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miRNA expression in cerebrospinal fluid of Alzheimer's disease patients
Poster Presentations: P2 patients were clearly less modulated by either iTBS or cTBS as compared to HCs (Fig 1). Differences of MEP amplitudes between AD patients and HCs at T15 were clearly significant. The discrepancy between the AD and HC group was most prominent after iTBS. While MEPs of AD patients did not change significantly post-iTBS (mean 1%), the HC group showed an increase in MEP amplitudes (mean 59%). On the other hand, both groups showed an inhibitory response to cTBS (AD mean -30%; HC mean -39%), whereas the inhibitory effect decreased faster in AD patients. Conclusions: TMS measures may be used as neurophysiologic biomarkers indicating functional/neuroplastic changes in AD patients.
Figure 1. Brain plasticity measures as assessed with cTBS over M1 in healthy controls and in AD patients.
P2-034
MIRNA EXPRESSION IN CEREBROSPINAL FLUID OF ALZHEIMER’S DISEASE PATIENTS
Argonde van Harten1, Joyce Mulders2, Philip Scheltens2, Wiesje Van der Flier2, Cees Oudejans2, 1Alzheimer Center VUmc, Amsterdam, Amsterdam, Netherlands; 2VU University Medical Center, Amsterdam, Netherlands. Background: MicroRNAs (miR) are small pieces of RNA that have the ability to post-transcriptionally regulate protein expression. Many miR have been shown to be differentially expressed in the cortex of AD patients and seemingly play a role in AD pathogenesis, rendering them a highly interesting target. In this study we aimed to evaluate whether miR can be measured in cerebrospinal fluid (CSF) and to ascertain whether miR are differentially expressed in CSF of Alzheimer’s disease (AD) patients using a data-driven qRT-PCR approach. Methods: RNA was isolated from 2 ml CSF of 19 AD patients and 19 age and sex-matched controls using the mirVana Paris isolation kit (Ambion). miR were quantified by means of qRT-PCR using Taqman Array MicroRNA Cards A and B (v3.0; Applied Biosystems) according to the megaplex protocol. Mean Ct value of all miR present in one plate was used as endogenous control. Relative quantification (RQ) was calculated as 2-ddCt. Results: On average 107 6 44 out of 776 miR (14%)were detected in CSF using megaplex array analysis. Abundance was very low with an average Ct value of 33 6 1 in all detected miR. Exploratory analysis showed a correlation between age and number of miR detected (r ¼ -0.41, P<0.05) and between age and mean Ct value of all detected miR (r ¼ 0.47, P <0.01). Because of these strong correlations, we performed separate between group comparisons for older and younger patients (cut-off 65 years). In the early-onset AD group 13 miR were differentially expressed (P <0.05). In the late-onset AD group 6 other miR were differentially expressed (P <0.05). Conclusions: We have shown that it is possible to measure miR in CSF, suggesting a potential future use as biomarkers in living patients. Several miR seem to be differentially expressed in CSF of AD patients. Further investigation is warranted, especially since accumulating evidence suggests miR expression plays an important role in AD pathogenesis. P2-035
PHOSPHOSPHINGOLIPID LEVELS ARE ASSOCIATED WITH COGNITIVE FUNCTION AND LEVEL OF EDUCATION IN HEALTHY SUBJECTS
Carla Ibrahim-Verbaas, Ayse Demirkan, Aaron Isaacs, Najaf Amin, John van Swieten, Ben Oostra, Cornelia van Duijn, Erasmus University Medical Center, Rotterdam, Netherlands.
