Monoclonal antibodies recognising cell surface molecules expressed by rat cerebellar interneurons

Journal q[ Neuroimmunologv, 6 (1984) 283-300

283

Elsevier JNI 00182

Monoclonal Antibodies Recognising Cell Surface Molecules Expressed by Rat Cerebellar Interneurons Michael Webb * and Peter L Woodhams M.I~C Det~dopawn:a!NourobiologyUni~ lnslituteof Neurdogv, 33Jo~nsMews,London WCI 2NS (GreatBritain)

(Received27 Augur, 1983) (Revised,m.~eived8 December,1983) (Acccpled8 December,t983)

Summary We describe monoclonal antibodies r~ognis~ng three cell surface antigens expressed by tat cere/~ellar granule neuroi3s. The anlibodies were produced after immunising mice wi,~h a cerebellar glycoprotein preparation, 6-1-3 recognises a glycoprotein with an apparent molecular weight of 144000 daltons, and gives an unusual staining pattern on cultured neurons. 7-81)2 reeognises postmitotic granule cells, hut not their precursors in the external granular layer. 'Ihe antigen was not detected in any brain region other than the cerebellum. 8-20-1 recognises a brain specific glycoprotein with an apperent molecular weight of 48 000 daltons, which appears on cerebellar granule cells only after the 10th postnatal day, Key words: Ce~ebellar development ~ Glyeoproteins- Monoclonal ,mzibodies - Neuronal cell surface

Introduction The complex pattern of tissue orsanisation and connectivity ~hibited by the mature nervous s~tem may arise in part through cell-cell interactions during

* Ih-esenl address: Mescular Dy~l~phy Laborai~'y, lnslimie of Ne~ok~, ~ . n huare, London WCIN 311G,Great Britain. " To whomall ¢ocresponder~shouldbe a d d ~ Abb~ons: BSA- bovine ~ n albe~n; ConA= coneanaveJinA; DAB= phosphate-buffered saline ¢onie/nin8 Ca~+ (0.9 raM) end Mg~+ (0.5 raM); EDTA•ethylene diamine tetra aceti~ acid; P = postnatalday; PAGEffip o l ~ d e 8el decu'ophoresis"$DS = sodiumdodecylsulphate.

284 development, leading to the preferential association between cells which ha,/e some selective chemical affinity with "each other (Sperry 1963). Most modern formulations of this hypothesis postulate that molecules exposed at the cell surface mediate these interactions (Moscona 1980; Gottlieb arid Giaser 1981). With the exception of the neural cell adhesion molecule (N-CAM) of chick (Rothbard et al. 1982) and rodent (Chuong et al. 1982) brain, little is known about the nature of the molecules that may be involved in these interactions. It may significant that N-CAM is a glycoprotein, as are most proteins exposed at the cell surface (Bretscher 1973; Steck 1974), e,s it has been suggested that the carbohydrate moieties of glyeoproteins may function as recognition signals in conjunction with membrane-located iectins (Barondes and Rosen 1976). Specific glycoproteins are concentrated at synapses (Cotman and Banker 1974; Pfenninger and Rees 1976), and a class of transient ConA binding glycoproteins in the parallel fibres of developing cerebellum has been suggested to be involved in the mechanism of synapse formation between the axons of granule neurons and the dendrites of Purkinje cells (Zanetta et al. 1978). A number of other systems have been described in which developmental changes in the profiles of expressed glycoproteiBs are observed, and in which a developmental significance for this changing profile is thought likely (Mintz ¢t al. 1981; Webb 1983). Experiments using lectins to alter or block the behaviour of cerebellar neurons in culture support the suggestion that cell membrane glycoproteins may be actively involved in the social behaviour of neurons (Trenkner and Sarkar 1977; Hatten and Messer 1978). in order to explore thes,." suggestions more rigorously, specific reagents able to recognise and define neuronal cell surface glycoproteins are required. Monoclonal antibodies are the reagents of choice for this task. in view of theiJ well known advantages over conventional sera. These include the possibility of producing monospecific reagents after immunising with a complex mixture of antigens, the unvarying nature of the antibody with time and from laboratory to laboratory, and the potential for producing unlimited amounts of pure reagent. Once produced, the antibodies can be used to study the distribution and development of their cognate antigens, to purify these antigens for detailed biochemical studies, and in studies designed to elucidate the function of the antigens by using the antibodies to block or perturb in vitro functional systems (We.bb et al. 1979; Greve and Gottlieb 1981: Rutishauser et al. 1983). A final advantage of monoclonal antibodies, which they share with conventional sera, is that the recognition and definition of new molecules is not dependent upcm them having a function which is already known. We recently pre.~ented evidence for the presence of a number of neuronal cell surface sialoglycoproteins which are expressed on cultured rat cerebellar interneatons, and which undergo reproducible changes in expression during culture maturation (Webb 1983). In r~rder to carry these observations further, we prepared lentil lectin binding glycoproteins from mature rat cerebellum, and used these to raise mouse monoclonal antibodies. We screened the antibodies produced against both cultured cerebellar neurons and eryostat tissue sections, to identify those antibodies recognising cerebellar granule cells. We describe.~here our preliminary chara,:terisation of three different antibodies, each of which recognises an antigen with features of particular interest, expressed by cerebellar granule neurons.

