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ORIGINAL ARTICLES

Alterations in Glutathone LevelsPadanson’s Disease andOther

Neurodegenerative Disorders AfKectmgBasal Gangha

in

Jeswinder Sian, BSc,” David T. Dexter, PhD,* Andrew J. Lees, FRCP,? Susan Daniel, MRCPath,?Yves Agid, MD, P hD J France Javoy-Agid, Ph D, I Peter Jenner, DSc,” and C. David Marsden, FRSi

Reduced glutathione (GSH) and oxidized glutathione (GSSG) levels were measured in various brain areas (substantianigra, putamen, caudate nucleus, globus pallidus, and cerebral cortex) from patients dying with Parkinson’s disease,progressive supranuclear palsy, multiple-system atrophy, and Huntington’s disease and from control subjects with noneuropathological changes in substantia nigra. GSH levels were reduced in substantia nigra in Parkinson’s diseasepatients (40 compared to contro l subjects) and GSSG levels were marginally (27 ) but insignificantly elevated;there were no changes in other brain areas. The only significant change in multiple-system atrophy was an increaseof GSH (176 ) coupled with a reduction of GSSG (60 ) in the globus pallidus. Th e only change in progressivesupranuclear palsy was a reduced level of GSH in the caudate nucleus (5 1 ). Th e only change in Huntington’s diseasewas a reduction of GSSG in the caudate nucleus (50 ). Despite profound nigral cell loss in the substantia nigra inParkinson’s disease, multiple-system atrophy, and progressive supranuclear palsy, the level of GSH in the substantianigra was significantly reduced only in Parkinson’s disease. This suggests that the change in GSH in Parkinson’sdisease is not solely due to nigral cell death, or entirely explained by drug therapy, for multiple-system atrophypatients were also treated with levodopa. The altered GSH/GSSG ratio in the substantia nigra in Parkinson’s diseaseis consistent with the concept of oxidative stress as a major component in the pathogenesis of nigral cell death inParkinson’s disease.

Sian J, Dexter DT, Lees AJ, Daniel S , Agid Y avoy-Agid F, Jenner P, Marsden CD.Alterations in glutathione levels in Parkinson’s disease and other neurodegenerative

disorders affecting basal ganglia. Ann Neurol 1994;36:348-355

The process underlying dopaminergic cell loss in thesubstantia nigra (SN) in patients with Parkinson’s dis-ease (P D) is unknown. Recent evidence from postmor-tem studies indicates that the induction of oxidativestress may play a role. Thus, in the SN of patientsdying with P D there are increased levels of total iron[ l , 23, decreased ferritin levels [3], increased lipid per-oxidation 14, 53, and a reduced activity of complex Iof t he mitochondria1 respiratory chain [63. Free radicaltoxicity normally is prevented by a range of antioxidant

vitamins and protective enzymes. Most of these appearto be intact in the SN in PD. Thus, levels of catalaseand glutathione peroxidase are normal or moderatelyreduced [7-93 while superoxide dismutase activitymay be increased [lo]. Normal levels of ascorbic acidand a-tocopherol are found in the SN in PD [2, 111.

Another important component protecting againstoxidative stress is reduced glutathione (G SH ), whichmay be depleted in PD. Glutathione plays a majorrole in the removal of peroxides, so preventing theformation of more damaging oxygen radicals, such asthe hydroxyl radical. Normally a high ratio of reducedG S H to oxidized glutathione (GSS G) is maintained1123. Glutathione is also important for the maintenanceof a-tocopherol and ascorbic acid in the reduced state1133. A disappearance of total glutathione and GSH

content in the SN of patients dying with PD was firstreported by Perry and colleagues 1141.However, thesefindings were criticized [ l 5 ] because of the high pro-portion of GSSG reported in control subjects and be-cause of the total absence of GSH in PD patients.Nevertheless, the concept of altered glutathione levels

From the “Parkinson’s Disease Society Experimental ResearchLabo-ratories, Pharmacology Gr oup , Biom edical Sciences Division, King’sCollege London, Lo ndon; Warkinson’s Disease Society Brain Ban k,University Departmentof Clinical Neurology, Instituteof Neurol-ogy, National Hospital, London, United Kingdom; and tLaboratoirede Medicine Experimental, I N S E M U289, HBpital de la Salpg-trii.re, Paris, France.

Received Jul 29, 1993, and in revised form Feb 14 and May 19,1994. Accepted for publication May 19, 1994.

