Poster Presentations P1 Background: The inability to form new memories is an early clinical sign of Alzheimer’s disease (AD) and the Ab peptide seems to play a key role in this process. Soluble, bio-derived oligomers of Ab are proposed as key mediators of synaptic and cognitive dysfunction. We investigated the effect of synthetic Ab1-42 oligomers on memory consolidation and retrieval in mice tested in the novel object recognition task; the ability of an anti-Ab antibody to prevent their detrimental action and if the deficit was reversible. Because recent data showed an involvement of PrPC in mediating oligomer action, we investigated their physical interaction and whether Ab oligomers would affect memory of PrPC knock-out (Prnp-/-) mice. Methods: Following intracerebroventricle injection of either Ab1-42 monomer, oligomers or fibrils (1.0 mM), mice underwent the object familiarization phase (day 1) and the memory test phase (day 2). Memory abilities were determined calculating the discrimination index based on the exploration time on either the novel or familiar object. To determine whether oligomers affect memory consolidation or retrieval one single injection was made either before familiarization or memory evaluation. The involvement of PrPC was determined injecting Ab oligomers in Prnp-/- mice, whereas the interaction between Ab oligomers and PrPC was verified using the surface plasmon resonance technique (SPR). Results: Our findings show that Ab1-42 oligomers impaired consolidation of long-term recognition memory, whereas mature Ab1-42 fibrils and freshly dissolved peptide did not. Similar results were obtained injecting Ab 1-40 oligomers. Retrieval of properly stored memories was not affected. The deficit induced by the oligomers was prevented by an anti-Ab antibody and fully recovered 10 days after the injection. We confirmed by SPR that Ab1-42 oligomers interact with PrPC, with nanomolar affinity, however PrP-expressing and Prnp-/- mice were equally susceptible to the cognitive deficits induced by Ab oligomer injection. Conclusions: Our data show that Ab oligomers are responsible for the induction of a reversible memory impairment by blocking memory consolidation. An Ab antibody can prevent such effects. Although we confirmed a binding between Ab oligomers and PrPC, our data suggest that PrPC is not required for their in vivo detrimental effects. P1-169
ROASTING PRODUCTS OF COFFEE (MELANOIDINS) AND THEIR ASSOCIATIONS WITH ALZHEIMER’S DISEASE
Tanja Sauer1,2, Monika Pischetsrieder1, Gerald Muench2, 1Chair of Food Chemistry, Erlangen, Germany; 2Chair of Pharmacology, School of Medicine, Sydney, Australia. Contact e-mail: Tanja.Sauer@lmchemie. uni-erlangen.de Background: Alzheimer‘s disease (AD) is a neurodegenerative disorder in which oxidative stress has been observed in affected brain regions. Interestingly, recent epidemiological studies showed that coffee consumption is inversely associated with the risk of AD. The beneficial effect of coffee is most likely due to the antioxidant activity of its phenolic compounds. We asked the question whether roasting products of coffee (melanoidins) which are known to have antioxidant activity might also contribute to its beneficial effects in AD. Consequently, redox-sensitive transcription factors like NF-kB and NRF2 were analyzed in this study. Methods: Besides coffee, a heated mixture containing ribose and lysine was used as a coffee-model. The NFkB/NRF2 activation was immunochemically detected as a nuclear translocation in different cell lines and tissues. Hydrogen peroxide was measured by the FOXPCA-Assay. Results: The coffee-extract as well as the coffee-model activated NF-kB and NRF2 in macrophages. Neither ribose or lysine, which were heated separately, nor the raw coffee-extract activated NF-kB and NRF2 which indicate that roasting products could act as a possible trigger for translocation. These results also demonstrate to be cell-unspecific as this effect could be verified in further cell lines as well as in human tissue. According to the literature both transcription factors can be activated by hydrogen peroxide. Hydrogen peroxide could be detected in the coffee-model and the coffee-extract. Finally, cells were stimulated with each sample in the presence of catalase. Catalase fully abolished the translocation of NF-kB. However in the case of NRF2, catalase could not inhibit the activation caused by the coffee-extract indicating a hydrogen peroxide independent mechanism. Moreover the extracellular hydrogen peroxide content during cell-incubation was measured. Interestingly, the amount of hydrogen peroxide
