γ-Secretase substrates APP, Notch1 and CD44 show distinct processing in NPC cells

Poster Presentations P1 solubilizing the above neuronal cells using mild detergents. The biotin-labeled ASPD-putative-ASPD-binding protein complexes were isolated using streptavidin beads. The isolated ASPD-binding proteins were separated by SDS-PAGE, visualized using silver stain. Results: We first examined whether ASPD-binding proteins are present or not in mature neuron-derived extracts using far-Western blotting. We could detect some bands that were specifically detected by anti-ASPD antibodies but not by an anti-Ab antibody using ASPDs as ligands. These bands were absent in extracts derived from immature neurons or from non-neuronal cells. We next attempted to isolate ASPD-binding proteins using biotin-labeled ASPDs as baits. We optimized the experimental conditions for isolating the biotin-labeled ASPD-putativeASPD-binding protein complexes from mature neuron-derived extracts and finally succeeded to detect the bands of the putative ASPD-binding proteins in SDS-PAGE analysis, some of which were identical to the bands detected by far-Western blotting. Conclusions: We thus isolated the candidates for the neurotoxic targets for ASPDs from mature neurons. Further analysis using mass spectrometry is proceeding to characterize the isolated proteins. P1-155

EXCLUSION OF PHOSPORYLATED APP CTFS FROM MEMBRANE REGION RICH IN ACTIVE g-SECRETASE

Takahide Matsushima, Tadashi Nakaya, Toshiharu Suzuki, Hokkaido University, Sapporo, Japan. Contact e-mail: [email protected]. ac.jp Background: In neuron, mature APP695 (mAPP, N- and O-glycosylated form) is phosphorylated at Thr668 within the motif 667-VTPEER-672 of cytoplasmic domain. Function of APP can be modified by cytoplasmic conformational change induced with the phosphorylation (reviewed in J. Biol. Chem. [2008] 283 29633-29637). Several reports indicated that phosphorylation or amino acid substitutions for Thr668 of APP could regulate the cleavage by b-secretase and/or g-secretase, or that phosphorylation at Thr668 has been up-regulated in AD brains. Contrary to these observation, a mutant mice carrying amino acid substitution of Ala for Thr668 (A/A mutant), which mimicked non-phosphorylated APP, did not influence in APP processing in brain. Thus the participation of phosphorylation in APP metabolism is still under consideration. We herein report the role of APP phosphorylation in g-site cleavage of APP. Methods: Levels of phosphorylated APP CTFs (pCTFs) and nonphosphorylated CTFs (nCTFs) in mice brain membrane were examined by Western blotting and their relative amounts were quantified. The brain samples were subjected to in vitro g-secretase assay and the cleavage of pCTFs was compared to that of nCTFs. Results: In CTFb, C99 and C89, the phosphorylated form was dominant compared to nonphosphorylated forms, while both pCTF and nCTF were even in quantity for CTFa, C83. When the membrane fraction including these CTFs was assayed for g-secretase cleavage, the cleavage of pCTFs was significantly lower compared to that of nCTFs in spite of kinetic equivalence of pCTFs and nCTFs for susceptibility to g-secretase. Conclusions: In vitro g-cleavage assay of CTFs indicates that phosphorylated and non-phosphorylated CTFs were kinetically equivalent substrates for g-secretase. However, more nCTFs were likely to be cleaved rather than pCTFs. This indicates that phosphorylation for Thr668 regulates the localization of CTFs instead of a direct inhibition of g-cleavage. The pCTFs may be located at a distance from the active g-secretase in the membrane, while non-phosphorylated CTFs are located close to the active enzyme. The model what we have proposed differes from the interpretation that phosphorylation of CTFs directly interferes or facilitates in cleavage by g-secretase. P1-156

THE PATHOGENIC I716F AMYLOID PRECURSOR PROTEIN (APP) MUTATION LEADS TO A REDUCED PROTEIN PROCESSING AND A VERY AGGRESSIVE EARLY-ONSET ALZHEIMER’S DISEASE

Cristina Guardia-Laguarta1, Marta Pera1, Jordi Clarimon1, Albert Llado2, Raquel Sa´nchez-Valle2, Jose Luis Molinuevo2, Teresa Go´mez-Isla1, Rafael Blesa1, Isidre Ferrer3, Alberto Lleo1, 1Hospital Sant Pau, Barcelona, Spain; 2Hospital Clinic, Barcelona, Spain; 3Hospital de Bellvitge, Barcelona, Spain. Contact e-mail: [email protected]

