Functional evidence for the role of axolemma in CNS myelination

Neuron,

Vol. 13, 473-485, August,

1994, Copyright

0 1994 by Cell Press

Functional Evidence for the Role of Axolemma in CNS Myelination Lucia M. Notterpek and Leonard H. Rome Department of Biological Chemistry and the Mental Retardation Research Center UCLA School of Medicine Los Angeles, California 900241737

Summary A direct role for neurons in CNS myelination has yet to be demonstrated. CNS myelination can be examined in cerebellar slice cultures, which faithfully reproduce both synthesis and wrapping of myelin. In an attempt to demonstrate a role for axolemma in this process, we generated more than 2000 axolemma-reactive monoclonal antibodies. One clone, C21.3, repeatedly blocked myelination in cerebellar slices, as documented by both biochemistry and morphology. The antibody caused a dramatic reduction in myelin lipid and protein synthesis. CNS white matter, sciatic nerve, and neuronal cultures were positively stained with G21.3, whereas oligodendrocytes and myelin were fully negative. The antibody identified a restricted number of proteins in purified axolemma. These results suggest a direct involvement of axons in CNS myelination. Introduction Unlike the PNS, in which axons are involved directly in myelination, a specific role for neurons in CNS myelination has yet to be demonstrated. Neuronal participation in PNS myelination was elucidated using Schwann cell cultures, whose ability to synthesize myelin requires neuronal contact (Bunge et al., 1982). In contrast, oligodendrocytes, the myelinating cells of the CNS, are able to produce myelin-like membranes in the absence of neurons (Bradel and Prince, 1983; Rome et al., 1986; Knapp et al., 1987). These observations suggest that some of the signals for myelin production reside within the oligodendrocyte itself or thattheycan be replaced byenvironmental cues present in the culture. Nevertheless, several investigators have reported neuronal influences on oligodendroglial proliferation and differentiation, both in vivo and in vitro (for review see Hardy and Reynolds, 1993). In vivo studies in the rat optic nerve indicated that proliferation of oligodendroglial precursors is dependent on electrical activity within the axons for the release of a glial mitogen (Barres and Raff, 1993). Moreover, the survival of developing oligodendroglia was promoted by neuronal cell surface or extracellular matrix (ECM)-bound neuronal signaling molecules (Barres et al., 1993). Similar results were obtained in vitro. In cell culture models of early development, dorsal root ganglion neurons were mitogenic for both embryonic and adult oligodendrocytes (Wood and Williams, 1984; Wood and Bunge, 1986). Furthermore,

conditioned media of cerebellar neurons (Levine, 1989) and neuronal cell lines (Bottenstein et al., 1988) also influenced the proliferation of oligodendrocyte precursors. Finally, adult axolemma-enriched fractions (DeVries, 1980) induced mitogenic effects on cultured IO-day-old oligodendrocytes (Chen and DeVries, 1989). Differentiation of oligodendroglia in the rat optic nerve has been shown to depend closely on axonal integrity (Valat et al., 1988). Gene expression for the major myelin proteins was modulated bythe presence of axons, as evidenced in normal developing CNS (Kidd et al., 1990) and under experimental conditions, such as nerve transection studies (McPhilemy et al., 1990). Furthermore, axons were required for the proper expression of specific developmentally regulated proteolipid protein (PLP) transcripts (Scherer et al., 1992). In vitro investigations also demonstrated that PLP and myelin basic protein (MBP) mRNA levels are elevated when oligodendrocytes are cocultured with spinal cord neurons (Macklin et al., 1986). Myelination in the CNS proceeds in a time- and region-specific pattern that may signify the importance of neuronal and environmental signals (Schwab and Schnell, 1989). Premyelinated axons appear to cluster large particles, proposed sodium channels, at the future nodes of Ranvier (Waxman and Ritchie, 1985), suggesting that they are destined to be myelinated. In fully wrapped fibers, internodal length and fiber diameter are highly correlated (Murray and Blakemore, 1980), indicating a precise relation between the axon and the myelin-producing cell. Furthermore, both nodal size and myelin thickness appear to be determined by specific local signals generated by the axon involved, since morphologically distinct axons can be myelinated bythesameoligodendrocyte(Waxman, 1987). In addition, an early presence of astroglial processes at the node of Ranvier is well documented, but their exact role in myelination is not known (Waxman, 1987; Hildebrand et al., 1993). Even less is understood about the influence of the ECM in the CNS, although an arginine-glycine-aspartic acid (RGD)containing ECM component has been implicated in oligodendrocyte adhesion and myelin synthesis in vitro (Cardwell and Rome, 1988a, 1988b; MalekHedayat and Rome, 1994). The presence of ECMderived proteins at the nodes of Ranvier has been documented, suggesting a role in node formation (Sanes, 1989). Based on these observations, it is reasonable to hypothesize that neurons are active participants in CNS myelination. Our approach to identify axonal components that are direct participants in myelination has involved isolation of monoclonal antibodies directed against axolemma. Similar strategies for the identification of functionally important antigens in CNS development have been successful (Valentine et al., 1985).

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well as glycoprotein-enriched axolemma fractions. From approximately 2000 axolemma-reactive clones, 800 were selected by a dot blot assay for their preferential reactivity toward axonal versus liver membranes, under nondenaturing conditions. All 800 clones were further screened by immunohistochemistry of frozen sections of rat brain. For the functional assay (see below), 60 of the 800 monoclonals were selected, based on their positive immunoreactivity against IO-day-old and adult rat cerebellar frozen sections. All 60 clones were highly reactive in adult white matter regions and in IO-day-old developing cerebellum; however, only the G21.3 and the LA-49 ciones had significant functional effects in the in vitro myelination assay (see below).

