P2X7-dependent, but differentially regulated release of IL-6, CCL2, and TNF-α in cultured mouse microglia.

ATP is an important regulator of microglia and its effects on microglial cytokine release are currently discussed as important contributors in a variety of brain diseases. We here analyzed the effects of ATP on the production of six inflammatory mediators (IL-6, IL-10, CCL2, IFN-γ, TNF-α, and IL-12p70) in cultured mouse primary microglia. Stimulation of P2X7 receptor by ATP (1 mM) or BzATP (500 µM) evoked the mRNA expression and release of proinflammatory cytokines IL-6, TNF-α, and the chemokine CCL2 in WT cells but not in P2X7(-/-) cells. The effects of ATP and BzATP were inhibited by the nonselective P2 receptor antagonists PPADs and suramin. Various selective P2X7 receptor antagonists blocked the P2X7-dependent release of IL-6 and CCL2, but, surprisingly, had no effect on BzATP-induced release of TNF-α in microglia. Calcium measurements confirmed that P2X7 is the main purine receptor activated by BzATP in microglia and showed that all P2X7 antagonists were functional. It is also presented that pannexin-1 hemichannel function and potential P2X4/P2X7 heterodimers are not involved in P2X7-dependent release of IL-6, CCL2, and TNF-α in microglia. How P2X7-specific antagonists only affect P2X7-dependent IL-6 and CCL2 release, but not TNF-α release is at the moment unclear, but indicates that the P2X7-dependent release of cytokines in microglia is differentially regulated.

The brain's best friend: microglial neurotoxicity revisited.

One long standing aspect of microglia biology was never questioned; their involvement in brain disease. Based on morphological changes (retracted processes and amoeboid shape) that inevitably occur in these cells in case of damage in the central nervous system, microglia in the diseased brain were called "activated." Because "activated" microglia were always found in direct neighborhood to dead or dying neuron, and since it is known now for more than 20 years that cultured microglia release numerous factors that are able to kill neurons, microglia "activation" was often seen as a neurotoxic process. From an evolutionary point of view, however, it is difficult to understand why an important, mostly post-mitotic and highly vulnerable organ like the brain would host numerous potential killers. This review is aimed to critically reconsider the term microglia neurotoxicity and to discuss experimental problems around microglia biology, that often have led to the conclusion that microglia are neurotoxic cells.

Microglia emerge from erythromyeloid precursors via Pu.1- and Irf8-dependent pathways.

Microglia are crucial for immune responses in the brain. Although their origin from the yolk sac has been recognized for some time, their precise precursors and the transcription program that is used are not known. We found that mouse microglia were derived from primitive c-kit(+) erythromyeloid precursors that were detected in the yolk sac as early as 8 d post conception. These precursors developed into CD45(+) c-kit(lo) CX(3)CR1(-) immature (A1) cells and matured into CD45(+) c-kit(-) CX(3)CR1(+) (A2) cells, as evidenced by the downregulation of CD31 and concomitant upregulation of F4/80 and macrophage colony stimulating factor receptor (MCSF-R). Proliferating A2 cells became microglia and invaded the developing brain using specific matrix metalloproteinases. Notably, microgliogenesis was not only dependent on the transcription factor Pu.1 (also known as Sfpi), but also required Irf8, which was vital for the development of the A2 population, whereas Myb, Id2, Batf3 and Klf4 were not required. Our data provide cellular and molecular insights into the origin and development of microglia.

Lithium enhances CRTC oligomer formation and the interaction between the CREB coactivators CRTC and CBP--implications for CREB-dependent gene transcription.

