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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, GABA A receptor (GABA A R) 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 GABA A R mediated inhibition. Our previous study has revealed that the coordinated downregulation of KCC2 and GABA A R 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., Competing Interests: Declaration of competing interest The authors report no conflicts of interest in relation to this work., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Aims: Although programmed cell death protein 1 (PD-1) typically serves as a target for immunotherapies, a few recent studies have found that PD-1 is expressed in the nervous system and that neuronal PD-1 might play a crucial role in regulating neuronal excitability. However, whether brain-localized PD-1 is involved in seizures and epileptogenesis is still unknown and worthy of in-depth exploration., Methods: The existence of PD-1 in human neurons was confirmed by immunohistochemistry, and PD-1 expression levels were measured by real-time quantitative PCR (RT-qPCR) and western blotting. Chemoconvulsants, pentylenetetrazol (PTZ) and cyclothiazide (CTZ), were applied for the establishment of in vivo (rodents) and in vitro (primary hippocampal neurons) models of seizure, respectively. SHR-1210 (a PD-1 monoclonal antibody) and sodium stibogluconate (SSG, a validated inhibitor of SH2-containing protein tyrosine phosphatase-1 [SHP-1]) were administrated to investigate the impact of PD-1 pathway blockade on epileptic behaviors of rodents and epileptiform discharges of neurons. A miRNA strategy was applied to determine the impact of PD-1 knockdown on neuronal excitability. The electrical activities and sodium channel function of neurons were determined by whole-cell patch-clamp recordings. The interaction between PD-1 and α-6 subunit of human voltage-gated sodium channel (Nav1.6) was validated by performing co-immunostaining and co-immunoprecipitation (co-IP) experiments., Results: Our results reveal that PD-1 protein and mRNA levels were upregulated in lesion cores compared with perifocal tissues of surgically resected specimens from patients with intractable epilepsy. Furthermore, we show that anti-PD-1 treatment has anti-seizure effects both in vivo and in vitro. Then, we reveal that PD-1 blockade can alter the electrophysiological properties of sodium channels. Moreover, we reveal that PD-1 acts together with downstream SHP-1 to regulate sodium channel function and hence neuronal excitability. Further investigation suggests that there is a direct interaction between neuronal PD-1 and Nav1.6., Conclusion: Our study reveals that neuronal PD-1 plays an important role in epilepsy and that anti-PD-1 treatment protects against seizures by suppressing sodium channel function, identifying anti-PD-1 treatment as a novel therapeutic strategy for epilepsy., (© 2023 The Authors. CNS Neuroscience & Therapeutics published by John Wiley & Sons Ltd.)

The GABA A receptor (GABA A R) is the main inhibitory receptor in the adult mammalian brain. GABA A R function is dependent on its expression, distribution, and the chloride (Cl - ) transmembrane gradient, which is determined by the potassium-chloride cotransporter 2 (KCC2) in the adult brain. KCC2 and GABA A R are downregulated in an activity-dependent manner during seizure induction. Functionally, KCC2 and GABA A R are closely related membrane proteins which modulate GABAergic inhibition. However, it remains unclear how their downregulation during seizure induction is coordinated. This study aimed to assess this interaction. Our results revealed that KCC2 and GABA A R were simultaneously downregulated in both in vivo and in vitro seizure models induced by the convulsant cyclothazide (CTZ), which was at least partly due to structural coupling in hippocampal neuronal membranes. Immunohistochemistry revealed colocalization of gephyrin with KCC2 and co-immunoprecipitation exhibited a direct coupling between GABA A R α1-subunit and KCC2 protein in hippocampal cell membranes. KCC2 specific short hairpin RNA (KCC2-shRNA) was employed to specifically reduce the expression of KCC2 in cultured hippocampal neurons. This resulted in a significant reduction in KCC2-independent GABAergic miniature inhibitory post-synaptic current (mIPSC) amplitude in shKCC2-transfected neurons. Further, pre-treatment with furosemide, a KCC2 inhibitor, during CTZ stimulation followed by washout significantly prevented convulsant stimulation-induced membrane KCC2 downregulation and significantly attenuated GABA A R downregulation concomitant with recovery of suppressed KCC2-independent GABAergic mIPSC amplitude. Our results suggest that the coordinated downregulation of KCC2 and GABA A R during seizure induction exerts a strong functional impact on GABA A R, highlighting an important regulatory mechanism in epilepsy., Competing Interests: Declaration of competing interest We declare that none of the work contained in this manuscript has been published in any language or is currently under consideration at any other journal, and there are no conflicts of interest to declare. All authors have contributed to, read, and approved this submitted manuscript in its current form., (Copyright © 2020 Elsevier Inc. All rights reserved.)