Reactivation of Dormant Relay Pathways in Injured Spinal Cord by KCC2 Manipulations.

Many human spinal cord injuries are anatomically incomplete but exhibit complete paralysis. It is unknown why spared axons fail to mediate functional recovery in these cases. To investigate this, we undertook a small-molecule screen in mice with staggered bilateral hemisections in which the lumbar spinal cord is deprived of all direct brain-derived innervation, but dormant relay circuits remain. We discovered that a KCC2 agonist restored stepping ability, which could be mimicked by selective expression of KCC2, or hyperpolarizing DREADDs, in the inhibitory interneurons between and around the staggered spinal lesions. Mechanistically, these treatments transformed this injury-induced dysfunctional spinal circuit to a functional state, facilitating the relay of brain-derived commands toward the lumbar spinal cord. Thus, our results identify spinal inhibitory interneurons as a roadblock limiting the integration of descending inputs into relay circuits after injury and suggest KCC2 agonists as promising treatments for promoting functional recovery after spinal cord injury.

Chloride transporters controlling neuronal excitability.

Synaptic inhibition plays a crucial role in regulating neuronal excitability, which is the foundation of nervous system function. This inhibition is largely mediated by the neurotransmitters GABA and glycine that activate Cl--permeable ion channels, which means that the strength of inhibition depends on the Cl- gradient across the membrane. In neurons, the Cl- gradient is primarily mediated by two secondarily active cation-chloride cotransporters (CCCs), NKCC1 and KCC2. CCC-mediated regulation of the neuronal Cl- gradient is critical for healthy brain function, as dysregulation of CCCs has emerged as a key mechanism underlying neurological disorders including epilepsy, neuropathic pain, and autism spectrum disorder. This review begins with an overview of neuronal chloride transporters before explaining the dependent relationship between these CCCs, Cl- regulation, and inhibitory synaptic transmission. We then discuss the evidence for how CCCs can be regulated, including by activity and their protein interactions, which underlie inhibitory synaptic plasticity. For readers who may be interested in conducting experiments on CCCs and neuronal excitability, we have included a section on techniques for estimating and recording intracellular Cl-, including their advantages and limitations. Although the focus of this review is on neurons, we also examine how Cl- is regulated in glial cells, which in turn regulate neuronal excitability through the tight relationship between this nonneuronal cell type and synapses. Finally, we discuss the relatively extensive and growing literature on how CCC-mediated neuronal excitability contributes to neurological disorders.

Evidence for Two Subpopulations of Cerebrospinal Fluid-Contacting Neurons with Opposite GABAergic Signaling in Adult Mouse Spinal Cord.

Spinal cerebrospinal fluid-contacting neurons (CSF-cNs) form an evolutionary conserved bipolar cell population localized around the central canal of all vertebrates. CSF-cNs were shown to express molecular markers of neuronal immaturity into adulthood; however, the impact of their incomplete maturation on the chloride (Cl-) homeostasis as well as GABAergic signaling remains unknown. Using adult mice from both sexes, in situ hybridization revealed that a proportion of spinal CSF-cNs (18.3%) express the Na+-K+-Cl- cotransporter 1 (NKCC1) allowing intracellular Cl- accumulation. However, we did not find expression of the K+-Cl- cotransporter 2 (KCC2) responsible for Cl- efflux in any CSF-cNs. The lack of KCC2 expression results in low Cl- extrusion capacity in CSF-cNs under high Cl- load in whole-cell patch clamp. Using cell-attached patch clamp allowing recordings with intact intracellular Cl- concentration, we found that the activation of ionotropic GABAA receptors (GABAA-Rs) induced both depolarizing and hyperpolarizing responses in CSF-cNs. Moreover, depolarizing GABA responses can drive action potentials as well as intracellular calcium elevations by activating voltage-gated calcium channels. Blocking NKCC1 with bumetanide inhibited the GABA-induced calcium transients in CSF-cNs. Finally, we show that metabotropic GABAB receptors have no hyperpolarizing action on spinal CSF-cNs as their activation with baclofen did not mediate outward K+ currents, presumably due to the lack of expression of G-protein-coupled inwardly rectifying potassium (GIRK) channels. Together, these findings outline subpopulations of spinal CSF-cNs expressing inhibitory or excitatory GABAA-R signaling. Excitatory GABA may promote the maturation and integration of young CSF-cNs into the existing spinal circuit.

