MECHANISMS OF BETA-AMYLOID IMPAIRMENT OF ERYTHROCYTE FUNCTION: IMPLICATIONS FOR VASCULAR ALTERATIONS

Poster Presentations: P3 12 University of New South Wales, Randwick-Sydney, Australia; 13Edith Cowan University, Perth, Australia; 14Edith Cowan University, Joondalup, Australia; 15National Institute on Aging, Bethesda, Maryland, United States; 16 Dementia Collaborative Research Centre, Sydney, Australia; 17King’s College London, London, United Kingdom; 18Icelandic Heart Association, Kopavogur, Iceland; 19Erasmus Medical Center, Rotterdam, Netherlands. Contact e-mail: [email protected]

Background: Genome-wide association studies have confirmed the CLU gene, coding for clusterin, as a genetic risk factor for late onset Alzheimer’s disease (LOAD). Clusterin, also called apolipoprotein J, has a role in clearance of Ab peptides from the brain and plasma clusterin levels have been associated with the severity of cognitive impairment in AD. We performed a genome-wide meta-analysis of plasma clusterin concentrations within the CHARGE consortium to test if genetic determinants of plasma clusterin could be identified and might provide insight into pathophysiological mechanisms underlying AD. Methods: Plasma clusterin levels were measured using various assays in 12101 participants from the Framingham, Age, Gene/Environment Susceptibility (AGES), Rotterdam, Older Australian Twins (OATS), Sydney Memory and Aging (MAS) and Hunter Community (HCS) studies. We used genetic data imputed to the 1000G Phase 1v3 reference panel, and linear regression models adjusted for age, sex, and population stratification. To account for heterogeneity between assays, we performed a pvalue-based meta-analysis using effective sample size as weights. We assigned SNPs reaching p-values below 10 -4 to genes and carried out enrichment analyses using WebGestalt. Results: Among the 8,245,500 meta-analyzed SNPs, we did not find genome-wide significant associations. Suggestive results (p < 5x10 -6) included 58 SNPs across 9 loci, near genes that are not known to be involved in determining risk of AD (see Table 1). Furthermore, no significant or suggestive association was found at the CLU locus. Enrichment analysis found an over-representation of genes involved in bipolar disorders (p¼6.04x10 -12) and schizophrenia (p¼2.88x10 -10). Conclusions: At this point, we have not identified any common variants associated with plasma clusterin levels at a genome-wide level of significance and have found no evidence that genetic determinants of plasma clusterin include genes known to be associated with the risk of AD. We are now planning to include an additional sample from the AddNeuroMed study and perform additional analyses to explain our results. Interesting results will be replicated in the Australian Imaging Biomarkers and Lifestyle (AIBL) aging study and the Baltimore Longitudinal Study of Aging (BLSA). Table 1 Top results from a plasma clusterin GWAS in the CHARGE consortium

SNP

Eff. Sample Chr. Alleles Freq Size

rs58697987 rs76789221 rs12173238 rs1434756 rs1110115 10:130609731:I rs2710306 rs117765134 rs184454536

1 3 5 8 8 10 12 12 16

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T/C T/C A/G T/G T/C I/R A/G T/C A/C

P-value

I2

Closest Genes (100kb)

0.14 6399.34 2.21x10-06 0 LOC643441 0.98 6564.67 4.26x10-06 51.9 OSTN 0.14 10469.17 3.83x10-06 70.4 PRELID2 0.73 9976.6 1.22x10-06 0 PREX2 0.59 10677.13 1.09x10-06 36.2 ATP6V1C1 0.33 9108.4 5.60x10-07 4.4 None 0.55 10570.14 3.30x10-06 0 ETV6, RNU6-19P 0.97 7886.73 2.50x10-06 0 PDZRN4 0.99 5919.68 1.65x10-06 6.9 TOX3

