Identification of myelin-associated glycoprotein as a major myelin-derived inhibitor of neurite growth

Neuron,

Vol. 13, 805-811, October,

1994, Copyright

0 1994 by Cell Press

Identification of Myelin-Associated G lycoprotein as a Major Myelin-berived Inhibitor * of Neurite G rowth n

1. McKerracher,* S. David,* D. 1. Jackson,* V. Kottis,+ R. j. Dunn,* and P. E. Braunt *Center for Research in Neuroscience The Montreal General Hospital Research Institute and McGill University Montreal, Quebec H3G IA4 Canada +Department of Biochemistry McGill University Montreal, Quebec H3G lY6 Canada

Summary Contact-dependent axon growth inhibitory activity is present in CNS myelin, but the inhibitory proteins have not been fully characterized. We report here that at least two peaks of inhibitory activity can be separated by fractionating solubilized CNS myelin proteins by DEAE chromatography. A major peakof inhibitory activity corresponded to the elution profile of myelin-associated glycoprotein (MAC). tmmunodepletion of MAC from these inhibitory fractions removed neurite growth inhibition, whereas recombinant MAC (ectodomain) was a potent inhibitor of neurite outgrowth. lmmunodepletion of MAG from total extracts of CNS myelin restored neurite growth up to 63% of control levels. These results establish that MAC is a significant, and possibly the major, inhibitor in CNS myelin; this has broad implications for axonal regeneration in the injured mammalian CNS. Introduction The regenerative neuronal response to injury is dependent upon the nonneuronal environment. Provided with the appropriate milieu, CNS neurons that fail to grow through adult CNS tissuecan regrowtheir injured axons (David and Aguayo, 1981; reviewed by Aguayo et al., 1991; Kromer et al., 1981). Factors derived from the nonneuronal environment that promote axon regrowth include neurotrophic factors (reviewed by Snider, 1994) and adhesion molecules (reviewed by Carbonetto and David, 1993) that, when lacking, could limit axon growth. However, a major barrier to axonal regeneration in the CNS of adult mammals is thought to be the presence of axon growth inhibitory molecules (reviewed by Schwab et al., 1993). Our understandingofthefunctions of these molecules is limited because only a few proteins with neurite growth inhibitory activity have been identified. Proteins with inhibitory properties are known to be involved in axon guidance decisions in development (Davies et al., 1990; Raper and Kapfhammer, 1990; Cox et al., 1990; Pini, 1993; Sretavan et al., 1994; Roskies and O ’Leary, 1994). A recently characterized inhibitory molecule, collapsin (Luo et al., 1993), is part

of a large family of proteins that are phtylogenetically conserved from insects to man (Kolodk.in et al., 1993). Members of this protein family (semaphorins) are required in development to restrict axons to their appropriate territories, as shown by blocking their function with monoclonal antibodies (Kolodkim et al., 1992, 1993). Other molecules with inhibitory activity that may serve a role in axon guidance or defining terminal innervation fields are CD44 (Sretavan et al., 1994) and tenascin (Faissner and Kruse, 1990; Steindler et al., 1989). The former inhibits axon growth from retinal explants in vitro, and in vivo, it appears to repel ipsilaterally projecting retinal axons as they enter the optic tract at the optic chiasm (Sretevan et #al., 1994). The cell-cell interactions mediated bythese molecules are complex and may involve both inhibitory and adhesive properties (Stamenkovic et al., 1991; Shimizu et al., 1989; Spring et al., 1989). In addition to molecules mediating negative cues for axon growth in development, some axon growth inhibitory activity is present in the adult mammalian CNS. A well documented inhibitory activity is that which is present in mammalian CNS mylelin. Two antigenitally related proteins, recognized by a monoclonal antibody designated IN-l, have been reported. to block neurite growth on differentiated oligodendrocytes and CNS myelin in vitro (Caroni and Schwab, 1988a, 1988b). The application of this antibody in vivo allows some axons to elongate long distances after CNS injury (Schnell and Schwab, 1990; Schnell et al., 1994). Furthermore, temporary suppression of myelination in the developing chick spinal colrd by application of anti-galactocerebroside and complement extends the permissive period for axonal regeneration after injury (Keirstead et al., 1992). Here, we provide evidence to show that several growth inhibitory proteins are present in mammalian CNS myelin. We identify one of these inhibitory proteins as rnyelin associated glycoprotein (MAC) and show that, in extracts of CNS myelin, it isthedominantcomponent involved in contact-mediated neurite growth inhibition. Results and Discussion Myelin Contains Several Inhibitory Components MAC Is a Prominent inhibitor of Neurite Growth To identify the growth inhibitory molecules present in myelin, NG108-15 cells were used in a24 hr bioassay to test substrates for neuritegrowth inhibition. These cells area neuroblastoma cell linewith motor neuronlike properties (Nelson et al., 1976; Chahary and Cheng, 1989). Purified bovine CNS myelin used as a tissueculturesubstrate permitted cell attachment but inhibited cyclic AMP-induced neurite outgrowth (Figures IA and IB). On a polylysine substrate, 70% + 2% (n = 26 experiments in triplicate) of the plated cells extended neurites, compared with 2% f 0.6% (n =

