neuroprotective effects of (±)-kavain in the mptp mouse model of parkinson's disease

Neuroprotective Effects of (6)-Kavainin the MPTP Mouse Model

of Parkinson’s DiseaseNICOLE SCHMIDT1 AND BORIS FERGER1,2*

1Institute of Pharmacology and Toxicology, Faculty of Pharmacy, University of Marburg, Marburg, Germany2Behavioural Neurobiology Laboratory, Swiss Federal Institute of Technology Zurich, Schwerzenbach, Switzerland

KEY WORDS dopamine; excitotoxicity; glutamate; neurodegeneration; substantianigra

ABSTRACT This is the first study to investigate the potential protective effects ofthe lipophilic kavapyrone (6)-kavain in the experimental MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) model of Parkinson’s disease (PD). Male C57BL/6 mice weretreated with (6)-kavain (50, 100, or 200 mg/kg i.p.) or vehicle 60 min before and 60 minafter a single administration of MPTP (30 mg/kg s.c.) or saline, respectively. Mice weresacrificed after 7 days and the neostriatum was analyzed for dopamine and its metab-olites using HPLC with electrochemical detection. Furthermore, nigral sections wereprocessed for tyrosine hydroxylase (TH) immunocytochemistry. To determine the effectsof (6)-kavain (200 mg/kg) on MPTP metabolism, HPLC analysis of striatal MPP1

(1-methyl-4-phenylpyridinium) levels was performed. MPTP treatment alone led to asignificant depletion of striatal dopamine levels to 12.61% of saline controls. The lowerdosages of (6)-kavain (50 and 100 mg/kg) showed only a nonsignificant attenuation ofMPTP-induced dopamine depletion, but a high dosage of (6)-kavain (200 mg/kg) signif-icantly antagonized the dopamine depletion to 58.93% of saline control values. Remark-ably, the MPTP-induced decrease of TH-immunoreactivity as well as the loss of nigralneurons was completely prevented by (6)-kavain (200 mg/kg). Striatal MPP1 levels werenot altered by (6)-kavain treatment. In conclusion, we found that MPTP metabolismwas not influenced by (6)-kavain and postulate the antiglutamatergic effects of (6)-kavain for its protective effects against MPTP toxicity. (6)-Kavain may be a novelcandidate for further preclinical studies in animal models of PD and other disorders withglutamatergic overactivity. Synapse 40:47–54, 2001. © 2001 Wiley-Liss, Inc.

INTRODUCTION

Kavain (see Fig. 1), a natural compound isolated fromthe pepper plant Piper methysticum Forst., is a highlylipophilic substance with CNS activity (Meyer andKretschmar, 1966). In animal models of epilepsy, anti-convulsive properties were demonstrated (Kretschmarand Meyer, 1969) and the kava extract has shown neu-roprotective effects against focal ischemia in mice (Back-haub and Krieglstein, 1992). More recently, (6)-kavainhas been shown to attenuate the veratridine-induced glu-tamate release measured by in vivo microdialysis (Fergeret al., 1998a). In vitro data from synaptosome prepara-tions indicate that (6)-kavain induces fast and specificinhibition of voltage-dependent sodium channels (Gleitzet al., 1995). As compounds which interfere with gluta-matergic neurotransmission might improve parkinso-nian symptoms and might prevent nigral degeneration,(6)-kavain was selected for the present study.

Parkinson’s disease (PD) is the most frequent neuro-logical disorder of the basal ganglia. PD is characterizedby the progressive degeneration of dopaminergic neuronsin the substantia nigra pars compacta (SNc), leading to adopamine depletion in the striatum (Ehringer andHornykiewicz, 1960; Riederer and Wuketich, 1976). Theetiology of PD is still unknown (Jenner, 1998), but severalpathogenetic factors such as oxidative stress, excitotoxic-ity, immunological processes, environmental toxins, andgenetic predisposition are under investigation (Olanowand Tatton, 1999; Larkin, 1999). Among them, we fo-cused our interest on glutamate-induced functional alter-ations and excitotoxicity, which seem to play an impor-

Contract grant sponsor: the German Research Foundation; Contract grantnumber: DFG FE 465/1-3.

