importantly, earlier reports demonstrated that 4- phenylpyridinium ion (MPP+), a toxic of MPTP, is more likely to be concentrated and particularly toxic in Western Blot striatal mitochondria than in nigral mitochondria [46–53]. The Western blot assays were performed as we described pre- viously [41, 55]. Striatal tissues were homogenized in lysis Preparation of Cytosolic and Mitochondrial Fraction buffer, containing 200 mM HCl (pH 6.8), 1 % SDS, for Western Blot and Analyses 5 mM ethylene glycol-bis(2-aminoethyl ether)-N,N,N’,N’- tetraacetic acid (EGTA), 5 mM ethylenediaminetetraacetic ac- The cytosolic and mitochondrial fractions were prepared as id (EDTA), 10 % glycerol, 1× phosphatase inhibitor cocktail I we described previously for Western blot analysis and the (Sigma-Aldrich), and 1× protease inhibitor cocktail (Sigma- neurochemical assay [41, 54, 55]. Mitochondria were isolated Aldrich). Lysate was centrifuged at 12,000×g for 30 min, and as described previously [56] with minor modifications [41, supernatant fraction was used for Western blot analysis. Mi- 57] for measurements of mitochondrial transmembrane poten- tochondrial and cytosolic fractions were prepared as described tial. Details of the procedure were provided in the Supplemen- above. Additional details regarding the procedure and anti- tary Information. bodies were provided in the Supplementary Information. 6256 Mol Neurobiol (2016) 53:6251–6269

Immunoprecipitation Fig. 2 Effect of MPPE or selegiline on changes in MAO-B activity,„ mitochondrial complex I activity, mitochondrial Mn-SOD (SOD-2) activ- Immunoprecipitation was performed as we described previ- ity and expression, and UCP-2 mRNA expression induced by MPTP. a, b MAO-B activity (a) and mitochondrial complex I (b) activity in the stri- ously [60] using protein G-sepharose (GE Healthcare, atum. c SOD-2 activity in the striatum. d, e Protein expression (d)and Piscataway, NJ, USA). Details regarding the procedure and immunoreactivity (e) of SOD-2 in the striatum. f SOD-2 activity in the antibodies were provided in the Supplementary Information. substantia nigra. g SOD-2 immunoreactivity in the substantia nigra. h UCP-2 mRNA expression in the striatum. Sal Saline, Sel Selegiline (0.25 mg/kg, i.p.), MPPE MPPE (0.25 mg/kg, i.p.), Veh Vehicle (5 % RT-PCR DMSO). Each value is the mean ± S.E.M. of six animals. *P<0.05, **P<0.01 vs. Veh + Sal; #P<0.05, ##P<0.01 vs. Veh + MPTP; Expression of uncoupling protein-2 (UCP-2) was assessed as &P<0.05 vs. Sel + MPTP (Two-way ANOVAwas followed by Fisher’s we described [61] using semi-quantitative RT–PCR to analyze LSD pairwise comparisons) messenger RNA (mRNA) level. Total RNA was isolated from striatal tissues using an RNeasy Mini Kit (Qiagen, Valencia, [66]. DNPH-labeled protein was detected by spectrophoto- CA, USA). Details regarding the primer sequences and pro- metric [41, 66]orslotblot[67] analysis. Details of the proce- cedure were given in the Supplementary Information. dure were provided in the Supplementary Information.

