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.

Anti-PD-1 treatment protects against seizure by suppressing sodium channel function.

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.

BDNF-TrkB signaling pathway-mediated microglial activation induces neuronal KCC2 downregulation contributing to dynamic allodynia following spared nerve injury.

Mechanical allodynia can be evoked by punctate pressure contact with the skin (punctate mechanical allodynia) and dynamic contact stimulation induced by gentle touching of the skin (dynamic mechanical allodynia). Dynamic allodynia is insensitive to morphine treatment and is transmitted through the spinal dorsal horn by a specific neuronal pathway, which is different from that for punctate allodynia, leading to difficulties in clinical treatment. K+-Cl- cotransporter-2 (KCC2) is one of the major determinants of inhibitory efficiency, and the inhibitory system in the spinal cord is important in the regulation of neuropathic pain. The aim of the current study was to determine whether neuronal KCC2 is involved in the induction of dynamic allodynia and to identify underlying spinal mechanisms involved in this process. Dynamic and punctate allodynia were assessed using either von Frey filaments or a paint brush in a spared nerve injury (SNI) mouse model. Our study discovered that the downregulated neuronal membrane KCC2 (mKCC2) in the spinal dorsal horn of SNI mice is closely associated with SNI-induced dynamic allodynia, as the prevention of KCC2 downregulation significantly suppressed the induction of dynamic allodynia. The over activation of microglia in the spinal dorsal horn after SNI was at least one of the triggers in SNI-induced mKCC2 reduction and dynamic allodynia, as these effects were blocked by the inhibition of microglial activation. Finally, the BDNF-TrkB pathway mediated by activated microglial affected SNI-induced dynamic allodynia through neuronal KCC2 downregulation. Overall, our findings revealed that activation of microglia through the BDNF-TrkB pathway affected neuronal KCC2 downregulation, contributing to dynamic allodynia induction in an SNI mouse model.

Deficit of PKHD1L1 in the dentate gyrus increases seizure susceptibility in mice.

Epilepsy is a chronic neurological disorder featuring recurrent, unprovoked seizures, which affect more than 65 million people worldwide. Here, we discover that the PKHD1L1, which is encoded by polycystic kidney and hepatic disease1-like 1 (Pkhd1l1), wildly distributes in neurons in the central nervous system (CNS) of mice. Disruption of PKHD1L1 in the dentate gyrus region of the hippocampus leads to increased susceptibility to pentylenetetrazol-induced seizures in mice. The disturbance of PKHD1L1 leads to the overactivation of the mitogen-activated protein kinase (MAPK)/extracellular regulated kinase (ERK)-Calpain pathway, which is accompanied by remarkable degradation of cytoplasmic potassium chloride co-transporter 2 (KCC2) level together with the impaired expression and function of membrane KCC2. However, the reduction of membrane KCC2 is associated with the damaged inhibitory ability of the vital GABA receptors, which ultimately leads to the significantly increased susceptibility to epileptic seizures. Our data, thus, indicate for the first time that Pkhd1l1, a newly discovered polycystic kidney disease (PKD) association gene, is required in neurons to maintain neuronal excitability by regulation of KCC2 expression in CNS. A new mechanism of the clinical association between genetic PKD and seizures has been built, which could be a potential therapeutic target for treating PKD-related seizures.

Furosemide prevents membrane KCC2 downregulation during convulsant stimulation in the hippocampus.

In adults, γ-aminobutyric acid (GABA) type A receptor (GABAAR)-mediated inhibition depends on the maintenance of low intracellular chloride anion concentration through neuron-specific potassium-chloride cotransporter-2 (KCC2). KCC2 has been widely reported to have a plasticity change during the course of epilepsy development, with an early downregulation and late recovery in neuronal cell membranes after epileptic stimulation, which facilitates epileptiform burst activity. Furosemide is a clinical loop diuretic that inhibits KCC2. Here, we first confirmed that furosemide pretreatment could effectively prevented convulsant stimulation-induced neuronal membrane KCC2 downregulation in the hippocampus in both in vivo and in vitro cyclothiazide-induced seizure model. Second, we verified that furosemide pretreatment rescued KCC2 function deficits, as indicated by E GABA depolarizing shift and GABAAR inhibitory function impairment induced via cyclothiazide treatment. Further, we demonstrated that furosemide also suppressed cyclothiazide-induced epileptiform burst activity in cultured hippocampal neurons and lowered the mortality rate during acute seizure induction. Overall, furosemide prevents membrane KCC2 downregulation during acute seizure induction, restores KCC2-mediated GABA inhibition, and interrupts the progression from acute seizure to epileptogenesis.

Coordinated downregulation of KCC2 and GABAA receptor contributes to inhibitory dysfunction during seizure induction.

The GABAA receptor (GABAAR) is the main inhibitory receptor in the adult mammalian brain. GABAAR 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 GABAAR are downregulated in an activity-dependent manner during seizure induction. Functionally, KCC2 and GABAAR 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 GABAAR 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 GABAAR 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 GABAAR downregulation concomitant with recovery of suppressed KCC2-independent GABAergic mIPSC amplitude. Our results suggest that the coordinated downregulation of KCC2 and GABAAR during seizure induction exerts a strong functional impact on GABAAR, highlighting an important regulatory mechanism in epilepsy.

Synergistic effect of TGF-beta superfamily members on the induction of Foxp3+ Treg.

TGF-beta plays an important role in the induction of Treg and maintenance of immunologic tolerance, but whether other members of TGF-beta superfamily act together or independently to achieve this effect is poorly understood. Although others have reported that the bone morphogenetic proteins (BMP) and TGF-beta have similar effects on the development of thymocytes and T cells, in this study, we report that members of the BMP family, BMP-2 and -4, are unable to induce non-regulatory T cells to become Foxp3+ Treg. Neutralization studies with Noggin have revealed that BMP-2/4 and the BMP receptor signaling pathway is not required for TGF-beta to induce naïve CD4+CD25- cells to express Foxp3; however, BMP-2/4 and TGF-beta have a synergistic effect on the induction of Foxp3+ Treg. BMP-2/4 affects non-Smad signaling molecules including phosphorylated ERK and JNK, which could subsequently promote the differentiation of Foxp3+ Treg induced by TGF-beta. Data further advocate that TGF-beta is a key signaling factor for Foxp3+ Treg development. In addition, the synergistic effect of BMP-2/4 and TGF-beta indicates that the simultaneous manipulation of TGF-beta and BMP signaling might have considerable effects in the clinical setting for the enhancement of Treg purity and yield.