- Email: [email protected]
Selective modulation of Ab42 by calcium channel antagonists. Relevance of calcium dynamics
Poster Presentations P3
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the oligomers of Ab was 10 kDa (equivalent to dimmer for Ab1-40 and decamers for Ab25-35) and that DHA dose-dependently reduced these decamers. Conclusions: DHA decreases the in vitro fibrillation of Ab by inhibiting the oligomeric amyloid species and, therefore, Ab-related neurotoxicity or behavioral impairment could be restrained by DHA. P3-261
CURRENT DRUG TARGET SITES IN CLINICAL PRACTICE OR CLINICAL TRIAL, EXTENT OF DISEASE MODIFICATION FOR ALZHEIMER’S DISEASE
Farhan Ul Haq Subhani, Bolam Medical College Quetta Balochistan Pakistan, Quetta, Pakistan. Contact e-mail: [email protected] Background: Alzheimer’s disease is most common form of dementia and one of complicated neurodegenerative diseases. Different therapeutic target sites are approved and number of drug target sites are under development for the treatment of Alzheimer’s disease. The disease modification is claimed by several drug developers for Alzheimer’s disease but the disease process has not been significantly affected by the available drugs either in clinical practice or clinical trials. In this review, discussion of the effects of different drug target sites on the disease modification will be compiled. Methods: It is a review to discuss in detail the efficacy of different drugs for the disease modifying effect of their respective target site. Results: Alzheimer’s disease have been know to scientifically community for almost 100 years and good therapeutic revaluation has been experienced during last 2 decades with respect to the treatment of this mysterious disease. Different drug target sites have been approved for the treatment of disease which includes cholinesterase inhibitors, NMDA receptor antagonist and products to elevate ketone bodies level in brain. Many drug target sites are in the process of development from discovery to phase III stage of clinical trail. These target sites are beta amyloid modulator, amyloid protein deposition inhibitors, nicotinic ACh modulator, muscarinic M1 agonist, muscarinic M2 antagonist, TrkA receptor modulator, proteasome inhibitor, AMPA receptor antagonist & modulator, anti-inflammatory cytokine synthesis inhibitor, caspase-3 inhibitor, PDE 4 inhibitor, tau aggregation inhibitor, beta & gamma-secretrase inhibitor, benzothiophene derivatives, COX-2 inhibitor, histamine H3 antagonist, antioxidant agents, imidazoline receptor antagonist, alpha 2 adrenoceptor antagonist, metal (Zn, Cu) chelating agent, free radical scavenger, MAO B inhibitor, 5-HT agonist and glycogen synthase kinase-3 inhibitor etc. Conclusions: In this review disease modification effect of few important drug targetsites for AD will be discussed. The modification process is not for sake of cure in all these target sites but we are being hopeful for having a definitive cure from this next generation of drug target sites in case of Alzheimer’s disease. P3-262
COGNITIVE AND PATHOLOGICAL BENEFITS OF Ab-SENSITIVE TH2 CELLS ARE INDEPENDENT OF ANTIGEN-SPECIFIC B CELL ACTIVATION
Douglas W. Ethell1, Chuanhai Cao2, Alexander Dickson3, Malgorzata B. Mamcarz3, Xiaoyang Lin2, Gary W. Arendash2,3, 1University of California Riverside, Riverside, CA, USA; 2Florida Alzheimer’s Disease Research Center, Tampa, FL, USA; 3Johnnie B. Byrd Alzheimer’s Center and Research Institute, Tampa, FL, USA. Contact e-mail: doug.ethell@ucr. edu Background: Boosting immune responses to Ab can reduce cognitive and pathology in mouse models of Alzheimer’s disease, but the role of Ab-specific antibodies in this process remains enigmatic. We have previously shown that Ab-specific T cells facilitate cognitive improvements without generating a measurable anti-Ab antibody responses. However, it is unclear whether Th1 and/or Th2 cells were necessary for this effect. Methods: Cognitively-impaired APP+PS1 mice received injections of Th2 cells isolated from Ab-vaccinated wild-type mice and re-stimulated in vitro with Ab, IL2 and IL-4. Mice were subjected to behavioral studies before and after receiving T cell injections, with extensive pathology and histology following the final behavioral studies. Results: APP+PS mice that received Ab-specific Th2 cells performed significantly better in working memory tasks, which
