Journal of Neuroscience Research 25486502 ( 1990)

Family of Human Neuronal External Surface Epitopes Defined by Torpedo Monoclonal Antibodies S. Wright, H. Sternberg, E.K. Bjornskov, D.T. Stephenson, and P.D. Kushner ALS and Neurornuscular Research Foundation, Pacific Presbyterian Medical Center. San Francisco

We are employing a library of monoclonal antibodies (MAbs) that were made to Torpedo cholinergic synaptosomes to identify conserved, physiologically vital epitopes of the neuronal surface. Our particular interest is in those epitopes that are present on some but not all neurons. In the present study we screened this library on different cell lines, the neuronal cell lines PC12, NG108, MC-IXC, and SY5Y, and the endocrine cell lines GH-3 and HIT. Of these cell lines, only SYSY cells bind MAbs that define neuronal surface subsets. Utilizing its parent cell line, SK-N-SH, we verified that six MAbs, Tor 25, Tor 103, Tor 190, Tor 201, Tor 219, and Tor 233, bind the external neuronal surface. The cytolocalization of all six MAbs is very similar: the membrane of the cell body and its processes are finely outlined in a punctate distribution. Western blot analyses of Torpedo electric organ homogenates, a highly enriched source of antigenic material, revealed that each MAb identifies multiple polypeptides, two of which have the relative mobilities of 180 kD and 67 kD. In a screen of peripheral nerves from cases of amyotrophic lateral sclerosis (ALS), we found that all these MAbs revealed surface alterations; some displayed a decrease in binding, while others displayed an increase. The combined data provide evidence that these epitopes belong to an important, complex family of polypeptides of the external neuronal surface.

cules of the neuronal cell surface UijeS MAbs that identify evolutionarily conserved determinants. These MAbs (designated as Tor) were derived from the synaptosome preparation of the Torpedo electromotor organ and many identify nervous system components (Kushner, 1984). This choice of immunogen is unique for two reasons: I ) the Torpedo electric organ itself is derived in development from motor neurons (Fox and Richardson, 1978), and 2) the synaptosome preparation is made up of purely cholinergic nerve terminals. These attributes mean that the Tor MAbs may be especially useful in identifying both novel molecules of motor neurons as well as molecules unique to cholinergic neurons. In the Torpedo the vast majority of the Tor MAbs recognize only nervous system elements (Kushner, 1984); and some, where they have been more thoroughly investigated, identify molecules of the nerve terminal, for instance, a glycosaminoglycan of synaptic vesicles (Carlson and Kelly, 1983; Kennedy, 1985; Trimble et al., 1988). Our data have revealed that several of the Tor MAbs bind the neuronal surface in Torpedo (Buckley et al., 1983; Kushner, 1984) and mammals (rat: Kushner, 1984; Kushner and Stephenson, 1984; cat: unpublished observations; human: Bjornskov et al., 1988). The evolutionary conservation of these epitopes in a surface location suggests an involvement in fundamental properties of neurons. One group of 13 MAbs recognizes the apparent membrane of human intramuscular nerve axons (BjornKey words: Torpedo synaptosomes, SK-N-SH, sur- skov et al., 1988). Some of these MAbs identify reface polypeptides, ALS stricted groups of CNS neurons (Kushner, 1984; Kush-

INTRODUCTION Many of the unique properties of neurons (e.g., pathfinding, synaptogenesis) appear to depend upon interactions carried out by polypeptides of neural cell surfaces. Antibody technology, particularly as represented by monoclonal antibodies (MAbs), is the most powerful route to discover molecules that reside on particular cellular structures. Our approach to characterize the mole0 1990 Wiley-Liss. Inc.

Received June 2, 1989; revised November 14. 1989: accepted November 15. 1989. Address reprint requests to P.D. Kushner, ALS and Neuromuscular Research Foundation, Pacific Presbyterian Medical Center, 235 1 Clay Street, Room 416. San Francisco. CA 94.1 15.

H. Sternberg is now at University of California. Berkeley Dept. of Physiology. Berkeley. CA 94720. E.K. Bjornskov is now at Oregon Health Sciences University, Dept. of Neurology, Portland. OR 47201.

Tor MAbs Bind Mammalian Cell Lines

ner and Stephenson, 1984). One MAb in particular. Tor 23, has been mapped extensively in the rat CNS, where it binds to thc surface of a rare and precise set of neurons that have a strong but incomplete overlap with cholinergic markers (acetylcholinesterase and choline acetyltransferase Stephenson and Kushner et al., 1986; unpublished observations) and with known primary motor neurons (Stephenson et al., 1987; Stephenson and Kushner, 1988). An elucidation of the molecular machinery on the neuronal surface can advance our understanding not only of basic neuroscience, but also of conditions of neuronal dysfunctions. An important complement to the investigation of novel molecules and especially of novel. rare molecules, is a cell line that expresses these molecules. We had previously identified a human teratocarcinoma cell line that binds several of the Tor MAbs (Kushner et al., 1987a), but these cultures require long differentiation and Tor MAb immunopositive cells are never more than a fraction of total cells. Thus, we have continued to screen the Tor MAbs on other cell lines and have now identified a human neuroblastoma cell line that seems particularly suited to study the Tor MAbs and their surface antigen(s). The second part of this study is an analysis of the distribution of the surface Tor MAbs on human intramuscular nerve fibers from cases of amyotrophic lateral sclerosis (ALS). a progressive, fatal disease of motor neurons whose etiology remains unknown. Molecular alterations in the surface of ALS peripheral nerve were identified by each of these Tor MAbs.

MATERIALS AND METHODS Monoclonal Antibodies The production and isolation of the Tor MAbs used in this study have been described previously (Kushner, 1984). Tor 23 and Tor 132 were considered separately in the group of 13 MAbs described in Bjornskov et al. (1988). During the course of the present work, we determined that Tor 132 and Tor 23 bind identically by all cytological and biochemical assays; Tor 132 and Tor 23 were therefore used interchangeably.

