Gamma—secretase modulators do not show a potency shift in high expressing model systems

Poster Presentations P1 lateralsclerosis (ALS) and from totemporal lobar degeneration (FTLD) with ubiquitin-positive inclusion bodies. Besides aggregation of TDP-43, ubiquitination, hyperphosphorylation, fragmentation and loss of nuclear localization was observed in diseases. However, it remains to be clarified whether TDP-43 aggregates are toxic or not, and how abnormality of TDP-43 mediates neuronal degeneration. Methods: We show here that the cells with TDP-43 inclusions suppressed cell-growth, using BrdU up-take analysis. And morphological relationship between TDP-43 inclusion and several transcription factors were detected in immunocytochemical analysis. Results: We report here that cell-growth is strongly suppressed in the cells with TDP-43 inclusions compared to the cells without inclusions. In these cells, RNA polymerase II and several transcription factors are co-localized with TDP-43 aggregates. Furthermore, accumulation of RNA polymerase II with phosphorylated TDP-43 inclusions was detected in FTLD brains. These results suggested that abnormal TDP-43 inclusions cause growth arrest in SH-SY5Y cells by recruiting general transcription factors and leading to toxicity or cellular dysfunction. Conclusions: In TDP-43 proteinopathy, transcriptional dysregulation may also contribute to neuronal degeneration.

P1-025

WHAT CAN HOMING PIGEONS TELL US ABOUT NAVIGATIONAL IMPAIRMENTS IN ALZHEIMER’S PATIENTS?

dent models of learning and memory. In the current study we investigated the effects of above mentioned drugs in rodent model of learning using the radial arm maze. Methods: Rats of 180-230g were habituated for 2 days in the radial arm maze. The rats, which were placed on restricted diet had to learn to solve the radial maze. The food cup in each arm was baited only once prior to the start of the experiment. Re-entry into an arm or entry followed by exit of the arm without eating the food pellet was considered as an error. The experiment was carried out over a period of 4 days. All the rats were challenged with scopolamine. The choice accuracy as well as total error was calculated. Results: It was observed that donepezil did not have any significant effect on choice accuracy and total error, while tacrine and rivastigmine was found to significantly improve acquisition in the radial arm maze by a significant increase in the choice accuracy and decrease in the total error. Memantine was found to be devoid of activity and was unable to reverse deficit induce by a scopolamine challenge. Conclusions: From the current study it can be concluded that not all the currently approved drugs for Alzheimer’s disease is useful in acquisition or learning in rodent models. While donepezil, which is, currently prescribed for mild to moderate AD did not show any effect in acquisition, tacrine and rivastigmine did show an effect. Memantine is likely to improve cognition in pathological condition involving glutaminergic dysregulation. However cholinesterase inhibitors are likely to show an effect in retention, which is likely to be evident in a clinical set up. P1-027

Paulo Jorge, ISPA-Iu, Lisbon, Portugal. Background: In humans, deficits in olfactory discrimination are generally associated with several forms of dementia and are among the first clinical signs of Alzheimer disease (AD). The link between olfactory dysfunction and AD is interesting in the present context because roughly half of patients in the earliest stages of AD and patients at risk for AD, exhibit a specific navigational impairment associated with loss of ability to discriminate radial optic flow cues, which in turn are necessary for precise determination of heading direction and therefore for correct integration of the path. In pigeons, large body of experiments has shown that olfactory deprivation causes decreases in the accuracy of homing orientation and/or homing success Methods: Here, we addressed the effect of olfactory deprivation on the correct integration of the displaced path in young homing pigeons. Young homing pigeons are known to preferentially use a path integration strategy to find their way home. During the outward journey to the release site, a control group was exposed to natural odors, a no odor group was exposed to synthetic bottled air (that contain no odors), and a novel odors group was exposed to synthetic bottled air with a fixed sequence of artificial odors. Results: Consistent with findings from other researchers, our young birds, deprived of olfactory information during displacement were disoriented. In contrast, birds exposed to natural odors, or to artificial/novel odors that contained no spatial information, were homeward oriented. Conclusions: These findings show that olfactory input activates non-olfactory path integration systems and, in the absence of olfactory stimulation, cues normally used by young pigeons for path integration, failed to produce homeward orientation. Similarly we suggest that loss of olfactory activation of path integrations systems could explain the navigational impairment in the early stages of Alzheimer disease patients. The striking parallel between the failure of young pigeons to utilize non-olfactory path integration cues when deprived of olfactory stimulation, and the occurrence of deficits in olfactory discrimination, radial optic flow discrimination, and navigational ability in early AD, suggests the diminished navigational ability in AD patients may be a direct result of the loss of olfactory activation. P1-026

