Research Article: N-truncated Aβ4–x peptides in sporadic Alzheimer’s disease cases and transgenic Alzheimer mouse models

Date Published: October 4, 2017

Publisher: BioMed Central

Author(s): Oliver Wirths, Susanne Walter, Inga Kraus, Hans W. Klafki, Martina Stazi, Timo J. Oberstein, Jorge Ghiso, Jens Wiltfang, Thomas A. Bayer, Sascha Weggen.

http://doi.org/10.1186/s13195-017-0309-z

Abstract

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.

The online version of this article (doi:10.1186/s13195-017-0309-z) contains supplementary material, which is available to authorized users.

Partial Text

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].

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 [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 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 [30]. 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 [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 Aβ4–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 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 [32]. Overall, the distribution of Aβ4–x peptides was also substantially different from the staining pattern reported for other N-truncated species, including Aβ2–x [7] 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 [33]. 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) [33].

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.

 

Source:

http://doi.org/10.1186/s13195-017-0309-z

 

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