285 Methods

Preparation o] cerebellar membrane glycoproteins The methods used were based on those described by Barclay c t a l . (1~75). ,All operations were carried out at 4°C in ~he presence of 1 mM phenyl-methyl sulphonyl fluoride to inhibit proteolysis. Tv'eive g of pooled cerebella derived from 30-35-day-old Porton rats were homogenised in 75 ml of 0.'42 M sucrose c~anlaiaing 0.9 mM Ca 2+ and 0.5 mM Mg 2+ . The homogenate was centrifuged zt 1¢_~0× g~,, for 10 min, the pellet was resuspended in 25 ml of the. ;;ame buffer, rehon~ogerLisedal~d centrifuged again at 1000 × g~v. Crude memb~'anes were pelleted from the pool~l supernatants by centrifugation at 100000 x g~v for 9~3rain. The membrane pellet was homogenised in 10 mM Tris-HCI pH 8.0 and the protein concentration was adjusted to 12 mg/ml. An equal volume of 4% deoxycholate in tile sz'.mebuffer was added, and the mixture was stirred overnight. Insoluble material w~s removed by centrifugation at 100000 × g~ for 75 rain, ar,d the pellet was r¢~-extracted in 2~ deoxycholate T~is-HCl pH 8.0 for 2 h. Af.'_er ccntrifhgation as above to remove insoluble residue, the dcoxychola~e soluble sx~pernatan',s were pooled. The solubi~ised membrane was subjected to affinity chromatography on a column of lentil lectin attached to Sepharose 4B (Pharmacia~. The de~ergeat-solubl¢ extract was applied at a flow rate of l0 m l / h to a 20 ml column of |ec~in gel which had previously been equilibrated in 0.5~ deoxycholate 10 mM Tris-HCi pH 8.0. The column was washed in starting buffer until the O.D. 280 of the eluale dropped to that of the s~arting buffer. Bound glycopro~eins were eluted with 0.5 M a-methyl mannoside in 1% deo×ycholate i~) mM Tris-HCI pH ~.0 and fractions contaiaing protein were pooled. The pooled fractions were passed over an affimty column of MRC OX7 antibody (a generous ~ift of Dr. A.F. Williams) to deplete them of Thy-I antigen, and the Thy-l-depleted preparatio~ was concentrated by ultrafihration in an Amicon pressure cell equil:,ped with a PM30 membrane. Aliquots of this preparation to be used in affinity chromatography were stored in solution at - 70°C. The preparation to be u,.,ed as immunogen was precipitated by the addi6on of 3 ,~olumes of absolute ethanol at - 2 0 ° C for 72 h, and the glycoprotems w~.~re recovered by centrifugation.

lmmunisation The antib~:lies described in this paper came from fusions using 3 different mice. Young adult female Balb/c mice were injected subcutaneously with 50 lag of glycoprotein emulsified in Freund's complete adjuvant. This was repeated after 1 week, and thereafter the mice were given weekly subcutaneous boosters of 50 izg of glyeoprotein ~n incomplete Freund's adjuvant. The total number of injections given varied from 6 to 9. Serum from the immunised mice was assayed by an indirect solid phase radioimmunoassay for binding to rat cerehellar homogenate. Three days prior to fusion, mice with an adequate titre of antibody received an intravenous injection of 100/~g of glycoprotein in phosphate-buffered saline.

Cell fusion and cell culture procedures Spleen c~lls from the immunised mice were fused with P3X63Ag8 myel'oma cells

