P19 Neurons Research Articles

P19-derived neuronal cells express H1, H2, and H3 histamine receptors: a biopharmaceutical approach to evaluate antihistamine agents

Histamine is a biogenic amine implicated in various biological and pathological processes. Convenient cellular models are needed to screen and develop new antihistamine agents. This report aimed to characterize the response of neurons differentiated from mouse P19 embryonal carcinoma cells to histamine treatment, and to investigate the modulation of this response by antihistamine drugs, vegetal diamine oxidase, and catalase. The exposure of P19 neurons to histamine reduced cell viability to 65% maximally. This effect involves specific histamine receptors, since it was prevented by treatment with desloratadine and cimetidine, respectively, H1 and H2 antagonists, but not by the H3 antagonist ciproxifan. RT-PCR analysis showed that P19 neurons express H1 and H2 receptors, and the H3 receptor, although it seemed not involved in the histamine effect on these cells. The H4 receptor was not expressed. H1 and H2 antagonists as well as vegetal diamine oxidase diminished the intracellular Ca2+ mobilization triggered by histamine. The treatment with vegetal diamine oxidase or catalase protected against mortality and a significant reduction of H2O2 level, generated from the cells under the histamine action, was found upon treatments with desloratadine, cimetidine, vegetal diamine oxidase, or catalase. Overall, the results indicate the expression of functional histamine receptors and open the possibility of using P19 neurons as model system to study the roles of histamine and related drugs in neuronal pathogenesis. This model is less expensive to operate and can be easily implemented by current laboratories of analysis and by Contract Research Organizations.

Exosomes induce neurogenesis of pluripotent P19 cells

Exosomes play a role in tissue/organ development and differentiation. Retinoic acid induces differentiation of P19 cells (UD-P19) to P19 neurons (P19N) that behave like cortical neurons and express characteristic neuronal genes such as NMDA receptor subunits. Here we report P19N exosome-mediated differentiation of UD-P19 to P19N. Both UD-P19 and P19N released exosomes with characteristic exosome morphology, size, and common protein markers. P19N internalized significantly higher number of Dil-P19N exosomes as compared to UD-P19 with accumulation in the perinuclear region. Continuous exposure of UD-P19 to P19N exosomes for six days induced formation of small-sized embryoid bodies that differentiated into MAP2-/GluN2B-positive neurons recapitulating RA-induction of neurogenesis. Incubation with UD-P19 exosomes for six days did not affect UD-P19. Small RNA-seq identified enrichment of P19N exosomes with pro-neurogenic non-coding RNAs (ncRNAs) such as miR-9, let-7, MALAT1 and depleted with ncRNAs involved in maintenance of stem cell characteristics. UD-P19 exosomes were rich with ncRNAs required for maintenance of stemness. P19N exosomes provide an alternative method to genetic modifications for cellular differentiation of neurons. Our novel findings on exosomes-mediated differentiation of UD-P19 to P19 neurons provide tools to study pathways directing neuron development/differentiation and develop novel therapeutic strategies in neuroscience.Graphical

NRF2 activation protects against valproic acid-induced disruption of neurogenesis in P19 cells

