Conn et al.

Identification of full length P-amyloid precursor protein in human neuronal and non-neuronal cell culture supernatant: a possible extracellular source for the generation of AD Kelly J . Conn', Gregoris Papastoitsist, Barbara Meckelein' and Carmela R. Abraham '

'Arthritis Center, Boston University School of Medicine, Boston, MA 02 1 18, USA 'Current address: Institute of Molecular Biology, Inc., Worcester, MA 01605, USA

KEY WORDS: ALZHEIMER'S DISEASE (AD), AMYLOID PRECURSOR PROTEIN (APPP). AMLOID BETA PROTEIN (A@, CELL CULTURE, LYTR~CELLULU PROCESSING, AMYLOID PRECLIRSOR-LIKE PROTEIN ( A P U )

Abstract Pprotein (AP) which is deposited in the amyloidplaques

and vasculature in brains ofAlzheimer's Disease (AD) pa- tients is a 39 to 43 amino acid peptide proteolytically de- rived from the amyloid precursor protein (APPP). Three major isoforms are expressed in the brain: APPP,J1 and APPP,,w which contain a Kunitz-like protease inhibitor domain 0, andAPPP,. To date it is still unknown which APPP isoforms are the precursors ofAB which proteolytic pathways are involved in its production, and ifthe process- ing occurs intracellularly and/or extracellularly. We now report the identification, by Western blot analysis, of an Apcontaining APPP protein which co-migrates with full length recombinant APPP,,, in the culture supernatant of two human neuroblastoma cell lines and in one human kid- ney cell line. This protein is recognized with six different antibodies towards APPP targeting intracellular; extracel-

Mar, and the Apregion ofAPPP The immunodetection of thb APprecursor is shown to be specific by absorption. The presence of full length APPP in culture supernatant strongly suggests that some processing ofAPPP may occur extracellularly. The recent identification of two soluble and1 or secretedproteases from AD and monkey brain both ca- pable of processing recombinant APPP in vitrolJ suggests that Approduction may occur extracellularly in vivo by an undescribed mechanism,

he extracellular accumulation of amyloid P protein (AP) in the brain parenchyma and vasculature of T patients with Alzheimer's disease (AD) is a

neuropathological hallmark of the d i s e a ~ e ' ~ . AP is the pro- teolytic product of a larger integral membrane protein, the amyloid precursor protein (APPP) of which there are three major isoforms present in the brain: APPP69s, APPP7S, and APPP,,, ranging in molecular weight from 1 10-135 kDa7n8.

Correspondence: Carmela R. Abraham, Ph.D., Department of Medicine, Boston University School of Medicine, 80 E. Concord Street, K-5, Boston, MA02118,USA Tel. 617-638-4310; Fax 617-638-5226

Submitted: August 30, 1994 Revision Accepted: October 6 , 1994

232

Am

yloi

d D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y Q

UT

Que

ensl

and

Uni

vers

ity o

f T

ech

on 1

1/22

/14

For

pers

onal

use

onl

y.

Arnyloid: Int. J. Exp. Clin. Invest. I , 232-239 (1994)

A number of mutations have been discovered in the APPP molecule in family members with autosomal domi- nant familial AD (FAD). Three of the FAD linked muta- tions convert the valine located three residues carboxyl to AD,, (Val,,, in APPP,,,,) to isole~cine~-’~, phenylalanineI3, or glycinel,. A fourth (“Swedish”) double mutation changes the lysine-methionine to asparagine-leucine at the -2 and -1 residues from the +1 aspartate (lys,,,-met,,, inAPPP,,,)”. The proximity of these mutations to AP suggest that al- tered enzymatic processing of APPP at the COOH and/or NH, terminus could be causative of AD.

Normal secretory processing ofAPPPI6 which occurs at AD,, by an enzyme termed a-secretase generates a non- amyloidogenic COOH-terminal fragment and a large NH,- terminal fragment, which is the main secreted form of APPP17,18. Alternative processing of APPP, by an enzyme termed p-secretase, results in a second secreted form of APPP whose COOH-terminus amino acid is thought to be Met, the amino acid at position -1 relative to the NH,-ter- minus 0fAp19. P-secretase cleavage also produces a COOH- terminal fragment of 99 residues which is believed to be a precursor to AP. A third type of cleavage, y-secretase, pro- cesses APPP at AD,,,.

