Poster Session P4: Molecular Mechanisms of Neurodegeneration - Presenilins

the y-secretase cleavage of many type I transmembrane proteins like APP, Notch, ErbB4 and cadherins. EphrinB ligands and EphB receptors are type I transmembrane proteins and the EphB receptors consist the largest family of tyrosine kinase receptors known today. The ephrinB-EphB system constitutes a bi-directional signaling system that transmits signals from both the ligand (ephrinB) and the receptor (EphB). The ephrinB-EphB interaction regulates important cellular functions like axon guidance, cell migration, angiogenesis and synaptic plasticity. Specifically, the ephrinB- EphB interaction modulates two forms of long-term synaptic plasticity that are used as models for information storage in the brain, the long-term potentiation (LTP) and the long-term depression (LTD). The ephrinB-EphB interaction induces the phosphorylation of Src kinase that acts as second messenger regulating various cellular functions like phosphorylation of cytoskeletal proteins, assembly of focal adhesions and memory formation. Objectives: To see if PS1/y-secretase system regulates ephrinB and EphB processing and the EpbB-mediated signaling. Methods: We tested the processing of ephirnB in brains and fibroblasts from normal (PSI+/+) and knockout (PS1-/-) mice on Western Blots. We also transfected PSI+/+ and PS1-/- mouse fibroblasts with EphB-GFP tagged receptor and we tested its processing on Western Blot. We stimulated PSI+/+ and PS1-/- fibroblasts with EphB receptor and we tested Src phosphorylafion in tile presence or absence of the y-secretase inhibitor L685,458 on Western Blots. Results and conclusions: We found that PS1/y-secrtase system regulates the proteolytic processing of both ephrinB ligands and their EphB receptors. Our data suggest that ephrinB and EphB are processed first by a metalloproteinase (MMP) producing a carboxy-terminal fragment termed CTF1, which is then cleaved by the PS1/y-secretase system producing a carboxy-terminal fragment termed CTF2, which affects gene expression. We also observed that the PS1/y-secretase system regulates the EphB-induccd phosphorylation of Src !dnase. Thus, PS1 may control synaptic structure and function by affecting the physiological processing of ephrinB ligands and EphB receptors as well as the ephrinB-EphB-mediated signaling.

~ THE ROLE OF CYCLOOXYGENASE (COX)-2 IN N141I MUTANT PRESENILIN-2 MEDIATED APOPTOSIS VIA WINGLESS INT (WNT)-2 SIGNAL TRANSDUCTION PATHWAY

Weiping Qin*, Giulio M. Pasinetti. Mount Sinai School of Medicine, New York, NY,, USA. Contact e-mail: giulio.pasinetti@mssm.edu

Most cases of early onset of familial Alzheimer's disease (AD) are caused by mutations in the presenilin (PS)- 1 and 2 genes. PS mutations lead to increased 13-amyloid formation and may induce apoptosis in some model systems. In this study using H4 neuroglioma cells stably transfected with wild-type (WT) or mutated (N141I) PS-2, we found that expression of N141I mutant PS-2 selectively induced apoptotic cell death, coincidental to elevation of (inducible) pro-inflammatory COX-2, but not (constitutive) COX-1 expression. Furthermore, we found that expression of N141I mutant PS-2 strongly promoted GSK-3~ activity and reduced the level of ~3-catenin translocation to the nucleus, in vitro. Most interestingly, we found that treatment of H4 cells expressing N141I mutant PS-2 with the preferential COX-2 inhibitor Nimesulide significantly protected H4 cells against N141I mutant PS-2 mediated apoptotic cell death. Interestingly, we found that the Nimesulide mediated inhibition of COX-2 activity assessed by inhibition of prostagladin (PG-E2) synthesis coincided with a down-regulation GSK-N3 activity and subsequent restoration of 13-catenin nuclear translocation to near control levels (in WT PS2 expressing H4 cells). Collectively, our study further supports a large body of evidence implicating COX-2 in AD, and indicates that N141I mutant PS2 may play an important role in AD pathogenesis via a COX-2 related mechanism. Supported by NIA grants AG13799, AG14239, AG14766 & Zenith Award from Alzheimer's Association to GMR