Valproic acid (VPA) is a commonly prescribed antiepileptic drug that causes fetal valproate syndrome (FVS) in developing embryos exposed to it. Symptoms of FVS include neural tube defects (NTDs), musculoskeletal abnormalities, and neurodevelopmental difficulties. One proposed mechanism of VPA-induced developmental toxicity is via oxidative stress, defined as the disruption of redox-sensitive cell signaling. We propose that redox imbalances caused by VPA exposure result in improper cellular differentiation that may contribute to FVS. In undifferentiated P19 mouse embryonal carcinoma cells treated with VPA, glutathione disulfide (GSSG) concentrations were higher and the glutathione (GSH)/GSSG redox potential (Eh) was more oxidizing compared to vehicle-treated control cells, both of which are indications of potential intracellular oxidative stress. Interestingly, VPA had no effect on GSH or GSSG levels in differentiated P19 neurons. Undifferentiated cells pretreated with 3H-1,2-dithiole-3-thione (D3T), an inducer of the nuclear factor erythroid 2-related factor 2 (NRF2) antioxidant response that combats cellular redox disruption, were protected from VPA-induced alterations to the GSH/GSSG system. To assess differential periods of susceptibility, P19 cells were exposed to VPA at various time points during their neuronal differentiation. Cells exposed to VPA early in the differentiation process did not undergo normal neurogenesis as measured by POU domain, class 5, transcription factor 1 (OCT4) and tubulin beta-3 chain (βIII-tubulin), markers of cell stemness and neuronal differentiation, respectively. Neurogenesis was improved with D3T pretreatments prior to VPA exposure. Furthermore, differentiating P19 cells treated with VPA exhibited increased protein oxidation that was diminished with D3T pretreatment. These findings demonstrate that VPA inhibits neurogenesis and propose NRF2-mediated redox homeostasis as a means to promote normal neuronal differentiation, thereby potentially decreasing the prevalence of FVS outcomes.

Atomic force microscopy reveals new biophysical markers for monitoring subcellular changes in oxidative injury: Neuroprotective effects of quercetin at the nanoscale.

Oxidative stress has been recognised as an important pathological mechanism underlying the development of neurodegenerative diseases. The biomarkers for assessing the degree of oxidative stress have been attracting much interest because of their potential clinical relevance in understanding the cellular effects of free radicals and evaluation of the efficacy of drug treatment. Here, an interdisciplinary approach using atomic force microscopy (AFM) and cellular and biological molecular methods were used to investigate oxidative damage in P19 neurons and to reveal the underlying mechanism of protective action of quercetin. Biological methods demonstrated the oxidative damage of P19 neurons and showed that quercetin improved neuronal survival by preventing H2O2-induced p53 and Bcl-2 down-regulation and modulated Akt and ERK1/2 signalling pathways. For the first time, AFM was employed to evaluate morphologically (roughness, height, Feret dimension) and nanomechanical (elasticity) properties in H2O2-induced neuronal damage. The AFM analysis revealed that quercetin suppressed H2O2-provoked changes in cell membrane elasticity and morphological properties, thus confirming its neuroprotective activity. The obtained results indicate the potential of AFM-measured parameters as a biophysical markers of oxidative stress-induced neurodegeneration. In general, our study suggests that AFM can be used as a highly valuable tool in other biomedical applications aimed at screening and monitoring of drug-induced effects at cellular level.

Neuroprotective effect of zolpidem against glutamate-induced toxicity is mediated via the PI3K/Akt pathway and inhibited by PK11195

Excitotoxicity is a pathological process in which neuronal dysfunction and death are induced by excessive glutamate stimulation, the major fast excitatory neurotransmitter in the mammalian brain. Excitotoxicity-induced neurodegeneration is a contributing factor in ischemia-induced brain damage, traumatic brain injury, and various neurodegenerative diseases. It is triggered by calcium overload due to prolonged over-activation of ionotropic N-methyl-d-aspartate (NMDA) receptors. Enhanced Ca2+ release results in neuronal vulnerability through several intertwined mechanisms, including activation of proteolytic enzymes, increased production of reactive oxygen species (ROS), mitochondrial dysfunction and modulation of intracellular signalling pathways. We investigated the neuroprotective effect of hypnotic zolpidem, a drug that exerts its central effects at the GABAA receptor complex, against glutamate-induced toxicity in P19 neurons. Zolpidem prevented death of P19 neurons exposed to glutamate, and abolished the glutamate-induced increase in ROS production, p53 and Bax expression, and caspase-3/7 activity. Zolpidem effects were mediated by marked over-activation of Akt kinase. The pro-survival effect, as well as the pAkt induction, were prevented in the presence of wortmannin, an inhibitor of phosphatidylinositol-3-kinase (PI3K) that functions upstream of Akt. The beneficial effect of zolpidem on neuronal survival was not prevented by flumazenil, a GABAA receptor antagonist. PK11195, a drug that modulates the mitochondrial translocator protein 18 kDa (TSPO) and F0F1-ATPase, prevented the beneficial effect of zolpidem, indicating that the mechanism of zolpidem action involves preservation of mitochondrial function and integrity. Zolpidem effects were further mediated by prevention of glutamate-induced increase in the expression of the NR2B subunit of NMDA receptor. The obtained results suggest the promising therapeutic potential of zolpidem against excitotoxic insults and highlight the importance of mitochondria and the Akt pathway as valuable targets for therapeutic interventions in glutamate-mediated neuropathological conditions.

