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.

Hypernegative GABAA Reversal Potential in Pyramidal Cells Contributes to Medial Prefrontal Cortex Deactivation in a Mouse Model of Neuropathic Pain.

Deactivation of the medial prefrontal cortex (mPFC) has been broadly reported in both neuropathic pain models and human chronic pain patients. Several cellular mechanisms may contribute to the inhibition of mPFC activity, including enhanced GABAergic inhibition. The functional effect of GABAA(γ-aminobutyric acid type A)-receptor activation depends on the concentration of intracellular chloride in the postsynaptic neuron, which is mainly regulated by the activity of Na-K-2Cl cotransporter isoform 1 (NKCC1) and K-Cl cotransporter isoform 2 (KCC2), 2 potassium-chloride cotransporters that import and extrude chloride, respectively. Recent work has shown that the NKCC1-KCC2 ratio is affected in numerous pathological conditions, and we hypothesized that it may contribute to the alteration of mPFC function in neuropathic pain. We used quantitative in situ hybridization to assess the level of expression of NKCC1 and KCC2 in the mPFC of a mouse model of neuropathic pain (spared nerve injury), and we found that KCC2 transcript is increased in the mPFC of spared nerve injury mice while NKCC1 is not affected. Perforated patch recordings further showed that this results in the hypernegative reversal potential of the GABAA current in pyramidal neurons of the mPFC. Computational simulations suggested that this change in GABAA reversal potential is sufficient to significantly reduce the overall activity of the cortical network. Thus, our results identify a novel pathological modulation of GABAA function and a new mechanism by which mPFC function is inhibited in neuropathic pain. Our data also help explain previous findings showing that activation of mPFC interneurons has proalgesic effect in neuropathic, but not in control conditions. PERSPECTIVE: Chronic pain is associated with the presence of depolarizing GABAA current in the spinal cord, suggesting that pharmacological NKCC1 antagonism has analgesic effects. However, our results show that in neuropathic pain, GABAA current is actually hyperinhibitory in the mPFC, where it contributes to the mPFC functional deactivation. This suggests caution in the use of NKCC1 antagonism to treat pain.

The epithelial polarity axis controls the resting membrane potential and Cl- co-transport in breast glandular structures.

The membrane potential (MP) controls cell homeostasis by directing molecule transport and gene expression. How the MP is set upon epithelial differentiation is unknown. Given that tissue architecture also controls homeostasis, we investigated the relationship between basoapical polarity and resting MP in three-dimensional culture of the HMT-3522 breast cancer progression. A microelectrode technique to measure MP and input resistance reveals that the MP is raised by gap junction intercellular communication (GJIC), which directs tight-junction mediated apical polarity, and is decreased by the Na+/K+/2Cl- (NKCC, encoded by SLC12A1 and SLC12A2) co-transporter, active in multicellular structures displaying basal polarity. In the tumor counterpart, the MP is reduced. Cancer cells display diminished GJIC and do not respond to furosemide, implying loss of NKCC activity. Induced differentiation of cancer cells into basally polarized multicellular structures restores widespread GJIC and NKCC responses, but these structures display the lowest MP. The absence of apical polarity, necessary for cancer onset, in the non-neoplastic epithelium is also associated with the lowest MP under active Cl- transport. We propose that the loss of apical polarity in the breast epithelium destabilizes cellular homeostasis in part by lowering the MP.

DNA demethylation in the hypothalamus promotes transcription of Agtr1a and Slc12a2 and hypertension development.