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• M O L E C U L A R MECHANISMS UNDERLYING M E M O R Y LOSS, SYNAPTIC PLASTICITY IMPAIRMENT AND NEURONAL DEGENERATION CAUSED BY LOSS OF PRESENILIN FUNCTION IN THE ADULT CEREBRAL CORTEX

Jie Shen* 1, Carlos Sanra 1, Se-Young Choi 2, Vassilios Beglopoulos 1, Seema Malkani 1, Dawei Zhang 1, B.S. Shankaranarayana Rao 3, Sumantra Chattarji 3 , Raymond J. Kelleher 4, Eric R. Kandel 5, Karen Duff 6, Alfredo Kirkwood 2. 1Harvard Medical School, Boston, MA, USA; 2Mind~Brain Institute, Johns Hopkins University, Baltimore, MD, USA; 3National Center for Biological Sciences, Bangalore, India; 4The Picower Center for Learning and Memory, MIT, Cambridge, MA, USA; 5Howard Hughes Medical Institute, Center for Neurobiology and Behavior, Columbia University, New York, NY,, USA; 6Nathan Kline Institute, Orangeburg, Ny USA. Contact e-mail: jshen@rics.bwh.harvard.edu

Background: Mutations in presenilins are the major cause of familial Alzheimer's disease. The pathogenic mechanism by which presenilin muta- tions cause memory loss and neurodegeneratiou remains unclear. Objective: We aim to elucidate the roles and identify the molecular targets ofpresenilins in the adult cerebral cortex, which is the most relevant experimental system for the investigation of the pathogenesis of Alzheimer's disease. Methods: We employed an advanced gene targeting approach to generate spatially- and temporarily-restricted conditional knockout mice lacking both presenilins in the postnatal forebrain. A combination of mouse behavioral, electrophysio- logical, biochemical, neuropathological and molecular biological techniques were then used to characterize the phenotypes of these mice at the sys- tems, cellular and molecular level. Conclusions: Conditional knockout mice lacking both presenilins in the postnatal forebrain exhibit impairments in hippocampal memory and synaptic plasticity prior to any neuropathological changes. These deficits are associated with specific reductions in responses of NMDA receptors, synaptic levels of aCaMKII, nuclear levels of CBP and expression of CREB/CBP target genes. With increasing age these mutant mice develop striking neurodegeueration of the cerebral cortex and worsening impairments of memory and synaptic function, accompanied by increased levels of the Cdk5 activator p25 and hyperphosphorylated tan. These results define essential roles and molecular targets of preseullins in synaptic plasticity, learning and memory, and neuronal survival in the adult brain. We propose that partial loss of presenilin function may contribute to memory loss and neurodegeneration in Alzheimer's disease.

~ 4 f ~ ABROGATION OF PRESENILIN FUNCTION INCREASES CAMP-DEPENDENT SYNAPTIC TRANSMISSION: INFLUENCE OF PRESENILIN-DEPENDENT PROTEOLYSIS OF DCC R EC EPTOR

Ang~le T. Parent* 1, Yoshihito Taniguchi 2, Natalie Y. Barnes 1, Sangram S. Sisodia 1 . 1 University of Chicago, Chicago, IL, USA; e Kyoto University School of Medicine, Kyoto, Japan. Contact e-mail: aparent@uchicago.edu

Background: Inheritance of mutant forms of Presenilin 1 (PSI) causes autosomal dominant, early-onset, familial Alzheimer's disease (FAD). PSI plays a role in multiple cellular functions including intramembranous gamma-secretase proteolysis of a set of type I membrane proteins, calcium homeostasis, and protein trafficking. FAD-linked PS 1 variants enhance the production of highly fibrillogenic Abetal-42 peptides. While the production and deposition of amyloid fibril correlate with the pathological cascades of FAD, we hypothesize that PSI dysfunction might also influence memory component(s) independent of amyloidogenesis. Objective(s): To assess the influence of PS 1 on cellular substrates of memory, we concentrated our efforts on glutamatergic syuaptic transmission and cAMP intracellular signaling cascades. We also explored the possibility that PS-dependent cleavage of deleted in colorectal cancer (DCC), a type I transmembrane protein known m play an essential role in taxon guidance and outgrowth, might influence the cAMP-dependent signafiug pathway. Methods: Primary cortical cultures were prepared from Wt and PS 1 knockout (KO) embryos. PS function was abrogated using highly selective inhibitors that impair

