Pharmacological enhancement of KCC2 gene expression exerts therapeutic effects on human Rett syndrome neurons and Mecp2 mutant mice

Abstract

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the methyl CpG binding protein 2 (MECP2) 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 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. 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 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. 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.

The enhancement of activity rescues the establishment ofMecp2null neuronal phenotypes

“…Similarly, Nifedipine abolished the reduction driven by CX546 on the expression of Rest, a gene that is generally upregulated in null samples (Abuhatzira et al, 2007). In good accordance with the functional data, in panel J we found that only CX546 increased the ratio of the expression levels of Kcc2 and Nkcc1, therefore indicating a faster rate of maturation of GABA signaling (Tang et al, 2019;Hinz et al, 2019).…”

“…From a functional perspective, CX546 rescued the responsiveness of null neuronal networks to stimuli, while decreasing their intracellular Cllevels, which suggests a restored responsiveness to GABA. This result fits with the recovered transcriptional levels of Kcc2, the neuron specific K + -Cl − co-transporter (KCC2) that is crucial for GABA signaling maturation (Ben-Ari et al, 2012) and its normalization in Rett syndrome currently represents a promising therapeutic approach (Tang et al, 2016;Tang et al, 2019;Lozovaya et al, 2019). Importantly, the simultaneous treatment of Mecp2 null neurons with CX546 and Nifedipine, a voltage gated calcium channels blocker, restricted the observed transcriptional rescues, therefore proving that most of the beneficial effects were due to enhanced neuronal activity.…”

Repurposing cell growth-regulating compounds identifies kenpaullone which ameliorates pathologic pain via normalization of inhibitory neurotransmission

“…In an elegant and very recently published study, small molecules were selected to enhance expression of Kcc2/KCC2 for effective rescue of a modeled Rett Syndrome phenotype (Tang et al, 2019).…”

Isolation and characterization of multi-protein complexes enriched in the K-Cl co-transporter 2 from brain plasma membranes

“…Deficits in Kcc2 activity are also believed to contribute to the development of temporal lobe epilepsy (17,18), in addition to other traumas including ischemia and neuropathic pain (19,20). Given the critical role that Kcc2 plays in determining the maturation of inhibitory neurotransmission, subtle changes in its function are also strongly implicated in autism spectrum disorders (ASD) (21), Down syndrome (22), fragile X syndrome (23), and Rett syndrome (24)(25)(26).…”