Reduction of calcineurin activity in brain by antisense oligonucleotides leads to persistent phosphorylation of τ protein at Thr181 and Thr231

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

Research output: Contribution to journalArticle

39 Scopus citations

Abstract

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

Original languageEnglish (US)
Pages (from-to)632-641
Number of pages10
JournalMolecular pharmacology
Volume55
Issue number4
StatePublished - 1999

All Science Journal Classification (ASJC) codes

  • Molecular Medicine
  • Pharmacology

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