Oligodendrocyte- and myelin-associated inhibitors of neurite outgrowth: Their involvement in the lack of CNS regeneration

EXPERIMENTAL

115,

NEUROLOGY

189-192

(19%‘)

Oligodendrocyte- and Myelin-Associated Inhibitors of Neurite Outgrowth: Their Involvement in the Lack of CNS Regeneration D. S. CADELLI, Brain

Research

Institute,

University

C. E. BANDTLOW, of Zurich,

AND M. E. SCHWAB

August-Forel-Str.

1, CH-8029

Zurich,

Switzerland

from developmental studies that neurite outgrowth is dependent on and regulated by extrinsic factors. Basically, neuronal cells respond to soluble trophic factors and substrate molecules. Neurotrophic and chemotropit factors as well as a number of adhesion and growth promoting substrate molecules are present in the developing CNS, and some of them also are detectable in the adult CNS (5-7). The existence of nonpermissive substrate effects was shown in a three-compartment chamber culture experiment; living or frozen peripheral and optic nerves were placed as bridges between culture chambers, and dissociated neurons plated in a central chamber had access to both the bridges (8). Neurons, central or peripheral, were grown with optimal trophic factor support. A drastic difference in neurite outgrowth was observed between the two bridges: the sciatic nerve explant contained several hundred axons while the optic nerve remained totally empty (8). These results pointed to a nonpermissive substrate effect of the CNS tissue. To further determine if it was topographically organized, slices of various CNS regions and of peripheral nerves were used as substrates for neuronal cultures. Neurons attached and grew very well on the PNS slices and to an extent on the CNS gray matter. However, adhesion was poor and neurite outgrowth was absent on the CNS white matter (9).

Until now central nervous system (CNS) neurites have been thought to have little capacity for regeneration following a lesion. When allowed to grow into peripheral nervous system (PNS) grafts, however, lesioned CNS axons are known to regenerate. Recently, an inhibitory substrate effect of CNS myelin and oligodendrocytes has been discovered which could be directly involved in the lack of regeneration. In culture, neurite growth cones were shown to specifically arrest their movement when contacting oligodendrocyte processes. The inhibitory components were characterized as two proteins of 35 and 250 kDa. A specific monoclonal antibody was generated (IN-l) that could neutralize these inhibitory effects. The role of the inhibitors in CNS regeneration was investigated in young rats receiving lesions of the corticospinal tract and implanted with a source of IN-1 mAB or control mAB. Results showed clear regeneration to over 10 mm in 2-5 weeks in IN-1 mAB-treated animals, while no fibers were detected further than 1 mm caudal to the lesion in controls. A similar, highly significant enhancement of regeneration was also found for the cholinergic septohippocampal pathway and for the optic nerve. These results show that lesioned CNS neurons can regenerate in CNS tissue when specific myelin components are neutralized, thus demonstrating that these inhibitory components play a crucial role in the lack of CNS regeneration. 0 1992 Academic Press, Inc.

INHIBITORY COMPONENT LOCALIZED ON OLIGODENDROCYTES

INTRODUCTION The lack of axon regeneration in the differentiated vertebrate central nervous system (CNS) was recognized and extensively described about a century ago (1). In contrast to the CNS, powerful regenerative processes take place in the peripheral nervous system (PNS), where they can lead to functional restoration. When given a peripheral nerve transplant to grow in, CNS neurons too are able to elongate over long distances (see, for example Refs. (2 and 3)) and even to successfully reinnervate the deafferented target region (4). Thus, the CNS tissue itself seemsto represent an unfavorable environment for neurite growth. It is known

To determine what cells were involved in this interaction, glial cell cultures containing astrocytes, oligodendrocytes and glial precursor cells, were prepared from perinatal rat optic nerve, and, after 2 days, peripheral ganglion or retinal neurons were added with appropriate amounts of trophic factors. Neurons grew well and after 2 weeks developed a dense neuritic network. Confirming previous observations, astrocytes were a good substrate for neurite outgrowth (see for instance Refs. (10 and 11)). Strikingly, however, neurites were avoiding a type of highly branched process bearing cells which, on the basis of antibody staining, was recognized as differentiated oligodendrocytes (12). To get a dynamic picture of these cell interactions,

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CADELLI,

BANDTLOW,

these co-cultures were continuously observed under video time lapse cinematography (13). As noted previously, perinatal astrocytes behaved as a good substrate and neurons grew over them with unchanged velocity. In contrast, as soon as a neurite growth cone touched an oligodendrocyte process, the growth cone collapsed and either remained immobile for hours or retracted toward the cell body. The other neurites of the affected neuron still continued to grow. Oligodendrocytes thus specifically exert a local inhibition of neuronal growth through a contact-mediated mechanism. BIOCHEMICAL INHIBITORY

