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Originally published In Press as doi:10.1074/jbc.M601498200 on June 27, 2006

J. Biol. Chem., Vol. 281, Issue 34, 24970-24978, August 25, 2006
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Identification of Neurite Outgrowth-promoting Domains of Neuroglycan C, a Brain-specific Chondroitin Sulfate Proteoglycan, and Involvement of Phosphatidylinositol 3-Kinase and Protein Kinase C Signaling Pathways in Neuritogenesis*

Keiko Nakanishi12, Sachiko Aono1, Kanako Hirano, Yoshiyuki Kuroda, Michiru Ida, Yoshihito Tokita, Fumiko Matsui, and Atsuhiko Oohira

From the Department of Perinatology, Institute for Developmental Research, Aichi Human Service Center, Kasugai, Aichi 480-0392, Japan

Received for publication, February 16, 2006 , and in revised form, June 23, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Neuroglycan C (NGC) is a transmembrane-type chondroitin sulfate proteoglycan that is exclusively expressed in the central nervous system. We report that the recombinant ectodomain of NGC core protein enhances neurite outgrowth from rat neocortical neurons in culture. Both protein kinase C (PKC) inhibitors and phosphatidylinositol 3-kinase (PI3K) inhibitors attenuated the NGC-mediated neurite outgrowth in a dose-dependent manner, suggesting that NGC promotes neurite outgrowth via PI3K and PKC pathways. The active sites of NGC for neurite outgrowth existed in the epidermal growth factor (EGF)-like domain and acidic amino acid (AA)-domain of the NGC ectodomain. The EGF-domain caused cells to extend preferentially one neurite from a soma, whereas the AA-domain caused several neurites to develop. The EGF-domain also enhanced neurite outgrowth from GABA-positive neurons, but the AA-domain did not. These results suggest that the EGF-domain and AA-domain have distinct functions in terms of neuritogenesis. From these findings, NGC can be considered to be involved in neuritogenesis in the developing central nervous system.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Proteoglycans (PGs)3 are a group of proteins with covalently attached sulfated glycosaminoglycan (GAG) chains. In the central nervous system (CNS), there are many PG species, some of which are classified into families with a domain structure common to their core proteins (for review, see Ref. 1). There is increasing evidence that PGs are involved in axonal outgrowth, synaptogenesis, neuronal cell differentiation, embryonic cell division, and repair of the CNS (1-4).

Neuroglycan C (NGC) is a membrane-spanning chondroitin sulfate proteoglycan (CSPG) with a core glycoprotein of 120 kDa that was originally found in the developing rat brain (5, 6). Its core protein is divided into five structurally different domains; an N-terminal domain to which a chondroitin sulfate chain is attached, an acidic amino acid (AA) cluster, a cysteinerich domain containing a single epidermal growth factor (EGF)-like module, a membrane-spanning segment, and a C-terminal cytoplasmic domain of 95 amino acids (5). NGC is a part-time PG, because most NGC molecules exist in a non-PG form without chondroitin sulfate (CS) in the mature cerebellum and retina (7, 8). Expression of NGC in the rat CNS is detected at embryonic day (E) 16, increases with a peak around the third postnatal week, then decreases to about a half of the peak level at adulthood (5, 8). Because neuritogenesis and synaptogenesis in the rat CNS actively occur in the late fetal and early postnatal periods, it is likely that NGC is involved in the formation of the neuronal circuit in the CNS. Immunohistochemical studies using an antibody against NGC show that NGC is expressed in association with neuronal structure such as dendrites or axons in the developing CNS (7-9). Immunostaining of cultured neuronal cells with anti-NGC antibody demonstrated that NGC is concentrated in budding neurites (8) or dendritic filopodia (10), which are considered to develop into neurites or postsynaptic spines. Schumacher et al. (9) have identified a chicken homolog of NGC named chicken acidic leucine-rich EGF-like domain containing brain protein (CALEB), and reported that Fab fragments of anti-NGC/CALEB antibodies interfere with the formation of neurites in chick tectal cells. Moreover, it has been shown that synapse function during postnatal development was impaired in CALEB-deficient mice (11). Thus, NGC is involved in neuritogenesis and/or synaptogenesis.

To study the function of NGC in neuritogenesis more directly, we produced a recombinant ectodomain of the NGC core protein and performed neurite outgrowth assays in primary cultured fetal rat neocortical neurons. We found that the EGF-domain and AA-domain of the NGC ectodomain enhanced the outgrowth of neurites from rat neocortical neurons, and that NGC-induced neurite outgrowth was mediated via phosphatidylinositol 3-kinase (PI3K) and protein kinase C (PKC) pathways.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—Neocortical neurons were cultured as described previously (12). Briefly, cerebral cortices of fetal (embryonic day 16) Wistar rats were digested with 0.02% papain and mechanically dissociated by trituration. The cells were plated on poly-L-lysine-coated 24-well plates at a density of ~5.3 x 103 cells/cm2. The cells were maintained in high glucose Dullbecco's modified Eagle's medium (Invitrogen) containing 0.02% heat-inactivated horse serum, 50 units/ml penicillin, 25 µg/ml streptomycin, 10 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES; Dojin Chemicals, Kumamoto, Japan), 25 µM 2-mercaptoethanol, and 10 µl/ml N2 supplement (Invitrogen), in a 95% air, 5% CO2, H2O-saturated atmosphere at 37 °C. For the experiment of immobilized NGC, culture plates precoated with poly-L-lysine were treated with recombinant NGC proteins at 4 °C overnight. The plates were washed three times with PBS before use. All of the animal experiments were performed with the approval of the Animal care and Experimentation Committee of our institution. Every effort was made to minimize animal suffering and reduce the number of animals used.

