The Journal of Neuroscience, February 1, 1996. 16(3):699-908

Amyloidogenic Processing of the Human Precursor Protein in Primary Cultures of Rat Hippocampal

Mikael Simons,1s2 Bart De Strooper, 2,s Gerd Multhaup,’ Pentti J. Tienari,l** Carlos G. Dot@* and Konrad Beyreutherl 1Center for Molecular Biology Heidelberg, University of Heidelberg, D-69 120 Heidelberg, Germany, 2Cell Biology Program, European Molecular Biology Laboratory, D-690 12 Heidelberg, Germany, and 3Center for Human Genetics, 3000 Leuven, Belgium

The aim of this study was to investigate the proteolytic pro- containing C-terminal fragments (1 O-l 2 kDa) intracellularly. cessing of the amyloid precursor protein (APP) in polarized Radiosequencing showed that these fragments were created at primary cultures of hippocampal neurons. We have used the a previously described p-secretase cleavage site and at a Semliki Forest virus (SFV) vector to express human APP695 in cleavage site 12 residues from the N terminus of the pA4 hippocampal neurons, sympathetic ganglia, and glial cells. The domain (ThFa4 of APP695), which we named S-cleavage. latter two cells secrete little or no APP, whereas hippocampal Based on the observation that mature hippocampal neurons neurons secrete two forms of APP695, which differ in sialic acid produce two potentially amyloidogenic fragments and secrete content and in their kinetic appearance in the culture medium. substantial amounts of pA4 when expressing human APP, our In addition, rat hippocampal neurons expressing human APP results strengthen the hypothesis that neurons play a central produced significant amounts of the 4 kDa peptide pA4. After 3 role in the process of PA4 deposition in cases of Alzheimer’s hr of metabolic labeling, the relative amount of PA4 peptide to disease and in aged primates. total cellular APP was 5.3%. Fibroblasts expressing APP695 using the same SFV vector mainly produced a related 3 kDa p3 Key words: Alzheimer’s disease;hippocampal neurons; SFV peptide, a nonamyloidogenic fragment. Remarkably, the hip- vector; amyloid precursor protein; p-amyloid peptide; amyloi- pocampal neurons also produced significant amounts of pA4- dogenic processing

