Embryonic Form Of Neural Cell Adhesion Molecule Research Articles

Embryonic form of N-CAM and development of the rat corticospinal tract; immuno-electron microscopical localization during spinal white matter ingrowth

The neural cell adhesion molecule N-CAM has been proposed to function in the guidance of outgrowing axons in the peripheral and central nervous system. Light microscopic observations have shown that the embryonic form of N-CAM (200–230 kDa) is present in the ventralmost part of the dorsal funiculus during corticospinal tract (CST) ingrowth in the first postnatal week (Joosten, Dev. Brain Res., 78 (1994) 226–236). Here, the subcellular localization of the embryonic form of N-CAM (E-NCAM) is determined by pre-embedding staining on vibratone sections and by postembedding immunogold-labelling on Epon embedded spinal cord sections. The electron microscopical observations indicate that E-NCAM is present on the outer membrane of CST growth cones as well as other unmyelinated axons which are present in the ventralmost part of the dorsal funiculus. Furthermore, E-NCAM is localized in an irregular patchy way on the outer side of the axonal membrane of small unmyelinated, later arriving CST axons. From these results it may be deduced that E-NCAM is involved in CST tract formation through guidance of outgrowing pioneer CST growth cones along other unmyelinated axons and through mediation of axon fasciculation of later arriving CST axons.

Developmental expression of N-CAM epitopes in the rat spinal cord during corticospinal tract axon outgrowth and target innervation

The neural cell adhesion molecule (N-CAM) is an integral membrane glycoprotein which mediates the adhesion of neurons to neurons and to other types of cells. During development, the N-CAM molecule is converted from a microheterogenous, highly sialylated, embryonic form (200–230 kDa) to several distinct, less sialylated but more adhesive, adult forms (120, 140 and 180 kDa). Monoclonal antibodies to epitopes of N-CAM (designated 5A5, 12F8, 5B8, 12F11 and 14E6) were used to investigate the spatial and temporal distribution of these neural cell adhesion molecules during the development of the corticospinal tract (CST) in rat spinal cord, from postnatal day 1 (P1) until adulthood. The light microscopical observations indicate that the embryonic form of N-CAM (200–230 kDa) recognized by 5A5 and 12F8 antibodies, respectively, is probably involved in the process of initial ingrowth of pioneer CST-fibers into the ventralmost part of the dorsal funiculus. The adult forms of N-CAM (120, 140, 180 kDa) recognized by 5B8, 12F11, and 14E6 antibodies, respectively, are present during later stages of CST white matter ingrowth and probably involved in fasciculation of the later arriving CST axons. During the period of CST target innervation (P4–P21), a gradual shift from embryonic (200–230 kDa) to adult (120, 140 and 180 kDa) forms of N-CAM occurs in spinal white matter and in the spinal gray matter adjacent to the ventral most part of the dorsal funiculus. The presence of embryonic N-CAM (200 kDa), with its low adhesive capacity in the CST outgrowth area may allow the CST axons to branch. If this branching is no longer desirable, only the higher affinity forms of N-CAM (120, 140 and 180 kDa) are expressed. The absence of N-CAM on CST target interneurons in the base of the dorsal horn and intermediate spinal gray matter strongly suggest that N-CAM is not involved in CST synapse formation. Together, these results suggest that various forms of N-CAM are involved in CST spinal white matter tract formation and subsequent target innervation, but not in the process of synapse formation.

Immunolocalization of different forms of neural cell adhesion molecule (NCAM) in rat taste buds

