Inhibition of PC12 Cell Attachment and Neurite Outgrowth by Detergent Solubilized CNS Myelin Proteins

European Journal of Neuroscience, Vol. 7, pp. 2524-2529, 199.5 0 European Neuroscience Association

SHORT COMMUNICATION Inhibition of PCI 2 Cell Attachment and Neurite Outgrowth by Detergent Solubilized CNS Myelin Proteins

Beatrix P. Rubin', Adrian A. Spillmann', Christine E. Bandtlow', Rainer Hillenbrand3, Flavio Kelles and Martin E. Schwab' 'Brain Research Institute, University of Zurich, August Forelstrasse 1 , 8029 Zurich, Switzerland *Present address: Laboratory of Neuroscience, Libero lstituto Universitario Campus Bio-Medico, 001 55 Roma, Italy 3Department of Neurobiology, Swiss Federal Institute of Technology, Honggerberg, 8093 Zurich, Switzerland

Keywords: regeneration, adhesion, substrate, myelin associated neurite growth inhibitors, mouse

Abstract

Adhesion and neurite outgrowth of PC12 cells, as well as the spreading of 3T3 fibroblasts, were inhibited in a dose dependent manner by detergent solubilized mouse central nervous system myelin proteins as a tissue culture substrate. These inhibitory effects could be neutralized by the monoclonal antibody IN-1 directed against the neurite growth inhibiting proteins NI-35 and NI-250. Separation of the detergent soluble proteins of bovine spinal cord by an anion exchange column showed that the peaks of inhibitory activity for the two cell lines overlapped, such that the PC12 cells were inhibited by a larger number of fractions comprising those inhibitory for 3T3 cells. Neurite outgrowth of PC12 cells was not influenced by the myelin associated glycoprotein, MAG.

Introduction In contrast to lesioned peripheral nerves, adult central nervous system (CNS) tissue is a non-permissive environment for neurite growth and regeneration (David and Aguayo, 1981; Schwab and Thoenen, 1985; reviewed in Schwab et al., 1993). If neuronal cells are seeded onto cryostat sections of CNS tissue in vitro, adhesion and neurite outgrowth are poor, especially on densely myelinated areas (Carbonetto et al., 1987; Crutcher, 1989; Savio and Schwab, 1989; Watanabe and Murakami, 1990; Sagot et al., 1991; Tuttle and Matthew, 1991). A strong inhibitory effect on adhesion and neurite outgrowth is exerted by cultured oligodendrocytes and by isolated CNS myelin membrane fractions which have been adsorbed onto culture dishes (Caroni and Schwab, 1988a; Schwab and Caroni, 1988; Fawcett et al., 1989; Bandtlow et al., 1990; McKerracher et al., 1994). It has been shown that, apart from primary neurons and neuroblastoma cells, 3T3 fibroblasts also respond by a lack of spreading to the presence of CNS myelin (Caroni and Schwab, 1988a; Schwab and Caroni, 1988). After separation of rat myelin proteins by SDS gel electrophoresis and reconstitution of the eluted proteins into liposomes, two fractions of mol. wt 35 and 250 kDa have been shown to inhibit neurite growth and to cause collapse of neuronal growth cones in vitro (Caroni and Schwab, 1988a; Bandtlow et al., 1993; Igarashi et al., 1993).

A monoclonal antibody (mAb), IN-I, was raised against the 250 kDa protein fraction and selected for its ability to neutralize the inhibitory effect of CNS myelin and the isolated protein fractions (NI-35/NI-250) on cell spreading and neurite outgrowth in vitro (Caroni and Schwab, 1988b). This antibody also allowed the regenera-

tion of lesioned nerve fibres in the adult spinal cord, brain and optic nerve over long distances (Schnell and Schwab, 1990, 1993; Cadelli and Schwab, 1991; Schnell et al., 1994; Weibel et al., 1994).

Here we demonstrate that NGF-primed PC12 cells were inhibited in their adhesion and in the production of neurites by myelin proteins which had been solubilized by a zwitterionic detergent. The inhibition of neurite outgrowth of the PC 12 cells was comparable to the inability of 3T3 fibroblasts to spread. Both types of inhibition could be neutralized to a large degree by the mAb IN-I. Hence PC12 cells can be used to test for the presence of inhibitory proteins during large-scale purification of the neurite growth inhibiting proteins using detergent extracts of CNS myelin as a starting material.

