Cultured PC12 Cells Research Articles

Leonurine promotes neurite outgrowth and neurotrophic activity by modulating the GR/SGK1 signaling pathway in cultured PC12 cells.

Depression is a common psychiatric disorder that affects almost 10% of children and adolescents worldwide. Numerous synthetic chemical antidepressants used to treat depression have adverse side effects. Therefore, new therapeutic approaches for depression treatment are urgently needed. Leonurus cardiaca has recently been shown to be effective for the treatment of nervous system diseases such as depression, but its mechanism is not clear. In this study, we aimed to reveal the mechanism underlying leonurine’s antidepressant activity. Leonurine was used to treat corticosterone-induced PC12 cells to examine its effect on neurite outgrowth and neurotrophic factors after treatment with the inhibitor of glucocorticoid receptor (GR) and serum-inducible and glucocorticoid-inducible kinase 1 (SGK1). Methyl thiazolyl tetrazolium assays were used to evaluate the viability of cells. High content analysis was used to detect cell area, total neurite length, maximum neurite length, and expression of GR, SGK1, brain-derived neurotrophic factor (BDNF), neurotrophic factor-3 (NT-3), and B-cell lymphoma-2 (BCL-2). The results showed that leonurine increased cell viability in a concentration-dependent manner, with the maximal prosurvival effect at 60 μM. Leonurine increased cell area, total neurite length, and maximum neurite length of corticosterone-induced PC12 cells, increased the expression of GR, BDNF, NT-3, and BCL-2, and decreased the expression of SGK1. After treatment with GR inhibitor RU486, the expressions of GR, BDNF, NT-3, and BCL-2 were significantly decreased and SGK1 was increased. In contrast, treatment with GSK650394 had the opposite effect of RU486. Our data indicate that leonurine promotes neurite outgrowth and neurotrophic activity in cultured PC12 cells, and its potential mechanism may involve the GR/SGK1 signaling pathway.

Neuroprotective effect of Apelin 13 on ischemic stroke by activating AMPK/GSK-3\u03b2/Nrf2 signaling

BackgroundPrevious studies had showed that Apelin 13 could protect against apoptosis induced by ischemic/reperfusion (I/R). However, the mechanisms whereby Apelin 13 protected brain I/R remained to be elucidated. The present study was designed to determine whether Apelin 13 provided protection through AMPK/GSK-3β/Nrf2 pathway.MethodsIn vivo, the I/R model was induced and Apelin 13 was given intracerebroventricularly 15 min before reperfusion. The neurobehavioral scores, infarction volumes, and some cytokines in the brain were measured. For in vitro study, PC12 cells were used. To clarify the mechanisms, proteases inhibitors or siRNA were used. Protein levels were investigated by western blotting.ResultsThe results showed that Apelin 13 treatment significantly reduced infarct size, improved neurological outcomes, decreased brain edema, and inhibited cell apoptosis, oxidative stress, and neuroinflammation after I/R. Apelin 13 significantly increased the expression of Nrf2 and the phosphorylation levels of AMPK and GSK-3β. Furthermore, in cultured PC12 cells, the same protective effects were also observed. Silencing Nrf2 gene with its siRNA abolished the Apelin 13’s prevention of I/R-induced PC12 cell injury, oxidative stress, and inflammation. Inhibition of AMPK by its siRNA decreased the level of Apelin 13-induced Nrf2 expression and diminished the protective effects of Apelin 13. The interplay relationship between GSK-3β and Nrf2 was also verified with relative overexpression. Using selective inhibitors, we further identified the upstream of AMPK/GSK-3β/Nrf2 is AR/Gα/PLC/IP3/CaMKK.ConclusionsIn conclusion, the previous results showed that Apelin 13 protected against I/R-induced ROS-mediated inflammation and oxidative stress through activating the AMPK/GSK-3β pathway by AR/Gα/PLC/IP3/CaMKK signaling, and further upregulated the expression of Nrf2-regulated antioxidant enzymes.

Integrating 3D Cell Culture of PC12 Cells with Microchip-Based Electrochemical Detection.

