Expression Of Cation-chloride Cotransporters Research Articles

ATN1-related infantile developmental and epileptic encephalopathy responding to Ketogenic diet

BackgroundResearch has shown gene ATN1 to be associated with the nuclear receptor signaling. Its mutations in an evolutionarily conserved histidine-rich motif may cause CHEDDA, short for congenital hypotonia, epilepsy, developmental delay and digital anomalies, a recently identified neurodevelopmental syndrome that could evolve into developmental and epileptic encephalopathy (DEE). Up to date, there have been reported less than 20 cases, whose clinical features and treatment are worth in-depth exploring. MethodsThe clinical characteristics and genetic data of an infant with CHEDDA and further DEE were analyzed, who carried a de novo ATN1 variant identified by trio whole-exome sequencing. The alike patients with such a neurodevelopmental syndrome and epileptic seizures were reviewed on the literature. ResultsThe infant harboring a de novo missense mutation in ATN1 (c.3155A>C; p.His1052Pro) held almost all features of CHEDDA and presented with drug-resistant epileptic spasms, differing from one case previously reported with the same gene variant exhibiting milder seizures controlled easily. We further reviewed 11 CHEDDA patients with epileptic seizures in the literature and compared the correlation between abnormal cerebral structure and the incidence of intractable epilepsy among CHEDDA patients. Fortunately, this patient's seizures decreased remarkably after administering ketogenic diet (KD). ConclusionCHEDDA patients have significant phenotypic differences, especially in the epilepsy severity and their drug resistance, even if they carry the same mutation hotspot. Ketogenic diet and other treatments like Topiramate should be recommended for ATN1-related refractory epilepsy based on their regulation on expression of cation-chloride cotransporters and cellular hyperpolarization.

Imbalanced expression of cation-chloride cotransporters as a potential therapeutic target in an Angelman syndrome mouse model

Angelman syndrome is a neurodevelopmental disorder caused by loss of function of the maternally expressed UBE3A gene. Treatments for the main manifestations, including cognitive dysfunction or epilepsy, are still under development. Recently, the Cl− importer Na+-K+-Cl− cotransporter 1 (NKCC1) and the Cl− exporter K+-Cl− cotransporter 2 (KCC2) have garnered attention as therapeutic targets for many neurological disorders. Dysregulation of neuronal intracellular Cl− concentration ([Cl−]i) is generally regarded as one of the mechanisms underlying neuronal dysfunction caused by imbalanced expression of these cation-chloride cotransporters (CCCs). Here, we analyzed the regulation of [Cl−]i and the effects of bumetanide, an NKCC1 inhibitor, in Angelman syndrome models (Ube3am−/p+ mice). We observed increased NKCC1 expression and decreased KCC2 expression in the hippocampi of Ube3am−/p+ mice. The average [Cl−]i of CA1 pyramidal neurons was not significantly different but demonstrated greater variance in Ube3am−/p+ mice. Tonic GABAA receptor-mediated Cl− conductance was reduced, which may have contributed to maintaining the normal average [Cl−]i. Bumetanide administration restores cognitive dysfunction in Ube3am−/p+ mice. Seizure susceptibility was also reduced regardless of the genotype. These results suggest that an imbalanced expression of CCCs is involved in the pathophysiological mechanism of Ube3am−/p+ mice, although the average [Cl−]i is not altered. The blockage of NKCC1 may be a potential therapeutic strategy for patients with Angelman syndrome.

