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

The Multifaceted Roles of KCC2 in Cortical Development

KCC2, best known as the neuron-specific chloride-extruder that sets the strength and polarity of GABAergic currents during neuronal maturation, isa multifunctional molecule that can regulate cytoskeletal dynamics via its C-terminal domain (CTD). We describe the molecular and cellular mechanisms involved in the multiple functions of KCC2 and its splice variants, ranging from developmental apoptosis and the control of early network events to the formation and plasticity of cortical dendritic spines. The versatility of KCC2 actions at the cellular and subcellular levels is also evident in mature neurons during plasticity, disease, and aging. Thus, KCC2 has emerged as one of the most important molecules that shape the overall neuronal phenotype.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

Expression and functions of KCC2 in the perinatal rodent cortex

KCC2 is the main neuronal K-Cl cotransporter, which lowers the intracellular Cl- concentration and thus maintains GABAA receptor-mediated inhibition during network activity in mature neurons. Independently of its function as an ion transporter, KCC2 was shown to be necessary for the formation of excitatory synapses, where it regulates the development of dendritic spines via modulation of the actin cytoskeleton, notably cofilin phosphorylation. KCC2 expression undergoes developmental up-regulation, which parallels the rostro-caudal CNS maturation in rodents, and underlies the robust negative shift in EGABA away from the depolarizing responses characteristic of immature neurons, as well as functionally regulates the intense spino- and synaptogenesis of cortical principal neurons. These findings posit KCC2 as a key molecule for coordinating the maturation and balance of excitatory and inhibitory neurotransmission during the brain growth spurt. While the role of KCC2 has been extensively studied during the first month of postnatal development in rodents, its functions in the perinatal cortex are only beginning to emerge. KCC2 knockout mice have altered hippocampal network activity already at embryonic day 18.5 when KCC2 is considered to be expressed at physiologically non-significant levels. Moreover, KCC2 has been found to regulate the migration of cortical interneurons by acting as a migratory stop signal in an ion transport-dependent way. On the other hand, in pyramidal neurons, KCC2-mediated ion-cotransport seems to be under kinetic suppression until around birth. Finally, the time window of the transition period of GABAergic signaling from depolarizing to hyperpolarizing, and the underlying upregulation of KCC2, coincides with the time when GABAAR modulators, notably general anesthetics, have been shown to induce lasting adverse effects on cognition and behavior in rodents. Thus far, it is not known why there is this defined time window of increased vulnerability to anesthetics, and the underlying molecular and cellular mechanisms warrant investigation. In this Thesis, the primary goal was to study the functional significance of early KCC2 expression in the developing neocortex and hippocampus of mice and rats and to assess the effects of genetic and pharmacological modulation of KCC2 expression on the formation of cortical networks. Particular focus was put on differentiating between the ion transport-dependent and -independent roles of KCC2. This work describes a novel ion transport-independent role of KCC2 in developmental neuroapoptosis, as well as ion transport-dependent functions of KCC2 in the regulation of hippocampal network events and developmental-stage dependent vulnerability to general anesthetics.KCC2-välitteinen kloridi-ekstruusio ylläpitää matalaa solunsisäistä kloriditasoa, ja vastaa siten GABAA-reseptorivälitteisestä inhibitiosta matuureissa hermosoluissa. Jyrsijöillä KCC2:n ilmentyminen aivokuoren hermosoluissa lisääntyy voimakkaasti vasta ensimmäisen postnataaliviikon jälkeen. KCC2 tasojen kasvaessa ja kloridi-ekstruusion tehostuessa varhaiskehitykselle tyypillliset depolaroivat GABAA välitteiset vasteet muuttuvat hermosoluja hyperpolarisoivaksi. Nopean, synaptisen inhibition lisäksi KCC2:lla on merkittävä rooli ärsyttävien hermoliitosten muodostumisessa, sillä se osallistuu dendriittien okahaarakkeiden aktiinirakenteiden säätelyyn. Tämä KCC2:n rakenteellinen rooli on ionikuljetuksesta riippumatonta. Vasta viime aikoina on ilmennyt, että KCC2:lla on vaikutusta myös sikiöaikaiseen aivojenkehitykseen. Aiemmin kuljettajan ilmentymistä on pidetty fysiologisesti merkityksettömänä hermosolujen varhaiskehityksessä. Tämän väitöskirjan osajulkaisuissa I-III tutkin, mikä merkitys varhaisella KCC2n ilmentymisellä on hiiren ja rotan kehittyvässä aivokuoressa sekä kuinka KCC2n ilmentymisen geneettinen tai farmakologinen muokkaus vaikuttavat hermoverkkojen muodostumiseen. Erityisenä painopisteenä työssäni on KCC2n ionisäätelyyn liittyvien ja siitä riippumattomien toimintojen eritteleminen. Väitöskirjani keskeinen uusi havainto on KCC2n ionisäätelystä riippumaton vaikutus sikiöaikaiseen neuroapoptoosiin sekä KCC2:n ionikuljetuksesta riippuvainen vaikutus hermoverkkoaktiivisuuteen ja hermosolujen rakenteeseen

K-Cl Cotransporter 2?mediated Cl? Extrusion Determines Developmental Stage?dependent Impact of Propofol Anesthesia on Dendritic Spines

Abstract Background General anesthetics potentiating γ-aminobutyric acid (GABA)–mediated signaling are known to induce a persistent decrement in excitatory synapse number in the cerebral cortex when applied during early postnatal development, while an opposite action is produced at later stages. Here, the authors test the hypothesis that the effect of general anesthetics on synaptogenesis depends upon the efficacy of GABA receptor type A (GABAA)–mediated inhibition controlled by the developmental up-regulation of the potassium-chloride (K-Cl) cotransporter 2 (KCC2). Methods In utero electroporation of KCC2 was used to prematurely increase the efficacy of (GABAA)–mediated inhibition in layer 2/3 pyramidal neurons in the immature rat somatosensory cortex. Parallel experiments with expression of the inward-rectifier potassium channel Kir2.1 were done to reduce intrinsic neuronal excitability. The effects of these genetic manipulations (n = 3 to 4 animals per experimental group) were evaluated using iontophoretic injection of Lucifer Yellow (n = 8 to 12 cells per animal). The total number of spines analyzed per group ranged between 907 and 3,371. Results The authors found a robust effect of the developmental up-regulation of KCC2–mediated Cl− transport on the age-dependent action of propofol on dendritic spines. Premature expression of KCC2, unlike expression of a transport-inactive KCC2 variant, prevented a propofol-induced decrease in spine density. In line with a reduction in neuronal excitability, the above result was qualitatively replicated by overexpression of Kir2.1. Conclusions The KCC2–dependent developmental increase in the efficacy of GABAA–mediated inhibition is a major determinant of the age-dependent actions of propofol on dendritic spinogenesis. </jats:sec