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

Influx of Calcium through L-type Calcium Channels in Early Postnatal Regulation of Chloride Transporters in the Rat Hippocampus

During the early postnatal period, GABAB receptor activation facilitates L-type calcium current in rat hippocampus. One developmental process that L-type current may regulate is the change in expression of the K+Cl− co-transporter (KCC2) and N+K+2Cl− co-transporter (NKCC1), which are involved in the maturation of the GABAergic system. The present study investigated the connection between L-type current, GABAB receptors, and expression of chloride transporters during development. The facilitation of L-type current by GABAB receptors is more prominent in the second week of development, with the highest percentage of cells exhibiting facilitation in cultures isolated from 7 day old rats (37.5%). The protein levels of KCC2 and NKCC1 were investigated to determine the developmental timecourse of expression as well as expression following treatment with an L-type channel antagonist and a GABAB receptor agonist. The time course of both chloride transporters in culture mimics that seen in hippocampal tissue isolated from various ages. KCC2 levels increased drastically in the first two postnatal weeks while NKCC1 remained relatively stable, suggesting that the ratio of the chloride transporters is important in mediating the developmental change in chloride reversal potential. Treatment of cultures with the L-type antagonist nimodipine did not affect protein levels of NKCC1, but significantly decreased the upregulation of KCC2 during the first postnatal week. In addition, calcium current facilitation occurs slightly before the large increase in KCC2 expression. These results suggest that the expression of KCC2 is regulated by calcium influx through L-type channels in the early postnatal period in hippocampal neurons

Neuronal carbonic anhydrase VII provides GABAergic excitatory drive to exacerbate febrile seizures

Peer reviewe

A variant of KCC2 from patients with febrile seizures impairs neuronal Cl- extrusion and dendritic spine formation

Genetic variation in SLC12A5 which encodes KCC2, the neuron‐specific cation‐chloride cotransporter that is essential for hyperpolarizing GABAergic signaling and formation of cortical dendritic spines, has not been reported in human disease. Screening of SLC12A5 revealed a co‐segregating variant (KCC2‐R952H) in an Australian family with febrile seizures. We show that KCC2‐R952H reduces neuronal Cl− extrusion and has a compromised ability to induce dendritic spines in vivo and in vitro. Biochemical analyses indicate a reduced surface expression of KCC2‐R952H which likely contributes to the functional deficits. Our data suggest that KCC2‐R952H is a bona fide susceptibility variant for febrile seizures.Peer reviewe

X-ray crystallographic analysis of homooligomeric flavin containing cysteine decarboxylases