Increased expression of angiotensin II AT1A receptor (encoded by Agtr1a) and Na+-K+-Cl- cotransporter-1 (NKCC1, encoded by Slc12a2) in the hypothalamic paraventricular nucleus (PVN) contributes to hypertension development. However, little is known about their transcriptional control in the PVN in hypertension. DNA methylation is a critical epigenetic mechanism that regulates gene expression. Here, we determined whether transcriptional activation of Agtr1a and Slc12a2 results from altered DNA methylation in spontaneously hypertensive rats (SHR). Methylated DNA immunoprecipitation and bisulfite sequencing-PCR showed that CpG methylation at Agtr1a and Slc12a2 promoters in the PVN was progressively diminished in SHR compared with normotensive Wistar-Kyoto rats (WKY). Chromatin immunoprecipitation-quantitative PCR revealed that enrichment of DNA methyltransferases (DNMT1 and DNMT3A) and methyl-CpG binding protein 2, a DNA methylation reader protein, at Agtr1a and Slc12a2 promoters in the PVN was profoundly reduced in SHR compared with WKY. By contrast, the abundance of ten-eleven translocation enzymes (TET1-3) at Agtr1a and Slc12a2 promoters in the PVN was much greater in SHR than in WKY. Furthermore, microinjecting of RG108, a selective DNMT inhibitor, into the PVN of WKY increased arterial blood pressure and correspondingly potentiated Agtr1a and Slc12a2 mRNA levels in the PVN. Conversely, microinjection of C35, a specific TET inhibitor, into the PVN of SHR markedly reduced arterial blood pressure, accompanied by a decrease in Agtr1a and Slc12a2 mRNA levels in the PVN. Collectively, our findings suggest that DNA hypomethylation resulting from the DNMT/TET switch at gene promoters in the PVN promotes transcription of Agtr1a and Slc12a2 and hypertension development.

Cation Chloride Cotransporter NKCC1 Operates through a Rocking-Bundle Mechanism.

The sodium, potassium, and chloride cotransporter 1 (NKCC1) plays a key role in tightly regulating ion shuttling across cell membranes. Lately, its aberrant expression and function have been linked to numerous neurological disorders and cancers, making it a novel and highly promising pharmacological target for therapeutic interventions. A better understanding of how NKCC1 dynamically operates would therefore have broad implications for ongoing efforts toward its exploitation as a therapeutic target through its modulation. Based on recent structural data on NKCC1, we reveal conformational motions that are key to its function. Using extensive deep-learning-guided atomistic simulations of NKCC1 models embedded into the membrane, we captured complex dynamical transitions between alternate open conformations of the inner and outer vestibules of the cotransporter and demonstrated that NKCC1 has water-permeable states. We found that these previously undefined conformational transitions occur via a rocking-bundle mechanism characterized by the cooperative angular motion of transmembrane helices (TM) 4 and 9, with the contribution of the extracellular tip of TM 10. We found these motions to be critical in modulating ion transportation and in regulating NKCC1's water transporting capabilities. Specifically, we identified interhelical dynamical contacts between TM 10 and TM 6, which we functionally validated through mutagenesis experiments of 4 new targeted NKCC1 mutants. We conclude showing that those 4 residues are highly conserved in most Na+-dependent cation chloride cotransporters (CCCs), which highlights their critical mechanistic implications, opening the way to new strategies for NKCC1's function modulation and thus to potential drug action on selected CCCs.

Cancer Stem Cells of Esophageal Adenocarcinoma are Suppressed by Inhibitors of TRPV2 and SLC12A2.

BACKGROUND: The potential of membrane transporters activated in cancer stem cells (CSCs) as new therapeutic targets for cancer is attracting increasing interest. Therefore, the present study examined the expression profiles of ion transport-related molecules in the CSCs of esophageal adenocarcinoma (EAC). METHODS: Cells that highly expressed aldehyde dehydrogenase 1 family member A1 (ALDH1A1) were separated from OE33 cells, a human Barrett's EAC cell line, by fluorescence-activated cell sorting. CSCs were identified based on the formation of tumorspheres. Gene expression profiles in CSCs were examined by a microarray analysis. RESULTS: Among OE33 cells, ALDH1A1 messenger RNA levels were higher in CSCs than in non-CSCs. Furthermore, CSCs exhibited resistance to cisplatin and had the capacity to redifferentiate. The results of the microarray analysis of CSCs showed the up-regulated expression of several genes related to ion channels/transporters, such as transient receptor potential vanilloid 2 (TRPV2) and solute carrier family 12 member 2 (SLC12A2). The cytotoxicities of the TRPV2 inhibitor tranilast and the SLC12A2 inhibitor furosemide were higher at lower concentrations in CSCs than in non-CSCs, and both markedly reduced the number of tumorspheres. The cell population among OE33 cells that highly expressed ALDH1A1 also was significantly decreased by these inhibitors. CONCLUSIONS: Based on the present results, TRPV2 and SLC12A2 are involved in the maintenance of CSCs, and their specific inhibitors, tranilast and furosemide, respectively, have potential as targeted therapeutic agents for EAC.

