Effect of protein synthesis inhibitors on the function of the neuronal K-Cl cotransporter KCC2

The Cl- and HCO3- electrochemical gradients across the plasma membrane dictate the electrical consequences of GABAA receptor (GABAAR) function and thereby play a significant role in neuronal GABA-mediated signalling. In adult pyramidal neurons, responses to GABA are maintained hyperpolarizing mainly by the action of K-Cl cotransporter isoform 2 (KCC2). KCC2 acts as a Cl- extrusion mechanism responsible for setting the intracellular Cl- concentration below the electrochemical equilibrium, a necessary condition for hyperpolarizing inhibition mediated by GABAARs. Recent evidence suggests that plasmalemmal KCC2 has a very high rate of turnover, pointing to a novel role for changes in KCC2 expression in diverse manifestations of neuronal plasticity. Some studies indicate that rapid down-regulation of KCC2 may be a general early response involved in various kinds of neuronal trauma. In this work, whole-cell patch-clamp was used to examine KCC2 function under a pharmacologically induced arrest of protein synthesis in living hippocampal brain slices from rat. The stability of KCC2 function was quantitatively assessed on the basis of the dendritic Cl- extrusion capacity in the presence of protein synthesis inhibitors cycloheximide and emetine. The parameter used for assessing extrusion capacity was a somato-dendritic Cl- gradient, which was imposed by a somatic Cl- load that resulted in a gradient of EGABA (ΔEGABA). The results of this study show that under general protein synthesis inhibitor-induced arrest of translation, KCC2 function persists unperturbed for at least 4 hours and hence that the cessation of mRNA translation cannot rapidly induce downregulation of KCC2-mediated Cl- extrusion. This finding precludes the use of protein synthesis inhibitors for rapid modulation of KCC2 function. Indirectly, the results presented here imply that the levels of KCC2 under pathophysiological conditions are primarily determined by the degradation rate and not by de novo synthesis.Gamma-aminovoihappo (GABA) on pääasiallinen hermoärsytystä ehkäisevä välittäjäaine aivoissa. GABA toimii sitoutumalla A tyypin GABA reseptoreihin (GABAAR), jotka ovat läpäiseviä kloridille (Cl-) ja bikarbonaatille. GABA-välitteinen nopea ehkäisy on riippuvainen hermosolukalvon yli vallitsevasta sisäänpäin suuntautuneesta Cl-:n sähkökemiallisesta gradientista, joka, GABA:n sitouduttua GABAAR:n, ajaa Cl- ioneja solun sisätilaan hyperpolaroiden solun lepokalvojännitteen. Neuronaalinen K-Cl-kuljettaja KCC2 poistaa aktiivisesti kloridia hermosoluista, mikä on välttämätön edellytys hyperpolaroivalle GABA-välitteiselle synaptiselle ehkäisylle. Rotan hippokampuksen neuroneissa tapahtuva ontogeneettinen GABAAR -vasteen muutos depolaroivasta hyperpolaroivaksi on riippuvainen KCC2:n ilmentämisen yksilönkehityksellisestä kasvusta. Tutkimukset viittaavat siihen, että solukalvolla olevan KCC2:n puoliaika on solukalvoproteiinille epätavallisen nopea. KCC2:n fosforylaatio proteiini kinaasi C:n avulla on osoitettu tärkeäksi KCC2:n posttranslationaaliseksi membraanivakauden säätelytekijäksi, mutta myös säätelyä proteiinisynteesin tasolla on ehdotettu mahdolliseksi nopean puoliajan mekanismiksi. Nopeat muutokset KCC2:n ilmentämisessä saattavatkin olla uusi hermosolujen muovautuvuuden mekanismi. Lisäksi, jotkin tutkimukset viittavat siihen, että nopea KCC2:n hajotus saattaa olla yleinen aikainen vaste monissa hermokudoksen vaurioissa. Tässä työssä, sähköfysiologisillä kokosolurekisteröineillä, tarkasteltiin KCC2 hajotusta, mittaamalla Cl-:n poiston tehokkuutta CA1 pyramidaalisolujen apikaalidendriiteistä farmakologisesti aiheutetun proteiinisynteesin eston aikana elävissä rotan hippokampus-alueen aivoleikkeissä. KCC2:n aktiivisuutta seurattiin mittaamalla hermosolun sisäistä Cl-:n pitoisuusgradienttiä, joka muodostui kokeellisesti aiheutetun somaattisen Cl- -kuorman ja dendriittisen Cl- -kuljetuksen seurauksena. Proteiinisynteesin estossa käytettiin kahta yhdistettä, emetiiniä ja sykloheksimidiä. Tämän työn tulokset osoittavat, että KCC2:n aktiivisuus pysyy muuttumattomana vähintään 4 tuntia farmakologisesti aiheutetun proteiinisynteesin eston aikana hermosolussa. Tästä seuraa, että pelkkä KCC2:n mRNA:n lukemisen esto ei kykene aiheuttamaan nopeaa Cl-:n poiston vähenemistä hermosolussa, eikä proteiinisynteesin estäjiä näin ollen ole mielekästä käyttää KCC2:n aktiivisuuden kokeelliseen säätelyyn. Epäsuorasti, tämän työn tulokset viittaavat siihen, että hermokudoksen patologisissa tiloissa, proteiinin hajotuksella on paljon suurempi rooli kuin synteesillä, KCC2:n proteiini- ja aktiivisuustasojen määräämisessä