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Figure 1. Neurite

Outgrowth

from

Dil-Labeled

NCIOB-15

Cells on Myelin

and MAC

Substrates

(A and B) Cells labeled with Dil and plated on polylysine showed extensive neurites (A), whereas rounded and did not extend neurites (B). (C) NClO&15 cells did not spread or extend neurites on recombinant MAC. (D) Heating of MAC at 8OT for 1 hr abolished its inhibitory properties. Scale bar, 100 urn.

17) on 8 ug and 4% + 1% (n = 15) on 4 ug of CNS myelin. Our goal was to assess and ultimately purify the myelin-associated growth inhibitors. To this end, we investigated a wide variety of agents and extraction conditions known to solubilize proteins and lipids with minimal denaturation and proteolytic degradation. In our hands, the most effective protocol was to employ 1% octylglucoside and moderate ionic strength. The extract normally contained 60% of the total myelin protein and had an electrophoretic polypeptide profile typical of myelin. The extract was strongly inhibitory, with only 14% f 2% (n = 12) of the cells extending neurites on 8 PLgof extracted protein. When an octylglucoside extract was chromatographed on a diethylaminoethyl (DEAE) anion exchange column, several peaks of inhibitory activity could be eluted with a 0.2-2 M salt gradient (Figure 2). The polypeptide profile obtained by SDS-polyacrylamide gel electrophoresis showed that the fractions eluting at low ionic strength containing the major inhibitory activity were enriched in a protein that migrated as a broad band at approximately 100 kDa.

cells grown

on 4 ug of myelin

were

These data led us to investigate whether MAG was present. Western blots with several different antiMAC antibodies (Nobile-Orazio et al., 1984) showed that the inhibitory peaks eluting in low salt corresponded to the elution profile for MAC (Figure 2A). To determine whether MAC in the first peak was responsible for inhibiting neurite growth, we removed it from the column eluant by immunodepletion and tested the residual proteins as growth substrates in comparison with undepleted fractions. Fractions containing MAC were pooled and incubated with Affigel 10 conjugated to anti-MAC antibody513(Johnsonetal.,1989a). Western blotsofthese samples confirmed the immunodepletion of MAC (Figure 38) and upon bioassay, demonstrated the loss of neurite growth inhibitory activity (Figure 3A), thereby establishing an association of MAC with growth inhibition. We then obtained direct evidence for the inhibitory effect of MAC on neurite outgrowth by testing recombinant MAC (extracellular domain) produced in insect cells (Johnson et al., 198913;Attiaet al., 1993). NG108-15 cells plated on recombinant MAC, as on myelin, re-

