Alterations in peptide levels in Parkinson's disease and incidental Lewy body disease

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1 Bran (1996), 119, Alteratons n peptde levels n Parknson's dsease and ncdental Lewy body dsease A. Fernandez, 1 M. L. de Ceballos, 1 S. Rose, 2 P. Jenner 2 and C. D. Marsden 3 1 Neurodegeneraton Group, Cajal Insttute, CSIC, Madrd, Span, the -Neurodegeneratve Dsease Research Centre, Pharmacology Group, Bomedcal Scences Dvson, Kng's College London and the 3 Unversty Department of Clncal Neurology, Insttute of Neurology, Natonal Hosptal for Neurology and Neurosurgery, London, UK Correspondence to: Professor P. Jenner, Pharmacology Group, Bomedcal Scences Dvson, Kng's College London, Manresa Road, London SW3 6LX, UK Summary he levels of the neuropeptdes Met- and Leu-enkephaln (ME-ENK, LEU-ENK), substance P and neurotensn were measured by a combned hgh performance lqud chromatography/radommunoassay (HPLC/RIA) method n postmortem samples of basal gangla from Parknson's dsease patents, ncdental Lewy body dsease patents (presymptomatc Parknson's dsease) and matched controls. Dopamne (DA) levels were reduced n the caudate nucleus and putamen n Parknson's dsease, but unaltered n ncdental Lewv body dsease. he levels of ME-ENK were reduced n the caudate nucleus, putamen and substanta ngra n Parknson's dsease. Met-enkephaln levels were reduced n the caudate nucleus and n the putamen n ncdental Lewy body dsease. Leu-enkephaln levels were decreased n the putamen and were undetectable n the substanta ngra n Parknson's dsease. Leu-enkephaln levels were unchanged n ncdental Lewy body dsease, although there was a tendency to a reducton n putamen. Substance P levels were reduced n the putamen n Parknson's dsease. No sgnfcant changes n substance P content were observed n ncdental Lewy body dsease. Neurotensn levels were ncreased n the substanta ngra n Parknson's dsease. Neurotensn levels n ncdental Lewy body dsease were not altered sgnfcantly, but tended to parallel the changes n Parknson's dsease. he changes n basal gangla peptde levels n ncdental Lewy body dsease generally followed a trend smlar to those seen n Parknson's dsease, but were less marked. hs suggests that they are an ntegral part of the pathology of the llness and not secondary to DA neuronal loss or a consequence of prolonged drug therapy. Keywords: Parknson's dsease; ncdental Lewy body dsease; basal gangla; neuropeptdes; hgh performance lqud chromatography Abbrevatons: DA = dopamne; HPLC = hgh performance lqud chromatography; L-dopa = L-3,4-dhydroxyphenylalanne; LEU-ENK = Leu-enkephaln; ME-ENK = Met-enkephaln; MPP = l-methyl-4-phenyl-l,2,3,6-tetrahydropyrdne; 6- OHDA = 6-hydroxydopamne; RIA = radommunoanalyss Introducton he destructon of the ngrostratal pathway wth the presence of Lewy bodes n remanng ngral neurons underles the motor defct whch characterzes Parknson's dsease (Gbb, 1987; Gbb and Lees, 1988). A marked reducton n stratal DA s the man bochemcal defct occurrng n Parknson's dsease. Replacement therapy wth L-3,4-dhydroxyphenylalanne (L-dopa) ntally reverses the motor symptoms. However, wth contnued treatment the therapeutc response dmnshes and motor complcatons (fluctuatons and dysknesas) usually appear. he nvolvement of neurotransmtter systems other than DA may contrbute to the symptomatology Oxford Unversty Press 1996 of Parknson's dsease. In partcular, a hghly regonal pattern of changes n neuropeptde levels n basal gangla occurs n Parknson's dsease (Agd and Javoy-Agd, 1985). In studes of post-mortem bran materal n Parknson's dsease, ME-ENK and LEU-ENK levels have been reported to be reduced n the caudate nucleus and putamen, and there s also a reducton of ME-ENK content n substanta ngra (aquet et al., 1983; Llorens-Cortes et al., 1984; Fernandez et al., 1992). Substance P levels have been reported to be decreased n the caudate nucleus, globus palldus and substanta ngra (Mauborgne et al., 1983; Fernandez et al.,

