Poster Session P4: Molecular Mechanisms of Neurodegeneration - Presenilins $551

trafficking or presentation of substrates to the "authentic" protease which maybe a molecule distinct from PS1. Objeetive(s): To determine if PSI alone can modulate y-secrtetase activity in insect cells. Methods: The baculovirus-insect expression system was used to co-express: (i) APP-C99; (ii) a pathogenic, constitutively- active mutant form of PS 1 lacking exon 9, (PS1AE9); (iii) nicastrin and (iv) tropomyosin in Spodoptera frugiperda (Sf9) cells. Results: Cells infected with APP-C99 alone produced an Alg- like species, and levels of this species were enhanced by the addition of baculovimses bearing the PS1AE9 mutation. The addition to APP-C99- infected cells of baculoviruses bearing nicastrin, also a transmembrane protein, had a neutral or inhibitory effect on the reaction; tropomyosin viruses had the same effect as nicastrin viruses. Conclusions: These results suggest that PS1AE9 molecules expressed in Sf9 cells retain the ability to modulate A~ levels. The baeuloviral-insect cell system can now be used to assess the ability of the other components of the y-secretase complex to modulate A[3 levels. Furthermore, generating point mutations in key residues of these candidate proteins and assessing y-secretase activity in insect cells will provide insight into the catalytic site (s) responsible for A[3 production.

~ BRAIN TRANSCRIPTOME IN CHANGES PRESENILIN-1 MUTANT M I C E

Karoly Mimics* 1, Zeljka Korade Mimics 2, Carmel F. Portugal a , David Terrano 3, Orly Lazarov 3 , Katherine C. Douglass 1 , David A. Lewis 1, Nina E Schor 2, Sangram S. Sisodia 3. 1Psychiatry, University of Pittsburgh, Pittsburgh, PA, USA; 2Pediatrics, Universitu of Pittsburgh, Pittsburgh, PA, USA; 3 Center for Mol. Neurobiology, University of Chicago, Chicago, IL, USA. Contact e-mail: karoly+@pitt.edu

Presenilins are highly homologous, ubiquitously expressed polytopic mem- brane proteins that play an essential role in intramembranous processing of amyloid precursor protein (APP), leading to the production of A~3 peptides. Familial forms of early-onset AD (FAD) are caused by the expression of mutant presenilin 1 (PSI) or presenilin 2 (PS2). Transgenic mice (TG) expressing the FAD-linked PS 1 Exon 9 deletion (AE9) variant fully "re- place" the endogenous mouse PS 1 and show enhanced production of A~42 peptides. Using DNA microarrays, we performed a transcription profiling of the hippocampus (HC) and frontal cortex (FC) of AE9 mutant mice and matched controls (TG mice that express human wild-type PS1). Of the >20,000 tested genes, we identified 93 genes that showed significant expression changes in both the HC and FC of AE9 TG mice. In parallel, we explored transcription profiles in brains of mice with conditional ablation of PS 1 expression in the forebrain. Comparing the HC and FC of the PS 1 KO mice to matched wild type controls led to the identification of 79 genes with significantly changed expression in both brain regions. When the AE9 TG and KO datasets were cross-compared, the vast majority of the common expression alterations were in changed in the opposite direction (16/19 for HC and 21/26 for FC). The most intriguing microarray-reported expression changes were verified by in situ hybridization. From the combined datasets we conclude that 1) the human mutant PS1 has a strong influence on the brain transcriptome; 2) PSi-dependent transcriptome changes show a high degree of similarity across HC and FC and 3) combining transcriptome data from PS 1 transgenic and PS 1 conditional KO animals is a useful strategy to identify transcript networks that are modulated by PS1. Supported by NARSAD Young Investigator Award (KM), RAC CHP and PIND (ZKM), Craumer Endowment of CHP (NFS) and EUison Medical Foundation (SSS, OL, DT).