Taste buds consist of approximately 100 taste cells, including three morphological types of short receptor cells which synapse on the peripheral gustatory nerves. Some of the receptor cells produce neural cell adhesion molecule (NCAM), which may play a role in formation of specific connections in this system. Antibodies directed against different forms of NCAM were utilized in an attempt to define not only the distribution, but also the type of NCAM within taste buds. Within each taste bud approximately 10% of the taste cells exhibit abundant immunoreactivity for 180 kD (ld) or 140 kD (sd) forms of NCAM (i.e., those with an intracellular domain) along virtually the entire surface of the cell. Ultrastructural analysis reveals that these abundantly immunoreactive taste cells are of the intermediate morphological type, although not all of the intermediate taste cells within any bud are immunoreactive. In addition, the ultrastructural studies show that punctate (ld/sd) NCAM-immunoreactivity occurs on the membranes of taste cells and nerve fibers throughout each taste bud. The embryonic form of NCAM (E-NCAM), rich in polysialic acid residues, is present only in association with nerve fibers and other unidentified elongate, thin profiles of a few taste buds. The nerve plexus beneath the gustatory epithelium is also rich in NCAM-immunoreactivity. These fibers occasionally reveal immunoreactivity indicative of only the 120 kD (ssd) form of NCAM, typical of glial cells.

Immunocytochemical localization of cell adhesion molecules in the developing and mature olfactory system.

The localization of Ca+(+)-independent cell adhesion molecules (CAMs) in the developing and mature olfactory epithelium and bulb is reviewed. The CAMs included in this article are the neural cell adhesion molecule (N-CAM), the 180 kD component of N-CAM (N-CAM 180), the embryonic form of N-CAM (E-N-CAM), L1 glycoproteins, J1 glycoproteins, and the adhesion molecule on glia (AMOG). In addition, the expression of the L2-HNK-1 carbohydrate epitope, shared by N-CAM, L1, J1 and myelin-associated glycoprotein (MAG) in the adult olfactory epithelium and bulb has also been documented. For the localization of these molecules at the light and electron microscopic levels, immunocytochemical techniques were used and are described in detail. During development and organogenesis, the olfactory system exhibits a pattern of CAM expression similar to the general pattern described for the developing nervous system. In the adult olfactory system, however, a significant retention of CAMs characteristic for developmental and morphogenetic processes, such as E-N-CAM, AMOG, as well as the high molecular weight components of J1 glycoproteins, can be observed. The retention of these embryonic features are most likely associated with the cell turnover and high plasticity of this system. Moreover, the predominance of N-CAM 180 with respect to other components of N-CAM, as well as the absence of the L2/HNK-1 carbohydrate epitope, are also particular traits of the primary olfactory system which could be associated with its exceptional properties.

Abnormal expression of embryonic neural cell adhesion molecule (N-CAM) in the developing mouse cerebellum after neonatal administration of cytosine arabinoside

We have previously found that neonatal administration of cytosine arabinoside (Ara-C) induces cerebellar malformation, including arrest of granule cell migration and destruction of glial palisade. In the present study, the expression of neural cell adhesion molecule (N-CAM) and its distribution were examined in the mouse malformed cerebellum induced by neonatal Ara-C administration. Western blot analysis demonstrated that, in the control group, the embryonic form of N-CAM (E-N-CAM) was expressed in the 7-day-old mouse (P7) cerebellum, but not in the P14 cerebellum which exhibited only the adult form of N-CAM (A-N-CAM). On the other hand, E-N-CAM was expressed in the Ara-C treated cerebellum not only at P7 but also at P14. Immunohistochemical studies revealed that E-N-CAM was observed in the area below the external granular layer (EGL) of the P7 cerebellum in both the control and Ara-C treated mice. In contrast, E-N-CAM immunoreactivity was found in the arrested EGL cells in the Ara-C treated cerebellum of P14 mice but not in the age-matched controls. Therefore, E-N-CAM was aberrantly expressed in the arrested EGL at P14. From the present and previous findings, it was speculated that the aberrant expression of E-N-CAM in the arrested EGL cells is attributed to abnormal cellular interactions probably arrested EGL cell-to-Bergmann glia.

