Genes In PC12 Cells Research Articles

Snake venom nerve growth factor-inspired designing of novel peptide therapeutics for the prevention of paraquat-induced apoptosis, neurodegeneration, and alteration of metabolic pathway genes in the rat pheochromocytoma PC-12cell.

Neurodegenerative disorders (ND), associated with the progressive loss of neurons, oxidative stress-mediated production of reactive oxygen species (ROS), and mitochondrial dysfunction, can be treated with synthetic peptides possessing innate neurotrophic effects and neuroprotective activity. Computational analysis of two small synthetic peptides (trideca-neuropeptide, TNP; heptadeca-neuropeptide, HNP) developed from the nerve growth factors from snake venoms predicted their significant interaction with the human TrkA receptor (TrkA). In silico results were validated by an in vitro binding study of the FITC-conjugated custom peptides to rat pheochromocytoma PC-12cell TrkA receptors. Pre-treatment of PC-12cells with TNP and HNP induced neuritogenesis and significantly reduced the paraquat (PT)-induced cellular toxicity, the release of lactate dehydrogenase from the cell cytoplasm, production of intracellular ROS, restored the level of antioxidants, prevented alteration of mitochondrial transmembrane potential (ΔΨm) and adenosine triphosphate (ATP) production, and inhibited cellular apoptosis. These peptides lack in vitro cytotoxicity, haemolytic activity, and platelet-modulating properties and do not interfere with the blood coagulation system. Functional proteomic analyses demonstrated the reversal of PT-induced upregulated and downregulated metabolic pathway genes in PC-12cells that were pre-treated with HNP and revealed the metabolic pathways regulated by HNP to induce neuritogenesis and confer protection against PT-induced neuronal damage in PC-12. The quantitative RT-PCR analysis confirmed that the PT-induced increased and decreased expression of critical pro-apoptotic and anti-apoptotic genes had been restored in the PC-12cells pre-treated with the custom peptides. A network gene expression profile was proposed to elucidate the molecular interactions among the regulatory proteins for HNP to salvage the PT-induced damage. Taken together, our results show how the peptides can rescue PT-induced oxidative stress, mitochondrial dysfunction, and cellular death and suggest new opportunities for developing neuroprotective drugs.

Systematic Analysis of tRNA-Derived Small RNAs Reveals the Effects of Xuefu-Zhuyu Decoction on the Hippocampi of Rats after Traumatic Brain Injury.

Background Traumatic brain injury (TBI) is one of the most common neurosurgical diseases and refers to brain function impairment or brain pathological changes induced by external causes. A traditional Chinese medicine, Xuefu-Zhuyu Decoction (XFZYD), has been indicated to harbor therapeutic properties against TBI. Transfer RNA (tRNA)-derived small RNAs, that is, tsRNAs (a group of small RNAs derived from tRNAs), are multifunctional regulatory noncoding RNAs generated under pressure and implicated in the progression of TBI. Methods A TBI model was successfully constructed using rats. We further performed sequencing and omics analyses to identify novel tsRNAs as drug targets for XFZYD therapy against TBI in the rat hippocampus. qPCR assays were used to further verify the experimental results. Gene Ontology (GO) was used to analyze the signaling pathways of downstream target genes of tsRNAs in the XFZYD-regulated TBI model. qPCR was used to detect the influence of overexpressed tsRNA mimics/inhibitors on their target genes in PC12 cells. Results Our RNA-Seq data illustrate that 11 tsRNAs were mediated by XFZYD. The experimental data revealed AS-tDR-002004 and AS-tDR-002583 as potential targets for XFZYD therapy and showed that they influenced TBI via the cadherin signaling pathway, cocaine addiction, circadian entrainment, and the nicotine pharmacodynamics pathway. We also confirmed that Pi4kb, Mlh3, Pcdh9, and Ppp1cb were target genes of 2 XFZYD-regulated tsRNAs in the hippocampus of a rat model and PC12 cells. Furthermore, biological function analysis revealed the potential therapeutic effects of tsRNAs, and the results showed that Mapk1 and Gnai1 were related genes for XFZYD therapy against TBI. Conclusion Our work successfully illuminates the efficiency of XFZYD in the treatment of TBI. The experimental data revealed AS-tDR-002004 and AS-tDR-002583 as potential targets for XFZYD therapy and showed that they influenced TBI via the cadherin signaling pathway, cocaine addiction, circadian entrainment, and the nicotine pharmacodynamics pathway in a TBI rat model.

