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Short communication

Monoclonal antibodies discriminating netrin-G1 and netrin-G2neuronal pathways

Kimie Niimi a,b, Sachiko Nishimura-Akiyoshi c, Toshiaki Nakashiba d, Shigeyoshi Itohara c,⁎

a Brain Science and Life Technology Research Foundation, 1-28-12 Narimasu, Itabashi, Tokyo 175-0094, Japanb Research Resources Center, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan

c Laboratory for Behavioral Genetics, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japand The Picower Institute for Learning and Memory, RIKEN-MIT Neuroscience Research Center, Massachusetts Institute of Technology,

Cambridge, Massachusetts 02139, United States

Received 4 June 2007; received in revised form 16 September 2007; accepted 17 September 2007

Abstract

Netrin-G1 and netrin-G2, belonging to a vertebrate-specific subfamily of the netrin family, distribute on axons of distinct neuronal pathways.To add to the array of molecular probes available for labeling unique neuronal circuits, we generated monoclonal antibodies against the netrin-G1and netrin-G2 proteins. The monoclonal antibody clones 171A18 and 30B15 differentially labeled specific neuronal circuits, the so-called netrin-G1 or netrin-G2 circuits in mice, respectively. Epitope mapping revealed linear epitopes for these monoclonal antibodies, which are commonamong splicing variants, and suggested that the anti-netrin-G1 monoclonal antibodies are applicable to various species including humans.© 2007 Elsevier B.V. All rights reserved.

Keywords: Netrin-G1; Netrin-G2; Monoclonal antibody; Neuronal pathway; Axon

1. Introduction

Netrin-G1 and netrin-G2 were identified in mice (Nakashibaet al., 2000, 2002) as novel members of the UNC-6/netrin familyknown as axon guidance molecules. These molecules are alsoknown as laminet-1 and laminet-2, respectively (Yin et al., 2002).Unlike classical netrins, netrin-Gs are predominantly linked tothe plasma membrane by a glycosyl phosphatidyl-inositol lipidanchor, preferentially expressed in the central nervous system invertebrates, and lack affinity to the known receptors for classicalnetrins (Nakashiba et al., 2000, 2002). Thus, netrin-G1 and netrin-G2 compose a vertebrate-specific subfamily, and their roles aredistinct from those of classical netrins (Nakashiba et al., 2000,2002, Nishimura-Akiyoshi et al., 2007). Intriguingly, netrin-G1and netrin-G2 preferentially distribute on distinct subsets of axonsin mice, such as thalamocortical, olfactory tract, temporoammo-

nic (perforant) and lateral perforant pathways for netrin-G1 andcortico-cortical, Schaffer collateral, and medial perforant path-ways for netrin-G2 (Nakashiba et al., 2002; Nishimura-Akiyoshiet al., 2007), and are thus suitable as molecular probes for iden-tifying specific neuronal circuits.

Recent studies indicate that netrin-G1 and netrin-G2 bind tothe netrin-G ligands NGL-1 (Lin et al., 2003) and NGL-2(Nishimura-Akiyoshi et al., 2007; Kim et al., 2006), respectively,in one-to-one relationships. Studies on netrin-G1 and netrin-G2knockout mouse demonstrated that the transneuronal interactionsof netrin-Gs and their receptors are important for establishingfunctional neuronal circuits in mice (Nishimura-Akiyoshi et al.,2007). Disruption of these functions might contribute to schizo-phrenia (Aoki-Suzuki et al., 2005) andRett syndrome (Borg et al.,2005; Archer et al., 2006).

Monoclonal antibodies (MAbs) that recognize netrin-G1 andnetrin-G2 will help to analyze the ligand-receptor interactions andserve as useful tools for selective labeling of specific neuronalcircuits. Here we describe mouse MAbs specific to netrin-G1 ornetrin-G2 that recognize common epitopes across splicing variantsand are applicable for biochemical and histochemical studies.

Journal of Neuroimmunology 192 (2007) 99–104www.elsevier.com/locate/jneuroim

⁎ Corresponding author. Tel.: +81 48 467 9724; fax: +81 48 467 9725.E-mail address: sitohara@brain.riken.jp (S. Itohara).

0165-5728/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.jneuroim.2007.09.026

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2. Materials and methods

2.1. Animals

Ntng1 knockout (KO) mice and Ntng2 KO mice (Nishimura-Akiyoshi et al., 2007) were used as recipients for immunization.Wild-type and KO mice were used for immunohistochemistry.All experimental protocols were approved by the RIKENInstitutional Animal Care and Use Committee.

