Phosphorylation-dependent regulation of axon fasciculation - PNAS

Proc. Nati. Acad. Sci. USA Vol. 88, pp. 10548-10552, December 1991 Neurobiology

Phosphorylation-dependent regulation of axon fasciculation (cell-adhesion molecules/cell-cell adhesion/axon outgrowth/phorbol esters/protein kinase C)

MELCHIORRE CERVELLO*t, VANCE LEMMONt, GARY

LANDRETHM§¶, AND URS RUTISHAUSER**II

Departments of *Genetics, tNeurosciences, and INeurology, and IThe Alzheimer Center, School of Medicine, Case Western Reserve University, Cleveland, OH 44106; and tInstituto di Biologia dello Sviluppo, Italian National Research Council, Palermo, Italy

Communicated by Fernando Nottebohm, August 8, 1991 (received for review February 5, 1991)

Axons often grow along other axons to proABSTRACT duce bundles called fascicles, and a number of cell adhesion molecules (CAMs) found on axon surfaces contribute to this process. The surprising observation that Fab fragments against individual CAMs can completely block fascicle formation suggests that the different axon-associated CAMs are functionally linked. The present studies investigate whether such a linkage might reflect intracellular regulatory mechanisms. Results obtained with chicken retinal explants in culture indicate that fasciculation is highly sensitive to cytoplasmic protein phosphorylation by means of a mechanism that does not alter levels of CAM expression. Moreover, the potent effect of individual Fabs on fasciculation disappears with enhanced phosphorylation. These observations suggest that growing axons possess a general regulatory process for the multiple CAMs that participate in fasciculation.

Over the past few years a number of cell-cell adhesion molecules (CAMs) have been identified on the surface of axonal processes in the vertebrate embryo (for review, see ref. 1). Not surprisingly, most of these molecules appear to contribute to the formation of axon bundles called fascicles. It has been shown that Fab fragments of antibodies specific to each CAM are potent inhibitors of overall axon fasciculation in vitro (2-7). This robust inhibition of an adhesion-related phenomenon is consistent with the proposal that these cell-surface molecules act as receptors in the formation of cell-cell bonds. However, the inhibition also represents an unexplained anomaly: if several of these different adhesion receptors are coexpressed on axons, why does addition of Fab against just one receptor almost completely inhibit bundling? The predicted result, which is, in fact, obtained in simple cell aggregation studies (8) and with axon growth on nonneuronal cells (9, 10) is partial inhibition by each Fab and strong inhibition only by a combination of antibodies. There is growing evidence that complex adhesion-related behaviors of growth cones, such as neurite outgrowth and fasciculation (11-13), can be influenced by nerve growth factor or through second-messenger systems. In this study, we have examined three different adhesion molecules involved in fasciculation, neural CAM (N-CAM) (1, 2), G4/L1 (3, 4), and F11 (4), in terms of the possibility that such anomalous Fab effects may be mediated through intracellular events. The results support the idea that cytoplasmic phosphorylation is a major determinant in neurite bundling and also underlies the ability of Fab fragments against one adhesion molecule to alter the overall state of fasciculation.

G4/Li were prepared against immunoaffinity-purified antigens; polyclonal anti-Fli was from F. Rathjen (Center for Molecular Biology, Hamburg, F.R.G.). Fab fragments were prepared by pepsin digestion and then reduced and alkylated (2). Cultures. Tissue-culture dishes (35 mm; Coming) were pretreated for 3-4 hr at 370C with poly(L-lysine) (Sigma) at 1.5 mg/ml in distilled water. After being washed extensively with calcium-magnesium-free phosphate buffer, pH 7.4, the dishes were incubated overnight at 370C with 1.5 ml of laminin solution at 5 pug/ml in the same buffer. Retinal explants from 7-day chicken embryos were flattened and attached to SM-nitrocellulose filters (Sartorius) (14). The retina-filter sandwich was cut into 0.35-mm strips perpendicular to the optic fissure, and the strips were placed with the ganglion cell layer in contact with the laminin substrate. Explants were incubated in Dulbecco's modified Eagle'sF-12 medium/10% fetal calf serum/2% chicken serum (GIBCO) at 37°C under 5% CO2. After 24-26 hr, fasciculation patterns were observed and photographed by using a Nikon inverted phase-contrast microscope. Antibodies and drugs were present throughout the culture of the retinal strips. Phorbol 12-myristate 13-acetate (PMA) was dissolved in dimethyl sulfoxide. Fab fragments and okadaic acid were dissolved in medium. Neurite outgrowth or fasciculation was not affected by the presence of nonimmune Fab fragments or dimethyl sulfoxide. Quantitation of Adhesion Molecules in Cultured Retina. Whole retinae were excised and placed in culture for 24 hr under otherwise identical conditions as the retinal strips, with and without 10 nM PMA. After being washed with phosphate-buffered saline, the tissue was solubilized in 60 ,ul of SDS/PAGE sample buffer, and 5 ,ul of each sample was loaded onto three slots of an SDS/7.5% PAGE gel. After electrophoresis, the proteins were transferred to nitrocellulose and immunostained with rabbit polyclonal anti-N-CAM, anti-L1, or anti-F11 by using a peroxidase-conjugated second antibody and 2-chloronaphthol. Blots were analyzed with a Shimadzu (Kyoto) densitometer.

