- Open Access
N-truncated Aβ4–x peptides in sporadic Alzheimer’s disease cases and transgenic Alzheimer mouse models
Alzheimer's Research & Therapy volume 9, Article number: 80 (2017)
The deposition of neurotoxic amyloid-β (Aβ) peptides in plaques in the brain parenchyma and in cerebral blood vessels is considered to be a key event in Alzheimer’s disease (AD) pathogenesis. Although the presence and impact of full-length Aβ peptides such as Aβ1–40 and Aβ1–42 have been analyzed extensively, the deposition of N-terminally truncated Aβ peptide species has received much less attention, largely because of the lack of specific antibodies.
This paper describes the generation and characterization of novel antibodies selective for Aβ4–x peptides and provides immunohistochemical evidence of Aβ4–x in the human brain and its distribution in the APP/PS1KI and 5XFAD transgenic mouse models.
The Aβ4–x staining pattern was restricted mainly to amyloid plaque cores and cerebral amyloid angiopathy in AD and Down syndrome cases and in both AD mouse models. In contrast, diffuse amyloid deposits were largely negative for Aβ4–x immunoreactivity. No overt intraneuronal staining was observed.
The findings of this study are consistent with previous reports demonstrating a high aggregation propensity of Aβ4–x peptides and suggest an important role of these N-truncated Aβ species in the process of amyloidogenesis and plaque core formation.
Alzheimer’s disease (AD) is a severe age-dependent neurodegenerative disorder accounting for the majority of dementia cases worldwide. It is characterized by two neuropathological hallmark lesions, namely the accumulation of amyloid-β (Aβ) peptides in the form of extracellular plaques and the formation of intracellular neurofibrillary tangles consisting of hyperphosphorylated tau protein . The formation of extracellular plaques is mechanistically linked to the amyloid cascade hypothesis, positioning the accumulation of Aβ peptides as a pivotal and triggering event in AD etiology .
The production of Aβ peptides depends on the sequential processing of the type I single transmembrane amyloid precursor protein (APP) by two proteases called β- and γ-secretase . In addition to full-length Aβ1–40 and Aβ1–42 peptides, a variety of peptides with shorter C-termini, such as Aβ1–37, Aβ1–38, or Aβ1–39, have been described [4, 5]. Furthermore, elongated amyloidogenic Aβ1–43 peptides have recently been reported . Similar heterogeneity has been observed at the Aβ N-terminus, and several N-terminally truncated Aβ forms, including Aβ2–x , AβpE3–x [8, 9], Aβ5–x [10, 11], and AβpE11–x [9, 12, 13], have been demonstrated in parenchymal or vascular deposits of human AD brains by immunohistochemical methods. An even more complex pattern of Aβ peptides has been reported by the use of mass spectrometry [14,15,16,17], which detected further variants such as Aβ7–42, Aβ8–42, or Aβ9–42. N-terminal truncations are believed to render Aβ peptides more neurotoxic and to promote enhanced peptide aggregation compared with the corresponding full-length peptides with an intact N-terminus .
Aβ4–x peptides appear to be among the most abundant Aβ species, but not much is known about the spatial distribution of these N-terminally truncated peptides in AD brains. Already in 1985, Masters and colleagues  reported the presence of a major Aβ peptide species starting with the phenylalanine (Phe) at position 4 of the Aβ sequence in both AD and Down syndrome (DS) cases, an observation that was subsequently verified . More recent mass spectrometric characterizations of Aβ isoform signatures in human brain samples confirmed that Aβ4–42 and Aβ1–42 represent highly abundant species in cortical samples of AD and control brains [14, 21]. Whether Aβ4–x peptides also occur within intracellular compartments is currently unclear. Likewise, the origin of Aβ4–x peptides remains unknown. In this respect, a recent in vitro study suggested that neurons predominantly secrete full-length Aβ peptides starting with the aspartate in position 1, whereas astrocytes and microglia in addition secrete measurable amounts of N-terminally truncated species, including Aβ4/5–x . The aim of the present work was to characterize newly generated Aβ4–x-specific antibodies as a tool to study Aβ4–x peptides in different AD transgenic mouse models and human patients with sporadic AD.
