WNK3 Maintains the GABAergic Inhibitory Tone, Synaptic Excitation and Neuronal Excitability via Regulation of KCC2 Cotransporter in Mature Neurons

The activation of chloride (Cl−)permeable gamma (γ)-aminobutyric acid type A(GABAA) receptors induces synaptic inhibition in mature and excitation in immature neurons. This developmental “switch” in GABA function controlled by its polarity depends on the postnatal decrease in intraneuronal Cl− concentration mediated by KCC2, a member of cation-chloride cotransporters (CCCs). The serine-threonine kinase WNK3 (With No Lysine [K]), is a potent regulator of all CCCs and is expressed in neurons. Here, we characterized the functions of WNK3 and its role in GABAergic signaling in cultured embryonic day 18 (E18) hippocampal neurons. We observed a decrease in WNK3 expression as neurons mature. Knocking down of WNK3 significantly hyperpolarized EGABA in mature neurons (DIV13–15) but had no effect on immature neurons (DIV6–8). This hyperpolarized EGABA in WNK3-deficient neurons was not due to the total expression of NKCC1 and KCC2, that remained unchanged. However, there was a reduction in phosphorylated KCC2 at the membrane, suggesting an increase in KCC2 chloride export activity. Furthermore, hyperpolarized EGABA observed in WNK3-deficient neurons can be reversed by the KCC2 inhibitor, VU024055, thus indicating that WNK3 acts through KCC2 to influence EGABA. Notably, WNK3 knockdown resulted in morphological changes in mature but not immature neurons. Electrophysiological characterization of WNK3-deficient mature neurons revealed reduced capacitances but increased intrinsic excitability and synaptic excitation. Hence, our study demonstrates that WNK3 maintains the “adult” GABAergic inhibitory tone in neurons and plays a role in the morphological development of neurons and excitability.

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KCC2 is required for the survival of mature neurons but not for their development

10.1101/2020.10.29.360875 ◽

2020 ◽

Author(s):

Georgina Kontou ◽

Josephine Ng ◽

Ross Andrew Cardarelli ◽

Jack Howden ◽

Catherine Choi ◽

...

Keyword(s):

Hippocampal Neurons ◽

Critical Role ◽

Dendritic Structure ◽

Subsequent Development ◽

Apoptotic Pathway ◽

Neuronal Viability ◽

Extrinsic Apoptotic Pathway ◽

Immature Neurons ◽

Mature Neurons ◽

Rapid Activation

ABSTRACTThe K+/Cl– co-transporter KCC2 (SLC12A5) allows mature neurons in the CNS to maintain low intracellular Cl− levels that are critical in mediating fast hyperpolarizing synaptic inhibition via type A γ-aminobutyric acid receptors GABAARs. In accordance with this, compromised KCC2 activity results in seizures but whether such deficits directly contribute to the subsequent changes in neuronal viability that lead to epileptogenesis, remains to be assessed. Canonical hyperpolarizing GABAAR currents develop postnatally which reflect a progressive increase in KCC2 expression levels and activity. To investigate the role that KCC2 plays in regulating neuronal viability and architecture we have conditionally ablated KCC2 expression in developing and mature neurons. Decreasing KCC2 expression resulted in the rapid activation of the extrinsic apoptotic pathway, in mature hippocampal neurons. Intriguingly, direct pharmacological inhibition of KCC2 in mature neurons resulted in the rapid activation of the extrinsic apoptotic pathway. In contrast, ablating KCC2 expression in immature neurons had no discernable effects on their subsequent development, arborization or dendritic structure. However ablating KCC2 expression in immature neurons was sufficient to prevent the subsequent postnatal development of hyperpolarizing GABAAR currents. Collectively, our results demonstrate that KCC2 plays a critical role in neuronal survival by limiting apoptosis, and mature neurons are highly sensitive to loss of KCC2 function. In contrast KCC2 appears to play a minimal role in mediating neuronal development or architecture.