RESULTS Fasciculation with Retinal Explants. Neurite fasciculation is a robust phenomenon in vivo and easily reproduced in tissue cultures. However, the degree of bundling is difficult to quantify, and differences are usually expressed in terms of a qualitative judgment of the overall neurite outgrowth pattern. To aid in this judgment, it is necessary to use a tissuesubstrate combination that produces consistent amounts of outgrowth and a moderate level of bundling. Thus, variability from explant to explant is diminished, and both increases and decreases in the size of fascicles are detectable. The culture

METHODS Materials. Okadaic acid was purchased from Diagnostic Chemicals, Oxford, CT; all other drugs were purchased from Sigma. Rabbit polyclonal antibodies against N-CAM and

of thin strips of defined regions of embryonic chicken retina Abbreviations: CAM, cell adhesion molecule; N-CAM, neural cell adhesion molecule; PMA, phorbol 12-myristate 13-acetate. "To whom reprint requests should be addressed at: Department of Genetics, Case Western Reserve University, 2040 Adelbert Road, Cleveland, OH 44106-4901.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Neurobiology: Cervello et al.

Proc. Natl. Acad. Sci. USA 88 (1991)

on a laminin-coated dish (6, 14) meets these criteria. Moreover, experimental perturbations can be compared by using adjacent retinal strips. With this system, reproducible results were obtained in three or more independent experiments and could be adequately represented by photomicrographs. Pharmacological Perturbation of Fasciculation. To investigate whether retinal axon fasciculation might be influenced by intracellular biochemical events, retinal tissue was cultured with a variety of drugs that affect major regulatory pathways. The most dramatic effects were a large increase in bundling with the phorbol ester PMA (5 nM), which activates protein kinase C (15), and okadaic acid (2 nM), which inhibits intracellular phosphoprotein phosphatases 1 and 2a (16) (Fig. 1). These effects were also seen with retinal explants grown on dishes coated with L1, another good substrate for neurite outgrowth (17) (data not shown). Treatment of explants with other agents that either increase or decrease intracellular protein phosphorylation gave consistent results. Thus, fasciculation was increased with dibutyryl-cAMP, decreased with the calcium-channel blocker verapamil, and partially inhibited by the protein kinase inhibitor H7 (data not shown). The thick fascicles produced in the presence of PMA or okadaic acid were also reduced in their length, relative to controls (Fig. 1). Such shortening, as produced by PMA or nerve growth factor, can be reversed by adding antibodies against adhesion molecules (refs. 11, 13; see also Fig. 3h). The rapid growth of the nonfasciculated neurites in the presence of PMA and Fabs argues that the pattern of outgrowth produced with 5 nM PMA does not reflect a primary inhibitory effect on elongation of individual neurites. Instead, the shorter outgrowth of fascicles is probably due to the fact