Address correspondence to p. Jenner, D S ~ , harmacology G ~ ~ ~Biomedical Sciences Division, King,s College London, Manresa

Road, London sw3G ~ ~ ,K ,

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in PD is s u p p o r t e d b y a more recen t r epo r t by Ried-e re r and co l l eagues (21 w h o s h o w e d t o ta l g lu t a t h io n elevels to be r e d u c e d b y 50 , i n t h e SN. I t r ema insu n c l e ar w h e t h e r a r e d u c t i o n i n GSH l eve ls do es occu rin SN, w h e t h e r t h i s is speci f ic to PD, or w h e t h e r itoccu r s i n other neu rodegene ra t i ve d i so rde r s a f f ect i ngt h e SN. I t is a l so u n k n o w n w h e t h e r a n y c h an g e i ng lu t a th ione i n SN i n PD occu r s as a resul t

ofl e v o d o p a

t r ea tmen t .Cons equen t ly , w e have ana lyzed GSH a n d GSSG

leve ls in a r a n g e of brain areas i n pa t i en t s with PD a n din pa t i en t s w i th o the r degene ra t i ve d i so rde r s , name lymul t i p l e - sys t em a t rophy MSA), prog re s s ive sup ranu -clear palsy (PSP), and Hunt ing ton ’s d i s ea se(HD), a n dc o m p a re d t h e m t o l ev e ls i nnormal individuals.

Materials and MethodsBrain tissue from patients dying with PD , PSP,or MSA andfrom con trol subjects dying of nonneurological disorders was

obtained from the Brain Bank ofINSERM U28 9, HBpitalde la Salp@trii.re,Paris, and the Parkinson’s Disease SocietyBrain Bank, London. Tissue from patients dying withHDwas supplied by D r Gavin Reynolds, De partme nt of Biomed-ical Science, Universityof Sheffield.

Tissue PreparationIn London, at autopsy, brains were removed and dividedmidsagittally. One-half of the brain was immediately frozenat -20”C, transported on Cardiceto the Brain Bank, andfrozen at 0°C until dissection. T he ot he r half of the brainwas placed in 10% form ol saline solution for at least6 weeksprior to neuropathological examination. In Paris, less than 2hours after autopsy, the brainstem was first separated fromthe rest of the brain. Subsequently, one hemisphere of thebrain was stored at 0°C until dissection, while the ot he rhalf was used fo r neuropathological examination.

T h e SN (total or zona compacta), putamen, caudate nu-cleus, globus pallidus, and cerebral cortex (Brodm ann area10) were dissected from frozen brain accordingto the tech-nique described by D ext er and co authors 141. Samples fromeach brain area were stored at - 0°C until the time of bio-

chemical analysis. Brain samples fr om patients with neurolog-ical disease were assayed at the same time as samples fromcont rol sub jects, which wer e matched as much as possiblefor age and postm ortem delay.For som e sub jects, insufficientmaterial was available for assaysto be undertaken in all re-gions of th e individual brains. Th e num bers of samples stud-ied from each brain area in each group are shown in Figures3 through 6. Whether totalSN or zona compacta alon e was

utilized is stated in the legends of the individual figures.Different control brains were used for comparison with

brains representing each of the neurodegenerative diseasesstudied. This was undertaken to ensure appropriate agematching and that postmortem handling, storage, and dissec-tion were as similar as possible in each grou p. This was partic-ularly important when brain material came from differentbrain banks and every effort was madeto obtain matchedcontrol samples from the same source of brain material.

Parkinson’s Disease GroupBrain tissue was obtained from 13 control subjects with noknown history of psychiatric or neurological disorder and

16 P D p atients (Table 1). Histological diagnosis of P D wasconfirmed in all the tissues utilized by the severe neuronalloss in the SN pars compacta and the presence of Lewy bod-ies in surviving cells. Th e control subjects and P D patientswere closely matched for age and postmortem delay (timebetween death and removal of brain). The caudate dopamineconten t was significantly lower in the P D patients (1.02*0.25 pg/g m) compared to the control subjects (2.84 0.40pg/gm; p < 0.05, Student’s test).

Multiple-System Atrophy GroupBrain tissue was obtained from10 control subjects withn oknown history of psychiatricor neurological disorder and 7MSA subjects (Table 1. Pathological diagnosis of MS A wasconfirmed by the presence of striatonigral degeneration withmarked gliosis and cellloss in the SN, caudate nucleus, andputamen. I n addition, there were pathological changes thatvaried in severity involving the olivo pon tocere bellar systemand preganglionic autonomic nuclei. Lewy bodies were notobserved in any brain area from M SA patients. C ontrol sub-jects and MSA patients were closely matched for age andpostm ortem delay.