The amyloid precursor protein (APP) is a widely expressed exclusively in tissue, which suggeststhat there are mech- loo-140 kDa integral membrane protein with a large anismsspecific to the brain that are responsiblefor the patho- N-terminal ectodomain and a small C-terminal cytoplasmic genesis.Among brain cells, neurons are strongly affected dur- domain (Kang et al., 1987). Although its physiological function ing AD by changes in their cytoarchitecture, giving rise to remains unknown, several findings suggest a central role for dystrophic neurites and neurofibrillary tangles. Neurons have APP in the pathogenesisof Alzheimer’s disease(AD) (Selkoe been proposed as a potential source of PA4 in the amyloid et al., 1994). APP is the parent molecule for a small, 39-43 plaques based on indirect evidence showing that neurons ac- residue amyloid peptide (PA4), which is the principal compo- cumulate APP in the neurites surrounding the amyloid deposits nent of the senile plaques and vascular amyloid found in the (Cork et al., 1990; Cras et al., 1991). Moreover, PA4 deposi- of AD patients (Glenner and Wong, 1984; Masters et al., tions in the brain appear to spreadalong neuronal projections, 1985). Furthermore, families with hereditary forms of AD, or supporting the idea that neurons play a causal role in the familial AD (FAD), carrying missensemutations in the APP pathogenesis(Hyman et al., 1990). have been identified (Mullan and Crawford, 1993), and Despite the great interest in neuronal APP processingin AD overexpression of such human APP mutants in transgenic mice research,APP processinghas been studied almost exclusivelyin causes amyloid deposition and plaque formation of similar non-neuronal cells. These studieshave demonstratedthat APP tinctorial properties such as those occurring in AD (Games et has three proteolytic cleavage sites. After transport from the al., 1995). Although APP is expressedubiquitously, pathologi- Golgi apparatus, APP can be cleaved within the PA4 region cal manifestations of AD such as PA4 deposits are found (a-cleavage) (Esch et al., 1990;Sisodia et al., 1990),leading to the Received June 21, 1995; revised Oct. 18, 1995; accepted Oct. 24, 1995. releaseof the large, 100 kDa ectodomain that is secreted.The This research was supported by SFB 317 (K.B., C.G.D.) and SFB 258, the amount of secretedprotein varies from cell to cell but doesnot Metropolitan Life Foundation, the Fonds der Chemischen Industrie, and the Fors- appearto exceed30% of totally synthesizedprotein (Weidemann chunesschwemunkt Baden-Wuertemberz (K.B.1. B.D.S. is a oostdoctoral fellow of et al., 1989; Caporaso et al., 1992; Selkoe et al., 1994). The the I&tional fronds for Scientific Rese&h‘(Nl%O, Belgium)‘and was supported by a short-term fellowship of EMBO. We thank Liane Meyn for the neuronal cultures. moleculesescaping a-cleavage can be internalized from the cell We are very grateful to the many investigators who provided probes and materials: surface, mediatedvia endocytosissignals in the cytoplasmictails B. Greenberg, T. Hartmann, A. Hemar, F. van L.euven, D. Selkoe, N. Robakis, H. Virta, and H. Wiesniewski. We thank H. Garoff and M.-L. Strand for help and advice (Haasset al., 1992a,b; Koo and Squazzo, 1994), and then pro- in optimizing the recombinant SFV stocks. cessedby P-cleavageat Met596(numbering after APP695), creat- Correspondence should be addressed to Dr. Konrad Beyreuther, Center for ing the N terminusof PA4 (AMPLY’).This scissionis followed by an Molecular Biology Heidelberg (ZMBH), University of Heidelberg, Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany. additional cleavage(-y-cleavage) in the region of Va1636to Thrwo, Copyright 0 1996 Society for Neuroscience 0270-6474/96/160899-10$05.00/O releasingPA4. APP processingis complicatedfurther in cells that 900 J. Neurosci., February 1, 1996, 76(3):899-908 Simons et al. l Neuronal APP Processing possess a polarized cell surface. In epithelial Madin-Darby canine C-terminal amino acids of APP (De Strooper et al., lYY5a); B716, a kidney (MDCK) cells, APP is transported to the basolateral polyclonal Ab raised against synthetic human l-40 PA4 peptide, which only recognizes the human l-16 sequence (B. De Stroopcr, unpublished domain from which its proteolytic fragments, the 100 kDa ectodo- observations); R 217, made against APPs (Lowcry ct al., 1991); Rl282, main and the 4 kDa PA4 peptidcs, arc secreted (Haass et al., raised against human synthetic l-40 PA4 peptide (Haass et al.. 1992a,b); 1994; Lo et al., 1994; De Strooper et al., 1995a). R48, raised against human synthetic PA4, 1-16 (Anderson ct al., 1992); Neurons have a polarized cell surface like epithelia and are and 4G8, an mAb against residues 17-24 of PA4 (Kim et al., IYHX). For differentiated into somatodendritic and axonal territories (Craig immunoprecipitation, each Ab was used at I:100 dilution except R217, which was used at 1:500, and B12/4, which was used at 1:lOOO. Ab 22C1 I and Banker, 1994). Little is known about APP processing in was used at l:lO,OOO for immunoblotting. polarized neurons; therefore, we decided to study the metabolism DNA cot~sflucts. From the plasmid PrcA4-pSP6S (Dyrks et al., 1988) of human APP695 in the hippocampal culture system. containing the APP695 sequence, the Spel site in the noncoding region of During culture in vitro, the hippocampal neurons undergo a se- APP695 was removed by using the Klenow fragment. The 3 kb &al quence of differentiation steps that leads to maturation into fully fragment containing the complete coding region of the APP695 cDNA was subcloned into the SnzuI site of pSFV I (Liljcstrom and Garoff, polarized cells with axons and dendrites forming synaptic net- 1991). works on the culture dish (Dotti et al., 1988). These neurons share Preparuriofl of recombinant SW The pSFV-APP6YS (human) and the many biological properties with their counterparts in the brain pSFV-helper 1 were linearized with &WI, and runoff‘ transcription was and, therefore, constitute an excellent in vitro model. It has been performed with SP6 RNA polymerase. Each APP transcription mix was shown that human APP695 is expressed in these neurons and that cotransfected with the helper-transcription mix to BHK cells using clec- troporation as described by Olkkonen et al. (1993). The culture super- it is delivered first to the axons and then transported to dendrites natant was collected after 20-24 hr incubation (5% CO?, 37°C). The by a mechanism similar to what has been termed “transcytosis” in virus-containing supernatant was titrated on BHK cells as dcscribcd by epithelial cells (Simons et al., 1995). Similar results have been Olkkonen et al. (1993). obtained with endogenous APP in neuron culture, confirming that Infection of the neurons and metabolic lahelirlg. Recombinant SFV diluted in conditioned maintenance medium was placed on 7- to l4-d-old the overexpression of APP via the virus vector mimics the nor- neurons. After allowing viral entry for I hr (5% CO,, 37”C), the virus mally observed situation (Yamazaki et al., 1995). solution was replaced by maintenance medium and the infection contin- To obtain sufficient levels of APP expression for biochemical ued for 2-3 hr. The medium then was exchanged by methionine-free analyses, we used recombinant Semliki Forest virus (SFV) coding MEM with l/IO N2 supplement containing 15 mgil mcthionine and 1’312 for human APP695 to infect primary cultures of rat hippocampal egg albumin. Metabolic labcling was performed for 3 hr with 200 &i/ml [“Slmethionine. In time-course experiments 3 hr after infection, cells neurons. This system has been used successfully to express heter- were pulsed for 10 min with methionine-free MEM containing 300 ologous proteins in neurons and offers the advantage of high $.Zi/ml [‘SS]methionine. Next, cells were washed twice with maintcnancc protein expression without affecting the polarized organization of medium, and fresh medium was added at 0, 30, 60, and I20 min after the cells during the early phase of infection (Ikonen et al., 1993; radioactive pulse. Culture medium from each time interval was processed immediately for immunoprccipitation. For in vko labcling with Olkkonen et al., 1993; De Hoop et al., 1994; Simons et al., 1995; [“Plorthophosphate, neurons were incubated 3 hr after infection in De Strooper et al., 1995b). phosphate-free MEM supplemented with l/IO N2 supplcmcnt containing 158 mg/l NaH>PO, for 15 min and then labeled with 1 mCi/ml H,“PO, MATERIALS AND METHODS for 3 hr. Extracellular medium was processed as described, cxccpt that Cell culture. Hippocampal neuron cultures were prepared from l&d-old orthovanadate (100 FM) and sodium fluoride (50 mM) were added. fetal rats as described by Goslin and Banker (1991). Briefly, after the Immutzc)precipitatior~, immunoblottirtg. gel electrophorcsis, urzd quuntifi- hippocampi were dissected and dissociated, the cells were plated on cation. After metabolic labeling, culture medium was harvested and cell either poly-o-lysine-coated plastic dishes (at a density of 4 X 10’ cells/b extracts were prepared in 1% Triton X-100, 1% sodium deoxycholate. cm dish) or glass covcrslips (at a density of 1.5 X 10’ cells/6 cm dish) and and 0.1% SDS with IO kg/ml chymostatin, leupeptin, anti-papain, and kept in minimum essential medium (MEM) supplemented with 10% pepstatin. Both culture medium and cell extracts were clcarcd first from horse serum. Cells were maintained in 5.0% CO2 at 36.5”C in MEM with precipitations, cell debris, and nuclei via 15 min centrifugation. The N2 supplement (“maintenance medium”) (Bottenstein and Sato, 1979). supernatant was incubated with Ab overnight at 4°C. Immune complexes Proliferation of non-neuronal cells was prevented by adding 5 pM cy- were recovered with protein A-Sepharose or protein G-agarose and tosine arabinoside. processed as described previously (De Strooper et al., lYY.3). In the case Glial cultures were prepared as described by Goslin and Banker (lYY1). of mAbs, polyclonal rabbit anti-mouse was used as capture Ab. For The cerebral hemispheres were removed from newborn rats and mechan- immunoblotting, cell extracts and extracellular medium were precipitated ically and chemically (trypsin) homogenized. Cells were cultured in MEM in 10% trichloroacetic acid (TCA), washed with acetone, and solubilized supplemented with 10% horse serum. To prevent neuronal contamina- in SDS sample buffer. TCA precipitates and immunoprccipitatcd proteins tion, only glial cultures that were passaged two to three times were used. were separated by 6% Tris-glycine, 4-20% Tris-glycine, or l&20% Superior cervical ganglia were dissected from lY- or 20-d-old fetal rats Tris-tricine polyacrylamide gels (Novex, San Diego, CA). We used low- and prepared as described by Peng et al. (1986). Explants were cultured range See Blue prestained standard (Novex) and high-range prestained for 14 d in N2-MEM supplemented with 10 /.LM Huorodeoxyuridine, 10 SDS-PAGE standard (Bio-Rad, Richmond, CA) as molecular weight FM uridine, 20 pg/ml tri-iodo+-threonine, and 10 @ml 2.5s nerve standards. Gels were enhanced by Intensify (DuPont NEN, Boston, MA), growth factor before use. dried, and exposed to Kodak XAR film (Rochester, NY). For immuno- Baby hamster kidney cells (BHK; CLLlO) were cultured in Glasgow’s blotting, membranes were blocked in PBS/S% milk and 0.2% Triton modified Eagle’s medium supplemented with 5% fetal calf serum (FCS), X-100 and incubated for 1 hr at room temperature with primary Ah 2 mM L-glutamine, 10% tryptose phosphate broth, IO0 U/ml penicillin, diluted in blocking buffer. Appropriate horseradish peroxidase-labeled and 100 gmiml streptomycin. secondary Ab (Bio-Rad) was added for 30 min. Blots were devclopcd by Primary fibroblasts from 13.5-d-old mouse embryos were isolated as enhanced chemiluminescence (Amersham Buchler, Braunschweig, Ger- described previously (Robertson, 1987) and maintained in DMEM sup- many), The intensity of radiolabeled bands was calculated using densito- plemented with 10% FCS, 2 mM glutamine, and antibiotics. metric scanning (Molecular Dynamics, Sunnyvale, CA). Antibodies. Antibodies (Abs) used in the experiments included poly- Enzymatic degiycosylution. For digestion with neuraminidase (Boeh- clonal anti-FdAPP raised against purified Escherichiu co/i FdAPP fusion ringer Mannheim, Indianapolis, IN), immunoprecipitates were made with protein consisting of the following: APP695 and the Fd fragment of 1% SDS and 1% /3-mercaptoethanol, boiled for 5 min, and diluted IO-fold murine IgM heavy chain (Weidemann et al., 1989); 22Cl1, a monoclonal to final concentrations of 50 mM sodium acetate and 4 mM calcium Ab (mAb) that recognizes a region between amino acids 66 and 81 of chloride, pH 5.5, 1% Nonidet P-40, 0.1% SDS, 10 &ml chymostatin, APP695 (Hilhich et al., 1993); Ab 692, a polyclonal Ab raised against leupeptin, anti-papain, pepstatin, and 10 mu/ml neuraminidase. After human PA4 synthetic peptide l-40; B1214, a polyclonal Ab against the 20 incubation at 37°C for 16 hr, samples were analyzed by SDS-PAGE. Simons et al. l Neuronal APP Processing J. Neurosci., February 1, 1996, 76(3):899-908 901