Combined molecular, structural and memory data unravel the destructive effect of tramadol on hippocampus

Tramadol is a synthetic analogue of codeine and stimulates neurodegeneration in several parts of the brain that leads to various behavioral impairments. Despite the leading role of hippocampus in learning and memory as well as decreased function of them under influence of tramadol, there are few studies analyzing the effect of tramadol administration on gene expression profiling and structural consequences in hippocampus region. Thus, we sought to determine the effect of tramadol on both PC12 cell line and hippocampal tissue, from gene expression changes to structural alterations. In this respect, we investigated genome‐wide mRNA expression using high throughput RNA-seq technology and confirmatory quantitative real‐time PCR, accompanied by stereological analysis of hippocampus and behavioral assessment following tramadol exposure. At the cellular level, PC12 cells were exposed to 600 μM tramadol for 48 hrs, followed by the assessments of ROS amount and gene expression levels of neurotoxicity associated with neurodegenerative pathways such as apoptosis and autophagy. Moreover, the structural and functional alteration of the hippocampus under chronic exposure to tramadol was also evaluated. In this regard, rats were treated with tramadol at doses of 50 mg/kg for three consecutive weeks. In vitro data revealed that tramadol provoked ROS production and caused the increase in the expression of autophagic and apoptotic genes in PC12 cells. Furthermore, in-vivo results demonstrated that tramadol not only did induce hippocampal atrophy, but it also triggered microgliosis and microglial activation, causing upregulation of apoptotic and inflammatory markers as well as over-activation of neurodegeneration. Tramadol also interrupted spatial learning and memory function along with long-term potentiation (LTP). Taken all together, our data disclosed the neurotoxic effects of tramadol on both in vitro and in-vivo. Moreover, we proposed a potential correlation between disrupted biochemical cascades and memory deficit under tramadol administration.

Panax notoginseng saponins protect PC12 cells against Aβ induced injury via promoting parkin-mediated mitophagy

Ethnopharmacological relevancePanax notoginseng (Burk) F. H. Chen is a well-known traditional Chinese medicine with a long history and is widely used in the treatment of cerebrovascular disease. Panax notoginseng saponins (PNS) are the main active ingredients in Panax notoginseng (Burk) F. H. Chen, and its injection is used to treat nerve damage caused by cerebral ischemia and other conditions. PNS is thought to alleviate cognitive impairment in patients with Alzheimer's disease; however, its mechanism of action is unclear. Aim of the studyWe elucidated the role of PNS in attenuating cellular mitochondrial damage caused by amyloid β (Aβ) protein and in protecting cell viability from the perspective of regulating autophagy. By investigating the effects of PNS on the targets regulating mitophagy, we wanted to reveal the autophagy related mechanism by which PNS attenuated Aβ damage in neuronal cells. Materials and methodsThe effect of PNS on the mitochondrial membrane potential of Aβ-injured PC12 cells was detected using flow cytometry, which reflected the alleviating effect of PNS on mitochondrial damage. Using mRFP-GFP-LC3-transfected PC12 cells, the effect of PNS on cellular autophagy flux was observed using laser confocal microscopy. Formation of the intracellular autophagosome was observed using transmission electron microscopy, which reflected the activation of autophagy by PNS. The siPINK1 lentivirus was used to silence the PINK1 gene in PC12 cells to obtain siPINK1-PC12 cells. The effects of PNS on the expression of the PINK1 gene and on the autophagy-related proteins LC3II/Ⅰ, p62, PINK1, parkin, NDP52, and OPTN were observed to reveal the possible targets of PNS in regulating autophagy. ResultsAfter PNS treatment, the viability of Aβ-injured PC12 cells improved and the mitochondrial membrane potential was restored. PNS treatment significantly enhanced the autophagy flux of damaged cells and increased the levels of LC3II/Ⅰ protein and decreased p62 protein, while significantly improving the structure and mitochondrial morphology of PC12 cells injured by Aβ. These changes led to more autophagosomes wrapping around the damaged mitochondria and promoting the depletion of OPTN, a mitophagy receptor. After silencing the PINK1 gene, PNS could not alter the PINK1 gene and protein levels, but could still increase LC3II/Ⅰ, decrease p62 and OPTN, and significantly increase the amount of parkin. ConclusionsPNS could enhance the autophagic activity of cells, alleviate mitochondrial damage caused by Aβ injury, and protect the activity of PC12 cells. It is possible that enhanced autophagy was achieved by promoting the recruitment of parkin protein to the mitochondrial receptors in a non-PINK1-dependent manner.

