antibodies to human caudate nucleus in huntington's chorea · removed by streptococcal...

Antibodies to Human Caudate Nucleus Neurons

in Huntington's Chorea

GUNNARHUSBY, LAWRENCELi, LARRYE. DAVIS, ELISABETH WEDEGE,EMREKOKMEN,and RALPHC. WILLIAMS, JR.

From the Departments of Medicine and Neurology, University of New Mexico School of Medicine,and the Bernalillo County Medical Center, Albuquerque, New Mexico 87108, and the Departmentof Neurology, University of Michigan Medical Center, Ann Arbor, Michigan 48100

A B S T RA C T Antibodies reacting with neuronal cyto-plasmic antigens present in normal human caudateand subthalamic nuclei were detected in 37 of 80 pro-bands afflicted with Huntington's disease (HD). IgGantibodies were detected by immunofluorescenceusing frozen sections of unfixed normal human and ratbrain. Specificity of IgG binding was confirmed usingpepsin F(ab')2 fragments of IgG isolated from positivesera. In vitro complement fixation of IgG antibodywas detected in 22 of 31 sera tested. Neuronal cyto-plasmic antigens reacting with positive HDsera werediminished after trypsin or RNAase treatment of tissuesections but were not removed by DNAase, neuramini-dase, EDTA, or dithiothreitol treatment. Antibodystaining of neurons could be removed after absorp-tion with isolated caudate nucleus neurons or byusing perchloroacetic acid extracts of caudate nucleus.

Prevalence of antibody reacting with neuronal cyto-plasm was 3% in 60 normal controls and 6% among awide variety of patients with diverse neurologicaldisorders. However, one-third of 33 patients withParkinson's disease showed presence of antineuronalantibody. Among patients with HD, a significant asso-ciation was noted between duration of clinical diseasegreater than 7 yr and titers of antibody of 1:2 orgreater (P < 0.001).

When 115 family members of HD probands weretested, 30% of unaffected spouses showed presenceof antineuronal antibody. 23.2% of first-degree rela-tives at risk for developing HD were also positive(P < 0.001). 10.5% of second-degree relatives showedpresence of antineuronal antibody. These data maysupport an environmental or infectious factor some-how involved in the ultimate expression of HD.

Received for publication 22 November 1976.

INTRODUCTION

Huntington's disease (HD)' is a degenerative diseaseof the central nervous system which appears to berelated to autosomal dominant genetic inheritance (1-5). Numerous family studies have documented thegenetic distribution of this disorder, however preciseinsight into its etiology is not yet available. Someevidence has been accumulated relating abnormalitiesin central nervous system receptor mechanisms in-volving reduced glutamic acid decarboxylase activityor gamma aminobutyric acid (6, 7) receptors to HD,but as yet no clear idea of genesis has been pre-sented. The present study arose from a chance observa-tion that several sera from patients with HD con-tained antibodies to neuronal cytoplasmic structureswhich were particularly prominent in human caudatenucleus. During the course of previous work, it wasfound that a substantial proportion of sera from chil-dren with Sydenham's chorea showed anticaudatenucleus neuronal antibody (8, 9). In the case of thesesera from children with rheumatic fever, antibodyactivity could be completely absorbed using purifiedmembranes from hemolytic group A streptococci. Alarge number of controls, including some patientswith diverse neurological diseases, was also pre-viously studied, and no significant increase in anti-neuronal antibodies was recorded (9). Subsequently,several sera from patients with HDwere also studiedand these sera showed striking staining of neuronsin frozen sections of human caudate nucleus whenindirect immunofluorescence technique was used.Antibody activity from sera of HD patients was not

I Abbreviations used in this paper: HD, Huntington'sdisease; PBS, phosphate-buffered saline; VCN, neuramini-dase.

The Journal of Clinical Investigation Volume 59 May 1977 - 922-932922

removed by streptococcal membrane absorption andit appeared that this reaction was distinct from thatpreviously recorded in Sydenham's chorea. Thepresent report presents findings on the prevalenceof antineuronal antibodies in a large group of sera

obtained from HDpatients. In addition, studies of un-

affected HD family members at risk as well as a

group of spouses of HD patients provide fascinat-ing possibilities regarding potential disease mech-anisms in this disorder.

