2 neural cell adhesion molecules

Neural Cell Adhesion Molecules

Kim Tieu and Peter H. Yu

1. Introduction

During development of the nervous system, cell migration, axonal outgrowth, axonal guidance, and selective cell adhesion and recognition are some of the crucial processes required for neural pattern formation. Numerous studies reveal that cells bind selectively to one another and that they segregate according to their tissue types. As early as 1907, Wilson demonstrated that dls- sociated cells from two different types of sponges, when mixed together, reassociated to form two organisms, each containing cells from one parent species. Similar results are also observed m coelenterates (Chalkey, 19451, chick embryonic cells (Moscona and Moscona, 1952) and amphibian embryonic cells (Holtfreter 1948a,b; Townes and Holtfreter, 1955). When the nervous system is in its developmental stages, neurons extend their axons over considerable distances to innervate their targets. Studies done in grasshopper (Bastiani et al, 1986; Bastiani and Goodman, 1986; du-Lac et al., 1986), zebrafish embryo (Kuwada, 1986,1992), and chick retinal ganglion cells (Silver and Sapiro, 1981) indicate that these connectivities are carried out in a highly stereospecific fashion. There are many theories regarding these selective interactions, and cell adhesion molecules have been shown to play a very significant role.

CAMS (cell adhesion molecules) have been shown in numerous studies to be essential in the processes of embryogenesis and hlstogenesis (for reviews, see Edelman, 1985a,b, 1986, 1988a,b; Edelman and Crossin, 1991). In addition to participating in these normal biological processes, CAMS are also linked to many patho- logical conditions such as osteoporosis, tumor spread, and ath-

From Neuromethods, vol 33 Cell Neurobrology Technrques Eds A A Boulton, G B Baker, and A N Bateson 0 Humana Press Inc

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erosclerosis. Based on the functions of these molecules, many bio- technology companies are developing new therapies for a wide variety of diseases (Travis, 1993).

NCAMs (neural cell adhesion molecules) were first isolated in 1977 (Brackenbury et al., 1977; Thiery et al, 1977) Many other types of CAMS have been subsequently identified and characterized. In general, they are grouped into four classes: cadherins, the immunoglobulin-like superfamily, integrins, and selectins (Hynes and Lander, 1992). The NCAM, a member of the Ig superfamily, is the most widely studied and characterized CAM. The focus of this chapter is to discuss different methods that have been used to isolate and study NCAM . However, prior to dis- cussing these methods, it is necessary to understand the classifi- cation, structure, expression, mechanism of binding, and possible functions of NCAMs in the central nervous system (CNS)

2. Neural Cell Adhesion Molecules

2.1. Structure and Isoforms of NCAMs

Early designations of NCAMs include D2 in the rat (Jorgensen, 1976,1980), NCAM in chick (Rustishauser et al., 19761, and NS-4 (Goridis et al, 1978) or BSP-2 in the mouse (Him et al, 1981). These molecules were later found to be identical and therefore were subsequently termed NCAMs. The NCAM is encoded by a single gene that is found on chromosome 9 in the mouse (D’Eustachio et al., 1985) and on chromosome 11 in humans (Nguyen et al., 1986). From this single gene, several transcripts are generated as a result of alternative splicing and polyadenylation (Owens et al., 1987; Goridis and Wille, 1988; Barbas et al., 1988; Santom et al., 1989, Thompson et al., 1989). Up to 192 possible isoforms of NCAM can be generated this way (Barthels et al., 1992). They can be further modified by posttranslational processes such phosphorylation and sialylation. In adult brain, three major forms of NCAM have been identified: NCAM-180, NCAM-140, and NCAM-120. These proteins appear in SDS-PAGE (sodium dodecylsulfate-polyacrylamide gels) as three bands with molecular weights of 180,140, and 120 kDa, respectively. They are also known as large cytoplasmic domain (Id), small cytoplasmic domain (sd), and small surface domain polypeptides (ssd) (Hemperly et al, 1986). As seen in Fig. 1, these three polypeptides have identical extracellular domains. They each have five immunoglobulin-like (Ig-like) domains fol-

Neural Cell Adhesion Molecules 105

COOH

Fig. 1. Structure of three major isoforms of NCAM: They all have five Ig-like (rectangles) and two fibronectin-like (circles) domains. The designated first Ig-like domain is closest to the N-terminal and the fifth one is connected to a fibronectin-like domain. Both NCAM-180 and NCAM-140 have one transmembrane segment, but NCAM-180 has a larger cytoplasmic domain (oval) than that of NCAM-140. NCAM-120 is anchored to the cell membrane via a phosphatidylinositol bond (jagged line). More detailed information may be found in relevant reviews (Rutishausher and Jessell, 1988; Edelman, 1988a; Edelman and Crossin, 1991; Goridis and Brunenet, 1992; Fields and Itoh, 19961,

lowing the extracellular N-terminal. These domains are connected to each other by disulfide bonds. The fifth Ig-like domain, which is connected to fibronectin-like domain, is the primary binding site of polysialic acid (PSA) (Rothbard, 1982; Finn et al., 1983; Nelson et al., 1995). PSA is a linear homopolymer of a2&linked sialic acid. The fourth domain is the site of insertion of an addi-

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tional sequence of 10 amino acids because of the presence of a VASE exon (variable alternative splice exon) in NCAM mRNA transcripts. (Small et al., 1988, Santoni et al., 1989; Small and Akeson, 1990; Barthels et al., 1992;). The roles of PSA and the VASE exon will be discussed in more detailed later. The third Q-like domain is suggested to be responsible for homophllic binding of NCAM (Rao et al., 1992); however, a recent study shows that this is not the only domain involved (Ranheim et al., 1996). The second domain binds heterophihcally to heparin-like polysaccharides and thereby mediates cell-cell and cell-extracellular matrix interactions (Cole and Akeson, 1989; see Daniloff et al., 1994, for functions of different components of NCAM). Another common feature of these three NCAM isoforms is the presence of two fibronectm type III domains.

The major differences between these polypeptides are the size of their cytoplasmlc domains and the mode of attachment to the cell membrane. NCAM-180 and NCAM-140 are integral membrane glycoproteins (Lyles et al., 1984). They each have a single transmembrane segment, but the cytoplasmic domain of NCAM-180 is 261 amino acids longer than that of NCAM-140 (Murray et al., 1986). In contrast to these two isoforms, NCAM-120 lacks intracellular domains and it is anchored to the cell membrane via phosphatidylinositol bond (Nybroe et al., 1985; He et al., 1986; Hempley et al., 1986). As a result, NCAM-120 can be released from the cell membrane because of hydrolytic cleavage (He et al., 1987). This soluble form of NCAM has been reported (Sadoul et al., 1986; Gower et al., 1988; Probstmeier et al., 1989). An actively secreted NCAM is also reported but it has not yet been characterized (Bock et al., 1987).

2.2. Expression of NCAM

Despite what its name implies, the expression of NCAM is not limited to neural tissues. NCAMs are also found m several cell types in nonneural tissues. Detailed studies reveal NCAM expression is regulated in a spatiotemporal fashion. During early embryogenesis, NCAMs appear in all three germ layers, but at later developmental stages, they are expressed mainly in cells of mesodermal and ectodermal origin (Crossin et al., 1985; Edelman 1986). The expression of different isoforms is developmental-stage and cell-type dependent. For example, in adult brain, NCAMs are synthesized in neurons (Prieto et al., 1989; Goldowitz et al., 1990)


whereas m immature brain, they are expressed in both neurons and glia (Langley et al., 1983; Hirn et al., 1983). NCAM-180 and NCAM-140 predominate in neurons (Gegelashvili et al., 1993) but in glial cells NCAM-120 is the major isoform (Nybroe et al., 1985; Noble et al., 1985). In embryonic stages, NCAMs contain high levels of PSA (Rothbard et al., 1982) but low quantities of the VASE exon (Small and Akeson, 1990). As the nervous system develops, the levels of PSA and VASE exon progressively decrease and increase, respectively (Rothbard et al., 1982; Small and Akeson, 1990). As the embryonic form of NCAMs disappear, NCAM-180, NCAM-140, and NCAM-120 appear (Chuong and Edelman, 1984). The developmental expression of NCAMs in vertebrate embryos has recently been found to be regulated by homeodomain genes (Wang et al., 1996).

In addition to genetic regulation, extracellular factors also affect the expression of NCAMs. In general, the effects of these agents are cell-type dependent and not very dramatic Nerve growth factor (NGF) has been shown to increase expression of NCAM in PC12 cells (Prentice et al., 1987; Doherty et al., 1988). Similarly, transforming growth factor-beta (TGF-8) increases NCAMs expression in fibroblast cell line (Roubin et al., 1990) as well as in embryonic or newborn mouse olfactory epithelium (Satoh and Takeuchi, 1995). TGF-/3, however, reduces levels of NCAMs in astrocytes (Saad et al., 1991). Thyroxin is another agent that affects NCAM expression. It decreases NCAM levels m skeletal muscle (Thompson et al., 1987), but increases this expression in Xenopus liver (Levi et al., 1990).