Tramadol exposure upregulated apoptosis, inflammation and autophagy in PC12 cells and rat’s striatum: An in vitro- in vivo approach

Aim and backgroundTramadol is a synthetic analogue of codeine, mostly prescribed for the alleviation of mild to moderate pains. It bears several side effects including emotional instability and anxiety. In this study, we focused on the alteration in expression of autophagic and apoptotic genes in PC-12 cells for our in vitro and structural and functional changes of striatum for our in vivo under chronic exposure of tramadol. MethodsFor in vitro side of the study, PC12 cells were exposed to tramadol (50 μM) and expression of apoptosis and autophagy genes were determined. In parallel, rats were daily treated with tramadol at doses of 50 mg/kg for three weeks for the in vivo side. Motor coordination, EMG, histopathology and gene expression were done. ResultsOur in vitro findings revealed that tramadol increased expression of apoptosis and autophagy genes in PC12 cells. Moreover, our in vivo results disclosed that tramadol not only provoked atrophy of rats’ striatum, but also triggered microgliosis along with neuronal death in the striatum. Tramadol also reduced motor coordination and muscular activity. ConclusionAltogether, our data indicated that tramadol induced neurotoxicity in the PC12 cells via apoptosis and autophagy and in striatum chiefly through activation of neuroinflammatory and apoptotic responses.

The Effect of Beta-Boswellic Acid on the Expression of Camk4 and Camk2α Genes in the PC12 Cell Line.

Purpose: Beta-boswellic acid (βBA) may play central roles in neural plasticity. Neural plasticity has significant implications for learning and memory which are governed by strict memoryrelated molecular pathways. To gain insight into the molecular mechanism by which βBA affects these pathways this study analyzed the expression patterns of Camk2α and Camk4 genes in PC12 cells treated with βBA. Methods: The cytotoxic effects of different βBA concentrations on PC12 cells were examined by MTT assay. For gene expression analysis, cells were treated with concentrations of 1 and 10 µM of βBA for 12, 24, 48, and 72 hours. Total RNA was purified by RNX-Plus solution and reverse transcribed into cDNA using Thermo Scientific Reverse Transcription reagents. The expression patterns of Camk2α and Camk4 genes were quantified by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Results: MTT assay indicated that βBA reduced PC12 cell viability in a time- and concentrationdependent manner. The 50% inhibitory concentrations for the 48 and 72 hours time points were 35 and 26 µM, respectively; while, the βBA concentrations up to 100 µM failed to kill 50% of the cells after 24 hours. According to the qRT-PCR data, the Camk2α variant is not expressed in either βBA-treated or untreated PC12 cells. However, a significant upregulation was observed inCamk4 after 12 hours of treatment with βBA, which followed by a significant downregulation after 24 hours and a persistent expression equal to the control until 72 hours. Conclusion: these findings indicate that PC12 cells not only does not express Camk2α but also its expression cannot be induced by βBA. However, βBA does modulate the expression of Camk4. This result provides further insight into the molecular mechanism by which βBA affects memory.

Cocaine- and amphetamine-regulated transcript promoter regulated by nicotine in nerve growth factor-treated PC12 cells.