Comparison of neurons derived from mouse P19, rat PC12 and human SH-SY5Y cells in the assessment of chemical- and toxin-induced neurotoxicity

BackgroundExposure to chemicals might be toxic to the developing brain. There is a need for simple and robust in vitro cellular models for evaluation of chemical-induced neurotoxicity as a complement to traditional studies on animals. In this study, neuronally differentiated mouse embryonal carcinoma P19 cells (P19 neurons) were compared with human neuroblastoma SH-SY5Y cells and rat adrenal pheochromocytoma PC12 cells for their ability to detect toxicity of methylmercury (MeHg), okadaic acid and acrylamide.MethodsRetinoic acid-treated P19 and SH-SY5Y cells and nerve growth factor-stimulated PC12 cells, allowed to differentiate for 6 days, were exposed to MeHg, okadaic acid and acrylamide for 48 h. Cell survival and neurite outgrowth were assessed with the calcein-AM assay and fluorescence detection of antibodies against the cytoskeletal neuron-specific protein βIII-tubulin, respectively. The effects of glutathione (GSH) and the potent inhibitor of GSH synthesis buthionine sulfoximine (BSO) on the MeHg induced-toxicity were assessed using the PrestoBlue™ cell viability assay and the TMRE mitochondrial membrane potential assay.ResultsDifferentiated P19 cells developed the most extensive neuronal network among the three cell models and were the most sensitive neuronal model to detect neurotoxic effects of the test compounds. MeHg produced a concentration-dependent toxicity in differentiated P19 cells and SH-SY5Y cells, with statistically significant effects at concentrations from 0.1 μM in the P19 neurons and 1 μM in the SH-SY5Y cells. MeHg induced a decrease in the cellular metabolic activity and mitochondrial membrane potential (ΔΨm) in the differentiated P19 cells and SH-SY5Y cells, that were attenuated by GSH. Okadaic acid and acrylamide also showed statistically significant toxicity in the P19 neurons, but not in the SH-SY5Y cells or the P12 cells.ConclusionsP19 neurons are more sensitive to detect cytotoxicity of MeHg, okadaic acid and acrylamide than retinoic acid-differentiated SH-SY5Y cells and nerve growth factor-treated PC12 cells. P19 neurons are at least as sensitive as differentiated SH-SY5Y cells to detect the loss of mitochondrial membrane potential produced by MeHg and the protective effects of extracellular GSH on MeHg toxicity. P19 neurons may be a useful model to study neurotoxic effects of chemicals.

Non-Serotonergic Neurotoxicity by MDMA (Ecstasy) in Neurons Derived from Mouse P19 Embryonal Carcinoma Cells.

