Humanin Blocks the Aggregation of Amyloid-β Induced by Acetylcholinesterase, an Effect Abolished in the Presence of IGFBP-3

Abstract

It is known that the humanin (HN) peptide binding to amyloid-beta (A beta) protects against its cytotoxic effects, while acetylcholinesterase (AChE) binding to A beta increases its aggregation and cytotoxicity. HN is also known to bind the insulin-like growth factor binding protein-3 (IGFBP-3). Here, we examined the regulation of A beta conformations by HN, AChE, and IGFBP-3 both in vitro and in the conditioned media from A549 and H1299 lung cancer cells. Our in vitro results showed the following: IGFBP-3 binds HN and blocks it from binding A beta in the absence or presence of AChE; HN and AChE can simultaneously bind A beta but not when in the presence of IGFBP-3; HN is unable to reduce the aggregation of A beta in the presence of IGFBP-3; and HN abolishes the aggregation of A beta induced by the addition of AChE in the absence of IGFBP-3. In the media, AChE and HN can simultaneously bind A beta. While both AChE and HN are detected when using 6E10 A beta antibodies, only AChE is detected when using the A beta 17-24 antibody 4G8, the anti-oligomer All, and the anti-amyloid fibril LOC antibodies. No signal was observed for IGFBP-3 with any of the anti-amyloid antibodies used. Exogenously added IGFBP-3 reduced the amount of HN found in a complex when using 6E10 antibodies and correlated with a concomitant increase in the amyloid oligomers. Immunodepletion of HN from the media of the A549 and H1299 cells increased the relative abundance of the oligomer vs the total amount of A beta, the A11-positive prefibrillar oligomers, and to a lesser extent the LOC-positive fibrillar oligomers, and was also correlated with diminished cell viability and increased apoptosis.