10549

that most growth cones in large bundles exert their pull on the fascicle rather than on the substrate (11). Effects of Fabs and PMA on Fasciculation. In Figs. 2 and 3 are illustrated typical effects on fasciculation produced by the separate actions of anti-N-CAM Fab, anti-Li Fab, or antiFli Fab each at 0.5 mg/ml as well as by a mixture of all three Fabs. Each anti-Fab condition included or excluded 5 nM PMA. These Fab concentrations are sufficient to maximally inhibit membrane-membrane adhesion. To minimize variations between retinas, each experimental set of four cultures (± Fab, ± PMA) used adjacent pieces of retina cut along the nasal/temporal axis. In addition, the naturally occurring shift in fascicle size along this axis (14) was exploited to match the level of fasciculation in controls. That is, to aid in comparing Fab effects with and without PMA, the effects of PMA alone were compensated for by using the more fasciculated nasal retina for all PMA- cultures and the less fasciculated temporal retina for PMA+ cultures. As expected from previous studies, each Fab without PMA almost completely inhibited fasciculation-that is, most outgrowth appeared as single fibers or thin bundles (Figs. 2 and 3, compare a with b and e withf). However, with PMA each Fab had little or no effect on fasciculation (c vs. d and g vs. h). Under these conditions, a strong decrease in bundling was obtained only by adding a mixture of all three Fabs (Fig. 3, e-h). Although use of nasal and temporal retina helped illustrate our findings, the key findings-that PMA negates individual Fabs, but not a mixture of Fabs-were all obtained with temporal retina. It is also important to note that the Fab mixture completely inhibited fasciculation of axons from both nasal and temporal retinae, indicating that the same adhesion systems are used in both regions of this tissue.

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Effect of PMA on CAM Expression. One mechanism by which PMA could enhance fasciculation would be to increase the levels of adhesion molecule expression. However, the amount of N-CAM, L1, or F11 in the retinal-strip cultures did not change detectably when PMA was added (Fig. 4). Furthermore, the level of N-CAM polysialylation, which has also been proposed to have a global regulatory effects on cell-cell interactions (18), was not changed. DISCUSSION The initial clues that perturbation of cellular biochemistry

could affect patterns of neurite outgrowth came from studies on the response of sensory ganglia to different concentrations of nerve growth factor (11, 19). More recently, these effects of nerve growth factor have been shown to be mimicked by PMA, suggesting that they involve intracellular protein phosphorylation (13) by means of protein kinase C activation (15). Our study begins by extending this pharmacology to nerve growth factor-unresponsive axons. Effects consistent with the action of PMA were seen by using other drugs that can indirectly affect levels of intracellular protein phosphorylation. Of particular relevance is the fact that okadaic acid, a specific inhibitor of intracellular serine/threonine phosphatases 1 and 2A (16), also increased fasciculation markedly.

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FIG. 2. Inhibition of fasciculation by Fabs, with and without PMA. Each horizontal set of four photographs illustrates representative examples from adjacent slices of retina cultured on laminin. With either antibodies against Li or N-CAM there is a strong inhibition of bundling without the drug but only a weak effect with 5 nM PMA. To provide a similar fasciculation level in the four ex-

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retina was used in a, b, e, f, and temporal (T) retina was used in c, d, g, h (see text). (Bar = 100 gm.)

The most unusual finding has been that PMA can neutralize the ability of Fab against one CAM to block overall fasciculation, whereas PMA does not affect the level of inhibition obtained with a mixture of Fabs against three different CAMs. Together, the drug and Fab effects suggest that there is a phosphorylation-dependent link between the function of different axon-associated adhesion systems. Three very different types of mechanisms could account for the Fab and PMA effects on fasciculation. The most obvious would be that PMA augments CAM expression to the extent that any one Fab can no longer overcome the enhanced adhesion. This possibility seems unlikely because there was no detectable change in the expression level of any of the three CAMs studied. A similar argument could be advanced that a decrease in polysialic acid content of N-CAM might enhance overall CAM function (18), but again no change in N-CAM sialylation was seen. A more complex alternative would be a nonlinear or threshold relationship between adhesion and neurite bundling plus activation of another and dominant fasciculation system by PMA. Thus, one Fab would decrease total adhesion below a critical threshold required for fasciculation, whereas PMA would increase adhesion above that threshold, even in the presence of the three Fabs. While nonlinearity