2 824 A. Fernandez et al. 1992). In contrast, neurotensn levels have been reported as ether unaltered throughout basal gangla (Bssette et al., 1985; Emson et al., 1985) or ncreased n substanta ngra (Fernandez et al., 1995). Immunocytochemcal studes show a dfferent pattern of change wth substance P and ME- ENK mmunostanng ether unaltered (Grafe et al., 1985; Waters et al., 1988) or even ncreased n stratum and globus palldus (Grafe et al., 1985; Goto et al., 1990) and unchanged n the substanta ngra (Waters et al., 1988). It s not known whether these peptde changes are part of the prmary pathology of Parknson's dsease, whether they occur secondary to the loss of DA neurons, or whether they are a result of prolonged drug therapy wth L-dopa. We have examned these questons by utlzng anmal models of Parknson's dsease such as the unlateral 6-hydroxydopamne (6-OHDA) lesoned rat (aylor et al., 1992), n combnaton wth L-dopa treatment for 6 months, or the l-methyl-4- phenyl-l,2,3,6-tetrahydropyrdne (MPP) treated parknsonan prmate (Jenner et al., 1986; aquet et al., 1988; aylor et al., 1991). Overall, the effects of ngrostratal degeneraton on basal gangla peptde levels dd not parallel the alteratons n peptde content observed n Parknson's dsease. hs suggested that they may be part of the prmary pathologcal process occurrng n Parknson's dsease. o confrm ths concluson usng post-mortem tssue from patents wth Parknson's dsease s dffcult. Bran materal from early or untreated parknsonans s not avalable. Consequently, we have nvestgated peptde content n basal gangla n Parknson's dsease and n bran materal from the 8-10% of normal ndvduals who at, post-mortem, are found to have evdence for ngral cell death and Lewy bodes (ncdental Lewy body dsease). Incdental Lewy body dsease s consdered to be the presymptomatc phase of Parknson's dsease (Forno and Alvord, 1971; Fearnley and Lees, 1991), snce the dstrbuton of pgmented cell loss observed n ncdental Lewy body dsease, although less severe, mrrors that of Parknson's dsease (Gbb, 1987; Fearnley and Lees, 1991). Patents and methods Patents Bran tssue was obtaned from the Parknson's Dsease Socety Bran Bank (London, UK) and from the Department of Pathology (Unversty of Innsbruck, Austra). he dssecton of all bran materal was undertaken by Dr F. R. Wells at the Parknson's Dsease Socety Bran Bank. he brans were dvded mdsagttally wth one-half mmerson fxed n 10% buffered formaln and the other frozen at -70 C. he bran tssue was dssected from frozen bran and stored at-70 C untl bochemcal analyss (Dexter et al., 1989). Controls conssted of fve males and one female (mean age±se, 68.3±5.7 years), who ded wthout any neurologcal or psychatrc dsease. Pathologcal examnaton of the brans dd not show cell loss or Lewy bodes n haematoxyln-eosn staned sectons of substanta ngra. Ffty control brans were screened to detect cases of ncdental Lewy body dsease. Incdental Lewy body dsease was dagnosed n sx apparently normal ndvduals (three males and three females; mean age±se, 68.2±5.8 years) whose brans exhbted cell loss n the substanta ngra and the presence of Lewy bodes n remanng neurons at post-mortem examnaton. Other basal gangla regons showed no overt pathologcal changes. However, other nucle such as the locus coeruleus and nucleus basals of Meynert were also nvolved, ndcatng that the regonal dstrbuton of the lesons was dentcal to that n Parknson's dsease. hese brans were pathologcally defned and valdated as ncdental Lewy body dsease cases (Gbb and Lees, 1988; Fearnley and Lees, 1991). Patents found to have ncdental Lewy body dsease were not known to have neurologcal symptoms or sgns n lfe and had not receved drug treatment. Parknson's dsease patents conssted of four males