Kainic acid-induced neurotrophic activities in developing cortical neurons

Using primary cultured cortical neurons from embryonic rat brains, we elucidated an alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)/kainic acid (KA) receptor-mediated neuroprotective mechanism through actions of nerve growth factor (NGF) in developing neurons. Neurotoxicity of KA in early days in vitro neurons was quite low compared with the mature neurons. However, pretreatment with anti-NGF antibody or TrkA inhibitor AG-879 profoundly raised KA toxicity. Furthermore, KA stimulation resulted in an increase of TrkA expression and phosphorylation, which was blocked not only by the AMPA/KA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione and AG-879, but also by the phospholipase C inhibitor U73122 and the intracellular calcium chelator BAPTA. A study of polyphosphoinositide turnover showed that KA-stimulated phospholipase C (PLC) activity was directly triggered by the AMPA/KA receptor activity, but not by the activity of TrkA or other excitatory amino acid receptor subtypes. Sources of KA-increased intracellular calcium levels were contributed by both extracellular calcium influx and intracellular calcium release and were partially sensitive to guanosine 5'-O-(2-thiodiphosphate). These results indicate that in developing cortical neurons, activation of AMPA/KA receptors by KA may induce expression, followed by activation of TrkA via PLC signaling and intracellular calcium elevation and hence increase reception of NGF on KA-challenged neurons. A G protein-coupled AMPA/KA receptor may be involved in these metabotropic events for neuronal protection.     

Roles of ionotropic glutamate receptors in early developing neurons derived from the P19 mouse cell line

We cultured a P19 mouse teratocarcinoma cell line and induced its neuronal differentiation to study the function of ionotropic glutamate receptors (GluRs) in early neuronal development. Immunocytochemical studies showed 85% neuronal population at 5 days in vitro (DIV) with microtubule-associated protein 2-positive staining. Thirty percent and 50% of the cells expressed the alpha-amino-3-hydroxy-5-methyl-4-isopropinonate (AMPA) receptor subunit, GluR2/3, and the kainate (kainic acid; KA) receptor subunit, GluR5/6/7, respectively. In Western blot analysis, the temporal expression of GluR2/3 began to appear at 3 DIV, whereas GluR5/6/7 was already expressed in the undifferentiated cells. P19-derived neurons began to respond to glutamate, AMPA and KA, but not to the metabotropic GluR agonist trans-1-aminocyclopentane-1,3-decarboxylic acid, by 5 DIV in terms of increases in intracellular calcium and phospholipase C-mediated poly-phosphoinositide turnover. Furthermore, KA reduced cell death of P19-derived neurons in both atmospheric and hypobaric conditions in a phospholipase C-dependent manner. The common AMPA/KA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione, but not the AMPA receptor antagonist, 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide disodium, profoundly increased hypobaric insult-induced neurotoxicity. In a flow cytometry study, the nerve growth factor-mediated antiapoptotic effect was facilitated by AMPA, with an induction of TrkA, but not p75(NTR) expression. Therefore, AMPA and KA receptors might mediate neurotrophic functions to facilitate neurotrophic factor signaling to protect neurons against hypoxic insult in early neuronal development.     

Knockdown of the aryl hydrocarbon receptor attenuates excitotoxicity and enhances NMDA-induced BDNF expression in cortical neurons

NMDA receptors play dual and opposing roles in neuronal survival by mediating the activity-dependent neurotrophic signaling and excitotoxic cell death via synaptic and extrasynaptic receptors, respectively. In this study, we demonstrate that the aryl hydrocarbon receptor (AhR), also known as the dioxin receptor, is involved in the expression and the opposing activities of NMDA receptors. In primary cultured cortical neurons, we found that NMDA excitotoxicity is significantly enhanced by an AhR agonist 2,3,7,8-tetrachlorodibenzo- p -dioxin, and AhR knockdown with small interfering RNA significantly reduces NMDA excitotoxicity. AhR knockdown also significantly reduces NMDA-increases intracellular calcium concentration, NMDA receptor expression and surface presentation, and moderately decreases the NMDA receptor-mediated spontaneous as well as miniature excitatory post-synaptic currents. However, AhR knockdown significantly enhances the bath NMDA application– but not synaptic NMDA receptor-induced brain-derived neurotrophic factor (BDNF) gene expression, and activating AhR reduces the bath NMDA-induced BDNF expression. Furthermore, AhR knockdown reveals the calcium dependency of NMDA-induced BDNF expression and the binding activity of cAMP-responsive element binding protein (CREB) and its calcium-dependent coactivator CREB binding protein (CBP) to the BDNF promoter upon NMDA treatment. Together, our results suggest that AhR opposingly regulates NMDA receptor-mediated excitotoxicity and neurotrophism possibly by differentially regulating the expression of synaptic and extrasynaptic NMDA receptors.     

