Ž .Brain Research 783 1998 109–114

Research report

Coenzyme Q attenuates the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine10

Ž .MPTP induced loss of striatal dopamine and dopaminergic axons in agedmice

M. Flint Beal a, Russell T. Matthews a, Alide Tieleman b,c, Clifford W. Shults b,c,)

a Neurology SerÕice, Massachusetts General Hospital, Boston, MA, USAb Neurology SerÕice, Veterans Affairs San Diego Health Care System, 3350 La Jolla Village DriÕe, San Diego, CA 92161, USA

c Dept. of Neurosciences, UniÕersity of California, San Diego, La Jolla, CA, USA

Received 17 June 1997; accepted 23 September 1997

Abstract

Ž .We investigated whether oral administration of coenzyme Q CoQ could attenuate 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine10 10Ž .MPTP neurotoxicity in one-year-old mice. Four groups of one-year-old, male C57BLr6 mice received a either standard diet or a diet

Ž .supplemented with CoQ 200 mgrkgrday for five weeks. After four weeks, one group that had received the standard diet and one10

group that had received the CoQ supplemented diet were treated with MPTP. The four groups continued on their assigned diets for an10

additional week prior to sacrifice. Striatal dopamine concentrations were reduced in both groups treated with MPTP, but they wereŽ .significantly higher 37% in the group treated with CoQ and MPTP than in the group treated with MPTP alone. The density of tyrosine10

Ž .hydroxylase immunoreactive TH–IR fibers in the caudal striatum was reduced in both MPTP-treated groups, but the density of TH–IRŽ .fibers was significantly 62% greater in the group treated with CoQ and MPTP than in the group treated with MPTP alone. Our results10

indicate that CoQ can attenuate the MPTP-induced loss of striatal dopamine and dopaminergic axons in aged mice and suggest that10

CoQ may be useful in the treatment of Parkinson’s disease. q 1998 Elsevier Science B.V.10

Keywords: Coenzyme Q ; Parkinson’s disease; Dopamine; Mitochondria10

1. Introduction

Ž .Parkinson’s disease PD is a progressive neurologicaldisorder in which the cardinal pathological feature is theloss of dopaminergic neurons in the substantia nigra pars

Ž .compacta SNpc and their axons, which project to thew xstriatum 9 . The cause of PD remains unknown, but our

understanding of mechanisms of nigral dopaminergic neu-ronal death was advanced by the discovery of 1-methyl-4-

Ž .phenyl-1,2,3,6-tetrahydropyridine MPTP , a neurotoxinthat selectively damages the nigrostriatal dopaminergicsystem and causes a parkinsonian syndrome in humansw x w x w x17 , monkeys 16 and mice 5 . The discovery that MPTPacts through inhibition of complex I of the electron trans-

w xport chain 28 stimulated study of mitochondrial functionin PD. Two groups have reported that the activity ofcomplex I of the electron transport chain is selectively

) Corresponding author. Fax: q 1-619-5527513; e-mail:cwshults@vapop.ucsd.edu.

w xreduced in the SN of patients with PD 13,20 . Complex Iactivity is also reduced in platelets from PD patientsw x3,15,19,34 and the reduction does not appear to be the

w xresult of debilitation or drug treatment 10,25 .The electron acceptor for complexes I and II of the

Ž .electron transport chain is coenzyme Q CoQ , and thew xpredominant form of CoQ in humans, is CoQ 7 . In10

addition to its function as a redox component of mitochon-dria, CoQ has also been shown to be a powerful antioxi-10

w xdant 7 . Substantial data have implicated mitochondrialdysfunction and excessive production of reactive oxygen

w xspecies in the pathogenesis of PD 8,14,18,21 . The centralrole of CoQ in two areas implicated in the pathogenesis10

of PD, mitochondrial dysfunction and oxidative damage,suggest that it may be useful as a treatment to slow theprogression of PD. Previous in vitro studies have demon-strated that CoQ can reduce the death of mesencephalic10

w xdopaminergic cells 1 and mitigate the age-associatedw xdecline in complex I function in rat skeletal muscle 32 . In

a previous study of MPTP in young mice, CoQ attenu-10

0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved.Ž .PII S0006-8993 97 01192-X

( )M.F. Beal et al.rBrain Research 783 1998 109–114110

ated the MPTP-induced reduction of dopamine in thestriatum, but the reduction was not statistically significantw x w x23 . Levels of CoQ in the brain 29 and various other10

w xtissues 7 decline with aging. The age related decline intissue levels of CoQ prompted us to reexamine the10

ability of oral CoQ to affect MPTP-induced damage to10Ž .the nigrostriatal system in middle aged one year old

mice.