Developing in vitro cell culture models that accurately mimic in vivo processes in a manner that also enables near real-time analysis of neurotransmitters is an important research area. New technologies being developed such as 3D scaffolds for cell culture and 3D printed microfluidics provide an opportunity for such advancements. In this work, PC12 cells were used as a model system and they were immobilized onto a 3D scaffold of polystyrene (PS) fibers. These fibers were created by electrospinning onto PS sheets, which were laser cut and, after cell seeding, inserted into a 3D printed microfluidic device. The 3D printed device was designed with threads for connecting commercial fittings (to integrate automated pumps and a 4-port injection system) and a steel pin for simple coupling with PDMS/polystyrene analytical devices. A straight PDMS channel was used for simple (and continuous) flow-based detection by sealing onto a PS base containing an embedded gold array working electrode and a platinum pseudo-reference. Electrochemical detection of stimulated catecholamine release was demonstrated. The insert-based system was then integrated with a bilayer valving PDMS device (for microchip electrophoresis) sealed onto a PS base (with electrodes for electrochemical detection). This base was embedded with a Pd decoupler (for grounding the separation voltage and adsorbing hydrogen) and a 33 µm carbon fiber working electrode for in-channel detection. PC12 cells were stimulated in the 3D cell culture device, and the valving/electrophoresis microchip was able to separate and detect dopamine and norepinephrine release. This work demonstrates the ability to integrate 3D cell scaffolds with microchip-based analysis for detection of multiple analytes released from cells.

Protective effect of Schisandra chinensis lignans on hypoxia-induced PC12 cells and signal transduction

ABSTRACTIt is well-known that hypoxia induces neuronal injury; however, the mechanisms underlying this observed effect remain to be determined. Schisandra chinensis lignans (SCL). The aim of this study was thus to examine the ability of Schisandra chinensis lignans (SCL) to prevent hypoxia-induced neuronal injury using a human adrenal pheochromocytoma cell line (PC12). Exposure to hypoxia significantly reduced cell survival rate in cultured PC12 cells. However, pretreatment with SCL at 10, 20 or 40 μmol/L followed by hypoxia prevented loss of cellular viability. Flow cytometry demonstrated that the apoptotic rate in PC12 cells following hypoxia was significantly increased. Pretreatment with SCL 20 or 40 μmol/L in hypoxia-exposed cells resulted in significantly reduced apoptotic rates compared to hypoxia. Immunocytochemical staining showed that protein expression of p-Akt was significantly diminished by hypoxia. Following pre-treatment with different concentrations of SCL, PC12 cells were markedly stimulated as evidenced by elevated protein expression of p-Akt in a concentration-dependent manner. The expression of p-Akt protein in the presence of PI3K/Akt signaling pathway inhibitor LY294002 and SCL was not markedly changed indicating that signal transduction was affected by this Chinese herb. There were no significant differences in total Akt protein expression following hypoxia or pretreatment with SCL. Western blot demonstrated that expression levels of caspase-3 protein were significantly increased while expression levels of Bcl-2 protein were decreased in hypoxic cells. Pretreatment with SCL followed by hypoxia significantly lowered expression levels of caspase-3 protein accompanied by elevated expression levels of Bcl-2 protein in a concentration-dependent manner. After co-incubation with LY29004 and SCL, down-regulation of expression of caspase-3 protein and up-regulation of the expression of Bcl-2 protein noted with SCL alone were suppressed. Data suggest that the protective effect exerted by SCL in hypoxia-induced PC12 cell injury involves enhanced cell proliferation and inhibition of apoptosis mediated by activation of PI3K/Akt signaling pathway. The increased protein Akt phosphorylation expression levels resulted in consequent reduced downstream caspase-3 expression and enhanced Bcl-2 expression.

Neuroprotective role of hyperforin on aluminum maltolate-induced oxidative damage and apoptosis in PC12 cells and SH-SY5Y cells

Many reports demonstrated that aluminum maltolate (Almal) has potential toxicity to human and animal. Our study has demonstrated that Almal can induce oxidative damage and apoptosis in PC12 cells and SH-SY5Y Cells, two in vitro models of neuronal cells. Hyperforin (HF) is a well-known antioxidant, anti-inflammatory, anti-amyloid and anti-depressant compound extracted from Hypericum perforatum extract. Here, we investigated the neuroprotective effect of HF against Almal-induced neurotoxicity in cultured PC12 cells and SH-SY5Y cells, mainly caused by oxidative stress. In the present study, HF significantly inhibited the formation of reactive oxygen species (ROS), decreased the level of lipid peroxide and enhanced the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) compared with Almal group in PC12 cells and SH-SY5Y cells. Additionally, HF suppressed the reduction of the mitochondrial membrane potential (MMP), cytochrome c (Cyt-c) release, activation of caspase-3, and the down-regulation of Bcl-2 expression and up-regulation of Bax expression induced by Almal in PC12 cells and SH-SY5Y cells. In summary, HF protects PC12 cells and SH-SY5Y cells from damage induced by Almal through reducing oxidative stress and preventing of mitochondrial-mediated apoptosis.