Table I Characteristics o f Control Subjects and Parkinson’s Disease (P D, and Multiple-System Atrophy( M S A )Patientsa

Contro l P D Contro l MSAPatient Details (n = 13) (n = 16) (n = 10) (n = 7 )

Age (yr) 73.5 2.9 77.1 * 1.8 68.3 5.7 63.4 3.0Sex

Female 1 4 1 2Male 12 12 7 5

Age at onset of disease (y r) 66 .8 -c 2.5 56.4 ? 2.8(59-7 ) (47- 72)

Duration of disease (yr ) 12.4-c 3.1 7.0 0.9

Levodop a dosage level at time of

Tim e between death and removal 15.6 2.2 13.4? 1.9 14.2 1.5 17.0 * 8.6

(5-24) (3-10)470 * 180 mglday 618 116 mglday

death (100- 1,000 mg) (330-1,100 mg)

of brain (hr)

‘Values are expressed as mean 5 standard error of mean. Rangesof values are indicatedin parentheses.

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Table 2. Characteristics of Control Subjects and Progressizle Supranuclear Palsy PSP) nd Huntington's Disease ( H D )Patients"

Patient Details ( n = 15) ( n = 11) ( n = 10) (n = 10)

Age (yr) 80.3 ? 1.9 70.0 2.2h 50.1 ? 4.7 52.9 ? 3.5Sex

Control PSP Control HD

Female 9 6 1 3Male 6 5 9 7

Duration of disorder yr) N A 9.4 ? 1.9Time between death and removal of brain (hr) 9.4 ? 1.8 11.8 t 1.9 36.0 ? 7.3 50.7 ? 10.7h

"Values are representedas mean standard erro rof mean.p < 0.03 as compared to control subjects, Student's t test.

N A = not available

Progressive Supranuclear Palsy GroupBrain tissue was obtained from 15 control subjects with n oknown history of psychiatricor neurological disorder and 11PSP patients (Ta ble 2). Pathological diagnosis of PSP wasconlirmed by nerve cell loss in the S N , globus pallidus, andbrainstem nuclei, including the locus ceruleus and superiorcolliculus, and th e presen ce of globos e neurofibrillary tanglesin surviving neurons in the pallidum, subthalamic nucleus,midbrain, and d entate nucleus. Contro l subjects and PSP pa-tients were closely matched for postmortem delay, but themean age of PSP patients was significantly lower than that ofcontrol subjects.

Huntington Diseau GroupBrain tissue was obtained from 10 control subjects withn oknown history of psychiatricor neurological disorder and 10HD patients (Table 2) . Morphological diagnosisof HD wasconfirmed by macroscopic atrophy with gliosis and markedneuronal loss in the caudate nucleus and putamen. The timebetween death and removal of the brain was longer for theHD patients than the control subjects.

Measurement of GfututhioiieLevelsBrain tissue was homogenized in6 volumes ice-cold 0.4 Mperchloric acid containing 0.1 mM diethylenetriaminepen-taacetic acid using a microsonic probe. The samples werecentrifuged at 4,000 rpm , 4°C for 1 5 minutes, and th e super-natant was analyzed for GSSG and G S H con tent immediatelyafter homogenization.

Measurement of Oxidized GfutathionrGSSG was measured by the enzymatic recycling proceduredescribed by Tietze [ 16) and Slivka and coauthors[ 15).

An aliquot (0.1 ml) of the tissue supernatant was addedto 1.35 ml of I1 mM N-ethylmaleimide (N EM ) in100 mMpotassium phosphate buffer containing 5 mM ethylene-diaminetetraacetic acid, p H 7 .5 . After a 20-minute incuba-tion period at room temperature, the reaction mixture waspassed th roug h Sep-P ak C- 18 cartridges (,Millipore, Wa tersAssociates, Watford, United Kingdom )to remove unreactedNEM. Preliminary experiments showed 99.6% (n= 6) ofthe unreacted NEM was retained by the cartridges.

Spectrophotom etric assays were performed using1.5 ml ofeluate to which 0.4 mM 5,5 -dithiobis-(L-nitrobenzoic cid)

(DTN B) , 0 .17 mM N AD PH , and 16 kg /mlof GSSG reduc-

case were added. The final assay volume was 2.0 ml. Thereaction was initiated by the addition of GSSG reductase.

Th e rate of reduction of DT N B to 5-thio-2-nitrobenzoate(T N B) was measured at 412 nm using a Shimadzu double-beam spectrophotometer at ambient temperature for 5 min-utes. A srandard curve was constructed using known amounts

of synthetic GS SG (10-100 ng). Th e standard curves toGSS G w ere l inear r = 0.994) ov er this concentration range.

