- Email: [email protected]
PLASMA AB40 AND AB42 DECLINE WITH PROGRESSION OF DEMENTIA
Oral Sessions: O4-10: Biomarkers: Blood-Based Abeta and Tau Markers
six scans. The primary analysis included 3388 hippocampal volume measures and investigated the association of A b 42/A b 40 (lowest vs. highest tertile) with rate of change in hippocampal volumes using a longitudinal linear mixed effects model. Rate of change was adjusted for age, sex, no. of APOE ε4 alleles, and years of education by including them as interaction terms with time; overall levels in these models were adjusted for the same variables plus mean total intracranial volume. Similar analyses were used for A b 42 and A b 40. Baseline association analyses were conducted with standard linear regression only in those who had an MRI within 6 months of their plasma sample (n¼911). Results: Patient characteristics are summarized in the table. There was marginal evidence of association between A b 42/A b 40 and hippocampal volume change (p¼0.079); for an 80 year old male, with no ε 4 alleles, 12 years of education, the fitted decline (per year) was 1.04% (95% CI: 0.82% to 1.25%) if their A b 42/ A b 40 was in the highest tertile, while it was 1.25% (95% CI: 1.03% to 1.47%) for the lowest tertile. Results for A b 42 were similar to those for the ratio (p¼0.077); there was no evidence of association for A b 40 (p¼0.71). Baseline hippocampal volumes were not associated with the A b measures. Conclusions: Although there is little potential for plasma A b to have clinical utility the findings are of biological interest and the results are in keeping with cognitive decline being associated with a low Ab42 and a low Ab42/Ab40 ratio. We plan to expand this study evaluating other MR measures such as grey matter volume loss. O4-10-02
PLASMA AB40 AND AB42 DECLINE WITH PROGRESSION OF DEMENTIA
Lawrence S. Honig1, Min-Suk Kang1, Ming-Xin Tang1, Jennifer J. Manly1, Richard Mayeux2, Nicole Schupf1, 1Columbia University Medical Center, New York, New York, United States; 2Columbia University Medical Center, New York, New York, United States. Contact e-mail: [email protected] Background: Beta-amyloid 1-40 (AB40) and 1-42 (AB42) are major components of the neuritic plaques seen neuropathologically in Alzheimer’s disease (AD). These peptides can also be measured in blood, although their origin may be cerebral. The relationship of changes in plasma levels of AB40 and AB42 to AD is still not well-established. This may relate to study of differing cohorts at different stages of disease and assay variations. Here we report the results of standardized measurements of AB40 and AB42 in the Washington Heights-Inwood Columbia Aging Project (WHICAP), a longitudinal multi-ethnic cohort study of community based elders, 65 years and older. Methods: We examined 810 participants of the WHICAP project who had three available plasma samples over time, and either had dementia (N ¼ 67; 94% mild and 97% possible or probable AD), or remained nondemented (N¼743), through the sampling period. These included 241 men (30%) and 569 women (70%), representing three race/ethnicity groups: 29% non-Hispanic whites, 29% with non-Hispanic blacks, 42% Hispanics. Mean education was 10.164.8(0-20)yr; 24% had 1 APOE4 allele. Mean age at first and last sampling were 76.565.5(range 66-95)yr, and 84.665.4(range 71-103)yr respectively. Mean interval between first and last blood sampling was 8.162.2(range 1-17)yr. AB40 and AB42 were measured in duplicate using the Innogenetics Luminex platform, as in the ADNI study, with mean COV of 3.5% for both. We used General Estimating Equations (GEE) to examine relationship of AB40 and AB42 changes over time, to dementia status during the sampling period. Results: At baseline, mean AB40 and AB42 were 113.0651.2 and 23.1612.1 pg/ml. Overall, both AB40 and AB42 increased with age. However, GEE analysis, adjusting for age, sex, ethnicity, education, and APOE genotype, showed that declines in both AB40 and AB42 occurred over time in persons with dementia, but not among those who remained nondemented during the blood sampling period (both p¼0.03). Conclusions: In this multi-ethnic community-based study, declining plasma AB40 and AB42 levels were associated with dementia. It is not certain which aspect of AD this might reflect. And, additional investigations are required to determine if plasma AB levels could be useful as diagnostic markers.