and two females (mean age±se, 78.7±2.1 years), who were clncally dagnosed and hstopathologcally defned by the presence of Lewy bodes and a characterstc pattern of neuronal loss n the substanta ngra. he age of onset of Parknson's dsease ranged between 59 and 73 years (mean±se, 69.2±3.1 years) and the duraton of the dsease ranged from 5 to 14 years (mean±se, 10.4±1.6 years). All the Parknson's dsease patents were recevng L-dopa (wth a perpheral dopa decarboxylase nhbtor) at the tme of death. he doses ranged between 200 and 1000 mg kg day-' (mean±se, 550±154 mg kg day" 1 )- he average tme between death and refrgeraton of the body dd not dffer between the three groups of subjects (mean±se, controls 2.9±0.2 h; ncdental Lewy body dsease 2.7±0.3 h and Parknson's dsease 2.8±0.4 h), and the average tmes between death and bran removal were smlar (mean±se, controls 14.3±1.5 h; ncdental Lewy body dsease 21.5±2.2 h and Parknson's dsease 16.6±1.7 h). Determnaton of levels of dopamne and ts metaboltes he levels of DA and ts metaboltes n samples from the caudate nucleus and putamen were measured by a standard HPLC technque wth electrochemcal detecton (Wagner et al., 1982). Peptde measurements For peptde measurements, frozen samples were weghed and boled for 15 mn n 100 volumes of a mxture of 1 N acetc acd and 0.02 N HC1 contanng 2-mercaptoethanol. Samples were homogenzed wth an Ultraturrax homogenzer ( r.p.m., 6 s) and centrfuged at g for 10 mn at 4 C. After centrfugaton, alquots of the supernatant were freeze-dred and stored at -20 C untl assayed. Met-enkephaln, LEU-ENK and neurotensn were measured by radommunoanalyss (RIA), followng HPLC

3 Peptde levels n Parknson's dsease 825 able 1 Levels of dopamne, dhydroxyphenylacetc acd, homovanllc acd and dopamne turnover rato n the caudate nucleus and putamen from normal subjects, ncdental Lewy body dsease patents and Parknson's dsease patents DA DOPAC HVA HVA + DOPAC/DA Caudate Control +Lb Parknson's dsease Putamen Control +Lb Parknson's dsease 3.09± ± ±0.23" 3.36± ± ±0.06*" 0.26± ± ± ± ± ±0.01* 3.48± ± ± ±0.38" 1.37± ± ±5.99*" 2.82± ± ±3.53*" Results are gven as mean±se expressed as mcrograms per gram wet weght of tssue (n = 6). DOPAC = dhydroxyphenylacetc acd; HVA = homovanllc acd; +Lb = Lewy body dsease patents. *P < 0.05; **P < 0.01; ***P < 0.001: compared wth control subjects (ANOVA, Student's / test). SN Fg. 1 Met-enkephaln levels n control subjects (C-Lb), ncdental Lewy body (C+Lb) and Parknson's dsease (PD) patents n basal gangla regons. CN = caudate nucleus; Put = putamen; GPL = globus palldus lateral segment; GPM = globus palldus medal segment; SN = substanta ngra. he results are means ±SE (n = 6). *P < 0.05 (ANOVA, Student's t test). separaton of the crude extracts, as prevously descrbed (de Ceballos et al., 1991). Substance P was quantfed n the crude extracts and levels correspond to mmunoreactve-lke materal. he dentty of the substance P mmunoreactvty detected by RIA was establshed by HPLC separaton of the extracts and shown to correspond to both authentc and oxdzed peptde. All the antsera used n the RIA were rased at the Cajal Insttute wth the excepton of ME-ENK antserum whch was purchased from Amersham Internatonal (Amersham, UK). he antsera dd not dsplay any cross-reactvty wth related or unrelated peptdes, wth the excepton of substance P antserum whch cross-reacted 50% wth oxdzed substance P, ME-ENK antsera whch cross-reacted 10% wth oxdzed ME-ENK, 6% wth LEU-ENK and 2% wth ME-ENK- Arg-Phe, and LEU-ENK antsera whch cross-reacted 3% wth ME-ENK. Alquots of' 25 I-peptdes (100 u.1; 7000 c.p.m.; Amersham Internatonal, UK) were ncubated wth 100 u.1 of standards or samples and 100 l of antserum n a fnal volume of 600 \x\ of RIA buffer (50 mm sodum phosphate buffer, Fg. 2 Leu-enkephaln levels n control subjects (C-Lb), ncdental Lewy body (C+Lb) and Parknson's