Poster Presentation: Molecular and Cellular Biology IV S?61

m INTRINSIC FERROXIDASE ACTIVITY IN THE ALZHEIMER’S AMYLOID PRECURSOR PROTEIN

Jack Timothy Rogers, Robert D Moir, Xoudong Huang, MA Gen Hosp. Boston/

Charlestown. MA: Amanda Venti, Nabeil Sarhan, Brigham and Women’s Hasp,

Boston, MA: William E Van Nostrand, SUNY at Stony Brook, Stonybrook, NY; Rudolf

Tunzi, Bush A, MA Gen Hasp, Boston/Churlestown, MA

Amyloid p/A4 protein precursor (APP) is a transmembrane protein existing in several different alternatively spliced isoforms. Cleavage of the precursor generates the APpeptida which is a constituent of the neuritic plaques of Alzheimer’s disease patients. The brains of AD patients also demonstrate a disruption of iron homeostasis which may lead to iron-catalyzed oxidative damage by the Fenton reaction. Ferritin is an important iron storage protein which prevents such damage by oxidizing Fe* ’ to the more inocuoua Fe”+ form, and is thus cytoprotectivr. A subregIon of APP is aucturally homologous to the catalytic ferroxidase site in the H-ferritin subunit. Thus we tested purified secreted APP (APP(a)) for iron oxidase activity. We found l’erroxidase activity in both full-length sAPP and a 22 amino acid peptide of APP spanning the homology domain. Several proteins found in nature, such as ceruloplaa- min and the yeast protein Fet3, are copper-dependent for their ferroxidase activity. and as APP has been shown to possess a copper-binding site for the reduction of Cu2+ to Cult, copper-loaded APP samples were aho assayed as positive for ferroxrdaae activity. Our results correlate well with the evidence regarding the ability of 17-mer peptides encompassing APP sequences to “promote neutite extension“, reverse ivzhemic damage to the rabbit rpinal cord?. and increase memory retention in rats.

IMMUNE COMPETENT MACROPHAGES PRODUCE NEURO- TROPHIC FACTORS

Ewdence ruggests that ~nononuclear phagocytes (MP) (perivascular and parenchymal macrophagea, microglia and/or monocytes) play both neurotoxic and neuroprotective roles in neurodegenerative diseases and in response to central nervous system trauma. To determine the mechanism(s) for MP neurotrophlc (those supporting neuronal and/or ghal survival and/or differentiation) responses we studied cultured fluids from MP following exposure to CD40 ligand (CD4OL) (Immunex Corp) and human peripheral nerve (PN). Laboratory models for neuronal viability/function were developed. Rat pheochromocytoma cells (PCl2) and primary rat cortical neuron\ (RCN) were treated with monocyte, peripheral blood mononuclear cell (PBMC). and monocyte-derived macrophage (MDM) conditioned neurobasal media and analyzed for cell survival (metabolism of Thiazolyl blue or MTT assay). PC12 cell viabihty Ggnificantly increased above controls following treatment with conditioned media from monocytes. MDM and PBMC (p<O.OOI 1. Cell viability correlated with MAPK pathway activation (ERKI/ERK2 kinase assay). Conditioned media from monocytea,

MDM and PBMC with or without immune stimulation (peripheral nerve, CD4OL) significantly increased viability of RCN as compared to controls (piO.001). To determine the effects of conditioned media on neuronal cells and astrocytes, RCN lyaates were prepared following exposure to conditioned media and aswyed for astrocyte- or neuron-Fpecific proteins by We&tern blot. Conditioned media signifi- cantly increased both neuronal and astrocytic proteins. EnLyme-linked immunosor- bent assays (ELISA) of culture fluids thawed detectable levels of brain derived neurotrophic factor (BDNF), nerve growth factor (NGF). and interleukin 6 (IL-6) with and wthout CD40L or PN stimulation. Taken together, our results suggest that MP secretions may support neuronal survival, regeneration and recovery. These findings could have implications for the neuropathogenesis of AlLheimer’s disease where MP activation is a central pan of the pathologic proce\\.

