Gephyrin Interacts with the K-Cl Cotransporter KCC2 to Regulate Its Surface Expression and Function in Cortical Neurons.

The K+-Cl- cotransporter KCC2, encoded by the Slc12a5 gene, is a neuron-specific chloride extruder that tunes the strength and polarity of GABAA receptor-mediated transmission. In addition to its canonical ion transport function, KCC2 also regulates spinogenesis and excitatory synaptic function through interaction with a variety of molecular partners. KCC2 is enriched in the vicinity of both glutamatergic and GABAergic synapses, the activity of which in turn regulates its membrane stability and function. KCC2 interaction with the submembrane actin cytoskeleton via 4.1N is known to control its anchoring near glutamatergic synapses on dendritic spines. However, the molecular determinants of KCC2 clustering near GABAergic synapses remain unknown. Here, we used proteomics to identify novel KCC2 interacting proteins in the adult rat neocortex. We identified both known and novel candidate KCC2 partners, including some involved in neuronal development and synaptic transmission. These include gephyrin, the main scaffolding molecule at GABAergic synapses. Gephyrin interaction with endogenous KCC2 was confirmed by immunoprecipitation from rat neocortical extracts. We showed that gephyrin stabilizes plasmalemmal KCC2 and promotes its clustering in hippocampal neurons, mostly but not exclusively near GABAergic synapses, thereby controlling KCC2-mediated chloride extrusion. This study identifies gephyrin as a novel KCC2 anchoring molecule that regulates its membrane expression and function in cortical neurons.SIGNIFICANCE STATEMENT Fast synaptic inhibition in the brain is mediated by chloride-permeable GABAA receptors (GABAARs) and therefore relies on transmembrane chloride gradients. In neurons, these gradients are primarily maintained by the K/Cl cotransporter KCC2. Therefore, understanding the mechanisms controlling KCC2 expression and function is crucial to understand its physiological regulation and rescue its function in the pathology. KCC2 function depends on its membrane expression and clustering, but the underlying mechanisms remain unknown. We describe the interaction between KCC2 and gephyrin, the main scaffolding protein at inhibitory synapses. We show that gephyrin controls plasmalemmal KCC2 clustering and that loss of gephyrin compromises KCC2 function. Our data suggest functional units comprising GABAARs, gephyrin, and KCC2 act to regulate synaptic GABA signaling.

Hippocampal and neocortical BRAF mutant non-expansive lesions in focal epilepsies.

OBJECTIVE: Mesial Temporal Lobe Epilepsy-associated Hippocampal Sclerosis (MTLE-HS) is a syndrome associated with various aetiologies. We previously identified CD34-positive extravascular stellate cells (CD34+ cells) possibly related to BRAFV600E oncogenic variant in a subset of MTLE-HS. We aimed to identify the BRAFV600E oncogenic variants and characterise the CD34+ cells. METHODS: We analysed BRAFV600E oncogenic variant by digital droplet Polymerase Chain Reaction in 53 MTLE-HS samples (25 with CD34+ cells) and nine non-expansive neocortical lesions resected during epilepsy surgery (five with CD34+ cells). Ex vivo multi-electrode array recording, immunolabelling, methylation microarray and single nuclei RNAseq were performed on BRAFwildtype MTLE-HS and BRAFV600E mutant non-expansive lesion of hippocampus and/or neocortex. RESULTS: We identified a BRAFV600E oncogenic variant in five MTLE-HS samples with CD34+ cells (19%) and in five neocortical samples with CD34+ cells (100%). Single nuclei RNAseq of resected samples revealed two unique clusters of abnormal cells (including CD34+ cells) associated with senescence and oligodendrocyte development in both hippocampal and neocortical BRAFV600E mutant samples. The co-expression of the oncogene-induced senescence marker p16INK4A and the outer subventricular zone radial glia progenitor marker HOPX in CD34+ cells was confirmed by multiplex immunostaining. Pseudotime analysis showed that abnormal cells share a common lineage from progenitors to myelinating oligodendrocytes. Epilepsy surgery led to seizure freedom in eight of the 10 patients with BRAF mutant lesions. INTERPRETATION: BRAFV600E underlies a subset of MTLE-HS and epileptogenic non-expansive neocortical focal lesions. Detection of the oncogenic variant may help diagnosis and open perspectives for targeted therapies.