*Correspondence to: PD Dr. Boris Ferger, Behavioural Neurobiology Labora-tory, Swiss Federal Institute of Technology Zurich, Schorenstrasse 16CH-8603,Schwerzenbach, Switzerland. E-mail: [email protected]

Received 5 July 2000; Accepted 7 September 2000

SYNAPSE 40:47–54 (2001)

© 2001 WILEY-LISS, INC.

tant role in the pathogenesis of neurodegenerativedisorders (Coyle and Puttfarken, 1993; Beal, 1995). Thestriatal dopamine depletion in PD provokes a cascade offunctional changes in the circuitry of the basal ganglialeading to overactivity of the glutamatergic subthalamo-pallidal and subthalamonigral pathways. There is evi-dence that glutamatergic overactivity participates in ni-gral degeneration and in the development of the motorsymptoms in PD (Vila et al., 1999). New invasive thera-peutic strategies using the reversible deep brain stimu-lation of the subthalamic nucleus (STN) (Limousin-Dow-sey et al., 1999) or the irreversible subthalamotomy aimto reduce this glutamatergic overactivity. However, theseinvasive approaches are not available to a great numberof PD patients and the long-term benefit is still unclear.Consequently, for most patients a pharmacological treat-ment is preferred, which is also probably accompanied bya lower risk.

There are presynaptic and postsynaptic pharmaco-logical intervention strategies which reduce glutama-tergic overactivity. On the presynaptic site the reduc-tion of glutamate release or the enhancement ofglutamate uptake by neurons and glia is important.MPP1 leads to enhanced glutamate release (Carboni etal., 1990) and glutamate uptake is impaired afterMPTP treatment (Hazell et al., 1997). In a chronicMPTP study in monkeys, parkinsonian motor abnor-malities were delayed by riluzole, which inhibits boththe release and the postsynaptic actions of glutamate(Bezard et al., 1998). Indeed, NMDA and AMPA gluta-mate receptor antagonists showed not only alleviationof the parkinsonian symptoms but also neuroprotectiveeffects (Turski et al., 1991; Klockgether et al., 1991;Santiago et al., 1992; Boireau et al., 1994). However,the protection with NMDA antagonists was not shownin all parkinsonian models (Kupsch et al., 1992; Son-salla et al., 1992). In addition, sodium channel blockerswhich were able to decrease glutamate release havebeen investigated in models of ischemia (Smith andMeldrum, 1995) and epilepsy (Meldrum, 1994) showingneuroprotective effects.

Since (6)-kavain has antiglutamatergic properties itseems worthwhile to study its effects in the MPTP(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) model

of PD. In the present study, we investigated the effectsof (6)-kavain on striatal dopamine (DA) and its metab-olites as well as on tyrosine hydroxylase-immunoreac-tivity (TH-IR) and cell loss in the SNc as neuroprotec-tive parameters. Furthermore, striatal levels of MPP1

(1-methyl-4-phenyl-pyridinium) were measured to ruleout that alterations in MPTP metabolism or uptakeaccount for the effects of (6)-kavain.

MATERIALS AND METHODSAnimals

Adult male C57BL/6 mice weighing 27–28 g (CharlesRiver, Sulzfeld, Germany) were housed in groups of3–4 under standardized conditions (temperature 23 62°C, relative humidity 55 6 5%, 12 h light/dark cycle,lights on at 7 AM) with free access to a standard diet(Altromint, Lage, Germany) and tap water. The exper-imental protocol was approved by the appropriate in-stitutional governmental agency (RegierungsprasidentGieben, Germany).

Experimental protocol

The experimental protocol consisted of eight groupswith 7–10 animals each. (6)-Kavain was suspended inneutral oil (Miglyolt) and given i.p. at dosages of 50,100, or 200 mg/kg. Two (6)-kavain injections were per-formed: one 60 min prior and another 60 min after s.c.injection of MPTP (30 mg/kg). Control animals receivedneutral oil instead of (6)-kavain and saline instead ofMPTP.

