Life Sciences, Vol . 24, pp . 809-816

Pergamon PressPrinted in the U.S .A .

HUNTINGTON'S DISEASE AND ITS ANIMAL MODEL :ALTERATIONS IN KAINIC ACID BINDING

Kevin Beawnont, Yves Maurln, Terry D . Reisine, Jeremy Z . FieldsErnest Spokes*, Edward D. Bird* and Henry I . Yamamura**

Department of Pharmacology, University of Arizona Health Sciences Center,Tucson, Arizona 85724 ; and*Addenbrookes Hospital, Cambridge, England .

(Received in final form January 24, 1979)

SUMMARY

The density of 3H-kainic acid (KA) binding was determined in sev-eral regions of Huntington's Diseased (HD) and control human brains .3H-Kainic acid binding was significantly reduced by 55X in the caudate nucleus and .by 53~ in the putamen of HD brains . In addition,9H-KA binding was determined in rat striatum at various intervalsfollowing lesion with KA, a procedure which produces an animal mod-el of HD . After KA lesion, 3H-KA binding in the rat striatum under-went a slow reduction, reaching 25~ of control after 6 weeks . Sev-eral properties of 3H-KA binding to rat brain membranes were alsoinvestigated, including inhibition by ions, regional distributionand displacement by various compounds . The findings confirm thevalidity of the KA-lesioned model for HD and suggest a post-synap-tic location for kainic acid receptors in the striatum .

INTRODUCTION

Kainic acid (KA), a cyclic analogue of L-glutamic acid, is an extremely po-tent neuronal depolarizing agent (1) . Upon injection into the rat striatum,KA causes the degeneration of neuronal cell bodies located at the site of in-jection, while axons passing through or terminating in the area of injectionare unaffected (2) . The neurotoxic effects of KA may be the result of excess-ive neuronal depolarization (3) mediated by synaptic glutamate receptors . Asynaptic site of action is supported by studies demonstrating that binding ofradiolabeled KA to rat brain is of high affinity, saturable, displaceable byL-glutamate, stereospecific, concentrated in synaptic membranes, and unevenlydistributed in rat brain regions (4) .

Histological and neurochemical alterations resulting from striatal injectionof KA (2,5,6) or L-glutamic acid (6 ,7) are remarkable similar to those occurringin Huntfington's Disease (HD), a hereditary disorder characterized behaviorallyby choreic movements and dementia and pathologically by shrinkage of the caudatenucleus and putamen, accompanied by neuronal degeneration and gliosis (8) .

Present addresses :KB :

Department of Neuropharmacology, Synthelabo-LERS, Bagneux, France .YM :

Laboratoire de Neurochimie, INSERM U 134, Hopital de la Salpetriere,Paris, France .

JZF : Department of Pharmacology, Chicago Medical School, Chicago, I11 .EDB : Department of Neuropathology, McLean Hospital, Boston, Mass .

to whom reprint requests should be sent

0024-3205/79/090809-0802 .00/0Copyright (c) 1979 Pergamon Press Ltd

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Consequently, striatal KA-induced degeneration serves as a model for investiga-tion of the etiology and therapy of HD (2) . We report here on the density ofthe KA binding site in HD and control human brain, and on the alterations ofKA binding produced by striatal KA lesions .

METHODS AND MATERIALS

Tissue : Two ug of KA in 1 ul saline were stereotaxically injected into thestriatum of pentobarbital-anesthetized adult male Sprague-Dawley rats (200-250gms) . Coordinates for the injection, determined using the atlas of Konig bKlippel (9), were 7 .8 mm anterior and 2 .6 mm lateral from the interaural lineand 4 .8 mm vertically from the surface of the brain . The solution was injectedthrough a 30-gauge needle over a period of 3 minutes, and the needle was leftin place for an additional 3 minutes before withdrawal . At various times afterlesioning, animals were sacrificed by decapitation, and left and right striatawere dissected and frozen . Pathological specimens of human brains were obtainedfrom 16 patients with HD, ages 42-74 years, (mean = 59 years) and from 18 con-trol individuals without infectious or malignant disease of the central nervoussystem, ages 17-88 years (mean = 57 years) .

HD patients were undergoing therapywith neuroleptics, diazepam, and morphine at time of death . Tissue was frozenat -70°C until the day of the assay . Both human and animal tissues werethroughly washed by centrifuging and resuspending four times in approximately150 volumes of fresh buffer prior to assay of 3H-KA binding . For studies in-volving displacement of 3H-KA by compounds or by ions, whole rat brain synapticmembranes were used . Synaptic membranes were prepared by the method of Zukinet al . (10) and were then frozen for a period of 1-7 days . Synaptic membraneswere thawed and washed four times by centrifuging and resuspending in freshbuffer prior to use .

