Background: Periventricular leukomalacia (PVL) is a frequent complication of preterm delivery. Proinflammatory cytokines, such as interferon-γ (IFN-γ) and tumor necrosis factor α (TNF-α) released from astrocytes and microglia activated by infection or ischemia have previously been shown to impair survival and maturation of oligodendrocyte progenitors and could thus be considered as potential factors contributing to the generation of this disease. The first goal of the present study was to investigate whether exposure of oligodendrocyte precursors to these cytokines arrests the maturation of ion currents in parallel to its effects on myelin proteins and morphological maturation. Secondly, in the search for agents, that can protect differentiating oligodendrocyte precursor cells from cytokine-induced damage we investigated effects of coapplications of corticosteroids with proinflammatory cytokines on the subsequent survival and differentiation of oligodendrocyte progenitor cells.

Key points• Hypertonicity-induced cation channels (HICCs) are key players in proliferation and apoptosis but their molecular identity has remained elusive.• We report that in HeLa cells, intracellular adenosine diphosphate ribose (ADPr) and cyclic ADPr (cADPr), as activators of TRPM2 cation channels, elicited currents that are identical to the osmotic activation of HICCs.• siRNA-mediated silencing of the expression of TRPM2 and CD38 (as the supposed source of ADPr and cADPr) inhibited HICC and nucleotide-induced currents and the osmotic volume response of cells.• Quantification of intracellular cADPr and extracellular application of nucleotides revealed that the outwardly directed gradient rather than the intracellular activity of ADPr and cADPr is the triggering factor for TRPM2.• Cloning of TRPM2 identified the C-splice variant as the molecular correlate of the HICC, which was supported by quantification of Ca 2+ selectivity.• Immunoprecipation and FRET/FLIM assays revealed the interaction of TRPM2 and CD38 and we propose a transport-related nucleotide export via CD38 as a novel mechanism of TRPM2 activation.Abstract Hypertonicity-induced cation channels (HICCs) are key-players in proliferation and apoptosis but their molecular correlate remains obscure. Furthermore, the activation profile of HICCs is not well defined yet. We report here that, in HeLa cells, intracellular adenosine diphosphate ribose (ADPr) and cyclic ADPr (cADPr), as supposed activators of TRPM2, elicited cation currents that were virtually identical to the osmotic activation of HICCs. Silencing of the expression of TRPM2 and of the ecto-enzyme CD38 (as a likely source of ADPr and cADPr) inhibited HICC as well as nucleotide-induced currents and, in parallel, the hypertonic volume response of cells (the regulatory volume increase, RVI) was attenuated. Quantification of intracellular cADPr levels and the systematic application of extra-vs. intracellular nucleotides indicate that the outwardly directed gradient rather than the cellular activity of ADPr and cADPr triggers TRPM2 activation, probably along with a simultaneous biotransformation of nucleotides. Cloning of TRPM2 identified the C-splice variant as the molecular correlate of the HICC, which could be strongly supported by a direct comparison of the respective Ca 2+ selectivity. Finally, immunoprecipitation and high-resolution FRET/FLIM imaging revealed the interaction of TRPM2 and K. Sato and J. Christmann contributed equally to this work. CD38 in the native as well as in a heterologous (HEK293T) expression system. We propose transport-related nucleotide export via CD38 as a novel mechanism of TRPM2/HICC activation. With the biotransformation of nucleotides running in parallel, continuous zero trans-conditions are achieved which will render the system infinitely sensitive. (

We have previously shown that treatment with the thyroid hormone T(3) increases the voltage-gated Na(+)current density (Nav-D) in hippocampal neurons from postnatal rats, leading to accelerated action potential upstrokes and increased firing frequencies. Here we show that the Na(+) current regulation depends on the presence of glial cells, which secrete a heat-instable soluble factor upon stimulation with T(3). The effect of conditioned medium from T(3)-treated glial cells was mimicked by basic fibroblast growth factor (bFGF), known to be released from cerebellar glial cells after T(3) treatment. Neutralization assays of astrocyte-conditioned media with anti-bFGF antibody inhibited the regulation of the Nav-D by T(3). This suggests that the up-regulation of the neuronal sodium current density by T(3) is not a direct effect but involves bFGF release and satellite cells. Thus glial cells can modulate neuronal excitability via secretion of paracrinely acting factors.

The aim of the present study has been to obtain high yields of oligodendrocyte precursor cells (OPCs) in culture. This is a first step in facilitation of myelin repair. We show that, in addition to factors, known to promote proliferation, such as basic fibroblast growth factor (FGF-2) and platelet derived growth factor (PDGF) the choice of the basal medium exerts a significant influence on the yield of OPCs in cultures from newborn rats. During a culture period of up to 9 days we observed larger numbers of surviving cells in Dulbecco's Modified Eagle Medium (DMEM), and Roswell Park Memorial Institute Medium (RPMI) compared with Neurobasal Medium (NB). A larger number of A2B5-positive OPCs was found after 6 days in RPMI based media compared with NB. The percentage of bromodeoxyuridine (BrdU)-positive cells was largest in cultures maintained in DMEM and RPMI. The percentage of caspase-3 positive cells was largest in NB, suggesting that this medium inhibits OPC proliferation and favors apoptosis. A difference between NB and DMEM as well as RPMI is the reduced Na + -content. The addition of equiosmolar supplements of mannitol or NaCl to NB medium rescued the BrdU-incorporation rate. This suggested that the osmolarity influences the proliferation of OPCs. Plating density as well as residual microglia influence OPC survival, BrdU incorporation, and caspase-3 expression. We found, that high density cultures secrete factors that inhibit BrdU incorporation whereas the presence of additional microglia induces an increase in caspase-3 positive cells, indicative of enhanced apoptosis. An enhanced number of microglia could thus also explain the stronger inhibition of OPC differentiation observed in high density cultures in response to treatment with the cytokines TNF-α and IFN-γ. We conclude that a maximal yield of OPCs is obtained in a medium of an osmolarity higher than 280 mOsm plated at a relatively low density in the presence of as little microglia as technically achievable.

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Mice lacking functional thyroid follicular cells, Pax8−/− mice, die early postnatally, making them suitable models for extreme hypothyroidism. We have previously obtained evidence in postnatal rat neurons, that a down-regulation of Na+-current density could explain the reduced excitability of the nervous system in hypothyroidism. If such a mechanism underlies the development of coma and death in severe hypothyroidism, Pax8−/− mice should show deficits in the expression of Na+ currents and potentially also in the expression of Na+/K+-ATPases, which are necessary to maintain low intracellular Na+ levels. We thus compared Na+ current densities in postnatal mice using the patch-clamp technique in the whole-cell configuration as well as the expression of three alpha and two beta-subunits of the Na+/K+-ATPase in wild type versus Pax8−/− mice. Whereas the Na+ current density in hippocampal neurons from wild type mice was upregulated within the first postnatal week, the Na+ current density remained at a very low level in hippocampal neurons from Pax8−/− mice. Pax8−/− mice also showed significantly decreased protein expression levels of the catalytic α1 and α3 subunits of the Na+/K+-ATPase as well as decreased levels of the β2 isoform, with no changes in the α2 and β1 subunits.

Im Interview: Die Leitung des GDA-Arbeitsprogramms „Krebserzeugende Gefahrstoffe“ über Hintergründe und Bedeutung des GDA Gefahrstoff-Checks.

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