The synaptic neuroplastin is involved in long-term potentiation at hippocampal CA1 synapses

K. -H. Smalla*†‡§, H. Matthies†§, K. Langna¨ se*§¶, S. Shabir§ʈ,T.M.Bo¨ ckers*,**, U. Wyneken*††, S. Staak*, M. Krug†, P. W. Beesley*ʈ, and E. D. Gundelfinger*‡‡

*Leibniz Institute for Neurobiology, Brenneckestrasse 6, 39118 Magdeburg, Germany; †Institute of Pharmacology and Toxicology, ‡Institute of Medical Neurobiology, Otto von Guericke University, Leipziger Strasse 44, 39120 Magdeburg, Germany; ʈSchool of Biological Sciences, Royal Holloway University of London, Egham, Surrey TW20 OEX, United Kingdom; **Institute of Anatomy, Westfa¨lische Wilhelms University, 48149 Mu¨nster, Germany; and ††Facultad de Medicina, Universidad de los Andes, San Carlos de Apoquindo 2200, Santiago, Chile

Edited by James L. McGaugh, University of California, Irvine, CA, and approved February 14, 2000 (received for review September 10, 1999) Neuroplastin-65 and -55 (previously known as gp65 and gp55) are nated gp65 and gp55—and contain two and three extracellular of the Ig superfamily that are enriched in rat fore- Ig domains, respectively. The 65-kDa, but not the 55-kDa, brain synaptic membrane preparations. Whereas the two-Ig do- isoform is enriched in rat forebrain postsynaptic density (PSD) main isoform neuroplastin-55 is expressed in many tissues, the preparations. Whereas np55 is expressed in most rat tissues, np65 three-Ig domain isoform neuroplastin-65 is brain-specific and en- is brain-specific (23, 26). Transcripts of both isoforms are widely riched in postsynaptic density (PSD) protein preparations. Here, we distributed in rat brain. However, whereas np55 is present in all have assessed the function of neuroplastin in long-term synaptic brain regions, np65 is concentrated in subpopulations of pre- plasticity. Immunocytochemical studies with neuroplastin-65-spe- dominantly forebrain and is enriched in neuropil regions cific differentially stain distinct synaptic neuropil re- (23, 24). gions of the rat hippocampus with most prominent immunoreac- Both proteins are highly glycosylated and occur as multiple tivity in the CA1 region and the proximal molecular layer of the glycoforms (23–26). Interestingly, a fraction of np65 carries dentate gyrus. Kainate-induced seizures cause a significant en- fucose ␣ (1–2)-linked to galactose in its carbohydrate moiety hancement of neuroplastin-65 association with PSDs. Similarly, (27). Fucosylated glycostructures have been implicated in long- long-term potentiation (LTP) of CA1 synapses in hippocampal slices term memory formation in different species (28–30) as well as enhanced the association of neuroplastin-65 with a detergent- in maintenance of hippocampal long-term potentiation (LTP) insoluble PSD-enriched protein fraction. Several antibodies against (31). Here, we assessed the role of neuroplastin in processes of the neuroplastins, including one specific for neuroplastin-65, in- synaptic plasticity. We provide strong evidence that the associ- hibited the maintenance of LTP. A similar effect was observed ation of np65 with the PSD fraction is regulated by synaptic when recombinant fusion protein containing the three extracellu- activity and that neuroplastin is involved in plasticity-dependent lar Ig domains of neuroplastin-65 was applied to hippocampal synaptic restructuring. slices before LTP induction. Microsphere binding experiments us- ing neuroplastin-Fc chimeric proteins show that constructs contain- Materials and Methods ing Ig1–3 or Ig1 domains, but not Ig2–3 domains mediate ho- Recombinant Proteins and Antibodies. A segment of the cDNA of mophilic adhesion. These data suggest that neuroplastin plays an np55 encoding residues Ϫ2 to 192 (23) was amplified essential role in implementing long-term changes in synaptic by PCR and cloned into the bacterial expression vector pQE30 activity, possibly by means of a homophilic adhesion mechanism. (Qiagen, Chatsworth, CA). Similarly, a np65 cDNA segment encoding amino acid residues 3–308 was introduced into pQE30. ell adhesion molecules (CAMs) are crucially involved in the From these constructs, recombinant proteins of either 26 kDa Cassembly and restructuring of synapses during development (Ig domains 2–3) or 40 kDa (Ig domains 1–3) containing six and synaptic plasticity. Members of various CAM families are N-terminal histidine residues were expressed (Fig. 1A). The first localized in synaptic junctions. These include: (i) members of the Ig domain of np65 (amino acids 3–118) was cloned into the Ig superfamily, e.g., NCAM (neural CAM)-180 (1) and, in pGEX␭1T vector (Amersham Pharmacia) to produce a 42-kDa Drosophila, Fasciclin 2 (2); (ii) N- and E-cadherins, (3, 4) and glutathione S-transferase fusion protein. Purified fusion proteins novel cadherin-related proteins (5); (iii) integrins (6, 7); and (iv) were used to produce the polyclonal rabbit antisera AS Ig1 the ␤-neurexin/neuroligin system (8, 9). Typically, synaptic against the first np65-specific Ig domain, AS Ig2–3 against Ig CAMs span the synaptic cleft by homophilic or heterophilic domains 2–3, and AS Ig1–3 against Ig domains 1–3. interaction and are anchored to distinct cytoskeletal elements on mAb SMgp65 was produced against ConA-binding SM gly- either side of the synapse (10–14). They are thus well placed to coproteins from rat brain (24). IgG fractions from antisera were mediate synapse formation and stabilization during develop- ment, and to participate in activity-induced synaptic plasticity, resulting in restructuring of synapses. Evidence for the latter is This paper was submitted directly (Track II) to the PNAS office. provided by the observation that the disturbance of synaptic cell Abbreviations: ACSF, artificial cerebrospinal fluid; CAM, ; NCAM, adhesion, e.g., in mutant animals or by the application of neural CAM; fEPSP, field excitatory postsynaptic potential; np65, neuroplastin-65; np55, antibodies or competitive peptides, in many cases interferes with neuroplastin-55; LTP, long-term potentiation; PSD, postsynaptic density; SM, synaptic membrane. long-term changes of synaptic plasticity. This has been reported § for Ig superfamily CAMs NCAM180 and L1 (15, 16), telen- K.-H.S., H.M., K.L., and S.S. contributed equally to this work. cephalin (17), Fasciclin 2 (18), and Aplysia apCAM (19), as well ¶Present address: Institute of Human Genetics, Otto von Guericke University, 39120 Mag- deburg, Germany. as for cadherins (20) and integrins (21, 22). ‡‡To whom reprint requests should be addressed. E-mail: gundelfinger@ifn-magde The neuroplastins are major glycoprotein components of burg.de. synaptic membrane (SM) preparations belonging to the Ig The publication costs of this article were defrayed in part by page charge payment. This superfamily (23). They originally were identified by using the article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. mAb SMgp65, which was generated against SM glycoprotein §1734 solely to indicate this fact.

