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Genistein (GEN) is the most abundant aglycone in soy isoflavones and is a key factor distinguishing soybeans from other food sources. It holds significant nutritional value and exhibits notable biological activity. However, the low water solubility of genistein limits its widespread application. To improve its water solubility, this study prepared γ-cyclodextrin (γ-CD)/GEN inclusion complexes and investigated their mechanism of action in alleviating hydrogen peroxide-induced oxidative stress damage in PC12 cells. The results showed that γ-CD/GEN could be uniformly and stably dispersed in the system. Cell experiments indicated that γ-CD/GEN significantly increased cell survival after treatment with organic hydrogen peroxide, and promoted the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and the expression of glutathione peroxidase 4 (GPx4). It also improved mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) levels, thereby alleviating cell damage caused by oxidative stress. Further Nrf2 knockdown experiments revealed that γ-CD/GEN failed to provide protection, and no protective effect was observed under RSL3 intervention. In conclusion, γ-CD/GEN exerts antioxidant effects by activating the Nrf2/GPx4 pathway, thereby reducing oxidative stress damage induced by organic hydrogen peroxide, providing a theoretical basis for the development of novel antioxidants. [Display omitted] • γ-CD/GEN inhibits organic hydrogen peroxide-induced oxidative stress in a dose-dependent manner in PC12 cells. • γ-CD/GEN evoked nuclear translocation of Nrf2 with concomitant activation of Nrf2 downstream antioxidant enzymes. • In Nrf2 and GPx4 knockdown PC12 cells, the protective effect of γ-CD/GEN against it was almost lost. [ABSTRACT FROM AUTHOR]

Latroeggtoxin-VI (LETX-VI), a proteinaceous neurotoxin mined from the egg transcriptome of spider L. tredecimguttatus , was previously found to promote the release of dopamine from PC12 cells. However, the relevant molecular mechanism has not been fully clear. Here LETX-VI was demonstrated to rapidly penetrate the plasma membrane of PC12 cells via the vesicle exocytosis/endocytosis cycle, during which vesicular transmembrane protein synaptotagmin 1 (Syt1) functions as a receptor, with its vesicle luminal domain interacting with the C-terminal region of LETX-VI. The C-terminal sequence of LETX-VI is the functional region for both entering cells and promoting dopamine release. After gaining entry into the PC12 cells, LETX-VI down-regulated the phosphorylation levels of Syt1 at T201 and T195, thereby facilitating vesicle fusion with plasma membrane and thus promoting dopamine release. The relevant mechanism analysis indicated that LETX-VI has a protein phosphatase 2A (PP2A) activator activity. The present work has not only probed into the Syt1-mediated action mechanism of LETX-VI, but also revealed the structure-function relationship of the toxin, thus suggesting its potential applications in the drug transmembrane delivery and treatment of the diseases related to dopamine release and PP2A activity deficiency. [ABSTRACT FROM AUTHOR]

Dopamine is an important neurotransmitter in the body and closely related to many neurodegenerative diseases. Therefore, the detection of dopamine is of great significance for the diagnosis and treatment of diseases, screening of drugs and unraveling of relevant pathogenic mechanisms. However, the low concentration of dopamine in the body and the complexity of the matrix make the accurate detection of dopamine challenging. Herein, an electrochemical sensor is constructed based on ternary nanocomposites consisting of one-dimensional Pt nanowires, two-dimensional MXene nanosheets, and three-dimensional porous carbon. The Pt nanowires exhibit excellent catalytic activity due to the abundant grain boundaries and highly undercoordinated atoms; MXene nanosheets not only facilitate the growth of Pt nanowires, but also enhance the electrical conductivity and hydrophilicity; and the porous carbon helps induce significant adsorption of dopamine on the electrode surface. In electrochemical tests, the ternary nanocomposite-based sensor achieves an ultra-sensitive detection of dopamine (S/N = 3) with a low limit of detection (LOD) of 28 nM, satisfactory selectivity and excellent stability. Furthermore, the sensor can be used for the detection of dopamine in serum and in situ monitoring of dopamine release from PC12 cells. Such a highly sensitive nanocomposite sensor can be exploited for in situ monitoring of important neurotransmitters at the cellular level, which is of great significance for related drug screening and mechanistic studies. [Display omitted] • Pt/MX binary composites by solvothermal synthesis. • Pt/MXPC ternary nanocomposites by mixing Pt/MX binary composites and PC. • Pt/MX/PC-modified SPCEs for electrochemical detection of dopamine. • In situ monitoring of dopamine released from PC12 cells by K+ stimulation. [ABSTRACT FROM AUTHOR]

Background Lidocaine was an anesthetic commonly used for analgesia, but the neurotoxicity could not be ignored. However, benzodiazepines could alleviate the toxicity when combined with other drugs. Purpose To explore the molecular mechanism of benzodiazepines in protecting nerve cells after the induction of lidocaine. Methods PC12 cells were induced by lidocaine (0 mM, 0.1 mM, 0.5 mM and 1 mM) first and then treated by benzodiazepines (0 μM–200 μM). RT-qPCR assays measured RNA expressions of epidermal growth factor receptor (EGFR) and microRNA-133a-3p (miR-133a-3p) in PC12 cell line, respectively. Western blot was for protein detections of EGFR and caspase-3. Flow cytometry assay assessed apoptosis and cellular viability was validated via Cell Counting Kit-8 (CCK-8) test. Bioinformatics analysis predicted the potential link between miR-133a-3p and EGFR and the binding was verified using the Dual luciferase reporter experiment. Results Benzodiazepines increased cellular viability of PC12 cells up to 100 μM while suppressed viability between 100 and 200 μM. Benzodiazepines (0 μM, 10 μM, 50 μM and 100 μM) did not regulate PC12 cell viability but promoted the viability of lidocaine-treated PC12 cells. Lidocaine downregulated miR-133a-3p RNA expression but facilitated EGFR mRNA expression, which was reversed after treated by benzodiazepines. MiR-133a-3p targeted and negatively regulated EGFR expressions in mRNA and protein levels. Furthermore, miR-133a-3p inhibitor and overexpressed EGFR transfection both restrained the decreased PC12 cell viability and prompted cell apoptosis caused by benzodiazepines. Conclusion Benzodiazepines restrained lidocaine-induced toxicity in PC12 cells which secured viability and reduced apoptosis via miR-133a-3p/EGFR pathway. [ABSTRACT FROM AUTHOR]

Abstract Ethnopharmacological relevance Zhi-zi-chi Decoction(ZZCD), a traditional Chinese medicine formula, has been reported its potential protective effect on psychological sub-health diseases. However, there still remains a lack of molecular mechanism interpretation. Aim of the study This study was aimed at investigating the mechanism of glutamate-induced toxicity in PC12 cells and the neuroprotective effect of ZZCD based on a novel strategy of the combination of cell metabolomics and pharmacology. Materials and methods The PC12 cells were treated with glutamate to simulate neurotoxic cell model. Gas chromatography coupled with mass spectrometry based on cell metabolomics approach was performed to comprehensively investigate the molecular mechanism of glutamate-induced toxicity The cell viability and cytotoxicity analysis, the determination of glutathione reductase(GR), superoxide dismutase(SOD) and reactive oxygen species(ROS), apoptosis analysis and western blot analysis were performed to evaluate the neuroprotection of ZZCD. Results Forty metabolites were identified as potential biomarkers in model cells by principal components analysis(PCA) and orthogonal partial least squares-discriminant analysis(OPLS-DA). Glutamate decreased the GR and SOD activities, increased the level of intracellular ROS, activated the apoptotic pathway, and induced the changes of energy metabolism, amino acid metabolism and lipid metabolism. In addition, the extract of ZZCD could reverse the disturbed metabolic pathways by regulating those potential biomarkers and exerted anti-oxidation and anti-apoptosis. Conclusion ZZCD has neuroprotective effect and the novel strategy can be applicable for other traditional Chinese medicine formulas. Graphical abstract Image 1 [ABSTRACT FROM AUTHOR]