In vitro and in vivo studies of electroactive reduced graphene oxide-modified nanofiber scaffolds for peripheral nerve regeneration

Graphene, as a promising biomaterial, has received great attention in biomedical fields due to its intriguing properties, especially the conductivity and biocompatibility. Given limited studies on the effects of graphene-based scaffolds on peripheral nerve regeneration in vitro and in vivo under electrical stimulation (ES), the present study was intended to systematically investigate how conductive graphene-based nanofibrous scaffolds regulate Schwann cell (SC) behavior including migration, proliferation and myelination, and PC12 cell differentiation in vitro via ES, and whether these conductive scaffolds could guide SC migration and promote nerve regeneration in vivo. Briefly, the reduced graphene oxide (RGO) was coated onto ApF/PLCL nanofibrous scaffolds via in situ redox reaction of the graphene oxide (GO). In vitro, RGO-coated ApF/PLCL (AP/RGO) scaffolds significantly enhanced SC migration, proliferation, and myelination including myelin-specific gene expression and neurotrophic factor secretion. The conditioned media of SCs cultured on AP/RGO scaffolds under ES could induce the differentiation of PC12 cells in a separate culture. In addition, PC12 cells cultured on the conductive AP/RGO scaffolds also showed elevated differentiation upon ES. In vivo implantation of the conductive AP/RGO nerve guidance conduits into rat sciatic nerve defects exhibited a similar healing capacity to autograft, which is the current gold standard in peripheral nerve regeneration. In view of the performance of AP/RGO scaffolds in modulating cell functions in vitro and promoting nerve regeneration in vivo, it is expected that the graphene-based conductive nanofibrous scaffolds would exhibit their potential in peripheral nerve repair and regeneration. Statement of significanceDespite the demonstrated capability of bridging the distal and proximal peripheral nerves, it remains a significant challenge with current artificial nerve conduits to achieve the desired physiological functions, e.g., the transmission of electrical stimuli. Herein, we explored the possibility of combining the conductive properties of graphene with electrospun nanofiber to create the electroactive biomimetic scaffolds for nerve tissue regeneration. In vitro and in vivo studies were carried out: (1) In vitro, the conductive nanofibrous scaffolds significantly promoted SC migration, proliferation and myelination including myelin specific gene expression and neurotrophicfactor secretion, and induced PC12 cell differentiation with electrical stimulation. (2) In vivo, the conductive nerve guidance conduit exhibited similar effects with the gold standard autograft. In view of the performance of this conductive scaffold in modulating the cell functions in vitro and promoting nerve regeneration in vivo, it is expected that the graphene-modified nanofibrous scaffolds will exhibit their potential in peripheral nerve repair and regeneration.

Different Fgfs have distinct roles in regulating neurogenesis after spinal cord injury in zebrafish