preparations (24, 25). They occur as two isoforms, neuroplas- Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073͞pnas.080389297. NEUROBIOLOGY tin-65 (np65) and neuroplastin-55 (np55)—previously desig- Article and publication date are at www.pnas.org͞cgi͞doi͞10.1073͞pnas.080389297

PNAS ͉ April 11, 2000 ͉ vol. 97 ͉ no. 8 ͉ 4327–4332 Downloaded by guest on September 28, 2021 killed 6 h later (35). Seizure development was assessed to distinguish control, kainate-treated nonseizure, and the kainate- treated seizure groups of rats. For preparation of synaptic proteins, cortices and hippocampi of three animals were pooled for one sample. All animal experiments were performed in accordance with the guidelines and regulations defined by the German Animal Welfare Act. Permission was obtained from the Regierungspraesidium Dessau.

Isolation and Analysis of Subcellular Protein Fractions. Synaptic proteins (PSD fraction) were prepared essentially as described by Carlin et al. (36) with modifications as detailed in ref. 37. For quantification of neuroplastin immunoreactivity in a PSD- enriched protein fraction after induction of LTP, hippocampal slices were stimulated as described (38). Two hours after teta- nization, 10 control or stimulated slices were pooled and ho- mogenized in 200 ␮l of PSD-extraction buffer (37) and kept for 1 h at 4°C to solubilize noncytoskeletal, non-PSD protein. Subsequently, samples were spun for1hat100,000 ϫ g, and the pellets were rehomogenized in extraction buffer and washed by centrifugation at 100,000 ϫ g. The resulting pellet is enriched in Fig. 1. Characterization of neuroplastin antibodies and fusion proteins. (A) PSD proteins. Map of np65 (np55 lacks the first Ig domain). The recombinant fragments to produce rabbit antisera AS Ig1, AS Ig2–3, and AS Ig1–3 are indicated below the Electrophysiology. LTP experiments were performed on trans- map. The epitope for mAb SMgp65 is located within the Ig2–3 region. The verse slices (400 ␮m) of the right hippocampus from 8-wk-old Ig1–3-Fc and Ig2–3-Fc fusion proteins produced in 293 cells are also shown. (B) male Wistar rats, strain Shoe, as described (32). Antibodies, Specificity of antibodies. Western blots of Triton X-100-solubilized membrane proteins from rat brain (lanes 1–3 and 6), np65-transfected 293 cells (lanes 4 recombinant protein, or appropriate control solutions were applied onto the CA1 stratum radiatum by using a microinfusion and 7), and np55-transfected 293 cells (lanes 5 and 8) were probed with the Ϸ ␮ following antibodies: AS Ig2–3 (lane 1), AS Ig1–3 (lane 2), AS Ig1 (lanes 3–5), pump (delivery rate of 0.75 l/min) for 80 min. After 60 min and mAb SMgp65 (lanes 6–8). Note, differential glycosylation of neuroplas- of infusion, LTP was induced by using three 100-Hz tins expressed by 293 cells results in size differences from neuroplastins of stimulus trains, each containing 50 pulses at double-pulse width brain membrane extracts (23, 26). mAb SMgp65 recognizes two glycoforms of with a 2-min interval between each train. Field excitatory np65 in transfected 293 cells (lane 7, and ref. 23). (C) Purification of the postsynaptic potentials (fEPSP) were recorded in 5- to 10-min Fc-fusion proteins. Affinity-purified fusion protein Ig1–3-Fc (lane 2) and hu- intervals for at least 3 h. LTP magnitude was calculated as man Fc alone (lane 3) were separated by SDS/PAGE and stained with Coomassie percent change of fEPSP as compared with