Development of Aβ4–x-specific polyclonal antibodies in guinea pigs
Antibodies were generated by the Peptide Specialty Laboratory (PSL; Heidelberg, Germany). In brief, immunization was carried out in two guinea pigs using the six-amino acid peptide (FRHDSG) corresponding to residues (4–9) of the Aβ peptide and coupled at its C-terminus via an additional cysteine residue to maleimide-activated keyhole limpet hemocyanin (KLH) (Thermo Fisher Scientific, Waltham, MA, USA). Antisera were generated by injection of peptides conjugated to KLH and emulsified with complete Freund’s adjuvant for a first injection, followed by injections 2, 3, and 4 at 2-week intervals with incomplete Freund’s adjuvant. Serum was collected prior to the first immunization (preimmune serum) and after the fourth immunization. The immune sera obtained after the final bleed were affinity-purified and named 029-1 and 029-2.
Antibody characterization by capillary isoelectric focusing immunoassay and Western immunoblot analysis
To assess the selectivity of purified polyclonal antibodies 029-1 and 029-2 for different N-terminal variants of Aβ, we employed a capillary isoelectric focusing (CIEF) immunoassay  and urea-bicine/bis-tris/Tris/sulfate sodium dodecyl sulfate (SDS)-PAGE followed by Western immunoblotting [22, 24]. For the CIEF immunoassay, synthetic Aβ peptides with different N-termini were separated on a Peggy Sue device (ProteinSimple, San Jose, CA, USA) by isoelectric focusing in microcapillaries and subsequently were probed with purified 029-1 and 029-2 antibodies. For comparison, the CIEF immunoassay was also performed with the mouse monoclonal antibodies IC16  and 6E10 (BioLegend, San Diego, CA, USA), which recognize several N-terminal variants of Aβ. The synthetic Aβ peptides Aβ1–40, Aβ2–40, Aβ3–40, pyroglutamate AβpE3–40, Aβ4–40, and Aβ5–40 were purchased from AnaSpec (Fremont, CA, USA). Peptide stock solutions (1 mg/ml) were prepared in dimethyl sulfoxide (DMSO) or, in the case of AβpE3–40, in 0.1% NH4OH and stored at −80 °C. Aliquots were thawed only once. The peptides were further diluted in 20 mM bicine, pH 7.6, with 0.6% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate and then mixed 1:4 with Premix G2 containing a pH 5–8 nested gradient, fluorescent pH standards, and a DMSO inhibitor mix (all reagents were obtained from ProteinSimple). The automated CIEF immunoassay on the Peggy Sue platform (ProteinSimple) was carried out as described previously for the NanoPro platform (ProteinSimple) . The final peptide concentration in the microcapillaries was 100 ng/ml per peptide. Assuming an internal capillary volume of roughly 0.5 μl, this corresponded to approximately 50 pg of peptide per capillary. Primary antibodies 029-1, 029-2, 6E10, and IC16 were diluted in antibody diluent (ProteinSimple). Detection was achieved with biotinylated anti-guinea pig immunoglobulin G (IgG; Dianova, Barcelona, Spain) in combination with streptavidin-horseradish peroxidase (HRP) (ProteinSimple) for antibodies 029-1 and 029-2 or with goat-anti-mouse-HRP secondary antibody (ProteinSimple) for 6E10 and IC16.