My&n-Derived 807

Growth

Inhibition

by MAC

Fraction

A

number

1 3 5 7 8 9 10 111213 14 151618 21 242729 ;+i

50 40 30 20 10 0

0~

4

8

12

16

20

24

28

Proteins

by DEAE Anion

Fraction number Figure 2. Analysis

of Growth

(A) Western blots of column (B) Neurite growth inhibition

Inhibition

after Separation

of Myelin

fractions probed with anti-MAC antibody. and protein profile present in the column

mained rounded and did not extend neurites after 24 hr (see Figure IC). Heating of recombinant MAC at 80°C for 1 hr destroyed this activity (see Figure ID) and restored neurite growth to control levels; 73% f 4% of neurons extended neurites on denatured MAG, which is not significantly different from 69% f 4% of neuronsextendingneuriteson bovineserumalbumin usedasacontrol (Figure4).Theseobservations,which show that neurite outgrowth is blocked by the MAG present in myelin as well as by poorly glycosylated recombinant MAC lacking the L2/HNK-1 carbohydrate epitope (Johnson et al., 1989b), suggest that the carbohydrate moieties are not the major components responsible for growth inhibitory properties of MAC. Although we report here that MAG has strong neurite growth inhibitory activity, its sequence homology with adhesion molecules of the immunoglobulin family (Arquint et al., 1987; Salzer et al., 1987) has led others to investigate a possible role for MAC in cell adhesion (Poltorak et al., 1987; Sadoul et al., 1990; Johnson et al., 1989a; Afar et al., 1991). How can our results on the growth inhibitory properties of MAC be reconciled with experimental evidence that the extracellular domain of MAC may mediate adhesion to axons? First, observations that MAC-containing liposomes bind to neurons (Poltorak et al., 1987) suggest a receptor-mediated interaction between neurons and MAC.

fractions

shown

Exchange

Chromatography

in (A).

However, such experiments do not indicate how MAC binding may affect neurite growth. More importantly, other recent experiments demonstrate that the neuronal cell type and developmental age determine the neuronal response to MAC (Mukhopadhyay et al., 1994). Non-MAG

Inhibitors

in CNS Myelin

CNS myelin extracts chromatographed on a DEAE anion exchange column consistently revealed several peaks of inhibitory activity. The inhibitolry activity that we identified in the high salt eluate was not enriched in MAC (see Figure2). This observation, togetherwith the retention of some inhibitory activity after MAC depletion from octylglucoside extracts of total myelin (see below), demonstrates that one or moreadditional inhibitors are present in CNS myelin. Although the molecular identity of the non-MAC inhibition present in our extracts is unknown, there are several possible candidates. Janusin, a protein of 160/180 kDa secreted by oligodendrocytes, is reported to have growth inhibitory properties (Pesheva et al., 1989; Fuss et al., 1993), although it is not expected to copurify with myelin. A more likely candidate is one of the myelin proteins recognized by the IN-I antibody that have been partiallycharacterized byschwaband collaborators (Caroni and Schwab, 1988a, 1988b; reviewed by Schwab et al., 1993). The epitope recognized by the

Neuron 808

B

1

60

2

70 60

T

50 40 30 20 10 0

Figure 4. Quantitative Analysis of Neurite NC108-15 Cells on Different Substrates

Figure 3. Removal of Inhibitory by lmmunodepletion

Activity

from Column

Fractions

The anti-MAC antibody513 was used to deplete MAC from fractions 9, 10, and 11 (Figure 2). (A)The inhibitory activity present in theoriginal pooled fractions and the MAC depleted fractions. The results are the mean of three experiments performed in duplicate + SEM. (B) Western blot of the pooled fractions (9, IO, and 11) before (lane 1) and after (lane 2) immunodepletion indicates that most of the MAC was removed.

Outgrowth

from

Comparison of cells grown on polylysine with cells grown on 4 pg of CNS myelin, recombinant MAC (MAC), denatured MAC (denat. MAC), or bovine serum albumin (BSA). Results are the mean of 23 experiments done in triplicate (2 experiments for denatured MAC).

theywill elongate their axons readilythrough the environment of a peripheral nerve graft (David and Aguayo, 1981; Aguayo et al., 1991). The robust regeneration of both CNS and PNS axons through a peripheral nerve environment, given that myelinating Schwann

IN-l antibody is present on oligodendrocytes and copurifies with myelin (Caroni and Schwab, 1988b). MAC Contributes Significantly to Growth Inhibition on Extracts of CNS Myelin To determine whether the MAC component of total CNS myelin protein contributed significantly to neurite growth inhibition, we immunodepleted MAC from

octylglucoside

extracts

of total

myelin

protein.

