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Chapter 46 Central nervous system regeneration: oligodendrocytes and myelin as non-permissive substrates for neurite growth
I) M . Gash and J . R . Sladek. J r . ( E d \ . ) I’ruRrers i n Brain Reseurdi, VoI. 7 8 i 198X E l x i i c r Sciencc Publishen B . V . (Biomedical Division)
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CHAPTER 46
Central nervous system regeneration: oligodendrocytes and myelin as non-permissive substrates for neurite growth P. Caroni, T. Savio and M.E. Schwab Institute f o r Brain Research, University of Zurich, August-Forel-Str. I , CH-8029 Zurich, Switzerland
Introduction During development of the nervous system neuronal processes grow out in a spatially and temporally highly coordinated fashion to produce the final, functional pattern of connections. Environmental cues are essential for initiation and guidance of neurite growth, for target recognition and for arrest of growth and synapse formation. A main goal of developmental neurobiology is the identification of the mechanisms and constituents responsible for these microenvironmental influences on developing neurons. Neurotrophic and neurotropic soluble factors, specific constituents of extracellular matrices and cell membranes serving as substrates for growing fibers or as ‘labels’ and recognition signals have been identified and probably act together in a complex manner during in vivo development. The capacity to repeat developmental processes following a lesion to axons is present in peripheral motor, sensory and autonomic neurons in higher vertebrates (Guth, 1956; Gorio et al., 1981). In sharp contrast, neurite regeneration and longdistance elongation is completely absent in the central nervous system (CNS). Transplantation experiments of pieces of peripheral nerve into the CNS have clearly demonstrated the ability of adult central neurons to repair and regrow their axons over long distances in a peripheral nerve microenvironment (Benfey and Aguayo, 1982; Richardson et al., 1984; So and Aguayo, 1985). Most remarkably, these axons stop growing almost im-
mediately when they encounter CNS tissue (David and Aguayo, 1981). Factors provoking and supporting neurite regeneration are produced by peripheral nerve Schwann cells in response to denervation. In the adult and lesioned CNS appropriate factors could be absent (Richardson and Ebendal, 1982; Abrahamson et al., 1986; Cajal, 1928). However, the presence of neurotrophic factors including nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) in the adult CNS of mammals has recently been demonstrated (Barde et al., 1982; Korsching et al., 1985; Shelton and Reichardt, 1986), and neurotrophic activities are released in increased amounts at sites of CNS lesions (Needels et al., 1986; Whittemore et al., 1987). Alternatively, particular substrate molecules important for neurite growth during development may be absent in the differentiated CNS (Liesi, 1985; Carbonetto et al., 1987). The experiments briefly summarized below lead us to postulate an additional hypothesis: the presence of distinct components which are non-permissive for neurite growth, expressed as specific membrane proteins by differentiated oligodendrocytes.
Optic vs. sciatic nerve explants as substrates for regenerating neurites in culture Dissociated neurons of newborn rat superior cervical ganglia, dorsal root ganglia, or embryonic day 17 retina were cultured in the narrow central chamber of a three-chamber Teflon ring. Explants
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of young adult rat optic nerves or sciatic nerves (meninges and epineurium removed; length 4 - 6 mm) were positioned under the Teflon ring connecting the middle chamber with one or the other of the side chambers, and sealed with Silicon grease (Schwab and Thoenen, 1985). NGF (sensory and sympathetic neurons) or BDNF (retina cells, Johnson et al., 1986) was added to the medium. After three to four weeks in culture, neurites emerging from the sciatic nerves in the side chambers and continuing their growth on the collagen substrate were observed in a number of cultures. Optic nerves showed no outgrowing neurites. Cultures were fixed after three to eight weeks and processed for electron microscopy. Up to several hundred neurites were found in the majority of the sciatic nerve explants. For retinal cells, the number of neurites per sciatic nerve was lower. Optic nerves in all these cultures were totally devoid of neurites (Schwab and Thoenen, 1985). Interestingly, the same results were found when the nerve explants were frozen and thawed three times before culturing. In frozen sciatic nerves, a preferential association of the neurites with basement membranes could be seen. This association was exclusive for the Schwann cell side of the basement membrane, a result which has also been observed in vivo (Ide et al., 1983; Schwab and Thoenen, 1985). In living nerves, neurites preferentially grew in contact with Schwann cells or, again, the Schwann cell basement membranes. These observations showed that the difference in the capacity to support neurite regeneration between central and peripheral nervous tissue was fully preserved under culture conditions. Various conclusions could be drawn from these results, e.g. the presence of high amounts of NGF or BDNF excludes the possibility of a lack of trophic factors in the CNS tissue as the primary cause of lacking neurite regeneration. Rather, a difference in the substrate properties between central and peripheral tissue can be postulated. Such a difference could consist of the lack of favorable, or the presence of non-permissive, constituents. The preferential association of regenerating neurites with Schwann cells and their basement membranes indicated the importance of favorable substrate conditions.