Treating Hyperexcitability in Human Cerebral Organoids Resulting from Oxygen-Glucose Deprivation.

Human cerebral organoids resemble the 3D complexity of the human brain and have the potential to augment current drug development pipelines for neurological disease. Epilepsy is a complex neurological condition characterized by recurrent seizures. A third of people with epilepsy do not respond to currently available pharmaceutical drugs, and there is not one drug that treats all subtypes; thus, better models of epilepsy are needed for drug development. Cerebral organoids may be used to address this unmet need. In the present work, human cerebral organoids are used along with electrophysiological methods to explore oxygen-glucose deprivation as a hyperexcitability agent. This activity is investigated in its response to current antiseizure drugs. Furthermore, the mechanism of action of the drug candidates is probed with qPCR and immunofluorescence. The findings demonstrate OGD-induced hyperexcitable changes in the cerebral organoid tissue, which is treated with cannabidiol and bumetanide. There is evidence for NKCC1 and KCC2 gene expression, as well as other genes and proteins involved in the complex development of GABAergic signaling. This study supports the use of organoids as a platform for modelling cerebral cortical hyperexcitability that could be extended to modelling epilepsy and used for drug discovery.

Age-appropriate potassium clearance from perinatal cerebrospinal fluid depends on choroid plexus NKCC1.

Regulation of the volume and electrolyte composition of the cerebrospinal fluid (CSF) is vital for brain development and function. The Na-K-Cl co-transporter NKCC1 in the choroid plexus (ChP) plays key roles in regulating CSF volume by co-transporting ions and mediating same-direction water movements. Our previous study showed ChP NKCC1 is highly phosphorylated in neonatal mice as the CSF K+ level drastically decreases and that overexpression of NKCC1 in the ChP accelerates CSF K+ clearance and reduces ventricle size [1]. These data suggest that NKCC1 mediates CSF K+ clearance following birth in mice. In this current study, we used CRISPR technology to create a conditional NKCC1 knockout mouse line and evaluated CSF K+ by Inductively Coupled Plasma Optical Emission spectroscopy (ICP-OES). We demonstrated ChP-specific reduction of total and phosphorylated NKCC1 in neonatal mice following embryonic intraventricular delivery of Cre recombinase using AAV2/5. ChP-NKCC1 knockdown was accompanied by a delayed perinatal clearance of CSF K+. No gross morphological disruptions were observed in the cerebral cortex. We extended our previous results by showing embryonic and perinatal rats shared key characteristics with mice, including decreased ChP NKCC1 expression level, increased ChP NKCC1 phosphorylation state, and increased CSF K+ levels compared to adult. Collectively, these follow up data support ChP NKCC1's role in age-appropriate CSF K+ clearance during neonatal development.

Bumetanide Attenuates Cognitive Deficits and Brain Damage in Rats Subjected to Hypoxia-Ischemia at Two Time Points of the Early Postnatal Period.

Neonatal hypoxia-ischemia (HI) is one of the main causes of tissue damage, cell death, and imbalance between neuronal excitation and inhibition and synaptic loss in newborns. GABA, the major inhibitory neurotransmitter of the central nervous system (CNS) in adults, is excitatory at the onset of neurodevelopment and its action depends on the chloride (Cl-) cotransporters NKCC1 (imports Cl-) and KCC2 (exports Cl-) expression. Under basal conditions, the NKCC1/KCC2 ratio decreases over neurodevelopment. Thus, changes in this ratio caused by HI may be related to neurological disorders. The present study evaluated the effects of bumetanide (NKCC cotransporters inhibitor) on HI impairments in two neurodevelopmental periods. Male Wistar rat pups, 3 (PND3) and 11 (PND11) days old, were submitted to the Rice-Vannucci model. Animals were divided into 3 groups: SHAM, HI-SAL, and HI-BUM, considering each age. Bumetanide was administered intraperitoneally at 1, 24, 48, and 72 h after HI. NKCC1, KCC2, PSD-95, and synaptophysin proteins were analyzed after the last injection by western blot. Negative geotaxis, righting reflex, open field, object recognition test, and Morris water maze task were performed to assess neurological reflexes, locomotion, and memory function. Tissue atrophy and cell death were evaluated by histology. Bumetanide prevented neurodevelopmental delay, hyperactivity, and declarative and spatial memory deficits. Furthermore, bumetanide reversed HI-induced brain tissue damage, reduced neuronal death and controlled GABAergic tone, maintained the NKCC1/KCC2 ratio, and synaptogenesis close to normality. Thereby, bumetanide appears to play an important therapeutic role in the CNS, protecting the animals against HI damage and improving functional performance.