Cation-chloride cotransporters and the polarity of GABA signalling in mouse hippocampal parvalbumin interneurons.

KEY POINTS: Cation-chloride cotransporters (CCCs) play a critical role in controlling the efficacy and polarity of GABAA receptor (GABAA R)-mediated transmission in the brain, yet their expression and function in GABAergic interneurons has been overlooked. We compared the polarity of GABA signalling and the function of CCCs in mouse hippocampal pyramidal neurons and parvalbumin-expressing interneurons. Under resting conditions, GABAA R activation was mostly depolarizing and yet inhibitory in both cell types. KCC2 blockade further depolarized the reversal potential of GABAA R-mediated currents often above action potential threshold. However, during repetitive GABAA R activation, the postsynaptic response declined independently of the ion flux direction or KCC2 function, suggesting intracellular chloride build-up is not responsible for this form of plasticity. Our data demonstrate similar mechanisms of chloride regulation in mouse hippocampal pyramidal neurons and parvalbumin interneurons. ABSTRACT: Transmembrane chloride gradients govern the efficacy and polarity of GABA signalling in neurons and are usually maintained by the activity of cation-chloride cotransporters, such as KCC2 and NKCC1. Whereas their role is well established in cortical principal neurons, it remains poorly documented in GABAergic interneurons. We used complementary electrophysiological approaches to compare the effects of GABAA receptor (GABAA R) activation in adult mouse hippocampal parvalbumin interneurons (PV-INs) and pyramidal cells (PCs). Loose cell-attached, tight-seal and gramicidin-perforated patch recordings all show GABAA R-mediated transmission is slightly depolarizing and yet inhibitory in both PV-INs and PCs. Focal GABA uncaging in whole-cell recordings reveal that KCC2 and NKCC1 are functional in both PV-INs and PCs but differentially contribute to transmembrane chloride gradients in their soma and dendrites. Blocking KCC2 function depolarizes the reversal potential of GABAA R-mediated currents in PV-INs and PCs, often beyond firing threshold, showing KCC2 is essential to maintain the inhibitory effect of GABAA Rs. Finally, we show that repetitive 10 Hz activation of GABAA Rs in both PV-INs and PCs leads to a progressive decline of the postsynaptic response independently of the ion flux direction or KCC2 function. This suggests intraneuronal chloride build-up may not predominantly contribute to activity-dependent plasticity of GABAergic synapses in this frequency range. Altogether our data demonstrate similar mechanisms of chloride regulation in mouse hippocampal PV-INs and PCs and suggest KCC2 downregulation in the pathology may affect the valence of GABA signalling in both cell types.

KCC2 Gates Activity-Driven AMPA Receptor Traffic through Cofilin Phosphorylation.

Expression of the neuronal K/Cl transporter KCC2 is tightly regulated throughout development and by both normal and pathological neuronal activity. Changes in KCC2 expression have often been associated with altered chloride homeostasis and GABA signaling. However, recent evidence supports a role of KCC2 in the development and function of glutamatergic synapses through mechanisms that remain poorly understood. Here we show that suppressing KCC2 expression in rat hippocampal neurons precludes long-term potentiation of glutamatergic synapses specifically by preventing activity-driven membrane delivery of AMPA receptors. This effect is independent of KCC2 transporter function and can be accounted for by increased Rac1/PAK- and LIMK-dependent cofilin phosphorylation and actin polymerization in dendritic spines. Our results demonstrate that KCC2 plays a critical role in the regulation of spine actin cytoskeleton and gates long-term plasticity at excitatory synapses in cortical neurons.

