THIRD INTERNATIONAL CONFERENCE ON ALZHEIMER’S DISEASE S83

327 COMPARISONS OF ALZHEIMER'S DISEASE !3A4 AMYLOID PRECURSOR PROTEIN FROM NORMAL AND DISEASED HUMAN BRAINS R.D. Muir, A.I. Bush, B.A. Rumble, K. Beyreuther*, and C.L. Masters. Department of Pathology, University of Melbourne and The Mental Health Research Institute of Victoria, Parkville 3052, Australia. *Center for Molecular Biology, University of Heidelberg, Germany.

The mechanism by which the amyloidogenic -4 protein is generated from the amyloid protein precursor (APP) in Alzheimer's disease (AD) is uncertain. We have been characterising the forms of APE' found in human brain and exploring the possibility that the APP expressed in AD patients has structural anomalies that result in aberrant catabolism of the molecule. Immunoblotting techniques identified two major forms of human brain APP with apparent molecular weights of 100-110 KDa and 120-130 KDa. Both the soluble and membrane fractions of human brain contained the forms in nearly equal amounts. Immunoprecipitation studies with carboxyl-terminus directed antibodies indicate that the soluble forms of APP are truncated. The forms and amounts of APP in normal and AD brains were not found to be significantly different.

A procedure has been developed for the complete purification of the human brain soluble and membrane 100-110 KDa and 120-130 KDa forms of APP under non-denaturing conditions. Membrane-associated forms of APP, once released from membranes by Triton X-100, were found to be soluble in detergent-free aqueous buffers. These findings suggest that potentially amyloidogenic forms of APP can adopt conformations that minimise hydrophobic interactions and allow these molecules to remain soluble outside membranes. Isoelectric focusing techniques indicate an unexpectedly low isoelectric point of 4.5 for the 110 KDa form of APP. The theoretical isoelectric point of APP fs 6-l.

The structure and properties of APP purified from the brains of normal and AD patients are currently being Investigated.

328 DISTINCT EXPRISSION PATTERNS Ol' j%ANYLOID PRNCDRSOR PROTEIN (BmD) IN PLhTLLSTS IN DO"N'S SI'ND~ "EMUS NOSORIUG SWJSCTS. ". Schlcaa~ch~r, 8. Oatasrewski, C. Eaaae and D. Selkoe. Canter for N.urologic Di#aaOea, Narvard Nadical School and Brigham and w’s Ecspital, Ncatcn, Iu. 02115 U.S.A.

To data, four distinct mutatione in tha t3-amyloid pr.cur*cr protein (@PP) g.n. hava ban ahom to ba the disease causing abnormality in saveral padigrma with familial Alzhaimrr’e diaeaaa (AD) or in hereditary carobral hamorehag* with amyloidoei# of Dutch typa. Tha parallels betraon p-amyloid protein &position in AD and 8yatMc amyloidcaea hava apurr& intorant in tha procawing of WP and ita dariratives in blood amus, plasma and tha ra*culature. Ha hara charactarizad isoforma of BAPP in plasma, platalats, lymphocyt*e and human umbilical rein ??ndoth*lial calla. “. datoct tha -140 *Da, N- plus 0-glyccaylatrd mature BAPP in mambranaa of emating and activatmd plateleta of Cc=1 aubjacta but only minute amcunta of the -122 kDa, N-glycosylated, immature pr.curwr; this auggweta vary littla 6 ncvc aynthaais of @PP in circulating control platalete. In contraat, manhbracae of purified plateleta from Down’s qr.droma (DS) patian contain similar amcunta of 140 and 122 *Da @PP polmptidas. The probable idatity of the augmented 122 *Da irroform in DS platalats .a the N-glyccaylatW3 pr.curaoe w.8 ??stahliahad by (1) We&am blotting following inmnmcpracipitation of platalat lyaatas with savaral distinct @PL’ ??ntibodiw; (2) co-migration with N-glycosylated PAPP of human kidnay 293 cello tran8fact~d with . @‘P,51~NA: and (3) deglyco~ylation ??rprrLunt* CC DS c control plateleta with neuraminidaaa or N-glycui&.sa. Our rmsults auggaat that circulatinp plate1ti.e of OS patienta ahow . higher constitutim BAPP ??rprea8ion than control plataleta, .s indicated by inc~~a8.d levels of tha iammturu, 122 kDa pnSP iacfcm. Purthar, "e purified platel&s by S*pharoae 28 and Imugard 10500 filtration to aliminata white blood call contwination and ~rfornud in vitro ~UbcliC labe11ing upuiJmnt8. Prmliainary results demonstrate tha biosynthaaia of BAPP in pwipheral platalata. In ??utoradiograna of immunoprecipitatea with ??ntibodims to $APPb,6_6Q5, “. dat*ct*d the innatura, N- glycoaylated 122 kDa ape&~, l*aaar amcunta of thr maturn, 140 kDa hcloprct*ia and amall amcunta of tha ccnetitutiva -10 *Da C- tati1 j-bP fragamnt. Thorn data prcvida ??ridmnc, that WP ia newly synthesirwl in circulating p1at*lata.

