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Characterization of New Polyclonal Antibodies Specific for 40 and 42 Amino Acid-Long Amyloid β Peptides: Their Use to Examine the Cell Biology of Presenilins and the Immunohistochemistry of Sporadic Alzheimer’s Disease and Cerebral Amyloid Angiopathy Cases

Characterization of New Polyclonal Antibodies Specific for 40 and 42 Amino Acid-Long Amyloid β... Characterization of New Polyclonal Antibodies Specific for 40 and Amino Acid-Long f3 Amyloid Peptides: Their Use to Examine the Cell Biology of Presenilins and the of Immunohistochemistry Sporadic Alzheimer's Disease and Cerebral Amyloid Angiopathy Cases Helene Barelli,' Anthony Lebeau,' Jean Vizzavona,7 Pia Delaere,3 Nathalie Chevallier,' Cyril Drouot, Philippe Marambaud,' Karime Ancolio,' Joseph D. Buxbaum,4 Olga Khorkova,5 Jeff Heroux,5 Sudhir Sahasrabudhe,5 Jean Martinez,2 Jean-Marie Warter,6 Michel and Mohr,7 Frederic Checlerl 'IPMC du CNRS, UPR411, Valbonne, France 2URA CNRS 1845, Montpellier, France 3Rhone-Poulenc Rorer, Vitry Alfortville, France 4The Rockefeller University, New York, New York, U.S.A. 5Hoechst-Marion-Roussel, Sommerville, New Jersey, U.S.A. 6Service des Maladies du Systeme Nerveux et du Muscle, Hopitaux Universitaires, Strasbourg, France 7Institut de Pathologie, Strasbourg, France ABSTRACT Background: In Alzheimer's disease (AD), the main monitored after metabolic and labeling immunoprecipi- histological lesion is a proteinaceous tation deposit, the senile procedures. Immunohistochemical was analysis plaque, which is mainly composed of a peptide called on brains of and performed cerebrovas- sporadic typical The aggregation process is thought to occur through cular AP3. cases. amyloid angiopathy (CAA) enhanced concentration of A(340 or increased produc- Results: Dot and blot Western indicate that analyses tion of the more readily aggregating 42 amino acid-long fractions IgG-purified of antisera native and recognize species. AP342 denaturated A,Bs. FCA3340 and FCA3542 display full Materials and Methods: Specificity of the antibodies for and specificity A,B42, Antibodies A(340 respectively. was assessed by dot blot, Western blot, ELISA, and im- their immunoprecipitate respective synthetic AP3 species munoprecipitation procedures on synthetic and but also endog- A,Bs and their related p3 counterparts endog- enous produced by secreted HK293 cells. A,B and AP3 p3 secreted transfected enously human 293 by kidney cells. production by wild-type and mutated presenilin 1-ex- This allowed us to show that mutations on 1 presenilin pressing cells transiently transfected with ,3APP751 was similar increased ratios of triggered and its A(342 p342 over total counterpart and ELISA p3. AP assays allow detection of about 25-50 of pg/ml and remain linear Af3s to 750 to 1500 without up pg/ml any cross-reactivity. FCA18 and FCA3542 label diffuse and mature of Address plaques correspondence and reprint requests to: Dr. Fred- AD case whereas eric sporadic FCA3340 reveals Checler, IPMC du only the CNRS, UPR411, 660 route des Luci- oles, 06560 mature lesions and Valbonne, France. Phone: labels their (33) 4 93 95 77 particularly central dense 60; Fax: (33) 4 93 95 77 08; e-mail: core. [email protected] In a CAA case, FCA18 and FCA3340 reveal lepto- © THE 1997, PICOWER INSTITUTE PRESS. All rights reserved. Molecular Medicine, Volume 3, Number 10, October 1997 695-707 696 Molecular Medicine, Volume 3, Number 10, October 1997 meningeal and cortical arterioles whereas FCA3542 only these mutations misroute the J3APP to a compartment such structures. where but not a-secretase, cleavages are faintly labels y-secretase, antibodies exclusively recog- antibodies should prove useful Conclusions: Polyclonal modified. Overall, these (FCA3340) or (FCA3542) were ob- and diagnostic approaches, as suggested nizing A,340 for fundamental A,B42 that FAD-linked presenilins for cell biological, and tained. These demonstrated by their usefulness biochemical, p342 and A,342, suggesting that immunohistochemical techniques. similarly affect both have two antibodies We developed polyclonal INTRODUCTION Af40 (FCA3340) or that specifically recognize ob- One of the main histopathological lesions Af42 and another one able to serve as (FCA3542) in disease-af- served the cortex of Alzheimer's for both species (FCA18). an aspecific probe A,B This pro- fected brains is the senile plaque (1). These novel antibodies are shown to be amenable to be mainly teinaceous deposit appears biochemical dot, and Western blots) and to (ELISA, amyloid 13 (A,B) peptide (2,3) composed of the cell biological (immunoprecipitation of condi- precur- that is derived from a large polypeptidic tioned Furthermore, we media) approaches. precursor protein (f3APP) sor, the ,B amyloid clearly establish by immunohistochemistry the na- approaches have clearly es- (4,5). Biochemical ture of the species observed in diffuse, mature, AP3 process ultimately tablished that the pathological and vascular deposits of a sporadic AD case and leading to the extracellular peptide aggregation is AD a cerebro- those of a presenile case with typical the concentration and/or the dependent upon vascular amyloid angiopathy (CAA). nature of the Af3 species generated (6,7). Muta- tions for the early-onset familial responsible forms of Alzheimer's disease (AD) apparently trigger a disturbance of 3APP processing. Consis- tent with this view is the finding that a double METHODS MATERIALS AND mutation taking place adjacent to the N-termi- Synthesis, Antigen Coupling, and Peptide nus of the sequence (double Swedish muta- AP Rabbit Immunization Procedures tion [8]) led to enhanced production of a 40 An- amino acid-long species (A1340; [9-1 1]). All were synthetized by classical solid peptides AO3 of other mutation located near the C-terminus (Boc strategy) with methyl benzhy- phase the sequence (12,13) appeared to increase resin means of a semiautomatic drylamine by AP3 a 42 acid the production of longer amino (Neosystem NPS 4000) and were pu- A,1 apparatus of have (14), the aggregation properties which rified as described previously (21). The purity More re- been shown to be exacerbated (6,7). and amino acid composition were confirmed by mass cently, two genes have been identified on chro- amino acid analysis and electrospray spec- 14 1 for most of the to the mosomes and that account trometry. The octapeptide corresponding familial forms of the disease of A(340 and 42 a agressive early-onset common N-terminus (displaying The gene products, presenilins 1 and 2, residue at its DAEFRHDS- (15-17). cysteine C-terminus, interfere with 13APP processing since the C- likely Cys) and octapeptides mimicking specific mutations in trigger in- terminal ends of and 42 (with an N-terminal pathological presenilins A,B40 creased A1342 over A340 ratios (18-20). and Cys-MVG- cysteine, Cys-GLMVGGVV [Af340] described Immunohistochemical studies have been GVVIA were as previ- [A,B42]) coupled conducted to establish the A,B content of the to either bovine serum albumin (BSA) ously (22) cerebrovascular neuro- diffuse, mature, and or keyhole limpet hemocyanin (KLH, Calbiochem) The nature of the species to m-maleidoben- pathological insults. that was previously conjugated AP3 the lesions is still somewhat contro- zoic acid KLH-cou- composing N-hydrosuccinimide (Sigma). 