Role of hyperpolarization-activated cyclic nucleotide-gated channel HCN2 in embryonic neural stem cell proliferation and differentiation.

Hyperpolarization-activated cyclic nucleotide-gated channels (HCN channels) are involved in spontaneous activity in many electrically active cell types such as cardiomyocytes and neurons. In this study, the role of HCN channels in proliferation and migration of Nestin and Sox2 expressing embryonic neural progenitor cells (NPC) originating from the subventricular zone (SVZ) was examined. Immunostaining and PCR data showed that the HCN2 subtype was highly expressed in these cells. Patch clamp recordings revealed a hyperpolarization-activated current, which was sensitive to inhibitors of HCN channels. Using the fluorescence dye bis-(1,3-dibutylbarbituric acid)-trimethineoxonol (DiBAC(4)(3)) we found that a prompt reduction of the extracellular K+ concentration, or exposing the cells to acute hypoxia, induced an instant hyperpolarization in the whole cell population. Recovery from low K+ induced hyperpolarization after extracellular calcium removal, or by re-oxygenation of hypoxic cells, was sensitive to ZD7288, a HCN channel inhibitor. Treatment of neurosphere cultures from the SVZ with ZD7288 caused a significant and reversible inhibition of neurosphere formation from single cells indicating that proliferation of progenitor cells was reduced. Furthermore, the migration of neuronal cells from neurospheres was considerably retarded in the presence of ZD7288. The results suggest that HCN2 channels are involved in controlling the proliferation of NPC and that HCN2 channel-induced spontaneous electrical activity may trigger the motility response of neurosphere-derived neurons in concert with other ion channels. Furthermore, the response to hypoxia suggests that HCN2 channels may trigger the chemotactic response of NPC to ischemic brain regions seen in many studies.

Acidic extracellular pH promotes accumulation of free cholesterol in human monocyte-derived macrophages via inhibition of ACAT1 activity.

BACKGROUND AND AIMS: In focal areas of advanced human atherosclerotic lesions, the intimal fluid is acidic. An acidic medium impairs the ABCA1-mediated cholesterol efflux from macrophages, so tending to increase their content of free cholesterol, which is then available for esterification by the macrophage enzyme ACAT1. Here we investigated whether low extracellular pH would affect the activity of ACAT1. METHODS: - Human monocyte-derived macrophages were first incubated with acetyl-LDL at neutral and acidic conditions (pH 7.5, 6.5, and 5.5) to generate foam cells, and then the foam cells were incubated with [3H]oleate-BSA complexes, and the formation of [3H]oleate-labeled cholesteryl esters was measured. ACAT1 activity was also measured in cell-free macrophage extracts. RESULTS: - In acidic media, ACAT1-dependent cholesteryl [3H]oleate generation became compromised in the developing foam cells and their content of free cholesterol increased. In line with this finding, ACAT1 activity in the soluble cell-free fraction derived from macrophage foam cells peaked at pH 7, and gradually decreased under acidic pH with a rapid drop below pH 6.5. Incubation of macrophages under progressively more acidic conditions (until pH 5.5) lowered the cytosolic pH of macrophages (down to pH 6.0). Such intracellular acidification did not affect macrophage gene expression of ACAT1 or the neutral CEH. CONCLUSIONS: Exposure of human macrophage foam cells to acidic conditions lowers their intracellular pH with simultaneous decrease in ACAT1 activity. This reduces cholesterol esterification and thus leads to accumulation of potentially toxic levels of free cholesterol, a contributing factor to macrophage foam cell death.

Neurotransmitters and Endothelins Acting on Radial Glial G-Protein-Coupled Receptors Are, Through Proteolytic NRG/ErbB4 Activation, Able to Modify the Migratory Behavior of Neocortical Cells and Mediate Bipolar-to-Multipolar Transition.