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Developmental-Dependent Action of Microtubule Depolymerization on the Function and Structure of Synaptic Glycine Receptor Clusters in Spinal Neurons

Journal of Neurophysiology ◽

10.1152/jn.00364.2003 ◽

2004 ◽

Vol 91 (2) ◽

pp. 1036-1049 ◽

Cited By ~ 27

Author(s):

Brigitte van Zundert ◽

Francisco J. Alvarez ◽

Juan Carlos Tapia ◽

Hermes H. Yeh ◽

Emilio Diaz ◽

...

Keyword(s):

Average Length ◽

Glycine Receptor ◽

Colchicine Treatment ◽

Morphological Properties ◽

Microtubule Depolymerization ◽

Spinal Neurons ◽

Functional Changes ◽

Microtubule Disruption ◽

Immature Neurons ◽

Mature Neurons

Microtubules have been proposed to interact with gephyrin/glycine receptors (GlyRs) in synaptic aggregates. However, the consequence of microtubule disruption on the structure of postsynaptic GlyR/gephyrin clusters is controversial and possible alterations in function are largely unknown. In this study, we have examined the physiological and morphological properties of GlyR/gephyrin clusters after colchicine treatment in cultured spinal neurons during development. In immature neurons (5-7 DIV), disruption of microtubules resulted in a 33 ± 4% decrease in the peak amplitude and a 72 ± 15% reduction in the frequency of spontaneous glycinergic miniature postsynaptic currents (mIPSCs) recorded in whole cell mode. However, similar colchicine treatments resulted in smaller effects on 10-12 DIV neurons and no effect on mature neurons (15-17 DIV). The decrease in glycinergic mIPSC amplitude and frequency reflects postsynaptic actions of colchicine, since postsynaptic stabilization of microtubules with GTP prevented both actions and similar reductions in mIPSC frequency were obtained by modifying the Cl- driving force to obtain parallel reductions in mIPSC amplitude. Confocal microscopy revealed that colchicine reduced the average length and immunofluorescence intensity of synaptic gephyrin/GlyR clusters in immature (approximately 30%) and intermediate (approximately 15%) neurons, but not in mature clusters. Thus the structural and functional changes of postsynaptic gephyrin/GlyR clusters after colchicine treatment were tightly correlated. Finally, RT-PCR, kinetic analysis and picrotoxin blockade of glycinergic mIPSCs indicated a reorganization of the postsynaptic region from containing both α2β and α1β GlyRs in immature neurons to only α1β GlyRs in mature neurons. Microtubule disruption preferentially affected postsynaptic sites containing α2β-containing synaptic receptors.

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Zinc Enhances the Inhibitory Effects of Strychnine-Sensitive Glycine Receptors in Mouse Hippocampal Neurons

Journal of Neurophysiology ◽

10.1152/jn.00500.2007 ◽

2007 ◽

Vol 98 (6) ◽

pp. 3666-3676 ◽

Cited By ~ 8

Author(s):

Hai Xia Zhang ◽

Liu Lin Thio

Keyword(s):