METHODS

Clinical material. 80 patients with clearly established HDconstituted the main group studied. 18 patients hospitalizedat the Albuquerque Veterans Administration Hospital or

Bemalillo County Medical Center were some of the sub-jects studied. An additional nine affected individualswere studied through samples kindly provided by theirresponsible physicians throughout the country. 25 HDsubjects were studied from clinical material available atAnn Arbor, Mich. and another 17 patients were drawnfrom patients followed through the Neurology service atthe University of Colorado Medical Center. Finally, 11HD subjects from Norway were also included in afflictedpropositi studied. Duration of overt disease was estimatedfrom the date of first recorded clinical symptoms and rangedfrom 1 to 28 yr. 115 blood samples from members of 31families of HD patients were also studied. First-degreerelatives were defined as parents, children, and siblings ofprobands with HD whereas second-degree relatives were

twice removed from the proband (i.e., grandparents, uncles,aunts, cousins, nieces, and nephews). An attempt was

made to study as many living relatives, both blood re-

lated and nonblood related, as were available. In addition,whenever possible, particular care was used to studyspouses of patients afflicted with HD. In all, a total of 20spouses were included in this phase of the study.

In one HD patient, simultaneous serum and cerebro-spinal fluid samples were available. Spinal fluid was con-

centrated 50 times to obtain an IgG concentration similarto that of the serum (11.0 mg/ml).

Control sera were drawn from a group of normal sub-jects and patients with diverse medical conditions as

previously noted (9). An additional new group of 198neurological controls was included in the present study.These were obtained from the in-patient and out-patientneurology services of the Bemalillo County Medical Center,Albuquerque Veterans Administration Hospital, and St.Josephs Hospital, Albuquerque, N. Mex. Sera were ob-tained from patients with a wide variety of neurologicaldiseases including Parkinson's disease, senile dementia,transverse myelitis, strokes, brain and spinal cord tumors,central nervous system trauma, multiple sclerosis, andmiscellaneous neurological conditions. A summary of thetypes of patients included among the neurological con-trols is shown in Table I.

Assay systems. The primary study technique utilized in-direct immunofluorescence and unfixed frozen sectionsof normal adult human brain as previously described (8, 9).Brain was obtained from the medical examiner's office fromindividuals who had sustained accidental death and wasstudied within 4-12 h of death. Small 2 x 2 x 5-mm blockswere obtained from caudate and subthalamic areas as wellas cerebral cortex, cerebellum, geniculate ganglion, and spinalcord, the latter to include large anterior horn cells in cross

HD

Neurological disease controlsDegenerative/vasculardisease:senile dementia, cerebralinfarction, cerebralhemorrhage, multiplecerebrovascular accidents

Primary brain tumors:meningioma, medullo-blastoma, astrocytoma,glioma, glioblastoma,pituitary adenoma sarcoma

CNStrauma:depressed skull fracture,subdural hematoma,epidural hemorrhage,quadriplegia, gunshotwounds

Miscellaneous neurologicalconditions:seizures, amyotrophiclateral sclerosis, myasthenia,transverse myelitis,peripheral neuritis, vestib-ular neuritis, Ticdouloureux, spinocerebellardegeneration

Parkinson's disease

Miscellaneous disease controls:pneumonia, chronic liverdisease, cancer, arterio-sclerotic heart disease,lymphoma, emphysema,peptic ulcer, inflammatorybowel disease

Normal controls

yr

60 80 37(46%)*

65 65

53 14

5

1

39 25 1

38 31

38 30165

69.0 33

2

1

10(6%)

11 (33.3%)

56 70 3*

30 60 2*

* Difference between prevalence of antibody in HDand nor-

mals by chi square, P < 0.0005 and difference between HDand miscellaneous disease controls, P < 0.0005.

section. Before frozen sections were prepared, brainsamples were carefully washed at 4°C for 2-24 h in coldphosphate-buffered saline (PBS) in an effort to remove

adsorbed blood or plasma proteins.4-6-jm frozen sections were cut in a cryostat and used

unfixed on slides. Indirect immunofluorescence proce-

Antineuronal Antibodies in Huntington's Chorea

TABLE IPrevalence of Antineuronal Antibody in HD

and Various Control Groups

Mean No. subjects No.age tested positive

923

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FIGURE IC Immunofluorescence reactions of antineuronal antibody again showing lightgreen specific fluorescence of entire neuronal cytoplasm. Bright orange-yellow autofluorescentlipofuscin granules (arrow) are noted within cells (x500).