KCC2 as a multifunctional protein in brain development and disease

Active extrusion of Cl- from the neuronal cytoplasm by the neuron-specific K-Cl co-transporter isoform KCC2 is necessary for the hyperpolarizing inhibitory Cl- currents mediated by the GABA receptors (GABAARs). Early in development and following cellular trauma or seizures, GABAAR-mediated signaling is often depolarizing and may even, in contrast to its classical inhibitory action, promote action potential firing. Developmental up-regulation of KCC2 is largely responsible for the shift from depolarizing to hyperpolarizing GABAAR-mediated signaling, and conditions associated with brain pathology often lead to loss of KCC2 and re-emergence of depolarizing GABAAR responses. The molecular mechanisms responsible for the up-regulation of KCC2 during development and those mediating its down-regulation, however, remain elusive. The present Thesis demonstrates that the low level of KCC2 protein in immature neurons is not a limiting factor for its functional activation. A single seizure episode induced with kainate triggers a fast transient enhancement of neuronal Cl- extrusion capacity paralleled by a large increase in surface-expressed but not total KCC2 protein in the hippocampus of neonatal rodents. This post-translational activation of KCC2 appears to be mediated by BDNF-TrkB signaling, as evidenced by its sensitivity to Trk inhibition and its absence in BDNF knockout mice. In contrast to these fast changes in functional expression of KCC2, no requirement for endogenous BDNF was observed for the developmental up-regulation of KCC2 protein. Another key finding of this work is that down-regulation and inactivation of KCC2 following intense NMDA receptor (NMDAR) activation is mediated via cleavage and truncation of KCC2 by the calcium-activated protease calpain. Importantly, the data obtained using inhibitors of protein degradation and protein synthesis indicate that the basal turn-over of KCC2 protein is slow and, consequently, down-regulation under pathological conditions is likely to result from enhanced degradation rather than from reduced de novo KCC2 synthesis. Together, the present findings highlight post-translational regulation as an important mediator of changes in the functional expression of KCC2 in response to conditions of enhanced neuronal activity, such as epileptic seizures. KCC2 has been traditionally regarded to have the most clearly defined physio-logical role of all the K-Cl cotransporters, as it is uniquely expressed in central neurons, and determines the neuronal response to activation of GABAA and glycine receptors. However, such a view has changed drastically following the unexpected observation that KCC2 has also a structural role in the morphological maintenance of dendritic spines, one that is independent of its ability to transport ions. The intimate temporal coincidence between the developmental onset of KCC2 expression and the most intense phase of synaptogenesis during the brain growth spurt points to a possible role for this protein in synapse formation. Importantly, whether KCC2 plays a role in spinogenesis i.e. in induction of spines during the brain growth spurt has not been investigated so far. The results of the present work demonstrate that expression of KCC2 is not only a necessary but also a sufficient condition for the induction of functional glutamatergic spines during the brain growth spurt. The results of this work support the idea of KCC2 as an important synchronizing factor in the functional development of glutamatergic and GABAergic signaling.KCC2 on solukalvolla sijaitseva kalium-kloridi-kuljettaja, joka yhdessä mm. natrium-kalium-kloridi-kuljettajan (NKCC1) kanssa säätelee neuronien solunsisäistä kloridipitoisuutta. Kloridipitoisuuden muutokset puolestaan määräävät ovatko hermosolun GABA- tai glysiinireseptoreiden vasteet solukalvoa depolaroivia vai hyperpolaroivia. Aivojen varhaiskehityksen aikana nämä vasteet ovat usein depolaroivia ja aivojen kehittyessä KCC2:n toiminnallisen ilmentymisen kasvun seurauksena nämä vasteet muuttuvat hyperpolaroiviksi. KCC2:lla on myös toinen, ioninkuljetustoiminnoista riippumaton rakenteellinen rooli hermosolujen synapsien ylläpitämisessä. Ei ole kuitenkaan tiedossa mikä on KCC2:n merkitys uusien synapsien synnyssä. Aivotrauman tai epileptisen hermoaktiivisuuden seurauksena GABA- tai glysiinireseptoreiden vasteet muuttuvat usein depolaroiviksi aikuisaivoissa. Syyt tähän ovat paljolti tuntemattomia, mutta seurauksena voi olla mm. epileptisen aktiivisuuden leviämistä estävien aivomekanismien eroosio sekä hyperpolarisoivia GABA vasteita hyödyntävien antiepileptisten lääkkeiden tehon heikkeneminen. Tässä väitöskirjatyössä tutkitaan epileptisen aktiivisuuden laadullisesti erilaista vaikutusta KCC2:n kloridinkuljetustoiminnon ilmentymiseen aivojen varhaiskehityksen ja myöhemmän kehityksen välillä. Työn tulokset osoittavat, että kalpaiini-niminen entsyymi vastaa KCC2:n toiminnallisesta alasajosta epileptisen aktiivisuuden seurauksena. Tämän väitöskirjatyön toinen tärkeä löydös on, että KCC2:n geneettisesti ohjattu ylituotanto (yliekspressio) johtaa uusien synapsirakenteiden syntyyn