Materials and methods Myelin fractions from mouse spinal cord were prepared according to the protocol of Colman et al. (1982), as described in Caroni and Schwab ( 1988a). Thz myelin fractions were subsequently centrifuged at 100 000 g for 1 h at 4°C. Extraction buffer (20 mM Tris-HCI pH 8.0, 60 mM CHAPS (3-[(3-cholamidopropyl)-dimethlammonio]- 1 - propane-sulfonate), 100 mM Na2S04, 1 mM EDTA and protease inhibitors: 2.5 mM iodacetamide, 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.1 pg/ml aprotinin, 2.5 pM betstatin, 1 pglml leupeptin, I pg/ml pepstatin A) was added at a ratio of 2:l to the pellet. The pellet was homogenized and extracted on a rotary shaker for 10 min at 4°C. The homogenate was centrifuged at 100 000 g for 1 h at 4°C

Correspondence to: B. P. Rubin

Received 31 May 1995, revised 4 August 1995, accepted 18 August 1995

Inhibition of PC12 cells by CNS myelin proteins 2525

to separate the soluble proteins in the supernatant from the insoluble proteins in the pellet. The latter were discarded. For the anion exchange chromatography, bovine spinal cord tissue was homogenized directly in extraction buffer at a ratio of 1: 10 (w/v) and centrifuged as described above. Affinity purified myelin associated glycoprotein (MAG) was prepared as described in Poltorak et al. (1987).

In a first separation step the soluble proteins were adsorbed to a Q-Sepharose anion exchange column (Pharmacia, Fine Chemicals, Uppsala, Sweden). Aliquots of the fractions which eluted with increasing salt concentrations (0-1 M NaCl in 20 mM Tris-HCI pH 8.0, 30 mM CHAPS) were taken directly and assayed for their biological activity: four well plates (well area corresponding to 1 cm2,

Greiner, Nuertingen, Germany) were coated either with the CHAPS extract at I , 5, 10 or 20 pg proteidwell or with the column fractions at a concentration of 10 pg/well. The wells were incubated at 4°C overnight in a humid chamber, and subsequently rinsed three times with Ca2+- and Mg2+-free Hank’s balanced salt solution (Hank’s CMF) (Gibco/BRL, Gaithersburg, MD) to wash off unbound protein. Control substrate consisted of culture dishes which had only been incubated with either the extraction buffer or the buffer used for the anion exchange column. For the treatment with the monoclonal antibodies IN-I (Caroni and Schwab, 1988b) and 01 (Sommer and Schachner, 1981) dishes were incubated, after the Hank’s CMF rinse, for 30 min at 37°C with hybridoma supematant, and aspirated; the

FIG. I . Neurite outgrowth of PC12 cells (A, C, E) and spreading of 3T3 cells (B, D, F) on CNS myelin protein coated substrates. K12 cells plated on tissue culture dishes pre-incubated with buffer showed extensive neurites within 24 h (A). On the same substrate, 3T3 cells were well spread I .5 h after plating (B). At 5 pg/well (I cm2) extracted myelin proteins PC12 cells showed a reduction in the number and length of their neurites (C). In parallel, 3T3 cells were inhibited in their spreading (D). At 20 kg/well (1 cm2) of myelin protein, PC12 cells showed very little outgrowth (E), and most 3T3 cells remained round (F). Scale bar represents 50 pm.

2526 Inhibition of PC12 cells by CNS myelin proteins

cells were then cultured in their regular medium containing 50% IN- 1 or 01 hybridoma supernatant.

