astroglial genes by MeCP2. On the other hand, in our culture conditions, MeCP2 deficiency did not impair the expression of GS transcripts in cultured astrocytes, but did affect the expression of GS protein. A very recent study has shown that defects in the AKT/mTOR pathway 5 Characterization of MeCP2-Deficient Astrocytes are responsible for altered translational control in MeCP2 mutant neuron. These findings suggest that a deficit in protein synthesis and/or turnover in the MeCP2-null astrocytes might influence the final levels of GS protein. Further studies are necessary to investigate whether MeCP2 deficiency impairs ” the synthesis and turnover of proteins in RTT. The most important finding in this study was that MeCP2 deficiency in astrocytes accelerates 19151731” Glu clearance. Consistent with this, RTT is associated with abnormalities in the Glu metabolism. Some studies have demonstrated increases in Glu levels in the cerebrospinal fluid of human RTT patients. On the other hand, in animal studies there have been instances of decreased Glu levels and/or Glu/Gln ratios, as determined by in MR spectroscopy. Furthermore, MeCP2-deficient microglia release an abnormally high level of Glu, causing excitotoxicity that may contribute to dendritic and synaptic abnormalities in RTT. These results clearly suggest that MeCP2 has the potential to regulate Glu levels in the brain under certain circumstances. Glu levels are altered in the RTT brain, but the mechanisms responsible for the changes in Glu metabolism are unknown. In light of our findings, we speculate that abnormal expression of Glu transporters and GS resulting from MeCP2 deficiency could lead to abnormal Glu clearance in astrocytes 
and in turn to altered levels of Glu in RTT brain. Additional studies are needed to determine the mechanisms underlying changes in Glu levels and Glu metabolism, and their role in the RTT brain. In conclusion, MeCP2 modulates Glu clearance through the regulation of astroglial genes in astrocytes. This study suggests a novel role for MeCP2 in astrocyte function; these findings may be useful in exploration of a new Relebactam chemical information approach for preventing the neurological dysfunctions associated with RTT. Materials and Methods Cell culture For each experiment, primary cultures were generated from individual MeCP2-null neonates and their wild-type littermates; tail snips from each neonate were obtained for genotyping, as described below. Enriched cultures of GFAP-expressing astroglial cells, which are virtually free of neurons and microglial cells, were established from the cerebral hemispheres of postnatal day 0 to P1 newborn mice, as previously described. In brief, pieces of dissected tissue were trypsinized for 10 min in Ca2- and Mg2-free phosphate-buffered saline supplemented with Characterization of MeCP2-Deficient Astrocytes 0.02% EDTA. Tissue samples were subsequently dissociated in Hank’s balanced salt solution containing 15% fetal calf serum by trituration though 10-ml plastic pipettes. Cells were pelleted at 1006g for 5 min, resuspended in Dulbecco’s modified Eagle’s medium containing 15% FCS, and seeded into 100-mm culture dishes previously coated with poly-D-lysine. Upon reaching confluency, cells were trypsinized and replated. Cells were used after the third passage in all experiments, and were seeded at 36104 cells/cm2 in 6-well plate dishes or 35-mm culture dishes. Cultures were assayed by immunochemical analysis using antibodies against GFAP, MAP2, and CD11b in order to