329 A NOVEL PLATELET MEMBRANE-ASSOCIATED FORM OF THE AMYLOID PROTEIN PRECURSOR (APP) OF ALZHEIMER'S DISEASE AND INCREASED EXPRESSION OF APP ON THE SURFACE OF THE ACTIVATED HUMAN PLATELET. Q.-X. Li*, A. I. Bush*, I. Mackenzie*, M. Berndt#, A. Friedhuber', B. Rumble*, J. Currie., K. Beyreuther" and C. L. Masters*. Department of Pathology, The University of Melbourne, and Neuropathology Laboratory, Mental Health Research Institute of Victoria, Australia*; The Baker Research Institute, Melbaurne#; Center for Molecular Biology, University of Heidelberg, Germany*'

PA4 protein (-4.kDa) is deposited in the brain in Alzheimer's Disease (AD), and is likely to be due to aberrant processing of membrane-associated amyloid protein precursor (APP). Normal processing of membrane-associated APP generates a truncated form which does not contain the intact DA4 sequence. The possible existence in the circulation of an AbP isoform with intact transmembrane and carboxyl terminus domains, referred as full-length, is of interest, as it may contribute to PA4 amyloidogenesis. We have previously shown that full- length APP is present in platelet-released material as a minor species (Bush et al, (1990) J. Bid. Chem. 265, 159771. In this study, we further characterized this form in platelet membrane and compared it to the membrane-bound form from human brain. The membranes were stripped with sodium carbonate. Under these conditions, most of the APP species associated with the platelet membrane can be released. However, this stripping did not release significant amounts of brain membrane-associated APP. The stripped membranes were immunaprecipitnted with antibodies raised against "native APP” or the cytoplasmic portion of APP. Western blots of inUnunoprecipitates were immunostained with monoclonal antibody (n@.b) 22Cll which recognizes the amino-terminal portion of APP. We found a novel 160 kDa species representins the full-length APP in both control and AD platelet membranes. The b&n had full length APP species with molecular weights of 110-145 kda. Electron microscopic inununogold studies using mAb 22Cll on the Intact platelets showed that gold particles were associated with a- granules as well as the cell surface. Labelled (12%) mAb 22Cll was used to demonstrate increased surface expression of APP during thrombin activation of washed platelets on control subjects. The surface APP species in platelet consists of the full-length APP as well as loo-130 kDa carboxyl-terminal truncated species. These data are consistent with human platelets having the full-length APP as a minor species, possibly distinct from the soluble form of APP which resides predominantly in the a-granules.

330 Precm Protein (APP) is svntbesized and meidlv trenmcrted bv &QQ~

genclicn cells. R.E.Fiie”, G.Huberl, R.J.Jobason’~‘. P.J.Morin’. J. H. SandellZ, G.Waloga’, and CR. Abmbem’,r. De@. of Biochemistry’, Medicine’, Anatomy’, Physiology’. Boston Umv. School of Medicine Bcstca, MA 02118 and GRECC. ENRVA Hospital’. Bedford, MA 01730.

We have begun to characterize the neuronal pmcessing of APP in tbe rabbit optic nerve. We beve found that the predominpot rapidly tmnqxted form is a 110 kD protein, with a minor component of -85kD being the only other immunoreactivity detected by an antisemm to the APP C-terminus (couricsy of Drs. D. 8elkce and H. Yamagucbi). Based on P previously published subcelluler fractionnticn protocol (Moth et al., 1991. J. Neumchem. 56:415-427). we find that the tramported APP is initially(Zb Post-injection) found in npid transport vesiclea and is subsequently (by 5h post-injection) found predominantly in amlemma. At steady state. the majority of APP is found in the uolem~~I region of P sucrose density gradient of axonal subfractions by Western blotting with tbe same antiserum. APP degmdiig ectivity capable of cleaving tbe protein at one of the sites required to generate the C-terminal, amyloidogaic A, fragment (Abmbem et al., 1990, Peptide Raearch 3:211-215), is found in the same region of the gmdieat; but only in the presence of 1% Tritcn X-100, indicating that this activity is within PII enclosed membrane.