CHARACTERIZATION COMPONENTS NI-35

AND

OF THE NI-250

The substrate properties of CNS myelin, the product of differentiated oligodendrocytes in uiuo, were tested by adsorbing adult rat spinal cord myelin on culture dishes: CNS myelin strongly impaired neurite outgrowth, while PNS myelin or membrane fractions from different nonneuronal tissues tested in the same way were good substrates (14). CNS myelin was subsequently used for biochemical studies. The inhibitory effect was abolished through protease treatment but not by lipid extraction. Myelin proteins were thus fractioned on SDS-PAGE, and the fractions were tested for their substrate quality upon reconstitution into liposomes and adsorption on culture dishes. Two highly inhibitory, minor protein fractions were isolated at 35 and 250 kDa; they were called the neurite growth inhibitors NI-35 and NI-250. These components have now been highly purified by HPLC from rat, bovine, and human white matter (Bandtlow, unpublished results). NI-250 appears to be a complex containing NI-35. Sequencing of the molecule is proceeding presently in the laboratory. NI-35/250 are tightly membrane-bound proteins and were not found in the PNS myelin nor in the trout or goldfish spinal cord myelin. They do not bind the lectin peanut agglutinin (Bandtlow, unpublished observations). Thus they are distinct from the repulsive components described in the chicken embryonic posterior tectum (15) and in the posterior half somite of chick embryos (16). NEUTRALIZING NEURITE

ANTIBODIES AGAINST GROWTH INHIBITORS

AND

SCHWAB

were seen to profusely grow in over several millimeters (17). In control optic nerves injected with an anti-galactocerebroside antibody no neurite ingrowth could be observed beyond 1 mm. INHIBITORS

AND

REGENERATION

Corticospinal Tract

The results mentioned above showed that the neurite inhibitors were the crucial components in the inhibitory substrate property of the CNS tissue and raised the question of their role in uiuo for the lack of regeneration in the CNS. The corticospinal tract (CST) was chosen because of its accessibility and its well-known anatomy. At 2-7 weeks of age rats received a transection of the dorsal two-thirds of the spinal cord; after a 2 to 4-week survival time CST axons were labeled by wheat germ agglutinin-horseradish peroxidase (WGA-HRP) injection into the frontal cortex. Two experimental models were designed: in a first series of experiments rat spinal cords were X-irradiated at birth preventing the development of oligodendrocytes while neurons and astrocytes were preserved (18). In a second series of experiments, IN-l antibody producing hybridoma cells were implanted into the brain where they developed small tumors secreting large amounts of the neutralizing antibody into the cerebrospinal fluid (19). In both cases complete longitudinal series of sections allowed a reliable reconstruction of the axonal growth. In both control and experimental rats, axons attempted to circumvent the lesion and many reached its distal edge. All regeneration distances were measured from this point in both groups. Control animals showed abortive regeneration reaching at most 1 mm caudal to the lesion. In X-irradiated or IN-l-treated rats, however, axons were able to regrow over long distances, elongating from 5-18 mm. The longer distances are close to or reach the total length of the original extent of these fibers. Despite the very different treatments, the same results were obtained with IN-l antibodies or X-irradiation. THE

ACETYLCHOLINESTERASE-POSITIVE SEPTOHIPPOCAMPAL TRACT

THE

Two monoclonal antibodies (IN-1 and IN-2) were produced against gel-purified NI-35 and NI-250 that could effectively neutralize both inhibitory activities (17). In particular, in the co-culture experiments antibodies (IN-l) successfully prevented the oligodendro@e-mediated inhibition and growth cones were able to extend processes and grow over them (13). In a further decisive experiment, the antibodies were injected into optic nerve explants, and co-cultured sensory neurons

This well characterized and accessible tract is of particular interest, because of its known regenerative potential and response to a trophic factor (NGF). A complete lesion of the septohippocampal tract was performed on 3-week-old rats by aspirating the fornix and fimbria. The septum was reconnected to the rostra1 tip of the hippocampus by a bridge of laminin-rich placenta basement membranes placed on a nitrocellulose filter support (20). NGF was present in the bridge, and the brains were implanted with IN-1 or anti-HRP antibody secreting hybridomas (20). After 2-5 weeks of survival,

