GLIA 24:415–427 (1998)
Intracellular Location, Temporal Expression, and Polysialylation Of Neural Cell Adhesion Molecule in Astrocytes in Primary Culture ˜ ANA,1 MARI´A SANCHO-TELLO,2 EVA CLIMENT,1 JOSE ´ M. SEGUI´,3 ROSA MIN JAIME RENAU-PIQUERAS,3 AND CONSUELO GUERRI1* 1Instituto Investigaciones Citolo ´ gicas, FVIB, Valencia, Spain 2Department Biologı ´a Celular, University de Valencia, Valencia, Spain 3Centro de Investigacio ´ n, Hospital la Fe, Valencia, Spain
KEY WORDS
NCAM; polysialic acid; sialyltransferase; astrocytes; primary culture; WGA; Golgi apparatus; development
ABSTRACT Neural cell adhesion molecules (NCAMs) constitute a group of cell surface glycoproteins that control cell-cell interactions and play important morphoregulatory roles in the developing and regenerating nervous system. NCAMs exist in a variety of isoforms differing in the cytoplasmic domain and/or their content in sialic acid. The highly sialylated form (PSA-NCAM) is expressed by neurons, whereas it is believed that the less sialylated NCAM forms are synthesised by astrocytes. Moreover, little is known about the molecular sequence of the events that contribute to its expression at the cell surface. Here we report that during the proliferation of cortical astrocytes, at 4 days in primary culture, these cells expressed PSA-NCAM as well as NCAM 180. Then, during cell differentiation these isoforms progressively disappeared and the NCAM 140 became predominant. By immunofluorescence and immunocytochemistry studies we also show that PSA-NCAM and NCAM are first observed in small cytoplasmic spots or vesicles, located in or near the Golgi apparatus, as demonstrated by their co-localization with labelled wheat germ agglutinin (WGA) in this cell organelle. Thereafter, immunostained cytoplasmic NCAM gradually disappeared and became detectable at the cell surface of differentiating astrocytes. We also describe for the first time sialyltransferase activity in these cells and report that the levels of this activity correlated with the decrease in PSA-NCAM expression during the differentiation of astrocytes. These results will contribute to our understanding of the PSA and NCAM intracellular transport pathways and their expression at the cell surface. Moreover, the presence of PSA-NCAM in astrocytes suggests their possible role in nerve branching, fasciculation, and synaptic plasticity. GLIA 24:415–427, 1998. r 1998 Wiley-Liss, Inc.
INTRODUCTION During the last decade research has demonstrated that glia-neuronal interactions are of fundamental importance to development and regeneration of nervous tissue (Abbott, 1991). In the immature CNS, neuronal cell body migration and neurite outgrowth along the glial surface are perhaps the best documented developmental processes mediated by cell-cell interactions. Neural migration is the basis of CNS pattern formation and is characterised by astroglia-guide transr 1998 Wiley-Liss, Inc.
location of nerve cells from neuroembryonic sites to adult location (Hatten et al., 1990; Rakic, 1990). How these cell-cell interactions occur is not currently known, although some evidence suggests that such contact is Contract grant sponsor: CICYT; Contract grant number: SAF96/0185; Contract grant sponsor: Generalitat Valenciana; Contract grant number: GV-D-VS-20–126– 96. *Correspondence to: Dr. Consuelo Guerri, Instituto Investigaciones Citolo´gicas, FVIB, Amadeo de Saboya 4, 46010-Valencia, Spain. E-mail:
[email protected] Received 2 March 1998; Accepted 15 April 1998
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mediated by a number of adhesion or recognition molecules that are expressed by both neurons and glial cells. Indeed, cell adhesion molecules (CAMs) are cell surface macromolecules that control cell-cell interactions during development and regeneration of the nervous system (Rakic et al.; 1994; Fields and Itoh, 1996). Among these molecules, neural cell adhesion molecules (NCAMs) have received particular attention during the last few years due to their important morphoregulatory role in the developing and regenerating nervous system (see review, Rougon, 1993). NCAMs constitute a group of closely related membrane glycoproteins belonging to the immunoglobulin superfamily that can promote axon growth, fasciculation, and cell adhesion by homophilic and heterophilic interactions. NCAM is encoded by a single gene, and three major protein isoforms (180, 140, and 120 kDa), differing mainly in the length of their cytoplasmic domain and their mode of attachment to the membrane, are produced by differential splicing. An additional regulatory mechanism of NCAM-mediated cell adhesion involves the attachment of large, negatively charged carbohydrate polymers of a-2,8-linked sialic acid polymer (PSA) to the fifth immunoglobulin domain of the molecule (see review, Edelman and Crossing, 1991). The modulation of adhesion arises from the length of the PSA, as this molecule has been shown to decrease NCAM homophilic binding and thereby attenuate cell adhesion. It has been proposed that this negative modulatory