dsease (PD) patents n basal gangla regons. CN = caudate nucleus; Put = putamen; GPL = globus palldus lateral segment; GPM globus palldus medal segment; SN = substanta ngra. he results are means±se (n = 6). *P < 0.05 (ANOVA, Student's t test). ph 7.4, contanng ether 0.2% gelatn for enkephalns or 0.3% bovne serum albumn for substance P and neurotensn). After h ncubaton at 4 C, bound and free peptde was separated usng actvated charcoal. Seral dlutons of the crude extracts or HPLC purfed samples gave competton curves parallel to those obtaned wth the synthetc peptdes. From these experments, the appropate dluton for each area and group of patents was selected. Senstvty of the RIA (15% of bound tracer) was 2 fmol for each peptde. Statstcal analyss Analyss of dfferences between groups was performed usng one-factor ANOVA followed by unpared Student's t test. Correlatons between ndvdual bochemcal levels wth dfferent ante- and post-mortem parameters were sought usng Pearson's coeffcent.

4 826 A. Fernandez et al CN Fg. 3 Substance P levels n control subjects (C-Lb), ncdental Lewy body (C + Lb) and Parknson's dsease (PD) patents n basal gangla regons. CN = caudate nucleus; Put = putamen; GPL = globus palldus lateral segment; GPM = globus palldus medal segment; SN = substanta ngra. he results are means±se (n = 6). *P < 0.05 (ANOVA, Student's / test). Fg. 4 Neurotensn levels n control subjects (C-Lb), ncdental Lewy body (C + Lb) and Parknson's dsease (PD) patents n basal gangla regons. GPL = globus palldus lateral segment; GPM = globus palldus medal segment; SN = substanta ngra. he results are means±se (n = 6). *P < 0.05 (ANOVA, Student's ; test). SN Basal gangla neuropeptde alteratons n Parknson's dsease and ncdental Lewy body dsease here was a parallel reducton (-50%) n ME-ENK levels n the caudate nucleus n Parknson's dsease and n ncdental Lewy body dsease compared wth controls (Fg. 1). here was a reducton n ME-ENK levels n putamen (90% decrease versus controls) and n substanta ngra (60% decrease versus controls) n Parknson's dsease. Met-enkephaln levels were also reduced by over 50% n the putamen (but not n substanta ngra) n ncdental Lewy body cases. Met-enkephaln concentratons were ncreased n both segments of the globus palldus n Parknson's dsease but the changes were not statstcally sgnfcant. A smlar ncrease was found n ncdental Lewy body dsease patents, but agan the dfferences dd not reach statstcal sgnfcance. Leu-enkephaln levels n the caudate nucleus and both segments of globus palldus were unaltered n Parknson's dsease and n ncdental Lewy body dsease compared wth controls (Fg. 2). In contrast, LEU-ENK concentratons were markedly reduced n the putamen and were undetectable n the substanta ngra n Parknson's dsease. here was a reducton of LEU-ENK content n the putamen n ncdental Lewy body dsease, but ths was not statstcal sgnfcant. here was a decrease n substance P concentratons n the putamen n Parknson's dsease (Fg. 3). Substance P content was non-sgnfcantly ncreased n the lateral and medal globus palldus n both Parknson's dsease and ncdental Lewy body dsease (Fg. 3). Substance P levels n the caudate nucleus and substanta ngra were unaltered n ether Parknson's dsease or ncdental Lewy body dsease compared wth controls. he small amount of tssue avalable and the low levels of neurotensn n stratal areas precluded the analyss of ths peptde n the caudate nucleus and putamen. Neurotensn levels were ncreased (two-fold) n the substanta ngra of Parknson's dsease patents (Fg. 4). Neurotensn concentratons n the ncdental Lewy body dsease group followed the same trends to those seen n Parknson's dsease but the changes dd not reach statstcal sgnfcance. Results Dopamne levels n Parkson's dsease and ncdental Lewy body dsease Dopamne levels were sgnfcantly reduced n the caudate nucleus (by 87%) and putamen (by 