Pseudohyperphosphorylation has Significant Effects on the Polymerization of Tau Isoforms

The microtubule-associated protein tau binds to and stabilizes microtubules in neurons in the human brain. The tau protein exists as six different isoforms that are generated by alternative splicing of a single gene. The isoforms differ by the presence or absence of three variable coding regions. Exons 2 and 3 are present in the amino-terminal of tau and exon 10 encodes a microtubule binding repeat.  The isoforms are 0N3R, 0N4R, 1N3R, 1N4R, 2N3R and 2N4R.  In Alzheimer's disease (AD) tau isoforms become hyperphosphorylated, forming insoluble straight and paired-helical filaments.  It has been hypothesized that tau hyperphosphorylation occurs early in AD and contributes significantly to neurodegeneration. To better understand how hyperphosphorylation affects tau, we previously created a pseudohyperphosphorylation mutant of the 2N4R isoform using site directed mutagenesis. Site directed mutagenesis allows the amino acids serine and threonine to be changed to the negatively charged glutamic acid and aspartic acid, thereby mimicking phosphorylation.  Seven AD-specific phosphorylation sites were chosen to be modeled.  We found that pseudohyperphosphorylation of 2N4R tau induced a SDS-resistant change in electrophoretic mobility, decreased microtubule binding and altered characteristics of polymerization into straight filaments.  We hypothesized that the difference in sequence of the isoforms would result in differential effects of pseudophosphorylation on isoform function. Preliminary results indicate that hyperphosphorylation of the isoforms has a similar effect as 2N4R tau, and in some cases a greater effect.  These results are consistent with the possibility that hyperphosphorylated isoforms are not equal and could have different toxicity in neurons in AD.