The phosphorylation state of tau in the developing rat brain is regulated by phosphoprotein phosphatases

The paired helical filaments (PHFs) in Alzheimer's disease neurofibrillary tangles are composed of PHF-tau which is thought to be hyperphosphorylated because several residues in postmortem samples of PHF-tau and human fetal tau are phosphorylated while the corresponding sites are not phosphorylated in autopsy-derived normal adult human brain tau. To determine how the phosphorylation of these sites is regulated, we isolated tau from rat brains at different embryonic and postnatal ages in the presence of okadaic acid to obtain tau in its most native in situ phosphorylation state. Fetal tau was highly phosphorylated from embryonic day 18 (E18) until postnatal day 11 (P11). Thereafter, the levels of fetal tau diminished as did its phosphorylation state concomitant with the appearance of the five adult tau isoforms. Several phosphorylation-dependent antibodies (i.e. AT270, AT8, AT180, T3P, and PHF1) that recognize PHF-tau also recognized these tau isoforms, albeit at reduced levels in the mature rat brain. This suggests that Thr172, Ser193, Thr222, Ser387, and Ser395 are normal sites of phosphorylation in rat brain tau. The inclusion of OK in the microtubule assembly buffers did not alter the ability of tau to bind microtubules at any age. However, phosphatases were activated and kinases were down-regulated in the rat brain after P12 since adult tau proteins were partially dephosphorylated at and beyond this time in the absence of OK. Protein phosphatase 2A (PP2A) and 2B (PP2B) activities in the adult rat brain extracts dephosphorylated tau efficiently, but protein phosphatases in extracts of the P6 rat brain did not have a similar effect. This suggests that the sensitivity of tau to OK after P12 may be regulated by the de novo induction of adult brain phosphatases. Finally, PP2A and/or PP2B in adult rat brain extracts dephosphorylated tau in a site-specific manner. Thus, PP2A and PP2B (or closely related phosphatases) may regulate the phosphorylation state of adult tau isoforms in vivo, and the generation of PHF-tau in the AD brain may result from the abnormal inactivation of similar phosphatases.