2.2. Antibodies

Rabbit polyclonal anti-netrin-G1 and anti-netrin-G2 weredescribed previously (Nakashiba et al., 2002; Nishimura-Akiyoshi et al., 2007). Other antibodies used were mouse MAbanti-c-myc (9E10; Roche, Indianapolis, IN), rabbit polyclonalanti-GFAP (DAKO, Glostrup, Denmark), horseradish peroxi-dase (HRP)-conjugated anti-mouse IgG (Jackson Immunore-search Laboratories, West Grove, PA), Alexa 546-conjugatedanti-rabbit IgG (Molecular Probes, Eugene, OR), andAlexa 488-conjugated anti-mouse IgG (Molecular Probes).

2.3. Immunization and fusion protocols

Recombinant Myc/His-tagged fusion proteins purified fromconditioned media of HEK293T cells stably secreting netrin-G1dor netrin-G2a (Nakashiba et al., 2000, 2002) were used asantigens. Ntng1 KO mice and Ntng2 KO mice were immunizedsubcutaneously four times at 2-wk intervals withMyc/His-taggednetrin-G1d and netrin-G2a, respectively, emulsifiedwithGERBUADJUVANT (GERBU Biotechnik GmbH, Gaiberg, Germany).The spleen cells of the immunized mice were fused with mousemyeloma cells (SP2/O) using 50% polyethylene glycol (HYBRI-MAX, Sigma, St Louis,MI) according to the procedure describedpreviously (Lane, 1985; Itohara et al., 1989). Seven days afterfusion, culture supernatants of each well were tested for immu-noreactivity by enzyme-linked immunosorbent assay (ELISA).The culture supernatants of selected clones and ascites recoveredfrom nude mice injected with the hybridomas were used for theexperiments. Subclasses of the MAbs were determined by a geldiffusion reaction (QED PermaType Antibody Subtyping Kit,QED Bioscience, San Diego, CA).

2.4. Immunoprecipitation

Either 0.5 μg of Myc/His-tagged netrin-G1d or 1.0 μg ofMyc/His-tagged netrin-G2a in binding buffer (1% Triton X-100, 2 mM EDTA, 100 mM NaCl, and 25 mM Tris–HCl, pH7.5) was incubated with the MAbs at 4 °C for 2 h. Theantigen–antibody complexes were collected on beads (ProteinG Sepharose 4 Fast Flow; Pharmacia Biotech, Wikstroms,Sweden). After washing three times with binding buffer, thebeads were mixed with sodium dodecyl sulfate-polyacryl-amide gel electrophoresis (SDS-PAGE) sample buffer, boiledfor 5 min, and centrifuged at 15,000 ×g for 1 min. Thesupernatant of the samples was fractionated by SDS-PAGEand blotted onto polyvinylidene difluoride membranes

(Immobilon-P; Millipore, Bedford, MA). After blocking themembrane with 5% skim milk in phosphate-buffered saline(PBS), it was incubated with mouse MAb anti-c-myc (1:400)for 1 h at room temperature. After washing three times withPBS supplemented with 0.05% Tween20 (PBSTween), theblots were incubated with HRP-conjugated anti-mouse IgG(1:50,000) for 40 min at room temperature, washed threetimes with PBSTween and developed in Western BlottingLuminol Reagent (Santa Cruz Biotechnology, Santa Cruz,CA).

2.5. Immunohistochemistry

Animals were deeply anesthetized with Avertin (2, 2, 2-tribromoethanol; Aldrich, Milwaukee, WI) and perfused trans-cardially with 4% paraformaldehyde in 0.1 M PB, pH 7.4. Thebrains were removed, post-fixed in the same fixative for 6 h at4 °C, and cryoprotected with 30% sucrose in 0.1 M PB at 4 °Cuntil they sank. Coronal cryosections (20 μm thick) werecut, blocked with M.O.M. mouse IgG blocking reagent (VectorLaboratories, Burlingame, CA) and 5% normal goat serum inPBSTween, and incubated with rabbit polyclonal anti-netrin-G1(1:2000), rabbit polyclonal anti-netrin-G2 (1:500), mouse MAbanti-netrin-G1 (1:500), or mouse MAb anti-netrin-G2 (1:500)overnight at 20 °C. For double immunostaining, the sections weresubsequently incubated with polyclonal anti-GFAP (1:200). Thesections were then washed five times with PBST, incubated with