)11971 THE LENGTH OF THE TRANSMEMBRANE DOMAIN OF APP IN

MICROSOMAL MEMBRANES IS SHORTER THAN PREDICTED.

Grziwu Kutharinn Beate, Heikr Subine Grimm. Joerg Thomnns Regulu. ZMBH, Univ

of Heidelbrrg. Heidelberg Grrmuny; Stefnn Friedrich Lichtenrhulcr. MGH, Dept

Molec Biology, Boston, MA

Proteoloytic processing of the Amyloid Precursor Protein (APP) by p-aecretase leads to CY9 which is further cleaved by the proteaae activity called y-secretase. y-secretaw cleavage occurs inside of the transmembrane domain (TMD) and generates the C-terminus of the AP-peptide. Previous studies suggested an influence of the length of the TMD on the preferred cleavage site of y-secretaae. However, so far the length of the TMD has been predicted by hydropathy algorithm\ and has never been biochemically defined. We determined the actual length and exact position of the TMD of APP in microaomal membranes in order to better understand the mechanism of y-secretase cleavage. We created 19 single cyst&e mutants of C99 with a cysteine placed at different positions within and close to the predicted TMD. Using in vitro

translation, the APP mutants were inserted into microsomal membrane,. By employ_ ing a membrane-unpermeable cysteme-specific labelling reagent, only tho,c mutants with 8 cysteme placed outside of the membrane were modified. Cyst&e n,odlflcation was analyzed on Tris-tricine and 2D-gels. We found that m mtcrosomal membranes, which are derived from the ER membrane, the TMD of APP is at both, its N. and C-terminal side shorter than predicted. The results obtained are in good agreement with the average length of the TMD of proteins typically residing m the ER. Additionally, we show that the middle of the experimentally defined TMD of APP inserted in microsomal membranes is positioned more C-terminally than predicted. The data show that APP inserted into thin membranes exposes some of the residues that were believed to be hidden in the membrane to potential binding partners and to cleavage in a hydrophilic environment. However, y-secretase cleavage sites AP40/42 were always buried inside of the membrane. The influence of the membrane thickness on the length and position of the TMD of APP and on y-sea&w cleavage specifity will be further analyzed.

STRUCTURAL DOMAIN REQUIREMENTS FOR PRESENILIN ENDOPROTEOLYSIS AND AB42 OVERPRODUCTION.

Jar Yoon Lrrm, Cnrlos Sauru. UniL, of Chrcago, Chicago. IL; Toi.wke Tomito,

Takeshr Iwatsubo, Univ of Tokvo, Tokw Jupan: Gopnl Thinakuran. Univ of Chicugo,

Chicago, IL

Pressnilin I(PSl), mutated in pedigrees of early-onset familial Alzheimer’s disease- (FAD), is a polytopic integral membrane protein that is endoproteolytically cleaved into stable N-terminal and C-terminal fragments. Little ia known about the proteolytic cleavage of PS and the domains of PS that are essential for the pathogenic function of mutant PS. We analyzed the structural domains of PSI that are essential for its metabohsm and the gain of function properties of FAD-linked PS variants, by generating a series of experimental deletion mutant\ We expressed each deletion mutant in stably transfected mouse N2a neuroblastoma cell lines and examined PSI endoproteolysis and replacement of mouse PSI derivativea. We also analyzed APP processing by I’%] methionine labeling followed by immunoprecipitation of APP metabolites with APP C-terminal and Apantibodies. The levels of A@40 and AP42 in the conditloned medium of cultured cell lines were quantified hy two-site ELISA assay. These analyses revealed that the introduction of pair-wise deletionc of the transmembrane(TM) domains within the N-terminal region Impairs PSI endoprote- olysis. Furthermor, A@42 overproduction by the corresponding FAD-linked variants wa\ al\o impaired hy TM deletions. Our studies of PSI deletion mutants also uncovered a dixordancc between ADproduction and the accumulation of APP C-termmal fragments. We are presently characterizing the subcellular localization of PSI deletion Inutantb. From these wdies, we conclude that the N-terminal TM domain\ are esxntial for PS rndoproteolysl\ and Ihe effect of Imutant PS on APP proce55ing.

(11991 WITHDRAWN