The suppressive effect of the specific KCC2 modulator CLP290 on seizure in mice.

Epilepsy is a chronic neurological disorder characterized by episodic dysfunction of central nervous system. The most basic mechanism of epilepsy falls to the imbalance between excitation and inhibition. In adults, GABAA receptor (GABAAR) is the main inhibitory receptor to prevent neurons from developing hyperexcitability, while its inhibition relies on the low intracellular chloride anion concentration ([Cl-]i). Neuronal-specific electroneutral K+-Cl- cotransporter (KCC2) can mediate chloride efflux to lower [Cl-]i for GABAAR mediated inhibition. Our previous study has revealed that the coordinated downregulation of KCC2 and GABAAR participates in epilepsy. According to a high-throughout screen for compounds that reduce [Cl-]i, CLP290 turns out to be a specific KCC2 functional modulator. In current study, we first confirmed that CLP290 could dose-dependently suppress convulsant-induced seizures in mice in vivo as well as the epileptiform burst activities in cultured hippocampal neurons in vitro. Then, we discovered that CLP290 functioned through preventing the downregulation of the KCC2 phosphorylation at Ser940 and hence the KCC2 membrane expression during convulsant stimulation, and consequently restored the GABA inhibition. In addition, while CLP290 was given in early epileptogenesis period, it also effectively decreased the spontaneous recurrent seizures. Generally, our current results demonstrated that CLP290, as a specific KCC2 modulator by enhancing KCC2 function, not only inhibits the occurrence of the ictal seizures, but also suppresses the epileptogenic process. Therefore, we believe KCC2 may be a suitable target for future anti-epileptic drug development.

M-Calpain Activation Facilitates Seizure Induced KCC2 Down Regulation.

Potassium chloride co-transporter 2 (KCC2), a major chloride transporter that maintains GABAA receptor inhibition in mature mammalian neurons, is down-regulated in the hippocampus during epileptogenesis. Impaired KCC2 function accelerates or facilitates seizure onset. Calpain, with two main subtypes of m- and µ-calpain, is a Ca2+-dependent cysteine protease that mediates the nonlysosomal degradation of KCC2. Although recent studies have demonstrated that calpain inhibitors exert antiepileptic and neuroprotective effects in animal models of acute and chronic epilepsy, whether calpain activation affects seizure induction through KCC2 degradation remains unknown. Our results showed that: (1) Blockade of calpain by non-selective calpain inhibitor MDL-28170 prevented convulsant stimulation induced KCC2 downregulation, and reduced the incidence and the severity of pentylenetetrazole (PTZ) induced seizures. (2) m-calpain, but not µ-calpain, inhibitor mimicked MDL-28170 effect on preventing KCC2 downregulation. (3) Phosphorylation of m-calpain has been significantly enhanced during seizure onset, which was partly mediated by the calcium independent MAPK/ERK signaling pathway activation. (4) MAPK/ERK signaling blockade also had similar effect as total calpain blockade on both KCC2 downregulation and animal seizure induction. The results indicate that upregulated m-calpain activation by MAPK/ERK during convulsant stimulation down regulates both cytoplasm- and membrane KCC2, and in turn facilitates seizure induction. This finding may provide a foundation for the development of highly effective antiepileptic drugs targeting of m-calpain.

Antinociceptive effects of novel epibatidine analogs through activation of α4ß2 nicotinic receptors.

The study of α4ß2 nicotinic receptors has provided new indications in the treatment of pain. Efforts have been made to explore new α4ß2 nicotinic receptor agonists, including TC-2559, as antinociceptive drugs. In this study, we discovered a set of novel epibatidine analogs with strong binding affinities to the α4ß2 nicotinic receptors. Among these compounds, C-159, C-163, and C-9515 attenuated formalin-induced nociceptive responses in mice; C-9515 caused the most potent analgesic effect, which was blocked by mecamylamine, a non-selective nicotinic receptor antagonist. Furthermore, C-9515 potently inhibited chronic constriction injury (CCI)-induced neuropathic pain in rats, which was sensitive to DHßE, a selective α4ß2 subtype antagonist, indicating that its analgesic effect was mediated by the activation of the α4ß2 nicotinic receptors. In conclusion, the epibatidine analog C-9515 was found to be a potent α4ß2 nicotinic receptor agonist with potent analgesic function, which demonstrated potential for the further exploration of its druggability.