Spatiotemporal expression patterns of genes coding for plasmalemmal chloride transporters and channels in neurological diseases.

Neuronal voltage changes which are dependent on chloride transporters and channels are involved in forming neural functions during early development and maintaining their stability until adulthood. The intracellular chloride concentration maintains a steady state, which is delicately regulated by various genes coding for chloride transporters and channels (GClTC) on the plasmalemma; however, the synergistic effect of these genes in central nervous system disorders remains unclear. In this study, we first defined 10 gene clusters with similar temporal expression patterns, and identified 41 GClTC related to brain developmental process. Then, we found 4 clusters containing 22 GClTC were enriched for the neuronal functions. The GClTC from different clusters presented distinct cell type preferences and anatomical heterogeneity. We also observed strong correlations between clustered genes and diseases, most of which were nervous system disorders. Finally, we found that one of the most well-known GClTC, SLC12A2, had a more profound effect on glial cell-related diseases than on neuron-related diseases, which was in accordance with our observation that SLC12A2 was mainly expressed in oligodendrocytes during brain development. Our findings provide a more comprehensive understanding of the temporal and spatial expression characteristics of GClTC, which can help us understand the complex roles of GClTC in the development of the healthy human brain and the etiology of brain disorders.

Cation-Chloride Cotransporters KCC2 and NKCC1 as Therapeutic Targets in Neurological and Neuropsychiatric Disorders.

Neurological diseases including Alzheimer's, Huntington's disease, Parkinson's disease, Down syndrome and epilepsy, and neuropsychiatric disorders such as schizophrenia, are conditions that affect not only individuals but societies on a global scale. Current therapies offer a means for small symptomatic relief, but recently there has been increasing demand for therapeutic alternatives. The γ-aminobutyric acid (GABA)ergic signaling system has been investigated for developing new therapies as it has been noted that any dysfunction or changes to this system can contribute to disease progression. Expression of the K-Cl-2 (KCC2) and N-K-C1-1 (NKCC1) cation-chloride cotransporters (CCCs) has recently been linked to the disruption of GABAergic activity by affecting the polarity of GABAA receptor signaling. KCC2 and NKCC1 play a part in multiple neurological and neuropsychiatric disorders, making them a target of interest for potential therapies. This review explores current research suggesting the pathophysiological role and therapeutic importance of KCC2 and NKCC1 in neuropsychiatric and neurological disorders.

Lateral Diffusion of NKCC1 Contributes to Chloride Homeostasis in Neurons and Is Rapidly Regulated by the WNK Signaling Pathway.

An upregulation of the Na+-K+-2Cl- cotransporter NKCC1, the main chloride importer in mature neurons, can lead to depolarizing/excitatory responses mediated by GABA type A receptors (GABAARs) and, thus, to hyperactivity. Understanding the regulatory mechanisms of NKCC1 would help prevent intra-neuronal chloride accumulation that occurs in pathologies with defective inhibition. The cell mechanisms regulating NKCC1 are poorly understood. Here, we report in mature hippocampal neurons that GABAergic activity controls the membrane diffusion and clustering of NKCC1 via the chloride-sensitive WNK lysine deficient protein kinase 1 (WNK1) and the downstream Ste20 Pro-line Asparagine Rich Kinase (SPAK) kinase that directly phosphorylates NKCC1 on key threonine residues. At rest, this signaling pathway has little effect on intracellular Cl- concentration, but it participates in the elevation of intraneuronal Cl- concentration in hyperactivity conditions associated with an up-regulation of NKCC1. The fact that the main chloride exporter, the K+-Cl- cotransporter KCC2, is also regulated in mature neurons by the WNK1 pathway indicates that this pathway will be a target of choice in the pathology.

Role of NKCC1 and KCC2 during hypoxia-induced neuronal swelling in the neonatal neocortex.

