Luminescence was measured with a luminescence counter

tive fold expression in cultured astrocytes. These results suggested that MeCP2 did not modify the expression of GS in the cultured astrocytes. Overall, the expression levels of GS mRNA did not differ between both strains of MedChemExpress BAY41-2272 astrocytes following treatment with Glu. Comparison of glutamate clearance between wild-type and MeCP2-null astrocytes bonitrile) and dihydrokainate are selective inhibitors for EAAT1 and EAAT2, respectively. To investigate the functional Glu transporters in our astrocyte cultures, we analyzed three Glu transporter blockers for their ability to alter the effects of Glu clearance. Glu clearance by the wild-type astrocytes was partially blocked by addition of TBOA and UCPH-101, but not DHKA. This suggests that EAAT1, but not EAAT2, plays a major role in Glu clearance under our astroglial culture conditions. Effects of glutamate on glutamine synthetase and EAAT1 protein in MeCP2-null astrocytes The initial set of experiments aimed to determine whether Glu modulate the translation of GS and EAAT1 protein. GS protein was expressed in both wild-type and MeCP2null astrocytes, and was significantly more abundant in MeCP2null astrocytes. After 12 h exposure to 0.011.0 mM Glu, wild-type astrocytes exhibited a dose-dependent increase in GS protein levels. Similar to its effect on the wild-type astrocytes, in the MeCP2-null astrocytes Glu exposure dose-dependently increased GS protein levels relative to untreated astrocytes. We then examined the effect 12829792 of 1.0 mM Glu on levels of GS protein, over a time course. GS expression was highest after 12 h exposure to 1.0 mM Glu, 10455325 decreasing slightly by 24 h in both wild-type and MeCP2-null astrocytes. Densitometric analysis of the bands in three independent experiments demonstrated that GS protein in MeCP2-null astrocyte cultures was higher than in wild-type astrocytes, 12 h but not 24 h after treatment. These results indicated that MeCP2 deficiency caused higher expression of GS protein in cultured astrocytes. We also asked whether treatment with 1.0 mM Glu altered expression of EAAT1 protein. EAAT1 protein was expressed in Characterization of MeCP2-Deficient Astrocytes both wild-type and MeCP2-null astrocytes, at levels that were similar in controls and MeCP2-null astrocytes. EAAT1 protein levels were altered in the wild-type astrocytes after treatment with 1.0 mM Glu. EAAT1 protein levels decreased significantly in the wild-type astrocytes, 24 h but not 12 h after treatment. In contrast, EAAT1 did not decrease in the MeCP2-null astrocytes, either 12 h or 24 h after treatment. In addition, the relative expression levels of EAAT1 24 h after treatment were lower in the wild-type than in the MeCP2-null culture, although the difference was not statistically significant. These results suggest that MeCP2 deficiency affects the expression of GS and EAAT1 protein, and that accelerated Glu clearance may result from dysregulation of GS and EAAT1 protein in MeCP2-null astrocytes. Discussion Recent studies suggest that glia, as well as neurons, cause neuronal dysfunction in RTT via non-cell-autonomous effects. Here, we have demonstrated that MeCP2 regulates the expression of astroglial marker transcripts, including GFAP and S100b in cultured astrocytes. In addition, MeCP2 is not essential for the cell morphology, growth, or viability; rather, it is involved in Glu clearance through the regulation of Glu transporters and GS in astrocytes. Altered astroglial gene expression and abnormal Glu clearanc