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within the media in presence of cells was much lower compared to control samples without cells. Conclusions: To summarize, these results spark interest in hydrogen peroxide generating roasting products of coffee as a neuroprotective therapy for AD. In the present study we demonstrated that the benefit of coffee can no longer only be seen as external supply of antioxidants but also as trigger for cellular antioxidant defence-system. P1-170
Aß DERIVATIVE VACCINATION IN OLD MOUSE LEMUR PRIMATES
Nadine Mestre-Frances1, Stephanie G. Trouche1, Allal Boutajangout2, Ayodeji Asuni2, Yoan Arribat1, Sylvie Rouland1, Thomas Wisniewski2, Blas Frangione2, Tangui Maurice1, Einar M. Sigurdsson2, Jean Michel Verdier1, 1INSERM U710, Montpellier, France; 2New York University School of Medicine, New York, NY, USA. Contact e-mail: nfrances@ univ-montp2.fr Background: Amyloid-b (Ab) accumulation in senile plaques is a hallmark of Alzheimer’s disease. Interventions focused on reducing Ab offer a promising approach to prevent or slow disease progression. Because of their phylogenetic proximity to humans, and their propensity to naturally accumulate Ab deposits, we evaluated the safety and efficacy of immunizing old mouse lemurs with the Ab derivative K6Ab1-30. Methods: Sixteen lemurs ranging from 6 to 9 years of age were immunized with the K6Ab1-30 peptide and 16 others received the alum adjuvant alone. Their immune response, cognition and histopathology were evaluated. Results: Old immunized microcebes showed a rather low IgG titer against Ab40 and 42 but significantly higher than in control animals. Five months after the last injection, the antibody response had returned to basal levels, with this reversibility being an important safety feature of the vaccine. There were limited improvements in cognitive function but immunized animals appeared to perform better than controls. Neuropathological analyses have shown no microhemorrhages or lymphocyte infiltration of the brains of old immunized lemurs. Both Ab burden (intra- and extracellular deposits) and microglial immunoreactivity were decreased in immunized animals. Pathological tau staining and astrocytosis were not affected in the immunized primates, whereas these animals had an increase in normal tau within pyramidal neurons. Conclusions: The mouse lemur model complements existing animal models. Because of its normal endogenous Ab levels, it is particularly useful to assess safety and efficacy of novel vaccines targeting Ab pathology prior to human clinical trials. P1-171
A NOVEL TISSUE CULTURE MODEL OF SPORADIC CREUTZFELDT-JAKOB DISEASE
Frances Prelli1, Ruben Vidal2, Thomas Wisniewski1, 1NYU School of Medicine, New York, NY, USA; 2Indiana University School of Medicine, Indianapolis, IN, USA. Contact e-mail:
[email protected] Background: The underlying pathogenesis of prion disease is the conformational change of the cellular prion protein (PrPC) to a pathological, protease resistant form, PrPRes. Many different animal and human forms of prion disease exist. It is possible to study some animal prion species in tissue culture such as scrapie 22L prions in mouse neuroblastoma cells (N2a). These infected cell lines retain stable and persistent replication of prions upon subpassage, and the newly formed PrPRes retains infectivity in bioassays. However, no tissue culture model of any human prion disease exists. We sought to develop a tissue culture model of sporadic Creutzfeldt-Jakob disease (CJD), the most common human prionosis. Such a model would be an alternative to lengthy bioassays in mice and enable efficient drug screening/development. Methods: N2a cells transfected with wild type human PrP (M at codon 129) were maintained in minimal essential medium (MEM). Aliquots of CJD brain tissue were homogenized (10% weight/volume) in cold phosphate buffered saline and 5% dextrose in sterile conditions. For infection the brain homogenate was further diluted to 2% in Opti-MEM in the presence of metal ions and added to sub confluent six-well plates. MEM in the presence of metal ions was added and the cells were incubated with infectious brain homogenate for an additional 12 h. The medium was replaced and the cells were grown until confluence and then they were split into 1:2 dilutions and transferred to 25 cm2 flasks (Corning). Cells were split 1:2 every 4-5 days to give rise to subsequent passages. The presence of PrPRes was followed by Western