For SDS-PAGE analysis, stock solutions of synthetic Aβ1–40, Aβ2–40, Aβ3–40, pyroglutamate Aβ3–40 (AβpE3–40), and Aβ4–40 were prepared in sample buffer (0.36 M bis-tris, 0.16 M bicine, 15% wt/vol sucrose, 1% wt/vol SDS, and 0.0075% wt/vol bromophenol blue) and stored at −80 °C. The Aβ peptides were further diluted in sample buffer, and aliquots containing 0.1, 1, and 10 ng of peptide per lane were separated by urea-bicine/bis-tris/Tris/sulfate SDS-PAGE and analyzed by immunoblotting as previously described . The blotting membranes were blocked in 2% electrochemiluminescence (ECL) advance blocking agent (GE Healthcare Life Sciences, Little Chalfont, UK) in PBS with 0.1% Tween 20 (PBS-T) for at least 4 h at room temperature (RT) and probed with the primary antibody 029-2 (1:300 in PBS-T) at 4 °C overnight. After three washing steps with PBS-T, biotinylated goat anti-guinea pig IgG (1:3000 in PBS-T; Life Technologies, Carlsbad, CA, USA) and streptavidin-HRP were employed as secondary reagents. Chemiluminescent signals obtained with ECL Prime Western Blotting Detection Reagent (GE Healthcare Life Sciences) were recorded. After three additional washing steps in PBS-T, the membrane was reprobed with 6E10 (1:1000 in PBS-T) and developed with biotinylated goat anti-mouse IgG (333 ng/ml in PBS-T; Vector Laboratories Ltd., Peterborough, UK) in combination with streptavidin-HRP complex (1:3000 in PBS-T).
Peptide competition assay
To further assess the selectivity of the Aβ4–x-specific polyclonal antibodies, a peptide competition experiment was performed. Antibody solution 029-2 was incubated with an excess of synthetic Aβ1–40 or Aβ4–40 for 5 h at RT with constant agitation, followed by centrifugation at 14,000 rpm for 5 minutes. The supernatant was used for immunohistochemical analysis of transgenic 5XFAD mouse brain tissues.
Human brain samples
Human brain samples from sporadic AD (n = 16, mean age 84.18 ± 8.04 years), nondemented control subjects (n = 6, mean age 84.17 ± 5.19 years), and DS (n = 2, mean age 61.0 ± 4.24 years) were obtained from the Netherlands Brain Bank (Table 1). The present study was approved by the ethics committee of the University Medical Center Göttingen.
Alzheimer mouse models
The generation of APP/PS1KI mice has been described previously . In this AD mouse model, human mutant APP751 containing the Swedish and London mutations is overexpressed under the control of the murine Thy-1 promoter, whereas murine presenilin-1 (PSEN1) with the M233T and L235P familial Alzheimer’s disease (FAD)-linked mutations is expressed under the control of the endogenous mouse PSEN1 promoter. APP/PS1KI mice were a generous gift of Dr. Laurent Pradier, Sanofi-Aventis, Paris, France. The analysis of 5XFAD mice (Tg6799) has also been reported previously [27, 28]. 5XFAD mice overexpress APP695 carrying the Swedish, Florida, and London mutations under the control of the murine Thy-1 promoter. In addition, human PS1 carrying the M146L and L286V mutations is also expressed under the control of the murine Thy-1 promoter. Male mice on a C57BL/6 × SJL genetic background were obtained from The Jackson Laboratory [strain B6SJL-Tg(AβPPSwFlLon,PSEN1*M146L*L286V)6799Vas/J; The Jackson Laboratory, Bar Harbor, ME, USA] and backcrossed for ten generations to C57BL/6 J wild-type mice to obtain an incipient congenic line on a C57BL/6 J genetic background. All animals were handled according to German guidelines for animal care.
Immunohistochemical and immunofluorescence analyses
Mice were killed by CO2 anesthetization followed by cervical dislocation. Brain samples were dissected and postfixed in 4% phosphate-buffered formalin at 4 °C. Immunohistochemistry was performed on 4-μm paraffin sections. The antibodies 029-1, 029-2 (1:250–500), D3E10 (against Aβ42, 1:1000; Cell Signaling Technology, Danvers, MA, USA), and IC16 (against the N-terminus of Aβ, 1:1000) were used for Aβ staining. Biotinylated secondary anti-guinea pig and anti-mouse antibodies (1:200) were purchased from Dianova and Dako (Glostrup, Denmark), respectively. Staining was visualized using the avidin-biotin complex method with a VECTASTAIN kit (Vector Laboratories) and diaminobenzidine (DAB) as the chromogen. Counterstaining was carried out with hematoxylin.
