N-truncated Aβ_4–x peptides in sporadic Alzheimer’s disease cases and transgenic Alzheimer mouse models

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 [1]. 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 [2].

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 [3]. In addition to full-length Aβ_1–40 and Aβ_1–42 peptides, a variety of peptides with shorter C-termini, such as Aβ_1–37, Aβ_1–38, or Aβ_1–39, have been described [4, 5]. Furthermore, elongated amyloidogenic Aβ_1–43 peptides have recently been reported [6]. Similar heterogeneity has been observed at the Aβ N-terminus, and several N-terminally truncated Aβ forms, including Aβ_2–x [7], Aβ_pE3–x [8, 9], Aβ_5–x [10, 11], and Aβ_pE11–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 [14‐17], which detected further variants such as Aβ_7–42, Aβ_8–42, or Aβ_9–42. 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 [18].

Aβ_4–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 [19] 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 [20]. More recent mass spectrometric characterizations of Aβ isoform signatures in human brain samples confirmed that Aβ_4–42 and Aβ_1–42 represent highly abundant species in cortical samples of AD and control brains [14, 21]. Whether Aβ_4–x peptides also occur within intracellular compartments is currently unclear. Likewise, the origin of Aβ_4–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 Aβ_4/5–x [22]. The aim of the present work was to characterize newly generated Aβ_4–x-specific antibodies as a tool to study Aβ_4–x peptides in different AD transgenic mouse models and human patients with sporadic AD.

In addition to full-length Aβ peptides starting with an aspartate in position 1 (Aβ_1–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 [18]. 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 Aβ_4–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 Aβ_4–x peptides and protected primary cortical neurons from the toxicity of Aβ_4–42 peptides in vitro [30]. However, in addition to monomeric and oligomeric Aβ_4–x peptides, NT4X-167 was shown to recognize Aβ_pE3–x peptides. Hence, this antibody is not suitable for accurate measurement of the abundance and distribution of Aβ_4–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 Aβ_4–x peptides was confirmed by CIEF immunoassay and urea SDS-PAGE, and no cross-reactivity for Aβ_1–40, Aβ_2–40, Aβ_3–40, Aβ_pE3–40, and Aβ_5–40 was observed. Furthermore, in immunohistochemical staining, the immunoreactivity could be entirely blocked by preabsorption with Aβ_4–40 but not Aβ_1–40 peptides. Two independent animals were immunized and yielded two antisera, 029-1 and 029-2, with nearly identical immunoreactivity, indicating that Aβ_4–9 might be a reliable immunogen to raise Aβ_4–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 Aβ_4–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 Aβ_4–x peptides was restricted largely to amyloid plaque cores and CAA, whereas diffuse amyloid deposits were negative. The presence of Aβ_4–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 [26]. 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 Aβ_4–x peptides [31]. 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 Aβ_4–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 Aβ_4–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 [32]. Overall, the distribution of Aβ_4–x peptides was also substantially different from the staining pattern reported for other N-truncated species, including Aβ_2–x [7] or Aβ_5–x [10, 11], which were not or less confined to cored neuritic plaques. Previous studies have demonstrated that Aβ_4–x peptides rapidly formed soluble oligomers and fibrillar higher-molecular-weight aggregates [33]. This biochemical property might explain not only the confined localization of Aβ_4–x peptides to amyloid cores but also their high neurotoxicity in vitro and in vivo. Short-term exposure of primary cortical neuron cultures to Aβ_4–40 and Aβ_4–42 peptides resulted in a concentration-dependent cytotoxic effect with comparable effect sizes to Aβ_1–42. Furthermore, the expression of Aβ_4–42 under the control of a neuronal promotor caused age-dependent behavioral deficits and hippocampal neuron loss in a transgenic mouse model (Tg4-42) [33].

Another important unresolved issue is the abundance of Aβ_4–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 Aβ_4–x antibodies for quantitative analysis, we combined the 029-2 antibody with a C-terminus-specific Aβ₄₀ antibody [29] in a sandwich ELISA. In the SDS-soluble brain fraction of 5-month-old heterozygous 5XFAD mice, this assay detected around 1 ng of Aβ_4–40 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 Aβ_1–40 and Aβ_1–42 peptides in the same brain extracts of the same animal cohort with a comparable ELISA system and IC16 as a capture antibody [28]. Combining the results from both studies indicates that Aβ_1–40 and Aβ_1–42 peptides are approximately 75-fold and 200-fold more abundant, respectively, than Aβ_4–40 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 Aβ_4–40 and Aβ_4–42 peptides were only a small fraction of the Aβ_1–40 and Aβ_1–42 peaks [5]. Taken together, these results indicate that, quantitatively, N-truncated Aβ_4–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 [19]. 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 [20]. In addition, later studies using mass spectrometry have generally supported that Aβ_1–42, Aβ_pE3–42, and Aβ_4–42 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 Aβ_4–x peptides and their distribution in soluble and insoluble brain fractions are clearly warranted. How Aβ_4–x peptides are generated remains entirely unclear. Although there is evidence that enzymatic activities can facilitate N-terminal Aβ truncations such as Aβ_2–x [37, 38], no enzymes have yet been identified that are able to generate Aβ_4–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 Aβ_3–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 Aβ_4–x peptides [39]. Our finding that Aβ_4–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 Aβ_4–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 Aβ_4–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 Aβ_4–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.