Eun-Joo Shin1& Yunsung Nam1

& Ji Won Lee1,2 & Phuong-Khue Thi Nguyen1&

Ji Eun Yoo1 & The-Vinh Tran1& Ji Hoon Jeong3 & Choon-Gon Jang4 & Young J. Oh5

&

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-hibitor with anti-Parkinsonian effects, but it is metabolized toamphetamines. Since another MAO-B inhibitor N-Methyl, N-propynyl-2-phenylethylamine (MPPE) is not metabolized toamphetamines, we examined whether MPPE induces behav-ioral side effects and whether MPPE affects dopaminergictoxic i ty induced by 1-methyl -4-phenyl -1 ,2 ,3 ,6-tetrahydropyridine (MPTP). Multiple doses of MPPE (2.5and 5 mg/kg/day) did not show any significant locomotoractivity and conditioned place preference, whereas selegiline(2.5 and 5 mg/kg/day) significantly increased these behavioralside effects. Treatment with MPPE resulted in significant at-tenuations against decreases in mitochondrial complex I activ-ity, mitochondrial Mn-SOD activity, and expression inducedby MPTP in the striatum of mice. Consistently, MPPE

significantly attenuated MPTP-induced oxidative stress andMPPE-mediated antioxidant activity appeared to be more pro-nounced in mitochondrial-fraction than in cytosolic-fraction.Because MPTP promoted mitochondrial p53 translocationand p53/Bcl-xL interaction, it was also examined whethermitochondrial p53 inhibitor pifithrin-μ attenuates MPTP neu-rotoxicity. MPPE, selegiline, or pifithrin-μ significantly atten-uated mitochondrial p53/Bcl-xL interaction, impaired mito-chondrial transmembrane potential, cytosolic cytochrome crelease, and cleaved caspase-3 in wild-type mice. Subsequent-ly, these compounds significantly ameliorated MPTP-inducedmotor impairments. Neuroprotective effects of MPPE ap-peared to be more prominent than those of selegiline. MPPEor selegiline did not show any additional protective effectsagainst 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 Kimkimhc@kangwon.ac.kr

1 Neuropsychopharmacology and Toxicology Program, College ofPharmacy, Kangwon National University, Chunchon 200-701,Republic of Korea

2 Hutecs Korea Pharm Co., Ltd., Osan 18111, Republic of Korea

3 Department of Pharmacology, College of Medicine, Chung-AngUniversity, Seoul 156-756, Republic of Korea

4 Department of Pharmacology, School of Pharmacy, SungkyunkwanUniversity, Suwon 440-746, Republic of Korea

5 Department of Systems Biology, Yonsei University College of LifeScience and Biotechnology, Seoul 120-749, Republic of Korea

6 Eve Topf Centers of Excellence for Neurodegenerative DiseasesResearch, Faculty of Medicine, Technion-Israel Institute ofTechnology, Haifa 31096, Israel

7 National Creative Research Initiative Center for Catalytic OrganicReactions, Department of Chemistry, Kangwon National University,Chunchon 200-701, Republic of Korea

8 Department of Regional Pharmaceutical Care and Sciences, GraduateSchool of Pharmaceutical Sciences, Meijo University,Nagoya 468-8503, Japan

9 NPO, Japanese Drug Organization of Appropriate Use and Research,Nagoya 468-8503, Japan

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

N-Methyl, N-propynyl-2-phenylethylamine (MPPE), a SelegilineAnalog, Attenuates MPTP-induced Dopaminergic Toxicitywith Guaranteed Behavioral Safety: Involvement of Inhibitionsof Mitochondrial Oxidative Burdens and p53 Gene-elicitedPro-apoptotic Change

p53 gene is a critical target for these compounds. Our resultssuggest that MPPE possesses anti-Parkinsonian potentialswith guaranteed behavioral safety and that the underlyingmechanism of MPPE requires inhibition of mitochondrial ox-idative stress, mitochondrial translocation of p53, and pro-apoptotic process.

Keywords N-Methyl,N-propynyl-2-phenylethylamine .

Mitochondria . Selegiline . Oxidative stress . Behavioralsafety . p53 gene knockoutmice . Pro-apoptosis . Parkinson’sdisease

Introduction

Selegiline, one of the propargylamine-based monoamine oxi-dase (MAO)-B inhibitors, has long been used as a monother-apy in early Parkinson’s disease (PD) or as an adjunctive ther-apy to levodopa in advanced PD [1]. In addition, it was shownthat 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-eficial effects of selegiline, psychiatric and cardiovascular ad-verse effects have presented a problem with its use [5–7], andearlier studies suggested that the metabolism of selegiline tomethamphetamine and amphetamine accounts for these ad-verse psychotropic effects [8–10].

N-Methyl, N-propynyl-2-phenylethylamine (MPPE) is apropargylamine-based MAO-B inhibitor [11], but it is notmetabolized to amphetamine derivatives [12]. Like otherpropargyl-containingMAO-B inhibitors possessing neuropro-tective properties, MPPE exerted neuroprotective effects inthe animal model of thiamine deficient encephalopathy [11].However, it has not been reported whether MPPE providesneuroprotection against any other neurotoxic conditions. Inaddition to the MAO-B inhibitory effect, several studies havesuggested that antioxidant and anti-apoptotic effects of pro-pargyl moiety are important for the neuroprotection providedby propargylamine-based MAO-B inhibitors [13–18].

p53, a tumor-suppressor gene, has been suggested to play akey role in the apoptotic processes found in various neurode-generative conditions, including PD [19–22]. Elevated proteinlevel of p53 was reported in the postmortem brain of PDpat ients [23, 24] or 1-methyl-4-phenyl-1 ,2 ,3 ,6-tetrahydropyridine (MPTP)-treated animal model of PD [25]as well. In addition to the well-known transcriptional regula-tion of pro-apoptotic and anti-apoptotic factors, p53 couldmediate apoptotic cell death via mitochondrial translocation[26–28]. Interaction of mitochondrial p53 with Bcl-2 or Bcl-xL impairs mitochondrial membrane integrity and inducesconsequent cytosolic release of cytochrome c [26, 29–31].Although it has been reported that p53 gene knockout [32]or pifithrin-α, a p53 transcription inhibitor [33], attenuates

MPTP-induced dopaminergic neurotoxicity, it remains un-known whether p53 mitochondrial translocation is involvedin its neurotoxic process.

In the present study, we examined the effect of MPPE onMPTP-induced neurotoxicity in comparison with selegiline.In addition, it was also investigated whether pifithrin-μ, amitochondrial p53 inhibitor, attenuates dopaminergic toxicityin this model. We found that MPPE significantly attenuatesdopaminergic toxicity induced by MPTP, that protective ef-fects of MPPE appear to be more pronounced than those ofselegiline, and that MPPE, selegiline, or pifithrin-μ do notsignificantly affect MPTP toxicity in p53 gene knockout[p53 (−/−)] mice. Importantly, we observed that locomotorfacilitation and conditioned place preference (CPP) are lesspronounced in mice treated with MPPE than in mice treatedwith selegiline, suggesting that MPPE possesses behavioralsafety. Furthermore, MPPE appeared to bemore effective thanselegiline against behavioral sensitization and CPP induced bymethamphetamine (MA).

Materials and Methods

Synthesis of MPPE

A solution of N-methylphenethylamine (676.0 mg, 5.0 mmol)in water (10.0 mL) added 1.0 M NaOH (6.0 mL, 6.0 mmol) atroom temperature. The mixture was stirred for 20 min, andpropargyl bromide (714.0mg, 6.0 mmol) was added. After themixture was stirred at room temperature for 2.5 h, the reactionmixture was quenched with water. The aqueous layer wasextracted with diethyl ether, and the combined layers werewashed with water, brine, and dried over anhydrous MgSO4.The crude product was purified by column chromatographyon silica gel (hexane:ethyl acetate, 1:1) to yield N-methyl-N-propargyl-2-phenylethylamine (533.1 mg, 62 %); 1H NMR(CDCl3, 300 MHz) δ 7.28–7.14 (m, 5H), 3.37 (d, J=2.4,2H), 2.78–2.72 (m, 2H), 2.69–2.63 (m, 2H), 2.35 (s, 3H),2.21 (t, J=2.4, 1H); 13C NMR (CDCl3, 75 MHz) δ 140.2,128.7, 128.4, 126.1, 78.5, 73.3, 57.4, 45.6, 41.8, 34.3.

A solution of N-methyl-N-propargyl-2-phenylethylamine(87.0 mg, 0.5 mmol) in dichloromethane (1.25 mL) added1.0 M HCl (1.0 mL, 1.0 mmol) at 0 °C. The reaction wasallowed to warm up to room temperature and stirred for2.5 h. After evaporation of the solvents in vacuo, N-methyl-N-propargyl-2-phenylethylamine∙HCl (91.0 mg, 87 %) wasproduced.

Animals

All animals were treated in accordance with the National In-stitutes of Health (NIH) Guide for the Humane Care and Useof Laboratory Animals (NIH Publication No. 85-23, 1985;

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www.dels.nas.edu/ila). The present study was performed inaccordance with the Institute for Laboratory Research(ILAR) guidelines for the care and use of laboratory animals.Mice were maintained under a 12-h light:12-h dark cycle andfed ad libitum. Breeding pairs of p53 gene heterozygous [p53(+/−)] mice with C57BL/6J background were obtained fromRIKEN BioResource Center (Tsukuba, Japan) [34]. p53 (−/−)mice were maintained as heterozygous breeding pairs, andneonates were genotyped by polymerase chain reaction(PCR) of DNA extracted from the tail, according to the infor-mation provided by the RIKEN BioResource Center. Addi-tional details regarding the gene characterization were givenin the Supplementary Information.

Conditioned Place Preference

CPP was examined as described previously [35–37]. The CPPapparatus was described in the Supplementary Information.As a control, mice received an i.p. injection of saline justbefore entering the white or black compartment. On days 1and 2, the mice were pre-exposed to the test apparatus for5 min. The guillotine doors were raised, and the mice wereallowed to move freely between the two compartments. Onday 3, the time that each mouse spent in each compartmentwas recorded for 15 min. On days 4, 6, 8, 10, 12, and 14, themice were injected with drugs before being confined to thewhite compartment, the non-preferred side, for 40 min. Ondays 5, 7, 9, 11, and 13, the mice were injected with salinebefore being confined to the black compartment, the preferredside, for 40 min. On day 15, the guillotine doors were raised.The mice were initially placed in the tunnel and the time spentby mice in each compartment was recorded for 15 min. Thescores were calculated from the differences in the time spent inthe white compartment between post-test and pre-test periods.Data were analyzed between 09:00 and 17:00 h.