Cell-cell communication plays a central role in the guidance of migrating neurons during the development of the cerebral cortex. Neuregulins (NRGs) are essential mediators for migration and maintenance of the radial glial scaffold. We show, in this study that soluble NRG reduces neuronal motility, causes transition of bipolar cells to multipolar ones, and induces neuronal mitosis. Blocking the NRG receptor, ErbB4, results in reduction of neuron-neuron and neuron-radial glial contacts and causes an increase in neuronal motility. Blocking the radial glial metabotropic glutamate receptor 5 (mGluR5), the nonselective cation channel transient receptor potential 3 (TRPC3), or matrix metalloproteinases (MMPs) results in similar effects as ErbB4 blockade. Soluble NRG counteract the changes in motility pattern. Stimulation of other radial glial G-protein-coupled receptors (GPCRs), such as muscarinic acetylcholine receptors or endothelin receptors counteract all the effect of mGluR5 blockade, but not that of ErbB4, TRPC3, and MMP blockade. The results indicate that neurotransmitters and endothelins acting on radial glial GPCRs are, through proteolytic NRG/ErbB4 activation, able to modify the migratory behavior of neurons.

Tannic acid inhibits electrogenic Na+/HCO3- co-transporter activity in embryonic neural stem cell-derived radial glial-like cells.

Self-renewing neural stem cells and progenitor cells are cell populations that generate radial glial cells and neurons through asymmetric division. Regulation of intracellular pH in stem cells with high metabolic activity is critical for both cell signaling and proliferation. We have recently found that a S0859-inhibitable electrogenic Na/HCO3 co-transporter (NBCe1, Slc4a4), is the primary pHi regulatory mechanism in stem cell-derived radial glial-like cells. Here we show, by using the voltage-sensitive fluorescent dye DiBAC4(3) and BCECF, a pH-sensitive dye, that an antioxidant, tannic acid (100 µM), can inhibit potassium- and calcium-dependent rapid changes in membrane potential and NBCe1 mediated pHi regulation in brain-derived glial-like cells in vitro. Furthermore, neural stem cell differentiation and neurosphere formation (proliferation) were completely inhibited by tannic acid. The present study provides evidence that tannic acid is a natural inhibitor of NBCe1. It is tempting to speculate that tannic acid or related compounds that inhibits NBCe1-mediated pHi regulation in glial-like cells may also have bearing on the treatment of glial neoplasms.

Regulation of intracellular pH by electrogenic Na+/HCO3- co-transporters in embryonic neural stem cell-derived radial glia-like cells.

A stroke causes a hypoxic brain microenvironment that alters neural cell metabolism resulting in cell membrane hyperpolarization and intracellular acidosis. We studied how intracellular pH (pHi) is regulated in differentiated mouse neural progenitor cells during hyperpolarizing conditions, induced by prompt reduction of the extracellular K+ concentration. We found that the radial glia-like population in differentiating embryonic neural progenitor cells, but not neuronal cells, was rapidly acidified under these conditions. However, when extracellular calcium was removed, an instant depolarization and recovery of the pHi, back to normal levels, took place. The rapid recovery phase seen in the absence of calcium, was dependent on extracellular bicarbonate and could be inhibited by S0859, a potent Na/HCO3 cotransporter inhibitor. Immunostaining and PCR data, showed that NBCe1 (SLC4A4) and NBCn1 (SLC4A7) were expressed in the cell population and that the pHi recovery in the radial glial-like cells after calcium removal was mediated mainly by the electrogenic sodium bicarbonate transporter NBCe1 (SLC4A4). Our results indicate that extracellular calcium might hamper pHi regulation and Na/HCO3 cotransporter activity in a brain injury microenvironment. Our findings show that the NBC-type transporters are the main pHi regulating systems prevailing in glia-like progenitor cells and that these calcium sensitive transporters are important for neuronal progenitor cell proliferation, survival and neural stem cell differentiation.

Endocannabinoid Signaling in Embryonic Neuronal Motility and Cell-Cell Contact - Role of mGluR5 and TRPC3 Channels.

Cell-cell communication plays a central role in the guidance of migrating neuronal precursor cells during the development of the cerebral cortex. Endocannabinoids (eCBs) have previously been shown to be one of the central factors regulating neuronal migration. In this study the effects of eCBs on different parameters, expected to affect embryonic cortical neuronal motility have been analyzed in neurosphere-derived neuroblasts using time-lapse microscopy. Increased endogenous production of the endocannabinoid 2-arachidonyl glycerol (2-AG) causes bursts of neuroblast motility. The neuroblasts move longer distances and show a low frequency of turning, and the number of neuron-neuron contacts are reduced. Similar changes occur interfering with the function of the metabotropic glutamate receptor 5 (mGluR5) or its transducer canonical transient receptor potential channel 3 (TRPC3) or the neuregulin receptor ErbB4. Blocking of 2-AG production reverses these effects. The data suggest that eCB-regulated neuronal motility is controlled by mGluR5/TRPC3 activity possibly via NRG/ErbB4 signaling.

