Immunoelectron microscopic localization of neural cell adhesion molecules (L1, N-CAM, and myelin-associated glycoprotein) in regenerating adult mouse sciatic nerve

The localization of the neural cell adhesion molecules L1, N-CAM, and the myelin-associated glycoprotein was studied by pre- and postembedding staining procedures at the light and electron microscopic levels in transected and crushed adult mouse sciatic nerve. During the first 2-6 d after transection, myelinated and nonmyelinated axons degenerated in the distal part of the proximal stump close to the transection site and over the entire length of the distal part of the transected nerve. During this time, regrowing axons were seen only in the proximal, but not in the distal nerve stump. In most cases L1 and N-CAM remained detectable at cell contacts between nonmyelinating Schwann cells and degenerating axons as long as these were still morphologically intact. Similarly, myelin-associated glycoprotein remained detectable in the periaxonal area of the degenerating myelinated axons. During and after degeneration of axons, nonmyelinating Schwann cells formed slender processes which were L1 and N-CAM positive. They resembled small-diameter axons but could be unequivocally identified as Schwann cells by chronical denervation. Unlike the nonmyelinating Schwann cells, only few myelinating ones expressed L1 and N-CAM. At the cut ends of the nerve stumps a cap developed (more at the proximal than at the distal stump) that contained S-100-negative and fibronectin-positive fibroblast-like cells. Most of these cells were N-CAM positive but always L1 negative. Growth cones and regrowing axons expressed N-CAM and L1 at contact sites with these cells. Regrowing axons of small diameter were L1 and N-CAM positive where they made contact with each other or with Schwann cells, while large-diameter axons were only poorly antigen positive or completely negative. 14 d after transection, when regrowing axons were seen in the distal part of the transected nerve, regrowing axons made L1- and N-CAM-positive contacts with Schwann cells. When contacting basement membrane, axons were rarely found to express L1 and N-CAM. Most, if not all, Schwann cells associated with degenerating myelin expressed L1 and N-CAM. In crushed nerves, the immunostaining pattern was essentially the same as in the cut nerve. During formation of myelin, the sequence of adhesion molecule expression was the same as during development: L1 disappeared and N-CAM was reduced on myelinating Schwann cells and axons after the Schwann cell process had turned approximately 1.5 loops around the axon. Myelin-associated glycoprotein then appeared both periaxonally and on the turning loops of Schwann cells in the uncompacted myelin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunoelectron microscopic localization of neural cell adhesion molecules (L1, N-CAM, and MAG) and their shared carbohydrate epitope and myelin basic protein in developing sciatic nerve

The cellular and subcellular localization of the neural cell adhesion molecules L1, N-CAM, and myelin-associated glycoprotein (MAG), their shared carbohydrate epitope L2/HNK-1, and the myelin basic protein (MBP) were studied by pre- and post-embedding immunoelectron microscopic labeling procedures in developing mouse sciatic nerve. L1 and N-CAM showed a similar staining pattern. Both were localized on small, non-myelinated, fasciculating axons and axons ensheathed by non- myelinating Schwann cells. Schwann cells were also positive for L1 and N-CAM in their non-myelinating state and at the onset of myelination, when the Schwann cell processes had turned approximately 1.5 loops. Thereafter, neither axon nor Schwann cell could be detected to express the L1 antigen, whereas N-CAM was found in the periaxonal area and, more weakly, in compact myelin of myelinated fibers. Compact myelin, Schmidt-Lanterman incisures, paranodal loops, and finger-like processes of Schwann cells at nodes of Ranvier were L1-negative. At the nodes of Ranvier, the axolemma was also always L1- and N-CAM-negative. The L2/HNK-1 carbohydrate epitope coincided in its cellular and subcellular localization most closely to that observed for L1. MAG appeared on Schwann cells at the time L1 expression ceased. MAG was then expressed at sites of axon-myelinating Schwann cell apposition and non-compacted loops of developing myelin. When compaction of myelin occurred, MAG remained present only at the axon-Schwann cell interface; Schmidt- Lanterman incisures, inner and outer mesaxons, and paranodal loops, but not at finger-like processes of Schwann cells at nodes of Ranvier or compacted myelin. All three adhesion molecules and the L2/HNK-1 epitope could be detected in a non-uniform staining pattern in basement membrane of Schwann cells and collagen fibrils of the endoneurium. MBP was detectable in compacted myelin, but not in Schmidt-Lanterman incisures, inner and outer mesaxon, paranodal loops, and finger-like processes at nodes of Ranvier, nor in the periaxonal regions of myelinated fibers, thus showing a complementary distribution to MAG. These studies show that axon-Schwann cell interactions are characterized by the sequential appearance of cell adhesion molecules and MBP apparently coordinated in time and space. From this sequence it may be deduced that L1 and N-CAM are involved in fasciculation, initial axon-Schwann cell interaction, and onset of myelination, with MAG to follow and MBP to appear only in compacted myelin. In contrast to L1, N- CAM may be further involved in the maintenance of compact myelin and axon-myelin apposition of larger diameter axons.     