CDKL5-mediated developmental tuning of neuronal excitability and concomitant regulation of transcriptome.

Cyclin-dependent kinase-like 5 (CDKL5) is a serine-threonine kinase enriched in the forebrain to regulate neuronal development and function. Patients with CDKL5 deficiency disorder (CDD), a severe neurodevelopmental condition caused by mutations of CDKL5 gene, present early-onset epilepsy as the most prominent feature. However, spontaneous seizures have not been reported in mouse models of CDD, raising vital questions on the human-mouse differences and the roles of CDKL5 in early postnatal brains. Here, we firstly measured electroencephalographic (EEG) activities via a wireless telemetry system coupled with video-recording in neonatal mice. We found that mice lacking CDKL5 exhibited spontaneous epileptic EEG discharges, accompanied with increased burst activities and ictal behaviors, specifically at postnatal day 12 (P12). Intriguingly, those epileptic spikes disappeared after P14. We next performed an unbiased transcriptome profiling in the dorsal hippocampus and motor cortex of Cdkl5 null mice at different developmental timepoints, uncovering a set of age-dependent and brain region-specific alterations of gene expression in parallel with the transient display of epileptic activities. Finally, we validated multiple differentially expressed genes, such as glycine receptor alpha 2 and cholecystokinin, at the transcript or protein levels, supporting the relevance of these genes to CDKL5-regulated excitability. Our findings reveal early-onset neuronal hyperexcitability in mouse model of CDD, providing new insights into CDD etiology and potential molecular targets to ameliorate intractable neonatal epilepsy.

Knockdown of calpain1 in lumbar motoneurons reduces spasticity after spinal cord injury in adult rats.

Spasticity, affecting ∼75% of patients with spinal cord injury (SCI), leads to hyperreflexia, muscle spasms, and cocontractions of antagonist muscles, greatly affecting their quality of life. Spasticity primarily stems from the hyperexcitability of motoneurons below the lesion, driven by an upregulation of the persistent sodium current and a downregulation of chloride extrusion. This imbalance results from the post-SCI activation of calpain1, which cleaves Nav1.6 channels and KCC2 cotransporters. Our study was focused on mitigating spasticity by specifically targeting calpain1 in spinal motoneurons. We successfully transduced lumbar motoneurons in adult rats with SCI using intrathecal administration of adeno-associated virus vector serotype 6, carrying a shRNA sequence against calpain1. This approach significantly reduced calpain1 expression in transduced motoneurons, leading to a noticeable decrease in spasticity symptoms, including hyperreflexia, muscle spasms, and cocontractions in hindlimb muscles, which are particularly evident in the second month post-SCI. In addition, this decrease, which prevented the escalation of spasticity to a severe grade, paralleled the restoration of KCC2 levels in transduced motoneurons, suggesting a reduced proteolytic activity of calpain1. These findings demonstrate that inhibiting calpain1 in motoneurons is a promising strategy for alleviating spasticity in SCI patients.

NMDA Receptors at Primary Afferent-Excitatory Neuron Synapses Differentially Sustain Chemotherapy- and Nerve Trauma-Induced Chronic Pain.

