Abbreviations used : GST, glutathione S-transferase ; PKC-γ, protein kinase C-γ.
Address correspondence and reprint requests to Dr. G. Kuwajima at Shionogi Institute for Medical Science, 2-5-1 Mishima, Settsu-shi, Osaka 566-0022, Japan.
Abstract : N-Copine is a novel protein with two C2 domains. Its expression is brain specific and up-regulated by neuronal activity such as kainate stimulation and tetanus stimulation evoking hippocampal CA1 long-term potentiation. We examined the localization and subcellular distribution of N-copine in mouse brain. In situ hybridization analysis showed that N-copine mRNA was expressed exclusively in neurons of the hippocampus and in the main and accessory olfactory bulb, where various forms of synaptic plasticity and memory formation are known to occur. In immunohistochemical analyses, N-copine was detected mainly in the cell bodies and dendrites in the neurons, whereas presynaptic proteins such as synaptotagmin I and rab3A were detected in the regions where axons pass through. In fractionation experiments of brain homogenate, N-copine was associated with the membrane fraction in the presence of Ca2+ but not in its absence. As a GST-fusion protein with the second C2 domain of N-copine showed Ca2+ -dependent binding to phosphatidylserine, this domain was considered to be responsible for the Ca2+ -dependent association of N-copine with the membrane. Thus, N-copine may have a role as a Ca2+ sensor in postsynaptic events, in contrast to the known roles of “double C2 domain-containing proteins,” including synaptotagmin I, in presynaptic events.
Recently, we identified a novel protein, N-copine, with two C2 domains (Nakayama et al., 1998). N-Copine shows high homology with human copine I, which was isolated as a member of a novel family of Ca2+ -dependent phospholipid-binding proteins (Creutz et al., 1998). Therefore, N-copine is considered to be a brain-specific member of the copine family. Although N-copine and copine I have two C2 domains, the sequences of their C2 domains are more similar to those of PLC-γ than to those of the double C2 domain proteins. Moreover, N-copine and copine I have C2 domains at the N terminus, whereas the double C2 domain proteins have C2 domains in the C terminus. Therefore, N-copine and copine I do not seem to be members of the double C2 domain protein family. These facts suggest that the neuronal roles of N-copine are different from those of the double C2 domain proteins, namely, the transport of synaptic vesicles during neurotransmitter release. In the previous study, we showed that the expression of N-copine mRNA in the hippocampus was up-regulated by kainate stimulation in vivo. We also showed that in acute hippocampal slices, tetanus stimulation evoking CA1 long-term potentiation enhanced the expression of N-copine mRNA in CA1 pyramidal cells. These results suggest the N-copine is involved in synaptic plasticity (Nakayama et al., 1998).
To further understand the neuronal roles of N-copine, we examined the distribution of N-copine expression in mouse brain and its association with the membrane fraction. Our results support the idea that N-copine has a role in postsynaptic events as a Ca2+ sensor, in contrast to the presynaptic roles of the double C2 domain proteins.
MATERIALS AND METHODS
All animals were treated ethically according to the rules of the Shionogi Animal Use and Care Committee.
Glutathione S-transferase (GST)-fusion proteins
cDNA fragments corresponding to three regions of mouse N-copine (C2A region, amino acid residues 4-161 ; C2B region, amino acid residues 141-270 ; C-terminal region, amino acid residues 240-428) were amplified by PCR and subcloned into the pGEX3T-3 vector for the expression of GST-fusion proteins (Pharmacia). The GST-fusion proteins (GST-C2A, GST-C2B, and GST-C terminus) were produced in E. coli (DH5α) by isopropylthiogalactoside induction and affinitypurified with glutathione-Sepharose 4B beads following the instruction manual for pGEX3T-3.
Phospholipid-binding assay of GST-fusion protein
Phosphatidylserine (Sigma) was sonicated in 50 mM HEPES-NaOH (pH 7.4) and 100 mM NaCl. It was then mixed with GST-C2A or GST-C2B in the same solution supplemented with 2 mM CaCl2 or 2 mM EGTA for 15 min at room temperature ; concentrations of phosphatidylserine and GST-fusion proteins in the mixture were 1.6 mg/ml and 20 μg/ml, respectively. Phosphatidylserine was precipitated by centrifugation at 10,000 g for 20 min at room temperature. Equal proportions of the supernatants and precipitates (corresponding to 2 μl of the reaction mixture) were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining.