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Background: Previous studies suggest that a mutation in the codon 716 of the amyloid precursor protein (APP) leads to familial early onset Alzheimer’s disease (EOAD) with an age of onset in the mid 50s. We report a novel mutation in APP (I716F) in a patient with an onset of 31 years. We investigate in detail the clinical, neuropathological and biochemical effects of this mutation. Methods: Neuropathological examination was performed using specific antibodies against amyloid-b , tau/phospho-tau, a -sinuclein, TDP43, and GFAP. cDNA constructs encoding APPV717I and APPI716F were generated by site-directed mutagenesis. CHO cells were transfected with wtAPP, APPV717I or APPI716F, and Ab 40 and Ab 42 levels were measured by ELISA. APP C-terminal fragments (CTFs) were measured by Western blot. We measured the APP intracellular domain (AICD) in an in vitro cell-free assay. Finally, g -secretase activity was measured by a fluorometric assay in cell lysates, and brain samples from the proband, AD patients and healthy controls. Results: The proband had a diagnosis of EOAD with an age of onset of 31 (age of death of 36). The father had developed EOAD with an age of onset of 35. The neuropathological examination of the proband showed abundant diffuse amyloid plaques, mainly stained for Ab42, and widespread neurofibrillary pathology corresponding to a stage VI of Braak. Lewy bodies and aberrant neurites were observed in the amygdala. CHO cells transfected with the APP I716F and V717I mutations showed a marked increase of the Ab42/40 ratio in the media compared to wtAPP. Both mutations led to a decrease in AICD production and reduced g-secretase activity. APP CTFs levels were increased in cells transfected with both mutations as well as in the proband’s brain sample. Conclusions: The APP I716F mutation is associated with a very early age of onset and aggressive neuropathological phenotype. Our data indicates that this mutation, as well as the V717I APP mutation, leads to an increased Ab 42/40 ratio but also to a reduced APP proteolysis by g -secretase. This suggests an additional mechanism by which mutations around g -secretase cleavage site may lead to AD. P1-157

g-SECRETASE SUBSTRATES APP, NOTCH1 AND CD44 SHOW DISTINCT PROCESSING IN NPC CELLS

Martina Malnar1, Harald Steiner2, Sven Lammich2, Silva Hecimovic1, 1 Rudjer Boskovic Institute, Zagreb, Croatia; 2Ludwig-Maximilians-University, Munich, Germany. Contact e-mail: [email protected] Background: It has been previously demonstrated that NPC1 dysfunction, that causes Niemann Pick type C disease (NPC), results in altered APP processing leading to increased levels of C99 and amyloid-b peptide (Ab). To elucidate whether the Ab increase upon NPC1 loss of function is due to a common mechanism of altered g-secretase processing, we analyzed the levels of b-like peptides and intracellular domains (ICDs) of APP, Notch1 and CD44 in wt (CHOwt) and NPC cells (CHO NPC1-null). Methods: The cells were transiently transfected with C99, Flag-Next or CD44DE-Flag constructs (all C-terminal myc tagged). In order to detect b-peptides, media were collected, cleared and immunoprecipitated using appropriate antibodies. Immunoprecipitates were subjected to SDS-PAGE on Scha¨gger gels and blotted onto nitrocellulose membranes. The membranes were probed with antibody FLAG-M2 (CD44-b and Notch1-b) or 6E10 (Ab). For detection of ICDs, cell lysates were subjected to SDS-PAGE, blotted on PVDF membranes and immunoblotted with antibody 9E10. Results: In contrast to the markedly increased levels of Ab, we did not observe an increase in Notch-b and CD44-b peptides in NPC compared to wt cells. Our observation that AICD levels were similar in wt and NPC cells, suggests that cholesterol accumulation upon NPC1 dysfunction may specifically affect g-secretase cleavage at g40/42-site and not g-secretase cleavage at the e-site. In addition, the levels of Notch1-ICD (NICD) and CD44-ICD were similar between wt and NPC cells, further supporting the hypothesis that g-secretase processing of Notch1 and CD44 is not altered in NPC cells. Conclusions: We found that NPC1 loss specifically affects Ab levels and not the levels of other g-secretase generated b-like peptides. These results suggest that there is not a common mechanism of g-secretase cleavage that would result in similar g-secretase processing of APP, Notch1 and CD44 in NPC cells. This finding supports a model of distinct processing pathways of g-secretase substrates, indicating that designing an

