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.

Polyphenol-rich Spondias mombin leaf extract abates cerebral ischemia/reperfusion-induced disturbed glutamate-ammonia metabolism and multiorgan toxicity in rats.

Introduction: This study investigated the protective properties of Spondias mombin leaf extract (SML), in cerebral ischemia/reperfusion (I/R) mediated toxicity in the brain, liver, and kidney of male Wistar rats. Materials and methods: Animals were subjected to 30 min of bilateral common carotid artery occlusion followed by 24 h of reperfusion (BCCAO/R). The animals were divided into sham, I/R, and I/R treated with SML (25, 50 and 100 mg/kg) or quercetin (20 mg/kg) groups. Animals were sacrificed after 24 h of reperfusion and markers of organ toxicity (urea creatinine, glutamine synthetase (GS), glutaminase (GA), aspartate aminotransferase (AST), alanine aminotransferase (ALT), acetylcholinesterase (AChE)) were measured in the brain regions (cortex, striatum, and hippocampus), liver, and kidney. Results and discussion: BCCAO/R significantly (p < 0.0001) inhibited the glutamate-glutamine cycle and mediated toxicity in the cerebral cortex, striatum, hippocampus, liver, and kidney of rats. Post-treatment with SML significantly (p < 0.0001) reversed glutamate-glutamine cycle inhibition and ameliorated cerebrohepatorenal toxicity in ischemic rats. Conclusion: Cerebral I/R significantly mediated cerebral, hepatic, and renal toxicity through the inhibition of glutamate-ammonia detoxification in rats, and SML protected against this post-ischemic glutamate-ammonia mediated multiorgan toxicity.

The role of SLC12A family of cation-chloride cotransporters and drug discovery methodologies.

The solute carrier family 12 (SLC12) of cation-chloride cotransporters (CCCs) comprises potassium chloride cotransporters (KCCs, e.g. KCC1, KCC2, KCC3, and KCC4)-mediated Cl- extrusion, and sodium potassium chloride cotransporters (N[K]CCs, NKCC1, NKCC2, and NCC)-mediated Cl- loading. The CCCs play vital roles in cell volume regulation and ion homeostasis. Gain-of-function or loss-of-function of these ion transporters can cause diseases in many tissues. In recent years, there have been considerable advances in our understanding of CCCs' control mechanisms in cell volume regulations, with many techniques developed in studying the functions and activities of CCCs. Classic approaches to directly measure CCC activity involve assays that measure the transport of potassium substitutes through the CCCs. These techniques include the ammonium pulse technique, radioactive or nonradioactive rubidium ion uptake-assay, and thallium ion-uptake assay. CCCs' activity can also be indirectly observed by measuring γ-aminobutyric acid (GABA) activity with patch-clamp electrophysiology and intracellular chloride concentration with sensitive microelectrodes, radiotracer 36Cl-, and fluorescent dyes. Other techniques include directly looking at kinase regulatory sites phosphorylation, flame photometry, 22Na+ uptake assay, structural biology, molecular modeling, and high-throughput drug screening. This review summarizes the role of CCCs in genetic disorders and cell volume regulation, current methods applied in studying CCCs biology, and compounds developed that directly or indirectly target the CCCs for disease treatments.

Study of the Functions and Activities of Neuronal K-Cl Co-Transporter KCC2 Using Western Blotting.

Potassium chloride cotransporters 2 (KCC2) is a member of the solute carrier family 12 (SLC12) of cation-chloride-cotransporters (CCCs), found exclusively in the neuron and is essential for the proper functioning of Cl- homeostasis and consequently functional GABAergic inhibition. Failure in proper regulation of KCC2 is deleterious and has been associated with the prevalence of several neurological diseases, including epilepsy. There has been considerable progress with regard to understanding the mechanisms involved in the regulation of KCC2, accredited to the development of techniques that enable researchers to study its functions and activities; either via direct (assessing kinase regulatory sites phosphorylation) or indirect (observing and monitoring GABA activity) investigations. Here, the protocol highlights how to investigate KCC2 phosphorylation at kinase regulatory sites - Thr906 and Thr1007- using western blotting technique. There are other classic methods used to directly measure KCC2 activity, such as rubidium ion and thallium ion uptake assay. Further techniques such as patch-clamp-electrophysiology are used to measure GABA activity; hence, indirectly reflecting activated and/or inactivated KCC2 as informed by the assessment of intracellular chloride ion homeostasis. A few of these additional techniques will be briefly discussed in this manuscript.

Neuroprotective effects of catechin and quercetin in experimental Parkinsonism through modulation of dopamine metabolism and expression of IL-1ß, TNF-α, NF-κB, IκKB, and p53 genes in male Wistar rats.