Nicotine and cocaine- and amphetamine-regulated transcripts (CART) have several overlapping functions, such as the regulation of reward, feeding behavior, stress response, and anxiety. Previous studies showed that nicotine regulates CART expression in various brain regions. However, the molecular mechanisms underlying this regulation are not known. This study investigated the regulatory effect of nicotine on promoter activity of the CART gene in PC12 cells, which were differentiated into a neuronal phenotype by nerve growth factor (NGF) treatment. Two vectors containing reporter genes (Gaussia luciferase or mCherry) and the 1,140-bp upstream of the transcriptional start site of the mouse CART gene are used to analyze the CART promoter activity. Transient transfection of PC12 cells with either vector displayed strong promoter activity in both undifferentiated and differentiated PC12 cells. CART promoter activity in the PC12 cell line is increased by forskolin or NGF treatment. In differentiated PC12 cells, exposure to 50nM nicotine for 6h increased CART promoter activity. However, treatment with higher nicotine doses for 6h and treatment with all nicotine doses for 24h showed no effect. A nicotine concentration of 50nM is comparable to brain nicotine levels experienced by chronic smokers over long periods of time. Taken together, these data indicate that nicotine may exert some of its actions through the regulation of CART transcription in the brain.

Inhibition of poly(ADP-ribose) polymerase-1 alters expression of mitochondria-related genes in PC12 cells: relevance to mitochondrial homeostasis in neurodegenerative disorders

Alzheimer's disease (AD) is characterized by the release of amyloid beta peptides (Aβ) in the form of monomers/oligomers which may lead to oxidative stress, mitochondria dysfunction, synaptic loss, neuroinflammation and, in consequence, to overactivation of poly(ADP-ribose) polymerase-1 (PARP-1). However, Aβ peptides are also released in the brain ischemia, traumatic injury and in inflammatory response. PARP-1 is suggested to be a promising target in therapy of neurodegenerative disorders.We investigated the impact of PARP-1 inhibition on transcription of mitochondria-related genes in PC12 cells. Moreover, the effect of PARP-1 inhibitor (PJ34) on cells subjected to Aβ oligomers (AβO) – evoked stress was analyzed.Our data demonstrated that inhibition of PARP-1 in PC12 cells enhanced the transcription of genes for antioxidative enzymes (Sod1, Gpx1, Gpx4), activated genes regulating mitochondrial fission/fusion (Mfn1, Mfn2, Dnm1l, Opa1, Fis1), subunits of ETC complexes (mt-Nd1, Sdha, mt-Cytb) and modulated expression of several TFs, enhanced Foxo1 and decreased Nrf1, Stat6, Nfkb1. AβO elevated free radicals concentration, decreased mitochondria membrane potential (MMP) and cell viability after 24h. Gene transcription was not affected by AβO after 24h, but was significantly downregulated after 96h. In AβO stress, PJ34 exerted stimulatory effect on expression of several genes (Gpx1, Gpx4, Opa1, Mfn2, Fis1 and Sdha), decreased transcription of numerous TFs (Nrf1, Tfam, Stat3, Stat6, Trp53, Nfkb1) and prevented oxidative stress. Our results indicated that PARP-1 inhibition significantly enhanced transcription of genes involved in antioxidative defense and in regulation of mitochondria function, but was not able to ameliorate cells viability affected by Aβ.

MAP Kinase Phosphatase 3 (MKP3) Preserves Norepinephrine Transporter Activity by Modulating ERK1/2 Kinase-Mediated Gene Expression.