1 mixed with an versial. This could be due to the nature of the pled antigens (about mg) were Freund then immunological tools, emphasizing the need for volume of complete adjuvant, equal antibodies able into New Zealand rabbits. the development of end-specific injected subcutaneously 1 and at the various Boosts were month later to fully discriminate among A,B spe- (0.5 mg) given sera cies 3-week intervals. Immunopositive generated. subsequent 697 H. Barelli et al.: Specific Antibodies to and A,f40 A1342 were monitored by dot blot (see with BSA- munological complexes were revealed with chlo- below) coupled antigens as screening ronaphtol as described previously (23). peptides. Purification of IgG Immunoprecipitation of Endogenous and The whole IgG fractions from various immune or Synthetic Species A,8 preimmune sera were obtained after treatment Transfected HK293 cells overexpressing the with octanoic acid according to the procedure Swedish mutated form of ,3APP751 were ob- previously described (23). tained, cultured, and metabolically labeled as previously described (25). Conditioned media (10 ml) were collected, diluted in one-tenth vol- Dot Blot Analysis ume of RIPA lOx buffer, incubated overnight BSA-coupled antigens (about 50 or various ng) with a 350-fold dilution of antibodies, then A,B species (resuspended in distilled water at a stirred for 4 hr in the presence of protein A- final concentration of 0.1 mg/ml) were dot-blot- sepharose (100 mg/ml, 100 Samples were gl). ted (1-2,g) onto nitrocellulose membranes (Hy- centrifuged, washed three times with radioim- bond-C, Amersham, les Ulis, France). Prior to the munoprecipitation assay (RIPA) 1 x, resus- immunochemical reaction, membranes were pended with loading buffer, electrophoresed on a treated in Tris buffer saline 140 mM (TBS: NaCl, 16.5% Tris-tricine gel, and radioautographed as 20 mM Tris-HCl, pH 7.5) containing 5% skim described elsewhere Stable (26). transfectants milk. Antibodies were then added for 4 hr at or overexpressing wild-type, mutated, delta various dilutions (primary screening of immun- Exon-9 presenilin 1 were (AE9PS1) transiently sera) or overnight at 40C at 1:500 (FCA18) and transfected with then la- ,BAPP75 1, metabolically 1:100 dilutions (FCA3340 and FCA3542) in the beled and analyzed for and secretion as p3 AP3 above x buffer. After rinsing (4 5 min) with TBS, described above. nitrocellulose sheets 1 were exposed for hr at Immunoprecipitations of synthetic and A,B40 room temperature to peroxidase-conjugated sec- Af42 (1 ,ug) were performed in 5 ml of RIPA 1 x ondary antibody (HRP-conjugated goat anti-rab- buffer with a 170-fold dilution of antibodies, bit from Immunotech or Promega, Marseille, then treated and analyzed as above. France). Nitrocellulose was finally rinsed in TBS as above and immunoreactive complexes were revealed by enhanced chemiluminescence ac- ELISA Assay cording to the recommendations of the manu- facturer (kit from Amersham). The ELISA procedure is based on an analytical electrochemiluminescence method. Capture and secondary antibodies were labeled with either Western Blot Analysis biotin or with the Origen TAG electrochemilu- Electrophoretic separation of the various A,B spe- minescent label (ECL) according to the manufac- cies was performed using a 16.5% SDS-poly- turer recommendations (IGEN, Gaithersburg, acrylamide gel with the Tris-tricine procedure USA). Labeled antibodies were separated from previously described (24). Briefly, dried samples unincorporated label using PD- 10 columns and standards (low molecular weight proteins (Pharmacia) and concentrated to 0.5-1 mg/ml from 31 kDa to 2.5 kDa from Promega) were Centricon-30 using (Amicon) concentrators. Be- in resuspended 30 ,ul of loading buffer (125 mM fore use, the antibodies were diluted to 4 ,ug/ml Tris/HCl, pH 8.45, containing 2% SDS, 20% with gelatin diluent buffer (IGEN), containing glycerol, and 5% 1,-mercaptoethanol). After 0.5% tween 20. In addition, M-280 paramag- heating for 5 min at 95°C, samples were electro- netic beads (IGEN) were diluted before use to 1.6 phoresed at room temperature for 90 min at 150 in mg/ml the same buffer. In each ELISA assay, V an using anode buffer at pH 8.9 and a cathode 25 ,lI of Aj3 were mixed at under samples 20°C buffer mM containing 100 tricine, pH 8.45. Pro- constant with 25 of stirring ,lI paramagnetic teins were then transferred 25 onto nitrocellulose beads, pul of biotinylated antibody, and 25 pul membranes C of (Hybond super, Amersham), then electrochemiluminescent-labeled antibody. antibody hybridization and After 2 hr, 200 plA of buffer was immunoreactivity assay (IGEN) were analysis performed as described above for added and the were read an IGEN samples using dot blot In analysis. some early experiments, im- Origen analyser. Standard curves were obtained 3, Number 10, October 1997 698 Molecular Medicine, Volume with concentrations of synthetic A,340 or known kDa A,B42 ranging between 25 and 1500 pg/ml. 31- 20.4/19.7 - 16.9- 14.4- Immunohistochemistry 8.1 6.2- The temporal neocortex of a patient affected by a 3.2 sporadic form of Alzheimer's disease (AD case of 2.5- [27] provided by Drs. C. the Charles Foix series Hopital de la Pitie- Duyckaerts and J. J. Hauw, Aj40 A42 Salpetriere, Paris) and of a patient neuropatho- as an AD case with typical logically diagnosed cerebrovascular amyloid angiopathy (CAA) were Paraffin-embedded specimens formalin fixed. AMOs" were (4-7 ,um thick), dewaxed, dehydrated, cut and pretreated with formic acid (80%). Samples 4 4 0 were rinsed in phosphate-buffered saline (PBS), Western blot analyses of FCA18 speci- FIG. 1. then incubated for 10 min with 3% hydrogen ficity peroxide to inhibit endogenous peroxidase. Non- One microgram of Af340 or A,B42 was electro- (A) specific sites were blocked by a 20-30 min expo- on a 16.5% Tris-tricine gel and transferred phoresed sure to 10% ovalbumin or BSA, then sections nitrocellulose sheets followed by hybridization to FCA were incubated with various dilutions of at with a 1:500 dilution of the IgG- overnight 4°C were re- antibodies. Immunological complexes fraction of FCA18 (in TBS containing 3% purified of 1/200 biotin- serum albumin as described in Mate- vealed by sequential application bovine [BSA]) rials and Methods. (B) One microgram of A1340 was IgG (Amersham), 1/400 ylated anti-rabbit nitrocellulose electrophoresed and transferred to (Amersham), streptavidin-peroxidase complex incubated at sheets as in A which were overnight (Sigma) as de- and 0,05% diaminobenzidine fraction with a 1:500 dilution of the FCA18 IgG 4°C Nuclei were counter- scribed previously (27). hr without or preincubated (for 8 at 4°C) (lane 1) to stained Harris hematoxylin. with 0.1 mM of synthetic peptide corresponding by 3), DesAsp- A31-7 (lane 2), acetyl A,B1-7 (lane the C-terminal heptapeptide of A,B1-7 (lane 4), or In A and nitrocellulose sheets APP,B (lane 5). B, Materials hr at room temperature) were then incubated (1 HRP-conjugated-IgG (1:1000 from Bale Bio- with goat-anti-rabbit A,B40 peptide was purchased in TBS containing 1% BSA); then immuno- dilution France. Aj343 peptide was chimie or Neosystem, complexes were revealed with chloronaphtol logical from U.S. was synthe- purchased Peptides. A,B42 as described in Materials and Methods. and the and the amino sized and purified, purity of Af342 were confirmed by acid composition mass and amino acid electrospray spectrometry analysis. to was able to for sponding -7) fully compete A,B1 A1340 FCA18 (Fig. 1B, lane 2). In labeling by the N-terminally acety- contrast, corresponding lane 3), Des-Asp lated heptapeptide (Fig. lB, RESULTS an hexapeptide (Fig. lane 4), or heptapeptide 1B, of FCA3340, and Specificity FCA18, end of mimicking the C-terminal APP,B (Fig. 1B, Polyclonal Antibodies Toward FCA3542 FCA18 of In lane 5) did not affect labeling A,B40. Aj8-Related Sequences did not addition, FCA18 recognize full-length interact FCA18 was theoretically designed to ,BAPP751 (not shown). Altogether, our data with of both and indicate that FCA18 interacts only with the N-terminus A,340 A,342 (see clearly of and Western blot analysis the N-terminus part A,3s. Materials Methods). show that FCA3340 illustrated in Figure LA shows that the IgG-puri- Western blot analyses with the dena- FCA3542 label denaturated fied fraction of FCA18 interacts and specifically both and 42. and A1842, respectively (Fig. 2). The spec- turated forms of peptides, A,B40 A,B40 AP3 documented has an absolute ificity of FCA3340 was further by Interestingly, FCA18 require- to ment for the free residue of A/3. Thus, the ability of the octapeptide corresponding aspartyl C-terminus to Af340 labeling by the intact N-terminal heptapeptide (corre- A,340 displace AP3 H. Barelli et al.: Specific Antibodies to A1340 and Af842 699 FCA3340 FCA3542 FCAI8 B A FCA352 kDa .r 31- 20.4/19.7 - 16.9- 14.4- 8.1 - 6.2 - A142 3.2- 2.5- AM43 ApO AS3 AM24= AM40 AM42 AM40 AM42 FIG. 4. FCA3542 specifically recognizes native FIG. 2. Western blot analyses of FCA3340 and and denaturated A1342 FCA3542 specificity (A) Two micrograms of A1342, and A,B43 were A,B40, One of or Aj342 was microgram electropho- A040 dot blotted, hybridized for 18 hr at 4°C with a 1:500 resed, Western blotted, and incubated with a 1:100 dilution of FCA18 or a 1:100 dilution of FCA3542; dilution of FCA3340 or FCA3542; then complexes then immunoreactive complexes were revealed by were revealed with chloronaphtol as described in ECL as described in Materials and Two Methods. (B) Materials and Methods. micrograms of Af342, or A,343 were electro- A,B40, phoresed