Fig. 1. Immunoprecipitation of netrin-G1 and netrin-G2 withMAbs 171A18 and30B15. A, Secreted netrin-G1d or netrin-G2a was immunoprecipitated withMAb 171A18. The netrin-G1d was specifically precipitated, while netrin-G2awas not. B, Secreted netrin-G1d or netrin-G2a was immunoprecipitated withMAb 30B15. Netrin-G2a, but not netrin-G1d, was specifically precipitated.MAbs 171A18 and 30B15 were not crossreactive. Pre, before precipitation; Sup,un-precipitated supernatants; Ppt, precipitated fractions. Open and closedarrowheads indicate netrin-G1 and netrin-G2, respectively. Arrows indicateimmunoglobulins used for immunoprecipitation.

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Alexa546-conjugated anti-rabbit IgG (1:1000) or Alexa488-conjugated anti-mouse IgG (1:2000) for 2 h at room temperature,and counterstained with DAPI (1:10,000, Molecular Probes), orwith NeuroTrace Fluorescent Nissl stain reagent (1:100, Molec-ular Probes).

For tracing of axons, 10% solution of biotinylated dextranamines (BDA-10,000; Molecular Probes) was injected into thelateral or medial entorhinal cortex by using a stereotaxic ap-paratus (0.2 μl per injection). Four days after the injection, brainswere fixed with 4% paraformaldehyde in 0.1 M PB for 2 h at4 °C, and coronal sections (40 μm) were cut on a freezingmicrotome. The sections were stained with MAb anti-netrin-G1or anti-netrin-G2 as described above, and then BDA-labeledaxons were visualized with Alexa-conjugated Avidin (5 μg/ml,Molecular Probes).

2.6. Epitope mapping

To determine the epitopes, MAb anti-netrin-G1 (171A18)was incubated with peptide arrays of netrin-G1a and Unknown

domain (Ukd) of netrin-G1d (Nakashiba et al., 2000); and MAbanti-netrin-G2 (30B15) was incubated with peptide arrays ofnetrin-G2a. Each spot was composed of 13 amino acids, and thespot arrays of netrin-G1a, Ukd of netrin-G1d, and netrin-G2acovered each entire amino acid sequence shifted by 3 aminoacids. The peptides were COOH-terminally covalently attachedto each spot of the cellulose membrane (Jerini AG, Berlin,Germany). After washing three times with PBST, themembranes were incubated with HRP-conjugated anti-mouseIgG (1:5000). The signals were detected by chemiluminescenceas described above for immunoprecipitation.

3. Results

Ntng1 KO mice and Ntng2 KO mice were immunized withthe recombinant Myc/His-tagged proteins purified from theconditioned media of HEK293T cells stably secreting netrin-G1d and netrin-G2a, respectively. After the last immunization,spleen cells of the immunized mice were fused with SP2/O cellsand hybridomas were screened by ELISA. By repeated limiting

Fig. 2. Epitope mapping. A, MAb 171A18 was incubated with membranes to which synthetic peptide arrays covering the entire sequences of netrin-G1a and Ukd ofnetrin-G1d shifted by 3 amino acids were attached. Four apparent signals, indicated by the box, were reproducibly detected. B, The amino acid sequences of netrin-G1aand Ukd of netrin-G1d. The putative dominant epitope ofMAb 171A18 is in domain V-1 (LE1), which is indicated by a hatched box. C,MAb 30B15 was reacted withthe membrane to which peptide arrays based on the amino acid sequence of netrin-G2a were attached. Two intense signals, indicated by the box, were reproduciblydetected. D, The amino acid sequence of netrin-G2a. The epitope is in domain C′, indicated by a hatched box.

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dilution of positive cells, we established clone 171A18 fornetrin-G1, and clone 30B15 for netrin-G2, respectively. Geldiffusion reaction revealed that both MAbs were IgG1.

