Cell-Derived Hexameric β-Amyloid: A Novel Insight Into Composition, Self-Assembly and Nucleating Properties

Abstract

A key hallmark of Alzheimer’s disease (AD) is the extracellular deposition of amyloid plaques composed primarily of the amyloidogenic amyloid-β (Aβ) peptide. The Aβ peptide is a product of sequential cleavage of the Amyloid Precursor Protein (APP), the first step of which gives rise to a C-terminal Fragment (C99). Cleavage of C99 by γ-secretase activity releases Aβ of several lengths and the Aβ42 isoform in particular has been identified as being neurotoxic. The misfolding of Aβ leads to subsequent amyloid fibril formation by nucleated polymerisation. This requires an initial and critical nucleus for self-assembly. Here, we identify and characterise the composition and self-assembly properties of cell-derived hexameric Aβ42 and show its nucleating properties which are dependent on the Aβ monomer availability. Identification of nucleating assemblies that contribute to self-assembly in this way may serve as therapeutic targets to prevent the formation of toxic oligomers.

Mechanism of Cellular Formation and In Vivo Seeding Effects of Hexameric β-amyloid Assemblies

“…1a). As previously reported [19,20], we detected in a reproducible manner a band at 28kDa in both C42 and C99-expressing cells with Aβ-speci c antibodies (W0-2 in Fig. 1b and 6E10 in Supplementary Fig.…”

“…1c). The use of this technique recently allowed for extensive biochemical analyses [20]. Among the collected observations, dot blotting with primary antibodies directed against the free C-terminal end of the two major Aβ isoforms (Aβ 40 , Aβ 42 ) was performed on the isolated ~28kDa assemblies and identi ed these to be composed of Aβ 42 (see Supplementary Fig.…”

“…Among the collected observations, dot blotting with primary antibodies directed against the free C-terminal end of the two major Aβ isoforms (Aβ 40 , Aβ 42 ) was performed on the isolated ~28kDa assemblies and identi ed these to be composed of Aβ 42 (see Supplementary Fig. S2 and [20]). Based on the observed size, these Aβ assemblies likely correspond to hexameric Aβ 42 assemblies, produced in a cellular context.…”

“…Identi cation of hexameric-like Aβ assemblies across a wide range of cell lines As Aβ self-assembly strongly depends on the context of its release, we sought to determine whether the assemblies of interest were produced particularly by CHO cells or commonly across other cell lines. Using the same procedure as described above [20], we assessed the Aβ pro le in transiently transfected mouse embryonic broblasts (MEF) (Fig. 2a) as well as two human immortalized models -human embryonic kidney (HEK293) cells (Fig.…”

“…We previously reported the presence of ~28kDa Aβ assemblies in Chinese hamster ovary (CHO) cells expressing amyloidogenic fragments of human APP [19]. Our recent biochemical studies have recently demonstrated that these assemblies likely correspond to Aβ 42 hexamers [20]. Here, we report the identi cation of similar, hexameric-like, Aβ assemblies across several cell lines -including a neuronal-like cell line -and, more importantly, in the 5xFAD mouse model of amyloid pathology [21] and the cerebrospinal uid of human AD patients.…”