Immunocytochemistry Determination of 4-hydroxynonenal

Immunocytochemistry was performed as described previous- The amount of peroxidation was determined by measur- ly [41]. Mice were perfused transcardially with 50 mL of ice- ing the level of 4-hydroxynonenal (HNE) using the TM cold PBS (10 mL/10 g body weight) followed by 4 % para- OxiSelect HNE adduct ELISA kit (Cell Biolabs, Inc., San ’ formaldehyde (20 mL/10 g body weight). Brains were re- Diego, CA, USA) according to the manufacturer smanual. moved and stored in 4 % paraformaldehyde overnight. Sec- Details of the procedure were provided in the Supplementary tions were subjected to immunostaining with primary anti- Information. body against SOD-2 [1:1000; kindly gifted by Dr. Kanefusa Kato at Aichi Prefectural Colony, Kasugai, Japan [62–64]or Determination of Mitochondrial ROS hydroxlase (TH) (1:500; Chemicon (EMD Millipore)). Details regarding immunocytochemistry and Determination of the formation of ROS was performed as quantitative analysis were presented in the Supplementary described previously [60, 68]. Mitochondrial fraction was in- information. cubated with 5 μM2’,7’-dichlorofluorescein diacetate (DCFH-DA, Molecular Probes, Eugene, OR, USA) for Stereological Analysis 15 min at 37 °C. The fluorescent intensity due to the ROS was measured at an excitation wavelength of 488 nm and Stereological analysis of the number of TH-immunoreactive emission wavelength of 528 nm using a fluorescent micro- cells in the substantia nigra (SN) was per- plate reader (Molecular Devices Inc.). formed as described previously [41, 57, 65]. Details of the procedure were provided in the Supplementary Information. Mitochondrial Transmembrane Potential

Superoxide Dismutase (SOD) Activity Mitochondrial transmembrane potential was measured as de- scribed previously [41, 56, 57] using 5,5′,6,6′-tetrachloro-1,1′, Striatal tissues were homogenized in 50 mM potassium phos- 3,3′-tetraethylbenzimidazolycarbocyanine iodide dye (JC-1; phate buffer (pH 7.8) and centrifuged at 13,000×g for 20 min. Molecular Probes), which exists as a green fluorescent mono- The resulting supernatant was used to measure SOD activity. mer at low membrane potential, but reversibly forms red fluo- SOD activity was determined on the basis of inhibition of rescent BJ-aggregates^ at polarized mitochondrial potentials. superoxide-dependent reactions as described previously [41]. Details of the procedure were provided in the Supplementary Details of the procedure were provided in the Supplementary Information. Information. Measurement of Level Determination of Protein Carbonyl The dopamine level was determined by HPLC coupled with The extent of protein oxidation was assessed by measuring the an electrochemical detector as described previously [41, 57, content of protein carbonyl group, which was determined with 69]. Details of the procedure were provided in the Supplemen- the 2,4-dinitrophenylhydrazine (DNPH)-labeling procedure tary Information. Mol Neurobiol (2016) 53:6251–6269 6257 6258 Mol Neurobiol (2016) 53:6251–6269

Statistical Analyses between MA and day (Supplementary Table 1). On the behav- ioral sensitization and conditioned place preference, two-way Data were analyzed using IBM SPSS ver. 21.0 (IBM, Chica- ANOVA showed significant effects of MA and pretreatment go, IL, USA). One-way analysis of variance (ANOVA) (treat- (Supplementary Table 1). A post hoc test revealed that pre- ment), two-way ANOVA (pretreatment × MA or MPTP), or treatment with selegiline (0.25 or 0.5 mg/kg, i.p.) or MPPE three-way ANOVA (p53 gene knockout × pretreatment × (0.25 or 0.5 mg/kg, i.p.) significantly attenuated behavioral MPTP) were employed for the statistical analyses. A sensitization (0.25 or 0.5 mg/kg of selegiline + MA vs. saline repeated-measures ANOVA (between-subjects factors: pre- +MA,P<0.05 or P<0.01, respectively; 0.25 or 0.5 mg/kg of treatment × MA; within-subjects factor: time) was conducted MPPE + MAvs. saline + MA, P<0.01) and conditioned place for the behavioral sensitization. Post hoc Fisher’s least signif- preference (0.5 mg/kg of selegiline + MA vs. saline + MA, icant difference pairwise comparisons tests were then con- P<0.05; 0.5 mg/kg of MPPE + MAvs. saline + MA, P<0.01) ducted. P values<0.05 were considered to be significant. induced by MA (1 mg/kg, i.p.). These attenuations appeared to be more