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Computational studies on the stability of C-terminal beta-amyloid dimer and mutants
P718
P4-237
Poster Presentation: P4
COMPUTATIONAL STUDIES ON THE STABILITY OF C-TERMINAL BETA-AMYLOID DIMER AND MUTANTS
Vidana Epa1, Victor Streltsov2, Jose Varghese2, 1CSIRO, Parkville, Australia; 2CSIRO, Melbourne, Australia. Background: Insoluble amyloid plaque deposits characterize the primary pathology of Alzheimer’s disease. The amyloid-beta (Ab) peptide, cleaved from the membrane-bound amyloid precursor protein (APP) by the action of b-secretase and g-secretase into 39-43 amino acid residue long fragments, is the major constituent of this plaque. It is now believed that a major source of the neurotoxicity in AD is due to the action of intermediate soluble Ab oligomers [1]. Recently, a deletion mutant at the Glu 22 position (D E22) of Ab was discovered in some Familial Alzheimer’s Disease (FAD) patients in Japan. This mutation, named the Osaka mutant, appears to cause the A b peptide to oligomerize and fibrillize much more rapidly compared to the wild type [2]. Methods: Streltsov et al. [3] have determined the x-ray crystal structure of Ab (18-41) within the framework of shark IgNAR (Ig New Antigen Receptor) single variable domain antibody CDR3 loop. The Ab portion of the crystal structure is observed to be a tetramer (or a dimer of dimers), and can provide valuable insight into the structure-function relationships of A b. In this work we perform in silico or computational studies in order to understand the stability and conformational dynamics of the Ab peptide and the Osaka mutant oligomers. Results: These simulations, employing molecular dynamics methodology in an explicit solvent environment, commence from the dimer present in the crystal structure. We observe the relative stability of the various secondary structure elements, inter-residue interactions and conformational transitions. Conclusions: The deletion mutation is located in an important turn region with formation and breaking of transient hydrogen bonds and ionic interactions contributing to the dynamical behaviour of the peptide. References: 1. Crouch, P.J., Harding, S.M., White, A.R., Camakaris, J., Bush, A.I., & Masters, C.L., (2008). International Journal of Biochemistry & Cell Biology, 40, 181198. 2. Inayathullah, M. & Teplow, D.P., (2011). Amyloid, 18(3), 98-107. 3. Streltsov, V.A., Varghese, J.N., Masters, C.L., & Nuttall, S.N. (2011). Journal of Neuroscience, 31, 1419 -1426. P4-238
O-GLCNACYLATION INCREASES THE TARGETING OF BETA-AMYLOID PRECURSOR PROTEIN TO PLASMA MEMBRANE
Yoon Sun Chung, Sungkwon Chung, Sungkyunkwan University School of Medicine, Suwon, South Korea. Background: b -amyloid precursor protein (APP) is transported to the plasma membrane, where it is sequentially cleaved by a-secretase and g -secretase. This pathway is called non-amyloidogenic pathway, since it precludes the production of hydrophobic b -amyloid peptide (Ab), the main culprit of Alzheimer’s disease (AD). Alternatively, once APP undergoes clathrin-dependent endocytosis, it can be sequentially cleaved by b-secretase and g-secretase, producing Ab (amyloidogenic pathway). O-GlcNAcylation is a novel type of O-linked glycosylation attaching the monosaccharide b-N-acetylglucosamine (GlcNAc) to serine and threonine residues. In fact, APP was the first plasma membrane protein shown to be O-GlcNAcylated. Recently, it is shown that O-GlcNAcylation of APP increases the non-amyloidogenic processing of APP and decreases the production of A b. Even though O-GlcNAcylation has been shown to modify the processing of APP, the role of O-GlcNAcylation in APP trafficking and endocytosis remains unknown. Methods: Levels of sAPPa and sAPP were measured from the conditioned media using specific ELISAs. Also, levels of Abs were measured from the conditioned media using specific ELISAs. O-GlcNAcylated APP was increased by using inhibitor of O-GlcNAcase, PUGNAc. Levels of O-GlcNAcylated APP were measured by using Western blotting. Biotinylation method was used to measure the levels of APP located in the plasma membrane. Results: When O-GlcNAcylated APP was increased by using inhibitor of O-GlcNAcase, PUGNAc, the level of APP in the plasma membrane was increased. Also, the level of sAPPa (a-secretase product) increased, while the level of sAPPb (b-secretase product) was concomitantly decreased,