Regulation of radial glial process growth by glutamate via mGluR5/TRPC3 and neuregulin/ErbB4.

Radial glial cells play an essential role through their function as guides for neuronal migration during development. Disruption of metabotropic glutamate receptor 5 (mGluR5) function retards the growth of radial glial processes in vitro. Neuregulins (NRG) are activated by proteolytic cleavage and regulate (radial) glial maintenance via ErbB3/ErbB4 receptors. We show here that blocking ErbB4 disrupts radial process extension. Soluble NRG acting on ErbB4 receptors is able to promote radial process extension in particular where process elongation has been impeded by blockade of mGluR5, the nonselective cation channel canonical transient receptor potential 3 (TRPC3), or matrix metalloproteases (MMP). NRG does not restore retarded process growth caused by ErbB4 blockade. Stimulation of muscarinic receptors restores process elongation due to mGluR5 blockade but not that caused by TRPC3, MMP or ErbB4 blockade suggesting that muscarinic receptors can replace mGluR5 with respect to radial process extension. Additionally, NRG/ErbB4 causes Ca2+ mobilization in a population of cells through cooperation with ErbB1 receptors. Our results indicate that mGluR5 promotes radial process growth via NRG activation by a mechanism involving TRPC3 channels and MMPs. Thus neurotransmitters acting on G-protein coupled receptors could play a central role in the maintenance of the radial glial scaffold through activation of NRG/ErbB4 signaling.

Calpain Activity Is Essential for ATP-Driven Unconventional Vesicle-Mediated Protein Secretion and Inflammasome Activation in Human Macrophages.

Extracellular ATP is an endogenous danger signal that is known to activate inflammatory responses in innate immune cells, including macrophages. Activated macrophages start to secrete proteins to induce an immune response, as well as to recruit other immune cells to the site of infection and tissue damage. In this study, we characterized the secretome (i.e., the global pattern of secreted proteins) of ATP-stimulated human macrophages. We show that ATP stimulation activates robust vesicle-mediated unconventional protein secretion, including exosome release and membrane shedding, from human macrophages. Pathway analysis of the identified secreted proteins showed that calpain-related pathways were overrepresented in the secretome of ATP-stimulated cells. In accordance with this, calpains, which are calcium-dependent nonlysosomal cysteine proteases, were activated upon ATP stimulation through a P2X purinoceptor 7 receptor-dependent pathway. Functional studies demonstrated that calpain activity is essential for the P2X purinoceptor 7 receptor-mediated activation of unconventional protein secretion. Unconventional protein secretion was followed by cell necrosis and NLRP3 inflammasome-mediated secretion of the mature form of the proinflammatory cytokine IL-1ß. Furthermore, ATP-driven NLRP3 inflammasome activation was also dependent on calpain activity. Interestingly, pro-IL-1ß and inflammasome components ASC and caspase-1 were released by ATP-activated macrophages through a vesicle-mediated secretion pathway. In conclusion, to our knowledge, we provide the first global characterization of proteins secreted by ATP-activated human macrophages and show a pivotal role for calpains in the activation of the inflammatory response during ATP exposure.

Transient receptor potential channels and their role in modulating radial glial-neuronal interaction: a signaling pathway involving mGluR5.

