Thyroid hormones reorganize the cytoskeleton of glial cells through Gfap phosphorylation and Rhoa-dependent mechanisms.

Thyroid hormones (3,5,3'-triiodo-L: -thyronine, T3; 3,5,3',5'-L: -tetraiodothyronine, T4; TH) play crucial roles in the growth and differentiation of the central nervous system. In this study, we investigated the actions of TH on proliferation, viability, cell morphology, in vitro phosphorylation of glial fibrillary acidic protein (GFAP) and actin reorganization in C6 glioma cells. We first observe that long-term exposure to TH stimulates cell proliferation without induce cell death. We also demonstrate that after 3, 6, 12, 18, and 24 h treatment with TH, C6 cells and cortical astrocytes show a process-bearing shape. Furthermore, immunocytochemistry with anti-actin and anti-GFAP antibodies reveals that TH induces reorganization of actin and GFAP cytoskeleton. We also observe an increased in vitro 32P incorporation into GFAP recovered into the high-salt Triton insoluble cytoskeletal fraction after 3 and 24 h exposure to 5 x 10(-8) and 10(-6) M T3, and only after 24 h exposure to 10(-9) M T4. These results show a T3 action on the phosphorylating system associated to GFAP and suggest a T3-independent effect of T4 on this cytoskeletal protein. In addition, C6 cells and astrocytes treated with lysophosphatidic acid, an upstream activator of the RhoA GTPase pathway, totally prevented the morphological alterations induced by TH, indicating that this effect could be mediated by the RhoA signaling pathway. Considering that IF network can be regulated by phosphorylation leading to reorganization of IF filamentous structure and that alterations of the microfilament organization may have important implications in glial functions, the effects of TH on glial cell cytoskeleton could be implicated in essential neural events such as brain development.

Resveratrol increases glutamate uptake and glutamine synthetase activity in C6 glioma cells.

Resveratrol, a phytoalexin found mainly in grapes, is a promising natural product with anti-cancer and cardio-protective activities. Here, we investigated, in C6 glioma cells, the effect of resveratrol on some specific parameters of astrocyte activity (glutamate uptake, glutamine synthetase and secretion of S100B, a neurotrophic cytokine) commonly associated with the protective role of these cells. Cell proliferation was significantly decreased by 8% and 26%, following 24h of treatment with 100 and 250 microM resveratrol. Extracellular S100B increased after 48 h of resveratrol exposure. Short-term resveratrol exposure (from 1 to 100 microM) induced a linear increase in glutamate uptake (up to 50% at 100 microM resveratrol) and in glutamine synthetase activity. Changes in these glial activities can contribute to the protective role of astrocytes in brain injury conditions, reinforcing the putative use of this compound in the therapeutic arsenal against neurodegenerative diseases and ischemic disorders.

Morphological alterations and induction of oxidative stress in glial cells caused by the branched-chain alpha-keto acids accumulating in maple syrup urine disease.

Maple syrup urine disease (MSUD) is an inherited neurometabolic disorder biochemically characterized by the accumulation of the branched-chain alpha-keto acids (BCKA) alpha-ketoisocaproic (KIC), alpha-keto-beta-methylvaleric (KMV) and alpha-ketoisovaleric (KIV) and their respective branched-chain alpha-amino acids in body fluids and tissues. Affected MSUD patients have predominantly neurological features, including cerebral edema and atrophy whose pathophysiology is not well established. In the present study we investigated the effects of KIC, KMV and KIV on cell morphology, cytoskeleton reorganization, actin immunocontent and on various parameters of oxidative stress, namely total antioxidant reactivity (TAR), glutathione (GSH) and nitric oxide concentrations, and on the activities of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) in C6 glioma cells. We initially observed that C6 cultivated cells exposed for 3 h to the BCKA (1 and 10 mM) changed their usual rounded morphology to a fusiform or process-bearing cell appearance, while 24 h exposure to these organic acids elicited massive cell death. Rhodamine-labelled phalloidin analysis revealed that these organic acids induced reorganization of the actin cytoskeleton with no modifications on total actin content. It was also observed that 3h cell exposure to low doses of all BCKA (1 mM) resulted in a marked reduction of the non-enzymatic antioxidant defenses, as determined by TAR and GSH measurements. In addition, KIC provoked a reduced activity of SOD and GPx, whereas KMV caused a diminution of SOD activity. In contrast, CAT activity was not modified by the metabolites. Furthermore, nitric oxide production was significantly increased by all BCKA. Finally, we observed that the morphological features caused by BCKA on C6 cells were prevented by the use of the antioxidants GSH (1.0 mM), alpha-tocopherol (trolox; 10 microM) and Nomega-nitro-L-arginine methyl ester (L-NAME; 500 microM). These results strongly indicate that oxidative stress might be involved in the cell morphological alterations and death, as well as in the cytoskeletal reorganization elicited by the BCKA. It is presumed that these findings are possibly implicated in the neuropathological features observed in patients affected by MSUD.