indicating the increased non-amyloidogenic processing of APP. Consistent with this results, the secreted levels of Ab species, Ab40 and Ab42, decreased by PUGNAc. These results strongly indicate that O-GlcNAcylation increases the plasma membrane targeting of APP, where it can be cleaved preferentially by a-secretase and g-secretase. Conclusions: O-GlcNAcylation of APP increased the non-amyloidogenic processing of APP reducing the generation of A b. Thus, O-GlcNAcylation of APP is implied as a potential therapeutic target for AD. P4-239
ABCA1 DEFICIENCY DIFFERENTIALLY AFFECTS THE AMYLOID PHENOTYPE OF APOE-ε3- AND APOE-ε4-TARGETED REPLACEMENT MICE
Iliya Lefterov1, Radosveta Koldamova2, Nicholas Fitz1, 1University of Pittsburgh, Pittsburgh, Pennsylvania, United States; 2University of Pittsburgh/GSPH, Pittsburgh, Pennsylvania, United States. Background: Inheritance of E4 allele of APOE (APOE4) is a major genetic risk factor for Alzheimer’s disease (AD), however the mechanisms underlying this association remain elusive. Detrimental effects of APOE4 on amyloid deposition might be due to its conformation. Alternatively, the higher incidence of AD and increased amyloid deposition in APOE4 carriers might be a result of lower APOE protein levels. We and others have reported that the deletion of Abca1 in APP expressing mice decreases APOE protein level in correlation with the increased amyloid deposition. In contrast, treatment of APP mice with LXR ligands increases ABCA1 and APOE protein level and decreases amyloid deposition. We hypothesize that the presence of functional ABCA1 is critical for the effects of APOE isoforms on amyloid deposition and cognitive decline. Methods: We examined the effects of Abca1 gene-dose on AD pathology and cognition in APP/PS1dE9 model mice expressing human APOE3 or APOE4 isoform. Results: Deletion of one copy of Abca1 gene in APP/PS1dE9 mice expressing APOE4 resulted in a significantly decreased performance in the radial water maze when compared to APOE4 mice expressing Abca1 wt. In contrast, lack of one copy of Abca1 did not exacerbate memory deficits of APP/APOE3. These changes in cognitive performance correlated to Ab plaques and insoluble Ab in the brain. Accordingly, amyloid pathology of APP/APOE3/Abca1 het mice did not differ significantly from APP/APOE3/Abca1wt which contrasted the effect of Abca1 deletion in APP/ APOE4 mice. We also found a significant difference between Ab level in serum of APP/APOE3 and APP/APOE4 mice lacking Abca1. Conclusions: Our study provides rationale to further investigate how APOE4 alleles interact with genes and environmental factors to contribute to AD phenotype. P4-240
EFFECT OF WHITE MATTER HYPERINTENSITY (WMH) LEVELS ON COGNITIVE FUNCTION ACCORDING TO APOLIPOPROTEIN E (APOE) GENOTYPE
Chang Hyung Hong1, Sang Joon Son2, Kang Soo Lee3, 1Ajou University School of Medicine, Suwon-si, Gyeonggi-do, South Korea; 2Ajou University, School of Medicine, Suwon-si, Gyeonggi-do, South Korea; 3Cha University, Seoul, South Korea. Background: The purpose of this study was to investigate the effect of White matter hyperintensities (WMH) severity on cognitive function according to the presence of apolipoprotein E (APOE) E4 allele. Methods: Of the participants in the hospital-based cohort study of dementia, 5077 elderly subjects who had available data for APOE genotype and WMH severity were studied. A standardized neuropsychological battery, containing tests of language, memory, attention and frontal/executive function was used for all subjects. Results: After multivariable adjustments, logistic regression analyses showed that higher odds of having cognitive impairment in both APOE E4 carrier and non-carrier when examining the effects of WMH on frontal/executive function tests such as Stroop test (E4 noncarrier, OR ¼ 2.36 (1.83-3.03); E4 carrier: OR ¼ 2.49 (1.65-3.76)). WMH severity was not even significantly associated with memory function in APOE E4 carriers (verbal memory test: E4 non-carrier, OR ¼ 1.44(1.131.84); E4 carrier, OR ¼ 1.36 (0.87-2.04)) (visuospatial memory test: E4 non-carrier, OR ¼ 1.86(1.45-2.37); E4 carrier, OR ¼ 1.35 (0.89-2.04)).
