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Detection of new anti-neutral glycosphingolipids antibodies and their effects on Trk neurotrophin receptors
FEBS Letters 580 (2006) 4991–4995
Detection of new anti-neutral glycosphingolipids antibodies and their effects on Trk neurotrophin receptors T. Miharaa, A. Uedaa, M. Hirayamab, T. Takeuchia, S. Yoshidad, K. Naitoc, H. Yamamotoa, T. Mutoha,* a
Department of Neurology, Fujita Health University School of Medicine, 1-98 Dengakugakubo, Kutsukake-cho, Toyoake, Aichi 470-1192, Japan b Department of Neurology, Kasugai Municipal Hospital, Kasugai, Aichi 486-8510, Japan c Department of Otolaryngology, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan d The Department of Internal Medicine, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan Received 4 August 2006; accepted 8 August 2006 Available online 17 August 2006 Edited by Sandro Sonnino
Abstract We screened sera from patients with various neurological disorders for the presence of anti-neutral glycosphingolipids antibodies and only found them in sera from relapsing polychondritis with limbic encephalitis patients. Neutral glycosphingolipids are resident in membrane lipid rafts where high affinity nerve growth factor (NGF) receptor, Trk is co-localized. Therefore, we examined whether these antibodies influence the action of NGF in NGF-responsive cells. The results strongly suggest that these antibodies enhance NGF-induced Trk autophosphorylation and neurite outgrowth as well as neurofilament M expression. These data strongly indicate that these anti-neutral glycosphingolipids antibodies have an functional impact on NGF-Trk-mediated intracellular signal transduction pathway. 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Neutral glycosphingolipids; Glucosylceramide; Galactosylceramide; Trk; Tyrosine phosphorylation; Nerve growth factor
1. Introduction There is an accumulating evidence that suggests the pathognomonic linkage between anti-gangliosides antibodies and acute motor axonal neuropathy and the cranial, bulbar and sensory variants of Guillain-Barre´ syndrome [1]. Inspite of so many publications at present, their real roles for the development of the neuropathies still remain to be elucidated. Gangliosides are acidic glycosphingolipids comprising a ceramide moiety embedded in the lipid bilayer and sialylated oligosaccharide core that is extracellularly displayed and capable of acting as an autoantibody target [2]. On the contrary, there is a few investigation on anti-neutral glycosphingolipids antibodies in neurological disorders. Major neutral glycosphingolipids include glucosylceramide (GlcCer), lactosylceramide (LacCer), globotriaosylceramide, globoside or galactosylceramide (GalCer). It has been only reported that
* Corresponding author. Fax: +81 562 93 1856. E-mail address: [email protected] (T. Mutoh).
anti-GalCer antibody is present in patients with GBS after Mycoplasma pneumoniae infection [3]. GlcCer is one of the cardinal neutral glycosphingolipids. It is located in the plasma membrane primarily in lipid rafts, a special membrane structure that also contains many signaling moieties such as receptor-type tyrosine kinases, cholesterol, sphingomyelin, and glycosphingolipids [4]. GlcCer is synthesized from ceramide through the enzymatic action of GlcCer synthase and constitutes the first step in the synthesis of all types of gangliosides. GlcCer is involved in the regulation of many biological events such as neuronal apoptosis and drug resistance to anti-cancer drugs [5,6]. Additionally, GlcCer induces neuronal growth and epidermal differentiation [7,8]. In this study, we screened sera from patients with various kinds of neurological disorders for the presence of an antibody against neutral glycosphingolipids and found novel anti-neutral glycosphingolipids antibodies against GlcCer and GalCer but not LacCer only in sera from the patients with relapsing polychondritis (RP) complicated by limbic encephalitis (LE)like symptoms. These antibodies were not found in sera from normal controls or patients with other neurological disorders to include uncomplicated RP. Then, we tried to characterize these anti-neutral glycosphingolipid antibodies using rat pheochromocytoma cell-line, PC12 cells. Unexpectedly, these novel antibodies enhanced the action of nerve growth factor (NGF) in cultured PC12 cells. This enhancement was mediated through NGF-induced Trk, a high affinity nerve growth factor receptor exhibiting tyrosine kinase activity, autophosphorylation response. The data clearly suggests that anti-neutral glycosphingolipids antibodies have an impact on the Trkmediated intracellular signaling pathway.
2. Materials and methods 2.1. Human sera After obtaining informed consent, sera were taken from various neurological patients (13 patients with viral encephalitis, 5 patients with paraneoplastic neurological syndrome, 5 patients with Guillain-Barre´ syndrome, 4 patients with Sjo¨gren syndrome, and 5 patients with Parkinson’s disease, 3 patients with RP complicated by LE and 2 patients with RP without LE) and 13 normal controls. The samples were stored at 20 C until use. All of the RP + LE patients exhibited psychiatric symptoms such as memory disturbance and delusions. The development of these symptoms coincided with the onset of canonical symptoms of RP such as ear swelling and erythema.