Activity-dependent regulation of the K/Cl transporter KCC2 membrane diffusion, clustering, and function in hippocampal neurons.

The neuronal K/Cl transporter KCC2 exports chloride ions and thereby influences the efficacy and polarity of GABA signaling in the brain. KCC2 is also critical for dendritic spine morphogenesis and the maintenance of glutamatergic transmission in cortical neurons. Because KCC2 plays a pivotal role in the function of central synapses, it is of particular importance to understand the cellular and molecular mechanisms underlying its regulation. Here, we studied the impact of membrane diffusion and clustering on KCC2 function. KCC2 forms clusters in the vicinity of both excitatory and inhibitory synapses. Using quantum-dot-based single-particle tracking on rat primary hippocampal neurons, we show that KCC2 is slowed down and confined at excitatory and inhibitory synapses compared with extrasynaptic regions. However, KCC2 escapes inhibitory synapses faster than excitatory synapses, reflecting stronger molecular constraints at the latter. Interfering with KCC2-actin interactions or inhibiting F-actin polymerization releases diffusion constraints on KCC2 at excitatory but not inhibitory synapses. Thus, F-actin constrains KCC2 diffusion at excitatory synapses, whereas KCC2 is confined at inhibitory synapses by a distinct mechanism. Finally, increased neuronal activity rapidly increases the diffusion coefficient and decreases the dwell time of KCC2 at excitatory synapses. This effect involves NMDAR activation, Ca(2+) influx, KCC2 S940 dephosphorylation and calpain protease cleavage of KCC2 and is accompanied by reduced KCC2 clustering and ion transport function. Thus, activity-dependent regulation of KCC2 lateral diffusion and clustering allows for a rapid regulation of chloride homeostasis in neurons.

The neuronal K-Cl cotransporter KCC2 influences postsynaptic AMPA receptor content and lateral diffusion in dendritic spines.

The K-Cl cotransporter KCC2 plays an essential role in neuronal chloride homeostasis, and thereby influences the efficacy and polarity of GABA signaling. Although KCC2 is expressed throughout the somatodendritic membrane, it is remarkably enriched in dendritic spines, which host most glutamatergic synapses in cortical neurons. KCC2 has been shown to influence spine morphogenesis and functional maturation in developing neurons, but its function in mature dendritic spines remains unknown. Here, we report that suppressing KCC2 expression decreases the efficacy of excitatory synapses in mature hippocampal neurons. This effect correlates with a reduced postsynaptic aggregation of GluR1-containing AMPA receptors and is mimicked by a dominant negative mutant of KCC2 interaction with cytoskeleton but not by pharmacological suppression of KCC2 function. Single-particle tracking experiments reveal that suppressing KCC2 increases lateral diffusion of the mobile fraction of AMPA receptor subunit GluR1 in spines but not in adjacent dendritic shafts. Increased diffusion was also observed for transmembrane but not membrane-anchored recombinant neuronal cell adhesion molecules. We suggest that KCC2, likely through interactions with the actin cytoskeleton, hinders transmembrane protein diffusion, and thereby contributes to their confinement within dendritic spines.

Targeting pathological cells with senolytic drugs reduces seizures in neurodevelopmental mTOR-related epilepsy.

Cortical malformations such as focal cortical dysplasia type II (FCDII) are associated with pediatric drug-resistant epilepsy that necessitates neurosurgery. FCDII results from somatic mosaicism due to post-zygotic mutations in genes of the PI3K-AKT-mTOR pathway, which produce a subset of dysmorphic cells clustered within healthy brain tissue. Here we show a correlation between epileptiform activity in acute cortical slices obtained from human surgical FCDII brain tissues and the density of dysmorphic neurons. We uncovered multiple signatures of cellular senescence in these pathological cells, including p53/p16 expression, SASP expression and senescence-associated ß-galactosidase activity. We also show that administration of senolytic drugs (dasatinib/quercetin) decreases the load of senescent cells and reduces seizure frequency in an MtorS2215F FCDII preclinical mouse model, providing proof of concept that senotherapy may be a useful approach to control seizures. These findings pave the way for therapeutic strategies selectively targeting mutated senescent cells in FCDII brain tissue.

