The Research Of The Neurotoxicity And The Interaction With Insulin Receptor Of Chemical Cross-linked Amyloid-β

Amyloid-β (Aβ) is a hallmark of Alzheimer’s disease (AD), and is considered as a major risk factor of AD. Aβ monomer forms aggregates of diverse molecular weight by self-assembly. The Aβ aggregates impair nervous system in different ways, in which the interactions between oligomeric AP and the receptors of important signaling pathways were demonstrated as a central mechanism of Aβ neurotoxicity. In Aβ studies, chemical cross-linking reactions are useful methods in the preparation of oligomeric Aβ. However, does chemical cross-linking alter the neurotoxicity, the interactions with major receptor and other physiological properties of AP or not, remain an unclear topic to be explained.1) We have achieved two kinds of AP aggregate of certain molecular weight from different cross-linking measurements by controlling reacting conditions. The metal ions were removed by chelation. We also produced Aβ aggregate with a traditional uncross-linking way. By tricine-SDS-PAGE, we found that different method result in different ratios of diverse oligomeric Aβs and monomers. We acquired maximum oligomeric Aβs content by PICUP and minimum oligomeric Aps content by uncross-linking. The Zeta potential of these three Aβs are in a range of-10～15mV, which inferring a trend of further aggregating. Cu2+oxidation cross-linked Aβ showed much lower ThT fluorescence intensity than the other two Aβs, demonstrating low P-sheet density and possible alternative neurotoxicity.2) We treated neurons of new-born SD rat with three kinds of Ap at a concentration gradient respectively, and determined neuronal survivals, neuronal apoptosis in different phases and key apoptotic protein activations by MTT assay, flow cytometry and western blotting. Cu2+oxidation cross-linked AP triggered minimum neuronal survival, induced neurons to highest levels of early/middle-late apoptosis, significantly upregulated the activation of apoptotic protein Caspase3, and powerfully promoted the transfer of cytochrome c from mitochondrial to cytosol. Nevertheless, it is hypothesized that Cu2+oxidation cross-linked Aβ regulates upsteam factors of Caspase9to enhance the recruitment of Caspase9, thus result in similar Caspase9activation level with the other two Aβs. Furthermore, the three Aβs caused upregulations of T-NOS and CAT activity, and supporting a complex mechanism of Cu2+oxidation cross-linked AP neurotoxicity.3) We successfully separated and enriched2-4mer oAβs by using electroelution and organic solvent precipitation, thus carried neuronal treatment with each oAβ respectively and observed the impact of neuronal IR. The result of immunocytofluorescence demonstrated that Aβ dimer mainly induced redistribution of neuronal IR, and Aβ trimer and tetramer tended to competitively bound to IR and caused downregulation of IR density.