0014-5793/$32.00 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2006.08.013
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T. Mihara et al. / FEBS Letters 580 (2006) 4991–4995
2.2. Anti-neutral glycosphingolipids antibodies To evaluate the patients’ sera for the presence of anti-neutral glycosphingolipids antibodies, purified GlcCer (a generous gift from Dr. Hirabayashi Y. Brain science Institute, RIKEN, Wako, Japan), GalCer (a generous gift from Dr. Furukawa K., Nagoya University School of Medicine, Nagoya, Japan), and LacCer (BioMol, USA) were processed by thin-layer chromatography (using a solvent of chloroform, methanol, and distilled water in a 55:20:3 vol/vol/vol ratio) and blotted onto a polyvinylidene difluoride membrane by an electrothermal blotter (ATTO Co. Ltd., Tokyo, Japan) as described previously [9]. This polyvinylidene difluoride (PVDF) membrane was probed with human sera (500· dilution) in blocking buffer (2% non-fat milk in the wash buffer phosphate-buffered saline (PBS) containing 1% Triton X-100). Following application of the second antibody, a positive band was detected with an enhanced chemiluminescence reagent (Perkin–Elmer Inc., Boston, MA). In some case, we also examined the presence of anti-GlcCer antibody using chemically synthesized pure GlcCer (from Dr. Y. Hirabayashi). 2.3. Cell culture and treatment Rat pheochromocytoma-derived PC12 cells and stable transfectants of human trk-complimentary DNA (PCtrk cells) were cultured as described [10]. The cells were cultured as monolayers in DMEM containing 7.5% FBS, 100 U/ml penicillin and 100 lg/ml streptomycin [11]. Cells were incubated at 37 C in a humidified 5% CO2 atmosphere. For morphological observation, cells were cultured in 24-well culture plates and examined using a phase contrast microscope [12]. Following photography, the cells were lysed in sodium dodecyl sulfate (SDS)sample buffer and evaluated by immunoblot analysis using an antineurofilament M antibody for neuronal differentiation as described previously [11].
Fig. 2. Immunoreactivity against chemically synthesized pure GlcCer. Chemically synthesized pure GlcCer were electrothermally blotted onto a polyvinylidene difluoride membrane. The membrane was probed with sera from patients with RP + LE (1) and from disease controls (2). Red asterisks indicate the positive bands corresponding to GlcCer. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
2.4. Immunoprecipitation and immunoblot analysis Because both neutral glycosphingolipids and Trk are located in the membrane lipid raft, a membrane structure that is also the site of transmembrane signaling, we examined the effect of anti-neutral glycosphingolipids antibodies on the high affinity NGF receptor, Trk. PCtrk cells were preincubated with sera (500· dilution) for 30 min and then stimulated with 50 ng/ml nerve growth factor (NGF) for 5 min. To evaluate receptor activity, Trk was immunoprecipitated with an anti-Trk antibody (a-Trk; Santa Cruz Biotechnology Inc., Santa Cruz, CA) and subjected to immunoblot analysis using either an anti-phosphotyrosine antibody (a-PY, 4G10, Upstate Biotechnol. Inc., Lake Placid, NY) or a-Trk antibody as described [10–12]. In some case, antibody-positive sera were precleared with protein-L agarose to remove all immunoglobulins for 4 h in the cold room with agitation.
Fig. 1. Immunoblot analysis for neutral glycosphingolipids. Neutral glycosphingolipids (GlcCer and LacCer (lanes a(1), b(1) and c(1)), GalCer (lanes a(2), b(2) and c(2)) were electrothermally blotted onto a polyvinylidene difluoride membrane. The membrane was probed with sera from the patients with RP + LE obtained before (a) and after (c) steroid hormone therapy and with sera from patients with only RP (b). The position of each neutral glycosphingolipids was determined on a high-performance thin-layer chromatography plate developed simultaneously without electrothermal blotting. Red asterisks indicate the positive bands corresponding to GlcCer () and to GalCer (). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 3. Effect of sera on Trk-tyrosine kinase activity in response to NGF. PCtrk cells were cultured on 15 cm in diameter culture dishes and were pretreated with serum-free medium for 1 h. (a) Then, cells were treated with sera (·200 diluted in final) (lane 3, patient with RP + LE; lane 4, another patient with RP + LE; same patient with RP + LE obtained after steroid hormone treatment) for 30 min and stimulated with 50 ng/ml NGF (lanes 2–5) or without (lane 1) for 5 min. (b) Cells were treated with sera (·200 diluted in final) (lanes 3, 4, patients with RP only; lane 5, disease control (Parkinson’s disease); lane 6, a normal control) for 30 min and stimulated with 50 ng/ml NGF (lanes 2–6) or without (lane 1) for 5 min. After stimulation, cells were collected with chilled PBS and subjected to the immunoprecipitation as described in ‘‘Section 2’’. These were performed at least three times with identical results. Arrows indicate position of the Trk protein. These precleared sera were subjected to Trk-immunoprecipitation. Detection of positive bands was performed using Western blot Chemiluminescence Reagent Plus (Perkin–Elmer Inc.) [8,10].