Samples treated with N-glycosidase F (10 U/ml, Boehringer Mannheim) were buffered in 50 mM sodium phosphate buffer, pH 7. For O-glycosidase (Boehringer Mannheim) treatment, immunoprecipitates were preincubated with 1 mU of neuraminidase at 37°C for 3 hr; 3 mU of 0glycosidase was used in sodium phosphate buffer, pH 7. As a control for unspecific degradation, parallel samples were incubated in the ab- sence of enzymes. Radiosequencing.After SDS-PAGE, [“S]methionine- and [3H]phenylalanine- labeled proteins were blotted into ProBlott membranes (Applied Biosys- terns, Foster City, CA). Membranes were subjected first to autoradiog- raphy, and strips were excised corresponding to the bands visualized by autoradiography. A modified cycle was used for protein sequencing on the Model 477A Protein Sequencer (Applied Biosystems). In the solvent- wash step, the Sl (n-heptane) delivery time was reduced from 100 to 25 sec. The S2 (ethyl acetate) delivery time was reduced to a total of 105 set divided into three 35 set deliveries alternated with two 5 set pause steps. In the anilinothiazolinone extraction step, the S3 transfer step (52 set) was split into three 25 set transfers. Each transfer was followed by a 5 set pause step and a 5 set transfer with argon. The solvent was directed entirely to the fraction collector, and each fraction was mixed with 3.5 ml of liquid scintillator (Ready Protein Cocktail, Beckman, Fullerton, CA) before counting for 10 min (Beckman LS 60001C). Immunofluorescence. Immunofluorescence was performed as described previously $imons et al., 1995). Cells were fixed with 4% p-formaldehyde in

PBS.II nH 7.4. auenched1 with 50 mM NH,Cl in PBS for 20 min. nermeabilized with increasing concentrations of ethanol, and blocked in 2% bovine serum albumin, 2% FCS, and 0.2% gelatin in PBS. The cells were incubated in the presence of primary Ab (anti-FdAPP and anti-MAP2) and fluoroscein- or rhodamine isothiocyanate-conjugated secondary Abs, as described by Simons et al. (1995). The coverslips were mounted in Moviol (Hoechst, B. Frankfurt, Germany), and the cells were viewed using an Axiophot (Zeiss, Jena, Germany). Photographs were taken using high-sensitivity film (Tmax 3200, Kodak).

