Effect of MAO-B Inhibitors on MPP+ Toxicity in Vivo

RUEY-MEEI WU,a,b RONG-CHI CHEN,c AND CHUANG C. CHIUEHd

aDepartment

of Neurology, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei, Taiwan of Neurology, En-Chu-Kong Hospital, Taipei, Taiwan

cDepartment dUnit

on Neurotoxicology and Neuroprotection, Laboratory of Clinical Science, National Institute of Mental Health, NIH, Clinical Center 10/3D-41, Bethesda, Maryland, USA

ABSTRACT: l-Deprenyl (Selegiline), a selective and irreversible type B monoamine oxidase inhibitor, has been used as an adjunct to levodopa therapy in Parkinsons disease. Recently, it is proposed as a putative neuroprotective agent in delaying the progression of cell death based on its capability of reducing the oxidative stress derived from the MAO-B dependent metabolism of dopamine, and blocking the development of MPTP-parkinsonism. However, a variety of experimental models suggest that l-deprenyl provides neuroprotection through multiple modes of mechanism other than the inhibition of MAOB. We have previously shown that l-deprenyl protects midbrain dopamine neurons from MPP+ toxicity by a novel antioxidant effect. In the present study we examined whether the protection against MPP + toxicity is also shared by other reversible or irreversible MAO-B inhibitors including (+)-deprenyl, Ro166491 and pargyline. Our data show that non of these MAO-B inhibitors changes the dopamine loss in the striatum induced by intranigral injection of MPP +. Our result suggests that l-deprenyl may possess a unique neuroprotective action on nigral neuron against MPP + toxicity independent of the MAO-B inhibition.

INTRODUCTION Monoamine oxidase (MAO) catalyzes the oxidative deamination of monoamine neurotransmitters and neuromodulators such as dopamine, noradrenaline, serotonin (5-hydroxytyramine), and -phenylethylamine (PEA), as well as some exogenous bioactive monoamines. Two types of MAO are existing in mammalian tissues, MAO-A and MAO-B, with different substrate and inhibitors specicity.1 MAO-A deaminates preferentially serotonin and is sensitive to selective inhibitors, such as clorgyline. MAO-B deaminates preferentially PEA and is sensitive to MAO-B inhibitors, such as l-deprenyl. Inhibitors of MAO-A have been proved to be effective antidepressant, while MAO-B blockers have been emphasized in the treatment of Parkinsons disease (PD).
bAddress for correspondence: Dr. Ruey-Meei Wu, Department of Neurology, National Taiwan University Hospital, No. 7, Chuang-Shan South Road, Taipei, 100, Taiwan. e-mail: rmwu@ha.mc.ntu.edu.tw

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l-Deprenyl (Selegiline), rst described in 1965 is a selective irreversible inhibitor of MAO-B.2 As an adjunct to levodopa therapy, it has been shown to provide mild antiparkinsonian effect and reduce motor uctuations in advanced PD.3,4 In addition to the symptomatic effect, greater interest has focused on its role as a neuroprotective agent to prevent neurons from degeneration. Recent clinical trials have shown that early treatment with l-deprenyl delays the progression of disability necessitating the introduction of levodopa therapy and also the symptoms and signs.5,6 The idea of ldeprenyl as a neuroprotective agent for patients with PD is based on the hypothesis that oxidative stress derived from the metabolism of dopamine,7,8 and/or exposure to a selective neurotoxin, such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) may contribute to the cell death in PD patients.9 MPTP, a neurotoxin causing selective destruction of dopaminergic neurons in the midbrain, has been shown to provide a striking pathological analogy to PD.911 l-Deprenyl blocks MPTP toxicity by inhibiting the conversion of MPTP to MPP+, the putative toxic metabolite,12 and suppressing the oxidative stress secondary to the metabolism of dopamine.7 However, recent laboratory studies suggest that l-deprenyl prevents neurodegeneration in various experimental models through a mechanism that does not depend on the inhibition of MAO-B activity. It has been shown that l-deprenyl enhances the activity of antioxidant defense enzymes such as superoxide dismutase and catalase in the striatum that in turn, may protect against MPP+ toxicity.1315 Tatton and colleagues reported that l-deprenyl can rescue neurons from MPTP toxicity and enhance the survival of motor neurons after axotomy by inducing trophic-like substance.16,17 Recently, they also demonstrated that l-deprenyl reduces apoptosis in cultured PC-12 cells through the enhancement of protein synthesis.18,19 Our previous studies have shown that l-deprenyl can suppress the hydroxyl radical formation induced by MPP+ in the striatum and thereby protect midbrain dopamine neurons against injury induced by MPP+ in vivo.20,21 Thus, a unique antioxidant effect of ldeprenyl, independent of the enzymatic inhibition of MAO was proposed to exert its neuroprotection.22 In the present study, we further examined whether the protection on dopaminergic neurons against MPP+ toxicity is shared by other MAO-B inhibitors such as (+)-deprenyl (a stereoisomer of l-deprenyl), RO16-6491, a moclobemide analog with reversible inhibition of MAO-B activity, and pargyline, or it is a pharmacological unique to l-deprenyl itself.