To evaluate whether selegiline or MPPE induces behavior-al side effects, selegiline (2.5 or 5.0 mg/kg, i.p.) or MPPE (2.5or 5 mg/kg, i.p.) dissolved in saline was administered imme-diately before mice were placed in the white compartment.Methamphetamine (MA; 0.5 or 1.0 mg/kg, i.p.) was used asa control drug. The experimental design was shown in Sup-plementary Fig. 1a. To examine the effect of selegiline orMPPE on CPP induced byMA, selegiline (0.25 or 0.5 mg/kg,i.p.) or MPPE (0.25 or 0.5 mg/kg, i.p.) was administered30 min before each MA treatment. MA (1 mg/kg, i.p.) dis-solved in saline was administered immediately before micewere placed in the white compartment. The experimental de-sign was shown in Supplementary Fig. 1c.

Locomotor Activity and Behavioral Sensitization

Locomotor activity was measured for 30 min as de-scribed previously [36, 38] using an automated video-

tracking system (Noldus Information Technology,Wagenin, The Netherlands). Eight test boxes (40×40×30 cm high) were operated simultaneously by an IBMcomputer. Animals were studied individually duringmeasurement of locomotion in each test box, where theywere adapted for 10 min before starting the recording.Data were collected and analyzed between 09:00 and17:00 h. To evaluate whether selegiline or MPPE in-duces behavioral side effects, mice received daily injec-tion of selegiline (2.5 or 5.0 mg/kg, i.p.) or MPPE (2.5or 5 mg/kg, i.p.) for seven consecutive days. MA (0.5or 1.0 mg/kg, i.p.) was used as a control drug. Imme-diately after each injection, mice were introduced intothe test box. The experimental schedule was shown inSupplementary Fig. 1b. To examine the effect ofselegiline or MPPE on the behavioral sensitization in-duced by MA, 40 min after the first (day 4), fourth(day 10), and seventh (day 16) injection of MA (i.e.,after the conditioning in the white compartment of CPPapparatus), locomotor activity was analyzed in a 30-minmonitoring period. After a withdrawal period for 6 days(day 22), mice received MA (1 mg/kg, i.p.), and loco-motor activity was measured for 30 min. Mice receivedselegiline (0.25 or 0.5 mg/kg, i.p.) or MPPE (0.25 or0.5 mg/kg, i.p.) every other day (i.e., day 18 and 20)during the MA withdrawal period and 30 min beforeMA treatment on day 22. The experimental designwas shown in Supplementary Fig. 1c.

Drug Treatment

MPTP (15 mg/kg, s.c.) was dissolved in sterile saline (1 ml/kg) immediately before use. MPPE (0.25 mg/kg/day, i.p.),selegiline (0.25 mg/kg/day, i.p.; Tocris Bioscience, Bristol,UK), or pifithrin-μ (2 mg/kg/day, i.p.; Sigma–Aldrich, St.Louis, MO, USA) were dissolved in dimethyl sulfoxide(DMSO) as a stock solution and then stored at 4 °C. Thesestock solutions were diluted in sterile saline (1 ml/kg) imme-diately before use, and the final DMSO concentration was 5%(v/v). The dose of selegiline or pifithrin-μ was determinedbased on previous studies [39, 40].

Mice received MPTP once daily for seven consecutivedays. MPPE or selegiline was administered once a day for28 days before MPTP treatment (day 1–28). During MPTPtreatment (day 29–35), MPPE, selegiline, or pifithrin-μ wereadministered 2 h prior to each MPTP treatment. One day afterthe final MPTP treatment, behavioral evaluation was per-formed, and then the mice were sacrificed. To examine mito-chondrial translocation of p53, cytosolic cytochrome c release,and subsequent caspase-3 cleavage, wild-type mice weretreated with MPTP as described above and sacrificed 1, 6,12, and 24 h after the final MPTP treatment. The experimentaldesign was shown in Supplementary Fig. 1d, e.

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Fig. 1 Changes in locomotoractivity and conditioned placepreference after repeatedtreatment with MPPE orselegiline and the effect of MPPEor selegiline on the locomotorfacilitation and conditioned placepreference induced by MA. aChemical structures of selegilineand MPPE. b–d Changes inlocomotor activity (b) andrepresentative locomotor patterns(c) and conditioned placepreference (d) after repeatedtreatment with MPPE (2.5 or5.0 mg/kg, i.p.) or selegiline (2.5or 5.0 mg/kg, i.p.).Methamphetamine (MA; 0.5 or1.0 mg/kg, i.p.) was used as acontrol drug. Representativelocomotor patterns show the trackof mouse movement during thefifth 5 min (i.e., 30–35 min afterthe last drug injection) oflocomotor recording afterrepeated treatment for sevenconsecutive days with each drug(c). Note the typical locomotortracing patterns (increase inmarginal activity [70]) afterrepeated treatment with MA orselegiline (c). e–f Effect of MPPE(0.25 or 0.5 mg/kg, i.p.) orselegiline (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 themean ± 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 ANOVAfor repeated measures; d, one-way ANOVA; e, two-wayANOVA for repeated measures; f,two-way ANOVA. Post hocFisher’s LSD pairwisecomparisons test followed)RL

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Behavioral Assessments After MPTP Treatment

Locomotor activity was measured for 30 min as describedabove. Rota-rod test was performed as described previously[41]. The apparatus (model 7650; Ugo Basile, Comerio, Va-rese, Italy) consisted of a base platform and a rotating rod witha non-slip surface. The rod was placed at a height of 15 cmabove the base. The rod, 30 cm in length, was divided intoequal sections by six opaque disks so that the animals wouldnot be distracted by one another. To assess motor perfor-mance, the mice were first trained on the apparatus for 2 minat a constant rate of 4 rpm. The test was performed 30 minafter training and an accelerating paradigmwas applied, whichis starting from a rate of 4 rpm to maximal speed of 40 rpm.The rotation speed was then kept constant at 40 rpm. Thelatency to fall was measured with a maximal cutoff time of300 s.

Brain Dissection

Mice were sacrificed by decapitation, and brains were rap-idly removed and placed on an ice-cold brain matrix (ASIInstruments, Warren, MI, USA). Coronal slices containingstriatum or substantia nigra were made at 1.1 to −0.1 mmor at −3.0 to −3.5 mm from bregma using chilled razorblades, according to the atlas of Franklin and Paxinos[42]. Dorsal striatum and substantia nigra were punchedbilaterally with a sample corer (2 mm inner diameter forstriatum, 1 mm inner diameter for substantia nigra; FineScience Tools Inc., Vancouver, Canada) and a plunger [43,44]. Dissected tissues were frozen in liquid nitrogen andstored at −80 °C until use.

Since it may be not available for preparing an enoughamount of mitochondrial fraction from substantia nigra, wehave mainly employed striatal tissue to investigate neuro-chemical changes in the mitochondrial fraction in this study[45]. Importantly, earlier reports demonstrated that 4-phenylpyridinium ion (MPP+), a toxic metabolite of MPTP,is more likely to be concentrated and particularly toxic instriatal mitochondria than in nigral mitochondria [46–53].

Preparation of Cytosolic and Mitochondrial Fractionfor Western Blot and Neurochemical Analyses

The cytosolic and mitochondrial fractions were prepared aswe described previously for Western blot analysis and theneurochemical assay [41, 54, 55]. Mitochondria were isolatedas described previously [56] with minor modifications [41,57] for measurements of mitochondrial transmembrane poten-tial. Details of the procedure were provided in the Supplemen-tary Information.

Monoamine Oxidase-B Activity

MAO-B activity was examined as described previously [39,58]. Striatal tissues were homogenized in 0.1 M potassiumphosphate buffer (pH 7.4), and 250 μL of homogenate wasadded to 1.25 mL of 0.5 mM kynuramine dissolved in potas-sium phosphate buffer. Then, 250 μL of 1 μM clorgyline wasadded to inhibit MAO-A activity. The reaction mixture wasincubated at 37 °C for 30min, and the reaction was terminatedby adding ice-cold 0.4 N perchloric acid. After centrifugationat 7500×g for 5 min, an equal volume of 0.1 N NaOH wasadded to the supernatant. Fluorescence intensity was recordedwith excitation and emission wavelengths of 315 and 350 nm,respectively. MAO-B activity was calculated using a standardcurve of 4-hydroxyquinoline, the resultant product of the re-action. The result was expressed as nanomoles 4-hydroxyquinoline formed per hour per milligram protein.

Complex I Activity

Complex I activity was examined as described previously[59]. Isolated mitochondrial sample (μL) was added to thereaction mixture containing 25 mM potassium phosphatebuffer (pH 7.8), 3.5 mg/mL bovine serum albumin,60 μM 2,6-dichloroindophenol, 70 μM decylubiquinone,and 1 μM antimycin A, and reaction mixture was incu-bated at 37 °C for 3 min. After adding NADH to the finalconcentration of 0.2 mM, the absorbance was recorded at60-s intervals for 4 min at 600 nm. Then, rotenone wasadded to the final concentration of 1 μM, and the absor-bance was recorded again at 60-s intervals for 4 min at600 nm. One unit of complex I activity was defined as1 μmol 2,6-dichloroindophenol reduced per minute, and itwas calculated based on the extinction coefficient for 2,6-dichloroindophenol of 19.1 mM/cm. The result wasexpressed as a percentage of the control group.

Western Blot

The Western blot assays were performed as we described pre-viously [41, 55]. Striatal tissues were homogenized in lysisbuffer, containing 200 mM Tris HCl (pH 6.8), 1 % SDS,5 mM ethylene glycol-bis(2-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), 5 mM ethylenediaminetetraacetic ac-id (EDTA), 10 % glycerol, 1× phosphatase inhibitor cocktail I(Sigma-Aldrich), and 1× protease inhibitor cocktail (Sigma-Aldrich). Lysate was centrifuged at 12,000×g for 30 min, andsupernatant fraction was used for Western blot analysis. Mi-tochondrial and cytosolic fractions were prepared as describedabove. Additional details regarding the procedure and anti-bodies were provided in the Supplementary Information.

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Immunoprecipitation

Immunoprecipitation was performed as we described previ-ously [60] using protein G-sepharose (GE Healthcare,Piscataway, NJ, USA). Details regarding the procedure andantibodies were provided in the Supplementary Information.