Measurement of Redui-ed GlutathioneG S H was measured using a minor modification of themethod described by Reed and coauthors 171. An aliquot(0.17 ml) of tissue supernatant was added to the internalstandard (1 mM cysteic acid) and 0.88 M iodoacetic acid.Excess sodium hydrogen carbonate was addedto precipitatesodium perchlorate. The samples were incubated at roomtempe rature in the dark for an hour. Subsequen tly, 0.5 mlof an alcoholic solution of 1.5% (vo l/vo l) 2,4-dinitro-fluorobenzene was added and the samples were incubatedfor a further 4 hours. Th en diethyl ether(1.0 ml) was addedto the samples, which were shaken and then centrifuged at2,000 rpm, room temperature, for2 0 minutes. T he aqueo usphase containing derivatized glutathione was separated andanalyzed by high -perfo rman ce liquid chromatography(HPLC).

Aliquots (10 ILL) were injected onto a Spherisorb S-5amino OD S column (25 x 4.6 m m, Phase Separations) andeluted with an ammonium acetate gradient in glacial aceticacid, methanol, and water (p H 5.05; flow rate 1.00-1.25 mlimin at 2,70 0 psi). G S H levels were measured using a Watersultraviolet de tector (m odel 441) a t a wavelength of 3 65 nm.Chromatographic peaks were integrated by a Waters 645data module. G S H derivatives were quantified in relationtothe internal standard (cysteic acid).

StatisticsGSH and GSSG levels in each brain region from controlsubjects and patients with PD , MSA, PSP,or HD were com-pared using an unpaired two-tailed Student'st test.

ResultsGlutathione Leiiels in Control SubjectsA high GSHIGSSG ratio (mean, 318: 1; range, 226-800: 1) was observed in control subjects fromall the

groups examined for the present study. Therewas no

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rp

t 0 a M ) M I S o w

Age < I N

B

F i g I Linea r regression analys is fo r A ) educed glutathione( G S H ) evels pniollgm wet weight) with age and ( B )oxidizedglutathione GSSG) evels (pmollgm wet weight) with ageinthe total substan tia nigra and pu tamen from control subjects.There was n o correlation between gluta thione letleis and patientage (regression coefficient< 0.01: n = 13-20),

obvious correlation betw een age and G S H or G SS Glevels in the SN and putamen of control subjects (Fig1). Similarly, there was no correlation between post-mortem delay (time berween death and removal ofbrain) and GSH (Fig 2A) or GSSG (Fig 2 B ) contentin the SN and putamen.

Glutathione Leuels in Parkin son's DiseaseTh ere was a significant reduction in G S H in theSN( 4 0 % ) from all P D patients cornparedto control sub-jects (P D: n = 16, 0.45 ? 0.03 +mol/gm wet weight;

control: n = 1 3 , 0.75 0.46; p < 0.01). This appliedto both the SN pars compacta and to the totalSN (Fig3 A ) . G S H levels in the cerebral cortex and putamenwere somewhat decreased com pared to contro l values,but these changes did not reach statistical significance.No alterations in G S H occu rred with o ther brain areasstudied.

In contrast, GS SG levels (Fig3 B )were not altered inPD patients compared to control subjects. Th e GS SGcontent in the SN from all PD patients was insignifi-cantly increased by 27% (PD: n = 13, 0.00310.0004 pmol/gm wet weight; control: n = 12, 0.0024

0.0004, p = 0.17, not significant ENS]).

Ghtathione Letleis i n Multiple-System A trophyThe G SH con tent of the lateral globus pallidus fromMSA patients was approximately double(1969%) thatfound in control subjec ts (Fig4A). There was a corre-sponding reduction (60 ) of the GSSG content in thelateral glo bus pallidus inMSA patients (Fig4B ) .Therewere no significant changesi n G S H or GSS G levels in

other brain areas examined.

Glutathione Leilels i n Progressive Supranuclear PalsyG S H levels in PSP patients were reduce d in the cau-date nucleus ( 5 1 % ) (Fig 5A). There were no alter-ations in G S H content in the cerebral cortex, putamen,or SN. GSSG content in PSP patients was not alteredin any of the brain areas examined (Fig5B) . (Th ere wasnot sufficient nigral tissue to measure GSSG levels.)

Glutathione Le?JeIs n Hun tingto n? DiseaseT he G S H conten t in cerebral cortex, caudate nucleus,

and SN zona compacta from HD patients was not al-tered compared to control values (Fig6A). Th ere wasa marked reduction in the GSS G( 5 0 % )content in thecaudate nucleus fromHD patients comparedto control

Fig 2 . Linear regression analysis fo r A) educed glutathione( G S H ) leziels(pmollgm u'et weight)u i t h postmortem delay(time between death and remcajalof brain) and (Bi oxidized glu-tathione GSSG) hels (pmollgtn urt weight) with postmortemde ay (hours) iti the total substantia nigra and putamen fromcontrol subjects. There was no correlation between glutathionefezels and poslmortem delay (regression coPfficient< 0.01n = 13-20).