BackgroundDespite conserved developmental processes and organization of the vertebrate central nervous system, only some vertebrates including zebrafish can efficiently regenerate neural damage including after spinal cord injury. The mammalian spinal cord shows very limited regeneration and neurogenesis, resulting in permanent life-long functional impairment. Therefore, there is an urgent need to identify the cellular and molecular mechanisms that can drive efficient vertebrate neurogenesis following injury. A key pathway implicated in zebrafish neurogenesis is fibroblast growth factor signaling.MethodsIn the present study we investigated the roles of distinct fibroblast growth factor members and their receptors in facilitating different aspects of neural development and regeneration at different timepoints following spinal cord injury. After spinal cord injury in adults and during larval development, loss and/or gain of Fgf signaling was combined with immunohistochemistry, in situ hybridization and transgenes marking motor neuron populations in in vivo zebrafish and in vitro mammalian PC12 cell culture models.ResultsFgf3 drives neurogenesis of Islet1 expressing motor neuron subtypes and mediate axonogenesis in cMet expressing motor neuron subtypes. We also demonstrate that the role of Fgf members are not necessarily simple recapitulating development. During development Fgf2, Fgf3 and Fgf8 mediate neurogenesis of Islet1 expressing neurons and neuronal sprouting of both, Islet1 and cMet expressing motor neurons. Strikingly in mammalian PC12 cells, all three Fgfs increased cell proliferation, however, only Fgf2 and to some extent Fgf8, but not Fgf3 facilitated neurite outgrowth.ConclusionsThis study demonstrates differential Fgf member roles during neural development and adult regeneration, including in driving neural proliferation and neurite outgrowth of distinct spinal cord neuron populations, suggesting that factors including Fgf type, age of the organism, timing of expression, requirements for different neuronal populations could be tailored to best drive all of the required regenerative processes.

Fe3+ Facilitates Endocytic Internalization of Extracellular Aβ1-42 and Enhances Aβ1-42-Induced Caspase-3/Caspase-4 Activation and Neuronal Cell Death.

Amyloid β (Aβ) peptide is a critical causative factor in Alzheimer's disease (AD) and of a variety of fragmented Aβ peptides Aβ1-42 thought to exhibit the most neurotoxic effect. The present study investigated the effects of Fe3+ on Aβ1-42 internalization and Aβ1-42-induced caspase activation and neurotoxicity using mouse hippocampal slices and cultured PC-12 cells. Extracellularly applied Aβ1-42 increased the cell-associated Aβ1-42 levels in a concentration-dependent manner, and the effect was enhanced by adding Fe3+. Fe3+-induced enhancement of the cell-associated Aβ1-42 levels was significantly inhibited by the endocytosis inhibitors dynasore and methyl-β-cyclodextrin. Aβ1-42 reduced PC-12 cell viability in a concentration-dependent manner, and further reduction of the cell viability was obtained with Fe3+. Aβ1-42-induced reduction of cell viability was not affected by A187, an antagonist of amylin-3 receptor. Aβ1-42 activated caspase-3, caspase-4, and caspase-8 to a variety of degrees and Fe3+ further enhanced Aβ1-42-induced activation of caspase-3 and caspase-4. Taken together, these results indicate that Fe3+ accelerates endocytic internalization of extracellular Aβ1-42, enhances Aβ1-42-induced caspase-3/caspase-4 activation, and promotes Aβ1-42-induced neuronal cell death, regardless of amylin receptor.

Expression of the Neuron-Related Genes in hNGF-β-Treated PC12 Cells

Human nerve growth factor-β (hNGF-β) plays a major role in the development and survival of the central and peripheral nervous systems; therefore, it can, in principle, be used as a therapeutic agent for the treatment of neurodegenerative diseases. As was shown, hNGF-β stimulates neuronal differentiation in vitro and regulates the expression of neuron-related genes. In our study, expression of the ATP1B1, Clusterin, MAO-A, and DUSP6 genes was evaluated in hNGF-treated PC12 cells. At first, hNGF-β protein was generated in a pET39b/BL21 (DE3) bacterial expression system, and biological activity of this protein was confirmed by differentiation of cultured PC12 cells. These cells were treated by the addition of recombinant hNGF-β protein in a final dose of 50 ng/ml diluted in RPMI 1640. Then total RNA was extracted from harvested PC12 cells at day 7 after treatment and used to analyze the expression levels of the above-mentioned genes by real-time PCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference gene. It was found that purified hNGF-β protein migrated as a clear band of ~37 kDa in SDS-PAGE. Moreover, differentiation of PC12 cells was observed 7 days after hNGF-β treatment. Expression of the ATP1B1 and DUSP6 genes was upregulated (230 and 193%), respectively after addition of hNGF-β protein. In contrast, the MAO-A and Clusterin genes showed practically no change in their expression, as compared to untreated cells. Generally, our study demonstrated that the treatment of PC12 cells with hNGF-β protein induces neural morphological modifications and differences in the expression of certain neuron-related genes. Changes in the expression of these genes will be applied in an experimental model.

