Data_Sheet_1_K+/Cl− cotransporter 2 (KCC2) and Na+/HCO3− cotransporter 1 (NBCe1) interaction modulates profile of KCC2 phosphorylation.PDF

K+/Cl− cotransporter 2 (KCC2) is a major Cl− extruder in mature neurons and is responsible for the establishment of low intracellular [Cl−], necessary for fast hyperpolarizing GABAA-receptor mediated synaptic inhibition. Electrogenic sodium bicarbonate cotransporter 1 (NBCe1) is a pH regulatory protein expressed in neurons and glial cells. An interactome study identified NBCe1 as a possible interaction partner of KCC2. In this study, we investigated the putative effect of KCC2/NBCe1 interaction in baseline and the stimulus-induced phosphorylation pattern and function of KCC2. Primary mouse hippocampal neuronal cultures from wildtype (WT) and Nbce1-deficient mice, as well as HEK-293 cells stably transfected with KCC2WT, were used. The results show that KCC2 and NBCe1 are interaction partners in the mouse brain. In HEKKCC2 cells, pharmacological inhibition of NBCs with S0859 prevented staurosporine- and 4-aminopyridine (4AP)-induced KCC2 activation. In mature cultures of hippocampal neurons, however, S0859 completely inhibited postsynaptic GABAAR and, thus, could not be used as a tool to investigate the role of NBCs in GABA-dependent neuronal networks. In Nbce1-deficient immature hippocampal neurons, baseline phosphorylation of KCC2 at S940 was downregulated, compared to WT, and exposure to staurosporine failed to reduce pKCC2 S940 and T1007. In Nbce1-deficient mature neurons, baseline levels of pKCC2 S940 and T1007 were upregulated compared to WT, whereas after 4AP treatment, pKCC2 S940 was downregulated, and pKCC2 T1007 was further upregulated. Functional experiments showed that the levels of GABAAR reversal potential, baseline intracellular [Cl−], Cl− extrusion, and baseline intracellular pH were similar between WT and Nbce1-deficient neurons. Altogether, our data provide a primary description of the properties of KCC2/NBCe1 protein-protein interaction and implicate modulation of stimulus-mediated phosphorylation of KCC2 by NBCe1/KCC2 interaction—a mechanism with putative pathophysiological relevance.</p

Inhibitors of protein synthesis preserve intracellular ATP-content during oxygen deprivation.

<p>Human neuronal SK-N-SH cells were cultured in an oxygen-free atmosphere for 12–72 h in the presence of 5 µg/ml cycloheximide (closed triangles), 0.1 mM thiopental (closed rhombi), 0.5 mM thiopental (asterisks), or left untreated (closed squares). Cells, cultured in a normoxic atmosphere (open squares) served as a control. ATP content of cells was measured in lysates by an ATP-driven luciferase assay. Determined relative light units (RLU) were normalized to protein content and represent the means ± standard deviations of three independent experiments. Experimental groups were statistically evaluated by performing two-way ANOVA followed by the Bonferroni’s <i>post hoc</i> test. Statistical differences of oxygen deprived SK-N-SH cells (closed squares) compared to oxygen-deprived cells treated with 5 µg/ml cycloheximide (^###, p<0.001), 0.5 mM thiopental (^{<>\raster(60%)="rg3"<>}, p<0.05; ^{<>\raster(60%)="rg3"<><>\raster(60%)="rg3"<>}, p<0.01), 0.1 mM thiopental (^¥¥, p<0.01), or untreated control cells (^§§, p<0.01; ^§§§, p<0.001) are shown.</p

Thiopental increases intracellular calcium levels but not cAMP content.