Abstract A facile, sensitive and selective electrochemical biosensor for the detection of dopamine (DA) released from nicotine-stimulated PC12 cells was fabricated by using a poly-celestine blue (CB) modified glassy carbon electrode (GCE). The poly-CB film was prepared by controlled electropolymerization and has about 5 nm thick on the GCE surface. In the optimal experimental condition, the poly-CB modified GCE showed excellent electrocatalytic activity toward the oxidation of DA. The response signal obtained by differential pulse voltammetry displayed a good linear relationship between the current response signal and the DA concentrations in range from 10 nmol L−1 to 0.7 μmol L−1 and 1 μmol L−1 to 10 μmol L−1 with a detection limit of 1.2 nmol L−1 (S/N = 3) and sensitivity of 17.0134 μA cm2 μM−1. In addition, the poly-CB modified GCE has successfully been applied to determine nicotine-induced dopamine released from PC12 cells with satisfactory recovery. The developed method held great potential for the detection of dopamine in biological samples and would bring great convenience to the diagnosis of diseases associated with deficiency in dopamine. Highlights • The nicotine-induced dopamine secreted by live PC12 cells was detected by a poly-celestine blue modified GCE. • The biosensor showed excellent electrocatalytic activity, selectivity and ultralow detection limit toward the DA. • The method held great potential for the clinical diagnosis of diseases related to deficiency of DA. [ABSTRACT FROM AUTHOR]

Background: Genistein is a soy-derived isoflavone and phytoestrogen with antioxidant and neuroprotective activity. Genistein has intrinsically low oral bioavailability that affects its dose-response activities.Purpose: Nanotechnologies were used to obtain the delivery of genistein to the brain: lipid-based nanovesicles, transfersomes, loaded with the phytoestrogen were developed as potential therapeutic or preventive strategy against neurodegenerative diseases by intranasal administration.Methods: Phosphatidylcholine from soybean and different edge activators were used to prepare transfersomes. The effect of selected nanovesicles on the oxidative damage was studied in PC12 cell line.Results: Suitable nanovesicles as carrier of genistein were obtained; their composition affects deformability, drug permeation behavior and cytotoxicity. In particular, the formulation containing Span 80, GEN-TF2, showed efficiency of internalization into the cell and it was able to attenuate ROS formation and to reduce the amount of apoptotic cells generated by H2O2 treatment compared to genistein.Conclusion: GEN-TF2 was able to reduce the oxidative damage suggesting a possible antioxidant role of this drug delivery system. These obtained data confer to GEN-TF2 a potential antioxidant activity and then it could be used as adjuvant therapy in oxidative stress-related neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Superparamagnetic iron oxide nanoparticles (SPIONs) have been widely used as theranostic drug-carrier and MRI contrast agent. Their potential effects are still in blank while SPIONs are used for brain. The present study aims to investigate SPIONs’ neurotoxicity in vitro and in vivo using stereotaxic technique. By co-incubating SPIONs with dopaminergic neuronal PC12 cells, we found that SPIONs had a dose-dependent cytotoxic in PC12 cells at 60–200 ug/mL but not at 10–50 ug/mL, it reduced cell viability, decreased the capacity of PC12 cells to extend neurites in response to nerve growth factor (NGF), induced a reduction of the tyrosine hydroxylase protein, while increasing PC12 cell apoptosis. Accordingly, the no-observed-adverse-effect level (NOAEL) of current SPIONs was 50 ug/mL in vitro, which would be useful for human health risk assessment. While directly injecting the SPIONs into the dorsal striatum or hippocampus, 7 and 14 days after surgery, nanoparticles decreased the TH + fiber density in both the dorsal striatum and the hippocampus. A behavioral evaluation demonstrated that SPIONs attenuated the animals’ motor coordination and spatial memory, as evaluated by the rotarod test and the Morris water maze. We further examined mitogen-activated protein kinase (MAPK) activation and found that c-Jun N-terminal kinase (JNK) was activated after SPIONs treatment. It suggests that the SPIONs-induced neurotoxicity might be mediated through the JNK signaling pathway. SPIONs could possibly induce neurotoxic effects on the dorsal striatum and hippocampus. [ABSTRACT FROM AUTHOR]

Clinical and experimental studies suggested that testosterone or estradiol alone supplementation may have antidepressant-like effects. However, the synergistic effect of testosterone/estradiol on the treatment of depression and the underlying mechanism remain largely unknown. Here, we hypothesized that an appropriate dosage of testosterone combined with estradiol may became a new therapeutic strategy in depression. Our results indicated that an appropriate testosterone/estradiol ratio of 10:1 (10 −8  mol/L testosterone and 10 −9  mol/L estradiol) was found, which has a significant synergistic anti-apoptotic effect on PC12 cells injury stimulated by corticosterone. We then confirmed the synergistic effect of the defined testosterone/estradiol ratio in PC12 cells mainly depend on the inhibitory activation of Traf6/TAK1 signaling pathway. Furthermore, we found that the defined testosterone/estradiol ratio could suppress the expression and ubiquitination of Traf6 protein through AR/ERα-mediated pathways. Finally, endogenous interactions of Traf6 with AR/ERα in PC12 cells were found using co-immunoprecipitation assays, and further verified how the defined testosterone/estradiol ratio plays a synergistic role in injury of PC12 cells. The current study demonstrated that an appropriate testosterone/estradiol ratio has a synergistic effect on PC12 cells injury induced by corticosterone through suppressing the activation of Traf6/TAK1 signaling pathway, suggesting that testosterone/estradiol might synergistically protect against neuronal apoptosis to ameliorate depressive symptoms. [ABSTRACT FROM AUTHOR]

Objective To investigate the role of LncRNA MALAT-1 in hypoxia-induced cell injury. Methods Pheochromocytoma-12 (PC12) cells were divided into seven groups: Control group, Hypoxia group (Cells treated with CoCl 2 ), MALAT-1 group (Hypoxic cells treated with MALAT-1), NC group (Hypoxic cells treated with empty plasmid), MALAT-1 siRNA group (Hypoxic cells treated with siRNA MALAT-1), SB203580 group (Hypoxic cells treated with p38MAPK inhibitor), and MALAT-1 + SB20358 group. The content of reactive oxygen species (ROS), malondialdehyde (MDA), super oxide dismutase (SOD) and lactate dehydrogenase (LDH) was determined. Cell viability was detected by MTT assay. Apoptotic cells were observed by Hoechst 33258 and TUNEL staining assay. Mitochondrial membrane potential (MMP) was measured using JC1 vital dye. Results The decreased cell viability and increased expressions of MALAT-1 and p-p38 were observed in hypoxic PC12 cells time-dependently ( P <  0.05). Besides, hypoxic PC12 cells had an elevation in p-p38, ROS, MDA and LDH with the increased apoptotic cells, but a reduction in SOD and MMP, and these similar changes were more obvious in those hypoxic cells treated with MALAT-1 when compared with Controls (all P <  0.05). However, the hypoxic PC12 cells treated with SB203580 and MALAT-1 siRNA led to opposite results compared with MALAT-1 group (all P  < 0.05). Importantly, SB203580 could reverse the function of MALAT-1 in aggravating the hypoxia injury of PC12 cells. Conclusion MALAT-1 can promote the apoptosis and oxidative stress of PC12 cells by activating p38MAPK pathway, thus aggravating the damage of PC12 cells induced by chemical hypoxia. [ABSTRACT FROM AUTHOR]