Uh\ha c g , ~\ < , ~ ? , . , l u > r = c ,11211

4 l'

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k - 1 44erebral cortex -

Putamen IT)

Globus pallidus IL)

-_ _ IGlobus pallidus (MI

I r I0.0 0 2 0.4 0 6 0 8 1.0 1 1 1.4

GSH pmalr./p we1 weigh1 human braln

Cerebral cortex

Caudate nucleus

Putamen 1.1

------I

-lobus pallidus (L)

Globus pallidus IM)

+-I4+ubstantia nigra IT)

I

0.WO 0 002 0.004 a.m 0.008

GSSC prnalesip n e t weight human brain

Fig 3 . Lecels of ( top) reduced ,&tathione (GSH) (pmo llgm wetweight, and (bottom) oxidized glutathione (GSSG) (pmollgrnwet weight) i n Parkinson's disease(hatched bars)and age-matched control (open bars)human autopsy brains. Values arerepresented as mean standa rd error of mean. Asterisk indi-cates p < 0.01 compared with controls, Student's t test. Numberof saniph is shown it2 each bar. T = total; L = lateral; M =medial: C = zana cornpacta.

subjects (Fig 6B). (There was not sufficient tissue tomeasure nigral GSSG levels.)

GSHIGSSGRatio in Szrbstantia Nigra i nParkinson's Disease and Multiple-System AtrophyThe nigral GSH IGS SG rat io tendedto be decreasedin both PD and MSA patients compared to controlsubjects (Fig 7). Th e reduct ion of the GSH IGS SG ra-tio in P D patients reached statistical significance p <0.05); that in MSA patients only just failed to meetstatistical significance( p = 0.051). No ratios could becalculated for PSP orHD patients since nigral GSSGlevels could not be measured d ueto the limited sampleavailable.

DiscussionMethodological h u e sWith the glutathione analysis method of Slivka andcolleagues [ 151,a high G SH IG SS G ratio was observedin the brains of all control subjects. This indicates that

little autolytic loss of glutathione had taken place as aresult of post mo rtem delay. Inde ed, in this study ther ewas no correlation betw een glutathio ne levels and post-morte m delay in control subjects. Th e great er part(>98 ) of total glutathione was in the reduced form(GS H) , which is consistent with the normal oxidativestate of the brain [12). Th e levels of G S H and GSS G

found were in accordance with those previously re-ported [ 151. The much higher levels of GSSG previ-ously repo rted by Perry and coauthors[ 141 must havebeen due to postmo rtem delayor methodological arti-facts. In the PSP group studied here, there was a sig-nificant difference in age between disease brains andcontrol brains. However, there was no correlation be-tween GSH or GSSG levels and age in any groupstudied.

Alterations in Glutathione in the Substantia NigraT he pre sent data indicate a selective reduc tionof G S H

in the SN of PD brains that occurs without a corre-

F i g 4. Lmels o ( t op ) reduced glu tathione (GSH) pmollgrn wetweight) and (bottom) oxidized glutathione(GSSG) pmol lgmwet weigh t) in multiple-system atrophy(hatched bars)and age-matched control (open bars)human autopsy brains. Values arerepresented as mean standard error of mean. Asterisk indicates p < 0.05 compared wi th controls. Stu de nti t test. The dzj-fereni-e or GSH in the sub3,tantia nigra compared t o controlswas not significant ( p > 0.1 . Number of samples is shown ineach bar. L = lateral; M = medial; T = total.

---II

Cerebral cortex

Globus pallidus M)

Globus pallidus 1,)E ---IFubstantia nigra

0.0 U.5 1.0 I s 2.0

csi i r rnocn/g wet urighl hunlan brain

Cerebral c o r t e x

3 4

__1utamen L )

Globus pallidus (M)

Globus pallidus IL)

Substantia nigra

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I

--ICaudate nucleus

Putamen (T)

I C I ASubstantia nigra c )

0.0 0 5 1.0 1.1 2 0

CSH pmdru g wet w r i g h l human brain

~ .A

ICerebral cortex

Caudate nucleus

Putamen TI

Substant ia n i p C) N

0.WO 0.001 0 002 0.003 0.W4 0.001 0 006

CSSC pmolcsig wet weight human brain

B

F i g 5 Levels of (A) reduced glutathione ( G S H ) (ynioflgm wetweighti and ( B )oxidized glutathione(GSSG)(ymollg m wetweighti in progressive supranuclear palsy (stippled bars) andage-matched control (op en bars) human autopsy brains. Valuesare represented as mean tandard error of mean. Asterisk in-

dicates p < 0.05 compared w ith controls. Student\ t test. ThediHerenc-e or GSH in substantia nigra compared t o controls wasnot significant (p > 0.31. Number of samples is shown in eachbar. T = total: C = compacta: N D = not determined.