The hlmover of APP in this system occurs rapidly &<5h). IO both the uolemmal fractions and the lateral geniculate nucleus (LGN), Iabelling of APP was maximal at Sb, dimioished to < 50% by IOh, and was essentially undetectable at 24h by immunoprsipitation with the anti-APP-C-terminus antiserum. In addition. higher h3, species of APP not pmaent in the axon or WN were detected in the Wina. Finally, C- terminus~onteinin8 fragments of APP or M. > 15 kD were not detected in my of these membrane preppmtions.

We have ncsntly used specific actibcdiea against the C-terminal end N-termmnl domeins of APP to stain tbe rabbit retina. These antibodies qpear to stain the Muller glill cells and pcasibly the retinal ganglion cells. while the photoreceptors and tbe interneurons are not staiaed.

Finally we have begun studies on the RB22 human retinoblastomn cells which are differentiated into neutxmaMke cells by CAMP nnrlogues and to glial-like cells by butynte. These cells express no detectable APP in tie nondifferentiated state, but upon differentiation expnss significant quantities of different APP isofomw es determined by Western blotting.

S84 THIRD INTERNATIONAL CONFERENCE ON ALZHEIMER’S DISEASE

We conclude that APP is synthesizexl in retinal ganglion cells and is rapidly transported as an 110 kD protein to both the axolemma and the nerve terminal. At thy locations APP is tuned over rapidly without accumulation of C-terminusantaining fragments of M, > 1OkD.

Supportedby AG_05894(REF), EY-O4777(GW),EY.O908(JHS), nod AJXDA IIRG 89-125, AG-O9905(CR4).

331 SPECIFIC AND SATURABLE BINDING OF THE AMYLOID PROTEIN PRECURSOR OF ALZHEIMER’S DISEASE BY ZINC(II). Ashley I. Bush’, Robert D. Moir’, Gerd Multhaup#, Timothy G. Wiiliamson’, Baden Rumble’, David H. Small*, Konrad Beyreuther# and Colin L. Masters* ‘Depalfrnenl of Pathology, University of Melbourne and Mental Health Research Institute of Victoria, Parkville, Australia. #Center for Molecular Biology. University of Heidelberg, Germany.

PA4 amyloid deposition in the brain, characteristic of Alzheimer’s disease (AD), results from the aberrant catabolism of the amyloid protein precursor (APP) which can occur from an overproduction of APP as in Down’s syndrome and in transgenic animals. The cause of sporadic AD is unknown. APP is expressed in neurons and we have found it to be abundant in the plasma and platelet fractions of human bloodf. In AD there is an elevation of 130 kDa APP levels in plasma following heparin-Sepharose chromatography2. The circulating forms of plasma APP are modified by a zinc-sensitive proteolytic mechanism2 and abnormalities of zinc metabolism have been described in Alzheimer’s disease3. For these reasons we studied the zinc binding properties of APP purified from the membrane fraction of human brain.

We found rapid and saturable binding of 65Zn2+ at a dissociation constant (KD) of 764 nM at pH 7.4, and 2.08 PM at pH 6.4. The binding of s5Zn2+ to APP was specific for all competing metal ions, including Ca2+ and Mgz+, with Co2+ able to compete off 70% of maximal binding. The stoichiometry of binding was 1 :l. A putative Znz+-binding site on APP was identified by enzymatic digestion of purified APP695- fusion protein coupled to Zn2+-chelating Sepharose. Studies of labeled APP binding to heparin indicate that zinc increases both the total binding and the proportion of high affinity binding. This effect is specific to zinc compared to other metal ions with Co2+ having a partial effect, as expected, and Al3+ causing a substantial increase in high affinity heparin binding alone.

Zinc binding to APP may modulate the physiological function or metabolism of the molecule, and a dysregulation of zinc metabolism in Alzheimer’s disease may therefore affect the catabolism of APP.

1. Bush Al. Martins RN, Moir RD, et al. (1990) J. No/. Chem., 265, 15977-15983. 2. Bush Al, Whyie S, wmas LD, et al. Ann. Neuro/., (in press). 3. Frederickson CJ. (1989) Int. Rev. Neurobiol.. 31: 145238.