averaged baseline brilliant blue. Molecular weight markers are shown in lane 1. responses measured during 30 min before drug application (mean Ϯ SEM). Statistical evaluation of the data was performed affinity-purified on GammaBind Plus Sepharose (Amersham with a two-tailed Mann–Whitney U test. Pharmacia) and buffer-exchanged into artificial cerebrospinal fluid (ACSF; ref. 32) on FPLC fast-desalting columns (Amer- Microsphere Binding Assays. Assays were carried out essentially as described (39). Briefly, anti-human Fc (Sigma) was passively sham Pharmacia). ␮ Eukaryotic recombinant proteins containing the Ig domains adsorbed onto 0.6- m diameter red covaspheres (Duke Scien- tific, Palo Alta, CA) or 1-␮m diameter yellow-green Fluoresbrite of np65 (amino acids 3–307) or np55 (amino acids 3- 191) fused microspheres (Polysciences) in PBS for 1 h. Microspheres were to the F region of human IgG were produced in 293 cells. The c washed for 2 min in PBS (three times) and pelleted. Subse- ORF were amplified by PCR from neuroplastin cDNAs as above quently, uncoupled sites were blocked by incubation in 5% FCS and cloned in-frame behind the BM40 signal peptide (33) into (GIBCO/BRL) for 1 h. After washing as above, beads were the eukaryotic expression vector pIgPlus (provided by P. incubated with appropriate Fc construct for 1 h, washed, and Doherty, King’s College, London). To express the Fc region of resuspended in PBS (50 ␮l). All incubations were at room human IgG alone, the BM40 signal sequence was fused directly ␮ temperature. Fc-coated microspheres were further diluted (10 l to the Fc sequence of pIgPlus. Cell lines stably expressing and into 50 ␮l PBS), sonicated on ice for 10 min, followed by secreting the fusion proteins were generated as described (23). incubation for 60 min at room temperature to allow aggregation. Fusion proteins were purified from culture supernatants by Samples (6 ␮l, three samples per time point) were taken at 15, affinity chromatography using GammaBind Plus Sepharose. For 30, 45, and 60 min and diluted in 1 ml of PBS. Three aliquots (100 LTP experiments, the buffer was exchanged as described for the ␮l per sample) were transferred to 96-well plates, and the plates antibodies. were centrifuged at 2,750 rpm for 10 min. Aggregation was monitored with a Leica fluorescent microscope. Images were Immunohistochemistry. Immunohistochemical experiments were captured and analyzed by using PHOTOLITE and IMAGE PROPLUS ␮ performed as described (34). Np65 was detected in 7- m frontal software (Media Cybernetics, Silver Spring, MD). For all exper- and sagittal sections by using AS Ig1 (1:300) as primary antibody iments, aggregation was determined as percent decrease in (22 h), followed by incubation with porcine anti-rabbit IgG nonaggregated beads relative to the number of nonaggregated (Dako) diluted 1:50 (30 min) and rabbit peroxidase- beads coated with Fc alone at time 0. antiperoxidase complex (Dako) diluted 1:100 (30 min). Immu- noreactivity then was visualized by using 3,3-diaminobenzidine Results (0.05%)/H2O2 (0.001%) (Sigma). Characterization of Neuroplastin Antibodies. Various antibodies directed against different extracellular domains of neuroplastin Kainate Treatment of Rats. Twelve-week-old male Wistar rats were were used to study the distribution and the function of these injected i.p. with kainate (13 mg/kg body weight) or saline and molecules in the rat hippocampus. Antibodies include rabbit