Cerebral cortex

I C I L

Substantia nigra

Caudate nuclems

Substantia nigra - NI r I@.ma @.Wl 0.wz 0 m 0.wJ 0.w.F l l . I y I

CSSD p m d c d g WCI weigh1 h u m a n brain

B

F i g 6 . Latiels of Ai reduced glutathione ( G S H ) ymol lgm wetuiezght) an d IBi oxidized glutathione GSSG) ymollgmwetweight) in Huntington's disease(stippled bars)and age-matched control (open bars) human autopsy brains. AsteriskindicateJ p < 0.05 compared with controls. Student's t test.Number of samples is shown iii each bar. N D = not deter-mined; C = zona compacta.

sponding significant increase in GSSG content. Therewere no other changes in glutathione content inPDbrains. However, the ratio GSH/GSSG in theSN ofP D brains was altered in favor of the oxidized formand in a manner consistent with the involvement ofoxidative stress in nigral cell loss in PD. Th e present

findings are in agreement with a recent report fromRiederer and colleagues which also showed a similardecrease of total glutathione 2 a nd G S H 1181 levelsin SN in PD.

T he m arked nigral depletion of GS H appearsto beselective to PD , since it was not observed in the SNOf MSA7 psp' Or HD although there was a

Fig 7 . Reduced-oxidized glutathione(GSHIGSSG)ratio i n thesubstantia nigra o f Parkinson's disease (PD, n = 16) and mul-

trend toward a reduction in and psp brains' Thisin GSH levels in PD are

not a general consequenceof neurodegeneration what-

tiple-jystmi atrophy(MSA,compared t o age-matched contra( n = 10-13, open bars) hu-man autopsy brains. Values are represented as mean

= 4 )patients (hatched bars)that the

Stan-ever the Cause, but are ~ ~ c hore evident in thepathological process underlying PD. There are equiva-

lent degrees of cell loss, around 70%, in the lateral

dard error of mean. Asterisk indicates p < 0.05 for PD com-pared wi th controls, Student? t test cfor M S Acompared to

controls. p = 0.051).

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ventral tier of the zona compacta of theSN in PD,MSA, and PSP 1191. Sup port for this conclusion come sfrom HD studies, where not only were nigral levels ofG S H unchanged but also there were n o alterations inGSH levels in the caudate nucleus despite the markedpathology found in this brain area[201. T he change inG SH in the SN in PD was unlikely to have been due

to L-dopa treatment. Patients with M SA also were tak-ing this drug in larger doses on average, yet there wasa smaller and insignificant fall in G S H c onte nt inMSAbrains. How ever, th ere was a fall in the ratio of G S Hto GS SG both in P D and MSA brains (although thatin MS A brains just failed to m ee t significance); this iscompatible with oxidative stress in both conditions,sowe cannot rule ou t a contribution from L-dopa therapy.

Alteratiom in Gbt athio ne in Other Brain RegionsThe re were som e interesting changes in G S H andGSSG content in other areas in MSA, PSP, andHD

brains, but these are difficult to interpret. The mostdramatic occurred in the lateral globus pallidus in MSAbrains whe re the re was an elevation of G S H levelscoupled to a reduction of GSSG content. This areaof the globus pallidus is specifically affected inMSA,showing a marked loss of myelinated fibers and gliosis[21]. This finding appears to signify an altered redoxstate that serves to maintain glutathione in its reducedform 1121. Why this should occur is not clear.

There was a reduction in GSH levels in the caudatenucleus in PSP brains but no equivalent change inGSSG in this area. This changeis similar to that ob-

served in the SN in P D brains, and may reflect thepresence of oxidative stress. How ever , the striatum inPSP brains exhibits only mild pathological changesC20). In HD brains there was a reduction in GSSG inthe caudate nucleus bu t no change in G S H in this area.T he reduction of GSSG in HD again is unexplained,but may be secondary to the macroscopic atrophy andloss of the GABAergic medium-sized spiny neuronsm.Possible Causes of Nigral Glutathione Defcieniyin Parkinson? Disease

