Interaction Of Amyloid-β Oligomer With Kinesin-1Determined By Fluorescence Cross-correlation Spectroscopy

Alzheimer’s disease (AD), also known as senile dementia, is the most common progressive neurodegenerative disorder of central nervous system. AD is mainly characterized by extracellular senile plaques (SPs) and intracellular neurofibrillar tangles (NFTs). Although the exact etiology and pathogenesis of AD are still unclear, the "amyloid hypothesis" of AD has dominated AD research for many years, which states that the overproduction and/or failure to clear of the A(3peptides may be the major pathogenic factors. Meanwhile, many lines of evidences point out to the neurotoxicity of A(3, especially A(342oligomers., are more neurotoxic than A(3monomers or insoluble Aβ fibrils. Deficiencies in axonal transport already have been found in AD patients and animal models. And kinesin plays an important role in axonal transport. Therefore, the interaction between Aβ oligomers and kinesin will help reveal the pathogenesis of AD, providing more theory and experimental evidences of AD.Dual-color fluorescence cross correlation spectroscopy (DC-FCCS) is a correlation analysis of fluctuation of the fluorescence intensity at the single molecule level. DC-FCCS evaluates the cross-correlation between two separate color channels, especially suitable for the study of biomolecular interactions. Although the interaction between amyloid precursor protein (APP) and kinesin has already been proved in vivo and in vitro, the direct interaction between Aβ oligomers and kinesin at the single molecule level is not reported yet. Here, we constructed recombinant plasmids and accomplished the expression and purification of two proteins, KLC-EGFP-His(KLC-E) and EGFP-KLC-His(E-KLC). Then we established a detection method to observe the interaction between Aβ42oligomers and KLC by DC-FCCS. The effects of stoichiometry and the EGFP tag position were also discussed.To construct plasmid pFastBacl-KLC-E and pFastBacl-E-KLC, we amplified the coding sequences of KLC gene and EGFP gene, and then inserted them into plasmid pFastBacl of the Bac-To-Bac Baculovirus expression system by different order respectively, adding His tag sequences in their C-terminus. Recombinant Baculovirus DNA was obtained in E.coli DH10Bac through transformation. After transfection with Cellfectin Ⅱ Reagent, recombinant Baculovirus was developed in Sf9cells. Then we used this viral stock to infect new Sf9cells to generate a high-titer baculoviral stock. After that, the baculoviral stock was used to infect Sf9cells for the expressions of KLC-E and E-KLC, and the two proteins were purified by Ni-NTA resin separately. The Aβ42monomers were labeled with Atto647N NHS ester of fluorescent dye and then AP42oligomers were prepared. At last, DC-FCCS was performed to observe the interaction between the A042oligomers and KLC. The results show that within0to4h, the binding efficiency between Aβ42oligomers and KLC-E, as well as Aβ42oligomers and E-KLC, is gradually strengthening. And both of KLC-E and E-KLC accelerate the oligomerization of Aβ42in solution either under high or low stoichiometry, while E-KLC facilitates the oligomerization of Aβ42a little bit more than that of KLC-E under low stoichiometry. The binding efficiencies between KLC-E and E-KLC to AP42oligomers under high stoichiometry are much higher than they are under low stoichiometry. Either under high or low stoichiometry, the binding efficiency of E-KLC to Aβ42oligomers is significantly higher than that of KLC-E to Aβ42oligomers.