4328 ͉ www.pnas.org Smalla et al. Downloaded by guest on September 28, 2021 Fig. 3. np65 content of synaptic protein fraction is regulated by synaptic activity. (A) Enhanced np65 immunoreactivity in the rat brain PSD fraction after kainate-induced seizure. Immunoblots of PSD proteins from untreated animals (lane 1), kainate-treated, nonseizure (lane 2), and kainate-treated seizure animals (lane 3) were developed with mAb SMgp65 or anti-tubulin antibodies. (B) Preparation of PSD-enriched fraction from hippocampal slices. Ten hippocampal slices were pooled and extracted with PSD-extraction buffer. Western blots of soluble fraction (S) and insoluble pellet fraction (P) were probed with mAb SMgp65, and antibodies against synaptophysin and SAP90/PSD-95. Note, a fraction of np65, but not np55, is detected in the PSD-enriched fraction. (C) LTP enhances np65 immunoreactivity in PSD- Fig. 2. Distribution of np65 in the forebrain detected with np65-specific enriched fraction. Western blots of PSD-enriched protein fractions from un- antibodies (AS Ig1). (A) Frontal section of rat forebrain. Intense immunoreac- treated and tetanized (2 h after tetanization) hippocampal slices were probed tivity is found in the cerebral cortex (Cx), the hippocampal formation (Hip), with SMgp65 and anti-tubulin antibody (control, lane 1; LTP, lane 2). Tetani- including dentate gyrus (DG), the putamen (Pu), and the amygdala (Am). Th, zation results in a 190 Ϯ 39% increase (n ϭ 6; Wilcoxon test, P Ͻ 0,03) of np65 thalamus. (B) Frontal section of somatosensory cortex; layering is indicated. in the PSD-enriched fraction. Arrowheads in A and B mark strongly stained barrel fields. (C) Sagittal section of the hippocampus. CA1, CA3, Ammon’s horn regions 1 and 3. (D–F) Enlarge- ments of the CA1 pyramidal cell layer (Py) and the granule cell layer (GC) of the DG indicate cell surface staining of these neurons (arrows). (Size bars: 2 mm in specific immunoreactivity were observed. The most prominent A; 200 ␮minB; 500 ␮minC;25␮minD and F; 100 ␮minE.) staining was found in the neuropil layers of the CA1 region and the dentate gyrus (Fig. 2 A and C–F). In CA1, the stratum oriens and the stratum radiatum display much stronger immunoreac- antisera raised against bacterial recombinant protein corre- tivity than the stratum lacunosum moleculare (Fig. 2C). A sponding to the np65-specific, the two common, or all three Ig high-power view (Fig. 2D) shows that the neuropil staining is domains (AS Ig1, AS Ig1–3, 2–3) (Fig. 1A), and mAb SMgp65, punctate and that somata of CA1 pyramidal neurons are sur- which recognizes both isoforms on immunoblots (24). In rat rounded by immunoreactivity. In contrast, the CA3 region shows brain membrane preparations, AS Ig2–3, AS Ig1–3, and mAb only moderate staining that is laminar in appearance (Fig. 2C). SMgp65 recognize two bands of 55 and 65 kDa (Fig. 1B, lanes Note the sharp boundary between the intensely immunoreactive 1, 2, and 6), whereas AS Ig1 detects a single band of 65 kDa (Fig. CA1 and moderately immunoreactive CA3 regions. In the 1B, lane 3). The specificity of AS Ig1 for np65 was further dentate gyrus, strong np65 immunoreactivity is found in the confirmed by showing that this antiserum detects an immuno- inner molecular layer, whereas the middle and outer molecular reactive band only on Western blots from np65-transfected but layers are less intensely stained (Fig. 2 C and E). The staining of not from np55-transfected 293 cells (Fig. 1B, lanes 4 and 5). the inner molecular layer is likely to come from projections of Consistent with previous data (23), mAb SMgp65 reacts with mossy cells of the hilar polymorphic layer (40). Granule cells, like polypeptides expressed by both cell lines (Fig. 1B, lanes 7 and 8). pyramidal cells, display significant cell surface staining (Fig. 2F).

