Int J Pept Res Ther DOI 10.1007/s10989-013-9379-3

Inhibition of Neurite Outgrowth by a Neuropilin-1 Binding Peptide Derived from Semaphorin 3A Masayuki Kohno • Koji Ohara • Tomohisa Horibe Koji Kawakami

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Accepted: 30 October 2013 Ó Springer Science+Business Media New York 2013

Abstract Semaphorin 3A (Sema3A), an axon guidance molecule, inhibits neurite outgrowth of sensory neurons. Recombinant Sema3A protein has also inhibited scratching behavior and improved skin inflammation in an atopic dermatitis model. In the present study, we investigated whether Sema3A-derived peptides could bind its receptor, neuropilin-1 (NRP1), to inhibit neurite outgrowth. Here, two candidate NRP1-binding (NPB) peptides, NPB7 and NPB15, were found to inhibit NGF-induced survival and neurite outgrowth of PC12 cells and rat primary neurons in serum-free medium. To investigate the preventive effect of the two NPB peptides in vivo, we assessed whether they could inhibit skin inflammation induced by repeated topical application of oxazolone in mice. NPB15 peptide, but not NPB7, inhibited ear swelling. The NPB15 peptide solution and Vaseline ointment groups showed slightly decreased epidermal nerve densities compared with controls. The combination of NPB15 peptide and Vaseline ointment increased the inhibitory effect of NPB15 peptide on epidermal nerve densities. These results suggest that Sema3Aderived peptides can bind to NRP1 and inhibit neurite outgrowth both in vitro and in vivo. Thus, these peptides may be potent candidates for the treatment of atopic dermatitis.

Electronic supplementary material The online version of this article (doi:10.1007/s10989-013-9379-3) contains supplementary material, which is available to authorized users. M. Kohno
K. Ohara
T. Horibe
K. Kawakami (&) Department of Pharmacoepidemiology, Graduate School of Medicine and Public Health, Kyoto University, Yoshidakonoecho, Sakyoku, Kyoto 606-8501, Japan e-mail: [email protected]

Keywords Semaphorin 3A
Peptide
Neurite outgrowth
Neuropilin-1
Atopic dermatitis Abbreviations AD Atopic dermatitis cGMP Cyclic guanosine monophosphate END Epidermal nerve density FBS Fetal bovine serum NGF Nerve growth factor NPB NRP1-binding NRP1 Neuropilin-1 OCD Oxazolone-induced chronic dermatitis rhNRP1 Recombinant human NRP1 rhSema3A Recombinant human semaphorin 3A/Fc chimera Sema3A Semaphorin 3A SPR Surface plasmon resonance

Introduction Semaphorin 3A (Sema3A) acts as a repellent for neurites by binding to a receptor complex composed of a binding subunit neuropilin-1 (NRP1) and a signaling transducer plexin A (Takahashi et al. 1999; Fan and Raper 1995). Sema3A is a secreted molecule that contains sema, plexin– semaphorin–integrin, and immunoglobulin-like domains, as well as a basic C-terminal tail. Nerve fiber outgrowth is regulated by several molecules, including nerve growth factor (NGF) and Sema3A, which induce retraction of NGF-sensitive neurons. NGF produced by keratinocytes is one of the major growth factors involved in the epidermal nerve invasion of atopic dermatitis (AD), and recombinant Sema3A protein was shown to alleviate skin inflammation