3,4-methylenedioxymethamphetamine (MDMA; ecstasy) is a commonly abused recreational drug that causes neurotoxic effects in both humans and animals. The mechanism behind MDMA-induced neurotoxicity is suggested to be species-dependent and needs to be further investigated on the cellular level. In this study, the effects of MDMA in neuronally differentiated P19 mouse embryonal carcinoma cells have been examined. MDMA produces a concentration-, time- and temperature-dependent toxicity in differentiated P19 neurons, as measured by intracellular MTT reduction and extracellular LDH activity assays. The P19-derived neurons express both the serotonin reuptake transporter (SERT), that is functionally active, and the serotonin metabolizing enzyme monoamine oxidase A (MAO-A). The involvement of these proteins in the MDMA-induced toxicity was investigated by a pharmacological approach. The MAO inhibitors clorgyline and deprenyl, and the SERT inhibitor fluoxetine, per se or in combination, were not able to mimic the toxic effects of MDMA in the P19-derived neurons or block the MDMA-induced cell toxicity. Oxidative stress has been implicated in MDMA-induced neurotoxicity, but pre-treatment with the antioxidants α-tocopherol or N-acetylcysteine did not reveal any protective effects in the P19 neurons. Involvement of mitochondria in the MDMA-induced cytotoxicity was also examined, but MDMA did not alter the mitochondrial membrane potential (ΔΨm) in the P19 neurons. We conclude that MDMA produce a concentration-, time- and temperature-dependent neurotoxicity and our results suggest that the mechanism behind MDMA-induced toxicity in mouse-derived neurons do not involve the serotonergic system, oxidative stress or mitochondrial dysfunction.

Recording axonal conduction to evaluate the integration of pluripotent cell-derived neurons into a neuronal network.

Stem cell transplantation is a promising therapy to treat neurodegenerative disorders, and a number of in vitro models have been developed for studying interactions between grafted neurons and the host neuronal network to promote drug discovery. However, methods capable of evaluating the process by which stem cells integrate into the host neuronal network are lacking. In this study, we applied an axonal conduction-based analysis to a co-culture study of primary and differentiated neurons. Mouse cortical neurons and neuronal cells differentiated from P19 embryonal carcinoma cells, a model for early neural differentiation of pluripotent stem cells, were co-cultured in a microfabricated device. The somata of these cells were separated by the co-culture device, but their axons were able to elongate through microtunnels and then form synaptic contacts. Propagating action potentials were recorded from these axons by microelectrodes embedded at the bottom of the microtunnels and sorted into clusters representing individual axons. While the number of axons of cortical neurons increased until 14 days in vitro and then decreased, those of P19 neurons increased throughout the culture period. Network burst analysis showed that P19 neurons participated in approximately 80% of the bursting activity after 14 days in vitro. Interestingly, the axonal conduction delay of P19 neurons was significantly greater than that of cortical neurons, suggesting that there are some physiological differences in their axons. These results suggest that our method is feasible to evaluate the process by which stem cell-derived neurons integrate into a host neuronal network.

CRISPR/Cas9-Mediated Genomic Deletion of the Beta-1, 4 N-acetylgalactosaminyltransferase 1 Gene in Murine P19 Embryonal Carcinoma Cells Results in Low Sensitivity to Botulinum Neurotoxin Type C.

Botulinum neurotoxins produced by Clostridium botulinum cause flaccid paralysis by inhibiting neurotransmitter release at peripheral nerve terminals. Previously, we found that neurons derived from the murine P19 embryonal carcinoma cell line exhibited high sensitivity to botulinum neurotoxin type C. In order to prove the utility of P19 cells for the study of the intracellular mechanism of botulinum neurotoxins, ganglioside-knockout neurons were generated by deletion of the gene encoding beta-1,4 N-acetylgalactosaminyltransferase 1 in P19 cells using the clustered regularly interspaced short palindromic repeats combined with Cas9 (CRISPR/Cas9) system. By using this system, knockout cells could be generated more easily than with previous methods. The sensitivity of the generated beta-1,4 N-acetylgalactosaminyltransferase 1-depleted P19 neurons to botulinum neurotoxin type C was decreased considerably, and the exogenous addition of the gangliosides GD1a, GD1b, and GT1b restored the susceptibility of P19 cells to botulinum neurotoxin type C. In particular, addition of a mixture of these three ganglioside more effectively recovered the sensitivity of knockout cells compared to independent addition of GD1a, GD1b, or GT1b. Consequently, the genome-edited P19 cells generated by the CRISPR/Cas9 system were useful for identifying and defining the intracellular molecules involved in the toxic action of botulinum neurotoxins.