Physiological release of endogenous tau is stimulated by neuronal activity

Propagation of tau pathology is linked with progressive neurodegeneration, but the mechanism underlying trans‐synaptic spread of tau is unknown. We show that stimulation of neuronal activity, or AMPA receptor activation, induces tau release from healthy, mature cortical neurons. Notably, phosphorylation of extracellular tau appears reduced in comparison with intracellular tau. We also find that AMPA‐induced release of tau is calcium‐dependent. Blocking pre‐synaptic vesicle release by tetanus toxin and inhibiting neuronal activity with tetrodotoxin both significantly impair AMPA‐mediated tau release. Tau secretion is therefore a regulatable process, dysregulation of which could lead to the spread of tau pathology in disease.     

Non-proteolytic neurotrophic effects of tissue plasminogen activator on cultured mouse cerebrocortical neurons

Most biological effects of tissue plasminogen activator (tPA), such as fibrinolysis, are mediated by its protease activity. Recent studies, however, have demonstrated that tPA also has several protease-independent effects such as: neuroprotection, microglial activation, and promoting LTP formation. In order to gain a better understanding of how tPA affects neurons, we examined neurite outgrowth and cell survival in low density cerebrocortical neuronal culture in the presence of tPA. tPA enhanced neurite elongation and neuronal survival. tPA protease inhibitors, PAI-1 or PMSF, did not alter either effect. Consistent with neurotrophic effects, tPA activated Raf-K/ERK, PKC and PI3-K/Akt, 5-60 min after treatment. In addition, specific inhibitors of these kinases reduced tPA-induced neurite outgrowth. Interestingly, survival-promoting effect of tPA was attenuated only by PI3-K inhibitors. Activation of signaling kinases suggests that tPA activates an upstream membrane receptor. Thus far, three membrane proteins, low density lipoprotein receptor-related protein (LRP), mannose receptor (MR), and annexin-II (AII), have been identified to bind tPA. While inhibiting LRP or MR did not change tPA-induced neurite outgrowth and cell survival, inhibiting AII blocked neurotrophic effects of tPA. Taken together, our results indicate that tPA has novel, non-proteolytic neurotrophic effects on cultured cortical neurons, which are likely mediated by AII.     

Brain-derived neurotrophic factor regulates AMPA receptor trafficking to post-synaptic densities via IP3R and TRPC calcium signaling

The change in the number of post-synaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type glutamatergic receptors (AMPARs) by neuronal activity is recognized as a molecular basis of synaptic plasticity. Here, we show that Ca(2+) transients evoked by brain-derived neurotrophic factor (BDNF) induce translocation of a subunit of AMPAR, GluR1, but not NMDAR, to the post-synaptic membrane in cultured cortical pyramidal neurons. Among BDNF-induced Ca(2+) transients, that dependent on IP3R was fully required, while store-operated calcium influx through the non-selective cation channel TRPC (transient receptor potential canonical) was partially required for the GluR1 up-regulation, suggesting that spatial and temporal calcium signaling regulate translocation of GluR1 to the polarized membrane domain.     

大鼠大脑皮质星形胶质细胞条件培养液促进小脑皮质神经元的生长

本研究从大鼠大脑皮质分离、纯化星形胶质细胞,再经培养后收集星形胶质细胞的无血清条件培养液。用盖玻片培养法与快速自动比色微量分析法研究了星形胶质细胞条件培养液对小脑皮质神经元生存以及神经元活力的影响。发现星形胶质细胞条件培养液能够明显提高小脑皮质神经元的体外存活率,增强神经元的活力。表明星形胶质细胞具有神经营养性作用。     

Epileptiform postsynaptic currents in primary culture of rat cortical neurons: Calcium mechanisms