2. Materials and method

2.1. Treatment of mice with CoQ and MPTP10

ŽFour groups of mice were studied: Standard diet con-. Ž .trol , ns11; CoQ supplemented diet CoQ , ns1210 10

Žtissue for measurement of dopamine and its metabolites.was available from 11 of these animals ; standard diet and

Ž . ŽMPTP treatment MPTP , ns11 3 animals in this groupdied following MPTP treatment, and only the remaining 8

.were included in the analyses ; CoQ supplemented diet10Ž .and MPTP treatment CoQ rMPTP , n s 11. Male10

C57BLr6 mice, which were one year old, were purchasedŽfrom the National Institute of Aging. CoQ Vitaline,10

.Ashland, OR was formulated into pellets of rat chow, andthe animals received a diet containing 200 mgrkgrday ofCoQ or standard rat chow for one month. Two groups10Ž .MPTP, CoQ rMPTP received MPTP. MPTP hydro-10

Ž .chloride Research Biochemicals was made up in normalŽ .saline pH 7.4 at 3 mgrml. Animals received intraperi-

toneal injections of either 15 mgrkg MPTP or an equiva-lent volume of saline every 2 h for five doses. The fourgroups of animals continued to receive their usual diets foran additional week and then were sacrificed.

2.2. Measurement of striatal leÕels of dopamine andmetabolites

The two striata were rapidly dissected on a chilled coldplate and placed in tubes containing ice-cold 0.1 M per-chloric acid. Tissue was subsequently sonicated andaliquots were taken for protein quantification using afluorometric assay. Dopamine, 3,4-dihydroxyphenylacetic

Ž . Ž .acid DOPAC and homovanillic acid HVA were quanti-fied by HPLC with 16-electrode electrochemical detectionw x2 .

2.3. Measurement of density of striatal dopaminergic fibers

The hindbrains were placed in a solution of 4% para-Ž .formaldehyde in phosphate buffered saline PBS . After

three days of immersion in the fixative, the brains weretransferred to PBS and shipped to the Univ. California,San Diego. Twenty-five micron sections were cut, and thebrains were immunolabelled for tyrosine hydroxylase as

w xpreviously described 26 . The caudal striatum was present

Žin the brains from 34 animals 11 control animals, 9 CoQ10.animals, 6 MPTP animals, and 8 CoQ rMPTP animals .10

The density of tyrosine hydroxylase–immunoreactiveŽ .TH–IR fibers in two regions of each side of the caudalstriatum was measured using an image analysis system.

Fig. 1. Levels of dopamine and ratios of DOPAC and HVA to dopamineŽ .in the striatum. A The striatal level of dopamine was 11% higher in the

Ž .animals that received the CoQ supplemented diet CoQ than the10 10Ž .animals that received the standard diet control , but the difference was

not significant. In the two groups of animals that received MPTP theŽ .group that also received CoQ CoQ rMPTP had a 37% higher level10 10

Ž .of striatal dopamine than the group that received MPTP only MPTP ;Ž . Ž .q, p-0.01 . B The ratio of DOPACrdopamine in the striatum didnot differ significantly among the four groups. The ratio of HVArdopa-mine in the striatum was significantly higher in the groups treated with

Ž) .MPTP , p-0.01 , but the ratio was significantly lower in theŽ .CoQ rMPTP than in the MPTP group q, p-0.01 .10

( )M.F. Beal et al.rBrain Research 783 1998 109–114 111

The system consists of a Nikon Optiphot microscope and aSony XC77RR camera connected to a Macintosh Quadra700 20r240 computer with a DT2255-60 Hz Mac II image

Ž .processing board Data Translation . The system utilizesŽ .the Image software—version 1.47 NIH . For measure-

ment of the density of TH–IR fibers, we used the 20=

objective and measured the density of TH–IR axons in aŽ 2 .rectangle approximately 76 800 mm in the four sites.