Neonatal hypoxia causes cytotoxic neuronal swelling by the entry of ions and water. Multiple water pathways have been implicated in neurons because these cells lack water channels, and their membrane has a low water permeability. NKCC1 and KCC2 are cation-chloride cotransporters (CCCs) involved in water movement in various cell types. However, the role of CCCs in water movement in neonatal neurons during hypoxia is unknown. We studied the effects of modulating CCCs pharmacologically on neuronal swelling in the neocortex (layer IV/V) of neonatal mice (post-natal day 8-13) during prolonged and brief hypoxia. We used acute brain slices from Clomeleon mice which express a ratiometric fluorophore sensitive to Cl- and exposed them to oxygen-glucose deprivation (OGD) while imaging neuronal size and [Cl-]i by multiphoton microscopy. Neurons were identified using a convolutional neural network algorithm, and changes in the somatic area and [Cl-]i were evaluated using a linear mixed model for repeated measures. We found that (1) neuronal swelling and Cl- accumulation began after OGD, worsened during 20 min of OGD, or returned to baseline during reoxygenation if the exposure to OGD was brief (10 min). (2) Neuronal swelling did not occur when the extracellular Cl- concentration was low. (3) Enhancing KCC2 activity did not alter OGD-induced neuronal swelling but prevented Cl- accumulation; (4) blocking KCC2 led to an increase in Cl- accumulation during prolonged OGD and aggravated neuronal swelling during reoxygenation; (5) blocking NKCC1 reduced neuronal swelling during early but not prolonged OGD and aggravated Cl- accumulation during prolonged OGD; and (6) treatment with the "broad" CCC blocker furosemide reduced both swelling and Cl- accumulation during prolonged and brief OGD, whereas simultaneous NKCC1 and KCC2 inhibition using specific pharmacological blockers aggravated neuronal swelling during prolonged OGD. We conclude that CCCs, and other non-CCCs, contribute to water movement in neocortical neurons during OGD in the neonatal period.

Expression patterns of NKCC1 in neurons and non-neuronal cells during cortico-hippocampal development.

The Na-K-2Cl cotransporter NKCC1 is widely expressed in cells within and outside the brain. However, our understanding of its roles in brain functions throughout development, as well as in neuropsychiatric and neurological disorders, has been severely hindered by the lack of reliable data on its developmental and (sub)cellular expression patterns. We provide here the first properly controlled analysis of NKCC1 protein expression in various cell types of the mouse brain using custom-made antibodies and an NKCC1 knock-out validated immunohistochemical procedure, with parallel data based on advanced mRNA approaches. NKCC1 protein and mRNA are expressed at remarkably high levels in oligodendrocytes. In immature neurons, NKCC1 protein was located in the somata, whereas in adult neurons, only NKCC1 mRNA could be clearly detected. NKCC1 immunoreactivity is also seen in microglia, astrocytes, developing pericytes, and in progenitor cells of the dentate gyrus. Finally, a differential expression of NKCC1 splice variants was observed, with NKCC1a predominating in non-neuronal cells and NKCC1b in neurons. Taken together, our data provide a cellular basis for understanding NKCC1 functions in the brain and enable the identification of major limitations and promises in the development of neuron-targeting NKCC1-blockers.

The loop diuretic torasemide but not azosemide potentiates the anti-seizure and disease-modifying effects of midazolam in a rat model of birth asphyxia.

Loop diuretics such as furosemide and bumetanide, which act by inhibiting the Na-K-2Cl cotransporter NKCC2 at the thick ascending limb of the loop of Henle, have been shown to exert anti-seizure effects. However, the exact mechanism of this effect is not known. For bumetanide, it has been suggested that inhibition of the NKCC isoform NKCC1 in the membrane of brain neurons may be involved; however, NKCC1 is expressed by virtually all cell types in the brain, which makes any specific targeting of neuronal NKCC1 by bumetanide impossible. In addition, bumetanide only poorly penetrates the brain. We have previously shown that loop diuretics azosemide and torasemide also potently inhibit NKCC1. In contrast to bumetanide and furosemide, azosemide and torasemide lack a carboxylic group, which should allow them to better penetrate through biomembranes by passive diffusion. Because of the urgent medical need to develop new treatments for neonatal seizures and their adverse outcome, we evaluated the effects of azosemide and torasemide, administered alone or in combination with phenobarbital or midazolam, in a rat model of birth asphyxia and neonatal seizures. Neither diuretic suppressed the seizures when administered alone but torasemide potentiated the anti-seizure effect of midazolam. Brain levels of torasemide were below those needed to inhibit NKCC1. In addition to suppressing seizures, the combination of torasemide and midazolam, but not midazolam alone, prevented the cognitive impairment of the post-asphyxial rats at 3 months after asphyxia. Furthermore, aberrant mossy fiber sprouting in the hippocampus was more effectively prevented by the combination. We assume that either an effect on NKCC1 at the blood-brain barrier and/or cells in the periphery or the NKCC2-mediated diuretic effect of torasemide are involved in the present findings. Our data suggest that torasemide may be a useful option for improving the treatment of neonatal seizures and their adverse outcome.