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Figure 1. The Effect of the C21.3 Monoclonal lipid Synthesis in Cerebellar Slice Cultures

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(A) The influence of a representative panel of white matter-reactive anti-axolemma monoclonal antibodies on myelin synthesis, as detected by the amount of 35S incorporation into sulfolipid. (6) Anti-GalC (CC) and G21.3 monoclonals were compared with control IgM at 10 and 20 pgiml. Sulfolipid synthesis levels were higher than in (A) because antibodies were prepared in a low sulfate medium. (C) Recovery of sulfolipid synthesis was observed in transiently treated cultures. The G21.3 monoclonal was added (+Ab) for the indicated days, followed by antibody washout for the indicated days (-Ab).

The myelination assay, which we recently described (Notterpek et al., 1993), utilizes cerebellar slices that can be reliably screened for antibodies which inhibit myelination.Theseculturesmyelinatewithan invivolike developmental profile, as evidenced by biochemical and morphological parameters. From over 2000 antibody-secreting, axolemma-reactive hybridomas, we identified 2 clones that repeatedly blocked myelination in the slice cultures. A characterization of 1 of these clones (G21.3) has led to functional evidence supporting a role for axons in CNS myelination. Results Identification of the G21.3 Monoclonal Antibody Over 4000 monoclonal antibodies were generated in BALB/c mice, using a number of different immunization protocols. Antigen preparations included axolemma from IO-day-old, 18-day-old, and adult rats, as

In Vitro Myelination Assay Priortotheir use in the myelination assay,the isotypes and the antibody titers of all 60 monoclonals were determined. The hybridomas were retested for antibody secretion after growth in low sulfate or sulfatefree medium, which was optimized to match the conditions of the in vitro myelination assay. Quadruplicate samples of cerebellar slices grown in 4 well plates were incubated with randomly assigned antibody (3-5 pg per well), from day 10 through day 21 in vitro. Myelin synthesis was measured at day 21 by the rate of ?5 incorporation into sulfolipid (see Experimental Procedures). G21.3 inhibited myelin synthesis by 60%-70%, when compared with a representative panel of monoclonals (Figure IAj. This result was confirmed in nine independent experiments, in which G21.3 consistently yielded 60%-80% inhibition of sulfolipid synthesis. These experiments included slices grown on type I collagen, poly-L-lysine, or an astroglial matrix (Rome et al., 1986), indicating that the functional effect of G21.3 was not due to interaction with the culture substratum. LA-49, a second interesting anti-axolemma clone, repeatedly inhibited sulfolipid synthesis by40%-60%. However, this monoclonal was not examined further. An anti-galactocerebroside (GalC) antibody (Ranscht et al., 1982) with a wellcharacterized myelin inhibitoryactivity(Ranscht et al., 1987) was included in each experiment as a positive control. This antibody invariably suppressed sulfolipid synthesis by 80%-90%. HNK-1 (Abo and Balch, 1981), a commercially available monoclonal IgM with a previously delineated developmental expression pattern (Yoshiharaet al., 1991), was also examined and found to suppress sulfolipid synthesis consistently by about 50%. All of the anti-axolemma monoclonals shown in Figure IA are of the IgM isotype. Under optimized conditions, it was observed subsequently that control !gM at 3-5 pg per well had no significant effect on myelination (Figure IB). For this experiment, hybridomas were grown in sulfate-free medium, which results in quantitatively greater incorporation of 35S into myelin sulfolipid. Attempts to purify the G21.3 monoclonal IgM by several methods have resulted in denaturation of the antigen-binding domain

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Figure 2. C21.3 Antibody

Treatment

inhibits

the Expression

of MBP in Cerebellar

Slices

Whole-mountcuituresweredoublelabeledwith monoclonalanti-NF(SMI 3l)and polyclonalanti-MBPantibody,followed byfluoresceinor rhodamine-conjugated secondary antibodies, respectively. Control samples exhibited bright NF (A) and MBP (B) immunoreactivity. C21.3antibodytreatment resultedin themaintenanceofNFimmunoreactivity(C),whereasadramaticdecreaseinMBPimmunoreactivity was observed (D). Peripheral oligodendrocytes in G21.3 antibody-treated cultures showed strong MBP immunoreactivity (E).

and low recovery. One protocol (Tatum, 1993) yielded a functionally active antibody that inhibited sulfatide synthesis by59%; however,theaffinityoftheantibody appeared to be decreased, since 5-fold greater amounts of antibody were needed for functional effect. Specific reactivity of the purified antibody was monitored using immunohistochemistry of cerebellar frozen sections. To determine whether the myelin inhibitory effect of G21.3 was reversible and not due to cell toxicity, slices were treated with antibody for various periods of time, followed by antibody removal (Figure IC).

For all of the standard functional assays, antibodies were applied for a total of 11 days, days IO-21 in vitro. Cultures that were antibody treated for only 3 days (days 10-13) and then allowed to recover for 8 days synthesized near control levels of sulfolipid. Slices treated for 7 or 9 days, prior to a 4 or 2 day recovery period, respectively, also showed a partial recovery (Figure 1C). The effect of the G21.3 antibody on myelin synthesis by isolated oligodendrocytes was also investigated. Hybridoma supernatant stocks, used at the same concentration that inhibited the slice cultures 60%-80%,