Effects of copper overload in P19 neurons: impairment of glutathione redox homeostasis and crosstalk between caspase and calpain protease systems in ROS-induced apoptosis.

Copper, a transition metal with essential biological functions, exerts neurotoxic effects when present in excess. The aim of the present study was to better elucidate cellular and molecular mechanisms of CuSO4 toxicity in differentiated P19 neurons. Exposure to 0.5mM CuSO4 for 24h provoked moderate decrease in viability, accompanied with barely increased generation of reactive oxygen species (ROS) and caspase-3/7 activity. Glutathione (GSH) and ATP contents were depleted, lactate dehydrogenase inactivated, and glyceraldehyde-3-phosphate dehydrogenase overexpressed. In severely damaged neurons exposed to only two times higher concentration, classical caspase-dependent apoptosis was triggered as evidenced by marked caspase-3/7 activation and chromatin condensation. Multifold increase in ROS, together with very pronounced ATP and GSH loss, strongly suggests impairment of redox homeostasis. At higher copper concentration protease calpains were also activated, and neuronal injury was prevented in the presence of calpain inhibitor leupeptin through the mechanism that affects caspase activation. MK-801 and nifedipine, inhibitors of calcium entry, and H-89 and UO126, inhibitors of PKA and ERK signaling respectively, exacerbated neuronal death only in severely damaged neurons, while ROS-scavenger quercetin and calcium chelator BAPTA attenuated toxicity only at lower concentration. In a dose-dependent manner copper also provoked transcriptional changes of genes involved in intracellular signaling and induction of apoptosis (p53, c-fos, Bcl-2 and Bax). The obtained results emphasize differences in triggered neuronal-death processes in a very narrow range of concentrations and give further insight into the molecular mechanisms of copper toxicity with the potential to improve current therapeutic approaches in curing copper-related neurodegenerative diseases.

GABAAergic stimulation modulates intracellular protein arginine methylation

Changes in cytoplasmic pH are known to regulate diverse cellular processes and influence neuronal activities. In neurons, the intracellular alkalization is shown to occur after stimulating several channels and receptors. For example, it has previously demonstrated in P19 neurons that a sustained intracellular alkalinization can be mediated by the Na+/H+ antiporter. In addition, the benzodiazepine binding subtypes of the γ-amino butyric acid type A (GABAA) receptor mediate a transient intracellular alkalinization when they are stimulated. Because the activities of many enzymes are sensitive to pH shift, here we investigate the effects of intracellular pH modulation resulted from stimulating GABAA receptor on the protein arginine methyltransferases (PRMT) activities. We show that the major benzodiazepine subtype (2α1, 2β2, 1γ2) is constitutively expressed in both undifferentiated P19 cells and retinoic acid (RA) differentiated P19 neurons. Furthermore stimulation with diazepam and, diazepam plus muscimol produce an intracellular alkalinization that can be detected ex vivo with the fluorescence dye. The alkalinization results in significant perturbation in protein arginine methylation activity as measured in methylation assays with specific protein substrates. Altered protein arginine methylation is also observed when cells are treated with the GABAA agonist muscimol but not an antagonist, bicuculline. These data suggest that pH-dependent and pH-independent methylation pathways can be activated by GABAAergic stimulation, which we verified using hippocampal slice preparations from a mouse model of fragile X syndrome.

HCS-Neurons: identifying phenotypic changes in multi-neuron images upon drug treatments of high-content screening