Immunocytological localization of the major peripheral nervous system glycoprotein P0 and the L2/HNK-1 and L3 carbohydrate structures in developing and adult mouse sciatic nerve

Immunocytological localization of the major glycoprotein of peripheral myelin P0 and its associated carbohydrate structures L2/HNK-1 and L3 was performed at the light- and electron-microscopic levels in mouse sciatic nerves at several developmental stages and in adulthood. P0 was first expressed on Schwann cells at the time that Schwann cells associated with axons on a 1:1 basis. P0 remains expressed at all times of myelin formation and in compact myelin. After cessation of myelination P0 is no longer detectable in the uncompacted parts of myelin, i.e., Schmidt-Lanterman incisures, paranodal loops, and outer and inner mesaxons. P0 is not detectable on basement membranes, interstitial collagens, and non-myelin-forming Schwann cells. The associated carbohydrate epitope L2 does not follow the expression of P0 at any developmental or adult stage. Until 21 days the L2 epitope is confined to nonmyelinated fibers. In sciatic nerves of mice older than 8 weeks, however, only a few nonmyelinated fibers remain L2-positive. L2 immunoreactivity is clearly seen in a subpopulation of compact myelin figures largely associated with motor fibers. The L3 epitope is never detectable on nonmyelinated fibers and becomes first visible when compact myelin is discerned. Unlike the L2 epitope L3 is present in most, if not all, compact myelin figures. These observations suggest that P0 may be involved in ensheathment of axons by Schwann cells at the decisive stages of initiation of myelination and later on, possibly in conjunction with the L3 carbohydrate structure, in maintenance of compact myelin. The appearance of the L2 carbohydrate epitopes in compact myelin of largely motor and fewer sensory nerve fibers at times when morphogenesis of myelin has ceased remains to be elucidated in functional terms.     

Regulation of neural cell adhesion molecule expression on cultured mouse Schwann cells by nerve growth factor.

Schwann cells from early postnatal mouse sciatic nerve were obtained as a homogenous population and shown by indirect immunofluorescence to express the neural cell adhesion molecules L1, N-CAM and J1 and their common carbohydrate epitope L2/HNK-1. L1 and N-CAM are synthesized in molecular forms that are slightly different from those expressed by small cerebellar neurons or astrocytes. As in astrocytes, the J1 antigen is expressed by Schwann cells in multiple forms generally ranging from 160 to 230 kd in the reduced state. J1 is secreted by Schwann cells in a 230-kd mol. wt form. Expression of L1 by Schwann cells can be regulated by nerve growth factor (NGF). L1 expression on the cell surface is increased 1.6-fold in the presence of NGF after 3 days of maintenance in vitro and 3-fold after 16 days. NGF does not change expression of N-CAM. The glia-derived neurite-promoting factor (GdNPF) increases L1 expression by a factor of 1.9 and decreases N-CAM expression by a factor of 0.4 after 3 days in vitro. J1 expression on Schwann cell surfaces remains unchanged in the presence of NGF or GdNPF. Antibodies to NGF abolish the influence of NGF on L1 expression. Addition of NGF antibodies to the Schwann cell cultures without exogenously added NGF decreases L1 expression, indicating that Schwann cells secrete NGF that may influence L1 expression by an autocrine mechanism. Our experiments show for the first time that cell adhesion molecule expression on a non-neuronal cell, the Schwann cell, can be directly regulated by the neurotrophic factor NGF. These observations indicate a considerable degree of 'plasticity' of peripheral glia in regulating cell adhesion molecule expression.     