Localization of np65 in the Rat Forebrain. The np65-specific AS Ig1 Synaptic Activity Regulates Association of np65 with the Synaptic was used to establish the distribution of this isoform in rat Protein (PSD) Fraction. As np65 is a putative PSD-associated forebrain. An overall view of a frontal section shows that np65 CAM, we investigated whether the association of this isoform is prominently expressed in the cerebral cortex, the hippocam- with the PSD is regulated by synaptic activity. Treatment of pus, the amygdala, and the striatum. Most intense staining within animals with kainate induces a widespread increase in synaptic the latter structure is found in the putamen (Fig. 2A). In the activity, which results in generalized tonic-clonic seizures (41, cortex, neuropil regions of layers II, III, and Vb/VI are promi- 42). As is well-established, kainate treatment induces seizures nently stained; other layers are moderately labeled (Fig. 2 A and (stages 5–6) in some, but not all, animals. The neuroplastin B). In the somatosensory cortex, barrel fields in layer IV exhibit content of the PSD fractions prepared from seizure, nonseizure, particularly strong staining (arrowheads, Fig. 2 A and B). Mod- and control groups of animals6hafterkainate treatment was erate staining also is observed in the medial and lateral thalamic determined by immunoblot analysis. The relative amount of nuclei. The overall distribution of the 65-kDa isoform of neu- np65 is significantly increased in the PSD fraction of seizure roplastin is consistent with the distribution of np65-specific animals 4- to 5-fold, as compared with nonseizure and control

transcripts (23). rats (Fig. 3A). The relative amount of tubulin in the PSD NEUROBIOLOGY In the hippocampus, significant regional differences in np65- fractions remains essentially unchanged. Light microscopy im-

Smalla et al. PNAS ͉ April 11, 2000 ͉ vol. 97 ͉ no. 8 ͉ 4329 Downloaded by guest on September 28, 2021 fraction (Fig. 3C). After 6 h, still an Ϸ1.3-fold increase is observed (not shown). No LTP-induced change was observed for tubulin (Fig. 3C).

Neuroplastin Antibodies and Recombinant Protein Block the Mainte- nance of LTP. Four different antibodies against various extracel- lular domains of neuroplastin (Fig. 1) were used to examine whether the protein is directly involved in LTP at CA1 synapses. Basal fEPSP responses recorded for 90 min were not affected by this treatment. Stimulation of the Schaffer collaterals with three subsequent 100-Hz-stimulus trains induced stable LTP for at least 3 h under control conditions, i.e., in the presence of ACSF or ACSF-containing control Igs (Fig. 4A). Application of neu- roplastin antibodies did not prevent induction of LTP, although fEPSPs in some cases appear to be somewhat reduced 5 min after stimulation. All four neuroplastin antibodies inhibited mainte- nance of LTP. Data are illustrated for the np65-specific antibody AS Ig1 (Fig. 4A) and summarized in Table 1. Ten minutes after tetanization, fEPSPs in experimental slices were not significantly different from controls. Thereafter, the potentiated fEPSPs declined. They became significantly lower than those in stably potentiated controls 30 min after tetaniziation and reached baseline within 1 h. In addition, the recombinant neuroplastin- Ig1–3-Fc caused rapid decay of potentiation between 10 and 30 min after tetanization and prevented maintenance of LTP (Fig. 4B and Table 1). Fc alone did not have this effect.

