Temperature and pharmacological rescue of a folding-defective, dominant-negative KV 7.2 mutation associated with neonatal seizures.

Benign familial neonatal seizures (BFNS) are a dominant epilepsy syndrome caused by mutations in the voltage-gated potassium channels K(V) 7.2 and K(V) 7.3. We examined the molecular pathomechanism of a BFNS-causing mutation (p.N258S) in the extracellular S5-H5 loop of K(V) 7.2. Wild type (WT) and mutant channels, expressed in both Xenopus laevis oocytes and CHO cells, were studied using electrophysiological techniques. The results revealed a pronounced loss-of-function with a dominant-negative effect of the mutant on WT K(V) 7.2 and K(V) 7.3 channels. Since single-channel recordings of K(V) 7.3-K(V) 7.2 and K(V) 7.3-N285S concatemers showed similar properties for both constructs, we hypothesized that the observed reduction in current amplitude was due to a folding and trafficking defect, which was confirmed by biochemical and immunocytochemical experiments revealing a reduced number of mutant channels in the surface membrane. Furthermore, rescuing experiments revealed that upon specific incubation of transfected CHO cells-either at lower temperatures of <30°C or in presence of the agonist retigabine (RTG)-the N258S-derived currents increased fivefold in contrast to the WT. The obtained results represent a first example of temperature and pharmacological rescue of a K(V) 7 mutation and suggest a folding and trafficking deficiency as the cause of reduced current amplitudes with a dominant-negative effect of N258S mutant proteins.

In Vitro Differentiated Human Stem Cell-Derived Neurons Reproduce Synaptic Synchronicity Arising during Neurodevelopment.

Neurons differentiated from induced pluripotent stem cells (iPSCs) typically show regular spiking and synaptic activity but lack more complex network activity critical for brain development, such as periodic depolarizations including simultaneous involvement of glutamatergic and GABAergic neurotransmission. We generated human iPSC-derived neurons exhibiting spontaneous oscillatory activity after cultivation of up to 6 months, which resembles early oscillations observed in rodent neurons. This behavior was found in neurons generated using a more "native" embryoid body protocol, in contrast to a "fast" protocol based on NGN2 overexpression. A comparison with published data indicates that EB-derived neurons reach the maturity of neurons of the third trimester and NGN2-derived neurons of the second trimester of human gestation. Co-culturing NGN2-derived neurons with astrocytes only led to a partial compensation and did not reliably induce complex network activity. Our data will help selection of the appropriate iPSC differentiation assay to address specific questions related to neurodevelopmental disorders.

TGFbeta-1 dependent fast stimulation of ATM and p53 phosphorylation following exposure to ionizing radiation does not involve TGFbeta-receptor I signalling.

BACKGROUND AND PURPOSE: It has been proposed that radiation induced stimulation of ATM and downstream components involves activation of TGFbeta-1 and that this may be due to TGFbeta-1-receptor I-Smad signalling. Therefore, the aim of this study was to clarify the distinct role of TGFbeta-1-receptor I-Smad signalling in mediating ATM activity following radiation exposure. MATERIALS AND METHODS: A549 cells were stably transfected with a conditionally regulatable TGFbeta-1 antisense construct (Tet-on-system) to test clonogenic activity following irradiation. Phosphorylation profile of ATM, p53, and chk2 was determined in non-cycling, serum-starved cells by immunoblotting. Likewise, A549 wild type cells were used to identify cell cycle distribution as a function of irradiation with or without pretreatment with CMK, a specific inhibitor of furin protease involved in activation of latent TGFbeta-1. Furthermore Western and immunoblot analyses were performed on serum-starved cells to investigate the dependence of ATM- and p53-stimulation on TGFbeta-1-receptor I-Smad signalling by applying a specific TGFbeta-1-receptor I inhibitor. RESULTS: Knock down of TGFbeta-1 by an antisense construct significantly increased clonogenic cell survival following exposure to ionizing radiation. Likewise, CMK treatment diminished the radiation induced G1 arrest of A549 cells. Moreover, both TGFbeta-1-knock down as well as CMK treatment inhibited the fast post-radiation phosphorylation of ATM, p53, and chk2. However, as shown by the use of a specific inhibitor TGFbeta-1-receptor I-Smad signalling was not involved in this fast activation of ATM and p53. CONCLUSIONS: We confirm that TGFbeta-1 plays a critical role in the stimulation of ATM- and p53 signalling in irradiated cells. However, this fast stimulation seems not to be dependent on activation of TGFbeta-1-receptor I-Smad signalling as recently proposed.

Site-directed mutagenesis of the ATM promoter: consequences for response to proliferation and ionizing radiation.

Although ATM, the protein defective in ataxia-telangiectasia (A-T), is activated primarily by radiation, there is also evidence that expression of the protein can be regulated by both radiation and growth factors. Computer analysis of the ATM promoter proximal 700-bp sequence reveals a number of potentially important cis-regulatory sequences. Using nucleotide substitutions to delete putative functional elements in the promoter of ATM, we examined the importance of some of these sites for both the basal and the radiation-induced activity of the promoter. In lymphoblastoid cells, most of the mutations in transcription factor consensus sequences [Sp1(1), Sp1(2), Cre, Ets, Xre, gammaIre(2), a modified AP1 site (Fse), and GCF] reduced basal activity to various extents, whereas others [gammaIre(1), NF1, Myb] left basal activity unaffected. In human skin fibroblasts, results were generally the same, but the basal activity varied up to 8-fold in these and other cell lines. Radiation activated the promoter approximately 2.5-fold in serum-starved lymphoblastoid cells, reaching a maximum by 3 hr, and all mutated elements equally blocked this activation. Reduction in Sp1 and AP1 DNA binding activity by serum starvation was rapidly reversed by exposure of cells to radiation. This reduction was not evident in A-T cells, and the response to radiation was less marked. Data provided for interaction between ATM and Sp1 by protein binding and co-immunoprecipitation could explain the altered regulation of Sp1 in A-T cells. The data described here provide additional evidence that basal and radiation-induced regulation of the ATM promoter is under multifactorial control.