RT-PCR

Expression of uncoupling protein-2 (UCP-2) was assessed aswe described [61] using semi-quantitative RT–PCR to analyzemessenger RNA (mRNA) level. Total RNAwas isolated fromstriatal tissues using an RNeasy Mini Kit (Qiagen, Valencia,CA, USA). Details regarding the primer sequences and pro-cedure were given in the Supplementary Information.

Immunocytochemistry

Immunocytochemistry was performed as described previous-ly [41]. Mice were perfused transcardially with 50 mL of ice-cold PBS (10 mL/10 g body weight) followed by 4 % para-formaldehyde (20 mL/10 g body weight). Brains were re-moved and stored in 4 % paraformaldehyde overnight. Sec-tions were subjected to immunostaining with primary anti-body against SOD-2 [1:1000; kindly gifted by Dr. KanefusaKato at Aichi Prefectural Colony, Kasugai, Japan [62–64] ortyrosine hydroxlase (TH) (1:500; Chemicon (EMDMillipore)). Details regarding immunocytochemistry andquantitative analysis were presented in the Supplementaryinformation.

Stereological Analysis

Stereological analysis of the number of TH-immunoreactivecells in the substantia nigra (SN) pars compacta was per-formed as described previously [41, 57, 65]. Details of theprocedure were provided in the Supplementary Information.

Superoxide Dismutase (SOD) Activity

Striatal tissues were homogenized in 50 mM potassium phos-phate buffer (pH 7.8) and centrifuged at 13,000×g for 20 min.The resulting supernatant was used to measure SOD activity.SOD activity was determined on the basis of inhibition ofsuperoxide-dependent reactions as described previously [41].Details of the procedure were provided in the SupplementaryInformation.

Determination of Protein Carbonyl

The extent of protein oxidation was assessed bymeasuring thecontent of protein carbonyl group, which was determinedwiththe 2,4-dinitrophenylhydrazine (DNPH)-labeling procedure

[66]. DNPH-labeled protein was detected by spectrophoto-metric [41, 66] or slot blot [67] analysis. Details of the proce-dure were provided in the Supplementary Information.

Determination of 4-hydroxynonenal

The amount of lipid peroxidation was determined by measur-ing the level of 4-hydroxynonenal (HNE) using theOxiSelectTM HNE adduct ELISA kit (Cell Biolabs, Inc., SanDiego, CA, USA) according to the manufacturer’s manual.Details of the procedure were provided in the SupplementaryInformation.

Determination of Mitochondrial ROS

Determination of the formation of ROS was performed asdescribed previously [60, 68]. Mitochondrial fraction was in-cubated with 5 μM 2’,7’-dichlorofluorescein diacetate(DCFH-DA, Molecular Probes, Eugene, OR, USA) for15 min at 37 °C. The fluorescent intensity due to the ROSwas measured at an excitation wavelength of 488 nm andemission wavelength of 528 nm using a fluorescent micro-plate reader (Molecular Devices Inc.).

Mitochondrial Transmembrane Potential

Mitochondrial transmembrane potential was measured as de-scribed previously [41, 56, 57] using 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolycarbocyanine iodide dye (JC-1;Molecular Probes), which exists as a green fluorescent mono-mer at low membrane potential, but reversibly forms red fluo-rescent BJ-aggregates^ at polarized mitochondrial potentials.Details of the procedure were provided in the SupplementaryInformation.

Measurement of Dopamine Level

The dopamine level was determined by HPLC coupled withan electrochemical detector as described previously [41, 57,69]. Details of the procedure were provided in the Supplemen-tary Information.

�Fig. 2 Effect of MPPE or selegiline on changes in MAO-B activity,mitochondrial complex I activity, mitochondrial Mn-SOD (SOD-2) activ-ity and expression, and UCP-2 mRNA expression induced byMPTP. a, bMAO-B activity (a) and mitochondrial complex I (b) activity in the stri-atum. c SOD-2 activity in the striatum. d, e Protein expression (d) andimmunoreactivity (e) of SOD-2 in the striatum. f SOD-2 activity in thesubstantia nigra. g SOD-2 immunoreactivity in the substantia nigra. hUCP-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 %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;&P<0.05 vs. Sel + MPTP (Two-way ANOVAwas followed by Fisher’sLSD pairwise comparisons)

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Statistical Analyses

Data were analyzed using IBM SPSS ver. 21.0 (IBM, Chica-go, IL, USA). One-way analysis of variance (ANOVA) (treat-ment), two-way ANOVA (pretreatment × MA or MPTP), orthree-way ANOVA (p53 gene knockout × pretreatment ×MPTP) were employed for the statistical analyses. Arepeated-measures ANOVA (between-subjects factors: pre-treatment × MA; within-subjects factor: time) was conductedfor the behavioral sensitization. Post hoc Fisher’s least signif-icant difference pairwise comparisons tests were then con-ducted. P values<0.05 were considered to be significant.

Results

Treatment With MPPE did not Significantly InducePsychotropic Locomotor Pattern and Conditioned PlacePreference: Comparison With Selegiline

In order to examine whether selegiline (Fig. 1a) or MPPE(Fig. 1a) induces psychotropic effects, we evaluated locomo-tor activity, locomotor pattern, and conditioned place prefer-ence (MAwas used as a control drug). One-way ANOVA forrepeated measures showed significant effects of treatment(between-subject factor) and day (within-subject factor), anda significant interaction between treatment and day on thelocomotor activity (Supplementary Table 1). On the condi-tioned place preference, one-way ANOVA indicated signifi-cant effect of treatment (Supplementary Table 1). A post hoctest revealed that repeated treatment with selegiline (2.5 or5.0 mg/kg/day, i.p.) resulted in significant increases in loco-motor activity (2.5 or 5.0 mg/kg of selegiline vs. saline,P<0.05) with psychotropic locomotor pattern (i.e., marginalactivity [70]) and conditioned place preference (2.5 or5.0 mg/kg of selegiline vs. saline, P<0.05 or P<0.01, respec-tively), although these increases were less pronounced thanthe MA case. However, MPPE (2.5 or 5.0 mg/kg/day, i.p.)did not induce significant change in locomotor activity, loco-motor pattern, or conditioned place preference (Fig. 1b–d).

Pretreatment With MPPE Attenuates MA-InducedBehavioral Sensitization and Conditioned PlacePreference: Comparison With Selegiline

As it was reported that selegiline attenuates behavioral(psychotropic) effects mediated by abusive drugs [2–4, 71],we examined whether selegiline analog MPPE is also effec-tive in response to the MA-induced behavioral sensitizationand conditioned place preference in the present study. On thelocomotor activity, two-way ANOVA for repeated measuresindicated significant effects of MA (between-subject factor)and day (within-subject factor) and a significant interaction

betweenMA and day (Supplementary Table 1). On the behav-ioral sensitization and conditioned place preference, two-wayANOVA showed significant effects of MA and pretreatment(Supplementary Table 1). A post hoc test revealed that pre-treatment with selegiline (0.25 or 0.5 mg/kg, i.p.) or MPPE(0.25 or 0.5 mg/kg, i.p.) significantly attenuated behavioralsensitization (0.25 or 0.5 mg/kg of selegiline + MAvs. saline+ MA, P<0.05 or P<0.01, respectively; 0.25 or 0.5 mg/kg ofMPPE +MAvs. saline +MA, P<0.01) and conditioned placepreference (0.5 mg/kg of selegiline + MA vs. saline + MA,P<0.05; 0.5 mg/kg ofMPPE +MAvs. saline +MA, P<0.01)induced by MA (1 mg/kg, i.p.). These attenuations appearedto be more pronounced in MPPE than those in selegiline(Fig. 1e–g).

MPPE Attenuates MPTP-Induced Changes in MAO-Band Complex I Activity in the Mitochondriaof the Striatum: Comparison With Selegiline

Two-way ANOVA revealed significant effects of MPTP(MAO-B and complex I activities) and pretreatment (MAO-B activity) and a significant interaction between MPTP andpretreatment (complex I activity) (Supplementary Table 2). Apost hoc test indicated that MPTP treatment significantly in-creased (P<0.01 vs. vehicle + saline) MAO-B activity. MPPE(P<0.05 vs. vehicle + MPTP) or selegiline (P<0.01 vs. ve-hicle + MPTP) significantly attenuated this increment. MPPEor selegiline also significantly decreased (P<0.05 vs. vehicle+ saline) MAO-B activity (Fig. 2a).

MPPE or selegiline did not significantly alter complex Iactivity. Treatment with MPTP resulted in a significant reduc-tion in complex I activity (P<0.01 vs. vehicle + saline). Thisreduction in complex I activity was significantly attenuated byMPPE (P<0.01 vs. vehicle + MPTP) or selegiline (P<0.05vs. vehicle + MPTP). This attenuation appeared to be moreevident in MPPE than selegiline (Fig. 2b).

MPPE upRegulates Mitochondrial Mn-SOD (SOD-2)Enzyme Activity, Protein Expression,and Immunoreactivity, and UCP-2 mRNA ExpressionInduced by MPTP in the Striatum: Comparison WithSelegiline

We, next, examined SOD-2 activity, protein expression, andimmunoreactivity afterMPTP treatment (Fig. 2c–e). Two-wayANOVA showed significant effects ofMPTP (SOD-2 activity,expression, and immunoreactivity) and pretreatment (SOD-2activity, expression, and immunoreactivity), and a significantinteraction between MPTP and pretreatment (SOD-2 activityand immunoreactivity) in the striatum (SupplementaryTable 2). A post hoc test revealed that MPTP treatment sig-nificantly decreased (P<0.01 vs. vehicle + saline) SOD-2-immunoreactivity. Treatment withMPPE (P<0.01 vs. vehicle

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+ MPTP) or selegiline (P<0.05 vs. vehicle + MPTP) signifi-cantly attenuated this reduction in SOD-2-immunoreactivityinduced by MPTP (Fig. 2e). This SOD-2-immunoreactivitywas consistent with the result obtained from Western blotanalysis (Fig. 2d). MPTP-induced decrease in SOD-2 activitywas attenuated by MPPE (P<0.01 vs. vehicle + MPTP) or

selegiline (P<0.05 vs. vehicle + MPTP) (Fig. 2c). Changes inSOD-2 activity and immunoreactivity in the striatum werecomparable to those in the substantia nigra (Fig. 2f, g).

