O3-02-08: Cerebrospinal fluid biomarkers correlate with slowing of EEG rhythm and cognitive speed in healthy elderly

T162 O3-02-07

Oral O3-03: Disease Mechanisms: Apolipoprotein E IDENTIFICATION OF MiRNA CHANGES IN ALZHEIMER’S DISEASE BRAIN AND CSF YIELDS PUTATIVE BIOMARKERS AND INSIGHTS INTO DISEASE PATHWAYS

Jill C. Richardson1, John P. Cogswell2, James Ward3, Ian A. Taylor3, Michelle Waters4, Yunling Shi5, Brian Cannon6, Kevin Kelnar6, Jon Kemppainen7, David Brown8, Caifu Chen9, Rab K. Prinjha10, Ann M. Saunders11, Allen D. Roses11, Cynthia A. Richards11, 1 Neurosciences CEDD, GlaxoSmithKline, Harlow, United Kingdom; 2 Department of Pharmacogenetics, GlaxoSmithKline, North Carolina, NC, USA; 3Department of Molecular Discovery Research, GlaxoSmithKline, Research Triangle Park, NC, USA; 4Department of Molecular Discovery Research, GlaxoSmithKline, Research Triangle Park, NC, USA; 5Department of Molecular Discovery Research, GlaxoSmithKline, Research Triangle Park, NC, USA; 6Asuragen Inc., Austin, TX, USA; 7Asuragen Inc., Austin, TX, USA; 8Asuragen, Inc.,, Austin, TX, USA; 9Applied Biosystems, Foster City, CA, USA; 10 Neurology CEDD, GlaxoSmithKline, Harlow, United Kingdom; 11 Department of Pharmacogenetics, GlaxoSmithKline, Research Triangle Park, NC, USA. Contact e-mail: [email protected] Background: Despite considerable progress in the development and validation of imaging and neuroelectrophysiological techniques, it remains difficult to conclusively diagnose Alzheimer’s disease (AD) in its early stages. There is an urgent medical need to develop novel biomarkers to inform the diagnosis, development and testing of novel therapeutic agents. MicroRNAs (miRNAs) are small regulatory RNAs that bind the 3’ untranslated region (UTR) of target genes and inhibit their expression post-transcriptionally. MiRNAs are more stably expressed than mRNAs and miRNAs can also be detected in human fluids. MiRNAs have essential functional roles in brain development and neuronal specification but their role in neurodegenerative diseases such as AD is unknown. We sought to investigate whether miRNAs were differentially expressed in brain and CSF between AD and non-affected controls. Methods: The expression of over 300 miRNAs was determined in hippocampus, medial frontal gyrus, and cerebellum from early and late stage AD compared to age-matched controls. CSF miRNA profiling was performed using RT and pre-amplification modifications of the TaqMan™ miRNA Assays (Applied Biosystems). 313-plex RT mix and miRNA PreAmp primers were obtained from Applied Biosystems. Results: We identified regional and stage-specific deregulation of miRNA expression in AD patient brains. We used experimental validation in addition to literature to reveal how the deregulated brain microRNAs are biomarkers for known and novel pathways in AD pathogenesis related to amyloid processing, neurogenesis, insulin resistance, and innate immunity. We additionally recovered miRNAs from cerebrospinal fluid and discovered AD-specific miRNA changes consistent with their role as potential biomarkers of disease. Conclusions: Our data provides the first genome scale description of miRNAs that are differentially expressed in Alzheimer’s disease brain and importantly further demonstrates their presence and disease-altered expression in cerebrospinal fluid. Through experimental follow up and literature mining of miRNA targets we could establish that the identified miRNAs are useful biomarkers of known and novel pathways contributing to AD pathogenesis. The quantitative changes in expression we have seen in CSF suggest that miRNAs have the potential to provide accessible biomarkers to aid diagnosis of AD. O3-02-08

CEREBROSPINAL FLUID BIOMARKERS CORRELATE WITH SLOWING OF EEG RHYTHM AND COGNITIVE SPEED IN HEALTHY ELDERLY

Erik Stomrud1, Oskar Hansson1, Lennart Minthon1, Kaj Blennow2, Ingmar Rose´n3, Elisabet Londos1, 1Clinical Memory Research Unit, Department of Clinical Sciences, Malmo¨, Lund University, Malmo¨, Sweden; 2 Institute of Neuroscience and Physiology, Department of Neurochemistry and Psychiatry, Sahlgrenska University Hospital, Gothenburg, Sweden; 3 Division of Clinical Neurophysiology, Department of Clinical Sciences, Lund University, Lund, Sweden. Contact e-mail: [email protected]