Action Potential ◽

Action Potentials ◽

Hippocampal Neurons ◽

Neuronal Excitability ◽

Hippocampal Slices ◽

Glycine Receptors ◽

Inhibitory Effects ◽

Action Potential Firing ◽

Embryonic Mouse ◽

Potential Firing

Although extracellular Zn2+ is an endogenous biphasic modulator of strychnine-sensitive glycine receptors (GlyRs), the physiological significance of this modulation remains poorly understood. Zn2+ modulation of GlyR may be especially important in the hippocampus where presynaptic Zn2+ is abundant. Using cultured embryonic mouse hippocampal neurons, we examined whether 1 μM Zn2+, a potentiating concentration, enhances the inhibitory effects of GlyRs activated by sustained glycine applications. Sustained 20 μM glycine (EC25) applications alone did not decrease the number of action potentials evoked by depolarizing steps, but they did in 1 μM Zn2+. At least part of this effect resulted from Zn2+ enhancing the GlyR-induced decrease in input resistance. Sustained 20 μM glycine applications alone did not alter neuronal bursting, a form of hyperexcitability induced by omitting extracellular Mg2+. However, sustained 20 μM glycine applications depressed neuronal bursting in 1 μM Zn2+. Zn2+ did not enhance the inhibitory effects of sustained 60 μM glycine (EC70) applications in these paradigms. These results suggest that tonic GlyR activation could decrease neuronal excitability. To test this possibility, we examined the effect of the GlyR antagonist strychnine and the Zn2+ chelator tricine on action potential firing by CA1 pyramidal neurons in mouse hippocampal slices. Co-applying strychnine and tricine slightly but significantly increased the number of action potentials fired during a depolarizing current step and decreased the rheobase for action potential firing. Thus Zn2+ may modulate neuronal excitability normally and in pathological conditions such as seizures by potentiating GlyRs tonically activated by low agonist concentrations.

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Regulation of Mineralocorticoid Receptor Expression during Neuronal Differentiation of Murine Embryonic Stem Cells

Endocrinology ◽

10.1210/en.2009-0753 ◽

2010 ◽

Vol 151 (5) ◽

pp. 2244-2254 ◽

Cited By ~ 19

Author(s):

Mathilde Munier ◽

Geri Meduri ◽

Say Viengchareun ◽

Philippe Leclerc ◽

Damien Le Menuet ◽

...

Keyword(s):

Neuronal Differentiation ◽

Mineralocorticoid Receptor ◽

Fluorescent Protein ◽

Morphological Changes ◽

Critical Role ◽

Embryonic Stem ◽

Embryoid Bodies ◽

Receptor Expression ◽

Neuronal Markers ◽

Mature Neurons

Mineralocorticoid receptor (MR) plays a critical role in brain function. However, the regulatory mechanisms controlling neuronal MR expression that constitutes a key element of the hormonal response are currently unknown. Two alternative P1 and P2 promoters drive human MR gene transcription. To examine promoter activities and their regulation during neuronal differentiation and in mature neurons, we generated stably transfected recombinant murine embryonic stem cell (ES) lines, namely P1-GFP and P2-GFP, in which each promoter drove the expression of the reporter gene green fluorescent protein (GFP). An optimized protocol, using embryoid bodies and retinoic acid, permitted us to obtain a reproducible neuronal differentiation as revealed by the decrease in phosphatase alkaline activity, the concomitant appearance of morphological changes (neurites), and the increase in the expression of neuronal markers (nestin, β-tubulin III, and microtubule-associated protein-2) as demonstrated by immunocytochemistry and quantitative PCR. Using these cell-based models, we showed that MR expression increased by 5-fold during neuronal differentiation, MR being preferentially if not exclusively expressed in mature neurons. Although the P2 promoter was always weaker than the P1 promoter during neuronal differentiation, their activities increased by 7- and 5-fold, respectively, and correlated with MR expression. Finally, although progesterone and dexamethasone were ineffective, aldosterone stimulated both P1 and P2 activity and MR expression, an effect that was abrogated by knockdown of MR by small interfering RNA. In conclusion, we provide evidence for a tight transcriptional control of MR expression during neuronal differentiation. Given the neuroprotective and antiapoptotic role proposed for MR, the neuronal differentiation of ES cell lines opens potential therapeutic perspectives in neurological and psychiatric diseases.

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RhoA/ROCK regulation of neuritogenesis via profilin IIa–mediated control of actin stability

The Journal of Cell Biology ◽

10.1083/jcb.200304021 ◽

2003 ◽

Vol 162 (7) ◽

pp. 1267-1279 ◽

Cited By ~ 156

Author(s):

Jorge Santos Da Silva ◽

Miguel Medina ◽

Cecilia Zuliani ◽

Alessia Di Nardo ◽

Walter Witke ◽

...

