Identification of KCC2 Expression Enhancer Compounds to Treat Autism Spectrum Disorders
A novel neuronal screen has identified small molecules effective in a mouse model of Rett Syndrome
Background
Whitehead Institute is seeking a licensee for intellectual property relating to targeting the K+/Cl- co-transporter 2 (KCC2) expression as a way to treat Rett Syndrome (RTT), a severe neurodevelopmental disorder caused by mutations in the X-linked gene Methyl CpG binding Protein 2 (MECP2). Due to a lack of neuron-based high-throughput screening assays, it has been challenging to discover small molecules that enhance the expression of the KCC2 gene. Whitehead researchers have developed a robust high-throughput drug screening platform that allows for rapid assessment of KCC2 gene expression in genome-edited human reporter neurons. The researchers have identified a group of small molecules that enhance KCC2 expression. Treatment with these identified compounds increases KCC2 expression in human wildtype and isogenic MECP2 mutant RTT neurons. Further, injection of two identified compounds into a Mecp2 mutant mouse model of RTT ameliorates the disease-associated respiratory and locomotion phenotypes.
Technology Overview
The scientists have used human embryonic stem (ES) cells in combination with CRISPR/Cas9 gene editing technology to identify small molecules that enhance KCC2 expression in neurons. They developed a robust HTS assay by inserting a 2A-luciferase reporter gene into the endogenous KCC2 locus in human ES cells, providing a convenient readout of KCC2 expression. By screening the KCC2 reporter human neurons, the researchers identified a number of hit KCC2 expression-enhancing compounds (KEECs) from ~900 small molecule compounds. Hit KEECs were validated by Western blot and quantitative RT-PCR experiments on cultured human WT and isogenic RTT neurons, as well as on organotypic mouse brain slices. The researchers have conducted pharmacological and molecular biology experiments to show that hit KEECs act through inhibition of the FLT3 or GSK3beta kinases, or activation of the SIRT1 or TRPV1 pathways. Treatment of RTT neurons with KEECs rescued disease-related deficits in the GABA functional switch, excitatory synapse development, as well as the reduced neuronal cell size. Finally, injection of the hit KEECs KW-2449 or Piperine into a Mecp2 mutant animal model of RTT ameliorated behavioral phenotypes including breathing pauses and reduced locomotion, which represent important preclinical data suggesting that the KEECs identified in this study may be effective to restore the impaired the E/I balance in the RTT brain and provide symptomatic RJ-1-02 treatment for RTT patients.
Benefits
Small molecule compounds identified can potentially benefit various brain diseases through a novel mechanism of enhancing KCC2 expression.
Applications
Screening tool for small molecules that modulate the level of KCC2 in neurons.