The decrease of GSH in PD might be the result ofdefective synthesis, excessive metabolism, or abnormalutilization. Activity of the rate-limiting synthetic en-zyme y-glutamylcysteine synthetase in theSN in PDremains to be determin ed, as does the enzyme mainlyresponsible for the translocation and breakdown ofboth GSH and GSSG, namely y-glutamyltranspepti-dase. Th e abilityto form mercapturate conjugates fromglutathion e appears unchanged since glutathione trans-ferase activity in the SN in P D reportedly is normal

Given the current concept of oxidative stress as a

contributory factor to nigral cell death in PD, changes

~ 2 2 3 .

in GS H utilization throug h its oxidative-reductivepathway might be relevant. However, there seems tobe no overall change in tissue levels of glutathioneperoxidase activity in the SN in PD [9]. Recently,however, a m arked elevation was found in glutathioneperoxidase-containing glial cells in theSN from par-kinsonian patients 1231. Under conditions of intense

oxidative stress, utilizationof GSH via glutathion e per-oxidase leads to an irreversible loss of intracellularG S H [24 ] . Despite the decreased GSH/GSSG ratioin the SN in PD, there was no absolute increase inGSSG levels that might argue against the decrease inG S H arising from oxidative stress. However, GS SG istransported from cells under conditions of oxidativestress as protection against its cytotoxic effects[25}.

T h e precise localization of altered G S H levels in theSN in P D is not known. Since neurons only constituteapproximately 1% of the total number of nigral cells,it is likely that the mark ed nigral G S H depletion found

in PD occurs in nonneuronal populations such as glia.Inde ed, histochemical studies have shown that G S H islocalized m ainly in glial cells and t he neuropil{26-28).This raises fundamental questions of the role playedby glial cells in the neuronal degeneration inSN thatcharacterizes PD.

Relationship Between Depletionof GSH avzd OtherIndices of Oxidutike Stress i n the Slrbstantia Nigr aThe deple tion of GS H in theSN in P D adds to thegrowing list of biochemical changes (increased lipidperoxidation, raised iron levels, inhibited complexI

activity) that suggest oxidative stressas a componentof the degene rative process[29]. Recently, we investi-gated t he same biochemical param eters in subjects withincidental Lewy body disease (pres ymp tomatic PD )1301. In the SN from these subjects, there was noalteration in iron levels and no significant decrease incomplex I activity but the levels of G S H w ere de-creased to the same extent as in advanced PD [31].This suggests that the reduction of GSH is the earliestindex of oxidative stressso far uncovered and that onlysubsequently do alterations in iron m etabolism a ndmi-tochondrial function become apparent. Interestingly,

depletion of G S H in brain using an inhibitor ofy-glutamylcysteine synthetase, namely buthionine sulfox-imine, can itself lead to mitochondrial damage [ 12).

There may, however, be the opposite connectionberween the depletion of GSH in the SN in P D andinhibition of complexI activity. Thus, hepatocytes ex-posed to 1-methyl-4-phenylpyridinium ion (MPP+and other mitochondrial toxins show a decrease inG S H content with no corresponding rise in GS SG lev-els, resulting in a fall in the GSHIGSSG ratio[32].This effect was attributed to a combination of de-creased cellular metabolism and an efflux of GS H from

the cells. So, even in incidental Lewy body disease

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there may be alterations in mitochondrial function thatremain to be detected but that leadto GSH depletion.

This study was supported by the Medical Research Council and theParkinson’s Disease Society and IN SERM .

References1. D exter D T, Carayon A, Javoy-Agid F, etal. Alterations in the

levels of iron, ferritin and other trace metals in Parkinson’s dis-ease and other neurodegenerative diseases affecting the basalganglia. Brain 1991;114:1953-1975

2. Riederer P, Sofic E, Rausch W-D , et al. Transition m etals, ferri-tin, glutathione and ascorbic acid in parkinsonian brains.J Neu-rochem 1989;52:515-520

3. Dexter DT , Carayon A, Vidaillhet M, etal. Decreased ferritinlevels in brain in Parkinson’s disease. J Ne uroc hem1990;55:16-20

4. Dexter D T, Carter CJ, WellsFR, et al. Basal lipid peroxidationin subsrantia nigra is increased in Parkinson’s disease.J Neuro-

chem 1989;52:381-3895. Dexter DT. Holley AE, Flitter WE, etal. lncreased levels of

lipid hyd roperoxides, bu t no evidence for increased free radicalformation, in the parkinsonian substantia nigra. Mov Disord1994;9:92-97

6. Schapira AHV , Cooper JM, DexterDT, e t al. Mitochondrialcomplex I deficiency in Park inson’s disease.J Neurochem 1990;54 323-82 7

