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Poster P2:: Monday Posters ments, found in neurofibrillary tangles, contain the hyperphosphorylated protein tau. Both senile plaques and neurofibrillary tangles have to coexist in the human brain to confirm the clinical diagnosis of AD. Nevertheless, the neuronal metabolism of their constitutive proteins, which could explain the formation of both amyloid deposits and paired helical filaments, remains poorly understood. In AD however, many serine and threonine residues of tau protein are phosphorylated by glycogen synthase kinase-3␤ (GSK3␤) and cyclin-dependent kinase-5 (Cdk5). Interestingly, GSK3␤ and Cdk5 are also able to phosphorylate APP on Thr668. Results: In rat cultured neurons expressing human APP, depolarization by 35 mM KCl induced an important increase in cytosolic calcium concentration. In these experimental conditions, a transient increased phosphorylation of human APP on Thr668 was observed, which was inhibited by nimodipine, a specific antagonist of L-type calcium channels. Roscovitine, a Cdk5 inhibitor, was also able to partly inhibit Thr668 phosphorylation. Concomitant with this increase in APP phosphorylation, a transient increased phosphorylation of endogenous tau was also demonstrated on serine 396/404, using the PHF1 antibody. These residues are known to be phosphorylated by GSK3␤. Following these transient hyperphosphorylations of both tau and APP, a progressive intraneuronal accumulation of A␤1-42 was measured. When human APP carrying the Thr668Ala mutation was expressed in rat cultured neurons, KCl-induced depolarisation did not allow the production of intraneuronal A␤1-42. Therefore, calcium mediated phosphorylation of APP on Thr668 leads to intraneuronal production of A␤1-42. Conclusions: In primary cultures of rat neurons, we conclude that high cytosolic calcium concentrations, induced by neuronal depolarization, trigger both tau hyperphosphorylation and the production of intraneuronal A␤1-42 resulting from APP phosphorylation on Thr668. Therefore, hyperphosphorylation could represent a biochemical link between the two main pathological lesions found in AD. P2-051

CATHEPSIN D, B AND L ARE DOWNREGULATED IN FIBROBLASTS FROM ALZHEIMER’S DISEASE PATIENTS

Aldo Orlacchio1, Lorena Urbanelli1, Carlo Massini1, Simona Mencarelli1, Giuseppe Pelicci2, Sandro Sorbi3, Andrei Hasilik4, Giorgio Bernardi5,6, Antonio Orlacchio5,6, Carla Emiliani1, 1Universita` di Perugia, Perugia, Italy; 2Instituto Europeo di Oncologia (IEO), Milan, Italy; 3Universita` di Firenze, Florence, Italy; 4PhilippsUniversity Marburg, Marburg, Germany; 5Centro Europeo di Ricerca sul Cervello (CERC) - Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Santa Lucia, Rome, Italy; 6Universita` di Roma ‘Tor Vergata’, Rome, Italy. Contact e-mail: [email protected] Background: Previous studies established that the population of neurons that frequently degenerate in Alzheimer’s Disease (AD) exhibit robust up-regulation of the lysosomal system. Objective(s): We investigated the expression of lysosomal proteases cathepsin D, B and L at peripheral level, using as cell model skin fibroblasts from AD patients affected either by sporadic or familial forms of the disease. Methods: Cell cultures, enzymatic assays, Western Blotting analysis. Results: By enzymatic assays, we observed a down-regulation of cathepsin D in 50% of AD patients. These decreased levels were consequent to regulation at transcriptional level. Western Blotting analysis confirmed the presence of decreased content of cathepsin D in AD fibroblasts. We extended the analysis to cathepsin B and cathepsin L and observed decreased content of these proteins in the same cells. A parallel increase of ras transcript and ras protein had been previously revealed in AD fibroblasts. To investigate the role of ras in the expression of cathepsin D, B and L, we over-expressed in primary fibroblasts a ras mutant (rasV12) known to induce premature senescence. Fibroblasts infection with a retrovirus encoding a constitutively active ras decreased cathepsin D levels but increased levels of cathepsin L and, to a lesser extent, of cathepsin B. Conclusions: These data demonstrate that decreased levels of cathepsins in AD fibroblasts are not correlated with ras activation. Overall data provide evidence that lysosomal dysfunction is not confined to CNS. In addition, decreased cathepsin levels and activities