dures followed those previously outlined (9, 10). Briefly,after incubation of tissue sections for 30 min with undilutedtest and control sera (heat inactivated at 560C for 30 min),sections were washed twice for 10 min in PBS, and fluores-cein-conjugated rabbit anti-human IgG, IgA, or IgM wasapplied with subsequent incubation and washing in PBS.Specificity of fluorescent staining was checked by standardblocking and absorption procedures (9). Sections were alsoexamined with fluoresceinated reagents alone withoutapplication of test sera. Most sections were read blindby two observers (G. H. and K. K.) and concordantpositive and negative results were recorded. To examinespecies specificity of the antineuronal antibodies, brain

P samples from rats were also obtained and were used inindirect immunofluorescence tests as described for the humanbrain samples. Rat brains were perfused with 60 ml ofcold PBS before removal.

Previous studies by Aarli et al. (11) have indicated thatsome nonspecific reactions between IgG and central

nervous system tissues may be due to Fc binding of IgGto myelin components. Accordingly, IgG from positive serawas isolated by ammonium sulfate precipitation or DEAEion-exchange chromatography and pepsin digested to pro-duce F(ab')2 fragments as previously described (9). Specificityof IgG binding to central nervous system structures wasthus studied by comparing immunofluorescence with wholeserum or isolated IgG and F(ab')2 fragments of the same IgG.

To examine the ability of the antineuronal antibodies tobind complement, sections were incubated with posi-tive sera and washed as described, thereafter overlaidwith fresh normal human serum as a source of comple-ment, washed, and finally treated with fluorescein isothio-cyanate-labeled antihuman C3. As a control, the normalhuman serum was heat inactivated at 56°C for 30 min be-fore use.

A search for specificity of antineuronal fluorescentstaining was made using various types of enzymatic andclinical treatments of frozen sections with modifications of

FIGURES IA and B (A) Immunofluorescence photomicrograph of positive staining patternsshowing antineuronal antibody. Staining of caudate nucleus cytoplasmic structures wasclearly seen with apparent relative spearing of nuclei. Bright dots (arrows) representedorange-yellow autofluorescence of lipofuscin granules within individual neurons. Diffuse lightgreen specific fluorescence was seen in medium and larger neurons (magnification x460). (B)Parallel section of human caudate nucleus to that shown in (A) stained with hematoxylin andeosin. This area is populated by nerve cell bodies of large and intermediate size (x460).

Antineuronal Antibodies in Huntington's Chorea 925

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several methods previously described (12-15). Frozen sec-tions were treated with trypsin, periodate, EDTA, sulfhydryl-active agents, DNAase, RNAase, neuraminidase, andlipase. Trypsin (Worthington Biochemical Corp., Freehold,N. J.) digestion utilized concentrations of 0.001 mg/mltrypsin in Tris buffer, at pH 8.6, for 60 min at 37°C. Aftertrypsin digestion, 30 iLg soya bean trypsin inhibitor wasapplied for 10 min to stop trypsin reactivity, followed bythree 4-min washes in PBS, pH 7.4. In some experiments,dithiothreitol was used in conjunction with trypsin (14). 2mMdithiothreitol was buffered to pH 8.6 with Tris buffer,and enzymatic digestion was carried out in the presence ofthe reducing agent. Neuraminidase (VCN) (Calbiochem, SanDiego, Calif.) was used at concentrations of 15, 25, 50,125, and 250 U/ml. VCNtreatment was carried out at 37°C inacetate buffer, at pH 5.1, for 30 min followed by a brief15-s wash and three 4-min washes of sections in PBS. Inaddition, a combination of VCN and 64 mMEDTA treat-ment at 37'C, pH 5.0, for 30 min was also utilized insome experiments. VCN was applied and washed off thesections as described, followed by incubation at roomtemperature in EDTA for 30 min. After three 4-min washesin PBS, slides were overlaid with test serum and fluorescenceprocedure was followed. This treatment was specificallyselected since previous reports by Woolley and Gommi(13) showed destruction of serotonin receptors by VCNplus EDTA but not by either reagent when used alone.Periodate treatment of tissue sections was performed for30 min at 22'C with concentrations of 64, 16, 4, 1, 0.25,and 0.06 mMusing methods described by Matre et al.(15). DNAase (Worthington Biochemical Corp.) treatmentof tissue sections used 0.005 mg/ml, pH 7.0, for 1 h at37'C and RNAase (Worthington) employed 4 Ag/ml of en-zyme on sections, at pH 7.4, for 1 h at 37°C. Lipase (Rhisopusdelemar 6,000 U/mg, Miles Laboratories, Inc., ResearchDiv., Kankakee, Ill.) was applied at concentrations of 1,200,600, 300, 150, and 30 U/ml in conjunction with 0.1 Mphosphate buffer, pH 5.8. After 52 min of incubation at 220C,tissue sections were washed three times in PBS for 4 min.For all enzymes or chemical treatments, two types of con-trols were used. Sections treated with PBS or base-linebuffers instead of enzymes or reagents served to test forthe effects of these reagents alone. Also enzyme-treatedsections were overlaid with normal human serum to moni-tor specificity of subsequent immunofluorescence reactions.