role of PSA helps the cells to detach from their neighbours, thus allowing them to respond to guidance or targeting cues, or to exhibit the plastic interactions required for motility (see review, Rutishauser and Landmesser, 1996). For example, in the developing brain, a highly polysialylated form (the so-called embryonic NCAM, E-NCAM or PSA-NCAM) is expressed during the period of growth that involves cell migration and neuritic extension. PSA-NCAM is gradually replaced by adult isoforms with a lower sialic acid content during later stages of brain maturation (Chuong and Edelman, 1984). These changes in the developmental conversion of the embryonic to the adult form of NCAM have been associated with stabilisation of the cellular interaction, terminal neuronal outgrowth, and synaptogenesis. In the adult brain, the highly sialylated form of NCAM or PSA-NCAM is retained only in certain brain areas undergoing structural reorganisation (Miragall et al., 1988; Theodosis and Poulain, 1993), in response to injury (Daniloff et al., 1986), and in regions capable of morphological or physiological plasticity (Cremer et al., 1994; Roullet et al., 1997; O’Connell et al., 1997). However, despite the information available on the structure-function relationship of PSA-NCAM, the mechanisms that control its level and the cellular and molecular sequence of events that contribute to its expression at the cell surface are not yet understood. In brain, the presence of a developmentally regulated sialyltransferase activity, which is required for the polysialylation of endogenous NCAM, has been demon-
strated in a smooth membrane fraction containing Golgi apparatus membranes (Breen et al., 1987). By immunocytochemistry, cell-surface-associated but not intracellular PSA-NCAM has been detected in embryonic kidney (Lackie et al., 1991) and rat brain (Zuber et al., 1992) and in neural cells (e.g., Miragall et al., 1988; Kiss et al., 1994). In neurons, for example, PSA-NCAM immunoreactivity was observed in specific areas of plasma membrane (e.g., dendrites, axons, and nerve terminals) (Kiss et al., 1994) but not intracellularly. Moreover, PSA-NCAM has not been detected in normal astroglia cells, although poorly sialylated isoforms of NCAM are synthesised by these cells (Noble et al., 1985). Peripheral nerve injury leads, however, to the expression of PSA-NCAM at the cell surface of activated astrocytes (Daniloff et al., 1986). These results suggest that astrocytes have the capacity to synthesise PSA-NCAM and raises the question of whether during normal development astrocytes could also synthesise PSA-NCAM. In this report, we provide evidence that during the proliferation of cortical astrocytes, PSA-NCAM and NCAM, as visualised by immunofluorescence, are first accumulated in small spots, probably vesicles, located in or near the Golgi apparatus. Then, during the differentiation period, the immunostained cytoplasmic NCAMs progressively disappear and NCAMs become detectable at the cell surface. This distribution of NCAMs was also observed by electron microscopy. We also detected the a-2,8 sialyltransferase (ST) activity in astrocytes in primary culture and found that the levels of this activity correlated with the decrease in PSANCAM expression during the differentiation of astrocytes.
MATERIALS AND METHODS Astrocyte Cultures Primary cultures of astrocytes from 21-day-old rat foetuses were prepared from brain hemispheres as previously described (Renau-Piqueras et al., 1989). Briefly, foetuses were obtained under sterile conditions, and the cerebral hemispheres were dissected free of meninges and mechanically dissociated (by pipetting 10 times with a 10-ml pipette) in Dulbecco’s modified Eagle’s medium (Life Technologies Barcelona, Spain). The cell suspension was vortexed at maximum speed (1 min) and filtered through a nylon mesh with a pore size of 80 µm. Cells were plated on 35-mm Nunc plastic tissue culture dishes (2.5 3 106 cells/dish, 5 ml/dish) and maintained in the same medium containing 20% foetal calf serum and 1% antibiotics. Cultures were grown in a humidified atmosphere of 5% CO2 and 95% air at 37°C. After 1 week of culture, the serum content was reduced to 10%. The medium was changed every 3 days. As demonstrated using [3H]thymidine and [3H]leucine incorporation and flow cytometry analysis, the cultures grow rapidly for 7–10 days (proliferative period), after which the proliferative capacity of the cells
EXPRESSION OF NCAM IN ASTROCYTES
decreases, changing towards a functional state corresponding to the differentiation period (Guerri et al., 1990). The purity of the astrocyte cultures was assessed by immunofluorescence using monoclonal or polyclonal anti-glial fibrillary acidic protein (anti-GFAP) antibodies. Possible contamination by neurons was assessed by immunofluorescence using an anti-MAP 2 (Boehringer Mannheim Barcelona, Spain, clone AP20). In our conditions, 95% of the cells were astrocytes. Neuronal cells and microglial cells were never detected, and occasionally some oligodendrocytes were observed.