correlated with higher plasma levels of soluble Ab, but no detectable Ab antibody titer using a polyvalent antibody that evaluates IgA, IgG, and IgM. Pathological analysis of the hippocampus revealed a 30% decrease of plaqueassociated microglia and less vascular amyloidosis in Th2-treated mice. The infusion of Ab-specific Th2 cells also reduced plasma levels of IFN-g, TNFa, GM-CSF, IL-2 and IL-4, which are elevated in untreated APP+PS1 mice. These results demonstrate that Ab-specific Th2 cells are sufficient to reverse cognitive impairment and provide multiple pathological benefits in an Alzheimer’s mouse model. Conclusions: We have found that a small number of purified Ab-specific T cells (Th2-biased) are sufficient to provide cognitive and pathological benefits in an APP+PS1 mouse model for Alzheimer’s disease, without the production of Ab-specific antibodies. Supported by NIH and the California Institute for Regenerative Medicine. P3-263
HYDROGEN SULFIDE, A NOVEL NEUROPROTECTANT IN THE NEURODEGENERATIVE DISEASES
Lifang Hu, Jinsong Bian, National University of Singapore, Singapore. Contact e-mail: [email protected] Background: Hydrogen sulfide (H2S) has recently been proposed to be a novel neuromodulator. The present study was designed to investigate its therapeutic value in the neurodegenerative diseases. Methods: We observed the effects of NaHS, an H2S donor, on neuroinflammation induced by betaamyloid in BV-2 microglial cells and cell injury induced by rotenone in human-derived dopaminergic neuroblastoma cell line (SH-SY5Y). Results: NaHS concentration-dependently suppressed beta amyloid -induced nitrite accumulation in BV-2 microglial cells and rotenone-induced cellular injury and apoptotic cell death in SH-SY5Y cells. It was found that NaHS prevented p38-MAPK phosphorylation-induced by neurotoxins in both BV-2 and SHSY5Y cells. In addition, NaHS also attenuated rotenone-mediated changes in Bcl-2/Bax levels, mitochondrial membrane potential (DJm) dissipation, cytochrome c release, caspase-9/3 activation and poly (ADP-ribose) polymerase (PARP) cleavage. 5-hydroxydecanoate (5-HD), a selective blocker of mitochondrial ATP-sensitive potassium (mitoKATP) channel, attenuated the protective effects of NaHS against rotenone-induced cell apoptosis. Conclusions: H2S protects against neurotoxins-induced cellular injury via p38MAPK pathway in both microglial and neuronal cells. MitoKATP channels may also contribute to the anti-apoptotic effect of H2S in neuronal cells. Our findings suggest that H2S may have important implications in the treatment of neurodegenerative diseases (e.g.Parkinson’s disease and Alzheimer’s disease). P3-264
SELECTIVE MODULATION OF AB42 BY CALCIUM CHANNEL ANTAGONISTS. RELEVANCE OF CALCIUM DYNAMICS
Antonello D’Arrigo, Elda Del Giudice, Michele Fabris, Maurizio Dalle Carbonare, Davide Colavito, Alberta Leon, Research & Innovation S.P.A., Padova, Italy. Contact e-mail: [email protected] Background: Evidence for a modulatory activity of dihydropyridines (DHPs) on beta-amyloid (Ab) production has been reported previously by our group in immortalised cells overexpressing a FAD-linked APP mutation (Facchinetti et al. Neurobiology of Aging 27: 218, 2006). In that report, nimodipine and other DHPs were described to affect the levels of Ab released into the extracellular medium. In the present study, the action exerted by a panel of calcium antagonists on amyloidogenesis was explored and further characterised employing mouse primary cortical neurons. Methods: Experiments were conducted on primary neurons from hippocampi and frontal cortices of embryonic-day 1618 (E1618) mice carrying the Swedish mutation of human amyloid precursor protein (APP), KM670/671NL. Neurons were maintained in culture for 7-8 days before the experiment. Both intracellular and extracellular beta-amyloid levels were assessed under different experimental conditions. Results: Among the tested compounds, nimodipine was found to specifically modulate Ab42 levels. Moreover, some DHPs, but not others, significantly altered Ab42/Ab40 ratio. These effects were not entirely accounted for by the role of these molecules as calcium channel antagonists. Furthermore, structurally unrelated blockers of