A new series of 1-phenyl-pyrrolo[1,2- b ]isoquinolin-3-one derivatives were designed, synthesized and demonstrated to act as antagonists for the glycine binding site of the NMDA receptor. These new derivatives protected PC12 cells against NMDA-induced injury and cell apoptosis in vitro , among which compound 13b exhibited excellent cytoneuroprotective potency and shown a dose-dependent prevention. The increased intracellular Ca2+ influx caused by NMDA in PC12 cells was reversed when pretreated with compound 13b. Furthermore, the interaction between compound 13b and the glycine binding site of the NMDA receptor was validated via MST assay. It was observed that the stereochemistry of compound 13b did not influence the binding affinity, which was consistent with the neuroprotective result. Molecular docking study confirmed the observed activity of compound 13b by virtue of their Pi-stacking, cation-Pi, H-bonding and Pi-electron interactions with the key amino acids in the glycine binding pocket. These results confirm the potential of 1-phenyl-pyrrolo[1,2- b ]isoquinolin-3-one derivatives as neuroprotective agents targeting the glycine binding site of the NMDA receptor. [Display omitted] • Synthesis of 25 novel 1-phenyl-pyrrolo[1,2- b ]isoquinolin-3-one derivatives. • Compound 13b has shown good neuroprotective effect on NMDA-induced injury in PC12 cells via Ca2+ antagonism. • Compound 13b acts as antagonist for the glycine binding site of the NMDA receptor. • Docking 13b into the glycine binding site to explore the structure-activity relationship. [ABSTRACT FROM AUTHOR]

Icariside II (ICS II), a phosphodiesterase 5 inhibitor (PDE 5-I), is a major ingredient of Epimedium brevicornum , with wide spectrum of neuroprotective properties. However, little is known about the potential beneficial effect of ICS II on neuronal cell proliferation, and its possible underlying mechanism remains still unclear. We hypothesized that the beneficial effect of ICS II on neuron-like highly differentiated rat pheochromocytoma (PC12) cell proliferation is correlated with the nitric oxide (NO) signaling pathway and its upstream of PI3K/AKT pathway. PC12 cells were treated with ICS II alone or together with L-NMMA, H89, KT-5823, and/or LY294002 (the inhibitor of NOS, PKA, PKG, PI3K, respectively). It was found that ICS II concentration-dependently promoted PC12 cells proliferation, and cell cycle analysis showed that the proportion of ICS II-treated PC12 cells in S phase was higher than that of control. Moreover, ICS II at the appropriate concentration (100 μM) not only increased nNOS expression, NO production, but also enhanced cGMP content and PKG activity. The addition of L-NMMA and KT-5 823 significantly inhibited the effects of ICS II on nNOS expression, NO production and PKG activity. Furthermore, LY294002 significantly decreased p -AKT level, NOS activity, NO production and nNOS expression, but it did not affect iNOS expression. These findings demonstrate that the beneficial effect of ICS II on neuronal cell proliferation, and its possible underlying mechanisms are, at least partly, through activating AKT/nNOS/NO/cGMP/PKG signaling pathway. [ABSTRACT FROM AUTHOR]

This study investigated the effects of fluoride exposure on the mRNA expression of Cav1.2 calcium signaling pathway and apoptosis regulatory molecules in PC12 cells. The viability of PC12 cell receiving high fluoride (5.0 mM) and low fluoride (0.5 mM) alone or fluoride combined with L-type calcium channel (LTCC) agonist/inhibitor (5 umol/L FPL6417/2 u mol/L nifedipine) was detected using cell counting kit-8 at different time points (2, 4, 6, 8, 12, 10, and 24 h). Changes in the cell configuration were observed after exposing the cells to fluoride for 24 h. The expression levels of molecules related to the LTCC were examined, particularly, Cav1.2, c-fos, CAMK II, Bax, and Bcl-2. Fluoride poisoning induced severe cell injuries, such as decreased PC12 cell activity, enhanced cell apoptosis, high c-fos, CAMKII, and Bax mRNA expression levels. Bcl-2 expression level was also reduced. Meanwhile, high fluoride, high fluoride with FPL64176, and low fluoride with FPL64176 enhanced the Cav1.2 expression level. In contrast, low fluoride, high fluoride with nifedipine, and low fluoride with nifedipine reduced the Cav1.2 expression level. Thus, Cav1.2 may be an important molecular target for the fluorosis treatment, and the LTCC inhibitor nifedipine may be an effective drug for fluorosis. [ABSTRACT FROM AUTHOR]

Controlling the cell-matrix interaction is a critical factor in the design and fabrication of tissue engineering scaffolds. A particular peptide sequence, Arg-Gly-Asp (RGD peptide), is often used as an adhesion ligand in the engineering of different types of tissues. While in some cases this has been adequate, the use of multiple ligands may be required for the successful engineering of some tissue types. We hypothesized that hydrogels presenting both the RGD peptide and the YIGSR peptide (Tyr-Ile-Gly-Ser-Arg) could successfully regulate the phenotype of PC12 cells, thereby providing a new platform for effective tissue engineering applications. We prepared alginate hydrogels modified with both RGD and YIGSR peptides at several different bulk ligand densities and determined the ways in which PC12 cells can respond to them in vitro . We demonstrate that alginate hydrogels presenting both RGD and YIGSR peptides successfully regulate the proliferation, morphological change, and neuronal differentiation of PC12 cells in vitro . Successful adhesion and proliferation of PC12 cells were dependent on the bulk density of RGD peptides, while neuronal differentiation was significantly enhanced by increasing the YIGSR density. These results suggest that hydrogels presenting multiple adhesion ligands offer many useful applications in tissue engineering approaches. [ABSTRACT FROM AUTHOR]

The fruit of Lycium ruthenicum Murray has been widely used as a precious Chinese herbal medicine. In this study, the major anthocyanin in L. ruthenicum fruit was obtained via an AKTA purifier system with YMC-Pack ODS-A column and identified by mass and nuclear magnetic resonance spectroscopy to be petunidin 3- O -[6- O -(4- O -( trans - p -coumaroyl)-α- l -rhamnopyranosyl)-β- d -glucopyranoside]-5- O -[β- d -glucopyranoside] (compound 1 ). Compound 1 exhibited higher scavenging activities against 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), 1,1-diphenyl-2-picrylhydra-zyl and superoxide radicals than the crude extract of anthocyanins. Moreover, compound 1 was demonstrated to have potential cytoprotective effect on hydrogen peroxide-induced intracellular reactive oxygen damage in neuronlike rat pheochromocytoma line cells by promoting cell proliferation, mitigating lipid peroxidation and regulating endogenous antioxidant enzymes (glutathione peroxidase, superoxide dismutase and catalase). [ABSTRACT FROM AUTHOR]

[Display omitted] • A neuro-specific substrate covered with lipid bilayer derived from neuronal differentiated cells was prepared. • Substrate-tethered lipid bilayers have anti-fouling effect on proteins, blood components and bacterial cells. • Cell membrane components of the lipid bilayer reduced affinity to astrocytes and macrophages. • Neuronal differentiated membranes of the lipid bilayer induced specific interactions with neuronal cells. The limitation of short-term usage of neural interfacing devices encouraged the development of neuro-specific biomaterials. Our hypothesis is that a biomaterial covered with the neuronal cell-derived membrane possessing neural cell adhesion molecule (L1CAM) can promote neuronal adhesion and activation and minimize immune responses at the condition of neural implantation. To demonstrate the hypothesis, we prepared the titanium surface-modified with a PC12 cell membrane-derived lipid bilayer, covalently tethered on the surface (PM-TLB). Anti-fouling studies informed us that PM-TLB was sufficiently resistant to the fouling of plasma proteins as well as the adhesion of blood components and bacteria. Cell studies demonstrated that PM-TLB is specific to neuronal cells and non-specific to astrocytes and macrophages, clearly shown in a normal condition and an inflammatory condition. The neuronal activation study supported that PM-TLB improves the outgrowth of neurites and activation stages more than the poly(l -lysine) polymer, which is the most used substrate for neuronal cells. These results conclude that PM-TLB is an efficient surface modification showing selective modulation against neurons and the immune system, promoting neuronal interaction and suppressing neuro-inflammatory responses for applications to neuro-implantable devices. [ABSTRACT FROM AUTHOR]