RESULTS To analyze processing and secretion of endogenous APP, neuro- 98 nal cultureswere labeledin the presenceof [“?S]methioninefor 3 hr, and cell lysate and extracellular medium were subjected to immunoprecipitation by a panel of different Abs againstC- and N-terminal epitopes of APP (see Materials and Methods). The Abs revealeda prominent band at 115kDa in the cell homogenate and a doublet of 120 and 100 kDa in the culture medium (Fig. L4). A C-terminal Ab recognizedthe 115 kDa band from the cell lysatebut not the APP cleavageproducts in the culture medium (data not shown), indicating that the latter forms had lost their cytoplasmic tails. The possibility that one of the bands in the (overexposed fig. B.) culture medium representsthe APP-like protein (APLP 1 or 2) was ruled out becauseboth bandsreacted with Ab R48 against Figure 1. Immunoprecipitation of [35S]methionine-labeled APP from cell epitope 1-16 of PA4 (not present in APLP) in immunoblots(Fig. homogenate and extracellular medium. A, Endogenous APP- and SFV- 24) and immunoprecipitations(data not shown). expressed APP695 (infected) were immunoprecipitated with Ab R217 from cell homogenate (C) and extracellular medium (M) of mature To analyze the processingand secretion of APP in more detail, primary hippocampal neuron cultures after a 3 hr metabolic labeling with we usedthe SFV-basedprotein expressionsystem (Liljestrom and [“‘Slmethionine. Note the comigration of endogenous and SFV-expressed Garoff, 1991). Neuronal cultures were infected by recombinant APP695. B, A hippocampal neuron (hippoc. neurons) culture (4 X 105 SFV carrying human APP695 and metabolically labeled with cells) and a glial culture (lo6 cells) (gliial cells) were infected with SFV- [35S]methioninefor 3 hr. Expression of human APP695 in hip- carrying APP695 and labeled for 3 hr with [35S]methionine. Immunopre- cipitations of cell homogenate (C) and extracellular medium (w were pocampalneurons led to secretion of the same 100 and 120 kDa performed with Ab R217. Glial cells show little APP expression compared bands(Fig. 1A), demonstratingthat the use of this vector system with hippocampal neuron cultures. Six cervical ganglion explants (gungfiaf led to processingsimilar to that observed for the endogenous cells) were infected with SFV-containing APP695 and metabolically la- APP. Also, the fact that the distribution of the endogenous beled for 8 hr. Resulting cell homogenate (C) and conditioned medium (M) were pooled and processed as above. C, Overexposed version of B. marker protein MAP2 remained polarized during virus infection Fluorogram was exposed for 3 weeks. Note the reduced secretion of glial supportsthe use of this expressionsystem for a detailed analysis cell cultures compared with neuronal cultures in B and the absence of of APP processing(Fig. 3B). detectable secretory APP in culture media of cervical ganglions. Primary neuronal cultures alwayscontain a low amount of glial cells (5-20%). To investigatethe possiblecontribution of infected glial cells to the APP produced by the neurons, a subconfluent superior cervical ganglia, as a sourcefor peripheral neurons,also homogenousculture of glial cells was infected and processedin was investigatedfor secretion (Fig. l&C). It is clear from Figure parallel, and levels of APP expressionwere compared in both the 1B that glial cells were infected much lessefficiently with recom- hippocampaland the glial cultures. In addition, an explant of binant SFV than were the hippocampalneurons under the exper- 902 J. Neurosci., February 1, 1996, 76(3):899-908 Simons et al. l Neuronal APP Processing