Soluble AD has been detected in the media of transfected cultured cells expressing ApPP and in normal biological fluids2k223. Transfected cell systems expressing the Swed- ish double mutation secrete 5 to 8 times more AP in vitro than those transfected with the wild type ~equence~~ . !~ , sug- gesting that these mutations increase the affinity of P- secretase for its substrate or that another protease which can cleave between Leu andAsp becomes a factor in APPP processing. Although the location and mechanism of the p-secretase cleavage has remained elusive, there has been increasing evidence that transfected cell systems process APPP intracellularly in the endosomal/lysosomal sys- tem26.27. Most recently, in a transfected cell culture system expressing the “Swedish” double mutation, AD has been located in intracellular compartments28.

Although transfected cell systems have been used ex- tensively for studying APPP metabolism and trafficking, very few groups have used non-transfected systems to study APPP metabolism. Here we report the identification of full length ApPP in the culture supernatant of non-transfected neuronal and non-neuronal cell lines. This finding suggests that an additional pathway may exist for APPP processing. The recent identification of a soluble and/or secreted zinc- metalloprotease AD brain!, which cleaves a synthetic sub- strate flanking the NH,-terminus of theAP at the p-secretase site and which is able to process recombinantAPPP in vitro to potentially amyloidogenic fragments, suggests that ex- tracellular processing of APPP could generate, to a certain extent, at least some soluble AP molecules that have been seen in cell media, CSF and in the AD brain2g.

Methods

Cell culture All cells were grown in“comp1ete” medium which con-

sists of Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, 5 units/ml peni- cillin G and 5 pg/ml streptomycin (Gibco, Gaithersburg, MD) and maintained in a humidified incubator at 37” C with 10% CO,. Human neuroblastoma (SK-N-MC and SK- N-SH) and human embryonal kidney cells (293) were pur- chased from ATCC and were grown in T-80 or T- 175 flasks (Marsh, Rochester, Ny; Falcon, Oxnard, CA) with com- plete media until nearly confluent. Cell viability was moni- tored by staining with 0.8% trypan blue and by measuring lactose dehydrogenase activity in the cell lysates and cul- ture supernatants using a commercially available diagnos- tic kit (Sigma, St. Louis, MO) and was greater than 98%.

Experimental assay 1 x lo5 - 5 x 10, cells (depending on cell and flask type)

were plated in T-80 or T-175 flasks and grown to near confluency. Cells were washed in 4 ml serum-free, antibi- otic-free DMEM (10 ml for T-175 flask) twice for 30 sec- onds and once for 30 minutes before being incubated for 12-16 hours in 4 ml fresh (10 ml for T-175 flask) serum- free, antibiotic-free DMEM. At the time of harvest, culture supernatant was collected and centrifuged at 100,000 x g for 60 minutes or 200,000 x g for 3 hours at 4°C. The su- pernatant was then concentrated 80-1 00 times using a Centricon- 10 microconcentrator (Amicon, Beverly, MA) and protein concentration was determined by the BCA method according to manufacturer’s instructions (Pierce, Rockford, IL).

Imm unoprecipitation Neuroblastoma cells (SK-N-MC) were grown to

confluency in a 60 mm dish (Corning) washed as above in 2 ml serum-free, antibiotic-free, methionine-free DMEM supplemented with 1% FCS before labeling with 200 pCi E~pre%’~s protein labeling mix (NEN, Boston, MA) in 2 ml of the same medium. Culture supernatant was centri- fuged at 100,000 x g and immunoprecipitated with 1:500 R1280 as described p r e v i ~ u s l y ~ ~ . APPP-transfected 293 culture supernatant, a generous gift from Dr. D. J. Selkoe, was described p rev i~us ly~~ .