Poster Presentations P3 calcium influx were completely uneffective on Ab levels. Experiments aiming at defining the impact of selected DHPs on APP cleavage as well as their ability to alter intracellular amyloid transport are currently ongoing. Conclusions: Results obtained to date emphasise the relevance of DHP-like structures as potential candidates in the development of an Ab-targeted pharmacology. P3-265
KMS88009 AND KMS88016 AS SMALL MOLECULE INHIBITORS FOR IN VIVO Ab FIBRIL FORMATION
YoungSoo Kim1, Ji Hun Byun1, Jiyeon Ryu1, Hana Hwang1, Ji Hoon Lee1, Jeiwon Cho1, HyeYun Kim1, Young-Gil Ahn2, Young Hoon Kim2, Maeng Sup Kim2, Gwan Sun Lee2, Inhee Mook-Jung3, Kyung Ho Yoo1, Dong Jin Kim1, 1Korea Institute of Science and Technology, Seoul, Republic of Korea; 2Hanmi Research Center, Hanmi Pharm. Co., Ltd., Seoul, Republic of Korea; 3Seoul National University College of Medicine, Seoul, Republic of Korea. Contact e-mail: [email protected] Background: Since cholinergic deficit in the central nervous system has been observed in AD patients, inhibitors for acetylcholine esterase (AchE) such as tacrin, donepezil, and rivastigmine are used for the clinical treatment of AD symptoms in current therapeutic approaches. Although these are the only FDA-approved AD therapies on the market, they cannot solve fundamental problems but can be only palliative solutions. Ab aggregates such as oligomers and fibrils deposit in the brain causing strong neuronal toxicity. In addition, it was recently reported that oligomers might be more toxic than fibrils, and the oligomers are becoming the target for the development of AD treatments. Methods: Here we report the synthesis and in vitro/vivo evaluations of a novel KMS88 series with potent inhibitory activities for Ab fibril formation. Thioflavin T assays, in vitro MTT cytotoxicity assays, transmission electron microscopy, pahrmacokinetics, behavior studies of transgenic mouse models, immunohistochemistry studies, and ex vivo brain imaging were performed to exam activities on Ab aggregation cascade in AD. Results: All 23 synthetic compounds in this study inhibited Ab(1-42) fibril formation near sub-mM range of IC50 (0.07-5.44 mM), better than curcumin (0.8 mM), without cytotoxicities on HT-22 neuronal cells by MTT assay. Then, KMS88009 (BA 64%, BBB >100%) and KMS88016 (BA 69%, BBB >100%) were selected as primary drug candidates for further in vivo studies. In the brains of AD transgenic mice, the amount of Ab fibrils were notably decreased after 7 months of oral administration. Prevention of cognitive impairment was also observed in behavior tests. Conclusions: KMS88009 and KMS88016 are promising AD therapeutics candidates which inhibit formation of Ab fibrils in AD brains and prevent cognitive impairment.
Background: The formation of Amyloid beta (Ab) oligomers and fibrils plays an important role in Alzheimer’s disease (AD). Among the wide variety of attempts to interpret amyloidosis mechanism, interactions of Ab and sulfate moieties of glycosaminoglycans (GAGs) or proteoglycans (PGs) have been reported as a critical factor in Ab oligomer/fibril formation. Negatively charged sulfate moieties of GAGs bind to Ab and induce a conformational change in Ab from a random coil to a b-sheet. Methods: Via thioflavin T (ThT) aggregation assays, SDS-PAGE with PICUP chemistry, and transmission electron microscopic studies, we examined effects of KMSB600 on Ab fibril formation. Results: As a result, KMSB600 inhibited the assembly Ab from monomers to oligomers/fibrils and also disaggregated preformed Ab fibrils. In addition, the excellent cell viability was observed due to KMSB600 at inhibits and reverses amyloidogenesis. A GAG mimicking organic acid compound, KMSB600 with a sulfate moiety, was found as a promising small molecule to inhibit Ab aggregation and to disaggregate Ab fibrils in vitro. Conclusions: Although the mechanism by KMSB600 on Ab fibril formation in vitro is still unclear, it could serve as a key scaffold for the development of more competent molecules to treat AD in vivo.
