N-Truncated Aβ 2-X Starting with Position Two in Sporadic Alzheimer’s Disease Cases and Two Alzheimer Mouse Models

Development of polyclonal antibodies in rabbits

The 14 amino acid peptide (AEFRHDSGYEVHHC) corresponding to residues (2–14) of the Aβ peptide plus an additional cysteine was synthesized (Clonestar spol. s r. o., Czech Republic) and coupled at its C-terminus to keyhole limpet hemocyanin (KLH) (Calbiochem) using Sulfo-SMCC (Sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate) (Thermo Scientific) and sulfo-MBS (m-maleimido benzoyl-N-hydoxysuccinimideester) (Thermo Scientific). Peptide-carrier conjugates were purified on PD-10 desalting columns (GE Healthcare). Antisera were generated by subcutaneous injection of peptide conjugates to KLH emulsified with Complete Freunds Adjuvant (for first injection) followed with incomplete Freunds adjuvant for injection 2, 3, and 4 (both Sigma, USA) into New Zealand White rabbits at 4 week intervals. Serum was collected prior to the first immunization (pre-immune serum) and after the fourth immunization. The immune sera obtained after the final bleed were affinity purified using SulfoLink kit (Pierce) according the manufacturer’s protocol.

Antibody characterization by capillary isoelectric focusing immunoassay and western immunoblot analysis

To assess the selectivity of purified polyclonal antibody 77 (pAb77) for different amino-terminal variants of Aβ, we employed the capillary isoelectric focusing immunoassay (CIEF-immunoassay) reported previously [14] and urea-Bicine/Bis-Tris/Tris/sulfate SDS-PAGE (1D-Aβ-PAGE) followed by western immunoblot [12, 15]. For the CIEF-immunoassay, combinations of synthetic Aβ peptides with different amino termini were separated by capillary isoelectric focusing and subsequently probed with purified pAb77 antibody. For comparison, detection was also performed with mAb 6E10 (Covance) that recognizes several amino-terminal variants of Aβ. The synthetic Aβ peptides Aβ_1 - 40, Aβ_2 - 40, Aβ_3 - 40, pyroglutamate Aβ_pE3 - 40, Aβ_4 - 40, and Aβ_5 - 40 were purchased from AnaSpec (Fremont, CA USA). Peptide stock solutions (1 mg/ml) were prepared in DMSO or, in the case of Aβ_pE3 - 40, in 0.1% NH₄OH and stored at −80°C. Aliquots were thawed only once. Peptides were diluted to a final concentration of 25 ng/ml in 20 mM bicine pH 7.6, 0.6 % CHAPS. Mixtures of a) Aβ_1 - 40 and Aβ_2 - 40, b) Aβ_3 - 40 and Aβ_4 - 40, c) Aβ_pE3 - 40 and Aβ_5 - 40 were subjected to automated capillary isoelectric focusing immunoassay on the NanoPro 1000 platform (ProteinSimple) as described previously [14]. Each capillary was loaded with approximately 12 pg per peptide. Primary detection antibodies pAb77 and mAb 6E10 (Covance) were diluted in antibody diluent (ProteinSimple) to final concentrations of 20.6 μg/ml and 2 μg/ml, respectively. For the detection the ProteinSimple amplification kit was used with anti-rabbit-biotin secondary antibody for purified pAb77 and anti-mouse-biotin secondary antibody for mAb 6E10, both in combination with streptavidin-HRP. In the last step of the automated assay, a mixture of luminol and peroxide (ProteinSimple) was loaded into each capillary and chemiluminescent signals were recorded after exposure times of 30, 60, 120, 240, 480, and 960 s. Although the antibody pAb77 was generated by using Aβ_2-14 as antigen, it also reacts with C-terminally longer Aβ peptides; therefore, we use the term Aβ_2-X for pAb77 reactivity throughout the text.

