P4-052

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Poster P4:: Wednesday Posters

proteins in their transmembrane domains (TMD). However, the precise mechanisms of enzyme-substrate interaction and the substrate requirements for ␥-secretase-dependent cleavage remain unclear. It is possible that substrate sequences distant from the cleavage sites are required for these actions. Objective(s): To elucidate the structural requirements for ␥-secretase recognition and cleavage of APP. Methods: We constructed a fusion protein of the Gal4 DNA-binding/VP16 transactivation (GVP) domain with the C99 form of APP, which is an immediate substrate for ␥-secretase in vivo. Upon transfection into HEK293 cells, the ␥-seceretase-dependent cleavage of this hybrid protein can be quantified with A␤ ELISAs and a luciferase reporter assay measuring APP intracellular domain (AICD) generation. Using this assay platform, we then performed mutagenesis studies to examine the functional role of APP luminal juxtamembrane domain (LJD) in substrate recognition and/or cleavage by ␥-secretase. Results: C99-GVP is a robust ␥-secretase substrate, as shown by the aforementioned assays. Major deletion of the LJD resulted in dramatic inhibition of its cleavage by ␥-secretase. In addition, replacement of the LJD of APP with corresponding regions from other known type I membrane proteins, such as Notch, amyloid precursor-like protein 2 (APLP2) and sterol regulatory element-binding protein (SREBP), also caused moderate to severe reduction in cleavage efficiency. Conclusions: Taken together, our results suggest an important role of the luminal juxtamembrane domain of APP in substrate recognition and/or cleavage by ␥-secretase. P4-050

ENDOGENOUS ABETA42 ACCUMULATION IN NEURONS IMPAIRS THE MULTIVESICULAR BODY SORTING PATHWAY

Claudia G. Almeida, Gunnar K. Gouras, Weill Medical College of Cornell University, New York, NY, USA. Contact e-mail: [email protected] Background: In Abeta precursor protein (APP) mutant transgenic mouse and in human Alzheimer’s disease (AD) brains, progressive intraneuronal accumulation of Abeta42 occurs especially in multivesicular bodies (MVBs). We reported early AD-like synaptic alterations in cultured APP mutant neurons; specifically, there are early reductions in post-synaptic PSD-95, accompanied by fewer glutamate receptors at the surface (Almeida et al., 2005). Objective(s): We hypothesized that Abeta42 accumulation in MVBs impairs the MVB sorting pathway. Methods: We utilized the trafficking of the transferrin (Tf) receptor (TfR), epidermal growth factor receptor (EGFR) and TrkB receptor to investigate the MVB sorting pathway in cultured neurons. By immuno-fluorescence confocal microscopy we studied Abeta42 colocalization with internalized EGF and Tf, markers of distinct endocytic pathways. We quantified EGF and Tf uptake by single cell analysis. By Western blot, we studied upon stimulation the degradation of EGFR (EGF or TGFalpha) and TrkB (BDNF) in APP mutant and wild-type neurons at 12 days in vitro (DIV). We also measured EGFR phosphorylation and ubiquitination in APP mutant and wild-type neurons. Since the MVB sorting pathway is regulated by the ubiquitin-proteasome system (UPS) we performed fluorimetric assays with substrates of deubiquitinating enzymes and of the proteasome. Results: We found that Abeta42 colocalizes more with MVB than with early/recycling endosome markers. Upon EGF stimulation, APP mutant neurons demonstrated impaired inactivation, degradation and ubiquitination of EGFR. EGFR degradation is dependent on translocation from MVB outer to inner membranes, which is regulated by the UPS. We provide evidence that Abeta accumulation in APP mutant neurons inhibits the activities of the proteasome and deubiquitinating enzymes. Conclusions: Our results suggest normal early endocytosis and an impaired late endocytosis/MVB pathway in APP mutant neurons. The inhibition of proteasome activity due to Abeta accumulation in MVBs appears to be responsible for an altered down-regulation of surface receptors in APP mutant neurons. These data suggest a mechanism whereby Abeta accumulation in neurons impairs the MVB sorting pathway via the UPS. We hypothesize that the inhibition of the UPS by Abeta is important for synaptic dysfunction in AD.

