Stimulation of GluN receptors decreases the surface density of GluN1/GluN2B subunits in cultured neocortical interneurons.

Changes in the density of NMDA (GluN) receptors in the neuronal membrane are critical for plasticity, whereas malfunction of precisely regulated GluN receptor activity may be involved in neurotoxicity. In cultured rat neocortical interneurons, we have studied the regulation of the surface density of GluN1, GluN2A and GluN2B subunits. Application of 5 µMol NMDA for 24 h followed by a washout period of 24 h decreased the response of GluN receptors for at least 2 days. The reduction was caused by a decrease in the surface density of GluN1/GluN2B subunits, whereas GluN2A subunits remained unaffected. Our data indicate that long but reversible low level activation of GluN receptors can cause long-term changes in their subunit composition in cultured interneurons.

GluA and GluN receptors regulate the surface density of GluN receptor subunits in cultured neocortical interneurons.

J. Neurochem. (2012) 121, 597-606. ABSTRACT: In cultured rat neocortical interneurons, we have studied the effect of long-term application of NMDA or AMPA on the surface density of the NMDA (GluN) receptor subunits GluN1 and GluN2B. Stimulation of Ca(2+) -permeable AMPA (GluA) receptors located on the interneurons decreased the response of GluN receptors. The reduction was caused by a decrease in the surface density of GluN1/GluN2B subunits. In contrast, stimulation of GluN receptors located on the interneurons enhanced the surface density of GluN1/GluN2B subunits. Both effects could be induced by network activation.

Vectors selected from adeno-associated viral display peptide libraries for leukemia cell-targeted cytotoxic gene therapy.

OBJECTIVE: For acute myeloid leukemia (AML), gene therapy may be used to treat patients refractory to conventional chemotherapy. However, availability of vectors sufficiently and specifically transducing this cell type is very limited. METHOD: Here we report the selection of capsid-modified adeno-associated viral (AAV) vectors targeting Kasumi-1 AML cells by screening random AAV displayed peptide libraries. RESULTS: The peptide inserts of the enriched capsid mutants share a common sequence motif. The same motif was selected in an independent library screening on HL-60 AML cells. Recombinant targeted vectors displaying the selected peptides transduced the target leukemia cells they have been selected on up to 500-fold more efficiently compared to AAV vectors with control peptide inserts. One of the selected clones (NQVGSWS) also efficiently transduced all members of a panel of four other AML cell lines. Binding and blocking experiments showed that NQVGSWS binding to leukemia cells is independent of the wild-type AAV-2 receptor heparin sulfate proteoglycan. Transduction assays on a panel of hematopoietic and nonhematopoietic cell lines showed that the NQVGSWS capsid was able to overcome resistance to AAV transduction, especially in hematopoietic cancer cells, whereas normal peripheral blood mononuclear cells and CD34(+) hematopoietic progenitor cells were not transduced. CONCLUSIONS: Consequently, recombinant targeted NQVGSWS AAV vectors harboring a suicide gene conferred selective killing to Kasumi-1 cells, but not to control cells. This suggests that the AAV mutant selected here may be used as a tool to target therapeutic genes to AML cells.

Phosphoinositide-3-kinases p110α and p110ß mediate S phase entry in astroglial cells in the marginal zone of rat neocortex.

In cells cultured from neocortex of newborn rats, phosphoinositide-3-kinases of class I regulate the DNA synthesis in a subgroup of astroglial cells. We have studied the location of these cells as well as the kinase isoforms which facilitate the S phase entry. Using dominant negative (dn) isoforms as well as selective pharmacological inhibitors we quantified S phase entry by nuclear labeling with bromodeoxyuridine (BrdU). Only in astroglial cells harvested from the marginal zone (MZ) of the neocortex inhibition of phosphoinositide-3-kinases reduced the nuclear labeling with BrdU, indicating that neocortical astroglial cells differ in the regulation of proliferation. The two kinase isoforms p110α and p110ß were essential for S phase entry. p110α diminished the level of the p27(Kip1) which inactivates the complex of cyclin E and CDK2 necessary for entry into the S phase. p110ß phosphorylated and inhibited glycogen synthase kinase-3ß which can prevent S-phase entry. Taken together, both isoforms mediated S phase in a subgroup of neocortical astroglial cells and acted via distinct pathways.