suppressed myelin synthesis by only IO%-30%, as measured by the incorporation of 35S into sulfolipid. This inhibition appeared to be due to nonspecific components in the hybridoma supernatant, since the purified IgM preparation of G21.3 that yielded 59% inhibition of myelin synthesis in the slices had no effect on the oligodendrocytes. Furthermore, other nonfunctional anti-axolemma monoclonal IgMs, secreted by mouse hybridomas, all showed similar levels of inhibition (IO%-30%) of sulfolipid synthesis when added to purified oligodendrocytes (data not shown). In contrast, the GalC monoclonal antibody caused a nearly complete block of myelin synthesis by the isolated oligodendrocytes (data not shown). The observed, apparently nonspecific negative effect of all the anti-axolemma hybridoma supernatants on isolated oligodendrocytes may be due to a factor that is secreted or shed by the hybridoma cells and/or to the activation of microglia, known contaminants of oligodendroglial cell cultures (Giulian and Baker, 1986). The morphology of oligodendrocytes was unaffected by the presence of the G21.3 monoclonal, as compared with control cells under phase microscopy, and immunocytochemical localization studies indicated a complete absence of the G21.3 antigen on the surface of cultured oligodendrocytes (see below). Morphological Evidence for the Inhibition of Myelination by G21.3 The extent of myelination in control and antibodytreated cerebellar slice cultures was also examined by immunostaining of paraformaldehyde-fixed, permeabilized whole-mount samples (Figure 2). A polyclonal antibody against MBP and a monoclonal antibody (SMI 31) against phosphorylated neurofilament (NF) were colocalized by the use of a rhodamineconjugated anti-rabbit IgG and a fluoroscein isothyocyanate (FITC)-conjugated anti-mouse IgG and IgM. Continuous bright MBP immunoreactivity (Figure 28) was noted along NF-positive (Figure 2A) axonal processes in control cultures. A staining pattern similar to that of MBP was obtained with antibody against GalC (data not shown). A marked decrease in MBP immunoreactivitywas noted in G21.3-treated cultures (Figure 2D). The small amount of MBP-positive membrane along neuronal processes appeared loose and detached. The intensity of this immunoreactivity was much lower than that in control samples, indicating a decrease in myelin synthesis. The intensity of NF labeling was comparable to that of the control cultures, and the number of NF-positive processes appeared unaffected by the antibody treatment (Figure 2C). The punctate reactivity along neuronal processes seen in Figure 2C is most likely due to the remaining G21.3 monoclonal, which would react with the antimouse IgG and IgM secondary antibody. The nuclear immunostaining observed in Figures 2A and 2C with the SMI 31 anti-NF monoclonal has been described previously (Sternberger, 1986; Schilling et al., 1989).

Peripheral oligodendrocytes that were not in contact with neurons maintained intense MBP immunoreactivity in antibody-treated slices (Figure 2E). These few cells, which migrated out from the core of the slice early in culture, served as an internal control for examination of non-neuron-associated oligodendrocytes, which appear to continue synthesizing myelin in the presenceof G21.3. In contrast, anti-GalC inhibits both axon-associated and peripheral, non-neuron-associated oligodendrocytes in these slice cultures (Notterpek et al., 1993). Slices were examined for the presence of myelinated fibers by transmission electron microscopy at day 21 in vitro, following IO days of incubation with antibodies. Control slices contained numerous wellcompacted myelinated profiles (Figure3A), previously documented in these cultures (Notterpek et al., 1993). The presence of NFs within the core region was used as an indication of axonal identity. The lack of myelin was consistently observed in CalC and G21.3 antibody-treated cultures (Figures 3B and 3C), whereas morphologicallydistinguishableoligodendrocytecell bodies were present. The absence of myelin in the micrographs of G21.3-treated cultures was even more pronounced than the biochemical inhibition levels (60%-80%; see above), suggesting that a nearly complete reduction of wrapping may occur, even in the presence of low levels of membrane synthesis. This hypothesis is supported bythe presence of occasional unwrapped myelin-like membranes in regions near oligodendroglial profiles. Most importantly, there were no signs of cytotoxicity after treatment with either C21.3 or GalC monoclonals. Furthermore, application of control IgMs had no effect on the abundance or quality of myelin profiles as compared with control cultures (data not shown). Biochemical Evidence for the Inhibition of Myelination Control and G21.3 antibody-treated slice cultures were homogenized in SDS gel sample buffer, and the same preparation was used for all three immunoblots shown in Figure 4. Protein levels of two myelin structural components, PLP and MBP, and an oligodendroglial cytoplasmic marker, 2’,3’-cyclic nucleotide 3’-phosphodiesterase (CNP), were investigated. CNP protein expression can be used as a rough estimation of oligodendroglial cell number, sinceonly low levels of CNP are found in compacted myelin (Hildebrand et al., 1993). Quantification of the CNP-immunoreactive bands revealed a moderate (22%) reduction due to G21.3 treatment, whereas MBP and PLP levels were substantially decreased (78% and 62% reduction, respectively; Figure 4). These data are consistent with the lackof myelin observed morphologically. Furthermore, they support a nonoligodendroglial target for inhibition bythe G21.3 monoclonal, sincethe number of oligodendrocytes in antibodytreated cultures was only slightly reduced, as indicated by the CNP levels.

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Figure 3. The Lack of Myelinated

Profiles

in Antibody-Treated

Slices

Thin sections of 22-day-old slices were examined by transmission electron microscopy. (A) Morphological organization of a control culture. Numerous myelinated profiles were seen, including simple elements (m,) and more complex elements with multiple well-compacted turns of myelin (mz). The axon cylinder (A) often contained membranous and fibrillar elements. (B) Anti-GalC-treated and (C) C21.3 monoclonal antibody-treated cultures. These cells, presumptive neurons (N) and oligodendroglia (O), did not show degenerative changes, yet the abundant myelin profiles seen in the control cultures were absent. All micrographs were printed at the same magnification, indicated in (C).

Purified myelin membranes were used as positive controls for the specificity of the immunoreactive bands in each Western blot (data not shown).