Keyword(s):

Neuronal Differentiation ◽

Hippocampal Neurons ◽

Morphological Changes ◽

Decisive Role ◽

Central Mechanism ◽

Actin Binding ◽

Actin Binding Protein ◽

Rhoa Activity ◽

Neuronal Soma ◽

Cultured Hippocampal Neurons

Neuritogenesis, the first step of neuronal differentiation, takes place as nascent neurites bud from the immediate postmitotic neuronal soma. Little is known about the mechanisms underlying the dramatic morphological changes that characterize this event. Here, we show that RhoA activity plays a decisive role during neuritogenesis of cultured hippocampal neurons by recruiting and activating its specific kinase ROCK, which, in turn, complexes with profilin IIa. We establish that this previously uncharacterized brain-specific actin-binding protein controls neurite sprouting by modifying actin stability, a function regulated by ROCK-mediated phosphorylation. Furthermore, we determine that this novel cascade is switched on or off by physiological stimuli. We propose that RhoA/ROCK/PIIa-mediated regulation of actin stability, shown to be essential for neuritogenesis, may constitute a central mechanism throughout neuronal differentiation.

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Effect of Lamotrigine on the Ca2+-Sensing Cation Current in Cultured Hippocampal Neurons

Journal of Neurophysiology ◽

10.1152/jn.2001.86.5.2520 ◽

2001 ◽

Vol 86 (5) ◽

pp. 2520-2526 ◽

Cited By ~ 18

Author(s):

Zhi-Gang Xiong ◽

Xiang-Ping Chu ◽

J. F. MacDonald

Keyword(s):

Action Potentials ◽

Hippocampal Neurons ◽

Neuronal Excitability ◽

Membrane Depolarization ◽

Slow Inward Current ◽

Calcium Sensing ◽

Cation Current ◽

Imaging Experiment ◽

Intracellular Ca ◽

Cultured Hippocampal Neurons

Concentrations of extracellular calcium ([Ca2+]e) in the CNS decrease substantially during seizure activity. We have demonstrated previously that decreases in [Ca2+]e activate a novel calcium-sensing nonselective cation (csNSC) channel in hippocampal neurons. Activation of csNSC channels is responsible for a sustained membrane depolarization and increased neuronal excitability. Our study has suggested that the csNSC channel is likely involved in generating and maintaining seizure activities. In the present study, the effects of anti-epileptic agent lamotrigine (LTG) on csNSC channels were studied in cultured mouse hippocampal neurons using patch-clamp techniques. At a holding potential of −60 mV, a slow inward current through csNSC channels was activated by a step reduction of [Ca2+]e from 1.5 to 0.2 mM. LTG decreased the amplitude of csNSC currents dose dependently with an IC50 of 171 ± 25.8 (SE) μM. The effect of LTG was independent of membrane potential. In the presence of 300 μM LTG, the amplitude of csNSC current was decreased by 31 ± 3% at −60 mV and 29 ± 2.9% at +40 mV ( P > 0.05). LTG depressed csNSC current without affecting the potency of Ca2+ block of the current (IC50 for Ca2+block of csNSC currents in the absence of LTG: 145 ± 18 μM; in the presence of 300 μM LTG: 136 ± 10 μM. n = 5, P > 0.05). In current-clamp recordings, activation of csNSC channel by reducing the [Ca2+]e caused a sustained membrane depolarization and an increase in the frequency of spontaneous firing of action potentials. LTG (300 μM) significantly inhibited csNSC channel-mediated membrane depolarization and the excitation of neurons. Fura-2 ratiometric Ca2+imaging experiment showed that LTG also inhibited the increase in intracellular Ca2+ concentration induced by csNSC channel activation. The effect of LTG on csNSC channels may partially contribute to its broad spectrum of anti-epileptic actions.

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Defective actin dynamics in dendritic spines: cause or consequence of age-induced cognitive decline?

