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

Abstract

According to the modified amyloid hypothesis, the key event in the pathogenesis of Alzheimer’s disease (AD) is the deposition of neurotoxic amyloid β-peptides (Aβs) in plaques and cerebral blood vessels. Additionally to full-length peptides, a great diversity of N-truncated Aβ variants is derived from the larger amyloid-β protein precursor (AβPP). Vast evidence suggests that Aβ_x-42 isoforms play an important role in triggering neurodegeneration due to their high abundance, amyloidogenic propensity and toxicity. Although N-truncated Aβ peptides and Aβ_x-42 species appear to be the crucial players in AD etiology, the Aβ_2-X isoforms did not receive much attention yet. The present study is the first to show immunohistochemical evidence of Aβ_2-X in cases of AD and its distribution in AβPP/PS1KI and 5XFAD transgenic mouse models using a novel antibody pAB77 that has been developed using Aβ_2-14 as antigen. Positive plaques and congophilic amyloid angiopathy (CAA) were observed in AD cases and in both mouse models. While in AD cases, abundant CAA and less pronounced plaque pathology was evident, the two mouse models showed predominantly extracellular Aβ deposits and minor CAA staining. Western blotting and a capillary isoelectric focusing immunoassay demonstrated the high specificity of the antibody pAb77 against Aβ-variants starting with the N-terminal Alanine-2.

Keywords

5XFAD AβPP/PS1KI Alzheimer’s disease immunohistochemistry mouse model N-truncated Aβ postmortem

INTRODUCTION

Alzheimer’s disease (AD) is the most common type of dementia worldwide. It is characterized by the accumulation of specific proteins, namely tau and amyloid-β protein (Aβ), in the brain. The amyloid hypothesis considers the accumulation of Aβ peptides as the central and triggering event in AD [1, 2]. The formation of neurotoxic oligomers and larger assemblies of Aβ is thought to be the product of an imbalance in its production and clearance [3]. Aβ is produced from the larger amyloid-β protein precursor (AβPP) by proteolytic cleavages executed by different secretase enzymes. The combined activities of β- and γ-secretase release Aβ peptides of various lengths [4]. The discovery that certain early-onset familial forms of AD may be caused by an enhanced production of Aβ peptides led to the hypothesis that amyloidogenic Aβ is causally involved in the AD pathogenic process [4]. Besides Aβ peptides starting with an aspartate at position 1, a variety of different N-truncated Aβ peptides have been identified in AD brains [5]. In support of the amyloid hypothesis, several autosomal dominant mutations in the AβPP and Presenilin (PSEN1, PSEN2) genes increase the production of Aβ_x-42 relative to Aβ_x - 40, boost the production of Aβ in general, or affect the aggregation propensity of Aβ by altering its amino acid sequence [6]. Additionally to the full-length Aβ peptides starting with N-terminal aspartate at position 1, different N-truncated isoforms have been demonstrated in human brain by amino acid sequencing or mass-spectrometric analyses [7 –10]. Some of these appear to be particularly abundant and to have neurotoxic properties due to their capacity to rapidly form stable aggregates (reviewed in [5]).

Not much is known about the presence of N-terminally truncated Aβ_2-X in the amyloid pathology of AD brains. Surface-enhanced laser desorption/ionization mass spectrometry was performed comparing AD and vascular dementia patients [11]. In AD, the authors found Aβ starting with amino acids Asp-1, Ala-2, pyrGlu-3, Phe-4, and Arg-5 in senile plaque extractions with Phe-4 to be the most prevalent one. In addition, Portelius and colleagues [10] carried out a mass spectrometric characterization of brain Aβ isoform signatures and reported the presence of Aβ_2 - 42 in cortex, hippocampus, and cerebellum of sporadic AD patients.