GAB2 GENE AND THE RISK FOR LOAD IN AN ELDERLY BRAZILIAN SAMPLE

Maria Aparecida Bicalho1, Renalice Vieira2, Marco Tulio Cintra1, 1  , Jonas Jardim de Paula3, Anna Luiza Souza1, Rafaela Avila Renato Braganc¸a1, Laiss Bertola1, Edgar Moraes1, Debora Miranda2, 1 UFMG, Belo Horizonte, Brazil; 2INCT Molecular Medicine, Belo Horizonte, Brazil; 3Universidade Federal de Minas Gerais, Belo Horizonte, Brazil. Contact e-mail: [email protected] Background: The late onset form of Alzheimer’s disease (LOAD) has a multifactorial etiology involving a complex interaction of genetics and envi-

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ronmental factors. The ε4 allele of APOE gene is a main genetic risk factor and is responsible for a dose-dependent increase in the risk about of 4-fold. More than 550 genes have been proposed as candidates for AD susceptibility although their roles in the pathogenesis of the disease have not been established yet. In a GWAS study, Reiman and colls (2007) suggested that GAB2 gene interacts with the ε4 allele modifying the risk and influencing Alzheimer’s neuropathology. The GAB2 protein is an intracellular scaffolding/docking molecule which participates in a variety of signaling pathways. Our aim was to investigate the impact of the GAB2 polymorphism rs2373115 on the risk of AD and the age-at-onset (AAO) in a sample of a Brazilian population. Methods: 269 LOAD patients and 114 controls (age  60 years) from the same outpatient geriatric center were recruited and were submitted to the same protocol. Genotyping of GAB2 rs2373115 and APOE were performed by Real-time PCR. The c 2 test was used for the comparison of genotypic and allelic frequencies. Clinical data were analyzed by c 2, t-Student and Mann Whitney tests. Kruskal-Wallis test was used to compare the AAO and a binary logistic regression was made to evaluate the risk effect. Results: The years of education between LOAD and controls were significant different (3.19 6 3.1 vs. 4.59 6 4.04 yr.,p ¼ 0.002). Higher frequencies of ε4 allele (52.8% vs. 24.8%, p < 0.001) was found in LOAD as well as an increased risk (OR ¼ 4.35, CI ¼ 2.54 - 7.46, p < 0.001). No statistical differences between the GAB2 genotypes and alleles were observed even when stratifying by APOE ε4 status (for the entire sample: OR ¼ 1.28 [0.78 - 2.08], p ¼ 0.330). No association was observed in the AAO (p ¼ 0.848). Conclusions: Conversely to the initial GWAS report, we did not find any significant association of the rs2373115 with LOAD. In our sample low levels of education seems to be a risk factor for LOAD. P3-073

MECHANISMS OF BETA-AMYLOID IMPAIRMENT OF ERYTHROCYTE FUNCTION: IMPLICATIONS FOR VASCULAR ALTERATIONS

Francesco Misiti1, Cristiana Carelli-Alinovi2, Bruno Giardina2, 1 University of Cassino and Southern Lazio, Cassino, Italy; 2School of Medicine, Catholic University, Rome, Italy. Contact e-mail: f.misiti@ unicas.it Background: It has well established that many abnormalities in vascular could be responsible for Alzheimer’s Disease (AD). In particular, the reduced deformability of erythrocytes and diminished vascular NO bioavailability are two of the most suspected events associated to vascular abnormalities in AD. At this regard, it is known that NO is a regulatory factor of RBC mechanical properties, and recently we have observed that amyloid beta peptide 1-42 (Abeta) inhibits red cell nitric oxide synthase (eNOS) activity (1). Protein kinase (PKC) and caspase 3 were considered the main candidates for eNOS regulation by Abeta. Methods: Blood samples were obtained from informed healthy donors, in accordance with the principles outlined in the Declaration of Helsinki. Enzyme activities and levels were evaluated by immune-assays. eNOS activity was measured indirectly by determination of main NO derived metabolites levels (NO3 /NO2 ). Red cell morphology was evaluated by optical microscopy. Results: Surprisingly, we observed that pretreatment of erythrocytes with the PKC inhibitor chelerythrine, prior to the addition of Abeta, blocks the appearance of the morphology alterations and the sustained decrease in nitrates and nitrites levels induced by Abeta. Inhibition of PKC does not affect Abeta mediated caspase-3 activation. On the other hand, pretreatment of erythrocytes with Z-DEVD-FMK, an inhibitor of caspase 3, prior to the addition of Abeta, partially reduces PKC activation, indicating the presence of some members of PKC family activated following to caspase 3 activation. Inhibition of caspase 3, furthermore lessens the Abeta induced-effects on the appearance of erythrocyte morphology alterations and it partially counteracts Abeta-mediated effects on NO derived metabolites levels. Conclusions: These data demonstrate that Abeta mediated alteration of red cell morphology, is mediated by a signaling pathway involving caspase 3 and specific isoforms of PKC, ultimately responsible for the diminished red cell eNOS activity and NO metabolic products. Thus, red blood cells/Abeta interaction probably contribute to the vascular alterations associated with AD, through