2.3. Mechanism of Binding

NCAMs have been shown to be involved in cell-cell mteractions between cell types such as neurons, astrocytes, oligoden- drocytes, Schwann cells, muscle cells, and fibroblasts (Rutishauser et al., 1983; Kellhauer et al., 1985; Bixby and Reichardt, 1987). The mechanism of this binding is calcium-independent and homophilic (i.e., NCAM on one cell binds directly to another NCAM on adjacent cell) (Rutishauser et al., 1982; Sadoul et al., 1983; Edelman, 1983; Hoffman and Edelman, 1983; Doherty and Walsh, 1992). This process is proposed to be mediated by the amino acid sequence in the third Ig-like domain (Rae et al., 1992); however, a recent study indicates that all five domains are involved and that they interact pairwise in an antiparallel orientation (Ranheim et al., 1996). The

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second Q-like domain has been found to bind heterophilically to heparin-like polysaccharides and thereby mediates cell-cell and cell-extracellular matrix interactions (Cole et al., 1989). This lat- ter type of binding has been shown in chick embryonic brain (Storms et al., 1996).

Changes in NCAM density (Sadoul et al., 1983), distribution on cell surfaces (Daniloff et al., 1986), or posttranslational modifica- tion (Hoffman and Edelman, 1983; Chuong and Edelman, 1984) may all affect the binding of NCAMs. A twofold increase in NCAM density leads to a more than 30-fold increase in binding rates (Hoffman and Edelman, 1983; Sadoul et al., 1983). Conversely, the steric effects of PSA reduce the binding of NCAM (Yang et al., 1992). Removal of PSA on NCAMs results in a fourfold increase in binding rates (Hoffman and Edelman, 1983). Presence of the VASE exon also influences interactions between cells (Chen et al., 1994). Cells without the VASE exon form aggregates which segregate to cells with the VASE exon. Finally, other surface molecules such as heparan sulfate proteoglycan, a functional analog of heparin, have also been shown to affect the binding of NCAMs. In some studies (Cole et al., 1986; Cole and Akeson, 1989), heparin inhibits NCAM binding, but in another study (Kadmon et al., 1990) heparin is required for this process. It is likely that heparin is essential for the binding of NCAM (Goridis and Brunet, 1992).

2.4. Roles of NCAM

2.4.1. Cell Adhesion, Migration, and Recognition

NCAMs are known to mediate cell-cell adhesion between numerous cell types in the nervous system and elsewhere. One way that NCAMs regulate the adhesion and migration of these cells is through the presence of PSA on these molecules. In vertebrate embryos, PSA seems to be confined to NCAMs (Tomasiewicz et al., 1993; Cremer et al., 1994). PSA is a linear homopolymer of a2,8-linked sialic acid (n = 8 to over loo), which is thought to be synthesized in the Golgi. Two different polysialyltransferase enzymes (PST and STX) have been identified and proposed to be responsible for adding PSA to NCAMs (Eckhardt et al., 1995; Nakayama et al., 1995; Scheidegger et al., 1995; Yoshida et al., 1995). A recent in vitro study demonstrated that PST alone was able to polysialate NCAMs (Nakayama and Fukuda, 1996). The level of PSA on NCAMs is regulated during development. The embryonic form of NCAMs has a higher degree of sialylation than

Neural Cell Adhesron Molecules 709

adult forms do. The steric hindrance from these large, negatively charged molecules reduces the adhesiveness of NCAMs. This effect partly explains how NCAMs are paradoxically involved in both cell adhesion and motility.

NCAMs play a significant role in morphogenesis and histogenesis. NCAM antibodies have been demonstrated to disrupt layer formation in the retina (Buskirk et al., 1980) and alter the mapping of the retina to the optic tectum (Fraser et al, 1984; 1988). PSA on NCAMs has been shown to be involved in axonal guidance, targeting (Tang et al., 1994; Yin et al., 1994) and cell migration (Tomasiewicz et al., 1993; Schwanzel-Fukuda et al., 1994; Lois et al., 1996) (see review, Rutishauser, 1996). Cell migration is involved in normal biological processes such as embryonic development, wound healing, and immune response and in patho- logical conditions such as tumor spread. Therefore, it is impor- tant that levels of NCAMs and their degrees of sialylation are tightly regulated throughout development as well as in adult life.

2.4.2. Synaptic Plasticity, Learning, and Memory In recent years, PSA has gained much attention because of its

various roles in synaptic plasticity, learning, and memory. CAMS have been proposed to be capable of either promoting (by increas- ing neurite outgrowth) or inhibiting (by stabilizing synaptic structures) nervous system plasticity. These effects of NCAMs depend on the isoform expressed. For instance, the presence of the VASE exon reduces neurite outgrowth (Doherty et al., 1992; Liu et al., 1993). At early stages of development, less than 3% of NCAM transcripts have the VASE exon, but this amount progressively increases to approx 50% in the adult CNS (Small and Akeson, 1990). In contrast to the VASE exon, PSA promotes neurite outgrowth (Landmesser et al., 1990; Doherty et al., 1992). During development, NCAMs are converted from PSA-rich (30% by weight) in embryonic form to PSA-poor (10%) in the adult form (Rothbard et al., 1982). In areas such as the olfactory bulb where neurogenesis and neurite outgrowth continue throughout life, the VASE exon is not expressed (Small and Akeson, 1990) and PSA- NCAM is retained (Miragall et al., 1988). The combined effect of increased VASE and decreased PSA results in the change of NCAM function from promoting to inhibiting synaptic plasticity.

Because of their influence on plasticity, NCAMs are proposed to affect learning and memory, and different studies have shown

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that NCAMs do affect LTP (long-term potentiation) (Luthl et al., 1994; Ronn et al., 1995; Muller et al., 1996). Intracerebral injection of antibodies against NCAMs has been shown to cause amnesia in chicks (Scholey et al., 1993; Rose, 1995). In another study, NCAM-deficient mice have been found to show defects in spatial learning (Cremer et al., 1994). Details on the involvement of NCAM in synaptic plasticity, learning, and memory are described in recent reviews (Doherty et al., 1995; Jorgensen, 1995; Fields and Itoh, 1996; Rutishauser and Landmesser, 1996).

2.4.3. Neuronal Regeneration

Since NCAM affects neuronal plasticity, it is proposed to be involved in neuronal regeneration as well. During regeneration of the spinal cord in the amphibian urodeles Pleurodeles waltl, embryonic NCAMs are re-expressed and increased intensely (Caubit et al., 1993). This form of NCAM is also reexpressed in rodent nerves in response to injury (Daruloff et al., 1986), and the rate of recovery is reduced when NCAM antibodies are applied to transected sciatic nerves in rats (Remsen et al., 1990). This topic of CAMS and neuronal regeneration has been recently reviewed (Damloff et al., 1994; Walsh and Doherty, 1996).

2.4.4. NCAMs and Diseases NCAM levels have been found to change in certain diseases.

For example, NCAM-120 was found to be significantly increased in the CSF of patients with schizophrenia (Poltorak et al. 1995). A postmortem study done by another group found that NCAM-PSA levels were reduced in the hippocampal region of schizophrenic patients (Barbeau et al., 1995). NCAM levels were also found to be increased in bipolar I and malor depression (Paltorak et al., 1996) as well as m the presence of fetal neural tube defects (Jorgenson, 1981). The expression of embryonic NCAMs, which are less adhesive than adult forms, has been found in certain forms of cancer such as Wilms tumor (Roth et al., 1988, Zuber and Roth, 1990), small cell lung carcinoma (Krbbelaar et al., 1989; Moolenaar et al., 1990), and human neuroblastoma (Moolenaar et al., 1990; Pate1 et al., 1989). NCAMs have been suggested as markers for some disorders such as multiple myeloma (Kaiser et al., 1994, 1996a,b; Ong et al., 1996) and small-cell lung cancer (Kibbelaar et al., 1989; Jaques et al., 1993). In general, it is uncertain at this moment whether the changes in NCAM levels are the cause or consequence of these diseases.


3. Methods

3.1. Cell Adhesion Assays

Cell adhesion assays are performed in order to see whether or not the cells of interest bind preferentially to each other. There are two types of adhesion assays: aggregation and binding. For aggregation assays, one studies aggregation of cells, plasma membrane fragments, liposomes, or beads bearing NCAMs. Similarly, in binding assays, one can study the binding of cells to cell monolayers, plasma membrane fragments, liposomes, or substrates bearing NCAMs. Each method has some advantages and disadvantages. For example, the study of aggregation of cells in suspension is ideal for a large number of samples, but sometimes the nonspecific binding is stronger than that from the molecules of interest. More detailed discussions of these different techniques and their advantages and disadvantages have been reviewed by Frazier and Glaser (1979) and Hoffman (1992).