The spinal dorsal horn contains vesicular glutamate transporter-2 (VGluT2)-expressing excitatory neurons and vesicular GABA transporter (VGAT)-expressing inhibitory neurons, which normally have different roles in nociceptive transmission. Spinal glutamate NMDAR hyperactivity is a crucial mechanism of chronic neuropathic pain. However, it is unclear how NMDARs regulate primary afferent input to spinal excitatory and inhibitory neurons in neuropathic pain. Also, the functional significance of presynaptic NMDARs in neuropathic pain has not been defined explicitly. Here we showed that paclitaxel treatment or spared nerve injury (SNI) similarly increased the NMDAR-mediated mEPSC frequency and dorsal root-evoked EPSCs in VGluT2 dorsal horn neurons in male and female mice. By contrast, neither paclitaxel nor SNI had any effect on mEPSCs or evoked EPSCs in VGAT neurons. In mice with conditional Grin1 (gene encoding GluN1) KO in primary sensory neurons (Grin1-cKO), paclitaxel treatment failed to induce pain hypersensitivity. Unexpectedly, SNI still caused long-lasting pain hypersensitivity in Grin1-cKO mice. SNI increased the amplitude of puff NMDA currents in VGluT2 neurons and caused similar depolarizing shifts in GABA reversal potentials in WT and Grin1-cKO mice. Concordantly, spinal Grin1 knockdown diminished SNI-induced pain hypersensitivity. Thus, presynaptic NMDARs preferentially amplify primary afferent input to spinal excitatory neurons in neuropathic pain. Although presynaptic NMDARs are required for chemotherapy-induced pain hypersensitivity, postsynaptic NMDARs in spinal excitatory neurons play a dominant role in traumatic nerve injury-induced chronic pain. Our findings reveal the divergent synaptic connectivity and functional significance of spinal presynaptic and postsynaptic NMDARs in regulating cell type-specific nociceptive input in neuropathic pain with different etiologies.SIGNIFICANCE STATEMENT Spinal excitatory neurons relay input from nociceptors, whereas inhibitory neurons repress spinal nociceptive transmission. Chronic nerve pain is associated with aberrant NMDAR activity in the spinal dorsal horn. This study demonstrates, for the first time, that chemotherapy and traumatic nerve injury preferentially enhance the NMDAR activity at primary afferent-excitatory neuron synapses but have no effect on primary afferent input to spinal inhibitory neurons. NMDARs in primary sensory neurons are essential for chemotherapy-induced chronic pain, whereas nerve trauma causes pain hypersensitivity predominantly via postsynaptic NMDARs in spinal excitatory neurons. Thus, presynaptic and postsynaptic NMDARs at primary afferent-excitatory neuron synapses are differentially engaged in chemotherapy- and nerve injury-induced chronic pain and could be targeted respectively for treating these painful conditions.

Cross-talk between zinc and calcium regulates ion transport: A role for the zinc receptor, ZnR/GPR39.

Zinc is essential for many physiological functions, with a major role in digestive system, skin health, and learning and memory. On the cellular level, zinc is involved in cell proliferation and cell death. A selective zinc sensing receptor, ZnR/GPR39 is a Gq-coupled receptor that acts via the inositol trisphosphate pathway to release intracellular Ca2+. The ZnR/GPR39 serves as a mediator between extracellular changes in Zn2+ concentration and cellular Ca2+ signalling. This signalling pathway regulates ion transporters activity and thereby controls the formation of transepithelial gradients or neuronal membrane potential, which play a fundamental role in the physiological function of these tissues. This review focuses on the role of Ca2+ signalling, and specifically ZnR/GPR39, with respect to the regulation of the Na+/H+ exchanger, NHE1, and of the K+/Cl- cotransporters, KCC1-3, and also describes the physiological implications of this regulation.

GABAergic system and chloride cotransporters as potential therapeutic targets to mitigate cell death in ischemia.

Gamma aminobutyric acid (GABA) is a critical inhibitory neurotransmitter in the central nervous system that plays a vital role in modulating neuronal excitability. Dysregulation of GABAergic signaling, particularly involving the cotransporters NKCC1 and KCC2, has been implicated in various pathologies, including epilepsy, schizophrenia, autism spectrum disorder, Down syndrome, and ischemia. NKCC1 facilitates chloride influx, whereas KCC2 mediates chloride efflux via potassium gradient. Altered expression and function of these cotransporters have been associated with excitotoxicity, inflammation, and cellular death in ischemic events characterized by reduced cerebral blood flow, leading to compromised tissue metabolism and subsequent cell death. NKCC1 inhibition has emerged as a potential therapeutic approach to attenuate intracellular chloride accumulation and mitigate neuronal damage during ischemic events. Similarly, targeting KCC2, which regulates chloride efflux, holds promise for improving outcomes and reducing neuronal damage under ischemic conditions. This review emphasizes the critical roles of GABA, NKCC1, and KCC2 in ischemic pathologies and their potential as therapeutic targets. Inhibiting or modulating the activity of these cotransporters represents a promising strategy for reducing neuronal damage, preventing excitotoxicity, and improving neurological outcomes following ischemic events. Furthermore, exploring the interactions between natural compounds and NKCC1/KCC2 provides additional avenues for potential therapeutic interventions for ischemic injury.

Gene replacement therapies for inherited disorders of neurotransmission: Current progress in succinic semialdehyde dehydrogenase deficiency.

