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Orexins are neuronal peptides that play a prominent role in sleep behavior and feeding behavior in the central nervous system, though their receptors also exist in peripheral organs, including the adrenal gland. In this study, the effects of orexins on catecholamine synthesis in the rat adrenomedullary cell line PC12 were investigated by focusing on their interaction with the adrenomedullary bone morphogenetic protein (BMP)-4. Orexin A treatment reduced the mRNA levels of key enzymes for catecholamine synthesis, including tyrosine hydroxylase (Th), 3,4-dihydroxyphenylalanie decarboxylase (Ddc) and dopamine β-hydroxylase (Dbh), in a concentration-dependent manner. On the other hand, treatment with BMP-4 suppressed the expression of Th and Ddc but enhanced that of Dbh with or without co-treatment with orexin A. Of note, orexin A augmented BMP-receptor signaling detected by the phosphorylation of Smad1/5/9 through the suppression of inhibitory Smad6/7 and the upregulation of BMP type-II receptor (BMPRII). Furthermore, treatment with BMP-4 upregulated the mRNA levels of OX1R in PC12 cells. Collectively, the results indicate that orexin and BMP-4 suppress adrenomedullary catecholamine synthesis by mutually upregulating the pathway of each other in adrenomedullary cells.

Beauvericin (BEA), a naturally occurring cyclic peptide with good pharmacological activity, has been widely explored in anticancer research. Although BEA is toxic, studies have demonstrated its antioxidant activity. However, to date, the antioxidant mechanisms of BEA remain unclear. Herein, we conducted a comprehensive and detailed study of the antioxidant mechanism of BEA using an untargeted metabolomics approach, subsequently validating the results. BEA concentrations of 0.5 and 1 μM significantly inhibited H 2 O 2 -induced oxidative stress (OS), decreased reactive oxygen species levels in PC-12 cells, and restored the mitochondrial membrane potential. Untargeted metabolomics indicated that BEA was primarily involved in lipid-related metabolism, suggesting its role in resisting OS in PC-12 cells by participating in lipid metabolism. BEA combated OS damage by increasing phosphatidylcholine, phosphatidylethanolamine, and sphingolipid levels. In the current study, BEA upregulated proteins related to the PI3K/AKT/mTOR pathway, thereby promoting cell survival. These findings support the antioxidant activity of BEA at low concentrations, warranting further research into its pharmacological effects., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Autophagy is a major clearance pathway for misfolded α-synuclein which promotes ferroptosis through NCOA4-mediated ferritin degradation. The regulation of these two processes to achieve improved neuroprotection in Parkinson's disease (PD) must be elucidated. Transcription factor EB (TFEB) is a master regulator of both autophagy and lysosome biogenesis, and lysosomes are important cellular iron storage organelles; however, the role of TFEB in ferroptosis and iron metabolism remains unclear. In this study, TFEB overexpression promoted the clearance of misfolded α-synuclein and prevented ferroptosis and iron overload. TFEB overexpression up-regulated transferrin receptor 1 (TfR1) synthesis and increased the localization of TfR1 in the lysosome, facilitating lysosomal iron import and transient lysosomal iron storage. TFEB overexpression increased the levels of cellular iron-safe storage proteins (both ferritin light and heavy chains). These functions in iron metabolism maintain the cellular labile iron at a low level and electrical activity, even under iron overload conditions. Notably, lower levels of cellular labile iron and the upregulation of ferritin light and heavy chains were reversed after TfR1 knockdown in cells overexpressing TFEB, indicating that TFEB regulates cellular labile iron and suppresses ferroptosis in a TfR1 dependent manner. Taken together, this evidence of the regulation of iron metabolism enriches our understanding of the function of TFEB. In addition, TFEB overexpression protects against ferroptosis and iron overload and provides a new direction and perspective for autophagy regulation in PD., Competing Interests: Declaration of competing interest None., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Alzheimer's disease (AD) is the most common type of dementia and has a complex pathogenesis with no effective treatment. Energy metabolism disorders, as an early pathological event of AD,have attracted attention as a promising area of AD research. Codonopsis pilosula Polysaccharides are the main effective components of Codonopsis pilosula, which have been demonstrated to regulate energy metabolism., Methods: In order to further study the roles and mechanisms of Codonopsis pilosula polysaccharides in AD, this study used an Aβ 1-40 -induced PC12 cells model to study the protective effects of Codonopsis pilosula polysaccharides and their potential mechanisms in improving energy metabolism dysfunction., Results: The results showed that Aβ 1-40 induced a decrease in PC12 cells viability, energy metabolism molecules (ATP, NAD+, and NAD+/NADH) and Mitochondrial Membrane Potential (MMP) and an increase in ROS. Additionally, it was found that Aβ 1-40 increased CD38 expression related to NAD+ homeostasis, whereas Silent Information Regulation 2 homolog1 (SIRT1, SIRT3), Peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) and SIRT3 activity were decreased. Codonopsis pilosula polysaccharides increased NAD+, NAD+/NADH, SIRT3, SIRT1, and PGC-1α related to NAD+, thus partially recovering ATP., Conclusion: Our findings reveal that Codonopsis pilosula polysaccharides protected PC12 cells from Aβ 1-40 -induced damage, suggesting that these components of the Codonopsis pilosula herb may represent an early treatment option for AD patients., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Neuritogenesis is the process underling nervous system regeneration; however, optimal extracellular signals that can promote neuronal regenerative activities require further investigation. Previously, we developed a novel method for inducing neuronal differentiation in rat PC12 cells using temperature-controlled repeated thermal stimulation (TRTS) with a heating plate. Based on neurogenic sensitivity to TRTS, PC12 cells were classified as either hyper- or hyposensitive. In this study, we aimed to investigate the mechanism of hyposensitivity by establishing two PC12-derived subclones according to TRTS sensitivity during differentiation: PC12-P1F1, a hypersensitive subclone, and PC12-P1D10, a hyposensitive subclone. To characterize these subclones, cell size and neuritogenesis were evaluated in subclones treated with nerve growth factor (NGF), bone morphogenetic protein (BMP), or various TRTS. No significant differences in cell size were observed among the parental cells and subclones. BMP4- or TRTS-induced neuritogenesis was increased in PC12-P1F1 cells compared to that in the parental cells, while no neuritogenesis was observed in PC12-P1D10 cells. In contrast, NGF-induced neuritogenesis was observed in all three cell lines. Furthermore, a BMP inhibitor, LDN-193189, considerably inhibited TRTS-induced neuritogenesis. These results suggest that the BMP pathway might be required for TRTS-induced neuritogenesis, demonstrating the useful aspects of these novel subclones for TRTS research.

Drug discovery from complex mixtures, like Chinese herbs, is challenging and extensive false positives make it difficult to obtain compounds with anti-Alzheimer's activity. In this study, a continuous method comprised of accelerated solvent extraction coupled with online two-dimensional countercurrent chromatography was developed for the efficient, scaled-up extraction and separation of six bioactive compounds from Citrus limon peels: neoeriocitrin, isonaringin, naringin, hesperidin, neohesperidin, and limonin. These active compounds were isolated and purified from the raw plant materials by two-dimensional countercurrent chromatography separation via two sets of an n-hexane/n-butanol/methanol/water solvent system: 0.23:1.00:0.25:1.13 and 0.47:1.00:0.38:1.46, v/v/v/v. The compounds were collected in yields of 0.22, 0.25, 0.10, 0.31, 0.29, and 0.28 mg/g, respectively, with purities of 95.79, 96.47, 97.69, 97.22, 98.11, and 98.82%, respectively. Subsequently, a simple and efficient in vitro method was developed for rapidly evaluating the acetylcholinesterase inhibitory activities of six bioactive components. Furthermore, the PC12 cell model and the in vitro metabolism of cytochromes P450 were employed to verify the monomers obtained from the continuous method. The results demonstrated that these six bioactive extracts from the C. limon peels were strong acetylcholinesterase inhibitors., (© 2020 Wiley-VCH GmbH.)