pronounced in MPPE than those in selegiline (Fig. 1e–g). Results MPPE Attenuates MPTP-Induced Changes in MAO-B Treatment With MPPE did not Significantly Induce and Complex I Activity in the Mitochondria Psychotropic Locomotor Pattern and Conditioned Place of the Striatum: Comparison With Selegiline Preference: Comparison With Selegiline Two-way ANOVA revealed significant effects of MPTP In order to examine whether selegiline (Fig. 1a) or MPPE (MAO-B and complex I activities) and pretreatment (MAO- (Fig. 1a) induces psychotropic effects, we evaluated locomo- B activity) and a significant interaction between MPTP and tor activity, locomotor pattern, and conditioned place prefer- pretreatment (complex I activity) (Supplementary Table 2). A ence (MA was used as a control drug). One-way ANOVA for post hoc test indicated that MPTP treatment significantly in- repeated measures showed significant effects of treatment creased (P<0.01 vs. vehicle + saline) MAO-B activity. MPPE (between-subject factor) and day (within-subject factor), and (P<0.05 vs. vehicle + MPTP) or selegiline (P<0.01 vs. ve- a significant interaction between treatment and day on the hicle + MPTP) significantly attenuated this increment. MPPE locomotor activity (Supplementary Table 1). On the condi- or selegiline also significantly decreased (P<0.05 vs. vehicle tioned place preference, one-way ANOVA indicated signifi- + saline) MAO-B activity (Fig. 2a). cant effect of treatment (Supplementary Table 1). A post hoc MPPE or selegiline did not significantly alter complex I test revealed that repeated treatment with selegiline (2.5 or activity. Treatment with MPTP resulted in a significant reduc- 5.0 mg/kg/day, i.p.) resulted in significant increases in loco- tion in complex I activity (P<0.01 vs. vehicle + saline). This motor activity (2.5 or 5.0 mg/kg of selegiline vs. saline, reduction in complex I activity was significantly attenuated by P<0.05) with psychotropic locomotor pattern (i.e., marginal MPPE (P<0.01 vs. vehicle + MPTP) or selegiline (P<0.05 activity [70]) and conditioned place preference (2.5 or vs. vehicle + MPTP). This attenuation appeared to be more 5.0 mg/kg of selegiline vs. saline, P<0.05 or P<0.01, respec- evidentinMPPEthanselegiline(Fig.2b). tively), although these increases were less pronounced than the MA case. However, MPPE (2.5 or 5.0 mg/kg/day, i.p.) MPPE upRegulates Mitochondrial Mn-SOD (SOD-2) did not induce significant change in locomotor activity, loco- Activity, Protein Expression, motor pattern, or conditioned place preference (Fig. 1b–d). and Immunoreactivity, and UCP-2 mRNA Expression Induced by MPTP in the Striatum: Comparison With Pretreatment With MPPE Attenuates MA-Induced Selegiline Behavioral Sensitization and Conditioned Place Preference: Comparison With Selegiline We, next, examined SOD-2 activity, protein expression, and immunoreactivity after MPTP treatment (Fig. –e). Two-way As it was reported that selegiline attenuates behavioral ANOVAshowed significant effects of MPTP (SOD-2 activity, (psychotropic) effects mediated by abusive drugs [2–4, 71], expression, and immunoreactivity) and pretreatment (SOD-2 we examined whether selegiline analog MPPE is also effec- activity, expression, and immunoreactivity), and a significant tive in response to the MA-induced behavioral sensitization interaction between MPTP and pretreatment (SOD-2 activity and conditioned place preference in the present study. On the and immunoreactivity) in the striatum (Supplementary locomotor activity, two-way ANOVA for repeated measures Table 2). A post hoc test revealed that MPTP treatment sig- indicated significant effects of MA (between-subject factor) nificantly decreased (P<0.01 vs. vehicle + saline) SOD-2- and day (within-subject factor) and a significant interaction immunoreactivity. Treatment with MPPE (P<0.01 vs. vehicle Mol Neurobiol (2016) 53:6251–6269 6259

+ MPTP) or selegiline (P<0.05 vs. vehicle + MPTP) signifi- selegiline (P<0.05 vs. vehicle + MPTP) (Fig. 2c). Changes in cantly attenuated this reduction in SOD-2-immunoreactivity SOD-2 activity and immunoreactivity in the striatum were induced by MPTP (Fig. 2e). This SOD-2-immunoreactivity comparable to those in the substantia nigra (Fig. 2f, g). was consistent with the result obtained from Western blot It has been reported that uncoupling protein-2 (UCP-2) is analysis (Fig. 2d). MPTP-induced decrease in SOD-2 activity important for protecting dopaminergic against was attenuated by MPPE (P<0.01 vs. vehicle + MPTP) or MPTP-induced mitochondrial oxidative stress and

