Neurochemical Research, Vol. 25, No. 4, 2000, pp. 503–509

5030364-3190/00/0400–0503$18.00/0 © 2000 Plenum Publishing Corporations

MPTP Decreases MT-I mRNA In Mouse Striatum

Patricia Rojas,1 Julio Rojas-Castañeda,2 Rosa María Vigueras,2 Sultan S. M. Habeebu,3

Carolina Rojas,2 Camilo Ríos,1 and Manuchair Ebadi4

(Accepted January 5, 2000)

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a drug that induces parkinsonism inhumans and non-human primates. Free radicals are thought to be involved in its mechanism ofaction. Recently, metallothionein has been proposed to play a role as a scavenger of free radi-cals. In the present work, we studied the effect of MPTP neurotoxicity on brain metallothionein-I (MT-I) mRNA expression. Male C-57 black mice were treated with MPTP (30 mg/kg, i.p.,daily) for 3 or 5 days. All animals were killed by cervical dislocation 7 days after the lastMPTP dose. The brains were removed quickly and immediately frozen, and quantitative in situhybridization was performed using MT-I cDNA probe. MT-I mRNA content in striatum, aregion which is known to be highly predisposed and sensitive to MPTP-induced oxidativestress, decreased by 30% (3 days) and 39% (5 days) respectively, after the last MPTP adminis-tration. These results suggest that MT-I gene expression is decreased in MPTP neurotoxicity. Itis suggested that the reduction of MT, an anti-oxidant and a free radical scavenger, in thestriatum by MPTP enables the neurotoxin to exert maximal oxidative damage to the striatum.

KEY WORDS: MPTP; Parkinson’s disease; metallothionein-I; MT-I mRNA; striatum; oxidative stress.

1 Laboratory of Neurotoxicology, Instituto Nacional de Neurologíay Neurocirugía, México City, México.

2 Laboratory of Histomorphology, Instituto Nacional de Pediatría,México City, México.

3 Department of Pharmacology, Toxicology and Therapeutics, Uni-versity of Kansas Medical Center, Kansas City, KS.

4 Department of Pharmacology, Physiology and Therapeutics, Uni-versity of North Dakota, School of Medicine and Health Sciences,Grand Forks, ND.

5 Address reprint request to: Patricia Rojas, Ph.D., Laboratory ofNeurotoxicology, Instituto Nacional de Neurología y Neuro-cirugía, Av. Insurgentes Sur No. 3877, Col. La Fama C.P. 14269,México D.F., México. Tel +52 5 606 4040; Fax +52 5 528 0095e-mail: rojas@buzon.main.conacyt.mx

1-methyl-4-phenylpyridinium ion (MPP+), its toxicmetabolite, by type-B monoamine oxidase (3). MPP+

is then accumulated into dopaminergic neurons by thehigh affinity dopamine uptake system (4). The mech-anism of MPP+ neurotoxicity is unknown, but its tox-icity has been related to MPP+ inhibition of site Imitochondrial respiration (5), and/or to induction ofoxidative stress (6). In previous studies, we foundenhanced lipid peroxidation after MPP+ administrationto mice (7), a process dependent on overproduction offree radicals, and also depletion of trace metals fol-lowing MPTP administration (8).

On the other hand, metallothioneins (MTs) arelow-molecular-weight, sulfur-rich, inducible proteinswhich lack aromatic amino acids, and exhibit highaffinity for cadmium, zinc and other heavy metals (9).The mammalian MT family contains four isoforms:MT-I and MT-II are expressed in many tissues includ-ing the brain (9); MT-III exists in zinc-containing neu-rons, and it is thought to play a role in the uptake ofzinc into neurons and transport of the metal within

INTRODUCTION

Administration of 1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine (MPTP) to mice, humans and non-humans primates, produces depletion of dopamine andcellular degeneration of the nigrostriatal pathway,mimicking that observed in Parkinson’s disease (1).This alteration is accompanied by a reduction of stri-atal dopamine content (2). MPTP is biotransformed to

neurons and their synaptic vesicles (10). MT-IV isexpressed in stratified squamous epithelia (11).