Highly sensitive and specific detection and monitoring of trace norepinephrine (NE) in biological fluids and neuronal cell lines is essential for the investigation of pathogenesis of certain neurological diseases. Herein, we constructed a novel electrochemical sensor for real-time monitoring of NE released by PC12 cells based on glassy carbon electrode (GCE) modified with honeycomb-like nickel oxide (NiO)-reduced graphene oxide (RGO) nanocomposite. The synthesized NiO, RGO and the NiO-RGO nanocomposite were characterized using X-ray diffraction spectrogram (XRD), Raman spectroscopy and scanning electron microscopy (SEM). The porous three-dimensional honeycomb-like structure of NiO and high charge transfer kinetics of RGO endowed the nanocomposite with excellent electrocatalytic activity, large surface area and good conductivity. The developed sensor exhibited superior sensitivity and specificity towards NE in a wide linear range from 20 nM to 14 μM and 14 μM–80 μM, with a low detection limit of 5 nM. The performances of the sensor in terms of excellent biocompatibility and high sensitivity allow it to be successfully employed in the tracking of NE release from PC12 cells under the stimulation of K+, providing an effective strategy for the real-time monitoring of cellular NE. An electrochemical sensor based on honeycomb-like NiO-reduced graphene oxide (RGO) nanocomposite was developed and applied for the real-time tracking of norepinephrine in neuronal cells. [Display omitted] • The synthesized NiO-RGO nanocomposite exhibited large surface area, high conductivity and electrocatalytic activity. • The NiO-RGO/GCE possessed a low detection limit for norepinephrine detection with excellent sensitivity. • The sensor was applied for the real-time monitoring of norepinephrine secreted by PC12 cells. [ABSTRACT FROM AUTHOR]

Perfluorohexanesulfonate (PFHxS), one of the major perfluoroalkyl compounds (PFCs), has been used in a variety of industrial and consumer applications and detected in serum in the general population. This raised a concern over its possible detrimental health effects, including neurotoxic effects. We have previously shown that PFHxS induced neuronal apoptosis via the NMDA receptor-mediated extracellular signal-regulated kinase (ERK) pathway. Recently, it has been reported that AMP-activated protein kinase (AMPK) acts as a key signal molecule in neuronal excitotoxicity as well as providing a neuroprotective function. In the present study, we have examined the involvement of AMPK in PFHxS-induced neuronal apoptosis using neuronal differentiated PC12 cells. PFHxS induced significant increases in intracellular [Ca 2+ ] via the NMDA receptor and the L-type voltage-gated calcium channel (L-VGCC). The inhibition of Ca 2+ loading by the NMDA receptor antagonist, MK801 and the L-VGCC blockers, nifedipine and diltiazem significantly reduced PFHxS-induced apoptosis. PFHxS induced sustained activation of AMPK and the inhibition of AMPK activation by compound C and AMPK siRNA significantly reduced PFHxS-induced caspase-3 activity. These results indicate the pro-apoptotic role of AMPK. The activation of AMPK was attenuated by MK801, nifedipine and diltiazem. However, the activation of AMPK was not affected by the ERK inhibitor, PD98059. Likewise, ERK activation was not affected by compound C but was substantially reduced by MK801, nifedipine or diltiazem. This suggests that the activation of AMPK and ERK is regulated by intracellular Ca 2+ loading in distinct pathways. Taken together, PFHxS-induced neuronal apoptosis is mediated by AMPK and ERK pathways, which are distinctly regulated by increased intracellular Ca 2+ via the NMDA receptor and L-VGCC. [ABSTRACT FROM AUTHOR]

Deoxynivalenol (DON) has broad toxicity in animals and humans. In this study the impact of DON treatment on apoptotic pathways in PC12 cells was determined. The effects of DON were evaluated on (i) typical indicators of apoptosis, including cellular morphology, cell activity, lactate dehydrogenase (LDH) release, and apoptosis ratio in PC12 cells, and on (ii) the expression of key genes and proteins related to apoptosis, including Bcl-2, Bax, Bid , cytochrome C (Cyt C), apoptosis inducing factor (AIF), cleaved-Caspase9, and cleaved-Caspase3. DON treatment inhibited proliferation of PC12 cells, induced significant morphological changes and apoptosis, promoted the release of Cyt C and AIF from the mitochondria, and increased the activities of cleaved-Caspase9 and cleaved-Caspase3. Bcl-2 expression decreased with increasing DON concentrations, in contrast to Bax and Bid , which were increased with increasing DON concentration. These data demonstrate that DON induces apoptosis in PC12 cells through the mitochondrial apoptosis pathway. [ABSTRACT FROM AUTHOR]

Epidemiology studies indicate that fruit and vegetable (FV) rich diets are associated with lower risk of Alzheimer's disease (AD), while, the underlying mechanism is not clear. The health promoting effects of diet-derived antioxidants (such as purified vitamin C, β-carotene, flavonoids) were indicated in dietary intervention experiments, However, the practical biological efficiency of FV consumed through diet could not be simply mimicked by purified antioxidants or their mixture. An in vitro neuron cell (PC12) culture experiment was carried out in the present study to observe the oxidative damage preventing effects of serum collected from FV juice treated rats. Our results showed that serum collected from FV juice treated rats antagonized the oxidative damage caused by H 2 O 2 treatment, and regulated the antioxidant gene expression. FV juice increased antioxidant biomarker levels and antioxidant enzyme activity in rat serum. Our results indicate that multiple mechanisms are involved in the neuron protective effects of FV-rich diet. [ABSTRACT FROM AUTHOR]

Astaxanthin, a carotenoid extensively found in marine organisms and increasingly used as a dietary supplement, has been reported to have neuroprotective effect. In this study, the neuroprotective effects and mechanisms of astaxanthin against glutamate-induced neurotoxicity were explored. The results showed that pretreatment with astaxanthin inhibited neuronal PC12 cell death induced by L-glutamate and reduced the production of reactive oxygen species (ROS) significantly. Astaxanthin prevented glutamate-induced apoptosis by depressing the elevation of Bax/Bcl-2 ratio, caspase-3 activation and Ca 2+ influx. Moreover, the loss of mitochondrial membrane potential (MMP) as well as the activation of ROS associated NF-κB and MAPK pathways were also inhibited upon treatment with astaxanthin. Therefore, the neuroprotective effects reflected the ability of astaxanthin in the regulation of multiple signaling pathways that protect mitochondria as well as cellular damage induced by L-glutamate. Thus, astaxanthin has the potential to be used as a prophylactic or remediation agent against neuronal disorders. [ABSTRACT FROM AUTHOR]

Ca2+-triggered neurotransmitter release involves complex regulatory mechanisms, including a series of protein-protein interactions. Three proteins, synaptobrevin (VAMP), synaptosomal-associated protein of 25 kDa (SNAP-25) and syntaxin, constitute the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) core complex that plays key roles in controlling vesicle fusion and exocytosis. Many other proteins participate in the regulation of the processes via direct and/or indirect interaction with the SNARE complex. Although much effort has been made, the regulatory mechanism for exocytosis is still not completely clear. Accumulated evidence indicates that the small GTPase Rab3 and synaptotagmin proteins play important regulatory roles during vesicle fusion and neurotransmitter release. This review outlines our present understanding of the two regulatory proteins, with the focus on the interaction of Rab3 with synaptotagmin in the regulatory process. [Display omitted] [ABSTRACT FROM AUTHOR]

Histone deacetylase (HDAC) inhibitors induce histone acetylation and gene expression by changing local chromatin structures. They can thereby influence various cells to proliferate or differentiate. It has been reported that trichostatin A (TSA) or valproic acid (VPA) can induce the neuronal differentiation of mouse embryonic neural stem cells and rat cerebellar granule cells. It is unclear however which gene is responsible for the neuronal differentiation induced by HDAC inhibitors. In this study, we investigated the contribution of immediate early gene (IEG) nur77 to the neuronal differentiation induced by TSA. We report that TSA induces neurite outgrowth in PC12 cells, and C646, an inhibitor of HAT (histone acetyl transferase) (p300), prevents TSA-induced neurite formation. The acetylation of the Lys14 residue of histone H3, and mRNA and protein expression of nur77 gene were found to be stimulated after treatment with TSA, but not in the presence of C646. A knock-down of nur77 inhibits the neurite outgrowth induced by TSA. Furthermore, the ectopic expression of nur77 significantly elicits neurite formation in PC12 cells. These results suggest that the expression of nur77 , which is up-regulated via the TSA-induced acetylation of Lys14 on histone H3, is essential for the neuronal differentiation in TSA-induced PC12 cells. [ABSTRACT FROM AUTHOR]