P275
Background: Phosphosphingolipids are implied in signal transduction and stress-related neuronal apoptosis. Sphingomyelin and ceramide levels are altered in mild cognitive impairment (MCI) and Alzheimer’s Dementia (AD). An earlier study by our group in healthy individuals associated several SNPs in the APOE region to sphingomyelin levels. In this project, we assessed the association of phosphosphingolipids to cognitive traits in a healthy cohort. Methods: Subjects were participants of the Erasmus Rucphen Family (ERF) study, a family-based cohort. Subjects with dementia, brain damage or sensory or mental impairments were excluded. Phenotyping included an extensive battery of cognitive tests. Phosphosphingolipid profiling was performed on venous blood. We performed partial correlation analyses between cognitive traits and phosphosphingolipid measures. Results: Full cognitive and phosphosphingolipid data were available for 709 subjects (age 18-89 years, mean age ¼ 51 years). The sphingomyelins that were associated to the APOE region, were borderline significantly associated (P ¼ 0.04) to DART (Dutch Adult Reading test, a proxy for intelligence) (spm22:0, spm24:0) and immediate recall (spm22:0). With a Bonferroni-corrected significance threshold of P ¼ 2.210 -4, 13 correlations were significant. Sphingomyelin SPM18:1 (P ¼ 0.0001) was correlated to recognition, and SPM20:1 (P ¼ 0.0000260) to fluency. Nine phosphatidylcholine traits (PCs) correlated to education (tophit PC36:2, P ¼ 0.0000265). Generally, the ethyl-esterized forms of the PCs associated to higher academic performance, non-ethylesterized forms to lower schooling. Also LPC18:0 (P ¼ 4.910 -6) and Cer20:0 (P ¼ 0.00016) correlated significantly to education. Conclusions: We identified correlations between phosphosphingolipids and fluency, recognition, immediate recall, DART and education in nondemented subjects. The associations to DART suggest that phosphosphingolipids are associated to intelligence. As phosphosphingolipid levels are also highly correlated with dietary factors, this association may be explained by socio-economic factors. Alternatively, the PCs which include the Omega 3 and 6 fatty acids may influence cognitive function. Beside replication efforts, our further explorations in this field will include Mendelian Randomization type disentanglement of the direction of causality by investigating associations of the genes related to cognitive function to phosphosphingolipid levels and vice versa. P2-036
DEVELOPMENT AND ANALYTICAL VALIDATION OF A NOVEL ASSAY FOR DETECTION OF HUMAN Ab1-42 PEPTIDE IN HUMAN CSF
Flora Berisha1, Jill Dunty2, Leonid Dzantiev2, Adam Simon3, Carol Gleason4, Oitak (Allen) Wong4, Mwanatumu Mbwana2, Sara Hapip2, Qian Ning2, Franklin Braffett2, Sarah Robles2, George Green4, Robert Neely5, Holly Soares6, James Wilbur2, Pankaj Oberoi2, Paul Rhyne4, David Stewart2, 1Bristol Myers Squibb, Lawrenceville, New Jersey, United States; 2Meso Scale Discovery, Gaithersburg, Maryland, United States; 3AJ Simon Enterprises LLC, Yardley, Pennsylvania, United States; 4 Bristol-Myers Squibb Company, Princeton, New Jersey, United States; 5 Bristol-Myers Squibb, Princeton, New Jersey, United States; 6 Bristol-Myers Squibb, Wallingford, New Jersey, United States. Background: Amyloid Beta 1 - 42 peptide (Ab 1-42) in human cerebrospinal fluid (CSF) has been a useful biomarker to study Alzheimer’s disease (AD). However, the majority of assays present challenges due to inter-lot variability and matrix intolerance. Here we describe the development and analytical validation of a novel A b 1-42 assay for analysis of human CSF using Fit-for-Purpose and CLSI principles. Elements of assay development are presented with results of multi-lot validation demonstrating robust performance and consistency. Methods: Antibodies against both the C-terminal and N-Terminal regions of A b 1-42 were selected based on sensitivity, specificity, physical properties, and CSF sample discrimination. A set of bioanalytical tests were performed on the critical reagents (antibodies, peptides, and controls) to ensure purity, integrity, performance, and lot-to-lot consistency. The assay was developed using MSD’s MULTI-ARRAYÒ technology and optimized to minimize CSF matrix effects and interferences. Analytical validation was performed across three independent kit lots to verify consistency, sensitivity, accuracy, and precision. Human CSF samples were used to establish the dynamic range of the assay and sample performance characteristics. Results: The A b 1-42 assay demonstrated excellent
Figure 1. Brain plasticity measures as assessed with cTBS over M1 in healthy controls and in AD patients.
