Tau phosphorylation in brain slices: Pharmacological evidence for convergent effects of protein phosphatases on tau and mitogen-activated protein kinase

Tau is a neuron-specific, microtubule-associated protein that forms paired helical filaments (PHFs) of Alzheimer's disease when aberrantly phosphorylated. We have attempted to elucidate the protein kinases and phosphatases that regulate tau phosphorylation. Incubation of rat, human, and rhesus monkey temporal neocortex slices with the phosphatase inhibitor okadaic acid induced epitopes of tau similar to those found in PHFs. Okadaic acid (1-20 μM) induced variant forms of tau at 60-68 kDa, which were recognized by the monoclonal antibodies Alz-50 (in humans only) and 5E2 and two polyclonal antipeptide antisera, OK-1 and OK-2. The phosphorylation- sensitive monoclonal antibody Tau-1 failed to recognize the slowest mobility forms of tau after okadaic acid treatment. FK-520 (1-10 μM), a potent inhibitor of calcineurin activity, was tested in brain slices and found not to alter tau mobility. However, combinations of FK-520 (5 μM) and okadaic acid (100 nM) caused tau mobility shifts similar to those seen after 10 μM okadaic acid treatment; similar results were seen using the calcineurin- selective inhibitor cypermethrin. Treatment of human slices with 10 μM okadaic acid decreased both protein phosphatase 2A and calcineurin activity; FK-520 inhibited only protein phosphatase 2B activity. A proposed tau- directed kinase, 42-kDa mitogen-activated protein kinase (p42(mapk)), was activated by okadaic acid (>100 nM) but not FK-520 (5 μM). Nerve growth factor (100 ng/ml) activated p42(mapk), particularly when used in combination with 100 nM okadaic acid; changes in tau mobility were seen when this kinase was activated. Forskolin (2 μM) antagonized the effects of nerve growth factor on both p42(mapk) activity and tau phosphorylation; forskolin alone had little effect on PHF-like tau formation induced by phosphatase inhibitors. These results outline complex interactions between tau-directed protein kinases and protein phosphatases and suggest potential sites for therapeutic intervention.