No abstract

Inhibitory GABA-ergic neurotransmission is of fundamental relevance for the adult vertebrate central nervous system and requires low chloride ion concentration in neurons. This basic ionic-homeostatic mechanism critically relies on expression and function of KCC2, a neuroprotective ionic transporter that extrudes neuronal chloride. Attenuated expression of KCC2 causes circuit malfunction in chronic pain and other neuropsychiatric illnesses. To find new analgesics, we screened 1057 cell growth-regulating compounds in cultured mouse primary cortical neurons for enhancement of Kcc2 gene expression. We identified kenpaullone (KP), which enhanced Kcc2/KCC2 expression and function in cultured rodent and human neurons by inhibiting GSK3ß. KP effectively reduced pathologic pain in preclinical mouse models. In nerve-injury pain, KP restored Kcc2 expression and GABA-evoked chloride reversal potential in the spinal cord dorsal horn. Delta-catenin, a phosphorylation-target of GSK3ß in neurons, activated the Kcc2 promoter via Kaiso transcription factor, and transient spinal transgenesis of delta-catenin mimicked KP's analgesic effects.1D), which is accompanied by increased KCC2 protein expression and increased expression of synaptophysin ( Fig. 1E-F). This latter finding suggests increased synaptic maturation. These data indicate that our rationally designed screen identified a GSK3/CDK kinase inhibitor, KP, that enhances Kcc2/KCC2 gene expression and not KCC2-mediated chloride extrusion in CNS neurons. KP functions as Kcc2/KCC2 gene expression enhancer in mammals including humans, where we document enhanced synaptic maturation and increased KCC2 expression and function. ============================= Fig. 1========================= Fig. 1. Compound screening for enhancers of Kcc2 expression in primary cortical neurons yields KenpaulloneA, primary mouse neurons, B-C: primary rat neurons. D-F: primary human neurons. A) Top panel: Screening paradigm using three rounds of primary screen based on luciferase (LUC) activity followed by secondary screen including Kcc2 RT-qPCR and Clomeleon chloride imaging. Bottom panel: Four compound "winners" including Kenpaullone (KP). Screening conducted in primary mouse neurons. B) Primary rat cortical neurons. Bar diagram: dose-dependent increase in Kcc2 mRNA expression after KP treatment, also reflected by increased protein expression as shown by KCC2 immuno-label (micrographs). Results represent the average mRNA expression of 4 independent neuronal cultures. p<0.05, one-way ANOVA C) Primary rat cortical neurons. Neuronal [Cl-]i, measured with ratiometric chloride indicator, clomeleon, is robustly and significantly reduced after KP treatment. Note that add-on treatment with KCC2-transport blocker, VU0240551, leads to a [Cl-]i ≥120mM, for both, vehicle-treated and KP-treated, indicating that KP's chloride lowering effect relies on KCC2 chloride extruding transport function. n≥75 neurons from 3 independent cultures; * p<0.01, t-test D) Primary human cortical neurons. KCC2 mRNA increases in a KP...