3.1.1. Preparation of Single Cells for Adhesion Assays

Single-cell suspensions can be prepared from cultured cells or tissues If tissues are to be used, embryonic tissues are a preferred source as it is more difficult to obtain viable cells from most tissues in older animals. Trypsin is commonly used to dissociate cells. The concentration of trypsin should be chosen according to the experiment. For example, cultured cells are more easily dissoci- ated than those from tissues, so, a lower concentration of trypsin should be used. The concentration of trypsin should be high enough to sufficiently produce single-cell suspensions yet not disrupt the integrity of the cell surface proteins of interest. Preliminary experiments should be done in order to determine trypsin levels. Up to 0 5% (w/v> of crude trypsin has been used (Brackenbury et al. 1977). If purified trypsin is used, 0.002-0.08% should be sufficient. The following is a general procedure used to prepare single-cell suspensions from tissues. More information can be obtained from other references (Moscona, 1952; Chuong et al., 1982, Brackenbury et al., 1981; Hoffman, 1992). 3.1 .I .I. MATERIALS

1. Trypsin (previously determined concentration). 2. 1 mM EDTA. 3. DNase I. 4. 3.5% bovine serum albumin (BSA) in HBSS.

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5. Ca2+ and Mg2+-free medium (Hanks’ balanced salt solution [HBSS] or phosphate-buffered saline [PBS], containing 1 mM EDTA, 20 mM HEPES, pH 7.4).

3.1.1.2. PROCEDURES

1. Wash the embryonic tissues once or twice with HBSS and then incubate with the appropriate concentration of trypsin in Ca2+/Mg2+-free medium such as HBSS at either 37°C for 20-30 minutes or room temperature for 60 min, with constant gentle shaking. DNase I (20 pg/mL of sample) is added to prevent cell aggregation caused by released DNA from damaged cells.

2. During this period of incubation, occasionally triturate the cells with decreasing tip-diameter, fire-polished, Pasteur pipets.

3. Add trypsm inhibitor to stop trypsin activity. 4. Remove large clumps of undissociated cells present by briefly

centrifuging the suspension (8OOg for 15 s). Keep the supernatant and discard the pellet.

5. Centrifuge the supernatant at 7008 for 3 min or 1008 for 15 min. Discard the supernatant and keep the pellet.

6. Ice-cold medium is required for the following steps. Debris and cells are separated by resuspendmg the pellet in HBSS containing DNase I (20 pg/mL). Gently layer this suspension over 3.5% BSA in HBSS. Centrifuge as in step 5 above. Again discard the supernatant and keep the pellet.

7. Wash the cells by gently pipetting them up and down with sufficient volume of medium. Centrifuge them as above.

8. Fmally, cells are resuspended in a desired volume of medium. Cell density and the outcome of single-cell suspension can be inspected microscopically using a hemocytometer. Cells should be maintained on ice until the adhesion assay is performed.

3.1.2. Preparation of Labeled Cells

In binding assays, cells are usually labeled prior to use. Vari- ous methods have been used for this purpose. In general, cells are labeled radioactively with 51Cr (Elite et al. 19901, 13Hl-leucine (McClay et al., 1981; Lotz et al., 1989), [35Sl-methionine (Cole et al., 19861, or fluorometrically with fluorescem diacetate (Bracken- bury et al., 19811, di1 and di0 (Friedlander et al., 1989). The chosen agent is incubated with the cells for a period of time and the unbound fraction is removed by washing with media If radioactive labels such as 13Hl-leucine or [35Sl-methionine are to be used,

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they should be incubated with cells in a medium free of these respective amino acids. Before these radioactive labels are added, preincubating cells in medium free of these amino acids for a period of time (a few hours) may be necessary.

The fluorescently labeled cells are preferred for small-scale experiments.. This method also allows one to inspect binding between cells microscopically and avoid potential exposure to radioactivity. However, if a large number of samples is involved, the radioactive method would be a better choice.

3.7.3. Aggregation Assay of Single-Cell Suspension

Aggregation of cells in suspension is determined by measuring the rate of decrease of particle numbers (Brackenbury et al. 1977) using a Coulter counter. Antibody inhibition is used as a control in these assays since nonspecific aggregation may occur.

3.1.3.1. MATERIALS 1. SME medium (Eagle’s minimum essential medium with spm-

ner salts containing HEPES pH 7.4 and 20 pg/mL of DNase. 2. Immune Fab’ in PBS (from IgG of immunized rabbits). 3. Nonimmune Fab’ in PBS (from IgG of unimmunized rabbits). 4. Glass screw-cap scintillation vials (28 x 61 mm). 5. 1% (v/v> glutaraldehyde in PBS.

3.1.3.2. PROCEDURE 1. Cells from the suspension prepared as described above are

resuspended in cold SME medium at a density of lo8 cells per ml. Aliquots (0.05 mL) are then preincubated at 4°C (or on ice) with 0.2-0.5 mL of cold PBS that contains 0.5-3 mg of either nonimmune or immune Fab’.

2. After 20 to 30 min, each sample above is diluted to 2 mL with prewarmed (37°C) SME medium. Mix to break up loose aggregates.

3. Transfer these samples to glass screw-cap scintillation vials (28 x 61 mm). Allow the cells to aggregate on a rotary shaker at 90 rpm, at 37°C.

4. At time points such as 0,20,40, and 60 min, 0.2 mL of each sample is removed and fixed with 1 mL 1% glutaraldehyde in PBS.

5. The number of free particles of different samples at each time point is counted by a Coulter counter. Results are expressed as percentage decrease in free particle number over each time

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3.1.4. Binding Assays of Labeled Single Ceils to Cell Monolayers

3.1.4.1. MATERIALS

1. SME medium (Eagle’s minimum essential medium with spin- ner salts containing HEPES pH 7.4 and 20 pg/mL of DNase).

2. Cell monolayer grown to confluence in a 35-mm dish. 3. Fluorescent-labeled cells.

3.1.4.2. PROCEDURES

1. Rinse the confluent cell monolayer that has been previously cultured in a 35-mm dish with SME.

2. Add 5 x lo6 fluorescently labeled cells as previously mentioned Use SME as medium. Incubate at 50 rpm on a rotary shaker for 30 min at room temperature.

3. Remove the unbound fraction of cells by gently replacing the medium several times with a pipet.

4. The binding of labeled cells to cell monolayer can be inspected and counted by usmg a fluorescence microscope. Cells may be fixed with 4% paraformaldehyde in PBS prior to being counted.

3.2. Detection of NCAM

NCAM antibodies are often used for the identification of NCAM expression. Both monoclonal and polyclonal NCAM antibodies have been used. Different types of antibodies have been produced to detect different forms of NCAMs. For example, meningococci group B share a2,8-PSA similar to NCAMs. The anti-MB monoclonal antibody is prepared by immunizing a mouse with meningoccocus B to detect embryonic NCAMs (Rougon et al, 1986). To detect total NCAMs, a polyclonal anti-NCAM antibody can be produced by immunizing rabbits with adult NCAM purified from bovine brain (Rougon and Marshak, 1986). NCAM antibodies are also available commercially from companies such as Sigma (St. Louis, MO), Chemicon (Temecula, MA), Cedalane (Horby, ON, Canada), and Endogen (Wabourn, MA). Immuno- histochemlstry and Western blots have been used to detect the expression of NCAMs. Western blot can provide both qualitative and quantitative measurements but this method does not reveal the anatomical localization of NCAMs. Immunohistochemistry does provide the anatomical localization of the protein of interest but it does not indicate whether a positive-staining cell has syn-


thesized the detected protein or acquired it by means of uptake from the extracellular space. This information can be obtained from 1~1 situ hybridization, The combination of immunohistochemistry and in situ hybridization can provide strong evidence about whether the cell of interest is indeed the site of synthesis or not.

3.2.1. lmmunohistochemistry

Briefly, general procedures for preparing tissue sections include fixing the tissues to be studied in paraformaldehyde at room temperature, soaking the tissues in 20-30% sucrose in PBS overnight at 4”C, freezing them, and sectioning them with cryostat. Please refer to relevant references regarding preparation of tissue sections (Aaron and Chesslet, 1989; Bonfanti et al., 1992; Bruses et al., 1995).

3.2.1 .I. MATERIALS

1. Prepared tissue sections to be studied. 2. Polyclonal anti-total NCAM (can be prepared as described by

Rougon and Marshak, 1986). 3. PBS. 4. 1% human serum albumin in PBS. 5. FITC-conjugated sheep antirabbit Ig or sheep antirabbit Ig. 6. 3,3’ diaminobenzidine. 7. H,O,. 8. Phenylenediamine.

3.2.1.2. PROCEDURES

1. Wash the sections twice in PBS then incubate them with 1% human serum albumin in PBS to block nonspecific sites.

2. Incubate sections with primary antibodies in PBS at optimal titer at 4°C for 24-72 h.

3. Wash the sections twice, 15 min each, in cold PBS containing 1% human serum albumin then incubate them with either FITC-conjugated sheep antirabbit immunoglobulins (Biosys, Compiegne, France) or sheep antirabbit immunoglobulms fol- lowed by rabbit peroxidase-antiperxidase.