Tissue preparation

Seven days after MPTP or saline administration,mice were sacrificed by cervical dislocation, brains rap-idly removed, and placed on an ice-cooled plate fordissection of the striatum. Immediately after dissec-tion, the striata were weighed and placed in a 2.0 mltube containing ice-cooled perchloric acid (500 ml, 0.4M), homogenized for 1 min at 20,000 rpm using anultra turrax and centrifuged for 15 min at 15,000g at4°C. The supernatant was passed through a 0.2 mmfilter and either immediately analyzed with HPLC andelectrochemical detection or frozen at –70°C until anal-ysis.

Determination of dopamine and metabolites

DA and its metabolites 3,4-dihydroxyphenylaceticacid (DOPAC) and homovanillic acid (HVA) as well asserotonin (5-HT) and its metabolite 5-hydroxyin-doleacetic acid (5-HIAA) were analyzed using reversedphase ion pair chromatography combined with electro-chemical detection under isocratic conditions as de-scribed earlier (Ferger et al., 1998b). In brief, the de-tector potential was set at 1750 mV using a glassycarbon electrode and an Ag/AgCl reference electrode.The mobile phase (0.6 mM 1-octanesulfonic acid, 0.27

Fig. 1. Chemical structure of (6)-kavain.

48 N. SCHMIDT AND B. FERGER

mM Na2 EDTA, 0.43 M triethylamine, and 35 ml ace-tonitrile/l, adjusted to pH 2.95 with H3PO4) was deliv-ered at a flow rate of 0.5 ml/min at 22°C onto thereversed phase column (125 3 3 mm with precolumn5 3 3 mm, filled with Nucleosil 120-3 C18; Knauer,Berlin, Germany). Twenty ml aliquots were injected byan autosampler with the cooling module set at 4°C.Data were calculated by an external standard calibra-tion.

Tyrosine hydroxylase immunoreactivity andNissl-staining

For tyrosine hydroxylase immunoreactivity (TH-IR),brainstems were postfixed in a solution containingmethanol (80%), formaldehyde (20%), and acetic acid(10%), and were cryoprotected in 25% sucrose/PBS.Coronal midbrain sections (30 mm) were cut using afreezing microtome. Nigral sections were immunocyto-chemically stained for TH using the free-floating tech-nique as described previously (Ferger et al., 1999). Inbrief, sections were rinsed three times in 0.1 M PBSand the endogenous peroxidase activity was sup-pressed by adding 3% H2O2 in 10% methanol. Follow-ing another three rinses in PBS, the sections werepreincubated in 5% normal horse serum (NHS) con-taining 3% Triton X-100 in PBS for 1 h and then trans-ferred to a 1:4,000 dilution of primary TH antiserumraised in mouse (Boehringer Mannheim, Germany) in2% NHS, 0.3% Triton X-100-PBS. Following overnightincubation at room temperature, sections were rinsedthree times in PBS and incubated in a 1:200 dilution ofbiotinylated antimouse immunoglobulin G raised inhorse (Vector, Burlingame, CA, USA), 0.3% TritonX-100-PBS for 1 h. Sections were then rinsed threetimes and transferred to a streptavidin solution (1:200in PBS, Dako, Denmark) for 1 h. For the visualizationreaction the sections were incubated in 0.05% 3,39-diaminobenzidine (Boehringer Mannheim) in 0.1 MPBS in the presence of 0.01% H2O2. Sections were thenmounted on gelatinized slides, left to dry overnight,dehydrated in ascending alcohol concentrations, andmounted on DePeX (Serva, Freiburg, Germany).

For Nissl-staining, nigral sections were stained withcresyl violet. Sections were then mounted on gelati-nized slides, left to dry overnight, dehydrated in as-cending alcohol concentrations, and mounted on De-PeX (Serva).

Microscopic analysis

Nucleated, process-bearing TH-immunoreactive sub-stantia nigra cells in the pars compacta (SNc) andNissl-positive cells were bilaterally counted underbright field illumination under blind conditions inthree sections per animal according to Kupsch et al.(1995). Quantitative analysis involved counting ofTH-IR neurons at the widest dimension of the SNc at

AP -3.16 (coordinates based on mouse atlas; Franklinand Paxinos, 1996) lateral to the roots of the thirdcranial nerve separating medial and lateral SNc at thelevel of the interpeduncular nucleus. Results are ex-pressed as mean TH-IR or Nissl positive cell counts persection.

