P1-090: Misfolded truncated tau-induced oxidative stress is mediated by specific subgroup of reactive oxygen species in a rat model of tauopathy

Poster Presentations P1 interactions between gender and genotype but not diet (p⫽0.13). Glutathione (see also poster by Choudhry et al, this meeting) and synaptophysin levels were unaffected by diet, gender and transgene expression. Levels of drebrin (here used as a marker of post-synaptic glutamatergic synapses) were significantly different by gender and genotype, and further altered by interactions between gender and diet, diet and genotype and gender, diet and genotype. VGLUT1 expression was significantly altered by genotype alone. Conclusions: These data suggest a pertubation in glutamatergic signalling in those trangsenic mice receiving this pro-oxidant diet. Moreover, these data suggest this transgenic model may be of use in testing and validation of biomarkers. P1-090

MISFOLDED TRUNCATED TAU-INDUCED OXIDATIVE STRESS IS MEDIATED BY SPECIFIC SUBGROUP OF REACTIVE OXYGEN SPECIES IN A RAT MODEL OF TAUOPATHY

Martin Cente1, Peter Filipcik1,2, Gabriela Krajciova1, Michal Novak1,2, 1 Institute of Neuroimmunology, SAS, Bratislava, Slovakia; 2AxonNeuroscience GmbH, Vienna, Austria. Contact e-mail: [email protected] Background: Oxidative stress has been implicated in pathogenesis of many neurodegenerative diseases including Alzheimer’s disease (AD). Tau protein inclusions as one of the most important pathological hallmarks of the disease were extensively studied, however the pathogenic pathways leading to their formation and links between intracellular tau protein modifications and oxidative stress are not completely understood. Therefore we have investigated the role of human misfolded truncated tau protein in association with oxidative stress, the most common risk factor of aging. Methods: All experiments were performed using primary cortico-hippocampal neurons prepared from transgenic rats expressing truncated human tau (AlzTau 151-391,4R). Neurons were cultured in vitro up to 20 days. Level of ROS and mitochondrial membrane depolarization was measured by fluorescent dichlorodifluorofluorescein and JC-1 probes respectively. Results: In this in vitro model we found that even at relatively low expression levels (20-50% of endogenous tau) human truncated tau protein induces oxidative stress in primary cortico-hippocampal neurons what was documented by increase of free oxygen radicals (⬃127% of ctrl, ***P⬍0.001), inhibition of mitochondrial transport (⬃67% of ctrl) and depolarization of mitochondrial membrane (⬃117% of ctrl, **P⬍0.01). As a consequence, increased sensitivity of diseased tau expressing neurons to induced oxidative stress was observed (after treatment with 3-morpholinosydnonimine or L-glutamic acid). Using two common antioxidants (vitamin C and E) we were able significantly eliminate misfolded tau-induced elevation of ROS. Vitamin C showed higher scavenging activity when compared to vitamin E. Conclusions: These data indicate that the elevated ROS level is a consequence of misfolded truncated tau activity. Accumulated ROS specifically comprised of water soluble oxidants and free radicals such as hydrogen peroxide, hydroxyl and superoxide radical (H2O2, .OH and O2.-). Furthermore, we have shown that oxidative stress is a very early event in neurodegeneration. These results suggest that an aberrant tau protein should be considered as a valid primary target for treatment of AD. Antioxidant strategy for slowing down of Alzheimer’s disease should be focused on elimination of water soluble oxidants and free radicals. P1-091

INVESTIGATION OF APP PATHOPHYSIOLOGY IN C. ELEGANS

Mary E. Wiese, Adam Antebi, Hui Zheng, Baylor College of Medicine, Houston, TX, USA. Contact e-mail: [email protected] Background: Mutations in APP leading to the development of Familial Alzheimer’s Disease (FAD) has been well established, however, the normal functional role of this protein within the neuron has proven elusive. We are using the model organism Caenorhabditis elegans to investigate the