P4-237
Poster Presentation: P4
COMPUTATIONAL STUDIES ON THE STABILITY OF C-TERMINAL BETA-AMYLOID DIMER AND MUTANTS
Vidana Epa1, Victor Streltsov2, Jose Varghese2, 1CSIRO, Parkville, Australia; 2CSIRO, Melbourne, Australia. Background: Insoluble amyloid plaque deposits characterize the primary pathology of Alzheimer’s disease. The amyloid-beta (Ab) peptide, cleaved from the membrane-bound amyloid precursor protein (APP) by the action of b-secretase and g-secretase into 39-43 amino acid residue long fragments, is the major constituent of this plaque. It is now believed that a major source of the neurotoxicity in AD is due to the action of intermediate soluble Ab oligomers [1]. Recently, a deletion mutant at the Glu 22 position (D E22) of Ab was discovered in some Familial Alzheimer’s Disease (FAD) patients in Japan. This mutation, named the Osaka mutant, appears to cause the A b peptide to oligomerize and fibrillize much more rapidly compared to the wild type [2]. Methods: Streltsov et al. [3] have determined the x-ray crystal structure of Ab (18-41) within the framework of shark IgNAR (Ig New Antigen Receptor) single variable domain antibody CDR3 loop. The Ab portion of the crystal structure is observed to be a tetramer (or a dimer of dimers), and can provide valuable insight into the structure-function relationships of A b. In this work we perform in silico or computational studies in order to understand the stability and conformational dynamics of the Ab peptide and the Osaka mutant oligomers. Results: These simulations, employing molecular dynamics methodology in an explicit solvent environment, commence from the dimer present in the crystal structure. We observe the relative stability of the various secondary structure elements, inter-residue interactions and conformational transitions. Conclusions: The deletion mutation is located in an important turn region with formation and breaking of transient hydrogen bonds and ionic interactions contributing to the dynamical behaviour of the peptide. References: 1. Crouch, P.J., Harding, S.M., White, A.R., Camakaris, J., Bush, A.I., & Masters, C.L., (2008). International Journal of Biochemistry & Cell Biology, 40, 181198. 2. Inayathullah, M. & Teplow, D.P., (2011). Amyloid, 18(3), 98-107. 3. Streltsov, V.A., Varghese, J.N., Masters, C.L., & Nuttall, S.N. (2011). Journal of Neuroscience, 31, 1419 -1426. P4-238
O-GLCNACYLATION INCREASES THE TARGETING OF BETA-AMYLOID PRECURSOR PROTEIN TO PLASMA MEMBRANE
Yoon Sun Chung, Sungkwon Chung, Sungkyunkwan University School of Medicine, Suwon, South Korea. Background: b -amyloid precursor protein (APP) is transported to the plasma membrane, where it is sequentially cleaved by a-secretase and g -secretase. This pathway is called non-amyloidogenic pathway, since it precludes the production of hydrophobic b -amyloid peptide (Ab), the main culprit of Alzheimer’s disease (AD). Alternatively, once APP undergoes clathrin-dependent endocytosis, it can be sequentially cleaved by b-secretase and g-secretase, producing Ab (amyloidogenic pathway). O-GlcNAcylation is a novel type of O-linked glycosylation attaching the monosaccharide b-N-acetylglucosamine (GlcNAc) to serine and threonine residues. In fact, APP was the first plasma membrane protein shown to be O-GlcNAcylated. Recently, it is shown that O-GlcNAcylation of APP increases the non-amyloidogenic processing of APP and decreases the production of A b. Even though O-GlcNAcylation has been shown to modify the processing of APP, the role of O-GlcNAcylation in APP trafficking and endocytosis remains unknown. Methods: Levels of sAPPa and sAPP were measured from the conditioned media using specific ELISAs. Also, levels of Abs were measured from the conditioned media using specific ELISAs. O-GlcNAcylated APP was increased by using inhibitor of O-GlcNAcase, PUGNAc. Levels of O-GlcNAcylated APP were measured by using Western blotting. Biotinylation method was used to measure the levels of APP located in the plasma membrane. Results: When O-GlcNAcylated APP was increased by using inhibitor of O-GlcNAcase, PUGNAc, the level of APP in the plasma membrane was increased. Also, the level of sAPPa (a-secretase product) increased, while the level of sAPPb (b-secretase product) was concomitantly decreased,