Immunofluorescence Astrocyte monolayers growing on 16-mm glass coverslips were used for double-immunofluorescence studies following procedures previously described (Cardoso et al., 1993; Sancho-Tello et al., 1995). Briefly, cells were washed with phosphate-buffered saline (PBS; 1.5 mM MgCl2 and 1 mM CaCl2) and fixed for 2 min in 220°C methanol. After blocking for 10 min in 3% bovine serum albumin (BSA) in PBS, cells were incubated simultaneously with anti-GFAP polyclonal antibody (1:500) and with either anti-NCAM monoclonal antibody (1: 500) or anti-PSA-NCAM monoclonal antibody (1:1000) in 1% BSA/PBS. The incubation was carried out at 37°C for 2 h in a moist chamber. After extensive washing with 0.1% Nonidet P-40 in PBS, cells were incubated for 1 h at room temperature with secondary antibodies: TRITC-conjugated goat anti-mouse IgG and FITCconjugated goat anti-rabbit IgG, diluted at 1:100 in 1% BSA/PBS. The coverslips were then washed with PBS and mounted in FA mounting fluid (Difco, Detroit, MI). For wheat germ agglutinin (WGA) studies, fixed cells were incubated with only one primary antibody (NCAM or PSA-NCAM) as above. Thereafter, cells were washed with Nonidet P-40 in PBS, and after a 30-min incubation with TRITC-conjugated goat anti-mouse IgG, FITCconjugated WGA (1:20) (Sigma Chemical Co., St. Louis, MO) was added and incubated for an additional 30 min. In sequential double staining (Kobayashi et al., 1997), live cells on coverslips were incubated with anti-PSA NCAM for 2 h, washed several times with 1% BSA/PBS, and then incubated with TRITC-conjugated goat antimouse IgG for 1 h. After several washings, the coverslips were fixed in 4% paraformaldehyde for 10 min, washed several times with 1% BSA/PBS, incubated with antiGFAP at 37°C for 1 h, washed, and incubated with FITC-conjugated goat anti-rabbit IgG at 37°C for 1 h. Astrocytes with double labelling were examined in a Zeiss fluorescence microscope with vertical illuminator for incident light (Ex 450–490/FT 510/LP 520 for FITC and Ex 510–560/FT 580 /LP 590 for TRITC). Analysis of double-labelled astrocytes with WGA and either NCAM or PSA-NCAM was also accomplished using confocal microscopy (ACAS 570 confocal laser photometer, Meridian Instruments, Okemos, MI). Cells were excited with an argon ion laser at 488 nm and 1.5 mW. FITC (WGA staining) and TRITC (NCAM or
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PSA-NCAM staining) fluorescence emissions were recorded at wavelengths between 515 and 545 nm (detector 1), and .605 nm (detector 2), respectively. The image scans were obtained with pixels of 0.3 µm2 using 4095 relative levels of fluorescence with a 1003 oil/NA 1.3 objective and an optical thickness of 0.99 µm with a pinhole of 400 µm. Approximately 100 cells (from three different cultures) were analysed per group.
Immunocytochemistry Astrocytes in primary culture were fixed as monolayers with 0.5% glutaraldehyde/4% formaldehyde in 0.1 M cacodylate buffer, pH 7.4, for 60 min at 4°C, detached from the plastic with a rubber policeman, washed in the buffer, incubated for 60 min in 50 mM NH4Cl, dehydrated in methanol, and embedded in Lowicryl K4M as previously described (Renau-Piqueras et al., 1989, 1997). Rat embryos at gestational day 18 (E18) and at postnatal day 6 (P6) were sacrificed, and their brains were removed by dorsal craniectomy, fixed by immersion, and embedded in Lowicryl K4M as described above. Location of PSA-NCAM and NCAM was performed with the immunogold procedure as previously described (Renau-Piqueras et al., 1989, Iborra et al., 1992). Ultrathin sections (80 nm) mounted on formvarcoated nickel grids were floated for 30 min on 0.1% BSA/Tris buffer (20 mM Tris/HCl, 0.9% NaCl, pH 7.4, containing 0.1% BSA, type V) supplemented with inactivated foetal calf serum (FCS), and then transferred to droplets of 0.1% BSA/Tris buffer containing 1% FCS and an anti-PSA-NCAM (1:300) or anti-NCAM (1:200) antibodies. The sections were incubated in a moist chamber for 180 min at 37°C. Although under these conditions the number of gold particles was reduced, this procedure results in a very specific, repetitive labelling for both antibodies. After three rinses with 0.1% BSA/Tris buffer for 10 min each, the grids were placed on droplets of 0.1% BSA/Tris buffer containing 0.5% Tween 20, 5% FCS, and an anti-mouse or rabbit IgG-gold complex (10 nm, 1:10 dilution; Sigma). The incubation time was 60 min at room temperature, as above. After two 30-min rinses with 0.1% BSA/Tris buffer and a rinse in bi-distilled water, the sections were air dried and finally counterstained with uranyl acetate. Controls were incubated without the first antibody. Control of clumping of gold particles in the anti-IgG-gold complexes was performed routinely. Astrocytes were recognised on the basis of morphological and immunocytochemical criteria. Serial coronal semi-thin sections of foetal brain, from olfactory bulbs through occipital poles, were used to classify the location of cells in the developing cerebral wall, and ultrathin sections from the dorsal domain were selected. In this domain the following zones can be observed: 1) germinal matrix or ventricular zone, 2) subventricular zone, 3) intermediate zone, 4) cortical zone, and 5) marginal zone. Sections were incubated
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simultaneously with an anti-GFAP antibody, polyclonal or monoclonal (to selected astroglial cells), and anti-PSANCAM or anti-NCAM antibodies as above. Occasionally a triple immunogold procedure using anti-GFAP, anti-NCAM, and anti-Rat 401 antibodies was used to differentiate astrocyte precursor cells (radial glia) in E18 brains. Micrographs were taken of germinal matrix and subventricular zone cells, both of which showed anti-GFAP and anti-NCAM labelling. Controls were carried out as described above.
