Background
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-beta protein (Aβ). In fact, these proteins are essential to confirm an AD diagnosis, given that the two major histopathological hallmarks are extracellular amyloid-β plaques surrounded by dystrophic neurites and intracellular neurofibrillary tangles. Furthermore, AD is characterized by neuronal loss, gliosis and congophilic angiopathy mainly affecting the cortex and the hippocampal formation [
1].
The amyloid hypothesis considers the accumulation of Aβ peptides as the central and triggering event in AD [
2,
3]. The formation of neurotoxic oligomers and larger assemblies of Aβ are thought to be the product of an imbalance in its production and clearance [
4]. Aβ derives from the larger amyloid precursor protein (APP) by proteolytic cleavage of different secretase enzymes. The combined activity of β- and γ-secretase activities releases Aβ peptides of various lengths [
5]. The γ-secretase comprises a high molecular weight complex that depends on presenilin-1 and -2 (PS1, PS2) activity to cleave within the transmembrane domain of APP to generate Aβ peptides and is composed of four integral membrane proteins: presenilin, nicastrin, Aph-1 and Pen-2 [
6]. Supporting the amyloid hypothesis, autosomal dominant mutations in APP, PS1 and PS2 genes cause familial early onset AD mainly by increasing the production of Aβ
x-42[
7]. However, advancing in age is considered the most prevalent risk factor for Aβ accumulation and most of the cases have a late onset. These cases are classified as sporadic AD.
Extracellular plaques are formed by Aβ peptides with different C–termini ranging from position 38 to 43 [
5]. Since the 42 amino acid isoforms Aβ
x-42 are highly susceptible to aggregate and to form oligomer and amyloid fibrils [
8], it is considered the main plaque component and the initiator of plaque formation in AD pathogenesis [
9]. In addition to the most prevalent species Aβ
x-40 and Aβ
x-42, other isoforms such as Aβ
x-38 has been reported in different mouse models, FAD cases due to mutations in APP and PS1 and in the vascular Aβ deposits of SAD cases [
10,
11]. Aβ species ending at position 43 have shown to be potently amyloidogenic and abundant [
12]. Additionally to the full length Aβ peptides starting with N-terminal aspartate at position 1, different N-truncated isoforms have been demonstrated to be as abundant as toxic due to their capacity to rapidly form stable aggregates [
13]. Not much is known about the N-terminally truncated Aβ
5-x present in the amyloid pathology of AD brains. It has been suggested that these Aβ variants are preferentially formed by an alternative cleavage of APP involving caspase activity [
14].
The aim of the present work was to characterize our recently generated antibody as a tool to study the presence of Aβ5-x in different AD transgenic mouse models and human cases, including sporadic AD and familial AD cases carrying either APP or PS1 mutations.
Discussion
Nowadays it is evident that a great heterogeneity of Aβ species exist that are deposited in AD brains including the full-length peptides, Aβ
1–40 and Aβ
1–42, and different C- and N- truncated isoforms (for example [
26‐
28]). Especially N-truncated Aβ peptides experienced a renaissance in the last decade with the development of new transgenic mouse models and novel Aβ N-terminal specific antibodies for AD research [
13,
23]. N-truncated Aβ
x–42 species have attracted much attention triggering the accumulation of Aβ into neurotoxic aggregates. Ancolio et al. [
29] was the first to show a selective and drastic increase of these species triggered by the mutation V715M APP770 in the APP gene, postulating that all Aβ
x–42 variants are main factors driving AD pathology.
The earliest evidence however for N-truncated peptides was observed by Masters et al. [
30] demonstrating that Aβ peptides starting with phenylalanine at position 4 are main components of amyloid plaques. Aβ
4–42 has been suggested to play an important role in the disease since it is deposited in early stages of the neuropathology before other isoforms appear [
31]. On the other hand, another prominent N-truncated variant, Aβ
pE3–42, was also suggested as a key player because of its limited degradation and remarkable stability [
32]. It is becoming more and more accepted that the most prevalent Aβ variants present in the brain regions affected by the disease are mainly the 42 ending variants Aβ
1–42, Aβ
pE3–42 and Aβ
4–42 (for example [
26,
28,
33]). These Aβ variants, particularly Aβ
4–42 and Aβ
pE3–42, are considered to be the most toxic due to their biochemical propensities to rapidly form stable oligomers [
23].
Despite the above mentioned importance of N-truncated peptides for AD, little is known about Aβ
5–X. It has been recognized to be present in Aβ deposits of SAD patients [
34]. Using mass spectrometry and Western blot of SAD cases and mass spectrometry of SAD and FAD cases (M146V PS1 or KM670/671NL APP), Aβ
5–40/42 was one of the detected N-truncated species [
26‐
28,
31]. Regarding transgenic mouse lines, mass spectrometry of immunoprecipitated Aβ peptides have also provided evidence of the presence of Aβ
5–42. Casas et al. [
20] reported Aβ
5–42 deposition in the APP/PS1KI mice. Our group detected Aβ
5–42 peptides in the 5XFAD mouse model [
35].