332 THE EXPRESSION OF 8 AMYLOID PRECURSOR PROTEIN mRNA IS NOT ALTERED BY NGF IN RAT BRAIN: IN V/V0 AND IN VITRO STUDIES, “G.L.Forloni, ON. Angeretti, OR. Del Bo, “R.Chiesa, ??N.Schiavo,and ??G. Vantini., “Istituto di Ricerche Farmacologiche “Mario Negri”, Milano, Italy. ??Fidia Research Laboratories, Abano Terme, Italy. The deposition of 8-amyloid in Alzheimer’s disease (AD) brain appears consequence of an aberrant proteolytic process combined with an alteration of amyloid precursor protein (APP) expression. NGF throphically affects the baso-cortical cholinergic neurons, degenerated in AD, and, on the bases of experimental data, has been proposed as a potential anti-dementia agent. Previous finding showed an increase of APP mRNA expression in septal region of hamsters treated with NGF. We investigated the influence of NGF on APP mRNA expression in rat septal/nucleus basalis system after chronically i.c.v. administration or in vitro exposure of cultured neurons. The rats were treated i.c.v. with mNGF (2.5.S, 8@pl) at 2, 4, 6, and 8 postnatal day (PD) and sacrificed on PD 10. Primary rat septallnucleus basalis neurons were dissociated at El7 and exposed to NGF (300 ng) for 10 days. Total RNA was extracted from cell cultures with phenol/chloroform and from brain tissue by caesium chloride method. Northern blot analysis was performed using a APP 32P-cDNA probe to detect the general APP sequence, cyclophiline probe (plbl5) was used as quantitative mRNA standard To verify the efficacy of NGF treatments the enzymatic activity of choline

acetyltransferase (ChAT) was measured in tissue culture and in rat striatum. NGF increased of 80% the ChAT activity in septal/nucleus basalis cells and of 24O%in homogenate from striatum. Although this clear evidence of NGF activity, we did not observe any significant change of APP mRNA expression neither in cells culture or in brain tissue. After in tiw treatment with NGF, APP mRNA expression was unchanged also in striatum and in pans-medulla. These data indicate that the neurothrophic effect of NGF on cholinergic neurons is not always associated with an alteration of APP expression.

333 AN ALTERNATIVE POLYADRNYLATION OF THE 6 AMYLOID PROTEIN PRECURSOR mRNA REGULATES THE TRANSLATION. J.N. OCTAVE and J.M. MALOTEWX. Labmatoire de Neurchhie, Universit.6 Catbolique de Louvain, UCL 1352. Avenue Hippoante 10, 51200 Ebussels, BELmJM.

The sequence of several cDNAs encoding the B amyloid protein precursor (6APP) showed that two polyadenylation sites of the mRNA are utilized; RNA blot analysis with different ribopro- has indicated that this explains the difference between the two major 3.2 and 3.4 kb mRNAs found in the human brain. These two mRNAs which contain all the sequence of the natural molecules were synthetized by in vitro transcription and transla- ted in Xenopus oocytes. The long mRNA using the second polyadenylatlon site produced higher amounts of protein as compared to short mRNA.

This stimulation of protein production by the 3’ untranslated region of the B APP mRNA is not influenced by the 5’ region of the mRNA, since the sequence contained within the two polyade- nylation sites of the B APP mRNA was also able to increase the production of the chicken lysozyme or the chloramphenicol acetyl transferase, as demonstrated by in vivo translation of different chimeric mRNAs obtained by in vitro transcription. This difference in protein production was also observed when chimeric cDNA constructs were transfected into CHO cells.

By measuring the stability of the lysozyme chimeric RNAs injected into oocytes, we have observed that the more efficiently translated mRNA is not more stable, indicating that the increased production of protein is not related to mRNA stability but to increased translation. As determined by computer aided analysis, the last 66 nucleotldes of the 3’ untranslated region found within the two utilized polyadenylation sites of the DAPP mRNA yield putative stem-loop structures which could be a translational regulatory element that governs BAPP production by interaction with cellular proteins present in Xenopus oocytes and CHO cells.

The alternative polyadenylation of the DAPP mRNA can regulate the translation, the 3.4 kb mRNA producing 3 times protein than the 3.2 kb mRNA. This factor of amplification might be of in viva significance for amyloid pathology in Alzheimer’s disease.

334 ASSOCIATION OF B-AMYLOID PRECURSOR WITH NEURITE CYTOSKELETON DURING NEURONAL DIFFERENTIATION OF NG-108 CELLS. A. Dagenais and J. Nalbantoglu, Dept Neurology and Neurosurgery, McGill Center for Studies in Aging, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada.

Aberrant neuritic sprouting is known to occur in Alzheimer disease (AD) brains. As the B-amyloid protein precursor LB-APP) has been detected in neurites surrounding senile plaques, we have investigated the possible role of I-APP in neurite extension. The expression and localization of p-APP was studied in NG-108 cells undergoing neuronal differentiation with dibutyryl CAMP (db CAMP). Northern blot analysis showed a gradual increase of p- APP mRNA which reached a 13 fold increase during maximum neuritic extension after 6 days with db CAMP. Immunoblotting with an antibody recognizing the extracellular domain of the protein showed a gradual increase Of the 103-140 kD fl-APP isoforms during differentiation. This increase was further confirmed by immunofluorescence. In non differentiated NG-108 cells. 8-APP had a faint perinuclear punctate pattern, consistent with an ER-Golgi localization. Upon neurOna differentiation, there was an increase in the