Preparation of Recombinant NGC Core Protein Peptides—Recombinant NGC proteins were prepared by the method of Yamauchi et al. (13) with a slight modification as follows. To produce recombinant rat NGC polypeptides as glutathione S-transferase (GST)-fused proteins, cDNA fragments encoding the entire ectodomain without the signal peptide (NGCect; amino acid 31-426), a domain containing the CS attachment site (NGCcs; 33-259), a domain containing the acidic amino acid (AA) cluster (NGCAA; 259-336), a domain containing the EGF module (NGCE; 336-426), and a domain containing both AA- and EGF-domains (NGCAE; 259-426) of rat NGC were subcloned into pGEX 4T-1 (Amersham Biosciences). The plasmids were introduced separately into Escherichia coli BL21. Expression of the fusion proteins was induced by addition of 1 mM isopropyl-beta-D-thiogalactopyranoside (Katayama Chemicals, Nagoya, Japan). Cells were lysed in BugBuster Protein Extraction Reagent (Novagen, Darmstadt, Germany). After centrifugation of the lysate at 10,000 x g for 20 min at 4 °C, the supernatant was applied to a glutathione-Sepharose 4B (Amersham Biosciences) column, and proteins bound to the beads were eluted with 50 mM Tris-HCl, pH 8.0, containing 10 mM glutathione. Affinity-purified GST-fused proteins were dialyzed against phosphate-buffered saline (PBS) at 4 °C overnight, and used for neurite outgrowth assays after sterilization by filtration.

Neurite Outgrowth Assay—Recombinant proteins were usually added to neuronal cell cultures 1 h after plating. For the assay with kinase inhibitors, first the kinase inhibitors were added 1 h after plating, then the recombinant proteins. Cells were fixed 24 h after plating with 3% paraformaldehyde in PBS containing 1% sucrose, 1 mM MgCl2, 0.1 mM CaCl2, and 0.1% glutaraldehyde for 1 h. After fixation, ten fields in each well were photographed using a digital camera (DXC-S500/OL; Olympus, Tokyo, Japan) through an inverted microscope (IX71, Olympus) at a x200 scale and analyzed. A neuron with neurites extending three somal diameters or longer was defined as a neuron with long neurites (Fig. 2B, panel a). The percentage of neurons with long neurites was calculated as the number with long neurites divided by the total number of neurons in 10 fields in each well, and the average from 4 wells was calculated. Neurite length was defined as the one-line distance from the center of the soma to the farthest tip of the longest neurite of individual neurons (Fig. 2B, panel b). The neurite length of all neurons in 10 photographs in each well was measured using the public domain NIH Image program. The average for 4 wells was calculated as the mean neurite length in each condition. We usually analyzed about 50-100 neurons per well. The number of neurites per neuron was determined as the number of primary processes from the soma. The histogram (Fig. 7) was made from all neurons with long neurites in 4 wells (total 40 fields). For the analysis of the percentage of {gamma}-aminobutyric acid (GABA)-positive neurons with long neurites, all GABA-positive neurons were examined in each well, since there were only a few GABA-positive neurons in each field (around 250-300 cells in each well, namely about one tenth of all neurons; Ref. 14). Data were plotted as the mean ± S.D. Statistical analysis was performed using analysis of variance followed by the Tukey-Kramer multiple-comparison test (InStat Ver. 3; Graph-Pad Software, San Diego, CA), and a p value of < 0.05 was considered significant.

Antibodies and other Chemicals—Mouse monoclonal antibodies against Tau-1 (Chemicon International, Temecula, CA), Microtubule-associated protein 2 (MAP2; Sigma-Aldrich), phosphotyrosine (PY-20; Sigma), beta-actin (Sigma), extracellular signal-regulated kinase (ERK; Sigma), and phospho-ERK (Sigma), and rabbit polyclonal antibodies against Tau (H-150; Santa Cruz Biotechnology, Santa Cruz, CA) and GABA (Sigma) were used. Staurosporine was purchased from Wako Chemicals (Osaka, Japan). Rp-adenosine 3',5'-cyclic monophosphorothioate triethylammonium salt (Rp-cAMPS), hypericin, U0126, calphostin C, and LY294002 were obtained from Sigma. Lavendustin A and bisindolylmaleimide I were purchased from Calbiochem (Darmstadt, Germany).

Immunocytochemistry—Freshly fixed neurons were first washed with PBS and blocked with 2% goat serum in PBS containing 0.1% Triton X-100 to reduce nonspecific antibody binding. Neurons were then incubated with a primary antibody at 4 °C overnight. After washing with PBS, they were incubated with a secondary antibody, either Cy3-conjugated anti-mouse IgG antibody (Jackson ImmunoResearch, West Grove, PA) or biotinylated anti-rabbit IgG antibody (Vector Laboratories, Burlingame, CA), at room temperature for 2 h, and in the latter case then incubated with streptavidin-conjugated Alexa Fluor 488 (Molecular Probes, Eugene, OR). For the double staining, samples were incubated with two kinds of antibody concurrently.