Polyclonal antibodies specific for N-copine
Antibodies specific for C2A domain and the C-terminal region of mouse N-copine (anti-C2A and anti-C terminus, respectively) were prepared. Antisera against two types of GST-fusion proteins, GST-C2A and GST-C terminus, were raised in New Zealand White rabbits. Antibodies specific for GST in antisera were removed with GST-conjugated Sepharose 4B. Next, the antibodies specific for N-copine C2A and C terminus were purified by affinity chromatography with Sepharose conjugated with the GST-fusion proteins. Protein and Sepharose were conjugated using cyanogen bromide-activated Sepharose 4B (Pharmacia). Affinity chromatography was performed according to the instruction manual from Pharmacia. Anti-C2A and anti-C terminus gave the same results in western blot and immunohistochemical analyses.
Fractionation of brain homogenate
Fractionation of mouse brain homogenate into three membrane fractions (P1, P2, and P3) and a cytosol fraction was performed as described previously (Kuwajima et al., 1992). An equal amount of each fraction (10 μg of protein) was analyzed by western blot. To study Ca2+ -dependent membrane association of N-copine, mouse brain was homogenized with a Teflon-Potter homogenizer in five volumes of a solution [20 mM Tris-HCl (pH 7.4), 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin] supplemented with 1 mM CaCl2 or 1 mM EGTA. After incubation at 4°C for 30 min, the homogenate was centrifuged at 40,000 g for 30 min at 4°C and fractionated into precipitant as membrane fraction and supernatant as cytosol fraction. An equal portion of each fraction was analyzed by western blot.
Western blot analysis
Proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Laemmli, 1970) and transferred to a polyvinylidene difluoride membrane (Millipore). After blocking with 5% skim milk in phosphate-buffered saline, the membrane was incubated with antibody against N-copine (anti-C2A and anti-C terminus : 70 ng/ml), Doc2α, or PKC-γ, and then with horseradish peroxidase-conjugated anti-rabbit IgG antiserum (1 : 2,000 dilution, Cappel). Peroxidase-coupled detection was performed with the enhanced chemiluminescence (ECL) system (Amersham). Antibody against PKC-γ was purchased from Zymed. Antibody against Doc2-α was a gift from G. Sakaguchi.
In situ hybridization
In situ hybridization was performed as described (Nakayama et al., 1995). The mice were killed by cervical dislocation. The brain was freshly frozen and cut into 8-μm-thick sections. Digoxigenin-labeled riboprobe was prepared as described in the instruction manual for SP6 and T7 polymerase (Boehringer) ; 1.5-kbp fragment (nucleotide residues 509-2,019) of mouse N-copine cDNA inserted into the pSPORT2 vector (GibcoBRL) was used as the template. The sections were hybridized with anti-sense riboprobe for 16 h at 55°C followed by washing with 2× saline-sodium citrate and 0.2× saline-sodium citrate at 55°C. The sections were incubated with alkali phosphatase-conjugated anti-digoxigenin antibody (Boehringer) and nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate as coloring substrate according to the instruction manual for the anti-digoxigenin antibody (Boehringer). As a negative control, the sections were incubated with solution containing sense probes ; no signal was obtained.
Mice were anesthetized with ether, and then 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) was perfused transcardially. The whole brain was dissected, dehydrated with alcohol, embedded in paraffin, and cut sagittally into 8-μm-thick sections. The sections were rehydrated, incubated in 0.3% H2O2/methanol, and equilibrated with phosphate-buffered saline. Immunostaining was performed as follows : blocking with 3% goat normal serum in phosphate-buffered saline ; incubation with anti-N-copine antibody (0.5-4 μg/ml), anti-synaptotagmin I antibody (Wako ; 5-15 μg/ml), or anti-rab3A antibody (Santa Cruz ; 0.6-2 μg/ml) ; incubation with biotinylated goat immunoglobulins against rabbit IgG (Dako) or mouse IgG (KPL) ; incubation with horseradish peroxidase-conjugated biotin-avidin complex (Vectastain Elite ; Vector) ; and color detection with 3,3′ -diaminobenzidine. For a negative control, sections were incubated with normal rabbit IgG (4 μg/ml) ; no staining was seen.