Sympathetic neurons and neuroblastoma cells cultured on brain sections interact differently with gray and white matter Optic nerves exclusively represent the white matter of the CNS. We, therefore, studied the substrate properties of white and gray matter, respectively. We have used frozen sections from different parts of the adult rat brain (spinal cord, cerebellum, forebrain) and also from sciatic and optic nerves as substrates for superior cervical ganglion neurons and neuroblastoma cells. Sections (20 pm thick) were dried on glass coverslips and washed with medium. Dissociated neurons of newborn rat superior cervical ganglia, or mouse neuroblastoma cells (line NB-2A) were cultured on the sections using an enriched L15 medium with 5 % rat serum and 100 ng/ml NGF (sympathetic neurons; Mains and Patterson 1973) or Dulbecco’s modified Eagle’s medium with 10% fetal calf serum (neuroblastoma cells). After two weeks (sympathetic neurons) or two days (neuroblastoma cells) cultures were fixed and stained with Cresyl violet or Coomassie blue. Both types of neuronal cells selectively adhered to the gray matter parts of the sections, highlighting the anatomical structure of cerebellum and spinal cord slices (Fig. la,b). In the case of superior cervical ganglion cells, bundles of neurites were seen to grow out and branch, again selectively on the gray matter (Fig. lb). Despite the presence of high amounts of NGF, the cells which are very rarely found on white matter had no visible axons. Bundles of neurites arising from neurons outside the brain slices were seen to approach spinal cord slices and follow their border without invasion of the white matter. Axons did, however, grow onto molecular layer areas of the cerebellum sections. Only very few neuroblastoma cells or sympathetic neurons adhered to the sections of the optic nerves, whereas evident fiber outgrowth occurred on sciatic nerve sections. These results confirmed the observations by Carbonetto et al. (1987) obtained with explants of chick sensory ganglia. They furthermore show that pronounced differences exist between adult brain gray and white matter areas in their substrate properties for
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Fig. 1. a . Neuroblastoma cells plated and cultured for two days on frozen sections of adult rat spinal cord. The darkly stained cells selectively adhere to the gray matter. x 20. b. Sympathetic neurons (rat superior cervical ganglia) cultured for two weeks on frozen sections of adult rat spinal cord adhere and extend processes selectively on gray matter. df, dorsal funiculi; arrow points to central canal. x 48.
neuronal adhesion and nerve fiber growth. Like optic nerve explants, CNS white matter in general seems to be non-permissive for neuronal adhesion and growth cone advancement. Gray matter areas, whether in spinal cord, cerebellum, or forebrain are permissive or favorable substrates.
Interaction of neurons and neuroblastoma cells with dissociated CNS glial cells
In order to further define a possible non-permissive substrate effect associated with central white matter, optic nerves of 7 - 12-day-old rats were dissociated and cultured on a polyornithine or polylysine substrate. Cell types were identified by antibody staining. The main cell types included differentiated oligodendrocytes (0, = galactocerebroside+ ; 04+; A2B5-), immature oligodendrocytes (0,- ; 0 4 + A2B,+), ; astrocytes (glial fibrillary acidic protein, G F A P f ) , and fibroblasts (Thy-1 + ) (Sommer and Schachner, 1981; Schnitzer and Schachner, 1982; Abney et al., 1983). Dissociated sympathetic, sensory or fetal retinal neurons were plated onto these cultures of non-neuronal cells and grown for two days to two weeks in the presence of the appropriate trophic factors (NGF, BDNF). Astrocytes and immature +
oligodendrocytes were rapidly contacted by neurons and represented a favorable substrate for neurite growth. In contrast, cells with a radial, highly branched and anastomosing process network and with antigenic characteristics of differentiated oligodendrocytes formed ‘windows’ in the network of neuronal processes (Fig. 2a,b). Neuronal cell bodies did not attach to these oligodendrocytes and their processes (Schwab and Caroni, in preparation). Similarly, mouse NB-2A neuroblastoma cells plated at high cell density onto optic nerve glial cultures did not associate with these highly branched oligodendrocytes. Fibers growing out from neuroblastoma cells in response to dibutyryl cyclic AMP strictly avoided the process network of oligodendrocytes. Likewise, 3T3 fibroblasts plated at high cell density onto oligodendrocyte-containing cultures rapidly attached, spread and formed monolayers leaving ‘windows’ around the oligodendrocytes (Fig. 2c,d). Clear-cut differences in the properties as substrates for neuronal attachement and neurite growth emerged from these experiments for the various types of central glial cells. Astrocytes are very favorable substrates for neuronal growth in culture, an observation which has been made by
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several investigators (Hatten et al., 1984; Noble et al., 1984; Fallon, 1985). Of significant interest in our search for the particular, non-permissive substrate quality of CNS white matter was the finding that differentiated oligodendrocytes seemed to exert a pronounced non-permissive substrate effect on neuronal attachment and neurite growth on a single cell-to-cell basis in culture. In contrast, immature oligodendrocytes did interact with neurons and neurites, a finding which may be of impor-
tance in view of the fact that during development oligodendrocyte precursors migrate into a preformed neurite fascicle and then start differentiation and formation of myelin. In the optic nerve, spinal cord, or corpus callosum myelination always follows axonal growth by several days (Rager, 1980; Hildebrand and Waxman, 1984; Looney and Elberger, 1986). Our experimental combination of growing axons with differentiated oligodendrocytes, therefore, does not correspond
Fig. 2. Highly branched oligodendrocytes and CNS myelin are non-permissive substrates for neurite extension and fibroblast spreading. a, b. Neurites of rat dorsal root ganglion neurons cultured in the presence of NGF avoid the territory of an oligodendrocyte (labeled with antibody 0, against galactocerebroside (b)). x 350. c, d. 3T3 fibroblasts cultured for three hours on ettablished optic glial cell culture. Spreading cells form monolayer interrupted by ‘windows’ around differentiated oligodendrocytes (galactocerebroside’ (d)). x 250. e, f . Rat superior cervical ganglion neurons growing in presence of NGF on meylin fractions from rat spinal cord (e) or from sciatic nerve (f). CNS myelin is a highly non-permissive substrate, in contrast to myelin from the PNS. Time in culture: 24 h. x 100.