487

mented with 5% FCS and 10% heat-inactivated horse serum. NG108 cells (Klee and Nirenberg, 1974) wcre a gift from Marshall Nirenberg and were grown in DMEH21 supplemented with 5% FCS. All culture media were supplemented with I % glutamine and 1% penicillin/ streptomycin. All cells were grown at 37°C. SK-N-SH and SY5Y cells were grown in 5% CO,; PC12 cells were grown in 7.5% CO,; NG108 cells wcre grown in 10% CO,. GH-3 cells (Furth, 1955) and HIT cells (Santerre et al., 1981) were obtained from Peter Kushner, University of California, San Francisco, and not grown but assayed as homogenates of cell pellets.

Human Biopsies Intercostal muscle biopsies were obtained from patients undergoing thoracic surgery for either pulmonary or cardiac conditions (serving as neurologically normal controls) and from ALS patients. For this study, early cases of ALS were defined as ones recently diagnosed with few physical limitations; advanced cases were defined as patients with major disabilities including respiratory muscle weakness. A total of 5 ALS cases were examined; three were early ALS and two were advanced ALS cases. A total of three control cases were examined. Radioimmune Assay (RIA) Homogenates of cells were prepared in phosphate buffered saline (PBS), 150 mM NaCl, 10 mM Na,PO,, 2 mM KCI, 1 mM KH,PO,, pH 7.4, and tested with solid phase RIA as described previously (Kushner et al., 1987b). Detecting antibodies were "'I-anti-mouse immunoglobulins (NEN-Dupont, Wilmington, DE). used at 50,000 countslminlwell.

Immunocytochemical Assays (ICA) To test cell lines, cells were grown to confluency and replated on 22 mm diameter coverslips for 1-3 days depending on plating density. For fixed cell staining, cultures were fixed for 30 min with cold 4% phosphate buffered paraformaldehyde solution (HCHO), rinsed several times with PBS, and incubated overnight at 4°C with a Tor MAb (1:lO dilution of hybridoma culture supernatant, diluted in PBS). Primary MAb binding was detected by applying fluorescein-conjugated anti-mouse Cell Lines immunoglobulins (high fluorescent product, diluted I : The SK-N-SH cell line, obtained from the Cell 100 in PBS. Antibodies, Inc., Davis, CA) for 2 hr at 22°C Culture Facility, University of California, San Fran- in the dark. To assay MAb binding to live cells. live cisco. and the SY5Y and MC-IXC cell lines, obtained cultures of SK-N-SH cells were incubated with a Tor from Paola Timeras, University of California, Berkeley, MAb (1: 10 hybridoma culture supernatant. diluted in are neuroblastomas of human origin (Biedler et al., culture medium) for 1 hr at 22°C. Cultures were subse1973). They were grown in RPMI with 10% fetal calf quently fixed by treatment with cold HCHO for 30 niin, serum (FCS). PC12 cells (Greene and Tischler, 1976) washed in PBS, and then incubated with fluoresceinwere from Louis Reichardt, University of California, conjugated anti-mouse immunoglobulins as for fixed culSan Francisco, and were grown in DME-H21 supple- ture staining. Coverslips were mounted onto microscope

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slides with 70% glycerol, sealed with nail polish, and examined with fluorescent optics on a Zeiss Ultraphot microscope. Muscle biopsies were assayed as described previously (Bjornskov et al., 1988). Briefly, muscle fascicles were mounted in gum tragacanth, flash-frozen in liquid nitrogen-cooled isopentane. cryosectioned ( I0 pm-thick sections), and mounted on gelatin-coated slides. For MAb localization, sections were hydrated for 5 min with PBS and incubated overnight at 4°C with a Tor MAb (hybridoma culture supernatant, diluted 1 : l O in PBS), and Tor MAb binding detected with fluorescein-conjugatcd antimouse immunoglobulins. Sections were mounted in 70% glycerol and examined as for cell staining. Controls for ICA included incubation of coverslips or sections with no primary antibody and with a Tor MAb that does not cross-react with the cell lines or with human tissue.

Colocalization of Neurofilament and Tor MAbs Monoclonal antibody to neurofilament subunits 70 kD and 200 kD (anti-NF, Biogenex, Dublin CA) was colocalized with Tor MAbs according to the technique of Lechago et al. ( 1 979). Intercostal muscle sections were incubated with anti-NF (overnight, 4"C), which was localized with human serum-adsorbed. peroxidase-conjugated anti-mouse immunoglobulins (Kirkegaard and Perry, Gaithersburg, MD) and developed with 0.05% diaminobenzidine (activated with 0.01% H,O,). The section was then incubated with a Tor MAb (overnight, 4°C) and subsequently treated with fluorescein-conjugated anti-mouse immunoglobulins. Sections were mounted in glycerol and viewed with tungsten and UV illumination. Controls included incubating near-adjacent sections with a single MAb to verify that one MAb did not block binding of the other. Biochemical Treatments of Human Intercostal Sections To test if the Tor MAbs bound to carbohydrate epitopes, intercostal muscle sections were treated with sodium m-periodate (NaIO,, Sigma, St. Louis, MO) according to a modified protocol of Basbaum et al. (1986). Sections were hydrated in PBS for 5 min and then the slides placed in Coplin jars containing either the experimental solution (50 mM NaIO, in PBS or 50 mM sodium acetate, pH 4.5) or the control solution (50 mM NaIO, + 0.1 M glucose in PBS or 50 mM sodium acetate, pH 4.5) and incubated for 2 hr at 4°C. The periodate solution was removed and sections were treated with 10 mM sodium borohydride (Sigma) in PBS for 30 min, to reduce any reactive aldehydes generated by the periodate cleavage. After a final rinse with PBS. sections were reacted as above with the Tor MAbs. As a

positive control for the removal of carbohydrate residues a section treated with both control and experimental solutions was incubated with fluorescein-conjugated concanavalin A (con A, Sigma). In both PBS and sodium acetate buffer, 2 hr effected near-complete loss of con A binding. We considered the loss of con A binding as indicating the removal of carbohydrate residues. To test if the Tor MAbs bound to lipids, intercostal muscle sections were treated with xylene to extract lipids and then tested for Tor MAb immunoreactivity. Frozen sections were hydrated in PBS for 5 min and the slides dehydrated through a graded series of alcohol solutions, consisting of 30%, 50%, 70%, 95%, 10096, 100% ethanol for 2 rnin each. The sections were then placed in xylene for 20 min and subsequently rehydrated through the alcohol solutions and into PBS. Sections were rinsed several times and immunostained with the Tor MAbs.

