P4-215: Mitochondrial transmembrane protein modulates APP processing

T734 P4-213

Poster Presentations P4: NOVEL GAMMA-SECRETASE MODULATORS (␥SecAs) INCREASE A␤ GENERATION BY ENHANCING ITS ENZYMATIC ACTIVITY

Jonghee Han1, Jiyeon Jang2, Yong-Keun Jung1, 1School of Biological Science/Bio-MAX, Seoul National University, Seoul 151-742, Korea, Seoul, Republic of Korea; 2Gwangju Institute of Science and Technology, Gwangju, Republic of Korea. Contact e-mail: [email protected] Background: Amyloid plaque is a major pathological feature of Alzheimer’s disease. This glutinous body is mainly composed of amyloid-beta(A␤) peptide which is cleaved from APP by two secretases, ␤ and ␥-secretase. A␤ has been considered as a underlying cause of the Alzheimer’s disease and is toxic in vitro and in vivo. In fact, enzymatic reaction which ␥-secretase works is the limiting step of A␤ generation. Thus, identification of genes that regulate it’s enzymatic activity is important for the treatment of Alzheimer’s disease. Methods: We isolated novel ␥-secretase activator (␥SecA) by using cell-based functional screening system that measures the ␥-secretase activity-dependent fluorescence. Then, AICDluciferase reporter assay reflecting ␥-secretase activity confirmed the stimulatory effects of the clones. Results: Ectopic expression of ␥SecA1 and ␥SecA3 increased ␥-secretase-mediated cleavage of CTF␤ in SY5Y-CTF␤ cells. In ELISA assay, ectopic expression of ␥SecA1 and ␥SecA3 increased the amount of A␤ peptides, which was significantly suppressed by the ␥-secretase inhibitor, L645.458. Enzymatic activities of ␥-secretase, but not ␤-secretase, were significantly enhanced by ␥SecA1 and ␥SecA3 in SY5Y-APPsw cells. Conversely, the enhanced ␥-secretase activity was suppressed by shRNA ␥SecA. More importantly, ␥SecA seems to not affect notch cleavage in the NICD-luciferase reporter assay. Conclusions: These results suggest that ␥SecA may regulate ␥-secretase activity and thus increase A␤ generation. P4-214

HUMAN HOMOLOGUE OF DROSOPHILA CRUMBS ATTENUATES SECRETION OF AMYLOID-BETA PEPTIDES

Masaki Nishimura1, Yachiyo Mitsuishi1, Hiroshi Hasegawa1, Akinori Matsuo1, Shinji Tagami2, Masayasu Okochi2, Masatoshi Takeda2, Ronald Roepman3, 1Shiga University of Medical Science, Shiga, Japan; 2Osaka University Graduate School of Medicine, Osaka, Japan; 3Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands Antilles. Contact e-mail: [email protected] Background: Gamma-secretase mediates intramembrane cleavage of type I transmembrane proteins such as amyloid-beta precursor protein (APP) and Notch receptor. Proteolytic processing of APP yields neurotoxic amyloid-beta peptides in Alzheimer’s brains, whereas gamma-cleavage of Notch releases the intracellular domain that is critical for the signal transduction. Recently, Herranz et al. have reported that Drosophila transmembrane protein Crumbs negatively regulates Notch signaling. Mammalian homologues of Crumbs are known as scaffold proteins controlling the formation of adherens junctions. Among the homologues, CRB2 is expressed in the brain at a higher level. Methods: Using cultured cells, we investigated a modulatory role of CRB2 on gamma-secretase cleavage of APP. Amyloid-beta levels were measured by ELISA, and protein interaction was assessed by coimmunoprecipitation. Results: When overexpressed in APP-expressing HEK293 cells, CRB2 suppressed secretion of amyloid-beta peptides. CRB2 knockdown using siRNA caused increased secretion of amyloid-beta from SH-SY5Y neuroblastoma cells. Analysis using the truncation mutants indicated that the N-terminal extracellular domain of CRB2 is dispensable for inhibition of gamma-secretase cleavage. Co-immunoprecipitation study suggested that CRB2 binds with presenilin. Conclusions: Our results suggest that CRB2 binds with presenilin complexes to negatively regulate gamma-secretase activity in mammalian cells.