Gel electrophoresis and immunoblotting Proteins were separated by SDS PAGE30. Ten to 30 pg

of total protein were loaded on either 4 2 0 % tris-tricine gradient gels (Novex, San Diego, CA) or 7.5% !is-glycine gels and transferred to Immobilon-P PVDF membranes (Millipore, Bedford, MA) as described previously31. Mem- branes were blocked with 5% non-fat dried milk in TBST

233

Am

yloi

d D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y Q

UT

Que

ensl

and

Uni

vers

ity o

f T

ech

on 1

1/22

/14

For

pers

onal

use

onl

y.

extracellular

COOH

FIGURE 1. Schematic map of the ApPP molecule, depicting the epitopes for the various antibodies used in this study. Antibodies to the intracellular domains of ApPP include C8 (targeting the last 20 amino acids) and IOEl (targeting APPP,,,, of APPP,,). Antibody R1280 is produced against AP 1-40. AblO recognizes APPP- of APPP,,,, while 22C11 targets an unidentified region of the N-terminus of APPP. 6E10 recognizes A&-,, and is specific for ApPP and does not recognize APLP.

(10 mM Tris-HC1 pH 7.9, 150 mM NaCl, 0.05% between 20) and incubated with primary antibody diluted in TBST for 60 minutes to 16 hours. Membranes were probed with alkaline phosphatase conjugated secondary antibody (Promega, Madison, WI), and bands were visualized with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (Sigma). As a control, a human recombinant full length APPP,,,, a gift from Drs. R. Siman and R. Scott, Cephalon, Inc, was used.

Antibodies against APPP The following antibodies (Abs) were used (Figure 1) at

the dilution indicated: C8, a rabbit polyclonal Ab raised against a synthetic peptide comprising the last 20 amino acids of APPP32, 1:500; a mouse monoclonal Ab 22C11, targeting the N-terminus of APPP (Boehringer Mannheim, Indianapolis, IN), 1500; Ab103), a rabbit polyclonal Ab recognizing APPP,,,, ofAPPP,,,, 1:300; 10E134, a rabbit polyclonal AD targeting APPP649472 of ADPP695, 1 :500; R12803,, a rabbit polyclonal Ab targeting amino acids 1- 40 of the AP molecule, 1 :500; and 6E103,, a mouse mono- clonal Ab recognizing Ap,-,, 1 :500. All primary antibodies (except 22C11 and 6E10) were pre-absorbed by incubat- ing 10 pg antigedpl antibody in 50-100 p1 TBST for 16 hours at 4°C.

Results Our initial identification, by Western blot, of a high

molecular weight APPP species in the culture supernatant of human neuroblastoma (SK-N-MC) cells using an anti- body to the C-terminus of APPP came as a surprise. To explore the possibility that these cells were releasing fidl length APPP into the culture supernatant, we tested the same culture supernatant with a variety of antibodies tar-

geting different epitopes of APPP. To this end, 10 ml of culture supematant was collected and centrifuged at 200,000 x g for 3 hours at 4°C. Soluble proteins were then concen- trated, electrophoresed, transferred and probed with six different antibodies directed towards various epitopes of APPP. For our study, we chose an antibody which targeted the very C-terminus of APPP, C8; one that recognized an intracellular domain, 10E1; an antibody to the entire AD region, R1280; two antibodies to extracellular domains, AblO and 22C11; and an antibody that was specific for APPP and not for APLP3’-39, 6E10 (Figure 1).

All the antibodies used in this study recognized a pro- tein from human neuroblastoma culture supernatant which co-migrated with human recombinant full length APPP,,, (rAPPP,,,). To determine if the immunodetection was spe- cific, four of the antibodies, for which we obtained the cor- responding antigen, were pre-absorbed for 16 hours at 4°C in a small volume and diluted to the appropriate concentra- tion before immunodetection by Western blot of culture supernatant proteins separated on a Tris-tricine 4-20% gra- dient gel. When each of the antibodies was pre-absorbed, immunodetection of the full length and other ADPP spe- cies was eliminated or greatly reduced (Figure 2). To de- termine if other non-transfected human cells produced a soluble high molecular weight AP-containing APPP spe- cies, we tested an additional human neuroblastoma cell line (SK-N-SH) and also a human kidney cell line (293). 4 ml of culture supernatant were collected from each cell type, centrifuged at 100,000 x g, concentrated and analyzed by Western blot as before. Figure 3 shows immunodetection of an AD-containing APPP species with antibodies AblO, which recognizes the N-terminus of the AD, and with 1 OE 1, which recognizes an intracellular region ofAPPP. All three human culture supernatants had an AP containing species of APPP that co-migrated with full length recombinant

234

Am

yloi

d D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y Q

UT

Que

ensl

and

Uni

vers

ity o

f T

ech

on 1

1/22

/14

For

pers

onal

use

onl

y.