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Poster Presentations: P3

two different mechanisms: i) reducing red cell deformability by altering cell shape and morphology and ii) reducing NO export, contributing to the reduced vascular NO bioavailability detected in AD patients. References: 1. Misiti F, et al. Cell Biochem Funct. 2012; 30(6):474-9 P3-074

VALIDATION OF A CASPASE-3 GENERATED TAU FRAGMENT AS A SERUM BIOMARKER OF BRAIN DAMAGE DUE TO STROKE: IMPLICATIONS FOR ALZHEIMER’S DISEASE

Dilek Inekci1, Thomas Linemann1, Daniela Ripova2, Kim Henriksen1, Ales Bartos2, 1Nordic Bioscience A/S, Herlev, Denmark; 2Prague Psychiatric Center, Prague, Czech Republic. Contact e-mail: din@ nordicbioscience.com Background: Stroke is an acute and massive degeneration of the brain and evolves over time. The objective of this study was to analyze whether clinical changes within one week are reflected in a caspase-3 cleaved fragment of the nervous-specific tau protein (Tau-C) measured in serial serum samples. Due to its smaller size the caspase-3 cleaved fragment of tau may be able to pass the blood-brain-barrier and be measured in serum compared to the full-length tau protein. Methods: Nineteen stroke patients (age 61613 years, 4 females) were evaluated by using the National Institutes of Health Stroke Scale (NIHSS) and serum samples were collected on admission (onset), 1 and 7 days later. The level of caspase-3 cleaved fragment of tau protein was measured using our in-house ELISA assay. The patients were divided into two subgroups according to changes from baseline to final examination using a NIHSS cut-off of 5 at day 7. Results: The NIHSS score of mild stroke patients improved (NIHSS change from 4 to 2), whereas the patients with poor outcome worsened (from 10 to 13). A significant difference in DTau-C level (p ¼ 0.0028) was found between patients with a good outcome (NIHSS  5) and patients with a poor outcome (NIHSS  6). A significant elevation (p < 0.0026) of Tau-C level was shown at day 7 for patients with a poor outcome (104.39 ng/mL 6 24.95 ng/mL) when compared with patients with a good outcome (42.86 ng/mL 6 6.53 ng/ mL). No significant difference (p ¼ 0.3716) was observed in Tau-C levels at day 0 between the patients with good outcome (42.3466.27) and poor outcome (30.8862.66). Conclusions: Tau-C is feasible to measure in serum even at onset of stroke event and its concentrations can reflect the clinical course and disability. This is also a validation of this nervous-specific protein as a potential biomarker in serum for brain damage of a various origin including Alzheimer’s disease. P3-075

ELEVATED SERUM COPPER LEVELS AND COGNITIVE DECLINE IN ALZHEIMER’S DISEASE

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DEVELOPMENT AND VALIDATION OF A RISK INDEX FOR COGNITIVE DECLINE BASED ON BLOOD-DERIVED RISK FACTORS

