The pattern of human tau phosphorylation is the result of priming and feedback events in primary hippocampal neurons

Tau, an axonal microtubule-associated protein, becomes hyperphosphorylated in several neurodegenerative diseases including Alzheimer disease (AD). In AD brain, tau is phosphorylated at pathological multiple-site epitopes recognized by the antibodies AT8 (S199/S202/T205), AT100 (T212/S214/T217), AT180 (T231/S235) and PHF-1 (S396/S404) and at individual sites such as S262 and S422. Although it is believed that the hyperphosphorylation of tau occurs in a precise cascade of phosphorylation events, this cascade remains to be demonstrated in mammalian neuronal cells. In the present study, human tau mutants in which disease-related sites associated with either an early (AT8, T231 and S262) or intermediate (T217) stage of tau pathology were mutated in alanine to inhibit their phosphorylation were overexpressed in primary hippocampal neurons to examine their impact on the phosphorylation of other disease-related sites. The mutation in alanine of S262 decreased the phosphorylation of the AT8 and PHF-1 epitopes and that of T217. When the sites included in the AT8 epitope were mutated in alanine, the phosphorylation of T217 and PHF-1 epitope was significantly reduced indicating that the decrease of AT8 phosphorylation was a key event in the impaired phosphorylation of T217 and PHF-1 by the S262 alanine mutant. Most interestingly, the mutation in alanine of T217 had a positive impact on the phosphorylation of the AT8 epitope, indicating the presence of a feedback loop between AT8 and T217 in rat hippocampal neurons. The phosphorylation of the AT180 epitope was increased when S262 and the sites forming the AT8 epitope were mutated in alanine. The mutation of the AT8 epitope also increased the phosphorylation of S422. All together, our data show that the sites forming the AT8 epitope could play a central role in regulating the phosphorylation of tau at disease-associated sites and that priming and feedback events take place to regulate the overall level of tau phosphorylation in rat hippocampal neurons.