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2 Department of Anatomy, Faculty of Medicine, Mie University, Tsu 514-8507, Japan
3 Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/Faculty of Medicine, Suita 565-0871, Japan
Address correspondence to Yoshimi Takai at the Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/Faculty of Medicine, Suita 565-0871, Osaka, Japan. Tel.: 81-66-879-3410. Fax: 81-66-879-3419. E-mail: ytakai{at}molbio.med.osaka-u.ac.jp
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Abstract |
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Key Words: nectin; afadin; cadherin; puncta adherentia junctions; synaptic junctions
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Introduction |
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Similarity in molecular mechanisms between junctions of synapses and epithelial cells has recently been substantiated by the findings that classical cadherins, neural (N)*- and epithelial (E)-cadherins, localize at both synaptic and puncta adherentia junctions in at least some brain regions (Yamagata et al., 1995; Fannon and Colman, 1996), and that N- and ß-catenins, cadherin-associated proteins, also localize at puncta adherentia junctions (Uchida et al., 1996). Furthermore, a novel type of cadherin, cadherin-related neuronal receptor, has been found to localize at synaptic junctions (Kohmura et al., 1998). These findings suggest that both synaptic and puncta adherentia junctions of synapses are cadherin-based cellcell junctions just as are typical adherens junctions in epithelial cells.
We have recently found a novel cellcell adhesion system at cadherin-based adherens junctions, consisting of at least two components, nectin and afadin (Mandai et al., 1997; Takahashi et al., 1999). Nectin is a Ca2+-independent cellcell adhesion molecule that belongs to the immunoglobulin superfamily (Aoki et al., 1997; Lopez et al., 1998; Takahashi et al., 1999; Miyahara et al., 2000; Satoh-Horikawa et al., 2000; Reymond et al., 2001; Sakisaka et al., 2001). Nectin comprises a family consisting of at least four members, nectin-1, -2, -3, and -4. Nectin-1 is identical to the poliovirus receptorrelated protein and has recently been shown to serve as the -herpes virus entry and cellcell spread mediator (Geraghty et al., 1998; Warner et al., 1998; Cocchi et al., 2000; Sakisaka et al., 2001). Each member of the nectin family forms a homo-cis-dimer, followed by formation of a homo-trans-dimer, causing cellcell adhesion (Lopez et al., 1998; Miyahara et al., 2000; Satoh-Horikawa et al., 2000; Reymond et al., 2001; Sakisaka et al., 2001). Nectin-3 furthermore forms a hetero-trans-dimer with either nectin-1 or -2, and the formation of each hetero-trans-dimer is stronger than that of each homo-trans-dimer (Satoh-Horikawa et al., 2000). Nectin-4 also forms a hetero-trans-dimer with nectin-1, and this formation is also stronger than that of the homo-trans-dimer (Reymond et al., 2001). Each member of the nectin family except nectin-4 has two or three splicing variants (Morrison and Racaniello, 1992; Eberlé et al., 1995; Lopez et al., 1995; Cocchi et al., 1998; Satoh-Horikawa et al., 2000). Most members have a COOH-terminal conserved motif of four amino acid residues (E/A-X-Y-V) that interacts with the PDZ domain of afadin (Takahashi et al., 1999; Satoh-Horikawa et al., 2000). Nectin-4 does not have the conserved motif, but interacts with the PDZ domain of afadin through its COOH terminus (Reymond et al., 2001). Afadin has at least two splicing variants, l- and s-afadin. l-Afadin, a larger splicing variant that connects nectin to the actin cytoskeleton, is an actin filamentbinding protein with one PDZ domain and three proline-rich domains. (Mandai et al., 1997; Takahashi et al., 1999). s-Afadin, a smaller splicing variant, has one PDZ domain, but lacks the actin filamentbinding domain and the third proline-rich domain (Mandai et al., 1997). Human s-afadin is identical to the AF6 protein, the gene of which is originally found to be fused to the ALL-1 gene in acute leukemia cases (Prasad et al., 1993).