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could lead to a decreased ability of proteolysis by the cell and might be related to the pathogenesis of the disease. P2-052

CHICKEN PRIMARY NEURONAL CELLS AS A POWERFUL MODEL FOR STUDYING TAU PHOSPHORYLATION, APP PROCESSING AND A␤ GENERATION

Juan Manuel Maler1, Hans W. Klafki1, Hermann Esselmann1, Nikolaus Kunz2, Sabine Paul1, Johannes Kornhuber1, Jens Wiltfang1, 1 University of Erlangen-Nuremberg, Erlangen, Germany; 2University of Ulm, Ulm, Germany. Contact e-mail: [email protected] Background: Neuritic Plaques containing aggregated beta-amyloid (A␤) peptides and neurofibrillary tangles composed of hyperphosphorylated tau protein represent the classical neuropathological hallmarks of Alzheimer’s disease (AD). Objectives: Understanding the cellular biology of key molecules such as APP, A␤ peptides, tau and related proteins may help to devise novel therapeutic strategies against AD. Methods: To address this question, a cell culture model based on primary chicken neurons was established. It allows the simultaneous assessment of A␤ peptide release from endogenous APP and the analysis of the phosphorylation state of tau protein under different experimental conditions. A␤ peptides were analysed by quantitative SDS-PAGE/immunoblot and surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS), tau proteins were assessed by Western blot. Results: A␤ peptides A␤140/42 plus three additional C-truncated species, namely A␤1-37/38/39 were constitutively released into the culture medium, strongly resembling the highly conserved pattern of 5 A␤ peptides present in human cerebrospinal fluid. Importantly, the chicken A␤ amino acid sequence is identical to human A␤, therefore, various commercial anti A␤ antibodies can be used for detection. Treating the cells with sulindac sulfide (100 ␮M) selectively decreased A␤1-42. In parallel, A␤1-38 and, to a lesser extent, A␤1 37 were increased, while A␤1-40 and the total amount of secreted A␤ peptides remained unchanged. Tau proteins prepared directly from embryonic chicken brains were highly phosphorylated at several sites such as Ser202/Thr205 (AT8 epitope), Ser422, Ser262. In contrast, tau proteins from primary chicken neurons (E8, 6th day in vitro) were unphosphorylated or showed only low levels of phosphorylation at these sites suggesting a significant change under cell culture conditions. Treating the cells with the phosphatase inhibitor okadaic acid induced PHF-like tau hyperphosphorylation as indicated by phosphospecific tau antibodies and a shift in electrophoretic mobility. Conclusion: Our results strongly support primary chicken neurons as a powerful experimental model for studying AD related proteins such as APP, A␤ peptides and tau simultaneously. P2-053

INCREASE OF THE PRODUCTION OF AMYLOID BETA-PEPTIDE BY LITHIUM CHLORIDE IS INDEPENDENT FROM ITS INHIBITION OF GSK3

Christine Feyt1, Pascal Kienlen-Campard1, Karelle Leroy2, Franscisca N’Kuli1, Bernadette Tasiaux1, Pierre J. Courtoy1, Jean-Pierre Brion2, Jean-Noel Octave1, 1Universite Catholique de Louvain, Bruxelles, Belgium; 2Universite Libre de Bruxelles, Bruxelles, Belgium. Contact e-mail: [email protected] Background: Alzheimer’s disease is characterized by the presence of senile plaques and neurofibrillary tangles in the brain. Senile plaques contain an amyloid core of A␤ peptide, while neurofibrillary tangles contain the hyperphosphorylated protein tau. Glycogen synthase kinase 3 (GSK3) is able to phosphorylate tau at many sites which are found to be phosphorylated in paired helical filaments in Alzheimer’s disease. Another substrate of GSK3 is ␤-catenin. The phosphorylation of ␤-catenin by GSK3 targets ␤-catenin for proteasomal degradation and prevents its translocation in the nucleus. Lithium chloride (LiCl) efficiently inhibits GSK3 and was reported to also decrease the production of amyloid-␤ peptide (A␤) from its precursor APP. Therefore, lithium has been proposed as a