BackgroundHigh-content screening (HCS) has become a powerful tool for drug discovery. However, the discovery of drugs targeting neurons is still hampered by the inability to accurately identify and quantify the phenotypic changes of multiple neurons in a single image (named multi-neuron image) of a high-content screen. Therefore, it is desirable to develop an automated image analysis method for analyzing multi-neuron images.ResultsWe propose an automated analysis method with novel descriptors of neuromorphology features for analyzing HCS-based multi-neuron images, called HCS-neurons. To observe multiple phenotypic changes of neurons, we propose two kinds of descriptors which are neuron feature descriptor (NFD) of 13 neuromorphology features, e.g., neurite length, and generic feature descriptors (GFDs), e.g., Haralick texture. HCS-neurons can 1) automatically extract all quantitative phenotype features in both NFD and GFDs, 2) identify statistically significant phenotypic changes upon drug treatments using ANOVA and regression analysis, and 3) generate an accurate classifier to group neurons treated by different drug concentrations using support vector machine and an intelligent feature selection method. To evaluate HCS-neurons, we treated P19 neurons with nocodazole (a microtubule depolymerizing drug which has been shown to impair neurite development) at six concentrations ranging from 0 to 1000 ng/mL. The experimental results show that all the 13 features of NFD have statistically significant difference with respect to changes in various levels of nocodazole drug concentrations (NDC) and the phenotypic changes of neurites were consistent to the known effect of nocodazole in promoting neurite retraction. Three identified features, total neurite length, average neurite length, and average neurite area were able to achieve an independent test accuracy of 90.28% for the six-dosage classification problem. This NFD module and neuron image datasets are provided as a freely downloadable MatLab project at http://iclab.life.nctu.edu.tw/HCS-Neurons.ConclusionsFew automatic methods focus on analyzing multi-neuron images collected from HCS used in drug discovery. We provided an automatic HCS-based method for generating accurate classifiers to classify neurons based on their phenotypic changes upon drug treatments. The proposed HCS-neurons method is helpful in identifying and classifying chemical or biological molecules that alter the morphology of a group of neurons in HCS.

Neuritogenic activity of coumarins from Clausena harmandiana

Coumarins have been identified from natural sources, especially green plants such as Clausena harmandiana. Antioxidant activity of the plant was assayed using 1,1-diphenyl-2-pricrylhydrazyl (DPPH) and Thiobarbituric Acid Reactive Substances (TBARS) methods while its neuritogenic effects was assessed on cultured P19 neurons. Nordentatin, isolated from the root bark of C. harmandiana, showed more potent antioxidant and inhibition of lipid peroxidation than its derivatives, dentatin and xanthoxyletin. Nordentatin with IC50 > 10 mM and viability of 82.74 ± 24.54% showed no neurotoxicity while dentatin and xanthoxyletin expressed neurotoxicity at 1 nM (viability 62.28 ± 12.11% and 43.02 ± 10.31%, respectively). Phase-contrast micrographs showed that nordentatin promoted the outgrowth of the neurites of the neurons. The neurons treated with the extracts displayed significantly more branching numbers of the neurites than the control (p < 0.05), indicating that nordentatin was a remarkable natural neuritogen.

Neural responses to electrical stimulation on patterned silk films

Peripheral nerve injury is a critical issue for patients with trauma. Following injury, incomplete axon regeneration or misguided axon innervation into tissue will result in loss of sensory and motor functions. The objective of this study was to examine axon outgrowth and axon alignment in response to surface patterning and electrical stimulation. To accomplish our objective, metal electrodes with dimensions of 1.5 mm × 4 cm, were sputter coated onto micropatterned silk protein films, with surface grooves 3.5 μm wide × 500 nm deep. P19 neurons were seeded on the patterned electronic silk films and stimulated at 120 mV, 1 kHz, for 45 min each day for 7 days. Responses were compared with neurons on flat electronic silk films, patterned silk films without stimulation, and flat silk films without stimulation. Significant alignment was found on the patterned film groups compared with the flat film groups. Axon outgrowth was greater (p < 0.05) on electronic films on days 5 and 7 compared with the unstimulated groups. In conclusion, electrical stimulation, at 120 mV, 1 kHz, for 45 min daily, in addition to surface patterning, of 3.5 μm wide × 500 nm deep grooves, offered control of nerve axon outgrowth and alignment.

