Abstracts

Rationale: K+/Cl− cotransporter 2 (KCC2), a neuron-specific chloride transporter, is a key determinant of the efficacy of GABAergic inhibition. The expression of KCC2 has been found to be reduced in many developmental and epileptic encephalopathy, including Rett syndrome (RTT), suggesting that KCC2 might play a role in the pathophysiology of these disorders.

Methods: To develop neuron-based high-throughput screening (HTS) assays to identify chemical compounds that enhance the expression of the KCC2 gene, we report the generation of a robust high-throughput drug screening platform that allows for the rapid assessment of KCC2 gene expression in genome-edited human reporter neurons.

Results: 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 3 (GSK3) 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 MECP2 mutant RTT neurons, and rescued electrophysiological and morphological abnormalities of RTT neurons. Injection of KEEC KW-2449 or piperine in Mecp2 mutant mice ameliorated disease-associated respiratory and locomotion phenotypes.

Conclusions: The small-molecule compounds described in our study may have therapeutic effects not only in RTT but also in other neurological disorders, such as many epileptic disorders, involving dysregulation of KCC2.

Funding: Please list any funding that was received in support of this abstract.: This research was supported by NIH grants HD 045022, R37-CA084198, NS088538, and MH104610 awarded to R.J.; MH085802 awarded to M.S.; a postdoctoral fellowship grant from the Simons Foundation to the Simons Center for the Social Brain at MIT awarded to X.T. (award no. 2389069); and a Rett Syndrome Research Trust (RSRT) grant awarded to R.J. (award no. 50-1873-0201). K.L. was supported by an International Rett Syndrome Foundation postdoctoral fellowship. X.S.L. was supported by a Damon Runyon Cancer Foundation postdoctoral fellowship. This work was also supported, in part, by the Koch Institute Support (core) grant P30-CA14051 from the National Cancer Institute.