3H-KA binding : 3H-KA binding was determined by a modification of the methoddescribed by Simon, et al . (4) . Throughly washed tissue homogenates (0 .3 - 0 .6mg protein/assay) were incubated in triplicate with 0 .5 - 150 nM 3H-KA (2 .3 Ci/mmole, New England Nuclear) in 4 ml of 0.05 M tris-citrate buffer, pH 7 .1 .After 30 minutes of incubation at 4°C, membranes were sedimented by centrifug-ing for 10 minutes at 48,000 x g . The pellets so obtained were rapidly surface-washed twice with 5 ml of ice cold distilled HZO and solubilized with NCS (Amer-sham/Searle) . Toluene-omnifluor scintillation cocktail was added and radio-activity determined by liquid scintillation spectrometry . Non-specific bindingwas determined in the presence of 1 mM L-glutamic acid and subtracted from thetotal bound to obtain specific binding . Protein concentrations were determinedby the method of Lowry et al . (11) .

Choline acetyltransferase (CAT) activity : 5 ul of unwashed tissue homogenate(3 .3% in 50 mM NaKPOy buffer) were added to 25 u l of an assay mixture contain-ing 0 .4 ml Na HPOg/NaH2 PO buffer (0 .2 M, pH 7 .4), 0 .17 ml eserine salicylate(0 .001 M), 0 .~6 m7 MgC7 2 ~0 .1 M), 0 .12 ml NaCI (3 M), 0 .12 ml choline chloride(0 .02 M), and 0 .1 ml 14 C-acetyl CoA (0 .02 mCi/ml) . After 20 minutes incubationat 37°C, 0 .1 ml of tetraphenylboron in 3-heptanone (50 mg/ml) was added and themixture was cooled in an ice bath for 5 minutes, then centrifuged for 2 minutesin a Beckman Microfuge B . The radioactivity (3H-acetylcholine fornied) in 50 ulof the organic layer was determined by liquid scintillation spectrometry .

RESULTS

Scatchard analysis of saturation isotherms for 3H-KA binding to washed wholerat brain homogenates revealed a binding site with a dissociation constant (Kp)of 5 .3 t 1 .8 nM and a receptor density (Bmax) of 182 ± 47 fmol/mg protein(n=5) .

The density of 3H-KA binding at .a concentration of 10

nM varled amongregions of rat brain, with the greatest density in the striatu~hippocampus>cerebellum ~ cerebral cortex>midbrain = pons-medulla . Several neuroexcitatoryamino acids displaced 3H-KA from whole rat brain homogenates (Table 1) .

Inhibition of 3H-KA binding to rat brain synaptic membranes .Values represent the concentration of the compound which inhibitsby 50~ the specific binding of 5 nM 3H-KA to whole rat brain synap-tic membranes and are the means of 2-4 experiments . Several com-pounds produced little or no inhibition at a concentration of100 uM, including GABA, muscimol, bicuculline, strychnine, gly-cine,taurine . histamine . adenosine . dopamine, atropine sulfate,serotonin, naltrexone, L-carnosine, L-cysteine, L-histamine, hemi-cholanium-3, sodium Phenobarbital, diazepam, metrazol, diphenyl-hydantion, clozapine, amitriptyline, theo.phylline, ouabain, 2,4-dinitrophenol, pyridoxal-5'-P0,� 2-mercaptoethanol, dithiothreitol,caffeine, ATP, AMP, GMIP, uradine monophosphate, inosine-5'-PO y ,inosine, cytosine, guanosine, L-ascorbic acid, 6-hydroxydopamine,6-hydroxyDOPA, o-phospho-L-serine, DL-a-aminopimelic acid, andY-hydroxybutyri c aci d .

Half-maximal displacement (ICSO ) of the specific binding of 5 nM 3H-KA occurredwith 0 .006 pM unlabeled KA, 0 .21 uM L-glutamate, and 12 uM D-glutamate . How-ever, L-aspartac acid, D-aspartic acid, and N-methyl-D-aspartic acid were con-siderable less potent, IC o's being greater than 0 .1 mM for all three compounds .Several inhibitors of L-g~utamate-induced excitations were relatively ineffec-tive at inhibiting 3 H-KA binding in our assay system . Thus, the ICso for dis-placing 5 nM 3H-KA was greater than 0 .1 mM for a-methyl-DL-glutamic acid, L-glutamic acid diethylester (L-GDEE), 2-amino-4-phosphonobutyric acid, and theglutamate synthetase inhibitor L-methionine-DL-sulfoxamane, as well as for theaspartate-antagonist (12), DL-a-aminoadipic acid . Several compounds producedlittle or no effect at a concentration of 0 .1 mM upon 3 H-KA binding to rat brainsynaptic membranes (Table 1, legend) .