Adenosine triphosphate (ATP) plays an important role in signal transmission via acting on P2X receptors. P2X 7 receptor is involved in pathophysiological changes of ischemic diseases. The PC12 cell line is a popular model system to study sympathetic neuronal function. In this study, the effects of P2X 7 on the viability or [Ca 2 + ] i in PC12 cells after exposure to oxygen–glucose deprivation (OGD) were investigated. The results showed that the viability of PC12 cells was decreased under the condition of OGD. BzATP, a P2X 7 agonist, decreased the viability, while P2X 7 antagonist oxATP or P2X 7 siRNA reversed the viability of PC12 cells under the condition of OGD. The expression levels of P2X 7 mRNA and protein in PC12 cells were up-regulated under the condition of OGD or BzATP treatment. The expression levels of P2X 7 mRNA and protein were significantly decreased in OGD PC12 cells, which were pretreated with oxATP or P2X 7 siRNA. It was also found that oxATP or P2X 7 siRNA effectively suppressed the increase of [Ca 2 + ] i induced by OGD. P2X 7 agonist ATP or BzATP enhanced the [Ca 2 + ] i rise induced by OGD in PC12 cells. The [Ca 2 + ] i peak induced by ATP or BzATP in OGD group was decreased by ERK inhibitor U0126. Therefore, P2X 7 antagonists or P2X 7 siRNA could depress the sympathetic neuronal damage induced by ischemia. [ABSTRACT FROM AUTHOR]

Abstract: Perfluorohexanesulfonate (PFHxS) is one of the major perfluoroalkyl compounds (PFCs) found in human blood and its possible neurotoxicity has been suggested. However, the neuronal responses to PFHxS are not much known. Many studies have demonstrated that the early exposure to environmental chemicals increases the risk of neurodegenerative diseases such as Parkinson's disease in later life. In this study, the effects of PFHxS on the neuronal cell death and the underlying mechanisms were examined using PC12 cells as a model of dopaminergic neuron. The treatment with PFHxS reduced cell viability in a dose-dependent manner. PFHxS increased cell apoptosis which was measured by caspase-3 activity and TUNEL staining. MK801, a NMDA receptor antagonist reduced PFHxS-induced apoptosis. PFHxS increased the activations of ERK1/2, JNK and p38 MAPK with different temporal activations. The treatment with PD98059, an ERK inhibitor, significantly reduced apoptosis, whereas SB203580, a p38 MAPK inhibitor, had no effect. JNK inhibition by SP600125 significantly increased apoptosis. PFHxS exposure also increased ROS formation, which was completely blocked by antioxidants, Trolox or N-acetylcysteine (NAC). However, neither Trolox nor NAC reduced PFHxS-increased apoptosis, suggesting that ROS may not be a critical mediator for PFHxS-induced apoptosis of cells. Moreover, ERK activation induced by PFHxS was blocked by MK801 but not antioxidants. Taken together, these results have demonstrated that PFHxS induces the apoptosis of dopaminergic neuronal cells, where NMDA receptor-mediated ERK pathway plays a pro-apoptotic role and JNK plays an anti-apoptotic role. Our results may contribute to understanding cellular mechanisms for PFHxS-induced neurotoxicity. [Copyright &y& Elsevier]

Abstract: In previous study, we have screened the effective fraction against Alzheimer’s disease (AD-EF) from the extracts of roots and rhizomes of Valeriana amurensis, based on which neuroprotective active constituents from AD-EF were investigated. Six new compounds 1–6, including four iridoids (xiecaoside A–C and xiecaoline A), one pinane-type monoterpeneglucoside (xiecaoside D), and one phenylpropanoid glycoside (xiecaoside E) were isolated together with 11 known compounds 7–17. The structures of 1–6 were elucidated by their spectroscopic data. The protective effects of compounds 1–17 on PC12 cells with neurotoxicity induced by amyloid-beta 1–42 (A?1–42) was also investigated, respectively. Consequently, compound 6 and lignans 11–17 were responsible for protecting against A?-induced toxicity in PC12 cells. [Copyright &y& Elsevier]

Highlights: [•] Long-term preexposure of PC12 cells to 1nM BPA inhibited neuronal differentiation. [•] Long-term preexposure of PC12 cells to 10nM NP inhibited neuronal differentiation. [•] The effects of BPA or NP were still observed even after a week withdrawal. [•] The effects of BPA or NP might be retained within PC12 cells. [ABSTRACT FROM AUTHOR]

Abstract: Gymnopilins are substances produced in fruiting bodies of the hallucinogenic mushroom, Gymnopilus junonius. Although, only a few biological effects of gymnopilins on animal tissues have been reported, it is believed that gymnopilins are a key factor of the G. junonius poisoning. In the present study, we found that gymnopilins inhibited ACh-evoked responses in neuronal cell line, PC12 cell, and determine the underlying mechanism. Gymnopilins were purified from wild fruiting bodies of G. junonius collected in Japan. Ca2+-imaging revealed that gymnopilins reduced the amplitude of ACh-evoked [Ca2+]i rises by about 50% and abolished the ACh responses remaining in the presence of atropine. Gymnopilins greatly reduced the amplitude of [Ca2+]i rises evoked by nicotinic ACh receptor agonists, 1,1-Dimethyl-4-phenylpiperazinium iodide (DMPP) and nicotine. In the whole-cell voltage clamp recording, gymnopilins inhibited the DMPP-evoked currents, but did not affect the voltage-gated Ca2+ channel currents. These results indicate that gymnopilins directly act on nicotinic ACh receptors and inhibit their activity. This biological action of gymnopilins may be one of the causes of the G. junonius poisoning. [Copyright &y& Elsevier]

Highlights: [•] Effect of oxidation of lipoproteins on neuritogenesis was examined in PC12 cells. [•] Either HDL or oxHDL induces a neurite retraction and subsequent neurite outgrowth. [•] S1P and LPA mediates HDL- and oxHDL-induced neurite retraction, respectively. [•] Neurite outgrowth activity is higher in ox-HDL than HDL and mediated by LPC. [Copyright &y& Elsevier]

Abstract: CART (cocaine- and amphetamine-regulated transcript) peptide is a neuropeptide with a powerful central anorexigenic effect. Specific CART peptide binding sites, most likely CART peptide receptors, have been found in PC12 cells. This study further characterizes the CART peptide binding sites in PC12 cells. After differentiation to a neuronal phenotype with nerve growth factor, the number of CART peptide binding sites in PC12 cells tripled. Following dexamethasone treatment, which transforms PC12 cells into chromaffin-like cells, the number of CART peptide binding sites substantially decreased. CART peptide did not affect the differentiation or acetylcholinesterase activity of PC12 cells, indicating that CART peptide does not participate in differentiation or neuronal activity. CART peptide increased the phosphorylation of SAPK/JNK (stress-activated protein kinase/c-Jun-amino-terminal kinase) and subsequent c-Jun protein expression. These effects were reversed by SP600125, a specific JNK-kinase inhibitor. CART peptide did not significantly affect ERK (extracellular signal-regulated kinase), CREB (cAMP responsive element binding protein), or p38 phosphorylation and c-Fos protein expression. Central administration of CART peptide into mice also resulted in increased c-Jun positive cells in dorsomedial hypothalamic nucleus and nucleus of the solitary tract, areas involved in food intake regulation. Activation of c-Jun by CART peptide might indicate a possible role of CART peptide in managing stress conditions rather than a role in cell proliferation or differentiation as well as the more complex and/or specific regulation ways by transcription factors in some nuclei involved in food intake regulation. The characteristics of stress that CART peptide potentially mediates should be further studied. [Copyright &y& Elsevier]

