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Poster Presentations P1

TTR was aged in the presence of Cl-, I-, ClO4-, NO3-, PO43-, SO42- and heparin, PO43-, SO42- and heparin showed greater effect during the nucleation phase and early elongation phase compared to the others, and the greatest effect was obtained by SO42-. However, TTR aggregated similarly in the presence of different cations. For L55P TTR, distinct structural features could be observed at different stages of aging, following the order of TTR tetramer, irregular aggregates, protofibrils and mature fibers along the aging process. On the other hand, we found TTR induced increase in intracellular Ca2⫹ within one minute of application (Ca2⫹max⫽40⫾6% and pD2⫽5.3⫾0.1 ␮M). In addition, studying the intracellular Ca2⫹ increase by L55P at different stages of ageing indicated that intracellular Ca2⫹ increase was significantly enhanced by TTR aged for 7 d compared to TTR aged for 0 and 3 d (P⬍0.01), while intracellular Ca2⫹ increase was significantly diminished by TTR aged for 14 d (P⬍0.05). Conclusions: TTR aggregation is mainly mediated by hydrophobic interactions. Anionic interactions are involved in TTR aggregation and proteoglycans might be an important factor in TTR aggregation and extracellular deposition. Disturbance of intracellular Ca2⫹ is an initial event in TTR-induced cytotoxicity. Protofibrils might be the most cytotoxic species followed by irregular aggregates and TTR tetramers, with mature fibers being the least cytotoxic. P1-382

PROTEOMIC ANALYSIS OF X11 INTERACTING PROTEINS

Boris Rogelj, Jacqueline C. Mitchell, Steven Lynham, Helen L. Byers, Malcolm Ward, Christopher C. Miller, Declan M. McLoughlin, Institute of Psychiatry, London, United Kingdom. Contact e-mail: [email protected] Background: The X11s (also known as munc18 interacting proteins, mints) are multi-domain adaptor proteins that are involved in the formation of multi-protein complexes. X11␣ and X11␤ are selective for neurons while X11␥ is ubiquitously expressed. Through interactions with other neuronal proteins, X11␣ and X11␤ are believed to be involved in regulation of neuronal signaling, trafficking and plasticity. Furthermore, through interacting with the cytoplasmic domain of APP, they have been shown to modulate APP processing so as to reduce cerebral A␤ production and deposition, making them potential therapeutic targets for Alzheimer’s disease (AD). Objective: The precise mechanisms by which the X11 proteins influence APP processing are not yet known but these can be modified by other X11-binding proteins. Methods: To further our understanding of X11␣ and X11␤ function, we have immunopurified X11s from the brains of transgenic mice overexpressing X11␣ or X11␤ and have used tandem mass spectrometry to identify interacting proteins that have been separated by 1-D gel electrophoresis. Results: In addition to confirming known interactions with CASK, APP and APPL1, we have identified 14 novel candidate interacting proteins. Some of these have now been confirmed by other methods such as co-immunoprecipitation of both transfected and endogenous proteins from neuronal and non-neuronal cells, GST pulldown assays and subcellular co-localization studies. The effects of these novel X11-interacting proteins upon X11-mediated reduction of A␤ production is being investigated. Conclusions: These findings further implicate X11 proteins in cellular signaling and trafficking events and may have relevance to the pathogenesis of Alzheimer’s disease. P1-383

DYRK1A UP-REGULATED BY A␤ AND OXIDATIVE STRESS, IS SIGNIFICANTLY OVEREXPRESSED IN THE BRAIN WITH ALZHEIMER’S DISEASE

Ryo Kimura, Kouzin Kamino, Takashi Kudo, Masatoshi Takeda, Osaka University Graduate School of Medicine, Suita, Osaka, Japan. Contact e-mail: [email protected] Background: DYRK1A (dual-specificity tyrosine regulated kinase 1A), mapped in the Down syndrome (DS) critical region of chromosome 21, was thought to be a candidate gene responsible for the mental retardation of patients with DS. Patients with DS over 40 years old develop neuropathological change to Alzheimer’s disease (AD). Transgenic mice over-