The effects of several ions upon 3H-KA bindin~ to whole rat brain synapticmembranes were determined . C1 - does not inhibit H-KA binding, since increasingthe concentration of tris-C1 buffer up to 200 mM does not inhibit 3H-KA binding .Therefore, the inhibition produced by a cation with C1 - as its counterion isprobably due solely to the cation . Monovalent cations, tested with C1 - as thecounterion, inhibit 3 H-KA binding in a dose-dependent manner . The inhibitorypotency of the alkali cations decreases with increasing molecular weight, Li+

Vol . 24, No . 9, 1979 Kainic

COMPOUND

Acid Binding in Huntington's Disease

TABLE IICso (MICROMOLAR)

KAINIC ACID 0 .006QUISQUALIC ACID 0.032DIHYDROKAINIC ACID 5

L-GLUTAMIC ACID 0 .21D-GLUTAMIC ACID 12DL-HOMOCYSTEIC ACID 13L-CYSTEIC ACID 16L-HOMOCYSTEIC ACID 18L-CYSTEINE SULFINIC ACID 18

L-GLUTAMINE 13

DL-a-AMINOADIPIC ACID 120D-ASPARTIC ACID 400L-ASPARTIC ACID 400N-METHYL-D-ASPARTIC ACID 850

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being the strongest inhibitor and Cs+ the weakest inhibitor (Table 2) . Na+ hasan ICsp of 61 mM when tested with C1 - as the counterion, which is similar tothe ICsp's of 50 mM and 66 mM obtained with Br- and I- , respectively, ascounterlons .

The divalent canons Ca m, Mn~, aüd Mgr are considerably morepotent inhibitors of 3H-KA binding than are the monovalent canons (Table 2) .

Inhibition of 3H-KA binding to rat brain membranes by ions .Values represent the concentration of ion necessary to inhibit by50% the binding of 5 nM 3H-KA to whole rat brain synaptic membranes .For experiments involving divalent cations, this -~HC1 buffer was usedrather than tris-citrate, in order to avoid chelation of divalentcations by citrate . Substitution of tris -HC1 buffer did not appre-ciably alter control binding values or binding data for monovalentcations . ICsp's were determined by log-probit analysis of the meanvalues from 2 to 5 experiments .

3H-KA binding site density and CAT activity in individual rat striate weredetermined at various intervals after striatal lesion with KA (Figure 1) . Thestriatum contralateral to the lesion served as control for each animal . Inaccordance with previous reports (5,6) CAT activity of lesioned striatum wasreduced to 30% of (control = 185 t 10 nmole/mg protein/hr) within 5 days oflesioning . In contrast, 3H-KA binding density was not significantly differentfrom control at 5 or 8 days after lesioning . However, 3H-KA binding was reducedto 78% of control after 14 days, decreasing to 25% of control at 48 days afterlesion .

Preliminary studies indicated that kinetic values for 3H-KA binding tothoroughly washed control hupen cerebral cortex (Kp - 7 nM, Bmax = 118 fmole/mgprotein) and hupen cerebellum (Kp = 11 nM, Bmax - 145 fmole/mg protein) aresimilar to kinetic values obtained with whole rat brain . In addition, thepotenc .y of L-gl utamate 1 n di s pl aci ng s H-KA from hupen cerebral cortex (ICsp -0 .2 uM) and hupen cerebellum (ICsp - 0 .6 uM) is similar to its potency in dis-placing 3 H-KA from whole rat brain membranes (ICSp - 0 .21 uM) .

The density of 3 H-KA binding was determined in human brain regions and foundto be significantly decreased in the caudate nucleus and putamen of H .D . brainsas compared to control human brains (Figure 2) . 3 H-KA binding density was sig-nificantly reduced by 55% in H .D . caudate nucleus, from a mean of 115 .5 ± 7 .7fmole/mg protein in control caudate nuclei (N - 12) to 51 .5 t 8 .2 fnale/mg pro-tein in H .D . caudate nuclei (N = 11), (pc0 .001 by two-tailed, unpaired "t"test) . The density of 3 H-KA binding was significantly reduced by 53% in H .D .putamen, from 128 .5 ± 8 .8 fmole/mg protein in control

utamen (N - 12) to60 .0 ±

5.8 finol e/mg protein i n H . D.

putamen (N - 11) ,

(ppc 0 .001

by two-tai 1 ed"t" test) .