The nectin-based cellcell adhesion has a potency to recruit the E-cadherincatenin complex through l-afadin, and is cooperatively involved with the cadherincatenin system during the formation of adherens junctions (Takahashi et al., 1999; Miyahara et al., 2000; Tachibana et al., 2000). In epithelial cells of afadin-/- mice and afadin-/- embryoid bodies, the proper organization of adherens junctions is severely impaired (Ikeda et al., 1999). In spermatozoa of nectin-2-/- mice, the nuclear and cytoskeletal morphology and mitochondrial localization are impaired (Bouchard et al., 2000). Thus, accumulating evidence demonstrates the important role of the nectinafadin system in the organization of adherens junctions.
s-Afadin/AF6 associates with the Eph B receptor tyrosine kinases, and has been shown to localize at postsynaptic densities of synaptic junctions in the CA1 area of adult rat hippocampus (Hock et al., 1998; Buchert et al., 1999). We have found that l-afadin colocalizes with N-catenin symmetrically at puncta adherentia junctions of the synapses between the mossy fiber terminals and dendrites of pyramidal cells in the CA3 area of adult mouse hippocampus (Nishioka et al., 2000). More recently, nectin-1 has been determined, by positional cloning, to be responsible for cleft lip/palateectodermal dysplasia, which is characterized by cleft lip/palate, syndactyly, mental retardation, and ectodermal dysplasia (skin, hairs, nails, teeth, and sweat glands) (Suzuki et al., 2000). Accumulating evidence suggests that the nectinafadin system, as well as the cadherincatenin system, plays an important role in the formation and remodeling of synaptic and puncta adherentia junctions. We have examined here whether the nectinafadin system is involved in the formation of synapses.
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Results |
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Immunoelectron microscopic analysis revealed that 90% of the immunogold particles detected with the antinectin-1 and antinectin-3 Abs that were raised against the cytoplasmic domains of nectin-1
and -3
, respectively, were concentrated in an asymmetrical manner on the plasma membranes of the mossy fiber terminals (presynaptic side) and dendrites (postsynaptic side) of pyramidal cells, respectively, at puncta adherentia junctions (Fig. 2, A and B). In contrast, the immunogold particles detected by the antil-afadin Ab were concentrated on both sides of the junctions (Fig. 2 C). It is therefore likely that at puncta adherentia junctions of these synapses, nectin-1 and -3 form a hetero-trans-dimer that is associated with l-afadin on both sides of the junctions. No signal for nectin-1, -3, or l-afadin was detected at synaptic junctions located on the spines of the dendrites (unpublished data).
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Localization of nectin-1 and -3 at developing synapses in cultured hippocampal neurons and impairment of formation of synapses by inhibition of the nectin-based adhesion
To obtain the evidence that the nectinafadin system is indeed involved in the formation of synapses, we added an inhibitor of nectin-1, glycoprotein D (gD), and examined the formation of synapses microscopically. gD is an envelope protein of herpes simplex virus type 1 that binds to nectin-1 and partially inhibits not only the formation of a homo-trans-dimer of nectin-1 but also the formation of a hetero-trans-dimer between nectin-1 and -3 (Sakisaka et al., 2001; unpublished data). We analyzed by immunofluorescence microscopy the localization of nectin-1 and -3, with synaptophysin as a synaptic vesicle marker protein (Navone et al., 1986) and PSD-95 as a postsynaptic marker protein (Cho et al., 1992) in primary cultured rat hippocampal neurons. After 10 d in culture when the number of cellcell contact sites increased, the nectin-1 signal localized at puncta and 70% of these puncta were immunoreactive for synaptophysin (Fig. 5 A). Nectin-3, furthermore, colocalized at two thirds of the puncta immunoreactive for both nectin-1 and synaptophysin. The puncta immunoreactive for nectin-1, synaptophysin, and nectin-3 likely correspond to synapses (Fig. 5 A, arrowheads in upper right insets). Consistently, most of the nectin-3 signals colocalized with PSD-95 (unpublished data). The puncta, which were immunoreactive for nectin-1 and synaptophysin but not for nectin-3, likely correspond to vesicle-filled axonal varicosities (Fig. 5 A, arrows in lower left insets), but it is not known whether these puncta correspond to cellcell contact sites. Addition of gD resulted in