SDS-PAGE and Immunoblotting—To confirm the production and purity of recombinant proteins, they were separated by SDS-PAGE on a 5-20% gradient gel (Bio Craft, Tokyo, Japan), and protein bands were stained with Coomassie Brilliant Blue.

For phosphorylation analysis, neocortical neurons were seeded on 6-cm dishes at a density of 4.2 x 104 cells/cm2. Fifteen minutes after treatment with NGCect (50 µg/ml) or GST (at a molar concentration equal to that of NGCect), the cells were washed with cold PBS and lysed in 200 µl of 50 mM HEPES, pH 7.5, containing 1% Triton X-100, 1 mM sodium orthovanadate, 100 mM NaF, 10 mM sodium pyrophosphate, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, and 5 µg/ml leupeptin. After 60 min of incubation on ice, cell lysates were centrifuged at 14,000 x g for 15 min, and the supernatants were used for Western blot analyses. Proteins precipitated from each sample by adding 3 volumes of 95% ethanol containing 1.3% potassium acetate at 0 °C were separated by SDS-PAGE on a 5-20% gradient gel (Bio Craft). The proteins in the gel were transferred electrophoretically to a Hybond ECL membrane (Amersham Biosciences). The membrane was incubated sequentially with primary antibodies and with adequate secondary antibodies, and immunoproducts were detected using the enhanced chemiluminescence (ECL) system (Amersham Biosciences). Immunoblotting using an anti-beta-actin antibody (Sigma) was done to estimate the yield of proteins after stripping of the membrane.


Figure 1
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  		FIGURE 1.
Preparation of recombinant NGC core polypeptides. A, NGC core protein contains several structurally unique domains such as an N-terminal signal peptide (SP) consisting of 30 amino acid residues, a chondroitin sulfate (CS) attachment site (arrow), an acidic amino acid cluster (AA), a single EGF-module (EGF), and a transmembrane domain (TM). Recombinant polypeptides containing one or more of these domains were produced in E. coli as GST-fused proteins; NGCect, the entire NGC ectodomain (amino acid no. 31-426 of rat NGC); NGCCS, a peptide containing the CS-attachment site (no. 33-259); NGCAA, a peptide containing AA (no. 259-336); NGCE, a peptide contaning EGF (no. 336-426); NGCAE, a peptide containing AA and EGF (no. 259-426). B, Coomassie Brilliant Blue staining of each GST-fused protein separated by SDS-PAGE. CS, NGCCS; AA, NGCAA; E, NGCE; AE, NGCAE; ect, NGCect; G, GST. The positions of molecular mass markers are indicated at the left of the panel.

 


Figure 2
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  		FIGURE 2.
Ectodomain of NGC core protein promotes outgrowth of neurites from neocortical neurons in culture. A, neocortical neurons from E16 fetal rats were cultured without any recombinant peptides (None) or with GST or NGCect for 24 h. Scale bar, 50 µm. B, quantification of neurite outgrowth activity. Panel a, percentage of cells with long neurites. A cell with long neurites was defined as a neuron with neurites extending three somal diameters or longer. Panel b, mean neurite length. Neurite length was defined as the one-line distance from the center of the soma to the farthest tip of the longest neurite of individual neurons. Data represent the mean ± S.D. (n = 4). ***, p < 0.001.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
A Recombinant Ectodomain of NGC Core Protein Promotes Neurite Outgrowth—The NGC core protein contains several structurally unique domains such as the CS-attachment site, an AA cluster, and a single EGF-like module as shown in Fig. 1A. To investigate domain-specific functions of the extracellular region of NGC, we produced recombinant polypeptides containing one or more of these domains in E. coli as GST-fused proteins (see "Experimental Procedures"). These recombinant polypeptides were purified separately by affinity column chromatography using a glutathione column, and then subjected to SDS-PAGE (Fig. 1B). Each sample contained a major band with the largest molecular size and a few minor bands, which might be synthetic precursors or degradation products of the major component. For control experiments, GST was also expressed in E. coli, and purified by affinity column chromatography. It was recovered by SDS-PAGE at a molecular size of 27 kDa, the expected size for GST (Fig. 1B, lane G).