MATERIALS AND METHODS Our previous study has shown that infusion of MPP+ 4.2 nmol into the substantia nigra produced around 50% reduction of dopamine level in the ipsilateral striatum of control and l-deprenyl provided a complete protection on nigral neurons against this injury.21 Thus, in this study, we used the same experimental paradigm to investigate the effects of tested MAO-B inhibitors on midbrain dopamine neurons against MPP+ toxictiy. Sprague-Dawley rats (male, 250350 g) were anesthetized with chloral hydrate (400 mg/kg, i.p.) and mounted in a stereotaxic apparatus (David Kopf Instruments) for intranigral infusion of drugs. MPP+, l-deprenyl, (+)-deprenyl, Ro 166491 and pargyline (RBI, Natic, MA) were dissolved in Ringer solution for infusion, individually. MPP+ 4.2 nmol/L was infused into one side of the substantia nigra to

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produce a moderate nigral injury. The contralateral striatum served as a control. To investigate the protective effect of MAO-B inhibitors against MPP+ toxicity, the other groups of animals received coadministration of MPP+ (4.2 nmol) with equal mole (4.2 nmol) of l-deprenyl, (+)-deprenyl, Ro 16-6491 or pargyline, individually into the substantia nigra. In the control study, some of the animals received Ringer solution (as sham control) or the tested MAO-B inhibitors solution (1 L) alone. The drug solution (1 L) was stereotaxically infused into the substantia nigra of rat (Paxinos & Watson, 1986; coordinates A 3.2, R 1.8, H 1.8) at an infusion rate of 0.2 L/ min via a 30-gauge needle. The injection needle was held in place for additional 3 min after the infusion. The body temperature of the animals was maintained at 37C using a thermo-regulator during the surgery. Four days after the surgery, rats were sacriced by decapitation. Their brains were removed immediately and dissected to obtain tissue from caudate nucleus. Tissue samples were homogenized in 0.1 N perchloride acid. After centrifuge, the clear supernatant (5 L) was injected into a high performance liquid chromatography (HPLC) coupled with electrochemical detector to assay the contents of dopamine and their metabolites. Values were expressed as mean SEM. Statistical analysis was conducted by Students t-test or ANOVA test for comparing the mean differences among groups, using p < 0.05 as signicant. RESULTS Intranigral infusion of Ringer solution (1 L), l-deprenyl (4.2 nmol in 1 L Ringer solution), (+)-deprenyl (4.2 nmol/L), Ro 16-6491(4.2 nmol/1 L), or pargyline (4.2 nmol/ 1 L) led to 9.6 2.9% (N = 3), 11.3 2.5% (N = 3), 10.2 2.2% (N = 3), 10.0 2.0% (N = 3), and 13.5 2.5% (N = 3) reduction in dopamine levels in the ipsilateral striatum four days after surgery, respectively. There was no signicant difference in the small decrease of striatal dopamine in the injected side induced by these agents (ANOVA test). Infusion of MPP+ 4.2 nmol into the substantia nigra produced a signicant reduction of dopamine levels to 53.4 3.1% (N = 10) of control in the ipsilateral striatum four days after the intranigral injection procedure. Coadministration of l-deprenyl (4.2 nmol) with MPP+ signicantly attenuated the dopamine loss caused by MPP+ to 88.0 3.5% of control (N = 8; p < 0.05). However, infusion of (+)-deprenyl (4.2 nmol), Ro 16-6491 (4.2 nmol) or pargyline (4.2 nmol) along with MPP+ into the substantia nigra did not signicantly change the MPP+induced dopamine depletion (FIG . 1), and the corresponding dopamine levels in the ipsilateral striatum were 48.7 3.6% (N = 8), 46.0 3.2% (N = 10) and 52.3 3.2% (N = 8) of control sides, respectively. The dopamine contents in the control sides of striatum were not signicantly different among the experimental groups (range: 40 54 pmol/mg wet weight of tissue; ANOVA test).

DISCUSSION Our present data showed that l-deprenyl provided signicant protection on midbrain dopamine neurons against MPP+ toxicity in vivo. None of the other MAOB inhibitors such as (+)-deprenyl, Ro-16-6491 and pargyline conferred similar

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FIGURE 1. Effects of l-deprenyl, (+)-deprenyl, Ro16-6491 and pargyline on the reduction of dopamine level induced by intranigral injection of MPP +. MPP+ (4.2 nmol in 1 L of Ringer solution) with or without MAO-B inhibitor was administered stereotaxically into one side of the substantia nigra of anesthetized rats. Four days after the intranigral injection, dopamine level in both sides of striatum were measured. Striatal dopamine content in the injected side was expressed as the percentage of control side. Values were expressed as mean SEM. p < 0.01 as compared with the MPP + group.