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to the situation found in normal development. Neuronal growth cones do, however, encounter mature oligodendrocytes and myelin under conditions of regeneration.
CNS myelin of higher vertebrates is a non-permissive substrate for neurite extension and fibroblast spreading Myelin is the product of differentiated oligodendrocytes and the major distinctive constituent of CNS white matter. We therefore tested rat CNS myelin fractions for their substrate properties in supporting NGF-induced neurite extension by superior cervical ganglion neurons in vitro. Rat CNS and peripheral nervous system (PNS) myelin fractions were isolated (Colman et al., 1982) and adsorbed to polylysine-coated tissue culture dishes. To allow for direct comparison of isolated fractions in vitro, droplets containing different substrates were adsorbed to separate regions of the same tissue culture dish. PNS myelin represented a good substrate for the growing neurites (Fig. 20, and PNS myelin/polylysine boundaries were apparently not detected by extending neurites. In contrast, CNS myelin boundaries were essentially never crossed. Neurons situated on CNS myelin did not extend isolated neurites (Fig. 2e). Thick neurite bundles occasionally connected closely spaced neurons. The non-permissive substrate effect of CNS myelin and oligodendrocytes was of a general nature, as it was observed for neuroblastoma cells in the presence of dibutyryl CAMP or glia-derived neurite-promoting factor (Guenther et al., 1986), as well as for the spreading and locomotion of 3T3 fibroblasts (Fig. 3a,b). For biochemical analysis, 3T3 cell spreading was routinely used to test substrate properties. Findings were confirmed with primary cultures of neurons and with neuroblastoma cells. Nonpermissiveness of CNS myelin was found to be due to protein, since myelin lipid fractions yielded permissive, artificial lipid vesicles and mild treatment with protease, e.g. with trypsin, abolished nonpermissiveness. Extraction experiments indicated that non-permissiveness is due to rnembranebound protein of myelin. Finally, adsorption of CNS myelin with high titer anti-myelin antiserum abolished non-permissiveness. In control ex-
Fig. 3. Identification of non-permissive substrate componentb of myelin as proteins. Spreading of 3T3 cells (four hours in culture) is pronounced on PNS myelin (a), but strongly impaired on CNS myelin (b). Extracted CNS myelin proteins reconstituted in liposomes (c) represent a very non-permissive substrate for 3T3 cell spreading. Pretreatment with protease converts these liposomes into a substrate allowing rapid fibroblast spreading (d). x 105.
periments, adsorption of the myelin with antigalactocerebroside antibody, or with antiproteolipid protein antibody did not affect the non-permissive substrate effects (Caroni and Schwab, in preparation).
CNS myelin contains potent inhibitor of neurite extension and of fibroblast spreading Solubilized rat CNS myelin protein was incorporated into artificial lipid vesicles by cholate solubilization followed by Sephadex G-50 chrorn-
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atography (Brunner et al., 1978). CNS myelin protein-containing liposomes adsorbed to tissue culture plastic were found to be a highly nonpermissive substrate for neurite extension and fibroblast spreading (Fig. 3c). Control liposomes from rat sciatic nerve myelin or from a number of non-neuronal rat tissues did not behave in a significantly different manner from protein-free lipid vesicles. As found for the original myelin fraction, non-permissiveness was completely abolished by protease treatment (Fig. 3d). Fractionation of solubilized myelin protein indicated that the non-permissive substrate property is due to rat CNS myelin proteins of 250 and 35 kDa. These proteins were also found in protein fractions from rat oligodendrocyte-containing cultures, but not from Schwann cell-containing cultures. Activity was not blocked by protease inhibitors and survived mild denaturing conditions. Liposomes containing 250 kDa protein mixed with excess rat liver homogenate at a ratio greater than 1:104, also prevented fibroblast spreading. Thus, addition of small amounts of inhibitory CNS myelin protein converted a neutral substrate into a non-permissive one. Selective removal of the 250 and 35 kDa regions (polyacrylamide gel electrophoresis) resulted in a CNS myelin protein fraction with favorable substrate properties for fibroblast spreading and neurite growth. We therefore conclude that rat CNS myelin contains proteins with inhibitory substrate properties. To determine the cellular location of the inhibitory proteins, monoclonal antibodies against gel-purified 250 kDa protein were produced and screened for inhibition-neutralization. Blocking antibodies bound to the surface of myelin-forming oligodendrocytes weakly but specifically. Such antibodies efficiently prevented the formation of ‘windows’ of excluded cells (3T3, neuroblastoma) in an optic nerve-derived glial culture. Thus, minor protein components of rat CNS myelin and of the surface of myelin-forming oligodendrocytes seem to be responsible for lack of adhesion and failure of neurite extension in vitro. Discussion When cultured neurons are given the choice between an optic nerve and a sciatic nerve explant as
