Diphenyl ditelluride induces hypophosphorylation of intermediate filaments through modulation of DARPP-32-dependent pathways in cerebral cortex of young rats.

We studied the effect of different concentrations of diphenyl ditelluride (PhTe)(2) on the in vitro phosphorylation of glial fibrillary acidic protein (GFAP) and neurofilament (NF) subunits from cerebral cortex and hippocampus of rats during development. (PhTe)(2)-induced hypophosphorylation of GFAP and NF subunits only in cerebral cortex of 9- and 15-day-old animals but not in hippocampus. Hypophosphorylation was dependent on ionotropic glutamate receptors, as demonstrated by the specific inhibitors 10 µM DL-AP5 and 50 µM MK801, 100 µM CNQX and 100 µM DNQX. Also, 10 µM verapamil and 10 µM nifedipine, two L-voltage-dependent Ca(2+) channels (L-VDCC) blockers; 50 µM dantrolene, a ryanodine channel blocker, and the intracellular Ca(2+) chelator Bapta-AM (50 µM) totally prevented this effect. Results obtained with 0.2 µM calyculin A (PP1 and PP2A inhibitor), 1 µM Fostriecin a potent protein phosphatase 2A (PP2A) inhibitor, 100 µM FK-506 or 100 µM cyclosporine A, specific protein phosphatase 2B inhibitors, pointed to PP1 as the protein phosphatase directly involved in the hypophosphorylating effect of (PhTe)(2). Finally, we examined the activity of DARPP-32, an important endogenous Ca(2+)-mediated inhibitor of PP1 activity. Western blot assay using anti-DARPP-32, anti-pThr34DARPP-32, and anti-pThr75DARPP-32 antibodies showed a decreased phosphorylation level of the inhibitor at Thr34, compatible with inactivation of protein kinase A (PKA) by pThr75 DARPP-32. Decreased cAMP and catalytic subunit of PKA support that (PhTe)(2) acted on neuron and astrocyte cytoskeletal proteins through PKA-mediated inactivation of DARPP-32, promoting PP1 release and hypophosphorylation of IF proteins of those neural cells. Moreover, in the presence of Bapta, the level of the PKA catalytic subunit was not decreased by (PhTe)(2), suggesting that intracellular Ca(2+) levels could be upstream the signaling pathway elicited by this neurotoxicant and targeting the cytoskeleton.