The Aβ4–x plaque load was quantified using 9-month-old hemi- and homozygous 5XFAD mice (n = 3–4) . In brief, three paraffin sections per animal were stained simultaneously with DAB. The relative Aβ4–x load in thalamus and cortex was evaluated using a BX51 microscope (Olympus, Center Valley, PA, USA) equipped with a Moticam Pro 282 camera (Motic, Wetzlar, Germany) and ImageJ1.47b software (http://imagej.nih.gov/ij). Representative images were captured and binarized to 8-bit black-and-white images using a fixed intensity threshold. Measurements were performed with analysis of the percentage of the area covered by DAB as previously described . Double-immunofluorescence staining was visualized using Alexa Fluor 488- and Alexa Fluor 594-conjugated secondary antibodies (Molecular Probes, Eugene, OR, USA) and 4′,6-diamidino-2-phenylindole using a Nikon C2+ confocal microscope equipped with 405 nm, 488-nm and 561-nm lasers (Nikon Instruments, Melville, NY, USA).
Enzyme-linked immunosorbent assay
The levels of Aβ4–40 peptides in 5-month-old heterozygous (n = 5) and homozygous (n = 6) 5XFAD mice were determined using a sandwich enzyme-linked immunosorbent assay (ELISA). Frozen brain hemispheres were weighed and sequentially extracted. First, brains were homogenized in 700 μl of Tris-buffered saline (120 mM NaCl, 50 mM Tris, pH 8.0, with cOmplete protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA) per 100 mg of tissue using a Dounce homogenizer (800 rpm). The resulting solution was centrifuged at 17,000 × g for 20 minutes at 4 °C. The pellet was dissolved in 800 μl of 2% SDS and sonicated, followed by a centrifugation step of 17,000 × g for 20 minutes at 4 °C. The supernatant containing SDS-soluble proteins was incubated while rotating with 1 μl of benzonase at RT for 10 minutes, followed by storage at −80 °C. The SDS brain fraction was used for ELISA analysis. To generate standard curves, synthetic Aβ4–40 peptides (PSL) were used. A C-terminal-specific antibody for Aβ40 (BAP-24)  served as a capture antibody and was incubated at 3 μg/ml in PBS, pH 7.2, on 96-well high-binding microtiter plates (Greiner Bio-One, Kremsmünster, Austria) overnight at 4 °C. After excess capture antibody was removed, freshly diluted brain samples (1:30) or Aβ peptide standards (31.25–750 pg/ml) in PBS containing 0.05% Tween 20 and 1% bovine serum albumin (BSA) were added. Then, the Aβ4–x-specific 029-2 antibody labeled with HRP using the Pierce EZ-LinkTM Plus Activated Peroxidase kit (Thermo Fisher Scientific) was diluted in PBS containing 0.05% Tween 20 and 1% BSA, added to each well, and incubated overnight at 4 °C. On the next day, plates were washed three times with PBS containing 0.05% Tween 20 and once with PBS. Subsequently, 50 μl of trimethylbenzidine ELISA peroxidase substrate (Interchim, Montluçon, France) was added and incubated for 1–5 minutes at RT in the dark. The reaction was terminated by adding 50 μl of 2 M H2SO4, and the absorbance was recorded using a Paradigm microplate reader (Beckman Coulter, Brea, CA, USA) at 450 nm.
Differences between groups were tested by unpaired t tests. All data were expressed as mean ± SD. Significance levels were as follows: ***p < 0.001; **p < 0.01; *p < 0.05. All calculations were performed using Prism version 6.07 for Windows software (GraphPad Software, La Jolla, CA, USA).