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Developmental Expression Pattern of the G21.3 Antigen in the Rat Cerebellum

Figure 4. Western Analysis G21.3-Treated Slice Cultures

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Oligodendrocyte

Proteins

in

Control and antibody-treated samples were analyzed on 10% SDS gels for the protein levels of MBP, PLP, and CNP (inserts). Quantification of the specific immunoreactive bands after 11 days of antibody treatment indicated 84% (MBP), 78% (PLP), and 22% (CNP). Identical samples were used for the three different blots, and each lane contains approximately 80 ug of total protein. Molecular masses are indicated in kilodaltons on the left of each gel.

lmmunolocalization in l-day-old developing cerebellum indicated that the G21.3 antigen was present in the external granular layer and the future Purkinje cell layer (Figure 5A). Additional reactivity was noted at day 10 in the internal granular layer and the future white matter (Figure 56). By postnatal day 18, the white matter contained the highest amount of G21.3-immunoreactive material (Figure 5C), whereas the Purkinje cell layer remained highlighted. In theadult, the G21.3 antigen was concentrated in the well-myelinated tracts of the cerebellum (Figure 5D), which contain efferent Purkinje cell axons, and in afferent myelinated projections from throughout the nervous system. The internal granular layer and the Purkinje cells showed decreased immunoreactivity compared with the IO- and ISday-old cerebella. Similar results were obtained in other areas of the developing CNS, in

Figure 5. In Vivo Cerebellar

Localization

of the C21.3 Antigen

Fresh frozen sections of developing and adult rat cerebellum were immunostained with the G21.3 monoclonal antibody, followed by an FITC-conjugated secondary antibody. The external granular layers of the l-day-old cerebellum (A) displayed strong immunoreactivity. In IO-day-old cerebellum, the Purkinje cell layer, external granular layer, and developing white matter showed intense immunoreactivity (B). The highest immunoreactivity was associated with white matter regions of 18.day-old (C) and adult (D) cerebella. All photographs were at the same magnification, as indicated by the bar in (D). wm, white matter; i, internal granular layer; p, Purkinje cell layer; m, molecular layer; e, external granular layer.

which expression of the G21.3 antigen along fiber tracts was found to precede that of myelin markers. In the adult brain, the most intense staining was localized to white matter regions. The frozen sections used for these studies were fixed and permeabilized by a method optimized for the identification of anti-axolemma antibodies (Bigbee et al., 1985). PNS and Nonneuronal Tissue Distribution of the G21.3 Antigen In addition to the CNS, the G21.3 antigen was found to be highly abundant in the PNS. The strongest immunoreactivity was observed in the basal lamina of Schwann cells (Figures 6A and 6B), a necessary cellular

componentfor PNS myelination(Bungeetal.,1982). In a cross-sectional view of the sciatic nerve, the antigen was also clearly detected at or near the periaxonal membrane (Figure 68, arrows), within the MBP-positive myelin (Figure 6C), suggesting a possible involvement of this molecule in axon-Schwann cell interaction. The presence of the G21.3 antigen was also examined in nonneuronal tissue. In comparison with the sciatic nerve, only a low level of G21.3 immunoreactivity was seen in the glomerular and tubular basal lamina of the kidney (Figure 6D). The pattern of G21.3 immunoreactivity was similar to that of the other known basal lamina markers, fibronectin (Figure 6E)

Role for Axolemma 479

in CNS Myelination

Figure 6. The G21.3 Localization

in the PNS and Basal Lamina

lmmunostaining of fresh frozen rat tissue sections. In longitudinal sections of the sciatic nerve, the basal lamina, including nodal regions (arrowheads), displayed strong C21.3 immunoreactivity (A). In cross-sectional view of the sciatic nerve, G21.3 immunoreactivity (B) was associated with the basal lamina (arrowheads) and the periaxonal region (arrows), whereas MBP immunoreactivity (C) indicated myelin surrounding the axon. The G21.3 antigen (D) was colocalized with fibronectin (E) in glomerular (g) and tubular(t) basal lamina of the kidney. Laminin staining (F) was found to distribute in a similar region as fibronectin. The level of G21.3 immunoreactivity was very low in liver (C). Magnification (A, B, and C), indicated in (C); magnification (D, E, F, and G), indicated in (C).

and laminin (Figure 6F). However, G21.3 did not colocalize preciselywith either of these proteins. Furthermore, neither anti-fibronectin nor anti-laminin antibody had a functional effect in the in vitro myelination assay. A similar low level staining was associated with the basal lamina of skeletal muscle (data not shown). Tissue sections of testis and pancreas were negative, whereas liver contained only a small amount of G21.3 immunoreactivity (Figure 6C). Cell Surface Staining of Cortical Neuron Cultures with the C21.3 Monoclonal Live and permeabilized neuron-enriched cultures were investigated for the presence of the G21.3 antigen. lmmunocytochemistry of live cells at 4°C revealed the presence of the C21.3 antigen on neuronal cell surfaces and processes (Figure 7A). Glial fibrillary acidic protein-positive astrocytes, present in neuronenriched cultures grown in serum-containing media, were not labeled with the antibody (data not shown). In permeabilized neuronal cultures, strong G21.3 immunoreactivity was associated with the cell soma (Fig-

ure 7B). The neuronal phenotype of the stained cells was confirmed by colocalization of antibody against neuron-specific enolase (NSE; Figure 7C) and with Neurotag Red (Raju and Dahl, 1982; data not shown). Similar data were observed in pure neuronal cultures grown in N2 serum-free medium (Bottenstein and Sato, 1979; data not shown). In fixed cultures, the immunoreactivity along neuronal processes was decreased, as compared with that of live cells, which may have resulted from loss of the antigen during the double labeling protocol. This finding may indicate a loose association of the G21.3 protein with neuronal processes in cultured cells, and it is supported by immunoprecipitation of the antigen from the media of neuronal cultures (see below). As mentioned above, the morphology of purified oligodendrocytes appeared unaffected by the presence of the G21.3 antibody in the culture media for 5 days (Figure 7D). This result is consistent with the lack of surface reactivity of the G21.3 antibody toward oligodendrocytes (Figure 7E). Oligodendroglial identity was confirmed by anti-GalC staining of a parallel culture (Figure 7F).

