Nongenomic actions of thyroxine modulate intermediate filament phosphorylation in cerebral cortex of rats.

The developmental effects of thyroid hormones (TH) in mammalian brain are mainly mediated by nuclear receptors regulating gene expression. However, there are increasing evidences of nongenomic mechanisms of these hormones associated with kinase- and calcium-activated signaling pathways. In this context, the aim of the present work was to investigate the signaling pathways involved in the mechanism of action of TH on cytoskeletal phosphorylation in cerebral cortex of 15-day-old male rats. Results showed that L-thyroxine (L-T4) increased the intermediate filament (IF) phosphorylation independently of protein synthesis, without altering the total immunocontent of these proteins. Otherwise, neither 3,5,3'-triiodo-L-thyronine (L-T3) nor neurotransmitters (GABA, ATP, L-glutamate or epinephrine) acted on the IF-associated phosphorylation level. We also demonstrated that the mechanisms underlying the L-T4 effect on the cytoskeleton involve membrane initiated actions through Gi protein-coupled receptor. This evidence was reinforced by the inhibition of cyclic adenosine 5'-monophosphate (cAMP) levels. Moreover, we showed the participation of phospholipase C, protein kinase C, mitogen-activated protein kinase, calcium/calmodulin-dependent protein kinase II, intra- and extracellular Ca2+ mediating the effects of L-T4 on the cytoskeleton. Stimulation of 45Ca2+ uptake by L-T4 was also demonstrated. These findings demonstrate that L-T4 has important physiological roles modulating the cytoskeleton of neural cells during development.

Effect of the branched-chain alpha-ketoacids accumulating in maple syrup urine disease on the high molecular weight neurofilament subunit (NF-H) in rat cerebral cortex.

In this study we investigated the effects of the branched chain alpha-ketoacids accumulating in maple syrup urine disease (MSUD) on the concentrations of the high molecular weight neurofilament subunit (NF-H) associated with the cytoskeletal fraction of the cerebral cortex of 12-day-old rats. Cortical slices were incubated with alpha-ketoisocaproic acid (KIC), alpha-keto beta-methylvaleric acid (KMV) and alpha-ketoisovaleric acid (KIV) at concentrations ranging from 0.5 to 1.0 mM. The cytoskeletal fraction was extracted and the immunoreactivity for phosphorylated and total NF-H was analyzed by immunoblotting. The in vitro 32P incorporation into NF-H was also determined. Results showed that treatment of tissue slices induced with KMV increased Triton-insoluble phosphorylated NF-H immunoreactivity, with no alteration in total NF-H immunoreactivity. Furthermore, KIC treatment drastically increased the total amount of NF-H, whereas KIV did not change either phosphorylated or total NF-H immunoreactivity. KMV also increased the in vitro 32P incorporation into NF-H, confirming the highly phosphorylated NF-H levels detected in the immunoblot. These findings demonstrate that KIC and KMV alter the dynamic regulation of NF-H assembly in the cytoskeletal fraction. Therefore we may suggest that cytoskeletal disorganization may be one of the factors associated with the neurodegeneration characteristic of MSUD disease.