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and scratching behavior in an AD model (Yamaguchi et al. 2008; Negi et al. 2012). Biopharmaceuticals, like antibodies and recombinant proteins, are expensive compared with conventional drugs due to the high manufacturing costs. Moreover, one of the main limitations for their usefulness is immunogenicity, and the development of neutralizing antibodies to biopharmaceuticals has an impact on their clinical efficacy (Alawadhi et al. 2012; Hesse and Sørensen 2007). Biopharmaceuticals are also larger than conventional drugs, which often necessitates the use of a suitable drug delivery system to overcome barriers to their application. For example, although some small molecules (\500 Da) are thought to penetrate the skin barrier, many proteins cannot (Bos and Meinardi 2000). Therapeutic peptides are becoming increasingly popular in a variety of applications, such as in anticancer and antimicrobial therapies, and drug delivery (Yeung et al. 2011). Peptide drugs are synthesized relatively easily, via either recombinant or solid-phase chemical synthesis techniques, and the production costs are generally cheaper than those of biopharmaceuticals. We previously designed a novel molecular-targeted drug termed a ‘‘hybrid peptide’’, which is composed of a target-binding peptide and a lytic peptide that act mainly by disintegrating the cancer cell membrane (Ueyama et al. 2011; Kohno et al. 2011; Kawamoto et al. 2011, 2013a, b; Yang et al. 2012; ). NRP1 is highly expressed in various cancers and a suitable target for cancer therapy (Parikh et al. 2003; Hong et al. 2007), and we previously examined the important sequence of Sema3A for binding to NRP1. Shirvan et al. (2000) showed that the specific antibody against the peptide at position 363–380 of Sema3A induced apoptosis of neuronal cells. Antipenko et al. (2003) showed that the recombinant Sema3A protein containing a deletion of the position 359–366 could not bind to NRP1. Teesalu et al. (2009) showed that peptides internalized into cells via NRP1 had the sequence of R/KXXR/K at the C-terminal (C-End rule). Based on the information as reported above, we confirmed that the NPB15 peptide at the position 363–377 could bind to NRP1 using Biacore system, and designed Sema3A-lytic hybrid peptide, in which NPB15 was combined with lytic peptide. We also recently developed a Sema3A-lytic hybrid peptide that targets NRP1-expressing cancer cells, and we demonstrated that the Sema3A-lytic peptide exerts cytotoxic activity against various pancreatic cancer cell lines (Ueyama et al. 2011). In this study, we selected NPB15 and NPB7 peptides containing C-End rule as a useful candidate of NRP1 agonist, and compared the ability of Sema3A-derived peptides to bind to NRP1 and inhibit neurite outgrowth to that of recombinant Sema3A protein. We also explored the possible use of Sema3A-derived peptides for treating AD.

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Materials and Methods Drugs and Materials 4-Ethoxymethylene-2-phenyl-2-oxazolin-5-one (oxazolone) was purchased from Sigma (USA) and dissolved in acetone as a 0.5 % (w/v) solution. Vaseline was purchased from WAKO Pure Chemicals (Japan). Recombinant human NRP1 (rhNRP1) was purchased from R&D Systems (USA). Recombinant human semaphorin 3A/Fc chimera (rhSema3A) and recombinant human b-NGF were purchased from Peprotech (USA). Peptide Synthesis NPB15 (NYQWVPYQGRVPYPR) and NPB7 (GRVPYPR) peptides were synthesized by Invitrogen (USA). The peptides were dissolved in water and then diluted in ethanol. Animals Female 5-week-old ICR mice were purchased from SLC Japan. Animal experiments were carried out in accordance with the guidelines of the Kyoto University School of Medicine. Biomolecular Interaction Analysis Surface plasmon resonance (SPR) experiments were performed with a Biacore T100 system (GE Healthcare, USA) as described previously (Frostell-Karlsson et al. 2000). All data analysis was performed with Biacore T100 Evaluation Software version 2.0.2. Cells and Cell Culture Conditions The PC12 cell was purchased from the Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer Tohoku University, Japan. PC12 cells were maintained in DMEM medium containing 10 % fetal bovine serum (FBS), 100 lg/ml penicillin, and 100 lg/ml streptomycin. WST Assay PC12 cells were plated onto 96-well plates at 3 9 103 cells per well in medium containing 1 % FBS (for cytotoxicity), or onto 96-well plates coated with poly-L-lysine at 1 9 104 cells per well in serum-free medium with or without 3 ng/ mL NGF (for cell survival). Cells were treated with rhSema3A and NPB peptides for 3 days. Cell viability was