95%) n patents wth Parknson's dsease compared wth controls (able 1). Levels of homovanllc acd and dhydroxyphenylacetc acd were unaltered n the caudate nucleus, but were reduced n the putamen n Parknson's dsease. Dopamne turnover measured by the rato of (homovanllc acd+dhydroxyphenylacetc acd):da was enhanced n both caudate and putamen n Parknson's dsease. In ndvduals wth ncdental Lewy body dsease, there was no change n the caudate or putamen DA content or n DA metabolte levels or DA turnover (able 1). Correlatons of peptde levels and wth ante- or post-mortem parameters here was no correlaton of ndvdual peptde levels wth ether sex, age. tme to refrgeraton or to autopsy (data not shown). here were sgnfcant postve correlatons between ME-ENK and LEU-ENK levels n the caudate nucleus, putamen and globus palldus. For example, n the putamen both peptdes were correlated n control subjects (0.903), n ncdental Lewy dsease cases (0.856) and n Parknson's dsease patents (0.904). Indvdual ME-ENK and substance P levels n medal globus palldus were correlated n ncdental Lewy dsease patents and Parknson's dsease patents. In agreement wth prevous studes, DA content n caudate

5 Peptde levels n Parknson's dsease 827 able 2 Summary of alteratons n peptde levels n Parknson's dsease and n unlateral 6-OHDA lesoned rats (untreated or treated wth L-dopa) Peptde Bran area Parknson's dsease ME-ENK LEU-ENK SP N Stratum Globus palldus Substanta ngra Stratum Globus palldus Substanta ngra Stratum Globus palldus Substanta ngra Stratum Globus palldus Substanta ngra I(+Lb) I/ /= =/ n /= = =/ 6-0 = /= 6-OHDA + L-dopa t Parknson's dsease alteratons n peptde content n prevous studes and n ths study. Data from lesoned rats (6-OHDA-lesoned rats untreated or treated wth L-dopa) are from Engber et al. (1991) and aylor el al. (1992). 6-OHDA = 6-hydroxydopamne; L-dopa = L-3,4-dhydroxyphenylalanne; stratum = caudate nucleus and/or putamen; SP = substance P; N = neurotensn. -I = decreased; = ncreased; = = unchanged, compared wth controls; = not determned. Stratal ME-ENK levels were also reduced n ncdental Lewy body dsease (+Lb). nucleus n Parknson's dsease was postvely correlated wth the age of onset of the llness (0.8644), but was negatvely correlated wth the duraton of the dsease ( ). here was no correlaton between ndvdual peptde levels n any regon and L-dopa dose. Dscusson he relatve dstrbuton of neuropeptde levels n control subjects was n general agreement wth those prevously reported (Buck et al., 1981; Cooper et al., 1981; Manberg et al., 1982; Mauborgne et al., 1983; aquet et al., 1983; Llorens-Cortes et al., 1984). However, n ths work the rato of enkephaln content n globus palldus lateral segment to ether caudate nucleus or putamen was not n accordance wth prevous studes (e.g. aquet et al., 1983; Llorens- Cortes et al., 1984). hs a consequence of the HPLC/ RIA technque used for the peptde measurements. As we prevously reported (de Ceballos et al., 1991), there s a dsparty between apparent peptde levels n crude extracts wth those of authentc peptdes as determned after HPLC purfcaton. Indeed, n recent studes usng another seres of controls and Parknson's dsease brans, we have shown that ME-ENK levels n globus palldus lateral segment are double those n caudate nucleus or putamen when measured n crude extracts, but are very smlar n HPLC purfed controls (de Ceballos et al., unpublshed observatons). Some neuropeptde alteratons occurrng n the brans of those wth establshed Parknson's dsease on L-dopa treatment are also present n ncdental Lewy body dsease. Snce these ndvduals were normal n lfe and dd not receve L-dopa therapy, and snce the extent of pathologcal change n the substanta ngra was small and was not assocated wth any change n caudate or putamen DA content, such alteratons n neuropeptde levels may reflect part of the prmary pathology