P4-051

INHIBITION OF ENDOCYTOSIS ACTIVATES ALTERNATIVE DEGRADATION PATHWAY OF ␤APP IN CULTURED CELLS

Tao Hong, Takashi Oguri, Masayasu Okochi, Akio Fukumori, Jingwei Jiang, Akiko Ikuta, Kanta Yanagida, Taisuke Nakayama, Yoshiko Ishizuka-Katsura, Kohji Mori, Takashi Morihara, Kojin Kamino, Toshihisa Tanaka, Takashi Kudo, Shinji Tagami, Masatoshi Takeda, Osaka University Graduate School of Medcine, Osaka, Japan. Background: ␤APP is sequentially endoproteolyzed by ␣/␤-secretase and ␥-cleavage. In the process, extracellular shedding by ␣-secretase (ADAM 9/10/17) or ␤-secretase (BACE 1/2) at position L17 or D1 (A␤ numbering) is prerequisite to generate P3 or A␤, respectively. In addition, several alternative extracellular cleavage sites in ␤APP were reported at position I-6, V-3, R5, E11, F20, and A21. Objective(s): Among these sites, position R5 is considered to be cleaved by ␣-secretase-like activity, whereas position E11, F20 and A21 are cleaved by ␤-secretase. Therefore, extracellular shedding of ␤APP is thought to be mediated exclusively by ␣/␤-secretase activities. However, so far the characteristics of cleavages at position V-3 and I-6 are not well understood. Methods: Combined immunoprecipitation and semi-quantitive MALDI-TOF MS was used. Results: The cleavage at position I-6 of ␤APP has characteristics distinct from that of ␣/␤-secretase, while the cleavage at V-3 seems to be mediated by ␤-secretase. Although inhibition of endocytosis enhances the cleavages at both V-3 and I-6, PMA, an ␣-secretase stimulator, treatment enhances neither of these cleavages. Interestingly, a ␤-secretase inhibitor, z-VLL-CHO, suppressed V-3 but not I-6 cleavage. Conclusions: Our data demonstrate that neither ␣ nor ␤-secretase undergoes extracellular shedding at I-6 of ␤APP. P4-052

ACCUMULATION OF SPHINGOLIPIDS INCREASES SECRETION OF THE AMYLOID ␤PEPTIDE BY STABILIZATION OF THE ␤AMYLOID PRECURSOR PROTEIN

Irfan Y. Tamboli1, Heike Hampel1, Konrad Sandhoff2, Jochen Walter1, Molecular Cell Bio, Neurology, University Clinic Bonn, Bonn, Germany; 2Kekule´-Institute for Organic Chemistry and Biochemistry, University of Bonn, Bonn, Germany. Contact e-mail: [email protected]

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Alzheimer’s disease is associated with extracellular deposits in the brain of amyloid-␤-peptides (A␤) that are generated by proteolytic processing of the ␤-amyloid precursor protein (APP). It has been shown that membrane lipids, including cholesterol and sphingolipids affect the subcellular transport of APP in the secretory pathway and its proteolytic processing. Previously, we also showed that the inhibition of glycosphingolipid (GSL) biosynthesis reduces the secretion of APP and A␤. Sphingolipids are highly enriched in the plasma membrane of cells. From here they are transported to late endosomal/lysosomal compartments where they are degraded. Inherited defects in degradation of these lipids cause sphingolipid storage disorders (SLSDs) marked by accumulation of GSLs in neurons associated with severe neurodegeneration. There is also a common defect in lipid transport along the endocytic pathway in SLSDs. Since processing of APP by secretases also occurs predominantly in postGolgi secretory and endocytic compartments, we investigated the effect of accumulation of sphingolipids on transport and processing of APP. We used cultured cells and experimentally increased GSL levels by incubation with bovine brain gangliosides. The proteolytic processing of APP and its derivatives was studied by the detection of full length and soluble APP, APP C-terminal fragments (CTFs) as well as A␤ and by pulse-chase experiments. Distribution of APP and its processing products was analyzed by iodixanol density gradient. The accumulation of GSLs markedly increased the secretion of endogenous APP and A␤. A strong increase in total APP-CTF levels upon addition of GSLs was also observed. Addition of sphingomyelin showed