T. Mihara et al. / FEBS Letters 580 (2006) 4991–4995 2.5. Measurement of NGF in serum The concentration of NGF concentrations in sera were measured by commercial ELISA kit (Promega, USA) according to the manufacturer’s instructions. To neutralize the effects of serum immunoglobulins, protein-L agarose was added and incubated with agitation for 2 h in the cold. Following agitation, the sera were centrifuged at 12 000 rpm for 2 min and resultant supernatants subject to evaluation. Each evaluation was performed in a triplicate.
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for an anti-neutral glycosphingolipid antibodies augmented NGF-induced Trk autophosphorylation (Fig. 3a). Sera negative for anti-neutral glycosphingolipid antibodies had no effect on NGF-induced Trk autophosphorylation (Fig. 3b). Intriguingly, sera from RP + LE patient obtained after treatment with steroid hormone and precleared with protein-L agarose did not reveal no augmentation of NGF-induced Trk autophosphorylation (compare Fig. 3a(4 and 5) and data not shown).
3. Results 3.1. Detection of anti-neutral glycosphingolipid antibodies A total of 50 serum samples were screened. Positive bands representing antibodies against GlcCer and GalCer were found in sera obtained from patients with RP + LE (Fig. 1a). These antibodies were not found in any of the patients with other neurological disorders to include RP in the absence of LE (Fig. 1b) or in the normal controls (data not shown). Intriguingly, after a course of steroids (pulse therapy followed by oral prednisolone), all of the RP + LE patients who were positive for anti-neutral glycosphingolipid antibodies had a remarkable improvement in their clinical symptoms that was accompanied by a disappearance of these autoantibodies (Fig. 1c). Furthermore, these anti-neutral glycosphingolipids antibodies in patients’ sera actually recognize chemically synthesized pure GlcCer (Fig. 2). 3.2. Effect on NGF-induced Trk autophosphorylation To evaluate the effects of the antibodies-positive sera on NGF-induced Trk autophosphorylation, PCtrk cells were pretreated with sera for 30 min followed by 5 min of NGF stimulation and immunoprecipitated the Trk protein. Sera positive
Fig. 5. NGF concentrations. Serum NGF concentration was measured by ELISA. RP + LE sera before the treatment (column 1, n = 3), with RP + LE after the treatment (column 2, n = 3), disease controls including RP without LE (column 3, n = 34), and normal controls (column 4, n = 13). The columns represent the means ± S.D.
Fig. 4. Morphological effects of RP + LE sera on NGF-induced neurite outgrowth in PCtrk cells. PCtrk cells were cultured on 24-well dish in the presence of 50 ng/ml of NGF (2–4, 7, 8) or absence of NGF (1, 5, 6) for 12 h. Before adding NGF, cells were pre-treated with patient’s serum taken before any treatment (3, 5) or after treatment (4, 6) and from RP patient (7) and normal control (8) for 30 min. We also examined the expression of neurofilament M in the total cell lysates prepared from those used for morphological studies by the immunoblot analysis with anti-neurofilament M antibody. Note that RP + LE patient’s serum before treatment strongly augmented NGF-induced neurite outgrowth in PCtrk cells but not taken after treatment nor disease and normal controls. These experiments were performed three times with identical results. Arrows indicate the position of neurofilament M.
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3.3. Effect on NGF-induced morphological differentiation To reinforce the previous findings, we also examined the effect of sera on NGF-induced morphological differentiation. In PCtrk cells, NGF-induced neurite outgrowth was enhanced by RP + LE patient sera when compared to the effect of NGF alone (Fig. 4). This enhancement was not observed with antineutral glycosphingolipid antibodies-negative sera from controls and other neurologic disorders to include LE negative RP. Additionally, the effect of sera on neurofilament M expression was also examined by immunoblot analysis. As was the case with neurite outgrowth, RP + LE sera taken before the treatment with steroid hormone enhanced neurofilament M expression but not taken after treatment nor disease and normal controls when compared to the effect of NGF alone (Fig. 4). Moreover, precleared sera from patients with RP + LE with protein-L agarose also did not induce such enhancement (data not shown).
3.4. Measurement of serum NGF concentration To test the possibility that serum NGF concentrations are increased in anti-neutral glycosphingolipids antibody-positive sera, NGF concentrations were measured. This is no difference in NGF concentrations between antibody positive and antibody negative sera (Fig. 5).