Care was taken to avoid areas in which there was signifi-cant density of blood vessels or white matter tracts. Thedensity of fibers from the 34 animals was determined intwo sessions, and a region of normal striatum from a ratwas used as the standard for calibration of the imageanalysis system to insure equivalent results between thetwo sessions. The mean of the densities of TH–IR materialin the four regions of the striatum was determined for eachanimal. The same evaluator determined the mean density asecond time for 10 animals, and the coefficient of correla-tion between the two sets of measurements was 0.99.

2.4. Determination of the number of TH–IR neurons

We counted TH–IR neurons in the substantia nigraŽ . Ž .SN and retrorubral nucleus RR because both nuclei

w xproject to the striatum 6,33 and both are affected byw xMPTP treatment 24 . Beginning at the first appearance of

the SN and continuing until the disappearance of the RR,

Ž .every fourth section 100 mm intervals was taken forcounting TH–IR neurons. Adequate sets of slides were

Žavailable from 39 animals 10 control animals, 11 CoQ10.animals, 8 MPTP animals, and 10 CoQ rMPTP animals .10

The cells were counted using the 10= objective and aneyepiece reticle. A cell was counted if the nucleus and atleast one process were clearly visible. The boundaries usedfor demarcation of the two nuclei were similar to those

w xpreviously used in the rat 26 . The Abercrombie correc-Ž wtion was applied according to the formula Nsn= tr t

x.qd , where N is the true number of cells, n is the totalnumber of counted cells, t is the thickness of the sectionŽ .25 mm and d is the average diameter of the counted cell.A separate evaluator counted the TH–IR neurons in 11animals. The coefficient of correlation between the tworaters was 0.98.

2.5. Statistics

All measurements were made by observers blinded tothe treatment group. Comparisons were made by applica-

Ž .tion of one factor analysis of variance ANOVA with posthoc Newman–Keuls test. In cases of inhomogeneity ofvariance, the data were transformed to the natural loga-rithm. Statistical analyses were performed with GBSTAT

Ž .5.4.6 Dynamic Microsystems, Silver Springs, MD .

Ž . Ž . Ž .Fig. 2. Representative sections of the caudal striatum immunolabelled for TH. A Standard diet, B CoQ supplemented diet, C standard diet with10Ž .MPTP treatment, D CoQ supplemented diet with MPTP. Although MPTP caused depletion of TH–IR fibers in the striatum, there was conspicuous10

preservation of the TH–IR fibers in the group treated with CoQ . Scale bars50 mm.10

( )M.F. Beal et al.rBrain Research 783 1998 109–114112

3. Results

ŽThe striatal level of dopamine in the CoQ group 60.210.ngrmg protein"7.5, mean"SD, 111% of control was

Ž .slightly higher than that in the control group 54.3"6.2 ,Žbut the difference was not statistically significant Fig.

.1A . As anticipated, the dopamine levels in the MPTPŽ .group 14.8"2.2, 27% of control and the CoQ rMPTP10

Ž .20.2"3.9, 37% of control were both significantly lowerŽthan that in either the control or the CoQ groups p-10

.0.01 . The striatal level of dopamine in the CoQ rMPTP10

group was 37% greater than that in the MPTP group, andŽ . Ž .the difference was highly significant p-0.01 Fig. 1A .

The ratios of DOPACrdopamine and HVArdopamineare considered to be indices of dopamine turnover andtypically increase with injury to the nigrostriatal dopamin-

w xergic system 11 . The ratio of DOPACrdopamine did notdiffer significantly among the four groups. The ratioHVArdopamine was significantly higher in the two groupstreated with MPTP than in those not lesioned. The ratiowas significantly lower in the group treated withCoQ rMPTP than in the group treated with MPTP alone10Ž .Fig. 1B .

The density of TH–IR fibers in the caudal striatumŽ .reflected the levels of dopamine Figs. 2 and 3 . Both of

the groups treated with MPTP had significantly less denseTH–IR fibers than either the control or CoQ groups.10

However, the density of TH–IR fibers in the CoQ rMPTP10

group was 62% and significantly greater than that in theŽ .MPTP group p-0.01 .

We counted the number of TH–IR neurons in the SNand RR, the mesencephalic dopaminergic nuclei that pro-ject to the striatum. Although the number of dopaminergic

Fig. 3. Density of TH–IR material in the striatum quantified using animage analysis system. The density of TH–IR material was significantly

Ž)reduced in both MPTP treated group , p-0.01 compared to control.and CoQ groups , but the CoQ rMPTP had significantly more TH–IR10 10

Ž .material than the MPTP group q, p-0.01 .