To examine whether the NGC ectodomain exerts any effect on the cellular events in neurons, we first added NGCect to rat neocortical neurons in culture. Twenty-four hours after plating, the neurites of neurons cultured in the presence of NGCect (50 µg/ml) were longer than those of control cells cultured without any NGC peptides (None) or with only GST (Fig. 2A). The percentage of cells which have neurites extending three somal diameters or longer was 66.6 ± 4.9 (mean ± S.D.) percent in the presence of NGCect. This was significantly high compared with the control value (None, 12.6 ± 7.0%; GST, 10.7 ± 3.9%; p < 0.001, Fig. 2B, panel a). The mean length of the longest neurite of individual neurons cultured in the presence of NGCect was 47.3 ± 3.3 µm, which was significantly longer than the control value (None, 22.1 ± 0.8 µm; GST, 22.4 ± 2.1 µm; p < 0.001, Fig. 2B, panel b). NGCect-mediated neurite outgrowth was also observed in cultures of hippocampal neurons from E16 fetal rats (data not shown). Furthermore, outgrowth-promoting activity was observed when we used pure NGCect prepared from NGCect by enzymatic removal of the GST portion (data not shown), indicating that the promotive activity of NGCect for neurite outgrowth was not due to the fusion of GST to the NGC ectodomain. When neocortical neurons were cultured on plates precoated with 50 µg/ml of NGCect, neurite elongation was also promoted significantly, but less than the cultures supplemented with NGCect to culture media (data not shown), suggesting that both soluble and substratum-immobilized NGC molecules exert neurite outgrowth-promoting activity.

Most neurites elongated by the NGCect treatment were immunopositive for tau (an axonal marker, Fig. 3A). Some elongated neurites were also immunopositive for MAP2 (a dendrite marker), although the intensity was low (Fig. 3B). Considering that the differentiation of neurites into axons or dendrites is determined after 3 days in culture (15), it is possible that the neurites elongated by NGCect treatment have not differentiated into axons completely by 24 h after plating.


Figure 3
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  		FIGURE 3.
NGCect-induced neurites are immunopositive for tau. A, neocortical neurons were cultured in the presence of NGCect for 24 h, and stained with a monoclonal antibody to tau, an axonal marker. B, double staining of neurons cultured for 24 h in the presence of NGCect with a monoclonal antibody to MAP2, a dendritic marker, and a polyclonal antibody to tau. Upper panels show photos of phase contrast. Scale bar, 50 µm.

 


Figure 4
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  		FIGURE 4.
NGC treatment reduced tyrosine phosphorylation of several proteins in neocortical neurons. Neocortical neurons were cultured for 20 h and then treated for 15 min with GST (G) or NGCect (N). A, each cell lysate was immunoblotted using antibody against phosphotyrosine (PY-20) or beta-actin. The positions of molecular mass markers are indicated at the left of the panel. Arrows indicate the bands with reduced tyrosine phosphorylation. B, immunoblotting with anti-phospho-ERK (anti-pERK) and anti-ERK antibodies of the cell lysates. G, GST-treated cells; N, NGCect-treated cells. Arrows indicate the positions of ERK1 and 2.

 
Treatment of Neurons with NGCect Reduces Tyrosine Phosphorylation—To find a cue of the intracellular mechanism for neurite elongation mediated by NGCect, we examined the tyrosine phosphorylation of neocortical neurons. Fifteen minutes after treatment with NGCect or GST, neurons were lysed and tyrosine phosphorylation was assessed by Western blotting using an antibody against phosphotyrosine (PY20). Treatment with NGCect reduced the tyrosine phosphorylation of about 34-, 37-40-, 46-56-, 92-, and 180-kDa proteins (Fig. 4A). The density of each tyrosine-phosphorylated band was compared between control and NGCect-treated sample after normalization to the density of beta-actin band using the public domain NIH Image program. Tyrosine phosphorylation was reduced by NGCect treatment to 74, 78, 64, 74, and 83% in 34-, 37-, 46-, 92-, and 180-kDa proteins, respectively. This result indicates that NGCect gives rise to a certain intracellular signal in cultured neurons.

Because the ERK pathway is involved in neurite outgrowth in some experimental systems (16, 17), we then examined whether ERKs were activated during NGCect-induced neuritogenesis. Neither the protein level nor the phosphorylation level of ERK1/2 was changed remarkably by NGCect treatment of neurons (Fig. 4B). Next, we examined the involvement of Src family tyrosine kinases whose molecular sizes are 50-60 kDa, because they have been demonstrated to be involved in the elongation of neurites in some cases (18-20). However, the level of c-Src phosphorylation was not changed in neurons treated with NGCect (data not shown). These results indicate that NGC-mediated neurite outgrowth is independent of MAPKs and Src kinase pathways.