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Poster P2:: Monday Posters

combined therapeutic agent, inhibiting both the hyperphosphorylation of tau and the production of A␤. Results: CHO cells and rat cultured neurons expressing human APP695 were treated with a nontoxic 5 mM concentration of LiCl. Nuclear translocation of ␤-catenin in both cellular models, as well as a decrease in tau phosphorylation in neurons was observed. In CHO cells, 5 mM LiCl increased the amount of extracellular A␤ and C-terminal ␤-cleaved fragment of APP (␤CTF). Increase in the ␤-cleavage did not result from a modification of the cellular distribution of APP and BACE1. Interestingly, the ␤-secretase activity of CHO cells was increased in the presence of LiCl. In primary culture of rat cortical neurons, GSK3 inhibition by LiCl induced a similar increase in both extracellular A␤ and ␤CTF. An increase in the amount of BACE1 protein was also observed. Since LiCl is not a specific inhibitor of GSK3 but can display a number of other activities, a more selective GSK3 inhibitor (SB415286) was utilized. At a nontoxic 25 ␮M concentration, SB415286 induced nuclear translocation of ␤-catenin and slightly reduced the production of extracellular A␤. LiCl and SB415286 had cumulative effects on extracellular A␤ production, indicating that LiCl-mediated increase in A␤ production is not related to GSK3 inhibition. Conclusions: Taken together, these results demonstrate that LiCl increases the production of extracellular A␤ by increasing the ␤-cleavage of APP. LiCl increases the production of A␤, independently of its inhibition of GSK3. P2-054

LACK OF PHOSPHORYLATION OF THE AMYLOID PRECURSOR PROTEIN ON THR668 RESIDUE INCREASES THE GAMMA-CLEAVAGE OF THE PROTEIN

Christine Feyt, Nathalie Pierrot, Joanne Van Hees, Jean-Noel Octave, Universite Catholique de Louvain, Bruxelles, Belgium. Contact e-mail: [email protected] Background: Senile plaques are one of the two hallmarks that characterize Alzheimer’s disease. The amyloid core of these extracellular lesions is composed of the amyloid beta peptide (A␤). A␤ peptide is released upon the sequential cleavage of the Amyloid Precursor Protein (APP) by ␤- and ␥-secretase activities. The C-terminal domain of APP can be phosphorylated at several serine, threonine and tyrosine residues. The phosphorylation of APP on threonine 668 (T668) seems to be specific to neuronal tissue and causes a structural change in the cytoplasmic domain of APP. Results: The influence of APP T668 mutation on extracellular A␤ generation was analyzed in two distinct cellular models. Using site directed mutagenesis, T668A or T668D substitution was introduced in the wild type APP695 sequence. In CHO cells, human APP was very slighlty phosphorylated on Thr668 and produced similar levels of extracellular A␤ as compared to CHO cells expressing APPT668A. On the other hand, a 25 % decrease of A␤ production was measured in the culture medium of CHO cells expressing APPT668D. In primary culture of rat cortical neurons, human APP expressed following infection by a recombinant adenovirus was phosphorylated on T668. When APPT668A was expressed in neurons, a 25 % increase in extracellular A␤ production was observed while the amount of C-terminal fragments of APP (CTF) decreased in the same proportion. Following inhibition of the ␥-cleavage by DAPT, neurons expressing APP or APPT668A produced similar amounts of CTF. Conclusions: It is concluded that absence of phosphorylation on Thr668 increases the ␥-cleavage of APP and the production of extracellular A␤. P2-055