Preparations rich in isolated neurons were obtainedfrom human caudate nucleus using the technique of Selingeret al. (16). These preparations were also used for ab-sorption studies (9). Lyophilized proteins extracted fromwhole caudate nucleus with perchloroacetic acid usingthe method previously described by Wedege (17) were alsoused for absorption of six selected sera at a concentra-tion of 10 mg/ml.

In some instances sera from HDpatients showing strongimmunofluorescent staining of human caudate nuclearneuronal cytoplasm were tested for possible specificity forgroup A streptococcal antigens utilizing group A mem-branes, cell walls, and carbohydrate kindly provided byDoctors I. van de Rijn and J. Zabriskie, The RockefellerUniversity, New York, and prepared as previously de-scribed (9).

TABLE IIAntibody Titration and Complement Fixation Tests of

Positive Antineuronal Antibody Sera fromPatients with HD

Anfibody titration* Complement fixation*

Titer No. of patients No. positive/no. tested

Neat 11 8/101:2 5 2/41:4 8 5/61:8 8 4/81:16 4 2/31:32 0NTxx 1

Total 37 21/31

* Determined by indirect immunofluorescence on frozensections from human caudate nucleus. xx not titrated.

To check for possible presence of antinuclear antibodies,mitochondrial antibodies, and microsomal antibodies, serawere tested by indirect immunofluorescence techniqueusing unfixed frozen sections from rat and human liveras substrate for nuclei, and human kidney as substratefor the two cytoplasmic antigens (10, 18, 19). In theseassays the sera were screened undiluted.

RESULTS

HD subjects. 37 of the 80 sera (46.3%) from HDsubjects studied showed positive staining reactionsfor neuronal cytoplasmic fluorescence (Table I, Fig.1). The titers of positive sera are shown in Table II.The neuronal cytoplasmic staining was much moreprominent with medium-sized neurons found in thecaudate nucleus, subthalamic, and basal ganglia areasthan with neurons of other comparable regions suchas cerebral cortex, cerebellum, geniculate ganglion,amygdala, or anterior horn cell areas of spinal cord.It was clear that neither cerebellar Purkinje cells norlarge motor anterior horn cell neurons of spinal cordwere stained by any of the positive sera. Thus, themost prominent fluorescent staining for antineuronalantibody consistently occurred with sections of caudatenucleus or subthalamic areas. Comparative resultsusing sections of human caudate nucleus, cerebralcortex, and trigeminal nucleus are shown in Table III.

Almost identical immunofluorescent findings wereobserved when corresponding brain samples fromrats were used instead of human material providing

FIGURES ID and E (D) Parallel hematoxylin and eosin section (to C) of human caudatenucleus showing types of large and intermediate neuronal bodies involved in specific immuno-fluorescence (x500). (E) Immunofluorescence of human caudate nucleus neurons again showingdiffuse cytoplasmic apple green fluorescence which spares nuclear structures. Arrow showslipofuscin autofluorescent granules (x520).