NGC-induced Neurite Outgrowth Is Mediated by PKC and PI3K—To identify the family of kinases responsible for NGCect signaling in neuritogenesis, we treated neocortical neurons with several kinase inhibitors in the presence (+) or absence (-)ofNGCect (N), and then examined neurite outgrowth (Fig. 5). We tested staurosporine (S, a broad spectrum protein kinase inhibitor, 50 nM), lavendustin A (L, an inhibitor of EGF receptor tyrosine kinase and p60c-src, 10 µM), Rp-adenosine 3',5'-cyclic monophosphorothioate triethylammonium salt (Rp-cAMPS; R, a protein kinase A inhibitor, 20 µM), hypericin (H, a protein kinase C inhibitor, 10 µM), and U0126 (U, a MAP kinase kinase (MEK) 1 and 2 inhibitor, 100 nM). Addition of NGCect increased the percentage of neurons with long neurites even in the presence of lavendustin A, Rp-cAMPS, and U0126 (L versus L + N, 5.6 ± 2.9 versus 40.6 ± 4.6%, p < 0.001; R versus R + N, 25.1 ± 1.8 versus 66.3 ± 4.8%, p < 0.001; U versus U + N, 32.3 ± 6.0 versus 72.5 ± 1.3%, p < 0.001, respectively) as well as in the absence of these inhibitors (GST (G) versus NGCect (N), 26.0 ± 1.4 versus 73.8 ± 4.0%, p < 0.001; Fig. 5A, panel b). The fact that U0126 did not block the NGC-mediated neurite outgrowth was consistent with the results of phosphorylation experiments shown in Fig. 4B. On the other hand, in the presence of staurosporine and hypericin, NGCect did not enhance neurite outgrowth (S and H in Fig. 5A, panel b). The mean neurite lengths in S + N and H + N conditions were not significantly different from those in S alone and H alone, respectively, while NGCect treatment increased the mean neurite length even in the presence of L, R, or U (L versus L + N, 23.6 ± 3.6 versus 44.3 ± 3.0 µm, p < 0.001; R versus R + N, 38.4 ± 2.3 versus 64.1 ± 4.7 µm, p < 0.001; U versus U + N, 40.2 ± 3.6 versus 65.1 ± 1.6 µm, p < 0.001; G versus N, 34.6 ± 1.3 versus 69.6 ± 4.8 µm, p < 0.001; Fig. 5A, panel c). Furthermore, a calmodulin kinase II inhibitor, KN62 (10 µM), did not prevent the NGCect-mediated neurite outgrowth either (data not shown).

Next, we tried a specific PKC inhibitor, bisindolylmaleimide I (B). Treatment with bisindolylmaleimide I inhibited the NGCect-mediated neurite outgrowth in a dose-dependent manner (the percentage of neurons with long neurites; 57.9 ± 5.1, 38.3 ± 1.3, 18.3 ± 3.7, and 7.2 ± 2.3% in 0, 1, 5, and 10 µM B + N, respectively, p < 0.001; the mean neurite length; 57.8 ± 2.0 µm, 40.9 ± 1.4 µm, 29.3 ± 2.6 µm, and 22.1 ± 1.8 µm, in 0, 1, 5, and 10 µM B + N, respectively, p < 0.001; Fig. 5B, panels a, b, and c). Another specific PKC inhibitor calphostin C (Cal; 100 nM) also blocked the NGCect-mediated neurite outgrowth (the percentage of neurons with long neurites; Cal + N, 3.0 ± 3.5%, p < 0.001 compared with the sample treated with NGCect alone: the mean neurite length; Cal + N, 13.8 ± 2.5 µm, p < 0.001 compared with the sample treated with NGCect alone; Fig. 5B, panels b and c). We also tested the involvement of PI3K in NGCect-mediated neurite outgrowth using a PI3K-specific inhibitor, LY294002 (LY). Treatment with LY294002 also inhibited NGCect-mediated neurite outgrowth dose-dependently (the percentage of neurons with long neurites; 20 µM LY + N, 18.8 ± 3.9%, and 100 µM LY + N, 2.1 ± 1.3%, p < 0.001 compared with the sample treated with NGCect alone: the mean neurite length; 20 µM LY + N, 27.0 ± 1.3 µm, and 100 µM LY + N, 13.9 ± 0.6 µm, p < 0.001 compared with the sample treated with NGCect alone; Fig. 5B, panels a, b, and c). These results suggest that both PKC and PI3K pathways are involved in neurite outgrowth mediated by NGCect.


Figure 5
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  		FIGURE 5.
PKC and PI3K inhibitors block NGCect-mediated neurite outgrowth. Neocortical neurons were cultured in the presence of various kinds of protein kinase inhibitor with (+) or without (-)50 µg/ml NGCect (N) for 24 h. A, effect of pretreatment with various protein kinase inhibitors. B, effect of pretreatment with PKC and PI3K inhibitors. Panel a, phase contrast photographs represented typical results. Panel b, percentage of neurons with long neurites. Panel c, mean neurite length. G, GST; N, NGCect; S, staurosporine; S + N, staurosporine + NGCect; R, Rp-cAMPS; R + N, Rp-cAMPS + NGCect; L, lavendustin A; H, hypericin; U, U0126; B10, bisindolylmaleimide I 10 µM; B10 + N, bisindolylmaleimide I 10 µM + NGCect; LY100, LY294002 100 µM; LY100 + N, LY294002 100 µM + NGCect; B, bisindolylmaleimide I; LY, LY294002; Cal, calphostin C. Scale bar, 50 µm. In A, panels b and c, ***, p < 0.001; n.s., not significant. In B, panels b and c, ***, p < 0.001 versus sample treated with NGCect alone.

