Pharmacological reactivation of inactive X-linked in cerebral cortical neurons of living mice.

Rett syndrome (RTT) is a genetic disorder resulting from a loss-of-function mutation in one copy of the X-linked gene methyl-CpG-binding protein 2 (). Typical RTT patients are females and, due to random X chromosome inactivation (XCI), ∼50% of cells express mutant MECP2 and the other ∼50% express wild-type MECP2. Cells expressing mutant MECP2 retain a wild-type copy of MECP2 on the inactive X chromosome (Xi), the reactivation of which represents a potential therapeutic approach for RTT. Previous studies have demonstrated reactivation of Xi-linked in cultured cells by biological or pharmacological inhibition of factors that promote XCI (called "XCI factors" or "XCIFs"). Whether XCIF inhibitors in living animals can reactivate Xi-linked in cerebral cortical neurons, the cell type most therapeutically relevant to RTT, remains to be determined. Here, we show that pharmacological inhibitors targeting XCIFs in the PI3K/AKT and bone morphogenetic protein signaling pathways reactivate Xi-linked in cultured mouse fibroblasts and human induced pluripotent stem cell-derived postmitotic RTT neurons. Notably, reactivation of Xi-linked corrects characteristic defects of human RTT neurons including reduced soma size and branch points. Most importantly, we show that intracerebroventricular injection of the XCIF inhibitors reactivates Xi-linked in cerebral cortical neurons of adult living mice. In support of these pharmacological results, we also demonstrate genetic reactivation of Xi-linked in cerebral cortical neurons of living mice bearing a homozygous XCIF deletion. Collectively, our results further establish the feasibility of pharmacological reactivation of Xi-linked as a therapeutic approach for RTT.