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Poster Presentations P1
blot. Results: N2a mouse neuroblastoma cells transfected with human PrP are permissive to infection with CJD PrPRes, which can be serially passaged. Critical factors to allow successful infection are: a CJD inoculum with a high level of PrPRes, matching of the codon 129 polymorphism between the inoculum/cell line and the presence of metal ions. Whether the cell passaged CJD PrPRes is infectious in human PrP expressing Tg mice is under study. Conclusions: We report a novel tissue culture model of sporadic CJD which can be utilized to study the pathogenesis of human prionoses and develop novel therapeutics. P1-172
OVEREXPRESSION OF WILD TYPE TDP-43 IN MOTONEURON OF NON-HUMAN PRIMATE
Takanori Yokota1, Azusa Uchida1, Hiroki Sasaguri1, Nobuyuki Kimura2, Toshiki Uchihara3, Hidehiro Mizusawa1, 1Tokyo Medical and Dental University, Tokyo, Japan; 2National Institute of Biomedical Innovation, Tokyo, Japan; 3Tokyo Metropolitan Institute for Neuroscience, Tokyo, Japan. Contact e-mail:
[email protected] Background: TDP-43 was found to be a major component of abnormal aggregation in the motoneuron in sporadic amyotrophic lateral sclerosis (ALS), and mutation of TDP-43 gene causes familial ALS. But the pathophysiology of wild-type TDP in sporadic ALS has not been clear. Methods: We injected an adeno-associated virus (AAV) vector expressing human wild-type TDP43 targeted to the spinal cord in non-human primate, cynomolgus. Results: Monkey developed motor paresis of upper limbs on the injected side, and motor nerve excitability was lost at the wrist on nerve conduction study in 4 weeks. TDP-43 was expressed in the cytoplasm as well as neuronal nuclei, and cyoplasmic staining was both diffuse and granular, indicative of preinclusion lesions.In these motoneurons with the cytoplasmic expression, immunoreactivity of endogenous cynomolgus TDP-43 was much decreased. Conclusions: The over-expression of wild-type TDP-43 caused cytoplasmic mislocalization of wild type TDP-43 with loss of nuclear TDP-43, mimicking features of the diseases. This monkey might be a non-human primate model of sporadic ALS. P1-173
THE LINK BETWEEN VASCULAR DISTURBANCES AND ALZHEIMER’S DISEASE
Karen J. Horsburgh, Michell Reimer, Philip Holland, Gillian Scullion, James McCulloch, Jill Fowler, University of Edinburgh, Edinburgh, United Kingdom. Contact e-mail:
[email protected] Background: Considerable evidence indicates that cerebrovascular dysfunction is a key feature of AD and could impact on the cognitive decline and degenerative processes which underlie AD. We are currently focussed on trying to elucidate the links between cerebrovascular dysfunction and cognitive decline and have focussed on the integrity of the brains ‘wiring’ within the white matter. Methods: We have developed a mouse model of chronic cerebral hypoperfusion (induced by application of microcoils to both common carotid arteries). Results: We have demonstrated that modest reductions in blood flow (chronic cerebral hypoperfusion) results in selective damage to specific components of the brains white matter, the myelinated axons, which we have been able to detect at both the cellular level (confocal imaging) and living whole brain (diffusion tensor imaging). These selective changes in white matter cause an impairment of working memory. We have also demonstrated that alterations in key components of the myelinated axon occur early in response to hypoperfusion at the onset of myelin damage. Furthermore, we have shown that severe (not modest) reductions in blood flow cause changes in AD pathology. Conclusions: Together the results highlight the vulnerability of white matter to modest reductions in blood flow which is sufficient to impair cognitive abilities. P1-174
EARLY DETECTION OF b-AMYLOID AGGREGATION IN IN VIVO AND IN VITRO MODELS OF ALZHEIMER’S DISEASE
Louise A. Scrocchi1, Elizabeth Karaskov1, Vivian Lee1, Hui Chen1, Melissa Osborne2, Marty T. Lehto1, Marni D. Uger1, Neil R. Cashman1, Bart Kus1, Cathleen M. Lutz2, Michael Sasner2, 1Amorfix Life Sciences Ltd.,