4. If peroxidase activity is used to reveal immunoreactivity, the sections can be incubated with 0.01% 3,3’diaminobenzidine and 0.01% H,O,.

5. After sections are washed twice with PBS, they are mounted in glycerol diluted with PBS (l:l), with 0.3 mg/mL phenylenediamine to prevent photobleaching.

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6. Peroxidase-labeled sections are examined with bright- and dark-field optics, but for FITC-labeled specimens, epi- fluorescence is observed with the appropriate filter.

3.2.2. In Situ Hybrrdlzatron

Tissue sections mounted on slides are hybridized with a labeled probe to determine the presence of mRNA. The hybridized samples are dipped in emulsion for cellular localization or autoradiographed on film for regional localization. Different probes have been used to detect the presence of mRNA of NCAM (Prieto et al., 1989; Goldowitz et al., 1990; Carbone et al., 1991; Lahr et al., 1993).

3.2.3. Western Blot

3.2.3.1. MATERIALS

1. Lysis buffer: 10 mM Tris-HCl (pH 7.4),0.15 M NaCl, 5 mM EDTA, l%(v/v) Triton X-100, protease inhibitors such as 10 uM leupeptin, 0.7 ug/mL pepstatin, 1 mM phenyl- methylsulfonyl fluoride, 0.23 unit/mL aprotinin, and 1 n-&I benzamidine.

2. SDS-polyacrylamide gel and different buffers used in Western blot method.

3. TBST 000 mM Tris, pH 7.5,0.9% NaCl, 0.1% Tween-20,0.1% BSA). 4. Primary and secondary antibodies. 5. ECL (Amersham). 3.2.3.2. PROCEDURES

1. Solubilize cells in lysis buffer for approx 10 min on ice. 2. Determine protein concentration in lysate with methods such

as the Bio-Rad (Bradford) protein assay. 3. If PSA is to be removed, treat sample with N-acetylneura-

minidase (0.5-l U/mL protein solution) and incubate for 30 min at 37°C.

4. The sample is mixed with an equal volume of SDS-PAGE sample buffer and boiled for 5 min.

5. Equal amounts of proteins are loaded into the wells of a 7.5% SDS-PAGE gel.

6. The separated proteins on SDS-PAGE are then electroblotted onto nitrocellulose membranes.

7. To block nonspecific binding sites, immerse the membrane with transferred proteins in 2% BSA in TBST (100 mM Trls,


pH 7.5, 0.9% NaCl, 0.1% Tween-20, 0.1% BSA). Incubate for 30-60 min with gentle agitation at room temperature or overnight at 4°C.

8. Incubate the membranes with properly diluted primary antibody against NCAM for 30-60 min with gentle agitation.

9. Wash with 3-4 changes of TBST with gentle agitation. 10. Transfer the membranes to a properly diluted second antibody

solution and incubate for 30-60 min. 11. Wash as in step 9. 12. Finally, different methods and substrates can be used to detect

the bands of NCAM on the membranes. An enhanced chemi- luminescence detection system (ECL, Amersham) is a popu- lar choice. With this method, the reacted bands are exposed to X-ray film.

3.3. Quantitation of NCAM

The amounts of NCAMs in tissues and cells have been measured by enzyme-linked immunosorbent assay (ELISA) (Prentice et al., 1987; Doherty et al., 1988; Rao et al., 1992) or an immunoelectrophoresis method (Jorgensen, 1976). The immunoelectro- phoretic method requires more brain tissue for each assay (0.5-1.0 mg) than ELISA does, but the procedures can be carried out in an accurate and reproducible fashion with low variance between samples (Jorgensen, 1995). Immunoblotting is also a common method that has been used (Chuong and Edelman, 1984). The antibody-excess assay is another alternative (Rodman and Akeson, 1981; Chen et al., 1994), but this method is not widely used.

3.3.1. ELISA Method

3.3.1 .I. MATERIALS

1. 96-well micro plates. 2. Cell suspension. 3. 4% paraformaldehyde or prechilled (-20°C) methanol. 4. PBS. 5. 1% BSA in PBS. 6. Rabbit anti-NCAM. 7. Peroxidase-conjugated sheep antirabbit Ig. 8. 0.2 % (w/v) o-phenylenediamine and 0.02% (v/v> H,O, in

citrate phosphate buffer. 9. 4.5 M H,SO,.

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3.3.1.2. PROCEDURES

1. Seed a density of approx 20,000 cells in 96-well plates for 18 h. 2. Carefully fix cells by exchanging 50% medium with 4%

paraformaldehyde (in corresponding medium used to culture cells) for 60 min at 20°C. Total medium is then changed with 4% paraformaldehyde for an additional 60 min. Alternatively, cells can be fixed with prechilled methanol (-20°C) for lo-15 min.

3. Wash cells 3 times with PBS. Incubate with PBS containing 1% BSA for 60 min to block nonspecific protein-binding sites.

4. Incubate cultures with primary antibodies (rabbit anti-NCAM) for 60 min at 20°C.

5. Wash three times with PBS containing 1% BSA. 6. Incubate for 60 min with peroxidase-conjugated sheep

antirabbit Ig. 7. Wash four times with PBS, twice with distilled water. 8. Color development is carried out by incubation of cultures with

50 uL of 0.2% (w/v) o-phenylenediamine and 0.02% (v/v) H,O, in citrate phosphate buffer for lo-30 mm

9. Stop reaction by adding 50 P-L of 4.5 M H,SO,. 10. Measure the samples at 492 nm.

3.3.2. Crossed lmmunoelectrophoresrs Method

Crossed immunoelectrophoresis was originally developed for studies of serum proteins (Laurell, 1965; Clarke and Freeman, 1968). Different from standard immunoelectrophoresis, crossed immunoelectrophoresis is both qualitative and quantitative Absolute quantitation is usually not practical with this method, but very good relative quantitation can be obtained by comparison of the test sample with a standard reference sample. This method has been routinely used by Jorgensen and colleagues to quantify NCAMs (Jorgensen, 1976; 1981; Bock and Braestrup, 1978). These followmg procedures are largely based on the studies done by this group.

3.3.2.1. MATERIALS

1. Homogenization buffer (2.7% w/v Triton X-100, 100 U/mL aprotinm, 73 mM-Tris, 24 mM barbital, 2 mM NaN,, at pH 8.6

2. First-dimensional gel (1% agarose, 0.6% Triton X-100,73 mM- Tris, 24 mM barbital, 2 mM NaN,, at pH 8.6)

3. Second-dimensional gel (similar to first dimensional gel but also contams appropriate concentration of NCAM antibodies).


3.3.2.2. PROCEDURES

1. The tissues or cells of interest are incubated with the homogenization buffer overnight at 4°C for solubilization. The protein concentration in the homogenate is measured by standard methods such as those of Lowry or Bradford.

2. The proteins m the sample are separated in the first-dimensional gel at 10 V/cm for 30 min at 15°C.

3. To force the antigen into the perpendicularly placed second- dimensional gel, turn the electric field 90” and continue electrophoresis at 2.5 V/cm for 18 h at 15°C.

4. The precipitates formed from the antigen-antibody complexes can then be stained with agent such as Coomassie brilliant blue R. The area under the peak is proportional to the concentration of NCAM and inversely proportional to the concentration of antibody used in second dimensional gel.

3.3.3. Western Blot

The procedures here are very similar to those described in Sec- tion 3.2.3., except that after the treatment of second antibodies, [‘251]-Protein A is added to bind to antibodies that reacted with proteins on the blot, The blot is autoradiographed. NCAMs can then be quantified in two ways* Areas of the nitrocellulose corresponding to the range of the weights of NCAM of interest are cut and counted in a gamma counter. A background radioactive count is obtained from a similar portion of nitrocellulose from the same blot. Alternatively, the intensity of the bands on the autoradiographs of the electrophoretic gels are analyzed by a densitometer.

3.3.4 Antibody-Excess Assay

This method can be used to calculate the number of NCAM molecules per cell. Briefly, the cells are incubated with a previously determmed excess of NCAM antibodies. Then they are incubated with a previously determined excess of [‘2511-Protein A. The cells are centrifuged and the pellets are counted in a gamma counter.

3.3.4.1. MATERIALS

1. Assay buffer 2. PBS (free of Ca*+ and Mg2+). 3. [‘251]-Protein A. 4. NCAM antibodies.

120 Tleu and Yu

3.3.4.2. PROCEDURES

1. Cells are collected and washed with PBS and then suspended in cold assay buffer

2. Use a hemocytometer to count the cells and incubate them with previously determined excess of NCAM antibodies in a final volume of 0.5 ml of assay buffer for 1 h at room temperature.

3. Wash the cells three times with assay buffer. 4. Resuspend and incubate the cell pellets in a final volume of

0.2 mL assay buffer containing a previously determined excess of [1251]-Protein A for 1 h at room temperature. Mix occasionally.