Amyloid: Int. J. Exp. Clin. Invest. I, 232-239 (1994)

FIGURE 2. Specific immunodetection with (even lane numbers) and without (odd lane numbers) absorption with the corresponding peptides of full length ApPP in the culture supernatant of human neuroblastoma (SK-N-MC) cells by four different antibodies. Lanes 1 and 2, antibody R1280; lanes 3 and 4, antibody C8; lanes 5 and 6, antibody 10E1; lanes 7 and 8, antibody AblO; lane 9, recombinant human (rh)APPP,,,.

APPP,,,. Note that Ab 10 also recognizes a lower molecu- lar weight APPP, most probably the secreted form which is released by action of a-secretase and contains at the C- terminus the first 16 amino acids of AP.

Once we had confirmed the presence of an AP contain- ing APPP species in the culture supernatant of three hu- man cell lines, we wanted to characterize fiuther this pro- tein using two additional antibodies. Neuroblastoma (SK- N-SH) culture supernatant was collected as before, centri- fuged at 200,000 x g, and blotted as before. To confirm that this protein was not an amyloid precursor-like protein (APLP) species, we immunobloted with antibodies 6E 10 (data not shown) and R1280, both of which recognize APPP but not APLP. Both antibodies recognized a protein which co-migrated with rApPP,5,. To determine more clearly if this species contains the NH,-terminus ofAPPP, we probed the culture supernatant with 22C11 which recognizes an epitope in the NH,-terminal region ofAPPP. Figure 4 shows the migration of the APPP species detected with 22C11 and R1280 on a 7.5% Tris-glycine gel. 22C11 detects the secreted NH,-terminus of APPP and also an APPP species which co-migrates with the rAPPP,,, control, as well as with the APPP detected by the R1280 antibody. R1280 immunoreacts extremely weakly with the secreted form of APPP (D. J. Selkoe, personal communication). This sug- gests that the APPP species not only contains the very C- terminus ofAPPP, as seen in Figure 2, but also that it has a substantial amount of the NH, terminus. Because the exact epitope of 22C11 is not known, we can not determine if this secreted species contains the complete NH, terminus.

In order to determine if this extracellular source of A0 could serve as a precursor, we wanted to determine first if the neuroblastoma (SK-N-SH) cells that we were working with produced and released AP into the culture superna- tant. AP is produced at such low levels in these cells that visualization by Western blot was not possible. Figure 5

shows metabolically labeled culture supernatant immuno- precipitated with R1280. AP is found in the culture super- natant in very low levels as compared with a transfected cell system when comparable amounts of radioactivity were loaded. The presence of AP confirms that APPP process- ing is occuning in these cells although the pathway of this processing has yet to be elucidated.

Discussion The association ofAP withAlzheimer's disease has been

well established40. The accumulation of sc'uble AP into fibrillar amyloid plaques is believed to be a primary event in the pathology of the disease. It is not yet known what causes the abundance of plaques in AD brain. However, studies with transfected cell systems bearing FAD muta- tions suggest that overproduction of AP or production of longer Awl could lead to an increase in plaque formation. Halting or reducing plaque formation, either by preventing the fibrillization of AD, or by reducing the amount of AP generated could potentially lead to therapy for the disease.

It is not yet fully understood which APPP isoforms are the precursors of AP, what cellular pathways are involved in APPP processing, or how soluble AD becomes fibrillar and precipitates in the brain. The use of transfected cell culture systems has suggested that one pathway for AP gen- eration occurs in the endosomatllysosomal compartments. However, to date, this type of experiment has not been car- ried out with non-transfected cell systems. This is in a large part due to the fact that non-transfected cells produce very low levels of AP compared with transfected cell systems (Figure 5). Our findings here, of an extracellular AP con- taining species of APPP, suggests that an additional path- way may exist for APPP processing in these human non- transfected cells.