286 (a gift from Dr. F Walsh, Institute of Neurology, London, by courtesy of Dr. C. Milstein) using the methods described by Kohler and Milstein (1975). The fused cells were plated in 5 × 96 well tissue culture plates above a feeder layer of mouse spleen cells in Dulbecco's modification of Eagle's medium containing 20~ foetal calf serum, 1 mM pyruvate, 10 -4 M hypoxanthine, 4 × 10 -7 M aminopterin and 1.6 x 10 -s M thymidine. Ceils were fed at 4-day intervals, and 12 days after fusion. wells ~ ere screened for antibody production using the solid phase assay mentioned above. Cells from positive wells were expanded and frozen in liquid nitrogen. Further screening was performed by immunofluorescence on either cryostat sections of ccre~,ellum or on cultured cerebellar neurons. Cell lines of interest were cloned by limiting dilution, and the cloned populations were frozen and injected into mice for the production of ascitic fluid. lmmunofluorescence on eryoMat sections For immunocytochemical studies, various brain regions were snap-frozen in isopentane in liquid nitrogen, and 10-/~m sections were cut with a cryostat. The unfixed sections were dried onto glass slides and incubated in either undiluted culture supernatant or ascitic fluid diluted 1 : 50 for 1 h. After washing they were incubated in fluorescein-conjugated goat anti-mouse serum (Miles-Yeda, 1 : 50) for 40 rain, washed again, fixed fer 5 rain in 4% formaldehyde in phosphate-buffered saline, and mounted in 50~ glycerol in phosphate-buffered saline. They were examined with a Leitz ortholux li microscope equipped for ¢pifluorescent illumination (filler block H) and photographed using a standard 45-second exposure. Immunofluorescenee on primary neuron cultures Primary cultures of cerebellar granule neurons or astrocytes were derived from young posthatal rats by the methods described previously (Wcbb 1983), except that th,~ cell suspension was plated on polylysine-treated glass coverslips for immunofluorescence studies. The cell-type composition and morphological maturation of these cultures is described in Webb (1983). Cultures were stained either live or after fix~:tion at room temperature for 10 rain in 4~ para~ormaldchyde in PBS. They were incubated in either culture supernatant (undiluted) or ascitic fluid (diluted 1:50 in DAB buffer) containing 10 mM sodium azide for 60 rain at room temperature. After washing, they were incubated in fluorescein-conjugated goat anti-mouse serum (Miles-Yeda) .:liluted ~ :50 in DAB containing 10~ foetal calf serum and 10 mM sodium azide for 60 rain at room temperature. Fixed cultures were mounted directly in 50~ glycerol in PBS, while live cells were post-fi:¢ed in 4~ paraformaldehyde prior to mounting. In .some experiments, astrocytes were stained live with the appropriate monoclonal antibody and a rhodamine-conju~ted sheep anti-mouse antibody (a Oft from Dr. F. Walsh). They were then fixed in 95% ethanol/~% acetic acid and stained with a rabbit anti-GFAP antibody and a fluorescein-conjugated swine anti-rabbit antibody. (;el eleclrophoresis and immu~iobiotting Affinity purified glycoprotein or homcz,enised membranes from various tissues

2g~ (prepared as described by Lakin and Fabre 1981) were ~:parated on 10~ polyacrylamide gels in the presence of SDS using the: buffer system d L x m m ~ (1970). The separated prot-ins were electrophoretically lransfevred to n ~ ~ sheets as described by fowbin et al. (1979), and residual protein binding s/tcs saturated by incubation with 3~g BSA in PBS. The' nitrocellulose ~ w~h transferred proteins were incubated for between 2 and 2~ h w;,th .%fokl concemra~| tissue ct~lture supernatant of the appropriate monocloaal antibody. ~ gn~ cubatior, s were carried out at 4°C, while shorter incubatic~ns were carried out a~ room temperature. After extensive washing in PBS/0.1~ Triton X-~00/I~ bound antibody was detected by incubating the sheets for 60 rain in [i:~ilsheep mouse Fab' fragment (Amer~ham International: 3 × 10 s cpm/ml in P B S / I ~ ~ followed by autoradiography.

Affinity chromatography of iodinated glycoproteins Affinity gels were prepared by coupling precipitated antibody from ascii: flm~ with cyanogen bromide-activated Sepharose 4B. Antiix,dies were precipitated from ascitic fluid by the addition of saturated ammonium salphate to 405 saturathm for 60 min at 20°C. The precipitate was washed once in 40~ ammonium sulphaa¢, then dissolved in 0.1 M NaHCO 3 pH 8.3, 0.5 M NaCI, and dialysed against the same buffer for 4g h. The protein was coupled to CNBr-activated Sepharos¢ 4B {Ph~. macia) according to the manufacturers instructions to give about 5 mg of p r ~ / m l of gel. One hundred/~g of freshly prepared glycoprotein fraction was iodinau~d w/ah I mCi 125I(Amersham International) in the presence of 1~ sodium deox~aolat¢ using 20/~g of ehlo~'amine T to catalyse the reaction. After 2 rain at room tenqaamw¢, the iodinated product was separated from unreacted iodine on a 5-ml column od[ Sephadex G-50 equilibrated wi'~h 1~ deoxycholate, lodinated glycoprotcm was stored in aliquots at - 70°C and used within 3 weeks of preparation. iodinated glycoprotein was passed over a 4-ml column of $¢pharos¢ 415 coupkd to a mouse monoelonal antibody of irrelevant specificity to remove mm-spccif'a:a~ bound components. An aliquot of the fraction recovered from this cohunn (5 × 10~ cpm) was then passed through a 200-tzl mini-column of aff'mity ~ which had previously been equilibrated in 1~ deoxycholate in 10 mM Tris-HC! pH 8.0. The column was washed sequentially with 10-ml aliquots of this buffer comainia$ respectively; 1 mM ED'I'A, 1 mg/ml BSA; 1 mM EDTA, 1 mg/ml BSA 0.1~ 0.5~ deoxycholate, 0.15 M NaC|. After a final 10-ml wash with 0.55 ~ I ¢ in "iris buffer, the beads were recovered and boiled for 10 rain in SDS sample buffer with or without reducing agent (5~ 2-mercaptoethanol 0.1 M dithiothrcitoi). $o$ab/lised material was stored at - 70°C and analysed by PAGE in $DS and autoradiography. Resets