The amygdala lesioning due to status epilepticus – Changes in mechanisms controlling chloride homeostasis

ObjectiveAmygdala has been demonstrated as one of the brain sites involved in the control of cardiorespiratory functioning. The structural and physiological alterations induced by epileptic activity are also present in the amygdala and reflect functional changes that may be directly associated with a sudden unexpected death. Seizures are always associated with neuronal damage and changes in the expression of cation-chloride cotransporters and Na/K pumps. In this study, the authors aimed to investigate if these changes are present in the amygdala after induction of status epilepticus with pilocarpine, which may be directly correlated with Sudden Unexpected Death in Epilepsy (SUDEP). MethodsPilocarpine-treated wistar rats 60 days after Status Epilepticus (SE) were compared with control rats. Amygdala nuclei of brain slices immunostained for NKCC1, KCC2 and α1-Na+/K+-ATPase, were quantified by optical densitometry. ResultsThe amygdaloid complex of the animals submitted to SE had no significant difference in the NKCC1 immunoreactivity, but KCC2 immunoreactivity reduced drastically in the peri-somatic sites and in the dendritic-like processes. The α1-Na+/K+-ATPase peri-somatic immunoreactivity was intense in the rats submitted to pilocarpine SE when compared with control rats. The pilocarpine SE also promoted intense GFAP staining, specifically in the basolateral and baso-medial nuclei with astrogliosis and cellular debris deposition. InterpretationThe findings revealed that SE induces lesion changes in the expression of KCC2 and α1-Na+/K+-ATPase meaning intense change in the chloride regulation in the amygdaloid complex. These changes may contribute to cardiorespiratory dysfunction leading to SUDEP.

Upregulation of MiRNA-149-5p Reduces the Infract Volume in Middle Cerebral Artery Occlusion Rats by Modulating Cation-Chloride Cotransporters Expressions

Brain ischemia often leads to the chloride gradient alternations, which affects volume regulation and neuronal survival. Increase in NKCC1 expression and reduction in KCC2 level under ischemic condition results in inflammation and neuronal death. In this study, we investigated the effect of mimic miRNA and coenzyme Q10 (CoQ10) on the expression of cation-chloride cotransporters (CCCs) (NKCC1 and KCC2) after cerebral ischemia. In this study, cerebral ischemia was modeled using the middle cerebral artery occlusion method. Rats were randomly divided into six groups: sham, model, negative control, vehicle, and the first and second treatments. In the Sham group, ischemia was not induced, and no treatment was performed. In the Model group, ischemia induction was performed, and other groups, in addition to ischemia induction, received Scramble miRNA, Ethanol, mimic miRNA-149-5p and CoQ10, respectively. Each group was divided into three subgroups to assess the volume of the tissue damage and neurological deficits scores (NDS) in subgroup 1, brain water content in subgroup 2, level of miRNA-149-5p and CCC expressions in subgroup 3. Our data suggested that the use of mimic miRNA and Q10 increased the level of miRNA-149 and KCC2 expression and decreased NDS, NKCC1 expression, brain water content, and infract volume. Findings of this study suggest that the mimic miRNA and Q10 may have neuroprotective effects through reducing infract volume and brain water content and modulating the expression of CCCs after brain ischemia.

Bumetanide, an Inhibitor of NKCC1 (Na-K-2Cl Cotransporter Isoform 1), Enhances Propofol-Induced Loss of Righting Reflex but Not Its Immobilizing Actions in Neonatal Rats.

Gamma-aminobutyric acid (GABA) has been shown to induce excitation on immature neurons due to increased expression of Na+-K+-2Cl- co-transporter isoform 1 (NKCC1), and the transition of GABAergic signaling from excitatory to inhibitory occurs before birth in the rat spinal cord and spreads rostrally according to the developmental changes in cation-chloride co-transporter expression. We previously showed that midazolam activates the hippocampal CA3 area and induces less sedation in neonatal rats compared with adolescent rats in an NKCC1-dependent manner. In the present study, we tested the hypothesis that propofol-induced loss of righting reflex (LORR) but not immobilizing actions are modulated by NKCC1-dependent mechanisms and reduced in neonatal rats compared with adolescent rats. We estimated neuronal activity in the cortex, hippocampus and thalamus after propofol administration with or without bumetanide, an NKCC1 inhibitor, by immunostaining of phosphorylated cyclic adenosine monophosphate-response element binding protein (pCREB). We studied effects of bumetanide on propofol-induced LORR and immobilizing actions in postnatal day 7 and 28 (P7 and P28) rats. The pCREB expression in the cortex (P = 0.001) and hippocampus (P = 0.01) was significantly greater in the rats receiving propofol only than in the rats receiving propofol plus bumetanide at P 7. Propofol-induced LORR or immobilizing effects did not differ significantly between P7 and P28. Bumetanide significantly enhanced propofol-induced LORR (P = 0.031) but not immobilization in P7 rats. These results are partially consistent with our hypothesis. They suggest that propofol may activate the rostral but not caudal central nervous system dependently on NKCC1, and these differential actions may underlie the different properties of sedative and immobilizing actions observed in neonatal rats.