Determination of MPP1

In an additional experiment, mice received a singlei.p. injection of (6)-kavain (200 mg/kg), selegiline (10mg/kg), or saline 5 min before administration of MPTP(30 mg/kg s.c.). Two hours after MPTP administrationthe mice were sacrificed by cervical dislocation andstriata were prepared as described above.

MPP1 was analyzed by reversed phase chromatog-raphy with UV-detection under isocratic conditions.The mobile phase (0.02 M potassium phosphate, aceto-nitrile:HPLC grade water (30:70) adjusted to pH 2.5with H3PO4) was delivered at a flow rate of 0.5 ml/minat 30°C onto the reversed phase column (250 3 4.6 mmwith precolumn 5 3 4 mm, filled with Nucleosil 100-C18; Knauer). The UV-detector was set at 295 nm.Twenty ml aliquots were injected using an autosam-pler. Data were calculated by an external standardcalibration.

Drugs and chemicals

MPTP-HCl was purchased from Research Biochem-icals International (RBI, Cologne, Germany) and(6)-kavain from Fa. Roth (Karlsruhe, Germany). DA-HCl, DOPAC, HVA, MPP1 iodide, and 1-octanesulfonicacid sodium salt were obtained from Sigma-AldrichChemie GmbH (Munich, Germany). HPLC-grade ace-tonitrile and perchloric acid were purchased fromMerck (Darmstadt, Germany). HPLC standards weredissolved in 0.1 M perchloric acid, (6)-kavain was sus-pended in neutral oil (Miglyolt), MPTP was dissolvedin saline. Injections were performed using a volume of10 ml/kg body weight. All doses are expressed in termsof the free base.

Statistics

All data were analyzed using ANOVA with subse-quent Bonferroni test for multiple comparisons. P ,0.05 was considered statistically significant.

RESULTSNeurochemical analysis of dopamine

and metabolites

The levels of DA and its metabolites were measured7 days after MPTP treatment and the following meanvalues 6 SEM of the Oil 1 Saline-treated control groupwere taken as 100%: DA 15.22 6 1.96; DOPAC 0.77 60.11; HVA 1.37 6 0.25; 5-HT 0.42 6 0.12; 5-HIAA0.31 6 0.02 ng/mg wet tissue weight. In the non-MPTP

KAVAIN PROTECTS AGAINST MPTP TOXICITY 49

treated groups (6)-kavain itself had no significant ef-fects on the dopamine (Fig. 2A) and metabolite levels(data not shown). MPTP treatment reduced the levelsof DA to 12.61%, of DOPAC to 31.25%, and of HVA to36.36% of control values. The high dose of (6)-kavain(200 mg/kg) significantly antagonized the decrease inDA to 58.93 6 5.39% and in its metabolites DOPAC to

54.48 6 8.96%, HVA to 96.85 6 11.13% compared tothe Oil 1 MPTP group (Fig. 2A). Moreover, the MPTP-induced increase of DA-turnover as determined by theDOPAC/DA and HVA/DA ratio was dose-dependentlyreduced by kavain (Fig. 2B,C). The serotonergic systemwas influenced neither by kavain nor by MPTP (datanot shown).

Fig. 2. Effects of (6)-kavain (Kav) (50, 100,or 200 mg/kg) or vehicle (Oil) on the levels ofDA (A) and DA turnover indicated byDOPAC/DA ratio (B) and HVA/DA ratio (C) 7days after administration of MPTP (30 mg/kg)or saline (Sal). Values of the Oil 1 Sal groupwere set at 100%. Mean values 6 SEM of n 57–10 mice are presented. ANOVA (DA:F(7,56) 5 39.72, P , 0.001); DOPAC/DA:F(7,56) 5 23.26, P , 0.001), and HVA/DA:(F(7,57) 5 17.70, P , 0.001) with subsequentBonferroni test for multiple comparisons:MPTP-treated groups compared with the cor-responding saline-treated groups (*P , 0.05,**P , 0.01, ***P , 0.001), kavain- and MPTP-treated groups compared with the Oil 1 MPTPgroup (##P , 0.01, ###P , 0.001).


