- Email: [email protected]
P2-367: The mode of action of an anti–β-secretase cleavage site antibody
T480
Poster Presentations P2:
identified the conformational epitope of B6-scFv and characterized their biological activity. Methods: Affinity selection of random peptide displaying phage library was performed using the anti-fibril scFv, B6 as a mold at 3-rounds biopanning. The isolated phage clones were analyzed their motif DNA sequences by the ABI Genetic Analyzer PRISM 3100. The motif sequences were chemically synthesized and tested their effect on A1-42 fibril formation by thioflavin T and precipitation assay (Yoshihara, et al., J. Biochem., 2, 2008). Results: We isolated phage clones from peptide phage display libraries and identified several different peptide motifs (B6L1, B6-L10 and B7-C15). The deduced amino acid sequences of these peptide motifs have little homology to A1-42 peptide. The synthetic peptides based on these phage motifs (B6-L1, B7-C15) had inhibitory effect on A1-42 fibrillation by binding to prefibrillar soluble oligomers. Conclusions: Taken these results together with the sequence homology analysis, it is suggested that B6-L1 and B7-C15 might be mimics of A1-42 fibril. To investigate this possibility, we characterize the specificity of immune response elicited with these peptides in mice.
ELISA. Results: Levels of sAPP␣ secreted into the media were unaffected suggesting that ␣-secretase cleavage was unaltered. Interestingly levels of sAPP in the media also appeared to be unaffected which would suggest that the effects of the antibody were not via steric hinderance. Levels of sAPP were, however, very low and any reduction may have been undetectable by this method. Support for the steric hinderance theory was provided by immunocytochemistry data showing time dependent entry of 2B12 into astrocytoma cells. Thus, 2B12 would appear not to affect APP internalisation. Results from a cell-free assay are presented to elucidate further the mode of action of our cleavage site antibody. Data are also presented relating to the effects of 2B12 on A42 levels and comparisons are also made between its effects on cells that constitutively express APP and transfected cell lines. Conclusions: In conclusion, while the mechanism of action of 2B12 is still unclear we have provided further evidence to support the use of such antibodies as a novel therapy. P2-368
P2-366
INHIBITION OF A PRODUCTION WITHOUT PERTURBING NOTCH AND NEUREGULIN SIGNALING
Shinichi Tatsumi, Masayasu Okochi, Shinji Tagami, Naohiro Itoh, Kouhei Nishitomi, Jingwei Jiang, Taisuke Nakayama, Kanta Yanagida, Takashi S. Kodama, Kohji Mori, Takashi Oguri, Masatoshi Takeda, Osaka University Graduate School of Medicine, Suita, Osaka, Japan. Contact e-mail: [email protected] Background: Inhibition of either of the secretases reduces A generation and is a fundamental strategy for the development of drugs to prevent Alzheimer’s disease. However, since typical compounds which inhibit A production affects BACE or PS/␥-secretase activity, it is considered that they should perturb Neuregulin and Notch signaling, which are physiological targets for these secretases. Methods: A search for inhibitors of A generation in a library of more than approximately 50,000 compounds by high-throughput screening identified compounds which inhibit neither BACE nor PS/␥-secretase. Results: We found a natural and physiological active compound which reduces A generation without affecting BACE1 or presenilin/␥-secretase activity. In agreement with this, it did not inhibit Notch signaling. Interestingly, it decreased -cleavage of APP, but did not affect extracellular shedding of Neuregulin1, which is associated with peripheral nerve myelination, by BACE1. Conclusions: Our data demonstrate that the compound induces a novel A reducing process, which could have fewer side-effects than secretase inhibitors. P2-367
THE MODE OF ACTION OF AN ANTI–SECRETASE CLEAVAGE SITE ANTIBODY
