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O5‐04‐06: Subcellular Targeting of Endogenous Amyloid‐B Oligomers in Human Primary Cells
Author(s) -
Meli Giovanni,
La Marca Valeria,
Manca Annalisa,
Scopa Chiara,
Ruggeri Federica,
Scardigli Raffaella,
Cattaneo Antonino
Publication year - 2016
Publication title -
alzheimer's and dementia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 6.713
H-Index - 118
eISSN - 1552-5279
pISSN - 1552-5260
DOI - 10.1016/j.jalz.2016.06.727
Subject(s) - endoplasmic reticulum , subcellular localization , microbiology and biotechnology , endogeny , biology , intracellular , cell culture , biochemistry , genetics , cytoplasm
Background: Amyloidb oligomers (AbOs) are crucial neurotoxic species in Alzheimer’s Disease (AD) but they are still difficult targets for therapy. We demostrated that recombinant antibody fragments can be exploited as intracellular antibodies to block or modulate the toxicity of endogenous AbOs in living cells. In this way, we established a new experimental paradigm of subcellularlocalized conformational-selective interference (CSI) (Meli et al., Nature Comm 2014). Methods:The intrabody-based CSI approach, previously established in mammalian fAD cell lines, was here developed through the use of new lentiviral systems, inducible or not, exploited in different murine and human primary cell systems. In detail, we investigated: i. primary neuronal stem cells (NSC) derived from neurogenic niches of the adult brain of AD mouse model Tg2576; ii. primary human fibroblasts from different AD patients (some of them carrying the V717I fAD mutation); iii. human iPS cells derived from primary fibroblasts. NSC were mantained in vitro as neurosphere cultures and differentiated in neurons or astrocytes. Results:We demostrated that subcellular AbOs are responsible for alterations on growth and differentiation of NSC, and for dysfunctions of mitochondria and of homeostatic mechanisms in primary cells. These alterations and dysfunctions are functionally rescued by our CSI approach, in which the anti-AbOs intrabodies were targeted to the Endoplasmic Reticulum and the early secretory pathway. Conclusions:The subcellular localization of the intrabody-based CSI for AbOs in the Endoplasmic Reticulum and the early secretory pathway, strongly suggests the role of these subcellular compartments in determining the toxic activity of endogenously produced AbOs. In mammalian fAD cell lines we found an AD-relevant functional link between the ER and mitochondria, which seems to be confirmed in human primary fibroblasts. Thus, the intrabody-based CSI for AbOs is an useful approach to study AbO actions inside living cells and it gives a new perspective for in vivo immunotherapy. Supported by Alzheimer’s Association NIRG-12-237751, Human Brain Project Neuroantibodies #604102.