7. Kish SJ, Moriro C, Hornykiewicz 0 Glutathione peroxidaseactivity in Parkinson’s disease brain. Neurosci Lett1985;58:

8. Ambani IM, Van Woert M H, Murphy S. Brain peroxidase andcatalase in Parkinson’s disease. Arch N eur ol 1975;3 2: 114-1 1 8

9. Marttila RJ, Lorentz H, Rinne UK. Oxygen toxicity protect-

ing enzym es in Parkinson ’s disease: increase of superoxidedismutase-like activity in the substantia nigra and basal nucleus.J Neurol Sci 1988;86:321-331

10. Saggu H, CookseyJ Dexte r DT, e t al. A selective increase inparticulate superoxide dismutase activity in parkinsonian sub-stanria nigra.J Neurochem 1989;53:692-697

11. Dexter D T, Ward RJ, Wells FR, et al. a-Tocopherol levels inbrain are not altered in Parkinson’s disease. A nn Ne urol 1992;32:591-593

12. Meister A, Anderson ME. Glutathione. Annu Rev Biochem1983;52:711-760

13. MarrenssonJ, ames EKL, Meister A. H igh affinity transport ofglutathione is a part of a multicom partment system essential formitochondrial function. Proc Natl Acad Sci USA 1990;87:7185-7 189

14. Perry TL, Godin DV , Han sen S. Parkinson’s disease: a disorder

343-346

due to nigral glutathione deficiency Neurosci Lett 1 982;33:

15. Slivka A, Spina MB , Coh en G . Reduced and oxidised glutarhi-one in human and monkey brain. Neurosci Lett 1987;74:112-118

16. Tietze F. Enzymatic method for quantitative determination ofnanogram amountsof total and oxidised glutathione. A nal Bio-chem 1969;27:502-522

17. Reed DJ, Babson JR, Beatty PW, etal. High performance liquidchromatography analysis of nanom ole levels of glutathione , glu-tarhione disulphide and related thiols and disulphides. Anal Bio-chem 1980;106:55-62

18. Sofic E, Lange KW, Jellinger K, et al. Reduced and oxidisedglutathione in the substantia nigra of patients with Parkinson’sdisease. Neurosci Lett 1992;142:128-130

19. Fearnley JM , Lees AJ. Ageing and Parkinson’s disease: subsran-ria nigra regional selectivity. Brain 1991;119:2283-2301

20. Gib b WR G. N europathology of th e subsrantia nigra. Eu rJ N eu-rol 1991;3 (suppl):48-59

2 1. Fearnley JM , Lees AJ. Striatonigral degeneration. B rain1990;

22. Perry TL, Yong VW. Idiopath ic Parkinson’s disease, progressiv esupranuclear palsy and glutathione m etabolism in the substantia

nigra of patients. Neurosci Lett 1986;67:269-27423. Dauvier P, Hirsch EC, Zhang P, etal. Glutathione peroxidase,

glial cells and P arkinson’s disease. Neuroscie nce 1093;52:1-624. UhligS, Wendel A. The physiological co nsequences of glutathi-

one variations. Life Sci 1992;5: 1083-109425. Griffiths O W , Meister A. Origin and turnover of mitochondrial

glutathione. Proc Natl Acad Sci USA 1985;82:4668-467226. Abramovitz M, H om ma H , IshigakiS, e t al. Characterization

and localization of glutathione-S -rransferase in rat brain andbinding of hormones, neurotransmitters and drugs.J Neuro-chem 1988;50:50-57

27. Slivka A, Mytilineou C, Coh enG . Hiscochemical evaluationofglutathione in brain. BrainRes 1987;409:275-284

28. Philbert MA, Beiswanger CM, Waters DK, etal. Cellular and

regional distribution of reduced glutathione in the nervous sys-tem of the rat: histochemical localisation by mercury orange andophthaldialdehyde-induced histo-fluorescence. Toxicol ApplPharmacol 1991;107:2 13-227

29. Jenn er P, Dexter D T, SianJ, et al. Oxidative stress as a causeof nigral cell death in Parkinson’s disease and incidental Lewybody disease. Ann Neurol 1992;35:S82-S87

30. Gi bb W RG , Lees AJ. The relevance of Lcwy bodyto the parho-genesis of idiopathic Parkinson’s disease. J Neu rol N eurosurgPsychiatry 1988;5 1:745-752

31, Dexter DT, Sian J, Rose S, et al. lndices of oxidative stressin individuals with incident al Lewy body disease. Ann N eu rol

32. Mithofer K, Sandy MS, Smith M T, et al. Mitochondrial poisonscause de pletion of reduc ed glutathione in isolated hepatocyres.Arch Biochem Biophys 1992;295:132-136

305-310

113: 1823- 1842

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