FOURTH INTERNATIONAL CONFERENCE ON ALZHEIMER’S DISEASE
or in early endosomal vesicles. We propose that several distinct a-secretascs are able to release APP which may account for the contrary findings reported before concerning the cellular compartment of a-secretase activity.
PRECUSOR-LIKE PRCYl’EiN 2 (APLP2) LS A
CHONDROITJN SULFATE PROTWGLYCAN:
IDENTIFICATION OF THE GLYC~AMINOGLYCAN MODIFICATION SITE. G. Thinakaran and S.S. Sisodia. Department of Pathology and the Neuropathology Laboratory, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.2196 USA.
266 POLARIZED SECRETION OF B-AMYLOID PRECURSOR PROTEIN AND AMYLOID-B PEPTIDE IN MDCK CELLS. C. Haass, E.H. Koo, D.B. Teplow and D.J. Selknc. Center for Nemologic Diseases, Brigham and Women’s Hospital, Harvard Medical School, Boston MA 02115, USA. The Ramyloid precursor protein (OAPP) is a widely expressed integral membrane protein which is proteolytically processed to yield several secreted derivatives, including soluble APP (APP,), the 4 kDa amyloid-l3 pcptide ([email protected]
and a related 3 kDa peptide (~3). To understand DAPP trafficking and processing, we attalyxcd the sorting of DAPP in Madin-Darby canine kidney (MDCK) cells, an epithelial cell known to possess physiologically distinct apical and basolatcral plasma membranes. Processing of LlAPP resulted in highly polarized secretion of APPs: more than 90% of APP, was detected in the basolateral compartment and less than 10% in the apical compartment. This was associated with a similar pmfcmntial localization of DAPP on the basolateral cell surface. Al3 and p3 peptides were also secreted predominantly basolaterally. In order to investigate cytoplasmic signals for basolateral sorting of MPP, we performed a C-terminal deletion analysis. Deletion of the last 32 amino acids (ending at aa 663) of the cytoplasmic tail of LIAPPggg,including the potential reintemalization signal NFfY, did not change the polarized sorting of OAPP and its protcolytic products. However, deleting the last 42 amino acids of the cytoplasmic tail (ending at position 653) resulted in increased localization of gAPP on the apical cell surface. Interestingly, this switch was not accompanied by any change in the basolateral secretion of APP,. We therefore postulate that most APPs molecules in MDCK cells arc generated before OAPP reaches the cell surface. Indeed, intracellular APPs was recovered by sodium carbonate extraction from isolated membrane vesicles. Therefore MDCK cells contain two populations of full-length DAPP molecules. The predominant population gives rise to intracellularly cleaved APPs which is subsequently secreted almost entirely basolaterally, whereas a minor second population of molecules results in cell-surface inserted OAPP, the polarized trafficking of which is regulated by signal(s) in the cytoplasmic tail.
Alzheimer’s disease is pathologically characterized by the deposition of Bamyloid in senile plaques and blood vessels. B-amyloid is a -4 kD peptide derived from larger amyloid precursor proteins (APP). Recent studies have indicated that APP is a member of a larger gene family that includes amyloid precursor-like proteins APLPl and APLPZ. We have examined the metabolism of mouse APLPZ in stably-transfected CHO cells and transiently-transfected COS-1 cells. The kinetics of maturation of APLP2 and secretion of soluble APLPZ-derivatives are consistent with trafftcking in the constitutive secretory pathway, similar to that described for APP. However, in sharp contrast to the posttranslational modification of APP in CHO and COS-1 cells, APLPL is modified by glycosaminoglycan (GAG). GAG-modified APLPZ is sensitive to digestion with chondroitinase AC indicating addition of chondroitin sulfate (CS) chains. Analysis of chimaeric APLP2 polypeptides revealed that CS GAG modification of APLP2 occurs in a region with little sequence homology to APP. Notably, this region contains a consensus CS GAG attachment site in the stretch ENEGSGMAEQ; the serine residue (amino acid 614 of APLPZ) was predicted to undergo CS GAG modification. We have documented that APLPZ polypeptides harboring a serlne to alanine substitution at position 614 fail to undergo CS GAG modification. Our observation that APLPZ and APP are modified by distinct biochemical pathways suggests that the two molecules may be functionally divergent. This work was supported by grants from the National Inst. of Health, the Robert L. and Clara G. Patterson Trust and the Adler Foundation.
TACRINE ALTERS THE PROCESSING OF ALZHEIMER’S BETAAMYLOID PRECURSOR PROTEIN IN CELL CULTURES. D.K.Lahiri, SLewis and M.R.Farlow. Laboratory of Molecular Neuro genetics, Institute of Psychiatric Research, Department of Psychiatry and Neurology, Indiana University School of Medicine, Indianapolis, IN 46202.