From Dr M. V. Chao, New York, we obtained a subclone of the PC12 cell line which grows neurites rapidly in the presence of NGF, but independently of laminin. These cells were passaged by mechanical detachment in Hank’s CMF and cultivated with RPMI 1640 medium (GibcoBRL) which contained 10% horse serum (Sera- lab, Sussex, UK), 5% fetal calf serum (Biological Industries, Kibbutz Beth Haemek, Israel) and 100 U/ml penicillin and 0.5 rng/ml streptomycin (PS) (GibcolBRL). Prior to the assay, PC12 cells were primed for 2 days by the addition of NGF (100 ng/rnl, 2.5 S, Harlan Bioproducts, Indianapolis, IN) to the culture medium. The PC12 cells were detached mechanically, trypsinized for 5 min at 37°C with 0.05% trypsin (Sigma, St Louis, MO) in Hank’s CMF and plated at a density of 3000-5000 cells/cm2 in culture medium with 100 ng/ml NGF. Attachment and neurite outgrowth were quantified in duplicate after 24 h. The number of cells which attached was counted in two longitudinal stripes corresponding to -30% of the area of the well. In six randomly chosen fields per well the percentage of PC12 cells with neurites longer than the diameter of the cell body was determined. About 150 cells were evaluated per well.

3T3 cells were maintained and passaged in Dulbecco’s modified Eagle medium (DMEM) with 10% FCS and PS. For the assay the cells were rinsed once in Hank’s CMF, detached with 0.1% Trypsin in phosphate buffered saline pH 7.5, with 0.025% EDTA, and seeded in culture medium at -5000-7000 cells/cm2. The percentage of cells which remained round, i.e. did not spread on the test substrate after 1.5 h, was determined by counting cells in five randomly chosen areas of the well. The assays were done in duplicate and -100 cells were evaluated per well.

Results The PC12 cells which had been primed with NGF for 2 days prior to the assay attached well and -80% of the cells produced neurites in 24 h on the control substrate (culture plastic preincubated with extraction buffer; Fig. IA). On the same substrate, within 1.5 h, 3T3 cells showed the typical spreading behavior, leading to a flat, round or polygonal, fibroblast morphology (Fig. IB). In contrast, on wells which had been coated with solubilized CNS myelin proteins, PC12 cells were inhibited in growing neurites and, at high concentrations, in their attachment (Fig. IC and E). The 3T3 cells were able to attach at the corresponding myelin protein concentrations, but remained round and were unable to spread (Fig. ID and F).

Attachment and neurite production of PC12 cells and spreading of 3T3 cells were quantified. PC 12 cells were inhibited in their adhesion in a dose dependent fashion at 5-20 pg/well ( I cm2 surface area) of detergent soluble myelin protein (Fig. 2A). The anti-adhesive effect was stable for 24 h. In contrast, 3T3 cells were able to attach within 1-2 h on myelin coated substrates (1-20 yg/well; Fig. ID and F). The production of neurites by the adherent PC12 cells was inhibited at 1-20 pg/well of extracted protein (Fig. 2B). 3T3 cells were also inhibited in spreading and in the formation of lamellipodia in a dose dependent fashion (Fig. 2C). In contrast to PC12 cells, the 3T3 cells were not inhibited at a concentration of 1 yg of extracted protein per well and completely overcame the inhibitory effect after 10 h.

To determine if the inhibition was caused by the inhibitory myelin proteins NI-35/NI-250, substrates were briefly preincubated with the mAb IN- 1 before the assay and the cells were cultured in its presence. In the presence of IN-I the neurite outgrowth inhibition of PC12 cells was strongly reduced (Fig. 2B). The inhibition of 3T3 cell

0 1 5 10 2 0

pg rnyelin proteins / well

0 1 5 10 2 0

pg myelin proteins / well

1 0 0 7

0 1 5 10 20

pg rnyelin proteins / well

FIG. 2. CNS myelin proteins inhibit attachment and neurite outgrowth of PC12 cells and spreading of 3T3 cells. (A) Quantification of attachment of PC12 cells on wells without and coated with 1-20 pg of detergent soluble proteins. (B and C ) Myelin proteins were assayed without addition or in the presence of the mAb IN-I or the mAb 01 (as a control) for their effect on neurite outgrowth (B) or 3T3 cell spreading (C). Results are the mean of three experiments done in duplicate: the standard deviation is indicated.