It has been reported that Aβ released from astrocytes and microglia included high proportions of N-terminally modified Aβ peptides, presumably including Aβ_2/3 - x and Aβ_4/5 - x [12]. The inhibition of BACE1 significantly reduced the level of Aβ_1 - 40 in cell culture supernatants while the level of the presumed Aβ_2 - 40 remained stable [12]. In the AβPP/PS1KI mouse model, Aβ_x-42 peptides represent the majority of Aβ species, with a complex pattern of N-truncated variants and dimers in an age-dependent manner, including Aβ starting with Ala-2 [13].

The aim of the present work was to characterize our recently generated antibody as a tool to study the presence of Aβ_2-X in two AD transgenic mouse models and human cases with sporadic AD.

MATERIAL AND METHODS

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.

RESULTS

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.

DISCUSSION

In a current review [5], we have discussed the amyloid hypothesis integrating N-truncated Aβ peptides, as there is mounting evidence that they might be of particular relevance for AD pathogenesis. It is now well accepted that besides Aβ peptides starting with an Asp at position 1 (Asp-1; Aβ_1 - x), a variety of different N-truncated Aβ peptides are found in AD brain including Ala-2, pyroglutamylated Glu-3, Phe-4, Arg-5, His-6, Asp-7, Ser-8, Gly-9, Tyr-10, and pyroglutamylated Glu-11 (reviewed in [5]). While the presence of Aβ_2 - 42 has been previously described in brain homogenates and cerebrospinal fluid from AD patients [15], to the best of our knowledge, so far, no study reported on the immunohistochemical staining pattern of Aβ_2-X (Ala-2) in brain tissue from sporadic AD and transgenic mouse models.

Miller et al. [19] compared the peptide compositions of the cerebrovascular and senile plaque core amyloid deposits in AD. Matrix-assisted, laser-desorption-time-of-flight mass spectrometry of plaque-Aβ revealed an array of peptides ending with Ala-42 of that sequence, while cerebrovascular Aβ began with Arg-1 ending at Val-40. The group verified that Phe-4 is the main component in plaques. Other N-termini reported were Asp-1, Ala-2, Arg-5, Asp-7, Ser-8, and Gly-9. Surface-enhanced laser desorption/ionization mass spectrometry was also used to compare AD and vascular dementia patients [11]. In AD, Aβ started predominantly with Phe-4, and lower amounts were found for Asp-1, Ala-2, pyrGlu-3, Phe-4, and Arg-5 in senile plaque extractions. Using immunoprecipitation in combination with mass spectrometric analysis in the three different brain regions analyzed from control, sporadic, and familial AD, the dominating Aβ isoforms were described as Aβ_1 - 42, Aβ_pE3 - 42, Aβ_4 - 42, and Aβ_1 - 40, with Aβ_1 - 42 and Aβ_4 - 42 being the dominant isoforms in hippocampus and cortex in all groups analyzed [10]. In AβPP/PS1KI mice, the combination of mass spectrometry and two-dimensional gel electrophoresis revealed a variety of N-truncated Aβ species [13]. In addition to full-length Aβ_1 - 42 peptides, Aβ_4/5 -42 and Aβ_{8/9/10/11 - 42} were detected at 2.5 months of age, followed by Aβ_2/3 -42 being detectable at four months of age. CNS and the cerebrospinal fluid from AβPP23 transgenic mice and AD brain were assessed by one- and two-dimensional gel electrophoresis, immunoblotting, and mass spectrometry [20]. Significant differences between the Aβ peptides extracted from the brains of AβPP23 mice and brain samples from sporadic AD cases (Braak stage V-VI) were observed in terms of the relative abundance of specific variants of Aβ peptides, such as pyroGlu-3, Aβ_1 - 42, and N-terminally truncated Aβ_2/3 -42. In the present study, we could show that pAb77 is a highly specific antibody reacting with the free N-terminus of Aβ_2-X, but not with other tested Aβ variants: Aβ_1 - 42, Aβ_1 - 40, Aβ_3 - 40, Aβ_pE3 - 40, Aβ_4 - 40, and Aβ_5 - 40. The antibody pAb77 reacted predominantly with plaque cores in AβPP/PS1KI and 5XFAD mouse models as well as in cases with sporadic AD, but also intraneuronal immunoreactivity could be detected in the transgenic lines. The presence of Aβ_2-X in somatodendritic compartments underscores previous observations demonstrating abundant intraneuronal Aβ in AβPP/PS1KI [21] or 5XFAD mice [17, 22]. In human cases, Aβ_2-X-like immunoreactivity was mostly confined to CAA with lower abundance in amyloid plaques in AD cases, which is in accordance with its reported relative low abundance in amyloid plaques collected by laser microdissection microscopy [8, 9].