<p>(A), SK-N-SH cells were left untreated or were incubated in the presence of 0.5 mM thiopental, 1 mM thiopental, or 40 µM thapsigargin in calcium containing (black bars) or calcium-free (white bars) RPMI medium for 1 h. For the last 30 min of the experiment cells were loaded with 2 µM fura-2 acetoxymethyl ester before intracellular calcium-complexes were excited at 340 nm and recorded at 510 nm. Values represent the means ± standard deviations. Experimental groups were statistically analyzed by performing one-way ANOVA followed by the Dunnett’s multiple comparisons test. ***, p<0.001 versus untreated control group, n = 3. In (B), SK-N-SH cells were left untreated or were incubated with 0.5 mM thiopental, 1 mM thiopental, or 100 µM forskulin for 15 min to 6 h before cell lysis. Cellular cAMP content was determined by a competitive cAMP immunoassay. Values represent the means ± standard deviations, n = 3. Experimental groups were statistically analyzed by performing two-way ANOVA followed by the Bonferroni’s <i>post hoc</i> test. Statistical differences versus untreated control cells are shown (***, p<0.001). In (C), immunoblots were performed using cells that were pretreated with 100 µM A484954, 10 µM camstatin, or 1 mM cAMPs-pR for 1 h before 0.5 mM thiopental were added for the last hour of the experiment. The immunoblots were analyzed with an anti-human phospho-eEF2 threonine 56 antibody or an anti-eEF2 antibody that detects endogenous levels of eEF2 independently of phosphorylation. Data are representative of three independent experiments.</p

Thiopental induces eEF2 phosphorylation.

<p>SK-N-SH cells were treated with 10 µM –2 mM thiopental for 6 h (A) or with 0.5 mM thiopental for 10 min to 12 h (B) and analyzed by immunoblotting with an anti-human phospho-eEF2 threonine 56 antibody (upper blots) or an eEF2 antibody that detects endogenous levels of eEF2 independently of phosphorylation (lower blots). Data are representative of four independent experiments.</p

Inhibition of hypoxic damage by thiopental is mediated by eEF2K.

<p>SK-N-SH cells were cultured in the presence or absence of 10 µM A484954 for 1 h before addition of 0.1 mM thiopental and exposure to hypoxia. After 72 h cellular damage was determined by an LDH-release assay. Values represent the mean ± standard deviations of four independent experiments. Statistical evaluation of experimental groups was performed by one-way ANOVA followed by the Bonferroni’s <i>post hoc</i> test. The statistically significant difference of thiopental treated, oxygen-deprived SK-N-SH cells in the presence or absence of A484954 is shown (***, p<0.001).</p

Inhibitors of protein synthesis reduce hypoxic neuronal damage.

<p>Cellular damage in human neuronal SK-N-SH cells was induced by oxygen deprivation (closed symbols) in an atmosphere containing 5% CO₂, 95% N₂ for 0–72 h and determined by an LDH release assay. Control cells were cultured in 5% CO₂, 21% O₂, and 74% N₂ (open symbols). In (A), cellular damage was measured in the presence (squares) or absence (rhombi) of fetal calf serum. In (B), 5 µg/ml cycloheximide (closed triangles) or 2 µg/ml actinomycin D (closed rhombi) were added to the cells in serum containing growth medium (squares). In (C), 0.1 mM thiopental (closed circles) or 0.5 mM thiopental (closed triangles) were added to the cells in serum containing growth medium (squares). Values represent the mean ± standard deviations of four separate experiments. Experimental groups were statistically analyzed by performing two-way ANOVA followed by the Bonferroni’s <i>post hoc</i> test. Statistically significant differences within groups shown for (A) are: serum treated oxygen deprived SK-N-SH cells versus serum treated normoxic control cells (***, p<0.001). Statistically significant differences within serum treated groups shown for (B) are: normoxic control cells versus oxygen deprived SK-N-SH cells (***, p<0.001); and oxygen deprived SK-N-SH cells versus oxygen-deprived cells treated with 2 µg/ml actinomycin D (^§§§, p<0.001) or versus oxygen-deprived cells treated with 5 µg/ml cycloheximide (^###, p<0.001). Statistically significant differences within serum treated groups shown for (C) are: normoxic control cells versus oxygen deprived SK-N-SH cells (***, p<0.001), versus oxygen-deprived cells treated with 0.1 mM thiopental (^{<>\raster(60%)="rg1"<>}, p<0.05; ^{<>\raster(60%)="rg1"<><>\raster(60%)="rg1"<><>\raster(60%)="rg1"<>}, p<0.001) or versus oxygen-deprived cells treated with 0.5 mM thiopental (^{<>\raster(60%)="rg2"<><>\raster(60%)="rg2"<><>\raster(60%)="rg2"<>}, p<0.001); and oxygen deprived SK-N-SH cells versus oxygen-deprived cells treated with 0.1 mM thiopental (^{<>\raster(60%)="rg3"<><>\raster(60%)="rg3"<><>\raster(60%)="rg3"<>}, p<0.001) or versus oxygen-deprived cells treated with 0.5 mM thiopental (^¥¥¥, p<0.001).</p

Thiopental inhibits protein synthesis, ameliorates hypoxic damage, and maintains energy balance during oxygen deprivation in primary cortical neurons.