23 5

Am

yloi

d D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y Q

UT

Que

ensl

and

Uni

vers

ity o

f T

ech

on 1

1/22

/14

For

pers

onal

use

onl

y.

M w 1 2 3 4 5 6

97 68

FIGURE 3. Detection of AD-containing ApPP species in the culture supernatant of two human neuroblastoma cell lines and a human kidney cell line. Lanes 1 and 2, neuroblastoma cells (SK-N-MC); lanes 3 and 4, kidney (293); lanes 5 and 6, neuroblastoma (SK-N-SH). Lanes 1 , 3 and 5 are probed with 10E1, lanes 2 , 4 and 6 are probed with antibody Abl 0.

Other extracellular C-terminal containing APPP proteins have been reported recently using systems different from the one described here. Such C-terminal containing APPP proteins were described in the brains of rats subjected to global cerebral ischemia (GCI) induced by cardiac arrest. The APPP proteins were labeled with antibodies to N-ter- minal, P-amyloid peptide and C-terminal domains of APPP4*. This study suggests that proteolytically cleaved fragments of the full-length APPP or the entire APPP mol- ecule accumulates extracellularly after GCI. Low levels of full length APPP have also been detected in the media of PC12 cell cultures43 and recent work by Goldgaber et a1 (this issue) provides evidence that transfected cell systems as well as primary cell cultures also produce extracellular soluble APPP. Our current study describes that extracellular APPP is

being produced by human, non-transfected cells and is not associated with cell death. Because these cells are tumor cell lines, the possibility exists that the APPP we see is not truly soluble but might be associated with lipid vesicles. It is known that a number of tumor cells frequently shed por- tions of their plasma membrane, as these cells attempt to evade the immune system by discarding major histocom- patibility complex (MHC) molecules". Such lipid vesicles could elude a 200,000 x g centrifugation, falsely suggest- ing the presence of soluble APPP. We are presently per- forming experiments to determine if this is the case or if the APPP we see is truly soluble.

The neuroblastoma cell line (SK-N-MC) in which we have done the majority of our work provides an excellent system for studying APPP processing. These human non- transfected cells not only produce extracellular fill1 length APPP but they also produce soluble AP (Figure 5). Fur- thermore, these cells secrete a zinc metalloprotease which

1 2 3

FIGURE 4. Detection of ApPP from neuroblastoma culture supernatant with 22Cl1, lane 2, and R1280, lane 3, after separation on a 7.5% gel. Note that 22Cll also detects secreted N-terminal fragments which migrate below the band detected with R1280 and the upper band detected with 22C11. Lane 1 is rhADPP,,,.

has been shown to be capable of processing rAPPP,Sl into potentially amyloidogenic fragments1. Future studies in- clude the evaluation of proteolytic products resulting from the incubation of purified APPP species described here with the purified metalloprotease.

Additionally, these cells could be used to identify can- didates for the y-secretase cleavage. p-secretase has been more widely studied than y-secretase because the cleavage site is more accessible than the cleavage site ofy-secretase, which is located in the membrane. Explaining how y-

23 6

Am

yloi

d D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y Q

UT

Que

ensl

and

Uni

vers

ity o

f T

ech

on 1

1/22

/14

For

pers

onal

use

onl

y.

Amyloid: Int. J Exp. Clin. Invest. 1. 232-239 (1994)

FIGURE 5. lmmunoprecipitation with R1280 of metabolically-labeled AP from transfected and non-transfected culture supernatant. Lanes 1 and 3 are iodinated 1-40 AD; lane 2 is culture supernatant from human neuroblastoma cells; lane 4 is human kidney (293) cells transfected with APPP,,, bearing the Swedish mutations. Lanes 1 and 2 were exposed 18 days while lanes 3 and 4 were exposed 7 days.

secretase cleavage occurs while the APPP transverses the membranes has been difficult. The identification of extra- cellular precursors of AP suggests that both the P and y cleavages could occur outside the cell. Processing ofAPPP extracellularly would enable a variety of enzymes to ac- cess the COOH-terminus of the AP molecule which would otherwise be inaccessible while APPP was in the membrane, either at the cell surface or in endosomaVlysosoma1 vesicles.

