Targeting subcellular pools of Amyloid-? oligomers in living cells through intrabodies: a new concept of conformational-selective interference to study the Alzheimer's disease pathogenesis

Amyloid-? oligomers (A?Os) are neurotoxic proteinaceous forms in Alzheimer's Disease (AD) but still unknown in terms of both molecular/structural composition and cellular generation and activity. Amyloid assembly states and dynamics are very difficult to study in living cells, also for the lack of selective tools of study. To this attempt, conformational-sensitive antibodies represent one of the more promising tools. 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 molecules. We generated, by an in vivo intracellular selection in yeast cells, a panel of conformation-sensitive antibody fragments selectively recognizing AD-relevant A?O conformers (Meli et al., J Mol Biol 2009). Recently, we expressed an anti-A?O single chain antibody fragment (scFv) as an intrabody, with the aim of intercepting A?Os 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) (Meli et al., Nature Comm 2014). We provided: i. a novel approach to selectively control levels and toxic conformations of biologically-active A?O in living cells; ii. a new dissection of cellular mechanisms of A?O generation, trafficking and actions. Indeed, by exploiting CSI, we demonstrate that intracellular A? can oligomerize into pathological forms, through critical conformations formed inside the endoplasmic reticulum (ER). Currently, we are investigating the functional effects mediated by the ER-localized CSI on some subcellular alterations and mitochondrial disfunctions, describing an altered link between ER and mitochondria as a probable subcellular mechanism of AD pathogenesis. We are also targeting the intrabody through different lentiviral systems in primary neuronal stem cells (NSC) derived from neurogenic niches of the adult brain of AD mouse models and in primary human fibroblasts from AD patients. As future perspective, the intrabodybased 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.