Expression of stress fibers in bullfrog mesothelial cells in situ

Summary Monoclonal antibodies (MABs) have been raised against acidic glycolipids extracted from the electric organ of Torpedo marmorata. One of these, designated L9, appears to recognize acidic glycolipids in adult T. marmorata electric organ, electromotor nerves and brain, adult rat sciatic nerve, and in embryonic and neonatal rat brain, starting at embryonic day (ED) 15 and disappearing by the 20th day of post-natal life. The epitope is present in growth cones isolated from 4-day-old rats; its proportion relative to total gangliosides is, however, no higher than that found in whole neonatal brain membranes. Desialidation of the acidic glycolipid fraction modifies neither the immunoreactivity nor the R_F value following thin-layer chromatography (TLC) of the antigen; it is concluded that the antigen is not a ganglioside. The MAB, HNK-1, recognizes the L9 antigen. Both HNK-1 and L9 recognize a sulphoglycolipid of the same R_F in TLC. The function of the L9 antigen is not known but its evolutionary conservation, presence in growth cones and its developmental regulation in the mammalian central nervous system indicate that it plays an important role in nervous system maturation.     

Studies of adhesion molecules mediating interactions between cells of peripheral nervous system indicate a major role for L1 in mediating sensory neuron growth on Schwann cells in culture

The involvement of the adhesion molecules L1, N-CAM, and J1 in adhesion and neurite outgrowth in the peripheral nervous system was investigated. We prepared Schwann cells and fibroblasts (from sciatic nerves) and neurons (from dorsal root ganglia) from 1-d mice. These cells were allowed to interact with each other in a short-term adhesion assay. We also measured outgrowth of dorsal root ganglion neurons on Schwann cell and fibroblast monolayers. Schwann cells (which express L1, N-CAM, and J1) adhered most strongly to dorsal root ganglion neurons by an L1-dependent mechanism and less by N-CAM and J1. Schwann cell-Schwann cell adhesion was mediated by L1 and N-CAM, but not J1. Adhesion of fibroblasts (which express N-CAM, but not L1 or J1) to neurons or Schwann cells was mediated by L1 and N-CAM and not J1. However, inhibition by L1 and N-CAM antibodies was found to be less pronounced with fibroblasts than with Schwann cells. N-CAM was also strongly involved in fibroblast-fibroblast adhesion. Neurite outgrowth was most extensive on Schwann cells and less on fibroblasts. A difference in extent of neurite elongation was seen between small- (10-20 microns) and large- (20-35 microns) diameter neurons, with the larger neurons tending to exhibit longer neurites. Fab fragments of polyclonal L1, N-CAM, and J1 antibodies exerted slightly different inhibitory effects on neurite outgrowth, depending on whether the neurites were derived from small or large neurons. L1 antibodies interfered most strikingly with neurite outgrowth on Schwann cells (inhibition of 88% for small and 76% for large neurons), while no inhibition was detectable on fibroblasts. Similarly, although to a smaller extent than L1, N-CAM appeared to be involved in neurite outgrowth on Schwann cells and not on fibroblasts. Antibodies to J1 only showed a very small effect on neurite outgrowth of large neurons on Schwann cells. These observations show for the first time that identified adhesion molecules are potent mediators of glia-dependent neurite formation and attribute to L1 a predominant role in neurite outgrowth on Schwann cells which may be instrumental in regeneration.     

Abnormal HNK-1 expression in the cerebellum of an N-CAM null mouse

Human natural killer antigen-1 (HNK-1) is a carbohydrate epitope associated with sulfoglucuronylglycolipids and glycoproteins. Biochemical analyses have demonstrated associations between the HNK-1 epitope and isoforms of the neural cell adhesion molecule (N-CAM) family. In the cerebellum, HNK-1 is prominently expressed in Purkinje cell dendrites and Golgi cells. Purkinje cell expression of HNK-1 reveals an array of parasagittal stripes and transverse zones. Interestingly, the parasagittal expression pattern of HNK-1 is different from those reported with several other markers such as zebrin II/aldolase C and the small heat shock protein HSP25. N-CAM null knockout mice were used to explore the possible role of the HNK-1/N-CAM interaction during the topographical organization of the cerebellar cortex. N-CAM null mice have no N-CAM immunoreactivity but otherwise the cerebellum appears morphologically normal. Further, in the N-CAM null HNK-1 immunoreactivity is abolished from Purkinje cell dendrites but is retained on Golgi cells and neurons of the cerebellar nuclei. Despite the absence of N-CAM/HNK-1, parasagittal stripes and transverse zones in the cerebellum as revealed by using zebrin II immunocytochemistry appear normal.     