a substrate for their neurites, they exclusively invade the sciatic nerves. Presence of high amounts of trophic factors in the medium and the fact that frozen optic nerves were equally unacceptable for the regenerating neurites suggested that local substrate properties, possibly of a non-permissive nature, could be responsible for this effect. The selective adhesion of neuroblastoma cells and sprouting sympathetic neurons to the gray matter areas of sections of various parts of the adult rat brain showed that poor neuronal adhesion and fiber outgrowth was restricted to CNS white matter areas. This result fits in well with the in vivo observations where fetal cholinergic or brain stem neurons transplanted into adult hippocampus or spinal cord were seen to regenerate neurites over distances of more than 12 mm, even though they were strictly confined to gray matter areas (Nornes et al., 1983; Bjorklund and Stenevi, 1984). In fact, no regeneration exceeding the sprouting distance of about 1 mm within CNS white matter has been reported up to now. Analyzing the cellular components of white matter (optic nerves) for their substrate properties, we found that differentiated oligodendrocytes represent a highly non-permissive substrate for adhesion of sympathetic, sensory or retinal neurons, neuroblastoma cells and 3T3 fibroblasts. This was in contrast to the favorable substrate effects exerted by astrocytes (Hatten et al., 1984; Noble et al., 1984; Fallon, 1985) and immature oligodendrocytes. Myelin, the product of oligodendrocytes, when isolated and adsorbed to tissue culture dishes likewise inhibited neurite outgrowth and fibroblast spreading. In contrast, Schwann cell myelin of the PNS favored neurite growth and 3T3 cell spreading and locomotion. The biochemical analysis of this non-permissive substrate effect of CNS showed us that the effect is associated with two defined protein bands having the characteristics of membrane proteins. Antibodies against these protein fractions neutralized the non-permissive substrate effects of isolated myelin as well as that of living oligodendrocytes. The studies briefly reviewed here lead us to the conclusion that CNS white matter could be refractory to neurite growth due to the presence of specific oligodendrocyte membrane components exerting non-permissive substrate effects. Since
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those effects can not be overcome by the presence of high doses of stimulators of fiber outgrowth in culture, it can be postulated that these nonpermissive substrate molecules could also play an important role in the lack of regeneration in higher vertebrates CNS in vivo. The possible roles of these components, e.g. during CNS development, remain to be investigated.
Acknowledgments This work was supported by the Swiss National Foundation for Scientific Research (Grant No. 3.043 - 0.84) and the Bonizzi-Theler-Foundation (Zurich).
References Abney, E.R., Williams, P.B. and Raff, M.C. (1983) Tracing the development of oligodendrocytes from precursor cells using monoclonal antibodies, fluorescence-activated cell sorting, and cell culture. Dev. Biol., 100: 166- 171. Abrahamson, I.K., Wilson, P.A. and Rush, R.A. (1986) Production and transport of endogenous trophic activity in a peripheral nerve following target removal. Dev. Brain Res., 27: 117-126. Barde, Y.-A,, Edgar, D. and Thoenen, H . (1982) Purification of a new neurotrophic factor from mammalian brain. EMBO J., 1: 549-553. Benfey, M. and Aguayo, A.J. (1982) Extensive elongation of axons from rat brain into peripheral nerve grafts. Nature, 296: 150- 152. Bjorklund, A. and Stenevi, U. (1984) Intracerebral neural implants: Neuronal replacement and reconstruction of damaged circuitries. Annu. Rev. Neurosci., 7: 279 - 308. Brunner, J . , Hauser, J . and Semenza, G. (1978) Single bilayer lipid-protein vesicles formed from phosphatidylcholine and small intestinal sucrase-isomaltase. J. Biol. Chem., 253: 7538 - 7546. Cajal, Ramon y. S. (1928) Degeneration and Regeneration of the Nervous System. English transl. and reprint 1959, Hafner, New York. Carbonetto, S., Evans, D. and Cochard, P . (1987) Nerve fiber growth in culture on tissue substrates from central and peripheral nervous systems. J . Neurosci., 7: 610- 620. Colman, D.R., Kreibich, G., Frei, A.B. and Sabatini, D.D. (1982) Synthesis and incorporation of myelin polypeptides into CNS myelin. J . Cell Biol., 95: 598 - 608. David, S . and Aguayo, A.J. (1981) Axonal elongation into peripheral nervous system ‘bridges’ after central nervous system injury in adult rats. Science, 214: 931 -933. Fallon, J.R. (1985) Preferential outgrowth of central nervous system neurites on astrocytes and Schwann cells as compared with nonglial cells in vitro. J . Cell Biol., 100: 198-207. Gorio, A , , Millesi, H. and Mingrino, S., Eds. (1981) Post-
traumatic Peripheral Nerve Regeneration, Raven Press, New York. Guenther, J . , Nick, H. and Monard, D. (1986) A glia-derived neurite-promoting factor with protease inhibitory activity. EMBO J., 4: 1963 - 1966. Guth, L. (1956) Regeneration in the mammalian peripheral nervous system. Physiol. Rev., 36: 441 -478. Hatten, M.E., Liem, R.K.M. and Mason, C.A. (1984) Two forms of cerebellar glial cells interact differently with neurons in vitro. J . Cell Biol.,98: 193-204. Hildebrand, C. and Waxman, S.G. (1984) Postnatal differentiation of rat optic nerve fibers: electron microscopic observations on the development of nodes of Ranvier and axoglial relations. J. Comp. Neurol., 224: 25 - 37. Ide, C., Tohyama, K., Yokata, R., Nitatori, T. and Onodera, S. (1983) Schwann cell basal lamina and nerve regeneration. Brain Res., 288: 61 -75. Johnson, J.E., Barde, Y.