CHONDROITIN SULPHATE PROMOTES NEUROPROTECTION AGAINST FREE RADICALINDUCED DAMAGE IN VITRO

Noelia Can˜as1, Teresa Valero1, Eulalia Montell2, Josep Verges2, Manuela G. Lo´pez1, 1Universidad Autonoma de Madrid, Madrid, Spain; 2 Bioiberica, Barcelona, Spain. Contact e-mail: [email protected] Background: Inflammatory response and generation of reactive oxygen species (ROS) are some of the factors that have been implicated in the pathobiology of AD. Is known that in AD, cortical areas rich in perineu-

ronal nets are less severely affected by neurofibrillary degeneration and net-associated neurons have no tangles. (Morawski et al. 2004). CS is a proteoglycan that forms part of the extracellular matrix of the perineuronal net; these nets act as a buffering system for ions such as those that are involved in generating oxidative stress. Also, several basic studies have shown that CS can protect articular chondrocytes by inhibiting molecules responsible for articular inflammation or by scavenging free radicals. Objective(s): To determine if CS, a physiological compound of the extracellular matrix, could exert neuroprotective effects against free radicals in SH-SY5Y cells. Methods: We have used the human neuroblastoma cell line SH-SY5Y. To induce free radical production we have used hydrogen peroxide (100 ␮M), as a source of exogenous ROS, and the combination of rotenone (10 ␮M) and oligomycin-A (0.1 ␮M), as a source of endogenous ROS generated at the mitochondria. Cell death was quantified as the release of LDH to the extracellular media and apoptosis was evaluated by analysing the cell cycle by flow cytometry. The fluorescent dye DCFH-DA was used as marker of generation of free radicals inside the cell. Results: CS protected SH-SY5Y cells in a concentration-dependent manner against hydrogen peroxide-induced damage; the highest protection was observed at 600 and 1000 ␮M. In the case of rotenone -oligomycin-A induced cell death, CS significantly protected at the concentrations of 10 and 60 ␮M. Regarding free radical generation measured as DCFH-DA fluorescence, 100 ␮M of CS reduced the fluorescence by 40% when ROS were generated by the combination of rotenone and oligomycin-A. The reduction of free radicals generated by hydrogen peroxide was also reduced by 40%, but, at the concentration of 600 ␮M of CS. Conclusions: These results suggest that chondroitin sulphate, a component of the extracellular matrix that forms part of the perineuronal net, can protect against the cell damage caused by exogenous or endogenous ROS. P2-056

HOMO-AND HETEROPHILIC INTERACTIONS OF APP-FAMILY-PROTEINS

Daniela Kaden1, Lisa M. Munter1, Philipp Voigt2, Michael Schaefer2, Gerd Multhaup1, 1FU Berlin, Berlin, Germany; 2UKBF, Berlin, Germany. Contact e-mail: [email protected] Background: The amyloid precursor protein (APP) of Alzheimer’s disease belongs to a protein super-family that shares similar functions. APLP1 structurally differs from APP and APLP2 insofar as it is exclusively expressed in brain and does not possess the KPI domain. We found that APP showed homophilic interaction (Scheuermann, JBC 2001) that possibly occurs through three identified contact sites in a zipper-like mechanism. Two contact sites are in the ectodomain and a third one is within the transmembrane sequence of A␤. So far, homo- and heterooligomerization of APP family members have not been correlated with their cellular and subcellular localization, which is a critical determinant for their biological function. Objective: To investigate the subcellular localization of APP and APLP homo- and heterodimers in living cells using YFP- and CFP-fusion proteins and to analyze the influence of secretase inhibitors on their cell surface distribution. Results: The subcellular localization of APP, APLP1 and APLP2 was analyzed in HEK293 cells. Surprisingly, confocal laserscanning microscopy of YFP-fusion proteins revealed striking differences between APLP1, APP and APLP2. APP exhibited a pronounced ER and Golgi staining and a much weaker cell surface staining. APLP1 was mainly localized to the cell surface. Interestingly, APLP2 showed a mixed form of localization between ER/Golgi and the cell surface. The cause of this distinctive distribution remains to be determined. When we used FRET (fluorescence resonance energy transfer) to analyze interactions of APPfamily-proteins we found that all three proteins can undergo homo- and heterophilic interactions. Upon application of inhibitors for alpha-, beta- or gamma-secretase activities, we showed (1) that the colocalization of APP and APLPs is clearly enhanced as revealed by increased FRET efficiencies and (2) that APP and APLP2 showed a cell surface staining resembling that of APLP1. Conclusion: All three proteins, APP, APLP1 and APLP2 can interact with each other on the cell surface in living cells. Both, homo- and heterodimers are substrates for alpha-, or beta- and gamma-secretases. This