EFFECT OF VARIOUS COGNITIVE ENHANCERS IN ACQUISITION TASK OF RADIAL MAZE

Pradeep Jayarajan, Ramakrishna Nirogi, Dhanalakshmi Shanmuganathan, Venkatesh Goura, Ramu Yaramusu, Kannadasan Marimuthu, Suven Life Sciences, Hyderabad. Background: Cholinesterase inhibitors like donepezil along with NMDA antagonist memantine are the current approved therapies for Alzheimer’s disease. It has been reported that not all of them are efficacious in all the ro-

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ANALYSIS OF IPS CELL MODELS OF ALZHEIMER’S DISEASE

Asa Abeliovich, Columbia University, New York, N. Y., United States. Background: Our goal is to generate accurate neuronal models of Alzheimer’s and Parkinson’s disease for the analysis of disease mechanism and for therapeutic development. No available gene-modified animal models fully recapitulate disease pathology. Furthermore, neuronal models of sporadic disease are limited by the difficulties in obtaining patient neurons. Our goal is to generate accurate neuronal models of Alzheimer’s and Parkinson’s disease for the analysis of disease mechanism and for therapeutic development. No available gene-modified animal models fully recapitulate disease pathology. Furthermore, neuronal models of ‘sporadic’ disease are limited by the difficulties in obtaining patient neurons. Methods: We are developing iPS cell-based models of Alzheimer’s and Parkinson’s. Briefly, patientderived skin cells are transduced with a cocktail of reprogramming factor genes using lentiviral vectors, as developed by Yamanakaand colleagues. Additional lines are derived from healthy age-matched controls. Results: We have succeeded in generating iPS cell models of familial and sporadic forms of Alzheimer’s disease. These lines have been validated using a number of standard criteria, and neurons can be generated by differentiation protocols. Conclusions: The generation of patient-derived iPS cells, and their differentiation into neurons, may allow for the analysis of cellular and molecular phenotypes relevant to the disease. Furthermore, the method may allow for the investigation of questions such as the role of genetic and epigenetic factors in ‘sporadic’ disease. P1-028

GAMMA—SECRETASE MODULATORS DO NOT SHOW A POTENCY SHIFT IN HIGH EXPRESSING MODEL SYSTEMS

Brian Bronk1, Barbara Tate1, Robyn Loureiro1, Wesley Austin1, Steffen Creaser1, Nathan Fuller1, Jed Hubbs1, 1Satori Pharmaceuticals, Cambridge, Massachusetts. Background: Modulation of gamma secretase activity is a promising therapeutic approach for the treatment of Alzheimer’s disease. Modulation of the enzyme reduces the production of the amyloidogenic Aß42peptide while sparing the production of other Aß species. Recent progress in the identification of small molecule modulators of gamma secretase (GSMs) provides tools for comparing the pharmacology of gamma modulation in both cellular and animal model systems. Methods: Data presented here compare the potency and pharmacology of diverse structural classes of GSMs when dosed to cells overexpressing human amyloid precursor protein (APP), a human neuroglioma cell line (H4), transgenic mice overexpressing human APP (Tg2576) and wild type mice. Effects on Aß38,40 and 42 were assayed