Acknowledgments We wish to thank Drs. D. J. Selkoe for the C8 and R1280

antibodies, for the human kidney cells (293) transfected with APPP,,, bearing the Swedish mutations and for the iodinated 1- 40 AP; E. Koo for AblO, and R. Siman for IOEl antibodies and the respective peptides used for absorption experiments and Drs. R. Siman and R. Scott for the gift of human recombinant APPP,,,. This study was supported by grants from NIH (AGO9905 and AGOOOOl), Cephalon, Inc. and the Zenith Award from the Alzheimer’s Association to C.R.A.

References

1 Papastoitsis G, Siman R, Scott Rand Abraham CR (1994). Identification of a metalloprotease from Alzheimer’s dis- ease brain able to degrade the P-amyloid precursor protein and generate amyloidogenic fragments. Biochemistry, 33, 192-199

2

3

4

5

6

7

8

9

Razzaboni BL, Papastoitsis G, Koo EH and Abraham CR ( 1 992). A calcium stimulated serine protease from monkey brain degrades the P-amyloid precursor protein. Brain Res,

Glenner GG and Wong CW (1984). Alzheimer’s disease and Down’s syndrome: sharing of a unique cerebrovascu- lar amyloid fibril protein. Biochem Biophp Res Commun,

Prelli F, Castano E, Glenner GG and Frangione B (1988). Differences between vascular and plaque core amyloid in Alzheimer’s disease and Down’s syndrome. J Neurochem, 51,648651 Masters CL, Simms G, Weinman NA, Multhaup G, McDonald BL and Beyreuther K (1 985). Amyloid plaque core protein in Alzheimer disease and Down syndrome. Proc Natl Acad Sci USA, 82,42454249 Selkoe DJ, Abraham CR, Podlisny MB and Duffy LK (1986). Isolation of low-molecular weight proteins from amyloid plaque fibers in Alzheimer’s disease. JNeumchem,

Muller-Hill B and Beyreuther K (1989). Molecular biol- ogy of Alzheimer’s disease. Ann Rev Biochem, 58, 287- 307 Selkoe DJ (1 99 I ) . Molecular pathology ofAlzheimer’s dis- ease. Neuron, 6,487-498 Goate A, Chartier-Harlin MC, Mullan M, Brown J, Crawford F, Fidani L, Giuffra L, Hayes A, Irving N, James L, Mant R, Newton P, Rooke K, Roques P, Talbot C, Pericak-Vance M, RosesA, Williamson R, Rossor M, Owen M and Hardy J (1991). Segregation of a missense mutation in the amyloid precursor protein gene with familial Alzheimer’s disease. Nature, 349, 704-706

589,207-216

122, 1131-1135

46, 1820-1834

237

Am

yloi

d D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y Q

UT

Que

ensl

and

Uni

vers

ity o

f T

ech

on 1

1/22

/14

For

pers

onal

use

onl

y.

Conn et al.

10

11

12

13

14

15

16

17

18

19

20

21

22

23

Yoshioka K, Miki T, Katsuya T, Ogihara T and Sakaki Y (1991). The 717 Val-Ile substitution in amyloid precursor protein is associated with familial Alzheimer’s disease re- gardless of ethnic groups. Biochem Biophys Res Commun,