DISCLOSURE STATEMENT

Authors’ disclosures available online (http://j-alz.com/manuscript-disclosures/15-0394r2).

References

Hardy

Selkoe

2002

The amyloid hypothesis of Alzheimer’s disease: Progress and problems on the road to therapeutics

Science 297 353 356

Hardy

Higgins

1992

Alzheimer’s disease: The amyloid cascade hypothesis

Science 256 184 185

Benilova

Karran

De Strooper

2012

The toxic Abeta oligomer and Alzheimer’s disease: An emperor in need of clothes

Nat Neurosci 29 349 357

Bayer

Wirths

Majtenyi

Hartmann

Multhaup

Beyreuther

Czech

2001

Key factors in Alzheimer’s disease: Beta-amyloid precursor protein processing, metabolism and intraneuronal transport

Brain Pathol 11 1 11

Bayer

Wirths

2014

Focusing the amyloid cascade hypothesis on N-truncated Abeta peptides as drug targets against Alzheimer’s disease

Acta Neuropathol 127 787 801

Bohm

Chen

Sevalle

Qamar

Dodd

Schmitt-Ulms

Fraser

St George-Hyslop

2015

Current and future implications of basic and translational research on amyloid-β peptide production and removal pathways

Mol Cell Neurosci 66 Pt A 3 11

Masters

Simms

Weinman

Multhaup

McDonald

Beyreuther

1985

Amyloid plaque core protein in Alzheimer disease and Down syndrome

Proc Natl Acad Sci U S A 82 4245 4249

Rufenacht

Guntert

Bohrmann

Ducret

Dobeli

2005

Quantification of the Abeta peptide in Alzheimer’s plaques by laser dissection microscopy combined with mass spectrometry

J Mass Spectrom 40 193 201

Güntert

Dobeli

Bohrmann

2006

High sensitivity analysis of amyloid-beta peptide composition in amyloid deposits from human and PS2APP mouse brain

Neuroscience 143 461 475

10.

Portelius

Bogdanovic

Gustavsson

Volkmann

Brinkmalm

Zetterberg

Winblad

Blennow

2010

Mass spectrometric characterization of brain amyloid beta isoform signatures in familial and sporadic Alzheimer’s disease

Acta Neuropathol 120 185 193

11.

Lewis

Beher

Cookson

Oakley

Piggott

Morris

Jaros

Perry

Ince

Kenny

Ballard

Shearman

Kalaria

2006

Quantification of Alzheimer pathology in ageing and dementia: Age-related accumulation of amyloid-β (42) peptide in vascular dementia

Neuropathol Appl Neurobiol 32 103 118

12.

Oberstein

Spitzer

Klafki

Linning

Neff

Knolker

Lewczuk

Wiltfang

Kornhuber

Maler

2015

Astrocytes and microglia but not neurons preferentially generate N-terminally truncated Abeta peptides

Neurobiol Dis 73 24 35

13.

Casas

Sergeant

Itier

Blanchard

Wirths

van der Kolk

Vingtdeux

van de Steeg

Ret

Canton

Drobecq

Clark

Bonici

Delacourte

Benavides

Schmitz

Tremp

Bayer

Benoit

Pradier

2004

Massive CA1/2 neuronal loss with intraneuronal and N-terminal truncated Abeta 42 accumulation in a novel Alzheimer transgenic model

Am J Pathol 165 1289 1300

14.