The guidance of developing neurons to the right position in the central nervous system is of central importance in brain development. Canonical transient receptor potential (TRPC) channels are thought to mediate turning responses of growth cones to guidance cues through fine control of calcium transients. Proliferating and 1- to 5-day-differentiated neural progenitor cells (NPCs) showed expression of Trpc1 and Trpc3 mRNA, while Trpc4-7 was not clearly detected. Time-lapse imaging showed that the motility pattern of neuronal cells was phasic with bursts of rapid movement (>60 µm/h), changes in direction, and intermittent slow phases or stallings (<40 µm/h), which frequently occurred in close contact with radial glial processes. Genetic interference with the TRPC3 and TRPC1 channel enhanced the motility of NPCs (burst frequency/stalling frequency). TRPC3-deficient cells or cells treated with the TRPC3 blocker pyr3 infrequently changed direction and seldom contacted radial glial processes. TRPC channels are also activated by group I metabotropic glutamate receptors (mGluR1 and mGluR5). As shown here, pyr3 blocked the calcium response mediated through mGluR5 in radial glial processes. Furthermore, 2-methyl-6-(phenylethynyl)pyridine, a blocker of mGluR5, affected the motility pattern in a similar way as TRPC3/6 double knockout or pyr3. The results suggest that radial glial cells exert attractant signals to migrating neuronal cells, which alter their motility pattern. Our results suggest that mGluR5 acting through TRPC3 is of central importance in radial glial-mediated neuronal guidance.

The role of glutamate and its receptors in the proliferation, migration, differentiation and survival of neural progenitor cells.

The mammalian central nervous system derives from multipotent neural progenitor cells (NPCs) of the developing brain. During development the progenitor cells have enormous potential. They proliferate actively and differentiate into all the three main cell types, i.e., neurons, astrocytes and oligodendrocytes, of the adult brain through a tightly regulated process that coordinates cell proliferation, survival, migration, differentiation and apoptosis. This process is regulated by multiple extracellular signals including neurotrophic factors, chemoattractants and neurotransmitters in a coordinated manner. The main excitatory neurotransmitter glutamate is involved in promoting and/or inhibiting the proliferation, survival, migration and differentiation of NPCs acting via ionotropic or metabotropic receptors. The role of glutamate in the regulation of cortical NPCs has been most extensively studied. Glutamate appears to have a similar role in hippocampal, striatal as well as adult neural progenitors. Ionotropic α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate (KA) receptors and metabotropic glutamate receptor 5 (mGluR5) are expressed early during embryonic development as well as in the neurogenic zones of the adult brain. Ca(2+)-permeable AMPA/KA receptors are initially of importance for cell proliferation and neuronal motility. At later stages of development N-methyl-D-aspartate (NMDA) receptors have a more prominent role. MGluR5, which is the main metabotropic glutamate receptor during early development, is expressed in early progenitors and radial glial cells. Activation of this receptor promotes the proliferation and survival of NPCs. MGluR5 is involved in the extension of radial glial processes and in regulation of the migration of early cortical neurons.

Extracellular acidosis is a novel danger signal alerting innate immunity via the NLRP3 inflammasome.

BACKGROUND: Local acidosis has been demonstrated in ischemic tissues and at inflammatory sites. RESULTS: Acidic extracellular pH triggers NLRP3 inflammasome activation and interleukin-1ß secretion in human macrophages. CONCLUSION: Acidic pH represents a novel danger signal alerting the innate immunity. SIGNIFICANCE: Local acidosis may promote inflammation at ischemic and inflammatory sites. Local extracellular acidification has been demonstrated at sites of ischemia and inflammation. IL-1ß is one of the key proinflammatory cytokines, and thus, its synthesis and secretion are tightly regulated. The NLRP3 (nucleotide-binding domain leucine-rich repeat containing family, pyrin domain containing 3) inflammasome complex, assembled in response to microbial components or endogenous danger signals, triggers caspase-1-mediated maturation and secretion of IL-1ß. In this study, we explored whether acidic environment is sensed by immune cells as an inflammasome-activating danger signal. Human macrophages were exposed to custom cell culture media at pH 7.5-6.0. Acidic medium triggered pH-dependent secretion of IL-1ß and activation of caspase-1 via a mechanism involving potassium efflux from the cells. Acidic extracellular pH caused rapid intracellular acidification, and the IL-1ß-inducing effect of acidic medium could be mimicked by acidifying the cytosol with bafilomycin A1, a proton pump inhibitor. Knocking down the mRNA expression of NLRP3 receptor abolished IL-1ß secretion at acidic pH. Remarkably, alkaline extracellular pH strongly inhibited the IL-1ß response to several known NLRP3 activators, demonstrating bipartite regulatory potential of pH on the activity of this inflammasome. The data suggest that acidic environment represents a novel endogenous danger signal alerting the innate immunity. Low pH may thus contribute to inflammation in acidosis-associated pathologies such as atherosclerosis and post-ischemic inflammatory responses.