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Figure 7. Dissociated Cortical Neurons Positively Stain with theMyelination Inhihitory Monoclonal C21.3 The G21.3 antigen was found to be present on the surface of live cortical neurons (A) by immunocytochemistry. In cultures that have been fixed and permeabilized, G21.3immunoreactive cell bodies (B) costained with anti-NSE polyclonal antisera (C). C21.3 antibody treatment for 5 days had no apparent effect on oligodendrocyte morphology (D) compared with controls (data not shown).TheG21,3antigenwasabsentfrom the surface of oligodendrocytes (E). AntiCalC staining of parallel cultures is shown in (F). Magnification (A, B, C, E, and F), indicated in (B). Magnification of the phase image in (D) is approximately 250 x.

Western Analysis of the G21.3 Antigen

kDa protein that appears to bind all IgMs regardless of their antigen specificity was identified in both axolemma and brain homogenates. An identical staining pattern, with a prominent >200 kDa protein and a less abundant 130 kDa protein, was obtained when

The presence of the G21.3 antigen in isolated myelin and axolemma from specific developmental stages and in total tissue homogenates was investigated by Western blots. In the course of these studies, a >200

Control MM 1

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Control IgM (lane 1) and the G21.3 monoclonal IgM (lanes 2-14) were used to detect immunoreactlve bands in several protein samples. Axolemma (IO-day-old) was reacted with a control IgM (lane 1) and C21.3 monoclonal (lane 2). C21.3-immunoreactive proteins were identified in Xl-day-old (lane 3) and adult (lane 4) axolemma. Purified myelin membranes (lane 5) did not contain G21.3.immunoreactive proteins. Total SDS sample buffer homogenates of spinal cord, brain stem, cortex, sciatic nerve, liver, and kidney were analyzed (lanes 6,8,9,10, and 11, respectively). All samples in lanes l-11 contained approximately 80 pg of total protein. Lanes 12-14 represent C21.3 Western blots of immunoprecipitates of neuronal culture media. The C21.3-immunoprecipitated sample (lane 14) contained proteins at 170 and 140 kDa, which comigrated with IQ-day-old axolemma components (lane 12). Nonspecific IgM-immunoprecipitated neuronconditioned media did not contain C21.3-immunoreactive proteins (lane 13). Proteins were separated on 5% polyacrylamide gels. Positions of the molecular mass standards of 200 and 116 kDa are indicated at the left of the figure. All other molecular masses were extrapolated from the positions of these standards on each gel (data not shown).

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a commercially available mouse control IgM (Figure 8, lane 1, arrowheads) or any of the nonfunctional anti-axolemma IgM clones were examined (data not shown). The high molecularweight protein was prominent in G21.3 Western blots (Figure 8, lanes 2, 3, 4, 7, 8,9, and 12); however, based on the results of the Western blots and the functional studies with control IgMs, we have concluded that this protein is not a specific target of the G21.3 antibody. Additional high molecular weight bands near the nonspecific band were also observed in some samples; however, these species were variable in their appearance and could have resulted from breakdown of the >200 kDa protein. The G21.3 monoclonal specifically identified a doublet at - 140 kDa and a single band at - 120 kDa in IO- and 18-day-old axolemma, (Figure 8, lanes 2 and 3). An additional faint 170 kDa band was present in freshly prepared IO-day-old axolemma (Figure 8, lane 12). The same protein bands were only faintly visible in adult axolemma (Figure 8, lane 4), suggesting developmental changes in theexpression of these proteins. Neither omittance of the reducing agent (B-mercaptoethanol) from the sample buffer nor N-glycosidase For 0-glycosidase treatment of the axolemma altered the observed gel migratory pattern (data not shown). In contrast to axolemma, myelin membranes contained no G21.3-immunoreactive material (Figure 8, lane 5). The 120and 140 kDa proteins were prominent in total tissue homogenates of adult rat spinal cord, cerebellum, cortex, and sciatic nerve (Figure 8, lanes 6,7,8, and 9, respectively) and wereexpressed at lower levels in liver and kidney homogenates (lanes 10 and 11). The 140 kDa band in these samples did not resolve into a doublet, as was seen in the axolemma; however, it was observed in other Western blots. Conditioned media from cortical neuron cultures were collected and immunoprecipitated with the G21.3 monoclonal. Western analysis of the immunoprecipitate revealed the same 140 kDa doublet and 170 kDa singlet (Figure 8, lane 14), as in IO-day-old axolemma (Figure 8, lane 12). Nonspecific IgM-precipitated, neuron-conditioned media were negative (Figure 8, lane 13). The presence of the G21.3 antigen in neuronconditioned media is in agreement with its basal lamina localization. All of the protein samples analyzed in FigureSwere prepared to minimize proteolytic degradation (see Experimental Procedures).