Developmental pharmacology of benzodiazepines under normal and pathological conditions.

Benzodiazepines are allosteric agonists of GABAA receptors (GABAAR), pentameric ligand-gated Cl(-) channels, which serve both an important neurodevelopmental role but are also the principal inhibitory system in the brain. However, their subunit composition, channel properties, and function, as well as their region-specific expression patterns, change through development. These processes have been extensively studied in rodents and to some extent confirmed in higher species. Specifically, GABAARs acquire faster kinetics with age and their pharmacology changes rendering them more sensitive to drugs that have higher affinity for α1 subunit-containing GABAARs, such as benzodiazepines, but also, their inhibitory function becomes more potent as they shift from having depolarising to hyperpolarising responses due to a shift in Cl(-) gradient and cation chloride cotransporter expression. Concerns have been raised about possible pro-apoptotic and paradoxical effects of benzodiazepines in the neonatal normal rat brain, although it is unclear, as yet, whether this extends to brains exposed to seizures. Growing evidence indicates that the pharmacology and physiology of GABAARs may be altered in the brain of rats or humans with seizures or epilepsy, or different aetiologies that predispose to epilepsy. These changes follow different paths, depending on sex, age, region, cell type, aetiology, or time-point specific factors. Identification of dynamic biomarkers that could enable these changes in vivo to be monitored would greatly facilitate the selection of more effective agonists with fewer side effects.

Expression of cation-chloride cotransporters in the spinal cord of rat of incision model

Objective To investigate the expression of sodium-potassium-chloride co-transporter 1 (NKCC 1) and potassiumchloride co-transporter 2 (KCC2) in the spinal dorsal horn and dorsal root ganglion (DRG) of the incisional rat.Methods Adult male Sprague-Dawley rats were randomly divided into 5 groups of 6,a control and 4 incision groups at 2 h,2 d,3 d,7 d following incision.Incision was made or only skin degerming under inhalation anesthesia.At the specific time,rats were perfused with fixative under anesthesia,then the L4-L5 spinal cord and bilateral L5 DRG removed,and NKCC1 and KCC2 expression estimated by immunohistochemistry.Results In the intact rats,NKCC1 was expressed in the DRG,and both NKCC1 and KCC2 was expressed in the dorsal horn with KCC2 predominated.NKCC 1 increased in the DRG (P=0.010) following incision.Compared to controls(18.0±2.8),the number of NKCC1-positive neurons was significantly higher in ipsilateral DRG on 2 h (40.7±2.0),2 d (54.7±9.8),3 d (45.3±8.6) following incision (P=0.026,0.001,0.008).NKCC1 also increased in the ipsilateral dorsal horn (P＜0.01),compared tocontrols (14.33±0.56),the number of NKCC1-positive neurons was significantly higher on 2 h (31.00±1.32),2 d (50.33± 1.80),3 d(38.67±3.04),7 d (32.33±0.21) and all P＜0.01.KCC2 decreased in the dorsal horn following incision (P<0.01).Compared to controls(42.7±2.6),the number of KCC2-positive neurons was significantly lower in the dorsal horn on 2 h(18.0±3.5),2 d(18.0±1.7),3 d(23.3±1.5) and 7 d (24.7±1.1) following incision (all P＜0.01).Conclusions NKCC1 increased in the DRG and dorsal horn while KCC2 decreased in the dorsal horn in the incision model of rat,which suggested that NKCC1 and KCC2 participated in the mechanism of hyperalgesia following incision. Key words: Sodium-potassium-chloride co-transporter 1 ; Potassium-chloride co-transporter 2; Postoperative pain; Hyperalgesia