INHIBITORS

OF

NEURITE

series of tissue sections were reacted for acetylcholinesterase (AChE). Regenerating fascicles of cholinergic fibers were seen to cross the bridge and enter the hippocampus, where they developed a dense fiber network in both control and experimental animals. In controls, however, the growth of the AChE-positive fibers remained limited to a maximum of 1 mm caudally and laterally from the point of entry into hippocampus. In contrast IN-l antibody-treated rats showed fibers elongating for 2-4 mm in both directions (20). The fibers were arranged in those layers and regions that normally receive a dense cholinergic innervation. Thus, they partly reconstituted their original anatomical distribution, a finding which is in agreement with previous observations after much longer periods of regeneration (21-23). This result shows that neurite growth inhibitors are crucially involved in regenerative processes in this system as well, and neutralizing these inhibitory factors considerably increases the regeneration distance or velocity. It has recently been shown that the regenerative response of this fiber system is also greatly enhanced through NGF infusions into the hippocampus (24). Thus, the septohippocampal system is illustrative in showing that axonal regeneration might be regulated by multiple factors. It will be highly interesting to try to maximize the regrowth by playing with a combination of these two or more regulatory factors. OPTIC

NERVE

In this strongly myelinated system, oligodendrocytes expressing the neurite growth inhibitors were eliminated through local X-irradiation of the optic nerve at birth. The chiasma was spared from irradiation because of the vital structures lying around it. Rats were then lesioned intracranially between 2 and 3 weeks of age. The optic nerve was visualized by aspiration of parts of the forebrain, and a complete cryolesion (three times, freezing with a liquid nitrogen-cooled probe) was performed. Basic fibroblast growth factor (bFGF) was supplied intraocularly and at the site of the lesion to prevent ganglion cell death (25). After l-2 weeks of survival, retinal ganglion cells were labeled through a WGA-HRP injection into the eye (Cadelli, Schnell, and Schwab, unpublished results). Regrowth of the labeled fibers was followed over complete series of longitudinal sections. Minimal regrowth responses, less than the lmm sprouting distance, could be seen in the unirradiated, normally myelinated control rats. In the irradiated, myelin- and inhibitor-free rats, however, regenerating fibers could be followed for 2.5-3.5 mm. Fibers were seen to stop at the chiasma, where immunofluorescence studies showed a preserved intense myelin staining. Results in this system show again the crucial role of the myelin-associated inhibitors and are very encouraging for future studies of target reinnervation.

191

GROWTH

CONCLUDING

NOTES

The question of the molecular characterization of NI35/250 should soon be answered through the sequencing and cDNA cloning of the molecules, which are under way. These results will show the possible homologies or relatedness of these molecules to other membrane proteins and other molecules with repulsive activities (15, 16). The evolutionary meaning of the neurite growth inhibitors will be of great interest since they appear in higher vertebrates. Moreover, in contrast to the other known inhibitory molecules (15, 16), they are not only repulsive but strongly inhibitory, and they are not only seen transiently during development but are permanently expressed. NI-35/250 appear late in development and may thus act as stabilizers of CNS structures. They also have been shown to play a guidance role, as “guard rails,” for a late growing CNS tract (26). Future trends in the field of regeneration will be to improve the inactivation of the neurite growth inhibitors (new antibodies or antibody fragments) and to further dissect and analyze the various factors that, in combination with the inhibitors, may regulate regeneration in the CNS. Four points deserve particular attention: neutralization of the NI-35/250 should be as complete as possible and long-lasting (e.g., infusion of antibody fragments); the intrinsic neuronal potentials to initiate and pursue regeneration that appear to differ in various tracts or subsets of neurons should be defined; the conditions at the lesion site like caverns, glial scars, and inflammatory cells have to be better studied, and finally trophic support for regenerating fibers, which clearly seems to be a limiting factor in some cases, should be investigated and optimized. ACKNOWLEDGMENTS This work is supported by grants from the Swiss National Science Foundation; the International Spinal Research Trust, Enfield/Middlesex, United Kingdom; the American Paralysis Association, Springfield, New Jersey; the Swiss Multiple Sclerosis Society; Regeneron Pharmaceuticals Inc., New York; and the Dr. Eric Slack-Gyr-Foundation.

REFERENCES RAMON Y CAJAL, S. 1928. Degeneration and Regeneration of the Nervous System (edited and translated by R. M. May, 1959), Hafner, New York. DAVID, S., AND A. J. AGUAYO. 1981. Axonal elongation into peripheral nervous system “bridges” after central nervous system injury in adult rats. Science 214: 931-933. BENFEY, M., AND A. J. AGUAYO. 1982. Extensive elongation of axons from rat brain into peripheral nerve grafts. Nature 296: 150-152. VIDAL-SANZ, M., M. B. GARTH, M. VILLEGAS-PERES, S. THANOS, AND A. J. AGUAYO. 1987. Axonal regeneration and syn-

192

5. 6. 7.

8.

9.

10.

11. 12.

13.

14.

15.