MiR-429 suppresses neurotrophin-3 to alleviate perineural invasion of pancreatic cancer

Perineural invasion (PNI) potentially increases the risk of relapse and abdominal pain in patients with pancreatic ductal adenocarcinoma (PDAC). However, the underlying mechanisms of PNI of PDAC is incompletely revealed. Our study aimed to investigate roles of miR-429 in modulating PNI in PDAC. We found that miR-429 was downregulated in PDAC cancer tissues and was profoundly decreased in tissues with PNI. It was reduced in nine of the ten examined pancreatic cancer cell lines. MiR-429 mimics restored its cellular expressions in MIA PaCa-2 and BxCP3 cells and significantly suppressed cell viability and invasion of the cancer cells. The online bioinformatic software predicted that neurotrophin-3 (NT-3) was a potential target gene of miR-429. It was showed that NT-3 mRNA elevated in PC cancer tissues, especially in patients presenting PNI. MiR-429 upregulation substantially suppressed the NT-3 mRNA and secretion in cancer cells. Also, the dual luciferase reporter assays confirmed the interaction between miR-429 and NT-3. When co-culturing the two PDAC cells with PC-12 cells, the invaded cell counts significantly increased comparing with the sole culture of cancer cells. However, miR-429 mimic transfection or NT-3 blocking retarded the cancer invasion in the co-culture system. Besides, we found that cancer cells conditioned medium (CM) treatment significantly increased the neurite outgrowth percentage in PC-12 cells, which was suppressed by culturing with CM from miR-429 mimics-transfected cells. In the CM cultured PC-12 cells, NT-3 receptor TrkC as well as pain-related proteins TRPV1 and TRPV2 significantly elevated. Collectively, miR-429 potentially suppressed neurotrophin-3 to alleviate PNI of PDAC.

Diazoxide induces endoplasmic reticulum stress-related neuroprotection mediated by p38 MAPK against Aβ25-35 insults.

The endoplasmic reticulum (ER) -resident caspase-12 was identified as a mediator of Aβ neurotoxicity. Recent evidence indicates that mitochondrial ATP-sensitive potassium (KATP) channel openers mediate their neuroprotective role by adjusting ER stress pathways, but the molecular details remain largely unknown and have been investigated. In this study, the protein expression levels of calreticulin (CRT) and caspase-12 activation and phosphorylated p38 MAPK were observed by immunoblotting in cultured PC12 cells from different groups: treatment with Aβ25-35 (group Aβ25-35), treatment with diazoxide (group diazoxide), pretreatment with diazoxide and then exposure to Aβ25-35 (group diazoxide + Aβ25-35), pretreatment with p38 MAPK inhibitor SB 203580 and then exposure to diazoxide and Aβ25-35 (group SB 203580 + diazoxide + Aβ25-35), and the control (group control). In response to the treatment with Aβ25-35 (10 µM) for 24 h, the protein expression levels of CRT and caspase-12 activation were increased and phosphorylated p38 MAPK was decreased significantly. Diazoxide reduced CRT overexpression and caspase-12 activation and increased the up-regulation of phosphorylated p38 MAPK. When SB 203580 was presented before exposure to diazoxide and Aβ25-35, CRT expression was markedly suppressed, and the inhibition effect of diazoxide on caspase-12 activation was almost eliminated. We showed that diazoxide induced ERS-related neuroprotection mediated by p38 MAPK against Aβ25-35 insults. From the clinical point of view, these results are of considerable importance for the understanding of AD pathogenesis. However, further studies are required to explore more detailed mechanisms of the observed effects.

Spinal protein phosphatase 1 constrains respiratory plasticity after sustained hypoxia

