1566 S. D´ıaz de Leo´n-Guerrero et al. Fig. 2. MeCP2 protein structure and common mutations associated with RTT. The MeCP2 protein domains, the most frequent mutations found in RTT and different phosphorylation sites are depicted. MeCP2 has different domains including a methyl CpG binding domain (MBD), a transcriptional repression domain (TRD), two nuclear localization signals (NLS) and a WW domain in the C-terminus. The black arrows (top) indicate the position of the eight most common mutations found in RTT; note that they locate preferentially to the MBD and TRD domains (Matijevic et al., 2009). Known phosphorylation sites of MeCP2 are shown (bottom) for CaMKII (S421) and homeodomain-interacting kinase 2 (S80; Zhou et al., 2006; Bracaglia et al., 2009), as well as predicted phosphorylation sites for cyclin dependent kinase 5 (CDK5), PKCa ⁄ b, AKT, PKCe and PKCd. Table 1. MeCP2 deﬁciency in different cells Cell type Neurons Astrocytes Microglia Effect Abnormal dendritic and axon development Unable to sustain proper neuron development Neurotoxic effect by excessive glutamate release Reference(s) Larimore et al. (2009) Ballas et al. (2009) Maezawa et al. (2009) Maezawa & Jin (2010) reduced in a time-dependent manner in WT astrocytes when co-cultured with MeCP2+ ⁄ ) astrocytes; additionally, astrocyte MeCP2 protein levels in MeCP2+ ⁄ ) mice show a signiﬁcant reduction with age. Inhibition of the gap junction communication resulted in a reduced spread of the MeCP2 deﬁciency and also rescued part of the phenotype. A more recent study by the same group showed that microglia also play an important role in the development of RTT (Maezawa & Jin, 2010). WT hippocampal neurons cultivated in conditioned media from very pure microglia cultures from MeCP2- mice had shorter and thinner dendrites, with stunted arborization, whereas those grown in WT microglia conditioned media showed a normal phenotype. Consistently, neurons grown in MeCP2-null microglia conditioned media showed a reduced staining for MAP2 (Microtubule-associated protein 2, a dendritic marker), acetylated tubulin (reduction indicates microtubule disruption), PSD95 (post- synaptic density-95, a post-synaptic marker) and glutamate receptor interacting protein 1 (a scaffold protein in the post-synaptic compart- ment). All of this suggests that MeCP2-null microglia conditioned media damages post-synaptic elements of excitatory synapses (Maezawa & Jin, 2010). MeCP2-null microglia did not present an activated morphology or enhanced proliferation, indicative that the neurotoxic effect is not dependent on microglia activation. The authors report that this neurotoxic effect results from an excess of glutamate release from the MeCP2-null microglia and that it is released through channels of the gap junction. As in Maezawa et al. (2009), blocking the gap junction channels reduced the levels of released glutamate and rescued neuron morphology. Contrary to previous results (Ballas et al., 2009), neurons grown in astrocytic conditioned media, from both WT and MeCP2-null mice, did not display damage in dendrites or synapses. For the effects seen in astrocytes, Maezawa & Jin (2010) suggest a loss of function in MeCP2-null astrocytes, which would be unable to support adequate dendritic development, whereas Ballas et al. (2009) suggest a gain of function in which astrocytes secrete a neurotoxic factor into the medium. Still further analysis would be necessary to determine the variability of soluble factors that are secreted by both astrocytes and microglia caused by an MeCP2 deﬁciency. Given that MeCP2 can regulate gene expression both positively and negatively (Chahrour et al., 2008; Ben-Shachar et al., 2009; Urdinguio et al., 2010) (see below), it is probable that loss of MeCP2 could lead to the overexpression of neurotoxic factors as well a reduction in soluble factors that are necessary for neuronal survival. Overall, these results point out a previously unconsidered important role of glial cells in the development of the RTT. Methyl-CpG binding protein 2 targets genes in the central nervous system Searching for possible MeCP2 targets to explain the RTT phenotype has not been an easy task. Until recently only two genes were ª 2011 The Authors. European Journal of Neuroscience ª 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd European Journal of Neuroscience, 33, 1563–1574