MT is one of the most important protein thioldefense mechanisms in mammalian cells (12). In thebrain, MT isoforms have been proposed to participate inthe transport, accumulation, and compartmentation ofzinc (see 13 and 14 for reviews and references). It hasalso been proposed that MT plays a role in adaptation tostress (15), and as an antioxidant agent (16–19).

Although liver is the organ most responsive to MTinduction, MT is also induced in other organs includ-ing brain, by various inducers such as heavy metals,glucocorticoid hormones, acute stress, some organicchemicals, and infection (20). Only MT-I and MT-IIisoforms are induced by these agents.

MT is a protein important in both oxidative stressand trace metals metabolism. In previous studies wefound that MT inducers protect against MPTP neu-rotoxicity (21). This prompted us to investigate thechanges in striatal MT protein levels in MPTP neuro-toxicity. In addition, we found that MPP+ decreases theconcentration of MT I+II proteins in the striatum (22).MPP+ damages dopaminergic neurons but MT I+IIproteins are expressed mainly in glial cells (23), whichare not known to be damaged by MPTP neurotoxicity.The present study was, therefore, designed to investi-gate how MPTP reduces striatal MT I+II proteins,using quantitative in situ hybridization to assess MT-ImRNA expression in the striatum.

EXPERIMENTAL PROCEDURE

Chemicals for In Situ Hybridization Studies. Restrictionenzymes were purchased from Promega (Madison, Wisconsin) orNew England Biolabs (Beverly, Massachusetts). Radiolabeled com-pounds were obtained from ICN ([32P] dCTP) or NEN ([35S] dCTP).mRNA was isolated using oligo [dT] chromatography (InvitrogenCorp.) RNA sizing ladders were from Life Technologies (Gaithers-burg, Maryland). High-efficiency hybridization solutions with orwithout 50% formamide were purchased from MRC (Cincinnati,Ohio). Proteinase K, T1 ribonuclease, and paraformaldehyde werefrom Sigma (St. Louis, Missouri). All other chemicals used for insitu hybridization were of molecular biology reagent grade.

Animals.All experiments were conducted on male C-57 blackmice (10–12 weeks of age) and they were maintained three per cage inair-conditioned rooms, subjected to 12 hr light-dark cycle, 21–22 °C,fed with Purina chow (Purina, México) and provided with tap waterad libitum.

MPTP Administration.MPTP hydrochloride was obtained fromResearch Biochemicals (Wayland, MA, U.S.A.). Mice were ran-domly assigned to one of three groups as follows: Control group,consisting of 8 mice given 0.9% NaCl solution (ip) daily for eitherthree or five days; MPTP 3-day group, consisting of 8 animals treatedwith MPTP (30 mg/kg, ip) daily for three days; and MPTP 5-daygroup, consisting of 8 animals treated with MPTP (30 mg/kg, ip)

daily for five days. All animals were killed by cervical dislocation7 days after the last MPTP or saline injection, and the brains were re-moved and immediately frozen in ice-cold isopentane solution in avessel kept on dry ice (<10 sec) and used for in situ hybridization.

In Situ Hybridization.Frozen sections were cut at 12 µm in thecoronal and horizontal planes with a Jung Frigocut 2800 N cryostat(Leica). Slides were processed for in situ hybridization studies usinga standard method as we described previously (24).

Preparation of Probes for mRNA Analysis andIn SituHybridization.Rat liver MT-I cDNA (p2A10), donated by Dr. RobertD. Andersen, was from a cDNA library generated from the liver ofCdCl2-treated rats (25). The probe (p2A10) is a nearly completecDNA copy of the rat MT-I mRNA sequence with extensive homol-ogy to mouse mRNA.