Lavender oil-potent anxiolytic properties via modulating voltage dependent calcium channels.

Recent clinical data support the clinical use of oral lavender oil in patients suffering from subsyndromal anxiety. We identified the molecular mechanism of action that will alter the perception of lavender oil as a nonspecific ingredient of aromatherapy to a potent anxiolytic inhibiting voltage dependent calcium channels (VOCCs) as highly selective drug target. In contrast to previous publications where exorbitant high concentrations were used, the effects of lavender oil in behavioral, biochemical, and electrophysiological experiments were investigated in physiological concentrations in the nanomolar range, which correlate to a single dosage of 80 mg/d in humans that was used in clinical trials. We show for the first time that lavender oil bears some similarities with the established anxiolytic pregabalin. Lavender oil inhibits VOCCs in synaptosomes, primary hippocampal neurons and stably overexpressing cell lines in the same range such as pregabalin. Interestingly, Silexan does not primarily bind to P/Q type calcium channels such as pregabalin and does not interact with the binding site of pregabalin, the α2δ subunit of VOCCs. Lavender oil reduces non-selectively the calcium influx through several different types of VOCCs such as the N-type, P/Q-type and T-type VOCCs. In the hippocampus, one brain region important for anxiety disorders, we show that inhibition by lavender oil is mainly mediated via N-type and P/Q-type VOCCs. Taken together, we provide a pharmacological and molecular rationale for the clinical use of the oral application of lavender oil in patients suffering from anxiety.

Ablation of Ca(V)2.1 voltage-gated Ca²âº channels in mouse forebrain generates multiple cognitive impairments.

Voltage-gated Ca(V)2.1 (P/Q-type) Ca²âº channels located at the presynaptic membrane are known to control a multitude of Ca²âº-dependent cellular processes such as neurotransmitter release and synaptic plasticity. Our knowledge about their contributions to complex cognitive functions, however, is restricted by the limited adequacy of existing transgenic Ca(V)2.1 mouse models. Global Ca(V)2.1 knock-out mice lacking the α1 subunit Cacna1a gene product exhibit early postnatal lethality which makes them unsuitable to analyse the relevance of Ca(V)2.1 Ca²âº channels for complex behaviour in adult mice. Consequently we established a forebrain specific Ca(V)2.1 knock-out model by crossing mice with a floxed Cacna1a gene with mice expressing Cre-recombinase under the control of the NEX promoter. This novel mouse model enabled us to investigate the contribution of Ca(V)2.1 to complex cognitive functions, particularly learning and memory. Electrophysiological analysis allowed us to test the specificity of our conditional knock-out model and revealed an impaired synaptic transmission at hippocampal glutamatergic synapses. At the behavioural level, the forebrain-specific Ca(V)2.1 knock-out resulted in deficits in spatial learning and reference memory, reduced recognition memory, increased exploratory behaviour and a strong attenuation of circadian rhythmicity. In summary, we present a novel conditional Ca(V)2.1 knock-out model that is most suitable for analysing the in vivo functions of Ca(V)2.1 in the adult murine forebrain.

Tetraspanin-13 modulates voltage-gated CaV2.2 Ca2+ channels.

Integration of voltage-gated Ca(2+) channels in a network of protein-interactions is a crucial requirement for proper regulation of channel activity. In this study, we took advantage of the specific properties of the yeast split-ubiquitin system to search for and characterize so far unknown interaction partners of CaV2 Ca(2+) channels. We identified tetraspanin-13 (TSPAN-13) as an interaction partner of the α1 subunit of N-type CaV2.2, but not of P/Q-type CaV2.1 or L- and T-type Ca(2+) channels. Interaction could be located between domain IV of CaV2.2 and transmembrane segments S1 and S2 of TSPAN-13. Electrophysiological analysis revealed that TSPAN-13 specifically modulates the efficiency of coupling between voltage sensor activation and pore opening of the channel and accelerates the voltage-dependent activation and inactivation of the Ba(2+) current through CaV2.2. These data indicate that TSPAN-13 might regulate CaV2.2 Ca(2+) channel activity in defined synaptic membrane compartments and thereby influences transmitter release.