$562 Poster Session P4: Molecular Mechanisms of Neurodegeneration - Presenilins

PS-mediated gamma-secretase cleavage. Level of synaptic contact was examined using the pre and postsynaptic markers, Bassoon and PSD-95, respectively, cAMP signaling cascade was assessed using an antibody that recognizes cAMP-dependent phosphorylation of PKA substrates. PS- dependent influence of DCC-mediated cellular changes was examined in transfected N2a neuroblastoma cells. Results: KO neurons, as well as Wt neurons treated with PS gamma-secretase inhibitors, exhibit higher frequency of miniature excitatory post-synaptic current compared to Wt neurons. This increase is accompanied by increases in the number of pre and postsynaptic contacts and the magnitude of cAMP-dependent phosphorylation of PKA substrates. We confirm that the transmembrane domain of endogenous DCC is cleaved by PS garnma-secretase activity in neurons. Interestingly, accumulation of the transmembrane portion of DCC receptor in N2a cells treated with PS gamma-secretase inhibitor activates cAMP-dependent phosphorylation of PKA substrates and neurite outgrowth. Conclusions: Our findings indicate that abrogation of PS function produces accumulation of DCC transmembrane domain in neurons that might directly contribute to enhance cAMP/PKA signaling, an essential component of synaptic transmission and memory processes. Supported by NIH and the Alzheimer's Association.

• IDENTIFICATION AND CHARACTERIZATION OF NOVEL CLEAVAGE-DEFECTIVE PRESENILIN-1 MUTANTS

Masaki Nishimura* 1, Yoshifumi Nakaya 1, Takuya Yamane 1, Hua-Qin Wang 1, Hirohisa Shiraishi 2, Hiroto Komano 2, Etsuro Matsnhara 3 , Mikio Shoji 3 , Bart De Strooper 4, Paul Fraser 5, Peter St George-Hyslop 5 . 1Shiga University of Medical Science, Shiga, Japan; 2National Institute for Longevity Science, Aichi, Japan; 3 0kayama University, Okayama, Japan; 4Center for Human Genetics, KUL, VIB, Leuven, Belgium; 5University of Toronto, Toronto, ON, Canada. Contact e-mail: mnishimu@belle.shiga-med.ae.jp

Background: Presenilin-1 (PS 1)undergoes endoproteolysis yielding N- and C-terminal fragments that are assembled into a macromolecular complex. PS 1 complex is considered to be gamma-secretase which mediates amyloid precursor protein (APP) cleavage to generate amyloid beta-peptide (A- beta). It has been proposed that PS1 constitutes the catalytic component of gamma-secretase complex and PS 1 endoproteolysis is an autocatalytic event. Objective(s): To clarify the effect of PS1 mutations on PS1 endoproteolysis and gamma-secretase activity. Results and conclusions: We screened a randomly mutagenized PSI cDNA library to identify mutants with defect of their endoproteolysis. 33 cleavage-defective mutants were identified, of which 6 mutants bore single missense mutations. Analysis using HEK293 cells stably expressing mutant PS1 and APP revealed that PS1 mutants formed stable complexes and that 5 of 6 mutants showed the similar profile of gamma-secretase activity. Epsflon-cleavage of APP was clearly suppressed in these mutant cells. A-beta ELISA disclosed that the 5 mutant cell lines secreted more A-beta42/43 than WT PSI cells, whereas the secretion of A-beta40 was reduced or not changed. Expression of these mutants in PS-null ceils restored the predominant secretion of longer A-beta. The enhanced secretion of A-beta42/43 from these cells was refractory to treatment with difiuoroketone or snhndac sulfide type of gamma-secretase inhibitors. Our results indicated that certain missense mutations of PSI perturbed its endoproteolysis and simultaneously enhanced the secretion of longer A-beta species.