Poster P4:: Wednesday Posters similar effects. However ␥-secretase activity was not affected by GSLs in in vitro assays. By biochemical and cell biological experiments, we demonstrate that the increased levels of cellular GSLs altered the distribution and stability of APP-CTFs. Similar results were also obtained in independent genetic cellular models of GSL storage. On the other hand, GSL deficient cells showed decreased levels of APP-CTFs. Together, these data demonstrate that GSLs enhance the secretion of A␤ likely by stabilizing APP-CTFs, thereby providing more substrate for ␥-secretases. Our studies suggest a novel role of GSLs in regulation of APP trafficking and A␤ generation along the endocytic pathway. Supported by Deutsche-Forschungsgemeinschaft. P4-053

JNK REGULATES APP PROCESSING

Alessio Colombo1, Sara Santambrogio1, Mariaelena Repici2, Catherine Centeno2, Cristina Ploia1, Gianluigi Forloni1, Tiziana Borsello1,2, 1Mario Negri Institute for Pharmacological Research, Milan, Italy; 2DBCM, University of Lausanne, Lausanne, Switzerland. Contact e-mail: [email protected] Background: The accumulation of Amyloid beta peptide (A␤) in senile plaques is widely believed to play a central role in Alzheimer’s disease (AD). A␤ peptides (␤1-40 and ␤1-42), derive from the proteolytic processing of the Amyloid Precursor Protein (APP). The exact mechanism by which these peptides trigger neuronal death is not well defined. However, the disturbance of the calcium homeostasis and the activation of caspases and c-Jun N-terminal kinase (JNK) are factors involved in the death process induced by A␤. It has recently been demonstrated that the JNK scaffold protein, JIP-1, interacts with the cytoplasmic domain of APP, suggesting that JIP-1 and JNK may play important roles in the metabolism of APP. It has also been established that APP is phosphorylated at Thr 668 by JNK. This phosphorylation in the cytoplasmic domain of APP may result in a regulation of the APP processing. Objective(s): We examined here the role of JNK in AD pathogenesis using a specific cell-permeable JNK inhibitor peptide, D-JNKI-1, and two different in vitro models: primary cortical neurons and H4-15x cells, stably transfected with human APP695 carrying Swedish mutation. Methods: Cortical neurons and H4 cells were treated with increasing concentrations of D-JNKI-1 for 24h and western blot analysis of secreted APP (APPs) was conducted on both culture media and cell lysates. Results: We observed a dose-response effect of D-JNKI-1, with a powerful reduction in APPs production. In fact, in cortical neurons, D-JNKI-1 (6␮M) reduced by about 80% the level of APPs in both lysates and media, and this also correlated with a decrease in the media of A␤ fragments (40%). In the H4-15x using 80␮M D-JNKI-1 we obtained 70% reduction in APPs production and 45% decrease of A␤ fragments. Conclusions: In both cellular models D-JNKI-1 prevented phosphorylation of APP at Thr 668 in a dose-dependent way. These data indicate an important role of JNK signalling pathway in the regulation of APP metabolism: in our models the proteolytic production of APPs and ␤1-40/1-42 was strongly reduced by the application of a specific JNK inhibitor that prevented the APP phosphorylation. P4-054

EFFECTS OF FOLATE, B12 AND B6 DEPRIVATION ON METHYLATION METABOLISM AND AMYLOID PROCESSING IN HUMAN NEUROBLASTOMA AND GLIOBLASTOMA CELLS

Andrea Fuso1, Fabrizio D’Anselmi1, Rosaria A. Cavallaro1, Paola Piscopo2, Annamaria Confaloni2, Sigfrido Scarpa1, 1University of Rome La Sapienza, Rome, Italy; 2Istituto Superiore di Sanita`, Rome, Italy. Contact e-mail: [email protected] Background: Several articles point the attention of AD researchers to the importance of Homocysteine as a risk factor for the onset of the sporadic form of the disease. Nevertheless, a causal explanation about the mechanism of Homocysteine toxicity is still missing. Homocysteine cycle is a