16% decrease in size and concomitant 109% increase in number of the puncta immunoreactive for nectin-1, -3, and synaptophysin (Fig. 6 A). Similar results were obtained with the puncta immunoreactive for PSD-95 (unpublished data). After 16 d in culture,
81% of the nectin-1positive puncta were immunoreactive for synaptophysin (Fig. 5 B). Nectin-3 colocalized at
74% of the puncta immunoreactive for both nectin-1 and synaptophysin. It is likely that nectin-1 and synaptophysin localize at the vesicle-filled presynaptic side and nectin-3 localizes at the postsynaptic side. Addition of gD resulted in
35% decrease in size and concomitant 149% increase in number of the puncta immunoreactive for nectin-1, -3, and synaptophysin (Fig. 6 B). These effects of gD were dose and time dependent. Similar results were obtained with the puncta immunoreactive for PSD-95 (unpublished data). Boiled gD (100°C for 10 min) had no significant effect on the formation of synapses at any concentrations tested in primary cultured hippocampal neurons (unpublished data). The effects of gD on the formation of synapses were not due to its cell toxicity, because the neurons treated with gD showed vesicle recycling as active as the control neurons, as estimated by measuring vesicle recycling in response to high K+ stimulation using a fluorescent dye (Fig. 7, AD). These results indicate that gD induces a decrease in size and a concomitant increase in number of synapses.
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Discussion |
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Our findings also shed new insight into the relationship between puncta adherentia and synaptic junctions. Although the relationship between the two types of junctions in general is still controversial, their structural relationship at the synapses between the mossy fiber terminals and dendrites of pyramidal cells in the CA3 area has extensively been studied by Amaral and Dent (1981) (Fig. 8). They have found that this type of synapse is so highly developed that puncta adherentia and synaptic junctions must have their own functions. Six puncta adherentia and eight synaptic junctions on average are present in a single nerve terminal. The former junctions locate almost exclusively at cellcell contact sites between the mossy fiber terminals and dendritic trunks of pyramidal cells, and the latter junctions are exclusively at the contact sites between synaptic vesiclecontaining active zones of the mossy fiber terminals and the heads of highly lobulated dendritic spines. In the mossy fiber terminals, the cytoplasmic face of puncta adherentia junctions are not covered with synaptic vesicles but with a row of mitochondria. Therefore, synaptic junctions are regarded as neurotransmitter release sites and puncta adherentia junctions as mechanical anchoring sites, and transformation from one type of junction to the other is unlikely, at least in this highly developed type of synapse. Our morphological results indicate that nectin-1 and -3 are specific for puncta adherentia junctions, at least between the mossy fiber terminals and dendrites of pyramidal cells in the CA3 area of adult mouse hippocampus.
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This neural membrane domain specialization is apparently similar to that found in the formation of the junctional complex, which mainly consists of adherens and tight junctions in epithelial cells, with respect to the order of junctional protein localization patterns. During the initial stage of the formation junctional complex in epithelial cells, primordial spot-like junctions are first formed at the tips of cellular protrusions radiating from adjacent cells (Yonemura et al., 1995; Adams et al., 1998; Ando-Akatsuka et al., 1999; Vasioukhin et al., 2000). The cadherincatenin and nectinafadin systems colocalize at the primordial junctions where claudin is not concentrated (Asakura et al., 1999; unpublished data). Claudin is a key cellcell adhesion molecule that forms tight junctions (Tsukita et al., 1999). As cellular polarization proceeds, claudin gradually accumulates at the spot-like junctions to form tight junctions, and the cadherincatenin and nectinafadin systems are sorted out from claudin to form adherens junctions.