5. Wash as in step 3. 6. Cell pellets are resuspended in scintillation fluid and counted

in a gamma counter. 7. Cpm obtained (after subtracting background cpm) are divided by

specific activity of the [1251]-Protein A to yield micrograms protein A molecules bound. After this result is converted to the number of protein A molecules bound, it is divided by the total number of cells in each sample to obtain the number of NCAM molecules per cell.

3.4. Purification of NCAM

The purification of NCAMs is difficult because of low basal concen- trations. Large quantities of tissue are required to produce sufficient amounts of NCAM for analysis. Brains from chick embryos (Hoffman et al., 1982; Rutishauser et al., 1982; Murray and Jensen, 1992; Storms et al., 1994,1996), and mice or cows (Chuong et al., 1982; Rougon and Marshak, 1986) have been used as sources of NCAMs for purification. Because they present as either transmembrane or membrane-bound proteins in these tissues, detergents are required to extract them. Neu- tral detergents such as octylthioglucoside, NP-40, Triton X-100, or zwitterionic detergents are recommended (Hoffman, 1992). Charged detergents are unsuitable since they tend to denature these cell-surface molecules. In the presence of detergent, standard chromatographic methods do not provide good separation, affinity chromatography is preferred (Hoffman, 1992). The purity of the product can be verified by using SDS-PAGE and immunoblot analysis.

3.4.1. Affinity Purrfication Method

3.4.1.1. MATERIALS

1. PBS. 2. An A pestle.


3. Dounce homogenizer. 4. EDTA. 5. Protease inhibitors. 6. NP-40 in PBS. 7. Monoclonal anti-NCAM IgG. 8. CNBr-activated Sepharose CL-2B (Pharmacia, Uppsala, Sweden). 9. PBS containing 50 mA4 diethylamine, 1 mM EDTA, 0.5%

Nonidet P-40.

3.4.1.2. PROCEDURES

1. Brains from 14-d chick embryos are removed and washed with cold PBS. Brain membranes are obtained by homogenizing brains on ice with an A pestle in a 7-mL Dounce homogenizer in PBS containing EDTA and a protease inhibitor such as aprotinin.

2. The homogenate is centrifuged in a one-step sucrose density gradient (0.8 M/2.25 M). The fraction from the interface is collected, washed and resuspended in PBS. These membranes can be stored at -20°C for later use.

3. Membranes collected above are extracted with Nonidet P-40 in PBS containing EDTA and then centrifuged at 25,OOOg for 15 min to remove particulate matter.

4. Monoclonal anti-NCAM IgG coupled to CNBr-activated Sepharose CL-2B (Pharmacia) is used for the affinity purification. NCAMs are eluted from the Sepharose with PBS containing 50 mM diethylamine, 1 mM EDTA, 0.5% Nonidet P-40.

5. Nonidet P-40 is removed by using Biobeads SM-2 and the protein is dialyzed against H,O, lyophilized, and stored at 4°C. All procedures are carried out at 4°C.

References

Aaron L. I and Chesselet M F (1989) Heterogeneous drstrrbution of polysialylated neuronal-cell adhesion molecule during post-natal development and m the adult an rmmunohrstochemrcal study m the rat brain Neuroscrence 28, 701-710

Barbas, J A , Charx, J C , Stemmetz, M., and Gorrdis, C (1988) Differential splrc- mg and alternative polyadenylation generates distmct NCAM transcrrpts and proteins in the mouse EMBO J 7,625-632

Barbeau, D , Liang, J J,, Robltallle, Y , Quiron, R., and Srwastava, L K (1995) Decreased expression of the embryonic form of the neural cell adhesion molecule m schizophrenic brains. Proc Nat1 Acad Sci USA 92,2785-2789

Barthels, D , Vopper, G , Boned, A., Cremer, H , and Wrlle, W (1992) High degree of NCAM diversity generated by alternative RNA splicing m brain and muscle Eur J Neuroscr 4,327-337

722 Tieuand Yu

Basham, M J , du-Lac, S , and Goodman, C S (1986) Guidance of neuronal growth cones m the grasshopper embryo I Recognition of a specific axonal pathway by the pCC neuron J Neuroscr 6,3518-3531

Bastram, M J and Goodman, C S (1986) Guidance of neuroanl growth cones m the grasshopper embryo III Recognition of specific ghal pathways J Neuroscz 6,3542-3551

Brxby, J L , and Reichardt, L F (1987) Effects of anhbodres to neural cell adhesion molecule (N-CAM) on the drfferentiahon of neuromuscular contacts between crhary ganglion neurons and myotubes ZM vitro Dev Bzol 119,363-372

Bock, E , Edvardsen, K, Gibson, A, Lmnemann, D , Lyles, J M , and Nybroe, 0 (1987) Characterrzatron of soluble forms of NCAM FEBS Lett 225,33-36

Bonfantl, L , Ohve, S., Poulam, D A, and Theodosis, D T (1992) Mapping of the distribution of polysralylated neural cell adhesion molecule throughout the central nervous system of the adult rat An lmmunohistochemical study Neuroscrence 49,419-436

Brackenbury, R I-l, Rutrshauser, U , and Edelman, G M (1981) Drstmct calcium-independent and calcium-dependent adhesion systems of chicken embryo cells Proc Nut1 Acad SCI USA 78,387-391

Brackenbury, R , Threry, J P , Rutlshauser, U , and Edelman, G M (1977) Adhe- sion among neural cells of the chick embryo I An immunological assay for molecules mvolved m cell-cell bmdmg J Bml Chem 252,6835-6840

Bruses, J. L., Oka, S , and Rutlshauser, LJ (1995) NCAM-associated polysiahc acid on cihary ganglion neurons IS regulated by Polysralyltransferase levels and mteractlon with muscle J Neuroscz 15,8310-8319

Buskrrk, D R , Thlery, J P , Rutishauser, U , and Edelman, G M (1980) Antr- bodies to a neural cell adhesion molecule disrupt histogenesis m cultured chick retmae Nature 285,488-489

Carbone, D I’, Koros, A M C , Lmnoila, R I, Jewett, P , and Gazdar, A F (1991) Neural cell adhesion molecule expression and messenger RNA sphcmg pattern m lung cancer lmes are correlated with neuroendocrme pheno- type and growth morphology Cancer Res 51,6142-6149

Caubit, X., Arsanto, J P , Figarella-Branger, D , and Thouveny, Y (1993) Expressron of polysialylated neural cell adhesion molecule (PSA-N-CAM) m developing, adult, and regenerating caudal spmal cord of the urodele amphibians lnt J Dev Brol 37‘327-36

Chalkley, H W (1945) Quantitatrve relation between the number of organized centers and tissue volume m regenerating masses of mmced body sections of hydra J Nat1 Cancer Inst 6, 191-195

Chen, A, Haines, S , Maxson, K , and Akeson, R A (1994) VASE exon expression alters NCAM-mediated cell-cell mteractrons J Neuroscl Res 38,483-492

Chuong, C M , McClam, D A , Street, P , and Edelman, G M (1982) Neural cell adhesion molecules m rodent brains isolated by monoclonal antibodies with cross-species reactivity Proc Nat1 Acad Set USA 79,4234-4238

Chuong, C M , and Edelman, G M (1984) Alterations in neural cell adhesion molecules during development of different regions of the nervous system J Neuroscl 4,2354-2368

Clark, H G M , and Freeman, T (1968) Quantitative immunoelectrophoresis of human serum proteins Clm Scz 35,403-413

Cole, G J , Loewy, A , and Glaser, L (1986) Neuronal cell-cell adhesion depends on mteractions of N-CAM with heparm-like molecules Nature 320,445-447


Cole, G J , and Akeson R (1989) Identification of a heparm binding domain of the neural cell adhesion molecule N-CAM using synthetic peptides Neuron 2,1157-1165

Cremer, H., Lange, R, Chnstoph, A Plomann, M, Vopper G., Roes, J , Brown, R., Baldwin, S., Kraemer, I’, Scheff, S , Barthels, D , Ralewsky, K , and Wille, W (1994) Inactivation of the N-CAM gene in mice results m size reduction of the olfactory bulb and deficits m spatial learning Nature 367,455-459

Crossm, K L., Chuong, C. M., and Edelman, G. M (1985) Expression sequences of cell adhesion molecules. Proc Natl Acad Scz USA 82,6942-6946

D’Eustachlo, I?, Owens, G , Edelman, G M , and Cunnmgham, B A (1985) Chro- mosomal location of the gene encoding to the neural cell adhesion molecule (N-CAM) in the mouse. PYOC NatJ Acad Ser. USA 82,7631-7635

Damloff, J , Levi, G , Grumet, M , Rieger, F , and Edelman, G (1986) Altered expression of neuronal cell adhesion molecules induced by nerve mlury and repair J Cell Blol 103,929-945

Damloff, J K , Moore, V , and Oliver, E H (1994) Activity of neural cell adhesion molecule (N-CAM) components’ a review. Cytobios 79, 97-106