Localisation of antigens in tissue sections The distrioution and developmental expression of the antigens rccognised b~" the

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anubodies 6-1-3. 7-8D2 and 8-20-1 was studied by immunofluorescence on 10-/tm coostat sections of cerebellum, During e:~rly screening of cloned supernatants. 7-8D2 was first recognised because of the intense staining it produced in the molecular layer of the mature rat, although with this relatively unconceutrated antibody source, cell bodies did not appear to be stained. However, when a more concentrated source of antibody was employed (ascitic fluid diluted 1:50). it was evident that the antigen was present on both the fibres and the cell bodies of granule cells. Both the molecular layer (consisting mainly of fasiculated granule cell axons) and the internal granular layer (consisting of tightly packed granule neuron cell bodies) were stained, although the intensity of staining in the internal granular layer was always weakeJ than that in the molecular layer (Fig. 1C). The antigen first became detectable, though only weakly, at P3 (Fig. 1A). and staining increased in intensity to P10. at which point adult levels of staining were observed (Fig. I B). It is of interest to note in Fig. 1B that. althougt] cells in the position ~>f migrating neurons appear to be fairly heavily stained, the exterual granular I~yet. which is the source of mitotic cells giving rise to granule neurons, is essentially uns!ained. This :trongly suggests that 7-8D2 is not expressed on proliferating neurobiasts, but is expressed once the cells become postmitotic. The an'!gen continues tc, be expressed in the mature cerebellum (Fig. 1C), No staining wes seen on other e~ IIs in the cerebellum: in particular, Purkinje cells and glial ceils appeared to be unst ,ined.

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Fig. 1. l()-izm ¢ryo~,tat ~eclions ~,taincd with "/-gD2 11/50 diluted ascitic fluid). A: P3 cerebellum; B: PlO cL.'rcbcilum; (': I'.',5 cerebellum: D: P35 brainstem. E ( ; L = external granule layer; I G L = intern,~l granule la~,cr: MI. r:~,~h,:ular layer; PC = Purkin.ie cell,,. Bar = ItX)/~m.

Fig. 2. 10-/tm cryostat sections stained with 8-20-1 (1/50 diluted as¢itic fluid..-~ and /$ ~~.~6-t-3 t t ~ : culture supernatant, C: 1/50 ascitic fluid, D). ,4:PI0 cerebellum: B: P35 cerebellum: ¢- PM"~ : ~ c ~ } ~ D: P35 cerebellum. Bar = 100/tm.

We e~amined other brain regions and found no staining of cortex, h i p p ~ : a m p u , . basal ganglia, hypothalamus, thalamus or brain stem. A repre~-emati~e ~ecta~r~ .~* brain stem showing no staining with 7-8D2 is shown in Fig. i D. li is t,f imere--,t t ~ both th,~ hiFpocampus and layer 11 of the cortex contain population-, ot - m J ~ granular neurons which were unstained by 7-8D2. Thus it appears that ex~,re,~,,~ ~q 7-8D2 antigen is highly specific to postmitotic cerebellar granule neuron--. 8-20-1 gave very intense staining of the granule cell bodies in the mterna! granular layer (Fig. 2B). The molecular layer w~ts also stained, though less inten:,e'~). N ~ staining was observed on other cell types, including other r.eurons: for e x a m p ~ . Purkinje cells appear in negative outline in Fig. 2B. ] h e antigen ~ a s exprex~d lat~:r in development than 7-8D2, and could not be detected in PIO cerebel!,,m. ,:~en though the granular layer is already partially formed at this age lF~g. 2A}. S~:~m~r~ was observed weakly after P10, and climbed slowly to reach adul~ levels b~ P2~. In adult rats, 8-20-1 labelled the ,,europil in the lateral brain stem ~ h d e ~ d ~ e m areas of white matter (e.g. the solitary tract. Fig. 3A) were complete~:, un,tz~mcd, f~ more medial parts of the brain stem ([~ig. 3B) the antibody stained snlal[ m.-ur~-. Large neurons in the magnocellular part of the lateral reticular nuck'us ~ c r c apparently unstained, and appeared in negative outli~ae. An interesting pat~cr~ ~ apparent on moving towards the forebrain. Weak diffuse s~aining v,a~ sce~ i.~ ~ " caudate putamen (Fig. 3C). and this st~tining was only apparent in contra~,t to ~ ¢