Fig. 3 Effect of MPPE or selegiline on MPTP-induced oxidative stress in the cytosolic and mitochondrial fractions of the striatum of mice. a–d Effect on cytosolic and mitochondrial formations (a, b) and expressions (c, d) of protein carbonyl induced by MPTP. e, f Effect on cytosolic and mitochondrial formations of 4-hydroxynonenal (HNE) induced by MPTP. g, h Effect on cytosolic and mitochondrial formations of reactive oxygen species (ROS) induced by MPTP. Sal Saline, Sel Selegiline (0.25 mg/kg, i.p.), MPPE MPPE (0.25 mg/kg, i.p.), Veh Vehicle (5 % DMSO). Each value is the mean ± S.E.M. of five animals. *P<0.01 vs. Veh + Sal; #P<0.05, ##P<0.01 vs. Veh + MPTP; &P<0.05 vs. Sel + MPTP (Two- way ANOVAwas followed by Fisher’s LSD pairwise comparisons) 6260 Mol Neurobiol (2016) 53:6251–6269 neurodegeneration [72, 73]. Thus, we evaluated UCP-2 SOD-2 activity, SOD-2 expression, SOD-2-immunoreac- expression in the striatum after MPTP treatment. Two- tivity, and UCP-2 mRNA expression induced by MPTP way ANOVA indicated significant effects of MPTP and (Fig. 2c–h). pretreatment and a significant interaction between MPTP and pretreatment (Supplementary Table 2). A post hoc MPPE Attenuates MPTP-Induced Oxidative Stress test showed that UCP-2 mRNA expression was signifi- (Mitochondria > ): Comparison With Selegiline cantly induced (P<0.05 vs. vehicle + saline) after the last MPTP treatment, and this induction was more sig- Next, we examined the effect of MPPE on the MPTP- nificantly potentiated by MPPE (P<0.01 vs. vehicle + induced oxidative stress in cytosolic and mitochondrial saline) or selegiline (P<0.05 vs. vehicle + saline) fractions. In the cytosolic fraction, two-way ANOVA (Fig. 2h). The effect of MPPE was more evident showed significant effects of MPTP (protein carbonyl (P<0.05) than that of selegiline against alterations in level and expression, 4-hydroxynonenal (HNE) level,

Fig. 4 Changes in the mitochondrial translocation of p53, mitochondrial p53/Bcl-xL interaction, cytosolic cytochrome c release, and cleaved caspase-3 expression 1, 6, 12, and 24 h after the final treatment with MPTP in the striatum of wild-type mice. a Representative bands from each time-point. b Quantification of p53 in the mitochondrial fraction, cytosolic fraction, and whole lysate. c Quantification of p53/ Bcl-xL interaction in the mitochondrial fraction. d Quantification of cytochrome c in the cytosolic fraction. e Quantification of cleaved caspase-3 in the whole lysate. Sal Saline. Each value is the mean ± S.E.M. of six animals. *P<0.05, **P<0.01 vs. Sal (One-way ANOVAwas followed by Fish- er’s LSD pairwise comparisons) Mol Neurobiol (2016) 53:6251–6269 6261 and reactive oxygen species (ROS) level) and pretreat- carbonyl level (the formation (cytosolic or mitochondrial frac- ment (protein carbonyl expression and ROS level) and tion: P<0.05 or P<0.01 vs. vehicle + MPTP, respectively) a significant interaction between MPTP and pretreat- and the expression (P<0.01 vs. vehicle + MPTP in both frac- ment (protein carbonyl expression and ROS level). In tions)), 4-HNE level (cytosolic or mitochondrial fraction: the mitochondrial fraction, significant effects of MPTP P<0.05 or P<0.01 vs. vehicle + MPTP, respectively), or and pretreatment and a significant interaction between ROS formation (P<0.01 vs. vehicle + MPTP in both frac- MPTP and pretreatment were shown on the protein tions) induced by MPTP. MPPE was more effective carbonyl level and expression, HNE level, and ROS (P<0.05 vs. selegiline + MPTP) than selegiline in attenuating level (Supplementary Table 3). A post hoc test indicated mitochondrial formations of protein carbonyl, HNE, and ROS that treatment with MPPE resulted in decreases in the protein (Fig. 3).