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complex biochemical pathway regulated by the presence of Folate, vitamin B12 and B6 (among other metabolites) and leading to the production of the methyl donor molecule S-adenosylmethionine. Having as a starting point the known alteration of S-adenosylmethionine and of methylation reactions in AD and in the elderly, we already demonstrated that PS1 and BACE could be regulated by DNA methylation. A deeper knowledge of this pathway seems necessary to apply in vivo the study of DNA methylation and to understand its relevance in AD onset. Objective(s): Human neuroblastoma and glioblastoma cell lines are used as a model to test the effects of Folate, B12 and B6 deprivation on methylation metabolism. Studying the connection between amyloid processing and methylation could explain the involvement of methylation in the AD onset and the possible differential role of neuronal and glial cells. Methods: SK-N-BE neuroblastoma cell line and A172 glioblastoma cell line were grown in F14 medium, either complete or deprived of Folate and Vitamin B12, of Vitamin B6 or of all three vitamins. Cells were also grown in the presence of S-adenosylmethionine in order to investigate the correlation between the vitamin deprivation and the methylation reactions. Cell extracts were prepared to analyze RNA expression (Real-Time PCR), protein synthesis (Western Blotting assay), beta-amyloid production (ELISA test), total DNA methylation and intracellular S-adenosylmethionine and S-adenosylhomocysteine content (HPLC assays). Results: Folate, B12 and B6 deprivation alter Homocysteine pathway causing a reduction of DNA methylation. Interestingly, this alteration affects PS1 and BACE expression only in neuroblastoma, but not in glioblastoma, with a consequent increase in amyloid production. Conclusions: These findings point out a possible different involvement of neural and glial cells in AD and provide a basis to study the effect of B vitamins deprivation in an animal model of AD. P4-055

EVIDENCE FOR A METHYLATION-BASED MOLECULAR MECHANISM OF HYPERHOMOCYSTEINEMIA IN AD ONSET LEADING TO INTRACELLULAR AMYLOID DEPOSITION IN MICE

Sigfrido Scarpa1, Vincenzina Nicolia1, Rosaria A. Cavallaro1, Fabrizio D’Anselmi1, Laura Ricceri2, Gemma Calamandrei2, Andrea Fuso1, 1University of Rome La Sapienza, Rome, Italy; 2Istituto Superiore di Sanita`, Rome, Italy. Contact e-mail: [email protected] Background: Sporadic AD seems to be a multi-factorial disease; among several risk factors, Homocysteine is not extensively studied because of the lack of causal correlation with the pathology. Homocysteine shares the same biochemical cycle with S-adenosylmethionine, the major methyl donor in eukaryotes, through a series of reactions regulated by Folate and vitamins B12 and B6. Moreover, S-adenosylmethionine and methylation are known to be altered in AD and in the elderly. On the basis of this knowledge, we can ascertain that hyperhomocysteinemia is an indicator of “methylation potential” and could exerts its action by the alterations of methylation reactions. Our previous works demonstrate that PS1 and BACE are regulated by methylation in vitro with increased amyloid production; this finding makes extremely interesting the study of the correlation between hyperhomocysteinemia, methylation and amyloid production in vivo. Objective(s): The use of transgenic mice with an accelerated amyloid deposition represent a good model to study the effect of Folate, B12 and B6 deprivation on Homocysteine, methylation reactions and amyloid production in vivo, in order to give a molecular model for the onset of AD. Methods: CRND8 mice, carrying a double mutated APP, and wild type mice were fed with a diet deficient in Folate, vitamin B12 and B6 or with a control diet. After 45 or 60 days of diet, the mice were sacrificed to analyze Homocysteine and S-adenosylmethionine levels, DNA methylation, expression of genes involved in AD, amyloid processing and deposition; moreover, the animals were analyzed for cognitive impairment. Results: Deprivation of Folate, B12 and B6 lead to hyperhomocysteinemia in vivo, together with an altered SAM/SAH ratio (i.e. altered methylation potential) both in plasma and in brain. This altered metabolism leads to the