[Expression of cation chloride cotransporter (NKCC1/KCC2) in brain tissue of children with focal cortical dysplasia type â ¡].

Objective: To investigate the expression of cation chloride cotransporter (NKCC1/KCC2) in the neurons from cerebral lesions of children with focal cortical dysplasia (FCD) type Ⅱ, to provide a morphological basis for revealing the possible mechanism of epilepsy. Methods: Eight cases of FCD type Ⅱ diagnosed at Beijing Haidian Hospital, Beijing, China and 12 cases diagnosed at Xuanwu Hospital, Capital Medical University, Beijing, China from February 2017 to December 2019 were included. The expression of NKCC1 and KCC2 in FCD type Ⅱa and FCD type Ⅱb was detected using immunohistochemistry and double immunohistochemical stains. The average optical density of NKCC1 in dysmorphic neurons and normal neurons was also determined using immunohistochemical staining in FCD type Ⅱa (10 cases). Results: The patients were all younger than 14 years of age. Ten cases were classified as FCD type IIa, and 10 cases as FCD type Ⅱb. NKCC1 was expressed in the cytoplasm of normal cerebral cortex neurons and KCC2 expressed on cell membranes. In dysmorphic neurons of FCD type Ⅱa, expression of NKCC1 increased, which was statistically higher than that of normal neurons (P<0.01). Aberrant expression of KCC2 in dysmorphic neurons was also noted in the cytoplasm. In the FCD Ⅱb type, the expression pattern of NKCC1/KCC2 in dysmorphic neurons was the same as that of FCD type Ⅱa. The aberrant expression of NKCC1 in balloon cells was negative or weakly positive on the cell membrane, while the aberrant expression of KCC2 was absent. Conclusions: The expression pattern of NKCC1/KCC2 in dysmorphic neurons and balloon cells is completely different from that of normal neurons. The NKCC1/KCC2 protein-expression changes may affect the transmembrane chloride flow of neurons, modify the effect of inhibitory neurotransmitters γ-aminobutyric acid and increase neuronal excitability. These effects may be related to the occurrence of clinical epileptic symptoms.

Chloride Homeostasis in Developing Motoneurons.

Maturation of GABA/Glycine chloride-mediated synaptic inhibitions is crucial for the establishment of a balance between excitation and inhibition. GABA and glycine are excitatory neurotransmitters on immature neurons that exhibit elevated [Cl-]i. Later in development [Cl-]i drops leading to the occurrence of inhibitory synaptic activity. This ontogenic change is closely correlated to a differential expression of two cation-chloride cotransporters that are the Cl- channel K+/Cl- co-transporter type 2 (KCC2) that extrudes Cl- ions and the Na+-K+-2Cl- cotransporter NKCC1 that accumulates Cl- ions. The classical scheme built from studies performed on cortical and hippocampal networks proposes that immature neurons display high [Cl-]i because NKCC1 is overexpressed compared to KCC2 and that the co-transporters ratio reverses in mature neurons, lowering [Cl-]i. In this chapter, we will see that this classical scheme is not true in motoneurons (MNs) and that an early alteration of the chloride homeostasis may be involved in pathological conditions.

Loss of NKCC1 function increases epithelial tight junction permeability by upregulating claudin-2 expression.