Discussion

The mechanism of myelin wrapping along selected axons in the CNS is largely unknown. Observations by several investigators suggest that neuronal, ECM, and perhaps astroglial signals are involved (for review see Waxman, 1987; Hildebrand et al., 1993; Hardy and Reynolds, 1993). W e have isolated and partiallycharacterized an anti-axolemma monoclonal antibody, designated G21.3, that inhibits myelination in cerebellar slice cultures, using a recently reported functional

assay (Notterpek et al., 1993). The myelin inhibitory effect of this antibody appears specific, since numerous other white matter- and axolemma-reactive clones do not display this functional effect. G21.3 antigen is ab sent from the cell surface of oligodendrocytes and from isolated myelin membranes, indicating that the antibody interferes with a nonoligodendroglial signal required for proper myelination. In this report, the function-blocking effect of the G21.3 monoclonal antibody is documented by several approaches. Biochemically, the measurement of 35S incorporation into sulfolipid was used. The inhibition byG21.3waslessthan that seenwithapositivecontrol monoclonal, anti-GalC, a known inhibitor of myelin production by oligodendrocytes (Dyer and Benjamins, 1988). This would be consistent for an antibody that does not bind directly to the myelin-forming cell, but instead interferes with an external signal that is required for membrane synthesis by axon-associated oligodendrocytes. lmmunocytochemical detection of MBP, a component that constitutes 3 0 % of all myelin proteins (Hildebrand et al., 1993), indicated a dramatic decrease in myelin along NF-positive processes and a preservation in areas in which oligodendrocytes were out of contact with neurons. This supports a mechanism whereby the G21.3 antibody interferes with a signal that is required for oligodendrocyte-axon association, followed by myelin production and deposition along internodal regionsof axons. Ultrastructural studies of the slice cultures indicated that the lack of myelination was not due to cell toxicity, since normal neuronal and oligodendrocytic profiles were clearly identifiable in the samples. In addition, the specificity of the inhibition of myelination by G21.3 was documented by Western blot analysis. Whereas MBP and PLP protein levels were severely reduced, there was considerably less effect on CNP. This finding is analogous to that in the shiverer mouse mutant, which has near normal levels of CNP but is severely deficient in compacted myelin (Mikoshiba et al., 1980). Given the functional significance of this antibody, we have begun to investigate the nature of the G21.3 antigen. The intense immunoreactivityin well-myelinated regions of the cerebellum suggests a function for the antigen in the maintenance of myelin, in addition to an early role in myelin deposition, indicated by the in vitro assay. The high abundance of the G21.3 antigen in highly proliferative regions of the cerebellum suggests a multifunctional molecule. Adhesion molecules with multiple functional domains involved in CNS development have been identified (Reichardt and Tomaselli, 1991), and the G21.3 antigen may be such a molecule. In vitro studies showed that the antigen is present on the surface of cultured neurons and is secreted into the culture media. A function-blocking antibody would be expected to bind a cell surface, secreted, or ECM molecule. The presence of the G21.3 antigen on neuronal membranes supports a model whereby axon-oligodendroglia contact regulates myelin synthesis and deposition. Our current data do

not rule out the possibility that the antigen present on neurons is made by other CNS cell types, such as astrocytes or microglia. However, in vivo immunocytochemical studies do not colocalize the G21.3 antigen with specific markers for either of these cell types (L. M. N. and L. H. R, unpublished data). The G21.3 Western blots identified four proteins in young axolemma, which are found at lower levels in adult axolemma but are completely absent from myelin. To date, a specific axonal molecule with a proven functional involvement in CNS myelination has not been identified. The G21.3-immunoreactive proteins in the IO-day-old axolemma could be such a component. It is interesting to note the low levels of the G21.3 antigen in adult axolemma (Figure 8, lane 4), since it is abundant in a total homogenate of the adult brain stem (lane 7). This observation suggests a change in the association of the antigen from the axonal membrane, where it appears to be present during development, to the ECM in the adult. This finding may indicate similarities between the G21.3 antigen and adhesion molecules that are immobilized in the ECM (e.g., N-CAM and myelin-associated glycoprotein; Sanes, 1989). Most of the G21.3 antigen can be extracted from adult white matter at physiological salt concentrations in the presence of 10 mM EDTA, in the absence of detergents (data not shown). This biochemical property might be expected from a loosely associated membrane protein and is consistent with the presence of the antigen in the media of neuronal cultures (Figure 8, lane 4). In vivo tissue distribution and biochemical properties of the G21.3 antigen do not support an identity with previously characterized ECM or adhesion molecules. For example, based on the lack of carbohydrate modifications susceptible to N- and 0-glycosidases, many of these moleculescan be ruled out. In addition, the G21.3 antigen is not recognized by the HNK-1 monoclonal, ruling out numerous proteins that express this common epitope. Furthermore, the antigen does not appear to form disulfide dimers, as has been documented for integrins and fibronectin. The in vivo tissue distribution of the G21.3 antigen and its regional specificity in the brain differ from that of any well-characterized adhesion or ECM molecule. The existence of novel yet unidentified ECM molecules in the brain has been proposed (Sanes, 1989). The functional effect of the G21.3 antibody is specific, and it has been established by four different approaches. G21.3 appears to be the first axolemmareactive monoclonal antibody that inhibits CNS myelination and suggests a direct involvement of axons in this process. A definitive role for the G21.3 antigen in the extracellular milieu would point to a role for the ECM in CNS myelination similar to that observed in the PNS. Experimental