Immunoblot and Immunogel Analyses Western transfers were performed according to the method described in Burnette (1981) and have been previously described (Kushner et a]., 1989b). Torpedo electroplax homogenates were prepared as in Kushner et al. ( I987b). Briefly, SDS-PAGE separated (Laemmli, 1970) Torpedo homogenates were electrophoretically transferred to nitrocellulose paper at constant voltage for 2-4 hr in transfer buffer (Burnette. 1981). After transfer, the nitrocellulose paper was incubated in blocking buffer (1-2 hr), the blot cut into strips, and strips incubated with the different Tor MAbs overnight at 22°C (diluted 1: 10 in PBS), on a rotator. A linker antibody was applied (rabbit anti-mouse, 1 : 1.000, Zymed, South San Francisco, CA) for 2 hr at 22"C, followed by iodinated Protein A (10 pCi. NEN) for 2 hr at 22°C. Strips were exposed to X-AR film (Kodak, Rochester, N Y ) with a Dupont Cronex lightning-plus inlensifying screen. After exposure, strips were reversibly stained for total protein with 0.5% Ponceau S in 1% acetic acid solution for 5 min and destained in distilled water for 2 min, as described in Salinovich and Montelaro (19861, to allow a direct comparison of the location of immunoreactive bands relative to that of total homogenate proteins and molecular weight standards (Biorad, Richmond, CA). Immunogel analyses were performed according to the technique of Burridge (1978) and have been previously described in detail (Kushner et al., 1987b). To control for the specificity of immunobands, lanes of separated proteins were treated in parallel with all reagents except the Tor MAb.

RESULTS Screens of Cell Lines Solid phase RIAs were performed to determine the binding spectrum for the library of Tor antibodies on cell

Tor MAbs Bind Mammalian Cell Lines TABLE I. Binding Profile of Cell Lines* PC12 SY5Y NGl08 MC-IXC HIT GH3 Tor MAbc

+

+

t

+

+

+

+ -

+

+

+

t

+

-

-

+

+

-

-

t

t

+

+ +

-

-

-

+

-

-

-

+

-

-

-

-

-

+

-

-

-

-

-

' ~

+

~

~

-

~

~

~

-

~

-

18. 19, 67, 81, 91, 95. 104, 106. 172, 174.244. 352 138 75". 85, 96, 182, 190" 16, 42. 72. 74" ', 83. 86h, 89, 98". I 15.'. 140h. 144h 102".h. 237 17. 21. 32. 40, 44, 49. 59, 68, 71, 122. 129. 196" 25. 28. 29. 55. 126. 170. 232, 233 33 38. 63. 99. 205. 269

~~

*Results from solid phase RlAa of Tor MAbs on cell lines. A signal of I.5 x background was the minimum considered as a positive signal. Signals ranged from 1.5 to 18 X background. "MAb binds HIT cell line also. hMAh binds GH-3 cell line also. 'Result confirmed in subsequent assays of PC 12. undifferentiated and differentiated (Fujii, personal communication).

489

binding and 2) specificity of this binding to the apparent plasma membrane. The MAbs Tor 25 and Tor 233 were applied to fixed cultures of SY5Y cells that had been grown on glass coverslips. Binding of Tor 25 and Tor 233 was to the apparent neuronal membrane (data not shown). At this point we decided to utilize the parent cell line of the SYSY, SK-N-SH cells, because the parent cell line may be more inclusive and the derivative cell line more restricted in phenotypic expression. Clonally derived SK-N-SH cells express two distinct cell phenotypes: a neuronal-appearing cell and a large flat cell. The neuronal cell has little cytoplasm and often forms extensive processes extending out from the cell body. SKN-SH cultures have biochemical properties of neuronal cells (Biedler et a]., 1978), and the two cell types can interconvert spontaneously (Ross et al., 1983). Figure I , a micrograph of a Nissl stained culture of SK-N-SH cells, illustrates the complex growth patterns of these cells.