Expression of alternative isoforms of the neural cell adhesion molecule (NCAM) on normal brain and a variety of brain tumours

A panel of monoclonal antibodies, including a reagent designated ERIC-1, have been characterized as binding to the human neural cell adhesion molecule (NCAM). These monoclonal antibodies bind in a relatively uniform manner to a variety of normal and neoplastic tissues arising from the neuroectoderm. However, multiple forms of the protein are known to arise from the differential splicing of exons within the NCAM gene located on chromosome 11 at q23. On human adult brain, four isoforms of 180, 170, 145 and 120 kDa have been identified. Here, we report the identification of another NCAM isoform of 95 kDa that is apparent on tissues following either N-glycanase or neuraminidase treatment to remove carbohydrate and sialic acid residues from the molecule respectively. NCAM expression is further complicated by differential post-translational modification of the molecule which is developmentally regulated. In general, fetal NCAM is more heavily polysialylated than the adult forms of the molecule. Human fetal brain has been shown to express the heavily sialylated embryonic form of NCAM, but following neuraminidase digestion, a similar pattern of NCAM expression is seen to that in adult brain. A variety of human brain tumours examined also show different patterns of NCAM expression, despite their uniform staining with monoclonal antibodies. The significance of these observations for designing new molecular and immunological approaches to the diagnosis of a variety of primary tumours is reviewed.

Olfactory neurons express a unique glycosylated form of the neural cell adhesion molecule (N-CAM).

mAb-based approaches were used to identify cell surface components involved in the development and function of the frog olfactory system. We describe here a 205-kD cell surface glycoprotein on olfactory receptor neurons that was detected with three mAbs: 9-OE, 5-OE, and 13-OE. mAb 9-OE immunoreactivity, unlike mAbs 5-OE and 13-OE, was restricted to only the axons and terminations of the primary sensory olfactory neurons in the frog nervous system. The 9-OE polypeptide(s) were immunoprecipitated and tested for cross-reactivity with known neural cell surface components including HNK-1, the cell adhesion molecule L1, and the neural cell adhesion molecule (N-CAM). These experiments revealed that 9-OE-reactive molecules were not L1 related but were a subset of the 200-kD isoforms of N-CAM. mAb 9-OE recognized epitopes associated with N-linked carbohydrate residues that were distinct from the polysialic acid chains present on the embryonic form of N-CAM. Moreover, 9-OE N-CAM was a heterogeneous population consisting of subsets both with and without the HNK-1 epitope. Thus, combined immunohistochemical and immunoprecipitation experiments have revealed a new glycosylated form of N-CAM unique to the olfactory system. The restricted spatial expression pattern of this N-CAM glycoform suggests a possible role in the unusual regenerative properties of this sensory system.

Expression of L1 and N-CAM cell adhesion molecules during development of the mouse olfactory system

The expression of the neural adhesion molecules L1 and N-CAM has been studied in the embryonic and early postnatal olfactory system of the mouse in order to gain insight into the function of these molecules during development of a neural structure which retains neuronal turnover capacities throughout adulthood. N-CAM was slightly expressed and L1 was not significantly expressed in the olfactory placode on Embryonic Day 9, the earliest stage tested. Rather, N-CAM was strongly expressed in the mesenchyme underlying the olfactory placode. In the developing nasal pit, L1 and N-CAM were detectable in the developing olfactory epithelium, but not in regions developing into the respiratory epithelium. At early developmental stages, expression of the so-called embryonic form of N-CAM (E-N-CAM) coincides with the expression of N-CAM, whereas at later developmental stages and in the adult it is restricted to a smaller number of sensory cell bodies and axons, suggesting that the less adhesive embryonic form is characteristic of morphogenetically dynamic neuronal structures. Moreover, E-N-CAM is highly expressed at contact sites between olfactory axons and their target cells in the glomeruli of the olfactory bulb. L1 and N-CAM 180, the component of N-CAM that accumulates at cell contacts by interaction with the cytoskeleton are detectable as early as the first axons extend toward the primordial olfactory bulb. L1 remains prominent throughout development on axonal processes, both at contacts with other axons and with ensheathing cells. Contrary to N-CAM 180 which remains detectable on differentiating sensory neuronal cell bodies, L1 is only transiently expressed on these and is no longer detectable on primary olfactory neuronal cell bodies in the adult. Furthermore, whereas throughout development L1 has a molecular form similar to that seen in other parts of the developing and adult central nervous systems, N-CAM and, in particular, N-CAM 180 retain their highly sialylated form at least partially throughout all ages studied. These observations suggest that E-N-CAM and N-CAM 180 are characteristic of developmentally active structures and L1 may not only be involved in neurite outgrowth, but also in stabilization of contacts among fasciculating axons and between axons and ensheathing cells, as it has previously been found in the developing peripheral nervous system.