Procedures

Monoclonal Antibody Antibody-producing

Production hybridomas

and Screening were generated

by the fusion

of immunized BALBlc mice splenocytes with X63-Ag8.653 myeloma cells (Milstein, 1980). Axolemma-enriched fractions from rat CNS white matter (DeVries, 1980) were used as antigens. Axolemma preparations were enriched in glycoproteins by chromatography on concanavalin A and wheat germ agglutinin affinity columns, as previously described (Hampson and Poduslo, 1987). Mice were immunized by repeated injections of loo-250 ftg of membrane preparation in phosphate-buffered saline (PBS) mixed I:1 with Freund’s adjuvant (Harlow and Lane, 1988). Serum antibody titers of each animal were monitored monthly by Western blots against the respective antigen. Only strongly reactive animals were used for fusions. Hybridoma supernatants were screened by dot blots against native rat liver and axonal membranes (Bigbee et al., 1985). Selected axolemma-reactive clones were rescreened by immunohistochemistry on fresh frozen rat brain sections (see below). White matter-reactive clones were subcloned and rescreened as before. lsotypes of the secreted antibodies were identified with Mouse-Typer Sub-lsotyping kit from Bio-Rad (Richmond, CA), according to the manufacturer’s instructions. Antibody titers were determined by ELISA (Bigbee et al., 1985). Ascites were produced in BALB/c mice for higher titer antibodies by using standard protocols (Harlow and Lane, 1988). In Vitro Myelination Assay Cetebellar slice cultures were established from newborn rat brains as previously described (Notterpek et al., 1993). Briefly, 400 Pm thick slices were cultured on coverslips coated with type I collagen, poly-L-lysine, or astroglial matrix (Rome et al., 1986) in4well plates.Theculturemediumwasmodulatedoverthefirst 11 days in vitro, to promote initial adhesion and proliferation, followed by differentiation. Myelin production was determined by the appearance of birefringent fiber tracts and by positive immunoreactivity of known myelin markers (see below). Quadruplicate cultures were randomly assigned antibodies or maintained as controls. Slices were incubated for days IO-21 in vitro with control hybridoma media (media that had not been exposed to the cells), with control antibody diluted in the same hybridoma media, or with experimental anti-axolemma supernatants at 1:5dilution (approximately equivalentto IO-20 pg/ml antibody protein, as determined by ELISA). Cultures were fed every other day, and antibodies were replenished every 24 hr. Sulfolipid Synthesis SliceandoligodendrocytecuIturesatday21 invitrowereassayed for myelin synthesis (Silberberg et al., 1972) by the incorporation of 5 PCi per well [35S]S0, (ICN, Costa Mesa, CA) into sulfolipid. Slices were incubated for 48 hr, whereas oligodendrocytes were incubated for 24 hr in complete low sulfate Dulbecco’s modified Eagle’s medium (DMEMYFIZ (Sigma, St. Louis, MO) with the radioisotope. Sulfolipids wereextracted and assayed as previously described (Cardwell and Rome, 198813). Anti-axolemma and antiGalC antibody supernatants (Ranscht et al., 1982) were added at I:5 dilution to the cultures, on days IO-21 in vitro, including the period of metabolic labeling. Control mouse IgM (Sigma) was diluted to concentrations identical to those of the experimental antibodies. For each condition, a minimum offourcultures were used, and experiments were repeated at least three times. We employthe term equivalent brain ageto mean thedays in culture plus the age of the rat pups at the time of their sacrifice (1 day in these studies). Morphological Studies at the Electron Microscopic Level Control and antibody-treated cerebellar slice cultures were processed as previously described (Notterpek et al., 1993). Samples werefixed for 12 hr in 2% paraformaldehyde, 2% glutaraldehyde, rinsed in 0.1 M Dulbecco’s PBS, osmicated, dehydrated in graded ethanols, cleared in propylene oxide, and embedded in EponAraldite. Small specimens from comparablewhite matter regions were dissected for subsequent flat embedding. After thin sectioning and poststaining in lead citrate, the specimens were observed by transmission electron microscopy on a ]EOL 100-E)! electron microscope. Ultrastructural observations were documented by photomicrographs.

