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P1‐273: Different mechanisms lead to the formation of neuritic and cell body amyloid‐β accumulations
Author(s) -
Muresan Zoia,
Muresan Virgil
Publication year - 2010
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.2010.05.825
Subject(s) - neurite , senile plaques , extracellular , soma , intracellular , amyloid (mycology) , microbiology and biotechnology , biology , chemistry , cell , neuroscience , pathology , alzheimer's disease , biochemistry , medicine , disease , botany , in vitro
Background: Increasing evidence supports the role of AbOligomers (AbOs) in the pathogenesis of Alzheimer’s Disease (AD). The use of specific conformational intracellular antibodies (intrabodies) allows to selectively target AbOs inside cells at different points for mechanistic as well as for interference purposes. We previously described the selection of conformation-sensitive and sequence-specific single chain Fv antibody fragments (scFvs), targeting AbOs (Meli et al., 2009). Now, we describe the use of these scFvs as intrabodies for selective subcellular targeting and intracellular knock-down/silencing of AbOs, in well established cellular models for AbOs production and secretion. Methods: Sensitive coimmunoprecipitation methods allowed verifying the binding of the recombinant scFvs to the natural AbOs derived from cell culture media or from human Cerospinal Fluid (CSF). The intracellular expression of the anti-AbOs intrabodies was performed in a well established cell model for AbOs production and secretion, referred to as 7PA2 cells (Walsh et al.,2000;2002), generating cells stably transfected with intrabodies in different formats.The in vivo binding and the modulation of AbOs generation were studied by experiments of subcellular fractionation on discontinuous iodixanol gradients and by intrabody-AbOs coimmunoprecipitation approaches. Results: The recombinant anti-AbOs scFvs selectively bind and immunoprecipitate the natural AbOs from 7PA2 conditioned media and from human Cerospinal Fluid (CSF). The effects of the intracellular expression of the anti-AbOs intrabodies on the Ab/AbOs formation and APP metabolism were studied by subcellular fractionation. Total microsomes fractionated from 7PA2 or 7PA2 expressing intrabodies showed differential distribution of Ab/AbOs. Moreover, a sensitive intrabody-AbOs co-immunoprecipitation approach allowed demonstrating a selective immunoprecipitation of specific forms of natural SDS-stable AbOs.The expression of an intrabody with an ERretention signal (KDEL) showed a significant reduction of AbOs secretion and selective modulation of AbOs accumulation in subcellular fractions. Conclusions: The binding ability of the anti-AbOs scFvs to the natural AbOs in vitro as well as in vivo (in 7PA2 living cells), validates their conformation-specificity and -selectivity. Moreover, the anti-AbOs intrabodies interfere with AbOs intracellular formation and secretion. The intrabody approach is therefore a promising way for interfering with the activity of different intracellular Ab assemblies (in ways that would not be possible by RNA-interference approaches) exploitable for new recombinant therapeutics.