Biological Chemistry ◽

10.1515/hsz-2015-0185 ◽

2016 ◽

Vol 397 (3) ◽

pp. 223-229 ◽

Cited By ~ 3

Author(s):

Till Georg Alexander Mack ◽

Patricia Kreis ◽

Britta Johanna Eickholt

Keyword(s):

Dendritic Spines ◽

Neuronal Excitability ◽

Replicative Senescence ◽

Morphological Changes ◽

Actin Dynamics ◽

Cellular Processes ◽

Filament Dynamics ◽

Neuronal Soma ◽

Deteriorating Process ◽

The Brain

Abstract Ageing is a complex deteriorating process that coincides with changes in metabolism, replicative senescence, increased resistance to apoptosis, as well as progressive mitochondria dysfunction that lead to an increase production and accumulation of reactive oxygen species (ROS). Although controversy on the paradigm of the oxidative damage theory of ageing exists, persuasive studies in Caenorhabditis elegans and yeast have demonstrated that manipulation of ROS can modify the process of ageing and influences the damage of proteins, lipids and DNA. In neurons, ageing impacts on the intrinsic neuronal excitability, it decreases the size of neuronal soma and induces the loss of dendrites and dendritic spines. The actin cytoskeleton is an abundant and broadly expressed system that plays critical functions in many cellular processes ranging from cell motility to controlling cell shape and polarity. It is thus hardly surprising that the expression and the function of actin in neurons is crucial for the morphological changes that occur in the brain throughout life. We propose that alterations in actin filament dynamics in dendritic spines may be one of the key events contributing to the initial phases of ageing in the brain.

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Striatal Chloride Dysregulation and Impaired GABAergic Signaling Due to Cation-Chloride Cotransporter Dysfunction in Huntington’s Disease

Frontiers in Cellular Neuroscience ◽

10.3389/fncel.2021.817013 ◽

2022 ◽

Vol 15 ◽

Author(s):

Melissa Serranilla ◽

Melanie A. Woodin

Keyword(s):

Huntington's Disease ◽

Huntington’S Disease ◽

Neurological Disorders ◽

Synaptic Inhibition ◽

Motor Impairments ◽

Immature Brain ◽

Amino Butyric Acid ◽

Mature Neurons ◽

Cation Chloride Cotransporters

Intracellular chloride (Cl–) levels in mature neurons must be tightly regulated for the maintenance of fast synaptic inhibition. In the mature central nervous system (CNS), synaptic inhibition is primarily mediated by gamma-amino butyric acid (GABA), which binds to Cl– permeable GABAA receptors (GABAARs). The intracellular Cl– concentration is primarily maintained by the antagonistic actions of two cation-chloride cotransporters (CCCs): Cl–-importing Na+-K+-Cl– co-transporter-1 (NKCC1) and Cl– -exporting K+-Cl– co-transporter-2 (KCC2). In mature neurons in the healthy brain, KCC2 expression is higher than NKCC1, leading to lower levels of intracellular Cl–, and Cl– influx upon GABAAR activation. However, in neurons of the immature brain or in neurological disorders such as epilepsy and traumatic brain injury, impaired KCC2 function and/or enhanced NKCC1 expression lead to intracellular Cl– accumulation and GABA-mediated excitation. In Huntington’s disease (HD), KCC2- and NKCC1-mediated Cl–-regulation are also altered, which leads to GABA-mediated excitation and contributes to the development of cognitive and motor impairments. This review summarizes the role of Cl– (dys)regulation in the healthy and HD brain, with a focus on the basal ganglia (BG) circuitry and CCCs as potential therapeutic targets in the treatment of HD.

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Astrocyte-Derived Small Extracellular Vesicles Regulate Dendritic Complexity through miR-26a-5p Activity

10.20944/preprints202002.0250.v1 ◽

2020 ◽

Author(s):

Alejandro Luarte ◽

Roberto Henzi ◽

Anllely Fernández ◽

Diego Gaete ◽

Pablo Cisternas ◽

...