Analysis of striatal MPP1 levels

In the control group, MPTP treatment led to striatalMPP1 levels of 21.89 6 0.64 ng/mg wet tissue weightmeasured 2 h after MPTP injection. Pretreatment with(6)-kavain (200 mg/kg) showed no significant alter-ation in MPP1 levels compared with controls. In con-trast, the MAO-B inhibitor selegiline (10 mg/kg), whichserved as positive control, decreased MPP1 levels sig-nificantly to 4.09 6 0.48 ng/mg wet tissue weight (Ta-ble I).

TH-immunoreactivity and Nissl-staining

MPTP administration alone led to a significant de-crease in the mean number of TH-immunoreactive andNissl-stained neurons in the SNc to 57% and 62%, re-spectively, compared with vehicle-treated controls. The

MPTP-induced reduction of TH-IR and Nissl-stainedneurons was significantly antagonized by (6)-kavain 200mg/kg. The lower dosages of (6)-kavain (50 and 100 mg/kg) showed a tendency to attenuate the decrease of TH-IRand Nissl-stained neurons, which, however, was not sig-nificant (Fig. 3A,B). The photomicrographs of represen-tative SNc sections clearly demonstrate that the applieddose of MPTP led to a pronounced loss of TH-IR and that(6)-kavain (200 mg/kg) was able to completely preventthe MPTP-induced decrease of TH-immunoreactivity(Fig. 4).

DISCUSSION

The results of the present study demonstrate for thefirst time that (6)-kavain is able to protect nigrostria-tal dopaminergic neurons against MPTP toxicity. TheMPTP-induced decrease in striatal DA levels, the de-crease in TH-immunoreactivity, as well as the loss ofnigral neurons were significantly antagonized bykavain at a high dose. The lower dosages of (6)-kavainshowed only a nonsignificant tendency toward protec-tion. These effects of (6)-kavain in the MPTP model ofPD may be either symptomatic by an enhancement ofdopaminergic neurotransmission, due to alterations in

TABLE I. Effects of saline, (6)-kavain (200 mg/kg), and selegiline(10 mg/kg) on striatal MPP1 levels

Treatment MPP1 levels [ng/mg wet tissue weight]

MPTP 1 vehicle 21.87 6 1.17MPTP 1 (6)-kavain 26.34 6 1.32MPTP 1 selegiline 4.09 6 0.48***

MPP1 levels were measured 2 h after MPTP treatment. Values are given asmean 6 SEM. ANOVA (F(2,11) 5 127.8) with subsequent Bonferroni test forcomparison with the MPTP 1 vehicle group (***P , 0.001).

Fig. 3. Effects of (6)-kavain (Kav) (50, 100,or 200 mg/kg) or vehicle (Oil) on TH-immuno-reactivity (A) and Nissl staining (B) 7 days afteradministration of MPTP (30 mg/kg) or saline(Sal). Values of the Oil 1 Sal group were set at100%. Mean values 6 SEM of n 5 7–10 mice arepresented. ANOVA (TH: F(7,57) 5 5.95, P ,0.001); Nissl: F(7,57) 5 6.21, P , 0.001) withsubsequent Bonferroni test for multiple com-parisons: MPTP-treated groups compared withthe corresponding saline-treated groups (*P ,0.05, **P , 0.01), (6)-kavain- and MPTP-treated groups compared with the Oil 1 MPTPgroup (#P , 0.05).


MPTP metabolism, or mediated by neuroprotectiveproperties.

A symptomatic effect of (6)-kavain seems unlikelysince (6)-kavain neither increases DA release nor in-hibits DA metabolism. In an in vivo microdialysis studyin freely moving rats, (6)-kavain produced only a non-significant decrease of extracellular DA levels (Baum etal., 1998). In a previous study with acute and chronic(6)-kavain administration, no alterations in DA or se-rotonin levels or turnover of both measured from stri-atal tissue was obtained (Boonen et al., 1998). Further-more, (6)-kavain was tested in various binding studiesand did not show any affinity to DA receptors (Dr. S.S.Chatterjee, pers. comm.).

