Conformational targeting of intracellular Aβ oligomers demonstrates their pathological oligomerization inside the endoplasmic reticulum

Abstract Aβ oligomers (AβOs) are crucially involved in Alzheimer’s Disease (AD). However, the lack of selective approaches for targeting these polymorphic Aβ assemblies represents a major hurdle in understanding their biosynthesis, traffic and actions in living cells. Here, we established a subcellularly localized conformational-selective interference (CSI) approach, based on the expression of a recombinant antibody fragment against AβOs in the endoplasmic reticulum (ER). By CSI, we can control extra- and intracellular pools of AβOs produced in an AD-relevant cell model, without interfering with the maturation and processing of the Aβ precursor protein. The anti-AβOs intrabody selectively intercepts critical AβO conformers in the ER, modulating their assembly and controlling their actions in pathways of cellular homeostasis and synaptic signalling. Our results demonstrate that intracellular Aβ undergoes pathological oligomerization through critical conformations formed inside the ER. This establishes intracellular AβOs as key targets for AD treatment and presents CSI as a potential targeting strategy. Introduction The proteolytic processing, misfolding and self-aggregation of several proteins are often associated with pathological conditions and conformational diseases. However, the identification, study and targeting of different conformations and multimeric states of a given protein in the complex context of subcellular compartments/microdomains of living cells remains a big challenge. A most relevant example of such a challenge is represented by the amyloid-β (Aβ) peptides, crucial players of Alzheimer’s disease (AD) pathogenesis1,2,3,4. In living cells, differently than in a test tube containing only synthetic peptides, the Aβ species are generated from the amyloid precursor protein (APP) by a complex process of regulated intramembrane proteolysis (RIP)5,6, and undergo a process of misfolding and aggregation, whose mechanism and subcellular localization(s) are still debated7. In particular, along the aggregation pathways, naturally occurring Aβ oligomers (AβOs) are still considered mysterious entities in terms of molecular and structural composition and activity8, even though they are recognized as the most neurotoxic proteinaceous forms in AD8,9. As an additional level of complexity, APP–RIP generates different proteolytic fragments and shares the secretases (both α, β and γ) with numerous different substrates5. Thus, the cellular biochemistry of Aβ is representative of two general mechanisms operating in living cells and organisms, namely RIP and amyloid formation (proteins enriched in cross β-sheet and prone to self-aggregation) and represents not only a pathologically relevant target, but also an ideal test case for the development of new approaches for studying these processes in cells. Conformational-sensitive antibodies are important tools for analysing amyloid assembly states and dynamics10. In particular, recombinant antibody fragments can be exploited as intracellular antibodies (intrabodies) for a subcellular-localized interference to block or modulate the function of target molecules11,12. In principle, if the intrabodies are intrinsically equipped with conformational-sensitive binding properties, they could be exploited for interference studies not currently feasible with nucleic acid targeting methods (that is, RNA interference or gene knockout), that can silence entire gene products (that is, APP or RIP machinery protein components) but not peculiar post-translational modification products (such as AβOs). Furthermore, even though new chemical modulators and inhibitors for APP–RIP or for Aβ assembly are intensively studied, their molecular selectivity and their precise subcellular delivery and actions remain not easy to control. In the current state of the art, while several conformation- and oligomeric-specific antibodies targeting the Alzheimer’s AβOs have been developed8, they are largely not exploitable for subcellular targeting and intracellular functional studies in living cells. We generated, by an in vivo intracellular selection in yeast cells, a panel of conformation-sensitive antibody fragments selectively recognizing AD-relevant AβO conformers13 ideal for the expression (as genes) in mammalian cells, as intrabodies targeted to different subcellular compartments. Here we expressed the anti-AβO single-chain antibody fragment (scFv) A13 (ref. 13) as an intrabody, with the aim of intercepting AβOs at subcellular sites of their putative formation, and of attempting their functional silencing. In this way, we established a new experimental paradigm of subcellular-localized and conformational-selective interference (CSI). The intrabody-based CSI besides providing a novel approach to selectively control biologically-active AβO conformers in living cells, allows a new dissection of cellular mechanisms of AβO generation, trafficking and actions. Indeed, by exploiting CSI, we demonstrate that intracellular Aβ can oligomerize into pathological forms, through critical conformations formed inside the endoplasmic reticulum (ER). The anti-AβOs intrabody selectively intercepts critical AβO conformers and controls their ‘toxic’ assembly in the ER, without interfering with the complex processes of maturation and processing of APP. Remarkably, the pool of targeted AβO conformers are involved in the deregulation of two independent pathways of cellular homeostasis and synaptic signalling. Our overall results firmly establish the ER as the site of formation of critical Aβ conformers and validate ER-formed intracellular AβOs as a key target for AD. For these reasons, the anti-AβOs CSI can be exploitable for in vivo therapeutic applications as well as to improve our understanding of the molecular and cellular processes of AD pathogenesis, thereby uncovering new targets for drugs development. Indeed, one of the earliest events in AD pathogenesis seems to be the intraneuronal Aβ oligomerization14,15 and the synaptic targeting of AβOs3,16 and current studies on the intracellular Aβ generation and oligomerization do not offer yet a common and conclusive opinion, also for the lack of adequate experimental tools of study. Results The anti-AβO scFvA13 expressed as an intrabody in mammalian cells This study is based on scFvA13, a recombinant antibody fragment generated by a direct intracellular selection of functional intrabodies in yeast cells against the in vivo misfolded antigen Aβ13. The scFvA13 selectively recognizes conformational Aβ epitopes, as attested by the preferential binding in vitro to synthetic oligomeric assemblies, such as the amyloid-derived diffusible ligands (ADDLs17), but not to the monomeric or fibrillar states13. Furthermore, the oligomeric-specific scFvA13-purified protein was characterized for its neutralizing properties against synthetic ADDLs13 or against endogenous Aβ species18 as well as for the recognition of pathological AβO de (...truncated)