Vol. 55, Issue 4, 632-641, April 1999

Reduction of Calcineurin Activity in Brain by Antisense Oligonucleotides Leads to Persistent Phosphorylation of  Protein at Thr¹⁸¹ and Thr²³¹

Timothy D. Garver,¹ Randall L. Kincaid, Richard A. Conn, and Melvin L. Billingsley

Department of Pharmacology, Pennsylvania State University College of Medicine, Milton S. Hershey Medical Center, Hershey, Pennsylvania (T.D.G., R.A.C., M.L.B.); and Veritas, Inc., Rockville, Maryland (R.L.K.)

Phosphorylation of  protein promotes stability of the axonal cytoskeleton; aberrant  phosphorylation is implicated in the biogenesis of paired helical filaments (PHF) seen in Alzheimer's disease. Protein kinases and phosphatases that modulate  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  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,  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 µM) and FK-520 (5 µM); these preparations showed enhanced  phosphorylation, consistent with a significant loss of calcineurin activity. Thus, we conclude that phosphorylation of at least two sites on  protein, namely, Thr¹⁸¹ and Thr²³¹, is regulated by calcineurin.