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.
Footnotes
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Send reprint requests to: Dr. Melvin L. Billingsley, Department of Pharmacology, Pennsylvania State University College of Medicine, Milton S. Hershey Medical Center, P.O. Box 850, Hershey PA 17033. E-mail mlb8{at}psu.edu
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↵1 Present address: Lineberry Research Associates, Research Triangle Park, NC 27709.
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This work was supported by grants from the American Health Assistance Foundation Alzheimer Research Program and a Targeted Research Grant from The Alzheimer’s Foundation (M.L.B.) and by an Advanced Predoctoral Fellowship from the Pharmaceutical Manufacturer’s Association Foundation (T.D.G.).
- Abbreviations:
- PHF
- paired helical filament
- ODN
- oligodeoxynucleotide
- aCSF
- artificial cerebrospinal fluid
- PP1
- protein phosphatase 1
- PP2
- protein phosphatase 2
- Received October 27, 1998.
- Accepted January 18, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
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