P3-267
DISAGGREGATION OF b-AMYLOID PLAQUES BY KMSB600
HyeYun Kim1, YoungSoo Kim1, GyoonHee Han2, Dong Jin Kim1, 1Korea Institute of Science & Technology (KIST), Seoul, Republic of Korea; 2Yonsei University, Seoul, Republic of Korea. Contact e-mail: cookietomato@ hotmail.com
REGULATION OF GENE EXPRESSION BY SAPPa AND RER DERIVATIVE IN DIFFERENTIATED NEUROBLASTOMA CELLS
Margaret M. Ryan, Gary Morris, Katie Bourne, Bruce G. Mockett, Wickliffe C. Abraham, Warren P. Tate, Joanna M. Williams, University of Otago, Dunedin, New Zealand. Contact e-mail: [email protected] Background: The amyloid precursor protein (APP) is cleaved by two opposing pathways, the most well understood of which yields the neurotoxic peptide, amyloid-b, while the second mutually exclusive pathway yields a secreted molecule, sAPPa, a 612 amino acid peptide. Infusion of sAPPa in vivo enhances the LTP-model of memory and inhibition of endogenous sAPPa synthesis in vivo by infusion of TAPI, a metalloprotease inhibitor, results in a reduced retention of spatial memories. This effect, however, can be rescued by co-infusion of recombinant sAPPa. LTP persistence and memory storage are dependent on new gene transcription and sAPPa is known to regulate gene expression, in particular it can up regulate expression of insulinlike growth factor-2 (IGF2) and IGF-binding protein-2 (IGFBP2). As short 3’ motifs of sAPPa have been shown to protect against amyloid-b-induced memory loss, we have investigated whether the RER-sAPPa motif can regulate gene expression in a manner similar to sAPPa. Methods: Retinoic acid-differentiated SH-SY5Y neuroblastoma cells were treated with either saline, recombinant sAPPa (1 or 10 nM) or RER (10 nM) for 2 h. The relative levels of IGF2 and IGFBP2 in the drug-treated vs. control groups were determined by extraction of total RNA from 6 x 105 cells, conversion to cDNA and a SYBR Green quantitative PCR assay, where HPRT levels were used as the normaliser. All assays were carried out in triplicate and the data presented is an average of three separate experiments (n¼3). Results: We found that sAPPa treatment resulted in an increase in the expression of both IGF2 (1 nM: 3 fold; 10 nM:1.5 fold) and IGFBP2 (1 nM: 3.6 fold; 10 nM: 1.7 fold) and preliminary results show that this effect was similar with the RER motif (IGF2: 7.5 fold; IGFBP2: 1.88 fold). Conclusions: These data show that the both sAPPa and the RER motif can induce gene expression changes in differentiated neuroblastoma cells. These results suggest that the RER motif may potentially be vital to the neuroprotective and neuromodulatory properties of sAPPa. P3-268
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P421
AMYLOID-BETA (Aß) 42 PREFIBRILLAR OLIGOMER-SPECIFIC MIMOTOPE-PEPTIDE WHICH INHIBITS Aß42 BUT NOT Aß40 FIBRIL FORMATION
Koichi Tanaka, Masaaki Nishimura, Sho Kitamoto, Sho Takiguchi, Makoto Yamaguchi, Hiroko Nagao, Shuhei Hashiguchi, Yuji Ito, Kazuhisa Sugimura, Kagoshima University, Kagoshima, Japan. Contact e-mail: [email protected] Background: Alzheimer’s disease (AD) is characterized by abnormal amyloid-ß (Aß) aggregation in brain including fibrils and oligomers. The Aß
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the oligomers of Ab was 10 kDa (equivalent to dimmer for Ab1-40 and decamers for Ab25-35) and that DHA dose-dependently reduced these decamers. Conclusions: DHA decreases the in vitro fibrillation of Ab by inhibiting the oligomeric amyloid species and, therefore, Ab-related neurotoxicity or behavioral impairment could be restrained by DHA. P3-261
CURRENT DRUG TARGET SITES IN CLINICAL PRACTICE OR CLINICAL TRIAL, EXTENT OF DISEASE MODIFICATION FOR ALZHEIMER’S DISEASE