Ferulic acid (FA) has been shown to have a neuroprotective effect on Alzheimer's disease induced by amyloid-beta (Aβ) neurotoxicity. This work aims to ascertain the structure-activity relationship of FA and its alkyl esters (FAEs) for evaluating the antioxidant activities in PC12 cells and Aβ 1-42 aggregation inhibitory activities in vitro, as well as the signaling mechanisms against oxidative stress elicited by Aβ 1-42 in PC12 cells. Our data showed that alterations in the subcellular localization and cytotoxicity of FAEs caused by the lipophilicity of FA were crucial when evaluating their antioxidant capacities. Pre-treating cells with butyl ferulate (FAC4) significantly attenuated Aβ 1-42 -evoked intracellular ROS formation. Besides, FAC4 exhibited the highest Aβ 1-42 aggregation inhibitory effectiveness. The molecular docking results showed that FAC4 binds to amide NH in Gln15 and Lys16 via a hydrogen bond. Notably, FAC4 could upregulate antioxidant defense systems by modulating the Keap1-Nrf2-ARE signaling pathway. Identification of the functions of FAEs could be useful in developing food supplements or drugs for treating AD.

The PC12 cell line is one of the most commonly used in neuroscience research, including studies on neurotoxicity, neuroprotection, neurosecretion, neuroinflammation, and synaptogenesis. Two types of this line are available in the ATCC collection: traditional PC12 cells grown in suspension and well-attached adherent phenotype. PC12 cells grown in suspension tend to aggregate and adhere poorly to non-coated surfaces. Therefore, it is necessary to modify the surface of culture vessels. This paper aims to characterise the use of two distinct variants of PC12 cells as well as describe their differentiation and neuronal outgrowth with diverse NGF concentrations (rat or human origin) on various surfaces. In our study, we evaluated cell morphology, neurite length, density and outgrowth (measured spectrofluorimetrically), and expression of neuronal biomarkers (doublecortin and NeuN). We found that the collagen coating was the most versatile method of surface modification for both cell lines. For adherent cells, the coating was definitely less important, and the poly-d-lysine surface was as good as collagen. We also demonstrated that the concentration of NGF is of great importance for the degree of differentiation of cells. For suspension cells, we achieved the best neuronal characteristics (length and density of neurites) after 14 days of incubation with 100 ng/mL NGF (change every 48 h), while for adherent cells after 3-5 days, after which they began to proliferate. In the PC12 cell line, doublecortin (DCX) expression in the cytoplasm and NeuN in the cell nucleus were found. In turn, in the PC12 Adh line, DCX was not expressed, and NeuN expression was located in the entire cell (both in the nucleus and cytoplasm). Only the traditional PC12 line grown in suspension after differentiation with NGF should be used for neurobiological studies, especially until the role of the NeuN protein, whose expression has also been noted in the cytoplasm of adherent cells, is well understood.

Zinc, a suspected potentiator of learning and memory, is shown to affect exocytotic release and storage in neurotransmitter-containing vesicles. Structural and size analysis of the vesicular dense core and halo using transmission electron microscopy was combined with single-cell amperometry to study the vesicle size changes induced after zinc treatment and to compare these changes to theoretical predictions based on the concept of partial release as opposed to full quantal release. This powerful combined analytical approach establishes the existence of an unsuspected strong link between vesicle structure and exocytotic dynamics, which can be used to explain the mechanism of regulation of synaptic plasticity by Zn 2+ through modulation of neurotransmitter release., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Neuronal cell death induced by amyloid-β (Aβ) oligomers is implicated in neuronal degeneration and is a leading cause of Alzheimer's disease (AD). Therefore, to identify effective therapeutic agents for AD, we investigated the neuroprotective effects of two naturally occurring retinoid X receptor (RXR) agonists (SPF1 and SPF2), isolated from the root of Sophora tonkinensis Gagnep., on the Aβ 25-35 -induced cytotoxicity against nerve growth factor-differentiated rat pheochromocytoma (PC12) cells. Pretreatment with SPFs significantly prevented Aβ 25-35 -induced apoptosis in PC12 cells, similarly to the synthetic RXR agonist bexarotene. These effects were blocked by the RXR antagonist PA452. When the effects of SPFs were studied in the presence of the liver X receptor (LXR) agonist T0901317, the protective effects of SPFs were enhanced, suggesting that RXR/LXR heterodimers may play a key role in the neuroprotective effects of SPFs. SPFs and T0901317 induced ATP-binding cassette transporter 1 (ABCA1) protein expression in PC12 cells when administered alone or in combination. Intriguingly, a functional inhibitor of ABCA1 cyclosporine A negated the neuroprotective effects of SPFs or T0901317. Taken together, these results demonstrate that the RXR agonists SPF1 and SPF2 protect PC12 cells from Aβ 25-35 -induced neurotoxicity in an RXR-dependent manner and that their effects are markedly enhanced by the LXR agonist T0901317, in part related to ABCA1 function. These results suggest a novel approach to the treatment or prevention of AD.

Objective: To investigate the regulation of AMPK-mTOR signal transduction pathway in paraquat-induced autophagy of pheochromocytoma cells (PC12) . Methods: The PC12 cell were treated with terminal concentrations of 0, 25, 50, 100, 200, 300 and 400 μmol/L PQ for 24 hours, and the cells were induced by 300 μmol/L PQ for different time (6, 12, 24, 48 h) . MTT was used to detect the relative survival rate of cells, and the dose/time-effect relationship was determined respectively. The cells were treated with PQ at concentrations of 0, 100, 200 and 300 μmol/L PQ for 24 hours, the lactate dehydrogenase (LDH) activity in the culture supernatant was detected by spectrophotometry. The expression and distribution of autophagic lysosomes were observed by MDC staining. The intracellular reactive oxygen species (ROS) was detected by dichlorofluorescein diacetate (DCFH-DA) . The expression of microtubule-related protein 1 light chain 3 (LC3) was measured by immunofluorescence. The protein level of LC3Ⅱ, p62, Beclin1 and p-AMPK, p-mTOR were detected by Western blot. Results: Compared with the control group, the cell survival rate of the 100, 200, 300, 400 μmol/L PQ group decreased significantly, and showed a dose-dependent pattern ( P <0.05) . The survival rate of cells treated with 300 μmol/L PQ decreased significantly with the prolongation of exposure time (12, 24, 48 h) ( P <0.05) . Compared with the control group, the activity of LDH in 100, 200, 300 μmol/L PQ-treated group were significantly higher while The fluorescence intensity of ROS was significantly increased ( P <0.05) . MDC staining showed the density of autophagic lysosomes and fluorescence intensity in PQ-treated group significantly decreased ( P <0.05) . Immunofluorescence results showed the LC3 fluorescence intensity of PQ-treated group decreased which was consistent with MDC staining results. Western blot showed that compared with the control group, the expression levels of autophagy related proteins LC3Ⅱand Beclin1 in PQ-treated group were significantly lower, while the expression level of p62 protein was higher ( P <0.05) . p-AMPK protein level decreased and p-mTOR protein expression increased in 200 and 300μmol/L PQ-treatd groups, with statistically significant difference ( P <0.05) . Conclusion: AMPK-mTOR signaling pathway played a regulatory role in PQ-induced decreased autophagy of PC12 cell.

Amphetamine (AMPH) abuse can influence neuropsychiatric disorders and cell apoptosis by interfering with the protein kinase B/ glycogen synthase kinase 3 beta (AKT/GSK3β) pathway. However, the mechanisms underlying this regulation are poorly understood. Using PC12 cells, we found that AMPH inhibited AKT and GSK-3β phosphorylation levels and increased total GSK-3β levels. Furthermore, AMPH caused an increase in the activity of protein phosphatase 2 (PP2A), a signaling protein upstream of AKT, which in turn inhibited phosphorylated AKT levels. Okadaic acid, a PP2A inhibitor, protected PC12 cells against AMPH-induced apoptosis. Together, our results suggest that the PP2A/AKT/GSK3β pathway plays an important role in AMPH-induced neurotoxicity.