expressing the DYRK1A gene exhibit neurodevelopmental delay and cognitive deficit. Objective(s): We try to examine whether DYRK1A is involved in the development of AD. Methods: DYRK1A mRNA expressional level was quantified in the brain of controls and AD, and we assessed whether A␤ and hydrogen peroxide in SH-SY5Y cell affected expression of DYRK1A. Furthermore, we examined the relation between the level of phosphorylated tau and overexpression of DYRK1A in HEK293 cell. Results: We found that expression of DYRK1A is increased in AD compared to that in control brains. A␤ and hydrogen peroxide induced the expression of DYRK1A in SH-SY5Y cell. Moreover, DYRK1A overexpression shows increased phosphorylation of tau at Thr212. Conclusions: This study suggests that DYRK1A may play a role as mediator between A␤ and tau in AD. P1-384

ASSOCIATION OF AD NEUROPATHOLOGICAL HALLMARKS WITH BIOLOGICAL METALS

Jean-Francois Monty1, Paul A. Adlard1, Irene Volitakis1,2, Mikhalina Cortes1, Qiao-Xin Li2, Katrina Laughton2, Catriona McLean2, Colin L. Masters1,2, Robert Cherny1, Victor L. Villemagne2, Ashley I. Bush3,4, 1Mental Health Research Institute, Parkville, Australia; 2Department of Pathology, The University of Melbourne, Parkville, Australia; 3Mental Health Research Institute and Department of Pathology, The University of Melbourne, Parkville, Australia; 4 Genetics & Aging Research Unit, Massachusetts General Hospital, Charlestown, MA, USA. Contact e-mail: [email protected] Background: Increasing evidence supports a role for Cu, Zn and Fe in Alzheimer’s disease (AD) pathophysiology. Zinc and copper, which interact with the amyloid precursor protein (A␤PP), ␤-amyloid (A␤), tau and phospho-tau to modify their structure, are enriched in amyloid deposits and tangle-bearing neurons. While Zinc concentrations in the AD neocortex are markedly increased, the cellular compartment involved is uncertain. Recent studies, however, have indicated a direct correlation between A␤ levels and zinc, with zinc levels increased in a compartment outside of the plaque, probably intracellularly. Objective(s): The primary aim of this study was to examine the association between the major neuropathological hallmarks of AD and metal content of brain tissue. Methods: We are in the process of surveying post-mortem frontal and temporal cortices matched for age and post-mortem interval, to assay metal levels (by inductively-coupled plasma mass spectrometry (ICPMS)) as well as histological and biochemical amyloid and tau content. A cohort of AD (n⫽12) and control (n⫽13) cases in addition to non-AD dementias, including cases of frontotemporal dementia (n⫽5) and dementia with Lewy bodies (n⫽5) have been obtained so far. In addition to assaying amyloid and neurofibrillary tangle burden by immunohistochemistry, we have also isolated PBS-soluble and insoluble fractions from brain homogenates of all cases for the quantitation of A␤ (Western blot and ELISA), tau/phospho-tau (Western blot) and metal (Cu, Zn, Fe, Mn, Al) content (ICPMS). Results and Conclusions: There is a marked elevation in brain zinc levels in AD, which preliminary data suggest correlates both with A␤ concentrations and phospho-tau. This would support a close biochemical relationship between A␤, phospho-tau and metals in the etiopathogenesis of AD. More detailed breakdown analysis will be presented at the conference. P1-385

METHYLATION REGULATES AMYLOID ␤ PRODUCTION: RELEVANCE OF SADENOSYLMETHIONINE/SADENOSYLHOMOCYSTEINE RATIO IN MICE FED WITH B VITAMIN DEPRIVED DIET

Rosaria A. Cavallaro1, Alessandro Zampelli1, Andrea Fuso1, Fabrizio D’Anselmi1, Claudia Desiderio2, Antonella De Rossi2, Sigfrido Scarpa1, 1University of Rome La Sapienza, Rome, Italy; 2CNR, Rome, Italy. Contact e-mail: [email protected] Background: Elevated plasma concentration of homocysteine (Hcy) is now a well recognized independent risk factor for cardiovascular dis-

