τ Phosphorylation in human, primate, and rat brain: Evidence that a pool of τ is highly phosphorylated in vivo and is rapidly dephosphorylated in vitro

The extent of τ phosphorylation is thought to regulate the binding of τ to microtubules: Highly phosphorylated τ does not bind to tubules, whereas dephosphorylated τ can bind to microtubules. It is interesting that the extent of τ phosphorylation in vivo has not been accurately determined. τ was rapidly isolated from human temporal neocortex and hippocampus, rhesus monkey temporal neocortex, and rat temporal neocortex and hippocampus under conditions that minimized dephosphorylation. In brain slices, we observed that τ isolated under such conditions largely existed in several phosphorylated states, including a pool that was highly phosphorylated; this was determined using epitope-specific monoclonal and polyclonal antibodies. This highly phosphorylated τ was dephosphorylated during a 120-min time course in vitro, presumably as a result of neuronal phosphatase activity. The slow-mobility forms of τ were shifted to faster-mobility forms following in vitro incubation with alkaline phosphatase. Laser densitometry was used to estimate the percent of τ in slow-mobility, highly phosphorylated forms. Approximately 25% of immunoreactive τ was present as slow-mobility (66- and 68-kDa) forms of τ. The percentage of immunoreactive τ in faster-mobility pools (42-54 kDa) increased in proportion to the decrease in content of 66- 68-kDa τ as a function of neuronal phosphatases or alkaline phosphatase treatment. These data suggest that the turnover of phosphorylated sites on τ is rapid and depends on neuronal phosphatases. Furthermore, τ is highly phosphorylated in normal-appearing human, primate, and rodent brain. The presence of a highly phosphorylated pool of τ in adult brain may modify the present hypotheses on how paired helical filaments of Alzheimer's disease are formed.