Molecular and functional expression of cation-chloride cotransporters in dorsal root ganglion neurons during postnatal maturation

GABA depolarizes and excites central neurons during early development, becoming inhibitory and hyperpolarizing with maturation. This "developmental shift" occurs abruptly, reflecting a decrease in intracellular Cl(-) concentration ([Cl(-)](i)) and a hyperpolarizing shift in Cl(-) equilibrium potential due to upregulation of the K(+)-Cl(-) cotransporter KCC2b, a neuron-specific Cl(-) extruder. In contrast, primary afferent neurons (PANs) are depolarized by GABA throughout adulthood because of expression of NKCC1, a Na(+)-K(+)-2Cl(-) cotransporter that accumulates Cl(-) above equilibrium. The GABA(A)-mediated depolarization of PANs determines presynaptic inhibition in the spinal cord, a key mechanism gating somatosensory information. Little is known about developmental changes in Cl(-) transporter expression and Cl(-) homeostasis in PANs. Whether NKCC1 is expressed in PANs of all phenotypes or is restricted to subpopulations (e.g., nociceptors) is debatable. Likewise, whether PANs express KCC2s is controversial. We investigated NKCC1 and K(+)-Cl(-) cotransporter expression in rat and mouse dorsal root ganglion (DRG) neurons with molecular methods. Using fluorescence imaging microscopy, we measured [Cl(-)](i) in acutely dissociated rat DRG neurons (P0-P21) loaded with N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide and classified with phenotypic markers. DRG neurons of all sizes express two NKCC1 mRNAs, one full-length and a shorter splice variant lacking exon 21. Immunolabeling with validated antibodies revealed ubiquitous expression of NKCC1 in DRG neurons irrespective of postnatal age and phenotype. As maturation progresses [Cl(-)](i) decreases gradually, persisting above equilibrium in >95% mature neurons. DRG neurons express mRNAs for KCC1, KCC3s, and KCC4, but not for KCC2s. Mechanisms underlying PANs' developmental changes in Cl(-) homeostasis are discussed and compared with those of central neurons.

Loss of Cation-Chloride Cotransporter Expression in Preterm Infants With White Matter Lesions: Implications for the Pathogenesis of Epilepsy

Epilepsy associated with preterm birth is often refractory to anticonvulsants. Children who are born preterm are also prone to cognitive delay and behavioral problems. Brains from these children often show diffuse abnormalities in cerebral circuitry that is likely caused by disrupted development during critical stages of cortical formation. To test the hypothesis that prenatal injury impairs the developmental switch of gamma-amino butyric acid (GABA)ergic synapses from excitatory to inhibitory, thereby disrupting cortical circuit formation and predisposing to epilepsy, we used immunohistochemistry to compare the expression of cation-chloride transporters that developmentally regulate postsynaptic GABAergic discharges in postmortem cerebral samples from infants born preterm with known white matter injury (n = 11) with that of controls with minimal white matter gliosis (n = 7). Controls showed the expected developmental expression of cation-chloride transporters NKCC1 and KCC2 and ofcalretinin, a marker of a GABAergic neuronal subpopulation. Samples from infants with white matter damage showed a significant loss of expression of both NKCC1 and KCC2 in subplate and white matter. By contrast, there were no significant differences in total cell number or glutamate transporter VGLUT1 expression. Together, these novel findings suggest a molecular mechanism involved in the disruption of a critical stage of cerebral circuit development after brain injury from preterm birth that may predispose to epilepsy.