TRAFFICKING AND F’ROCBggING OF AMYLOID PRECURf4OR CELLS. A.C.Y. Lo’, PRUIEIN (APP) IN POIARIZED m CL. Chen s, C. Haass I, D. Teplow r, and S.S. Sisodia ‘. Departments of Pathology’ and Neuroscience’, The Johns Hopkins School of Medicine, Baltimore, Maryland 21205, and Center for Neurologic Diseases’, Harvard Medical School, Brigham & Women’s Hospital, Boston, MA 02115. Alzheimer’s Disease (AD), a progressive neurodegenemtive disease, is pathologically characterized by the presence of senile plaques (SP) in neoconex, amygdala, and hippocampus. The principal component of SP is Aa. derived from APP. In earlier studies, we documented that APP is rapidly transported in rat peripheral nerves, suggesting that APP is selectively trafftcked in highly polarized cells. Since neurons are experimentally intractable to most cellular and biochemical investigations, we examined the trafficking and processing of APP in a polarized epithelial cell line (MDCK). We examined APP maturation in MDCK cells stably tmnsfected with either wild type human APP-695 cDNA (line APPWT-1) or APP695 cDNA that harbors the Swedish double mutation associated with early-onset AD (line APPSWE-1). We document that >9C% of soluble APP derived from APP695-WI is secreted into the basolateral compartment following cleavage at the a-serretase site. On the other hand, we document that a fraction of soluble APP dedvatives from APPSWE-1 cells is cleaved near, or at, the N-terminus of A8 (presumably at the g-secretase site) and that these forms are predominantly secreted into the apical compartment. Furthermore, a membrane-bound C-terminal fragment that contains the entire ABpeptide is elevated in lysates of APPSWE-1 cells. Using domain-specific biotinylation and release assays, we failed to detect &cleaved soluble APP derivatives generated from cell surface APPs in the medium of APPSWE-1 cells. Our data is consistent with S-secretase cleavage occuring in an intracellular compartment. Finally, we demonstrate that AB is preferentially secreted into the basolateml compartment from APPSWE-1 cells. Remarkably, while previous studies indicated that the majority of &related species secreted from APPWT-1 cells contains an N-terminal arginine (+5), the majority of A&related molecule derived from APP695-SWE contains an N-terminal aspartate residue (+l).
The predominant features of Alzheimer’s disease (AD) am the presence of B-amyloid-containing plaques in the cerebral cortex and the loss of basal forebrain cholinergic neurons. Amyloid g-protein (Mr. -4.5 kDa) is derived from a family of large (Mr.-110-140 kDa) g-amyloid precursor proteins (APP) which are integral membrane glycoprotems. Soluble derivatives of APP lacking the cytoplasmic tail, trammembrane domain and a small portion of the extracellular domain are generated by APP secretases. Using cell cultures, we investigated the possibility that APP processing can be regulated by a centrally active cholinesterase inhibitor, tacrine (9amino-1,2,3.4-tetrahydroacridine) which was reported to improve memory and cognitive functions in patients with AD. We analyzed the level of APP in glial, fibroblast, pheochromocytoma (PCl2) and neuroblastoma cells by immunoblotting cell lysates and conditioned media. Normal levels of secretion of soluble APP derivatives by cells into conditioned media were severely inhibited by treating cells with tacrine. A similar decrease after treatment with tacrine was observed when neuroblastoma and PC12 cells were pretreated with either growth factors, phorbol ester or retinoic acid. To determine whether the effect of tacrine on APP levels was specific or a more general phenomenon affecting other proteins, we measured the level of heat shock protein-70 (HSP-70) and another secretory protein, protease nexin-1 (PN-1). Tacrine treatment did not alter the level of HSMO in cell extracts and tacrine affected mildly the secretion of PN-1. Thus, the processing of HSP and PN-1, unlike APP, was not severely affected by treating cells with tacrine. Our results suggest that tacrine may inhibit an acetylcholmesteraseassociated moteolvtic activitv involved in the secretion of APP. which results in less secietion of so1uble’APP into the conditioned media from tacrine treated cells. The effect of tacrine along with cholinergic agonists and antagonists on the secretion of APP has also been studied. These results demonstrate that tacrine regulates APP secretion in cell cultures and suggest the oossibilitv that tacrine therauv of AD disease mav have effects on the process of B-&nyloid deposition.’ . We.gratefully acknowledge Dr. Steve Wagner and Mr. Jeff Farrow (SIBIA) for their assistance in mAbP2-1 immunoblotting and tz51-tbmmbiiPN-1 binding assays. These studies were supported by grants from the NIH to DKL and ME