Narsue S, Igarashi S, Kobayashi H, Aoki K, Inuzuka T, Kaneko K, Shimizu T, Iihara K, Kojima T, Miyatake T and Tsuji S (1991). Mis-sense mutation Val-Ile in exon 17 of amyloid precursor protein gene in Japanese familial Alzheimer’s disease. Lancet, 337,978-979 Hardy J (Alzheimer’s Disease Research Group) (1 99 1). Molecular classification of Alzheimer’s disease. Lancet, 337, 1342 Murrel J, Farlow M, Ghetti B and Benson MD (1991). A mutation in the amyloid precursor protein associated with hereditary Alzheimer’s disease. Science, 254,97-99 Chartier-Harlin MC, Crawford F, Houlden H, Warren A, Hughes D, Fidani L, Goate A, Rossor M, Roques P, Hardy J and Mullan M (1991). Early-onset Alzheimer’s disease caused by mutations at codon 717 of the P-amyloid pre- cursor protein gene. Nature, 353,844-846 Mullan M, Crawford F, Axelman K, Houlden H, Lilius L, Winblad B and Lannfelt L (1992). A pathogenic mutation for probable Alzheimer’s disease in the APPP gene at the N-terminus of P-amyloid. Nature Genet, 1, 345-347 Weidemann A, Konig G, Bunke D, Fischer P, Salbaum M, Masters C and Beyreuther K (1989). Identification, bio- genesis, and localization of precursors of Alzheimer’s dis- ease A4 amyloid protein. Cell, 7, 115-126 Esch FS, Keim PS, Beattie EC, Blacher RW, Culwell AR, Oltersdorf T, McClure D and Ward PJ (1990). Cleavage of amyloid beta protein during constitutive processing of its precursor. Science, 248, 1122-1 124 Sisodia SS, Koo EH, Beyreuther K, UnterbeckA and Price DL (1990). Evidence that amyloid protein in Alzheimer’s disease is not derived by normal processing. Science, 248.

Seubert P, OltersdorfT, Lee MG, Barbour R, Blomquist C, Davis DL, Bryant K, Fritz LC, Galasko D, Thai LJ, Lieberburg I and Schenk DB (1993). Secretion of beta- amyloid precursor protein cleaved at the amino terminus of the beta-amyloid peptide. Nature, 361, 26C263 Haass C, Schlossmacher MG, Hung AY, Vigo-Pelfrey C, Mellon A, Ostaszewski BL, Lieberburg I, Koo EH, Schenk D, Teplow DB and Selkoe DJ (1992). Amyloid beta-pep- tide is produced by cultured cells during normal nietabo- lism. Nature, 359,322-325 Seubert P, Vigo-Pelfrey C, Esch F, Lee M, Dovey H, Davis D, Sinha S, Schlossmacher M, Whaley J, Swindlehurst C, McCormack R, Wolfert R and Selkoe DJ (1992). Isolation and quantification of soluble Alzheimer’s P-peptide from biological fluids. Nature, 359, 325-327 Shoji M, Golde TE, Ghiso J, Cheung TT, Estus S, Shaffer LM, Cai XD, McKay DB, Tintner R, Frangione B and Younkin SG (1992). Production of the Alzheinier aniyloid beta protein by normal proteolytic processing. Science, 258, 126-129 Busciglio J, Gabuzda D, Matsudeira P and Yankner B (1993). Generation of 0-amyloid in the secretory pathway