Rhian Thomas, Eryl Liddell, Emma Kidd, Welsh School of Pharmacy / School of Biosciences, Cardiff University, Cardiff, United Kingdom. Contact e-mail: [email protected] Background: Proteolytic cleavage of APP by - and ␥-secretases results in the production of Amyloid  (〈) which accumulates in the brains of sufferers of Alzheimer’s Disease. We have raised an antibody, 2B12, that binds in the vicinity of the -secretase cleavage site but not within the A region of APP. We have demonstrated that this antibody is capable of inhibiting A40 production in neuroblastoma and astrocytoma cells expressing native APP. We hypothesised that this antibody enters the cell by binding to APP when it is at the cell surface and then becomes internalised with the protein. It could then inhibit cleavage of APP by -secretase via steric hinderance. Alternatively it might prevent APP internalisation or promote APP degradation after internalisation and thus reduce downstream production of A. Methods: Cells were incubated with antibodies (2B12, anti-N-term APP or control IgG) at 10g/ml for two days. Media was then removed and either immunoprecipated with antibodies to A40 and subsequently tested in an ELISA, or tested directly in an ELISA for sAPP␣ and sAPP. Cells were then lysed and A42 levels determined in an
GETO REDUCES A PRODUCTION BY GAMMASECRETASE INHIBITION
Jinzhou Tian1,2, Jing Shi1, Junxiang Yin3, Shuli Sheng4, Yi Xu1, Leiming Zhang1, Rong Wang5, Chongshun Song3, Pengwen Wang3, Yongyan Wang6, 1BUCM Neurology Centre, Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, China; 2Department of Preclinical Medicine, Hubei College of Chinese Medicine, Wuhan, China; 3Institute of Geriatrics, Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, China; 4Lab. Of Neurobiochemistry, Xuanwu Hospital, CUMS, Beijing, China; 5Lab. of Neurobiochemistry, Xuanwu Hospital, CUMS, Beijing, China; 6China Academy of Chinese Medical Sciences, Beijing, China. Contact e-mail: [email protected] Background: Although a recent study opens perspectives for therapy of AD by gamma-secretase inhibition (Dewachter et al. J Neurosci. 2002; 22:3445-53), but it has not been reported for herbal therapy in inhibiting gamma-secretase substrate (PS1) related to production of Abeta production. This study aimed to investigate effects of GETO, a combination of herbal active components, on Abeta production and beta-secretase substrate (BACE1) and gamma-secretase substrate (PS1). Methods: Thirty APPV717I transgenic mice aged 3 months were randomly divided into a APP group which was given distilled water, a APP⫹donepezil group which was administered donepezil (0.92mg/kg/d), and three APP⫹GETO groups which were separately administered with a small dose (0.075g/kg/ d), a middle dose (0.15g/kg/d), a large (0.3g/kg/d) per day for 8 months. Six vehicle mice of C57BL/6J were given distilled water. Immunohistochemistry and Western blot analysis were used in the determining Abeta, BACE1 and PS1 in the hippocampus of mice. Results: Compared to APP or APP⫹donepezil group, expression of total Abeta level in the large dose of GETO was significantly decreased, which closes to the level in C57BL/6J mice. Western blot by antibodies against Abeta protein showed that the large dose had normalized Abeta band density. There was significantly increased expression of PS1 in the large dose, which also closes to those in C57BL /6J mice and lower than APP or APP⫹donepezil group. Although expression of BACE1 in the large dose was lower than those in APP group but much higher than those in C57BL/6J mice. The decreased BACE1 level in APP⫹donepezil was still greater than those in C57BL/6J mice. The decreased BACE1 level in APP⫹donepezil was also greater than those in C57BL/6J mice. Conclusions: This data suggests that GETO reduces Abeta production in the brain of APPV717I mice perhaps by inhibiting PS1 activity rather than BACE1. The large dose of GETO has better benefit in the inhibition of PS1 than middle or small dose. This study promises a potential strategy for preventing the development of AD. This work was supported by The 973 Project (Grant No. 2003CB517104), The 111 Project (Grant No. B08006), The NSFC Project (Grant No. 30672693), and Chutian Scholars Project.