Doherty, P , Mann, D A, and Walsh, F S. (1988) Comparison of the effects of NGF, activators of protein kinase C, and a calcium ionophore on the expression of Thy-l and N-CAM m PC12 cell cultures J Cell BioJ 107,333-340

Doherty, P , Moolenaar, C E C K, Ashton, S A., Michahdes, R J A M and Walsh, F S (1992) Use of the VASE exon down regulates the neurite growth promotmg activity of NCAM 140 Nature 356,791-793

Doherty, P and Walsh, F S (1992) Cell adhesion molecules, second messen- gers and axonal growth CUYY Open Neurobiol. 2,595-601

Dohert,y P , Fazeh, M S , and Walsh, F S (1995) The neural cell adhesion molecule and synaptic plasticity J Neuroblol 26, 437-446

du-Lac, S , Bastiam, M J , and Goodman, C S. (1986) Guidance of neuronal growth cones in the grasshopper embryo. II Recognition of a specific axonal pathway by the aCC neuron J Neuroscr 6,3532-3541

Eckhard, M , Muhlenhoff, M , Bethe, A, Koopman, J., Frosch, M., and Gerardy- Schachn, R (1995) Molecular characteristics of eukaryotic polysialtransferase-1. Nature 373,715-718

Edelman, G M (1983) Cell adhesion molecules Scrence 219,450-457 Edelman, G M (1985a) Cell adhesion and the molecular processes of morpho-

genesis Ann Rev Blochem 54,135-169 Edelman, G M (1985b) Specific cel1 adhesion m histogenesis and morphogen-

em, in The Cell WI Contact Adkeslons and Juncttons as Morpkogenetic Detem- nants (Edelman, G. M and Thiery, J P , eds.), Wiley, New York, pp 139-169

Edelman, G M (1986) Cell adhesion molecules m the regulation of animal form and tissue pattern Ann Rev BioJ 2, 81-116

Edelman, G M (1988a) Morphoregulatory molecules. Btockemzstry 27,3533-3543 Edeman, G M (198813) Topoblology An mtroduction to molecular embryology

Basic, New York Edelman, G M. and Crossm, K L (1991) Cell adhesion molecules implications

for a molecular histology Ann Rev Blockem 60, 155-190. Ehces, M J , Osborn, L , Takada, Y , Crouse, C , Luhowskyl, S , Hemler, M E ,

and Lobb, R. (1990) VCAM-1 on activated endothelmm interacts with the leukocyte mtegrm VLA-4 at site distmct from the VLA-4/frbronectm bmdmg site Cell 60,577-584

124 Tleu and Yu

Fields, R D and Itoh, K (1996) Neural cell adhesion molecules m activity- dependent development and synaptic plasticity Trends Neuroscz 19,473-480

Finn, J , Finn, U , Deagostuu-Bazin, H , and Goridis, C (1983) Occurrence of a2- 8 linked polysialosyl units m a neural cell adhesion molecule Bzochem Biophys Res Commun. 112,482-487.

Fraser, S. E , Murray, B. A, Chuong, C M , and Edelman, G M (1984) Alter- ation of the retmotectal map m Xenopus by antibodies to neural cell adhesion molecules PYOC Nat1 Acad Scz USA 81,4222-4226

Fraser, S. E , Carhart, M S., Murray, B A, Chuong,C M , and Edelman, G. M (1988) Alterations m the retinotectal prelection by antibodies to Xenopus N- CAM Dev BzoJ 129,217-230

Frazier, W and Glaser L (1979) Cell surface components mvolved m cell recognition Annu Rev Btochem 48,491-527

Friedlander, D R , Mege, R. M , Cunnmgham, B A, and Edelman, G M (1989) Cell sorting-out is modulated by both the specificity and amount of different cell adhesion molecules (CAMS) expressed on cell surfaces Proc N&J Acad SCI USA 86,7043-7047

Gegelashvlh, G , Anderson, A M , Schousboe, A, and Bock, E (1993) Charac- terrzation of NCAM diversity m cultured neurons, FEBS Lett 324,337-340

Goldowrtz, D , Barthels, D , Lorenzon, N , Jungblut, A, and Wille, W (1990) NCAM gene expression during the development of cerrebellum and dentate gyrus in the mouse Dev Bram Res 52,151-160.

Gorrdrs, C , Joher, M. A, Hirsch, M., and Schachner, M (1978) Cell surface proteins of cultured brain cells and then recognition by anti-cerebellum (anti- NS-4) antiserum J Neuuochem. 31,531-539

Goridis, C , and Wille, W (1988) The three size classes of mouse NCAM proteins arise from a single gene by a combmatron of alternative spllcmg and use of different polyadenylation sites Neurochem Inf 12, 269-272

Goridis, C., and Brunet, J F (1992) NCAM structuraly diversity, function and regulation of expression Cell BtoJ 3,189-197

Gower, H. J , Barton, C H , Elsom, V L., Thompson, J , Moore, S E , Dickson, G , and Wash, F. S (1988) Alternative sphcmg generates a secreted form of N-CAM m muscle and brain Cell 55,955-964.

He, H T., Barbet, J , Charx, J C , and Goridls, C (1986) Phosphatidylmositol is involved in the membrane attachment of NCAM-120, the smallest component of the neural cell adhesion molecule. EMBO J 5,2489-2494

He, H T , Fmne, J , and Goridis, C (1987) Biosynthesis, membrane association, and release of N-CAM-120, a phosphatidylmositol-linked form of the neural cell adhesion molecule J Cell BtoJ 105,2489-2500

Hemperly, J J., Edelman, G. M , and Cunningham, B A. (1986) cDNA clones of the neural cell adhesion molecule (N-CAM) lacking a membrane-span- ning region consistent with evidence for membrane attachment via a phosphatidylmositol intermediate Proc N&J Acad Scr USA 83,9822-9826

Him, M , Pierres, M, Deagostml-Bazin, H , Hirsch, M , and Goridis, C (1981) Monoclonal antibody against cell surface glycoprotem of neurons Brazn Res 214,433-439

Hirn, M., Ghandour, M S , Deagostmi-Bazm, H , and Gorrdrs, C (1983) Mo- lecular heterogeneity and structural evolution during cerebellar ontogeny detected by monoclonal antibody of the mouse cell surface antigen BSP-2 Bram Res 265, 87-100


Hoffman, S (1992) Assays of cell adhesion, m Cell-Cell Inferactzon. (Stevenson, B R., Gallm, W. J , and Paul, D L , eds 1, Oxford University Press, New York, pp 1-29

Hoffman, S., Sorkin, B C , White, P C , Brackenbury, R., Mallhammer, R , Rutlshauser, U , Cunningham, B A, and Edelman, G M (1982) Chemical characterlzatlon of a neural cell adhesion molecule purified from embryonic brain membranes ] Biol Chem 257,7720-7729

Hoffman, S and Edelman, G M (1983) Kinetics of homophlhc binding by embryonic and adult forms of the neural cell adhesion molecule. Proc Natl Acad SCI USA 80,5762-5766

Holtfreter, J (1948a) Concepts on the mechamsm of embryomc mductlon and tts relation to parthenogenesis and malignancy Symp Sot , Exp Biol II, 17-49

Holtfreter, J (194813) Slgmflcance of the cell membrane in embryonic processes Ann NY Acad Sci. 49,709-760

Hynes, R 0. and Lander, A. D. (1992) Contact and adhesive speciflcltles m the associations, migrations, and targetting of cells and axons. Cell 68,303-322

Jaques, G , Auerback, B., Pntsch, M , Wolf, M., Madry, N , and Havemann, K (1993) Evaluation of serum neural adhesion molecule as a new tumor marker m small cell lung cancer Cancer 72,418-425

Jorgensen, 0 S (1976) Localization of the antigens Dl, D2 and D3 m the rat brain synaptic membranes ] Neurochem. 27,1223-1227

Jorgensen, 0 S. (1981) Neuronal membrane D2-protein during rat brain ontogeny J Neurochem 37,939-946

Jorgensen, 0. S (1995) Neural cell adhesion molecule (NCAM) as a quantlta- tlve marker m synaptic remodeling Neurochem Res 20,533-547

Jorgensen, 0 S , DeLouvee, A, Thlery, J P., and Edelman, G M (1980) The nervous system specific protein D2 is involved m adhesion among neurltes from cultured rat gangha FEBS Lett 11, 39-42.

Kadmon, G , Kowltz, A, Altevogt, P , and Schachner, M (1990) The neural cell adhesion molecule N-CAM enhances Ll-dependent cell-cell interactions I Cell Bfol 110, 193-208

Kaiser, U , Jaques, G , Havemann, K , and Auerbach, B (1994) Serum NCAM A potential new prognostic marker for multiple myelom. Blood 83,871-873 (letter)

Kaiser, U , Auerbach, B , and Oldenburg, M. (1996a) The neural cell adhesion molecule NCAM In multiple myeloma Leukemia Lymphoma 20,389-395

Kaiser, U , Oldenburg, M , Jaques, G , Auerbach, B , and Haveman, K (1996b) Soluble CD56 (NCAM) a new differential diagnostic and prognostic marker Ann Hemato 73,121-126

Kellhauer, G , Falssner, A, and Schachner, M (1985) Differential inhibition of neurone-neurone, neurone-astrocyte and astrocyte-astrocyte adhesion by Ll, L2 and N-CAM antibodies. Nature 316,728-730.