Conditions that cause the loss of epithelial barrier integrity are often accompanied by dysregulation of tight junction protein expression and/or localization. Recently, we have reported that patients with mutations in SLC12A2, the gene encoding the basolateral Na+-K+-2Cl- cotransporter (NKCC1), suffer from severe gastrointestinal deficits, including chronic gastrointestinal inflammation, gastrointestinal hemorrhage, intestinal obstruction, and constipation. Although the intestinal inflammation observed in patients with loss of NKCC1 function may or may not be due to tight junction dysfunction, we investigated whether the loss of NKCC1 function affects paracellular ion transport and epithelial barrier function. Wild-type HT29-MTX-E12 and CRISPR/Cas9-mediated NKCC1 knockout (KO) HT29 clones were tested for tight junction protein expression and localization. Tightness of epithelial cell monolayer was assessed by measurement of transepithelial electrical resistance and permeability of molecular tracers in transwell filters. Tight junction protein localization was assessed by immunofluorescence. Loss of NKCC1 expression strongly increases the expression of claudin-2 and occludin in epithelial cell monolayers. Loss of NKCC1 significantly reduces the transepithelial electrical resistance (TER) indicating an increase in paracellular ions flux, consistent with upregulation of the cation-selective and channel-forming claudin-2. In addition, NKCC1-KO monolayers showed a significant increase in the paracellular flux of small molecules like fluorescein (0.33 kDa), whereas the permeability of higher molecular weight TRITC-Dextran (4 kDa and 70 kDa) remained unchanged. Thus, NKCC1 regulates tight junction protein expression and loss of NKCC1 function affects epithelial barrier integrity.

A Wholistic View of How Bumetanide Attenuates Autism Spectrum Disorders.

The specific NKCC1 cotransporter antagonist, bumetanide, attenuates the severity of Autism Spectrum Disorders (ASD), and many neurodevelopmental or neurodegenerative disorders in animal models and clinical trials. However, the pervasive expression of NKCC1 in many cell types throughout the body is thought to challenge the therapeutic efficacy of bumetanide. However, many peripheral functions, including intestinal, metabolic, or vascular, etc., are perturbed in brain disorders contributing to the neurological sequels. Alterations of these functions also increase the incidence of the disorder suggesting complex bidirectional links with the clinical manifestations. We suggest that a more holistic view of ASD and other disorders is warranted to account for the multiple sites impacted by the original intra-uterine insult. From this perspective, large-spectrum active repositioned drugs that act centrally and peripherally might constitute a useful approach to treating these disorders.

Ptpn20 deletion in H-Tx rats enhances phosphorylation of the NKCC1 cotransporter in the choroid plexus: an evidence of genetic risk for hydrocephalus in an experimental study.

BACKGROUND: Congenital hydrocephalus occurs with some inheritable characteristics, but the mechanisms of its development remain poorly understood. Animal models provide the opportunity to identify potential genetic causes in this condition. The Hydrocephalus-Texas (H-Tx) rat strain is one of the most studied animal models for investigating the causative genetic alterations and analyzing downstream pathogenetic mechanisms of congenital hydrocephalus. METHODS: Comparative genomic hybridization (CGH) array on non-hydrocephalic and hydrocephalic H-Tx rats was used to identify causative genes of hydrocephalus. Targeted gene knockout mice were generated by CRISPR/Cas9 to study the role of this gene in hydrocephalus. RESULTS: CGH array revealed a copy number loss in chromosome 16p16 region in hydrocephalic H-Tx rats at 18 days gestation, encompassing the protein tyrosine phosphatase non-receptor type 20 (Ptpn20), a non-receptor tyrosine phosphatase, without change in most non-hydrocephalic H-Tx rats. Ptpn20-knockout (Ptpn20-/-) mice were generated and found to develop ventriculomegaly at 8 weeks. Furthermore, high expression of phosphorylated Na-K-Cl cotransporter 1 (pNKCC1) was identified in the choroid plexus (CP) epithelium of mice lacking Ptpn20 from 8 weeks until 72 weeks. CONCLUSIONS: This study determined the chromosomal location of the hydrocephalus-associated Ptpn20 gene in hydrocephalic H-Tx rats. The high level of pNKCC1 mediated by Ptpn20 deletion in CP epithelium may cause overproduction of cerebrospinal fluid and contribute to the formation of hydrocephalus in Ptpn20-/- mice. Ptpn20 may be a potential therapeutic target in the treatment of hydrocephalus.