Hydroxylated polychlorobiphenyls (OH-PCBs) are major metabolites of PCBs that are widely distributed in the environment. While the effects of penta- to hepta-chlorinated OH-PCBs on neuronal differentiation have been widely reported, those of lower chlorinated OH-PCBs have not been extensively studied. To investigate the effects of lower chlorinated OH-PCBs on neuronal development, we studied the effects of mono- to hexa-chlorinated OH-PCBs on PC12 cells. Morphological changes were examined using an automatic system IN Cell Analyzer. Seventeen of the 20 OH-PCBs investigated promoted neuronal elongation in an OH-PCB concentration-dependent manner, while three OH-PCB congeners suppressed neuronal elongation based on Dunnett's analysis. In particular, the top five OH-PCBs (4OH-PCB2, 4'OH-PCB3, 4'OH-PCB25, 4'OH-PCB68, and 4'OH-PCB159), which have hydroxyl groups at the para-position and chlorine substitutions at the 2, 4, or 3' positions, significantly promoted neuronal elongation. Moreover, these neuronal elongations were suppressed by U0126, and phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 was observed in PC12 cells treated with 4OH-PCB2, 4'OH-PCB25, and 4'OH-PCB159. Taken together, our results indicate that the effect of OH-PCB on neuronal development is not dependent on the number of chlorine groups but on the chemical structure, and the mitogen-activated kinase kinase (MEK)-ERK1/2 signaling pathway is involved in this process.

Levodopa (L-dopa) remains the best treatment for Parkinson's disease (PD). However, long-term L-dopa treatment induces dyskinesia. The mechanism of L-dopa-induced dyskinesia (LID) is not fully understood. Enhanced activity of protein kinase A (PKA) and pulsatile dopamine (DA) stimulation plays an important role in LID. Tyrosine hydroxylase (TH) is the rate-limiting enzyme for DA synthesis. Decreased TH activity causes reduced pulsatile DA stimulation, which in turn reduces LID. Moreover, TH is a substrate of CaMKII. However, it is unknown whether inhibition of CaMKII reduces LID by downregulating the activity of TH. In this study, we found that CaMKII antagonist KN-93 reduced DA released in PC12 cells; in the meantime, KN-93 reduced phosphorylated levels of CaMKIIα and TH at Ser 40. Intrastriatal administration of KN-93 reduced LID without affecting the antiparkinsonian effect of L-dopa in PD mice. Mechanistically, KN-93 treatmentreduced phosphorylated CaMKIIα levels and subsequently downregulated phosphorylated TH at Ser 40 expression. Consequently, extracellular DA efflux was reduced andthe activation threshold of the PKA pathway was lowered. Moreover, KN-93 treatment reduced the expression of Arc and Penk, two immediate early genes, induced by chronic L-dopa. These data indicate that inhibition of CaMKIIα decreases LID at least partially by suppressing TH activity and subsequently reducing extracellular DA efflux and the activity of the PKA pathway, suggesting that CaMKIIα may be an alternative target for the treatment of LID., (Copyright © 2018 Elsevier B.V. All rights reserved.)

This study aimed to explore the efficacy of propofol to treat malignant pheochromocytoma (PCC) in vitro and in vivo. In vitro, PC12 cells were treated with different concentrations of propofol (0, 1, 5, and 10 μg/mL) for specific times followed by a MTT assay to detect cell proliferation. Transwell assays were performed to assess the function of propofol on the migration and invasion of PC12 cells, and flow cytometry to analyze cell apoptosis and cell cycle progression. Quantitative real-time polymerase chain reaction was carried out to analyze the expression level of mRNA (Bcl-2, Bax, and CyclinE). The levels of Bcl-2, Bax, CyclinE, FOXO1, FOXO3, Bim, procaspase-3, and active caspase-3 were determined by western blotting. In vivo, the effects of propofol on PCC tumor growth were detected by transplanted mouse model. Transferase dUTP nick-end labeling was performed to detect tissue cell apoptosis. The results indicated that propofol inhibited PC12 cell proliferation, prevented cell migration and invasion, and induced the apoptosis of PC12 cells in a dose- and time-dependent manner. Propofol treatment increased the expression of Bax and decreased that of Bcl-2. In addition, propofol significantly induced the G1/S phase arrest in PC12 cells, and the expression of Cyclin E was reduced. Moreover, the levels of FOXO1, FOXO3, Bim, procaspase-3, and active caspase-3 were enhanced by propofol treatment. In vivo, propofol treatment significantly reduced the PCC tumor growth and induced tissue cell apoptosis. In conclusion, propofol has potent anti-PCC activity in vitro and in vivo, and is a potential small-molecule drug for treating malignant PCC.

Huntington's disease (HD) is a monogenic inherited polyglutamine-mediated neurodegenerative disorder for which effective therapies are currently unavailable. Neuropeptide Y (NPY) has been implicated as a potential therapeutic target in several neurodegenerative diseases, including HD. However, its mechanisms of action in the context of HD pathology remain unknown. Here, we investigated the beneficial effects of Y2 receptor (Y2R) activation with NPY or Y2R selective agonist NPY 13-36 in the R6/2 mouse and PC12 cell models of HD. Also, we explored the effects of selective pharmacological blockage of Y2R using selective non-peptide small molecule Y2R antagonist SF31 in vivo and in vitro. Our results showed that activation of Y2R with intranasal NPY or NPY 13-36 led to an improved motor function in R6/2 mice as revealed by rotarod performance, vertical pole test, and hindlimb clasping behaviour. Also, intranasal NPY or NPY 13-36 led to a decrease in aggregated mHtt and mediated increase in dopamine and cAMP-regulated phosphoprotein, 32kDa (DARPP-32), brain-derived neurotrophic factor (BDNF), and activated extracellular signal-regulated protein kinases (pERK1/2) levels in R6/2 mice. Intranasal NPY or NPY 13-36 had no effect on body weight but showed positive effects on survival in R6/2 mice. Furthermore, intranasal NPY or NPY 13-36 attenuated induction of proinflammatory cytokine and inflammatory mediators in R6/2 mice. In contrast, antagonizing by using SF31 exacerbates phenotypic severity in R6/2 mice and treatment effects with either intranasal NPY or NPY 13-36 were significantly blocked . In vitro, using inducible PC12/Htt Q103-EGFP cells, treatment with NPY or NPY 13-36 protected against mHtt-mediated neuromorphological defects (neurite length and soma area) and neurotoxicity but had no effect on mHtt inclusion body formation. Conversely, co-treatment with SF31 significantly inhibited these effects. Together, our findings extend previous evidence of the beneficial effects of NPY in R6/2 mice, and more importantly, suggest that targeted activation of Y2R receptor might be a promising disease-modifying target for HD and other neurodegenerative diseases., (Copyright © 2018 Elsevier Inc. All rights reserved.)

6'- O -galloylpaeoniflorin (GPF), a galloylated derivative of paeoniflorin isolated from peony root, has been proven to possess antioxidant potential. In this present study, we revealed that GPF treatment exerted significant neuroprotection of PC12 cells following OGD, as evidenced by a reduction of oxidative stress, inflammatory response, cellular injury, and apoptosis in vitro. Furthermore, treatment with GPF increased the levels of phosphorylated Akt (p-Akt) and nuclear factor-erythroid 2-related factor 2 (Nrf2), as well as promoted Nrf2 translocation in PC12 cells, which could be inhibited by Ly294002, an inhibitor of phosphoinositide 3-kinase (PI3K). In addition, Nrf2 knockdown or Ly294002 treatment significantly attenuated the antioxidant, anti-inflammatory, and antiapoptotic activities of GPF in vitro . In vivo studies indicated that GPF treatment significantly reduced infarct volume and improved neurological deficits in rats subjected to CIRI, as well as decreased oxidative stress, inflammation, and apoptosis, which could be inhibited by administration of Ly294002. In conclusion, these results revealed that GPF possesses neuroprotective effects against oxidative stress, inflammation, and apoptosis after ischemia-reperfusion insult via activation of the PI3K/Akt/Nrf2 pathway.