It has been reported that uncoupling protein-2 (UCP-2) isimportant for protecting dopaminergic neurons againstMPTP-induced mitochondrial oxidative stress and

Fig. 3 Effect of MPPE orselegiline on MPTP-inducedoxidative stress in the cytosolicand mitochondrial fractions of thestriatum of mice. a–d Effect oncytosolic and mitochondrialformations (a, b) and expressions(c, d) of protein carbonyl inducedby MPTP. e, f Effect on cytosolicand mitochondrial formations of4-hydroxynonenal (HNE)induced by MPTP. g, h Effect oncytosolic and mitochondrialformations of reactive oxygenspecies (ROS) induced by MPTP.Sal Saline, Sel Selegiline(0.25 mg/kg, i.p.), MPPEMPPE(0.25 mg/kg, i.p.), Veh Vehicle(5 % DMSO). Each value is themean ± 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 byFisher’s LSD pairwisecomparisons)

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neurodegeneration [72, 73]. Thus, we evaluated UCP-2expression in the striatum after MPTP treatment. Two-way ANOVA indicated significant effects of MPTP andpretreatment and a significant interaction between MPTPand pretreatment (Supplementary Table 2). A post hoctest showed that UCP-2 mRNA expression was signifi-cantly induced (P<0.05 vs. vehicle + saline) after thelast MPTP treatment, and this induction was more sig-nificantly potentiated by MPPE (P<0.01 vs. vehicle +saline) or selegiline (P<0.05 vs. vehicle + saline)(Fig. 2h). The effect of MPPE was more evident(P<0.05) than that of selegiline against alterations in

SOD-2 activity, SOD-2 expression, SOD-2-immunoreac-tivity, and UCP-2 mRNA expression induced by MPTP(Fig. 2c–h).

MPPE Attenuates MPTP-Induced Oxidative Stress(Mitochondria > Cytosol): Comparison With Selegiline

Next, we examined the effect of MPPE on the MPTP-induced oxidative stress in cytosolic and mitochondrialfractions. In the cytosolic fraction, two-way ANOVAshowed significant effects of MPTP (protein carbonyllevel and expression, 4-hydroxynonenal (HNE) level,

Fig. 4 Changes in themitochondrial translocation ofp53, mitochondrial p53/Bcl-xLinteraction, cytosolic cytochromec release, and cleaved caspase-3expression 1, 6, 12, and 24 h afterthe final treatment with MPTP inthe striatum of wild-type mice. aRepresentative bands from eachtime-point. b Quantification ofp53 in the mitochondrial fraction,cytosolic fraction, and wholelysate. c Quantification of p53/Bcl-xL interaction in themitochondrial fraction. dQuantification of cytochrome c inthe cytosolic fraction. eQuantification of cleavedcaspase-3 in the whole lysate. SalSaline. Each value is the mean ±S.E.M. of six animals. *P<0.05,**P<0.01 vs. Sal (One-wayANOVAwas followed by Fish-er’s LSD pairwise comparisons)

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and reactive oxygen species (ROS) level) and pretreat-ment (protein carbonyl expression and ROS level) anda significant interaction between MPTP and pretreat-ment (protein carbonyl expression and ROS level). Inthe mitochondrial fraction, significant effects of MPTPand pretreatment and a significant interaction betweenMPTP and pretreatment were shown on the proteincarbonyl level and expression, HNE level, and ROSlevel (Supplementary Table 3). A post hoc test indicatedthat treatment with MPPE resulted in decreases in the protein

carbonyl level (the formation (cytosolic or mitochondrial frac-tion: P<0.05 or P<0.01 vs. vehicle + MPTP, respectively)and the expression (P<0.01 vs. vehicle + MPTP in both frac-tions)), 4-HNE level (cytosolic or mitochondrial fraction:P<0.05 or P<0.01 vs. vehicle + MPTP, respectively), orROS formation (P<0.01 vs. vehicle + MPTP in both frac-tions) induced by MPTP. MPPE was more effective(P<0.05 vs. selegiline + MPTP) than selegiline in attenuatingmitochondrial formations of protein carbonyl, HNE, and ROS(Fig. 3).

Fig. 5 Effect of pifithrin-μ,selgiline, or MPPE on themitochondrial translocation ofp53, mitochondrial p53/Bcl-xLinteraction, mitochondrialtransmembrane potential,cytosolic cytochrome c release,and cleaved caspase-3 expression6 h after the final treatment withMPTP in the striatum ofwild-type(WT) or p53 gene knockout[p53(−/−)] mice. a Representativebands of mitochondrial andcytosolic p53 and mitochondrialp53/Bcl-xL interaction. b Effecton mitochondrial translocation ofp53 induced by MPTP. c Effecton mitochondrial p53/Bcl-xLinteraction induced by MPTP. dEffect on decrease inmitochondrial transmembranepotential induced by MPTP. eEffect on cytosolic cytochrome crelease induced byMPTP. f Effecton cleaved caspase-3 induced byMPTP. Sal Saline, PFTμPifithrin-μ (2 mg/kg, i.p.), SelSelegiline (0.25 mg/kg, i.p.),MPPE MPPE (0.25 mg/kg, i.p.),Veh Vehicle (5 % DMSO). Eachvalue is the mean ± S.E.M. of 5–7animals. *P<0.01 vs. corre-sponding Veh + Sal; #P<0.05,##P<0.01 vs. WT mice treatedwith Veh + MPTP (two-wayANOVA (b, c) or three-wayANOVA (d–f) was followed byFisher’s LSD pairwisecomparisons)

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Mitochondrial Translocation of p53, p53/Bcl-xLInteraction, Cytosolic Release of Cytochrome c,and Cleavage of Caspase-3 Induced by MPTP

As previous reports demonstrated that binding of mitochon-drial p53 with Bcl-xL can induce cytochrome c release andconsequent pro-apoptotic changes [26, 29–31], we examinedwhether MPTP activates mitochondrial translocation of p53and the interaction between mitochondrial p53 and Bcl-xL.One-way ANOVA indicated a significant effect of time onthe mitochondrial and cytosolic p53 expression, mitochondri-al p53/Bcl-xL interaction, cytosolic cytochrome c release, andcleaved caspase-3 expression (Supplementary Table 4). Apost hoc test revealed that mitochondrial p53 translocation(Fig. 4a, b) or p53/Bcl-xL interaction (Fig. 4a, c) was signif-icantly increased (1 and 6 h, P<0.01; 12 h, P<0.05) after thelast treatment of MPTP, and these changes were most evident6 h later. Consistently, cytosolic cytochrome c release (1 and12 h, P<0.05; 6 h, P<0.01) and caspase-3 cleavage (1 and12 h, P<0.05; 6 h, P<0.01) were most pronounced 6 h afterthe final MPTP treatment (Fig. 4d–e), respectively. Total p53protein expression in whole lysate was not significantly al-tered in the entire range of time-course, suggesting that proteinexpression may not be changed in these early time-points ofour experimental condition (Fig. 4).

MPPE Attenuates MPTP-Induced MitochondrialTranslocation of p53 and p53/Bcl-xL Interaction:Comparison With Selegiline or Pifithrin-μ

Since MPTP-induced mitochondrial p53 translocation andp53/Bcl-xL interaction were most pronounced 6 h later(Fig. 4a–c), we focused on this time-point to examine theeffect of MPPE or selegiline. Additionally, the effect ofpifithrin-μ, a specific inhibitor of p53/Bcl-xL interaction,was examined. Two-way ANOVA showed significant effectsof MPTP and pretreatment on the mitochondrial and cytosolicp53 expression and mitochondrial p53/Bcl-xL interaction(Supplementary Table 5). A post hoc test revealed thatMPTP-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), andthe effect of MPPE was more pronounced than that ofselegiline (Fig. 5a–c).

MPPE Attenuates MPTP-Induced Changesin Mitochondrial Transmembrane Potential, CytosolicRelease of Cytochrome c, and Cleavage of Caspase-3:Comparison With Selegiline, Pifithrin-μ, or GeneticInhibition of p53

As MPTP-induced mitochondrial p53 translocation and p53/Bcl-xL interaction were significantly attenuated byMPPE, we

extended our finding by examining the effect of MPPE onchanges in mitochondrial transmembrane potential inducedby MPTP in WT- and p53 (−/−)-mice. Three-way ANOVAshowed significant effects of MPTP and pretreatment and asignificant interaction between p53 gene knockout and MPTPon the mitochondrial transmembrane potential, cytosolic cy-tochrome c release, and cleaved caspase-3 6 h after the finalMPTP treatment. p53 gene knockout also exerted significanteffect on the cytosolic cytochrome c release (SupplementaryTable 5). A post hoc test indicated that mitochondrial trans-membrane potential was significantly reduced (P<0.01) 6 hafter the final MPTP treatment inWTmice, and this reductionwas significantly attenuated by MPPE (P<0.01), selegiline(P<0.05), pifithrin-μ (P<0.05), or p53 gene depletion(P<0.01) (Fig. 5d). Consistently, MPTP-induced cytosoliccytochrome c release (P<0.01) and subsequent increase incleaved caspase-3 (P<0.01) were significantly attenuated byMPPE (P<0.01), selegiline (P<0.05), pifithrin-μ (P<0.01),or p53 gene depletion (P<0.01) (Fig. 5e, f). However, MPPEor selegiline did not show any additional protective effectsagainst p53 gene knockout-mediated attenuation, suggestingthat p53 is a critical target for either compound (Fig. 5d–f).

MPPE Attenuates MPTP-Induced Decreases in TyrosineHydroxylase (TH) Expression and Dopamine Level,and Increase in Dopamine Turnover Rate: ComparisonWith Selegiline, Pifithrin-μ, or Genetic Inhibition of p53

Next, we examined the effect of MPPE on the MPTP-induceddopaminergic toxicity. Three-way ANOVA showed signifi-cant effects of MPTP and pretreatment (nigrostriatal TH-immunoreactivities and striatal TH expression) and a signifi-cant interaction between p53 gene knockout and MPTP(nigrostriatal TH-immunoreactivities and striatal TH expres-sion) (Supplementary Table 6). As shown in Fig. 6a–c, a posthoc test indicated that striatal TH-immunoreactivity (TH-IR)and TH expression were significantly decreased (P<0.01) in

�Fig. 6 Effect of pifithrin-μ, selgiline, or MPPE on MPTP-induceddopaminergic loss in wild-type (WT) and p53 gene knockout[p53(−/−)] mice. a–c Effect on decreases in TH-immunoreactivity(a, b) and TH expression (c) induced by MPTP in the striatum. d, eEffect on decreases in TH-immunoreactivity induced by MPTP in thesubstantia nigra. Dashed lines indicate substantia nigra pars compactafor quantitative analysis. f, g Effect on decrease in dopamine level (f)and increase in dopamine turnover rate (g) induced by MPTP in thestriatum. 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 five animals. *P<0.05,**P<0.01 vs. corresponding Veh + Sal; #P<0.05, ##P<0.01 vs. WTmice treated with Veh + MPTP; &P<0.05 vs. WT mice treated withSel + MPTP (three-way ANOVA was followed by Fisher’s LSDpairwise comparisons). Scale bar=500 μm

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the striatum of WT mice. MPTP-induced decreases in TH-IRand TH expression were significantly attenuated by MPPE(P<0.01), selegiline (P<0.05), pifithrin-μ (TH-IR, P<0.01;TH expression, P<0.05) or p53 gene depletion (TH-IR,P<0.01; TH expression, P<0.05). The results from the stria-tum are comparable to those from the nigral area (Fig. 6d, e).