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pathophysiology of APP. This system is relevant to the mammalian system as both APP and key synaptic transmission pathways are highly conserved. C. elegans are particularly attractive for the study of APP because the amyloid precursor-like protein 1 (apl-1) is the only homolog of APP in the worm, while mammals contain two others, APLP1 and APLP2. The genetic redundancies from the APP homologs make analysis of APP function in mice challenging, however, in worms null mutation in the apl-1 gene leads to a lethal larval arrest, allowing direct assessment of the apl-1/APP structure and function by genetic rescue. In addition to physiological investigation, C. elegans also offer distinct advantages to study neuronal and aging pathways relevant to AD pathogenesis. Methods: To investigate the role of apl-1/APP in the regulation of cholinergic neurotransmission RNAi knockdown of apl-1 was performed followed by drug testing on the acetylcholine esterase inhibitor aldicarb. Classical epistasis experiments were performed to determine where apl-1 operates in synaptic transmission. In addition, we created transgenic worms expressing human APPC99 in neurons in order to generate a model with which to study the pathogenesis of A␤. Results: Consistent with an essential role for APP family of proteins in the regulation of cholinergic neurotransmission in the mouse neuromuscular junction, we show that RNAi knockdown of apl-1 leads to hypersensitivity to aldicarb. Through epistasis we have found that apl-1 operates after the action of the synaptic vesicle docking protein unc-18. In worms expressing APPC99, genetic crosses reveal that trafficking of this molecule is kinesin-dependent. Conclusions: Loss of apl-1/APP leads to excessive secretion of acetylcholine. apl-1/APP acts at the pre-synaptic compartment after the vesicle docking stage of synaptic vesicle exocytosis. Currently, we are performing structural/functional studies to map the domains required for apl-1/APP function as well as evaluating the processing of APPC99, A␤ pathology and effects of aging on pathology. P1-092

IMPACTS OF PARTIAL REDUCTION OF BACE1 ON SYNAPTIC AND MEMORY DYSFUNCTION IN ALZHEIMER’S TRANSGENIC MICE

Masuo Ohno, Ryoichi Kimura, Nathan Kline Institute, New York University School of Medicine, Orangeburg, NY, USA. Contact e-mail: [email protected] Background: The leading hypothesis of Alzheimer’s disease (AD) pathogenesis holds that the pivotal event is cleavage of amyloid precursor protein (APP), which is initiated by the beta-secretase enzyme BACE1 and results in releasing neurotoxic amyloid-beta (Abeta) peptides. Remarkably, our previous studies have demonstrated that BACE1 gene deletion (BACE1-/-) reduces cerebral Abeta levels, prevents pathological changes such as amyloidosis, gliosis and neuron loss, and rescues memory deficits associated with AD in transgenic mouse models. Our results not only strongly support the amyloid cascade hypothesis but also have important implications for validating therapeutic potential of BACE1 inhibition for the treatment of AD. Objective(s): To test partial inhibition of BACE1, as expected for AD treatments with appropriate dosage of inhibitors, this study was designed to determine whether heterozygous BACE1⫹/- mutation can impact Abeta production and improve synaptic/cognitive deficits in a mouse model of AD. Methods: BACE1-/- mutant (Jackson Laboratory: C57Bl/6 background) or C57Bl/6 control mice were crossbred to AD transgenic mice that overexpress human APP and PS1 harboring five familial AD mutations (5XFAD: B6/SJL hybrid). We applied hippocampal slice electrophysiology and behavioral assays to four genotypic groups of mice (wild-type, BACE1⫹/-, APP/PS1⫹/-, and BACE1⫹/-;APP/PS1⫹/-) in the resultant F1 progeny. Results: 5XFAD APP/PS1 transgenic mice at 6 months of age, but not at ⬍4 months of age, showed impairments in basal synaptic transmission (AMPA components) and NMDA receptor-dependent long-term potentiation (LTP) induced by three and ten theta-burst tetanic stimulation at Schaffer collateral-CA1 synapses. Paired pulse facilitation with different interstimulus intervals (20-500 ms) was normal in APP/PS1 mice at both ages. Consistent with the synaptic failure, APP/PS1 mice were significantly impaired at 6 (but not ⬍4) months of age in hippocampus-dependent contextual fear conditioning and spontaneous al-