TABLE IIIComparative Staining Reactions Using Serafrom 10 Patients

with Huntington's Chorea and Frozen Sections ofNormal Human Brain

Patient no. Catidate ntcletis Cerebral cortex Trigeminal nucleus

1 ++* (1:8)1 +* (1:2)t +* (1:2)2 + (1:4) + (1:2) + (1:2)3 0 0 04 + (1:2) + +5 + (1:4) + (1: 1) + (1: 1)6 + (1:1) + +7 0 0 08 0 0 09 0 0 0

10 0 0 0

* Relativeserum.

strength of immunofluorescence with undiluted

t Figures in parentheses refer to titer of positive staining inimmunofluorescence.

evidence that the antineuronal antibodies werenot species specific.

Concomitant screening of test sera for the presenceof antinuclear antibody as well as mitochondrial andmicrosomal antibodies was performed to insureneuronal cytoplasmic specificity of the immunofluores-cent staining. Five of the positive sera from HD pa-tients showed antinuclear reactivity. However, inthese five sera, immunofluorescent staining was alsolocalized to neuronal cytoplasmic structures clearlyidentified by the concomitant finding of autofluorescentgranules of lipofuscin known to be present in the cyto-

1: 16 r * 0

plasm of such cells (Fig. 1). None of the sera wasshown to contain antimitochondrial or antimicrosomalreactivity when tested on rat and human kidneysections.

An attempt was next made to examine whether thepresence of positive reactions for antineuronal anti-body correlated in any way with clinical durationof disease. Two patients included as presumptiveHDhad not as yet manifested clinical symptoms buthad one parent and one child already involved withovert HD. These subjects (both negative for anti-neuronal antibody) were not included in this analysis.In 74 patients, where accurate clinical data were avail-able about actual initial onset of disease, it appearedthat a larger proportion of patients with duration ofdisease greater than 7 yr (21/36) showed positive anti-body reactions than did subjects with disease dura-tion of 7 yr or less (16/38) (chi-square, 2.82). How-ever, some exceptions to these data were observed.These data are shown graphically in Fig. 2. It canbe seen that sera showing higher titers of anti-neuronal antibody were more prominent in patientswith disease duration of over 7 yr. If patients withtiter of antibody of 1:2 or greater in subjects withless than 7 yr of clinical disease were compared tothose with 7 yr duration or greater, chi-square was9.06 (P < 0.001).

In one patient where both serum and spinal fluidwere available, concentrated spinal fluid showed onlya weak (±) and immunofluorescence reactionwhereas serum produced stronger staining (2+) andwas positive to a dilution of 1:16.

A diverse group of control patients was also examined

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-L- *

.1 I- 1 L..S. 1 as t1 2 3 4 5 6 7 8 9

IL I I I I di 1. I

10 11 12 13 14 15 16 17 B 19 2) 21 28

Duration of Clinical Symptoms (years)

FIGURE 2 Graph showing relationship between presence and titer of antineuronal antibodyand duration in years of clinical manifestations of HD in 74 patients.

928 G. Husby, L. Li, L. Davis, E. Wedege, E. Kokmen, and R. Williams, Jr.

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for the presence of antineuronal antibody. Resultsare summarized in Table I. It can be seen that alow overall incidence of antineuronal antibody wasnoted among normal subjects (3.3%) and controlpatients with various miscellaneous medical dis-orders (4.2%). A slightly higher general prevalence(6%) of antineuronal antibody was recorded in theneurological control group. Of particular interest wasthe presence of the antibody in one-third of 33 pa-tients with Parkinson's disease, with titers comparableto those of HD patients (Table II). Patients withintracranial tumors, strokes, or miscellaneous neuro-logical conditions showed a lower overall prevalenceof positive reactions for antineuronal antibody.

Assay for complement binding. Sections sequen-tially incubated with positive HD sera, fresh nor-mal human serum, and fluorescein isothiocyanate-labeled anti-C3 showed cytoplasmic staining similar tothat of the antineuronal antibodies in 22 of the 31sera tested. There was apparently no correlation be-tween the antineuronal antibody titer and the capac-ity for complement fixation (Table II). When thefresh human serum was heat inactivated in controlexperiments, no fluorescent staining was observed.These experiments clearly demonstrated that anti-neuronal antibodies present in HDsera were capableof complement fixation. In contrast, none of theeight positive sera tested from patients with Parkin-son's disease showed complement fixation when in-direct immunofluorescence technique was used.

