Abstract 2097 Comparative Analysis of Wild-Type and Pseudophosphorylated Tau Protein Variants

Tau protein facilitates assembly, organization, and stability of microtubules found in neuronal axons. Hyperphosphorylation of tau results in a loss of tau function from decreased microtubule binding affinity; such a loss of function is prominently observed in neurodegenerative diseases like Alzheimer's Disease. The polymerization of tau also plays a role in maintaining neuronal integrity, and its disruption is a hallmark of several neurodegenerative conditions. The current study employed 7-Phos - a tau mutant with seven specific site pseudophosphorylation mutations - to mimic the characteristic changes found in hyperphosphorylation. The primary objective was to analyze and compare the biophysical properties of Wild-Type tau proteins with 7-Phos, assessing changes in polymerization and structural conformation. 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 characteristic of phosphorylated tau and 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 (AFM) was used to measure the length of 7-Phos tau polymers over several days. 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. AFM imaging revealed that 7-Phos isoforms form longer polymers than their wild-type analogs. This study introduces novel AFM-based measurements to characterize the biophysical properties of tau. The 7-Phos isoforms exhibited distinct polymerization behavior compared to WT. These differences highlight the effectiveness of 7-Phos as a model mutant for understanding the mechanistic changes in tau protein behavior induced by phosphorylation. NIH grant #1R15NS108245-01A1.