Nidogen is a prosurvival and promigratory factor for adult Schwann cells

Schwann cells provide a favorable microenvironment for successful regeneration of the injured peripheral nerve. Even though the roles of extracellular matrix proteins in the Schwann cell physiology have long been studied, the precise function of nidogen, a ubiquitous component of the basal lamina, in Schwann cells is unknown. In this study, we show that the protein and mRNA messages for nidogens are up-regulated in the sciatic nerve after sciatic nerve transection. We demonstrate that recombinant nidogen-1 increased the process formation of Schwann cells cultured from adult rat sciatic nerves and that nidogen-1 prevented Schwann cells from serum-deprivation-induced death. In addition, nidogen-1 promoted spontaneous migration of Schwann cells in two-independent migration assays. The Schwann cell responses to the recombinant nidogen-1 were specific because the nidogen-binding ectodomain of tumor endothelial marker 7 inhibited the nidogen responses without affecting Schwann cell response to laminin. Finally, we found that beta1 subunit-containing integrins play a key role in the nidogen-induced process formation, survival, and migration of Schwann cells. Altogether, these results indicate that nidogen has a prosurvival and promigratory activity on Schwann cells in the peripheral nerve.     

Laminin A, B1, and B2 Chain Gene Expression in Transected and Regenerating Nerves: Regulation by Axonal Signals

Abstract: Laminin A, B1, and B2 chain mRNA levels in degenerating and regenerating mouse sciatic nerves were examined using northern blot analysis. In normal intact nerves, B1 and B2 mRNA steady-state levels were high, but when the nerves were crushed, the steady-state levels of B1 and B2 mRNA per milligram wet tissue weight of the distal segments of the nerves increased five- to eightfold over that of control levels as the total RNA and β-actin mRNA levels increased, suggesting that these increases were the consequence of Schwann cell proliferation after axotomy. When the steady-state levels of B1 and B2 mRNA were normalized as the ratio to total RNA or β-actin mRNA levels, however, they drastically decreased to about 20% of the normal nerve levels in the nerve segments distal to both the crush and transaction sites 1 day after injury. In the crushed nerves, B1 and B2 mRNA levels gradually increased as the regenerating nerves arrived at the distal segments and reestablished normal axon–Schwann cell contact, and then returned to normal levels on the 21 st day. In the transected nerves, where Schwann cells continued to be disconnected from axons, both B1 and B2 mRNA levels remained low. Cultured Schwann cells expressed detectable levels of B1 and B2 chain mRNA which significantly increased when the cells were cocultured with sensory neurons. However, mRNA for A chain was not detectable in the normal, axotomized nerves or in cultured Schwann cells. These data indicate that Schwann cells express laminin B1 and B2 chain mRNA that are up-regulated by axonal or neuronal contact, but they do not express A chain mRNA.     

Demonstration of immunochemical identity between the nerve growth factor-inducible large external (NILE) glycoprotein and the cell adhesion molecule L1.

The nerve growth factor-inducible large external (NILE) glycoprotein and the neural cell adhesion molecule L1 were shown to be immunochemically identical. Immunoprecipitation with L1 and NILE antibodies of [3H]fucose-labeled material from culture supernatants and detergent extracts of NGF-treated rat PC12 pheochromocytoma cells yielded comigrating bands by SDS-PAGE. NILE antibodies reacted with immunopurified L1 antigen, but not with N-CAM and other L2 epitope-bearing glycoproteins from adult mouse brain. Finally, by sequential immunoprecipitation from detergent extracts of [35S]methionine-labeled early post-natal cerebellar cell cultures or [3H]fucose-labeled NGF-treated PC12 cells, all immunoreactivity for NILE antibody could be removed by pre-clearing with L1 antibody and vice versa.     