1 T

0 5 10 purified MAG pghell

FIG. 3. Affinity purified MAG is not inhibitory for neurite outgrowth of PC12 cells. MAG was incubated at 5 and 10 pg/well ( I cm’); as a control, wells were incubated with buffer. The results shown are the mean of three experiments, and the standard deviation is given.

spreading was completely neutralized by IN-I at myelin protein concentrations of up to 10 pg/well (Fig. 2C). The mAb 01 , which is directed against an abundant, myelin specific glycolipid, had no effect (Fig. 2B and C).

MAG was recently shown to have neurite growth inhibitory effects in culture for young cerebellar granule cells, adult dorsal root ganglion (DRG) neurons, and the neuroblastomacell line NG 108 (McKerracher et al., 1994; Mukhopadhyay et al., 1994). To compare the effects of MAG with those of CNS myelin protein extracts in our assay, affinity purified MAG was adsorbed on culture dishes at a concentration of 5 or 10 pg/well. No inhibitory effect on neurite outgrowth of PC12 cells was observed (Fig. 3). In addition, MAG did not influence the adhesion of PC12 cells and, if at all, had a slightly beneficial effect on the spreading of 3T3 cells (data not shown). The MAG preparations used were biologically active; they promoted neurite outgrowth from perinatal rat DRG and inhibited outgrowth from adult DRG neurons, very similar to the observations described by Mukhopadhyay et al. (1994) (C. E. Bandtlow, unpublished observations).

In order to investigate the possibility that the assays described above could serve to monitor for the presence of proteins with inhibitory activity during a biochemical purification procedure, the detergent soluble proteins of bovine spinal cord homogenate were separated on an anion exchange column. An example of such a separation is given in Figure 4. The different fractions were tested for their inhibitory activity on the spreading of 3T3 cells and the neurite outgrowth of PC12 cells. An enrichment of inhibitory activity for neurite outgrowth of PC12 cells was found in a broad peak eluting at an intermediate salt concentration, which comprised that of inhibitory activity for 3T3 cell spreading (Fig. 4).

Discussion PC12 cells were inhibited in a dose dependent fashion in adhesion and neurite outgrowth, and 3T3 cells were inhibited in cell spreading by detergent solubilized myelin proteins as a substrate. After separation of detergent soluble proteins of bovine spinal cord by anion exchange chromatography, the inhibitory activity for PC12 and 3T3 cells eluted

-- PC12 - 3T3

0.8

zi c C

0.6 .a, U

0)

0.4 3 F -

0.2

0.1

0 10 20 30 40 50 60

fraction number

FIG. 4. Inhibitory activity for neurite outgrowth of PC12 cells and spreading of 3T3 cells eluted as overlapping peaks from an anion exchange column. Detergent extracted proteins from bovine spinal cord homogenate were loaded on a Q-Sepharose column and eluted with the indicated salt gradient (straight line). The eluted protein was monitored by UV absorbance at 290 nm (bold line). The inhibitory activities were detected by the number of PC12 cells without neurites and by the number of 3T3 cells inhibited in cell spreading on 10 pg/weII (an2) protein.

as overlapping peaks, whereby a larger number of fractions was found to be inhibitory for the PC12 cells than the 3T3 cells. The inhibitory effects could be neutralized by the mAb IN-1 directed against the myelin associated, neurite growth inhibiting proteins NI-35 and NI-250 (Caroni and Schwab, 1988b). For both cell lines, MAG was a growth permissive substrate.

In response to the detergent soluble myelin proteins, PC12 cells showed a reduced adhesion which was not overcome for 24 h. In contrast, 3T3 cells were able to attach at the same protein concentrations and to overcome the inhibitory effect on spreading and lamellipodia formation within a time span of -10 h. These differences could be due to a different sensitivity of the two cell types towards the inhibitory myelin proteins; indeed, at 50 pg of detergent solubilized myelin proteidwell, the 3T3 cells also showed a reduction in attachment (data not shown). Alternatively, 3T3 cells might produce extracellular matrix andor proteases which could allow them to overcome the inhibitory substrate effects.