Highlights: [•] 7,8-DHF as a selective TrkB agonist is neuroprotective. [•] 7,8-DHF has been proved to have antioxidant activity. [•] 7,8-DHF could protect PC12 cells against 6-OHDA-induced toxicity. [•] The protection by 7,8-DHF is due to eliminating ROS and improving SOD activity. [ABSTRACT FROM AUTHOR]

Highlights: [•] Toxicological effect of UV wavelengths at different exposure doses was evaluated. [•] 250 and 310nm dose showed the highest and lowest toxic effects on PC12 cells. [•] Among 4-wavelengths, 290nm exhibited marked DNA repair ability to its LD50. [•] Incidence of toxicity on cells relies on wavelengths and exposure doses of UV. [•] Lower wavelength and higher exposure time have prominent toxic effect on cells. [Copyright &y& Elsevier]

Abstract: In applications of silicon nanowire field-effect transistors (SiNW-FETs) as biosensors, the SiNW-FETs conventionally are all area modified (AAM), with receptors covering not only the minute SiNW surface area but also the relatively large surrounding substrate area. In this study, using a bottom-up technique, we successfully fabricated selective surface modified (SSM) SiNW-FETs with the receptors on the SiNW sensing surface only. In this approach, the strategy was to modify the SiNWs with a chemical linker of 3-aminopropyltrimethoxysilane (APTMS) prior to photolithographic fabrication of the device. The APTMS molecules modifying the SiNWs survived the harsh photolithographic processes, including coating with photoresist, washing with organic solvent, and thermal annealing. These SSM SiNW-FETs also exhibited desirable electrical characteristics such as ohmic contact and high transconductance. Using the biotin–avidin binding system, we showed that the faster response time and smaller sample requirements of the SSM SiNW-FETs, relative to the conventional AAM SiNW-FETs, clearly show that restricting the surface modification of the SiNW-FETs substantially improves their detection sensitivity. Detection with a SSM boronic acid-modified SiNW-FET of the dopamine released under high-K+ buffer stimulation from living PC12 cells also demonstrates that SiNW-FETs can serve as highly sensitive biosensors for biomedical diagnosis. In binding affinity measurements with SiNW-FETs, the dissociation constants (K d) of the biotin–avidin and dopamine–boronic acid complexes were determined to be 15±1fM and 33±8fM, respectively. [Copyright &y& Elsevier]

Abstract: Purpose: There is an increasing amount of experimental evidence that oxidative stress has a central role in the neuropathology of neurodegenerative diseases. It has been suggested that the loss of cell function results from the increased oxidative damage to proteins and DNA. Herein, we investigated the effect of a natural neuroprotective flavonoid, calycopterin, on H2O2-induced disruption of phase II detoxifying enzyme system and cAMP response element binding protein (CREB) phosphorylation. Methods: PC12 cells were treated with 25, 50 and 100μM of calycopterin for 3h, followed by adding H2O2 (150μM) for 24h. The extent of apoptosis was assessed by comet assay. The level of phosphorylated CREB, nuclear factor erythroid 2-related factor 2 (Nrf2), glutamylcysteine synthetase (γ-GCS) and heme oxygenase 1 (HO-1) were measured by western blot method. The concentration of glutathione (GSH) was determined in whole cell lysate using dithionitrobenzoic acid method. Superoxide dismutase (SOD) activity was measured by colorimetric assay. Result: Morphological analysis of protection induced by calycopterin, determined by comet assay, showed that calycopterin reduced DNA in tail. We found that H2O2 decreased mitochondrial membrane potential (MMP), while, calycopterin prevented this decrease in MMP in presence of H2O2. In H2O2-treated cells, calycopterin also suppressed cytochrome C release to cytosol that is necessary for maintaining mitochondrial homeostasis in survived cells. Moreover, calycopterin, in presence of H2O2 inhibited the decrease caused by oxidative stress in stress-sensing transcription factors, CREB and Nrf2, which play an important role in antioxidant capacity of the cell. There was also an increase in γ-GCS and HO-1 levels in calycopterin pretreated cells. In the presence of H2O2, calycopterin inhibited decrease in GSH level and SOD activity. Conclusion: We provided documentation of neuroprotective effect of a natural flavone, calycopterin, against H2O2-induced oxidative stress in differentiated PC12 cells by modulating the level of CREB phosphorylation and Nrf2 pathway. [Copyright &y& Elsevier]

Abstract: In this work we tried to identify mechanisms that could explain how chemical inhibition of heat-shock protein 90 reduces nerve growth factor signaling in rat pheochromocytoma PC12 cells. Geldanamycin is an antibiotic originally discovered based on its ability to bind heat-shock protein 90. This interaction can lead to the disruption of heat-shock protein 90-containing multimolecular complexes. It can also induce the inhibition or even degradation of partner proteins dissociated from the 90kDa chaperone and, eventually, can cause apoptosis, for instance, in PC12 cells. Before the onset of initial apoptotic events, however, a marked decrease in the activity of extracellular signal-regulated kinases ERK 1/2 and protein kinase B/Akt can be observed together with reduced expression of the high affinity nerve growth factor receptor, tropomyosine-related kinase, TrkA, in this cell type. The proteasome inhibitor MG-132 can effectively counteract the geldanamycin-induced reduction of TrkA expression and it can render TrkA and ERK1/2 phosphorylation but not that of protein kinase B/Akt by nerve growth factor again inducible. We have found altered intracellular distribution of TrkA in geldanamycin-treated and proteasome-inhibited PC12 cells that may, at least from the viewpoint of protein localization explain why nerve growth factor remains without effect on protein kinase B/Akt. The lack of protein kinase B/Akt stimulation by nerve growth factor in turn reveals why nerve growth factor treatment cannot save PC12 cells from geldanamycin-induced programmed cell death. Our observations can help to better understand the mechanism of action of geldanamycin, a compound with strong human therapeutical potential. [Copyright &y& Elsevier]

Highlights: [•] TCDD induces the apoptosis of neurons in rat brain and cultured neural cells. [•] TCDD-induces the activation of GSK-3β and the decrease of β-catenin in neural cells. [•] Inhibition of GSK-3β activity blocks TCDD-induced down-regulation of β-catenin. [•] Inhibition of GSK-3β activity can block TCDD-induced neural apoptosis. [Copyright &y& Elsevier]

Abstract: Cancer cells are more sensitive to oxidative stress due to higher levels of reactive oxygen species. Therefore, the ability of anti-cancer agent combretastatin A-4 (CA-4) and triazole analogues to induce reactive oxygen species may be important for selectivity against cancer cells. The purpose of the present study was to investigate the structural requirements for reactive oxygen species production by CA-4 and the triazole analogues Ana-2, Ana-3 and Ana-4. Ana-2 and Ana-3 mimic the cis configuration in CA-4; Ana-3 lacks the phenolic hydroxyl group, while Ana-4 mimics a trans configuration. The rat pheochromocytoma cancer cell line PC12 was used as model system. CA-4 and Ana-2 were highly toxic; Ana-3 was less toxic, whereas Ana-4 was non-toxic. The probe dihydroethidium detected reactive oxygen species production from CA-4, Ana-2, and Ana-3. CA-4 and Ana-2 also induced oxidation of the reactive oxygen species probe dihydrorhodamine and activation of caspase-3. Thus, the phenolic hydroxyl group in CA-4 and Ana-2 was necessary for dihydrorhodamine oxidation, caspase-3 activation, and increased cytotoxicity. [Copyright &y& Elsevier]