Developmental Expression of HNK-1-Reactive Antigens in Rat Cerebral Cortex and Molecular Heterogeneity of Sulfoglucuronylneolactotetraosylceramide in CNS Versus PNS

Monoclonal antibody HNK-1 reacts with a carbohydrate epitope present in proteins, proteoglycans, and sulfoglucuronylglycolipids (SGGLs). On high-performance TLC plates, SGGLs of the CNS from several species migrated consistently slower than those from the PNS, a result indicating possible differences in the structures. The structural characteristics of the major SGGL, sulfoglucuronylneolactotetraosylceramide (SGGL-1), from CNS was compared with those of SGGL-1 from PNS. Although the composition, sequence, and linkages of the carbohydrate moiety of the SGGL-1 species were identical, SGGL-1 from CNS contained mainly short-chain fatty acids, 16:0, 18:0, and 18:1, amounting to 85% of the total fatty acids, whereas SGGL-1 from PNS contained large proportions (59%) of long-chain fatty acids (greater than 18:0). These differences in the fatty acid composition accounted for the different migration pattern observed. The developmental expression of SGGLs and HNK-1-reactive proteins was studied in rat cerebral cortex between embryonic day (ED) 15 to adulthood. SGGLs in the rat cortex were maximally expressed around ED 19 and almost completely disappeared by postnatal day (PD) 20. This expression was contrary to their increasing expression in the cerebellum and sciatic nerve with postnatal development. Six to eight protein bands with a molecular mass of greater than 160 kDa were HNK-1 reactive in the rat cerebral cortex at different ages. The major HNK-1 reactivity to the 160-kDa protein band seen in ED 19 to PD 10 cortex decreased and completely disappeared from the adult cortex, whereas several other proteins remained HNK-1 reactive even in the adult. Western blot analyses of the neural cell adhesion molecules (N-CAMs) during development of the rat cortex with a polyclonal anti-N-CAM antibody showed that the major HNK-1-reactive protein bands were not N-CAMs. Between PD 1 and 10, 190-200-kDa N-CAM was the major N-CAM, and between PD 15 to adulthood, 180-kDa N-CAM was the only N-CAM present in the rat cortex.     

Chemical and structural changes of neurofilaments in transected rat sciatic nerve

The sequence of changes occurring in transected rat sciatic nerve was examined by electron microscopy and by sodium dodecyl sulfate (SDS) polyacrylamide disc gel electrophoresis. Representative segments of transected nerves were processed for ultrastructural examinations between 0 and 34 days after the transection of sciatic nerves immediately below the sacro-sciatic notch. The remainder of the transected nerves and the intact portions of sciatic nerves were desheathed and immediately homogenized in 1 percent SDS containing 8 M urea and 50 mM dithioerythritol. Solubilized proteins were analyzed on 12 percent gels at pH 8.3 in a discontinuous electrophoretic system. Initial changes were limited to the axons of transected nerve fibers and were characterized by the loss of microtubules and neurofilaments and their replacement by an amorphous floccular material. These changes became widespread between 24 and 48 h after transection. The disruption of neurofilaments during this interval occurred in parallel with a selective loss of 69,000, 150,000 and 200,000 mol wt proteins from nerve homogenates, thus corroborating the view that these proteins represent component subunits of mammalian neurofilaments. Furthermore, the selective changes of neurofilament proteins in transected nerves indicate their inherent lability and suggest their susceptibility to calcium-mediated alterations. Electrophoretic profiles of nerve proteins during the 4-34-day interval after nerve transection reflected the breakdown and removal of myelin, the proliferation of Schwann cells and the deposition of endoneurial collagen. A marked increase of intermediate-sized filaments within proliferating Schwann cell processes was not accompanied by the appearance of neurofilamentlike proteins in gels of nerve homogenates.     