290 completely unstained white matter of the internal capsule, In the cortex, no staining ~:t all was seen with 8-20-1 (Fig. 3DL It lhus appears that there is a posterior-anterior gradient in the expression of the antigen, such that cerei~ellum ( I G L ) > brain st,.~m > forebrain (basal ganglia) > cortex. In all regions where staining was observe~l, it was confined to small neuronal elements. Glial structures and white matter were never stained by the antibody, 8-20-1 is therefore :~ neuron-specific marker, ahhough it recognises a less limited subset of neurons than 7,8D2, and is not region-specific, 6-1-3 gave patterns of staining that were difficult to interpret, In original screening of the cloned supernatants, a pattern of dots and rings was f,,!md, which was considered at that stage to be artifactual (Fig, 2CL When more concentrated atdibody was used, bright fluorescence was observed, but it could not be clearly k~calised to any particular cellular or structural feature of the cerebellum (Fig. 2D). Tiffs point will be discussed in more detail later.

Immtotofht~rescence on ctdllo'ed ce,~ls We studied the expression of the antigens rt.n:ognised by the monoclonal antibodies on cultures of cerebellar neurons and astmcytes from posmatal cerebellum. The <

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Fig. 31 IO-gn~ cryostat sectl;.~nsof various adult rat brain regionsstained with 8,20-1 (!/50 dilul~t ascidc fk,id}. A: Lateral region of brain stem, Note staining of neuropit but absence of staining of white matter ~ract.,, (WML S~I ~ :~ solitary tract. B; Magnt~etlular region of lateral reticular nucl~u.',. Neuropil corre~pL~t'tdingt~ small neurons i~ slaiued, but large neurons (LN} are apparently uastalned. C: ('audale pulamen. Weak staining of Ihe neuropil is only apparent in conl~tsl to the completely unstained x~hire realtor (WM) of the internal capsu}e. D: Cerebral cortex. No staining is ob,~r,.~'d in ~=n~-slruc~ures, Bar ~ IfJl) #m.

291 neuronal cultures consist of about 95% small cerebeltar intert~euri=ns contaminated with a low and variable proportion of morphologically distinct astrt~cytes, fibroblasts and endothelial cells (Webb 19831, The5' undergo a certain degree of differel~tiation in culture, and we were interested in knowing whether any of the at~tigens ~howed developmental changes in expression. This question was of pa~ticttt~r interest in view of the observation lhal some of the surface sialoglycoproleins change in amount or structure te, g. by desialidation) during the development of these cells in culture (Webb 19831, The staining of live cells is also a criterion of surface l~calisalion of the antigen in question, Cultures were prepared from P8 rat cerebella, and stained at various ages. Both 6-1-3 and 8-20-t were expressed en these neurons as early as 24 h after initiating the culture (Fig. 4). 8-20,1 gave typical surface fluorescence on these live cells, with a ring of fluorescence around the cell body, and nascent fibres were also stained, though less intensely. 6-l-3 gave a highly unusual pattern of fluorescence on the live cells, Although the antigen was clearly restrict~xl to the outer surface of the cells, it appeared in discrete patches, both on the cell bodies and on the fibres. Some regions

Fig, 4. Cerebeltarneurons stained after culture for 24 h. Live ~lls were stained with 1/50 diluted asci~es of 8-20-1 (~, B) or 6-l-3 (C, D). Identical fieldswere ph:~tographedunder phase (A, C) and fluorescence ( ~ D I optics. Arr~.'~wsindicate parts of cells under phase optics which correspond wid~ intense 6~Id staining under fluorescenceoptics, Bar = 100/~m.

~.u2 ,ff the ",lamed cells appeared completely free of fluorescence, while other regions "+uore,,ced brightl.',, Similarly. some fibre regions failed to express antigen, while >,reakx fluorescence was detected on other regions of the same fibre. This result is ,n,,i~,tentl 5 obtained when 6-1-3 is used t o stain these cultures. Both 6-1-3 and 8-2(1-1 continued to be expressed on older cultures, in order to

I Ig 5 ('++'1¢b¢!l~11 ncurom. ~,tamed after 12 days (,.I 1?. I:'. [ ' ) or 4 days (C, D ) in culture. Fixed cells were ,I,.,m.,..d ',~.+lh ], ~tl diluted ~ln~.'lt¢:+.,,ff ~-20-1 ( .4. B) or (~+]+.~ ( ( ' [')..,L t.'. E. phase; B. D. I.'¢:orresptmdinB fhlol~.'nk'u'll~.'¢ Optics. I~ll I00 t£111. ~0|¢ difl~'renl ~,~llu' ill (" lind 1),