Differentiation-regulated loss of the polysialylated embryonic form and expression of the different polypeptides of the neural cell adhesion molecule by cultured oligodendrocytes and myelin.

The expression of the neural cell adhesion molecule (N-CAM) on cultured murine oligodendrocytes, their precursors, and myelin was examined by indirect immunofluorescence, biosynthetic radiolabeling followed by immunoprecipitation and Western blot analysis, using antibodies specific for various forms of the molecule. In all culture systems studied, whether the oligodendrocytes were cultured as an enriched fraction containing precursor cells or in the presence of astrocytes and neurons, a similar differentiation-stage-related expression of N-CAM was seen. At early developmental stages many tetanus toxin receptor- and A2B5 antigen-positive putative oligodendrocyte precursors with bipolar morphology were seen and found to express N-CAM in its embryonic form. Of the 04 antigen-positive immature oligodendrocytes with few slender processes most expressed N-CAM, but few the embryonic form of N-CAM. The more mature 01 or 010 antigen-positive oligodendrocytes were found to express exclusively the adult form of N-CAM. Oligodendrocytes synthesized the 120 and 140 kD forms of N-CAM (N-CAM 120 and N-CAM 140), but not N-CAM 180, although with differentiation, N-CAM 120 predominated in oligodendrocytes and also in pure myelin. N-CAM 120 could be released from oligodendrocytes and myelin by phosphatidylinositol-specific phospholipase C, suggesting that in both oligodendrocytes and myelin N-CAM 120 is inserted into the membrane by covalent linkage to phosphatidylinositol.

Monoclonal antibodies to polysialic acid reveal epitope sharing between invasive pathogenic bacteria, differentiating cells and tumor cells

Monoclonal antibodies (mAb) for rapid diagnosis and detection of invasive bacteria and identification of pathogenic factors in infectious disease are equally important in medical microbiology and clinical pathology and may even provide a breakthrough in basic medical and cell biology research. Such a situation evolved from the application of a unique mAb against the poorly immunogenic homopolymers of alpha 2,8-linked sialic acid of Escherichia coli K1 and meningococci group B capsules which could be derived from immune-hyperreactive NZB-autoimmune mice. The cross-reactivity of this mAb with identical polysialic acid (polySA) units of the neural cell adhesion molecule (N-CAM) revealed antigenic mimicry as the basis for the escape of the above-mentioned bacteria from host immune response and immune defense. The mAb proved to be a specific and sensitive diagnostic reagent as well as a very efficient therapeutic agent in experimental E. coli K1 and meningococcal group B infections in mice. Furthermore, the mAb was found to react exclusively with long-chain polySA units characteristic of the embryonic form of N-CAM. This led to the discovery that the embryonic form of N-CAM is present outside neural tissue in the mesodermally derived kidney where it is specifically expressed during embryonic organ differentiation and reexpressed under conditions of malignant growth in nephroblastoma. Therefore, the embryonic form of N-CAM represents an onco-differentiation antigen in kidney.

Release of the 120 kDa component of the mouse neural cell adhesion molecule N-CAM from cell surfaces by phosphatidylinositol-specific phospholipase C

To study the membrane anchoring of the 120 kDa component of the neural cell adhesion molecule N-CAM, the smallest form lacking a transmembrane domain, cultured mouse neural cells were treated with phosphatidylinositol-specific phospholipase C from Staphylococcus aureus. When live cultures of astrocytes and neurons are treated with phosphatidylinositol-specific phospholipase C, N-CAM 120 is released into the supernatant. Under these conditions N-CAM 140 and N-CAM 180 are not released. Phospholipase C from Bacillus cereus or Clostridium perfringens does not release N-CAM 120. The embryonic form of N-CAM on astrocytes migrating as a broad band between 120 and 180 kDa is also partially released by phosphatidylinositol-specific phospholipase C as a band migrating between 120 and 160 kDa. These observations suggest novel mechanisms in regulation of N-CAM 120 expression on the cell surface and in modulation of N-CAM-mediated cell adhesion.