O4-10-03
P271
AN ASSAY FOR SEEDED PROTEIN AGGREGATION DETECTS ABETA SEEDS IN SERUM
Damian Christopher Crowther, Liisa van Vliet, Sue Kuhaudomlarp, Fabrice Gielen, Jing Yan, Mayida Azhar, Marie-Pierre Hinault, Florian Hollfelder, University of Cambridge, Cambridge, United Kingdom. Contact e-mail: [email protected] Background: Many common neurodegenerative disorders are characterised by progressive protein aggregation. In Alzheimer’s disease the principal pathologies of interest are amyloid plaques composed of the amyloid-beta peptide (Ab) and tangles comprised of tau. The spread of pathology within the brain is thought to be mediated by a prionlike seeding process that accelerates the aggregation of susceptible polypeptides in the neighbourhood of existing deposits. Soluble seeds of Ab may travel widely: between neurones or even to the CSF and peripheral circulation. Methods: We measure seeded Ab aggregation in brain and blood extracts from animal models of Alzheimer’s disease. Firstly we examine brains from Drosophila melanogaster expressing the Ab peptide versus non-expressing controls. Secondly we assess brain and serum from aged CRND8 mice and compare them to their non-transgenic littermate controls. Brains are homogenised in PBS followed by dilution to give a protein concentration of 3 mg/ml. Serum is diluted to 3 mg/ml in PBS.Our assay uses microfluidic apparatus to compartmentalise these crude biological extracts into aqueous droplets (50 mm diameter) containing fluorescent-labelled Ab42 peptide along with molten agarose (37 C). The droplets are cooled to room temperature, whereupon the agarose forms a gel, and are then incubated for up to 3 hr. Following this incubation the oil phase is removed and the uncoated agarose microbeads are washed to remove unincorporated fluorescent peptide. Results: Seeded aggregates are marked by their having incorporated fluorescent-labelled Abeta during the incubation step. Aggregates that are larger than MW 1000 kDa are retained within the agarose matrix and are detected and quantified by fluorescence activated cytometry (FACS). Over 10,000 beads are assessed for each assay.In the brain extracts we see significantly higher fluorescence signals in microbeads derived from AD-affected organisms as compared to controls. Remarkably this higher fluorescence signal is also seen in the serum samples from affected mice. Conclusions: These data indicate that Ab-seeding activity can be detected in the brain and serum of models of Alzheimer’s disease. We are now testing human clinical samples as this assay may provide novel prognostic and/or diagnostic tools in humans.
Figure. Agarose beads alone have a low intrinsic fluorescence (grey). In the presence of fluorescent labelled Ab peptide (blue) the agarose beads retain a background level of fluorescence. Healthy murine serum resulted in increased fluorescence labelling of beads (orange) but the strongest signal was seen for serum derived from CRND8 mice (green).
six scans. The primary analysis included 3388 hippocampal volume measures and investigated the association of A b 42/A b 40 (lowest vs. highest tertile) with rate of change in hippocampal volumes using a longitudinal linear mixed effects model. Rate of change was adjusted for age, sex, no. of APOE ε4 alleles, and years of education by including them as interaction terms with time; overall levels in these models were adjusted for the same variables plus mean total intracranial volume. Similar analyses were used for A b 42 and A b 40. Baseline association analyses were conducted with standard linear regression only in those who had an MRI within 6 months of their plasma sample (n¼911). Results: Patient characteristics are summarized in the table. There was marginal evidence of association between A b 42/A b 40 and hippocampal volume change (p¼0.079); for an 80 year old male, with no ε 4 alleles, 12 years of education, the fitted decline (per year) was 1.04% (95% CI: 0.82% to 1.25%) if their A b 42/ A b 40 was in the highest tertile, while it was 1.25% (95% CI: 1.03% to 1.47%) for the lowest tertile. Results for A b 42 were similar to those for the ratio (p¼0.077); there was no evidence of association for A b 40 (p¼0.71). Baseline hippocampal volumes were not associated with the A b measures. Conclusions: Although there is little potential for plasma A b to have clinical utility the findings are of biological interest and the results are in keeping with cognitive decline being associated with a low Ab42 and a low Ab42/Ab40 ratio. We plan to expand this study evaluating other MR measures such as grey matter volume loss. O4-10-02
PLASMA AB40 AND AB42 DECLINE WITH PROGRESSION OF DEMENTIA