HDfamily studies. 115 sera from 31 families con-taining at least one HD proband were studied forpresence of antineuronal antibody. These sera in-cluded 20 husband-wife spouse pairs in which one(husband or wife) had established HD. 6 of 20 un-affected spouses (30%) of subjects with HD showedpresence of positive reactions for antineuronal anti-body. One spouse showed a titer of 1:16 which wasamong the highest recorded. Her HD-affected husbandwas also positive with 1:4 titer. In addition, 13 of 56first-degree HDfamily members (23.2%) clearly at riskfor eventual development of HD(children of marriageswhere one parent had established HD) showed pres-ence of antineuronal antibody. By contrast, only 10.5%or 2 of 19 nonblood-related relatives where suchfamily members were married to spouses at risk forHDwere positive for antineuronal antibody. In addi-tion, 19 second-degree relatives indirectly at riskfor HD (grandparents with HD) showed a 10.5%incidence of antibody. Analysis of these data by the chi-square method showed a highly significant differencebetween spouses and first degree relatives directly atrisk and normal controls (P < 0.001), Table IV. Illustra-tive examples of representative family distribution ofantineuronal antibody are shown in Fig. 3.

Attempts to characterize HD serum-staining anti-

TABLE IVPrevalence of Antineuronal Antibody in HDFamilies

Family members tested No. tested No. positive

Probands with HD 80 37

Spouses of probands with HD 20 6*

First-degree HDrelatives 56 131Second-degree HDrelatives 19 2§Nonblood related relatives 19 2

(excluding spouses)

Normal controls 60 2*t§* Difference between prevalence of antibody in normal con-trols and spouses of HDprobands, P < 0.001.t Difference between normal controls and first-degree rela-tives by chi square, P < 0.001.§ Chi square difference between normals and second-degreerelatives NS.

gen. Frozen tissue sections were treated withDNAase and RNAase and examined with a panel ofstrongly positive sera. DNAase digestion of sectionsproduced no change in staining, however slightdiminution was recorded after RNAase digestion inseveral instances. Trypsin digestion was also associatedwith diminution in positive staining of neuronal cyto-plasm. No significant changes in staining patternswere noted after lipase, VCN, or EDTAafter lipase,neuraminidase, or EDTA treatment. Some increasein immunofluorescent staining occurred after periodatetreatment; however this appeared to be nonspecificsince parallel control observations using previouslynegative normal sera also showed an increased generaltendency for binding to periodate-treated sections.Similar results using this reagent have been previouslyrecorded (12).

Studies of specificity. 31 of the 37 HD sera posi-tive for antineuronal antibodies were also tested usingflourescein isothiocyanate-labeled rabbit antibodiesspecific for IgG, IgM, and IgA, respectively. In allcases only antineuronal antibodies of the IgG classwere detected.

Binding of HD IgG antibody to neuronal tissueswas assessed before and after IgG digestion by pepsin.Serum and isolated IgG from positive sera werestudied in parallel with pepsin F(ab')2 fragments ofIgG in immunofluorescence reactions. Clear evi-dence of antibody combining-site binding using F(ab')2fragments as well as whole IgG preparations was ob-tained in all positive sera tested (Table V).

In addition, careful absorption of HD sera withgroup A streptococcal cell walls, membranes, andpurified carbohydrate, utilizing methods previouslydescribed (9), produced no diminution or significant


(+)I

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A

II

(-) (+)

HD8yr

B

(-) (-) (-)

HDt t

*~*(-) HDNT (+)

*Froternl

twins

FIGURE 3 Diagrams of representative familial constellationsof HDand the occurrence of antineuronal antibody. Designa-tions in parentheses (+) or (0) indicate positive or negativetests for antineuronal antibody. Ages of family membersare shown by numbers inside circles or squares. (A) FamilyX shows positive tests in nonblood-related spouse of probandas well as in as yet unaffected daughter aged 29. FamilyXIII shows three affected siblings with HD all negativefor antibody, however positive tests in three as yet non-affected children ages 26, 35, and 22. Family XX shows posi-tive antibody in a 65-yr-old as yet nonaffected son withnegative tests in his two siblings. (B) Family XVIII showsa negative serum antibody test in the 37-yr-old affected pro-band with positive tests in her husband and negative testsin the three as yet nonaffected children. Family XVI showsa family containing fraternal twins. The affected male twinwas not available for testing but the female as yet non-affected twin was positive for antineuronal antibody.