The norepinephrine transporter (NET) mediates the clearance of norepinephrine (NE) from the extracellular space and is a target of therapeutic antidepressants and psychostimulants. Previously we identified a MAP kinase phosphatase 3 (MKP3), as an important modulator of protein kinase C (PKC) mediated internalization of the related dopamine transporter (DAT). Here we show that MKP3 decreases PKC-mediated down regulation of NET expressed in PC12 cells. We demonstrate that this process involves a PKC-stimulated decrease of NET surface expression that is dependent on dynamin. Surprisingly, MAP kinase inhibitors have no effect on the PKC-mediated regulation of NET activity, suggesting that, like PKC-mediated regulation of the DAT, the acute activation of MAP kinases is not likely to be involved. To elucidate potential mechanisms we used a substrate trap-based assay to identify extracellular-signal-regulated kinase (ERK)1/2 as the predominant substrate of MKP3. Furthermore we also established that brief chemical stabilization of a modified destabilized MKP3 does not alter PKC-mediated down regulation of NET. Finally, the expression of a dominant negative version of H-Ras, an upstream activator of ERK1/2, abolishes phorbol 12-myristate 13-acetate (PMA)-mediated down regulation of NET in a manner similar to MKP3. Taken together we propose that chronic MKP3 expression regulates surface NET through the sustained inhibition of ERK1/2 MAP kinase signaling that alters gene expression in PC12 cells. This is supported by gene expression data from naïve and MKP3-expressing PC12 cells that reveal robust decreases in gene expression of several genes in the MKP3-tranfected cells. Interestingly, caveolin-1, a protein with a critical role in membrane protein trafficking is down regulated by MKP3 expression. We further show that selective silencing of the caveolin-1 gene in naïve PC12 cells attenuates PKC-mediated downregulation of NET activity, consistent with a potential role for caveolins in regulating NET surface expression. In summary, these results suggest that chronic MKP3 expression alters the expression of genes in PC12 cells that are involved in the regulation of NET surface expression.

Characterization of the Rat GAL2R Promoter: Positive Role of ETS-1 in Regulation of the Rat GAL2R Gene in PC12 Cells.

Galanin receptor 2 (GAL2R) is a G protein-coupled receptor for the neuropeptide galanin that regulates many important physiological functions and pathological processes. To investigate the molecular mechanism governing GAL2R gene transcription, the rat GAL2R promoter was isolated and analyzed. We found that the region from -320 to -300 of the GAL2R promoter contains two putative ETS-1 elements and plays an important role in regulating GAL2R promoter activity. We also showed that transcription factor ETS-1 bound to this region in vitro and in vivo. Overexpression of ETS-1 significantly increased GAL2R promoter activity and transcription of the GAL2R gene, whereas knockdown of ETS-1 produced the opposite effects. In addition, we showed that ETS-1 recruited co-activator p300 to the GAL2R promoter. These data indicate a role for ETS-1 in the control of the GAL2R gene expression and provide a basis for understanding the transcriptional regulation of the GAL2R gene.

Apigenin mediated protection of OGD-evoked neuron-like injury in differentiated PC12 cells.

Ischemic stroke has been confirmed to cause neuronal injury due to its insufficient supply of glucose and oxygen to brain tissue. Previous research has shown that oxidative stress, a result of excessive accumulation of reactive oxygen species (ROS), relates to pathophysiology of ischemic stroke, and causes oxidative damage to biomolecules, eventually leading to programmed cell death. Meanwhile, apigenin has been shown to exhibit antioxidant, anti-inflammatory, anti-cancer properties and neuroprotective action. Hence, this study was to investigate the potential mechanisms underlying the neural protection of apigenin on oxygen and glucose deprivation/reperfusion (OGD/R) induced neuronal injury in differentiated PC12 cells. Cells were pretreated with apigenin for 6 h, and then subjected to OGD for 12 h followed by reperfusion for 24 h. The results showed that OGD/R significantly decreased cell viability, mitochondrial membrane potential, mRNA levels of antioxidant and detoxifying enzymes and Nrf2 protein expression, while elevated the release of LDH, cell apoptosis, intracellular ROS level, P53 protein expression and upregulated its downstream genes in PC12 cells. However, apigenin effectively inhibited these undesirable changes induced by OGD/R. Our findings demonstrate that this compound attenuates OGD/R induced neuronal injury mainly by virtue of its anti-apoptosis and antioxidative properties via affecting the expression of Nrf2 and P53, and their downstream target gene transcription.