Presynaptic but not postsynaptic GABA signaling at unitary mossy fiber synapses.

Dentate gyrus granule cells have been suggested to corelease GABA and glutamate both in juvenile animals and under pathological conditions in adults. Although mossy fiber terminals (MFTs) are known to express glutamic acid decarboxylase (GAD) in early postnatal development, the functional role of GABA synthesis in MFTs remains controversial, and direct evidence for synaptic GABA release from MFTs is missing. Here, using GAD67-GFP transgenic mice, we show that GAD67 is expressed only in a population of immature granule cells in juvenile animals. We demonstrate that GABA can be released from these cells and modulate mossy fiber excitability through activation of GABAB autoreceptors. However, unitary postsynaptic currents generated by individual, GAD67-expressing granule cells are purely glutamatergic in all postsynaptic cell types tested. Thus GAD67 expression does not endow dentate gyrus granule cells with a full GABAergic phenotype and GABA primarily instructs the pre- rather than the postsynaptic element.

Vezatin is essential for dendritic spine morphogenesis and functional synaptic maturation.

Vezatin is an integral membrane protein associated with cell-cell adhesion complex and actin cytoskeleton. It is expressed in the developing and mature mammalian brain, but its neuronal function is unknown. Here, we show that Vezatin localizes in spines in mature mouse hippocampal neurons and codistributes with PSD95, a major scaffolding protein of the excitatory postsynaptic density. Forebrain-specific conditional ablation of Vezatin induced anxiety-like behavior and impaired cued fear-conditioning memory response. Vezatin knock-down in cultured hippocampal neurons and Vezatin conditional knock-out in mice led to a significantly increased proportion of stubby spines and a reduced proportion of mature dendritic spines. PSD95 remained tethered to presynaptic terminals in Vezatin-deficient hippocampal neurons, suggesting that the reduced expression of Vezatin does not compromise the maintenance of synaptic connections. Accordingly, neither the amplitude nor the frequency of miniature EPSCs was affected in Vezatin-deficient hippocampal neurons. However, the AMPA/NMDA ratio of evoked EPSCs was reduced, suggesting impaired functional maturation of excitatory synapses. These results suggest a role of Vezatin in dendritic spine morphogenesis and functional synaptic maturation.

Input-specific learning rules at excitatory synapses onto hippocampal parvalbumin-expressing interneurons.

Hippocampal parvalbumin-expressing interneurons (PV INs) provide fast and reliable GABAergic signalling to principal cells and orchestrate hippocampal ensemble activities. Precise coordination of principal cell activity by PV INs relies in part on the efficacy of excitatory afferents that recruit them in the hippocampal network. Feed-forward (FF) inputs in particular from Schaffer collaterals influence spike timing precision in CA1 principal cells whereas local feedback (FB) inputs may contribute to pacemaker activities. Although PV INs have been shown to undergo activity-dependent long term plasticity, how both inputs are modulated during principal cell firing is unknown. Here we show that FF and FB synapses onto PV INs are endowed with distinct postsynaptic glutamate receptors which set opposing long-term plasticity rules. Inward-rectifying AMPA receptors (AMPARs) expressed at both FF and FB inputs mediate a form of anti-Hebbian long term potentiation (LTP), relying on coincident membrane hyperpolarization and synaptic activation. In contrast, FF inputs are largely devoid of NMDA receptors (NMDARs) which are more abundant at FB afferents and confer on them an additional form of LTP with Hebbian properties. Both forms of LTP are expressed with no apparent change in presynaptic function. The specific endowment of FF and FB inputs with distinct coincidence detectors allow them to be differentially tuned upon high frequency afferent activity. Thus, high frequency (>20 Hz) stimulation specifically potentiates FB, but not FF afferents. We propose that these differential, input-specific learning rules may allow PV INs to adapt to changes in hippocampal activity while preserving their precisely timed, clockwork operation.