A role of the nectinafadin system in formation of synapses
We have provided here the evidence that the nectinafadin system plays an important role in the formation of synapses in cultured rat hippocampal neurons by the use of gD, an inhibitor of nectin-1 (Sakisaka et al., 2001). We have found that gD induces a decrease in synapse size and a concomitant increase in synapse number. The exact mechanism of the decrease in synapse size remains to be clarified, but nectin-1, -3, l-afadin, N-catenin, and N-cadherin colocalize at immature synapses during development, indicating the presence of clusters of the nectinafadin and cadherincatenin systems at the contact sites. We have previously shown that the nectinafadin system organizes adherens junctions cooperatively with the cadherincatenin system in nonneuronal cells (Ikeda et al., 1999; Takahashi et al., 1999; Miyahara et al., 2000; Tachibana et al., 2000). Therefore, the decrease in synapse size may be due to the partial inhibition of the hetero-trans-dimer between nectin-1 and -3, which may affect the cadherin-mediated cellcell adhesion and result in smaller synapses. The exact mechanism of the increase in synapse number in the presence of gD is also unknown, but may be due to either failure of determination of the proper positions of synapses or compensation for functionally less competent smaller synapses. Our present results indicate that the formation of the hetero-trans-dimer between nectin-1 and -3 plays an important role in determining the position and size of synapses.
It has been reported that inhibition of the N-cadherin function by its Abs results in the formation of synapses at the regions beyond the appropriate target cells in the chick retinotectal tract, suggesting that N-cadherin plays a role in the formation of synapses (Yamagata et al., 1995). Addition of antiN- and antiE-cadherin Abs and cadherin peptides has been shown to induce suppression of long-term potentiation in mouse hippocampus (Tang et al., 1998). Moreover, in mutant mice lacking cadherin-11, long-term potentiation in the CA1 area of mouse hippocampus is enhanced (Manabe et al., 2000). Our present results, together with the earlier observations, suggest that the nectinafadin system, as well as the cadherincatenin system, plays an important role in the formation of synapses. This role of the nectinafadin system is consistent with the recent finding that nectin-1 is a gene responsible for cleft lip/palateectodermal dysplasia, which is characterized by mental retardation in addition to ectodermal dysplasia (Suzuki et al., 2000).
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Materials and methods |
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Immunofluorescence microscopy
Adult mice (ddy strain) were deeply anesthetized by ether, and perfused with freshly prepared 2% paraformaldehyde in PBS for 15 min. Brains were dissected out and cut with a razor into several coronal sections (2-mm thickness), which were soaked with the same fixative at 4°C for 2 h. For cryoprotection, sections were placed into 20% sucrose solution for 2 h and 25% sucrose solution overnight. The sections were frozen using liquid nitrogen. Serial 10-µm-thick sections were cut in a cryostat. The samples were incubated with the primary Abs described above, followed by incubation with a secondary Ab (Amersham-Pharmacia Biotech) coupled with fluorescein, Texas red, or Cy5. After being washed with PBS, they were embedded and viewed with a confocal imaging system (BioRad Laboratories; MRC-1024).
Immunoelectron microscopy
Immunoelectron microscopy, using the silver-enhanced immunogold method, was performed as previously described (Mizoguchi et al., 1994). The 10-µm-thick sections were incubated with the primary Abs described above, followed by incubation with a secondary Ab coupled with 1.4-nm gold particles (Nanoprobes Inc.). The sample-bound gold particles were silver-enhanced by the HQ-silver kit (Nanoprobes Inc.) at 18°C for 12 min. The samples were again washed and postfixed with 0.5% osmium oxide in a buffer containing 100 mM cacodylate buffer, pH 7.3. They were dehydrated by passage through a graded series of ethanol (50, 70, 90, and 100%) and propylene oxide, and embedded in epoxy resin. From this sample, ultrathin sections were cut, stained with uranyl acetate and lead citrate, and then observed with an electron microscope (JEM-1200EX; JEOL).