293 improve the morphology of these older cultures, which become fragile with increasing age, they were fixed in 4~ paraformaldehyde prior to staining. 8-20-1 continued to show bright staining over the entire surface of cell bodies, and fibres were more intensely stained than in the younger cultures (Figs. 5A. B). 6-1-3 continued to .,,how the same patchy distribution as was observed after culture for 24 h. The stained regions of cell bodies or fibres were not in any way obviously different from unstained regions when the cells were examined under phase contrast (Fig. 5. C - F ) . The patterns of staining observed are not consistent with trivial explanations of the phenomenon, e.g. that the antibody is only staining debris. We attempted to stain both 24 h (live) and older (fixed) cultures with 7-8D2, but found only exceedingly weak fluorescence (data not shown). The possible reasons for this finding are discussed later. During the course of these studies, we also examined cultured cerebellar astrocytes, but found no staining of these cells with any of the antibodies (Fig. 6). Other flat cells in ~hese cultures which were GFAP-negative. and are probably fibroblasts or endothelial cells, were also unstained by any of the antibodies.

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Fig. 6. Cerebellar astrocyte cultures -;rained with monoclonalantibodies and rabbit anti-GFAP antibody. Live cells were stained with 1/50 di',uted ascites of the appropriate monoclonal antibody followedby a

rhodamineoconjugatedsheep anti-mouse ar.tibody. They were then fixed in 95% ethanol/5ff acetic acid and stained overnight with a 1/200 diluted rabbit anti-GFAP serum, followed by a fluorescein-conjugated swine anti-rabbit serum. Preparations were photographed under phase (A, D. G). fluoreseein (B. E. H) or rhodamine (C. b. I) optics. No staining of any flat cells with any of the monoclonal antibodies was observed. The GFAP-negative flat cells are probably a mixture of fibroblasts and endothelial cells. A - C : cells stained with o-1-3; D- F: cells stained with 7-8D2, G - I, ~ens ~tained with 8-20-1. Bar = 100/~m.

294

Characterisation of the atttigens recognised by 6-1-3 and 8-20-1 We used immunoblotting techr, iques and affinity chromatography to carry out a prelhninary characterisation of the antigens recognised by the monoclonal antibodies, Figure 7 shows the result of electrophoresing a constant a m o u n t (60 /Lg) of various tissue hcmogenates, transferring the separated proteins to nitrocellulose paper, and staining with concentrated tissue culture supernatant from 8-20-1. A single major band at 48 kilodaltons is found in "tracks loaded with homogenate of cerebellum (Fig. 7, track 1), forebrain (track 3) or spinal cord (not shown). In some gels, a doublet of b a n d s was observed, the heavier m e m b e r of the pair at 48K. the lighter at about 46K. The same pa~lern was observed when purified glycoproteins, 20/~g, prepared from either juvenile (track 10) or adult (track 11) cerebellum were used as sample. Track 2 shows the re.~ult of omitting reducing agent from the sample buffer in which ¢erebellar homogenate was solubilised. The same b a n d pattern is

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Fig. 7. Identification of the 8-20-1 antigen by immunoblotting. Crude homogenates from var;ous tissues md purified cerebellar glycoproteins were electrophoretically separated in a 10~ polyacrylamlde gel and transferred to a nitrocellulosesheet as described in Methods. The sheet was stained with 5 x concentrated g-20 1 ti.~sueculture :.upernatant and bound antibody was detected by autoradiography after incubation ill I~1 second antibody. Sixty ,ag of homogenate was applied to each of tracks i-9. Tissue identifications are (1) cerebellum. (2) cerebellum prepared in aon-reducing sample buffer, (3) forebrain, (4) heart, {5) lung. (6) liver, (7) kidney, (8) spleen. (9) thymt.,s. Twenty ag ol cerebellar glycoproteins from juvenile Ipoolcd P6-PI5) (track 10) or adult (P35) (track 11) were aKo analy~d on the same gel.