<p>In (A), cortical neurons were treated with 10 µM −2 mM thiopental for 30 min and analyzed for phosphorylation of eEF2 and AMPK by immunoblotting. In (B), cortical neurons were left untreated or were exposed to 0.1–2 mM thiopental for 6 h and then pulse labeled with 200 µCi of [³⁵S]methionine for an additional 2 h. Cellular lysates were separated by 10% SDS-PAGE and the amounts of newly synthesized proteins were detected by autoradiography on dried electrophoresis gels. In (C/D), cortical neurons were exposed to hypoxia for 48 h in the presence or absence of 0.5 mM thiopental. Cellular damage was determined by an LDH-release assay (C). The relative intracellular ATP-content was measured by an ATP-driven luciferase assay (D). Values represent the mean ± standard deviations of three independent experiments. Statistical evaluation of experimental groups was performed by one-way ANOVA followed by the Bonferroni’s <i>post hoc</i> test. The statistically significant difference of oxygen-deprived cortical neurons in the presence or absence of thiopental is shown (***, p<0.001).</p

Thiopental inhibits global protein synthesis.

<p>SK-N-SH cells were left untreated or were exposed to 0.5 mM thiopental, 1 mM thiopental, or 5 µg/ml cycloheximide for 6 h and then pulsed with [³⁵S]methionine for an additional 2 h. Cell lysates were separated by SDS-PAGE. The amounts of newly synthesized proteins were detected by autoradiography on dried electrophoresis gels (A) and quantified by densitometry (B). Values represent the means ± standard deviations. Statistical differences between experimental groups were determined by performing one-way ANOVA followed by the Dunnett’s multiple comparisons test. ***, p<0.001 versus untreated control group (first bar). The results shown are representative of three independent experiments.</p

Hypoxic neuronal damage is independent of caspase-3.

<p>SK-N-SH cells were cultured in the presence (grey bars) or absence (black bars) of 0.5 mM thiopental without oxygen for 0–72 h before caspase-3 like activity was assessed by a fluorogenic caspase-3 activity assay. Values are expressed as relative light units (RLU)/min and were normalized to protein content. As a control, apoptosis was induced by 40 µM thapsigargin in normoxic SK-N-SH cells for 30 min (white bar). Statistic evaluation of experimental groups was performed by one way ANOVA followed by the Dunnett’s multiple comparisons test. ***, p<0.001 versus untreated control cells (black bar, 0 h hypoxia). The results shown are representative of three independent experiments.</p

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<p>Structural and functional plasticity of synapses are critical neuronal mechanisms underlying learning and memory. While activity-dependent regulation of synaptic strength has been extensively studied, much less is known about the transcriptional control of synapse maintenance and plasticity. Hippocampal mossy fiber (MF) synapses connect dentate granule cells to CA3 pyramidal neurons and are important for spatial memory formation and consolidation. The transcription factor Bcl11b/Ctip2 is expressed in dentate granule cells and required for postnatal hippocampal development. Ablation of Bcl11b/Ctip2 in the adult hippocampus results in impaired adult neurogenesis and spatial memory. The molecular mechanisms underlying the behavioral impairment remained unclear. Here we show that selective deletion of Bcl11b/Ctip2 in the adult mouse hippocampus leads to a rapid loss of excitatory synapses in CA3 as well as reduced ultrastructural complexity of remaining mossy fiber boutons (MFBs). Moreover, a dramatic decline of long-term potentiation (LTP) of the dentate gyrus-CA3 (DG-CA3) projection is caused by adult loss of Bcl11b/Ctip2. Differential transcriptomics revealed the deregulation of genes associated with synaptic transmission in mutants. Together, our data suggest Bcl11b/Ctip2 to regulate maintenance and function of MF synapses in the adult hippocampus.</p