Neurite formation of PCI 2 cells was inhibited down to concentrations of 1-5 pg/well of extracted myelin protein, similar to the inhibitory effect observed for the neuroblastoma cells NG I08 (McKerracher et al., 1994). Video timelapse microscopy has shown that the encounter between a motile growth cone and an oligodendrocyte or the application of CNS myelin or the purified inhibitory proteins leads to collapse and arrest of the growth cone through the involvement of specific second messenger mechanisms (G-proteins, calcium) (Fawcett et al., 1989; Bandtlow er al., 1990, 1993; Igarashi et al., 1993). These mechanisms may be responsible for the reduction of neurite initiation and outgrowth by PC12 cells observed here.

To determine the contribution of the inhibitory myelin proteins NI-35 and NI-250 to the inhibitory effects observed in the present

2528 Inhibition of PC12 cells by CNS myelin proteins

assays, the functionally active mAb IN-I. raised against the inhibitory proteins (Caroni and Schwab, 1988b), was applied. The strong neutralizing effect observed with regard to neurite outgrowth of PCI 2 and spreading of 3T3 cells shows that a major part of the inhibitory activity was caused by the IN-I antigens. As seen in the example of the chromatographic separation in Figure 4 and the previous figures, the ease and reproducibility of the PC12 assay make it a useful tool for the characterization and purification of neurite growth inhibitory activities from extracts of mouse CNS myelin and bovine spinal cord homogenate. The PC12 cells seemed to be inhibited by the same fractions as the 3T3 cells, but also by additional fractions eluting at a slightly higher salt concentration which were not inhibitory for the 3T3 cells. This could reflect either a higher sensitivity of the PC12 cells towards the inhibitory proteins or the presence of activities with a differential effect on the two cell types in the fractions eluting at higher salt concentrations. Additional steps of purification will be necessary to determine the exact number of peaks of inhibitory activity for PC12 cells and 3T3 cells contained in detergent extracts of bovine spinal cord.

MAG has been shown to be inhibitory for neurite outgrowth of cerebellar neurons, adult, but not newborn DRG neurons, and the neuroblastoma cell line NG 108 (McKerracher et al., 1994; Mukhopadhyay et al., 1994). Hence it was of interest to determine its effect on the PC12 and the 3T3 cells in our assay system. We did not find an inhibitory effect on cell attachment of PC12 cells or on spreading of 3T3 cells (data not shown). This is in agreement with previous findings which show that primary neurons do not react in their adhesive behaviour to membrane bound MAG (Mukhopadhyay e ta / . , 1994). PC12 cells were also not inhibited in neurite outgrowth by affinity purified MAG, a result which resembles the behaviour of early postnatal DRG neurons which are promoted in neurite outgrowth by MAG (Poltorak et a/ . , 1987; Mukhopadhyay et a/ . , 1994).

In conclusion, the presence of myelin proteins with potent inhibitory substrate effects can be demonstrated if CNS myelin proteins are solubilized by a zwitterionic detergent and adsorbed to tissue culture plastic. PC12 cells and 3T3 cells react in a dose dependent fashion to the inhibitory activity which is found in a broad peak after anion exchange chromatography. Most of the inhibitory activity is due to the neurite growth inhibitory proteins neutralized by the mAb IN-I . MAG, a potential inhibitor of neurite growth for some neuronal cell types, is not found to be inhibitory, either for the PC12 or for the 3T3 cells.

Acknowledgements The expert technical assistance of Barbara Niederost is gratefully acknow- ledged. We thank Roland Schob for his help with photography. We are grateful to Dr Moses Chao for providing us with PC12 cells. This work was supported by grants from the Swiss National Science Foundation (no. 31-29981.90). the International Research Institute for Paraplegia (Zurich), the Schiller Foundation (Zurich), the Union Bank of Switzerland and Regeneron Pharmaceuticals, Tarrytown (New York).

Abbreviations CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-I-propane-

sulfonate) CNS central nervous system DMEM Dulbecco’s modified Eagle medium DRG dorsal root ganglion Hank’s CMF Ca*+- and Mg2+-free Hank’s balanced salt solution

MAG myelin associated glycoprotein PMSF phenylmethylsulfonyl fluoride PS penicillin and streptomycin

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Inhibition of PC12 Cell Attachment and Neurite Outgrowth by Detergent Solubilized CNS Myelin Proteins

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