3H-KA binding was not significantly correlated with age at death,for either HD or control individuals, and was not correlated with duration ofH .D . symptoms . In cerebellum (N s 3), frontal cortex (N = 7), and globuspallidus (N = 5), the density of 3H-KA binding in HD brains did not differ sig-nificantly from controls (data not shown) .

ION (mMg

TABLE II

ION (mMg

LiCI 32 CaC1 2 2 .3NaCI 61 MnC1 2 2 .8KC1 149 MgC1 2 '24NH,, C1 171RbCI 192 NaBr 50CsCI 197 NaI 66

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1Cainic Acid Binding in Huntington's Disease

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1001

3H-KAINIC ACID &ND~Iß

10 20 30 40 50

DAYS AFTER LESION

FIGURE 1

Time course of alterations of 3H-KA binding and CAT activity ofrat striatum following KA lesion . For each animal, values for 3H-KAbinding (finales/mg protein) and CAT activity (males acetylcholinesynthesized/mg protein/hour) were determined for the lesioned stri-atum and compared to the contralateral control striatum . Each pointrepresents the mean t S .E .M . for 4-10 animals .

DISCUSSION

We have found a significant decrease in 3 H-KA binding in both HD and KA-lesloned striatum . In our hands, the dissociation constant of the rat brainbindiny site for 3H-KA is 5 nM, which is a 10-fold higher affinity than that(59 nM) reported previously by Simon, et al . (4) . This discrepancy may be dueto the more extensive washing procedure that we utilized to remove the largequantities of endogenous glutamate, canons, and other possible unknown inhibi-tors of 3H-KA binding present in brain tissue . In support of this possibilitywe found that aliquots of the supernatants of the first three tissue washessignificantly inhibit 3H-KA binding to thoroughly (4X) washed brain tissue (datanot shown) . The regional distribution of 3H-KA binding in rat brain by our de-termination is quite similar to that reported by Simon et al (4), which indi-cates that we are probably measuring the same binding site .

We have confirmed previous findings (4) that the ability of certain aminoacids to produce neuroexcitation does not clearly parallel their potency indisplacing 3H-KA binding .

In particular, the lack of affinity of L- and Daspartate and of N-methyl-D-aspartate for the 3H-KA binding site contrasts with

1

75

11

111

_I

1

O111

H 50 1 1zO 11_____

~~ CAT ACTIVRY

25

814

Kainic Acid Binding in Huntington's Disease

Vol . 24, No . 9, 1979

100

100

Oo

PUTIUAEN

CAUDATE NUCLEUS

"~S

FI GORE 2

CONTIIOL HD

CONTROL ND

Density of 3H-KA binding in the caudate nucleus and putamen of HDand control human brains . Specific 3H-KA binding was determinedat a concentration of 10 nM, as described in Materials and Methods .Each point represents a different brain . Bars represent the meanvalue for each area .

their high neuroexcitatory potencies . This finding is consistent with neurophy-siological evidence that "aspartate-preferring" receptors are distinct from"glutamate-preferring" receptors (12,13), the latter having a greater sensiti-vity to KA . Several amino acids with widely varying potencies in exciting mam-malian central neurons (D-glutamic acid, DL-homocysteic acid, L-cysteic acid,L-cysteine sulfinic acid) as well as L-glutamate, which is without neuroexcita-tory effects in the cat spinal cord (14,15), have similar affinities for the3H-KA binding site, all yielding ICsos within the range of 10-20 uM . The aff-inity of these neuroexcitatory amino acids for the 3 H-KA binding site is 100-fold less than the affinity of KA itself . Therefore, the site mediating KA-induced neuroexcitation most likely represents only one of several receptorsmediating amino acid-induced excitations . The low potency of the glutamateantagonists L-GDEE, a-methyl-DL-glutamate, and 2-amino-4-phosphonobutyric acidat displacing 3H-KA may indicate that 1) these antagonists act elsewhere thanthe amino acid recognition site to block neuronal excitation, 2) antagonistsbind to a different conforTnation of receptors than do agonists, or 3) KA actsat a subpopulation of excitatory receptors that is not sensitive to these inhi-bitors . This third possibility is supported by the recent finding that KA-induced depolarization of rat thalamic neurons is not blocked by concentrations

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Kainic Acid Binding in Huntington's Disease