Farhan Ul Haq Subhani, Bolam Medical College Quetta Balochistan Pakistan, Quetta, Pakistan. Contact e-mail: [email protected] Background: Alzheimer’s disease is most common form of dementia and one of complicated neurodegenerative diseases. Different therapeutic target sites are approved and number of drug target sites are under development for the treatment of Alzheimer’s disease. The disease modification is claimed by several drug developers for Alzheimer’s disease but the disease process has not been significantly affected by the available drugs either in clinical practice or clinical trials. In this review, discussion of the effects of different drug target sites on the disease modification will be compiled. Methods: It is a review to discuss in detail the efficacy of different drugs for the disease modifying effect of their respective target site. Results: Alzheimer’s disease have been know to scientifically community for almost 100 years and good therapeutic revaluation has been experienced during last 2 decades with respect to the treatment of this mysterious disease. Different drug target sites have been approved for the treatment of disease which includes cholinesterase inhibitors, NMDA receptor antagonist and products to elevate ketone bodies level in brain. Many drug target sites are in the process of development from discovery to phase III stage of clinical trail. These target sites are beta amyloid modulator, amyloid protein deposition inhibitors, nicotinic ACh modulator, muscarinic M1 agonist, muscarinic M2 antagonist, TrkA receptor modulator, proteasome inhibitor, AMPA receptor antagonist & modulator, anti-inflammatory cytokine synthesis inhibitor, caspase-3 inhibitor, PDE 4 inhibitor, tau aggregation inhibitor, beta & gamma-secretrase inhibitor, benzothiophene derivatives, COX-2 inhibitor, histamine H3 antagonist, antioxidant agents, imidazoline receptor antagonist, alpha 2 adrenoceptor antagonist, metal (Zn, Cu) chelating agent, free radical scavenger, MAO B inhibitor, 5-HT agonist and glycogen synthase kinase-3 inhibitor etc. Conclusions: In this review disease modification effect of few important drug targetsites for AD will be discussed. The modification process is not for sake of cure in all these target sites but we are being hopeful for having a definitive cure from this next generation of drug target sites in case of Alzheimer’s disease. P3-262
COGNITIVE AND PATHOLOGICAL BENEFITS OF Ab-SENSITIVE TH2 CELLS ARE INDEPENDENT OF ANTIGEN-SPECIFIC B CELL ACTIVATION
Douglas W. Ethell1, Chuanhai Cao2, Alexander Dickson3, Malgorzata B. Mamcarz3, Xiaoyang Lin2, Gary W. Arendash2,3, 1University of California Riverside, Riverside, CA, USA; 2Florida Alzheimer’s Disease Research Center, Tampa, FL, USA; 3Johnnie B. Byrd Alzheimer’s Center and Research Institute, Tampa, FL, USA. Contact e-mail: doug.ethell@ucr. edu Background: Boosting immune responses to Ab can reduce cognitive and pathology in mouse models of Alzheimer’s disease, but the role of Ab-specific antibodies in this process remains enigmatic. We have previously shown that Ab-specific T cells facilitate cognitive improvements without generating a measurable anti-Ab antibody responses. However, it is unclear whether Th1 and/or Th2 cells were necessary for this effect. Methods: Cognitively-impaired APP+PS1 mice received injections of Th2 cells isolated from Ab-vaccinated wild-type mice and re-stimulated in vitro with Ab, IL2 and IL-4. Mice were subjected to behavioral studies before and after receiving T cell injections, with extensive pathology and histology following the final behavioral studies. Results: APP+PS mice that received Ab-specific Th2 cells performed significantly better in working memory tasks, which