Cell Surface Staining of the SK-N-SH Cell Line SK-N-SH cells were tested by ICA with each of the 13 Tor MAbs that had been found to bind components of human intramuscular nerves (Bjornskov et al., 1988). Six Tor MAbs, Tor 25. Tor 103, Tor 190,Tor 201, Tor lines: neuronal cells PC12, NG108, SYSY, and MC-IXC 219. and Tor 233, bound the neuronal cells of the SKand endocrine cells HIT and GH-3. Table I details which N-SH cultures, while seven MAbs, Tor 23, Tor 58, Tor Tor MAbs bound each cell line. In total. 60 out of 136 70,Tor 75, Tor 132, Tor 145, and Tor 232, did not bind Tor-MAbs tested cross-reacted with at least one cell line. these cultures. Fine punctate immunostaining was The number of Tor MAbs bound relative to the total present all over the cell bodies and their processes. To number of MAbs tested for the individual cell lines was verify unambiguously which MAbs bound externally ori451136 (PC12), 411136 (SYSY), 191136 (NG108), 221 ented surface epitopes, live cultures of SK-N-SH were 117 (MC-IXC) 161136 (GH-3). and 201136 (HIT). tested for MAb binding. By this assay, all six of the Twelve MAbs bound all of the cell lines. Many of these positive MAbs bound the external surface of neuronal 12 MAbs are likely either to recognize common proteins cells of SK-N-SH cultures. Figures 2 and 3 present mior to bind proteins non-specifically: four of these MAbs crographs of SK-N-SH cells incubated with Tor MAbs, bound homogenates of the Torpedo liver in the original while cells were alive (Figs. 2A,C, 3) and incubated with screens (Kushner, 1984); eight displayed a high binding Tor MAbs after cells had been fixed (Fig. 2B,D). All six signal when tested with RIA on a mixture of common antibodies positive on the live cell cultures stained the proteins (data not shown). Other Tor MAbs bound spe- external plasma membrane in a similar manner: they discific cell lines solely or in combinations, although no played a brightly fluorescent outline along the perimeter MAbs bound only the endocrine cell lines or only all of of the cell soma and its processes. There was often more the neuronal cell lines. Twelve MAbs were identified intense staining where cells adjoined. At higher power that recognize only PC12 cells. Five MAbs bound only this staining was fine and punctate over both the cell the MC-IXC cell line. Among the MAbs that only bound soma (en face) and the processes. Staining on fixed cells the SYSY cells were Tor 25 and Tor 233, 2 of the 13 was brighter (compare Fig. 2A and B), and all MAbs that MAbs that had been shown previously to cross-react with bound fixed cells also bound live cells. In summary. the the apparent surface of human intramuscular nerves six Tor MAbs bind the veritable external surface and in (Bjornskov et al., 1988). MAbs that identify molecules combination with Tor-23 (Kushner et al., 1987a) comof the axolemma of human neurons are of specific inter- pose a group of 7 Tor- MAbs that identify molecules of est to us; therefore, the SYSY cell line was selected for the surface of human neurons. further studies. To verify the specificity of surface staining. we As an extension of the RIA analysis, MAb binding tested Tor 244, which binds a component internal to the was tested with ICA. ICA served to confirm 1) Tor MAb muscle endplate (Bjornskov et al., 1988). In cultures of

490

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Fig. I . Nissl stained culture of SK-N-SH demonstrates the two cell types present: a large epithelial-like cell (small arrows) and a smaller, spiny, neuronal-like cell (large arrows). For the most part the neuronal cells are stained darkly and the flat cells are stained lightly. Both cell types grow individually and in clus-

ters. The neuronal cell type extends fine branched processes (large arrowheads). In contrast the flat cells have large bulbous extensions that sometimes end in fine processes (small arrowheads). Scale bar = 10 km. The magnification is the same for Figs. 1-3.

SK-N-SH cells, Tor 244 recognized only fixed and not live cells (Fig. 2C,D). This result further substantiated the specificity of the surface binding seen with the other six Tor MAbs. The micrograph of Tor 244 on live cells also serves to illustrate the absence of second antibody binding and hence the specificity of the primary antibody in these experiments (Fig. 2C). Because the SK-N-SH cells express two cell types (see Fig. I ) , binding to cells with neuronal versus non-

Biochemical Analyses of the Tor Antigens

neuronal phenotypes was assessed. The large, flat, nonneuronal cells did not appear to stain with any of the surface-binding Tor MAbs. In contrast, most. if not all, of the neuronal cells on a coverslip were positive for binding of the surface MAbs (see Figs. 2A, 3). As control for the specificity of imniunostaining to particular cell phenotypes, Tor 244 in the fixed cell preparation stained the cytoplasm and processes of both cell types although the flat cell was stained less intensely than the neuronal cell.

Immunoblot and immunogel analyses were performed to determine whether the seven surface MAbs

Fig. 2. Fluorescent Tor MAb localization on live (A,C) and fixed (B,D) SK-N-SH cells. A: Tor. 219 binding to live cells in the whole mount finely outlines the perimeter of the cell soma and its processes; binding appears punctate en face. ln cultures, most of the neuronal cells are positive and staining is often brighter where two cells adjoin. B : Tor 219 staining of fixed cells is brighter and more punctate than in the live cell preparation and is present all over the cell body and its processes. Cytoplasmic staining is above that found in the live cell preparation; the nuclei are not immunostained. C: Tor 244, a MAb that binds human muscle. does not stain live cultures. D: Tor. 244 binds intracellularly to both cell types in fixed SK-N-SH cultures .

Tor MAbs Bind Mammalian Cell Lines

491

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Wright et al.

Screen of ALS Intramuscular Nerve bound polypeptide antigens. For this analysis, the Torpedo electric organ was used as a highly enriched source of antigen. Each of the seven Tor MAbs that bound the external surface of the SK-N-SH cells recognized multiple polypeptide bands of SDS-PAGE separated Torpedo electric organ homogenates. Figure 4 presents an autoradiogram of an iinmunoblot whose individual lanes were probed with different Tor MAbs. These seven Tor MAbs all bound two polypeptide bands at 180 kD and 67 kD, which are the molecular weights previously determined for polypeptide antigens identified by Tor 23 (Kushner et al.. 1987b). Tor 23 and Tor 25 bound only those two bands, although with Tor 23 the 180 kD band strongly predominated, whereas with Tor 25 the two bands were more equal (see Fig. 4, lanes 1 and 7, respectively). Tor 103 bound the two common bands with the relative density seen with Tor 23; in addition Tor 103 identified a third band, less intensely, at 94 kD (data not shown). Tor 190, Tor 201, Tor 219, and Tor 233 recognized the two common bands, another band at approximately 41 kD, and an additional broad band that ran from below the 180 kD band to above the 67 kD band (Fig. 4, lanes 2-5). By these analyses Tor 23 and Tor 25 have the most restricted polypeptide specificity. The band at 67 kD we have identified as a presynaptic form of Torpedo acetylcholinesterase (Kushner et al., 1987b). The identity of the 180 kD band is not yet known, although it does share specific biophysical properties with the 67 kD species (Kushner et al.. 1987b). The identity of the other bands has not been established. The fact that each of the MAbs identify bands at 180 and 67 kD is evidence in favor of the antigen(s) being related. To determine whether the epitopes defined by the Tor MAbs in human nerves are carbohydrates or lipids, sections of human intramuscular nerve were treated with sodium periodate to effect a cleavage of virtually all carbohydrates and with xylene to remove lipids. Treated sections were then immunostained with the Tor MAbs. Binding of the MAbs, Tor 23, Tor 25, Tor 103. Tor 190, Tor 20 I , Tor 2 19,and Tor 233, persisted after treatment with sodium periodate with the same intensity and distribution as with no treatment (data not shown). Since no differences in MAb distribution were found after periodate treatment, there were no epitopes obscured by carbohydrate residues that were present elsewhere (e.g., on myofibers). Delipidification by xylene treatment caused some morphological alterations in the nerve; all MAbs, however, continued to bind with this treatment although all appeared to have an increased binding (data not shown). By these criteria the epitopes within human nerves defined by the above Tor MAbs are not of a carbohydrate or a lipid nature and thus must be proteinous.