Lawrence S. Honig1, Min-Suk Kang1, Ming-Xin Tang1, Jennifer J. Manly1, Richard Mayeux2, Nicole Schupf1, 1Columbia University Medical Center, New York, New York, United States; 2Columbia University Medical Center, New York, New York, United States. Contact e-mail: [email protected] Background: Beta-amyloid 1-40 (AB40) and 1-42 (AB42) are major components of the neuritic plaques seen neuropathologically in Alzheimer’s disease (AD). These peptides can also be measured in blood, although their origin may be cerebral. The relationship of changes in plasma levels of AB40 and AB42 to AD is still not well-established. This may relate to study of differing cohorts at different stages of disease and assay variations. Here we report the results of standardized measurements of AB40 and AB42 in the Washington Heights-Inwood Columbia Aging Project (WHICAP), a longitudinal multi-ethnic cohort study of community based elders, 65 years and older. Methods: We examined 810 participants of the WHICAP project who had three available plasma samples over time, and either had dementia (N ¼ 67; 94% mild and 97% possible or probable AD), or remained nondemented (N¼743), through the sampling period. These included 241 men (30%) and 569 women (70%), representing three race/ethnicity groups: 29% non-Hispanic whites, 29% with non-Hispanic blacks, 42% Hispanics. Mean education was 10.164.8(0-20)yr; 24% had 1 APOE4 allele. Mean age at first and last sampling were 76.565.5(range 66-95)yr, and 84.665.4(range 71-103)yr respectively. Mean interval between first and last blood sampling was 8.162.2(range 1-17)yr. AB40 and AB42 were measured in duplicate using the Innogenetics Luminex platform, as in the ADNI study, with mean COV of 3.5% for both. We used General Estimating Equations (GEE) to examine relationship of AB40 and AB42 changes over time, to dementia status during the sampling period. Results: At baseline, mean AB40 and AB42 were 113.0651.2 and 23.1612.1 pg/ml. Overall, both AB40 and AB42 increased with age. However, GEE analysis, adjusting for age, sex, ethnicity, education, and APOE genotype, showed that declines in both AB40 and AB42 occurred over time in persons with dementia, but not among those who remained nondemented during the blood sampling period (both p¼0.03). Conclusions: In this multi-ethnic community-based study, declining plasma AB40 and AB42 levels were associated with dementia. It is not certain which aspect of AD this might reflect. And, additional investigations are required to determine if plasma AB levels could be useful as diagnostic markers.
O4-10-03
P271
AN ASSAY FOR SEEDED PROTEIN AGGREGATION DETECTS ABETA SEEDS IN SERUM
Damian Christopher Crowther, Liisa van Vliet, Sue Kuhaudomlarp, Fabrice Gielen, Jing Yan, Mayida Azhar, Marie-Pierre Hinault, Florian Hollfelder, University of Cambridge, Cambridge, United Kingdom. Contact e-mail: [email protected] Background: Many common neurodegenerative disorders are characterised by progressive protein aggregation. In Alzheimer’s disease the principal pathologies of interest are amyloid plaques composed of the amyloid-beta peptide (Ab) and tangles comprised of tau. The spread of pathology within the brain is thought to be mediated by a prionlike seeding process that accelerates the aggregation of susceptible polypeptides in the neighbourhood of existing deposits. Soluble seeds of Ab may travel widely: between neurones or even to the CSF and peripheral circulation. Methods: We measure seeded Ab aggregation in brain and blood extracts from animal models of Alzheimer’s disease. Firstly we examine brains from Drosophila melanogaster expressing the Ab peptide versus non-expressing controls. Secondly we assess brain and serum from aged CRND8 mice and compare them to their non-transgenic littermate controls. Brains are homogenised in PBS followed by dilution to give a protein concentration of 3 mg/ml. Serum is diluted to 3 mg/ml in PBS.Our assay uses microfluidic apparatus to compartmentalise these crude biological extracts into aqueous droplets (50 mm diameter) containing fluorescent-labelled Ab42 peptide along with molten agarose (37 C). The droplets are cooled to room temperature, whereupon the agarose forms a gel, and are then incubated for up to 3 hr. Following this incubation the oil phase is removed and the uncoated agarose microbeads are washed to remove unincorporated fluorescent peptide. Results: Seeded aggregates are marked by their having incorporated fluorescent-labelled Abeta during the incubation step. Aggregates that are larger than MW 1000 kDa are retained within the agarose matrix and are detected and quantified by fluorescence activated cytometry (FACS). Over 10,000 beads are assessed for each assay.In the brain extracts we see significantly higher fluorescence signals in microbeads derived from AD-affected organisms as compared to controls. Remarkably this higher fluorescence signal is also seen in the serum samples from affected mice. Conclusions: These data indicate that Ab-seeding activity can be detected in the brain and serum of models of Alzheimer’s disease. We are now testing human clinical samples as this assay may provide novel prognostic and/or diagnostic tools in humans.
Figure. Agarose beads alone have a low intrinsic fluorescence (grey). In the presence of fluorescent labelled Ab peptide (blue) the agarose beads retain a background level of fluorescence. Healthy murine serum resulted in increased fluorescence labelling of beads (orange) but the strongest signal was seen for serum derived from CRND8 mice (green).