 


Figure 6
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  		FIGURE 6.
Polypeptides containing the acidic and/or EGF-like domains promote neurite outgrowth. A, neocortical neurons were cultured for 24 h in the presence of each recombinant GST-fused polypeptide (GST, NGCect, NGCE, NGCCS, NGCAA, or NGCAE) represented in Fig. 1A. Scale bar, 50 µm. B, quantification of neurite outgrowth-promoting activity of these polypeptides. Panel a, Percentage of cells with long neurites. Panel b, mean neurite length. G, GST; ect, NGCect; E, NGCE; CS, NGCCS; AA, NGCAA; AE, NGCAE. Data represent mean ± S.D. (n = 4). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

 
The Active Site of NGCect for Neurite Outgrowth Exists in the Region Containing the Acidic and EGF-like Domains—To examine which region of NGCect has promotive activity for neurite elongation, we added separately the five GST-fused polypeptides (NGCect, NGCCS, NGCAA, NGCE, and NGCAE) shown in Fig. 1A as well as GST to culture medium of neocortical neurons at an equal molar concentration; 50 µg/ml for NGCect, 38 µg/ml for NGCCS, 26 µg/ml for NGCAA, 27 µg/ml for NGCE, 33 µg/ml for NGCAE, and 20 µg/ml for GST. As shown in Fig. 6A, the addition of NGCAA, NGCE, or NGCAE apparently promoted neurite elongation 24 h after plating as did the addition of NGCect, but the addition of NGCCS or GST did not. The percentages of neurons with neurites extending three somal diameters or longer were 75.6 ± 7.1, 72.4 ± 12.7, 50.5 ± 9.8, and 62.6 ± 9.4 in NGCect-, NGCE-, NGCAA-, and NGCAE-treated cultures, respectively. These values were significantly higher than those in GST- and NGCCS-treated cultures (GST, 19.4 ± 11.1%; NGCCS, 22.9 ± 5.0%; Fig. 6B, panel a). The mean lengths of neurites were 54.7 ± 3.6, 50.5 ± 5.7, 41.9 ± 5.1, and 48.7 ± 6.4 µm in NGCect-, NGCE-, NGCAA-, and NGCAE- treated cultures, respectively. These were significantly longer than those in GST- and NGCCS-treated cultures (GST, 26.3 ± 4.6 µm; NGCCS, 28.6 ± 2.4 µm; Fig. 6B, panel b). These findings indicate that both NGCAA and NGCE have a promotive effect on the outgrowth of neurites from neocortical neurons.

According to our careful observation, neurons in the presence of NGCE appeared to extend one neurite, whereas those in NGCAA-treated cultures developed several neurites. Then, we counted the number of neurites elongating from a soma in each condition. As shown in Fig. 7, neurons with one long neurite dominated in the presence of NGCE, whereas many of the neurons treated with NGCAA had several neurites. Neurons treated with NGCect or NGCAE tended to develop an intermediate number of neurites. These results suggest that NGCE and NGCAA have somewhat different functions in neurite outgrowth.


Figure 7
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  		FIGURE 7.
NGCE preferably promotes elongation of one neurite, whereas NGCAA promotes elongation of several neurites. The number of neurites extending from a soma (primary processes) of each neuron was counted and represented in the histogram in each culture condition. Only neurons with neurites extending three somal diameters or longer were counted. The polypeptide added to the culture is shown in the upper right of each panel. #; number.

 
NGCE, but Not NGCAA, Promotes Neurite Outgrowth in GABAergic Neurons—It is generally accepted from anatomical studies using Golgi or immunohistochemical techniques that the majority (about 80%) of layer IV neurons in the mouse or rat barrel cortex are spiny (presumably excitatory) neurons, whereas about 20% are sparsely spiny or aspiny (presumably inhibitory) neurons (21-23). Therefore, the data shown in Figs. 2 and 6 indicate that both NGCE and NGCAA promote the elongation of neurites from excitatory neurons. It is of interest to examine whether they also promote the elongation of neurites from inhibitory neurons. To examine this, we performed double immunofluorescence labeling of cultured neurons treated with each fusion protein using antibodies against MAP2 (a neuronal marker) and GABA (a main inhibitory neurotransmitter in neocortex). GABA-positive neurons accounted for 10-15% of all MAP2-positive neurons in our culture at 1 day of cultivation (14). The addition of NGCect or NGCE increased the number of GABA-positive neurons with long neurites, while the addition of GST, NGCCS, or NGCAA did not (Fig. 8A). The percentages of GABA-positive neurons with neurites extending three somal diameters or longer were 24.6 ± 3.7 and 25.4 ± 1.8% in the presence of NGCect and NGCE, respectively. These values were significantly higher than those in the presence of GST, NGCCS, and NGCAA (GST, 8.9 ± 2.0%; NGCCS, 6.5 ± 1.9%; and NGCAA, 11.9 ± 1.9%; p < 0.001; Fig. 8B, panel a). The mean lengths of neurites were 35.7 ± 1.6 and 43.7 ± 4.8 µmin the presence of NGCect and NGCE, respectively, which were significantly longer than those in the presence of GST, NGCcs, and NGCAA (GST, 22.2 ± 2.2 µm; NGCCS, 21.2 ± 0.8 µm; and NGCAA, 23.7 ± 3.0 µm; p < 0.001; Fig. 8B, panel b). Thus, NGC promotes neurite outgrowth in about 25% of GABAergic neurons, and this percentage is small compared with the overall proportion of neurons extending neurites (about 60-70%; see Fig. 2). Because neocortical GABAergic neurons consist of many kinds of interneurons (24), NGC seems to affect a particular subclass of GABAergic neurons. NGCE, but not NGCAA, promotes neurite outgrowth in GABAergic neurons, suggesting again that these two domains of the NGC core protein exert neurite-promoting activity via distinct mechanisms.