A delineation of molecules at the surface of peripheral motor nerves in ALS may reveal specific, molecular alterations responsible for motor neuron dysfunction. Thus, the binding of the surface Tor MAbs was analyzed on human intramuscular nerves from cases of ALS. These Tor MAbs, Tor 23, Tor 25, Tor 103, Tor 190, Tor 201, Tor 2 19, and Tor 233, displayed alterations in binding to ALS nerve fibers relative to the distribution present in normal control tissue. The most striking alteration in ALS peripheral nerve was reduced or lost immunoreactivity, observed with three of the MAbs, Tor 23, Tor 25, Tor 103. Four of the MAbs, Tor 190, Tor 201, Tor 219, Tor 233, displayed an apparent increase in immunoreactivity. In ALS, as in normal tissue (Bjornskov et al., 1988), binding was never observed on any component of muscle. including myofibers or the myotendinous junction. Tor 23 binding in ALS nerve relative to neurologically normal control tissue is presented in Figure 5 . The ALS nerve bundles displayed many unstained, dark holes that appeared to be areas of absent nerve fibers or fibers that did not express the Tor 23 epitope. On fibers that bind Tor 23, binding was present along the surface of the Schwann cell (Fig. 5C) and was sometimes but not always present on the axon (Fig. 5A). To ascertain if alterations in peripheral nerve could be detected prior to respiratory impairment (with presumably little intercostal nerve degeneration), we examined tissue from several cases of ALS taken at the time of diagnosis (early ALS). Within the same tissue sample, the distribution of the Tor 23 epitope was both normally appearing in some nerve bundles and abnormally appearing in others (data not shown). The bundles that were stained abnormally manifested changes similar to those present in the advanced cases, i.e., fibers appeared to be missing and there was an absence of binding to the axolemma. An alteration observed in early ALS not seen in advanced ALS was an increase of immunoreactivity inside a few fibers; this increase occurred primarily in medium-sized bundles from the early ALS cases that often contained some fibers that appeared swollen or enlarged. In summary, Tor 23 binding to peripheral nerve bundles of both early and advanced ALS cases displayed the general morphological staining characteristics seen in tissue from normal control cases but specific alterations of binding were present. These alterations were more dramatic in tissue from advanced cases of ALS. MAb Tor 25 bound similarly to Tor 23 in all cases tested. Tor 103 also displayed a diminution of binding in nerve fibers from ALS cases similar to that of Tor 23 (Fig. 6A). Tor 103 binding was unique in the following respects: 1 ) in all cases binding was more intense and

Tor MAbs Bind Mammalian Cell Lines

Fig. 3. Immunofluorescent localization of Tor 233 (A), Tor 190 (B), Tor 103 (C), and Tor 25 (D) on live SK-N-SH cultures. Immunopositive cells display fluorescence that outlines the perimeter of cells and at close observation appears punctate. All MAbs that are positive stain with a similar pattern and on the same cell type, namely the neuronal cell.

493

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Fig. 4. Immunoblot analysis of the surface Tor MAbs. Homogenates of Torpedo electroplax were separated electrophoretically on a 9% polyacrylamide minigel and transferred to nitrocellulose. Lane 1: Tor 23. Lane 2: Tor 233. Lane 3: Tor 219. Lane 4: Tor 201. Lane 5: Tor 190. Lane 6: culture medium without antibody. Lane 7: Tor 25. All Tor MAbs recognize bands at 180 kD and 68 kD (filled arrows). For Tor

23 the major band is 180 kD. Tor 23 and Tor 25 recognize only those two bands, while Tor 190. Tor 201, Tor 219, Tor 233 identify another band at 4 I kD (open arrow) and a broad area between the 180 kD and 68 kD bands. The latter density could represent degradative products of the 180 kD species. Mobilities of molecular weight standards are indicated at the left. Lanes 2-5 are a shorter exposure of the autoradiogram.

more irregular with Tor 103 than with Tor 23 (compare Figs. 5A and 6A); 2) in advanced ALS the endoneurial region, which was negative with Tor 23, had some Tor 103 immunoreactivity (Fig. 6A); and 3) in early ALS cases Tor 103 binding to nerve bundles was similar to normal control tissue while Tor 23 binding was altered (data not shown). As a contrast to Tor 103 binding, Figure 6B presents an ALS nerve in cross section stained with Tor 2 19, an antibody whose binding was increased in advanced ALS cases. In advanced ALS, Tor 219 binding was distinctly increased in the endoneurial region of both large and small nerve bundles (Fig. 6B). The contrast between Tor 2 I9 and Tor 103 in advanced ALS was all the more striking because in normal tissue (Bjornskov et al., 1988) and in early ALS (data not shown), the distribution of the two MAbs was similar if not the same. Tor 233 bound like Tor 219. The MAbs Tor 190 and Tor 20 1 had an even greater increase of binding in nerves from advanced ALS cases than did Tor 219 and Tor 233. There was an accumulation of Tor 190 immunoreactivity especially in large fibers at the edge of the nerve bundle (Fig. 6C). Tor 201 bound with increased density on the Schwann cell but often with an absence of the internal axonal stain (Fig. 6D). There were regions within a bundle that lacked