The stinging test is an in vivo protocol that evaluates sensitive skin using lactic acid (LA). A soothing sensation of cosmetics or ingredients can be also appreciated through a decrease in stinging score. To predict the soothing sensation of a product before in vivo testing, we developed a model based on an LA test and substance P (SP) release using a co-culture of human keratinocytes and NGF-differentiated PC12 cells. A bacterial fucose-rich polysaccharide present in Fucogel ® was evaluated as the soothing molecule in the in vivo stinging test and our in vitro model. Excluding toxic concentrations, the release of SP was significant from 0.2% of lactic acid for the PC12 cells and from 0.1% of lactic acid for the keratinocytes. When the pH was adjusted to approximately 7.4, LA did not provoke SP release. At these concentrations of LA, 0.1% of polysaccharide showed a significant decrease in SP release from the two cellular types and in co-cultures without modifying the pH of the medium. In vivo, a stinging test using the polysaccharide showed a 30% decrease in prickling intensity vs the placebo in 19 women between the ages of 21 and 69. Our in vitro model is ethically interesting and is adapted for cosmetic ingredients screening because it does not use animal experimentation and limits human volunteers. Moreover, Fucogel ® reduced prickling sensation as revealed by the in vivo stinging test and inhibits the neurogenic inflammation as showed by our new in vitro stinging test based on SP release., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Decursin, the major bioactive component of Angelica gigas Nakai, exhibited neuroprotective properties. Our previous studies showed that decursin conferred neuroprotective effects in PC12 cells induced by Amyloid-β (Aβ) 25-35 via antiapoptosis and antioxidant. In this study, the antiinflammatory effects of decursin against PC12 cells injury stimulated by Aβ 25-35 were assessed. Our results demonstrated that decursin suppressed the expression of cyclooxygenase-2 protein and prostaglandin E2 content which was stimulated by Aβ 25-35 in PC12 cells. Meanwhile, the nuclear translocation of nuclear factor-κB in Aβ 25-35 -treated PC12 cells was also inhibited by decursin. In addition, decursin suppressed phosphorylation of the two upstream pathway kinases, p38 and c-Jun N-terminal kinase. Overall, our findings indicate that decursin exerts protective effects against neuroinflammation stimulated by Aβ 25-35 in PC12 cells by abolishing cyclooxygenase-2 protein expression through inactivation of nuclear factor-κB via the upstream kinases including p38 and c-Jun N-terminal kinase. This work provides a new insight into the pharmacological mode of decursin and should facilitate its therapeutic application in treatment of inflammatory disorders., (Copyright © 2017 John Wiley & Sons, Ltd.)

Neurodegenerative diseases (NDD) are typically associated with neuron loss in nervous system areas. Interventions with related death mechanisms may ameliorate NDD progression. Oxidative stress plays an important role in NDD cell death routines. However, tert-butylhydroperoxide (t-BHP), a widely used oxidative stress stimulus, induces neural cell death through a mechanism that remains elusive. In our study, the ferroptosis marker events occurred after co-treatment with 100 μM t-BHP for 1 h, all of which were reversed in the presence of the ferroptosis inhibitor ferrostatin-1 (Fer-1) and the iron chelator deferoxamine, implying the occurrence of ferroptosis. Moreover, mitochondrial dysfunction accompanied by a decreased in membrane potential and ATP production, increased mitochondrial ROS generation. Furthermore, this mitochondrial dysfunction could be reversed by Fer-1. In addition, JNK1/2 and ERK1/2 were activated upstream of the ferroptosis and mitochondrial dysfunction. In summary, these data suggest that ferroptosis, coupled with mitochondrial dysfunction, was involved in t-BHP-induced PC12 death. JNK1/2 and ERK1/2 played important roles in t-BHP-induced cell death. Overall, this study might provide clues to the oxidative stress-based strategies for cell protection in NDD.

Diffusible amyloid-β (Aβ) oligomers are currently presumed to be the most cytotoxic Aβ assembly and held responsible to trigger the pathogenesis of Alzheimer's disease (AD). Thus, Aβ oligomers are a prominent target in AD drug development. Previously, we reported on our solely D-enantiomeric peptide D3 and its derivatives as AD drug candidates. Here, we compare one of the most promising D3 derivatives, ANK6, with its tandem version (tANK6), and its head-to-tail cyclized isoform (cANK6r). In vitro tests investigating the D-peptides' potencies to inhibit Aβ aggregation, eliminate Aβ oligomers, and reduce Aβ-induced cytotoxicity revealed that all three D-peptides efficiently target Aβ. Subsequent preclinical pharmacokinetic studies of the three all-D-peptides in wildtype mice showed promising blood-brain barrier permeability with cANK6r yielding the highest levels in brain. The peptides' potencies to lower Aβ toxicity and their remarkable brain/plasma ratios make them promising AD drug candidates.

Background/aim: Most of the previous investigators have used various types of media for the culture of nerve cells. In order to optimize the culture conditions, we compared the growth rate and amino acid consumption by two popular neuron models, rat PC12 and human SH-SY5Y, grown in DMEM or DMEM: Ham's F-12 (1:1): non-essential amino acids, supplemented with 10% fetal bovine serum (referred to DMEM and Mix, respectively)., Materials and Methods: Cell growth was monitored by the MTT method. Amino acids in the culture medium were quantitated by amino acid analysis after deproteinization., Results: Efficient cell attachment could be achieved even if PC12 cells were inoculated at extreme lower cell density in a non-coated plain dish, without addition of its condition medium. Both PC12 and SH-SY5Y cells proliferated up to slightly higher cell density in DMEM than in Mix. Approximately 2-fold higher utilization rate of glutamine and essential amino acids was observed in DMEM. Amyloid peptides such as Aβ 1-42 and Aβ 25-35 suppressed their growth nearly by 50%., Conclusion: The present study suggests the usefulness of DMEM for the study of searching neuroprotective substances, based on its favorable effects on cell attachment, cell growth and amino acid utilization as well as amyloid peptide sensitivity., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Inflammation plays critical roles in the pathogenic mechanisms of several neurodegenerative disorders including Alzheimer's disease (AD). Previous study revealed that CoQ10 augmented cellular antioxidant defense capacity, thereby protecting PC12 cells from oxidative neurotoxicity. However, the mechanism by which CoQ10 inhibits inflammation remains unknown. In this study, we aim to examine the effects of CoQ10 on Aβ 25-35 -induced inflammatory in PC12 cells and the underlying molecular mechanism of its neuroprotective action. CoQ10 suppressed the protein expression of COX-2 and the level of PGE2 in Aβ 25-35 -injured PC12 cells. These inhibitions appeared to correlate with the suppression of NF-κB activation by CoQ10, as pretreating PC12 cells with CoQ10 blocked the translocation of NF-κB into the nuclear compartment and degradation of the inhibitory subunit IκB. Overall, these results implied that CoQ10 attenuated neuroinflammatory responses through the inactivation of NF-κB dependent inflammatory pathways in Aβ 25-35 -induced PC12 cells. Therefore, CoQ10 may have therapeutic potential for neurodegenerative diseases by inhibiting pro-inflammatory mediators production., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Silymarin (SM) is a well-known antioxidant, anti-inflammatory and anti-cancer compound extracted from the milk thistle. Here, we investigated the protective effect of SM against acrylamide (AA)-induced neurotoxicity, mainly caused by oxidative stress, via activation of the nuclear transcription factor E2-related factor 2 (Nrf2) signalling pathway in PC12 cells. The MTT reduction assay was used to measure cell viability in various drug-treated groups and demonstrated that SM could increase cell viability in AA-treated PC12 cells. We then measured the reactive oxygen species (ROS) levels by the peroxide-sensitive fluorescent probe DCFH-DA and intracellular glutathione (GSH) and malondialdehyde (MDA) levels by absorption spectrophotometry. Our data revealed that SM could reduce ROS and MDA levels and increase GSH levels in AA-induced PC12 cells. To identify a potential mechanism for SM-induced protection, we measured the mRNA and protein expression levels of Nrf2 and its downstream target antioxidants glutathione peroxidase (Gpx), glutamate cysteine ligase catalytic subunit (GCLC) and glutamate cysteine ligase modifier subunit (GCLM) by quantitative real-time PCR and Western blot, respectively. The results suggested that SM could activate Nrf2 signalling and increase the expression of Nrf2, Gpx, GCLC and GCLM in AA-treated PC12 cells. In conclusion, SM can effectively alleviate AA-induced neurotoxicity in PC12 cells., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

We have reported recently that the proliferation of PC12 cells exposed to micromolar concentrations of Tl(I) or Tl(III) has different outcomes, depending on the absence (EGF - cells) or the presence (EGF + cells) of epidermal growth factor (EGF) added to the media. In the current work, we investigated whether EGF supplementation could also modulate the extent of Tl(I)- or Tl(III)-induced cell apoptosis. Tl(I) and Tl(III) (25-100 μM) decreased cell viability in EGF - but not in EGF + cells. In EGF - cells, Tl(I) decreased mitochondrial potential, enhanced H 2 O 2 generation, and activated mitochondrial-dependent apoptosis. In addition, Tl(III) increased nitric oxide production and caused a misbalance between the anti- and pro-apoptotic members of Bcl-2 family. Tl(I) increased ERK1/2, JNK, p38, and p53 phosphorylation in EGF - cells. In these cells, Tl(III) did not affect ERK1/2 and JNK phosphorylation but increased p53 phosphorylation that was related to the promotion of cell senescence. In addition, this cation significantly activated p38 in both EGF - and EGF + cells. The specific inhibition of ERK1/2, JNK, p38, or p53 abolished Tl(I)-mediated EGF - cell apoptosis. Only when p38 activity was inhibited, Tl(III)-mediated apoptosis was prevented in EGF - and EGF + cells. Together, current results indicate that EGF partially prevents the noxious effects of Tl by preventing the sustained activation of MAPKs signaling cascade that lead cells to apoptosis and point to p38 as a key mediator of Tl(III)-induced PC12 cell apoptosis.