For one-dimensional urea-Bicine/Bis-Tris/Tris/ sulfate SDS-PAGE (1D-Aβ-PAGE), stock solutions of synthetic Aβ were prepared in 0.1% NH₄OH and stored at −80°C. The peptides were further diluted in sample buffer (0.36 M Bis–Tris, 0.16 M Bicine, 15% w/v sucrose, 1% w/v SDS, and 0.0075% w/v bromphenol blue), and aliquots corresponding to 100, 250, or 500 pg per lane were separated by 1D-Aβ-PAGE and detected by western blotting. The blots were probed with the primary antibodies pAb 77 (1:1000 in Roti ^®-Block; Carl Roth, Karlsruhe, Germany) or mAb 6E10 (1:1000 in Roti ^®-Block) at 4°C overnight. As secondary reagents, we employed peroxidase-conjugated horse anti-rabbit IgG (1:10000 in PBS-T; Calbiochem #401315, Frankfurt, Germany) or biotinylated goat anti-mouse IgG (333 ng/ml in PBS-T; Vector Laboratories Ltd., Peterborough, United Kingdom) in combination with streptavidin–horseradish peroxidase complex (1:3000 in PBS-T; Amersham Pharmacia, Germany) for 60 min at room temperature. Chemiluminescent detection was achieved with ECL Prime Western Blotting Detection Reagent (GE Healthcare, München, Germany).

Immunoprecipitation of Aβ peptides from TBS and SDS-brain extracts from 5XFAD mice

TBS- and SDS-soluble proteins were extracted from brain samples of 5XFAD mice as previously described [16]. Protein concentrations were measured using a Roti-Quant universal protein assay kit (Carl Roth) according to the instructions of the supplier. Aβ peptides were immunoprecipitated from brain extracts from 5XFAD mice with mAb6E10 (Covance) covalently immobilized on sheep-anti-mouse IgG dynabeads M-280 [14]. Aliquots of brain extracts prepared in TBS or SDS and containing approximately 300 μg of total protein were adjusted to a volume of 800 μl with water. To each sample, 200 μl of a fivefold concentrated IP-detergent buffer was added to yield final concentrations of 50 mM HEPES, pH 7.4, 150 mM NaCl, 0.5% (v/v) Igepal C630 (“Nonidet-P40”), 0.25% (w/v) sodium deoxycholate, 0.05% (w/v) SDS. After addition of 30 μl of 6E10-coated magnetic bead and overnight incubation at 4°C, the Aβ-peptides were eluted for 5 min at 95°C in sample buffer and analyzed by urea-bicine/bistris/tris/sulfate SDS-PAGE, essentially as described above. The plaque material from 5XFAD brain is completely soluble in TBS and after centrifugation in SDS. No ultracentrifugation step is required.

Human brain samples

The Netherlands Brain Bank provided human brain samples with a mean age of 87.3±4.35 (11 female and 3 male cases). The Ethical Committee of the University Medicine Göttingen approved the project.

Alzheimer mouse models

The generation of AβPP/PS1KI mice has been described previously [13]. In brief, human mutant AβPP751 containing the Swedish and London mutations is overexpressed under the control of the murine Thy-1 promoter, whereas murine PS1 with the M233T and L235P FAD-linked mutations is expressed under the control of the endogenous mouse PS1 promoter. All mice designated PS1KI were homozygous for PS1 knock-in mutations, in comparison to the AβPP/PS1KI mice that had one additional hemizygous AβPP751SL transgene. AβPP/PS1KI mice are a generous gift of Dr. Laurent Pradier, Sanofi-Aventis, France. The generation of 5XFAD mice (Tg6799) has been described previously [17]. 5XFAD overexpress the 695 amino acids isoform of the human AβPP (AβPP695) carrying the Swedish, Florida, and London mutations under the control of the murine Thy1-promoter. In addition, human Presenilin-1 (PS1) carrying the M146L and L286V mutations mice is expressed also under the control of the murine Thy1-promoter. Male mice on a C57Bl/6 × SJL genetic background were obtained from Jackson Laboratories (strain: B6SJL-Tg(AβPPSwFlLon,PSEN1*M146L* V)6799Vas/J) and backcrossed for 10 generations to C57Bl/6J wildtype (WT) mice to obtain an incipient congenic line on a C57Bl/6J genetic background. All animals were handled according to German guidelines for animal care.

