Juliana Karl Frizzo

S100B-mediated inhibition of the phosphorylation of GFAP is prevented by TRTK-12.

S100B belongs to a family of calcium-binding proteins involved in cell cycle and cytoskeleton regulation. We observed an inhibitory effect of S100B on glial fibrillary acidic protein (GFAP) phosphorylation, when stimulated by cAMP or Ca2+/calmodulin, in a cytoskeletal fraction from primary astrocyte cultures. We found that S100B has no direct effect on CaM KII activity, the major kinase in this cytoskeletal fraction able to phosphorylate GFAP. The inhibition of GFAP phosphorylation is most likely due to the binding of S100B to the phosphorylation sites on this protein and blocking the access of these sites to the protein kinases. This inhibition was dependent on Ca2+. However, Zn2+ could substitute for Ca2+. The inhibitory effect of S100B was prevented by TRTK-12, a peptide that blocks S100B interaction with several target proteins including glial fibrillary acidic protein. These data suggest a role for S100B in the assembly of intermediate filaments in astrocytes.

Guanosine Enhances Glutamate Transport Capacity in Brain Cortical Slices

Summary1.The effect of guanosine on L-[3H] glutamate uptake was investigated in brain cortical slices within physio-pathological range of glutamate(1–1000 μ M). In these conditions, glutamate uptake was significantly enhanced in slices treated with 100 μ M guanosine only at 100 and 300 μ M glutamate (44 and 52%, respectively).2.Evaluation of kinetic parameters showed that guanosine affected significantly only uptake Vmax (23%).3.The guanosine withdrawal did not abolish its significant effect on glutamate uptake when 100 or 300 μ M glutamate were used (an increase of 66 and 35%, respectively).4.These results support the hypothesis of a protective role for guanosine during excitotoxic conditions when glutamate levels are enhanced (e.g. brain ischemia and seizures), possibly by activating glutamate uptake. Moreover, our results may contribute to understand the antiexcitotoxic mechanism of guanosine on glutamate transport, giving new information concerning its mechanism of action.

β-Hydroxy-butyrate alters the extracellular content of S100B in astrocyte cultures

Astrocytes have a variety of roles in maintaining neural tissue physiology, including energetic support, uptake and metabolism of glutamate and secretion of neurotrophic factors. Glutamate toxicity has been implicated in neurodegenerative disorders associated with conditions related to energy failure, and to elevation of glutamate extracellular levels in brain. Glucose is the main energetic substrate for brain cells but, in some circumstances, the ketone bodies are used as a supplementary source and have been suggested to be neuroprotective agents against seizure disorders. Here, we investigate some possible biochemical changes in astrocyte cultures induced by beta-hydroxy-butyrate, the predominant blood ketone body. Its effect upon S100B secretion, astrocyte morphology and glutamate uptake was particularly investigated. S100B, a calcium-binding protein expressed and secreted by astrocytes, has neurotrophic activity and a possible role in epileptogenesis. Cell morphology was investigated by phase-contrast microscopy and immunocytochemistry for actin, GFAP and S100B. Our data show that beta-hydroxy-butyrate induces dramatic changes in astrocyte morphology and, independent of this, causes changes in the extracellular content of S100B. We observed an increment in S100B 1 h after beta-hydroxy-butyrate addition and a decrease 24 h later. No changes were observed in glutamate uptake. These astrocytic modifications may be associated with reduced neuronal excitability observed in the ketogenic condition.

S100B protein stimulates calcineurin activity.

S100B is a calcium binding protein from astrocytes that regulates protein phosphorylation by binding to substrates and protein kinases. S100B might also regulate protein phosphatases and this was investigated for protein phosphatase 2B (calcineurin). The results indicate that S100B (5–10 μM) increased the activity of both purified and cytoskeletal calcineurin in a Ca2+-dependent manner. This effect was blocked by a specific inhibitor of calcineurin activity, but not by TRTK-12 (an inhibitor of S100B binding to other protein targets). The present results and the known co-localization of S100B and calcineurin in the astrocyte cytoskeleton suggest that S100B may play a role in the phosphorylation state of cytoskeletal proteins.

Involvement of the S100B in cAMP-induced cytoskeleton remodeling in astrocytes: a study using TRTK-12 in digitonin-permeabilized cells.

Abstract1. Stellation of astrocytes in culture involves a complex rearrangement of microfilaments, intermediate filaments, and microtubules, which reflects in part the plasticity of these cells observed during development or after injury.2. An astrocytic calcium-binding protein, S100B, has been implicated in the regulation of plasticity due to its ability to interact with cytoskeletal proteins.3. We used digitonin-permeabilized astrocytes to introduce TRTK-12, a peptide that binds to the C-terminal of S100B and blocks its interaction with cytoskeletal proteins.4. TRTK-12 was able to block cAMP-induced astrocyte stellation and this effect was dependent on the concentration of the peptide. These results support the idea that S100B has a modulatory role on astrocyte morphology.