Neurodevelopment is a highly organized and complex process involving lasting and often irreversible changes in the central nervous system. Inherited disorders of neurotransmission (IDNT) are a group of genetic disorders where neurotransmission is primarily affected, resulting in abnormal brain development from early life, manifest as neurodevelopmental disorders and other chronic conditions. In principle, IDNT (particularly those of monogenic causes) are amenable to gene replacement therapy via precise genetic correction. However, practical challenges for gene replacement therapy remain major hurdles for its translation from bench to bedside. We discuss key considerations for the development of gene replacement therapies for IDNT. As an example, we describe our ongoing work on gene replacement therapy for succinic semialdehyde dehydrogenase deficiency, a GABA catabolic disorder.

Restoring neuronal chloride extrusion reverses cognitive decline linked to Alzheimer's disease mutations.

Disinhibition during early stages of Alzheimer's disease is postulated to cause network dysfunction and hyperexcitability leading to cognitive deficits. However, the underlying molecular mechanism remains unknown. Here we show that, in mouse lines carrying Alzheimer's disease-related mutations, a loss of neuronal membrane potassium-chloride cotransporter KCC2, responsible for maintaining the robustness of GABAA-mediated inhibition, occurs pre-symptomatically in the hippocampus and prefrontal cortex. KCC2 downregulation was inversely correlated with the age-dependent increase in amyloid-ß 42 (Aß42). Acute administration of Aß42 caused a downregulation of membrane KCC2. Loss of KCC2 resulted in impaired chloride homeostasis. Preventing the decrease in KCC2 using long term treatment with CLP290 protected against deterioration of learning and cortical hyperactivity. In addition, restoring KCC2, using short term CLP290 treatment, following the transporter reduction effectively reversed spatial memory deficits and social dysfunction, linking chloride dysregulation with Alzheimer's disease-related cognitive decline. These results reveal KCC2 hypofunction as a viable target for treatment of Alzheimer's disease-related cognitive decline; they confirm target engagement, where the therapeutic intervention takes place, and its effectiveness.

Neuronal K+-Cl- cotransporter KCC2 as a promising drug target for epilepsy treatment.

Epilepsy is a prevalent neurological disorder characterized by unprovoked seizures. γ-Aminobutyric acid (GABA) serves as the primary fast inhibitory neurotransmitter in the brain, and GABA binding to the GABAA receptor (GABAAR) regulates Cl- and bicarbonate (HCO3-) influx or efflux through the channel pore, leading to GABAergic inhibition or excitation, respectively. The neuron-specific K+-Cl- cotransporter 2 (KCC2) is essential for maintaining a low intracellular Cl- concentration, ensuring GABAAR-mediated inhibition. Impaired KCC2 function results in GABAergic excitation associated with epileptic activity. Loss-of-function mutations and altered expression of KCC2 lead to elevated [Cl-]i and compromised synaptic inhibition, contributing to epilepsy pathogenesis in human patients. KCC2 antagonism studies demonstrate the necessity of limiting neuronal hyperexcitability within the brain, as reduced KCC2 functioning leads to seizure activity. Strategies focusing on direct (enhancing KCC2 activation) and indirect KCC2 modulation (altering KCC2 phosphorylation and transcription) have proven effective in attenuating seizure severity and exhibiting anti-convulsant properties. These findings highlight KCC2 as a promising therapeutic target for treating epilepsy. Recent advances in understanding KCC2 regulatory mechanisms, particularly via signaling pathways such as WNK, PKC, BDNF, and its receptor TrkB, have led to the discovery of novel small molecules that modulate KCC2. Inhibiting WNK kinase or utilizing newly discovered KCC2 agonists has demonstrated KCC2 activation and seizure attenuation in animal models. This review discusses the role of KCC2 in epilepsy and evaluates its potential as a drug target for epilepsy treatment by exploring various strategies to regulate KCC2 activity.

Regulation of Neuronal Chloride Homeostasis by Pro- and Mature Brain-Derived Neurotrophic Factor (BDNF) via KCC2 Cation-Chloride Cotransporters in Rat Cortical Neurons.