In this study we demonstrate that the primary culture of rat cortical neurons is a convenient model for investigations of epileptogenesis mechanisms and specifically, of the postsynaptic epileptiform currents (EC) reflecting periodical asynchronous glutamate release. In particular, we have revealed that in primary culture of cortical neurons EC can appear spontaneously or can be triggered by the withdrawal of magnesium block of NMDA receptor channels or by shutting down GABAergic inhibition. EC were found to depend on intracellular calcium oscillations. The secondary calcium release from intracellular stores was needed for EC synchronization. EC were suppressed by the influences causing either neuronal calcium overload or decrease of intracellular calcium concentration. Calcium entry into neurons in the case of NMDA receptor hyperactivation or in the case of calcium ionophore ionomycin treatment eliminated EC. The suppression of EC also occurred after a decrease of intracellular calcium concentration induced by BAPTA loaded into the neurons or by stimulation of calcium removal from cells via Na⁺/Ca²⁺ exchanger by 1 nM ouabain. Partial dependence of EC on action potential generation was found. Thus, EC in neurons are activated by intracellular periodic calcium waves within a limited concentration window.     

Calcium signal-initiated early activation of NF-κB in neurons is a neuroprotective event in response to kainic acid-induced excitotoxicity

We demonstrate that activation of nuclear factor κB (NF-κB) in neurons is neuroprotective in response to kainic acid (KA)-induced excitotoxicity. Combination of Western blotting, immunocytochemistry, and electrophoresis mobility shift assay showed that KA exposure induced a fast but transient nuclear translocation of the NF-κB p65 subunit and increased DNA-binding activity of NF-κB in primary cultured cortical neurons. The transient NF-κB activity was associated with upregulation of antiapoptotic Bcl-xL and XIAP gene products revealed by real-time PCR. Knockdown of p65 decreased neuronal viability and antiapoptotic gene expression. In addition, we showed that KA-stimulated DNA-binding activity of NF-κB was associated with reactive oxygen species and calcium signals, using AMPA/KA receptor antagonist, calcium chelator, and antioxidant. These results suggest that the fast and transient activation of NF-κB initiated by calcium signals is one of the important proximal events in response to KA-induced excitotoxicity, which has neuroprotective effect against KA-induced apoptosis.     

(−)1-(Benzofuran-2-yl)-2-propylaminopentane Shows Survival Effect on Cortical Neurons Under Serum-Free Condition Through Sigma Receptors

SUMMARY 1. The rapid cell death of cortical neurons in serum-free culture was rescued by the condition medium from the high-density culture, but not by brain-derived neurotrophic factor or basic fibroblast growth factor.2. Similar rescue was observed by the addition of (–)BPAP, an impulse enhancer, and (+)-pentazocine, a sigma receptor agonist. These actions were blocked by BD1063, a sigma receptor antagonist.3. (–)BPAP showed a weak displacement activity in the [³H]pentazocine binding to synaptic membranes from rat cerebral cortex.4. These findings suggest that (–)BPAP and (+)-pentazocine have unique survival activity on cortical neurons through sigma receptors.     

Evidence for glutamate-mediated activation of hippocampal neurons by glial calcium waves

Communication from astrocytes to neurons has recently been reported by two laboratories, but different mechanisms were thought to underlie glial calcium wave activation of associated neurons. Neuronal calcium elevation by glia observed in the present report is similar to that reported previously, where an increase in neuronal calcium was demonstrated in response to glial stimulation. In the present study hippocampal neurons plated on a confluent glial monolayer displayed a transient increase in intracellular calcium following a short delay after the passage of a wave of increased calcium in underlying glia. Activated cells displayed action potentials in response to glial waves and showed antineurofilament immunoreactivity. Finally, the N-methyl-D -aspartate glutamate receptor antagonist DL -2-amino-5-phosphonovaleric acid and the non-NMDA glutamate receptor antagonist 6,7-dinitroquinoxaline-2,3-dione significantly reduced the responsiveness of neurons to glial calcium waves. Our results indicate that hippocampal neurons growing on hippocampal or cortical astrocytes respond to glial calcium waves with elevations in calcium and increased electrical activity. Furthermore, we show that in most cases this communication appears to be mediated by ionotropic glutamate receptor channels. © 1995 John Wiley & Sons, Inc.     

