Identification of novel molecules by which Mecp2 knock-out astrocytes exert a synaptotoxic action on neurons

Background. Over 95% of Rett syndrome (RTT) cases are given by mutations in the X-linked methyl-CpG-binding protein 2 (MECP2) gene. Although initial studies supported a role for MeCP2 exclusively in neurons, several recent data indicate the involvement also of astrocytes, which can affect neuronal maturation through non-cell autonomous mechanisms. Nevertheless, many aspects of astrocyte dysfunctions in RTT remain still unknown. Objectives: According to the crucial role of astrocytes in promoting synapse formation and functioning, and the profound synaptic alterations in RTT, we investigated the influence of Mecp2 null astrocytes on synaptic maturation and we explored the involvement of two molecules in the occurrence of synaptic defects. Methods: To reproduce in vitro the heterozygous condition of RTT brains, we tested the effect of null astrocytes or their conditioned medium (ACM) on WT neurons. By immunufluorescence we analysed pre- and post-synaptic densities in cultured neurons. RNA sequencing was used to gain insight into the involved molecular mechanisms. Results: We demonstrated that Mecp2 null astrocytes dramatically affect synaptogenesis and synaptic functionality, and molecular analyses highlighted in null astrocytes both the activation of an inflammatory pathway and a concomitant reduction in cholesterol metabolism. As validation of these underscored molecular mechanisms, we proved that Mecp2 null astrocytes, when in culture with WT neurons, express and secrete excessive levels of Interleukin-6 (IL-6), which exerts a synaptotoxic action. Indeed, we found that the recombinant IL-6 causes synaptic defects in WT neurons and, coherently, a neutralizing IL-6 antibody rescues KO astrocyte-mediated synaptic alterations. In parallel, we observed a reduced cholesterol metabolism in Mecp2 null astrocytes and demonstrated that cholesterol supplementation reverts synaptic defects. Conclusion: These in vitro data constitute the rationale for studying the pathogenic role of IL-6 and cholesterol in Mecp2 deficient mice and explore novel therapeutic strategies targeting these two factors to improve RTT symptoms.