Klbbelaar, R E., Moolenaar, C E, C., Michalides, R. J. A M , Bitter-Suermann, D , Addis, B J., and Mool, W J (1989) Expression of the embryonal neural cell adhesion molecule N-CAM m lung carcinoma Diagnostic usefulness of monoclonal antlbody 735 for the distinction between small cell lung cancer and non-small cell lung cancer I Pathol 159,23-28

Kuwada, J Y (1986) Cell recognition by neuronal growth cones m a simple vertebrate embryo Science 233,740-746

Kuwada, J Y. (1992) Growth cone guidance m the zebrafish central nervous system Curr Open Neurobzol 2, 31-35

126 Tieu and Yu

Lahr, G , Mayerhofer, A, Bucher, S , Barthels, D , Wille, W , and Gratzl, M (1993) Neural cell adhesion molecules m rat endocrme tissues and tumor cells distribution and molecular analysis Endocrinology 132, 1207-1217

Landmesser, L., Dahm, L , Tang, J C , and Rutishauser, U (1990) Polysiahc acid as a regulator of mtramuscular nerve branchmg during embryonic development. Neuron 4,655-667

Langley, 0 K , Gombos, G , Him, M , and Goridis, C (1983) Distribution of the neural antigen BSP-2 m the cerebellum durmg development Int I Dev Neurosci 1,393-401

Laurell, C B (1965) Antigen-antibody crossed electrophoresis Analyf Bmchem 10,358-361

Levi, G , Broders, F , Dunon, D , Edelman, G M , and Threry, J P (1990) Modu- lation of the expressron of the neural cell adhesion molecule NCAM durmg metamorphosis of Xenopus haevis Development 109,681-692

Liu, L , Hames, S , Shaw, R , and Akeson, R A (1993) Axon growth IS enhanced by NCAM lacking the VASE exon when expressed m either the growth sub- strate or the growing axon J Neuroscl Res 35,327-345

LOIS, C., Garcia-Verdugo, J M , and Alvarez-Buylla, A (1996) Chain migration of neuronal precursors Scrence 271,978-981

Lotz, M , Burdsal, C , Erickson, H , and McClay, D (1989) Cell adheison of fibronectm and tenascm quantitative measurements of initial bmdmg and subsequent strengthenmg response 1. Cell BIOI 109,1795-1805

Luthl, A, Laurent, J I’, Figurov, A, Muller, D., and Schachner, M (1994) Hip- pocampal long-term potentiation and neural cell adhesion molecules Ll and NCAM Nature 372,777-779

Lyles, J M , Lmnemann, D , and Bock, E (1984) Biosynthesis of the D2-cell adhesion molecule post-translational modifications, intracellular transport, and developmental changes 1 Cell Blol 99,2082-2091

McClay, D R , Wessel, G M., and Marchase, R B (1981) Intercellular recognmon quantitation of initial bmdmg events Proc Nat1 Acad Sn USA 78,4975-4979

Miragall, F , Kadmon, G , Husmann, M, and Schachner, M (1988) Expression of cell adhesion molecules m the olfactory system of the adult mouse presence of the embryonic form of N-CAM Dev Bzol 129,516-531

Moolenaar, C E. C., Muller, E J , Schol, D J , Figdor, C G , Bock, E , Bitter- Suermann, D , and Michahdes, R J A M (1990) Expression of neural-cell- adhesion-molecule-related sialoglycoprotein m small-cell lung-cancer and neuroblastoma cell lines H69 and CHP-212 Cancer Res 50,1102-1106

Moscona, A (1952) Cell suspensions from organ rudiments of chick embryos Exp Cell Res 3,535-539

Moscona, A and Moscona, H (1952) Dissociation and aggregation of cells from organ rudiments of the early chick embryos 1 Anaf 86,287-301

Muller, D , Wang, C , Skibo, G , Tom, N , Cremer, H , Calaora, V , Rougon, G , and Kiss, J Z (1996) PSA-NCAM is required for activity-induced synaptic plasticity Neuron 17,413-422

Murray, B A., Hemperly, J J , Predrger, E A, Edelman, G M , and Cunnmgham, B A (1986) Alternatively spliced mRNA code for different polypeptide chams of the chicken neural cell adhesion molecule (N-CAM) y Cell Blol 102,189-193

Murray, B A and Jensen, J (1992) Evidence for heterophilic adhesion of embryonic retinal cells and neuroblastoma cells to substratum-adsorbed ncam y Cell Brol 117,1311-1320


Nakayama, J and Fukuda, M. (1996) A human polysialyltransferase directs rn zlztro synthesis of polysralic acid. J Brol. Chem 271, 1829-1832.

Nakayama, J , Fukuda, M N , Fredette, B J., Ranscht, B., and Fukuda, M (1995) Expression clonrng of a human polysralytransferase that forms the polysralylated neural cell adhesion molecule present in embryonic bram Proc Nat1 Acad Sci USA 92,7031-7035

Nelson, R W , Bates, P A., and Rutishauser, U (1995) Protein determinants for specific polysialatron of the neural cell adhesion molecules J B~ol Chem 270, 17,171-17,179.

Nguyen, C., Matter, M G , Matter, J F., Santonr, M. J , Goridrs, C , and Jordan, B R (1986) Localization of the human NCAM gene to band q23 of chromosome 11 The thud gene coding for a cell interaction molecule mapped to the distal portion of the long arm of chromosome 11 J Cell Brol 102,711-715

Noble, M , Albrechtsen, M , Moller, C , Lyles, J., Bock, E , Goridis, C., Watanabe, M , and Rutishauser, U (1985) Glial cells express N-CAM/D2-CAM-like polypeptrdes in vitro Nature 316,725-728

Nybroe, 0, Albrechtsen, M , Dahlin, J., Linnemann, D , Lyles, J M , Moller, C J., and Bock, E (1985) Biosynthesis of the neural cell adhesion molecule characterrzatron of polypeptrde c J Cell Brol 101,2310-2315

Ong, F , Kaiser, U , Seelen, P J , Hermans, J , Wijermans, P W , de Krevret, W , Jaques, G , and Kluin-Nelemans J C (1996) Serum neural cell adhesion molecule differentiates multiple myeloma from paraprotememras due to other causes Blood 87,712-716

Owens, G C , Edelman, G M , and Cunningham, 8. A (1987) Organrzatron of the neural cell adhesion molecule (N-CAM) gene Alternative exon usage as the basis for different membrane-associated domains Proc Nat1 Acad Scz USA 84,294-298

Patel, K , Moore, S. E , Dickson, G , Rossell, R J , Beverley, P C , Kernshead, J T , and Walsh, F S (1989) Neural cell adhesion molecule (NCAM) IS the antigen recognrzed by monoclonal antibodies of similar specrfrcrty m small- cell lung carcinoma and neuroblastoma Int J Cancer 44, 573-578

Poltorak, M., Khoja, I., Hemperly, J. J, Willrams, J R , el-Mallakh, R., and Free, W. J (1995) Disturbances m cell recognition molecules (N-CAM and Ll antigen) m the CSF of patients with schrzophrenra Exp Neural 131,266-272

Poltorak, M , Frye, M. A, Wright, R., Hemperly J J., George, M. S., Pazzaglia, P J , Jerrels, S A, Post, R M , and Freed, W. J. (1996) Increased neural cell adhesion molecule in the CSF of patients with mood disorder / Neurochem 66,1532-1538.