A human mutation in Gabrg2 associated with generalized epilepsy alters the membrane dynamics of GABAA receptors.

Neuronal activity modulates the membrane diffusion of postsynaptic γ-aminobutyric acid (GABA)(A) receptors (GABA(A)Rs), thereby regulating the efficacy of GABAergic synapses. The K289M mutation in GABA(A)Rs subunit γ2 has been associated with the generalized epilepsy with febrile seizures plus (GEFS+) syndrome. This mutation accelerates receptor deactivation and therefore reduces inhibitory synaptic transmission. Yet, it is not clear why this mutation specifically promotes febrile seizures. We show that upon raising temperature both the number of GABA(A)Rs clusters and the frequency of miniature inhibitory postsynaptic currents decreased in neurons expressing the K289M mutant but not wild-type (WT) recombinant γ2. Single-particle tracking experiments revealed that raising temperature increases the membrane diffusion of synaptic GABA(A)Rs containing the K289M mutant but not WT recombinant γ2. This effect was mediated by enhanced neuronal activity as it was blocked by glutamate receptor antagonists and was mimicked by the convulsant 4-aminopyridine. Our data suggest the K289M mutation in γ2 confers GABA(A)Rs with enhanced sensitivity of their membrane diffusion to neuronal activity. Enhanced activity during hyperthermia may then trigger the escape of receptors from synapses and thereby further reduce the efficacy of GABAergic inhibition. Alteration of the membrane diffusion of neurotransmitter receptors therefore represents a new mechanism in human epilepsy.

Silencing KCC2 in mouse dorsal hippocampus compromises spatial and contextual memory.

Delayed upregulation of the neuronal chloride extruder KCC2 underlies the progressive shift in GABA signaling polarity during development. Conversely, KCC2 downregulation is observed in a variety of neurological and psychiatric disorders often associated with cognitive impairment. Reduced KCC2 expression and function in mature networks may disrupt GABA signaling and promote anomalous network activities underlying these disorders. However, the causal link between KCC2 downregulation, altered brain rhythmogenesis, and cognitive function remains elusive. Here, by combining behavioral exploration with in vivo electrophysiology we assessed the impact of chronic KCC2 downregulation in mouse dorsal hippocampus and showed it compromises both spatial and contextual memory. This was associated with altered hippocampal rhythmogenesis and neuronal hyperexcitability, with increased burst firing in CA1 neurons during non-REM sleep. Reducing neuronal excitability with terbinafine, a specific Task-3 leak potassium channel opener, occluded the impairment of contextual memory upon KCC2 knockdown. Our results establish a causal relationship between KCC2 expression and cognitive performance and suggest that non-epileptiform rhythmopathies and neuronal hyperexcitability are central to the deficits caused by KCC2 downregulation in the adult mouse brain.

Epilepsy related to focal neuronal lipofuscinosis: extra-frontal localization, EEG signatures and GABA involvement.

Focal neuronal lipofuscinosis (FNL) is an uncommon epileptic disorder related to an excess of lipofuscin accumulation within dysmorphic-appearing neurons (DANs), whose epileptogenic mechanisms are still poorly understood. It shares some clinical and neuroimaging similarities with focal cortical dysplasia of type IIb (FCDIIb), but it represents a different pathological entity. Here, we identified two patients with FNL among a 10-year cohort of 323 patients who underwent neurosurgery for a focal pharmacoresistant epilepsy. We describe the electroclinical, metabolic and neuropathological features of both patients with FNL who benefited from a comprehensive presurgical investigation. While the previous reports showed frontal lobe localization of the lesion, FNL was identified in the temporal lobe, in one of our patients. EEG investigations in both patients showed striking focal and rich interictal activity resembling that described in FCDIIb. Besides focal intraneuronal lipofuscin accumulation, the neuropathological analysis demonstrated that somata of DANs were surrounded by a large amount of GABAergic presynaptic buttons, suggesting the involvement of interneurons in the epileptogenicity of FNL. To further explore the role of GABAergic transmission in the generation of epileptiform activity in FNL, we performed in vitro multi-electrode array recordings on the post-surgery tissue from one patient. Spontaneous interictal-like discharges (IILDs) were identified only in the restricted area displaying the highest density of lipofuscin-containing DANs, suggesting a close correlation between the density of lipofuscin-containing neurons and epileptogenicity. Moreover, IILDs were blocked by the GABAA receptor antagonist gabazine. All together, these findings showed how GABA signaling may contribute to the generation of interictal-like activity in FNL tissue.