of Parknson's dsease. In agreement wth prevous studes ME-ENK content was decreased n the caudate nucleus (Fernandez et al., 1992; Svam, 1991), putamen and substanta ngra n Parknson's dsease (aquet et al., 1983; Llorens-Cortes et al., 1984). In ncdental Lewy body dsease a sgnfcant reducton n ME-ENK levels was also observed n the caudate nucleus and the putamen. In 6-OHDA lesoned rats or n MPP treated monkeys, there were ncreased stratal ME-ENK levels (ha et al., 1983; Svam et al., 1987; Dacko and Schneder, 1991; aylor et al , 1992). herefore, the decrease observed n Parknson's dsease and n ncdental Lewy body dsease does not appear to be a consequence of ngrostratal pathway degeneraton. In contrast, n both segments of the globus palldus, ME-ENK content tended to be ncreased n both Parknson's dsease (Grafe et al., 1985; Goto et al., 1990; de Ceballos et al ) and ncdental Lewy body dsease. hs change s smlar to that seen n 6-OHDA and MPP lesoned anmals (ha et al., 1983; Svam et al., 1987; aylor et al., 1991), so may be a reflecton of ngrostratal loss. he changes observed n ncdental Lewy body dsease cannot be ascrbed to L-dopa therapy, but may reflect the underlyng dsease process (able 2). In 6-OHDA lesoned rodents and n MPP treated prmates, the leson nduced ncrease n ME-ENK levels s a consequence of ncreased preproenkephaln gene expresson (Angulo et al., 1986; Young et al., 1986; Svam et al., 1987; Augood et al., 1989; Gerfen et al., 1990). It has been recently reported that preproenkephaln gene expresson s enhanced n parknsonan caudate nucleus and putamen (Nsbet et al., 1995). Although ths mght explan the tendency to an ncrease n ME-ENK levels n globus palldus, the man projecton area of the stropalldal enkephaln pathway, s dffcult to reconcle wth the observaton of a decrease n stratal levels of ME-ENK n Parknson's dsease. However.

6 828 A. Fernandez et al. ncreased bosynthess accompaned by decreased levels may be a result of ncreased release of ME-ENK. Changes n LEU-ENK levels partly paralleled those of ME-ENK. hus, LEU-ENK levels were reduced n putamen (aquet et al., 1983) and substanta ngra of patents wth Parknson's dsease. he marked correlaton between the levels of ME-ENK and LEU-ENK strongly suggests that both peptdes derve from the same precursor, preproenkephaln (Gublerefa/., 1982). here was no statstcally sgnfcant changes n LEU-ENK levels n ncdental Lewy body dsease, although those n putamen were consderably reduced. Changes n LEU-ENK levels appear to be characterstc of Parknson's dsease snce a ngrostratal leson does not alter stratal LEU-ENK levels (Jenner et al., 1986; aylor et al., 1991, 1992). Furthermore, although there s a leson-nduced decrease n LEU-ENK levels n substanta ngra, ths was reversed by L-dopa, such that t ncreased seven-fold compared wth values for control rats (aylor et al., 1992). In the present study, substance P levels were decreased n the putamen (Mauborgne et al., 1983), tended to ncrease n globus palldus (Grafe et al., 1985; Goto et al., 1990; de Ceballos et al., 1993) and were unchanged n substanta ngra (Grafe et al., 1985; Waters et al., 1988) n Parknson's dsease n agreement wth some prevous fndngs. In 6- OHDA lesoned rats, decreased stratal (Lndefors et al., 1989) and ngral substance P content and decreased preprotachyknn mrna has been reported (Young et al., 1986; Svam et al., 1987; Gerfen et al., 1990; Engber et al., 1991; aylor et al., 1992). L-Dopa treatment completely reversed the reducton n substance P content n substanta ngra n such anmals (Engber et al., 1991; aylor et al., 1992) (able 2). hus, the absence of change n ngral substance P levels n Parknson's dsease (Grafe et al., 1985; Waters et al., 1988) may be vewed as a combned effect of ngostratal pathway degeneraton and L-dopa treatment. Indeed, pre-protachyknn mrna levels are also unaltered n