Recombinant np65-Fc Fusion Proteins Display Homophilic Adhesion. Many Ig superfamily adhesion molecules, including NCAM and L1, mediate homophilic binding (43, 44). Microsphere binding assays were carried out to determine whether this is the case for np65 and np55. Fluorescent microspheres coated with Fc chi- meric proteins containing all three (Ig1–3-Fc) or the two com- mon Ig (Ig2–3-Fc) domains were used to test this possibility. The results show that minimal aggregation (1.25 Ϯ 0.31%; n ϭ 9) was observed for beads coated with the Fc portion of human Ig alone (Fig. 5A). As expected reproducible aggregation was observed for microspheres coated with chimeric NCAM-human Fc protein (Fig. 5B). Aggregation also was observed for beads coated with Ig1–3-Fc, but not Ig2–3-Fc (Fig. 5 D and C). The percent aggregation observed with the NCAM and Ig1–3-Fc constructs Ϯ ϭ Ϯ ϭ Fig. 4. Antibodies against np65 (A) and recombinant neuroplastin extracellular were 43.61 0.55% (n 9) and 37.28 0.31% (n 9), domains (B) prevent maintenance of LTP at CA1 synapses in hippocampal slices. respectively. The specificity of the aggregation observed with (A) Ensemble average of fEPSPs of all experiments (n ϭ 7) with slices superfused Ig1–3-Fc was tested by carrying out the binding assay in the with antibody AS Ig1 (■) or with ACFS containing rabbit Igs (n ϭ 8; E). (B) presence of SMgp65 antibody or by heat-denaturing the con- Ensemble average of fEPSPs of all experiments (n ϭ 7) with slices perfused with struct in the presence of 2-mercaptoethanol before carrying out ■ ϭ E recombinant fusion protein Ig1–3-Fc ( ) or with the Fc fragment alone (n 6; ). the assay (Fig. 5 E and F). In both cases, aggregation was blocked Superimposed representative samples of fEPSPs taken 10 min before and 120 min and did not differ significantly from that observed for the after tetanus are inserted in A and B. (Scale bar: 2 mV, 2 ms.) Fc-coated beads. These data suggest that the Ig-1 domain is involved in the homophilic binding of np65. Indeed, aggregation (34.58 Ϯ 0.10%, n ϭ 9) of beads coated with Ig1-Fc is observed munohistochemistry revealed no major redistribution of np65 (Fig. 5G). This aggregation is significantly reduced (23.30 Ϯ distribution after kainate treatment (not shown). 0.63%, P Ͼ 0.001, n ϭ 9) by inclusion of the np65-specific In another series of experiments, we tested whether the antiserum AS Ig1 in the assay medium. association of np65 with synaptic protein fractions is regulated by LTP in acutely isolated hippocampal slices. Because of the Discussion limited amount of tissue, it was not practical to prepare con- Neuroplastin has long been known as a synaptic glycoprotein ventional PSD fractions from individual slices. Therefore, a (24); however, its functions have been unknown. Here, we detergent-insoluble PSD-enriched fraction was isolated. PSD provide strong evidence that np65 is involved in plasticity at enrichment in this fraction was confirmed by the PSD marker hippocampal synapses. First, np65 is highly and differentially protein SAP90/PSD95 and by the absence of the synaptic vesicle expressed in the hippocampus with most prominent appearance protein synaptophysin in the pellet (Fig. 4B). As expected (24), in the synaptic neuropil region of CA1. Second, a significant a fraction of np65, but not np55, is found in the PSD-enriched increase in np65 immunoreactivity is detected in PSD-enriched fraction (Fig. 3B). and PSD fractions after LTP induced in hippocampal slices or Immunoblots of PSD-enriched fractions prepared from after seizures induced by kainate, respectively. Recent studies untreated and tetanized (2 h after tetanization) hippocampal have shown that changes of synaptic efficacy in the brain, e.g., as slices were probed with neuroplastin antibodies. Tetanization induced by ischemia, can induce changes in synaptic structure results in a Ϸ2-fold increase of np65 in the PSD-enriched and in the protein composition of PSDs (45, 46). Kainate is

