Effects Of Metal Ions And Inhibitors On The Aggregation And Cytotoxicity Of Amyloid-β Peptide

Alzheimer’s disease(AD)is the leading cause of dementia,accounting for about 60 percent to 80 percent of the cases.AD is the fourth leading causes of death in industrialized societies,preceded only by heart disease,cancer and stroke.AD brings not only serious social problems but also a heavy burden on economic and social development.The primary pathological hallmarks of AD are the deposition of amyloid plaques in the medial temporal lobe and cortex of AD-affected brains and neurofibrillary tangles formed by hyperphosphorylated tau.Although the etiology of AD remains unclear,many genetic and pathological evidence supports that amyloid-β(Aβ)peptide,the main component of the amyloid plaque,is the most important pathogenic factor of AD.AP aggregation causes a cascade of pathological changes including the formation of neurofibrillary tangles,increased intracellular oxidative stress,synaptic injury,inflammation and degeneration of neurons,which ultimately lead to clinical manifestations of dementia in AD patients.Aβ monomer that present in healthy body has normal physiological function.Experimental evidence indicated that many factors derived from body and environment could lead to Aβ aggregation and toxicity.PM2.5 related epidemiological and toxicological studies indicated that many metal ions absorbed on PM2.5 could be risk factors that lead to Aβaggregation.The studies of the mechanism underlying PM2.5-induced neurodegeneration have already become the hot spots of current researches.Therefore,the present thesis investigated the effects of seven PM2.5 related metal ions on Aβ aggregation and cytotoxicity.For this purpose,in vitro studies of aggregation kinetics,structure,morphology and cytotoxicity were performed.Curcumin and HSA were indicated to ameliorate Aβ-induced neurotoxicity.However,the effects of these two inhibitors on Aβ aggregation are still largely subjects of debate.Therefore,effects of these two inhibitors on different stages of Aβ aggregation were also studied.The present thesis might contribute to the understanding of the role of influencing factors and inhibitors in Aβ aggregation and provide some implications in prevention and therapeutics of AD.In the second chapter,the effects of Mg2+,Mn2+,Ni2+,Cr2+,Co2+,Pb2+ and Cu2+ on Aβ40 aggregation kinetics,structure and cytotoxicity were first studied.The results indicated that the effects of these divalent metal ions on Aβ40 aggregation were different.Ni2+,Cr2+,Co2+,Pb2+and Cu2+ suppressed the formation and growth process of Aβ40 fibril.Further turbidity test and TEM examination showed that despite inhibiting the Aβ40 fibril formation and growth process,Ni2+,Cr2+,Co2+,Pb2+ and Cu2+ induced the formation of amorphous aggregates by acting on monomeric Aβ40 aggregation.ThT assay and ANS assay demonstrated that Ni2+,Cr2+,Co2+,Pb2+ and Cu2+ inhibited Aβ40 fibrillation through inhibiting the formation of β-sheet structures and hydrophobic structures.When SH-SY5Y cells were treated with Aβ40 monomers together with Ni2+,Cr2+,Co2+,Pb2+ or Cu2+,the resulted cytotoxicity was lower than that induced by the growth of Aβ40 fibrils.Ni2+,Cr2+,Co2+,Pb2+ and Cu2+ also reduced the cytotoxicity induced by the growth of Aβ40 fibrils.In contrast,Mg2+ and Mn2+ did not significantly affect the aggregation and cytotoxicity of Aβ40.A lot of experimental evidence demonstrated that Aβprotofibril and fibril could act as aggregate seeds capable of promoting Aβ fibrillation.The seeding-mediated aggregation also leads to significant cytotoxicity.Nevertheless,it is still unknown that whether metal ions-induced non-fibrillar,amorphous aggregates could act as aggregate seeds to promote A(3 fibrillation or not.Therefore,in the third chapter,the seeding ability and related cytotoxicity of Mg2+,Mn2+,Ni2+,Cr2+,Co2+,Pb2+ and Cu2+-induced Aβ40 aggregates were studied.The results indicated that different metal ions-induced Aβ40 aggregates could act as aggregate seeds to promote AP40 fibrillation.Aβ40 fibrils and these metal ions-induced Aβ40 aggregates could produce significant cytotoxicity through seeding-mediated aggregation.Experimental evidence indicated that seeding-mediated aggregation of Aβ aggregates could lead to significant cytotoxicity.Therefore,inhibitors directed against this process might be beneficial for AD therapeutics.In the fourth and fifth chapters,the effects of curcumin and HSA on Aβ fibrillation were studied,respectively.In the fourth chapter,because curcumin could interfere with ThT fluorescence assay,QCM-D was used to investigate the effect of curcumin on the growth of Aβ40 fibrils.The results showed that,compared with the control,addition of curcumin promoted the deposition of Aβ40 monomer on the A(340 fibrils to form the flexible structured Aβ40 aggregates.The curcumin-induced Aβ40 aggregates were less able to grow.In the fifth chapter,the results indicated that HSA could inhibit Aβ42 monomer fibrillation and the growth of AP42 protofibrils and fibrils.The inhibitory effects of HSA on the growth of Aβ42 protofibrils and fibrils were more significant than that of HSA on Aβ42 monomer fibrillation.SPR measurement showed that the KD of HSA from its reaction with Aβ42 monomers,protofibrils and fibrils were 17.5,3.3 and 0.3 μM,respectively.Therefore,the results indicated that the increased affinity of HSA with Aβ42 protofibrils and fibrils could account for the potent inhibitory effect of HSA on the growth of Aβ aggregates.Moreover,kd of HSA from its reaction with Aβ42 monomers,protofibrils and fibrils were 1.2×10-2s-1、0.4×10-2s-1、0.1 ×10-2s-1,respectively.The results indicated that the complex formed by HSA and Aβ42 monomer was unstable compared to which formed by HSA and A(342 protofibril/fibril.HSA could inhibit the seeding-mediated cytotoxicity of Aβ42 aggregates in a dose-dependent manner.In summary,the present in vitro study might contribute to the in-depth understanding of the effects of metal ions and inhibitors on Aβ aggregation and cytotoxicity and provide some implications for future AD-related studies.