178, 1141-1 146

1122-1 124

24

25

26

27

28

29

30

31

32

33

34

35

36

37

in neuronal and non-neuronal cells. Proc NatlAcad Sci USA,

Citron M, Oltersdorf T, Haass C, McConlongue 1, Hung AY, Seubert P, Vigo-Pelfrey C, Lieberburg I and Selkoe DJ (1 992). Mutation of the P-amyloid precursor protein in fa- milial Alzheimer’s disease increases P-protein production. Nature, 360,672474 Cai X, Golde T and Younkin S (1993). Release of excess amyloid P protein from a mutant amyloid P protein precur- sor. Science, 259, 5 14-5 16 Golde TE, Estus S, Younkin LH, Selkoe DJ and Younlun SG (1992): Processing of the amyloid protein precursor to potentially amyloidogenic derivatives. Science, 255, 728- 730 Haass C, Koo EH, Mellon A, Hung AY and Selkoe DJ (1992b). Targeting of cell-surface beta-amyloid precursor protein to lysosomes: alternative processing into amyloid- bearing fragments Nature, 357, 50CL503 Sisodia SS and Thinakaran G (1 994). Trafficking and me- tabolism of the “Swedish” APPP variant. Neurobiol Ag- ing, 15, S76 Tabaton M, Nunzi MG, Xue R, Autilio-Gambetti L and Gambetti P (1994). Soluble amyloid P-protein (AP) is de- tected in Alzheimer but not normal brains. Neurobiol Ag- ing, 15, S79 Laemmli UK (1970). Cleavage of structural proteins dur- ing the assembly of the head of bacteriophage T4. Nature, 227, 68&685 Abraham CR, Selkoe DJ and Potter H (1988). Immu- nochemical identification of the serine protease inhibitor a- 1 -antichymotrypsin in the brain amyloid deposits of Alzheimer’s disease. Cell, 52, 487-501 Podlisny MB, Tolan D and Selkoe DJ (1991). Homology of the amyloid P-protein precursor in monkey and human supports a primate model for P-amyloidosis in Alzheimer’s disease. Am JPathol, 138, 1423-1435 Buxbaum JD, Gandy SE, Ciccheti P, Ehrlich ME, Czemik AJ, Fracasso RP, Ramabhadran TV, Unterbeck AJ and Greengard P (1 990). Processing of Alzheimer P/A4 pre- cursor protein: Modulation by agents that regulate protein phosphorylation. Proc Natl Acad Sci USA, 87,60036006 Siman R, Mistretta S, Durkin JT, Savage MJ, LohT, Trusko S and Scott RW (1993). Processing of the beta-amyloid precursor. Multiple proteases generate and degrade poten- tially amyloidogenic fragments. JBiol Chem, 268, 16602- 16609 Tamaoka A, Kalaria RN, Lieberburg I and Selkoe DJ (1992). Identification of a stable fragment of the Alzheimer amy- loid precursor containing the beta-protein in brain microvesseles. Proc Natl Acad Sci USA, 89, 1345-1349 Kim KS, Wen GY, Bancher C, Chen CMJ, Sapienza VJ, Hong H and Wisniewski HM (1990). Detection and quan- tification of amyloid P-peptide with 2 monoclonal anti- bodies. Neurosci Res Commun, 7, 113-122 Wasco W, Bupp K, Magendantz M, Gusella JF, Tanzi RE and Solomon F (1992). Identification of a mouse brain cDNA that encodes a protein related to the Alzheimer’s disease-associated amyloid beta protein precursor. Proc Nut1 Acad Sci USA, 89, 10758-10762

90,2092-2096

23 8

Am

yloi

d D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y Q

UT

Que

ensl

and

Uni

vers

ity o

f T

ech

on 1

1/22

/14

For

pers

onal

use

onl

y.

Amyloid: Int. J. Exp. Clin. Invest. 1, 232-239 (1994) ~

38

39

40

41

Sprecher CA, Grant FJ, G r i m G, O’Hara PJ, Norris F, Norris K, Foster DC (1993). Molecular cloning of the cDNA of a human amyloid precursor protein homolog: evidence for a multigene family. Biochem, 32,44814486 YanYC, Bai Y, Wang LF, Miao SY and Koide SS (1990). Characterization of cDNA encoding a human sperm mern- brane protein related toA4 amyloid protein. Pmc NutlAcud Sci USA, 87,2405-2408 Selkoe DJ (1994). Alzheimer’s disease: A central role for amyloid. J Neuroputhol Exp Neurol, 53,438447 Suzuki N, CheungTT, Cai XD, OdakaA, Otvos L, Eckman C, Golde TE and Younkin SG (1994). An increased per- centage of long amyloid P protein secreted by familial

42

43

44

amyloid p protein precursor (PAPP,,,) mutants. Science, 264, 1136-1 139 Pluta R, Kida E, Lossinsky AS, GolabekAA, Mossakowski MJ and Wisniewski HM (1994). Complete cerebral ischemia with short-term survival in rats induced by car- diac arrest. I. Extracellular accumulation of Alzheimer’s P-arnyloid protein precursor in the brain. Bruin Res, 649,

Ripellino JA, Vassilacopoulou D and Robakis NK (1994). Solubilization of full-length amyloid precursor proteins from PC12 cell membranes. JNeumsci Res, 39,211-218 Anichini A, Mortarini R and Parmiani G (1993). The role of cytokines in the modulation of cell surface antigens of human melanoma. Internut J Biol Murk, 8, 15 1-1 54

323-328

239

Am

yloi

d D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y Q

UT

Que

ensl

and

Uni

vers

ity o

f T

ech

on 1

1/22

/14

For

pers

onal

use

onl

y.