• FUNCTIONAL ANALYSIS OF NICASTRIN AND PEN-2 IN y-SECRETASE COMPLEX

Isoo Nofiko* 1,2, Tsurnoka Makiko 1 , Miyagishi Makoto 3, Taira Kazunari s , Tsuji Shoji 2, Tomita Talsuke 1 , Iwatsubn Takeshi 1 . tDepartment of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan; 2Department of Neurology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan; 3Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Tokyo, Japan. Contact e-mail: isoon-tky@umin.ac.jp

y-Secretase is an aspartyl protease that catalyzes intramembrane proteolysis of various type I transmembrane proteins, including ~-amyloid precursor protein (APP) to liberate amyloid-~3 peptide (A[3) that gets deposited in Alzheimer's disease (AD) brains. Four membrane proteins, presenilin (PS), nicastrin (NCT), APH-1 and PEN-2, are known as components of the protease complex. Overexpression of these components is sufficient for the reconstitution of the active y-secretase complex. We previously demonstrated the "stabilization and activation" model in y-secretase assembly: NCT and APH-1 stabilizes PS, and PS/NCT/APH-1 complex is activated by PEN-2 that confers the y-secretase activity and facilitates the formation of PS fragments. However, the precise functions of each component in y-secretase activity still remain unknown. To elucidate the molecular functions of NCT and PEN-2 in y-secretase, we focused on the domains conserved beyond species, and generated cDNAs encoding human NCT harboring amino acid substitutions in luminal domain and human PEN-2 carrying mutations within transmembrane domains. To abolish the effect of endogenous proteins that may affect those of mutant proteins, we established N2a cell lines stably expressing short interfering RNA (siRNA) targeted against the UTR region that down-regulate the expression of endogenous mouse NCT or PEN-2. The levels of endogenous proteins as well as PS 1 fragments were decreased and APP-stubs were accumulated in these cell lines. The analysis of the effects of mutant NCT or PEN-2 on the stabilization, localization and activity of y-secretase complex will be presented.

~ P S 1 SUPPRESSES APOPTOSIS AND PREVENTS TAU HYPERPHOSPHORYLATION VIA CADHERIN/PI3K SIGNALING; EFFECTS OF PS1 FAD MUTATIONS

Lia Bald* 1, Junichi Shioi 1, Paul Wen l, Zhiping Shao 1, Miguel Gama-Sosa 1 , Alexander Schwarzman 2, Rachel Neve 3 , Nikolaos K. Robakis 1 . 1Mount Sinai School of Medicine, New York, NY,, USA; 2State University of New York at Stony Brook, Stony Brook, NY,, USA; 3Harvard University, Belmont, MA, USA. Contact e-mail: lia.baki@mssm.edu

Background: The PI3K/Aki signaling pathway controls differentiation, communication and survival in a wide variety of cell types, including neurons. We obtained data showing that PS1, a protein involved in familial Alzheimer's disease (FAD), promotes cell survival by activating PI3K/Akt cell signaling: absence of PS1 correlates with impaired activation of the PI3K/Akt survival pathway and density-dependent apoptosis, whereas re-introduction of PS1 into PS1 null cells stimulates PI3K-dependent phosphorylation of Akt and rescues cells from apoptosis. This PS1 function is unaffected by y-secretase inhibitors but is abolished upon inhibition of PI3K activity, suggesting that PI3K is the mediator of the survival effect of PS 1. Objective(s): Explore the mechanism(s) and the consequences of this new y-secretase-independent function of PS 1 and determine the effect of PS1 FAD mutations on PI3K/Aki signaling. Methods: Fibroblasts derived from PSI+/+ and PSI- / - mouse embryos were used for transfection and infection experiments in the presence and absence of various inhibitors. Lysates were analyzed by co-immunoprecipitation or Western blotting. Results: Pharmacological and genetic approaches revealed that PS1 acts at a step at or upstream of PI3 kinase. Co-immunoprecipitation experiments revealed that PS1 forms a complex with the p85 regulatory subunit of PI3K and promotes its association with E- and N-cadhefins. Anti-cadherin function blocking antibodies that prevent cadherin/p85 association abolished the PSI-induced activation of PI3K/Akt signaling. Thus, PS1 stimulates PI3K/Akt signaling by promoting cadherin/PI3K association. To identify