Immunocytological localization of the HNK-1 carbohydrate in murine cerebellum,hippocampus and spinal cord using monoclonal antibodies with different epitope specificities

The HNK-1 carbohydrate, an unusual 3′-sulfated glucuronic acid epitope characteristic of many neural recognition molecules, serves as a ligand in neural cell interactions and is differentially expressed in the quadriceps and saphenous branches of the femoral nerve in the PNS of adult mice. Based on these observations, we investigated the possibility that the HNK-1 carbohydrate may be differentially distributed in neurons and fiber tracts also in the CNS thereby contributing to different targeting and guidance mechanisms. We have used antibodies with different HNK-1 epitope specificities to probe for subtle differences in expression patterns. In the adult mouse cerebellum the HNK-1 carbohydrate is detectable in stripe-like compartments in the molecular and Purkinje cell layers, whereas N-CAM and its associated α2,8 polysialic acid does not show this compartmentation. In the adult hippocampus, the HNK-1 carbohydrate localizes to perineuronal nets of inhibitory interneurons and marks the inner third of the molecular layer of the dentate gyrus. In the adult spinal cord, HNK-1 labeling is most pronounced in gray matter areas. White matter enriched regions show differential labeling with regard to fiber tracts and antibody specificity. Whereas the different antibodies do not show differences in staining in the cerebellum and the hippocampus, they show differences in staining pattern of fiber tracts and motoneurons in the spinal cord. The HNK-1 expression pattern also differed in the adult spinal cord from that observed at embryonic day 14 and postnatal day 14. Our observations suggest a functional role in the specification of functionally discrete compartments in different areas of the CNS and during development.     

04 and A007-sulfatide antibodies bind to embryonic Schwann cells prior to the appearance of galactocerebroside; regulation of the antigen by axon-Schwann cell signals and cyclic AMP

In the rat sciatic nerve, the relationship between Schwann cells, axons, the extracellular matrix and perineurial sheath cells undergoes extensive modification between embryo day 15 and the onset of myelination during the first postnatal day. Little is known about molecular changes in Schwann cells in this important prenatal period. In the present paper, we use immunofluorescence to study the prenatal development and postnatal regulation of the antigen(s) recognized by the 04 monoclonal antibody and a well-characterized rat monoclonal antibody to sulfatide, A007. We show that, in a series of immunochemical tests, the 04 antibody recognizes only sulfatide in neonatal and adult rat nerves. Both antibodies first bind to Schwann cells in the sciatic nerve at embryo day 16-17, and all Schwann cells bind both antibodies at birth. In the adult nerve, both nonmyelin-forming and myelin-forming cells are labelled with the antibodies. Schwann cells dissociated from embryo day 15 nerves and cultured in the absence of axons develop neither 04 nor A007 binding on schedule, and 04-positive and A007-positive Schwann cells from postnatal nerves lose the ability to bind these antibodies during the first few days in culture. Schwann cells in the distal stump of transected nerves also sharply down-regulate cell surface binding of 04. High numbers of 04-positive or A007-positive Schwann cells reappear in cultures treated with agents that mimic or elevate intracellular cAMP. We conclude that two anti-sulfatide antibodies 04 and A007, recognize an antigen, probably sulfatide, that appears very early in Schwann cell development (one to two days prior to galactocerebroside) but is nevertheless subject to upregulation by axonal contact or elevation of intracellular cAMP.     

Expression of several adhesive macromolecules (N-CAM, L1, J1, NILE, uvomorulin, laminin, fibronectin, and a heparan sulfate proteoglycan) in embryonic, adult, and denervated adult skeletal muscle

Levels of the neural cell adhesion molecule N-CAM in muscle are regulated in parallel with the susceptibility of muscle to innervation: N-CAM is abundant on the surface of early embryonic myotubes, declines in level as development proceeds, reappears when adult muscles are denervated or paralyzed, and is lost after reinnervation (Covault, J., and J. R. Sanes, 1985, Proc. Natl. Acad. Sci. USA, 82:4544-4548). Here we used immunocytochemical methods to compare this pattern of expression with those of several other molecules known to be involved in cellular adhesion. Laminin, fibronectin, and a basal lamina-associated heparan sulfate proteoglycan accumulate on embryonic myotubes after synapse formation, and their levels change little after denervation. L1, J1, nerve growth factor-inducible large external protein, uvomorulin, and a carbohydrate epitope (L2/HNK-1) shared by several adhesion molecules are undetectable on the surface of embryonic, perinatal, adult, or denervated adult muscle fibers. Thus, of the molecules tested, only N-CAM appears on the surface of muscle cells in parallel with the ability of the muscle cell surface to accept synapses. However, four antigens--N-CAM, J1, fibronectin, and a heparan sulfate proteoglycan--accumulate in interstitial spaces near denervated synaptic sites; regenerating axons traverse these spaces as they preferentially reinnervate original synaptic sites. Of particular interest is J1, antibodies to which block adhesion of central neurons to astrocytes (Kruse, J., G. Keihauer, A. Faissner, R. Timpl, and M. Schachner, 1985, Nature (Lond.), 316:146-148). J1 is associated with collagen and other fibrils in muscle and thus may be an extracellular matrix molecule employed in both the central and peripheral nervous systems.     