The neurobehavioral, brain redox-stabilizing and neurochemical modulatory properties of catechin and quercetin in rotenone-induced Parkinsonism, and the involvement of NF-κB-mediated inflammation, were investigated. Male Wistar rats subcutaneously administered with multiple doses of 1.5 mg/kg rotenone were post-treated with 5-20 mg/kg catechin or quercetin. This was followed by neurobehavioral evaluation, biochemical estimations, and assessment of neurotransmitter metabolism in the striatum. Expression of genes involved in the canonical pathway for the activation of NF-κB mediated inflammation (IL-1ß, TNF-α, NF-κB, and IκKB) and the pro-apoptotic gene, p53, in the striatum was determined by RT-qPCR. Catechin and quercetin mitigated neurobehavioral deficits caused by rotenone. Both flavonoids attenuated striatal redox stress and neurochemical dysfunction, optimized disturbed dopamine metabolism, and improved depletion of neuron density caused by rotenone toxicity. While administration of catechin produced a more pronounced attenuating effect on IL-1ß, TNF-α, and p53 genes, the attenuating effect of quercetin (20 mg/kg) was more pronounced on NF-κB and IκKB gene expressions when compared to the group administered with rotenone only. Comparatively, quercetin demonstrated superior protection against rotenone neurotoxicity. It is concluded that catechin and quercetin have potential relevance in Parkinson's disease therapy through amelioration of redox stress, optimization of dopamine metabolism, and modulation of anti-inflammatory and anti-apoptotic pathways.

Role of the cation-chloride-cotransporters in the circadian system.

The circadian system plays an immense role in controlling physiological processes in our body. The suprachiasmatic nucleus (SCN) supervises this system, regulating and harmonising the circadian rhythms in our body. Most neurons present in the SCN are GABAergic neurons. Although GABA is considered the main inhibitory neurotransmitter of the CNS, recent studies have shown that excitatory responses were recorded in this area. These responses are enabled by an increase in intracellular chloride ions [Cl-]i levels. The chloride (Cl-) levels in GABAergic neurons are controlled by two solute carrier 12 (SLC12) cation-chloride-cotransporters (CCCs): Na+/K+/Cl- co-transporter (NKCC1) and K+/Cl- co-transporter (KCC2), that respectively cause an influx and efflux of Cl-. Recent works have found altered expression and/or activity of either of these co-transporters in SCN neurons and have been associated with circadian rhythms. In this review, we summarize and discuss the role of CCCs in circadian rhythms, and highlight these recent advances which attest to CCC's growing potential as strong research and therapeutic targets.

Targeting the WNK-SPAK/OSR1 Pathway and Cation-Chloride Cotransporters for the Therapy of Stroke.

Stroke is one of the major culprits responsible for morbidity and mortality worldwide, and the currently available pharmacological strategies to combat this global disease are scanty. Cation-chloride cotransporters (CCCs) are expressed in several tissues (including neurons) and extensively contribute to the maintenance of numerous physiological functions including chloride homeostasis. Previous studies have implicated two CCCs, the Na+-K+-Cl- and K+-Cl- cotransporters (NKCCs and KCCs) in stroke episodes along with their upstream regulators, the with-no-lysine kinase (WNKs) family and STE20/SPS1-related proline/alanine rich kinase (SPAK) or oxidative stress response kinase (OSR1) via a signaling pathway. As the WNK-SPAK/OSR1 pathway reciprocally regulates NKCC and KCC, a growing body of evidence implicates over-activation and altered expression of NKCC1 in stroke pathology whilst stimulation of KCC3 during and even after a stroke event is neuroprotective. Both inhibition of NKCC1 and activation of KCC3 exert neuroprotection through reduction in intracellular chloride levels and thus could be a novel therapeutic strategy. Hence, this review summarizes the current understanding of functional regulations of the CCCs implicated in stroke with particular focus on NKCC1, KCC3, and WNK-SPAK/OSR1 signaling and discusses the current and potential pharmacological treatments for stroke.

Toxicological and pharmacological assessment of a multiherbal phytopharmaceutical on Triton X-1339-induced hyperlipidemia and allied biochemical dysfunctions.

This study investigated the safety and therapeutic effect of a multiherbal tea (MHT) on Triton X-1339-induced hyperlipidemia and associated biochemical and tissue dysfunctions. An infusion of the MHT was assessed for phytoconstituents, proximate and mineral composition, and antioxidant activity. Wistar rats administered 200 mg/kg Triton X-1399 were post-treated with MHT for 14 days followed by biochemical estimations in serum, heart, liver, and kidney of animals. Hematological and histopathological evaluations of the blood, and liver, respectively, were also performed. Different phytochemicals were detected in MHT, toxic metals were absent and antioxidant activity was appreciable. Disturbances in glucose level and redox homeostasis, alterations in liver, kidney, and heart function markers, and imbalances in hematological parameters precipitated by triton toxicity were mitigated by posttreatment with MHT. Multiherbal tea also ameliorated triton-induced hepatic histoarchitectural abnormalities. These results suggest that MHT is apparently an effective antilipemic tea with minimal or no side effects. PRACTICAL APPLICATIONS: Hyperlipidemia is one of the core risk factors for arteriosclerosis and a major contributor to other adverse health conditions. The prevalence of hyperlipidemia has increased drastically in the last few decades. Plant and plant products have been extensively used in the management of dyslipidemia and many plant-based antilipemic products with poorly defined toxicity and pharmacological profiles abound in the market. The results of this study demonstrated the protective effects of a MHT against triton-induced hyperlipidemia, atherogenic tendency, and dysfunction of key organs in rats and lent credence to its therapeutic relevance in the management of hyperlipidemia and related diseases.