Application of aurintricarboxylic acid for the adherence of mouse P19 neurons and primary hippocampal neurons to noncoated surface in serum-free culture

Dissociated primary neuron culture has been the most widely used model systems for neuroscience research. Most of these primary neurons are cultured on adhesion matrix-coated surface to provide a proper environment for cell anchorage under serum-free conditions. In this study, we provide an alternative technique to promote the adhesions of these neurons using aurintricarboxylic acid (ATA), a nonpeptide compound, without surface manipulations. We first demonstrated that ATA could promote Chinese hamster ovary cell attachment and proliferation in serum-free medium in a dosage-dependent manner. We later showed that ATA significantly enhanced the attachment of the retinoic acid differentiated P19 mouse embryonal carcinoma (P19) neurons, with an optimal concentration around 30 μg/mL. A similar result was seen in primary hippocampal neurons, with an optimal ATA concentration around 15 μg/mL. Further morphological assessments revealed that the average neurite length and neuronal polarization were almost identical to that obtained using a conventional method with poly-L-lysine surface. The advantages of using the ATA treatment technique for immunochemical analysis are discussed.

Quercetin supplementation: insight into the potentially harmful outcomes of neurodegenerative prevention

Dietary antioxidant supplements have been considered for the prevention of neuronal oxidative injury and death. Recent studies indicate that excessive antioxidants could exert adverse effects, thereby questioning the safety of prolonged supplementation. The aim of our study was to investigate the effects of quercetin (up to 150 μM), the ubiquitous plant-derived flavonoid and highly potent scavenger of reactive oxygen species (ROS) on healthy P19 neurons, in order to assess the efficacy and safety of its long-term use in neurodegenerative prevention. Although exposure for 24 h to quercetin did not compromise neuronal survival, morphological examination revealed diminished neuronal branching, a finding probably related to an observed decrease in lactate dehydrogenase activity. Using 2',7'-dichlorofluorescin diacetate and dot-blot analysis, we found reduced basal levels of ROS and 4-hydroxy-2-nonenal, a biomarker of lipid peroxidation, confirming the antioxidative mechanism of quercetin action. Unexpectedly, quercetin also depleted intracellular glutathione content. Reverse transcriptase PCR and western blot analysis showed depletion of total RNA amount and changes in the expression of cell survival regulating genes Bcl-2, p53, and c-fos. Nuclear condensation and caspase-3/7 activity, phenomena related to programmed cell death cascade, were not affected. The potential risk of observed changes indicates that quercetin-enriched supplements should be taken with caution. The diversity of quercetin effects and complexity of possible intracellular interactions between affected genes pointed out the necessity for additional pharmacological and toxicological studies in order to better elucidate the mechanisms of quercetin action and to recognize its potential side effects at higher doses and during long-term administration.

P19 embryonal carcinoma cells exhibit high sensitivity to botulinum type C and D/C mosaic neurotoxins

Botulinum neurotoxins (BoNTs) inhibit neurotransmitter release at peripheral nerve terminals. They are serologically classified from A to G, C/D and D/C mosaic neurotoxins forming further subtypes of serotypes C and D. Cultured primary neurons, as well as neuronal cell lines such as PC12 and Neuro-2a, are often utilized in cell-based experiments on the toxic action of botulinum toxins. However, there are very few reports of the use of neural cell lines for studying BoNTs/C and D. In addition, the differentiated P19 neuronal cell line, which possesses cholinergic properties, has yet to be tested for its susceptibility to BoNTs. Here, the responsiveness of differentiated P19 cells to BoNT/C and BoNT/DC is reported. Both BoNT/C and BoNT/DC were shown to effectively bind to, and be internalized by, neurons derived from P19 cells. Subsequently, the intracellular substrates for BoNT/C and BoNT/DC were cleaved by treatment of the cells with the toxins in a ganglioside-dependent manner. Moreover, P19 neurons exhibited high sensitivity to BoNT/C and BoNT/DC, to the same extent as cultured primary neurons. These findings suggest that differentiated P19 cells possess full sensitivity to BoNT/C and BoNT/DC, thus making them a novel susceptible cell line for research into BoNTs.