Keyword(s):

Extracellular Vesicles ◽

Hippocampal Neurons ◽

Morphological Changes ◽

Morphological Differentiation ◽

Total Content ◽

Neuronal Morphology ◽

Cultured Astrocytes ◽

Neuronal Proteins ◽

Dendritic Complexity ◽

Mirna Content

In the last decades, it has been established that astrocytes play key roles in the regulation of neuronal morphology. However, the contribution of astrocyte-derived small extracellular vesicles (sEVs) to morphological differentiation of neurons has only recently been addressed. Here, we showed that cultured astrocytes expressing a GFP tagged version of the stress-regulated astrocytic enzyme Aldolase C (Aldo C-GFP) release small extracellular vesicles (sEVs) which are transferred into cultured hippocampal neurons. Surprisingly, Aldo C-GFP-containing sEVs (Aldo C-GFP sEVs) displayed an exacerbated capacity to reduce the dendritic complexity in developing hippocampal neurons compared to sEVs derived from control (i.e. GFP-expressing) astrocytes. Using bioinformatics and biochemical tools, we found that the total content of overexpressed Aldo C-GFP correlates with an increased content of endogenous miRNA-26a-5p in both total astrocyte homogenates and sEVs. Notably, neurons magnetofected with a nucleotide sequence that mimics endogenous miRNA-26a-5p (mimic 26a-5p) not only decreased the levels of neuronal proteins associated to morphogenesis regulation and also reproduced morphological changes induced by Aldo-C-GFP sEVs. Furthermore, neurons magnetofected with a sequence targeting miRNA-26a-5p (antago 26a-5p) were largely resistant to Aldo C-GFP sEVs. Our results support a novel and complex level of astrocyte-to-neuron communication mediated by astrocyte-derived sEVs and the activity of their miRNA content.

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Perinatal fentanyl exposure leads to long-lasting impairments in somatosensory circuit function and behavior

10.1101/2020.09.19.304352 ◽

2020 ◽

Author(s):

Jason Alipio ◽

Catherine Haga ◽

Megan E Fox ◽

Keiko Arakawa ◽

Rakshita Balaji ◽

...

Keyword(s):

Cortical Neurons ◽

Morphological Changes ◽

Anterior Cingulate ◽

Preclinical Model ◽

Sensory Stimuli ◽

Synaptic Excitation ◽

Molecular Abnormalities ◽

Postsynaptic Currents ◽

And Behavior

One consequence of the opioid epidemic are lasting neurodevelopmental sequelae afflicting adolescents exposed to opioids in the womb. A translationally relevant and developmentally accurate preclinical model is needed to understand the behavioral, circuit, network, and molecular abnormalities resulting from this exposure. By employing a novel preclinical model of perinatal fentanyl exposure, our data reveal that fentanyl has several dose-dependent, developmental consequences to somatosensory function and behavior. Newborn male and female mice exhibit signs of withdrawal and sensory-related deficits that extend at least to adolescence. As fentanyl exposure does not affect dams' health or maternal behavior, these effects result from the direct actions of perinatal fentanyl on the pups' developing brain. At adolescence, exposed mice exhibit reduced adaptation to sensory stimuli, and a corresponding impairment in primary somatosensory (S1) function. In vitro electrophysiology demonstrates a long-lasting reduction in S1 synaptic excitation, evidenced by decreases in release probability, NMDA receptor-mediated postsynaptic currents, and frequency of miniature excitatory postsynaptic currents, as well as increased frequency of miniature inhibitory postsynaptic currents. In contrast, anterior cingulate cortical neurons exhibit an opposite phenotype, with increased synaptic excitation. Consistent with these changes, electrocorticograms reveal suppressed ketamine-evoked γ oscillations. Morphological analysis of S1 pyramidal neurons indicate reduced dendritic complexity, dendritic length, and soma size. Further, exposed mice exhibited abnormal cortical mRNA expression of key receptors and neuronal growth and development, changes that were consistent with the electrophysiological and morphological changes. These findings demonstrate the lasting sequelae of perinatal fentanyl exposure on sensory processing and function.

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