Reduction of calcineurin activity in brain by antisense oligonucleotides leads to persistent phosphorylation of tau protein at Thr181 and Thr231.
Phosphorylation of tau protein promotes stability of the axonal cytoskeleton; aberrant tau phosphorylation is implicated in the biogenesis of paired helical filaments (PHF) seen in Alzheimer's disease. Protein kinases and phosphatases that modulate tau phosphorylation have been identified using in vitro techniques; however, the role of these enzymes in vivo has not been determined. We used intraventricular infusions of antisense oligodeoxynucleotides (ODNs) directed against the major brain isoforms of the Ca2+/calmodulin-dependent phosphatase calcineurin to determine how reduced activity of this enzyme would affect tau dephosphorylation. Five-day infusions of antisense ODNs (5 and 10 nmol/day) in rats decreased immunoreactive levels and activity 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 protein remained phosphorylated as determined by using the phosphorylation-sensitive monoclonal antibodies AT-180 (Thr231) and AT-270 (Thr181). In contrast, AT-180 and AT-270 sites were completely dephosphorylated during incubation of neocortical slices from vehicle-infused controls and sense ODN-treated rats. Neocortical slices from antisense-treated rats were incubated with the phosphatase inhibitors okadaic acid (100 nM; 10 microM) and FK-520 (5 microM); these preparations showed enhanced tau phosphorylation, consistent with a significant loss of calcineurin activity. Thus, we conclude that phosphorylation of at least two sites on tau protein, namely, Thr181 and Thr231, is regulated by calcineurin.
Garver
T D
TD
Department of Pharmacology, Pennsylvania State University College of Medicine, Milton S. Hershey Medical Center, Hershey, Pennsylvania 17033, USA.
Kincaid
R L
RL
Conn
R A
RA
Billingsley
M L
ML
D000818:Animals; D001921:Brain / Q000187:drug effects* / Q000378:metabolism; D019703:Calcineurin / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D066298:In Vitro Techniques; D008297:Male; D019579:Neocortex / Q000187:drug effects / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D013912:Threonine / Q000378:metabolism; D016875:tau Proteins / Q000378:metabolism*
D016376:Oligonucleotides, Antisense; D016875:tau Proteins; D013912:Threonine; D019703:Calcineurin