Jasmine Nettiksimmons1, Hilsa Ayonayon2, Tamara Harris3, Caroline L. Phillips4, Caterina Rosano5, Suzanne Satterfield6, Kristine Yaffe2, 1UCSF, San Francisco, California, United States; 2 University of California San Francisco, San Francisco, California, United States; 3National Institute on Aging, Bethesda, Maryland, United States; 4 National Institute on Aging, Bethesda, Maryland, United States; 5 University of Pittsburgh, Pittsburgh, Pennsylvania, United States; 6 University of Tennessee, Memphis, Tennessee, United States. Contact e-mail: [email protected] Background: We developed and validated a risk index for cognitive decline in older adults based on blood-derived risk factors. Previous studies have focused on blood marker differences between dementia and control groups; we developed an index in non-demented older adults to identify individuals at high risk for cognitive decline. Methods: The included eight risk factors previously associated with cognitive aging: APOEε4 status, plasma amyloid-b 42/40 ratio, telomere length, cystatin C, fasting glucose, C-reactive protein, interleukin-6, and albumin. Our cohort was comprised of 2,184 older adults (baseline average age 73.5). The outcome was person-specific cognitive slope (Modified Mini-Mental Status Examination-3MS) from 11 years of follow-up. An index created with a weighted sum of dichotomized risk factors was divided into low, medium, and high risk categories based on the slope distribution at each level of the index in the development sample (2/3 of the cohort, n¼1632). Risk categories were validated with the remaining sample (n¼833) using linear regression models. Amyloid was measured on a subsample (n¼865), and was only included in a secondary index. Results: The risk categories showed significant differences from each other and were predictive of prospective cognitive decline in the validation sample, even after adjustment for age and baseline cognitive score: the low risk group (risk score: 0-1, 24.8%) declined 0.32 points/year (-0.46, -0.19), the medium risk group (risk score: 2-3, 58.7%) declined 0.55 points/year (-0.65, -0.45), and the high risk group (risk score: 4-7, 16.6%) declined 0.69 points/year (-0.85, -0.54). Using the secondary index which included amyloid-b 42/40 (validation N ¼ 279) the low risk group (risk score: 0-2, 26.9%) declined 0.20 points/year (-0.42, 0.01), the medium risk group (risk score: 3-5, 61.3%) declined 0.55 points/year (-0.72, -0.38), and the high risk group (risk score: 6-9, 11.8%) declined 0.83 points/year (-1.14, -0.51). Conclusions: A risk index based on 8 blood-based risk factors was able to predict cognitive decline over an 11 year follow-up period. Further validation in other cohorts is necessary. This index could be used to enrich study samples with individuals at high risk for cognitive decline.

Dong-Woo Lee1, Jun-Hyun Park2, 1Sanggye Paik Hospital, Seoul, South Korea; 2Sanggye Paik Hospital, Seoul, South Korea. Contact e-mail: [email protected] Background: Copper takes part in a variety of biological reduction - oxidation (redox) processes, and is an important cofactor of many redox enzymes.In our previous study, serum copper levels were significantly negatively correlated with scores on cognitive test subscores. We explored whether a deregulation of the serum copper pool has correlation with worsening of follow up MMSE score. Methods: We assessed serum copper, ceruloplasmin and free-copper levels in 89 patients with Alzheimer’s disease (AD) (mean age, 77.83 years; 41 men, 48 women). 41 patients were completed first year follow up MMSE and 15 patients for second year follow up MMSE. Results: In first year, there were no significant correlation between follow up MMSE with baseline serum copper level(r¼0.133, p¼0.406) or baseline serum free copper level(r¼-0.002, p¼0.989). However in second year, there were significant negative correlation between follow up MMSE scores with baseline serum copper level(r¼-0.692, p¼0.004) and baseline serum free copper level(r¼-0.649, p¼0.009). Conclusions: These results suggest an association between serum copper and cognitive decline in Alzheimer disease. Further research is needed to establish whether copper is an independent risk factor for cognitive decline.

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CEREBROSPINAL FLUID BIOMARKERS ARE NOT INFLUENCED BY GRAVITY DRIP OR ASPIRATION EXTRACTION METHODOLOGY

Alan Rembach1, Sarah Maher2, Francis Mooney2, Tabitha Nash1, Lesley Vidaurre1, Qiao-Xin Li1, Chris Fowler1, Kelly Pertile1, Brett Trounson1, Rebecca Rumble1, Stephanie Rainey-Smith3, Lisbeth Evered4, Brendan Silbert5, Simon Matthew Laws6, Kevin Taddei7, Lance Macaulay8, David Darby9, Ralph Martins7, Steven Collins1,