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measured with WST-8 solution (Cell Count Reagent SF; Nacalai Tesque, Japan). Primary Cultures Cortical neurons were cultured as described previously (Sasaki et al. 1998). Briefly, pregnant Sprague–Dawley rats were killed by CO2 exposure, and embryos (at embryonic day 17) were immediately removed from the uterus. The presumptive embryonic cortex was dissected in cold L15 medium and digested for 10 min at 37 °C in Accumax (PHOENIX Flow Systems, USA). The tissue was then triturated to a single-cell suspension and plated on poly-Llysine-coated dishes in Neurobasal Media containing B27 (Invitrogen). Neurite Outgrowth Assay PC12 cells were plated onto dishes coated with poly-Llysine in serum-free medium and primed with 25 ng/mL of NGF for 3 days. The PC12 cells or primary neurons were then plated onto 96-well plates coated with poly-L-lysine at 3 9 103 cells per well in serum-free medium with or without 3 ng/mL NGF, and treated with rhSema3A and NPB peptides for 3 days. The cells were fixed with 1 % glutaraldehyde and then stained with 0.2 % crystal violet. In each of the triplicate wells, the neurite outgrowth number from the cells was counted (Kato et al. 2002). Oxazolone-Induced Chronic Contact Hypersensitivity Response The experimental scheme is shown in Online Resource 1. The ICR mice were sensitized and challenged with oxazolone as described previously (Tamura et al. 2004). The right ear was sensitized by a single application of 10 ll (5 ll each for the inner and outer ear) of 0.5 % oxazolone in acetone 6 or 7 days before the first challenge (day 0). Ten microliters of 0.5 % oxazolone in acetone were applied repeatedly to the sensitized right ear 2 or 3 times per week. In the non-sensitized animals, acetone alone was applied to the right ear. NPB7 or NPB15 peptides were applied topically at 40 or 25 lg/ear day, respectively, to the right ear in a volume of 10 ll/ear (5 ll each for the inner and outer ear). In the NPB15 ointment treatment, NPB15 peptide was mixed with Vaseline on paraffin film immediately before application. Each drug was administered 2 or 4 times per oxazolone challenge. On the day of the oxazolone challenge, each drug was administered 30 min before the challenge. The changes in the ear thickness were measured with a dial caliper. Two days after the final challenge, the animals in each group were sacrificed to remove the ears.

Measurement of Scratching Behavior The scratching behavior of the mice was recorded with a digital video camera (Olympus, Japan) for 30 min at 30 min after the oxazolone challenge on day 7. The number of scratching actions was counted from the video playback. One scratching action was considered to be a series of scratch movements on the right ear with the hind paws, as reported previously (Tsukumo et al. 2010). Immunohistochemical Staining Harvested right ear samples were fixed in 4 % neutralbuffered paraformaldehyde, embedded in paraffin, and sectioned. The 5-lm-thick paraffin sections were deparaffinized with lemosol and graded ethanol. Endogenous peroxidases were quenched with 0.5 % hydrogen peroxidase in PBS for 30 min at room temperature. After washing with PBS, sections were incubated for 30 min at room temperature with 5 % skimmed milk (Nacalai Tesque). Sections were incubated at 4 °C overnight with a polyclonal rabbit antibody against PGP 9.5 (1:400; UltraClone, UK). After washing with PBS, the sections were incubated with a HRP-conjugated anti-rabbit IgG secondary antibody (GE Healthcare). The peroxidase reaction was visualized by incubating the sections in diaminobenzidine (Dako). Sections were then counterstained with hematoxylin, washed, and mounted. The areas of the epidermis and the numbers of immunoreactive (PGP 9.5-positive) nerve fibers in the epidermis were quantified with imaging software (Image JÒ, NIH), and the epidermal nerve density (END) was then calculated (Okawa et al. 2012). Statistical Analysis Statistical differences were determined using the Student’s t test. P values less than 0.05 were considered statistically significant.

Results NRP1-Binding Peptides Derived from Sema3A are Highly Conserved Among Mammals and Bind Recombinant Human NRP1 In our previous study, we designed an NPB15 peptide containing 15 amino acids that we confirmed to be an NRP1-binding peptide based on crystal structure analysis of the sema domain of Sema3A (Ueyama et al. 2011). This region was shown by sequence analysis to be highly conserved among human, mouse, and rat (Fig. 1a). Two arginine residues contained in the NPB15 peptide are

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A Human (AA353-387) LGPYAHRDGPNYQWVPYQGRVPYPR PGTCPSKTFG Mouse (AA353-387) LGPYAHRDGPNYQWVPYQGRVPYPR PGTCPSKTFG Rat (AA353-387) LGPYAHRDGPNYQWVPYQGRVPYPR PGTCPSKTFG NPB15 NYQWVPYQGRVPYPR NPB7 GRVPYPR

outgrowth was counted. We found that NGF-induced neurite outgrowth was inhibited by rhSema3A and NPB peptides (Fig. 2d). NPB Peptides Inhibit NGF-Induced Neurite Outgrowth in Rat Primary Neurons