Parknson's dsease patents treated wth L-dopa (Nsbet et al., 1995). Neurotensn content n substanta ngra was ncreased n Parknson's dsease (Fernandez et al., 1995), and no statstcally sgnfcant changes were observed n globus palldus. In another seres of Parknson's dsease patents, neurotensn levels were shown to be ncreased n both zona compacta and zona retculata of the substanta ngra (Fernandez et al., 1995). In ncdental Lewy body dsease, neurotensn levels only tended to smlar changes n these structures. Followng a ngrostratal leson there are ncreased stratal and palldal neurotensn levels, whch are not modfed by L-dopa treatment. However, ngral neurotensn levels, whch were unchanged followng a 6-OHDA leson, were ncreased by L-dopa treatment (aylor et al., 1992). hus, changes n neurotensn levels appear to be secondary to DA neuron loss n combnaton wth prolonged drug therapy. he fact that n ncdental Lewy body dsease patents only tendences were observed, further support ths concluson. Hypoknetc dsorders, such as Parknson's dsease, have been postulated to result from the selectve changes n the dfferent strofugal pathways (Albn et al., 1989), whch contan varous neuropeptdes as cotransmtters. Actvty n the pathway from stratum to lateral globus palldus (colocalzng enkephalns) s thought to be ncreased, whle actvty n the pathway from stratum to medal globus palldus and substanta ngra (colocalzng tachyknns) s thought to be decreased n Parknson's dsease. In ths study some of the observed alteratons n peptde levels are compatble wth ths scenaro. However, the statonary levels of a gven peptde n a partcular structure may not ndcate actvty n a neuronal pathway. hus, as argued above, the reduced levels of ME-ENK and LEU-ENK n the stratum n Parknson's dsease, especally n the putamen, and the smlar but less marked trends n ncdental Lewy body dsease, may reflect ncreased release of the peptdes due to overactvty of such neurons contanng enkephalns. hs s supported by the fndng of ncreased proenkephaln mrna message n the stratum n Parknson's dsease (Nsbet et al., 1995). Wth regard to substance P, the matter s more complex, for L-dopa treatment reverses the decrease n preprotachyknn mrna message n expermental anmals wth lesons of the dopamnergc pathway {see above), and such message s unchanged n Parknson's dsease stratum (Nsbet et al., 1995). hs mght explan why substance P levels were not sgnfcantly altered n medal globus palldus and especally n substanta ngra n Parknson's dsease. he most mportant fndng of the present study s that some neuropeptde alteratons occurrng n Parknson's dsease are also observed n ncdental Lewy body dsease. Most convncng are the reductons n ME-ENK levels n stratum n both dsorders, whch contrast wth the ncrease found n 6-OHDA lesoned rats or MPP treated monkeys. From these results several conclusons can be drawn. Frst, such changes n peptde levels may be characterstc of the dsease and not just secondary to DA neuron loss. Secondly, snce smlar changes are observed n ncdental Lewy body dsease, they may be an early component of the pathologcal process rather than beng secondary bochemcal alteratons resultng from loss of the ngrostratal pathway or a drug nduced event. Addtonal nsght nto the nvolvement of these basal gangla peptde systems n the pathophysology of Parknson's dsease could provde new therapeutc strateges for the llness. Acknowledgements We wsh to thank Dr D. Dexter for hs help, Dr F. R. Wells for neuropathologcal examnaton of all the bran tssues, J. Hrschborn and A. Arnedo for excellent techncal assstance and Dr Guaza for helpful dscussons on the statstcal analyss of the data. hs work was supported by the EEC, Scherng Espafa S.A., he Medcal Research Councl and the Parknson's Dsease Socety. A.F. receved a grant from Scherng Espafa S.A.

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