on a 16.5% Tris-tricine and Western gel and nitrocellulose sheets were blotted, hybridized for 24 hr at 4°C with a 1:100 dilution of the IgG-puri- fied fraction of FCA3542 (in TBS 5% containing skim milk). were revealed Immunological complexes as above. FCA3340 (Fig. 3A lane whereas the 1), peptide mimicking the C-terminal sequence of was A,B42 unable to affect the immunolabeling (Fig. 3A, lane 2). Dot blot analysis illustrates the ability of AB40 W FCA3340 to recognize native A,B40. This interac- tion was abolished by the C-terminal octapeptide of A,340 (Fig. 3B, lane 1) but not of Af42 1 2 lane (Fig. 3B, 2) or A,B43 (Fig. 3B, lane 3). Com- B plete specificity of FCA3542 for Af342 was also observed dot blot and (Fig. 4). Thus, (Fig. 4A) Western blot demonstrate that (Fig. 4B) analyses C 1 2 3 FCA3542 labeled both native and denaturated but failed to detect and A,B42 A,B40 A1343. FIG. 3. FCA3340 native specifically recognizes and denaturated Af340 Two of Aj340 was (A) micrograms electrophoresed Immunoprecipitation of Synthetic and on a 16.5% Tris-tricine gel and Western blotted, and Endogenous A18-Related Sequences by nitrocellulose sheets were incubated for 24 hr at 4°C FCA18, FCA3340, and FCA3542 with a 1:100 dilution of the IgG-purified fraction of FCA3340 (in TBS skim containing 5% milk) prein- of Af340 and 42 Immunoprecipitation synthetic cubated 24 hr at without or (for 4°C) (lane C) with indicated that FCA18 precipitates both spe- AP3 0.1 mM of to synthetic octapeptides corresponding cies whereas FCA3340 and FCA3542 re- only Af333-40 (lane 1) or Af335-42 (lane 2); then immu- vealed Af340 and respectively (Fig. 5A). In Af342, nological complexes were revealed by ECL as de- scribed in and In 2 of Materials Methods. B, ,ug order to examine whether FCA3340 could be AP3 were dot then for 18 hr at blotted, hybridized 4°C used to the a- and precipitate y-secretase-de- with a 1:100 dilution of FCA3340 for preincubated rived we la- p340 fragment (4), metabolically 24 hr at 4°C without (lane or with the C) synthetic beled human 293 cells the kidney overexpressing peptides (0.1 mM) to A/333-40 corresponding (lane since this mutation Swedish mutated Af335-42 or ,3APP75 1, 1), (lane 2), A,B35-43 (lane 3). Immu- noreactivities were revealed as above. is to exacerbate the of both thought production 700 Molecular Medicine, Volume 3, Number 10, October kl BDta Ap42 Af40 A040 A042 . - _. 46 _1 il~ kD 46wc _l_ 30_ 30_ a _ 21.5_ e _ _ _ _ |_ 14.3 2|4 :M. 3:4 __I_l|. _ _ _ lWP' _.4 _ | _ _P A181 _5 33! 18 35 3*4 SWS 2.4 .r3t 1 2 3 FIG. 5. Immunoprecipitation of synthetic and endogenous A3s by FCA antibodies (A) Immunoprecipitations of synthetic A/340 and Af342 (1 ,ug) were in 5 ml 1 x performed of RIPA buffer with a 170-fold dilution of the IgG-purified fraction of the indicated FCAs. Immunoprecipitates were washed, electropho- resed on a 16.5% Tris-tricine gel, Western blotted, and probed with a 1:500 dilution of FCA18. Immunoreactivities were revealed with chloronaphtol as described in Materials and Methods. (B) Transfected HK293 cells overexpress- ing the Swedish mutated form of J3APP were obtained, cultured, and metabolically labeled as in described Materi- als and Methods. Conditioned media (10 ml) were collected, diluted in a one-tenth volume of RIPA lOX buffer, incubated overnight with a 350-fold dilution of FCA18 (lanes 1), FCA3340 (lane 2), or FCA3542 (lane 3), then stirred for 4 hr in the presence of protein A-sepharose (100 mg/ml, 100 ,l). Samples were centrifuged, washed three times with RIPA lx, resuspended with loading buffer, electrophoresed on a 16.50% Tris-tricine gel, anld ra- dioautographed as described earlier (25). Arrow indicates the and p3 fragments. AO3 species (see Introduction). FCA3340 not only 0 3- AO immunoprecipitates endogenous A,B40 but also 0,2 allows the recovery of a low molecular weight product C I that did not interact with FCA18, indi- cating lack of the N-terminus of A,B (Fig. <I°' 5B). The fact that this 3-kDa product increased upon -'U B 0,33 phorbol ester treatment of the cells (not shown), I 0-61 M60 mown-E as expected for an ca-secretase-derived product 0- Cy) 0,2 I-E (28-31), strongly suggests that it could be gen- CM uine p340. FCA3542 interacted with A,B42 and 0- CY) 0,1 its p3 counterpart, the generation of which ap- pears clearly lower than those of A/340 and p340 8 28 9 21 15 12 40 1, 5B1). M 145 (Fig. Immunoprecipitation of A,B and Their p3 FIG. 6. Counterparts Generated by Stable Immunoprecipitation of AfBs and p3 from wild-type and mutated PS1-expressing Transfectants Overexpressing Wild-Type HK293 cells and Mutated Presenilin 1 Stable transfectants overexpressing PS1 or the indi- Figure 6 shows that HK293 cells overexpressing cated mutated PS1 were transiently transfected with wild-type presenilin 1 (PS 1) transiently trans- 2 of wild-type f3APP751. Two days after jig transfec- tion, cells were metabolically labeled for 6 fected with wild-type ,BAPP751 hr; then secreted A,B40 conditioned media were submitted to a two-step imi- and A,B42, the latter species for accounting about munoprecipitation by FCA3340 and FCA3542 (350- of total 10% recovered. Independent clones AP3 fold dilution). Immunoprecipitated proteins were mutated or expressing AE9-PS1 secreted highly on electrophoresed a 16.5% Tris-tricine gel, radioau- increased amounts of Af342, leading to aug- tographed, and scanned as described in Materials mented and Methods. Panel A illustrates the ratio of ratios of over total A/342 A,342 (Fig. 6A). AP3 over total A: + Af40) and panel B indicates (Af42 Interestingly, the quantification of the various p3 the ratio of over + p342 total p3 (p342 p340) ob- counterparts indicated increased ratios of p342 served with the indicated independent numbered over total p3 secreted that (Fig. 6B) fully paral- clones. Valtues are the means S.E.M of three + de- leled those observed for terminations. AP3. H. Barelli et al.: Specific Antibodies to A1340 and A1142 701 -1-I- FIG. 7. ELISA assays (A and B) 750 pg of A1340 or I0000 AfA42 (in 25 was mixed with jil) beads elec- paramagnetic (25 il), trochemiluminescent-labeled _ FCA3340 or FCA3542 (25 -------(ECL) O I-_ ulI, and biotinylated- jig/ml), M M me .!a or in the con- 4G8 (A) (B) 6E10 PE CLO4 in Materials and ditions described Methods. After hr, samples were read using an IGEN Origen analyser. Bars are the means ± 20_- 30OND, SEM of independent determina- tions. Standard curves for A340 and Af342 were obtained (C) (D) 200M. with various concentrations of AIs (in 25 p1), ECL- synthetic 1.1- FCA3340 (filled circles) or ECL- 10O' FCA3542 (open circles) (25 p1, 1 AM , Z 4G8 biotinylated (25 ,ul) v --Iq - a ,u~g/rnl), 0 m and paramagnetic beads (25 m no ,lI). 100n io00 as de- Signals were monitored UI above. scribed AM4pg ELISA Assays radic form of Alzheimer's disease was examined the fractions of FCA18, using IgG-purified The novel ELISA procedure described here em- FCA3340, or FCA3542. As shown in Figure 8, ployed, as capture primary antibody, 4G8 and FCA18 intensely detected a huge amount of dif- 6E10 monoclonals and FCA3340 and FCA3542 and fuse and mature senile plaques (Fig. 8A, C, as detecting antibodies. The two capture antibod- 8B indicates that FCA1 8 D). Furthermore, Figure ies were biotinylated whereas the FCAs were The treatment of also labeled a vascular deposit. labeled with the TAG electrochemiluminescent serum or omission the slice with the preimmune moiety (ECL). Figure 7 shows that biotinylated antibodies led to a total absence of of FCA18 in combination 6E10 (Fig. 7A) and 4G8 (Fig. 7B) labeling (not shown). Treatment of the same or FCA3542 with either ECL-labeled FCA3340 with FCA3340 revealed an intense la- specimen allow for detection of or specific A,B40 A,42, of mature but failed to beling plaques (Fig. 9A) We further examined the sensitivity respectively. detect diffuse Figure 9B indicates that plaques. of the ELISA by means of 4G8 as the primary the central core of senile plaques is in- only 7C shows that FCA3340 capture antibody. Figure whereas the tensely labeled by FCA3340 periph- could allow the detection of 25 to 50 pg/ml of not stained. eral halo of degenerating fibers was A,B40. The standard curve was linear at concen- labels both diffuse and ma- FCA3542 intensely trations of A,B40 up to 750 pg/ml, without inter- lesions and also stains the de- ture (Fig. IOA-D) curves ference from A,342. Standard performed located in the vascular wall of a cerebral posits