Regulation of intracellular innate immune amyloid beta (aβ) clearance

Abstract

AbstractBackgroundInnate immune Aβ clearance may be involved in Alzheimer’s disease (AD) etiology and treatment response following anti‐amyloid immunotherapy. Regulation of intracellular Aβ clearance is not measured in AD risk‐ or diagnostic assessments, and pathways and mechanisms are insufficiently mapped. We present assays for Aβ catalysis in myeloid cells (THP‐1 cells and human iPSC microglia).MethodTHP‐1 cells were differentiated toward macrophages, and treated with various inhibitors including IDE inhibitor (6bK, Tocris),BACE1 inhibitor (LY2886721, Tocris), Nep inhibitor (Phosphoramidon, Tocris), Bafilomycin A1(Sigma)(1h) and Rapamycin (Sigma) (18h) before adding 100 ng/ml Aβ40 (2h), followed by 4h chase. For AD patient derived microglia, reprogrammed iPSCs were differentiated through a myeloid progenitor stage. Mature microglia were characterized by qPCR, Immunofluorescence and phagocytosis assay. Two immunoassays were developed on the Simoa platform using proprietary sheep monoclonal antibodies (Pre Diagnostics, Norway). Both assays employ an Aβ c‐terminal antibody for capture (AB.4D7). Assay 20‐x employs a specific Aβ20’ cut point antibody (NH2‐20.2B8) and assay x‐40 a linear mid‐domain antibody (AB.2A9) binding full length Aβ as detector.Result1) Innate immune cells (THP1, microglia) catabolize Aβ 1‐40 producing 20‐x fragments. 2) Inhibition of Nep and BACE1 reduces the production of 20‐x fragment of Aβ in THP1 cells. 3) Autophagy inhibitor BafA1 increases both 1‐40 and 20‐x fragments indicating the role of the endo‐lysosomal system in the degradation of full length and 20‐x Aβ. 4) Autophagy inducer, Rapamycin reduces both 1‐40 and 20‐x fragments indicating the role of mTOR mediated autophagy and the endo‐lysosomal system in the degradation of full length and 20‐x Aβ.Conclusion1) We demonstrate a novel assay for Aβ degradation by Innate immune cells including THP‐1 and iPSC derived microglia. 2) Inhibition of Nep and BACE1 reduces 20‐x peptide generation indicating this as a potential marker for Aβ. 3) Modulation of autophagy affects 20‐x peptide generation indicating the potential of targeting endo‐lysosomal and autophagic pathway against AD.