Effect of glutamate receptor antagonists on migrating neural progenitor cells.

Neurotransmitters such as glutamate are potential regulators of neurogenesis. Interference with defined glutamate receptor subtypes affects proliferation, migration and differentiation of neural progenitor cells. The cellular targets for the actions of different glutamate receptor ligands are less well known. In this study we have combined calcium imaging, measurement of membrane potential, time-lapse imaging and immunocytochemistry to obtain a spatial overview of migrating mouse embryonic neural progenitor cell-derived cells responding to glutamate receptor agonists and antagonists. Responses via metabotropic glutamate receptor 5 correlated with radial glial cells and dominated in the inner migration zones close to the neurosphere. Block of metabotropic glutamate receptor 5 resulted in shorter radial glial processes, a transient increase in neuron-like cells emerging from the neurosphere and increased motility of neuron-like cells. α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors are present on the majority of migrating neuronal cells, which with time accumulate at the outer edge of the migration zone. Blocking these receptors leads to an enhanced extension of radial glial processes and a reduced motility of neuron-like cells. Our results indicate that functional glutamate receptors have profound effects on the motility of neural progenitor cells. The main target for metabotropic glutamate receptor 5 appears to be radial glial cells while AMPA/kainate receptors are mainly expressed in newborn neuronal cells and regulate the migratory progress of these cells. The results suggest that both metabotropic glutamate receptor 5 and AMPA/kainate receptors are of importance for the guidance of migrating embryonic progenitor cells.

Role of low voltage activated calcium channels in neuritogenesis and active migration of embryonic neural progenitor cells.

The central role of calcium influx and electrical activity in embryonic development raises important questions about the role and regulation of voltage-dependent calcium influx. Using cultured neural progenitor cell (NPC) preparations, we recorded barium currents through voltage-activated channels using the whole-cell configuration of the patch-clamp technique and monitored intracellular free calcium concentrations with Fura-2 digital imaging. We found that NPCs as well as expressing high-voltage-activated (HVA) calcium channels express functional low-threshold voltage-dependent calcium channels in the very early stages of differentiation (5 h to 1 day). The size of the currents recorded at -50 versus -20 mV after 1 day in differentiation was dependent on the nature of the charge carrier. Peak currents measured at -20 mV in the presence 10 mM Ca2+ instead of 10 mM Ba2+ had a tendency to be smaller, whereas the nature of the divalent species did not influence the amplitude measured at -50 mV. The T-type channel blockers mibefradil and NNC 55-0396 significantly reduced the calcium responses elicited by depolarizing with extracellular potassium, while the overall effect of the HVA calcium channel blockers was small at differentiation day 1. At differentiation day 20, the calcium responses were effectively blocked by nifedipine. Time-lapse imaging of differentiating neurospheres cultured in the presence of low-voltage-activated (LVA) blockers showed a significant decrease in the number of active migrating neuron-like cells and neurite extensions. Together, these data provide evidence that LVA calcium channels are involved in the physiology of differentiating and migrating NPCs.

Effects of acute hypoxia/acidosis on intracellular pH in differentiating neural progenitor cells.

The response of differentiating mouse neural progenitor cells, migrating out from neurospheres, to conditions simulating ischemia (hypoxia and extracellular or intracellular acidosis) was studied. We show here, by using BCECF and single cell imaging to monitor intracellular pH (pH(i)), that two main populations can be distinguished by exposing migrating neural progenitor cells to low extracellular pH or by performing an acidifying ammonium prepulse. The cells dominating at the periphery of the neurosphere culture, which were positive for neuron specific markers MAP-2, calbindin and NeuN had lower initial resting pH(i) and could also easily be further acidified by lowering the extracellular pH. Moreover, in this population, a more profound acidification was seen when the cells were acidified using the ammonium prepulse technique. However, when the cell population was exposed to depolarizing potassium concentrations no alterations in pH(i) took place in this population. In contrast, depolarization caused an increase in pH(i) (by 0.5 pH units) in the cell population closer to the neurosphere body, which region was positive for the radial cell marker (GLAST). This cell population, having higher resting pH(i) (pH 6.9-7.1) also responded to acute hypoxia. During hypoxic treatment the resting pH(i) decreased by 0.1 pH units and recovered rapidly after reoxygenation. Our results show that migrating neural progenitor cells are highly sensitive to extracellular acidosis and that irreversible damage becomes evident at pH 6.2. Moreover, our results show that a response to acidosis clearly distinguishes two individual cell populations probably representing neuronal and radial cells.