Phosphodiesterase 2 negatively regulates adenosine-induced transcription of the tyrosine hydroxylase gene in PC12 rat pheochromocytoma cells

Adenosine induces expression of the tyrosine hydroxylase (TH) gene in PC12 cells. However, it is suggested that atrial natriuretic peptide (ANP) inhibits expression of this gene. Using real-time PCR and luciferase reporter assays we found that ANP significantly decreases the adenosine-induced transcription of the TH gene. Results of measurements of cyclic nucleotide concentrations indicated that ANP-induced accumulation of cGMP inhibits the adenosine-induced increase in cAMP level. Using selective phosphodiesterase 2 (PDE2) inhibitors and a synthetic cGMP analog activating PDE2, we found that PDE2 is involved in coupling the ANP-triggered signal to the cAMP metabolism. We have established that ANP-induced elevated levels of cGMP as well as cGMP analog stimulate hydrolytic activity of PDE2, leading to inhibition of adenosine-induced transcription of the TH gene. We conclude that ANP mediates negative regulation of TH gene expression via stimulation of PDE2-dependent cAMP breakdown in PC12 cells.

Baicalin protects PC-12 cells from oxidative stress induced by hydrogen peroxide via anti-apoptotic effects

Primary objective: To examine the neuroprotection of baicalin, a flavonoid compound derived from the dried root of Scutellaria baicalensis Georgi, on neurons.Research design: A rat PC12 cell line was used to study the neuroprotection and possible mechanisms of baicalin on H2O2-induced neuron damage.Methods: Three anti- and one pro-apoptosis genes in PC12 cells were examined. Cell apoptosis was induced by H2O2 and apoptotic rate was obtained by flow cytometry. MTT for cell viability, immunofluorescence microscopy for promoter activity and western blot for gene expression were also employed.Results: Data of MTT reduction assay and flow cytometry revealed that viability loss and apoptotic rate were reduced by pre-treatment of PC12 cells with baicalin for 24 hours. Baicalin was also found to increase SOD, GSH-Px activities and to decrease MDA level. Results from Western blot and immunofluorescence microscopy showed baicalin increased the expressions of survivin, Bcl-2 and p-STAT3 and decreased caspase-3 expression which were attenuated by AG-490.Conclusions: The results point to the possibility of the neuroprotective effects of baicalin on neuronal apoptosis induced by oxidative stress and indicate that activation of the JAK/STAT signalling pathway might involve the anti-apoptotic effect of baicalin.

Divalent Cation Transport by Vesicular Nucleotide Transporter

The vesicular nucleotide transporter (VNUT) is a secretory vesicle protein that is responsible for the vesicular storage and subsequent exocytosis of ATP (Sawada, K., Echigo, N., Juge, N., Miyaji, T., Otsuka, M., Omote, H., and Moriyama, Y. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 5683-5686). Because VNUT actively transports ATP in a membrane potential (Δψ)-dependent manner irrespective of divalent cations such as Mg(2+) and Ca(2+), VNUT recognizes free ATP as a transport substrate. However, whether or not VNUT transports chelating complexes with divalent cations remains unknown. Here, we show that proteoliposomes containing purified VNUT actively took up Mg(2+) when ATP was present, as detected by atomic absorption spectroscopy. The VNUT-containing proteoliposomes also took up radioactive Ca(2+) upon imposing Δψ (positive-inside) but not ΔpH. The Δψ-driven Ca(2+) uptake required ATP and a millimolar concentration of Cl(-), which was inhibited by Evans blue, a specific inhibitor of SLC17-type transporters. VNUT in which Arg-119 was specifically mutated to alanine, the counterpart of the essential amino acid residue of the SLC17 family, lost the ability to take up both ATP and Ca(2+). Ca(2+) uptake was also inhibited in the presence of various divalent cations such as Mg(2+). Kinetic analysis indicated that Ca(2+) or Mg(2+) did not affect the apparent affinity for ATP. RNAi of the VNUT gene in PC12 cells decreased the vesicular Mg(2+) concentration to 67.7%. These results indicate that VNUT transports both nucleotides and divalent cations probably as chelating complexes and suggest that VNUT functions as a divalent cation importer in secretory vesicles under physiological conditions.