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

Objective: To study the effect of methamphetamine (METH) exposure on S-nitrosylation of protein disulphide isomerase and the neurotoxicity of METH in PC12 cells., Methods: PC12 cells were exposed to different concentrations of METH, and the cell viability was assessed using the cell-counting kit-8. PC12 cells exposed to METH in the presence of the NOS inhibitor N-nitro-L-arginine (L-NNA) were examined for cell viability and S-nitrosylation of protein disulphide isomerase using the biotin-switch method, and the changes in cell morphology were examined with HE staining., Results: METH exposure obviously decreased the cell viability and increased S-nitrosylation of protein disulphide isomerase, and the effect of METH was obviously inhibited by L-NNA treatment., Conclusion: METH can cause obvious neurotoxicity and promote S-nitrosylation of protein disulphide isomerase in PC12 cells.

We investigated the effects of 25Mg-Porphyrin-Fullerene nanoparticles, (25MgPMC16) smart ferroporphyrin nanoparticles, on PC12 cells exposed to oxygen-glucose deprivation/reperfusion. In order to explore its effect on cells under oxygen-glucose deprivation conditions, the cultures were pretreated with 25MgPMC16 24 hours prior to oxygen-glucose deprivation/reperfusion. To initiate the oxygen-glucose deprivation/reperfusion, the cell culture medium was replaced with a glucose-free medium and the cells were transferred to a humidified incubation chamber in a mixture of 95% N2 and 5% CO2 at 37° C for 30, 60 and 120 min. Cell viability was assessed by MTT assay. Exposure of PC12 cells to 30, 60 and 120 min oxygen-glucose deprivation significantly decreased the cell viability. Pretreatment of the cultures with 25MgPMC16 significantly increased cell viability in a concentration-dependent manner. Pretreatment, the cultures with MK-801 (10 µM), a non-competitive NMDA antagonist, has attenuated the cell death after 30 min oxygen-glucose deprivation. We concluded that 25MgPMC16 could protect PC12 cells against oxygen-glucose deprivation/reperfusion-induced cell injury in a concentration-dependent manner. That could be due to the effect of 25MgPMC16 on ATP synthesis and the antioxidant effects of its components.

Mg(2+) is an essential cation to maintain cellular functions, and intracellular Mg(2+) concentration ([Mg(2+)]i) is regulated by Mg(2+) channels and transporters. In our previous study, we demonstrated that MPP(+) elicits Mg(2+) influx across the cell membrane and Mg(2+) mobilization from mitochondria, and the resulting [Mg(2+)]i is an important determinants of the cell viability in MPP(+) model of Parkinson's disease (PD). It indicates that cellular Mg(2+) transport is one of the important factors to determine the progress of PD. However, whether the expression levels of Mg(2+) transport proteins change in the progress of PD has still been obscure. In this study, we estimated the mRNA expression levels of Mg(2+) transport proteins upon the exposure to MPP(+). In thirteen Mg(2+) transport proteins examined, mRNA expression level of SLC41A2 was increased and that of ACDP2, NIPA1 and MMgT2 were decreased. Knockdown of SLC41A2, ACDP2 or NIPA1 accelerated the MPP(+)-induced cell degeneration, and overexpression attenuated it. The decrease in the mRNA expression levels of NIPA1 and MMgT2 were also elicited by rotenone, H2O2 and FCCP, indicating that mitochondrial dysfunction related to this down-regulation. The increase in that of SLC41A2 was induced by an uncoupler, FCCP, as well as MPP(+), suggesting that it is an intrinsic protection mechanism against depolarized mitochondrial membrane potential and/or cellular ATP depletion. Our results shown here indicate that alteration of Mg(2+) transport proteins is implicated in the MPP(+) model of PD, and it affects cell degeneration., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Neuronal death is a pathophysiological process that is often caused by hypoxia/ischemia. However, the causes of hypoxia/ischemia-induced neuronal death are debated, and additional experimental data are needed to resolve this debate. In the present study, we applied oxygen-glucose deprivation (OGD) to PC12 cells and hippocampal neurons to establish a hypoxia/ischemia model. We evaluated the effects of OGD on cell death/apoptosis and on the levels of two excitatory amino acid neurotransmitters, aspartic acid and glutamic acid, in both hippocampal neurons and the medium used to culture the hippocampal neurons. We also evaluated GluR2 expression in hippocampal neurons as well as the effects of OGD on whole-cell potassium currents in PC12 cells and hippocampal neurons. Our experimental results showed that OGD significantly decreased cell viability and markedly enhanced apoptosis in PC12 cells and hippocampal neurons. OGD treatment for 3 h increased the levels of Asp and Glu in the medium used to culture hippocampal neurons, but decreased both the levels of Asp and Glu and GluR2 expression in hippocampal neurons. Furthermore, OGD altered the electrophysiological properties of voltage-dependent potassium channels in PC12 cells and hippocampal neurons in different ways; OGD decreased the voltage-dependent potassium current in PC12 cells, but increased this current in hippocampal neurons. On the basis of these results, we concluded that OGD enhanced neuronal cell death/apoptosis in addition to altering neuronal excitatory amino acid neurotransmitter signaling and whole-cell voltage-dependent potassium currents.

Background: Propofol (2,6-diisopropylphenol) is one of the most frequently used anesthetic agents. One of the main side effects of propofol is to reduce blood pressure, which is thought to occur by inhibiting the release of catecholamines from sympathetic neurons. Here, the authors hypothesized that propofol-induced hypotension is not simply the result of suppression of the release mechanisms for catecholamines., Methods: The authors simultaneously compared the effects of propofol on the release of norepinephrine triggered by high K-induced depolarization, as well as ionomycin, by using neuroendocrine PC12 cells and synaptosomes. Ionomycin, a Ca ionophore, directly induces Ca influx, thus bypassing the effect of ion channel modulation by propofol., Results: Propofol decreased depolarization (high K)-triggered norepinephrine release, whereas it increased ionomycin-triggered release from both PC12 cells and synaptosomes. The propofol (30 μM)-induced increase in norepinephrine release triggered by ionomycin was dependent on both the presence and the concentration of extracellular Ca (0.3 to 10 mM; n = 6). The enhancement of norepinephrine release by propofol was observed in all tested concentrations of ionomycin (0.1 to 5 μM; n = 6)., Conclusions: Propofol at clinically relevant concentrations promotes the catecholamine release as long as Ca influx is supported. This unexpected finding will allow for a better understanding in preventing propofol-induced hypotension.

Lead (Pb)-induced neurodegeneration and its link with widespread neurobehavioral changes are well documented. Experimental evidences suggest that ethanol could enhance the absorption of metals in the body, and alcohol consumption may increase the susceptibility to metal intoxication in the brain. However, the underlying mechanism of ethanol action in affecting metal toxicity in brain cells is poorly understood. Thus, an attempt was made to investigate the modulatory effect of ethanol on Pb intoxication in PC12 cells, a rat pheochromocytoma. Cells were co-exposed to biological safe doses of Pb (10 μM) and ethanol (200 mM), and data were compared to the response of cells which received independent exposure to these chemicals at similar doses. Ethanol (200 mM) exposure significantly aggravated the Pb-induced alterations in the end points associated with oxidative stress and apoptosis. The finding confirms the involvement of reactive oxygen species (ROS)-mediated oxidative stress, and impairment of mitochondrial membrane potential, which subsequently facilitate the translocation of triggering proteins between cytoplasm and mitochondria. We further confirmed the apoptotic changes due to induction of mitochondria-mediated caspase cascade. These cellular changes were found to recover significantly, if the cells are exposed to N-acetyl cysteine (NAC), a known antioxidant. Our data suggest that ethanol may potentiate Pb-induced cellular damage in brain cells, but such damaging effects could be recovered by inhibition of ROS generation. These results open up further possibilities for the design of new therapeutics based on antioxidants to prevent neurodegeneration and associated health problems.