Purified polyclonal antibodies 029-1 and 029-2 show high selectivity for N-truncated Aβ4–x peptides
The selectivity of the affinity-purified polyclonal antibodies 029-1 and 029-2 for different N-terminal variants of Aβ was studied by three independent methods. First, a CIEF immunoassay demonstrated that both antibodies detected synthetic Aβ peptides starting with the Phe residue in position 4 without any noticeable cross-reactivity to other tested Aβ peptides (Fig. 1b and Additional file 1). In contrast, control antibody IC16 detected the N-terminus of Aβ with clear signals for Aβ1–40, Aβ2–40, and Aβ3–40. Reactivity to AβpE3–40 and Aβ4–40 was very low, whereas Aβ5–40 was not detected (Fig. 1a). Antibody 6E10 served as an additional control and detected all synthetic peptides applied (Additional file 1: Figure S1) as previously shown . Second, urea bicine/bis-tris/Tris/sulfate SDS-PAGE followed by Western immunoblotting was performed for 029-2 and indicated that this antibody selectively recognized Aβ peptides starting with Phe in position 4 in amounts as low as 1 ng. Again, no cross-reactivity was detected against any of the other synthetic Aβ peptides tested, namely Aβ1–40, Aβ2–40, Aβ3–40, and AβpE3–40 (Fig. 1c). Reprobing of the membrane with antibody 6E10 allowed the detection of all tested Aβ peptides (Fig. 1d). Third, importantly, preabsorption of 029-2 with synthetic Aβ4–40 peptides completely blocked 029-2 immunoreactivity in formalin-fixed, paraffin-embedded brain tissue from 5XFAD mice. In contrast, preabsorption with full-length Aβ1–40 peptides did not result in reduced immunoreactivity, and Aβ4–x-positive extracellular deposits were still detected (Fig. 2).
Aβ4–x in sporadic AD cases
On tissue slides from human patients with sporadic AD, 029-2 labeled extracellular amyloid deposits as well as blood vessels (Fig. 3). In contrast to IC16, which detects full-length Aβ peptides and N-truncated variants and decorates either cored plaques or more diffuse extracellular Aβ deposits (Fig. 3a, c, e), 029-2 staining was restricted mainly to compact neuritic plaques (Fig. 3f) and plaque cores (Additional file 2) as well as vascular deposits (Fig. 3b, d). Table 1 summarizes a semiquantitative analysis of 029-2 versus IC16 staining in sporadic AD and nondemented control specimens, in which the immunoreactivity in the form of cerebral amyloid angiopathy (CAA) and extracellular amyloid deposition was assessed. Double-labeling and confocal microscopy using IC16 (red) and 029-2 (green) confirmed the presence of Aβ4–x peptides predominantly in cored neuritic plaques, whereas more diffuse extracellular deposits showed only IC16 immunoreactivity (Fig. 4a). Confocal analysis of blood vessels revealed partial colocalization of Aβ4–x peptides with IC16 immunoreactivity in the vessel wall. However, in a minor subset of vessels, the vessel walls showed either IC16 or 029-2 immunoreactivity and a nonoverlapping staining pattern (Fig. 4b, arrowheads).