The Ability of Diphenylpiperazines to Prevent Neuronal Death in Dorsal Root Ganglion Neurons In Vitro After Nerve Growth Factor Deprivation and In Vivo After Axotomy

Abstract: The mechanism of neuroprotection by the calcium channel antagonist flunarizine against neuronal death is unknown. We investigated the ability of other calcium channel antagonists (cinnarizine, nimodipine, nicardipine, diltiazem, and verapamil), calmodulin antagonists, and calpain inhibitors to prevent neuronal death in rat dorsal root ganglion neurons in vitro after nerve growth factor (NGF) deprivation and the ability of cinnarizine and diltiazem to protect in vivo after axotomy. In vitro, only neurons treated with cinnarizine or flunarizine were protected from death after withdrawal. In vivo, cinnarizine, but not diltiazem, protected dorsal root ganglion neurons in rats after unilateral sciatic nerve crush. Intracellular calcium concentration ([Ca²⁺],) was evaluated with fura 2 after NGF deprivation In vitro. Neurons "committed to die" 24 h after NGF deprivation displayed a decline in [Ca^a+], before visible morphological deterioration consistent with cell death. The influx of extracellular calcium was not necessary to produce neuronal death. Neurons deprived of NGF gradually lost the ability to respond to elevated external potassium with an increase in [Ca²⁺], during the first 24 h after trophic factor deprivation. After 24 h, neurons deprived of NGF could not be rescued by readministration of NGF. Neurons protected from cell death with diphenylpiperazines maintained their response to high external potassium, suggesting continued membrane integrity. We speculate that diphenylpiperazines may protect sensory neurons via an unknown mechanism that stabilizes cell membranes.     

Sprouty1 regulates neuritogenesis and survival of cortical neurons

In multicellular organisms, receptor tyrosine kinases (RTKs) control a variety of cellular processes, including cell proliferation, differentiation, migration, and survival. Sprouty (SPRY) proteins represent an important class of ligand-inducible inhibitors of RTK-dependent signaling pathways. Here, we investigated the role of SPRY1 in cells of the central nervous system (CNS). Expression of SPRY1 was substantially higher in neural stem cells than in cortical neurons and was increased during neuronal differentiation of cortical neurons. We found that SPRY1 was a direct target gene of the CNS-specific microRNA, miR-124 and miR-132. In primary cultures of cortical neurons, the neurotrophic factors brain-derived neurotrophic factor (BDNF) and Basic fibroblast growth factor (FGF2) downregulated SPRY1 expression to positively regulate their own functions. In immature cortical neurons and mouse N₂A cells, we found that overexpression of SPRY1 inhibited neurite development, whereas knockdown of SPRY1 expression promoted neurite development. In mature neurons, overexpression of SPRY1 inhibited the prosurvival effects of both BDNF and FGF2 on glutamate-mediated neuronal cell death. SPRY1 was also upregulated upon glutamate treatment in mature neurons and partially contributed to the cytotoxic effect of glutamate. Together, our results indicate that SPRY1 contributes to the regulation of CNS functions by influencing both neuronal differentiation under normal physiological processes and neuronal survival under pathological conditions.     

A novel anti-epileptic agent, perampanel, selectively inhibits AMPA receptor-mediated synaptic transmission in the hippocampus

Perampanel is a non-competitive AMPA receptor antagonist that is under development as an anti-epileptic therapy. Although it is known to reduce calcium flux mediated by AMPA receptors in cultured cortical neurons, there are no studies of its selectivity in synaptic transmission in more intact systems. In the present study using hippocampal slices, perampanel (0.01-10μM) has been tested on pharmacologically isolated synaptic responses mediated by AMPA, NMDA or kainate receptors. Perampanel reduced AMPA receptor-mediated excitatory postsynaptic field potentials (f-EPSPs) with an IC(50) of 0.23μM and a full block at 3μM. This compares with an IC(50) of 7.8μM for GYKI52466 on these responses. By contrast, perampanel at 10μM had no effect on responses mediated by NMDA or kainate receptors, which were completely blocked by 30μM D-AP5 and 10μM NBQX respectively. The concentrations of perampanel required to reduce AMPA receptor-mediated responses are not dissimilar to those in plasma following anti-convulsant doses and are consistent with AMPA receptor antagonism being its primary mode of action.     

The neurotrophic effects of PACAP in PC12 cells: control by multiple transduction pathways

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are closely related members of the secretin superfamily of neuropeptides expressed in both the brain and peripheral nervous system, and they exhibit neurotrophic and neurodevelopmental effects in vivo. Like the index member of the Trk receptor ligand family, nerve growth factor (NGF), PACAP promotes the differentiation of PC12 cells, a well-established cell culture model, to investigate neuronal differentiation, survival and function. Stimulation of catecholamine secretion and enhanced neuropeptide biosynthesis are effects exerted by PACAP at the adrenomedullary synapse in vivo and on PC12 cells in vitro through stimulation of the specific PAC1 receptor. Induction of neuritogenesis, growth arrest, and promotion of cell survival are effects of PACAP that occur in developing cerebellar, hippocampal and cortical neurons, as well as in the more tractable PC12 cell model. Study of the mechanisms through which PACAP exerts its various effects on cell growth, morphology, gene expression and survival, i.e. its actions as a neurotrophin, in PC12 cells is the subject of this review. The study of neurotrophic signalling by PACAP in PC12 cells reveals that multiple independent pathways are coordinated in the PACAP response, some activated by classical and some by novel or combinatorial signalling mechanisms.     