Plasticity is an important aspect of the neural control of breathing. One well-studied form of respiratory plasticity is phrenic long-term facilitation (pLTF) induced by acute intermittent but not sustained hypoxia. Okadaic acid-sensitive protein phosphatases (PPs) differentially regulate phrenic nerve activity with intermittent vs. sustained hypoxia, at least partially accounting for pLTF pattern sensitivity. However, okadaic acid inhibits multiple serine/threonine phosphatases, and the relevant phosphatase (PP1, PP2A, PP5) for pLTF pattern sensitivity has not been identified. Here, we demonstrate that sustained hypoxia (25 min, 9-10.5% O2) elicits phrenic motor facilitation in rats pretreated with bilateral intrapleural injections of small interfering RNAs (siRNAs; Accell-modified to preferentially transfect neurons, 3.33 μM, 3 days) targeting PP1 mRNA (48 ± 14% change from baseline, n = 6) but not PP2A (14 ± 9% baseline, n = 6) or nontargeting siRNAs (4 ± 10% baseline, n = 7). In time control rats (no hypoxia) treated with siRNAs ( n = 6), no facilitation was evident (-9 ± 9% baseline). siRNAs had no effect on the hypoxic phrenic response. Immunohistochemistry revealed PP1 and PP2A protein in identified phrenic motoneurons. Although PP1 and PP2A siRNAs significantly decreased PP1 and PP2A mRNA in PC12 cell cultures, we were not able to verify "knockdown" in vivo after siRNA treatment. On the other hand, PP1 and PP2A siRNAs significantly decreased PP1 and PP2A mRNA in PC12 cell cultures, verifying the intended siRNA effects. In conclusion, PP1 (not PP2A) is the relevant okadaic acid-sensitive phosphatase constraining phrenic motor facilitation after sustained hypoxia and likely contributing to pLTF pattern sensitivity. NEW & NOTEWORTHY This study demonstrates that the relevant okadaic acid-sensitive Ser/Thr protein phosphatase (PP) constraining facilitation after sustained hypoxia is PP1 and not PP2A. It suggests that PP1 may be critical in the pattern sensitivity of hypoxia-induced phrenic motor plasticity.

Dimethyl fumarate ameliorates chemotherapy agent-induced neurotoxicity in vitro

Chemotherapy agents such as oxaliplatin, cisplatin, paclitaxel, and bortezomib frequently cause severe peripheral neuropathy and there is currently no effective strategy to prevent this. Dimethyl fumarate (DMF) is a new oral drug for the treatment of multiple sclerosis, and has neuroprotective effects via up-regulation of the nuclear factor-erythroid-2-related factor 2 (Nrf2)-dependent antioxidant response. In this study, we investigated the effect of DMF on chemotherapy agent-induced neurodegenerations in cultured cells. We found that DMF and its metabolite monomethyl fumarate (MMF) attenuated oxaliplatin-, cisplatin-, and bortezomib- (but not paclitaxel-) induced inhibition of neurite outgrowth, but had no effect on cell death as a result of these agents in cultured PC12 cells and primary cultured rat dorsal root ganglion (DRG) neurons. Furthermore, Nrf2 DNA binding activity was increased by DMF and MMF in PC12 cells. These findings suggest that DMF, which activates Nrf2 pathway, has a potential protective action against chemotherapy-induced neurotoxicity, particularly neurite impairments.

Extracellular Bio-imaging of Acetylcholine-stimulated PC12 Cells Using a Calcium and Potassium Multi-ion Image Sensor.

In biochemistry, Ca2+ and K+ play essential roles to control signal transduction. Much interest has been focused on ion-imaging, which facilitates understanding of their ion flux dynamics. In this paper, we report a calcium and potassium multi-ion image sensor and its application to living cells (PC12). The multi-ion sensor had two selective plasticized poly(vinyl chloride) membranes containing ionophores. Each region on the sensor responded to only the corresponding ion. The multi-ion sensor has many advantages including not only label-free and real-time measurement but also simultaneous detection of Ca2+ and K+. Cultured PC12 cells treated with nerve growth factor were prepared, and a practical observation for the cells was conducted with the sensor. After the PC12 cells were stimulated by acetylcholine, only the extracellular Ca2+ concentration increased while there was no increase in the extracellular K+ concentration. Through the practical observation, we demonstrated that the sensor was helpful for analyzing the cell events with changing Ca2+ and/or K+ concentration.

Growing Neural PC-12 Cell on Crosslinked Silica Aerogels Increases Neurite Extension in the Presence of an Electric Field.