Primary Antibodies Polyclonal anti-GFAP (rabbit IgG) was from Sigma. The working dilutions were 1:300 for immunofluorescence and 1:50 for immunocytochemistry. Monoclonal anti-GFAP (clone G-A-5) was from Boehringer Mannheim). The working dilutions were 1:500 for immunofluorescence and 1:50 for immunocytochemistry. Monoclonal (clone H28–123–16, Boehringer Mannheim) or polyclonal NCAM (Miragall et al., 1988) was used at a working dilution of 1:500 for Western blots and 1:2000 for immunocytochemistry. These antibodies react with the three polypeptide chains of NCAM (180, 140, and 120 kDa) (Gennarini et al, 1984). Monoclonal antiNCAM (clone OB11, Sigma). was used at a working dilution of 1:500 for both immunofluorescence and Western blot. This antibody recognises the intracytoplasmatic domain of 180-kDa NCAM. To detect PSANCAM, two antibodies were used: a mouse IgM that recognised specifically a-2–8-linked PSA with a chain length of more than 12 residues (Rougon et al., 1986), which was used at a 1:1000 dilution for immunofluorescence studies, and the monoclonal antibody 735, which specifically binds to a polysialic acid with eight or more residues (Frosch et al., 1985; Miragall et al., 1988). Both antibodies were used for immunofluorescence (1:500) and immunocytochemical (1:300) studies.
Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis and Western Blot Astrocytes were washed with PBS, harvested from tissue culture wells, homogenised in extraction buffer (PBS, 2% Nonidet P-40, 1.25 mM phenylmethylsulfonyl fluoride, 40 µM leupeptin, aprotinin 10µg/ml, 1 mM sodium orthovanadate), and incubated on ice for 15 min. Cell lysates were frozen and thawed three times and then centrifuged for 20 min at 15,000 3 g. The resulting supernatants were mixed with 6X SDS sample buffer (1X 0.125 M Tris/HCl, pH 6.8, 2% SDS, 2 mM EDTA, 5% (vol/vol) 1-mercaptoethanol) and boiled for 3 min. Proteins were separated in SDS-polyacrylamide slab gels using the discontinuous gel and buffer system of Laemmli (1970), employing 7% (wt/vol) polyacrylamide in the separation gel. After electrophoresis, proteins were transferred to nitrocellulose paper using a semidry electroblotter apparatus. The nitrocellulose
paper was incubated overnight with NCAMs (monoclonal antibodies, clones H28–123–16; OB 11) and then incubated for 1 h with anti-rat IgG-alkaline phosphatase conjugate (Promega, Madison, WI). After 10–20 min of colour development, the nitrocellulose sheets were washed and photographed. Microsomal Preparation Astrocytes were washed with PBS and scraped from the dishes with a rubber policeman. Cells were homogenised in 10 mM sodium cacodylate, pH 7.0, using a Potter-Elvehjem system with a Teflon pestle. The homogenate was centrifuged for 15 min at 9000 3 g, and the resulting supernatant was then centrifuged for 60 min at 120,000 3 g. The pellet was rehomogenised in buffer to yield a concentration of 4–10 mg of protein per ml. These preparations were stored frozen at 220°C until used. Sialyltransferase Assay Poly a-2–8-ST activity in astrocytes was determined by the procedure described by Easton et al. (1995), using colominic acid as an exogenous acceptor for ST. Briefly, a microsomal suspension from astrocytes (0.1– 0.2 mg of protein/assay) or from 15-day-old rat brain (0.3–0.5 mg of protein/assay) (Easton et al., 1995) was incubated in a total volume of 50 µl containing 10 mg of colominic acid (Calbiochem, La Jolla, CA), 25 nmol of CMP-[3H]NeuAc (3.8 Ci/mol; Du Pont-NEN, Boston, MA), 5 µmol of sodium cacodylate buffer, pH 6.5, 0.25 µmol of MnCl2, and 0.15 µl of Triton X-100. Incubations were carried out for 16 h at 37°C. Reactions were stopped by the addition of 200 µl of 0.05 M ammonium acetate, pH 5.4, and cooling on ice. Samples were then centrifuged in an Eppendorf centrifuge for 4 min at maximum speed. Pellets were washed once with the same amount of buffer and centrifuged again. The combined supernatants were passed over a column (0.7 3 50 cm) of Bio-Gel P-4 (200–400 mesh) equilibrated with 0.05 M ammonium acetate, pH 5.4, at a flow of 10 ml/h at room temperature. Fractions of 0.5 ml were collected and assayed for radioactivity by liquid scintillation counting. Incorporation was calculated from the sum of counts in the colominic-acid-containing fractions. The values were corrected for the incorporation into endogenous acceptors by running incubations lacking colominic acid and subtracting the counts found in these samples. RESULTS Immunofluorescence of PSA-NCAM and NCAM in Proliferating and Differentiating Astrocytes in Primary Culture To investigate whether cortical astrocytes express PSA-NCAM and other isoforms of NCAM during their