Ž .neurons was reduced in the both the MPTP 1163"214Ž .and CoQ rMPTP groups 972"236 , the number of10

TH–IR neurons in the two MPTP-treated groups did notŽdiffer significantly with that in the control group 1286"

.385 .

4. Discussion

Ours is the first demonstration that CoQ alone can10

reduce the damage to the mesostriatal dopaminergic sys-tem in an animal model of PD. Treatment of mice withCoQ before and after MPTP treatment resulted in the10

relative preservation of striatal dopamine, a lowerHVArdopamine ratio and attenuation of the loss of TH–IRfibers. Although our study was not designed to examinesurvival, the fact that three of the 11 animals in the MPTPgroup died but none of the animals in the CoQ rMPTP10

group died further supports a protective role for CoQ .10

Since we did not achieve significant neuronal loss withŽMPTP treatment similar to the experience of others

w x.12,35 , we do not have data regarding the ability ofCoQ to protect against neuron loss. We were surprised10

that treatment with MPTP did not increase theDOPACrdopamine ratio, as it did for the HVArdopamineratio. The reason for this difference is unclear.

MPTP treatment of aged mice, is particularly relevantto PD because it incorporates many of the cardinal bio-

Žchemical and anatomical features reduction of striatal.dopamine and mesencephalic dopaminergic neurons and

Žthe clinical features predominant occurrence in older sub-.jects of the disorder.

Studying both the dopamine levels and the density ofTH–IR fibers in the striatum allowed us to examine twoaspects of the MPTP-injury to striatal dopaminergic axons.One might predict that one week after MPTP treatment,the striatal concentration of dopamine, which is the prod-uct of a number of steps including the enzymatic action ofTH and aromatic acid decarboxylase and storage of thecatecholamine, would be affected to a greater extent thanthe level of only TH–IR material. This is what we found.The dopamine level was 27% and the density of TH–IRmaterial was 37% in MPTP-treated animals as comparedto control animals. This difference may simply reflect thecontinued presence of TH in dying axons, which stillcontained TH but no longer could produce dopamine.However, the greater preservation of TH–IR material in

Ž .the CoQ rMPTP animals 60% of control as compared10Ž .to the MPTP-treated animals 37% of control may suggest

that the CoQ rMPTP animals had greater preservation of10

the components involved in dopamine synthesis and agreater potential for long term recovery at times beyondone week.

We presume that CoQ acted by enhancement of mito-10

chondrial function andror by reduction of the levels of

( )M.F. Beal et al.rBrain Research 783 1998 109–114 113

reactive oxygen species in the damaged tissue. MPTPw ximpairs complex I activity 28 and increases the produc-

w xtion of reactive oxygen species 4,30 . CoQ increases the10

activity of the mitochondrial electron transport chain bothw xin vitro 22 and attenuates the age-associated decline in

w xmitochondrial activity in vivo 32 . In addition, CoQ has10w xbeen shown to be an effective antioxidant 31 .

The greater effectiveness of CoQ in aged animals, as10w xcompared to that in young mice 23 , probably is the result

of the decline in brain levels of CoQ with aging. These10

observations and our study underscore the obvious, butoften overlooked, fact that models of neurodegenerativedisorders of the aged are most accurately assessed in agedanimals.

Recent work from our group has suggested that CoQ10

may play a role in the development of PD. We hadpreviously reported that the activities of complexes I andIIrIII from platelet mitochondria were significantly re-duced in patients with early untreated PD when comparedto agersex-matched neurologically normal control subjectsw x10 . We recently found that the level of CoQ was10

significantly lower in mitochondria from the parkinsonianpatients than in mitochondria from agersex-matched con-trol subjects and that the levels of CoQ and the activities10

of complex I and complex IIrIII were significantly corre-w xlated 27 .

In summary, we found that oral treatment of aged micewith CoQ caused a significant attenuation of the loss of10

striatal dopamine and dopaminergic axons caused byMPTP. Our finding indicates that CoQ may be a useful10

therapy to ameliorate the progression of PD.

Acknowledgements

The work was supported by NS32365, NS31579 andPO1 AG12992 grants to M.F.B. and a Merit Review Grantfrom the Dept. of Veterans Affairs and a Center of Excel-lence Award from the National Parkinson Fndn. to C.S.Coenzyme Q was generously supplied by Vitaline, Ash-10

land, OR.

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