-A,, Schwab, M.E. and Thoenen, H. (1986) Brain-derived neurotrophic factor supports the survival of cultured rat retinal ganglion cells. J . Neurosci., 6: 3031 - 3038. Korsching, S., Auburger, G., Heumann, R., Scott, J. and Thoenen, H. (1985) Levels of nerve growth factor and its mRNA in the central nervous sytem of the rat correlate with cholinergic innervation. EMBO J . , 4: 1389- 1393. Liesi, P. (1985) Laminin-immunoreactive glia distinguish regenerative adult CNS systems from non-regenerative ones. EMBO J., 4: 2505-2511. Looney, G.A. and Elberger, A . J . (1986) Myelination of the corpus callosum in the cat: time course, topography, and functional implications. J . Comp. Neurol., 248: 336- 347. Mains, R.E. and Patterson, P.H. (1973) Primary cultures of dissociated sympathetic neurons. J . Cell Biol., 59: 329 - 345. Needels, D.L., Nieto-Sampedro, M. and Cotman, C.W. (1986) Induction of a neurite-promoting factor in rat brain following injury or deafferentation. Neuroscience, 18: 517 - 526. Noble, M., Fok-Seang, J . and Cohen, J . (1984) Glia are a unique substrate for the in vitro growth of central nervous system neurons. J. Neurosci., 4: 1982 - 1903. Nornes, H., Bjorklund, A. and Stenevi, U . (1983) Reinnervation of the denervated adult spinal cord of rats by intraspinal transplants of embryonic brain stem neurons. Cell Tissue Res., 230: 15-35. Rager, G.H. (1980) Development of the retinotectal projection in the chicken. Adv. Anat. Embryol. Cell Biol., 63: 1-92. Richardson, P.M. and Ebendal, T. (1982) Nerve growth activities in rat peripheral nerve. Brain Res., 246: 57-64. Richardson, P.M., Issa, V.M.K. and Aguayo, A.J. (1984) Regeneration of long spinal axons in the rat. J . Neurocytol., 13: 165-182. Schnitzer, J . and Schachner, M. (1982) Cell type specificity of a neural cell surface antigen recognized by the monoclonal antibody A2B5. Cell Tissue Res., 224: 625-636. Schwab, M.E. and Thoenen, H. (1985) Dissociated neurons regenerate into sciatic but not optic nerve explants in culture irrespective of neurotrophic factors. J . Neurosci., 5: 2415-2423. Shelton, D.L. and Reichardt, L.F. (1986) Studies on the expression of the p nerve growth factor (NGF) gene in the central nervous system: level and regional distribution of NGF
370 mRNA suggest that NGF functions as a trophic factor for several distinct populations of neurons. Proc. Nurl. Acud. Sci. USA, 83: 2714-2718. So, K . F . and Aguayo, A.J. (1985) Lengthy regrowth of cut axons from ganglion cells after peripheral nerve transplantation into the retina of adult rats. Bruin Res., 328: 349- 354. Sommer, I . and Schachner, M . (1981) Monoclonal antibodies ( 0 , to 0,) to oligodendrocyte cell surface: an immuno-
cytological study in the central nervous system. Dev. Biol., 83: 311-327. Whittemore, S.R., Larkfors, L., Ebendal, T., Holets, V.R., Ericsson, A. and P e r s o n , H. (1987) Increased &nerve growth factor messenger RNA and protein levels in neonatal rat hippocampus following specific cholinergic lesions. J . Neurosci., 7: 244 - 25 1.
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Central nervous system regeneration: oligodendrocytes and myelin as non-permissive substrates for neurite growth P. Caroni, T. Savio and M.E. Schwab Institute f o r Brain Research, University of Zurich, August-Forel-Str. I , CH-8029 Zurich, Switzerland
Introduction During development of the nervous system neuronal processes grow out in a spatially and temporally highly coordinated fashion to produce the final, functional pattern of connections. Environmental cues are essential for initiation and guidance of neurite growth, for target recognition and for arrest of growth and synapse formation. A main goal of developmental neurobiology is the identification of the mechanisms and constituents responsible for these microenvironmental influences on developing neurons. Neurotrophic and neurotropic soluble factors, specific constituents of extracellular matrices and cell membranes serving as substrates for growing fibers or as ‘labels’ and recognition signals have been identified and probably act together in a complex manner during in vivo development. The capacity to repeat developmental processes following a lesion to axons is present in peripheral motor, sensory and autonomic neurons in higher vertebrates (Guth, 1956; Gorio et al., 1981). In sharp contrast, neurite regeneration and longdistance elongation is completely absent in the central nervous system (CNS). Transplantation experiments of pieces of peripheral nerve into the CNS have clearly demonstrated the ability of adult central neurons to repair and regrow their axons over long distances in a peripheral nerve microenvironment (Benfey and Aguayo, 1982; Richardson et al., 1984; So and Aguayo, 1985). Most remarkably, these axons stop growing almost im-
mediately when they encounter CNS tissue (David and Aguayo, 1981). Factors provoking and supporting neurite regeneration are produced by peripheral nerve Schwann cells in response to denervation. In the adult and lesioned CNS appropriate factors could be absent (Richardson and Ebendal, 1982; Abrahamson et al., 1986; Cajal, 1928). However, the presence of neurotrophic factors including nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) in the adult CNS of mammals has recently been demonstrated (Barde et al., 1982; Korsching et al., 1985; Shelton and Reichardt, 1986), and neurotrophic activities are released in increased amounts at sites of CNS lesions (Needels et al., 1986; Whittemore et al., 1987). Alternatively, particular substrate molecules important for neurite growth during development may be absent in the differentiated CNS (Liesi, 1985; Carbonetto et al., 1987). The experiments briefly summarized below lead us to postulate an additional hypothesis: the presence of distinct components which are non-permissive for neurite growth, expressed as specific membrane proteins by differentiated oligodendrocytes.