Expression of the adhesion molecules L1, N-CAM and J1/tenascin during development of the murine small intestine

We have previously studied the immunohistological localization of the three adhesion molecules L1, N-CAM and J1/tenascin in adult mouse small intestine and shown that L1 expression in epithelial crypt cells underlies the adhesion of these cells to one another [63]. To obtain further insight into the functional roles of L1, N-CAM and J1/tenascin in this organ we studied their expression starting at embryonic day 14 during embryonic and early postnatal morphogenesis and during epithelial cell migration in the adult. Expression of L1 was restricted to neural cells until approximately postnatal day 5, when L1 started to be detectable on crypt but not on villus cells, predominantly on the basolateral membrane infoldings. As in brain, L1-specific mRNA was approximately 6 kb in size. L1 from intestine appears to differ from the brain-derived equivalent in possessing a higher level of glycosylation. N-CAM was detectable from embryonic day 14 onward in neural and also in mesenchymal cells. Expression by smooth muscle cells decreased during development. In the villus core, N-CAM was strongly detectable at contact sites between smooth muscle cells forming the cellular scaffold of the villus. From embryonic day 14 onward, N-CAM appeared in both 180- and 140-kDa forms. J1/tenascin was present in both neural and mesenchymal cells from embryonic day 14 onward. Starting at embryonic day 17, J1/tenascin appeared concentrated at the boundary between mesenchyme and epithelium in an increasing gradient from the crypt base to the villus top. From embryonic day 14 onward J1/tenascin consisted of the 190- and 220-kDa components. J1/tenascin from intestine differed from brain-derived J1 in its carbohydrate composition. These observations show that the three adhesion molecules are expressed by distinct cell populations and may serve as cell-type-specific markers in pathologically altered intestinal tissue.     

Detection of the L2/HNK-1 Carbohydrate Epitope on Glycoproteins and Acidic Glycolipids of the Insect Calliphora vicina

The same or a very similar carbohydrate determinant, as represented by some sulfated, glucuronic acid-containing glycosphingolipids of human peripheral nerve, occurs on several adhesion molecules in the mammalian nervous system. In the present study, the occurrence of this epitope on glycoproteins and glycolipids of the fly, Calliphora vicina, was investigated by Western blot analysis and thin-layer chromatogram immunostaining. Several monoclonal antibodies recognizing an epitope on various neural cell adhesion molecules, designated L2 (334, 336, 349, and 412); the monoclonal antibody HNK-1 (recognizing an epitope on human natural killer cells); and a human IgM M-protein were found to react by Western blot analysis with various glycoproteins from larval and adult brains, although the intensity of staining of bands recognized by each antibody varied. Acidic glycolipids from pupae were also recognized, but only by the L2 antibody 334 and IgM M-protein. After desulfation of the acidic glycolipid fraction, the immunostaining pattern remained the same, an observation suggesting that the L2/HNK-1 epitope on insect acidic glycolipids contains a nonsulfated, glucuronic acid moiety. These observations indicate that the L2/HNK-1 carbohydrate structure occurs not only in vertebrates but also in insects on both glycoproteins and glycolipids, a finding suggesting a high degree of phylogenetic stability of this functionally important carbohydrate.     