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Fig. 1 NPB peptides derived from Sema3A are highly conserved between humans and mice and are able to bind NRP1 protein. a The sequences of the sema domain (amino acid residues 353–387) of Sema3A protein from human, mouse, and rat were aligned by using ClustalW, and are shown in conjunction with the sequences of the NPB15 (363–377) and NPB7 (371–377) peptides. b The binding abilities of the NPB15 and NPB7 peptides were compared by biosensor analysis. The biosensor response (RU/Da) at 13 lM of each peptide concentration was calculated as described in the ‘‘Materials and methods’’ section

important for its interaction with NRP1 (Ueyama et al. 2011). To confirm the ability of an NPB7 peptide containing these two arginines to bind to NRP1, rhNRP1 protein was immobilized on sensor chips, and its interaction with NPB7 and NPB15 peptide was analyzed by the Biacore system. As shown in Fig. 1b, biophysical analysis demonstrated that the NPB7 peptide also had the ability to interact with rhNPR1 immobilized on the sensor chip and that the binding activity was higher than that of NPB15 peptide. Thus, we decided to use these two peptides in subsequent experiments. NPB Peptides Inhibit NGF-Induced Neurite Outgrowth in PC12 Cells We next assessed the effect of rhSema3A and NPB peptides on the cell viability of PC12 cells in medium containing 1 % FBS. Neither rhSema3A nor NPB peptides exhibited a cytotoxic activity in PC12 cells (Fig. 2a). We examined the inhibitory effect of rhSema3A and the NPB peptides on NGF-induced cell survival of PC12 cells. NGF-induced cell survival was inhibited by rhSema3A and the NPB peptides in a concentration-dependent manner (Fig. 2b). We then assessed the inhibitory effect of rhSema3A and the NPB peptides on the NGF-induced neurite outgrowth of PC12 cells with crystal violet staining. NGF promoted cell attachment and neurite outgrowth in PC12 cells (Fig. 2c). Based on these staining results, neurite

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We attempted to confirm the effect of the NPB peptides on rat primary neurons prepared from embryos before conducting the in vivo experiments. We assessed the inhibitory effect of rhSema3A and the NPB peptides on NGF-induced neurite outgrowth of rat primary neurons with crystal violet staining, finding that NGF-induced neurite outgrowth was inhibited by rhSema3A and the NPB peptides (Fig. 3a, b). Effect of NPB Peptides on Skin Inflammation Induced by Repeated Topical Application of Oxazolone in Mice To compare the preventive effect of the two NPB peptides on skin inflammation in vivo, we examined whether these peptides could inhibit the inflammation induced by repeated topical application of oxazolone in mice. In the oxazolone-challenged group, the ear thickness significantly increased throughout the experimental period (from day 2 onward). The NPB15 peptide suppressed the increase in ear thickness at days 7 and 9. In contrast, the NPB7 peptide failed to block the increase in ear thickness (Fig. 4a). We then assessed topical application of the NPB15 peptide (mixed with Vaseline in the form of an ointment) in the oxazolone-induced dermatitis model as a means to enhance the treatment efficacy of NPB15. Treatment with the NPB15 solution (group III) and NPB15 ointment (group IV) blocked the increase in ear thickness at days 7 and 9 (Fig. 4b). In the control group (II), the ear weight significantly increased (by 2.4-fold) compared with that of the acetone-treated group (I) at day 9 (Fig. 4c). Treatment with the NPB15 solution and NPB15 ointment suppressed the increase in ear weight by 47.4 and 45.0 %, respectively. To assess the effect of the NPB15 peptide on END, we performed immunohistochemical analysis of nerve fibers with an antibody against PGP9.5, which is expressed specifically by neurons. In the dermatitis model caused by repeated exposure to oxazolone, ear thickness significantly increased in a challenge-dependent manner. Furthermore, the epidermis was thicker and the END of the oxazolonetreated group was lower than that of the acetone-treated group (data not shown). Among the four oxazoloneexposed groups, the NPB15 solution (III) and Vaseline ointment (V) slightly decreased the END compared with the control group. The combination of the NPB15 peptide and the Vaseline ointment (IV) enhanced the inhibitory effect of the NPB15 peptide (Fig. 4d).