Optic vs. sciatic nerve explants as substrates for regenerating neurites in culture Dissociated neurons of newborn rat superior cervical ganglia, dorsal root ganglia, or embryonic day 17 retina were cultured in the narrow central chamber of a three-chamber Teflon ring. Explants
364
of young adult rat optic nerves or sciatic nerves (meninges and epineurium removed; length 4 - 6 mm) were positioned under the Teflon ring connecting the middle chamber with one or the other of the side chambers, and sealed with Silicon grease (Schwab and Thoenen, 1985). NGF (sensory and sympathetic neurons) or BDNF (retina cells, Johnson et al., 1986) was added to the medium. After three to four weeks in culture, neurites emerging from the sciatic nerves in the side chambers and continuing their growth on the collagen substrate were observed in a number of cultures. Optic nerves showed no outgrowing neurites. Cultures were fixed after three to eight weeks and processed for electron microscopy. Up to several hundred neurites were found in the majority of the sciatic nerve explants. For retinal cells, the number of neurites per sciatic nerve was lower. Optic nerves in all these cultures were totally devoid of neurites (Schwab and Thoenen, 1985). Interestingly, the same results were found when the nerve explants were frozen and thawed three times before culturing. In frozen sciatic nerves, a preferential association of the neurites with basement membranes could be seen. This association was exclusive for the Schwann cell side of the basement membrane, a result which has also been observed in vivo (Ide et al., 1983; Schwab and Thoenen, 1985). In living nerves, neurites preferentially grew in contact with Schwann cells or, again, the Schwann cell basement membranes. These observations showed that the difference in the capacity to support neurite regeneration between central and peripheral nervous tissue was fully preserved under culture conditions. Various conclusions could be drawn from these results, e.g. the presence of high amounts of NGF or BDNF excludes the possibility of a lack of trophic factors in the CNS tissue as the primary cause of lacking neurite regeneration. Rather, a difference in the substrate properties between central and peripheral tissue can be postulated. Such a difference could consist of the lack of favorable, or the presence of non-permissive, constituents. The preferential association of regenerating neurites with Schwann cells and their basement membranes indicated the importance of favorable substrate conditions.
Sympathetic neurons and neuroblastoma cells cultured on brain sections interact differently with gray and white matter Optic nerves exclusively represent the white matter of the CNS. We, therefore, studied the substrate properties of white and gray matter, respectively. We have used frozen sections from different parts of the adult rat brain (spinal cord, cerebellum, forebrain) and also from sciatic and optic nerves as substrates for superior cervical ganglion neurons and neuroblastoma cells. Sections (20 pm thick) were dried on glass coverslips and washed with medium. Dissociated neurons of newborn rat superior cervical ganglia, or mouse neuroblastoma cells (line NB-2A) were cultured on the sections using an enriched L15 medium with 5 % rat serum and 100 ng/ml NGF (sympathetic neurons; Mains and Patterson 1973) or Dulbecco’s modified Eagle’s medium with 10% fetal calf serum (neuroblastoma cells). After two weeks (sympathetic neurons) or two days (neuroblastoma cells) cultures were fixed and stained with Cresyl violet or Coomassie blue. Both types of neuronal cells selectively adhered to the gray matter parts of the sections, highlighting the anatomical structure of cerebellum and spinal cord slices (Fig. la,b). In the case of superior cervical ganglion cells, bundles of neurites were seen to grow out and branch, again selectively on the gray matter (Fig. lb). Despite the presence of high amounts of NGF, the cells which are very rarely found on white matter had no visible axons. Bundles of neurites arising from neurons outside the brain slices were seen to approach spinal cord slices and follow their border without invasion of the white matter. Axons did, however, grow onto molecular layer areas of the cerebellum sections. Only very few neuroblastoma cells or sympathetic neurons adhered to the sections of the optic nerves, whereas evident fiber outgrowth occurred on sciatic nerve sections. These results confirmed the observations by Carbonetto et al. (1987) obtained with explants of chick sensory ganglia. They furthermore show that pronounced differences exist between adult brain gray and white matter areas in their substrate properties for
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Fig. 1. a . Neuroblastoma cells plated and cultured for two days on frozen sections of adult rat spinal cord. The darkly stained cells selectively adhere to the gray matter. x 20. b. Sympathetic neurons (rat superior cervical ganglia) cultured for two weeks on frozen sections of adult rat spinal cord adhere and extend processes selectively on gray matter. df, dorsal funiculi; arrow points to central canal. x 48.
neuronal adhesion and nerve fiber growth. Like optic nerve explants, CNS white matter in general seems to be non-permissive for neuronal adhesion and growth cone advancement. Gray matter areas, whether in spinal cord, cerebellum, or forebrain are permissive or favorable substrates.