Endogenous brain-sparing responses in brain pH and PO2 in a rodent model of birth asphyxia

Abstract Aim To study brain-sparing physiological responses in a rodent model of birth asphyxia which reproduces the asphyxia-defining systemic hypoxia and hypercapnia. Methods Steady or intermittent asphyxia was induced for 15-45 min in anesthetized 6- and 11-days old rats and neonatal guinea pigs using gases containing 5% or 9% O2 plus 20% CO2 (in N2). Hypoxia and hypercapnia were induced with low O2 and high CO2, respectively. Oxygen partial pressure (PO2) and pH were measured with microsensors within the brain and subcutaneous (?body?) tissue. Blood lactate was measured after asphyxia. Results Brain and body PO2 fell to apparent zero with little recovery during 5% O2 asphyxia and 5% or 9% O2 hypoxia, and increased more than twofold during 20% CO2 hypercapnia. Unlike body PO2, brain PO2 recovered rapidly to control after a transient fall (rat), or was slightly higher than control (guinea pig) during 9% O2 asphyxia. Asphyxia (5% O2) induced a respiratory acidosis paralleled by a progressive metabolic (lact)acidosis that was much smaller within than outside the brain. Hypoxia (5% O2) produced a brain-confined alkalosis. Hypercapnia outlasting asphyxia suppressed pH recovery and prolonged the post-asphyxia PO2 overshoot. All pH changes were accompanied by consistent shifts in the blood-brain barrier potential. Conclusion Regardless of brain maturation stage, hypercapnia can restore brain PO2 and protect the brain against metabolic acidosis despite compromised oxygen availability during asphyxia. This effect extends to the recovery phase if normocapnia is restored slowly, and it is absent during hypoxia, demonstrating that exposure to hypoxia does not mimic asphyxia.Peer reviewe

GABA actions and ionic plasticity in epilepsy

Concepts of epilepsy, based on a simple change in neuronal excitation/inhibition balance, have subsided in face of recent insights into the large diversity and context-dependence of signaling mechanisms at the molecular, cellular and neuronal network level. GABAergic transmission exerts both seizure-suppressing and seizure-promoting actions. These two roles are prone to short-term and long-term alterations, evident both during epileptogenesis and during individual epileptiform events. The driving force of GABAergic currents is controlled by ion-regulatory molecules such as the neuronal K-Cl cotransporter KCC2 and cytosolic carbonic anhydrases. Accumulating evidence suggests that neuronal ion regulation is highly plastic, thereby contributing to the multiple roles ascribed to GABAergic signaling during epileptogenesis and epilepsy.Peer reviewe

KCC2-Mediated Cl- Extrusion Modulates Spontaneous Hippocampal Network Events in Perinatal Rats and Mice