EXPRESSION OF NCAM IN ASTROCYTES
differentiation in primary culture, double-labelling immunofluorescence was carried out at different days of culture (4, 7, and 14) using anti-GFAP (an astroglial marker) and either anti-PSA-NCAM or anti-NCAM antibodies. To detect PSA-NCAM in astrocytes, the monoclonal antibody that specifically reacts with PSA was used in most of the immunofluorescence studies (Rougon et al., 1986). After 4 days in culture, GFAP staining was located in the intermediate filaments of astrocytes (Fig. 1B,D). In live cells, PSA-NCAM was located as a punctate staining over some areas of the cell surface (Fig. 1A). In contrast, in fixed cells, PSANCAM was seen in the cell cytoplasm and appeared in small aggregates (Fig. 1C). In some fixed cells, staining was also observed on the cell surface. A similar labelling pattern was obtained when astrocytes were incubated with either monoclonal antibodies that recognise NCAM 180 kDa (clone OB11) (Fig. 1C) or all of the NCAM isoforms (data not shown), respectively. To determine whether the NCAM-stained spots correspond to a sorting of glycoprotein-containing vesicles, we double labelled astrocytes with anti-PSA-NCAM or anti-NCAM and with FITC-conjugated WGA. WGA is a lectin that binds to sialic acid and N-acetylglucosaminyl residues, and it has been shown that WGA binding is predominant in the trans-Golgi network elements (Tartakoff and Vesally, 1983; Guasch et al., 1995; RenauPiqueras et al., 1997). As shown in Fig. 1, WGA staining appeared in the cell body of astrocytes and mainly within the perinuclear region and Golgi apparatus (Fig. 1F). A similar pattern was observed when cells were labelled with anti-PSA-NCAM (Fig. 1E) or anti-NCAM, although some punctate staining was also observed in the astrocyte processes. The possible association of WGA and NCAM labelling, was assessed using laser confocal microscopy. This analysis revealed a high degree of co-localisation between the pattern of labelled WGA and the immunoreactivity of either anti-PSANCAM (see Fig. 4A) or anti-NCAM (data not shown). At 7 days of culture, PSA-NCAM appeared in the Golgi apparatus and in the cytoplasm as small spots in the perinuclear region of some cells (Fig. 2A). Moreover, this pattern of labelling was similar to that of WGA staining, which, in addition, labelled tubular structures, probably rough endoplasmic reticulum (Fig. 2B). In contrast, in some cells, PSA-NCAM immunolabelling was also located in the astroglial processes that were not stained with WGA (data not shown). A similar staining pattern was observed when cells were labelled with anti-NCAM (Fig. 3C,E). However, NCAM epitope was also associated with some stained structures appearing at the cell surface or at the contacts between cells (Fig. 3C,E). These structures, which were not labelled with WGA (Fig. 3C), have previously been defined as clusters (Bloch, 1992) and were never observed in cells at day 4 of culture. Analysis by confocal laser microscopy of 7-day-old astrocytes revealed a certain degree of co-localization between labelled WGA and PSA-NCAM (Fig. 4B) or NCAM (data not shown) immunoreactivity patterns.
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At 15 days of culture or during the astrocyte cell differentiation period (Guerri et al., 1990), PSA-NCAM immunoreactivity was scarce. In contrast, cells incubated with anti-NCAM displayed abundant brightly labelled clusters (Fig. 3A,C,E). At this stage of astrocyte development, NCAM and WGA labelling showed important differences in the two staining distribution patterns, as demonstrated by immunofluorescence (Fig. 3A,B) and confocal microscopy (Fig. 4C).
