Poster Session P4: Molecular Mechanisms of Neurodegeneration - Presenilins $563

the consequences of PSI-mediated activation of PI3K/Akt signaling we examined the effects of wild-type and mutant PS1 on Akt-dependent phosphorylation of glycogen synthase kinase-3 (GSK-3) isoforms alpha and beta, and on GSK-3-dependent phopshorylation of tau. Our data show that PS1 promotes Akt-dependent phosphorylation/inactivation of both glycogen synthase kinase-3 (GSK-3) isoforms and suppresses GSK- 3-dependent phosphorylation of tau at residues overphosphorylated in AD. In contrast, PS1 FAD mutations inhibit the PSI-dependent activation of the PI3K/Akt pathway, thus preventing GSK-3~3 phosphorylation and promoting tan overphosphorylation and caspase-3 activation. Conclusions: Our data show that PS1 may prevent development of AD pathology by promoting cadherin/PI3K association and activating the PI3K/Akt signaling pathway. In contrast, FAD mutations may promote AD pathology by inhibiting this pathway

~ T H E C-TERMINAL RESIDUES OF PEN-2 MODULATE THE PROTEIN INTERACTION IN GAMMA-SECRETASE COMPLEX

Hiroshi Hasegawa*. CRND, University of Toronto, Toronto, ON, Canada. Contact e-mail: hiroshi.hasegawa@utoronto.ca

Background: PEN-2 identified as a modifier of sel-12 or hop-1 by the genetic screening in C. elegans is revealed to be involved in the presenilin complex. In addition, PEN-2 plays pivotal roles in the maturation of the presenilin complex, such as the nicastrin maturation and presenilin endo- proteolysis, as well as gamma-secretase activities. However, the mechanism of the interaction and the functional domains remain unclear. Objective(s): To investigate the domains in PEN-2 responsible to the protein interaction and the maturation of the complex Method(s): The successive deletion mutants at the C-terminus were studied with immunoprecipitation and glyc- erol velocity gradient fractionation for the protein interaction analysis. In addition, the effect of the mutants on the maturation of the components was analyzed by the co-transfection with presenilin, nicastrin, APH-1 and either wild-type PEN-2 or mutant PEN-2 cDNA. Results: The deletion analysis at the C-terminus of PEN-2 in transient or stable transfectants revealed the critical sequence for the interaction with presenilin, nicastrin or APH-1. The substitution mutant to alanine and the deletion mutant at this domain also modulated the interaction significantly. In the glycerol velocity gradient analysis of the stable transfectant, these mutations affected the fracfiona- tion pattem. On the other hand, both mutant and wild-type PEN-2 were localized in Golgi apparatus and ER by the iodixanol fractionation. Thus, the subcellular localization is unlikely to affect the protein interaction. In addition, the co-transfection of four components including wild-type PEN-2 showed the abundant nicastrin maturation and the increase in the presenilin fragments, while this maturation process was significantly modulated in PEN-2 mutants. Conclusions: The C-terminal residues of PEN-2 play an important role in the interaction with the components in the presenilin complex and the maturation of both nicastrin and presenilin.

•P4f• PRESENILIN 2 FAMILIAL ALZttEIMER'S DISEASE MUTATIONS DECREASE A[~ AND CTF), WHILE DIFFERENTIALLY AFFECTING NICD PRODUCTION

Emily S. Walker*, Maribel Martinez, Anne Brunkan, Silva Hecimovic, Jun Wang, Alison Goate. Washington University, St. Louis, MO, USA. Contact e-mail: eswalker@ icarus, wustl, edu

Background: Certain mutations in the presenilin genes (PS 1 and PS2) are associated with early-onset familial Alzheimer's disease (FAD). To date 133 familial Alzheimer's disease (FAD) mutations have been identified in PS1 and 9 FAD mutations have been identified in PS2. The presenilins are necessary for the y-secretase cleavage of Notch to N[3 and NICD, and amyloid precursor protein (APP) to A~3 and CTFy. The major A~ isoform is 40 amino acids long, however, a small percentage is 42 amino acids long, and FAD mutations increase the ratio of A~342/AI340. Objective: Our objective was to determine if FAD mutations in PS2 affect production of