Fig. 5 Effect of pifithrin-μ, selgiline, or MPPE on the mitochondrial translocation of p53, mitochondrial p53/Bcl-xL interaction, mitochondrial transmembrane potential, cytosolic cytochrome c release, and cleaved caspase-3 expression 6 h after the final treatment with MPTP in the striatum of wild-type (WT) or p53 gene knockout [p53(−/−)] mice. a Representative bands of mitochondrial and cytosolic p53 and mitochondrial p53/Bcl-xL interaction. b Effect on mitochondrial translocation of p53 induced by MPTP. c Effect on mitochondrial p53/Bcl-xL interaction induced by MPTP. d Effect on decrease in mitochondrial transmembrane potential induced by MPTP. e Effect on cytosolic cytochrome c release induced by MPTP. f Effect on cleaved caspase-3 induced by MPTP. Sal Saline, PFTμ Pifithrin-μ (2 mg/kg, i.p.), Sel Selegiline (0.25 mg/kg, i.p.), MPPE MPPE (0.25 mg/kg, i.p.), Veh Vehicle (5 % DMSO). Each value is the mean ± S.E.M. of 5–7 animals. *P<0.01 vs. corre- sponding Veh + Sal; #P<0.05, ##P<0.01 vs. WT mice treated with Veh + MPTP (two-way ANOVA (b, c) or three-way ANOVA (d–f) was followed by Fisher’s LSD pairwise comparisons) 6262 Mol Neurobiol (2016) 53:6251–6269

Mitochondrial Translocation of p53, p53/Bcl-xL extended our finding by examining the effect of MPPE on Interaction, Cytosolic Release of Cytochrome c, changes in mitochondrial transmembrane potential induced and Cleavage of Caspase-3 Induced by MPTP by MPTP in WT- and p53 (−/−)-mice. Three-way ANOVA showed significant effects of MPTP and pretreatment and a As previous reports demonstrated that binding of mitochon- significant interaction between p53 gene knockout and MPTP drial p53 with Bcl-xL can induce cytochrome c release and on the mitochondrial transmembrane potential, cytosolic cy- consequent pro-apoptotic changes [26, 29–31], we examined tochrome c release, and cleaved caspase-3 6 h after the final whether MPTP activates mitochondrial translocation of p53 MPTP treatment. p53 gene knockout also exerted significant and the interaction between mitochondrial p53 and Bcl-xL. effect on the cytosolic cytochrome c release (Supplementary One-way ANOVA indicated a significant effect of time on Table 5). A post hoc test indicated that mitochondrial trans- the mitochondrial and cytosolic p53 expression, mitochondri- membrane potential was significantly reduced (P<0.01) 6 h al p53/Bcl-xL interaction, cytosolic cytochrome c release, and after the final MPTP treatment in WT mice, and this reduction cleaved caspase-3 expression (Supplementary Table 4). A was significantly attenuated by MPPE (P<0.01), selegiline post hoc test revealed that mitochondrial p53 translocation (P<0.05), pifithrin-μ (P<0.05), or p53 gene depletion (Fig. 4a, b) or p53/Bcl-xL interaction (Fig. 4a, c) was signif- (P<0.01) (Fig. 5d). Consistently, MPTP-induced cytosolic icantly increased (1 and 6 h, P<0.01; 12 h, P<0.05) after the cytochrome c release (P<0.01) and subsequent increase in last treatment of MPTP, and these changes were most evident cleaved caspase-3 (P<0.01) were significantly attenuated by 6 h later. Consistently, cytosolic cytochrome c release (1 and MPPE (P<0.01), selegiline (P<0.05), pifithrin-μ (P<0.01), 12 h, P<0.05; 6 h, P<0.01) and caspase-3 cleavage (1 and or p53 gene depletion (P<0.01) (Fig. 5e, f). However, MPPE 12 h, P<0.05; 6 h, P<0.01) were most pronounced 6 h after or selegiline did not show any additional protective effects the final MPTP treatment (Fig. 4d–e), respectively. Total p53 against p53 gene knockout-mediated attenuation, suggesting protein expression in whole lysate was not significantly al- that p53 is a critical target for either compound (Fig. 5d–f). tered in the entire range of time-course, suggesting that protein expression may not be changed in these early time-points of our experimental condition (Fig. 4). MPPE Attenuates MPTP-Induced Decreases in (TH) Expression and Dopamine Level, MPPE Attenuates MPTP-Induced Mitochondrial and Increase in Dopamine Turnover Rate: Comparison Translocation of p53 and p53/Bcl-xL Interaction: With Selegiline, Pifithrin-μ, or Genetic Inhibition of p53 Comparison With Selegiline or Pifithrin-μ Next, we examined the effect of MPPE on the MPTP-induced Since MPTP-induced mitochondrial p53 translocation and dopaminergic toxicity. Three-way ANOVA showed signifi- p53/Bcl-xL interaction were most pronounced 6 h later cant effects of MPTP and pretreatment (nigrostriatal TH- (Fig. 4a–c), we focused on this time-point to examine the immunoreactivities and striatal TH expression) and a signifi- effect of MPPE or selegiline. Additionally, the effect of cant interaction between p53 gene knockout and MPTP pifithrin-μ, a specific inhibitor of p53/Bcl-xL interaction, (nigrostriatal TH-immunoreactivities and striatal TH expres- was examined. Two-way ANOVA showed significant