295 found as in the reduced samples, and there is no shift in the apparent molecular weigbt of the band, suggesting that the native polypeplide is not associated via disulphide bonds with any other component. Under the conditions employed for detecting the antigen in samples of brain homogenate, amigen wa.~ not detected in the same weight of homogenised lung, liver, spleen, thymus, heart or kidney. lmmunobiotting was not successful i~ identifying the antigen recognised by 6-1-3. We therefore used affinity chromatography of iodinated glycoproteins to identify the cognate antigen. The glycoproteins used for affinity chromatography were freshly prepared and iodinated in the presence of deoxycholate as described in Methods. Iodinated sample was first passed over a 4-ml column of irrevelant mouse monoclonal antibody to deplete the preparation of non-specifically bound components, and then passed over a 200-/~1 minicolumn of 6-1-3 coupled to Sepharose 4B. After extensive washing, bound components ~vere eluted by boiling the ge~ in SDS sample

Fig. 8. Identificationof the 6-1-3 antigen by affinity chromatography,lodinated cerebellarglycoproteins were subjectedto affinity chromatographyas described in lVlethods.The bound material was analysedon a 10~opolyacrylamidegel and detected by autoradiography.O) 7-8D2column,(2) 6-1-3column eluted in reducingconditions,(3) 6-1-3 column eluted in non-reducingconditions,(4) 8-18C5 control column.

296 buffer in reducing (5~ 2-mercaptoethano]/0.1 M dithiothreitol) or non-reducing conditions. A single band at 144 kilodaltons was observed when samples recovered from 6-1-3 columns were analysed on SDS gels. This band had an identical mobility in both reducing and non-reducing conditions (Fig. 8, tracks 2 and 3). As a control on the specificity of the columns, Fig. 8 shows the results obtained when the same glycoprotein samples were applied to two other monoclonal antibody affinity columns. No bands were observed in sample eluted from a 7-8D2 column, and a completely different pattern was observed in sample eluted from a column of an antibody called 8-18C5. (8-I8C5 recognises a myelin glycoprotein, and gives the same pattern on immunobiotting as it does after affinity chromatography: Linnington, Webb and Woodhams, submitted for publication.) These observations suggest that the band at 144 K is the true 6-1-3 antigen~ and that the antigen has a simple subunit structure, lacking disulphide bonds with other polypeptides. We attempted to use the same techniques to identify the 7-8D2 antigen, but without success. No bands at all were found after staining immunoblots, and no bands were seen on gel analysis of sample eluted from the affinity column (Fig. 8). Possible reasons for this failure are discussed later.

Discussion A number of monocional antibodies recognising molecules displayed a! the neuronal cell surface have been described. Eisenbarth et al. (1979) describo:! an antibody, A285, recognising a ganglioside expressed on all neurons. Cohen and Selvendren (1981) and Vulliamy et al. (1981) described a pair of antibodies which between them distinguish peripheral and central neurons, although the nature of the antigens was not investigated. Him et al. (1981) adopted the strategy of immunising with glycoproteins to produce monoclonal antibodies against a previously ci~aracterised glycoprotein of the neuronal cell surface, and Lakin and Fabre (198!) have produced r~onoclonal antibodies against human brain glycoproteins. We were interested in producing monoclonal antibodies against previously unknown or ill-defined neuronal membrane glycoproteins in order to extend our preliminary observations on the glycoproteins expressed by cultured rat cerebeliar neurons. Each of the antibodies we describe here has some particular feature of interest. 7-8D2 and 8-20-1 are both neuron-specific antigens. 7-8D2 has been found only on cerebellar granule cells, and it thus constitutes a marker for a very specific neuron subset. In contrast, 8-20-1 is neuron-specific, but is found in several different brain regions. A gradi,ent of expression of the antigen was found, and the intensity of staining diminished in more anterior regions of the brain, until the antigen was undetectable in the cortex. In all regions that express 8-20-1, the staining appears to be c,~nfined to small intrinsic neurons. Although there are limitations oti the interpretation of cell surface fluorescence on 10-1am sections, our impression L,; that large neurons are unstained in regions that do express the antigen (e.g. Purkini, cells in the cerebellum, Fig. 2B, and !arge neurons in the magnoceilular r e , o n ~,f the latera| reticular nucleus, Fig. 3B).

