P1-468: Amyloid oligomers mobilize fyn kinase to lipid rafts

Poster Presentations P1 Background: Elevated levels of ␤-secretase activity, the rate limiting enzyme for A␤ production, have been detected in Alzheimer’s disease (AD) post-mortem (pm) brain tissue. ␤-site amyloid precursor protein cleaving enzyme (BACE1) is a key source of ␤-secretase activity. We have compared BACE1 mRNA expression in AD and control pm brains, and characterised the inter-region variations in expression between the cerebellum, medial temporal, superior occipital superior parietal and frontal gyrus. Quantitative real-time reverse transcription PCR (RT qPCR) is increasingly used to analyse gene expression in neurodegeneration. However, identification of valid reference genes requires careful analysis to ensure that their expression is not altered by the disease process. We have therefore analysed reference gene expression in the pm brain tissue to identify stable reference genes for RT qPCR data normalisation. Methods: Snap frozen pm brain tissue was obtained from control (n⫽5) and AD (n⫽30) individuals. Total RNA was extracted, quantified and used as a template for cDNA synthesis. RT qPCR analyses of BACE1 and a range of potential reference genes were carried out using TaqMan® Gene Expression Assays from Applied Biosystems on an Applied Biosystems 7500 Real Time PCR System. Mean cycle threshold (Ct) values for each sample and gene of interest were transformed to raw, non-normalised quantities prior to analysis in geNorm, where reference gene stability was defined as the average pairwise variation of a particular gene with all other candidate genes. Relevant endogenous reference genes were then included for the subsequent normalisation of RT qPCR data. Neuronal and glial-specific gene expression was used to indicate the cell populations present. Results: This study identified valid reference genes for RT qPCR expression studies in AD pm brain tissue. Using these, we have examined BACE1 expression in the brain regions described above in AD and non-demented controls. Conclusions: Identification of reference genes which are stably expressed across AD and control pm brain tissue is invaluable for RT qPCR data analysis. Accurate quantification of BACE1 expression levels in AD may improve our understanding of the increased ␤-secretase activity observed in the disease.

Background: A characteristic hallmark of Alzheimer’s disease (AD) is the extracellular depositions of the amyloid ␤-peptide (A␤) in senile plaques. A␤ is produced from the amyloid precursor protein (APP) by sequential proteolytic cleavage by ␤-secretase and ␥-secretase. ␤-Secretase cleaves APP on the extracellular side, generating soluble APP and a 99-residue membrane bound fragment (C99). Subsequent cleavage of C99 by ␥-secretase in the transmembrane domain results in the release of A␤ from APP. Two major forms of A␤ are produced, 40 (A␤40) and 42 (A␤42) residues long. The most abundant form of A␤ is A␤40, but A␤42 is more hydrophobic and thus more prone to aggregate, and the species that is of particular importance in the early plaque formation. Thus, the length of the hydrophobic C-terminal tail seems very important for the oligomerization and neurotoxic properties of the A␤ peptide. Longer A␤-variants, for example A␤43, are even more hydrophobic than A␤42. It might be possible that the formation of amyloid plaques is seeded by trace amounts of A␤1-42 or A␤1-43. However, no extensive study regarding longer A␤ variants in plaque cores or amyloid preparations from human brain exists so far. Methods: Here, we studied whether longer A␤ species are deposited in AD brains by examining 6 AD brains including one familial Alzheimer’s disease case. We purified plaque cores from these brains and cyanogen bromide was used to generate C-terminal fragments of suitable length. The protocol for plack core purification is efficient, to monitor the yield of plack cores during the many steps, 30 ␮l of the material is viewed under polarized light in every step throughout the purification procedure. The A␤-fragments were analyzed by LC-MS/MS. We used synthetic peptides as standards for quantifications and used the standard curves generated from these to get an expression of the amount of A␤ as pmol A␤/g of wet brain tissue. Results: We have detected the longer A␤ variant, A␤43, in all the brains analyzed. Conclusions: A␤42 was readily detected, and A␤43 was present in all cases. No variants longer than A␤43 could be observed. P1-470