correlated with higher plasma levels of soluble Ab, but no detectable Ab antibody titer using a polyvalent antibody that evaluates IgA, IgG, and IgM. Pathological analysis of the hippocampus revealed a 30% decrease of plaqueassociated microglia and less vascular amyloidosis in Th2-treated mice. The infusion of Ab-specific Th2 cells also reduced plasma levels of IFN-g, TNFa, GM-CSF, IL-2 and IL-4, which are elevated in untreated APP+PS1 mice. These results demonstrate that Ab-specific Th2 cells are sufficient to reverse cognitive impairment and provide multiple pathological benefits in an Alzheimer’s mouse model. Conclusions: We have found that a small number of purified Ab-specific T cells (Th2-biased) are sufficient to provide cognitive and pathological benefits in an APP+PS1 mouse model for Alzheimer’s disease, without the production of Ab-specific antibodies. Supported by NIH and the California Institute for Regenerative Medicine. P3-263
HYDROGEN SULFIDE, A NOVEL NEUROPROTECTANT IN THE NEURODEGENERATIVE DISEASES
Lifang Hu, Jinsong Bian, National University of Singapore, Singapore. Contact e-mail: [email protected] Background: Hydrogen sulfide (H2S) has recently been proposed to be a novel neuromodulator. The present study was designed to investigate its therapeutic value in the neurodegenerative diseases. Methods: We observed the effects of NaHS, an H2S donor, on neuroinflammation induced by betaamyloid in BV-2 microglial cells and cell injury induced by rotenone in human-derived dopaminergic neuroblastoma cell line (SH-SY5Y). Results: NaHS concentration-dependently suppressed beta amyloid -induced nitrite accumulation in BV-2 microglial cells and rotenone-induced cellular injury and apoptotic cell death in SH-SY5Y cells. It was found that NaHS prevented p38-MAPK phosphorylation-induced by neurotoxins in both BV-2 and SHSY5Y cells. In addition, NaHS also attenuated rotenone-mediated changes in Bcl-2/Bax levels, mitochondrial membrane potential (DJm) dissipation, cytochrome c release, caspase-9/3 activation and poly (ADP-ribose) polymerase (PARP) cleavage. 5-hydroxydecanoate (5-HD), a selective blocker of mitochondrial ATP-sensitive potassium (mitoKATP) channel, attenuated the protective effects of NaHS against rotenone-induced cell apoptosis. Conclusions: H2S protects against neurotoxins-induced cellular injury via p38MAPK pathway in both microglial and neuronal cells. MitoKATP channels may also contribute to the anti-apoptotic effect of H2S in neuronal cells. Our findings suggest that H2S may have important implications in the treatment of neurodegenerative diseases (e.g.Parkinson’s disease and Alzheimer’s disease). P3-264
SELECTIVE MODULATION OF AB42 BY CALCIUM CHANNEL ANTAGONISTS. RELEVANCE OF CALCIUM DYNAMICS
Antonello D’Arrigo, Elda Del Giudice, Michele Fabris, Maurizio Dalle Carbonare, Davide Colavito, Alberta Leon, Research & Innovation S.P.A., Padova, Italy. Contact e-mail: [email protected] Background: Evidence for a modulatory activity of dihydropyridines (DHPs) on beta-amyloid (Ab) production has been reported previously by our group in immortalised cells overexpressing a FAD-linked APP mutation (Facchinetti et al. Neurobiology of Aging 27: 218, 2006). In that report, nimodipine and other DHPs were described to affect the levels of Ab released into the extracellular medium. In the present study, the action exerted by a panel of calcium antagonists on amyloidogenesis was explored and further characterised employing mouse primary cortical neurons. Methods: Experiments were conducted on primary neurons from hippocampi and frontal cortices of embryonic-day 1618 (E1618) mice carrying the Swedish mutation of human amyloid precursor protein (APP), KM670/671NL. Neurons were maintained in culture for 7-8 days before the experiment. Both intracellular and extracellular beta-amyloid levels were assessed under different experimental conditions. Results: Among the tested compounds, nimodipine was found to specifically modulate Ab42 levels. Moreover, some DHPs, but not others, significantly altered Ab42/Ab40 ratio. These effects were not entirely accounted for by the role of these molecules as calcium channel antagonists. Furthermore, structurally unrelated blockers of
