Formation and activity of subcellular pools of Alzheimer's Abeta oligomers in living cells

Critical conformations of some proteins and peptides involved in different neurodegenerative disorders are difficult to study, especially in living cells and at the subcellular level. The development of conformational-sensitive antibodies can help the study of amyloid assembly states and dynamics. In detail, recombinant small antibody fragments can be exploited as intracellular antibodies (intrabodies) in living cells for a subcellular-localized interference to block or modulate the function of target molecules. Abeta oligomers (A?Os) are considered the most neurotoxic proteinaceous forms in Alzheimer's Disease (AD) but they are still mysterious entities, existing in a huge number of different assembly states and conformations. In the attempt to study the processes of Abeta oligomerization in living cells, we firstly generated, by an in vivo intracellular selection in yeast cells1, some conformation-sensitive antibody fragments selectively recognizing AD-relevant AbetaO conformers1. Then, we expressed one of the anti-AbetaO antibody fragments as an intrabody, with the aim of intercepting AbetaOs at subcellular sites of their putative formation, and of attempting their functional silencing. In this way, we recently established a new experimental paradigm of subcellular-localized and conformational-selective interference (CSI)2 demonstrating also that the endoplasmic reticulum (ER) plays a crucial role in the oligomerization of Abeta into pathological forms. Thus our study in living cells provides: i. a novel approach to selectively control levels and toxic conformations of biologically-active AbetaO and ii. a new way to dissect cellular mechanisms of AbetaO generation, trafficking and actions. Currently, we are investigating the functional effects mediated by the intrabody targeted in the ER on some subcellular alterations and mitochondrial dysfunctions, describing a new link between ER and mitochondria. We are also targeting the intrabody through different lentiviral systems in primary neuronal stem cells derived from neurogenic niches of the adult brain of AD mouse models and in primary human fibroblasts from AD patients. As future perspective, the intrabody-based 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.