Interaction of neurons and neuroblastoma cells with dissociated CNS glial cells
In order to further define a possible non-permissive substrate effect associated with central white matter, optic nerves of 7 - 12-day-old rats were dissociated and cultured on a polyornithine or polylysine substrate. Cell types were identified by antibody staining. The main cell types included differentiated oligodendrocytes (0, = galactocerebroside+ ; 04+; A2B5-), immature oligodendrocytes (0,- ; 0 4 + A2B,+), ; astrocytes (glial fibrillary acidic protein, G F A P f ) , and fibroblasts (Thy-1 + ) (Sommer and Schachner, 1981; Schnitzer and Schachner, 1982; Abney et al., 1983). Dissociated sympathetic, sensory or fetal retinal neurons were plated onto these cultures of non-neuronal cells and grown for two days to two weeks in the presence of the appropriate trophic factors (NGF, BDNF). Astrocytes and immature +
oligodendrocytes were rapidly contacted by neurons and represented a favorable substrate for neurite growth. In contrast, cells with a radial, highly branched and anastomosing process network and with antigenic characteristics of differentiated oligodendrocytes formed ‘windows’ in the network of neuronal processes (Fig. 2a,b). Neuronal cell bodies did not attach to these oligodendrocytes and their processes (Schwab and Caroni, in preparation). Similarly, mouse NB-2A neuroblastoma cells plated at high cell density onto optic nerve glial cultures did not associate with these highly branched oligodendrocytes. Fibers growing out from neuroblastoma cells in response to dibutyryl cyclic AMP strictly avoided the process network of oligodendrocytes. Likewise, 3T3 fibroblasts plated at high cell density onto oligodendrocyte-containing cultures rapidly attached, spread and formed monolayers leaving ‘windows’ around the oligodendrocytes (Fig. 2c,d). Clear-cut differences in the properties as substrates for neuronal attachement and neurite growth emerged from these experiments for the various types of central glial cells. Astrocytes are very favorable substrates for neuronal growth in culture, an observation which has been made by
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several investigators (Hatten et al., 1984; Noble et al., 1984; Fallon, 1985). Of significant interest in our search for the particular, non-permissive substrate quality of CNS white matter was the finding that differentiated oligodendrocytes seemed to exert a pronounced non-permissive substrate effect on neuronal attachment and neurite growth on a single cell-to-cell basis in culture. In contrast, immature oligodendrocytes did interact with neurons and neurites, a finding which may be of impor-
tance in view of the fact that during development oligodendrocyte precursors migrate into a preformed neurite fascicle and then start differentiation and formation of myelin. In the optic nerve, spinal cord, or corpus callosum myelination always follows axonal growth by several days (Rager, 1980; Hildebrand and Waxman, 1984; Looney and Elberger, 1986). Our experimental combination of growing axons with differentiated oligodendrocytes, therefore, does not correspond
Fig. 2. Highly branched oligodendrocytes and CNS myelin are non-permissive substrates for neurite extension and fibroblast spreading. a, b. Neurites of rat dorsal root ganglion neurons cultured in the presence of NGF avoid the territory of an oligodendrocyte (labeled with antibody 0, against galactocerebroside (b)). x 350. c, d. 3T3 fibroblasts cultured for three hours on ettablished optic glial cell culture. Spreading cells form monolayer interrupted by ‘windows’ around differentiated oligodendrocytes (galactocerebroside’ (d)). x 250. e, f . Rat superior cervical ganglion neurons growing in presence of NGF on meylin fractions from rat spinal cord (e) or from sciatic nerve (f). CNS myelin is a highly non-permissive substrate, in contrast to myelin from the PNS. Time in culture: 24 h. x 100.
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to the situation found in normal development. Neuronal growth cones do, however, encounter mature oligodendrocytes and myelin under conditions of regeneration.
CNS myelin of higher vertebrates is a non-permissive substrate for neurite extension and fibroblast spreading Myelin is the product of differentiated oligodendrocytes and the major distinctive constituent of CNS white matter. We therefore tested rat CNS myelin fractions for their substrate properties in supporting NGF-induced neurite extension by superior cervical ganglion neurons in vitro. Rat CNS and peripheral nervous system (PNS) myelin fractions were isolated (Colman et al., 1982) and adsorbed to polylysine-coated tissue culture dishes. To allow for direct comparison of isolated fractions in vitro, droplets containing different substrates were adsorbed to separate regions of the same tissue culture dish. PNS myelin represented a good substrate for the growing neurites (Fig. 20, and PNS myelin/polylysine boundaries were apparently not detected by extending neurites. In contrast, CNS myelin boundaries were essentially never crossed. Neurons situated on CNS myelin did not extend isolated neurites (Fig. 2e). Thick neurite bundles occasionally connected closely spaced neurons. The non-permissive substrate effect of CNS myelin and oligodendrocytes was of a general nature, as it was observed for neuroblastoma cells in the presence of dibutyryl CAMP or glia-derived neurite-promoting factor (Guenther et al., 1986), as well as for the spreading and locomotion of 3T3 fibroblasts (Fig. 3a,b). For biochemical analysis, 3T3 cell spreading was routinely used to test substrate properties. Findings were confirmed with primary cultures of neurons and with neuroblastoma cells. Nonpermissiveness of CNS myelin was found to be due to protein, since myelin lipid fractions yielded permissive, artificial lipid vesicles and mild treatment with protease, e.g. with trypsin, abolished nonpermissiveness. Extraction experiments indicated that non-permissiveness is due to rnembranebound protein of myelin. Finally, adsorption of CNS myelin with high titer anti-myelin antiserum abolished non-permissiveness. In control ex-
Fig. 3. Identification of non-permissive substrate componentb of myelin as proteins. Spreading of 3T3 cells (four hours in culture) is pronounced on PNS myelin (a), but strongly impaired on CNS myelin (b). Extracted CNS myelin proteins reconstituted in liposomes (c) represent a very non-permissive substrate for 3T3 cell spreading. Pretreatment with protease converts these liposomes into a substrate allowing rapid fibroblast spreading (d). x 105.
periments, adsorption of the myelin with antigalactocerebroside antibody, or with antiproteolipid protein antibody did not affect the non-permissive substrate effects (Caroni and Schwab, in preparation).