The strength of inhibitory neurotransmission depends on intracellular neuronal chloride concentration, primarily regulated by the activity of cation-chloride cotransporters NKCC1 (Sodium-Potassium-Chloride Cotransporter 1) and KCC2 (Potassium-Chloride Cotransporter 2). Brain-derived neurotrophic factor (BDNF) influences the functioning of these co-transporters. BDNF is synthesized from precursor proteins (proBDNF), which undergo proteolytic cleavage to yield mature BDNF (mBDNF). While previous studies have indicated the involvement of BDNF signaling in the activity of KCC2, its specific mechanisms are unclear. We investigated the interplay between both forms of BDNF and chloride homeostasis in rat hippocampal neurons and in utero electroporated cortices of rat pups, spanning the behavioral, cellular, and molecular levels. We found that both pro- and mBDNF play a comparable role in immature neurons by inhibiting the capacity of neurons to extrude chloride. Additionally, proBDNF increases the endocytosis of KCC2 while maintaining a depolarizing shift of EGABA in maturing neurons. Behaviorally, proBDNF-electroporated rat pups in the somatosensory cortex exhibit sensory deficits, delayed huddling, and cliff avoidance. These findings emphasize the role of BDNF signaling in regulating chloride transport through the modulation of KCC2. In summary, this study provides valuable insights into the intricate interplay between BDNF, chloride homeostasis, and inhibitory synaptic transmission, shedding light on the underlying cellular mechanisms involved.

Molecular Signaling Effects behind the Spontaneous Soleus Muscle Activity Induced by 7-Day Rat Hindlimb Suspension.

The elimination of ground reaction force (support withdrawal) vastly affects slow postural muscles in terms of their regulation and structure. One of the effects of support withdrawal in this study was an immediate postural muscle inactivation, followed by the daily gradual development of spontaneous activity of the slow postural soleus muscle in response to rat hindlimb suspension to mimic space flight. The origin of this activity is somewhat akin to muscle spasticity after spinal cord injuries and is the result of KCC2 content decline in the spinal cord's motor neurons. However, the physiological consequences of unloading-induced spontaneous activity remain unexplored. We have conducted an experiment with the administration of a highly specific KCC2 activator during 7-day unloading. For this experiment, 32 male Wistar rats were divided into 4 groups: C+placebo, C+CLP-290 (100 mg/kg b w), 7HS+placebo, and 7HS+CLP-hindlimb-suspended group with CLP-290 administration (100 mg/kg b w). The soleus muscles of the animals were dissected and analyzed for several proteostasis- and metabolism-related parameters. CLP-290 administration to the unloaded animals led to the upregulation of AMPK downstream (p-ACC) and mTOR targets (p-p70S6k and p-4E-BP) and an enhanced PGC1alpha decrease vs. the 7HS group, but neither prevented nor enhanced atrophy of the soleus muscle or myofiber CSA.

High Doses of ANA12 Improve Phenobarbital Efficacy in a Model of Neonatal Post-Ischemic Seizures.

Phenobarbital (PB) remains the first-line medication for neonatal seizures. Yet, seizures in many newborns, particularly those associated with perinatal ischemia, are resistant to PB. Previous animal studies have shown that in postnatal day P7 mice pups with ischemic stroke induced by unilateral carotid ligation, the tyrosine receptor kinase B (TrkB) antagonist ANA12 (N-[2-[[(hexahydro-2-oxo-1H-azepin-3-yl)amino]carbonyl]phenyl]-benzo[b]thiophene-2-carboxamide, 5 mg/kg) improved the efficacy of PB in reducing seizure occurrence. To meet optimal standards of effectiveness, a wider range of ANA12 doses must be tested. Here, using the unilateral carotid ligation model, we tested the effectiveness of higher doses of ANA12 (10 and 20 mg/kg) on the ability of PB to reduce seizure burden, ameliorate cell death (assessed by Fluoro-Jade staining), and affect neurodevelopment (righting reflex, negative geotaxis test, open field test). We found that a single dose of ANA12 (10 or 20 mg/kg) given 1 h after unilateral carotid ligation in P7 pups reduced seizure burden and neocortical and striatal neuron death without impairing developmental reflexes. In conclusion, ANA12 at a range of doses (10-20 mg/kg) enhanced PB effectiveness for the treatment of perinatal ischemia-related seizures, suggesting that this agent might be a clinically safe and effective adjunctive agent for the treatment of pharmacoresistant neonatal seizures.

Gephyrin Interacts with the K-Cl Cotransporter KCC2 to Regulate Its Surface Expression and Function in Cortical Neurons.

