Pharmacological enhancement of gene expression exerts therapeutic effects on human Rett syndrome neurons and mutant mice.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the methyl CpG binding protein 2 () gene. There are currently no approved treatments for RTT. The expression of K/Cl cotransporter 2 (KCC2), a neuron-specific protein, has been found to be reduced in human RTT neurons and in RTT mouse models, suggesting that KCC2 might play a role in the pathophysiology of RTT. To develop neuron-based high-throughput screening (HTS) assays to identify chemical compounds that enhance the expression of the gene, we report the generation of a robust high-throughput drug screening platform that allows for the rapid assessment of gene expression in genome-edited human reporter neurons. From an unbiased screen of more than 900 small-molecule chemicals, we have identified a group of compounds that enhance KCC2 expression termed KCC2 expression-enhancing compounds (KEECs). The identified KEECs include U.S. Food and Drug Administration-approved drugs that are inhibitors of the fms-like tyrosine kinase 3 (FLT3) or glycogen synthase kinase 3β (GSK3β) pathways and activators of the sirtuin 1 (SIRT1) and transient receptor potential cation channel subfamily V member 1 (TRPV1) pathways. Treatment with hit compounds increased KCC2 expression in human wild-type (WT) and isogenic mutant RTT neurons, and rescued electrophysiological and morphological abnormalities of RTT neurons. Injection of KEEC KW-2449 or piperine in mutant mice ameliorated disease-associated respiratory and locomotion phenotypes. The small-molecule compounds described in our study may have therapeutic effects not only in RTT but also in other neurological disorders involving dysregulation of KCC2.