effects sion) (Supplementary Table 6). As shown in Fig. 6a–c, a post of MPTP and pretreatment on the mitochondrial and cytosolic hoc test indicated that striatal TH-immunoreactivity (TH-IR) p53 expression and mitochondrial p53/Bcl-xL interaction and TH expression were significantly decreased (P<0.01) in (Supplementary Table 5). A post hoc test revealed that MPTP-induced mitochondrial p53 translocation and p53/ Bcl-xL interaction were significantly attenuated by MPPE (P<0.01), selegiline (P<0.05), or pifithrin-μ (P<0.01), and Fig. 6 Effect of pifithrin-μ, selgiline, or MPPE on MPTP-induced„ dopaminergic loss in wild-type (WT) and p53 gene knockout the effect of MPPE was more pronounced than that of [p53(−/−)] mice. a–c Effect on decreases in TH-immunoreactivity selegiline (Fig. 5a–c). (a, b) and TH expression (c) induced by MPTP in the striatum. d, e Effect on decreases in TH-immunoreactivity induced by MPTP in the MPPE Attenuates MPTP-Induced Changes substantia nigra. Dashed lines indicate substantia nigra pars compacta for quantitative analysis. f, g Effect on decrease in dopamine level (f) in Mitochondrial Transmembrane Potential, Cytosolic and increase in dopamine turnover rate (g) induced by MPTP in the Release of Cytochrome c, and Cleavage of Caspase-3: striatum. Sal Saline, PFTμ Pifithrin-μ (2 mg/kg, i.p.), Sel Selegiline Comparison With Selegiline, Pifithrin-μ,orGenetic (0.25 mg/kg, i.p.), MPPE MPPE (0.25 mg/kg, i.p.), Veh Vehicle (5 % * Inhibition of p53 DMSO). Each value is the mean ± S.E.M. of five animals. P<0.05, **P<0.01 vs. corresponding Veh + Sal; #P<0.05, ##P<0.01 vs. WT mice treated with Veh + MPTP; &P<0.05 vs. WT mice treated with As MPTP-induced mitochondrial p53 translocation and p53/ Sel + MPTP (three-way ANOVA was followed by Fisher’sLSD Bcl-xL interaction were significantly attenuated by MPPE, we pairwise comparisons). Scale bar=500 μm Mol Neurobiol (2016) 53:6251–6269 6263 6264 Mol Neurobiol (2016) 53:6251–6269 the striatum of WT mice. MPTP-induced decreases in TH-IR MPPE-mediated antioxidant efficacy in mitochondrial frac- and TH expression were significantly attenuated by MPPE tion was more pronounced than that in cytosolic fraction. (P<0.01), selegiline (P<0.05), pifithrin-μ (TH-IR, P<0.01; MPPE treatment positively modulated MPTP-induced neuro- TH expression, P<0.05) or p53 gene depletion (TH-IR, toxic alterations in mitochondrial Mn-SOD activity and ex- P<0.01; TH expression, P<0.05). The results from the stria- pression, mitochondrial translocation of p53, mitochondrial tum are comparable to those from the nigral area (Fig. 6d, e). transmembrane potential, cytosolic cytochrome c release, Consistent with the result of TH, three-way ANOVA cleaved caspase-3, and dopaminergic system. Neuroprotec- showed significant effects of p53 gene knockout (dopamine tion offered by MPPE appeared to be more prominent than level in the striatum), MPTP (dopamine level and dopamine that offered by selegiline. turnover rate in the striatum), and pretreatment (dopamine Selegiline-induced behavioral side effects have been well level and dopamine turnover rate in the striatum) and signifi- known to be caused by its , methamphetamine, cant interactions between p53 gene knockout and MPTP (do- and amphetamine [10, 74, 75]. However, it was demonstrated pamine level in the striatum) or p53 gene knockout and pre- that MPPE does not metabolize to amphetamines [12]. As treatment (dopamine level in the striatum) (Supplementary expected, repeated treatment with selegiline in significant be- Table 6). A post hoc test revealed that MPTP treatment sig- havioral sensitization and conditioned place preference, al- nificantly decreased (P<0.01) dopamine (DA) level and sig- though it is much less pronounced than in the case of meth- nificantly increased (P<0.01) DA turnover rate in the stria- amphetamine. However, repeated treatment with MPPE did tum. These changes were significantly reversed by MPPE not significantly induce behavioral sensitization or (both, P<0.01), selegiline (DA level, P<0.05; DA turnover rate, P<0.05), pifithrin-μ (both, P<0.01), or genetic deple- tion of p53 (both, P<0.01) (Fig. 6f, g). The level of DOPAC and HVA was presented in Supplementary Fig. 2.MPPE, selegiline, or pifithrin-μ did not provide additional protective effects against attenuation mediated by genetic depletion of p53 (Fig. 6).