4. Discussion The present study clearly demonstrates the presence of novel anti-neutral glycosphingolipid antibodies in the sera of patients with RP + LE. These autoantibodies were not found in normal controls or in patients with a variety of other neurological disorders. It is particularly intriguing that all of LE-negative RP patients do not exhibit these antibodies. In all RP + LE patients, following a therapeutic course of steroids these autoantibodies were no longer present and this absence coincided with clinical recovery and resolution of MRI findings. There is only few case reports disclosing the complication of LE in RP patients [13]. Therefore, it is quite difficult to collect sera from such patients at present, although all of RP + LE patients in the present study exhibit anti-neutral glycosphingolipids antibodies. The specificity of these antibodies in relation to other neurological disorders should be examined on further patients. What is the molecular mechanisms for the development of the present autoantibodies to neutral glycosphingolipids? Interestingly, auricular cartilage contains large amounts of neutral glycosphingolipids [14]. Patients with RP demonstrate marked auricular inflammation and induration, particularly so in the patients in this study. Therefore, we speculate that the abundance of these neutral glycosphingolipids in areas subject to destruction by the inflammatory process would make them preferred targets for an autoimmune response, although we have to examine further this hypothesis. In this regard, it is quite interesting that a recent report by Yamaguchi et al. demonstrates that autoantibodies to GM2 ganglioside are produced in the rat model of GM2 ganglioside-storage disease [15], GM2 gangliosidosis, although GM2 ganglioside is a normal constituent of membrane lipids. These new anti-neutral antibodies have an impact on Trkmediated intracellular signaling pathway as shown in this
T. Mihara et al. / FEBS Letters 580 (2006) 4991–4995
study. NGF is a cardinal neuroprotective, neurotrophic factor that promotes both neuronal survival and differentiation [16]. The initial step in the intracellular signal transduction of NGF is the activation of Trk-associated tyrosine kinase activity through the autophosphorylation of tyrosine residues [17]. Therefore, we speculated that these antibodies have a negative effects on Trk-mediated signaling pathway, because it seems these anti-neutral glycosphingolipids antibodies as a pathognomonic factor for the development of LE in RP. Unexpectedly, these antibodies enhances NGF-induced Trk autophosphorylation in PCtrk cells. This result suggests that these antibodies might promote Trk receptor clustering, because both are located in the same membrane structure, lipid rafts on the membrane platform. We have already observed similar phenomena where a specific ligand for GM1 ganglioside that is membrane raft-resident glycosphingolipids enhances NGF-induced Trk autophosphorylation [18]. These enhancing effects of the antibodies-positive sera were also observed in the experiments of NGF-induced neurite outgrowth and neurofilament M expression. The sera that is precleared with protein-L agarose to remove serum immunoglobulins do not exhibit such enhancing effects anymore, suggesting that immunoglobulins in antibodies-positive sera are the responsible molecules for such enhancement. This interpretation was supported by the data on NGF concentration in sera. As described earlier, NGF concentrations in antibodies-positive sera were not different in those of antibodies-negative sera. Currently, there are no reliable antibodies directed against GlcCer and GalCer. These autoantibodies may provide a solution to this problem and provide new tools for basic research on neutral glycosphingolipids in neurons. Acknowledgements: The present study was partly supported by the grant-in-aid on Center of Excellence (COE), a priority area on Functional Glycomics, and High-Tech research from the Ministry of education, culture, sports, science and technology of Japan. We deeply thank Dr. Y. Hirabayashi for his kind supply of purified GlcCer and chemically synthesized pure GlcCer.
References [1] Willison, H.J. and Yuki, N. (2002) Peripheral neuropathies and anti-glycolipid antibodies. Brain 125, 2591–2625. [2] Ledeen, R.W. and Wu, G. (1992) Ganglioside function in the neuron. Trends Glycosci. Glycotech. 4, 174–187. [3] Kusunoki, S., Shiina, M. and Kanazawa, I. (2001) Anti-Gal-C antibodies in GBS subsequent to mycoplasma infection: evidence of molecular mimicry. Neurology 57, 736–738. [4] Masserini, M., Palestini, P. and Pitto, M. (1999) Glycolipidenriched caveolae and caveolae-like domains in the nervous system. J. Neurochem. 73, 1–11. [5] Yamashita, T., Wada, R., Sasaki, T., Deng, C., Bierfreund, U., Sandhoff, K. and Proia, R.L. (1999) Vital role for glycosphingolipid synthesis during development and differentiation. Proc. Natl. Acad. Sci. USA 96, 9142–9147. [6] Morjani, H., Aouali, N., Belhoussine, R., Veldman, R.J., Levade, T. and Manfait, M. (2001) Elevation of glucosylceramide in multidrug-resistant cancer cells and accumulation in cytoplasmic droplets. Int. J. Cancer. 94, 157–165. [7] Schwarz, A.D. and Futerman, T.M. (1997) Distinct roles for ceramide and glucosylceramide at different stages of neuronal growth. J. Neurosci. 17, 2929–2938. [8] Watanabe, R., Wu, K., Paul, P., Marks, D.L., Kobayashi, T., Pittelkow, M.R. and Pagano, R.E. (1998) Up-regulation of glucosylceramide synthase expression and activity during human keratinocyte differentiation. J. Biol. Chem. 273, 9651–9655.