• FUNCTIONAL ANALYSIS OF CAENORHABDITIS ELEGANS SPE-4 EXPRESSED IN HUMAN CELLS

Aya Yamasald *1 , Masayasu Okochi 2, Chi'istian Haass 1 Harald Steiner 1. 1Adolf-Butenandt-lnstitut, Department of Biochemistry, Laboratory for Alzheimer's and Parkinson's Disease Research, Ludwig Maximilians University, Munich, Germany; 2Department of Post-Genomics and Diseases, Division of Psychiatry and Behavioral Proteomics, Osaka University Graduate School of Medicine, Osaka, Japan. Contact e-mail: Aya. Yamasaki@pbm. med. uni-muenchen.de

Background: y-Secretase is an aspartyI protease complex that catalyzes the intramembrane cleavage of APP and other type I transmembrane proteins. Reconstitution studies have demonstrated that the active y-secretase complex consists of presenilin (PS), which carries the active site, nicastrin (NCT), APH-1 and PEN-2. Among the three PS homologues in C. elegans, SPE-4 is the most distant one and implicated in protein transport or sorting during spermatogenesis. Despite its limited sequence identity with PSI, the active site aspartates in transmembrane domains (TMD) 6 and 7 are conserved indicating a proteolytie function for SPE-4. Interestingly, when expressed in human cells, SPE-4 is not incorporated into a y-secretase complex. Objeetive(s): ]b address the question whether SPE-4 is a protease and to map functional domains of PS1 required for y-secretase complex formation and activity we exchanged domains of SPE-4 with those of PS 1. Methods: We have generated HEK293 cells stably expressing SPE-4/PS1 chimera. Results: Since the PSI C-terminus is required for y-secretase complex formation, we first generated SPE-4/PSlc, in which the SPE- 4 C-terminus was exchanged with that of PSI. Although expression of SPE-4/PSlc led to replacement of endogenous PS, it allowed only partial y-secretase complex formation and thus inhibited ¥-secretase activity. SPE- 4/PS1¢ co-immunoprecipitated with immature NCT and APH-1 but not with PEN-2, which was dramatically downregulated. To restore this defect we substituted the N-terminus of SPE-4 up to the fifth TMD with the corresponding region of PS1 thus generating SPE-4/PS1Ns/C. Expression of SPE-4/PSINs/C restored normal PEN-2 levels and NCT maturation. Despite normal v-secretase complex formation, v-secretase activity was still inhibited. Since the chimeric proteins were not endoproteolyzed we additionally exchanged the SPE-4 large loop domain with that of PS1, generating PS1/SPE-46/7, a chimera with TMDs 6 and 7 as the sole SPE-4 domains. PS 1/SPE-46/7 underwent endoproteolysis and displayed normal V- secretase activity. Conclusions: Our results suggest that first, the C-terminus of PS1 is required for binding to NCT and APH-1, second, the N-terminal domain of PS1 is required to interact with PEN-2, third, activation of the y-secretase complex i"eqaires PS endoproteolysis and finally the SPE-4 active site is functionally conserved.

• PRESENILIN MODULATES PEN-2 LEVELS POST-TRANSLATIONALLY BY PROTECTING IT FROM PROTEASOMAL DEGRADATION

Adam S. Crystal*. University of Pennsylvania School of Medicine, Philadelphia, PA, USA. Contact e-mail: acrystal@mail.med.upenn.edu

Background: The y-secretase complex is composed of four requisite components: presenilin (PS), Pen-2, Aph-1 and nicastrin. Together these proteins function to cleave various substrates including Notch, APP and N- cadherin. The y-secretase complex is therefore central to the pathogenesis of Alzheimer's disease, as well as normal development and physiology. Understanding the role of each protein will advance the understanding of y-secretase function, and thus a variety of pathogenic and physiological processes. The role and regulation of Pen-2 in y-secretase assembly, maturation and function is unclear. However, in the absence of PS, Pen- 2 protein levels are significantly and uniquely reduced. Objective: To determine the mechanism(s) by which PS regulates Pen-2 protein levels. Methods: We studied the effect of presenilin expression levels, as well as proteasome inhibition, on Pen-2 protein in cell culture and a mouse model. We used semi-quantitative rtPCR to measure the effect of the genetic absence of PS on Pen-2 mRNA levels. We have begun to analyze mutated Pen-2 proteins in an attempt to understand the critical domains and