Externally applied electrical stimulation (ES) has been shown to enhance the nerve regeneration process and to influence the directionality of neurite outgrowth. In addition, the physical and chemical properties of the substrate used for nerve-cell regeneration is critical in fostering regeneration. Previously, we have shown that polyurea-crosslinked silica aerogels (PCSA) exert a positive influence on the extension of neurites by PC-12 cells, a cell-line model widely used to study neurite extension and electrical excitability. In this work, we have examined how an externally applied electric field (EF) influences the extension of neurites in PC-12 cells grown on two substrates: collagen-coated dishes versus collagen-coated crosslinked silica aerogels. The externally applied direct current (DC) bias was applied in vitro using a custom-designed chamber containing polydimethysiloxane (PDMS) embedded copper electrodes to create an electric field across the substrate for the cultured PC-12 cells. Results suggest orientation preference towards the anode, and, on average, longer neurites in the presence of the applied DC bias than with 0 V DC bias. In addition, neurite length was increased in cells grown on silica-crosslinked aerogel when compared to cells grown on regular petri-dishes. These results further support the notion that PCSA is a promising material for nerve regeneration.

Ferroptosis is newly characterized form of neuronal cell death in response to arsenite exposure

Ferroptosis is a novel iron-dependent form of cell death implicated in brain pathology. However, whether arsenite is an inducer of ferroptosis in the neuron remains completely unknown. In this study, the seven-week-old healthy C57BL/6 J male mice were treated with environmental related doses (0.5, 5 and 50 mg/L) of arsenite for 6 months via drinking water, and the ferroptosis-related indicators were further determined. Our results demonstrated for the first time that, arsenite exposure significantly reduced the number of neuron and caused the pathological changes of mitochondria in the cerebral cortex of mice. We further revealed that arsenite induced ferroptotic cell death in neuron by accumulation of reactive oxygen species and lipid peroxidation products, disruption of Fe2+ homeostasis, depletion of glutathione and adenosine triphosphate, inhibition of cysteine/glutamate antiporter, activation of mitogen-activated protein kinases and mitochondrial voltage-dependent anion channels pathways, up-regulation of endoplasmic reticulum stress, all of which were involved in the process of ferroptosis. These findings were also verified in the cultured PC-12 cells by using ferropotosis inhibitor, desferoxamine. Taken together, our results not only reveal a novel mechanism that chronic arsenite exposure may trigger the new form of cell death, ferroptosis, but also shed a new light on a potential clue for the intervention and prevention against arsenite-related neurodegenerative diseases.

Gualou Guizhi Granule Protects Against Oxidative Injury by Activating Nrf2/ARE Pathway in Rats and PC12 Cells.

Stroke involves numerous pathophysiological processes and oxidative stress is considered as a main cellular event in its pathogenesis. The nuclear factor erythroid-2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway plays a key role in inducing phase II detoxifying enzymes and antioxidant proteins and is now considered as a interesting therapeutic target for the treatment of stroke. The objective of this study is to investigate the protective effect of Gualou Guizhi granule (GLGZG) against oxidative stress and explore the protective mechanism of the Nrf2/ARE pathway. In vivo, administration of GLGZG in a rat model of focal cerebral ischemia significantly suppressed oxidative injury by increasing the activity of superoxide dismutase and glutathione level and decreasing reactive oxygen species and malondialdehyde levels. Western blot analysis showed that GLGZG induced nuclear translocation of Nrf2, and combined with real-time PCR results, which indicated that GLGZG up-regulated the Nrf2/ARE pathway. In addition, in cultured PC12 cells, GLGZG protected against H2O2 induced oxidative injury and activated the Nrf2/ARE pathway. All the results demonstrated that GLGZG in the management of cerebral ischemia and H2O2 induced oxidative injury may be associated with activation of Nrf2/ARE signaling pathway.

Genistein, a Phytoestrogen in Soybean, Induces the Expression of Acetylcholinesterase via G Protein-Coupled Receptor 30 in PC12 Cells.