two secretory APP products also were observed. The 120 kDa form appeared first, and its secretion was almost complete 120 min after the pulse,whereas the 100 kDa form was observedonly after 60 min and was still being secretedafter 180 min of chase. This was confirmed in four independent experiments with two different Abs. Quantitation of the kinetic differencesis shown in Figure 4B. Becausein other cellsAPP695 usuallyyields one singlesecreted APP with M, - 100 kDa (Weidemann et al., 1989; Esch et al., 1990;Oltersdorf et al., 1990;Golde et al., 1992),the nature of two solubleAPPs in hippocampalcultures wasexplored further. Pro- teolysis of the higher molecular weight form by present in the extracellular medium was excluded, becauseincubation of culture supernatantat 37°C during 3 hr in the absenceof protein- ase inhibitors did not change the ratio between the two forms (data not shown). Furthermore, both proteins reacted in immu- noblots with Abs that recognize the C- and N-terminal ends of secretoryAPP (Fig. 24), confirming that proteolysisin the culture medium was not involved in the generation of the two bands. Therefore, other post-translational modifications were investi- gated. Both proteinswere weakly phosphorylatedto a similarlevel (Fig. 24), excluding the possibility that one band is phosphory- lated and the other is not. Glycosylation was explored using carbohydrate-cleavingenzymes. Endoglycosidase F decreasedthe molecular weight of both bandsby -2 kDa, indicating similar levelsof N-glycosylation (Fig. 2B). Treatment with neuraminidase Figure 2. Characterization of secreted APP695. A: Lefi, Immunoblotting causedthe disappearanceof the upper band and comigrationof of endogenous secreted APP (endogenous) and SFV-expressed APP695 the upper band with the lower band (Fig. 2C). Treatment with (infected) with Ab R48 specific for PA4 1-16 from conditioned media of O-glycosidaseand neuraminidaseled to the sameresult (data not cultured hippocampal neurons (R 488;lo-fold more medium was used from shown). These resultsdemonstrate that the heterogeneityof se- noninfected neuronal cultures). Middle, Immunoblotting of culture me- dium from SFVJAPP695 neurons infected with Ab 22CII. Right, Immu- creted APP695 in hippocampalneurons is caused by a higher noprecipitation of cultured media from infected SFV/695 and content of sialicacids in the upper band, causingan increasedM, [32P]phosphate-labeIed neurons (phosphorylation) with Ab R217. B, C, (-20 kDa). Endoglycosidase F and C (N-Glycosidase F) and neuraminidase treatment One of the issuesin AD that is still unresolvedis to what extent of immunoprecipitated SFV-expressed secretory APP695 from culture medium of hippocampal neurons. In B and C, the leji lanes contain control neuronsare able to produce PA4 and other low molecularweight samples that were incubated in the absence of enzyme. peptides, including p3. The p3 peptide arisesfrom cleavage by a-secretasewithin the PA4 domain and comprisesPA4 residues 17-40. imental conditions. Interestingly, glial cells secreted only minor One dish containing -4 X 10’ hippocampalneurons was in- amounts of APPs, detectable only after overexposure of the gels fected with recombinant SFVlAPP695 and labeledfor 3 hr with (Fig. lC), which confirms previous investigations (Haass et al., 200 &i/ml [35S]methionineto analyzewhether PA4 wassecreted. 1991). Because the glial cultures contained at least lo-fold more Immunoprecipitationwas carried out on the culture mediumwith glial cells than the hippocampal cultures routinely used, we con- the well characterized antiserum R1280 (Haass et al., 1992b), cluded that contaminating glial cells did not contribute signifi- which precipitates both PA4 and p3 fragments in other cell cantly to the pool of APP observed in our experiments. Although cultures. A major 4 kDa protein wasobserved after 24 hr exposure these experiments clearly demonstrate secretory processingof of the gel (Fig. 5A), whereasa very weak p3 signal wasobserved APP by hippocampalneurons, significant secretory processingof only after longer exposures(data not shown).For comparison,we APP in the ganglion explant was not observed,even though the expressedAPP695 in primary mouse fibroblasts via the SFV cellswere infected by the virus and expressedAPP695 (Fig. lB,C). vector (Fig. 5B). The ratio of PA4 to p3 was reversed,demon- To determinethe time courseof APP secretion, one dish (4 X strating that hippocampal neurons differ in their processing of lo5 cells)of infected hippocampalneurons was pulse-labeledfor APP695. The 4 kDa peptide secreted from the neurons was 10 min, and medium was assayed for secreted APP after 30, 60, identified as authentic PA4 peptide because it reacted with both 120, and 180 min of chase (Fig. 4). At each time point, the mAb 4G8 against amino acids 17-24 and polyclonal Ab B7/6, complete medium was collected and replaced with an equai which is specificfor amino acids l-16 of the PA4 sequence(Fig. amount of fresh medium. Figure 4 representsthe fractional ac- 5A). Radiosequencingof the protein confirmed that the peptide cumulation of the pulse-labeledAPP into the culture medium. was indeed pA4, starting at Asp’ of the P-amyloid sequence(Fig. Secretion of APP in hippocampalneurons was slow, and even 5C). Densitometric scanning analysisshowed that the relative after 3 hr APP from the pool synthesizedduring the 10 min pulse amount of secretedPA4 peptide to total cellular APP (corrected labeling continued to appear in the culture medium. Moreover, for methioninecontent) was already 5.3 t 1.9% (n = 6) after 3 hr the overall kineticsof this processwas not only somewhatdifferent of labeling. from that of other cells (Weidemannet al., 1989;Oltersdorf et al., Finally, we investigatedthe C-terminalAPP fragmentsproduced 1990), but intriguing kinetic differencesin the appearanceof the by hippocampalneurons. After infection and metaboliclabeling, the Simons et al. . Neuronal APP Processing J. Neurosci., February 1, 1996, 76(3):899-908 903

MAP2. whereas the axon is labeled with anti-APP. The axon is marked with arrows, and the dendrites are marked with arrowheads. 904 J. Neurosci., February 1, 1996, 76(3):899-908 Simons et al. l Neuronal APP Processing