Consistent with the result of TH, three-way ANOVAshowed significant effects of p53 gene knockout (dopaminelevel in the striatum), MPTP (dopamine level and dopamineturnover rate in the striatum), and pretreatment (dopaminelevel and dopamine turnover rate in the striatum) and signifi-cant interactions between p53 gene knockout and MPTP (do-pamine level in the striatum) or p53 gene knockout and pre-treatment (dopamine level in the striatum) (SupplementaryTable 6). A post hoc test revealed that MPTP treatment sig-nificantly decreased (P<0.01) dopamine (DA) level and sig-nificantly increased (P<0.01) DA turnover rate in the stria-tum. These changes were significantly reversed by MPPE(both, P<0.01), selegiline (DA level, P<0.05; DA turnoverrate, 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 DOPACand HVA was presented in Supplementary Fig. 2. MPPE,selegiline, or pifithrin-μ did not provide additional protectiveeffects against attenuation mediated by genetic depletion ofp53 (Fig. 6).

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

Three-way ANOVA indicated significant effects of p53 geneknockout (rota-rod performance), MPTP (locomotor activityand rota-rod performance), and pretreatment (rota-rodperformance) and a significant interaction between p53 geneknockout and MPTP (rota-rod performance) (SupplementaryTable 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 p53gene depletion (P<0.01) resulted in a significant attenuationagainst MPTP-induced hypolocomotor activity. However,MPPE, selegiline, or pifithrin-μ did not affect the locomotoractivity in MPTP-treated p53 (−/−) mice (Fig. 7a). The effectof MPPE, selegiline, pifithrin-μ, or p53 gene knockout onlocomotor activity paralleled that on rota-rod performance(Fig. 7b).

Discussion

The present study shows that MPPE provides neuroprotectionwith behavioral safety (as assessed by locomotor activity, lo-comotor pattern [70], and conditioned place preference).

MPPE-mediated antioxidant efficacy in mitochondrial frac-tion was more pronounced than that in cytosolic fraction.MPPE treatment positively modulated MPTP-induced neuro-toxic alterations in mitochondrial Mn-SOD activity and ex-pression, mitochondrial translocation of p53, mitochondrialtransmembrane potential, cytosolic cytochrome c release,cleaved caspase-3, and dopaminergic system. Neuroprotec-tion offered by MPPE appeared to be more prominent thanthat offered by selegiline.

Selegiline-induced behavioral side effects have been wellknown to be caused by its metabolites, methamphetamine,and amphetamine [10, 74, 75]. However, it was demonstratedthat MPPE does not metabolize to amphetamines [12]. Asexpected, repeated treatment with selegiline in significant be-havioral sensitization and conditioned place preference, al-though it is much less pronounced than in the case of meth-amphetamine. However, repeated treatment with MPPE didnot significantly induce behavioral sensitization or

Fig. 7 Effect of pifithrin-μ, selgiline, or MPPE on the behavioralimpairments induced by MPTP in wild-type (WT) and p53 geneknockout [p53(−/−)] mice. a Effect on hypolocomotor activity inducedby MPTP. b Effect on reduction in rota-rod performance induced byMPTP. 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 10–12 animals. *P<0.01 vs.corresponding Veh + Sal; #P<0.05, ##P<0.01 vs. WT mice treated withVeh + MPTP; &P<0.05 vs. WT mice treated with Sel + MPTP (three-way ANOVAwas followed by Fisher’s LSD pairwise comparisons)

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conditioned place preference in this study. Interestingly, it hasbeen suggested that selegiline possesses therapeutic potentialsfor management of drug abuse [2–4]. In these studies, it wasproposed that the reduction of dopamine metabolism or thenormalization of glucose utilization in the limbic system isinvolved in selegiline-mediated inhibition of cocaine-induced self-administration or subjective euphoria. In addi-tion, Davidson et al. [76] showed that selegiline treatmentattenuates total AMPA GluR1 levels and its phosphorylationinduced by chronic methamphetamine in the prefrontal cortex,which has been known to play a role in behavioral sensitiza-tion or drug-seeking behavior. Consistently, we observed thatboth MPPE and selegiline attenuate behavioral sensitizationand conditioned place preference induced by the non-toxicdose (1 mg/kg, i.p.) of methamphetamine in mice, althoughattenuation by MPPE was more evident than that byselegiline. Thus, it remains to be determined whether MPPEaffects dopaminergic degeneration induced by the toxic dose[41, 57, 67, 77, 78] of MA.

MAO-B is mainly located in the outer membrane of mito-chondria and primarily metabolizes dopamine in brain [79].We showed here that treatment with MPTP resulted in a sig-nificant increase of MAO-B activity in the striatum, and ourresult is consistent with previous report [80]. Since hydrogenperoxide is produced during MAO-B-mediated oxidation ofmonoamine neurotransmitters, it could be postulated that ele-vated MAO-B activity might induce mitochondrial oxidativestress and, possibly, consequent inhibition of complex I activ-ity. This postulation is supported by previous studies usingMAO-B transgenic mice [81–83]. In addition, it has beenreported that MAO-B activity increases gradually with agingin human brain [84, 85], which may contribute to the predis-position to Parkinson’s disease [86]. As reflected by our find-ing, it is possible that MPPE- or selegiline-mediated MAO-Binhibition plays a positive role in attenuating oxidative stressinduced by MPTP.

It is well documented that mitochondrial complex I inhibi-tion mediated by MPP+, an active metabolite of MPTP, is oneof the main mechanisms in the neuropathology induced byMPTP [87]. Mitochondrial complex I inhibition induces in-creases in ROS production and mitochondrial oxidative stress,which in turn lead to further irreversible inhibition of mitochon-drial complex I in a positive feedback manner [88–90]. In thisstudy, MPPE or selegiline mitigated MPTP-induced complex Iinhibition andmitochondrial oxidative stress. Restoration of theexpression and activity of mitochondrial Mn-SOD (SOD-2)may contribute to the MPPE-mediated antioxidant defense.

It has been reported that UCP-2 provides neuroprotectionin various neurodegenerative processes via suppressing thefree radical generation, mitochondrial calcium influx, andcaspase-3 activation [91–93]. In addition, previous studiesshowed that MPTP-induced dopaminergic cell death and ox-idative stress were more pronounced in UCP-2 gene knockout

mice, while less pronounced in UCP-2 transgenic mice thanwild-type mice [72, 73]. As MPPE upregulated UCP-2mRNA expression after MPTP treatment, the enhancementof UCP-2 may be important for MPPE-mediated inhibitionof mitochondrial oxidative stress and mitochondrial dysfunc-tion, although underlying mechanism remains to be explored.

p53 is a tumor-suppressor gene, and it has been suggestedas one of the redox-sensitive transcription factors [94]. It iswell known that p53 induces apoptosis and oxidative stressthrough transcription-dependent and transcription-independent mechanisms [28, 95, 96] and that mitochondrialtranslocation of p53 is a key event during the transcription-independent apoptosis mediated by p53 [28, 95]. In this study,we observed significant increases in mitochondrial p53 trans-location and concomitant p53/Bcl-xL interaction as early as1 h after the final treatment with MPTP, and these levelspeaked at the 6 h time-point. These changes were followedby an impaired mitochondrial transmembrane potential, cyto-solic cytochrome c release, and capase-3 cleavage. Our find-ings are in agreement with previous studies [26, 27] showingthat mitochondrial p53 translocation is important for rapidpro-apoptotic responses. Endo et al. [26] showed that mito-chondrial p53 began to increase 1 h after cerebral ischemiain vivo, but nuclear p53 was first observed 72 h later, whendelayed neuronal death had been already observed. In addi-tion, Erster et al. [27] suggested that mitochondrial p53 trig-gers a rapid wave of caspase-3 activation and amplifies theslower transcriptional responses mediated by nuclear p53.This study is the first investigation on the role of mitochon-drial p53 translocation in MPTP-induced neurotoxicity.

In this study, MPTP-induced interaction between mito-chondrial p53 and Bcl-xL was significantly attenuated bypifithrin-μ, a mitochondrial p53 inhibitor. MPPE or selegilinealso significantly inhibited mitochondrial p53 translocationand consequent p53/Bcl-xL interaction in our study. p53/Bcl-xL interaction has been demonstrated to dissociate Bcl-xL and pro-apoptotic Bax or Bak, which in turn leads to mi-tochondrial membrane permeabilization and cytosolic releaseof cytochrome c [28, 31]. Furthermore, Zhao et al. [97] dem-onstrated that mitochondrial p53 physically binds to mito-chondrial Mn-SOD and suppresses its superoxide scavengingactivity after 12-O-tetradecanoylphorbol-13-acetate (TPA)treatment in vitro. Therefore, it may be possible that MPPE-induced inhibition of mitochondrial translocation of p53 is aprerequisite for exerting antioxidant and anti-apoptotic activ-ities. This is clearly supported by the results obtained fromp53 (−/−) mice. MPTP-induced mitochondrial dysfunction,cytochrome c release, caspase-3 cleavage, and dopaminergicneurotoxicity were significantly less pronounced in p53 (−/−)mice than in WT mice. Moreover, MPPE (or selegiline) didnot show any additional protective effect on the attenuation bygenetic depletion of p53, suggesting that the p53 gene is acritical target for either compound.