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, is a 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.

The metabolic signaling of the nucleoredoxin-like 2 gene supports brain function.

The nucleoredoxin gene NXNL2 encodes for two products through alternative splicing, rod-derived cone viability factor-2 (RdCVF2) that mediates neuronal survival and the thioredoxin-related protein (RdCVF2L), an enzyme that regulates the phosphorylation of TAU. To investigate the link between NXNL2 and tauopathies, we studied the Nxnl2 knockout mouse (Nxnl2-/-). We established the expression pattern of the Nxnl2 gene in the brain using a Nxnl2 reporter mouse line, and characterized the behavior of the Nxnl2-/- mouse at 2 months of age. Additionally, long term potentiation and metabolomic from hippocampal specimens were collected at 2 months of age. We studied TAU oligomerization, phosphorylation and aggregation in Nxnl2-/- brain at 18 months of age. Finally, newborn Nxnl2-/- mice were treated with adeno-associated viral vectors encoding for RdCVF2, RdCVF2L or both and measured the effect of this therapy on long-term potential, glucose metabolism and late-onset tauopathy. Nxnl2-/- mice at 2 months of age showed severe behavioral deficiency in fear, pain sensitivity, coordination, learning and memory. The Nxnl2-/- also showed deficits in long-term potentiation, demonstrating that the Nxnl2 gene is involved in regulating brain functions. Dual delivery of RdCVF2 and RdCVF2L in newborn Nxnl2-/- mice fully correct long-term potentiation through their synergistic action. The expression pattern of the Nxnl2 gene in the brain shows a predominant expression in circumventricular organs, such as the area postrema. Glucose metabolism of the hippocampus of Nxnl2-/- mice at 2 months of age was reduced, and was not corrected by gene therapy. At 18-month-old Nxnl2-/- mice showed brain stigmas of tauopathy, such as oligomerization, phosphorylation and aggregation of TAU. This late-onset tauopathy can be prevented, albeit with modest efficacy, by recombinant AAVs administrated to newborn mice. The Nxnl2-/- mice have memory dysfunction at 2-months that resembles mild-cognitive impairment and at 18-months exhibit tauopathy, resembling to the progression of Alzheimer's disease. We propose the Nxnl2-/- mouse is a model to study multistage aged related neurodegenerative diseases. The NXNL2 metabolic and redox signaling is a new area of therapeutic research in neurodegenerative diseases.

KCC2 membrane diffusion tunes neuronal chloride homeostasis.

Neuronal Cl- homeostasis is regulated by the activity of two cation chloride co-transporters (CCCs), the K+-Cl- cotransporter KCC2 and the Na+-K+-Cl- cotransporter NKCC1, which are primarily extruding and importing chloride in neurons, respectively. Several neurological and psychiatric disorders including epilepsy, neuropathic pain, schizophrenia and autism are associated with altered neuronal chloride (Cl-) homeostasis. A current view is that the accumulation of intracellular Cl- in neurons as a result of KCC2 down-regulation and/or NKCC1 up-regulation may weaken inhibitory GABA signaling and thereby promote the development of pathological activities. CCC activity is determined mainly by their level of expression in the plasma membrane. Furthermore, CCCs undergo "diffusion-trapping" in the membrane, a mechanism that is rapidly adjusted by activity-dependent post-translational modifications i.e. phosphorylation/dephosphorylation of key serine and threonine residues. This represents probably the most rapid cellular mechanism for adapting CCC function to changes in neuronal activity. Therefore, interfering with these mechanisms may help restoring Cl- homeostasis and inhibition under pathological conditions. This article is part of the special issue entitled 'Mobility and trafficking of neuronal membrane proteins'.