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Fig. 2 NPB peptides inhibit NGF-induced neurite outgrowth in PC12 cells. a Effect of rhSema3A and NPB peptides on cell viability. b Inhibitory effect of rhSema3A and NPB peptides on NGF-induced cell survival of PC12 cells in serum-free medium. c Crystal violet staining of NGF-induced neurite outgrowth. Scale bar, 200 lm.

d Inhibitory effect of rhSema3A and NPB peptides on NGF-induced neurite outgrowth. The results are presented as the mean ± SEM. (bars) of triplicate determinations and the assay was repeated three times

The number of scratch attempts was measured from 30 to 60 min after the last oxazolone challenge. Repeated challenges with oxazolone increased the number of scratches. The NPB15 solution, Vaseline ointment, and their combination could not suppress scratching frequency compared with the control (Fig. 4e).

responses from repulsion to attraction in the presence of cGMP (Song et al. 1998). Low-dose Sema3A was found to rapidly induce desensitization of growth cone collapse via clathrin-mediated endocytosis, which inhibited growth cone collapse by high-dose Sema3A (Piper et al. 2005). It is reported that the short peptide including adjacent two arginines at C-terminal was internalized via NRP1 (Teesalu et al. 2009). To achieve a stable inhibitory effect of NPB peptides on neurite outgrowth, further trials are required. Sema3A is expressed in activated dendritic cells, T cells, and some tumor cells. In general, Sema3A negatively regulates immune responses. Since Sema3A suppressed T cell proliferation by inhibiting actin cytoskeleton reorganization (Lepelletier et al. 2006; Catalano et al. 2006), and Sema3A regulated the entry of dendritic cells into the lymphatic system (Takamatsu et al. 2010), the inhibitory effect of NPB15 peptide on ear swelling may involve other mechanisms that are distinct from those of neurons, such as via regulation of the immune system. The PC12 cell line is a rat pheochromocytoma cell line that has been used widely as a model of neuronal

Discussion The NPB7 peptide exerted a stronger inhibitory effect on neurite outgrowth in vitro than that of NPB15 peptide (Figs. 1, 2, 3). However, in the in vivo study, though the NPB15 peptide inhibited oxazolone-induced ear swelling, the NPB7 peptide increased the swelling, and the NPB15 peptide could not inhibit oxazolone-induced scratching behavior (Fig. 4). It is possible that the dichotomic or multiple functions of Sema3A influence these results. Although Sema3A was initially identified based on its growth cone collapse-inducing activity (Luo et al. 1993), it was subsequently found to convert neuronal growth cone

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Fig. 3 Inhibitory effect of rhSema3A and NPB peptides on NGFinduced neurite outgrowth of rat primary neurons. a Rat primary neurons were cultured with or without 3 ng/ml NGF in serum-free medium for 3 days and stained with crystal violet. Scale bar, 200 lm. b The number of neurites of the primary neurons were counted. The results are presented as the mean ± SEM. (bars) of triplicate determinations and the assay was repeated three times

differentiation in response to NGF and other agents instead of primary culture neurons (Rukenstein et al. 1991). In addition, it is known that NGF induces cell survival and neurite outgrowth of PC12 cells in serum-free medium (Rukenstein et al. 1991), and we used PC12 cells and rat primary neurons in vitro studies. In this study, we used the repeated oxazolone-induced chronic dermatitis (OCD) model to assess the efficacy of NPB peptides in inhibiting neurite outgrowth in vivo. Increased END and NGF production in the lesioned skin, as well as a severe scratching behavior, were previously observed in response to the oxazolone challenge in this model (Tamura et al. 2004; Kakurai et al. 2006). The AD model of NC/Nga mice is often used, but its incidence of AD-like lesions under conventional condition is low and the first sign of skin changes occurs after 6–8 weeks, unless an expensive Dermatophagoides farinae body ointment is used (Negi et al. 2012; Shiohara et al. 2004). On the other hand, in the OCD model, the incidence of ear dermatitis is very high, quantitative and sequential data of ear pachyderma is easily obtained, the experimental period is short,

