American Journal ofPathology, Vol. 149, No. 2, August 1996 Copyright t Amenican Societyfor Investigative Pathology p3 -Amyloid Peptide Has a Unique and Potentially Pathogenic Immunohistochemical Profile in Alzheimer's Disease Brain Linda S. Higgins,* Greer M. Murphy Jr.,t lism.13'14 The enzymology of 1-amyloid formation re- Lysia S. Forno,* Rosanne Catalano,* and mains unclear except that two proteolytic processing Barbara Cordell* steps are required to liberate 13-amyloid from the From Scios Nova Inc.,* Mountain View, California, and the backbone of 1-APP. The proteinase(s) that gener- Departments ofPsychiatry and Behavioral Sciencest and ates the amino terminus of the peptide is referred to Pathology,t Stanford University School ofMedicine, as 1-secretase and that producing the carboxy ter- Stanford, and the Veterans Administration Medical Center,f minus is referred to as y-secretase. Palo Alto, California Another 13-APP processing activity exists, a-secre- tase. This single processing event releases the bulk of the precursor from its membrane-bound state The presence of f-amyloid in brain tissue is such that it can be secreted into extracellular com- characteristic ofAlzbeimer's disease (AD). A nat- partments.1-17 The a-secretase cleavage occurs uraly occurring derivative ofthe -amyloidpep- near the transmembrane domain of 13-APP within the tide, p3, possesses aU of the structural determi- 13-amyloid domain. The primary a-secretase site of nants required for fibril assembly and proteolysis has been mapped between residues 16 neurotoxicity. p3-specific antibodies were used and 17 of the amyloid domain,18 although minor to examine the distribution of this peptide in cleavages have also been observed adjacent to this brain. p3 reactivity was absent or sparse in aged major site.13 A by-product of a-secretase action is an non-AD brains but was prevalent in selected ar- 8.0-kd fragment consisting of the carboxy-terminal and in a eas of AD brain in diffuse deposits portion of the 13-amyloid domain followed by the cy- subset ofdystrophic neurites. p3-reactive dystro- toplasmic domain and carboxy terminus of 13-APP. phic neurites werefound both independent in the Although the a- and 1-secretase cleavage steps neuropil and associated with plaques. Little or proteolytic frag- no reactivity was observed to amyloid cores in originate independently, producing classical plaques or to amyloid in the cerebral ments of 8.0 kd and larger amyloidogenic carboxy- all of these vasculature. The exclusive appearance ofp3 re- terminal fragments, respectively, frag- activity in AD brainplus the selective localization ments are substrates for y-secretase14 (Figure 1). As of p3 reactivity to abnormal structures in the a consequence, an amino-terminally truncated 3-kd temporal lobe limbic system suggests thatp3 may peptide, p3, is generated together with the complete be a contributingfactor to AD pathology. (AmJ 13-amyloid peptide.10'12'14 19 Both 4-kd 13-amyloid Pathol 1996, 149:585-596) and p3 peptides are secreted from cultured cells and both are identical from residues -17 to 39 to 43 10,12 The deposition of f3-amyloid is a central and early The biological and physical properties of 13-amy- event in the pathogenesis of AD. Genetic evidence, loid have been under intensive investigation. Be- both human1' 2 and transgenic,3-5 supports this con- cause the peptide can be chemically synthesized, tention, as do histological studies of AD brain tissue primarily from individuals with Down's syndrome.6-9 Supported by Marion Merrell Dow, the National Institute of Mental The production of ,B-amyloid has been shown to Health, and the Department of Veteran's Affairs Medical Research result from normal cellular metabolism,10-12 and re- Program. cently we have demonstrated that this peptide is a Accepted for publication April 3, 1996. non-obligatory minor degradative by-product of Address reprint requests to Dr. Barbara Cordell, Scios Nova Inc., ,B-amyloid precursor protein (13-APP) catabo- 2450 Bayshore Parkway, Mountain View, CA 94043. 585 586 Higgins et al AJP August 1996, Vol. 149, No. 2 tions have revealed that f-amyloid peptides (fre- H2N N _COH quently ,B-amyloid 25 to 35) can induce neuronal apoptosis, 36,37 can increase the vulnerability of neu- rons to excitotoxic damage38'39 and to injury by glu- ~~~~~~1 117 2r, 36 - \43 DAEFRHDSGYEVHHQKLVFFAEDGSNKGAQII OGIAT1. cose deprivation,40 and can participate in microglial T t activation resulting in secretion of neurotoxic sub- ez1% y secretasesktes p3 stances.41 These biological activities of the (-amy- loid peptide have been interpreted to explain the 4 kDa LaMyloid neuronal dysfunction and loss observed in AD. Y The p3 peptide possesses the sequences critical Figure 1. Schematic representation of 3-APP-derived (3-amyloid and p3 peptides. The amino acid sequence of the /3-amyloid domain is for peptide aggregation and toxicity to neurons; how- shown with a-, (-, and y-secretase processing sites indicated by ever, the potential pathogenicity of this naturally