Role for Axolemma 483

in CNS Myelination

Dissociated Cortical Cultures Cortical neuronal cultures were established from day 14-18 embryonic rat brains by the method of Aizenman and de Vellis (1987). Cells were maintained in 10% serum containing DMEMl F12 or in NZ-supplemented (GIBCO BRL, Grand Island, NY; Bottenstein and Sato, 1979) serum-free DMEM/F12 media. The addition of 15 m M KCI to the growth medium greatly enhanced the survival of neurons (unpublished data). Cultures in the serumcontaining media were treated with 20 Rg/ml 5-fluoro-2’-deoxyuridine to decrease glial proliferation (Wuarin and Sidell, 1991). From embryonic day 21, the serum content was progressively changed from all fetal calf to all calf serum. Samples were used for immunocytochemistry at postnatal days 5-14. Immunoprecipitation of neuron-conditioned media was performed as described (Scarpa et al., 1992). G21.3 ascites or supernatant was added after preclearing with nonspecific IgM (Sigma) and protein A Sepharose CL4B (Pharmacia LKB, Sweden) conjugated to anti-mouse immunoglobulin from rabbit (ICN Biochemicals, Inc., Costa Mesa, CA). Specific immunoprecipitate was collected in the same manner, substituting the G21.3 monoclonal for the nonspecific IgM. Samples were washed 4-6 times with immunoprecipitation buffer and analyzed on SDS gels. C21.3-immunoreactive proteins were identified by subsequent Western analysis (see below). Oligodendrocyte cultures were established as previously described (Rome et al., 1986). lmmunocytochemical Studies For all immunocytochemical studies, the same secondary antibodies, goat anti-rabbit IgC, L-rhodamine conjugate (Boehringer Mannheim Corp., Indianapolis, IN), and goat anti-mouse IgG and IgM fluorescein conjugate (Boehringer Mannheim; both at dilutions of 1:250) were used. Samples were incubated with the secondary antibodies for 2 hr at room temperature in the dark. Observations were documented by photomicrographs using a Nikon Microphot-FXA microscope. Cerebellar S/ice Cultures Slices, as whole mounts, were examined as previously described (Notterpek et al., 1993). Briefly, after fixation in 4% paraformaldehyde, followed by blocking and permeabilization, samples were incubated with primary antibodies. Polyclonal anti-MBP (gift of Dr. Anthony Campagnoni, UCLA) and monoclonal anti-NF (SMI 31; Sternberger Monoclonals, Inc., Baltimore, MD) antibodies were added overnight at 4OC in PBS. The anti-MBP antibody was preabsorbed against fixed astrocytes to decrease cross-reactivity toward glial fibrillary acidic protein. lmmunoreactivity was revealed by incubations of fluorochromeconjugated secondary antibodies (see above). Samples were mounted in glycerol, PBS and photographed. Control samples without the primary antibodies were completely negative. In Vitro Cultures Neurons and oligodendrocytes were immunostained with the G21.3 monoclonal antibody. Live samples were rinsed with cold PBS and incubated for 16 hr at 4OC with G21.3 hybridoma supernatant at l:l-I:20 or ascites at 1:200. After four 5 min rinses in PBS, cells were fixed in freshly prepared 4% paraformaldehyde for 10 min at room temperatureand blocked with 5% goat serum in PBS for 30 min. Secondary antibody incubations were performed as above. Parallel cultures for intracellular antigen detection were rinsed, fixed, rinsed, blocked, and permeabilized, prior to the primary antibody incubation. In addition to the G21.3 monoclonal, a polyclonal antiserum to NSE (Polysciences, Inc., Warrington, PA) at I:1000 was added. Live oligodendroglial cultures were immunolabeled with anti-CalC (Ranscht et al., 1982). Samples were washed in PBS, and secondary antibodies were used as above. Samples incubated with control mouse IgM (Sigma)orwithout primary antibodies werecompletely negative. Fmzen Tissue Sections Fresh frozen sections were cut at 5-10 urn thickness and dried onto SuperfrostIPlus microscope slides (Fisher Scientific). After a l-2 min quick fixation in alcoholic paraformaldehyde (90% ethanol, 1% paraformaldehyde) (Bigbee et al., 1985), sections were rinsed for 10 min in two changes of PBS and blocked in 10% goat serum in PBS for 30 min at room temperature. Monoclonal

C21.3 supernatant or ascites were used as above, followed by washes and secondary antibody incubation. For the colocalization studies, polyclonal anti-laminin and anti-fibronectin (GIBCO BRL), both at l:lOOO, and anti-MBP (as above) were used, followed by secondary antibody incubation, as above. Slides were mounted in glycerol, PBS, sealed with nail polish, and photographed. Biochemical Procedures Axolemma and myelin prepared by the method of DeVries (1980) were resuspended in 10 m M TES (pH 7.5) containing 2 ug/ml aprotinin, 500 uM benzamidine, 2 ug/ml chymostatin, 5 uM leupeptin , 5 GM pepstatin, and 1 m M phenylmethylsulfonyl fluoride (all from Boehringer Mannheim) and were solubilized in 10% SDS. Fresh tissue and in vitro slice cultures were directly homogenized in SDS sample buffer. Protein was measured by the BCA protein assay (Pierce, Rockford, IL; Smith et al., 1985). Sampleswere boiled for5 min, in the presenceof 2% B-mercaptoethanol, and SDS-polyacrylamide gel electrophoresis was performed according to Laemmli (1970). Gels were transferred to nitrocellulose membranes overnight (Towbin et al., 1979) and briefly stained in Ponceau S to confirm protein levels. Membranes were blocked for l-2 hr at room temperature in 10% nonfat milk in PBS. Primary antibodies were added overnight at 4OC, in 5% sheep serum containing PBS. Polyclonal anti-MBP (gift of Dr. Anthony Campagnoni, UCLA) at 1:2000, polyclonal anti-PLP (generously provided by Dr. Wendy Macklin, UCLA) at 14000, and polyclonal anti-CNP (gift of Dr. David Colman) at I:1000 were used. Monoclonal C21.3 ascites at I:200 and mouse IgM (Sigma) at l-2 Rg/ml were diluted in 5% sheep serum containing PBS. Blots were washed and incubated in sheep anti-rabbit IgC-POD immunoglobulin (Boehringer Mannheim, Federal Republic of Germany) at I:2000 or sheep anti-mouse immunoglobulin horseradish peroxidase conjugate at I:4000 (Amersham,Arlington Heights, IL) in 5% sheepserum containing PBS, for 2 hr at room temperature. After extensive washes, blots were incubated in ECL Western blot detection reagents (Amersham) according to the manufacturer’s instructions. Immunoreactive bands were quantitated by densitometry. Samples processed without primary antibodies were completely negative. N-glycosidase F (PNCase) and 0-glycosidase (Boehringer Mannheim) treatment of IO-day-old axolemma was performed according to the manufacturer’s instructions. Acknowledgments The authors thank P. N. Bullock for help with the slice preparations, D. Baybridge and H. Roseboro for their invaluable assistance with the monoclonal antibody production, and 1. Asai for expert technical assistance with the electron microscopy samples. We thank Drs. R. H. Edwards, S. P. Hamilton, and C. Weinmaster for critical reading of the manuscript. This work was sup ported by U. S. Public Health Service grant HD-06576 and by a grant from the National Multiple Sclerosis Foundation (RC 2200A-l). L. M. N. is a University of California dissertation year fellow and was supported in part by a Share Traineeship Award from the UCLA Mental Retardation Research Center. All correspondence should be addressed to L. H. R. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

November

11,1993;

revised

May 4,1994.

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