Mississauga, ON, Canada; 2The Jackson Laboratory, Bar Harbor, ME, USA. Contact e-mail:
[email protected] Background: The accumulation of b-amyloid (Ab) is the hallmark of Alzheimer’s Disease (AD) in humans, and a number of mouse models have been developed to mimic AD pathology. Changes in the levels of Ab (both total and aggregated Ab) are used as biomarkers for evaluating therapeutic efficacy in pre-clinical animal trials. The A4 (Amorfix Aggregated Ab Assay) was developed to provide a quantitative method for detection of aggregated species of Ab. It eliminates the need for formic acid extraction, and detects the presence of aggregated Ab several months prior to detection with immunohistochemistry. This is critical as ideally therapies could be developed that would prevent, rather than repair, synaptic and neuronal damage. Methods: Aggregated Ab was isolated from samples using an affinity-based enrichment method. Ab was released with disaggregants and detected using an ultra-sensitive immunoassay that makes use of the N-terminal antibody 4G10 and C-terminal antibodies 1F8 and/or 2H12. Results: Aggregated Ab was detected in brain homogenates from several transgenic mouse models of AD starting as early as 1 month of age. The kinetics of accumulation of aggregated Ab varied depending on the animal model with the slowest rate of accumulation observed in mice with a single mutation in hAPP (Amyloid Precursor Protein), and the most aggressive accumulation in mice with multiple hAPP mutations or hAPP mutations combined with PS1 (Presenilin 1) mutations. Aggregates were also detected in plasma from two AD models (hAPPswe and hAPPswePS1dE9). The A4 has also been used for the specific quantification of aggregated Ab 1-42 in the presence of Ab 1-40 in tissue culture media. Conclusions: The A4 provides an immunochemical method for early detection of aggregated Ab in transgenic mouse brain or tissue culture. The A4 quantitatively measures changes in aggregated Ab following treatment with therapeutics without the need for immunohistochemistry, and provides a means to screen lead compounds earlier in disease progression. This should enable the reduction of the overall time and cost for pre-clinical trials and improve the chances of therapeutic efficacy. The identification of aggregated Ab in AD mouse plasma supports future development of the aggregated peptide as a biomarker for diagnosis and clinical monitoring in humans. P1-175
GENETIC DEFICIENCY OF RAGE PROTECTS AGAINST Ab ACCUMULATION IN AN ALZHEIMER MOUSE MODEL VIA MODULATING BETA AND GAMMA SECRETASE ACTIVITY
Fang Fang, Shiqiang Yan, Hung L. Nguyen, Shirley Shidu Yan, Columbia University, New York, NY, USA. Contact e-mail:
[email protected] Background: Receptor for Advanced Glycation Endproducts (RAGE) has been demonstrated to play an important role in the pathogenic mechanisms of Alzheimer’s disease (AD). Homozygous RAGE null (-/-) mice were crossed with mAPP mice overexpressing mutant APP/Ab to generate double transgenic mAPP/RO(-/-) mice. Methods: Ab1-40 and Ab1-42 ELISA, amyloid beta plaque immunostaining, radial arm water maze study, AchE immunostaining, b-secretase and g-secretase activity and GSK-3b signal transduction were performed in Tg mice and non transgenic littermates. Results: Tg mAPP/RO(-/-) mice demonstrated decreased Ab1-40 and Ab1-42 level and reduced amyloid beta plaque accumulation in the brains, preservation of spatial learning/memory ,diminished neuropathologic changes at 12 months of age as compared with Tg mAPP mice. These effects were associated with reduced levels of b-secretase and g-secretase activity and decreased GSK-3b activity (increased phosphorylation of GSK-3b). Consistent with this result, Tg mAPP mice expressing neuronal RAGE deficient cytosolic domain responsible for RAGE-mediated signal transduction displayed reduced Ab accumulation and decreased b and g secretase activity. To gain insight into the molecular mechanism whereby RAGE modulates APP processing, we evaluated b-secretase and g-secretase activity in rat neuroblastoma B103 cells stably expressing human wild-type (wt) APP. We found that b and g secretase activity was regulated via GSK-3b pathway in B103-wt APP cells. The inhibition of GSK activity with LiCl significantly inhibit b and g secretase activity, decrease Ab1-40 and Ab1-42 level in the conditioned medium of cultured B103-wtAPP cells. Knockdown of RAGE