P1-468

AMYLOID OLIGOMERS MOBILIZE FYN KINASE TO LIPID RAFTS

Ritchie Williamson, Alessia Usardi, Diane P. Hanger, Brian H. Anderton, MRC Centre for Neurodegeneration Research, Institute of Psychiatry, King’s College London, London, United Kingdom. Contact e-mail: [email protected] Background: Soluble amyloid beta and tau has been reported to accumulate in the lipid rafts of a transgenic Alzheimer’s disease mouse model through an as yet unknown mechanism. Furthermore, amyloid has been reported to bind to components of neuronal lipid rafts. Methods: Confocal immunofluorescence was used to measure amyloid accumulation on the neuronal membrane. Post-amyloid exposure, lipid rafts were isolated from primary hippocampal cultures by sucrose gradient centrifugation. Results: Here we report a novel action of amyloid on neuronal plasma membranes. Exogenously applied soluble amyloid binds to neuronal membranes and triggers an immediate fyn-dependent alteration in the protein composition of lipid rafts. These rafts recruit additional fyn and tau. This is followed by a recruitment and redistribution of amyloid to lipid raft domains where it accumulates causing further alterations in lipid raft distribution and composition. Redistribution of amyloid and subsequent cell death was found to be fyn-dependent and accompanied by widespread alterations in the tyrosine phosphorylation state of lipid raft proteins. Conclusions: These results identify lipid rafts as key targets of soluble amyloid action that leads to subsequent fyn-dependent neuronal cell death and defines a mechanism by which amyloid oligomers and tau accumulate in lipid rafts. P1-469

LONG AMYLOID-〉 PEPTIDE VARIANTS IN ALZHEIMER’S DISEASE BRAINS

Hedvig Welander, Bengt Winblad, Lars O. Tjernberg, Karolinska Institutet, Stockholm, Sweden. Contact e-mail: [email protected]

T357

ESSENTIAL ROLE OF AIMP IN AMYLOID-␤1-42 NEUROTOXICITY AND TAU HYPERPHOSPHORYLATION

Taein Kam, Sungmin Song, Yong-Keun Jung, School of Biological Science/Bio-MAX, Seoul National University, Seoul, Republic of Korea. Contact e-mail: [email protected] Background: Accumulation of amyloid-␤ (A␤) in the brain is the primary influence driving Alzheimer’s disease (AD) pathogenesis. Methods: By using the DNA microarray analysis, we isolated a novel A␤-interacting membrane protein (AIMP), which is essentially involved in A␤1-42 neurotoxicity. Results: AIMP expression is strongly increased in A␤-positive neurons in the brain of Tg2576 mice and AD patients. AIMP-deficient cortical neurons are resistant to A␤1-42 neurotoxicity. Also, A␤1-42 potently binds to AIMP in vitro and A␤ neurotoxicity is blocked by synthetic peptides of AIMP interfering the interaction of AIMP and A␤1-42. Further, we isolated three AIMP-associated kinases (AAK1, 2, 3) which mediated the cytotoxicity caused by A␤1-42 and AIMP agonistic antibody (K1). In addition, forced expression of AIMP or treatment with K1 antibody induced the accumulation of hyperphosphorylated Tau, a major component of neurofibrillary tangles, via the AAK1. Conclusions: AIMP might be a neuronal receptor for A␤1-42 that plays an important role in A␤ neurotoxicity and Tau hyperphosphorylation. P1-471

ALZHEIMER-ASSOCIATED A␤ PEPTIDES ARE IMPORTED INTO MITOCHONDRIA VIA THE TOM IMPORT MACHINERY

Camilla A. Hansson1, Nyosha Alikhani2, Homira Behbahani1, Birgitta Wiehager1, Pavel Pavlov1, Bengt Winblad1, Elzbieta Glaser2, Maria Ankarcrona1, 1Karolinska Institutet, Stockholm, Sweden; 2 Stockholm University, Stockholm, Sweden. Contact e-mail: [email protected]