could not be separated from each other and, therefore, were radio- 30’ 60’ 120’ 180’ (min) sequenced as a mixture. [3H]Phenylalanine radioactivity peaks were obtained at cycles 3,4,5,6,9, and 10 (Fig. 60). These results confirm that the fragments in these bands are heterogeneous, likely consisting of C-terminal fragments starting at Leu” of the classical a-secretase sAPP695a ---) sAPP6956 + site and at two other positions, i.e., Glu” and Gin” of the PA4 sequence. For simplicity, we will call the two additional sites (Y’- secretase sites, although the site at position Gin” likely is also amyloidogenic, because an important fraction of peptides in amyloid plaques of AD patients starts exactly at that position (Masters et al., ,” 1985; Naslund et al., 1994). DISCUSSION n 120kLhAPPs Although the metabolismof APP has been studied in detail in fz 1OOkDSAPPS many cell types, information about the processingof APP in the 30 T cells that are affected in AD, i.e., hippocampalneurons, is absent. The reasonfor this is the difficulty in obtaining sufficient amounts of material from cultured neuronsfor biochemicalanalyses. We circumvented this problem by expressinghuman APP695 via the SFV-expressionsystem. This vector is ideally suited for this pur- pose becausea wave of newly synthesizedAPP is transported through the biosynthetic pathway after infection; therefore, post- translational modifications can be monitored easily. All of our controls in this study (Figs. 1, 3) and in previous studies(Simons et al., 1995; Yamazaki et al., 1995) suggestthat the membrane- trafficking systemsin theseneurons perform normally during the time courseof the study. O-30 30-60 60-120 120-180 min Particularly important was the observation that rat hippocam- pal neurons are able to produce PA4 peptide in relatively high F@re 4. Time course of APP695 secretion in hippocampal neuron amountscompared with the p3 peptide when expressinghuman cultures. A, SFViAPP6954nfected hippocampal neurons were pulse- APP695. We evaluated this by using the widely usedAb R1282. labeled for 10 min with [“%]methionine, and culture media were collected at the times indicated (see also Materials and Methods). Secreted APP695 This Ab usually precipitates more p3 (residues17-40 of PA4) was immunoprecipitated with Ab R217 at each time interval. B, Quanti- than PA4 from a variety of cells, including stably transfected tative analysis of four independent experiments performed as in A. Filled human 293 kidney cells and MDCK cells, Chinesehamster ovary columns indicate 120 kDa APPs; hashed colum?zsindicate 100 kDa APPs. cells, and human umbilical vein endothelial cells (Haasset al., 1992b,1994). In contrast, we found that rat hippocampalneurons cell lysates were immunoprecipitated with Ab B1214 against the 20 produce substantialamounts of /3A4, whereasp3 wasvisible only C-terminal amino acid residues of the cytoplasmic tail of APP. In faintly, even after prolonged exposure of the fluorographs from lo-20% of the Tris-tricine polyacrylamide gels, four C-terminal R1282 immunoprecipitations.A reverse ratio was observed in fragments appeared between 8 and 12 kDa (Fig. 6A). The two upper fibroblasts(Fig. SB). Becausethe small amountsof contaminating bands reacted with Ab B7/6, indicating that they contained the entire glial cells in the cultures did not contribute significantly to APP PA4 peptide (data not shown). By densitometric scanning analysis, production after SFV infection (Fig. l&C), our conclusionis that the relative amount (corrected for methionine content) of the the hippocampalneurons are the sourceof the PA4 peptide. C-terminal fragments compared with that of full-length APP was Previously, PA4 production has been studied in a number of shown to be 13.4 + 5.25% (n = 8) after 3 hr of labeling. The brain-derivedcell types, including rat astrocytes,human astrocytes, PACcontaining (potentially) amyloidogenic fragments represented rat cortical and hippocampalneuronal cultures, and humanmixed- -67% of the C-terminal fragments. brain cultures (Haasset al., 1992b; Busciglioet al., 1993). These The sequence of the 8, 8.5, 10, and 12 kDa C-terminal deriva- studiesfound that mostly p3 was produced from rodent neuronal tives was identified by radiosequencing. [3’S]Methionine or cultures,whereas human astrocytes and humanmixed-brain cultures [“Hlphenylalanine metabolically radiolabeled C-terminal frag- produced the highestlevels of pA4. The discrepancywith our find- ments were isolated from lo-20% of the Tris-tricine gels and ings could be attributable to the difficulty in detecting significant subjected to N-terminal radiosequence analysis (Fig. 6B-D). The amountsof PA4 in rodent neuronsbecause of reducedaffinity of the 12 kDa [““Slmethionine-labeled fragment revealed a radioactive anti-PA4 Abs to the rodent /3A4 peptide.Another possibilityis that signal at cycle 12. Alignment with the known sequence of APP rodent neuronsindeed produce only smallamounts of endogenous indicated that cleavage of APP had occurred at the 12 amino acids PA4. Only after human APP wasintroduced into thesecells would N-terminal to PA4 (Fig. 6B). The results show that neuronal cells they start secretingflA4. Recent resultsfrom our laboratoriesdem- are capable of generating a fourth cleavage at the N-terminal side onstrate that rodent APP expressedvia the SFV vector produces of the P-cleavage site, which we propose to name the S-cleavage much smalleramounts of PA4 comparedwith the amountsobserved site, which is generated in neurons by an unidentified 6-secretase. after expressinghuman APP. Severalfamilial APP mutants,how- Radiosequencing of the [35S]methionine-labeled 10 kDa fragment ever, produced more PA4 wild-type APP695 (De Strooper et al., caused ?S peaks in cycle 4 and cycles 19-20, indicating that this band 1995b).These data are in agreementwith the resultsobtained with was generated by P-cleavage (Fig. 6C). The 8 and 8.5 kDa fragments the recent transgenicmouse model expressinghuman APP with Slmons et al. l Neuronal APP Processing J. Neurosci., February 1, 1996, 76(3):899-908 905

C

k -cpm 400,

-16

-6 -4

DAEFRHDSQYEVHHQKLVFFAEDVQSN

cycle 1-27

Frgure 5. PA4 secretion from primary cultures of hippocampal neurons. A, Immunoprecipitation of PA4 from culture medium of SFV/APP695infected neurons after a 3 hr [“Slmethionine labeling with the polyclonal Ab RI282 (against PA4 l-40), the monoclonal Ab 4G8 (mAb4G8) (against PA4 17-24), and the polyclonal Ab B716 (against PA4 1-16). B, Comparison of PA4ip3 ratio in hippocampal neurons (hippoc. neurons) andjibroblusfs. APP695 was expressed via the SFV vector, and cells were labeled with 1”Slmethionine for 3 hr. Medium was immunonrecioitated with Ab 692. A highYG BA4/o3. was observed in hippocampal neurons. The ratio was reversed in fibroblasts. C, Radiosequencing of a [“H]phenylalanine-labeled 4 kDa peptide from culture medium of hippocampal neurons.