Aβ4–x in transgenic mouse models
With antibody 029-2 against Aβ4–x, abundant extracellular amyloid plaques were detectable in 10-month-old 5XFAD (Fig. 5b) and 8-month-old APP/PS1KI mice (Fig. 5j). Compared with staining with the Aβ42-selective antibody D3E10 (Fig. 5a), 029-2 immunoreactivity was restricted to the plaque core (Fig. 5b) in aged 5XFAD mice. Double-labeling with an APP antibody (red) confirmed the localization of Aβ4–x (green) in the plaque core, whereas APP was detected largely within dystrophic neurites in the vicinity of the amyloid plaque (Fig. 5c–e). This was further supported by double-labeling with D3E10 and 029-2, clearly indicating that Aβ4–x immunoreactivity was restricted to the central plaque core (Fig. 5f–h). Similar results were obtained for the APP/PS1KI line, in which D3E10 displayed a more diffuse and dissipated staining pattern (Fig. 5i), whereas 029-2 labeled only the central plaque core (Fig. 5j). A quantitative analysis of extracellular Aβ4–x-positive deposits using antibodies 029-1 and 029-2 was carried out in the cortex, subiculum, and thalamus of 9-month-old heterozygous and homozygous 5XFAD mice (Fig. 6a and Additional file 3). For both antibodies, a significantly increased plaque load could be detected in all analyzed areas in homozygous compared with heterozygous 5XFAD mice (all p < 0.05). In addition, to evaluate the suitability of the 029-2 antibody for ELISA quantification, Aβ4–40 levels were determined in the SDS-soluble brain fraction of 5-month-old heterozygous and homozygous 5XFAD mice. As expected, significantly increased Aβ4–40 levels were detected in homozygous 5XFAD mice (p < 0.001), supporting a gene dose-dependent increase in insoluble Aβ4–40 peptide levels (Fig. 6b).
In addition to full-length Aβ peptides starting with an aspartate in position 1 (Aβ1–x), a variety of N-terminally truncated and posttranslationally modified Aβ peptides have been detected in human AD brains [14, 16, 17]. N-terminal truncations were shown to promote the aggregation propensity of Aβ peptides . However, the presence of N-truncated peptides has been demonstrated mainly by mass spectrometry following immunoprecipitation with generic Aβ antibodies such as 4G8 or 6E10 [14, 15, 21], and their genesis in vivo is mostly unresolved. Consequently, the functional role of N-truncated Aβ peptides in the pathogenesis of AD has remained unclear, including fundamental questions about the abundance and distribution of N-truncated isoforms. This shortcoming has been particularly obvious for the Aβ4–x peptides starting with Phe in position 4, which have been proposed to be an abundant Aβ species in AD brains but for which no specific antibodies have been generated to date. We have previously reported a monoclonal antibody, NT4X-167, that preferentially detected Aβ4–x peptides and protected primary cortical neurons from the toxicity of Aβ4–42 peptides in vitro . However, in addition to monomeric and oligomeric Aβ4–x peptides, NT4X-167 was shown to recognize AβpE3–x peptides. Hence, this antibody is not suitable for accurate measurement of the abundance and distribution of Aβ4–x peptides. We have now raised polyclonal antibodies by immunizing guinea pigs with the six-amino acid peptide (FRHDSG) corresponding to residues 4–9 of the Aβ peptide sequence. The specificity of these antibodies for Aβ4–x peptides was confirmed by CIEF immunoassay and urea SDS-PAGE, and no cross-reactivity for Aβ1–40, Aβ2–40, Aβ3–40, AβpE3–40, and Aβ5–40 was observed. Furthermore, in immunohistochemical staining, the immunoreactivity could be entirely blocked by preabsorption with Aβ4–40 but not Aβ1–40 peptides. Two independent animals were immunized and yielded two antisera, 029-1 and 029-2, with nearly identical immunoreactivity, indicating that Aβ4–9 might be a reliable immunogen to raise Aβ4–x-specific antibodies in guinea pigs. Compared with the well-established antibody IC16, which preferentially detects full-length Aβ peptides, the immunohistochemical staining patterns of the newly generated Aβ4–x-specific antibodies were quantitatively and qualitatively different. In brain sections of both patients with sporadic AD and two AD mouse models, the distribution of Aβ4–x peptides was restricted largely to amyloid plaque cores and CAA, whereas diffuse amyloid deposits were negative. The presence of Aβ4–x peptides in amyloid plaque cores raises the question whether these truncated species are critical in the very early stages