Genistein, 4′,5,7-trihydroxyisoflavone, is a major isoflavone in soybean, which is known as phytestrogen having known benefit to brain functions. Being a common phytestrogen, the possible role of genistein in the brain protection needs to be further explored. In cultured PC12 cells, application of genistein significantly induced the expression of neurofilaments (NFs), markers for neuronal differentiation. In parallel, the expression of tetrameric form of proline-rich membrane anchor (PRiMA)-linked acetyl-cholinesterase (G4 AChE), a key enzyme to hydrolyze acetylcholine in cholinergic synapses, was induced in a dose-dependent manner: this induction included the associated protein PRiMA. The genistein-induced AChE expression was fully blocked by the pre-treatment of H89 (an inhibitor of protein kinase A, PKA) and G15 (a selective G protein-coupled receptor 30 (GPR30) antagonist), which suggested a direct involvement of a membrane-bound estrogen receptor (ER), named as GPR30 in the cultures. In parallel, the estrogen-induced activation of GPR30 induced AChE expression in a dose-dependent manner. The genistein/estrogen-induced AChE expression was triggered by a cyclic AMP responding element (CRE) located on the ACHE gene promoter. The binding of this CRE site by cAMP response element-binding protein (CREB) induced ACHE gene transcription. In parallel, increased expression levels of miR132 and miR212 were found when cultured PC12 cells were treated with genistein or G1. Thus, a balance between production and destruction of AChE by the activation of GPR30 was reported here. We have shown for the first time that the activation of GPR30 could be one way for estrogen or flavonoids, possessing estrogenic properties, to enhance cholinergic functions in the brain, which could be a good candidate for possible treatment of neurodegenerative diseases.

Suppression of HDAC2 in Spinal Cord Alleviates Mechanical Hyperalgesia and Restores KCC2 Expression in a Rat Model of Bone Cancer Pain

Epigenetic modulation participates in the mechanism of multiple types of pathological pain, so targeting the involved regulators may be a promising strategy for pain treatment. Our previous research identified the analgesic effect of the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) on mechanical hyperalgesia in a rat model of bone cancer pain (BCP) via restoration of μ-opioid receptor (MOR) expression. However, the specific types of HDACs contributing to BCP have not been explored. The present study investigated the expression pattern of some common HDACs and found that HDAC2 was up-regulated in a time-dependent manner in the lumbar spinal cord of BCP rats. TSA application suppressed HDAC2 expression in cultured PC12 cells and reversed the augmented HDAC2 in BCP rats. An RNA-interfering strategy confirmed the essential role of HDAC2 in the modulation of mechanical hyperalgesia following tumor cell inoculation, and we further examined its possible downstream targets. Notably, HDAC2 knock-down did not restore MOR expression, but it robustly reversed the down-regulation of potassium-chloride cotransporter 2 (KCC2). The impaired KCC2 expression is a vital mechanism of many types of pathological pain. Therefore, our results demonstrated that HDAC2 in spinal cord contributed to the mechanical hyperalgesia in BCP rats, and this effect may be associated with KCC2 modulation.

Triphenylphosphonium Moiety Modulates Proteolytic Stability and Potentiates Neuroprotective Activity of Antioxidant Tetrapeptides in Vitro.

Although delocalized lipophilic cations have been identified as effective cellular and mitochondrial carriers for a range of natural and synthetic drug molecules, little is known about their effects on pharmacological properties of peptides. The effect of triphenylphosphonium (TPP) cation on bioactivity of antioxidant tetrapeptides based on the model opioid YRFK motif was studied. Two tetrapeptide variants with L-arginine (YRFK) and D-arginine (YrFK) were synthesized and coupled with carboxyethyl-TPP (TPP-3) and carboxypentyl-TPP (TPP-6) units. The TPP moiety noticeably promoted YRFK cleavage by trypsin, but effectively prevented digestion of more resistant YrFK attributed, respectively, to structure-organizing and shielding effects of the TPP cation on conformational variants of the tetrapeptide motif. The TPP moiety enhanced radical scavenging activity of the modified YRFK in a model Fenton-like reaction, whereas decreased reactivity was revealed for both YrFK and its TPP derivative. The starting motifs and modified oligopeptides, especially the TPP-6 derivatives, suppressed acute oxidative stress in neuronal PC-12 cells during a brief exposure similarly with glutathione. The effect of oligopeptides was compared upon culturing of PC-12 cells with CoCl2, L-glutamic acid, or menadione to mimic physiologically relevant oxidative states. The cytoprotective activity of oligopeptides significantly depended on the type of oxidative factor, order of treatment and peptide structure. Pronounced cell-protective effect was established for the TPP-modified oligopeptides, which surpassed that of the unmodified motifs. The protease-resistant TPP-modified YrFK showed the highest activity when administered 24 h prior to the cell damage. Our results suggest that the TPP cation can be used as a modifier for small therapeutic peptides to improve their pharmacokinetic and pharmacological properties.