T. Mihara et al. / FEBS Letters 580 (2006) 4991–4995 [9] Mihara, T., Mutoh, T., Yoshikawa, T., Yano, S., Asano, Y. and Yamamoto, H. (2005) Postinfectious myeloradiculoneuropathy with cranial nerve involvements associated with human herpesvirus 7 infection. Arch. Neurol. 62, 1755–1757. [10] Mutoh, T., Tokuda, A., Hamano, T. and Kuriyama, M. (2000) Unglycosylated Trk protein does not co-localize nor associate with ganglioside GM1 in a stable clone of PC12 cells overecpressing Trk (PCtrk cells). Glycoconj. J. 17, 233–237. [11] Mutoh, T., Tokuda, A., Miyadai, T., Hamaguchi, M. and Fujiki, N. (1995) Ganglioside GM1 binds to the Trk protein and regulates receptor function. Proc. Natl. Acad. Sci. USA 92, 5087–5091. [12] Mutoh, T., Tokuda, A., Inokuchi, J. and Kuriyama, M. (1998) Glucosylceramide synthase inhibitor inhibits the action of nerve growth factor in PC12 cells. J. Biol. Chem. 273, 26001–26007. [13] Fujiki, F., Tsuboi, Y., Hashimoto, M., Nakajima, M. and Yamada, T. (2004) Non-herpetic limbic encephalitis associated
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with relapsing polychondritis. J. Neurol. Neurosurg. Psychiat. 75, 1646–1647. Fukaya, N., Ito, M., Iwata, H. and Yamagata, T. (1989) Characterization of the glycosphingolipids of pig cortical bone and cartilage. Biochim. Biophys. Acta 1004, 108–116. Yamaguchi, A., Katsuyama, K., Nagahama, K., Takai, T., Aoki, I. and Yamanaka, S. (2004) Possible role of autoantibodies in the pathophysiology of GM2 gangliosidoses. J. Clin. Invest. 113, 200– 208. Barbacid, M. (1995) Neurotrophic factors and their receptors. Curr. Opin. Cell. Biol. 7, 148–155. Kaplan, D.R., Martin-Zanka, D. and Parada, L.F. (1991) Tyrosine phosphorylation and tyrosine kinase activity the Trk proto-oncogene product induced by NGF. Nature 350, 158–160. Mutoh, T., Tokuda, A., Guroff, G. and Fujiki, N. (1993) The effect of the B subunit of cholera toxin on the action of nerve growth factor on PC12 cells. J. Neurochem. 60, 150–1547.
Detection of new anti-neutral glycosphingolipids antibodies and their effects on Trk neurotrophin receptors T. Miharaa, A. Uedaa, M. Hirayamab, T. Takeuchia, S. Yoshidad, K. Naitoc, H. Yamamotoa, T. Mutoha,* a
Department of Neurology, Fujita Health University School of Medicine, 1-98 Dengakugakubo, Kutsukake-cho, Toyoake, Aichi 470-1192, Japan b Department of Neurology, Kasugai Municipal Hospital, Kasugai, Aichi 486-8510, Japan c Department of Otolaryngology, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan d The Department of Internal Medicine, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan Received 4 August 2006; accepted 8 August 2006 Available online 17 August 2006 Edited by Sandro Sonnino
Abstract We screened sera from patients with various neurological disorders for the presence of anti-neutral glycosphingolipids antibodies and only found them in sera from relapsing polychondritis with limbic encephalitis patients. Neutral glycosphingolipids are resident in membrane lipid rafts where high affinity nerve growth factor (NGF) receptor, Trk is co-localized. Therefore, we examined whether these antibodies influence the action of NGF in NGF-responsive cells. The results strongly suggest that these antibodies enhance NGF-induced Trk autophosphorylation and neurite outgrowth as well as neurofilament M expression. These data strongly indicate that these anti-neutral glycosphingolipids antibodies have an functional impact on NGF-Trk-mediated intracellular signal transduction pathway. 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Neutral glycosphingolipids; Glucosylceramide; Galactosylceramide; Trk; Tyrosine phosphorylation; Nerve growth factor
1. Introduction There is an accumulating evidence that suggests the pathognomonic linkage between anti-gangliosides antibodies and acute motor axonal neuropathy and the cranial, bulbar and sensory variants of Guillain-Barre´ syndrome [1]. Inspite of so many publications at present, their real roles for the development of the neuropathies still remain to be elucidated. Gangliosides are acidic glycosphingolipids comprising a ceramide moiety embedded in the lipid bilayer and sialylated oligosaccharide core that is extracellularly displayed and capable of acting as an autoantibody target [2]. On the contrary, there is a few investigation on anti-neutral glycosphingolipids antibodies in neurological disorders. Major neutral glycosphingolipids include glucosylceramide (GlcCer), lactosylceramide (LacCer), globotriaosylceramide, globoside or galactosylceramide (GalCer). It has been only reported that
* Corresponding author. Fax: +81 562 93 1856. E-mail address: [email protected] (T. Mutoh).
