ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2008, Vol. 34, No. 1, pp. 43–48. © Pleiades Publishing, Inc., 2008. Original Russian Text © O.M. Vol’pina, T.D. Volkova, M.A. Titova, Yu.G. Gershovich, N.I. Medvinskaya, A.N. Samokhin, A.V. Kamynina, V.S. Shalgunov, D.O. Koroev, M.P. Filatova, M.B. Obosnaya, and N.V. Bobkova, 2008, published in Bioorganicheskaya Khimiya, 2008, Vol. 34, No. 1, pp. 50–55.
New Approaches to the Immunotherapy of Alzheimer’s Disease with the Synthetic Fragments of a7 Subunit of the Acetylcholine Receptor O. M. Vol’pinaa,1, T. D. Volkovaa, M. A. Titovaa, Yu. G. Gershovichb, N. I. Medvinskayab, A. N. Samokhinb, A. V. Kamyninaa, V. S. Shalgunova, D. O. Koroeva, M. P. Filatovaa, M. B. Obosnayaa, and N. V. Bobkovab a
Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow, 117997 Russia b Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, Moscow oblast, 142290 Russia Received February 13, 2007; in final form, May, 30, 2007
Abstract—The effect of immunization with the synthetic fragments of the α7 subunit of the acetylcholine nicotine receptor on the spatial memory of mice subjected to olfactory bulbectomy, which causes the development of the neurodegenerative disease of Alzheimer’s type, was studied. NMRI mice were immunized with the KLH conjugates of two peptide fragments of the N-terminal fragment of the α7 subunit extracellular fragment, subjected to olfactory bulbectomy to cause the development of the neurodegenerative disease of Alzheimer’s type, and then the state of the spatial memory was evaluated. It was shown that 20% of bulbectomized mice immunized with N-terminal 1-23 fragment exhibited good spatial memory after training. Immunization with the peptide construct (159–167)–(179–188) consisting of two hydrophilic exposed regions of α7-subunit induced good spatial memory in 50% of bulbectomized mice, while in the control group, which received only KLH, none of animals were learned. Thus, the development of immunotherapy with peptide (159–167)–(179–188) seems to be a promising approach to prophylaxis and treatment of Alzheimer’s disease. Key words: acetylcholine receptor, α7 subunit; Alzheimer’s disease; olfactory bulbectomy; spatial memory; synthetic peptides DOI: 10.1134/S1068162008010056
One of the recent hypotheses assumes that βÄ-(1–42), when binding to the ACR α7-subunit, forms a complex that penetrates into the cells, is accumulated there, and launches the cascade of the reactions resulting in the cell death [7–9]. Thus, the ACR α7-subunit could be a new target in the AD therapy [10]. We have assumed that the antibodies against the ACR α7-subunit could hinder its binding to βÄ. The use of synthetic fragments of the ACR α7-subunit, which induce the generation of the antibodies specific to the receptor in question, as immunogens seems to be promising. Bulbectomized NMRI mice with behavior, morphological, and biochemical symptoms of neurodegenerative AD-type process, including the loss of spatial memory, deficiency of acetylcholinergic systems, and the increased level of brain βÄ, developed long date after the removal of olfactory bulbs were used as a model of the sporadic form of AD [11–13]. Therefore, the aim of this work was to study the effect of immunization with various fragments of α7subunit on the spatial memory of BE mice.
INTRODUCTION Alzheimer’s disease is a heavy neurodegenerative disease resulting in memory loss, marked cognitive distortions, and inevitable death. The mass neuron destruction in the specific brain structures responsible for the storage and processing of information is the final result of the of the pathology progress [1].2 β-Amyloid peptides, in particular, βÄ-(1–42) have been shown to play a leading role in the mechanism of neuron destruction at AD [2–5]. On the other hand, AD development is known to be accompanied by a decrease of the content of the neuronal α7-type ACR, which is mainly localized in neurons of the central nervous system [6]. 1
Corresponding author; phone: +7 (495) 336-5777; e-mail:
[email protected]. 2 Abbreviations: ACR, acetylcholine receptor; AD, Alzheimer’s disease; BE animals, animals (mice) subjected to olfactory bulbectomy; CFA, complete Freund’s adjuvant; Fmoc, 9-fluorenylmethoxycarbonyl; IFA, incomplete Freund’s adjuvant; KLH, keyhole limpet hemocyanin; and PBS, phosphate buffered saline, 0.15 M NaCl in 0.01 M NaH2PO4 (pH 7.4).
43
44
VOL’PINA et al.
RESULTS AND DISCUSSION We had previously studied the ability of some synthetic peptide fragments corresponding to the exposed variable sites of the N-terminal extracellular domain of the α7-subunit of human ACR to induce the generation of antibodies specifically binding to the recombinant N-terminal extracellular domain of the human ACR α7-subunit [14, 15]. The epitope mapping of the anti(1–23)
peptide antibodies was performed using of the shortened fragments; the four sites of the protein, being the targets for these antibodies, were revealed [15]. In this study, the two peptide fragments, peptide (I) corresponding to the 1–23 N-terminal α-helix site of the ACR α7-subunit and peptide (II) involving the two hydrophilic sites 159–167 and 179–188 exposed were chosen.
1
23
GEFQRKLYKELVKNYNPLERPVA (I) 159
167 179
188
(159–167)–(179–188) QMQEADISG–VPGKRSERFY
The peptides were synthesized on the p-alkoxybenzyl polymer by the solid-phase method according to the Fmoc-scheme and were used for immunization of animals after HPLC purification. The first stage of the study included the investigation of the immunogenic activity of the free peptide fragments and their mixtures in NMRI mice used for AD modeling. The use of free peptides nonconjugated with the high-molecular carrier seemed to be especially attractive for obtaining the strictly directed immune answer without impurities of antibodies to the carrier protein. However, the immune answer for the free peptides in NMRI mice appeared to be too low or unstable (Table 1). After immunization with peptide (I), the titers of the antipeptide antibodies significantly differed from animal to animal; their values varied from 4.0 to