Abstract: Since resveratrol is considered to exert a unique dual effect, protective for normal cells but toxic to tumor cells, its action on undifferentiated (original) and differentiated PC12 cells was analyzed, because undifferentiated cells are tumorigenic and differentiated ones are neuronal in nature. Compared to resveratrol-untreated cells in both undifferentiated and differentiated cell groups, cells treated with different doses of resveratrol, at dosages of 1, 10 and 100μM, showed the following alterations. Dying/dead cells were significantly increased in a dose-dependent manner in undifferentiated cells, but they were unchanged at doses of up to 10μM resveratrol in differentiated cells. In living cells, neurites were short in undifferentiated cells, but drastically elongated with an increased number in differentiated cells. The expression of SIRT1 was drastically reduced in undifferentiated cells, but stable in differentiated cells. SIRT3 was significantly enhanced in a dose-dependent manner at resveratrol doses of up to 10μM in both cells, with reduction and more enhanced at a dosage of 100μM in undifferentiated and differentiated cells, respectively. Mitochondrial number and ATP synthase β subunit expression was unaltered at doses of up to 10μM and were significantly reduced at doses of 100μM in undifferentiated cells, but they were significantly increased in a dose-dependent manner, with a slight reduction in the ATP synthase at doses of 100μM, in differentiated cells. In a dose-dependent manner, the number of autophagosomes and the LC3-II/LC3-I ratio were significantly less in undifferentiated cells and greater in differentiated cells. Also, in a dose-dependent manner, the expression of phosphorylated AMP-activated kinase (AMPK) was significantly less in undifferentiated cells and greater in differentiated cells. Resveratrol-induced AMPK suppression and activation, possibly through the modulation of SIRT protein activity, may thus be related to the inhibition and promotion of mitochondrial and autophagic functions, leading to cell death and survival in undifferentiated and differentiated cells, respectively. [Copyright &y& Elsevier]

Abstract: Aim: It is well known that neural stem/progenitor cells (NS/PC) are an ideal cell type for the treatment of central nervous system (CNS) disease. However, ethical problems have severely hampered fetal NS/PC from being widely used as a source for stem cell therapy. Recently, it has been demonstrated that autologous bone marrow mesenchymal stem cells (BMSC) can transdifferentiate into neural progenitor cells (NPC). The biological function of BMSC derived NPC (MDNPC) in neuronal systems remains unknown. In the present study, we aimed to investigate whether MDNPC can promote in vitro neural regeneration, a process comprising mainly the generation of neurons and neurotransmitters. Main methods: We co-cultured BMSC, MDNPC or fetal NS/PC with PC12 cells and studied their roles on proliferation, neurite formation and dopamine release from PC12 cells. Furthermore, we also explored the mechanisms by which MDNPC regulate dopamine secretion from PC12 derived neural cells using Western blot. Key findings: We found that both MDNPC and NS/PC had similar morphologies and there were no significant differences between MDNPC and NS/PC in promoting PC12 cell proliferation, neurite outgrowth, and dopamine release. We also demonstrated that NS/PC induced dopamine secretion was associated with an upregulation of dopamine transporter (DAT) levels. Significance: In summary, MDNPC were comparable to NS/PC in promoting neural regeneration, indicating that MDNPC are a promising candidate source of neural stem cells for the treatment of neurological diseases. [Copyright &y& Elsevier]

Abstract: A great number of the neurites interconnect neuronal cells in a brain to form the complicate neural circuits, whose structures are dynamically changed with changing the numbers and destinations of the neurites. Fabricating a model of neural network in vitro is one of the promising methods to precisely assay the signal transmission and processing within the circuit as well as to examine behaviors of individual cells. In this study, aiming to fabricate the dynamically alterable neural network in vitro, the chemically modified surface with the photo-reactive self-assembled monolayer was applied to navigate the neurite outgrowth activities of differentiated PC12 cell in the spatially and temporally controlled manner. Numbers of the cell soma were effectively adhered and simultaneously arrayed according to the 25μm square patterns, which were easily fabricated with a single shot of the 365-nm ultraviolet (UV) irradiation and pre-coated with the extracellular matrix (ECM) protein. Narrow neurites were successively guided along the 5μm line patterns drawn on the surface by stepwise irradiation of the UV light in the intended designs and at appropriate timing. Sprouting number, elongating direction, bending, branching, and formation of autapse-like structure were controllable. The rate of neurite elongation was dependent on the ECM species, that were pre-coated beneath the cell soma, suggesting the ECM stimulated the basal side of the cell soma and affected the outgrowth process of the neurite. Navigation of the neurite elongation along the microline pattern for a primary rat brain cortex neuron was also achieved. [Copyright &y& Elsevier]

Abstract: Once nanoparticles enter the central nervous system (CNS), they immediately encounter a complex environment of resident microglial immune cell and neurons. In the present study, NPs of 20–60nm in diameter (SiO2-NPs, TiO2-NPs, HAP-NPs and Fe3O4-NPs) were evaluated for their ability to induce microglia-mediated neurodegeneration. The microglia were directly exposed to NPs. The results showed that both TiO2-NPs and HAP-NPs induced significant iNOS expression, resulting in NO release from the microglia. The expression levels of MCP-1 and MIP-1α were also upregulated. These activation effects were accompanied by the activation of the transcription factor NF-κB. In addition, the secretion levels of TNF-α, IL-1β and IL-6 were variably increased by all four NPs. Subsequently, the cell-free supernatants from microglia monocultures were harvested and tested for their ability to stimulate PC12 cells. The results demonstrated that microglia-derived soluble factors induced by TiO2-NPs suppressed Th gene expression, and those by TiO2-NPs and HAP-NPs caused cytotoxicity in PC12 cells. Taken together, these results suggest that the NPs induced microglial activation and subsequently caused the release of proinflammatory factors that contributed to the dysfunction and cytotoxicity in PC12 cells. [Copyright &y& Elsevier]

Abstract: Four kinds of chitosan conduits with longitudinal multi-channels and controlled internal microstructures were prepared using a special mold and a freeze-drying method. One of the conduits was fabricated from a chitosan solution (ab NC), while the other three groups were made from a pre-gelled chitosan solution using genipin as a chemical cross-linker (ab gNC), dibasic sodium phosphate as a physical cross-linker (ab pNC) or a combined ionic and covalent co-cross-linker (ab gpNC), respectively. The porosity of the chitosan conduits ranged from 88 to 90%. The gpNC showed highly interconnected and uniformly distributed pores compared to NC, the gNC and pNC. In contrast, the gNC and gpNC showed about 10% of the volume swelling ratio in 37°C PBS solution, although the gpNC scaffold''s water uptake was the highest, at more than 17 times its original mass. Compressive tests showed that gpNC had significant elasticity and maintained its physical integrity even after compressing them down to 20% of their original height. The elastic modulus of gpNC reached 80kPa, which was more than twice that of the other groups. Adhesion and proliferation of PC12 cells on chitosan gpNC scaffolds showed excellent properties by MTT and SEM observation, which indicated the potential of gpNC scaffolds for nerve tissue engineering applications. [Copyright &y& Elsevier]

Abstract: Luteolin, a 3′, 4′, 5, 7-tetrahydroxyflavone, is an active compound in Rosmarinus officinalis (Lamiacea), and has been reported to exert several benefits in neuronal cells. However cholinergic-induced activities of luteolin still remain unknown. Neuronal differentiation encompasses an elaborate developmental program which plays a key role in the development of the nervous system. The advent of several cell lines, like PC12 cells, able to differentiate in culture proved to be the turning point for gaining and understanding of molecular neuroscience. In this work, we investigated the ability of luteolin to induce PC12 cell differentiation and its effect on cholinergic activities. Our findings showed that luteolin treatment significantly induced neurite outgrowth extension, enhanced acetylcholinesterase (AChE) activity, known as neuronal differentiation marker, and increased the level of total choline and acetylcholine in PC12 cells. In addition, luteolin persistently, activated extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt; while the addition of pharmacological MEK/ERK1/2 inhibitor (U0126) and PI3k/Akt inhibitor (LY294002) attenuated luteolin-induced AChE activity and neurite outgrowth in PC12 cells. The above findings suggest that luteolin induces neurite outgrowth and enhanced cholinergic activities, at least in part, through the activation of ERK1/2 and Akt signaling. [Copyright &y& Elsevier]