anti-GalCer antibody is present in patients with GBS after Mycoplasma pneumoniae infection [3]. GlcCer is one of the cardinal neutral glycosphingolipids. It is located in the plasma membrane primarily in lipid rafts, a special membrane structure that also contains many signaling moieties such as receptor-type tyrosine kinases, cholesterol, sphingomyelin, and glycosphingolipids [4]. GlcCer is synthesized from ceramide through the enzymatic action of GlcCer synthase and constitutes the first step in the synthesis of all types of gangliosides. GlcCer is involved in the regulation of many biological events such as neuronal apoptosis and drug resistance to anti-cancer drugs [5,6]. Additionally, GlcCer induces neuronal growth and epidermal differentiation [7,8]. In this study, we screened sera from patients with various kinds of neurological disorders for the presence of an antibody against neutral glycosphingolipids and found novel anti-neutral glycosphingolipids antibodies against GlcCer and GalCer but not LacCer only in sera from the patients with relapsing polychondritis (RP) complicated by limbic encephalitis (LE)like symptoms. These antibodies were not found in sera from normal controls or patients with other neurological disorders to include uncomplicated RP. Then, we tried to characterize these anti-neutral glycosphingolipid antibodies using rat pheochromocytoma cell-line, PC12 cells. Unexpectedly, these novel antibodies enhanced the action of nerve growth factor (NGF) in cultured PC12 cells. This enhancement was mediated through NGF-induced Trk, a high affinity nerve growth factor receptor exhibiting tyrosine kinase activity, autophosphorylation response. The data clearly suggests that anti-neutral glycosphingolipids antibodies have an impact on the Trkmediated intracellular signaling pathway.
2. Materials and methods 2.1. Human sera After obtaining informed consent, sera were taken from various neurological patients (13 patients with viral encephalitis, 5 patients with paraneoplastic neurological syndrome, 5 patients with Guillain-Barre´ syndrome, 4 patients with Sjo¨gren syndrome, and 5 patients with Parkinson’s disease, 3 patients with RP complicated by LE and 2 patients with RP without LE) and 13 normal controls. The samples were stored at 20 C until use. All of the RP + LE patients exhibited psychiatric symptoms such as memory disturbance and delusions. The development of these symptoms coincided with the onset of canonical symptoms of RP such as ear swelling and erythema.
0014-5793/$32.00 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2006.08.013
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T. Mihara et al. / FEBS Letters 580 (2006) 4991–4995
2.2. Anti-neutral glycosphingolipids antibodies To evaluate the patients’ sera for the presence of anti-neutral glycosphingolipids antibodies, purified GlcCer (a generous gift from Dr. Hirabayashi Y. Brain science Institute, RIKEN, Wako, Japan), GalCer (a generous gift from Dr. Furukawa K., Nagoya University School of Medicine, Nagoya, Japan), and LacCer (BioMol, USA) were processed by thin-layer chromatography (using a solvent of chloroform, methanol, and distilled water in a 55:20:3 vol/vol/vol ratio) and blotted onto a polyvinylidene difluoride membrane by an electrothermal blotter (ATTO Co. Ltd., Tokyo, Japan) as described previously [9]. This polyvinylidene difluoride (PVDF) membrane was probed with human sera (500· dilution) in blocking buffer (2% non-fat milk in the wash buffer phosphate-buffered saline (PBS) containing 1% Triton X-100). Following application of the second antibody, a positive band was detected with an enhanced chemiluminescence reagent (Perkin–Elmer Inc., Boston, MA). In some case, we also examined the presence of anti-GlcCer antibody using chemically synthesized pure GlcCer (from Dr. Y. Hirabayashi). 2.3. Cell culture and treatment Rat pheochromocytoma-derived PC12 cells and stable transfectants of human trk-complimentary DNA (PCtrk cells) were cultured as described [10]. The cells were cultured as monolayers in DMEM containing 7.5% FBS, 100 U/ml penicillin and 100 lg/ml streptomycin [11]. Cells were incubated at 37 C in a humidified 5% CO2 atmosphere. For morphological observation, cells were cultured in 24-well culture plates and examined using a phase contrast microscope [12]. Following photography, the cells were lysed in sodium dodecyl sulfate (SDS)sample buffer and evaluated by immunoblot analysis using an antineurofilament M antibody for neuronal differentiation as described previously [11].
Fig. 2. Immunoreactivity against chemically synthesized pure GlcCer. Chemically synthesized pure GlcCer were electrothermally blotted onto a polyvinylidene difluoride membrane. The membrane was probed with sera from patients with RP + LE (1) and from disease controls (2). Red asterisks indicate the positive bands corresponding to GlcCer. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
2.4. Immunoprecipitation and immunoblot analysis Because both neutral glycosphingolipids and Trk are located in the membrane lipid raft, a membrane structure that is also the site of transmembrane signaling, we examined the effect of anti-neutral glycosphingolipids antibodies on the high affinity NGF receptor, Trk. PCtrk cells were preincubated with sera (500· dilution) for 30 min and then stimulated with 50 ng/ml nerve growth factor (NGF) for 5 min. To evaluate receptor activity, Trk was immunoprecipitated with an anti-Trk antibody (a-Trk; Santa Cruz Biotechnology Inc., Santa Cruz, CA) and subjected to immunoblot analysis using either an anti-phosphotyrosine antibody (a-PY, 4G10, Upstate Biotechnol. Inc., Lake Placid, NY) or a-Trk antibody as described [10–12]. In some case, antibody-positive sera were precleared with protein-L agarose to remove all immunoglobulins for 4 h in the cold room with agitation.