A~3, CTFy and NICD in addition to affecting the ratio of A~42/A[340. Methods: We tested 8 reported PS2 FAD mutations (R62H, T122P, $130L, N141I, V1481, M239V, M239I, D439A) and two PS1 FAD mutations at the residue homologous to PS2 M239 (PS1 M233T/V). All experiments were performed in PS 1/2 knock-out mouse embryonic fibroblasts transiently transfected with PS2 and APP or Notch cons~ucts. Results: PS2 R62H, $130L, V148I and D439A had no effect on the A~42/AI340 ratio while T122P, N141I, M239V and M239I increased the ratio greatly. These latter four PS2 FAD mutations caused decreases in both A~ and CTFy production. While most of the mutations tested had a similar effect on CTFy and NICD production, the PS2 M239 and PS 1 M233 mutants had a differential affect. Conclusions: It is unlikely that R62H, S130L, V148I or D439A are pathogenic FAD mutations since they do not affect the A[542/A~40 ratio significantly. Interestingly, most PS2 FAD mutations we examined severely affected AI3, CTF 7 and NICD production. This is in contrast to most PS1 FAD mutations we have studied which have little affect on CTFy or NICD levels - this suggests that PS2 FAD mutations have a more severe effect on function. Finally, FAD mutations at PS2 M239 and PS1 M233 are the only PS mutations we have studied or seen published that decrease CTFy production without affecting NICD production. Tl~is residue could be important for an interaction with APE

~ R O L E OF PRESENILINI-fl-CATENIN INTERACTION IN EMBRYONIC DEVELOPMENT

Li Liu* 1, Joanna Jankowsky 2, Anna Kordylewska 1 , Margaret Veselits 1, Manuel Utset 1, David Borchelt 2, Gopal Thinakaran 1.1Department of Neurobiology, Pharmacology and Physiology, The University of Chicago, Chicago, IL, USA," 2Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, MD, USA. Contact e-mail: liliu @ bsd. uchicago, edu

Background: Mutations in the gene encoding presenilin 1 (PS1) account for the majority of early-onset familial Alzheimer's disease cases. PS1 comprises eight transmembrane domains and a large hydrophilic cytoplasmic loop (HL) domain. PS1 is an essential component of y-secretase, which generates [5-amyloid peptide by proteolysis of APP and releases Notch intracellular domain by proteolysis of Notch receptor. In addition, PS1 also interacts with ~-eatenin via its HL domain, and absence of PS1 expression leads to stabilization of free cytosolic f3-catenin. ~-catenin is a key component in Wnt-signaling that is essential in mammalian embryonic development. However, due to early developmental defects associated with loss of Notch function, it was not possible to study the role of PSl-~-catenin interaction in embryonic development using PS1 null mice. Objective: To investigate the role of PSl-[5-catenin interaction in mammalian embryonic development. Methods: We have generated transgenic mice overexpressing a human PS 1 deletion mutant (PS 1AHL) lacking the HL domain (Glu304- Gly371) under the control of tTA responsive promoter. The morphology of PS1AHL/tTA transgenie and non-transgenic embryos was studied at various stages of embryogenesis by using histological analyses and whole- mount in situ hybridization. Results: Our preliminar3r studies show that embryonic expression of PS1AHL transgene leads to perinatal lethality in the PS1AHL/tTA transgenic mice. In contrast, transgenic offsprings are viable when PS1AHL expression is suppressed during development by inclusion of doxycycline in the diet. Gross morphological examination did not show any differences between PS1AHL/tTA transgenic and non- transgenic embryos in the length of body axis and tail. Normal patterning of axial skeleton, and normal somite segmentation were revealed by histological analyses in PS1AHL/tTA transgenic embryos by El9. In addition, none of the PS 1AHL/tTA transgenic embryos examined showed intracranial hemorrhage. Together, none of the characteristic Notch-related developmental defects reported in PS1 - / - mice was found in PS1AHL/tTA transgenic embryos. Conclusion: Results of our investigation support a Notch-independent role for PS 1 during late stages of mammalian embryonic development, in which PS 1-[3-catenin interaction may be involved.