Prentrce, H M , Moore, S E , Drckson, J G., Doherty, P , and Walsh, F S (1987) Nerve grow factor-induced changes m neural cell adhesion molecule (N-CAM) m PC12 cells EMBO J 6,1859-1863

Prreto, A L, Crossm, K L , Cunningham, B A, and Edelman, G. M. (1989) Localrzatron of mRNA for neural cell adhesion molecule (N-CAM) polypeptides m neural and nonneural tissues by rn srtu hybridization Proc Natl Acad Scl USA 86,9579-9583

Probstmerer, R , Kuhn, K , and Schachner, M (1989) Binding properties of the neural cell adhesron molecule to different components of the extracellular matrix 1 Neurochem 53,1794-1801

Ranhelm, T S, Edelman, G M, and Cunningham, B A (1996) Homophilic adhesion mediated by the neural cell adhesion molecule mvolves multiple rmmunoglobulm domains Neurobrology 93,4071-4075

128 Tleu and Yu

Rao, Y , Wu, X F , Gariepy, J , Rutishauser, U , and Sm, C H (1992) Identifrca- bon of a peptide sequence mvolved m homophillc bmdmg m the neural cell adhesion molecule NCAM J Cell Bzol 118,937-949

Remsen, L., Strain, G , Newman, M , Satterlee, N , and Damloff, J (1990) Anh- bodies to the neural cell adhesion molecule disrupt functional recovery m mlured nerves Expl Neurol 110,268-273

Rodman, J S and Akeson, R (1981) A new antigen common to the rat nervous and immune systems. II Molecular characterization y Neuroscz Res 6,179-192

Rernn, L. C. B , Bock, E , Lmnemann, D , and Jahnsen, H. (1995) NCAM-antibodies modulate induction of long-term potentiation m rat hippocampal CA1 Brain Res 677, 145-151

Rose, S P (1995) Glycoprotems and memory formation Behav Brazn Res 66, 73-78

Roth, J, Zuber, C., Wagner, P , Taat]es, D J , Weisgerber, C , Heitz, P U , Goridis, C , and Bitter-Suermann, D (1988) Re-expression of polysiahc acid units of the neural-cell-adhesion molecule m Wilms’ tumor Proc Natl Acad SCI USA 85,2999-3003

Rothbard, J B , Brackenbury, R , Cunningham, B. A , and Edelman, G M (1982) Differences m the carbohydrate structures of neural cell-adhesion molecules from adult and embryonic chicken brains ] Blol Chem 257,11064-11069

Roubm, R , Deagostmi-Bazm, H , Hirsch, M R , and Goridis, C (1990) Modula- tion of NCAM expression by transforming growth factor-beta, serum and autocrme factors J Cell Biol 111,673-684.

Rougon, G , Dubois, C , Buckley, N , Magnam, J. L , and Zollmger, W (1986) A monoclonal antibody against menmgococcus group B polysaccharides dis- tmguishes embryomc from adult N-CAM J Cell Bzol 103,2429-2437

Rougon, G. and Marshak, D (1986) Structural and immunological characterization of the ammo terminal domain of mammalian neural cell adhesive molecules J Brol Chem. 261,3396-3401

Rutishauser, U (1996) Polysiallc acid and the regulation of cell mteractions Curr Open Cell Blol 8, 679-684.

Rutishauser, U , Threry, J P , Brackenbury, R , Sela, B A, and Edelman, G M (1976) Mechanisms of adhesion among cells from neural tissues of the chick embryo. Proc Nat1 Acad Scz USA 73,577-581

Rutshauser, U , Hoffman, S , and Edelman, G M (1982) Bmdmg properties of a cell adhesion molecule from neural tissue Proc Natl Acad Scz USA 79,685-689

Rutishauser, U., Grumet, M., and Edelman, G. M. (1983) Neural cell adhesion molecule mediates initial interactions between spinal cord neurons and muscle cells m culture J Cell Blol 97, 145-152

Rutmhauser, U and Jessell, T. M (1988) Cell adhesion molecules m vertebrate neural development Phys~ol Rev 68,819-857

Runshauser, U. and Landmesser, L. (1996) Polysialic acid m the vertebrate nervus system a promoter of plasticity m cell-cell mteractions. Trends Neuroscz 19,422-427

Saad, B , Constam, D B , Ortmann, R., Moos, M , Fontana, A, and Schachner, M (1991) Astrocyte-derived TGF-beta 2 and NGF differentially regulate neural recognition molecule expression by cultured astrocytes J Cell Brol 115,473-484

Sadoul, R , Hun, M , Deagostmi-Basin, H , Rougon, G , and Goridis, C (19831 Adult and embryonic mouse neural cell adheison molecules have different bmdmg properties Nature 304,347-349.


Sadoul, K , Meyer, A., Low, M. G , and Schachner, M. (1986) Release of the 120 kDa component of the mouse neural cell adhesion molecule N-CAM from cell surfaces by phosphatldylinosltol-specific phospholipase C Neuroscz Lett. 72,341-346

Santoni, M. J , Barthels, D , Vopper, G , Boned, A, Gonda, C , and Wllle, W (1989) Differential exon usage involving an unusual splicing mechanism generates at least 8 types of NCAM cDNA in mouse brain EMBO ] t&385-392.

Satoh, M and Takeuchi, M (1995) Induction of NCAM expression m mouse olfactory keratm-positive basal cells m vitro Dev Bran Res 87,111-119

Scheldegger, P. E., Sternberg, L R., Roth, J , and Lowe, J. B (1995) A human STX cDNA confers polysiahc acid expression in mammalian cells. J Bml. Chem 270,22685-22688

Scholey, A B., Rose, S P , Zamaru, M R , Bock, E , and Schachner, M. (1993) A role for the neuronal cell adhesion molecule m a late, consolidatmg phase of glycoprotein synthesis SIX hours followmg passive avoidance training of the young chick. Neuroscience 55,499509.

Schwanzel-Fukuda, M , Remhard, G R , Abraham, S , Crossm, K L , Edelman, G. M., and Pfaff, D. W (1994) Antibody to neural cell adhesion molecule can disrupt the migration of luteinizmg hormone-releasing hormone neurons mto the mouse bram I Comp Neural 342,174-185

Silver, J and Sapiro, J. (1981) Axonal guidance during development of the optic nerve the role of pigmented epithelia and other extrmslc factors J Comp Neural 202,521-538

Small, S J , Hames, S L , and Akeson, R A. (1988) Polypeptlde varlatlon m an NCAM extracellular ~mmunoglobulm-like fold is developmentally regulated through alternative sphcmg Neuron 1,1007-1017.

Small, S J. and Akeson, R (1990) Expression of the unique NCAM VASE exon 1s independently regulated m distinct tissues during development J Cell Bzol. 111,2089-2096

Storms, S D , Jensen, J J , Yaghmai, D , and Murray, B A (1994) Multiple mechanisms of N2A and CHO cell adhesion to NCAM purified from chick embryonic brain and retina Exp Cell Res 214,100-112.

Storms, S D , Anvekar, V. M , Adams, L. D., and Murray, B. A (1996) Heterophlhc NCAM mediated cell adhesion to proteoglycans from chick embryonic brain membranes. Exp Cell Res. 223,385-394

Tang, J , Rutishauser, U., and Landmesser, L (1994) Polyslahc acid regulates growth cone behavior during sorting of motor axons m the plexus region Neuron 13,405-414

Thlery, J P , Brackenbury, R , Rutlshauser, U , and Edelman, G M. (1977) Adhesion among neural cells of the chick embryo II Purlflcatlon and characterization of a cell adheison molecule from neural retina. 1. Biol Chem 252, 6841-6845.

Thompson J , Moore, S. E , and Walsh, F S (1987) Thyroid hormones regulate expression of the neural cell adhesion molecule in adult skeletal muscle. FEBS Left 219,135-138

Thompson, J., Dickson, G., Moore, J E , Gower ,H J , Putt, W, Kemmer, J G., Barton, H C., and Walsh, F S. (1989) Alternative splicing of the neural cell adhesion molecule gene generates variant extracellular domam structure m skeletal muscle and brain Genes Dev 3,348-357.

Tieu and Yu

Tomasiewicz, H , Ono, K , Yee, D , Thompson, C , Goridis, C , Rutishauser, U , and Magnuson, T (1993) Genetic deletion of a neural cell adhesion molecule variant (NCAM 180) produces distinct defects m the central nervous system Neuvon 11,1163-1174

Townes, P. L and Holtfreter, J (1955) Directed movements and selective adhesion of embryonic amphibian cells ] Exp Zoo1 128,53-120

Travis, J (1993) Biotech gets a grip on cell adhesion Science 260,906-908 Walsh, F S, and Doherty, P. (1996) Cell adhesion molecules and neuronal

regeneration Curr Open Neurobrol 8,707-713 Wang ,Y , Jones, F S , Krushel, L A, and Edelman, G. M (1996) Embryonic

expression patterns of the neural cell adhesion molecule gene are regulated by homeodomam binding sites Proc Nat1 Acad Su USA 93,1892-1896

Wilson, H. V ( 1907) On some phenomena of coalescence and regeneration m sponges. 1 EXQ Zoo1 5,245-258

Yang, I’, Yin, X , and Rutishauser, LJ (1992) Intercellular space IS affected by the polyslahc acid content of NCAM ] Cell Blol 116,1487-1496

Ym, X , Cal, X , and Rutrshauser, U (1994) Effect of polysiahc acid on the behavior of retinal ganglion cell axons during growth mto the optic tract and tectum Development 121,3439-3446

Yoshida, Y , Kolima, N , Kurosawa, N , Hamamato, T, and Tsu~i, S (1995) Molecular clonmg of Siacr2,3GalBl,4GlcNAca2,8-sialyltransferase from the mouse brain J Bzol Chem 270,14628-14633

Zuber, C and Roth, J (1990) The relatronshlp of polysiahc acid and the neural cell adhesion molecule N-CAM m Wilms tumor and their subcellular dlstri- butions Euu J Cell Bzol 51,313-321

2 neural cell adhesion molecules

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