Cytokine Regulation of Nerve Growth Factor-Mediated Cholinergic Neurotrophic Activity Synthesized by Astrocytes and Fibroblasts

The neurotrophic activity of astrocytes and fibroblasts and its regulation by various cytokines were investigated. Astrocyte conditioned medium (ACM) enhanced the survival of neurons and the proliferation of astrocytes in embryonic cortical cultures grown in serum-free defined medium. However, these results were not affected by acidic fibroblast growth factor, interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF alpha), and transforming growth factor-beta 1. In contrast, ACM induced choline acetyltransferase expression in septal cholinergic neurons via nerve growth factor (NGF)-dependent and -independent mechanisms. However, neither acidic nor basic fibroblast growth factor is involved in this biological activity in ACM. The cytokines listed above mainly stimulate NGF-mediated cholinergic neurotrophic activity in ACM. A combination of IL-1 beta and TNF alpha significantly enhanced choline acetyltransferase activity in septal neurons co-cultured with astrocytes, and this effect was found to be mediated by NGF produced by activated astrocytes. Effects of astrocytes on GABAergic neurons were also examined. ACM was found to increase glutamate decarboxylase activity in neuronal cultures from septum in the presence of Ara-C. However, the cytokines did not enhance this activity in ACM. Moreover, a combination of IL-1 beta and TNF alpha had no effect on glutamate decarboxylase activity in septal neurons co-cultured with astrocytes. In a final set of experiments, cholinergic neurotrophic activity in skin-derived fibroblast conditioned medium (FCM) was examined. FCM was found to possess biological activity similar to that of ACM on septal neurons grown in serum-free defined medium with Ara-C. The cytokines also enhanced NGF-mediated cholinergic neurotrophic activity in FCM. Astrocytes and fibroblasts were found to possess NGF-type and non-NGF-type cholinergic neurotrophic activity, and various cytokines were found to regulate the NGF-type cholinergic neurotrophic activity in both types of cells. NGF produced by astrocytes and fibroblasts that are activated by cytokines is likely to be important for development and regeneration of NGF-sensitive neurons in the central and peripheral nervous systems.     

Basic Fibroblast Growth Factor Selectively Increases AMPA-Receptor Subunit GluR1 Protein Level and Differentially Modulates Ca2+ Responses to AMPA and NMDA in Hippocampal Neurons

Abstract: The excitatory neurotransmitter glutamate is believed to play important roles in development, synaptic plasticity, and neurodegenerative conditions. Recent studies have shown that neurotrophic factors can modulate neuronal excitability and survival and neurite outgrowth responses to glutamate, but the mechanisms are unknown. The present study tested the hypothesis that neurotrophic factors modulate responses to glutamate by affecting the expression of specific glutamate-receptor proteins. Exposure of cultured embryonic rat hippocampal cells to basic fibroblast growth factor (bFGF) resulted in a concentration-dependent increase in levels of α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-receptor subunit GluR1 protein as determined by western blot, dot-blot, and immunocytochemical analyses. In contrast, bFGF did not alter levels of GluP2/3, GluR4, or the NMDA-receptor subunit NR1. Nerve growth factor did not affect GluR1 levels. Calcium-imaging studies revealed that elevation of [Ca²⁺]_i, resulting from selective AMPA-receptor activation, was enhanced in bFGF-pretreated neurons. On the other hand, [Ca²⁺]_i responses to NMDA-receptor activation were suppressed in bFGF-treated neurons, consistent with previous studies showing that bFGF can protect neurons against NMDA toxicity. Moreover, neurons pretreated with bFGF were relatively resistant to the toxicities of glutamate and AMPA, both of which were shown to be mediated by NMDA receptors. These data suggest that differential regulation of the expression of specific glutamate-receptor subunits may be an important mechanism whereby neurotrophic factors modulate activity-dependent neuronal plasticity and vulnerability to excitotoxicity.