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Youdim and colleagues [13, 15, 18] suggested that propar-gyl moiety plays a critical role in the neuroprotective and anti-apoptotic activity provided by propargylamine-basedMAO-Binhibitors. In their studies, propargylamine-based MAO-B in-hibitors upregulated neurotrophic factors [98, 99] and main-tainedmitochondrial membrane integrity via inhibition of pro-apoptotic factors (e.g., Bax and Bad) by induction of anti-apoptotic factors (e.g., Bcl-2 and Bcl-xL) [18, 100] in neuro-toxic conditions. Thus, we cannot rule out the possibility thatpropargyl moiety of MPPE is also important for its anti-apoptotic potentials.

Combined, we showed that MPPE does not significantlyfacilitate locomotor activity or conditioned place preference.Either compound attenuates MA-induced behavioral side ef-fects, although MPPE is more effective than selegiline inblocking the behavioral effects. MPPE attenuated MPTP-induced oxidative stress (mitochondrial > cytosol), mitochon-drial translocation of p53, mitochondrial p53/Bcl-xL interac-tion, impaired mitochondrial transmembrane potential,proapoptotic change, and consequent dopaminergic neurotox-icity (including motor impairment). Since MPPE or selegilinedid not provide any additional protection on the attenuation byp53 gene knockout, it is proposed that the p53 gene is a criticaltarget for either compound. Finally, the results of the presentstudy would seem to establish MPPE as a potential drug de-velopment candidate for the treatment of PD.

Acknowledgments This study was supported by a grant(14182MFDS979) from the Korea Food and Drug Administration andpartially by Basic Science Research Program through the National Re-search Foundation of Korea (NRF) funded by the Ministry of Science,ICT and Future Planning (no. NRF-2013R1A1A2060894 and no. NRF-2013R1A1A1007378), Republic of Korea, and by the National ResearchFoundation of Korea (NRF) grant funded by the Korea government(MSIP) (2011–0018355). Yunsung Nam and The-Vinh Tran were sup-ported by the BK21 PLUS program, National Research Foundation ofKorea, Republic of Korea. Equipment at the Institute of New Drug De-velopment Research (Kangwon National University) was used for thisstudy. The English in this document has been checked by at least twoprofessional editors, both native speakers of English (Beverly Hills En-glish, Los Angeles, CA90024, USA).

Compliance with Ethical Standards

Conflict of Interest The authors declare that they have no competinginterests.

References

1. Teo KC, Ho SL (2013) Monoamine oxidase-B (MAO-B) inhibi-tors: implications for disease-modification in Parkinson’s disease.Transl Neurodegener 2(1):19. doi:10.1186/2047-9158-2-19

2. HoMC, Cherng CG, Tsai YP, Chiang CY, Chuang JY, Kao SF, YuL (2009) Chronic treatment with monoamine oxidase-B inhibitors

decreases cocaine reward in mice. Psychopharmacology (Berl)205(1):141–149. doi:10.1007/s00213-009-1524-5

3. Bartzokis G, Beckson M, Newton T, Mandelkern M, Mintz J,Foster JA, Ling W, Bridge TP (1999) Selegiline effects oncocaine-induced changes in medial temporal lobe metabolismand subjective ratings of euphoria. Neuropsychopharmacology20(6):582–590. doi:10.1016/S0893-133X(98)00092-X

4. Houtsmuller EJ, Notes LD, Newton T, van Sluis N, Chiang N,Elkashef A, Bigelow GE (2004) Transdermal selegiline and intra-venous cocaine: safety and interactions. Psychopharmacology(Berl) 172(1):31–40. doi:10.1007/s00213-003-1616-6

5. Eisler T, Teravainen H, Nelson R, Krebs H, Weise V, Lake CR,Ebert MH, Whetzel N et al (1981) Deprenyl in Parkinson disease.Neurology 31(1):19–23. doi:10.1212/WNL.31.1.19

6. Heinonen EH, Myllyla V (1998) Safety of selegiline (deprenyl) inthe treatment of Parkinson’s disease. Drug Saf 19(1):11–22. doi:10.2165/00002018-199819010-00002

7. Lees A (2005) Alternatives to levodopa in the initial treatment ofearly Parkinson’s disease. Drugs Aging 22(9):731–740. doi:10.2165/00002512-200522090-00002

8. Engberg G, Elebring T, Nissbrandt H (1991) Deprenyl(selegiline), a selective MAO-B inhibitor with active metabolites;effects on locomotor activity, dopaminergic neurotransmissionand firing rate of nigral dopamine neurons. J Pharmacol ExpTher 259(2):841–847

9. Kwon YS, Ann HS, Nabeshima T, Shin EJ, Kim WK, Jhoo JH,Jhoo WK, Wie MB et al (2004) Selegiline potentiates the effectsof EGb 761 in response to ischemic brain injury. Neurochem Int45(1):157–170. doi:10.1016/j.neuint.2003.10.005

10. Okuda C, Segal DS, Kuczenski R (1992) Deprenyl alters behaviorand caudate dopamine through an amphetamine-like action.Pharmacol Biochem Behav 43(4):1075–1080. doi:10.1016/0091-3057(92)90484-W

11. KwanE, Baker GB, Shuaib A, Ling L, ToddKG (2000) N-methyl,N-propargyl-2-phenylethylamine (MPPE), an analog of deprenyl,increases neuronal cell survival in thiamin deficiency encephalop-athy. Drug Development Research 51(4):244–252. doi:10.1002/ddr.5

12. Rittenbach K, Sloley BD, Ling L, Coutts RT, Shan J, Baker GB(2005) A rapid, sensitive electron-capture gas chromatographicprocedure for analysis of metabolites of N-methyl, N-propargylphenylethylamine, a potential neuroprotective agent. JPharmacol Toxicol Methods 52(3):373–378. doi:10.1016/j.vascn.2005.07.001

13. Bar-Am O, Amit T, Weinreb O, Youdim MB, Mandel S (2010)Propargylamine containing compounds as modulators of proteo-lytic cleavage of amyloid-beta protein precursor: involvement ofMAPK and PKC activation. J Alzheimers Dis 21(2):361–371. doi:10.3233/JAD-2010-100150

14. Chen JJ, SwopeDM (2005) Clinical pharmacology of rasagiline: anovel, second-generation propargylamine for the treatment ofParkinson disease. J Clin Pharmacol 45(8):878–894. doi:10.1177/0091270005277935

15. Maruyama W, Youdim MB, Naoi M (2001) Antiapoptotic prop-erties of rasagiline, N-propargylamine-1(R)-aminoindan, and itsoptical (S)-isomer, TV1022. Ann N Y Acad Sci 939:320–329.doi:10.1111/j.1749-6632.2001.tb03641.x

16. Trudler D, Weinreb O, Mandel SA, Youdim MB, Frenkel D(2014) DJ-1 deficiency triggers microglia sensitivity to dopaminetoward a pro-inflammatory phenotype that is attenuated byrasagiline. J Neurochem 129(3):434–447. doi:10.1111/jnc.12633

17. Villaran RF, Tomas-Camardiel M, de Pablos RM, Santiago M,Herrera AJ, Navarro A, Machado A, Cano J (2008) Endogenousdopamine enhances the neurotoxicity of 3-nitropropionic acid inthe striatum through the increase of mitochondrial respiratory

6266 Mol Neurobiol (2016) 53:6251–6269

inhibition and free radicals production. Neurotoxicology 29(2):244–258. doi:10.1016/j.neuro.2007.11.001

18. Weinreb O, Bar-Am O, Amit T, Chillag-Talmor O, Youdim MB(2004) Neuroprotection via pro-survival protein kinase C isoformsassociated with Bcl-2 family members. FASEB J 18(12):1471–1473. doi:10.1096/fj.04-1916fje

19. Camins A, Pallas M, Silvestre JS (2008) Apoptotic mechanismsinvolved in neurodegenerative diseases: experimental and thera-peutic approaches. Methods Find Exp Clin Pharmacol 30(1):43–65. doi:10.1358/mf.2008.30.1.1090962

20. Checler F, Alves da Costa C (2014) p53 in neurodegenerativediseases and brain cancers. Pharmacol Ther 142(1):99–113. doi:10.1016/j.pharmthera.2013.11.009

21. Sawa A (2001) Mechanisms for neuronal cell death and dysfunc-tion in Huntington’s disease: pathological cross-talk between thenucleus and the mitochondria? J Mol Med (Berl) 79(7):375–381.doi:10.1007/s001090100223

22. Sugrue MM, Tatton WG (2001) Mitochondrial membrane poten-tial in aging cells. Biol Signals Recept 10(3–4):176–188. doi:10.1159/000046886

23. Kang H, Shin JH (2014) Repression of rRNA transcription byPARIS contributes to Parkinson’s disease. Neurobiol Dis 73C:220–228. doi:10.1016/j.nbd.2014.10.003

24. Mogi M, Kondo T, Mizuno Y, Nagatsu T (2007) p53 protein,interferon-gamma, and NF-kappaB levels are elevated in the par-kinsonian brain. Neurosci Lett 414(1):94–97. doi:10.1016/j.neulet.2006.12.003

25. Mandir AS, Simbulan-Rosenthal CM, Poitras MF, Lumpkin JR,Dawson VL, Smulson ME, Dawson TM (2002) A novel in vivopost-translational modification of p53 by PARP-1 in MPTP-induced parkinsonism. J Neurochem 83(1):186–192. doi:10.1046/j.1471-4159.2002.01144.x

26. Endo H, Kamada H, Nito C, Nishi T, Chan PH (2006)Mitochondrial translocation of p53 mediates release of cyto-chrome c and hippocampal CA1 neuronal death after transientglobal cerebral ischemia in rats. J Neurosci 26(30):7974–7983.doi:10.1523/JNEUROSCI.0897-06.2006

27. Erster S, Mihara M, Kim RH, Petrenko O, Moll UM (2004)In vivo mitochondrial p53 translocation triggers a rapid first waveof cell death in response to DNA damage that can precede p53target gene activation. Mol Cell Biol 24(15):6728–6741. doi:10.1128/MCB.24.15.6728-6741.2004

28. Speidel D (2010) Transcription-independent p53 apoptosis: analternative route to death. Trends Cell Biol 20(1):14–24. doi:10.1016/j.tcb.2009.10.002

29. Chipuk JE, Kuwana T, Bouchier-Hayes L, Droin NM, NewmeyerDD, Schuler M, Green DR (2004) Direct activation of Bax by p53mediates mitochondrial membrane permeabilization and apopto-sis. Science 303(5660):1010–1014. doi:10.1126/science.1092734

30. Mihara M, Erster S, Zaika A, Petrenko O, Chittenden T, PancoskaP, Moll UM (2003) p53 has a direct apoptogenic role at the mito-chondria. Mol Cell 11(3):577–590. doi:10.1016/S1097-2765(03)00050-9