4330 ͉ www.pnas.org Smalla et al. Downloaded by guest on September 28, 2021 Table 1. Antibodies against different Ig domains of neuroplastin and the recombinant fusion protein Ig1-3-Fc prevent maintenance of hippocampal CA1 LTP % Increase in fEPSP (mean Ϯ SEM)

5 min 10 min 30 min 60 min 120 min

Rabbit IgG fraction (n ϭ 8) 180.2 Ϯ 30.2 172.2 Ϯ 21.9 188.2 Ϯ 18.2 208.5 Ϯ 15.5 180.0 Ϯ 14.1 AS Ig1 (n ϭ 7) 121.9 Ϯ 24.8 153.6 Ϯ 24.6 124.5 Ϯ 20.6** 102.6 Ϯ 18.4** 101.8 Ϯ 9.5** AS Ig1–3 (n ϭ 8) 159.0 Ϯ 18.5 166.3 Ϯ 20.1 138.8 Ϯ 14.8** 124.6 Ϯ 11.3** 100.9 Ϯ 4.1** AS Ig2–3 (n ϭ 8) 132.5 Ϯ 13.5 154.4 Ϯ 13.8 128.2 Ϯ 12.8* 100.9 Ϯ 10.0** 104.4 Ϯ 6.1**

Control IgG fraction (n ϭ 7) 163.3 Ϯ 9.3 162.7 Ϯ 5.4 181.8 Ϯ 16.8 187.0 Ϯ 19.0 175.2 Ϯ 39.0 SMgp65 (n ϭ 7) 139.7 Ϯ 17.6 160.2 Ϯ 31.9 146.8 Ϯ 28.7* 139.2 Ϯ 21.6* 108.1 Ϯ 8.4**

hum IgG-Fc (n ϭ 6) 135.3 Ϯ 5.3 137.4 Ϯ 8.2 140.0 Ϯ 7.7 142.8 Ϯ 10.4 145.9 Ϯ 29.4 † †† †† Ig1–3Fc (n ϭ 7) 139.8 Ϯ 15.3 150.9 Ϯ 15.0 113.6 Ϯ 7.8 105.4 Ϯ 13.0 91.3 Ϯ 14.3

Antibodies, recombinant protein, or appropriate control solutions were applied from 60 min before to 20 min after high-frequency stimulation. Data represent percent change of the fEPSP as compared to averaged baseline responses measured during 30 min before application (mean Ϯ SEM). *, P Ͻ 0.05; **, P Ͻ 0.02 as compared to ACSF containing IgG fraction from rabbit normal serum; *, P Ͻ 0.05; **, P Ͻ 0.02 as compared to IgG fraction from hybridoma culture medium; †, P Ͻ 0.05; ††, P Ͻ 0.02 as compared to human IgG-Fc.