MAb binding as seen in Tor 23 stained sections. Alterations of binding were all the more apparent when neighboring sections were compared. Figures 6A-D are micrographs of near-adjacent sections. To evaluate the specificit,y of the Tor MAb alterations, Tor MAb distribution was analyzed relative to Fig. 5. Fluorescent Tor 23 localization in peripheral nerve bundles of intercostal muscle from an advanced case of ALS (A,C) and from neurologically normal (B,D) cases, in cross ( A , B ) and longitudinal (C.D) sections. A: Tor 23 binding in ALS peripheral nerve is completely absent from some fibers and is present only on the external Schwann cell surface on others. lmmunopositive fibers stain unevenly. B: In contrast. Tor 23 binding to control nerve bundles precisely outlines each fiber in a double ring of stain with binding being somewhat brighter on the outer ring of stain than the internal ring. C: An ALS nerve bundle cut in a partial longitudinal aspect confirms the uneven distribution of Tor 23 and reveals that MAb binding is present all along individual immunopositive fibers (arrows). D: In a section of control peripheral nerve cut longitudinally. Tor 23 binding appears evenly distributed along the Schwann cell surface of individual fibers and a presumptive node of Ranvier (arrow). E: A section of ALS peripheral nerve incubated with an antibody that does not bind large nerve bundles (Tor58) demonstrates the binding specificity of Tor 23. Yellow deposits are autofluorescent lipofuscin. Scale bar 10 p n .

Tor MAbs Bind Mammalian Cell Lines

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TABLE 11. Binding Profile of Tor MAbs* Tor MAb

SK-N-SHIICA Control PNllCA ALS PNIICA MW

23

25

103

-

+

+

I

1

II

Dec

Dec

Dec

A

A

B

190

201

219

233

+

+

+

+

111

Ill

II

11

Inc

Inc

Inc

Inc

C

C

C

C

*Summary of the binding profile of the Tor MAbs. Results from the biochemical and ininiunocytochemical analyses are presented for each of the seven Tor MAbs examined. Imniunocytochemical analysis (ICA) of SK-N-SH cells indicates two groups of MAbs: those that bind the cells ( + ) and one that is negative ( - ) . ICA ot' control peripheral nerve (PN) indicates three general staining patterns: I. uniform stain on an inner and outer ring of nerve fibers: 11, like I but in addition stains the presumptive Schwann cell nucleus of some fibers; and I I I , stains the Schwann cell predominantly. ICA of ALS PN indicates two subdivisions. MAbs that have decreased binding (Dec) and MAbs that have increased binding (Inc). Interestingly. MAbs in group 1 are all Dec in ALS PN. MAbs in group Ill are all Inc. whereas group 11 had one MAb that is Dec and two MAbs that are Inc. Molecular weight analysis ( M W ) demonstrates three groups: A , the most restrictive recognition (binding only 2 bands): B. identification of 3 bands; and C . identification of multiple bands. By these analyses, Tor 23. Tor 2 5 , Tor 103. Tor 190. and Tor 201 are unique; Tor 219 and Tor 233 could be the s a n e MAb.

morphology. Individual nerve fibers were viewed with Nomarski optics and the morphology examined and cornpared to Tor MAb binding. Figure 7A.B present micrographs of the same tissue section photographed with Nomarski optics t o visualize the myelin sheaths (A) and with fluorescent optics to reveal Tor 23 binding (B). Myelin was seen to be present and normal appearing in at least some fibers that completely lacked Tor MAb binding. Secondly. as a monitor of axon integrity. anti-NF was colocalized with the Tor MAbs on the same section and the distribution of each compared. Tor 23 colocalized with anti-NF demonstrated that Tor 23 binding was lost in some areas (Fig. 7C,E) where neurofilament immunoreactivity was present (Fig. 7D,E). This result verified the specificity of the loss of Tor 23 immunoreactivity . To assess the overall integrity of the ALS nerve bundle. sections colabelled with anti-NF and the Tor MAbs were cxamined and the number of fibers that were doubly and singly labelled counted. A percentage of coincidence was calculated by dividing the number of fibers containing both markers by the estimated number of total fibers present. The MAbs used for quantitative colocalizations were Tor 23, Tor 103. and Tor 201 ; the first two are MAbs that were decreased in binding in advanced ALS; and the third, one that was increased. Each of these MAbs was analyzed on sections from control,

early ALS, and advanced ALS cases. Results from this analysis are presented in Figure 8. In control nerve bundles most fibers were positive -for the Tor MAbs and anti-NF. In early ALS the number of nerve fibers with coincident binding was somewhat decreased with all three Tor MAbs. In advanced ALS, as expected, the coincident binding was markedly reduced with Tor 23 and Tor 103. Coincident binding with Tor 201 likewise was reduced but less dramatically. 'Thus, all Tor MAbs displayed a loss of binding relative to the presence of NF. There were regions with dense NF staining and no Tor MAb binding (Tor 23, Fig. 7E). The existence of these areas accounted for the reduced coincident staining observed in the advanced ALS cases.

Summary Table I1 suminarizes the characterization of the binding of the Tor MAbs. Data collected within this table provide evidence that these MAbs identify at least six unique epitopes; Tor 219 and Tor 233 may identify the same epitope.