Lithium salts are important drugs to treat bipolar disorder. Previous work showed that lithium by enforcing the interaction between the transcription factor CREB and its coactivator CRTC1 enhanced cAMP-stimulated CREB-dependent gene transcription. Both CREB and CRTC have been implicated in neuronal adaptation, which might underlie lithium's therapeutic action. In the present study the mechanisms of lithium action on cAMP-induced CREB-dependent gene transcription were further elucidated. Transient transfection assays revealed that all three CRTC isoforms conferred lithium responsiveness to CREB whereas their intrinsic transcriptional activities remained unchanged by lithium, suggesting a conformational change of CREB or CRTC by lithium. In in vitro protein-protein interaction assays lithium enhanced the interaction between CREB and both coactivators CRTC and CBP. Furthermore, lithium enforced the oligomerization of CRTC, a prerequisite for CREB interaction. For further evaluation it was investigated whether lithium competes with magnesium, which coordinates the conformation of the CREB basic region leucine zipper (bZip). Mutational analysis of the magnesium coordinating lysine-290 within the bZip, in vitro and intracellular interaction assays and luciferase reporter-gene assays revealed that the effect of lithium on the CREB-CRTC interaction or on the transcriptional activity, respectively, was not affected by the mutation, thus excluding a magnesium-lithium competition. However, the CREB-CRTC interaction was strongly increased in lysine-290-mutants thereby extending the CRTC-CREB interaction domain. Taken together the results exclude a competition between lithium and magnesium at the bZip, but suggest that lithium by enforcing the CRTC-oligomer formation and the interaction of CREB-CBP-CRTC enhances cAMP-induced CREB-dependent gene transcription.

Neuroprotective function for ramified microglia in hippocampal excitotoxicity.

BACKGROUND: Most of the known functions of microglia, including neurotoxic and neuroprotective properties, are attributed to morphologically-activated microglia. Resting, ramified microglia are suggested to primarily monitor their environment including synapses. Here, we show an active protective role of ramified microglia in excitotoxicity-induced neurodegeneration. METHODS: Mouse organotypic hippocampal slice cultures were treated with N-methyl-D-aspartic acid (NMDA) to induce excitotoxic neuronal cell death. This procedure was performed in slices containing resting microglia or slices that were chemically or genetically depleted of their endogenous microglia. RESULTS: Treatment of mouse organotypic hippocampal slice cultures with 10-50 µM N-methyl-D-aspartic acid (NMDA) induced region-specific excitotoxic neuronal cell death with CA1 neurons being most vulnerable, whereas CA3 and DG neurons were affected less. Ablation of ramified microglia severely enhanced NMDA-induced neuronal cell death in the CA3 and DG region rendering them almost as sensitive as CA1 neurons. Replenishment of microglia-free slices with microglia restored the original resistance of CA3 and DG neurons towards NMDA. CONCLUSIONS: Our data strongly suggest that ramified microglia not only screen their microenvironment but additionally protect hippocampal neurons under pathological conditions. Morphological activation of ramified microglia is thus not required to influence neuronal survival.

Telomere shortening reduces Alzheimer's disease amyloid pathology in mice.

Alzheimer's disease is a neurodegenerative disorder of the elderly and advancing age is the major risk factor for Alzheimer's disease development. Telomere shortening represents one of the molecular causes of ageing that limits the proliferative capacity of cells, including neural stem cells. Studies on telomere lengths in patients with Alzheimer's disease have revealed contrary results and the functional role of telomere shortening on brain ageing and Alzheimer's disease is not known. Here, we have investigated the effects of telomere shortening on adult neurogenesis and Alzheimer's disease progression in mice. The study shows that aged telomerase knockout mice with short telomeres (G3Terc-/-) exhibit reduced dentate gyrus neurogenesis and loss of neurons in hippocampus and frontal cortex, associated with short-term memory deficit in comparison to mice with long telomere reserves (Terc+/+). In contrast, telomere shortening improved the spatial learning ability of ageing APP23 transgenic mice, a mouse model for Alzheimer's disease. Telomere shortening was also associated with an activation of microglia in ageing amyloid-free brain. However, in APP23 transgenic mice, telomere shortening reduced both amyloid plaque pathology and reactive microgliosis. Together, these results provide the first experimental evidence that telomere shortening, despite impairing adult neurogenesis and maintenance of post-mitotic neurons, can slow down the progression of amyloid plaque pathology in Alzheimer's disease, possibly involving telomere-dependent effects on microglia activation.

Interaction of the general transcription factor TnrA with the PII-like protein GlnK and glutamine synthetase in Bacillus subtilis.