with that the FCA3542- A,B42 (Fig. 7D) indicated vessel (Fig. 1OD). derived remained linear up to 1500 pg/ml signal without cross-reactivity from Af340. of a Presenile Immunohistochemistry of a Alzheimer's Disease Case with Typical Immunohistochemistry Sporadic Alzheimer's Disease Case Cerebral Amyloid Angiopathy (CAA) tem- A case of Alzheimer's disease with The immunohistochemical labeling of the presenile typ- affected a CAA was examined. FCA18 reveals an in- poral neocortex of a patient by spo- ical 702 Number Molecular Medicine, Volume 3, 10, October FIG. 8. Immunohistochemical analysis of the temporal neocortex of a sporadic case of AD by FCA18 IgG-purified fraction The temporal neocortex of a patient with sporadic Alzheimer's disease was examined by immunohistochemistry a 500-fold using dilution of the IgG-purified fraction of FCA18. Immunological complexes were revealed by the biotin-streptavidin-peroxidase procedure described in Materials and Methods. White arrowheads in A, C, and D indicate mature senile plaques while black arrows (A, D) indicate diffuse plaques. Note in B (arrowhead) the label- ing of a cortical arteriole. X Magnification: A-C, 100; D, X200. tense of both labeling leptomeningeal and corti- iopathological importance. Genetic and cell bio- cal arterioles 1 FCA3340 (Fig. lA). also labels the logical approaches combined with histochemical wall of cortical but it also arterioles, reveals nu- studies should bring their own clues to recon- merous senile plaques (Fig. struct the of 11B). By contrast, puzzle the molecular events altered FCA3542 only faintly stains the vascular lesions in Alzheimer's disease. These distinct approaches whereas numerous immature and mature necessitate the of useful tools development that plaques are still detectable I1 (Fig. C). could serve as molecular probes. Of particular interest are those probes capable of discriminat- ing between the various 'physiological" and po- of tentially "pathogenic" species AP3. DISCUSSION We have obtained an antiserum (FCA18) The mechanisms the that underlying production and can be used as a tool for ubiquitous recog- deposition of in the brain of Alzheimer's dis- all The of nizing A,3 species. characterization the A,B ease-affected patients are likely of central FCA18 indicates that phys- epitope recognized by it 703 H. Barelli et al.: Specific Antibodies to A1340 and A1342 respectively. These antibodies recognize A1342, and denaturated forms of A,3 and can both native in dot and Western blot approaches. This be used us to establish the content of the various allowed species in micropunched tissues from spo- A,3 radic AD and Down syndrome brains as well as lemurian brains (manuscripts in in microcebus The two antibodies also appear po- preparation). for cell biological approaches tentially useful immunoprecipitate their respective since they as well as their p3-related fragments. species AP3 Particularly interesting was the observation that mutations or deletion on the pre- the pathogenic senilin 1 not only affected the recovery of A,B42 but also, to a very similar extent, that of its p342 that the occurrence of counterpart. This suggests trigger phenotypic alter- such mutations likely not the a-secretase cleavages. ations of the y- but assay described here allows the The ELISA of 25-50 pg/ml of A,B40 and A,342. It detection be noted that these levels of sensitivity should the quantification of the various A,3s in permit biological fluids of patients affected by the the disease. There are divergent views concerning the nature of in parenchymal deposits. Mori and 9. Immunohistochemical analysis of the FIG. AP3 of a sporadic case of AD by temporal neocortex co-workers reported on a major Af340 species FCA3340 IgG-purified fraction showed a major contribution (32), while others The temporal neocortex (sporadic AD case) was in- Our IgG-purified fractions of of Af342 (33,34). cubated with a 250-fold dilution of the IgG-purified particularly adapted to immuno- FCAs appeared fraction of FCA3340 and immunoreactivity was re- studies, as underlined by the spe- histochemistry vealed by the biotin-streptavidin-peroxidase method. of amyloid deposits as well as the cific labeling Arrows in A label the central dense core of mature background staining. Clearly, plaques without revealing diffuse lesions as also very low aspecific shown in B. Magnification: A, X 100; B, X400. of the Af3 species present in the neo- the nature of a sporadic AD case varies according to cortex the of lesion. Diffuse plaques were not la- type beled by FCA3340 but were detected by FCA18 and FCA3542, indicating that A,342 was likely form observed in early lesions. the prominent included the first aspartyl residue of This AP. well with other studies of AD and not recognized when it is blocked or This agrees residue is brains (35,36), as well as of in a bond, as shown by the Down's syndrome engaged peptidyl mammalian brains (37,38), documenting of FCA18 recognition of acetylated as- other absence fact that A,B42 formation likely preceeds residue (see Results) or full-length /3APP the partyl deposition. All antibodies labeled the ma- shown). FCA18 immunoprecipitates an in- (not A,B40 ture but FCA3340 stained only their 12-kDa product characterized as the plaques, tracellular central core as previously reported (39). It is p12 fragment (25), indicat- ,-secretase-derived important to note that the study of Iwatsubo and that FCA1 8 can interact with the N-terminus ing was carried out with A/342-directed products. Further- co-workers of all and AP3-containing AP3 antibodies which also recognize (36). In was used to immunoprecipitate A,B A,B43 more, FCA18 this context, it is interesting that FCA3542 does from human kidney HK293 stably secreted Aj343 Results), ruling out the not recognize (see transfected cells overexpressing the wild-type contribution of this species to the label- forms of /3APP (26). possible and Swedish mutated of lesions. now developed two other poly- ing We have also exist concern- Discordant observations clonal antibodies, FCA3340 and FCA3542, that of the vascular deposits, since the nature display clearcut specificity toward A/340 and ing Molecular Medicine, Volume 3, Number 10, October 1997 FIG. 10. Immunohistochemical analysis of the temporal neocortex of a sporadic case of AD by FCA 3542 IgG-purified fraction The temporal neocortex (sporadic AD case) was incubated with a 250-fold dilution of the IgG-purified fraction of FCA3542, then immunoreactivity was revealed by the biotin-streptavidin-peroxidase method. White arrowheads in plaques while black arrow in B indicates an immature plaque. Note in D the label- A, B, and C indicate mature of a cortical arteriole. Magnification: A, B, and D, X200; C, X400. ing of the vascular wall Roher et al. (40) reported on virtually equal cell biological, and immunochemi- biochemical, amounts of A,B40 and A,B42, which is in dis- cal to identify and/or quantify vari- approaches be of with other studies (41,42). Our ex- ous species. These tools should great agreement AP3 in the amination of a typical CAA case indicates an help investigating dysfunctions AP-related and in Alzheimer's disease intense labeling of leptomeningeal vessels that take place neuropa- cortical arterioles by FCA18 and FCA3340, thology. whereas a weak was observed very staining with FCA3542. The observed in this pattern CAA case suggests a major contribution of ACKNOWLEDGMENTS Af340 in leptomeningeal and cortical vessels, is in with a previous study We wish to Drs. Thinakaran and which agreement acknowledge the identified in Sisodia for showing that major species (John Hopkins Institute, Baltimore) AP3 cerebrovascular lesions ended at valine 40 res- providing us with the AE9-PS1 cDNA. We sin- A. and C. Cucumel for idue (33). cerely thank Drs. Cupo novel on We thank J. D. In conclusion, our study describes their advice antigen coupling. A,3 antibodies that can be used in Bardes for animal care and blood sampling, F. end-specific H. 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Dif- normal aging has the same characteristics as ferences between vascular and plaque core that in Alzheimer's disease. Am. J. Pathol. amyloid in Alzheimer's disease. J. Neuro- 148: 259-265. chem. 51: 648-651. Communicated by P. Greengard. Accepted August 11, 1997. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Molecular Medicine Springer Journals