The 452 bp insert cDNA sequence was cleaved from pBR322vector by digestion with Pstl, gel purification, and labeling with[32P]dCTP (for mRNA analysis) or [35S] dCTP (for in situ hy-bridization) by random hexanucleotide primed synthesis usingKlenow fragment (Boehringer-Mannheim, Richmond, California).The unincorporated 32P- and 35S-labeled nucleotides were removedby G-50 column chromatography (Bio-spin, Bio-Rad, Indianapolis,Indiana). The specific activities of the 32P- and 35S-probes were108–109 cpm/µg each.

Prehybridization Events and Hybridization Procedures.Priorto hybridization, the sections were rehydrated by passing themthrough graded concentrations of ethanol, and the slides were thenplaced on the rack in a humidity-controlled incubation chamber.Forty-five microliters of prehybridization buffer (1% sodium do-decyl sulfate [SDS], 1 M NaCl) were applied to each section, andthe slides were incubated under parafilm coverslips in an incuba-tion chamber at 42°C for 2–3 hr. After this prehybridization step, thecoverslips were carefully removed and a 45 µl aliquot of high-efficiency hybridization solution containing 50% formamide (Mol-ecular Research Center, Inc., Cincinnati, Ohio) and 10 mM dithio-threitol, was added. For hybridization, 1 µl of 35S-labeled DNA probe(per 24 mm2 area) was applied to the sections, and they were thenincubated under parafilm coverslips at 42°C for 24 hr in a humidifiedincubation chamber. After hybridization, the slides were immersedin 1x standard saline citrate, 50% formamide, and the coverslipswere removed. The slides were then washed in the same solutionat 45°C for 3 hr, with several changes of solution. Subsequently,the sections were dipped briefly in H2O followed by 80% ethanol,and then air dried.

Procedure for Autoradiography.Film autoradiography wasperformed by apposition of tissue sections to X-ray film (Kodak) for6 weeks at −80°C. Film images were analyzed on an MC1D imageanalysis system (Imaging Research, Inc., St. Catherines, Ontario,Canada). Commercial radioactivity standards (ARC 146B; Ameri-can Radiolabeled Chemicals, St Louis, MO) were coexposed withtissue sections and were used to calibrate digitized images to opti-cal density (OD) standards (Kodak, Rochester, NY) for regionalquantitation of hybridization signals.

Emulsion autoradiography was performed by dipping slides inKodak NTB-2 Nuclear Tract liquid emulsion:H2O (1:1) at 42°C, asdescribed previously (26,27). Emulsion-coated slides were stored inlight-tight containers with desiccant at 4°C for 25 days. Slides werethen developed in Kodak Dektol developer/H2O (1:1) for 3 minutes,rinsed in H2O for 30 seconds, fixed in Kodak Rapid Fix for 4 min-utes, and washed in running water for 20–30 minutes. Sections werethen counterstained with 0.5% cresyl violet in H2O for 2 minutes,rinsed briefly in H2O, differentiated in 70% EtOH, and dehydratedthrough graded alcohols, cleared in xylene, and mounted with Per-mount (from Sigma St. Louis, Missouri).

504 Rojas et al.

Quantitation of Emulsion Autoradiograms.Emulsion autoradio-graphy was analyzed under brightfield illumination with ReichertUltrastar microscope. Control and experimental groups were exam-ined using a calibrated ocular grid. The number of labeled cells andthe number of grains in each labeled cell falling within the grid weredetermined. Grains over neuropil areas were also analyzed. Back-ground was evaluated by counting grains over similar-sized areas andvalues were substracted from the grain densities measured in differentbrain regions. The various elements were located in twenty randomlyselected fields per slide, in 3 or 4 slides per mouse brain. Grain countswere obtained and analyzed “blind” by two independent observers.