The current report aims to provide a better understanding of a possible contribution of Aβ
5–X in three different transgenic amyloid mouse models, SAD and FAD cases. Previous papers reported on a rabbit polyclonal [
34] and a mouse monoclonal antibody [
14] specific to the N-terminal end of Aβ
5–40/42, which bind to the epitope Aβ
5–12 (NH
2-RHDSGYEVC-COOH). We have generated a rabbit polyclonal antibody, which specifically binds to Aβ
5–x as shown by dot blot and Western blot. The AB5-3 strongly detects Aβ
5–42 low molecular weight oligomers showing its specificity for peptides starting with an arginine at position 5, while other Aβ variants starting at position 1, 3 (pyroglutamate) and 4 did not cross-react. While the polyclonal antibody AB5-3 reacted specifically with the free N-terminus of Aβ
5–42, we cannot exclude that it also may recognize Aβ
6-X variants.
In agreement with the observations of the previously published polyclonal antibody [
34], we detected parenchymal and vascular deposits in SAD. Vascular Aβ
5-x seems to be present in most (80%) of the cases. Confirming published observations [
34], the vascular Aβ
5-x deposits often showed a stronger signal than aggregation in plaques of SAD cases. Immunostainings with the AB5-3 confirmed the presence of the Aβ
5–x in extracellular deposits, however at a minor degree corroborating previous studies.
For the first time we present evidence that Aβ
5-x immunoreactivity is abundant in extracellular plaques and vascular deposits in different FAD cases. The cases studied included subjects expressing the APP Arctic and Swedish mutations, as well as the PS1 delta exon 9 mutation. Different patterns of Aβ
5–X aggregation were found among the different FAD cases. Previous data obtained from patients carrying the APP Arctic mutation reported ring-like plaques with Aβ peptides showing an accentuation of the contour and negative or weakly stained centers [
36]. According to Nilsberth et al. [
16] the Arctic mutation leads to an increase of Aβ protofibril formation, however the underlying molecular mechanism is still unknown.
Intraneuronal Aβ is a major risk factor in AD pathology triggering neuron loss [
37‐
40]. During the last years, intraneuronal accumulation has been reported in several mouse models including APP
SDLPS1
M146L[
41], APP
SLPS1
M146L[
42], Tg2576 [
43], 3xTg-AD [
19], APP
Arc[
44,
45], 5XFAD [
21], APP
T714I[
46], APP
SL/PS1
M146L[
47], APP/PS1KI [
20,
48,
49], TBA2 mice expressing pyroglutamate modified Aβ
3–42[
50] and in Tg4-42 expressing Aβ
4–42[
23]. Furthermore, early intraneuronal Aβ accumulations have been detected in the homozygous 5XFAD mouse model at 1.5 months of age immediately preceding extracellular plaque deposition occurring at the age of 2 months [
51]. Abundant intraneuronal Aβ has been demonstrated to correlate with a reduced number of neurons irrespectively of the extracellular Aβ aggregates in APP/PS1KI and in 5XFAD mice [
40].
Using the AB5-3 no Aβ
5-x intraneuronal immunoreactivity was found in young mice APP/PS1KI and in 5XFAD mice. This observation indicates that Aβ
5-x may not contribute to trigger neuronal degeneration and appears late in AD pathology. Despite the fact that Aβ
5–x is a scarce variant in AD brains it was present in almost all sporadic cases and in virtually all the familial AD cases tested with a different degree regarding CAA and plaque deposition. Contrasting previous findings with abundant intraneuronal N-truncated Aβ
4-x in homozygous 5XFAD mice [
51], Aβ
5–x appeared late in the amyloid cascade as there was no intraneuronal staining in young APP/PS1KI and homozygous 5XFAD mice. A variety of different N-truncated Aβ peptides besides Aβ
5-x (Arg-5) have been identified in AD brains including Ala-2, pyroglutamylated Glu-3, Phe-4, His-6, Asp-7, Ser-8, Gly-9, Tyr-10 and pyroglutamylated Glu-11 [
26‐
31,
52‐
55]. We cannot rule out however that the polyclonal antibody AB5-3 may cross-react with other N-truncated Aβ variants not studied here that may be more prevalent than Arg-5 in amyloid plaques.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
EAG performed experiments and wrote the paper, YB, BCR and OW contributed to experiments, LL, MI, AP and AV-A contributed with neuropathological and clinical expertise, TAB designed the study and wrote the paper. All authors read and approved the final manuscript.