Characterization of New Polyclonal Antibodies Specific for 40 and 42 Amino Acid-Long Amyloid β Peptides: Their Use to Examine the Cell Biology of Presenilins and the Immunohistochemistry of Sporadic Alzheimer’s Disease and Cerebral Amyloid Angiopathy Cases

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Springer Journals
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Abstract

Characterization of New Polyclonal Antibodies Specific for 40 and Amino Acid-Long f3 Amyloid Peptides: Their Use to Examine the Cell Biology of Presenilins and the of Immunohistochemistry Sporadic Alzheimer's Disease and Cerebral Amyloid Angiopathy Cases Helene Barelli,' Anthony Lebeau,' Jean Vizzavona,7 Pia Delaere,3 Nathalie Chevallier,' Cyril Drouot, Philippe Marambaud,' Karime Ancolio,' Joseph D. Buxbaum,4 Olga Khorkova,5 Jeff Heroux,5 Sudhir Sahasrabudhe,5 Jean Martinez,2 Jean-Marie Warter,6 Michel and Mohr,7 Frederic Checlerl 'IPMC du CNRS, UPR411, Valbonne, France 2URA CNRS 1845, Montpellier, France 3Rhone-Poulenc Rorer, Vitry Alfortville, France 4The Rockefeller University, New York, New York, U.S.A. 5Hoechst-Marion-Roussel, Sommerville, New Jersey, U.S.A. 6Service des Maladies du Systeme Nerveux et du Muscle, Hopitaux Universitaires, Strasbourg, France 7Institut de Pathologie, Strasbourg, France ABSTRACT Background: In Alzheimer's disease (AD), the main monitored after metabolic and labeling immunoprecipi- histological lesion is a proteinaceous tation deposit, the senile procedures. Immunohistochemical was analysis plaque, which is mainly composed of a peptide called on brains of and performed cerebrovas- sporadic typical The aggregation process is thought to occur through cular AP3. cases. amyloid angiopathy (CAA) enhanced concentration of A(340 or increased produc- Results: Dot and blot Western indicate that analyses tion of the more readily aggregating 42 amino acid-long fractions IgG-purified of antisera native and recognize species. AP342 denaturated A,Bs. FCA3340 and FCA3542 display full Materials and Methods: Specificity of the antibodies for and specificity A,B42, Antibodies A(340 respectively. was assessed by dot blot, Western blot, ELISA, and im- their immunoprecipitate respective synthetic AP3 species munoprecipitation procedures on synthetic and but also endog- A,Bs and their related p3 counterparts endog- enous produced by secreted HK293 cells. A,B and AP3 p3 secreted transfected enously human 293 by kidney cells. production by wild-type and mutated presenilin 1-ex- This allowed us to show that mutations on 1 presenilin pressing cells transiently transfected with ,3APP751 was similar increased ratios of triggered and its A(342 p342 over total counterpart and ELISA p3. AP assays allow detection of about 25-50 of pg/ml and remain linear Af3s to 750 to 1500 without up pg/ml any cross-reactivity. FCA18 and FCA3542 label diffuse and mature of Address plaques correspondence and reprint requests to: Dr. Fred- AD case whereas eric sporadic FCA3340 reveals Checler, IPMC du only the CNRS, UPR411, 660 route des Luci- oles, 06560 mature lesions and Valbonne, France. Phone: labels their (33) 4 93 95 77 particularly central dense 60; Fax: (33) 4 93 95 77 08; e-mail: core. [email protected] In a CAA case, FCA18 and FCA3340 reveal lepto- © THE 1997, PICOWER INSTITUTE PRESS. All rights reserved. Molecular Medicine, Volume 3, Number 10, October 1997 695-707 696 Molecular Medicine, Volume 3, Number 10, October 1997 meningeal and cortical arterioles whereas FCA3542 only these mutations misroute the J3APP to a compartment such structures. where but not a-secretase, cleavages are faintly labels y-secretase, antibodies exclusively recog- antibodies should prove useful Conclusions: Polyclonal modified. Overall, these (FCA3340) or (FCA3542) were ob- and diagnostic approaches, as suggested nizing A,340 for fundamental A,B42 that FAD-linked presenilins for cell biological, and tained. These demonstrated by their usefulness biochemical, p342 and A,342, suggesting that immunohistochemical techniques. similarly affect both have two antibodies We developed polyclonal INTRODUCTION Af40 (FCA3340) or that specifically recognize ob- One of the main histopathological lesions Af42 and another one able to serve as (FCA3542) in disease-af- served the cortex of Alzheimer's for both species (FCA18). an aspecific probe A,B This pro- fected brains is the senile plaque (1). These novel antibodies are shown to be amenable to be mainly teinaceous deposit appears biochemical dot, and Western blots) and to (ELISA, amyloid 13 (A,B) peptide (2,3) composed of the cell biological (immunoprecipitation of condi- precur- that is derived from a large polypeptidic tioned Furthermore, we media) approaches. precursor protein (f3APP) sor, the ,B amyloid clearly establish by immunohistochemistry the na- approaches have clearly es- (4,5). Biochemical ture of the species observed in diffuse, mature, AP3 process ultimately tablished that the pathological and vascular deposits of a sporadic AD case and leading to the extracellular peptide aggregation is AD a cerebro- those of a presenile case with typical the concentration and/or the dependent upon vascular amyloid angiopathy (CAA). nature of the Af3 species generated (6,7). Muta- tions for the early-onset familial responsible forms of Alzheimer's disease (AD) apparently trigger a disturbance of 3APP processing. Consis- tent with this view is the finding that a double METHODS MATERIALS AND mutation taking place adjacent to the N-termi- Synthesis, Antigen Coupling, and Peptide nus of the sequence (double Swedish muta- AP Rabbit Immunization Procedures tion [8]) led to enhanced production of a 40 An- amino acid-long species (A1340; [9-1 1]). All were synthetized by classical solid peptides AO3 of other mutation located near the C-terminus (Boc strategy) with methyl benzhy- phase the sequence (12,13) appeared to increase resin means of a semiautomatic drylamine by AP3 a 42 acid the production of longer amino (Neosystem NPS 4000) and were pu- A,1 apparatus of have (14), the aggregation properties which rified as described previously (21). The purity More re- been shown to be exacerbated (6,7). and amino acid composition were confirmed by mass cently, two genes have been identified on chro- amino acid analysis and electrospray spec- 14 1 for most of the to the mosomes and that account trometry. The octapeptide corresponding familial forms of the disease of A(340 and 42 a agressive early-onset common N-terminus (displaying The gene products, presenilins 1 and 2, residue at its DAEFRHDS- (15-17). cysteine C-terminus, interfere with 13APP processing since the C- likely Cys) and octapeptides mimicking specific mutations in trigger in- terminal ends of and 42 (with an N-terminal pathological presenilins A,B40 creased A1342 over A340 ratios (18-20). and Cys-MVG- cysteine, Cys-GLMVGGVV [Af340] described Immunohistochemical studies have been GVVIA were as previ- [A,B42]) coupled conducted to establish the A,B content of the to either bovine serum albumin (BSA) ously (22) cerebrovascular neuro- diffuse, mature, and or keyhole limpet hemocyanin (KLH, Calbiochem) The nature of the species to m-maleidoben- pathological insults. that was previously conjugated AP3 the lesions is still somewhat contro- zoic acid KLH-cou- composing N-hydrosuccinimide (Sigma). 