protection. The MAO-B inhibitors (+)-deprenyl, Ro-166491 and pargyline investigated in this study possess some pharmacological properties different from those of l-deprenyl. (+)-Deprenyl is a stereoisomer of l-deprenyl. It is mostly metabolized to (+)-amphetamine and (+)-methamphetamine. l-Deprenyl is metabolized into () -desmethylselegiline (DES) and 1-()-methamphetamine, which can both be converted to 1-()-amphetamine in the liver.23 The principal pharmacological action of l- or d-amphetamine is the release of catecholamine from the presynaptic neurons. On higher concentration, uptake of catecholamine may take place. The dopamine releasing effect of d-form amphetamine is 10 times potent than that of l-form.24 Moreover, d-amphetamine is a more potent MAO-A inhibitor than l-amphetamine. Thus, the d-amphetamine is more potent in producing elevation of blood pressure and stereotypic locomotor behavior in rats.23 Additionary, d-deprenyl and d-amphetamine have also been shown to exhibit a more potent inhibitory effect on amine uptake than l-derpenyl did.25 In contrast to l-deprenyl and d-deprenyl, Ro 16-6491 is a reversible, highly potent and selective MAO-B inhibitor.26 Also unlike both form of deprenyl, it is not metabolized to potentially active compounds such as amphetamine and methamphetamine, and without the inhibitory effect on dopamine uptake.25 Its analog, lazabemide (Ro19-6327), a very potent and selective MAO-B inhibitor, has been shown to improve daily living activities in early Parkinsons disease.27 Hence, The ndings that d-deprenyl, Ro16-6491 and pargyline (a classic MAO-A and MAO-B inhibitor) did not change the dopamine depletion in the striatum induced by MPP+ suggest that l-deprenyl may possess a unique neuroprotective effect on

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mesencephalic dopamine neurons against MPP+ toxicity in vivo. The mechanism of neuroprotection is independent of the MAO-B inhibition and probably unrelated to other pharmacological properties of l-deprenyl, such as amphetamine effect and amine-uptake inhibition. There is compelling evidence suggesting that the neuroprotective mechanisms of l-deprenyl against MPP+ toxicity are not dependent on inhibition of MAO-B. Our previous studies have demonstrated that l-deprenyl may act like an antioxidant to suppress the generation of hydroxyl radical elicited by MPP+ in vivo without altering MAO-B.20 Moreover, supplementary treatment with l-deprenyl after single injection of MPP+ in the substantia nigra can rescue midbrain dopamine neurons against severe injury induced by MPP+ in vivo.21 Tatton and Greenwood have also observed the rescue effect of l-deprenyl which protects dopaminergic neurons from MPTP even when it is administered 72 hours afterward and in a dose too small to block MAO activity.16 Our recent studies have shown that intrastriatal infusion of MPP+ increases uorescent products of lipid peroxidation.28 The potent inhibitor of lipid peroxidation U-78517F, and hydroxyl radical scavenger dimethylsulfoxide can protect nigral neurons against MPP+ toxicity.28 Accordingly, l-deprenyl may suppress hydroxyl radical formation and therefore cease the propagation of peroxidation of polyunsaturated fatty acids induced by MPP+ in vivo. The ndings that l-deprenyl suppressed the lipid peroxidation induced by NADPH and ascorbic acid in rat brain homogenate were consistent with this hypothesis.29 Furthermore, the protection of l-deprenyl against MPTP toxicity may be also associated with the up-regulation of endogenous antioxidant enzymes and antiapoptotic molecules such as superoxidase dismutase,catalase, glutathione, Bcl-2 and BclXL.14,30 Over-expression of SOD or Bcl-2 in the transgenic mice were reported to protect against MPTP toxicity.31,32 Thus, it appears that l-deprenyl can protect midbrain dopamine neurons against the oxidative injury induced by MPTP through multiple modes of action. In addition to MPTP toxicity, l-deprenyl has been shown to provide neuroprotective effects against 6-OHDA and the noradrenergic neurotoxin DSP-4.33,34 l-Deprenyl, but not other MAO-B inhibitors, has been reported to augment the CNTF gene expression17 and reduce PC12 cell apoptosis.18 More recently, Mytilineou and colleagues reported that l-deprenyl can prevent neurodegeneration induced by excitotoxicity or reduced glutathione secondary to administration of buthionine sulfoximine (BSO), in which the nonselective MAO-B inhibitor pargyline or dopamine uptake inhibitor mazindol did not diminish BSO toxicity.35,36 Thus, it is clear from these laboratory data that l-deprenyl can exert its unique neuroprotection via the mechanisms more than MAO-B inhibition. To the extent, these laboratory data might also provide benecial evidence for l-deprenyl as a neuroprotective agent in the treatment of Parkinsons disease. In conclusion, the present study demonstrated that l-deprenyl, but not other MAO-B inhibitors such as (+)-deprenyl, Ro 16-6491 or pargyline, signicantly attenuates the striatal dopamine loss caused by intranigral injection of MPP+ in vivo. These ndings suggest that l-deprenyl may protect against MPP+ toxicity through a mechanism independent of MAO-B inhibition.

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ACKNOWLEDGMENT This study was supported from the grant of National Science Council, Taiwan, R.O.C. (NSC 84-2331-B002-249).
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