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and a low amount of drug is required. OCD is reported to be associated with a shift in the local cytokine pattern from the Th1- to the Th2-type profile and the increased infiltration of inflammatory cells into the lesioned skin (Tamura et al. 2004). As described above, because the symptoms in the OCD model are also observed in AD patients, this OCD model would also be an appropriate model for human AD. Itching and scratching are troublesome and inexorable symptoms of AD and markedly reduce the quality of life of affected patients (Koblenzer 1999; Ikoma et al. 2006). Itchassociated scratching injures the skin and aggravates the inflammation, which in turn induces a stronger itching that is associated with a hedonic sensation. Thus, the itchscratch cycle develops easily and aggravates the skin condition. For the treatment of itching, antihistamines are commonly used as the drugs of first choice, but they often fail to suppress the itching in AD patients (Klein and Clark 1999). The sprouting of epidermal nerve fibers has been found in AD patients (Urashima and Mihara 1998), and this phenomenon is possibly induced by NGF that is mainly released from keratinocytes (Ikoma et al. 2006). Anti-NGF antibody inhibited scratching behavior and epidermal nerve fibers on the established dermatitis of the NC/Nga mice (Takano et al. 2005). However, in the clinical application of protein drugs such as Sema3A and anti-NGF antibody for AD patients, there are various obstacles to overcome. Patients with severe AD need to apply drugs to vast areas of the skin of the entire body every day, and abundant protein drugs are needed for these patients. Use of protein drugs instead of steroids for the treatment of AD would be difficult, because protein drugs have a number of problems. For example, biopharmaceuticals cost much more per patient on average than conventional pharmaceuticals because manufacturing plants must have a sufficient capability for quality-controlled large-scale production and the purification of protein drugs to remove potential contaminants such as endotoxins and viral particles (Ahmed et al. 2012). In addition, most protein drugs induce greater immune responses than small compounds. It is not only monoclonal antibodies, but also interferons and a soluble tumor necrosis factor receptor, Etanercept, that show high immunogenicity (Alawadhi et al. 2012; Hesse and Sørensen 2007; Kessler et al. 2006). The development of neutralizing antibodies to these protein drugs is associated with a reduced efficacy, increased instances of dose escalation, and, ultimately, increased discontinuation of treatment (Alawadhi et al. 2012). Many proteins also show a low stability, due to degeneration and aggregation at room temperature, which complicates the development of protein drug-containing ointments. On the other hand, many short peptides have a reduced immunogenicity and a lower price than protein drugs (Krumpe and Mori 2006). In clinical applications of

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Fig. 4 Effect of NPB peptides on skin inflammation induced by repeated topical application of oxazolone in mice. a Comparison of treatment efficacy between NPB7 and NPB15 peptides. NPB7 (40 lg/ mouse day) and NPB15 (25 lg/mouse day) were applied topically under the preventive treatment protocol (Fig. S1), as described in the Materials and methods section. Each point represents the mean ± S.E.M. of three mice. b–e Comparison of the treatment efficacy of NPB15 between an ethanol solution and Vaseline ointment. NPB15

(25 lg/mouse day) was dissolved in 75 % ethanol or mixed with Vaseline and applied topically according to the treatment protocol. b Ear swelling. c Ear weights on day 9. d Epidermal nerve fibers. e Scratching behavior on day 7. OX oxazolone, I Acetone, II OX?ethanol, III OX?NPB15 solution, IV OX?NPB15 & Vaseline ointment, V OX?Vaseline ointment. Bar in D and E, mean. Each point indicates the mean ± SEM. of 3–4 mice (I–IV, n = 4; V, n = 3). *P \ 0.05, significantly different compared to group II

antipruritic drugs for AD, small compounds or peptides would be a better replacement for conventional ointments such as topical corticosteroids and immunosuppressants than proteins. Thus, the findings of this study will aid the further development of peptide-based treatments for AD that act by targeting NRP1.

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Acknowledgments We thank Keiko Shimoura and Kumi Kodama (Department of Pharmacoepidemiology, Kyoto University) for technical assistance. Conflict of interest The authors declare that they have no conflict of interest. All institutional and national guidelines for the care and use of laboratory animals were followed.

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