Poster Presentations P3 calcium influx were completely uneffective on Ab levels. Experiments aiming at defining the impact of selected DHPs on APP cleavage as well as their ability to alter intracellular amyloid transport are currently ongoing. Conclusions: Results obtained to date emphasise the relevance of DHP-like structures as potential candidates in the development of an Ab-targeted pharmacology. P3-265
KMS88009 AND KMS88016 AS SMALL MOLECULE INHIBITORS FOR IN VIVO Ab FIBRIL FORMATION
YoungSoo Kim1, Ji Hun Byun1, Jiyeon Ryu1, Hana Hwang1, Ji Hoon Lee1, Jeiwon Cho1, HyeYun Kim1, Young-Gil Ahn2, Young Hoon Kim2, Maeng Sup Kim2, Gwan Sun Lee2, Inhee Mook-Jung3, Kyung Ho Yoo1, Dong Jin Kim1, 1Korea Institute of Science and Technology, Seoul, Republic of Korea; 2Hanmi Research Center, Hanmi Pharm. Co., Ltd., Seoul, Republic of Korea; 3Seoul National University College of Medicine, Seoul, Republic of Korea. Contact e-mail: [email protected] Background: Since cholinergic deficit in the central nervous system has been observed in AD patients, inhibitors for acetylcholine esterase (AchE) such as tacrin, donepezil, and rivastigmine are used for the clinical treatment of AD symptoms in current therapeutic approaches. Although these are the only FDA-approved AD therapies on the market, they cannot solve fundamental problems but can be only palliative solutions. Ab aggregates such as oligomers and fibrils deposit in the brain causing strong neuronal toxicity. In addition, it was recently reported that oligomers might be more toxic than fibrils, and the oligomers are becoming the target for the development of AD treatments. Methods: Here we report the synthesis and in vitro/vivo evaluations of a novel KMS88 series with potent inhibitory activities for Ab fibril formation. Thioflavin T assays, in vitro MTT cytotoxicity assays, transmission electron microscopy, pahrmacokinetics, behavior studies of transgenic mouse models, immunohistochemistry studies, and ex vivo brain imaging were performed to exam activities on Ab aggregation cascade in AD. Results: All 23 synthetic compounds in this study inhibited Ab(1-42) fibril formation near sub-mM range of IC50 (0.07-5.44 mM), better than curcumin (0.8 mM), without cytotoxicities on HT-22 neuronal cells by MTT assay. Then, KMS88009 (BA 64%, BBB >100%) and KMS88016 (BA 69%, BBB >100%) were selected as primary drug candidates for further in vivo studies. In the brains of AD transgenic mice, the amount of Ab fibrils were notably decreased after 7 months of oral administration. Prevention of cognitive impairment was also observed in behavior tests. Conclusions: KMS88009 and KMS88016 are promising AD therapeutics candidates which inhibit formation of Ab fibrils in AD brains and prevent cognitive impairment.
Background: The formation of Amyloid beta (Ab) oligomers and fibrils plays an important role in Alzheimer’s disease (AD). Among the wide variety of attempts to interpret amyloidosis mechanism, interactions of Ab and sulfate moieties of glycosaminoglycans (GAGs) or proteoglycans (PGs) have been reported as a critical factor in Ab oligomer/fibril formation. Negatively charged sulfate moieties of GAGs bind to Ab and induce a conformational change in Ab from a random coil to a b-sheet. Methods: Via thioflavin T (ThT) aggregation assays, SDS-PAGE with PICUP chemistry, and transmission electron microscopic studies, we examined effects of KMSB600 on Ab fibril formation. Results: As a result, KMSB600 inhibited the assembly Ab from monomers to oligomers/fibrils and also disaggregated preformed Ab fibrils. In addition, the excellent cell viability was observed due to KMSB600 at inhibits and reverses amyloidogenesis. A GAG mimicking organic acid compound, KMSB600 with a sulfate moiety, was found as a promising small molecule to inhibit Ab aggregation and to disaggregate Ab fibrils in vitro. Conclusions: Although the mechanism by KMSB600 on Ab fibril formation in vitro is still unclear, it could serve as a key scaffold for the development of more competent molecules to treat AD in vivo.