The K+-Cl- cotransporter KCC2, encoded by the Slc12a5 gene, is a neuron-specific chloride extruder that tunes the strength and polarity of GABAA receptor-mediated transmission. In addition to its canonical ion transport function, KCC2 also regulates spinogenesis and excitatory synaptic function through interaction with a variety of molecular partners. KCC2 is enriched in the vicinity of both glutamatergic and GABAergic synapses, the activity of which in turn regulates its membrane stability and function. KCC2 interaction with the submembrane actin cytoskeleton via 4.1N is known to control its anchoring near glutamatergic synapses on dendritic spines. However, the molecular determinants of KCC2 clustering near GABAergic synapses remain unknown. Here, we used proteomics to identify novel KCC2 interacting proteins in the adult rat neocortex. We identified both known and novel candidate KCC2 partners, including some involved in neuronal development and synaptic transmission. These include gephyrin, the main scaffolding molecule at GABAergic synapses. Gephyrin interaction with endogenous KCC2 was confirmed by immunoprecipitation from rat neocortical extracts. We showed that gephyrin stabilizes plasmalemmal KCC2 and promotes its clustering in hippocampal neurons, mostly but not exclusively near GABAergic synapses, thereby controlling KCC2-mediated chloride extrusion. This study identifies gephyrin as a novel KCC2 anchoring molecule that regulates its membrane expression and function in cortical neurons.SIGNIFICANCE STATEMENT Fast synaptic inhibition in the brain is mediated by chloride-permeable GABAA receptors (GABAARs) and therefore relies on transmembrane chloride gradients. In neurons, these gradients are primarily maintained by the K/Cl cotransporter KCC2. Therefore, understanding the mechanisms controlling KCC2 expression and function is crucial to understand its physiological regulation and rescue its function in the pathology. KCC2 function depends on its membrane expression and clustering, but the underlying mechanisms remain unknown. We describe the interaction between KCC2 and gephyrin, the main scaffolding protein at inhibitory synapses. We show that gephyrin controls plasmalemmal KCC2 clustering and that loss of gephyrin compromises KCC2 function. Our data suggest functional units comprising GABAARs, gephyrin, and KCC2 act to regulate synaptic GABA signaling.

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.

SLC12A5 as a novel potential biomarker of glioblastoma multiforme.

BACKGROUND: Glioblastoma multiforme (GBM) is the most prevalent and malignant intracranial tumor with significant features of dismal prognosis and limited therapeutic solutions. Consequently, the present studies are committed to exploring potential biomarkers through bioinformatics analysis, which may serve as valuable prognostic predictors or novel therapeutic targets and provide new insights into the pathogenesis of GBM. METHODS: We filtered overlapping differentially expressed genes (DEGs) based on expression profilings from three GBM microarray datasets (GSE116520, GSE4290 and GSE68848) and combined RNA sequencing data from The Cancer Genome Atlas and the Genotype-Tissue Expression databases. Hub genes were prioritized from DEGs after performing protein-protein interaction (PPI) network analysis and weighted gene co-expression network analysis (WGCNA). This was followed by survival analysis to identify potential biomarkers among hub genes. Ultimately, the distributions of gene expressions, genetic alterations, upstream regulatory mechanisms and enrichments of gene functions of the identified biomarkers were analysed on public databases. QRT-PCR, immunohistochemical staining and western blotting was also used to confirm the gene expression patterns in GBM and normal brain tissues. CCK-8 assay clarified the effects of the genes on GBM cells. RESULTS: A total of 322 common DEGs were determined and nine genes were subsequently considered as hub genes by the combination of PPI network analysis and WGCNA. Only SLC12A5 had prognostic significance, which was deficient in GBM whereas especially enriched in normal neural tissues. SLC12A5 overexpression would inhibit cell proliferation of U251MG. Genetic alterations of SLC12A5 were rarely seen in GBM patients, and there was no apparent association existed between SLC12A5 expression and DNA methylation. SLC12A5 was prominently involved in ion transport, synapse and neurotransmitter. CONCLUSION: SLC12A5 shows promise to function as a novel effective biomarker for GBM and deserves further systematic research.

Inhibiting with-no-lysine kinases enhances K+/Cl- cotransporter 2 activity and limits status epilepticus.