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Ab-specific g-secretase inhibitor might be feasible. Alternatively, Ab increase in NPC cells might be due to defective Ab degradation. This work was funded by the grants: Bilateral project between Germany and Croatia (H.S. and S.H.) and Ministry of Science of the Republic of Croatia 098-0982522-2525 (S.H.). P1-158

AMYLOIDOGENIC METABOLISM OF HUMAN APP IN X11-LIKE DEFICIENT MICE BRAIN

Maho Kondo1, Megumi Sakuma1, Yuhki Saito1, Tadashi Nakaya1, Masahiro Maeda2, Toshiharu Suzuki1, 1Hokkaido University, Sapporo, Japan; 2IBL Co. Ltd, Fujioka, Japan. Contact e-mail: komaho@pharm. hokudai.ac.jp Background: X11-like (X11L), which is neuronal adaptor protein containing a phosphotyrosine binding (PTB) and two PDZ domains, interacts with the cytoplasmic 681GYENPTY687 motif of APP695 through its PTB domain and regulates APP metabolism (reviewed in J. Biol. Chem. [2008] 283, 29633). In brain hippocampus of X11L-KO mice, b-site cleavage of murine endogenous APP was facilitated and amounts of Ab increased (J. Biol. Chem. [2006] 281, 37853). Further analysis revealed that X11L can function to anchor mature APP outside of detergent-resistant membrane (DRM) where band g-secretases are rich in activity (J. Biol. Chem. [2008] 283, 35763). Our observations propose an idea that malfunction of X11L in the regulation of APP metabolism may facilitate amyloidogenic metabolism of APP in AD. But the effects of X11L deficiency on metabolism of human APP expressing at physiological level in mouse brain have not been revealed along with the regulatory mechanism of APP-X11L interaction. Here we tried to understand these issues. Methods: By crossing X11L-KO mice and transgenic mice expressing human APP at physiological level (APP-Tg), we newly generated APP-Tg/X11L-KO mice. APP C-terminal fragments (CTF) in APP-Tg and APP-Tg/X11L-KO mice brain were analyzed by immunoblotting. The levels of human Ab40 and Ab42 were measured by sELISA system. To identify the regulatory region of X11L in APP-X11L interaction, we prepared epitopetagged and GST-fused X11L proteins including various region deletions and amino acid mutations. The interaction of APP-X11L was examined by co-immunoprecipitation in cells and GST pull-down assay in vitro. Results: APP-Tg/X11L-KO mice showed significant increase of human APP CTFb and Ab compared to APP-Tg mice. We identified the region of X11L that regulates association with APP in outside of, and amino-terminal to, the PTB domain. Conclusions: X11L deficiency induces the amyloidgenic metabolism of human APP as does endogenous APP. This result supports the function of X11L which anchors APP outside of DRM and suppress the pathogenic metabolism of APP. Modification of the region that regulates APP association may induce a conformational change within PTB domain and alter APP-binding ability. Some qualitative change of X11L such as decreased association with APP may lead to onset sporadic AD. P1-159

REGULATORY MECHANISMS OF APP AND ALCADEIN TRANSPORT BY KINESIN-1

Masahiko Araseki, Takanori Kawano, Keiko Furukori, Tohru Yamamoto, Toshiharu Suzuki, Laboratory of Neuroscience, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan. Contact e-mail: [email protected] Background: APP and Alcadein (Alc) are an evolutionarily conserved type I membrane protein. Both proteins associate in their cytoplasmic domain through the interaction with X11-like (X11L) neuron-specific adaptor protein. Thus, APP and Alc are subject to a coordinated metabolism and an identical neural localization along with a similar function as cargo-receptor (J. Biol. Chem. [2004] 279, 24343- 24354; EMBO J. [2007] 26, 1475-1486). APPand Alc-cargos are independently transported with same motor, kinesin-1, but at different velocities, indicating that the regulatory systems of vesicle transport can distinguish between APP and Alc cargoes. Indeed, APP associates to kinesin-1 through the interaction with JIP1b, while Alc can associate directly to kinesin-1. Here we analyzed transport system of APP and Alc by kinesin-1 in detail. Methods: EGFP-APP, Alc, KLC, and KHC are expressed in primary cultured neurons derived from wild-type or mutant mice brain or differentiating neuronal cultured-cell lines. Transport of APP- and Alc-cargos