Poster Presentations P2:
identified the conformational epitope of B6-scFv and characterized their biological activity. Methods: Affinity selection of random peptide displaying phage library was performed using the anti-fibril scFv, B6 as a mold at 3-rounds biopanning. The isolated phage clones were analyzed their motif DNA sequences by the ABI Genetic Analyzer PRISM 3100. The motif sequences were chemically synthesized and tested their effect on A1-42 fibril formation by thioflavin T and precipitation assay (Yoshihara, et al., J. Biochem., 2, 2008). Results: We isolated phage clones from peptide phage display libraries and identified several different peptide motifs (B6L1, B6-L10 and B7-C15). The deduced amino acid sequences of these peptide motifs have little homology to A1-42 peptide. The synthetic peptides based on these phage motifs (B6-L1, B7-C15) had inhibitory effect on A1-42 fibrillation by binding to prefibrillar soluble oligomers. Conclusions: Taken these results together with the sequence homology analysis, it is suggested that B6-L1 and B7-C15 might be mimics of A1-42 fibril. To investigate this possibility, we characterize the specificity of immune response elicited with these peptides in mice.
ELISA. Results: Levels of sAPP␣ secreted into the media were unaffected suggesting that ␣-secretase cleavage was unaltered. Interestingly levels of sAPP in the media also appeared to be unaffected which would suggest that the effects of the antibody were not via steric hinderance. Levels of sAPP were, however, very low and any reduction may have been undetectable by this method. Support for the steric hinderance theory was provided by immunocytochemistry data showing time dependent entry of 2B12 into astrocytoma cells. Thus, 2B12 would appear not to affect APP internalisation. Results from a cell-free assay are presented to elucidate further the mode of action of our cleavage site antibody. Data are also presented relating to the effects of 2B12 on A42 levels and comparisons are also made between its effects on cells that constitutively express APP and transfected cell lines. Conclusions: In conclusion, while the mechanism of action of 2B12 is still unclear we have provided further evidence to support the use of such antibodies as a novel therapy. P2-368
P2-366
INHIBITION OF A PRODUCTION WITHOUT PERTURBING NOTCH AND NEUREGULIN SIGNALING
Shinichi Tatsumi, Masayasu Okochi, Shinji Tagami, Naohiro Itoh, Kouhei Nishitomi, Jingwei Jiang, Taisuke Nakayama, Kanta Yanagida, Takashi S. Kodama, Kohji Mori, Takashi Oguri, Masatoshi Takeda, Osaka University Graduate School of Medicine, Suita, Osaka, Japan. Contact e-mail: [email protected] Background: Inhibition of either of the secretases reduces A generation and is a fundamental strategy for the development of drugs to prevent Alzheimer’s disease. However, since typical compounds which inhibit A production affects BACE or PS/␥-secretase activity, it is considered that they should perturb Neuregulin and Notch signaling, which are physiological targets for these secretases. Methods: A search for inhibitors of A generation in a library of more than approximately 50,000 compounds by high-throughput screening identified compounds which inhibit neither BACE nor PS/␥-secretase. Results: We found a natural and physiological active compound which reduces A generation without affecting BACE1 or presenilin/␥-secretase activity. In agreement with this, it did not inhibit Notch signaling. Interestingly, it decreased -cleavage of APP, but did not affect extracellular shedding of Neuregulin1, which is associated with peripheral nerve myelination, by BACE1. Conclusions: Our data demonstrate that the compound induces a novel A reducing process, which could have fewer side-effects than secretase inhibitors. P2-367
THE MODE OF ACTION OF AN ANTI–SECRETASE CLEAVAGE SITE ANTIBODY