First-in-line benzodiazepine treatment fails to terminate seizures in about 30% of epilepsy patients, highlighting a need for novel anti-seizure strategies. It is emerging that impaired K+/Cl- cotransporter 2 (KCC2) activity leads to deficits in GABAergic inhibition and increased seizure vulnerability in patients. In neurons, the with-no-lysine (WNK) kinase-STE20/SPS1-related proline/alanine-rich (SPAK) kinase signalling pathway inhibits KCC2 activity via T1007 phosphorylation. Here, we exploit the selective WNK kinase inhibitor WNK463 to test the effects of pharmacological WNK inhibition on KCC2 function, GABAergic inhibition, and epileptiform activity. Immunoprecipitation and western blotting analysis revealed that WNK463 reduces KCC2-T1007 phosphorylation in vitro and in vivo. Using patch-clamp recordings in primary rat neurons, we further observed that WNK463 hyperpolarized the Cl- reversal potential, and enhanced KCC2-mediated Cl- extrusion. In the 4-aminopyridine slice model of acute seizures, WNK463 administration reduced the frequency and number of seizure-like events. In vivo, C57BL/6 mice that received intrahippocampal WNK463 experienced delayed onset of kainic acid-induced status epilepticus, less epileptiform EEG activity, and did not develop pharmaco-resistance to diazepam. Our findings demonstrate that acute WNK463 treatment potentiates KCC2 activity in neurons and limits seizure burden in two well-established models of seizures and epilepsy. In summary, our work suggests that agents which act to increase KCC2 activity may be useful adjunct therapeutics to alleviate diazepam-resistant status epilepticus.

Time-dependent cortical plasticity during moderate-intensity continuous training versus high-intensity interval training in rats.

The temporal pattern of cortical plasticity induced by high-intensity interval training (HIIT) and moderate-intensity continuous training (MICT) is required to clarify their relative benefits to prevent neurological disorders. The purpose of this study is to define the time-dependent effects of work-matched HIIT and MICT on cortical plasticity, endurance, and sensorimotor performances over an 8-week training period in healthy rats. Adult healthy rats performed incremental exercise tests and sensorimotor tests before and at 2, 4, and 8 weeks of training. In parallel, cortical markers related to neurotrophic, angiogenic, and metabolic activities were assessed. Results indicate that HIIT induced an early and superior endurance improvement compared to MICT. We found significant enhancement of speed associated with lactate threshold (SLT) and maximal speed (Smax) in HIIT animals. MICT promoted an early increase in brain-derived neurotrophic factor and angiogenic/metabolic markers but showed less influence at 8 weeks. HIIT upregulated the insulin-like growth factor-1 (IGF-1) as well as neurotrophic, metabolic/angiogenic markers at 2 and 8 weeks and downregulated the neuronal K-Cl cotransporter KCC2 that regulates GABAA-mediated transmission. HIIT and MICT are effective in a time-dependent manner suggesting a complementary effect that might be useful in physical exercise guidelines for maintaining brain health.

Region-Specific KCC2 Rescue by rhIGF-1 and Oxytocin in a Mouse Model of Rett Syndrome.

Rett syndrome (RTT) is characterized by dysfunction in neuronal excitation/inhibition (E/I) balance, potentially impacting seizure susceptibility via deficits in K+/Cl- cotransporter 2 (KCC2) function. Mice lacking the Methyl-CpG binding protein 2 (MeCP2) recapitulate many symptoms of RTT, and recombinant human insulin-like growth factor-1 (rhIGF-1) restores KCC2 expression and E/I balance in MeCP2 KO mice. However, clinical trial outcomes of rhIGF-1 in RTT have been variable, and increasing its therapeutic efficacy is highly desirable. To this end, the neuropeptide oxytocin (OXT) is promising, as it also critically modulates KCC2 function during early postnatal development. We measured basal KCC2 expression levels in MeCP2 KO mice and identified 3 key frontal brain regions showing KCC2 alterations in young adult mice, but not in postnatal P10 animals. We hypothesized that deficits in an IGF-1/OXT signaling crosstalk modulating KCC2 may occur in RTT during postnatal development. Consistently, we detected alterations of IGF-1 receptor and OXT receptor levels in those brain areas. rhIGF-1 and OXT treatments in KO mice rescued KCC2 expression in a region-specific and complementary manner. These results suggest that region-selective combinatorial pharmacotherapeutic strategies could be most effective at normalizing E/I balance in key brain regions subtending the RTT pathophysiology.