Enhanced early expression of membrane receptors with the Rous sarcoma virus promoter in baculovirus-infected insect cells.

The effect of the Rous sarcoma virus (RSV) long terminal repeat enhancer/promoter on expression levels of complementary DNAs (cDNAs) encoding seven transmembrane receptors was studied using the baculovirus expression vector system. Expression of the human α(2B)-adrenoceptor (AR) cDNA under the control of the polyhedrin (POL) promoter produced up to 7.6 pmol/mg protein at 28 H post infection (p.i.) in Sf9 cells. The addition of the RSV promoter increased the expression to 11.6 pmol/mg protein. Dramatic increases in expression levels at early times were also obtained with the α(2A)-AR, the M1 and M4 muscarinic receptors, and the orexin OX1 receptor. Analysis of the time-dependent expression revealed that expression driven by the RSV promoter reaches almost maximum 24 H p.i. and that this promoter is superior to the often used POL promoter at early times p.i. when functional studies need to be performed. Functional enhancement of signaling as a result of early expression is demonstrated with the α(2B)-AR and the OX1 receptor. Finally, enhanced green fluorescent protein fluorescence in living cells was used to monitor expression by various viral promoters. The results verified the early transcriptional activity of the RSV promoter, whereas the cytomegalovirus promoter was found to be poorly active in Sf9 cells.

Inhibiting TRPA1 ion channel reduces loss of cutaneous nerve fiber function in diabetic animals: sustained activation of the TRPA1 channel contributes to the pathogenesis of peripheral diabetic neuropathy.

Peripheral diabetic neuropathy (PDN) is a devastating complication of diabetes mellitus (DM). Here we test the hypothesis that the transient receptor potential ankyrin 1 (TRPA1) ion channel on primary afferent nerve fibers is involved in the pathogenesis of PDN, due to sustained activation by reactive compounds generated in DM. DM was induced by streptozotocin in rats that were treated daily for 28 days with a TRPA1 channel antagonist (Chembridge-5861528) or vehicle. Laser Doppler flow method was used for assessing axon reflex induced by intraplantar injection of a TRPA1 channel agonist (cinnamaldehyde) and immunohistochemistry to assess substance P-like innervation of the skin. In vitro calcium imaging and patch clamp were used to assess whether endogenous TRPA1 agonists (4-hydroxynonenal and methylglyoxal) generated in DM induce sustained activation of the TRPA1 channel. Axon reflex induced by a TRPA1 channel agonist in the plantar skin was suppressed and the number of substance P-like immunoreactive nerve fibers was decreased 4 weeks after induction of DM. Prolonged treatment with Chembridge-5861528 reduced the DM-induced attenuation of the cutaneous axon reflex and loss of substance P-like immunoreactive nerve fibers. Moreover, in vitro calcium imaging and patch clamp results indicated that reactive compounds generated in DM (4-hydroxynonenal and methylglyoxal) produced sustained activations of the TRPA1 channel, a prerequisite for adverse long-term effects. The results indicate that the TRPA1 channel exerts an important role in the pathogenesis of PDN. Blocking the TRPA1 channel provides a selective disease-modifying treatment of PDN.

Glycosylphosphatidylinositol (GPI)-anchoring of mamba toxins enables cell-restricted receptor silencing.

Muscarinic toxins (MTs) are snake venom peptides found to selectively target specific subtypes of G-protein-coupled receptors. In here, we have attached a glycosylphosphatidylinositol (GPI) tail to three different toxin molecules and evaluated their receptor-blocking effects in a heterologous expression system. MT7-GPI remained anchored to the cell surface and selectively inhibited M(1) muscarinic receptor signaling expressed in the same cell. To further demonstrate the utility of the GPI tail, we generated MT3- and MTα-like gene sequences and fused these to the signal sequence for GPI attachment. Functional assessment of these membrane-anchored toxins on coexpressed target receptors indicated a prominent antagonistic effect. In ligand binding experiments the GPI-anchored toxins were found to exhibit similar selection profiles among receptor subtypes as the soluble toxins. The results indicate that GPI attachment of MTs and related receptor toxins could be used to assess the role of receptor subtypes in specific organs or even cells in vivo by transgenic approaches.