Northern Blot Analysis of MT-I mRNA.The mRNA from wholebrain was isolated by using an mRNA isolation kit (Invitrogen). Equalamounts of mRNA (1 µg) were loaded, electrophoresed on 1.5%agarose, 2.2 M formaldehyde gels, and blotted onto nylon membranes(MSI, Westboro, MA) by capillary transfer. After 2–3 hr incubationwith prehybridization buffer (1 M NaCl, 1% SDS) at 65°C,hybridization was performed using the 32P-labeled rat MT-I cDNAprobe at 65°C in high-efficiency hybridization solution, for 14–24 hr.Unbound MT-I probe was removed by washing with 0.1x standardsaline citrate, containing 0.1% SDS at 65°C for 5–6 hr. The membranewas exposed to XAR-5 film (Kodak) as described previously.

Striatal Estimation of MT Proteins.The MT proteins concen-tration of each tissue sample was estimated by the silver-saturationmethod (28). This method measures all MT isoforms (MT I, II, IIIand IV). Mice were killed by cervical dislocation 7 days after MPTPtreatment for 5 consecutive days as describe above. MT proteinswere analyzed in corpus striatum, as follows: all laboratory glass-ware, polypropylene tubes and disposable micropipette tips wereimmersed for several hours in 5% v/v concentrated HNO3/H2O,thoroughly rinsed in deionized water, and nitrogen gas-dried beforeuse, to avoid any possible contamination. Stainless steel dissectionmaterial was cleaned by rapid immersion in 3% v/v nitric acid, andthoroughly rinsed with deionized water prior to its use. Tissue samplesof corpus striatum were homogenized in 500 µl of ice-cold 0.05 Mphosphate buffer (pH 7.0) containing 0.015 M NaCl and 0.145 MKCl. Silver ion aqueous solution (20 mg/L, 250 µl) was added to thetissue extract (500 µl) to bind cytosolic proteins and other ligandsincluding MT proteins. Rat hemoglobin (200 µl) was added andsamples boiled for 2 minutes to precipitate silver excess. Only silverbound to MT proteins is left in the supernatant after two hemoglobin-boiling treatments. MT proteins concentration was calculated fromsilver concentration in the supernatant as determined by atomicabsorption analysis using a Perkin-Elmer 360 Atomic absorptionSpectrophotometer with HGA-2200 graphite furnace. A deuteriumarc background corrector was used to compensate for backgroundsignal. For both samples and standards, a 20 µl aliquot of each finaldiluted solution was injected into the furnace. Furnace program wasoptimized by measuring the standards signal height at severaltemperature settings, for both char and atomization stages, accord-ing to the procedure of Welz (29). MT proteins detection limit wasabout 5 µg MT/g wet tissue.

Statistical Evaluation of Data.Data are expressed as mean ±SEM. Data were analyzed statistically using one way ANOVA fol-lowed by Tukey’s post-hoc test.

RESULTS

The specificity of the MT-I cDNA hybridizationprobe was determined by Northern blot analysis ofmRNA samples obtained from brains of rats receiving

ZnSO4. Equal amounts of brain mRNA (1 µg) wereanalyzed. In zinc-treated brains a single band of ap-proximately 570 nucleotides, hybridized to the rat liverMT-I cDNA probe, whereas in saline-treated brains,only a trace of MT-I mRNA was detected (Fig. 1).Having confirmed that the probe (p2A10) specificallydetects MT-I mRNA, and it is a nearly completecDNA to sequence homologous to mouse mRNA, theprobe was utilized to investigate the effects of MPTPon the brain expression of MT-I.

Using zinc-treated mice liver as positive controland RNase-treated brain and RNase-treated liver asnegative controls, the topographic distribution ofMT-I mRNA in the brain was determined by in situhybridization histochemistry. To enhance the reliabil-ity of the results, brains from eight different mice wereused for each determination. MT-I mRNA exhibited auniform distribution in zinc-treated mice liver, but wasabsent from the RNase-treated mice liver and Rnase-treated brain (data not shown).