The specificities of MAbs 171A18 and 30B15 were ex-amined by immunoprecipitation. These MAbs were incubatedwith Myc/His-tagged netrin-G1d and Myc/His-tagged netrin-

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G2a, and successful immunoprecipitation was detected byWestern blotting using anti-c-myc antibodies. MAb 171A18specifically precipitated netrin-G1d (Fig. 1A, lane3), but notnetrin-G2a (Fig. 1A, lane 6). Similarly,MAb 30B15 precipitatednetrin-G2a (Fig. 1B, lane 6), but not netrin-G1d (Fig. 1B, lane3). Furthermore, immunocytochemistry was performed withcells stably expressing one of the isoforms, such as netrin-G1a,-G1c,-G1d,-G1e, and-G2a. MAb 171A18 reacted with cells ex-pressing netrin-G1a,-G1c,-G1d, and -G1e, but not with netrin-G2a (data not shown).MAb 30B15 reacted with cells expressingnetrin-G2a, but not with any of the other cells (data not shown).None of these MAbs were applicable for Western blot analyses.These results indicate that MAbs 171A18 and 30B5 selectivelyrecognize epitopes representing native forms of netrin-G1 andnetrin-G2, respectively, with no crossreactivity, and that MAb171A18 has no isoform selectivity.

Recent studies indicate that netrin-G1 and netrin-G2 interactselectively with NGL-1 and NGL-2 at their extracellulardomains, respectively (Lin et al., 2003; Kim et al., 2006;Nishimura-Akiyoshi et al., 2007). Co-immunoprecipitationexperiments were performed to examine if the MAbs are ableto access the ligand-receptor complex. The netrin-G/NGLcomplexes were incubated with the MAbs, and precipitateswere examined with anti-FLAG and anti-c-myc antibodies forNGLs and netrin-Gs, respectively. MAb 171A18 specificallyimmunoprecipitated netrin-G1/NGL-1 complexes (data notshown). Similarly,MAb 30B15 specifically immunoprecipitatednetrin-G2/NGL-2 complexes (data not shown). These resultssuggest that epitopes for these antibodies are sufficiently distantfrom the ligand-receptor interaction sites.

We used peptide arrays to directly determine the epitopes.MAb 171A18 or 30B15 was incubated with membranes towhich synthetic peptide arrays covering the entire sequences ofnetrin-G1a, netrin-G2a, or Ukd of netrin-G1d shifted by 3 aminoacids were attached. HRP-conjugated anti-mouse IgG wasreacted with the membranes and the signals were detected bychemiluminescence (Fig. 2). In the case of MAb 171A18, threeindependent experiments reproducibly exhibited four apparentsignals in domain V-1 (LE1) of netrin-G1 (Fig. 2A). Otherscattered signals in Fig. 2A are likely non-specific, because theywere not reproducible. No signals were detected in the Ukd ofnetrin-G1d. The results indicated that four amino acids (KNYQ;positions 331-334; Fig. 2B) constitute a linear epitope. 30B15produced intense signals for two peptides in domain C′(Fig. 2C). The results indicate that 10 amino acids (PRCDLAD-DAG; positions 490–499; Fig. 2D) constitute a linear epitope.Comparison between mouse netrin-G1 and netrin-G2 sequencesalso supported the non-crossreactivities of MAbs 171A18 and30B15 to these molecules.

One of the most intriguing features of netrin-G1 and netrin-G2is that they distribute in neuronal circuits in most brain areaswithout overlap (Nakashiba et al., 2002; Nishimura-Akiyoshiet al., 2007), and thus provide valuable molecular tags for thesecircuits. To examine whether the MAbs generated here can beused for identifying specific neuronal circuits, we examinedmouse brains immunohistochemically with these MAbs. BothMAbs showed the same patterns of immunoreactivity as those ofspecific rabbit polyclonal antibodies (Fig. 3; Nishimura-Akiyoshiet al., 2007). For example, anti-netrin-G1 antibodies stained thelayers I and IV of the neocortex (Fig. 3A, B), layer Ia of thepiriform cortex (Fig. 3 D, E), and stratum lacunosum-moleculareof CA1 and the outer one-third of the molecular layer of thedentate gyrus in hippocampus (Fig. 3G, H). These immunesignals represent the thalamocortical axons, mitral cell axons,temporoammonic pathway originated from layer III of the en-torhinal cortex (EC), and lateral perforant pathway originatedfrom layer II of the lateral EC, respectively. Anti-netrin-G2antibodies stained the middle one-third of the molecular layer ofthe DG in hippocampus (Fig. 3J, K), representing the medialperforant pathway originated from layer II of the medial EC. Weobserved no immune signals in brain sections from knockoutmice lacking either netrin-G1 or netrin-G2 molecules (Fig. 3C, F,I, L), revealing specificity of these MAbs as well as polyclonalantibodies. To confirm axonal distribution of netrin-G1 andnetrin-G2 in specific neuronal pathways, we carried out doublestaining experiments using fluorescent Nissl stain, anti-GFAP,and BDA tracer together with anti-netrin-G1 and anti-netrin-G2MAbs. The immune signals of both netrin-G1 and netrin-G2 didnot represent neuronal nor astrocytic cell bodies (Fig. 3 M–P).The immune signals of netrin-G1 and netrin-G2 overlapped withDBA-labeled temporoammonic pathway and medial perforantpathway (Fig. 3Q, R), respectively.