oc- arrows; the large arrows represent major cleavage sites and the smaller arrows represent minor sites. The p3 peptide and (3-amyloid curring ,B-amyloid derivative has not been ad- peptides are denoted in conjunction with their sequences. Afrequently dressed. To begin to understand whether or not p3 used neurotoxic synthetic peptide is indicated by the boxed sequence (residues 25 to 35) and the amyloid fibril nucleation and assembly contributes to the disease process, we generated sequence (residues 25 is 43) is denoted by the dashed underline. The antibodies that selectively recognize p3 but not p3 and 4.1 antibody epitopes, forp3 and (3-amyloidpeptides, respec- tively, are each designated with antibody symbols. The epitope recog- f-amyloid. These p3-specific antibodies were used nized by 4.1 MAb was described in Higgins et al.4 to survey AD and age-matched control brain tissue using immunohistochemistry. We report that p3 im- many studies have focused on identifying the struc- munoreactivity is associated with AD pathology and tural determinants required for self-aggregation and that the profile of stained structures is distinct from insolubility. Numerous investigations have demon- those reacting with f-amyloid antibodies. strated that a variety of different synthetic f-amyloid peptides can form fibrils in vitro.20'21 Although both amino- and carboxy-terminal ,B-amyloid synthetic fragments have been shown capable of fibril forma- Materials and Methods tion,22 28 other important features of amyloid were Production and Characterization ofp3 often absent such as insolubility and fibril stability. More detailed and rigorous analyses of subdomains Antisera within the ,B-amyloid sequence have subsequently Three rabbits were repeatedly immunized with a syn- identified the hydrophobic carboxy-terminal domain thetic peptide containing residues 17 to 28 of the as the critical region for seeding amyloid aggrega- ,B-amyloid sequence. A cysteine residue was added tion,29 for forming a ,3-sheet structure,3031 and for to the carboxy terminus of the peptide to enable insolubility.31 These physical features of amyloid can conjugation to keyhole limpet hemocyanin using be recreated with synthetic peptide spanning amino N-maleimido-6-aminocaproyl ester of 1-hydroxy-2- acids 26 to 422931 and with a minimal sequence nitro-4-benzenesulfonic acid (Bachem California, encompassing residues 25 to 35.21 It should be Torrance, CA). Samples of sera were collected be- noted that these biophysical features are derived fore peptide immunization and at 10-day intervals from in vitro analyses and the relationship of these after each immunization. The sera were analyzed by parameters to those occurring in vivo has not been enzyme-linked immunosorbent assay for reactivity elucidated. with a set of synthetic peptides, including ,B-amyloid The ,B-amyloid peptide has also been shown to 1 to 28, 17 to 28, and 18 to 28. All peptides were possess biological activity. Yankner et a132 first iden- prepared using an Applied Biosystems peptide syn- tified the neurotoxic effects of synthetic ,B-amyloid thesizer 430A (Foster City, CA), purified by high peptides using cultured hippocampal rat neurons. A pressure liquid chromatography, and characterized carboxy-terminal peptide fragment containing resi- using both amino acid composition and amino-ter- dues 25 to 35 was demonstrated to possess the toxic minal microsequence analyses. All peptides were effects for neurons when applied at micromolar con- solubilized in H20 containing 20% isopropanol. Se- centrations. Subsequent to this original study, the rial dilutions of each serum sample were made and neurotoxic effects of B-amyloid have been exten- reacted with 100 ,tl of a 1 ,umol/L solution of each sively characterized in vitro and in vivo. It has been peptide. Bound antibody was detected with horse- determined that the neurotoxic effect elicited in vitro radish-peroxidase-conjugated anti-rabbit secondary was highly dependent on the aggregated fibrillar antibody and the chromogenic substrate solution 0- state of the amyloid peptide.21,33-35 Other investiga- phenylenediamine and H202 in citric phosphate p3 Peptide in Alzheimer's Brain 587 AJPAugust 1996, Vol. 149, No. 2 buffer, pH 5. For immunohistological competition ex- as appropriate. Double-labeling reactions were per- periments, each peptide was incubated at a final formed as previously described42 using both perox- concentration of 30 ,umol/L with a 1:1000 dilution of idase antiperoxidase and avidin-biotin complex p3 antibody 2 or 3 in phosphate-buffered saline con- methods. The tau mouse monoclonal antibody taining 0.2% gelatin. After incubation for 60 minutes (clone tau-1) was obtained from Boehringer Mann- at 370C and overnight at 40C, the antibody-peptide heim (Indianapolis, IN). Silver staining was per- solutions were used in the standard single-antibody formed according to standard methods. All stained immunohistological protocol using AD brain sec- sections were assessed by three investigators who tions.