Luteolin and apigenin are dietary flavones and exhibit a broad spectrum of biological activities including antioxidant, anti-inflammatory, anti-cancer and neuroprotective effects. The lipid peroxidation product 4-hydroxy-2-nonenal (4-HNE) has been implicated as a causative agent in the development of neurodegenerative disorders. This study investigates the cytoprotective effects of luteolin and apigenin against 4-HNE-mediated cytotoxicity in neuronal-like catecholaminergic PC12 cells. Both flavones restored cell viability and repressed caspase-3 and PARP-1 activation in 4-HNE-treated cells. Luteolin also mitigated 4-HNE-mediated LC3 conversion and reactive oxygen species (ROS) production. Luteolin and apigenin up-regulated 4-HNE-mediated unfolded protein response (UPR), leading to an increase in endoplasmic reticulum chaperone GRP78 and decrease in the expression of UPR-targeted pro-apoptotic genes. They also induced the expression of Nrf2-targeted HO-1 and xCT in the absence of 4-HNE, but counteracted their expression in the presence of 4-HNE. Moreover, we found that JNK and p38 MAPK inhibitors significantly antagonized the increase in cell viability induced by luteolin and apigenin. Consistently, enhanced phosphorylation of JNK and p38 MAPK was observed in luteolin- and apigenin-treated cells. In conclusion, this result shows that luteolin and apigenin activate MAPK and Nrf2 signaling, which elicit adaptive cellular stress response pathways, restore 4-HNE-induced ER homeostasis and inhibit cytotoxicity. Luteolin exerts a stronger cytoprotective effect than apigenin possibly due to its higher MAPK, Nrf2 and UPR activation, and ROS scavenging activity.

Phosphorylation and lipidation provide posttranslational mechanisms that contribute to the distribution of cytosolic proteins in growing nerve cells. The growth-associated protein GAP43 is susceptible to both phosphorylation and S-palmitoylation and is enriched in the tips of extending neurites. However, how phosphorylation and lipidation interplay to mediate sorting of GAP43 is unclear. Using a combination of biochemical, genetic, and imaging approaches, we show that palmitoylation is required for membrane association and that phosphorylation at Ser-41 directs palmitoylated GAP43 to the plasma membrane. Plasma membrane association decreased the diffusion constant fourfold in neuritic shafts. Sorting to the neuritic tip required palmitoylation and active transport and was increased by phosphorylation-mediated plasma membrane interaction. Vesicle tracking revealed transient association of a fraction of GAP43 with exocytic vesicles and motion at a fast axonal transport rate. Simulations confirmed that a combination of diffusion, dynamic plasma membrane interaction and active transport of a small fraction of GAP43 suffices for efficient sorting to growth cones. Our data demonstrate a complex interplay between phosphorylation and lipidation in mediating the localization of GAP43 in neuronal cells. Palmitoylation tags GAP43 for global sorting by piggybacking on exocytic vesicles, whereas phosphorylation locally regulates protein mobility and plasma membrane targeting of palmitoylated GAP43., (© 2014 Gauthier-Kemper et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Alterations in gut microbiome composition have an emerging role in health and disease including brain function and behavior. Short chain fatty acids (SCFA) like propionic (PPA), and butyric acid (BA), which are present in diet and are fermentation products of many gastrointestinal bacteria, are showing increasing importance in host health, but also may be environmental contributors in neurodevelopmental disorders including autism spectrum disorders (ASD). Further to this we have shown SCFA administration to rodents over a variety of routes (intracerebroventricular, subcutaneous, intraperitoneal) or developmental time periods can elicit behavioral, electrophysiological, neuropathological and biochemical effects consistent with findings in ASD patients. SCFA are capable of altering host gene expression, partly due to their histone deacetylase inhibitor activity. We have previously shown BA can regulate tyrosine hydroxylase (TH) mRNA levels in a PC12 cell model. Since monoamine concentration is known to be elevated in the brain and blood of ASD patients and in many ASD animal models, we hypothesized that SCFA may directly influence brain monoaminergic pathways. When PC12 cells were transiently transfected with plasmids having a luciferase reporter gene under the control of the TH promoter, PPA was found to induce reporter gene activity over a wide concentration range. CREB transcription factor(s) was necessary for the transcriptional activation of TH gene by PPA. At lower concentrations PPA also caused accumulation of TH mRNA and protein, indicative of increased cell capacity to produce catecholamines. PPA and BA induced broad alterations in gene expression including neurotransmitter systems, neuronal cell adhesion molecules, inflammation, oxidative stress, lipid metabolism and mitochondrial function, all of which have been implicated in ASD. In conclusion, our data are consistent with a molecular mechanism through which gut related environmental signals such as increased levels of SCFA's can epigenetically modulate cell function further supporting their role as environmental contributors to ASD.

Acrylamide is a neurological and reproductive toxicant in humans and laboratory animals; however, the neuron developmental toxicity of acrylamide remains unclear. The aims of this study are to investigate the cytotoxicity and neurite outgrowth inhibition of acrylamide in nerve growth factor (NGF)- or fibroblast growth factor 1 (FGF1)-mediated neural development of PC12 cells. MTS assay showed that acrylamide treatment suppresses NGF- or FGF1-induced PC12 cell proliferation in a time- and dose-dependent manner. Quantification of neurite outgrowth demonstrated that 0.5 mM acrylamide treatment resulted in significant decrease in differentiation of NGF- or FGF1-stimulated PC12 cells. This decrease is accompanied with the reduced expression of growth-associated protein-43, a neuronal marker. Moreover, relative levels of pERK, pAKT, pSTAT3 and pCREB were increased within 5-10 min when PC12 cells were treated with NGF or FGF1. Acrylamide (0.5 mM) decreases the NGF-induced activation of AKT-CREB but not ERK-STAT3 within 20 min. Similarly, acrylamide (0.5 mM) decreases the FGF1-induced activation of AKT-CREB within 20 min. In contrast to the NGF treatment, the ERK-STAT3 activation that was induced by FGF1 was slightly reduced by 0.5 mM acrylamide. We further showed that PI3K inhibitor (LY294002), but not MEK inhibitor (U0126), could synergize with acrylamide (0.5 mM) to reduce the cell viability and neurite outgrowth in NGF- or FGF1-stimulated PC12 cells. Moreover, acrylamide (0.5 mM) increased reactive oxygen species (ROS) activities in NGF- or FGF1-stimulated PC12 cells. This increase was reversed by Trolox (an ROS scavenging agent) co-treatment. Together, our findings reveal that NGF- or FGF1-stimulation of the neuronal differentiation of PC12 cells is attenuated by acrylamide through the inhibition of PI3K-AKT-CREB signaling, along with the production of ROS.