Background: Alzheimer’s disease (AD) is a progressive disease where neuropathology is thought to begin decades before development of cognitive decline. Cerebrospinal fluid (CSF) biomarkers are suggested to be early markers for the disease and specific changes on quantitative EEG are visible already in the MCI state for individuals progressing to AD. How these AD-associated biomarkers correlate early in the disease development is however still largely unknown. Hereby, the aim of this study was to investigate the relationship of CSF biomarkers and qEEG in cognitively healthy elderly. Methods: Analysis of CSF biomarkers ␤-amyloid1-42 (A␤42), total tau protein (T-tau) and phosphorylated tau protein (P-tau), frequency analysis of qEEG, and memory testing were performed on 33 individuals with normal cognition over a period of 4.5 years. Results: Increased relative theta power on qEEG correlated with high CSF P-tau levels (p ⬍0.01) and a high CSF P-tau/A␤42 ratio (p ⬍0.001). The correlations were even stronger with increased relative theta power located in the right posterior quadrant of the head (p ⬍10-4). T-tau correlated in a similar pattern with relative theta power, however to a lesser extent. Furthermore, high CSF P-tau/A␤42 ratio and increased relative theta power correlated with slowing of cognitive speed on memory tests (p ⬍0.01). Conclusions: AD-associated patterns of change in CSF biomarkers, qEEG rhythm and cognitive speed appear to be associated already in cognitively healthy individuals, before any symptoms of cognitive disease. The results hereby might strengthen the role of CSF biomarkers as preclinical markers for neurodegenerative changes in the brain. TUESDAY, JULY 29, 2008 ORAL O3-03 DISEASE MECHANISMS: APOLIPOPROTEIN E O3-03-01

APOLIPOPROTEIN E PROTECTS CEREBROVASCULAR CELLS AND ASTROCYTES FROM A␤-MEDIATED CELL DEATH

Ilona B. Bruinsma1, Micha M. M. Wilhelmus2, Matthijs Kox2, Miel van Hout2, Rob Veerhuis3, Robert M. W. de Waal2, Marcel M. Verbeek2, 1Donder Centre for Neuroscience, Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands; 2Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands; 3Free University Medical Centre, Amsterdam, Netherlands. Contact e-mail: [email protected] Background: Previously, we described that in cerebrovascular cells (CBVs) amyloid beta (A␤) induced cytotoxicity and apolipoprotein E (apoE) production is dependent on their apoE genotype. Cells carrying two copies of the apoE ⑀4 allele (apoE 4/4 cells) were more vulnerable for A␤-induced cytotoxicity and produced also lower amounts of apoE than apoE 3/3 or 3/4 cells. We also demonstrated that apoE produced by CBVs inhibited A␤-induced cytotoxicity in a dose-dependent manner. Methods: Astrocytes are considered to be the most abundant cells in the brain and are regarded as the major source of apoE in the brain. In this study, we compared apoE production and effects of apoE on A␤-induced toxicity in cultured primary human astrocytes with CBVs. Results: ApoE production in astrocytes was approximately 10 times lower than in CBVs (0.1 vs. 10.0 ng/␮g protein/day) and was not dependent on apoE genotype, in contrast to in CBVs. Despite their low ApoE production levels, astrocytes were less vulnerable to A␤-induced cytotoxicity than CBVs (25 % vs. 42 %), suggesting a more important role for apoE in regulating A␤-mediated cell death of CBVs than in astrocytes. In contrast, when we co-incubated either astrocytes or CBVs with conditioned medium containing high amounts of apoE either cell type was protected from A␤-induced cytotoxicity. This suggests that a minimum level of ApoE is needed to protect cells from A␤. In contrast to CBVs, we did not observe a relation between apoE genotype and A␤-mediated cell death in astrocytes. Previously, we have suggested that A␤ internalization protects CBVs from A␤-induced cytotoxicity. Internalization leads to a decrease in cell surface accumulation of A␤, which