31. Wolff S, Erster S, Palacios G, Moll UM (2008) p53’s mitochon-drial translocation and MOMP action is independent of Puma andBax and severely disrupts mitochondrial membrane integrity. CellRes 18(7):733–744. doi:10.1038/cr.2008.62

32. Trimmer PA, Smith TS, Jung AB, Bennett JP Jr (1996) Dopamineneurons from transgenic mice with a knockout of the p53 generesist MPTP neurotoxicity. Neurodegeneration 5(3):233–239. doi:10.1006/neur.1996.0031

33. DuanW, ZhuX, LadenheimB,YuQS, Guo Z, Oyler J, Cutler RG,Cadet JL et al (2002) p53 inhibitors preserve dopamine neuronsand motor function in experimental parkinsonism. Ann Neurol52(5):597–606. doi:10.1002/ana.10350

34. Tsukada T, Tomooka Y, Takai S, Ueda Y, Nishikawa S, Yagi T,Tokunaga T, Takeda N et al (1993) Enhanced proliferative poten-tial in culture of cells from p53-deficient mice. Oncogene 8(12):3313–3322

35. Shin EJ, Nabeshima T, Suh HW, Jhoo WK, Oh KW, Lim YK,Kim DS, Choi KH et al (2005) Ginsenosides attenuatemethamphetamine-induced behavioral side effects in mice via ac-tivation of adenosine A2A receptors: possible involvements of thestriatal reduction in AP-1 DNA binding activity andproenkephalin gene expression. Behav Brain Res 158(1):143–157. doi:10.1016/j.bbr.2004.08.018

36. Shin EJ, Bing G, Chae JS, Kim TW, Bach JH, Park DH, YamadaK, Nabeshima T et al (2009) Role of microsomal epoxide hydro-lase in methamphetamine-induced drug dependence in mice. JNeurosci Res 87(16):3679–3686. doi:10.1002/jnr.22166

37. Shin EJ, Bach JH, Nguyen TT, Jung BD, Oh KW, Kim MJ, JangCG, Ali SF et al (2011) Gastrodia Elata Bl attenuates cocaine-induced conditioned place preference and convulsion, but not be-havioral sensitization in mice: Importance of GABA(A) receptors.Cu r r Neu r oph a rmaco l 9 ( 1 ) : 26–29 . do i : 1 0 . 2 174 /157015911795017326

38. ZhangW, Shin EJ, Wang T, Lee PH, Pang H, Wie MB, KimWK,Kim SJ et al (2006) 3-Hydroxymorphinan, a metabolite of dextro-methorphan, protects nigrostriatal pathway against MPTP-eliciteddamage both in vivo and in vitro. FASEB J 20(14):2496–2511.doi:10.1096/fj.06-6006com

39. Muralikrishnan D, Samantaray S, Mohanakumar KP (2003) D-deprenyl protects nigrostriatal neurons against 1-methyl-4-phe-nyl-1,2,3,6-tetrahydropyridine-induced dopaminergic neurotoxic-ity. Synapse 50(1):7–13. doi:10.1002/syn.10239

40. Venna VR, Verma R, O’Keefe LM, Xu Y, Crapser J, Friedler B,McCullough LD (2014) Inhibition of mitochondrial p53 abolishesthe detrimental effects of social isolation on ischemic brain injury.Stroke 45(10):3101–3104. doi:10.1161/STROKEAHA.114.006553

41. Shin EJ, Shin SW, Nguyen TT, Park DH, Wie MB, Jang CG, NahSY, Yang BW et al (2014) Ginsenoside Re rescuesmethamphetamine-induced oxidative damage, mitochondrial dys-function, microglial activation, and dopaminergic degeneration byinhibiting the protein kinase Cdelta gene. Mol Neurobiol 49(3):1400–1421. doi:10.1007/s12035-013-8617-1

42. Franklin KBJ, Paxinos G (2008) The mouse brain in stereotaxiccoordinates, 3rd edn. Academic, San Diego

43. Borgkvist A, Usiello A, Greengard P, Fisone G (2007) Activationof the cAMP/PKA/DARPP-32 signaling pathway is required formorphine psychomotor stimulation but not for morphine reward.Neuropsychopharmacology 32(9):1995–2003. doi:10.1038/sj.npp.1301321

44. Mijatovic J, Airavaara M, Planken A, Auvinen P, Raasmaja A,Piepponen TP, Costantini F, Ahtee L et al (2007) Constitutive Retactivity in knock-in multiple endocrine neoplasia type B miceinduces profound elevation of brain dopamine concentration viaenhanced synthesis and increases the number of TH-positive cellsin the substantia nigra. J Neurosci 27(18):4799–4809. doi:10.1523/JNEUROSCI.5647-06.2007

45. Tran TV, Nam Y, Mai HN, Shin EJ, Kim HC (2014) MPPE, aselegiline analog, attenuates MPTP-induced dopaminergic toxici-ty with negligible behavioral side effects. Brain Conference 2014(abstract P-137). Seoul. The Korean Society of Brain and NeuralScience, Asian Society for Neuropathology, and the KoreanSociety for Neurodegenerative Disease. pp. 199

46. Freed C, Revay R, Vaughan RA, Kriek E, Grant S, Uhl GR, KuharMJ (1995) Dopamine transporter immunoreactivity in rat brain. JComp Neurol 359(2):340–349. doi:10.1002/cne.903590211

47. Hoffman AF, Lupica CR, Gerhardt GA (1998) Dopamine trans-porter activity in the substantia nigra and striatum assessed by

Mol Neurobiol (2016) 53:6251–6269 6267

high-speed chronoamperometric recordings in brain slices. JPharmacol Exp Ther 287(2):487–496

48. Sun J, Xu J, Cairns NJ, Perlmutter JS,Mach RH (2012) DopamineD1, D2, D3 receptors, vesicular monoamine transporter type-2(VMAT2) and dopamine transporter (DAT) densities in aged hu-man brain. PLoS One 7(11):e49483. doi:10.1371/journal.pone.0049483

49. Miller GW, Gainetdinov RR, Levey AI, Caron MG (1999)Dopamine transporters and neuronal injury. Trends PharmacolSci 20(10):424–429. doi:10.1016/S0165-6147(99)01379-6

50. Davey GP, Clark JB (1996) Threshold effects and control of oxi-dative phosphorylation in nonsynaptic rat brain mitochondria. JNeurochem 66(4):1617–1624. doi:10.1046/j.1471-4159.1996.66041617.x

51. Davey GP, Peuchen S, Clark JB (1998) Energy thresholds in brainmitochondria. Potential involvement in neurodegeneration. J BiolChem 273(21):12753–12757. doi:10.1074/jbc.273.21.12753

52. Perier C, Vila M (2012) Mitochondrial biology and Parkinson’sdisease. Cold Spring Harb Perspect Med 2(2):a009332. doi:10.1101/cshperspect.a009332

53. Dauer W, Przedborski S (2003) Parkinson’s disease: mechanismsand models. Neuron 39(6):889–909. doi:10.1016/S0896-6273(03)00568-3

54. Gogvadze V, Orrenius S, Zhivotovsky B (2003) Analysis of mi-tochondrial dysfunction during cell death. Curr Protoc Cell BiolChapter 18:Unit 18.5. doi:10.1002/0471143030.cb1805s19

55. NamY,WieMB, Shin EJ, Nguyen TT, Nah SY, Ko SK, Jeong JH,Jang CG et al (2015) Ginsenoside Re protects methamphetamine-induced mitochondrial burdens and proapoptosis via genetic inhi-bition of protein kinase C delta in human neuroblastoma dopami-nergic SH-SY5Y cell lines. J Appl Toxicol 35(8):927–944. doi:10.1002/jat.3093

56. Xiong Y, Gu Q, Peterson PL, Muizelaar JP, Lee CP (1997)Mitochondrial dysfunction and calcium perturbation induced bytraumatic brain injury. J Neurotrauma 14(1):23–34. doi:10.1089/neu.1997.14.23

57. Nguyen XK, Lee J, Shin EJ, Dang DK, Jeong JH, Nguyen TT,Nam Y, Cho HJ et al (2015) Liposomal melatonin rescuesmethamphetamine-elicited mitochondrial burdens, pro-apoptosis,and dopaminergic degeneration through the inhibition PKCdeltagene. J Pineal Res 58(1):86–106. doi:10.1111/jpi.12195

58. Dhingra NK, Raju TR, Meti BL (1997) Selective reduction ofmonoamine oxidase A and B in the frontal cortex of subordinaterats. Brain Res 758(1–2):237–240. doi:10.1016/S0006-8993(96)01477-1

59. Janssen AJ, Trijbels FJ, Sengers RC, Smeitink JA, van den HeuvelLP, Wintjes LT, Stoltenborg-Hogenkamp BJ, Rodenburg RJ(2007) Spectrophotometric assay for complex I of the respiratorychain in tissue samples and cultured fibroblasts. Clin Chem 53(4):729–734. doi:10.1373/clinchem.2006.078873

60. Tran HY, Shin EJ, Saito K, Nguyen XK, Chung YH, Jeong JH,Bach JH, Park DH et al (2012) Protective potential of IL-6 againsttrimethyltin-induced neurotoxicity in vivo. Free Radic Biol Med52(7):1159–1174. doi:10.1016/j.freeradbiomed.2011.12.008

61. Shin EJ, Jeong JH, Bing G, Park ES, Chae JS, Yen TP, Kim WK,Wie MB et al (2008) Kainate-induced mitochondrial oxidativestress contributes to hippocampal degeneration in senescence-accelerated mice. Cell Signal 20(4):645–658. doi:10.1016/j.cellsig.2007.11.014

62. KimHC, JhooWK, KimWK, Suh JH, Shin EJ, Kato K, Ho Ko K(2000) An immunocytochemical study of mitochondrialmanganese-superoxide dismutase in the rat hippocampus afterkainate administration. Neurosci Lett 281(1):65–68. doi:10.1016/S0304-3940(99)00969-6

63. Kim HC, Yamada K, Nitta A, Olariu A, Tran MH, Mizuno M,Nakajima A, Nagai T et al (2003) Immunocytochemical evidence

that amyloid β (1–42) impairs endogenous antioxidant systemsin vivo. Neuroscience 119(2):399–419. doi:10.1016/S0306-4522(02)00993-4