297 7-8D2 does not appear to be heavily expressed on cerebellar granule ceil~ until after the last mitosis, but it is expressed on migrating neurons which are only just postmitotic. 8-20-1 appears later in cerebellar development and is not detectable at PI0. It is interesting that the antigen can be demonstrated in 1-day-old neuron cultures of P8 cerebellum, when the neuron:~ are chronologically younger than the el rliest age at which the antigen can be found in vivo. It seems likely that the tissue culture conditions force the neurons in the direction of rapid differentiation, with the consequent early expression of 'mature' neuron markers. It should be noted, however, that the neurons do not achieve their full biochemical developmerJt after 24 h in vitro, as similar cultures have been observed to show progressive biochemical changes up to 7-8 days in vitro (Gallo et al. 1982, Webb 1983). In vivo, it seems that the granule neurons immediately after mitosis are 7-8D2 + 8-20-1-, and att~n the mature phenotype 7-8D2 + 8-20-1 + only after the 15th postnatal day. Although 8-20-1 could be demonstrated on young and mature neuronal cultures, 7-8D2 gave only very weak staining. In vit.w of the widespread distribution of 8-20-1 antigen on neurons throughout the brain and spinal cord, it may be that the antigen mediates a function essential to the cells that bear it, and is obligately expressed by these cells in vitro. In contrast, 7-8D2 is so specific to the cerebellar granule neurons that it does not seem likely that it mediates any basic cellular activity required for neuronal survival or activity. Such a region-specifi~ antigen would seem more likely to be involved in some aspect of the generation or maintenance of histotypic order in the cerebellum. If this is the case, we suggest that this is a 'luxury' function, similar to the differentiated functions of permanent cell lines, which can be lost by the cells without prejudicing their survival in culture. It may be that some specific cue is provided by the in vivo environment to ,~'ause expression of 7-8D2, which is lacking in vitro. 6-1-3 gave a highly unusual staining pattern on the cultured neurons. The antigen appeared to be concentrated in discrete patches on both the cell bodies and fibres in both young and mature cultures. This pattern was not artifactually induced by capping of the antigen by antibody because it was also observed in cells that were fixed prior to staining. Equally, it was not a fixation artifact, as neurons stained live after 24 h in culture gave the same pattern. A similar streaky distrib::~ion of fluorescence is seen when cells ¢xpressing fibronectin are stained (e.g. see Bayne et ai. 1983, p. 263). Although 6-1-3 antigen is not fibronectin (the 6-1-3 antigen has an apparent molecular weight of 144 I~, whereas that of fibronectin i:~ 220 K, and neurons have not been observed to express fibronectin), it may be a similar type of molecule that belongs more to the extracellular matrix than to the cell membrane proper. This would also account in part for the confusing picture given wher~ tissue sections are stained with tht~ antibody. Of all three antigens described in this paper, 6-1-3 is the least known in terms of its distribution, both throughout the brain, on different tissues, and on c',ifferent cell types. Thus, while we are confident in describing 7-8D2 and 8-20-] as neuron-specific markers, 6-1-3 is an antigen that is clearly expressed by neuron~ but it may also be on other cell types. We attempted a preliminary characterisation of the antigens recognised by these antibodies. Immunoblotting identified the 8-20-1 antigen as a 48 K polypeptide

298 expressed in cerebellum, forebrain and spinal cord. The antigen does not appear to be linked via disulphide bonds to any other polypeptide, as its migration in gels was unaffected by the presence or absence of reducing agent. 8-20-1 was enriched in a glycoprotein fraction specifically eluted from a lentil lectin column by a-methyl glucoside. This observation strongly suggests that the antigen is a glyeoprotein bearing glucose/mannose in the carbohydrate moiety. In some gels, a double band was observed. Since this was no'~ always seen, and since it was most prominent in glycoprotein fractions which had taken about 2 days to prepare, we think that the lower molecdar weight band represents a limited proteolytic breakdown product of the 48 K band. 6-1-3 antigen was identified as a 144 K polypeptide by chromatography of iodinated glycoproteins on a 6-1-3 affinity gel. Once again, the lack of effect of omitting reducing agent suggests that disulphide bands are not involved in flaking the antigen polypeptide with any other component. The fact that the antigen was detected in purifie~l glycoproteins suggests that it is a glucose/mannose containing glycoprotein. We were unable to identify the 7-8D2 antigen using these techniques. No bands at all were observed on immunoblotting or in gels of material eluted from a 7-8D2 affinity column. The failure of blotting may be because the antigen does not transfer well to nitrocellulose, but it is more likely that the molecule is denatured by the conditions of electrophoresis, and loses antigenicity. Similarly, the failure of the affinity column may be due to a loss of antigenicity in detergent. Alternatively, the antigen may not be a glycoprotein, and hence was not present in the original sample. This would seem an unlikely possibility, as at least some 7-8D2 must have been present in the original immunogen which was prepared in the same way. Future studies ,qhould addrcsz the solubility and antigenicity of 7-8D2 in various detergents as a preliminary to further attempts to identify the antigen. In conclusion, the approach of immunising with a glycoprotein fraction has yielded a number of interesting antibodies which have considerable potential for further work. In future studies, the antibodies will be of use in the more detailed biochemical characterisation of the antigens they recognise. It may also be useful to use these and other similar antibodies in attempts to perturb the in vitro behaviour of cultured cells, which may give some indication of the functions of the cognate molecules. This approach has been attempted with some success for molecules with previously Hnknown functions in lymphoid cells (Webb et al. ~979; Gallatin et al. !983), muscle ce!is (Greve and Gottlieb 1981) and in chick neurons (Rutishauser et al. 1983).

Acknowledgements We thank Dr. F. Waish for his advice and encouragement, Mr. J, McGovern for technical help, and Mrs. M. Cohen for typing the manuscript.

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