To study the effects of kavain in vivo we used thedopaminergic neurotoxin MPTP in the widely acceptedmodel of PD (Gerlach and Riederer, 1996). MPTPpenetrates the blood–brain barrier and is enzymati-cally metabolized to MPP1 via MAO-B. Theoretically,(6)-kavain could decrease MPTP toxicity due to aninhibition of MPTP metabolism, as MPTP toxicity isdirectly related to the formation of MPP1 (Langston etal., 1984). Indeed, the MAO-B inhibitor selegiline pre-vented MPTP toxicity (Heikkila et al., 1985). In thepresent study, we demonstrated that (6)-kavain didnot significantly alter MPP1 levels. It was reported

that the kava extract shows some MAO-B inhibitingactivity in vitro, but the same group demonstrated that(6)-kavain itself did not exhibit relevant MAO-B inhi-bition (Uebelhack et al., 1998). (6)-Kavain and thekava extract often differ in their action profiles, as theextract consists of several constituents with structuraland pharmacological variability (Uebelhack et al.,1998).

We postulate that MPTP uptake into the brain andMPTP metabolism are not influenced by (6)-kavain,and thus other mechanisms are responsible for theprotective effect.

MPP1 is selectively transported into dopaminergicnerve terminals by the DA transporter and inducesmitochondrial impairment by a direct inhibition ofcomplex I activity with interruption of oxidative phos-phorylation and energy depletion (Tipton and Singer,1993). A consequence is the partial neuronal depolar-ization with secondary activation of NMDA receptorsand inhibition of glutamate uptake (Beal, 1995). Ele-vated extracellular glutamate levels increase calciuminflux, which in turn leads to an enhanced formation ofreactive oxygen species, activation of proteases andlipases, resulting in membrane damage and finally inneuronal death (Beal, 1995). (6)-Kavain might be ableto interrupt this cascade, as it has been shown to re-

Fig. 4. Photomicrographs of representative SNc sections stained immunocytochemically againsttyrosine hydroxylase (TH) at the level of the third cranial nerve of mice treated with oil 1 saline (A),(6)-kavain (200 mg/kg) 1 saline (B), oil 1 MPTP (30 mg/kg) (C), and (6)-kavain (200 mg/kg) 1 MPTP(30 mg/kg) (D). Scale bar 5 200 mm.


duce glutamate release in vitro (Gleitz et al., 1996) andin vivo (Ferger et al., 1998a). The mechanism of kavainto protect against MPTP toxicity may be due to itsability to block sodium channels (Gleitz et al., 1995),intracellular calcium influx (Gleitz et al., 1996), anddue to the subsequent reduction of excessive glutamaterelease. Excessive glutamate release was demon-strated after local MPP1 administration in rat stria-tum (Carboni et al., 1990). The reduction of glutamaterelease may also be of importance in the glutamatergicprojection from the subthalamic nucleus (STN) to thesubstantia nigra pars reticulata to exert an antiexcito-toxic, neuroprotective effect of (6)-kavain.

Nigrostriatal DA depletion leads to a compensatoryglutamatergic overactivity which is evident in the STNand subsequently inhibits the thalamocortical path-way, leading to hypomotility and akinesia (Sian et al.,1999). Kavain significantly attenuated the MPTP-in-duced hypomotility (data not shown), which indicatesalso functional improvement against MPTP toxicity.However, other effects of (6)-kavain which producefunctional recovery may also lead to an improvement ofMPTP-induced motor dysfunction. It would be interest-ing to find out if (6)-kavain is able to normalize theglutamatergic overactivity in the STN. Therefore, ad-ditional electrophysiological and neurochemical exper-iments are necessary to characterize the antiglutama-tergic properties in more detail.

In conclusion, this study demonstrates for the firsttime the efficacy of (6)-kavain to reduce MPTP-neuro-toxicity in vivo. (6)-Kavain or its derivatives might beof interest to develop novel drugs for the treatment ofPD. The sodium and calcium channel blockade withsubsequent inhibition of glutamate release may ac-count for the neuroprotective effects and may also leadto functional improvement of parkinsonian symptoms.

ACKNOWLEDGMENTS

We thank Mrs. Beate Ullrich for excellent technicalassistance, Dr. Dorothee Ferger for helpful discussion,and Dr. Christopher D. Earl for linguistic revision ofthe manuscript.

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neuroprotective effects of (±)-kavain in the mptp mouse model of parkinson's disease

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