815

of L-GDEE which are effective against L-glutamate-induced depolarization (16) .The discrepancy between the neuroexcitatory potency of several compounds

and their ability to displace 3H-KA from brain membranes raises the possibilitythat the 3H-KA binding site measured in vitro does not correspond to the recep-tor that mediates KA-induced neuroexcitation in vivo . However, the findingsof Simon et al . (4) that the binding site is localized exclusively in neuronaltissue and is most concentrated in synaptic membranes strongly indicates thatthe binding site is relevant to the neuroexcitatory actions of KA . Furthermore,we have found that the close analogue dihydrokainic acid, which is a very weakneuronal excitant (13), has a 1000-fold lower affinity for the 3H-KA bindingsite than does KA . This finding also indicates that under these conditions 3H-KA is not binding to a neuronal L-glutamate uptake site, since dihydrokainicacid is a more potent inhibitor of L-glutamate uptake into rat striatal synap-tosomes than is KA, which is itself very weak in this respect (17) . The likeli-hood that the 3H-KA binding site measured in vitro corresponds to the site med-iating toxicity in vivo is also supported by t~studies of Campochiaro andCoyle (18) which demonstrate that during the maturation of the rat striatun,the increase in susceptibility to KA neurotoxicity correlates temporally withthe increase in 3H-KA binding .

Several canons inhibit 3H-KA binding, some at physiological concentrations .Divalent canons are more potent than monovalent cations, although the inhibi-tion by divalent canons may be partially attributable to complex formationwith KA. At their IC , Na and Mn~ produced a lowering in affinity with nothan e in Bmax of 3H-~ binding to rat brain synaptic membranes (unpublisheddata . Thus, although the affinity of KA for its receptor in vitro is in thenanomolar range, considerably higher concentrations may be required to producehalf-maximal binding in vivo due to competitive inhibition by canons .

The relatively slow decrease in 3H-KA binding following KA lesion was un-expected since dendrites and cell bodies, which would be expected to bear theglutamate receptors, are destroyed within 2-3 days of lesioning (19) . However,Olney and de Gubareff (20) have recently reported that postsynaptic densitiesremain adhering to the presynaptic terminals at 21 days after KA lesion . Thepostsynaptic densities are thought to contain receptors for neurotransmitters,and their continued presence after dendrites and cell bodies have degeneratedmay account for the relatively slow decrease in KA receptor density afterlesion . The eventual 75% decrease in KA binding following lesion suggests thata majority of striatal KA receptors are located on neurons with cell bodiesintrinsic to the striatum, possibly at sites post-synaptic to corticostriatalgl utamatergic afferents .

3H-KA binding was found to be significantly reduced by 55% in the caudatenucleus and by 53% in the putamen of HD brains as compared to control humanbrains . This 3H-KA binding site measured in human brain is similar to thebinding site of rat brain by several criteria, including dissociation constant,binding density, and affinity for L-glutamate . Drugs present in brain sampleswere most likely removed prior to assay by the thorough tissue washing proce-dure . In any case, representatives of each of the classes of drugs used by HDpatients do not significantly effect 3H-KA binding at a concentration of 100 u M .The finding that 3 H-KA binding is decreased in HD caudate and putamen but not infrontal cortex, cerebellum, or globus pallidus provides a preliminary indicationthat generalized destruction of KA sensitive sites, which may be postsynaptic toglutamate-releasing neurons, does not occur in this disease . In addition, thereduced binding of 3H-KA in HD and KA-lesioned striatum supports the validityof the KA-lesion animal model for HD.

ACKNOWLEDGEMENTS

We would like to acknowledge the technical assistance of David Chapman andThomas McManus and the secretarial assistance of Cathy Kousen . We thank Dr .Ante Padjen for donating samples of quisqualic acid and Dr . Michael Schmidt for

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for providing samples of dihydrokainic acid . We would also like to thank Dr .Peter C . Johnson for providing histological verification of the lesions .

REFERENCES

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0~, J .W . F .~Rhee andO .L . Ho, (1974) Brain Res . _77 : 507-512 .4 . Simon, J .R ., J .F . Contrera, and M .J . Kuhar, 97~j,J Neurochem . _26 : 141-147.5 . Coyle, J .T . and R . Schwarcz, (1976) Nature Lond 2 3 : 517- 19 .6 . McGeer, E .G . and P.L . McGeer, (1976) Naturé

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Olney, J .W . and T . de Gubareff, (1978

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- 0.18 . Campochiaro, P . and J .T . Coyle,(1978

Proc Nat . Acad .Sci . 75 : 2025-2029 .19 . Hattori, Y . and E .G . McGeer,(197~, Bra n Res .

-20 . Olney, J .W . and T . de Gubareff,(197

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