Role of reactive oxygen species in the neural and hormonal regulation of the PNMT gene in PC12 cells

In adrenergic cells norepinephrine is N‐methylated by the enzyme phenylethanolamine N‐methyltransferase (PNMT) to produce epinephrine. Prior studies have demonstrated that the PNMT gene in adrenal chromaffin cells may be regulated hormonally via the hypothalamic‐pituitary‐adrenal axis and neurally via stimulation of the splanchnic nerve. Further, hypoxia has been shown to play a key role in the regulation of PNMT. The purpose of this study was to examine the impact of reactive oxygen species (ROS) produced by the hypoxia mimetic agent CoCl2 on the hormonal and neural stimulation of PNMT. RT‐PCR analyses show inductions of the PNMT intron‐retaining and intronless mRNA splice variants by CoCl2 (3.0‐ and 1.76‐fold respectively). Transient transfection assays of cells treated simultaneously with CoCl2 and dexamethasone show increased promoter activity (18.5‐fold), while mRNA levels of both splice variants do not demonstrate synergistic effects. Similar results were observed when investigating the effects of CoCl2‐induced ROS on the neural stimulation of PNMT. Our findings demonstrate that CoCl2‐induced ROS have synergistic effects on the hormonal and neural activation of the PNMT promoter and suggest post‐transcriptional regulation of PNMT following hormonal or neural activation during hypoxia. This study was funded by the Natural Sciences and Engineering Research Council of Canada.

Role of Reactive Oxygen Species in the Neural and Hormonal Regulation of the PNMT Gene in PC12 Cells

The stress hormone, epinephrine, is produced predominantly by adrenal chromaffin cells and its biosynthesis is regulated by the enzyme phenylethanolamine N-methyltransferase (PNMT). Studies have demonstrated that PNMT may be regulated hormonally via the hypothalamic-pituitary-adrenal axis and neurally via the stimulation of the splanchnic nerve. Additionally, hypoxia has been shown to play a key role in the regulation of PNMT. The purpose of this study was to examine the impact of reactive oxygen species (ROS) produced by the hypoxia mimetic agent CoCl2, on the hormonal and neural stimulation of PNMT in an in vitro cell culture model, utilizing the rat pheochromocytoma (PC12) cell line. RT-PCR analyses show inductions of the PNMT intron-retaining and intronless mRNA splice variants by CoCl2 (3.0- and 1.76-fold, respectively). Transient transfection assays of cells treated simultaneously with CoCl2 and the synthetic glucocorticoid, dexamethasone, show increased promoter activity (18.5-fold), while mRNA levels of both splice variants do not demonstrate synergistic effects. Similar results were observed when investigating the effects of CoCl2-induced ROS on the neural stimulation of PNMT via forskolin. Our findings demonstrate that CoCl2-induced ROS have synergistic effects on hormonal and neural activation of the PNMT promoter.

Enhancement of tyrosine hydroxylase expression by Cordyceps militaris

Abstract Cordyceps militaris is a popular medicinal mushroom, and has received extensive attention for medical application because of its various physiological activities. However, there is limited information about the function of Cordyceps militaris on dopaminergic system. This study has attempted to evaluate the effect of cultured fruiting bodies of Cordyceps militaris extract (CME) on the expression of the tyrosine hydroxylase (TH) gene in PC12 cells and rat brain and stomach. Related mRNA levels were determined by the RT-PCR. Protein levels were measured by Western blot and immunohistochemistry. Our results demonstrated CME induced TH gene expression both in vitro and in vivo. Treatment of 10 µg/ml and 20 mg/kg CME to PC12 cells and rat cells yielded significant increases of TH protein levels. Significantly, TH immunoreactive neurons were detected not only in the brain but also in the stomach. TH-immunohistochemical staining was markedly enhanced in animals treated with CME compared to those in the untreated control. These results suggest that CME can upregulate the dopaminergic (DArgic) system, and may contribute to neuroprotection in neurodegenerative diseases.