or vesicle-associated kinesin-1 motor was analyzed using a total internal reflectance fluorescence (TIRF) microscopy system. Results: Association of Alc with KLC could form a functional kinesin-1 motor and induce anterograde transport of Alc-cargo, while Association of APP and JIP1b with KLC failed to activate kinesin-1 motor. We identified a minimum region of Alc, which can activate kinesin-1, together with identification of KLC region that Alc binds. Conclusions: Our results indicate that APP and Alc are transported by same motor system under the different regulation of kinesin-1 activation. Because metabolism of APP including Ab generation is involved in the vesicular transport system, malfunction of kinesin-1 anterograde transport system to APP and Alc may induce aberrant production of Ab. P1-160

ABETA42-TO-ABETA40- AND ANGIOTENSINCONVERTING ACTIVITIES IN DIFFERENT DOMAINS OF ANGIOTENSIN-CONVERTING ENZYME

Kun Zou1, Tomoji Maeda1, Ryutaro Oba2, Makoto Michikawa3, Hiroto Komano1, 1Iwate Medical University, Yahaba, Japan; 2BML, INC., Kawagoe, Japan; 3National Institute for Longevity Sciences, National Center for Geriatrics and Gerontology, Obu, Japan. Contact e-mail: [email protected] Background: Amyloid b-protein 1-42 (Ab42) is believed to play a causative role in the development of Alzheimer’s disease (AD), although it is a minor part of Ab. In contrast, Ab40 is the predominant secreted form of Ab and recent studies have suggested that Ab40 has neuroprotective effects and inhibits amyloid deposition. We have reported that angiotensin-converting enzyme (ACE) converts Ab42 to Ab40 and its inhibition enhances brain Ab42 deposition (Zou et al. J Neurosci, 2007). ACE is commonly targeted by ACE inhibitors for the treatment of hypertension in elderly populations and it has two homologous domains, each having a functional active site. Methods: We identified the domain of ACE which is responsible for converting Ab42 to Ab40 using N-domain, C-domain and both N- and C-domain recombinant proteins of ACE. Results: Interestingly, we found that the Ab42-to-Ab40-converting and the angiotensin-converting activities are located in different domains of ACE. Conclusions: ACE domains select substrates and these results suggest that ACE inhibitors could be designed to specifically target the angiotensin-converting domain, without inhibiting the Ab42-to-Ab40-converting activity of ACE. P1-161

THE ROLE OF APP PROCESSING IN OXIDANTINDUCED DAMAGE IN CULTURED NEUROBLASTOMA CELLS

Lin Zheng1, Nodi Dehvari2, Katarina Ka˚gedal3, Eirikur Benedikz2, Richard Cowburn4, Alexei Terman5, Jan Marcusson6, 1Linko¨ping University, Linko¨ping, Sweden; 2Karolinska Institute, Stockholm, Sweden; 3Pathology II, Linko¨ping University, Linko¨ping, Sweden; 4AstraZeneca R&D, So¨derta¨lje, Sweden; 5Department of Clinical Pathology and Cytology, Karolinska University Hospital, Stockholm, Sweden; 6Geriatric Department, Linko¨ping University, Linko¨ping, Sweden. Contact e-mail: [email protected] Background: Increasing evidence shows the toxicity of intracellular amyloid b-protein (Ab) to neurons and the involvement of lysosomes in this process in Alzheimer disease (AD). In our previous study, we showed that oxidative stress enhances autophagy and leads to intralysosomal accumulation of Ab in cultured neuroblastoma cells. Furthermore, we found that oxidative stress can induce neuronal death through autophagy of Ab and consequent lysosomal membrane permeabilization in cultured HEK293 cells. Methods: To further investigate the mechanisms of possible autophagy activation and APP processing in neuron like cells, we compared the effects of hyperoxia (40% ambient oxygen) in retinoic acid differentiated neuroblastoma SH-SY5Y cells that were transfected with vector, or APPwt, or APPswe. Results: Exposure to hyperoxia for five days increased the number of cells with Ab-containing lysosomes, as well as the number of apoptotic cells compared to normoxic conditions. The rate of apoptosis in all three cell lines demonstrated dependence on intralysosomal Ab content (Vector