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Poster Presentations P1

using a sensitive plate based ELISA system (MSD). Results: Unlike gamma secretase inhibitors, which demonstrate a “potency shift" when the concentration of substrate is very high as in systems overexpressing APP (Burton et al 2008), the potency of GSMs does not demonstrate a systematic shift in overexpressing systems. The pattern of modulation of Aß peptides was consistent across model systems and specific to each structurally distinct GSM, with some demonstrating a decrease in Aß42 and an increase in Aß38 and others demonstrating a decrease in both Aß42 and 38. Conclusions: The physiological significance of these alterations in cleavage site is not well understood, but appears to be driven by unique interactions of GSMs with gamma secretase. P1-029

A COMPARISON OF CONVENTIONAL VERSUS PUSH-PULL MICRODIALYSIS FOR THE DETECTION OF AMYLOID b IN THE MOUSE BRAIN

Ruth Motter1, Isaac Veinbergs2, Thomas Cremers3, Marieke Van der Hart3, Lisa Yu3, Harm Kooijker3, Gunnar Flik3, Robert Freije3, Robert Brendza1, Pearl Tanaka1, 1Elan, South San Francisco, California; 2Elan Pharmaceuticals, South San Francisco, California; 3Brains On-Line, South San Francisco, California. Background: Strong evidence supports a central role for beta-amyloid protein (Aß) in the pathogenesis of Alzheimer’s disease (AD). Much attention has been focused on toxic forms of soluble and insoluble Aß. Gaining an understanding of the soluble species of Aß that are released from neurons may provide greater insight into the pathophysiology of AD. Previous work has demonstrated the ability to measure extracellular concentrations of soluble Aß in the brains of freely moving animals using in vivo microdialysis (Cirrito et al., 2003). Methods: We have compared two distinct microdialysis methods, conventional microdialysis and push-pull microdialysis, to monitor Aß levels in the brain interstitial fluid (ISF) of AD transgenic animals. In addition, we examined the effects of gamma secretase inhibition with ELN44989 on the levels of Aß (1-x) and Aß (1-40). Conventional microdialysis is limited by the membrane cut-off size and generally allows measurement of molecules smaller than 30-60 KDa. Conversely, push-pull microdialysis allows for detection of molecules up to 1-3 MDa offering the advantage of detecting a broader range of soluble oligomeric Aß species. Both methods were employed in this study, followed by ELISA measurement of Aß (1-x) and Aß (1-40). Results: Aß (1-x) and Aß (1-40) in dialysates from conventional microdialysis were 243 pg/ml and 125 pg/ml, respectively. Using push pull microdialysis, levels of Aß (1-x) and Aß (140) were notably higher. In both cases, ISF Aß levels were significantly reduced following systemic administration of ELN44989. Conclusions: We conclude that push-pull microdialysis provides distinct advantages over conventional microdialysis for the detection of Aß species in the rodent brain, and can provide greater insight into role of multiple Abeta species (including high molecular weight) to pathological and functional endpoints. P1-030

EVALUATION OF TRANSLATIONAL ALZHEIMER’S DISEASE BIOMARKERS IN THE AGED DOG: THE EFFECTS OF AGE AND BACE INHIBITION ON CSF AMYLOID

Joseph A. Araujo1, Guy Higgins2, Christina de Rivera3, Norton Milgram4, 1 Intervivo Solutions Inc; 2InterVivo Solutions, Toronto, Ontario; 3VivoCore, Toronto, Ontario; 4CanCog Technologies, Toronto, Ontario. Background: Aged dogs demonstrate both cognitive decline and neuropathology that models the spectrum of successful aging through early Alzheimer’s disease (AD). The present study sought to further refine the aged canine model by characterizing the effects of age and BACE inhibition on CSF Aß42. Methods: CSF was obtained from young (2-4 y), middle-aged (6-8 y) and old (>10 y) Beagle dogs, and was frozen until Aß42 was measured by ELISA. Each of the three age groups were then divided randomly into 3 dose groups (3, 10 and 30 mg/kg) and received a proprietary BACE inhibitor two days following the baseline CSF draw. CSF samples were collected 3 hours following dosing. Results: Aß42 levels in the old group were significantly reduced compared to both young and middle-aged dogs. Aß42