Immunocytochemical Studies Analysis of anti-PSA-NCAM binding sites in proliferating astrocytes in culture revealed that gold particles appeared scattered over the plasma membrane, Golgi apparatus cisternae, some vesicles and, mainly, over processes (Fig. 5). Quantitative analysis of gold particle distribution in the cell body indicates that 72% of labelling corresponded to the cytoplasm and vesicles and 23% to plasma membrane, and 4% was over the Golgi apparatus. A similar distribution was observed in astroglial cells (GFAP-positive cells) from the germinal matrix or ventricular zone and subventricular zone in E18 and P6 brains (Fig. 5B). In all of the cases, there was very little background. Polyclonal anti-NCAM, which recognises the different NCAM isoforms, was present in all of the astrocytic cells examined in vitro and in vivo, and labelling was localised over the cytoplasm and plasma membrane of both the cell body and processes (Fig. 5D). Immunostaining was limited in the Golgi apparatus, and the distribution of gold particles in the cell body of astrocytes in culture was approximately 80% over plasma membrane and 20% over cytoplasm, including vesicles and Golgi apparatus.
Western Blot Analysis of NCAMs in Cortical Astrocytes To verify the presence of PSA-NCAM in astrocytes and to determine the temporal expression pattern of the NCAM isoforms in cortical astrocytes, Western blot analysis of astrocytes at 4, 7, and 14 days in culture was carried out. As shown in Fig. 6, at 4 days in culture, a strong immunoreactive band was present between 200 and 180 kDa, although NCAM-140 was weakly detected (Fig. 6). The 200-kDa band corresponds to the described PSA-NCAM, which migrates in the same position as the PSA-NCAM observed in postnatal brain (Fig. 6). However, as astrocytes differentiated, the 200and 180-kDa bands progressively disappeared, and the immunoreactive band corresponding to 140 kDa increased in intensity. On day 14 of culture, NCAM-140 was the only isoform in the astrocytes (Fig. 6). It is noteworthy that under our conditions and with the antibodies we used, we could not detect the NCAM 120-kDa isoform.
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Fig. 1. Micrographs showing immunofluorescence of astrocytes at 4 days of culture double labelled with anti-PSA-NCAM (A and C) and anti-GFAP (B and D) antibodies. In A, cells were not fixed before incubating with PSA-CAM. E and F correspond to cells simultaneously labelled with anti-PSA-NCAM (E) and WGA (F). As shown, in live cells (A), PSA-NCAM is distributed over some areas of the cell
surface whereas in fixed cells (C) staining is located in the cytoplasm as small spots. E and F illustrate the high degree of co-localization between WGA and PSA-NCAM staining. Note that in both cases the Golgi apparatus was also stained (arrowheads). A–D, 3900; E and F, 3410.
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Fig. 2. Double immunofluorescence of 7-day-old astrocytes in primary culture showing the intracellular distribution of PSA-NCAM (A) or NCAM (C) and WGA (B and D). E and F correspond to cells double stained with anti-NCAM (E) and anti-GFAP (F). As illustrated, fluorescence corresponding to WGA appears located over the Golgi apparatus (arrows) and tubular structures (probably rough endoplasmic reticulum) and as spots distributed mainly in the perinuclear area
near the Golgi apparatus. A shows the fluorescence corresponding to PSA-NCAM, which co-localises with positive WGA, structures such as Golgi apparatus, and perinuclear spots (B). In C and E, anti-NCAM labelling appears associated following a pattern in clusters (arrows), which does not correspond with that of WGA (D). A–D, 3900; E and F, 3410.
ST Activity in Astrocytes
astrocytes. We also found that ST activity was high in astrocytes at 4 days of culture, but in cells at 14 days, the activity decreased by approximately one-half. The levels of ST activity in 14-day-old astrocytes was approximately 1/20 that found in brain microsomal preparations of 14 day-old rats (0.49 6 0.07 nmol/mg of protein/h; Fig. 7).
Microsomal membrane preparations from astrocytes on different days (4, 7, and 14 days) of culture were assayed for ST using a recently described procedure (Easton et al., 1995). With this procedure, we were able to detect for the first time the presence of ST in
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Fig. 3. Micrographs of immunofluorescence from simultaneously labelled 15-day-old astrocytes showing the distribution of NCAM (A,C, and E) versus WGA (B) or GFAP (D and F). As shown, NCAM appears distributed in cluster (arrows), and cells display important differences between WGA and NCAM staining patterns A–F, 3900.