MPPE Attenuates MPTP-Induced Behavioral Impairments: Comparison With Selegiline, Pifithrin-μ, or Genetic Inhibition of p53

Three-way ANOVA indicated significant effects of p53 gene knockout (rota-rod performance), MPTP (locomotor activity and rota-rod performance), and pretreatment (rota-rod performance) and a significant interaction between p53 gene knockout and MPTP (rota-rod performance) (Supplementary Table 7). A post hoc test revealed that MPTP-induced dopa- minergic toxicity was accompanied by a significant reduction (P<0.01) in locomotor activity. Treatment with MPPE (P<0.01), selegiline (P<0.05), pifithrin-μ (P<0.05), or p53 gene depletion (P<0.01) resulted in a significant attenuation against MPTP-induced hypolocomotor activity. However, MPPE, selegiline, or pifithrin-μ did not affect the locomotor activity in MPTP-treated p53 (−/−)mice(Fig.7a). The effect of MPPE, selegiline, pifithrin-μ, or p53 gene knockout on locomotor activity paralleled that on rota-rod performance (Fig. 7b). Fig. 7 Effect of pifithrin-μ, selgiline, or MPPE on the behavioral impairments induced by MPTP in wild-type (WT) and p53 gene knockout [p53(−/−)] mice. a Effect on hypolocomotor activity induced by MPTP. b Effect on reduction in rota-rod performance induced by Discussion MPTP. Sal Saline, PFTμ Pifithrin-μ (2 mg/kg, i.p.), Sel Selegiline (0.25 mg/kg, i.p.), MPPE MPPE (0.25 mg/kg, i.p.), Veh Vehicle (5 % – * The present study shows that MPPE provides neuroprotection DMSO). Each value is the mean ± S.E.M. of 10 12 animals. P<0.01 vs. corresponding Veh + Sal; #P<0.05, ##P<0.01 vs. WT mice treated with with behavioral safety (as assessed by locomotor activity, lo- Veh + MPTP; &P<0.05 vs. WT mice treated with Sel + MPTP (three- comotor pattern [70], and conditioned place preference). way ANOVAwas followed by Fisher’s LSD pairwise comparisons) Mol Neurobiol (2016) 53:6251–6269 6265 conditioned place preference in this study. Interestingly, it has mice, while less pronounced in UCP-2 transgenic mice than been suggested that selegiline possesses therapeutic potentials wild-type mice [72, 73]. As MPPE upregulated UCP-2 for management of drug abuse [2–4]. In these studies, it was mRNA expression after MPTP treatment, the enhancement proposed that the reduction of dopamine metabolism or the of UCP-2 may be important for MPPE-mediated inhibition normalization of glucose utilization in the is of mitochondrial oxidative stress and mitochondrial dysfunc- involved in selegiline-mediated inhibition of cocaine- tion, although underlying mechanism remains to be explored. induced self-administration or subjective . In addi- p53 is a tumor-suppressor gene, and it has been suggested tion, Davidson et al. [76] showed that selegiline treatment as one of the -sensitive transcription factors [94]. It is attenuates total AMPA GluR1 levels and its phosphorylation well known that p53 induces apoptosis and oxidative stress induced by chronic methamphetamine in the , through transcription-dependent and transcription- which has been known to play a role in behavioral sensitiza- independent mechanisms [28, 95, 96] and that mitochondrial tion or drug-seeking behavior. Consistently, we observed that translocation of p53 is a key event during the transcription- both MPPE and selegiline attenuate behavioral sensitization independent apoptosis mediated by p53 [28, 95]. In this study, and conditioned place preference induced by the non-toxic we observed significant increases in mitochondrial p53 trans- dose (1 mg/kg, i.p.) of methamphetamine in mice, although location and concomitant p53/Bcl-xL interaction as early as attenuation by MPPE was more evident than that by 1 h after the final treatment with MPTP, and these levels selegiline. Thus, it remains to be determined whether MPPE peaked at the 6 h time-point. These changes were followed affects dopaminergic degeneration induced by the toxic dose by an impaired mitochondrial transmembrane potential, cyto- [41, 57, 67, 77, 78]ofMA. solic cytochrome c release, and capase-3 cleavage. Our find- MAO-B is mainly located in the outer membrane of mito- ings are in agreement with previous studies [26, 27]showing chondria and primarily metabolizes dopamine in brain [79]. that mitochondrial p53 translocation is important for rapid We showed here that treatment with MPTP resulted in a sig- pro-apoptotic responses. Endo et al. [26] showed that mito- nificant increase of MAO-B activity in the striatum, and our chondrial p53 began to increase 1 h after cerebral ischemia result is consistent with previous report [80]. Since hydrogen in vivo, but nuclear p53 was first observed 72 h later, when peroxide is produced during MAO-B-mediated oxidation of delayed neuronal death had been already observed. In addi- monoamine , it could be postulated that ele- tion, Erster et al. [27] suggested that mitochondrial p53 trig- vated MAO-B activity might induce mitochondrial oxidative gers a rapid wave of