1 mixed with an versial. This could be due to the nature of the pled antigens (about mg) were Freund then immunological tools, emphasizing the need for volume of complete adjuvant, equal antibodies able into New Zealand rabbits. the development of end-specific injected subcutaneously 1 and at the various Boosts were month later to fully discriminate among A,B spe- (0.5 mg) given sera cies 3-week intervals. Immunopositive generated. subsequent 697 H. Barelli et al.: Specific Antibodies to and A,f40 A1342 were monitored by dot blot (see with BSA- munological complexes were revealed with chlo- below) coupled antigens as screening ronaphtol as described previously (23). peptides. Purification of IgG Immunoprecipitation of Endogenous and The whole IgG fractions from various immune or Synthetic Species A,8 preimmune sera were obtained after treatment Transfected HK293 cells overexpressing the with octanoic acid according to the procedure Swedish mutated form of ,3APP751 were ob- previously described (23). tained, cultured, and metabolically labeled as previously described (25). Conditioned media (10 ml) were collected, diluted in one-tenth vol- Dot Blot Analysis ume of RIPA lOx buffer, incubated overnight BSA-coupled antigens (about 50 or various ng) with a 350-fold dilution of antibodies, then A,B species (resuspended in distilled water at a stirred for 4 hr in the presence of protein A- final concentration of 0.1 mg/ml) were dot-blot- sepharose (100 mg/ml, 100 Samples were gl). ted (1-2,g) onto nitrocellulose membranes (Hy- centrifuged, washed three times with radioim- bond-C, Amersham, les Ulis, France). Prior to the munoprecipitation assay (RIPA) 1 x, resus- immunochemical reaction, membranes were pended with loading buffer, electrophoresed on a treated in Tris buffer saline 140 mM (TBS: NaCl, 16.5% Tris-tricine gel, and radioautographed as 20 mM Tris-HCl, pH 7.5) containing 5% skim described elsewhere Stable (26). transfectants milk. Antibodies were then added for 4 hr at or overexpressing wild-type, mutated, delta various dilutions (primary screening of immun- Exon-9 presenilin 1 were (AE9PS1) transiently sera) or overnight at 40C at 1:500 (FCA18) and transfected with then la- ,BAPP75 1, metabolically 1:100 dilutions (FCA3340 and FCA3542) in the beled and analyzed for and secretion as p3 AP3 above x buffer. After rinsing (4 5 min) with TBS, described above. nitrocellulose sheets 1 were exposed for hr at Immunoprecipitations of synthetic and A,B40 room temperature to peroxidase-conjugated sec- Af42 (1 ,ug) were performed in 5 ml of RIPA 1 x ondary antibody (HRP-conjugated goat anti-rab- buffer with a 170-fold dilution of antibodies, bit from Immunotech or Promega, Marseille, then treated and analyzed as above. France). Nitrocellulose was finally rinsed in TBS as above and immunoreactive complexes were revealed by enhanced chemiluminescence ac- ELISA Assay cording to the recommendations of the manu- facturer (kit from Amersham). The ELISA procedure is based on an analytical electrochemiluminescence method. Capture and secondary antibodies were labeled with either Western Blot Analysis biotin or with the Origen TAG electrochemilu- Electrophoretic separation of the various A,B spe- minescent label (ECL) according to the manufac- cies was performed using a 16.5% SDS-poly- turer recommendations (IGEN, Gaithersburg, acrylamide gel with the Tris-tricine procedure USA). Labeled antibodies were separated from previously described (24). Briefly, dried samples unincorporated label using PD- 10 columns and standards (low molecular weight proteins (Pharmacia) and concentrated to 0.5-1 mg/ml from 31 kDa to 2.5 kDa from Promega) were Centricon-30 using (Amicon) concentrators. Be- in resuspended 30 ,ul of loading buffer (125 mM fore use, the antibodies were diluted to 4 ,ug/ml Tris/HCl, pH 8.45, containing 2% SDS, 20% with gelatin diluent buffer (IGEN), containing glycerol, and 5% 1,-mercaptoethanol). After 0.5% tween 20. In addition, M-280 paramag- heating for 5 min at 95°C, samples were electro- netic beads (IGEN) were diluted before use to 1.6 phoresed at room temperature for 90 min at 150 in mg/ml the same buffer. In each ELISA assay, V an using anode buffer at pH 8.9 and a cathode 25 ,lI of Aj3 were mixed at under samples 20°C buffer mM containing 100 tricine, pH 8.45. Pro- constant with 25 of stirring ,lI paramagnetic teins were then transferred 25 onto nitrocellulose beads, pul of biotinylated antibody, and 25 pul membranes C of (Hybond super, Amersham), then electrochemiluminescent-labeled antibody. antibody hybridization and After 2 hr, 200 plA of buffer was immunoreactivity assay (IGEN) were analysis performed as described above for added and the were read an IGEN samples using dot blot In analysis. some early experiments, im- Origen analyser. Standard curves were obtained 3, Number 10, October 1997 698 Molecular Medicine, Volume with concentrations of synthetic A,340 or known kDa A,B42 ranging between 25 and 1500 pg/ml. 31- 20.4/19.7 - 16.9- 14.4- Immunohistochemistry 8.1 6.2- The temporal neocortex of a patient affected by a 3.2 sporadic form of Alzheimer's disease (AD case of 2.5- [27] provided by Drs. C. the Charles Foix series Hopital de la Pitie- Duyckaerts and J. J. Hauw, Aj40 A42 Salpetriere, Paris) and of a patient neuropatho- as an AD case with typical logically diagnosed cerebrovascular amyloid angiopathy (CAA) were Paraffin-embedded specimens formalin fixed. AMOs" were (4-7 ,um thick), dewaxed, dehydrated, cut and pretreated with formic acid (80%). Samples 4 4 0 were rinsed in phosphate-buffered saline (PBS), Western blot analyses of FCA18 speci- FIG. 1. then incubated for 10 min with 3% hydrogen ficity peroxide to inhibit endogenous peroxidase. Non- One microgram of Af340 or A,B42 was electro- (A) specific sites were blocked by a 20-30 min expo- on a 16.5% Tris-tricine gel and transferred phoresed sure to 10% ovalbumin or BSA, then sections nitrocellulose sheets followed by hybridization to FCA were incubated with various dilutions of at with a 1:500 dilution of the IgG- overnight 4°C were re- antibodies. Immunological complexes fraction of FCA18 (in TBS containing 3% purified of 1/200 biotin- serum albumin as described in Mate- vealed by sequential application bovine [BSA]) rials and Methods. (B) One microgram of A1340 was IgG (Amersham), 1/400 ylated anti-rabbit nitrocellulose electrophoresed and transferred to (Amersham), streptavidin-peroxidase complex incubated at sheets as in A which were overnight (Sigma) as de- and 0,05% diaminobenzidine fraction with a 1:500 dilution of the FCA18 IgG 4°C Nuclei were counter- scribed previously (27). hr without or preincubated (for 8 at 4°C) (lane 1) to stained Harris hematoxylin. with 0.1 mM of synthetic peptide corresponding by 3), DesAsp- A31-7 (lane 2), acetyl A,B1-7 (lane the C-terminal heptapeptide of A,B1-7 (lane 4), or In A and nitrocellulose sheets APP,B (lane 5). B, Materials hr at room temperature) were then incubated (1 HRP-conjugated-IgG (1:1000 from Bale Bio- with goat-anti-rabbit A,B40 peptide was purchased in TBS containing 1% BSA); then immuno- dilution France. Aj343 peptide was chimie or Neosystem, complexes were revealed with chloronaphtol logical from U.S. was synthe- purchased Peptides. A,B42 as described in Materials and Methods. and the and the amino sized and purified, purity of Af342 were confirmed by acid composition mass and amino acid electrospray spectrometry analysis. to was able to for sponding -7) fully compete A,B1 A1340 FCA18 (Fig. 1B, lane 2). In labeling by the N-terminally acety- contrast, corresponding lane 3), Des-Asp lated heptapeptide (Fig. lB, RESULTS an hexapeptide (Fig. lane 4), or heptapeptide 1B, of FCA3340, and Specificity FCA18, end of mimicking the C-terminal APP,B (Fig. 1B, Polyclonal Antibodies Toward FCA3542 FCA18 of In lane 5) did not affect labeling A,B40. Aj8-Related Sequences did not addition, FCA18 recognize full-length interact FCA18 was theoretically designed to ,BAPP751 (not shown). Altogether, our data with of both and indicate that FCA18 interacts only with the N-terminus A,340 A,342 (see clearly of and Western blot analysis the N-terminus part A,3s. Materials Methods). show that FCA3340 illustrated in Figure LA shows that the IgG-puri- Western blot analyses with the dena- FCA3542 label denaturated fied fraction of FCA18 interacts and specifically both and 42. and A1842, respectively (Fig. 2). The spec- turated forms of peptides, A,B40 A,B40 AP3 documented has an absolute ificity of FCA3340 was further by Interestingly, FCA18 require- to ment for the free residue of A/3. Thus, the ability of the octapeptide corresponding aspartyl C-terminus to Af340 labeling by the intact N-terminal heptapeptide (corre- A,340 displace AP3 H. Barelli et al.: Specific Antibodies to A1340 and Af842 699 FCA3340 FCA3542 FCAI8 B A FCA352 kDa .r 31- 20.4/19.7 - 16.9- 14.4- 8.1 - 6.2 - A142 3.2- 2.5- AM43 ApO AS3 AM24= AM40 AM42 AM40 AM42 FIG. 4. FCA3542 specifically recognizes native FIG. 2. Western blot analyses of FCA3340 and and denaturated A1342 FCA3542 specificity (A) Two micrograms of A1342, and A,B43 were A,B40, One of or Aj342 was microgram electropho- A040 dot blotted, hybridized for 18 hr at 4°C with a 1:500 resed, Western blotted, and incubated with a 1:100 dilution of FCA18 or a 1:100 dilution of FCA3542; dilution of FCA3340 or FCA3542; then complexes then immunoreactive complexes were revealed by were revealed with chloronaphtol as described in ECL as described in Materials and Two Methods. (B) Materials and Methods. micrograms of Af342, or A,343 were electro- A,B40, phoresed on a 16.5% Tris-tricine and Western gel and nitrocellulose sheets were