P3-267
DISAGGREGATION OF b-AMYLOID PLAQUES BY KMSB600
HyeYun Kim1, YoungSoo Kim1, GyoonHee Han2, Dong Jin Kim1, 1Korea Institute of Science & Technology (KIST), Seoul, Republic of Korea; 2Yonsei University, Seoul, Republic of Korea. Contact e-mail: cookietomato@ hotmail.com
REGULATION OF GENE EXPRESSION BY SAPPa AND RER DERIVATIVE IN DIFFERENTIATED NEUROBLASTOMA CELLS
Margaret M. Ryan, Gary Morris, Katie Bourne, Bruce G. Mockett, Wickliffe C. Abraham, Warren P. Tate, Joanna M. Williams, University of Otago, Dunedin, New Zealand. Contact e-mail: [email protected] Background: The amyloid precursor protein (APP) is cleaved by two opposing pathways, the most well understood of which yields the neurotoxic peptide, amyloid-b, while the second mutually exclusive pathway yields a secreted molecule, sAPPa, a 612 amino acid peptide. Infusion of sAPPa in vivo enhances the LTP-model of memory and inhibition of endogenous sAPPa synthesis in vivo by infusion of TAPI, a metalloprotease inhibitor, results in a reduced retention of spatial memories. This effect, however, can be rescued by co-infusion of recombinant sAPPa. LTP persistence and memory storage are dependent on new gene transcription and sAPPa is known to regulate gene expression, in particular it can up regulate expression of insulinlike growth factor-2 (IGF2) and IGF-binding protein-2 (IGFBP2). As short 3’ motifs of sAPPa have been shown to protect against amyloid-b-induced memory loss, we have investigated whether the RER-sAPPa motif can regulate gene expression in a manner similar to sAPPa. Methods: Retinoic acid-differentiated SH-SY5Y neuroblastoma cells were treated with either saline, recombinant sAPPa (1 or 10 nM) or RER (10 nM) for 2 h. The relative levels of IGF2 and IGFBP2 in the drug-treated vs. control groups were determined by extraction of total RNA from 6 x 105 cells, conversion to cDNA and a SYBR Green quantitative PCR assay, where HPRT levels were used as the normaliser. All assays were carried out in triplicate and the data presented is an average of three separate experiments (n¼3). Results: We found that sAPPa treatment resulted in an increase in the expression of both IGF2 (1 nM: 3 fold; 10 nM:1.5 fold) and IGFBP2 (1 nM: 3.6 fold; 10 nM: 1.7 fold) and preliminary results show that this effect was similar with the RER motif (IGF2: 7.5 fold; IGFBP2: 1.88 fold). Conclusions: These data show that the both sAPPa and the RER motif can induce gene expression changes in differentiated neuroblastoma cells. These results suggest that the RER motif may potentially be vital to the neuroprotective and neuromodulatory properties of sAPPa. P3-268
P3-266
P421
AMYLOID-BETA (Aß) 42 PREFIBRILLAR OLIGOMER-SPECIFIC MIMOTOPE-PEPTIDE WHICH INHIBITS Aß42 BUT NOT Aß40 FIBRIL FORMATION
Koichi Tanaka, Masaaki Nishimura, Sho Kitamoto, Sho Takiguchi, Makoto Yamaguchi, Hiroko Nagao, Shuhei Hashiguchi, Yuji Ito, Kazuhisa Sugimura, Kagoshima University, Kagoshima, Japan. Contact e-mail: [email protected] Background: Alzheimer’s disease (AD) is characterized by abnormal amyloid-ß (Aß) aggregation in brain including fibrils and oligomers. The Aß