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(20) and threshold effects (21) have been observed for CAM function in aggregation and neurite-outgrowth assays, the anomalous Fab behavior described here is not seen in such systems (8-10). Moreover, although intracellular protein phosphorylation can alter the effectiveness of known adhesion systems (see below), there is no evidence to suggest that a new and dominant cell-cell adhesion system has been induced by PMA. The third and most interesting explanation is that the function of CAMs on growing axons is regulated by a common phosphorylation-dependent event that can be influenced by binding of a ligand, in this case polyclonal Fab. There is experimental precedent for both Fab-induced changes in intracellular biochemistry and the ability of phosphorylation to affect neurite behavior. Polyclonal Fabs against N-CAM and Li on PC-12 cells have been shown to reduce levels of inositol phosphates, lower intracellular pH, and increase intracellular Ca2+ (22). Conversely, drugs that alter intracellular protein phosphorylation can influence the outgrowth of neurites (12, 13, 23). It may also be relevant that cell surface molecules with extracellular homology to

N-CAM can have cytoplasmic domains with phosphoprotein phosphatase activity (24). Despite these arguments, the vast range of effects that can be attributed to intracellular phosphorylation, plus the molecular and cellular complexity of fasciculation, preclude a definitive mechanistic interpretation of the phenomena reported here. Nevertheless, the possible relationship of our observations to an interesting and biologically useful regulatory mechanism for growing axons prompts further comment. In this spirit, Fig. 5 illustrates a scheme by which intracellular phosphorylation could regulate fasciculation. From the known actions of protein kinase C (15) and phosphatases 1 and 2A (16), it is likely that phosphorylation of serine/threonine residues of'cytoplasmic polypeptides is involved. The apparent ability of protein phosphorylation to influence several CAMs simultaneously is the most intriguing aspect of our studies. The most direct regulatory mechanism would be that each CAM has a phosphorylation-sensitive cytoplasmic domain that influences its own adhesive function. Alternatively, phosphorylation could affect an additional cytoplasmic component(s) that interacts less directly

10552


Neurobiology: Cervello et al.

which is based on the assumption that polyclonal Fab fragments can mimic the natural complementary receptors for CAMs, is also motivated by biological considerations. In migrating along other neurites, growth cones have to adhere to and pull on the substrate to effect motion and stabilize position but also must detach from it to allow translocation. In considering our present findings, it is interesting to speculate that the migrating growth cone uses phosphorylationregulated adhesion to produce a "caterpillar drive," attaching at its leading edge and detaching at its trailing edge. This mechanism would be analogous to the rapid make-and-break adhesions of T lymphocytes, which are facilitated by a protein kinase-dependent negative feedback loop triggered by adhesion itself (31). Finally, in the larger context of neural tissues, the ability to detach adhesions actively could represent an important element of plasticity in the formation and remodeling of pathway and innervation patterns.

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FIG. 4. Effect of PMA on expression of adhesion molecules by retinal explants in culture. Densitometric scans of SDS/PAGE immunoblots of retinal extracts after staining with polyclonal antibodies against F11, L1, and N-CAM. Upper and lower lines of each pair represent tissues cultured without and with PMA, respectively. The scans, with pairs offset vertically for easier visualization, are essentially identical for each CAM, indicating that PMA has no effect on CAM expression or on polysialylation of N-CAM.

and more generally with CAM function. Intracellular portions of N-CAM and Li are known to be phosphorylated (25-27). Although these observations are consistent with regulation by phosphorylation of CAMs, F11 is an extrinsic membrane protein linked to the membrane by means of phosphatidyl inositol (28), which would argue for a less direct mechanism of control. The basis for regulation of adhesive function also remains an open question. Effects on cell-cell adhesion could reflect changes in receptor affinity by means of cytoplasmic phosphorylation (29, 30). On the other hand, these effects could also occur through changes in overall avidity produced by a phosphorylation-dependent receptor redistribution, possibly involving cytoskeletal components. Also included in

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FASCICLE ---' FIG. 5. Hypothetical model for phosphorylation-dependent regulation of fasciculation. Formation of fascicles is depicted as requiring both receptor-binding activity and intracellular phosphorylation of as-yet-unidentified protein(s). Solid arrows represent experimental results documented in this report. Dashed arrows represent the proposal that Fabs against individual adhesion receptors can inhibit overall neurite-neurite interaction by means of inhibition of the phosphorylation event and that this inhibition mimics a natural negative feedback of fasciculation by means of the same pathway.