Creatine and antioxidant treatment prevent the inhibition of creatine kinase activity and the morphological alterations of C6 glioma cells induced by the branched-chain alpha-keto acids accumulating in maple syrup urine disease.

Accumulation of the branched-chain alpha-keto acids (BCKA), alpha-ketoisocaproic acid (KIC), alpha-keto-beta-methylvaleric acid (KMV), and alpha-ketoisovaleric acid (KIV) and their respective branched-chain alpha-amino acids (BCAA) in tissues and biological fluids is the biochemical hallmark of patients affected by the neurometabolic disorder known as maple syrup urine disease (MSUD). Considering that brain energy metabolism is possibly altered in MSUD, the objective of this study was to determine creatine kinase (CK) activity, a key enzyme of energy homeostasis, in C6 glioma cells exposed to BCKA. The cells were incubated with 1, 5, or 10 mM BCKA for 3 h and the CK activity measured afterwards. The results indicated that the BCKA significantly inhibited CK activity at all tested concentrations. Furthermore, the inhibition caused by the BCKA on CK activity was totally prevented by preincubation with the energetic substrate creatine and by coincubation with the N-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, indicating that deficit of energy and nitric oxide (NO) are involved in these effects. In contrast, other antioxidants such as glutathione (GSH) and trolox (soluble Vitamin E) were not able to prevent CK inhibition. In addition, we observed that the C6 cells changed their usual rounded morphology when exposed for 3 h to 10 mM BCKA and that creatine and L-NAME prevented these morphological alterations. Considering the importance of CK for brain metabolism homeostasis, it is conceivable that inhibition of this enzyme by increased levels of BCKA may contribute to the neurodegeneration of MSUD patients.

Branched-chain alpha-keto acids accumulating in maple syrup urine disease induce reorganization of phosphorylated GFAP in C6-glioma cells.

In this study we investigate the effects of the branched-chain keto acids (BCKA) alpha-ketoisocaproic (KIC), alpha-ketoisovaleric (KIV), and alpha-keto-beta-methylvaleric (KMV) acids, metabolites accumulating in maple syrup urine disease (MSUD), on the in vitro phosphorylation of glial fibrillary acidic protein (GFAP) and cytoskeletal reorganization in C6-glioma cells. We observed that after 3 h treatment with KIC, KIV, or KMV cells showed retracted cytoplasm with bipolar processes containing packed GFAP filaments as revealed by immunocytochemistry. Western Blot analysis by anti-GFAP monoclonal antibody demonstrated that BCKA were not able to alter GFAP immunocontent in total cell homogenate, but the immunocontent as well as the in vitro (32)P incorporation into GFAP recovered into the high salt Triton-insoluble cytoskeletal fraction were significantly increased. Western Blot using monoclonal antiphosphoserine antibody showed that BCKA induced increased immunocontent of phosphoserine-containing amino acids in several proteins in total cell homogenate. In addition, the immunocontent of phosphoserine-containing amino acids was also greatly increased in GFAP recovered in the high-salt Triton insoluble cytoskeletal fraction, corresponding to the polymerized intermedite filament (IF) proteins present in the cell. In conclusion, our results indicate that KIC, KIV, or KMV increased the serine/threonine in vitro phosphorylation of GFAP leading to increased Triton-insoluble GFAP immunocontent and cytoskeletal reorganization. Considering IF networks can be regulated by phosphorylation of polypeptide subunits leading to reorganization of the IF filamentous structure, we could suppose that GFAP hyperphosphorylation and disorganization of cellular structure could be involved in the brain damage characteristic of MSUD patients.