Molecular docking studies, molecular dynamics and ADME/tox reveal therapeutic potentials of STOCK1N-69160 against papain-like protease of SARS-CoV-2.

SARS-CoV-2 is a new strain of Coronavirus that caused the pneumonia outbreak in Wuhan, China and has spread to over 200 countries of the world. It has received worldwide attention due to its virulence and high rate of infection. So far, several drugs have experimented against SARS-CoV-2, but the failure of these drugs to specifically interact with the viral protease necessitates urgent measure to boost up researches for the development of effective therapeutics against SARS-CoV-2. Papain-like protease (PLpro) of the viral polyproteins is essential for maturation and infectivity of the virus, making it one of the prime targets explored for SARS-CoV-2 drug design. This study was conducted to evaluate the efficacy of ~ 50,000 natural compounds retrieved from IBS database against COVID-19 PLpro using computer-aided drug design. Based on molecular dock scores, molecular interaction with active catalytic residues and molecular dynamics (MD) simulations studies, STOCK1N-69160 [(S)-2-((R)-4-((R)-2-amino-3-methylbutanamido)-3-(4-chlorophenyl) butanamido) propanoic acid hydrochloride] has been proposed as a novel inhibitor against COVID-19 PLpro. It demonstrated favourable docking score, the free energy of binding, interacted with key amino acid residues necessary for PLpro inhibition and also showed significant moderation for parameters investigated for ADME/tox (Adsorption, distribution, metabolism, excretion and toxicological) properties. The edge of the compound was further established by its stability in MD simulation conducted for 30 ns employing GROMACS software. We propose that STOCK1N-69160 is worth further investigation for preventing SARS-CoV-2.

The Therapeutic Potential of Neuronal K-Cl Co-Transporter KCC2 in Huntington's Disease and Its Comorbidities.

Intracellular chloride levels in the brain are regulated primarily through the opposing effects of two cation-chloride co-transporters (CCCs), namely K+-Cl- co-transporter-2 (KCC2) and Na+-K+-Cl- co-transporter-1 (NKCC1). These CCCs are differentially expressed throughout the course of development, thereby determining the excitatory-to-inhibitory γ-aminobutyric acid (GABA) switch. GABAergic excitation (depolarisation) is important in controlling the healthy development of the nervous system; as the brain matures, GABAergic inhibition (hyperpolarisation) prevails. This developmental switch in excitability is important, as uncontrolled regulation of neuronal excitability can have implications for health. Huntington's disease (HD) is an example of a genetic disorder whereby the expression levels of KCC2 are abnormal due to mutant protein interactions. Although HD is primarily considered a motor disease, many other clinical manifestations exist; these often present in advance of any movement abnormalities. Cognitive change, in addition to sleep disorders, is prevalent in the HD population; the effect of uncontrolled KCC2 function on cognition and sleep has also been explored. Several mechanisms by which KCC2 expression is reduced have been proposed recently, thereby suggesting extensive investigation of KCC2 as a possible therapeutic target for the development of pharmacological compounds that can effectively treat HD co-morbidities. Hence, this review summarizes the role of KCC2 in the healthy and HD brain, and highlights recent advances that attest to KCC2 as a strong research and therapeutic target candidate.

Catechin, quercetin and taxifolin improve redox and biochemical imbalances in rotenone-induced hepatocellular dysfunction: Relevance for therapy in pesticide-induced liver toxicity?

Hepatotoxicity occurs as a result of adverse effects of some xenobiotics on the liver, which is often the target tissue of toxicity for environmental chemicals. Rotenone, used as a natural pesticide, is an environmental poison reported to cause organ toxicity. This study investigated the protective effect of three flavonoids, catechin, quercetin and taxifolin (2,3-Dihydroquercetin) in rotenone-induced hepatotoxicity. Male Wistar rats were administered rotenone for 10 days followed by post treatment with catechin (5, 10 and 20 mg/kg), quercetin (5, 10 and 20 mg/kg) or taxifolin (0.25, 0.5 and 1 mg/kg), respectively, for 3 days. Bioindices of oxidative stress and hepatocellular injury were measured in serum and tissue homogenate of animals. Rotenone intoxication produced liver damage in rats as reflected in alterations to activities/levels of enzymic and non-enzymic oxidative stress markers and enzymes linked with inflammation, as well as the transaminases, gamma glutamyl transpeptidase, bilirubin, and lactate dehydrogenase. Catechin, quercetin and taxifolin post treatment significantly attenuated these (p < 0.0001) rotenone-induced imbalances. Comparatively, quercetin displayed the best apparent ameliorative activity. It clearly showed superior activity to catechin. However, taxifolin appeared to show comparable activity to quercetin and better activity than catechin in some of the assays despite being administered at considerably lower doses. The results provide insight on the relative efficacy and structure-activity relationships of the selected flavonoids in ameliorating liver damage and also indicate that additional structural and metabolic factors may be involved in the structure-activity relationships of flavonoids.