Functional α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors in differentiating embryonic neural progenitor cells.

Glutamate-responsive α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors are considered to play a significant role in neurogenesis. We have studied the functional expression of these receptors in migrating embryonic neural progenitor cells (NPCs). The majority of neurosphere-derived NPCs express AMPA receptors already during the first day of differentiation, based on mRNA quantification, immunocytochemistry, and Ca²+ imaging. The expression of GluR1 mRNA was significantly increased at 5 days of differentiation. The AMPA receptor subunits coexpressed with neuronal markers and were present in all cells at the outer periphery of the migration zone. In migrating NPCs, most of the AMPA receptors were philantotoxin sensitive and Ca²+-permeable, suggesting that in addition to their role in plasticity, the receptors are of importance in NPC differentiation.

BDNF and TrkB in neuronal differentiation of Fmr1-knockout mouse.

Fragile X syndrome (FXS) is a common cause of inherited mental retardation and the best characterized form of autistic spectrum disorders. FXS is caused by the loss of functional fragile X mental retardation protein (FMRP), which leads to abnormalities in the differentiation of neural progenitor cells (NPCs) and in the development of dendritic spines and neuronal circuits. Brain-derived neurotrophic factor (BDNF) and its TrkB receptors play a central role in neuronal maturation and plasticity. We studied BDNF/TrkB actions in the absence of FMRP and show that an increase in catalytic TrkB expression in undifferentiated NPCs of Fmr1-knockout (KO) mice, a mouse model for FXS, is associated with changes in the differentiation and migration of neurons expressing TrkB in neurosphere cultures and in the developing cortex. Aberrant intracellular calcium responses to BDNF and ATP in subpopulations of differentiating NPCs combined with changes in the expression of BDNF and TrkB suggest cell subtype-specific alterations during early neuronal maturation in the absence of FMRP. Furthermore, we show that dendritic targeting of Bdnf mRNA was increased under basal conditions and further enhanced in cortical layer V and hippocampal CA1 neurons of Fmr1-KO mice by pilocarpine-induced neuronal activity represented by convulsive seizures, suggesting that BDNF/TrkB-mediated feedback mechanisms for strengthening the synapses were compromised in the absence of FMRP. Pilocarpine-induced seizures caused an accumulation of Bdnf mRNA transcripts in the most proximal segments of dendrites in cortical but not in hippocampal neurons of Fmr1-KO mice. In addition, BDNF protein levels were increased in the hippocampus but reduced in the cortex of Fmr1-KO mice in line with regional differences of synaptic plasticity in the brain of Fmr1-KO mice. Altogether, the present data suggest that alterations in the BDNF/TrkB signaling modulate brain development and impair synaptic plasticity in FXS.

Selective interference with TRPC3/6 channels disrupts OX1 receptor signalling via NCX and reveals a distinct calcium influx pathway.

TRPC channels play significant roles in the regulation of neuronal plasticity and development. The mechanism by which these nonselective cation channels exert their trophic actions appears to involve entry of Ca(2+) into the cells. Using a neuronal cell model (differentiated human IMR32 neuroblastoma cells), we demonstrate a central role for sodium entry via TRPC3/6 channels in receptor-mediated increases in intracellular calcium. These Na(+)-dependent Ca(2+) influxes, which were observed in a subpopulation of cells, were efficiently blocked by protein kinase C activation, by the Na(+)/Ca(2+) exchanger inhibitors, and by molecular disruption of TRPC3/6 channel function. On the other hand, another subpopulation of cells showed a Na(+)-independent Ca(2+) entry upon stimulation of the same receptors, orexin/hypocretin and bradykinin receptors. This second type of response was not affected by the above mentioned treatments, but it was sensitive to polyvalent cations, such as ruthenium red, spermine and Gd(3+). The data suggest that a NCX-TRPC channel interaction constitutes an important functional unit in receptor-mediated Ca(2+) influx in neuronal cells.