Cell-type specific distribution of chloride transporters in the rat suprachiasmatic nucleus

The suprachiasmatic nucleus (SCN) is a circadian oscillator and biological clock. Cell-to-cell communication is important for synchronization among SCN neuronal oscillators and the great majority of SCN neurons use GABA as a neurotransmitter, the principal inhibitory neurotransmitter in the adult CNS. Acting via the ionotropic GABAA receptor, a chloride ion channel, GABA typically evokes inhibitory responses in neurons via Cl− influx. Within the SCN GABA evokes both inhibitory and excitatory responses although the mechanism underlying GABA-evoked excitation in the SCN is unknown. GABA-evoked depolarization in immature neurons in several regions of the brain is a function of intracellular chloride concentration, regulated largely by the cation-chloride cotransporters NKCC1 (sodium/potassium/chloride cotransporter for chloride entry) and KCC1-4 (potassium/chloride cotransporters for chloride egress). It is well established that changes in the expression of the cation-chloride cotransporters through development determines the polarity of the response to GABA. To understand the mechanisms underlying GABA-evoked excitation in the SCN, we examined the SCN expression of cation-chloride cotransporters. Previously we reported that the K+/Cl− cotransporter KCC2, a neuron-specific chloride extruder conferring GABA's more typical inhibitory effects, is expressed exclusively in vasoactive intestinal peptide (VIP) and gastrin-releasing peptide (GRP) neurons in the SCN. Here we report that the K+/Cl− cotransporter isoforms KCC4 and KCC3 are expressed solely in vasopressin (VP) neurons in the rat SCN whereas KCC1 is expressed in VIP neurons, similar to KCC2. NKCC1 is expressed in VIP, GRP and VP neurons in the SCN as is WNK3, a chloride-sensitive neuron-specific with no serine–threonine kinase which modulates intracellular chloride concentration via opposing actions on NKCC and KCC cotransporters. The heterogeneous distribution of cation-chloride cotransporters in the SCN suggests that Cl− levels are differentially regulated within VIP/GRP and VP neurons. We suggest that GABA's excitatory action is more likely to be evoked in VP neurons that express KCC4.

Cell specificity of altered cation–chloride cotransporter expression and GABAergic innervation in the epileptic cerebral cortex

Evaluation of: Aronica E, Boer K, Redeker S et al.: Differential expression patterns of chloride transporters, Na+–K+–2Cl-cotransporter and K+–Cl- cotransporter (KCC2), in epilepsy-associated malformations of cortical development. Neuroscience 145(1), 185–196 (2007). The study by Aronica and colleagues used immunocytochemistry to investigate the changes in the expression and distribution of cation–chloride cotransporters in the cerebral cortex of epileptic patients when compared with age-matched controls. The aim was to determine whether or not the deregulation of cation–chloride cotransporter expression might contribute to the hyperexcitability that leads to seizures. The authors studied brain tissue from patients with medically intractable epilepsy associated with different types of malformations, including focal cortical dysplasia type IIB, hemimegalencephaly and ganglioglioma. They found weak neuronal and glial expression of the Na+–K+–2 Cl- cotransporter (NKCC1) in the normal control adult cortex, whereas NKCC1 was strongly expressed in different cell types from most patients, including large dysplastic neurons, balloon cells and astrocytes. In addition, the K+–Cl- cotransporter (KCC2) was reported to be diffusely distributed in the neuropil of control tissue, although more intense somatic KCC2 immunostaining was associated with large dysplastic neurons but not balloon cells in epileptic patients. The intense expression of NKCC1 in dysplastic neurons and the altered subcellular distribution of KCC2 were compared with the situation in the immature normal cortex. The authors concluded that altered expression of cation–chloride cotransporters in epileptic patients, in conjunction with malformations in cortical development, may contribute to epileptogenicity.