Western blots before and after immunodeIpletion show complete removal of MAC (Figure 5A). Coomassie blue-stained gels (not shown) of these samples produced identical polypeptide profiles, except for loss of the band in the region of the gel to which MAC migrates. Neurite growth after MAC depletion was restored to about 63% of control level (Figure 5B). Therefore, MAC is a major inhibitor of neurite outgrowth, but other proteins (Caroni and Schwab, 198813) also contribute to growth inhibition on CNS myelin. Implication of MAC Inhibitory Activity for Axon Regeneration in the CNS and PNS of Adult Mammals Neurons in the adult mammalian CNS show little tendency to regenerate through adult CNS tissue, but

1

2

Figure 5. Removal by lmmunodepletion from Total Extract of CNS Myelin

of Inhibitory

Activity

(A) Western blot showing MAC present in the octylglucoside extract of CNS myelin (lane 1) and the immunodepletion of MAC from the extract (lane 2). Each lane was loaded with 50 &g of protein. (B) Comparison of neurite growth on substrates prepared from the buffer control (ctl), 8 pg of octylglucoside extract from whole myelin (El), and 8 pg of MAC-depleted extract (dep El). Results are the mean of three experiments done in triplicate it SEM.

Myelin-Derived 809

Growth

Inhibition

by MAC

cells express MAC, may be due to the rapid removal of myelin debris after peripheral nerve damage. This rapid removal contrasts with the slow removal of myelin debris in the injured CNS, which maytake several weeks or months to complete (Stoll et al., 1989; David et al., 1990). We have demonstrated that MAC present in PNS myelin is capable of inhibiting neurite growth, but that nonmyelin contaminants present in peripheral nerve myelin preparations can override growth inhibition (unpublished data). Other data showing that axon growth inhibitory activity is present in peripheral nerves in vivo come from studies on the PNS of Ola mouse mutants, in which there is a marked delay in Wallerian degeneration (including the removal of myelin debris) after peripheral nerve injury (Lunnetal.,1989;Perryetal.,1991). Inthesemice,axon regeneration does not occur in the well myelinated phrenic (Brown et al., 1994) and facial nerves (Chen and Bisby, 1993) if the distal stump does not degenerate but occurs in nerves in which unmyelinated Schwann cells are present (Brown et al., 1992,1994). Additionally, unfixed cryostat sections of normal adult rat peripheral nerve, which should contain MAC, promote neurite growth from embryonic neurons (Sandrock and Matthew, 1987; Carbonetto et al., 1987) but not from neurons from adult mammals (Bedi et al., 1992). A difference between the ability of developing and adult neurons to grow on MAC has been demonstrated by Mukhopadhyay et al. (1994). These findings are in accordance with the extensive longdistance axon growth of fetal rodent and human neurons through myelinated tracts of adult host rats (Wictorin and Bjorklund, 1992; Wictorin et al., 1992; Davies et al., 1994) and with the failure of adult neurons to show similar growth after CNS injury (reviewed by Schwab et al., 1993). We show that MAG is a major component of the multiple neurite growth inhibitory proteins present in CNS myelin. Our data suggestthatMAG,which is relativelyabundant(-1%) in CNS myelin (Trapp, 1990; Quarles et al., 1992), may significantly contribute to the failure of injured axons to regenerate within the adult mammalian CNS. Experimental Procedures Bioassayfor Inhibition of Neurite Growth Bovine CNS myelin or recombinant MAG at 4 or 8 pg of protein was added to polylysine-coated 96 well plates, dried, and washed with phosphate-buffered saline before plating the cells. Neurite growth was assessed with NGlO&15 cells that have motor neuron-like properties, express acetylcholine, and are capable of making synapses on muscle cells or with other NG108-15 cells (Nelson et al., 1976; Chaharyand Cheng, 1989). Cells maintained in Dulbecco’s minimal essential medium containing 10% fetal bovine serum, 1% hypoxanthine-aminopterin-thymidine, and 1% penicillin/streptomycin weretransferred to mediacontaining 5% fetal bovine serum and were primed with 1 m M cyclic AMP for 2 days prior to labeling with the fluorescent marker l,l’dioctadecyl-3,3,3’,Y-tetramethylindocarbocyanine perchlorate (Dil). Cells were incubated for l-2 hr in the presence of Dil (3 &ml) and then then washed and cultured for another day before use in the neurite growth assays. After removing the cells from the flasks with trypsin/EDTA, they were plated at 1000 cells per well in 96 well plates in triplicate and cultured in Dulbecco’s minimal