Rat pheochromocytoma PC 12 cells are known to develop features of dopaminergic neurons upon treatment with nerve growth factor. They express in part estrogen receptors α and β, and G-protein coupled receptor 30. Estrogens promote development of these cells and exert neuroprotective effects. Here we treated differentiated PC 12 cells with physiological concentrations of 17-β-estradiol. We observed with immunocytochemistry cytoplasmic staining for SHBG in a portion of these cells Double immunostaining for estrogen receptor-β revealed that some PC 12 cells contained both antigens. Numbers of estrogen receptor-β positive cells were significantly higher after estradiol treatment; an effect that was not altered by pretreatment of cultures with tamoxifen. With reverse transcriptase polymerase chain reaction we observed sex hormone binding globulin encoding transcripts indicating intrinsic expression of the steroid binding globulin. We conclude that estrogen treatment induces SHBG expression in differentiated PC12., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Polyglutamine (polyQ) amyloid fibrils are observed in disease tissue and have been implicated as toxic agents responsible for neurodegeneration in expanded CAG repeat diseases such as Huntington's disease. Despite intensive efforts, the mechanism of amyloid toxicity remains unknown. As a novel approach to probing polyQ toxicity, we investigate here how some cellular and physical properties of polyQ amyloid vary with the chirality of the glutamine residues in the polyQ. We challenged PC12 cells with small amyloid fibrils composed of either L- or D-polyQ peptides and found that D-fibrils are as cytotoxic as L-fibrils. We also found using fluorescence microscopy that both aggregates effectively seed the aggregation of cell-produced L-polyQ proteins, suggesting a surprising lack of stereochemical restriction in seeded elongation of polyQ amyloid. To investigate this effect further, we studied chemically synthesized D- and L-polyQ in vitro. We found that, as expected, D-polyQ monomers are not recognized by proteins that recognize L-polyQ monomers. However, amyloid fibrils prepared from D-polyQ peptides can efficiently seed the aggregation of L-polyQ monomers in vitro, and vice versa. This result is consistent with our cell results on polyQ recruitment but is inconsistent with previous literature reports on the chiral specificity of amyloid seeding. This chiral cross-seeding can be rationalized by a model for seeded elongation featuring a "rippled β-sheet" interface between seed fibril and docked monomers of opposite chirality. The lack of chiral discrimination in polyQ amyloid cytotoxicity is consistent with several toxicity mechanisms, including recruitment of cellular polyQ proteins., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Alzheimer's disease (AD) is a common neurodegenerative disorders, in which oxidative stress plays an important role. The present study investigated the effect of eicosapentaenoic acid-enriched phospholipids (EPA-enriched PL) from the sea cucumber Cucumaria frondosa on oxidative injury in PC12 cells induced by hydrogen peroxide (H2O2) and tert-butylhydroperoxide (t-BHP). We also studied the effect of EPA-enriched PL on learning and memory functions in senescence-accelerated prone mouse strain 8 (SAMP8) in vivo. Pretreatment with EPA-enriched PL resulted in an enhancement of survival in a dose-dependent manner in H2O2 or t-BHP damaged PC12 cells. EPA-enriched PL pretreatment could also reduce the leakage of lactate dehydrogenase (LDH), and increase the intracellular total antioxidant capacity (T-AOC) and superoxide dismutase (SOD) activity compared with the H2O2 or t-BHP group. The down-regulated Bcl-2 mRNA level and up-regulated Bax, Caspase-9, and Caspase-3 mRNA expression induced by H2O2 or t-BHP could be restored by EPA-enriched PL pretreatment. These results demonstrated that EPA-enriched PL exhibited its neuroprotective effects by virtue of its antioxidant activity, which might be achieved by inhibiting the mitochondria-dependent apoptotic pathway. The neuroprotective effect of EPA-enriched PL was also verified in vivo test: the EPA-enriched PL administration prevented the development of learning and memory impairments in SAMP8 mice. Our results indicated that EPA-enriched PL could offer an efficient and novel strategy to explore novel drugs or functional food for neuronprotection and cognitive improvement., (Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Western blot analysis demonstrated that PC-12 cells express monomeric and dimeric forms of serine racemase (m-SR, d-SR) and that 1321N1 cells express m-SR. Quantitative RT-PCR and functional studies demonstrated that PC-12 cells express homomeric and heteromeric forms of nicotinic acetylcholine receptors (nAChR) while 1321N1 cells primarily express the α7-nAChR subtype. The effect of nAChR agonists and antagonists on SR activity and expression was examined by following concentration-dependent changes in intracellular d-Ser levels and SR protein expression. Incubation with (S)-nicotine increased d-Ser levels, which were attenuated by the α7-nAChR antagonist methyllycaconitine (MLA). Treatment of PC-12 cells with mecamylamine (MEC) produced a bimodal reduction of d-Ser reflecting MEC inhibition of homomeric and heteromeric nAChRs, while a unimodal curve was observed with 1321N1 cells, reflecting predominant expression of α7-nAChR. The nAChR subtype selectivity was probed using α7-nAChR selective inhibitors MLA and (R,S)-dehydronorketamine and α3β4-nAChR specific inhibitor AT-1001. The compounds reduced d-Ser in PC-12 cells, but only MLA and (R,S)-dehydronorketamine were effective in 1321N1 cells. Incubation of PC-12 and 1321N1 cells with (S)-nicotine, MEC and AT-1001 did not affect m-SR or d-SR expression, while MLA and (R,S)-dehydronorketamine increased m-SR expression but not SR mRNA levels. Treatment with cycloheximide indicated that increased m-SR was due to de novo protein synthesis associated with phospho-active forms of ERK1/2, MARCKS, Akt and rapamycin-sensitive mTOR. This effect was attenuated by treatment with the pharmacological inhibitors U0126, LY294002 and rapamycin, which selectively block the activation of ERK1/2, Akt and mTOR, respectively, and siRNAs directed against ERK1/2, Akt and mTOR. We propose that nAChR-associated changes in Ca(2+) flux affect SR activity, but not expression, and that MLA and (R,S)-dehydronorketamine bind to allosteric sites on the α7-nAChR and promote multiple signaling cascades that converge at mTOR to increase m-SR levels., (Published by Elsevier Inc.)

Intracellular Ca(2+) levels are tightly regulated in the neuronal system. The loss of Ca(2+) homeostasis is associated with many neurological diseases and neuropsychiatric disorders such as Parkinson's, Alzheimer's, and schizophrenia. We investigated the mechanisms involved in intracellular Ca(2+) signaling in PC-12 cells. The stimulation of NGF-differentiated PC-12 cells with 3μM ATP caused an early Ca(2+) release followed by a delayed Ca(2+) release. The delayed Ca(2+) release was dependent on prior ATP priming and on dopamine secretion by PC-12 cells. Delayed Ca(2+) release was abolished in the presence of spiperone, suggesting that it is due to the activation of D2 dopamine receptors (D2R) by dopamine secreted by PC-12 cells. This was shown to be independent of PKA activation but dependent on PLC activity. An endocytosis step was required for inducing the delayed Ca(2+) release. Given the importance of calcyon in clathrin-mediated endocytosis, we verified the role of this protein in the delayed Ca(2+) release phenomenon. siRNA targeting of calcyon blocked the delayed Ca(2+) release, decreased ATP-evoked IP3R-mediated Ca(2+) release, and impaired subsequent Ca(2+) oscillations. Our results suggested that calcyon is involved in an unknown mechanism that causes a delayed IP3R-mediated Ca(2+) release in PC-12 cells. In schizophrenia, Ca(2+) dysregulation may depend on the upregulation of calcyon, which maintains elevated Ca(2+) levels as well as dopamine signaling., (© 2013.)

Real-time observation of intracellular process of signal transduction is very useful for biomedical and pharmaceutical applications as well as for basic research work of cell biology. The conventional methods used to observe intracellular reactions have not been convenient with several steps such as labeling and washing steps prior to the readout. Consequently, there is a critical need for label-free observation techniques for monitoring intracellular reactions. For feasible and reagentless observation of intracellular alterations in real time, we examined the use of a high-resolution two-dimensional surface plasmon resonance (2D-SPR) imager for monitoring of intracellular signal transduction that was mainly translocation of protein kinase C via local refractive index change in PC12 cells adhered on a gold sensor slide without any indicator reagent. PC12 cells were stimulated with KCl and phorbol-12-myristate-13-acetate (PMA, a protein kinase C [PKC] activator) at different concentrations in order to induce intracellular PKC translocation. 2D-SPR signal (reflection intensity change) is very consistent with the cellular response normally detected for these stimulants. Our results suggest that complex intracellular reactions could be real-time monitored and characterized by the 2D-SPR imager. It is further expected that signal transmission that was followed by the translocation of signaling proteins could be observed at the single cell level with the high-resolution 2D-SPR imager., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Rotenone, an inhibitor of mitochondrial complex I, has been widely regarded as a neurotoxin because it induces a Parkinson's disease-like syndrome. The fruit and root bark of Lycium chinense Miller have been used as traditional medicines in Asia to treat neurodegenerative diseases. In this study, we examined the neuroprotective effects of Lycium chinense Miller extracts in rotenone-treated PC12 cells. Treatment with rotenone reduced PC12 cell viability and cellular ATP levels. Conversely, caspase 3/7 activity, the ratio of Bax:Bcl-2 expression levels, mitochondrial superoxide level, and intracellular calcium (Ca(2+)) concentration were elevated. Pretreatment with Lycium chinense Miller extracts significantly increased cell viability and ATP levels. Additionally, they attenuated caspase activation, mitochondrial membrane depolarization and mitochondrial superoxide production. Moreover, confocal microscopy showed that the mitochondrial staining pattern was restored from that of extracts treated cells and that the increase in intracellular Ca (2+) level was blunted by treatment with the extracts. Our results suggest that Lycium chinense Miller extracts may have the possible beneficial effects in Parkinson's disease by attenuating rotenone induced toxicity.