FAD 717 (FAD mutant; mutation in codon 717) which produced tation of these results is that the secretory processing of APP in typical AD-associated amyloid plaques (Games et al., 1995). neurons is a complex process taking place at different stations In contrast to previous studies, which concluded that secretory during APP trafficking in the neurons. We do not know whether processing of APP in neurons was less prominent (Haass et al., 1991; the latter form is produced by desialylation of the 120 kDa form, Hung et al., 1992) or nonexistent (Alliquant et al., 1994), we found e.g., after endocytosis and subsequent release by recycling, or appreciable a-secretase processing of APP695 in both uninfected whether the 100 kDa is derived from a pool of APP following and infected hippocampal neurons. In our opinion, the reasons for another transport route through the biosynthetic pathway. We this apparent discrepancy between our findings and those of others have observed that there is a pool of APP in hippocampal neurons most likely are to be found at the level of the neuronal culture that never achieves full glycosylation within the time span during systems that were used. The hippocampal neuron culture used here which we followed the cells (M. Simons and B. De Strooper, yields well differentiated neurons that elaborate many interneuronal unpublished observations). That the desialylation occurs by se- contacts (Goslin and Banker, 1991). These intemeuronal contacts creted sialidases after secretion is virtually excluded, however, may be important in this respect because we found that neurons because prolonged incubation of the two proteins in conditioned derived from cervical sympathetic ganglion cells, which do not form medium at 37°C did not change the relative amounts of the two synaptic contacts in vitro, did not secrete APP in detectable amounts forms. The function of APP processing in neurons is still not (Fig. l&C). The alternative possibility that secretory processing of known, but we assume that a-secretory processing of APP has an APP is a particular characteristic of hippocampal neurons [as op- important physiological role in the central nervous system, which posed to cortical neurons used in the other study (Alliquant et al., is in agreement with other studies (Nitsch et al., 1992). 1994) and sympathetic neurons used here] cannot be ruled out at this The study of the intracellular C-terminal remnants of APP was time. particularly informative. Typically, the nonamyloidogenic frag- Secretory processing of APP in hippocampal neurons is some- ments are the primary cleavage products in many cell types what slower than that of non-neuronal cells and, in contrast to (Oltersdorf et al., 1990; Caporaso et al., 1992; Golde et al., 1992; other cells (Weidemann et al., 1989; Oltersdorf et al., 1990) it Haass et al., 1992a; Lo et al., 1994). In neurons, however, we find yielded two APP forms of -100 and 120 kDa in the extracellular high amounts of PA4-containing C-terminal fragments (Fig. 6) medium. Both forms are phosphorylated and N-glycosylated, but compared with nonamyloidogenic fragments, emphasizing the they contain different amounts of sialyl residues because they both possibility that neurons have an intrinsic tendency to catabolize migrate together as a single band in SDS-PAGE after treatment APP in an amyloidogenic manner. Another interesting finding in with neuraminidase (Fig. 2C). Intriguingly, a short pulse (10 min) this study was the identification by radiosequencing of a of metabolic labeling caused secretion of labeled APPs for >3 hr. C-terminal APP fragment starting at Asns8” of the APP695 se- The sialic acid-rich form of 120 kDa started to appear in the quence. This fragment was detected reproducibly in all of our medium after 30-60 min, whereas the 100 kDa form appeared experiments and is an authentic product of APP processing in only after 60 min and beyond (Fig. 4). The most likely interpre- neurons. Because this cleavage product is found in equal amounts 906 J. Neurosci., February 1, 1996, 76(3):899-908 Simons et al. l Neuronal APP Processing

120

100

80

eo

40

20

64 - 0 NIKTEEISEVKNDAEFRNDSQYEVH

32 - 16 -

6-

LVPFAEDVQSNXQAIIQLWVQQVVI QKLVFFAhDVQSNNQAIIQLNVQQV BVRHQKLVFFAEDVQSNKQAIIQLN Cy&Sl-25

Figure 6. Analysis of APP695derived C-terminal fragments in hippocampal neurons. A, Hippocampal neurons were infected with SFV/APP695 and metabolically labeled with [35S]methionine or [“Hlphenylalanine. Cells were solubilized, and detergent-soluble lysates were immunoprecipitated with Ab B12/4. Immunoprecipitates were separated on a lo-20% Tris-tricine gel. Radiosequence analysis of the [35S]methionine-labeled sample is shown for the 12 kDa fragment (B). Radioactivity obtained at each cycle for the [ Hlphenylalanine-labeled samples is graphed for the 10 kDa (C) and 8-8.5 kDa C-terminal fragments (D).

compared with those of the the (Y- and P-cleaved fragments, it N-terminal heterogeneity, yielding three major products. One representsa significant intermediate in APP metabolism in these fragment started at residue Leu”, which is at the classical cells. Whether this intermediate servesas a precursor for PA4 a-secretasesite (Esch et al., 1990). The other two fragmentsare production is under further investigation.A complex set of 8-12 slightly longer, with N termini likely starting at positionsGln15 kDa C-terminal fragments also has been isolated from brain and Glu” of the PA4 sequence,suggesting that ru-secretasein homogenates,which suggeststhat these fragments also are pro- neuronscleaves APP at three different positions.The alternative ducedin vivo (Estuset al., 1992). Expressionof /3A4-bearingAPP explanation, that a-secretaseprocessing in neuronsis performed C-terminal fragmentshas shownthat thesefragments have a high by a set of different enzymes,is more likely. The latter possibility tendencyto aggregateboth in vitro (Dyrks et al., 1988)and in cells was advancedrecently by other investigators(Zhong et al., 1994) (Wolf et al., 1990). Therefore, PA4-containing fragments may basedon studiesin fibroblasts.Multiple a-secretasesalso would function not only as precursorsfor flA4, but they also could form explain the extremely relaxed specificity of cy-secretase(Sisodia et intracellular aggregatesduring the pathogenesisof AD. al., 1992;Maruyama et al., 1991;De Strooper et al., 1993)and the Although radiosequencingof the 10 kDa C-terminal fragment problem of inhibiting its activity by a panel of different (Fig. 6B) showed that it was generated by a single type of inhibitors (De Strooper et al., 1992). P-cleavageyielding a fragment starting at Asp’ of the PA4 se- Of particular relevance for AD is that one of the alternative quence, the 8-8.5 kDa C-terminal fragments displayed o-cleaved fragmentsstarted exactly at a position identified previ- Simons et al. l Neuronal APP Processing J. Neurosci., February 1, 1996, 76(3):899-908 907