Hybridization of the probe to brain sectionsshowed that MT-I mRNA is expressed throughout thebrain. Several features of its relative abundance areshown in Fig. 2A and 2B. High levels of hybridiza-tion occured in regions such as the cerebellum(orange>>yellow) in control groups but this pattern didnot change significantly in MPTP-treated mice. Mod-erate levels of MT-I mRNA (light blue>dark blue>darkpink) were expressed in other regions of the brain incontrol group, including cortex and striatum. An exam-ination of autoradiographs obtained from the MPTP-treated brains revealed significant reduction of the MT-ImRNA expression in the striatum, suggesting that MT-ImRNA is decreased by MPTP in the neuronal and glialelements of this brain region. However, the constitu-tive MT levels in various cells of the striatum have not

MT-I mRNA Expression after MPTP Administration 505

Fig. 1.Northern blot analysis of mRNA expressed in control (panel A)and zinc-treated rat brain (panel B). Rats were injected with 4 µl of100 µM ZnSO4 icv and decapitated 6 hr post zinc treatment. Equalamounts of mRNA (1 µg) from control and zinc-treated brains wereelectrophoresed on 1.5% agarose and formaldehyde gels, blotted, andhybridized with 32P-labeled rat MT-I cDNA probe A 0.16–1.77KbRNA ladder was used for determination of the size of the brainMT-I mRNA.

been investigated quantitatively, and remain to be de-termined in future studies.

In situ hybridization histochemistry and autoradio-graphic analysis of brains after 3 and 5 days of MPTPtreatment (Fig. 2A and 2B), showed that MT-I mRNA

concentration decreased only in the striatum (darkpink<dark blue<light blue) but not in other brain regions(p < 0.05).

Finally, hybridization signals were found to beconcentrated mainly over cell bodies by microscopic

506 Rojas et al.

Fig. 2A. A typical pseudocolor image of horizontal sections of the mouse brain demonstrating the regional localization of MT-I mRNA and theeffects of MPTP on the level of MT-I mRNA detected by in situ hybridization histochemistry and autoradiography. The colors of the imagescorrespond to the relative optical densities (RODs) obtained following digitization; RODs were converted to color as shown at the right sideof figure. Note that the reduction in MT-I mRNA hybridization signals is mostly in the striatum (St). Fig. 2B. A typical pseudocolor image ofcoronal sections of the mouse brain demonstrating a reduction in MT-I mRNA expression in striatum (St.) in MPTP neurotoxicity at day 3 andday 5 of MPTP administration. The regional distribution of MT-I mRNA following MPTP treatment compared with saline treatment, wasanalyzed, and gave similar results in eight separate sets of experiments.

examination of grain densities on sections processedwith emulsion autoradiography (Fig. 3) as expected forspecific hybridization to mRNA.

A detailed analysis of cellular localization of MT-I mRNA showing the density of emulsion grains/area

(Table I) revealed that MPTP only reduced the con-centration of MT-I mRNA in cells of striatum by 30%and 39% 7 days after repeated MPTP administrationdaily for 3 and 5 days, respectively. No differenceswere found in other brain regions.

We also determined the concentration of MT pro-teins. Mice exposed to MPTP for 5 days showed a sig-nificant decrease (49% vs control group values) intheir striatal MT proteins concentration, as previouslydescribed (Fig. 4; 21).

DISCUSSION

The present study shows that MPTP, which gen-erates free radicals, and specifically damages dopamin-ergic nigrostriatal pathway, reduces MT-I mRNAconcentration in the striatum in a dose-dependentmanner as well as reducing striatal total MT proteinconcentration. This suggests that MT-I protein is beingdown-regulated in striatal cells by MPTP.

Our findings in the present study suggest that thereduction in striatal MT protein concentration is due, atleast in part, to reduced MT-I mRNA expression. MT-I is preferentially expressed in glial cells (23) thus, theneuronal cell death associated with MPTP action is notlikely to be the cause of the MT-I mRNA reductionobserved in this study.