A wide range of growth factors encode information into specific temporal patterns of MAP kinase (MAPK) and CREB phosphorylation, which are further decoded by expression of immediate early gene products (IEGs) to exert biological functions. However, the IEG decoding system remain unknown. We built a data-driven based on time courses of MAPK and CREB phosphorylation and IEG expression in response to various growth factors to identify how signal is processed. We found that IEG expression uses common decoding systems regardless of growth factors and expression of each IEG differs in upstream dependency, switch-like response, and linear temporal filters. Pulsatile ERK phosphorylation was selectively decoded by expression of EGR1 rather than c-FOS. Conjunctive NGF and PACAP stimulation was selectively decoded by synergistic JUNB expression through switch-like response to c-FOS. Thus, specific temporal patterns and combinations of MAPKs and CREB phosphorylation can be decoded by selective IEG expression via distinct temporal filters and switch-like responses. The data-driven modeling is versatile for analysis of signal processing and does not require detailed prior knowledge of pathways.

α-Synuclein (α-syn) is a presynaptic protein that is widely implicated in the pathophysiology of Parkinson's disease (PD). Recently, four α-syn isoforms that are produced by alternative splicing have been described, they are α-syn140, α-syn126, α-syn112, and α-syn98. The stable cell lines which expressed the four α-syn isoforms respectively were obtained, and the aggregation formation of these α-syn isoforms and their associated toxicity to PC12 cell were investigated. The results of this study indicate that over-expression of α-syn isoforms alone in dopaminergic cells have no effect on the formation of α-syn oligomeric species and cell viabilities. When exposed to rotenone, these cell lines which over expressed exon 5-lacking form of α-syn isoforms showed the formation of oligomeric species and toxicity to PC12 cells., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Ionic liquids have recently received considerable attention due to their negligible vapor pressure and substitute for conventional organic solvents. However, their solubility in water and a great deal of literature regarding their toxicity on aquatic organisms have caused much concern in recent years. This study aims to evaluate the cytotoxicity of 1-octyl-3-methylimidazolium chloride ([C(8)mim][Cl]) on the rat pheochromocytoma (PC12) cell line by cell viability assay and to determine the cytological alterations and damages in PC12 cells after 24h of exposure at the concentrations of 0.07, 0.14, and 0.28 mM of [C(8)mim][Cl]. The results show that [C(8)mim][Cl] inhibits PC12 cell growth and decreases their viabilities in a remarkable dose-dependent manner, and the 24h EC(50) of [C(8)mim][Cl] for PC12 cells is calculated to be around 0.56 mM. Our results also reveal that [C(8)mim][Cl]-exposure induces DNA damage, sustained increase of intracellular Ca(2+), overproduction of reactive oxygen species, gradually exhausted cellular ATP, mitochondrial permeability transition, and apoptosis in PC12 cells. We suppose that mitochondrial permeability transition and mitochondrial dysfunction maybe the major cytotoxicity mechanism of [C(8)mim]Cl for PC12 cells., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The activation of ATP-sensitive potassium (KATP) channels in PC12 cells play a pivotal role in protection against the neurotoxic effect of rotenone. However, it remains unclear why rotenone seems to preferentially affect activation of KATP channels and if this could affect its physiological activity. In this study, we sought to determine how the different energy states caused by various doses of rotenone affect the KATP opening state and whether the KATP opening state influences the expression of tyrosine hydroxylase (TH) which is related with DA synthesis. With patch clamp technology, results showed that treatment of PC12 cells with rotenone (0.2-1 µg/ml) for 15 min can cause KATP channel opening with significantly increased intracellular ROS production. Treatment with rotenone (2-16 ng/ml) for 24 h also caused the channels to open with gently increased ROS. In order to study if the rather long-term action on KATP channel opening states could affect the specified function of PC12 cells, the KATP channel opener pinacidil and the inhibitor glibenclamide were used to treat cells for 24 h, and the expression of TH was detected. Our results showed that treatment of PC12 cells with glibenclamide for 24 h can notably promote TH expression and can also enhance the expression of TH which were reduced by rotenone. These data indicate that the energy states in PC12 induced by various doses of rotenone could significantly influence the opening states of KATP channels. However long-term energy stress may raise the opening rate and opening sensitivity of this channel. In addition, our results demonstrate for the first time that activation of plasma membrane KATP channels induced by rotenone inhibits TH expression which influences DA synthesis in PC12 cells.

Background Aims: Immunoregulation of mesenchymal stromal cells (MSC) is more efficient at restoring biologic function of injured tissue than transdifferentiation during transplantation. However, the exact mechanisms and characteristics of MSC regarding immunomodulation are still unknown, especially in the damaged niche after hypoxic-ischemic insult. We investigated the anti-apoptotic actions of MSC against the neurotoxicity of hydrogen peroxide (H(2)O(2)) on pheochromocytoma (PC12) cells., Methods: To mimic hypoxic-ischemic brain injury in vivo, a relatively high H(2)O(2) concentration and short period were used to treat PC12 cells. MSC were co-cultured directly with the injured PC12 cells, and 5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazoly)-3-(4-sulfophenyl)tetrazolium, inner salt (MTS), lactose dehydrogenase (LDH) and nitric oxide (NO) assays, intracellular Ca(2+) and resting membrane potential (RMP) were analyzed. Apoptotic-associated genes and cytokine releases were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA)., Results: After exposure to H(2)O(2), the viability of PC12 cells was significantly decreased, while the levels of LDH and NO increased, resulting in intracellular Ca(2+) accumulation and cell apoptosis. Co-culture of MSC with the H(2)O(2)-treated PC12 cells sustained viability of the PC12 cells, reduced LDH levels and NO release, and improved Ca(2+) influx and cell apoptosis. The injured PC12 cells exhibited lower RMP following co-culture with MSC compared with the injured PC12 cells alone. The mRNA expression levels of Bcl-2 were up-regulated and caspase-3 levels down-regulated in the MSC co-culture groups. The release of interleukin (IL)-10 was gradually reduced by co-cultured MSC, while the release of IL-6 was sharply increased following MSC co-culture., Conclusions: These findings indicate that MSC have neuroprotective effects on suppressing cell apoptosis via regulation of the H(2)O(2)-impaired microenvironment, partly through IL-6 and IL-10 cytokine production.

Currently, there is no completely effective therapy available for metastatic phaeochromocytomas (PCCs) and paragangliomas. In this study, we explore new molecular targeted therapies for these tumours, using one more benign (mouse phaeochromocytoma cell (MPC)) and one more malignant (mouse tumour tissue (MTT)) mouse PCC cell line - both generated from heterozygous neurofibromin 1 knockout mice. Several PCC-promoting gene mutations have been associated with aberrant activation of PI3K/AKT, mTORC1 and RAS/RAF/ERK signalling. We therefore investigated different agents that interfere specifically with these pathways, including antagonism of the IGF1 receptor by NVP-AEW541. We found that NVP-AEW541 significantly reduced MPC and MTT cell viability at relatively high doses but led to a compensatory up-regulation of ERK and mTORC1 signalling at suboptimal doses while PI3K/AKT inhibition remained stable. We subsequently investigated the effect of the dual PI3K/mTORC1/2 inhibitor NVP-BEZ235, which led to a significant decrease of MPC and MTT cell viability at doses below 50 nM but again increased ERK signalling. Accordingly, we next examined the combination of NVP-BEZ235 with the established agent lovastatin, as this has been described to inhibit ERK signalling. Lovastatin alone significantly reduced MPC and MTT cell viability at therapeutically relevant doses and inhibited both ERK and AKT signalling, but increased mTORC1/p70S6K signalling. Combination treatment with NVP-BEZ235 and lovastatin showed a significant additive effect in MPC and MTT cells and resulted in inhibition of both AKT and mTORC1/p70S6K signalling without ERK up-regulation. Simultaneous inhibition of PI3K/AKT, mTORC1/2 and ERK signalling suggests a novel therapeutic approach for malignant PCCs.