DISCUSSION Previous work has shown that PSA-rich NCAM isoforms are predominantly expressed in neurons, whereas astroglial cells, under normal conditions, synthesise NCAM forms with a lower PSA content (NCAM 140, NCAM 120) (Noble et al., 1985). In the present study, we demonstrate that during the proliferation and differ-
entiation periods, cortical astrocytes in primary culture express the PSA-NCAM as well as the 180-kDa and 140-kDa NCAM isoforms. In addition, the presence and temporal pattern of sialyltransferase activity are also demonstrated for the first time in astrocytes. We show that during the proliferation of astrocytes, PSA-NCAM appeared in the cytoplasm as a granular staining. Afterwards, during cell differentiation, these aggre-
EXPRESSION OF NCAM IN ASTROCYTES
Fig. 4. Confocal microscopy images of astrocytes at 4 (A), 7 (B), and 14 (C) days of culture stained with PSA-NCAM (A and B) or NCAM (C) and WGA. The line scan mode was done by scanning laser light through the marked white line (left side of the figure) with a step size of 0.1 µm. The graphs indicate the fluorescence intensity profiles from the line scan. The abscissa indicates the distance between the beginning and the end of the scanning line (query length), and the ordinate indicates the fluorescence intensity (arbitrary units). Fluorescence recorded by detector 1 is shown in filled circles (PSA-NCAM in A and B and NCAM in C) and by detector 2 in black (WGA).
gates disappeared and NCAM gradually appeared on the cell surface or at the cell-cell contacts as clusters. It is well established that NCAM isoforms are glycoproteins, and experimental evidence indicates that the biosynthesis of PSA-NCAM occurs by post-translational modifications. Indeed, in vivo and in vitro experiments (Lyles et al., 1984; Alcaraz and Goridis, 1991)
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have shown that high-mannose residues are incorporated into the cis-Golgi apparatus before polysialylation occurs, which suggests that addition of PSA takes place in an intracellular compartment at or beyond the medial or trans-Golgi apparatus network. However, little is known about the regulation of PSA-NCAM or the sorting and targeting mechanisms involved in the polysialylation of NCAM. The present immunofluorescence and immunocytochemical results support the view that during proliferation of astrocytes, NCAM is polysialylated in the Golgi apparatus, stored, and sorted in small vesicles throughout the trans-Golgi apparatus network to the periphery of the cell (Farquar and Hauri, 1997), where NCAM would then exert its adhesion properties. This suggestion is supported by the following findings. 1) PSA-NCAM and NCAM immunoreactivity is abundant in the cytoplasm of proliferating astrocytes (4 days of culture) (Guerri et al., 1990), where it extensively co-localizes with WGA binding sites. WGA binding has been shown to be predominantly in the trans-Golgi network (Tartakoff and Vesalli, 1983; Guasch et al., 1995). 2) An increase in surface NCAM cluster staining and a decrease in cytoplasmic granular immunoreactive PSA-NCAM or NCAM were associated with a decrease in the degree of WGA co-localization at 7 days of culture. This suggests that, although some NCAM is still in vesicles in the cell cytoplasm, NCAM has largely been transported to the cell periphery. 3) In differentiated astrocytes (15 days of culture) (Guerri et al., 1990), NCAM labelling appeared only on the cell surface as patches, and this pattern is the most common in mature astrocytes (data not shown). At this stage of development, the NCAM staining distribution pattern was not associated with the WGA labelling. This view is consistent with the finding that when transport from the Golgi apparatus to plasma membrane is inhibited, de novo PSA synthesis becomes detectable in the Golgi apparatus (Scheidegger et al., 1994). We do not know, as yet, whether the PSA-NCAM or NCAM labelling observed in the cytoplasm of astrocytes corresponds to newly synthesised PSA-NCAM or NCAM and/or to the NCAM pool originated from the plasma membrane recycling process. The fact that NCAM labelling consistently appeared over the Golgi apparatus and was less pronounced on the cell surface suggests that an important amount of this labelling corresponds to the biosynthesis-dependent process. Indeed, some experimental evidence indicates that PSANCAM is regulated primarily at the level of synthesis (Scheidegger et al., 1994; Rutishauser and Landmesser, 1996). It has been suggested that the biosynthesisindependent process may result in rapid changes in PSA-NCAM surface expression, whereas changes in biosynthesis are observable only after a longer period of time (Kiss and Rougon, 1997). Our results show that the time course of NCAM appearance over the cell surface of astrocytes is very long, approximately 7–15 days. This raises the question about what type of
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Fig. 5. Electron microscopy immunocytochemistry showing the anti-PSA-NCAM (A–C) and anti-NCAM (D) binding sites on 7-day-old astrocytes in primary culture (A,C, and D) and on astroglial cells from the ventricular zone (B) from brain postnatal cortex (P6). As shown, PSA-NCAM appears over or near the plasma membrane (arrows),
scattered on the cytoplasm (arrowheads), and over cisternae of the Golgi apparatus (C). N-CAM is located mainly over plasma membrane. Small-size gold particles (circle) correspond to anti-GFAP. A, 3105,000 (scale bar: 0.25 mm); B, 380,000 (scale bar: 0.25 mm); C, 3105,000 (scale bar: 0.2 mm); D, 380,000 (scale bar: 0.25 mm).