Abstract: An endocrine disrupter, bisphenol-A is widely used in the production of plastics and coatings. Recently, it was reported that bisphenol-A affected neurotransmitters in the mammalian brain. On the basis of these reports, it was considered that bisphenol-A affected neuronal differentiation. In this study, the morphological changes in nerve growth factor (NFG)-induced differentiation caused by bisphenol-A were confirmed using a PC12 cell system. When a low concentration of bisphenol-A was added to medium containing NGF, it inhibited neurite extension. In addition, to clarify whether bisphenol-A affects the early and late stages of the NGF-signaling pathway in cell differentiation, changes of phosphorylation of MAP kinases and cAMP-response element binding protein (CREB) in PC12 cells treated with and without BPA in medium containing NGF were investigated using western blot analysis. As results, bisphenol-A significantly inhibited phosphorylation of CREB and ERK1/2 MAPK. [Copyright &y& Elsevier]

Abstract: Neuroprotective agents have been in the focus of attention in the treatment of ischemic stroke. Salidroside, a phenylpropanoid glycoside isolated from Rhodiola rosea L., possessed a wide range of biological activities, especially neuroprotection. In an attempt to improve neuroprotective effects of new salidroside analogs for ischemic stroke, a series of novel aralkyl alcoholic 2-acetamido-2-deoxy-β-d-pyranosides were synthesized and their protective activities against the hypoglycemia and serum limitation induced cell death in rat pheochromocytoma cells (PC12 cells) were studied. Most compounds showed strong neuroprotective effects, especially for 4g and 4h, which exhibited a great potency superior to salidroside. MTT assay, Hoechst 33342 staining, and flow cytometry with annexin V/PI staining collectively showed that pretreatment with 4g and 4h attenuated cell viability loss and apoptotic cell death in cultured PC12 cells. Caspase-3 colorimetric assay and Rhodamine 123 staining revealed the changes in expression levels of caspase-3 and mitochondrial membrane potential in PC12 cells on exposure to hypoglycemia and serum limitation with and without 4g and 4h pretreatment, respectively. All the results suggested that 4g and 4h protects the PC12 cells against hypoglycemia and serum limitation induced apoptosis possibly by modulation of apoptosis-related gene expression and restoration of the mitochondrial membrane potential. Therefore, these novel findings may provided a new framework for the design of new aralkyl alcoholic 2-acetamido-2-deoxy-β-d-pyranosides as neuroprotective agents for treating cerebral ischemic stroke and neurodegenerative diseases. [Copyright &y& Elsevier]

Abstract: In this study, we determined the protective potential of trans resveratrol against oxygen–glucose deprivation (OGD) induced reactive oxygen species mediated apoptotic damages in PC12 cells. In vitro model of ischemic cerebral stroke was created by keeping cells in an OGD condition for 6h followed by 24h reoxygenation. Cells received biologically safe doses (5, 10, and 25μM) of trans resveratrol in the following schedules for 24h prior to OGD; during 6h of OGD; for 24h post OGD and whole treatment group which starts from 24h before OGD and lasted to 24h post OGD. Anti-ischemic potential of trans resveratrol was assessed by measuring the regulation of lipid peroxidation, reactive oxygen species production, glutathione content, and expression (mRNA and protein) of apoptotic markers such as Bax, Bcl2 and Caspase-3. Hypoxia inducible factor-1α (HIF-1α) was also assessed to correlate the changes with ischemic injuries. Significant (P<0.05) restoration in lipid peroxidation, reactive oxygen species, and glutathione content were observed following the treatment of trans resveratrol in cells receiving OGD and re-oxygenation. Changes induced by trans resveratrol could be correlated well with alterations in the expression of Bax, Bcl2, Caspase-3 and HIF-1α. These results indicate that trans resveratrol administration attenuates free radical formation and mitochondria mediated apoptosis perhaps by regulating the expressions of Bax, Bcl2, and Caspase-3 in PC12 cells receiving OGD and re-oxygenation insult. [Copyright &y& Elsevier]

Abstract: Acid-sensing ion channels (ASICs) are ligand-gated cation channels that respond to acidic stimuli. They are expressed throughout the mammalian nervous system. Complex subunit combinations and lack of specific blockers of native receptors result in the difficulty of resolving the functions of ASICs. In this study, we showed that rat pheochromocytoma cells (PC12 cells) functionally express ASICs with the activity of endogenous proton-gated conductance. PF is the principal active ingredient extracted from the root of Paeoniae alba, a Chinese herb commonly used to treat neurodegenerative disorders, especially PD. It was found that PF significantly up regulated the expression of LC3-II, which is specifically associated with autophagic vacuole membranes. PF also reduced the MPP+ and acidosis-induced accumulation of α-synuclein, the major component of Lewy bodies. Moreover, PF was highly efficacious in modulating ASICs activity and protein expression. In addition, the data showed that PF was able to protect PC12 cells against MPP+ and acidosis-induced cytotoxicity. In summary, these findings demonstrate for the first time that PF could enhance the autophagic degradation of α-synuclein by regulating the expression and activity of ASICs and thus produces protective effects against cytotoxicity. It also offers the experimental evidence for the potential role of ASICs in the pathogenesis of PD. [Copyright &y& Elsevier]

Abstract: The purpose of this study was to develop the modulation methods for the attachment and detachment of specifically positioned adhesive animal cells cultured on an electrode surface with the application of a weak electrical potential. A patterned indium tin oxide (ITO) optically transparent working electrode was placed on the bottom of a chamber slide with a counter-(Pt) and reference (Ag/AgCl) electrode. The ITO patterning was formed by a reticulate ITO region and arrayed square glass regions of varying size. Using the 3-electrode culture system, the author succeeded in modulation of the attachment and detachment of animal cells on the working electrode surface. Animal cells suspended in serum or sera containing medium were drawn to and attached on a reticulate ITO electrode region to which a +0.4-V vs. Ag/AgCl-positive potential was applied. Meanwhile, the cells were successfully placed on the square glass regions by −0.3-V vs. Ag/AgCl-negative potential application. Animal cells were detached not only from the ITO electrode but also from the square glass regions after the application of a ±1.0-V vs. Ag/AgCl, 3-MHz rectangular wave potential in PBS(−) for 30–60min. Rectangular wave potential-induced cell detachment is almost completely noncytotoxic, and no statistical differences between trypsinization and the high frequency wave potential application were observed in HeLa cell growth. The electrical modulation of the specifically positioned cell attachment and detachment techniques holds potential for novel optical microscopic cell sorting analysis in lab-on-chip devices. [Copyright &y& Elsevier]

Abstract: Identifying small molecules that suppress apoptosis is promising for the therapy of brain diseases. We recently showed that autocrine bone morphogenetic protein (BMP) signaling involves the effects of cholesterol myristate present in traditional Chinese medicine on mesenchymal stem cells. The present study evaluated the effects of cholesterol myristate on the apoptosis and BMP signaling of PC12 cells. PC12 cells transfected by the inhibitor of differentiation (Id1) promoter reporter construct target gene of BMP4 signaling; cholesterol myristate increases the activity of Id1 promoter. However, structurally related steroids such as cholesterol, β-sitosterol and cholesten-3-one, lack of the myristate, did not affect the activity of Id1 promoter, suggesting that myristate is essential for the effect of cholesterol myristate. These effects depend on BMP signaling. Apoptosis analysis indicated that cholesterol myristate inhibited the apoptosis of PC12 cells induced in serum-free condition. Cholesterol myristate significantly increases the expression of BMP4, BMPRIA, p-Smad1/5/8, Id1 and its antiapoptotic target gene Bcl-xL in PC12 cells treated in serum-free condition. Moreover, BMP antagonist reduced the anti-apoptotic effect of cholesterol myristate. Thus, this study is to provide evidence that BMP-Id pathway targeted by cholesterol myristate suppresses the apoptosis of PC12 cells. Our findings are therefore of considerable therapeutic significance and provide the potential of newly exploiting cholesterol myristate and clinically in brain disease therapies. [Copyright &y& Elsevier]