Fig. 1. Immunoblot analysis for neutral glycosphingolipids. Neutral glycosphingolipids (GlcCer and LacCer (lanes a(1), b(1) and c(1)), GalCer (lanes a(2), b(2) and c(2)) were electrothermally blotted onto a polyvinylidene difluoride membrane. The membrane was probed with sera from the patients with RP + LE obtained before (a) and after (c) steroid hormone therapy and with sera from patients with only RP (b). The position of each neutral glycosphingolipids was determined on a high-performance thin-layer chromatography plate developed simultaneously without electrothermal blotting. Red asterisks indicate the positive bands corresponding to GlcCer () and to GalCer (). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 3. Effect of sera on Trk-tyrosine kinase activity in response to NGF. PCtrk cells were cultured on 15 cm in diameter culture dishes and were pretreated with serum-free medium for 1 h. (a) Then, cells were treated with sera (·200 diluted in final) (lane 3, patient with RP + LE; lane 4, another patient with RP + LE; same patient with RP + LE obtained after steroid hormone treatment) for 30 min and stimulated with 50 ng/ml NGF (lanes 2–5) or without (lane 1) for 5 min. (b) Cells were treated with sera (·200 diluted in final) (lanes 3, 4, patients with RP only; lane 5, disease control (Parkinson’s disease); lane 6, a normal control) for 30 min and stimulated with 50 ng/ml NGF (lanes 2–6) or without (lane 1) for 5 min. After stimulation, cells were collected with chilled PBS and subjected to the immunoprecipitation as described in ‘‘Section 2’’. These were performed at least three times with identical results. Arrows indicate position of the Trk protein. These precleared sera were subjected to Trk-immunoprecipitation. Detection of positive bands was performed using Western blot Chemiluminescence Reagent Plus (Perkin–Elmer Inc.) [8,10].
T. Mihara et al. / FEBS Letters 580 (2006) 4991–4995 2.5. Measurement of NGF in serum The concentration of NGF concentrations in sera were measured by commercial ELISA kit (Promega, USA) according to the manufacturer’s instructions. To neutralize the effects of serum immunoglobulins, protein-L agarose was added and incubated with agitation for 2 h in the cold. Following agitation, the sera were centrifuged at 12 000 rpm for 2 min and resultant supernatants subject to evaluation. Each evaluation was performed in a triplicate.
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for an anti-neutral glycosphingolipid antibodies augmented NGF-induced Trk autophosphorylation (Fig. 3a). Sera negative for anti-neutral glycosphingolipid antibodies had no effect on NGF-induced Trk autophosphorylation (Fig. 3b). Intriguingly, sera from RP + LE patient obtained after treatment with steroid hormone and precleared with protein-L agarose did not reveal no augmentation of NGF-induced Trk autophosphorylation (compare Fig. 3a(4 and 5) and data not shown).
3. Results 3.1. Detection of anti-neutral glycosphingolipid antibodies A total of 50 serum samples were screened. Positive bands representing antibodies against GlcCer and GalCer were found in sera obtained from patients with RP + LE (Fig. 1a). These antibodies were not found in any of the patients with other neurological disorders to include RP in the absence of LE (Fig. 1b) or in the normal controls (data not shown). Intriguingly, after a course of steroids (pulse therapy followed by oral prednisolone), all of the RP + LE patients who were positive for anti-neutral glycosphingolipid antibodies had a remarkable improvement in their clinical symptoms that was accompanied by a disappearance of these autoantibodies (Fig. 1c). Furthermore, these anti-neutral glycosphingolipids antibodies in patients’ sera actually recognize chemically synthesized pure GlcCer (Fig. 2). 3.2. Effect on NGF-induced Trk autophosphorylation To evaluate the effects of the antibodies-positive sera on NGF-induced Trk autophosphorylation, PCtrk cells were pretreated with sera for 30 min followed by 5 min of NGF stimulation and immunoprecipitated the Trk protein. Sera positive
Fig. 5. NGF concentrations. Serum NGF concentration was measured by ELISA. RP + LE sera before the treatment (column 1, n = 3), with RP + LE after the treatment (column 2, n = 3), disease controls including RP without LE (column 3, n = 34), and normal controls (column 4, n = 13). The columns represent the means ± S.D.
