Abstract
We have recently reported on the preparation of a membrane-associated β-barrel Pore-Forming Aβ42 Oligomer (βPFOAβ42). It corresponds to a stable and homogeneous Aβ42 oligomer that inserts into lipid bilayers as a well-defined pore and adopts a specific structure with characteristics of a β-barrel arrangement. As a follow-up of this work, we aim to establish βPFOAβ42's relevance in Alzheimer's disease (AD). However, βPFOAβ42 is formed under dodecyl phosphocholine (DPC) micelle conditions—intended to mimic the hydrophobic environment of membranes—which are dynamic. Consequently, dilution of the βPFOAβ42/DPC complex in a detergent-free buffer leads to dispersion of the DPC molecules from the oligomer surface, leaving the oligomer without the hydrophobic micelle belt that stabilizes it. Since dilution is required for any biological test, transfer of βPFOAβ42 from DPC micelles into another hydrophobic biomimetic membrane environment, that remains associated with βPFOAβ42 even under high dilution conditions, is a requisite for the validation of βPFOAβ42 in AD. Here we describe conditions for exchanging DPC micelles with amphipols (APols), which are amphipathic polymers designed to stabilize membrane proteins in aqueous solutions. APols bind in an irreversible but non-covalent manner to the hydrophobic surface of membrane proteins preserving their structure even under extreme dilution conditions. We tested three types of APols with distinct physical-chemical properties and found that the βPFOAβ42/DPC complex can only be trapped in non-ionic APols (NAPols). The characterization of the resulting βPFOAβ42/NAPol complex by biochemical tools and structural biology techniques allowed us to establish that the oligomer structure is maintained even under high dilution. Based on these findings, this work constitutes a first step towards the in vivo validation of βPFOAβ42 in AD.
Highlights
Amyloid-β (Aβ) oligomers have been proposed as the Aβ species responsible for the neurotoxicity observed in Alzheimer’s disease (AD) (Haass and Selkoe, 2007)
Under optimized dodecyl phosphocholine (DPC) micelle conditions, we showed that Aβ42 assembles into a stable and homogeneous oligomer that inserts into lipid bilayers as a well-defined pore and adopts a structure with characteristics of a β-barrel arrangement
As mentioned in the introduction, due to the dynamic nature of detergent micelles, the βPFOAβ42/DPC complex was not expected to be stable under high dilution conditions
Summary
Amyloid-β (Aβ) oligomers have been proposed as the Aβ species responsible for the neurotoxicity observed in Alzheimer’s disease (AD) (Haass and Selkoe, 2007). This heterogeneity and transient nature have prevented a consensus as per the specific Aβ oligomer form responsible for AD neurotoxicity (Benilova et al, 2012) To resolve this issue, many laboratories worldwide have developed in vitro conditions to obtain as homogeneous and stable Aβ oligomer preparations as possible with which to subsequently establish the links between the specific features of the Aβ oligomer under study and AD neurotoxicity (Lambert et al, 1998; Galeazzi et al, 1999; Bitan et al, 2003; Barghorn et al, 2005; Jan et al, 2010; Sandberg et al, 2010; O’Malley et al, 2014). The second one involves generating antibodies against the Aβ oligomer of interest to establish the oligomer presence in AD human brain tissue (Barghorn et al, 2005; Lambert et al, 2007; Lasagna-Reeves et al, 2011)
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