Poster Presentations P1 ease and Alzheimer’s Disease (AD). This may be a sign of a complex interaction of genetic, physiologic and pathologic factors. Deficiencies of folate, vitamin B12 and vitamin B6 are important because of the alteration of S-adenosylmethionine (SAM)/Hcy metabolism. This could lead to the increase of Hcy and S-adenosylhomocysteine (SAH) levels, and the decrease of SAM /SAH ratio, known as “methylation potential”, SAM being the main methyl donor in living organisms. Furthermore, low SAM levels and low B vitamin status are often observed in AD and in aging subjects. It has already been shown that gene methylation is involved in amyloid ␤-protein precursor (APP) processing and amyloid ␤ production through the regulation of PS1 and BACE expression. Thus, it was intriguing to study the relationships between homocysteine, SAM/SAH ratio in plasma and tissues, the DNA methylation status and amyloid ␤ production. Objective(s): In order to achieve a molecular model for AD, we used transgenic mice that have an accelerated amyloid accumulation; these mice are a good model to study the metabolic alterations caused by folate, vitamin B12 and vitamin B6 deprivations and the effects on DNA methylation and amyloid ␤ production. Methods: CRND8 mice, with a double mutated APP, together with wild type mice were fed with a deprived diet (without folate, vitamin B12, and vitamin B6) or with a control diet, for 45 or 60 days. Then they were sacrificied to analyze plasma Hcy, SAM and SAH, brain and liver SAM and SAH by HPLC technique, brain DNA methylation, amyloid ␤ accumulation. Results: The diet deprived of folate, vitamin B12 and vitamin B6 led to a marked hyperhomocysteinemia, to an accumulation of SAH (strong inhibitor of SAM-dependent methyl transferases), and to a decrease in SAM/SAH ratio in plasma, brain and liver. This is associated with brain DNA hypomethylation, with liver hypertrophy and with amyloid plaque deposition. Conclusions: These results are important because they correlate SAM/SAH ratio (methylation potential) with amyloid ␤ accumulation. P1-386

ANALYSIS OF THE INVOLVEMENT OF DJ-1 (PARK-7) IN AD, PICK’S AND PSP

Ravindran Kumaran1, Tammaryn Lashley2, Ann Kingsbury2, Alan Pittman1, David Mann3, Andrew Lees1, Rohan de Silva1, Rina Bandopadhyay1, 1Reta Lila Weston Institute of Neurological Studies, London, United Kingdom; 2Queen Square Brain Bank, London, United Kingdom; 3Greater Manchester Neurosciences Centre, Manchester, United Kingdom. Contact e-mail: [email protected] Background: Deletion and point mutations of the DJ-1 gene are responsible for some autosomal recessive forms of Parkinson’s disease but its implication in disease pathogenesis is unknown. DJ-1 has been shown to be expressed in significant amounts in both normal and brains with various neurodegenerative diseases. Additionally, DJ-1 has been shown to co-localize with tau positive inclusions in Alzheimer’s disease (AD) and Pick’s disease (PiD). Objective(s): We analyzed the relationship between DJ-1 and tau in protein inclusions of various neurodegenerative disorders in an independent cohort of cases. We have also analyzed the DJ-1 gene in a progressive supranuclear palsy (PSP) series and compared that to control. Methods: Standard immunohistochemistry protocols have been used for DJ-1 (polyclonal) and tau (AT-8, RD3 and RD4) monoclonal antibodies. Co-localization has been demonstrated by confocal microscopy. Biochemical isolates of insoluble protein from brain tissue have been analyzed by immunoblotting. DJ-1 gene analysis (SNP analysis) has been performed by PCR followed by automated sequencing. Results: We observed robust DJ-1-immunoreactivity (DJ-1 IR) in neurofibrillary tangles (NFTs) and neuropil threads (NTs) in AD brains. Some DJ-1 IR neurites were also associated with amyloid plaques. However, the plaques themselves remained unstained. Confocal microscopy confirmed the presence of DJ-1 and tau proteins in NFTs and NTs. A number of Pick bodies from our PiD cases showed variable labelling with DJ-1 antibody. DJ-1 IR was seen in a small number of NFTs and NTs and also in a few DJ-1 glial inclusions (coiled bodies) in basal pons in PSP. No DJ-1 was detected in ubiquitin