EXPRESSION OF NCAM IN ASTROCYTES
Fig. 6. Western blot analysis of NCAM (200, 180, and 140 kDa) in cortical astrocytes at 4, 7, and 14 days of culture. Each line contains 500 µg of total protein from the cell lysate. MW, molecular weight standard; P5, brain homogenate from 5-day-old rat.
Fig. 7. Changes in CMP-NeuAC:(NeuAca2=8)n (colominic acid) sialyltransferase activity in cortical astrocytes in primary culture throughout its differentiation. Each point represents the average 6 SD of four astrocyte microsomal preparations from different cultures.
signals or external clues could influence or modulate the PSA-NCAM or NCAM surface appearance in cultured astrocytes. Experimental evidence indicates that the cell electrical activity and the target interactions are important factors involved in the appearance of PSA-NCAM at the cell surface (Kiss et al., 1994). Therefore, it may be that during brain development or adult plasticity, the neuronal electrical activity (Fryer
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and Hockfield, 1996) or activity-dependent communication between neurons and glial cells (e.g., glutamate, Ca21) (Pasti et al., 1997; Hansson and Ronnback, 1995) accelerates or modulates the PSA-NCAM or NCAM surface expression in astrocytes and neurons. Moreover, as these molecules act mainly by homophilic binding, NCAM-mediated interactions may well be involved in the control of glia cell number, migration, differentiation, and plasticity. Indeed, a recent report demonstrated that NCAMs do inhibit astroglial cell proliferation, which suggests that NCAM homophilic binding may be involved in the control of glia proliferation (Sporns et al., 1995). As we pointed out above, recent experimental evidence indicates that NCAM polysialylation is primarily regulated at the biosynthetic level and that this process involves the Golgi enzyme ST (Eckhardt et al., 1995; Nakayama et al., 1995). These studies have shown that the levels of PSA expression are closely correlated with ST activity. Indeed, during the course of brain development, the levels of both ST activity (Breen et al., 1987) and its mRNA (Nakayama et al., 1995) parallel the production of PSA-NCAM. Our results demonstrating that the levels of PSA-NCAM expression in astrocytes decrease with the ST activity throughout the course of astrocyte proliferation and differentiation are consistent with these findings. The importance of astrocytes and their differentiation state have been clearly demonstrated in both the developing brain (Rakic, 1991) and in response to CNS injury. Thus, transplantation of astrocytes into lesioned mature CNS demonstrates that whereas immature astrocytes provide support and guidance for developing neurites, transplantation of mature astrocytes is accompanied by significantly less neurite outgrowth-promoting activity (Krobert et al., 1997). Although the molecular basis of this developmental switch is unknown, the release of growth factors (Valles et al., 1994) and expression of some adhesion molecules, including PSANCAM, in immature astrocytes may be involved in these neurite outgrowth-promoting effects. In fact, in brain injuries where astroglia proliferation occurs (Hatten et al., 1991), high levels of PSA-NCAM are also expressed by glial cells (Kiss et al., 1993; Le Gal et al., 1992). The polysialylated form of NCAM has also been shown to be expressed in astrocytes of some discrete areas of the adult brain that retain the capacity to undergo morphological changes, such as the olfactory system (Miragall et al., 1988; Franceschini and Barnet, 1996), optic nerve (Kobayashi et al., 1997), and hypothalamic-neurohypophysial system (Theodosis et al., 1991). Therefore, the expression of PSA-NCAM in astroglial cells observed in some adult brain areas or during brain injury may resemble that which occurs under normal conditions during brain development. The presence of PSA in proliferating astrocytes may affect glia-neuron interaction, facilitating neuronal developmental plasticity and potential to change. Thereafter, as the astroglia matures and the synaptic contacts have been estab-
˜ ANA ET AL. MIN
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lished, the degree of sialylation of NCAM in neurons and astroglial cells would decrease, leading to an increase in cell adhesion. In conclusion, our results suggest that NCAM is polysialylated in the Golgi apparatus and then transported to the cell periphery where it could exert its adhesion properties. These results will contribute to an understanding of the PSA-NCAM and NCAM intracellular transport pathways and their regulation in astrocytes. Moreover, the presence of PSA-NCAM in astrocytes suggests their possible role in nerve branching, fasciculation, and synaptic plasticity.
ACKNOWLEDGMENTS We gratefully acknowledge the generosity of Dr. Rougon (Laboratoire de Ge´ne´tique et Physiologie du De´velopment, Marseille, France) and Dr. Miragall (Institut fu¨r Anatomie, Universita¨t Regensburg, Germany) for their antibodies to PSA-NCAM and NCAM. We also thank M. March for technical assistance. This work was supported by CICYT (SAF96/0185), Generalitat Valenciana (GV-D-VS-20–126–96) and Fundacio´n Ramo´n Areces. R. Min˜ana is a fellow of Generalitat Valenciana.
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