Pharmacological reversal of synaptic and network pathology in human MECP2‐KO neurons and cortical organoids

Duplication or deficiency of the X‐linked MECP2 gene reliably produces profound neurodevelopmental impairment. MECP2 mutations are almost universally responsible for Rett syndrome (RTT), and particular mutations and cellular mosaicism of MECP2 may underlie the spectrum of RTT symptomatic severity. No clinically approved treatments for RTT are currently available, but human pluripotent stem cell technology offers a platform to identify neuropathology and test candidate therapeutics. Using a strategic series of increasingly complex human stem cell‐derived technologies, including human neurons, MECP2 ‐mosaic neurospheres to model RTT female brain mosaicism, and cortical organoids, we identified synaptic dysregulation downstream from knockout of MECP2 and screened select pharmacological compounds for their ability to treat this dysfunction. Two lead compounds, Nefiracetam and PHA 543613, specifically reversed MECP2‐ knockout cytologic neuropathology. The capacity of these compounds to reverse neuropathologic phenotypes and networks in human models supports clinical studies for neurodevelopmental disorders in which MeCP2 deficiency is the predominant etiology. Synopsis Deficiency of the X‐linked MECP2 gene profoundly impairs neurodevelopment. Clinically, mutations in MECP2 most commonly present as the severe and untreatable disease Rett syndrome. Innovative human pluripotent stem cell (PSC) technology enables the investigation of therapeutic candidates. Neurons differentiated from human MECP2 ‐KO PSCs showed altered expression of synapse‐relevant genes, synaptic morphology, and decreased calcium and network activities compared to control neurons. An in silico neural network simulation affirmed that synaptic structural parameters link with neural network activity, supporting our approach of rescuing synaptic structure and increasing activity. Strategic screening of select drug candidates with mechanisms of action that counteract MECP2 ‐KO deficiencies isolated two lead compounds, Nefiracetam and PHA 543613, for subsequent validation in 3D human cell models. MECP2 ‐mosaic neurospheres, a novel model of Rett female brain mosaicism, showed altered calcium activity that could be reversed by Nefiracetam and/or PHA 543613. Nefiracetam and/or PHA 543613 increased synaptic gene expression and reversed network pathology in human MECP2 ‐KO cortical organoids, arguing for their clinical trial in patients with MeCP2‐deficient neurodevelopmental disorders. Graphical Abstract