blotted, hybridized for 24 hr at 4°C with a 1:100 dilution of the IgG-puri- fied fraction of FCA3542 (in TBS 5% containing skim milk). were revealed Immunological complexes as above. FCA3340 (Fig. 3A lane whereas the 1), peptide mimicking the C-terminal sequence of was A,B42 unable to affect the immunolabeling (Fig. 3A, lane 2). Dot blot analysis illustrates the ability of AB40 W FCA3340 to recognize native A,B40. This interac- tion was abolished by the C-terminal octapeptide of A,340 (Fig. 3B, lane 1) but not of Af42 1 2 lane (Fig. 3B, 2) or A,B43 (Fig. 3B, lane 3). Com- B plete specificity of FCA3542 for Af342 was also observed dot blot and (Fig. 4). Thus, (Fig. 4A) Western blot demonstrate that (Fig. 4B) analyses C 1 2 3 FCA3542 labeled both native and denaturated but failed to detect and A,B42 A,B40 A1343. FIG. 3. FCA3340 native specifically recognizes and denaturated Af340 Two of Aj340 was (A) micrograms electrophoresed Immunoprecipitation of Synthetic and on a 16.5% Tris-tricine gel and Western blotted, and Endogenous A18-Related Sequences by nitrocellulose sheets were incubated for 24 hr at 4°C FCA18, FCA3340, and FCA3542 with a 1:100 dilution of the IgG-purified fraction of FCA3340 (in TBS skim containing 5% milk) prein- of Af340 and 42 Immunoprecipitation synthetic cubated 24 hr at without or (for 4°C) (lane C) with indicated that FCA18 precipitates both spe- AP3 0.1 mM of to synthetic octapeptides corresponding cies whereas FCA3340 and FCA3542 re- only Af333-40 (lane 1) or Af335-42 (lane 2); then immu- vealed Af340 and respectively (Fig. 5A). In Af342, nological complexes were revealed by ECL as de- scribed in and In 2 of Materials Methods. B, ,ug order to examine whether FCA3340 could be AP3 were dot then for 18 hr at blotted, hybridized 4°C used to the a- and precipitate y-secretase-de- with a 1:100 dilution of FCA3340 for preincubated rived we la- p340 fragment (4), metabolically 24 hr at 4°C without (lane or with the C) synthetic beled human 293 cells the kidney overexpressing peptides (0.1 mM) to A/333-40 corresponding (lane since this mutation Swedish mutated Af335-42 or ,3APP75 1, 1), (lane 2), A,B35-43 (lane 3). Immu- noreactivities were revealed as above. is to exacerbate the of both thought production 700 Molecular Medicine, Volume 3, Number 10, October kl BDta Ap42 Af40 A040 A042 . - _. 46 _1 il~ kD 46wc _l_ 30_ 30_ a _ 21.5_ e _ _ _ _ |_ 14.3 2|4 :M. 3:4 __I_l|. _ _ _ lWP' _.4 _ | _ _P A181 _5 33! 18 35 3*4 SWS 2.4 .r3t 1 2 3 FIG. 5. Immunoprecipitation of synthetic and endogenous A3s by FCA antibodies (A) Immunoprecipitations of synthetic A/340 and Af342 (1 ,ug) were in 5 ml 1 x performed of RIPA buffer with a 170-fold dilution of the IgG-purified fraction of the indicated FCAs. Immunoprecipitates were washed, electropho- resed on a 16.5% Tris-tricine gel, Western blotted, and probed with a 1:500 dilution of FCA18. Immunoreactivities were revealed with chloronaphtol as described in Materials and Methods. (B) Transfected HK293 cells overexpress- ing the Swedish mutated form of J3APP were obtained, cultured, and metabolically labeled as in described Materi- als and Methods. Conditioned media (10 ml) were collected, diluted in a one-tenth volume of RIPA lOX buffer, incubated overnight with a 350-fold dilution of FCA18 (lanes 1), FCA3340 (lane 2), or FCA3542 (lane 3), then stirred for 4 hr in the presence of protein A-sepharose (100 mg/ml, 100 ,l). Samples were centrifuged, washed three times with RIPA lx, resuspended with loading buffer, electrophoresed on a 16.50% Tris-tricine gel, anld ra- dioautographed as described earlier (25). Arrow indicates the and p3 fragments. AO3 species (see Introduction). FCA3340 not only 0 3- AO immunoprecipitates endogenous A,B40 but also 0,2 allows the recovery of a low molecular weight product C I that did not interact with FCA18, indi- cating lack of the N-terminus of A,B (Fig. <I°' 5B). The fact that this 3-kDa product increased upon -'U B 0,33 phorbol ester treatment of the cells (not shown), I 0-61 M60 mown-E as expected for an ca-secretase-derived product 0- Cy) 0,2 I-E (28-31), strongly suggests that it could be gen- CM uine p340. FCA3542 interacted with A,B42 and 0- CY) 0,1 its p3 counterpart, the generation of which ap- pears clearly lower than those of A/340 and p340 8 28 9 21 15 12 40 1, 5B1). M 145 (Fig. Immunoprecipitation of A,B and Their p3 FIG. 6. Counterparts Generated by Stable Immunoprecipitation of AfBs and p3 from wild-type and mutated PS1-expressing Transfectants Overexpressing Wild-Type HK293 cells and Mutated Presenilin 1 Stable transfectants overexpressing PS1 or the indi- Figure 6 shows that HK293 cells overexpressing cated mutated PS1 were transiently transfected with wild-type presenilin 1 (PS 1) transiently trans- 2 of wild-type f3APP751. Two days after jig transfec- tion, cells were metabolically labeled for 6 fected with wild-type ,BAPP751 hr; then secreted A,B40 conditioned media were submitted to a two-step imi- and A,B42, the latter species for accounting about munoprecipitation by FCA3340 and FCA3542 (350- of total 10% recovered. Independent clones AP3 fold dilution). Immunoprecipitated proteins were mutated or expressing AE9-PS1 secreted highly on electrophoresed a 16.5% Tris-tricine gel, radioau- increased amounts of Af342, leading to aug- tographed, and scanned as described in Materials mented and Methods. Panel A illustrates the ratio of ratios of over total A/342 A,342 (Fig. 6A). AP3 over total A: + Af40) and panel B indicates (Af42 Interestingly, the quantification of the various p3 the ratio of over + p342 total p3 (p342 p340) ob- counterparts indicated increased ratios of p342 served with the indicated independent numbered over total p3 secreted that (Fig. 6B) fully paral- clones. Valtues are the means S.E.M of three + de- leled those observed for terminations. AP3. H. Barelli et al.: Specific Antibodies to A1340 and A1142 701 -1-I- FIG. 7. ELISA assays (A and B) 750 pg of A1340 or I0000 AfA42 (in 25 was mixed with jil) beads elec- paramagnetic (25 il), trochemiluminescent-labeled _ FCA3340 or FCA3542 (25 -------(ECL) O I-_ ulI, and biotinylated- jig/ml), M M me .!a or in the con- 4G8 (A) (B) 6E10 PE CLO4 in Materials and ditions described Methods. After hr, samples were read using an IGEN Origen analyser. Bars are the means ± 20_- 30OND, SEM of independent determina- tions. Standard curves for A340 and Af342 were obtained (C) (D) 200M. with various concentrations of AIs (in 25 p1), ECL- synthetic 1.1- FCA3340 (filled circles) or ECL- 10O' FCA3542 (open circles) (25 p1, 1 AM , Z 4G8 biotinylated (25 ,ul) v --Iq - a ,u~g/rnl), 0 m and paramagnetic beads (25 m no ,lI). 100n io00 as de- Signals were monitored UI above. scribed AM4pg ELISA Assays radic form of Alzheimer's disease was examined the fractions of FCA18, using IgG-purified The novel ELISA procedure described here em- FCA3340, or FCA3542. As shown in Figure 8, ployed, as capture primary antibody, 4G8 and FCA18 intensely detected a huge amount of dif- 6E10 monoclonals and FCA3340 and FCA3542 and fuse and mature senile plaques (Fig. 8A, C, as detecting antibodies. The two capture antibod- 8B indicates that FCA1 8 D). Furthermore, Figure ies were biotinylated whereas the FCAs were The treatment of also labeled a vascular deposit. labeled with the TAG electrochemiluminescent serum or omission the slice with the preimmune moiety (ECL). Figure 7 shows that biotinylated antibodies led to a total absence of of FCA18 in combination 6E10 (Fig. 7A) and 4G8 (Fig. 7B) labeling (not shown). Treatment of the same or FCA3542 with either ECL-labeled FCA3340 with FCA3340 revealed an intense la- specimen allow for detection of or specific A,B40 A,42, of mature but failed to beling plaques (Fig. 9A) We further examined the sensitivity respectively. detect diffuse Figure 9B indicates that plaques. of the ELISA by means of 4G8 as the primary the central core of senile plaques is in- only 7C shows that FCA3340 capture antibody. Figure whereas the tensely labeled by FCA3340 periph- could allow the detection of 25 to 50 pg/ml of not stained. eral halo of degenerating fibers was A,B40. The standard curve was linear at concen- labels both diffuse and ma- FCA3542 intensely trations of A,B40 up to 750 pg/ml, without inter- lesions and also stains the de- ture (Fig. IOA-D) curves ference from A,342. Standard performed located in the vascular wall of a cerebral posits with that the FCA3542- A,B42 (Fig. 7D) indicated vessel (Fig. 1OD). derived remained