Poster Session P4: Molecular Mechanisms of Neurodegeneration - Presenilins $553 e•-•2] PRESENILIN- RELATED PUTATIVE INTRAMEMBRANE ASPARTYL-PROTEASES Evgeny I. Rogaev* 1.2 Yuri K. Moliaka 3, Anastasia Grigorenko 4,5.1 Univ. of Massachusetts, BNR1, Worcester, MA, USA; 2Research Center of Mental Health, Fac. of Bioengineering and Bioinformatics, Lomonosow Moscow State Univ., Moscow, Russian Federation; 3 Univ. of Massachusetts Medical School, BNRI, Worcester, MA, USA; 4Research Center of Mental Health, Academy of Medical Sciences, Moscow, Russian Federation; 5BNRI, Univ. of Massachussets Medical School, Worcester, MA, USA. Contact e-mail: evgeny.Rogaev @umassmed.edu Background: We and others described presenilin-related families of pro- teins. We designated this abundant type of polytopic proteins in enkaryotes as IMPAS (IMP) (Rogaev et al, 2001; Grigorenko et al, 2002), termed by others also as PSH or SPP/SPPL (Ponting et al, 2002; Weihofen et al, 2002). The SPP/IMP1 cleaves short signal peptide remnants, but the function of IMPs in vivo is unknown. Objective(s): We searched for putative substrates of IMP proteolytic activity and functional, structural and pharmacological identities between IMPs and homologous PSs (presenilins). The role of IMPs in AD has to be examined. Methods and Results: First, we found that vertebrate IMPAS is a more diverse protein family than PSs. C.elegans imp-l, imp-2 are orthologous to different members of human IMPs and imp-3 is a more distant IMP-prototype. Second, we cloned and expressed in mammalian cells five paralogous IMP1-5 genes. The proteins are de- tected by immunoblots as modified holoproteins co-interacting also in high molecular-weight associates. We identified three domains with invariant structural signature between all eukaryotic IMPs, PSs and Archaea and bac- terial peptidases D-6GxGD-PxL. Missense-mutations were introduced in the conserved residues of IMPI (SPP) gene. To identify substrates of IMP1 the cellular assay with co-transfected proteins was employed. We identified that IMP1, but not IMP4, induced cleavage of wild and mutant isoforms of PS1 holoprotein in this assay. The proteolytic activity was modulated by some mutations in IMP1; it was completely abolished by mutations in D265A, D219A and P317L, but not G264 (AD-mimic mutation). Specific gamma- secretases suppressed IMPl-induced cleavage of PS1. The genetic role of SNPs in two IMP genes in AD have been examined. Conclusions: The data suggest IMPI is a bi-aspartyl-like protase which is affected by mutations and some inhibitors in a manner similar to PSs. The multipass transmemhrane proteins may be potential substrates for IMP- cleavage. The potential role of such proteolysis in vivo has to be studied. IMP1,2,3,4 genes have overlapped pattern of expression in human tissues (retroposon-like IMP5 is modestly expressed in pancreas only) and similar protein characteristics. Nevertheless, these proteins presumably have diverse functions and proteolytic activities. ~-3] ANALYSIS OF PRESENILIN COMPLEX BY BLUE NATIVE ELECTROPHORESIS AND MASS SPECTROMETRY REVEALS LINKAGE TO CYTOSKELETAL ELEMENTS Janetta G. Culvenor* 1,2, Shane B. Reeve 3, A.I. Smith 3, Colin L. Masters 1,2.1Th e University of Melbourne, Parkville, Vic, Australia; 2Mental Health Research Institute of Victoria, Parkville, Vic, Australia; 3Baker Heart Research Institute, Melbourne, Vic, Australia. Contact e-mail: janetta @ unimelb, edu.au The presenilin (PS) complex is a multi-protein protease critical in generation of the amyloid beta peptide by controlled intramemhrane cleavage of the amyloid precursor protein. We have applied Blue Native gel electrophoresis (BN-PAGE) to analysis of the complex from brain membranes. We reported that the complex from mouse and human brain migrates at 400 kDa after solubilization in 0.5% dodecyl-maltoside. Following gel excision, electroe- lution, and second dimension denaturing electrophoresis, we detected PSI, PS2, APH-lb, and PEN-2 from the complex by western blotting with specific antibodies [1]. More recently we have used BN-PAGE for semi-purification of the complex from mouse embryonic brain. Preparative BN-PAGE was used to isolate the PS complex gel region, proteins were