The consequences of this reduction in MT-ImRNA are relevant to the mechanism of action ofMPTP, considering that MT-I has antioxidant proper-ties (see 18). MT-I’s down-regulation in the striatumby MPTP, renders the striatum susceptible to highdegrees of MPTP-induced oxidative damage.

As MT-I consists of at least, 30% thiol aminoacid residues per molecule (9), the reduction in thiol-related defenses against free radicals becomes highlyimportant. How MPTP reduces striatal MT-I mRNAexpression remains to be investigated. One possibleexplanation implicates certain promoter sites of MT-I synthesis already described: the catecholamine site,the transition metal site and the steroid site (30,31).As both dopamine and transition metals such as totalcopper (bound and free) have been found to be de-creased as a consequence of MPTP action (8), areduction in promoter-induced MT-I expression is tobe expected.

Oxidative stress is the result not only of overpro-duction of reactive oxygen species, but also, it is aconsequence of decreases in the cell’s antioxidant pro-tective mechanisms (32). MPTP administration de-creases the level of reduced glutathione (33) and striatal

MT-I mRNA Expression after MPTP Administration 507

Fig. 3. Cellular localization of MT-I mRNA in brains of control andMPTP treated mice, using 35S-labeled cDNA probe. In striatum, 35S-labeling was localized mainly to the cells of control (A) and MPTP-treated (B, C) mice brain. Following 3 days of MPTP treatment (B),the labeling on the cells was decreased significantly (30%)compared to that in control mice (A). After 5 days of MPTPtreatment (C) the labeling was decreased further (39%) compared tothe control group (A).

MT content (21), thereby decreasing the cell’s thiol-related defenses against free radicals.

As a result of the high concentration of free ironand free copper (prooxidants) and low concentration offerritin (iron binding protein) in the striatum, it is pre-sumed that this region is highly vulnerable to oxidativestress. This vulnerability is increased by the reductionin MT-I concentration (21) following MPTP adminis-tration, as occurs in 6-hydroxydopamine neurotoxicity(24). Furthermore, free copper ions, normally chelatedby MT, can participate in a Fenton-like reaction to

promote production of hydroxyl radicals, a well-knownfeature related to MPTP action (8).

It has been suggested that oxygen-derived freeradical species can directly interact with MT to oxi-datively modify the protein (34) and its expression.MT may act therefore, as a sacrificial scavenger forhydroxyl and superoxide radicals. In addition, MPTPinduces a threefold increase in superoxide dismutaseactivity in the striatum (35); this may reflect a com-pensatory change after MT reduction.

These results suggest that MT-I gene expression isaltered in MPTP neurotoxicity. It is suggested that theelimination of MT, an anti-oxidant and a free radicalscavenger, in the striatum by MPTP enables MPTP toexert maximal oxidative damage to the striatum.

ACKNOWLEDGMENTS

This study was supported in part by CONACyT 28605-M andUSPHS NS 34566-06 grants. We thank to Norma Serrano for hertechnical assistance.

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Table I. The Effects of MPTP on the Level of MT I mRNA (Grains/100 µm2) in Various Regions of Mouse Brain

Grains/100 µm2 Mean ± SEM

Selected areas of the brain Subregions Saline MPTP 3 days MPTP 5 days

Hippocampus Dentate gyrus 418.70 ± 9.28 382.1 ± 35.4 433.9 ± 11.9CA1-Field pyramidal 228.90 ± 24.1 190.3 ± 11.4 215.6 ± 9.7CA1-Field molecular 58.20 ± 5.72 44.3 ± 5.8 46.1 ± 6.0CA2-Field pyramidal 233.50 ± 19.9 204.6 ± 12.70 220.0 ± 15.3CA2-Field molecular 49.5 ± 4.33 43.0 ± 6.17 47.0 ± 7.03CA3-Field pyramidal 135.75 ± 32.04 116.5 ± 15.37 117.3 ± 19.95CA3-Field molecular 51.80 ± 9.06 43.5 ± 10.98 44.2 ± 4.43

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MT-I mRNA Expression after MPTP Administration 509