Die Familie der homooligomeren Flavin-enthaltenden Cystein-Decarboxylasen (HFCD) kann in zwei Unterfamilien eingeteilt werden. Die in dieser Arbeit untersuchten Proteine EpiD und MrsD gehören zusammen mit MutD zu den LanD-Proteinen. Diese katalysieren einen wichtigen Reaktionsschritt, eine oxidative Decarboxylierung eines C-terminalen Cysteins, in der Biosynthese der Lantibiotika Epidermin, Mersacidin und Mutacin III. Zwei weitere Mitglieder der HFCD-Proteine, das bakterielle Dfp und HAL3 aus Arabidopsis thaliana (AtHAL3) katalysieren die nicht oxidative Decarboxylierung von (R)-4'-Phospho-N-Panthenoylcystein zu 4'-Phosphopantethein in der Biosynthese des bei allen Organismen sehr wichtigen Coenzyms A (CoA). Die Kristallstrukturen der Proteine EpiD und MrsD sowie der inaktiven Mutante EpiD-H67N im Komplex mit dem Pentapeptid DSYTC wurden gelöst. Die Homododekamere der untersuchten Proteine bilden Partikel mit einer 23-Punktsymmetrie, mit auf den Spitzen eines Tetraeders lokalisierten Trimeren. Die Monomere besitzen eine Rossmann-artige Tertiärstruktur, wobei sich die Wechselwirkungen dieser Tertiärstruktur, die häufig von dinukleotidbindenden Proteinen verwendet wird, mit den Kofaktoren FMN und FAD bei den HFCD-Proteinen deutlich von denen anderer Flavoproteine unterscheiden. Die beiden charakteristischen Sequenzmotive dieser Proteinfamilie, PASANT und PXMNXXMW, sind an der Kofaktorbindung, Wechselwirkungen mit dem Substrat und wichtigen strukturellen Eigenschaften beteiligt. Die Struktur des Komplexes von EpiD-H67N mit DSYTC und ein modellierter Komplex von MrsD mit den fünf C-terminalen Aminosäuren des Vorläuferpeptides von Mersacidin (MrsA) erklären die unüblich breite Substratspezifität von EpiD und die Unterschiede zu derjenigen von MrsD. Die von den Proteinen der HFCD-Familie katalysierte Decarboxylierung eines C-terminalen Cysteins zeigt einen neuartigen Reaktionsmechanismus, der sich deutlich von den bisher bekannten Decarboxylierungen und Dehydrierungen unterscheidet. Als ersten Reaktionsschritt kann man eine Oxidation der Thiolgruppe annehmen, da nur diese Kontakt mit dem Kofaktor hat. Die entstehende beta-Thioaldehyd-Carbonsäure decarboxyliert danach analog zu den beta-Keto-Carbonsäuren wahrscheinlich spontan.The family of homooligomeric flavin containing cystein decarboxylases (HFCD) can be divided into two subfamilies. The proteins investigated in this work, EpiD and MrsD belong together with MutD to the so-called LanD proteins. They catalyse an important reaction step, an oxidative decarboxylation of a C-terminal cysteine, during the biosynthesis of the lantibiotics epidermin, mersacidin and mutacin III, respectively. Two further members of the HFCD proteins, the bacterial Dfp and HAL3 from Arabidopsis thaliana (AtHAL3) catalyse the non-oxidative decarboxylation of (R)-4'-phospho-N-panthenoylcysteine to 4'-Phosphopantetheine during the biosynthesis of coenzyme A, which is very important for all organisms. The crystal structure of the proteins EpiD and MrsD, as well as the inactive mutant EpiD-H67N complexed with the pentapeptide DSYTC could be solved. The homododecamers of the investigated proteins build up particles of 23 point symmetry, with trimers localised at the vertices of a tetrahedron. The protomers show a typical Rossmann fold but the interactions of this tertiary structure, which is used often by dinucleotide binding proteins, with the cofactors FMN and FAD within the HFCD family differs clearly from that of other flavoproteins. The two characteristic sequence motifs PASANT and PXMNXXWM of this family are involved in binding of the cofactor and substrate as well as important structural features. The structure of the complex of EpiD with DSYTC and a modeled complex of MrsD with the five C-terminal amino acids of the precursor of mersacidin MrsA explain the unusually broad substrate specificity of EpiD and the differences to MrsD. The catalysed decarboxylation of a C-terminal cysteine shows a novel mechanism, which differs clearly from the known decarboxylations and dehydrogenations. As a first step one could suggest the oxidation of the thiol group, because it is the only one which is in contact with the cofactor. The evolving beta-thioaldehyd might decarboxylate spontaneously like the analogous beta-ketocarbonic acids

A variant of KCC2 from patients with febrile seizures impairs neuronal Cl- extrusion and dendritic spine formation

Genetic variation in SLC12A5 which encodes KCC2, the neuron‐specific cation‐chloride cotransporter that is essential for hyperpolarizing GABAergic signaling and formation of cortical dendritic spines, has not been reported in human disease. Screening of SLC12A5 revealed a co‐segregating variant (KCC2‐R952H) in an Australian family with febrile seizures. We show that KCC2‐R952H reduces neuronal Cl− extrusion and has a compromised ability to induce dendritic spines in vivo and in vitro. Biochemical analyses indicate a reduced surface expression of KCC2‐R952H which likely contributes to the functional deficits. Our data suggest that KCC2‐R952H is a bona fide susceptibility variant for febrile seizures.Peer reviewe