TnrA is a master transcription factor regulating nitrogen metabolism in Bacillus subtilis under conditions of nitrogen limitation. When the preferred nitrogen source is in excess, feedback-inhibited glutamine synthetase (GS) has been shown to bind TnrA and disable its activity. In cells grown with an energetically unfavorable nitrogen source such as nitrate, TnrA is fully membrane-bound via a complex of AmtB and GlnK, which are the transmembrane ammonium transporter and its cognate regulator, respectively, originally termed NrgA and NrgB. The complete removal of nitrate from the medium leads to rapid degradation of TnrA in wild-type cells. In contrast, in AmtB-deficient or GlnK-deficient strains, TnrA is neither membrane-bound nor degraded in response to nitrate depletion. Here, we show that TnrA forms either a stable soluble complex with GlnK in the absence of AmtB, or constitutively binds to GS in the absence of GlnK. In vitro, the TnrA C-terminus is responsible for interactions with either GS or GlnK, and this region appears also to mediate proteolysis, suggesting that binding of GlnK or GS protects TnrA from degradation. Surface plasmon resonance detection assays have demonstrated that GS binds to TnrA not only in its feedback-inhibited form, but also in its non-feedback-inhibited form, although less efficiently. TnrA binding to GlnK or GS responds differentially to adenylate nucleotide levels, with ATP weakening interactions with both partners.

Stimulation by lithium of the interaction between the transcription factor CREB and its co-activator TORC.

Lithium salts are clinically important drugs used to treat bipolar mood disorder. The mechanisms accounting for the clinical efficacy are not completely understood. Chronic treatment with lithium is required to establish mood stabilization, suggesting the involvement of neuronal plasticity processes. CREB (cAMP-response-element-binding protein) is a transcription factor known to mediate neuronal adaptation. Recently, the CREB-co-activator TORC (transducer of regulated CREB) has been identified as a novel target of lithium and shown to confer an enhancement of cAMP-induced CREB-directed gene transcription by lithium. TORC is sequestered in the cytoplasm and its nuclear translocation controls CREB activity. In the present study, the effect of lithium on TORC function was investigated. Lithium affected neither the nuclear translocation of TORC nor TORC1 transcriptional activity, but increased the promoter occupancy by TORC1 as revealed by chromatin immunoprecipitation assay. In a mammalian two-hybrid assay, as well as in a cell-free GST (glutathione transferase) pull-down assay, lithium enhanced the CREB-TORC1 interaction. Magnesium ions strongly inhibited the interaction between GST-CREB and TORC1 and this effect was reversed by lithium. Thus our results suggest that, once TORC has entered the nucleus, lithium as a cation stimulates directly the binding of TORC to CREB, leading to an increase in cAMP-induced CREB target-gene transcription. This novel mechanism of lithium action is likely to contribute to the clinical mood-stabilizing effect of lithium salts.

Inactivation of the general transcription factor TnrA in Bacillus subtilis by proteolysis.

Under conditions of nitrogen limitation, the general transcription factor TnrA in Bacillus subtilis activates the expression of genes involved in assimilation of various nitrogen sources. Previously, TnrA activity has been shown to be controlled by protein-protein interaction with glutamine synthetase, the key enzyme of ammonia assimilation. Furthermore, depending on ATP and 2-oxoglutarate levels, TnrA can bind to the GlnK-AmtB complex. Here, we report that upon transfer of nitrate-grown cells to combined nitrogen-depleted medium, TnrA is rapidly eliminated from the cells by proteolysis. As long as TnrA is membrane-bound through GlnK-AmtB interaction it seems to be protected from degradation. Upon removal of nitrogen sources, the localization of TnrA becomes cytosolic and degradation occurs. The proteolytic activity against TnrA was detected in the cytosolic fraction but not in the membrane, and its presence does not depend on the nitrogen regime of cell growth. The proteolytic degradation of TnrA as a response to complete nitrogen starvation might represent a novel mechanism of TnrA control in B. subtilis.

Chronic lithium salt treatment reduces CRE/CREB-directed gene transcription and reverses its upregulation by chronic psychosocial stress in transgenic reporter gene mice.

