The Study Of β-Amyloid(1-42) Oligomers Preparation, Identification And Neurotoxicity

Objective: 1. To investigate the effect of different preparation conditions on aggregated Aβ oligomers, and to study the different aggregation morphology of β-amyloid(Aβ_1-42) using atomic force microscopy（AFM）. 2. To study the effect of Aβ oligomers on memory impairment and neurotoxicity in rats.Methods: 1. Preparation and observation of Aβ Oligomers: Aβ_1-42 peptide wasdiluted into PBS at 4℃ for 1d, 4℃ for 7d and 37℃ for 1d. The Aβ oligomers distribution was observed by Western blot. Aβ_1-42 morphological structure was observed by the tapping mode and phase imaging technology. 2. Study of the memory impairment and neurotoxicity of Aβ oligomers in rats: Alzheimer disease（AD） models were established by injecting Aβ_1-42 oligomers（1μg / μl each） into lateral cerebral ventricle with micro-injection system; Morris water maze test was used to evaluate the changes of learning and memory abilities in AD rats. Morphologic changes of cortex and hippocampus were observed by HE staining under light microscope; 3. Using SPSS19.0 statistical software for statistical analysis. Results: 1. Aβ oligomers presented mainly dimer and trimer（between 6-15Kd） at 4 ℃ for 1d, pentamer（about 19Kd） and ten dimer（between 49-64Kd） mainly at 4 ℃ for 7d, trimer（about 15Kd） and hexamer（about 30Kd） and fourteen-mer（about 70Kd） mainly at 37 ℃ for 1d. 2.Different morphous were observed, including globular oligomers with diameters varying from 2 to 6 nm, ring-like structures, protofibrillar structures. Primary protofibrils appear to contain bead by bead assemblies of multiple globular structures, and ripe protofibrils like tight rope-like structures. 3. The effect of Aβoligomers on memory impairment: The escape latency of Aβ oligomers group and Aβ fibers group after modeling were prolonged than those of before modeling（P <0.05）; Compared with Aβ fibers group, the escape latency of Aβoligomers group was significantly longer（P <0.05）. 4.The influence of Aβoligomer on the cortex and hippocampus in rats: there were visible neuron damage both in Aβ oligomer group and fiber group,especially in Aβ oligomer.Hippocampal neuron disorder, reducing the number of cells, a large number of cytoplasmic vacuolization, nuclear condensation, stained. Conclusions: 1.Incubation temperature and time are important conditions for the degree of polymerization of the oligomer, the longer the incubation time, the higher the incubation temperature and the higher the degree of polymerization of Aβoligomers. 2. AFM is a good tool to study protein aggregation process, Aβaggregation continuous process are observed, comprising a spherical oligomers,the cyclic structure, elementary fibrils, mature fibrils, fibers and other bodies. 3.Relatively the neurotoxicity of high molecular weight Aβ oligomers are stronger than the fibers.