was also reduced significantly following treatment with the BACE inhibitor. A significant interaction between treatment and age was also found, which reflected both lower basal levels of Aß42 in old dogs, as well a non-significant treatment effect in the aged dogs. By contrast, Aß42 levels were reduced by BACE inhibitor treatment in both young and middle-aged dogs. Analysis of percent change revealed a significant effect of dose, which was due to the largest reductions under the high dose. Conclusions: These findings demonstrate that CSF levels of Aß42 are affected by both age and BACE inhibition. The proprietary BACE inhibitor dose dependently lowered CSF Aß42 in the same dogs and was well tolerated, demonstrating the aged dog is a robust model system for examining therapeutics targeting Aß. This effect, however, was least robust in old dogs, which may be due to the lower basal levels of CSF Aß42 in this age group. These results reveal a cross-sectional biomarker pattern that parallels that seen in the clinical progression to AD. This observation is further supported by elevated CSF phospho-tau in aged compared to young dogs. Further longitudinal examination of various translational biomarkers in the dog is underway including CSF biomarkers and FDG-PET. The present results further supports use of the aged beagle dog as a natural model for efficacy and safety evaluation of novel AD therapeutics. P1-031

EVALUATION OF COLOR PREFERENCE IN ZEBRAFISH: A POSSIBLE POTENTIAL MODEL FOR LEARNING AND MEMORY DISORDERS

Avdesh Avdesh1, Mathew Martin-Iverson2, Mengqi Chen3, David Groth4, Alinda Mondal5, Newman Morgan6, Michael Lardelli7, Ralph Martins8, Giuseppe Verdile9, 1Centre of Excellence for Alzheimer’s disease Research and Care, ECU, Joondalup, WA, Australia; 2UWA, Mount Claremont; 3 ECU, Joondalup, Australia; 4Curtin University of Technology, Perth; 5 ECU, Perth; 6The University of Adelaide, Adelaide, South Australia; 7 Zebrafish Genetics Laboratory, The University of Adelaide, Adelaide, South Australia; 8Edith Cowan University, Perth; 9ECU, Perth. Background: There is growing interest in using zebrafish (Daniorerio) to model neurodegenerative disorders such as Alzheimer’s disease. A zebrafish model of tauopathies has recently been developed and characterised in terms of its neuropathological hallmarks (i.e. neurofibrillary tangles) and cell death. However, it is also necessary to validate these models for function by assessing learning and memory. The majority of tools to assess learning and memory in animal models involve visual stimuli, including colour preference. To validate the zebrafish as a model for colour-associated-learning and memory it is necessary to evaluate its natural preference for any pre-existing bias towards a particular colour. Methods: In the present study we have used four different colours (red, yellow, green and blue) to test natural colour preference of the zebrafish using two procedures: place preference (PP) and T-maze. Results: Results from both experiments indicate that zebrafish exhibit a strong aversion towards blue colour relative to all other colours (red, yellow and green) when tested in combinations. No preferences were found amongst reds, yellows and greens using place preference. However, when the zebrafish were assessed using the T-maze, red and green were equally pleasant and both were preferred over yellow. Conclusions: The results from the present study show a strong aversion towards blue colour compared to red, green and yellow, with yellow being less preferred relative tored and green. The findings from this study may underpin any further designing of colour-based learning and memory paradigms or experiments involving aversion, anxiety or fear in the zebrafish. P1-032

EFFECTS OF CANNABINOIDS ON THE SHORT AND LONG-TERM MEMORY FUNCTIONS IN MICE

Avdesh Avdesh1, Ralph Martins2, Mathew Martin-Iverson3, Yikae Hoe3, 1 Centre of Excellence for Alzheimer’s disease Research and Care, ECU, Joondalup, WA, Australia; 2ECU; 3UWA, Mount Claremont, Washington. Background: Evidence from rodent studies support the view that agonists of cannabinoid CB1 receptors impair tasks that involve memory, while CB1antagonists improve performance on such tasks. However, any one behavioural procedure to assess memory is open to influences from a variety of