The molecular mechanism of action of the mood stabilizer lithium is assumed to involve changes in gene expression leading to neuronal adaptation. The transcription factor CREB (cAMP-responsive element binding protein) regulates the expression of many genes and has been implicated in important brain functions and the action of psychogenic agents. We here investigated the effect of lithium on cAMP-responsive element (CRE)/CREB-mediated gene transcription in the brain, using transgenic reporter mice that express the luciferase reporter gene under the control of four copies of the rat somatostatin gene promoter CRE. Chronic (21 days) but not acute (24 h) treatment with lithium (7.5 mmol/kg) significantly decreased CRE/CREB-directed gene expression in hippocampus, cortex, hypothalamus, and striatum to 60-70%, and likewise reduced CREB phosphorylation. As bipolar disorder is also considered as a stress-related disorder, the effect of lithium was determined in mice submitted to a paradigm for chronic psychosocial stress. As shown before, stress for 25 days significantly increased CRE/CREB-directed gene expression in several brain regions by 100-150%. Treatment of stressed mice with lithium decreased stress-induced CRE/CREB-directed gene expression to control levels in nearly all brain regions and likewise reduced CREB phosphorylation. Chronic lithium treatment induced beta-catenin accumulation and decreased cAMP levels, indicating an inhibitory effect of lithium on glycogen synthase kinase 3 and the adenylate cyclase/protein kinase A signalling cascade, which are known to modulate CREB activity. We here for the first time show that lithium regulates CRE/CREB-directed gene transcription in vivo and suggest CREB as a putative mediator of the neuronal adaptation after chronic lithium treatment.

Interaction of the membrane-bound GlnK-AmtB complex with the master regulator of nitrogen metabolism TnrA in Bacillus subtilis.

PII proteins are widespread and highly conserved signal transduction proteins occurring in bacteria, Archaea, and plants and play pivotal roles in controlling nitrogen assimilatory metabolism. This study reports on biochemical properties of the PII-homologue GlnK (originally termed NrgB) in Bacillus subtilis (BsGlnK). Like other PII proteins, the native BsGlnK protein has a trimeric structure and readily binds ATP in the absence of divalent cations, whereas 2-oxoglutarate is only weakly bound. In contrast to other PII-like proteins, Mg2+ severely affects its ATP-binding properties. BsGlnK forms a tight complex with the membrane-bound ammonium transporter AmtB (NrgA), from which it can be relieved by millimolar concentrations of ATP. Immunoprecipitation and co-localization experiments identified a novel interaction between the BsGlnK-AmtB complex and the major transcription factor of nitrogen metabolism, TnrA. In vitro in the absence of ATP, TnrA is completely tethered to membrane (AmtB)-bound GlnK, whereas in extracts from BsGlnK- or AmtB-deficient cells, TnrA is entirely soluble. The presence of 4 mm ATP leads to concomitant solubilization of BsGlnK and TnrA. This ATP-dependent membrane re-localization of TnrA by BsGlnK/AmtB may present a novel mechanism to control the global nitrogen-responsive transcription regulator TnrA in B. subtilis under certain physiological conditions.

The Synechococcus elongatus P signal transduction protein controls arginine synthesis by complex formation with N-acetyl-L-glutamate kinase.

This communication identifies, for the first time, a receptor protein for signal perception from the P(II) signal transduction protein in the cyanobacterium Synechococcus elongatus. P(II), a phosphoprotein that signals the carbon/nitrogen status of the cells, forms a tight complex with the key enzyme of the arginine biosynthetic pathway, N-acetylglutamate (NAG) kinase. In complex with P(II), the catalytic activity of NAG kinase is strongly enhanced. Complex formation does not require the effector molecules of P(II), 2-oxoglutarate and ATP, but it is highly susceptible to modifications at the phosphorylation site of P(II), Ser-49. Stable complexes were only formed with the non-phosphorylated form of P(II) but not with Ser-49 mutants. In accordance with these data, NAG kinase activity in S. elongatus extracts correlated with the phosphorylation state of P(II), with high NAG kinase activities corresponding to non-phosphorylated P(II) (nitrogen-excess conditions) and low activities to increased levels of P(II) phosphorylation (nitrogen-poor conditions), thus subjecting the key enzyme of arginine biosynthesis to global nitrogen control.