known to strongly affect synaptic activity in limbic structures, binding partners. This has been reported e.g., for integrins (21, including pyramidal neurons of the hippocampus (41, 42), and 22) and cadherins (20), as well as for the Ig superfamily member synaptic restructuring was described to take place after LTP telencephalin (17). (47–49). The observed enhanced association of np65 with syn- Currently, the mechanism by which neuroplastin affects syn- aptic structures may reflect an involvement of the molecule in aptic plasticity is unknown. Our interaction studies in vitro these plasticity-induced restructuring processes. Third, the per- provide clear evidence that np65, but not np55, mediates ho- turbation experiments with antibodies and recombinant mophilic binding, and that the interaction is blocked by antineu- polypeptide that almost completely suppress the maintenance of roplastin antibody, which also blocks LTP. The 65-kDa isoform LTP at CA1 synapses provide the most direct evidence for the has an interaction potential similar to that of other Ig super- involvement of neuroplastin in synaptic plasticity. One of the family adhesion molecules, including NCAM and L1, which are applied antibodies exclusively recognizes np65, further support- known to mediate homophilic binding (43, 44). Both CA3 and ing the view that this isoform is crucially involved in synaptic CA1 pyramidal cells express np65 transcripts (23), suggesting effects of neuroplastin. The most plausible explanation of anti- that the protein could be present on both sides of synapses body-mediated suppression of long-term changes of synaptic between Schaffer collaterals and CA1 pyramidal cell dendrites. strength is that they interfere with existing or newly established Thus, a homophilic interaction across the synaptic cleft is protein–protein interactions mediated by the antigens. A similar conceivable, assuming a width of the cleft of 20–30 nm (50) and competitive disruptive effect may be achieved by the use of an extension into the cleft of np65 of about 13–16 nm, as peptides or recombinant protein domains, which mimic natural suggested by molecular modeling of its three Ig domains (C. Reissner and E.D.G., unpublished data). However, in con- trast to NCAM, where multiple Ig domains may be involved in homophilic interaction and bending of the protein has to be invoked to fit into the synaptic cleft (43), in np65, primarily the first isoform-specific Ig domain may mediate physical interac- tion. This is supported by the observation that Ig1-Fc mediates clustering of microspheres, and this response is significantly blocked by the np65-specific antiserum. Nonetheless, it must be stressed that the role of np55 in neuronal plasticity processes is not excluded by our experiments, as no tools specific for this isoform are available. Interestingly, normal synaptic transmission appears essentially unaffected by the neuroplastin antibodies and Ig1–3-Fc fusion protein, and, also, short-term potentiation is possible in the presence of these agents. Thus, neuroplastin appears to be involved in long-term synaptic changes only. Similar observa- tions have been made for other CAMs found to be involved in synaptic plasticity, including L1 and NCAM (15, 16, 51), telen- cepahlin (17), integrins (21, 22), cadherins (20), amyloid pre- cursor protein (52, 53), and N-syndecan (54). Fig. 5. Homophilic aggregation assay. Microspheres were coated with: (A)5 The time course of decay of synaptic potentiation produced by ␮ ␮ ␮ ␮ gFc alone; (B)4 g NCAM-Fc;(C)5 g Ig2–3-Fc;(D)5 g Ig1–3-Fc;(E) Ig1–3-Fc neuroplastin antibodies is reminiscent of that reported for ␮ ␮ in the presence of mAb SMgp65 (10 l hybridoma supernatant per 50- l assay protein kinase C (PKC) inhibitors (55, 56). Interestingly, the volume); (F) Ig1–3-Fc denatured by boiling for 5 min in the presence of 1% ␥ (vol/vol) ␤-mercaptoethanol. (G) Fluoresbrite microspheres coated with 5 ␮g distribution of PKC immunoreactivity in the hippocampus (57) Ig1-Fc;(H) Ig1-Fc (5 ␮g)-coated microspheres in the presence of AS Ig1 (5 ␮l). In is remarkably similar to that of np65. Two Ig superfamily CAMs,

all panels, aggregation is shown after 60 min assay time. Note that NCAM (B), NCAM and L1, have been implicated in PKC-dependent sig- NEUROBIOLOGY Ig1–3-Fc (D), and Ig1-Fc (G), but not Ig2–3-Fc (C) mediate aggregation. naling processes in growth cones (58). Thus, it will be interesting

Smalla et al. PNAS ͉ April 11, 2000 ͉ vol. 97 ͉ no. 8 ͉ 4331 Downloaded by guest on September 28, 2021 to find out whether the observed effect of neuroplastin on LTP ponents. Regulation mechanisms may include activity-driven maintenance also involves PKC. regulation of CAM expression (62–64) as well as activity- Recent evidence indicates that rapid changes in synaptic regulated recruitment of NCAM180 (65) or np65 into the plasticity are accompanied by rapid ultrastructural modifications synapse to locally enhance physical interaction or to enlarge the to or an altered number of synapses. Thus, the occurrence of contact area of the synaptic membranes and in turn cause dendritic protrusions and spine-like structures has been visual- strengthening of synapses. ized in living hippocampal neurons in culture 30–60 min after LTP induction (59, 60). Furthermore, electron microscopic We thank R. Grimm and W. Tischmeyer for providing kainate-treated analyses of calcium precipitates in spines following thetaburst animals and K. Richter, C. Otto, K. Zobel, K. Schulzeck, and R. stimulation revealed that LTP promotes formation of new Mummery for expert technical assistance. The pIgPlus vector was a gift mature synapses contacting the same presynaptic terminal 1 h from Prof. P. Doherty. This study was supported by the Deutsche after tetanization (61). Such structural changes associated with Forschungsgemeinschaft (to M.K., S.S., and E.D.G.), the Volkswagen- long-term synaptic plasticity may depend on the action of a Stiftung (to E.D.G. and U.W.), the Wellcome Trust (to P.W.B.), and a variety of very different CAMs and extracellular matrix com- North Atlantic Treaty Organization travel grant (to P.W.B. and E.D.G.).

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