Fig. 4. Morphological effects of RP + LE sera on NGF-induced neurite outgrowth in PCtrk cells. PCtrk cells were cultured on 24-well dish in the presence of 50 ng/ml of NGF (2–4, 7, 8) or absence of NGF (1, 5, 6) for 12 h. Before adding NGF, cells were pre-treated with patient’s serum taken before any treatment (3, 5) or after treatment (4, 6) and from RP patient (7) and normal control (8) for 30 min. We also examined the expression of neurofilament M in the total cell lysates prepared from those used for morphological studies by the immunoblot analysis with anti-neurofilament M antibody. Note that RP + LE patient’s serum before treatment strongly augmented NGF-induced neurite outgrowth in PCtrk cells but not taken after treatment nor disease and normal controls. These experiments were performed three times with identical results. Arrows indicate the position of neurofilament M.
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3.3. Effect on NGF-induced morphological differentiation To reinforce the previous findings, we also examined the effect of sera on NGF-induced morphological differentiation. In PCtrk cells, NGF-induced neurite outgrowth was enhanced by RP + LE patient sera when compared to the effect of NGF alone (Fig. 4). This enhancement was not observed with antineutral glycosphingolipid antibodies-negative sera from controls and other neurologic disorders to include LE negative RP. Additionally, the effect of sera on neurofilament M expression was also examined by immunoblot analysis. As was the case with neurite outgrowth, RP + LE sera taken before the treatment with steroid hormone enhanced neurofilament M expression but not taken after treatment nor disease and normal controls when compared to the effect of NGF alone (Fig. 4). Moreover, precleared sera from patients with RP + LE with protein-L agarose also did not induce such enhancement (data not shown).
3.4. Measurement of serum NGF concentration To test the possibility that serum NGF concentrations are increased in anti-neutral glycosphingolipids antibody-positive sera, NGF concentrations were measured. This is no difference in NGF concentrations between antibody positive and antibody negative sera (Fig. 5).
4. Discussion The present study clearly demonstrates the presence of novel anti-neutral glycosphingolipid antibodies in the sera of patients with RP + LE. These autoantibodies were not found in normal controls or in patients with a variety of other neurological disorders. It is particularly intriguing that all of LE-negative RP patients do not exhibit these antibodies. In all RP + LE patients, following a therapeutic course of steroids these autoantibodies were no longer present and this absence coincided with clinical recovery and resolution of MRI findings. There is only few case reports disclosing the complication of LE in RP patients [13]. Therefore, it is quite difficult to collect sera from such patients at present, although all of RP + LE patients in the present study exhibit anti-neutral glycosphingolipids antibodies. The specificity of these antibodies in relation to other neurological disorders should be examined on further patients. What is the molecular mechanisms for the development of the present autoantibodies to neutral glycosphingolipids? Interestingly, auricular cartilage contains large amounts of neutral glycosphingolipids [14]. Patients with RP demonstrate marked auricular inflammation and induration, particularly so in the patients in this study. Therefore, we speculate that the abundance of these neutral glycosphingolipids in areas subject to destruction by the inflammatory process would make them preferred targets for an autoimmune response, although we have to examine further this hypothesis. In this regard, it is quite interesting that a recent report by Yamaguchi et al. demonstrates that autoantibodies to GM2 ganglioside are produced in the rat model of GM2 ganglioside-storage disease [15], GM2 gangliosidosis, although GM2 ganglioside is a normal constituent of membrane lipids. These new anti-neutral antibodies have an impact on Trkmediated intracellular signaling pathway as shown in this
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study. NGF is a cardinal neuroprotective, neurotrophic factor that promotes both neuronal survival and differentiation [16]. The initial step in the intracellular signal transduction of NGF is the activation of Trk-associated tyrosine kinase activity through the autophosphorylation of tyrosine residues [17]. Therefore, we speculated that these antibodies have a negative effects on Trk-mediated signaling pathway, because it seems these anti-neutral glycosphingolipids antibodies as a pathognomonic factor for the development of LE in RP. Unexpectedly, these antibodies enhances NGF-induced Trk autophosphorylation in PCtrk cells. This result suggests that these antibodies might promote Trk receptor clustering, because both are located in the same membrane structure, lipid rafts on the membrane platform. We have already observed similar phenomena where a specific ligand for GM1 ganglioside that is membrane raft-resident glycosphingolipids enhances NGF-induced Trk autophosphorylation [18]. These enhancing effects of the antibodies-positive sera were also observed in the experiments of NGF-induced neurite outgrowth and neurofilament M expression. The sera that is precleared with protein-L agarose to remove serum immunoglobulins do not exhibit such enhancing effects anymore, suggesting that immunoglobulins in antibodies-positive sera are the responsible molecules for such enhancement. This interpretation was supported by the data on NGF concentration in sera. As described earlier, NGF concentrations in antibodies-positive sera were not different in those of antibodies-negative sera. Currently, there are no reliable antibodies directed against GlcCer and GalCer. These autoantibodies may provide a solution to this problem and provide new tools for basic research on neutral glycosphingolipids in neurons. Acknowledgements: The present study was partly supported by the grant-in-aid on Center of Excellence (COE), a priority area on Functional Glycomics, and High-Tech research from the Ministry of education, culture, sports, science and technology of Japan. We deeply thank Dr. Y. Hirabayashi for his kind supply of purified GlcCer and chemically synthesized pure GlcCer.
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