Reduction of calcineurin activity in brain by antisense oligonucleotides leads to persistent phosphorylation of tau protein at Thr(181) and Thr(231)

Phosphorylation of tau protein promotes stability of the axonal cytoskeleto n; aberrant tau phosphorylation is implicated in the biogenesis of paired h elical filaments (PHF) seen in Alzheimer's disease. Protein kinases and pho sphatases that modulate tau phosphorylation have been identified using in v itro techniques; however, the role of these enzymes in vivo has not been de termined. We used intraventricular infusions of antisense oligodeoxynucleot ides (ODNs) directed against the major brain isoforms of the Ca2+/calmoduli n-dependent phosphatase calcineurin to determine how reduced activity of th is enzyme would affect tau dephosphorylation. Five-day infusions of antisen se ODNs (5 and 10 nmol/day) in rats decreased immunoreactive levels and act ivity of calcineurin throughout the brain; sense ODNs, scrambled ODNs, and infusion vehicle alone had no effect. When neocortical slices were prepared from antisense ODN-treated rats and incubated for 1 to 2 h in vitro, tau p rotein remained phosphorylated as determined by using the phosphorylation-s ensitive monoclonal antibodies AT-180 (Thr(231)) and AT-270 (Thr(181)). In contrast, AT-180 and AT-270 sites were completely dephosphorylated during i ncubation of neocortical slices from vehicle-infused controls and sense ODN -treated rats. Neocortical slices from antisense-treated rats were incubate d with the phosphatase inhibitors okadaic acid (100 nM; 10 mu M) and FK-520 (5 mu M); these preparations showed enhanced tau phosphorylation, consiste nt with a significant loss of calcineurin activity. Thus, we conclude that phosphorylation of at least two sites on tau protein, namely, Thr(181) and Thr(231), is regulated by calcineurin.