In situ hybridization analysis of N-copine expression in mouse brain
Figure 1 shows an in situ hybridization analysis of the expression of N-copine mRNA in mouse brain. Signals were highly restricted to the main and accessory olfactory bulbs and hippocampus (Fig. 1A, C, and D). In the hippocampus, strong signals were observed in pyramidal cells of the CA1-CA3 regions and granule cells of the dentate gyrus (Fig. 1C). Among them, pyramidal cells of CA3 showed the strongest signals. In contrast, the signals of pyramidal cells of CA2 were weaker (Fig. 1C). In the main and accessory olfactory bulbs, granule cells showed strong signals (Fig. 1D). Signals were also detected in the mitral cell layer. Neurons in the cerebral cortex layer II were also positive, although not strongly (Fig. 1B). Some neurons in the brainstem and spinal cord were also positive (data not shown). We did not detect any signals in glial cells. Thus, we concluded that N-copine mRNA was expressed specifically in neurons.
Immunohistochemical analysis of N-copine expression in mouse brain
We prepared two antibodies against mouse N-copine. Both antibodies recognized a single band of ~62 kDa in mouse brain homogenate by western blot, showing that they were specific for N-copine (Fig. 2). Using these antibodies, we investigated immunohistochemical localization of N-copine in mouse brain. Both antibodies showed the same results ; the regions and cells showing positive staining were almost the same as those showing positive signals in the in situ hybridization analysis (Fig. 3). In the hippocampus, pyramidal cells, granule cells, and neurons in the dentate gyrus showed strong immunoreactivities (Fig. 3A and C). Strong staining was also seen in the stratum radiatum in CA1 and CA3 and the inner molecule layer of the dentate gyrus (Fig. 3C). The immunoreactivities in the stratum oriens in CA1-CA3, through which axon bundles from pyramidal cells pass, were weak (Fig. 3A and C). At higher magnification, staining of the cell bodies and dendrites of CA1-CA3 pyramidal cells was detected (Fig. 3D and F). In the accessory and main olfactory bulbs, granule cells showed positive staining (Fig. 3D and E). The cell bodies and dendrites of granule cells were strongly stained (Fig. 3E). These results suggest that N-copine is present mainly in the cell body and dendrites of certain neurons. In addition, the fimbria hippocampi were also stained (Fig. 3A), suggesting that N-copine is also present in restricted parts of the axons. For comparison, we investigated the immunohistochemical localization of synaptotagmin I (Südhof and Rizo, 1996), a member of the double C2 domain protein family, and rab3A (Johnston et al., 1991). Both proteins are known to be localized presynaptically and to associate with synaptic vesicles (Li et al., 1994 ; Ullrich et al., 1994). The staining patterns of these proteins were quite similar to each other but different from that of N-copine ; antibodies against synaptotagmin I and rab3A strongly stained the stratum oriens and the stratum radiatum. However, the cell bodies and dendrites of pyramidal cells in the CA1 region of the hippocampus were not stained with these antibodies (Fig. 3G and H). Thus, localization of N-copine in brain neurons was quite different from that of synaptic vesicle-associated proteins such as synaptotagmin I and rab3A.
Ca2+-dependent association of N-copine with membrane
In fractionation experiments of mouse brain homogenate, N-copine was enriched in the cytosol and P2 fractions (Fig. 2). We examined whether the association of N-copine with the membrane was dependent on Ca2+. We prepared a homogenate of whole mouse brain in the presence or absence of Ca2+, and fractionated it into the cytosol and the membrane fractions. In the absence of Ca2+, N-copine was detected mainly in the cytosol fraction (Fig. 4). In contrast, in the presence of Ca2+, N-copine was present mostly in the membrane fraction. PKC-γ and Doc2α, whose C2 domains are known to show Ca2+-dependent phospholipid binding, also showed association with the membrane in the presence of Ca2+. Thus, N-copine was associated with the membrane fraction in the presence of Ca2+.
We examined the phospholipid-binding properties of the two C2 domains in N-copine by using their fusion proteins with GST. We incubated the GST-fusion proteins with phosphatidylserine and centrifuged the mixture. GST-C2B coprecipitated with phosphatidylserine in the presence of Ca2+ but not in its absence (Fig. 5). In contrast, GST-C2A coprecipitated with phosphatidylserine in the presence and absence of Ca2+. GST by itself did not precipitate. Thus, the C2B domain of N-copine showed Ca2+-dependent phospholipid binding but the C2A domain showed Ca2+-independent phospholipid binding.