P2-034
MIRNA EXPRESSION IN CEREBROSPINAL FLUID OF ALZHEIMER’S DISEASE PATIENTS
Argonde van Harten1, Joyce Mulders2, Philip Scheltens2, Wiesje Van der Flier2, Cees Oudejans2, 1Alzheimer Center VUmc, Amsterdam, Amsterdam, Netherlands; 2VU University Medical Center, Amsterdam, Netherlands. Background: MicroRNAs (miR) are small pieces of RNA that have the ability to post-transcriptionally regulate protein expression. Many miR have been shown to be differentially expressed in the cortex of AD patients and seemingly play a role in AD pathogenesis, rendering them a highly interesting target. In this study we aimed to evaluate whether miR can be measured in cerebrospinal fluid (CSF) and to ascertain whether miR are differentially expressed in CSF of Alzheimer’s disease (AD) patients using a data-driven qRT-PCR approach. Methods: RNA was isolated from 2 ml CSF of 19 AD patients and 19 age and sex-matched controls using the mirVana Paris isolation kit (Ambion). miR were quantified by means of qRT-PCR using Taqman Array MicroRNA Cards A and B (v3.0; Applied Biosystems) according to the megaplex protocol. Mean Ct value of all miR present in one plate was used as endogenous control. Relative quantification (RQ) was calculated as 2-ddCt. Results: On average 107 6 44 out of 776 miR (14%)were detected in CSF using megaplex array analysis. Abundance was very low with an average Ct value of 33 6 1 in all detected miR. Exploratory analysis showed a correlation between age and number of miR detected (r ¼ -0.41, P<0.05) and between age and mean Ct value of all detected miR (r ¼ 0.47, P <0.01). Because of these strong correlations, we performed separate between group comparisons for older and younger patients (cut-off 65 years). In the early-onset AD group 13 miR were differentially expressed (P <0.05). In the late-onset AD group 6 other miR were differentially expressed (P <0.05). Conclusions: We have shown that it is possible to measure miR in CSF, suggesting a potential future use as biomarkers in living patients. Several miR seem to be differentially expressed in CSF of AD patients. Further investigation is warranted, especially since accumulating evidence suggests miR expression plays an important role in AD pathogenesis. P2-035
PHOSPHOSPHINGOLIPID LEVELS ARE ASSOCIATED WITH COGNITIVE FUNCTION AND LEVEL OF EDUCATION IN HEALTHY SUBJECTS
Carla Ibrahim-Verbaas, Ayse Demirkan, Aaron Isaacs, Najaf Amin, John van Swieten, Ben Oostra, Cornelia van Duijn, Erasmus University Medical Center, Rotterdam, Netherlands.