CNS myelin contains potent inhibitor of neurite extension and of fibroblast spreading Solubilized rat CNS myelin protein was incorporated into artificial lipid vesicles by cholate solubilization followed by Sephadex G-50 chrorn-
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atography (Brunner et al., 1978). CNS myelin protein-containing liposomes adsorbed to tissue culture plastic were found to be a highly nonpermissive substrate for neurite extension and fibroblast spreading (Fig. 3c). Control liposomes from rat sciatic nerve myelin or from a number of non-neuronal rat tissues did not behave in a significantly different manner from protein-free lipid vesicles. As found for the original myelin fraction, non-permissiveness was completely abolished by protease treatment (Fig. 3d). Fractionation of solubilized myelin protein indicated that the non-permissive substrate property is due to rat CNS myelin proteins of 250 and 35 kDa. These proteins were also found in protein fractions from rat oligodendrocyte-containing cultures, but not from Schwann cell-containing cultures. Activity was not blocked by protease inhibitors and survived mild denaturing conditions. Liposomes containing 250 kDa protein mixed with excess rat liver homogenate at a ratio greater than 1:104, also prevented fibroblast spreading. Thus, addition of small amounts of inhibitory CNS myelin protein converted a neutral substrate into a non-permissive one. Selective removal of the 250 and 35 kDa regions (polyacrylamide gel electrophoresis) resulted in a CNS myelin protein fraction with favorable substrate properties for fibroblast spreading and neurite growth. We therefore conclude that rat CNS myelin contains proteins with inhibitory substrate properties. To determine the cellular location of the inhibitory proteins, monoclonal antibodies against gel-purified 250 kDa protein were produced and screened for inhibition-neutralization. Blocking antibodies bound to the surface of myelin-forming oligodendrocytes weakly but specifically. Such antibodies efficiently prevented the formation of ‘windows’ of excluded cells (3T3, neuroblastoma) in an optic nerve-derived glial culture. Thus, minor protein components of rat CNS myelin and of the surface of myelin-forming oligodendrocytes seem to be responsible for lack of adhesion and failure of neurite extension in vitro. Discussion When cultured neurons are given the choice between an optic nerve and a sciatic nerve explant as
a substrate for their neurites, they exclusively invade the sciatic nerves. Presence of high amounts of trophic factors in the medium and the fact that frozen optic nerves were equally unacceptable for the regenerating neurites suggested that local substrate properties, possibly of a non-permissive nature, could be responsible for this effect. The selective adhesion of neuroblastoma cells and sprouting sympathetic neurons to the gray matter areas of sections of various parts of the adult rat brain showed that poor neuronal adhesion and fiber outgrowth was restricted to CNS white matter areas. This result fits in well with the in vivo observations where fetal cholinergic or brain stem neurons transplanted into adult hippocampus or spinal cord were seen to regenerate neurites over distances of more than 12 mm, even though they were strictly confined to gray matter areas (Nornes et al., 1983; Bjorklund and Stenevi, 1984). In fact, no regeneration exceeding the sprouting distance of about 1 mm within CNS white matter has been reported up to now. Analyzing the cellular components of white matter (optic nerves) for their substrate properties, we found that differentiated oligodendrocytes represent a highly non-permissive substrate for adhesion of sympathetic, sensory or retinal neurons, neuroblastoma cells and 3T3 fibroblasts. This was in contrast to the favorable substrate effects exerted by astrocytes (Hatten et al., 1984; Noble et al., 1984; Fallon, 1985) and immature oligodendrocytes. Myelin, the product of oligodendrocytes, when isolated and adsorbed to tissue culture dishes likewise inhibited neurite outgrowth and fibroblast spreading. In contrast, Schwann cell myelin of the PNS favored neurite growth and 3T3 cell spreading and locomotion. The biochemical analysis of this non-permissive substrate effect of CNS showed us that the effect is associated with two defined protein bands having the characteristics of membrane proteins. Antibodies against these protein fractions neutralized the non-permissive substrate effects of isolated myelin as well as that of living oligodendrocytes. The studies briefly reviewed here lead us to the conclusion that CNS white matter could be refractory to neurite growth due to the presence of specific oligodendrocyte membrane components exerting non-permissive substrate effects. Since
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those effects can not be overcome by the presence of high doses of stimulators of fiber outgrowth in culture, it can be postulated that these nonpermissive substrate molecules could also play an important role in the lack of regeneration in higher vertebrates CNS in vivo. The possible roles of these components, e.g. during CNS development, remain to be investigated.
Acknowledgments This work was supported by the Swiss National Foundation for Scientific Research (Grant No. 3.043 - 0.84) and the Bonizzi-Theler-Foundation (Zurich).
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