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inclusions in frontotemporal dementia with ubiquitin inclusions. DJ-1 IR was present in biochemical isolates of insoluble tau from PSP and PiD. The DJ-1 gene analysis data from our PSP series and control cases will be presented. Conclusions: DJ-1 specifically associates with tau inclusions in AD and PiD and could play a prominent role in the pathogenesis of these diseases. P1-387

COMPARATIVE ANALYSIS OF HEPARAN SULFATE CO-LOCALIZATION WITH A␤40 AND A␤42 IN DIFFERENT A␤ DEPOSITS OF APP TRANSGENIC MICE AND ALZHEIMER’S DISEASE PATIENTS

Paul O’ Callaghan1, Hong Yu2, Lars N. G. Nilsson1, Martin Ingelsson1, Bradley T. Hyman3, Jin-Ping Li4, Ulf Lindahl4, Lars Lannfelt1, Xiao Zhang1, 1Department of Public Health and Caring Science, Division of Molecular Geriatrics, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden; 2Department of Disease Biology, Local Discovery Research Area, CNS and Pain Control, AstraZeneca R&D, So¨derta¨lje, Stockholm, Sweden; 3Alzheimer’s Disease Research Laboratory, Massachusetts General Hospital, Charlestown, MA, USA; 4 Department of Medical Biochemistry and Microbiology, Biomedical Center Uppsala University, Uppsala, Sweden. Contact e-mail: [email protected] Background: Heparan sulfate (HS) is a universal component of amyloid deposits, including ␤-amyloid (A␤) plaques in Alzheimer’s disease (AD). It has been suggested that HS plays a facilitory role in the development of amyloid deposits in vivo and amyloid fibril formation in vitro. The A␤ composition of various plaque types has demonstrated differences with respect to levels of A␤40 or A␤42, however little is known of HS association with these A␤ species, particularly in the human APP transgenic mouse model. Objective: The aim of this study was to compare the pattern of HS co-localization with A␤40 and A␤42 in ␤-amyloid plaques of the Tg2576 mouse, carrying the human amyloid precursor protein 670/671 mutation with that observed in human AD brain. Methods: Adjacent brain sections of Tg2576 mice and hippocampal sections from human AD cases were immunostained with 10E4 antibody for HS, 6E10 antibody for total A␤ and species specific antibodies against A␤40 and A␤42. HS and A␤ deposits, with ␤-sheet secondary structure, were also stained using sulfated alcian blue (SAB) and Congo red (CR) respectively. Results: In the Tg2576 mice brain, all A␤40 positive plaques and blood vessels were congophilic and positive for HS, localized in dense components of the plaque. Conversely A␤42 plaques, while congophilic, showed incomplete HS colocalization. In the AD brain, A␤40 positive blood vessels were both congophilic and HS-positive. However A␤40 positive plaques demonstrated limited HS co-localization as observed in scattered dense deposits of diffuse neuritic plaques. In contrast, A␤42 neuritic plaques presented with HS positive deposits central to the dense core. Conclusion: Our data indicate differences in HS co-localization with different forms of A␤ deposits between Tg2576 mouse and AD patients. P1-388

FAILURE OF CYTOPLASMIC-NUCLEAR TRANSPORT IN ALZHEIMER’S DISEASE

Hyoung-Gon Lee, Masumi Ueda, Xiongwei Zhu, George Perry, Mark A. Smith, Case Western Reserve University, Cleveland, OH, USA. Contact e-mail: [email protected] Background: Many proteins including various cyclins and cyclindependent kinases together with their cognate inhibitors have been reported to be ectopically localized in the cytoplasm rather than the nucleus in the vulnerable neurons in Alzheimer’s Disease (AD). Since nuclear localization is critical to function and the cytoplasmic-nuclear transportation of large molecules, such as proteins, is actively regulated, the aberrant localization of nuclear proteins to the cytoplasm in AD could be caused by the failure of the cytoplasmic-nuclear transpor-