Characterization of pseudophosphorylated tau protein.

Abstract

Tau protein facilitates assembly, organization, and stability of microtubules found in neuronal axons. Hyperphosphorylation results in a loss of tau function from decreased microtubule binding affinity which is prominent in Alzheimer's Disease. The current study employed 7-Phos—a tau mutant with seven specific site pseudophosphorylation mutations—to mimic the characteristic changes found in hyperphosphorylation. In vivo, tau has six naturally occurring isoforms that vary in the length of the N-terminal projection domains, three of which were investigated in this study. Biophysical characterization of these isoforms was accomplished with several techniques. SDS-PAGE was used to identify an upward band shift, a feature of 7-Phos. A thioflavin fluorescence assay was performed to compare the arachidonic acid induced polymerization of the 7-Phos variants with their wild-type counterparts. Atomic force microscopy was used to measure the length of 7-Phos tau polymers over several days and to analyze the intermolecular interactions between molecules of 7-Phos. We determined that the total amount of polymerization measured by thioflavin fluorescence of the two longer 7-Phos isoforms, 1N4R and 2N4R, were similar to their corresponding wild-type proteins. The total amount of polymerization of the shortest isoform, 0N4R, was greater than its wild-type. Atomic force microscopy (AFM) imaging revealed that 7-Phos isoforms form longer polymers than their wild-type. Atomic force microscopy of immobilized tau layers allowed comparison of projection domain lengths between 7-Phos and wild-type. These results demonstrate some novel AFM-based measurements to characterize tau polymerization, projection domain extension/conformation and intermolecular interactions.