essential medium, 5% fetal bovine serum, 1% hypoxanthineaminopterin-thymidine, and 1 m M cyclic AMP. After 24 hr, the cultures were fixed with 4% paraformaldehyde, 0.1% glutaraldehyde, and 0.1 M phosphate buffer, and then washed; finally, the percentage of cells with neurites of one cell body diameter or longer was determined. Equal volumes of fractions obtained from chromatography experiments were added to 96 well plates, dried, and washed. NG108-15 cells were plated on these substrates, and after 24 hr, the cultures were fixed and stained with 0.5% cresyl violet. The percentage of cells with neurites of one cell lbody diameter or longer was determined. Percent inhibition was calculated as (C - E/C) x 100, where C was the average number of cells with neurites on polylysine substrates and E was the average number of cells with neurites on the test substrate.

Preparation of Myelin and DEAE Chromatography Bovine corpus callosum was homogenized in 0.32 M sucrose with a Dounce homogenizer. Nuclei and large tissue aggregates were removed by centrifugation; myelin was purified by density gradient centrifugation, two rounds of osmotic shock, and removal of small membrane fragments, followed by discontinuous gradient centrifugation (Norton and Poduslo, 1973). Bovine brain myelin was extracted for 2 hr at 20°C with 1% octylglucoside (I ml per milligram of protein) inI 0.2 M phosphate buffer (pH 6.8) containing 0.1 M Na2S0,, 1 m M EDTA, 1 m M dithiothreitol, and a cocktail of protease inhibitors. The extract was clarified by centrifugation at 400,000 g x min and applied to a column of DEAE-Sepharose (Pharmacia; 1.3 cm x 10 cm). After several washes, elution was effected with a NaCl gradient (0.2-2.0 M) containing 1% octylglucoside, 50 rnM Tris-HCI (pH 7), and 1 m M dithiothreitol. Fractions (2.0 mI)werecollected, and protein concentrations were estimated by the Bradford protein assay (BioRad, Toronto, Canada). Proteins were prepared for SDS-polyacrylamide gel electrophoresis by precipitation with trichloracetic acid, removal of detergent with acetone, and dispersion in boiling sample buffer. Aliquots of 70 ~1 were tested in culture.

lmmunodepletion Approximately 2 mg of purified anti-MAC antibody (513) (Johnson et al., 1989a) was conjugated to 1 ml of Affigel 10 (BioRad) according to the protocol of the supplier. After antibody conjugation, the beads were treated with 0.5 M ethanolamine HCI (pH 7.5) for 45 min to block nonspecific reactive groups and washed in phosphate-buffered saline. Immunodepletion of MAG from the pooled fractions (g-II), or from total extract of myelin (El), wasdone byincubating8OOmIof thissamplewith theanti-MAGAffi-Gel beads in a 50 ml polypropylene tube for 2-3 hr at 4OC. The protein sample was then removed by centrifugation. Polypeptides present in aliquots of the pooled samples before and after incubation with anti-MAC-Affi-Gel beads were separated by SDS-polyacrylamidegel electrophoresisand Western blotted with anti-MAC antibody to determine the efficilency of immunodepletion.

Preparation of Recombinant MAC The baculovirus system was used to express a modified form of MAC in S. frugiperda (Sf 9) cells, as previously described (Johnson et al., 1989b; Attia et al., 1993). In brief, this was achieved by truncating MAC in such a way as to remove the transmembrane and cytoplasmic domains, thereby permitting synthesis and secretion of the soluble ectodomain. Complete purification was achieved by affinity chromatography on a column of immobilized anti-MAC (513) antibody.

We gratefully acknowledge financial support from the Canadian Network for the Study of Neural Regeneration and Functional Recovery. We thank Bharatkumar Patel, Arthur Roach, Andres Lozano, Elsa Horvath, and Dao Ly for providing help at different stages of this project.

Neuron 810

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement”in accordance with 18 USC Section 1734 solely to indicate this fact. Received

August

26,1994.

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