Rhian Thomas, Eryl Liddell, Emma Kidd, Welsh School of Pharmacy / School of Biosciences, Cardiff University, Cardiff, United Kingdom. Contact e-mail: [email protected] Background: Proteolytic cleavage of APP by - and ␥-secretases results in the production of Amyloid  (〈) which accumulates in the brains of sufferers of Alzheimer’s Disease. We have raised an antibody, 2B12, that binds in the vicinity of the -secretase cleavage site but not within the A region of APP. We have demonstrated that this antibody is capable of inhibiting A40 production in neuroblastoma and astrocytoma cells expressing native APP. We hypothesised that this antibody enters the cell by binding to APP when it is at the cell surface and then becomes internalised with the protein. It could then inhibit cleavage of APP by -secretase via steric hinderance. Alternatively it might prevent APP internalisation or promote APP degradation after internalisation and thus reduce downstream production of A. Methods: Cells were incubated with antibodies (2B12, anti-N-term APP or control IgG) at 10g/ml for two days. Media was then removed and either immunoprecipated with antibodies to A40 and subsequently tested in an ELISA, or tested directly in an ELISA for sAPP␣ and sAPP. Cells were then lysed and A42 levels determined in an
GETO REDUCES A PRODUCTION BY GAMMASECRETASE INHIBITION
Jinzhou Tian1,2, Jing Shi1, Junxiang Yin3, Shuli Sheng4, Yi Xu1, Leiming Zhang1, Rong Wang5, Chongshun Song3, Pengwen Wang3, Yongyan Wang6, 1BUCM Neurology Centre, Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, China; 2Department of Preclinical Medicine, Hubei College of Chinese Medicine, Wuhan, China; 3Institute of Geriatrics, Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, China; 4Lab. Of Neurobiochemistry, Xuanwu Hospital, CUMS, Beijing, China; 5Lab. of Neurobiochemistry, Xuanwu Hospital, CUMS, Beijing, China; 6China Academy of Chinese Medical Sciences, Beijing, China. Contact e-mail: [email protected] Background: Although a recent study opens perspectives for therapy of AD by gamma-secretase inhibition (Dewachter et al. J Neurosci. 2002; 22:3445-53), but it has not been reported for herbal therapy in inhibiting gamma-secretase substrate (PS1) related to production of Abeta production. This study aimed to investigate effects of GETO, a combination of herbal active components, on Abeta production and beta-secretase substrate (BACE1) and gamma-secretase substrate (PS1). Methods: Thirty APPV717I transgenic mice aged 3 months were randomly divided into a APP group which was given distilled water, a APP⫹donepezil group which was administered donepezil (0.92mg/kg/d), and three APP⫹GETO groups which were separately administered with a small dose (0.075g/kg/ d), a middle dose (0.15g/kg/d), a large (0.3g/kg/d) per day for 8 months. Six vehicle mice of C57BL/6J were given distilled water. Immunohistochemistry and Western blot analysis were used in the determining Abeta, BACE1 and PS1 in the hippocampus of mice. Results: Compared to APP or APP⫹donepezil group, expression of total Abeta level in the large dose of GETO was significantly decreased, which closes to the level in C57BL/6J mice. Western blot by antibodies against Abeta protein showed that the large dose had normalized Abeta band density. There was significantly increased expression of PS1 in the large dose, which also closes to those in C57BL /6J mice and lower than APP or APP⫹donepezil group. Although expression of BACE1 in the large dose was lower than those in APP group but much higher than those in C57BL/6J mice. The decreased BACE1 level in APP⫹donepezil was still greater than those in C57BL/6J mice. The decreased BACE1 level in APP⫹donepezil was also greater than those in C57BL/6J mice. Conclusions: This data suggests that GETO reduces Abeta production in the brain of APPV717I mice perhaps by inhibiting PS1 activity rather than BACE1. The large dose of GETO has better benefit in the inhibition of PS1 than middle or small dose. This study promises a potential strategy for preventing the development of AD. This work was supported by The 973 Project (Grant No. 2003CB517104), The 111 Project (Grant No. B08006), The NSFC Project (Grant No. 30672693), and Chutian Scholars Project.