DISCUSSION This study reports on a group of epitopes highly conserved in evolution that by biochemical analyses are on polypeptides and by cytolocalizations reside on the external neuronal surface. The comparison of MAb binding to different types of mammalian cell lines constitutes a screen that can reveal the specificity of the MAb. In this regard. we tested a combination of neuronal and endocrine cell lines. We determined that nearly one-half of the Tor MAbs from the original Tor library bound at least one of the niammalian cell lines tested. Those MAbs that in our screens bound all cell lines correspond to those that by other assays have the least specificity for the nervous system. Other MAbs bound specific cell lines solely or in combinations. These MAbs are of particular interest. For example, the 12 Tor MAbs that recognize only PC 12 cells, a cell line widely used in neural investigations, have obvious potential applications. Likewise, those MAbs that bind only the MC-IXC, a human neuroblastonia with cholinergic properties (Biedler et al., 1978). may serve to identify features of cholinergic metabolism. Several Tor MAbs that localize to the apparent neuronal surface in the mammalian nervous system bound the SYSY cell line. Therefore. this line and its parent cell line, SK-N-SH, were used for further studies. By the assay of live cell staining, 6 of the 13 MAbs that previously had been found to bind the apparent surface of human peripheral nerves (Bjornskov et al., 1988) bind the external portion of the plasma membrane. In combination with our previous finding that Tor 23 binds the external surface of neurons in cultures of a

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human teratocarcinoma (Kushner et al., 1987a), a total of 7 MAbs of the 13 that recognize components of human peripheral nerve bind the plasma membrane on its external surface. Given our postulate of evolutionary conservation, it is perhaps significant that the cells we deemed the most important to focus on are of human origin and are phylogenetically far removed from the class of our immunogen, elasmobranch. In the cultures of SK-N-SH cells the neuronal specificity of Tor MAb binding was confirmed. The cell type that was stained was the neuronal cell and not the flat cell. Likewise in our previous study of the complex mixture of teratocarcinoma cells, staining was to neuronal cells (Kushner, et al., 1987a). Thus these Tor MAbs are of interest only for their evolutionary cross-reactivity and surface localization but also for their neuronal specificity. We found it unusual that Tor 23 does not bind any of the cell lines. We did find, however, that Tor 23 does bind neuronal cells in the teratocarcinoma cultures after extensive differentiation (Kushner et al., 1987a). Interestingly, we have recently identified parameters of differentiation whereby Tor 23 also binds neuronal cells of SK-N-SH cultures (Kushner et al., 1989a). These experiments suggest that the expression of the Tor 23 antigen depends upon a high degree of neuronal differentiation and confirm that the SK-N-SH cell line is particularly valuable for the investigation of the Tor family of neuronal surface antigens. Evidence that this group of Tor MAbs recognizes a family of surface polypeptides is that 1) each of these MAbs binds cytologically to externally oriented epitopes of the plasma membrane and 2) each of the MAbs recognizes two common polypeptides by immunoblot analyses. Importantly, each of the epitopes recognized by the seven MAbs is proteinous, as determined by analyses conducted on tissue sections. The Tor MAbs do not identify the same epitope as evidenced by the different bio-

Fig. 6. Tor MAb binding in near-adjacent sections of a nerve bundle from an advanced ALS case. A: Tor 103. B: Tor219. C: Tor 190.D: Tor 201. A: Tor 103 staining is decreased both in the number of immunopositive fibers and in the loss of axolemma binding. There are regions of faint staining in the endoneurium (arrow). B: Tor 219 staining is increased; staining around individual nerve fibers is less distinct than in control tissue, but there is increased staining in the endoneurium (arrow). c: Tor 190 binding reveals a heavier deposition of stain around the Schwann cell as well as an increase in punctate stain in the endoneurium (arrow). D: Tor 201 binding is also increased around the Schwann cell; the axolemma appears, however, to have lost immunoreactivity and a few fibers appear to be completely immunonegative (arrow). Scale bar = 10 p m .

chemical and histochemical binding patterns (Table 11). The cumulative data indicate that none of the antigen(s) is any of the known molecules of peripheral nerve, e.g., neural cell adhesion molecule (N-CAM) (Cashman et al., 1987), laminin (Sanes, 1982), or heparin sulfate proteoglycan (Eldridge et al., 1986). Future studies will employ the SK-N-SH cell line to study this polypeptide family. The conserved cross-reactivity , from cartilaginous fish to humans, implies that this polypeptide family is vital to neuronal function. The alterations in MAb binding we find in ALS nerves support this notion. These alterations in ALS are of two kinds, a reduction in MAb binding and an increase in MAb binding. This result clearly illustrates the complexity of this polypeptide family. Furthermore, our findings indicate that 1) the loss of motor neurons in ALS involves a loss of integrity of the nerve membrane; 2) the loss of nerve membrane integrity in ALS is not simply a global loss but rather a selected loss, whereby some components are lost, while others persist or even are amplified; and 3) this particular molecular family defined by these Tur MAbs is involved in the disease process, although the specificity of these alterations to ALS has not yet been addressed. In ALS, the Tor MAbs bind exclusively to nerve fibers, unlike NCAM, which also localizes to muscle fibers (Cashman et al., 1987). Alterations in ALS peripheral nerve bundles have been described previously (Wohlfart, 1957; Bradley et al., 1983); we have documented a loss of surface epitopes even when the axon is apparently intact, as judged by the presence of neurofilaments, both individually on nerve fibers and statistically on whole nerve bundles. In a portion of fibers Zor MAb binding was present on the Schwann cell while absent from the axon. This result provides further confirmation that the primary defect in ALS is neuronal. Our study provides a descriptive framework to analyze tissue from other conditions of peripheral nerve degeneration when such tissue becomes available. One human tissue we would particularly like to test is from cases of ALS prior to any severe motor symptoms or from relatives of patients with familial ALS, since subtle alterations in Tor MAb binding that we have seen present in early stages of the disease may exist even prior to clinical symptoms. Animal systems to model regenerative and degenerative processes would be helpful. Ideally we would use the rat, which we have used in CNS investigations. These Tor MAbs are murine-derived and unfortunately do not appear to cross-react with rat peripheral nerve (unpublished observations). Therefore, to complement our in vitro analyses with the SK-N-SH cells, we are screening other anim.als for Tor MAb crossreactivity in the periphery to develop an in situ model system.