NGC-mediated Neurite Outgrowth Appears to Be EGF-independent—NGC has an EGF-like motif (5) and EGF-like activity for the proliferation of breast cancer cell lines (25). Thus, there is a possibility that NGC induces neurite outgrowth via its EGF activity. Then, we examined whether or not the activity of NGCE is dose-responsive. Fig. 9 shows that NGCE promotes the elongation of neurites in cultured neocortical neurons in a dose-dependent manner, with an EC50 of around 1-5 µg/ml. It is generally considered that most growth factors such as FGFs and EGFs have a proliferating effect on various cells including neural progenitor cells at a dose of 10-20 ng/ml. If the neurite outgrowth-promoting activity of NGCE is mediated via EGF receptors (ErbB family molecules), NGC would promote neuritogenesis at a dose similar to that promoting cell proliferation. However, the fact that a conspicuously high concentration of NGCE (1-5 µg/ml) was required to promote neurite outgrowth suggests that this effect of NGCE is independent of its EGF activity. Actually, we examined the neurite outgrowth-promoting activity of some EGF family proteins such as neuregulin, HB-EGF, and EGF (all purchased from Sigma), and found that none of these factors promote neurite outgrowth at a dose of either 4 µg/ml or 20 ng/ml (data not shown). In addition, the putative NGC receptor has been reported to be ErbB3 (25), but the expression of ErbB3 mRNA in the E16 rat brain is restricted to around the third ventricle and is not detected in neurons of the neocortex (26). Our result that an EGF receptor tyrosine kinase inhibitor, lavendustin A, did not block NGC-mediated neurite outgrowth (Fig. 5A) also supports this idea. Thus, it is unlikely that NGCE mediates neurite outgrowth via the EGF receptor pathway.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Active Sites of NGCect for Promotion of Neurite Outgrowth—The work presented here directly demonstrated that a recombinant ectodomain of the NGC core protein promotes neurite outgrowth from rat neocortical neurons in culture. Its active sites existed within the EGF-domain and AA-domain (Fig. 6). These two domains are likely to function via different mechanisms, because addition of the EGF-domain caused one neurite to elongate from individual neurons and promoted neurite outgrowth in GABAergic as well as excitatory neurons, while addition of the AA-domain caused several neurites to develop and did not promote GABAergic neurites (Figs. 7 and 8). Actually, NGC/CALEB binds to the fibrinogen-like domain of tenascin-C or -R (members of the tenascin family of extracellular matrix proteins) via the AA-domain (27). Tenascin-C promotes neurite outgrowth through neuronal {alpha}7beta1 integrin (28) or via activation of the cell adhesion molecule F3/contactin (29). It is possible that the AA-domain of NGC promotes neurite outgrowth in cooperation with other extracellular matrix macromolecules such as tenascins. The EGF-domain of NGC also had strong neurite outgrowth-promoting activity. NGC has EGF-like activity and its putative receptor is ErbB3 (25). However, the neurite outgrowth-promoting activity of NGC seems to be independent of the EGF activity, since none of the EGF family members promote neurite outgrowth and an EGF receptor tyrosine kinase inhibitor did not block the NGC-mediated neurite outgrowth (see "Results"). Thus, NGC would bind receptor(s) other than ErbB3 on neocortical neurons for neurite outgrowth. It is necessary to identify binding partners of NGCect on neuronal cells to fully understand the mechanism of NGC-mediated neurite outgrowth.


Figure 8
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  		FIGURE 8.
NGCE, but not NGCAA, promotes neurite outgrowth in GABA-positive neurons. A, neocortical neurons were cultured for 24 h in the presence of each recombinant polypeptide (GST, NGCect, NGCE, NGCCS, and NGCAA) represented in Fig. 1A, and then immunostained with antibodies against MAP2 (a neuronal marker) and GABA. Scale bar, 50 µm. B, quantification of neurite outgrowth-promoting activity of these polypeptides in GABA-positive neurons. Panel a, percentage of GABA-positive neurons with long neurites. Note that NGCE, not NGCAA, promotes the elongation of neurites from only a small proportion (about 25%) of GABA-positive neurons. Panel b, mean neurite length of GABA-positive neurons. G, GST; ect, NGCect; E, NGCE; CS, NGCCS; AA, NGCAA. Data represent the mean ± S.D. (n = 4). ***, p < 0.001.

 


Figure 9
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  		FIGURE 9.
Dose dependence of the neurite outgrowth-promoting activity of NGCE. Neurons were cultured in the presence of NGCE at various concentrations ranging from 0 to 27 µg/ml. Percentage of cells with long neurites (A) and mean neurite length (B) was plotted. Data represent the mean ± S.D. (n = 4).