ously as the first residue of PA4 (ll-42), a major component of De Hoop MJ, Huber LA, Stenmark H, Williamson E, Zerial M, Parton AD plaques (Masters et al., 1985; Naslund et al., 1994). This RG, Dotti CG (1994) The involvement of the small GTP-binding pro- finding, therefore, corroborates our conclusion that neurons are tein RabSa in neuronal endocytosis. Neuron l3:l l-22. De Strooper B, Craessaerts K, Dewachter I, Moechars D, Greenberg B, the primary producers of amyloidogenic peptides in AD. Van Leuven F, Van Den Berghe H (199Sa) Basolateral secretion of When differences in APP processing in neuronal and non- amyloid precursor protein in MDCK cells is disturbed by alterations of ncuronal cells are considered, it is also important to compare APP intracellular pH and by introducing a mutation associated with familial trafficking in various cell types. Recent studies have shown that Alzheimer’s disease. J Biol Chem 270:4058-4065. APP is sorted to the basolateral side in epithelial MDCK cells De Strooper B, Simons M, Multhaup G, Van Leuven F, Beyreuther K, Dotti CG (1995b) Production of intracellular amyloid-containing frag- (Haass et al., 1994; Lo et al., 1994; De Strooper et al., 1995a), ments in neurons expressing mutants of human amyloid precursor where its secretory products are released. Less PA4 appears to be protein causing Alzheimer’s disease and protection against amyloido- produced in MDCK cells (Haass et al., 1994) compared with genesis by subtle amino acid substitutions in the rodent sequence. hippocampal neurons. Usually, basolateral proteins are routed to EMBO J 14:4932-493X. the dendrites in neurons and apical proteins are transported to De Strooper B, Umans L, Van Leuven F, Van Den Berghe H (1993) Study of the synthesis and secretion of normal and artificial mutants of the axon (Craig and Banker, 1994). The reason APP does not murine amyloid precursor protein (APP): cleavage of APP occurs in a follow this scheme is not yet known. It may be that the basolateral late compartment of the default secretion pathway. J Cell Biol sorting signals in APP are inactivated by post-translational mod- 12 1:295-304. ifications in neurons. One such possible modification is phosphor- DC Stroopcr B, Van Leuven F, Van den Berghe H (1992) a2- ylation of the basolateral signal (Mostov et al., 1992). Alterna- Macroglobulin and other proteinase inhibitors do not interfere with the secretion of amyloid precursor protein in mouse neuroblastoma cells. tively, the protein might interact with basolateral proteins in FEBS Lett 308:50-53. MDCK cells and be carried piggyback to the basolateral plasma Dotti CC, Sullivan CA, Banker GA (1988) The establishment of polarity membrane. Another possibility is that the sorting machinery in by hippocampal neurons in culture. J Neurosci 8:1454-1468. neurons and epithelial cells differs, and that this leads to different Dyrks T, Weidemann A, Multhaup G, Salbaum JM, Lemaire HG, Kang J, sorting behavior. Future work will have to address these impor- Mullcr HB, Masters CL, Beyreuther K (1988) Identification, trans- membrane orientation and biogenesis of the amyloid A4 precursor of tant issues and pinpoint the subcellular localization of the pro- Alzheimer’s disease. EMBO J 7:949-957. cessing events. Esch FS, Keim PS, Beattie EC, Blather RW, Culwell AR, Oltersdorf T, In conclusion, our results support the working hypothesis that McClure D, Ward PJ (1990) Cleavage of peptide during the intracellular trafficking of APP is related to its metabolism, constitutive processing of its precursor. Science 24X:1 122-l 124. and our data emphasize the necessity to investigate the metabo- Estus S, Goldc TE, Kunishita T, Blades D, Lowery D, Eisen M, Usiak M, Qu XM, Tabira T, Greenberg BD, Younkin SG (1992) Potentially lism of APP in polarized neurons. The finding of several new amyloidogenic, carboxy-terminal derivatives of the amyloid protein proteolytic cleavage sites in neurons supports this contention and precursor. Science 255:726-728. has encouraged us to analyze the metabolism of the familial Games D, Adams D, Alessandrini R, Barbour R, Berthelette P, Blackwell mutants of APP in detail in this system as well (De Strooper et al., C, Carr T, Clemens J, Donaldson T, Gillespie F, Guido T, Hagopian S, 1995b). We anticipate that the current approach of expressing Johnson-Wood K, Khan K, Lee M, Leibovitz P, Lieberburg I, Little S, heterologous proteins in fully differentiated neurons using the Masliah E, McConlogue L, Montoya-Zavala M, Mucke L, Paganini L, Penniman E, Power M, Schenk D, Seubcrt P, Snyder B, Soriano F, Tan SFV expression system will prove useful for the study of many H, Vitalc J, Wadsworth S, Wolozin B, Zhao J (1995) Alzheimer-type other central nervous diseases and will be widely applicable. In neuropathology in transgenic mice overexpressing V717F beta-amyloid addition, this system might serve as an informative in vitro model precursor protein. Nature 373:523-527. for AD because of the high production of PA4 and amyloidogenic Glenner G, Wong C (1984) Alzheimer’s disease: initial report of the purification and characterization of a novel ccrehrovascular amyloid APP fragments. protein. Biochem Biophys Rcs Commun 120:885-X90. 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