indicating the increased non-amyloidogenic processing of APP. Consistent with this results, the secreted levels of Ab species, Ab40 and Ab42, decreased by PUGNAc. These results strongly indicate that O-GlcNAcylation increases the plasma membrane targeting of APP, where it can be cleaved preferentially by a-secretase and g-secretase. Conclusions: O-GlcNAcylation of APP increased the non-amyloidogenic processing of APP reducing the generation of A b. Thus, O-GlcNAcylation of APP is implied as a potential therapeutic target for AD. P4-239
ABCA1 DEFICIENCY DIFFERENTIALLY AFFECTS THE AMYLOID PHENOTYPE OF APOE-ε3- AND APOE-ε4-TARGETED REPLACEMENT MICE
Iliya Lefterov1, Radosveta Koldamova2, Nicholas Fitz1, 1University of Pittsburgh, Pittsburgh, Pennsylvania, United States; 2University of Pittsburgh/GSPH, Pittsburgh, Pennsylvania, United States. Background: Inheritance of E4 allele of APOE (APOE4) is a major genetic risk factor for Alzheimer’s disease (AD), however the mechanisms underlying this association remain elusive. Detrimental effects of APOE4 on amyloid deposition might be due to its conformation. Alternatively, the higher incidence of AD and increased amyloid deposition in APOE4 carriers might be a result of lower APOE protein levels. We and others have reported that the deletion of Abca1 in APP expressing mice decreases APOE protein level in correlation with the increased amyloid deposition. In contrast, treatment of APP mice with LXR ligands increases ABCA1 and APOE protein level and decreases amyloid deposition. We hypothesize that the presence of functional ABCA1 is critical for the effects of APOE isoforms on amyloid deposition and cognitive decline. Methods: We examined the effects of Abca1 gene-dose on AD pathology and cognition in APP/PS1dE9 model mice expressing human APOE3 or APOE4 isoform. Results: Deletion of one copy of Abca1 gene in APP/PS1dE9 mice expressing APOE4 resulted in a significantly decreased performance in the radial water maze when compared to APOE4 mice expressing Abca1 wt. In contrast, lack of one copy of Abca1 did not exacerbate memory deficits of APP/APOE3. These changes in cognitive performance correlated to Ab plaques and insoluble Ab in the brain. Accordingly, amyloid pathology of APP/APOE3/Abca1 het mice did not differ significantly from APP/APOE3/Abca1wt which contrasted the effect of Abca1 deletion in APP/ APOE4 mice. We also found a significant difference between Ab level in serum of APP/APOE3 and APP/APOE4 mice lacking Abca1. Conclusions: Our study provides rationale to further investigate how APOE4 alleles interact with genes and environmental factors to contribute to AD phenotype. P4-240
EFFECT OF WHITE MATTER HYPERINTENSITY (WMH) LEVELS ON COGNITIVE FUNCTION ACCORDING TO APOLIPOPROTEIN E (APOE) GENOTYPE
Chang Hyung Hong1, Sang Joon Son2, Kang Soo Lee3, 1Ajou University School of Medicine, Suwon-si, Gyeonggi-do, South Korea; 2Ajou University, School of Medicine, Suwon-si, Gyeonggi-do, South Korea; 3Cha University, Seoul, South Korea. Background: The purpose of this study was to investigate the effect of White matter hyperintensities (WMH) severity on cognitive function according to the presence of apolipoprotein E (APOE) E4 allele. Methods: Of the participants in the hospital-based cohort study of dementia, 5077 elderly subjects who had available data for APOE genotype and WMH severity were studied. A standardized neuropsychological battery, containing tests of language, memory, attention and frontal/executive function was used for all subjects. Results: After multivariable adjustments, logistic regression analyses showed that higher odds of having cognitive impairment in both APOE E4 carrier and non-carrier when examining the effects of WMH on frontal/executive function tests such as Stroop test (E4 noncarrier, OR ¼ 2.36 (1.83-3.03); E4 carrier: OR ¼ 2.49 (1.65-3.76)). WMH severity was not even significantly associated with memory function in APOE E4 carriers (verbal memory test: E4 non-carrier, OR ¼ 1.44(1.131.84); E4 carrier, OR ¼ 1.36 (0.87-2.04)) (visuospatial memory test: E4 non-carrier, OR ¼ 1.86(1.45-2.37); E4 carrier, OR ¼ 1.35 (0.89-2.04)).