P275
Background: Phosphosphingolipids are implied in signal transduction and stress-related neuronal apoptosis. Sphingomyelin and ceramide levels are altered in mild cognitive impairment (MCI) and Alzheimer’s Dementia (AD). An earlier study by our group in healthy individuals associated several SNPs in the APOE region to sphingomyelin levels. In this project, we assessed the association of phosphosphingolipids to cognitive traits in a healthy cohort. Methods: Subjects were participants of the Erasmus Rucphen Family (ERF) study, a family-based cohort. Subjects with dementia, brain damage or sensory or mental impairments were excluded. Phenotyping included an extensive battery of cognitive tests. Phosphosphingolipid profiling was performed on venous blood. We performed partial correlation analyses between cognitive traits and phosphosphingolipid measures. Results: Full cognitive and phosphosphingolipid data were available for 709 subjects (age 18-89 years, mean age ¼ 51 years). The sphingomyelins that were associated to the APOE region, were borderline significantly associated (P ¼ 0.04) to DART (Dutch Adult Reading test, a proxy for intelligence) (spm22:0, spm24:0) and immediate recall (spm22:0). With a Bonferroni-corrected significance threshold of P ¼ 2.210 -4, 13 correlations were significant. Sphingomyelin SPM18:1 (P ¼ 0.0001) was correlated to recognition, and SPM20:1 (P ¼ 0.0000260) to fluency. Nine phosphatidylcholine traits (PCs) correlated to education (tophit PC36:2, P ¼ 0.0000265). Generally, the ethyl-esterized forms of the PCs associated to higher academic performance, non-ethylesterized forms to lower schooling. Also LPC18:0 (P ¼ 4.910 -6) and Cer20:0 (P ¼ 0.00016) correlated significantly to education. Conclusions: We identified correlations between phosphosphingolipids and fluency, recognition, immediate recall, DART and education in nondemented subjects. The associations to DART suggest that phosphosphingolipids are associated to intelligence. As phosphosphingolipid levels are also highly correlated with dietary factors, this association may be explained by socio-economic factors. Alternatively, the PCs which include the Omega 3 and 6 fatty acids may influence cognitive function. Beside replication efforts, our further explorations in this field will include Mendelian Randomization type disentanglement of the direction of causality by investigating associations of the genes related to cognitive function to phosphosphingolipid levels and vice versa. P2-036
DEVELOPMENT AND ANALYTICAL VALIDATION OF A NOVEL ASSAY FOR DETECTION OF HUMAN Ab1-42 PEPTIDE IN HUMAN CSF
Flora Berisha1, Jill Dunty2, Leonid Dzantiev2, Adam Simon3, Carol Gleason4, Oitak (Allen) Wong4, Mwanatumu Mbwana2, Sara Hapip2, Qian Ning2, Franklin Braffett2, Sarah Robles2, George Green4, Robert Neely5, Holly Soares6, James Wilbur2, Pankaj Oberoi2, Paul Rhyne4, David Stewart2, 1Bristol Myers Squibb, Lawrenceville, New Jersey, United States; 2Meso Scale Discovery, Gaithersburg, Maryland, United States; 3AJ Simon Enterprises LLC, Yardley, Pennsylvania, United States; 4 Bristol-Myers Squibb Company, Princeton, New Jersey, United States; 5 Bristol-Myers Squibb, Princeton, New Jersey, United States; 6 Bristol-Myers Squibb, Wallingford, New Jersey, United States. Background: Amyloid Beta 1 - 42 peptide (Ab 1-42) in human cerebrospinal fluid (CSF) has been a useful biomarker to study Alzheimer’s disease (AD). However, the majority of assays present challenges due to inter-lot variability and matrix intolerance. Here we describe the development and analytical validation of a novel A b 1-42 assay for analysis of human CSF using Fit-for-Purpose and CLSI principles. Elements of assay development are presented with results of multi-lot validation demonstrating robust performance and consistency. Methods: Antibodies against both the C-terminal and N-Terminal regions of A b 1-42 were selected based on sensitivity, specificity, physical properties, and CSF sample discrimination. A set of bioanalytical tests were performed on the critical reagents (antibodies, peptides, and controls) to ensure purity, integrity, performance, and lot-to-lot consistency. The assay was developed using MSD’s MULTI-ARRAYÒ technology and optimized to minimize CSF matrix effects and interferences. Analytical validation was performed across three independent kit lots to verify consistency, sensitivity, accuracy, and precision. Human CSF samples were used to establish the dynamic range of the assay and sample performance characteristics. Results: The A b 1-42 assay demonstrated excellent