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

Abstract

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.

Signaling Mechanisms and Disrupted Cytoskeleton in the Diphenyl Ditelluride Neurotoxicity

“…This is in line with previous evidence pointing dysregulation of Ca 2+ homeostasis as an important event in driving different neuropathologies, such as in the expression of the malignant phenotypes [191] and in neurodegenerative conditions [192]. Interestingly, (PhTe) 2 provoked different responses in the cerebral cortex and hippocampus [182], reinforcing that different types of cells can respond in a different way to the same extracellular signal molecule. Taking into account the compelling evidence of its ability in disrupting signaling mechanisms, ditelluride can be used as a tool to induce molecular changes similar to those found in different pathologies of the brain.…”

“…Acute brain slices exposed to (PhTe) 2 showed PP1-mediated hypophosphorylation of GFAP and NF subunits in cerebral cortex of 9- and 15-day-old rats but not in hippocampus at this developmental period [182]. Hypophosphorylation was dependent on ionotropic glutamate receptors, L-VDCC, and ryanodine channels.…”

“…Decreased cAMP and PKA catalytic subunit support that (PhTe) 2 disrupted the cytoskeletal associated phosphorylating/dephosphorylating system of neurons and astrocytes through PKA-mediated inactivation of DARPP-32, promoting PP1 release and hypophosphorylation of IF proteins of those neural cells (Figure 4). Buffering the intracellular Ca 2+ by the Ca 2+ chelator Bapta-AM showed that it is upstream of cAMP and PKA modulation [182]. During artificial or agonist-induced Ca 2+ oscillations, Willoughby and Cooper [195] detected fast, periodic changes in type 8 adenylyl cyclase (AC8) with subsequent PKA-mediated phosphodiesterase 4 (PDE4) activity in human embryonic kidney (HEK293) cells.…”

NMDA Receptors and Oxidative Stress Induced by the Major Metabolites Accumulating in HMG Lyase Deficiency Mediate Hypophosphorylation of Cytoskeletal Proteins in Brain From Adolescent Rats: Potential Mechanisms Contributing to the Neuropathology of This Disease

“…In line with this, hypophosphorylated NF proteins have been associated with the neurotoxicity of quinolinic acid and tellurium (Heimfarth et al 2012a(Heimfarth et al , b, 2013aPierozan et al 2014).…”

Intermediate Filaments as a Target of Signaling Mechanisms in Neurotoxicity

“…Depending on the site of phosphorylation, DARPP-32 is able to produce opposing biochemical effects, that is, inhibition of PP1 activity or inhibition of protein kinase A (PKA) activity [17]. Decreased cAMP and PKA catalytic subunits support that (PhTe) 2 disrupts the cytoskeletal-associated phosphorylating/dephosphorylating system of neurons and astrocytes through PKA-mediated inactivation of DARPP-32, promoting PP1 release and hypophosphorylation of IF proteins of those neural cells [18]. Regarding neurons, hypophosphorylation of IF proteins could be associated with cell dysfunction since decreased phosphorylation of KSP repeats in the carboxyl-terminal domains of NF-M and NF-H correlates with impaired axonal transport and increased NF-packing density.…”