CADELLI,

BANDTLOW,

apse formation in the superior colliculus by retinal ganglion cells in the adutt rat. J. Neurosci. 7(S): 2894-2909. BARDE, Y-A. 1989. Trophic factors and neuronal survival. i?euron 2: 1525-1534. JESSEL, T. M. 1988. Adhesion molecules and the hierarchy of neural development. Neuron 1: 3-13. TESSIER-LAVIGNE, M., M. PLACZEK, A. G. S. LUMSDEN, J. DODD, AND T, M, JESSEL. 1988. Chemotropic guidance of developing axons in the mammalian CNS, Nature 336: ‘775-778. SCHWAB, M. E., AND H. THOENEN. 1985. Dissociated neurons regenerate into sciatic but not optic nerve explants in culture irrespective of neurotrop~c factors, J. Neurosci. c(9): 24152423. SAV~O, T., AND M. E. SCHWAB. 1989. Rat CNS white matter, but not gray matter, is nonpermissive for neuronal cell adhesion and fiber outgrowth. J. Neurosci. 9: 1126-1133. LINDSAY, R. M. 1979. Adult rat brain astrocytes support survival of both NGF-sensitive and -insensitive neurons. Nature 282: 80-82. HAITEN, M. E., LIEM, R. K. M., AND C. A. MASON. 1984. Two forms of cerebellar glial cells interact differently with neurons in vitro. J. Cell Bid. 98: 193-204. SCHWAB, M. E., AND P. CARONI. 1988. Oligodendrocytes and CNS myelin are non-permissive substrates for neurite growth and fibroblast spreading in vitro. J. Neurosci. 8: 2381-2393. BANDTLOW, C. E., T. ZACHLEDER, AND M. E. SCHWAB. 1990. Oligodendrocytes arrest neurite growth by contact inhibition. J. Neurosci. 10:3837-3848. CARONI, P., AND M. E. SCHWAB. 1988. Two membrane protein fractions from rat central myelin with inhibitory properties for neurite growth and fibroblast spreading. J. CeZZBiol. 106: 12811288. WALTER,J., R. KERN-VEITS,J. Hw,B. STOLZE, AND F. BONHOEFFER. 1987. Recognition of position-specific properties of tectal cell membranes by retinal axons in vitro. Development 101:685-696.

AND SCHWAB 16. K~ZYNES,R. J., AND C. D. STERN. 1984. Segmentation in the vertebrate nervous system. Nature 310: 786-787, 17. CARONI, P., AND M. E. SCHWAB. 1988. Antibody against myelinassociated inhibitor of neurite growth neutralizes nonpermissive substrate properties of CNS white matter. Neuron 1: 85-96. 18. SAVIO, T., AND M. E. SCHWAB. 1990. Lesioned corticospinal tract axons regenerate in myelin-free rat spinal cord. Proc. Natl. Acad. Sci. USA 87: 4130-4133. 19. SCHNELL, L., AND M. E. SCHWAB. 1990. Axonal regeneration in the rat spinal cord produced by an antibody against myelin-associated neurite growth inhibitors. Nature 343: 269-272. 20. CADELLI, D., AND M. SCHWAB. 1991. Regeneration of lesioned septo-hippocampal acetylcholinesterase-positive axons is improved by antibodies against the myelin-associated neurite growth inhibitors NI-35/250. Eur. J. Neurosci. 3: 825-832. 21. SEGAL, M., U. STJZNJXV~,AND A. BJBRKLUND. 1981. Reformation in adult rats of functional septo-hippocampal connections by septal neurons regenerating across an embryonic hippocampal tissue bridge. Neurosci. Lett. 27: 7-12. 22. BJ~RKLIJND, A., AND U. STENEVI. 1984. Intracerebral neural implants: neuronal replacement and r~onst~~io~ of damaged circuitries. Annu. Rev. Neurosei. 7: 279-308. 23. NILSSON, 0. G., D. J. CLARKE, P. BRUNDIN, AND A. BJGRKLUND. 1988. Comparison of growth and reinnervation properties of cholinergic neurons from different brain regions grafted to the hippocampus. J. Comp. Neural. 268: 204-222. 24. HAGG, T., H. L. VAHLSING, M. MANTHORPE, AND S. VARON. 1990. Nerve growth factor infusion into the denervated adult rat hippocampal formation promotes its choline& reinnervation. J. Neurosci. 10(S): 3087-3092. 25. S~~RS,J.,B.HAUSM~N,K.UNSIC~R,A~~.~ERRY.~~~~. Fibroblast growth factors promote the survival of adult rat retinal ganglion cells after transection of the optic nerve. Neurosci. Lett. 76: 157-162. 26. SCHWAB, M. E., AND L. SCHNELL. 1991. Channeling of developing rat corticospinal axons by myelin-associated neurite growth inhibitors. J. Neurosci. 1 l(3): 709-721.