of the pathology. We have not yet conducted a comprehensive longitudinal study comparing different animal ages and time points before or after the onset of amyloid deposition. However, using two-dimensional Western blotting combined with mass spectrometry, N-terminally truncated Aβ peptide species starting at position 4 or 5 have already been detected at 2.5 months of age in the APP/PS1KI line, indeed indicating a very early appearance of these truncated species . In good agreement and using a similar experimental approach, Sergeant and colleagues reported that Aβ aggregates at the first stages of amyloid deposition in nondemented individuals with amyloid and tau pathologies are composed predominantly of N-truncated variants, including Aβ4–x peptides . Antibodies raised in guinea pigs are especially useful for colocalization studies because most high-quality antibodies against other Aβ species or APP fragments have previously been generated in either mice or rabbits. Indeed, double-immunofluorescence staining demonstrated Aβ4–x-positive amyloid plaque cores decorated by APP-positive dystrophic neurites with no overlap in the fluorescent signals. In line with this observation, no intraneuronal staining was observed for Aβ4–x peptides in mice at the ages of 8–10 months. However, it could be worth studying younger animals because intraneuronal Aβ accumulation is most prominent in young mice prior to amyloid plaque formation . Overall, the distribution of Aβ4–x peptides was also substantially different from the staining pattern reported for other N-truncated species, including Aβ2–x  or Aβ5–x [10, 11], which were not or less confined to cored neuritic plaques. Previous studies have demonstrated that Aβ4–x peptides rapidly formed soluble oligomers and fibrillar higher-molecular-weight aggregates . This biochemical property might explain not only the confined localization of Aβ4–x peptides to amyloid cores but also their high neurotoxicity in vitro and in vivo. Short-term exposure of primary cortical neuron cultures to Aβ4–40 and Aβ4–42 peptides resulted in a concentration-dependent cytotoxic effect with comparable effect sizes to Aβ1–42. Furthermore, the expression of Aβ4–42 under the control of a neuronal promotor caused age-dependent behavioral deficits and hippocampal neuron loss in a transgenic mouse model (Tg4-42) .
Another important unresolved issue is the abundance of Aβ4–x peptides in relation to full-length Aβ peptides in both AD and transgenic mouse models of the disease. To start to address this issue and to evaluate the novel Aβ4–x antibodies for quantitative analysis, we combined the 029-2 antibody with a C-terminus-specific Aβ40 antibody  in a sandwich ELISA. In the SDS-soluble brain fraction of 5-month-old heterozygous 5XFAD mice, this assay detected around 1 ng of Aβ4–40 per milligram of tissue with approximately fivefold higher levels in homozygous 5xFAD mice of the same age. In a previous study, we had determined the levels of full-length Aβ1–40 and Aβ1–42 peptides in the same brain extracts of the same animal cohort with a comparable ELISA system and IC16 as a capture antibody . Combining the results from both studies indicates that Aβ1–40 and Aβ1–42 peptides are approximately 75-fold and 200-fold more abundant, respectively, than Aβ4–40 peptides in the 5XFAD mouse model at 5 months of age. This also fits with a matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of formic acid brain extracts of 7-month-old 5XFAD mice. Although peptide concentrations cannot be deduced from peak heights in mass spectra, this analysis showed that the signal intensities generated by the Aβ4–40 and Aβ4–42 peptides were only a small fraction of the Aβ1–40 and Aβ1–42 peaks . Taken together, these results indicate that, quantitatively, N-truncated Aβ4–x peptides are a minor Aβ species in the 5XFAD mouse model. However, evidence from human studies suggests that the proportions of N-truncated to full-length Aβ peptides might be substantially different in the brains of patients with AD. Already more than 30 years ago, it was shown by N-terminal sequencing analysis of Aβ peptides purified from amyloid plaque cores that only around 10% of peptides displayed an intact N-terminus, whereas > 60% started with the Phe residue in position 4 . Another sequencing study confirmed that Aβ peptides starting with Phe represented the major component of plaques, whereas full-length Aβ starting with Asp was detected predominantly in