linear up to 1500 pg/ml signal without cross-reactivity from Af340. of a Presenile Immunohistochemistry of a Alzheimer's Disease Case with Typical Immunohistochemistry Sporadic Alzheimer's Disease Case Cerebral Amyloid Angiopathy (CAA) tem- A case of Alzheimer's disease with The immunohistochemical labeling of the presenile typ- affected a CAA was examined. FCA18 reveals an in- poral neocortex of a patient by spo- ical 702 Number Molecular Medicine, Volume 3, 10, October FIG. 8. Immunohistochemical analysis of the temporal neocortex of a sporadic case of AD by FCA18 IgG-purified fraction The temporal neocortex of a patient with sporadic Alzheimer's disease was examined by immunohistochemistry a 500-fold using dilution of the IgG-purified fraction of FCA18. Immunological complexes were revealed by the biotin-streptavidin-peroxidase procedure described in Materials and Methods. White arrowheads in A, C, and D indicate mature senile plaques while black arrows (A, D) indicate diffuse plaques. Note in B (arrowhead) the label- ing of a cortical arteriole. X Magnification: A-C, 100; D, X200. tense of both labeling leptomeningeal and corti- iopathological importance. Genetic and cell bio- cal arterioles 1 FCA3340 (Fig. lA). also labels the logical approaches combined with histochemical wall of cortical but it also arterioles, reveals nu- studies should bring their own clues to recon- merous senile plaques (Fig. struct the of 11B). By contrast, puzzle the molecular events altered FCA3542 only faintly stains the vascular lesions in Alzheimer's disease. These distinct approaches whereas numerous immature and mature necessitate the of useful tools development that plaques are still detectable I1 (Fig. C). could serve as molecular probes. Of particular interest are those probes capable of discriminat- ing between the various 'physiological" and po- of tentially "pathogenic" species AP3. DISCUSSION We have obtained an antiserum (FCA18) The mechanisms the that underlying production and can be used as a tool for ubiquitous recog- deposition of in the brain of Alzheimer's dis- all The of nizing A,3 species. characterization the A,B ease-affected patients are likely of central FCA18 indicates that phys- epitope recognized by it 703 H. Barelli et al.: Specific Antibodies to A1340 and A1342 respectively. These antibodies recognize A1342, and denaturated forms of A,3 and can both native in dot and Western blot approaches. This be used us to establish the content of the various allowed species in micropunched tissues from spo- A,3 radic AD and Down syndrome brains as well as lemurian brains (manuscripts in in microcebus The two antibodies also appear po- preparation). for cell biological approaches tentially useful immunoprecipitate their respective since they as well as their p3-related fragments. species AP3 Particularly interesting was the observation that mutations or deletion on the pre- the pathogenic senilin 1 not only affected the recovery of A,B42 but also, to a very similar extent, that of its p342 that the occurrence of counterpart. This suggests trigger phenotypic alter- such mutations likely not the a-secretase cleavages. ations of the y- but assay described here allows the The ELISA of 25-50 pg/ml of A,B40 and A,342. It detection be noted that these levels of sensitivity should the quantification of the various A,3s in permit biological fluids of patients affected by the the disease. There are divergent views concerning the nature of in parenchymal deposits. Mori and 9. Immunohistochemical analysis of the FIG. AP3 of a sporadic case of AD by temporal neocortex co-workers reported on a major Af340 species FCA3340 IgG-purified fraction showed a major contribution (32), while others The temporal neocortex (sporadic AD case) was in- Our IgG-purified fractions of of Af342 (33,34). cubated with a 250-fold dilution of the IgG-purified particularly adapted to immuno- FCAs appeared fraction of FCA3340 and immunoreactivity was re- studies, as underlined by the spe- histochemistry vealed by the biotin-streptavidin-peroxidase method. of amyloid deposits as well as the cific labeling Arrows in A label the central dense core of mature background staining. Clearly, plaques without revealing diffuse lesions as also very low aspecific shown in B. Magnification: A, X 100; B, X400. of the Af3 species present in the neo- the nature of a sporadic AD case varies according to cortex the of lesion. Diffuse plaques were not la- type beled by FCA3340 but were detected by FCA18 and FCA3542, indicating that A,342 was likely form observed in early lesions. the prominent included the first aspartyl residue of This AP. well with other studies of AD and not recognized when it is blocked or This agrees residue is brains (35,36), as well as of in a bond, as shown by the Down's syndrome engaged peptidyl mammalian brains (37,38), documenting of FCA18 recognition of acetylated as- other absence fact that A,B42 formation likely preceeds residue (see Results) or full-length /3APP the partyl deposition. All antibodies labeled the ma- shown). FCA18 immunoprecipitates an in- (not A,B40 ture but FCA3340 stained only their 12-kDa product characterized as the plaques, tracellular central core as previously reported (39). It is p12 fragment (25), indicat- ,-secretase-derived important to note that the study of Iwatsubo and that FCA1 8 can interact with the N-terminus ing was carried out with A/342-directed products. Further- co-workers of all and AP3-containing AP3 antibodies which also recognize (36). In was used to immunoprecipitate A,B A,B43 more, FCA18 this context, it is interesting that FCA3542 does from human kidney HK293 stably secreted Aj343 Results), ruling out the not recognize (see transfected cells overexpressing the wild-type contribution of this species to the label- forms of /3APP (26). possible and Swedish mutated of lesions. now developed two other poly- ing We have also exist concern- Discordant observations clonal antibodies, FCA3340 and FCA3542, that of the vascular deposits, since the nature display clearcut specificity toward A/340 and ing Molecular Medicine, Volume 3, Number 10, October 1997 FIG. 10. Immunohistochemical analysis of the temporal neocortex of a sporadic case of AD by FCA 3542 IgG-purified fraction The temporal neocortex (sporadic AD case) was incubated with a 250-fold dilution of the IgG-purified fraction of FCA3542, then immunoreactivity was revealed by the biotin-streptavidin-peroxidase method. White arrowheads in plaques while black arrow in B indicates an immature plaque. Note in D the label- A, B, and C indicate mature of a cortical arteriole. Magnification: A, B, and D, X200; C, X400. ing of the vascular wall Roher et al. (40) reported on virtually equal cell biological, and immunochemi- biochemical, amounts of A,B40 and A,B42, which is in dis- cal to identify and/or quantify vari- approaches be of with other studies (41,42). Our ex- ous species. These tools should great agreement AP3 in the amination of a typical CAA case indicates an help investigating dysfunctions AP-related and in Alzheimer's disease intense labeling of leptomeningeal vessels that take place neuropa- cortical arterioles by FCA18 and FCA3340, thology. whereas a weak was observed very staining with FCA3542. The observed in this pattern CAA case suggests a major contribution of ACKNOWLEDGMENTS Af340 in leptomeningeal and cortical vessels, is in with a previous study We wish to Drs. Thinakaran and which agreement acknowledge the identified in Sisodia for showing that major species (John Hopkins Institute, Baltimore) AP3 cerebrovascular lesions ended at valine 40 res- providing us with the AE9-PS1 cDNA. We sin- A. and C. Cucumel for idue (33). cerely thank Drs. Cupo novel on We thank J. D. In conclusion, our study describes their advice antigen coupling. A,3 antibodies that can be used in Bardes for animal care and blood sampling, F. end-specific H. 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Dif- normal aging has the same characteristics as ferences between vascular and plaque core that in Alzheimer's disease. Am. J. Pathol. amyloid in Alzheimer's disease. J. Neuro- 148: 259-265. chem. 51: 648-651. Communicated by P. Greengard. Accepted August 11, 1997.

Journal

Molecular Medicine – Springer Journals

Published: Oct 1, 1997

Keywords: Presenilin (PS1); Cerebral Amyloid Angiopathy Type; Cortical Arterioles; Mature Plaques; Synthetic Aβ

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