Preparation of gD
gD, an envelop protein of herpes simplex virus type 1, an -herpes virus, was prepared as previously described (Sakisaka et al., 2001). In brief, a baculovirus transfer vector for gD (285t) (amino acids 1285) (Rux et al., 1998) was constructed as follows: pFastBac1-Msp-Fc was first constructed by subcloning the inserts encoding the honeybee melittin signal peptide (Tessier et al., 1991) and the human IgG Fc into pFastBac1 (GIBCO BRL). A cDNA fragment of gD (285t) was then inserted into pFastBac1-Msp-Fc to express the chimeric protein fused with the NH2-terminal signal peptide and the COOH-terminal IgG Fc. A baculovirus bearing this cDNA was prepared according to the manufacturer's protocol. High Five insect cells (Invitrogen) were grown in serum-free medium EX-CELL 400 (JRH Biosciences), infected with the baculovirus, and cultured at 26°C for 72 h. The culture supernatant was collected and applied to a protein ASepharose column (Amersham Pharmacia Biotech.), and then eluted with 20 mM glycine buffer, pH 2.5. The eluted protein was immediately neutralized with 1 M Tris and dialyzed against PBS. The chimeric protein of gD (285t) was used as gD in this paper.
Primary culture of rat hippocampal neurons
Rat hippocampal neurons were cultured as previously described (Takeuchi et al., 1997). In brief, hippocampi were isolated from rat embryos (20-d gestation), dissociated, plated on poly-L-lysinecoated glass coverslips, and cultured in MEM with 10% horse serum. After 4 d in culture, the medium was replaced with MEM supplemented with N2 supplement, 1 mg/ml of ovalbumin, 1 mM pyruvate, and 5 mM cytosine arabinoside in the presence of various concentrations (0.06, 0.6, 3.0, 8.75, 35, and 70 µg/ml) of intact gD or gD boiled at 100°C for 10 min. After 10 d in culture, the neurons were fixed with 2% paraformaldehyde in PBS for 4 h. Alternatively, the medium was again replaced with a fresh medium in the presence of the same concentration of gD or boiled gD as added after 4 d in culture. The neurons were cultured for another 6 d and fixed with 2% paraformaldehyde in PBS for 4 h. The neurons were subjected to immunofluorescence microscopic analysis with the rabbit polyclonal antinectin-1, rat monoclonal antinectin-3, mouse monoclonal anti-synaptophysin, and mouse monoclonal antiPSD-95 Abs. By the confocal imaging system, puncta immunoreactive for nectin-1, -3, and synaptophysin were counted as synapses, and their sizes were measured. At least 1,000 puncta per culture were measured. Puncta immunoreactive for PSD-95 were also counted and their sizes were measured. This quantitative analysis was performed using the public domain NIH image program v1.61.
Labeling with FM1-43
To determine the viability of cultured rat hippocampal neurons, the neurons were loaded with FM1-43 (Molecular Probes) by the addition of 50 mM KCl as previously described (Becherer et al., 2000; Schikorski and Stevens, 2001). In brief, after 16 d in culture, the neurons were incubated with the medium containing 50 mM KCl and 20 µM FM1-43 for 90 s. After being washed with HBSS, the neurons were incubated with HBSS at 37°C for 15 min. They were fixed with 4% paraformaldehyde in PBS at 37°C for 15 min. After being washed with PBS, the neurons were examined with the confocal imaging system.
Other procedures
Protein concentrations were determined with BSA as a reference protein (Bradford, 1976). SDS-PAGE was performed as previously described (Laemmli, 1970).
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Footnotes |
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* Abbreviations used in this paper: Ab, antibody; CSM, crude synaptic membrane fraction; CSV, crude synaptic vesicle fraction; E-cadherin, epithelial cadherin; gD, glycoprotein D; Ho, the homogenate fraction; N-cadherin, neural cadherin; P1, nuclear pellet fraction; P2, crude synaptosome fraction; P3, microsome fraction; P4, -7, and -14, postnatal day 4, 7, and 14; P2A, myelin fraction; P2B, ER and Golgi complex fraction; P2C, synaptosome fraction; P2D, mitochondria fraction; PSD, postsynaptic density fraction; S, soluble cytosol fraction; SM1SM3, synaptic membrane fraction; SM4, intrasynaptosomal mitochondria fraction; SS, synaptic soluble fraction.
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Acknowledgments |
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The work at Kyoto and Mie Universities was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology and from the Ministry of Health and Welfare, Japan (2000, 2001). The work at Osaka University was supported by Grants-in-Aid for Scientific Research and for Cancer Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (2000, 2001).
Submitted: 30 April 2001
Revised: 2 January 2002
Accepted: 2 January 2002
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