the vasculature . In addition, later studies using mass spectrometry have generally supported that Aβ1–42, AβpE3–42, and Aβ4–42 belong to the Aβ peptide species with a high prevalence in AD brains [14, 15, 21, 34]. Finally, comparative biochemical studies of amyloid plaques isolated from human AD brains and APP transgenic mouse models have also shown that N-truncated Aβ peptides are much more prevalent in patients with AD, and it has been proposed that the greater abundance of N-truncated Aβ peptides is at least in part responsible for the substantially lower solubility of amyloid plaque material from humans [35, 36]. In any case, additional studies in human AD brains using genuinely quantitative methods to determine the abundance of Aβ4–x peptides and their distribution in soluble and insoluble brain fractions are clearly warranted. How Aβ4–x peptides are generated remains entirely unclear. Although there is evidence that enzymatic activities can facilitate N-terminal Aβ truncations such as Aβ2–x [37, 38], no enzymes have yet been identified that are able to generate Aβ4–x peptides. Nonenzymatic generation of truncated Aβ peptides has also been proposed, and it has been shown that full-length Aβ peptides can spontaneously decompose into shorter N- and C-terminally truncated isoforms in vitro in the absence of proteases [39, 40]. With respect to N-terminal truncations, spontaneous decomposition of full-length to Aβ3–x peptides with subsequent cyclization of the N-terminal Glu to pGlu has been demonstrated, but there is only very limited evidence that a similar process could produce Aβ4–x peptides . Our finding that Aβ4–x peptides are confined largely to amyloid cores supports an important role of these N-truncated Aβ species in the process of amyloid plaque formation. Beyond that, further understanding of the pathological relevance of Aβ4–x peptides will likely require clarification of their origin and the subsequent generation of genetic loss-of-function models.
Antibodies developed in the present study selectively detect Aβ4–x species and might represent useful research tools for immunohistochemical or biochemical analyses of the occurrence or distribution of these peptides. Our analysis reveals that Aβ4–x is restricted mainly to amyloid plaque cores and CAA in AD and DS cases, as well as in the 5XFAD and APP/PS1KI transgenic mouse lines of AD. This is in line with previous reports demonstrating the high aggregation propensity of these N-terminally truncated Aβ variants.
Amyloid precursor protein
Bovine serum albumin
Cerebral amyloid angiopathy
Capillary isoelectric focusing
Enzyme-linked immunosorbent assay
Familial Alzheimer’s disease
Keyhole limpet hemocyanin
Nondemented control subject
Phosphate-buffered saline with 0.1% Tween 20
Peptide Specialty Laboratory
Sodium dodecyl sulfate
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The authors gratefully acknowledge financial support provided by the Alzheimer Forschung Initiative (grant 16013 to OW), Gerhard-Hunsmann Stiftung (to OW), Stiftung VERUM (to SWe), and the Forschungskommission of the Medical Faculty of the Heinrich-Heine-University Düsseldorf (grant 9772513 to SWe). JW is supported by an Ilídio Pinho professorship and iBiMED (UID/BIM/04501/2013) at the University of Aveiro, Portugal. We thank Drs. Karlheinz Baumann and Manfred Brockhaus (F. Hoffmann-La Roche Ltd., Basel, Switzerland) for providing the carboxyl terminus-specific Aβ antibody BAP-24.
Ethics approval and consent to participate
All procedures involving human tissue were approved by the ethics committee of the University Medical Center Göttingen (protocol number 12/1/15). All tissues were received from the Netherlands Brain Bank (NBB), and all materials and data collected by the NBB are obtained on the basis of written informed consent. All experimental procedures in the study that involved animals were approved by the institutional animal care and use committee at University Medicine Göttingen.
Consent for publication
All authors gave consent for publication.
The authors declare that they have no competing interests.
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CIEF electropherograms for antibodies 6E10 and 029-1. (PDF 184 kb)
Quantification of extracellular 029-1 positive plaque load in cortex, subiculum, and thalamus in heterozygous and homozygous 9-month-old 5XFAD mice. (PDF 119 kb)
Predominant plaque core staining using 029-2 in a patient with sporadic AD. (PDF 183 kb)