We thank Wendy Elmslie, Denice Major, and David Starke for excellent technical assistance. This work was supported by National Institutes of Health Grants HD18369 and EY06107 to U.R., EY05285 to V.L., GM34908 and National Science Foundation Grant BNS%302 to G.L. 1. Rutishauser, U. & Jessell, T. (1988) Physiol. Rev. 68, 819-857. 2. Rutishauser, U., Gall, W. E. & Edelman, G. M. (1978) J. Cell Biol. 79, 382-393. 3. Stallcup, W. B. & Beasley, L. (1985) Proc. Nat!. Acad. Sci. USA 82, 1276-1280. 4. Rathjen, F. G., Wolff, J. M., Frank, R., Bonhoeffer, F. & Rutishauser, U. (1987) J. Cell Biol. 104, 343-353. 5. Ruegg, M. A., Stoeckli, E. T., Lanz, R. B., Streit, P. & Sonderegger, P. (1989) J. Cell Biol. 109, 2363-2378. 6. Drazba, J. & Lemmon, V. (1990) Dev. Biol. 138, 82-93. 7. Rathjen, F. G., Wolff, J. M., Chang, S., Bonhoeffer, F. & Raper, J. A. (1987) Cell 51, 841-849. 8. Rathjen, F. G. & Rutishauser, U. (1984) EMBO J. 3, 461-465. 9. Bixby, J. L., Pratt, R. S., Lilien, J. & Reichardt, L. F. (1987) Proc. Natl. Acad. Sci. USA 84, 2555-2559. 10. Neugebauer, K. M., Tomaselli, K. J., Lilien, J. & Reichardt, L. F. (1988) J. Cell Biol. 107, 1177-1187. 11. Rutishauser, U. & Edelman, G. M. (1980) J. Cell Biol. 87, 370-378. 12. Bixby, J. L. (1989) Neuron 3, 287-297. 13. Hsu, L. (1989) Anat. Embryol. 179, 511-518. 14. Halfter, W., Newgreen, D. F., Sauter, J. & Schwarz, U. (1983) Dev. Biol. 95, 56-64. 15. Heasley, L. E. & Johnson, G. L. (1989) J. Biol. Chem. 264, 8646-8652. 16. Haystead, T. A. J., Sim, A. T. R., Carling, D., Honnor, R. C., Tsukitani, Y., Cohen, P. & Hardie, D. G. (1989) Nature (London) 337, 78-81. 17. Lagenauer, C. & Lemmon, V. (1987) Proc. Nat!. Acad. Sci. USA 84, 7753-7757. 18. Rutishauser, U., Acheson, A., Hall, A. K., Mann, D. M. & Sunshine, J. (1988) Science 240, 53-57. 19. Levi-Montalcini, R. & Angeletti, P. U. (1968) Physiol. Rev. 48, 534-569. 20. Hoffman, S. & Edelman, G. M. (1983) Proc. Nat!. Acad. Sci. USA 80, 5762-5766. 21. Doherty, P., Fruns, M., Seaton, P., Dickson, G., Barton, C. H., Sears, T. A. & Walsh, F. S. (1990) Nature (London) 343, 464-466. 22. Schuch, U., Lohse, M. J. & Schachner, M. (1989) Neuron 3, 13-20. 23. Mattson, M. P., Taylor-Hunter, A. & Kater, S. B. (1988) J. Neurosci. 8, 1704-1711. 24. Hunter, T. (1989) Cell 58, 1013-1016. 25. Sadoul, R., Kirchoff, F. & Schachner, M. (1989) J. Neurochem. 53, 1471-1478. 26. Mackie, K., Sorkin, B. C., Nairn, A. C., Greengard, P., Edelman, G. M. & Cunningham, B. A. (1989) J. Neurosci. 9, 1883-18%. 27. Salton, S. R., Shelanski, M. L. & Greene, L. A. (1983) J. Neurosci. 3, 2420-2430. 28. Wolff, J. M., Brummendorf, T. & Rathjen, F. G. (1989) Biochem. Biophys. Res. Commun. 161, 931-938. 29. Sibley, D. R., Benovic, J. L., Caron, M. G. & Lefkowitz, R. J.

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