4. Discussion

We generated mouse MAbs to netrin-G1 and netrin-G2.Immunocytochemistry and immunoprecipitation analyses re-vealed the specificity of MAbs 171A18 and 30B15 to netrin-G1and netrin-G2, respectively. There were no crossreactivity be-tween MAbs 171A18 and 30B15 in these analyses. These anti-bodies, however, cannot be used for Western blot analysis. Theresults suggest that both MAbs recognize epitopes representingnative forms of proteins. These MAbs did not interfere withbinding between netrin-Gs and their receptors, and thus will serveas useful tools for future biochemical studies of netrin-Gs andNGLs.

Epitope mapping using peptide arrays revealed that MAb171A18 recognizes a linear epitope (KNYQ; positions 331–

Fig. 3. Immunohistochemistry with MAbs 171A18 and 30B15. A–I, Immunostaining of netrin-G1. Wild-type brain samples were stained with polyclonal anti-netrin-G1antibody (A, D, G; red) and with 171A18 (B, E, H; green). Corresponding brain areas of Ntng1 KO mice were used to reveal the specificity of 171A18 (C, F, I). J–L,Immunostaining of netrin-G2. Wild-type brain sample was stained with polyclonal anti-netrin-G2 antibody (J; red) and with 30B15 (K; green). Corresponding brain area ofNtng2 KO mice was used to reveal the specificity of 30B15 (L). M, Hippocampas stained with 171A18 (green) and Fluorescent Nissl (red). N, Hippocampas stained with30B15 (green) and Fluorescent Nissl (red). O, Hippocampas stained with 171A18 (green) and anti-GFAP (red). P, Hippocampas stained with 30B15 (green) and anti-GFAP(red). Q, The temporoammonic pathway labeled with BDAwas immunoreactive to 171A18. R, The medial perforant pathway labeled with BDAwas immunoractive to30B15. DG, dentate gyrus; MML,middle molecular layer; OML, outer molecular layer; SLM, stratum lacunosum-moleculare; TA, temporoammonic pathway; MPP, medialperforant pathway. Bar: 200 μm (A–F, M–P); 400 μm (G–L); 100 μm (Q, R).

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334) in domain V-1 (LE1) of netrin-G1, which is common to allisoforms. This was consistent with findings that MAb 171A18reacted with all cell lines tested that express one of the variousnetrin-G1 splicing variants. Similar experiments reproduciblyindicated that MAb 30B15 recognizes a linear epitope(PRCDLADDAG; positions 490–499) in domain C′. Thoughwe did not directly examine its reactivity to splicing variants,the results suggest that MAb 30B15 recognizes netrin-G2b and-G2c, as well as netrin-G2a.

Alignments of the amino acid sequences of mouse netrin-G1a and orthologues of other species indicate that the linearepitope of MAb 171A18 is conserved in rat, human, dog, andchicken, but not in zebrafish (data not shown). Thus, MAb171A18 can be used for various species, including humans.Similar study suggests that MAb 30B15 can be applied at leastfor rats (data not shown).

In this study, we obtained MAbs that distinguish netrin-G1and netrin-G2. They bind each target protein specificallyregardless of the isoform, recognize the native conformations ofthe proteins, react in paraformaldehyde-fixed tissue, and do notinterrupt ligand-receptor binding. These antibodies are usefulfor biochemical analyses of netrin-G1 and netrin-G2, andprovide valuable tools for labeling specific neuronal circuitsin vivo.

Acknowledgements

We thank C. Itakura for encouragement. This work wassupported in part by the Special Postdoctoral Researchers Pro-gram of RIKEN (S.N.-A.), a Grant-in-Aid for Young Scientistsfrom the Ministry of Education, Culture, Sports, Science, andTechnology (to S.N.-A.), and the Strategic Programs for Researchand Development from RIKEN (S.I.).

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