It is generally thought that hippocampal neurons of perinatal rats and mice lack transport-functional K-Cl cotransporter KCC2, and that Cl- regulation is dominated by Cl- uptake via the Na-K-2Cl cotransporter NKCC1. Here, we demonstrate a robust enhancement of spontaneous hippocampal network events (giant depolarizing potentials [GDPs]) by the KCC2 inhibitor VU0463271 in neonatal rats and late-gestation, wildtype mouse embryos, but not in their KCC2-null littermates. VU0463271 increased the depolarizing GABAergic synaptic drive onto neonatal CA3 pyramidal neurons, increasing their spiking probability and synchrony during the rising phase of a GDP. Our data indicate that Cl- extrusion by KCC2 is involved in modulation of GDPs already at their developmental onset during the perinatal period in mice and rats.Peer reviewe

BDNF is required for seizure-induced but not developmental up-regulation of KCC2 in the neonatal hippocampus

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Forebrain-independent generation of hyperthermic convulsions in infant rats

Febrile seizures are the most common type of convulsive events in children. It is generally assumed that the generalization of these seizures is a result of brainstem invasion by the initial limbic seizure activity. Using precollicular transection in 13-day-old rats to isolate the forebrain from the brainstem, we demonstrate that the forebrain is not required for generation of tonic-clonic convulsions induced by hyperthermia or kainate. Compared with sham-operated littermate controls, latency to onset of convulsions in both models was significantly shorter in pups that had undergone precollicular transection, indicating suppression of the brainstem seizure network by the forebrain in the intact animal. We have shown previously that febrile seizures are precipitated by hyperthermia-induced respiratory alkalosis. Here, we show that triggering of hyperthermia-induced hyperventilation and consequent convulsions in transected animals are blocked by diazepam. The present data suggest that the role of endogenous brainstem activity in triggering tonic-clonic seizures should be re-evaluated in standard experimental models of limbic seizures. Our work sheds new light on the mechanisms that generate febrile seizures in children and, therefore, on how they might be treated.Peer reviewe

Loss of non-canonical KCC2 functions promotes developmental apoptosis of cortical projection neurons

KCC2, encoded in humans by the SLC12A5 gene, is a multifunctional neuron-specific protein initially identified as the chloride (Cl-) extruder critical for hyperpolarizing GABA(A) receptor currents. Independently of its canonical function as a K-Cl cotransporter, KCC2 regulates the actin cytoskeleton via molecular interactions mediated through its large intracellular C-terminal domain (CTD). Contrary to the common assumption that embryonic neocortical projection neurons express KCC2 at non-significant levels, here we show that loss of KCC2 enhances apoptosis of late-born upper-layer cortical projection neurons in the embryonic brain. In utero electroporation of plasmids encoding truncated, transport-dead KCC2 constructs retaining the CTD was as efficient as of that encoding full-length KCC2 in preventing elimination of migrating projection neurons upon conditional deletion of KCC2. This was in contrast to the effect of a full-length KCC2 construct bearing a CTD missense mutation (KCC2(R952H)), which disrupts cytoskeletal interactions and has been found in patients with neurological and psychiatric disorders, notably seizures and epilepsy. Together, our findings indicate ion transport-independent, CTD-mediated regulation of developmental apoptosis by KCC2 in migrating cortical projection neurons.Peer reviewe

An Ion Transport-Independent Role for the Cation-Chloride Cotransporter KCC2 in Dendritic Spinogenesis In Vivo

The neuron-specific K-Cl cotransporter, KCC2, is highly expressed in the vicinity of excitatory synapses in pyramidal neurons, and recent in vitro data suggest that this protein plays a role in the development of dendritic spines. The in vivo relevance of these observations is, however, unknown. Using in utero electroporation combined with post hoc iontophoretic injection of Lucifer Yellow, we show that premature expression of KCC2 induces a highly significant and permanent increase in dendritic spine density of layer 2/3 pyramidal neurons in the somatosensory cortex. Whole-cell recordings revealed that this increased spine density is correlated with an enhanced spontaneous excitatory activity in KCC2-transfected neurons. Precocious expression of the N-terminal deleted form of KCC2, which lacks the chloride transporter function, also increased spine density. In contrast, no effect on spine density was observed following in utero electroporation of a point mutant of KCC2 (KCC2-C568A) where both the cotransporter function and the interaction with the cytoskeleton are disrupted. Transfection of the C-terminal domain of KCC2, a region involved in the interaction with the dendritic cytoskeleton, also increased spine density. Collectively, these results demonstrate a role for KCC2 in excitatory synaptogenesis in vivo through a mechanism that is independent of its ion transport functio

Developmental Expression Patterns of KCC2 and Functionally Associated Molecules in the Human Brain

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