caspase-3 activation and amplifies the stress and, possibly, consequent inhibition of complex I activ- slower transcriptional responses mediated by nuclear p53. ity. This postulation is supported by previous studies using This study is the first investigation on the role of mitochon- MAO-B transgenic mice [81–83]. In addition, it has been drial p53 translocation in MPTP-induced neurotoxicity. reported that MAO-B activity increases gradually with aging In this study, MPTP-induced interaction between mito- in [84, 85], which may contribute to the predis- chondrial p53 and Bcl-xL was significantly attenuated by position to Parkinson’sdisease[86]. As reflected by our find- pifithrin-μ, a mitochondrial p53 inhibitor. MPPE or selegiline ing, it is possible that MPPE- or selegiline-mediated MAO-B also significantly inhibited mitochondrial p53 translocation inhibition plays a positive role in attenuating oxidative stress and consequent p53/Bcl-xL interaction in our study. p53/ induced by MPTP. Bcl-xL interaction has been demonstrated to dissociate Bcl- It is well documented that mitochondrial complex I inhibi- xL and pro-apoptotic Bax or Bak, which in turn leads to mi- tion mediated by MPP+, an active metabolite of MPTP, is one tochondrial membrane permeabilization and cytosolic release of the main mechanisms in the neuropathology induced by of cytochrome c [28, 31]. Furthermore, Zhao et al. [97]dem- MPTP [87]. Mitochondrial complex I inhibition induces in- onstrated that mitochondrial p53 physically binds to mito- creases in ROS production and mitochondrial oxidative stress, chondrial Mn-SOD and suppresses its superoxide scavenging whichinturnleadtofurtherirreversibleinhibitionofmitochon- activity after 12-O-tetradecanoylphorbol-13-acetate (TPA) drial complex I in a positive feedback manner [88–90]. In this treatment in vitro. Therefore, it may be possible that MPPE- study, MPPE or selegiline mitigated MPTP-induced complex I induced inhibition of mitochondrial translocation of p53 is a inhibition and mitochondrial oxidative stress. Restoration of the prerequisite for exerting antioxidant and anti-apoptotic activ- expression and activity of mitochondrial Mn-SOD (SOD-2) ities. This is clearly supported by the results obtained from may contribute to the MPPE-mediated antioxidant defense. p53 (−/−) mice. MPTP-induced mitochondrial dysfunction, It has been reported that UCP-2 provides neuroprotection cytochrome c release, caspase-3 cleavage, and dopaminergic in various neurodegenerative processes via suppressing the neurotoxicity were significantly less pronounced in p53 (−/−) free generation, mitochondrial calcium influx, and mice than in WT mice. Moreover, MPPE (or selegiline) did caspase-3 activation [91–93]. In addition, previous studies not show any additional protective effect on the attenuation by showed that MPTP-induced dopaminergic cell death and ox- genetic depletion of p53, suggesting that the p53 gene is a idative stress were more pronounced in UCP-2 gene knockout critical target for either compound. 6266 Mol Neurobiol (2016) 53:6251–6269

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Kwan E, Baker GB, Shuaib A, Ling L, Todd KG (2000) N-methyl, Acknowledgments This study was supported by a grant N-propargyl-2-phenylethylamine (MPPE), an analog of deprenyl, (14182MFDS979) from the Korea Food and Drug Administration and increases neuronal cell survival in thiamin deficiency encephalop- partially by Basic Science Research Program through the National Re- athy. Drug Development Research 51(4):244–252. doi:10.1002/ search Foundation of Korea (NRF) funded by the Ministry of Science, ddr.5 ICT and Future Planning (no. NRF-2013R1A1A2060894 and no. NRF- 12. Rittenbach K, Sloley BD, Ling L, Coutts RT, Shan J, Baker GB 2013R1A1A1007378), Republic of Korea, and by the National Research (2005) A rapid, sensitive electron-capture gas chromatographic Foundation of Korea (NRF) grant funded by the Korea government procedure for analysis of metabolites of N-methyl, N- (MSIP) (2011–0018355). Yunsung Nam and The-Vinh Tran were sup- propargylphenylethylamine, a potential neuroprotective agent. J ported by the BK21 PLUS program, National Research Foundation of Pharmacol Toxicol Methods 52(3):373–378. doi:10.1016/j. Korea, Republic of Korea. Equipment at the Institute of New Drug De- vascn.2005.07.001 velopment Research (Kangwon National University) was used for this 13. Bar-Am O, Amit T, Weinreb O, Youdim MB, Mandel S (2010) study. The English in this document has been checked by at least two Propargylamine containing compounds as modulators of proteo- professional editors, both native speakers of English (Beverly Hills En- lytic cleavage of amyloid-beta protein precursor: involvement of glish, Los Angeles, CA90024, USA). MAPK and PKC activation. J Alzheimers Dis 21(2):361–371. doi: 10.3233/JAD-2010-100150 Compliance with Ethical Standards 14. 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