Study On The Preparation, Properties And Mechanism Of Neurotoxicity Of Amyloid A (Aβ) Mutant

Alzheimer’s disease(AD), a kind of neurodegenerative diseases often found in old people, was first reported by a German scientist Alois Alzheimer in1907. There are about30million AD patients worldwide, and the number of the patients is increasing every year, which threats the people’s health seriously. The clinical manifestations of AD include cognitive decline, behavioral abnormalities and reduced activity of daily life. The characteristic pathological features of Alzheimer’s disease are senile plaques, neurofibrillary tangles, the loss of neurons, and granulovacuolar degeneration. The mechanism of AD is extremely complicated, and may be the result of the interaction of multiple factors. However, the exact mechanism of the AD remains elusive.Amyloid beta peptide is the major component of the senile plaques in the AD brain and occupies a central position in the pathogenesis of AD. Aβ is released into synaptic clefts after generation, and interacts with free metal ions there. Copper and zinc may promote aggregation of Aβ and reduced to product reactive oxygen species(ROS), contributing to the progression in AD. Compared with human amyloid beta amino acid sequence, three amide acid alternations are found in mouse amyloid beta amino acid sequence, namely R5G, Y10F and H13R. The senile plaques are absent in the brain of the adult mouse, which may be associated with these three amide acids. Previous study showed that Y10F Aβ1-42attenuates the neurotoxicity of amyloid beta, indicating that tyrosine10is playing an important role in causing neurotoxicity. In addition to the amyloid peptide aggregation in the brain, the pathological features of AD cells include heme metabolic disorders. It is reported that chemical synthesis of Aβ can bind with heme through Arg5and His13, and form a complex with peroxidase activity. By comparing the properties and neurotoxicity between hAβ and mAβ, we try to find the reason and molecular mechanism that why there are no senile plaques in the brain of adult brain. We first got the prokaryotic expressed hAβ1-42and mAβ1-42with no extra amino acids. We detect the aggregation ability between hAβ1-42and mAβ1-42by CD, ThT fluorescence assay, ANS fluorescence assay and transmission electron microscope. The cellular toxicity was investigated by ROS fluorescent detector and SHSY5Y cell viability. We also detected the ability of binding with heme and the peroxidase activity of mAβ1-42-Heme and hAβ1-42-Heme. Our study indicated that mAβ1-42was less prone to form fibrils and has a weaker hydrophobicity. The TEM suggested that mAβ1-42can form long, narrow fibrils. Besides, mAβ1-42had a weaker neurotoxicity, which may be due to the reduced generation of ROS. Compared with hAβ1-42-Heme complex, mAβ1-42-Heme didn’t has a Soret peak at412nm and had a weaker perocidase acitivity.There are some amino acid mutations of amyloid beta in some populations, which leads to the AD at early age. Compared with amino acid sequence of wide type amyloid beta, E22Q was found in some Dutch AD patients and△E22was found in some Osaka AD patients. We got the E22Q-Aβ1-42and△E22Aβ1-42with high purification. We detect the aggregation ability of E22Q-Aβ1-42and△E22Aβ1-42by CD, ThT fluorescence assay, ANS fluorescence assay and transmission electron microscope. The cellular toxicity were investigated by ROS fluorescent detector and SHSY5Y cell viability. Our result indicated that, compared with wide type Aβ1-42, E22Q-Aβ1-42and△E22Aβ1-42was less prone to form fibrils and has a weaker hydrophobicity. Besides, E22Q-Aβ1-42and△E22Aβ1-42had a stronger neurotoxicity than wide type Aβ1-42, which may be due to the increased generation of ROS.SXAS(small X-ray angle scattering) can be used to detect the particle with a diameter of1to100nm. As the amyloid beta can assemble to form large aggregates, we use the SXAS to study the aggregation of amyloid beta in the absence or in the presence of Cu2+/Zn2+. The SXAS indicated that the radius is getting larger and larger as the amyloid beta aggregates. After adding metals, especially Zn2+, the amyloid beta assembles much faster.The research finding in this paper provides theory basis for understanding the molecular mechanism of AD and preventing AD.