Neuronal cell adhesion molecules and cytotactin are colocalized at the node of Ranvier

Immunocytochemical methods were used to show that Ng-CAM (the neuron-glia cell adhesion molecule), N-CAM (the neural cell adhesion molecule), and the extracellular matrix protein cytotactin are highly concentrated at nodes of Ranvier of the adult chicken and mouse. In contrast, unmyelinated axonal fibers were uniformly stained by specific antibodies to both CAMs but not by antibodies to cytotactin. Ultrastructural immunogold techniques indicated that both N-CAM and Ng-CAM were enriched in the nodal axoplasm and axolemma of myelinated fibers as well as within the nodal regions of the myelinating Schwann cell. At embryonic day 14, before myelination had occurred, small-caliber fibers of chick embryos showed periodic coincident accumulations of the two CAMs but not of cytotactin, with faint labeling in the axonal regions between accumulations. Cytotactin was found on Schwann cells and in connective tissue. By embryonic day 18, nodal accumulations of CAMs were first observed in a few medium- and large-caliber fibers. Immunoblot analyses indicated that embryonic to adult conversion of N-CAM and a progressive decrease in the amount of Ng-CAM and N-CAM occurred while nodes were forming. Sciatic nerves of mouse mutants with defects in cell interactions showed abnormalities in the distribution patterns and amount of Ng-CAM, N-CAM, and cytotactin that were consistent with the known morphological nodal disorders. In trembler (+/Tr), intense staining for both CAMs appeared all along the fibers and the amounts of N-CAM in the sciatic nerve were found to be increased. In mice with motor endplate disease (med/med), Ng-CAM and N-CAM, but not cytotactin, were localized in the widened nodes. Both trembler and med/med Schwann cells stained intensely for cytotactin, in contrast to normal Schwann cells which stained only slightly. All of these findings are consistent with the hypothesis that surface modulation of neuronal CAMs mediated by signals shared between neurons and glia may be necessary for establishing and maintaining the nodes of Ranvier.     

Histologic study of the outcome of components of nerves of human corpses grafted in nude mice (a model of chimeric intercellular communication)

Human corpse nerves are microsurgically grafted in nude mouse and biopsied from 1 to 152 days after graft. Schwann cells ever survived and probably produce angiogenic factors stimulating mice vessels. Others nerves are grafted end to end with mouse sciatic nerve. Mouse's neurites invading grafts are surrounded by human Schwann cells and set up normal chimeric nerve.     

Altered expression of neuronal cell adhesion molecules induced by nerve injury and repair

Peripheral nerve injury results in short-term and long-term changes in both neurons and glia. In the present study, immunohistological and immunoblot analyses were used to examine the expression of the neural cell adhesion molecule (N-CAM) and the neuron-glia cell adhesion molecule (Ng-CAM) within different parts of a functionally linked neuromuscular system extending from skeletal muscle to the spinal cord after peripheral nerve injury. Histological samples were taken from 3 to 150 d after crushing or transecting the sciatic nerve in adult chickens and mice. In unperturbed tissues, both N-CAM and Ng-CAM were found on nonmyelinated axons, and to a lesser extent on Schwann cells and myelinated axons. Only N-CAM was found on muscles. After denervation, the following changes were observed: The amount of N-CAM in muscle fibers increased transiently on the surface and in the cytoplasm, and in interstitial spaces between fibers. Restoration of normal N-CAM levels in muscle was dependent on reinnervation; in a chronically denervated state, N-CAM levels remained high. After crushing or cutting the nerve, the amount of both CAMs increased in the area surrounding the lesion, and the predominant form of N-CAM changed from a discrete Mr 140,000 component to the polydisperse high molecular weight embryonic form. Anti-N-CAM antibodies stained neurites, Schwann cells, and the perineurium of the regenerating sciatic nerve. Anti-Ng-CAM antibodies labeled neurites, Schwann cells and the endoneurial tubes in the distal stump. Changes in CAM distribution were observed in dorsal root ganglia and in the spinal cord only after the nerve was cut. The fibers within affected dorsal root ganglia were more intensely labeled for both CAMs, and the motor neurons in the ventral horn of the spinal cord of the affected segments were stained more intensely in a ring pattern by anti-N-CAM and anti-Ng-CAM than their counterparts on the side contralateral to the lesion. Taken together with the previous studies (Rieger, F., M. Grumet, and G. M. Edelman, J. Cell Biol. 101:285-293), these data suggest that local signals between neurons and glia may regulate CAM expression in the spinal cord and nerve during regeneration, and that activity may regulate N-CAM expression in muscle. Correlations of the present observations are made here with established events of nerve degeneration and suggest a number of roles for the CAMs in regenerative events.(ABSTRACT TRUNCATED AT 400 WORDS)