electroeluted under native conditions, and fractionated by SDS Tris-tricine PAGE. After silver staining, bands were digested with trypsin, and analysed by MALDI-TOF mass spectrometry for mass peptide fingerprinting. Information to date in- dicates further evidence for cytoskeletal linkage. We have identified [3-actin, c~-tubulin, [3-tubulin and KIF3A with the complex. KIF 3A is a component of the kinesin-II motor complex associated with intracellular transport of membrane-bound organelles and anterograde axonal transport in neurons and has been reported to accumulate with PS 1 proximal to the neuronal cell body following axonal ligation in vitro [2]. This is consistent with previous evidence of PS interaction with cytoskeletal associated proteins namely CLIP-170 (linking membrane organelles to microtubules), and ABP-280 (cross-linker of actin and between actin and glycoproteins) [3]. Supported by the Australian NHMRC. References: [1] Culvenor J.G. et al., (2004) Eur. J. Biochem. 271,375. [2] Kamal A. et al., (2001) Nature, 414, 643. [3] Van Gassen G., et al., (2000) Neurobiol. Dis.7,135. ~2-~ GENE EXPRESSION PROFILING IN THE HIPPOCAMPUS OF PRESENILIN 1 KNOCKOUT (PS1/KO) MICE J.G. Cui* 1, y. Zhao 1 P.E. Fraser 2, P. St George-Hyslop 2, D. Westaway 2, W.J. Lukiw I 1Neuroscience and Ophthalmology, Louisiana State Unh:ersity Health Sciences Center, New Orleans, LA, USA; 2Centre for Research into Neurodegenerative Disease, University of Toronto, Toronto, ON, Canada. Contact e-mail: wlukiw@lsuhsc.edu Background: Mutations in presenilin-I (PSI) genes are a major cause of early-onset Alzheimer's disease (AD). Within lipid raft domains of neural membranes, PS1 in concert with nicastrin and beta amyloid precursor protein (A[3PP), orchestrate cleavage of A~PP into ragged amyloidogenic A[5 peptides. Objectlve(s): To further investigate the roles of PS1 gene expression in brain function, the hippocampus of 6 month old PS 1/KO mice, were collected and analyzed using DNA arrays containing oligonucleotide targets for 12488 murine genes and expressed sequence tags. Methods: Hippocampi derived from 129/Sv x C57BL/6J PS 1/KO strains were rapidly processed and total RNA was extracted using phenol-guanidine isothio- cyanate reagents and profiled using RNA 6000 Nano LabChips (Caliper Technologies, Mountainview, CA; Agilent Technologies, Palo Alto, CA). eDNA and biotinylated antisense cRNA were synthesized using the Super- script Choice System (Invitrogen, Carlsbad, CA) and Enzo Biorray High Yield RNA Transcript Labeling kits (Affymetrix, Santa Clara, CA). After hybridization against U74Av2 GeneChips (Affymetrix), DNA arrays were scanned at 570 um and features were extracted using Data Mining Tool 3.0 (Affymetrix) and Genespring 4.1.5 (Silicon Genetics, Redwood City, CA) bioinformaties algorithms. Results: Total brain RNA exhibited very high spectral quality with A260/A2s 0 and 28S/18S typically > 1.9 and > 1.4, respectively. Multiple changes in highly significant (p<0.05) neuro-specific gene expression levels were quantified. Strongly up-regulated genes in- cluded glial-specific markers such as glial fibrillary acidic protein (GFAP; Target/GenBank 94144g_at/X02801), glycerol phosphate dehydrogenase (GPDH; 92592_at/M25558) and S100[~ protein (101467_at/L22144). A[3PP (93063_at/U82624) gene expression was found to be down-regulated. Con- clusions: Gene expression patterns consistent with loss of control of glial cell division, expansion of glial-specific gene markers and compromised AI3PP expression are implicated in adult PS1/KO mice. This suggests roles of PSI in neuronal-glial cell-cell contact and in the regulation of glial cell proliferation, both of which may initiate or exacerbate pathological changes associated with AD brain. Supported in part by the Canadian Institutes of Health Research and NIA AG18031. • APH-1A IS REQUIRED FOR PRESENILIN-DEPENDENT y-SECRETASE ACTIVITY DURING MOUSE EMBRYOGENESIS Guojun Ma*, Tong Li, Donald L. Price, Philip C. Wong. Johns Hopkins University, School of Medicine, Baltimore, MD, USA. Contact e-maiL" gma2 @jhmi.edu Recent studies indicate that Aph-1 along with nicastrin and Pen-2 are essential components of the presenilin-dependent y-secretase complex.