Expression changes of dopaminergic system-related genes in PC12 cells induced by manganese, silver, or copper nanoparticles

Nanoparticles have received a great deal of attention for producing new engineering applications due to their novel physicochemical characteristics. However, the broad application of nanomaterials has also produced concern for nanoparticle toxicity due to increased exposure from large-scale industry production. This study was conducted to investigate the potential neurotoxicity of manganese (Mn), silver (Ag), and copper (Cu) nanoparticles using the dopaminergic neuronal cell line, PC12. Selective genes associated with the dopaminergic system were investigated for expression changes and their correlation with dopamine depletion. PC12 cells were treated with 10 μg/ml Mn-40 nm, Ag-15 nm, or Cu-90 nm nanoparticles for 24 h. Cu-90 nanoparticles induced dopamine depletion in PC12 cells, which is similar to the effect induced by Mn-40 shown in a previous study. The expression of 11 genes associated with the dopaminergic system was examined using real-time RT-PCR. The expression of Txnrd1 was up-regulated after the Cu-90 treatment and the expression of Gpx1 was down-regulated after Ag-15 or Cu-90 treatment. These alterations are consistent with the oxidative stress induced by metal nanoparticles. Mn-40 induced a down-regulation of the expression of Th; Cu-90 induced an up-regulation of the expression of Maoa. This indicates that besides the oxidation mechanism, enzymatic alterations may also play important roles in the induced dopamine depletion. Mn-40 also induced a down-regulation of the expression of Park2; while the expression of Snca was up-regulated after Mn-40 or Cu-90 treatment. These data suggest that Mn and Cu nanoparticles-induced dopaminergic neurotoxicity may share some common mechanisms associated with neurodegeneration.

Identification of neuronal target genes for CCAAT/Enhancer Binding Proteins

CCAAT/Enhancer Binding Proteins (C/EBPs) play pivotal roles in the development and plasticity of the nervous system. Identification of the physiological targets of C/EBPs (C/EBP target genes) should therefore provide insight into the underlying biology of these processes. We used unbiased genome-wide mapping to identify 115 C/EBPβ target genes in PC12 cells that include transcription factors, neurotransmitter receptors, ion channels, protein kinases and synaptic vesicle proteins. C/EBPβ binding sites were located primarily within introns, suggesting novel regulatory functions, and were associated with binding sites for other developmentally important transcription factors. Experiments using dominant negatives showed C/EBPβ to repress transcription of a subset of target genes. Target genes in rat brain were subsequently found to preferentially bind C/EBPα, β and δ. Analysis of the hippocampal transcriptome of C/EBPβ knockout mice revealed dysregulation of a high percentage of transcripts identified as C/EBP target genes. These results support the hypothesis that C/EBPs play non-redundant roles in the brain.

The MAPK pathway is required for depolarization‐induced “promiscuous” immediate‐early gene expression but not for depolarization‐restricted immediate‐early gene expression in neurons

Depolarization, growth factors, neurotrophins, and other stimuli induce expression of immediate early genes (IEGs) in neurons. We identified a subset of IEGs, IPD-IEGs, which are induced preferentially by depolarization, but not by neurotrophins or growth factors, in PC12 cells. The "promiscuous" IEGs Egr1 and c-fos, induced by growth factors and neurotrophins, in addition to depolarization, require activation of the MAP kinase signaling pathway for induction in response to KCl depolarization in PC12 cells; MEK1/2 inhibitors block KCl-induced Egr1 and c-fos expression. In contrast, MEK1/2 inhibition has no effect on KCl-induced expression of the known IPD-IEGs in PC12 cells. Additional "candidate" IDP-IEGs were identified by a microarray comparison of genes induced by KCl in the presence vs. the absence of an MEK1/2 inhibitor in PC12 cells. Northern blot analyses demonstrated that representative newly identified candidate IPD-IEGs, as with the known IPD-IEGs, are also induced by a MAP kinase- independent pathway in response to depolarization, both in PC12 cells and in rat primary cortical neurons. Nerve growth factor and epidermal growth factor are unable to induce the expression of the Crem/Icer, Nur77, Nor1, Rgs2, Dusp1 (Mkp1), and Dscr1 genes in PC12 cells, validating their identification as IPD-IEGs. Inhibiting calcium/calmodulin-dependent kinase II (CaMKII), calcineurin, or protein kinase A (PKA) activity prevents KCl-induced IPD-IEG mRNA accumulation, suggesting that the IPD-IEG genes are induced by depolarization in neurons via a combination of calcineurin/PKA- and CaMKII-dependent pathways.