P4-215

MITOCHONDRIAL TRANSMEMBRANE PROTEIN MODULATES APP PROCESSING

Hiroshi Hasegawa, Masaki Nishimura, Shiga University of Medical Science, Otsu, Japan. Contact e-mail: [email protected] Background: Amyloid beta peptides (Abeta) are thought to be generated in mainly in cell surface, endosome and Trans-Golgi network by presenilin complexes with gamma-secretase activities. However, significant amount of Abeta is produced in other subcellular compartments, like early secretary pathway or mitochondria. Gamma-cleavage of amyloid precursor protein in early secretary pathway more preferentially occurs at the position 42 to generate Abeta42 that is toxic to neurons. Thus, in addition to the secreted Abeta, intracellular Abeta may play an important role in the neuronal cell death in Alzheimer’s disease. Mitochondria is reported to have essential components of gamma-secretase complexes, presenilin, nicastrin, APH-1 and PEN-2 and work as a mature enzymatic complex in vitro. However, the detail remains unknown.Mitochondria are one of the vulnerable organelles to aging. A lot of proteins in mitochondria are modified and differently translated by the long-lasting stresses, such as an oxidative stress. In addition, recent paper showed the importance of the modulator protein, TMP21, in gamma-secretase complexes. Thus, we tried to find a mitochondrial protein that modulates gamma-secretase activities. Methods: Modulator activities of mitochondrial proteins in generation were screened by the knock-down of specific proteins using a siRNA method and by over-expression. To clarify the mechanism of the modulation, the interaction with the components of presenilin complexes, gammaand epsilon-secretase activities were assessed. Results: One mitochondrial protein was identified to positively modulate secretase activities. Overexpression of the protein showed more than fourfold increase in both Abeta40 and Abeta42 generated from the whole cells. AICD generation was also increased using a reporter assay. The artificial tagging at the C-terminus of the protein loses the modulating functions, suggesting that C-terminus is likely to be essential to the modulating function. Direct interaction with the components of presenilin complexes was not detected. Moreover, in vitro gamma-secretase assay showed no significant modulation. Conclusions: This mitochondrial protein indirectly modulates APP processing through the C-terminus. P4-216

THE CADHERIN-BINDING FACTOR P120 PROMOTES THE ␥-SECRETASE PROCESSING OF CADHERINS BUT INHIBITS APP PROCESSING AND A␤ PRODUCTION

Junichi Shioi, Zen Kouchi, Julie Chan, Nikolaos K. Robakis, Mount Sinai School of Medicine, New York, NY, USA. Contact e-mail: [email protected] Background: Protein p120 contains tandem repeats of Armadillo domains and interacts with both presenilin 1 (PS1) and cadherins. We have reported that cadehrins bind to PS1 and are substrates of the PS/␥-secretase system. Here we examine the role of p120 in the ␥-secretase processing of cadherins and APP. Methods: Protein p120 was either knocked down in HEK293 cells using siRNA or it was introduced by trasduction into SW48 cells which lack p120. APP and cadherin processing was evaluated in vitro by cell-free assays. Secreted A␤⫻-40 and ⫻-42 in conditioned media was examined by ELISA assays. Results: To examine whether p120 is necessary for the PS1-mediated N-cadherin processing, we knocked down p120 KD in HEK293 cells. In in vitro assay, production of both of a 33 kDa and a 25 kDa fragment of N-cadherin, corresponding to the metalloproteinase (MP) product CTF1 and ␥-secretase product CTF2, respectively, were severely impaired compared to control cells. We also examined production of these cadherin fragments in cell extract of SW48 cells transduced with or without p120. Whereas mock-transduced cells produced some amount of CTF1 and CTF2 after 2 hrs of incubation, cells transduced with p120 produced more CTF1 and CTF2, the latter of which was produced prominently. APP processing was also analyzed using anti-APP antibody to detect APP/CTF␣␤ and AICD. Interestingly, cells transduced with p120