Mutations in SLC12A5 in epilepsy of infancy with migrating focal seizures

The potassium-chloride co-transporter KCC2, encoded by SLC12A5, plays a fundamental role in fast synaptic inhibition by maintaining a hyperpolarizing gradient for chloride ions. KCC2 dysfunction has been implicated in human epilepsy, but to date, no monogenic KCC2-related epilepsy disorders have been described. Here we show recessive loss-of-function SLC12A5 mutations in patients with a severe infantile-onset pharmacoresistant epilepsy syndrome, epilepsy of infancy with migrating focal seizures (EIMFS). Decreased KCC2 surface expression, reduced protein glycosylation and impaired chloride extrusion contribute to loss of KCC2 activity, thereby impairing normal synaptic inhibition and promoting neuronal excitability in this early-onset epileptic encephalopathy

Carbonic anhydrase seven bundles filamentous actin and regulates dendritic spine morphology and density

Intracellular pH is a potent modulator of neuronal functions. By catalyzing (de)hydration of CO2, intracellular carbonic anhydrase (CA(i)) isoforms CA2 and CA7 contribute to neuronal pH buffering and dynamics. The presence of two highly active isoforms in neurons suggests that they may serve isozyme-specific functions unrelated to CO2-(de)hydration. Here, we show that CA7, unlike CA2, binds to filamentous actin, and its overexpression induces formation of thick actin bundles and membrane protrusions in fibroblasts. In CA7-overexpressing neurons, CA7 is enriched in dendritic spines, which leads to aberrant spine morphology. We identified amino acids unique to CA7 that are required for direct actin interactions, promoting actin filament bundling and spine targeting. Disruption of CA7 expression in neocortical neurons leads to higher spine density due to increased proportion of small spines. Thus, our work demonstrates highly distinct subcellular expression patterns of CA7 and CA2, and a novel, structural role of CA7.Peer reviewe

Endogenous neurosteroids influence synaptic GABA_Areceptors during post-natal development

GABA plays a key role in both embryonic and neonatal brain development. For example, during early neonatal nervous system maturation, synaptic transmission, mediated by GABA A receptors (GABA ARs), undergoes a temporally specific form of synaptic plasticity to accommodate the changing requirements of maturing neural networks. Specifically, the duration of miniature inhibitory postsynaptic currents (mIPSCs), resulting from vesicular GABA activating synaptic GABA ARs, is reduced, permitting neurones to appropriately influence the window for postsynaptic excitation. Conventionally, programmed expression changes to the subtype of synaptic GABA AR are primarily implicated in this plasticity. However, it is now evident that, in developing thalamic and cortical principal- and inter-neurones, an endogenous neurosteroid tone (eg, allopregnanolone) enhances synaptic GABA AR function. Furthermore, a cessation of steroidogenesis, as a result of a lack of substrate, or a co-factor, appears to be primarily responsible for early neonatal changes to GABAergic synaptic transmission, followed by further refinement, which results from subsequent alterations of the GABA AR subtype. The timing of this cessation of neurosteroid influence is neurone-specific, occurring by postnatal day (P)10 in the thalamus but approximately 1 week later in the cortex. Neurosteroid levels are not static and change dynamically in a variety of physiological and pathophysiological scenarios. Given that GABA plays an important role in brain development, abnormal perturbations of neonatal GABA AR-active neurosteroids may have not only a considerable immediate, but also a longer-term impact upon neural network activity. Here, we review recent evidence indicating that changes in neurosteroidogenesis substantially influence neonatal GABAergic synaptic transmission. We discuss the physiological relevance of these findings and how the interference of neurosteroid-GABA AR interaction early in life may contribute to psychiatric conditions later in life. </p