abolition of positive staining. The absence of absorp-tion by group A streptococcal antigens clearly differ-entiated the antineuronal antibody of HD from thatassociated with rheumatic fever and Sydenham'schorea (9). Preparations of isolated caudate nucleusneurons obtained by the Selinger technique (16) wereused for absorption in conjunction with isolatedhuman hepatocytes or extracts of human liver andkidney. Clear absorption of antineuronal antibodywas accomplished by preparations of isolated neurons.Furthermore, absorption of positive HD sera withperchloroacetic acid extracts from normal humancaudate nucleus (17) containing extractable cyto-plasmic proteins also completely removed immuno-fluorescent staining.

DISCUSSION

This study demonstrates the presence of serum IgGantibodies reacting with antigens localized in neuronalcytoplasm in 46% of 80 patients afflicted with HD.Although neurons showing reactivity were alsopresent in other areas of the central nervous system,strong concentration of the antigen appeared to bepresent in subthalamic and caudate nucleus areasof normal brain. Antineuronal antibodies in HDsera apparently showed different specificity fromthose previously described in the sera of childrenwith rheumatic fever and Sydenham's chorea (8, 9)since they were not absorbed with group A hemolyticstreptococcal membranes. Absorption of antibody was,however, complete using isolated neuronal prepara-tions. The antigen in neuronal cytoplasm reactingwith HD antineuronal antibodies has not yet beenfully characterized but appears to be relatively resistantto digestion with VCN, lipase, DNAase, and treat-ment with EDTA. Definite reduction in positivestaining patterns was recorded after trypsin digestionof frozen sections or after RNAase treatment. Theeffective absorption of antineuronal antibodies withperchloroacetic acid extracts from normal caudate nu-cleus indicates that the antigens(s) involved belongto the group of proteins extractable with this reagent(17). Further characterization of subcellular neuronalcytoplasmic fractions eventually employing immuno-logical electron microscope techniques will be neces-sary before the exact cellular localization of theneuronal HD-reactive antigen is clear.

Spinal cord anterior hom motor neurons and cere-bellar Purkinje cells were not stained and neuronsin the amygdala and locus ceruleus also showed nega-tive or very weak staining. Absence of staining ofPurkinje cells which possess gamma aminobutyricacid receptors was of particular interest.

Antineuronal antibodies in HD subjects appearedto show some relation to previous duration of clini-


cal disease in that the prevalence of antibody as wellas mean titer was higher in subjects with clinicaldisease manifestations over 7 yr. The serum antibodydetected in these studies was restricted to the IgGclass. Unfortunately only one sample of cerebrospinalfluid has been available for study. Since 46% of 80HD subjects studied showed antibody, it is con-ceivable that a different spectrum or prevalence of anti-body might be present in spinal fluid if the primarygenesis of antibody production were in the centralnervous system itself. Such a situation has beenamply demonstrated in the case of subacute scleros-ing panencephalitis where oligoclonal restriction ofcerebrospinal fluid antibody to measles-like antigenshas been demonstrated (20). The prevalence of anti-body reacting with neuronal antigens was relativelylow among miscellaneous neurological controlsincluding those with demyelinating disorders, avariety of strokes, brain tumors, or previous traumato central nervous system. A striking exception to thiswas recorded among patients with Parkinson's diseasewhere the incidence of positive tests for antibody was33.3%. The high proportion of positive antineuronalantibody reactions among subjects with Parkinson'sdisease may represent a fascinating parallel findingto the current study of HD. Both disorders repre-sent physiological disturbance of neuronal func-tion in basal ganglia structures. Indeed HD can bepresent in a rigid form clinically suggesting Parkin-son's disease (21, 22). In both, damage to neuronsand subsequent alteration of native autologousneuronal antigens might be capable of inducing syn-thesis of antibodies directed against damagedneuronal structures. The increased titer and preva-lence with duration of disease in HD might indeedfit with such an hypothesis. However, it is con-ceivable that antineuronal antibody in both condi-tions might be directed against virally induced anti-gens somehow associated with expression of thedisease. Postinfluenzal Parkinsonism has in the pastbeen attributed to long-term viral sequelae and itstill remains a possibility in terms of the etiology ofthe whole disease spectrum itself (23, 24).