Neuroprotective Effect of Quercetin Against Hydrogen Peroxide-induced Oxidative Injury in P19 Neurons

Oxidative stress is implicated in neuronal death in a variety of neurodegenerative diseases. In the present study, P19 neurons obtained by the differentiation procedure from mouse teratocarcinoma P19 cells were used to investigate the ability of quercetin, a plant-derived flavonoid, to prevent neuronal death induced by exposure to 150 μM or 1.5 mM hydrogen peroxide (H(2)O(2)) for 24 h. Quercetin treatment improved viability of P19 neurons exposed to both types of oxidative injury. During the modest oxidative stress, quercetin diminished generation of reactive oxygen species (ROS) and prevented H(2)O(2)-induced nuclear condensation, increase in caspase 3/7 activity and rise in poly(APD-ribose) polymerase expression. Expression of Bcl-2 family members Bax and Bcl-2 was not affected by quercetin treatment at both the transcriptional and translational levels. During the severe oxidative injury, quercetin prevented H(2)O(2)-induced rise in ROS accumulation and changes in plasma membrane integrity and nuclear morphology. The obtained results suggest that neuroprotective effects of quercetin are related to its antioxidative action and prevention of events associated with programmed cell death cascade. In the light of these findings, one might assume beneficial effects of quercetin for the prevention of oxidative stress-driven neuronal loss in human aging and age-related neurodegenerative diseases.

PH uniquely modulates protein arginine methylation

Protein arginine methyltransferases (PRMTs) function in the alkaline milieu of the nucleus and at neutral pH of the cytosol. Accordingly, several PRMTs are broadly active over a range of pHs. We investigated the effect altering pH had on protein arginine methylation using a variety of defined substrates, recombinant PRMTs and cell extracts.We demonstrate that pH-induced alterations in the extent of methylation and the methyl-product formed depend both on the particular substrate assayed and the PRMT that modifies it. We also find that transient intracellular alkalinization of mouse embryonic P19 neurons by NH4Cl results in sustained changes in substrate methylation. Altogether our results are consistent with a hypothesis in which altered substrate methylation resulting from pH-induced changes of PRMT activities coupled with low levels of demethylation may provide the long-term tag(s) necessary for the formation and maintenance of “molecular memory”. Key words: Arginine methylation, protein arginine methyltransferase, pH, P19 cells, insect cell lysate, SmD1 peptide.

Ceruloplasmin-induced aggregation of P19 neurons involves a serine protease activity and is accompanied by reelin cleavage

The cytoarchitectural organization of the nervous system depends partly on extracellular serine proteases, including reelin. This 400K protein, which also exists as the N-terminally-derived 300K and 180K fragments, acts through binding to the lipoprotein receptors apolipoprotein E receptor 2 (ApoER2) and very low-density lipoprotein receptor (VLDLR). Ceruloplasmin (CP), a multifunctional protein found in the circulation and also expressed on glial cells, was shown to bind to, and induce aggregation of neurons newly differentiated from P19 embryonic stem cells. This indicated a potential developmental role of CP in neuronal organization, possibly in relation with reelin and other extracellular serine proteases. Therefore, we analysed the effect of cell-impermeant, large spectrum, serine protease inhibitors on CP-induced neuroaggregation and studied reelin expression. Soybean trypsin inhibitor and aprotinin (SBTI+Apro) inhibited CP neuroaggregative action. Undifferentiated and neurally-differentiating cultures secreted the 400K reelin. The 180K fragment was present during and after differentiation whereas the 300K species was barely detectable. However, CP stimulated generation of the 300K in the differentiated neuronal cultures, and SBTI+Apro abolished this CP effect. Time course profiles and function-blocking antibody indicated that neuroaggregation does not depend on the generation of the 300K fragment or on reelin action. CP neuroaggregative action thus involves a pericellular serine protease, different from reelin. On the other hand, the CP stimulation of reelin cleavage is in line with a possible role of CP in nervous system development. Since P19 cells express ApoER2 and VLDLR, they can help understanding relationships existing between CP, reelin and intervening protease(s).