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Tor 132

90

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Tor 103

' i i -

X

I 70

tI

-

f

I

X

50

-

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Fig. 8. Coincidence of Tor MAb positive fibers and anti-NF binding. A histogram of the percentage of coincidence of Tor MAb inmunopositive fiber5 and anti-NF binding on peripheral nerve from control, early. and advanced (late) ALS cases. On sections that were costained, nerve bundles in cross section were examined. and the fibers immunopositive for both antiNF and Tor MAb were counted. A value for total number of

fibers present was obtained by adding coincident fibers to fibers that were only Tor positive and fibers that were only anti-NF positive. Percentage values were calculated by dividing the number of coincident fibers by the number of total fibers. X=data points; .=mean of all points. The vertical line represents the standard deviation. Each point represents up to five separate determinations.

Fig. 7. Localization of Tor 23 on sections of a nerve bundle from an advanced ALS case compared to other morphological parameters. A and B are micrographs from the same section stained with Tor 23 viewed under Nomarski optics (A) to reveal myelin sheaths and under fluorescent optics (B) to reveal MAb immunoreactivity. Of two neighboring myelinated fibers (small arrows, A) one displays Tor 23 binding while the other does not (small arrows. B). C (Tor 23), D (anti-NF), and E (double exposure of Tor 23 and anti-NF) present colocalization of Tor 23 and anti-NF on the same section of peripheral nerve stained with anti-NF with a peroxidase label and secondly for Tor binding with a fluorescent label. Sections were viewed with fluorescent (C), Koehler (D), and combined optics (E). Tor 23 staining (C.E) on peripheral nerve reveals regions of dark unstained holes. Anti-NF localized to the same section (D.E) is present in large axons and in dense clusters of smaller axons. The double exposure (E) shows areas of coincident binding (filled arrow) in addition to areas of either Tor binding alone (curved arrow) or N F binding alone (open arrow). A and B are the same magnification; C-E are the same magnification. Scale bar = 10 k m .

As a final note, we have seen MAb binding to the surface of the axon and Schwann cell with most of these MAbs. The presence of surface epitopes on both the neuron and its ensheathing Schwann cell supports the current idea of a complex and intimate interaction between these two classes of cells. While the axon and Schwann cell must have molecular specializations that serve their separate roles, they also have some features in common, including ion channels (rev. by Gray and Ritchie, 1985), releasable transmitters (Dennis and Miledi, 1974; Minchin and Iversen, 1974), and nerve growth factor receptor. present on Schwann cells during development (Scarpini et al., 1988), after axotomy (Taniuchi et al., 1986, 1988), and in human axonal neuropathies (Sobue et al., 1988). These findings of neuronal specializations of glia are indicative of an important, potentiating role of glia in neural activity. Interestingly, we have observed that this phenomenon of shared epitopes between neuronal and glial cells is not limited to PNS tissue but is also seen in the CNS. In a

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study of the distribution of Tor 23 in the human isocortex we have determined that this MAb binds the apparent limiting membrane of rare and select neurons of the human isocortex as well as a discrete population of astrocytes within the subcortical white matter (Kushner et al., 1989b). Further analysis of the cellular function of the antigens recognized by the Tor surface MAbs could lead to a better understanding of 1 ) interactions between glial and neuronal cells and 2) what roles these molecules play in neurodegenerative disorders, especially those involving cholinergic neurons, ALS and Alzheimer’s disease.

ACKNOWLEDGMENTS This project was supported by the State of California, Department of Health Services; the ALS and Neuromuscular Research Foundation, San Francisco; the ALS Society of America; and the NFL Charities, New York. The authors gratefully acknowledge Dr. Forbes H. Norris and Evelyn Nave Garrett for helpful discussions and criticisms.

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Scarpini E, Ross AH, Rosen JL, Brown MJ, Rostami A, Koprowski H , Lisak RP (1988): Expression of nerve growth factor receptor during human peripheral nerve development. Dev Biol 125: 301-3 10. Sobue G, Yasuda T, Mitsuma T. Ross AH, Pleasure D (1988): Expression of nerve growth factor receptor in human peripheral neuropathies. Ann Neurol 24:64-72. Stephenson DT, Kushner PD (1986): A set of limbic and motor neurons in the rat brain defined by Tor 23. Neurosci Abst 12906. Stephenson DT, Kushner PD (1988): An atlas of a rare neuronal surface antigen in the rat central nervous system. J Neurosci 83035 -3056. Stephenson DT. St John PA. Barker JL, Kushner PD (1987): A cell surface rpitope which is present on some but not all motor neurons. Neurosci Abst 13: 1396.

Taniuchi M. Clark HB, Johnson EM, Jr (1986): Induction of nerve growth factor receptor in Schwann cells after axotomy. Proc Natl Acad Sci USA 83:4094-4098. Taniuchi M, Clark HB. Schweitzer JB. Johnson EM. Jr (1988): Expression of nerve growth factor receptors by Schwann cells of axotomired peripheral nerves: lJhdstructural location, suppression by axonal contact, and binding properties. J Neurosci 8:664-68 I . Trimble WS, Cowan DM. Scheller RH (1988): VAMP-I: A synaptic vesicle-associated integral membrane protein. Proc Natl Acad Sci USA 85:4538-4541. Wohlfart G ( 1957): Collateral regeneration from residual motor nerve fibers in amyotrophic lateral sclerosis. Neurology 7: 124-134.

Family of human neuronal external surface epitopes defined by Torpedo monoclonal antibodies.

We are employing a library of monoclonal antibodies (MAbs) that were made to Torpedo cholinergic synaptosomes to identify conserved, physiologically v...
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