N-Copine is a novel two-C2 domain-containing protein that shows brain-specific expression (Nakayama et al., 1998). Because of its high homology with human copine I (Creutz et al., 1998), N-copine is considered to be a brain-specific member of the copine family. The expression of N-copine mRNA is upregulated by kainate stimulation in vivo and tetanus stimulation evoking CA1 long-term potentiation in acute hippocampal slices. These results suggest that N-copine could be involved in synaptic plasticity. To understand its roles in the CNS, we examined the expression of N-copine in mouse brain. In situ hybridization and immunohistochemical analyses showed that the expression of N-copine was highly restricted to neurons of the main and accessory olfactory bulbs and hippocampus. The hippocampus is considered to be important for learning and memory and shows various forms of long-term synaptic changes (Ben-Ari and Represa, 1990 ; Bliss and Collingridge, 1993 ; Bear and Malenka, 1994). The accessory olfactory bulb has a role in the memory for pheromone recognition (Brennan et al., 1990 ; Kaba and Nakanishi, 1995). Thus, the results about the localization of N-copine support the idea that it plays a role in synaptic plasticity and memory formation.
Although N-copine does not have a signal sequence or a transmembrane region, it was detected in both the cytosolic and the membrane fractions. Its association with the membrane fraction was dependent on Ca2+. These results suggest that N-copine is translocated from cytosol to membrane with an increase in the intracellular Ca2+ concentration. This property must be due to its C2B domain because GST-C2B but not GST-C2A showed Ca2+-dependent binding to phosphatidylserine. Furthermore, the C2B domain of N-copine conserves five Asp residues that are considered to be critical for Ca2+ binding by other C2 domain-containing proteins (Shao et al., 1996). Therefore, N-copine may be translocated between cytosol and membrane as a Ca2+ sensor. In contrast to the C2B domain, the C2A domain of N-copine showed Ca2+-independent binding to phospholipid. This property may not be very important, because N-copine itself did not bind to the membrane fraction without Ca2+. The C2A domain of N-copine may have other yet unknown roles.
Several proteins, such as synaptotagmin, rabphilin-3A (Shirataki et al., 1993), and Doc2 (Orita et al., 1995 ; Sakaguchi et al., 1995), have two C2 domains and construct a family designated the double C2 domain protein family (Südhof and Rizo, 1996). Most of the double C2 domain proteins in neurons are localized presynaptically and are involved in the transport of synaptic vesicles during neurotransmitter release (Geppert et al., 1994 ; Li et al., 1994 ; Orita et al., 1996). Although N-copine and copine I have two C2 domains, they are not likely to be members of the double C2 domain protein family, considering their structures and possible functions. In the present study, we found different localization of N-copine and synaptotagmin I in mouse brain from immunohistochemical analyses. Especially in the hippocampal CA1-CA3 region, N-copine-positive staining was detected in the cell bodies and dendrites of pyramidal cells but not in the stratum oriens, where axons of pyramidal cells pass through. In contrast, synaptotagmin I and another synaptic vesicle-specific protein, rab3A, were detected in the stratum oriens but not in the cell bodies and dendrites of pyramidal cells. Another member of the double C2 domain protein family, rabphilin-3A, which is known to be localized exclusively in nerve terminals, shows localization similar to synpatotagmin I (Li et al., 1994). These results suggest that N-copine is mainly present in the dendrites and cell bodies and plays a postsynaptic role, in contrast to the presynaptic roles of synaptotagmin and other double C2 domain proteins. In the previous study, we showed that tetanus stimulation to Schaffer commissural/collateral fibers induced the upregulation of N-copine expression in postsynaptic neurons-CA1 pyramidal cells (Nakayama et al., 1998). These results also support a postsynaptic role of N-copine.
In summary, we revealed the expression of N-copine in mouse brain and its Ca2+-dependent association with the membrane fraction. The results suggest that N-copine has a role in postsynaptic events as a Ca2+ sensor and is involved in synaptic plasticity. However, its role is mostly unclear, yet. Also, the roles of its family proteins, including copine I, remain to be clarified. Investigation of the roles and functions of N-copine and its family proteins should offer new insights into the intracellular Ca2+-signaling system and help to clarify the molecular mechanism of the synaptic plasticity of the CNS.