Two novel CLCN2 mutations accelerating chloride channel deactivation are associated with idiopathic generalized epilepsy.

Heterozygous mutations in the CLCN2 gene encoding the voltage-gated chloride channel CLC2 have been identified in patients with idiopathic generalized epilepsy (IGE). Yet the involvement of CLCN2 in epilepsy remains controversial. To investigate the involvement of CLCN2 in another independent sample, we screened 52 unrelated patients from IGE families and 23 patients with Doose syndrome for mutations in CLCN2. No mutations were found in patients with Doose syndrome. In three unrelated IGE families, we identified two novel missense mutations, p.Arg235Gln and p.Arg577Gln, which were absent in large ethnically-matched control populations, and one novel p.Arg644Cys variant, which was also found in five Indian controls. Functional characterization of mutant channels using heterologous expression in mammalian cells and whole-cell patch-clamp recordings revealed faster deactivation kinetics as the major phenotype of both missense mutations. This finding predicts a loss of function that may contribute to intracellular chloride accumulation or neuronal hyperexcitability. However, the incomplete segregation of the mutations among affected members and the transmission by unaffected parents suggests that these CLCN2 mutations alone are not sufficient to induce epilepsy. They may instead represent susceptibility factors among other so far undetected genetic alterations in the respective families.

Reciprocal Regulation of KCC2 Trafficking and Synaptic Activity.

The main inhibitory neurotransmitter receptors in the adult central nervous system (CNS) are type A γ-aminobutyric acid receptors (GABAARs) and glycine receptors (GlyRs). Synaptic responses mediated by GlyR and GABAAR display a hyperpolarizing shift during development. This shift relies mainly on the developmental up-regulation of the K+-Cl- co-transporter KCC2 responsible for the extrusion of Cl-. In mature neurons, altered KCC2 function-mainly through increased endocytosis-leads to the re-emergence of depolarizing GABAergic and glycinergic signaling, which promotes hyperexcitability and pathological activities. Identifying signaling pathways and molecular partners that control KCC2 surface stability thus represents a key step in the development of novel therapeutic strategies. Here, we present our current knowledge on the cellular and molecular mechanisms governing the plasma membrane turnover rate of the transporter under resting conditions and in response to synaptic activity. We also discuss the notion that KCC2 lateral diffusion is one of the first parameters modulating the transporter membrane stability, allowing for rapid adaptation of Cl- transport to changes in neuronal activity.

KCC2 Regulates Neuronal Excitability and Hippocampal Activity via Interaction with Task-3 Channels.

KCC2 regulates neuronal transmembrane chloride gradients and thereby controls GABA signaling in the brain. KCC2 downregulation is observed in numerous neurological and psychiatric disorders. Paradoxical, excitatory GABA signaling is usually assumed to contribute to abnormal network activity underlying the pathology. We tested this hypothesis and explored the functional impact of chronic KCC2 downregulation in the rat dentate gyrus. Although the reversal potential of GABAA receptor currents is depolarized in KCC2 knockdown neurons, this shift is compensated by depolarization of the resting membrane potential. This reflects downregulation of leak potassium currents. We show KCC2 interacts with Task-3 (KCNK9) channels and is required for their membrane expression. Increased neuronal excitability upon KCC2 suppression altered dentate gyrus rhythmogenesis, which could be normalized by chemogenetic hyperpolarization. Our data reveal KCC2 downregulation engages complex synaptic and cellular alterations beyond GABA signaling that perturb network activity thus offering additional targets for therapeutic intervention.