Haussmann

Jahn

Linning

Janssen

Liepold

Portelius

Zetterberg

Bauer

Schuchhardt

Knolker

Klafki

Wiltfang

2013

Analysis of amino-terminal variants of amyloid-beta peptides by capillary isoelectric focusing immunoassay

Anal Chem 85 8142 8149

15.

Wiltfang

Esselmann

Cupers

Neumann

Kretzschmar

Beyermann

Schleuder

Jahn

Ruther

Kornhuber

Annaert

De Strooper

Saftig

2001

Elevation of beta-amyloid peptide 2-42 in sporadic and familial Alzheimer’s disease and its generation in PS1 knockout cells

J Biol Chem 276 42645 42657

16.

Huttenrauch

Baches

Gerth

Bayer

Weggen

Wirths

2015

Neprilysin deficiency alters the neuropathological and behavioral phenotype in the 5XFAD mouse model of Alzheimer’s disease

J Alzheimers Dis 44 1291 1302

17.

Jawhar

Trawicka

Jenneckens

Bayer

Wirths

2012

Motor deficits, neuron loss, and reduced anxiety coinciding with axonal degeneration and intraneuronal Abeta aggregation in the 5XFAD mouse model of Alzheimer’s disease

Neurobiol Aging 33 196.e129 196.e140

18.

Bouter

Dietrich

Wittnam

Rezaei-Ghaleh

Pillot

Papot-Couturier

Lefebvre

Sprenger

Wirths

Zweckstetter

Bayer

2013

N-truncated amyloid beta (Abeta) 4-42 forms stable aggregates and induces acute and long-lasting behavioral deficits

Acta Neuropathol 126 189 205

19.

Miller

Papayannopoulos

Styles

Bobin

Lin

Biemann

Iqbal

1993

Peptide compositions of the cerebrovascular and senile plaque core amyloid deposits of Alzheimer’s disease

Arch Biochem Biophys 301 41 52

20.

Schieb

Kratzin

Jahn

Mobius

Rabe

Staufenbiel

Wiltfang

Klafki

2011

Beta-amyloid peptide variants in brains and cerebrospinal fluid from amyloid precursor protein (APP) transgenic mice: Comparison with human Alzheimer amyloid

J Biol Chem 286 33747 33758

21.

Breyhan

Wirths

Duan

Marcello

Rettig

Bayer

2009

APP/PS1KI bigenic mice develop early synaptic deficits and hippocampus atrophy

Acta Neuropathol 117 677 685

22.

Oakley

Cole

Logan

Maus

Shao

Craft

Guillozet-Bongaarts

Ohno

Disterhoft

Van Eldik

Berry

Vassar

2006

Intraneuronal beta-amyloid aggregates, neurodegeneration, and neuron loss in transgenic mice with five familial Alzheimer’s disease mutations: Potential factors in amyloid plaque formation

J Neurosci 26 10129 10140

23.

Braak

1991

Neuropathological staging of Alzheimer-related changes

Acta Neuropathol 82 239 259

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

Abstract

Keywords

INTRODUCTION

MATERIAL AND METHODS

Development of polyclonal antibodies in rabbits

Antibody characterization by capillary isoelectric focusing immunoassay and western immunoblot analysis

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

Human brain samples

Alzheimer mouse models

Immunohistochemistry and immunofluorescence of Aβ

RESULTS

The purified polyclonal antibody pAb77 shows high selectivity for truncated Aβ2-X

Aβ2-X in transgenic mouse models

Aβ2-X in sporadic AD cases

DISCUSSION

DISCLOSURE STATEMENT

References

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

Aβ_2-X in transgenic mouse models

Aβ_2-X in sporadic AD cases