The species nonspecific nature of the antineuronalantibodies provides interesting possibilities for thedevelopment of an experimental model of HD in ani-mals, however it is difficult to understand how the anti-body during life could directly react with cytoplasmicantigen localized within individual neurons.

The demonstrated ability of the antineuronal anti-bodies to fix complement, along with binding ofpepsin F(ab')2 antibody fragments, provides furthersupport for the antibody nature of this reaction.Whether or not this is involved directly in pathogenesisor in the tissue lesions themselves must await direct

TABLE VComparative Antineuronal Antibody Reactions Using Whole

Serum, IgG, and F(ab')2 Fragments fromRepresentative HDSamples

Patient serum studied Whole serum IgG F(ab')2

Male, 50yr 1:8* 1:4 1:4Female, 56 yr 1:4 1:4 1:4Male, 58 yr 1:16 1:16 1:8

* Titer of antineuronal antibody staining by indirect immuno-fluorescence.

parallel studies of the lesions themselves in HDcerebral tissues.

Several previous reports have documented the pres-ence of antineuronal antibodies in sera from patientswith carcinomatous neuropathy (23-27). It is notclear what relationship these reactions have to theantineuronal antibodies documented in the presentreport.

The family studies recorded here provide fascinat-ing preliminary data which appear to link an environ-mental influence or conceivably an infectious agent tothe expression of HD. Most telling in this regardwere the studies of the 20 spouse pairs where 30%of unaffected spouses showed presence of antineuronalantibody. First-degree relatives directly at riskshowed a prevalence of 23% as against the prevalenceof 3% in normal controls. As degree of direct rela-tion to HDparent or grandparents declined, the preval-ence of positive antineuronal antibody fell to 10.5%.Since HD has been previously clearly shown to berelated to an autosomal dominant inheritance pat-tern, these data considered together suggest that anti-neuronal antibody may reflect exposure to some agentor common environmental factor. Such exposure, if itwere a virus or slow viral infection (28, 29), mightbe magnified in spouses of afflicted patients. However,to actually develop clinical manifestations and pro-gression of the disease, one would theoreticallyhave to possess a specific HD-reactive IR gene (30,31). Such a hypothesis might explain the findingsrecorded in the current report. Additional studiesare now needed to attempt to characterize the antigenreactive with HD antineuronal antibody and tofurther explore the prevalence of such antibody inkindreds as well as spouses of patients with Parkin-son's disease.

ACKNOWLEDGMENTSThe authors are indebted to the many physicians who assistedin providing serum samples from their patients with Hunting-ton's disease. In particular, Dr. William J. Kimberling,Denver, Colo., Dr. Ruth Atkinson, Albuquerque, New Mex.,Dr. Charles Terry, Waco, Tex., Dr. Elmo Anderson, Al-


buquerque, New Mex., Dr. John Hester, Fort Lions, Colo.Dr. L. J. Barber, Roswell, NewMex., Dr. William Rosenzweig,Tulsa, Okla., Dr. 0. Devarajan, Newton, Conn., Dr. J.Vaughan, Las Vegas, New Mex., Dr. E. Sagedal of EegHospital, Kristiansand, Norway, and Dr. B. Bandvik, Oslo,Norway were of great help in collecting serum samples fromHD patients. It is also a pleasure to acknowledge thesuperb technical help of Ms. Kathy Kilpatrick and theexpert secretarial assistance of Ms. Bernadette Marquez. Weare also indebted to Dr. Mario Komfeld for help withneuropathological dissections and photomicrographsof neural tissue and Dr. Betty Skipper for advice with statisti-cal analysis.

This research was supported in part by grants 13824-07and TOIAI0053 from the U. S. Public Health Service and inpart by a grant from the Kroc Foundation.

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antibodies to human caudate nucleus in huntington's chorea · removed by streptococcal...

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