 
Involvement of PKC and PI3K Pathways in NGC-mediated Neurite Outgrowth—We found that NGCect-mediated neurite outgrowth was dependent on PKC and PI3K pathways. PKC phosphorylates serine and threonine residues of target proteins in many organs including the CNS, and is involved in various biological processes such as development, differentiation, proliferation, carcinogenesis, and memory (30-32). The PKC family can be divided into three groups; a group of conventional (c) PKC isoforms ({alpha}, beta, {gamma}), a group of novel (n) PKC isoforms ({delta}, {epsilon}, {eta}, {theta}, µ), and a group of atypical (a) PKC isoforms ({zeta}, {iota}, {kappa}) (30-32). cPKCs and nPKCs can be inhibited by bisindolylmaleimide I, with an IC50 value within 10-100 nM. The fact that a higher dose (5-10 µM) of bisindolylmaleimide I was required for the inhibition of NGC-induced neurite outgrowth suggests the involvement of aPKCs rather than cPKCs and nPKCs. PKC{zeta}, a member of the aPKC subfamily, is regulated by PI3K, which produces PIP3 from PI-4, 5-bisphosphate in response to various growth factors (33, 34). Shi et al. (35) have reported that hippocampal neuronal polarity is controlled by the polarized distribution of mPar3 (mammalian homolog of a Caenorhabditis elegans polarity protein Par3)/mPar6/aPKC protein complex and the PI3K signaling pathway. The accumulation of PIP3 mediated by PI3K is reported to be important for neuronal polarization and the formation of axons (36). Our preliminary immunocytochemical study demonstrated the existence of PKC{zeta} in neurites extending from NGCect-treated neurons. Therefore, we tried to examine the involvement of PKC{zeta} using a cell-permeable myristoylated PKC{zeta}-specific pseudosubstrate peptide inhibitor in NGC-induced neurite outgrowth, but the pretreatment with this myristoylated drug was toxic to neurons under our culture conditions. NGCect may activate the PI3K-aPKC pathway to promote the extension of neurites in a way similar to that for neuronal polarity.

Core Protein and Chondroitin Sulfate (CS) Chain of NGC—There is increasing evidence that chondroitin sulfate proteoglycans (CSPGs) are the major axon growth inhibitory components of the glial scar that blocks successful regeneration (37-41). Because removing CS chains with CS-degrading enzymes promoted regeneration of corticospinal axons, this axonal inhibitory effect of CSPGs is believed to be caused by their CS chains in many cases (40, 41). Then, what roles do core proteins of CSPGs play in the development and regeneration of the CNS? Core proteins appear to have an activity different from that of the GAG side chains even in terms of neuritogenesis. For example, both versican V1 and V2 isoforms bear CS chains, but only V1 isoform promotes neurite outgrowth from hippocampal neurons (42), suggesting that the activity is associated with a part of the V1 core protein. Similarly, phosphacan short isoform, a non-proteoglycan variant of phosphacan, also promotes neurite outgrowth from cortical neurons (43). On the other hand, the inhibitory activity of phosphacan for neurite extension from the dorsal root ganglion explants is associated with its core protein, not with the CS side chains (44). The core protein of NG2, a large transmembrane CSPG, also inhibits neurite outgrowth from neonatal cerebellar granule cells in culture (45). Our present work showed that the acidic and EGF-like domains of the NGC core protein have neurite outgrowth-promoting activity. Thus, it is likely that core proteins of CSPGs exert their own effects, apart from their CS side chains, on neuritogenesis in the developing CNS.

A Soluble Form of NGC—A soluble form of NGC, or the NGC ectodomain, can be detected not only in the developing brain (5) but also in cultures of neurons (13). In addition, neuronal depolarization of chick retinal cells in culture facilitates the proteolytic conversion of CALEB, a chick homolog of NGC, into a truncated transmembrane form (11). Because NGC is concentrated in budding neurites or dendritic filopodia (8, 10), that are considered to be largely differentiated into postsynapses, the NGC ectodomain shed from the filopodia would act on the tip of a growing axon to promote its extension toward the filopodia. When the axon tip meets a dendritic filopodium, it is differentiated into the presynapse to form a complete synapse with a dendritic filopodium. Versican is known to act on the tip of a growing axon to promote enlargement of presynaptic varicosities in retinal axons (46). Taken together, both a truncated transmembrane form of NGC on a postsynaptic site and a soluble form on a presynaptic site may be involved in synapse formation in the CNS. The observation that synapse function during postnatal development was impaired in CALEB-deficient mice (11) appears to support the above-mentioned function of NGC in synaptogenesis.


   FOOTNOTES
 
* This work was supported in part by grants-in-aid for Scientific Research from the Ministry of Education, Science, Culture and Sports of Japan, and from the Japan Society for the Promotion of Science. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Both authors contributed equally to this work. Back

2 To whom correspondence should be addressed: Dept. of Perinatology, Inst. for Developmental Research, Aichi Human Service Center, Kasugai, Aichi 480-0392, Japan. Tel.: 81-568-88-0811; Fax: 81-568-88-0829; E-mail: nakanishi{at}inst-hsc.jp.

3 The abbreviations used are: PGs, proteoglycans; CALEB, chicken acidic leucine-rich EGF-like domain-containing brain protein; CNS, central nervous system; CS, chondroitin sulfate; CSPG, chondroitin sulfate proteoglycan; EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; FGF, fibroblast growth factor; GABA, {gamma}-aminobutyric acid; GAG, glycosaminoglycan; GST, glutathione S-transferase; MAP2, microtubule-associated protein 2; MAPK, mitogen-activated protein kinase; NGC, neuroglycan C; PBS, phosphate-buffered saline; PI3K, phosphatidylinositol 3-kinase; PIP3 phosphatidylinositol 3,4,5-triphosphate; PKC, protein kinase C; AA, acidic amino acid. Back



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 DISCUSSION
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