64. Kurobe N, Kato K (1991) Sensitive enzyme immunoassay for ratMn superoxide dismutase:Tissue distribution and developmentalprofiles in the rat central nervous tissue, liver, and kidney. BiomedRes 12(2):97–103. doi:10.2220/biomedres.12.97

65. Vijitruth R, Liu M, Choi DY, Nguyen XV, Hunter RL, Bing G(2006) Cyclooxygenase-2 mediates microglial activation and sec-ondary dopaminergic cell death in the mouse MPTP model ofParkinson’s disease. J Neuroinflammation 3:6. doi:10.1186/1742-2094-3-6

66. Oliver CN, Ahn BW, Moerman EJ, Goldstein S, Stadtman ER(1987) Age-related changes in oxidized proteins. J Biol Chem262(12):5488–5491

67. Shin EJ, Duong CX, Nguyen XK, Li Z, Bing G, Bach JH, ParkDH, Nakayama K et al (2012) Role of oxidative stress inmethamphetamine-induced dopaminergic toxicity mediated byprotein kinase Cdelta. Behav Brain Res 232(1):98–113. doi:10.1016/j.bbr.2012.04.001

68. Lebel CP, Bondy SC (1990) Sensitive and rapid quantitation ofoxygen reactive species formation in rat synaptosomes.Neurochem Int 17(3):435–440. doi:10.1016/0197-0186(90)90025-O

69. Wang Q, Shin EJ, Nguyen XK, Li Q, Bach JH, Bing G, KimWK,Kim HC et al (2012) Endogenous dynorphin protects againstneurotoxin-elicited nigrostriatal dopaminergic neuron damageand motor deficits in mice. J Neuroinflammation 9:124. doi:10.1186/1742-2094-9-124

70. Jhoo WK, Shin EJ, Lee YH, Cheon MA, Oh KW, Kang SY, LeeC, Yi BC et al (2000) Dual effects of dextromethorphan oncocaine-induced conditioned place preference in mice. NeurosciLett 288(1):76–80. doi:10.1016/S0304-3940(00)01188-5

71. He S, Grasing K (2006) L-methamphetamine and selective MAOinhibitors decrease morphine-reinforced and non-reinforced be-havior in rats; Insights towards selegiline’s mechanism of action.Pharmacol Biochem Behav 85(4):675–688. doi:10.1016/j.pbb.2006.10.022

72. Andrews ZB, Horvath B, Barnstable CJ, Elsworth J, Yang L, BealMF, Roth RH, Matthews RT et al (2005) Uncoupling protein-2 iscritical for nigral dopamine cell survival in a mouse model ofParkinson’s disease. J Neurosci 25(1):184–191. doi:10.1523/JNEUROSCI.4269-04.2005

73. Lu M, Zhao FF, Tang JJ, Su CJ, Fan Y, Ding JH, Bian JS, Hu G(2012) The neuroprotection of hydrogen sulfide against MPTP-induced dopaminergic neuron degeneration involves uncouplingprotein 2 rather than ATP-sensitive potassium channels. AntioxidRedox Signal 17(6):849–859. doi:10.1089/ars.2011.4507

74. WuWR, Zhu XZ (1999) The amphetamine-like reinforcing effectand mechanism of L-deprenyl on conditioned place preference inmice. Eur J Pharmacol 364(1):1–6. doi:10.1016/S0014-2999(98)00831-0

75. Yasar S, Schindler CW, Thorndike EB, Szelenyi I, Goldberg SR(1993) Evaluation of the stereoisomers of deprenyl foramphetamine-like discriminative stimulus effects in rats. JPharmacol Exp Ther 265(1):1–6

76. Davidson C, Chen Q, Zhang X, Xiong X, Lazarus C, Lee TH,Ellinwood EH (2007) Deprenyl treatment attenuates long-termpre- and post-synaptic changes evoked by chronic methamphet-amine. Eur J Pharmacol 573(1–3):100–110. doi:10.1016/j.ejphar.2007.06.046

77. Krasnova IN, Cadet JL (2009) Methamphetamine toxicity andmessengers of death. Brain Res Rev 60(2):379–407. doi:10.1016/j.brainresrev.2009.03.002

78. Ares-Santos S, Granado N, Espadas I, Martinez-Murillo R,Moratalla R (2014) Methamphetamine causes degeneration of

6268 Mol Neurobiol (2016) 53:6251–6269

dopamine cell bodies and terminals of the nigrostriatal pathwayevidenced by silver staining. Neuropsychopharmacology 39(5):1066–1080. doi:10.1038/npp.2013.307

79. Shih JC, Chen K, Ridd MJ (1999) Monoamine oxidase: fromgenes to behavior. Annu Rev Neurosci 22:197–217. doi:10.1146/annurev.neuro.22.1.197

80. Kang YK, Oh HS, Cho YH, Kim YJ, Han YG, Nam SH (2010)Effects of a silkworm extract on dopamine and monoamineoxidase-B activity in an MPTP-induced Parkinsons disease mod-el. Laboratory Animal Research 26(3):287–292

81. Lieu CA, Chinta SJ, Rane A, Andersen JK (2013) Age-relatedbehavioral phenotype of an astrocytic monoamine oxidase-Btransgenic mouse model of Parkinson’s disease. PLoS One 8(1):e54200. doi:10.1371/journal.pone.0054200

82. Mallajosyula JK, Kaur D, Chinta SJ, Rajagopalan S, Rane A,Nicholls DG, Di Monte DA, Macarthur H et al (2008) MAO-Belevation in mouse brain astrocytes results in Parkinson’s pathol-ogy. PLoS One 3(2):e1616. doi:10.1371/journal.pone.0001616

83. Siddiqui A,Mallajosyula JK, RaneA, Andersen JK (2010) Abilityto delay neuropathological events associated with astrocyticMAO-B increase in a Parkinsonian mouse model: implicationsfor early intervention on disease progression. Neurobiol Dis40(2):444–448. doi:10.1016/j.nbd.2010.07.004

84. Fowler JS, Volkow ND, Wang GJ, Logan J, Pappas N, Shea C,MacGregor R (1997) Age-related increases in brain monoamineoxidase B in living healthy human subjects. Neurobiol Aging18(4):431–435. doi:10.1016/S0197-4580(97)00037-7

85. Saura J, Andres N, Andrade C, Ojuel J, Eriksson K, Mahy N(1997) Biphasic and region-specific MAO-B response to agingin normal human brain. Neurobiol Aging 18(5):497–507. doi:10.1016/S0197-4580(97)00113-9

86. Kumar MJ, Andersen JK (2004) Perspectives onMAO-B in agingand neurological disease: where do we go from here? MolNeurobiol 30(1):77–89. doi:10.1385/MN:30:1:077

87. Nicklas WJ, Vyas I, Heikkila RE (1985) Inhibition of NADH-linked oxidation in brain mitochondria by 1-methyl-4-phenyl-pyr-idine, a metabolite of the neurotoxin, 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine. Life Sci 36(26):2503–2508. doi:10.1016/0024-3205(85)90146-8

88. Cleeter MW, Cooper JM, Schapira AH (1992) Irreversible inhibi-tion of mitochondrial complex I by 1-methyl-4-phenylpyridinium:evidence for free radical involvement. J Neurochem 58(2):786–789. doi:10.1111/j.1471-4159.1992.tb09789.x

89. Dai DF, Chiao YA, Marcinek DJ, Szeto HH, Rabinovitch PS(2014) Mitochondrial oxidative stress in aging and health span.Longev Healthspan 3:6. doi:10.1186/2046-2395-3-6

90. Zhang Y, Marcillat O, Giulivi C, Ernster L, Davies KJ (1990) Theoxidative inactivation of mitochondrial electron transport chaincomponents and ATPase. J Biol Chem 265(27):16330–16336

91. Diano S, Matthews RT, Patrylo P, Yang L, Beal MF, BarnstableCJ, Horvath TL (2003) Uncoupling protein 2 prevents neuronaldeath including that occurring during seizures: a mechanism forpreconditioning. Endocrinology 144(11):5014–5021. doi:10.1210/en.2003-0667

92. Mattiasson G, Shamloo M, Gido G, Mathi K, Tomasevic G, Yi S,Warden CH, Castilho RF et al (2003) Uncoupling protein-2 pre-vents neuronal death and diminishes brain dysfunction after strokeand brain trauma. Nat Med 9(8):1062–1068. doi:10.1038/nm903

93. Sullivan PG, Dubé C, Dorenbos K, Steward O, Baram TZ (2003)Mitochondrial uncoupling protein-2 protects the immature brainfrom excitotoxic neuronal death. Ann Neurol 53(6):711–717. doi:10.1002/ana.10543

94. Seemann S, Hainaut P (2005) Roles of thioredoxin reductase 1and APE/Ref-1 in the control of basal p53 stability and activity.Oncogene 24(24):3853–3863. doi:10.1038/sj.onc.1208549

95. Chi SW (2014) Structural insights into the transcription-independent apoptotic pathway of p53. BMB Rep 47(3):167–172. doi:10.5483/BMBRep.2014.47.3.261

96. Liu D, Xu Y (2011) p53, oxidative stress, and aging.Antioxid Redox Signal 15(6):1669–1678. doi:10.1089/ars.2010.3644

97. Zhao Y, Chaiswing L, Velez JM, Batinic-Haberle I, Colburn NH,Oberley TD, St Clair DK (2005) p53 translocation to mitochon-dria precedes its nuclear translocation and targets mitochondrialoxidative defense protein-manganese superoxide dismutase.Cancer Res 65(9):3745–3750. doi:10.1158/0008-5472.CAN-04-3835

98. Sagi Y, Mandel S, Amit T, Youdim MB (2007) Activation oftyrosine kinase receptor signaling pathway by rasagiline facilitatesneurorescue and restoration of nigrostriatal dopamine neurons inpost-MPTP-induced parkinsonism. Neurobiol Dis 25(1):35–44.doi:10.1016/j.nbd.2006.07.020

99. Weinreb O, Amit T, Bar-Am O, Youdim MB (2007) Induction ofneurotrophic factors GDNF and BDNF associated with the mech-anism of neurorescue action of rasagiline and ladostigil: new in-sights and implications for therapy. Ann N YAcad Sci 1122:155–168. doi:10.1196/annals.1403.011

100. Mandel S, Weinreb O, Amit T, YoudimMB (2005) Mechanism ofneuroprotective action of the anti-Parkinson drug rasagiline and itsderivatives. Brain Res Brain Res Rev 48(2):379–387. doi:10.1016/j.brainresrev.2004.12.027

Mol Neurobiol (2016) 53:6251–6269 6269