Immunohistochemistry and immunofluorescence of Aβ

Mice were sacrificed via CO₂ anesthetization followed by cervical dislocation. Brain samples were dissected and post-fixed in 4% phosphate-buffered formalin at 4°C. Immunohistochemistry was performed on 4 μm paraffin sections. The antibodies pAb77 (1:200–1:500), 24311 (against pan-Aβ [18]; 1:1000) and 4G8 (against Aβ_17 - 24; 1:1000) were used for Aβ staining. Biotinylated secondary anti-rabbit and anti-mouse antibodies (1:200) were purchased from DAKO. Staining was visualized using the ABC method, with a Vectastain kit (Vector Laboratories) and diaminobenzidine as chromogen. Counterstaining was carried out with hematoxylin. Fluorescence staining was visualized using AlexaFluor488- and AlexaFluor594-conjugated secondary antibodies (Molecular Probes). As a control, pAb77 was replaced with immuno-absorbed pAb77. Immunoabsorption of pAb77 was carried using Aβ_2 - 40 (3 μg peptide per 5 μl of antibody), incubation at room temperature for 5 h with rotation, followed by centrifugation at 14,000 g for 5 min.

The purified polyclonal antibody pAb77 shows high selectivity for truncated Aβ_2-X

The selectivity of the affinity purified polyclonal antibody pAb77 for different amino-terminal variants of Aβ was studied by several independent methods. Both, capillary isoelectric focusing immunoassay and urea-Bicine/Bis-Tris/Tris/sulfate SDS-PAGE (1D-Aβ-PAGE) followed by western immunoblotting indicated that pAb77 preferentially recognizes Aβ peptides starting at Ala at position two. Only minor cross-reactions (if any) were observed with other synthetic Aβ peptides including Aβ_1 - 40, Aβ_3 - 40, Aβ_pE3 - 40, Aβ_4 - 40, and Aβ_5 - 40 (Fig. 1). Furthermore, pre-absorption of pAb77 with synthetic Aβ_2 - 40 efficiently reduced pAb77-immunoreactivity in formalin-fixed and paraffin-embedded brain tissues from AβPP/PS1KI mice (Fig. 2A). Finally, Aβ peptides and sAβPPα were immunoprecipitated with mAb 6E10 from TBS- and SDS-soluble fractions from 7- and 12-month-old 5XFAD mice and analyzed on western blots with pAb77. A single peptide band, presumably corresponding to Aβ_2 - 42, was detected in samples from 5XFAD but not in WT lysates. Reprobing the same blot with mAb6E10 showed a prominent signal comigrating with Aβ_1 - 42, as well as a faster migrating band, likely representing Aβ_2 - 42. In addition, several other bands with lower electrophoretic mobilities were detected (Fig. 2B). Those protein bands that were observed in both 5XFAD and WT mice most probably included immunoglobulin heavy- and light chains derived from the 6E10 immunoprecipitation step.

Aβ_2-X in transgenic mouse models

Using pAb77 against Aβ_2-X, abundant extracellular amyloid plaques were detectable in 12-month-old AβPP/PS1KI and 5XFAD mice (Fig. 3A, B). Staining was most abundant in the plaque core (Fig. 3B) with a few Aβ_2-X-positive blood vessels (Fig. 3C, G). In addition to extracellular Aβ_2-X immunoreactivity, cell-associated intraneuronal staining was detected in the CA1 region of the hippocampus and in cortical areas (Fig. 3D–F, H). Occasionally, pAb77-positive plaque-independent axonal spheroids were detected (Fig. 3I).

Aβ_2-X in sporadic AD cases

In tissue slides from human sporadic AD brain, pAb77 against Aβ_2-X decorated predominantly blood vessels and showed a substantially lower reactivity with amyloid plaques (Fig. 4). Plaque-associated immunoreactivity was mostly found in the plaque core, whereas 4G8 (recognizing generic Aβ) showed intense labeling of both the central and peripheral plaque region (Figs. 4 and 5). Table 1 demonstrates a semiquantitative analysis of pAb77 staining versus pan-Aβ (polyclonal antibody 24311 [18]) in sporadic AD and non-demented control specimen. Scores were given for the following categories of Aβ protein deposits detectable in the tissues: CAA (congophilic amyloid angiopathy) and cored plaques, defined as Aβ protein deposits with a compacted center circled by less structured protein aggregates. Remarkably, pAb77 decorated mostly CAA and minor plaque pathology.